Clostridium butyricum-fermented Chinese herbal medicine enhances the immunity by modulating the intestinal microflora of largemouth bass (Micropterus salmoides)

Probiotic-fermented Chinese herbal medicine has been shown to stimulate immunity and modulate gut microbiota. It has been considered to be an ideal alternative to antibiotics in a variety of species. In this study, Clostridium butyricum was used as a fermentation strain, while a mixture of licorice root, Astragalus membranaceus and Acanthopanax senticosus was used as a substrate for solid-state fermentation. The fermented product (F-CHM) was mixed with basic feed at levels of 0 (NC), 5% (S-5%), 10% (S-10%) and 20% (S-20%) to feed largemouth bass for 56-day. The results showed that no significant effect of F-CHM was observed on weight gain, but the resistance to A.hydrophila infection was improved. The activity of alkaline phosphatase and lysozyme in plasma was increased with F-CHM addition, and the content of malondialdehyde was significantly decreased. In the spleen, a higher expression of the inflammatory factor TGF-β and apoptosis related genes (Caspase-3 and p53) was detected after the addition of F-CHM. With the addition of F-CHM, the midgut villus height of largemouth bass was significantly increased, and the expression of the intestinal barrier related genes, ZO-1 and Claudin-4 was upregulated. Furthermore, the concentration of short-chain fatty acids was increased in the S-20% F-CHM addition group. Sequencing results showed that the intestinal flora abundance and diversity were dramatically increased in the S-10% group. In addition, the development of harmful bacteria was restrained, and the colonization of beneficial flora was increased. Collectively, dietary F-CHM can be applied as a largemouth bass feed supplement to enhance fish immunity, regulate the intestinal flora and improve the ability of the host to protect against pathogenic bacteria.