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Relationships amongst phenyltio-carbamide sensitivity, body composition, coffee and tea consumption

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Relationships amongst phenyltio-carbamide sensitivity, body composition, coffee and tea consumption

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

Received: January 2022 - Accepted: March 2022


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


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

1. Summary

Polymorphisms of TAS2R38 gene responsible for bitter taste perception elicit a bimodal receptor response in the population upon the detection of phenylthiocarbamide and 6-n-propylthiouracil, respectively. Genetic differences in sensitivity to phenylthiocarbamide and 6-n-propylthiouracil may affect body composition, food preferences, and frequency of consuming different food types. To date, no publication has been published in Hungary on the joint study of these factors.

The aim of the present research is to find correlations between phenylthiocarbamide taster status and body composition, and the frequency of consumption of different bitter-tasting foods.

In the study, a taster status survey of participants (n = 170), a bioimpedance-based body composition analysis (n = 96) and completed a food frequency questionnaire of bitter foods (n = 170) were conducted.

Descriptive statistical methods, cross-tabulation analysis, multiple correspondence analysis, and Mann-Whitney test were used for data analysis at 5% significance level.

The proportions of the taster and non-taster categories proved to be the same as reported by international literature (70%/30% respectively). There were no significant correlations among taster status and the other examined parameters, however, based on the multiple correspondence analysis, the observed trends are in accordance with the international literature. There were significant correlations among gender, body composition and some variables describing food preference.

Based on the literature data and our own results, there can be a relationship between genotype and body composition, and genotype and food choice. Further analyses with large-sample size and representative research are needed to substantiate these assumptions.

Abbreviations: PTC: phenylthiocarbamide; PROP: propylthiouracil; SNP: Single Nucleotid Polymorphism; GPCR: G Protein Coupled Receptor; PAV: Proline-Alanine-Valine; AVI: Alanine-Valine-Isoleucine; AAI: Alanine-Alanine-Isoleucine; PAI: Proline-Alanine- Isoleucine; PVI: Proline-Valine-Isoleucine; AAV: Alanine-Alanine-Valine; FFQ: Food Frequency Questionnaire; BIA: Bioelectrical Impedance Analysis; BMI: Body Mass Index; PBF: Percentage of Body Fat; VFA: Visceral Fat Area; MCA: Multiple Correspondence Analysis.

2. Introduction

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

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

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

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

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

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

Figure 1. The chemical structure of phenyl-thiocarbamide (PTC) and 6-n-propylthiouracil

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

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

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

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

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

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

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

Table 1. Polymorphisms of TAS2R38 gene, and the amino acids of the coded protein based on [16, 17]

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

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

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

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

3. Scope

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

4. Methods

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

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

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

Table 2. Merging of the food frequency questionnaire categories

5. Statistical analyses

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

6. Results

6.1. Demographic parameters

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

6.1.1. Taster status

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

Table 3. Results of the taster status survey according to genders and in total (number of individuals, n=170) Results of investigation of body composition analysis and its relation to the taster status

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

Table 4. Averaged values of the body composition data (average ± standard deviation, n=96)

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

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

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

Figure 2. Results of multiple correspondence analysis for taster status, gender, and body composition parameters (n = 96, p=0,05). Abbreviations: BMI = Body Mass Index; PBF = Percent Body Fat; VFA = Visceral Fat Area Relationship of coffee consumption and taster status

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

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

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

Figure 3. Relationships between coffee consumption and taster status and gender (n = 143, p = 0.05) Abbreviations: ‘Milk ‘= flavored with milk, milk replacer, dairy product, ‘Sweetened’ = flavored with any sweetener (sugar, artificial and natural sweeteners), ‘Type of coffee’ - Assorted: consumed alternately or as a blend (Arabica) Relationship among taster status and tea consumption

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

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

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

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

Figure 4. Correlations of tea consumption with taster status and gender (n = 156, p=0,05) Abbreviations: ’Tea type’ – Several types including black tea: consumption of several tea types, including black tea; ’Tea type – Several types, but no black tea’: consumption of several tea types, except black tea; ’Sweetened: with any sweeteners’ (sugar, artificial and natural sweeteners); ’Flavouring – Variegated’: occasionally different flavouring (sometimes sweetened and / or lemon, sometimes unflavoured); ’Flavouring - More items’: flavouring with both sweetener and lemon.

7. Discussion

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

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

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

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

8. Conclusions

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

9. Statements

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

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

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

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

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Initial microbiological experience in small-scale fruit beer product development

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Initial microbiological experience in small-scale fruit beer product development

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

Received: December 2021 – Accepted: March 2022


1 Novel Food Kft.
2 Eurofins Food Analytica Kft.


Beer, bottling machine hygiene, small-scale brewing, lactic acid bacteria, Enterobacteriaceae, Bacillus, Pectinatus, Megasphera, wild yeast, fungi, CIP cleaning system, alpha acids

1. Summary

The market share of small-scale breweries in the total Hungarian beer market was 3 percent in 2020 [1]. The goal of the law affecting the sale of small-scale beers (the so-called “Beer Act”, published in Issue 275 of 2020 of the Hungarian Gazette on December 11, 2020) is to create an opportunity for small-scale breweries to gain a better market position [2]. The measure is expected to have a positive effect on the trends that have been going on in Hungary for years, such as the increase in the number of market participants, the expansion of the product range and the increase in consumer interest.

In addition to the above encouraging trends, as consumers, we find that, on average, small-scale breweries lag behind large-scale producers in terms of producing a constant high quality and in ensuring the stability and shelf life of the bottles of beers. The quality deficit mentioned is mainly due to the deficiencies in the quality management systems of small breweries, the inadequate level of expertise available and the specific sales conditions.

In this article, small-scale product development of a fruit beer representing one of the most sensitive product categories in terms of packaged product stability because of its low alcohol content and, at the same time, high sugar content is presented. Mainly the experience related to the achievement of microbiological stability is summarized in the paper, while also dealing with the development of the manufacturing environment, summarizing the most important sources of danger and possibilities for failure, and drawing the attention of existing and future manufacturers to the possibility that the certificates of conformity of manufacturers of brewing equipment do not always guarantee their proper functioning, and in many cases they may have to be reviewed and modified. The microbiological relationships mentioned in our manuscript are based on our own observations. The product-specific test methods used in the course of the project are also presented in detail.

2. Introduction

2.1. Small-scale brewing

In legal terms, breweries that produce less than 200,000 hectoliters of beer per year has been called small-scale breweries in Hungary since 2017. Regulation before 2017 drew the line at 8,000 hectoliters, under which breweries benefited from a 50% excise tax rebate [3].

2.2. Fruit beers

The category fruit beer includes beers that are made with some kind of fruit or a combination of several fruits. Here the word fruit is used in a culinary rather than a botanical sense: fleshy, seed-associated plant structures that are sweet or sour and can be eaten raw. These include, for example, pome fruits (apples, pears, quinces), stone fruits (cherries, plums, peaches, apricots, mangoes, etc.), as well as fruits that have “berry” in their English names (strawberries, raspberries, blueberries), currants, citrus fruits, dried fruits (dates, prunes, raisins, etc.), tropical fruits (banana, pineapple, guava, papaya, fig, pomegranate, cactus figs, etc.) [7].

Flavored beer: Beer for which other flavoring substances may be used instead of or in addition to hops to create a flavor effect. The detailed characteristics of these products are recorded in the product data sheet.

In the case of flavored beers, the flavoring substances are added to the wort or the beer during the brewing operations, at the latest during maturation or filtration. As a result of the flavoring substance added during maturation or filtration, the original gravity of the finished beer may not increase by more than 1/3 [8].

2.3. Launching production in a small-scale brewery

Ideally, the main sub-processes of launching production in a small-scale brewery are as follows:

  1. Conducting official licensing procedures
  2. Construction of the production plant and auxiliary facilities
  3. Product development
  4. Installation of brewing technology
  5. Product manufacturing
  6. Product sales

It is important to emphasize that during an efficient and cost-optimized production launch, product development precedes the acquisition of brewing technology, a sub-process specifically based on the experience of the former. This sequence can be achieved with the involvement of a service organization specializing in product development, which has the professional and technological background required for the process.

2.4. Technological equipment involved in our project

For the small-scale production of fruit beer, the following main technological equipment were installed:

  • Malt mill
  • Mash house equipment
  • Combined mashing/filtering tub
  • Universal hop kettle - Whirlpool tub
  • Electric control panel for the brewing process
  • Wort cooling and recuperation equipment
  • Heat supply equipment
  • Mash house auxiliary equipment
  • Fermentation area equipment
  • Barrel washing and filling equipment
  • Diatomaceous earth filter
  • Beer pasteurization equipment
  • Bottling machine
  • Compressed air supply equipment
  • Refrigeration technology equipment
  • Brewery ancillary equipment

In the above list, equipment which is specifically used to ensure or improve the microbiological stability of beer or have an above-average effect on it are highlighted.

3. Microbiological production control

3.1. Microbiological stability of beer

The biological stability of beer is compromised by any microorganism that is able to multiply in beer, cause turbidity or form bottom sediments, and damage the beer through its metabolites. The number of these microorganisms is small, as only lactic acid bacteria and yeasts are able to grow under the given anaerobic conditions due to the alcohol content, carbon dioxide content, bitterness and low pH of beer. There is a certain period of time between the infection and turbidity caused by these microorganisms and the appearance of the bottom sediment, the length of which depends on the degree of infection, the virulence of the organisms, the quality of the beer, the access of oxygen and the storage temperature.

Microbiological stability can be ensured by the use of biologically sound adjusting yeasts with high fermentation potential, the concentrated culture of which and the thorough washing, cleaning and disinfection of the tanks, lines and equipment have been checked.

Automatic cleaning equipment deserves special attention. Sharp filtration, together with pumping with the exclusion of ambient air and the use of containers cleaned with sufficient thoroughness, allows the beer to be dispensed without pasteurization. Close microbiological control is required at each stage, such as fermentation, maturation, filtration and pumping. [4]

3.2. Factors influencing the microbiological stability of beer

From a microbiological point of view, beer is a relatively stable beverage. The beer parameters that contribute to this stability are as follows:

  • Ethanol content (up to 10%, sometimes even higher): exposure to 5% ethanol has been shown to increase the permeability of the cell membrane and thus to interfere with the proton-driving force across the membrane (which is important for energy production). This means that most microbes do not survive or multiply in beer at this alcohol level.
  • Carbon dioxide content (~0.5% v/v): dissolved CO2 creates an anaerobic environment, preventing the growth of microorganisms that cause aerobic deterioration.
  • Low pH (pH 3.8-4.7): many microorganisms are unable to grow at low pH (pH<5) because they cannot maintain intracellular pH homeostasis at these low pH values.
  • Iso-alpha acids (15-100 µg/L, the concentration may be different from this): iso-alpha acids exert an antimicrobial effect by increasing the permeability of bacterial cell membranes.
  • Decreased nutrient availability (most fermentable sugars are metabolized by yeast): many important nutrients, such as carbohydrates, amino acids and some vitamins B are present in very low concentrations in beer as they are consumed by the yeast during fermentation. Any increased nutrient levels (e.g., carbohydrates in low-alcohol beers) pose a risk of proliferation of microorganisms that cause spoilage.
  • Low oxygen content (preferably below 0.1 µg/L): anaerobic conditions reduce the risk of potential growth of microorganisms that cause aerobic spoilage.

In modern brewing, a number of techniques are used to prevent the entry of microbiological contaminants or their survival during the brewing process, as well as during filling/packaging, in order to increase microbiological stability. Some examples:

  • Boiling the mash, pasteurization, or sterile filtration before packaging.
  • Well-designed brewing equipment that resists aggressive hygiene practices, such as CIP (Clean-In-Place) cleaning.
  • Elimination of many traditional (and microbiologically risky) production processes (e.g., spontaneous fermentation or open fermentation vessels).

3.3. Causes of infection

Pediococcus cerevisiae in the form of mono- and diplococci, or tetracocci, clouds the beer and gives it an acidic, diacetyl taste reminiscent of butter.

Lactic acid bacteria produce lactic acid, formic acid and acetic acid. They also cause turbidity and, in part, form bottom sediment.

Wild yeasts are rare. They make the beer cloudy, form a juicy bottom sediment and also impart a mostly aromatic, distinct, partly coarsely bitter taste.

Cultured yeasts cause turbidity, bottom sediment, or only separate yeast colonies in the pumped off beer. Even if they remain only imperfectly in the beer after filtration, they can multiply after the rich oxygen uptake during pumping, especially if there is a large difference between the final degree of fermentation of the beer and the dispensed degree of fermentation [4].

3.4. Spoilage microorganisms

3.4.1. Microorganisms most often associated with brewing and beer

Each raw material (e.g., malt, hops, water or additives) carries its own specific microorganisms. The proliferation of these microorganisms during one of the brewing steps results in the formation of metabolites that cause aftertastes. In the event that these microorganisms survive all steps of the brewing process, including pasteurization, if used, they may be present in the bottled beer as potential spoilage agents. Yeast used for the fermentation can also be a source of contamination.

It has been observed that during yeasting, the yeast can be contaminated with small amounts of bacteria and wild yeast. To avoid this, proper treatment of brewer’s yeast is required.

Additional sources of contamination can be the brewing equipment (vessels, lines) if they are not properly cleaned and maintained. Until packaging is completed, the final steps in the manufacturing process (after fermentation) may also be prone to contamination by microorganisms that are airborne or present in the filling apparatus (e.g., due to high humidity).

Spoilage microorganisms most often found in breweries and in beer are listed in Figure 1.

Figure 1. Most common beer spoilage microorganisms during the various steps of the brewing process and in the finished product. Orange arrows indicate the steps of the manufacturing process where the microbial load is reduced by heat treatment (wort boiling and pasteurization). [2]

Contaminating bacteria in beer are mostly lactic acid bacteria belonging to the genera Lactobacillus and Pediococcus (accounting for more than 80% of the bacterial infections in beer), but other anaerobic bacteria such as Pectinatus and Megasphaera are sometimes also found in spoiled beer. [2] Lactic acid bacteria [6]

Lactic acid bacteria are strictly fermentative, facultative anaerobic Gram-positive, non-spore-forming rods or cocci that belong to the order Lactobacillales. Most Gram-positive bacteria are inhibited by iso-alpha acids however some are resistant to these antibacterial compounds. The two most common lactic acid bacteria in beer are Lactobacillus brevis and Pediococcus damnosus. These bacteria produce acetic acid and lactic acid, and also compounds responsible for various aftertastes, such as diacetyl (“buttery” flavor). Pediococcus in particular is known to produce large amounts of vicinal diketones. Pediococcus also has a relatively high alcohol tolerance: it can proliferate even at ethanol concentrations above 10%. In addition, lactic acid bacteria also produce exopolysaccharides (EPS), which cause so-called silky turbidity in beer due to the increased viscosity and mucous appearance.

The most important Lactobacillus species are L. brevis and L. lindneri; less common are L. rossiae, L. buchneri, L. coryniformis, L. casei and L. backii. L. brevis often develops longer, parallel-walled, single or double rods with a round end (0.7 × 4 μm), with the double rods often being bent. It does not form cell chains, but extremely long rods (up to 50 μm) can sometimes be found in beer. Common characteristics of L. brevis are (hetero-fermentative) gas formation, fermentation of pentoses and melibioses, as well as the ability to cleave arginine. This is the most common beer spoilage bacterium, causing turbidity and sediment, while also lowering the pH perceptibly, which in turn gives beer an acidic taste. However, it does not produce diacetyl. It often appears as a secondary contaminant.

L. bucherni is able to ferment melisitose, unlike L. brevis. L. lindneri forms short, slightly irregular or coccoid cells that are arranged in longer chains. Sometimes long rods are formed. Heterofermentative species mainly ferment glucose and maltose and do not cleave arginine. Mild sedimentation and turbidity may be observed in the beer however taste defects do not usually occur. This is a typical primary contaminant that is often found in yeast factories or in the fermentation area, but can also pass through the filters, being very small cells.

L. rossiae has similar properties and is mucus-forming. Facultative heterofermentative species, such as L. casei, L. coryniformis and L. plantarum form shorter rods that are often arranged in chains. They are mostly found in weaker hop beers (e.g., wheat beer) and cause obvious taste defects due to diacetyl formation. They often appear only as secondary contaminants. The obligate homofermentative beer spoilage L. backii ferments mannose, mannitol and sorbitol. It also differentiated from the other species by the absence of fermentation of maltose and gluconate.

P. damnosus is characterized by the formation of tetrads. It is typically a primary contamination that often occurs in cultured yeast and unfiltered beer. The cells can also be transferred to the bottled beer through the filter. Contamination results in strong diacetyl formation (buttery taste) and a decrease in pH, and beers are often slightly turbid and exhibit noticeable sedimentation. Two other Pediococcus species that cause beer spoilage, P. inopinatus and P. claussenii, behave similarly, although both species are less common. The latter causes mucus formation in the beer. Enterobacteriaceae [6]

Enterobacteriaceae is a facultative anaerobic Gram-negative bacterial family. The two genera commonly associated with brewing are Citrobacter and Rahnella (most likely to be introduced with the water used for brewing). These bacteria are responsible for the production of a number of compounds causing aftertastes, such as VDKs (e.g., diacetyl), 2,3-butanediol, DMS, acetaldehyde and lactic acid. These compounds are produced at the beginning of the fermentation. Bacillus [6]

Gram-positive, facultative anaerobic, spore-forming bacteria. Due to spore formation, they survive heat treatment, including pasteurization. Bacillus also poses a risk because it can reduce nitrate to nitrite, which can lead to the formation of N-nitrosamines (classified as carcinogenic, teratogenic and mutagenic substances). Since certain Bacillus species are able to produce large amounts of lactic acid, they can also cause acidification. Most Bacillus species (but not their spores) are susceptible to the iso-alpha acids from hops. Pectinatus [6]

These Gram-negative, strictly anaerobic bacteria can produce large amounts of acetic acid and acetoin, and hydrogen sulfide production (rotten egg aroma) has also been reported.

Pectinatus cerevisiiphilus and P. frisingensis are also strictly anaerobic, catalase-negative, Gram-negative bacteria, and have similar negative effects as the species listed so far. The cells are slender (0.8 × 4 μm), parallel-walled with a pointed end, slightly bent or serpentine or corkscrew-like, and serially flagellated on one side. Similarly to M. cerevisiae, they grow in the range of 15 to 40 °C (with the optimum being between 28 and 32 °C). They ferment various sugars, sugar alcohols and organic acids (mainly pyruvate and lactate). The primary metabolites are propionic acid, acetic acid, pyruvic acid, acetoin and CO2. Beers contaminated with them (pH above 4.3, alcohol content below 5% vol.) exhibit not only serious sedimentation and turbidity problems, but also unpleasant odor and taste defects (sewage odor). Similarly to M. cerevisiae, these are typically secondary contaminants that occur primarily in the bottling area. Megasphaera

Megasphaera species can appear as Gram-negative, strictly anaerobic contaminants in both wort and finished beer. They cause turbidity in beer and produce large amounts of hydrogen sulfide and a number of short-chain fatty acids (“cheesy” aroma). [2]

Catalase-negative, strictly anaerobic, Gram-negative Megasphaera cerevisiae forms large oval or round cells (1.2 – 1.6 μm) that exist in the form of diplococci and short chains. They ferment fructose, pyruvic acid and lactic acid, in particular. [6]

The primary metabolites are butyric acid, acetic acid, propionic acid, valeric acid, as well as CO2 and hydrogen gas. Only a slight turbidity is exhibited by the beer; however, due to the above-mentioned metabolites, there can be significant odor and taste defects (sewage odor). The species is sensitive to alcohol (below 5% vol.) and prefers a higher pH value (above 4.4). The secondary contaminant that is present primarily in the vicinity of the bottling equipment are typically favorable to these bacterial species [6]. Wild yeast

Any strain of yeast, except the selected Saccharomyces yeast, is a contaminant. These yeast contaminants are usually referred to by brewers as wild yeast, which may be Saccharomyces cerevisiae or non-Saccharomyces strains, such as Brettanomyces bruxellensis, Candida or Pichia. Proliferation of wild yeast can carry a safety risk: the alcohol content can increase due to the metabolism of the infecting yeast. These wild yeasts are sometimes able to ferment dextrins and starch into ethanol (so-called superattenuation). Along with the production of ethanol, the CO2-content, and thus the bottle pressure increases, which can pose a safety risk due to the bursting of the bottles. In addition, wild yeast can ruin the beer through the production of ester or phenolic aftertaste (e.g., 4-vinylguaiacol), as well as turbidity or sediment formation. It is important to note that acid washing of the yeast cells does not remove these wild yeast contaminants [5]. Fungi

Field infestation by Fusarium fungi poses a serious food safety risk to cereals. Almost all parts of the plant (germ, root, stalk, stem, leaf pod, leaf, ear and grain) can become affected. Severely infected plants produce less and lower quality crops, toxins are produced in the diseased grains, their germination vigor is reduced. The pathogens that cause the disease are different Fusarium species with various infectivity and toxin production, which can be greatly related to environmental factors, such as temperature and humidity. The toxins can be present throughout the entire brewing process, up to the bottled finished product. Certain Aspergillus species can also produce mycotoxins. Both Fusarium and Aspergillus species produce hydrophobic compounds, which are small surfactant proteins that cause foaming [6].

4. Cleaning, disinfection

Cleaning and disinfection of the lines, tanks and equipment is key in a brewery. All surfaces and equipment must be clean, the presence of contaminating bacteria, yeasts and fungi must be eliminated.

Examples of possible contaminants in a brewery:

  • Beer left over from previous brewing
  • Microbiological contaminants (yeasts, bacteria, fungi)
  • Hop residues
  • Calcium oxalate (beer stone that can be removed with acids)
  • Lipids, proteins (removal with bases)
  • Mineral deposits in the water circuit

In this regard, it is important to distinguish between cleaning agents and disinfectants.

Cleaning agents remove product residues and deposits, such as lipids and proteins. Depending on their pH value, these cleaning agents can be classified as alkaline, acidic or neutral cleaning agents. In order to further increase the cleaning capacity, additives, e.g., surfactants can be added. These are water-soluble molecules that reduce the surface tension of water, making it easier to remove contaminants.

Disinfectants are used to destroy most microbial contaminants. Here again, it is important to point out that bacterial spores are very difficult to destroy, which is why this process is called disinfection, not sterilization. Examples of disinfectants:

  • Halogenated disinfectants, for example NaOCl (sodium hypochlorite). NaOCl is a commonly used product, but unstable above 40 °C (with increased risk of corrosion).
  • Oxidizing agents, such as H2O2.
  • Quaternary ammonium compounds (often called quats). Quats are cationic surfactants. Despite their good properties, quats are not used in breweries very often because they typically from foams and difficult to rinse, which endangers the quality of the beer, e.g., may impair the stability of the foam.
  • Steam disinfection.
  • Critical points include the so-called dead spaces (pipe ends, branches in the lines, sampling points, poor welding, etc.). Lines and tanks are best cleaned with an integrated CIP system.

5. Laboratory tests

5.1. General experience

During the microbiological testing of the sour cherry beer samples and the sour cherry concentrates, we found the following:

  • Filtering of the sour cherry beer samples was not possible due to the high fiber content.
  • Also, when testing the beers, in the case of the plate casting process, when covering 10 ml of the sample with a suitable layer thickness of 3 to 5 mm of PCA (for microbial count test) or DRBC (for yeast count test) culture medium in a large Petri dish (140 x 14.8 mm), the agar did not gel because of the low pH of the sample. Therefore, in these tests, the test volume was first reduced from 10 ml to 1 ml.
  • Then the microbiological study of the sour cherry concentrates was started, due to the expected sterility of the raw material, using our own method for the detection of presence/absence.
  • This method was further modified to include the testing of beers. As the issue was not the sterility of the beer but its practical shelf life, as a final, modified solution, the presence/absence of reproducible microorganisms was tested by an enrichment method in both cases, with the same amount of inhibitor as prescribed in the recipe of the finished beer (0.02 g/L potassium sorbate). In this way, it was practically modelled whether the microorganisms that may be present in the beer can reproduce at a high nutrient content.

5.2. Description of the test methods of sour cherry beers

5.2.1. Microbial count, plate casting, colony counting (MSZ EN ISO 4833-1:2014, accredited method)

The stock suspension and the decimal dilutions are prepared from the sample according to the international standard MSZ EN ISO 6887. Using a sterile pipette, 1 ml of the sample (for liquid samples) or stock suspension is added to two Petri dishes. The procedure is repeated with additional dilutions, if necessary. 12 to 15 ml of 44 to 47 °C PCA agar is added to each Petri dish. The Petri dishes are inverted and incubated in a thermostat at 30 °C for 72±3 hours.

5.2.2. Yeast count, surface spreading, colony counting, water activity >0.95 (MSZ EN ISO 21527-1:2013, accredited method)

The stock suspension and the decimal dilutions are prepared from the sample according to the international standard MSZ EN ISO 6887. 1 ml of the sample (for liquid samples) or stock suspension is added evenly in 3 portions to the surface of the DRBC agar filled in Petri dishes, and the sample portions are spread on the surface of the agar. The procedure is repeated with an additional degree of dilution and, if necessary, with additional dilutions. The dishes are inverted after 15 minutes and incubated in a thermostat at 25 °C for 3 to 5 days.

5.2.3. Presence/absence detection of reproducible microbes, enrichment technique (own method)

In the case of a 0.33-liter bottle of beer, the sample is divided into 3 equal portions and the sample portions are incubated for 72 hours at 30 C° in 3 x 100 ml stock broth containing 0.02 g/liter of potassium sorbate. At the end of the culture period, 1 µl of each enriched sample portion is applied to a PCA plate, and the plates are incubated for 72 hours at 30 °C. If no increase in the colonies is observed on the plate, the result is reported as ‘negative/100 ml’, while if colonies do form, the result is reported as ‘positive/100 ml’.

Composition of the PCA (Plate Count Agar) culture medium for microbial count determination:

  • tryptone 5 g/l
  • yeast extract 2.5 g/l
  • glucose 1 g/l
  • agar 9 g/l
  • pH: 7.0±0.2 (25 °C)

5.2.4. Presence/absence detection of reproducible yeast, enrichment technique (own method)

In the case of a 0.33-liter bottle of beer, the sample is divided into 3 equal portions and the sample portions are incubated for 72 hours at 25 °C in 3 x 100 ml of malt broth containing 0.02 g/liter of potassium sorbate. At the end of the culture period, 1 µl of each enriched sample portion is applied to a DRBC agar plate, and the plates are incubated for 72 hours at 25 °C. If no increase in the colonies is observed on the plate, the result is reported as ‘negative/100 ml’, while if colonies do form, the result is reported as ‘positive/100 ml’.

Composition of the DRBC (Dichloran Rose-Bengal Chloramphenicol) agar:

  • enzymatically digested animal and plant tissues 5 g/l
  • glucose 10 g/l
  • potassium dihydrogen phosphate 1 g/l
  • magnesium sulfate 0.5 g/l
  • dichloran (2,6-dichloro-4-nitroaniline) 0.002 g/l
  • Rose Bengal 0.025 g/l
  • agar 15.0 g/l
  • pH: 75.6±0.2 (25 °C)

Composition of the Takács stock broth (MSZ 3640/13-76):

  • tryptone 4 g/l
  • meat extract 4 g/l
  • yeast extract 2 g/l
  • sodium chloride 2 g/l
  • disodium hydrogen phosphate 2 g/l
  • pH 7.2-7.4 (25 °C)

Laboratory tests were carried out by the laboratory of EUROFINS Food Analytica Kft.

6. Relationships between the microbiological state of the finished product and the bottling machine

In all cases, the control tests applied during product development and subsequent production were extended to the examination of the microbiological condition of the technological equipment. At the same time, the possible effects of the microbiological condition of the equipment on product quality were explored.

In the course of the project, by examining the bottling machine, manufacturing defects of the machine were brought to light, which were later acknowledged and eliminated by the manufacturer based on the results of our tests.

  • The manufacturer’s CIP (Cleaning-In-Place) cleaning and disinfection program was set on the pneumatic branch lines of the taps with insufficient exposure time
  • The foaming water supply was not connected to the CIP system
  • The beer druck tank could not be cleaned adequately due to the dead spaces it contained
  • The CO2 inlet branch line of the beer druck tank was not connected to the CIP system

The results of the comprehensive microbiological testing of the bottling machine used in our project before conversion are summarized in Table 1, while the results of the microbiological testing of the beer bottled using this machine are summarized in Table 2. The microbiological tests were performed by the laboratory of EUROFINS Food Analytica Kft., a testing laboratory accredited by NAH (National Accreditation Authority) under reg. no. NAH-1-1582/2021. Objectionable results are highlighted in the table in red.

Table 1. Comprehensive microbiological testing of the bottling machine before conversion

Test method – yeast count: MSZ ISO 21527-1:2013 [9]
Test method – microbial count: MSZ EN ISO 4833-1:2014 [10]

Table 2. Microbiological testing of small-scale fruit beer before conversion

Test method – yeast count: MSZ ISO 21527-1:2013 [9]
Test method – microbial count: MSZ EN ISO 4833-1:2014 [10]

Prior to the conversion, the quality defects of bottled fruit beer that could be attributed to its microbiological condition were: taste defects (ester aftertaste), increase in CO2 content and thus in bottle pressure, gushing (foaming beer squirting when the bottle is opened).

At our suggestion, the following modifications were made to the bottling machine by the manufacturer:

  • Increased operating time of the CIP program on the pneumatic branch lines of the taps
  • Connection of the foaming water supply to the CIP system
  • Elimination of the dead spaces of the beer druck tank
  • Connection of the CO2 inlet line of the beer druck tank to the CIP system

The results of the comprehensive microbiological testing of the bottling machine used in our project after conversion are summarized in Table 3, while the results of the microbiological testing of the beer bottled using this machine are summarized in Table 4.

Table 3. Comprehensive microbiological testing of the bottling machine after conversion

Test method – yeast count: MSZ ISO 21527-1:2013 [9]
Test method – microbial count: MSZ EN ISO 4833-1:2014 [10]

Table 4. Microbiological testing of small-scale fruit beer after conversion

Test method – yeast count: MSZ ISO 21527-1:2013 [9]
Test method – microbial count: MSZ EN ISO 4833-1:2014 [10]

Following the conversion, the hygienic condition of the bottling machine became satisfactory (Table 3), and the previously observed quality defects of the bottled fruit beer related to its microbiological condition were eliminated, as shown by the test results in Table 4. Due to the achieved microbiological stability, the preservation of the quality of the product could be ensured.

7. References

[1] Növekedes.hu (2020): A kisüzemi sörfőzdék piaci részesedése 5-6 százalékra nőhet az új törvény hatására. (Hozzáférés: 2021. 10.16.)

[2] 2020. évi CXL. törvény a kereskedelemről szóló 2005. évi CLXIV. törvény módosításáról

[3] A Tanács 92/83/EGK Irányelve (1992. október 19.) az alkohol és az alkoholtartalmú italok jövedéki adója szerkezetének összehangolásáról. 1. szakasz, 4. cikk

[4] Ludwig Narziss (1981): A sörgyártás. Mezőgazdasági Kiadó, Budapest.

[5] KULeuvenX (2021): Beer: the science of brewing. BrewingX. KU Leuven, Leuven, Belgium

[6] Hans Michael Eßlinger (2009): Handbook of Brewing: Processes, Technology, Markets. WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

[7] Beer Judge Certification Program, Inc. (BJCP) (2015): 2015 Style Guidelines

[8] Magyar Élelmiszerkönyv Bizottság (2013): 2-702 számú irányelv (régi 2-96 számú irányelv) Sör. Magyar Élelmiszerkönyv - Codex Alimentarius Hungaricus. Magyar Élelmiszerkönyv Bizottság, Budapest

[9] MSZ ISO 21527-1:2013 Élelmiszerek és takarmányok mikrobiológiája. Horizontális módszer az élesztők és a penészek számlálására. 1. rész: Telepszámlálásos technika a 0,95-nél nagyobb vízaktivitású termékekre (Microbiology of food and animal fedding stuffs. Horizontal method for teh enumeration of yeasts and moulds. Part 1: Colony count technique in products with water activity greater than 0,95)

[10] MSZ EN ISO 4833-1:2014 Az élelmiszerlánc mikrobiológiája. Horizontális módszer a mikroorganiz-musok számlálására. 1. rész: Telepszámlálás 30 °C-on lemezöntéses módszerrel (ISO 4833-1:2013) (Microbiology of the food chain. Horizontal method for the enumeration of microorganisms. Part 1: Colony count at 30 degrees C by the pour plate technique (ISO 4833-1:2013)


Production of Single Cell Protein by the fermentation biotechnology for Animal Feeding

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Production of Single Cell Protein by the fermentation biotechnology for Animal Feeding

DOI: https://doi.org/10.52091/EVIK-2022/2-3-ENG

Received: October 2021 - Accepted: March 2022


1 Széchenyi István University, Faculty of Agricultural and Food Sciences, Department of Water and Environmental Sciences
2 SISAF Nanotechnology Drug Delivery, Ulster University
* Corresponding Author: Judit Molnár: Széchenyi István University, Faculty of Agricultural and Food Sciences, Department of Water and Environmental Sciences


Kwashiorkor, single cell protein, food by-products, animal feeding, fermentation, biotechnology

1. Summary

Background: Fermentation is a sort of biotechnology that uses microorganisms to produce animal food through chemical process. In ancient times, wastes were treated with chemicals, but now companies convert wastes to valuable food, food ingredients or feed products such as single cell oils or single cell protein. The most used substrate is molasses and corn steep liquor which is a part of the fermentation process.

Aim: The aims of the manuscript is to provide an overview of the yeast strains and food by-products used in production of single cell proteins by fermentation process. Furthermore, the manuscript summarizes the role of single cell protein in animal feed.

Methods: Electronic searches were conducted on Google Scholar database Medline and PubMed. A further search was conducted on the Food and agricultural organisation FAO research article database.

Results: Single cell protein produced by these substrates and different microorganisms (algae, yeast, bacteria) play an important role in animal feeding. Furthermore, SCP is a high-quality protein, unsaturated fatty acids, vitamins and minerals sources for animals.

Conclusion: Production of single cell of protein through the fermentation has several significant benefits including sustainability, health and production efficacy.

2. Introduction

In ancient times, wastes were treated by various chemicals, but this method wasn’t the best. As the worldwide population grows, over recent decades, both animal and dairy production have been increasing steadily. The world now produces more than 350 million tonnes of animal-derived protein, and this value will rise up to around 1250 million tonnes by 2050, to meet global demand for animal-based protein [1]. Now, a lot of company convert various wastes into useful food, food ingredients or feed products for human nutrition and animal feeding. These products are also environment friendly and healthy such as biogas, biofuels, bioenergy. Therefore, different methods and techniques are providing opportunity to develop these products as single cell oils, single cell protein, chemicals, enzymes and many others.

Following the carbohydrate and fat, protein is the major macronutrient, which the body requires in large amount. It is an essential factor for growth, repair of the body and maintenance of health. All of the proteins are made up of the 20 amino acids, and they determine the nutrition values of protein. Some of amino acids cannot be synthesized by humans but are still essential (valine, leucine, isoleucine, phenylalanine, tryptophan, lysine, histidine, methionine and threonine) and must be obtained from our diet. The general structure of amino acids is shown in the Figure 1.

Protein digestion begins in the stomach and continues in the lumen of the intestine and so the proteins are degraded into mono and di amino acids. Those amino acids are absorbed by specific transporters in the intestines, and then released into the blood for use by other tissues, that are considered as the fundamental building blocks of proteins in the body, and they serve as the nitrogenous backbones for compounds like neurotransmitters, enzymes and hormones [2, 3]. Although, both the plant and animal proteins are similar in components, both contain the nearly the same amino acids, but the animal protein contains all the essential amino acids [4].

In general, the human body needs between 1.0 g to 1.5g of protein for each kilogram of weigh in children and adults respectively [5]. If there is insufficient protein in diet chronically that could cause kwashiorkor disease, which is a severe form of malnutrition [6].

Figure 1. General formula for an amino acid: amino group (-NH2), carboxyl group (-COOH) and replaceable group (-R) [7]

Single cell protein (SCP) is one of the high qualities and valuable dietary products from wastes [8, 9, 10, 11, 12]. SCP is a biomass which is produced by different microorganisms and it can also be termed as bio-protein, microbial protein or biomass. These microorganisms can be used as protein-rich ingredients in human and animal diet as well [8]. Furthermore, the SCP can be a good alternative to plant protein sources, and it can be produced throughout the year. In addition, they don’t emit greenhouse gases. The most important thing is the selection of cheap and suitable substrates or agro-industrial by-products and valuable microorganisms to produce protein and reduce the production cost of single cell proteins [8, 13, 14, 15, 16, 17]. In order to achieve this, different substrates were used as apple pomace, yam peels, citrus pulp, potato peels, pineapple waste, papaya waste [8]. However, the most used by-products are molasses and corn steep liquor. It is also important to choose microorganisms for research and industrial purpose as well.

This manuscript focuses on single cell proteins produced by microorganisms (algae, yeast, bacteria) as an alternative protein source. Due to the favorable content values of the single cell protein produced by fermentation (protein, vitamin, mineral), it can be used in digestible form for human nutrition, especially with vitamin supplementation and this contributes to the protection and treatment of malnutrition as a functional food and functional food ingredient [10].

3. Material and method

Electronic searches were conducted on Google Scholar database, Medline and PubMed. A further search was conducted on internet. The search items included, nutrition, dietary, protein, single cell protein, immune system. This review was conducted to analyse the recent literature to show the impact of nutrition, and single cell protein on the dietary system.

4. Result

4.1. Single cell protein produced by fermentation

Single cell protein (SCP) is a protein from cultivated microbial biomass and it can be used for protein supplementation. The SCP fermentation process can be seen in Figure 2. Agricultural and industrial wastes used as substrate to yield SCP. Algae, fungi and bacteria are all the main sources of microbial protein that can be utilized as SCP (Table 1) [18]. In addition, the acceptability of species as food depends on the growth rate, substrate used, contamination, associated toxins. The produced biomass is rich in proteins, amino acids as lysine and methionine, unsaturated fatty acids, vitamins and minerals. Therefore, these are used as food, food supplements [18] and animal feed in the world.

Figure 2. Producing single cell protein by fermentation technology (Modified scheme [8])
Table 1. Single cell protein (biomass) production from microorganisms and different substrates

4.2. Use of food by-products for the production of biomass, in particular molasses and corn steep liquor

Food loss and waste reduction is an important way to reduce costs of production, increase the food system capacity and is also a way to join the environmental sustainability campaign. Food waste also contains several biodegradable components for pathogenic microorganisms that can cause communicable diseases. Thus, food loss and waste reductions also have a positive effect on the well-being and health of the consumers. Therefore, the European Union (EU) is promoting the reduction of food wastes and these food by-products from vegetables, fruits, beverages, sugar, meat, aquaculture and seafood also contain functional or bioactive components. The food by-products can be used in nutraceutical or pharmaceutical industries. These can be transformed by fermentation biotechnology into animal feed products [30]. One of the most used food by-products are molasses and corn steep liquor. Molasses (M) is a by-product of sugar cane and it contains several compounds for fermentation for example vitamins, minerals, sucrose and organic compounds. In addition, corn steep liquor (CSL) is a by-product of the corn wet milling industry and it is rich in several components such as vitamins, minerals, amino acids and proteins. Furthermore, the CSL is also an important source of nitrogen [31]. The used molasses and corn steep liquor as a substrate in the fermentation process can be seen in Table 2

Table 2. Summary of literature references of the beneficial effects of molasses and corn steep liquor

4.3. Role of single cell protein produced by fermentation in animal feeding

The high quality and high protein rich human food and animal feed important to increase with the global population grows. Single cell protein (SCP) products based on microbial biomass, have a potential ingredient to this need [42]. The SCP contains high quality omega-3 fatty acids, vitamins, micronutrients, protein and other useful component for animal body. These valuable components can be seen in Table 3.

Table 3. Valuable components in single cell protein from different microorganisms [42]

Single cell proteins in animal feed supplement protein requirements well in addition to conventional feeds. This can also affect the quality of products of animal origin. The role of single cell proteins in animal feed is confirmed by several manuscripts, which are shown in Table 4.

Table 4. The role of single cell proteins in animal feed

7. References

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[18] Anupama, Ravindra P. (2000): Value-added food: Single cell protein. Biotechnology Advances.18. pp. 459-479. DOI

[19] Patelski P., Berlowska J., Dziugan P., Pielechprzybylska K., Balcerek M., Dziekonska U., Kalinowska H. (2015): Utilisation of sugar beet bagasse for the biosynthesis of yeast SCP. Journal of Food Engineering. 167. pp. 32-37. DOI

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[22] Coca M., Barrocal V. M., Lucas S., Gonzálezbenito G., García-Cubero M. T. (2015): Protein production in Spirulina platensis biomass using beet vinasse-supplemented culture media. Food and Bioproducts Processing. 94. pp. 306-312. DOI

[23] Hanh V., Kim K. (2009): High-Cell-Density Fed-Batch Culture of Saccharomyces cerevisiae KV-25 Using Molasses and Corn Steep Liquor. Journal of Microbiology and biotechnology.19. pp. 1603-1611. DOI: DOI

[24] Zepka L. Q., Jacob-Lopes E., Goldbeck R., Souzasoares L. A., Queiroz M. I. (2010): Nutritional evaluation of single-cell protein produced by Aphanothece microscopica Nägeli. Bioresource Technology. 101. pp. 7107-7111. DOI: DOI

[25] Rajoka M. I., Khan S. H., Jabbar M. A., Awan M. S., Hashmi A. S. (2006): Kinetics of batch single cell protein production from rice polishings with Candida utilis in continuously aerated tank reactors. Bioresource Technology. 97. pp. 1934-1941. DOI: DOI

[26] Yadav J. S. S., Bezawada J., Ajila C. M., Yan S., Tyagi R. D., Surampalli R. Y. (2014): Mixed culture of Kluyveromyces marxianus and Candida krusei for single-cell protein production and organic load removal from whey. Bioresource Technology. 164. pp. 119-127. DOI

[27] De Gregorio, A., Mandalari, G., Arena, N., Nucita, F., Tripodo, M. M., Lo Curto, R. B. (2002): SCP and crude pectinase production by slurry-state fermentation of lemon pulps. Bioresource Technology. 83. pp. 89-94. DOI

[28] Lo Curto, R. B., Tripodo M. M. (2001): Yeast production from virgin grape marc. Bioresource Technology. 78. pp. 5-9. DOI

[29] Fontana J. D., Czeczuga B., Bonfim T. M. B., Chociai M. B., Oliveira B. H., Guimaraes M. F., Baron M. (1996): Bioproduction of carotenoids: the comparative use of raw sugarcane juice and depolymerized bagasse by Phaffia Rhodozyma. Bioresource Technology. 58. pp. 121-125. DOI

[30] Socas-Rodríguez B., Álvarez-Rivera G., Valdés A., Ibánez E. (2021): Food by-products and food wastes: are they safe enough for their valorization? Trends in Food Science & Technology. 114. pp. 133-147. DOI

[31] Amado I. R., Vázquez J. A., Pastrana L., Teixeira J. A. (2017): Microbial production of hyaluronic acid from agro-industrial by-products: Molasses and corn steep liquor. Biochemical Engineering Journal. 117. pp. 181-187. DOI

[32] Palmonari A., Cavallini D., Sniffen C. J., Fernandes L., Holder P., Fagioli L., Fusaro I., Biagi G., Formigoni A., Mammi L. (2020): Short communication: Characterization of molasses chemical composition. Journal of Dairy Science. 103. pp. 6244-6249. DOI

[33] Wang J., Chen L., Yuan X.-J., Guo G., Li J.-F., Bai Y.-F., Shao T. (2017): Effects of molasses on the fermentation characteristics of mixed silage prepared with rice straw, local vegetable by-products and alfalfa in Southeast China. Journal of Integrative Agriculture. 16. pp. 664-670. DOI

[34] Sarka E., Bubnik Z., Hinkova A., Gebler J., Kadlec P. (2012): Molasses as a by-product of sugar crystallization and a perspective raw material. Procedia Engineering. 42. pp. 1219-1228. DOI

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[36] Siverson A., Vargas-Rodriguez C. F., Bradford B. J. (2014): Short communication: Effects of molasses products on productivity and milk fatty acid profile of cows fed diets high in dried distillers grains with solubles. Journal of dairy Science. 97. pp. 3860-3865. DOI

[37] Karigidi K. O., Olaiya C. O. (2020): Antidiabetic activity of corn steep liquor extract of Curculigo pilosa and its solvent fractions in streptozotocin-induced diabetic rats. Journal of Traditional and Complementery Medicine. 10. pp. 555-564. DOI

[38] Li X., Xu W., Yang J., Zhao H., Xin H., Zhang Y. (2016): Effect of different levels of corn steep liquor addition on fermentation characteristics and aerobic stability of fresh rice straw silage. Animal Nutrition. 2. pp. 345-350. DOI

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Application of an in vitro test system for the selection of probiotic bacterial strains

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Application of an in vitro test system for the selection of probiotic bacterial strains

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

Received: March 2022 - Accepted: May 2022


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


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

1. Summary

The aim of our studies was to evaluate in vitro methods for the simple and efficient selection of putative probiotic bacterial strains. Of the possible methods, the following were tested: culturing on selective media, Gram staining, catalase assay, hemolytic, clonality and aggregation ability, gastric acid tolerance and bile acid tolerance. A total of 217 bacterial strains isolated from raw sheep’s milk, curdled milk and sheep’s cheese samples produced in Transylvania were included in our experiments. Isolates with hemolytic activity, as well as those exhibiting Gram-negative or catalase-positive phenotypes not characteristic of probiotics were excluded from our studies. Based on the results of RAPD-PCR studies suitable for the detection of individual-level polymorphisms, a total of 34 clone classes and 57 strains with unique RAPD patterns were identified. From each of the 34 clone classes thus narrowed, one strain was selected and tested for its aggregation ability, as well as its gastric acid and bile acid tolerance. High aggregation values above 70%, typical of probiotic strains, were measured in the case of a total of six isolates. In the course of the presence-absence studies conducted on the surface of solid media supplemented with acid or bile acid, it was possible to select several strains specifically tolerant to acid or bile acid. Based on our results, isolates to be included in further tests, e.g., in antibiotic resistance and antimicrobial activity assays, were selected.

2. Bevezetés

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

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

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

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

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

3. Materials and methods

3.1 Isolation, culturing, preservation and storage of bacterial strains

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

Restoration and culturing conditions of the isolated and control bacterial strains included in the experiments

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

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

3.2. Selective culture conditions, its media and their preparation

3.2.1. Physiological saline solution

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

3.2.2. Phosphate buffer solution (PBS)

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

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

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

3.2.4. MRS agar (pH = 5.4)

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

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

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

3.2.6. CASO agar

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

3.2.7. Anaerobic culturing

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

3.3. Classical microbiological tests

3.3.1. Examination of colony morphology

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

3.3.2. Gram staining

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

3.3.3. Catalase test

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

3.3.4. Hemolysis test

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

3.4. Clonality test

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

Table 2. Parameters of the RAPD-PCR method

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

3.5. Investigation of autoaggregation

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

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

[1 − (Ameasurement time / A0) × 100],

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

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

3.6. Analysis of acid and bile acid tolerance

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

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

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

3.6.2. Strain restoration and optical density (OD) measurement

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

3.6.3. Presence-absence test

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

3.6.4. Process of bile acid and hydrochloric acid treatment

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

Figure 1. Flow chart of bile acid and hydrochloric acid treatment

3.6.5. Inoculation and viable cell count determination

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

4. Results and evaluation

4.1. Classical microbiological tests

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

Table 3. Main characteristics of strains based on the results of classical microbiological tests

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

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

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

4.2. Clonality test

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

Figure 2. Clonality test of members of clone class 34 (samples: E211–E217, positive control: Lactobacillus acidophilus LA-5, negative control: distilled water, molecular marker: WM; Gene Ruler 1 kb Plus DNA Ladder)
Figure 3. Gel photograph of several clone classes and individual strains (samples: E149–E194, positive control: E31, negative control: distilled water, molecular marker: WM; Gene Ruler 1 kb Plus DNA Ladder)

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

4.3. Examination of autoaggregation

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

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

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

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

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

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

Figure 4. Results of autoaggregation studies of our own isolates and control strains after 5 hours of incubation [Data represent mean ± standard deviation of 2 biological x 3 technical replicates; the horizontal red line allows the visualization of well-aggregating (>70%) strains]
Figure 5. Results of autoaggregation studies of our own isolates and control strains after 5 and 24 hours of incubation [Data represent mean ± standard deviation of 2 biological x 3 technical replicates; the horizontal red line allows the visualization of well-aggregating (>80%) strains ]

4.4. Examination of acid and bile acid tolerancea

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

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

Table 4. Results of the presence-absence test performed on the surface of solid culture medium supplemented with acid*

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

Table 5. Results of the presence-absence test performed on the surface of solid culture medium supplemented with bile acid*

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

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

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

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

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

5. Conclusions

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

6. Acknowledgment

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

7. Literature

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

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

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

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

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

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

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

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Effect of a compound bio-preservative on microbiological indicators and shelf life of fresh pork chops

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Effect of a compound bio-preservative on microbiological indicators and shelf life of fresh pork chops

DOI: https://doi.org/10.52091/EVIK-2022/2-5-ENG

Received: December 2021 – Accepted: March 2022


1 South Ural State University
2 South Ural State Agrarian University
3 LLC "Antey"


preservative, antioxidant, fresh meat products, total viable count, yeast, shelf life, storage, microbiology

1. Summary

The article deals with the study of the effect that a compound preservative produces on microbiological indicators and shelf life of fresh pork products. The effect of various preservatives on the total viable count and yeast growth in fresh meat during storage was studied. Experimental studies have shown that the compounds of additive a preservative mixture* actively inhibits microorganism growth during the fresh pork chops storage. In the control sample, the number of microorganisms on the seventh day of storage was 12*104 CFU/g, and, in the sample with the compound additive preservative mixture added, it amounted to 0.1*104 CFU/g. The usage of the ready to use preservative mixture allows actively suppressing the yeast reproduction during long-term storage (seven days) of coarsely chopped fresh pork products (250 CFU/g). The optimal method for applying the preservative to fresh pork chops has been determined. Applying the preservative to coarsely chopped fresh meat by simply mixing and massaging (for example, together with spices or marinades) is the most rational method for this product type. Primary and secondary lipid degradation products are considered, and the peroxide and acid numbers of fresh meat products during 30-day storage are determined. After 30 days of storage, a noticeable increase in oxidative processes in the control sample is observed, whereby the end point of the shelf life of coarsely chopped fresh pork products has been chosen.

*We have to negligate the trade mark name of the preservative mixture by the law of advertisement (The Ed.)

2. Introduction

The problem of efficient preservation of food and raw materials at all stages of their production, storage, transportation and trade, including home food preservation, appears highly relevant today. According to some estimates, up to 25% of the world’s food produced is susceptible to the damaging effects of mold alone [1].

Current methods for preserving food products and preventing their microbiological spoilage are divided into three groups: physical, chemical, and biological ones. Physical methods include temperature (thermal and refrigeration) exposure, drying, vacuuming, etc. Chemical methods comprise salting, smoking, brining, the use of preservatives, etc. Biological ones consist in the treatment with starter and bioprotective cultures, the use of bactericides, enzyme preparations, etc [2, 3]. Each of these methods has certain limitations in the production of a particular product due to their impact on organoleptic properties and nutritional values as well as technical feasibility (for example, need for the required equipment, scarcity of the substances or preparations used). Of all the known methods for preventing microbiological spoilage, chemical preservatives are considered the most easily applicable, quickly feasible, not requiring special equipment and/or changing the manufacturing method [4, 5]. However, the meat industry is rather conservative in terms of the use of food additives, due to the fact, that chemical preservatives are allowed in the production of meat products only in limited quantities, mainly in the manufacture of jellied products and for surface treatment [6]. In addition, consumers overwhelmingly have a negative attitude to meat, labelled as containing preservatives. In this regard, the use of chemical preservatives in the production of meat products is significantly limited and cannot be regarded as universal means to prevent microbiological spoilage.

In Russia, the consumption level and production of pork has been growing rapidly recently. At that, the meat industry is dominated by pork nowadays. Pork production increased by 23% in 2020.

Pork is also a source of complete animal protein and has a high nutritional value. In addition, pork meat contains vitamins, macro- and microelements necessary for a comprehensive development of the human body [7].

For all health benefit properties to be maximally preserved, the rules of processing, transportation and storage of meat have to be observed. According to the Sanitary Rules and Norms SanPiN and Technical Regulations of the Customs Union TRCU 034/2013 “On the safety of meat and meat products”, pork belongs to the category of perishable goods.

If the storage conditions and terms are violated, the growth and reproduction of microorganisms in fresh pork significantly accelerates, which leads to an increase in bacterial contamination. Under favorable conditions, microorganisms accumulate on the surface and gradually penetrate deep into the meat, causing the product spoilage. During storage, meat loses its positive properties, its organoleptic, physical and chemical parameters deteriorate significantly, and the risk of harm to human health increases due to the vital activity of pathogenic microbial flora. There are several types of meat spoilage: putrefaction, slime production, mold formation, acid fermentation (meat souring), etc. The intensity of these processes depends on temperature, relative humidity, microorganism type, and the degree of initial meat contamination [8].

Putrid spoilage is most often found when storage conditions are violated. Putrefactive microflora causes meat spoilage. Putrefactive microorganisms can be both aerobic and anaerobic. They are able to secrete protease enzymes that break down proteins. These microorganisms include aerobic bacilli (B. pyocyaneum, B. mesentericus, B. subtilis, B. megatherium), anaerobic clostridia (Cl. putrificus, Cl. histolyticus, Cl. perfringens, Cl. sporogenes) and facultative anaerobic cocci. The end products of aerobic putrefaction are ammonia, carbon dioxide, hydrogen sulfide, and mercaptans. Each of these compounds can cause harm to a human body, which manifests as a serious intoxication [9].

Anaerobic putrefaction of pork can occur without oxygen. Therefore, even vacuum packing will not protect the meat from spoilage if storage temperature requirements are violated. The end products of anaerobic putrefaction are the products of decarboxylation of amino acids causing the formation of off-odour substances, such as indole, skatol, phenol, cresol, diamines. Their derivatives are cadaveric poisons (cadaverine, putrescine, etc.); they are toxic to humans and can cause death [10].

Slime production is a result of slime-forming microorganisms (lactic acid bacteria, yeast, and micrococci) proliferating and partially dying off on the pork meat surface. The meat storage at a temperature of 18 to 25 oC and high humidity contribute to slime production. However, some microorganisms that cause slime formation can develop even at sub-zero temperatures. During sliming, the meat surface becomes sticky, acquires a gray-green hue and a stale off-odour, the pH of the meat surface layers is 5.2 to 5.3. It is important to distinguish between slime production and the initial stage of putrefaction, as each is caused by a completely different microflora [11].

Another equally dangerous type of meat spoilage is mold formation, which occurs when microscopic fungi develop on the surface during long-term storage of the product. When mold grows, the meat quality decreases because of protein hydrolysis and deamination of amino acids. The fungi most often found on the meat surface are Mucor, Penicillium, Aspergillus and Cladosporium. They are able to grow at low temperatures (in the refrigerators). These fungi produce mycotoxins, cause food spoilage, allergic reactions and various diseases in humans [12, 13].

The goal of this paper is investigating the effect of the compounds of food additives in the preservative mixture (detailed in the section 3.1.) on the resistance of fresh pork to microbiological spoilage during storage.

3. Materials and methods

3.1. Research objects

The research objects in this paper are the follow items:

  • Coarsely chopped fresh pork (with a fat content of not more than 15% by weight)
  • The compounds of food additive preservative mixture. The content of ready to use mixture are potassium sorbate (E202), sodium acetate (E262), sodium benzoate (E211), glycerin (E422), carboxymethylcellulose (E466) and an antioxidant (dihydroquercetin). The additive is manufactured by a research and manufacturing association Russia
  • Lactic acid
  • Acetic acid
  • Sodium acetate (E262)

3.2. Research methodology

The total viable count and the amount of yeast were determined by plating the product onto agar plates with culture media, allowing microorganisms to grow and counting all individual colonies.

The peroxide value and acid value were found using the standard methods [14, 15]. Method for determining the peroxide value is based on the reaction of the oxidation products of animal fats (peroxides and hydroperoxides) with potassium iodide in a solution of acetic acid and isooctane or chloroform, followed by quantitative determination of the released iodine with a solution of sodium thiosulfate using a titrimetric method. The method for determining the acid value is based on the dissolution of a sample in a mixed solvent, and titration of free fatty acids with a solution of potassium hydroxide.

All analyses were repeated in triplicate unless otherwise stated and the average values were calculated. The results are expressed as the mean value ± standard deviation. Significant differences between the mean values at significance level p < 0.05 were identified using the one-way analysis of variance and Student’s test. Microsoft Excel version 2010 was used as the statistical analysis software.

4. Results and discussion

To identify the functional properties of preservative mixture, tests were carried out on chilled pork in comparison with control samples and the most common substances having a preservative effect (lactic acid, acetic acid and sodium acetate). A comparative assessment of microbiological indicators in meat products was carried out, wherefore the quantities of mesophilic aerobic and facultative anaerobic microorganisms (MAFAM) were monitored for 7 days at a temperature of 8 to 10 oC. The experimental results are shown in Figure 1.

Figure 1. Effect of various preservatives on the total viable count of fresh pork chops during storage

The most common indicator of meat chops spoilage is natural acid fermentation. As a rule, acid fermentation develops in muscle tissue rich in glycogen. The main signs of the process are a sour off odour, gray or greenish hue, a decrease in the tissue elasticity and, as a result, a loose consistency. The causative agents of the defect are psychrotrophic lactic acid bacteria and yeast fungi, which ferment carbohydrates to form organic acids, as well as gases (carbon dioxide and, in some cases, hydrogen). In addition to meat carbohydrates, chopped meat products also contain carbohydrates that come from onions, marinades and other ingredients. These carbohydrates located in the brine between pork cuts are a favorable medium for pathogens of acid fermentation to develop.

Our experimental studies have shown that the selected preservatives actively inhibit microbial growth during storage. Thus, on day 7, the microbial content in the control sample was 12·104 CFU/g, in the sample with lactic acid added was 2·104 CFU/g, in the sample with acetic acid added was 1.8·104 CFU/g, in the sample with sodium acetate added was 0.7·104 CFU/g, and in the sample with the compounds of preservative mixture added was 0.1·104 CFU/g.

Preventing the yeast development in meat chops is an important component of raw meat manufacturers’ success, as it directly relates to the shelf life of the product and guarantees its safety for the consumer [16]. The contamination of meat products results from contaminated workers’ hands, storage containers, unsterilized spices and onions. Figure 2 shows the effect that various preservatives produce on the yeast growth in raw meat during storage.

Figure 2. Effect of various preservatives on the yeast growth in fresh pork chops during storage

According to the research results, the classical preservatives (lactic acid, acetic acid and sodium acetate) have a weak effect on the growth and reproduction of yeast during raw pork storage. The fast growth of yeast in fresh pork chops starts on day 2 and reaches its peak value of 1600 CFU/g (control sample) on day 7. However, the use of the compounds of the preservative mixture allows actively suppressing the yeast reproduction during long-term storage of fresh pork products (250 CFU/g).

The data obtained confirm that the preservative mixture not only effectively inhibits the growth of yeast, but also exhibits a clear antimicrobial activity against a wide range of microorganisms.

On studying the microbiological indicators of coarsely chopped fresh pork, it was found that the yeast content on the brined meat cuts surface is hundreds of times higher than in the internal tissue. Given this fact, it is obviously the meat surface as well as the ingredients in the brine that should mostly be exposed to preservatives. In order to verify this statement, the microbiological indicators of raw pork prepared according to the same recipe, but using different methods for applying the preservative, were compared. In one sample, the preservative was applied with a syringe solution; in another one, it was added in a liquid form, being mixed with onions and marinade and then massaged. The control sample was prepared without any preservatives. The experimental results are shown in Figure 3.

Figure 3. Effect of the method of applying a preservative on the yeast growth in fresh pork chops

Massaging was carried out in a meat tumbler “Metat master” in accordance with the tumbling program selected. Tumbling parameters are shown in Table 1.

Table 1. Tumbling parameters

Thus, applying a preservative to raw meat chops by simply mixing and massaging (for example, together with spices or marinades) is the most rational method for this product type. In this case, two positive effects are achieved simultaneously: the preservative concentration in the area affected by yeast increases and the total amount of the preservative in the product decreases, which provides advantages from the points of view of both product safety for consumer health and economic benefit.

The study of the increase in the shelf life of the product preserved using the preservative mixture was carried out at various time periods (from 5 to 30 days), every 5 days the amounts of primary and secondary lipid degradation products being measured and peroxide and acid numbers being determined [17].

The food additive was applied with different mass concentrations (0.1%, 0.4%, 0.6% and 1%).

Based on the data obtained, the dependence of the peroxide number (PN) values of the samples on the product storage duration was determined (Figure 4).

Depending on the dosage of the preservative mixture, the PN values vary, but in all options the increasing dynamics is observed over time. The highest rate is detected in the control sample. The lowest oxidative processes are observed in the samples containing 0.6% and 1% of the preservative and having significant differences with the control sample.

Figure 4. Accumulation of primary oxidation products in fresh pork chops during storage

On day 10, all samples containing preservative mixture exhibit a sharp increase in the PN values (1.6 times on average), which causes the interaction of acetic acid contained in the marinade with the antioxidant (dihydroquercetin), accompanied by a shift in acidity towards the alkaline side. On day 15 of storage, the PN value continues to gradually increase, and the active substance of the food additive begins inhibiting lipid peroxidation throughout the storage period, demonstrating significant differences with the control sample. After 30 days of storage, a noticeable increase in oxidative processes in the control sample is observed, whereby the end point of the shelf life of fresh meat has been chosen. However, the antioxidant activity allows increasing the product storage stability.

Similar dynamics are observed in the acid number variation (Figure 5).

Figure 5. Acid number variation over the entire product shelf life

Oxygenated products with excessive acidity reduce meat quality due to moisture loss. In turn, moderate acidity of meat products reduces the product quality to a lesser degree, so it remains juicy. In addition, the antioxidant contained in preservative mixture allows increasing the shelf life.

5. Conclusions

The novelty of this research is theoretically justified, and a high performance of the compounds of food additive preservative mixture in manufacturing raw pork is experimentally confirmed. Applying the additive allows reducing losses during heat treatment and storage of the meat products, increasing yield and improving consistency, as well as reducing the cost of production and increasing the shelf life of up to 30 days. This result is ensured by the use of the latest broad-spectrum antimicrobial preparation of preservative mixture. The preparation has a significant bactericidal effect and inhibits the growth and development of yeast. The most rational way to apply the preparation into a meat system is to add it to the product by simply mixing and then massaging (for example, with spices or marinades). As the preservative contains the natural antioxidant dihydroquercetin, the preparation can be used to good advantage for products with a high fat content to prevent the lipid fraction oxidation during storage. The optimal concentrations of the preparation in the meat system is from 0.6% to 1% by weight. A higher concentration will lead to a higher price of the end product. Meanwhile, a concentration of the bio-preservative less than 0.6% by weight will reduce the product storage stability and its resistance to microbial spoilage.

6. Conflicts of interest

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

7. Acknowledgement

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

8. References

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[2] Huffman, R. D. (2002): Current and future technologies for the decontamination of carcasses and fresh meat. Meat Science, 62, pp. 285–294. DOI

[3] Zhang, H. Z., Wu, J., Guo, X. (2015): Effects of antimicrobial and antioxidant activities of spice extracts on raw chicken meat quality. Food Science and Human Wellness, 5, pp. 39-48. DOI

[4] Chen, J. H., Ren, Y., Seow, J., Liu, T., Bang, W. S., Yuk, H. G. (2012): Intervention Technologies for Ensuring Microbiological Safety of Meat: Current and Future Trends. Comprehensive Reviews in Food Science and Food Safety, 11, pp. 119–132.

[5] Naveena, B. M., Sen, A. R., Vaithiyanathan, S., Babji, Y., Kondaiah, N. (2008): Comparative efficacy of pomegranate juice, pomegranate rind powder extract and BHT as antioxidants in cooked chicken patties. Meat Science, 80, pp. 1304–1308, DOI

[6] Aymerich, T., Picouet, P. A., Monfort, J. M. (2008): Meat decontamination technologies for meat products. Meat Science, 78, pp. 114–129. DOI

[7] Lucera, A., Costa, C., Conte, A., Del Nobile, M. A. (2012): Food applications of natural antimicrobial compounds. Frontiers in Microbiology, 3, pp. 1–13. DOI

[8] Russell, S. M. (2009): Understanding poultry spoilage. Last accessed 14th April 2017.

[9] Doulgeraki, A. I., Ercolini, D., Villani, F., Nychas, G. E. (2012): Spoilage microbiota associated to the storage of raw meat in different conditions. The International Journal of Food Microbiology, 157, pp. 130–141. DOI

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[11] Thomas, C. J., O’Rourke, R. D., McMeekin, T. A. (1987): Bacterial penetration of chicken breast muscle. Food Microbiology, 4(1), pp. 87–95. DOI

[12] Scallan, E., Hoekstra, R. M., Angulo, F. J., Tauxe, R. V., Widdowson, M. A., Roy, S. L., Jones, J. L., Griffin, P. M. (2011): Foodborne illness acquired in the United States-major pathogens. Emerging Infectious Diseases, 17, pp. 7–15. DOI

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[17] Aminzare, M., Hashemi, M., Ansarian, E., Bimkar, M., Azar, H. H., Mehrasbi, M. R., Daneshamooz, S., Raeisi, M., Jannat, B., Afshari, A. (2019): Using Natural Antioxidants in Meat and Meat Products as Preservatives: A Review. Journal of Animal and Veterinary Advances, 7, pp. 417–426.


News of the MTA Working Committee of Food Analysis and Classification Short summaries of the presentations of 2nd quarter of 2022 Analysis and classification of carbohydrates.

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News of the MTA Working Committee of Food Analysis and Classification
Short summaries of the presentations of 2nd quarter of 2022
Analysis and classification of carbohydrates.

Characterization of fiber and small molecule carbohydrate composition of cereals opportunities and challenges from the perspective of separation techniques

Eszter Schall, Marietta Klaudia Juhászné Szentmiklóssy, Sándor Tömösközi
(Budapest University of Technology and Economics, Department of Applied Biotechnology and Food Science, Research Group of Cereal Science and Food Quality):

In our plant-based foods, carbohydrates play an important role not only in terms of nutrition (energy source, food safety), but also in sensory and technological sense (structure, texture, stocking, way of production, etc.). In our research group we deal intensively with the composition of dietary fibre in cereals. But more detailed information about its quantitative and qualitative characterization, variability, and role in technological properties is only available in the case of common wheat. In addition, the characterization of FODMAPs (fermentable oligo-, di-, monosaccharides and polyols) among short-chain carbohydrates is gaining more and more attention in research due to their role in gastrointestinal disorders (irritable bowel syndrome). The analysis of both fibres and small molecular weight carbohydrates is a serious challenge, partly due to their diverse structure and their small amount. In the recent years, our research group has focused on the quantitative and qualitative characterization of arabinoxylans, the quantification of β-glucans, their quantitative and qualitative variability in cereals and understanding of their technological role. In addition, we have successfully adapted chromatographic and enzymatic methods for the quantitative characterization of FODMAP components. They can be used to obtain information about the typical amount in cereals, their variability between varieties and species, and any changes during processing. In the future, we intend to expand our equipment by adapting and developing chromatographic methods for the quantitative and qualitative characterization of β-glucans and arabinogalactan peptides.

Recent applications and possibilities of VIS, NIR and MIR spectroscopy in carbohydrate analysis

János Slezsák, András Salgó, Szilveszter Gergely
(Budapest University of Technology and Economics, Department of Applied Biotechnology and Food Science, Research Group of Cereal Science and Food Quality):

Vibration spectroscopy provides a fast, non-destructive way to study food industrial raw materials, as well as intermediates and end products. The first analytical applications of infrared spectroscopy were based primarily on the mid infrared (MIR) range. [1] However in recent decades, the near infrared (NIR) range was dominant in the study of food analysis (almost the entire agricultural and food vertical), as it has higher energy, so it can be used better than MIR in case of complex matrices of biological origin, and in contrast to the visible (Vis) range, significant chemical information can be obtained from the NIR spectra. Following the traditions of our research group [2], we have recently carried out comparative studies in the field of carbohydrate analysis with respect to different electromagnetic ranges (Vis, NIR, MIR) and optical configurations. This included the construction of spectral libraries, in which the spectra of several pure mono-, di- and polysaccharides were recorded with dispersive (DS), Fourier transform (FT) and diode-array (DA) NIR spectrophotometers to obtain information on the absorption characteristics of different carbohydrates. Based on the results, NIR techniques provide a good opportunity to identify pure carbohydrates, in several cases it is possible to distinguish highly similar compounds (e.g. identification of anhydrous or hydrated carbohydrates, determination of botanical origin in the case of starch). In addition to the study of pure systems, we have developed mathematical models for the qualification of various powder mixtures, including the quantification of carriers for commercially available flavorings (e.g. maltodextrin). One of the unusual segments of our research is the qualification of powdered samples (either in homogeneous or mixed form) through different packaging materials, either for identification or quantification. Based on the results of these experiments, in several cases it seems possible to develop NIR based techniques that allows the qualitative or quantitative determination of different carbohydrates without breaking the packaging. [3-4] Based on international trends, the Vis and MIR ranges are also receiving increasing attention due to advances in spectrum processing and evaluation. The implementation of advances in computer science (artificial intelligence based techniques, machine learning, data fusion) in the evaluation of analytical results will be an important task in the coming years. In addition, our objectives include a comparative study of miniaturized infrared devices, which are becoming more widespread today but have questionable reproducibility.


[1] Yakov M. Rabkin (1987): Technological Innovation in Science The Adoption of Infrared Spectroscopy by Chemists. Isis 78:1, pp. 31-54.

[2] Tömösközi S., Lásztity R, Salgó A., Vértessy G. B (2021): 100+10 év a felsőfokú élelmiszertudományi oktatás és a kutatás szolgálatában. Magyar Kémikusok Lapja 76: 10, pp. 286-292.

[3] Slezsák J., Szabó É., Gergely S., Salgó A. (2018): Measuring of food industrial raw materials via polyethylene packages by NIR spectrophotometers using different optical arrangements. Acta Alimentaria: An International Journal Of Food Science 47:1 pp. 104-112.

[4] Slezsák J., Szabó É., Besenyő G., Salgó A., Gergely S. (2019): Developing a model system for NIR based identification and quantitative analysis of food additives measured via polyethylene foils [konferencia-előadás]. NIR 2019, Gold Coast, Ausztrália.

Identification of the role of carbohydrates in the development of techno-functional and organoleptic properties of cereal-based food matrices

Renáta Németh, Edina Jaksics, Alexandra Farkas, Dávid Fekete, Sándor Tömösközi
(Budapest University of Technology and Economics, Department of Applied Biotechnology and Food Science, Research Group of Cereal Science and Food Quality)

Since ancient times, cereal grains have been the main source of energy in our diet, due to their high carbohydrate content (60-80%). The main constituents of carbohydrates in cereals are polysaccharides (70-80%), of which starch is of primary importance as a primary nutrient reserve. In addition, cell wall-forming polysaccharides such as arabinoxylans, beta-glucan, cellulose, etc., which are collectively referred to as non-starch polysaccharides, are present in significant amounts and function as dietary fibre. From a technological point of view, starch and non-starch polysaccharides are considered to be the most significant, as their molecular size and structure largely determine their solubility, hydration, gelling properties and thus the properties and quality of the intermediate and final products. Cereals also contain 1-2% of smaller carbohydrates, oligo-, di- and monosaccharides, which can also affect processing to some extent (e.g., fermentation). Several specific instruments can be applied to study the technological (e.g. mixing, stretching, gelling) behavior of grain flours, especially wheat. Examples include the rapid visco analyzer (RVA) and Mixolab techniques, as well as amylographic analysis and falling number measurement, which can be applied well to study the role of carbohydrate components. The mentioned instruments test the gelatinisation and gelling properties in a dilute flour-water suspension. An exception is Mixolab, by which mixing and viscous behavior together in a complex dough matrix can be investigated.

Our research on starch properties also confirms that the composition of starch (e.g., amylose-amylopectin ratio) fundamentally influences the gelatinisation properties (gelatinisation temperature, peak viscosity, degree of liquefaction) and gel formation (final viscosity). It can also be noted that isolated starches behave slightly different than flours, which may suggest interactions between flour constituents. The properties of non-starch polysaccharides, primarily arabinoxylans (AX), have also been investigated in our research works several times. Due to their structure, crosslinks can occur or decompose between arabinoxylan molecules through ferulic acid groups under redox conditions. Our results showed that using an appropriate oxidative enzyme (e.g., peroxidase, pyranose oxidase) crosslinks can form between AXs, increasing the consistency and viscosity of the matrix (suspension, dough). All this has proved to be suitable, for example, for improving the consistency of gluten-free dough matrices. In contrast, hydroxyl radical oxidation led to depolymerization of AXs, deteriorating the technological properties of the dough system. The study of the role of each constituents in a model system (fractioned into constituents and then reconstituted) provides an opportunity to identify deeper relationships. Our experiments in wheat-based model dough have shown that the reduction and re-oxidation of gluten network suggest the incorporation of added AX into the gluten structure, which is an important information for understanding the structural role of AXs in the dough and end product matrices.

There are still many unanswered questions about the compositional and structural variability of carbohydrates in cereals and their understanding of their closely related nutritional and technological role. All this will require further research and methodological improvements in order to promote conscious food development and the provision of scientifically sound, credible information to consumers.


2022/2 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 Marc 2022 to May 2022

13.060 Water quality

MSZ EN ISO 20236:2022 Water quality. Determination of total organic carbon (TOC), dissolved organic carbon (DOC), total bound nitrogen (TNb) and dissolved bound nitrogen (DNb) after high temperature catalytic oxidative combustion (ISO 20236:2018) – which has withdrawn the MSZ EN 12260:2004 –

MSZ EN ISO 16266-2:2022 Water quality. Detection and enumeration of Pseudomonas aeruginosa. Part 2: Most probable number method (ISO 16266-2:2018)

67 Food technology

67.040 Food products in general

MSZ ISO/TS 22002-6:2022 Prerequisite programmes on food safety. Part 6: Feed and animal food production

67.050 General methods of tests and analysis for food products

MSZ EN 15662:2018 Foods of plant origin. Multimethod for the determination of pesticide residues using GC- and LC-based analysis following acetonitrile extraction/partitioning and clean-up by dispersive SPE. Modular QuEChERS-method

67.200 Edible oils and fats. Oilseeds

MSZ EN ISO 665:2020 Oilseeds. Determination of moisture and volatile matter content (ISO 665:2020)

67.220 Spices and condiments. Food additives

MSZ EN ISO 6571:2022 Spices, condiments and herbs. Determination of volatile oil content (hydrodistillation method) (ISO 6571:2008 + Amd 1:2017) CONSOLIDATED VERSION

Withdrawn national standards from Marc 2022 to May 2022

67.100 Milk and milk products

MSZ 2713-5:1988 Chemical and physical test for butter. Determination of water dispersion value

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


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