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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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(Figure 6B with error bars) Growth inhibition assay showing the activity of Listeria monocytogenes 39-2 (R2) treated with culture supernatants from: None (R2 Cont.), Lb. delbrueckii 4797-2 (Bac−), Lb. curvatus FS47, P. acidilactici Bac3, Lb. curvatus Beef3, En. faecium FS56-1, En. thailandicus FS92, and Lc. lactis FLS1. Growth inhibition assay using neutralized culture supernatants. Data points represent the means of triplicate replications and error bars represent the standard deviations from the means (error bars were not used for all curves in the main paper to prevent clutter). Treatments with different lowercase letters are significantly different (p < 0.05); letters in brackets are for entire group of graph lines.
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biomolecules-05-01178-f008: (Figure 6B with error bars) Growth inhibition assay showing the activity of Listeria monocytogenes 39-2 (R2) treated with culture supernatants from: None (R2 Cont.), Lb. delbrueckii 4797-2 (Bac−), Lb. curvatus FS47, P. acidilactici Bac3, Lb. curvatus Beef3, En. faecium FS56-1, En. thailandicus FS92, and Lc. lactis FLS1. Growth inhibition assay using neutralized culture supernatants. Data points represent the means of triplicate replications and error bars represent the standard deviations from the means (error bars were not used for all curves in the main paper to prevent clutter). Treatments with different lowercase letters are significantly different (p < 0.05); letters in brackets are for entire group of graph lines.

Mentions: Another observation in the study was the tailing of most of the growth curves in the microplate assays (Figure 4, Figure 5 and Figure 6). Although the reason was not ascertained, it is likely due to a slow lysis of the bacterial cells as bacteriocins form membrane pores in susceptible cells and/or the result of a change in cellular morphology upon nutrient depletion during extended stationary phase which may have an effect on optical density. Figure 5B with error bars has been published as an Appendix Figure A1; likewise, Figure 6B with error bars has been published as an Appendix Figure A2.


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

Vijayakumar PP, Muriana PM - Biomolecules (2015)

(Figure 6B with error bars) Growth inhibition assay showing the activity of Listeria monocytogenes 39-2 (R2) treated with culture supernatants from: None (R2 Cont.), Lb. delbrueckii 4797-2 (Bac−), Lb. curvatus FS47, P. acidilactici Bac3, Lb. curvatus Beef3, En. faecium FS56-1, En. thailandicus FS92, and Lc. lactis FLS1. Growth inhibition assay using neutralized culture supernatants. Data points represent the means of triplicate replications and error bars represent the standard deviations from the means (error bars were not used for all curves in the main paper to prevent clutter). Treatments with different lowercase letters are significantly different (p < 0.05); letters in brackets are for entire group of graph lines.
© Copyright Policy
Related In: Results  -  Collection

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

biomolecules-05-01178-f008: (Figure 6B with error bars) Growth inhibition assay showing the activity of Listeria monocytogenes 39-2 (R2) treated with culture supernatants from: None (R2 Cont.), Lb. delbrueckii 4797-2 (Bac−), Lb. curvatus FS47, P. acidilactici Bac3, Lb. curvatus Beef3, En. faecium FS56-1, En. thailandicus FS92, and Lc. lactis FLS1. Growth inhibition assay using neutralized culture supernatants. Data points represent the means of triplicate replications and error bars represent the standard deviations from the means (error bars were not used for all curves in the main paper to prevent clutter). Treatments with different lowercase letters are significantly different (p < 0.05); letters in brackets are for entire group of graph lines.
Mentions: Another observation in the study was the tailing of most of the growth curves in the microplate assays (Figure 4, Figure 5 and Figure 6). Although the reason was not ascertained, it is likely due to a slow lysis of the bacterial cells as bacteriocins form membrane pores in susceptible cells and/or the result of a change in cellular morphology upon nutrient depletion during extended stationary phase which may have an effect on optical density. Figure 5B with error bars has been published as an Appendix Figure A1; likewise, Figure 6B with error bars has been published as an Appendix Figure A2.

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