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A Microplate Growth Inhibition Assay for Screening Bacteriocins against Listeria monocytogenes to Differentiate Their Mode-of-Action.

Vijayakumar PP, Muriana PM - Biomolecules (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Animal Science, Oklahoma State University, Monroe Street, Stillwater, OK 74078, USA. paul.v@uky.edu.

ABSTRACT
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.

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Treatment of culture supernatants produced by Lb. delbrueckii 4797-2 (Bac−) and Bac+ strains: Lb. curvatus FS47, P. acidilactici Bac3, Lb. curvatus Beef3, En. faecium FS56-1, En. thailandicus FS92, and Lc. lactis FLS1. (A) Comparison of bacteriocin activity after filter sterilization or heat pasteurization; (B) The pH of MRS broth media before inoculation and after overnight growth of various strains used in this study; (C) Bacteriocin activity titers of cell-free supernatants against L. monocytogenes 39-2, before and after neutralization. Treatments with the same culture supernatant that have different lower case letters are significantly different (p < 0.05); treatments with the same lowercase letter are not significantly different (p > 0.05). No differences were found in duplicate replications of samples in (A) and (C).
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biomolecules-05-01178-f001: Treatment of culture supernatants produced by Lb. delbrueckii 4797-2 (Bac−) and Bac+ strains: Lb. curvatus FS47, P. acidilactici Bac3, Lb. curvatus Beef3, En. faecium FS56-1, En. thailandicus FS92, and Lc. lactis FLS1. (A) Comparison of bacteriocin activity after filter sterilization or heat pasteurization; (B) The pH of MRS broth media before inoculation and after overnight growth of various strains used in this study; (C) Bacteriocin activity titers of cell-free supernatants against L. monocytogenes 39-2, before and after neutralization. Treatments with the same culture supernatant that have different lower case letters are significantly different (p < 0.05); treatments with the same lowercase letter are not significantly different (p > 0.05). No differences were found in duplicate replications of samples in (A) and (C).

Mentions: Bacteriocin culture preparations were examined for heat resistance, both as a potential replacement for filter sterilization and as an indication that bacteriocin preparations would survive applications in heated foods. We observed no loss in activity when centrifuged/heat treated bacteriocin preparations were compared to filter-sterilized preparations, nor any differences between duplicate samples (Figure 1A). In subsequent heating trials, we increased the temperature to 85 °C for 15–20 min, because of the use of larger samples, obtaining similar results. The ability to survive high heat treatment without loss of activity demonstrates that these bacteriocins may be added to foods that will be heated and still retain activity. The bacteriocins used in this study demonstrate thermal stability under conditions simulating pasteurization and confirm heat resistance observed in other bacteriocins [10].


A Microplate Growth Inhibition Assay for Screening Bacteriocins against Listeria monocytogenes to Differentiate Their Mode-of-Action.

Vijayakumar PP, Muriana PM - Biomolecules (2015)

Treatment of culture supernatants produced by Lb. delbrueckii 4797-2 (Bac−) and Bac+ strains: Lb. curvatus FS47, P. acidilactici Bac3, Lb. curvatus Beef3, En. faecium FS56-1, En. thailandicus FS92, and Lc. lactis FLS1. (A) Comparison of bacteriocin activity after filter sterilization or heat pasteurization; (B) The pH of MRS broth media before inoculation and after overnight growth of various strains used in this study; (C) Bacteriocin activity titers of cell-free supernatants against L. monocytogenes 39-2, before and after neutralization. Treatments with the same culture supernatant that have different lower case letters are significantly different (p < 0.05); treatments with the same lowercase letter are not significantly different (p > 0.05). No differences were found in duplicate replications of samples in (A) and (C).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4496717&req=5

biomolecules-05-01178-f001: Treatment of culture supernatants produced by Lb. delbrueckii 4797-2 (Bac−) and Bac+ strains: Lb. curvatus FS47, P. acidilactici Bac3, Lb. curvatus Beef3, En. faecium FS56-1, En. thailandicus FS92, and Lc. lactis FLS1. (A) Comparison of bacteriocin activity after filter sterilization or heat pasteurization; (B) The pH of MRS broth media before inoculation and after overnight growth of various strains used in this study; (C) Bacteriocin activity titers of cell-free supernatants against L. monocytogenes 39-2, before and after neutralization. Treatments with the same culture supernatant that have different lower case letters are significantly different (p < 0.05); treatments with the same lowercase letter are not significantly different (p > 0.05). No differences were found in duplicate replications of samples in (A) and (C).
Mentions: Bacteriocin culture preparations were examined for heat resistance, both as a potential replacement for filter sterilization and as an indication that bacteriocin preparations would survive applications in heated foods. We observed no loss in activity when centrifuged/heat treated bacteriocin preparations were compared to filter-sterilized preparations, nor any differences between duplicate samples (Figure 1A). In subsequent heating trials, we increased the temperature to 85 °C for 15–20 min, because of the use of larger samples, obtaining similar results. The ability to survive high heat treatment without loss of activity demonstrates that these bacteriocins may be added to foods that will be heated and still retain activity. The bacteriocins used in this study demonstrate thermal stability under conditions simulating pasteurization and confirm heat resistance observed in other bacteriocins [10].

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Animal Science, Oklahoma State University, Monroe Street, Stillwater, OK 74078, USA. paul.v@uky.edu.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus