Purpose: To determine antagonistic activity of secondary metabolites produced by a Bacillus subtilis strain against Salmonella in vitro and during alfalfa seeds sprouting.
Methods: A Bacillus subtilis strain isolated from biofertilized soil of blackberry crops in Queretaro, Mexico showing antagonistic capacity was grown in tryptic soy broth (4 days at 35°C, 180 rpm). Supernatant was recovered and used to determine antagonist effect against five strains of Salmonella using well diffusion test on tryptic soy agar (TSA) by triplicate. Secondary metabolites were precipitated by acidification with HCl (pH 2) for 24 h, resuspended in sterile water and used to irrigate alfalfa seeds inoculated with a Salmonella cocktail (~6 log CFU/g) during germination for 5 days. Aerobic mesophilic bacteria (AMB) and Salmonella were enumerated periodically in TSA and TSA supplemented with rifampicin, respectively.
Results: In vitro test of supernatant showed inhibition halos against all Salmonella strains (range 11.17 - 19.91 mm). After five days, alfalfa sprouts irrigated with secondary metabolites, Salmonella population was reduced by 5.26 ± 0.09 log CFU/g, whereas sprouts irrigated with sterile water only showed a 1.21 ± 0.05 log CFU/g reduction. AMB did not showed significance differences (Tukey, P < 0.05). Additionally, growth of alfalfa was promoted with secondary metabolites showing a final length of 13.8 cm ± 0.8 compared to 7.13 cm ± 0.63 in those sprouts irrigated with water.
Significance: Application of secondary metabolites produced by this Bacillus subtilis strain could be a strategy to eliminate Salmonella on alfalfa sprouts and reducing sprouting time.