In vitro compatibility screening of Bacillus pumilus and Bacillus altitudinis strains with selected candidate prebiotics for in ovo application of synergistic synbiotics

Bacillus species have demonstrated beneficial effects on intestinal health, production parameters, and immune function in poultry under both standard and disease-challenged conditions. Previously, we found that several novel ingredients (beta-glucan, vegetable protein hydrolysate, and liquid seaweed extract) demonstrated growth stimulation effects on Bifidobacterium lactis and Lactobacillus plantarum, Here, we extended this approach to examine in vitro synbiotic combinations of five novel marine-derived candidate probiotic Bacillus strains to assess their potential for in ovo applications. Beta-glucan enhanced the growth of all candidate Bacillus probiotic strains compared to a glucose control (p ≤ 0.05), suggesting a broad-spectrum modulatory role over a 24-h period, with variable magnitudes of response observed between strains. Species specificity was also observed, with lentinus stimulating the Bacillus pumilus but not the Bacillus altitudinis strains. A seaweed extract consistently stimulated the growth of one of the B. altitudinis strains (p ≤ 0.05), which, like all of the strains evaluated here, is seaweed-derived. This suggests potential ecological adaptation in substrate utilization. The shared environmental origin may influence substrate specificity and metabolic complementarity between strains and prebiotic candidates. Both B. altitudinis strains also exhibited enhanced growth at almost all time points (p ≤ 0.05) when cultured with vegetable protein hydrolysate. Based on these findings, we evaluated the effect of a potential synbiotic formulation comprising one of the B. altitudinis strains and vegetable protein hydrolysate in chickens, in ovo. The components were administered intra-amniotically at embryonic development day 18.5, utilizing a standard vaccination protocol. The hatchability of the chickens was not affected, thereby demonstrating the established dose as safe and applicable for further investigation. KEY POINTS: • Shared origin of bioactive compounds may enhance probiotic-prebiotic compatibility in vitro • Protein hydrolysate offers a novel alternative to carbohydrate prebiotics • In ovo delivery of Bacillus-based synbiotic formulations offers potential as an early microbiome programming strategy.