Intestinal Delivery of Probiotic Bacteria in Dairy Calves Using Monophosphorylated Starch-Based Hydrogel Carrier

Gastrointestinal diseases in calves remain a serious challenge in livestock production, necessitating the search for effective alternatives to antibiotic therapy. This study compared two formulations of the probiotic Bacillus subtilis—a liquid suspension and a hydrogel tablet based on monophosphorylated starch—in terms of fecal recovery, clinical efficacy, and effects on the composition of the culturable microbiota. A kanamycin-resistant strain of B. subtilis was used to prepare both formulations. The starch carrier was chemically modified by monophosphorylation to confer protective and pH-responsive properties. The release kinetics of bacteria from the tablets were evaluated in vitro using sequential buffer solutions simulating the gastrointestinal tract of calves (pH 6.0, 2.2, and 7.6). The in vivo experiment was conducted on 20 calves with diarrhea, divided into control, suspension, and hydrogel groups. Fecal consistency was assessed using a 4-point scale, bacterial titers were determined by plate counting, and Candida spp. DNA content was quantified by real-time PCR. The hydrogel formulation provided prolonged bacterial detection in feces: by day 14, the titer in the suspension group had significantly decreased, whereas viable cells in the hydrogel group were maintained at a significantly higher level (p = 0.023). Both probiotic formulations contributed to the normalization of fecal consistency by day 3 after treatment withdrawal (p = 0.013 for the suspension and p = 0.005 for the hydrogel), while no improvement was observed in the control group (p = 0.057). Candida spp. DNA levels significantly decreased in the treated groups by days 3 and 14 (p < 0.001), indicating suppression of fungi by B. subtilis. The in vitro modeling confirmed the protective properties of the starch matrix, with only minimal reduction in bacterial titer at pH 2.2. Thus, the hydrogel formulation based on monophosphorylated starch ensures prolonged retention of B. subtilis in the intestine, enhances the therapeutic effect against diarrhea, and contributes to the reduction in Candida spp. in feces. These findings support the potential of this formulation as a promising probiotic delivery system for veterinary practice.