Production of histamine and tyramine by lactic acid bacteria isolated from vacuum-packed sugar-salted fish

The incidence of histamine- or tyramine-producing lactic acid bacteria was examined in several products of vacuum-packed sugar-salted fish (salmon, halibut, mackerel). No histamine-producing isolates were observed, whereas the majority of tyramine-producing isolates were identified as Carnobacterium spp. These organisms were shown to be important members of the microbial flora during storage of vacuum-packed sugar-salted salmon at 5°C. The amount of tyramine produced was reduced by lowering the temperature from 9°C to 4°C for all of five strains of carnobacteria or lactobacilli. The majority of tyramine was produced during the exponential growth phase for Carnobacterium piscicola N 5 and Lactobacillus viridescens N 69. The ability of these bacteria to produce tyramine may be used as an index of microbial quality/acceptability of stored vacuum-packed sugar-salted fish.     

Isolation and characterization of biogenic amine-producing bacteria in fermented soybean pastes

Biogenic amines (BAs) are produced primarily by microorganisms found in fermented foods and are often implicated in food poisoning. BA-producing bacteria found in fermented soybean pastes were isolated and characterized using a decarboxylating medium and multiplex PCR analysis. Two BA-producing bacteria were isolated from traditional soybean pastes: one was a histamine-producing Clostridium strain, and the other was a tyramine-producing Pseudomonas strain. The Clostridium strain was determined to be a potent histamine producer among the cultures tested. Synthesis of tyramine by Pseudomonas sp. T1 was observed for the first time in this study.     

Isolation, properties and behaviour of tyramine-producing lactic acid bacteria from wine

Wines containing high levels of biogenic amines were investigated for the presence of tyramine-producing strains. Two different Lactobacillus brevis (IOEB 9809 and IOEB 9901) able to produce the amine were isolated. None of the isolated strains identified as Oenococcus oeni formed tyramine. In addition, other Lact. brevis and Lact. hilgardii strains from our collection (IOEB) and the American Type Culture Collection (ATCC) were strong tyramine producers. Lactobacillus brevis IOEB 9809 and Lact. hilgardii IOEB 9649 were found to produce tyramine and phenylethylamine simultaneously. The conditions that can influence tyramine formation in wine were evaluated for three strains of Lact. brevis (IOEB 9809 and IOEB 9901) and Lact. hilgardii (IOEB 9649). Tyrosine was the major factor affecting tyramine formation and was enhanced by the presence of sugars, mainly glucose. Tyrosine decarboxylase (TDC) activity greatly depended on the presence of the precursor, which suggested that tyrosine induced the TDC system. These results indicate that Lactobacillus could be the lactic acid bacteria responsible for tyramine production in wine.     

Factors affecting tyramine production in <Emphasis Type="Italic">Enterococcus durans</Emphasis> IPLA 655

The decarboxylation of tyrosine by certain lactic acid bacteria leads to the undesirable presence of tyramine in fermented foods. Tyramine is the most frequent biogenic amine found in cheese and is also commonly found in other fermented foods and beverages. The tyramine-producing strain Enterococcus durans IPLA 655 was grown in a bioreactor under different conditions to determine the influence of carbon source, tyrosine and tyramine concentrations, and pH on tyramine production. The carbon source appeared to have no significant effect on the production of tyramine. In contrast, tyrosine was necessary for tyramine production, while the presence of tyramine itself in the growth medium inhibited such production. pH showed by far the greatest influence on tyramine synthesis; tyramine was produced in the greatest quantities at pH 5.0, although this was accompanied by a reduced growth rate.     

Tyramine degradation and tyramine/histamine production by lactic acid bacteria and Kocuria strains

Of 53 strains of lactic acid bacteria and Kocuria, screened for production or degradation of biogenic amines, 29 Kocuria varians and four strains of Enterococcus faecalisproduced tyramine and, at lower concentrations, histamine. In contrast, Lactobacillus strains that did not possess amino acid decarboxylase activity degraded tyramine. The greatest tyramine oxidase activity was present in the strains L. casei CRL705 (98% degradation) and CRL678 (93%) as well as in L. plantarum CRL681 (69%) and CRL682 (60%).     

Biogenic amine production by lactic acid bacteria isolated from cider

AIMS: To study the occurrence of histidine, tyrosine and ornithine decarboxylase activity in lactic acid bacteria (LAB) isolated from natural ciders and to examine their potential to produce detrimental levels of biogenic amines. METHODS AND RESULTS: The presence of biogenic amines in a decarboxylase synthetic broth and in cider was determined by reversed-phase high-performance liquid chromatography (RP-HPLC). Among the 54 LAB strains tested, six (five lactobacilli and one oenococci) were biogenic amine producers in both media. Histamine and tyramine were the amines formed by the LAB strains investigated. Lactobacillus diolivorans were the most intensive histamine producers. This species together with Lactobacillus collinoides and Oenococcus oeni also seemed to produce tyramine. No ability to form histamine, tyramine or putrescine by Pediococus parvulus was observed, although it is a known biogenic amine producer in wines and beers. CONCLUSIONS: This study demonstrated that LAB microbiota growing in ciders had the ability to produce biogenic amines, particularly histamine and tyramine, and suggests that this capability might be strain-dependent rather than being related to a particular bacterial species. SIGNIFICANCE AND IMPACT OF THE STUDY: Production of biogenic amines by food micro-organisms has continued to be the focus of intensive study because of their potential toxicity. The main goal was to identify the microbial species capable of producing these compounds in order to control their presence and metabolic activity in foods.     

The capacity of Enterobacteriaceae species to produce biogenic amines in cheese

The amino acid decarboxylating activity and production of biogenic amines by 104 cheese-associated Enterobacteriaceae species (58 Enterobacter, 18 Serratia, eight Escherichia, seven Hafnia, six Arizona, four Citrobacter and three Klebsiella) were investigated. All strains could decarboxylate at least two amino acids in M?ller's broth and in Niven's medium, and the decarboxylase activity was strain specific. In a laboratory medium containing all free amino acids, all strains could produce more than 100 ppm cadaverine, putrescine was produced by 96% of strains. Tyramine and histamine were produced in the lowest concentrations. A positive correlation existed between cadaverine concentration and Enterobacteriaceae counts in cheese, that may have caused the increase in decarboxylase content. This study suggests that it is possible to limit the presence of cadaverine in cheese, thereby controlling the Enterobacteriaceae counts, a sign of contamination during cheese making and/or storage.     

青藏高原垂穗披碱草青贮饲料中乳酸菌的多样性及低温发酵菌株的筛选

【目的】探究青藏高原垂穗披碱草青贮饲料中乳酸菌的多样性,筛选在低温条件下(10、15和25°C)生长性能较好的优良菌株。【方法】将垂穗披碱草青贮饲料中分离纯化的乳酸菌进行形态特征观察及16S r RNA基因测序鉴定;用MRS液体培养基在10、15和25°C条件下分离、初筛乳酸菌,选取高吸光度值的菌株作为优势菌株。用绿汁发酵液在10、15和25°C条件下培养测定其p H值,选取低p H值菌株作为优势菌株,并综合MRS培养基筛选结果确定优良菌株。【结果】从不同温度和发酵阶段的垂穗披碱草青贮饲料中共分离得到108个乳酸菌菌株,它们分属于6个属、18个种。其中,清酒乳杆菌LS-24在15°C条件下发酵液p H值显著降低(P0.05),戊糖片球菌PP-63在发酵初期生长速度较快,植物乳杆菌LP-21在15°C条件下发酵液p H值降至3.9且有最大活菌数。【结论】在青藏高原垂穗披碱草青贮饲料中发现的乳酸菌属基本涵盖了前人在常温青贮饲料中发现的所有属,但种数略少;在108株菌中,清酒乳杆菌LS-24、戊糖片球菌PP-63和植物乳杆菌LP-21在低温条件下均表现出较好的繁殖和发酵特性,可作为青贮饲料低温发酵的备选菌株。     

Identification of a novel enzymatic activity from lactic acid bacteria able to degrade biogenic amines in wine

The main objectives of this study were the search for enzymatic activities responsible for biogenic amine (BA) degradation in lactic acid bacteria (LAB) strains isolated from wine, their identification, and the evaluation of their applicability for reducing BAs in wine. Fifty-three percent of the 76 LAB cell extracts showed activity against a mixture of histamine, tyramine, and putrescine when analyzed in-gel. The quantification of the degrading ability for each individual amine was tested in a synthetic medium and wine. Most of the bacteria analyzed were able to degrade the three amines in both conditions. The highest percentages of degradation in wine were those of putrescine: up to 41 % diminution in 1 week. Enzymes responsible for amine degradation were isolated and purified from Lactobacillus plantarum J16 and Pediococcus acidilactici CECT 5930 strains and were identified as multicopper oxidases. This is the first report of an efficient BA reduction in wine by LAB. Furthermore, the identity of the enzymes involved has been revealed.     

产生物胺乳酸菌的筛查与检测

目的对上海市场上食品和药品中分离出的20株乳杆菌,13株链球菌,3株乳球菌和3株肠球菌的产生物胺能力进行检测,以揭示其潜在的安全性问题。方法检测的生物胺共包括6种：分别为酪胺、精胺、尸胺、组胺、腐胺和色胺。利用添加了前体氨基酸的氨基酸脱羧酶筛选培养基对各菌株的产胺能力进行初筛,通过培养基中指示剂的颜色变化判定产胺能力。结果在检测的39株菌中,8株菌具有产酪胺的能力,7株菌具有产精胺的能力,1株菌具有产组胺的能力,1株菌具有产腐胺的能力。尤其是精胺和酪胺的产量较为引人关注。结论生物胺的危害水平取决于个体解毒的能力,但在筛选食品药品用菌株时应运用规范的方法来检测其产生物胺的能力,以保障相应食品药品的安全问题。     

Improved multiplex-PCR method for the simultaneous detection of food bacteria producing biogenic amines

This study describes a simple and rapid multiplex-PCR method to determine the ability to produce histamine, tyramine and putrescine by bacteria. The assay is an improved method based on an assay designed for lactic acid bacteria. This improved method includes a pair of primers based on sequences from histidine decarboxylases from Gram-negative bacteria. Under the optimised conditions, the assay yielded a 367-bp DNA fragment from histidine decarboxylases of Gram-positive bacteria, 534-bp fragment from histidine decarboxylases of Gram-negative bacteria, 924-bp from bacterial tyrosine decarboxylases, and 1446-bp fragment from bacterial ornithine decarboxylases. The method was successfully applied to several biogenic amine-producing bacterial strains, even when DNAs of several target organisms were included in the same reaction. This simple method could be easily incorporated in food control laboratories to detect potentially biogenic amine-producing bacteria in foods.     

富锌产γ-氨基丁酸乳酸菌的筛选及初步鉴定

用含0.2%-0.25%Zn^2＋的MRS培养基从发酵酸菜、酸奶中分离出6株具富锌能力细菌,经形态和生理生化特性鉴定,初步判断菌株为乳酸菌。采用纸层析检测MRSG培养基培养的6株菌上清液后,初步确定有4株能产γ-氨基丁酸。以此4菌株基因组为模板PCR扩增到540 bp的片段,经测序和序列分析,证明是谷氨酸脱羧酶基因高度保守区域,初步证明谷氨酸脱羧酶基因的存在。此4株菌发酵液经HPLC检测和分析后,证实2号菌株能转化谷氨酸钠生成GABA,产量为4.8 g/L,在应用方面具有很大的潜力。     

Distribution of membrane-bound monoamine oxidase in bacteria.

The distribution of membrane-bound monoamine oxidase in 30 strains of various bacteria was studied. Monoamine oxidase was determined by using an ammonia-selective electrode; analyses were sensitive and easy to perform. The enzyme was found in some strains of the family Enterobacteriaceae, such as Klebsiella, Enterobacter, Escherichia, Salmonella, Serratia, and Proteus. Among strains of other families of bacteria tested, only Pseudomonas aeruginosa IFO 3901, Micrococcus luteus IFO 12708, and Brevibacterium ammoniagenes IAM 1641 had monoamine oxidase activity. In all of these bacteria except B. ammoniagenes, monoamine oxidase was induced by tyramine and was highly specific for tyramine, octopamine, dopamine, and norepinephrine. The enzyme in two strains oxidized histamine or benzylamine. Correlations between the distributions of membrane-bound monoamine oxidase and arylsulfatase synthesized in the presence of tyramine were discussed.     

Distribution of membrane-bound monoamine oxidase in bacteria.

The distribution of membrane-bound monoamine oxidase in 30 strains of various bacteria was studied. Monoamine oxidase was determined by using an ammonia-selective electrode; analyses were sensitive and easy to perform. The enzyme was found in some strains of the family Enterobacteriaceae, such as Klebsiella, Enterobacter, Escherichia, Salmonella, Serratia, and Proteus. Among strains of other families of bacteria tested, only Pseudomonas aeruginosa IFO 3901, Micrococcus luteus IFO 12708, and Brevibacterium ammoniagenes IAM 1641 had monoamine oxidase activity. In all of these bacteria except B. ammoniagenes, monoamine oxidase was induced by tyramine and was highly specific for tyramine, octopamine, dopamine, and norepinephrine. The enzyme in two strains oxidized histamine or benzylamine. Correlations between the distributions of membrane-bound monoamine oxidase and arylsulfatase synthesized in the presence of tyramine were discussed.     

The tyrosine decarboxylation test does not differentiate Enterococcus faecalis from Enterococcus faecium

According to the current edition of the Bergey's Manual of Systematic Bacteriology [11] the tyrosine decarboxylation test allows the differentiation of enterococci. Tyrosine is decarboxylated to the biogenic amine tyramine by E. faecalis and not by E. faecium strains. In the present study we sequenced the16S rDNA of two tyramine-producing strains, BIFI-56 and BIFI-58, presumptively classified as E. faecalis. Their 16S rDNA were identical to the same fragment from the E. faecium type strain. Several E. faecium strains were then checked for their ability to decarboxylate tyrosine and also a putative tyrosine decarboxylase-coding gene was PCR amplified from these strains. All the strains confirmed as E. faecium produced tyramine and possessed a DNA fragment coding for a putative tyrosine decarboxylase. The concordance of the two methods allows us to conclude that the tyrosine decarboxylase test cannot be used in the differentiation of E. faecalis from E. faecium since at least some E. faecium strains are tyramine producers.     

Lactic acid bacteria from chicken carcasses with inhibitory activity against Salmonella spp. and Listeria monocytogenes

This study was conducted to isolate psychrotrophic lactic acid bacteria (LAB) from chicken carcasses with inhibitory activity against strains of Salmonella spp. and Listeria monocytogenes. A total of 100 broiler samples were examined for the presence of LAB. Ninety-two LAB isolates that showed antimicrobial effects against Salmonella spp. and L. monocytogenes were further analysed to examine their LAB (Gram-positive, catalase negative, oxidase negative) and psychrotrophic characteristics (ability to grow at 7 °C). Fifty isolates were further selected and identified initially using standard biochemical tests in miniature (Micro-kits API CH 50) and then by sequencing of the 16s-23s rRNA gene boundary region (Intergenic Spacer Region). By molecular identification, these isolates were classified into 5 different LAB species: Lactobacillus salivarius, Lactobacillus reuteri, Lactobacillus johnsonii, Pediococcus acidilactici, and Lactobacillus paralimentarius. None of the isolates produced tyramine or histamine.     

Inhibition of food-borne bacterial pathogens by bacteriocins from lactic acid bacteria isolated from meat.

Ten strains of bacteriocin-producing lactic acid bacteria were isolated from retail cuts of meat. These 10 strains along with 11 other bacteriocin-producing lactic acid bacteria were tested for inhibitory activity against psychotrophic pathogens, including four strains of Listeria monocytogenes, two strains of Aeromonas hydrophila, and two strains of Staphylococcus aureus. Inhibition due to acid, hydrogen peroxide, and lytic bacteriophage were excluded. The proteinaceous nature of the inhibitory substance was confirmed by demonstration of its sensitivity to proteolytic enzymes. Eight of the meat isolates had inhibitory activity against all four L. monocytogenes strains. Bacteriocin activity against L. monocytogenes was found in all of the strains obtained from other sources. Activity against A. hydrophila and S. aureus was also common.     

Inhibition of food-borne bacterial pathogens by bacteriocins from lactic acid bacteria isolated from meat

Ten strains of bacteriocin-producing lactic acid bacteria were isolated from retail cuts of meat. These 10 strains along with 11 other bacteriocin-producing lactic acid bacteria were tested for inhibitory activity against psychotrophic pathogens, including four strains of Listeria monocytogenes, two strains of Aeromonas hydrophila, and two strains of Staphylococcus aureus. Inhibition due to acid, hydrogen peroxide, and lytic bacteriophage were excluded. The proteinaceous nature of the inhibitory substance was confirmed by demonstration of its sensitivity to proteolytic enzymes. Eight of the meat isolates had inhibitory activity against all four L. monocytogenes strains. Bacteriocin activity against L. monocytogenes was found in all of the strains obtained from other sources. Activity against A. hydrophila and S. aureus was also common.     

Safety evaluation <Emphasis Type="Italic">in vitro</Emphasis> of <Emphasis Type="Italic">Enterococcus durans</Emphasis> from Tibetan traditional fermented yak milk

Despite its ubiquity in fermented dairy products, the safety of lactic acid enterococcal bacteria remains controversial. In this study, five Enterococcus durans strains — A1, A2, B1, B2, and C1 — were isolated from traditional fermented yak milk from Tibet. To evaluate the strains’ safety, biogenic amine production, antibiotic resistance and presence of known virulence determinants were investigated. Strain A1 can produce biogenic amines for histamine, spermine, and spermidine (mean values: 8.64, 8.31, and 0.30 mg/L, respectively). Polymerase chain reaction amplification for Strain A1 found genes involved in expression of gelatinase (gleE), cytolysin (cylA, cylB, and cylM), sex pheromones (ccf and cpd) and cell wall adhesion (efaA). Strain A2 showed sensitivity or intermediate resistance to all tested antibiotics, and no virulence determinants except gelE and ccf, but did produce tyramine at a relatively high level (912.02 mg/L). Both strains B1 and B2 could produce histamine (10.43 and 10.56 mg/L, respectively), and showed vancomycin resistance; B1 also produced tyramine (504.02 mg/L). Strain C1 could produce all five biogenic amines tested in the study -putrescine, histamine, tyramine, spermine, and spermidine; concentrations were 6.51, 9.59, 205.85, 5.55, and 5.39 mg/L, respectively. All E. durans strains found in Tibetan traditional fermented yak milk thus offer potential risk.     

Evaluation of the fermentability of oat fractions obtained by debranning using lactic acid bacteria

Aims: The overall kinetics of the fermentation of four oat fractions obtained by debranning using three potentially probiotic lactic acid bacteria were investigated. The main objective was to study the suitability of these fractions as fermentation media for the growth and the metabolic production of bacteria isolated from human intestine. Methods and Results: The cell growth, lactic acid production and substrate uptakes of the three lactobacilli was monitored for 30 h. An unstructured mathematical model was used to describe and fit the experimental data. In the medium from fraction B (1–3% pearlings or β-glucan-rich fraction) all strains reached the highest cell populations, maximum growth rates and maximum lactic acid productions. This could be because of the high levels of total fibre and β-glucan of this fraction. Limited growth and lactic acid formation was found in medium A (0–1% pearlings or bran-rich fraction). Conclusions: Medium B (1–3% pearling fraction) is the most suitable for fermentation and produces considerably higher probiotic cell concentrations. Significance and Impact of the Study: Debranning technology could be used to separate fractions from cereal grains for the production of functional formulations with higher probiotic levels than the ones that were obtained with the whole grain.