In this study, the nutritional components of Hongjundouzha inoculated with Neurospora crassa at different fermentation times were analyzed. RNA sequencing (RNA-seq), quantitative real-time polymerase chain reaction (real-time PCR) and ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS)-based non-target metabolomics were used to find out the main differentially expressed genes (DEGs), key metabolites and metabolic pathways affecting the fermentation of this strain, and the transcription and expression of cellulase, an important gene, was explored. The results showed that the second day of fermentation was the key period for the transformation of nutrients in Hongjundouzha and the most active period of gene expression, where cellulase expression was the highest. Carbohydrates in okara were metabolized through the starch and sucrose metabolism and carbon metabolism pathways to produce D-arabinose, sorbitol, D-xylose, L-fucose, linoleic acid, gamma-linolenic acid and arachidonic acid. On the third day, amino acid metabolism occurred. Proteins in okara were metabolized into glutamate, alanine, valine and other amino acids through the amino acid biosynthesis pathway. On the fourth day, secondary metabolites were produced. Daidzein, genistein and genistin in okara were transformed into neohesperidin, strychnine, squamolone and other substances with special physiological activities through the secondary metabolite biosynthesis pathway. The mycelia began to age, lysophosphatidyl choline (LPC) 18:4/18:1 and 18:1, lipids indicating food spoilage, appeared. In general, the second day of fermentation was the most active period of nutrient transformation in Hongjundouzha. The product matured on the third day of fermentation, and began to age and rot on the fourth day.

This study shed light on the hepatoprotective mechanism of β-glucan from Ganoderma sinense produced in Wuping, China on mice with acute alcoholic liver injury (AALI) through studies on the correlation between the gut microbiota and its metabolites. A total of 180 specific pathogen-free (SPF)-grade mice were randomly assigned to six groups: two control groups (CK and A, gavaged with distilled water and carboxymethyl cellulose sodium (CMC-Na), respectively), two model groups (C and D, gavaged with distilled water and CMC-Na, respectively), an experimental group (D, gavaged with 200 mg/kg of G. lucidum β-glucan), and a positive control group (E, gavaged with 300 mg/kg of diphenyl diester). After one week of continuous administration, serum and liver biochemical indices were measured in each group and histopathological observation of liver sections was studied. Meanwhile, the differences in the composition of the intestinal microbiota and metabolites were analyzed among groups. The results indicated that oral administration of 200 mg/kg of G. sinense β-glucan ameliorated liver function in AALI mice. Compared with the model groups, the experimental group exhibited significantly reduced levels of alanine aminotransferase (ALT), triglyceride (TG), total cholesterol (TC), malondialdehyde (MDA) and alcohol dehydrogenase (ADH) (P < 0.05) and significantly increased levels of total superoxide dismutase (T-SOD) and reduced glutathione (GSH) (P < 0.05). Histopathological observation confirmed that β-glucan from G. lucidum significantly attenuated liver cell and lobular lesions, thereby exerting a hepatoprotective effect. G. sinense β-glucan improved the gut microbiota and substance metabolism in mice. Specifically, compared with the model group, the ratio of Firmicutes/Bacteroidoota (F/B) increased in the experimental group, and the relative abundance of beneficial bacteria such as GCA-900066575 and Roseburia in Firmicutes significantly increased as well. Additionally, G. sinense β-glucan altered the metabolism of lipids and lipid-like molecules such as cholesterol, DHA ethyl ester, and oleic acid methyl ester through affecting Bacteroides in the Bacteroidota phylum and altered propionic acid metabolism through affecting Lactobacillus in the Firmicutes phylum, thereby alleviating AALI. Therefore, the protective mechanism of G. sinense polysaccharides against AALI in mice might be related to the regulation of amino acid metabolism, lipid and lipid-like molecule metabolism, and propionic acid metabolism by improving the composition of the gut microbiota.