Phenotypic and technological diversity of lactic acid bacteria and staphylococci isolated from traditionally fermented sausages in southern Greece

The physicochemical and microbiological characteristics of spontaneously fermented sausages made by two medium-sized enterprises (MSE) located in southern Greece have been studied. A total of 300 lactic acid bacteria and 300 staphylococcal strains have been isolated and identified by their physiological characteristics. Lactobacillus plantarum strains were found to dominate the lactic acid bacteria microbiota in most of the cases with L. sakei strains prevailing in some of them and L. rhamnosus strains occasionally accompanying the dominant lactic acid bacteria microbiota. On the other hand, S. saprophyticus strains were found to dominate the staphylococcal microbiota in all spontaneously fermented sausages with of S. simulans, S. xylosus, S. gallinarum and S. cohnii cohnii strains being sporadically present. Following the identification, an evaluation of their technological properties, namely proteolytic and lipolytic capacities as well as production of biogenic amines and antimicrobial compounds, took place. None of the lactic acid bacteria and staphylococci was found to possess lipolytic activity whereas a total of 6 lactic acid bacteria and 51 staphylococci strains were found to be able to hydrolyse either the sarcoplasic, myofibrillar or both protein fractions. Furthermore, only one L. sakei strain and 185 staphylococci strains were found to possess decarboxylase activity against lysine, tyrosine, ornithine or histidine. Finally none of the staphylococcal microbiota and 3 lactic acid bacteria strains were found to be able to produce antimicrobial compounds of proteinaceous nature against Listeria monocytogenes.     

白鱼腐败菌产生物胺能力分析

为了探讨白鱼（Anabarilius）腐败菌产生物胺的能力,釆用聚合酶链式反应技术检测腐败菌的组氨酸、赖氨酸、鸟氨酸和酪氨酸的脱羧酶基因,应用反相高效液相色谱技术测定接种腐败菌培养基和无菌白鱼鱼块中生物胺含量,并对主要腐败菌的生物胺产量因子进行分析。结果表明：8 种腐败菌均含有至少1 种氨基酸脱羧酶基因;接种不同腐败菌的液体培养基和无菌白鱼鱼块均检测到多种生物胺,其中Morganella sp.wf-1和Morganella sp.wf-2具有很强的产腐胺、尸胺和组胺能力,使得接种这两种腐败菌的无菌白鱼鱼块中腐胺、尸胺和组胺含量、单胺、二胺、生物胺指数和总生物胺含量等指标在腐败过程中均显著高于对照组和接种其他腐败菌的无菌白鱼鱼块。同时,Morganella sp.wf-1和Morganella sp.wf-2的腐胺、尸胺、组胺、单胺、二胺、生物胺指数和总生物胺的产量因子均高于其他腐败菌。结果表明,Morganella sp.wf-1和Morganella sp.wf-2是白鱼腐败过程中产生物胺的主要腐败菌。     

Production of biogenic amines by lactic acid bacteria: screening by PCR, thin-layer chromatography, and high-performance liquid chromatography of strains isolated from wine and must

Biogenic amines are frequently found in wine and other fermented food. We investigated the ability of 133 strains of lactic acid bacteria isolated from musts and wines of different origins to produce histamine, tyramine, and putrescine. We detected the genes responsible for encoding the corresponding amino acid decarboxylases through PCR assays using two primer sets for every gene: histidine decarboxylase (hdc), tyrosine decarboxylase (tdc), and ornithine decarboxylase (odc); these primers were taken from the literature or designed by us. Only one strain of Lactobacillus hilgardii was shown to possess the hdc gene, whereas four strains of Lactobacillus brevis had the tdc gene. None of the Oenococcus oeni strains, the main agents of malolactic fermentation, was a biogenic amine producer. All PCR amplicon band-positive results were confirmed by thin-layer chromatography and high-performance liquid chromatography analyses.     

Improved screening procedure for biogenic amine production by lactic acid bacteria

An improved screening plate method for the detection of amino acid decarboxylase-positive microorganisms (especially lactic acid bacteria) was developed. The suitability and detection level of the designed medium were quantitatively evaluated by confirmation of amine-forming capacity using an HPLC procedure. The potential to produce the biogenic amines (BA) tyramine, histamine, putrescine, and cadaverine, was investigated in a wide number of lactic acid bacteria (LAB) of different origin, including starter cultures, protective cultures, type strains and strains isolated from different food products. Also, several strains of Enterobacteriaceae were examined. Modifications to previously described methods included lowering glucose and sodium chloride concentrations, and increasing the buffer effect with calcium carbonate and potassium phosphate. In addition, pyridoxal-5-phosphate was included as a codecarboxylase factor for its enhancing effect on the amino acid decarboxylase activity. The screening plate method showed a good correlation with the chemical analysis and due to its simplicity it is presented as a suitable and sensitive method to investigate the capacity of biogenic amine production by LAB. Tyramine was the main amine formed by the LAB strains investigated. Enterococci, carnobacteria and some strains of lactobacilli, particularly of Lb. curvatus. Lb. brevis and Lb. buchneri, were the most intensive tyramine formers. Several strains of lactobacilli, Leuconostoc spp., Weissella spp. and pediococci did not show any potential to produce amines. Enterobacteriaceae were associated with cadaverine and putrescine formation. No significant histamine production could be detected for any of the strains tested.     

Amino acid-decarboxylase activity of bacteria isolated from fermented pork sausages.

The occurrence of amino acid-decarboxylase activity in 92 strains of lactic acid bacteria, coagulase-negative staphylococci, and Enterobacteriaceae isolated from Spanish fermented pork sausages was investigated. The presence of biogenic amines in a decarboxylase synthetic broth was determined by ion-pair high performance liquid chromatography with o-phtalaldehyde post-column derivatization. Among the 66 lactic acid bacteria strains tested, 21 lactobacilli (in particular, Lactobacillus curvatus) and all 16 enterococci were amine producers. Tyramine was the main amine produced by these bacteria, although they also produced phenylethylamine, tryptamine, and/or the diamines putrescine and cadaverine. None of the lactic acid bacteria produced histamine. Coagulase-negative staphylococci were found to be negative amine-producers. Aromatic monoamines, apart from histamine, were not formed by Enterobacteriaceae. This family was responsible for cadaverine and putrescine production. The results obtained for biogenic amine production by bacteria in a synthetic medium suggest that amino acid-decarboxylase activity is strain dependent rather than being related to specific species.     

葡萄酒中生物胺的产生与工艺控制

生物胺存在于多种发酵食品中，人体吸收过量的生物胺后会引起不良的生理反应。在葡萄酒苹果酸—乳酸发酵(MLF)过程中，有些乳酸菌能够对氨基波脱投产生生物胺。利用PCR与DNA探针技术能够快速检测葡萄酒中的组胺产生菌。工艺上采用接种法进行MLF，并在MLF完成后对乳酸菌进行有效清除、可以显著降低葡萄酒中生物胺的含量。     

Volatile and biogenic amines, microbiological counts, and bacterial amino acid decarboxylase activity throughout the salt-ripening process of anchovies (Engraulis encrasicholus)

Chemical and microbiological parameters were studied during the industrial production of salt-ripened anchovies (Engraulis encrasicholus). Gradual acidification and increases in the proteolysis index and in total volatile basic nitrogen were observed. At the end of the maturing process, the values reached pH 5.55 +/- 0.03, 21.33 +/- 5.82%, and 44.06 +/- 12.47 mg/ 100 g, respectively. In the three studied anchovy batches, the biogenic amines tyramine, histamine, putrescine, cadaverine, and agmatine increased during ripening. The highest values were found in the batch where initial microbial load was highest (batch 1), especially for enterobacteria and enterococci. Tyramine was the most abundant amine, reaching values from nondetectable to 90 mg/kg, whereas histamine did not surpass 20 mg/kg. Among the microorganisms isolated, Enterobacter cloacae, Aerococcus viridans, Kocuria varians, and Staphylococcus chromogenes were able to decarboxylate amino acids and produce biogenic amines in vitro. Most (70.59%) of the microorganisms identified were able to produce histamine, 23.53% were able to produce the diamines putrescine and cadaverine, and only 11.76% were able to produce tyramine, although this substance was the major biogenic amine found in anchovy samples.     

Evidence for proteolytic activity and biogenic amines production in Lactobacillus curvatus and L. homohiochii

After screening 372 strains of Lactobacillus spp. isolated from a Portuguese traditional dry fermented sausage, two Lactobacillus strains, a Lactobacillus homohiochii and a L. curvatus were selected, because they were positive for tyrosine and ornithine decarboxylase activities. Evidence for extracellular proteolytic activity was also demonstrated for the two Lactobacillus strains, with some strain variation in terms of specific activities towards different substrates. Proteolytic activity was shown to be maximal in the early exponential growth. This proteolytic activity was higher when cells were grown in a peptide-rich medium such as MRS, when compared to skim milk. A study using several protease inhibitors showed that this activity is associated with metalloproteases in the case of the L. curvatus strain, but for L. homohiochii besides metalloproteases, serine-type proteases are also involved. In proteinaceous substrates, like dry fermented sausages, the formation of the biogenic amines putrescine and tyramine cannot be excluded when ongoing proteolysis leads to their precursors, as it is the case in the presence of these proteolytic Lactobacillus strains. Their ability to produce biogenic amines may be used as an index of microbial quality of the fermented meat product.     

PCR detection of foodborne bacteria producing the biogenic amines histamine, tyramine, putrescine, and cadaverine

This study describes an easy PCR method for the detection of foodborne bacteria that potentially produce histamine, tyramine, putrescine, and cadaverine. Synthetic oligonucleotide pairs for the specific detection of the gene coding for each group of bacterial histidine, tyrosine, ornithine, or lysine decarboxylases were designed. Under the conditions used in this study, the assay yielded fragments of 372 and 531 bp from histidine decarboxylase-encoding genes, a 825-bp fragment from tyrosine decarboxylases, fragments of 624 and 1,440 bp from ornithine decarboxylases, and 1,098- and 1,185-bp fragments from lysine decarboxylases. This is the first PCR method for detection of cadaverine-producing bacteria. The method was successfully applied to several biogenic amine-producing bacterial strains.     

葡萄酒中生物胺含量的影响因素研究

研究了葡萄酒生产过程中影响生物胺产生量的主要因素,结果表明,葡萄酒酒精发酵和苹果酸．乳酸发酵过程均有牛物胺的产生,影响葡萄酒中生物胺产生的主要因素是乳酸菌中氨基酸脱羧酶的活性,本研究添加了氨基酸脱羧酶辅酶的样品与对照相比,生物胺含量提高了90．6％。酵母菌和乳酸菌种类、酒精发酵温度,生物胺前体物质也是影响生物胺含量的主要因素,发酵温度越高、生物胺产生量越;在一定范围内,氨基酸的浓度越大产生物胺越多。但酵母菌和乳酸菌的添加量对生物胺浓度的影响较小。     

Production of Biogenic Amines by Lactic Acid Bacteria Isolated from Uzicka Sausages

The aim of this study was to monitor production of seven biogenic amines (Cadaverine – CAD, Putrescine – PUT, Spermine – SPE, Spermidine – SPD, Histamine – HIS, Tyramine – TYR and Tryptamine – TRY) in selected 24 lactic acid bacteria (LAB) strains. The decarboxylase activity of the microorganisms was studied in growth medium after 24 h cultivation. The ability of 24 LAB isolates cultivated in MRS broth and M17 broth supplement with 0.5% glucose to produce biogenic amines was assessed using liquid chromatography tandem mass spectrometry (LC-MS/MS). The investigation showed that LAB isolated from Uzicka sausage are not significant producers of biogenic amines in vitro.     

酒酒球菌中生物胺相关基因的检测

酒酒球菌在葡萄酒苹果酸-乳酸发酵过程中可通过脱羧基作用将氨基酸转化为生物胺.该研究采用PCR技术对27株酒酒球菌中与生物胺产生相关的基因-组氨酸脱羧酶基因、鸟氨酸脱羧酶基因、酪氨酸脱羧酶基因进行了检测,研究了这些菌株产生生物胺的特性.结果显示,所有菌株均不含有组氨酸脱羧酶基因、鸟氨酸脱羧酶基因、酪氨酸脱羧酶基因,不具有产生组胺、腐胺和酪胺的能力,因而具有较高的生物胺代谢安全性.     

Screening of biogenic amine production by lactic acid bacteria isolated from grape must and wine

The potential to produce the biogenic amines tyramine, histamine and putrescine, was investigated for lactic acid bacteria (LAB) of various origin, including commercial malolactic starter cultures, type strains and 78 strains isolated from Spanish grape must and wine. The presence of biogenic amines in a decarboxylase synthetic broth was determined by reverse-phase high performance liquid chromatography (RP-HPLC). Tyramine was the main amine formed by the LAB strains investigated. Leuconostoc strains were the most intensive tyramine formers. No potential to form biogenic amines was observed in Oenococcus oeni strains. Two strains of Latobacillus buchneri were associated with putrescine formation. None of the lactic acid bacteria produced histamine. According to these in vitro results, the commercial starter bacteria analyzed did not produce histamine, tyramine and putrescine.     

Formation of biogenic amines by proteolytic enterococci during cheese ripening

Six strains of Enterococcus faecalis and three strains of E faecium were investigated for their proteolytic activity and ability to decarboxylate the amino acids lysine, histidine, tyrosine, ornithine and phenylalanine. All strains tested displayed cell wall‐associated proteolytic activities, and three E faecalis strains displayed additionally extracellular proteolysis. None of the strains tested was able to form putrescine, histamine or cadaverine. Eight strains decarboxylated tyrosine, and two strains of E faecalis and one strain of E faecium formed phenylethylamine. Gouda cheese was produced from milk supplemented with two strains of E faecalis (10⁷ cfu ml⁻¹). Both strains were proteolytic and potent tyramine formers. Final viable counts of 10⁹ cfu g⁻¹ and tyramine concentrations of 477 mg kg⁻¹ were detected after a 12 week ripening period. © 1999 Society of Chemical Industry     

黄酒前酵中生物胺生成规律的研究

本研究对黄酒前酵工序的生物胺生成规律及影响因素进行了分析探讨。采用反相高效液相色谱技术,改进了生物胺定量检测法,该检测体系准确可靠,样品峰型对称分离度好且缩短了检测时间。分析了前酵工序中主要微生物,氨基酸,发酵醪酸度、糖度、酒精度、p H对生物胺生成的影响。发现前酵工序对成品酒生物胺的影响度为77.67%,第一次开耙阶段生物胺总量增幅最大,达到7.63 mg/L。研究发现主要酿造微生物中乳酸菌总数与生物胺总量成正相关,乳酸菌在搭窝期间生长速率最大,为7.13×106CFU/(m L·h)。在前酵工序中生物胺总量与发酵醪酸度、酒精度、p H呈显著正相关,与糖度呈负相关;前酵过程中主要生物胺与前体氨基酸也呈明显正相关。本研究分析了黄酒前酵中生物胺生成规律,有助于建立降低黄酒生物胺含量的更安全、科学的工艺。     

Tyramine production of technological important strains of <Emphasis Type="Italic">Lactobacillus</Emphasis>, <Emphasis Type="Italic">Lactococcus</Emphasis> and <Emphasis Type="Italic">Streptococcus</Emphasis>

The aim of this paper was to study the biogenic amines (histamine, tyramine, putrescine, cadaverine, agmatine, spermine and spermidine) production of selected technological important lactic acid bacteria (strains of the genera Lactococcus, Lactobacillus and Streptococcus). Three methods (ion-exchange chromatography (IEC), PCR and cultivation method with pH indicator) were used. Within the 39 strains of lactic acid bacteria tested, the production of tyramine (formed by tyrosine decarboxylase) was detected in eight strains (3 strains of Lactococcus lactis subsp. lactis, three strains of Lactococcus lactis subsp. cremoris, 1 strain of Streptococcus thermophilus and 1 strain of Lactobacillus delbrueckii subsp. bulgaricus). The other tested biogenic amines were not detected. Cultivation in decarboxylation broth seems to be the least accurate method for the detection of biogenic amines due to enhanced risk of false-positive reactions. Therefore, in order to detect bacteria producing biogenic amines, the combination of PCR and chromatographic methods (e.g. IEC) can be recommended.     

高效降解生物胺乳酸菌的筛选、鉴定及特性研究

为获得高效降解生物胺的乳酸菌，以鱼露为试验原料，从鱼露中分离纯化并筛选具有高效降解4种常见生物胺活性的菌株，通过形态观察、碳源利用和分子生物学鉴定，该菌株，并研究其生长和生物胺降解特性。结果表明，共筛选得到8株生物胺降解菌株，其中最佳生物胺降解菌为FSCBAD033，该菌株被鉴定为发酵柠檬乳杆菌（Limosilactobacillus fermentum）。该菌株可以降解86.4%腐胺、78.5%尸胺、72.3%组胺和100%酪胺，且在含有前体氨基酸的培养基中不积累该四种生物胺。菌株FSCBAD033的最适生长温度、初始pH值和NaCl含量分别为40 ℃、6和3%，其在温度30～40 ℃、初始pH值5～7、NaCl含量不超过6%的范围内降解生物胺能力较高（四种生物胺降解率均＞50%）。     

生物胺降解乳酸菌的筛选与特性研究

为筛选出高效降解生物胺的优良乳酸菌,该研究以实验室前期筛选的41株乳酸菌作为研究对象,运用显色培养、高效液相色谱（HPLC）法对菌株生物胺产生和降解能力进行筛选,并对筛选出的菌株进行耐盐及耐酸实验。结果表明,筛选得到5株高效降解生物胺菌株,其中以植物乳杆菌（Lactobacillus plantarum）30的生物胺降解能力最好,其对尸胺、组胺、酪胺的降解率分别为62.42%、74.32%、89.97%。植物乳杆菌30能在含盐量0～9%和pH为4.5～8.5环境中较好的生长繁殖,该菌株无生物胺生成活性,并同时具备生物胺降解能力和耐盐耐酸能力,可作为蛋白质类发酵食品的发酵剂运用于食品中降低生物胺毒性。     

Identification and characterization of Enterococcus spp. in Greek spontaneous sausage fermentation

A total of 108 enterococcal strains previously isolated from spontaneously fermented sausages were identified using phenotypic traits, pulsed-field gel electrophoresis (PFGE), and sequencing of the 16S rRNA gene. The proteolytic and lipolytic activities of these isolates and their ability to decarboxylate lysine, tyrosine, ornithine, and histidine and to produce antimicrobial compounds also were assessed. All strains were identified as Enterococcus faecium, and a lack of correlation between data derived from phenotypic and those derived from genotypic techniques was evident. Wide strain diversity was revealed by both phenotypic properties and PFGE strain typing results. Few strains were present in all batches, suggesting a possible persistence in the respective production plants. Neither proteolytic nor lipolytic activities were detected, and none of the strains decarboxylated lysine, tyrosine, ornithine, or histidine. A total of 42 E. faecium strains inhibited in vitro growth of Listeria monocytogenes, which suggests possible contribution of these strains to the safety of the end product and possible utilization of these strains as protective cultures.