Evaluation of bioprotective potential of Bacillus species cell-free supernatant against Listeria species in cultured ingredient for application to cottage cheese

ABSTRACT: Listeria monocytogenes is a foodborne pathogen problematic for the dairy industry, prompting dairy manufacturers to seek antilisterial additives to reduce the risk in dairy products that support the survival and growth. Bacillus spp. have been explored as being potential producers of antilisterial metabolites to inhibit Listeria spp. Bacillus subtilis and Bacillus pumilus were cultured in a food-grade medium mimicking tryptic soy broth supplemented with yeast extract (FG-TSBYE) and processed into foamed cell-free supernatant (CFS-F) to produce a cultured ingredient. This cultured ingredient was evaluated for antilisterial activity in a 96-well broth assay against Listeria target strains (n = 3). Bacillus subtilis Y487 was found to completely inhibit growth of all 3 Listeria target strains, whereas B. pumilus strains (HD49A and VF0409D) produced inhibitory activity against 2 out of 3 strains. Bacillus CFS-F showed good reproducibility with no significant difference found between independent cultures on the same plate. Biological reproducibility was variable for Listeria strains in response to Bacillus CFS-F. Listeria monocytogenes WRLP42 showed some consistency of technical replication (same plate) and good biological replication (across plates), whereas Listeria innocua WRLP438 and L. monocytogenes WRLP96 showed high variability among technical replicates (same plate) and high variability among biological target replication (across plates). Cottage cheese was formulated with freeze-dried CFS-F (CFS-F-FD) and inoculated with Listeria target strains (n = 3) and stored at 7°C for 25 d. The CFS-F-FD from B. pumilus VF0409D and B. subtilis Y487 were effective in delaying the lag phase of L. monocytogenes WRLP96 in cottage cheese by 7 d; however, CFS-F-FD induced changes in the texture and appearance of the cottage cheese. This proof-of-concept study demonstrates the efficacy of Bacillus spp. cell-free supernatant as a potential Listeria control measure in dairy foods. Further research is necessary to optimize antilisterial production and cell-free supernatant processing to minimize product changes.