DOI

Received: May 2022 – Accepted: July 2022

¹ Semmelweis University, Faculty of Health Sciences, Department of Dietetics and Nutritional Sciences

Keywords

polyphenolic compounds, diabetes, medicinal food, estimated glomerular filtration rate (eGFR), DPPH, ABTS, ORAC, FRAP, CUPRAC, Folin-Ciocalteu methods, gallic acid equivalent, catechin equivalent

2. Introduction

Bees have been around for 125 million years, and their evolutionary success has allowed them to become a perennial species that can utilize virtually every habitat on Earth. This ability to survive is largely due to the chemical composition and application of the special products they produce (honey, beeswax, venom, propolis, pollen and royal jelly). Propolis, the bees’ remedy against pathogenic microorganisms, has been used by mankind as a medicine since ancient times [1].

Propolis, known in Hungarian as bee glue, is a sticky, resinous substance, produced by bees (Apis mellifera L.) from beeswax, saliva and sap from the bark, buds and leaves of trees [2, 3]. They collect mainly from poplar, but also from birch, willow, horse chestnut, pine, oak, elm and alder [4]. The composition of propolis is mostly made up of plant resins, waxes, essential oils and pollen. In addition to these, it also contains smaller amounts of other substances, such as compounds partially produced by bees [2].

Bees use propolis in the hive many ways, including for disinfection, construction and maintenance of the hive, and for protection [2, 4, 5, 6], as well as to keep the humidity and temperature in the hive stable throughout the year, to seal holes and cracks and the inner wall of the hive. Propolis is also an important element of the so-called social immune system of honeybees, which, thanks to its antipathogenic (antimicrobial) properties, provides a certain general protection to the entire bee family against infections and parasites [8, 9].

3. Characteristics of propolis

The physical and chemical components of propolis, its quality and the possibilities of using it for physiological and medicinal purposes depend on the origin of the propolis, i.e., the climate, the botanical source and the species of the bees [4, 10]. The color of the product also depends on the origin, it is usually brown, but at the same time all the shades from yellow to black appear in it, in many cases with a reddish or greenish hue. The smell of propolis is aromatic, with notes of honey, resin, wax and vanilla mixed in it. Its taste is very characteristic [4].

Raw propolis typically consists of 50% plant resin, 30% wax, 10% essential and aromatic oils, 5% pollen, and 5% of other organic matter. More than 300 components have been identified in propolis, which differ depending on the source [9].

The compounds found in propolis include polyphenolic compounds (phenolic acids, flavonoids and their esters, e.g., caffeic acid phenyl ester), diterpenes, sesquiterpenes, lignans, aromatic aldehydes, alcohols, amino acids, fatty acids, organic acids, hydrocarbons, vitamins and minerals [9, 11].

The main bioactive components of propolis are flavonoids, which greatly contribute to the pharmacological effects of propolis. The amount of flavonoids is used as a criterion when evaluating the quality of temperate climate propolis. Flavonoids have a wide spectrum of biological properties, such as antibacterial, antiviral and anti-inflammatory effects [9].

Although volatile substances make up only 10% of the components of propolis, they are responsible for the characteristic resinous smell and contribute of the beneficial effects of propolis on health. Volatile substances are dominated by terpenoids, which play an important role in distinguishing good quality propolis from poor quality or counterfeit propolis, and also exhibit antioxidant, antimicrobial and other biological effects [9].

Although different bee species prefer different plants, even the chemical profile of propolis produced by the same species is not always the same. The composition of propolis varies by bee colony, location and season, and this makes it difficult to study it and make consistent health claims [12]. The protective properties of the bioactive substances found in propolis can also provide significant benefits in maintaining human health [5].

In recent years, several studies have confirmed that different propolis samples can be completely different in terms of chemical composition and biological activity [1, 7].

4. Propolis-containing products

A significant number of propolis-containing products are available on the market: medical and over-the-counter preparations, foods and drinks that help maintain health [7].

Propolis tincture is an extract of raw propolis made with a solvent (most often a mixture of water and ethanol). According to our knowledge, there are practical and application questions related to propolis tincture that should be answered and uniform regulations should be applied:

• Various preparation recipes are known;
• Soaking raw propolis for different lengths of time results in different tinctures;
• Differences in the extraction solvent (different amount and ethanol concentration) affect the composition of the preparations;
• The relationship between raw propolis and the composition of the tincture is not known.

In addition to tinctures, other propolis-containing foods are also available, such as lozenges, propolis honey, capsules filled with propolis extract [3].

In some countries, standardized propolis products with a constant bioactive substance concentration are already available [13].

5. Dose and safety

Clinical studies on mice and humans report that propolis and its constituents are generally well tolerated and non-toxic, except when used in very large amounts [5].

Determining the exact dose of propolis, on the basis of the studied population, the dosage regimen, compliance (accurate taking of the substance) and the purity of the product, faces significant difficulties, since the phenolic compounds found in propolis vary according to geographical origin, the bioactivity can also differ significantly, which makes it difficult to determine the correct dosage [14]. According to a particular study, based on previous animal experiments and applying a margin of safety, the safe dose of propolis for healthy humans is 70 mg/day [14].

Egy tanulmány szerint a korábbi állatkísérletek alapján és egy biztonsági tartalékot alkalmazva az egészséges emberek számára a propolisz biztonságos dózisa 70 mg/nap [15].

6. Physiological and therapeutic effects of propolis

Propolis has received increasing attention in recent years due to its beneficial effects on the human body. It is increasingly accepted as a preventive and therapeutic agent. However, the bioavailability of the useful substances found in propolis varies, which is also influenced by individual physiological conditions. According to a study, as a result of the consumption of propolis, its active ingredients can also be detected in the blood plasma [16].

6.1. Fighting infections, the immune system

Propolis can be considered as a potential medicinal food (“nutraceuticals”). Propolis extracts have anti-bacterial, antiviral and antifungal effects [3]. The immunoprotective and antioxidant properties of propolis are explained by its bioactive phytochemical components, regardless of its origin. A 2019 review cited immune system support as a health benefit of propolis [5].

The effect of propolis supplementation has also been studied among patients infected with the COVID-19 virus. In a recent, high quality (double-blind, placebo-controlled) study conducted in 2020, the effect of propolis on clinical symptoms was investigated. Infection was confirmed with a PCR test in participants aged 18 to 75. Members of the intervention group (n=40) received tablets containing 300 mg of Iranian green propolis extract three times a day for 2 weeks, while the control group (n=40) received no such treatment. The main result of the study was that the clinical symptoms of the disease improved faster in the group receiving propolis in terms of the duration and severity of the initial symptoms [17].

6.2. Cancerous diseases

Propolis has an antioxidant effect, which can be beneficial for the body in terms of neutralizing free radicals formed in excess [3], thus it can contribute to the regulation and control of inflammatory processes, tumor formation and aging processes. Its anti-inflammatory properties have been demonstrated in connection with propolis samples of Brazilian, Chinese and Malay origin. Its antitumor effect has been proven not only in in vitro, but also in vivo experiments taking place in living organisms) [3].

According to the results of another research, Brazilian red propolis had antioxidant properties and significantly reduced the percentage of survival of human tumor cells under laboratory conditions [10]. Alcoholic extracts of Turkish propolis also exhibited an inhibitory effect on the growth of tumor cells against human tumor cells (liver, colon, breast, cervix, prostate) [18].

The study, the aim of which was to find out whether propolis and the polyphenolic/flavonoid compounds contained in it can have an inhibitory effect on the growth of human bladder tumors in a cell culture, ended with promising results. Based on this, propolis may be suitable for auxiliary treatment of the disease in addition to surgery, to reduce or prevent the chance of tumor recurrence [19].

6.3. Diabetes

In relation to the effect of propolis on the human body, the reduction of blood sugar levels has also been studied [2]. According to the reliable, aggregated, comprehensive analysis of the results of several studies with similar objectives, the use of propolis reduced the fasting blood sugar level by 0.8 mmol/l compared to the subjects who did not receive treatment. In addition, taking propolis also reduced the value of HbA1c (A subunit of hemoglobin. The Ed.), which indicates the evolution of the blood sugar level of the examined person in retrospect over a period of 1 to 3 months. It is interesting to mention that the treatment did not affect the insulin level, so it follows that the drop in blood sugar level was not due to the effect of insulin. Almost 400 diabetic patients took part in the study, who were treated with 226 to 1,500 mg of propolis per day for 56 to 180 days. According to the authors, despite the positive results, further research is still needed regarding the type (composition) and dosage of propolis. This is so because the dose ranges were wide and the places of origin of the propolis samples used were varied [2]. In the studies related to propolis, it was stated that it is important to know the geographical and botanical origin, because they can affect the biological activity of the propolis, its effect and the composition of its organic components [3].

The objective of another study was to investigate the effect of Brazilian green propolis on type 2 diabetes patients through changes in blood test data. 80 people participated in the study, of which 39 received a placebo. The 41 people in the other group received 226.8 mg Brazilian green propolis per day during the 8-week period. The results indicate that Brazilian green propolis used in the aforementioned quantity and frequency can reduce then deterioration of uric acid levels and eGFR (estimated Glomerular Filtration Rate) values, which indicate kidney complications, in patients with type 2 diabetes [20].

In connection with the healing of diabetic leg ulcers, a favorable effect was reported in the study of Australian propolis samples. A favorable wound-healing role was also mentioned in connection with Chinese propolis extracts [3].

6.4. Cardiovascular diseases

In a human study published in 2017, changes in blood lipid levels were investigated as a result of oral application of propolis solutions. In the double-blind, placebo-controlled clinical trial, 35 of the 67 subjects received propolis, while 32 were given a placebo supplement (without propolis). In the propolis group, a significant increase in HDL (High Density Lipoprotein) was observed after 90 days. This effect may contribute to the reduction of the risk of cardiovascular diseases [21].

A 2019 review paper mentions lowering blood pressure as one of the health benefits of propolis. In this literature review, a total of 63 publications were reviewed, the majority of which were reports on animal experiments, but some key human studies were also included. According to the results, propolis can be an effective antioxidant and anti-inflammatory agent. Based on this, it is presumably effective against various chronic diseases, e.g., in preserving the health of the cardiovascular system, reducing atherosclerosis and reducing high blood pressure [5].

6.5. The skin and the nervous system

The components of propolis can be widely used to heal wounds and the human skin itself, and can also contribute to reducing the symptoms of some nervous system diseases (Alzheimer’s disease, Parkinson’s disease) [5].

In addition to research related to nervous system diseases, the protective effects of propolis on retinal cells have also been reported [22]. Propolis can also be used to prevent various eye diseases, such as macular degeneration in the aging population and myopia in the younger generation, but further studies are needed to prove this [5].

The range of commercially available propolis-containing skin care products is expanding, with creams and body lotions predominating. According to the advertisements, the majority of skin care products have a „soothing, moisture-rich, anti-aging” effect, and are also effective against eczema [23].

6.6. Alimentary canal

When examining the beneficial properties of various propolises, in the case of Brazilian green propolis, the stimulation of the functioning of the intestinal system was mentioned, as well as its beneficial effect in the treatment of gastric ulcers, while the liver protective function of propolis was proven in animal experiments [3].

The polyphenols in propolis can support the development and maintenance of a healthy intestinal flora by limiting the growth of pathogenic bacteria and, in addition, prevent their adhesion to human intestinal cells [24]. The possible therapeutic effect of propolis on inflammatory bowel diseases is still being investigated today, but many experiments still need to be performed before clinical application can begin [5].

6.7. Allergizing effect

In addition to its many beneficial physiological effects, propolis can also trigger allergic reactions (swelling, dermatitis, hives) in susceptible individuals. This is most common among beekeepers, but it also depends on individual sensitivity [3]. Therefore, it is recommended that the therapeutic use of propolis products is always carried out under medical supervision [5].

6.8. Summary of physiological and therapeutic effects

Several studies have proven that the observed beneficial physiological effects are not the result of a single prominent compound, but rather the combined effect of the complex components of propolis [9].

Overall, it can be stated that as a natural substance with good medicinal properties, propolis and its components can be used in a wide range of ways, including wound and skin healing, and in the treatment of some neurological diseases and atherosclerosis. Interest in the health effects of propolis and the number of publications have been continuously increasing in the last 30 years. However, even more human clinical studies are needed to confirm the beneficial effect of propolis for specific population groups. Preclinical studies support the antioxidant and anti-inflammatory effect of propolis, which can prevent or slow the progression of various chronic diseases, including heart disease, diabetes, high blood pressure, tumors and neurodegenerative diseases (e.g., Alzheimer’s disease) [5].

7. New areas of application of propolis

One of the areas of use of propolis can be to improve the growth performance and productivity of farm animals. Based on our knowledge so far, it can be said that propolis has a beneficial effect on the normal laboratory values, growth and productivity of the animals included in the studies. In addition, it is considered as a possible alternative to antibiotics in the production of animal feed, because it has the advantage that it does not induce resistance in microorganisms [25].

Another area of intensive research in the last few years have been the application of propolis in food preservation. Food preservatives primarily include antimicrobial and antioxidant agents. Antimicrobial agents added to foods serve two purposes: to stop the natural spoilage of food and to avoid/control contamination by microorganisms, including pathogenic microorganisms. Antioxidants are used to extend shelf life and prevent spoilage. Propolis favorably combines antioxidant and antimicrobial properties. However, its large-scale use as a food preservative has not yet been realized, as this would require proper standardization of the product [7].

8. Antioxidant properties of propolis

The antioxidant properties of propolis are manly determined by the bioactive components found in it, primarily the phenolic compounds, depending on the botanical and geographical origin. The phenolic compound profile of propolis is slightly different from that of honey. While in the former, the profile is mainly determined by the botanical origin, and the dominant flavonoids are quercetin, myricetin, chrysin, apigenin, luteolin, pinocembrin and pinobanksin, and of phenolic acids, p-hydroxybenzoic acid, p-coumaric acid, cinnamic acid, gallic acid, ferulic acid and caffeic acid, in propolis, which typically comes from poplar and birch in Central Europe, chrysin, kaempferol, apigenin, pinocembrin and pinobanksin are the most characteristic and, in addition to phenolic acids, their esters (e.g., caffeic acid and ferulic acid esters) also occur. Among the latter, the phenylethyl ester of caffeic acid is outstanding in terms of tumor prevention properties (although its effect also depends on the synergistic effect of other accompanying phenolic compounds). The polyphenols in propolis have been proven to inhibit the formation of amino, oxide and peroxide type free radical, as well as the formation of complexes between free radicals and transition metals, and also lipid peroxidation [26].

In addition to the differences depending on the origin of propolis, the literature is not uniform regarding the extraction method of the antioxidant compounds, and the differences can significantly influence the extraction results.

Based on the available data, extraction was mainly carried out with different mixtures of ethanol and water in the experiments, but extraction with methanol and other solvent also occurs. Regarding the methods for determining the antioxidant properties, only the results of experiments based on in vitro spectrophotometry have been reported, including the determination of radical scavenging properties (DPPH – 2,2-diphenyl-1-picrylhydrazyl, ABTS – 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), ORAC (Oxygen Radical Absorbance Capacity), as well as total polyphenol content (Folin-Ciocalteu method) and total flavonoid content. Although in the case of propolis extracts from different locations, polyphenol contents of the same order of magnitude as in the case of honey were measured (typically in the 18-500 mg gallic acid equivalent/ml range), in the case of a Turkish sample there was also a value above 19,000 mg gallic acid equivalent/ml, and values above 1,000 mg gallic acid equivalent/ml were also measured for Brazilian samples. The situation is similar with regard to the total flavonoid content, where the majority of the samples stayed in the range typical of honey (1-25 mg catechin equivalent/ml), however, there were also extremely high values: nearly 5,000 mg catechin equivalent/ml in the case of an Algerian propolis, and a value over 29,000 mg catechin equivalent/ml in the case of a Turkish propolis. As for the radical scavenging ability, once again, values in the range of honey are reported by researchers (e.g., 50-80% inhibition in the case of DPPH radicals), but extreme values are also typical here (e.g., 90.7–99.34% inhibition in the case of a Malay honey). Similar to honey, numerous studies have confirmed in the case of propolis its effectiveness in the case of tests carried out on various animal and human bodily fluids and cell cultures, in terms of antioxidation properties.

9. Synergistic interaction of propolis and honey

Propolises of different origins can show a synergistic interaction not only with each other, but also when mixed with honey. Following the mixing of propolis extracts from Iraq, it was possible to prove a synergistic effect against various pathogens (E. coli, S. aureus, C. albicans) in microbiological tests. Similarly, in animal experiments, the extent of the wound healing effect (reepithelization) was increased in the case of a propolis mixture [27].

Due to the deterioration of sensory characteristics, propolis is typically mixed with honey in a proportion of no more than 1%. Even at this concentration, a four- to fivefold increase in the amount of phenolic compounds, phenolic acids and flavonoids was measured, and the anthocyanin and carotenoid content of the mixture also increased several times. Of the flavonoids, especially the amount of galangin, chrysin, pinocembrin and pinobanksin, while of phenolic acids, the amount of ferulic acid, caffeic acid and p-coumaric acid increased. The radical scavenging (ABTS, DPPH) and metal ion reducing capacity (FRAP – Ferric Reducing Antioxidant Power, CUPRAC – Cupric Reducing Antioxidant Capacity), measured by various in vitro methods, also showed multiple increases [26].

The synergistic interaction of propolis and honey was also confirmed in antimicrobial tests. In the research, in the case of antibiotic-resistant strains of E. coli, S. aureus and C. albicans, honey strengthened the effect of propolis both in cultures of individual strains and their mixtures [29].

10. Acknowledgment

Az anyag összeállításához Bencsik Boglárka demonstrátor hallgató is hozzájárult.

11. References

[1] Bankova, V. (2005): Recent trends and important developments in propolis research, eCAM; 2 (1) pp. 29–32 DOI

[2] Csupor, D. (2020): A propolisz és a cukorbetegség: mítosz vagy valóság? [online] PirulaKalau. (Hozzáférés 2021. 06. 05.)

[3] Soós, Á. (2020): Nyers és extrahált propoliszok elemtartalmi vizsgálata és földrajzi eredet szerinti azonosítása. Doktori (PhD) értekezés, Debreceni Egyetem, Kerpely Kálmán Doktori Iskola.

[4] Pedrotti, W. (2009): A szépítő, gyógyító méz, propolisz és társaik. pp. 48-52. Ventus Libro Kiadó.

[5] Braakhuis, A. (2019): Evidence on the Health Benefits of Supplemental Propolis. Nutrients, 11, p. 2705. DOI

[6] Cornara, L.; Biagi, M.; Xiao, J.; Burlando, B. (2017): Therapeutic properties of bioactive compounds from different honeybee products. Front. Pharmacol. 2017, 8, p. 412.

[7] Bankova V., Trusheva P.B. (2016): New emerging fields of application of propolis, Maced. J. Chem. Chem. Eng. 35 (1), pp. 1–11.

[8] Simone M., Evans J. D., Spivak M. (2009): Resin collection and social immunity in honey bees, Evolution 63, pp. 3016–3022. DOI

[9] Huang S., Zhang CP., Wang K., Li GQ., Hu F.L. (2014): Recent Advances in the Chemical Composition of Propolis, Molecules, 19, pp. 19610-19632; DOI

[10] de Mendonça, I., Porto, I., do Nascimento, T., de Souza, N., Oliveira, J., Arruda, R., Mousinho, K., dos Santos, A., Basílio-Júnior, I., Parolia, A. & Barreto, F. (2015): Brazilian red propolis: phytochemical screening, antioxidant activity and effect against cancer cells. BMC Complementary and Alternative Medicine, 15 (1).

[11] Batista, L.L.V.; Campesatto, E.A.; Assis, M.L.B.d.; Barbosa, A.P.F.; Grillo, L.A.M.; Dornelas, C.B. (2012): Comparative study of topical green and red propolis in the repair of wounds induced in rats. Rev. Col. Bras. Cir. 2012, 39, pp. 515–520.

[12] Anjum, S.I.; Ullah, A.; Khan, K.A.; Attaullah, M.; Khan, H.; Ali, H.; Bashir, M.A.; Tahir, M.; Ansari, M.J.; Ghramh, H.A. (2018): Composition and functional properties of propolis (bee glue): A review. Saudi J. Biol. Sci. 2018

[13] Berretta, A., Silveira, M., Cóndor Capcha, J. & De Jong, D. (2020): Propolis and its potential against SARS-CoV-2 infection mechanisms and COVID-19 disease: Running title: Propolis against SARS-CoV-2 infection and COVID-19. Biomed Pharmacother., 131, 110622, DOI

[14] Farooqui, T.; Farooqui, A.A. (2012): Beneficial effects of propolis on human health and neurological diseases. Front. Biosci. 2012, 4, pp. 779–793.

[15] Alkis, H.E.; Kuzhan, A.; Dirier, A.; Tarakcioglu, M.; Demir, E.; Saricicek, E.; Demir, T.; Ahlatci, A.; Demirci, A.; Cinar, K.; et al. (2015): Neuroprotective effects of propolis and caffeic acid phenethyl ester (CAPE) on the radiation-injured brain tissue (Neuroprotective effects of propolis and CAPE). Int. J. Radiat. Res. 2015, 13, pp. 297–303.

[16] Yesiltas, B.; Capanoglu, E.; Firatligil-Durmus, E.; Sunay, A.E.; Samanci, T.; Boyacioglu, D. (2014): Investigating the in-vitro bioaccessibility of propolis and pollen using a simulated gastrointestinal digestion System. J. Apic. Res. 2014, 53, pp. 101–108.

[17] Miryan, M., Soleimani, D., Dehghani, L., Sohrabi, K., Khorvash, F., Bagherniya, M., Sayedi, S. & Askari, G. (2020): The effect of propolis supplementation on clinical symptoms in patients with coronavirus (COVID-19): A structured summary of a study protocol for a randomised controlled trial. Trials, 21.

[18] Turan, I., Demir, S., Misir, S., Kilinc, K., Mentese, A., Aliyazicioglu, Y. & Deger, O. (2015): Cytotoxic Effect of Turkish Propolis on Liver, Colon, Breast, Cervix and Prostate Cancer Cell Lines. Tropical Journal of Pharmaceutical Research, 14(5), pp. 777-782.

[19] Štajcar D. (2009): Propolis and its flavonoid compounds cause cytotoxicity on human urinary bladder transitional cell carcinoma in primary culture, Period biol, Vol 111, No 1, 2009.

[20] Fukuda, T., Fukui, M., Tanaka, M., Senmaru, T., Iwase, H., Yamazaki, M., Aoi, W., Inui, T., Nakamura, N. & Marunaka, Y. (2015): Effect of Brazilian green propolis in patients with type 2 diabetes: A double-blind randomized placebo-controlled study. Biomedical Reports, 3(3), pp. 355-360.

[21] Mujica, V., Orrego, R., Pérez, J., Romero, P., Ovalle, P., Zúñiga-Hernández, J., Arredondo, M. & Leiva, E. (2017): The Role of Propolis in Oxidative Stress and Lipid Metabolism: A Randomized Controlled Trial. Evidence-Based Complementary and Alternative Medicine, 2017, Article ID 4272940. DOI

[22] Nakajima, Y.; Shimazawa, M.; Mishima, S.; Hara, H. (2007): Water extract of propolis and its main constituents, caffeoylquinic acid derivatives, exert neuroprotective effects via antioxidant actions. Life Sci. 2007, 80, pp. 370–377

[23] New Zealand Medicines and Medical Devices Safety Authority, Eczema Cream. 2014. (Hozzáférés 2018.03.10.)

[24] Alkhaldy, A.; Edwards, C.A.; Combet, E. (2018) The urinary phenolic acid profile varies between younger and older adults after a polyphenol-rich meal despite limited differences in in vitro colonic catabolism. Eur. J. Nutr. 2018.

[25] Silva-Carvalho R., Baltazar F., Almeida-Aguiar C. (2015) Propolis - A complex natural product with a plethora of biological activities that can be explored for drug development, Evidence-Based Complementary and Alternative Medicine, Article ID 206439, 29 pages,. DOI

[26] Habryka, C., Socha, R., Juszczak, L. (2020) The Effect of Enriching Honey with Propolis on the Antioxidant Activity, Sensory Characteristics, and Quality Parameters, Molecules, 25, 1176. DOI

[27] Al-Waili, N. Mixing two different propolis samples potentiates their antimicrobial activity and wound healing property: A novel approach in wound healing and infection, Veterinary World, EISSN: 2231-0916, 1188.

[28] Martinello, M, Mutinelli, F. (2021) Antioxidant Activity in Bee Products: A Review, Antioxidants 10, 71. DOI

[29] Al-Waili, N., Al-Ghamdi, A., Ansari, M. J., Al-Attal, Y., Salom, K. (2012), Synergistic Effects of Honey and Propolis toward Drug Multi-Resistant Staphylococcus Aureus, Escherichia Coli and Candida Albicans Isolates in Single and Polymicrobial Cultures, Int. J. Med. Sci. 2012, 9, 793.

DOI

Received: May 2022 – Accepted: July 2022

¹ University of Debrecen, Institute of Food Science

Keywords

spices, bread, fortification, macroelement, nutrient reference value (NRV)

2. Introduction

Conscious food consumers have recognized and accepted that the consumption of “healthier” foods can prevent certain diseases. (The term “healthier” food can be misleading, because according to EU laws, “unhealthy” food cannot be placed on the market. In the present case, I accept that this term represents a comparative. The Ed.) In addition to researchers, industry also strives to develop and produce „healthier” foods [1, 2]. Bread and bakery products play an important role in the human diet. Wheat bread is generally an efficient source of energy and contains irreplaceable nutrients. The fortification of these products with functional components is widespread in order to improve health protection [3]. Examples of such components are spices and herbs [1, 2], as well as byproducts of cereals, pseudo-cereals, vegetable or fruit products [3].

Several publications have reported on the fortification of breads with various substances, which was also detailed by Varga-Kántor et al. [4].

Fortified breads are more valuable than plain bread from a nutritional physiology point of view, as they contain ingredients that have a beneficial effect on health. Spices and herbs are examples of this.

These plants, which are equally important in the pharmaceutical industry and in gastronomy, have been used by mankind for a long time. They have a strong, concentrated smell and taste, so consuming large amounts of herbs may even have an adverse sensory effect [5]. The spices we used and their active ingredients are applied in the treatment of several diseases. Many scientific books and studies have reported on their use in this area.

A detailed description of the spices used and measured in our experimental program can be found in the following sources: Pushpagadan [6], Kurian [7], Peter [8], Charles [9], Gupta [10], Kintzios [11], Chen [12], Sasikumar [13], Pandey [14]. These works describe the origin of the spices, their physiological effects on humans, and their history.

Spices that contain compounds with potent antioxidant and disease-preventing effects have a high element content, which is important for a balanced diet and lifestyle. Table 1 contains the measurement results of other authors for these parameters.

While Barin et al. [15] and the USDA [21] measured a calcium concentration of 22,000 mg/kg in basil, the values reported by other authors were between 10,000 and 15,000 mg/kg. In the case of dill, the results of Rahmatollah and Mahbobeh [17] and the USDA [21] were similar, while lower concentrations were measured by Özcan [16]. The USDA database [21] had a higher calcium content for oregano than Barin et al. [15] and Öczan [16]. The calcium content of caraway was similar [16, 21], while in the case of chives, there was a 1,000 mg/kg difference [15, 21]. When looking at rosemary, the results show that two authors obtained similar results of about 8,000 mg/kg [18, 19], but in the other two cases, higher calcium contents were determined [16, 21]. In the case of garlic granules, there was no significant difference between the measured concentrations [20, 21].

In the case of potassium content, the highest concentration was measured in dill. Potassium contents close to 34,000 mg/kg were determined by two authors [16, 21], but the values of Rahmatollah and Mahbobeh [17] were twice as high. In the case of basil, concentrations above 24,000 mg/kg were measured by three authors [16, 18, 21], however, Ozygit et al. [19] only measured a potassium content of 8,000 mg/kg. In the case of oregano, the measured values of this parameter were between 12,000 and 19,000 mg/kg. In the case of caraway, the results obtained differed significantly. The USDA database [21] described a potassium content of more than 26,000 mg/kg in chives. The potassium content of rosemary was similar in two cases [16, 21]. Ozygit et al. [19] measured a lower value than this, while the value measured by Özcan [16] was approximately 2,000 mg/kg higher. There was no significant difference in the results of garlic granules.

Looking at the magnesium content results, there were significant differences for all spices between the concentrations measured and published by the researchers. The determined values were in the order of thousands, with the exception of garlic granules.

A similar trend can be observed for the sodium content, as the measured concentrations differ significantly in the various studies.

In the case of phosphorus, similar values were obtained by the authors for oregano and garlic granules. In the case of the other herbs, there were significant differences between the results of the authors, often in the order of thousands.

Regarding the sulfur content of the spices, it can be seen that very different concentrations were determined in dill, and very high values were obtained for the garlic granules.

3. Materials and methods

3.1. Preparation of the breads

In this study, the macroelement content of seven dried spices (basil, dill, oregano, caraway, chives, rosemary and garlic granules) and 42 fortified breads was determined.

The raw materials for the products were purchased in a supermarket in Debrecen. After the analysis of the spices, the breads were prepared based on the recipe of Varga-Kántor et al. [4] and Kántor et al. [20].

These samples contained different concentrations of dried spices (0, 2, 4, 6, 8, 10 and 12 g). The additional ingredients were wheat flour (BL 55, 500 g), 10% vinegar (8 g), sunflower oil (44 g), salt (5 g), granulated sugar (5 g), yeast (30 g), milk (2.8% fat, 150 ml) and 25 °C water (100 ml). The ingredients were stored at room temperature, in their original packaging, in the dark or in a refrigerator until the products were prepared. After kneading, the leavening time was 1 hour at room temperature. The next step was the shaping of the loaves, followed by resting for 10 minutes. The breads were baked in a convection oven at 210 ⁰C and 95% humidity for 15 minutes (RXB 606, convection oven, Budapest, Hungary). After baking, the products were left in the oven for 6 minutes.

3.2. Determination of element content

In the case of spices, the samples purchased in the store were not dried, but the breads were dried according to standard MSZ 20501-1 [22]. Sample preparation was carried out based on the method of Kovács et al. [23]. After measuring the bread into a digestion tube, 10 ml of nitric acid (69% v/v; VWR International Ltd., Radnor, USA) was added to the sample and it was left to stand overnight. Predigestion was carried out at 60 °C for 30 minutes. After cooling, before the main digestion, 3 ml of hydrogen peroxide (30% v/v; VWR International Ltd., Radnor, USA) was used, and then the sample was kept at 120 °C for 90 minutes. After cooling, it was diluted with high purity water (Millipore SAS, Molsheim, France) and the mixture was filtered on filter paper (388, Sartorius Stedim Biotech SA, Gottingen, Germany). The element content was determined using an ICP-OES (Inductively coupled plasma optical emission spectrometer, Thermo Scientific iCAP 6300, Cambridge, UK) instrument. The wavelengths used were 315.8 nm (Ca), 769.8 nm (K), 280.2 nm (Mg), 818.3 nm (Na), 185.9 nm (P) and 180.7 nm (S).

3.3. Statistical analysis

To determine the mean, standard deviation and statistically verifiable differences, one-factor analysis of variance (Tukey and Dunnett’s T3 test) was used with SPSS statistical software (version 13; SPSS Inc. Chicago, Illinois, USA). Measurements were carried out in triplicate.

3.4. Calculation of the daily intake value from the nutrient reference value (NRV)

NRV values are contained in Regulation (EU) No 1169/2011 of the European Parliament and of the Council [24] and the EFSA scientific bulletin [25]. Data are presented as a percentage for 100 g of product, which means the consumption of approximately 1.5 slices of bread.

NRV (%) = (element content of the bread/daily reference intake) x100

In the case of sodium, the daily reference intake is 2,000 mg [25], while no relevant data were found for sulfur.

4. Results and evaluation

4.1. Measurement results of the element content of the spices

The results of the macroelement content measurements of the herbs examined by us are presented in Table 2. The values are given on an as received basis.

The highest calcium concentration was measured in the case of basil, followed by chives. The measured values were similar in dill and oregano. A value of more than 10,000 mg/kg was measured in rosemary, while in the case of caraway, the concentration was higher than 6,000 mg/kg. The lowest calcium content was measured in garlic granules.

In the case of the potassium content, basil and dill exhibited outstanding values. In caraway, chives and garlic granules, the concentrations were above 10,000 mg/kg. The herbs oregano and rosemary showed the lowest values among the plants analyzed.

The highest magnesium content was measured in basil, which had twice the concentration of dill, which also had a high value compared to the other samples tested. The values for oregano, caraway, chives and rosemary were between 2,000 and 3,000 mg/kg. The lowest magnesium content was found in garlic granules.

An outstanding sodium content was measured in dill, but the concentrations were very low on the other samples. Values higher than 100 mg/kg were obtained for the basil and garlic granule samples. In the other cases, the measured values were below 100 mg/kg.

In case of the phosphorus content, the concentration in caraway was the highest, followed by the garlic granules. Values between 3,000 and 4,000 mg/kg were measured for basil, dill and chives. Rosemary had the lowest concentration.

During the determination of the sulfur content, concentrations of more than 1,000 mg/kg were measured in each sample. Similarly outstanding values were obtained for dill and garlic granules, followed by chives with a concentration of more than 3,000 mg/kg. For the other spices, with the exception of basil, sulfur contents between 1,000 and 2,000 mg/kg were detected.

Comparing the results of Table 1 with the concentration measured by us, it can be stated that in the course of our analyses, higher values were obtained for the calcium content of chives and the sulfur content of dill and caraway, while lower values were measured for the potassium content of dill, oregano and chives and the phosphorus content of chives. However, from the measured concentrations it can be concluded that the results obtained are similar to the values mentioned in the other studies, except for the data regarding the sodium content. In this case, results that are significantly different from the literature data can be seen.

4.2. Measurements results of the breads fortified with spices

The breads were prepared based on a predetermined recipe [4, 20], and samples without spices were also prepared. Based on the results, it was determined that the measured parameters of the control breads were similar to the literature data (Ca: 476; K: 2,200; Mg: 260; Na: 2,585; P: 1,478 and S: 1,008 mg/kg [4]; Ca: 510; K: 2,418; Mg: 285; Na: 3,180; P: 1,512 and S: 948 mg/kg [20]), except for the sodium content.

The results are reported on a dry matter basis (Tables 3, 4 and 5). In the tables, significant deviations from the control samples are marked with the letter „a” in each column.

4.2.1. Calcium content results

The calcium contents of the fortified breads are presented in Table 3. In most cases, the addition of spices increased the element content of the fortified breads. The biggest increase was experienced in the case of basil breads. In this case, the difference compared to the control sample was more than 500 mg/kg. Breads containing dill, chives or rosemary showed a difference of about 300 mg/kg between the control bread and the breads fortified with 12 g of spices. The additional value of breads fortified with oregano and caraway was smaller, around 100-200 mg/kg.

Although the calcium content of oregano exceeded 10,000 mg/kg, the increase experience in fortified breads was not as large as when using spices with similar calcium content.

The lowest calcium content was determined in the bread containing 12 g of garlic granules. The other samples showed significant differences compared to the control sample.

4.2.2. Potassium content results

The potassium contents of the samples analyzed are also shown in Table 3. Based on the results, it appears that the addition of basil and dill increased the potassium concentration of the breads the most. In the case of the breads fortified with 12 g of caraway, a difference of about 300 mg/kg was found, compared to the control sample. In the other cases, the difference was barely more than 200 mg/kg.

In terms of potassium content, the greatest increase was exhibited by the breads with basil and dill, followed by the product samples with caraway, oregano and garlic granules. In all cases, the lowest values were measured in breads with rosemary and chives. This difference is probably due to the difference already present in the control breads.

4.2.3. Magnesium content results

Data on the magnesium content of the breads are presented in Table 4. In the spices, the highest values were measured in the case of basil and dill, which affected the magnesium content of the breads. When examining the samples, the highest magnesium content was determined in the product fortified with basil. This was the largest difference (200 mg/kg) between the control bread and the a sample containing 12 g of spice. This result was followed by breads fortified with dill. Products with caraway and rosemary showed a similar trend, with a maximum difference of 60 mg/kg between the sample containing the most spice and the control bread. In the case of breads with oregano, the increase was 40 mg/kg in the bread containing the most spice compared to the control product. For those spices where the magnesium content was below 2,000 mg/kg, there was no significant difference in the fortified breads. When looking at samples with the same amount of spices, the highest values were measured in the basil breads in all cases. The lowest concentrations were detected in breads with garlic granules and chives.

4.2.4. Sodium content results

Data on the sodium content of the products prepared can be seen in Table 4. Regarding the samples, the measured values were between 2,400 and 3,100 mg/kg. The sodium content of the spices was low compared to the other macronutrients, except for dill. There was no statistically proven difference in the results of the products with basil, oregano, caraway and garlic granules. In the case of the samples with dill, the reason for the increase was probably the sodium content of the spice, which affected the element content of the final products.

In the case of chives and rosemary, the sodium content of the spices was below 100 mg/kg. Therefore, the decrease in one case and the increase in the other cannot be explained. When considering the same amount of spices, the highest sodium content was measured in breads with rosemary, dill and basil. This tendency was also observed in the case of the control breads. Since the breads were made by hand, it is possible that the distribution of table salt was not uniform in all cases, and this may also cause differences.

4.2.5. Phosphorus content measurement results

The phosphorus content results of the samples are presented in Table 5. Based on the results, the phosphorus content of the breads was similar. In most cases, there was no statistically verifiable difference between the samples. Smaller differences were measured in the products with caraway and garlic granules, which is due to the phosphorus content of the spices. The phosphorus content of these spices exceeded 4,000 mg/kg. For the other spices, concentrations below 4,000 mg/kg were determined in all other cases.

The highest phosphorus content was measured in the products with caraway, followed by breads with dill, garlic granules and basil. The lowest concentration was measured in the products flavored with chives, however, low phosphorus contents were measured in the breads with rosemary and oregano as well.

4.2.6. Sulfur content results

Table 5 shows the sulfur content of the breads. Based on the concentrations obtained, larger differences were measured in the products with basil and garlic granules, and smaller differences were measured in the case of the other fortifications when increasing the amount of spices. Analyzing the spices, the highest sulfur content was determined in dill and garlic granules (more than 7,000 mg/kg), however, even the addition of larger amounts of spices to the breads did not increase the measured concentrations. It can be seen that in the other cases the value of the measured parameter did not increase with the increase in the amount of spices. Minor differences could be observed, but the sulfur content of the spices had no significant effect on the sulfur content of the final products.

Daily intake contribution results calculated from the nutrient reference value (NRV)

Tables 3, 4 and 5 show the daily contribution values for (Ca, K), (Mg, Na) and (P) per 100 g of product, respectively.

In the case of calcium content, the consumption of 100 g of control bread per day covers 5 to 6% of the daily calcium intake. By increasing the amount of spices in the samples, these values also increased. The highest contribution was calculated for the breads with basil, followed by the products with rosemary, dill, and chives.

In the case of the potassium content, the contribution of the control breads was between 10 and 11%. When different amounts of spices were added, a smaller increase was calculated tan in the case of the calcium content. For the breads with the most spices, the increase in daily contribution was even as high as 3% (samples with basil and dill) compared to the control products.

The magnesium content of the control breads is responsible for approximately 7% of the daily magnesium intake. In this case, once again, the most significant differences were observed in the bread with basil. With 12 g of spice, the increase was more than 5% compared to the control sample.

The sodium intake values of all samples were around 12 to 13%. In the case of the products with dill and rosemary, the values were higher.

In terms of phosphorus content, all of the reads covered more than 20% of the daily phosphorus intake. The contribution of the samples with caraway showed a minimal increase. For the breads fortified with 12 g of spices, the increase was around 2% compared to the control products.

5. Conclusions

As the results show, the spices themselves have a high macronutrient content. In terms of calcium, potassium, magnesium and sodium, basil exhibited outstanding values. High values were also measured in dill, chives, oregano and garlic granules.

The content of calcium, potassium and magnesium in fortified breads increased. In the case of calcium, the biggest difference was found in the products with basil. A clear increase was also observed for the other samples as well, except for the application of garlic granules.

Outstanding results were also achieved in terms of the potassium content of products with basil and dill. A difference of almost 600 mg/kg was measured between the control sample and the bread with 12 g of spice.

There was no significant difference in the magnesium content. A greater increase in concentration was only observed for the products with basil.

The samples with rosemary and dill showed a slight increase in sodium content, which can also be observed with the same amount of spices.

No significant differences were found in the phosphorus and sulfur contents; similar values were measured.

Based on the results, the largest daily contribution of macronutrients was provided by the breads with basil, followed by the breads with dill. In the case of the sodium content of the breads, the daily intake contributions of the products with dill and rosemary were the largest.

Overall, it was possible to prepare products whose element content in most cases differed significantly from that of the control breads, so the contribution of the products to the daily reference values also increased.

6. Acknowledgment

This research was financed by the Higher Education Institution Excellence Program of the Hungarian Ministry of Innovation and Technology (NKFIH-1150-6/2019), within the framework of the 4th thematic program of the University of Debrecen.

7. References

[1] Balestra F., Cocci E., Pinnavaia G., Romani S. (2011): Evaluation of antioxidant, rheological and sensorial properties of wheat flour dough and bread containing ginger powder. LWT- Food Science and Technology 44 (3) pp. 700-705. DOI

[2] Gawlik-Dziki U., Swieca M., Dziki D., Baraniak B., Tomiło J., Czyz J. (2013): Quality and antioxidant properties of breads enriched with dry onion (Allium cepa L.) skin. Food Chemistry 138 (2-3) pp. 1621-1628. DOI

[3] Dziki D., Rozy1o R., Gawlik-Dziki U., Swieca M. (2014): Current trends in the enhancement of antioxidant activity of wheat bread by the addition of plant materials rich in phenolic compounds. Trends in Food Science and Technology 40 (1) pp. 48-61. DOI

[4] Varga-Kántor A., Alexa L., Topa E., Kovács B, Czipa N. (2021): Szárított bazsalikommal dúsított kenyerek vizsgálata és eredményeinek értékelése. Élelmiszervizsgálati közlemények. LXVII (4) pp. 3665-3671. DOI

[5] Gibson, M. (2018). Food Science and the Culinary Arts. Academic Press is an imprint of Elsevier

[6] Pushpagadan P., George V. (2012): Basil. In: Peter KV (ed) Handbook of Herbs and Spices. Volume 1. Second edition. Woodhead Publishing Limited

[7] Kurian, A. (2012): Health benefits of herbs and spices. In: Peter KV (ed) Handbook of Herbs and Spices. Volume 2. Second Edition. Woodhead Publishing Limited.

[8] Peter K.V. (2012): Introduction to herbs and spices: medicinal uses and sustainable production. In: Peter KV (ed) Handbook of Herbs and Spices. Volume 2. Second Edition. Woodhead Publishing Limited.

[9] Charles D.J. (2013): Antioxidant Properties of Spices, Herbs and Other Sources. Springer Science+Business Media New York.

[10] Gupta R. (2012): Dill. In: Peter KV (ed) Handbook of Herbs and Spices. Volume 1. Second edition. Woodhead Publishing Limited.

[11] Kintzios S.E. (2012): Oregano. In: Peter KV (ed) Handbook of Herbs and Spices. Volume 2. Second Edition. Woodhead Publishing Limited.

[12] Chen H. (2012): Chives. In: Peter KV (ed) Handbook of Herbs and Spices. Volume 1. Second edition. Woodhead Publishing Limited.

[13] Sasikumar B. (2012): Rosemary. In: Peter KV (ed) Handbook of Herbs and Spices. Volume 1. Second edition. Woodhead Publishing Limited.

[14] Pandey U.B. (2012): Garlic. In: Peter KV (ed) Handbook of Herbs and Spices. Volume 1. Second edition. Woodhead Publishing Limited.

[15] Barin J.S., Pereira J.S.F., Mello P.A., Knorr C.L., Moraes D.P., Mesko M.F., Nóbrega J.A., Korn M.G.A., Flores E.M.M. (2012): Focused microwave-induced combustion for digestion of botanical samples and metals determination by ICP OES and ICP-MS. Talanta 94 pp. 308-314. DOI

[16] Özcan M. (2004): Mineral contents of some plants used as condiments in Turkey. Food Chemistry 84 (3), pp. 437-440. DOI

[17] Rahmatollah R., Mahbobeh R. (2010): Mineral contents of some plants used in Iran. Pharmacognosy Researh 4 pp. 267-270. DOI

[18] Özcan M.M., Akbulut M. (2007): Estimation of minerals, nitrate and nitrite contents of medicinal and aromatic plants used as spices, condiments and herbal tea. Food Chemistry 106 (2) pp. 852-858. DOI

[19] Ozyigit I.I., Yalcin B., Turan S., Saracoglu I.A., Karadeniz S., Yalcin I.E., Demir G. (2018): Investigation of Heavy Metal Level and Mineral Nutrient Status in Widely Used Medicinal Plants’ Leaves in Turkey: Insights into Health Implications. Biological Trace Element Research 182 pp. 387-406. DOI

[20] Kántor A., Fischinger L.Á., Alexa L., Papp-Topa E., Kovács B., Czipa N. (2019): Funkcionális kenyér, avagy a fokhagyma és készítményei hatása a kenyér egyes paramétereire/Functional bread, or the effects of garlic and its products on certain parameters of bread. Élelmiszervizsgálati közlemények/Journal of Food Investigation 65 (4) pp. 2704-2714.

[21] USDA (2011): USDA National Nutrient Database for Standard References. United States Department of Agriculture/Agriculture Research Service, Washington DC.

[22] Magyar Szabványügyi Testület (MSzT) (2007): Sütőipari termékek vizsgálati módszerei. Magyar Szabvány MSz 20501-1. Magyar Szabványügyi Testület, Budapest.

[23] Kovács B., Győri Z., Csapó J., Loch J., Dániel P. (1996): A study of plant sample preparation and inductively coupled plasma emission spectrometry parameters. Communication in Soil Science and Plant Analysis 27 (5-8) pp. 1177-1198. DOI

[24] REGULATION (EU) No 1169/2011 OF THE EUROPEAN PARLIAMENT AND OF THE COUNCIL (2011)

[25] EFSA (2019): Dietary reference values for sodium. EFSA Journal. DOI

DOI

Received: August 2022 – Accepted: September 2022

¹ University of Szeged, Faculty of Engineering, Institute of Food Engineering

Keywords

flexitarian, omnivore, vegetarian, vegan, plant-based, sustainability, sustainable food consumption

2. Food is a source of nutrients

Food is a source of vital macro- and micronutrients, vitamins. Foodstuffs, including water are sources of life, necessary and unavoidable for the functioning of our body and to maintain good health. The foods we eat also have influence on the composition of our microbiota. But foods are not only sources of energy, protein, fat and carbohydrates, but they are also a source of enjoyment by providing good taste and smell. Foodstuffs either eaten raw or cooked are part of our social life and our culture.

3. Our diet varies

Our diet varies depending on our geographical location, societal status, economical buying power, our education and cultural background. Mediterranean countries provide a more favourable environment for the production of a wide range of vegetables and fruits allowing a varied diet. Whether and lifestyle have an influence on the gastronomic culture. Seasonality would also influence the availability of foods. Religion, ethical, moral and animal welfare issues motivate consumers, as well. (Jewish, Muslim, Hindu and other religious restrictions not allowing the consumption of pork, beef and certain other types of foods are well-known for a long time.) Some societies are more conservative than others, high level of neophobia would be an obstacle in the acceptance of food innovation and that of novel products. Information, especially the lack of evidence-based information and fake news via social media have a major role in consumers’ decisions. On one hand, consumers are becoming more conscious, mainly health-conscious, more and more environment-conscious requesting healthy, ’natural’, clean label and sustainably produced foodstuffs to be marketed. On the other hand they follow trends as much as they set up those.

4. Planetary Health – the EAT-Lancet Report (2019) [1]

Food is the single strongest lever to optimize human health and environmental sustainability on Earth. An immense challenge facing humanity is to provide a growing world population with healthy diets from sustainable food systems.

Transformation to healthy diets by 2050 will require substantial dietary shifts. Global consumption of fruits, vegetables, nuts and legumes will have to double, and consumption of foods such as red meat and sugar will have to be reduced by more than 50%. A diet rich in plant-based foods and with fewer animal source foods confers both improved health and environmental benefits. Thus, the EAT-Lancet Report urges a radical transformation of the global food system.

As the goal set up in the EAT-Lancet Report is to achieve „Planetary Health Diets” for nearly 10 billion people by 2050, the Commission would continue its work and publish another report in 2024.

5. Different food consumption patterns – Omnivores, vegetarians, flexitarians and anything in between

The most relevant diets are summarized in Table 1. providing different definitions and data for the prevalence and consumption.

Varied diets – unless restricted by environmental, economic and social-cultural factors – allow the moral, ethical and spiritual approach of people being reflected.

We are mainly omnivores in Europe (72.3% based on a survey conducted in 2021 in six EU Member States) [2], such as North Americans (66% in 2019) [3], regularly consuming meats (pork, beef, mutton, goat, chicken and other poultry), but mainly red meat. An omnivore diet does not exclude any foods or food groups, unless the given consumer has food allergy, intolerance or other food-related health issue.

A small proportion of consumers are vegetarians (ovo-, lacto or ovo-lacto vegetarians) or vegans but they strictly follow their choice of diet, they are persistent and consistent in their decision to follow a meat-free, plant-based (e.g. vegetables, fruits, legumes, cereals etc.) diet. On average, 4.6% of Europeans are vegetarians, but it varies, 5-7% in the United Kingdom, 4.6% in Germary, 4.1 in Italy and Austria, 4.0% in AUT, 3.6% in Switzerland and as low as 2.1% in Estonia (see Table 1.), to name a few.

Vegans, who follow a more strict diet by excluding all meat, dairy, eggs and honey (all meat-based ingredients), form a small group of people. Data on the proportion of vegans in different countries are provided in Table 1. The production process must not use animal-derived products either, such as gelatine for clarifying juice or wine, or animal-based glue for product packaging.

Do we need definitions for vegetarian and vegan diets at all? Maybe not. However, in case food business operators (food processors and retailers) are willing to label foods as being suitable for vegetarian and vegan consumers, for example as „vegan food”, than we have to have a clear definition in order to be able to control the labelling. Furthermore, it would be useful to have an (and only one) internationally used, clear and harmonised logo for vegan foods. A symbol for labelling vegan and vegetarian products and services called „V-Label” exists. It was registered in 1996. [4]

Until today, there is no official definition for vegetarian and vegan diets. Despite the very detailed and comprehensive EU food legislation, there is no definition for vegetarianism and veganism, thus labelling rules for suitable food products have not been set up. In 2019, the European Commission (EC) began to define the concept of vegetarian and vegan food following the authorization given by a law passed in 2011. The EU Food Information Regulation stipulated that the EC is to issue an implementing act defining requirements for “information related to suitability of a food for vegetarians or vegans” (Article 36(3)(b) Regulation (EU) No 1169/2011). The European Vegetarian Organization (EVU is the umbrella organisation of vegan and vegetarian associations ad societies throughout Europe, „representing plant-based interests in the EU”, as they claim) together with FoodDrinkEurope (FDE is a food industry confederation in the European Union), have prepared proposals [5] for possible names. They point out, that the Commission has failed to act upon this responsibility since 2011 and does not consider the matter to be of high priority.

The proposed definition for food suitable for vegans is as follows: „Foods that are not products of animal origin and in which, at no stage of production and processing, use has been made of or the food has been supplemented with - ingredients (including additives, carriers, flavourings and enzymes), or - processing aids, or - substances which are not food additives but are used in the same way and with the same purpose as processing aids, that are of animal origin.

5.1. Vegetarian foods

Foods are belonging to this group, which are meet the requirements of vegan foods, with the difference that in their production and processing milk and dairy products, colostrum, eggs, honey, beeswax, propolis, or wool grease (including lanolin derived from the wool of living sheep or their components or derivatives) may be added or used.

Dedicated vegans usually start as vegetarians. According to the VeganZ study [2] conducted in six EU member states, 67.3% of vegans reported initially being vegetarian. In addition, 83% of vegetarians (FR) can imagine only buying plant-based products. As such, one can expect a proportion of vegetarian study participants to not only give up eating meat and fish in the future, but also to give up all animal-derived products. So, it is interesting to note that there is a trend towards veganism among vegetarians.

Besides that, 12.1% of omnivores are not opposed to a vegan diet, while 28.2% can imagine going vegetarian.

There are numerous variations between the omnivore and the vegan diets, such as – including but not limited to – reducetarian, flexitarian, semi-vegetarian, pescetarian (who exclude (red) meat from their diet, but eat fish), pesce-pollotarian, pollotarian diets, not to mention the ovo-, lacto- and ovo-lacto-vegetarian eating habits (Table 1.).

6. The flexitarian diet

6.1. Flexitarians

Consumers who are reducing their consumption of meat are also referred to in the literature as ’meat reducers’, ’low meat-eaters’ or ’semi-vegetarians’. [6]

Flexitarians deliberately aim to reduce animal products in their diet, but do not strictly exclude any meat. Flexitarian is a marriage of two words: flexible and vegetarian. The term was coined more than a decade ago by D. J. Blatner in her 2009 book “The Flexitarian Years to Your Life.” Blatner says you don’t have to eliminate meat completely to reap the health benefits associated with vegetarianism – you can be a vegetarian most of the time, but still enjoy a burger or steak when the urge hits. By eating more plants and less meat, it’s suggested that people who follow the diet will not only lose weight but can improve their overall health, lowering their rate of heart disease, diabetes and cancer, and live longer as a result.

According to the German Society for Nutrition, you can also call „flexitarians” „flexible vegetarians”. Even though they consume meat and fish, they do it less frequently than traditional omnivores. [7] Flexitarians are also known as casual vegetarians or vegivores. The flexitarian diet can be generally defined as a semi-vegetarian, plant-forward diet. It is a flexible eating style that emphasizes the addition of plant or plant-based foods and encourages meat to be consumed less frequently and/or in smaller portions.

Flexitarians, consumers reducing their consumption of meat are also referred to as „meat reducers” or „low meat-eaters”.

As the terms flexitarian and semi-vegetarian (even called earlier as partial- and pseudo-vegetarian) are often used as synonyms, neither vegetarian nor flexitarian have definitions, so it is rather difficult to compare these groups and to study their proportion. So in order to clearly differentiate them, they are arranged in Table 2. according to their attitude towards and consumption of meat.

Calories in the flexitarian diet mostly come from nutrient-rich foods such as fruits, legumes, whole grains and vegetables. When it comes to protein, plant-based foods (e.g., soy foods, legumes, nuts and seeds) are the primary source. Protein also comes from eggs and dairy, with lesser amounts coming from meat, especially red and processed meats. Due to the emphasis on nutrient-dense foods, the flexitarian diet encourages limiting one’s intake of saturated fat, added sugars and sodium. [8] Whether the latter is true or not, could be further studied. Following a flexitarian diet might not necessarily ensure a healthier nutrition, than that of omnivores. The interpretation of the term flexitarian is so diverse and its composition might differ so much, that we should be aware of the type of the food of animal origin and the frequency of its consumption to be able to judge.

The term flexitarian has been criticized by some vegetarians and vegans as an oxymoron because people following the diet are not vegetarians but omnivores as they still consume the flesh of animals. [9]

As there is no consensus regarding the definition of flexitarianism, it is rather difficult to measure or estimate the number and proportion of flexitarian consumers. Some consumers think of themselves as flexitarian when they cut meat consumption by half, only for one day, reduce it to 4 days/week, or even less. This discrepancy might have led to the following classification: „heavy flexitarian” (1 or 2 times per week meat for dinner), „medium flexitarian” (half of the week a meatless dinner) and „light flexitarian” (meat consumption frequency 5 or 6 times per week) [10]. This classification helps to overcome the huge differences in the interpretation of the term „flexitarian”.

Whether the classification of flexitarian consumers is based on a self-reported weekly meat consumption frequency or based on the measurement of the food consumption pattern by other means, it may lead to very different data. So we have to handle data on the proportion of flexitarians by care.

Even if the number of vegans and vegetarians has risen, most of the population is still consuming meat and other products of animal origin: on average 18.3% of Europeans consider themselves flexitarians. Their number is higher in Germany (27.3%) and Austria (25.8%) and lower in Spain (13.1%) and in Italy (12.1%). [2] (See Table 1. for more data.)

More than 50% of non-vegans in Germany intend to reduce their consumption of animal-derived products in the future. [2]

15.3% of flexitarians can imagine going vegan, while 54.8% would switch to a vegetarian diet.

Looking at all those, who actively reduce or fully exclude at least some animal products, including vegetarians, pescetarians and flexitarians, the group in total represents 30.8% of the population: 10 to 30 % of Europeans no longer consider themselves full meat-eaters anymore. [11].

7. Environmental concerns – plant-based solutions

In contrast to vegans and vegetarians, flexitarians attribute their main reasons for reduced meat consumption to the environment and sustainability (72.1%). [2]

Some authors [12, 13, 14] refer explicitly to a flexitarian diet as an important dietary change that significantly contributes to reducing the environmental footprint of the food system and providing more healthy eating patterns and nutritional benefits to food consumers. These studies define a flexitarian dietary pattern as predominantly plant-based complemented with modest amounts of animal foods (meat, dairy, fish). [10]

More and more people in Europe choose plant-based products over animal-based nutrition, occasionally or permanently. Almost all big supermarket chains list veggie meat and dairy alternatives.

Flexitarianism or ‘casual vegetarianism’ is an increasingly popular, plant-based diet that claims to reduce your carbon footprint and improve your health with an eating regime that’s mostly vegetarian yet still allows for the occasional meat dish. The rise of the flexitarian diet is a result of people taking a more environmentally sustainable approach to what they eat by reducing their meat consumption in exchange for alternative protein sources. [15]

Reducing meat and dairy consumption could cut greenhouse gas emissions by between 0.7-8 billion tons of CO₂eq annually by 2050 — that’s roughly between 1 percent and 16 percent of current emissions. But the Intergovernmental Panel on Climate Change (IPCC) is clear that in many poorer societies, it’s hard to find alternatives to animal protein. The EU has avoided policy that encourages citizens to cut meat eating, fearing political backlash. [16]

Another term should be mentioned here: „demitarian diet”. „Demitarianism” is the practice of making a conscious effort to reduce meat consumption largely for environmental reasons. The term was devised in 2009 in Barsac (France) at a workshop of environmental agencies, where they developed “The Barsac Declaration: Environmental Sustainability and the Demitarian Diet”. [17]

8. Plant-based diets

As there is an increasing need for alternative proteins, plant-based diets are gaining momentum. Plant-based diets have been praised for their benefit to our health and the environment. There is neither an official definition nor consensus on what defines a plant-based diet. It is used to describe a variety of dietary patterns, from the Mediterranean diet to Vegetarian and Vegan diets. The descriptions of plant-based diets mainly focus on the promotion of healthy plant foods, such as fruits, vegetables, bean, pulses, nuts etc., and they do not necessarily exclude the consumption of meat and dairy products, so these are not expecting the total avoidance of products of animal origin. [18, 19]

Although a plant-based diet is often used to describe a plant-only or vegan diet, it is not about the complete avoidance of animal products. Plant-based diets should be thought of as plant-forward diets or ‘flexitarian’ approaches, which emphasise eating healthy plant foods. While meat and dairy products are not necessarily avoided altogether, the frequency and portions that they are consumed will be reduced and most of the nutrients should come from healthy plant foods.

According to a Harvard Business Review [20] flexitarian consumers are the biggest market for plant-based products (accounting for 70% of sales in some categories [21], and 30% of overall shoppers [22]).

9. Food and Health

As mentioned before, in contrast to vegans and vegetarians, flexitarians attribute their main reasons for reduced meat consumption to the environment and sustainability. However, there are also health reasons and societal concerns pushing consumers to change their dietary habits. The health issues, the high prevalence of Non-Communicable Diseases (NCDs) is well-known. Whether it is hidden hunger, obesity or CVDs, tumors or other health issues in relation to food consumption, the non-balanced diet has long-term consequences. Short term changes, such as following fashion-diets are not appropriate in case we wanted to avoid the negative health consequences of our diet.

Consumers are becoming increasingly aware of the relationship between food and health and are changing their purchasing behaviour accordingly.

79% of Belgian respondents (n=17.000 (2021)) actively seek information on healthy living, and they expect regulators to play a stronger role in promoting health and environmental sustainability. BE consumers eat more fruit (51%) and vegetables (57%) than previously. [23]

10. Societal problems

The importance of societal problems – besides of health-related and environmental issues – should also be emphasized, as the increasing amount of non-evidence-based information spread most efficiently via social media and by bloggers and other influencers would undermine the reliability and trustfulness of science and its golden rules.

Another phenomenon is, when dogmas are being built. Numerous food-related dogmas were built in the last decades. These also endanger trust.

Consumers may also lose their trust in the food system due to greenwashing and similar attempts. When food companies are aiming to overdo and mimic environmental-friendly practices, consumers become most disappointed when the reality becomes evident.

11. Trend or fad?

An increasing group of food consumers are purposefully reducing their meat intake, without totally eliminating meat from their diet. They have no intention to become vegetarian or vegan, but for health and environmental reasons they are flexible and reduce their meat consumption.

The demand for vegan and vegetarian food products including alternatives to meat, milk, or eggs, has expanded considerably during recent years in Europe. [24]

Being a high-flying trend, a major innovation in the current decade, but will plant-based meat analogues continue to rise and generate enormous income for investors and for the time being, or is it going to be a fad?

„It is unlikely that plant-based meat will continue to grow as rapidly as it has the past few years. While it is certainly not a short-term fad, steep growth-rates will certainly cool down before 2025.” [25]

It was found that the percentage of heavy flexitarians (see definitions in Table 1. and above) decreased from more than 15 per cent in 2011 to less than 10 per cent in 2019, while the percentage of light flexitarians increased from 36 per cent in 2011 to 41 per cent in a Dutch survey. Such figures contribute to a slightly higher average in the number of days in which meat was eaten at dinner: from 4.6 days a week (2011) to 4.8 days a week (2019). And this outcome could be reconciled with the fact that per capita meat consumption in the Netherlands has been stable between 2011 and 2019 at approximately 39 kg. All this suggests that flexitarianism has made little progress in the past 10 years – at least, when it comes to overt behaviour. [10].

12. Generational differences

A recent US survey [26] examined the food priorities and buying power of Generation Z, how more Americans are concerned about environmental sustainability. The 17^th annual 2022 Food & Health Survey, conducted online (n=1,005, ages 18 to 80) oversampled Gen Z consumers (ages 18-24), who showed strong interest in the environment. When asked whether they believed their generation was more concerned about the environmental impacts of their food choices than other generations, Gen Z was the most likely to say yes at 73%, followed by millennials at 71%. Among all age groups, 39% said environmental sustainability had an impact on their purchasing decisions for foods and beverages, which was up from 27% in 2019.

13. Sustainable diets

The United Nations Food and Agriculture Organization (FAO) defines sustainable diets as having a low environmental impact, while meeting current nutritional guidelines, all while remaining affordable, accessible and culturally acceptable. [27]

Cultural and historical background, gastronomy, consumer habits and the role food plays in our culture have an immense effect on the way how and what we eat.

Consumer habits are rather difficult to change. Besides, it is widely known, that there can be large discrepancies between consumers’ self-perception and their actual behaviour, for example between the number of self-declared flexitarians and their actual meat consumption (frequency).

Despite all scientific evidence and scholarly consensus about what a healthy and sustainable dietary pattern consists of, in current practice mostly only small minorities of food consumers turn out to be able to meet such dietary recommendations. This indicates clearly that it must be expected that moving to a flexitarian diet style in which meat intake is limited to some degree is considered a dramatic dietary shift to many people. This implies that irrespective of the consensus about what a sustainable diet generally is, it is much less clear and uncontroversial how willing and helpful consumers could be to drive the transition to meat-restricted diets and dishes. [10]

Throughout human history, consumers abstained from eating meat on a regular basis, even if it was not a question of buying power or poverty, but a religious reason (see „Friday Fish” or „meat-free-Fridays”) or others.

We should not underestimate the role of meat in our diet, its sensory and nutritional value, its role in the national cuisine (see the examples of Germany, Switzerland and Hungary), how it is associated with wealth and power, traditional foods and tradition which might be an obstacle to innovation and novelty. The role animal husbandry plays in the economy, mainly in agricultural countries and numerous other factors would influence the way we relate to foods.

In case we will have a growing interest and commitment to increase our vegetable and fruit consumption, to reduce the meat intake than, with or without plant-based meat analogues, we may achieve healthier life for ourselves and for our fellow human beings.

14. References

[1] Lancet (2019): Healthy Diets from Sustainable Food Systems. Food Planet Health. EAT-Lancet Commission Summary Report.

[2] Veganz (2022): Veganz Nutrition Report 2021.

[3] IFIC (2020): A Consumer Survey on Plant Alternatives to Animal Meat. January 30, 2020. International Food Information Council.

[4] V-Label

[5] EVU (2019): Definitions of “vegan” and “vegetarian” in accordance with the EU Food Information Regulation. EVU Position Paper. European Vegetarian Union. July 2019.

[6] Malek, L. & Umberger W.J. (2021): Distinguishing meat reducers from unrestricted omnivores, vegetarians and vegans: A comprehensive comparison of Australian consumers. Food Quality and Preference, 88 (2021), Article 104081

[7] Deutsche Gesellschaft für Ernahrung (2022): Flexitarier — die flexiblen Vegetarier. German Society for Nutrition.

[8] Pike, A. (2021): What is the Flexitarian Diet? Food Insight.

[9] Wikipedia

[10] Dagevos, H. (2021). Finding flexitarians: Current studies on meat eaters and meat reducers. Trends in Food Science and Technology, 114, 530-539. DOI

[11] EIT Food (2021): Plant-based for the Future. Insights on European consumer and expert opinions. White Paper. A qualitative study funded by EIT Food and conducted by the University of Hohenheim. 12 Feb. 2021. pp.: 1-13.

[12] Hedenus, F. et al. (2014): The importance of reduced meat and dairy consumption for meeting stringent climate change targets. Climate Change, 124 (2014), pp.: 79-91

[13] Springmann, M. et al. (2018): Options for keeping the food system within environmental limits. Nature, 562 (2018), pp.: 519-525

[14] IPCC (2019): Climate Change and land: An IPCC special report on climate change, desertification, land degradation, sustainable land management, food security, and greenhouse gas fluxes in terrestrial ecosystems. Chapter 5: Food security. Intergovernmental Panel on Climate Change, Geneva (2019), pp.: 1-200

[15] BBC (2018): What is a ‚flexitarian’ diet? BBC GoodFood.

[16] Politico (2022): Vegan fact check. In: SANTE Press Review. 06-09-22. Polish MEP calls for vegan food in EU canteens. September 6. 2022.

[17] „The Barsac Declaration: Environmental Sustainability and the Demitarian Diet” (2009)

[18] Bánáti D. (2020): Veggie burgers, vegan meats? The ruling of the European Parliament paved the way for meat substitutes with meat denominations. Journal of Food Investigation. Vol. 66. No. 4. / LXVI. évf. 4. szám, pp.: 3166-3174.

[19] EUFIC (2021): What is a plant-based diet and dies it have any benefits? European Food Information Council.

[20] Spenner, P. and Freeman, K. (2021): To keep your customers, keep it simple. Harvard Business Review. (last accessed 06.12.2021).

[21] ABP EatWell Research, interviewed by ProVeg, September 2021.

[22] Smart Protein Project (2021): What consumers want: A survey on European consumer attitudes towards plant-based foods. Country specific insights. European Union’s Horizon 2020 research and innovation programme (No 862957). Available at (last accessed 09.12.2021).

[23] Deloitte (2021): The Future of Food. Challenges & opportunities: Perspectives from consumers and food companies. Deloitte Belgium.

[24] EIT Food (2020): The V-PLACE – Enabling consumer choice in Vegan or Vegetarian Food Products.

[25] FoodNavigator (2021): Do plant-based search trends point to category slowdown? ’The data is predictive of decreased trial’. 01 Sept. 2021.

[26] IFIC (2022): 2022 Food & Health Survey: Diets, Food Prices, Stress and the Power of Gen Z. International Food Information Council. May 18, 2022.

[27] Burlingame, B. (2012): Sustainable diets and biodiversity. Directions and solutions for policy, research and action. IOM Sustainable Diets.

[28] Koch, F. et al. (2019): Meat consumers and non-meat consumers in Germany: A characterisation based on results of the German National Nutrition Survey II. Journal of Nutritional science. Volume 8. The Nutrition Society.

[29] Latvala, T. et al. (2012): Diversifying meat consumption patterns: Consumers’ self-reported past behaviour and intentions for change. Meat Science, 92 (2012), pp.: 71-77

[30] Vanhonacker, F. et al. (2013): Flemish consumer attitudes towards more sustainable food choices. Appetite, 62 (2013), pp.: 7-16

[31] Hielkema, M.H. & Lund, T.B. (2021): Reducing meat consumption in meat-loving Denmark: Exploring willingness, behavior, barriers and drivers. Food Quality and Preference, 93 (2021), Article 104257

[32] Malek, L. et al. (2019): Committed vs. uncommitted meat eaters: Understanding willingness to change protein consumption. Appetite, 138 (2019), pp.: 115-126

[33] Hagmann, D. et al. (2019): Meat avoidance: Motives, alternative proteins and diet quality in a sample of Swiss consumers. Public Health Nutrition, 22 (2019), pp.: 2448-2459

[34] Webster, J. et al. (2022): Risk of hip fracture in meat-eaters, pescatarians, and vegetarians: results from the UK Women’s Cohort Study. BMC Medicine 20, Article number: 275 (2022). DOI

[35] Ipsos Mori (2018): What does it mean to consumers? Ipsos MORI Global Advisor Survey. August 2018 An exploration into diets around the world. pp.: 1-14.

[36] ABC (2019): Vegans a 1 per cent minority in a country of meat eaters, survey finds. 25 Oct 2019.

[37] Askew, K. (2022): Vegetarians often have lower intakes of nutrients linked with bone and muscle health. Foodnavigator.com.

[38] Kateman, B. (Ed.) (2017): Introduction. In: B. Kateman (Ed.): The reducetarian solution: How the surprisingly simple act of reducing the amount of meat in your diet can transform your health and the planet, TarcherPerigee, New York (2017) pp.: xv-xviii

[39] Neff, R.A. et al. (2018): Reducing meat consumption in the USA: A nationally representative survey of attitudes and behaviours. Public Health Nutrition, 21 (2018), pp.: 1835-1844

[40] Rosenfeld, D.L. et al. (2019): Mostly vegetarian, but flexible about it: Investigating how meat-reducers express social identity around their diets. Social Psychological and Personality Science, 194855061986961.

[41] Anon. (2012): Thomson Reuters–NPR Health Poll: Meat Consumption 2012, March 2012. (accessed February 2018). In: R.A. Neff et al. (2018): Reducing meat consumption in the USA: A nationally representative survey of attitudes and behaviours. Public Health Nutrition, 21 (2018), pp.: 1835-1844

[42] Barclay, E. & Aubrey, A. (2016): Eat less meat, we’re told. But Americans’ habits are slow to change. The Salt, 26 February. (accessed February 2018). In: R.A. Neff et al. (2018): Reducing meat consumption in the USA: A nationally representative survey of attitudes and behaviours. Public Health Nutrition, 21 (2018), pp. 1835-1844

[43] FGI Research Inc. (2014): FGI Survey Report 2014 Monday Effect Online Panel. Durham, NC: FGI Research. In: R.A. Neff et al. (2018): Reducing meat consumption in the USA: A nationally representative survey of attitudes and behaviours. Public Health Nutrition, 21 (2018), pp.: 1835-1844

[44] Lacroix, K. & Gifford, R. (2019): Reducing meat consumption: Identifying group-specific inhibitors using latent profile analysis. Appetite, 138 (2019), pp.: 233-241

[45] Lacroix, K. & Gifford, R. (2020): Targeting interventions to distinct meat-eating groups reduces meat consumption. Food Quality and Preference, 86 (2020), Article 103997

[46] Lentz, G. et al. (2018): Gauging attitudes and behaviours: Meat consumption and potential reduction. Appetite, 127 (2018), pp.: 230-241

[47] Salehi, G. (2020): Consumers’ switching to vegan, vegetarian and plant-based (Veg*an) diets: A systematic review of literature. Conference paper. 19th International Congress on Public and Nonprofit Marketing Sustainability: new challenges for marketing and socioeconomic development. DOI

[48] The Flexitarian (2022): What To Eat Now? Welcome to The Flexitarian.

[49] Healthline (2022): The Flexitarian Diet: A Detailed Beginner’s Guide.

[50] U.S.News: The Flexitarian Diet.

[51] Malek, L. & Umberger, W.J. (2021): How flexible are flexitarians? Examining diversity in dietary patterns, motivations and future intentions. Cleaner and Responsible Consumption. Volume 3, December 2021, 100038., DOI

[52] Onwezen, M. et al. (2020): Consumers more inclined to eat ‘alternative’ proteins compared to 2015. Wageningen Economic Research, Wageningen (2020)

[53] Cordts, A. et al. (2013): Consumer Response to Negative Information on Meat Consumption in Germany. International Food and Agribusiness Management Review Volume 17 Special Issue A, 2014 In.

[54] Estell, M. et al. (2021): Plant protein and plant-based meat alternatives: Consumer and nutrition professional attitudes and perceptions. Sustainability, 13 (2021), p. 1478

[55] The Free Library

[56] Wikipedia

[57] Urban Dictionary

[58] Ruby, M.B. (2012): Vegetarianism: A blossoming field of study. Appetite, 58 (2012), pp.: 141-150, 10.1016 / j.appet.2011.09.019

[59] Barr, S.I. & Chapman, G.E. (2022): Perceptions and practices of self-defined current vegetarian, former vegetarian, and non-vegetarian women. Journal of the American Dietetic Association, 102 (2002), pp.: 354-360, 10.1016 / S0002-8223(02)90083-0

[60] Willetts, A. (1997): Bacon sandwiches got the better of me. In: P. Caplan (Ed.), Food, health, and identity, Routledge, New York, NY (1997), pp.: 111-131

[61] Krizmanic , J. (1992): Here’s who we are. Vegetarian Times, 182 (1992), pp.: 78-80

[62] Gossard, M.H. & York, R. (2003): Social structural influences on meat consumption. Human Ecology Review, 10 (2003), pp.: 1-9

[63] Statista (2022): Share of vegetarian and vegan individuals in Italy between 2014 and 2022. Aug 26, 2022.

[64] Demoskop (2014): One in ten Swedes is vegetarian or vegan, according to study. 24 March 2014. Independent.

[65] Statista (2021): Share of Hungarians following a special diet 2019, by type. Apr 19, 2021.

[66] Harris Poll (2019): How many people are vegan? How many eat vegan when eating out? Asks the Vegetarian Resource Group. The Harris Poll.

[67] IBOPE (2018): Pesquisa do IBOPE aponta crescimento histórico no número de vegetarianos no Brasil. Sociedade Vegetariana Brasileira. 20 Mai 2018.

[68] El Milenio (2020): ¿Cuántos Veganos y vegetarianos hay en Argentina? 5 noviembre, 2020.

[69] Max Rubner-Institut (MRI) (2008): Nationale verzehrsstudie II. Ergebnisbericht teil 1 [nationale consumption study II]. Retreived (2008)

[70] Mensink, GBM et al. (2016): Prevalence of persons following a vegetarian diet in Germany. J. Health Monit. 1, pp.: 2-14. DOI

[71] Pfeiler, T.M. & Egloff, B. (2018): Examining the ‘Veggie’ personality: results from a representative. German sample. Appetite 120, pp.: 246–255.

[72] Kunst, A. (2022): Statistica. Feb, 3. 2022.

[73] Ipsos Mori (2018): An exploration into diets around the world. Ipsos MORI Global Advisor Survey. August 2018.

[74] Rosenfeld, D.L. & Burrow A.L. (2017): The unified model of vegetarian identity: A conceptual framework for understanding plant-based food choices. Appetite, 112 (2017), pp. 78-95, 10.1016 / j.appet.2017.01.017

[75] Díaz, E. M. (2017): El veganismo como consumo ético y transformador: un análisis de la intención de adoptar el veganismo ético. PhD dissertation. Universidad Pontificia Comillas. In: G. Salehi (2020): Consumers’ switching to vegan, vegetarian and plant-based (Veg*an) diets: A systematic review of literature. Conference paper. 19th International Congress on Public and Nonprofit Marketing Sustainability: new challenges for marketing and socioeconomic development. DOI

[76] The Vegan Society. (1979): Definition of veganism. Accessed 12 June 2019 In: G. Salehi (2020): Consumers’ switching to vegan, vegetarian and plant-based (Veg*an) diets: A systematic review of literature. Conference paper. 19th International Congress on Public and Nonprofit Marketing Sustainability: new challenges for marketing and socioeconomic development. DOI

[77] NewNutrition Business (2019): 10 Key Trends in Food, Nutrition & Health 2020. In: Vegan olio (2021): How many vegans and vegetarians are in the world today?

[78] Cliceri, D. et al. (2018): The influence of psychological traits, beliefs and taste responsiveness on implicit attitudes toward plant- and animal-based dishes among vegetarians, flexitarians and omnivores. Food Quality and Preference. Vol. 68, September 2018, pp.: 276-291. DOI

DOI: https://doi.org/10.52091/EVIK-2022/2-1-ENG

Received: January 2022 - Accepted: March 2022

¹ Institute of Food Science and Technology, Hungarian University of Agriculture and Life Sciences
² Pécsi Brewery
³ Department of Dietetics and Nutritional Sciences, Semmelweis University, Faculty of Health Sciences

Keywords

taste perception, single nucleotide polymorphism, electric impedance, Body Mass Index, food preferences

2. Introduction

Humans perceive their environment and its relation to them through their sense organs and senses. Five major senses are distinguished: sight, hearing, touch, olfaction, and gustation. There are further channels of sensations also known, e.g. balance, hunger, thirst, pain or discomfort [1]. Perception of taste and flavours are related to the oral and nasal area, including the sense of smell and the trigeminal sensation through the chemosensory system. It belongs to the chemical senses, and it focuses on the perception of the chemicals in our environment. Taste receptors detect the chemicals in the consumed food, which are generally called tastants. These are usually water-soluble molecules, which provide information on the quality and safety of food [2].

Taste perception is a direct contact process, which takes place in the oral cavity. The receptors can be found on the surface of the tongue, in the pharynx, on the palate and in the upper part of the oesophagus. Receptors are organized in the taste buds, which are located in the taste papillae. The sensory information is transferred through the VII; IX. and X. cranial nerves, then the brainstem’s and the thalamus’ nuclei and finally arrives to the frontal operculum and the gustatory cortex of the insula. These areas and the nuclei of tractus solitarius in the brainstem are linked with the hypothalamus and the amygdala, thus probably influencing hunger and satiety, homeostatic reactions to eating and any emotions linked to eating [2, 3].

Bitter taste often triggers a rejection, which is an innate human reaction. On one hand, this aversive reaction is due to the fact, that many bitter tasting compounds (secondary plant metabolites, e.g. alkaloids, some inorganic and synthetic compounds, and in case of food the rancid fat) are toxic, thus consuming them might be harmful, or life-threatening [4].

On the other hand, several bitter tasting compounds are known, which have beneficial effect from the pharmacological or nutritional point of view. These compounds are for example the glucosinolates and their decomposition products, the isocyanates, which are found in cabbage, broccoli or brussels sprouts (all belong to the Brassicaceae family). Coffee, tea, and cocoa contains methylated xanthine derivatives, like caffeine, theophylline, and theobromine; in beers we find the alpha-acids, which originate from the hop and are mainly responsible for the bitter taste. In case of the vegetable species, the bitter taste note might trigger rejection, in the case of the latter products; bitterness is an expected part of their sensory character [5, 6, 7].

In the field of taste perception, five basic tastes are distinguished: sweet, salty, sour, bitter and umami. This last one was accepted as a basic taste following the discovery of its specialized taste receptor in 2002 [8]. Among the five basic quality, the detection of bitter taste is the most complex; the TAS2R gene family, which consists of 25 functional genes, performs its regulation. These genes are coding the TAS2Rs receptors, which structurally bind to given bitter taste compounds (ligands), however in case of several receptors, their ligands is not identified yet [7].

Phenylthiocarbamide (PTC, also known as 1-phenylthiourea) and the 6-n-propylthiouracil (PROP) are colourless or white, crystalline, bitter tasting organic compounds: both have sulphur containing (SCN) functional group. Their use is different: phenylthiocarbamide is used as an industrial additive, colorant, while the propylthiouracil is applied as an antithyroid agent in case of hyperthyroidism [9, 10]. The structure of PTC and PROP shown in the figure 1.

Peculiarity of these two compounds, that they trigger a bimodal reaction in humans: a part of the population is able to perceive their bitter taste, while others not. Its discovery is linked to the chemist Arthur Fox. In 1931, Fox working in a laboratory of the DuPont chemical company accidentally released some fine crystalline PTC to the atmosphere of the room. A colleague working nearby complained on perceiving bitter taste. Fox did not perceive any bitter taste, despite the fact that he directly contacted the fine dust. After this occasion, he tested his family and friends, and categorized the individuals as ‘taster’ or ‘non-taster’. Laurence Hasbrouck Synder geneticist, who identified that the inheritance of the non-taster status is a recessive phenomenon according to the Mendelian genetics [11], strengthened his results.

In the 1960’s the issue of changing of PTC to PROP has risen, because of the strong, sulphuric odour of PTC. In the 1980’s toxicological information also questioned the use of PTC, so researchers started to work with PROP after the comparison of the two compounds and measuring the threshold concentration of PROP [12].

Bartoshuk and co-workers discovered in 1991, that the non-taster group gives relatively homogenous responses, while the reaction of tasters were much more different, and one of their subgroups perceived the bitter taste of PROP much more intensively. Individuals, belonging to that subgroup were called supertasters. The supertaster status is not influenced by the genotype responsible for the taster status, but this discovery resulted in a third type of classification label, medium taster [13].

Taster status is defined by some variations of the genetic domain; in this case the single nucleotid polymorphisms (SNP). SNP’s are DNA sequence variations that affect one nucleotide, which are identified between the genetic domains of two individuals belonging to the same species. Each human genome has a unique SNP pattern, but these changes might be called SNP, if they show up at least in 1% of the total population. SNP’s are usually the results of errors during the DNA replication, or caused by DNA damage. They might be located in genes (both in coding and in non-coding sections), and between genes (intergenetically), thus might cause change in structure or in functions [14].

A database (dbSNP) is collecting these SNP’s, was created in 1999 by the American National Centre for Biotechnology Information and the National Human Genome Research Institute. The number of discovered SNP’s was dramatically increased by the Human Genome Project, which mapped the whole humane genome in 2003, thus resulting a total number of more than 650 million SNP’s in the database up to date (ncbi.nlm.nih.gov/sn/) [15].

In case of PTC or PROP sensitivity the SNP’s of TAS2R28 gene (responsible for bitter taste perception) define, whether the individual perceives bitter taste or not. This gene codes a heptahelical (including seven transmembrane domain), G-protein coupled bitter taste perceiving receptor, which binds to the N-C=S group of the compounds. In this case, the gene contains 1002 nucleotides, from which 3 are functionally missense-coding SNP’s, which cause a non-synonym changes, thus modifying the structure of the coded protein.

The amino acid sequence of this protein is shown in Table 1.

The two most frequent haplotype are the PAV and AVI. Individuals having dominant PAV/PAV, or PAV/AVI diplotype are usually belong to the taster group, while the recessive AVI/AVI diplotypes are non-tasters. With a much lower occurrence (1-5%), AAI, PAI, PVI and AAV haplotypes also occur in some ethnics and populations. In case of PVI and AAV the two status is usually balanced. Based on the studies it might can be concluded that the occurrence of the taster status varies between 55% and 85%, depending on the investigated population [16, 17, 18].

In Hungary, György Forray paediatrician and György Bánkövi mathematician performed a survey on children aged 7-15, in Budapest in 1967. During their study, they applied the Harris-Kalmus method with PTC solutions in order to measure the taste threshold of the children, and thus they concluded their taster status. According to their results 67.8% of the children belonged to the taster group, but they did not find a significant correlation between gender and taster status. They have published their research in the journal Orvosi Hetilap [19].

From the anatomic point of view the polymorphysm has a relationship with the number of taste buds: tasters have more fungiform papillae and more taste pores [12].

The study of PTC and PROP sensitivity’s effect on other factors have started in the 1960’s. The psycho-pharmacologist researcher Roland Fischer (born in Hungary) was the first, who assumed that there might be a relationship between taste perception and food preference [20]. Even until now several researchers study the relationship between taster status (and its haplo- and diplotypes) and body mass index [17, 21], food preference and frequencies of different food consumption (e.g. alcoholic drinks [22, 23], vegetables, especially the Brassicales [24, 25], coffee, tea [26], sweeteners [27]), and some diseases (e.g. Parkinson- disease, gastrointestinal tumours, chronic rhinosinusitis) and their symptoms [28, 29, 30].

3. Scope

The scope of the current research is to investigate correlations between taster status, body composition and the consumption frequency of bitter tasting foods. To achieve that we have performed PTC status survey, bioimpedance-based body composition measurement and used a food frequency questionnaire focused on bitter tasting foods.

4. Methods

Data collection took place in February and March of 2019, participants were volunteers from the Food Science Faculty of Szent István University, and Faculty of Health Sciences, Semmelweis University (students and staff), altogether 170 people. In the taster status survey 170 people participated, in the body composition study we had 96 participants, the food frequency questionnaire (FFQ) was filled out by 170 individuals. All data were recorded anonymously. To link the different type of data, all participants received an individual code. Participants were informed on the data handling according to the general GDPR guidelines (Regulation (EU) 2016/679).

Taster status was defined with PTC-impregnated paper strips (Precision Europe, Northampton, United Kingdom). PTC is present at 20 micrograms per strip. Individuals were assigned to the taster or non-taster category based on their responses after tasting the paper strips.

The body composition was measured with an InBody 770 (InBody USA, Cerritos, California) device, which works based on bioelectric impedance analysis (BIA). This method relies on the different levels of conductivity of the human body’s tissues. The measurement is simple and non-invasive, which provides accurate data for several anthropometric parameters, e.g. percentage of body fat, and its distribution [31]. From the recorded data set we have used the body mass index (BMI, kg/m2), the body fat percentage (PBF, %) and the visceral fat area (VFA, cm2) for further analysis [32, 33, 34]. The FFQ questionnaire involves a list of specific foods or food types, and respondents have to indicate the consumption frequency of these items [35]. Our questionnaire was assembled including bitter tasting food types, consumption frequencies were measured with category scales. The final forms were implemented through the Google Forms platform, data recording was performed online. From the recorded data in this study we report the values concerning coffee and tea consumption, not only the frequency indices, but its type and flavorings also. In order to provide transparent data, the FFQ categories were merged into three major categories (see Table 2).

5. Statistical analyses

To analyse the recorded datasheet, we applied descriptive statistical methods (mean, standard deviation, percentages). Afterwards, data were transformed into category variables, thus suitable for contingency table analysis, multiple correspondence analysis (MCA) and Mann-Whitney test at 5% significance level [36]. XLStat 2020.1.3. and Microsoft® Office Excel® 2016 softwares were used for data analysis.

6. Results

6.1. Demographic parameters

55 males and 115 females participated in this study, so the ratio of genders are 32.5% male and 67.65% female. The youngest respondent was 19 years old, while the eldest was 40 years old, the average age was 23.85±3.05 years. Based on their residence 44.70% lived in the capital of Hungary (Budapest), 55.30% lived in other locations. In the latter group 24.46% lived in Pest County (relating it to the total data that was 13.53%). There were only two Hungarian counties (Zala and Csongrád-Csanád) which were not indicated in any of the respondents.

6.1.1. Taster status

The distribution of taster status data (Table 3.) showed that 72.94% of the respondents were tasters, while 27.06% were non-tasters. The ratio of non-tasters among males was 23.63%, while in case of females it was 28.69%. Based on the results of the contingency table analysis there is no significant relationship between the gender and the taster status (χ²(1, n=170)=0.483, p=0.48).

6.1.1.1. Results of investigation of body composition analysis and its relation to the taster status

Body composition analysis was performed in case of 23 males and 73 females, altogether on 96 individuals. The averaged data of these values are listed in Table 4.

BMI data showed that among the males 11 individuals were obese (BMI from 25.0 to 29.9) and three individuals were overweight (BMI > 30.0). The percentage of body fat values showed obesity in case of 6 people (PBF > 27%), while the visceral fat are was higher than the upper limit of 100 cm2 value in the case of 5 people.

Among the females the BMI showed undernourishment for 5 individuals (BMI < 18.5), 7 were obese, and 3 were overweight. The percentage of body fat data showed that 18 people was obese, and the visceral fat area was higher than 100 cm2 for 15 participants.

Based on the statistical evaluations we did not find significant relationships in case of any of the obesity-indicating parameters and the taster status (BMI: χ²(3, n=96)=0.42, p=0.93; PBF: χ²(1, n=96)=0.45, p=0.50; VFA: χ²(1, n=96)=0.01, p=0.90). The multiple correspondence analysis results on Figure 2 shows that the obesity indicating parameters have relationships with each other. The patterns show that non-tasters are positioned closer to the categories of normal body composition and body weight. Outcomes of the contingency table analysis showed that on the basis of BMI values the ratio of overweight individuals (compared to the normal weighted ones) were significantly higher among males, than among females (χ²(3, n=96)=21.52, p<0.0001).

6.1.1.2. Relationship of coffee consumption and taster status

Among the FFQ respondents, 27 individuals do not consume coffee, so their data was removed from the analysis. Flavouring categories were the following: ‘with milk’ (referring to the use of milk, dairy products, or milk substitutes) and ‘with sweetener’ (referring to the use of any sweeteners (sugar, natural or artificial sweeteners). The ‘mixed’ coffee variety indicated the consumption of both Arabica and Robusta (individually or as a blend). From the 143 consumers 24 individuals drink their coffee black (without sweetener, milk, or milk substitute).

Based on the contingency table analysis there is no significant relationship among taster status and coffee consumption (χ²(1, n=170)=0.02, p=0.88), consumption frequency (χ²(1, n=143)=2,57, p=0,10) and the consumed type of coffee (χ²(3, n=143)=4.21, p=0.24). Similarly there was no significant relationship between the type of consumption, like black (χ²(1, n=143)=0.60, p=0.43), with milk (χ²(1, n=143)=0.28, p=0.59) or sweetened (χ²(1, n=143)=0.17, p=0.67) and the taster status.

The patterns of multiple correspondence analysis (Figure 3) shows that non-tasters consume coffee less frequently than the tasters, and they are unable to specify the type of coffee they consume. When the non-tasters consume coffee, they prefer the sweetened way. Tasters use Arabica type, and they usually do not add sweetener to it. Even if they add milk, it is not necessarily means the addition of sweetener. There is a clear distinction among genders: there are significantly more coffee consumers among women (χ²(1, n=143)=3.65, p=0.05), furthermore females have their coffee with milk and sweetener, while males prefer to drink it without milk (black). This is supported with the outcomes of contingency analysis (drinking coffee black: χ²(1, n=143)=3.46, p=0.05; with milk: χ²(1, n=143)=6.51, p=0.01).

6.1.1.3. Relationship among taster status and tea consumption

Fourteen respondents reported that they do not consume tea, so their results were not analysed. The major categories were ‘Several types including black tea’ (consuming several tea types, including black tea); ‘Several types, but no black tea’ (consuming regularly other type of tea than black). The ‘Sweetened’ label refers to the use of any sweeteners (sugar, artificial and natural sweeteners) for tea consumption.

The ‘Flavouring – Variegated’ category means the use of several ways of flavouring (sometimes with sugar, with lemon and sometimes without sugar), while the ‘Flavouring – More items’ refers to the use of sweetener and lemon. Among the 156 tea consumers 57 individuals drink their tea without flavouring (no sweetener, no lemon added).

During our analysis we did not find significant relationship among taster status and tea consumption (χ²(1, n=170)=1.26, p=0.26), its frequency (χ²(1, n=156)=0.95, p=0.32), the consumed tea types (χ²(5, n=156)=2.57, p=0.76) and the flavouring types of the tea (χ²(4, n=156)=5.13, p=0.27). There were also no significant differences among genders.

Pattern of the multiple correspondence analysis (Figure 4) shows that females and tasters consume tea more frequently, especially black teas and herbal infusions, both flavoured, or non-flavoured. Males and non-tasters consume tea less frequently, they prefer green tea, flavoured with lemon and sweetener, or only with lemon. It was not typical among the respondents that they might consume only fruit infusions.

7. Discussion

The ratio of tasters and non-tasters in our study is in accordance with those reported in the literature, namely 70% vs. 30% in the American and Caucasian population [6, 37]. We did not find relationship between taster status and BMI value, similarly to previous studies [17, 38]. Contrary to these results, some researchers were able to find significant correlations among these parameters [39]. Generally, the results on this field are controversial; there is no consensus among the researchers. Our new outcomes did not show relationships between taster status, body fat percentage and visceral fat area. However, our results showed significant differences between the genders in the overweight BMI category. The reason behind this is the muscle weight of the two genders: the BMI does not differentiate between fat tissue and non-fat tissue and does not take into consideration the distribution of body fat. Therefore, the BMI value’s specificity is high, but its sensitivity is low [40]. In case of the male participants the skeletal muscle mass was significantly higher (Mann-Whitney U=1664, n1 =23, n2 = 73, p<0.0001, two-sided), so more of these individuals were put into the overweight category.

Although we did not find significant relationships in case of coffee consumption, we have observed trends, patterns according to the multiple correspondence analysis. Non-tasters consume coffee less frequently, and they are unable to specify its exact type. These two factors are probably related to each other, since those people who are less interested in coffee consumption, are also less interested in the exact type of coffee. When these individuals consume coffee, they usually add sweeteners, this is less typical in case of tasters, which is supported with literature data [41]. The difference among genders in flavouring or not flavouring the coffee might be related to a social expectation, that the espresso shot is more masculine, while the latte type drinks (e.g. milk espresso) is more feminine [42].

In case of tea consumption, we did not find significant relationships, but several trends were recognized, which are in accordance with the international literature, stating that tasters prefer green tea in a smaller extent [43, 44].

The limitation of our study, that it was not representative from the demographic point of view. During the tests, we have worked with commercially available paper strips, while using PTC or PROP solutions might lead to results that are more precise.

8. Conclusions

Both literature data and our own results show that there might be some level of relationships among genotype, body composition and food choice. It is very likely, that not the genotype, but the phenotype (taster - non-taster) will be the factor which indirectly, through the food choice and food preferences might contribute to obesity, and its related diseases. Since eating habits and food preferences are influenced by other factors (like sociodemographic or psychological ones), these effects might overwrite the expected consequences of the phenotype (preference or aversion toward bitter taste). Furthermore, representative studies with larger sample size are necessary to confirm these hypotheses.

9. Statements

Financial support: The project was supported by the grant EFOP-3.6.3-VEKOP-16-2017-00005. It was also supported by the Ministry of Innovation and Technology grant number ÚNKP-19-3-I-SZIE-65 New National Excellence Program. The authors thank the support of the National Research, Development, and Innovation Office of Hungary (OTKA, contracts No FK 137577).

Contribution of authors: Experimental design: BB, AL, MVB, AG; Data acquisition: BB, DK, AL, MVB, ZK; Data analysis: BB, AG; Preparation of manuscript: BB, AG, ZK; Supervision and approval of manuscript: BB, AG, DK, AL, MVB, ZK.

Conflicts of interest: The authors have no conflicts of interest.

Acknowledgements: Barbara Biró thanks the support of the Hungarian University of Agriculture and Life Sciences, Doctoral School of Food Science. Attila Gere thanks the support of the Premium Postdoctoral Program and the National Research, Development and Innovation Office (project number K134260). The authors thank the cooperation of the test participants.

10. References

[1] Miller-Keane, O’Toole M. (2003): Miller-Keane Encyclopedia & Dictionary of Medicine, Nursing & Allied Health, 7th ed. Saunders, Philadelphia.

[2] Purves D; Augustine G. J; Fitzpatrick D; et al. (2004): Neuroscience, 3rd ed. Sinauer Associates, Sunderland.

[3] Gottfried J. A. (2011): Neurobiology of Sensation and Reward, 1st ed. CRC Press, Boca Raton. DOI

[4] Meyerhof W; Behrens M; Brockhoff A; et al. (2005): Human bitter taste perception. Chemical Senses, 30 (Suppl 1) pp. 14-15. DOI

[5] Wieczorek M. N; Walczak M; Skrzypczak-Zielińska M; et al. (2017): Bitter taste of Brassica vegetables: the role of genetic factors, receptors, isothiocyanates, glucosinolates and flavor context. Critical Reviews in Food Science and Nutrition, 58 (18) pp. 3130-3140. DOI

[6] Tepper B. J. (2008): Nutritional Implications of Genetic Taste Variation: The Role of PROP Sensitivity and Other Taste Phenotypes. Annual Review of Nutrition, 28 pp. 367-388. DOI

[7] Beckett E. L; Martin C; Yates Z; et al. (2014): Bitter taste genetics - the relationship to tasting, liking, consumption and health. Food and Function, 5 (12) pp. 3040-3054. DOI

[8] Kurihara K. (2009): Glutamate: From discovery as a food flavor to role as a basic taste (umami). American Journal of Clinical Nutrition, 90 (3) pp. 1-3. DOI

[9] National Center for Biotechnology Information, PubChem Database. Phenylthiourea, CID=676454. (Hozzáférés: 2020. 05. 20.)

[10] National Center for Biotechnology Information, PubChem Database. Propylthiouracil, CID=657298. (Hozzáférés: 2020. 05. 20.)

[11] Trivedi B. P. (2012): The finer points of taste. Nature, 486 S2-S3. DOI

[12] Bartoshuk L. M; Duffy V. B; Miller I. J. (1994): PTC/PROP Tasting: Anatomy, Psychophysics, and Sex Effects. Physiology and Behavior, 56 (6) pp. 1165-1171. DOI

[13] Hayes J. E; Keast R. S. J. (2011): Two decades of supertasting: Where do we stand? Physiology and Behavior, 104 (5) pp. 1072-1074. DOI

[14] Brookes A. J. (1999): The essence of SNPs. Gene, 234 (2) pp. 177-186. DOI

[15] National Center for Biotechnology Information and U.S. National Library of Medicine Database of Single Nucleotide Polymorphisms (dbSNP). (Hozzáférés: 2020. 05. 20.)

[16] Kim U. K; Drayna D. (2005): Genetics of individual differences in bitter taste perception: Lessons from the PTC gene. Clinical Genetics, 67 (4) pp. 275-280. DOI

[17] Deshaware S; Singhal R. (2017): Genetic variation in bitter taste receptor gene TAS2R38, PROP taster status and their association with body mass index and food preferences in Indian population. Gene, 627 pp. 363-368. DOI

[18] Campbell M. C; Ranciaro A; Froment A; et al. (2012): Evolution of functionally diverse alleles associated with PTC bitter taste sensitivity in Africa. Molecular Biology and Evolution, 29 (4) pp. 1141-1153. DOI

[19] Forrai Gy; Bánkövi Gy. (1967): Phenylthiocarbamid-ízlelőképesség vizsgálata budapesti gyermekpopulációban. Orvosi Hetilap, 108 (36) pp. 1681-1687. DOI

[20] Fischer R; Griffin F; England S; et al. (1961): Taste Thresholds and Food Dislikes. Nature, 191 pp. 1328. DOI

[21] Carta G; Melis M; Pintus S; et al. (2017): Participants with Normal Weight or with Obesity Show Different Relationships of 6-n-Propylthiouracil (PROP) Taster Status with BMI and Plasma Endocannabinoids. Scientific Reports, 7 (1) pp. 1-12. DOI

[22] Choi J. H; Lee J; Yang S; et al. (2017): Genetic variations in taste perception modify alcohol drinking behavior in Koreans. Appetite, 113 pp. 178-186. DOI

[23] Yang Q; Dorado R; Chaya C; et al. (2018): The impact of PROP and thermal taster status on the emotional response to beer. Food Quality and Preference, 68 pp. 420-430. DOI

[24] Shen Y; Kennedy O. B; Methven L. (2016): Exploring the effects of genotypical and phenotypical variations in bitter taste sensitivity on perception, liking and intake of brassica vegetables in the UK. Food Quality and Preference, 50 pp. 71-81. DOI

[25] Mezzavilla M; Notarangelo M; Concas M. P; et al. (2018): Investigation of the link between PROP taste perception and vegetables consumption using FAOSTAT data. International Journal of Food Sciences and Nutrition, 70 (4) pp. 484-490. DOI

[26] De Toffoli A; Spinelli S; Monteleone E; et al. (2019): Influences of Psychological Traits and PROP Taster Status on Familiarity with and Choice of Phenol-Rich Foods and Beverages. Nutrients, 11 (6) pp. 1329. DOI

[27] Yang Q; Kraft M; Shen Y; et al. (2019): Sweet Liking Status and PROP Taster Status impact emotional response to sweetened beverage. Food Quality Preference, 75 pp. 133-144. DOI

[28] Cossu G; Melis M; Sarchioto M; et al. (2018): 6-n-propylthiouracil taste disruption and TAS2R38 nontasting form in Parkinson’s disease. Movement Disorders, 33 (8) pp. 1331-1339. DOI

[29] Choi J; Kim J. (2019): TAS2R38 Bitterness Receptor Genetic Variation and Risk of Gastrointestinal Neoplasm: A Meta-Analysis. Nutrition and Cancer - An International Journal, 71 (4) pp. 585-593. DOI

[30] Dżaman K; Zagor M; Sarnowska E; et al. (2016): The correlation of TAS2R38 gene variants with higher risk for chronic rhinosinusitis in Polish patients. Otolaryngologia Polska - The Polish Otolaryngology, 70 (5) pp. 13-18. DOI

[31] Dubiel A. (2019): Bioelectrical impedance analysis in medicine. World Scientific News, 125 pp. 127-138.

[32] WHO (2000): Obesity: Preventing and managing the global epidemic. WHO Technical Report Series 894, Geneva.

[33] American Council on Exercise (2020): Percent Body Fat Norms for Men and Women. ACE - Tools & Calculators. Hozzáférés: 2020. 06. 18.

[34] InBody USA. InBody 770 Result Sheet Interpretation. (Hozzáférés: 2020. 06. 18.)

[35] Welch A. A. (2013): Dietary intake measurement: Methodology. In: Caballero B. (ed.): Encyclopedia of Human Nutrition, 3rd ed; vol. 2. Academic Press, Oxford, pp. 65-73. DOI

[36] Greenacre M. (2017): Correspondence Analysis in Practice, 3rd ed. Chapman and Hall/CRC, New York. DOI

[37] Tepper B. J. (1999): Does genetic taste sensitivity to PROP influence food preferences and body weight? Appetite, 32 (3) pp. 422. DOI

[38] Yackinous C. A; Guinard J. (2002): Relation between PROP (6-n-propylthiouracil) taster status, taste anatomy and dietary intake measures for young men and women. Appetite, 38 (3) pp. 201-209. DOI

[39] Choi S. E; Chan J. (2015): Relationship of 6-n-propylthiouracil taste intensity and chili pepper use with body mass index, energy intake, and fat intake within an ethnically diverse population. Journal of the Academy of Nutrition and Dietetics, 115 (3) pp. 389-396. DOI

[40] Adab P; Pallan M; Whincup P. H. (2018): Is BMI the best measure of obesity? BMJ, 360 pp. 15-16. DOI

[41] Masi C; Dinnella C; Monteleone E; et al. (2015): The impact of individual variations in taste sensitivity on coffee perceptions and preferences. Physiology and Behavior, 138 pp. 219-226. DOI

[42] Reitz J. K. (2007): Espresso. Food, Culture and Sociology, 10 (1) pp. 7-21. DOI

[43] Chamoun E; Mutch D. M, Allen-Vercoe E; et al. (2018): A review of the associations between single nucleotide polymorphisms in taste receptors, eating behaviors, and health. Critical Reviews in Food Science and Nutrition, 58 (2) pp. 194-207. DOI

[44] Pasquet P; Oberti B; El Ati J; et al. (2002): Relationships between threshold-based PROP sensitivity and food preferences of Tunisians. Appetite, 39 (2) pp. 167-173. DOI

DOI: https://doi.org/10.52091/EVIK-2022/2-4-ENG

Received: March 2022 - Accepted: May 2022

¹ Hungarian Dairy Research Institute Ltd.
² Széchenyi István University, Faculty of Agricultural and Food Sciences, Department of Food Science
³ Széchenyi István University, Wittmann Antal Multidisciplinary Doctoral School in Plant, Animal, and Food Sciences

Keywords

probiotic, Lactobacillus, bile acid, gastric acid, RAPD-PCR, autoaggregation

2. Bevezetés

Probiotics are living organisms that, when used in appropriate amounts, have a beneficial effect on the health of the host organism [1, 2]. They must meet a number of conditions in order to be allowed to display the probiotic designation. Among other things, they need to have an increased tolerance to various body fluids (gastric acid, bile acids, digestive enzymes) and they must stabilize the intestinal microbiota by binding to intestinal epithelial cells through their ability to adhere [3].

There is a growing worldwide demand for probiotic products that have a beneficial effect on health, both in terms of human consumption and the feeding of farm animals. The use of antibiotics for yield enhancement has been banned in the European Union since 2006 [4], and the focus has been even more on probiotics since that date.

Year after year, a large number of bacterial strains are isolated in order to exploit their beneficial effects on health. Complex and costly animal studies must be preceded by a selection system of in vitro experiments that allows the simple, rapid and cost-effective selection of strains, from hundreds or even thousands of isolates, that will hopefully prove probiotic in in vivo studies [5, 6, 7].

Based on the above, the aim of our work was to develop and evaluate in vitro measurement methods that can be used to quickly and efficiently investigate the classical microbiological characteristics, clonality, aggregation ability, as well as the resistance to gastric acid and bile acid of bacterial strains. We sought to answer the question whether the large number of isolates studied by us included clones and strains with potentially beneficial (even probiotic) properties that should be included in further in vitro studies. It was also examined whether the test system was working well or whether it was necessary to optimize the individual steps, as well as what acid and bile acid concentrations the different strains were able to tolerate, and whether there was any correlation between the results of the aggregation test and the acid and bile acid tolerance. Accordingly, of the in vitro tests, the results obtained by the following test methods are presented in our publication:

• Classical microbiological tests (culturing on selective media and determination of colony morphology, Gram staining and subsequent microscopic examination, catalase test, hemolysis test)
• Clonality assay by RAPD-PCR method
• Autoaggregation test
• Presence-absence studies conducted on the surface of solid media supplemented with acid or bile acid

3. Materials and methods

3.1 Isolation, culturing, preservation and storage of bacterial strains

Our studies were conducted with bacterial strains isolated from raw sheep’s milk, curdled milk and sheep’s cheese samples produced in Transylvania. The products had a natural microbiota and no commercially available starter cultures were used for their production. For the preparation of the cheeses, rennet was made by the shepherds from veal stomachs. The goal was to isolate highly efficient probiotic strains that would be later used in the development of probiotic products. Restoration and culturing of the 217 isolates and the control strains were performed under the conditions listed in Table 1.

* De Man–Rogosa–Sharpe agar supplemented with clindamycin and ciprofloxacin

Isolates were preserved and stored in glycerol stock solutions. A strain taken from the surface of MRS–CC agar or MRS pH 5.4 agar was washed into 3 ml of broth with an inoculating loop, and then it was incubated according to the needs of the strain. 300 µl of the grown culture was added to a cryo (freezer) tube, 900 µl of a 60% glycerol solution was added, it was vortexed and frozen in liquid nitrogen for ca. 30 seconds. Storage was conducted at -80 °C in an ultra-freezer.

3.2. Selective culture conditions, its media and their preparation

3.2.1. Physiological saline solution

For the preparation of the diluent used to prepare the decimal dilution series, 8.5 g of NaCl and 1 g of tryptone were weighed and dissolved in 1 L of distilled water. 9.3 ml was added to the test tubes, and they were sterilized in an autoclave at 121 °C for 15 minutes.

3.2.2. Phosphate buffer solution (PBS)

For one liter of distilled water, the following substances were weighed on an analytical balance: 80 g of sodium chloride, 2 g of potassium chloride, 14.4 g of disodium hydrogen phosphate dodecahydrate and 2.4 g of potassium dihydrogen phosphate. Dissolution was aided by a magnetic stirrer and when the solution became particle-free, it was sterilized in an autoclave at, 121 °C for 15 minutes. The solution thus prepared corresponds to PBS with a tenfold concentration, i.e., for further use it has to be diluted as follows: 100 ml of 10× PBS solution is added to 900 ml of distilled water. After proper mixing, the 1× PBS solution is ready for use.

3.2.3. De Man–Rogosa–Sharpe (MRS) agar and broth (pH = 6.2)

Of commercially available MRS broth (VWR, Radnor, PA, USA) or MRS agar (VWR), the quantity recommended by the manufacturer was weighed to analytical accuracy and ten dissolved in distilled water, using a magnetic stirred until dissolved. The pH was adjusted to the desired value (6.2 ± 0.2) with 1 M HCl. Following this, the culture media were sterilized in an autoclave at 121 °C for 15 minutes.

3.2.4. MRS agar (pH = 5.4)

MRS agar (VWR) was prepared according to the manufacturer’s instructions, its pH value was adjusted to 5.4 with 1 M HCl, and then it was sterilized in an autoclave under standard conditions (121 °C, 15 minutes).

3.2.5. MRS agar supplemented with clindamycin and ciprofloxacin (MRS–CC)

In addition to the basic MRS agar, MRS–CC agar also contained two antibiotic stock solutions that could not be sterilized in an autoclave. For the preparation of one of the stock solutions, 2.0 mg of clindamycin hydrochloride (Sigma Aldrich, St. Louis, MO, USA) was dissolved in 10 ml of distilled water, while for the other, 20.0 mg of ciprofloxacin hydrochloride (Sigma Aldrich) was dissolved in 10 ml of distilled water. The antibiotic stock solutions were then filtered through a 0.22 μm pore size membrane filter (Millipore, Burlington, MA, USA) into sterile screw-capped Erlenmeyer flasks. 0.1 ml of clindamycin and 1.0 ml of ciprofloxacin stock solutions were added to the MRS agar cooled to 45 °C under aseptic conditions, using sterile, disposable pipettes (Greiner Bio-One Hungary, Mosonmagyaróvár, Hungary). Thus, the final concentration of clindamycin in the basic MRS agar was 0.1 mg/l, while that of ciprofloxacin was 10.0 mg/l.

3.2.6. CASO agar

CASO agar (VWR) and CASO broth (VWR) were prepared according to the manufacturer’s instructions. Sterilization was performed in an autoclave under standard conditions, at 121 °C for 15 minutes.

3.2.7. Anaerobic culturing

Anaerobic conditions in the course of our studies were ensured as follows: agar plates were incubated in an AnaeroPack Rectangular jar (Merck, Darmstadt, Germany), with the addition of GENbox anaer anaerobic salt (bioMériux, Marcy-l’Étoile, France). Information on the existence of anaerobic conditions was provided by the color change of the Microbiologic Aerotest indicator (Merck) from white to blue.

3.3. Classical microbiological tests

3.3.1. Examination of colony morphology

Macromorphological characteristics of the restored strains were recorded. Among other things, the size, color, surface properties (glossy, matte) of the colonies, as well as the design of the edges of the colonies (regular, irregular, jagged) were observed.

3.3.2. Gram staining

One drop of distilled water, in which a solitary colony was suspended, was added to a degreased slide. The dried smear was stained with crystal violet solution for 2 minutes, then it was treated with lugol solution for 1 minute. Following this, the sample was rinsed with distilled water, then treated with a decolorizing solution for half a minute, which extracted the dye from the Gram-negative cells but not the Gram-positive ones. After another rinsing with distilled water, contrast staining was carried out with safranin for 1 minute. This was followed by rinsing with distilled water, the smears were allowed to dry, and then they were examined under a light microscope (Axio Scope, Carl Zeiss, Oberkochen, Germany) at various magnifications. Performing Gram staining is important because lactic acid bacterial strains with potential probiotic properties are among Gram-positive microbes.

3.3.3. Catalase test

There are microorganisms that produce catalase enzymes that can break down toxic hydrogen peroxide into water and oxygen (2 H₂O₂ = 2 H₂O + O₂). In order to confirm the catalase production of our isolates, colonies of fresh cultures were placed on slides using an inoculation loop, and a drop of 3% H₂O₂ was added. In positive cases, colonies began to visibly bubble. S. aureus strain ATCC 49775 was used as a positive control, which indicated catalase activity with strong effervescence. Catalase-positive strains are not suitable as probiotics for sure.

3.3.4. Hemolysis test

In the coarse of our hemolysis studies, one colony of each freshly restored strain was transferred to Columbia blood agar (Biolab Zrt., Budapest, Hungary). Results were evaluated after 24 hours of anaerobic incubation at 37 °C. S. aureus, which exhibits β-hemolysis on 5% sheep blood culture medium, was again used as a positive control.

3.4. Clonality test

Bacterial DNA was isolated from the bacterial strains using Chelex 100 Resin (Bio-Rad, Hercules, CA, USA), according to the protocol provided by the manufacturer. For the polymerase chain reaction, the reaction mixture containing the Red Taq 2 mM MgCl₂ Master Mixet (VWR), the primer named 1254 chosen by us (Bio-Science, Budapest, Hungary), molecular biology grade AccuGENE water (Lonza, Basel, Switzerland) and the sample (DNA template of the bacterial strains) were measured into a 1.5 ml Eppendorf tube. The samples were analyzed by RAPD-PCR, using the RAPD_03 program of a Mastercycler PCR (Eppendorf, Hamburg, Germany) instrument, the parameters of which are shown in Table 2.

Steps 2 through 4 were carried out 40 times. Following the completion of the program, the amplified DNA molecules were made visible and evaluated by gel electrophoresis. A 1% agarose gel was prepared for the gel electrophoresis. 0.6 g of agarose (VWR) was weighed and dissolved in 60 ml of 1×TBE TRIS-boroacetic acid solution. The solution was boiled until completely homogenized. It was cooled to lukewarm temperature and 6 µl of DNS ECO Safe dye solution (Pacific Image Electronics, Torrance, CA, USA) was added. Meanwhile, it was agitated on a magnetic stirrer, and then the gel was poured. The cooled gel with the dye was poured into the tray. After setting, the tray was placed in the electrophoresis tank, previously filled with gel electrophoresis buffer (1×TBE solution), then the gel comb was removed. The RAPD-PCR reaction products were then added to the individual pockets.

3.5. Investigation of autoaggregation

The test method used was based on the research of Del Re et al. [8], with minor modifications. Our own isolates and control strains were incubated at 37 °C for 18 hours under anaerobic conditions in MRS broth at pH 6.2. The samples were then centrifuged (Eppendorf Centrifuge 5804 R) at 2426 × g for 6 minutes.

The supernatant was discarded, 50 ml of 1×PBS solution was measured onto the bacterial pellets, and they were vortexed (10 sec). They were centrifuged again the supernatant was discarded and the pellet was redissolved in 1×PBS solution. After vortexing, 900 µl of 1×PBS and 100 µl of cell suspension were measured into semi-micro cuvettes (Greiner Bio-One Hungary). Optical density was measured at a wavelength of 600 nm with a BioMate 160 UV-VIS spectrophotometer (Thermo Fisher Scientific; Waltham, MA, USA), and the OD₆₀₀ values were standardized to 0.2 for each sample for the measurement results to be comparable. The set values were checked by OD₆₀₀ measurements. In the case of appropriate values, 4 ml each of bacterial suspension was dispensed into sterile Wassermann tubes, labeled A, B and C for each sample, to ensure three technical replicates. The samples thus prepared were aerobically incubated in Wassermann tubes at room temperature during the assay. Optical density measurements were performed at 0, 5 and 24 hours. At each measurement time point, 200 µl was removed from the top of the bacterial suspension with a wide-tip pipette tip (Axygen, Union City, CA, USA), and it was diluted with 800 µl of 1×PBS solution in a semi-micro cuvette. At each of the three measurement times, the OD₆₀₀ value of each lettered sample was measured three times and the percentage of aggregation was calculated according to the formula given by García-Cayuela et al. [9]

[1 − (A_{measurement time} / A_{0) × 100],}

where: A_{measurement time}: the absorbance value of the cell suspension at the given measurement time (5 h, 24 h); A0: the absorbance value of the cell suspension at time 0 h.

Currently, there is no uniform system for the assessment of autoaggregation. In the course of their studies, Del Re et al. [8] rated strains with an aggregation value of >80% as well aggregating isolates, while strains with a value of <10% were considered non-aggregating.

3.6. Analysis of acid and bile acid tolerance

3.6.1. Acid and bile acid culture media required for the test

To test for acid tolerance, MRS culture medium (VWR) was prepared as described, and it was sterilized in an autoclave at 121 °C for 15 minutes. Next, the pH was adjusted with 1 M HCl under aseptic conditions to the following values: 6.0; 5.5; 5.0; 4.0; 3.0. The sterile culture media were cooled back to 45 °C, and plates were poured into square Petri dishes (Greiner Bio-One Hungary). The MRS culture medium with a pH of 6.0 served as the untreated medium.

Too test for bile acid tolerance, the MRS culture medium (VWR) was prepared according to the manufacturer’s instructions. After sterilization (at 121 °C, 15 min), sterile-filtered porcine bile (Sigma Aldrich) was added to the basic agar cooled back to 45 °C, using a 0.45 μm pore size membrane filter (Thermo Fisher Scientific). Supplementation was performed to achieve final bile concentrations of 0%, 0.1%, 0.2% and 0.5% in the culture medium. MRS agar containing no bile served as a negative control.

3.6.2. Strain restoration and optical density (OD) measurement

Bacterial strains were restored in a pH 6.2 MRS broth as a result of anaerobic incubation at 37 °C for 18 hours. The multiplied cultures formed more or less pellets at the bottom of the Falcon tube, which was evaluated. The cultures were centrifuged (2426 × g, 6 min, room temperature) (Eppendorf Centrifuge 5804 R). The supernatant was discarded, and the samples were redissolved in 1×PBS solution. After a short (10 sec) vortexing, centrifugation was repeated, and the supernatant was discarded again. After redissolution in 1×PBS solution, vortexing was performed for 10 sec, and the optical density of a 10-fold dilution of the suspension was measured with a BioMate 160 UV-VIS spectrophotometer (Thermo Fisher Scientific) at 600 nm. Following the measurement, suspensions with a uniform OD₆₀₀ value of 0.5 were prepared. For accuracy, the OD₆₀₀ values of the suspensions with adjusted cell densities were remeasured.

3.6.3. Presence-absence test

Of the cell suspensions with an OD₆₀₀ = 0.5, 18 (9 technical × 2 biological replicates) × 10 µl were applied to the surface of culture media with different pH values and bile acid contents, and then the plates were incubated at 37 °C for 48 hours, as described in Section 3.2.7.

3.6.4. Process of bile acid and hydrochloric acid treatment

The tested bacterial strains were treated with bile acid and hydrochloric acid, according to the agents added to the culture media. MRS culture media with a pH of 6.0 with no bile or hydrochloric acid served as negative controls. The procedure of the tests is illustrated in Figure 1.

3.6.5. Inoculation and viable cell count determination

Decimal dilution series were prepared from the cultures of both our own isolates and the control bacterial strains, and then 100 µl of each dilution member was spread on the surface of MRS agar plates with a pH value of 6.0. The plates thus prepared were incubated at 37 °C for 72 hours under anaerobic conditions. At the end of the incubation period, the colonies were counted.

4. Results and evaluation

4.1. Classical microbiological tests

The aim of classical microbiological tests was to select Gram-negative, catalase-positive and hemolyzing strains. Using these methods, we were able to eliminate out of the 217 isolates those that did not meet the criteria for probiotics. Table 3 shows a non-exhaustive list of strains with appropriate characteristics based on the results of classical microbiological tests, which were included in subsequent studies (aggregation, acid tolerance and bile acid tolerance studies).

*Colony morphology was examined with strains developed on MRS agar adjusted to a pH value of 6.2.

Of the 217 isolates, 25 catalase-positive and 29 Gram-negative strains were identified. These were also excluded from the clone classes and from individual strains that did not fit into the clone classes after the clonality test. None of the strains hemolyzed on blood agar, so although this test did not help to narrow down the large sample number, it was absolutely necessary to perform it to assess the safety of the probiotic strains.

According to the practice of our group, Sedlačková et al. Also included only Gram-positive, rod-shaped and catalase-negative isolates in their further in vitro studies [10]. In their study, a total of 59 Gram-positive and catalase-negative strains were isolated, of which 7 were isolated from raw milk and 12 from cheese prepared from raw cow’s milk. The colony morphology was found to be similar to that of the colonies of L. acidophilus LA-5.

4.2. Clonality test

RAPD-PCR assays were carried out in parallel with classical microbiological tests. Based on the unique RAPD patterns, the 217 strains were classified into 34 clone classes, of which a gel photograph of clone class 34 is shown in Figure 2; Figure 3 shows several clone classes and individual strains.

In the course of our studies, 57 individual strains were found, which could not be classified into clone classes, so the range of isolates was narrowed down to 91 based on the results of the clonality tests. Gram-negative and catalase-positive isolates were excluded by classical microbiological methods, leaving a total of 34 clone classes and 37 individual strains that could not be classified into clone classes, reducing the starting number to 71 isolates. This greatly aided preselection, as less than one third (32.7%) of the strains remained. As the results of the RAPD-PCR assays are highly dependent on laboratory conditions, precise execution of the method is of paramount importance for the reproducibility of the results [11]. The 1254 primer used allowed the comparison of isolates with similar patterns in the course of the RAPD-PCR assays. This is consistent with the statement of Torriani et al. [12] that primer 1254 is eminently suitable for detecting polymorphisms among L. delbrueckii strains.

4.3. Examination of autoaggregation

In our further studies, the 37 individual strains that could not be classified into clone classes were not included, so the in vitro test series were continued by selecting one bacterial strain from each of the 34 clone classes for the examination of autoaggregation. Our goal was to find well-aggregating (>70% after 5 hours of incubation, >80% after 24 hours of incubation) and non-aggregating (<25%) strains, which then could be included in further acid and bile acid tolerance experiments. It was hypothesized that well-aggregating strains would be more likely to be probiotic, and thus they may also be able to better tolerate acid and bile acid treatment.

Aggregation assay measurements were carried out after 0, 5 and 24 hours. It was decided to perform measurements after 5 hours on the basis of the results of Kos et al. [13], who found that L. acidophilus M92 was already highly autoaggregated after 5 hours. The authors cultured their test strains in MRS broth to preserve some of the cell surface proteins that allow aggregation [13].

The 34 strains were tested in two biological duplicates. Isolates with an aggregation value over 70% were found after 5 hours of treatment, namely the following six E15, E66, E92, E173, E198 and E216. L. acidophilus LA-5 and ATCC 4356 strains used as positive controls also aggregated well (78.2% and 72.1%, respectively) (Figure 4).

It should be mentioned that the well-aggregating strains formed pellets visible to the naked eye at the bottom of the Wassermann tubes, and the upper part of the suspension became clear. The same finding was made by García-Cayuela et al. [9], who isolated 126 L. plantarum strains from cheese samples made from raw milk and carried out preliminary evaluation of the aggregation (sedimentation) ability of the strains in MRS broth with the naked eye, on the basis of which the appearance of snowflake-like aggregates has been reported. Fourteen strains were included in the autoaggregation study, and optical density measurements were performed after 2, 6, 20 and 24 hours. The highest autoaggregation values (28.5-59.5%) were observed after 1 day. Values increased over time, however, they varied from strain to strain. Compared to the aggregation percentages reported by them, we measured higher values (>75%) after 5 hours of incubation.

Xu et al. [14] tested the ability of probiotic and pathogenic strains to self-aggregate. The results obtained after 2 hours of incubation showed that three strains (Bifidobacterium longum B6, L. rhamnosus GG and L. brevis KACC 10553) performed well, with aggregation percentages between 40 and 50%. Tuo et al. [15] examined the aggregation ability of 22 Lactobacillus strains after 5 hours of incubation at 37 °C, and values of 24.2 to 41.4% were obtained. They used L. rhamnosus GG as a positive control, which proved to be the best performing strain with an aggregation value of 41.4%.

Cumulative results of the autoaggregation study of Transylvanian and control strains (after 5 and 24 hours of incubation) are shown in Figure 5. It can be stated that each strain achieved a higher value after 24 hours compared to its result after 5 hours. The probiotic L. acidophilus LA-5 used as a control and L. acidophilus ATCC 4356, which has a well-aggregating phenotype, performed excellently after 24 hours, as reported in the literature (94.1% and 93.5%, respectively). Of the strains belonging to the 34 clone classes, 19 aggregated above 80%. This means that the method developed by us proved to be suitable to distinguish between well and poorly aggregating isolates. In a 24-hour autoaggregation study of Lactobacillus strains isolated from yogurts, Prabhurajeshwar and Chandrakanth [16] measured values that were lower than our results (39.4-52.0%).

4.4. Examination of acid and bile acid tolerancea

Acid and bile acid tolerance was studied using 6 strains (E15, E66, E92, E173, E198, E216) that aggregated well after 5 hours of incubation, and L. acidophilus LA-5 was used as a positive control, the latter strain being probiotic, having adequate aggregation indices and having displayed excellent properties in similar studies in the past [17]. In addition, from our own isolates, strain E10 with less favorable aggregation ability (22.7%) was also included in our studies in order to determine whether there is a correlation between good aggregation and between acid and bile acid tolerance.

Results were recorded after 48 hours of incubation. At the site of the bacterial suspension droplets with a volume of 10 µL inoculated onto the surface of the culture medium, colony growth or the absence of proliferation was observed. It was judged with the naked eye whether the strains were able to visibly proliferate on the surface of the culture media supplemented with acid or bile acid, as well as on the surface of the control culture media (presence-absence test). On the one hand, we were looking to answer the question what acid and bile acid concentrations the individual strains were able to tolerate and, on the other hand, whether there is a correlation between the aggregating ability and the acid or bile acid tolerance. Our results are shown in Tables 4 and 5.

* n = 18 (9 parallels × 2 replicates).
0: no proliferation, +: poor proliferation, ++: moderate proliferation, +++: clearly visible, strong proliferation.

* n = 18 (9 párhuzamos × 2 ismétlés).
0: nincs szaporodás, +: gyenge szaporodás, ++: közepes mértékű szaporodás, +++: jól látható, erőteljes szaporodás.

It can be seen that L. acidophilus LA-5 grew well on MRS culture media with pH values of 6.0, 5.5, 5.0 and 4.0, as well as on MRS culture media containing 0.1% and 0.2% bile acid, thus it proved to be well tolerant of acid and moderately tolerant of bile acid. The control strain showed only a week growth on culture media containing 0.5% bile acid. Neither the control, nor the Transylvanian strains formed colonies on the most acidic (pH = 3.0) MRS culture medium, so solid culture media with pH values of 4.0 and 3.0 proved to be suitable for pre-selection.

Pan et al. [18] maintained a L. acidophilus NIT strain isolated from infant feces in a glycine–hydrochloric acid buffer (pH: 2.0; 3.0; 4.0) for 1, 2 or 3 hours. After the treatment, the bacterial pellet was resuspended, and 20 µL of the suspension of the appropriate dilution members was spread on the surface of MRS agar plates. It was found that after 3 hours of treatment, only 10% of L. acidophilus cells survived. Although our studies were not performed in the same experimental setup, the results may explain why L. acidophilus did not form colonies on a culture medium with a pH value of 3.0. By the addition of 3% whey protein isolate, Vargas et al. [19] achieved that Streptococcus thermophilus ST-M5 and L. delbrueckii subsp. bulgaricus LB-12 survived acid treatment in maximum numbers. The aim of Valente et al. [20] was to assess the in vitro and in vivo probiotic potential of lactic acid bacterial strains (L. plantarum B7 and L. rhamnosus D1) isolated from traditional Brazilian cheeses. Both strains were moderately tolerant of 0.3% of ox bile after 12 hours of incubation. Both isolates B7 and D1 have been shown to be resistant to artificial digestive juices (pH: 2.0 and 3 g/L pepsin) [20].

The physiological concentration of bile acid salts varies between 0.3% and 0.5% in the gastrointestinal tract [21], this is why 0.5% was chosen as the highest bile concentration. The author mentioned also added to his culture medium 0.3, 0.5, 1.0 or 2.0% bile acid salt, and then 10 µL of the stock culture was applied to the surface of the culture medium. Although he worked not with Lactobacillus, but with Lactococcus strains isolated from raw cow’s and goat’s milk and from traditional kefir, his experimental system was similar to ours. Lactococcus lactis strains did not tolerate any of the bile acid concentrations used.

Based on our results, it was found that the selected isolates generally well tolerated the presence of 0.5% bile acid, which in turn was not true for strain E10, which barely proliferated even at the lowest (0.1%) bile concentration. The poor aggregation ability of isolate E10 was accompanied by good acid tolerance and poor bile acid tolerance. The control strain L. acidophilus LA-5, although poorly, but still proliferated on the culture medium containing 0.5% bile. During the procedure used, the strains were exposed to the destructive ingredients not only for a few hours, but they were in contact with them for 48 hours. It is worth mentioning that the negative effects of the destructive agents can be mitigated by the addition of whey protein powder to the culture medium [19]. Presence-absence testing on the surface of the solid culture medium supplemented with acid or bile acid can be considered a relatively fast method, because the required culture media can be prepared easily, dropping onto the surface of the culture medium can be performed quickly, so the results are available in a short time.

5. Conclusions

Our efforts to develop some elements of an in vitro test system for the selection of probiotic bacterial strains have proven to be successful. The steps presented here do not necessarily need to be further refined, because they are already capable of the pre-selection of large sets of isolates. Although primer 1254 has been shown to be a good choice, it will be worth performing the RAPD-PCR reaction with other primers in subsequent clonality assays. There was a positive correlation between the results of the aggregation studies and those of the acid and bile acid tolerance tests, however, to factually establish the probiotic properties of the isolated strains, further in vitro studies and in vivo animal experiments are needed. In order to have an even more efficient selection than at present, it seems worthwhile to supplement the test system with other elements, e.g., antibiotic resistance or antimicrobial activity assays.

6. Acknowledgment

The authors would like to thank the financial support of the project titled “Innovative scientific workshops in Hungarian agricultural higher education”, ID no. EFOP-3.6.3-VEKOP-16-2017-00008, and of the project titled “Developments for intelligent specialization in cooperation between the University of Veterinary Medicine and the Faculty of Agricultural and Food Sciences of Széchenyi István University”, ID no. EFOP-3.6.1-16-2016-00024.

7. Literature

[1] Hill, C., Guarner, F., Reid, G., Gibson, G.R., Merenstein, D.J., Pot, B., Morelli, L., Canani, R.B., Flint, H.J., Salminen, S., Calder, P.C., Sanders, M.E. (2014): The International Scientific Association for Probiotics and Prebiotics consensus statement on the scope and appropriate use of the term probiotic. Nature Reviews Gastroenterology and Hepatology 11 pp. 506-514. DOI

[2] Fijan, S., Frauwallner, A., Varga, L., Langerholc, T., Rogelj, I., Lorber, M., Lewis, P., Povalej-Bržan, P. (2019): Health professionals’ knowledge of probiotics: an international survey. International Journal of Environmental Research and Public Health 16 pp. 3128. DOI

[3] Szakály, S. (2004): Probiotikumok és Humánegészség. Vissza a Természethez! Magyar Tejgazdasági Kísérleti Intézet, Mosonmagyaróvár.

[4] European Parliament, Council of the European Union (2003): Regulation (EC) no. 1831/2003 of the European Parliament and of the Council of 22 September 2003 on additives for use in animal nutrition. Official Journal of the European Union L268 pp. 29-43.

[5] Papadimitriou, K., Zoumpopoulou, G., Foligné, B., Alexandraki, V., Kazou, M., Pot, B., Tsakalidou, E. (2015): Discovering probiotic microorganisms: in vitro, in vivo, genetic and omics approaches. Frontiers in Microbiology 6 pp. 58. DOI

[6] Williams, C.F., Walton, G.E., Jiang, L., Plummer, S., Garaiova, I., Gibson, G.R. (2015): Comparative analysis of intestinal tract models. Annual Review of Food Science and Technology 6 pp. 329-350. DOI

[7] Antal, O., Némethné Szerdahelyi, E., Takács, K. (2020): In vitro humán emésztési modellek alkalmazása a táplálkozástudomány területén (Application of in vitro human digestion models in the field of nutrition science). Élelmiszervizsgálati Közlemények - Journal of Food Investigation 66 pp. 3141-3157.

[8] Del Re, B., Sgorbati, B., Miglioli, M., Palenzona, D. (2000): Adhesion, autoaggregation and hydrophobicity of 13 strains of Bifidobacterium longum. Letters in Applied Microbiology 31 pp. 438-442. DOI

[9] García-Cayuela, T., Korany, A.M., Bustos, I., de Cadiñanos, L.P.G., Requena, T., Peláez, C., Martínez-Cuesta, M.C. (2014): Adhesion abilities of dairy Lactobacillus plantarum strains showing an aggregation phenotype. Food Research International 57 pp. 44-50. DOI

[10] Sedláčková, P., Horáčková, Š., Shi, T., Kosová, M., Plocková, M. (2015): Two different methods for screening of bile salt hydrolase activity in Lactobacillus strains. Czech Journal of Food Sciences 33 pp. 13-18. DOI

[11] Pereszlényi, K. (2019): Tejsavbaktériumok genetikai azonosságának vizsgálata molekuláris markerekkel. Szakdolgozat. Széchenyi István Egyetem, Mosonmagyaróvár.

[12] Torriani, S., Zapparoli, G., Dellaglio, F. (1999): Use of PCR-based methods for rapid differentiation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis. Applied and Environmental Microbiology 65 pp. 4351-4356. DOI

[13] Kos, B., Šušković, J., Vuković, S., Šimpraga, M., Frece, J., Matošić, S. (2003): Adhesion and aggregation ability of probiotic strain Lactobacillus acidophilus M92. Journal of Applied Microbiology 94 pp. 981-987. DOI

[14] Xu, H., Jeong, H.S., Lee, H.Y., Ahn, J. (2009): Assessment of cell surface properties and adhesion potential of selected probiotic strains. Letters in Applied Microbiology 49 pp. 434-442. DOI

[15] Tuo, Y.F., Yu, H.L., Ai, L.Z., Wu, Z.J., Guo, B.H., Chen, W. (2013): Aggregation and adhesion properties of 22 Lactobacillus strains. Journal of Dairy Science 96 pp. 4252-4257. DOI

[16] Prabhurajeshwar, C., Chandrakanth, K. (2019): Evaluation of antimicrobial properties and their substances against pathogenic bacteria in vitro by probiotic lactobacilli strains isolated from commercial yoghurt. Clinical Nutrition Experimental 23 pp. 97-115. DOI

[17] Süle, J., Varga, L., Varga, K., Hatvan, Z., Kerényi, Z. (2022) Probiotikus baktériumtörzsek szelektálására alkalmas kísérleti rendszer egyes elemeinek kidolgozása (Developing basic elements of an experimental system for selection of probiotic bacterial strains). Magyar Állatorvosok Lapja 144 (közlésre benyújtva).

[18] Pan, X.D., Chen, F.Q., Wu, T.X., Tang, H.G., Zhao, Z.Y. (2009): The acid, bile tolerance and antimicrobial property of Lactobacillus acidophilus NIT. Food Control 20 pp. 598-602. DOI

[19] Vargas, L.A., Olson, D.W., Aryana, K.J. (2015): Whey protein isolate improves acid and bile tolerances of Streptococcus thermophilus ST-M5 and Lactobacillus delbrueckii ssp. bulgaricus LB-12. Journal of Dairy Science 98 pp. 2215-2221. DOI

[20] Valente, G.L.C., Acurcio, L.B., Freitas, L.P.V., Nicoli, J.R., Silva, A.M., Souza, M.R., Penna, C.F.A.M. (2019): Short communication: In vitro and in vivo probiotic potential of Lactobacillus plantarum B7 and Lactobacillus rhamnosus D1 isolated from Minas artisanal cheese. Journal of Dairy Science 102 pp. 5957-5961. DOI

[21] Yerlikaya, O. (2019): Probiotic potential and biochemical and technological properties of Lactococcus lactis ssp. lactis strains isolated from raw milk and kefir grains. Journal of Dairy Science 102 124-134. DOI

DOI: https://doi.org/10.52091/EVIK-2022/1-3-ENG

Received: October 2021 – Accepted: January 2022

¹ University of Debrecen, Faculty of Agricultural and Food Sciences and Environmental Management, Institute of Nutritional Science
² University of Debrecen, Doctoral School of Nutritional and Food Sciences

Keywords

lentils, rice, beans, nutrient content, mineral content, sulfur-nitrogen

2. Introduction

The lentil, rice and dried bean varieties commercially available in Hungary show great variety in terms of their county of origin. Shoppers can choose from products from five continents on the shelves of a supermarket. This variability is also reflected in the range of raw materials supplied for communal catering. In order to design the right menu, which can even meet special nutritional needs, it is essential to know the nutrient content with sufficient accuracy, which also includes the mineral content. Food labeling only provides information on the main nutrients, but not on the mineral content. In the course of the study, the nutrient and mineral content of crops originating from different countries and appearing in the wholesale and retail trade in Hungary was determined.

3. General characterization of the studied crops

3.1 Jasmine rice

Rice (Orzyza sativa L.) has been a food since the Neolithic. It reached Europe through the ancient Greeks, Romans and then the Mohammedan peoples [1]. It was first categorized by Carl von Linné in the Species Plantarium in 1753 [2]. The geographical boundaries of current rice production are the latitudes of 53o north and 40o south. In 2018, the world’s total rice production was 782 million tonnes. The largest rice-producing countries are China with 214.08 million tonnes/year, India with 172.58 million tonnes/year and Indonesia with 83 million tonnes/year. Rice production in Hungary was 55-68.5 thousand tonnes/year in the 1970s, 30-47 thousand tonnes/year in the 1980s, 10 thousand tonnes/year since the 1990s [3]. The 1000-grain weight of rice grains is between 12 and 54 g. The quality of rice can also be characterized by the profile index. This parameter characterizes the length and width of the grain, based on which it can be slender (3.0<), medium (3.0-2.1), hemispherical (2.1-1.1) and round (1.0>). Rice is a valuable and popular crop which is well reflected in its more than 8,000 varieties.

Outstanding among the varieties is the long-grained jasmine rice, which, when ready for cooking, has a soft texture and a pleasant aroma. Jasmine rice (KDML 105) produced in the northern and northeastern growing areas of Thailand has an outstanding aroma content [4] and has been bred from the Khao Dow Mali 105 and Kor Kho 15 varieties [5]. Its characteristic is that it grows only once a year, in the rainy season. As a result, the crop ripens at the same time, it is harvested at the same time, and the crop is placed on the market at the same time, resulting in a depressed commercial price. The producer can choose to store his crop (which results in storage costs) or sell it immediately at a lower profit. The nutrient content of jasmine rice is different from that of other rice varieties. According to the database of the US Department of Agriculture, Agricultural Research Service, Food Data Central, it has an energy content of 356 kcal, a protein content of 6.67 g/100g, a fat content of practically zero, and a carbohydrate content of 80 g/100 g [6]. According to the measurements of Chee-Hee Se et al., its energy content is 349 kcal, protein content is 6.98±0.16, carbohydrate content is 79.6±0.30, while the fat content is 0.26±0.07 g/100 g [7]. University of Arkansas student Mills and the instructor Wang in 2020 examined samples from nine varieties native to Thailand but grown in the USA [8].

Their nutrient content measurement results were as follows.

• Protein content (g/100 g): 7.61±0.01; 7.65±0.01; 8.39±0.02; 10.89±0.15; 6.99±0.03; 7.87±01; 9.09±0.02; 6.87±0.00; 8.41±0.13;
• Fat content (g/100 g): 0.015±0.00; 0.19±0.00; 0.56±0.02; 0.54±0.01; 0.31±0.01; 0.43±0.01, 0.4±0.01; 0.26±0.01; 0.45±0.01.

The mineral content of rice varieties measured by other authors is shown in Table 1.

3.2. Lentils

The lentil (LensCulinarisMedik. SSP. Culinaris) is one of the oldest cultivated plants of mankind. It was already cultivated in Central Europe during the Stone Age [9]. It is also mentioned in the Bible, in the first book of Moses (Moses 25:27-34), but stable carbon isotope studies have shown that it was also an important part of the diet in ancient Egypt [10]. Its botanical description in 1787 was carried out by Friedrich Kasimir Medikus, a German physicist and botanist [11]. It is currently grown on five continents, in several countries, including Hungary. According to the United Nations Food and Agriculture Organization (UN FAO), it was grown on about 4.3 million hectares between 2012 and 2014, with an annual world lentil production of 5 million tonnes. In 2017, the size of growing area has already reached 6.5 million hectares [12]. The world’s largest lentil producers are Canada, India and the United States, but Australia is also among the emerging countries. In Europe, the largest lentil-producing countries are Russia, Spain and France. Canada accounts for 40% of world production, India is second with 22% and Turkey is third with 8.1%.

Several varieties of lentils are known. They can be distinguished on the basis of the size of the seed: large, medium and small seed, but also on the basis of the color variation of the seed: brown, yellow, red, black or green lentils. Some varieties have outstanding nutrient content. Masooregy is an Indian large seed red lentil variety. Cultivated by Bahauddin Zakariya University in Pakistan, Masoor 85 has a protein content of 30.41 g/100 g, while the protein content of NIAB Masoor is 30.6 g/100 g, which are outstanding values [13].

The types of lentils commercially available in Hungary are distinguished according to the size and color of the lentil seeds.

In terms of nutrient content, lentils are a protein-rich crop. Comparing the measurement results of several authors, its protein content shows variability. Based on electronic data collection by Ganesan and Bajoun in 2017 from the database of the Department of Agriculture, Agricultural Research Service, Food Data Central operated by the government of the USA, the protein content of lentils is 24.44-25.71 g/100 g [14]. According to the New Nutrient Table (2005) edited by Imre Rodler, the protein content of lentils is 26 g/100 g, its carbohydrate content is 53 g/100 g, and the fat content is 1.9 g/100 g [15].

In 2004, Wang and Daun examined lentil samples grown by several randomly selected Western Canadian producers. The average protein content of the large seed brown lentils examined by them was 27.3 g/100 g, its carbohydrate content was 44 g/100 g, and the fat content was 1.2 g/100 g, while the average protein content of the medium seed brown lentils was 25.9 g/100 g, its carbohydrate content was 44.8 g/100 g, and the fat content was 1.0 g/100 g [16]. The mineral content of lentils measured by other authors is shown in Table 2.

3.3. Beans

Among legumes, the most important plants for the food industry belong to the Fabaceae family. These are peas, beans, lentils, lupine and peanuts.

Beans (Phaseolus vulgaris L.) belong to the family of Papilionaceae. They are native land is considered to be the areas of Mexico and Guatemala 500-1,800 m above sea level, and they came to Europe after the discovery of the New World. The oldest bean finds are almost 10,000 years old and were found in Peru [17]. They are characterized by a great richness of form, and there are several variants within the species. Their flowers have a well-developed, zygomorphic, characteristic butterfly shape with bilateral symmetry. The fruit is a multi-seeded, flattened or cylindrical pod. The pods contain 4 to 8 seeds, depending on the variety. The color of the seed is varied.

In Hungary, two species are grown: common beans, also known as garden beans (Phaseolus vulgaris L.), and creeper beans or butter beans (Phaseolus coccineus L.). World bean production (Phaseolus vulgaris L.) was 11.23 million tonnes in 1961 and 30.43 million tonnes in 2018, which means a nearly threefold increase. In 2018, the world’s largest bean-producing country was India with 6.22 million tonnes, followed by Brazil with 2.62 million tonnes. The volume produced in Hungary has decreased significantly in the last 50 years: while in 1962 the amount of beans produced was nearly 31 thousand tonnes, by 1990 this number had decreased to 3,546 tonnes. The low point was 2010 with 545 tonnes. From 2014 to the present, the average production has been 1,500 to 1,700 tonnes/year [3]. The amount of nutrients found in beans depends on the variety, the climate, the growing area and the cultivation technology. Beans can be stored for years under appropriate conditions without damage [18].

In terms of nutrient content, the most valuable component of ripe beans is protein. Bean proteins are made up of valuable essential amino acids such as lysine, methionine, cysteine and tryptophan.

The nutrient and mineral content of beans measured by other authors is shown in Table 3.

3.4. Sulfur-nitrogen ratio

The sulfur content of foods is not very often determined, although its amount is an important indicator of sulfur-containing amino acids. Sulfur occurs I the soil in organic and inorganic forms. The most important sulfides in the soil are FeS2 (pyrite) and FeS, and the most important sulfates are gypsum (CaSO4·2H2O) and anhydrite (CaSO4). The amount of organically bound sulfur varies in direct proportion to and is strongly correlated with the humus content of the soil: r=0.84. The organic sulfur content of the soil varies from soil type to soil type [31]: in chernozem soils it is 75%, while in podzolic soils it is approximately 50%. The sulfur replenishment in different soil types also depends on air pollution and on industrial sulfur emission. Between 1972 and 1974, the amount of sulfur precipitating from the air due to air pollution in the central parts of Great Britain reaches 50 kg/year/ha [38]. In 1980, A. Martin compared the results measured by several authors over a period of 20 years and found that the amount of sulfur precipitating from the air varied by geographical area and season [39]. In 1988, J. Archer calculated the amount of sulfur in agricultural production areas in East England as generally at least 30 kg/year/ha, based on several measurements carried out on the 1970s [36]. In the United Kingdom, sulfur dioxide emissions have been steadily declining for the last 50 years. Emissions today are about 3% of those measured in the 1970s [40]. Plants usually absorb most of the sulfur through the roots in the form of sulfate, or through the stomata of the leaves. The absorbed sulfate is reduced in several steps. It first reacts with ATP to form adenosine phosphosulfate (APS), while inorganic phosphate (Pan) is released from ATP:

SO₄^2- + ATP → APS + P_an

With the help of ATP, APS is phosphorylated a second time to phosphoadenosine phosphosulfate. The sulfate thus bound is reduced to sulfite by an enzyme carrying a hydrogen atom, then it is then further reduced by NADPH to sulfide-S (S2-), which reacts with serine to form cysteine [32].

Sulfur occurs in plants in both inorganic and organic forms. There is no sharp boundary between the two fractions, sulfate is the S reserve of the plant. If the sulfur supply of crops is increased, the inorganic sulfur content will increase primarily, and organically bound sulfur to a lesser extent. The absorbed sulfur is stored by the plant in the form of sulfate, which is reduced to an organic form as needed. First, the plant meets its organic sulfur demand, only then the absorbed sulfur is stored [33]. The greatest significance of sulfur is that it is a constituent of peptides, proteins and lipids, and a building block of sulfur-containing amino acids. Of the sulfur compounds, the amount of cysteine and methionine is significant. The presence of these is essential in various food and feed raw materials. The specific role of sulfur is manifested in enzymes and coenzymes containing the SH group. 90% of SH groups are linked to proteins in plants. In the case of sulfur deficiency, the protein synthesis of the plant is disturbed, the amount of soluble nitrogen compounds increases and the protein content decreases [20]. Then relationship between the elements can be demonstrated by statistical methods. In studies on bread wheat, a correlation of r=0.73 (α=0.01) was measured in the relationship between the sulfur and nitrogen content [21]. In Poland, studies on beans (Phaseolus vulgaris L.) have been carried out for several years, during which the protein content of the crop was increased by 13.6% with adequate sulfur supply [37]. In Northern Germany, in a study on rapeseed, the nitrogen uptake of the plant was increased by 40% with adequate sulfur supply [34].

As no comprehensive studies had been found in the literature by us regarding the composition of the individual crops, we consider it important to provide basic data on this element for the food raw materials studied as well.

4. Materials and methods

4.1. Raw materials

Samples were purchased in December 2020, by random subjective selection in various retail stores in Hungary. The selection criteria was for the samples to differ according to their country of origin or distributor. Seven types of brown lentils from five different distributors and countries of origin, four types of jasmine rice from four different distributors and three countries of origin, and four types of white beans from four different distributors and three countries of origin were analyzed and their nutrient contents were determined. Summary tables of the results, descriptive statistical analyzes and graphs were prepared in Microsoft Excel.

The samples analyzed are listed in Table 4 based on their crop characteristics.

4.2. Analytical method

Analytical tests were performed on the basis of the food analysis guidelines of the Hungarian Standards Institution (HSI) and the Hungarian Food Codex at the Faculty of Agriculture, Food Science and Environmental Management Instrument Center of the University of Debrecen. Analytical methods are listed in Table 5. To determine the protein content, the amount of nitrogen measured was multiplied by 6.25.

Note: “MSZ” means “Magyar Szabvány = Hungarian Standard”

Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES) is a quantitative elemental analysis method, for which samples were prepared according to a study published by professors and lecturers of the University of Debrecen [35].

4.3. Statistical method

Statistical analysis was performed using descriptive statistical analysis, and regression analysis was performed using Microsoft Excel.

5. Results and evaluation

5.1. Results of rice samples and their evaluation

The results of the nutrient analysis of the rice samples are shown in Table 6, and the descriptive statistical evaluation of the data is presented in Table 7. The measurement results and their statistical evaluation of the mineral analysis are summarized in Table 8 and 9, the measurement results demonstrated in Figures 1 and 2.

*Moderately variable

The protein (6.47-7.04 m/m%), carbohydrate (77.49-78.94 m/m%) and dietary fiber (4.52-4.91 m/m%) content of the rice samples was homogeneous. In the case of the samples tested, the mineral content exhibited moderate or high variability. The highest variability was observed when measuring the Na (CV%=27.19) and Fe (CV%=26.43) content. It is worth noting that the Na content was highest in the sample from Cambodia and lowest in the samples from Thailand, while the Fe content was highest in one of the samples from Thailand and lowest in the sample from Cambodia and another sample from Thailand.

5.1.1. Sulfur-nitrogen ratio

The relative S/N ratios of the rice samples are shown in Table 10.

The fifth row of the table is based on the lowest ratio (R2) and shows the percentage difference from it.

The strongest correlation is found between samples R2 and R4; their country of origin is Thailand, but the final value is also close for sample R1. The largest deviation was found in the case of sample R3, with its country of origin being Vietnam. The correlation indicates the similar agrochemical characteristics of the soil and the cultivation area.

5.2. Results of lentil samples and their evaluation

The results of the nutrient analysis of the lentil samples are shown in Table 11, and the descriptive statistical evaluation of the data is presented in Table 12. The results and statistical evaluation of the mineral analysis are summarized in Tables 13 and 14 and Figures 3 and 4.

*Moderately variable

*Moderately variable

In the case of the samples tested, several values showed moderate variability in terms of mineral content. The protein (19.91-24.05 m/m%), carbohydrate (53.46-56.86 m/m%) and dietary fiber (18.76-20.14 m/m%) content of the lentil samples was found to be statistically homogeneous, but there was a 15% difference between the lowest and highest values in percentage terms. Of minerals, phosphorus (CV%=13.3) and copper (CV%=10.67) exhibited moderate variability. The other minerals were statistically homogeneous. It is important to note that the amounts of Mg, Mn, Na, S and Zn were statistically homogeneous, but the values were in the upper part of the statistical range (CV%~10). The protein, carbohydrate and dietary fiber contents were all homogeneous.

The amount of phosphorus had the highest coefficient of variation. This value was lowest for the samples from Russia and Poland, while it was highest for the produce grown in Ukraine. In general, lentils grown in Canada and Poland had the highest mineral content, while it was lowest in the lentils grown in Russia and Ukraine. The relative S/N ratios of the lentil samples are shown in Table 15.

In the case of medium seed samples (L1, L2, L5, L6, L7), the values for samples L1, L2 and L7 were closest to each other. These samples came from Ukraine and Russia. In the case of samples L4 and L5, the cultivation area was the same, but sample L4 was large seed brown lentils, while sample L5 was medium seed lentils, the values of which were well separated from the values of other cultivation areas. Sample L3 (Canada) was also large seed lentils, with an S/N ratio different from the other values.

5.2.1. Regression analysis of sulfur-nitrogen ratio

Regression analysis of the amount of sulfur and nitrogen was performed only in the case of lentils, given the larger number of samples. Our regression statistics measurement data are shown in Table 16, the line characteristic of the correlation and the equation of the line are shown in Figure 5.

The correlation between sulfur and nitrogen content can also be measured in wheat studies, and the correlation is r=0.7515 [25], which affects the amount of cystine as a gluten component, and thus the quality o the finished product [26].

5.3. Results of dried bean samples

The results of the nutrient analysis of the bean samples are shown in Table 17, and the descriptive statistical evaluation of the data is presented in Table 18. The results of the mineral analysis and their statistical evaluation are summarized in the Tables 19 and 20 and demonstrated in Figures 6 and 7.

*Moderately variable / **Highly variable

*Moderately variable / **Highly variable

The protein (18.8-19.96 m/m%), carbohydrate (57.55-58.14 m/m%) and dietary fiber (23.27-24.33 m/m%) content of the white bean samples was statistically homogeneous, but with the exception of magnesium, the results showed moderate or high variability in terms of the amount of minerals. Moderately variable were the phosphorus (CV%=16.67), sulfur (CV%=15.55), iron (CV%=14.84), manganese (CV%=16.02) and zinc (CV%=19.26). Highly variable were calcium (CV%=27.41), copper (CV%=21.44), potassium (CV%=21.15) and sodium (CV%=22.44).

The highest mineral content was measured in the case of beans grown in Hungary, while the lowest was measured in the case of beans grown in Ethiopia and Ukraine.

5.4. Comparison of the measured values with the reference values

The measured data were compared with the values in the New Nutrient Table edited by Imre Rodler [15]. Percentage differences in the nutrient and mineral contents are shown in Table 21 for rice, Table 22 for lentils and Table 23 for beans.

*Results with different orders of magnitude.

The amounts of copper, iron and manganese differ by orders of magnitude from the values of the New Nutrient Table (data highlighted in brick red in Table 21). After comparing the values in the New Nutrient Table with the results in Table 1, measured by other authors (Cu=2.6-9.96 mg/kg, Fe=1.83-31.5 mg/kg and Mn=0.07-10.73 mg/kg), it can be stated that the difference is several orders of magnitude compared to the results found in international literature. Because of these differences, it is necessary and recommended to update available basic data periodically.

In the case of the samples, all samples had a lower protein content than the reference value, while all but one sample had a higher carbohydrate content than the reference value [15]. In terms of minerals, the amount of calcium was significantly higher, while the amounts of potassium, magnesium, sodium, phosphorus and zinc were less than the reference values [15].

In the case of the lentil samples, the protein content was significantly lower, while the carbohydrate content was higher. Of minerals, the amounts of calcium, copper and iron were significantly higher, while the amounts of magnesium and sodium were significantly lower than the reference values [15].

The protein content of the bean samples was on average 13.1% lower, and the carbohydrate content was slightly reduced. Of minerals, the amount of calcium was significantly higher, the amounts of iron, magnesium, zinc and phosphorus were higher, while the amounts of manganese and sodium were lower than the reference values [15].

6. Summary, conclusions

In our measurements, on average, the protein content of the crops was lower and their carbohydrate content was higher than the corresponding reference values [15]. With respect to macronutrients, the change is the same as the change in the nutrient content of crops measured by other authors and associated with the climate change of Earth [22, 23, 24]. Strong variability was measured for several minerals. Based on our measurements, our hypothesis was accepted that the significant diversity of the crops by country of origin is reflected in their nutrient content. In the case of lentils, a correlation was found between the amounts of S and N (r=0.88). The S/N ratios observed were almost the same within countries or for neighboring countries, but were different for samples from different cultivation areas. Comparing the results of our measurements with the data in the New Nutrient Table, orders of magnitude differences were found [15].

Based on our work, it is recommended that the variability of the nutrient and mineral contents is taken into account. Adequate nutrient knowledge of the raw materials is essential for accurate menu planning. Providing adequate nutrition for short- and long-term tasks, or for long-term health and availability, can be of great or even strategic importance to those performing work accompanied by high physical or mental strain (such as those working in law enforcement or members of the armed forces). The nutrients needed for these stresses can be provided by a natural diet, but knowledge and availability of accurate data is also a prerequisite.

It is recommended that changes in nutrient content according to the place of origin are taken into account already in the planning and execution phase of raw material procurement procedures.

7. References

[1] Tanács L. (2003): Élelmiszeripari nyersanyag és áruismeret, ISBN: 963 482 612 1, Szegedi Tudományegyetem, Szegedi Élelmiszeripari Főiskolai kar. pp. 41-45.

[2] Linneaei, C. (1753): Species Plantarum. p. 333.

[3] Food and Agriculture Organization of the United Nations (2020).

[4] Yoshihashi, T., Nguyen, T., T., H., Kabaki, N. (2004): Area Dependency of 2-Acetyl-1-Pyrroline Content, Aromatic Rice Variety, Khao Dawk Mali 105. Food Technology 38 (2) pp.105-109. DOI

[5] Pitiphunpong, S., Champangern, S., Suwannaporn, P. (2011): The Jasmine Rice (KDML 105 Variety) Adulteration Detection Using Physico-Chemical Properties. Chiang Mai Journal of Science 38 (1) pp.105-115.

[6] US Department of Agriculture, Agricultural research Service (2019): Food Data Center Search Results. FDC ID: 1827323.

[7] Se C-H., Chuah, K., A., Mishra, A., Wickneswari, R., Karupaiah, T. (2016): Evaluating Crossbred Red Rice Variants for Postprandial Glucometabolic Responses: A Comparison with Commercial Varieties. Nutrients 8 (5) p. 308. DOI

[8] Mills, A. K., Wang, Y-J. (2020): Characterization of jasmine rice cultivars grown in the United States. Discovery, The Student Journal of Dale Bumpers College of Agricultural, Food and Life Sciences 21 (1) pp. 59-68.

[9] Borsos J., Pusztai P., Radics L., Szemán L., Tomposné L. V., (1994): Szántóföldi növénytermesztéstan, Kertészeti és Élelmiszeripari Egyetem, Kertészeti Kar, Budapest.

[10] Touzeau, A., Amiot, R., Blichert-Toft, J., Flandrois, J-P., Fourel, F., Grossi, V., Martineau, F., Richardin, P., Lécuyer, C. (2014): Diet of ancient Egyptians inferred from stable isotope systematics Journal of Archeological Science 46 pp.114-124. DOI

[11] Medikus, F., K. (1787): Vorlesungender Churpfälzischenphysicalisch-ökonomischen Gesellschaft. 2. Bd., p. 361.

[12] Food and Agriculture Organization of the United Nations (FAO), (2019): The Global Economy of Pulses, Rome.

[13] Zia-Ul-Haq, M., Ahmad, S., Aslam, Shad, M., Iqbal, S., Qayum, M., Ahmad, A., Luthria D., L, Amarowicz R. (2011): Compositional studies of lentil (Lens culinaris Medik.) cultivars commonly grown in Pakistan. Pakistan Journal of Botany 43 (3) pp. 1563-1567.

[14] Ganesan, K., Xu., B.: (2017): Polyphenol-Rich Lentils and Their Healt Promoting Effects. International Journal of Molecular Sciences 18 (11) p. 2390. DOI

[15] Rodler I. (2005): Új tápanyagtable, ISBN:963-226-009-0 Medicina Könyvkiadó Rt. Budapest pp.251-258.

[16] Wang, N., Daun, J., K. (2005): Effects of variety and crude protein content on nutrients and anti-nutrients in lentils (Lens culinaris). Food Chemistry 95 (3) pp. 493–502. DOI

[17] Gentry, H., S. (1969): Origin of the common bean, Phaseolus vulgaris. Economic Botany 23 pp. 55-69. DOI

[18] Tanács L. (2003): Élelmiszeripari nyersanyag- és áruismeret, 74-78.

[19] U.S. Department of Agriculture, Agricultural Research Service (2016): Food Data Central Search Results. FDC ID: 747441.

[20] Loch J., Nosticzius Á. (2004): Agrokémia és növényvédelmi kémia, Mezőgazda kiadó.

[21] Liu, Y., Ohm, J-B., Harelend, G., Wiersma, J., Kaiser, D. (2011): Sulfur, Protein Size Distribution, and Free Amino Acids in Flour Mill Streams and Their Relationship to Dough Rheology and BreadmakingTraits. Cereal Chemistry 88 (2) pp. 109–116. DOI: DOI

[22] Myers, S., Zanobetti, A., Kloog, I., Huybers, P., Leakey, A., Bloom, A., Carlisle, E., Dietterich, L., Fitzgerald, G., Hasegawa, T., Holbrook, N., Nelson, R., Ottman, M., Raboy, V., Sakai, H., Sartor, K., Schwartz, J., Seneweera, S., Tausz, M., Usui, Y. (2014): Increasing CO2 threatens human nutrition. Nature 510 (7503) pp. 139-142. DOI

[23] Zhu, C., Kobayashi, K., Loladze, I., Zhu, J., Jiang, Q., Xu, X., Liu, G., Seneweera, S., Ebi, K., L., Drewnowski, A., Fukagawa ,N.. K., Ziska, L.. H. (2018): Carbon dioxide (CO2) levels this century will alter the protein, micronutrients, and vitamin content of rice grains with potential health consequences for the poorest rice-dependent countries. Science Advances 4 (5) p. 1012. DOI

[24] Dong, J., Gruda, N., Lam, S.. K., Li, X., Duan, Z. (2018): Effects of Elevated CO2 on Nutritional Quality of Vegetables: A Review. Frontiers in Plant Science 9 (924). DOI: DOI

[25] Győri Z. (2005): Sulphur content of winter wheat grain in long term field experiments. Communications in Soil Science and Plant Analysis 36 (1-3) pp. 373-382. DOI: DOI

[26] Moss, H., J., Wrigley, C., W., Macrithie, F., Randall, P., J. (1981): Sulphur and nitrogen fertilizer effects on wheat. II. Influence on grain quality. Australian Journal of Agricultural Research 32 (2) 213–226. DOI

[27] Verma, D., K., Srivastav P., P. (2017): Proximate Composition, Mineral Content and Fatty Acids Analyses of Aromatic and Non-Aromatic Indian Rice. Science Direct, Rice Science 24 (1): pp. 21-31. DOI

[28] Jiang, S., L., Wu, J., G., Thang, N., B., Feng, Y., Yang, X., E., Shi, C., H. (2008): Genotypic variation of mineral elements contents in rice (Oryza sativa L.). European Food Research and Technology  228 pp. 115-122. DOI

[29] Vunain, E., Chirambo, F., Sajidu, S., Mguntha, T., T. (2020): Proximate Composition, Mineral Composition and Phytic Acid in Three Common Malawian White Rice Grains. Malawi Journal of Science and Technology 12 (1).

[30] Timoracká, M., Vollmannová, A., Ismael, D., S. (2011): Minerals, Trace Elements And Flavonoids Content In White And Coloured Kidney. Potravinarstvo 5 (1 ). DOI

[31] Grunwaldt, H., S. (1961): Untersuchungen zum Schwefelhaushaltschleswig-holsteinischer Böden. Diss. d Landw. Fakultätder Christian-Albrechts-Universität Kiel.

[32] Kylin, A. (2006): The effect of light, carbon dioxide, and nitrogen nutrition on the incorporation of S from external sulphate into different S-contaning fractions in Scenedesmus, with special reference to lipid S. PhysiologiaPlantarum 19 (4) pp.883-887 DOI

[33] Saalbach, E., Kessen, G., Judel, G., K. (1961): Über den Einfluß von Schwefel auf den Ertag und die Eiweißqualtät von Futterpflanzen. Z. Pflanzenernähr Düng budenkunde 93 (17). DOI

[34] Schnug, E., Haneklaus, S., and Murphy, D. (1993): Impact of sulphur fertilization on fertilizer nitrogen efficiency. Sulphur in Agriculture; The Sulphur Institute Washington, DC; 16,. pp.31–34.

[35] Kovács B., Győri Z., Prokisch,J., and Daniel, P. (1996): A study of plant sample preparation and inductively coupled plasma atomic emission spectrometry parameters. Communications in Soil Science and Plant Analysis 27 (3–4). pp.207–215. DOI

[36] Archer, J. (1988): Crop Nutrition and Fertilizer Use; Farming Press Ltd., Ipswich, pp.57-64.

[37] Głowacka, A., Gruszecki, T., Szostak, B., Michałek, S. (2019): The Response of Common Bean to Sulphur and Molybdenum Fertilization, International Journal of Agronomy 2019 Article ID 3830712, 8. DOI

[38] OECD (1977). The OECD programme on long range transport of air pollutants. Paris, OECD.

[39] Martin, A. (1980): Sulphur in air and deposited from air and rain over Great Britain and Ireland, Environmental Pollution (Series B) I pp.177-193

[40] Department for Enviroment Food & Rural Affairs (United Kingdom) (2020): Emissions of air pollutants in the UK – Sulphur dioxide (SO2) (Hozzáférés 2021. 09. 10.)

DOI: https://doi.org/10.52091/EVIK-2022/1-5-ENG

Received: September 2021 – Accepted: December 2021

¹ South Ural State Agrarian University, Troitsk, Russian Federation
² South Ural State University (national research university), Chelyabinsk, Russian Federation

Keywords

feed ration; stinging nettle; rabbit meat; nutritional value; biochemical indicators.

2. Introduction

Recently, the production of new improved food products providing a person with complete proteins, essential nutrients, micronutrients and vitamins has become increasingly important worldwide. At the same time, the production of cheap, dietary meat and meat products enriched with vitamins has become very relevant. One of the ways to obtain them is a perpetual modification through adjusting animal rations [1, 2, 3].

Most countries have recently experienced a sharp increase in the rabbit meat production. Great importance is attached to the development of rabbit breeding in Russia as one of the sources of providing the population with dietary meat [4]. Rabbit meat can be compared to chicken meat by its juiciness, softness, taste and digestibility. Rabbit meat is low in fat, connective tissue, cholesterol and sodium salts, it is fine-fibred and highly digestible [5, 6]. One of the possible ways of a perpetual modification of rabbit meat is the introduction of stinging nettle (Urtica dioica) into the ration of rabbits [2].

Nettle as a weedy plant is widespread throughout the European part of Russia, the Caucasus and Western Siberia, and is found in Eastern Siberia, the Far East and Central Asia. Nettle belongs to high-yielding plants, it is a good source for obtaining highly nutritious grass meal containing many nutrients. The chemical composition of grass, hay, and grass meal from stinging nettle is presented in Table 1 [7, 8, 9, 10, 11, 12, 13, 14, 15]. In early spring, nettle contains twice more vitamin C than oranges and lemons, and it contains as much provitamin-A as carrots and has much vitamin K – up to 400 IU/kg. Notably, large quantities of ascorbic acid are contained in fresh leaves and stalks of nettle (up to 269 mg/kg), when nettle is dried, it is destroyed, and its amount decreases markedly [11, 16, 17].

Many authors recommend using young nettle in raw, scalded, or boiled form, in the form of infusions, extracts, hay, grass meal or powders as an additive to the ration of pigs, cattle and poultry to increase their resistance, vitality and productivity, as well as to accumulate vitamin A and mineral elements in processed products [18, 19, 20].

The purpose of the research was to study the influence of the supplementary feeding with the stinging nettle hay on the balanced ration, biochemical indicators, nutritional value, and keeping quality of rabbit meat.

3. Materials and methods

The objects of the research were: fodder base, live animals, and carcasses of rabbits of the Soviet chinchilla breed. This breed is the most widespread and promising in Russia among the combined rabbits, it is characterized by a high plasticity and good adaptability to various climatic and feed conditions [21].

The studies covered 30 rabbits aged from 3 to 6.5 months. 3 groups of animals were formed: control and two experimental groups, 10 animals each. The rabbits of the control group received a ration consisting of oats, wheat bran, carrots, cabbage, cereal-and-legume hay and natural land grass (in the summer months) [22]. 5% of the coarse fodder in terms of nutritional value were replaced with stinging nettle hay for the rabbits of experimental group I, and 25% were replaced for experimental group II.

The rabbits were selected by the principle of pairs of analogues [23, 24], and were kept in group cages in identical conditions. All the animals were clinically healthy. The feeding rations for all the rabbit groups were balanced by all nutrients according to the current standards [25]. To make rations, a comprehensive zootechnical analysis of the used fodder was carried out with the help of the IR-4500 infrared analyzer. The content of basic nutrients in the fodder was determined as follows: nitrogen – by Kjeldahl method, fiber – by Kebenerg and Shtoman method, sugar – by the ebuliostatic method (method for the determination of sugars based on the reduction of copper; Ed.), calcium – by the trilonometric method (complex formation titrimetric method using murexide indicator; Ed.), phosphorus – by the colorimetric method, ash – by the dry ashing method [26].

To prepare nettle hay, young nettle was mowed in May-June and dried in the shade to a moisture content of 12.16%, because rabbits usually do not eat freshly cut nettle [27, 28].

Control weighing of the animals was carried out once a week. The rabbits were slaughtered at the age of 6.5 months after fasting for 24 hours. After stunning, the carcasses were bled white by cutting off the heads. The skins were cased, the extremities were removed along the carpal and tarsal joints, the carcasses were eviscerated and trimmed. The meat was left at a temperature of 15±5 °C for 18 hours for maturation.

When assessing biochemical indicators and nutritional value of the rabbit meat, we determined the content of moisture, fat, protein, and ash, including macronutrients, vitamin C and amino acids. The moisture content was determined in the rabbit meat by drying to a constant weight in an oven at a temperature of 150±2 °C. Meat fat was determined using a Soxhlet extraction apparatus. The amount of protein was determined by mineralization of a meat sample with sulfuric acid according to Kjeldahl, distillation into a solution, followed by titration. The total amount of ash was found by burning organic matter with a free air access. The content of iron, copper, zinc, cobalt, magnesium, manganese and lead in the rabbit meat was determined by dry mineralization followed by atomic absorption spectrophotometry. The content of vitamin C in the meat extract was determined by titration with 2,6-dichlorophenolindophenol. Ion exchange chromatography on an amino acid analyzer was used to examine amino acids in the rabbit meat [29].

The nutritional, energy, and biological value of the studied rabbit meat was calculated according to the generally accepted methods [30, 31].

Studying the keeping quality of the meat when stored for 3 months at –18 °C, we investigated a combination of organoleptic, physico-chemical and microbiological indicators. The amount of volatile fatty acids was determined by distillation of the meat in the presence of sulfuric acid, followed by titration of the distillate with potassium hydroxide. The method for determining ammonia and ammonium salts is based on the ability of ammonia and ammonium salts to form a yellow-brown substance with Nessler’s reagent. The essence of determining the primary protein breakdown products in the broth lies in the deposition of proteins by heating and the formation of copper sulfate complexes with the products of the primary breakdown of the depositing proteins in the filtrate. The acid index characterizing the degree of fat spoilage was found by alkali titration of molten fat [32].

Statistical processing of the research results was carried out according to a regulated method [33] using the Microsoft Excel XP and Statistica 8.0 software suites. The dependencies in the experimental data were searched using the variance analysis [34].

4. Results and discussion

4.1. Studying the rabbit ration balance

All the experimental animals received the same fodder during the experiment (with the exception of nettle hay), taking into account their age and live weight. The rabbits received oats, grass-and legume hay, natural land grass in summer; carrots and cabbage were added to the ration three times a week. The animals of the control group did not receive stinging nettle hay, 5% of the coarse fodder in terms of nutritional value were replaced with the nettle hay for the rabbits of experimental group I, and 25% were replaced for experimental group II. The rations were compiled taking into account the age of the animals – for the animals aged 90-120 days and for the rabbits older than 120 days (Table 2).

The rations of all the experimental rabbits aged 90-120 days were balanced by the main nutrients, except for the high fiber content (1.6-1.7 times more than the norm). The rations of the experimental groups (for 1 animal per day), as opposed to the control group, contained slightly less feed units (-1 and -6 g of feed units*) and, accordingly, less energy value (-0.01 and -0.07 MJ), but significantly more raw protein (+1.2 and +5.4 g per 100 g of feed units) and digestible protein +5.8 and +26.7 g per 100 g of feed units), and carotene (+0.5 and +2.0 mg per 100 g of feed units).

* 1 feed unit: energy content of 1kg of medium dried oats

The rations for the older rabbits (1 animal per day), similar to the rations for the young rabbits, were characterized by a high fiber content – by 1.4-1.5 times. The rations of the experimental groups contained more raw protein (+1.2 and +7,0 g per 100 g) and digestible protein (+5.9 and +33.8 g), carotene (+0.5 and +2.6 mg) and slightly less energy value (-0.01 and -0.06 MJ) than in the control group. The increased content of crude and digestible protein, carotene, and vitamin E in the rations of the experimental groups throughout the entire experiment was preconditioned by the addition of the stinging nettle hay rich in these substances.

Note: The two values in parentheses always refer to the two nettle portions: 5% and 25%, respectively.

However, due to the lower energy value of the stinging nettle hay than the grass-and-legume hay, we observed a decrease in the nutrition value in the rations of the experimental groups as compared to the control group.

The ration structure for the rabbits aged 90-120 days contained coarse fodder – 29-31%, succulent fodder – 2-3%, green fodder – 27-28%, concentrates – 39-41%. The ration for the rabbits older than 120 days contained coarse fodder – 32-34%, succulent fodder – 21-22%, concentrates – 45-46%, there was no green fodder.

As it can be seen from the consumption of fodders over the entire experiment, breeding of the rabbits with the introduction of 5% (per 0.13 kg of fed units) and 25% (per 0.05 kg of fed units) of the stinging nettle hay in terms of nutritional value of coarse fodders as compared to the content in the traditional ration was characterized by the lest feeds per 100 g of the gain by feeding 25% nettle.

4.2. Studying the biochemical indicators and nutritional value of rabbit meat

Rabbit meat is close to chicken by its dietary indicators and surpasses it by the content of protein. There is no significant difference in the chemical composition of rabbit meat of different breeds. The chemical composition of meat depends more on the animal age and the feeding level [5, 6].

The content of basic nutrients was determined in the muscle tissue of matured rabbit meat (Table 3).

*P<0,05; **P<0,001

It was established that there was less water in the meat of the animals from experimental group I than in the control group (-10,38%, P<0.001) and experimental group II (by 6.66%, P<0.001). The mass fraction of protein in the rabbit meat of experimental group I is larger than in the rabbit meat of the control group by 0.81% (P<0.05), and experimental group II – by 1.30% (P<0.01). The fat content of the muscle tissue in the rabbits of the control group and experimental group I did not differ significantly, while in experimental group II this indicator was lower than in the control group by 0.4% (P<0.05). The content of vitamin C and ash in all the samples was out of statistical control.

The data of the variance analysis covering the chemical composition of the boneless rabbit meat are presented in Table 4.

*P<0.05; **P<0.01; ***P<0.001

It was determined that the introduction of nettle had the maximum influence on the water content; the amount of protein and fat in the muscle tissue of the rabbit meat 2.1 and 3.6 times less depended on the supplementary feeding with nettle feeding than the water content of the meat.

Based on the chemical composition, we calculated the energy value of the rabbit meat ignoring perinephric fat (Table 5).

It was revealed that the caloric density of the muscle tissue in the rabbits of the control group and experimental group I differed insignificantly (by +4.187 kJ/g i.e., +0.7%), while the muscles of the rabbits in the control group contained more amount of fat, and experimental group I – more protein. The reduced nutrient value of the muscle tissue of the rabbits of experimental group II (by -20.93 and -25.12 kJ/g i.e., -3.4 and -4.1%) is preconditioned by the low content of protein and fat in the muscles. The increased caloric density of the boneless meat and bone meat in experimental group I (+75.36 kJ/g i.e., +9.6%; +62,80 kJ/g i.e., +10.6%) and experimental group II (+20.93 kJ/g i.e., +2.9%; +12.56 kJ/g i.e., +2.1%) was determined by large deposits of fat on the shoulders and groin.

Note: The two values in parentheses always refer to the two nettle portions: 5% and 25%, respectively.

Based on the aforesaid, it follows that the introduction of 5% of the nettle hay into the rabbit ration resulted in a decrease in the moisture content and an increase in the protein content in the rabbit meat, and the introduction of 25% – ensured a lower fat content of the rabbits’ muscle tissue. The energy value of the rabbit meat increased in proportion to the nettle dosage in the ration due to a larger deposition of fat on the shoulders and groin.

The mineral composition of the rabbit meat samples is shown in Table 6.

*P<P0,05; **P<0,01

It was established that the meat samples of the rabbits in experimental group I was distinguished by a high content of iron and zinc. There is 1.27 mg/kg more (20.66%) iron in it as compared to the meat of the control rabbits, and 0.83 mg/kg (12.61%) more than in the meat of experimental group II, and it has more zinc by 4.20 mg kg (51.33%; P<0.01) and 1.27 mg/kg (11.41%), respectively. The samples of the rabbit meat from experimental group II contain 2.93 mg/kg (35.83%; P<0.01) more zinc than the control group. The highest copper content was observed in the rabbit meat of experimental group II – by 0.07 mg/kg (48.61%) as compared to the control group, and by 0.04 mg/kg (19.16%) as compared to experimental group I.

The least cobalt content was found in the meat of the rabbits of the experimental groups: in the samples of group II this indicator is less than in the control group by 0.14 mg/kg (32.73%), and in the meat of group I – by 0.03 mg/kg (5.91%).

The proportion of magnesium was the same in all the rabbit meat samples, and the proportion of manganese was 2.2 times higher in the meat of experimental group II (P<0.01), and 0.09 mg/kg more (85.85%; P<0.05) in the meat of experimental group I than in the control group. As compared to the meat of the control animals, the lead content in the rabbit meat of experimental group II decreased by 0.10 mg/kg (19.31%), of experimental group I – by 0.07 mg/kg (13.41%).

The results of the variance analysis covering the mineral composition of the rabbit meat are shown in Table 7.

*P<0,05

We can see from the obtained data that the addition of nettle to a larger extent influenced the content of zinc and manganese. In contrast, the effect of nettle is approximately 4 times less on the content of iron and copper and 5-6 times less – on the amount of cobalt, lead and magnesium.

Thus, the introduction of nettle into the rabbit ration increased the content of zinc, manganese, iron and copper in the meat. Moreover, the content of zinc and iron was higher at a dosage of 5% of the nutritional value of coarse fodder than at a 25% dosage, and the amount of manganese and copper grew with an increase in the concentration of nettle in the ration. There was less cobalt and lead in the rabbit meat proportional to the share of nettle in the fodder.

The biological value of rabbit meat is judged by the content of complete and incomplete proteins and their amino acid composition. With the animals ageing, the content of complete proteins in rabbit meat increases, while the content of incomplete proteins decreases. The meat of animals aged 4-5 months may considered to be most complete [6].

To assess the protein quality, we carried out an amino acid analysis of the rabbit meat, the results of which are shown in Table 8.

It was determined that the content of such amino acids as threonine, serine, proline, alanine, valine, and lysine in the meat was practically the same. As compared to the control rabbit meat, the meat of the rabbits of experimental group I contained slightly more methionine (+9.77 g/kg i.e., +40.79%), isoleucine (+8.27 g/kg i.e., 7.22 times more), phenylalanine (+13.54 g/kg i.e., 6.37 times more), glutamic acid (+6.84 g/kg i.e., 62.40%), glycine (+0.29 g/kg i.e., +16.23 %) and histidine (+3.08 g/kg i.e., 24.38%). The rabbit meat of experimental group II had a higher amount of the same amino acids as compared to the control group: methionine (+2.1 g/kg i.e., 8.77%), isoleucine (+2.81 g/kg i.e., 3.1 times more), phenylalanine (+6.76 g/kg i.e., 3.68 times more), glutamic acid (+6.03 g/kg i.e., 55.01%), glycine (+0.13 g/kg i.e., 7.39%) and histidine (+7.82 g/kg i.e., 61.91%). The amount of some amino acids varied randomly; both high and low indices were present in the groups. This concerned aspartic acid, tyrosine and leucine, while arginine was found only in one sample from the control group and experimental group I.

Note: The two values in parentheses always refer to the two nettle portions: 5% and 25%, respectively.

The amino acid content in the rabbit meat samples was subjected to the variance analysis (Table 9).

*P<0.05

Judging by the indicator of the nettle’s power of influence on the amino acid content of meat, the amount of phenylalanine, isoleucine, glutamic acid, tyrosine, leucine, methionine and arginine changed most of all due to feeding with nettle.

As a result of the amino acid analysis, we revealed a tendency of prevailing such essential amino acids as methionine, isoleucine and phenylalanine, as well as non-essential amino acids – glutamic acid and glycine in the meat of the rabbits grown on the ration with the introduction of 5% of nettle of the nutritional value of coarse fodder as compared to the 25% dosage and the control group. The histidine content increased in proportion to the concentration of nettle in the rabbit ration.

4.3. Studying the keeping quality of meat

All the frozen rabbit meat samples corresponded to fresh meat by the organoleptic indicators. The surface of the carcasses had a pink drying crust, the fat tissue was yellowish white, the muscles in the section were slightly moist, leaving slight moisty spots on the filter paper (which is typical of frozen meat), pale pink with a reddish tint. The muscles are dense, elastic, the body hole is typical of fresh rabbit meat, the broth is transparent, and its smell was acceptable.

During the chemical analysis of rabbit freshness, we assessed such indicators as the content of ammonia and ammonium salts, the content of primary protein breakdown products in the broth, the amount of volatile fatty acids (VFA), and the fat acidity value in the adipose tissue.

When determining ammonia and ammonium salts, after adding Nessler’s reagent, the meat extract from all the samples remained transparent and acquired a greenish-yellow color, which corresponded to the requirement of fresh meat. The rabbit meat broth from all the samples remained transparent after the addition of copper sulfate, which indicated the absence of primary protein breakdown products in the meat and, therefore, the meat freshness. The amount of volatile fatty acids (VFA) in the muscle tissue and the fat acidity value of the rabbit meat samples are shown in Table 10.

* According to Pronin and Fisenko (2018), **P<0.05

As it can be seen from the above data, the content of VFA in all the rabbit meat samples corresponded to fresh meat, but the differences between the groups were unreliable in terms of this indicator. However, the following tendency was observed: VFA in the meat of experimental group I is 0.22 mg KOH (-6.16%) less, and in experimental II it is 0.23 mg KOH (+3.36%) more than in the meat of the control group. As for the acidity value, the fat of the rabbits from all the groups corresponded to the premium-grade fresh fat. The fat acidity value in the rabbit meat of experimental group I and control group did not differ significantly, while in the rabbit meat of experimental group II this indicator was 0.24 mg KOH (-28.16%, P<0.05) lower than in the control group. The influence of the addition of the stinging nettle hay into the rabbit ration on the amount of VFA and the fat acidity value of the meat is shown in Table 11.

*P<0.05

It was established that feeding with nettle did not influence the amount of VFA in the rabbit meat after 3 months storage, and the change in the fat acidity value reliably depended on the supplementary feeding with nettle.

Thus, the introduction of nettle into the rabbit ration had a positive effect on the keeping quality of the rabbit meat when stored for 3 months at a temperature of -18 °C. With an increase in the proportion of nettle in the ration, the rabbits’ fat acidity value decreased, i.e., its food safety is increased. A 5% dosage of the nettle hay in the rabbit ration of the nutritional value of coarse fodder resulted in a slight decrease in VFA in the meat as compared to a 25% dosage of nettle. This allowed us to suggest that the lower dosage of nettle in the ration had a better effect on the safety of the muscle tissue in the rabbit meat than the higher dose.

5. Conclusions

The introduction of the studied dosages of the stinging nettle hay into the ration led to an increase in the content of crude (+3.5 and +20.3%), digestible protein (+4.4 and +22.8%) and carotene (+3.3 and +22.7%). In this case, growing rabbits with a dosage of 5% (per 0.13 kg of feed units) and 25% (per 0.05 kg of feed units) of the stinging nettle hay of the nutritional value of coarse fodders was characterized by the least feeds per 10 g of the gain as compared to the content in the traditional ration (1.17 kg of feed units). The introduction of 5% of the nettle hay into the rabbit ration as compared to the control group: influenced a decrease in the moisture content (the power of effect is -10,38%), an increase in the content of protein (the power of influence of +34.2%), zinc (the power of influence of +35.6%) and manganese (the power of influence of +34.2%) in the rabbit meat; we revealed a tendency of prevailing essential amino acids: methionine, isoleucine, phenylalanine, as well as non-essential amino acids – glutamic acid and glycine in the meat.

The introduction of 25% of the nettle hay into the ration resulted in a lower fat content (the power of effect is -19.7%) and a higher manganese content (the power of effect is +34.2%) in the muscle tissue of rabbits.

We revealed a positive influence of the supplementary feedings with nettle on the keeping quality of meat when stored for 3 months at -18 °C due to slightly smaller amounts of volatile fatty acids (-6.2%) and the fat acidity value (-28.2%) than the control samples.

Note: The two values in parentheses always refer to the two nettle portions: 5% and 25%, respectively.

6. Conflicts of interest

We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the content of this paper.

7. Acknowledgement

The work was supported by Act 211 of the Government of the Russian Federation, contract No. 02.A03.21.0011.

8. References

[1] Tsaregorodtseva, E. V. (2015): The creation of meat products with a given level of quality, nutritional and biological value. Bulletin of Mari State University. Series: Agricultural Sciences. Economic Sciences, 2(2), pp. 63-67.

[2] Lisitsyn, A. B., Chernukha, I. M., Lunina, O. I., Fedulova, L. V. (2016): Legal framework and scientific principles for creating functional meat-based food products. Bulletin of Altai State Agrarian University, 12(146), pp. 151-158.

[3] Zolotareva, E. L. (2018): The global meat market: current development trends and prospects for Russia’s participation. Bulletin of Kursk State Agricultural Academy, 3, pp. 167-171.

[4] Velkina, L. V. (2019): Global rabbit breeding trends. Agricultural Economics of Russia, 3, pp. 93-98.

[5] Komlatsky, V. I. (2016): Rabbit meat based on the modern profitable technology. Animal Breeding of the South of Russia, 5(15), pp. 2.

[6] Ruleva, T. A. (2016): Rabbit meat as a dietary product. Its chemical composition and organoleptic characteristics. Innovation Science, 3-4, pp. 61-64.

[7] Evdokimova, R. S., Yutkina, I. S., Karimova, A. Z. (2014): The distribution of some elements in the soil and tissues of stinging nettle (Urtica dioica L.). Volga Scientific Bulletin, 11-1 (39), pp. 23-25.

[8] Trineeva, O. V., Safonova, E. F., Slivkin, A. I. (2014): Determination of fat-soluble vitamins in plant objects by the TLC method. Sorption and Chromatographic Processes, 14, pp. 144-149.

[9] Trineeva, O. V., Slivkin, A. I. (2015): A study of the micronutrient composition of stinging nettle leaves. Scientific news of Belgorod State University. Series: Medicine. Pharmacy, 22(219), pp. 169-174.

[10] Trineeva, O. V., Slivkin, A. I., Dmitrieva, A. V. (2015): Determination of the amount of free amino acids in the leaves of stinging nettle. Questions of Biological, Medical and Pharmaceutical Chemistry, 5, pp. 19-25.

[11] Yutkina, I. S., Evdokimova R. S., Karimova, A. Z. (2014): The distribution of micronutrients and ascorbic acid in the soil and tissues of stinging nettle (Urtica dioica). Science and Modernity, 32-1, pp. 68-74.

[12] Balagozian, E. A., Pravdivtseva, O. E., Orekhova, A. D., Kurkin, V. A. (2016a): A comparative phytochemical analysis of raw materials of stinging nettle and its main impurities. Questions of Biological, Medical and Pharmaceutical Chemistry, 12, pp. 15-18.

[13] Balagozian, E. A., Pravdivtseva, O. E., Orekhova, A. D., Kurkin, V. A. (2016b): A comparative phytochemical analysis of raw materials of stinging nettle and its main impurities. Questions of Biological, Medical and Pharmaceutical Chemistry, 12, pp. 15-18.

[14] Pekh, A. A. (2019): The content of micronutrients in stinging nettle depending on the habitat in the Republic of North Ossetia-Alania. News of the Mountain State Agrarian University, 2, pp. 38-41.

[15] Tatvidze, M. L., Kupatashvili, N. N. (2018): A study of some biologically active substances of dry leaves of stinging nettle. Theoretical and Applied Science, 6 (62), pp. 157-161. DOI

[16] Trineeva, O. V., Safonova, E. F., Slivkin, A. I. (2017): The validation of the method for determining ascorbic acid using high performance thin-layer chromatography. Sorption and Chromatographic Processes, 3, pp. 414-421.

[17] Guskov, A. A., Rodionov, Yu. V., Anokhin, S. A., Glivenkova, O. A., Plotnikova, S. V. (2018): The technology of the vacuum-pulse extraction of soluble substances from nettle and hops. Innovative Engineering and Technology, 2(15), pp. 23-27.

[18] Kalinkina, O. V., Sychev, I. A. (2017): The influence of stinging nettle polysaccharide on blood and blood formation. Bulletin of Tver State University. Series: Biology and Ecology, 1, pp. 62-68.

[19] Korzh, L. (2017): Enriching the rations of laying hens. Animal Breeding of Russia, 4, pp. 17.

[20] Filippova, O. B., Frolov, A. I., Maslova, N. I. (2019): The biological basis for the stimulation of the resistance of calves using the modern technology for dairy cattle breeding. Science in Central Russia, 1(37), pp. 61-70.

[21] Zhitnikova, Yu. Zh. (2004): Rabbits: breeds, breeding, management, care. Rostov-on-Don, Fenix, pp. 256.

[22] Ryadchikov, V. G. (2012): The basics of nutrition and feeding of farm animals. Krasnodar, Kuban State Agrarian University, pp. 328.

[23] Viktorov, P. I., Menkin, V. K. (1991): Methodology and organization of livestock experiments. Moscow, Agropromizdat, pp. 112.

[24] Zabelina, M. V. (2014): Research methods in private zootechnics. Saratov, Saratov State Agrarian University, pp. 60.

[25] Kalashnikova, A. P., Fisinina, V. I., Scheglova V. V., Kleimenova, N. I. (2003): Norms and rations of feeding farm animals. Reference manual. 3rd revised and enlarged edition. Moscow, Russian Agricultural Academy, pp. 456.

[26] Kirilov, M. P., Makhaev, E. A., Pervov, N. G., Puzanova, V. V., Anikin, A. S. (2008): Methodology for calculating the exchange energy in fodders based on the content of crude nutrients. Dubrovitsy, All-Russia Research Institute for Animal Husbandry of the Russian Agricultural Academy, pp. 382.

[27] Balakirev, N. A., Nigmatulin, R. M., Sushentsova, M. A. (2015): Fodders and feeding rabbits. Moscow, Kazan, Nauchnaya Biblioteka Publishing House, pp. 268.

[28] Kahikalo, V. G., Nazarchenko, O. V., Balandin, A. A. (2019): A practical guide to fur farming and rabbit breeding. St. Petersburg, Lan Publishing House, pp. 328.

[29] Antipova, L. V., Glotova, I. A., Rogov, I. A. (2001): Methods of studying meat and meat products. Moscow, Kolos, pp. 376.

[30] Gotsiridze, N., Tortladze, L. (2001): Determination of the biological value of rabbit meat. Zootechnics, 8, pp. 31-32.

[31] Martinchik, A. N., Maev, I. V., Yanushevich, O. O. (2005): General nutritionology. Moscow, Medicine, pp. 392.

[32] Pronin, V. V., Fisenko, S. P. (2018): Veterinary and sanitary expertise with the basics of technology and standardization of animal breeding products. St. Petersburg, Lan Publishing House, pp. 240.

[33] Vasilieva, L. A. (2007): Statistical methods in biology, medicine and agriculture. Novosibirsk, Novosibirsk State University, pp. 320.

[34] Yudenkov, V. A. (2013): Variance analysis. Minsk, Business offset, pp. 76.

DOI: https://doi.org/10.52091/EVIK-2021/4-1-ENG

Received: September 2021 – Accepted: November 2021

¹ Hungarian University of Agriculture and Life Sciences, Buda Campus, Institute of Food Science and Technology, Food Science Research Group

Keywords

microalgae, protein, composition, feeding, food use, climate change, carbon footprint

2. Introduction

By 2050, the population of Earth will grow to nearly 10 billion, an increase of about 25% compared to today’s population. In addition, the significant depletion of our Earth’s water resources also makes it necessary to restructure our diet, as the amount of water needed to produce 1 kg of food is 13,000 liters for cattle, 5,520 liters for chicken, while only 50 liters for peas or lentils. All this means that we can expect a significant increase in the price of foods of animal origin, which will mean that we will have to reduce the proportion of them in our diet significantly.

The different plant protein sources make a positive contribution to the protection of the environment and the fight against climate change due to their more efficient use of water. On the other hand, legumes require 30 to 70% less synthetic fertilizers due to their nitrogen-binding properties, they increase soil power and have a positive effect on soil biology. It is also a known fact that due to nutrient transformation losses, the production of 1 kg of animal protein requires at least 6 to 16 times more cultivable area. In addition, the carbon dioxide footprint of the production of foods, especially of those based on beef, is about 10 times more than that of plant-based foods.

The structure of food consumption in Europe is characterized by a 59% proportion of the daily protein intake coming from protein sources of animal origin (meat, fish, milk), with proteins of plant origin representing only 41% of the total. More than 50% of the latter is wheat protein. As a result, a few cereals (wheat, corn, rice) could become staple foods, leading to geographical homogeneity of foods, dietary monotony and nutritional imbalances, increasing the risk of micronutrient deficiencies, overweightness and chronic obesity, as well as NCDs (Non Communicable Diseases), including cardiovascular diseases, stroke, cancer and diabetes.

Based on all this, it is becoming increasingly important to identify and investigate alternative plant and other protein sources, in addition to the protein sources mentioned above, which can contribute to meeting the protein needs of an increasing human population, as well as to addressing the unbalanced nutritional status of them.

An important group of protein crops are legumes (e.g., dried beans, runner beans, chickpeas, horse beans, lentils, grass peas, black-eyed peas, dried peas, autumn and spring peas) with a high protein content of 20 to 40% on average. Dry leguminous seeds are rich in protein and lysine, but low in sulfur-containing amino acids. At the same time, field crops with good nutritional values but low protein content (e.g., sunflower, canola, corn, sorghum, rice, wheat) are low in lysine and rich in sulfur-containing amino acids. Taking into account the positive nutritional values of the two plant groups, products containing complete plant protein can be developed by their combined use.

The protein content of various crops (Table 1) may show significant variability not only between species but also within a given species. In addition, protein content can also be altered by environmental factors and the food processing technology.

Other alternative protein sources include single cell proteins (SCPs) produced by fermentation technologies, seaweeds living in saltwater, duckweed species living in freshwater and various insect species. Protein content values of the different sources can vary widely depending on the species, the cultivation technology and the nutrient supply (Table 2).

3. Characterization and occurrence of microalgae

Algae, also called seaweeds, are eukaryotes capable of photosynthesis. Algae represent one of the oldest life forms on Earth, having existed on our planet for about 3 billion years. They produce one-third of Earth’s living matter and about 50% of its organic carbon [1]. These plants have survived all geological epochs and climate changes. Algae still account for 90% of Earth’s oxygen production. These organisms have allowed life to form on Earth, and they use the power of sunlight to produce organic food from inorganic materials through photosynthesis. In many respects, algae are the most diverse living things in the world. They have the simplest structure and are closely related to bacteria. The most complex ones, Charophyceae species, resemble kelp to the point of confusion. The smallest algae are picoalgae with a size of 0.5 μm, while the largest are 50 to 100 m long Macrocystis species (Phaeophyceae). They occur under the most extreme conditions in fresh and salt water, hot springs, on snow and ice surfaces, in the soil and in the upper layer of some rocks [2]. Algae are mostly eukaryotes, typically classified as „lower” plants that have no true stems, roots and leaves, and are generally capable of photosynthesis. Algae are widely classified into Rhodophyta (red algae), Phaeophyta (brown algae) and Chlorophyta (green algae) and are classified as macroalgae and microalgae by size. Macroalgae (seaweeds) are multicellular, large size algae that are visible to the naked eye, while microalgae are microscopic single-celled organisms and may be prokaryotes, similar to cyanobacteria (Chloroxybacterium), or eukaryotes, similar to green algae (Chlorophyta).

Microalgae, as excellent sources of various organic carbon compounds, can be used in the manufacture of health supplements, drugs and cosmetics [2]. they can also be used in wastewater treatment, atmospheric CO₂ reduction and the production of biofuels. A wide range of bio-products can be extracted from microalgae, such as polysaccharides, lipids, pigments, proteins, vitamins, bioactive compounds and antioxidants [3]. In addition to all this, they are playing an increasingly important role in the feed and food industries (Figure 1).

4. General composition of microalgae

As with all other higher plants, the chemical composition of algae varies depending on the type of cultivation, such as environmental parameters, temperature, illumination, pH and mineral content of the medium, CO₂ supply and mixing speed: 9-77% protein, 6-54% carbohydrate, 4-74% lipid (Table 3)

4.1. Protein and amino acid content of microalgae

Based on research results it can be stated that algae are a source of protein with an amino acid composition similar to that of plant proteins. Examination of the net protein utilization, i.e., of the proper amino acid composition, digestibility and biological value, also led to a similar result.

Several microalgae species produce large amounts of various essential amino acids and proteins that can be used in foods and feeds, one of the main reasons they occupy a prominent place among alternative proteins. Certain species of microalgae can produce as much protein as other rich protein sources, e.g., eggs, meat and milk [6].

In addition, the amino acid pattern of almost all algal species is very similar to the protein pattern of many foods. Of the amino acids, they are only low in cysteine and lysine. Since cells are able to synthesize almost all amino acids, they can be used to provide the essential amino acid needs of both humans and animals [7]. The composition of amino acids synthesized by microalgae, especially the amount and composition of free amino acids, varies greatly, depending on the species, growth conditions and the growth phase [8].

In addition, microalgae proteins are easily digestible and have a relatively high nutritional value. Microalgae produce 2.5 to 7.5 tons/ha/year protein [9], for example, the green microalgae Chlorella is a rich source of different types of marketed proteins. Another protein-rich microalga is Arthrospira. Proteins from microalgae lower cholesterol levels by activating cholecystokinin. They also have other important enzymatic effect [10]. For example, the microalga called Lyngbya majuscula produces microcholine-A, a protein with immunosuppressive effects [11]. The microalga Nostoc produces a protein called cyanovirin, which is known to have an antiviral activity against both HIV and the influenza virus [12]. At the same time, Anabaena and Porphyridium species produce the enzyme SOD (superoxide dismutase), which protects against oxidative damage, while Isochrysis galbana produces the enzyme carbonic anhydrase, which plays a key role in the conversion of CO₂ to carbonic acid and bicarbonate. Microcystis aeruginosa produces a number of amino acids, including proline, serine, glycine and valine.

4.2. Fatty acids

Polyunsaturated fatty acids play an important role in tissue protection and have a beneficial effect on health. Omega-3 and omega-6 fatty acids are especially important for humans, but the human body is unable to produce these fatty acids. Therefore, intake from an external source, such as various foods, is essential. Docosahexaenoic acid (DHA), linoleic acid, eicosapentaenoic acid (EPA), arachidonic acid and gamma-linolenic acid have been shown to lower cholesterol levels, delay aging, protect membrane integrity and prevent cardiovascular diseases [13,14]. Several species of microalgae capable of synthesizing these valuable fatty acids have been studied. These studies have shown that Pavlova lutheri produces large amounts of polyunsaturated fatty acids [15], Arthrospira platensis mainly produces and accumulates stigmasterol, sitosterol and γ-linolenic acid [16], while Porphyridium produces arachidonic acid, Nannochloropsis, Phaeodactylum, Nitzschia, Isochrysis and Diacronema species produce eicosapentaenoic acid and Crypthecodinium and Schizochytrim microalgae produce docosahexaenoic acid in significant amounts [17, 18, 19].

The polyunsaturated EPA and DHA are also pharmaceutically very important omega-3 fatty acids. They play a key role in the treatment of inflammatory diseases, heart problems, arthritis, asthma and headaches, among other things [20, 21, 22].

4.3. Polysaccharides

Polysaccharides are widely used in the food industry, primarily as gelling and thickening agents. Many polysaccharides used in the food industry, such as agar, alginates and carrageenans are extracted from macroalgae, e.g., Laminaria, Gracilaria and Macrocystis species [8]. One of the most promising microalgal species, the unicellular red alga Porphyridium cruentum produces a galactan exopolysaccharide, that can replace carrageenan in many cases. Chlamydomonas mexicana also produces significant amounts of polysaccharides, and it is used in the United States as a soil improver. Sulfated algal polysaccharides also have pharmacological properties and they play a prominent role in stimulating the human immune system [23].

4.4. Photosynthetic pigments

It can be said in general that each species of algae has a specific pigment combination that creates its characteristic colour. In addition to chlorophylls, the primary photosynthetic pigments, supplementary or secondary pigments, such as phycobilins and a number of carotenoids are also produced by microalgae. These natural pigments have the ability to improve the efficiency of light energy utilization and provide protection for algae from the harmful effects of solar radiation. They are used preferentially when added to foods and feeds as natural antioxidants and colourants [24].

4.4.1. Carotenoids

Carotenoids are naturally occurring pigments that play a role in the formation of the colour of fruits, vegetables and other plants [25]. They are typically isoprenoid polyene pigments derived from lycopene, ranging in colour from yellow to red, and are produced by de novo photosynthetic organisms and some other microorganisms [8]. Carotenoids ingested with foods or feeds are either accumulated or metabolized by the body. Carotenoids can be found in the meat of various animals, eggs, fish skin (trout, salmon), crustaceans (shrimp, lobster, Antarctic krill, crab) and subcutaneous fat, skin, egg yolk, liver and the feather of birds (e.g., poultry) [26].

In algae, carotenoids primarily play the role of sunscreen and light collector, i.e., they protect the photosynthetic apparatus from light damage [24]. They also play a role in phototropism and phototaxis. Some microalgae undergo carotenogenesis in response to various environmental effects (e.g., light, temperature, salts, nutrients). During this, algae stop their growth dramatically alter their carotenoid metabolism, resulting in the accumulation of secondary carotenoids [27].

There are more than 600 carotenoids in nature, about 50 of which show provitamin A activity. These include α-carotene, β-carotene and β-cryptoxanthin [28]. A β-Carotene protects membrane lipids from peroxidation, thus preventing and reducing the development of many serious and fatal diseases, such as cancer, cardiovascular disease, Parkinson’s disease and atherosclerosis [29, 30, 31].

Relatively few carotenoids are used in the food and feed industries: β-carotene and astaxanthin, lutein, zeaxanthin, lycopene, etc. Among microalgae, the main carotenoid-producing species are Dunaliella salina and Haematococcus pluvialis which produce significant amounts of β-carotene and astaxanthin, respectively. The microalga Dunaliella salina produces β-carotene in an amount that accounts for 10 to 14% of its dry matter content [32].

β-Carotene serves as an essential nutrient, mainly as a food colouring, and is increasingly used in various dietary supplements due to its health-protective effects, but is also used preferentially by the cosmetics industry [33].

In the food industry, β-carotene is also used regularly in various soft drinks, cheeses, butter or margarines due to its beneficial physiological effect, as it possesses a provitamin activity [34].

Astaxanthin has a number of beneficial properties, including improving eye health, muscle strength and endurance, protecting the skin, reducing premature aging, inflammation and damage caused by UVA radiation. It also plays an important role in animal feeding, as it promotes growth and reproduction, improves vision, has an immunostimulatory effect and also aids in post-injury regeneration [35, 36].

Numerous studies have shown that daily intake of astaxanthin protects cells and tissues from oxidative effects and that its effect on free radicals is significantly, about 500 times more intense than that of vitamin E. The microalga Haematococcus pluvialis produces 4-5% astaxanthin on the basis of dry biomass [37], so its dried biomass is marketed as a rich source of astaxanthin and is sold on the market at a price of about 2,500 US $/kg.

4.4.2. Chlorophyll

Each alga contains one or more chlorophylls. Their primary photosynthetic pigment is chlorophyll-a, and it is also the only chlorophyll in Cyanobacteria (blue-green algae) and red algae (Rhodophyta). Like all higher plants, Chlorophytes (true green seaweeds) and Euglenophytes (flagellate seaweeds) also contain chlorophyll-b; chlorophyll-c, -d and -e are found in many other marine algae. The quantity of chlorophylls is usually up to 0.5-1.5% of the dry matter content [38].

In addition to being used as a food and pharmaceutical colourant, chlorophyll derivatives also have a health-protective effect. They are also traditionally used because of their wound healing and anti-inflammatory properties [39]. Epidemiological studies conducted in the Netherlands (Cohort Study) have shown a significant correlation between chlorophyll consumption and a reduction in the risk of colon cancer [40].

4.4.3. Phycobilins

In addition to chlorophyll and carotenoid lipophilic pigments, Cyanobacteria (blue-green algae), Rhodophytes (red algae) and Cryptophyta algae also contain so-called phycobilins, which are coloured, fluorescent pigments. Like chlorophylls, they bind to proteins (phycobiliproteins) which, contrary to chlorophyll-protein complexes located in the membranes, are water-soluble proteins and are important components of the photochemical system. Significant amounts of phycobilins, mainly blue phycocyanin and red phycoerithrin are found in Spirulina algae and Porphyridium, respectively.

The use of phycobilins is quite widespread. In addition to being widely used in clinical immunofluorescence studies to detect fluorescently labeled antibodies as a fluorescent marker [38], phycocyanin is currently used in Japan and China as a natural colourant in foods, such as chewing gums, candies, dairy products, jellies, ice creams and soft drinks. It is also widely used in the cosmetics industry, for example, in lipsticks, eyeliners and eyeshadows [41].

According to a study, phycocyanin is one of the most versatile blue colourant, providing a bright blue colour to various jellies and coated soft candies [42], while a number of pharmacological properties are also attributed to phycocyanin, including antioxidant, anti-inflammatory, neuroprotective and hepatoprotective effects [43, 44, 45].

4.5. Tocopherols and sterols

Tocopherols are widespread in nature, occurring in both lower and higher plants as parts of the photosynthetic system.

Research in this area has revealed that Euglena has the highest content of tocopherols among the various microalgae species [46].

The sterols produced by plants are called phytosterols. Microalgae can make a major contribution to the production of phytosterols, they can be considered as efficient and promising sources for their large-scale production. Some microalgae contain high levels of sterols. Microalgae sterols have health-protective, cholesterol lowering and anti-inflammatory properties, and are effective in the treatment of certain neurological diseases, such as Parkinson’s disease [47, 48], and are increasingly used in the food industry as dietary supplements and food ingredients [49, 50]. Some microalgae, such as species belonging to the genera Pavlova and Thalassiosira, are rich in sterols [51, 52].

4.6. Vitamins, minerals

Microalgae biomass is a valuable source of almost all essential vitamins, as it contains vitamins B₁, B₂, B₃, B₅, B₆, B₁₂, C, E and H, among other things, and its mineral content (e.g., Na, K, Ca, Mg, Fe, Zn and trace elements) is also significant [53]. The vitamin B₁₂ and iron content of some microalgae, such as Spirulina species, is particularly high, therefore, they are often used in foods and dietary supplements made for vegetarians.

The vitamin content of algae depends on the genotype, as well as the stage of the growth cycle, the nutrition of the algae and the light intensity. Thus, their vitamin content can be increased by selecting the right species, choosing the right culture conditions and/or by genetically modifying them. However, the vitamin content of the cells can be significantly reduced by using inappropriate environmental conditions, harvesting and biomass drying methods [54].

4.7. Antioxidants

Microalgae are photoautotrophic organisms, that is, organisms that depend on light as the energy source to produce organic molecules from inorganic molecules. This process is known as photosynthesis, and the food chain is usually based on these organisms. During their development, these organisms have developed an effective defense system against various abiotic effects affecting them, such as high levels of free radicals and reactive oxygen species [23]. Due to the high antioxidant content of certain algae species (e.g., Isochrysis galbana, Chlorella vulgaris, Nannochloropsis oculata, Tetraselmis tetrathele, Chaetoceros calcitrans), their use has been increasing in some cosmetics (e.g., sunscreens) and in functional foods.

The research of Natrah et al. [55] has shown that the methanolic extract of some fresh/untreated microalgae exhibits an antioxidant activity that is higher than that of α-tocopherol, but lower than that of the synthetic antioxidant BHT (butylhydroxytoluene). However, the latter and BHA (butylhydroxyanisole) being synthetic antioxidants, their safe use if questionable, as their use in high doses may be carcinogenic and tumorigenic [56, 57].

4.8. Other biologically active components

Microalgae are undoubtedly a large repository of versatile compounds with significant biological activity, as well as unique and interesting structure and function [58].

In recent decades, marine microorganisms, especially Cyanobacteria, have become the focus of medical research aimed at developing new drugs and antibiotics. Data published up to 1996 revealed about 208 Cyanobacterial compounds exhibiting biological activity. By 2001, the number rose to 424. The compounds identified include various lipoproteins (40%), alkaloids, amides, etc. [59], many of which have cytotoxic, antitumor, antimicrobial (antibacterial, antifungal), antiviral (e.g., anti-HIV) activities, as well as biomodulatory, for example, immunosuppressive and anti-inflammatory effects [59, 60].

Numerous studies have shown that microalgae may also contain compounds that are effective in treating cancer and tumors by inhibiting angiogenesis. Angiogenesis is a physiological process during which new blood vessels emerge from existing blood vessels. Although angiogenesis is a normal process, pathological conditions can develop under certain conditions, such as cancer, atherosclerosis, arthritis, diabetic retinopathy and ischemic stroke. Pathological angiogenesis promotes the development and growth of tumors [61, 62]. Fucoxanthin and fucoxanthinol, found in many species of microalgae, have been shown to inhibit the process of angiogenesis in the aortic ring of rats by reducing the formation and growth of microvessels [63]. Fucoxanthin has also been shown to protect DNA from photooxidation [64]. Microalgae, especially blue-green algae, are currently considered to be potential sources of active ingredients that can be used in the treatment of cancer, as several studies have demonstrated their anti-cancer effects [65].

5. Some major microalgal species

Although many indigenous microalgal populations have been used for various purposes for centuries, their large-scale cultivation has only begun in the last few decades [66]. Of the assumed roughly 30,000 species of microalgae, only a few thousands are kept in stock collections [67, 68], of which a few hundred are considered more important due to their chemical composition, and very few are grown in industrial quantities [69].

The biotechnologically most relevant microalgae include green algae (Chlorophyta), such as Chlorella vulgaris, Haematococcus pluvialis, Dunaliella salina and Spirulina maxima, which belongs to the phylum of Cyanobacteria. Their cultivation, marketing and use are very significant, mainly as dietary supplements and animal feed additives.

5.1. Spirulina species

Spirulina (Arthrosphira) algae are a tiny, filamentous, freshwater, spiral-shaped, blue-green algae species that is abundant in the alkaline lakes of Mexico and Africa and has been consumed by the local population since ancient times [59]. Its characteristic feature is that its cell membrane is very weak, and this makes it easy to utilize. It is also one of its important physiological features that it can easily become a colloidal solution when exposed to moisture and is very easy to digest. Spirulina is widely grown all over the world (3,000 tons/year) and is used as a food and feed supplement due to its high protein content (60 to 70%, including 18 amino acids, 8 of which are essential) and its excellent nutritional value. For example, its γ-linoleic acid content is remarkably high [70, 71]. Its digestibility and absorption are superior to both animal and plant proteins. It contains the vitamins important to the body (C, B₁, B₂, B₅, B₆, B₉, B₁₂, A, E), trace elements, of which iron, iodine, calcium, sodium, potassium, copper, magnesium, manganese, zinc, phosphorus, selenium, chromium and vanadium are the most significant. It is a particularly good source of iodine and potassium. It stimulates the immune system greatly due to its high content of β-carotene, chlorophyll and γ-linolenic acid. Its polyunsaturated fatty acid content is significantly higher than that of marine fish. Spirulina contains high levels of GLA (gamma-linolenic acid), which is found in greater amounts only in breast milk. Spirulina has a number of health-protecting effects: it lowers high blood fat levels, cholesterol levels, high blood pressure, elevated blood sugar levels, is suitable for treating kidney failure, and promotes the growth of probiotics, such as Lactobacilli, in the gut [19]. It is the main source of natural phycocyanin, used as a natural blue colourant in foods and cosmetic products, and also as a biochemical tracer in immunoassays [70, 71, 72].

5.2. Chlorella vulgaris

This species of algae is one of the oldest, simplest plants on Earth. Its nearly 4% chlorophyll content, strong cell wall and high pigment and cellulose content make the detoxifying effect of Chlorella unique. It binds and removes heavy metals from the body, cleanses the intestinal flora. By improving liver function, it helps to remove other contaminants, in addition to heavy metals, and to detoxify the body.

Its physiological effects are similar to those of Spirulina: it is high in protein, contains all the essential amino acids, and it is a storehouse of various vitamins, trace elements and minerals. Chlorella vulgaris has been used in the Far East since ancient times in alternative medicine, as well as in the preparation of various traditional foods. It is widely cultivated and used, primarily in animal feeding, aquaculture and as a dietary supplement, in many countries, including China, Japan, Europe and the United States. The health-protecting effects of Chlorella are manifested, for example, in the rapid healing of stomach ulcers and other wounds, and it is useful in the treatment of constipation, anemia, hypertension, diabetes, infant malnutrition and neurosis. The prophylactic role of the glycolipids found in Chlorella against the development of atherosclerosis and hypocholesterolemia has been demonstrated by research [58]. However, one of the most important substances in Chlorella is ß-1,3-glucan, which is an active immunostimulant, it binds free radicals and reduces the amount of blood fats [19].

The γ-linolenic acid (GLA) content of Spirulina and Chlorella is very high. The role of GLA in the functioning of the body is extremely diverse. On the one hand, it is important for the proper functioning of the immune system, and on the other hand, it has an anti-inflammatory effect, lowers blood pressure and improves blood circulation. It prevents platelets from sticking together, thus reducing the risk of formation of blood clots. It has a positive effect on cholesterol levels, thus reducing the risk of atherosclerosis. It improves nervous system function and eliminates excess fluid from the body.

5.3. Haematococcus pluvialis

This freshwater microalgae, with a size of barely 0.1 mm, attracted the interest of researchers early on. Haematococcus pluvialis is the plant with the highest astaxanthin content (1.5-3.0% dry weight) based on previous research. This carotenoid pigment has a very strong radical scavenging effect that exceeds the antioxidant properties of β-carotene, vitamin C and vitamin E. The astaxanthin production of the algae is a natural reaction to environmental stress. Thanks to the protective functions of astaxanthin, in a state of deep sleep, these algae can survive without food and water for more than 40 years, so they can easily survive the heat of summer or the icy cold of winter. They will only wake up again and regain their original green, active state when living conditions are right again. As a result, algae defied the harshest environmental conditions even at the early stages of Earth’s history. The ability of certain algae species to survive both droughts and ice ages is due to their astaxanthin shield. Astaxanthin is a bioactive antioxidant that has been shown to be effective against Alzheimer’s disease and Parkinson’s disease, as well as macular degeneration in both animal and human experiments. In some cosmetics, the astaxanthin used can slow down the aging process of the skin. In addition, the immune-boosting and anti-inflammatory effects of astaxanthin have been reported, as well as its beneficial effects on the development of cardiovascular diseases and atherosclerosis.

Haematococcus pluvialis is currently a natural source of this pigment, its commercial utilization is outstanding, especially in aquaculture (salmon and trout farming) [73]. There is another natural source of astaxanthin, however, the yeast Xanthophyllomyces dendrorhous requires large amounts of expensive nutrients for proper pigmentation [36].

5.4. Dunaliella salina

Dunaliella salina is a halotolerant microalgae, its natural habitats being salt lakes. It is able to accumulate large amounts of β-carotene, which is why this species of algae is sought after mainly as a food colourant. Research has shown that the Dunaliella salina community in Pink Lake, Victoria, Australia, can produce up to 14% carotenoids [74] and some Dunaliella algae can contain up to 10% in cultivated cultures.

Higher β-carotene content can be achieved with adequate nutrient supply under high salt and light conditions [75, 76]. Similarly to Haematococcus algae, Dunaliella contains significant amounts of astaxanthin. However, Haematococcus is a freshwater algae that is difficult to grow in outdoor cultures because it is easily infected, requiring a closed system, and the extraction of astaxanthin is more complicated than in the case of Dunaliella, as Haematococcus has a thick cell wall that has to be disrupted by physical methods.

6. Use for animal feed

Today, many species of microalgae (e.g., Chlorella, Tetraselmis, Spirulina, Nannochloropsis, Nitzchia, Navicula, Chaetoceros, Scenedesmus, Haematococcus, Crypthecodinium) are used to feed farm animals, pets and fish.

Even a small amount of microalgae biomass has an immunostimulatory effect, which results in growth stimulation, disease resistance, has antiviral and antibacterial effects, improves absorption and the colonization stimulation of probiotic cultures such as Lactobacilli, and thus results in an increase in reproductive performance and weight [77]. By providing feeds that contain algae, the appearance of the animals improves visibly, which is manifested in a healthy skin and a shiny coat, both in the case of farm animals (poultry, cows, breeding bulls) and in the case of pets (cats, dogs, rabbits, ornamental fish and birds) [78].

Feed is the main exogenous factor influencing animal health and accounts for a significant part of the major cost of animal husbandry, and so it is very important to identify high quality, chemical and toxic substance free alternative protein sources that can replace or complement traditional protein sources [26]. The results of a large number of nutritional and toxicological evaluations have demonstrated the suitability of algal biomass as a valuable feed supplement [38]. Currently, about 30% of the global algae production is sold for feed purposes [53].

Becker et al [53] performed feeding experiments on broiler chickens, in which conventional proteins were replaced with species of different microalgae, namely Chlorella, Euglena, Oocystis, Scenedesmus, Spirulina, usually at a rate of 10%. In laying hens, no differences were found in egg production and egg quality (size, weight, shell thickness, egg solids, albumin index, etc.) and in feed conversion efficiency between birds fed with algae-containing feeds and the control birds.

However, Haematococcus microalgae can also be used as a natural colourant in the feeding of broiler chickens, making the skin of the birds yellower and the egg yolk more orange [79]. Studies were performed on chickens that were fed the biomass (5% or 10%) of red microalgae (Porphyridium species). Although no difference was found in the body weight and egg count of the chickens, the composition of the meat and eggs showed decreased cholesterol levels (by 10%) and healthier fatty acid composition and increased linoleic acid and arachidonic acid levels (by 29% and 24%, respectively). In addition, the colour of the egg yolk was darker due to a carotenoid content that was 2.4 times higher than average [80]. At the same time, it was observed that chickens fed with algal biomass consumed 10% less food in the case of feeds containing either 5% or 10% algae, and had serum cholesterol levels significantly lower (by 11% and 28%, respectively) than that of the control group.

Microalgae biomass is a feed with excellent nutritional values and is eminently suitable for breeding pigs. They can be used to replace traditional proteins such as soy flour or fish meal, and their acceptance is not difficult for the animals [38].

It is hypothesized that algae may be an excellent food source for ruminants, as these animals are able to digest even the cell wall of unprocessed algae. However, only a limited number of experiments have been performed with these animal species, as these procedures are expensive and large amounts of algae are required to perform appropriate feeding experiments. However, some experiments have shown that sheep, lambs and cattle were unable to digest the carbohydrate fraction efficiently when fed certain algal species (e.g., Chlorella, Scenedesmus obliquus and Scenedesmus quadricauda) [81, 82]. Better digestibility was achieved when Spirulina accounted for 20% of the total sheep feed, and it was observed that in calves fed a diet containing Scenedesmus algae, there were only minimal differences when compared to animals fed the control feed [83].

Microalgae feeds are currently used mainly to supplement and replace zooplankton used for the breeding of fish, fish fry and other aquatic animals (crustaceans, etc.) [84, 85]. The species most often used in aquaculture are Chlorella, Tetraselmis, Isochrysis, Pavlova, Phaeodactylum, Chaetoceros, Nannochloropsis, Skeletonema and Thalassiosira [86, 87].

Microalgae contain nutrients that are essential for aquatic animals, and these determine the quality, growth, health and disease resistance of the farmed animals. Mixed microalgae cultures have been shown to be useful for animal growth to provide adequate protein composition, vitamin content and high levels of polyunsaturated fatty acids (mainly EPA, AA and DHA), which are vital for the survival and growth of many freshwater and marine animals in the early stages of life [88]. One of the beneficial effects of algae is attributed to the fact that they increase the food intake of marine fish offspring, which enhances their growth and survival, and also improves the quality of fish meat [89]. In addition, the presence of algae in the breeding tanks of European sea bass larvae has been shown to increase digestive enzyme secretion [90]. When many aquatic species, such as Salmonidae (salmon and trout), shrimp, lobster, marine vertebrates, goldfish and koi carp are kept under intensive conditions, carotenoid colourants are added to their feed to achieve their characteristic muscle colour. Carotenoids, such as astaxanthin and canthaxanthin, have beneficial effects on animal health, growth and reproduction, by promoting the development of larvae [33].

7. Food use

In the early 1950s, microalgae were used to replace certain foods and were often used as single cell proteins in the diets of malnourished children and adults. Today, in human nutrition, microalgae are marketed in the form of various dietary supplement pills, capsules and liquids [91].

Gross et al conducted research in which Scenedesmus obliquus algae was added to the normal diet of children (5 g/day) and adults (10 g/day) during a four-week test period. Blood panel, urine composition, serum protein and uric acid concentration and weight change were measured, but the parameters analyzed did not deviate from normal values, only a slight increase in body weight was observed.

The same authors subsequently performed a three-week study in both slightly (Group I) and severely (Group II) malnourished four-year-old children. Four-year-old children in Group I (10 g algae/day) showed a significant weight gain (27 g/day) compared to children in the control group who received a normal diet, and no adverse symptoms were experienced. Group II was fed a diet enriched with 0.87 g algae/kg body weight, replacing only 8% of the total protein with algae protein, and the daily weight gain was approximately seven times that of children in the control group, while all anthropogenic parameters were normal. The authors concluded that the significant improvement in health can be attributed not only to algae protein but also to other important health-protective and immune-enhancing components [92].

Large-scale production of microalgae suitable for human consumption has been growing worldwide. There are many forms of microalgae and other health-protective dietary supplements on the market, such as various pills, powders, capsules, lozenges and liquids [23, 93]. Microalgae are also used in the preparation of various foods, such as algae pastries, biscuits, breads, snacks, candies, yogurts and soft drinks, which also provide the health and immunomodulatory effects associated with microalgae biomass [94].

Despite some reluctance on the part of consumers over novel foods in recent decades, there is a growing consumer demand for natural, health-enhancing foods today. Thus, functional foods containing microalgae biomass are also becoming increasingly popular. These products are also proving to be very attractive and varied from a sensory point of view, and their consumption also brings health benefits, satisfying consumer needs in all respects [23].

8. Acknowledgement

We are grateful for the funding of the NKTH, TKP2020-NKA 24 "Thematic Excellence Program”.

9. References

[1] Field, C.B., Behrenfeld, M.J., Randerson, J.T., Falkowski, P. (1998): Primary production of the biosphere: integrating terrestrial and oceanic components. Science, 281, pp. 237-240. DOI

[2] regi.tankonyvtar.hu

[3] Brennan, L., Owende, P. (2010): Biofuels from microalgae- a review of technologies for production, processing, and extractions of biofuels and co-products. Renew Sustain Energy Review, 14, pp.557–77. DOI

[4] Khan, M. I., Shin, J. H., Kim, J. D. (2018): The promising future of microalgae: current status, challenges, and optimization of a sustainable and renewable industry for biofuels, feed, and other products. Microbial Cell Factories, 17, DOI

[5] de Medeiros, V.P.B., da Costa, W.K.A, da Silva, R.T., Pimentel, T.C., Magnani, M. (2021): Microalgae as source of functional ingredients in new-generation foods: challenges, technological effects, biological activity, and regulatory issues. Critical Review in Food Science and Nutrition, DOI

[6] Gouveia, L., Batista, A.P., Sousa, I., Raymundo, A., Bandarra, N. (2008): Microalgae in novel food products. In: Konstantinos N, Papadopoulos PP, editors. food chemistry research development. New York: Nova Science Publishers; pp. 75–112.

[7] Guill-Guerrero, J.L., Navarro-Juárez, R., López-Martínez, J.C., Campra-Madrid, P., Rebolloso-Fuentes, M.M. (2004): Functional properties of the biomass of the three microalgal species. Journal of Food Engineering, 65, pp. 511-517. DOI

[8] Borowitzka, M.A. (1988): Vitamins and fine chemicals from micro-algae. In M.A. Borowitzka, and L.J. Borowitzka (Eds), Micro-algal biotechnology pp. 153-196. Cambridge, UK: Cambridge University Press

[9] Stephen, B., Hayes M., (2017): Algal Proteins.:Extraction, application, and challenges concerning production. Foods.6, pp.33. DOI

[10] Smee, D.F., Bailey, K.W., Wong, M.H., Keefe, B.R.O., Gustafson, K.R., Mishin, V.P., Gubareva, V.L. (2008): Treatment of influenza A (H1N1) virus infections in mice and ferrets with cyanovirin-N. Antiviral Research. 80, pp. 266–71. DOI

[11] Arya V, Gupta VK. (2001): A review on marine immunomodulators. International Journal of Pharmacy and Life Sciences. 2, pp. 751-758.

[12] Zappe, H., Snell. M.E., Bossard. M.J. (2008): PEGylation of cyanovirin-N, an entry inhibitor of HIV. Advances Drug Delivivery Review. 60(1), pp. 79–87. DOI

[13] Hu, F.B., Bronner, L., Willett, W.C., Stampfer, M.J., Rexrode, K.M., Albert, C.M. (2002): Fish and omega-3 fatty acid intake and risk of coronary heart disease in women. JAMA. 287, pp. 1815–1821. DOI

[14] Guedes, A.C.A. (2010): Production, extraction and characterization of selected metabolites from microalgae and cyanobacteria. Ph.D. Thesis Porto,: Escola Superior de Biotecnologia, Universidade Católica Portuguesa

[15] Santhosh, S., Dhandapani, R., Hemalatha, R. (2016): Bioactive compounds from Microalgae and its different applications—a review. Advances in Applied Science Research. 7(4), pp. 153–158.

[16] Bandarra, N.M., Pereira, P.A., Batista, I., and Vilela, M.H. (2003). Fatty acids, sterols and – tocopherol in Isochrysis galbana. Journal of Food Lipids, 18, 25-34. DOI

[17] Donato, M., Vilela, M.H., and Bandarra, N.M. (2003). Fatty acids, sterols, α-tocopherol and total carotenoids composition of Diacronema vlkianum. Journal of Food Lipids, 10, 267-276. DOI

[18] Spolaore, P., Joannis-Cassan, C., Duran, E., and Isambert, A. (2006). Commercial applications of Microalgae- review. Journal of Bioscience and Bioengineering, 101, 87-96. DOI

[19] Hamilton M, Haslam R, Napier J, Sayanova O. Metabolic engineering of microalgae for enhanced production of omega-3 long chain polyunsaturated fatty acids. Metab Eng. 2014;22:3–9. DOI

[20] Draaisma RB, Wijffels RH, Slegers PM, Brentner LB, Roy A, Barbosa MJ. Food commodities from microalgae. Curr Opin Biotechnol. 2013;24:169–77. DOI

[21] Koller M, Muhr A, Braunegg G. Microalgae as versatile cellular factories for valued products. Algal Res. 2014;6:52–63. DOI

[22] Pulz, O., and Gross, W. (2004). Valuable products from biotechnology of microalgae. Applied Microbiology and Biotechnology, 65, 635-648. DOI

[23] Van den Berg, H, Faulks, R., Granado, H.F., Hirschberg, J., Olmedilla, B., Sandmann, G., Southon, S., and Stahl, W. (2000). The potential for the improvement of carotenoid levels in foods and the likely systemic effects. Journal of the Science of Food and Agriculture, 80, 880-912. DOI

[24] Ben-Amotz, A., and Fishler, R. (1998). Analysis of carotenoids with emphasis on 9-cis-β-carotene in vegetables and fruits commonly consumed in Israel. Food Chemistry, 62, 515-520. DOI

[25] Breithaupt, D.E. (2007). Modern application of xanthophylls in animal feeding - a review. Trends in Food Science and Technology, 18, 501-506. DOI

[26] Bhosale, P. (2004). Environmental and cultural stimulants in the production of carotenoids from microorganisms. Applied Microbiology and Biotechnology, 63, 351-361. DOI

[27] Faure, H., Fayol, V., Galabert, C., Grolier, P., Moel, G.L., Steghens, J., Kappel, A.V., Nabet, F. (1999). Carotenoids: 1. Metabolism and physiology. Annales de Biologie Clinique, 57,169-183.

[28] Raja, R., Hemaiswarya, S., and Rengasamy, R. (2007). Exploitation of Dunaliella for β-carotene production. Applied Microbiology and Biotechnology, 74, 517-523. DOI

[29] Lobo V, Patil A, Phatak A, Chandra N. Free radicals, antioxidants and functional foods: impact on human health. Pharmacogn Rev. 2010;4(8):118–26. DOI

[30] Uttara B, Singh AV, Zamboni P, Mahajan R. Oxidative stress and neurodegenerative diseases: a review of upstream and downstream antioxidant therapeutic options. Curr Neuropharmacol. 2009;7(1):65–74. DOI

[31] Pham-Huy LA, He H, Pham-Huy C. Free radicals, antioxidants in disease and health. Int J Biomed Sci. 2008;4(2):89–96.

[32] Sathasivam R, Juntawong N. (2013):Modified medium for enhanced growth of Dunaliella strains. Int J Curr Sci.;5:67–73.

[33] Baker, R., and Gunther, C. (2004). The role of carotenoids in consumer choice and the likely benefits from their inclusion into products for human consumption. Trends in Food Science and Technology, 15, 484-488. DOI

[34] Tsuchiya, M., Scita, G., Freisleben, H.L., Kagan, V.E., and Packer, L. (1992). Antioxidant radical-scavenging activity of carotenoids and etinoids compared to β-tocopherol. Methods of Enzymology, 213, 460 – 472. DOI

[35] Beckett, B.R., and Petkovich, M. (1999). Evolutionary conservation in retinoid signalling and metabolism. American Zoology, 39, 783-795. DOI

[36] Dufossé, L., Galaup, P., Yaron, A., Arad, S.M., Blanc, P., Murthy, K.N.C., and Ravishankar, G.A. (2005). Microorganisms and microalgae as sources of pigments for food use: ascientific oddity or an industrial reality?. Trends in Food Science and Technology, 16, 389-406. DOI

[37] Sathasivam R, Radhakrishnan R, Hashem A, Abd_Allah EF. Microalgae metabolites: a rich source for food and medicine. Saudi J Biol Sci. 2017. DOI

[38] Becker, E.W. (1994). Microalgae: biotechnology and microbiology. Cambridge University Press.

[39] Ferruzi, M.G., and Blakeslee, J. (2007). Digestion, absorption, and cancer preventive activity of dietary chlorophyll derivatives. Nutrition Research, 27, 1-12. https://doi.org/10.1016/j.nutres.2006.12.003">DOI

[40] Balder, HF, Vogel, J., Jansen, M.C., Weijenberg, M.P., van den Brandt, P.A., Westenbrink, S., van der Meer, R., and Goldbohm, R.A. (2006). Heme and chlorophyll intake and risk of colorectal cancer in the Netherlands cohort study. Cancer Epidemiology Biomarkers and Prevention, 15,717-725. DOI

[41] Sekar, S., and Chandramohan, M. (2007). Phycobiliproteins as a commodity: trends in applied research, patents and commercialization. Journal of Applied Phycology, DOI

[42] Jespersen, L., Strømdahl, L.D., Olsen, K., and Skibsted, L.H. (2005). Heat and light stability of three natural blue colorants for use in confectionery and beverages. European Food Research and Technology, 220, 261–266. DOI

[43] Romay, C.H., Gonzalez, R., Ledon, N., Remirez, D., and Rimbau, V. (2003). Phycocyanin: a biliprotein with antioxidant, anti-inflammatory and neuroprotective effects. Current Protein and Peptide Science, 4, 207-216. DOI

[44] Benedetti, S., Benvenuti, F., Pagliarani, S., Francogli, S., Scoglio, S., and Canestrari, F. (2004). Antioxidant properties of a novel phycocyanin extract from the blue-green alga Aphanizomenon flos-aquae. Life Sciences, 55, 2353-2362. DOI

[45] Bhat, V.B., and Madyastha, K.M. (2000). C-Phycocyanin: a potent peroxyl radical scavenger in vivo and in vitro. Biochemical and Biophysical Research Communications, 275, 20-25. DOI

[46] Kusmic, C., Barsacchi, R., Barsanti, L., Gualteri, P., and Passarelli, V. (1999). Euglena gracilis as a source of the antioxidant vitamin E. Effects of culture conditions in the wildstrain and in the natural mutant WZSL. Journal of Applied Phycology, 10, 555-559. DOI

[47] Devaraj S, Jialal I. Vega-Lopez. Plant sterol-fortified orange juice effectively lowers cholesterol levels in mildly hypercholesterolemic healthy individuals. Arterioscler Thromb Vasc Biol. 2004;24:25–8.

[48] Kim HJ, Fan X, Gabbi C, Yakimchuk K, Parini P, Warner M. Liver X receptor β (LXRβ): a link between β-sitosterol and amyotrophic lateral sclerosis—Parkinson’s dementia Proc. Natl Acad Sci USA. 2008;105(6):2094–9.

[49] Fernandes P, Cabral JM. Phytosterols: applications and recovery methods. Bioresour Technol. 2007;98(12):2335–50.

[50] Srigley CT, Haile EA. Quantification of plant sterols/stanols in foods and dietary supplements containing added phytosterols. J Food Compos Anal. 2015;40:163–76. DOI

[51] Luo X, Su P, Zhang W. Advances in microalgae-derived phytosterols for functional food and pharmaceutical applications. Mar Drugs. 2015;13(7):4231–54. DOI

[52] Volkman JK. A review of sterol markers for marine and terrigenous organic matter. Org Geochem. 1996;9:83–99. DOI

[53] Becker, E.W. (2004). Microalgae in human and animal nutrition. In A. Richmond (Ed), Handbook of microalgal culture (pp. 312-351). Oxford: Blackwell. DOI

[54] Brown, M.R., Mular, M., Miller, I., Farmer, C., and Trenerry, C. (1999). The vitamin content of microalgae used in aquaculture. Journal of Applied Phycology, 11, 247-255. DOI

[55] Natrah, F., Yosoff, F.M. Shariff, M., Abas, F., and Mariana, N.S. Screening of Malaysian indigenous microalgae for antioxidant properties and nutritional value. Journal of Applied Phycology 19, 711-718. DOI

[56] Schilderman, P.A.E.L., ten Vaarwerk, F.J., Lutgerink, J.T., Van der Wurff, A., ten Hoor, F., and Kleinjans, J.C.S. (1995). Induction of oxidative DNA damage and early lesions in rat gastro-intestinal epithelium in relation to prostaglandin H synthese-mediated metabolism of butylated hydroxyanisole. Food and Chemical Toxicology, 33, 99-109. DOI

[57] Aruoma, O.I. (2003). Methodological considerations for characterizing potential antioxidant actions of bioactive components in plant foods. Mutation Research, 523, 9-20. DOI

[58] Yamaguchi, K. (1997). Recent advances in microalgal bioscience in Japan, with special reference to utilization of biomass and metabolites: a review. Journal of Applied Phycology, 8, 487-502. DOI

[59] Burja, A.M., Banaigs, B., Abou-Mansour, E., Burgess, J.G., Wright, P.C. (2001). Marine cyanobacteria - a prolific source of natural products. Tetrahedron, 57, 9347-9377. DOI

[60] Singh, S., Kate, B.N., and Banerjee, U.C. (2005). Bioactive compounds from Cyanobacteria and Microalgae: na overview. Critical Reviews in Biotechnology, 25, 73-95. DOI

[61] Armstrong AW, Voyles SV, Armstrong EJ, Fuller EN, Rutledge JC. Angiogenesis and oxidative stress: common mechanisms linking psoriasis with atherhosclerosis. J Dermatol Sci. 2011;63:1–9. DOI

[62] Cherrington JM, Strawn LM, Shawver LK. New paradigms for the treatment of cancer: the role of anti-angiogenesis agents. Adv Cancer Res. 2000;79:1–38. DOI

[63] T, Matsubara K, Akagi R, Mori M, Hirata T. Antiangiogenic activity of brown algae fucoxanthin and its deacetylated product, fucoxanthinol. Agric Food Chem. 2006;54:9805–10. DOI

[64] Heo SJ, Jeon YJ. Protective effect of fucoxanthin isolated from Sargassum siliquastrum on UV-B induced cell damage. J Photochem Photobiol B. 2009;95:101–7. DOI

[65] Russo P, Cesario A. New anticancer drugs from marine cyanobacteria. Curr Drug Targets. 2012;13(8):1048–53. DOI

[66] Borowitzka, M.A. (1999). Commercial production of microalgae: ponds, tanks, tubes and fermenters. Journal of Biotechnology, 70, 313-321. DOI

[67] Chaumont, D. (1993). Biotechnology of algal biomass production: a review of systems for outdoor mass culture. Journal of Applied Phycology, 5, 593-604. DOI

[68] Radmer, R.J., and Parker, B.C. (1994). Commercial applications of algae: opportunities and constraints. Journal of Applied Phycology, 6, 93-98. DOI

[69] Olaizola, M. (2003). Commercial development of microalgal biotechnology: from the test tube to the marketplace. Biomolecular Engineering, 20, 459-466. DOI

[71] Shimamatsu, H. (2004). Mass production of Spirulina, an edible microalga. Hydrobiologia, 512, 39-44. DOI

[70] Ötles, S., and Pire, R. (2001). Fatty acid composition of Chlorella and Spirulina microalgae species. Journal of AOAC International, 84, 1708-1714. DOI

[72] Kato, T. (1994). Blue pigment from Spirulina. New Food Industry, 29, 17-2.

[73] Lorenz, R.T., and Cysewski, G.R. (2000). Commercial potential for Haematococcus microalgae as a natural source of astaxanthin. Trends in Biotechnology, 18, 160-167. DOI

[74] Aasen, A.J., Eimhjellen, K.E., and Liaaen-Jensen, S. (1969). An extreme source of β-carotene. Acta Chemica Scandinavica, 23, 2544-2545. DOI

[75] Ben-Amotz, A., and Avron, M. (1980). Glicerol, β-carotene and dry algal meal production by commercial cultivation of Dunaliella. In G. Shelef, and C.J. Soeder (Eds), Algae Biomass (pp. 603-610). Amsterdam: Elsevier/North Holland Biomedical Press.

[76] Oren, A. (2005). A hundred years of Dunaliella research: 1905-2005. Saline Systems, 1, 2 DOI

[77] Harel, M., and Clayton, D. (2004). Feed formulation for terrestrial and aquatic animals. US Patent 20070082008 (WO/2004/080196)

[78] Certik, M., and Shimizu, S. (1999). Biosíntesis and regulation of microbial polyunsaturated fatty acid production. Journal of Biosciences and Bioengineering, 87, 1-14. DOI

[79] Waldenstedt, L., Inborr, J., Hansson, I., and Elwinger, K. (2003). Effects of astaxanthin-rich algal meal (Haematococcus pluvialis) on growth performance, caecal campylobacter and clostridial counts and tissue astaxanthin concentration of broiler chickens. Animal Feed Science and Technology, 18, 119-132. DOI

[80] Ginzberg, A., Cohen, M., Sod-Moriah, U., Shany, S., Rosenshtrauch, A., and Arad, S. (2000). Chickens fed with biomass of the red microalga Porphyridium sp. have reduced blood cholesterol level and modified fatty acid composition in egg yolk. Journal of Applied Phycology, 12, 325-330. DOI

[81] Hintz, H.F., Heitmann, H., Weird, W.C., Torell, D.T., and Meyer, J.H. (1966). Nutritive value of algae grown on sewage. Journal of Animal Science, 25, 675-681. DOI

[82] Davis, I.F., Sharkey, M.J., and Williams, D. (1975). Utilization of sewage algae in association with paper in diets of sheep. Agriculture and Environment, 2, 333-338. DOI

[83] Calderon, C.J.F., Merino, Z.H., and Barragán, M.D. (1976). Valor alimentico del alga espirulina (Spirulina geitleri) para ruminants. Tecnica Pecuaria en Mexico, 31, 42-46.

[84] Benemann, J.R. (1992). Microalgae aquaculture feeds. Journal of Applied Phycology, 4, 233-245. DOI

[85] Chen, Y.-C. (2003). Immobilized Isochrysis galbana (Haptophyta) for long-term storage and applications for feed and water quality control in clam (Meretrix lusoria) cultures. Journal of Applied Phycology, 15, 439-444. DOI

[86] Apt, K.E., and Behrens, P.W. (1999). Commercial developments in microalgal biotechnology. Journal of Phycology, 35, 215-226. DOI

[87] Muller-Feuga, A. (2000). The role of microalgae in aquaculture: situation and trends. Journal of Applied Phycology, 12, 527-534. DOI

[88] Volkman, J.K., Jeffery, S.W., Nichols, P.D., Rogers, G.I., and Garland, C.D. (1989). Fatty acid and lipid composition of 10 species of microalgae used in mariculture. Journal of Experimental Marine Biology and Ecology, 128, 219-240. DOI

[89] Naas, K.E., Naess, T., and Harboe, T. (1992). Enhanced 1st feeding of Halibut Larvae (Hippoglossus hippoglossus L) in green water. Aquaculture, 105, 143-156. DOI

[90] Cahu, C.L., and Zambonino-Infante, J.L. (1998). Algal addition in sea bass (Dicentrarchus labrax) larvae rearing: effect on digestive enzymes. Aquaculture, 161, 479-489. DOI

[91] Spolaore, P., Joannis-Cassan, C., Duran, E., and Isambert, A. (2006). Commercial applications of Microalgae- review. Journal of Bioscience and Bioengineering, 101, 87-96. DOI

[92] Gross, R., Gross, U., Ramirez, A., Cuadra, K., Collazos, C., and Feldheim, W. (1978). Nutritional tests with green Scenedesmus with health and malnourished children. Archiv fur Hydrobiologie, Beihefte Ergebnisse der Limnologie, 11, 161-173.

[93] Hallmann, A. (2007). Algal transgenics and biotechnology. Transgenic Plant Journal, 1, 81-98.

[94] Belay, A. (1993). Current knowledge on potential health benefits of Spirulina platensis. Journal of Applied Phycology, 5, 235-240. DOI

DOI: https://doi.org/10.52091/EVIK-2021/4-2-ENG

Received: July 2021 – Accepted: October 2021

¹ University of Debrecen, Institute of Food Science

Keywords

fruit, lyophilization, freeze drying, Total phenolic content (TPC), flavonoid, vitamin C

2. Introduction

Over the past few decades, there has been a steady increase in interest in research into the antioxidant effects of fruits, especially red fruits, as they support the proper functioning of the human body through their prominent role in nutritional physiology [1, 2]. Fruits are extremely rich in phenolic compounds, such as tannins, anthocyanins and flavonoids, and are considered a very good source of vitamins. They are high in sugar, contain dietary fiber and organic acids (oxalic acid, malic acid, citric acid, fumaric acid), while low in calories and fat [11]. These plant substances are present in higher concentrations in small fruits (blueberries, blackberries, strawberries, sour cherries and raspberries) [13], thus having a positive effect on the health and performance of the human body, and may provide protection against, for example, digestive, cardiovascular and other chronic diseases [3, 4, 5, 6, 7, 8].

Phenolic compounds present in fruits form a very large group of plant metabolites and exert their defense mechanisms over a very wide range [9, 14]. These compounds also affect the organoleptic properties and quality of the fruits [11, 12]. Flavonoids are secondary plant metabolites that have a protective function in fruits, against dehydration, infections, mechanical damage, etc. [15]. Vitamin C is a water-soluble vitamin that is essential for the human body, as it plays an important role in the defense against scurvy, as well as in maintaining healthy skin, gums and blood vessels, among other things [16]. Not only bioactive compounds, but also minerals may be responsible for the antioxidant effect. Exogenous antioxidants, such as vitamins C and E, flavonoids, carotenoids and elements with antioxidant effects, such as selenium, zinc, manganese, etc., are also key to the functioning of the human body’s defense mechanism. Red fruits contain higher amounts of the elements that are essential for the healthy functioning of the human body. For example, several studies have reported high potassium, calcium and magnesium contents in such fruits, as well as their low sodium content [17, 18, 19, 20].

In their fresh state, the fruits spoil in a short time, and their shelf life can be extended by reducing their moisture content, i.e., by drying. The production of such fruits is a major challenge for the food industry, as some drying processes can damage the antioxidant effects of the plants [10]. Therefore, it may be interesting to assess how freeze drying (as a gentle method) affects the bioactive content and antioxidant effects of the fruits.

3. Materials and methods

The fruits examined by us were strawberries (Fragaria x ananassa), raspberries (Rubus idaeus), sour cherries (Prunus cerasus), blackberries (Rubus) and blueberries (Cyanococcus). Fresh fruits were obtained from the same commercial unit, their place of cultivation was the north-eastern region of Hungary. The tests were started by examining the total polyphenol, flavonoid, acid and vitamin C contents of the fresh fruits. Following this, fresh fruits were lyophilized using a Heto Powerdry PL 9000 lyophilizer at -45 °C for 24 to 48 hours, and then the above tests were again carried out on the freeze-dried samples. Element content was tested only in the case of fresh samples, as neither drying ovens nor lyophilization has not an effect on the element content of the plants.

3.1. Determination of dry matter content

In the case of fresh fruits, the dry matter content was determined using a drying oven (Memmert UF 75 Universal Oven, Memmert GmbH+Co. KG, Schwabach, Germany). Samples were dried at 55 °C to constant weight for 12 hours, and then the moisture and dry matter content of the samples was determined using a formula. As lyophilization is a freeze-drying method, no further drying was performed on the lyophilized samples.

3.2. Total phenolic content (TPC)

The total phenolic content was determined using Folin-Ciocalteu reagent according to the method described by Singleton et al. [21]. After homogenization, the samples were soaked in an 80:20 mixture of methanol (Scharlab S. L., Spain) and distilled water, then they were filtered through fluted filter paper (Sartorius Stedim Biotech S.A., Gottingen, Germany). 0.5 ml of the samples was pipetted into a test tube, followed by the addition of 2.5 ml of Folin-Ciocalteu reagent (VWR International S.A.S., France) and 2 ml of 75 g/l sodium carbonate (Scharlab S. L., Spain) solution. For the formation of the colored compound, the samples were allowed to stand for 2 hours at room temperature in a light-protected area, and then the absorbance of the samples was measured in a 1 cm cuvette at 760 nm using a spectrophotometer (Evolution 300 LC, Thermo Electron Corporation, England). The calibration solution used in the determination of the total phenolic content was prepared from a stock solution of gallic acid (Alfa Aesar GmbH&Co. KG, Karlsruhe, Germany), so the results were obtained in mg GAE/100 g (Gallic Acid Equivalent).

3.3. Determination of flavonoid content

A spectrophotometric method was used to determine the total flavonoid content. Samples were once again soaked in an 80:20 mixture of methanol (Scharlab S. L., Spain) and distilled water, then they were filtered through fluted filter paper (Sartorius Stedim Biotech S.A., Gottingen, Germany). 1 ml of the filtered samples was pipetted into test tubes containing 4 ml of a 20:80 methanol:distilled water mixture and 0.3 ml 5% of sodium nitrite (Scharlau Chemie S.A., Spain), then 5 minutes were allowed to pass. At the end of the waiting time, 0.3 ml 10%of aluminum chloride (Scharlab S.L., Spain) and 2 ml of 1 M sodium hydroxide (Sigma-Aldrich Chemie GmbH, Germany) solution were pipetted to the samples, and the volume was filled to 10 ml using a methanol: distilled water mixture. Finally, the absorbance of the samples was measured in a 1 cm cuvette using a spectrophotometer (Evolution 300 LC, Thermo Electron Corporation, England) at 510 nm. A stock solution of catechin (Cayman Chemical Company, USA) was used for the calibration solutions, and the results were obtained in mg CE/100 g (Catechin Equivalent) [22].

3.4. Determination of vitamin C content

The vitamin C content of the samples was determined using a metaphosphoric acid solution [23]. To 5 g of the samples was added 100 ml of a 3% metaphosphoric acid (Thermo Fischer GmbH, Germany) solution, then it was mixed. It was then washed into a 250 ml volumetric flask and an additional 50 ml of metaphosphoric acid was added. The mixture was filtered through fluted filter paper (Sartorius Stedim Biotech S.A., Gottingen, Germany). 50 ml of the filtrate was pipetted into a titration flask, then 30 ml of distilled water, 5 ml of 2% hydrochloric acid (VWR International S.A.S, France), 5 ml of 1% potassium iodide (Sigma-Aldrich Chemie GmbH, Germany) and 1 ml of starch indicator (VWR International S.A.S., France) were added. The resulting solution was finally titrated with a 0.004 M potassium iodate (Sigma-Aldrich Chemie GmbH, Germany) solution. Results are given in mg/100 g.

3.5. Determination of total acid content

Acid content was determined according to the method described by Czipa (2014) [23]. Fresh samples were homogenized, lyophilized samples were pulverized, and then 20 g was weighed into an Erlenmeyer flask and 150 ml of distilled water was added. After thorough stirring, it was heated on a water bath at 85-95 °C for 30 minutes, and then it was allowed to cool to room temperature. The mixture was filtered through cotton wool and then made up to the mark with distilled water in a 250 ml volumetric flask. 25 ml of the resulting filtrate was pipetted out and made up to 100 ml with distilled water ml. Titration was carried out in the presence of a few drops of phenolphthalein indicator with 0.1 M sodium hydroxide (Sigma-Aldrich Chemie GmbH, Germany). Results are given in %.

3.6. Determination of element content

Sample preparation was performed according to the method of Kovács et al. [24]. During the test, 3 g of the sample was weighed into a 100 ml digestion tube. Concentrated nitric acid (10 ml) was added to the samples, they were allowed to stand overnight, and then were heated at 60 °C for 30 minutes. Following this, hydrogen peroxide (3 ml) was added to the samples and they were heated again at 120 °C for 90 minutes. At the end of this time, the samples were made up to 50 ml with high purity distilled water (Milli-Q water purification system; Millipore SAS, Molsheim, France), and filtered through 388 filter-paper (Sartorius Stedim Biotech S.A., Gottingen, Germany). Element content was measured with an ICP-OES (Inductively Coupled Plasma Optical Emission Spectrometer) instrument (Thermo Scientific iCAP 6300, Cambridge, UK). The elements were measured at the following wavelengths: Ca (317.9 nm), K (766.4 nm), Mg (279.5 nm), Na (589.5 nm), P (185.9 nm), S (180.7 nm), Mn (259.3 nm), Zn (213.8 nm). The Rf power of the ICP instrument was set to 1200 W.

3.7. Statistics

Analytical testing of the samples was performed in triplicate in each case. SPSS software (version 13; SPSS Inc. Chicago, Illinois, USA) was used for the evaluation of the results. Using the program, the mean and standard deviation were determined, and then Tukey and Dunnett’s T3 test (one-way analysis of variance) was used to determine statistically significant differences between the results.

4. Results

4.1. Dry matter content

Among the fruits we examined, strawberries and sour cherries had the lowest dry matter content (12.6%), while the highest results were obtained for blackberries (16.8%). In order to make the results of the different tests comparable, the values are given on a dry matter basis in each case.

4.2 Total phenolic compounds (TPC)

The total phenolic content of the fresh and lyophilized fruits is summarized in Figure 1. Very high values (945-1363 mg GAE/100 g) were obtained for all samples. Higher values were measured for the lyophilized samples than for the fresh fruits. In our opinion, this may be due to the fact that lyophilization for these compounds is a more gentle drying method than using an oven.

As shown in the figure, there is no significant difference between fresh blackberries (967 mg GAE/100 g) and fresh blueberries (945 mg GAE/100 g). In the case of these fruits, the phenolic content is not significantly higher even after lyophilization (1037-1061 mg GAE/100 g). In contrast, for strawberries, raspberries and sour cherries, values between 1,145 and 1,363 mg GAE/100 g were obtained. No significantly different values were obtained for fresh strawberries and fresh sour cherries (1,145 and 1,150 mg GAE/100 g), and for lyophilized strawberries and lyophilized sour cherries (1,306 and 1,283 mg GAE/100 g). These differences could not be verified statistically.

4.3. Flavonoid content

The flavonoid content of the tested samples is shown in Figure 2. It is clear that there is no significant difference between the fresh and lyophilized samples, i.e., lyophilization does not significantly affect the presence of these compounds in the samples. In contrast to the phenolic compounds content, raspberries had the lowest flavonoid content (309-340 mg CE/100 g), while blueberries had the highest values (647-707 mg CE/100 g). In the case of strawberries and blackberries, almost the same results were obtained, and the differences were statistically verifiable in each case.

4.4. Total acid content

The total acid content is shown in Figure 3. It can be clearly seen that lyophilization did not have a beneficial effect on these compounds, as significantly lower results were obtained for all samples compared to the fresh fruits. Very high acid contents were measured for fresh raspberries and fresh sour cherries (54.4-54.5%). As a result of lyophilization, these values decreased by two-thirds (16.6-16.7%). In the case of the other fruits, the acid content did not even reach 30%. Statistically verifiable differences were obtained in all cases, except for lyophilized sour cherries and lyophilized raspberries (P=0.167).

4.5. Vitamin C content

The vitamin C content of the fruits is shown in Figure 4. In this case, lyophilization did not have a large effect on the vitamin C content, since, as shown in the figure, the results after lyophilization were lower for all samples. The highest values (236 and 242 mg/100 g) were obtained for strawberries. Similar results were obtained for raspberries and blackberries (172-199 mg/100 g), and for sour cherries and blueberries (102-128 mg/100 g). Significant results were obtained in almost all cases, except for fresh strawberries with fresh raspberries, fresh raspberries with fresh blackberries, and for fresh sour cherries with fresh blueberries.

4.6. Element content

The element contents of the samples are shown in Table 1. Although several elements were measured, only the most important results are highlighted. The calcium content of the fruits was between 240 and 2302 mg/kg. Among the results, the calcium content of blueberries was extremely low compared to the other samples (240 mg/kg). There was no statistically verifiable difference between strawberries and blackberries (P=0.096).

The potassium content was highest in the case of strawberries (12,693 mg/kg). In contrast, blueberries have an extremely low potassium content of 3,765 mg/kg. Values between 6,582 and 9,521 mg/kg were obtained for the other samples. The differences were statistically verifiable in all cases. The magnesium content of the fruits was between 195 and 1,383 mg/kg. Once again, blueberries exhibited an extremely low value of 195 mg/kg. At the same time, the magnesium content of strawberries was 1,383 mg/kg. Significant differences were obtained in almost all cases, with the exception of strawberries-raspberries and sour cherries-raspberries. The sodium content values of the fruits was between 5.15 and 23.1 mg/kg. Compared to the other samples, the sodium content of strawberries and blackberries was very high (22.9 and 23.1 mg/kg). Phosphorus content results were between 863 and 2,024 mg/kg. Similarly to calcium, potassium and magnesium, blueberries had the lowest result for phosphorus (863 mg/kg) as well. Significant results were obtained for all samples. In the case of sulfur, values between 445 and 695 mg/kg were measured.

The lowest result was obtained for blueberries, while the highest was obtained for blackberries. Significant results were obtained in all cases except for blueberries-strawberries-sour cherries [25].

4.7. Conclusions

The nutritional parameters of different red fruits were examined. Our aim was to compare the examined parameters (total phenolic content, flavonoid, acid and vitamin C content) in the fresh state of the fruits and after lyophilization. In addition, the major element contents (calcium, potassium, magnesium, sodium, phosphorus, sulfur) of the fresh samples were also determined. In terms of the total phenolic content and the flavonoid content, higher results were obtained for all fruits after lyophilization. The reason for this may be that lyophilization does not have as adverse an effect on these compounds as the use of a drying oven. Positive results were also obtained for vitamin C. The presence of this vitamin was slightly reduced in these samples by lyophilization. In contrast, much lower acid content results were obtained after lyophilization. In terms of their element content, blueberries had the lowest values, while the highest values were obtained in the case of strawberries. Based on the results obtained, it can be stated that in the case of the parameters examined (except for the acid content), freeze drying, also known as lyophilization, is a much more gentle drying method than the use of an oven.

5. References

[1] Fu L., Xu B.-T., Xu X.-R., Gan R.-Y., Zhang Y., Xi E.-Q., & Li H.-B. (2011): Antioxidant capacities and total phenolic contents of 62 fruits. Food Chemistry 129 (2) 345–350. pp. DOI

[2] Imeh U., Khokhar S. (2002): Distribution of Conjugated and Free Phenols in Fruits: Antioxidant Activity and Cultivar Variations. Journal of Agricultural and Food Chemistry 50 (22) 6301–6306. pp. DOI

[3] de Souza V. R., Pereira P. A. P., da Silva, T. L. T., de Oliveira Lima L. C., Pio R., Queiroz F. (2014): Determination of the bioactive compounds, antioxidant activity and chemical composition of Brazilian blackberry, red raspberry, strawberry, blueberry and sweet cherry fruits. Food Chemistry 156 362–368. pp.

[4] Denardin C. C., Hirsch G. E., da Rocha R. F., Vizzotto M., Henriques A. T., Moreira J. C. F., Emanuelli T. (2015): Antioxidant capacity and bioactive compounds of four Brazilian native fruits. Journal of Food and Drug Analysis 23 (3) 387–398. pp. DOI

[5] Moo-Huchin V. M., Moo-Huchin M. I., Estrada-León R. J., Cuevas-Glory L., Estrada-Mota I. A., Ortiz-Vázquez E., Sauri-Duch E. (2015): Antioxidant compounds, antioxidant activity and phenolic content in peel from three tropical fruits from Yucatan, Mexico. Food Chemistry 166 17–22. pp.  DOI

[6] Yildiz H., Ercisli S., Hegedus A., Akbulut M., Topdas E. F., Aliman J. (2014): Bioactive content and antioxidant characteristics of wild (Fragaria vesca L.) and cultivated strawberry (Fragaria × ananassa Duch.) fruits from Turkey. Journal of Applied Botany and Food Quality 87 274–278. pp. DOI

[7] Slatnar A., Jakopic J., Stampar F., Veberic R., Jamnik P., (2012): The Effect of Bioactive Compounds on In Vitro and In Vivo Antioxidant Activity of Different Berry Juices. PLoS ONE 7 (10) 1-8. pp.

[8] Namiesnik J., Vearasilp K., Nemirovski A., Leontowicz H., Leontowicz M., Pasko P., Martinez-Ayala A.L., González-Aguilar G.A., Suhaj M., Gorinstein S. (2014): In vitro studies on the relationship between the antioxidant activities of some berry extracts and their binding properties to serum albumin. Applied Biochemistry and Biotechnology 172 2849–2865. pp.

[9] Baiano A. (2014): Influence of genotype, pedoclimatic conditions, viticultural practices and ripening on the phenolic composition of grapes. A review. In J. S. Cámara (Ed.), Grapes: Production, phenolic composition and potential biomedical effects. Food and Beverage Consumption and Health Series. New York, NY: Nova Science 1–26. pp. ISBN: 978-1-63321-410-1

[10] Lutz M., Hernández J., Henríquez C. (2015): Phenolic content and antioxidant capacity in fresh and dry fruits and vegetables grown in Chile. CyTA - Journal of Food. Taylor & Francis. 1–7. pp.

[11] Aly A., Maraei R., El-Leel O. A. (2019): Comparative study of some bioactive compounds and their antioxidant activity of some berry types. Slovak Journal of Food Sciences. Potravinarstvo Slovak Journal of Food Sciences 13 (1) 515–523. pp. DOI

[12] Lachowicz S., Kolniak-Ostek J., Oszmianski J., Wisniewski R. (2017): Comparison of phenolic content and antioxidant capacity of bear garlic (Allium ursinum L.) in different maturity stages. Journal Food Processing and Preservation 41 (1) 1–10. pp. DOI

[13] Toledo-Martín E., García-García M., Font R., Moreno-Rojas J., Salinas-Navarro M., Gómez P., del Río-Celestino M. (2018): Quantification of Total Phenolic and Carotenoid Content in Blackberries (Rubus Fructicosus L.) Using Near Infrared Spectroscopy (NIRS) and Multivariate Analysis. Molecules 23 (12) 3191. p. DOI

[14] Zapata, P. J., Martínez-Esplá, A., Gironés-Vilaplana, A., Santos-Lax, D., Noguera-Artiaga, L., Carbonell-Barrachina, Á. A. (2019): Phenolic, volatile, and sensory profiles of beer enriched by macerating quince fruits. LWT – Food Science and Technology 103 139–146. pp. DOI

[15] Oliveira K. G., Queiroz V. A. V., Carlos L. A., Cardoso L. M., Pinheiro-Sant’Ana H. M., Anunciacao P. C., Menezes C. B., Silva E. C., Barros F. (2017): Effect of the storage time and temperature on phenolic compounds from sorghum grain and flour. Food Chemistry 216 390–398. pp. DOI

[16] Rekha C., Poornima G., Manasa M., Abhipsa V., Pavithra Devi J., Vijay Kumar H. T., Prashith Kekuda T. R. (2012): Ascorbic acid, total phenol content and antioxidant activity of fresh juices of four ripe and unripe citrus fruits. Chem Sci Trans. 1 (2) 303–310. pp. DOI

[17] Nour V., Trandafir I., Ionica M. E. (2011): Ascorbic acid, anthocyanins, organic acids and mineral content of some black and red currant cultivars. Fruits 66 353–362. pp. DOI

[18] Plessi M., Bertelli D., Albasini A., (2007): Distribution of metals and phenolic compounds as a criterion to evaluate variety of berries and related jams, Food Chemistry 100 419–427. pp.

[19] Nile, S. H., & Park, S. W. (2014). Edible berries: Bioactive components and their effect on human health. Nutrition 30 (2) 134–144. pp. DOI

[20] Rodler I. 2008. Élelmezés- és táplálkozás-egészségtan. Medicina Könyvkiadó, Budapest.

[21] Singleton, V. L., Orthofer, R., Lamuela Raventos, R. M. (1999): Analysis of Total Phenols and Other Oxidation Substrates and Antioxidants by Means of Folin Ciocalteu reagent. Methods in Enzymology 299 265–275. pp.

[22] Kim, D.O, Jeong, S.W., Lee, C.Y. (2003): Antioxidant capacity of phenolic phytochemicals from various cultivars of plums. Food Chemistry 81 321–326. pp.

[23] Czipa, N. (2014): Élelmiszeranalitika gyakorlati jegyzet élelmiszermérnök BSc III. évfolyam részére. Debreceni Egyetem, Debrecen.

[24] Kovács, B., Győri, Z., Csapó, J., Loch, J., Dániel, P. (1996): A study of plant sample preparation and inductively coupled plasma emission spectrometry parameters. Communication in Soil Science and Plant Analysis 27 (5-8) 1177–1198. pp.

[25] Słupski, J., Lisiewska, Z., Kmiecik, W. (2005): Contents of macro and microelements in fresh and frozen dill (Anethum graveolens L.). Food Chemistry 91 (4) 737–743. pp. DOI

DOI: https://doi.org/10.52091/EVIK-2021/3-1-ENG

Received: June 2021 – Accepted: August 2021

¹ University of Debrecen, Faculty of Economics and Business, Institute of Marketing and Commerce

Keywords

netnography, functional foods, Consumer Style Inventory Test (CSI test), transtheoretical model

2. Introduction, literature review

2.1. Risk factors for health loss

Parts of health behavior are all health-related behaviors that manifest themselves as components of a healthy lifestyle, and as behaviors resulting from health motivations and health needs [1]. In Hungary, according to the NEFI (National Institute for Health Development) [2] 80% of the risks of health loss can be attributed to behavioral factors, of which a sedentary lifestyle and inadequate nutrition stand out.

Physical inactivity is responsible for 10% of cancers, has a serious impact on coronary heart disease, type 2 diabetes and osteoporosis, results in depressive symptoms, and last but not least, is responsible for 5 million deaths worldwide each year [3,4]. Physical activity and active sporting activities are separate conceptual categories. Activities related to physical activity can be divided into four groups according to their medium and way of implementation. Based on this, work-related, transport-related, household-related and leisure-time physical activities can be distinguished [5]. 53% of Hungarian never participates in any sporting activity and roughly half of the population does not engage in even moderate physical activity [6].

The concept of a sedentary lifestyle is important in the study of health behavior, as it has become a typical way of life in developed societies in recent decades. Any activity during waking hours where the metabolic equivalent (1 MET = 3.5 ml/min/kg body weight oxygen consumption) is less than 1.5 is considered sitting. A sedentary lifestyle has extremely negative effects on health in the long run [7,8]. Nearly half of the adult population of Hungary spends more than 5 hours 31 minutes sitting daily, and 10% work more than 8 hours 31 minutes sitting [9].

It si well known that obesity is a risk factor fro many chronic diseases. In 2008, there were 1.5 billion overweight people [10], in 2014, the number was more than 2.1 billion, and half of humanity is projected to be overweight by 2030 [11]. Results of the latest surveys are depressing, as they indicate that 58% of the adult population is overweight or obese based on their body mass index [12].

2.2. Health foods

The problems outlined are global and pose significant challenges for the food industry, among others. Improvements are needed which, due to their beneficial health effects, can slow down the spread of diseases of civilization and increase life expectancy spent in health [13]. Health foods have been created to treat the deficiencies caused by an unbalanced diet, to restore energy balance and to maintain health. Their names are extremely varied (e.g., healthy food, designer food, functional food, pharmafood), and the term functional food is most commonly used in the literature [14].

Foods with special health protection effects are not officially categorized and defined in Hungary, but the term functional food is widely used in the international literature. Based on the internationally accepted definition of ILSI², this includes foods that, due to their bioactive ingredients, in addition to normal nutrition, have health benefits [15]. The main groups of functional food ingredients are vitamins and minerals, proteins, peptides, antioxidants, fatty acids and phytochemicals, and pre- and probiotics [14]. In the early 2000s, the most popular functional foods were energy and sports drinks, probiotic dairy products, ”heart-friendly” products and ready-to-eat cereals [16]. According to the 2016 statistics of Google Food Trends, within the category of functional foods, ”healthy ingredients”, such as turmeric, apple cider vinegar and avocado oil, as well as bitter melon and kefir proved to be the most popular among consumers [17]. Between January 1990 and June 2018, the most studied functional foods and ingredients were prebiotics, probiotics and antioxidants, according to the bibliometric assessment of Yeung et al. (2018), who analyzed the most cited and sought for items in the literature [18] Among the factors influencing consumers’ willingness to buy, the most significant are health effects, taste, quality, value for money, and their knowledge about functional foods [19,20]. Consumption of health foods and a healthy diet can be considered cornerstones of health behavior.

2.3. Examining health behavior

To study health behavior, a number of models are used by researchers. The transtheoretical model of behavior change, hereinafter TTM³, was originally introduced as an integration of different theoretical concepts in clinical psychology [21, 22]. Prochaska and Prochaska [23], in order for professionals to be able to have a significant and lasting impact on health-threatening behaviors, have developed a model that can be applied to study the health behavior of not only the minority who is motivated for change, but the entire population. TTM encompasses process-oriented variables to predict and explain how and when subjects change their behavior [24]. Behavior change is a process that takes place over a long period of time and goes through a defined series of stages [25]. The model can be used to examine exactly where a person/group is in the transition to sustainable health behavior. Based on this, five stages are distinguished [26]:

• Precontemplation,
• Contemplation,
• Preparation,
• Action and
• Maintenance.

In the precontemplation stage, the individual is unaware of the consequences of risk behavior, does not seek information and is not interested in changing health behavior in a positive direction. In the contemplation stage, the individual weighs the benefits of the change and compares them to the costs of change. They are aware of the need for change, but if the costs are considered to be excessive, further steps are not taken. In the preparation stage, the individual is already prepared to take certain steps and possesses an action plan. In the action stage, he individual takes specific steps to protect their health. As awareness increases, the chances of returning to past behavior decrease. Real behavior change can be achieved in the maintenance stage, after at east six months. At this point, the new form of behavior becomes a natural part of the individual’s life and there is no need for reinforcements from the environment either [14, 26].

In the primary research outlined in the present study, TTM was used by our group to investigate the transition to a healthy diet.

From an economic point of view, the elements of health behavior that manifest themselves in behavior are shopping and consumption. Consumers approach the market with basic decision styles. These can be defined as mental shopping orientations that characterize consumer choices [27]. To measure the diversity of decision styles, the Consumer Style Inventory, hereinafter referred to as the CSI test [28]. CSI has been validated in many countries around the world (e.g., the United Kingdom, New Zealand, South Korea, Germany, Singapore, China, Malaysia, India, Turkey, USA) and is widely used [29]. CSI has been used in the past in general commerce [30, 31], in the monitoring of online behavior [28], and in organic food buying [32], among others. With regard to health/functional foods, no research has been carried out so far in which CSI has also been incorporated, and this was attempted by our group in our quantitative studies. In addition to exploring the decision-making styles and attitudes related to the purchase and consumption of health foods, it was also considered important to carry out a study in the online space, as this is one of the most relevant information source and communication interfaces today.

³ Transteorethical Model

2.4. Health communication nowadays – obtaining information online

According to 2021 data, there are approximately 5.16 billion active Internet users worldwide [33], and 4.48 billion of them use social media [34]. In recent years, social media has changed people’s interactions, including health-related communication [35]. Benetoli et al. [36] identified convenient and quick access, improved health knowledge and a sense of social and emotional support as the benefits of obtaining health information through social media. Disadvantages of social media included questionable credibility, information overload and the increased time spent online, among others. Johns et al. [37] classified studies published between 2000 and 2016 in terms of changes in health behavior and the impact of social media. As a result of their research, it was found that social media had no effect on giving up smoking or weight loss, but had an effect on increased physical activity.

As an axiom, it can be stated that digital communication is an integral part of of the advanced societies of today. Research in the online space is a useful addition to a type of marketing research that are group has been working on. Netnography is a qualitative research method that adapts the techniques of netnographic studies to examine the culture of online communities [38]. It can be used to understand the mindset and decision-making mechanisms of online consumer groups [39]. Ten years ago, Dörnyei and Mitev [40] recorded the basic forms of online communication: instant messengers, e-mail lists, game interfaces, chat applications, blogs, search engines, forums, social media sites. In terms of their usage, these channels have undergone a radical change. While blogs and forums flourished in 2010, today consumers barely use these platforms at all. Today’s most popular, almost exclusive virtual communication interfaces are content and video sharing sites under the umbrella of social media, of which Facebook stands out, with 2.853 billion users wolrdwide [34].

3. Materials and methods

In our research, the goal was to examine consumer attitudes towards health foods, which we analyzed in the common subset of physical activity and the diet. The research took place between April and November of 2019, and then a follow-up was performed in April 2021 by repeating our netnographic analyses. Our studies were carried out in three stages.

In the first step of primary data collection, a netnographic research was conducted with data recorded in a search engine on the one hand and the content analysis of of posts and comments made groups of publicly available social sites on the other hand. The interests of consumers present in the online space, as well as changes in them were detected in terms of health-conscious eating and physical activity.

In the second stage of our research, two focus group interviews were conducted. The impact of regular exercise on the purchase and consumption of health foods was examined, and the implications of this in developing and maintaining a diet considered to be healthier by consumers. 7 people each were included in the studies, based on preliminary criteria. The conditions for inclusion in the groups were as follows:

• The subject is over 18 years of age;
• The subject does not work in the fields of journalism, marketing, advertising, PR or market research;
• The subject has not participated in a market research survey related to the topic of physical activity and/or health-conscious eating in the previous year;
• The subject has not participated in a focus group discussion in the previous year;
• The subject does not have a milk protein allergy;
• First group: The subject regularly engages in physical activity;
• Second group: The subject does not engage in physical activity.

The differentiating factor for inclusion in the groups was performing regular physical activity, so an active and a passive group were created. At the start of the study, participants introduced themselves one by one, and then they had a conversation for a few minutes under the guidance of the moderator, creating group cohesion and an atmosphere of trust. The first part of the scenarios examined the factors that play a role in the development of a healthy lifestyle. In the second block, buying and consumption habits related to health foods were explored. The transtheoretical model of behavior change was incorporated in the scenarios, and it was examined with respect to the topic of healthy eating. In both cases, group discussions took place in an informal style and lasted an hour and a half. Minutes and audio recordings of the discussions were taken, which allowed for accurate analysis.

In the third stage of our research, an online questionnaire survey was conducted, which was shared several social media groups with the help of dietitians. A total of 378 people completed the questionnaire. To ensure representativeness, the sample was adjusted so that it reflects the composition of the Hungarian population in terms of gender distribution. As a result, mathematical-statistical analyses were performed on a sample of 300 people. In addition to key demographic data, based on Eurobarometer [9] surveys, statements related to physical activity and sedentary lifestyle were formulated, which were classified as background variables in the analysis. The questionnaire included the Hungarian translation of the Consumer Style Inventory (CSI), which was adapted and modified for health foods based on the research of Prakash et al. [32]. Items with Cronbach’s alpha values above 0.7 were included in our own research, and one dimension, statements related to environmentally conscious consumption, was omitted. As a result, 25 statements were formulated that respondents had to rate on a Likert scale of 1 to 5. The transtheoretical model of behavior change was incorporated into the questionnaire and it was examined in relation to healthy eating. Based on the focus group discussions, expansion of the TTM statements was considered to be justified, so the 6-point ordinal scale of Szabó [41] was incorporated in the questionnaire. This essentially separates the 5 stages defined in the literature by dividing the action phase into two subcategories. The main goal of our quantitative research was to identify consumer attitudes in the CSI adapted to health foods. To achieve this, in the first step the normal distribution of the variables was tested, and then the reliability of the scales was analyzed, in each case obtaining good or excellent reliability. Following this, factor analysis was performed using the CSI variables. After running several possible procedures, principal component analysis was finally applied with Varimax rotation and Kaiser normalization. The KMO criterion of factor analysis was met, exhibiting an almost excellent value (0.853). During the analysis, three variables were excluded from the CSI scale, as they distorted both reliability and the results of factor analysis (”Typically, I buy health foods at a discount price.”, ”I usually choose lower priced products.”, ”I’d rather buy well-known, domestic brand products.”). As a result, the explanatory power of the model has improved. A total of four factors were created to form differentiated attitude structures. In the next step, the reliability of the factors obtained was checked by calculating Cronbach’s alpha values, and then sample segmentation was carried out. The analysis was performed using the K-means clustering procedure, during which four well-separated, homogeneous groups were identified, based on consumer attitudes in the CSI. Characterization of each cluster was performed by cross-tabulation analysis and analysis of variance.

4. Results and their evaluation

4.1. Results of the netnographic study

Our study was conducted in line with today’s trends, using a search engine on the one hand and by content analysis of social media sites on the other hand. Different search engines and browsers have been optimized for different terms, and so search results and and hit lists may differ from each other. We used the Google search engine through the Google Chrome browser both in 2019 and 2021. As a first step, our results were compared to those of Gál et al. [42] in terms of search hits of nutrition-related keywords. Then the changes in search hits were identified for terms related to healthy eating and regular exercise over the intervening two years. Changes in nutrition-related keyword search results are shown in Figure 1.

Gál et al. [42] included the terms “healthy diet” and “healthy eating” as synonyms for ”health-conscious eating”. It can be seen that both in 2017 and 2019, the leading hits were generated by the term healthy eating, but in 2021 an explosive growth of the term “healthy diet” can be observed. The number of hits has increased nearly tenfold in four years. Search results for terms related to both nutrition and sports are shown in Figure 2.

The number of hits for search engine terms shows a variable rate increase. Of the key terms provided by us, ”regular sporting activities” and ”healthy eating and sports” proved to be the most sought after. ”Healthy eating and sports”, although the second most common content among search terms, shows a declining trend compared to 2019. At the same time, content on the topic ”healthy eating and sports” has tripled, yielding more than 6.5 million hits in 2021.

After defining the keywords, posts from publicly accessible pages were analyzed, and this was followed up by monitoring in 2021. The popularity of forum portals continued to show a declining trend, so those sites were not investigated. However, it is important to note that there was a periodic activity in the case of forum portals (e.g. hoxa.hu, gyakorikerdesek.hu) in 2020. It is assumed that this can be attributed to the quarantine caused by the pandemic. But the explosive growth of social media groups has now almost completely overridden the activity of forum portals.

In Hungary, of the social media sites, currently the trinity of Facebook, Instagram and YouTube is the most popular among active internet users. The “hashtag” is an international communication tool for navigating between and orientation on the surfaces. Hashtags allow us to get to the type of content that interests us on any of the social media giants’ websites (YouTube videos; photo-based short Instagram posts / users / pages; typed text based Facebook posts / users / groups / pages). In addition to the three basic pillars, also appearing are Tik-Tok, which is mainly used by young people, and Twitter, which is less popular in Hungary but more popular internationally. Of social media, the analysis of Facebook pages and groups was chosen, because nowadays most of the internet user community communicates on this interface. All open and closed groups, as well as pages, with at least 3,000 members and followers were examined. Only Hungarian groups and pages were analyzed. In addition to keywords, their hashtag variations were also used (e.g., #regularsports; #healthyeating) to facilitate more accurate content analysis. Four main topics were identified during the analysis of the groups and pages, and these are illustrated in Figure 3.

Based on our analyses, when weighing nutrition and exercise, the topic of healthy eating clearly appears in a more pronounced way in the interest of consumers. Healthy foods and dishes are most commonly associated with the terms ”free” and ”reduced” in public awareness, such as the terms sugar-free, chemical-free, reduced salt, reduced carbohydrate. In addition, there is an increasing emphasis on gluten- and dairy-free eating and different types of diets. This confirms the previous research results of Gál et al. [42], according to which a health-conscious diet and lifestyle is associated with some kind of diet or weight loss program by the majority of people.

The most common content in Facebook groups or pages in the common subset of diet and exercise is a combination of workout plans and recipes. These include short videos or photos that offer some kind of recipe along with a form of exercise or workout plan, typically using ”reduced” or ”free” ingredients. Workout plans are typically “challenges” over a longer period of time (e.g., broken down for a month), or forms of movement presented in a short video. Particularly popular contents are home exercises that can be performed without any aids or with minimal use of aids (e.g., dumbbells).

The content encountered second most frequently is requests for suggestions on a health problem or a change in lifestyle or diet. This is most noticeable regarding the topic of eating, less content requesting suggestions is found on exercise and physical activity.

The third most common content is providing advice or information. In the case of this type of content, mainly articles and stories, often with questionable authenticity, from associated sites appear on the pages, as well as short videos and infographics. Advice on the topic of physical activity is typically about how to start exercising regularly, what pitfalls and difficulties one might encounter. In the field of nutrition, the most common are discussion initiating contents related to gluten and sugar consumption, as well as dairy products and caffeine. This is followed by the presentation and promotion of “healthy products” and posts emphasizing the importance of fruit and vegetable consumption.

Other major type of content is the presentation of motivational examples. In this content, photos of “transformations” that occur as a result of some diet, dietary change or regular exercise are typically uploaded by users. Motivational examples often include presentations of “own stories” about restoring health. In these stories, people who share the content report a positive change in health as a result of a diet considered to be healthy and/or regular physical activity.

Overall, it can be stated that healthy eating and physical activity are popular activities among internet users. In the common subset of diet and exercise, the emphasis was typically on issues related to nutrition in the media examined by us. The most popular types of content are personal in nature and have a community-building power.

4.2. Results of focus group studies

4.2.1. Factors that play a role in the development of a healthy lifestyle

When describing the results of our focus group research, groups are referred to as ”active” and ”passive” ones, revealing the attitudes and peculiar characteristics of the given group. In the first block of the scenario, we sought to answer the question what similarities and differences could be detected between the groups in the topic of health. Regarding the groups, it can be generally said that in the subjective assessment of health, the active group considers their lifestyle to be healthy, while the passive group considers it to be unhealthy. In the development of a healthy lifestyle, the groups studied unanimously thought that the right amount and quality of sleep, proper nutrition, mental health and regular exercise were vital.

Following this, the groups had to rank 15 factors according to the influence of each component. Based on the ranking thus developed, the 5 most important factors according to each group are listed in Table 1.

Regarding the second most important features of the groups, 2 factors were raised to the same level by each group, as in neither case were they able to reduce the ranking to 1 component at this level. Nutrition and exercise were considered to be relevant by both groups, but it should be emphasized that information, accessibility and adequate financial situation were the most important for the passive group. Regarding the transition to a healthy lifestyle, the groups studied had to make arguments as to why it could be easy, as well as what would be difficult in the process. Overall, the same factors were listed both as pros and cons. The groups attached similarly great importance to the influence of the social environment, which they believed had a strong impact on the individual’s health behavior.

4.2.2. Customer and consumer habits and motivations related to health foods

There are differences between the categories of food most often purchased and consumed by the two groups. The passive group consumed a higher proportion of meat products, quick-frozen and processed foods. The active group preferred seasonal fruits and vegetables, dairy products and fresh bakery products. Members of the active group, according to their own statements, plan their purchases in advance, while impulse buying is more common among the passive group.

Prior to examining consumer attitudes towards health foods, their concept as clarified with group members: ”They are foods that have one or more nutritional biological benefits in addition to excellent taste. These advantages include lower energy content, mainly through the reduction of fat content or the omission of sugars, enrichment in certain minerals (Ca, Se, Mg), depletion in others (Na), addition of multivitamins or the use of probiotic lactic acid bacteria in different foods.” All of the subjects in the study bought and consumed health foods. For members of the active group, ”being free of something” was important, which manifested itself mainly in the avoidance of fat, salt and sugar. Members of the passive group typically preferred products ”fortified with something”. The active group bought more types of health food more often than the passive group.

It was characteristic of both groups that subjects had changed their eating habits over the previous year. The reason for this was the development of some kind of sensitivity/allergy, as well as the need to change lifestyles and to try new diets. Nutritional trends affected the active group, but they usually researched a diet before trying it. Members of the passive group are generally said to be uninterested in different trends, as well as dietary recommendations.

Regarding the purchase and consumption of food, the active group considers the healthiness of food to be the most important factor, while for the passive group it is value for money. Members of the active group attach particular importance to nutrient composition, to products that are ”free of something”. For the passive group, in addition to easy availability, previous positive experience has an impact on food buying habits.

4.2.3. Differences in the transition to a healthy diet based on the TTM

Using the statements translated by Soós et al. [26] based on the TTM, it was examined where the groups were in the transition to what they considered to be a healthier diet. Stages in the behavior change are described below, with examples of the statements made:

• Precontemplation: In the next six months, I do not intend to switch to a diet I consider healthier;
• Contemplation: I feel a strong urge to switch to a diet I consider healthier;
• Preparation: Over the next month, I will be taking steps to switch to a diet I consider healthier;
• Action: Over the past six months, I have switched to a diet I consider healthier;
• Maintenance: I have been eating healthier for over six months now.

In the transition to a diet that is considered healthy, 30% of the active group was in the action stage, while 70% was in the maintenance stage. In contrast, 70% of the passive group was in the precontemplation or contemplation stage, while 30% was in the preparation stage. Based on this, it can be concluded that the passive group is less open to developing and maintaining a health-conscious diet.

Overall, it can be stated that great emphasis is placed on the consumption of health foods among the group who perform physical activity regularly. Purchases are planned more purposefully by the active group and, according to their own statements, their diet is more based on awareness.

4.3. Results of the questionnaire survey

4.3.1. Presentation of the sample

Our quantitative study was conducted in the online space. The gender distribution of our sample reflects the composition of the Hungarian population, however, our results are more exploratory, as the sampling took place in a specific medium. The sample was made up of people who follow the online work and activity of dietitians, and themselves spend time regularly in the online space. The distribution of the sample according to different background variables is shown in Tables 2 and 3.

Examining the age distribution, it can be stated that our sample is representative of internet users, i.e., the 18 to 49 age group is typically represented. Compared to the demographic composition of the Hungarian population, the proportion of people with higher education is much higher in our sample. Nearly half of the respondents consider themselves mostly health-conscious, engage in physical activity regularly, and 41.7% of these people spend 31 to 60 minutes with exercise daily. Two thirds of the sample spend between 2 hours 31 minutes and 8 hours 30 minutes a day sitting, with an additional 17% spending even more. This rate is higher than the Hungarian data measured by the Eurobarometer [9].

The transition to a healthier diet shows a more positive picture compared to the overall data of the Hungarian population. It must be added that, based on our representative national surveys, the proportion of people in the precontemplation stage is decreasing, while the proportion of people in the preparation, action and maintenance stages is increasing. In 2014 and 2019, 48% and 41% of the population was in the precontemplation stage, respectively, while the proportion of people maintaining a diet considered to be healthier has increased from 17.4% to 23.6% [43]. Development of the transition to a diet considered to be healthier in our sample is shown in Table 4.

4.3.2. Results of the factor analysis

According to our results, CSI’s attitudes towards health foods are determined by four factors in our sample. The factor structure of the CSI test is illustrated in Table 5.

The second factor is the Recreational, hedonistic value dimension, where the explained variance is 11.834%. This attitude is driven by the joy of of shopping, which has a decisive influence. Once again, high factor weights can be seen in the analysis, so this attitude is significantly separated from the other factors. This factor is skewed to the right (Skewness = 0.275), which means that in the whole sample, respondents do not really consider this attitude to be characteristic of themselves.

The third factor is the Uncertain, confused value dimension, in which the explained variance is 11.308%. It is characteristic of this attitude that the individual has difficulties making a decision about where to buy, as well as about what brand to choose, they feel that purchases should be planned more carefully. This factor is slightly skewed to the right (Skewness = 0.049), suggesting that the respondents who completed the questionnaire consider this attitude to be less characteristic of themselves.

The fourth factor is the Devoted, brand-loyal value dimension (explained variance: 10.872%). This attitude is characterized by brand loyalty and identifies quality with a higher price. The factor is significantly skewed to the left (Skewness = -0.882), so this type of behavior appears with a positive sign in the mindset of the respondents in the sample.

Method: Principal Component Analysis, Rotational method: Varimax with Kaiser Normalization. KMO=0,849

Prior to the cluster analysis of the CSI adapted for health foods, it was considered necessary to validate the list of claims. The Health- and self-conscious value dimension includes nine items, with a Cronbach’s alpha index of 0.922. There are four elements in the Recreational, hedonistic value dimension. Two of these items are considered inverted (”Shopping is not a pleasant activity for me”, ”I make my shopping trips fast”), after the recoding of which the scale has a Cronbach’s alpha index of 0.720. The Uncertain, confused value dimension contains five elements, with a Cronbach’s alpha index of 0.701. The fourth value dimension is Devoted, brand-loyal, which contains four statements. The Cronbach’s alpha index of the scale is 0.673. By deleting elements, there was no significant improvement in the Cronbach’s alpha index in any of the value dimensions. Based on our results, the list of statements is suitable for the characterization of the examined dimensions.

4.3.3. Segmentation results

The results of the factor analysis confirmed that the obtained factors are suitable for the cluster analysis, so in the next step the segmentation of the sample was performed. The analysis was performed using the K-means clustering procedure, and four groups were separated along the 22 factors. The value dimensions characteristic of the clusters are illustrated in Figure 4.

Following this, the socio-demographic background of each segment was characterized by cross-tabulation analysis and the deviations from the mean were examined by analysis of variance. Finally, the differences between the groups in the areas of physical activity, sedentary lifestyle and the transition to a diet considered to be healthier were examined.

4.3.3.1. Uninterested (cluster 1)

For those in the Uninterested group, the impact of foods on health is less important, they make no effort to buy good quality health food. Of the clusters, they show the least propensity to consume health foods, but cannot be considered dismissive. They do not identify the price of products with quality. They do not have favorite brands, and if they find a brand they like, they are not loyal to it. They want to finish shopping as soon as possible, since it is not a pleasant activity for them at all, they make quick decisions in the choice of both the store and the product. Typically, all statements are undervalued by Uninterested people compared to the other clusters.

The first cluster is the smallest group, making up 14.3% of the sample. In this cluster, men are inordinately overrepresented (72.1%), and the youngest age group, 18-29 is prominent (41.9%). This group has the highest proportion of people with high school diplomas (37.2%). Uninterested people has the highest proportion of respondents who are not health-conscious at all (16.3%) or mostly not health-conscious (34.9%). 60.5% of the group do not intend to switch to a diet they consider healthier in the next 6 months. They perform physical activity occasionally (32.6%) or infrequently (27.9%), spending on it 30 minutes or less (55.8%). Members of the group typically spend between 5 hours 31 minutes and 8 hours 30 minutes sitting daily (41.9%).

4.3.3.2. Health-oriented (cluster 2)

For Health-oriented people, it is extremely important to buy high quality health foods, and they are making a special effort to do so. They believe less that the price of a product determines its quality. For the sake of variety, they shop in several stores and always have health foods in their kitchen. Shopping is not one of the favorite activities in their lives, they like to get it done quickly. They have a few favorite brands and typically buy these. When they buy health foods, they usually do so in the same store. Of the clusters, they consider themselves the most health-conscious. In order to maintain their health, they choose foods very carefully and intend to make efforts in the near future to buy health foods.

The second cluster accounts for nearly one-third of the sample (32.6%). In this group, we find almost equal numbers of women (49%) and men (51%). Based on age, the majority belong to the 30-39 (25.3%) and 40-49 (18.4%) age groups, who typically have college degrees. Those in the group consider themselves mostly (54.1%) or very health conscious (27.6%). According to their own statements, almost one-third of the cluster (29.6%) have been eating healthily for at least six months, while one-fifth (20.4%) have always been eating healthily. This group contains the highest proportion of those who engage in physical activity regularly (76.5%). They typically spend 31-90 minutes on active movement. Members of the cluster mostly spend between 2 hours 31 minutes and 5 hours 30 minutes sitting daily (40.8%).

4.3.3.3. Variety seekers (cluster 3)

Variety seekers are less likely to identify product quality with high price. A higher than average proportion of them is reported to be looking for new types of health foods for purchase. Shopping is a decidedly pleasant and fun experience for them, members of the group believe that it is one of the really enjoyable activities of their lives that they spend a significant amount of time on. Compared to the sample average, they are more likely to have health foods at home. They are less loyal to brands, much more interested in novelty and variety.

The third cluster makes up 28% of the sample. Two-thirds of the group are women (64.3%). In this cluster, people in the 18-29 and 30-39 age groups make up two-thirds of the group (67.8%). A quarter of the group (26.2%) say they are partially health-conscious, while 20.2% feel a strong urge to switch to a diet they consider to be healthier. Two-thirds of the cluster performs physical activity on a regular basis, spending on average 31-60 minutes on it daily. Variety seekers typically (38.1%) spend between 5 hours 31 minutes and 8 hours 30 minutes sitting daily.

4.3.3.4. Uncertain brand choosers (cluster 4)

It is important for Uncertain brand choosers people to buy high quality health foods, they are the ones who clearly identify product quality with a high price. Compared to the sample average, they are more likely to look for new types of health foods to buy, but these are not accumulated in their homes. They consider shopping less enjoyable and usually do this activity quickly. They have a few favorite brands that they are loyal to. They believe they should plan their shopping more carefully. For them, it takes time to choose carefully for the best possible purchase, many times it is even difficult to choose the store where they want to shop. It presents a great difficulty for them when they have to choose from a number of brands, a large selection confuses them. Compared to the sample average, they consider themselves less health-conscious, but they would like to make an effort to buy health foods in the near future.

The fourth cluster makes up one-fourth of our sample (25%). This group is also characterized by the majority of women (54.7%). Age groups 50-59 (16%) and over 60 years (18.7%) dominate this group. This cluster contains a higher proportion of partially health-conscious respondents (28%) who would like to take steps in the near future to switch to a diet they consider healthier (22.7%). Uncertain brand-loyal people tend to engage in occasional physical activity (32%), spending less than 30 minutes on it. On average, they spend between 2 hours 31 minutes and 5 hours 30 minutes (32%) sitting daily.

The four clusters show distinctly different socio-demogpraphic characteristics and represent different value dimensions in relation to attitudes towards health foods. They are at different stages in the transition to a diet that is considered healthy. Significant differences can be detected in our sample regarding the regularity and duration of physical activity and the diet considered to be healthier. However, a sedentary lifestyle cannot be considered determinant of attitudes towards health foods.

5. Summary

In the online space, when balancing nutrition and exercise, consumers clearly place more emphasis on healthy eating, based on our netnographic survey. Search engine hit lists show a variable rate growth from year to year in the area of healthy eating and exercise. For the keyword healthy eating, a nearly tenfold increase was observed between 2017 and 2021. In social media, in the common subset of diet and exercise, four main types of content can be distinguished, the most popular of which are posts dealing with a combination of training plans and recipes. Our focus group studies have highlighted that different consumer preferences can be observed for health foods depending on whether we are talking about active or passive groups. According to the active group, the most important feature of a healthy lifestyle is the consumption of health foods, while the passive group believes that the most important thing to be informed about what is healthy and what is not. The active group characterizes health foods with being free of something, while the passive group characterizes them with fortification with something. Those who engage in physical activity regularly are more open to consuming health foods and are more affected by diet-related trends. Based on our quantitative research, attitudes towards health foods are determined by four value dimensions. In the Health- and self-conscious attitude, consumption of health foods is of paramount importance. The Recreational, hedonistic attitude is characterized by the joy of shopping. In the Uncertain, confused value dimension, uncertainty and indecisiveness stand out, which is reflected in both store and brand choices. The Devoted, brand-loyal attitude identifies quality with a high price, and both store and brand selection are carried out along definite ideas. Following factor analysis, sample segmentation was performed, resulting in the identification of four major groups. Among the clusters, Uninterested people represent the smallest proportion of our sample, and they undervalue all statements. The Uninterested group does not want to switch to a diet they consider healthier, for them it is not important to consume health foods. They perform physical activity infrequently and for a short period of time. The Health-oriented cluster is characterized by the exact opposite set of values. For them, it is important to buy health foods and they make an effort to do so. Shopping itself is not a pleasant activity for them. Members of the cluster perform physical activity regularly and for longer periods of time, and they have the highest proportion of actors and maintainers among the stages of the diet they consider healthy. For Variety seekers, quality is not related to a high price. They are looking for new types of health foods, however, they are more motivated by the joy of shopping. they are not loyal to a particular store or brand type. They engage in physical activity regularly and feel a strong urge to switch to a diet they consider healthier. In contrast, Uncertain brand choosers people stick to one brand type, but are confused by a large selection of brands. They consider themselves less health-conscious, but in the future they would like to strive to buy health foods and also to switch to a diet they consider healthier. Typically, they perform physical activity occasionally and for shorter periods of time.

According to the results of our research, the purchase and consumption of health foods and attitudes towards health foods are related to the diet and physical activity of the individuals, however, independent of the time consumers spend sitting daily.

6. Acknowledgment

This publication was supported by the project titled Debrecen Venture Catapult Program, No. EFOP-3.6.1-16-2016-00022. The prject was supported by the European Union and co-financed by the European Social Fund.

7. References

[1] Szakály, Z. (2016): Egészségmagatartás, viselkedésváltozás és személyre szabott táplálkozás: az élethosszig tartó egészség koncepciója, in Fehér, A., Kiss, V. Á., Soós, M., Szakály, Z. (szerk.): Hitelesség és Értékorientáció a Marketingben. Debreceni Egyetem Gazdaságtudományi Kar, Debrecen, pp. 5-25.

[2] NEFI (2017): Egészségjelentés 2016. Információk a népegészségügyi beavatkozások célterületeinek azonosításához a nem fertőző betegségek és az egészségmagatartási mutatók elemzése alapján. Nemzeti Egészségfejlesztési Intézet, Budapest.

[3] Griera, J.L., Manzanares, J.M., Barbany, M., Contreras, J., Amigó, P., Salas-Salvadó, J. (2007): Physical activity, energy balance and obesity. Public Health Nutrition. 10 (10A) pp. 1194-1199. DOI

[4] Chaput, J.P., Saunders, T.J., Mathieu, M.È., Henderson, M., Tremblay, M.S., O’Loughlin, J., Tremblay A. (2013): Combined associations between moderate to vigorous physical activity and sedentary behaviour with cardiometabolic risk factors in children. Appl Physiol Nutr Metab. 38 pp. (5) 477-483. DOI

[5] Csányi, T. (2010): A fiatalok fizikai aktivitásának és inaktív tevékenységeinek jellemzői. Új pedagógiai szemle. 60 (3-4) pp. 115–129.

[6] Ács, P., Prémusz, V., Morvay-Sey, K., Kovács, A., Makai, A., Elbert, G. (2018): A sporttal, testmozgással összefüggésben lévő mutatók változása Magyarországon és az Európai Unióban az elmúlt évek eredményeinek nyomán. Sport- és egészségtudományi füzetek. 2 (1) pp. 61-76.

[7] Biswas, A., Oh, P. I., Faulkner, G. E., Bajaj, R. R., Silver, M. A., Mitchell, M. S., Alter, D. A. (2015): Sedentary Time and Its Association With Risk for Disease Incidence, Mortality, and Hospitalization in Adults. Annals of Internal Medicine. 162 (2) pp. 123-132. DOI

[8] Marshall, A., Miller, Y., Burton, N., Brown, W. (2009): Measuring Total and Domain-Specific Sitting. Medicine & Science in Sports & Exercise. 42 (6) pp. 1094-1102. DOI

[9] EUROBAROMETER (2018): Sport and physical activity (Hozzáférés: 2021.03.21.)

[10] Ádány, R. (2011): Megelőző orvostan és népegészségtan, Medicina Könyvkiadó Zrt, Budapest

[11] Dobbs, R., Sawers, C., Thompson, F., Manyika, J.,  Woetzel, J., Child P., Mckenna, S., Spatharou, A. (2014): How The World Could Better Fight Obesity. McKinsey&Company (Hozzáférés: 2021.03.27.)

[12] KSH (2020): Tehetünk az egészségünkért; Társadalomstatisztikai összefoglaló kiadványok (Hozzáférés: 2021.02.22.)

[13] Szakály, Z. (2017): Táplálkozásmarketing. In: Szakály, Z. (szerk.): Élelmiszer-marketing. Akadémiai Kiadó, Budapest, 487–439. ISBN: 978-963-454-061-8

[14] Szakály Z. (2011): Táplálkozásmarketing. Mezőgazda Kiadó, Budapest

[15] Papp-Bata, Á., Csiki, Z., Szakály, Z. (2018): Az egészségvédő élelmiszerekkel kapcsolatos fogyasztói magatartás - A hiteles tájékoztatás szerepe. Orvosi Hetilap 159 (30) pp. 1221-1225. DOI

[16] Weststrate, J. A., Poppel, G. Van, Verschuren, P. M. (2002): Functional foods, trends and future. British Journal of Nutrition 88 (2) pp. 233–235 DOI

[17] GOOGLE (2016): 2016 Food Trends from Google Search Data: The Rise of Functional Foods (Hozzáférés: 2021.02.25.)

[18] Yeung, A.W.K., Mocan, A., Atanasov, A.G. (2018): Let food be thy medicine and medicine be thy food: a bibliometric analysis of the most cited papers focusing on nutraceuticals and functional foods. Food Chem. 269 pp. 455–465. DOI

[19] Urala, N., Lähteenmäki, L. (2003): Reasons behind consumers’ functional food choices, Nutrition & Food Science, 33 (4), pp. 148-158. DOI

[20] Lau, T.-C. (2019): Regulations, opportunities, and key trends of functional foods in Malaysia. Nutraceutical and Functional Food Regulations in the United States and Around the World, 561–573. DOI: https://doi.org/10.1016/B978-0-12-816467-9.00034-4

[21] Prochaska, J.O., Diclemente, C.C. (1982): Transtheoretical therapy: Toward a more integrative model of change. Psychotherapy: Theory, Research and Practice 19 (3) pp. 276-288. DOI

[22] Prochaska, J.O., Diclemente, C.C., Norcross, J.C. (1992): In search of how people change: Applications to addictive behaviors. American Psychologist 47 (9) pp. 1102-1114. DOI

[23] Prochaska J.O., Rochaska, J. M. (2011): Behavior change. In D. B. Nash, J. Reifsnyder, R. J. Fabius, V. P. Pracilio (Eds.), Population Health: Creating a culture of wellness pp. 23-41.

[24] Johnson, S.S., Paiva, A.L., Cummins, C.O., Johnson, J.L., Dyment, S.J., Wright, J.A. (2008): Transtheoretical model-based multiple behavior intervention for weight management: Effectiveness on a population basis. Preventive Medicine, 46 (3) pp. 238-246. DOI

[25] Czeglédi, E. (2012): A viselkedésváltozás transzteoretikus modelljének alkalmazási lehetőségei az elhízás kezelésében. Mentáhigiéné és Pszichoszomatika 13 (4) pp. 411-434. DOI

[26] Soós, M., Kovács, B., Szakály, Z. (2016): A viselkedésváltozás szintjein a testtömeg-menedzselés folyamatában – élelmiszerfogyasztás és fizikai aktivitás. Táplálkozásmarketing 3 (2) pp. 19-28. DOI

[27] Sproles, G. B., Kendall, E. L. (1986): A methodology for profiling consumers’ decision making styles. The Journal of Consumer Affairs 20 (2) pp. 267–279.

[28] Sam, K. M., Chatwin, C. (2015): Online consumer decision-making styles for enhanced understanding of Macau online consumer behavior. Asia Pacific Management Review 20 (2) pp. 100–107. DOI

[29] Nayeem, T., Casidy, R. (2015): Australian consumers’ decision-making styles for everyday products. Australian Marketing Journal 23 pp. 67-74. DOI

[30] Lysonski, S., - Durvasula, S. (2013): Consumer decision making styles in retailing: Evolution of mindsets and psychological impacts. Journal of Consumer Marketing 30 (1) pp. 75–87 DOI

[31] Eun Park, J., Yu, J., Xin Zhou, J. (2010): Consumer innovativeness and shopping styles. Journal of Consumer Marketing 27 (5) pp. 437–446. DOI

[32] Parakash, G., Pankaj, K. S., Rambalak, Y. (2018): Application of consumer style inventory (CSI) to predict young Indian consumer’s intention to purchase organic food products. Food Quality and Preference 68 pp. 90–97. DOI

[33] Internet World Stats (2021): World Internet Usage and Population Statistics 2021 Year-Q1 Estimates (Hozzáférés: 2021.03.12.)

[34] Datareportal (2021): Global Media Stats

[35] Farmer, A. N. D., Bruckner Holt Cem, Cook M.J., Hearing S. D. (2009): Social networking sites: a novel portal for communication. Postgraduate Medical Journale 85 (1007) pp. 455–459. DOI

[36] Benetoli A., Chen T. F., Aslani P. (2019): Consumer perceptions of using social media for health purposes: Benefits and drawbacks. Health Informatics Journal 25 (4) pp. 1661–1674 DOI

[37] Johns, D. J., Langley, T. E., & Lewis, S. (2017): Use of social media for the delivery of health promotion on smoking, nutrition, and physical activity: a systematic review. The Lancet 390. DOI

[38] Kozinets, R. V. (2002): The Field Behind the Screen: Using Netnography for Marketing Research in Online Communities. Journal of Marketing Research 39 pp. 61-72. DOI

[39] Dörnyei, K. (2008): Bioélelmiszer fogyasztási szokások. Marketing & Menedzsment 42 (4) pp. 34-42.

[40] Dörnyei, K., Mitev, A. (2010): Netnográfia, avagy on-line karosszék-etnográfia a marketingkutatásban. Vezetéstudomány 41 (4) pp. 55-68.

[41] Szabó, S. (2016): Egészségorientált táplálkozási szokások és a fogyasztói magatartás kapcsolata. Doktori (PhD) értekezés. Kaposvári Egyetem Gazdaságtudományi Kar

[42] Gál, T., Soós, M., Szakály, Z. (2017): Egészségtudatos táplálkozással kapcsolatos fogyasztói insight-ok feltárása netnográfiával – esettanulmány. Vezetéstudomány 48 (4) pp. 46-54. DOI

[43] Szakály, Z., Nábrádi, Zs. (2021): Az egészségtudatosság és a fogyasztók ismeretei. In: Kukovics, S. (szerk.): A hús szerepe a humán táplálkozásban (megjelenés alatt)

DOI: https://doi.org/10.52091/EVIK-2021/3-2-ENG

Received: March 2021 – Accepted: June 2021

¹ University of Debrecen, Faculty of Economics and Business, Institute of Marketing and Commerce

Keywords

food industry developments, food nanotechnology, consumer acceptance, willingness to buy, food industrial use of titanium dioxide

2. Introduction – nanotechnology

One of the most dynamically developing disciplines today is the research of nanoscale materials. Research and application of nanotechnology is one of the great scientific, developmental and technical challenges of the 21^st century.

Nanotechnology means the production, of materials, devices and systems that use artificially formed nanoparticles, i.e., particles of material that do not exceed 100 nanometers in size [1]. Nanostructured materials are also found in nature (e.g., clays, zeolites), but can be produced artificially as well.

Many nanotechnological applications are known in practice. Examples include highly resistant materials used in construction; lightweight, elastic clothing and sports equipment made of materials resistant to physical stress; self-cleaning paints that protect buildings from, for example, the harmful effects of smog and other contaminants; nanosensors that enable efficient and economical quality control in the food industry; highly miniaturized electronic devices; antibacterial coatings for industrial equipment and household appliances; selective release and high bioavailability drugs; innovative tools for the remediation of contaminated soils and waters. However, in addition to the benefits, nanotechnology poses risks to the environment and human health that are difficult to assess. Scientific research, while still proving to be scarce, suggests that nanoparticles are more reactive and mobile than larger particles and can therefore be toxic to humans and the environment. Little is known about the fate of nanoparticles in the environment. In the human body, nanoparticles may be able to cross the cell membrane and reach internal organs. Some studies have shown that many types of nanoparticles cause greater oxidative stress at the cellular level, increasing the risk of degenerative diseases [1].

2.1. Nanotechnology in the food industry

Due to their special properties, the use of nanostructured materials can also be promising in many food applications [2].

Foods containing nanoparticles should be considered as novel foods under Regulation (EC) No 258/97, as foods or food ingredients produced by such technology were not consumed in significant quantities in the European Union before May 15, 1997; thus, their placing on the market is preceded by an authorization procedure accompanied by a rigorous safety assessment [2]. As part of the authorization process, EU regulation has recently required food ingredients derived from the use of nanotechnologies to undergo a safety assessment before they can be placed on the market, and only then can they be authorized [3]. Related to this, the term nanofood has emerged to refer to foods that are produced, processed or packaged using a nanotechnology technique or device, or to which a nanomaterial is added and/or is enriched with a nanomaterial [4].

Nanotechnologies aimed at improving food quality or safety can theoretically be diverse, but their practical application is still in its infancy. Since food nanotechnology is also a new field for food science, nanotechnology is also a major challenge for the food economy, including food security and safety, traceability, certain areas of food processing and packaging, some new opportunities for nutrient intake, longer food shelf life and many other aspects of consumer protection, from agricultural production to the consumers’ tables [2].

The use of nanoparticles in food processing can contribute to the improvement of nutritional quality, taste, color and stability or to increasing shelf life and, in the case of liquid foods, to the improvement of flow properties. An additional benefit of nanotechnology may be that it can contribute to the development of foods with lower fat, sugar and salt content, thereby reducing the incidence of food-related diseases [5].

Currently, these products are available in four categories:

• nanostructured food ingredients and substances, such as nano-titanium dioxide, which is used as an anti-caking agent or pigment;
• nanostructured delivery systems that improve the bioavailability of bioactive compounds in fortified foods and supplements;
• novel packaging materials designed to strengthen the protective function of the product;
• and the use of food contact materials for food processing and storage, such as nano-silver, which is used for its antimicrobial properties [6, 7, 8, 9].

Nanotechnology is currently considered to be the most widespread among food industrial commercial applications in the packaging process [2, 10]. Several types of use of nanomaterials in packaging materials can be distinguished. In the case of nanocomposites, advantageous properties (mechanical or functional, e.g., gastightness, temperature / humidity stability) are achieved by adding nanoparticles to the plastic.

A similar effect can be achieved with nanocoatings applied to the surface of the packaging material. Aluminum coatings applied with the help of vacuum are now widespread mainly in the packaging of snacks, confectionery and coffee. For example, if the thickness of the aluminum layer applied as a coating does not exceed 50 nm, the coating metal can be considered a nanomaterial [11]. In addition to the above, there are several applications that are still in the research phase [12, 13, 14, 15], such as newly developed food packaging capable of detecting the presence of pathogens and contaminants.

Although this technology offers consumers unprecedented benefits such as higher added value, longer shelf life and increased food safety, nanofoods also pose health, environmental, economic, social and political risks [16, 17]. According to Berekaa, despite the huge benefits that nanoparticles can bring to the food industry, the public is very concerned about their toxicity and potential negative environmental impact. Due to the health consequences of the nanoparticles entering the human body, their potential risks to human health need to be assessed without delay [5]. In his paper, Halliday points out that EU regulations on food and food packaging require a specific risk assessment before nanomaterials are placed on the market [18].

In the course of our research, it was examined to which extent the concept of nanotechnology in the food industry has spread in the public consciousness, i.e., presumably how many people are aware of this technology and its potential application in the food industry. Following this, it was assessed how receptive consumers were about the technology, how they saw its future, and whether they would be willing to buy nanofoods. In our work, the potential dangers of nanotechnology were analyzed, and also the areas in which they may occur, as well as how attitudes, consumer acceptance and willingness to buy change in the light of this.

2.1.1. Foods and packaging materials produced using nanotechnology – some examples [1]

2.1.1.1. Creamier ice cream with unchanged fat content

When making ice cream that is creamier than traditional ones, titanium dioxide consisting of nano-sized grains is added to the raw material of ice cream to increase its creaminess and improve its taste, while keeping its fat content the same as that of traditional ice creams. In its nano form, titanium dioxide is thought to be cytotoxic, however, no data have been found in the scientific literature on the mechanism of absorption of nano TiO2 from the intestinal tract.

2.1.1.2. Table salt and sugar that do not form lumps with moisture

Nano-sized particles of titanium dioxide are added to table salt and sugar as anti-caking agent. For toxicological aspects see Section 2.1.1.1.

2.1.1.3. Fruit juices enriched with bioactive molecules

Bioactive molecules such as phytosterols, vitamins and antioxidants are added to fruit juices by the way of nanoencapsulation to improve them. Nanoencapsulation is not known to have adverse health effects.

2.1.1.4. Bread enriched with omega-3 fatty acids

Omega-3 fatty acids are added to bread by nanoencapsulation; this way the unpleasant taste of the fatty acids is not felt, and thus the fortified bread retains its traditional taste. Nanoencapsulation is not known to have adverse health effects.

2.1.1.5. Plastic bottles for beer

Beer bottles with a modified composition are produced by adding a nanocomposite material containing clay particles. The purpose of clay-polymer nanocomposites is to minimize carbon dioxide loss and oxygen uptake to extend the shelf life of carbonated beverages. The toxicological effects of the nanolayer are unknown; it has not yet been demonstrated that nanoparticles can be released from the packaging material.

2.1.1.6. Antimicrobial food packaging for meat and other foods

Food packaging materials containing active nano-silver inhibit the growth of microbes and help to prevent possible bacterial contamination. Nanoparticle-sized silver is presumably cytotoxic. It has not yet been demonstrated that nanoparticles can be released from the packaging material.

3. Materials and methods

To answer the research questions, online questionnaire interviews involving 200 people were conducted. During the sampling, the snowball method was used, i.e., the selection of the sample was not random, but in this way we were able to reach a wide range of respondents. Under these conditions, the survey cannot be considered representative, the results obtained can only be applied to the actual respondents. Background variables of the questionnaire included gender, age, place of residence, education and average income.

In the course of the questionnaire survey, consumer attitudes towards nanotechnology in the food industry were assessed using 17 closed-ended questions. Then, in order to be able to analyze them in depth, two focus group studies were conducted. Consumers’ attitudes towards the topic were determined in advance by screening questions, based on which they were classified into one of the two focus groups. The first group included consumers who rejected nanotechnology based on the screening questions, while participants in the second group viewed this technology favorably. During the formation of the two groups, we sought to ensure that the consumers interviewed were included in the research in an equal distribution with regard to gender. In terms of age, people between the ages of 20 and 65 participated in the interviews.

Due to the pandemic situation at the time of the research, the two groups of eight people each were interviewed via an online platform.

At the beginning of the focus group interviews, participants were asked to briefly introduce themselves, and then two passages, taken from Sodano et al.’s communication and translated into Hungarian [1], were read aloud in the first half of the discussions. The first text introduced nanotechnology in general, while the second part described six products that had been made by some kind of nanotechnological process, but only the advantageous properties of the products have been emphasized in the description. The first half of the interview questions concerned the awareness and acceptance of nanotechnology in the food industry, but group members also had to answer questions related to the texts they had heard.

In the second half of the focus group discussion, the part of the text that highlights the potential risks and negative impacts associated with the technology and, thus, the products was read aloud. Following this, once again participants were asked questions, this time focusing on the risks, and it was examined how much their attitude towards the topic had changed.

4. Results and evaluation

In this chapter, the most important results of the primary research are presented, in the order they took place.

4.1. Results of the questionnaire survey

The first question of the questionnaire focused on factors considered important when purchasing food. This was important because, after this, the backbone of the research was the examination of the acceptance of nanotechnology in the food industry, taking into account the categories mentioned here. As can be seen from Figure 1, of the factors listed, taste was mentioned first, i.e., for 76.0% of the respondents taste was the most important consideration when purchasing or selecting a food. Based on the comparison with the background variables, it was revealed that men in the sample had a significantly (p=0.014) higher proportion (80.0%) who considered taste important than women (61.2%), and also that consumers who, according to their own statements, live in better-than-average financial conditions (live well on their income and can also save some money) also consider taste to be an important criterion when choosing (88.9%).

Slightly behind, high quality (68.5%) and price (63.5%) was second and third in terms of purchasing considerations. As had been expected previously, for these categories, 86.2% of those with a sound financial background rated high quality as an important aspect, while in terms of price, this proportion fell to 47.3%.

High food safety was considered important even less than one half of the respondents (47.0%), which may be due to the fact that they were not aware of the specific meaning of the term.

Respondents considered added value (for example, higher omega-3 fatty acid content) to be the least important aspect, with this factor ranking last of the listed ones with 17.5%. Only 20.0% of men and 16.4% of women consider this category when purchasing food. In terms of financial status, this criterion was least important for consumers with below-average income (7.0%).

In the following, the proportion of respondents with knowledge on nanotechnology in the food industry (spontaneous recall) was examined. The innovative and novel nature of the technology is also supported by the fact that only a quarter of respondents have heard of it.

When the four categories of nanotechnology currently available in the food industry were also listed [6, 7, 8, 9] (supported knowledge), only 62.0% of consumers still answered that they had not yet heard of the new technology in question (Figure 2). Of the entire sample, there was only one person who had heard of all the categories listed. Of the four categories, packaging materials made using nanotechnology were the best known (28.5%). 11.5% each of the participants in the survey have already heard of nanostructured food ingredients and materials, as well as the use of food contact nanomaterials, respectively. Respondents were least familiar with nanostructured delivery systems, the proportion in this case was not even 5.0%. Consumers who have heard of this category had some kind of college degree.

In the following, the acceptance of nanotechnology in the food industry was examined using the aspects listed in the first question that were considered important at the time of purchase. The results are shown in Table 1.

Based on the results obtained (for the sample), it can be said in general that the majority is open to the new technology if it has a beneficial effect on one of the properties of the food purchased. 71.9% of respondents would buy food made with nanotechnology if its organoleptic properties were better. Of the aspects considered important when buying food, taste finished first: 76% of respondents chose this factor. It should be noted that a greater willingness to buy due to more favorable sensory properties was an expected outcome. The older age group gave the highest proportion of affirmative answers to this characteristic (89.5%, p=0.047), and there was no significant relationship to the other background variables. In order to have a positive effect on the texture of foods, 68.8% of the consumers in the sample would buy a product made with a nanotechnological process. In the hope of better texture, 85.2% of respondents aged 56-65 would be open to buying products made with the new technology. A significant increase in the shelf life and use-by date of foods due to the nanotechnology process had an incentive effect on shopping for 62.5% of respondents. In the case of this question, a significantly higher proportion of women answered yes than men (women: 70.0%, men: 46.3%). 78.6% of the respondents to the questionnaire would buy food made with some kind of nanotechnology process if it increased food safety. 90% of the older age group and 78.6% of women were represented in the „yes” answers in this regard. 63.0% of respondents answered „yes” to the question of whether they would buy a food produced with nanotechnology development if it has added value such as a higher omega-3 fatty acid content. This represents an exceptionally high proportion considering that added value as a purchase criterion finished last in terms of importance with 17.5%. Thus, although it is typically not an important factor for the consumers in the sample that the food has some added value, they would still choose a product manufactured with nanotechnology that is richer in omega-3 fatty acids. Finally, 78.1% of respondents were open to food packaging produced with a new method that guarantees safer storage. In this case as well, women and those aged 55-65 had the highest proportion of „yes” answers.

Figure 3 illustrates how many percents more respondents would be willing to pay for a food that has been produced or modified using some kind of nanotechnology process. Typically, the additional cost consumers in the sample considered most acceptable was between 0% (i.e., they would not pay more at all for a product manufactured with this technology) and 5-10% (30.7% and 30.7% of respondents, respectively). 22.4% would pay 0-5% more and 15.6% would pay 10-20% more for this type of food. The proportion of respondents willing to assume an additional cost of more than 20% did not even reach one percent. Consumers who would be willing to pay 0-5% more for a product manufactured with nanotechnology are those who have a lower-than-average monthly net income, while respondents who say they live in better-than-average financial conditions would be willing to pay 5-10%, 10-15%, 15-20%, or even more than 20% more for such foods.

In our research, it was also addressed how respondents felt about the possible adverse consequences of nanotechnology in the food industry. Based on the results obtained, it was found that more than half of the questionnaire respondents (53.6%) believed that foods made with the nanotechnology process carry unknown hazards. In this case, in terms of proportions, men can be said to be the most skeptical, with 74.3% saying that nanotechnology in the food industry could pose a risk.

Figure 4 shows the probability of the occurrence of the different hazards in the opinion of the respondents in percentage distribution. 71.4% of respondents who consider the technology to be risky believed that foods made with the nanotechnology process pose mainly health risks. This was followed by environmental risks (56.3%). In this case, almost twice as many women believed that nanotechnology in the food industry could cause environmental damage (p=0.020). Consumers in the sample considered negative economic and social impacts to be the least probable. For these two categories, typically women were also in the significant majority (p=0.001). However, it can be said for all categories that respondents with higher education represented a higher proportion.

4.2. Results of the focus group studies

Since the main objective of our research was to examine nanotechnology in the food industry from a consumer perspective and to explore the expected rate of acceptance and possible rejection of the technology, after examining the quantitative results of the online questionnaire, it was considered appropriate to analyze the responses received in more depth using a qualitative method, therefore, focus group interviews were conducted to facilitate interpretation.

4.2.1. Results of the focus group study of people accepting nanotechnology in the food industry

Our discussion began with an association game designed to resolve any anxieties of the interviewees. Members of the group were asked to say positive and/or negative words and phrases that come to mind in connection with the topic. The following words were mentioned: innovation, invention, new opportunities, interesting, sci-fi, foods of the future, possible solutions to many problems.

The next question was whether they had already encountered any of the listed categories of nanotechnology applications or something similar (creamier ice cream with the same fat content; salt and sugar that do not form lumps with moisture; fruit juices enriched with bioactive molecules; bread enriched with omega-3 fatty acids; plastic bottles for beer; antimicrobial food packaging for meat and other foods). All of the respondents had already met soft drinks and beers packed in special PET bottles. Fruit juices enriched with various vitamins, minerals and antioxidants were mentioned by several people, and one person saw bread enriched with omega-3 fatty acids in a store while shopping (he didn’t remember exactly which store it was). In addition to the categories read aloud, they have seen eggs that contained excess omega-3 fatty acids, known various dietary supplements to which vitamins, minerals or antioxidants were added, and a participant had read on the internet about an intelligent packaging material that recognizes contaminants. He did not remember whether the packaging material had been made with nanotechnology in the food industry, but he believed that this category fit exactly into this topic.

Following this, those present were asked to express their views and evaluate how they perceived the six categories described above. Positive thoughts were associated with the products by everyone. They were thought to be useful in many ways, and it was thought to be a good idea to add such extra values to foods that allow people to get vitamins and other minerals without having to take separate capsules into their body. According to the participants, the facts that the use of nanotechnology can make food storage safer and increase shelf life can also be advantages. When asked if they would like to buy this type of food, all participants answered in the affirmative. One person stated that he was somewhat averse to nanotechnology-modified ice cream, while two people said the same thing about bread enriched with omega-3 fatty acids, but they could not specifically explain why.

This was followed by solving a task together, in which members of the group were asked to jointly establish an order for the six products based on which they considered to be the most sympathetic and which the least. The popularity of the products is illustrated by the data in Table 2.

The group unanimously agreed with the assumption that in the future we would encounter many of these or similar products on store shelves. It was thought that foods produced with nanotechnology were likely to become more widespread if the pace of food industry developments remained the same. One of our interviewees said that due to the overpopulation of the Earth and the constant decline of arable land, it will be necessary to deploy such tools in order to avoid an increasing rate of hunger and malnutrition, and to prevent people from suffering from the lack of certain nutrients. Everyone has accepted the vision that foods produced with such technology and other similar developments will become more popular and accessible, provided, of course, that they will be available at affordable prices. Intelligent food packaging that recognizes bacteria and contaminants has been found to be especially useful and practical.

According to them, basic foods (dairy products, pasta, flours, cereal flakes) could also be enriched with added values (vitamins, minerals, antioxidants).

In the second half of the focus group interview, the part of the texts was read aloud that described the potential risks involved in using the technology. Following this, it was assessed whether participants’ opinions, attitudes and willingness to buy changed as a result of what they had heard. The majority believed that if it were not safe to consume a product, it would ultimately not be able to be marketed. According to another opinion, while it sounded a little scary, and so he would think twice about buying this type of product, he still would not reject the technology.

Finally, participants were asked to reconsider, in light of the information they had learned, the order established above, as to which category they would be most likely to purchase. For better comparability, the order before describing potential hazards and the new order are listed in the same table. The results are shown in Table 3.

Although the final order was changed at several points, the opinions and willingness to buy of the group members did not change significantly after the exploration of possible dangers.

4.2.2. Results of the focus group study of people rejecting nanotechnology in the food industry

The study scenario in this case was the same as it was for the previous group. Presentation of the first part of the text was followed by an association game, the essence of which was that participants had to say adjectives and expressions, whether positive or negative, that came tom mind about nanotechnology. This time, compared to the interviews with the accepting group, the opinions (answers) were much more mixed: innovative, dangerous, bizarre, this is the future, foods made in a laboratory, unnatural. One of our interviewees also noted that these products were likely to be very expensive.

Of the six products made with nanotechnology in the food industry, half of the group had already encountered fruit juices enriched with bioactive molecules, and everyone was familiar with the special PET bottles. As similar products, sports drinks and dietary supplements fortified with vitamins and minerals were mentioned, which had already been encountered by them in retail trade, and one person had already read online about packaging materials made with nanotechnology, and another participant cited a scientific paper on artificial meat as an example.

Following this, once again, members of the group were asked to share their views on the six products which had been introduced at the beginning of the interview. Someone thought it was extremely scary to hear about these, while others thought that they would be very unhealthy for sure. Many people felt that it was unnecessary to enrich fruit juices with such substances when they were already full of vitamins anyway. The idea of bread enriched with omega-3 fatty acids was specifically thought to be „crazy”. One participant did not consider packaging to be a bad idea, and two of them also commented favorably on PET bottles.

When asked whether they would buy these products, the answer was clearly no. The group was less prone to rejection in the case of the PET bottles, with 4 people inclined to buy, and one person said the same about antimicrobial packaging.

Subsequently, the group rejecting nanotechnology also had to jointly establish an order for the six products, based on acceptability (in this case, we cannot speak of popularity, as the members of the group reject nanotechnology in the food industry). The results are shown in Table 4.

Regarding the vision for the future, participants believed that the trend of developments suggests that more and more products of this kind will be available commercially. There was also a remark in this regard that „the world is not moving in the right direction”. One person added that he was confident that we would stick to natural food sources. Several people agreed with the statement that if it is not the food industry that works with such technology, but the construction or textile industry, it may even be useful. When asked whether they would like more of these products to be available in the future, the group’s response was a clear and consistent no.

The final chapter of the focus group interview concentrated on the potential risks of nanotechnology. After discussing the potential dangers of nanotechnology with participants, their opinions were asked. Their position did not change much after what they had heard, since, as they said, they had not considered it to be a good idea, and it only strengthened their belief that such a technology could have negative consequences. The unanimous opinion of the group was that they would continue to not buy such products as they are sure that they are harmful not only to human health but also to the environment.

As a final task, participants were asked to, in possession of all the information, jointly establish a new, final order as to which category they would consider most acceptable and which least acceptable. Compared to the previous one, the order did not change much, and the result was as follows. The orders before and after the description of the risks (new order) are shown in Table 5.

5. Conclusions

Despite the fact that 74.5% of the respondents were not previously familiar with nanotechnology and its application possibilities, and almost half of the respondents believed that it involved some risk, the survey of knowledge of nanotechnology and the examination of consumers’ willingness to buy revealed that the degree of acceptance of the technology and the willingness to buy can be said to be very favorable. If, through this technology, food quality is expected to change in a positive direction, acceptance exceeds 60%.

The most important aspect when buying foods was taste, while added value finished last with 17.5%. Nevertheless, 63.0% of those who completed the questionnaire replied that they would buy a product made with a nanotechnology process if the product thus contained some kind of added value.

The focus group interview revealed that the group of acceptors, as expected, was extremely positive about the technology, and even after the description of the potential risks, neither their opinion, nor their willingness to buy typically changed.

Reaffirming Berekaa’s claim that the public is very concerned about toxicity and potential negative environmental effects [5], in the case of the group of rejectors, participants unanimously stated that the technology is extremely risky and dangerous to both the environment and humans. However, they also added that in their view and based on the trends, the proliferation of commercially available such products will be inevitable in the future. In their case it can be said that although they do not prefer the possibilities of using nanotechnology, their rejection was less pronounced for those categories of application of the technology that do not specifically change the properties of foods, but their peripherals (such as packaging).

In Chapter 3 of our paper, the statements of Sodano were already quoted, according to which the willingness to buy nanofoods for the six categories examined (creamier ice cream with the same fat content; salt and sugar that do not form lumps with moisture; fruit juices enriched with bioactive molecules; bread enriched with omega-3 fatty acids; plastic bottles for beer; antimicrobial food packaging for meat and other foods) depends to a large extent on the assessment of the perceived risks and benefits [1]. Our results obtained in the course of our research support this, as the willingness to buy of consumers who already had a positive attitude towards the technology is also very favorable, while rejectors showed the opposite consumer behavior.

6. Acknowledgment

This publication was prepared with the professional support of the New National Excellence Program of the Ministry of Innovation and Technology, code number ÚNKP-20-3-I-DE-404, financed from the National Research, Development and Innovation Fund.

7. References

[1] Sodano, V., Gorgitano, M.T., Verneau, F. (2015): Consumer acceptance of food nanotechnology in Italy. British Food Journal 118 (3) pp. 714-733

[2] Zentai A., Frecskáné Csáki K., Szeitzné Szabó M., Farkas J., Beczner J. (2014): Nanoanyagok felhasználása az élelmiszeriparban. Magyar Tudomány 175 (8) pp. 983-993

[3] Cubadda, F., Aureli, F., D Amato, M., Raggi, A., Mantovani, A. (2013): Nanomaterials in the food sector: new approaches for safety assessment. Rapporti ISTISAN 13/48.

[4] Joseph, T. and Morrison, M (2006): Nanoforum report: nanotechnology in agriculture and food. (Hozzáférés: 2014. 06. 12.).

[5] Berekaa, M. M. (2015): Nanotechnology in food industry; Advances in Food processing, Packaging and Food Safety. International Journal of Current Microbiology and Applied Sciences 4 (5) pp. 345-357

[6] Chaudhry, Q., Scotter, M., Blackburn, J., Ross, B., Boxall, A., Castle, L. y and Watkins, R. (2008): Applications and implications of nanotechnologies for the food sector. Food Additives and Contaminants 25 (3) pp. 241-258

[7] Cushen, M., Kerry, J., Morris, M., Cruz-Romero, M. and Cummins, E. (2012): Nanotechnologies in the food industry. Trends in Food Science & Technology 24 (1) pp. 30-46

[8] Weir, A., Westerhoff, P., Fabricius, L., Hristovski, K. and von Goetz, N. (2012): Titanium dioxide nanoparticles in food and personal care products. Environmental Science & Technology 46 (4) pp. 2242-2250 DOI

[9] Mura, S., Seddaiu, G., Bacchini, F., Roggero, P.P. and Greppi, G.F. (2013): Advances of nanotechnology in agro-environmental studies. Italian Journal of Agronomy 8 (18) pp. 127-140

[10] Chaudhry, Q., Castle, L., Watkins, R. (2010): Nanotechnologies in Food. Royal Society of Chemistry Publishers, Cambridge, UK.

[11] Bradley, E. L., Castle, L., Chaudhry, Q. (2011): Applications of Nanomaterials in Food Packaging with a Consideration of Opportunities for Developing Countries. Trends in Food Science & Technology 22 pp. 604-610

[12] Sozer, N. and Kokini, J.L. (2009): Nanotechnology and its applications in the food sector. Trends in Biotechnology, 27 (2) pp. 82-89.

[13] Neethirajan, S. and Jayas, D.S. (2011): Nanotechnology for the food and bioprocessing industries. Food and Bioprocess Technology 4 (1) pp. 39-47

[14] Cushen, M., Kerry, J., Morris, M., Cruz-Romero, M., Cummins, E. (2012): Nanotechnologies in the food industry. Trends in Food Science & Technology 24 (1) pp. 30-46

[15] Qureshi, M.A., Karthikeyan, S., Karthikeyan, P., Khan, P.A., Uprit, S. and Mishra, U.K. (2012): Application of nanotechnology in food and dairy processing: an overview. Pakistan Journal of Food Sciences 22 (1) pp. 23-31

[16] Cockburn, A., Bradford, R., Buck, N., Constable, A., Edwards, G., Haber, B., Hepburn, P., Howlett, J., Kampers, F., Klein, C., Radomski, M., Stamm, H., Wijnhoven, S. and Wildermann, T. (2012): Approaches to the safety assessment of engineered nanomaterials (ENM) in food. Food and Chemical Toxicology 50 (6) pp. 2224-2242

[17] Hubbs, A.F., Sargent, L.M., Porter, D.W., Sager, T.M., Chen, B.T., Frazer, D.G. and Battelli, L.A. (2013): Nanotechnology toxicologic pathology. Toxicologic Pathology 41 (2) pp. 395-409

[18] Halliday, J. (2007): EU Parliament votes for tougher additives regulation. FoodNavigator.com (Hozzáférés: 2014. 06. 12.).

DOI: https://doi.org/10.52091/EVIK-2021/3-3-ENG

Received: February 2021 – Accepted: June 2021

¹ University of Szeged, Faculty of Engineering, Institute of Engineering Management and Economy

Keywords

packaging material, polymers, plastics, bioplastics, degradable plastics, plastic types, consumer behavior, consumer demographics, eco-awareness

2. Introduction

Packaging technology research is one of today’s rapidly evolving disciplines, with innovative implications for many other disciplines, such as the food industry. The advent of plastic packaging materials has opened up new perspectives in improving the shelf life of foods. The history of plastics goes back only 155 years, while the use of bioplastics has a history of only a few decades. Nevertheless, application of the latter has been increasing in recent times at a rate which is significantly higher rate than that of conventional plastics.

In recent years, there has been a significant increase in interest in natural polymers on the part of both industry and academia, which is presumably related to the difficulties in the field of waste management and the relevant regulation. A further incentive for the development of bioplastics may be the declining amount of fossil raw materials available for the industry.

2.1. Aim of the study

In my study, I sought to answer the following questions:

• What do consumers think about packaging in general?
• How do they see the need to package food products?
• Do they know environmentally friendly packaging materials?
• Are the properties of the packaging material taken into account when purchasing?
• What characteristics are considered important when choosing packaging material?

3. Literature review

3.1. Position, definition and properties of plastics

Plastics are macromolecules composed of monomers that are made artificially, entirely or in part [1]. Polymers (from the Greek, meaning many parts) are mainly composed of eight chemical elements: C, H, O, N, Cl, F, S, Si. These atoms are linked to each other by covalent bonds to form molecules. The small molecules used in polymer production are traditionally produced from petroleum. Today, significant research is being conducted to be able to produce these from renewable raw materials [2].

Plastics can also be referred to as the materials of the 21^st century, without which we could hardly imagine our lives today. On the one hand, artificial polymers can be produced economically and, on the other hand, they allow technical solutions that would not otherwise be possible [3]. The impact of plastics and plastic packaging materials on our environment is the subject of an extensive debate among both professional and lay communities.

Campaigns in recent years have been directed primarily against the use of plastics, although in practice, the use of only a relatively small proportion of them, plastics used for packaging may be responsible for damage to the environment. The plastic waste pollution of the environment is mainly due to the fact that plastic packaging materials can be produced relatively cheaply, they are not of great value after use, so unfortunately they end up as not recycled waste, even if this is not justified [4].

3.2. Plastic packaging and food packaging

Foods are biologically sensitive substances. Their original freshness and shelf life depend on the intrinsic properties of the product and on external conditions. Intrinsic properties are the microbiological state of the food, its composition, water activity and pH. External conditions include processing hygiene, the optimum gas or gas mixture, the packaging machine, the packaging material, and the temperature during processing and storage [5].

The most significant plastic packaging material type is polyethylene. The different polyethylene types are members of the simplest synthetic polymer family produced in the largest amount, polyolefins. The most common types of plastics are polyethylene (01 - PET), high density polyethylene (02 - HDPE), polyvinyl chloride (03 - PVC), low density polyethylene (04 - LDPE), polypropylene (05 – PP) and polystyrene (06 - PS). In parentheses are the conventional codes and abbreviations of the different plastics. The code for other plastics no listed here is 07 [6].

3.3. Bioplastics

Bioplastics are made from raw materials from renewable sources, while degradable plastics are mixtures of plastics made from conventional raw materials and additives that aid degradation. The best known bioplastics discovered in the 20^th century are starch-based ones, polylactic acid, poly(hydroxyalkanoate) and polybutylene succinate adipate, and their use has been increasing significantly in recent years.

Life cycle analyses have shown that, compared to conventional plastics, the use of bioplastics can reduce greenhouse gas emissions by 30 to 50% annually [7].

3.4. Consumer behavior, trends

By the concept of consumer behavior we mean the processes and activities that are aimed at obtaining, using and evaluating a given product. In its examination, a significant distinction should be made according to the product group to which the goods to be acquired belong, the so-called nondurable or durable consumer goods [8].

Factors influencing consumer behavior can be grouped as follows [9]:

Cultural factors

• Culture
• Subculture
• Social classes

Social factors

• Reference group
• Family
• Social statuses

Personal factors

• Age, family, life cycle
• Economic conditions
• Occupation
• Lifestyle
• Personality

Psychological factors

• Motivation
• Perception
• Learning
• Beliefs, attitudes

According to the introductory text of the website of Dr. Törőcsik Kft., „The trend is the intensification and spread in society of certain phenomena and processes taking place in the market, which has a significant impact on the behavior and habits of consumers in the foreseeable future” [10].

Among the trends of 2019, environmental awareness has emerged, manifesting itself in Plastic Free July and Straw Free August.

Plastic Free July started in Australia back in 2011 and has since spread around the world. In Hungary, it was first announced in 2018, but became well-known only in 2019 [11].

Hungarian environmental organizations have also embarked on an active campaign, as an image of a turtle drowning because of a plastic straw posted on the internet has made the public realize that animals see plastic waste thrown away by many people as food [12].

4. Materials and methods

In order to achieve the research goal, both secondary and primary information collection were carried out.

During the secondary research, to form the basis for my primary research work, the available Hungarian and international surveys conducted earlier and related to the topic were reviewed.

In the primary data collection, of the marketing research methods, the quantitative procedure was chosen, more specifically the questionnaire survey. In this type of research, due to the large sample size, it is essential to use mathematical-statistical methods, and the results of the research are reported in a quantified way, taking into account the requirement system for statistical reliability tests [13].

The questionnaire was prepared in July 2020, and it was completed through an electronic platform. Online completion was chosen because, over the last en years, online quantitative research has become one of the most important data collection channels for market research. Both researchers and their clients are now convinced that online research not only offers more in terms of speed and cost-effectiveness than personal or telephone data collection, but the reliability and authenticity of the data are also unquestionable [14].

The questionnaire can be divided into two main parts:

1. Packaging knowledge, opinions, habits related to food products;
2. Demographic questions (gender, age, education, economic status).

The questionnaire was completed by the respondents between July 20 and 31, 2020.

For the completion, the following two methods were used:

1. Quota sampling, in which the population was divided into subgroups (based on age groups), and the elements were selected from these; followed by the
2. Snowball method, which means that the individuals selected previously were asked to forward the link to the questionnaire to people they know who belong to a similar age group.

When designing the research, the goal was to reach 500 people. This goal was slightly exceeded, so the final size of the sample was 513 people.

When processing the data, the program TIBCO Statistica™ Trial Download for Windows Version 13.5.0.17 was used. In most cases, the results obtained were rounded to 2 decimal places, according to the rules of rounding. Where this method was not used (e.g., standard deviation), it is indicated in the paper.

During the evaluation, frequency was examined, cross-tabulation analyses were performed, and descriptive statistical analysis was carried out. Figures were prepared using the 2021 version of Microsoft Excel.

5. Results and evaluation

Basic demographic characteristics of the persons completing the questionnaires are summarized in Table 1.

In addition, education and economic status were also examined. 67% had college degrees and 29% had high school diplomas. Approximately 60% of the respondents considered themselves and their family to be in an average economic situation, while roughly 30% classified themselves as having a situation more favorable than average.

Question 1 of the questionnaire was in fact a task. Respondents were asked to describe what comes to mind when they hear the word packaging. About 16% of questionnaire respondents mentioned the word plastic, while only 14% first thought of the term protection. In addition to these two major categories, marketing, paper, waste and garbage were also mentioned.

Question 2 concerned the opinion on the viability of food packaging. Statements were listed and respondents had to decide how much they agreed with them. During the evaluation, the arithmetic mean was calculated, and the statements are arranged in Table 2 in the order of their decreasing value. Means were rounded to 2 decimal places, while the standard deviation was left with the decimal places calculated by the Statistica program.

The statements, based on the arithmetic mean showing the agreement, remained in the order they were in the questionnaire. Respondents associated the role of packaging with protection. This is in agreement with the outcome of the association task. These values are well indicated by the median, while the modus decreased from 5 to 1 at the last statement. The degree of the standard deviation changes inversely with the value of the arithmetic mean of the agreement: the average degree of agreement decreases, while the degree of deviation from the mean increases.

In Question 3, the answer was sought whether the research participant had already encountered foods with biodegradable packaging. The answers of the questionnaire respondents are illustrated in Figure 1.

Based on this, it was found that more than half of the respondents had already encountered this type of packaging, approximately one-fifth had not, while roughly 1/3 of them admitted that they did not know whether or not they had encountered degradable packaging.

Examining the responses in view of demographic variables, the following results were obtained (Table 3).

Remark: Within each category, high values are highlighted in red and bold. The sample number in the age group over 65 is low, so their answers are indicated, but the data were not taken into account in the calculations.

Based on the statistical analysis, it was found that among the subjects interviewed by me, biodegradable packaging had been encountered primarily by individuals meeting the following criteria:

• Men;
• Aged 18 to 45;
• With a college degree

Míg azok, akik nem találkoztak ilyen csomagolással jellemzően:

• Women;
• Aged 46 to 65;
• With a college degree

Although higher education is included in both categories, this is not a contradiction, because the other two groups in terms of education are present in high proportions at the statement Don’t know.

However, answers to Question 4 paint a somewhat more nuanced picture. I tried to eliminate „non-truth tellers”. The question was whether the person in question had a habit of inspecting the food packaging at the time of purchase. Overall, ¾ of the respondents do not examine the food product for the type of packaging, and only ¼ do so occasionally or in all cases.

According to my calculations, only 28.8% of those who answered yes to Question 4 said that they usually inspect the type of packaging in the case of foods, and only 5.84% claimed that they always do so. In contrast, 64.96% usually do not or never do so.

It has been proven that the Hungarian population is very eco-conscious and environmentally friendly in theory, but they are not necessarily so in reality.

In the case of Question 5, respondents were further asked what packaging materials they chose most often when buying food. The frequency distribution is shown in Figure 2.

My previous statement can be supported by the figure, according to which the majority of customers (about 75%) do not check the packaging of the product. This 75% is the sum of those answering Don’t know and What is available. This ratio is the same as the value calculated above. The term Nothing means the following: Nothing, I take packaging with me.

Roughly 10% of the subjects interviewed said they chose products with degradable packaging. Their main demographic characteristics are summarized in Table 4.

Based on the results, those people who actually buy food in degradable packaging belong to the following main demographic groups:

• Women;
• Aged 46 to 65;
• With a college degree;
• With average income

With uestion 6, the answer was sought what kind of packaging consumers preferred for different types of food. Various product groups were listed, including the meat products to be examined later. Three possibilities were offered to choose from:

• Pre-packaged product;
• Unpackaged goods or goods from the counter;
• I do not usually buy such product

While almost all respondents (93.37%) choose unpackaged goods in the case of fruits and vegetables, the proportion is only about 78.00% in the case of bakery products. The cause for this may be that the increasingly popular specialty bakery products (diet, high-fiber, seeded, etc.) are often sold in a pre-packaged form. In the cases of cheeses and dairy products, the proportion of those choosing pre-packaged products is exceptionally high (75.83%). In the case of meat products, the groups of those choosing pre-packaged and nonpackaged goods are more evenly distributed. The proportions are 47.00% and 46.00% in the case of sliced goods, while they are 41.00% and 49.00% in the case of dry goods sold in the forms of bars, respectively. It is worth noting that, compared to the other product groups, the proportions of those answering I do not usually buy such products are the highest in these two cases (roughly 7% and 10%).

Question 7 again was a scale question. Respondents were asked to indicate the importance of packaging material characteristics listed in the questionnaire on a scale of 1 to 5 already used. The following characteristics had to be assessed:

• Quality;
• Thickness;
• Transparency;
• Environmentally friendly nature;
• Recyclability;
• Degradable nature

From the analysis (Table 5) it can be concluded that the most important parameter according to the respondents is quality, followed by environmental friendliness and recyclability. Each of these received an average value above 4.00. Respondents therefore consider environmental protection to be important.

6. Conclusions

Based on my research, the following were found:

• Most of the respondents associated the word packaging with plastics, and this was followed by the term “protection”.
• Participants in the research agreed with the following statement to the greatest extent regarding the purpose of packaging: We protect the product from external damage and contamination.
• ¾ of the respondents do not inspect food products with respect to the type of packaging, and only ¼ do so occasionally or always.
• It has been proven that the Hungarian population is very eco-conscious and environmentally friendly in theory, but not necessarily in practice.

• People who actually buy foods in degradable packaging can be characterized by the following major demographic data:

  • Women;
  • Aged 46 to 65;
  • With a college degree;
  • With an average income.

7. Acknowledgment

The author thanks the tender titled „Improving the competitiveness of traditional PICK products through innovative solutions applied at different stages of the food chain”, No. GINOP-2.2.1-15-2017-00101, for its help in writing this article.

8. References

[1] Miskolczi, N. (2012). Műanyagok kémiája és technológiája. Digitális Tankönyvtár, Pannon Egyetem. (Hozzáférés: 2021.02.10.)

[2] Lente, G. (2020): Ezeregynél is több molekula meséi Akadémiai Kiadó, Budapest DOI

[3] Náray-Szabó, G. (2016): Kémia, Akadémiai Kiadó, Budapest DOI

[4] Romhány, G. (2018): Polimer anyagismeret műszaki menedzsereknek Akadémiai Kiadó, Budapest DOI

[5] Szalai, M., Tanninen, T. (1998): Élelmiszerek módosított légterű, ún. védőgázos csomagolására alkalmas fóliák és azok előállítása. XXVII Óvári Tudományos Napok. 4. 883-886. Mosonmagyaróvár.

[6] Molnár, K. (2019): Anyagismereti alapok. Budapest. (Hozzáférés: 2021.01.10.)

[7] Bagi, I. (2013): Műanyag és Gumiipari Évkönyv. Budapest: BB Press.

[8] Bauer, A., Berács , J., Kenesei , Z. (2016). Marketing alapismeretek. Budapest: Akadémiai Kiadó DOI

[9] Kotler, P., Keller, K. (2012). Marketingmenedzsment. Budapest: Akadémiai Kiadó DOI

[10] Trend Inspiráció (dátum nélkül): Trendek. Dr. Törőcsik Marketing Inspiráció Fogyasztói Magatartás Kutató Intézet Kft. (Hozzáférés: 2021.01.21.)

[11] Index. (2019): Idén is lesz műanyagmentes július (Hozzáférés: 2020.01.09.)

[12] Viland, G. (2019): A tudatos vásárlás lehet az új trend. Magyar Hírlap. Augusztus 10. (Hozzáférés: 2020.02.09.)

[13] Boncz, I. (2015): Kutatásmódszertani alapismeretek. Pécs: Pécsi Tudományegyetem (Hozzáférés: 2021.01.13.)

[14] Hoffmann, M., Kozák, Á., Veres, Z. (2016): Bevezetés a piackutatásba. Budapest: Akadémiai Kiadó DOI

DOI: https://doi.org/10.52091/EVIK-2021/3-4-ENG

Received: 2020. November – Accepted: 2021. March

¹ South Ural State University (national research university), Chelyabinsk, Russian Federation

Keywords

semi-finished products from meat of broiler chickens, freeze-dried ground apples, Brazil nuts

2. Introduction

Poultry meat is a dietary product with a high content of easily digestible proteins, low content of fat and cholesterol, it costs less than other meat, takes little time to cook and suits well for daily consumption [1]. However, today consumers tend to prefer “healthy” products, which makes producers expand the range of foods enriched with nutrients. This explains the relevance of using plant-based natural additives in meat processing industry, because they improve the quality characteristics of raw meat, and also increase nutritional and biological value of finished products [2].

It is a known fact that apple powder is rich in vitamins, organic and phenol carboxylic acids, monosaccharides, pectins, and dietary fiber, while the Brazil nut is considered a great source of complete protein, such mineral nutrients as Se, Cu, Mn, I, and fatty acids [3, 4, 5, 6, 7]. That is why, these plant raw materials are separately used in cakes, bread, chocolate, cutlets, curd cheese, cereal bars, nut and seed butters [8, 9, 10, 11, 12, 13, 14] to increase their nutrient density. The aim of our research was to study the possibility of combined use of freeze-dried ground apples and Brazil nut kernels in the technology of stuffed meat products with increased nutritional value.

3. Materials and methods

The following was used as materials of the research:

• Chilled broiler chicken legs manufactured by OAO Turbaslinskiye Broilery (Republic of Bashkortostan, Blagoveshchensk) in accordance with GOST 31962-13;
• Freeze-dried ground apples manufactured by PAO Sibirskiy Gostinets (Pskov Region, Moglino) in accordance with TU 10.39.25-001-34457722-18;
• Kernels of Brazil nuts of Bolivian origin manufactured by OOO Komservis (Moscow Region, Mytishchi) in accordance with TU 9760-002-76440635-16;
• Letniy Sad food additive manufactured by OOO Kulmbakh-D (Moscow Region, Krasnoarmeysk) in accordance with TU 10.89.19-008-58251238-20. Ingredients: dill, garlic, mustard, table salt, maltodextrin, dextrose, E621, dill extract, caraway extract, E100;
• Chicken pockets with butter and herbs cooked according to TU 9214-013-64474310-12 by way of baking stuffed broiler chicken legs at 200 ˚C for 20 minutes.

Control samples were cooked according to a traditional recipe (Table 1), test samples were cooked adding 7% dried ground apples, 5% crushed Brazil nut kernels and 4% less butter.

The dosages of the plant raw materials were chosen taking into account the known data published in a number of scientific papers [8, 9, 10, 11, 12, 13, 14] The test samples of chicken pockets were cooked using deboned chicken legs with skin, flat in shape, with a longitudinal cut in the form of a pocket filled with butter, mixed herbs, ground dried apples, and Brazil nut kernels. The cut was joined with skewers.

The plant raw materials were tested for the content of protein and fat according to MU 4237-86, sugar – GOST 8756.13-87, table salt – GOST 15113.7-77, starch – using standard approach [15]. The meat and meat products were tested for protein according to GOST 25011-2017, fat – GOST 23042-2015, moisture – GOST 9793-2016, table salt – GOST 9957-2015. Sensory evaluation of the laboratory samples was carried out according to GOST 9959-2015. The content of dietary fiber in all samples was determined using the traditional approach [15], content of organic acids – according to М 04-47-12, mineral elements – using iCAP 7200 DUO emission spectrometer.

All measurements were carried out in three replications. Statistical analysis was performed using Microsoft Excel XP and Statistica 8.0 software package. The statistical error of the data did not exceed 5% (at 95% confidence level).

4. Results and discussions

Analyzing the nutritional composition of the non-traditional plant raw materials in comparison with poultry meat (Table 2), it was found that Brazil nut kernels contained a relatively high amount of lipids (11 times more), which made it possible to reduce the amount of butter in the recipe, and hence to decrease cholesterol content in the test samples.

Apple powder proved to have relatively high levels of sugars, dietary fiber, and organic acids, in comparison with both raw meat and other plant components. It is well known that non-volatile acids in fruits not only determine taste and aroma of finished products, but also contribute to the production of gastric juice and have a choleretic effect [16], while insoluble (lignin, cellulose, chitin) and soluble (pectin, inulin) dietary fiber is able to effectively bind heavy metal ions and organic substances [17]. All these factors a priori suggest that this new component in the chicken pockets recipe should have a positive effect on the human organism.

The amino acid content in Letniy Sad food additive was due to sodium glutamate (E621) in its composition, while the presence of table salt at the level of 34.9 ± 2.2% allowed not to introduce any more of it.

The mineral composition of all plant components turned out to be richer than that of broiler chicken legs in terms of the number of elements (Table 3). In terms of the content of micronutrients, which have great physiological importance for the human organism, the Brazil nut contained 12 times more Ca, 7.4 times more Fe, 7.2 times more Se, 6.3 times more Mg, 3.6 times more P and Zn, but the Cu, Mn and Co content were also higher than in the poultry meat. Similarly, the dried ground apple powder contained 2.4 time more Fe, 2 times more Ca and 2.7 times more Si, additionally it’s Ag, Au, B, Be, Cu, Ga, Mn, Mo contain were also higher, than the content of poultry meat. Considering 0.5% dosage of Letniy Sad food additive as per the recipe, its contribution to the total mineral value of ready chicken pockets can be considered significant only in terms of Na content, which was 38 times more than in raw meat.

The levels of heavy metals in nuts – As, Cd, Pb, not found in semi-finished meat products, did not exceed the regulated norms of TR CU 021/2011.

Chilled chicken legs had a relatively high content of K, Si, as well as Na.

Thus, it was proved efficient to use such plant components in the technology of baked meat products in order to increase their nutritional value.

Tasting of the laboratory samples of chicken pockets established that apple and nut raw materials in the specified ratio had a positive effect on the consumer characteristics of the product. At the same time, the control sample did not have outstanding taste and aromatic properties, with creamy tones predominant, leveling the characteristics of a meat product. The mixture of the plant materials accounted for the formation of apple and nut notes in the smell and a slight sour-sweet tone in the taste of the products. The color on the cut acquired a caramel shade. The appearance, consistency, and juiciness of all samples were consistently high.

When testing physical and chemical indicators, it was found that the samples under study did not differ significantly in moisture, fat, and sodium chloride content (Table 4). However, the test samples contained slightly more protein (by 2.1 %), as well as dietary fiber and organic acids, which is a benefit from the standpoint of modern nutritional science.

The study of the mineral composition of the laboratory samples revealed that the test samples exceeded the control ones in terms of the amount of most macro- and microelements (Figures 1, 2). Specifically, as for macronutrients, baked samples with a modified recipe contained more Ca (1.7 times), Mg (35.4 %), and P (20 %); as for microelements – more Mo (473 times), Au (132 times), Cu (56 times), B and Mn (28 times), W (20 times), Be (17 times), Sn (15.8 times), Fe and Ti (1.5-1.6 times), Se (1.4 times), Zn (23.1 %), etc.

Furthermore, the amounts of microelements established according to MR 2.3.1.2432-08 satisfy the daily demand of an adult in Mo by 30.4 %, Cu - by 4.3%, Mn - by 2.1 % if one eats 100 g of baked poultry meat products with the added apple powder and Brazil nut.

Minerals are essential for the human body. They are a part of tissues, hormones, enzymes, intracellular fluid. They are needed for the formation of blood and bone cells, functioning of the nervous system, regulation of muscle tone, processes of energy generation, growth and recovery of the body [18, 19].

5. Conclusions

The nutrient composition of the raw materials and finished products was studied. We found that it is possible to use freeze-dried ground apples (in an amount of 7%) and Brazil nut kernels (in an amount of 5 %) together in the recipe of stuffed meat products. Modifying the recipe for chicken pockets, we obtained a product with improved consumer properties, increased nutrition value, and a decrease in the amount of butter by 4%.

6. Acknowledgement

The work was supported by Act 211 of the Government of the Russian Federation, contract № 02.A03.21.0011.

7. References

[1] Denisyuk, E. A., Tyurina, E. O. (2019): Effect of spinach on food value and economic efficiency of poultry meat semi-finished products production in conditions of LLC “Pervy Myasokombinat”. Bulletin of the Nizhny Novgorod State Agricultural Academy, 4 (24), pp. 28-32.

[2] Asfondyarova, I. V., Sagaidakovskaia, E. S. (2018): Meat semi-finished products of high nutritional and biological value. XXI Century: Resumes of the Past and Challenges of the Present, 7(43), pp. 87-92.

[3] Kishtikov, Kh. B., Dzhappueva, Zh.R. (2017): Chemical composition and curative, dietary, and preventative functions of fruit and vegetable powders added to bakery goods made of wheat flour. Alley of Science, 4(9), pp. 789-796.

[4] Pyanikova, E. A., Cheremushkina, I.V., Kovaleva, E.A., et al. (2020): The effect of apple powder on the consumption of crispbread. Bulletin of Voronezh State University of Engineering Technology. 82(1), pp. 157-163. doi.org/10.20914/2310-1202-2020-1-157-163

[5] Kantoroeva, A. K. (2019): Analysis of the development of the world market for nut crops. Economics and Management: Problems, Solutions. 2(3), pp. 147-154.

[6] Klimova, E. V. (2008): Comparative study of total oil content, fatty acid profile, peroxide value, concentration of tocopherol, phytosterol and squalene in the kernels of Brazil nuts, pecans, pine nuts, pistachios and cashews. Food and processing industry. Abstract journal. 2, p. 369.

[7] Martins, M., Klusczcovski, A.M., Scussel, V.M. (2014): In vitro activity of the brazil nut (bertholletia excelsa h. b. k.) oil in aflatoxigenic strains of aspergillus parasiticus. European food research and technology. 239(4), pp. 687-693.

[8] Nurgalieva, A. A., Pusenkova, L. I. (2017): Use of apple powder in baked confectionery products. Alley of Science. 3(10), pp. 241-248.

[9] Perfilova, O. V. (2019): Development of a new method for preparing white flour dough using apple and pumpkin powder. New Technologies. 1(47), pp. 141-148. doi.org/10.24411/2072-0920-2019-10114.

[10] Linovskaya, N. V. (2019): Development of chocolate with finely ground additions. Scientific works of the Kuban State Technological University” electronic network polythematic journal. 9, pp. 114-123.

[11] Mogilniy, M. P. (2017): Evaluation of the biological value of minced meat products with fruit fillings. Modern Humanities Success). 2(6), pp. 57-62.

[12] Ukkonen, T. I., Belozerova, M. S. (2017): Development of curd cheese with increased selenium content. Materials of the VIII International Scientific and Technical Conference «Low-temperature and food technologies in the XXI century». pp. 264-267.

[13] Patent No. 2706159 RF. Cereal bar for nutrition of those working with harmful compounds of arsenic and phosphorus. Kazan National Research University. Gumerov T. Yu., Gabdukaeva L. Z., Shvink K. Yu. Application dd. 14.05.2019; published 14.11.2019.

[14] Patent No. 2603892 RF. Method for preparing nut-like mass. Rodionova N. S., Popov E. S., Alekseeva T. V., Sokolova O. A., Shakhov A. S. Application dd. 01.07.2015; published 10.12.2016.

[15] Skurikhin, I.M., Tutelyan, V.A. (1998): A guide to the methods of analyzing food quality and safety. Moscow, Brandes, Medicine, p. 342.

[16] Nechaev, A. P., Traubenberg, S. E., Kochetkova, A. A., et al. (2012): Food Chemistry: 5th edition, revised and expanded. – SPb.: Giord, p. 670.

[17] Nikiforova, T. E., Kozlov, V. A., Modina, E. A. (2010): Solvation-coordination mechanism of sorption of heavy metal ions by cellulose-containing sorbent from aqueous media. Chemistry of plant raw material. 4, pp. 23-30.

[18] Dydykina, I. S., Dydykina, P. S., Alekseyeva, O. G. (2013): Trace elements (copper, manganese, zinc, boron) and healthy bone: prevention and treatment of osteopenia and osteoporosis. Effective Pharmacotherapy. 38, pp. 42-49.

[19] Krutenko, V. V. (2013): A close look at the role of gold trace element in the human body. Bulletin of problems of biology and medicine. 2(3), pp. 19-24.

DOI: https://doi.org/10.52091/JFI-2021/1-1-ENG

Received: October 2020 – Accepted: January 2021

¹ Department of Nutritional Science and Production Technology, Faculty of Agricultural and Environmental Sciences, Szent István University, Kaposvár Campus
² Department of Physics and Control, Faculty of Food Science, Szent István University, Budapest Campus
³ Adexgo Kft., Balatonfüred, Hungary
^* Corresponding author: bazar@agrilab.hu

Keywords

Fructose (fruit sugar), sugars, °Brix, NIR-spectroscopy (near-infrared), adverse physiological effects of fructose, metabolic disorder, cardio-vascular diseases, pre-treatment of spectra, valence vibration, harmonic vibration, statistical spectra analysis

2. Introduction

Food sweeteners have become the most widely used additives in the food processing industry, especially in the production of beverages and other products such as desserts and yoghurts. One of the oldest sweetener to have been documented in history is honey [1]. This, and some of the traditional sweeteners such as maple syrup, carob, and agave, consumed for decades are largely made up glucose, fructose, sucrose, minerals and other compounds [1]. Glucose is almost always present in foods and plays an essential role in the regulation of metabolism in human. It can be ingested either as free available sugar (glucose powder) or bonded in polymers, in the case of starch, dextrin, and maltodextrins. Glucose could also be bonded in disaccharides, like in the case fructose bond to glucose in sucrose [2].

For some time now, concerns about the form and levels of sweeteners used in the food industry, and the °Brix value of processed foods, have been topical due to the health implications of the consumers. This is mainly because of the risk of developing metabolic abnormality (diabetes) associated with high intake of sugar, especially sugar of high fructose content. Consumers are therefore becoming more conscious of what they consume, and will at times prefer a reduction of the caloric levels of processed foods, consequently reducing sugar intake [3].

High fructose intake was found to be associated with a high risk of metabolic syndrome [4], obesity, diabetes and an increase in blood triglyceride concentrations and insulin resistance compared with high glucose intake [5], [6], [7]. High risk of cardiovascular diseases and even malignant tumors in body tissues may be related to excessive fructose intake [8], and also dyslipidemia and kidney diseases [9].

Over the years, the application of near-infrared spectroscopy (NIR) to analyze the forms of sugar in food sweeteners, has been found to be easier, faster and cost-efficient [10] compared with tedious and reagent involving methods, such as gas chromatography (GC), high-performance liquid chromatography (HPLC) and enzymatic analysis [11], [12]. The HPLC is the most frequently used method for assessing free fructose, free glucose, sucrose, maltose, and lactose content [13].

The NIR spectral region is found between 800 to 2500 nm (12500–4000 cm−1) range, with absorptions representing overtones and combinations which are associated with –CH, –OH, –NH, and –SH functional groups [14]. In the case of glucose, 1st overtone of O–H stretching corresponds to absorption bands at 1195, 1385, 1520, 1590, 1730 nm, 1st overtone of O–H stretching of fructose and sucrose at 1433 nm, and O–H combination band of sucrose, glucose and fructose at 1928 nm [14].

Mono- and disaccharides, such as glucose, fructose, sucrose, lactose, were also analyzed in aqueous solutions [15]. Although the same molar concentrations of all the concerned sugars were dissolved, the mass that those represented differed considerably due to the differences in molecular weights of mono- and disaccharides. When quantifying sugars in mixtures, the molar concentration of the sugar solutions gave less accurate calibration models compared with those fitted on weight per volume concentration. Since the spectral information is mostly the light absorbance of chemical bonds during excitation, this information is more proportional with the number of chemical bonds and atoms in the aqueous solution, than with the number of molecules. Regression coefficient vectors of calibration models for each of the sugars also revealed the spectral regions holding the highest importance in the quantitative analysis of the sugars. Regression vectors of the 1100-1800 nm interval, associated with signals of O–H and C–H bonds, showed the significance of characteristic spectral regions of water and the dissolved sugars. The calibration on the concentration of the sugars within the mixtures showed accurate validation performance even at low concentration levels (0.0018 – 0.5243 g/cm3), R2CV of 0.841 and 0.961, SECV of 0.024 g/cm3 and 0.012 g/cm3 for glucose and fructose, respectively. This showed possible quantification of a specific sugar in a mixture of sugars in a solution using NIR spectroscopy [15].

In related studies [10], [16], [17], glucose, fructose and sucrose were quantified in different fruit juices using NIR, and accurate partial least square regression (PLSR) models were reported (R2 > 0.854, 0.963, 0.953 for glucose, fructose, sucrose, respectively). Good PLRS models were reported for predicting glucose within 900-2200 nm wavelength range [18], whereas the 900-1650 nm interval was reported to be good for the discrimination of organic sugar and conventional brown sugar using partial least squares discriminant analysis (PLS-DA) models [19]. In a study, the concentration of glucose in an aqueous mixture of glucose, albumin and phosphate was quantified using NIR and reported accurate PLRS models [20]. The possibility to predict the glucose, fructose and sucrose content in Morindae officinalis extracts utilizing NIR was also reported [21].

The Hungarian food industry is flooded with many sweeteners for food processing. However, there are three major sweeteners: K-syrup LDX and K-sweet F55, which are two commonly used isosugars, and D-sucrose. K-syrup LDX is a sweet, viscous, quickly crystallizing syrup often used in food and pharmaceutical industry as a raw material for fermentation. It contains high amount of glucose or dextrose (93%), and small amount of fructose (0.5%) and viscous liquid [22]. K-sweet F55, however, is a high caloric isosugar consisting of glucose and fructose, where the fructose content is higher (55%) than the glucose (45%) [23], and the third sweetener is D-sucrose or refined sugar, which is increasingly being replaced with K-syrup LDX and K-sweet F55.

This study aimed to determine the applicability of NIR spectroscopy to quantify glucose, fructose, sucrose content and °Brix of aqueous solutions of the widely used sweeteners, D-sucrose, K-syrup LDX, and K-sweet F55.

3. Materials and Methods

3.1. Sample preparation

Three kinds of sugars were used with brand names: D-sucrose (Carl Roth GmbH, Karlsruhe, Germany): 100% sucrose; K-Syrup LDX (KALL Ingredients Kft., Tiszapüspöki, Hungary): 93% glucose + 0.5% fructose + 6.5% water; K-Sweet F55 (KALL Ingredients Kft., Tiszapüspöki, Hungary): 45% glucose + 55% fructose. Aqueous solutions were prepared at 10 different concentrations for each of the three sugars, separately. A total sample of 30 samples was prepared, 100 ml of each.

3.2. Laboratory measurement

°Brix was measured with Hanna HI96801 Digital Refractometer, and recorded as reference for subsequent NIRS calibrations. Glucose, fructose and sucrose concentration of the respective sugar solutions was calculated based on the mass of sweetener added to the solutions and the percentages of the individual sugars within the sweeteners. The following formulas were used for the calculation of glucose and fructose in K-sweet F55 and K-syrup LDX solutions:

1. Glucose in K-syrup LDX solution = 93/100*amount of K-syrup in solution (g/100g)
2. Fructose in K-syrup LDX solution = 0.5/100*amount of K-syrup in solution (g/100g)
3. Glucose in K-Sweet F55 solution = 45/100*amount of K-sweet F55 in solution (g/100g)
4. Fructose in K-sweet F55 solution = 55/100* amount of K-sweet F55 in solution (g/100g)

Accordingly, each of the 30 samples was described with °Brix, and concentrations of total sugar, glucose, fructose and sucrose, as listed in Table 1.

D-sucrose: 100% sucrose; K-syrup LDX: 93% glucose+0.5% fructose; K-sweet F55: 45% glucose+ 55% fructose; SD: standard deviation; Max: maximum value; Min: minimum value

3.3. NIRS measurement

The samples were scanned at room temperature (25 °C) using a FOSS NIRSystems 6500 (FOSS NIRSystems, Inc, Laurel, MD, USA) spectrometer, operated with WinISI v1.5 software (InfraSoft International, Port Matilda, PS, USA). The scanning was done in transmission mode after measuring 1 ml sugar solution into a quartz cuvette having 1 mm pathlength. Two rounds of scanning of each sample were done randomly, and the subsequent sample was used to wash the cuvette three times between each sample scanning. Sixty spectral data were obtained and the spectra of the two rounds were averaged resulting in 30 spectra.

3.4. Spectral pre-processing and multivariate data analysis

The Unscrambler v9.7 (CAMO Software AS, Oslo, Norway) software was used for the analysis of the NIR data, while the MS Excel 2013 was used to calculate the descriptive statistics for the variables measured and calibrated for °Brix, glucose, fructose, and sucrose concentration.

For scatter correction of spectra, and to obtain accurate and robust calibration models, several spectral types of preprocessing were performed: standard normal variate (SNV), multiplicative scatter correction (MSC) and gap-segment second derivative (2nd order derivative, gap of 5 data points, segment of 5 data points).

Using multivariate data analyses, both the separation of the solutions prepared with different sweeteners and the calibration on the targeted quantitative parameters was performed. Principal component analysis (PCA) [24] was used to investigate the multidimensional pattern of the spectra data and to identify differences among the three groups of the sweetener solutions. The spectral data within the NIR range (1100-1850nm) were calibrated with the laboratory data as the reference, using partial least squares regression (PLSR) models [24]. The optimum number of latent variables (LV) used for the PLSR modelling was determined by full (leave-one-out) cross-validation, when in a 30-step iterative process each of the 30 samples was left out of the calibration once and was used for validating the model [24].

Evaluation of PLSR models was done by comparing the calibration statistics with that of the cross-validation. The determination coefficient of calibration (R2C) and cross-validation (R2CV), and the root mean square error of calibration (RMSEC) and cross-validation (RMSECV) were compared, where larger R2 value and smaller RMSE value represent the better model. During the model optimization processes, RMSECV values were minimized.

4. Results and discussions

The recorded raw spectra show the typical NIR absorption of water, with a major peak at 1450 nm, representing the 1st overtone region of O–H bond (Figure 1). The small peak around 1780 nm represents the 1st overtone of C–H bonds. The second derivative spectra were calculated with the gap-segment derivative function, where both gap and segment were set to 5 data points to avoid noise enhancement of the derivative function, still keeping the useful signals within the pretreated data.

The negative peaks of the 2nd derivative spectra (Figure 2) indicate the locations and relative amplitude of the original overlapping absorptions appearing as one in the raw spectra. This shows the well-described phenomenon that major peak of the raw spectrum at 1450 nm is formed by at least two underlying absorptions of water at 1416 nm and 1460 nm, representing less and more H-bonded water, respectively [15].

The applied spectral pretreatments (2nd derivative, or SNV, or MSC) did not allow visual differentiation of solutions with different sweeteners, while the gradual changes of the water absorption peaks indicated the effect of the increasing concentration of dissolved sugars on the structure of water [15].

Figure 3 shows the 3D plot of the PCA performed with 2^nd derivative spectra of all the 30 solutions. The solutions of the three types of sweeteners are indicated with different colors and numbers. The two plots show the same result from different angles, highlighting that 4th principal component (PC4) is responsible for the separation of K-Sweet F55 from D-sucrose and K-Syrup LDX, and PC2 is responsible for the separation of D-sucrose from K-Syrup LDX and K-Sweet F55. Thus, PC2, as new latent variable covering approximately 2% of the variance of the original NIR data, describes the difference between the disaccharide and monosaccharide solutions, while PC4, covering less than 1% of the variance of the original NIR data, describes the difference between the solutions of high fructose syrup and that of the other sweeteners. The combination of PC2 and PC4 describes the differences between glucose solutions and others.

Figure 4 shows the loading vectors of PC2 and PC4. The wavelength regions having the largest deviation from zero are the most responsible for score values of the principal components, thus, the assigned peaks indicate the absorptions causing the difference between the sugar solutions. The band assignments are in good harmony with previous findings [14,15], i.e. peaks in the 1300-1600 nm interval refer to the molecular changes of water caused by the dissolved sugars, while the peaks in the 1600-1850 nm interval represent characteristic C–H bands.

The results of the calibration models developed using PLS regression on the measured °Brix and calculated fructose, glucose, sucrose concentrations are presented in Table 2 and Figure 5.

LV: number of latent variables, R²_C: determination coefficient of calibration, RMSEC: root mean square error of calibration, R²_CV: determination coefficient of cross-validation, RMSECV: root mean square error of cross-validation, MSC: multiplicative scatter correction, SNV: standard normal variate, 2D5G5S: 2nd order derivative with 5-point gap and 5-point segment

The best results for °Brix were achieved with no spectral pretreatment. The RMSE of °Brix remained around 1 °Bx, which was almost third of the standard deviation of the measured reference values. The RMSE of the sugar concentrations was similarly low. The least accurate model was achieved for fructose, which is caused by the group of samples with fructose content below 0.05% - for these samples the model performed worse than in the higher concentration regions (Figure 5. (b)). Second derivative pretreatment gave the best result for glucose and sucrose, while the best models for fructose were achieved without pretreatment of the NIR spectra.

The calibration and cross-validation regression lines (Y-fit) of the best °Brix, fructose, glucose and sucrose models are shown in Figure 5. The black diagonal line shows the optimal Y-fit, while blue and red lines show the calibration and cross-validation Y-fits. The blue dots show the NIR predicted composition values of samples during calibration in the function of the laboratory reference values, and red dots show the NIR predicted values at cross-validation testing, again, in the function of the reference values measured. The closer the dots are to the regression line and the less the regression line deviates from the optimal Y-fit, the better the calibration model is. In most of the cases, the achieved Y-fits are hitting the optimum, meaning that the NIR predicted values are almost equal to the actual laboratory reference values. The calibration and cross-validation results of this study are in agreement with the previously cited results achieved with sugar solutions and fruit juices. These results confirm that, after a proper calibration process, NIR spectroscopy is a useful and effective tool for easy, rapid and accurate measurement of individual sugars in mixed solutions.

5. Conclusions

The results of this study performed with widely used sweeteners confirm the previously published findings that NIR spectroscopy is a useful and powerful technology to detect and quantify individual sugar types even in mixture solutions. Since NIR spectrometers have not only reached the portable size but have become extremely small as a fingernail-sized chip, the importance of this technology in everyday food qualification seems to be underestimated. Wide aspects of applications should be tested and used for monitoring products and warrant food safety and quality. Among these applications, checking and certifying the fructose content of beverages and foods would advantage consumers’ health, as this constituent has been proven to raise the risk of several diseases of modern times. NIR spectroscopy as secondary correlative analytical technology will likely remain to be unsuitable for detecting and quantifying fructose in a complex liquid of completely unknown composition, but may be suitable for indicating the excessive presence of fructose in a known liquid meant to be containing no or only a certain amount of fructose. The usability of NIR tools is limited and they should not be considered as subtituents of classical analytical methods, however, by rational use of opportunities, useful applications can be developed for practice.

6. References

[1] Edwards, C. H., Rossi, M., Corpe, C. P., Butterworth, P. J., & Ellis, P. R. (2016): The role of sugars and sweeteners in food, diet and health: Alternatives for the future. Trends in Food Science and Technology, 56, pp. 158-166.
https://doi.org/10.1016/j.tifs.2016.07.008

[2] White, E., McMahon, M., Walsh, M., Coffey, J. C., & O’Sullivan, L. (2014): Creating Biofidelic Phantom Anatomies of the Colorectal Region for Innovations in Colorectal Surgery. Proceedings of the International Symposium on Human Factors and Ergonomics in Health Care, 3 (1), pp. 277-282.
https://doi.org/10.1177/2327857914031045

[3] Gardner, C., Wylie-Rosett, J., Gidding, S. S., Steffen, L. M., Johnson, R. K., Reader, D., & Lichtenstein, A. H. (2012): Nonnutritive sweeteners: Current use and health perspectives: A scientific statement from the American heart association and the American diabetes association. Circulation, 126 (4), pp. 509-519.
https://doi.org/10.1161/CIR.0b013e31825c42ee

[4] Taskinen, M. R., Packard, C. J., & Borén, J. (2019): Dietary fructose and the metabolic syndrome. Nutrients, 11 (9), pp. 1-16.
https://doi.org/10.3390/nu11091987

[5] Bray, G. A. (2013): Energy and fructose from beverages sweetened with sugar or high-fructose corn syrup pose a health risk for some people. Advances in Nutrition, 4 (2), pp. 220-225.
https://doi.org/10.3945/an.112.002816

[6] Malik, V. S., & Hu, F. B. (2015): Fructose and Cardiometabolic Health What the Evidence from Sugar-Sweetened Beverages Tells Us. Journal of the American College of Cardiology, 66 (14), pp. 1615-1624.
https://doi.org/10.1016/j.jacc.2015.08.025

[7] Rizkalla, S. W. (2010): Health implications of fructose consumption: A review of recent data. Nutrition and Metabolism, 7, pp. 1-17.
https://doi.org/10.1186/1743-7075-7-82

[8] Biró, G. (2018): Human biological characteristics of fructose. Journal of Food Investigation, 64 (1), pp. 1908-1916.

[9] Collino, M. (2011): High dietary fructose intake: Sweet or bitter life? World Journal of Diabetes, 2 (6), pp. 77.
https://doi.org/10.4239/wjd.v2.i6.77

[10] Liu, Y., Ying, Y., Yu, H., & Fu, X. (2006): Comparison of the HPLC method and FT-NIR analysis for quantification of glucose, fructose, and sucrose in intact apple fruits. Journal of Agricultural and Food Chemistry, 54 (8), pp. 2810-2815.
https://doi.org/10.1021/jf052889e

[11] Giannoccaro, E., Wang, Y. J., & Chen, P. (2008): Comparison of two HPLC systems and an enzymatic method for quantification of soybean sugars. Food Chemistry, 106 (1), pp. 324-330.
https://doi.org/10.1016/j.foodchem.2007.04.065

[12] Yuan, H., Wu, Y., Liu, W., Liu, Y., Gao, X., Lin, J., & Zhao, Y. (2015): Mass spectrometry-based method to investigate the natural selectivity of sucrose as the sugar transport form for plants. Carbohydrate Research, 407, pp. 5-9.
https://doi.org/10.1016/j.carres.2015.01.011

[13] International Association of Analytical Chemistry, A. (1990): Official Methods of Analysis of the AOAC. Arlington VA

[14] López, M. G., García-González, A. S., & Franco-Robles, E. (2017): Carbohydrate Analysis by NIRSChemometrics. In K. G. Kyprianidis & S. Jan (Eds.), Developments in Near-Infrared Spectroscopy pp. 81-95
https://doi.org/10.5772/67208

[15] Bázár, G., Kovacs, Z., Tanaka, M., Furukawa, A., Nagai, A., Osawa, M., Itakura, Y., Sugiyama, H., Tsenkova, R. (2015): Water revealed as molecular mirror when measuring low concentrations of sugar with near infrared light. Analytica Chimica Acta, 896, pp. 52-62.
https://doi.org/10.1016/j.aca.2015.09.014

[16] Xie, L., Ye, X., Liu, D., & Ying, Y. (2009): Quantification of glucose, fructose and sucrose in bayberry juice by NIR and PLS. Food Chemistry, 114 (3), pp. 1135-1140.
https://doi.org/10.1016/j.foodchem.2008.10.076

[17] Rodriguez-Saona, L. E., Fry, F. S., McLaughlin, M. A., & Calvey, E. M. (2001): Rapid analysis of sugars in fruit juices by FT-NIR spectroscopy. Carbohydrate Research, 336 (1), pp. 63-74.
https://doi.org/10.1016/S0008-6215(01)00244-0

[18] Mekonnen, B. K., Yang, W., Hsieh, T. H., Liaw, S. K., & Yang, F. L. (2020): Accurate prediction of glucose concentration and identification of major contributing features from hardly distinguishable near-infrared spectroscopy. Biomedical Signal Processing and Control, 59, pp. 101923.
https://doi.org/10.1016/j.bspc.2020.101923

[19] De Oliveira, V. M. A. T., Baqueta, M. R., Março, P. H., & Valderrama, P. (2020): Authentication of organic sugars by NIR spectroscopy and partial least squares with discriminant analysis. Analytical Methods, 12, pp. 701-705.
https://doi.org/DOI: 10.1039/C9AY02025J

[20] Khadem, H., Eissa, M. R., Nemat, H., Alrezj, O., & Benaissa, M. (2020): Classification before regression for improving the accuracy of glucose quantification using absorption spectroscopy. Talanta, 211 (January), pp. 120740.
https://doi.org/10.1016/j.talanta.2020.120740

[21] Hao, Q., Zhou, J., Zhou, L., Kang, L., Nan, T., Yu, Y., & Guo, L. (2020): Prediction the contents of fructose, glucose, sucrose, fructo-oligosaccharides and iridoid glycosides in Morinda officinalis radix using near-infrared spectroscopy. Spectrochimica Acta - Part A: Molecular and Biomolecular Spectroscopy, pp. 234
https://doi.org/10.1016/j.saa.2020.118275

[22] KALL, I. (2020): K-syrup LDX. Retrieved from http://kallingredients.hu/en/products/2/14/k-syrup-ldx

[23] KALL, I. (2020): K-sweet F55. Retrieved from http://kallingredients.hu/en/products/2/12/k-sweet

[24] Naes, T., Isaksson, T., Fearn, T., & Davies, T. (2002): A user-friendly guide to multivariate calibration and classification. Chichester, UK: NIR Publications

DOI: https://doi.org/10.52091/JFI-2021/1-4-ENG

Received: June 2020 – Accepted: October 2020

¹ South Ural State University (national research university), Chelyabinsk, Russian Federation

Keywords

mayonnaise sauce, pine nut oil cake, protein concentrate, functional food product, β-carotene

2. Bevezetés

Mayonnaise sauces, like all mayonnaise products, are among the most popular everyday consumer goods. Main components for mayonnaise sauces involve natural products with high biological value and health-promoting properties. In this regard, the development of mayonnaise product formulas can be viewed as a promising line of research [1, 2].

Hydrocolloids and protein-polysaccharide complexes, plant extracts, vitamin and mineral complexes, dietary fiber, polyunsaturated fatty acids and protein concentrates are the most valuable functional ingredients in the production of emulsion foods for particular nutritional uses. These biologically active components allow you to structure a person’s diet in order to improve metabolism, immunity, nervous and endocrine systems, functioning of individual organs and body systems [3, 4].

Currently, protein concentrates are widely used in production of various sauces, pastes, dairy, and confectionery products. Such popularity of protein concentrates is due to the protein deficiency that more than 60% of people suffer from to a varying degree [5].

At the same time, every year scientists all over the world come up with new sources and methods of protein isolation to create new functional foods enriched with protein concentrates. It has been established that regular consumption of such products helps to increase body’s resistance to harmful factors, strengthen immunity, and improve metabolism [6].

Pine nut oil cake, obtained by extracting oil from pine nut kernels, is a secondary raw material, but it is of great importance as an additional source of complete protein, easily digestible carbohydrates, vitamins, and minerals. With the right choice of the method of its extraction and purification, it is possible to obtain a protein-rich concentrate that can be added to various foods in order to give them functional properties.

The composition of the protein of pine nut oil cake is determined by the composition of the protein of the kernels of pine nuts.

The content of essential amino acids in the protein composition of pine nut kernels ranges from 36 to 40%.

The content of some individual essential amino acids in pine nut protein is specific, which is characteristic of all types of plant materials. It should be noted that in terms of amino acid composition, namely, the content of phenylalanine, tyrosine, histidine, tryptophan, arginine, pine nut oil cake protein is as good as the protein of the major grain and oilseed crops. It is close to dairy protein in terms of tryptophan content, surpassing it in terms of arginine and histidine content.

The composition of the lipid fraction of pine nut oil cake is characterized by a quantitative predominance of polyunsaturated fatty acids – linoleic and γ-linolenic –, belonging to the ω-6 family.

The vitamin and mineral value of pine nut oil cake depends both on the initial chemical composition of the processed nut and on the residual oil content in the oil cake after pressing.

Pine nut oil cake has a high content of tocopherols (11.8 mg/100 g of product), thiamine (0.6 mg/100 g of product), and riboflavin (1.83 mg/100 g of product).

Pine nut oil cake is a concentrate of biologically valuable food substances like proteins, lipids, carbohydrates [7].

3. Materials and methods

The following was used as the material for research:

• ground cake of pine nut kernels, produced in accordance with TU 9146-001-53163736-06 (by “Siberian Product”, supplied by “Altai Dar LLC”, Altai Territory, Barnaul, Russia) [14];
• Beta-carotene 30% os, plant-based, liquid, oil-soluble (by “NATEC”, Moscow);
• reference and test samples of mayonnaise sauces.

Organoleptic characteristics of mayonnaises and mayonnaise sauces must comply with the requirements of GOST 31761-2012 “Mayonnaises and mayonnaise sauces. General specifications” [8]. Testing of organoleptic characteristics was carried out at (20±2) °C after at least 12 hours after production.

Organoleptic indicators were determined in the following sequence: texture, appearance, colour, smell, taste.

The mass fraction of protein was determined by the Kjeldahl titration method.

The stability of the emulsion was determined by centrifugation.

The intact emulsion stability was determined by centrifuging the emulsion for 5 minutes at 1500 rpm.

The dynamic viscosity of the samples was determined using “Reostat-2” rotational viscometer (Germany) at 20 ºС.

The degree of oxidative deterioration was determined by the peroxide number of the oil phase using the iodometric method and calculating the degree of oxidative deterioration of the product [9-11].

All measurements were carried out in three replications. Statistical analysis was performed using Microsoft Excel XP and Statistica 8.0 software package. The statistical error of the data did not exceed 5% (at 95% confidence level).

4. Results and discussion

Mayonnaise sauce is a finely dispersed emulsion product with a fat content of not less than 15%, produced from refined deodorized oil, water, with or without dairy by-products, food additives and other food ingredients (GOST 31761-2012 “Mayonnaises and mayonnaise sauces. General specifications”) [8].

The ingredients of the obtained mayonnaise sauce included refined deodorized cooking oil, egg powder, mustard flour, granulated sugar, table salt, 80% acetic acid, as well as a protein concentrate made of pine nut oil cake, natural β-carotene and water. Introducing β-carotene to the formula of the mayonnaise sauce increased the stability of its fatty phase to oxidation and extended its shelf life [12].

The production technology of the functional mayonnaise sauce was based on the “classic” mayonnaise sauce production technology.

The specified amount of water of 35–40 °C (not taking into account the water used to prepare the acetic acid solution) was poured into the mixer with a steam-water jacket. The mixer was turned on, and dry components – granulated sugar, salt, pine nut oil cake – were heated and added to the mixer. The mass was mixed intensively at 70-80 rpm and heated to 80-85 °C for 25-30 minutes. Then, the resulting suspension was cooled to 35-40 °C, egg powder and mustard flour were added, after which the emulsion was heated to 55-60 °C during 15-20 minutes.

After heating, the emulsion was again cooled to 25-30 °C, the number of revolutions was reduced to 30-40 rpm, and oil with pre-dissolved β-carotene was introduced. Following that, after adding acetic acid solution into the sauce, it was subject to stirring for another 3-5 minutes and subsequently homogenized at a pressure of 0.9-2.5 MPa.

The use of pine nut oil cake made it possible to reduce the content of egg products in the sauce formula, to lower the cholesterol, and to increase the protein content in the finished product.

The use of β-carotene in the sauce formula enhanced the color of natural egg products.

The use of pine nut oil cake not only simplified the mayonnaise sauce production process, but also allowed to obtain a colloidal system consisting of finely dispersed particles of cell walls. Intensive mixing ensured a complete interaction of proteins, fats and carbohydrates with other components, which facilitated emulsion stability, as finely dispersed cell walls of pine nut oil cake formed a solid three-dimensional structure, enhancing the emulsifying and stabilizing effect.

Reducing the mass fraction of egg powder in the formula to less than 1% made it difficult to obtain a stable emulsion, which led to a decrease in the viscosity of the finished product. The consistency of the finished product became watery, its organoleptic characteristics were low [13]. Therefore, we chose 1%, 2% and 3% dosages of pine nut oil cake to be introduced instead of egg powder.

Formulas of mayonnaise sauces are given in Table 1.

Test samples of mayonnaise sauces with pine nut oil cake were tested for organoleptic characteristics (Table 2).

The appearance of mayonnaise sauces is shown in Figure 1.

Physical and chemical indicators are given in Table 3.

The use of pine nut oil cake increased the overall protein content in the finished product. Pine nut oil cake is an effective emulsifier, and in combination with a conventional emulsifier – egg powder – ensured a good, smooth consistency of the sauce and high stability of the emulsion. It allowed to obtain a finished product with a viscosity that meets consumer requirements for compatibility with other ingredients of a dish or food systems.

In the next stage of research we studied how the quality of the mayonnaise sauce changed during storage.

Storing the samples at 20 ºC provoked the oxidation without changing the mechanism of the process and violating the colloidal stability of the product. The dynamics of the peroxide number of the oil phase of mayonnaise sauce samples during the storage at 20 ºC is shown in Figure 2.

The oxidation of the samples during storage was caused by exposure to light. When stored for more than four weeks, the peroxide number of the reference sample exceeded the level of 11 mmol of active oxygen/kg, and as for the test samples, it did not reach the level of 6 mmol of active oxygen/kg.

The use of β-carotene (0.2%) in the mayonnaise sauce can significantly increase the oxidation stability of the product without adding a preservative, as well as enrich the mayonnaise with biologically active substances of plant origin.

Based on all types of studies, it can be concluded that 3% was the most preferable dosage of pine nut oil cake in the formula of the mayonnaise sauce.

5. Conclusions

Using pine nut oil cake, which possesses good emulsifying properties, in the amount of 3%, alongside a conventional egg powder emulsifier, increased the viscosity of the finished product, ensured a smooth texture of the sauce and high stability of the emulsion. The use of pine nut oil cake made it possible to reduce the content of egg products in the sauce formula and lower the amount of cholesterol in the finished product. Besides, the introduction of pine nut oil cake to the sauce formula increased its protein content. The use of β-carotene (0.2%) in the mayonnaise sauce can significantly increase the oxidation stability of the product without adding a preservative and enrich the mayonnaise with biologically active substances of plant origin.

Thus, pine nut oil cake obtained by processing kernels of pine nuts is a promising functional additive. This material is a suitable to produce fat emulsions, including the reduced fat content products, due to the protein and carbohydrate content of oil cake, thus can provide the necessary rheological structure for low-fat products too.

6. Acknowledgement

The work was supported by Act 211 of the Government of the Russian Federation, contract № 02.A03.21.0011.

7. References

[1] Chung, C., Degner, B., McClements, D. J. (2014): Development of reduced-calorie foods: Microparticulated whey proteins as fat mimetics in semi-solid food emulsions. Food Research International, 56, pp. 136–145. http://doi.org/10.1016/j.foodres.2013.11.034.

[2] Emadzadeh, B., Ghorani, B. (2015): Oils and fats in texture modification. In J. Chen, A. Rosenthal (Eds.), Modifying food texture pp. 99–112. Woodhead Publishing.

[3] Cheung, I., Gomes, F., Ramsden, R., Roberts, D. G. (2002): Evaluation of fat replacers Avicel™, N Lite S™ and Simplesse™ in mayonnaise. International Journal of Consumer Studies, 26 (1), pp. 27–33. http://doi.org/10.1046/j.1470-6431.2002.00207.x.

[4] Ma, Z., Boye, J. I. (2013): Advances in the design and production of reduced-fat and reduced-cholesterol salad dressing and mayonnaise: A review. Food and Bioprocess Technology, 6 (3), pp. 648–670.

[5] Sikora, M., Badrie, N., Deisingh, A. K., Kowalski, S. (2008): Sauces and Dressings: A Review of Properties and Applications. Critical Reviews in Food Science and Nutrition, 48 (1), pp. 50-77. http://doi.org/10.1080/10408390601079934.

[6] Diftis, N. G., Biliaderis, C. G., Kiosseoglou, V. D. (2005): Rheological properties and stability of model salad dressing emulsions prepared with a dry-heated soybean protein isolate–dextran mixture. Food Hydrocolloids, 19 (6), pp. 1025–1031. http://doi.org/10.1016/j.foodhyd.2005.01.003

[7] Gómez-Ariza, J.L., Arias-Borrego, A., García-Barrera, T. (2006): Multielemental fractionation in pine nuts (Pinus pinea) from different geographic origins by size-exclusion chromatography with UV and inductively coupled plasma mass spectrometry detection. Journal of Chromatography, 1121 (2), pp. 191-199. http://doi.org/10.1016/j.chroma.2006.04.025.

[8] GOST 31761-2012. Mayonnaises and mayonnaise sauces. General specifications. Moscow, 2013. pp. 1-13.

[9] Skurikhin, I.M., Tutelyan, V.A. (1998): A guide to the methods of analyzing food quality and safety. Moscow, Brandes, Medicine, pp. 110–115.

[10] Karas, R., Skvarča, M., Žlender, B. (2002): Sensory quality of standard and light mayonnaise during storage. Food Technology and Biotechnology, 40, pp. 119–127.

[11] Calligaris, S., Manzocco, L., Nicoli, M. C. (2007): Modelling the temperature dependence of oxidation rate in water-in-oil emulsions stored at sub-zero temperatures. Food Chemistry, 101 (3), pp. 1019–1024. http://doi.org/10.1016/j.foodchem.2006.02.056

[12] Cortez, R., Luna-Vital, D. A., Margulis, D., Mejia, E. G. (2017): Natural pigments: stabilization. 6th International Conference on Agriproducts processing and Farming. IOP Conf. Series: Earth and Environmental Science. 422, (20), IOP Publishing. http://doi:10.1088/1755-1315/422/1/012090.

[13] Kishk, Y. F. M., Elsheshetawy, H. E. (2013): Effect of ginger powder on the mayonnaise oxidative stability, rheological measurements, and sensory characteristics. Annals of Agricultural Sciences, 58 (2), pp. 213–220. http://doi.org/10.1016/j.aoas.2013.07.016.

[14] Oil industry by-products. TU catalog. Number: TU 9146-001-53163736-2006. Name: Pine nut kernel cake. Siberian product "; 656055, Altai kr., Barnaul, st. A. Petrova, 1886. http://92.243.65.78/techdocs/kgs/tu/679/info/126955/ (Hozzáférés: 2020. 06. 11.)

DOI: https://doi.org/10.52091/EVIK-2021/1-5-ENG

Received: November 2020 – Accepted: January 2021

¹ University of Debrecen, Faculty of Agricultural and Food Sciences and Environmental Management, Institute of Food Technology
² University of Debrecen Doctoral School of Nutrition and Food Sciences

Keywords

brewer’s spent grain, inactive malt, byproduct, sustainability, fiber

2. Introduction

Brewer’s spent grain is a byproduct of the brewing technology (Figure 1), which is usually utilized as animal feed, but in many cases it is transported from manufacturing plants as waste. With our experiments, we were looking to answer the question whether brewer’s spent grain can be reintroduced into the food industry, and whether enrichment with it has a proven positive effect on the nutrition characteristics of medallions made from wheat flour.

3. Brewery byproducts

The brewing industry uses various grains to produce malt. In addition to the usual and most often used barley (Hordeum vulgare L.) and wheat (Triticum aestivum L.), other grains such as maize (Zea mays L.), rice (Oryza sativa L.), oats (Avena sativa L.), millet (Pancium miliaceum L.), rye (Secale cereale L.), sorghum (Sorghum bicolor L.), spelt (Triticum spelta L.), quinoa (Chenopodium quinoa Willd.), buckwheat (Fagopyrum esculentum Moench) and amaranths (Amaranthaceae) are used more and more often as sources of starch [2, 4, 36, 37, 39].

In order to achieve the targeted organoleptic and chemical properties of the different recipes, malt blends are commonly used, which not only affects the properties of the final product, i.e., beer, but at the same time also affects the byproducts [5, 26, 29].

In the brewing process, the goal is to obtain the maximum extract content from malt and the additives during mashing. The byproduct left behind after the filtration of the mash is called brewer’s spent grain, also known as inactive malt [3, 10, 38, 40].

Brewer’s spent grain accounts for about 85% of the byproducts generated during the brewing process [25, 34]. According to some studies, the disposal of brewer’s spent grain as waste may be of environmental concern, which is why one possible use of brewer’s spent grain in aquaculture feed is being addressed. In feed intended for fish, it can effectively replace soybean meal at a rate of 50% as a potential source of protein [8, 12, 13].

Other brewery byproducts include malt germ, hot lees, brewer’s yeast and other gases, such as carbon dioxide [11, 33, 35].

3.1. Nutritional parameters of brewer’s spent grain

Brewer’s spent grain is a valuable source of nutrients. Data on the average nutrient content per 1000 g dry matter are shown in Table 1. It is a useful source of protein and fiber, rich in vitamins (mainly B1, B2 and B6) and minerals, especially calcium, phosphorus, magnesium, potassium and sodium [1].

3.2. Enrichment possibilities of bakery products with brewer’s spent grain

The regulations for bakery products can be found in prescription 1-3/16-1 of the Hungarian Food Codex (HFC) [17]. According to the definition of the HFC, enriched foods are products that contain a significant amount of one or more complementary food components. These products are not necessarily developed for general consumption, but are targeted at a specific target group [7, 32]. In the case of brewer’s spent grain added to the dough of bakery products, it can also be called an enrichment, since following drying and crushing/grinding, brewer’s spent grain can also be used in bakery products in the form of flour.

According to the literature, one of the most practical uses of brewer’s spent grain is composting, but it can also be used as an enrichment agent in the production of foods, such as baking bread, in a proportion of 5 to 10% [40]. When brewer’s spent grain is used in a higher proportion, the crumb of the bread may be sticky [15]. As a result of the enrichment, the dietary fiber content of the finished product increases. Dietary fiber has a beneficial effect on the functioning of the stomach, the small intestine and the colon [14, 41]. According to literature data, the consumption of dietary fiber by the Hungarian population is only 20-25 grams, compared to the recommended 30-35 grams per day. Enrichment with brewer’s spent grain would not only increase fiber intake, but also protein intake [9, 28, 30]. Due to its easy digestibility, barley malt in the inactive form is also used in many cases in products for small children, and its infusion has a digestion stimulating effect [27, 31].

4. Materials and methods

4.1. Preparation of products enriched with brewer’s spent grain

During our experiments, our control medallion recipe was compiled as defined in the Hungarian Food Codex [17]. In the case of the enriched products, light (barley) and dark (a 1:1:1 mixture of Chateau black dyeing malt and barley malt roasted to chocolate color and to black color) malts were used in different concentration relative to the weight of the flour. The medallions were prepared with both malts at 10%, 25% and 50% enrichment levels. After adequate mixing, the dough of the medallions was prepared from the ingredients in the recipe (Figure 2), the balls of 4-5 cm diameter were formed from the dough, and the products were prepared by baking the balls at 150 oC for 45 seconds using an electric medallion oven (Figure 3).

Names and abbreviations of the samples prepared:

• C: Control malt
• LM 10%: Light malt, 10% enrichment
• LM 25%: Light malt, 25% enrichment
• LM 50%: Light malt, 50% enrichment
• DM 10%: Dark malt, 10% enrichment
• DM 25%: Dark malt, 25% enrichment
• DM 50%: Dark malt, 50% enrichment

4.2. Chemical characteristics of the medallions enriched with brewer’s spent grain

Laboratory analyses were carried out in triplicate in the laboratories in the Institute of Food Technology and the Institute of Food Science of the Faculty of Agricultural and Food Sciences and Environmental Management of the University of Debrecen. The test were performed according to the relevant standards and methods (Table 2).

4.2.1. Total polyphenol content

In terms of the total polyphenol content of medallions enriched with brewer’s spent grain, higher values were recorded in each case compared to the control sample (Figure 4). The medallion enriched with light malt at a concentration of 50% (LM 50%) had the highest total polyphenol content of 85.17 mg GAE/100 g. Of the raw materials, the test was also performed on the light and dark malt. Dark malt had a higher total polyphenol content (132.18 mg GAE/100 g) than light malt (102.22 mg GAE/100 g).

MSZ: Hungarian Standard; MÉ: Codex Alimentarius Hungaricus

4.2.2. Flavonoid content

Regarding the flavonoid content of the medallions, it was found that enrichment with brewer’s spent grain resulted in an increase in the flavonoid content. Compared to the control sample, higher values were observed in this case (Figure 5). DM 50% medallion enriched with brewer’s spent grain had the highest flavonoid content, with a value of 27.32 mg CE/100 g.

4.2.3. Dry matter content, moisture content

In the case of the dry matter content (Figure 6), only the medallion with the code DM 10% had a higher value, 93.52%, compared to the control sample. We found that in the case of samples made from light and dark malt, the products enriched with smaller amounts of brewer’s spent grain had a higher dry matter content. With respect to the average of dry matter content values, higher values were measured in the samples enriched with dark malt, but the difference of only a few tenths of a percent did not prove to be significant.

4.2.4. Crude protein content

In terms of protein content (Figure 7), higher values were obtained for all of our enriched products compared to the control sample. Medallion with the code LM 50% had the highest protein content (13.04%). The average protein content of the products enriched with light malt, 11.88%, was higher than the average of the medallions enriched with dark malt (11.56%).

4.2.5. Fat content

During the examination of the fat content, higher values were measured in all cases compared to the control sample. As the enrichment concentration increased, the fat content of the medallions increased as well, both in the case of samples enriched with light malt and dark malt (Figure 8). The average value of the products with different light malt enrichment was 21.15%, while in the case of dark malt, the value was 23.76%. For all products enriched with dark malt, a higher fat content was measured compared to the products enriched with light malt (LM 10% - 19.55%; LM 25% - 20.49%; LM 50% - 23.4% and DM 10% - 19.7%; DM 25% - 23.72%; DM 50% - 27.87%).

4.2.6. Carbohydrate content

Of the data for total carbohydrate content (Figure 9), the highest value, 57.7%, was obtained for the control medallion, of which sugar accounted for 0.7%. This characteristic was found to be 57.23% for the sample coded DM 10%. It was true for all products enriched either with light or dark malt that the carbohydrate content decreased with the increasing rate of enrichment. Sample LM 50% had the highest sugar content of 2.62%.

4.2.7. Dietary fiber content

The dietary fiber content of the medallions was higher than that of the control medallion without enrichment for all enriched products (values ranged from 10 to 40%). The dietary fiber content increased with the rate of enrichment for the medallions enriched with both types of malt, however, the values of LM 10% (17.4%) and LM 25% (19.2%), as well as those of DM 10% (15.6%) and DM 25% (18.5%) were similar to each other, as opposed to the medallions enriched with 50% malt. The highest value was obtained for medallion DM 50% (38.9%), followed by the dietary fiber content of sample LM 50% (27.9%). The outstanding value is almost double of the value of the control sample (Figure 10).

4.2.8. Common salt content

When measuring the salt content of each medallion (Figure 11), the highest value was obtained for the control medallion (2.5%). This was followed by the products with an enrichment of 10% (LM 10% 2.28% and DM 10% 2.36%), then the products with an enrichment of 25% (LM 25% and DM 25%), and finally the medallions with an enrichment of 50% (samples LM 50% and DM 50%). The medallions enriched with dark malt always exhibited higher values (2.36%; 1.73%; 1.41%) than their light counterparts (2.28%, 1.52%, 1.18%).

4.2.9. Energy content

During the study, the energy content of the medallions was also determined (Figure 12). The energy content value of the control medallion (1984 kJ/100 g, 474 kcal/100 g) was exceeded in all cases by the enriched medallions. The medallion enriched with 50% dark malt had the highest energy content of 2324 kJ/100 g (555 kcal/100 g). In terms of energy content, the data were almost identical, showing only small differences compared to the control sample and also to each other.

4.2.10. Organoleptic analysis

In April 2019, 20 judges were asked to evaluate, by tasting and completing a questionnaire, the following four organoleptic characteristics: appearance, smell, taste, texture. They were able to express their opinions using a scale from 1 to 5, where 1 meant very bad and 5 meant delicious.

As a result of the sensory examinations, it was found that enrichment with brewer’s spent grain deteriorated the properties of the products in all cases (Figure 13). Irrespective of the malt type, there was only a slight difference between the 10% and 25% enrichments, while the 50% enrichment resulted in a large decrease. All parameters of the products with 10 and 25% enrichment with light malt fell into the good category (with values above 4.0), so we definitely would like to continue our research with these two products.

5. Summary and recommendations

In terms of the total polyphenol, flavonoid, protein, fat, dietary fiber and energy content, higher values were measured in all cases compared to the control sample, whereas a decrease was found for three of the parameters analyzed: dry matter, carbohydrate and common salt content. This effect can be considered advantageous in the case if parameters with reduced values, especially because of the reduced carbohydrate content, while among the chemical components with increased values, the increase in fiber content is particularly important. It is possible to introduce the utilization of brewer’s spent grain in the baking industry, and the enrichment of wheat flour medallions with brewer’s spent grain had a positive effect on their nutritional values. However, as a result of the enrichment, based on the data of organoleptic analysis, a certain unfavorable change in the properties of the medallions (apeearance, smell, taste, texture) could be observed, but the results of the enrichment with light brewer’s spent grain show that an edible product can be prepared by further developments aimed at improving the organoleptic properties.

6. References

[1] Agrocrop Kft. (2013): Sörtörköly. http://agrocropkft.com/soripari-mellektermekek/sortorkoly/ (Hozzáférés: 2019. 10. 29.)

[2] Alexa L., Kántor A., Kovács B., Czipa N. (2018): Determination of micro and trace elements of commercial beers. Journal of Microbiology, Biotechnology and Food Sciences. 7 (4) pp. 432-436. DOI: https://doi.org/10.15414/jmbfs.2018.7.4.432-436

[3] Arendt, E. K., Moroni, A., Zannini, E. (2011): Medical nutrition therapy: Use of sourdough lactic acid bacteria as a cell factory for delivering functional biomolecules and food ingredients in gluten free bread, Microbial Cell Factories 10 (1) S15 DOI: https://doi.org/10.1186/1475-2859-10-S1-S15

[4] Baloghné Nyakas A. (2013): Mezőgazdasági növénytan alapjai. Debreceni Egyetemi Kiadó, Debrecen. pp. 223

[5] Ciosek, A., Nagy V., Szczepanik, O., Fulara, K., Poreda, A. (2019): Wpływ nachmielenia brzeczki na bakterie kwasu mlekowego (The Effect of Wort Hopping on Lactic Acid Bacteria). Przemysl Fermentacyjny i Owocowo-Warzywny (Fermentation- and Fruit- & Vegetable Processing Industry) 12/2019 pp. 4-8. DOI: http://dx.doi.org/10.15199/64.2019.12.1

[6] Czipa N. (2014): Élelmiszeranalitika gyakorlati jegyzet. Debreceni Egyetem Élelmiszertudományi Intézet, Debrecen. pp. 68.

[7] Csapó J., Albert Cs. (2018): Funkcionális élelmiszerek. Scientia Kiadó, Kolozsvár. pp. 282

[8] Csapó J., Csapóné Kiss Zs. (2003): Élelmiszer-kémia. Mezőgazda Kiadó, Budapest. pp. 468

[9] Horváth P. (2007): Táplálkozástan. Képzőművészeti Kiadó, Budapest. pp. 195

[10] Jackson, M. (2007): Eyewitness Companions Beer. Dorling Kindersley Publishers Ltd, London. pp. 288

[11] Jankóné J. (2006): Élelmiszeripari technológiák. Jegyzet, Szeged. pp. 240

[12] Jayant, M., Hassan, M. A., Srivastava, P. P., Meena, D. K., Kumar, P., Wagde, M. S. (2018): Brewer’s spent grains (BSGs) as feedstuff for striped catfish, Pangasianodon hypophthalmus fingerlings: An approach to transform waste into wealth. Journal of Cleaner Production 199 pp. 716-722 DOI: https://doi.org/10.1016/j.jclepro.2018.07.213

[13] Kaur, V. I., Saxena, P. K. (2004): Incorporation of brewery waste in supplementary feed and its impact on growth in some carps. Bioresource Technology 91 (1) pp. 101-104 DOI: https://doi.org/10.1016/s0960-8524(03)00073-7

[14] Kovácsné Kalmár K. (2012): Sütőipari termékelőállítás. Nemzeti Agrárszaktanácsadási. Képzési és Vidékfejlesztési Intézet, Budapest. pp. 356

[15] Lakatos E. (2013): Élelmiszeripari technológiák I. Malom-, Sütő- és Édesipar. Palatia Nyomda és Kiadó Kft., Mosonmagyaróvár. pp. 118

[16] Lásztity R., Törley D. (1987): Élelmiszer Analitika Elméleti alapjai I. 3.7.2.3. fejezet – Szénhidrát (m/m) %, fenolkénsavas módszer pp. 620

[17] Magyar Élelmiszerkönyv Bizottság: Magyar Élelmiszerkönyv (MÉ) 1-3/16-1 számú előírás a sütőipari termékekről

[18] Magyar Élelmiszerkönyv Bizottság: Magyar Élelmiszerkönyv (MÉ) 3-2-2008/1. sz. irányelv 1. sz. melléklet – Élelmi rost (m/m) %, enzimes hidrolízis

[19] Magyar Szabványügyi Testület (MSzT) (2007): Fehérje (m/m) %, Kjeldahl módszer. Magyar Szabvány MSZ 20501-1:2007 7. fejezet. Magyar Szabványügyi Testület, Budapest.

[20] Magyar Szabványügyi Testület (MSzT) (2007): Konyhasó (m/m) %, titrálás, Mohr szerint. Magyar Szabvány MSZ 20501-1:2007 3.2. szakasz. Magyar Szabványügyi Testület, Budapest.

[21] Magyar Szabványügyi Testület (MSzT) (2018): Sütőipari termékek vizsgálati módszerei. 2. rész: Kenyerek és vajaskifli érzékszervi vizsgálata. Magyar Szabvány MSZ 20501-2:2018 Magyar Szabványügyi Testület, Budapest.

[22] Magyar Szabványügyi Testület (MSzT) (2007): Szárazanyag (m/m) %, tömegmérés. Magyar Szabvány MSZ 20501-1:2007 2. fejezet. Magyar Szabványügyi Testület, Budapest.

[23] Magyar Szabványügyi Testület (MSzT) (2007): Szénhidrát tartalomból cukor (m/m) %, titrálás Bertrand szerint. Magyar Szabvány MSZ 20501-1:2007 8.1 szakasz. Magyar Szabványügyi Testület, Budapest.

[24] Magyar Szabványügyi Testület (MSzT) (2007): Zsírtartalom (m/m) %, extrakció, tömegmérés. Magyar Szabvány MSZ 20501-1:2007 4. 1. szakasz. Magyar Szabványügyi Testület, Budapest.

[25] Mahmood, A. S. N., Brammer, J. G., Hornung, A., Steele, A., Poulston, S. (2013): The intermediate pyrolysis and catalytic steam reforming of Brewers spent grain. Journal of Analitycal and Applied Pyrolysis 103 pp. 328-342 DOI: https://doi.org/10.1016/j.jaap.2012.09.009

[26] Nagy V. (2019): Sörgyártás alapanyagainak és melléktermékének hasznosítási lehetőségei a sütőiparban. Harmadik SÁNTHA-FÜZET. A 2018/2019-es tanév Tudományos Kerekasztal előadásainak absztraktkötete. Debreceni Egyetem, Debrecen. pp. 123-124

[27] Pedrotti, W. (2008): Gabonafélék: Legfőbb energiaforrásaink. Kossuth Kiadó, Budapest. pp. 125

[28] Pollhamer E. (2001): Táplálkozzunk egészségesebben, gabona alapú termékekkel. Szaktudás Kiadó Ház, Budapest. pp. 107

[29] Poreda, A., Zdaniewicz, M. (2018): Advances in brewing and malting technology. Uniwersytet Rolniczy im. Hugona Kollataja w Krakowie, Kraków. pp. 453

[30] Rigó J. (2007): Dietetika. Medicina Könyvkiadó Zrt., Budapest. pp. 328

[31] Rodler I. (2006): Élelmiszercélok. Az egészséges táplálkozás ajánlásai. pp. 73-76. In: Új tápanyagtáblázat. (Szerk. RODLER I. – ZAJKÁS G.) Medicina Könyvkiadó Zrt., Budapest.

[32] Rodler I. (2008): Élelmezés- és táplálkozás-egészségtan. Medicina Könyvkiadó Zrt. Budapest. pp. 548

[33] Schmidth J. (2003): A takarmányozás alapjai. Mezőgazda Kiadó, Budapest. pp. 452

[34] Shen, Y., Abeynayake, R., Sun, X., Ran, T., Li, J., Chen, L., Yang, W. (2019): Feed nutritional value of brewers’ spent grain residue resulting from protease aided protein removal. Journal of Animal Science and Biotechnology 10 (78) pp. 1-10 DOI: https://doi.org/10.1186/s40104-019-0382-1

[35] Szabó S. (1998): Söripari technológia. Agrárszakoktatási Intézet, Budapest. pp. 288

[36] Tanács L. (2005): Élelmiszer-ipari nyersanyagismeret. Szaktudás Kiadó Ház, Budapest. pp. 387

[37] Tarko, T., Jankowska, P., Duda-Chodak, A., Kostrz, M. (2018): Value of some selected cereals and pseudocereals for beer production. In: Advances in brewing and malting technology. (Edited by Poreda, A., Zdaniewicz, M.) Uniwersytet Rolniczy im. Hugona Kollataja w Krakowie, Kraków. pp. 303-319

[38] Tóth N., Murányi I., Bódi Z. (2009): Az árpa söripari tulajdonságainak vizsgálata. Növénytermelés. (Szerk. NAGY J.) 58. (1) pp. 93-111. DOI: https://doi.org/10.1556/novenyterm.58.2009.1.9

[39] Trummer, J. (2018): Grains usable for malting and brewing: A practical overview. In: Advances in brewing and malting technology. (Edited by Poreda, A., Zdaniewicz, M.) Uniwersytet Rolniczy im. Hugona Kollataja w Krakowie, Kraków. pp. 67-87.

[40] Vogel W. (2015): Házi sörfőzés. Mezőgazda Kiadó, Budapest. pp. 128

[41] Werli J. (2011): Sütőipari technológia II. VM Vidékfejlesztési, Képzési és Szaktanácsadási Intézet, Budapest. pp. 198

[42] 1169/2011/EU rendelet: Az Európai Parlament és a Tanács 1169/2011/EU rendelete (2011. október 25.) a fogyasztók élelmiszerekkel kapcsolatos tájékoztatásáról.