Abstract: To explore the lactic acid bacteria fermentation characteristics of Flammulina velutipes and its mechanism in alleviating hyperlipidemia, a mixed-strain starter suitable for fermenting F. velutipes was selected from 8 species of lactic acid bacteria. A mouse model of hyperlipidemia was established, and the hyperlipidemic and normal animals were gavaged with fermented F. velutipes (FFV). The results showed that FFV prepared with a mixed starter of Lacticaseibacillus rhamnosus, Lactobacillus delbrueckii subsp. bulgaricus, and Lactobacillus acidophilus (2:3:1, m/m) had a sensory score of 84.60, a pH of 3.46, a total acid production of 0.78 g/100 g, and a total viable count of 2.7 × 1010 CFU/mL. FFV reduced liver wet mass and index and improved hepatic antioxidant capacity. It also significantly reduced the levels of serum total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C) and the activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in hyperlipidemic mice, showing potent hypolipidemic effects. FFV significantly reduced the Firmicutes/Bacteroidetes ratio in the mouse gut microbiota and increased the relative abundances of Akkermansia, Faecalibacterium, Dubosiella, Lactobacillus, Bifidobacterium, and Bacteroides, while reducing the levels of Helicobacter and Desulfovibrio. Moreover, the changes in the microbiota were correlated with the levels of serum lipids, transaminases, and liver oxidative stress. A total of 4 354 metabolites belonging to 21 major categories were identified in the mouse gut contents, among which amino acids and their derivatives accounted for the largest proportion (39.98%). After being induced by a high-fat diet, the gut microbial metabolites of mice changed significantly. FFV improved the composition of gut microbial metabolites in hyperlipidemic mice by regulating the contents of polypeptides, fatty acyls, organic acids, and alkaloids produced by gut microbiota metabolism. This study confirms that FFV alleviates hyperlipidemia, modulates the gut microbiota and rectifies metabolic disturbances in mice, providing a theoretical basis for the development of functional foods from FFV.

Key words: Flammulina velutipes; lactic acid bacteria; fermentation characteristics; lowering blood lipids; gut microbiota; metabolites