The <Emphasis Type="Italic">dnaK</Emphasis> gene as a molecular marker for the classification and discrimination of the <Emphasis Type="Italic">Lactobacillus casei</Emphasis> group

It is hard to accurately identify specific species of the Lactobacillus casei group using phenotypic techniques alone. Some strains of this species group are considered to be probiotic and are widely applied in the food industry. In this study, we compared the use of two phylogenetic markers, the 16S rRNA and dnaK genes, for species discrimination of the members of the L. casei group using sequencing and RFLP. The results showed that L. casei, Lactobacillus paracasei, Lactobacillus zeae and Lactobacillus rhamnosus could be clearly distinguished based on the dnaK gene. The average sequence similarity for the dnaK gene (87.8%) among type strains was significantly less than that of the 16S rRNA sequence (99.1%). Therefore, the dnaK gene can be proposed as an additional molecular phylogenetic marker for L. casei that provides higher resolution than 16S rRNA. Species-specific RFLP profiles of the Lactobacillus strains were obtained with the enzyme ApoI. Our data indicate that the phylogenetic relationships between these strains are easily resolved using sequencing of the dnaK gene or RFLP assays.     

Species-specific FISH analysis of cecal microflora in rats administered with lactic acid bacteria

Summary We investigated the effects of lactic acid bacterial (LAB) cells in rats using fluorescence in situ hybridization (FISH) targeting 16S rRNA to identify the cecal microbial community. We designed a novel species-specific 16S rDNA probe to detect Lactobacillus rhamnosus (Lrham454). Subtractive technique using the LAC722 probe (Sakai et al. 2004 Journal of Bioscience and Bioengineering 98, 48) under different hybridization stringency (LAC722(L-H)) was applied to identify Lactococcus lactis. We also applied Lplan447 and LAC722(L) to detect Lactobacillus plantarum and a wide range of LAB (total LAB), respectively. We optimized the hybridization and washing conditions and then quantified L. rhamnosus, L. plantarum, and L. lactis cells in rat cecal contents. We monitored increases in individual bacterial populations and in total LAB caused by the administration of the corresponding LAB cells. Growth, food efficiency and internal disorders did not significantly differ among the rats administered with LABs. Rats administered with polydextrose (POL) developed diarrhea, which decreased the total numbers of cecal bacteria, whereas the simultaneous administration of POL and L. rhamnosus KY-3 eased this symptom, and recovered the numbers of total LAB and of L. rhamnosus.     

Identification and characterization of the dominant lactic acid bacteria isolated from traditional fermented milk in Mongolia

Five samples of Airag and 20 of Tarag (both in Mongolia) were collected from scattered households. One hundred strains of lactic acid bacteria (LAB) were isolated and identified from these samples according to phenotypic characterization and 16S rRNA gene sequence analysis. Eighty-five isolates belonged to the genus Lactobacillus, 15 being classified as coccoid LAB. All isolates belonged to 5 genera and 11 to different species and subspecies. Lactobacillus (Lb.) helveticus was predominant population in Airag samples, Lb. fermentum and Lb. helveticus were the major LAB microflora in Tarag.     

Rapid Identification of <Emphasis Type="Italic">Lactobacillus</Emphasis><Emphasis Type="Italic">acidophilus</Emphasis> by Restriction Analysis of the 16S–23S rRNA Intergenic Spacer Region and Flanking 23S rRNA Gene

A rapid molecular approach was developed for the initial identification of Lactobacillus acidophilus strains which are difficult to identify using a single biochemical test. The 16S–23S rRNA intergenic spacer regions and flanking 23S rRNA genes of 19 strains of lactobacilli were amplified and the nucleotide sequences and restriction site polymorphisms were analyzed. AluI was the most useful of the restriction enzymes analyzed and produced reproducible digestion profiles in the L. helveticus, L. plantarum, and L. casei groups, as well as in L. acidophilus. This restriction fragment length polymorphism method may be useful for the identification of L. acidophilus strains in dairy products.     

Genetic diversity of the genus <Emphasis Type="Italic">Lactobacillus</Emphasis> bacteria from the human gastrointestinal microbiome

The species and strain genetic diversity of bacterial cultures belonging to the genus Lactobacillus, which were isolated from the gastrointestinal microbiome of the human population living in the former Soviet Union in the years 1960–1980, was studied. The bacteria demonstrated probiotic characteristics. Phylogenetic analysis of sequences of the gene coding for 16S rRNA detected earlier by us, showed that the gene found in bacteria isolated from the intestinal content of healthy adultsand represented by species L. plantarum, L. helveticus, L. casei/paracasei, L. rhamnosus, and L. fermentum has high homology (97–100%) with this gene in type representatives of the species. The genotypic and strain diversity of cultures was studied using RAPD-PCR and nonspecific primers. This method with the use of the ERIC-1 primer gave reliable and reproducible results as compared that using with M13 and MSP primers and allowed the identification of examined bacteria belonging to the genus Lactobacillus at the level of species and certification at the strain level.     

Supplementation of the Diet with High-Viscosity Beta-Glucan Results in Enrichment for Lactobacilli in the Rat Cecum

BBn (BioBreeding) rats were fed casein-based diets supplemented with barley flour, oatmeal flour, cellulose, or barley β-glucans of high [HV] or low viscosity [LV] in order to measure the prebiotic effects of these different sources of dietary fiber. The dietary impact on the composition of the cecal microbiota was determined by the generation of denaturing gradient gel electrophoresis (DGGE) profiles of PCR-amplified 16S rRNA gene sequences. The DGGE profiles produced from the cecal microbiota of rats within each dietary group were similar, but consensus profiles generated from pooled bacterial DNAs showed differences between rat groups. Animals fed HV glucans (HV-fed rats) had DGGE consensus profiles that were 30% dissimilar from those of the other rat groups. A 16S rRNA gene fragment that was more conspicuous in the profiles of HV-fed animals than in those of cellulose-fed rats had sequence identity with Lactobacillus acidophilus. Measurements of L. acidophilus rRNA abundance (DNA-RNA hybridization), the preparation of cloned 16S rRNA gene libraries, and the enumeration of Lactobacillus cells (fluorescent in situ hybridization) showed that lactobacilli formed a greater proportion of the cecal microbiota in HV-fed rats. In vitro experiments confirmed that some lactobacilli utilize oligosaccharides (degree of polymerization, 3 or 4) present in β-glucan hydrolysates. The results of this study have relevance to the use of purified β-glucan products as dietary supplements for human consumption.     

<Emphasis Type="Italic">Lactobacillus rhamnosus</Emphasis> JCM 2771: Impact on Metabolism of Isoflavonoids in the Fecal Flora from a Male Equol Producer

Many beneficial effects of probiotics have been reported; however, few have focussed on the effects of Lactobacillus, a probiotic, on the bioconversion of isoflavonoids. We hypothesized that Lactobacillus rhamnosus will modify the metabolism of isoflavone. In an in vitro incubation, L. rhamnosus JCM 2771 produced daidzein from daidzin along with genistein. However, daidzin and genistein were not detected in the incubation solution of daidzein with L. rhamnosus. In the fecal suspension from a male equol producer with daidzein, equol was detected in the presence of a low or high concentration of L. rhamnosus. In the fecal incubation with daidzin, the equol concentration increased with an increasing concentration of L. rhamnosus JCM 2771. L. rhamnosus affected the equol production in the in vitro incubation of daidzein with fecal flora from a male equol producer. We demonstrated for the first time that L. rhamnosus JCM 2771 could produce genistein from daidzin and affect the equol production of fecal flora from a male equol producer in vitro.     

Molecular Identification and Typing of Putative Probiotic Indigenous Lactobacillus plantarum Strain Lp91 of Human Origin by Specific Primed-PCR Assays

In the present scenario, it is now well documented that probiotics confer health benefits to the host and the purported probiotic effects are highly strain specific. Hence, accurate genotypic identification is extremely important to link the strain to the specific health effect. With this aim, specific primed-PCR assays were developed and explored for the molecular identification and typing of a putative indigenous probiotic isolate Lp91 of human faecal origin. PCR with specific primers targeting 23S rRNA gene of genus Lactobacillus and 16S rRNA gene of species L. plantarum resulted positive for Lp91. In addition, BLAST analysis of 16S rRNA gene sequence of Lp91 and multiple sequence alignment of 16S rRNA gene variable (V2-V3) regions along with the reference sequences revealed it as L. plantarum with a sequence identity of more than 99%. Furthermore, resolution of 16S rRNA gene sequences was sufficient to infer a phylogenetic relationship amongst Lactobacillus species. In order to determine strain-level variations, randomly amplified polymorphic DNA (RAPD) banding profiles of Lp91 obtained with OPAA-01, OPAP-01 and OPBB-01 primers were compared with those of reference strains of Lactobacillus spp., and Lp91 could be delineated as a distinct strain. Apart from this, presence of probiotic markers viz. bile salt hydrolase (bsh) and collagen-binding protein (cbp) encoding genes in Lp91 genome could be attributed to its exploitation as a potential probiotic adjunct in the development of indigenous functional foods. Lactobacillus isolates/or strains from the gastrointestinal system, fermented products and other environmental niches could be identified and characterized by employing the PCR methods developed in this study; they are rapid, reproducible and more accurate than the conventional methods based on the fermentation profiles.     

The microbiology of Bandji,palm wine of Borassus akeassii from Burkina Faso: identification and genotypic diversity of yeasts,lactic acid and acetic acid bacteria

Aim

To investigate physicochemical characteristics and especially genotypic diversity of the main culturable micro‐organisms involved in fermentation of sap from Borassus akeassii, a newly identified palm tree from West Africa.

Methods and Results

Physicochemical characterization was performed using conventional methods. Identification of micro‐organisms included phenotyping and sequencing of: 26S rRNA gene for yeasts, 16S rRNA and gyrB genes for lactic acid bacteria (LAB) and acetic acid bacteria (AAB). Interspecies and intraspecies genotypic diversities of the micro‐organisms were screened respectively by amplification of the ITS1‐5.8S rDNA‐ITS2/16S‐23S rDNA ITS regions and repetitive sequence‐based PCR (rep‐PCR). The physicochemical characteristics of samples were: pH: 3·48–4·12, titratable acidity: 1·67–3·50 mg KOH g^?1, acetic acid: 0·16–0·37%, alcohol content: 0·30–2·73%, sugars (degrees Brix): 2·70–8·50. Yeast included mainly Saccharomyces cerevisiae and species of the genera Arthroascus, Issatchenkia, Candida, Trichosporon, Hanseniaspora, Kodamaea, Schizosaccharomyces, Trigonopsis and Galactomyces. Lactobacillus plantarum was the predominant LAB species. Three other species of Lactobacillus were also identified as well as isolates of Leuconostoc mesenteroides, Fructobacillus durionis and Streptococcus mitis. Acetic acid bacteria included nine species of the genus Acetobacter with Acetobacter indonesiensis as predominant species. In addition, isolates of Gluconobacter oxydans and Gluconacetobacter saccharivorans were also identified. Intraspecies diversity was observed for some species of micro‐organisms including four genotypes for Acet. indonesiensis, three for Candida tropicalis and Lactobacillus fermentum and two each for S. cerevisiae, Trichosporon asahii, Candida pararugosa and Acetobacter tropicalis.

Conclusion

fermentation of palm sap from B. akeassii involved multi‐yeast‐LAB‐AAB cultures at genus, species and intraspecies level.

Significance and Impact of the Study

First study describing microbiological and physicochemical characteristics of palm wine from B. akeassii. Genotypic diversity of palm wine LAB and AAB not reported before is demonstrated and this constitutes valuable information for better understanding of the fermentation which can be used to improve the product quality and develop added value by‐products.     

Rapid discrimination and classification of the <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> group based on a partial <Emphasis Type="Italic">dnaK</Emphasis> sequence and DNA fingerprinting techniques

The Lactobacillus plantarum group comprises five very closely related species. Some species of this group are considered to be probiotic and widely applied in the food industry. In this study, we compared the use of two different molecular markers, the 16S rRNA and dnaK gene, for discriminating phylogenetic relationships amongst L. plantarum strains using sequencing and DNA fingerprinting. The average sequence similarity for the dnaK gene (89.2%) among five type strains was significantly less than that for the 16S rRNA (99.4%). This result demonstrates that the dnaK gene sequence provided higher resolution than the 16S rRNA and suggests that the dnaK could be used as an additional phylogenetic marker for L. plantarum. Species-specific profiles of the Lactobacillus strains were obtained with RAPD and RFLP methods. Our data indicate that phylogenetic relationships between these strains are easily resolved using sequencing of the dnaK gene or DNA fingerprinting assays.     

A comparative study of the effect of probiotics on cariogenic biofilm model for preventing dental caries

Dental caries is induced by oral biofilm containing Streptococcus mutans. Probiotic bacteria were mainly studied for effect on the gastrointestinal tract and have been known to promote human health. However, the information of probiotics for oral health has been lack yet. In this study, we investigated influence of various probiotics on oral bacteria or cariogenic biofilm and evaluated candidate probiotics for dental caries among them. The antimicrobial activity of the spent culture medium of probiotics for oral streptococci was performed. Probiotics were added during the biofilm formation with salivary bacteria including S. mutans. The oral biofilms were stained with a fluorescent dye and observed using the confocal laser scanning microscope. To count bacteria in the biofilm, the bacteria were plated on MSB and BHI agar plates after disrupting the biofilm and cultivated. Glucosyltransferases (gtfs) expression of S. mutans and integration of lactobacilli into the biofilm were evaluated by real-time RT-PCR. Among probiotics, Lactobacillus species strongly inhibited growth of oral streptococci. Moreover, Lactobacillus species strongly inhibited formation of cariogenic biofilm model. The expression of gtfs was significantly reduced by Lactobacillus rhamnosus. The integration of L. rhamnosus into the biofilm model did not exhibit. However, L. acidophilus and L casei integrated into the biofilm model. These results suggest that L. rhamnosus may inhibit oral biofilm formation by decreasing glucan production of S. mutans and antibacterial activity and did not integrate into oral biofilm, which can be a candidate for caries prevention strategy.     

Identification of Lactobacillus Isolates from the Gastrointestinal Tract, Silage, and Yoghurt by 16S-23S rRNA Gene Intergenic Spacer Region Sequence Comparisons

Lactobacillus isolates were identified by PCR amplification and sequencing of the region between the 16S and 23S rRNA genes (spacer region). The sequences obtained from the isolates were compared to those of reference strains held in GenBank. A similarity of 97.5% or greater was considered to provide identification. To check the reliability of the method, the V2-V3 region of the 16S rRNA gene was amplified and sequenced in the case of isolates whose spacer region sequences were less than 99% similar to that of a reference strain. Confirmation of identity was obtained in all instances. Spacer region sequencing provided rapid and accurate identification of Lactobacillus isolates obtained from gastrointestinal, yoghurt, and silage samples. It had an advantage over 16S V2-V3 sequence comparisons because it distinguished between isolates of Lactobacillus casei and Lactobacillus rhamnosus.     

Highly Heterogeneous Probiotic Lactobacillus Species in Healthy Iranians with Low Functional Activities

Background

Lactic acid bacteria (LAB) have been considered as potentially probiotic organisms due to their potential human health properties. This study aimed to evaluate both in vitro and in vivo, the potential probiotic properties of Lactobacillus species isolated from fecal samples of healthy humans in Iran.

Methods and Results

A total of 470 LAB were initially isolated from 53 healthy individual and characterized to species level. Of these, 88 (86%) were Lactobacillus species. Biochemical and genetic fingerprinting with Phene-Plate system (PhP-LB) and RAPD-PCR showed that the isolates were highly diverse consisted of 67(76.1%) and 75 (85.2%) single types (STs) and a diversity indices of 0.994 and 0.997, respectively. These strains were tested for production of adhesion to Caco-2 cells, antibacterial activity, production of B12, anti-proliferative effect and interleukin-8 induction on gut epithelial cell lines and antibiotic resistance against 9 commonly used antibiotics. Strains showing the characteristics consistent with probiotic strains, were further tested for their anti-inflammatory effect in mouse colitis model. Only one L. brevis; one L. rhamnosus and two L. plantarum were shown to have significant probiotic properties. These strains showed shortening the length of colon compared to dextran sulfate sodium and disease activity index (DAI) was also significantly reduced in mouse.

Conclusion

Low number of LAB with potential probiotic activity as well as high diversity of lactobacilli species was evident in Iranian population. It also suggest that specific strains of L. plantarum, L. brevis and L. rhamnosus with anti-inflammatory effect in mouse model of colitis could be used as a potential probiotic candidate in inflammatory bowel disease to decrease the disease activity index.     

Processing Environment and Ingredients Are Both Sources of Leuconostoc gelidum,Which Emerges as a Major Spoiler in Ready-To-Eat Meals

Mesophilic and psychrotrophic organism viable counts, as well as high-throughput 16S rRNA gene-based pyrosequencing, were performed with the aim of elucidating the origin of psychrotrophic lactic acid bacteria (LAB) in a ready-to-eat (RTE) meal manufacturing plant. The microbial counts of the products at the end of the shelf life were greatly underestimated when mesophilic incubation was implemented due to overlooked, psychrotrophic members of the LAB. Pseudomonas spp., Enterobacteriaceae, Streptococcaceae, and Lactobacillus spp. constituted the most widespread operational taxonomic units (OTUs), whereas Leuconostoc gelidum was detected as a minor member of the indigenous microbiota of the food ingredients and microbial community of the processing environment, albeit it colonized samples at almost every sampling point on the premises. However, L. gelidum became the most predominant microbe at the end of the shelf life. The ability of L. gelidum to outgrow notorious, spoilage-related taxa like Pseudomonas, Brochothrix, and Lactobacillus underpins its high growth dynamics and severe spoilage character under refrigeration temperatures. The use of predicted metagenomes was useful for observation of putative gene repertoires in the samples analyzed in this study. The end products grouped in clusters characterized by gene profiles related to carbohydrate depletion presumably associated with a fast energy yield, a finding which is consistent with the fastidious nature of highly competitive LAB that dominated at the end of the shelf life. The present study showcases the detrimental impact of contamination with psychrotrophic LAB on the shelf life of packaged and cold-stored foodstuffs and the long-term quality implications for production batches once resident microbiota are established in the processing environment.     

Lactobacillus Strain Diversity Based on Partial hsp60 Gene Sequences and Design of PCR-Restriction Fragment Length Polymorphism Assays for Species Identification and Differentiation

A phylogenetic tree showing diversities among 116 partial (499-bp) Lactobacillus hsp60 (groEL, encoding a 60-kDa heat shock protein) nucleotide sequences was obtained and compared to those previously described for 16S rRNA and tuf gene sequences. The topology of the tree produced in this study showed a Lactobacillus species distribution similar, but not identical, to those previously reported. However, according to the most recent systematic studies, a clear differentiation of 43 single-species clusters was detected/identified among the sequences analyzed. The slightly higher variability of the hsp60 nucleotide sequences than of the 16S rRNA sequences offers better opportunities to design or develop molecular assays allowing identification and differentiation of either distant or very closely related Lactobacillus species. Therefore, our results suggest that hsp60 can be considered an excellent molecular marker for inferring the taxonomy and phylogeny of members of the genus Lactobacillus and that the chosen primers can be used in a simple PCR procedure allowing the direct sequencing of the hsp60 fragments. Moreover, in this study we performed a computer-aided restriction endonuclease analysis of all 499-bp hsp60 partial sequences and we showed that the PCR-restriction fragment length polymorphism (RFLP) patterns obtainable by using both endonucleases AluI and TacI (in separate reactions) can allow identification and differentiation of all 43 Lactobacillus species considered, with the exception of the pair L. plantarum/L. pentosus. However, the latter species can be differentiated by further analysis with Sau3AI or MseI. The hsp60 PCR-RFLP approach was efficiently applied to identify and to differentiate a total of 110 wild Lactobacillus strains (including closely related species, such as L. casei and L. rhamnosus or L. plantarum and L. pentosus) isolated from cheese and dry-fermented sausages.     

Identification of phenotypically and genotypically related <Emphasis Type="Italic">Lactobacillus</Emphasis> strains based on nucleotide sequence analysis of the <Emphasis Type="Italic">groEL,rpoB, rplB</Emphasis>, and <Emphasis Type="Italic">16S rRNA</Emphasis> genes

Sixty-eight cultures of lactic acid bacteria were isolated and identified from national fermented milk drinks (airan, koumiss, kurunga, shubat) home-made in different regions of the Republic of Kazakhstan and the Buryat Republic of Russia. The cultures of lactic acid bacteria of the genus Lactobacillus were identified as L. paracasei and L. rhamnosus related to the L. casei group and as L. brevis, L. buchneri, L. diolivorans, and L. parabuchneri (the L. buchneri group) using the classical microbiological methods and on the basis of the 16S rRNA gene sequence analysis. The polymorphism of the nucleotide sequences of the genes groEL, rpoB, and rplB encoding specific proteins was studied for intraspecific differentiation of the lactobacilli. The analysis of these genes allowed a more accurate identification of the lactobacilli that are genetically and phenotypically related to the L. casei group as L. paracasei subsp. paracasei and L. paracasei subsp. tolerans. The gene nucleotide sequences of all the genotyped strains were deposited in the GenBank database.     

Molecular Cloning, Expression of minD Gene from Lactobacillus acidophilus VTCC-B-871 and Analyses to Identify Lactobacillus rhamnosus PN04 from Vietnam Hottuynia cordata Thunb.

The minD gene encoding an inhibitor cell division MinD homolog from Lactobacillus acidophilus VTCC-B-871 was cloned. We showed that there were 97 % homology between minD genes of L. acidophilus VTCC-B-871 and Lactobacillus rhamnosus GG and Lactobacillus rhamnosus Lc705. Based on the analysis of the DNA sequence data from the L. rhamnosus genome project and sequenced minD gene of L. acidophilus VTCC-B-871, a pair of primers was designed to identified the different minD genes from L. acidophilus ATCC 4356, L. rhamnosus ATCC 11443. Besides, the polymerase chain reaction product of minD gene was also obtained in L. rhamnosus PN04, a strain was isolated from Vietnamese Hottuynia cordata Thunb. In addition, we performed a phylogenetic analysis of the deduced amino acid sequence of MinD homologs from L. acidophilus VTCC-B-871 with the other strains and compared the predicted three-dimension structure of L. acidophilus VTCC-B-871 MinD with Escherichia coli MinD, there are similarity that showed evolution of these strains. The overexpression of L. acidophilus VTCC-B-871 MinD in E. coli led to cell filamentation in IPTG and morphology changes in different sugar stresses, interestingly. The present study is the first report characterizing the Lactobacilus MinD homolog that will be useful in probiotic field.     

Lactobacillus oligofermentans sp. nov., Associated with Spoilage of Modified-Atmosphere-Packaged Poultry Products

Unidentified lactic acid bacterium (LAB) isolates which had mainly been detected in spoiled, marinated, modified atmosphere packaged (MAP) broiler meat products during two previous studies, were identified and analyzed for their phenotypic properties and the capability to produce biogenic amines. To establish the taxonomic position of these isolates, 16S rRNA gene sequence analysis, numerical analysis of ribopatterns, and DNA-DNA hybridization experiments were done. Unexpectedly for a meat-spoilage-associated LAB, the strains utilized glucose very weakly. According to the API 50 CHL test, arabinose and xylose were the only carbohydrates strongly fermented. None of the six strains tested for production of histamine, tyramine, tryptamine, phenylethylamine, putrescine, and cadaverine were able to produce these main meat-associated biogenic amines in vitro. The polyphasic taxonomy approach showed that these strains represent a new Lactobacillus species. The six isolates sequenced for the 16S rRNA encoding genes shared the highest similarity (95.0 to 96.3%) with the sequence of the Lactobacillus durianis type strain. In the phylogenetic tree, these isolates formed a distinct cluster within the Lactobacillus reuteri group, which also includes L. durianis. Numerical analyses of HindIII-EcoRI ribotypes placed all isolates together in a cluster with seven subclusters well separated from the L. reuteri group reference strains. The DNA-DNA hybridization levels between Lactobacillus sp. nov. isolates varied from 67 to 96%, and low hybridization levels (3 to 15%) were obtained with the L. durianis type strain confirming that these isolates belong to the same species different from L. durianis. The name Lactobacillus oligofermentans sp. nov. is proposed, with strain LMG 22743^T (also known as DSM 15707^T or AMKR18^T) as the type strain.