The behavior of Listeria monocytogenes on fully ripened Greek Graviera cheese was evaluated. Three batches (A, B, and C) were tested. Batches A and C were prepared with a commercial starter culture, while in batch B the starter culture was combined with an enterocin-producing Enterococcus faecium Graviera isolate. Cheese pieces were surface inoculated with a five-strain cocktail of L. monocytogenes at ca. 3 log CFU/cm2, packed under air or vacuum conditions, stored at 4, 12, or 25 degrees C, and analyzed after 0, 3, 7, 15, 30, 60, and 90 days. L. monocytogenes did not grow on the cheese surface, regardless of storage conditions. However, long-term survival of the pathogen was noted in all treatments, being the highest (P < 0.05) at 4 degrees C under vacuum conditions. Overall, the lower the storage temperature, the higher and longer the survival of L. monocytogenes was. Although enterocin A-specific PCR products were detected in situ in cheese batch B, inhibition of L. monocytogenes by the enterocin-producing strain was not enhanced compared with batches A and C, which also contained enterocin A, but in lower amounts. Additionally enterocins B, P, L50A, and L50B; lactococcin G; and plantaricin A genes were detected in all batches, suggesting that indigenous bacteriocin-producing lactic acid bacteria might contribute to Listeria inhibition in cheese. In conclusion, Graviera cheeses that may be accidentally contaminated in retail at the European Union maximal allowable level of 100 CFU/cm2 or g are at low risk regarding a potential outgrowth of L. monocytogenes, which, however, may survive for a long period during cheese storage.
The current microbiological regulatory criteria in the European Union specify a maximum Listeria monocytogenes population of 100 CFU/g allowable in ready-to-eat foods provided the product will not exceed this limit throughout its shelf life. The aim of this study was to validate the manufacturing method for traditional Greek Graviera cheese produced from thermized milk. Initial challenge experiments evaluated the fate of inoculated L. monocytogenes (ca. 4 log CFU/ml, three-strain cocktail) in thermized Graviera cheese milk (TGCM; 63 degrees C for 30 s) in the presence and absence of a product-specific starter culture (SC) in vitro. Milk samples were incubated for 6 h at 37 degrees C and then for 66 h at 18 degrees C. Experiments were conducted to evaluate the fate of a cocktail of three nonpathogenic L. monocytogenes and L. innocua indicator strains inoculated (ca. 3 log CFU/g) in Graviera cheeses commercially manufactured from TGCM+SC. Cheeses were brined, ripened at 18 degrees C and 90% relative humidity for 20 days, and stored at 4 degrees C for up to day 60 under vacuum. In TGCM, L. monocytogenes increased by ca. 2 log units, whereas in TGCM+SC L. monocytogenes growth was retarded (P < 0.05) after a ca. 1-log increase within 6 h at 37 degrees C. Populations of Listeria indicator strains did not grow in TGCM+SC cheeses at any stage; they declined 10-fold in fresh cheeses within 5 days and then survived with little death thereafter. Thus, growth inhibition but not inactivation of potent natural Listeria contaminants at levels below 100 CFU/g occurs in the core of traditional Greek Graviera cheese during fermentation, ripening, and storage.
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).
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