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Examination of skin-fermented natural wines

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Examination of skin-fermented natural wines


Received: May 2022 – Accepted: July 2022


1 Tokaj-Hegyalja Egyetem, Lorántffy Intézet, Szőlészeti és Borászati Tanszék
2 Pannon Egyetem, Soós Ernő Kutató- Fejlesztő Központ, Víztechnológiai Kutatócsoport


amphora, qvevri, ceramic egg, organic production, antioxidants, NMR analysis, quercetin, procyanidins, catechins, caffeic acid, p-coumaric acid, galacturonic acid, succinic acid, caftaric acid, tartaric acid, malic acid, hydroxycinnamic acid

1. Summary

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:

  1. 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;
  2. The grapes intended for winemaking may only be harvested by hand;
  3. Only spontaneous fermentation processes may be used;
  4. The use of additives is prohibited;
  5. No modification of the composition of the grapes (increase in acid or alcohol content) is allowed;
  6. No procedures classified as “rough” are allowed (e.g., filtration, tangential filtration, flash pasteurization, heat treatment, reverse osmosis);
  7. Addition of sulfur before or during fermentation is prohibited;
  8. 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”;
  9. 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).

Figure 1. Plain amphora [21]

Most important characteristics of amphora use:

  • 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.

7. Irodalom

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[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.

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Determination of fat-soluble vitamins A, D2, D3, E and K3 by isotope dilution and LC-MS/MS instrument assembly

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Determination of fat-soluble vitamins A, D2, D3, E and K3 by isotope dilution and LC-MS/MS instrument assembly


Received: February 2022 – Accepted: June 2022


1 Bálint Analitika Kft.


water-soluble and water-insoluble vitamins, vitamers, coenzymes, cofactors, recommended daily intake (RDI), isotope dilution

1. Summary

The purpose of our publication is the determination of the total amount (of natural origin and added) of fat-soluble vitamins A, D2, D3 and E in low amounts in foods (wheat flour, soft drinks, effervescent tablets) and dietary supplements using a liquid chromatography tandem mass spectrometry (LC-MS/MS) method. The samples were diluted with isotope-labeled derivatives of the target components (vitamin A-d6, vitamin D2-d3, vitamin D3-d3, vitamin E-d6), and after extraction and saponification, they were purified by liquid-liquid extraction. After a solvent exchange, the concentration of the vitamins was determined on a C8 HPLC column using acidic mobile phases (0.1% formic acid in water/methanol) and LC-MS/MS technique. In dietary supplements, the analysis of the fat-soluble vitamin K3 may also be important, because the use of vitamin K3 is currently not approved in human formulations. During the determination of vitamin K3, saponification is not necessary, due to its structure, alkaline hydrolysis would lead to the decomposition of vitamin K3, so this component was analyzed by a method different from the one used for the other vitamins. LC-MS/MS analysis of small amounts of vitamin K3 is more complicated than that of other vitamins due to the low sensitivity of the MS instrument to vitamin K3. The determination of vitamin K3 was therefore carried out after chemical derivatization with L-cysteine as a derivatizing reagent, also with isotope dilution and LC-MS/MS technique. After intralaboratory validation, the methods were successfully used in domestic and international proficiency tests in infant formulas and liquid vitamin preparations.

2. Introduction

Vitamins are organic molecules that are essential for the functioning of the human and animal body. They are necessary for the growth and maintenance of the cell population, for the proper functioning of certain organs, and for the maintenance of normal metabolism [1]. Vitamins are complex organic molecules whose structure and function in the body are very different from each other, so it is easiest to group them on the basis of their solubility. Based on this, water-soluble and fat-soluble vitamins are distinguished [1]. Vitamins B and C are water-soluble vitamins. These vitamins cannot be stored by the body for a long time, they are mostly excreted from the body in the urine, so it is necessary to replace them in the right amount every day. Vitamins A, D, E and K are fat-soluble vitamins (Table 1).

Table 1. Structure of the vitamins A, D2, D3, E and K3 investigated, their trivial names and most important physicochemical characteristics

Unlike water-soluble vitamins, the body can store these vitamins for months. Vitamin intake can be accomplished through a varied diet.

We can distinguish between natural vitamins found in foods and synthetic vitamins added to foods. Unfortunately, the latter ones cannot be utilized in the same way as their natural counterparts, and they also reduce the utilization of other nutrients and put a strain on the kidneys. This is because the vitamins added to the samples are not accompanied by the enzymes, coenzymes and cofactors necessary for absorption, as opposed to the vitamins naturally occurring in foods. Based on Annex XIII of Regulation (EU) No 1169/2011 of the European Parliament and of the Council, the recommended daily vitamin and mineral intake reference values for adults are 800 µg/day for vitamin A, 5 µg/day for vitamin D, 12 mg/day for vitamin E and 75 µg/day for vitamin K [2]. Vitamin K3 is used in animal husbandry and is added to feed, but vitamin K3 cannot be mixed with food intended for human consumption and cannot occur in dietary supplements either [3].

In the case of vitamin analysis, it is important to indicate whether it is the analysis of the vitamin added to the food or the determination of the total vitamin content. Naturally occurring vitamins B and vitamers are often present in the sample in a bound form, from which they can be released by hydrolysis or enzymatic sample preparation [4]. In the case of fat-soluble vitamins, however, sample preparation always includes a saponification step, during which the vitamins are released from their bound form, thus making it possible to determine the total vitamin content [5],[6],[7],[8],[9]. In the current paper, the determination of fat-soluble vitamins is discussed.

According to the relevant standard, vitamin analysis must be carried out using a liquid chromatography (HPLC) method with an optical detector (HPLC-UV). These standards contain the determination of vitamins occurring in high concentrations (>mg/100 g). The analysis of vitamins occurring in lower concentrations (µg/100 g) requires a longer or more complicated sample preparation, during which a high degree of sample purification and enrichment is performed (e.g., using preparative HPLC) [5],[6],[7],[8],[9], or we are forced to use a measurement technique that enables the selective determination of the target components even during the examination of complex matrices. One of these coupled techniques is liquid chromatography – tandem mass spectrometry (LC-MS/MS), which can be used with isotope dilution to determine the concentration of the tested compounds with high accuracy. In the course of isotope dilution, isotopically labeled (2H, 13C, 15N, 18O) analogs of the target components as internal standards (ISTD) are added to the samples and they are mixed homogeneously. These ISTDs compensate for the losses of target components during both sample preparation and instrumental analysis [4]. Our laboratory is committed to the use of isotope-labeled ISTDs, therefore we have developed an LC-MS/MS method for the determination of small amounts of fat-soluble vitamins, which contains all isotope-labeled analogs (deuterated compounds of vitamins). The purpose of our publication is to determine fat-soluble vitamins (A, D2, D3 and E) in wheat flour, soft drinks, effervescent tablets and dietary supplements using an LC-MS/MS instrument assembly, and also the validation and application of the method. Another goal was to develop an LC-MS/MS method for the determination of vitamin K3 in dietary supplements, during which chemical derivatization was attempted to achieve appropriate sensitivity.

3. Materials and methods

3.1. Materials and instruments used

Analytical grade standards of the vitamins (A, D2, D3, E and K3) and of the isotope-labeled analogs vitamin D2-d3, vitamin D3-d3, vitamin E-d6 and vitamin K3-d8, as well as L-cysteine, ascorbic acid, sodium hydroxide, Ascentis Express C8 (100 x 3 mm, 2.7 µm) HPLC column, HPLC grade solvents and formic acid were purchased from Sigma-Merck Kft. (Budapest, Hungary). Vitamin A-d6 was ordered from Cambridge Isotope Laboratories (Andover, MA, USA). Standards (with the exception of vitamin A) and internal standards vitamin E-d6 and vitamin K3-d8 were dissolved in ethyl alcohol so that their concentration was 1 mg/mL. The solutions were stored in a refrigerator at +4 °C for up to half a year. Labeled standards vitamin D2-d3 (100 µg/mL in methanol) and vitamin D3-d3 (1000 µg/mL in methanol) were obtained in a solution form. Vitamin A and vitamin A-d6 were dissolved in methanol containing 0.1% (m/v) butylated hydroxytoluene (BHT) (1 mg/mL) and stored at -18 °C for up to half a year. For calibration, a 10 µg/mL standard mixture (A, D2, D3, E) and a 10 µg/mL individual K3 standard solution were prepared in methanol and it was stored in a refrigerator at +4 °C for a maximum of 3 months. Of internal standards, an ISTD standard mixture of 20 µg/mL (vitamin A-d6, vitamin D2-d3, vitamin D3-d3, vitamin E-d6) and an individual ISTD standard solution of 10 µg/mL of vitamin K3-d8 were prepared in methanol and the were stored in a refrigerator at -18 °C for a maximum of 3 months.

For the LC-MS/MS studies, a Shimadzu Nexera UHPLC LC-30AD liquid chromatography system was used, which included a SIL-30AC autosampler, a CTO-20AC column thermostat and a CBM-20A communications bus module (Shimadzu Corporation, Kyoto, Japan). The triple quadrupole mass spectrometer coupled with the UHPLC was an AB Sciex 6500+ QTRAP with an IonDrive Turbo V Source ion source and a 6500 QTRAP instrument with a Turbo V Source ion source (the two systems were used alternately). The measuring software was Analyst (1.7.1) and the software used for quantification was MultiQuant (3.0.3) (Sciex; Warrington, Cheshire, UK).

The shaker used for extraction was a CAT S50 flask shaker (M. Zipperer GmbH, Ballrechten-Dottingen, Germany). A TurboVap II (Biotage, Uppsala, Sweden) type evaporator was used for the evaporation of the samples. Liquid vitamin dietary supplement proficiency testing samples and breakfast cereal quality control (QC) samples were ordered from FAPAS (Food Analysis Performance Assessment Scheme, Sand Hutton, UK), while the infant formula proficiency testing sample was ordered from the National Food Chain Safety Office (NÉBIH, Budapest, Hungary).

3.2. Sample preparation for the determination of vitamins A, D2, D3 and E

The analyses of wheat flour, soft drinks, effervescent tablets and dietary supplements were performed. 1.00 g of a homogeneous sample was measured into a 60 mL glass tube and 50 µL of a 20 µg/mL ISTD solution (vitamin A-d6, vitamin D2-d3, vitamin D3-d3, vitamin E-d6) was pipetted onto it, then 20 mL of ethanol and 5 mL of distilled water were added. Following this, 0.5 g of ascorbic acid and 5 mL of 12.5 M sodium hydroxide solution were measured onto the sample. The sample was mixed at 60 °C on a magnetic stirrer for one and a half hours. After the extraction/saponification, the sample was allowed to cool to room temperature and then 5 mL of distilled water and 5 mL of n-hexane was added. The sample was shaken for 1 hour (700 rpm), and then the liquid phases were allowed to separate for 10 minutes. 1.0 mL of the hexane phase was pipetted into a glass evaporating tube and it was evaporated to dryness at 40 °C under a stream of nitrogen. The sample residue was redissolved in 1.0 mL of methanol and it was filtered into an HPLC vial using a PTFE syringe filter (Gen-lab Kft., Budapest, Hungary). During sample preparation, there was a fivefold sample dilution.

3.3. LC-MS/MS method for the determination of vitamins A, D2, D3 and E

Vitamins were separated on a C8 HPLC column by linear and binary gradient elution (Figure 1). The aqueous mobile phase (eluent A) was 0.1% (v/v) formic acid in water, the organic mobile phase (eluent B) was 0.1% (v/v) formic acid in methanol. In the solvent gradient, the ratio of eluent B increased from 80% to 100% between 0 and 3 minutes, the ratio of eluent B was 100% between 3 and 10 minutes, the ratio of eluent B decreased to 80% between 10 and 10.1 minutes and it remained 80% until minute 14. The flow rate was 0.5 mL/min, analysis time was 14 minutes, injection volume was 5 µL, and the column thermostat temperature was 30 °C. MS/MS detection conditions are listed in Table 2. The settings of the ion source were as follows: sheath gas: 45 units, gas 1 (nebulizer gas): 40 units, gas 2 (drying gas): 40 units, drying gas temperature: 350 °C, capillary voltage: +5,500 V.

Table 2. MRM ion transitions of vitamins A, D2, D3 and E and the corresponding voltage values. Ion transitions used for quantitative evaluation are marked in bold.

3.4. Sample preparation for the determination of vitamin K3 in dietary supplements

1.00 g of a homogeneous sample was measured into a 60 mL glass tube and 100 µL of a 10 µg/mL vitamin K3-d8 ISTD solution was pipetted onto it, then 20 mL of ethanol and 5 mL of distilled water was added. The sample was extracted at room temperature for 1 hour (700 rpm), and then 15 mL of distilled water and 5 mL of n-hexane was added. The sample was shaken for 1 hour (700 rpm), and then the liquid phases were allowed to separate for 10 minutes. 1.0 mL of the hexane phase was pipetted into a glass evaporating tube and it was evaporated to dryness at 40 °C under a stream of nitrogen. The sample residue was redissolved in 0.5 mL of methanol and 0.5 mL of a freshly prepared 0.2% (v/v) solution of L-cysteine (1 mg/mL) with formic acid was added. After vortexing, the sample was allowed to stand at room temperature for half an hour until the reaction took place, and after another round of mixing, the sample was filtered into an HPLC vial using a hydrophilic PTFE syringe filter (Gen-lab Kft., Budapest, Hungary). During sample preparation, there was a fivefold sample dilution.

3.5. LC-MS/MS method for the determination of vitamin K3

After derivatization, vitamin K3 was separated on a C8 HPLC column by linear and binary gradient elution (Figure 2). The aqueous mobile phase (eluent A) was 0.1% (v/v) formic acid in water, the organic mobile phase (eluent B) was 0.1% (v/v) formic acid in methanol. In the solvent gradient, the ratio of eluent B was 20% between 0 and 1 minute, the ratio of eluent B increased from 20% to 70% between 1 and 5 minutes, the ratio of eluent B was 95% between 5.1 and 8 minutes, then the ratio of eluent B decreased to 20% at 8.1 minutes and it remained 20% until minute 12. The flow rate was 0.45 mL/min, analysis time was 12 minutes, injection volume was 10 µL, and the column thermostat temperature was 30 °C. MS/MS detection conditions are listed in Table 3. The settings of the ion source were as follows: sheath gas: 45 units, gas 1 (nebulizer gas): 40 units, gas 2 (drying gas): 40 units, drying gas temperature: 350 °C, capillary voltage: +5,500 V.

Table 3. MRM ion transitions of derivatized vitamin K3 and the corresponding voltage values. Ion transitions used for quantitative evaluation are marked in bold.

3.6. Optimizing ion transitions

1 µg/mL individual standard solutions diluted with 0.1% (v/v) formic acid in methanol were delivered from an infusion syringe to the mass spectrometer using a syringe pump, and with the help of the automatic optimization software, a minimum of 2 ion transitions were set for each component, except for vitamin K3. In the case of vitamin K3, 0.5 mL of the standard solution (10 µg/mL) was derivatized with 0.5 mL of L-cysteine solution and the derivative was optimized with 6 ion transitions in the mass spectrometer in order to find the transitions which the matrix compounds present in the sample do not possess within the retention time window of the K3 derivative.

3.7. Method validation

The determination of fat-soluble vitamins A, D2, D3 and E in wheat flour, soft drink, effervescent tablet and dietary supplement samples was validated by intralaboratory validation. The analytical performance characteristics examined were as follows: selectivity, identification (ion ratios), recovery at 0.5 and 5 mg/kg levels by the analysis of 10 parallel samples at each level, repeatability and reproducibility. The limit of quantification (LOQ) was determined from the signal-to-noise ratio. The determination of vitamin K3 in dietary supplements was validated on the basis of the same procedure at 0.1 and 1.0 mg/kg levels with 8 repetitions at each level. Calibration was checked by fitting a seven-point calibration curve where the points were 0.01 µg/mL, 0.05 µg/mL, 0.10 µg/mL, 0.50 µg/mL, 1.0 µg/mL, 5.0 µg/mL and 10.0 µg/mL. The concentration of ISTDs was 0.2 µg/mL.

4. Results and evaluation

4.1. LC-MS/MS method for the determination of fat-soluble vitamins

Due to their apolar nature, fat-soluble vitamins can be analyzed using atmospheric pressure chemical ionization (APCI) as an ion source during LC-MS measurements [10]. At the same time, the instrument used by us also ionized vitamins with high sensitivity using an electrospray ionization (ESI) source, so APCI was not necessary. Following ion transition optimization, chromatographic separation was attempted on a C8 HPLC column, because vitamins A, D2, D3 and E, due to their lipophilic nature (Table 1), show too high retention on a C18 column. In addition, many samples contain large amounts of natural and/or added beta-carotene, whose hydrophobicity is even greater, so it can only be eluted from a C18 column after a long wash. The retention of vitamins was significantly reduced on the C8 column compared to that exhibited on a C18 column (Figure 1). The use of eluents with an acidic pH was chosen because of the positive ionization mode.

Figure 1. Separation of vitamins A, D2, D3 and E (1 µg/mL) on a C8 HPLC column.

Compared to vitamins K1 and K2, vitamin K3 is difficult to ionize in its native form, so it is hard to analyze with LC-MS. Yuan et al. recommended the chemical derivatization of vitamin K3, after which this vitamin can be detected with sufficient sensitivity using an MS instrument [11]. The derivatization applied by us is based on the method of Yuan et al., in which vitamin K3 is reacted with cysteamine under identical conditions, during which a Michael addition reaction takes place [11]. We performed the reaction not with cysteamine, but with L-cysteine. After the introduction of cysteine, the hydrophobicity of the derivative is much lower than that of native vitamin K3 (Table 4) and thus its retention on the C8 column is also reduced (Figure 2).

Table 4. Parent ion (m/z 294.1) and daughter ions of derivatized vitamin K3 recorded with an LC-ESI(+)-MS/MS instrument assembly.
Figure 2. Separation of derivatized vitamin K3 (1 µg/mL) on a C8 HPLC column.

Completion of the derivatization reaction between vitamin K3 and L-cysteine was confirmed by recording a mass spectrum. Based on what was described in Section 3.4., a 5 µg/mL derivatized solution was prepared and the mass spectrum of the derivative was recorded in Q1 scan mode, scanning the 200–400 m/z range (Figure 3). The [M+H]+ monoisotopic mass of the quasi molecular ion (protonated molecule) of the assumed derivative is 294.1 Da, the signal of which appears in the spectrum (Figure 3).

Figure 3. Mass spectrum of derivatized vitamin K3 (5 µg/mL).

Therefore, the reaction presumably also took place with L-cysteine, which was confirmed by recording the product ion spectrum (Figure 4).

Figure 4. Product ion spectrum of derivatized vitamin K3 (5 µg/mL).

In the product ion spectrum, the m/z 294.1 ion was fragmented with a collision energy of 15 V, the fragments are listed in Table 4. m/z 115.1 and m/z 205.1 ions clearly belong to vitamin K3, confirming the structures of vitamin K3 fragments reported by Yuan et al. [11]. The m/z 173.1 fragment corresponds to the protonated molecule of vitamin K3, while the m/z 122.1 fragment is the protonated molecule of L-cysteine.

4.2. Method validation, proficiency testing

During the validation of the methods, there were no interfering signals in the blank samples within the retention window of the target components and the ion ratios of the target components detected in the samples were the same as the ion ratios calculated for the calibration solutions, thus the condition for MS/MS identification was met. Calibration was linear between 0.01 and 1.0 µg/mL concentration, above which (1.0–10.0 µg/mL) the curve became quadratic in nature. Relative recovery values corrected with the ISTD fulfilled the 80-120% criterion and the precision values (RSD%) did not exceed 10% (Tables 5-9).

Table 5. Reproducibility analysis of vitamins A, D2, D3 and E in wheat flour at 0.5 and 5.0 mg/kg levels.
Table 6. Reproducibility analysis of vitamins A, D2, D3 and E in soft drinks at 0.5 and 5.0 mg/kg levels.
Table 7. Reproducibility analysis of vitamins A, D2, D3 and E in effervescent tablets at 0.5 and 5.0 mg/kg levels.
Table 8. Reproducibility analysis of vitamins A, D2, D3 and E in dietary supplements at 0.5 and 5.0 mg/kg levels.
Table 9. Reproducibility analysis of vitamin K3 in dietary supplements at 0.1 and 1.0 mg/kg levels.

The limit of quantification (LOQ) was defined as the lower calibration point, which corresponds to 0.05 mg/kg due to the fivefold dilution of the sample. The LOQ could be further reduced by a lower sample dilution or by increasing the injection volume. The accuracy of the method was verified by participation in domestic and international proficiency tests. In the program organized by NÉBIH, infant formula contained vitamins A and E; the values assigned to the sample for vitamins A and E were 0.495 and 13.6 mg/100 g. The values detected by us were 0.465 and 13.6 mg/100 g, corresponding to Z-scores of -0.3 and 0.0. The condition for a successful proficiency test is -2 ≤ Z ≤ 2. Organized by FAPAS, the second proficiency test sample was a liquid vitamin dietary supplement in which the vitamin D3 content was analyzed and a vitamin D3 concentration of 0.206 mg/100 g was detected. The target value was 0.211 mg/100 g, for which the calculated Z-score is -0.2, so it was acceptable. Our proficiency test results are summarized in Table 10.

Table 10. Proficiency testing results.

5. Conclusions

The goal of this paper was to develop a new LC-MS/MS method for the determination of fat-soluble vitamins in food and dietary supplement samples. By combining the analysis with isotope dilution, it was possible to develop a method with great accuracy and high precision, which was validated within the laboratory and was successfully applied in domestic and international proficiency tests.

6. References

[1] Zempleni, J., Suttie, J.W., Gregory III, J.F., Stover, P.J. (2013): Handbook of Vitamins, 5th Edition, CRC Press, Boca Raton, FL, USA.

[2] Az Európai Parlament és a Tanács 1169/2011/EU rendelete (2011): a fogyasztók élelmiszerekkel kapcsolatos tájékoztatásáról, az 1924/2006/EK és az 1925/2006/EK európai parlamenti és tanácsi rendelet módosításáról és a 87/250/EGK bizottsági irányelv, a 90/496/EGK tanácsi irányelv, az 1999/10/EK bizottsági irányelv, a 2000/13/EK európai parlamenti és tanácsi irányelv, a 2002/67/EK és a 2008/5/EK bizottsági irányelv és a 608/2004/EK bizottsági rendelet hatályon kívül helyezéséről. Az Európai Unió Hivatalos Lapja L 304/18.

[3] FDA (2021), Vitamin K Substances and Animal Feed

[4] Tölgyesi, Á. (2021): Gyakorlati példák a folyadékkromatográfiával kapcsolt hármas kvadrupol rendszerű tandem tömegspektrometria élelmiszer-, bio- és textilanalitikai alkalmazására, Kromatográfus különszám, Gen-lab Kft., Budapest, Magyarország

[5] MSZ EN 12822:2014. Élelmiszerek. Az E-vitamin meghatározása nagy hatékonyságú folyadékkromatográfiával. Az alfa-, béta-, gamma- és delta-tokoferol mérése.

[6] MSZ EN 12823-1:2014. Élelmiszerek. Az A-vitamin meghatározása nagy hatékonyságú folyadékkromatográfiával. 1. rész: Az all-E-retinol és 13-Z-retinol mérése.

[7] MSZ EN ISO 6867:2001. Takarmányok. Az E-vitamin-tartalom meghatározása. Nagy hatékonyságú folyadékkromatográfiás módszer (ISO 6867:2000).

[8] MSZ EN 12823-2:2000. Élelmiszerek. Az A-vitamin meghatározása nagy hatékonyságú folyadékkromatográfiával. 2. rész: A béta-karotin mérése.

[9] MSZ EN 12821:2009. Élelmiszerek. A D-vitamin meghatározása nagy hatékonyságú folyadékkromatográfiás módszerrel. A kolekalciferol (Dˇ3^-vitamin) vagy az ergokalciferol (Dˇ2^-vitamin) mérése.

[10] Arachchige, G.R.P., Thorstensen, E.B., Coe, M., McKenzie, E.J., O’Sullivan, J.M., Pook, C.J. (2021): LC-MS/MS quantification of fat soluble vitamers – A systematic review, Anal. Biochem. 613,113980. DOI

[11] Yuan, T.-F., Wang, S.-T. Li, Y. (2017): Quantification of menadione from plasma and urine by a novel cysteaminederivatization based UPLC–MS/MS method, J. Chromatogr. B 1063 p.107-111. DOI

[12] Az Európai Parlament és a Tanács 1925/2006/EK rendelete (2006. december 20.) a vitaminok, ásványi anyagok és bizonyos egyéb anyagok élelmiszerekhez történő hozzáadásáról / Regulation (EC) No 1925/2006 of the European Parliament and of the Council of 20 December 2006 on the addition of vitamins and minerals and of certain other substances to foods


Evolution of the mineral content of winter wheat in Hungary based on 30 years of measurement results

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Evolution of the mineral content of winter wheat in Hungary based on 30 years of measurement results


Received: March 2022 – Accepted: June 2022


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 Nutrition Science


grain, winter wheat, years of cultivation, mineral content, phosphorus, potassium, magnesium, calcium, manganese, zinc, copper, changes in composition by year, boxplot diagram

1. Summary

Nowadays, research related to food science and nutrition places more and more emphasis on the examination of the chemical, feeding and nutrition-physiology quality of various products of plant origin and the evolution of the changes. The attention of many researchers is focused on these quality issues related to nutritional values, and on how these characteristics have changed over the past few decades, thanks to the intensive agrotechniques applied and the available varieties.

In terms of our eating habits, cereals have been playing a central role in our everyday lives for thousands of years. In addition to providing energy thanks to their carbohydrate content, cereals can also be seen as a source of proteins, fibers, vitamins and, last but not least, minerals, as there are traditions of making and eating bread made from cereals both worldwide and in Hungary. Grain-based food production is part of a nation’s culture. By preparing our manuscript, our goal was to analyze the large number of samples available from different growing areas to obtain an answer to a question that arises in many people, how the mineral content of winter wheat, which is a staple food in our diet, has changed over the past decades. Thus, by demonstrating the combined effect of changing ecological conditions, the applied agrotechniques and biological foundations, we intend to provide an accurate picture of the evolution of the mineral content of cereals.

2. Introduction

Overall, wheat grain is a significant source of nutrients for mankind [1]. A significant part of the mineral intake of humans comes from cereals [2]. At the same time, it is also worth considering during utilization that the distribution of minerals in the wheat grain is not uniform, minerals are mostly contained in the husk parts (bran), but in many countries, only the endosperm, which is much poorer in mineral elements, is utilized [3, 4]. Accordingly, even though whole-grain flours and products made from them have a higher mineral content, in this case, the possibility of mycotoxins entering the food chain also has to be considered. That is why a balanced and varied diet, as with all other food groups, is extremely important when consuming cereal-based products.

Reviewing literature data, a rather diverse picture is obtained regarding the mineral composition of cereals, data range in a wide interval, as evidenced by the literature data collected in Table 1.

Table 1. Evolution of the mineral content of winter wheat, taking into account of different literature sources [1, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14]

In connection with this, several researchers [15, 16] have pointed out in the last few decades that the lack of certain micronutrients is a real problem worldwide, and as a result, the proportion of those suffering from some kind of nutrient deficiency within the population is getting higher and higher. At the same time, other researchers [17, 18] report that the amount of certain minerals in food also decreased. According to them, iron, iodine and zinc are the mineral elements that are most lacking in our daily diet. This is also confirmed by the 1996 and 2002 WHO surveys [19, 20], according to which about half of the world’s population may be affected by a lack of iron and zinc. According to some literature sources [21], supplementing wheat flour with minerals may be a solution to this problem.

Starting from the 1960s, during cultivation, the emphasis was on the use of varieties that could really contribute to overcoming global food shortage by ensuring adequate yields, but in the meantime, the evolution of the amount of minerals, which are also of outstanding importance from a nutrition physiology point of view, received much less attention [22]. Increasing yields can be accompanied by a decrease in the mineral content of the wheat grain [23], but this is not easy to judge, since as we know, the quality of plant products, including the mineral content, is evolves as a result of the combined effect of many factors. The composition of the crops may also depend on the biological characteristics of the plant, the applied agrotechnical factors and the existing ecological conditions [24, 25].

Taking into consideration all this knowledge and performing a comparative analysis with literature data, element content data collected over 30 years were processed and statistically analyzed, in order to provide researchers and those interested in the profession with a realistic picture of the mineral composition of cereals, including winter wheat, and its evolution over time.

3. Materials and methods

The winter wheat samples that form the basis of our study come from the period between 1974 and 2004. The size of the sample set differs by year, but overall thousands of samples were analyzed in the above-mentioned time range. The test samples were primarily taken from agrotechnical experiments, in which the effects of various crop areas were examined under different ecological conditions, and the yield results were evaluated.

During sample preparation, the samples were ground with a Retsch Sk-1 or Sk-3 mill. To determine the amount of minerals, initially an ashing digestion method was used [26], while later a wet digestion procedure was used [27]. According to the latter method, 1 g of the sample was measured into a digestion tube, after which the digestion was carried out with nitric acid and hydrogen peroxide at the appropriate temperature. The wet digestion procedure carried out in a closed space allows the digestion of the total element content of the sample. Following digestion, an SP 90 PYE UNICAM atomic absorption spectrophotometer was used to determine the element content until 1998, and in the subsequent period until 1998, a LABTAM 8440 (LABTAM Ltd., Australia) and then an OPTIMA 3300 DV (Perkin-Elmer Ltd, USA) inductively coupled plasma optical emission spectrometer (ICP-OES) was used. The measurements were carried out at the Debrecen University of Agriculture (DATE), and then at its legal successor, the University of Debrecen, in the Instrument Center of the Institute of Food Science of the Faculty of Agricultural and Food Sciences and Environmental Management. In order to check our test methods, we took part in domestic and international proficiency tests using certified reference materials, and in the case of both flour quality and mineral content analyses, we used a BCR CRM 189 (European Reference Material) certified whole grain wheat sample. Data are reported on a dry matter basis.

To evaluate the results obtained, the SPSS 22.0 statistical program package was used, and the mean, standard deviation and relative standard deviation values were determined. To represent our measurement results, a boxplot was used, which is a graphical analysis method in which the location of the interquartile range provides information about the distribution of the analytical data, and this type of diagram also informs us about the evolution of outlying values. With the help of the boxplot diagram, we have an opportunity to separate the extreme outliers within the data set, which is important because during the processing of such a large number of measurement data, the appearance of such values among the measurement results is inevitable, and they are omitted from the evaluation during statistical processing.

4. Results

Our measurements covered a sample set of 30 years. The measurement results of about 4,200 samples were processed. The phosphorus content of the samples ranged from 1.5 to 5.6 g/kg, the potassium content from 1.6 to 5.8 g/kg, the calcium content from 200 to 780 mg/kg, the magnesium content from 600 to 2,000 mg/kg, the zinc content from 6.00 to 79.0 mg/kg, the copper content from 1.7 to 10.4 mg/kg, and the manganese content from 13.0 to 69.1 mg/kg. These data (Table 2) fit well into the ranges of the indicated literature data, the values in the table show the extreme values and the interquartile ranges, with the latter data expressing the range in which the middle 50% of the measured values fluctuate.

Table 2. Measurement results of winter wheat samples by tested element, processing 30 years of data

Examining the available data element by element, the results of the statistical processing are illustrated in the form of a boxplot diagram in Figure 1.

In the case of phosphorus and potassium, the deviation values covered a relatively wide range, but examining the interquartile values, it can be concluded that our results typically fall in the range of 2.9 to 4.0 g/kg. In terms of the average of the years studied, the range is between 2.9 and 3.4 g/kg for phosphorus and between 3.3 and 3.9 g/kg for potassium. These values are already in good agreement with the data reported in the literature. With regard to the concentration of these two macroelements, no decrease was found over the 30 years examined.

The values of the calcium content of the test samples also fall within a wide interval in terms of the 30-year period. By summarizing our measurement results, values between 200 and 780 mg/kg were determined. In the average of 30 years, the interquartile range of the data obtained during the statistical analysis covers a much narrower range. The middle 50% of the analytical values lies between 336 and 395 mg/kg. In the case of the lower quartile, the lowest value was measured in 1977. The average of the samples analyzed this year showed a Ca content of 252 mg/kg. The highest upper quartile was measured in 2003. In that year, an average Ca content of 530 mg/kg was detected.

No significant decrease in the calcium content can be reported during this period either in the case of the samples analyzed by us.

The magnesium content results fell in a wide range of 600 to 2,000 mg/kg, in accordance with the literature data. The interquartile range gives values between 1,029 and 1,213 mg/kg in the average of the years of the study, with significant differences between the year in this case as well, as the 25% percentile value was the lowest in 1978 at 622 mg/kg, while the 75% percentile value was outstanding in 1980 at 1,703 mg/kg.

When examining our data, a relative standard deviation of nearly 20% was determined for the evolution of the manganese content. The standard deviation values are well illustrated in the boxplot diagram. Our results ranged from 13.0 to 69.1 mg/kg, but the range between the 25 and 75% percentiles is only 33.0 to 42.9 mg/kg, i.e., 50% of the analytical data is located in this narrow range. However, statistical analysis also indicates several outliers in the diagram.

In the case of microelements, such as zinc and copper, relative standard deviation values even higher than those found for manganese were encountered. In the average of the study years, a relative standard deviation of 25.8% was calculated for zinc and the result was 22.8% for copper. The boxplot diagram clearly shows that the measurement data range widely, there is a large standard deviation, and there are many outliers among the individual results. Looking at the results of the boxplot diagram, the values of the 25 and 75% percentile range for zinc are between 19.9 and 28.5 mg/kg, while in the case of copper the values of this range are between 3.7 and 4.9 mg/kg.

Figure 1. Evolution and distribution of mineral content in winter wheat test samples

5. Conclusions

Among cereals, wheat is the dominant arable crop in the world, accounting for a significant part of the plant-derived products made from it, thus providing the human body with important minerals as a whole. Nowadays, many researchers raise the question of how, in the case of field crops grown for the production of various food raw materials, the amount of minerals and their relative proportions have changed over the decades, while there have been a change in the varieties available, in ecological conditions and, of course, the applied agrotechniques, i.e., all of the factors that simultaneously influence the quality of a plant product and its nutritional value.

Our test results came from cultivation years between 1974 and 2004 and from different growing areas. In the investigated production areas, the effectiveness of different agrotechnical treatments was investigated, typically under different ecological conditions, with different varieties.

Looking at the average of the years, of the elements analyzed in the samples, lower relative standard deviation data were found for phosphorus, potassium and magnesium, with values of 11.7%, 11.0% and 13.0%, respectively. The standard deviation of our measurement results was much more hectic in the case of copper and zinc. The standard deviation value was 22.8% for copper and 25.8% for zinc.

In terms of the 30 years examined, no decrease in the mineral content was experienced. At the same time, it should be emphasized that reliable conclusions and authentic timeline findings can only be drawn on the basis of simultaneous measurements of archived samples. We intend to continue our work in this spirit.

6. References

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Characteristics and uses of propolis

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Characteristics and uses of propolis*

* Current European legislation food information shall not attribute to any food the property of preventing, treating or curing a human disease, nor refer to such properties. Results on the effects of propolis on human health are published as scientific information. The Editor.


Received: May 2022 – Accepted: July 2022


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


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

1. Summary

Propolis (bee glue) is an extremely valuable “byproduct” of beekeeping. Its ingredients include many bioactive substances that have a beneficial effect on the human body, which is why propolis has been used by mankind for thousands of years, mainly for medicinal and occasionally cosmetic purposes. Many medicinal and cosmetic products are still produced from the substance today. Its composition varies considerably depending on the geographical location and the health of the producing bees. Its most important components are polyphenolic compounds (phenolic acids, flavonoids, flavonoid esters, diterpenes, sesquiterpenes), lignans, aromatic aldehydes, alcohols, amino acids, fatty acids, organic acids, hydrocarbons, vitamins and minerals. Propolis can be considered a medicinal food. Extracts made from it possess antibacterial, antiviral and antifungal effects. Propolis, in limited quantities, is also suitable for human consumption. The safe dose of propolis for healthy people is 70 mg/day.

With our manuscript, we intend to provide a brief review of the literature on the beneficial effects of propolis on human health.

2. Introduction

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

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

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

3. Characteristics of propolis

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

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

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

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

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

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

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

4. Propolis-containing products

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

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

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

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

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

5. Dose and safety

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

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

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

6. Physiological and therapeutic effects of propolis

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

6.1. Fighting infections, the immune system

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

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

6.2. Cancerous diseases

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

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

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

6.3. Diabetes

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

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

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

6.4. Cardiovascular diseases

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

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

6.5. The skin and the nervous system

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

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

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

6.6. Alimentary canal

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

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

6.7. Allergizing effect

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

6.8. Summary of physiological and therapeutic effects

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

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

7. New areas of application of propolis

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

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

8. Antioxidant properties of propolis

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

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

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

9. Synergistic interaction of propolis and honey

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

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

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

10. Acknowledgment

Az anyag összeállításához Bencsik Boglárka demonstrátor hallgató is hozzájárult.

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

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

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

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

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

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

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Determination of the macroelement content of breads fortified with different spices and their contribution to the nutrient reference value

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Determination of the macroelement content of breads fortified with different spices and their contribution to the nutrient reference value


Received: May 2022 – Accepted: July 2022


1 University of Debrecen, Institute of Food Science


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

1. Summary

Many studies are published on food fortification, as the production, testing and consumption of functional foods has become a central issue these days. Bread is one of our important staple foods, and we also regularly eat various spices. Bread may also contain spices. In the course of our work, bread recipes containing different spices in different quantities were developed. In this study, the macroelement content of seven spices (basil, dill, oregano, caraway, chives, rosemary and garlic granules) and 42 fortified breads were determined using an inductively coupled plasma optical emission spectrometer (ICP-OES), and their contribution to the nutrient reference value (NRV) was calculated. Based on the measured concentrations, higher element contents were measured in the spices used by us compared to the values of other studies. Outstanding results values were determined in basil, dill, oregano and chives.

In the case of breads, the calcium, potassium and magnesium content of the products made with the above-mentioned spices was higher than the data found in the literature. Taking into account the results, it was possible to produce macroelement-containing products that contribute to the body’s daily macroelement needs more than usual.

2. Introduction

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

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

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

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

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

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

Table 1. Spices’ element content is other studies (mg/kg))

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

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

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

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

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

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

3. Materials and methods

3.1. Preparation of the breads

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

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

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

3.2. Determination of element content

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

3.3. Statistical analysis

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

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

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

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

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

4. Results and evaluation

4.1. Measurement results of the element content of the spices

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

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

Table 2. Element content of the spices for original matter (mg/kg)

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

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

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

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

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

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

4.2. Measurements results of the breads fortified with spices

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

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

4.2.1. Calcium content results

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

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

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

4.2.2. Potassium content results

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

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

Table 3. Calcium and potassium content of the enriched breads (mg/kg) and their NRVs (%) for 100 g products (p=0,01%, a-the marking shows the significant differences from the control per column)

4.2.3. Magnesium content results

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

Table 4. Magnesium and sodium content of the enriched breads (mg/kg) and their NRVs (%) for 100 g products (p=0,01%, a-the marking shows the significant differences from the control per column)

4.2.4. Sodium content results

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

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

Table 5. Phosphorus and sulphur content of the enriched breads (mg/kg) and their NRVs (%) for 100 g products (p=0,01%, a-the marking shows the significant differences from the control per column)

4.2.5. Phosphorus content measurement results

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

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

4.2.6. Sulfur content results

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

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

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

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

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

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

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

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

5. Conclusions

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

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

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

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

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

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

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

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

6. Acknowledgment

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

7. References

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Flexitarianism – the sustainable food consumption?

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Flexitarianism – the sustainable food consumption?


Received: August 2022 – Accepted: September 2022


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


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

1. Summary

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.

Table 1. Eating habits and preferred diets from unrestricted omnivore via flexitarian to vegan (The codes in the table are the ISO codes of the name of the countries)

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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2022/3 Review of national standardization

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Review of national standardization


  • Anna Szalay1

1 Hungarian Standards Institution

The following Hungarian standards are commercially available at MSZT (Hungarian Standards Institution, H-1082 Budapest, Horváth Mihály tér 1., phone: +36 1 456 6893, fax: +36 1 456 6841, e-mail: kiado@mszt.hu, postal address: H-1450 Budapest 9., Pf. 24) or via website: www.mszt.hu/webaruhaz.

Published national standards from June 2022 to August 2022

07.100.30 Food microbiology

MSZ EN ISO 4833-1:2013/A1:2022 Microbiology of the food chain. Horizontal method for the enumeration of microorganisms. Part 1: Colony count at 30 °C by the pour plate technique. Amendment 1: Clarification of scope (ISO 4833-1:2013/Amd 1:2022) – which is amendment of MSZ EN ISO 4833-1:2014 –

MSZ EN ISO 4833-2:2013/A1:2022 Microbiology of the food chain. Horizontal method for the enumeration of microorganisms. Part 2: Colony count at 30 °C by the surface plating technique. Amendment 1: Clarification of scope (ISO 4833-2:2013/Amd 1:2022) – which is amendment of MSZ EN ISO 4833-2:2014 –

MSZ EN ISO 20836:2022 Microbiology of the food chain. Polymerase chain reaction (PCR) for the detection of microorganisms. Thermal performance testing of thermal cyclers (ISO 20836:2021)

13.020.55 Biobased products

MSZ EN 17399:2020 Algae and algae products. Terms and definitions

13.060 Water quality

MSZ EN ISO 5667-1:2022 Water quality. Sampling. Part 1: Guidance on the design of sampling programmes and sampling techniques (ISO 5667-1:2020) – which has withdrawn the MSZ EN ISO 5667-1:2007 –

MSZ EN ISO 10304-4:2022 Water quality. Determination of dissolved anions by liquid chromatography of ions. Part 4: Determination of chlorate, chloride and chlorite in water with low contamination (ISO 10304-4:2022) – which has withdrawn the MSZ EN ISO 10304-4:2000 –

MSZ EN ISO 13163:2022 Water quality. Lead-210. Test method using liquid scintillation counting (ISO 13163:2021) – which has withdrawn the MSZ EN ISO 13163:2019 –

MSZ EN 14614:2021 Water quality. Guidance standard for assessing the hydromorphological features of rivers

65 Agriculture

65.120 Animal feeding stuffs

MSZ EN 15784:2022 Animal feeding stuffs: Methods of sampling and analysis. Detection and enumeration of Bacillus spp. used as feed additive – which has withdrawn the MSZ EN 15784:2010 –

MSZ EN 15786:2022 Animal feeding stuffs: Methods of sampling and analysis. Detection and enumeration of Pediococcus spp. used as feed additive – which has withdrawn the MSZ EN 15786:2010 –

MSZ EN 15787:2022 Animal feeding stuffs: Methods of sampling and analysis. Detection and enumeration of Lactobacillus spp. used as feed additive – which has withdrawn the MSZ EN 15787:2010 –

MSZ EN 15788:2022 Animal feeding stuffs: Methods of sampling and analysis. Detection and enumeration of Enterococcus (E. faecium) spp. used as feed additive – which has withdrawn the MSZ EN 15788:2010 –

MSZ EN 15789:2022 Animal feeding stuffs: Methods of sampling and analysis. Detection and enumeration of Saccharomyces cerevisiae used as feed additive – which has withdrawn the MSZ EN 15789:2010 –

MSZ EN 16936:2017 Animal feeding stuffs: Methods of sampling and analysis. Screening on the antibiotics tylosin, virginiamycin, spiramycin, bacitracin-zinc and avoparcin at sub-additive levels in compound feed by a microbiological plate test

MSZ EN 16967:2017 Animal feeding stuffs: Methods of sampling and analysis. Predictive equations for metabolizable energy in feed materials and compound feed (pet food) for cats and dogs including dietetic food

MSZ EN 17517:2022 Animal feeding stuffs: Methods of sampling and analysis. Determination of mineral oil saturated hydrocarbons (MOSH) and mineral oil aromatic hydrocarbons (MOAH) with on-line HPLC-GC-FID analysis

MSZ EN 17547:2022 Animal feeding stuffs: Methods of sampling and analysis. Determination of vitamin A, E and D content. Method using solid phase extraction (SPE) clean-up and high performance liquid chromatography (HPLC)

MSZ EN 17550:2022 Animal feeding stuffs: Methods of sampling and analysis. Determination of carotenoids in animal compound feed and premixtures by high performance liquid chromatography. UV detection (HPLC-UV)

67 Food technology

67.050 General methods of tests and analysis for food products

MSZ EN 17254:2020 Foodstuffs. Minimum performance requirements for determination of gluten by ELISA

67.100 Milk and milk products

MSZ EN ISO 24223:2022 Cheese. Guidance on sample preparation for physical and chemical testing (ISO 24223:2021)

67.200 Edible oils and fats. Oilseeds

MSZ EN ISO 18363-1:2022 Animal and vegetable fats and oils. Determination of fatty-acid-bound chloropropanediols (MCPDs) and glycidol by GC/MS. Part 1: Method using fast alkaline transesterification and measurement for 3-MCPD and differential measurement for glycidol (ISO 18363-1:2015)

MSZ EN ISO 18363-3:2022 Animal and vegetable fats and oils. Determination of fatty-acid-bound chloropropanediols (MCPDs) and glycidol by GC/MS. Part 3: Method using acid transesterification and measurement for 2-MCPD, 3-MCPD and glycidol (ISO 18363-3:2017)

Corrected national standards from June 2022 to August 2022

67.120 Meat, meat products and other animal produce

MSZ ISO 23776:2021 Meat and meat products. Determination of total phosphorous content

67.200 Edible oils and fats. Oilseeds

MSZ EN ISO 18363-2:2019 Animal and vegetable fats and oils. Determination of fatty-acid-bound chloropropanediols (MCPDs) and glycidol by GC/MS. Part 2: Method using slow alkaline transesterification and measurement for 2-MCPD, 3-MCPD and glycidol (ISO 18363-2:2018)

MSZ EN ISO 18363-4:2021 Animal and vegetable fats and oils. Determination of fatty-acid-bound chloropropanediols (MCPDs) and glycidol by GC/MS. Part 4: Method using fast alkaline transesterification and measurement for 2-MCPD, 3-MCPD and glycidol by GC-MS/MS (ISO 18363-4:2021)

For further information please contact Ms Anna Szalay, sector manager on food and agriculture, e-mail: a.szalay@mszt.hu


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