This study aims to examine the effects of the mixture of Bacillus cereus G1-11 and Exiguobacterium acetylicum G1-33, isolated from the gut of hybrid groupers (Epinephelus fuscoguttatus ♀ × E. lanceolatus ♂), on the host. The hybrid groupers were divided into a control (C, without any probiotics), B. cereus (BC, 10¹⁰ cfu/g), E. acetylicum (EA, 10⁸ cfu/g), compound (mix, a 1:1 mixture of B. cereus and E. acetylicum), and positive reference group (P, Lactobacillus acidophilus, 5 × 10⁸ cfu/L). Each group had four replicates, with 30 fish per replicate (53.30 ± 0.50 g), and were fed for 60 days. The results showed that adding probiotics to the feed significantly improved the weight gain, weight growth rate, specific growth rate, and digestive enzyme activities of hybrid groupers compared to the C group. The compound group was the most significant. In addition, composite probiotics added to feed significantly upregulated the expression levels of several growth-related genes in the liver and muscles. The activities of alkaline phosphatase, catalase, glutathione peroxidase, glutathione transferase, lysozyme, and total antioxidant capacity in the serum and liver were significantly influenced through mixed probiotic feeding. Moreover, the expression levels of several immune-related genes in the liver, spleen, and head kidney were significantly enhanced by adding single and mixed probiotics to feed, with the synergy of mixed probiotics being the best. An analysis of the gut microbiota showed that adding composite bacteria enhanced the richness and diversity of the gut microbiota, significantly increasing the relative abundance of potential probiotics (Cetobacterium and Microbacterium) while decreasing the presence of potential pathogens (Mycoplasma). Overall, our findings highlighted the efficacy of mixed probiotics (B. cereus and E. acetylicum) in enhancing growth performance, nutritional value of hybrid grouper feed, antioxidant capacity, immune response, and intestinal health, in finding the best combination of functional feed additives.

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