Lactic acid bacteria (LAB) have historically been used in food fermentations to preserve foods and are generally-recognized-as-safe (GRAS) by the FDA for use as food ingredients. In addition to lactic acid; some strains also produce bacteriocins that have been proposed for use as food preservatives. In this study we examined the inhibition of Listeria monocytogenes 39-2 by neutralized and non-neutralized bacteriocin preparations (Bac+ preps) produced by Lactobacillus curvatus FS47; Lb. curvatus Beef3; Pediococcus acidilactici Bac3; Lactococcus lactis FLS1; Enterococcus faecium FS56-1; and Enterococcus thailandicus FS92. Activity differences between non-neutralized and neutralized Bac+ preps in agar spot assays could not readily be attributed to acid because a bacteriocin-negative control strain was not inhibitory to Listeria in these assays. When neutralized and non-neutralized Bac+ preps were used in microplate growth inhibition assays against L. monocytogenes 39-2 we observed some differences attributed to acid inhibition. A microplate growth inhibition assay was used to compare inhibitory reactions of wild-type and bacteriocin-resistant variants of L. monocytogenes to differentiate bacteriocins with different modes-of-action (MOA) whereby curvaticins FS47 and Beef3, and pediocin Bac3 were categorized to be in MOA1; enterocins FS92 and FS56-1 in MOA2; and lacticin FLS1 in MOA3. The microplate bacteriocin MOA assay establishes a platform to evaluate the best combination of bacteriocin preparations for use in food applications as biopreservatives against L. monocytogenes.

Bacteriocin-producing (Bac+) lactic acid bacteria (LAB) comprising selected strains of Lactobacillus curvatus, Lactococcus lactis, Pediococcus acidilactici, and Enterococcus faecium and thailandicus were examined for inhibition of Listeria monocytogenes during hotdog challenge studies. The Bac+ strains, or their cell-free supernatants (CFS), were grouped according to mode-of-action (MOA) as determined from prior studies. Making a mixture of as many MOAs as possible is a practical way to obtain a potent natural antimicrobial mixture to address L. monocytogenes contamination of RTE meat products (i.e., hotdogs). The heat resistance of the bacteriocins allowed the use of pasteurization to eliminate residual producer cells for use as post-process surface application or their inclusion into hotdog meat emulsion during cooking. The use of Bac+ LAB comprising 3× MOAs directly as co-inoculants on hotdogs was not effective at inhibiting L. monocytogenes. However, the use of multiple MOA Bac+ CFS mixtures in a variety of trials demonstrated the effectiveness of this approach by showing a >2-log decrease of L. monocytogenes in treatment samples and 6–7 log difference vs. controls. These data suggest that surface application of multiple mode-of-action bacteriocin mixtures can provide for an Alternative 2, and possibly Alternative 1, process category as specified by USDA-FSIS for control of L. monocytogenes on RTE meat products.

Bacteriocin-producing (Bac+) lactic acid bacteria (LAB) were isolated from a variety of food products and animal sources. Samples were enriched in de Man, Rogosa, and Sharpe (MRS) Lactocilli broth and plated onto MRS agar plates using a “sandwich overlay” technique. Inhibitory activity was detected by the “deferred antagonism” indicator overlay method using Listeria monocytogenes as the primary indicator organism. Antimicrobial activity against L. monocytogenes was detected by 41 isolates obtained from 23 of 170 food samples (14%) and 11 of 110 samples from animal sources (10%) tested. Isolated Bac+ LAB included Lactococcus lactis, Lactobacillus curvatus, Carnobacterium maltaromaticum, Leuconostoc mesenteroides, and Pediococcus acidilactici, as well as Enterococcus faecium, Enterococcus faecalis, Enterococcus hirae, and Enterococcus thailandicus. In addition to these, two Gram-negative bacteria were isolated (Serratia plymuthica, and Serratia ficaria) that demonstrated inhibitory activity against L. monocytogenes, Staphylococcus aureus, and Enterococcus faecalis (S. ficaria additionally showed activity against Salmonella Typhimurium). These data continue to demonstrate that despite more than a decade of antimicrobial interventions on meats and produce, a wide variety of food products still contain Bac+ microbiota that are likely eaten by consumers and may have application as natural food preservatives.

Fruits and vegetables are high in nutrients that are essential for a healthy lifestyle. However, they also harbor an extensive array of microorganisms such as bacteria, which can be beneficial, neutral, or pathogenic. Foodborne pathogens can contaminate produce at any stage from the farm to the consumer’s table. Appropriate washing techniques using sanitizers can reduce the risk of pathogen contamination. Issues related to maintaining concentration, efficacy, and other problems have been a challenge for the food industry and, when left unresolved, have led to different outbreaks of foodborne illnesses. In this study, the efficacy of a lytic bacteriophage cocktail was examined for its ability to infect and reduce the contamination of Escherichia coli O157:H7 (E. coli O157:H7), in media with a high organic load, using a microplate technique. The study was conducted for 3 h to determine if the bacteriophage cocktail could reduce the pathogen in the presence of a high organic load. A significant (p < 0.05) reduction in the population of E. coli O157:H7 was observed, representing a 99.99% pathogen reduction at the end of 3 h. Fresh spinach leaves were washed in sterile potable or organic water (~9000 ppm organic load) containing E. coli O157:H7 and a bacteriophage cocktail to study the effectiveness of bacteriophages against the foodborne pathogen. Results indicated that the bacteriophage significantly (p < 0.05) reduced the contamination of E. coli O157:H7 in both situations. The study also demonstrated the bacteriophages’ ability to infect and reduce the pathogen in an organic-rich environment. This characteristic differs from commercially available sanitizers that have demonstrated a tendency to bind with the available organic load. Thus, these studies highlight the advantage of employing bacteriophages during produce wash to eliminate foodborne pathogen contamination on fruits and vegetables.

The increasing concern for food safety has created a need to evaluate novel techniques to eliminate or control pathogens, resulting in safe food. In this study, four bacteriophages of bovine origin, specific to E. coli O157:H7, were successfully isolated and characterized. A microplate reader assay demonstrated the efficacy of the bacteriophage (phage) cocktail against E. coli O157:H7 resulting in a significant reduction (p < 0.01) in the target pathogen population. The phage cocktail demonstrated significant efficacy (p < 0.05) against E. coli O157:H7 in the presence of the most utilized sanitizers in the United States, namely 100 parts per million (ppm) free chlorine and 100-ppm peroxyacetic acid. Survival in the sanitizer concentrations demonstrates the potential use of phage cocktail and sanitizer synergistically to enhance sanitation operations in the food industry.