Occurrence of biogenic amine-forming lactic acid bacteria in wine and cider

A collection of 810 lactic acid bacteria (LAB) strains isolated from wine and cider was screened for potential biogenic amine (BA) producers by combining molecular and phenotypic approaches. A newly developed multiplex PCR method allowed for the simultaneous detection of four genes involved in the production of histamine (histidine decarboxylase, hdc), tyramine (tyrosine decarboxylase, tyrdc) and putrescine (via either ornithine decarboxylase, odc, or agmatine deiminase, agdi) while TLC and HPLC analysis allowed for BA-production determination. One hundred and fifty-eight LAB strains were monitored by the molecular/phenotypic double approach and revealed a good correlation between genotypic and phenotypic data. Eighteen per cent of the tested strains were positive for at least one BA target gene with up to three detected simultaneously, in particular amongst Lactobacillus brevis and Lactobacillus hilgardii isolates for the tyrdc and agdi genes. The most frequent gene corresponded to the agdi gene detected in 112 strains (14% of all LAB strains) of 10 different LAB species. The tyrdc gene was detected in 67 strains represented by 7 different LAB species (8% overall), especially those isolated from wine. Lower levels of hdc⁺ (2% of strains) and especially odc⁺ (0.5% of strains) strains were observed. Interestingly, species that have never been described to carry BA-producing pathway genes were identified in this study. Furthermore, only one cadaverine-producer was detected and corresponded to Lactobacillus 30a, a collection strain not found in fermented beverages, although cadaverine is commonly detected in wines.     

Biodiversity of lactic acid bacteria in French wheat sourdough as determined by molecular characterization using species-specific PCR

The lactic acid microflora of nine traditional wheat sourdoughs from the Midi-Pyrénées area (South western France) was previously isolated and preliminary characterized using conventional morphological and biochemical analysis. However, such phenotypic methods alone are not always reliable and have a low taxonomic resolution for identification of lactic acid bacteria species. In the present study, a total of 290 LAB isolates were identified by PCR amplification using different sets of specific primers in order to provide a thorough characterization of the lactic flora from these traditional French sourdoughs. Overall, the LAB isolates belonged to 6 genera: Lactobacillus (39%, 8 species), Pediococcus (38%, 1 species), Leuconostoc (17%, 2 species), Weissella (4%, 2 species), Lactococcus (1%, 1 species) and Enterococcus (< 1%, 1 species) and 15 different species were detected: L. plantarum, L. curvatus, L. paracasei, L. sanfranciscensis, L. pentosus, L. paraplantarum, L. sakei, L. brevis, P. pentosaceus, L. mesenteroides, L. citreum, W. cibaria, W. confusa, L. lactis and E. hirae. Facultative heterofermentative LAB represent more than 76% of the total isolates, the main species isolated herein correspond to L. plantarum and P. pentosaceus. Obligate heterofermentative lactobacilli (L. sanfranciscencis, L. brevis) represent less than 3% of the total isolates whereas Leuconostoc and Weissella species represent 21% of the total isolates and have been detected in eight of the nine samples. Detection of some LAB species was preferentially observed depending on the isolation culture medium. The number of different species within a sourdough varies from 3 to 7 and original associations of hetero- and homofermentative LAB species have been revealed. Results from this study clearly confirm the diversity encountered in the microbial community of traditional sourdough and highlight the importance of LAB cocci in the sourdough ecosystem, along with lactobacilli.     

The inhibitory effects of wine phenolics on lysozyme activity against lactic acid bacteria

The lysozyme of hen's egg white is used in winemaking to control spontaneous lactic acid bacteria (LAB). A total of eight LAB strains, isolated from grape must and wine, were used to assess the inhibitory effects of wine phenolics on lysozyme activity. The presence of phenolics, extracted from grape pomace, in growth medium reduced the mortality rate due to the lysozyme activity. This effect was especially clear in the case of strains belonging to Lactobacillus uvarum, Pediococcus parvulus and Oenococccus oeni, which are more sensitive to lysozyme than L. plantarum and L. hilgardii strains. Cell lysis assays carried out on four strains sensitive to lysozyme and Micrococcus lysodeikticus ATCC 4698, used as a reference strain, confirmed the inhibition of grape pomace phenolics on the muramidase. There was no interference from non-flavonoids, flavanols and flavonol compounds, when they were tested individually, on the lysozyme activity against the strains. Anthocyanins extracted from grape skins slightly inhibited the activity only against M. lysodeikticus. However, proanthocyanidins extracted from seed berries, strongly inhibited the lysozyme. In this extract, dimers were the predominant oligomers of flavan-3-ol. The study demonstrated that the effectiveness of lysozyme against LAB in red winemaking is related to the amount of low molecular weight proanthocyanidins that are released when the grapes are macerating.     

聚合酶链反应检测乳酸菌酪氨酸脱羧酶基因

酪氨酸脱羧酶与乳酸菌发酵食品中酪胺的产生密切相关。根据从GenBank中检索到的酪氨酸脱羧酶基因序列设计一对特异性引物，采用PCR技术对乳酸菌的基因组DNA片段进行扩增，以此建立以酪氨酸脱羧酶基因为靶的产酪胺乳酸菌的分子生物学检测方法。结果表明，供试12株乳酸菌中有9株菌扩增出1133bp DNA片段对其中3株菌的扩增产物进行DNA序列测定，测序结果采用国际互联网上NCBI的BLAST工具进行同源性检索分析，发现它与已知的Enteroeoeeus faecalis，Enterococcus faecium，Canmbacterium divergens的酪氨酸脱羧酶基因序列均高度同源，其中PLP(5’-磷酸吡哆醛)的结合位点高度保守，证明该扩增产物是酪氨酸脱羧酶的基因片段。应用该法与常规平板检测法比较显示，二者检测结果基本一致，表明本研究建立的PCR方法可作为一种快速、高度特异的检测产酪胺乳酸菌菌株的新方法。     

Genetic screening of lactic acid bacteria of oenological origin for bacteriocin-encoding genes

A total of 330 lactic acid bacteria isolated from South African red wines during alcoholic and malolactic fermentations and 9 commercial malolactic bacteria starter cultures were screened for antimicrobial activity. Of the entire screened isolates, 26 strains, belonging to the species Lactobacillus plantarum, Lactobacillus paracasei, Lactobacillus hilgardii and Oenococcus oeni, showed activity towards various wine-related and non-wine-related indicator strains. A PCR-based screening revealed the presence of the plantaricin encoding genes plnA, plnEF, plnJ and plnK in five selected Lb. plantarum strains. Furthermore, a co-culture experiment with Lb. plantarum and Enterococcus faecalis was performed. A complete inhibition of cell growth of Ent. faecalis was observed within 72 h. Four putative bacteriocin-encoding genes in the genome of O. oeni were identified and sequenced.     

Potential of wine-associated lactic acid bacteria to degrade biogenic amines

Some lactic acid bacteria (LAB) isolated from fermented foods have been proven to degrade biogenic amines through the production of amine oxidase enzymes. Since little is known about this in relation to wine micro-organisms, this work examined the ability of LAB strains (n = 85) isolated from wines and other related enological sources, as well as commercial malolactic starter cultures (n = 3) and type strains (n = 2), to degrade histamine, tyramine and putrescine. The biogenic amine-degrading ability of the strains was evaluated by RP-HPLC in culture media and wine malolactic fermentation laboratory experiments. Although at different extent, 25% of the LAB isolates were able to degrade histamine, 18% tyramine and 18% putrescine, whereas none of the commercial malolactic starter cultures or type strains were able to degrade any of the tested amines. The greatest biogenic amine-degrading ability was exhibited by 9 strains belonging to the Lactobacillus and Pediococcus groups, and most of them were able to simultaneously degrade at least two of the three studied biogenic amines. Further experiments with one of the strains that showed high biogenic amine-degrading ability (L. casei IFI-CA 52) revealed that cell-free extracts maintained this ability in comparison to the cell suspensions at pH 4.6, indicating that amine-degrading enzymes were effectively extracted from the cells and their action was optimal in the degradation of biogenic amines. In addition, it was confirmed that wine components such as ethanol (12%) and polyphenols (75 mg/L), and wine additives such as SO₂ (30 mg/L), reduced the histamine-degrading ability of L. casei IFI-CA 52 at pH 4.6 by 80%, 85% and 11%, respectively, in cell suspensions, whereas the reduction was 91%, 67% and 50%, respectively, in cell-free extracts. In spite of this adverse influence of the wine matrix, our results proved the potential of wine-associated LAB as a promising strategy to reduce biogenic amines in wine.     

定量PCR技术检测杀菌型产品中乳酸菌

目的利用定量PCR(quantitativePCR,qPCR)技术检测杀菌型产品中乳酸菌,并与标签标示相比较,查探产品中各类菌种的异同性及数量。方法以市售的发酵乳及含乳饮料作为研究对象,设计可区分产品中常见的6种乳酸菌特异性引物及探针,利用qPCR技术对方法的特异性、灵敏度进行验证,并建立标准扩增曲线;利用模拟样品对方法进行检验,最后对市场上购买的实际样品进行检测。结果除去干酪乳杆菌,其他5种引物都能特异性扩增出其目标菌,并且对其他的乳酸菌均无扩增现象;DNA检测的灵敏度可达到2～4pg;单一标准菌种扩增曲线线性较好,相关系数r2值在0.954～0.992之间;对模拟样品的检测,与平板法差异性比较,P值均大于0.05,无显著性差异。对市售样品进行检测,产品中标记的干酪乳杆菌、双歧杆菌和嗜热链球菌与检测结果存在不匹配现象。结论本研究建立的qPCR方法可快速、准确地对产品中德氏乳杆菌保加利亚亚种、嗜热链球菌、乳酸乳球菌、嗜酸乳杆菌、双歧杆菌的鉴定和定量的分析。     

Isolation and identification of cultivable lactic acid bacteria in traditional fermented milk of Tibet in China

One hundred and seventy‐one strains of lactic acid bacteria were isolated from 44 traditional fermented milk samples from different region of Tibet. Among these samples, the concentrations of lactic acid bacteria varied from 10 ⁵ to 10 ⁹ colony forming unit/mL. The ratio of rod bacteria (71.3%) was higher than that of cocci (28.7%), and was considered to be the major population. Lactobacillus fermentum (31%) and Lactobacillus casei (28%) were the most predominant species among all these isolates. This study systematically analysed the microbial composition of traditional fermented milk in China, which may become a valuable source for further starter selection.     

DGGE and real-time PCR analysis of lactic acid bacteria in bacterial communities of the phyllosphere of lettuce

Food associated indigenous microbial communities exert antagonistic effects on pathogens and may routinely deliver health relevant microorganisms to the GI tract. By using molecular, culture independent methods including PCR-DGGE of 16S rDNA-coding regions and real-time PCR (RT-PCR) as well as BIOLOG metabolic fingerprinting, microbial communities on lettuce were analyzed in samples from fields, from supermarkets and soil. Amplified 16S rRNA gene sequences (57.7%) could be assigned to species previously reported as typical for the phyllosphere including Pantoea agglomerans, Pseudomonas flavescens, Moraxella spp., and Mycobacterium spp. 71.8% of the sequences obtained represented so far undescribed taxa. Principal component analysis of BIOLOG metabolic profiles indicated a seasonal variation in the lettuce phyllosphere microbial community structure. Various lactic acid bacteria were detected including several Lactobacillus and Leuconostoc species in particular on lettuce from organic farming. By RT-PCR lactobacilli were found with a range of abundances from 1x10(4 )to 1x10(5 )copies/g lettuce. Considering the importance of salad in many diets lettuce may contribute to a constant supply with LAB.     

Effect of phenolic acids on glucose and organic acid metabolism by lactic acid bacteria from wine

The influence of phenolic (p-coumaric, caffeic, ferulic, gallic and protocatechuic) acids on glucose and organic acid metabolism by two strains of wine lactic acid bacteria (Oenococcus oeni VF and Lactobacillus hilgardii 5) was investigated. Cultures were grown in modified MRS medium supplemented with different phenolic acids. Cellular growth was monitored and metabolite concentrations were determined by HPLC-RI. Despite the strong inhibitory effect of most tested phenolic acids on the growth of O. oeni VF, the malolactic activity of this strain was not considerably affected by these compounds. While less affected in its growth, the capacity of L. hilgardii 5 to degrade malic acid was clearly diminished. Except for gallic acid, the addition of phenolic acids delayed the metabolism of glucose and citric acid in both strains tested. It was also found that the presence of hydroxycinnamic acids (p-coumaric, caffeic and ferulic) increased the yield of lactic and acetic acid production from glucose by O. oeni VF and not by L. hilgardii 5. The results show that important oenological characteristics of wine lactic acid bacteria, such as the malolactic activity and the production of volatile organic acids, may be differently affected by the presence of phenolic acids, depending on the bacterial species or strain.     

大豆蛋白水解物促进乳酸发酵的作用

通过研究添加大豆蛋白水解物对保加利亚乳杆菌培养过程中体系的酸度、粘度、细菌生长量的影响，验证了大豆蛋白水解产物对保加利亚乳杆菌增殖的促进作用。     

Molecular identification of lactic acid bacteria in Chinese rice wine using species-specific multiplex PCR

Chinese rice wine (CRW), a national unique and traditional food, has more than 4,000 years of history and is very popular in China. Lactic acid bacteria (LAB) play important roles in the fermentation of CRW because of its strong lactic acid production and high acid tolerance. It would be of practical interest to isolate the LAB species from CRW and to develop a simple and effective method for its accurate identification. In this study, 11 CRW-related LAB strains, frequently occurring in the fermentation of CRW, were isolated or collected and analyzed with the nested specifically amplified polymorphic DNA (nSAPD)-PCR technique. Eleven species-specific sequence characterized amplified region (SCAR) markers were generated from 11 polymorphic nSAPD bands. Three multiplex PCR assays were further established for the joint use of the 11 species-specific SCAR markers. Thus, a simple rapid molecular method was developed based on the application of nSAPD, SCAR, and multiplex PCR techniques, which allowed a rapid and simultaneous detection of 11 CRW-related LAB species. The simple rapid PCR-based molecular method can be used as a useful tool for the identification of LAB species in the traditionally fermented foods, especially in the rapid developing CRW industry in China.     

乳酸菌产胞外多糖的研究

综述了乳酸菌产胞外多糖的研究进展，包括产胞外多糖的菌株、影响生物合成的因素等，并介绍了乳酸菌产胞外多糖的开发及应用。     

乳酸菌液态发酵兔肉的研究

采用乳酸菌液态发酵法泡制免肉，研制出一种具有泡菜风味的兔肉食品。     

Evolution and identification of lactic acid bacteria isolated during the ripening of Sardinian sausages

Lactic acid bacteria (LAB) were isolated during the production and the ripening of Sardinian sausage, a typical Italian dry fermented sausage. Samples were taken at different stages, and 112 strains were isolated. The isolates were characterized using the micromethod proposed by Font de Valdez et al. [Font de Valdez, G., Savoy de Giori, G., Oliver, G., & De Ruiz Holgado, A. P. (1993). Development and optimization of an expensive microsystem for the biochemical characterization of lactobacilli. Microbiologie Aliments Nutrition, 11, 215–219]. Schillinger and Lücke’s [Schillinger, U., & Lücke, F. K. (1987). Identification of lactobacilli from meat and meat products. Food Microbiology. (4), 199–208] scheme and the biochemical patterns given by Bergey’s Manual of Systematic Bacteriology [Bergey’s Manual of Systematic Bacteriology (1986). Baltimore: William and Wilkins] were used for preliminary identification. A PCR-based method was then used to confirm the results. LAB were the dominant flora during ripening. They consisted mainly of homofermentative mesophilic rods. Lactobacillus sakei (43,3%), Lactobacillus plantarum (16,6%) and Lactobacillus curvatus (13,3%) were the main isolates. The results of the biochemical identification methods agreed well with those of PCR-based identification (91% agreement).     

乳酸菌发酵代谢合成叶酸的影响因素

对嗜酸乳杆菌以及乳酸乳球菌发酵合成叶酸的影响因素进行了研究。结果表明,乳酸菌代谢合成叶酸的产率为17~100μg/L,菌种、培养时间、pH值、对氨基苯甲酸(PABA)质量浓度会影响乳酸菌合成叶酸的产量。与乳酸乳球菌乳酸亚种相比,嗜酸乳杆菌CH-2生成的叶酸产量要高。不同菌株生成叶酸的能力与pH值有关,嗜酸乳杆菌在pH值为4.2叶酸产率明显下降,乳酸乳球菌乳酸亚种产叶酸的能力则不受pH值影响。添加PABA可以显著提高乳酸菌的叶酸产率。选择适宜的乳酸菌菌株,优化发酵工艺参数可以提高乳及相关食品中叶酸的质量浓度,达到生物方式强化叶酸的效果。     

乳酸菌冷冻干燥保护剂的筛选

通过单因素比较和正交试验设计,确定适合保加利亚乳杆菌和嗜热链球菌的最佳冻干保护剂配方。通过对15种冻干保护剂单因素保护效果的比较,以及对选定的4种保护剂的正交试验结果,可以确定嗜热链球菌最佳冻干保护剂配方为脱脂乳粉12%、海藻糖1%、甘油3%、谷氨酸钠1%,保加利亚乳杆菌最佳冻干保护剂配方为脱脂乳粉8%、海藻糖1%、甘油2%、谷氨酸钠1.5%。     

Exopolysaccharides production as affected by lactic acid bacteria and fermentation time

The aim of this work was to examine the ropiness of Lactobacillus helveticus BCRC14030, L. helveticus BCRC14076, and Streptococcus thermophilus BCRC14085, and evaluate the effect of fermentation time on exopolysaccharides (EPS) production by the ropy strain. Each of the three strains was inoculated in skim milk medium and incubated in a fermenter for 0–84 h at pH 5, 37 °C. The fermented samples, containing a net volume of 300 ml skim milk, were withdrawn at intervals of 0, 12, 16, 24, 32, 36, 48, 60, 72, and 84 h for determinations of ropy condition, EPS yield, and molecular mass. EPS with ropiness values of 11.3–21.0 mm, produced from L. helveticus BCRC14030 at 32–60 h demonstrated the ropy nature of the strain. Those EPS were composed of high molecular mass of 26,500 kDa. The highest EPS yield of 0.73 g l⁻¹ from this strain was observed (P < 0.05) at less favourable fermentation condition of 60 h. In addition, a relationship between the presence of high molecular mass and the ropiness of EPS from L. helveticus BCRC14030 was revealed.     

Differentiation of mixed lactic acid bacteria communities in beverage fermentations using targeted terminal restriction fragment length polymorphism

Lactic acid bacteria (LAB) are an important group of bacteria in beer and wine fermentations both as beneficial organisms and as spoilage agents. However, sensitive, rapid, culture-independent methods for identification and community analyses of LAB in mixed-culture fermentations are limited. We developed a terminal restriction fragment length polymorphism (TRFLP)-based assay for the detection and identification of lactic acid bacteria and Bacilli during wine, beer, and food fermentations. This technique can sensitively discriminate most species of Lactobacillales, and most genera of Bacillales, in mixed culture, as indicated by both bioinformatic predictions and empirical observations. This method was tested on a range of beer and wine fermentations containing mixed LAB communities, demonstrating the efficacy of this technique for discriminating LAB in mixed culture.     

抑制金黄色葡萄球菌的乳酸菌抗菌肽基因筛选及表达鉴定

以具有抑菌效果的L2、L16、L19等7株乳酸菌为模板,通过PCR扩增验证部分菌株中含有PlnF、PlnE、PlnN、PlnJ、PlnK等抗菌肽基因,以pET28a为载体并在抗菌肽基因两端引入Nco I和Xho I酶切位点,经双酶切和T4连接酶反应构建pET28a-抗菌肽重组质粒,转化E.coli BL21(DE3),培养至OD_(600)=0.6时,加入0.5mmol/L的IPTG诱导6h,菌体在400 W、超声4s、间歇5s条件下破碎后以8mol/L尿素进行变性处理和Ni柱纯化透析复性后的蛋白与相对应未纯化的蛋白相比,仅PlnF纯化蛋白对金黄色葡萄球菌具有抑菌效果[抑菌圈直径为(13.43±0.21)mm],而其他蛋白几乎无抑菌活性。进一步通过Tricine-SDS-PAGE电泳和nanoLC-ESI-MS/MS验证,目的蛋白与PlnF抗菌肽具有97.6%的同源性。