Biogenic amine production by lactic acid bacteria, acetic bacteria and yeast isolated from wine

Biogenic amines are undesirable in all foods and beverages because if consumed at too high concentration, they may induce food-borne intoxications. The biogenic amine production by 155 strains of lactic acid bacteria, 40 strains of acetic bacteria and 36 strains of yeast isolated from wine were analysed in wine, grape must and synthetic media by HPLC. We did not observe biogenic amine production by acetic bacteria and yeast; however, we found production of histamine, tyramine, phenylethylamine and putrescine by LAB. A correlation of 100% was observed between biogenic amine production in synthetic medium and wine and between activity and presence of gene. With the results expose in this paper, we can consider than the lactic acid bacteria are the microorganisms responsible of histamine, tyramine and phenylethylamine production in wine. However, we cannot consider the microorganisms analysed in this work to be those responsible for tryptamine, cadaverine and putrescine levels in wine. These results could lead to future applications for preventing excessive amounts of histamine, tyramine and phenylethylamine forming during vinification and storage.     

Biogenic amines content in traditional dry fermented sausage Petrovská klobása as possible indicator of good manufacturing practice

The aim of this study was to determine the content of nine biogenic amines (tryptamine, phenylethylamine, putrescine, cadaverine, histamine, serotonin, tyramin, spermidine and spermine) and total biogenic amines in traditional dry fermented sausage Petrovská klobása at the end of drying period, as possible indicators of good manufacturing practice (GMP).Chromatographic separation of dansyl chloride derivatizated amines was completed in 8 min. Analyses were performed by HPLC-DAD on Eclipse XDB-C18 column. Tryptamine and spermine were determined in all analyzed samples, while histamine, serotonin and spermidine were not detected in any samples. Average content of individual biogenic amine ranged from 5.54 mg/kg (putrescin) to 75.1 mg/kg (tryptamine) and total content of biogenic amines varied from 77.8 mg/kg to 174 mg/kg.The results obtained in this study provided no evidence for deviations from good manufacturing practice in Petrovská klobása production.     

Inhibitory effects of garlic and other spices on biogenic amine production in Myeolchi-jeot,Korean salted and fermented anchovy product

This study was carried out to reduce biogenic amine contents in Myeolchi-jeot, Korean salted and fermented anchovy (Engraulis japonicus). The effects of a variety of spices including ginger, garlic, green onion, red pepper, clove and cinnamon, on biogenic amine production were determined by HPLC. The greatest inhibitory effect on biogenic amine production was observed in the culture treated by garlic extract. In the culture, the contents of putrescine, cadaverine, histamine, tyramine and spermidine were reduced by up to 11.2%, 18.4%, 11.7%, 30.9% and 17.4%, respectively, compared to control. The other spice extracts tested showed less or no effect in reducing biogenic amine contents. In addition, the extract of garlic showed the highest antimicrobial activity against the amine producers tested. The extract of garlic at a concentration of 5% (weight basis) was finally applied to the ripening of Myeolchi-jeot in situ, and then overall production of biogenic amines in Myeolchi-jeot was found to be reduced by up to 8.7%, compared to control. Consequently, it is expected that the findings of this study might be helpful for enhancing the safety of Myeolchi-jeot.     

Genetic screening of biogenic amines production capacity from some lactic acid bacteria strains

There is an increasing interest for using lactic acid bacteria (LAB) as a starter and adjunct cultures for producing novel foods with particular functional traits. The ability of the starter to produce biogenic amines (BA) should be taken into account wherein protective starters should be selected to avoid hygienic and toxicological risks. This work aimed to study the possibilities of forming BA (histamine, putrescine, agmatine and tyramine) from thirty two LAB strains belonging to species of the genera Lactobacillus and Streptococcus that used in food products as well as strains isolated from healthy breast-fed infants. The analytical protocol involved using polymerase chain reaction (PCR) and thin layer chromatography (TLC) techniques to determine the ability of LAB strains to form BA. The presence of key genes involved in the biosynthetic pathways of the BA was also assessed by PCR. Six LAB strains gave positive results for putrescine production wherein the maximum level was 14.6 mg/kg. Six strains gave positive results for histamine production (maximum level was 31.7 mg/kg) and were positive for the presence of histidine decarboxylase (HDC) gene. Seven strains exhibited positive results for tyramines production (maximum level was 2.85 mg/kg) and were positive for the presence of tyrosine decarboxylase (TDC) gene. Eight strains gave positive results for agmatine production (maximum level was 174.5 mg/kg) and were positive for the presence of dihydrolase (deiminase) gene that responsible for agmatine formation. It could be concluded that the microorganisms used in food and dairy production should be screened carefully by PCR for their ability to produce BA.     

Biogenic amines in commercial fish and fish products sold in southern China

Eight biogenic amines (histamine, tryptamine, putrescine, 2-phenylethylamine, cadaverine, tyramine, spermidine and spermine) were determined in 13 species of fish and 49 fish products commonly consumed in southern China. The biogenic amines were analyzed by reverse phase HPLC with a fluorescence detector following pre-column derivation with dansyl chloride. In the fish samples, the total level of biogenic amine (BA) ranged from 5.03 mg/kg to 156.17 mg/kg, with a mean value of 44.17 mg/kg. Histamine levels were less than 21.85 mg/kg. The BA levels detected were not high enough to indicate fish decomposition. Low BA levels were also found in most fish product samples. However, several samples of fermented fish products and packaged fish products showed higher levels (484.42 mg/kg of BA in lightly cured horse mackerel and 166.45 mg/kg of BA in packaged eel). Specifically, lightly cured horse mackerel, which is a traditional fish product in southern China, exhibited relatively high levels of 2-phenylethylamine (57.61 mg/kg), cadaverine (244.41 mg/kg) and tyramine (62.85 mg/kg). Such samples with relatively high BA content require careful monitoring to ensure their safety for human consumption.     

The potentials of Bacillus licheniformis strains for inhibition of B. cereus growth and reduction of biogenic amines in cheonggukjang (Korean fermented unsalted soybean paste)

The aim of this study was to control Bacillus cereus growth and biogenic amines formation by using a starter in cheonggukjang fermentation. We used the four selected B. licheniformis strains for cheonggukjang fermentation and investigated whether the strains are effective in reducing both B. cereus level and biogenic amines content. We confirmed that the selected strains are able to reduce B. cereus level to below l log CFU/g after cheonggukjang fermentation from the initial level of 3–4 log CFU/g (artificial contamination). Tryptamine, β-phenylethylamine, putrescine, cadaverine, tyramine, spermidine, and spermine were detected whereas histamine, serotonin, noradrenaline, and dopamine were not detected in the cheonggukjang samples. Most of biogenic amines contents were lower than those of previous studies, indicating that the strains produced the low level of biogenic amines in cheonggukjang fermentation. The acceptable levels of amino-type nitrogen, free amino acids, and isoflavones were contained in the cheonggukjang samples. These results demonstrate that the selected strains are not only effective for reduction of both B. cereus contamination and biogenic amines but also suitable for preparation of cheonggukjang with good quality.     

Effect of rose polyphenols on oxidation,biogenic amines and microbial diversity in naturally dry fermented sausages

Rose polyphenols (RPs) have antioxidant and antibacterial activities. In order to evaluated the application of RPs in meat products, the effect of RPs on the safety of fermented sausages was investigated. In the present study, RPs inhibited the increase of pH. Phenylethylamine, putrescine, cadaverine, tyramine and spermidine can be detected in sausages, while spermine and tryptamine were not observed. Sausages containing RPs had lower thiobarbituric acid reactive substance values and biogenic amines than the control. Moreover, RPs decreased the total bacterial count but increased growth rate of lactic acid bacteria. The effect of RPs on oxidation, biogenic amines and bacterial growth is dependent on the concentration. High-throughput DNA sequence analysis revealed that operational taxonomical units (OTUs) of samples without PRs were higher than those containing 3 mg/g RPs, and 323 OTUs shared between two batches. Especially, a higher level of Lactobacillales OTUs was observed in the 3 mg/g batches. These results indicated that RPs could be used to improve the safety of fermented sausage by inhibiting lipid oxidation, biogenic amines formation and spoilage bacterial growth, and increasing growth rate and richness of lactic acid bacteria.     

Sequencing-based screening of functional microorganism to decrease the formation of biogenic amines in Chinese rice wine

As a traditional alcoholic beverage, Chinese rice wine with a unique flavor and high nutritional value has been popular in China for thousands of years. The biogenic amines in Chinese rice wine and other fermented food are attracting much more attention than ever. There are numerous microorganisms accompanying the fermentation of Chinese rice wine. Based on the next-generation sequencing technology, an in vivo screening process was carried out to find the strains without biogenic amines formation, and then these strains were screened and back-added into the fermentation mash. It was found that the Lactobacillus might not produce biogenic amines by the correlation analysis of bacteria community succession and biogenic amines change. After that, three Lactobacillus strains without biogenic amines formation were screened and identified as Lactobacillus plantarum and L. hilgardii. The L. plantarum JN01 showed high lactic acid production, low acetic acid production, and high ethanol tolerance which was preferable for Chinese rice wine brewing. When 0.001 gDCW/t of L. plantarum JN01 was used to inoculate the fermentation mash, the biogenic amines concentration was significantly decreased 24%. Although the formation of biogenic amines was further inhibited by increasing the dose of L. plantarum JN01, the total acid and the residue sugar would increase resulting an incomplete fermentation. In this paper, we firstly developed an effective method to decrease the formation of biogenic amines in traditional fermentation foods based on the next-generation sequencing technology. This research provided new strategy to decrease biogenic amines in fermentation food.     

Preliminary evaluation of biogenic amines content in Chilean young varietal wines by HPLC

Biogenic amines are organic nitrogenous compounds produced in wines mainly during malolactic fermentation. Its presence is a health risk for sensitive individuals and it could negatively affect the wine quality. The objective of this work was to determine the biogenic amines content in Chilean young varietal wines. For that, a liquid chromatographic method with pre-column derivatization was optimized and validated to achieve a reliable quantification of the most important biogenic amines in wines, i.e. histamine, tyramine, spermine, spermidine, putrescine, cadaverine and phenylethylamine. Using a face-centered central composite design derivatization and chromatographic conditions were optimized. Derivatization with dansyl chloride showed an optimum yield for all biogenic amines at 60 °C during 60 min. These derivatives were optimally separated with a C₁₈ YMC-Pack ODS-A column set a 40 °C using a mobile phase composed of acetonitrile and water under gradient conditions. An effective separation and quantification of the biogenic amines was achieved in 40 min using ultraviolet detection at 254 nm. Validation was established according to ICH guidelines, calibrations in matrix fitted a linear regression model with R² > 0.997. Repeatability (n = 6) and intermediate precision (n = 3) in matrix showed RSD values <2.34% and <3.16%, respectively. This method was applied to determine for the first time the biogenic amines content in Chilean wines. Twenty-seven samples of young varietal wines, i.e. Cabernet Sauvignon (n = 9), Merlot (n = 8) and Carménère (n = 10) were analyzed finding a biogenic amines content from 18.12 to 39.84 mg L^-1, where putrescine showed the highest concentration in all grape varieties. No significant difference was found between grapes varieties regarding total biogenic amines content (P = 0.7842, df = 26). Along with putrescine, histamine and tyramine were the most abundant biogenic amines but similar levels were also observed with spermidine.     

Reduction of the biogenic amine contents in low salt-fermented soybean paste by gamma irradiation

Irradiation effects on biogenic amines of low salt-fermented soybean paste were investigated during fermentation at 25 °C for 12 weeks. Low salt-fermented soybean paste was prepared with 6% and 8% salt, and a 12% salted one was used as a control. The prepared fermented soybean pastes were irradiated with doses of 5, 10, and 15 kGy. Non-irradiated samples (6% and 8% salt) showed optimum pH to produce biogenic amines during fermentation. The biogenic amines detected were putrescine, cadaverine, β-phenylethylamine, spermidine, spermine, tryptamine, histamine, tyramine, and agmatine. Most biogenic amines, except β-phenylethylamine and tyramine, were significantly reduced by irradiation treatment during fermentation. Lower salt conditions produced more biogenic amines than higher ones. However, gamma irradiation of low salt-fermented soybean paste could reduce the biogenic amine contents during fermentation.     

Induction of plantaricin MG under co-culture with certain lactic acid bacterial strains and identification of LuxS mediated quorum sensing system in Lactobacillus plantarum KLDS1.0391

A bacteriocin named plantaricin MG was produced by Lactobacillus plantarum KLDS1.0391 which was isolated from “jiaoke”, a traditional, naturally fermented cream from Inner Mongolia in China. Bacteriocin production was increased significantly when L. plantarum KLDS1.0391 co-cultured with certain lactic acid bacteria (LAB) strains. Among 76 strains belonging to the genera Lactobacillus, Lactococcus, Leuconostoc and Streptococcus, four strains namely Lactobacillus helveticus KLDS1.9207, Enterococcus faecium KLDS4.0352, Lactobacillus reuteri KLDS1.0737 and Enterococcus faecalis KLDS4.0313 were shown to induce bacteriocin production of L. plantarum KLDS1.0391. Cell numbers of L. plantarum KLDS1.0391 were greatly enhanced when co-cultured with four bacteriocin-inducing strains. Bacteriocin production was not induced by autoclaved cultures and cell-free supernatants (CFS) of inducing strains, indicating that living cells of inducing strains might be necessary for enhancement of bacteriocin production. The presence of plnD and luxS genes was detected by polymerase chain reaction (PCR), and the existence of plNC8HK gene was detected by single oligonucleotide nested PCR (SON-PCR).     

Elaboration of Tempranillo wines at two different pHs. Influence on biogenic amine contents

The aim of this work was to study the effect of pH on biogenic amine formation during the elaboration and conservation (7 months) of Tempranillo wines. Grapes at two pHs (3.4 and 3.7) were vinified to achieve this purpose. After alcoholic fermentation the wines were either inoculated with a commercial malolactic starter or not inoculated. Identification and clonal distribution of lactic acid bacteria and amino acid concentration were determined in an attempt to explain the biogenic amine generation. The results showed that the pH of the must influenced the clonal distribution of the Oenococcus oeni strains which conducted the malolactic fermentation and also the concentration of amino acids in the wines after alcoholic fermentation. These aspects could account for the higher biogenic amine formation in wines with the lowest pH during malolactic fermentation. In these wines, inoculation with a malolactic starter was favourable since it produced a lower biogenic amine concentration after malolactic fermentation. Furthermore, the pH also influenced the concentration of amino acids after malolactic fermentation and the lactic acid bacteria distribution during the conservation of the wines. These aspects allow explaining the greater formation of histamine, tyramine and putrescine in the wines with the lowest pH vinified via lactic acid bacteria inoculation after seven months of conservation.     

Monitoring the contents of biogenic amines in sufu by HPLC with SPE and pre-column derivatization

Sufu is a nutritious fermented food whose consumption is currently increasing in many countries. The purpose of this study was to develop an HPLC method with SPE and pre-column derivatization for monitoring the content of biogenic amine in sufu and to understand the differences in the contents of biogenic amines with different processing technologies. Biogenic amines in sufu were extracted with 6% trichloroacetic acid, derivatized with 4-chloro-3,5-dinitrobenzotrifluoride, purified with Waters Oasis MCX cartridge, separated by HPLC using gradient elution, and detected by spectrophotometric UV at 254 nm. The method linearity, calculated for each biogenic amine, has a correlation coefficient higher than 0.9915. Detection limits of the biogenic amines were ranged from 0.016 mg/kg for putrescine to 0.067 mg/kg for spermine. Spiking experiments showed that the overall recoveries ranged between 83.6% and 103.5% with relative standard deviations of 1.26–5.24%. Of the seven biogenic amines under study, at least four biogenic amines were detectable in different kinds of sufu. Tryptamine, putrescine, histamine, and tyramine were the most prevailing biogenic amines in all sufu samples. The mean values of histamine, tryptamine, phenylethylamine, tyramine, putrescine, spermidine and spermine determined in samples were found to be 18.2 mg/kg, 39.0 mg/kg, 1.1 mg/kg, 21.7 mg/kg, 34.6 mg/kg, 11.1 mg/kg, and 8.7 mg/kg, respectively. The total contents of biogenic amines in bacteria fermented sufu samples (129.0 mg/kg) were slightly lower than those in enzymatically ripened sufu samples (137.7 mg/kg). The concentrations of biogenic amines detected in sufu samples were below the maximum permissible limits.     

Monitoring of the quality of modified atmosphere packaged broiler chicken cuts stored in different temperature conditions: B. Biogenic amines as quality-indicating metabolites

The formation of biogenic amines was studied in broiler chicken cuts stored in modified atmosphere (MA) packages. Also, the applicability of biogenic amines for the quality control of MA-packaged broiler chicken cuts was evaluated in various constant and variable temperature schemes. It was found that temperature dependent changes took place in the concentrations of tyramine, putrescine and cadaverine. The changes in the concentrations of these amines were in accordance with the microbiological quality of broiler chicken cuts. On the basis of the results it was concluded that tyramine, putrescine and cadaverine could serve as quality indicating tools for MA-stored poultry.     

Biogenic amine content and aromatic profile of Salama da sugo,a typical cooked fermented sausage produced in Emilia Romagna Region (Italy)

Salama da sugo is a fermented sausage from Ferrara tradition, subjected to a long ripening period (4–6 months). It can be consumed after cooking, served with its sugo, i.e. the liquid extracted by cooking process. Besides to pork meat and fat, also NaCl, curing salts, sugar, spices and almost 15% of red wine are added. The aim of this research was the microbial and aromatic profile characterization of Salama da sugo during ripening. The product was sampled in order to study chemico-physical parameters, microbial population composition (Enterobacteriaceae, lactobacilli, enterococci, micrococci and staphylococci, including coagulase positive, fungi, total mesophilic bacteria), biogenic amine content and aromatic profile. The results showed that this traditional product was characterized by a complex aroma.Moreover, the biogenic amine concentration was low (less than 200 mg/kg for tyramine, about 50 mg/kg for cadaverine and putrescine while histamine was below the detection limit).     

Factors influencing biogenic amine production by a strain of Oenococcus oeni in a model system

Biogenic amines are undesirable compounds produced in foods mainly through bacterial decarboxylase activity. No legal limits are given by international institutions but the presence of these substances is a fundamental quality parameter for alcoholic beverages. This work evaluated the interactive effect of some variables on the biogenic amine production of the strain Oenococcus oeni T56. At higher pH values biogenic amines were produced in considerable amount whereas high ethanol concentrations and low concentration of pyridoxal-5-phosphate reduced their accumulation, such as.The roles of arabinose and SO₂ are more complex and involve specific pathways rather than whole cellular activity.     

Biogenic amine inhibition and quality protection of Harbin dry sausages by inoculation with Staphylococcus xylosus and Lactobacillus plantarum

This study was conducted to evaluate the effects of inoculation with Staphylococcus xylosus, Lactobacillus plantarum, or a mixture of strains (L. plantarum + S. xylosus) on the formation of biogenic amines (BAs) and quality characteristics in Harbin dry sausage. Microbial analysis shows that total aerobic bacteria and lactic acid bacteria (LAB) counts were higher in the inoculated sausages, especially in those inoculated with a mixture of strains, but the growth of enterobacteriaceae was inhibited (P < 0.05). A sharp decrease in the pH value of the sausages was observed, and the moisture content and water activity were significantly decreased during fermentation. Sausages inoculated with a mixture of bacterial strains had the lowest pH, moisture content and water activity (P < 0.05). Inoculation of dry sausages with S. xylosus or L. plantarum, especially a mixture of strains (L. plantarum + S. xylosus), significantly delayed lipid oxidation, improved sensory characteristics, and inhibited BA accumulation. Six types of BAs (cadaverine, putrescine, tryptamine, 2-phenylethylamine, histamine, and tyramine) were inhibited by the presence of L. plantarum and S. xylosus, and a mixture of them had the most inhibitive effect. Correlation analysis showed that the BA concentrations correlated well with enterobacteriaceae counts, and some BAs were negatively correlated with LAB counts. These results demonstrate that S. xylosus and L. plantarum could be used as starter cultures in Harbin dry sausage production to inhibit BA accumulation and improve quality characteristics.     

A new fear in wine: Isolation of Staphylococcus epidermidis histamine producer

Biogenic amines (BAs) are undesirable compounds in all foods and beverages because they may induce food-borne intoxications when consumed at high concentrations. The aim of this study was to identify the organism responsible for the synthesis of histamine in a Tempranillo red wine from Ribera de Duero D.O. (Denomination of origin), Spain. Bacteria present in wines after malolactic fermentation were isolated, identified and typed. Four strains of the species Oenococcus oeni and two strains of Staphylococcus epidermidis were found. None of the O. oeni strains produced histamine, cadaverine or putrescine, but one of the S. epidermidis strains (Pa) was able to produce all three in synthetic medium and grape must, although not in wine. From the data obtained in this work, histamine present in Tempranillo wine B was produced by the S. epidermidis strain Pa. This is the first report in which the presence of S. epidermidis has been reported in wine and whose ability to produce histamine, putrescine and cadaverine has been demonstrated in grape must.     

Effect of starter cultures on microbial ecosystem and biogenic amines in fermented sausage

The microbial ecology of fermented sausages inoculated with different starter cultures (Pediococcus pentosaceus + Staphylococcus xylosus, batch A; Lactobacillus farciminis + Staphylococcus saprophyticus, batch B) was studied by culture-dependent and independent methods. The concentration of biogenic amines, pH and a_w (water activity) were followed. The starter B can inhibit indigenous bacteria effectively judged by traditional microbiological method and PCR-DGGE fingerprint. There was no statistical difference in pH between batches A and B at 2 days when substantial amounts of BAs were produced, while the amounts of histamine, putrescine, cadaverine and tyramine in batch B was lower than in batch A significantly (P < 0.001). As a result, the starter cultures which can complete, grow well and inhibit amine-positive bacteria play an important role in reducing the production of BAs during ripening of fermented sausage.     

Occurrence of biogenic amines in beers from Chilean market

The presence of tryptamine, phenylethylamine, putrescine, cadaverine, histamine, tyramine, spermidine and spermine was determined for the first time in beers commercialized in Chilean market. Dansylated amines were separated on a Zorbax-XDB C₁₈ column using a binary mobile phase composed of acetonitrile and 20 mmol L⁻¹ ammonium formate, pH 5.5. Chromatographic conditions were optimized using an experimental design, and validation was carried out following ICH recommendations. Calibration data fitted a linear regression model with R² > 0.996. Repeatability (n = 6) and intermediate precision (n = 3) in matrix showed RSD values lower than 5.00% and 5.21%, respectively. Recoveries at three different concentrations ranged from 75.50 to 96.48%. The proposed method was applied to determine the biogenic amines content in 101 beer samples produced by macro- (n = 65) and microbreweries (n = 36) using low (n = 63) and high fermentations (n = 38). Biogenic amines content in beer samples ranged from 0.53 to 85.04 mg L⁻¹, from which beers produced by microbreweries (19.13 ± 16.92 mg L⁻¹) showed a significant higher biogenic amines content (P = 0.0021) than beers produced by macrobreweries (9.65 ± 4.50 mg L⁻¹). Putrescine was the principal biogenic amine found in all kind of beers regardless origin, and the type of fermentation and brewery. Only 2 samples presented relevant levels of histamine and tyramine, but both below the limits reported by the European Food Safety Authority (EFSA). Thus, it can be concluded that beers commercialized in Chile are not a serious toxicological risk (e.g. hypertensive crisis) regarding the type and content of biogenic amines.