Behavior of artificial listerial contamination in model Greek Graviera cheeses manufactured with the indigenous nisin A‐producing strain Lactococcus lactis subsp. cremoris M104 as costarter culture

Abstract

Growth of Listeria monocytogenes during processing of traditional Greek Graviera cheese is inhibited, but the pathogen may survive ripening. Therefore, this study used Lactococcus lactis subsp. cremoris M104, a nisin A‐producing (NisA+) raw milk isolate, to enhance inactivation of a nonpathogenic Listeria cocktail contaminated in model Graviera mini cheeses. Cheeses were manufactured from thermized milk with a commercial starter culture (CSC) or the CSC plus strain M104 (CSC + M104), ripened at 18 °C and 90% RH for 20 days and stored at 4 °C in vacuum for 60 days. Listeria populations declined 10‐fold in all fermenting cheeses but then survived with little death during ripening and storage. NisA+ M104 colonies were prevalent and the nisA gene was detected, but nisin activity was weak to undetectable, in all CSC + M104 cheeses. Thus, Graviera cheese processing should be optimized to ensure nisA gene expression by strain M104 at levels sufficient to increase inactivation of L. monocytogenes. Practical applications This study provided important preliminary evidence that the in situ NisA+ antilisterial activity of L. lactis subsp. cremoris M104 during Graviera cheese fermentation increased by reducing the curd cooking temperature from 48 °C to 42 °C. This indicated the feasibility to increase nisin production and activity by suitably modifying selected technological factors during traditional cooked hard cheese processing. Apart from its bioprotective potential, this NisA+ L. lactis subsp. cremoris genotype (represented by the indigenous strains M104 and M78) possesses desirable sugar fermentation capabilities and enzymatic activities similar to those of the best performing industrial L. lactis or L. cremoris strains in commercial starters (Parapouli et al., ). Since 2015 this novel lactococcal genotype has been applied as hand‐made, costarter culture for the production of commercial Greek Graviera cheeses characterized by specific flavor characteristics and improved textural properties than counterpart cheeses produced with CSCs only (Pappas Bros. collaborating cheese plant, Epirus; private communication).

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

“…Two virulent L. monocytogenes strains of cheese origin, ISS G79 (serotype 1/2b) and ISS G185 (serotype 1/2a) plus L. monocytogenes no.10 (serotype 4ab), an avirulent reference strain, were used in accordance with previous studies [44],[46],[49]. Frozen (−30 °C) stock cultures of each strain in tryptic soy broth with 0.6% yeast extract (Lab M, Heywood, UK) plus 20% glycerol were activated twice (30 °C, 24 h) in 10 mL of brain heart infusion (BHI) broth (Lab M) before use.…”

“…The flasks were then returned in the 37 °C incubation chamber for another 3 h and then were shifted at 22 °C in a cooling incubator for additional 66 h. The excessive whey that was separated above the broken granular curd in the CSC + M78-RT flasks only was not drained out to maintain equal cultured milk volumes in all challenge treatments. After 72 h, 2% edible sea salt was added to each flask and incubation continued at 12 °C for 5 days (120 h) to simulate the brining process at the usual salt level of ripened Graviera cheeses [20],[46]. All samples were analyzed at inoculation (0 h), after 3 h at 37 to 42 °C, another 3 h at 37 °C, 24 and 72 h at 22 °C, and 120 h at 12 °C.…”

“…Recent studies of the Dairy Research Institute focused on the commercial application of an indigenous NisA+ L. lactis subsp. cremoris rare genotype (strains M78 and M104) isolated from Greek raw milk [44],[45] as novel, craft-made, costarter adjunct culture in traditional Greek cooked hard cheese manufacture [46]. With regard to the NisA+ activity of strains M78 and/or M104, it was strong in pure culture in model sterile cheese milk [47] as well as in commercially thermized milks [48].…”

“…With regard to the NisA+ activity of strains M78 and/or M104, it was strong in pure culture in model sterile cheese milk [47] as well as in commercially thermized milks [48]. Both strains, however, failed to exhibit similar strong NisA+ activity levels in thermized Graviera cheese milks (TGCM) and model Graviera minicheeses manufactured with addition of CSCs containing Streptococcus thermophilus as the primary cheese acidifier [46],[49]. These findings prompted to conduct quantitative real-time PCR (qRT-PCR) studies, which revealed that the NisA+ costarter strain M78 was capable of promoting sufficient growth and nisA gene expression in the core of commercial CSC-inoculated Graviera cheeses (14–15 kg each) for at least one week after manufacture [50].…”

Risk assessment of Listeria monocytogenes in Gouda cheese

“…In such cases, nisin can inhibit growth of L. monocytogenes in cheese Samelis et al, 2017), but due to proteolysis during ripening, its effect can be reduced over time ).…”