Effect of Pressure, Reconstituted RTE Meat Microbiota, and Antimicrobials on Survival and Post-pressure Growth of Listeria monocytogenes on Ham

Abstract

Pressure treatment of ready-to-eat (RTE) meats extends the shelf life and reduces risks associated with Listeria monocytogenes. However, pressure reduces numbers of Listeria on ham by less than 5 log (CFU/g) and pressure effects on other meat microbiota are poorly documented. This study investigated the impact of pressure and RTE meat microbiota, with or without nisin and rosemary oil, on survival of Listeria after refrigerated storage. Ham was inoculated with a 5-strain cocktail of L. monocytogenes alone or with a cocktail of RTE meat microbiota consisting of Brochothrix thermosphacta, Carnobacterium maltaromaticum, Leuconostoc gelidum, and Lactobacillussakei. Products were treated at 500 MPa at 5°C for 1 or 3 min, with or without rosemary extract or nisin. Surviving cells were differentially enumerated after pressure treatment and after 4 weeks of refrigerated storage. After 4 weeks of storage, products were also analyzed by high throughput sequencing of 16S rRNA amplicons. Pressure treatment reduced counts of Listeria by 1 to 2 log (CFU/g); inactivation of RTE meat microbiota was comparable. Counts of Listeria increased by 1-3 log (CFU/g) during refrigerated storage. RTE meat microbiota did not influence pressure inactivation of Listeria but prevented growth of Listeria during refrigerated storage. Rosemary extract did not influence bacterial inactivation or growth. The combination of nisin with pressure treatment for 3 min reduced counts of Listeria and meat microbiota by >5 log (CFU/g); after 4 weeks of storage, counts were below the detection limit. In conclusion, pressure alone does not eliminate Listeria or other microbiota on RTE ham; however, the presence of non-pathogenic microbiota prevents growth of Listeria on pressure treated ham and has a decisive influence on post-pressure survival and growth.

Keywords: Lactobacillus sakei; Leuconostoc gelidum; Listeria monocytogenes; antimicrobials; high pressure processing; meat microbiota; nisin; ready-to-eat meat.

FIGURE 1

Effect of pressure on survival and post-pressure growth of L. monocytogenes and RTE meat microbiota on ham treated with rosemary extract or an equivalent volume of 0.5% ethanol as solvent control. Shown are cell counts of ham inoculated with a 5-strain cocktail of L. monocytogenes (A) or RTE meat microbiota (B) after treatment at 500 MPa for 1 or 3 min without (white) or with (gray) rosemary extract at ∼325 μg/cm². Cell counts of L. monocytogenes and other strains were monitored immediately after pressure treatment (solid columns) and after storage for 4 weeks at 4°C (hatched columns). Surviving cells were enumerated by surface plating on non-selective TS (L. monocytogenes) or APT agar (RTE meat microbiota). Data are shown as means ± standard deviations of triplicate independent experiments. Treatment means (solid columns) within each panel with different letters are significantly different (P < 0.05). The interception point of abscissa and ordinate represents the highest detection limit.

FIGURE 2

Effect of pressure and RTE meat microbiota on survival and post-pressure growth of L. monocytogenes and RTE meat microbiota on ham treated with 0.5% ethanol (solvent control) or an equivalent volume of rosemary extract in 0.5% ethanol. Shown are total cell counts (A) on non-selective agar and cell counts of L. monocytogenes (B) enumerated on selective PALCAM (B) agar. Samples were treated at 500 MPa for 1 or 3 min without (white) or with (gray) rosemary at ∼325 μg/cm². Cell counts were enumerated immediately after pressure treatment (solid columns) and after storage for 4 weeks at 4°C (hatched columns). Data are shown as means ± standard deviations of triplicate independent experiments. Treatment means (solid columns) within each panel with different letters are significantly different (P < 0.05). The interception point of abscissa and ordinate represents the highest detection limit.

FIGURE 3

Effect of pressure on survival and post-pressure growth of L. monocytogenes and RTE meat microbiota on ham. Ham was inoculated with a 5-strain cocktail of L. monocytogenes (A) or RTE meat microbiota (B) and treated at 500 MPa for 1 or 3 min with 0.02 N HCl (solvent control) or an equivalent volume of a nisin preparation in 0.02 N HCl to achieve a concentration of 2 μg/ cm², corresponding to 0.05 μg/cm² pure nisin. Cell counts of L. monocytogenes and RTE meat microbiota were monitored immediately after pressure treatment (solid columns) and after storage for 4 weeks at 4°C (hatched columns). Surviving cells were enumerated on non-selective TS (L. monocytogenes) or APT agar (other strains). Data are shown as means ± standard deviations of triplicate independent experiments. Treatment means (solid columns) within each panel with different letters are significantly different (P < 0.05). The interception point of abscissa and ordinate represents the highest detection limit.

FIGURE 4

Effect of pressure and RTE meat microbiota on survival and post-pressure growth of L. monocytogenes and RTE meat microbiota on ham treated with 0.02 N HCl (solvent control) or an equivalent volume of a nisin preparation in 0.02 N HCl to achieve a concentration of 2 μg/cm², corresponding to 0.05 μg/cm² pure nisin. Shown are total cell counts (A) on non-selective agar and cell counts of L. monocytogenes (B) enumerated on selective PALCAM (B) agar. Samples were treated at 500 MPa for 1 or 3 min without (white) or with (gray) nisin. Cell counts ere enumerated immediately after pressure treatment (solid columns) and after storage for 4 weeks at 4°C (hatched columns). Data are shown as means ± standard deviations of triplicate independent experiments. Treatment means (solid columns) within each panel with different letters are significantly different (P < 0.05). The interception point of abscissa and ordinate represents the highest detection limit.

FIGURE 5

Principal component analysis (PCA) of the % abundance of 16S rRNA sequences on ham containing a nisin preparation, rosemary extract, or without additions after storage for 4 weeks at 4°C. Ham was inoculated with 5-strain cocktails of L. monocytogenes and RTE meat microbiota, treated for 1 or 3 min at 500 MPa, or left untreated. The percentage of variation explained by the plotted principal coordinates is indicated on the axes. Each symbol represents a type of sample and each color represents a treatment: no addition (), or controls with 0.5% ethanol (•) or 0.02N HCl (), rosemary extract (), nisin (); no pressure treatment (blue), 1 min at 500 MPa (red), 3 min at 500 MPa (black).