Efficacy of Lactobacillus salivarius (L28) to control foodborne pathogens in a variety of matrices

Listeria monocytogenes (L. monocytogenes), Salmonella, and Escherichia coli O157:H7 (E.coli O157:H7) all pose a significant threat to food safety across the United States (U.S). Policies in place help diminish cross-contamination onto foodstuffs, but prevalence of these organisms has not diminished. Lactic acid bacteria (LAB) have been regarded as Generally Recognized as Safe by the U.S. Food and Drug Administration (FDA), allowing them as food additives. The antimicrobial properties of LAB are well known, but innovative ways to utilize LAB strains in the food industry need to be further explored. The objective of this study was to assess Lactobacillus salivarius (L28)’s ability to inhibit foodborne pathogens in a variety of matrices, which include stainless steel, dry pet food, and human colorectal epithelial adenocarcinoma cells (Caco-2). The experiment was divided into three parts; 1.) Reduction of L. monocytogenes attachment on stainless steel utilizing L28 at different temperatures and concentrations and its efficacy as a pre-treatment to prevent future attachment, 2.) Examination of the fate of Salmonella spp. present in raw chicken fat for pet kibble coating in tandem with L28 biocontrol agent, and 3.) Examination of the displacement of Salmonella and E. coli O157:H7 on caco-2 cells by L28.
During the first phase of the project, stainless steel coupons were inoculated with L. monocytogenes for 24h at 12°C and 25°C, the coupons were treated with various concentrations of L28 for 24h. Consequently, L28 significantly (P<0.05) inhibited L. monocytogenes attachment at both temperatures and at most concentrations. After the reduction was noted, stainless steel coupons were pre-treated with L28 at the same concentrations and temperatures, then inoculated with L. monocytogenes for an hour. All pre-treatment concentrations significantly prevented L. monocytogenes attachment at 25°C, only the high concentration overnight cultures were able to provide significant inhibition at 12°C. For the second phase of the project, raw chicken fat was inoculated with a five-strain cocktail of Salmonella to mimic Salmonella contamination. The Salmonella inoculated chicken fat was assigned to one of two treatments in which half was inoculated with L28, and the other half served as the control. The chicken fat was applied onto the kibble and homogenized in a tumble drum, the target concentration of L28 was 106 CFU/lb of kibble. The kibble was allowed to dry for 4h, then the samples were stored at 25°C for 72h. Time points were plated for Salmonella enumeration at 0h, 4h, 24h, 48h, and 72h for the chicken fat and 0h, 4h, and 72h for the pet kibble. L28 was unsuccessful at inhibiting Salmonella in raw chicken fat 25°C during the 72h period. Salmonella counts per gram of chicken fat at the zero hour timepoint did not differ statistically (P>0.05) from the seventy-two hours timepoint for the control or the treatment. L28 was effective at inhibiting Salmonella on dry pet kibble stored at 25°C. At each timepoint, 0h, 4h, and 72h L28 treatments showed significant Salmonella reduction (P<0.05) when compared to their respective control. Lastly, human epithelial adenocarcinoma ATCC cell line, caco-2, were grown to confluency and seeded into 24-well plates. The target density for the caco-2 cells was 5.0 x 104 cells/well. The treatment groups consisted of Salmonella control, E. coli O157:H7 control, L28 control, L28 and Salmonella co-inoculation and L28 and E. coli O157:H7 co-inoculation. Samples were incubated at 37°C for 30 min, the caco-2 cells were rinsed, detached and lysed. Samples were then plated for enumeration. Bacterial counts were reported as number of bacterial cells per 100 caco-2 cells. Results showed significant differences (P<0.05) between the Salmonella control and treatment means, showing significant reduction from our L28 treatment. Escherichia coli O157:H7 attachment to caco-2 cells did not differ between the control and treatment groups (P>0.05). Lactobacillus salivarius attachment onto caco-2 cells was not significantly impacted (P>0.05) by the presence of Salmonella or E. coli O157:H7. Overall, L28 was successful at inhibiting and preventing L. monocytogenes attachment onto stainless steel at both 12°C and 25°C. L28 was also successful at inhibiting Salmonella on dry pet food coated with chicken fat when applied in tandem. Furthermore, L28 was successful at inhibiting Salmonella but not E. coli O157:H7 on caco-2 cells. The results of these projects suggest potential for Lactobacillus salivarius (L28) as a biocontrol agent to further improve food safety directly or indirectly.