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This study utilized high-throughput sequencing and SEM observation to elucidate the microbial composition of a Tibetan herder's homemade kefir grain named TKG-Y. Subsequently, S. warneri KYS-164 was isolated from TKG-Y, which can produce mixed protein substances with antibacterial activity, namely bacteriocin-like inhibitory substances (BLIS). BLIS can significantly reduce the growth rate of Escherichia coli 366-a, Staphylococcus aureus CICC 10384 and mixed strains at low concentrations (1 × MIC). The presence of the warnericin-centered gene cluster in KYS-164 may explain the antibacterial properties of the BLIS. Pepsin and an acidic environment can reduce the number of colonies of KYS-164 by 2.5 Log 10 CFU mL -1 within 1 h, and reduce the antibacterial activity of BLIS by 21.48%. S. warneri KYS-164 showed no antibiotic resistance and biological toxicity after 80 subcultures, while BLIS produced by 40 generations of the strain retained their inhibitory efficacy against pathogenic bacteria. After 48-hour fermentation of milk with KYS-164, volatile compounds such as aldehydes, phenols, esters, and alcohols, giving it a floral, fruity, milky, oily, and nutty aroma, were released, enriching the sensory characteristics of dairy products. This study not only revealed the bacterial colony composition information of home-made kefir grain TKG-Y but also discovered and proved that S. warneri KYS-164 has the potential to inhibit bacteria and ferment dairy products. This will provide a basis for subsequent applied research on KYS-164.

Tibetan kefir grains (TKGs) are a complex protein-lipid-polysaccharide matrix composed of various microorganisms. Microorganisms have the benefit of being effective, secure, and controllable when used for selenium enrichment. In this study, selenium-enriched Tibetan kefir grains (Se-TKGs) were made, and the microbiology composition was analyzed through a metagenomic analysis, to explore the influence of selenium enrichment. The microbial composition of TKGs and Se-TKGs, as well as the probiotic species, quorum sensing system (QS) and functional genes were compared and evaluated. Lactobacillus kefiranofaciens was the most abundant microbial species in both communities. Compared with TKGs, Se-TKGs had a much higher relative abundance of acetic acid bacteria. Lactobacillus helveticus was the most common probiotic species both in TKGs and Se-TKGs. Probiotics with antibacterial and anti-inflammatory properties were more abundant in Se-TKGs. QS analysis revealed that Se-TKGs contained more QS system-associated genes than TKGs. Moreover, Kyoto Encyclopedia of Genes and Genomes analysis revealed that the pathway for human disease ko01501 had the greatest relative abundance in both TKGs and Se-TKGs. Compared with TKGs, Se-TKGs demonstrated a greater relative abundance of different drug resistance-related metabolic pathways. Additionally, linear discriminant analysis effect size was used to examine the biomarkers responsible for the difference between the two groups. In this study, we focused on the microbiological structure of TKGs and Se-TKGs, with the aim of establishing a foundation for a more thorough investigation of Se-TKGs and providing a basis for exploring potential future use., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The authors report no conflicts of interest to declare., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Tibetan kefir (TK) is a good source of probiotics and has antioxidative, anti-inflammatory, and gut microbiota-regulating properties. Ochratoxin A (OTA) causes liver injury through the gut microbiota. It is unclear whether TK has differential effects on the gut-liver axis and whether these effects can alleviate liver injury induced by OTA. Herein, we investigated the hepatoprotective effect of TK in OTA-fed mice and its underlying mechanisms in the context of the gut. The results indicate that microorganisms in TK bound to OTA and decreased its gastrointestinal absorption. TK improved the intestinal barriers, and suppressed oxidative stress and inflammatory responses. Moreover, TK supplementation alleviated the gut microbiota dysbiosis by enriching beneficial microbiota such as Akkermansia and Lachnospiraceae UCG-006 , and reducing harmful bacteria. Importantly, TK supplementation reversed the liver injury by reducing gut leakage of Bacteroides and lipopolysaccharides. Furthermore, untargeted metabolomics revealed that TK reversed the perturbation of metabolites involved in glycerophospholipid and bile acid metabolism, and regulated metabolites that are pertinent to the citrate cycle and tryptophan metabolism. TK also increased the production of short-chain fatty acids and anti-inflammatory metabolites. These data are consistent with the protective effects of TK against OTA-induced liver injury by maintaining the intestinal barrier and modulating the gut microbiota and metabolites. These findings will facilitate therapeutic interventions for mycotoxicosis.

Ochratoxin A (OTA) is reported to cause intestinal damage following ingestion of contaminated foods. Tibetan kefir (TK) is a fermented dairy product that possesses antioxidant, anti-inflammatory, and gut microbiota-regulating properties. However, it is not clear if TK can alleviate OTA-associated intestinal toxicity. Here, we investigated whether TK can prevent OTA-induced intestinal barrier disruption in mice. To this end, OTA-fed mice were treated with sterile water (control) or TK by oral gavage once daily, for 3 weeks. The histological changes of ceca, the expression of tight junction proteins and mucins, and the levels of oxidative stress, inflammatory response, and gut microbiota were then assessed. Results revealed that treatment with TK reversed OTA-driven histopathological changes in the ceca, and was associated with increased cecal mucin levels. TK administration to OTA-treated mice significantly elevated the expression levels of tight junction proteins (claudin-1, zonula occludens-1, and occludin). Additionally, TK supplementation suppressed OTA-induced oxidative stress and reduced inflammation via the NF-κB signaling pathway in the ceca. Moreover, TK supplementation depleted harmful bacteria (e.g., Turicibacter and Desulfovibrio), while supporting short-chain fatty acids (SCFAs)-producing bacteria (e.g., Lachnospiraceae, Blautia, and Ruminococcus), which maintained the SCFAs levels. Taken together, our findings indicate that TK may emerge as a viable dietary strategy to prevent intestinal toxicity-based injuries., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

This study aimed to investigate the alleviative effects of Lactobacillus kefiranofaciens JKSP109 (LK) and Saccharomyces cerevisiae JKSP39 (SC) isolated from Tibetan kefir grain on colon inflammation and colorectal carcinogenesis. Azoxymethane (AOM) and dextran sulfate sodium (DSS) were used to establish a mouse model of colorectal cancer (CRC). The treatment group mice were administered with LK, SC, or the combination of LK and SC for five days per week from the day of receiving AOM. The composition of the gut microbiota was assessed using internal transcribed spacer 2 and 16S rRNA gene high-throughput sequencing. Furthermore, the biomarkers associated with gut barrier integrity, inflammation, regulators of cell proliferation, and apoptosis were evaluated. The results showed that the administration of LK, SC, and their combination increased the body weights and decreased the disease activity index (DAI) score and tumor multiplicity. As compared to the CRC model group, the three treatment groups positively regulated the gut microbiota. Meanwhile, the three treatments also enhanced the gut barrier, decreased the expression of proinflammatory cytokines and oncocyte proliferation indicators, and increased the expression of terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)-positive tumor epithelial cells and content of short chain fatty acids in fecal samples. All these results indicated that the LK and SC alleviated the inflammation and colorectal carcinogenesis in AOM/DSS-induced CRC mouse models, and the majority of tested indexes in the combination group were superior to single strain groups.

Tibetan kefir grains (TKGs) are distinctive and complex mixtures with protein-lipid-polysaccharide matrices and multiple microorganism species. The objective of this study was to evaluate the microflora composition, probiotic species and functional genes within TKGs. Metagenomic analysis was used to evaluate communities of three TKGs, revealing the presence of 715 species, with Lactobacillus kefiranofaciens as the most dominant species. The relative abundances of acetic acid bacteria and yeast significantly differed among the three TKGs (acetic acid bacteria: p < 0.01; yeast: p < 0.05), and the dominant yeast species also varied across three TKGs. Lactobacillus helveticus was the most abundant listed probiotic species, and its abundance did not significantly differ across three TKGs. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that ko01501 was the most abundant pathway that related to human disease. There are 16 different KOs (KEGG Orthology) in the ko01501 pathway were annotated in TKGs, which helps to resist β-lactam. This study provided a new insight into the microbial community structures and the presence of probiotic species within TKGs and provides a foundation for further targeted studies., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Tibetan kefir grains (TKG) are multi-functional starter cultures used in foods and have been applied in various fermentation systems. This study aimed to investigate the microbial community composition of TKG, the detoxification abilities of TKG and their isolates towards common mycotoxins, and the potential for applying TKG and their associated microbial populations to avoid mycotoxin contamination in dairy products. Cultivation-independent high-throughput sequencing of bacterial and fungal rDNA genes indicated that Lactobacillus kefiranofaciens and Kazachstania turicensis were the most abundant bacterial and fungal taxa, respectively. In addition, 27 total isolates were obtained using cultivation methods. TKG removed more than 90% of the Ochratoxin A (OTA) after 24 h, while the isolate Kazachstania unisporus AC-2 exhibited the highest removal capacity (~46.1%). Further, the isolate exhibited good resistance to acid and bile salts environment. Analysis of the OTA detoxification mechanism revealed that both adsorption and degradation activities were exhibited by TKG, with adsorption playing a major detoxification role. Furthermore, the addition of OTA did not affect the microbial community structure of TKG. These results indicate that TKG-fermented products can naturally remove mycotoxin contamination of milk and could potentially be practically applied as probiotics in fermentation products., (Copyright © 2021. Published by Elsevier Ltd.)