Hurdle factors minimizing growth of Listeria monocytogenes while counteracting in situ antilisterial effects of a novel nisin A-producing Lactococcus lactis subsp. cremoris costarter in thermized cheese milks.

John Samelis,Athanasia Kakouri

doi:10.3934/microbiol.2018.1.19

Abstract

The capacity of growth, survival, and adaptive responses of an artificial contamination of a three-strain L. monocytogenes cocktail in factory-scale thermized (65 °C, 30 s) Graviera cheese milk (TGCM) was evaluated. Bulk TGCM samples for inoculation were sequentially taken from the cheese making vat before process initiation (CN-LM) and after addition of a commercial starter culture (CSC), the CSC plus the nisin A-producing (NisA+) costarter strain Lactococcus lactis subsp. cremoris M78 (CSC + M78), and all ingredients with the rennet last (CSC + M78-RT). Additional treatments included Listeria-inoculated TGCM samples coinoculated with the NisA+ costarter strain M78 in the absence of the CSC or with the CSC in previously sterilized TGCM to inactivate the background microbiota (CSC-SM). All cultures were incubated at 37 to 42 °C for 6 h, followed by additional 66 h at 22 °C, and 48 h at 12 °C after addition of 2% edible salt. L. monocytogenes failed to grow and declined in all CSC-inoculated treatments after 24 h. In contrast, the pathogen increased by 3.34 and 1.46 log units in the CN-LM and the CSC-SM treatments, respectively, indicating that the background microbiota or the CSC alone failed to suppress it, but they did so synergistically. Supplementation of the CSC with the NisA+ strain M78 did not deliver additional antilisterial effects, because the CSC Streptococcus thermophilus reduced the growth prevalence rates and counteracted the in situ NisA+ activity of the costarter. In the absence of the CSC, however, strain M78 predominated and caused the strongest in situ nisin-A mediated effects, which resulted in the highest listerial inactivation rates after 24 to 72 h at 22 °C. In all TGCM treatments, however, L. monocytogenes displayed a “tailing” survival (1.63 to 1.96 log CFU/mL), confirming that this pathogen is exceptionally tolerant to cheese-related stresses, and thus, can't be easily eliminated.

Highlights

Most traditional cooked-hard, hard or semi-hard cheeses, such as the Greek Graviera [11,12], the French Cantal [13], and the popular varieties Edam, Gouda [14,15] and Cheddar [16,17] usually have pH and aw values higher than the EC regulatory ―safe-value‖ ranges, they are not supportive of L. monocytogenes growth [11,12,13,14,15,16,17]
Bulk TGCM samples for inoculation were sequentially taken from the cheese making vat before process initiation (CN-LM) and after addition of a commercial starter culture (CSC), the CSC plus the nisin A-producing (NisA+) costarter strain Lactococcus lactis subsp. cremoris M78 (CSC + M78), and all ingredients with the rennet last (CSC + M78-RT)
We showed that the primary hurdle factors which appeared to reduce the growth and in situ nisin A production and activity of this novel L. lactis subsp. cremoris costarter culture were responsible for the simultaneous growth suppression of the artificial L. monocytogenes contamination in factory-scale prepared TGCMs and in simulating Graviera cooked cheese curd environments

Summary

Introduction

Most traditional cooked-hard, hard or semi-hard cheeses, such as the Greek Graviera [11,12], the French Cantal [13], and the popular varieties Edam, Gouda [14,15] and Cheddar [16,17] usually have pH and aw values higher than the EC regulatory ―safe-value‖ ranges, they are not supportive of L. monocytogenes growth [11,12,13,14,15,16,17]. One of the most promising measures to enhance inactivation of L. monocytogenes in cheese may be the addition to the processed milk of novel bacteriocin-producing (Bac+) strains of lactic acid bacteria (LAB) as starter, costarter or adjunct bioprotective cultures [24,25,26]. L. lactis strains that produce nisin A (NisA+), nisin Z or lacticins 3147 and 481, singly or in combinations, have been evaluated as antagonistic costarter strains, supplementing the primary starter culture, in cheese and other dairy products [25,31,32,33]. The direct use of Bac+ (Nis+) starter or adjunct cultures in fermented dairy products appears to be more advantageous than addition of nisin (e.g., Nisaplin) or other bacteriocins because it may solve several problems associated with the in situ loss of activity or degradation of the active peptides by irreversible

Methods

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Conclusion