Optimization of lyophilization medium for Vibrio harveyi phage and application in oral delivery to groupers

Vibrio harveyi is a major pathogen in grouper aquaculture, and the rise of antibiotic resistance necessitates the development of new control strategies. Phage therapy is considered a promising alternative, but formulation stability limits its practical application. This study aimed to optimize the lyoprotectant formulation for the V. harveyi phage V-YDF132 and to evaluate its immunomodulatory effects as a feed additive in grouper, as well as its prophylactic efficacy against V. harveyi infection. Among nine candidate composite protectants screened, formula P9 (consisting of inulin, skim milk, gelatin, and sodium L-glutamate) was identified as the optimal formulation. The resulting preparation exhibited good stability: the phage titer decreased by 0.51 log immediately after lyophilization; after 21 d at 37 °C in an accelerated stability test, the titer decreased by only 0.90 log; and after 9 months at 4 °C, the titer decreased by 0.51 log. During a 14-d feeding trial, there were no differences in growth performance between the phage-fed and control groups. Feeding phage-supplemented diets significantly increased the activities of serum alkaline phosphatase (AKP), nitric oxide synthase (NOS), and lysozyme (LYZ), and significantly upregulated the expression of immune-related genes (CD4, CD8, IgM, and TCRβ) and inflammatory cytokines (IL-6 and IL-8) in the spleen. Challenge tests showed that the phage-fed group achieved a relative percent survival (RPS) of 28.6%. In summary, the lyophilized phage V-YDF132 formulation can serve as a potential immunomodulator and, when administered orally, enhances grouper resistance to V. harveyi.