Microbial diversity associated with Tibetan kefir grains and its protective effects against ethanol-induced oxidative stress in HepG2 cells

Tibetan kefir grains (TKGs) and their products are multi-functional health supplements. This study aimed to explore the diversity of microorganisms in Tibetan kefir grains (TKGs) and the protective effect of isolates on ethanol-treated HepG2 cells. Metagenomic sequencing showed that Lactobacillus Kefiranofaciens was the most abundant bacterial taxon, while Kluyveromyces marxianus was the most abundant fungal taxum in TKGs. Thirteen isolates belonging to the genera Lactobacillus (3), Fructobacillus (1), Lactococcus (1), Pediococcus (1), Acetobacter (3), and Kluyveromyces (4), were obtained by cultivation methods. KEGG pathway analysis revealed that K. marxianus contributed four KEGG pathways related to hepatic disease. LC-MS analysis revealed CFEs are rich sources of peptides amino acids, lipids and amino acids. Cell-free extracts (CFEs) prepared from K. marxianus protected HepG2 cells from ethanol-induced injury. Treatment with CFEs from isolates YG-4 and YK-6 significantly increased cell viability and reduced ethanol cytotoxicity. Also, YG-4 and YK-6 decreased reactive oxygen species, malondialdehyde and alanine aminotransferase, increased superoxide dismutase and catalase, and reduced apoptosis and loss of potential in mitochondria. In addition, CFEs, especially YG-4-derived CFEs, decreased ethanol-induced inflammatory cytokines (IL-6, IL-17 and TNF-α), while YG-4 and YK-6 CFEs upregulated the regulatory cytokine IL-4. Thus, YG-4 had the most significant anti-inflammatory effects by reducing ethanol-induced oxidative stress. These isolates’ properties may provide new avenues to use K. maxianus in alcoholic liver disease.