Optimization of fermentation process for Sihuang compound with Bacillus subtilis and its efficacy against Escherichia coli infections and growth performance in broilers

Sihuang (SH) compound is a traditional herbal formula used to treat chicken colibacillosis, while its antimicrobial activity is relatively poor. To enhance its therapeutic efficacy and broaden its biological activities, an in vitro probiotic-fermented Sihuang (FSH) was established. The response surface methodology was applied to optimize the fermentation conditions, and the physiological characteristics and therapeutic efficacy of FSH for avian pathogenic Escherichia coli (APEC)-challenged broilers were investigated and compared with the unfermented SH both in vitro and in vivo. Consequently, Bacillus subtilis (B. subtilis) was identified as the optimal probiotic, leading to significant increases in total flavonoids (247.32 %), baicalin (47.63 %) and berberine (10.25 %), while reducing pH from 6.31 to 5.32 (P < 0.0001). Meanwhile, FSH markedly increased the total antioxidant capacity (T-AOC), immune function, the activities of glutathione peroxidase (GPx) and catalase (CAT) in APEC-challenged broilers (P < 0.01). In terms of intestinal health, FSH increased the abundance of Lactobacillus in the cecum and intestinal α-amylase activity, and also significantly enhanced the expression levels of tight junction proteins (TJPs) related genes (ZO-1, Mucin-2, Occludin, Claudin-1 and Claudin-4) (P < 0.01). 1 g/L of FSH improved the growth performance, as evident by inhibiting APEC-induced decrease in feed conversion ratio (FCR) (day14-21), while increasing the average daily gain (ADG) and FCR (day 21-35) (P < 0.01). Collectively, these findings demonstrate that FSH offers superior therapeutic efficacy and growth performance at half the recommended dose of SH, making it a promising antibiotic substitute for poultry industry.