自然发酵辣椒酱中乳酸菌的分离与鉴定

从自然发酵的辣椒酱中分离出产酸量高、生长良好的菌株,经过形态学鉴定、生理生化特性及发酵性能试验,最终选定Lact.1和Lact.2两株适用于发酵辣椒试验的乳酸菌。鉴定结果表明Lact.1为植物乳酸杆菌(Lactobacillusplanetarium),Lact.2为肠膜明串珠菌(Leuconostocmesenteroides)。试验结果表明两株乳酸菌产酸速度快,最适生长温度在30℃到40℃之间,最适生长pH 6.0,当食盐浓度在7%以下能生长。     

发酵香肠中分离纯化的三株乳酸菌产酸特性研究

为了研究从发酵香肠中分离纯化的3株乳酸菌粪肠球菌(Enterococcus faecalis)、戊糖片球菌(Pediococcus pentosaceus)和肠膜明串珠菌(Leuconostoc mesenteroides)的产酸性能,对这3株菌在不同pH、温度、NaCl、NaNO2条件下的生长情况及产酸情况进行了测定。研究结果显示:这3株菌中,肠膜明串珠菌和戊糖片球菌的生长特性较好;粪肠球菌的生长特性虽不如肠膜明串珠菌和戊糖片球菌,但产酸能力最好,戊糖片球菌耐盐性最好、肠膜明串珠菌耐亚硝酸盐的特性最好;在不同温度和pH条件的测试中,肠膜明串珠菌的生长能力最好,粪肠球菌次之。这3株乳酸菌在发酵肉制品中均产生乳酸。总之,这3株菌均具有用于发酵制备乳酸的能力。     

PCR-DGGE法分析西藏传统发酵乳制品中乳酸菌的多样性

目的：运用聚合酶链式反应和变性梯度凝胶电泳（polymerase chain reaction- denatured gradient gelelectrophoresis,PCR-DGGE）技术分析西藏传统发酵乳制品中乳酸菌的生物多样性。方法：从西藏8个牧区采集19份样品,提取样品总DNA,用巢式和降落PCR扩增16S rRNA的V3区段,对扩增产物做变性梯度凝胶电泳,用NTsys 2.10e软件分析条带的相似性,切胶回收条带并测序,鉴定菌种并构建系统进化树、分析优势菌种。结果：19份样品中的乳酸菌菌群组成包括Lactobacillus paracasei、Lactobacillus helveticus、Lactobacillus fermentum、Lactobacillus crispatus、Lactobacillus delbrueckii、Lactobacillus buchneri、Lactococcus raffinolactis、Leuconostocmesenteroide、Lactobacillus plantarum、Pediococcus pentosaceus、Lactococcus lactis、Streptococcus thermophilus。综合样品和牧区的乳酸菌分布情况,确定Lactobacillus delbrueckii为优势菌种。结论：PCR-DGGE技术能够有效分析西藏地区发酵乳制品中乳酸菌的多样性。     

Classification of ropy slime-producing lactic acid bacteria based on DNA-DNA homology, and identification of Lactobacillus sake and Leuconostoc amelibiosum as dominant spoilage organisms in meat products.

The classification of lactic acid bacteria able to cause ropy slime on vacuum-packed cooked meat products was carried out based on DNA-DNA homology. The ropy slime-producing lactobacilli were identified as strains of Lactobacillus sake and the ropy slime-producing leuconostocs, such as Leuconostoc amelibiosum and Leuconostoc mesenteroides.     

Souring and breakdown of cyanogenic glucosides during the processing of cassava into akyeke

The population and composition of the lactic acid bacteria microbiota as well as the content of cyanogenic glucosides occurring at various stages of fermentation and subsequent processing of cassava roots into akyeke, a steamed sour cassava meal, were investigated. The number of lactic acid bacteria and percentage titratable acidity increased during 5 days of fermentation, but decreases were observed in the subsequent operations of 'washing' the dough with water followed by partial drying and steaming. In field and laboratory samples, Lactobacillus plantarum accounted for 59.3% and 52.3%, Lactobacillus brevis 23.3% and 22.8% and Leuconostoc mesenteroides subsp. cremoris 14.5% and 15.8%, respectively, of all lactic acid bacteria isolated at various stages of fermentation and processing. A reduction of about 98% occurred in the total cyanogens (CN) content of cassava roots during processing, from 69.3 to 1.4 and 110.3 to 2.8 mg CN equivalent/kg dry weight for laboratory and field samples of akyeke, respectively.     

云南传统腌制食品可培养乳酸菌多样性及其发酵特性研究

以云南传统腌制食品为研究对象,采用纯培养分离、形态学观察及分子生物学鉴定方法进行乳酸菌多样性考察,并对分离菌株进行了发酵特性初步研究。结果表明,云南传统特色腌制食品中可培养乳酸菌多样性强,从11个样本中分离得到10株优势乳酸菌,涵盖乳杆菌属（Lactobacillus）、明串球菌属（Leuconostoc）、肠球菌属（Enterococcus）的菌株,分别为短乳杆菌（Lactobacillus brevis）1097b、哈尔滨乳杆菌（Lactobacillus harbinensis）1119b、干酪乳杆菌（Lactobacillus casei）1128b、戊糖乳杆菌（Lactobacillus pentosus）1133b、植物乳杆菌（Lactobacillus plantarum）1116b、1125b、1126b、1127b、肠系膜明串珠菌（Leuconostoc mesenteroides）1089b、粪肠球菌（Enterococcus faecium）1129b;另外,不同分离源的乳酸菌发酵特性差异显著,乳酸发酵类型多样性丰富。     

Direct detection and identification of lactic acid bacteria in a food processing plant and in meat products using denaturing gradient gel electrophoresis

We established a novel system using denaturing gradient gel electrophoresis (DGGE) to quickly identify bacteria known to be responsible for spoilage in meat processing plants and meat products. We extracted bacterial DNA from swabbed samples at various locations in the plant and from meat products and performed PCR amplification, targeting 16S rDNA from the dominant organisms. The amplification products were subjected to DGGE, and the contaminating bacteria in the meat products and the plant were analyzed. This analysis indicated that lactic acid bacteria and spoilage-causing bacteria are widely distributed within the meat processing plant. We developed molecular size markers to identify the dominant organisms obtained from the plant and meat products. The establishment of the present method allows quick and simple identification of bacteria causing the possible deterioration of products and contamination and thus permits constant monitoring of any harmful bacteria within meat processing plants.     

Evaluation of lactic acid bacteria,isolated from lightly preserved fish products,as starter cultures for new fish-based food products

Lactic acid bacteria suitable for fish fermentation were selected and their capacity as starter cultures in fish raw material was evaluated. Of 23 strains of lactic acid bacteria which were isolated from lightly preserved herring products and identified by the Biolog system mainly as Lactobacillus and Leuconostoc spp., three strains, which grew well at 5–10°C in the presence of food preservatives, were chosen for evaluation. Qualitative changes in minced fish, inoculated with lactic acid bacteria, and stored at 10°C for 7 weeks, were examined by organoleptic, bacteriological and chemical methods, and were found to be strongly strain specific. Inhibition of the competitive flora was more significant in the samples inoculated with Lactobacillus plantarum and Lactobacillus mesenteroides than in others. The best organoleptic and chemical results were obtained with Leuconostoc mesenteroides; this strain looks promising as a starter culture for fish fermentation and for the development of new fish products, which organoleptically resemble meat products.     

Succession of dominant and antagonistic lactic acid bacteria in fermented cucumber: insights from a PCR-based approach

The goal of the investigation was to study the succession of major groups of lactic acid bacteria (LAB) and their antagonism in salt-fermented cucumber using PCR. In a direct detection method as well as a short enrichment process, PCR enabled detection of Leuconostoc and Lactobacillus during early hours of fermentation. Subsequently, Lactobacillus and Pediococcus emerged as the dominant genera. Nucleic acid sequence of culture-independent clones confirmed the detection of Pediococcus as a dominant genera emerging during late stages of fermentation. PCR also revealed time-dependent emergence of mesentericin, pediocin and plantaricin A producers and accounted for the LAB succession in the fermenting samples. A total of 328 LAB isolates were obtained collectively from 30 cucumber samples, of which PCR could identify an overwhelming 186 Lactobacillus isolates followed by 113 Pediococcus and 29 Leuconostoc isolates, respectively. Based on antimicrobial assay against target strain Leuconostoc mesenteroides NRRL B640, 28% of the LAB were bacteriocin producers, of which pediocin producers were substantial, followed by plantaricin A and mesentericin producers. The bacteriocins elaborated by the isolates were active against a large number of Gram-positive target LAB strains and pathogenic bacteria including Bacillus cereus, Enterobacter aerogenes, Enterococcus faecalis, Escherichia coli, Listeria monocytogenes and Staphylococcus aureus.     

啤酒有害菌的PCR检测和SYBR Green实时PCR定量

啤酒有害菌是一些能在啤酒中存活并使啤酒的外观和风味发生改变的细菌,对其进行快速检测和定量是啤酒生产急待解决的问题。我们从华润雪花啤酒（中国）有限公司各工厂提供的样品中分离到28株啤酒有害菌,16S rDNA序列的系统进化分析表明,其中26个菌株属于乳杆菌属（Lactobacillus spp．）、1个菌株为明串珠菌属（Leuconostoc spp．）,1个菌株为片球菌属（Pediococcu sp．）。根据酒花（hop）抗性基因horA、horB和horC的保守序列设计了扩增这3个基因的PCR引物,用这些引物对28株啤酒有害菌进行了常规PCR检测,检出率分别为89％、79％和75％,用hor A—horC双引物进行检测,检出率为100％。用SYBR Green实时定量PCR技术,以horA基因为靶序列,建立了对啤酒有害菌的细胞数进行快速定量的新方法,用该方法测定的污染啤酒样品中有害菌的浓度与平板培养法相近。     

Effects on the development of blown pack spoilage of the initial numbers of Clostridium estertheticum spores and Leuconostoc mesenteroides on vacuum packed beef

Beef steaks were inoculated with Clostridium estertheticum spores and Leuconostoc mesenteroides cells at all combinations of numbers of 0, 10, 100 or 1000/cm(2) for each organism. After vacuum packaging the steaks were stored at 4, 1, or -1.5°C. Pack volumes were determined by water displacement at suitable intervals. Irrespective of L. mesenteroides numbers, for packs containing meat inoculated with 0, 10, 100 or 1000 spores/cm(2), 60, 16, 3 and 1 of 60 packs did not swell. The times of onset of swelling were twice as long at -1.5 as at 4°C, but they were not affected by the inoculated numbers of L. mesenteroides. Rates of pack swelling increased with increasing storage temperature and number of spores, but decreased with increasing numbers of inoculated L. mesenteroides. Lactic acid bacteria can apparently prevent development of blown pack spoilage of vacuum packs containing meat contaminated with low numbers of C. estertheticum.     

水牛乳中可培养乳酸菌多样性分析

采用传统分离培养方法,从三品杂交生水牛奶混合样品中,分离出105株乳酸菌,通过形态、生理生化、API细菌鉴定系统及16S rDNA基因序列分析方法对各菌株属种进行鉴定。16S rRNA序列分析结果显示,105株菌共分为5个属8个种,呈现较为丰富的乳酸菌多样性,具体数量分布为乳酸乳球菌21株,植物乳杆菌19株,格氏乳球菌17株,乳明串珠菌13株,食窦魏斯氏菌11株,肠膜明串珠菌8株,类肠膜魏斯氏菌6株,嗜热链球菌5株,糊精乳杆菌5株。由此可知,水牛乳中可培养乳酸菌优势菌群的主次关系为：乳酸乳球菌（Lactococcus lactis）＞植物乳杆菌（Lactobacillus plantaru）＞格氏乳球菌（Lactococcus garvieae）＞乳明串珠菌（Leuconostoc lactis）＞食窦魏斯氏菌（Weissella cibaria）,此为后续开发水牛乳中优势乳酸菌资源提供了良好的理论基础。     

自然发酵豆酱中明串珠菌的分离鉴定

分离筛选传统发酵豆酱中的明串珠菌,研究其益生特性。以采集自东北6 个地区的56 份自然发酵豆酱为研究对象,用稀释涂布平板方法对明串珠菌进行分离筛选,然后对疑似菌株进行形态学特征、生理生化特征和16S rRNA基因序列进行分析,确定菌株种属序列,然后对鉴定得到的明串菌株进行耐酸耐胆盐性能比较。结果表明：从56?份豆酱样品中共分离到118?株疑似乳酸菌,从中得到6?株明串珠菌,鉴定3?株为乳酸明串珠菌,3?株为肠膜明串珠菌肠膜亚种。综合比较6?株明串珠菌耐酸耐胆盐性,发现菌株FX6益生性最好,在pH?3.0环境培养3?h后存活率可达85.16%,在含0.3 g/100 mL胆盐环境培养6?h后存活率高达96.07%。因此,菌株FX6可用于进一步科研以及工业应用,其在豆酱发酵过程的作用机理仍需深入研究。     

Monitoring the lactic acid bacterial diversity during shochu fermentation by PCR-denaturing gradient gel electrophoresis

The presence of lactic acid bacteria (LAB) during shochu fermentation was monitored by PCR-denaturing gradient gel electrophoresis (DGGE) and by bacteriological culturing. No LAB were detected from fermented mashes by PCR-DGGE using a universal bacterial PCR primer set. However, PCR-DGGE using a new primer specific for the 16S rDNA of Lactococcus, Streptococcus, Tetragenococcus, Enterococcus, and Vagococcus and two primers specific for the 16S rDNA of Lactobacillus, Pediococcus, Leuconostoc, and Weissella revealed that Enterococcus faecium, Lactobacillus casei, Lactobacillus fermentum, Lactobacillus nagelii, Lactobacillus plantarum, Lactococcus lactis, Leuconostoc citreum, Leuconostoc mesenteroides, and Weissella cibaria inhabited in shochu mashes. It was also found that the LAB community composition during shochu fermentation changed after the main ingredient and water were added during the fermentation process. Therefore, we confirmed that PCR-DGGE using all three primers specific for groups of LAB together was well suited to the study of the LAB diversity in shochu mashes. The results of DGGE profiles were similar to the results of bacteriological culturing. In conclusion, LAB are present during shochu fermentation but not dominant.     

Evaluation of exopolysaccharide production by Leuconostoc mesenteroides strains isolated from wine

ABSTRACT:  Exopolysaccharide (EPS)-producing lactic acid bacteria are responsible for the alteration of wine and other fermented beverages. The potential to produce EPS was investigated for Leuconostoc mesenteroides strains isolated from Spanish grape must and wine. Most strains were able to produce EPS from sucrose containing media. Based on their EPS-producing phenotype and on their EPS monosaccharide composition, the L. mesenteroides strains analyzed could be arranged in 2 groups. One group comprises mucoid strains producing a glucan polymer, and the other group includes strains producing a fructan polymer. The presence of a glucosyltransferase encoding gene in the glucan producing L. mesenteroides strains was assayed by PCR. Two primer sets, PF1-PF8 and GTFF-GTFR, were used to amplify internal fragment of known glucosyltransferase genes. None of the glucan-producing strains gave a positive amplicon by the primer sets used. Therefore, new tools need to be developed to broaden the range of potentially spoiling agents detected by PCR in fermented beverages.     

The metabolism of several carboxylic acids by lactic acid bacteria

The anaerobic metabolism of citrate, fumarate, gluconate, malate, 2-oxoglutarate and pyruvate by 137 strains of 23 species of lactic acid bacteria was investigated. The bacteria were from various sources (plant material, meat and dairy products, dough and wine) and belonged to the genera Lactobacillus, Leuconostoc, Pediococcus, and Streptococcus. The ability of metabolize the acids was determined by thin layer chromatography or by enzymatic analysis after growth of the strains in a glucose-containing medium. All strains metabolized pyruvate and only 12 mainly heterofermentative strains were malate negative. These strains were also unable to decompose citrate. This acid was fermented by 23 strains, all of which metabolized malate. Many lactic acid bacteria reduced 2-oxoglutarate to hydroxyglutarate. The strains of Lactobacillus plantarum did not metabolize 2-oxoglutarate whereas all strains of Leuconostoc oenos decarboxylated this acid and formed 4-hydroxybutyrate and succinate. Gluconate was fermented by 52 mainly heterofermentative strains. No correlation was observed between the ability to ferment citrate, malate or gluconate.     

Intraspecific diversity of Lactobacillus curvatus, Lactobacillus plantarum, Lactobacillus sakei, and Leuconostoc mesenteroides associated with vacuum-packed meat product spoilage analyzed by randomly amplified polymorphic DNA PCR

The intraspecific diversity of Leuconostoc mesenteroides, Lactobacillus curvatus, Lactobacillus sakei, and Lactobacillus plantarum was analyzed by randomly amplified polymorphic DNA (RAPD) PCR with universal primers M13 and T3. The study included 100 reference strains and 210 isolates recovered from two vacuum-packed Spanish meat products, fiambre de magro adobado and morcilla, previously identified by rDNA-restriction fragment length polymorphism profiles. The RAPD-M13 profiles identified isolates at species level in L. plantarum and L. mesenteroides, while RAPD-T3 provided profiles in L. sakei. The combination of RAPD-M13 and RAPD-T3 fingerprints revealed a total of 17 profiles in L. mesenteroides, 6 in L. sakei, 12 in L. plantarum, and 6 in L. curvatus. Of these, six profiles corresponding to L. mesenteroides and one corresponding to L. sakei were found in both products. The Shannon-Weaver diversity index (H'), calculated according to RAPD-M13 and RAPD-T3 profiles during storage, revealed that most profiles appeared only in single samplings in both products, indicating a high strain substitution rate during chilled storage of vacuum-packed meat products. When bloating appeared, only one profile corresponding to L. mesenteroides subsp. dextranicum was present throughout the storage period.     

Diversity of lactic acid bacteria isolated from AOC Salers cheese

The objective of this work was to describe the diversity of lactic acid bacteria in traditional raw milk Salers cheeses at the species and strain levels. The characterization of 381 strains isolated during ripening and various strain collections was investigated using physiological analysis and molecular techniques: Rep-PCR, species and genus specific amplifications and the sequence analysis of 16S rDNA for strain typing and taxonomic identification. The strains belonged to Lactobacillus plantarum, Lactobacillus paracasei, Lactococcus lactis, Lactococcus garviae, Enterococcus faecalis, Enterococcus faecium, Leuconostoc mesenteroides, Leuconostoc pseudomesenteroides, Streptococcus salivarius, Streptococcus millieri, Streptococcus macedonicus and Pediococcus pentosaceus. A wide phenotypic and genomic heterogeneity was observed within the different species (Lactobacillus plantarum, Lactobacillus paracasei and Leuconostoc mesenteroides) according to the origin and the time of ripening. The natural microflora was different from strain collection and each method must be combined to identify and characterize natural microflora. This study revealed the low selectivity of selective media used for the isolation of different groups of lactic acid bacteria except the Facultatively Heterofermentative lactobacilli medium selecting mesophile lactobacilli and SB medium selective for Enterococcus. The study reveals, for the first time, the microbial lactic acid bacteria community of Salers cheese and its diversity. A better knowledge of microbial flora will be useful to improve understanding of sensory quality of cheeses.     

假肠膜明串珠菌产细菌素复配抑菌液的筛选及保鲜效果研究

目的 筛选出最优的假肠膜明串珠菌（Leuconostoc mesenteroides, LM）产细菌素复配抑菌液,同时探究抑菌液对冷鲜鸡肉的保鲜效果。方法 以冷鲜鸡肉及其主要腐败菌为研究对象,研究假肠膜明串珠菌产细菌素的最佳生长周期,并利用假肠膜明串珠菌产细菌素抑菌液、乳酸链球菌素抑菌液、假肠膜明串珠菌产细菌素和柚子精油复配抑菌抑菌液以及假肠膜明串珠菌产细菌素和茶多酚复配抑菌液分别浸泡鸡肉,从肉的pH、汁液流失率、色度、菌落总数、挥发性盐基氮（total volatile basic nitrogen, TVB-N）、感官评定等评价其对鸡肉冷藏品质的影响,比较以上几种抑菌液对鸡肉的保鲜效果,筛选出最佳的抑菌液。结果 假肠膜明串珠菌产细菌素的生长周期与其抑菌活性呈一定正相关。实验组的4种抑菌液均能延缓冷鲜鸡肉劣变,但1.5%假肠膜明串珠菌产细菌素抑菌液、40 mg/L乳酸链球菌素抑菌液对冷鲜鸡肉保鲜效果有限。而1.5%假肠膜明串珠菌产细菌素和0.2%茶多酚复配抑菌液、1.5%假肠膜明串珠菌产细菌素和10%柚子精油复配抑菌液的保鲜效果较好,尤其在以1.5%假肠膜明串珠菌产细菌素和10%柚子精油复配抑菌液液处理时,对冷鲜鸡肉的保鲜作用最强。结论 1.5%假肠膜明串珠菌产细菌素和10%柚子精油复配抑菌液对冷鲜鸡肉具有最佳的保鲜效果。