The ancient white wine making technology, the “qvevri”, is gaining more and more attention among consumers, not only because it is unique and special, but also because sustainability and closeness to nature are fundamental characteristics of this winemaking process. All of this is demonstrated by the fact that this ancient Georgian process using traditional clay vessels was added to the UNESCO List of Intangible Cultural Heritage of Humanity in 2013, and in 2020, The International Organisation of Vine and Wine (OIV) included skin-fermented white wine in the category of special wines. This wave is also present in Hungary, since “natural” wine and “orange wine” have already appeared in a 2021 law as „Other restricted terms”. The essence of the winemaking process is skin-contact fermentation and microoxidation, for which a number of vessels can be used: amphoras or qvevris, ceramic eggs or spin barrels, as a function of which the chemical composition of the wines may vary, as well as the formation of the precursor compounds of the aroma components. In this study, natural wines produced in the Tokaj wine region, using amphoras and ceramic egg vessels were examined.
2. Introduction
Today, the philosophy of natural winemaking has grown into a movement, finding producers and consumers in many countries. According to their philosophy, winemaking society had never before used so many pesticides to protect grapes, so many winemaking aids and preservatives, as today, which is extremely harmful to both wildlife and flora, and this is not sustainable farming. It is necessary to return to the roots, to the winemaking practice of ancient times, when winemaking was an art and the wines produced this way had a soul, combining the spirit of the place of production with the artistic world of the winemaker. This is especially true for the world of amphora wines made in the South Caucasus [1].
The counterargument that often arises against these products is that, on the one hand, they are not microbiologically stable, since no technological operations are carried out that would reduce the amount of microorganisms entering from the grapes and proliferating in the must and wine and, on the other hand, there is no adequate plant protection activity in the grapes against pathogens (e.g., black rot) that lead to a changed chemical composition and may produce mycotoxins. Another factor of concern that only a limited amount of test results is available on the migration properties of the various storage vessels.
3. Literature review
3.1. The concept of natural wine, the peculiarities of its production
The roots of the natural winemaking movement can be traced back to 1978, when Marcel Lapierre and Julet Chauvet first made wines free of sulfur and additives in Beaujolais, France [2].
Commonly used names for natural wines include low-intervention wine, naked wine and raw wine, which refer to the rules used during their production.
In March 2020, a Charter formulating the regulation for natural wines and the official name „vin méthode nature” were adopted by the French ministry of Agriculture, the INAO (Institut national de l’origine et de la qualité, the National Institute of Origin and Quality) and the DGCCRF (Direction générale de la concurrence, de la consommation et de la répression des fraudes, the General Directorate for Competition Policy, Consumer Affairs and Fraud Control), together with the Association of Natural Wines.
3.1.1. Most important characteristics of natural wines:
Must be produced from grapes that are certified organic (EU or Nature&Progrés) or come from a vineyard that is at least in the second year of transition;
The grapes intended for winemaking may only be harvested by hand;
Only spontaneous fermentation processes may be used;
The use of additives is prohibited;
No modification of the composition of the grapes (increase in acid or alcohol content) is allowed;
No procedures classified as “rough” are allowed (e.g., filtration, tangential filtration, flash pasteurization, heat treatment, reverse osmosis);
Addition of sulfur before or during fermentation is prohibited;
Depending on the use of sulfur, producers can use two types of logos on the labels: „without added sulfur” or „less than 30 mg/l sulfur added”;
Lots that are not considered natural wines must be clearly distinguishable (differentiated labeling), thus avoiding consumer deception [2].
3.2. Skin-fermented white wines
A special category of natural wines is skin-fermented white wines, often called qvevri, amphora, amber or orange wines. As a result of changing trends, older, traditional styles are starting to appear among winemakers as well. The popularity of skin-fermented white wines is constantly increasing, similarly to the ever-increasing demand for natural wines. Additionally, orange wines represent a special category because, due to skin-contact fermentation, they simultaneously carry the flavors typical of white wines and the texture and tannins characteristic of red wines [3]. Consumers especially like it when the flavor of the wine is enriched with a special aroma range by the storage vessel, so more and more winemakers use ceramic eggs and amphoras. This technology has many followers in France, Portugal, the USA, Italy, Slovenia and Austria [4, 5, 6, 7, 8]. The most important distinguishing features are different color (from deep yellow to amber), increased polyphenol content [9, 10, 11], the formation of volatile compounds (vanilla, roasted peanuts, walnuts) [12, 7] and the appearance of mineral notes [13, 14].
The duration of contact with the skin plays a particularly important role not only during fermentation, but also during the subsequent maturation. A long contact time with the skin promotes the dissolution of both phenolic and mineral substances. Procyanidins and catechins, which are important from an oenological point of view, occur in the skin, the seeds and the stem, while simple phenols (caffeic acid, p-coumaric acid) are found in the highest concentration in the berry flesh. As a result of skin-contact soaking for as long as possible, increasing alcohol concentration and the continuously increasing temperature during fermentation, the proportion of tannins in the wine from the seeds also increases. This process may be related to the improved permeability and/or rupture of the cells that contain the phenolic substances. If the fermented new wine is kept on the skin for a longer time after the completion of the fermentation, tannins from the seeds become dominant in its composition and the proportion of polymeric pigments increases [15, 16]. Several research have been published on the effect of the place of production [17], the grape variety [18] and the grapevine load [19] on the phenolic composition of musts and wines.
Among polyphenols, quercetin and shikimic acid are of outstanding importance. Quercetin is found in amounts of 10 to 20 mg/l and shikimic acid in amounts of 30 to 50 mg/l in white wines. The head of the Wine and Health Committee of the International Organisation of Vine and Wine drew attention to this after it had become known that these two compounds are the main active ingredients of the drug Tamiflu, which is used as an antidote to avian flu and is made from Chinese star anise extract. This was another argument for the beneficial effect of white wine consumption [20].
3.3. Special storage vessels
3.3.1. Amphora
They are made in many places all around the world, each master potter uses a unique process and raw material, and the shapes are often different. In Hungary, the works of a domestic potter are the most widespread, the raw material of his amphoras is a fire-resistant material, which is made thinner with chamotte made from its own material. They are solid, with a shell-like fracture surface, the raw materials are fire-resistant clays that burn to color and which, after firing at 1,200 to 1,250 °C, turn into pots resistant to acids and alkalis, with a water absorption of less than 4% (Figure 1).
As opposed to metal containers, microoxidation takes place in the amphora;
While wooden barrels leave a strong mark on the aroma and taste of the wines, the character of the grape variety and the terroir prevails in the amphoras;
In the amphoras, the specific characteristics of the grape variety which are otherwise covered by conventional winemaking processes (e.g., the herbal flavor of the Furmint grape variety) become more prominent;
Terracotta amphoras are made from minerals that are similar in composition to that of the vine soil, and which are absorbed by the vines during their life, which means that during fermentation and maturation the grapes end up in a chemical environment similar to the one they were in while on the vine; making wine in amphoras thus enhances the mineral notes in the wines;
The effective thermal insulation of the amphora continuously ensures that the fermentation process takes place under balanced temperature conditions.
3.3.2. Ceramic egg
The ceramic egg is an egg-shaped vessel based on a cement material that is widespread in Australia. An Australian company that sells its products for wine fermentation and storage worldwide has gained a good reputation among ceramic egg manufacturers. The Australian vessels have a wall thickness of 11-12 mm, a volume of 675 liters and a tare weight of 180 kg. They are fired at 1,285 oC for 42 hours, which ensures the special microporous structure of the vessel’s wall. The shape of the inverted egg ensures a special material flow, which guarantees the beneficial mixing of the fermenting must stored in it (Figure 2).
Figure 2. Ceramic egg in a winery in Tállya (Source: own photo)
4. Materials and methods
4.1. Comparative analysis of natural wines of the same vintage when using ceramic eggs and clay amphoras
Table 1 contains the data on the origin of the examined wines. In the winery operating in Tállya, natural winemaking technology is used for the preparation of the wines. The grape growing areas are located on the border of Tállya and Mád in eight vineyards, with Furmint and Hárslevelű varieties cultivated in integrated farming. They strive to use as little pesticides as possible and use no absorbable active ingredients at all. Their wines undergo spontaneous fermentation, no wine processing agents are used, and the wines are made and bottles without sulfur. For fermentation, the Australian ceramic eggs described above are used.
Furmint wine was made from organic grapes in a winery in Bodrogkeresztúr. Fermentation was carried out in a black clay amphora from Hungary (Figure 3).
Figure 3. Anthracite amphora in a Tokaj winery (Source: own photo)
One of the characteristic white grape varieties of the Savoie wine region in France is Roussette de Savoie (named after the French word for „rust”), which shows many similarities with the Furmint grape variety in terms of its ampelographic properties. Genetic tests have not confirmed the familial relationship, but in recent years the Altesse variety has appeared all over Europe in various wine regions famous for their sweet wines. The raw material which was processed at the Tokaj Wine Region’s Research Institute for Viticulture and Oenology and fermented in a clay amphora comes from the Lencsés vineyard in Tokaj.
Table 1. Origin of the wine samples used in the analysis
The chemical composition was examined with large instrument analysis (NMR - Nuclear Magnetic Resonance) in the Szerencs laboratory of Diagnosticum Zrt.
H NMR technique [22]: H NMR spectra were recorded at 26.85 °C with a Bruker AVANCE 400 spectrometer and a 400’54 ASCEND magnet system (Bruker, Karlsruhe, Germany) in proton NMR mode at a frequency of 400.13 MHz. For targeted analysis, sample preparation and analytical parameters were as follows: pH adjustment to pH 3.1 with an automatic BTPH system, addition of deuterium and tetramethylsilane, relaxation delay 4 s, sampling time 3.98 s, spectral width: 8223.68 Hz.
For the statistical analysis of the data, MANOVA and independence tests and IBM Corp. 2016 SPSS Statistics for Windows, Version 23.0. Armonk, NY (USA) software were used.
5. Analytical results
5.1. NMR analysis of natural wines made in ceramic eggs and amphoras
The results are shown in Table 2.
Table 2. Chemical composition of the wine samples and the data of the relevant NMR reference database compared to white wines produced in a conventional way
In comparison with the analytical values of the white wines included in the database of Bruker BioSpin GmbH and made with the normal white wine making process, it can be stated that the examined skin-fermented white wines had a lower content of tartaric acid and a higher content of citric acid, galacturonic acid, succinic acid and caftaric acid. Tartaric acid, malic acid and citric acid come from the grapes, while galacturonic acid and succinic acid are formed during fermentation. The results show that by the end of the fermentation, a greater part of tartaric acid is removed in the form of tartar than in the case of a normal white wine, and malic acid can also break down due to the presence of the natural lactic acid bacterial flora. Shikimic acid, to which a beneficial physiological effect is attributed, turned out to be characteristic of the variety, because a significant concentration difference compared to the other wine samples could only be measured in the case of the Altesse amphora wine. Caftaric acid (caffeoyltartaric acid) is a derivative of hydroxycinnamic acid and the ester of caffeic acid and tartaric acid, and is one of the most important phenolic compounds in the flesh of the grape berry. As a result of prolonged soaking and fermentation on the skins, higher values can be detected in skin-fermented white wines compared to normal white wines, with five times higher values measured in ceramic eggs. If reduced glutathione (GSH) is present in the must, caftaric acid-ortho-quinone reacts with this first, forming 2-glutathionylcaftaric acid (grape reaction product, GRP). GRP is colorless, does not react with polyphenol oxidase and no browning occurs.
Comparing amphora and ceramic egg wines using NMR analysis and the MANOVA statistical method, the following findings can be presented:
Measurement data from the individual wine samples, which apparently show no difference, have been omitted. The other parameters were evaluated by group, since one of the conditions of MANOVA is that the number of variables examined together cannot be higher than the number of observations (that is, more than 3, because this was the number of observations per vessel type).
In addition, however, the variables met the other conditions of multivariate analysis of variance: the residues are normally distributed and their standard deviation is homogeneous with two exceptions where it is slightly affected: in the case of fumaric acid and methylbutanol. There are no extremes or outliers in one dimension (there is a suitable exchange in 4 cases) and, based on the Mahalanobis distance, in several dimensions, there is no multicollinearity between the final groups, however, due to multicollinearity, fumaric acid, galacturonic acid and 2-methylpropanol were not examined separately, because it would not have given a new, evaluable result compared to the other variables examined in the given group.
No differences were found in the quantity of monovalent, non-higher alcohols (ethanol, methanol) depending on the storage vessel type (F(2;3)=2.681; p=0.641).
In the case of organic acid content of grape origin (tartaric acid, malic acid, citric acid), when examined together, there is no significant difference between the wines by storage vessel type (F(2;3)=6.856; p=0.130). However, when looking at tartaric acid (F(2;3)=23.115; p<0.05) or malic acid (F(2;3)=36.914; p<0.05) alone, there is a difference: wines stored in ceramic eggs have a higher tartaric acid content and a lower malic acid content compared to amphora batches.
In the case of organic acids formed during fermentation (lactic acid, acetic acid, succinic acid), when examined together, there is no significant difference between the wines by storage vessel type (F(2;3)=2.064; p=0.343). However, when looking at lactic acid (F(2;3)=11.755; p<0.05) or succinic acid (F(2;3)=10.814; p<0.05) alone, there is a difference: wines stored in ceramic eggs have a lower content of lactic acid and succinic acid compared to amphora batches. When examined outside of the model, the amount of fumaric acid does not differ (t(4)=4.303; p=0.238), while the amount of galacturonic acid differs (t(4)=4.303; p<0.05) by storage vessel type, it being lower in the case of ceramic eggs.
Regarding fermentation byproducts (acetoin, acetaldehyde), a significant difference was found when examining the factors together (F(2;3)=36.718; p<0.05). The acetaldehyde content was found to be lower in the ceramic egg (F(2;3)=36.718; p<0.05). The same can be said for the amount of acetoin, which was close to the significance limit (F(2;3)=6.852; p=0.059).
When higher alcohols (2,3-butanediol, 2-phenylethanol, 3-methylbutanol) were examined together, there was no difference (F(2;3)=6.826; p=0.130), while when butanediol was examined independently, the result was close to the significance limit (F(2;3)=7.383; p=0.053), it being lower in ceramic eggs.
When polyphenols (shikimic acid, trigonelline, caftaric acid) were examined together, no significant differences were detected (F(2;3)=13.,606; p=0.069), but the amount of caftaric acid was significantly higher in ceramic eggs, if the values were assessed individually (F(2;3)=36.977; p<0.05).
A statistically verifiable difference was found in the amount of proline based on an independence test, it being lower in ceramic eggs (t(4)=2.770; p<0.05). It is characteristic of free amino acids that proline is present in wines in almost 50%, the proportion of arginine is 10%, this ratio remains the same in amphora wines, but in ceramic eggs the proportion typical of Tokaj wines (30-25%) can be observed [23].
6. Conclusions
Natural winemaking technology is the representation in wine of an approach that demonstrates, on the one hand, the close-to-nature dedication of its maker, and on the other hand, the imprint of the characteristics of the vineyard soil. Hygiene plays a very important role, without which the use of a chemical-free technology becomes impossible. The insistence on naturalness and sustainability can justify trying out the possibilities offered by different storage vessels and endows the wines produced in this way with added value. Each storage vessel adds to and shapes the chemical composition of the wine. They can also be important factors in market positioning, not only because they are special and unique, but also because the ideological values associated with them (the grape harvest, separated from mother earth, can complete its life journey of becoming wine in a similar environment) can endow these types of wine with a distinctive character.
[4] Mandal, K. (2010): Genetische Charakterisierung von Wildhefe-Referenzstämmen mit geeigneten Markern. Wissensbericht 2010. Klosterneuburg, Austria, Institut für Weinbau Klosterneuburg:235-236.
[7] Martins,N., Garcia, R., Mendes, D., Costa Freitas, A.M., da Silva, M.G., Cabrita, M.J. (2018): An ancient winemaking technology: Exploring the volatile composition of amphora wines. LWT 96 288-295.
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[10] Rossetti, F. & Boselli, E. (2017): Effects of in-amphorae winemaking on the chemical and sensory profile of Chardonnay wine. Scientia Agriculturae Bohemica, 48 (1) 39-46.
[11] Bene ZS. & Kállay M. (2019): Polyphenol contents of skin-contact fermented white wines. Acta Alimentaria 48 515-524.
[12] Baiano, a., Mentana, A., Quinto, m., Centonze, D., Longobardi, F., Ventrella A., Agostiano, A., Varva, G., De Gianni, A., Terracone, C. (2015): The effect of in-amphorae aging on oenological parameters, phenolic profile and volatile composition of Minutolo white wine. Food Res. Int. 74 294-305.
[13] Diaz, C., Laurie, V.F., Molina, A.-M., Bücking, M. & Fisher, R. (2013): Characterization of selected organic and mineral components of kvevri wines. Am. J.Enol.Vitic. 64 532-537.
[14] Diaz, C. (2014): Investigation of traditional winemaking methods with a focus on spontaneous fermentation and the impact on aroma. Doktorin dissertation, RWTH Aachen University, Aachen, Németország
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[16] Bene ZS. & Kállay M. (2018): A szőlő fenolos vegyületeinek borokra gyakorolt hatása a héjonerjesztés során. In: szerk. Dankó L.: Narancsbor-Fejezetek a gasztronómiai újdonságok témaköréből. Bodrogkeresztúr. Tokajbor-Bene Kft. Kiadó. pp.18-25.
[17] Gambelli, L.& Santaroni, G.P. (2004) Polyphenols content in some Italian red wines of different geographical origins. Journal of Food Composition and Analysis. 17 (5) 613–618.
[18] Landrault, N., Poucheret, P., Ravel, P., Gasc, F., Cros, G., Teissedre, P.L. (2001): Antioxidant capacities and phenolics levels of french wines from different varieties and vintages. J. Agric. Food Chem. 49 (7) 3341–3348.
[19] Leskó, A. (2011): A tőketerhelés hatása a szőlőbogyó, a must és a bor összetételére. PhD-értekezés, BCE, Budapest
[21] Légli A. (2015): A Légli Kőagyag Amfora. https://www.legli.hu/amfora (Hozzáférés: 27.12.2021)
[22] Godelmann, R., Fang, F., Humpfer, E., Schutz, B., Bansbach, M., Schafer, H., Spraul, M. (2013): Targeted and Nontargeted Wine Analysis by H-1 NMR Spectroscopy Combined with Multivariate Statistical Analysis. Differentiation of Important Parameters: Grape Variety, Geographical Origin, Year of Vintage. Journal of Agricultural and Food Chemistry 61 (23) 5610-5619.
[23] Csomós E. (2003): Magyar fehér- és vörösborok összehasonlító vizsgálata a szabad aminosav és a biogén amin tartalom alapján. PhD-értekezés, BMGE, Budapest
Flexitarians became the largest dietary group after omnivores, they play a significant role when it comes to effectively reducing the consumption of meat and other animal-derived products and thus in fighting climate change.
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. However, there are substantial differences in the proportion of consumers considering themselves and/or categorised as flexitarian. Furthermore, the lack of a definition or at least a wide consensus on what to be considered a flexitarian diet makes it even more difficult to estimate the size of this consumer group.
Why could the classification of flexitarianism still be useful and support a sustainable food consumption? Instead of following strict rules, strengthening consumers’ efforts to pursue a more sustainable diet according to their own intention (such as following a flexitarian eating pattern) may be more effective.
Different food consumption patterns are described in this article from omnivores via reducetarians, flexitarians, vegetarians to vegans, where possible definitions and data are provided on the proportion of consumers following such diet patters.
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.
Table 2. Consumption of certain food groups in different types of diets – with special regard to meat consumption
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 17th 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.
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1 South Ural State University (national research university), Chelyabinsk, Russian Federation 2 South Ural State Agrarian University, Troitsk, Russian Federation 3 LLC „Antey”
Keywords
adulteration, fruit and berry raw materials, chemical composition of fruits, organic acids profile, mineral elements.
1. Summary
We studied organoleptic, physical, chemical parameters, and nutrient composition of strawberry, raspberry, and melon powders and identified their profile of organic acids and mineral composition produced by a Russian company. It was found that the color and flavor ranges of the studied materials were uncharacteristic of the initial raw materials. The actual protein and lipids levels did not correspond to the ones declared by the manufacturer in the labeling, and were uncharacteristic of the processed raw materials. In all powder samples the sugars were represented by sucrose in 80-97%. This high level of sucrose content indicated the addition of 40.4-52.3% white sugar. The amount and ratio of organic acids did not correspond to the profile of natural raw materials. Thus, the strawberry powder lacked oxalic and tartaric acids, the raspberry raw material lacked malic acid, and the melon material – citric acid. The strawberry powder above the detection limit did not contain such essential macro- and microelements as Ca, Mg, B, Co, the amount of Si, Fe, K was at trace level. The raspberry powder was devoid of detectable amount of Co and K, and B, Ca, Cu, Mg, Mn, Si important for plant life were present in residual amounts. The “obligatory” amount of K, Fe, Ca, Co, Cu, Mg, Mn were absent in the melon powder, which did not correspond to the fundamental laws of the plant physiology. The results obtained allowed to conclude about misinformation and qualitative adulteration of the materials. Currently, there are practically no studies aimed at determining quality and chemical composition of fruit and berry powders in order to identify adulteration, though this type of survey would be great practical interest both for producers and consumers.
2. Introduction
The modern consumer market of edible raw materials and foods is extremely important strategic part of the modern economy of the Russian Federation. In recent years, the spread of adulterated goods there has reached such a level that it threatens Russia’s national security. Adulteration of agricultural raw materials should be regarded as one of the most dangerous types of fraudulent practices, because it creates favorable conditions for unfair competition, leading to stagnation, loss of export potential of domestic food producers and, consequently, to the decrease in the investment appeal of the industry.
Fresh juicy berries and fruits are natural sources of biologically active substances. However, these are seasonal, perishable products. So, to level the seasonal nature of consumption, increase the shelf life of the finished product and reduce the transportation and storage costs, they are often processed and dried [1, 2].
Strawberry (Fragaria x ananassa, D.) is known as a berry with high content of organic acids (citric, malic, quinic, salicylic, as well as succinic and traces of shikimic and glycolic upon ripening), vitamins C, PP, E, B1, B2, B6, B9, K, carotene, pectin and other substances. Strawberry is rich in phenolic compounds which have antioxidant, anti-inflammatory, and anticancer action [3, 4]. Ripe raspberry (Rubus іdaeus L.) contains free organic acids (citric, malic, salicylic), minerals (Co, Cu, K, Na, Fe, Ca, Mg, P) [1, 5], vitamins (B-group, PP, C, provitamin A), tanning substances [6]. Raspberry has diuretic, choleretic, anti-anemic effect, helps strengthen the walls of blood vessels and promotes intestinal health [14]. Melon fruits (Cucumis melo) contain proteins, carbohydrates (sugars, starch, fiber), organic acids, vitamins (B-group, PP, A, C, β-carotene), minerals (K, Na, Fe, Ca, Mn, Mg, Zn). Melon is especially recommended in case of exhaustion, anemia, atherosclerosis, and some other cardiovascular diseases. Melon enhances the effect of antibiotics reducing their toxicity [7].
Rich chemical composition of dried fruit and berry raw materials allows to use them in the production of dairy and baked goods, confectionery, snacks, salads, ketchups, seasonings in order to enrich them with vitamins, minerals, organic acids, fiber, etc. [8]. Knowing the chemical composition of fruit and berry raw materials, identifying components forming the organoleptic characteristics not only constitutes a prerequisite for the production of competitive products, but also makes it possible to identify adulteration. The purpose of the research was to assess the quality and to identify the chemical composition of fruit and berry powders. Research objectives were to study organoleptic properties, physical and chemical parameters, as well as nutrient composition of fruit and berry powders comparing them with commonly known data; to identify the profile of organic acids and mineral composition of the plant material under study.
3. Materials and methods
The investigated products were fruit powders of strawberry, raspberry and melon produced by a Russian company. According to the declaration of the manufacturer, the composition of these powders is 100% corresponding natural raw materials containing no preservatives, dyes, or artificial flavorings.
Organoleptic characteristics of the fruit powders were studied according to GOST 8756.1-2017. Moisture content was determined according to GOST 33977-2016, fat and protein content – according to MU 4237-86 guidelines, non-volatile acids – according to M 04-47-2012, sugars – according to M 04-69-2011, metal and foreign impurities, contamination with grain pests – according to GOST 15113.2-77, food fibers – using the generally accepted method [9], minerals – according to MUK 4.1.1482-03 and MUK 4.1.1483-03 guidelines. All measurements were carried out in three replications.
4. Results and discussion
Sensory evaluation of the quality of the studied materials showed the following: in appearance, the samples of processed strawberries, raspberries, and melons were finely ground homogeneous loose odorless powders, which is uncharacteristic of each type of the original natural raw material. The colour was identified as intense, uniform throughout the mass of the powders, uncharacteristic of dried products, with the following tones: pink with a gray hue for the strawberry powder, light burgundy for the raspberry powder, and light yellow for the melon powder. A sweet taste was noted in the strawberry and melon, and a sour taste in the raspberry material.
According to the results of physical and chemical study of plant materials, no deviations were found from the normal values. Thus, the moisture content of the powders under study was within the range of 4.2-5.1% (in various literature data, the range is 4-12% [1], no infestation with grain pests or presence of metallic and foreign impurities were found.
Fruits and berries have rich chemical composition, which makes them unique elements of a healthy diet [5]. In this regard, we investigated the main nutrients contained in the studied samples of fruit and berry powders.
To begin with, we compared the obtained test results with the information on the product packaging. We found that the actual levels of protein and lipids content did not correspond to the ones stated in the labeling, which indicates misinformation of the consumers. Thus, the amount of proteins and fats in the strawberry powder was 26 and 3.5 times lower, in the raspberry powder – 8 and 60 times higher, respectively, in the melon powder, contrary, it was slightly higher, as for protein in particular – by 55% (Table 1) than the labelling of the products.
Taking into account the fact that drying significantly increases the concentration of dry substances and, consequently, biologically active components [1, 2], it was determined that not all samples of the plant powders contained protein and fat even within the generally known range for fresh raw materials. For example, the amount of protein and lipids in the strawberry powder should be 7.0 g/100 g and 1.0 g/100 g, respectively [1]. The obtained results were far below.
Table 1. Nutrient Composition of Fruit and Berry Powders
Note: *content indicated on the packaging of fruit and berry powders, **in terms of dry matter. a Karkh et al., 2014, / b Akimov et al., 2020, / c Akimov et al., 2021, / d Sannikova, 2009, / e Erenova, 2010, / f Dulov, 2021, / g Pochitskaya et al., 2019, / h Baygarin et al., 2015, / i Medvedkov et al., 2015.
The most important indicator of the quality of fruits and berries is their sugar content, which depends on both the characteristics of a certain variety and weather conditions in the period of crop formation [5, 7]. It is known that for fresh raspberries, the content of sugars is 4-10 %, for dried berries - 34.5-42.2% [5]. Fresh strawberries contain 7.3-11.7% of sugars, which, as in raspberries, are represented mainly by fructose, glucose, and sucrose; their amount varies from 5.9 to 8.9 % [3, 4]. In the fruits of cultivated melon, the level of sugars is 7.0-21.0% [7, 10].
It was found that the ratio of mono- and disaccharides in the studied raw materials did not correspond to the data obtained by a number of scientists in practical studies [5, 6, 10, 11, 12, 13]. As for sugar content in strawberries, fructose should prevail significantly, in melon – sucrose, whereas in raspberries fructose and glucose content should be equivalent. It was revealed that in all samples of plant materials sugars were 80-97% represented by sucrose, and its high level indicated 40.4-52.3% addition of white sugar. In addition, the quantitative levels of monosaccharides in the strawberry powder did not even fall within the lower limits of their content established for fresh berries.
Plant material is distinguished first of all by the presence of dietary fiber, regular consumption of which contributes to the prevention of overweight and obesity, gastrointestinal, cancer, and cardiovascular diseases.
It was determined that by the content of dietary fiber, the studied samples of vegetable material were closer to the levels of characteristic of fresh juicy berries and fruits, since it is known, for example, that the amount of dietary fiber in dried chopped strawberries is not less than 8.0 g/100 g [5]. In our case the dietary fiber content of our samples were only 3.91±0.20 g/100 g.
It is well known that berry and fruit raw materials are characterized by a specific profile of organic acids and macronutrients, and the analysis of their content allows to determine adulteration or to prove its natural character [8]. So, these characteristics were studied in more detail. According to a number of authors, citric acid predominates in raspberry, while the content of malic acid is significantly lower. Salicylic acid in raspberries, which has bactericidal, antipyretic, and analgesic action, is of particular importance [5, 6]. Strawberries contain malic, benzoic, citric, tartaric, oxalic, succinic, and salicylic acids with the predominance of citric and malic ones [11]. Organic acids in cultivated varieties of melon are represented by malic and succinic acids, whereas citric and glucuronic acids appear during storage [10]. According to the test results, the amount and ratio of organic acids in the studied fruit powders did not correspond to the profile of natural raw materials (Table 2). Thus, oxalic and tartaric acids were absent in the strawberry powder, malic acid – in the raspberry raw material, and citric acid – in the melon material (their concentration stayed below the limit of detection).
Table 2. Profile of Organic Acids and Mineral Elements of Fruit and Berry Powders
Notes: *according to TR CU 021/2011, ** in terms of dry matter.
a Stepanov et al., 2013, / b Karkh et al., 2014, / c Akimov et al., 2020, / d Akimov et al., 2021, / e Sannikova, 2009, / f Erenova, 2010, / g Dulov, 2021, / h Pochitskaya et al., 2019 / i Medvedkov et al., 2015
Strawberries and raspberries are known to be rich in macro- and micronutrients. Thus, 100 g of strawberries cover 330% of the daily demand in Si, 264% in B, 40% in Co; 100 g of raspberries – 120% of the daily demand in Si, 250% in B [11]. Si is involved in the metabolism of most mineral elements and vitamins. It’s lack leads to the decrease of digestibility of Ca, Fe, Co, Mn and metabolic disturbance. B plays an important role in the prevention and treatment of bone disease.
Co is a coenzyme of many enzymes, it activates the metabolism of fats and synthesis of folic acid [11]. The berries also contain Fe, Zn, Mn, Cu, Mo etc. It was determined that the strawberry powder under study did not contain in detectable amount of intrinsic essential macro- and microelements, namely Ca, Mg, B, Co, the amount of Si, Fe, K was at the trace level, indicating that the material was not natural. The raspberry powder turned out to be devoid of Co, K, whereas the amount of B, Ca, Cu, Mg, Mn, Si, important for the plant life, was residual. The mineral composition of melon fruit includes K, Ca, Mg, P, Nа, Fe. K is of extreme importance in the mineral nutrition of melon. The higher level of potassium nutrition increases productivity, disease resistance, accumulation of ascorbic acid and sugars [15]. The content of Fe, which plays a leading role in the formation of red blood cells – carriers of oxygen – is 17 times higher in melon than in milk [16]. When testing the mineral profile of the melon powder, it was found that it lacked the plant physiologically “obligatory” amount of K, Fe, Ca, Co, Cu, Mg, Mn, which does not correspond to the fundamental laws of physiology of the plant itself. The results allowed us to conclude about the qualitative adulteration of this plant material.
5. Conclusions
The results of physical and chemical tests of the studied raw materials showed deviations from the norms. Studying the levels of proteins and fats of products of strawberry, raspberry, and melon powders confirmed the fact of adulteration. The data obtained during organoleptic evaluation of quality and identification of profile of sugars, organic acids, and mineral elements allowed us to conclude that the powders under study were not natural fruit and berry raw materials.
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. Thanks
The work was supported by Act 211 of the Government of the Russian Federation, contract No. 02.A03.21.0011.
8. References
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1 South Ural State Agrarian University, Troitsk, Russian Federation 2 South Ural State University (national research university), Chelyabinsk, Russian Federation
The article presents the results of studying the influence of the supplementary feeding with stinging nettle hay on the ration balance, biochemical indicators, nutritional value, and keeping quality of rabbit meat. It was established that the replacement of 5% and 25% of coarse fodder with stinging nettle hay resulted in an increase in the content of crude (by 3.5-20.3%), digestible protein (by 4.4-22.8%) and carotene (by 3.3-22.7%) in terms of nutritional value. Growing rabbits with the introduction of a dosage of 5% and 25% 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/day). 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 influence of -10,38%, P<0.001), an increase in the content of protein (the power of influence of 34.2%, P<0.01), zinc (the power of influence of 35.6%, P<0.01) and manganese (the power of influence of 34.2%, P<0.01) in the rabbit meat.
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].
Table 1. The chemical composition and nutritional value of stinging nettle hay fodders
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).
Table 2. The consumption of fodders by the animals during the experiment (day/animal)
* 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).
Table 3. The chemical composition of the muscle tissue of the rabbit meat (¯X±S¯x, n=10)
*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.
Table 4. The influence of the supplementary feeding with the stinging nettle hay on the chemical composition of the muscle tissue of the rabbit meat (n=10)
*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).
Table 5. Nutrition value of the rabbit meat ignoring perinephric fat, kJ/100 g
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.
Table 6. The mineral composition of the rabbit meat (¯X±S¯x, n=10)
*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.
Table 7. The influence of the supplementary feeding with the stinging nettle hay on the mineral composition of the rabbit meat (n=10)
*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.
Table 8. Amino acid composition of the rabbit meat, g/kg (¯X±S¯x, n=5)
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).
Table 9. The influence of the supplementary feeding with the stinging nettle hay on the amino acid composition of the rabbit meat (n=10)
*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.
Table 10. The amount of VFA and the fat acidity value of the rabbits (¯X±S¯x, n=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.
Table 11. The influence of the supplementary feeding with the stinging nettle hay on the rabbit meat freshness indicators (n=10)
*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.
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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
1. Summary
In our research, the aim was to examine consumer attitudes related to health foods, and these were analyzed in terms of physical activity and diet. Our studies were carried out in three stages. First, a netnographic analysis (a study of social interactions in the contemporary digital communication environment – Editor) was performed with data recorded in a search engine on the one hand and with the content analysis of posts and comments made in groups of publicly available social media sites on the other hand. The interest and its changes of consumers present in the online space were detected in the common subset of health-conscious eating and physical activity. While the number of hits shows a variable rate growth from year to year, the contetns are concentrated in relatively stable groups. Based on this, four main topics can be distinguished in the online space in the common subset of healthy eating and exercise:
Training plans with recipes,
Requests for recommendations,
Providing advice,
Motivational examples.
During teh second stage of our research, focus group interviews were conducted. The impact of regular exercise on the purchase and consumption of health foods was examined, and also the implications of this in developing and maintaining a diet perceived to be healthier by the consumers. 7 people were included in each study, based on preliminary criteria. The differentiating factor in joining the groups was the performance of regular physical activity, so an active and a passive group was formed. The identification of differences and characteristics was fundamental to the design of our quantitative research. During the third stage of our research, we were the first in Hungary to adapt the Consumer Style Inventory (CSI)1 test for health foods, the final version of which contains 25 items. In adition, differences in the way people transition to a healthy diet were examined. Based on the Eurobarometer survey, statements related to physical activity and sedentary lifestyle were formulated, which were classified as background variables in the analysis. The survey includes a gender-representative sample of 300 people. In our exploratory research, attitudes appearing in CSI were identified by principal component analysis, and then groups were formed by K-means cluster analysis. Based on this, four homogeneous consumer groups were identified in terms of attitudes towards health foods:
Uninterested,
Health-oriented,
Variety seekers,
Uncertain brand choosers.
Our results show that a sedentary lifestyle has no effect, while a diet considered healthy, as well as the regularity and duration of physical activity have significant effects on attitudes toward health foods.
1 A method designed for the measurement of consumer decision-making style
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.
Figure 1. Changes in nutrition-related keyword search results
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.
Figure 2. Search results for nutrition and sports terms
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.
Figure 3. Social media content on nutrition and physical activity
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.
Table 1. The most important features of a healthy lifestyle
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.
Table 2. The distribution of the sample according to the main background variablesTable 3. The distribution of the sample according to the other background variables
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.
Table 4. The evolution of switching to a nutrition considered healthier
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.
Table 5. Factor structure of the Consumer Style Inventory test
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.
Figure 4. Clusters formed from CSI value adapted for health foods, based on the factors developed
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.
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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
1. Summary
Today, food industry developments are driven by two megatrends: global warming and the need to address nutrition-related adverse health consequences (diseases of civilization, obesity, hunger and an aging society). As a result, consumer preferences have also changed, as „everyday” needs such as the acceptable price, pleasant taste and safe consumption of foods, as well as for the food to satisfy physiological needs, have become essential requirements and do not represent a demonstrable market advantage. The market presence of a product is expected to be successful if, in addition to the above, its ingredients and physiological effects can be demonstrated to improve or increase consumer well-being, their state of health or physical performance.
One of the fastest growing disciplines today is nanotechnology, which has many applications in the food industry. Even though this technology brings unprecedented benefits to consumers and may be able to solve many global problems, nanofoods also carry many risks and dangers. Although nanotechnology is still unknown to many, the willingness to buy is very high among those interviewed if the technology improves some of the properties of the food. Based on their attitudes, consumers can be divided into two well-distinguishable groups: those who see potential advantages and disadvantages in radically different ways.
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 21st 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%).
Figure 1. Aspects considered important when buying food (N=200)
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.
Figure 2. Knowledge of the four categories of food nanotechnology among respondents (N=200)
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.
Table 1. Willingness to buy food produced by nanotechnological development, taking into account certain aspects (N=200)
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.
Figure 3. Willingness to pay extra for foods made with nanotechnology (N=200)
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.
Figure 4. Probability of occurrence of potential risks of foods made by nanotechnology according to the respondents (N=107)
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.
Table 2. Order of listed categories of food nanotechnology by popularity among acceptors
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.
Table 3. Order of the listed categories of food nanotechnology according to preference before and after the description of the potential risks among the acceptors
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.
Table 4. The order of the listed categories of food nanotechnology based on consumer acceptance among rejectors
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.
Table 5. Order of listed categories of food nanotechnology according to acceptance before and after description of potential risks among rejectors
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.
[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
Packaging technology is one of today’s rapidly evolving disciplines, with innovative implications for many other disciplines, such as the food industry. Plastics can also be referred to as the materials of the 21st century, without which we could hardly imagine our lives today. 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. In my qualitative, online study, 513 people answered my questions about what the main function of packaging is, what characteristics a packaging material should possess, foods in which packaging are preferred, whether they had ever encountered environmentally friendly packaging materials. In addition to a lot of useful information, it turned out that Hungarian people are typically eco-conscious on paper, but in reality they do not pay enough attention to it. It is primarily college graduate women between the ages of 46 and 65 who also take environmental and ecological considerations into account when buying food.
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 21st 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 20th 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:
Packaging knowledge, opinions, habits related to food products;
The questionnaire was completed by the respondents between July 20 and 31, 2020.
For the completion, the following two methods were used:
Quota sampling, in which the population was divided into subgroups (based on age groups), and the elements were selected from these; followed by the
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.
Table 1. Number and distribution of research participants based on demographic data (N=513)
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.
Table 2. Extent of agreement with statements concerning the viability of packaging, and other related statistical indicators (N=513)
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.
Figure 1. Distribution of respondents based on their answers to Question 3 (%, N=513)
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).
Table 3. Distribution of research participants in view of their answers to Question 3, based on demographic criteria (%, N=513)
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.
Figure 2. Distribution of research participants based on their answer to Question 5 (%, N=513)
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.
Table 4. Main demographic characteristics of those choosing foods with degradable packaging (%, N=513)
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
Table 5. Average and other statistical indicators showing the importance of packaging parameters (N=513)
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.
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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
1. Summary
The results of studying the combined use of freeze-dried ground apples (in an amount of 7%) and Brazil nut kernels (in an amount of 5 %) in the technology of baked poultry products are presented. The modification of the recipe made it possible to obtain stuffed meat products with improved consumer properties (apple and nut notes in the smell, slight sourish-sweetish tone in the taste, caramel shades in the color) and increased nutritional value (content of dietary fiber, mineral elements Mo, Au, Cu, B, Mn, W, Be, Sn, Fe, Ca, Mg, P, organic acids, protein) alongside a decrease in the amount of butter by 4%.
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.
Table 1. Recipe for Laboratory Samples of Chicken Pockets
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.
Table 2. Nutrient Composition of Materials under Study
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.
Table 3. Mineral Composition of Materials Under Study
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.
Table 4. Nutrient Composition of Laboratory Samples of Chicken Pockets
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.
Figure 1. Macroelement Composition of Laboratory Samples of Chicken PocketsFigure 2. Microelement Composition of Laboratory Samples of Chicken Pockets
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
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[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.
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1 Ministry of Agriculture, Department of Food Economics and Quality Policy 2 Hungarian University of Agriculture and Life Sciences 3 National Institute of Pharmacy and Nutrition 4 University of Veterinary Medicine 5 National Food Chain Safety Office, Directorate for Risk Management
Keywords
Food labeling, nutrition labeling, voluntary labeling, mandatory labeling, Codex Alimentarius Commission, harmonization of food law, Big 8, Big 4, Traffic light, Battery, Nordic Keyhole, Nutri Score, GDA (Guideline Daily Amount)
1. Summary
Food labeling is one of the most diverse areas of food law, and special attention is paid to nutrition labeling within this area. This is not a coincidence, as modern nutrition science is evolving year by year, and legal changes must also keep pace with this. Nutrition labeling is particularly important for those who struggle with obesity or certain metabolic diseases or have special nutritional needs for other reasons. In a somewhat unusual way, the regulation of nutrition labeling has not appeared primarily in regulations at the national level, but its development began within an international framework, with the first breakthrough being the Codex Alimentarius and the expert work carried out within it. Hungary has been participating in this work since the beginning, so the Hungarian regulation, regardless of historical periods, has been relatively harmonized with the current best labeling practices in the world, with complete harmonization taking place by the time Hungary was on the verge of joining the European Union. In this study, we look back at the most important international, EU and Hungarian steps in the development of the regulation, not only presenting legal changes, but also comparing them to the changing requirements of the various periods. In addition to the current regulatory environment and challenges for nutrition labeling, key voluntary labeling schemes are also included in this communication.
2. Introduction
At the international level, the cornerstone of nutrition labeling decree was laid in 1985 by the Codex Alimentarius established by the FAO/WHO, in the form of a guide to nutrition labeling. Nutrition labeling decree was based both in the European Union and Hungary on the Codex Alimentarius (Figure 1).
Figure 1. Regulatory relationships of the nutrition labeling of foods
* MÉM-SZEM: former Ministry of Agriculture and Food – former Ministry of Social Affairs and Health
** MÉ: Codex Alimentarius Hungaricus
*** FM-NM-IKM: former Ministry of Agriculture – former Ministry of Public Health – former Ministry of Industry and Trade
Nutrition labeling was first regulated by the Council of the European Community in 1990 with the adoption of Directive 90/496/EEC. Compliance with the directive was voluntary and it applied to all foods intended for normal public consumption (Figure 2).
In Hungary, indicating the “essential” elements of nutrition labeling of foods was voluntary until the mid-1970s and 1980s, then, from 1988, indication of the energy content became mandatory. From 1996, rules for the nutrition labeling of foods had been defined by the Hungarian Food Codex (Codex Alimetarius Hungaricus), with a definite content but still on a voluntary basis [1], and this remained in force until December 13, 2014.
Prior to December 13, 2014, nutrition labeling was a mandatory element on the packaging only if the manufacturer, using today’s regulatory terminology, made a nutrition or health claim, or such a claim was published in relation to the product, or if it was a food for people with special nutritional needs (e.g., baby food) [2].
In the meantime, more and more countries have introduced mandatory nutrition labeling at the international level, mainly for public health purposes, in order to reduce obesity and to prevent certain chronic diseases [3]. Recognizing the growing public interest in the link between the diet and health [4] and also because solutions were needed to the health challenges related to overweightness and obesity [5,6], it has become clear that the creation of harmonized rules at the EU level was urgent and essential to ensure adequate consumer information. In light of this, Regulation (EU) No 1169/2011 on the provision of food information to consumers was adopted, which defines the general principles, requirements and obligations for the labeling of foods, and also makes it mandatory to indicate the nutrient content of foods. The primary purpose of nutrition labeling is to provide information to consumers about the nutritional composition of foods, helping them to make informed decisions [7].
Figure 2. Chronological summary of laws governing labeling of nutrition value
* MÉM: former Ministry of Agriculture and Food
** MÉM-SZEM: former Ministry of Agriculture and Food – former Ministry of Social Affairs and Health
*** FM-NM-IKM: former Ministry of Agriculture – former Ministry of Public Health – former Ministry of Industry and Trade
Of course, food labeling alone is not enough. In order for information to achieve its purpose, it is also necessary to motivate consumers and for them to know the principles of good nutrition. Education and informing consumers for educational purposes are indispensable for consumers to better understand food information and thus incorporate the given foods correctly into their own diets [8, 9, 10, 11, 12, 13, 14].
In this article, the development of the European Union and Hungarian regulations regarding the nutrition labeling of foods intended for normal public consumption are described, as well as the related practices and experiences. Due to the complexity of the topic, laws on foods for special dietary uses and on foods containing claims are not discussed in detail in this publication.
3. Nutrition labeling at the international level (Codex Alimentarius)
3.1. Operation and purpose of the Codex Alimentarius Commission
The main purpose of the Codex Alimentarius Commission (hereinafter referred to as the: Codex), operating within the framework of the specialized agencies of the United Nations FAO and WHO, is to develop food standards, guidelines and other related documents in order to achieve global harmonization, which also facilitates international trade. Behind all this is the protection of consumer health and also the establishment of fair practices in the food chain. It can be said that the Codex seeks international agreement and therefore shows suitable flexibility. It allows individual countries to incorporate Codex standards and guidelines into their own laws and recommendations. This is also the case with the Codex guideline on nutrition labeling. The Codex operates within the framework of committees specialized for certain areas, and the documents drawn up and adopted by it are finalized with the approval of the main committee [15, 16].
3.2. The Codex and nutrition labeling
With regard to nutrition labeling, two specialized committees play key roles, one of which is the Codex Committee on Nutrition and Foods for Special Dietary Uses (CCNFSDU) which, through its activities in this field, contributes, among other things, to the enforcement of scientific and professional basis and to the determination of dietary intake reference values. The other such specialized committee is the Codex Committee for Food Labelling (CCFL), which finalizes the information on nutrient composition related to food labeling in this area. A guide on nutrition and health claims has been developed within the framework of a similar collaboration. One of the objectives of the guidelines is to provide consumers with an understanding of the labels on the products and to provide them with sufficiently detailed information [17].
The basic requirements for nutrition labeling were first defined in guideline CXG 2-1985 in 1985 as a voluntary labeling element (except for food intended for specific groups, for which nutrition labeling was already mandatory at that time and was regulated by a separate standard that is CODEX STAN 146-1985), which applied to both prepacked and non-prepacked foods. The guideline is still being developed and refined to this day, and in this spirit there have been complete revisions in 1993 and 2011, and nine amendments between 2003 and 2017. Initially, nutrition labeling was voluntary, however, with a modification in 2012, it became mandatory for prepacked foods. In 2011, an annex defining the general principles of the Nutrient Reference Values (NRVs) for the population over 36 months of age was added to the guideline, which was revised four times between 2013 and 2017.
At international level, the general guideline for claims (CXG-1-1979) was adopted by the Codex in 1979. The principles of nutrition and health claims were defined for guidance in 1997, supplemented by terms such as „low fat”, „high fat”, etc. (CXG-23-1997).
The Codex guideline makes the data in Table 1 mandatory, but if a nutrition or health claim is made on food, the labeling should be supplemented with the nutrient claimed or the other substances with physiological effects, e.g. caffeine content. When there is a claim related to fatty acids, the amounts of the different fatty acids (saturated, monounsaturated, polyunsaturated) and, where required by member state regulation, the trans fatty acid content, in addition to the mandatory elements. The amounts of vitamins and minerals may be indicated if the product contains significant amounts of them.
It also allows for the voluntary indication of additional nutrients if, for example, required by national regulation, formulated by national recommendations or simply considered to be useful by the producer of the food. In all cases (mandatory, voluntary), the data must be expressed for 100 g weight or 100 ml volume, or portion, and it may be supplemented by the percentage of the Nutrition Reference Value (NRV). Regarding the presentation (font size, order of energy and nutrients, etc.), general principles have been formulated in the recommendations [18].
In addition to consumer education programs, the Codex guideline provides the opportunity to use other forms of voluntary expression through eye-catching graphic elements or symbols. These can help the consumer to get to know and understand the given nutrition declaration, and thus the nutrient content of the food, more easily.
Table 1. Content elements of the nutrition labeling of foods (=mandatory) in various laws
4. Regulation of nutrition labeling in the European Union
4.1. Antecedents of legal harmonization
The basic objective of the regulation of food labeling, and thus of nutrition labeling, is to properly inform the consumer. In 1979, Council Directive 79/112/EEC on the labeling of foodstuffs in the European Union [19, 1], did not yet cover the topic of nutrition labeling. Nutrition labeling was first regulated in 1990 by Council Directive 90/496/EEC as a voluntary labeling option, following the Codex Alimentarius guidelines on nutrition labeling. An exception was the regulation of foods for special dietary uses. At that time, however, it was agreed among food legislators that food business operators, especially small and medium-sized enterprises, should be encouraged to gradually introduce nutrition labeling [20].
Council Directive 90/496/EEC provided two options for nutrition labeling, the elements of which are shown in Table 1. Quantities could be indicated per 100 g weight or 100 ml volume, or per portion, provided that the number of portions in the package was also indicated. There were specific rules for their display: they had to be indicated in a tabular form or in a linear, quantity-by-quantity manner, in a clearly visible way, depending on the space available (at that time, the applicable minimum font size had not yet been determined). Mandatory elements of the label showed the quantities of energy, protein, carbohydrate, fat or energy, protein, carbohydrate, sugars, fat, saturates, dietary fiber and sodium.
The nutrition labeling may have included one or more of the following: starch, polyols, mono-unsaturates, polyunsaturates, cholesterol. Vitamins and minerals present in significant amounts could also be indicated. The annex to the directive also contained the recommended daily allowances for some vitamins and minerals, as well as the definitions of significant amounts (when determining the significant amount, 15% of the recommended intake in this annex should normally be taken into account for each 100 grams, 100 milliliters or one package of the food, if the package contains only one portion). Graphic display was allowed, but special rules were not defined.
The calculation of nuritional value could be based on the results of the tests performed by the food manufacturer, or on calculations based on known or actual average values of the ingredients used, or on calculations based on generally established and accepted data.
Nutrition and health claims appeared more and more frequently on food labels throughout the European Union. Member state regulations were quite diverse, therefore harmonization was necessary, resulting in Regulation (EC) No 1924/2006 on nutrition and health claims made on foods, which specifies which claims (e.g., low energy, energy reduced, source of protein, etc.) may appear on the label and under what conditions. The foods on which a claim is made can have an effect on dietary habits and overall nutrient intake, therefore consumers should be aware of their nutrient content. This goal can be achieved by the mandatory nutrition labeling of such foods [21]. Nutrition labeling is also mandatory in case of addition of vitamins, minerals and certain other substances to foods (Regulation (EC) No 1925/2006).
According to a 2003 study by the DG SANCO (Directorate-General Health & Consumer Protection), 35 to 85% of pre-packaged products in EU member states bore nutrition labeling. The survey pointed out that consumers are interested in nutrition labeling, especially in the case of processed foods, but the majority only requires it, but do not actually use this information.
The results of a consultation in member states in 2003 drew attention to the fact that the voluntary nutrition labeling system was not working satisfactorily and that a legal change was inevitable. Mandatory nutrition labeling was required. The mode of display was particularly important, because the use of small font sizes and multilingual labels made labels confusing, and there was also a need to define exceptions (e.g. packaging materials with small surface areas, non-prepacked products, alcohols etc.). Legislators have recognized that the obligation to provide nutrition labeling may present a problem to businesses because of the additional costs, to which a long transition period and the development of guidelines could be a solution. It was found that alternative nutrition labeling could also be useful, however, if there are too many labeling methods on the market, a great variety can also confuse consumers and the functioning of the internal market. As a result of the survey, the options „Big 4” (energy, protein, carbohydrate, fat) and „Big 8” („Big 4” supplemented by saturated fatty acids, sugar, fiber and salt) were proposed by member states. It was noted that consumers do not understand the indication of the amount of sodium, so it is necessary to use the term salt (table salt). It was also judged that providing the energy content in kJ was not understandable for everyone, therefore the introduction of the use of Kcal was also on the agenda [22, 23].
Regarding the use of other alternative forms of nutrition labeling (in addition to the nutrition labeling) there was a consensus that it should be clear and easy to understand. They also agreed that GDA (guideline daily amount) is a useful and easy to understand form of expression for all stakeholders of the food chain, but it can only be successful if it is harmonized at the EU level and developed by EFSA (European Food Safety Authority) or another independent scientific body [23].
A 2005 consumer survey by BEUC (The European Consumers’ Organisation), conducted in five countries (Germany, Denmark, Spain, Hungary and Poland), showed that nutrition labeling is of paramount importance to respondents; 74 to 84% of those interviewed stated that nutrition labeling was necessary. However, price, date of minimum durability/shelf-life and brand name are the most sought after information, nutrition labeling is read by only a few people, but the amount of fat and portion size are read by 50% of respondents. They spoke in support of other simplified forms of display. They also found that nutrition claims attract consumers’ attention and influence their purchases. 80% of respondents stated that nutrition labeling was easy to find and 70% thought it was easy to understand, while for 50% this information was also reliable. Survey data have shown that the marketing value of claims is markedly high [24].
The Commission’s 2007 White Paper on nutrition, overweight and obesity related health issues noted that the number of overweight and obese people in the European Union, especially children, had risen significantly over the previous three decades. Although the individual is primarily responsible for their own and their children’s lifestyles, it is an indisputable fact that the environment also effects behavior. Also, only a well-informed consumer is able to make rational decisions. Finally, an optimal outcome in this area can only be achieved if the different policy areas (horizontal approach) and the various levels of action (vertical approach) complement each other and are integrated.
It pointed out the need to think about making nutrition labeling mandatory and the regulation of the simplified labeling used on the front side of packaging.
The Commission’s findings in the White Paper, growing consumer interest in the relationship between the diet and health, as well as the need to select a diet that meets the individual’s needs have necessitated the implementation of a nutrition labeling systems that is uniform and mandatory throughout the European Union [25, 26].
EU rules on food labeling, pertaining to all foods, were laid down by Directive 2000/13/EC, most of which reached back to the regulatory principles that emerged in 1978, while Council Directive 90/496/EEC had become obsolete, therefore it was time to amend it [27, 7].
4.2. Legal harmonization
Based on the findings of the White Paper and the results of the surveys, Regulation (EU) No 1169/2011 (hereinafter referred to as: the Regulation) on the provision of food information to consumers, which ensures a high level of consumer protection, the free movement of goods and a level playing field, was established. The Regulation contains detailed rules on the labeling of prepacked foods, but also covers the labeling of non-prepacked foods to some extent. Since mandatory nutrition labeling imposes a significant burden on food business operators, the regulation allowed stakeholders a five-year preparation time, i.e., nutrition labeling on prepacked foods became mandatory from December 13, 2016 [7]. The goal of the legislation was to enable food information to reach the average consumer and to help them make a decision, despite their limited nutritional knowledge, while not creating barriers to trade [22, 25].
Nutrition labeling according to the Regulation must be applied to all foods. Exceptions are food supplements and natural mineral waters. Unlike before, the new type of nutrition labeling prioritizes as mandatory elements energy content and nutrients whose excessive intake carries a health risk. Exceptions to this are carbohydrates and protein, which have become mandatory items due to the increasing frequency of diabetes and the resulting kidney disease. The elements in yellow in Table 2 are mandatory, but it is possible to provide additional elements (marked in blue) on a voluntary basis. Nutrition labeling is also mandatory for the use of nutrition and health claims (on the packaging in the case of prepacked foods, while it does not have to displayed on non-prepacked foods, but the information should be available). Vitamins and minerals present in significant amounts may also be indicated, in accordance with the rules on specific values.
Certain foodstuffs are exempt from labeling in accordance to Annex V to Regulation (EU) No 1169/2011.
Table 2. Mandatory and voluntary elements of nutrition labeling of foods in Regulation (EU) No 1169/2011
The information may be given per 100 g weight or 100 ml volume but may also be expressed per portion or unit of consumption (for specific portion/packaging unit or characteristic unit of consumption due to the nature of the food). The amounts of vitamins and minerals referred to in Part A of Annex XIII to the Regulation should also be expressed as a percentage of the nutrient reference value (NRV) per 100 grams or 100 milliliters of the product. The energy content and the amounts of nutrients may also be expressed as a percentage of the nutrient reference value, expressed per 100 g weight or 100 ml volume, or per portion or unit of consumption. For nutrient reference values expressed per 100 grams 100 milliliters, the following information should also be provided: „Reference intake of an average adult (8400 kJ / 2000 kcal).
In terms of presentation, the Regulation is quite precise and clear; the elements of the nutrition labeling shall be presented in a specific order, preferably in a tabular form (if this is not possible, then continuously, without interruption) following each other, in the same field of vision, in a specific font size. The nutrition labeling is a closed list to which, in the case of foods for normal public consumption, additional elements cannot be added within the list, only to the end of the list (e.g. the amount of lactose should not be included with the sugars, it can only be displayed following the table).
The calculation of nutritional value could be based on the results of the tests performed by the food manufacturer, or on calculations based on known or actual average values of the ingredients, or on calculations based on generally established and accepted data.
Tolerance limits for nutrition labeling are important because, due to the natural variations in the composition of the raw materials and the effects of production and storage, it is not possible to determine the nutrient content of foods accurately within the analytical error.
However, the values given on the label must not deviate from the actual values to such a significant extent as to mislead or harm consumers. In relation to this, a guide has been developed under the coordination of the European Commission to help establish tolerance limits for nutrition values displayed on food labels.
According to the Regulation, specific elements of the nutrition labeling can be repeated in the main field of vision in two ways:
energy, or
energy, fat, saturates, sugars, salt.
In addition to the mandatory display, the Regulation also allows the use of graphic forms and symbols.
There are many voluntary graphic expressions and representations of nutrition labeling in the European Union. These display formats differ from each other. These display categories are not comparable, as they are based on completely different principles and have different uses.
Currently, we can basically distinguish four categories (Table 3).
Table 3. Examples of voluntary nutrition labeling of foods
5. Legal environment in Hungary
From the middle of the 19th century, the authorities of developed European countries began to adopt food laws. The first legal regulation of food in Hungary was Act XLVI of 1895 (on the prohibition of counterfeiting agricultural produce, products and articles) [29].
In the first decades of the 20th century, severe food crises occurred on the continents, from malnutrition to overnutrition. Over time, overeating in Europe started to pose an increasing health risk, leading to obesity and other health disorders. As a result, health organizations in developed and developing countries have become increasingly concerned with the regulated satisfaction of human nutritional needs. They were looking for the amount of energy, protein, fat, vitamins etc. which was absolutely necessary to maintain health, but at the same time they also studied the excessive intake of these nutrients and its consequences.
Starting from 1949, the Institute of Food Science (former name of the National Institute of food and Nutrition Science (OÉTI) regularly examined the diet of the Hungarian population and continuously modified the domestic nutrient requirements and created nutrient tables [30].
Statutory order no. 27 of 1958 was the first legal act that regulated the production and distribution of foods and beverages and ordered the establishment of the Hungarian Food Codex [31]. Nutrition labeling did not appear as such in this order, but the importance of the diet and nutritional health was already emphasized for the „health of our people”. As a result of joining the work of the Codex Committee (1963), the ideas and current issues appeared in food regulation in Hungary as well [32].
As regards nutrition labeling, MÉM decree 25/1976 (VII. 11.) on the implementation of Act IV of 1976 on foodstuffs [33] provided that „…where possible, essential nutrients should also be indicated on the packaging of the food to promote modern nutrition”[34]. The concept of “essential nutrient” was not defined in the decree, however, the nutrient table based on the work of OÉTI and edited by Dr. Róbert Tarján and Dr. Károly Lindner names them: energy content, carbohydrate, protein, fat [30]. At that time, laws did not define every detail and, as a result, individual professional decisions, evaluations and authorizations in connection with the given product played important roles.
Hungary recognized the importance of communicating nutrition labeling to consumers and, accordingly, MÉM decree 25/1976 (VII. 11.) provided the opportunity for voluntary nutrition labeling. During this period, a Codex document on the subject did not yet exist.
Strict rules applied to the fortification of foods with vitamins (e.g. only vitamins that also occurred in the food naturally were allowed to be added as fortification to the food). The name of the vitamin and its amount in the food had to be indicated and, in case of „diet” foods, the amounts of the important nutrients, in addition to the otherwise mandatory general labeling data had to be added.
For certain products/product groups, salt, fat, protein, starch, carbohydrate and energy content were also mentioned in the standards as quality criteria, but their indication was not mandatory in all cases. For example, in case of breads containing whole wheat flour, the carbohydrate content (per 100 g of product) had to be indicated in addition to the energy content (expressed in kJ) (MSZ-08-1377-86).
MÉM (former Ministry of Agriculture and Food) decree 25/1976 (VII. 11.) was replaced in 1988 by MÉM-SZEM (former Ministry of Agriculture and Food – former Ministry of Social Affairs and Health) decree 10/1988 (VI. 30.), which required the mandatory indication of the energy content per 100 grams (cm3) of the product, expressed in kJ, in case of prepacked foods. Among other things, the decree included the main types of „diet foods”, e.g. the categories of energy reduced foods; low energy; energy free; reduced sodium content and low purine, and their criteria. Nutrition labeling was mandatory on these foods, i.e., the energy content and the amounts of the nutrients that provided the energy, as well as the nutrients characteristic to the food and , possibly vitamins had to be indicated. Foods were allowed to be fortified or supplemented with certain vitamins (retinol, calciferol tocopherol, thiamine, pyridoxine, pantothenic acid, folic acid, cobalamin, ascorbic acid) [35].
This regulation provided that the product information sheet and the certificate of analysis must include the nutritional composition of the food „(protein, fat, carbohydrate, etc.) and other characteristics, energy content (per 100 grams or 100 cm3)”. On the packaging of the food had to be indicated: „the name of the food as specified in the standard, manufacturing authorization, product data sheet or other specification (e.g., marketing authorization in case of imported foods), and other mandatory information specified in the relevant standard (e.g., dry matter content, fat content, etc.)”.
Hungary’s application for membership of the European Union, submitted in April 1, 1994, made it necessary to prepare for legal harmonization. Council Directive 90/496/EEC on nutrition labeling was incorporated into Regulation 1-1-90/496 of the Hungarian Food Codex. Joint FM-NM-IKM decree 1/1996 (I. 9.) on foodstuffs stated that the energy content of foods must be given according to the Hungarian Food Codex. In addition to the data required for a given type of food, foods for special dietary uses and foods with claims (today these are called nutrition and health claims) had to bear the nutrition labeling required by the Hungarian Food Codex [36, 37, 38].
Before 1996, about five thousand kinds of food could be bought, but this number increased sevenfold by the turn of the millennium, because in the meantime new requirements, consumer needs and expectations appeared. Food production had shifted towards the manufacture of higher quality foods, and for this reason, as well as in preparation for accession, the creation of a new legal framework became necessary [29].
The elaboration of Act No. LXXXII of 2003 (the fifth Hungarian food act) was necessitated by Hungary’s membership in the European Union. The basic ideas of the law included the protection of the interests and health of consumers, the protection of the environment, and the promotion of fair competition and the free movement of goods [39]. In Hungary, during the preparation period between 1995 and 2004, the regulations of the European Union were gradually adopted into the food act and ministerial decrees, however, with the accession to the EU on May 1, 2004, these transitional legal acts became obsolete [36, 29].
In 2004, Directive 2000/13/EC on the labeling of foodstuffs was transposed in accordance with the specifications of joint FVM-ESzCsM-GKM (former Ministry of Agriculture and Rural Development – Ministry of Health Social and Family Affairs – former Ministry of Economy and Trade Affairs) decree 19/2004 (II. 26.), so the legal harmonization of food labeling had been completed. Nutrition labeling remained a voluntary labeling element (with the exception of foods with claims, fortified foods and foods for special dietary uses) until the entry into force and mandatory application of Regulation (EU) No 1169/2011.
6. Conclusions and the future of nutrition labeling
Nutrition labeling of foods has come a long way in the European Union, creating the opportunity for consumers to enjoy uniform and detailed information in all member states of the Community. EU and national legislators still face a number of challenges. From health and environmental points of view, our current food consumption habits are receiving increasing criticism. Average energy intake, the consumption of sugars, salt and fats remains above recommended levels, while the consumption of whole grains, fruits and vegetables, legumes and nuts is low [40]. The increase in the incidence of overweight and obesity is critical, so this trend needs to be reversed according to the guidance of FAO and WHO, which requires a shift towards a plant-based diet. The consumption of more fruits and vegetables could also reduce the risk of diet-related diseases and, according to some calculation, the environmental impact of the human diet [41]. The regulation of food labeling must therefore continue to follow scientific developments and provide consumers with the information that can form the basis for a balanced and sustainable consumption of food that meets individual needs in the most comprehensible way possible.
[4] Shine A., O’Reilly S., O’Sullivan K. (1997): Consumer use of nutrition labels. British Food Journal 99 (8) pp. 290-296. https://doi.org/10.1108/00070709710188390
[5] Swinburn B.A., Caterson I., Seidell J.C., James W.P.T. (2004): Diet, nutrition and the prevention of excess weight gain and obesity. Public Helath Nutrition 7 (1a) pp. 123-146 https://doi.org/10.1079/PHN2003585
[8] Gill C., Lynn S. (2005): Consumer understanding and use of nutrition labelling: a systematic review. Public Health Nutrition 8 (1) pp. 21-28 https://doi.org/10.1079/PHN2004666
[9] WHO (2018): Global nutrition policy review 2016-2017: country progress in creating enabling policy environments for promoting healthy diets and nutrition. https://www.who.int/publications-detail/9789241514873 (Hozzáférés: 2020. 04. 20.)
[10] Szakos D., Ózsvári L., Kasza Gy. (2020): Perception of Older Adults about Health-Related Functionality of Foods Compared with Other Age Groups. Sustatinability, 12 (7), pp. 2748. https://doi.org/10.3390/su12072748
[11] Plasek B., Lakner Z., Kasza Gy., Temesi Á. (2019): Consumer evaluation of the role of functional food products in disease prevention and the characteristics of target groups. Nutrients, 12 (1), pp. 69 https://doi.org/10.3390/nu12010069
[12] Szente V., Szabó S., Varga Á., Szakály Z. (2013): Az egészségre vonatkozó jelölések fogyasztói megítélése. Élelmiszer, Táplálkozás és Marketing 9 (1), pp. 85-90.
[13] Németh A., Szabó E., Kasza Gy., Ózsvári L. (2020): Development of lactose free, functional dairy foods based on consumer survey. GRADUS 7 (1), pp. 26-29 http://gradus.kefo.hu/archive/2020-1/
[14] Kiss A., Pfeiffer L., Popp J., Oláh J., Lakner Z. (2020): A Blind Man Leads a Blind Man? Personalised Nutrition-Related Attitudes, Knowledge and Behaviours of Fitness Trainers in Hungary. Nutrients, 12 (3), pp. 663. https://doi.org/10.3390/nu12030663
[18] World Health Organization Food and Agriculture Organization of the United Nations (2007): Food Labelling http://www.fao.org/3/a1390e/a1390e00.htm (Hozzáférés: 2020. 05. 10.)
[23] Summary of result for the consultation document on: “Labelling: competeteviness, consumer information and better regulation for the EU” Directorate E-Safety of the Food Chain, Unit E4-Food law, nutrition and labelling. pp. 5-20.
[40] Willett W. et al (2019): ‘Food in the Anthropocene: the EAT-Lancet Commission on healthy diets from sustainable food systems’, in Lancet, 393, pp. 447-92. https://doi.org/10.1016/S0140-6736(18)31788-4
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
The utilization of food industry byproducts is one of today’s important environmental and economic tasks. Byproducts that form during food production are typically used for feed purposes, but in many cases these materials can also be used in the production of human foods. The brewer’s spent grain left behind after brewing beer is a byproduct with favorable nutrition parameters, with low sugar and high fiber and protein contents. The main objective of our experiments was the reintroduction of brewer’s spent grain into the food industry, with a focus on innovation and sustainable development, by utilizing it in commercially available bakery products (salty medallions / wafers) formulated and regulated in the Hungarian Food Codex. Brewer’s spent grain consists of vegetable proteins and fibers (inactive malt), which may improve the compositional characteristics when preparing bakery products. In the course of our research, medallions enriched with brewer’s spent grain were prepared, of the beneficial parameters of which its high dietary fiber content should be highlighted, which can contribute to the realization of a health-conscious diet for consumers. A diet rich in dietary fiber, combined with an adequate amount of exercise, can reduce the risk of developing certain diseases (e.g., cancer and cardiovascular diseases).
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.
Figure 1. Brewer’s spent grain (BSG)
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].
Table 1. Chemical composition of Brewer’s Spent Grain (BSG) [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
Figure 2. Components of the medallion snacksFigure 3. Baked medallion snacks
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).
Table 2. Methods of determination
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).
Figure 4. Total polyphenol content of the enriched products
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.
Figure 5. Flavonoid content of the enriched products
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.
Figure 6. Dry matter content of the enriched products
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%).
Figure 7. Crude protein content of the enriched products
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%).
Figure 8. Fat content of the enriched products
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%.
Figure 9. Carbohydrate content of the enriched products
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).
Figure 10. Dietary fiber content of the enriched products
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%).
Figure 11. Common salt content of the enriched products
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.
Figure 12. Energy content of the enriched products
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.
Figure 13. Organoleptic analysis of the enriched 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.
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1 National Food Chain Safety Office, System Management and Supervision Directorate 2 University of Veterinary Medicine, Digital Food Chain Education, Research, Development and Innovation Institute 3 University of Debrecen, Doctoral School of Nutrition and Food Sciences
Keywords
deterministic exposure estimation, chronic aflatoxin M1 exposure, consumer groups at risk, carcinogenic effect, risk of liver cancer
1. Summary
The mycotoxin contamination of foods also appears in the food chain. Aflatoxin is metabolized in animals and its aflatoxin M1 (AFM1) metabolite, which is similarly, but ten times less genotoxic and carcinogenic than aflatoxin B1 (AFB1), is also present in milk, liver and eggs. Of these, the most significant food safety risk is posed by the contamination of milk with AFM1. In our article, the deterministic exposure estimation of Hungarian consumers is presented, based on the AFM1 contamination of milk and dairy products. The results indicate that the exposure of children under three years of age clearly poses a health risk, while the exposure of the 3 to 6 year old age group is borderline. The exposure of older age groups in ng/kg body weight does not pose an immediate health risk due to the increasing body weight. However, it needs to be emphasized that the presence of carcinogenic compounds should be kept to a minimum in all age groups. To this end, we propose an amendment to the regulation regarding the factory inspection of milk.
2. Introduction
In our previous paper on mycotoxin contamination, we presented the mycotoxin contamination of foods and feeds, the legal regulation of their tolerable maximum concentrations, the limitations of sampling procedures, and the experiences of current domestic practice were analyzed [1]. Using the data of the food consumption survey conducted in 2009 and measurement results available from the period 2010-2018, the exposure of Hungarian consumers to DON and aflatoxin M1 was estimated. Based on our preliminary estimates, it was determined that for some consumers, exposure to aflatoxin M1 and DON may exceed the toxicological reference values from time to time due to the yearly variation in food contamination levels, which may pose a risk to human health.
In our present paper, the results of our calculations carried out by a deterministic method and using the latest analytical measurement results and the data of the Hungarian food consumption survey performed in 2018-2019 using the methodology uniformly applied in Europe are presented, which provides information on chronic AFM1 exposure of different consumer age groups.
Intensive research is induced by the expected spread of mycotoxin-producing fungi due to global warming, the increase in food and feed contamination levels caused by the toxins they produce and the health problems and economic damages attributed to them. The large number of reports on the results are made more manageable by research area by the regularly published review articles, such as those on the interaction of mycotoxin-producing Aspergillus species with soil microorganisms [2], on human physiological effects of mycotoxin exposure [3], on the application of biocontrol technologies to reduce aflatoxin contamination, on the effect of silage production technology and the microbiota on aflatoxin contamination [4], on the sources, occurrence and regulation of mycotoxin contamination [5, 6] and on its detection methods [7]. A special edition of the journal Frontiers in Microbiology, containing 22 of the latest research articles and summaries, has also been published in the form of a book [8].
In view of the above reviews, only literature publications closely related to the objective of our paper are summarized below.
2.1. Occurrence of aflatoxins
Aflatoxins and other mycotoxins that occur in raw agricultural products (mainly peanuts, maize, rice, nuts, figs, spices and dried fruits) and feeds enter the food chain and can be detected in milk [9, 10, 11, 12], eggs, meat and offal [6, 13]. Aflatoxicol has been detected in the liver, kidney and meat of broiler and laying hens [14, 15]. Compared to the concentration of AFB1 in the feed, >5700-, >4600- and >3800-fold concentrations were measured in the livers of hens, in egg yolk and in egg white, respectively [16].
In addition to the exposure of the animals to AFB1 (µg/kg body weight), the concentration of AFM1 entering the milk from the feed depends on a number of factors, such as the health status of the cow, its milk yield, the lactation period, etc. [17]. The transmission rate is higher in specimens with higher milk yields [18]. The results of several studies have been reported in the literature, according to which the rounded transmission rate varied from 0.35 to 6%. Lower transmission rates (0.08%-0.33%) were observed in sheep [19].
There is less research on the transmission of aflatoxin to the liver, meat and eggs, but these report significantly lower transmission rates compared to milk, making milk still the most significant source of aflatoxin among foods of animal origin [4, 20].
When a food contaminated with AFB1 is consumed, AFM1 is excreted in breast milk to a similar extent as in cow’s milk [21, 22, 23, 24, 25, 26]. Infants and young children who are fed formula or milk drinks based on cow’s milk may also be exposed to AFM1. The results of European surveys indicate much lower levels than African or Asian publications [27].
As with all mycotoxins, aflatoxins show significant annual fluctuations in their levels depending on the weather conditions affecting fungal growth and toxin production [28].
For its most recent risk assessment [29], EFSA used the results of aflatoxin M1 measurements reported by Member States after 2013. Statistical data for some of the major food categories are summarized in Table 1.
Table 1. AFM1 mean and 95th percentile concentration values based on the 2013-2020 Member State data of EFSA.
Comment:
N: number of measurement results; % LCD (left censored data): ratio of results below the detection/quantification limit; P95: 95th percentile; LB: lower bound – result of substitution with the lowest concentration value; UB: upper bound – result of substitution with the highest concentration value; Other: foods for infants and young children.
2.2. Health effects of aflatoxins
Aflatoxins (especially AFB1, AFG1 and AFM1) proved to be extremely potent carcinogenic, kidney and liver damaging, genotoxic, malformative, reproductive capacity decreasing, immunosuppressive and nervous system damaging compounds in all experimental animal species, such as fish, ducks, mice, rats and monkeys [30]. A recently published study showed that the spores of pathogenic fungi cause severe, fatal infections in various birds [31].
High levels of AFB1, both in humans and animals, can cause fast-action, acute poisoning, during which severe hepatitic failure can lead to death, however, human risk of this in developed countries is negligible. Of aflatoxins, aflatoxin B1 is the most potent carcinogenic and genotoxic compound, and it is the one most commonly found in foods and feeds. Most often, it causes hepatocellular carcinoma (HCC), which is why AFB1 has been classified as a Group 1 human carcinogen by the IARC. After consumption of feed contaminated with AFB1, its hydroxy metabolite, aflatoxin M1, which is also a carcinogenic compound, although with a toxicity that is about one tenth of that of AFB1, is excreted by dairy cows in milk [32, 33].
Aflatoxins are rapidly and extensively adsorbed in the small intestine and, once in the liver, the metabolism of aflatoxin is catalyzed by the cytochrome P450 enzyme system found there. AFB1, AFG1 and AFM1 are converted to a reactive electrophilic epoxide that is capable of covalently binding to both DNA and proteins. Glutathion S-transferases (GST) are able to form a conjugative link with the 8,9-exo epoxide of AFB1, which is no longer able to enter harmful reactions in the body, and is excreted through bile and the kidneys. Polymorphisms among individuals result in high variability in enzymatic processes, and thus sensitivity to aflatoxin also varies from individual to individual [30, 34, 35]. In optimal cases, most aflatoxin metabolites are excreted within a few days, however, they have been observed to be present in protein-bound form over a longer period of time (e.g., in the case of aflatoxin-albumin adducts), with a half-life of 30 to 60 days in peripheral circulation [36].
Aflatoxins also damage liver cells directly, as well as indirectly, by altering the expression of genes involved in lipid metabolism. Increased cholesterol, triglyceride and lipoprotein production can cause the disintegration of hepatocytes. Hepatocyte death may lead to acute hepatitis, which can result in liver failure and, in more severe cases death. The disrupted metabolism of hepatitis patients can lead to malnutrition, which indirectly contributes to a general decrease in the antioxidant capacity of hepatocytes, to a loss of liver tissue regeneration capacity and, ultimately, liver failure [3].
Based on the opinion of EFSA experts, a key point in the risk assessment of aflatoxins is the evaluation of the role these toxins play in the development of liver cancer. From this point of view, children are particularly sensitive to aflatoxins, because, due to their low body weight, have a higher intake of food per kg body weight, and the risk of developing liver cancer is also higher in individuals infected with the hepatitis B (or C) virus and in the elderly. In people living in areas where both hepatitis B virus (HBV) infection and aflatoxin exposure are common, hepatocellular carcinoma (HCC) samples show a mutation hotspot (G-T transformation) at codon 249 of the p53 gene, which mutation is considered to be a signature of aflatoxin-induced HCC [37]. The possible reason for this is that hepatitis infection of the liver alters the expression of genes encoding aflatoxin detoxification enzymes, resulting in, for example, the induction of CYP enzymes or a decreased GST activity, thereby preventing the body from adequately eliminating aflatoxins [35]. Due to the immunosuppressive effect of aflatoxins, elderly people with chronic diseases are at particular risk, because in their case the efficiency of cell-level repair mechanisms is inferior, so the elimination of aflatoxins is also less effective. It should be emphasized that aflatoxins are able to cross the placenta, so aflatoxin exposure of pregnant women can also endanger the fetus [38].
2.3. The effect of processing on the aflatoxin content in foods
The common feature of aflatoxins is that they are stable, resistant to processing and heat effects. As a consequence, their presence must also be taken into account in the case of processed foods. Certain processing steps, such as sorting, refining, grinding, cooking, baking, frying in oil, roasting, preservation, flocculating, alkaline cooking, nixtamalisation, extrusion and fermentation, can reduce the concentration of mycotoxins in crops and processed foods, but they are not adequate enough to eliminate all contaminants, so the role of prevention at the very beginning of the food chain is of paramount importance [20]. For example, in terms of AFM1 contamination, it is important to reduce the AFM1 contamination of feeds using pre- and post-harvest biotechnological methods as well as toxin binders [4, 17].
Of heat treatment processes, conventional cooking and baking have little effect on mycotoxin contamination, while methods performed at higher temperatures, or possibly using dry heat [39], are more efficient. The breakdown of mycotoxins is enhanced by the presence of sugars, e.g., glucose, during heat treatment [40].
During the wet milling of cereals, such as corn, aflatoxin is distributed among the milling fractions in the following proportions: soaking water: 39–42%, fiber: 30–38%, gluten: 13–17%, germ, 6–10% and starch: 1%. Thus, the total aflatoxin level in the processed products decreases with the proportion remaining in the soaking water. After the dry milling of corn, the groats, bran and flour fractions contain only 6 to 10% of the original aflatoxin content, with most of the aflatoxin entering the germ and husk fractions [20].
Contamination of rice with aflatoxin most often occurs due to improper harvest and storage conditions. Mycotoxins are found primarily in the rice husk and bran layers. Husked brown rice and white rice obtained by polishing are gradually less contaminated [41].
The various heat treatment processes, pasteurization and freezing do not have a significant effect on the aflatoxin content of milk and dairy products [42]. The reduction effect of some heat treatment processes on AFM1 expressed in numerical values are as follows: pasteurization: 7.6%-12.9%, boiling: 14.5-23.9% [43], UHT treatment: 32% [44].
Different physical and chemical methods have been used with good efficiency to reduce the AFM1 content of milk or other liquid products: microwave irradiation (52%) [43], membrane filtration (81%) [45], biofiltration (81%) [46] combination of centrifugation and filtration (83%) [45], ozone treatment [47], the use of adsorbents (85-90%) [48, 49].
Intensive research is underway on the use of microorganisms. Encouraging results for the reduction of AFM1 contamination in milk have been obtained using Saccharomyces cerevisiae (90-93%) [50], S. cerevisise + L. rhamnosus, L. delbrueckii spp. bulgaricus, B. lactis (100%) [49], the mixture of different yeasts (65-69%) [51], heat-treated L. plantarum (94,5%) [44], L. bulgaricus (58%) [52] and in yogurt using S. thermophilus, L. bulgaricus and L. plantrium strains [53]. It remains to be seen how (in the case of using live microbes) changes in organoleptic properties can be eliminated if non-conventional cultures are used, and how the lactic acid bacterium-AFM1 complex formed can be removed from the product [44].
3. Data used to estimate consumer exposure
Exposure (g/kg body weight or ng/kg body weight) is calculated by multiplying the amount of food consumed (g/kg body weight) and the contaminant concentrations measured in it (ng/kg). In the deterministic method, we multiply the mean (median), or sometimes an upper percentile (95.0, 97.5) value. This calculation results in a point estimate giving a specific value [54, 55]. A more subtle estimate is obtained by probabilistic methods [56, 57], in which the distribution of input data is taken into account and thus a distribution is also obtains for exposure. Care should be taken when considering test results below the limit of quantification (LOQ). If the proportion of samples below the LOQ is between 50 and 80%, a maximum likelihood estimate (MLE) gives the best results [58].
Whichever method is used for the estimation, it is important to take into account the uncertainty of each calculation step, their magnitude, and to evaluate the results obtained in light of their cumulative effect [59]. The calculated uncertainty interval includes the true value with a certain level of confidence, i.e., with a certain degree of certainty [60]. The amount of contaminant entering the consumer’s body (EDI) is compared to the toxicological reference value(s) to determine the expected health risk.
For both short-term and long-term exposure estimation, it is worth examining the consumer groups that are particularly affected by the consumption of the given food/contaminant combination, and comparing the exposure of average consumers and „large consumers” [61].
3.1. Reference values for exposure assessment
Depending on the specific properties of the contaminant, the reference value may be the Acceptable Daily Intake (ADI), the Provisionally Tolerable Weekly/Monthly Intake (PTW/MI) or the Acute Reference Dose (ARfD). For food contaminants, the reference value is usually the tolerable daily intake (TDI). The benchmark dose (BMD) is the smallest dose that is estimated from the fitted dose-response curve at which a preselected effect level (benchmark response – BMR) can be observed, usually an increase or decrease of 5 or 10% compared to the control group. The lower confidence value of the BMD is the BMDL [62]. In the case of aflatoxins, Margin of Exposure (MoE) analysis is used to characterize the risk, as no TDI or other toxicological reference value can be established. In such a case, the value of the BMDL, adjusted by the uncertainty factor, is compared to the estimated exposure. The risk attributed to a contaminant can also be expressed as the ratio of the exposure to other reference values, the Hazard Quotient (HQ) or the Hazard Index (HI), which is the sum of the hazard ratios of substances acting on the same target organ or organ system, usually used for cumulative estimates [63].
EFSA recommends the use of 4 μg/kg body weight/day as the BMDL10 value as a benchmark for AFM1 risk characterization [29]. The results obtained are considered to be of concern below 10,000, with an MoE of 10,000 or greater indicating little risk to public health.
To characterize the risk of AFM1, the safe dose recommended by Kuiper-Goodmann (0.2 ng/kg body weight/day) can also be used to calculate the hazard index (HI), which is a quotient of a tumor-causing dose in 50% of animals and a safety factor of 50,000 [64].
According to the 2018 calculations of the JECFA [20], with an average daily intake of 1 ng/kg body weight AFB1, the probability of developing liver cancer is on average 0.269 per 100,000 persons per year, with the upper limit of the 95% confidence interval of the estimate being 0.562/100,000 persons/year in HBsAg+ (positive for hepatitis B surface antigen) individuals. For HBsAg- (negative for hepatitis B surface antigen) individuals, the mean value was 0.017 cancers/year/100,000 persons, with the upper limit of the 95% confidence interval of the estimate being 0.049/100,000 persons/year. The estimated mean values for AFM1 are one order of magnitude lower: 0.027/100,000 persons for HBsAG+, and 0.002/100,000 persons for HbsAg- individuals [20].
JECFA estimated the risk of hepatocellular carcinoma (HCC) associated with aflatoxin exposure using Equation 1:
Ri = [(PHBV+ × HBV+) + (PHBV− × (1–HBV+))] x AF bevitel (1),
where Ri is the HCC risk for region i, HBV+ is the prevalence of chronic hepatitis B in the study population, PHBV+ is the probability of developing liver cancer in this fraction of the population and PHBV- is the probability of developing liver cancer in the rest of the population.
3.2. Food consumption data
The calculations were performed using data from two representative Hungarian food consumption surveys conducted 10 years apart. The three-day survey of 2009 provided food consumption data for 4,992 individuals for a total of 14,976 consumption days, processed by dietitians and broken down into raw materials for the characterization of food consumption habits [65]. The ratio of milk and dairy product consumption days is shown in Figure 1.
Of the 14,976 consumption days in the 2009 survey, the consumption frequency [%] of milk, sour cream and cream, cheese and kefir or yogurt was 75.2, 52.8, 46.3 and 19.1, respectively.
The 2018-2020 survey was conducted within the framework of EFSA’s Europe-wide EU MENU or “What’s on the table in Europe?” project, in accordance with the recommended, uniform methodology [66, 67]. Participating persons were selected from the households participating in the Hungarian Central Statistical Office Household Budget and Living Conditions survey. During the program, two consumption days of 2,657 individuals between the ages of 1 and 74 were recorded, with the help of dietitians. On the 5,314 consumption days, the consumption frequencies [%] of milk, sour cream and cream, cheese and kefir or yogurt were 96.8, 54, 60.6 and 24. The ratio of milk and dairy product consumption days is shown in Figure 2.
Figure 1. The proportion of milk and dairy product consumption days by food group in the 2009 surveyFigure 2. The proportion of milk and dairy product consumption days by food group in the 2018-2020 survey
The distribution of consumers by age group is shown in Table 2.
Table 2. Age groups of the 2009 and 2018-2020 food consumption surveys and the number and proportion of consumers of dairy products by age group
Changes in the frequency of consumption of milk and various dairy products were compared using the milk and dairy product consumption days of the 2009 and 2018-2020 food consumption surveys. The numbers of consumption days of the different foods were compared to the total consumption days of the given survey (Figure 3). The frequencies of consumption of the different foods during the survey periods are characterized by the figure. Among the food categories studied, the consumption frequency of milk and milk-based desserts increased by more than 20%. The consumption frequency of cheeses shows an increase of 14%. The consumption frequencies of sour milk products (kefir, yogurt, sour cream), cream and flavored milks remained almost constant (with the former increasing slightly and the latter decreasing to a small degree). The consumption frequencies of condensed milk and milk powder has decreased significantly. Overall, it can be stated that the consumption frequencies of milk and dairy products has increased slightly over the last 10 years.
Based on the change in consumption frequencies over 10 years, an increase in aflatoxin exposure could be expected, however, this effect was offset by the change in the amounts consumed. The average consumption in milk equivalent, calculated with the median value of processing and enrichment factors, was 310.7 g/day in 2009, and this value decreased to 295.3 g/day in 2018-2020.
Figure 3. Proportion of consumption days to total consumption days; changes in consumption frequencies of various food groups based on the results of the 2009 and 2018-2020 food consumption surveys
Very little data were available on AFM1 concentrations in processed dairy products, so a database of AFM1 processing and enrichment factors for sour milk products (e.g., kefir, yogurt, sour cream) and various cheeses (hard, semi-hard, soft and processed cheeses, fresh cheeses) was compiled on the basis of the latest literature data, and consumer exposure was calculated with the milk equivalent of the consumed quantities of these products.
3.3. Aflatoxin concentration data
AFM1 analytical data are partly derived from NÉBIH’s 2011-2020 Hungarian monitoring survey (1,288 data). 40% of the samples contained measurable amounts of AFM1. Most of the measurements were performed by and HPLC methods on samples taken from the milk of dairy farms or private producers and, to a small extent, from commercially available mixed milk. In addition to the large number of items exhibiting contamination below the LOQ (60%), there were also items with very high contamination compared to the average. Values above 100 ng/kg were: 110, 122, 141, 149, 150, 190, 238, 240, 252, 260, 292, 376, 513, 740 and 860 ng/kg, respectively. We were unable to check the correctness of the results, but we saw no reason to omit them either, so the full data set was used in our further calculations. Another 1,177 samples were analyzed by January 2021 within the framework of the joint project of the University of Debrecen and NÉBIH („Determining of the short- and long-term aflatoxin exposure of Hungarian consumers in the dairy product chain and establishing risk management measures”). In the latter case, milk samples taken directly from the transport tankers by the staff of the dairy company at the 9 dairy farms participating in the project were analyzed between 2019 and 2021 by the ELISA method in the laboratory of the Instrument Center of the University of Debrecen. In samples with concentrations above the 20 ng/kg „action level”, exact AFM1 concentrations were confirmed by HPLC in the laboratory of NÉBIH. The number of samples with concentrations above the LOQ was 672 (57.1%). In the case of samples with concentrations above 20 ng/kg, the dairy farm was notified and it was recommended that appropriate precautionary measures be taken. As a result of this intervention, it was possible to stop the increase in milk contamination, and the AFM1 contamination of the milk produced was kept below the 50 ng/kg level. Detailed results will be published in the final report of the project.
The number of milk samples examined, broken down by year, is shown in Figure 4.
Figure 4. Yearly test sample numbers from the Hungarian survey of NFCSO (NÉBIH) and from dairy farms participating in the joint project
To refine our estimate and to compensate for the large number of values below the LOQ, instead of the usual LOQ, LOQ=0 and LOQ/2 approximations, the values of concentration data below the LOQ were also taken into account with the values of data generated with the help of a distribution with an element number identical to that of the number of measurement results. To measurement results above the LOQ, different distributions were fitted using the GAMLSS and GAMLSS.dist packages of the R statistical software using maximum likelihood estimation, then we used the parameters describing the goodness of the fit (AIC – Akaike’s Information Criterion, BIC – Bayesian Information Criterion and Global Deviance) to select the distributions that gave the optimal fit. The adequacy of the fits was also evaluated by visual comparison of the histogram made from the data and the distribution obtained, as well as by examining the normality of the differences and using a Q-Q plot. The two best-fit distributions were the two-parameter lognormal (Figure 6) and the four-parameter Box-Cox t-distribution (BCT) (Figure 7), which is suitable for the modeling of slowly decaying, continuously distributed data with positive or negative distortion similar to those of aflatoxins [68, 69]. Exposure calculations were performed with a lognormal distribution generated with the assumption of LOQ=5. The selected distributions were then fitted to the entire AFM1 data set, and the evaluation was performed again. Given that a positive change was observed in the parameters describing the goodness of the fit, the distribution chosen were considered to be acceptable.
Descriptive statistics for AFM1 test results and the fitted distribution are summarized below.
Table 3. Descriptive statistics of AFM1 test results (ng/kg) used for the calculations
The relative frequency distributions of NÉBIH and DE test results are shown in Figure 5.
Figure 5. Relative frequency distribution of the AFM1 contamination of milk samples taken in the NÉBIH monitoring program and in the framework of the DE-NÉBIH cooperation
With the exception of the outstanding NÉBIH measurement result values (indicated with blue-red asterisk) in the 10-15 ng/kg range, the frequency of AFM1 concentrations in the LOQ-70 ng/kg range was very similar in the two series of measurements and this justifies the joint evaluation of the measurement results. The relative frequency of samples containing AFM1 in concentrations above 70 ng/kg was <0.5% in the NÉBIH study.
A limiting factor in the risk assessment of aflatoxins was the lack of contamination data. According to the recommendation of EFSA [29], food categories for which the number of positive samples does not exceed 25 or for which the proportion of samples below the limit of quantification is greater than 80% should be excluded. In terms of AFM1 results, only the testing of milk met this criterion (Table 4), the number of tests for processed dairy products proved to be very small.
Table 4. Number of samples tested and % of >LOQ values
1: Formula: infant formula and other baby food 2: Milk-based food
Figure 6. Lognormal distribution fitted to AFM1 concentration results measured in milkFigure 7. Box-Cox t-distribution fitted to AFM1 concentration results measured in milk
4. Consumer exposure
In the case of food consumption data, the Observed Individual Means (OIM) method recommended for long-term estimation was used. First, all milk and dairy product consumption data were converted to milk equivalent, using the enrichment and processing factors specific to the given food category (Equations 2 and 3).
The intake of foods e1, ..., ej expressed in g/kg body weight (B) on a given consumption day (n), expressed in milk equivalent is
where
me is the mass (g) of food e on consumption day ni,
F is the processing (e.g., enrichment) factor characteristic of food e,
kg body weight is the body weight of the person belonging to the given consumption day,
where e is the AFM1 concentration in the milk used for the preparation of food e and is the AFM1 concentration in the processed food.
Fe is the value calculated from the min., med. and max. results obtained in the experiments.
By multiplying the amounts consumed in g/kg body weight/day by the average AFM1 concentration (ng/kg) calculated from the values of the fitted distribution functions, the exposure values for each consumption day were obtained (ng/kg body weight/day). The intake values of the 2 (2018-2020 survey) or 3 (2009 survey) consumption days of the participating persons were averaged. The results were aggregated by consumer age group and consumer exposure was calculated using the data from both food consumption surveys.
First, the effects of the minimum (Fmin), median (Fmed) and maximum (Fmax) values of the processing factors on the result of the exposure estimation were examined. The calculation was performed with the fitted lognormal AFM1 mean data, as well as with the mean (EDIátl) and 97.5 percentile (EDI0,975) milk consumption values of the 2018-2020 survey. The results are summarized in Table 5. The table illustrates the differences between the mean calculated using the deterministic method and the 97.5 percentile results, based on the 2018-2020 (EU MENU) survey.
Table 5. Estimated combined daily milk and milk product consumption of the various age groups as a function of dairy product processing factors
Taking into account the minimum-median-maximum values of the processing factors did not notably affect the results. There were significant differences in the mean values of the toddler age group when considering the minimum and median factors, therefore, the values calculated with the median of the processing and enrichment factors are used below to present the different exposure estimation results.
The exposure of the various age groups was calculated based on the P0.05, mean, median P0.975 percentile estimated daily intake values (EDI) of the 2009 food consumption survey, median processing factors and mean AFM1 concentration data. The exposures of the different consumer age groups were compared on the basis of the calculated EDI.
Figure 8. Estimated daily intakes (ng/kg/day) deterministic estimation; Comparison of the P0.025, mean, median and P0.95 EDI values of the different age groups, based on the 2009 consumption data
The estimated daily intake values of the age groups of the 2018-2020 survey show a trend similar to that of the 2009 data (Figure 9).
Figure 9. Estimated daily intakes, deterministic estimation; Comparison of the P0.025, mean, median and P0.95 EDI values of the different age groups, based on the 2009 and 2018-2020 consumption data
Comparing both the mean and the 97.5 percentile estimated daily intake values, it is clear that the exposure of each age group has been found mostly constant over the past 10 years. The only noticeable differences are in the mean values of the toddler age group and the 97.5 percentile values of the children age group, but the differences are not significant. The number of items in the toddler age group in the 2009 survey is very low (90 people) compared to the 2018-2020 survey (482 people). Values calculated with a smaller number of elements are burdened with a greater uncertainty.
4.1. Assessment of consumer exposure
Based on the obtained exposure values, the Margin of Exposure (MoE) approach (Equation 3), the hazard index (HI) (Equation 4) and the probability increase of liver cancer attributable to AFM1 intake were used to assess the risk of the Hungarian population. For the MoE method, the BMDL10 value for AFM1 of 4 μg/kg body weight/day was taken into consideration:
Consumer exposure is considered to be risky if the value of MoE is <10,000. The MoE values of exposure calculated by the deterministic estimation from the consumption data of the 2018-2020 food consumption survey are shown in Table 6.
Table 6. MoE values of average and large consumers (97.5 percentile) by age group
The health risk threshold (10,000) is reached or approached only by the “large consumers” (97.5 percentile) of the toddler and children age groups. In the case of the other age groups, no significant risk can be identified using this risk characterization methodology.
For the calculation of the hazard index, the safe dose recommended by Kuiper-Goodmann [59] was used (0.2 ng/kg body weight):
When calculating the hazard index (HI), the degree of risk is directly proportional to the EDI values and is considered to be of concern when the value is 1 or higher. As an example, the results of deterministic estimates using the consumption data of the 2018-2020 food consumption survey by age group are shown in Table 7.
Table 7. Hazard indices (HI) calculated from the EDI values of the different age groups
Note: HI values that pose a health risk are indicated by bold numbers
HI values calculated from the mean and 97.5 percentile values of the estimated daily intakes of the age groups indicate that the risk from exposure of the adolescent, adult and elderly age groups is not considered to be of concern. However, for toddlers and children, in the case of the 97.5 percentile values (large consumers), exposure is well above levels considered to be safe. One of the most important of the above results is the HI value of 1, characterizing the average intake of toddlers, as it suggests that a significant proportion of this age group is exposed to large amounts of AFM1, which is of great health concern.
Assuming a Hungarian hepatitis B prevalence of 0,7% [70], the incidence of liver cancer associated with aflatoxin was calculated using Equation 1. Calculations were also performed with the mean and upper 95% confidence values for the likelihood of developing liver cancer:
Átlag RMo= [(0,0269 × 0,007) + (0,0017× 0,993)] × EDI,
CI,.95 RMo= [(0,0562 × 0,007) + (0,0049× 0,993)] × EDI.
The values of hepatocellular carcinoma (HCCi) attributable to aflatoxin exposure derived from the mean and 97.5 percentile results of AFM1 exposure values calculated from the consumption data of the 2018-2020 food consumption survey by deterministic estimation (DET) (illness/100,000 persons/year) are summarized by age group in Table 8.
Table 8. Incidence of liver cancer as a function of EDI by age group
The risk of developing HCC is increased many times by aflatoxin exposure in the presence of chromic hepatitis B. As the prevalence of hepatitis B is low in Hungary (and in Europe in general), the increase in HCCi induced by aflatoxin is not high either. Although the numerical value of the estimated incidence of liver cancer proved to be very low, their relative values show in this case as well the high risk of “large consumers” of toddlers and children compared to the other age groups.
5. Situation assessment, recommendations
Chronic exposure to AFM1 calculated by the deterministic method and compared to various reference values consistently indicates that the exposure of children aged 1<3 is the highest in the studied age groups. The lowest exposure values are observed for the oldest age groups. Due to a lack of data, exposure in infants aged <1 could not be studied. However, the correlation is not directly between age and intake amounts, but between the change (typically increase) in body weight of increasingly older age groups and the intake amounts.
Given that the toxicity of aflatoxins primarily poses a health risk to developing organisms, special attention should be paid to reducing their exposure and keeping it to a minimum. However, it should be emphasized that the presence of carcinogenic compounds should be kept to a minimum in all age groups.
The body is burdened not only with the AFM1 contamination of breast milk and other milks or milk-based products, but also with the AFB1 taken with other foods and which is 10 times more toxic than AFM1. Since their mechanism of action is the same, the effects of aflatoxins and AFM1 add up. Therefore, we need to pay attention to the quality of our food and the storage conditions of products with open packaging. Products with a musty smell and traces of mold should not be consumed, even after cooking or baking.
Milks, analyzed during the annual monitoring inspections and exhibiting contamination levels that are 10 to 15 times higher than the maximum tolerable level allowed by the law are also marketed. Particularly at risk are those individuals who regularly consume milk from the same source where the animals are fed aflatoxin-contaminated feed.
To date, there is no routine and industry-wide large scale process that can reliably and completely eliminate the aflatoxin content of foods, so the focus remains on preventing contamination. This is a complex task that requires the involvement of all stakeholders in the food chain, starting with the application of good agricultural practices, and the proper preparation and management of arable land. This is followed by the selection of hybrids resistant to mold, and then a series of measures taken during the harvesting, transport and storage of crops that can prevent the growth of molds (setting appropriate temperature and humidity levels, sorting, peeling and physical treatment of the crops). Last but not least, the appropriate storage and treatment cereals, silage or other processed feed products intended for animal feed, checking their aflatoxin levels and their physical, chemical or biological detoxification, if necessary [4].
The success of prevention and the adequacy of milk shipments can also be checked at the level of dairy farms and dairy plants. With a sampling plan developed for the detection of the aflatoxin M1 content of raw milk and an early warning system, and by applying the 20 ng/kg action threshold already proven to be useful in Italy, an increase in the level of contamination can be predicted effectively. Based on the warning, the dairy farm can prevent the maximum tolerable AFM1 concentration (50 ng/kg) from being reached in accordance with local conditions, for example, by modifying the composition of the feed or by using toxin binders. This can reduce the use of contaminated milk batches in primary and secondary milk processing and, consequently, reduce consumer exposure [1, 10, 71].
It should also be noted that, due to the uncertainty of the detection, ELISA kits set to indicate an AFM1 concentration of 50 ng/kg may still classify batches of milk with contaminations of ≤ 65-70 ng/kg as adequate in 50% of the cases.
In order to protect infants and young children who are most exposed to AFM1 and who are also the most vulnerable, but also to protect the health of the entire population, it is recommended that the regulation of dairy plant inspections is amended in a way that ensures that if the AFM1 contamination of the milk delivered from a farm is ≥20 ng/kg, the plant is obligated to notify the dairy farm and NÉBIH, and thereafter to monitor the effectiveness of the dairy farm measures taken to reduce the contamination by daily monitoring of the contamination of the milk delivered from the farm.
It is also recommended that the warning level used in the self-inspection of dairy farms is set to 20 ng/kg instead of the current 50 ng/kg. ELISA kits for the detection of AFM1 at a concentration of 5-10 ng/kg are available for both dairy plant and producer monitoring, so there is no methodological obstacle to the establishment of a new warning threshold.
6. Acknowledgment
The authors would like to thank to Judit Sali and Katalin Csizmadia for providing the relevant data of the 2018-2020 NÉBIH consumption survey, the staff of the DE Instrument Center and NÉBIH for the AFM1 analysis of the milk samples, Attila Nagy and Gabriella Miklós of NÉBIH for their useful suggestions, Béla Béri, who participated in the DE-NÉBIH project, and to the managers and employees of Alföldi Tej and the dairy farms for their cooperation.
Our research program was supported by the National Research, Development and Innovation Fund in the 2018-1.2.1-NKP foundation system with the designation 2018-1.2.1-NKP-2018-00002 (AA, KK).
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The effect of lactation number and lactation stage on milk yield, on the composition and on the microbiological properties of raw cow milk in a Hungarian dairy farm
1 University of Debrecen, Faculty of Agricultural and Food Sciences and Environmental Management, Institute of Food Science 2 University of Debrecen, Faculty of Agricultural and Food Sciences and Environmental Management, Institute of Animal Science, Biotechnology and Environmental Protection, not independent Faculty of Animal Husbandry 3 University of Debrecen, Doctoral School of Animal Science
Changes in the composition and hygienic properties of milk affect producer price, so it is essential for the responsible dairy farmer to collect information on changes in these parameters due to various factors. In their study, the authors seek to answer the question whether there is a fluctuation in the daily milk yield of cows and in the composition (fat and protein content) and microbiological properties (somatic cell count, total plate count, coliform and S. aureus count) of raw cow’s milk in primiparous and multiparous cows or at different stages of their lactation. Based on the data of a Hungarian large-scale dairy farm, it was found that there was no difference in the fat and protein content of the milk, but the daily milk yield was higher in the case of multiparous cows and, compared to the milk of primiparous cows, their milk had a higher somatic cell count and larger amounts of coliform bacteria. The daily milk yield decreased in the successive stages of lactation, but the fat and protein content of the milk increased, which is presumably due to the concentrating effect of the decreasing milk yield. No significant change was observed in the colony count of microorganisms at the different stages of lactation.
2. Introduction
Cow’s milk has a high nutritional value; it contains fats, proteins, carbohydrates, vitamins and minerals, among other things [1]. Examination of the composition of milk is a routine practice on dairy farms for monitoring the hygiene, nutritional and health aspects of dairy cows [2]. The composition of milk can be influenced by a number of factors, such as the number of lactations, the stage of lactation, the season and the feeding technology [3, 4, 5]. Thus, the composition of milk may vary during lactation, and as a result of the interactions of different environmental factors, there may be differences between the different dairy farms [6]. According to Dürr et al., the creation of databases in order to determine the causes and consequences of differences in milk yield and milk composition is of paramount importance. A database should also include records related to these parameters, as well as to lactation-related events and to the individual cows [7].
Due to its nutritional value, high water activity and neutral pH, milk serves as an excellent medium for various microorganisms, which may include pathogenic organisms such as Campylobacter jejuni, Salmonella spp., Staphylococcus aureus, Listeria monocytogenes, Yersinia enterocolitica, etc. [8, 9]. During the primary infection of milk, sick animals themselves are the sources of infection. In case of dairy animals suffering from a systemic disease accompanied by the spread of pathogens, the pathogens may be excreted in the milk. During secondary contamination, the contamination of milk is of environmental origin. Due to improper milking hygiene, milk can be contaminated by the faeces of the animals or the equipment used in milking (milking machines, milk lines, milk storage tanks), among other things [10]. As the milk enters the teat cistern and then the teat canal, it can become infected with various microorganisms, of environmental origin, so the bacterial infection of the first milk jets is remarkably high. At the beginning of milking, it is therefore advisable to separate the first jets of milk from milk that is milked subsequently, and to ensure that they are destroyed [11]. To reduce the number of bacteria in milk, heat treatment is most commonly used. The initial microbiological state of the milk is not only important from a food safety point of view, but can also affect the quality of the dairy products made from it [12].
Coliform bacteria can cause mastitis in dairy animals. Mastitis caused by coliform bacteria can reduce milk production in dairy animals, causing economic losses to dairy farms [13]. The presence of these bacteria in the environment is common, so their presence in food may indicate environmental contamination [14, 15].
Raw milk can be contaminated with S. aureus from a number of sources, such as the environment, milkers’ hands, milking equipment, and so on [16]. The economic damage due to mastitis caused by S. aureus comes from a decrease in the quantity and quality of the milk produced, an increase in the number of somatic cells measured in it, a reduction in the purchase price of lower quality milk, which also leads to a decrease in the turnover of dairy farmers [17]. Prevention is an effective means of controlling S. aureus infection. It can be prevented by adhering to appropriate housing and milking technologies, more frequent fly extermination, pre- and post-disinfection of the teats, the use of disposable udder wipers and regular laboratory testing [18].
External and internal factors can affect the composition of milk, as well as the microbiological state of raw milk. The latter is mostly determined by the hygienic condition of the surfaces that come into direct contact with the milk [19]. Research by Peles et al. found that different husbandry and milking methods at dairy farms with different cow numbers had influencing effects on the microbiological quality of milk [20]. Tessema sought to find a correlation between breed, the age of the animals, the number of lactations and the stage of lactation, as well as the likelihood of S. aureus occurrence.
In his study, he found a significant difference in the prevalence of S. aureus in milk for the two cattle species studied. S. aureus was found in higher proportion of crossbred cows and in older individuals [21]. Examining the milk of different varieties, Bytyqi et al. found no difference in the colony counts obtained [22].
Although fewer Hungarian studies have been conducted on the subject, several international publication have examined whether the number of lactations and the stage of lactation have an influence on the daily milk volume of the animals, the composition of the milk and its microbiological parameters. According to Tessema, there is a significant difference in the prevalence of S. aureus, depending on how many lactations the animals have been through. In his study, he found that S. aureus was more common in the milk of cows that had calved more than twice and produced a positive California Mastitis Test [21]. Tenhagen et al. also found that the incidence of S. aureus increases with the age of the animals [23]. This may be related to the fact that the milking machine may damage the teats during milking, allowing microorganisms to enter the udder from the environment [24]. Another possible reason is that the health status of dairy animals may deteriorate with advancing age, which may have an adverse effect on the somatic cell count of milk [25].
The objective of our study was to determine at a Hungarian large-scale dairy farm whether there is a difference in the daily milk yield of primiparous and multiparous cows, as well as cows at different stages of lactation, and also in the composition (fat and protein content) and microbiological parameters (somatic cell count, total plate count, coliform and S. aureus count) of primiparous and multiparous cows, as well as cows at different stages of lactation.
3. Materials and methods
3.1. Place and time of sampling
A dairy farm in Hajdú-Bihar county (Hungary) was the site of our studies. 440–450 Holstein-Friesian cows are milked on the farm. The farm uses deep litter livestock-keeping and monodiet feeding. Milking takes place in a milking parlor, and no post-disinfection is carried out after milking.
The data on the daily amount of milk, fat and protein content and somatic cell count used for the calculations are derived from the milking results, i.e., from the examination results of the milk samples collected monthly by the Állattenyésztési Teljesítményvizsgáló Kft. All milking results of 38 individuals between May 2015 and January 2020 were used in the calculations. During the calculations, data on the first lactation of the cows (n=387), data on the 2nd to 5th lactation of the cows (n=446), as well as data on the early (under 100 days; n=275), middle (100 to 200 days; n=249) and late (over 200 days; n=309) stages of the lactation of the cows were summarized for the abovementioned time period.
Microbiological tests were performed between May 2018 and October 2019. A total of 62 milk samples were taken from 38 randomly selected, clinically healthy individuals for the microbiological tests. According to which lactation cycle the animals were in and in which stage of lactation during the sampling, the samples taken from the individual cows were classified as follows: 26 samples were taken from 15 primiparous cows and 36 samples were taken from 23 multiparous (2 to 5 calvings) Holstein-Friesian cows. Of the total 62 samples taken, 23 were taken in the early stage of lactation, 21 samples in the middle stage of lactation and 18 samples were taken from cows in the late stage of lactation.
Following pre-disinfection of the teats, wiping them dry using paper towels and milking of the first milk jets, samples were taken from all four udder quarters into sealable sterile plastic sampling vessels with a capacity of 50 ml. The vessels were transported in coolers to the microbiology laboratory of the Institute of Food Science of the University of Debrecen within two hours of sampling. Samples were processed within 24 hours of sampling.
3.2. Microbiological tests
Preparation of the milk samples and the subsequent microbiological tests were performed according to the procedure described by Petróczki et al. [26]. Sample preparation was carried out according to standard MSZ EN ISO 6887-1:2017 [27], the samples were stored at 4 °C until the beginning of the analysis, and they were homogenized by shaking before processing. To prepare the dilution series, peptone water was used which was prepared by dissolving 8.5 g of sodium chloride (VWR International Kft., Hungary) and 1.0 g of peptone (Merck Kft., Hungary) in 1,000 ml of distilled water.
After weighing the appropriate amounts (9 ml each) into test tubes, the diluent was sterilized in a pressure cooker at 120 °C for 30 minutes, then it was cooled and the decimal dilution series was prepared.
The total plate count was determined according to standard MSZ EN ISO 4833-1:2014 [28], which prescribes the use of a tryptone-glucose-yeast agar (Plate Count Agar, PCA) culture medium (Biolab Zrt., Hungary) supplemented with milk powder. After performing the prescribed plate casting method, the plates were incubated at 30 °C for 72 hours.
The amount of coliform bacteria was determined by the plate casting method according to standard ISO 4832:2006 [29], using sterile crystal violet-bile-lactose agar (Violet Red Bile Lactose, VRBL, Biolab Zrt., Hungary). The incubation period was 24 hours at 30 °C.
The determination of S. aureus was performed according to standard MSZ EN ISO 6888-1:2008 [30] by the spreading method, for which Baird-Parker agar (BPA, Biolab Zrt., Hungary) supplemented with egg yolk-tellurite emulsion (LAB-KA Kft., Hungary). The incubation period was 48 hours at 37 °C. S. aureus was isolated from other Staphylococcus species using a latex agglutination test (Prolex Staph Xtra Kit, Ferol Kft., Hungary).
3.3. Statistical analysis
For the analysis of our experimental results, for the calculation of descriptive statistics, for the logarithmic transformation of the amount of microorganisms and for performing t-tests and analysis of variance, the SPSS v.22.0 [31] software was used.
In the case of lactation number, variables were compared using unpaired t-tests and non-parametric Mann-Whitney tests, while in the case of lactation stage, the comparison was performed by one factor analysis of variance and non-parametric Kruskal-Wallis tests. Since the total plate count, the coliform count and the somatic cell count were not found to be normally distributed variables in several cases, a logarithmic transformation was used for these parameters. During statistical analyses, a value of P<0.05 was considered to indicate a significant difference.
4. Results
4.1. Effect of lactation number on milk yield, raw milk composition and its microbiological parameters
Mean and standard deviation values of the daily milk yield of primiparous and multiparous cows, as well as those of the fat and protein content, somatic cell count, total plate count, coliform and S. aureus count of the milk produced by them are shown in Table 1. Cows selected for the study were classified into groups based on the number of lactations (and calvings) into groups of primiparous and multiparous individuals. The average milk yield was 25.67 kg/day for primiparous cows, while in the case of multiparous cows it was 31.04 kg/day. The difference is significant (P<0.05), which means that our experiments confirmed the findings of Bondan et al. and Yang et al., that the daily amount of milk produced by multiparous cows is higher than that of primiparous cows [5, 32]. In the case of crossbred Holstein-Friesian cows, Gurmessa and Melaku studied, inter alia, the effect of calving number on milk yield, but found no difference between the daily milk yield of primiparous and multiparous cows [33]. Pratap and his research group also found no difference in the average daily milk yield of primiparous and multiparous cows (6.43±1.39 and 5.89±2.37 l/day, respectively) [34].
The average fat content of milk during the first lactation of cows selected during our research was 3.74±0.40%, while the average fat content of milk samples taken during the second or later lactation was 3.75±0.36%. The difference was not found to be significant (P>0.05). Similarly to our own results, no difference was found between the average fat content of the milk of primiparous and multiparous crossbred Holstein-Friesian cows by Gurmessa and Melaku, or Pratap et al. [33, 34]. On the other hand, it was found by Bondan and his group that the number of lactations did have an effect on the fat content of milk in Holstein-Friesian cows. While the fat content was 3.47±0.67% in the case of cows lactating for the first time, it was 3.43±0.68% and 3.41±0.67% for cows lactating for the 2nd or 3rd time and for cows that had calved at least 4 times, respectively [5]. In their study in Sudan, Shuiep and his working group examined changes in the fat content of milk with the number of lactations in local and crossbred cows. In the case of local cows, cows in their fourth lactation period had a lower milk fat content (4.82±0.55%) than cows with fewer calvings (1:5.16±0.32; 2:5.22±0.34; 3:5.14±0.34). No difference was observed in the case of crossbred cows [6]. In contrast, Yang et al. found in their research that Holstein-Friesian cows lactating for the first time had a lower milk fat content (3.88%) [32]. Based on the varied results of our study and other literature references, we came to the conclusion that the fat content of milk may be influenced by other factors besides the lactation number.
During the first lactation of the Holstein-Friesian cows selected by us, the average protein content of the milk was 3.24±0.19%, while the average protein content of milk samples taken during the second or later lactation was 3.31±0.16%, with the difference being not significant (P>0.05). This is in line with the findings of Gurmessa and Melaku, as well as those of Pratap et al., as these authors did not find any difference between the average protein content of the milk of primiparous and multiparous cows [33, 34]. In contrast, the research group of Bondan found that the lactation number affected the protein content of milk in Holstein-Friesian cows. While the protein content was 3.24±0.37% in the case of cows lactating for the first time, it was 3.23±0.38% and 3.19±0.37% in the case of cows lactating for the 2nd or 3rd time and for cows that had calved at least 4 times, respectively [5]. Changes in the protein content of milk with the lactation number was also studied by the research group of Shuiep. In the case of local cows, the protein content of milk was lower in the case of cows in their fourth lactation period (3.67±0.19%), than in the case of cows with fewer calvings (1:4.01±0.11; 2:3.82±0.12; 3:3.84±0.12). No difference was observed in crossbred cows [6].
According to our results, the average somatic cell count [242.2×103 (5.12±0.42 lg) cell/ml] in the milk of primiparous cows is less (P<0.05) then in the milk of multiparous cows [356.3×103 (5.39±0.39 lg) cell/ml]. In none of the cases did the averages exceed the limit value [M=400.0×103 (5.60 lg) cfu/ml] laid down in Regulation (EC) No 853/2004 [35]. The results obtained at the Hungarian dairy farm are in line with relevant literature data. According to Mikó et al., as the lactation number increases, the somatic cell count of milk may also increase [25]. This finding was also found to be true by Yang and his group [32]. Sheldrake et al. observed a significant relationship between the calving number and the somatic cell count. They found that with the increase in calving number, there was a smaller change in the udder quarters of healthy animals, however, in the case of udder quarters infected with S. aureus, the somatic cell count increased significantly [36]. The research team of Bondan found that as the lactation number of the Holstein cows studied increased, so did the somatic cell count in milk. While the average somatic cell count in primiparous cows was 4.83±1.73 lg cell/ml, it was 5.31±1.72 és 5.84±1.62 lg cell/ml in cows that had calved 2 or 3 times and cows that calved 4 or more times, respectively [5].
In the milk samples taken from Holstein-Friesian cows that only calved once, i.e., cows in their first lactation period, the average total plate count was 5.1×103 (3.36±0.58 lg) cfu/ml, while it was 4.6×103 (3.30±0.59 lg) cfu/ml in the milk samples taken from multiparous cows, however, the difference was not significant (P>0.05).
However, the average coliform count [1.1×103 (1.35±1.20 lg) cfu/ml] in the milk of multiparous cows was more (P<0.05) than the average coliform count [1.1×101 (0.65±0.61) lg) cfu/ml] measured in the milk of primiparous cows. Tenhagen et al. also included clinically healthy cows in their study, which found that although coliform bacteria were found in higher quantities in the milk of multiparous cows, the difference was not significant [23].
S. aureus was present in only one of the 26 milk samples taken from primiparous cows, in an amount of 5.0×101 (1.70 lg) cfu/ml, while it could be detected in eight of the 36 milk samples taken from multiparous cows. The mean S. aureus count in these samples was 1.5×102 (1.92±0.56 lg) cfu/ml. The values do not exceed the limit value [M=5.0×102 (2.70 lg) cfu/ml] specified by EüM decree 4/1998 (XI. 11.) [37]. In the case of the individuals in whose milk S. aureus could be detected during the microbiological tests, the average somatic cell count was between 44.3×103 (4.65 lg) cell/ml and 357.2×103 (5.55 lg) cell/ml on the basis of milking data. In his study, Tessema also found that the prevalence of S. aureus was higher in the case of multiparous cows (i.e., who had calved more than twice) (who produced a positive California Mastitis Test) [21]. According to Tenhagen et al., the incidence of S. aureus increases with the age and lactation stage of the animals [23].
Table 1. Milk yield of primiparous and multiparous cows and compositional and microbiological properties of milk collected from them
a, b The values marked with different letters in the same rows of the table differ significantly (P<0.05)
4.2. Effect of lactation stage on milk yield, raw milk composition and its microbiological parameters
Mean and standard deviation values of the daily milk yield of cows in the early, middle and late stages of lactation, as well as those of the fat and protein content, somatic cell count, total plate count, coliform and S. aureus count of the milk produced by them are shown in Table 2. The cows selected for the study were classified into groups of early, middle and late lactation stage individuals, based in their stage of lactation. When examining the changes in the daily milk yield of the individuals with the stage of lactation, the finding in the literature that the daily milk yield decreases towards the end of lactation was confirmed. While the average daily milk yield of cows in the early stage of their lactation was 32.10±4.73 kg/day, that of cows in the middle stage of their lactation was 29.08±5.09 kg/day, while that of cows in the late stage of lactation was 23.36±3.63 kg/day. The difference was significant (P<0.05). Bondan and his group came to a similar conclusion in their study. The average milk yield between days 6 and 60 of the lactation of the Holstein-Friesian cows studied by them was 29.4±8.72 l/cow/day; for cows between days 61 and 120 of their lactation, it was 29.2±8.66 l/cow/day; for cows between lactation days 121 and 220, the milk yield was 26.2±8.01 l/cow/day, and at the end of the lactation (>220 days), it was 22.0±7.49 l/cow/day [5]. Gurmessa and Melaku, as well as Pratap et al. also found that the daily milk yield of the animals was higher at the beginning of lactation (6.81±1.45 l/day) than at the end of lactation (5.48±0.05 l/day). In their studies, the daily milk yield of crossbred Holstein-Friesian cows in the middle stage of their lactation was the highest (7.17±0.05 liter) [33, 34]. According to Auldist et al., the effect of lactation stage on milk yield (e.g., a decrease) may be due to a change in the number and activity of the secretory cells within the mammary gland because of physiological reasons [2].
The fat content of the milk of the individuals studied shows a change as we progress towards the end of lactation. The average fat content of cows in the early and middle stages of lactation was 3.65±0.43% and 3.59±0.41%, respectively, which were lower (P<0.05) than the fat content of the milk of cows in the late stage of lactation (3.99±0.47%). The increase in milk fat concentration at the end of lactation may be associated with a decrease in milk yield as lactation progresses, as a decrease in milk yield may have a concentrating effect on milk composition [2]. The fat content of milk also varies at the three stages of lactation according to the publication of Gurmessa and Melaku. For cows in the early or late stages of lactation, the average fat content of the milk (4.46±1.44% and 4.46±1.44%, respectively) was significantly higher than that of cows in the middle stage of lactation (3.70±0.89%) [33]. The publication of Bondan et al. states that the fat content of milk at the end of lactation (>200 days) is higher (3.55±0.67%) than at earlier stages of lactation. However, they also found that the measured average fat content (3.30±0.66%) was lowest between days 61 and 120 of the lactation of the cows. Between days 6 and 60, and between days 121 and 220, average fat contents of 3.40±0.65% and 3.40±0.66% were measured, respectively [5]. Shuiep et al. studied changes in the fat content of milk of local and crossbred cows in Sudan with lactation stage. In the case of the local breed, there was no difference in the fat content between the early (5.31±0.51%), middle (4.67±1.56%) and late (5.28±0.75%) stages of lactation. However, in the case of crossbred cows, the fat content was higher at the end of lactation (4.45±1.43%) than at the beginning of the lactation (3.41±1.09%) or in the middle stage of lactation (3.33±1.05%) [6].
Similar to the fat content, a change in protein content can also be observed as we progress towards the end of lactation. The average protein content measured at the beginning of lactation was 3.08±0.15%, it was 3.20±0.19% in the middle of lactation and 3.56±0.20% at the end of lactation, the difference being significant (P<0.05). Bondan et al. came to a similar conclusion: a higher protein content (3.41±0.36%) was measured at the end of lactation (>200 days) than at earlier stages of lactation. It was also found that the measured average protein content (3.03±0.31%) was the lowest between days 61 and 120 of the lactation of the cows. The measured average protein content between days 6 and 60 of the lactation and between days 121 and 220 were 3.05±0.36% and 3.18±0.32%, respectively [5]. Gurmessa and Melaku, as well as the research group of Pratap did not find any difference in protein content between cows in the early (3.55±1.43%), middle (3.17±0.15%) and late (3.33±0.16%) stages of their lactation [33, 34]. In the case of local and crossbred cows in Sudan, changes in milk protein content with the lactation stage were investigated by Shuiep et al. In the case of the local breed, the protein content of the milk of the animals was higher at the beginning (3.87±0.52%) and in the middle (3.91±0.18%) of lactation than at the end of lactation (3.67±0.17%). In the case of crossbred cows, there was no difference in protein content at the beginning (3.67±0.17%), middle (3.69±0.16%) and end (3.63±0.22%) of lactation [6].
The average somatic cell count in the early stage of lactation of the cows selected for our research was 195.1×103 (5.07±0.43 lg) cell/ml, it was 370.6×103 (5.28±0.50 lg) cell/ml in the middle of lactation, and it was 336.4×103 (5.33±0.41 lg) cell/ml in the late stage of lactation. The somatic cell count was higher (P<0.05) in the milk of individuals in the late stage of lactation than in the case of cows at the beginning of lactation. As lactation progresses, somatic cell counts also show an increasing trend according to Bondan et al. While in the milk of Holstein-Friesian cows between days 6 and 60 of their lactation the average somatic cell count was 4.79±1.90 lg cell/ml, between days 61 and 120 it was 4.89±1.90 lg cell/ml, between days 121 and 220 it was 5.21±1.75 lg cell/ml, and in the case of lactations lasting more than 220 days, this parameter was 5.53±1.53 lg cell/ml in the milk of the cows studied [5].
Total plate counts were also determined during the different stages of lactation of Holstein-Friesian cows. At the beginning of lactation, the average total plate count was 6.8×103 (3.42±0.67 lg) cfu/ml, in the middle of lactation it was 4.4×103 (3.39±0.46 lg) cfu/ml, while at the end of lactation it was 2.7×103 (3.13±0.56 lg) cfu/ml. No significant difference was found between the total plate count values obtained (P>0.05).
Regarding the number of coliform bacteria, the highest count [1.3×103 (1.30±1.23 lg) cfu/ml] was measured in samples taken at the beginning of the lactation of the cows, while the lowest average coliform count [2.2×101 (0.76±0.79 lg) cfu/ml] was detected in samples taken in the middle stage of lactation. The average coliform count in the samples taken at the end of lactation was 1.50×102 (0.90±0.94 lg) cfu/ml. There was no significant difference between the results (P>0.05).
S. aureus was present in a total of 9 samples (14.52%) out of 62 individual milk samples, with an average count of 1.4×102 (1.89±0.53 lg) cfu/ml. In the six samples (9.68%) taken from cows in the early stage of lactation, the average S. aureus count was 1.2×102 (1.81±0.56 lg) cfu/ml, for the single animal in the middle of lactation (1.61%) the S. aureus count was 8.2×101 (1.91 lg) cfu/ml, while for the two cows at the end of lactation (3.23%) it was 2.4×102 (2.15±0.70 lg) cfu/ml.
Table 2. Milk yield of cows in their early, mid and late lactation stages and compositional and microbiological properties of milk from them
a, b, c The values marked with different letters in the same rows of the table differ significantly (P<0.05)
5. Conclusions
In the course of our research work in a Hungarian dairy farm, it was proven that, under large-scale husbandry conditions, for primiparous cows the average daily milk yield is significantly lower (P<0.05) than in the case of multiparous cows. This is probably due to the fact that cows in their first lactation need more amino acids and fat to develop their bodies compared to animals that have already undergone several lactation periods [38].
Regarding the fat and protein content of the milk, there was no significant difference between primiparous and multiparous cows. Since one can find in the literature findings that both support and contradict our results, we assume that the fat and protein contents of milk are also influenced by other factors (e.g., breed, season, etc.), but mapping out of these factors was not the objective of our study. For example, Shuiep et al. reported in their paper different results for two different cattle breeds when examining the changes in the fat and protein contents of milk with the lactation number [6].
Based on our measurement results, we found that the somatic cell count, as well as the coliform count and the S. aureus count were significantly higher (P<0.05) in the milk samples taken from multiparous cows compared to the milk samples taken from primiparous cows. The reason why microorganisms can be measured in larger amounts in milk samples taken from multiparous cows is probably that the teats may have been damaged during the lactations (due to, for example, the milking machine), which may have increased the chances of microorganisms entering the udder [24]. Another reason may be that as the age progressed or the number of lactations increased, the condition of the dairy animals may have deteriorated, which may have had an adverse effect, for example, on the somatic cell count of the milk [25].
It was also proven that, in the stages of lactation, a decreasing trend in daily milk yield can be observed, but the fat and protein contents show an increase. This is presumably due to the concentrating effect of the decreasing milk yield.
There was no difference in milk samples taken at different stages of lactation in terms of total plate count and coliform count, however, in the late stage of lactation, the somatic cell count showed an increase.
6. Acknowledgment
This publication was supported by the project EFOP-3.6.3-VEKOP-16-2017-00008. The project was supported by the European Union and co-financed by the European Social Fund.
We are grateful to the management and staff of the dairy farm for their helpful assistance.
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Characterization of Serratia species and qualitative detection of Serratia marcescens in raw and pasteurized milk by an analytical method based on polymerase chain reaction
1 Hungarian Dairy Research Institute Ltd, Mosonmagyaróvár 2 Széchenyi István University, Wittmann Antal Multidisciplinary Doctoral School in Plant, Animal, and Food Sciences, Mosonmagyaróvár 3 Széchenyi István University, Faculty of Agricultural and Food Sciences, Department of Food Science, Mosonmagyaróvár
Serratia species are opportunistic pathogenic microorganisms primarily known as nosocomial infectious agents, which can also cause food quality problems. The appearance of the extracellular pigment-producing Serratia marcescens in cow’s milk causes its red discoloration, posing a challenge to the dairy industry and food certification laboratories. The detection of the bacterium by conventional procedures based on microbiological methods is time-consuming and labor-intensive, and in many cases does not lead to satisfactory results due to the competitive inhibitory effect of the accompanying microflora. Following the analysis of the relevant literature, the published endpoint PCR methods and the primers used for the detection of S. marcescens were evaluated in in silico and in vitro assays, and then the procedure was tested on farm milk samples. Using the method, a total of 60 raw and pasteurized milk samples were analyzed, more than half of which (i.e., 32) were identified as S. marcescens positive. The significance of our work is mainly represented by the application of the published test methods in food industry practice. Our results highlight to the importance of detecting this bacterial species.
2. Introduction and literature review
Nowadays, the impeccable quality and long shelf life of foods is a basic requirement of consumers. Accordingly, there is a growing demand for ever faster, more accurate and more reliable food diagnostic procedures. In this context, molecular diagnostic methods are gaining ground, for example in the rapid detection of pathogenic microorganisms. Polymerase chain reaction (PCR)-based diagnostic kits suitable for the identification of pathogenic microbes are produced by many manufacturers, and these are also used successfully in Hungarian food testing laboratories. These molecular biological tests are mainly suitable for the detection of microbes hose presence poses a high risk to public health (e.g., Escherichia coli, Salmonella Typhimurium, Listeria spp.). Less attention is paid to pathogens that are not required to be tested by law, such as Serratia species present in raw and pasteurized milk.
Serratia species are found in many places in our environment [1]. They are saprophytes or opportunistic pathogens [2], facultative anaerobic, biofilm-forming organisms [1, 3]. S. marcescens grows particularly well in phosphorus-containing environments (e.g., soaps, shampoos) and is also resistant to certain disinfectants [4, 5], so it can cause various nosocomial diseases [6, 7, 8]. Increasing antibiotic resistance of S. marcescens has also been reported in the literature [8, 9, 10]. The bacterium therefore survives and grows easily, so it may find its way into foods under inadequate hygienic conditions. Presumably, it can enter drinking milk as a result of violating hygiene rules, it can grow there and degrade the quality of food [1, 11, 12]. For some species, spoilage is indicated by a characteristic red hue.
In the case of the Hungarian dairy sector, accurate data are not available on the extent of the prevalence of Serratia species and S. marcescens, and on which species cause the infections and degrade milk quality. Nor is there a Hungarian survey on the extent of Serratia contamination of dairy farms. With the exception of a few publications, the available information on the exposure of the dairy industry to Serratia is also lacking at the international level. Such exceptions are a scientific article on the epidemic of mastitis caused by S. marcescens at Finnish diary farms [1], and an older report discussing the role played by pigment-forming Serratia species in mastitis [13].
The following Serratia species may be responsible for the red discoloration of milk: S. marcescens, S. rubidaea, S. plymuthica and S. nematodiphila (Table 1). According to their incidence, S. marcescens is of greater importance. Their characteristic pigment is the red prodigiosin, a water-insoluble secondary metabolite that is produced under specific environmental conditions [14, 15, 16, 17] (Figure 1). The typical red colonies appearing on the culture medium alone do not provide sufficient information to identify Serratia, as certain species of many other genera, not belonging to Enterobacteria, may also produce prodigiosin [14, 18].
Table 1. Characterization of Serratia species and their pigment production [19–22]Figure 1. Pure culture of Serratia marcescens on tryptone-soy agar (TSA) (30 °C, 48 h)
There is currently no ISO standard for the detection of Serratia species in foods. In their 2006 book chapter [9], Grimont and Grimont discuss the characteristics of the genus Serratia, as well as aspects of their isolation and identification. However, identification by classical microbiological methods is rather cumbersome and often ineffective due to the inhibitory effect of the accompanying flora, despite the fact that the pink discoloration of the milk sample is clearly visible to the naked eye. Although culture media are available for the selective growth of the bacterium [47], in practice their use does not provide a satisfactory solution. In addition, conventional methods are time and labor intensive.
There are commercially available rapid methods for the determination of S. marcescens, for example the miniaturized test kit from bioMérieux called Rapid ID 32 E, which satisfies the requirements of standard ISO 7218 [48]. However, a colony growing on a culture medium is required to perform the test. Diagnostic tests based on the PCR method, as mentioned before, could provide a solution to overcome the difficulties of detection. At present, however, only the Genesig product of Primerdesign can be mentioned as a molecular diagnostic kit for the detection of S. marcescens[49].
The literature relevant for the food industry and, in particular, the dairy industry, is rather poor on the detection of Serratia species, including S. marcescens, by either endpoint PCR or real-time PCR methods. Hejazi et al. [50] carried out the serotyping of S. marcescens by the RAPD-PCR technique. Serological samples from patients in need of hospital care were used in their study. Iwaya et al. [6] also tested blood samples for S. marcescens strains using a real-time PCR method. Zhu et al. [51] performed molecular characterization of S. marcescens strains by RFLP and PCR methods, while Joyner et al. [2] detected S. marcescens strains in marine and other aquatic environmental samples (e.g., coral mucus, sponge pore water, sediment, sewage, wastewater and diluted wastewater) by real-time PCR. A study of Bussalleu and Althouse, published in 2018, reports a conventional endpoint PCR technique suitable for the identification of S. marcescens that effectively detects the presence of the microorganism in wild boar semen [52].
Our goal was the set up a classical PCR method suitable for the detection of S. marcescens in milk. The significance of our work lies in the fact that PCR-based methods described in the literature and the primers used were analyzed, then the procedure deemed appropriate was adopted to food hygiene analytical practice. In our experiments, qualitative determination of the possible S. marcescens contamination underlying the discoloration of factory, raw and pasteurized milk samples was performed.
3. Materials and methods
3.1. In silico studies
Based on the literature, three primer pairs (Table 2) were selected, which were evaluated by computer modeling, by so-called in silico analysis, as well as in vitro experiments in order to find the most suitable one for subsequent PCR assays.
Table 2. Serratia marcescens-specific primer pairs used in this study
In our in silico studies, the specificity of the a primer sequences was verified by comparison with a DNA database (NCBI BLAST) [54]. Comparison with the database allows for homology search (“blasting”). Following this, the suitability of the primers, i.e., whether a possible PCR reaction takes place with the selected genomes, was tested with a molecular biology software (SnapGene 5.1.5.) [55]. In the latter case, positive and negative control genomes were downloaded from the NCBI database, and then the SnapGene software was used, in an in silico way, to investigate whether the PCR reaction would take place with the primer pairs. The positive and negative controls used for reference purposes were whole chromosome genomes (Table 3).
Table 3. Genomes of bacterial strains used as positive and negative controls in in silico analyses and their reactions to primer pairs
* Primerek: A. Fpfs1 és Rpfs2; B. FluxS1 és RluxS2; C. Serratia2-for és Serratia2-rev.
Jelmagyarázat:
3.2. In vitro experimental studies
To confirm the results of the in silico studies, in vitro were performed in which the selected primer pairs were tested in laboratory PCR analyses on genomic DNA samples of selected strains of bacteria (several S. marcescens strains were used as positive control and Lactobacillus delbrueckii subsp. delbrueckii, Streptococcus thermophilus, Enterococcus faecalis and Micrococcus luteus were used as negative controls). The microorganisms were bacterial strains belonging to the collection of MTKI Kft. and coming from factory environment, determined by genetic identification.
When putting together the components required for the PCR reaction, 5.2 µL of PCR grade sterile water, 10 µL of DreamTaq Green 2× PCR Master Mix (Thermo Fisher Scientific, Waltham, Massachusetts, USA), 0.4 µL (10 pmol/µl) primer and 4 µL of isolated bacterial genomic DNA were used for each reaction. The negative control of the reactions was PCR grade sterile water. The program parameters of the PCR instrument (Mastercycler Nexus Gradient; Eppendorf International, Hamburg, Germany) were as follows: 95 °C for 1 minute, then for 40 cycles 95 °C for 15 seconds, 59.5 °C for 15 seconds, 72 °C for 10 seconds and, finally, 72 °C for 7 minutes [52].
For size separation of the DNA segments formed during the PCR reaction, a 10 µL sample was analyzed on a 2% agarose gel [TBE buffer (Tris-borate-EDTA) (10×), Thermo Fisher Scientific; Agarose DNA Pure Grade, VWR International, Debrecen, Hungary; ECO Safe Nucleic Acid Staining Solution 20.000×, Pacific Image Electronics, Torrance, California, USA]. The DNA size marker was the GeneRuler Low Range DNA Ladder (Thermo Fisher Scientific). Gel documentation was performed using the Gel Doc Universal Hood II gel documentation equipment and software (Bio-Rad, Hercules, California, USA).
3.3. analysis of raw and pasteurized milk samples
On the one hand, we used in our study factory raw and pasteurized milk samples in the case of which S. marcescens contamination was suspected due to their pink discoloration. On the other hand, factory raw and pasteurized milk samples that arrived at the laboratory together with the above samples but not exhibiting discoloration were also tested.
For the DNA digestion and purification process, the NucleoSpin Microbial DNA kit (Macherey-Nagel, Düren, Germany) was used according to the manufacturer’s instructions. The reaction tubes containing the eluted DNA were stored in a freezer at -20 °C.
Next, the suitability of DNA isolation and the amplifiability of the samples were checked by 16S rDNS polymerase chain reaction, using primers 27f (5’-AGAGTTGATCMTGGCTCAG-3’) and 1492r (5’-TACGGYTACCTTGTTACGACTT-3’). The total volume of the PCR reaction for 1 sample was 5.6 µL of PCR grade sterile water, 10 µL DreamTaq Green 2× PCR Master Mix, 0.2 µL (10 pmol/µl) of the primers and 4 µL of isolated bacterial genomic DNA. The negative control of the reactions was PCR grade sterile water. The program parameters of the PCR instrument were as follows: 95 °C for 4 minutes, then for 40 cycles 95 °C for 20 seconds, 54 °C for 30 seconds, 72 °C for 1 minute and, finally, 72 °C for 5 minutes.
For the separation of the DNA segments formed during the PCR reaction, a 5 µL sample was analyzed on a 1% agarose gel. The DNA size marker was the GeneRuler 1 kb Plus DNA Ladder (Thermo Fisher Scientific). The DNA sample tested was judged to be suitable for further PCR analysis if the length of the copies of the amplified DNA fragment was as expected (~1500 bp).
In the next step, samples were subjected to S. marcescens-specific PCR analysis and gel electrophoresis as described in subsection IN VITRO EXPERIMENTAL STUDIES. The results were evaluated on the basis of the presence/absence principle.
In order to check the suitability of the method, PCR results of the milk samples were compared with the few available API (bioMérieux, Budapest, Hungary) test results in a control test. The method was then used to detect the presence of S. marcescens in raw and pasteurized milks.
4. Results
In our in silico studies, when examining the homology of the primers, they showed similarity primarily to S. marcescens chromosome genomes. However, matches were also found in the case of S. rubidaea and S. nematodiphila strains and some non-Serratia species. These results were taken into account during the selection of reference genomes designed for our SnapGene software studies. The need for further investigation was justified by the fact that appropriate homology or the matching of the basis do not automatically mean that the PCR reaction will take place, because the direction of the primers, their melting temperature and the size of the PCR product formed are also critical, among other things.
In the SnapGene test, PCR reactions were predicted with the following parameters: our analyses were performed with at least 15 bases matching and the exclusion of single isolated mismatches. The minimum melting temperature was 50 °C and the maximum length of the fragment obtained as the result of the amplification was 3 kbp.
As shown in Table 3, when matched with the S. marcescens genomes, the primer pair Serratia2-for and Serratia2-rev showed amplification in all cases. The PCR reaction generally resulted in six or seven amplicons on the 16S rDNA sections. The adhesion site of the Fpfs1–Rpfs2 and FluxS1–RluxS2 primer pairs is located outside the 16S rDNA in most S. marcescens strains, but in some cases they did not show in silico amplification, so their sensitivity did not prove to be adequate. In the negative control genomes, the completion of a PCR reaction was predicted by the primer pair Serratia2-for and Serratia2-rev in some cases for certain S. rubidaea and S. nematodiphila strains. Using primers Fpfs1–Rpfs2, the PCR reaction would take place in the case of a S. nematodiphila strain. Primers FluxS1–RluxS2 did not predict the occurrence of a reaction on any of the selected negative control genomes (Table 3).
In S. marcescens genomes selected as positive controls in in vitro experiments, all three primer pairs gave signals according to the expected fragment size, and none gave a signal on the negative controls. The analysis carried out with the primer pair Serratia2-for and Serratia2-rev is shown in Figure 2. In the case of negative samples, the weak signals at around 50 bp are caused by the accumulation of the byproduct aspecific DNA fragments, primer dimers.
Based on the results of in silico analyses and in vitro studies, primers Serratia2-for and Serratia2-rev were considered to be suitable for further work, despite the fact that their specificity was not perfect. The decision was based on the probable frequency of occurrence of S. marcescens on the one hand and the importance of avoiding samples with false negative results on the other.
In order to check the suitability of the method that had been set up, factory milk samples were tested in a control study. Some of the milk samples (n=10) exhibited pink discoloration. Using our test method, nine samples were found to be positive for the microbe sought. We also had API test results for four of the samples. The four API-positive samples were also found to be positive in the PCR assay. The method was then used to detect S. marcescens in raw and pasteurized milks.
Some of the milk samples showed peach-pink discoloration (Figure 3), but it was not clear in many cases due to the pale or yellowish tint. A total of 60 samples were analyzed. Of these, 32 (53.3%) gave positive results and 28 (46.7%) gave negative results for the presence of S. marcescens.
Figure 4 shows the result of one of our assays, the separation by gel electrophoresis. It can be clearly seen that the positive control strain gave a positive signal, while the negative control sample gave a negative signal, and positive signals were obtained for three test samples. The weak signals appearing in the case of negative samples are again caused by the accumulation of primer dimers.
Figure 2. Results of PCR analysis with Serratia2-for and Serratia2-rev primers on the genome of selected bacterial strains. Lanes: 1. Serratia marcescens 551R; 2. Serratia marcescens 1911; 3. Lactobacillus delbrueckii subsp. delbrueckii 0801; 4. Streptococcus thermophilus 1102; 5. Enterococcus faecalis 1101; 6. Micrococcus luteus CLTB1; 7. Negative control (sterile water); M: Molecular weight markerFigure 3. Milk samples. Left sample is netive and right sample is positive for Serratia marcescens, based on the result of PCR testFigure 4. Gel electrophoresis image of Serratia marcescens-specific PCR assay. Lanes 1 to 7: Milk samples; K+: Positive control (genomic DNA from Serratia marcescens); K-: Negative control (sterile water); M: Molecular weight marker
5. Discussion
When evaluating our results, it is important to take into account that the PCR analysis is a method suitable for the amplification and detection of the target DNA in the sample, based on which it is not possible to determine whether the amplified S. marcescens-specific DNA comes from viable, dead or so-called VBNC cells. In the VBNC (“viable but not culturable”) state, the cells are viable, metabolically active, but cannot be propagated by classical culture methods. This condition is reversible.
The objective of our work was to establish a classical PCR method for the detection of S. marcescens. Using the test procedure applied, qualitative determination of the S. marcescens contamination responsible for the discoloration of milk samples can be carried out.
Although the experiments presented here focused on the detection of pigment-producing S. marcescens, a future genus-level study could identify all 20 Serratia species (Table 1). The significance of the detection of other Serratia species is evidenced by the fact that, although the genus Pseudomonas is the main cause of the spoilage of chilled raw milk, the dangers of Serratia species in this respect are also known [56]. In addition to Pseudomonas strains, Serratia strains have also been identified in many cases as causes of milk spoilage. Members of the genus Serratia have been detected in dairy plants [3, 12], in raw milk samples stored at 4° C [56, 57, 58] and in milk containers [59]. It was noted by Grimont and Grimont[9] already a decade and a half ago that raw milk lots can occasionally be contaminated with Serratia species, and the species most often occurring in diary products are S. liquefaciens and S. grimesii.
The presence of psychotrophic Serratia species (e.g., S. liquefaciens) in raw milk can cause spoilage even after heat treatment. Baglinière et al. found that the thermally stable Ser2 protease produced by S. liquefaciens may be a significant factor in the destabilization of UHT milk [11, 60].
In conclusion, it can be stated that a genus-level study would be an interesting research project that would fill a gap, and which would allow the monitoring of raw milk in this respect, the wide detection of Serratia species. Presumably, the results would provide useful information not only to the stakeholders of the dairy economy and the dairy industry, but could also have an impact on Hungarian regulatory and monitoring practice.
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1 Szent István University, Faculty of Mechanical Engineering, Institute of Process Engineering, Gödöllő (Since 01. Feruary 2021: Hungarian University of Agriculture and Life Sciences, Institute of Technology) 2 Szent István University, Faculty of Mechanical Engineering , Institute of Machinery and Informatics, Gödöllő (Since 01. Feruary 2021: Hungarian University of Agriculture and Life Sciences, Institute of Technology)
Keywords
fruit damage, TTF (time to failure), rheological testing of fruits, viscoelastic models, time-dependent deformation, loading and unloading curves, dissipated energy, biological yield point, biological rupture point, damage limit value, damage resistance, creep curve, deformation
1. Summary
One of the most significant phenomena in the processing of horticultural crops, leading to the damaging of the fruit, is fatigue due to repeated mechanical stress, which endangers the integrity of the produce, especially during transport. In the event of such damages, the immediate environment of the damaged fruit, or even the entire batch of crops may be in danger, as the biological processes leading to spoilage are not limited to the individual crop damaged. In the case of repeated effects, a force less than the static limit value is sufficient to cause spoilage, but in addition to the load, the material properties of the given crop, as well as the energy balance observed during damage play important roles in determining the mechanical resistance. Accordingly, in our work, a description of the spoilage process is built on the material models most characteristic of the selected crops, on the dissipated energy indicators measured during repeated loads, and on the definition and determination of the spoilage time. In the experiments, the fatigue indices of Golden Delicious apples, making up most of the apple production of the European Union, and of long shelflife Packham pears are compared by setting up linear regression models.
2. Introduction
When sorting produce, not only the size and shape, but also the extent of a possible damage or, in many cases, the fact of the damage itself is the basis for the selection. Automated machine recognition, which in most cases is performed by spectral imaging methods, today can effectively separate damaged crop tissues from healthy ones and finding damages under the surface which are not visible to the naked eye does not pose a problem to the technology either [1, 2]. Reliability depends on the hardware design (i.e., the accuracy of the equipment used) on the one hand, and on the algorithms used [3]. In addition to sorting, the method also uses camera monitoring, which can take into account the ripeness of tomatoes with the help of the appropriate software, and which allows the fully automated operation of the harvesting robots [4].
Although with effective detection the damaged crops can be easily removed from the processing chain, in addition to screening, the objective of getting as many healthy goods as possible to customers after the harvest, and the necessary treatment processes must also be kept in mind. Since international surveys show that a significant proportion of crops does not reach consumers in the market due to losses at different stages of processing [5, 6, 7], in addition to the precise detection of injuries, prevention must also play a key role. This also requires destructive testing of the crops and the direct observation of spoilage processes.
The material properties of various agricultural and horticultural crops can be described using viscoelastic models, consisting partly of elastic and partly of viscous components. Complex material structures can also be built from basic elements connected in a serial or parallel way, and of the three-element systems, the Poynting-Thomson model has been used several times in previous research to characterize Maloideae [8, 9, 10]. In the case of viscoelastic systems, deformation due to mechanical interactions depends not only on the magnitude of the stress, but also on the speed of the load, and creep and relaxation are an important part of the load and deformation process: while in the case of the former, a constant load results in increasing deformation, in the case of the latter phenomenon, a constant deformation results in a continuous decrease in stress [11].
The reaction of a given crop to a mechanical impact is shown on the load-deformation curve which, in addition to the creep and relaxation parameters, provides information on the total amount of energy generated in the load process: the area enclosed by the load and unload curves also serves as the basis for dissipated energy calculations in other fields [12, 13], and it is closely related to the viscoelastic properties of the test material and, in the case of crops, to the mechanical resistance and the susceptibility to damages [14].
The load limit that leads to microscopic damage to the cell structure, which can also cause crop spoilage, is called the biological yield point. Although as biological materials, different crops may be capable of healing or even complete regeneration, mechanical impacts applied during processing should be kept below the biological yield point. The limit value can also be indicated by a damage visible to the naked eye and affecting a larger area, which is called the rupture point in the literature. In the case of such damages, the crop is very likely to spoil [15, 16]. There is usually a significant variance between damage limits (even in the case of the same exact load), which is also affected by the ripeness of the given crop, as well as the conditions provided during storage and processing.
In additions to collisions resulting from improper handling, most damages are caused by vibrations during transport. Unfortunately, the observation of processes that end in damages by destructive tests is not an area that today’s research focuses on, although the mapping of fatigue due to repeated loads is also essential in fruits [17].
During transport simulations, the frequencies causing the greatest damage have already been unanimously identified [18, 19, 20], so in the case of destructive tests with repeated loads, experience shows that it is advisable to set the frequency range below 10 Hz.
Multivariate regression models that take into account different test parameters are often used to describe the mechanical properties of fruits and vegetables [21, 22]. The objective of our research was to study the less discussed phenomenon of fatigue in crops, and to determine the relationship between damage limit values (biological yield point or rupture point) and related factors (energy balance, material properties). The goal was to establish a linear equation for the damage limit value, which is determined by considering the parameters that can be measured during repetitive compressive load.
3. Materials and methods
3.1. Measuring instrument and the securing of the fruits
Destructive tests were performed with the instrument called DyMaTest, provided by the Hungarian Institute of Agricultural Engineering of NAIK. The instrument applies a load to the fruit with a cylindrical (flat-faced) pressure pin, and the pressure force can be adjusted arbitrarily using the software interface developed for the instrument [23]. The deformation of the crop can be registered with a laser sensor that detects the movement of the measuring pin, and the force can be registered with a special measuring cell designed for the instrument. Tests were performed after setting a sinusoidal pressure force up to the fruit failure limit.
To perform the measurements, the crops were secured in a sand bed. To check that the creep of the sand applied did not affect the results obtained, control measurements were performed using a completely inelastic bearing ball with a diameter of 32 mm. During the compressive loads, there was no detectable displacement in the measuring range of the photoelectric sensor, so the deformation of the sand does not appear on the load curves of the fruits at all. Prior to testing, sand preparation consisted of wetting, sieving and compaction operations [24].
3.2. Crop deformation curves
For the tests with repetitive loads, a cyclic waveform was used, which can be characterized by the following function:
Fm = Fmax(1-cos(ωt))
where Fmax is the peak value of the periodic load function [N] and ω is the angular velocity of the load [s-1].
The resulting deformation due to periodic loading is also periodic. Figure 1.a shows the time function of the deformation of a Golden apple, while Figure 3.b shows the force-deformation curve. Typical deformation curves for Packham pears are shown in Figures 3.c and 3.d.
As a result of the cyclic load with a constant amplitude, the deformation changes continuously, and this can be noticed in the increase of the envelope (or the mean). Since these envelopes increase similarly to the creep curves observed under static loading, this process is called dynamic creep [25].
The response function to the cyclic load can be described by the following equation:
Wm = β+Wmax(1-cos(ωt-δ))
where w is the deformation [mm], β is the creep term, wmax is the peak value of the periodic deformation function [mm], ω is the angular velocity [s-1], and δ is the phase shift between the load and deformation time functions.
To characterize creep (in this case, to give ), the literature generally uses a linear approximation. Although this approximation may be appropriate for a significant region of the creep in most cases, the initial and failure sections of the curve cannot be linearized, so the method carries inaccuracies when considering the entire creep process. In order to avoid this, numerical solutions were used in the data management processes related to deformation, in which the operations were performed not by approximation, but by direct processing of the data series.
In the case of the curves shown in Figure 1, the damage limit of the fruits, in this case the rupture point, has already been determined, and the data after this point have been removed from the diagrams. By analyzing the curves obtained this way, we can actually obtain information about the energy conditions taking place until failure, as well as about the material properties experienced this far.
Since the rupture point cannot be distinguished clearly during the analysis of the diagrams in many cases, especially in the case of loads that take place rapidly and the concomitant sharply changing deformation processes, accurate determination was therefore performed by high frame rate video surveillance (Figure 2). The camera used recorded 240 frames per second, and the rupture point sought was the first frame of the failure phase, when the pressure pin visibly exits the slowly increasing deformation range during the creep phase and causes damage to the crop tissue that is visible from the outside by breaking the skin. In this case, both the skin and the flesh are damaged, so the material behavior is approximated by the modeling of not a structure with a homogeneous composition, but of a „structure”.
Figure 1. Time vs. deformation (a) and force vs. deformation (b) functions of a Golden Delicious apple, and time vs. deformation (c) and force vs. deformation (d) functions of a Packham pear Deformation – Time – ForceFigure 2. Determination of the rupture point by analyzing high frame rate recording Force - Deformation
The sampling frequency of the DyMaTest is 2 kHz, which is 8.3 times higher than that of the video recordings of the rupture point. The absolute error of the frame analysis compared to the data collected by the material testing instrument is 4.16 milliseconds, which is the lowest resolution unit of the camera. Figure 2.b illustrates the error range for the rupture point. The rupture point as a test parameter is hereinafter denoted by the notation , which refers to the term time to failure.
3.3 Viscoelastic material properties
To determine the material properties of fruits, the three-element Poynting-Thomson model was used, which had already been used in previous research projects on apples. The coupling of the model is shown in Figure 3, and it can be characterized by the following equation:
where E1 and E2 are the elastic components of the mechanical model [N mm-1] and ƞ is the viscous element [Ns mm-1]. Fm is the compressive force recorded during the measurements [N] and wm is the deformation obtained during the measurements [mm].
Figure 3. Identification of the computer mathematical model DyMaTest material testing equipment - Investigated crop - Mathematical model – Measured compressive load – Measured deformation – Calculated deformation
The block-oriented writing of the equation was performed in a Matlab Simulink environment, where the model was identified with the force and deformation data obtained during the measurements (Figure 3). The values of the elastic and viscous coefficients were determined for the calculated curve (w) that best fit the measured results ()wm). To minimize the difference between the two data sets, we used a procedure based on the least square method:
After running the minimum search process, the model coefficients E1, E2 and ƞ were recorded and were used as test parameters. The approximations carried out with the presented mathematical system provided R2 values between 0.967 and 0.998.
3.4. Analysis of the hysteresis curves
The force vs. deformation diagrams in Figures 1.b, 1.d and 4 show recurrent hysteresis processes where the area enclosed by the load and unload curves is closely related to the energy indices of the crop for the given cycle. The horizontal axis shows that the curve does not close after unloading, so a wM permanent deformation occurs in the material in each cycle until the next compressive load, and the wR elastic deformation of the given crop is due to the difference between the load peak and the permanent deformation (the sum of the two gives the total magnitude of the deformation in the given cycle).
Figure 4. Force vs. deformation curve of a single load cycle (a) and the force vs. deformation curve until failure of a crop (b) for a Golden Delicious apple Load – Unloading - Deformation
If we examine the areas between the curves, by subtracting the energy associated with the elastic deformation (ER) from the total work (E), the dissipated energy of the cycle (ED) is obtained. This energy loss can be calculated by determining the area between the curves:
where twM is the time elapsed between the start of the loading process and the end of the unloading [s] and F is the load function produced by the test equipment [N].
Since the area calculation was performed by the numerical integration of the force and deformation data over time, the previously mentioned approximation functions and their inaccuracies associated with them can be avoided.
Although the calculation of energy losses is included in several studies that describe the damage mechanism, only a portion of the dissipated energy that can be determined from the hysteresis curve is related to material damage and the failure process [13]. In other fields, such as the rheological description of pavement asphalt layers, calculation methods have also been developed that point directly to the moment of failure using the dissipated energy data. These include the so-called dissipated energy quotient, which can be calculated by the following equation [26]:
where EDi is the total energy loss up to the given cycle [N mm] and EDn is the energy loss of the given cycle [N mm].
When the dissipated energy quotient is plotted as a function of the number of cycles (Figure 5), it can hint at two damage indicators: the onset of the cracking process of the given asphalt is indicated by a 10% drop in the ramp-up slope of the curve, and the fracture seen at the peak is the fatigue failure [26].
In the course of our experiments on fruits, the said drop in the slope cannot be observed so clearly in most cases, which is probably a consequence of the rapid load settings. However, the internal rupture point clearly appears in our own results as well. In addition to the time elapsed until the rupture point and the viscoelastic model coefficients, this data is also used to construct the equations describing the damage process.
Figure 5. Internal rupture point indicating fatigue based on the quotient calculated from dissipated energies for a Golden Delicious (a) and a Packham (b) produce Ratio of dissipated energy – Internal breaking point – Number of load cycles
3.5. Test parameters, load settings
Our objective was to describe, using parameters related to the damage process, the time to failure (TTF), which will be a dependent variable of the resulting equations. When characterizing failure, we aim to establish linear regression equations.
Compressive loads were applied to 25 Golden Delicious apples and 25 Packham pears (i.e., the number of replicates for each crop was 25), and six different measurement frequencies were used for each fruit. These frequencies fall into the range considered to be the most dangerous in transportation research, mainly in the range below 10 Hz, and taking into account the setting options of our instrument, they were 2.5, 3.7, 5, 7.5, 10 and 11.6 Hz. Thus, a total of 300 compressive loads were applied, and from the force, deformation and time data obtained during the loads, the E1, E1 and η coefficients of the material model were determined in each case, as well as the TTF time to failure and the EDRmax internal damage index, using the methods detailed above. In addition, it is also taken into account whether the process was influenced by the test frequencies.
Because of the different load resistance of the Golden and Packham crops, different compressive forces had to be applied: in the case of Packham pears, failure was already reached in one of the first cycles at certain values of the frequency range, while Golden apples were much more resistant, so considering the compatibility of the damage times and dissipated energy values to be detailed later, a load of 4 N to pears and a load of 14 N was applied to apples. In practice this means that at settings greater than 4 N, for most of the frequency values investigated, immediate destruction occurred in pears, and in the case of settings below 14 N, load processes orders of magnitude longer would have to be run to visibly damage the apples.
Results
4.1. Times to failure and energy indicators hinting at internal damage
Average and standard deviation values of the ties to failure for each frequency setting are shown in Table 1. Figure 6.a shows a chart of the average values of Golden apples, while for Packham pears, the results are shown in Figure 6.b. In the case of apples, the rupture points occurred as expected, i.e., irreversible damage occurred earlier at higher frequencies, while there was a deviation from this in the average values obtained for pears, as at settings above 5 Hz, there is an increasing trend can be seen in time to failure.
Table 1. Average times to failure and standard deviations of the results
In the case of the Golden apples, larger standard deviation values can be found at lower frequency settings, while at higher frequencies, the extreme values of the error ranges move closer to the average values. The endpoints of the standard deviation range show a similar trend to the frequency dependence of the average in Golden apples, but in the case of pears, the minimum values of the standard deviation range no longer represent the change in the average values, thus different characteristics are observed for pears between the 25 measurements.
Figure 6. Frequency dependence of the times to failure for Golden Delicious apples (a) and for Packham pears (b) Failure time - Frequency
Since the standard deviation is quite significant for both apples and pears (Table 1), the role of the additional parameters considered in the study (viscoelastic model coefficients, as well as energy indices) is particularly important when considering their effect on the damage process during the description of time to failure.
Figure 7 shows the peak values of the energy loss quotient calculated from the dissipated energy, and the results for 25 crops each were also averaged for each frequency setting.
In the case of the Golden apples examined, both the energy loss values recorded for each cycle and the maximum quotient values indicating internal rupture show a decreasing trend towards higher frequency settings, however, in the case of pears, this process is reversed, and the trend describing the frequency dependence also has a different nature.
Figure 7. Frequency dependence of the average values of accumulated dissipated energy Maximum of dissipated energy ratio
4.2. Evaluation of viscoelastic model parameters
The frequency dependence of the elastic (E1' E2) and viscous (ƞ) material properties of the crops is shown in Figure 8, where the values of each series of measurements are displayed averaged at each frequency setting. Numerical results are summarized in Tables 2 and 3.
Table 2. Average values and standard deviations of viscoelastic model parameters at each load frequency for Golden Delicious applesTable 3. Average values and standard deviations of viscoelastic model parameters at each load frequency for Packham pearsFigure 8. Averages of elastic and viscous model parameters for Golden apples (a, c) and Packham pears (b, d) Viscous element - Frequency
The elastic coefficients in the case of Golden apples do not exhibit an apparent frequency dependence, while a slight decrease can be detected in the case of the E1 parameters when higher test frequencies are used. In previous experiments, apples tended to behave more rigidly at higher load velocities [25]: if a higher load velocity corresponds to a higher frequency in the present case, then this reaction is consistent with the earlier experience.
However, in the case of pears, there is a clear increase when component E1 is examined, and this result may explain the obtained time to failure data: at the frequencies above 5 Hz, a more elastic, softer surface is formed near the load zone in the pears examined, and the increased elasticity provides a more favorable mechanical resistance for the crops. Thus, in the most dangerous frequency range, higher values do not necessarily carry the most significant damage potential. The E2 elastic coefficient is constant in the studied range for both Golden apples and Packham pears.
By plotting the viscous parameters, a clear frequency dependence is obtained for both Golden apples and Packham pears. The curve obtained for apples shows a similarity to the frequency dependence of a dynamic viscosity factor presented in a previous research [27], while in the case of pears, also the frequency around 5 Hz breaks the downward trend, this may also be related to the rupture point in the frequency curve of the times to failure.
The error ranges showing the standard deviations are wider in the case of pears, the widest range of standard deviation was recorded at the 2.5 Hz setting. One of the reasons for this is that with this setting, several pears were already destroyed in the first loading phase of the first cycle.
Table 4. Analysis of variance of viscoelastic model parameters
The degree of frequency dependence was checked by analysis of variance (ANOVA) and the results are summarized in Table 4. In the case of Golden apples, a significant correlation can only be detected for coefficient η (p<<0.05), and this confirms the conclusion that can be drawn from the diagrams, which were reached in the case of coefficients E1 and E2: the elastic elements and the frequency in the studied range are not detectably related. In the case of Packham pears, however, in addition to η, the frequency dependence of the elastic coefficient E1 can also be detected, which plays a significant role in the mechanical resistance experienced above 5 Hz.
4.3. Lineáris tönkremeneteli modellek
Using the results of the tests presented and the values of the load frequencies, the possibility of four different failure modes for Golden Delicious apples is suggested, according to the following search function:
TTF = A+Bη+CEDRmax+Df+KE1+JE2'
where A, B, C, D, K are E constants. The different versions are described in Table 5. These include the elastic and viscous material properties of the crops, as well as the peak value of the dissipated energy, but not the frequency settings.
Table 5. Linear models that can be created from the measured parameters for Golden apples
In the curves showing the model parameters and as the result of the analysis of variance, there was no significant relationship between the elastic coefficients and the frequency, but the elasticity for the Golden apples had a clear effect on the failure process, resulting in a detectable increase. While the elastic coefficient E1 is a defining part of the equation, E2 contributes only negibly to the accuracy of the fit, so we chose the third equation for the simplest description of the failure of Golden apples:
TFF = 0,533+2,736η+0,141EDRmax-0,261E1
The models applicable to Packham pears are summarized in Table 6. In these versions, the load frequency appears as well, playing an important role in the description of the time to failure.
Table 6. Linear models that can be created from the measured parameters for Packham pears
(a) variable: EDRmax
(b) variables: EDRmax, η
(c) variables: EDRmax, η, f
(d) variables: EDRmax, η, f, E2
Although the parameter E1 was related to the frequency, failure is not affected by this coefficient, but E2 connected in parallel with the viscous component. Since both the frequency and the elastic factor E2 contribute significantly to the accuracy of the linear approximation, a fourth equation was written for Packham pears:
TTD = 0-091+0,788η+0,085EDRmax-0,103f+1,524E2.
The results of the analysis of variance checking the validity of the equations are shown in Table 7. Since the F values obtained are considered to be significant (p<0.05), the approximations described are valid.
Table 7. Analysis of variance of the approximation equations
Time to failure results (TTFsz) of the models generated after substitution, as well as their relationships to the measured results (TTFm) are shown in Figure 9 over the entire study range. Averaged results by frequency of the approximation applied to Golden Delicious apples were between 1.54% and 3.85% relative error, while the results averaged by crop were between 1.01% and 31.13%. For Packham pears, averaging the results obtained for each frequency setting, the relative errors were between 2.42% and 6.22%, while the deviations of the values calculated for individual crops were between 0.04% and 34.51% compared to the measured time to failure. The higher error values were not related to the given frequency settings, but to the different mechanical resistance and material properties of each crop.
Figure 9. Relationship between measured and calculated times to failure for Golden Delicious apples (a) and Packham pears (b), evaluating all measurement results Faliure time (measured) - Faliure time (calculated)
5. Conclusions
Repetitive loading during fruit processing and transport procedures causes significant damage, so in our work we investigated failure caused by fatigue, and to this end we developed multivariate linear regression models based on the most important material properties and energy indices related to the failure process, and which can predict the damage resistance of the tested Maloideae (Golden Delicious apples and Packham pears).
In some cases, the rupture point indicating failure cannot be evaluated from the deformation data obtained during the measurements, in which case limit values determined by rapid filming and frame analysis may be helpful during the analyses. The accuracy of this depends on the frame refresh rate of the cameras used, and this, together with image resolution, is constantly evolving in mobile devices, so these devices are also becoming suitable for similar measurement tasks, and their use in the monitoring of the deformation of fruits is no longer unprecedented.
Observing internal damage on the basis of energy calculations may represent a new research direction in the study of fruit damages, as environmental impacts in processing procedures need to be addressed accordingly (limitation or modification of handling, dropping and vibration limit values). However, a precise definition of the phenomenon in order to describe the damage process in the cellular structure in more detail is still awaiting microlevel investigation and confirmation.
6. Acknowledgment
The authors would like to thank the Institute of Agricultural Mechanization of NAIK for providing the DyMaTest material testing instrument. We would also like to thank Dr. László Földi for his help in computer modeling and Dr. László Székely for his help in establishing the multivariate equations.
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