Occurrence of Biogenic Amines in Wines from the Central European Region (Zone B) and Evaluation of Their Safety.

The decarboxylation of the corresponding amino acids by microorganisms leads to the formation of biogenic amines (BAs). From a toxicological point of view, BAs can cause undesirable physiological effects in sensitive individuals, particularly if their metabolism is blocked or genetically altered. The current study aimed to monitor and evaluate the content of eight biogenic amines (BAs) in 232 samples of wines (white, rosé, red) produced in the Central European region (Zone B). White wines (180 samples), rosé wines (17 samples), and red wines (35 samples) were analyzed. High-performance liquid chromatography equipped with a ultraviolet-visible diode array detector (UV/VIS DAD) was applied to identify and quantify the BAs present in wines. In general, histamine (HIS), tyramine (TYM), putrescine (PUT), cadaverine (CAD), phenylethylamine (PEA), spermine (SPN) and spermidine (SPD) were detected in all tested wine samples. Tryptamine (TRM) was not present in any of the samples examined. In white and red wines, SPD, TYM, and PUT were most often detected. Regarding rosé wines, the three major BAs were SPN, TYM, and CAD. The BA content in red wines was generally higher than in rosé and white wines. However, HIS concentrations above the recommended limit of 10 mg/L were detected in 9% of the red wine samples. In addition, alarming levels of PUT, HIS, TYM, and PEA, with serious potential impact on consumer health, were recorded in two red wine samples. On the whole, the presence and concentrations of BAs in wine should be constantly evaluated, primarily because alcohol intensifies the hazardous effects of BAs.

Effect of Selected Factors Influencing Biogenic Amines Degradation by Bacillus subtilis Isolated from Food.

Modern food technology research has researched possible approaches to reducing the concentration of biogenic amines in food and thereby enhance and guarantee food safety. Applying adjunct cultures that can metabolise biogenic amines is a potential approach to reach the latter mentioned goal. Therefore, this study aims to study the crucial factors that could determine the decrease in biogenic amines concentration (histamine, tyramine, phenylethylamine, putrescine and cadaverine) in foodstuffs using Bacillus subtilis DEPE IB1 isolated from gouda-type cheese. The combined effects of cultivation temperature (8 °C, 23 °C and 30 °C) and the initial pH of the medium (5.0, 6.0, 7.0 and 8.0) under aerobic and also anaerobic conditions resulted in the decrease of the tested biogenic amines concentration during the cultivation time (another factor tested). Bacillus subtilis was cultivated (in vitro) in a medium supplemented with biogenic amines, and their degradation was detected using the high-performance liquid chromatography equipped with UV-detector. The course of biogenic amines degradation by Bacillus subtilis DEPE IB1 was significantly influenced by cultivation temperature and also the initial pH of the medium (p < 0.05). At the end of the cultivation, the concentration of all of the monitored biogenic amines was significantly reduced by 65-85% (p < 0.05). Therefore, this strain could be used for preventive purposes and contributes to food safety enhance.

Changes in the Quality Attributes of Selected Long-Life Food at Four Different Temperatures over Prolonged Storage.

This study reports the development of selected indicators affecting changes in food quality and safety of selected long-life canned (Szeged goulash, canned chicken meat, pork pâté, canned tuna fish) and dehydrated (instant goulash soup) food during a two-year storage experiment at four different temperatures. The storage temperatures were selected to represent Arctic (−18 °C), temperate (5 °C), subtropical (25 °C) and tropical (40 °C) climatic zones where such food is likely to be stored during, for example, humanitarian and military missions. Microorganism amounts below the detection limit (p < 0.05), regardless of the storage temperature (p ≥ 0.05), were monitored in canned samples. The contents of dry matter, fat and proteins did not change during storage, regardless of the storage temperature (p ≥ 0.05). During the 24-month storage, all food showed an increase in the level of ammonia (p < 0.05) and the TBARS-value (p < 0.05), whereas the rate of increase in both parameters was significantly higher at higher storage temperatures (p < 0.05). The losses of individual amino acids during storage ranged from 5% rel. calculated on the amino acid contents in Month "0" up to 15% rel. (p < 0.05). With storage temperatures above the freezing point, the hardness values decreased with the increase in the storage temperature (p < 0.05) and prolongation of the storage period (p < 0.05). Moreover, with temperatures of −18 °C, the development of hardness, measured as the "decrease rate", was significantly higher compared to the absolute values.

Detection and relative quantification of amine oxidase gene (yobN) in Bacillus subtilis: application of real-time quantitative PCR.

Degradation of undesirable biogenic amines (BAs) in foodstuffs by microorganisms is considered one of the most effective ways of eliminating their toxicity. In this study, we design two sets of primers for the detection and quantification of the amine oxidase gene (yobN) and endogenous (housekeeping) gene (gyrB) in Bacillus subtilis. Moreover, these sets can be used for relative quantification of yobN by real-time PCR (qPCR). We also tested the degradation of BAs by three bacterial strains (B. subtilis strains: IB1a, CCM 2216, CCM 2267) in a mineral medium over a two-day period. Their degradation abilities were verified by high performance liquid chromatography with UV detection (HPLC/UV). According to the results, two strains significantly (P < 0.05) reduced histamine, tyramine, putrescine, and cadaverine. Moreover, our results indicate that the degradation ability of B. subtilis strains could be limited by sporulation because the gene encoding amine oxidase (yobN) is no longer expressed in the spores.

The impact of cell-free supernatants of Lactococcus lactis subsp. lactis strains on the tyramine formation of Lactobacillus and Lactiplantibacillus strains isolated from cheese and beer.

Tyramine is one of the most toxic biogenic amines and it is produced commonly by lactic acid bacteria in fermented food products. In present study, we investigated the influence of selected nisin-producing Lactococcus lactis subsp. lactis strains and their cell-free supernatants (CFSs) on tyramine production by four Lactobacillus and two Lactiplantibacillus strains isolated from cheese and beer. Firstly, we examined the antimicrobial effect of the CFSs from twelve Lactococcus strains against tested tyramine producers by agar-well diffusion assay. Six Lactococcus strains whose CFSs showed the highest antimicrobial effect on tyramine producers were further studied. Secondly, we investigated the influence of the selected six Lactococcus strains and their respective CFSs on tyramine production by tested Lactobacillus and Lactiplantibacillus strains in MRS broth supplemented with 2 g.L-1 of l-tyrosine. Tyramine production was monitored by HPLC-UV. The tyramine formation of all tested Lactobacillus and Lactiplantibacillus strains was not detected in the presence of Lc. lactis subsp. lactis CCDM 71 and CCDM 702, and their CFSs. Moreover, the remainder of the investigated Lactococcus strains (CCDM 670, CCDM 686, CCDM 689 and CCDM 731) and their CFSs decreased tyramine production significantly (P < 0.05) - even suppressing it completely in some cases - in four of the six tested tyramine producing strains.

Occurrence of Biogenic Amines Producers in the Wastewater of the Dairy Industry.

Out of six samples of wastewater produced in the dairy industry, taken in 2017 at various places of dairy operations, 86 bacterial strains showing decarboxylase activity were isolated. From the wastewater samples, the species of genera Staphylococcus, Lactococcus, Enterococcus, Microbacterium, Kocuria, Acinetobacter, Pseudomonas, Aeromonas, Klebsiella and Enterobacter were identified by the MALDI-TOF MS and biochemical methods. The in vitro produced quantity of eight biogenic amines (BAs) was detected by the HPLC/UV-Vis method. All the isolated bacteria were able to produce four to eight BAs. Tyramine, putrescine and cadaverine belonged to the most frequently produced BAs. Of the isolated bacteria, 41% were able to produce BAs in amounts >100 mg L-1. Therefore, wastewater embodies a potential vector of transmission of decarboxylase positive microorganisms, which should be taken into consideration in hazard analyses within foodstuff safety control. The parameters of this wastewater (contents of nitrites, nitrates, phosphates, and proteins) were also monitored.

Application of qPCR for multicopper oxidase gene (MCO) in biogenic amines degradation by Lactobacillus casei.

Degradation of undesirable biogenic amines (BAs) in foodstuffs by microorganisms is considered one of the most effective ways of eliminating their toxicity. In this study, we designed two sets of primers for the detection and quantification of the multicopper oxidase gene (MCO), which encodes an enzyme involved in BAs degradation, and endogenous (glyceraldehyde-3-phosphate dehydrogenase) gene (GAPDH) in Lactobacillus casei group by real-time PCR (qPCR). We tested 15 Lactobacillus strains in the screening assays (thus, MCO gene possessing assay (PCR) and monitoring of BAs degradation by HPLC-UV), in which Lactobacillus casei CCDM 198 exhibited the best degradation abilities. For this strain, we monitored the expression of the target gene (MCO) in time (qPCR), the effect of redox treatments (cysteine, ascorbic acid) on the expression of the gene, and the ability to degrade BAs not only in a modified MRS medium (MRS/2) but also in a real food sample (milk). Moreover, decarboxylase activity (ability to form BAs) of this strain was excluded. According to the results, CCDM 198 significantly (P < 0.05) reduced BAs (putrescine, histamine, tyramine, cadaverine), up to 25% decline in 48 h. The highest level of relative expression of MCO (5.21 ± 0.14) was achieved in MRS/2 media with cysteine.

Modelling biogenic amines in fish meat in Central Europe using censored distributions.

The aim of the study is to model concentrations of selected biogenic amines in various fish species (Atlantic salmon, Atlantic cod, striped catfish) bought in retail stores in Central Europe. Since the data contains non-detectable values, statistical methods for left-censored values from the exponential and Weibull distributions are applied and used to evaluate and compare the amount of biogenic amines in fish samples. Moreover, a risk of exceeding certain limits of biogenic amine concentrations to protect human health is determined. There are relatively high concentrations of putrescine, cadaverine and histamine in almost all fish species. Moreover, there was a significant difference in mean concentrations (distributions of concentrations, respectively) of histamine, tyramine and spermidine among the species.

Hydrogen Sulfide Effects on the Survival of Lactobacilli with Emphasis on the Development of Inflammatory Bowel Diseases.

The gut microbiota is a complex component of humans that depends on diet, host genome, and lifestyle. The background: The study purpose is to find relations between nutrition, intestinal lactic acid bacteria (LAB) from various environments (human, animal intestine, and yogurt) and sulfate-reducing microbial communities in the large intestine; to compare kinetic growth parameters of LAB; and to determine their sensitivity to different concentration of hydrogen sulfide produced by intestinal sulfate-reducing bacteria. METHODS: Microbiological (isolation and identification), biochemical (electrophoresis), molecular biology methods (DNA isolation and PCR analysis), and statistical processing (average and standard error calculations) of the results were used. THE RESULTS: The toxicity of hydrogen sulfide produced by sulfate-reducing bacteria, the survival of lactic acid bacteria, and minimal inhibitory concentrations (MIC) were determined. The measured hydrogen sulfide sensitivity values were the same for L. paracasei and L. reuteri (MIC > 1.1 mM). In addition, L. plantarum and L.fermentum showed also a similar sensitivity (MIC > 0.45 mM) but significantly (p < 0.05) lower than L.reuteri and L. paracasei (1.1 > 0.45 mM). L. paracasei and L. reuteri are more sensitive to hydrogen sulfide than L. fermentum and L. plantarum. L. pentosus was sensitive to the extremely low concentration of H2S (MIC > 0.15 mM). CONCLUSIONS: The Lactobacillus species were significantly sensitive to hydrogen sulfide, which is a final metabolite of intestinal sulfate-reducing bacteria. The results are definitely helpful for a better understanding of complicated interaction among intestinal microbiota and nutrition.

The Sulfate-Reducing Microbial Communities and Meta-Analysis of Their Occurrence during Diseases of Small-Large Intestine Axis.

Sulfate-reducing bacteria (SRB) are often isolated from animals and people with ulcerative colitis and can be involved in the IBD development in the gut-intestine axis. The background of the research consisted of obtaining mixed cultures of SRB communities from healthy mice and mice with colitis, finding variation in the distribution of their morphology, to determine pH and temperature range tolerance and their possible production of hydrogen sulfide in the small-large intestinal environment. The methods: Microscopic techniques, biochemical, microbiological, and biophysical methods, and statistical processing of the results were used. The results: Variation in the distribution of sulfate-reducing microbial communities were detected. Mixed cultures from mice with ulcerative colitis had 1.39 times higher production of H2S in comparison with samples from healthy mice. The species of Desulfovibrio genus play an important role in diseases of the small-large intestine axis. Meta-analysis was also used for the observation about an SRB occurrence in healthy and not healthy individuals and the same as their metabolic processes. Conclusions: This finding is important for its possible correlation with inflammation of the intestine, where the present of SRB in high concentration plays a major part. It can be a good possible indicator of the occurrence of IBD.

Formulation, Characterization and Properties of Hemp Seed Oil and Its Emulsions.

The formulation, characterization, and anticipated antibacterial properties of hemp seed oil and its emulsions were investigated. The oil obtained from the seeds of Cannabis sativa L. in refined and unrefined form was characterized using iodine, saponification, acid values, and gas chromatography, and was employed for the preparation of stable oil-in-water emulsions. The emulsions were prepared using pairs of non-ionic surfactants (Tween, Span). The effects of the emulsification method (spontaneous emulsification vs. high-intensity stirring), hydrophilic lipophilic balance (HLB), type and concentration of surfactant, and oil type on the size and distribution of the emulsion particles were investigated. It was found that the ability to form stable emulsions with small, initial particle sizes is primarily dependent on the given method of preparation and the HLB value. The most efficient method of emulsification that afforded the best emulsions with the smallest particles (151 ± 1 nm) comprised the high-energy method, and emulsions stable over the long-term were observed at HBL 9 with 10 wt % concentration of surfactants. Under high-intensity emulsification, refined and unrefined oils performed similarly. The oils as well as their emulsions were tested against the growth of selected bacteria using the disk diffusion and broth microdilution methods. The antibacterial effect of hemp seed oil was documented against Micrococcus luteus and Staphylococcus aureus subsp. aureus. The formulated emulsions did not exhibit the antibacterial activity that had been anticipated.

Sensitivity to Enterocins of Biogenic Amine-Producing Faecal Enterococci from Ostriches and Pheasants.

Enterococci are widespread bacteria forming the third largest genus among lactic acid bacteria. Some possess probiotic properties or they can produce beneficial proteinaceous antimicrobial substances called enterocins. On the other hand, some enterococci produce biogenic amines (BAs), so this study is focused on the sensitivity to enterocins of biogenic amine-producing faecal enterococci from ostriches and pheasants. Altogether, 60 enterococci isolated from faeces of ostriches and pheasants were tested for production of BAs. This target of the identified enterococci involved 46 strains selected from 140 ostriches and 17 from 60 pheasants involving the species Enterococcus hirae, E. faecium, E. faecalis, and E. mundtii. Although BAs histamine, cadaverine, putrescine, and tryptamine were not detected in the enterococci tested, in general high BA production by the tested enterococci was noted. The species E. hirae formed the majority of the enterococcal strains from ostrichs faeces (34 strains). High production of tyramine (TYM) was measured with an average amount of 958.16 ± 28.18 mg/ml. Among the enterococci from pheasants, the highest was production of TYM compared to phenylethylamine, spermidine, and spermine. Enterococci featured high BA production; however, they were sensitive to seven enterocins with inhibition activity ranging from 100 up to 25,600 AU/ml.

Decarboxylase-positive Enterococcus faecium strains isolated from rabbit meat and their sensitivity to enterocins.

BACKGROUND: The objective of the study was to determine sensitivity of Enterococcus faecium strains from rabbit meat to enterocins. RESULTS: Twenty-five decarboxylase-positive strains (from rabbit meat) allotted to the species E. faecium by genotypization and by MALDI TOF MS spectrometry identification (evaluation score value range 2.104-2.359; in the range for highly probable species identification-score value 2.300-3.000 and secure probable species identification/probable species identification-2.000-2.299) were studied. Seventeen strains were gelatinase positive. Although they did not produce histamine (HIS), spermidine, and spermine, they produce at least one among seven tested biogenic amines (BAs) in small amounts (2-10 mg/L) or up to very high amounts (>1000 mg/L). Putrescine was produced by two strains. These decarboxylase-positive strains were sensitive to enterocins (Ents). All strains were sensitive to Ent 2019 and Ent 55 (inhibitory activity from 200 to 819 200 AU/mL). Twenty-two strains were inhibited by Ent A(P) and Ent 4231; 20 strains were sensitive to Ent M. CONCLUSION: Our results have spread the basic knowledge related to inhibitory spectrum of enterocins showing sensitivity of decarboxylase-positive strains to enterocins. Protective possibilities of enterocins in meat processing were also indicated.

Effect of lantibiotic gallidermin against biogenic amine-producing faecal staphylococci from ostriches and pheasants.

In ostriches and pheasants, there is still limited information relating to staphylococci and their properties. Biogenic amines (BAs) are nitrogenous low-molecular-weight substances with biological functions in animals, plants and microorganisms. In this study, we focused on BA production by targeted faecal staphylococci from ostriches and pheasants and their sensitivity to lantibiotic bacteriocin gallidermin. Gallidermin belongs in a group of polycyclic proteinaceous antimicrobial substances. Thirty-six faecal staphylococci (24 strains from 140 ostriches, 12 from 60 pheasants) comprising different species were tested. Staphylococci from ostriches and pheasants did not produce tryptamine-TRYP, putrescine-PUT, cadaverine-CAD or histamine-HIS. Production of tyramine-TYM, phenylethylamine-PEA was high or very high (100-1000 mg/L). Production of spermine-SPM and spermidine-SPD by staphylococci was very low or low although in the case of staphylococci from pheasants medium production of SPM was found. Because of the risk posed by BAs for consumers, the control of BA-producing bacteria is important from the points of view not only of safety assessment of food-producing animals but also of human health safety. The sensitivity to gallidermin in biogenic amine-producing staphylococci from ostriches and pheasants detected here is the most promising indication for further application of gallidermin for veterinary purposes. The novelty of our study lies in testing the ability of faecal staphylococci from ostriches and pheasants to produce BAs and in their treatment with gallidermin which has so far not been tested in this way.

Microflora of processed cheese and the factors affecting it.

The basic raw materials for the production of processed cheese are natural cheese which is treated by heat with the addition of emulsifying salts. From a point of view of the melting temperatures used (and the pH-value of the product), the course of processed cheese production can be considered "pasteurisation of cheese." During the melting process, the majority of vegetative forms of microorganisms, including bacteria of the family Enterobacteriaceae, are inactivated. The melting temperatures are not sufficient to kill the endospores, which survive the process but are often weakened. From a microbiological point of view, the biggest contamination problem of processed cheese is caused by gram-positive spore-forming rod-shaped bacteria of the genera Bacillus, Geobacillus, and Clostridium. Other factors affecting the shelf-life and quality of processed cheese are mainly the microbiological quality of the raw materials used, strict hygienic conditions during the manufacturing process as well as the type of packaging materials and storage conditions. The quality of processed cheese is not only dependent on the ingredients used but also on other parameters such as the value of water activity of the processed cheese, its pH-value, the presence of salts and emulsifying salts and the amount of fat in the product.

Effects of different strains Penicillium nalgiovense in the Nalzovy cheese during ripening.

BACKGROUND: The present study examined the influence of eight selected strains of Penicillium nalgiovense as secondary culture on the ripening of soft mould cheese. Ion-exchange chromatography, SDS-PAGE and sensory analysis were used for comparison of P. nalgiovense with common secondary mould culture (Penicillium camemberti). RESULTS: Proteolytic activity was more intensive in the case of the strains of P. nalgiovense in comparison with the control strains of P. camemberti. Some differences in the proportion and amount of free amino acids were observed between the strains of P. nalgiovense during ripening. Lower proteolytic activity of P. nalgiovense was more desirable for sensory properties of cheese, especially in case sample N2. CONCLUSION: This study provides information on the usability of selected strains of P. nalgiovense that could support the development of sensory properties of mould-ripened soft cheese. The cheese with the culture N2 had the best flavour after 35 days of storage. Moreover, this strain showed the lowest bitterness in comparison with the other used P. nalgiovense cultures. The intensity of bitterness of sample N2 was comparable to that of the control sample with P. camemberti. © 2015 Society of Chemical Industry.

Biogenic amine production by Lactococcus lactis subsp. cremoris strains in the model system of Dutch-type cheese.

The aim of this study was to compare the biogenic amine production of two starter strains of Lactococcus lactis subsp. cremoris (strains from the Culture Collection of Dairy Microorganisms - CCDM 824 and CCDM 946) with decarboxylase positive activity in a model system of Dutch-type cheese during a 90-day ripening period at 10°C. During ripening, biogenic amine and free amino acid content, microbiological characteristics and proximate chemical properties were observed. By the end of the ripening period, the putrescine content in both samples with the addition of the biogenic amine producing strain almost evened out and the concentration of putrescine was >800mg/kg. The amount of tyramine in the cheeses with the addition of the strain of CCDM 824 approached the limit of 400mg/kg by the end of ripening. In the cheeses with the addition of the strain of CCDM 946 it even exceeded 500mg/kg. In the control samples, the amount of biogenic amines was insignificant.

Effects of temperature, pH and NaCl content on in vitro putrescine and cadaverine production through the growth of Serratia marcescens CCM 303.

The aim of this study was to evaluate the combined effect of temperature (10, 20 and 37°C), pH (4, 5, 6, 7 and 8), and NaCl content (0, 1, 3, 4, 5 and 6% w/v) on the growth and putrescine and cadaverine production of Serratia marcescens CCM 303 under model conditions. The decarboxylase activity of S. marcescens was monitored in broth after cultivation. The cultivation medium was enriched with selected amino acids (ornithine, arginine and lysine; 0.2% w/v each) serving as precursors of biogenic amines. Levels of putrescine and cadaverine in broth were analysed by high-performance liquid chromatography after pre-column derivatisation with o-phthalaldehyde reagent. S. marcescens produced higher amounts of putrescine (up to 2096.8 mg L(-1)) compared to cadaverine content (up to 343.3 mg L(-1)) in all cultivation media. The highest putrescine and cadaverine concentrations were reached during cultivation at 10-20°C, pH 5-7 and NaCl content 1-3% w/v. On the other hand, the highest BAs production of individual cell (recalculated based on a cell; so called "yield factor") was observed at 10°C, pH 4 and salt concentration 3-5% w/v as a response to environmental stress.

The effect of long-term storage on the quality of sterilized processed cheese.

The aim of this work is to evaluate the effect three different storage temperatures (6, 23 and 40 °C) on the sterilized processed cheese quality during 24-month storage. Sterilized processed cheese (SPC) is a product with extended shelf life (up to 2 years). The samples of SPC were subjected to basic chemical analyses, i.e. pH-values, dry matter, fat, crude protein and ammonia content, and microbiological analyses, i.e. total number of microorganisms, number of coliforms, colony forming units of yeasts and/or moulds and spore-forming microorganisms. Furthermore, amino acid content (ion-exchange chromatography), protein profile (SDS-PAGE) and fat globules size (image analysis of microscopic technique) were monitored and sensory analysis (scale test and pair comparative test) was implemented, too. Increasing storage temperature and length evoked decrease of total amino acid content and protein nutrition value, increase of ammonia amount, protein changes, enlargement of fat globule size and deterioration of sensory properties of SPC. All the changes grew expressive with increasing storage temperature and time.

The influence of fat and monoacylglycerols on growth of spore-forming bacteria in processed cheese.

Highly undesirable microbial contaminants of processed cheese are endospore-forming bacteria of the genera Bacillus and Clostridium. Survival of Bacillus subtilis, B. cereus, Clostridium butyricum and C. sporogenes was examined in model processed cheese samples supplemented with monoacylglycerols. In processed cheese samples, monoacylglycerols of undecanoic, undecenoic, lauric and adamantane-1-carboxylic acid at concentration of 0.15% w/w prevented the growth and multiplication of both Bacillus species throughout the storage period. The two species of Clostridium were less affected by monoacylglycerols in processed cheese samples and only partial inhibition was observed. The effect of milk fat content on microbial survival in processed cheese was also evaluated. The growth of Bacillus sp. was affected by the fat level of processed cheese while population levels of Clostridium sp. did not differ in processed cheese samples with 30, 40 and 50% fat in dry matter.