Specific enumeration of lactic acid bacteria in ragi tape by colony hybridization with specific oligonucleotide probes

Lactic acid bacteria (LAB) are among the integral microflora of ragi tape, a dry starter of Balinese rice wine, brem. The species diversity and population level of LAB present in different types of ragi tape were studied by colony hybridization using 16S and 23S rDNA targeted oligonucleotide probes. These probes were DB6, Lbc, Wgp, and Rpt, which were specific for Enterococcus faecium, Lactobacillus curvatus, Weissella spp., and Pediococcus pentosaceus, respectively. Results revealed that P. pentosaceus and Weissella spp. were the predominant LAB in ragi tape, whereas L. curvatus and E. faecium were associated specific to types of ragi tape. A 21-mer species-specific oligonucleotide probe, Rpt, that targets the 16S rDNA of P. pentosaceus was developed in this study and found to be highly specific to be used as an effective tool to enumerate population of this species in ragi tape and its population changes during rice wine production. It was detected that LAB showed active growth during the early stage of brem fermentation. A succession of growth of LAB population during the fermentation was observed in which the heterofermentative LAB, Weissella spp., grew first, followed by the proliferation of P. pentosaceus.     

Flour carbohydrate catabolism and metabolite production by sourdough lactic acid bacteria

The aim of this study was to assess the mode of carbohydrate catabolism by lactic acid bacteria isolated from traditional sourdoughs, as well as to study their effect on the metabolites produced. For this purpose, single cultures of the heterofermentative lactic acid bacteria Lactobacillus sanfranciscensis, Lactobacillus brevis, Weissella cibaria, and the homofermentative Lactobacillus paralimentarius and Pediococcus pentosaceus were grown in liquid media containing glucose, fructose, maltose and sucrose, either as a single carbon source or in combination with glucose. Carbon catabolism and the production of metabolites were determined by HPLC analysis. W. cibaria could ferment all carbon sources, L. sanfranciscensis, L. paralimentarius and P. pentosaceus could not ferment sucrose, while L. brevis could only ferment maltose. The presence of glucose did not influence the utilization of fructose and maltose by L. sanfranciscensis, while it repressed the fermentation of fructose, maltose and sucrose by W. cibaria, and fructose and maltose by L. paralimentarius and P. pentosaceus. Moreover, L. sanfranciscensis and L. brevis could obtain extra ATP through the reduction of fructose to mannitol, which favored the production of acetic acid against ethanol. The utilization of fructose as an electron acceptor has a decisive effect on the prevailing of L. sanfranciscensis and L. brevis in spontaneously fermented sourdough and in the scarce appearance of the other lactic acid bacteria studied.     

Preliminary characterization of lactic acid bacteria isolated from Zlatar cheese

AIMS: Isolation, characterization and identification of lactic acid bacteria (LAB) from artisanal Zlatar cheese during the ripening process and selection of strains with good technological characteristics. METHODS AND RESULTS: Characterization of LAB was performed based on morphological, physiological and biochemical assays, as well as, by determining proteolytic activity and plasmid profile. rep-polymerase chain reaction (PCR) analysis and 16S rDNA sequencing were used for the identification of LAB. PCR analysis was performed with specific primers for detection of the gene encoding nisin production. Strains Lactobacillus paracasei subsp. paracasei, Lactobacillus plantarum, Lactobacillus brevis, Lactococcus lactis subsp. lactis, Enterococcus faecium and Enterococcus faecalis were the main groups present in the Zlatar cheese during ripening. CONCLUSIONS: Temporal changes in the species were observed during the Zlatar cheese ripening. Mesophilic lactobacilli are predominant microflora in Zlatar cheese. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study we determined that Zlatar cheese up to 30 days old could be used as a source of strains for the preparation of potential starter cultures in the process of industrial cheese production. As the Serbian food market is adjusting to European Union regulations, the standardization of Zlatar cheese production by using starter culture(s) based on autochtonous well-characterized LAB will enable the industrial production of this popular cheese in the future.     

Lactic acid bacteria in fermented foods in Thailand

Traditional fermented foods (fish, meat and vegetable products), produced by many different processes, are eaten in many parts of Thailand. Lactic acid bacteria are responsible for the souring and ripening of these foods. Homofermentative strains of Lactobacillus pentosus, L. plantarum and Pediococcus pentosaceus are dominant in foods with low salt concentrations whereas P. halophilus strains are present in foods containing high salt. Strains of Lactobacillus sake, other Lactobacillus spp., P. acidilactici and P. urinaeequi are frequently found. Heterofermentative strains of L. brevis, L. confusus, L. fermentum, L. vaccinostercus, other Lactobacillus spp., and of Leuconostoc spp. are distributed as minor bacteria and strains of Staphylococcus, Enterococcus and Halobacterium are occasionally isolated.S. Tanasupawat is with the Department of Microbiology, Faculty of Pharmaceutical Sciences. Chulalongkorn University, Bangkok 10330, Thailand; K. Komagata is with the Department of Agricultural Chemistry, Tokyo University of Agriculture, Sakuragaoka 1-1-1, Setagaya-ku, Tokyo 156, Japan.     

Identification and succession of lactic acid bacteria during fermentation of 'urutan', a Balinese indigenous fermented sausage

'Urutan' is a Balinese traditional fermented sausage, which is made of lean pork and fat mixed with spices, sugar, and salt. The mixture is stuffed into cleaned pig intestine and fermented under uncontrolled condition during sun drying for 5 days. The investigation showed that lactic acid bacteria (LAB) were the dominating bacteria during 'urutan' fermentation. Among the 71 isolates obtained, lactobacilli dominated by 77.5% and the other 22.5% were pediococci. Based on physiological characteristics, the isolates were classified into 13 groups: nine belonged to the lactobacilli and the other four were pediococci. One isolate representing each group was chosen randomly, and then was identified by 16S rDNA sequence comparison. Phylogenetic relationship positioned three groups to Lactobacillus plantarum and four groups were closely related to L. farciminis. Two groups were identified as obligate heterofermentative lactobacilli: one was L. fermentum and the other was distantly related to L. hilgardii. Two groups belonging to the pediococci were strains of Pediococcus acidilactici and the other two were closely related to P. pentosaceus. A dramatic succession occurred during fermentation of 'urutan'. Three species mainly dominated the process wherein the initial growth was started by L. plantarum then followed by the growth of P. acidilactici, and finally, L. farciminis was found to be predominant at the last stage of fermentation.     

The characterization of lactic acid producing bacteria from the rumen of dairy cattle grazing on improved pasture supplemented with wheat and barley grain

Aims:  To identify and characterize the major lactic acid bacteria in the rumen of dairy cattle grazing improved pasture of rye grass and white clover and receiving a maize silage and grain supplement with and without virginiamycin.
Methods and Results:  Eighty-five bacterial isolates were obtained from the rumen of 16 Holstein-Friesian dairy cows. The isolates were initially grouped on the basis of their Gram morphology and by restriction fragment length polymorphism analysis of the PCR amplified 16S rDNA. A more definitive analysis was undertaken by comparing the 16S rDNA sequences. Many of the isolates were closely related to other previously characterized rumen bacteria, including Streptococcus bovis, Lactobacillus vitulinus , Butyrivibrio fibrisolvens , Prevotella bryantii and Selenomonas ruminantium . The in vitro production of l - and/or d -lactate was seen with all but five of the isolates examined, many of which were also resistant to virginiamycin.
Conclusion:  Supplementation of grain with virginiamycin may reduce the risk of acidosis but does not prevent its occurrence in dairy cattle grazing improved pasture.
Significance and Impact of the Study:  This study shows that lactic acid production is caused, not only by various thoroughly researched types of bacteria, but also by others previously identified in the rumen but not further characterized.     

Biological and genetic classification of canine intestinal lactic acid bacteria and bifidobacteria

To investigate the distribution of lactic acid bacteria (LAB) inhabiting canine intestines, a total of 374 gram-positive LAB and bifidobacteria (BF) isolated from large intestinal contents in 36 dogs were classified and identified by phenotypic and genetic analyses. Based on cell morphological sizes, these isolates were divided into seven biotypes containing the genera Lactobacillus, Bifidobacterium, Enterococcus, and Streptococcus. The LAB and BF isolates were classified into 38 chemotypes based on SDS-PAGE protein profile analysis of whole cells. Furthermore, partial 16S rDNA sequencing analysis demonstrated the presence of 24 bacterial species in the 38 chemotypes from 36 dogs. The identified species consisted of ten species belonging to the genus Lactobacillus (78.8%), seven species to the genus Bifidobacterium (6.8%), five species to the genus Enterococcus (11.6%), one species of Streptococcus bovis (2.0%), and one species of Pediococcus acidilactici (0.8%). In particular, the most predominant species in canine intestines were L. reuteri, L. animalis, and L. johnsonii and were found in the high frequency of occurrence of 77.8, 80.6, and 86.1%, respectively. Besides these, Enterococcus faecalis, Bifidobacterium animalis subsp. lactis, Pediococcus acidilactici, and Streptococcus bovis were also isolated in the present study. The sequences of the isolates also showed high levels of similarity to those of the reference strains registered previously in the DDBJ and the similarity was above 97.2%. Their partial 16S rRNA genes were registered in the DDBJ.     

Isolation and characterization of a novel lactic acid bacterium for the production of lactic acid

We isolated a novel lactic acid bacterium from a Korean traditional fermented food, soybean paste. The newly isolated strain, dubbed RKY2, grew well on glucose, sucrose, galactose, and fructose, but it could not utilize xylose, starch, or glycerol. When the partially amplified 16S rDNA sequence (772 bp) of the strain RKY2 was compared with 10 reference strains, it was found to be most similar toLactobacillus pentosus JCM 1588^T, with 99.74% similarity. Therefore, the strain RKY2 was renamedLactobacillus sp. RKY2, which has been deposited in the Korean Collection for Type Cultures as KCTC 10353BP.Lactobacillus sp. RKY2 was found to be a homofermentative lactic acid bacterium, because its end-product from glucose metabolism was found to be mainly lactic acid. It could produce more than 90 g/L of lactic acid from MRS medium supplemented with 100 g/L of glucose, with 5.2 g L⁻¹ h⁻¹ of productivity and 0.95 g/g of lactic acid yield.     

Vaginal lactic acid bacteria in the mare: evaluation of the probiotic potential of native Lactobacillus spp. and Enterococcus spp. strains

Lactic acid bacteria (LAB) are important members of the human vaginal microbiota and their presence is considered beneficial. However, little is known about native vaginal bacteria in other animal species such as the horse. The aim of this work was to quantify the vaginal lactic acid bacteria and lactobacilli of mares and to establish if selected equine vaginal lactic acid bacteria, particularly Lactobacillus and Enterococcus spp. strains, could exhibit potential as probiotics. The vaginal lactic acid bacteria and lactobacilli of 26 mares were quantified by plate counts. Five strains (three Lactobacillus spp. and two Enterococcus spp.) were characterised and adhesion to vaginal epithelial cells, antimicrobial activity and ability to form biofilms were evaluated. Lactic acid bacteria were recovered from the 26 samples and lactobacilli counts were detected in 18 out of 26 mares (69%). Probiotic properties tested in this study varied among the isolates and showed promising features for their use as equine probiotics.     

Isolation and characterization of fructophilic lactic acid bacteria from fructose-rich niches

Fourteen strains of fructophilic lactic acid bacteria were isolated from fructose-rich niches, flowers, and fruits. Phylogenetic analysis and BLAST analysis of 16S rDNA sequences identified six strains as Lactobacillus kunkeei, four as Fructobacillus pseudoficulneus, and one as Fructobacillus fructosus. The remaining three strains grouped within the Lactobacillus buchneri phylogenetic subcluster, but shared low sequence similarities to other known Lactobacillus spp. The fructophilic strains fermented only a few carbohydrates and fermented d-fructose faster than d-glucose. Based on the growth characteristics, the 14 isolates were divided into two groups. Strains in the first group containing L. kunkeei, F. fructosus, and F. pseudoficulneus grew well on d-fructose and on d-glucose with pyruvate or oxygen as external electron acceptors, but poorly on d-glucose without the electron acceptors. Strains in this group were classified as “obligately” fructophilic lactic acid bacteria. The second group contained three unidentified strains of Lactobacillus that grew well on d-fructose and on d-glucose with the electron acceptors. These strains grew on d-glucose without the electron acceptors, but at a delayed rate. Strains in this group were classified as facultatively fructophilic lactic acid bacteria. All fructophilic isolates were heterofermentative lactic acid bacteria, but “obligately” fructophilic lactic acid bacteria mainly produced lactic acid and acetic acid and very little ethanol from d-glucose. Facultatively fructophilic strains produced lactic acid, acetic acid and ethanol, but at a ratio different from that recorded for heterofermentative lactic acid bacteria. These unique characteristics may have been obtained through adaptation to the habitat.     

16S-ARDRA, a tool for identification of lactic acid bacteria isolated from grape must and wine

Lactic acid bacteria (LAB) are found in a great variety of habitats, including grape must and wines. There is a close relationship between the species of LAB which develop during fermentation and the eventual quality of the wine. For these reasons analytical techniques allowing fast and reliable identification of wine LAB are needed. In this work a simple and accurate protocol for identifying species of LAB isolated from grape must and wine is presented. This protocol is based on the amplification, directly from colony, of 16S rDNA and later digestion with one of the following restriction enzymes BfaI, MseI and AluI. A sequential use of the three enzymes is proposed to simplify LAB wine identification, first MseI, then BfaI and finally, if necessary, AluI digestion. The technique was able to discriminate 32 of the 36 LAB reference species tested and allowed the identification of 342 isolates from musts and wines. The isolates belonged to the species: Lactobacillus brevis, L. collinoides, L. coryniformis, L. bilgardii, L. mali, L. paracasei, Leuconostoc mesenteroides, Oenococcus oeni, Pediococcus parvulus and P. pentosaceus.     

Biochemical characterisation of the esterase activities of wine lactic acid bacteria

Esters are an important group of volatile compounds that can contribute to wine flavour. Wine lactic acid bacteria (LAB) have been shown to produce esterases capable of hydrolysing ester substrates. This study aims to characterise the esterase activities of nine LAB strains under important wine conditions, namely, acidic conditions, low temperature (to 10°C) and in the presence of ethanol (2–18% v/v). Esterase substrate specificity was also examined using seven different ester substrates. The bacteria were generally found to have a broad pH activity range, with the majority of strains showing maximum activity close to pH 6.0. Exceptions included an Oenococcus oeni strain that retained most activity even down to a pH of 4.0. Most strains exhibited highest activity across the range 30–40°C. Increasing ethanol concentration stimulated activity in some of the strains. In particular, O. oeni showed an increase in activity up to a maximum ethanol concentration of around 16%. Generally, strains were found to have greater activity towards short-chained esters (C2–C8) compared to long-chained esters (C10–C18). Even though the optimal physicochemical conditions for enzyme activity differed from those found in wine, these findings are of potential importance to oenology because significant activities remained under wine-like conditions.     

Pediocin production by recombinant lactic acid bacteria

Production of the anti-listerial bacteriocin, pediocin, by lactic acid bacteria (LAB) transformed with the cloning vector pPC418 (Ped⁺, 9.1 kb) was influenced by composition of media and incubation temperature. Maximum pediocin production, tested against Listeria innocua, by electrotransformants of Lactococcus lactis ssp. lactis was measured in tryptone/lactose/yeast extract medium after 24 h growth at 30 °C, while incubation at 40 °C was optimum for Ped⁺ transformants of Streptococcus thermophilus and Enterococcus faecalis. The amount of pediocin produced by S. thermophilus in skim milk and cheese whey supplemented with 0.5% yeast extract was estimated as 51000 units ml^–1 and 25000 units ml^–1, respectively. Pediocin production remained essentially unchanged in reconstituted skim milk or whey media diluted up to 10-fold. The results demonstrate the capacity of recombinant strains of LAB to produce pediocin in a variety of growth media including skim milk and inexpensive cheese whey-based media, requiring minimum nutritional supplementation.     

乳酸菌低温菌株的复合诱变选育

【目的】采用复合诱导的方法,选育出能在低温条件下性状稳定、生长良好且具有工业生产价值的低温乳酸菌菌株。【方法】使用紫外照射和亚硝基胍处理的方法对分离的Q1菌株进行诱变处理,将选育得到的低温菌株Q1-4-6与出发菌株Q1在15°C、20°C、37°C下,比较其生长速率、产酸量和糖降解量。【结果】从玛曲牧民自制酸奶中分离得到一株乳酸菌Q1,根据其形态学特征、生理生化特性,同时结合16SrDNA序列分析结果,将其鉴定为肠球菌属棉籽糖肠球菌(Enterococcus raffinosus)。经过多轮诱变选育出一株能在低温下良好生长、遗传性状稳定的菌株Q1-4-6,将出发菌株与诱变菌株在15°C、20°C比较其生长、产酸和糖降解量发现,诱变菌株均好于出发菌株。在37°C的自然条件下,诱变菌株和出发菌株的生长速率差异不大,但均略高于出发菌株。在15°C培养发现,诱变菌株生长速率、产酸量均高于购买菌株干酪乳杆菌(CL04)和嗜热链球菌(CL05)。【结论】通过UV+NTG复合诱变,最终选育出一株在低温下生长良好、遗传性状稳定的菌株Q1-4-6。     

泡菜、传统腊肠中降胆固醇乳酸菌的筛选及鉴定

摘要：【目的】筛选具有降胆固醇功能的乳酸菌菌株,为乳酸菌体外、体内的降胆固醇生理特性和机理研究奠定基础。【方法】以碳酸钙-MRS选择性培养基(Calcium Carbonate-Man Rogosa and Sharp Medium)从中国传统食品泡菜、腊肠中筛选乳酸菌,应用改良的胆固醇筛选培养基筛选具有较高降胆固醇能力的乳酸菌菌株,并研究其耐酸性,耐胆盐活性,生长曲线及产酸特性;结合菌落形态学、接触酶反应、革兰氏染色、碳水化合物微量鉴定管及16SrRNA寡核苷酸碱基序列分析鉴定菌株。【结果】筛选得到的两     

降解尿酸乳酸菌复合菌系的筛选与菌株组合提升降解效果

【背景】高尿酸症由血液中尿酸含量明显升高而导致,利用乳酸菌对人体的益生作用缓解高尿酸血症越来越受到关注。【目的】获得具有降解尿酸能力的乳酸菌复合菌系与纯培养菌株。【方法】以泡菜为样品来源,以尿酸为底物,采用MRS培养基筛选降解尿酸的乳酸菌复合菌系,通过高效液相色谱法测定复合菌系对尿酸的降解能力。【结果】得到一组乳酸菌复合菌系,当培养温度为37 °C、pH值为6.20、静置培养72 h后复合菌系对尿酸的降解率为12.08%;通过优化培养条件,当该菌系在以牛肉膏为单一氮源、初始pH值为5.00、温度为35 °C的条件下培养72 h,尿酸降解率上升至17.19%,降解率比优化前提高了42.3%;从该菌系中分离出两株具有尿酸降解能力的菌株UA-1与UA-2,它们的尿酸降解率分别为10.85%和8.65%;通过形态学观察和16S rRNA基因序列分析,经鉴定两株菌均为布氏乳杆菌(Lactobacillus buchneri)。将两株单菌组合降解尿酸试验发现,UA-1与UA-2比例为2:1的尿酸降解率为20.2%,比原复合菌系的降解能力提高了67.22%。【结论】研究证明了乳酸菌复合菌系对尿酸的降解能力优于单个菌株,为后续利用乳酸菌复合菌系应用提供了数据支持。     

Barriers to application of genetically modified lactic acid bacteria

To increase the acceptability of food products containing genetically modified microorganisms it is necessary to provide in an early stage to the consumers that the product is safe and that the product provide a clear benefit to the consumer. To comply with the first requirement a systematic approach to analyze the probability that genetically modified lactic acid bacteria will transform other inhabitants of the gastro-intestinal (G/I) tract or that these lactic acid bacteria will pick up genetic information of these inhabitants has been proposed and worked out to some degree. From this analysis it is clear that reliable data are still missing to carry out complete risk assessment. However, on the basis of present knowledge, lactic acid bacteria containing conjugative plasmids should be avoided. Various studies show that consumers in developed countries will accept these products when they offer to them health or taste benefits or a better keepability. For the developing countries the biggest challenge for scientists is most likely to make indigenous fermented food products with strongly improved microbiological stability due to broad spectra bacteriocins produced by lactic acid bacteria. Moreover, these lactic acid bacteria may contribute to health.     

一株产共轭亚油酸乳酸菌的鉴定及其特性

从酸菜汁中分离筛选到一株产共轭亚油酸(CLA)能力较高的乳酸菌。经鉴定,确定为植物乳杆菌Lactobacliius plantarum。微氧条件可提高CLA的产量,催化亚油酸(LA)生成CLA的酶受着LA的诱导。37℃对细胞生长和CLA生成最为有利。对数生长期为6～12h,18h后进入稳定期。在14～22h,CLA生成量快速增加,24h时达到最高值。该菌的培养物经萃取、甲酯化后,进行了气相色谱分离,生成的CLA产物为c9/t9,c11-CLA和t10,c12-CLA异构体的混合物。     

Genomic organization of lactic acid bacteria

Current knowledge of the genomes of the lactic acid bacteria, Lactococcus lactis and Streptococcus thermophilus, and members of the genera Lactobacillus, Leuconostoc, Pediococcus and Carnobacterium is reviewed. The genomes contain a chromosome within the size range of 1.8 to 3.4 Mbp. Plasmids are common in Lactococcus lactis (most strains carry 4–7 different plasmids), some of the lactobacilli and pediococci, but they are not frequently present in S. thermophilus, Lactobacillus delbrueckii subsp. bulgaricus or the intestinal lactobacilli. Five IS elements have been found in L. lactis and most strains carry multiple copies of at least two of them; some strains also carry a 68-kbp conjugative transposon. IS elements have been found in the genera Lactobacillus and Leuconostoc, but not in S. thermophilus. Prophages are also a normal component of the L. lactis genome and lysogeny is common in the lactobacilli, however it appears to be rare in S. thermophilus. Physical and genetic maps for two L. lactis subsp. lactis strains, two L. lactis subsp. cremoris strains and S. thermophilus A054 have been constructed and each reveals the presence of six rrn operons clustered in less than 40% of the chromosome. The L. lactis subsp. cremoris MG1363 map contains 115 genetic loci and the S. thermophilus map has 35. The maps indicate significant plasticity in the L. lactis subsp. cremoris chromosome in the form of a number of inversions and translocations. The cause(s) of these rearrangements is (are) not known. A number of potentially powerful genetic tools designed to analyse the L. lactis genome have been constructed in recent years. These tools enable gene inactivation, gene replacement and gene recovery experiments to be readily carried out with this organism, and potentially with other lactic acid bacteria and Gram-positive bacteria. Integration vectors based on temperate phage attB sites and the random insertion of IS elements have also been developed for L. lactis and the intestinal lactobacilli. In addition, a L. lactis sex factor that mobilizes the chromosome in a manner reminiscent to that seen with Escherichia coli Hfr strains has been discovered and characterized. With the availability of this new technology, research into the genome of the lactic acid bacteria is poised to undertake a period of extremely rapid information accrual.