Qiuhua Bao

Diversity of lactic acid bacteria associated with traditional fermented dairy products in Mongolia

Spontaneous milk fermentation has a long history in Mongolia, and beneficial microorganisms have been handed down from one generation to the next for use in fermented dairy products. The objective of this study was to investigate the diversity of lactic acid bacteria (LAB) communities in fermented yak, mare, goat, and cow milk products by analyzing 189 samples collected from 13 different regions in Mongolia. The LAB counts in these samples varied from 3.41 to 9.03 log cfu/mL. Fermented yak and mare milks had almost identical mean numbers of LAB, which were significantly higher than those in fermented goat milk but slightly lower than those in fermented cow milk. In total, 668 isolates were obtained from these samples using de Man, Rogosa, and Sharpe agar and M17 agar. Each isolate was considered to be presumptive LAB based on gram-positive and catalase-negative properties, and was identified at the species level by 16S rRNA gene sequencing, multiplex PCR assay, and restriction fragment length polymorphism analysis. All isolates from Mongolian dairy products were accurately identified as Enterococcus faecalis (1 strain), Enterococcus durans (3 strains), Lactobacillus brevis (3 strains), Lactobacillus buchneri (2 strains), Lactobacillus casei (16 strains), Lactobacillus delbrueckii ssp. bulgaricus (142 strains), Lactobacillus diolivorans (17 strains), Lactobacillus fermentum (42 strains), Lactobacillus helveticus (183 strains), Lactobacillus kefiri (6 strains), Lactobacillus plantarum ssp. plantarum (7 strains), Lactococcus lactis ssp. lactis (7 strains), Leuconostoc lactis (22 strains), Leuconostoc mesenteroides (21 strains), Streptococcus thermophilus (195 strains), and Weissella cibaria (1 strain). The predominant LAB were Strep. thermophilus and Lb. helveticus, which were isolated from all sampling sites. The results demonstrate that traditional fermented dairy products from different regions of Mongolia have complex compositions of LAB species. Such diversity of LAB provides useful information for further studies of probiotic strain selection and starter culture design, with regard to the industrial production of traditional fermented milk.

Selection of potential probiotic lactobacilli for cholesterol-lowering properties and their effect on cholesterol metabolism in rats fed a high-lipid diet

The objectives of this study were to screen probiotic characteristics of lactobacilli isolated from traditionally homemade koumiss products in Xinjiang and Inner Mongolia of China, and to determine and compare the effect of the Lactobacillus strains in vivo on lipid metabolism in rats fed with a high-lipid diet. Three out of 68 strains of Lactobacillus isolated from the koumiss were screened for bile-salt resistance, acid tolerance, and different hypocholesterolemic properties. The cholesterol-lowering effects of the 3 screened strains were estimated in rats fed a high-lipid diet by determination of serum lipids, liver, and fecal cholesterol, fecal total bile acids, and short-chain fatty acids. After a 4-wk feeding period, in comparison with the control group, the groups LIP-1 and MG9-2 had a significant reduction in serum total cholesterol, triglyceride and low-density lipoprotein cholesterol, and significantly increased high-density lipoprotein, the group E7301 had similar effects on serum lipids, but the change was not significant. The groups LIP-1, MG9-2 and E7301 had a significant reduction in liver cholesterol content and an increase in fecal cholesterol content compared with the control group. Total bile acid excretion was significantly higher in rats fed MG9-2 than the other groups. Rats fed diets containing lactic acid bacteria strains had significantly higher propionic acid and butyric acid concentrations in the feces compared with the control. Results indicated that the 3 screened Lactobacillus strains were able to lower cholesterol in vitro, and reduce cholesterol effectively in vivo. The mechanisms behind the hypocholesterolemic effect of 3 strains are likely to be diverse and will need further investigation.

Isolation and identification of lactic acid bacteria from Tarag in Eastern Inner Mongolia of China by 16S rRNA sequences and DGGE analysis

Tarag is a characteristic fermented dairy product with rich microflora (especially lactic acid bacteria), developed by the people of Mongolian nationality in Inner Mongolia of China and Mongolia throughout history. One hundred and ninety-eight samples of Tarag were collected from scattered households in Eastern Inner Mongolia, and total of 790 isolates of lactic acid bacteria (LAB) were isolated by traditional pure culture method. To identify these isolates and analyze their biodiversity, 16S rRNA gene sequences analysis and PCR-DGGE were performed respectively. The results showed that 790 isolates could be classified as 31 species and subspecies. Among these isolates, Lactobacillus helveticus (153 strains, about 19.4%), Lactococcus lactis subsp. lactis (132 strains, about 16.7%) and Lactobacillus casei (106 strains, about 11.0%) were considered as the predominated species in the traditional fermented dairy products (Tarag) in Eastern Inner Mongolia. It was shown that the biodiversity of LAB in Tarag in Inner Mongolia was very abundant, and this traditional fermented dairy product could be considered as valuable resources for LAB isolation and probiotic selection.

Complete nucleotide sequence of plasmid plca36 isolated from Lactobacillus casei Zhang

The complete 36,487 bp sequence of plasmid plca36 from Lactobacillus casei Zhang was determined. Plca36 contains 44 predicted coding regions, and to 23 of them functions could be assigned. For the first time, we identified a relBE toxin-antitoxin (TA) locus in a Lactobacillus genus, perhaps indicating a potential role for plca36 in host survival under extreme nutritional stress. A region encoding a cluster of conjugation genes (tra) was also identified. The cluster showed high similarity and co-linearity with tra regions of pWCFS103 and pMRC01 from Lactobacillus plantarum and Lactococcus lactis, respectively. Comparative gene analysis revealed that plasmids from the genus Lactobacillus may have contributed to the environmental adaptation mainly by providing carbohydrate and amino acid transporters. In addition, two chromosome-encoded relBE systems in Lactobacillus johnsonii and Lactobacillus gasseri were identified.

Investigation of bacterial and fungal diversity in tarag using high-throughput sequencing

This is the first study on the bacterial and fungal community diversity in 17 tarag samples (naturally fermented dairy products) through a metagenomic approach involving high-throughput pyrosequencing. Our results revealed the presence of a total of 47 bacterial and 43 fungal genera in all tarag samples, in which Lactobacillus and Galactomyces were the predominant genera of bacteria and fungi, respectively. The number of some microbial genera, such as Lactococcus, Acetobacter, Saccharomyces, Trichosporon, and Kluyveromyces, among others, was found to vary between different samples. Altogether, our results showed that the microbial flora in different samples may be stratified by geographic region.

Transcriptome analysis of probiotic Lactobacillus casei Zhang during fermentation in soymilk

Lactobacillus casei Zhang is a widely recognized probiotic bacterium, which is being commercially used in China. To study the gene expression dynamics of L. casei Zhang during fermentation in soymilk, a whole genome microarray was used to screen for differentially expressed genes when grown to the lag phase, the late logarithmic phase, and the stationary phase. Comparisons of different transcripts next to each other revealed 162 and 63 significantly induced genes in the late logarithmic phase and stationary phase, of which the expression was at least threefold up-regulated and down-regulated, respectively. Approximately 38.4% of the up-regulated genes were associated with amino acid transport and metabolism notably for histidine and lysine biosynthesis, followed by genes/gene clusters involved in carbohydrate transport and metabolism, lipid transport and metabolism, and inorganic ion transport and metabolism. The analysis results suggest a complex stimulatory effect of soymilk-based ecosystem on the L. casei Zhang growth. On the other hand, it provides the very first insight into the molecular mechanism of L. casei strain for how it will adapt to the protein-rich environment.

Phylogenetic study of Lactobacillus acidophilus group, L. casei group and L. plantarum group based on partial hsp60, pheS and tuf gene sequences

The partial nucleotide sequences encoding elongation factor Tu (tuf gene), 60-kDa heat shock protein (hsp60 gene) and phenylalanyl-tRNA synthase (pheS gene) were determined to assess the suitability as phylogenetic markers for discriminating the closely related species in Lactobacillusacidophilus group, L. casei group and L. plantarum group. A total of 234 lactobacilli were chosen from traditional fermented dairy products that were not exactly assigned to species based on biochemical tests and 16S rRNA gene sequences. The sequencing of partial tuf, hsp60 and pheS gene of all strains was performed, and then, the phylogenetic trees were constructed by neighbor-joining method. Phylogenetic tree revealed three genes provided better resolution of each Lactobacillus species than 16S rDNA, and all of strains were clearly identified as L. casei (63 strains), L. plantarum (58 strains) and L. helveticus (113 strains) by comparison of sequences with the type strains. From our results, the partial sequences of three genes had a higher discriminatory power than 16S rRNA gene sequences and were an alternative molecular tool for the taxonomical analysis of L. casei group, L. plantarum group and L. acidophilus group.

A survey of the bacterial composition of kurut from Tibet using a culture-independent approach.

Kurut (fermented yak milk) made by natural fermentation is a very important dairy food for the local people in Tibet (China). It is important to fully understand the bacterial composition of kurut for quality improvement and industrial production. Because more than 99% of prokaryotes cannot be cultured and identified by methods currently used in taxonomy, we applied a culture-independent approach to explore the microbial biodiversity of this traditional food. In this study, a bacterial 16S rRNA gene clone library, including 460 clones, was constructed using total DNA extracted from 30 samples of kurut. After screening by restriction fragment length polymorphism (RFLP) analysis, 56 operational taxonomic units (OTU) with unique RFLP patterns were obtained. Then, 1 representative sequence of every OTU was sequenced and phylogenetically analyzed. The representative phylotypes were affiliated with 5 groups, including Lactococcus lactis ssp. lactis, Lactobacillus helveticus, Streptococcus thermophilus, Lactobacillus delbrueckii ssp. bulgaricus, and Acetobacter. In addition, nearly one-third of the representative clones (132 clones) had low similarity to species in GenBank (<97%), and these phylotypes were regarded as unknown bacteria. The characteristics of kurut are determined not only by lactic acid bacteria well known by the culture-dependent approach but also by bacteria that have not yet been identified.

Multilocus sequence typing of Streptococcus thermophilus from naturally fermented dairy foods in China and Mongolia

BackgroundStreptococcus thermophilus is a major dairy starter used for manufacturing of dairy products. In the present study, we developed a multilocus sequence typing (MLST) scheme for this important food bacterium. Sequences of 10 housekeeping genes (carB, clpX, dnaA, murC, murE, pepN, pepX, pyrG, recA, and rpoB) were obtained for 239 S. thermophilus strains, which were isolated from home-made fermented dairy foods in 18 different regions of Mongolia and China.MethodsAll 10 genes of S. thermophilus were sequenced, aligned, and defined sequence types (STs) using the BioNumerics Software. The nucleotide diversity was calculated by START v2.0. The population structure, phylogenetic relationships and the role of recombination were inferred using ClonalFrame v1.2, SplitsTree 4.0 and Structure v2.3.ResultsThe 239 S. thermophilus isolates and 18 reference strains could be assigned into 119 different STs, which could be further separated into 16 clonal complexes (CCs) and 38 singletons. Among the 10 loci, a total of 132 polymorphic sites were detected. The standardized index of association (IAS = 0.0916), split-decomposition and ρ/θ (relative frequency of occurrence of recombination and mutation) and r/m value (relative impact of recombination and mutation in the diversification) confirms that recombination may have occurred, but it occurred at a low frequency in these 10 loci. Phylogenetic trees indicated that there were five lineages in the S. thermophilus isolates used in our study. MSTree and ClonalFrame tree analyses suggest that the evolution of S. thermophilus isolates have little relationship with geographic locality, but revealed no association with the types of fermented dairy product. Phylogenetic analysis of 36 whole genome strains (18 S. thermophilus, 2 S. vestibularis and 16 S. salivarius strains) indicated that our MLST scheme could clearly separate three closely related species within the salivarius group and is suitable for analyzing the population structure of the other two species in the salivarius group.ConclusionsOur newly developed MLST scheme improved the understanding on the genetic diversity and population structure of the S. thermophilus, as well as provided useful information for further studies on the genotyping and evolutionary research for S. thermophilus strains with global diversity.

Multilocus sequence typing of Lactobacillus casei isolates from naturally fermented foods in China and Mongolia

Lactobacillus casei is a lactic acid bacterium used in manufacturing of many fermented food products. To investigate the genetic diversity and population biology of this food-related bacterium, 224 Lb. casei isolates and 5 reference isolates were examined by multilocus sequence typing (MLST). Among them, 224 Lb. casei isolates were isolated from homemade fermented foods, including naturally fermented dairy products, acidic gruel, and Sichuan pickles from 38 different regions in China and Mongolia. The MLST scheme was developed based on the analysis of 10 selected housekeeping genes (carB, clpX, dnaA, groEL, murE, pyrG, pheS, recA, rpoC, and uvrC). All 229 isolates could be allocated to 171 unique sequence types, including 25 clonal complexes and 71 singletons. The high index of association value (1.3524) and standardized index of association value (0.1503) indicate the formation of an underlying clonal population by all the isolates. However, split-decomposition, relative frequency of occurrence of recombination and mutation, and relative effect of recombination and mutation in the diversification values confirm that recombination may have occurred, and were more frequent than mutation during the evolution of Lb. casei. Results from Structure analyses (version 2.3; http://pritch.bsd.uchicago.edu/structure.html) demonstrated that there were 5 lineages in the Lb. casei isolates, and the overall relatedness built by minimum spanning tree showed no clear relationship between the clonal complexes with either the isolation sources or sampling locations of the isolates. Our newly developed MLST scheme of Lb. casei was an easy and valuable tool that, together with the construction of an MLST database, will contribute to further detailed studies on the evolution and population genetics of Lb. casei from various niches.