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This study investigated the community composition of lactic acid bacteria (LAB) from yaks' milk (YM) Tibetan yellow cattle milk (TM) and their fermented products from different counties in the Nyingchi Prefecture, Tibet using Pacific Biosciences (PacBio) single-molecule real-time (SMRT) sequencing. Sequencing revealed 26 genera and 94 species from 71 dairy samples; amongst these Lactobacillus delbrueckii (36.17%), Streptococcus thermophilus (19.46%) and Lactococcus lactis (18.33%) were the predominant species. This study also identified the main factors influencing LAB community composition by comparing amongst samples from different locations, from different milk types, and from different altitudes. The LAB communities in YM and TM were more diverse than in fermented yaks' milk (FYM) and fermented Tibetan yellow cattle milk (FTM) samples. Similarly, whether milk was fermented or not accounted for differences in LAB species composition while altitude of the dairy products had very little effect. Milk source and production process were the most likely causes of drastic shifts in microbial community composition. In addition, fermented dairy products were enriched in genes responsible for secondary metabolic pathways that were potentially beneficial for health. Comprehensive descriptions of the microbiota in different dairy products from the Nyingchi Prefecture, Tibet might help elucidate evolutionary and functional relationships amongst bacterial communities in these products.

Bifidobacteria are health-promoting human gut inhabitants, but accurate species-level composition of the gut bifidobacteria and their responses to probiotic intervention have not been fully explored. This was a follow-up work of our previous study, in which 104 volunteers from six different Asiatic regions (Singapore, Indonesia, Xinjiang, Gansu, Inner Mongolia, Mongolia) were recruited. The gut microbiota and their responses towards Lactobacillus casei Zhang (LCZ) intervention were characterized (at days 0, 7, and 14; 14 days after stopping probiotic intake), and region-based differential responses were observed after LCZ intervention. This study further investigated changes in the species-level gut bifidobacteria by PacBio small-molecule real-time sequencing (SMRT) using bifidobacteria-specific primers. Firstly, this study found that Bifidobacterium adolescentis (42.58%) and Bifidobacterium breve (26.34%) were the core species across the six Asiatic regions. Secondly, principal coordinate analysis of probiotic-induced changes in the gut bifidobacterial microbiota (represented by weighted UniFrac distances) grouped the six regions into two clusters, namely northern (Xinjiang, Gansu, Inner Mongolia, and Mongolia) and southern (Singapore, Indonesia) regions. Thirdly, LCZ intervention induced region-based differential responses of gut bifidobacterial microbiota. The relative abundance of Bifidobacterium animalis in subjects from northern but not southern region substantially increased after LCZ intervention. Moreover, LCZ intervention significantly increased the weighted UniFrac distances in the southern but not northern subjects 7 days after LCZ intervention. The gut B. adolescentis correlated significantly and negatively with the weighted UniFrac distances of the baseline gut bifidobacterial microbiota in subjects of northern but not southern region, suggesting a possible homeostatic effect of LCZ on the gut bifidobacterial population of northern but not southern subjects. Collectively, our study found that probiotic-induced responses of the gut bifidobacterial microbiota varied with subjects' geographic origins, and B. adolescentis might play a role in maintaining the overall stability of the gut bifidobacterial population. KEY POINTS: • The core species in the six Asiatic regions are Bifidobacterium adolescentis and Bifidobacterium breve. • The gut bifidobacterial microbiota in people from various geographic origins showed different responses on probiotic administration.

This study aimed to investigate the effects and underlying molecular mechanisms of icariin (ICA) and curcumol on autophagy, ferroptosis, and lipid metabolism in prostate cancer (PCa), in vitro and in vivo. Normal prostate epithelial cells RWPE-1 and PCa cell lines DU145 and PC-3 were treated with ICA and curcumol. Ferrostatin-1 (Fer-1) or 3-MA was added to treat DU145 and PC-3 cells. In addition, we knocked down miR-7. The mechanism of ICA and curcumol in PCa cells after the knockdown of miR-7 was verified by in vitro nude mice tumorigenesis experiments. ICA and curcumol had no significant effect on the viability of RWPE-1 cells, but there was a significant difference between DU145 and PC-3 cells. After treatment with ICA and curcumol, the proliferation of PCa cells was inhibited, apoptosis, reactive oxygen species (ROS) levels, and miR-7 expression were increased. The combined treatment of ICA and curcumol had a more significant effect. ICA and curcumol treatment induced autophagy and ferroptosis in PCa cells, and si-miR-7 reversed the effects of ICA and curcumol on autophagy and ferroptosis. MiR-7 targeted mTOR and regulated the expression of the mTOR/SREBP1 pathway in PCa cells. ICA and curcumol may affect the lipid metabolism of PCa cells by affecting SREBP1. In addition, the effects and mechanisms of ICA and curcumol on autophagy, ferroptosis, and lipid metabolism in PCa cells were verified in vivo. ICA and curcumol synergistically regulated the miR-7/mTOR/SREBP1 pathway to induce autophagy and ferroptosis in PCa cells and affected lipid metabolism.

Ultra-performance liquid chromatography coupled with a quadrupole-time-of-flight mass spectrometryElevated Energy was used to investigate changes in the metabolite profile of brown milk and fermented brown milk produced using Streptococcus thermophilus S10. Samples were analyzed in both positive and negative electron ionization modes. Data were analyzed by multivariate statistical methods for biomarker metabolites that were differentially abundant in brown milk and fermented brown milk. We identified 43 differentially abundant metabolites based on mass spectrophotometry fragmentation patterns. These metabolites included peptides, AA, fatty acids and related metabolites, carbohydrate metabolites, vitamins, and nucleosides. Some of these metabolites are known to alter the sensorial quality of fermented dairy products. Thus, it is likely that some of the currently identified differentially abundant metabolites also contribute to the unique flavor, taste, and aroma of fermented brown milk. The bitterness and astringency of fermented brown milk are likely to be due to some of these peptides, whereas the sweetness and sourness could be a result of changes in carbohydrate levels. No previous study has analyzed metabolomics changes during fermentation of brown milk. Thus, our data are a valuable reference for future development and improvement of fermented brown milk products.

Streptococcus thermophilus is an important bacterium used in the production of fermented dairy products. Yogurt with good flavor is preferred by consumers; thus, variation in flavor-formation characteristics among isolates is attracting attention. Here, acetaldehyde production characteristics of 30 isolates were evaluated in parallel with genotyping and multilocus sequence typing of key functional genes involved in acetaldehyde production. The results showed that isolates could be divided into 3 phenotypically distinct groups: high-acetaldehyde-yielding isolates (>10 mg/L), medium-acetaldehyde-yielding isolates (5-10 mg/L) and low-acetaldehyde-yielding (

The antiphage ability is an important feature of fermentation strains in the dairy industry. Our previous work described the bacteriophage exclusion (BREX) system in the probiotic strain, Lactobacillus casei Zhang. The function of L. casei Zhang pglX gene in mediating 5'-ACRC

As an important part of ecosystem, microbes are widely distributed in various habitats. In recent years, more and more attention has been paid to the study on gut microbiota. The gut microbiota and their metabolites influence human and animal nutrition processing, metabolic balance, immune function, gastrointestinal development and other physiological activities. With the deepening of studies on human and animal gut microbiota, it has been found that some factors, such as diet, age, gender, and living environment, impacting on the composition of gut microbiota, while the differences among animal species have more significant influence on the gut microbiota composition. In relatively primitive grassland ecosystems, soil microbes interact with human and animal activities. On the one hand, microbes in the soil environment are the driving forces for the transformation and circulation of organic matter and nutrients. The improvement of soil microbial community diversity is beneficial for the soil fertility. On the other hand, human and animal activities will affect the diversity of soil microbial community. Although there are a lot of researches on soil microbiota, animal and human gut microbiota, their differences in diversity and composition within the same environment have not been studied. The advent of sequencing technology provides an effective mean for the accurate and comprehensive understanding of microbes, especially for the study of uncultivable microorganisms. The PacBio single-molecule real-time (SMRT) technology is advantageous in producing long sequence reads with high accuracy. Based on sequencing the full length 16S rRNA genes, the microbiota composition can be identified to the species level. Therefore, it is an effective approach for studying microbial diversity. We collected 56 stool and soil samples from Xilinguole, including 6, 10, 11, 9, and 10 stool samples from human, goat, cattle, horse, and sheep, respectively, as well as 10 soil samples. Genomic DNA was extracted from the samples. After DNA extraction and quality check, the 16S rRNA genes of all samples were amplified from the genomic DNA. The PCR products were sequenced using the PacBio RS II instrument. The QIIME software (V1.7) was used to analyze the sequencing data, and the R software (version 3.5.0) was used to further analyze and visualize the results. Firstly, it was found that the gut microbiota diversity of human was significantly lower than other samples ( P 0.01). The overall composition of the human and animal gut microbiota were dominated by the Firmicutes and Bacteroidetes phyla. However, the soil microbiota was dominated by Proteobacteria and Acidobacteria. At the genus level, the sheep, goat and cattle gut microbiota were dominated by Clostridium , Bacteroides , and Oscillibacter , while the horse gut microbiota was mainly composed of Clostridium , Eubacterium , and Treponema . The soil microbiota was composed mainly of Blastocatella and Bacillus . The human gut microbiota comprised much of Veillonella , Clostridium , Escherichia/Shigella . At the species level, the human gut microbiota mainly contained Escherichia/Shigella , dysenteriae , Streptococcus salivarius . The sheep, goat, cattle, and horse gut microbiota were dominated by Oscillibacter valericigenes and Eubacterium coprostanoligenes . The major species in soil were Blastocatella fastidiosa and Bacillus longiquaesitum . Moreover, principal coordinate analysis (PCoA) and hierarchical clustering analysis showed some differences in the microbiota structure among human gut, animal gut and soil samples. The gut microbiota structure was similar among cattle, goats and sheep. They were more different from the samples collected from human, horse and soil. We classified all samples into four clusters. Cluster 1 only included human samples; cluster 2 comprised the horse samples; cluster 3 was consisted of the sheep, goat, and cattle samples; while cluster 4 contained only the soil samples. Lastly, we identified the discriminatory OTUs and assigned them taxonomically to the species level. In conclusion, there were significant differences between animal gut microbiota and soil microbiota. The soil microbiota was more complex. As an omnivore, human gut microbiota diversity was significantly lower than other herbivorous animal (namely cattle, goat, horse and sheep). Although cattle, sheep, goat and horses are all herbivorous animals, the distinct features of the digestive systems could contribute to the difference in gut microbiota composition of horse from those of sheep, goat and cattle. This study revealed the differences of gut microbiota diversity between human and other animals, as well as from the soil microbial community. This work has laid a theoretical foundation for further studies on microbial diversity in different habitats.

Streptococcus (S.) thermophilus is an important dairy starter in the production of fermented dairy products has important significance, from natural fermentation in the past to industrial production today. While the genetic architecture underlying S. thermophilus traits and phenotypes is largely unknown. Here, we sequenced 185 S. thermophilus strains, which isolated from natural fermented dairy products of China and Mongolia and using 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.

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.

Streptococcus thermophilus is widely used in the dairy industry to produce fermented milk. Gas chromatography-ion mobility spectrometry-based metabolomics was used to discriminate different fermentation temperatures (37°C and 42°C) at 3 time points (F0: pH = 6.50 ± 0.02; F1: pH = 5.20 ± 0.02; F2: pH = 4.60 ± 0.02) during S. thermophilus milk fermentation, and differences of fermentation physical properties and growth curves were also evaluated. Fermentation was completed (pH 4.60) after 6 h at 42°C and after 8 h at 37°C; there were no significant differences in viable cell counts and titratable acidity; water-holding capacity and viscosity were higher at 37°C than at 42°C. Different fermentation temperatures affected volatile metabolic profiles. After the fermentation was completed, the volatile metabolites that could be used to distinguish the fermentation temperature were hexanal, butyraldehyde, ethyl acetate, ethanol, 3-methylbutanal, 3-methylbutanoic acid, and 2-methylpropionic acid. Specifically, at 37°C of milk fermentation, branched-chain AA had higher levels, and leucine, isoleucine, and valine were involved in growth and metabolism, which promoted accumulation of some short-chain fatty acids such as 3-methylbutanoic acid and 2-methylpanprooic acid. At 42°C, at 3 different time points during fermentation, ethanol from glycolysis all presented higher levels, including acetone and 3-methylbutanal, producing a more pleasant flavor in the fermented milk. This work provides detailed insight into S. thermophilus fermented milk metabolites that differed between incubation temperatures; these data can be used for understanding and eventually predicting metabolic changes during milk fermentation.

The bacteriophage exclusion (BREX) system is a novel prokaryotic defense system against bacteriophages. To our knowledge, no study has systematically characterized the function of the BREX system in lactic acid bacteria. Lactobacillus casei Zhang is a probiotic bacterium originating from koumiss. By using single-molecule real-time sequencing, we previously identified N6-methyladenine (m6A) signatures in the genome of L. casei Zhang and a putative methyltransferase (MTase), namely, pglX. This work further analyzed the genomic locus near the pglX gene and identified it as a component of the BREX system. To decipher the biological role of pglX, an L. casei Zhang pglX mutant (ΔpglX) was constructed. Interestingly, m6A methylation of the 5′-ACRCAG-3′ motif was eliminated in the ΔpglX mutant. The wild-type and mutant strains exhibited no significant difference in morphology or growth performance in de Man-Rogosa-Sharpe (MRS) medium. A significantly higher plasmid acquisition capacity was observed for the ΔpglX mutant than for the wild type if the transformed plasmids contained pglX recognition sites (i.e., 5′-ACRCAG-3′). In contrast, no significant difference was observed in plasmid transformation efficiency between the two strains when plasmids lacking pglX recognition sites were tested. Moreover, the ΔpglX mutant had a lower capacity to retain the plasmids than the wild type, suggesting a decrease in genetic stability. Since the Rebase database predicted that the L. casei PglX protein was bifunctional, as both an MTase and a restriction endonuclease, the PglX protein was heterologously expressed and purified but failed to show restriction endonuclease activity. Taken together, the results show that the L. casei Zhang pglX gene is a functional adenine MTase that belongs to the BREX system. IMPORTANCELactobacillus casei Zhang is a probiotic that confers beneficial effects on the host, and it is thus increasingly used in the dairy industry. The possession of an effective bacterial immune system that can defend against invasion of phages and exogenous DNA is a desirable feature for industrial bacterial strains. The bacteriophage exclusion (BREX) system is a recently described phage resistance system in prokaryotes. This work confirmed the function of the BREX system in L. casei and that the methyltransferase (pglX) is an indispensable part of the system. Overall, our study characterizes a BREX system component gene in lactic acid bacteria.

Objective: To evaluate the effect of the probiotic, Lactobacillus rhamnosus Probio-M9 (Probio-M9), on the quality of life in stressed adults. Methods: Twelve postgraduate student volunteers were recruited. Six volunteers received oral Probio-M9 for 21 days, while the remaining six received a placebo instead. Fecal samples were collected from the volunteers before and after the intervention. Metagenomic sequencing, nontargeted metabonomics, and quality-of-life follow-up questionnaires were used to evaluate the impact of Probio-M9 consumption on the gut microbiota and life quality of the volunteers. Results: Probio-M9 improved the psychological and physiological quality-of-life symptoms significantly in stressed adults (p &lt, 0.05). The probiotic intervention was beneficial in increasing and maintaining the diversity of gut microbiota. The abundance of Barnesiella and Akkermansia increased in the probiotics group. The feature metabolites of pyridoxamine, dopamine, and 5-hydroxytryptamine (5-HT) were positively correlated with Barnesiella and Akkermansia levels, which might be why the mental state of the volunteers in the probiotic group improved after taking Probio-M9. Conclusions: We identified that oral Probio-M9 can regulate the stability of gut microbiota and affect the related beneficial metabolites, thereby affecting the quality of life (QoL) of stressed adults. Probio-M9 might improve the psychological and physiological quality of life in stressed adults via the gut-brain axis pathway. The causal relationship should be further explored in future studies.

Koji is a kind of Japanese traditional fermented starter that has been used for centuries. Many fermented foods are made from koji, such as sake, miso, and soy sauce. This study used the single molecule real-time sequencing technology (SMRT) to investigate the bacterial and fungal microbiota of 3 Japanese koji samples. After SMRT analysis, a total of 39121 high-quality sequences were generated, including 14354 bacterial and 24767 fungal sequence reads. The high-quality gene sequences were assigned to 5 bacterial and 2 fungal plyla, dominated by Proteobacteria and Ascomycota, respectively. At the genus level, Ochrobactrum and Wickerhamomyces were the most abundant bacterial and fungal genera, respectively. The predominant bacterial and fungal species were Ochrobactrum lupini and Wickerhamomyces anomalus, respectively. Our study profiled the microbiota composition of 3 Japanese koji samples to the species level precision. The results may be useful for further development of traditional fermented products, especially optimization of koji preparation. Meanwhile, this study has demonstrated that SMRT is a robust tool for analyzing the microbial composition in food samples.

Koumiss plays an important role in income generation for herdsmen and the daily nutritional intake of local people. In this study, the pH, protein, fat, lactose, lactic acid, amino acid, and vitamin content of koumiss were investigated during the entire fermentation period. The results showed that the protein content of fresh mares’ milk and koumiss ranged from 1.86 ± 0.07% to 2.22 ± 0.08%; fat content ranged from 0.74 ± 0.16% to 1.480 ± 0.08%; VB₁ concentration ranged from 1.48 ± 0.12 to 4.86 ± 0.42 µg/100 g; VB₂ concentration ranged from 2.48 ± 0.12 µg/100 g to 4.86 ± 0.32 µg/100 g; and VE concentration ranged from 99.90 ± 0.05 to 44.63 ± 0.63 µg/100 g. There were significant differences between fresh mares’ milk and koumiss in relation to VE content. The content of 17 amino acids ranged from 2.40 ± 0.11 µg/100 g (for MET) to 39.64 ± 0.01 µg/100 g (for GLU). Fresh mares’ milk and aged koumiss had high nutritional value; however, unripe koumiss (i.e., with a shorter fermentation time) had low nutritional value. Based on the data from this study, an optimized manufacture process for koumiss is proposed. PRACTICAL APPLICATIONS: The dynamic changes of chemical and nutritional composition of koumiss were evaluated in this present paper. The nutrient content of koumiss is the highest at the point of 12 hr fermentation. The optimized manufacture process for koumiss is proposed based on the result of this study. This paper will provide the basic information for koumiss industrial application.

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 lactic acid bacterium Streptococcus thermophilus is a major starter culture for the production of dairy products. In this study, the physiochemical characteristics of milk fermented by the MGA45-4 isolate of S. thermophilus were analyzed. Our data indicate that milk fermented using S. thermophilus MGA45-4 maintained a high viable cell count (8.86 log10 colony-forming units/mL), and a relatively high pH (4.4), viscosity (834.33 mPa·s), and water holding capacity (40.85%) during 14 days of storage. By analyzing the volatile compound profile using solid-phase microextraction and gas chromatography/mass spectrometry, we identified 73 volatile compounds in the fermented milk product, including five carboxylic acids, 21 aldehydes, 13 ketones, 16 alcohols, five esters, and 13 aromatic carbohydrates. According to the odor activity values, 11 of these volatile compounds were found to play a key role in producing the characteristic flavor of fermented milk, particularly octanal, nonanal, hexanal, 2,3-butanedione, and 1-octen-3-ol, which had the highest odor activity values among all compounds analyzed. These findings thus provide more insights in the chemical/molecular characteristics of milk fermented using S. thermophilus, which may provide a basis for improving dairy product flavor/odor during the process of fermentation and storage.

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.

The levels of resistance in 17 Lactobacillus casei isolates and 15 Lactobacillus plantarum isolates to 10 antibiotics were determined using a standardized macrodilution method and the presence/absence of 20 genes implicated in antibiotic resistance in these isolates was determined by polymerase chain reaction (PCR) using gene-specific primers; 11 isolates possessed one or more of these genes but they were not always associated with phenotypic resistance. L. plantarum isolates had the widest spectrum of MIC values for streptomycin ranging from 16 to 512 μg/mL. In particular, two isolates of L. plantarum IMAU60045 and IMAU80091 both possessed aadA and ant(6) genes implicated in resistance to streptomycin but varied in their tolerance to streptomycin as evidenced by their minimum inhibitory concentration (MIC) of 16 and 256 μg/mL, respectively. Selection of high streptomycin resistance of L. plantarum IMAU60045 was performed over a 30 day period using serial passage with regular increases in streptomycin concentration to reflect the changes in resistance levels. Final MIC value of 16,384 μg/mL was recorded which was 1024-fold higher than the original parental isolate. Furthermore, associated variable degrees of increase in the MIC value for gentamicin, kanamycin and neomycin illustrated that, under the challenge of streptomycin, cross-resistance to other structurally related antibiotics of the same class developed. The relative quantity of gene expression (RQ) for the streptomycin resistance gene aadA was 3.35 times greater after passage in increasing concentrations of streptomycin than the original parental isolate. This was greater than the increase in the RQ value for the streptomycin resistance gene ant(6) after passage.

Naturally fermented yak milk (NFYM) is a traditional Tibetan fermented product that contains a rich microbial community. This study used high-throughput pyrosequencing to investigate the bacterial and fungal community diversity of 16 NFYM samples from the villages Geda and Ningzhong in Tibet. Pyrosequencing produced a total of 112,173 high quality bacterial 16S rRNA gene sequences and 90,980 fungal high quality reads from the NFYM. Sequence analysis grouped the high quality gene sequences into eleven bacterial and five fungal phyla, of which Firmicutes and Ascomycota dominated. At the genus level, Lactobacillus and Saccharomyces were the dominating bacteria and fungi, respectively. Principal coordinate analysis (PCoA) based on weighted and unweighted UniFrac values revealed no significant differences in the structure of bacterial and fungal microbiota. However, multivariate analysis of variance (MANOVA) based on unweighted UniFrac distances showed significant differences (P

Food deterioration is largely related to contamination by undesirable microorganisms; it can cause economical loss and serious health-related problems. Owing to increased awareness of health issues by consumers, biological food preservation using antibacterial Lactobacillus species is being increasingly studied. Here, we make a preliminarily assessment of the antibacterial activity of 347 Lactobacillus plantarum isolates against five common food-borne pathogens (Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, Salmonella typhimurium, and Shigella flexneri). Nine of these isolates had broad-spectrum antibacterial activity and were selected for further experimentation. Subsequent assays based on physical, chemical, and sensory evaluations of yoghurts fermented in the presence or absence of each of the selected isolates and persistence of antibacterial activity following protease challenge revealed that L. plantarum isolate IMAU10216 was superior to the other isolates. L. plantarum IMAU10216 also exhibited high in vitro gastrointestinal tract tolerance. To conclude, we have identified a novel bioactive antibacterial L. plantarum isolate with potential for use as a probiotic in the production of fermented milk or other related dairy products. Further in vivo studies are now required.

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.

Koji is a kind of Japanese traditional fermented starter that has been used for centuries. Many fermented foods are made from koji, such as sake, miso, and soy sauce. This study used the single molecule real-time sequencing technology (SMRT) to investigate the bacterial and fungal microbiota of 3 Japanese koji samples. After SMRT analysis, a total of 39121 high-quality sequences were generated, including 14354 bacterial and 24767 fungal sequence reads. The high-quality gene sequences were assigned to 5 bacterial and 2 fungal plyla, dominated by Proteobacteria and Ascomycota, respectively. At the genus level, Ochrobactrum and Wickerhamomyces were the most abundant bacterial and fungal genera, respectively. The predominant bacterial and fungal species were Ochrobactrum lupini and Wickerhamomyces anomalus, respectively. Our study profiled the microbiota composition of 3 Japanese koji samples to the species level precision. The results may be useful for further development of traditional fermented products, especially optimization of koji preparation. Meanwhile, this study has demonstrated that SMRT is a robust tool for analyzing the microbial composition in food samples.

A total of 29 strains of Bifidobacterium were isolated from 18 samples of human feces in different ethnic minority regions of China. All isolates were identified as Bifidobacterium longum (9 strains) and Bifidobacterium pseudocatenulatum (20 strains) based on 16S rRNA gene sequencing and phylogenetic analysis. These strains were preliminarily tested for their suitability to become probiotics by assessing their ability to survive adequately at low pH conditions and their tolerance of different concentrations of bile salts and simulated gastrointestinal juices. In vitro tests were sequentially used to predict the survival of these strains in the simulated conditions in the human gastrointestinal tract. These strains were first exposed to pH 2.5 for 3h, and 7 out of the 29 strains were discriminated from the others by their high survival rates. Out of these 7 strains, 4 were found to grow and survive well at an even lower pH of 2.0 and in high bile salt concentration. Apart from the gastrointestinal survival capacity, both fermentation efficiency and storage characteristics are important criteria for selecting for suitable potential probiotic strains. Therefore, the fermentation efficiency in bovine milk and the bacterial viability during the storage in the resultant fermented milk were also evaluated for these 4 selected strains. In this study, we isolated and identified 29 novel Bifidobacterium strains. Based on our initial evaluation, at least 4 of them may serve as valuable resources for further dairy probiotic strain selection.

Lactic acid bacteria (LAB) are industrially important bacteria that are widely used in the fermented food industry, especially in the manufacture of yogurt. Characteristic flavors are produced by LAB during fermentation and storage that affect the quality and acceptability of fermented milk products. In this study, the volatile compounds in milk fermented by Streptococcus thermophilus IMAU80842 alone, Lactobacillus delbrueckii ssp. bulgaricus IMAU20401 alone, or both species together were identified using solid-phase microextraction methods coupled with gas chromatography-mass spectrometry. A total of 53, 43, and 32 volatile compounds were identified in milk fermented by S. thermophilus alone, L. delbrueckii ssp. bulgaricus alone, or both species together, respectively. The presence of some important flavor compounds was confirmed: acetic acid, acetaldehyde, acetoin, 2,3-butanedione, ethanol, and 1-heptanol. Our results demonstrate that the composition of the volatile compounds in fermented milk depends on the species of LAB used and whether they are used alone or in combination. This is important for the selection of appropriate starter cultures for the production of different types of fermented milk product with particular flavors.

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.

One hundred and two strains of Lactobacillus plantarum, all isolated from traditional dairy products of minority nationalities, were evaluated for in vitro probiotic properties, including acid and bile tolerance, aggregation activity, and antibacterial activity. Of these, 12 strains with a high tolerance to simulated gastric juice (pH 2.5, 3 h of incubation) were selected, of which eight of these also showed a good tolerance to bile salt. All selected 12 strains showed a high autoaggregation percentage after being incubated at room temperature for 20 h, and nine of these significantly inhibited growth of the five indicator intestinal pathogens. Six L. plantarum strains (IMAU10120, IMAU10156, IMAU40126, IMAU70004, IMAU60042, and IMAU60171) were selected for further evaluation of their fermentation characteristics, sensory quality, and viable counts, both during the fermentation process of soymilk and following a 28-day storage of the soymilk. The six strains had an acid-producing ability at the refrigerated temperature. All of the fermented soymilk samples had a viable count above log 8.45 CFU/ml, and viability in the samples was maintained during storage. Based on both the probiotic properties and fermented characteristics, L. plantarum IMAU10120 was the best potential probiotic strain of those tested for the production of fermented functional soymilk.

Tolerance to acid is an important feature for probiotic bacteria during transition through the gastrointestinal tract. Proteomics analysis of a new probiotic bacterium, Lactobacillus casei Zhang, was performed upon 30-min exposure to low acid stress (pH 2.5 vs. pH 6.4) using two-dimensional electrophoresis. Out of 33 protein spots that showed changes of expression between the two pHs, 22 showed 1.5-fold higher expression at pH 2.5 than at pH 6.4, whereas five spots had expression decreased by 1.5-fold at pH 2.5. There were also six protein spots that were exclusively present on different pH maps. Further analysis showed that eight of the enhanced proteins, NagA, NagB, PGM, GlmM, LacC, TDP, GALM and PtsI, were involved in carbohydrate catabolism. Moreover, quantitative RT-PCR showed that the mRNA expression levels of dnaK, nagB, galm, estC, tuf and luxS were consistent with changes in protein expression. We postulate that there might be some relationship between differentially expressed proteins and acid tolerance in L. casei Zhang.

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.