In-vitro digestive assay to understand the effect of probiotics and a prebiotic and essential oil blend on Salmonella Enteritidis

This study evaluated the effect of probiotics, a prebiotic and essential oil blend and their combination on the growth of Salmonella Enteritidis (SE) in a digestive assay mimicking three digestive compartments of the poultry digestive tract: crop, proventriculus, and intestine. A poultry commensal Lactobacillus reuteri (LR) and SE strain were utilized. The prebiotic product was included at 0.025% (w/w) and probiotic product, a combination of 3 strains of Bacillus spp., at 2 × 10⁶ cfu/g in the feed. This experiment was a completely randomized design with the treatments: Positive control (PC); PRE = PC + prebiotic; PRO = PC + probiotic; and PRE + PRO. In the crop, 5 g of peaking layer feed (4.5% Ca), 1 ml each of SE (2 × 10⁶ cfu/ ml) and LR (2 × 10⁶ cfu/ml), and 0.03 M hydrochloric acid (HCL) were added. pH was adjusted to 5.2 with HCL as needed and tube mixed by shaking vigorously and incubated for 30 min at 40°C. The proventriculus was simulated by adding pepsin and 1.0 M HCL to reduce the pH to 3.0 and incubated for 45 min at 40°C. Intestinal conditions were simulated by adding pancreatin and sodium bicarbonate to adjust the pH to 6.4 to 6.8 and incubated for 2 h at 40°C. Total bacterial count changes were recorded for each compartment by spread plate technique and incubated at 37°C for 48 h. XLT4 and MRS were used for SE and LR and incubated aerobically and anaerobically, respectively. Heat shock was applied at 80°C for 10 mins before enumerating Bacillus on TSA agar aerobically. Data were logtransformed and subjected to ANOVA using the PROC GLM procedure of SAS (V9.4), means were separated using least-squares means. An interaction between compartment and product occurred for total counts of SE (P< 0.001). In the intestine, PRE, PRO, or PRE+PRO significantly reduced the total count of SE compared to the PC (P< 0.05). Bacillus and LR total counts increased from crop to proventriculus to intestine (P< 0.001). PRE and PRO products were able to reduce growth of SR in-vitro but did not show additive effect. The amount of HCL was more than what has been previously used in similar digestibility assays. Follow-up in-vitro experiments are needed to validate if the efficacy of these products differs when using broiler or layer diets due to dietary Ca buffering capacity.

Key Words: Prebiotic, Probiotic, Digestive assay, in-vitro, Salmonella