Search

Streptococcus (S.) thermophilus, an indispensable dairy starter, has been used in autochthonous as well as industrial milk fermentation. However, the genetic architecture underlying S. thermophilus traits and phenotypes is largely unknown. Here, we sequenced 185 S. thermophilus strains, isolated from natural fermented dairy products of China and Mongolia and used comparative genomic and genome wide association study to provide novel point for genetic architecture underlying its traits and phenotypes. Genome analysis of S. thermophilus showed association of phylogeny with environmental and phenotypic features and revealed clades with high acid production potential or with substantial genome decay. A few S. thermophilus isolated from areas with high chloramphenicol emissions had a chloramphenicol-resistant gene CatB8. Most importantly, we defined a growth score and identified a missense mutation G1118698T located at the gene AcnA that were both predictive of acidification capability of S. thermophilus. Our findings provide novel insight in S. thermophilus genetic traits, antibiotic resistant and predictive of acidification capability which both may had huge help in culture starter screening.

Goat milk powder is a nutritious and easy-to-store product that is highly favored by consumers. However, the presence of contaminating bacteria and their metabolites may significantly affect the flavor, solubility, shelf life, and safety of the product. To comprehensively and accurately understand the sanitary conditions in the goat milk powder production process and potential threats from bacterial contamination, a combination of Pacific Biosciences single molecule real-time sequencing and droplet digital PCR was used to evaluate bacterial contamination in seven goat milk powder samples from three dairies. Ten phyla, 119 genera, and 249 bacterial species were identified. Bacillus, Paenibacillus, Lactococcus, and Cronobacter were the primary genera. Bacillus cereus, Lactococcus lactis, Alkaliphilus oremlandii, and Cronobacter sakazakii were the dominant species. With droplet digital PCR, 6.3 × 104 copies per g of Bacillus cereus and 1.0 × 104 copies per g of Cronobacter spp. were quantified, whic...

The gut microbes of dairy cows are strongly associated with their health, but the relationship between milk production and the intestinal microbiota has seldom been studied. Thus, we explored the diversity of the intestinal microbiota during peak lactation of dairy cows. The intestinal microbiota of nine dairy cows at peak lactation was evaluated using the Pacific Biosciences single-molecule real-time (PacBio SMRT) sequencing approach. A total of 32,670 high-quality 16S rRNA gene sequences were obtained, belonging to 12 phyla, 59 families, 107 genera, and 162 species. Firmicutes (83%) were the dominant phylum, while Bacteroides (6.16%) was the dominant genus. All samples showed a high microbial diversity, with numerous genera of short chain fatty acid (SCFA)-producers. The proportion of SCFA producers was relatively high in relation to the identified core intestinal microbiota. Moreover, the predicted functional metagenome was heavily involved in energy metabolism. This study provided novel insights into the link between the dairy cow gut microbiota and milk production.

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.

Koumiss is a traditional fermented dairy product and a good source for isolating novel bacteria with biotechnology potential. In the present study, we applied the single-cell amplification technique in the metagenomics analysis of koumiss. This approach aimed at detecting the low-abundant bacteria in the koumiss. Briefly, each sample was first serially diluted until reaching the level of approximately 100 cells. Then, three diluted bacterial suspensions were randomly picked for further study. By analyzing 30 diluted koumiss suspensions, a total of 24 bacterial species were identified. In addition to the previously reported koumiss-associated species, such as Lactobacillus (L.) helveticus. Lactococcus lactis. L. buchneri, L. kefiranofaciens, and Acetobacter pasteurianus, we successfully detected three low-abundant taxa in the samples, namely L. otakiensis. Streptococcus macedonicus, and Ruminococcus torques. The functional koumiss metagenomes carried putative genes that relate to lactose metabolism and synthesis of typical flavor compounds. Our study would encourage the use of modern metagenomics to discover novel species of bacteria that could be useful in food industries.

Background In Kazakhstan, traditional artisanal cheeses have a long history and are widely consumed. The unique characteristics of local artisanal cheeses are almost completely preserved. However, their microbial communities have rarely been reported. The current study firstly generated the Single Molecule, Real-Time (SMRT) sequencing bacterial diversity profiles of 6 traditional artisanal cheese samples of Kazakhstan origin, followed by comparatively analyzed the microbiota composition between the current dataset and those from cheeses originated from Belgium, Russian Republic of Kalmykia (Kalmykia) and Italy. Results Across the Kazakhstan cheese samples, a total of 238 bacterial species belonging to 14 phyla and 140 genera were identified. Lactococcus lactis (28.93%), Lactobacillus helveticus (26.43%), Streptococcus thermophilus (12.18%) and Lactobacillus delbrueckii (12.15%) were the dominant bacterial species for these samples. To further evaluate the cheese bacterial diversity of Kazakhstan cheeses in comparison with those from other geographic origins, 16S rRNA datasets of 36 artisanal cheeses from Belgium, Russian Republic of Kalmykia (Kalmykia) and Italy were retrieved from public databases. The cheese bacterial microbiota communities were largely different across sample origins. By principal coordinate analysis (PCoA) and multivariate analysis of variance (MANOVA), the structure of the Kazakhstan artisanal cheese samples was found to be different from those of the other geographic origins. Furthermore, the redundancy analysis (RDA) identified 16 bacterial OTUs as the key variables responsible for such microbiota structural difference. Conclusion Our results together suggest that the diversity of bacterial communities in different groups is stratified by geographic region. This study does not only provide novel information on the bacterial microbiota of traditional artisanal cheese of Kazakhstan at species level, but also interesting insights into the bacterial diversity of artisanal cheeses of various geographical origins. Electronic supplementary material The online version of this article (doi:10.1186/s12866-016-0911-4) contains supplementary material, which is available to authorized users.

Lactobacillus casei Zhang is a new probiotic strain isolated from koumiss in Inner Mongolia of China, which has been commercially used as starter in the manufacture of dairy products. To study gene expression profile of L. casei Zhang during growth in milk, a whole-genome microarray was used. Compared to L. casei Zhang grown to the late logarithmic phase in milk, 61 genes were significantly up-regulated (>5 fold) in the stationary phase, whereas 26 genes were down-regulated. Collectively, these data showed that the majority of the identified genes were involved in carbohydrate metabolism and energy production, followed by genes involved in nucleotide metabolism, inorganic ion transport, amino acid transport and metabolism, chaperones, etc. This study demonstrates the fundamental effects of cultural conditions on the transcriptome of L. casei Zhang. Moreover, it improves the understanding of growth and survival mechanism of the bacterium during the late stage of milk fermentation.

Fermented soymilk was produced with the single culture of the six probiotic strains, including Lactobacillus casei Zhang, Bifidobacterium animalis ssp. lactis V9, Lactobacillus acidophilus NCFM, Lactobacillus rhamnosus GG, Bifidobacterium animalis Bb12, and Lactobacillus casei Shirota, and their fermentation characteristics were evaluated. The free amino nitrogen of soymilk fermented with the four probiotic strains, L. casei Zhang, B. animalis V9, L. acidophilus NCFM and L. rhamnosus GG, increased greatly. Significant increases in viable counts were observed among the six probiotic strains. All the fermented soymilk had a viable count above 8.69 log CFU/g at the end of fermentation, especially the four probiotic strains of L. casei Zhang, L. acidophilus NCFM, B. animalis Bb12, and L. casei Shirota, which were all over 9.00 log CFU/g. The content of glucose and fructose decreased for all the six probiotic strains after the fermentation, but along with the reduction in the content of stachyose and raffinose only for B. animalis V9, L. rhamnosus GG, and B. animalis Bb12. Our results indicated that the six probiotic strains can grow well and increase the contents of bioactive substances in soymilk, including γ-aminobutyric acid, vitamin B6, and total isoflavone aglycone, and deserves further research on utilization as potential starters for soymilk.

Various traditional fermented yak milk and raw milk foods could be considered as an abundant resource for obtaining novel lactic acid bacteria (LAB) with unique properties. Eighty-eight samples of yak milk products were collected from Gansu Province in China. Three hundred and nineteen strains of LAB isolated from these samples were identified by phenotypic methods, 16S rRNA gene sequence analysis and PCR-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) technology. Among the isolates, one hundred and sixty-four isolates (51.41% of the total) were classified under Lactobacilli, and one hundred and fifty-five (48.59%) belonged to cocci. All the isolates were classified to six genera (Lactobacillus, Lactococcus, Leuconostoc, Streptococcus, Enterococcus and Weissella) and twenty-one species. Lactobacillus helveticus (87 strains), Leuconostoc mesenteroides subsp. mesenteroides (49 strains), Streptococcus thermophilus (39 strains), Lactobacillus casei (31 strains) and Lactococcus lactis subsp. lactis (19 strains) were considered as the predominant populations in the yak milk products. The results showed that there were abundant genus and species LAB existing in yak milk products in Gansu Province in China. The obtained LAB pure cultures may be a valuable source for further starter selection.

Sour congee is a popular food in the western regions of Inner Mongolia in China. It has a complex microbial population, which contributes to its unique flavor and functional properties. In this study, we chose 28 sour congee samples that were collected from different areas in Inner Mongolia for analysis of the microbial community of lactic acid bacteria (LAB) by classical biochemical tests, 16S rRNA gene sequencing, multiplex PCR assay of recA gene and restriction fragment length polymorphism (RFLP) analysis of the tuf gene (encoding elongation factor Tu). The results revealed that all the isolates were identified as Lactobacillus (L.) paracasei (38 strains), L. fermentum (28 strains), L. plantarum (7 strains), L. brevis (4 strains), L. reuteri (2 strains), L. amylolyticus (1 strain), Enterococcus (E.) faecalis (3 strains), E. italicus (2 strains) or Lactococcus lactis subsp. lactis (1 strain). The predominant LAB were L. casei and L. fermentum in sour congee samples. The diversity of LAB derived from sour congee could offer useful information for further research on sour congee, and the results demonstrated that the combination of tuf gene and RFLP patterns can be considered as a useful tool for differentiation of the L. casei group.

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 105to 109colony 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.

A total of thirty-three strains of Bacillus were isolated from sixteen samples of naturally fermented congee in Inner Mongolia of China and identified by 16S rDNA sequence analysis. Probiotic properties including acid, bile tolerance and artificial gastrointestinal juice resistance as well as inhibition on pathogenic bacteria were used for screening of Bacillus. After the preliminary selection, four strains including Bacillus licheniformis IMAUB1002, Bacillus subtilis IMAUB1011, Bacillus amyloliquefaciens IMAUB1014 and Bacillus amyloliquefaciens IMAUB1034 showed high tolerance to simulated gastric juice at pH 2.0 for 3 h with survival rate all above 92%. And then through gastrointestinal transit, survival rates of these four strains were above 90%. Furthermore, Bacillus licheniformis IMAUB1002 performed well in tolerance to bile salt (0.6%) and inhibitory activity to five food-borne pathogens among four strains of Bacillus. The results suggested that Bacillus licheniformis IMAUB1002 should be considered as a potential probiotics. Further study will be focused on evaluation of these porbiotics properties in vivo and clarification of its other functional properties so as to use it in functional foods production in future.

Forty-three samples of kurut, a kind of traditional naturally-fermented yak milk, were collected in Qinghai, China. One hundred and forty-eight lactic acid bacterial (LAB) strains were isolated and identified from the kurut samples according to phenotypic characterization and 16S rRNA gene sequence analysis. Among them, 52 isolates belonged to the Lactobacillus strains. Ninety-six isolates were resolved to coccoid LAB. The results showed that these isolates belonged to five genera and thirteen different species and subspecies. Moreover, Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus were the predominant population in these samples. This paper systematically studied the composition of LAB in kurut, which may provide raw data for further study involving probiotics strain selection and starter culture design for industrialization production of traditional fermented milk in the future.

Among thirtyLactobacillus strains, which were isolated and identified from the home-made koumiss in Mongolia,Lactobacillus helveticus MG2-1 was screened out for the best acid tolerance in artificial gastric juice at pH 3.0 after incubating for 3 h with the survival rate 96.15±4.22%. Furtherin vitro assessment revealed MG2-1 showed high tolerance to the artificial gastrointestinal juice and bile salts. Moreover, other desired probiotic properties, including the ability of sodium taurocholate deconjugation (with the amount of cholic acid liberated 2.95±0.11 μM), cholesterol removal (cholesterol removal rate 51.74±0.04%) and adhesion to Caco-2 cells (the adhesive rate 1.51±0.11%), were also observed. The results highlighted the probiotic potential of MG2-1, which deserved further investigation inin vivo studies to elucidate its health benefits.

Chen X., Yang M., Sun Z., Liu W., Sun T., Meng H., Zhang H. (2009): Molecular cloning and characterisation of alpha subunit of H + -ATPase in Lactobacillus casei Zhang. Czech J. Food Sci., 27: 49-54. Lactic acid bacteria as potential probiotics . H + -ATPase is considered a key gene in several bacteria with the ability of acid tolerance . We cloned and sequenced the full length cDNA of alpha subunit of H + -ATPase gene in Lactobacillus casei Zhang, which had been isolated from traditional home-made koumiss in Inner Mongolia of China. The results showed that the respective cDNA sequence is composed of 1530 nucleotides and codes a putative protein including 509 amino acids. In addition, we also reconstructed the phylogenic trees for H + -ATPase gene based on amino acids sequences of diverse strains of Lactic acid bacteria.

Twenty-nine traditional fermented yak milk (kurut) and 15 traditional fermented Mongolian cow milk (FMCM) samples were collected from three regions of Tibet in China, and their chemical composition and microbiological properties were analyzed. Average levels of the major composition of kurut and FMCM, respectively, were as follows: total solid, 16.91+/-1.30 g/100 g and 12.65+/-1.14 g/100 g; fat, 6.83+/-1.18 g/100 g and 3.92+/-0.92 g/100 g; total protein, 4.93+/-0.36 g/100 g and 3.33+/-0.71 g/100 g; free amino acid, 8.57+/-3.22 mmol/l and 2.23+/-0.62 mmol/l; calcamin, 117.26+/-9.66 mg/100 g and 86.51+/-7.55 mg/100 g; and phosphorus, 109.28+/-7.97 mg/100 g and 78.86+/-6.99 mg/100 g. The average bacteria count of lactic acid bacteria in kurut and FMCM were 7.66+/-0.71 and 8.03+/-0.68 log colony-forming units (cfu)/ml, respectively. Moreover, the average yeast counts of these two samples were 6.48+/-0.81 and 5.92+/-0.52 log cfu/ml, respectively. The result showed that the major nutritional composition content of kurut was significantly higher than that of FMCM, which indicated that kurut can be considered an invaluable fermentation product for human being.

Thirty-two batches of samples of kurut (naturally fermented yak milk) were collected at four locations in Qinghai, China, and their chemical and microbiological composition was analyzed. Average levels of major components were: total solids 14.3 ± 1.52 g/100 ml, fat 5.37 ± 1.04 g/100 ml, total protein 5.44 ± 1.06 g/100 ml, lactose 2.34 ± 0.172 g/100 ml, ash 0.860 ± 0.080 g/100 ml and titratable acidity 158 ± 16.3 °T. Results showed that the kurut had richer nutrients than the yoghurt. The HP (kurut samples from Plateau yaks in the northwest of Qinghai) had better chemical nutrients density than the MH (kurut samples from Huanhu yaks in the south of Qinghai) and LH (kurut samples from Huanhu yaks in the east of Qinghai). Sodium dodecyl sulfate–PAGE and densitometry analysis results demonstrated that kurut was rich in casein, immunoglobulins, serum albumin, β-lactoglobulin, α-lactalbumin, and two unknown fractions in part samples. Microbiological enumeration revealed that the kurut contained larger lactic acid bacteria and yeast than those of the traditional fermented milk referred, with the average counts of lactic acid bacteria of 9.18 ± 0.851 log cfu/ml and the yeast 8.33 ± 0.624 log cfu/ml. The lactic acid bacteria dominated in the HP and LH kurut while the yeast dominated in MH kurut. The kurut was judged nutritional and indicates the latent value for people’s health.

Animal husbandry is a traditional industry with regional characteristic in the Inner Mongolia of China. Recent years, animal breeding has been one of the main pollution sources in this area, followed by domestic sewage and industrial wastewater. The pollution of livestock farm feces may accelerate the development of pathogens and antibiotic resistance genes which pose health risks to humans and animals. In present research, culture-independent molecular ecological methods based on DGGE combined with qPCR were used to investigate the pollution to surrounding environment with different degrees of livestock farm. The cluster analysis of DGGE patterns showed that the livestock farm feces from point pollution source flowed with wastewater discharge has resulted in an impacted range of at least 3000 m, but it did not cause pollution to residential water delivered from upstream of sewage drain outlet. qPCR results revealed that 5 common pathogens (Escherichia coli, Enterococcus, Staphylococcus aureus, Shigella, and Salmonella) presented decreased trend as the sampled distance from point pollution source increased. Also, qPCR assays of 10 common antibiotic resistance genes (tetO, tetL, rpp, rpoB, sul2, sulA, floR, yidY, mphA, and ermC) which cause resistance to tetracycline, rifampicin, fluoroquinolone, quinolone, and erythromycin have been found in the environmental samples. This study clearly indicates the livestock farm discharge pollutants contaminated to the surrounding environment. Our data have provided important information to pollution control in the future.

Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus are key factors in the fermentation process and the final quality of dairy products worldwide. This study was performed to investigate the effects of the proportions of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus isolated from traditionally fermented dairy products in China and Mongolia on the profile of volatile compounds produced in samples. Six proportional combinations (1:1, 1:10, 1:50, 1:100, 1:1000, and 1:10,000) of L. delbrueckii subsp. bulgaricus IMAU20401 to S. thermophilus ND03 were considered, and the volatiles were identified and quantified by solid-phase microextraction and gas chromatography–mass spectrometry (SPME-GC-MS) against an internal standard. In total, 89 volatile flavor compounds, consisting of aldehydes, ketones, acids, alcohols, esters, and aromatic hydrocarbons, were identified. Among these, some key flavor volatile compounds were identified, including acetaldehyde, 3-methylbutanal, acetoin, 2-heptanone, acetic acid, butanoic acid, and 3-methyl-1-butanol. The of L. delbrueckii subsp. bulgaricus IMAU20401 to S. thermophilus ND03 influenced the type and concentration of volatiles produced. In particular, aldehydes and ketones were present at higher concentrations in the 1:1000 treatment combination than in the other combinations. Our findings emphasize the importance of selecting the appropriate proportions of L. delbrueckii subsp. bulgaricus and S. thermophilus for the starter culture in determining the final profile of volatiles and the overall flavor of dairy products.

Lactobacillus helveticus strain H10 was isolated from traditional fermented milk in Tibet, China. We sequenced the whole genome of strain H10 and compared it to the published genome sequence of Lactobacillus helveticus DPC4571.

Lactobacillus delbrueckii subsp. bulgaricus strain ND02 is a Chinese commercial dairy starter used for the manufacture of yoghurt. It was isolated from naturally fermented yak milk in Qinghai, China. Here, we report the main genome features of ND02 and several differences with two other published genomes of Lactobacillus delbrueckii subsp. bulgaricus strains.

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.

Fermented milk is a very delicious and nutritional food distributed in the international markets. Rapid preliminary identification of lactic acid bacteria to the species level is an important issue for the fermentation industry. One hundred seventy-one strains of lactic acid bacteria (LAB) were isolated from twenty-nine kurut samples and fifteen traditional fermented mongolian cattle milk (FMCM) samples in Tibet, China. All of the strains were isolated and divided into bacilli or cocci by phenotype, and then differentiated by restriction fragment length polymorphism (RFLP) analysis using a set of restriction enzymes, AluI, HaeIII, BsmaI, TspRI and HinfI. Restriction pattern analyses indicated that the lactobacilli could be clearly identified at the species level and the LAB cocci at the genera level by the five restriction enzymes. Further studies on 16S rDNA sequences of representative and undefined strains showed that the combination of RFLP analysis of polymerase chain reaction (PCR) for amplifying 16S rDNA and 16S rDNA sequence analysis is rapid, easy to perform, and effective for large-scale preliminary identification of LAB.

Bifidobacterium animalis subsp. lactis strain V9 is a Chinese commercial bifidobacteria with several probiotic functions. It was isolated from a healthy Mongolian child in China. We present here the complete genome sequence of V9 and compare it to 3 other published genome sequences of B. animalis subsp. lactis strains. The result indicates the lack of polymorphism among strains of this subspecies from different continents.

Background Streptococcus 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. Methods All 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. Results The 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. Conclusions Our 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. Electronic supplementary material The online version of this article (doi:10.1186/s12866-015-0551-0) contains supplementary material, which is available to authorized users.