Screening of Strategies for Enhancing and Preserving Antibacterial Efficacy: Optimizing Inactivation and Desiccation of Black Soldier Fly Larvae Against Avian Enteric Pathogens

Chicken manure is widely used for rearing black soldier fly larvae (BSFL). The effects of chicken manure-borne pathogens ( Escherichia coli and Salmonella pullorum ) and subsequent larval processing treatments on antimicrobial peptide expression in BSFL remain poorly clarified. Here, BSFL were exposed to single or combined inoculation of Escherichia coli (E. coli) and Salmonella pullorum (S. p). Compared with the control group, all pathogen-inoculated groups showed significant increases in larval weight and body length. Each group was further divided by stimulation type into three sub-treatments: unstimulated, heat-stimulated (Hs), and Microwave-stimulated (Ws). At 6 h after stimulation, the expression of defensin and cecropin, as well as antibacterial activity were determined. Under unstimulated conditions, 11 of 16 infection groups exhibited lower gene expression fold changes than uninfected control. No sample inhibited E. coli, while 7 of 16 suppressed S. p. Both heat and microwave stimulation markedly upregulated defensin and cecropin expression. All stimulated samples strongly inhibited S. p; most also showed antibacterial activity against E. coli (16 Ws, 14 Hs). Receiver operating characteristic (ROC) analysis indicated that defensin ΔCt (sample Ct minus reference Ct) was negatively correlated with the inhibition diameter of E. coli (r = −0.4381), and could effectively discriminated antibacterial efficacy against both pathogens. A unified cutoff value was identified at 2.43. Larval pretreatment with 1% starch-5% sucrose-0.1% Tween-80 before drying optimally retained anti-S. p activity, with a maximum inhibition diameter of 2.65 mm in the Sp2E4 — Ws treatment. This study provides theoretical and technical guidance for BSFL-antibacterial-protein production.