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Mixed thermophilic and mesophilic commercial starter cultures (CSCs), particularly those including Streptococcus thermophilusas a primary milk acidifier, have been found to reduce growth and counteract in situ nisin A (NisA+) antilisterial effects by the novel, indigenous Lactococcus lactissubsp. cremorisM78 costarter in traditional Graviera thermized milk cheese curds. Therefore, this model challenge study evaluated growth and in situ NisA+ activity of strain M78 in coculture with S. thermophilusST1 singly in sterilized raw milk (SRM). Strain ST1, derived from a CSC for cheese, was challenged at two inoculation levels (5 and 7 log CFU/mL) in SRM against 6 and 3 log CFU/mL of strain M78 and Listeria monocytogenes,respectively. Pure cultures of each strain and cocultures of strain ST1 with the CSC L. lactisLL2, in replacement of strain M78, served as controls. At the high (7-log) inoculation level, the rapid, competitive growth (>9.3 log CFU/mL) of S. thermophilusST1 reduced growth of both L. lactisby at least 10-fold; the industrial strain LL2 retained slightly higher relative population densities (7.4 to 9.1%) than the wild NisA+ strain M78 (3.8 to 5.6%) after 6 h at 37°C, followed by an additional 66 h of incubation at 22°C. In full contrast, at the low (5-log) inoculation level, S. thermophilusST1 failed to predominate in SRM at 6 h; thus, the starter lactic acid bacteria populations were reversed in favor of L. lactis. Notably, strain M78 retained higher relative population densities (83.0 to 90.1%) than the CSC strain LL2 (80.3 to 85.2%) at 22°C. Moreover, at the 5-log ST1 level, the direct and deferred in situ NisA+ activities of strain M78 were at similar levels with its pure culture with L. monocytogenesin SRM, whereas at the 7-log ST1 level, the respective NisA+ effects were counteracted. Hence, 10- to 100-fold lowered inoculation levels of CSC S. thermophilusare required to enhance the performance of the M78 costarter in traditional Greek cheese technologies.

This study evaluated in situ expression of the nisAgene by an indigenous, nisin A–producing (NisA+) Lactococcus lactissubsp. cremorisraw milk genotype, represented by strain M78, in traditional Greek Graviera cheeses under real factory-scale manufacturing and ripening conditions. Cheeses were produced with added a mixed thermophilic and mesophilic commercial starter culture (CSC) or with the CSC plus strain M78 (CSC+M78). Cheeses were sampled after curd cooking (day 0), fermentation of the unsalted molds for 24 h (day 1), brining (day 7), and ripening of the brined molds (14 to 15 kg each) for 30 days in a fully controlled industrial room (16.5°C; 91% relative humidity; day 37). Total RNA was directly extracted from the cheese samples, and the expression of nisAgene was evaluated by real-time reverse transcription PCR (qRT-PCR). Agar overlay and well diffusion bioassays were correspondingly used for in situ detection of the M78 NisA+ colonies in the cheese agar plates and antilisterial activity in whole-cheese slurry samples, respectively. Agar overlay assays showed good growth (>8 log CFU/g of cheese) of the NisA+ strain M78 in coculture with the CSC and vice versa. The nisAexpression was detected in CSC+M78 cheese samples only, with its expression levels being the highest (16-fold increase compared with those of the control gene) on day 1, followed by significant reduction on day 7 and almost negligible expression on day 37. Based on the results, certain intrinsic and mainly implicit hurdle factors appeared to reduce growth prevalence rates and decrease nisAgene expression, as well as the nisin A–mediated antilisterial activities of the NisA+ strain M78 postfermentation. To our knowledge, this is the first report on quantitative expression of the nisAgene in a Greek cooked hard cheese during commercial manufacturing and ripening conditions by using a novel, rarely isolated, indigenous NisA+ L. lactissubsp. cremorisgenotype as costarter culture.

Enterococcus faeciumKE82, isolated from traditional Greek Graviera cheese, was identified in pure broth cultures in vitro as a multiple enterocin–producing bacterial strain possessing the structural entA, entB, and entPenterocin genes. E. faeciumKE82 was further assessed for in situ antilisterial activity in raw milk (RM) and commercially thermized milk (TM; 63°C for 30 s) in the presence of the indigenous microbiota and in sterile raw milk (SRM; 121°C for 5 min) with or without the addition of two commercial starter culture (CSC) strains Streptococcus thermophilusand Lactococcus lactis. Growth of Listeria monocytogeneswas completely inhibited in RM incubated at 37°C for 6 h, whereas the pathogen was significantly inactivated in RM+KE82 samples during further incubation at 18°C for 66 h. In contrast, L. monocytogeneslevels increased by approximately 2 log CFU/ml in TM, but in TM+KE82 samples, pathogen growth was retarded during the first 6 h at 37°C followed by growth cessation and partial inactivation at 18°C. After 48 to 72 h, growth of L. monocytogenesin SRM+CSC samples decreased by 4 to 5 log CFU/ml compared with the SRM control, whereas additional 10-fold decreases in the pathogen were observed in SRM+CSC+KE82 samples. Reverse transcription PCR analysis of SRM+KE82 and SRM+CSC+KE82 samples confirmed that the entAand entBgenes were transcribed, but entPgene transcription was not detected. All RM and SRM samples inoculated with E. faeciumKE82 displayed strong in situ inhibitory activity against L. monocytogenesin well diffusion bioassays, whereas activity was weaker to undetectable in comparable or additional TM+KE82 samples; no milk sample without E. faeciumKE82 had activity against L. monocytogenes. The findings of this study indicate that E. faeciumKE82 is an antilisterial agent that could be used in traditional dairy foods because it concomitantly produces enterocins A and B in situ in milk.

This study was conducted to evaluate the behavior of Staphylococcus aureusduring processing, ripening, and storage of traditional Greek Graviera cheese in accordance with European Union Regulation 1441/2007 for coagulase-positive staphylococci in thermized milk cheeses. Lactococcus lactissubsp. cremorisM104, a wild, novel nisin A-producing (NisA+) strain, also was evaluated as an antistaphylococcal adjunct. A three-strain cocktail of enterotoxigenic (Ent+) S. aureusincreased by approximately 2 log CFU/ml when coinoculated (at approximately 3 log CFU/ml) in thermized Graviera cheese milk (TGCM; 63°C for 30 s) with commercial starter culture (CSC) and/or strain M104 at approximately 6 log CFU/ml and then incubated at 37°C for 3 h. However, after 6 h at 37°C, significant retarding effects on S. aureusgrowth were noted in the order TGCM+ M104 > TGCM+ CSC = TGCM+ CSC+ M104 > TGCM. Additional incubation of TGCM cultures at 18°C for 66 h resulted in a 1.2-log reduction (P <0.05) of S. aureuspopulations in TGCM+ M104. The Ent+ S. aureuscocktail did not grow but survived during ripening and storage when inoculated (at approximately 3 log CFU/g) postcooking into Graviera mini cheeses prepared from TGCM+ CSC or TGCM+ CSC+ M104, ripened at 18°C and 90% relative humidity for 20 days, and stored at 4°C in vacuum packages for 2 months. A rapid 10-fold decrease (P <0.05) in S. aureuspopulations occurred within the first 24 h of cheese fermentation.Reductions of S. aureuswere greater by approximately 0.4 log CFU/g in CSC+ M104 than in CSC only cheeses, concomitantly with the presence of NisA+ M104 colonies and nisin-encoding genes in the CSC plus M104 cheeses and their corresponding microbial consortia only. A high level of selective survival of a naturally nisin-resistant EntC+ S. aureusstrain from the cocktail was noted in CSC+ M104 cheeses and in coculture with the NisA+ M104 strain in M-17 broth. In conclusion, although S. aureusgrowth inhibition is assured during Graviera cheese ripening, early growth of the pathogen during milk curdling and curd cooking operations may occur. Nisin-resistant S. aureusstrains that may contaminate Graviera cheese milks postthermally may be difficult to control even by the application of the NisA+ L. lactissubsp. cremorisstrain M104 as a bioprotective adjunct culture.

The behavior of Listeria monocytogeneson 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 faeciumGraviera isolate. Cheese pieces were surface inoculated with a five-strain cocktail of L. monocytogenesat ca. 3 log CFU/cm2, packed under air or vacuum conditions, stored at 4, 12, or 25°C, and analyzed after 0, 3, 7, 15, 30, 60, and 90 days. L. monocytogenesdid 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°C under vacuum conditions. Overall, the lower the storage temperature, the higher and longer the survival of L. monocytogeneswas. Although enterocin A–specific PCR products were detected in situ in cheese batch B, inhibition of L. monocytogenesby 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 Listeriainhibition in cheese. In conclusion, Graviera cheeses that may be accidentally contaminated in retail at the European Union maximal allowable level of 100 CFU/cm2or g are at low risk regarding a potential outgrowth of L. monocytogenes, which, however, may survive for a long period during cheese storage.

A polyphasic taxonomic approach was used to identify a major atypical group of gas-forming, arginine-negative lactic acid bacteria associated with spoilage of whole (nonsliced) refrigerated (4°C) cooked hams produced in two Greek industrial meat plants. Biochemical characterization revealed that the ham isolates shared their phenotypic properties with Leuconostoc carnosum, Weissella viridescens, and Weissella hellenica. However, gas chromatographic analysis of cellular fatty acids clearly differentiated the ham isolates from the Weissellaspp. None of the isolates contained eicosenoic acid (n-C20:1), which is typically synthesized by W. viridescens, but all strains contained high amounts of C19cycl acid, which is absent in W. hellenicaand has been found in trace amounts in W. viridescens. All strains had similar cellular fatty acid profiles, which were qualitatively similar to those of the cellular fatty acids of L. carnosum. In addition to the phenotypic and chemotaxonomic tests, three representative isolates were studied using a lactic acid bacteria database, which employs 16S and 23S HindIII restriction fragment length polymorphism patterns as operational taxonomic units in a numerical analysis. The isolate patterns were identical to those of the L. carnosumtype strain, NCFB 2776T. Based on the polyphasic taxonomic approach, the dominating lactic acid bacteria group was identified as L. carnosum.