Athanasia Kakouri

Evaluation of the extent and type of bacterial contamination at different stages of processing of cooked ham

In an attempt to determine the composition and origin of the spoilage flora of refrigerated vacuum‐packed cooked ham, the changes in microbial numbers and types were followed along the processing line. Results revealed Lactobacillus sake and Leuconostoc mesenteroides ssp. mesenteroides as the major causative agents of spoilage of sliced ham stored at 4 °C and 12 °C, due to recontamination in the cutting room. On the contrary, the progressive deterioration of whole ham under the same storage conditions was associated with a non‐identifiable group of leuconostoc‐like bacteria. Except for lactic acid bacteria, no other organism grew in vacuum packs of either sliced or whole ham. Although atypical leuconostocs could not be detected among isolates recovered from freshly produced whole ham, they appeared to survive cooking and proliferate during storage. Neither these organisms however, nor Lact. sake and Leuc. mesenteroides were important in curing and tumbling as carnobacteria, mainly Carnobacterium divergens, and Brochothrix thermosphacta dominated at this stage. A progressive inversion of the ham microflora from mostly Gram‐negative at the beginning of processing to highly Gram‐positive prior to cooking was noted. Listeria monocytogenes cross‐contaminated ham during tumbling. However, the pathogen was always absent from the vacuum‐packed product provided that heating to a core temperature of 70 °C occurred and recontamination during slicing and packing was prevented. The percentage distribution of different species of lactic acid bacteria as well as the uncommon phenotypic characteristics of some strains were discussed.

The spoilage microflora of cured, cooked turkey breasts prepared commercially with or without smoking

Lactobacillus sakei subsp. carnosus was predominant in the spoilage flora of sliced, vacuum-packed, smoked, oven-cooked turkey breast fillets which developed mild, sour spoilage flavors after 4 weeks storage at 4 degrees C. In contrast, Leuconostoc mesenteroides subsp. mesenteroides predominated in the spoilage flora of sliced, vacuum-packed, unsmoked, boiled turkey breast fillets from the same plant which were also stored at 4 degrees C. The spoilage flora of the unsmoked breasts grew faster than that of the smoked breasts and was more diverse. Lactobacillus sakei, Weissella viridescens and an atypical group of leuconostoc-like bacteria were also members of the unsmoked turkey breasts flora. Consequently, the unsmoked breasts spoiled after 2 weeks at 4 degrees C: the packs swelled and the meat developed strong sour odors and flavors and abundant slime. Except for the unidentified leuconostocs, which apparently survived boiling of the unsmoked turkey, all the spoilage organisms contaminated the meats during the slicing and vacuum packaging operations. From their biochemical reactions and cellular fatty acid profiles, the atypical leuconostocs were more closely related to Leuconostoc carnosum than W. viridescens. Carnobacteria and Brochothrix thermosphacta were present in relatively large numbers on the raw turkey, but were not numerous in the spoilage flora of the cooked, vacuum-packed meat products.

Changes in the microbial composition of raw milk induced by thermization treatments applied prior to traditional Greek hard cheese processing.

The microbiological quality, safety, and composition of mixtures of ewes and goats milk (90:10) used for cheesemaking were evaluated before and after thermization at 60 and 67 degrees C for 30 s. Such mild thermal treatments are commonly applied to reduce natural contaminants of raw milk before processing for traditional hard Greek cheeses. Raw milk samples had an average total bacterial count of 7.3 log CFU/ml; most of these bacteria were lactic acid bacteria (LAB) and pseudomonads. The LAB flora of raw milk was dominated by enterococci (40.8%), followed by lactococci (20.4%), leuconostocs (18.4%), and mesophilic lactobacilli (10.2%). Enterococcus faecalis (30.1%) and Enterococcus faecium (13.7%) were the most common LAB isolates, followed by Enterococcus durans, Lactococcus lactis subsp. lactis, Lactobacillus plantarum, and Leuconostoc lactis. Thermization at 60 degrees C for 30 s was effective for reducing raw milk contamination by enterobacteria (5.1 log CFU/ml), coagulase-positive staphylococci (3.3 log CFU/ml), and Listeria (present in 25-ml samples) to safe levels, but it also reduced mesophilic lactococci, leuconostocs, lactobacilli, and selected enterococci (72.0%) in thermized milk. Thermization at 67 degrees C for 30 s had a major inactivation effect on all bacterial groups. Two nisin-producing L. lactis subsp. lactis strains (M78 and M104) were isolated from raw milk, but neither nisin-producing nor other bacteriocin-producing LAB strains were isolated from thermized milk. Thus, thermization treatments control harmful bacteria but also may have a negative impact on milk quality by reducing desirable LAB and the biodiversity of raw milk bacteria overall, inactivating potentially protective LAB strains and enhancing the ability of potentially pathogenic enterococci to grow in fresh cheese curds.

FTIR-based polyphasic identification of lactic acid bacteria isolated from traditional Greek Graviera cheese

This study used a combination of phenotypic, physical (Fourier Transformed Infra-Red [FTIR] spectroscopy) and molecular (RFLP and SSCP analysis of 16S rRNA genes) methods to identify the lactic acid bacteria (LAB) flora present in traditional Greek Graviera cheese after five weeks of ripening. A total of 300 isolates collected from high dilution plates of TSAYE (incubated at 30 °C), M-17 (22 °C) and M-17 (42 °C) agar media were clustered by FTIR and then representative strains of each cluster were cross-identified blindly by all methods. Based on their FTIR spectra, 282 isolates were LAB grouped in 28 clusters. The LAB species identified and their prevalence in the cheese samples were: Lactobacillus casei/paracasei (68.8%), Lactobacillus plantarum (19.5%), Streptococcus thermophilus (8.9%), Enterococcus faecium (2.1%), and Lactococcus lactis (0.7%). Also, Staphylococcus equorum (11 isolates), Corynebacterium sp. (5 isolates) and Brevibacterium sp. (1 isolate) were recovered from TSAYE. Comparative identification results showed that phenotypic and molecular methods were in mutual agreement as regards the LAB species identified. The present polyphasic identification approach based on rapid FTIR screening of 10-fold more isolates than a previous classical identification approach allowed or improved detection of few sub-dominant species; however the predominant LAB species in the cheese samples were the same with both approaches.

Use of ionizing radiation doses of 2 and 4 kGy to control Listeria spp. and Escherichia coli O157:H7 on frozen meat trimmings used for dry fermented sausage production

This study evaluated survival of Listeria spp. (four-strain mixture of Listeria innocua plus a non-virulent Listeria monocytogenes strain) and Escherichia coli O157:H7 strain ATCC 43888 during fermentation and ripening of Greek dry sausages formulated from meat and pork fat trimmings previously inoculated with ca. 6logcfug(-1) of the target bacteria and then irradiated in frozen (-25°C) blocks at doses of 0 (control), 2 or 4kGy. Irradiation of the trimmings at 2kGy reduced initial contamination of the sausage batter with Listeria and E. coli O157:H7 by 1.3 and 2.0 logcfug(-1), respectively, while the corresponding reductions at 4kGy were 2.4 and 5.5 logcfug(-1), respectively. In fact, E. coli O157:H7 was eliminated by 4kGy at formulation (day 0) as compared to 7 and 21 days of ripening in samples treated at 2 and 0kGy, respectively. Despite the fact that irradiation assisted in faster declines of listeriae during fermentation, these bacteria showed a strong tailing during ripening, which was more pronounced in sausages irradiated at 4kGy. As a consequence, survival of Listeria in 28-day sausages irradiated at 2 or 4kGy was ca. 2 logcfug(-1) and similar (P>0.05) to that in non-irradiated samples. Irradiation showed promise for controlling E. coli O157:H7 and, to a lesser extent, L. monocytogenes in fermented sausages.

Fate of Listeria monocytogenes on Fully Ripened Greek Graviera Cheese Stored at 4, 12, or 25°C in Air or Vacuum Packages : In Situ PCR Detection of a Cocktail of Bacteriocins Potentially Contributing to Pathogen Inhibition

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.

Microbial stability and safety of traditional Greek Graviera cheese: characterization of the lactic acid bacterial flora and culture-independent detection of bacteriocin genes in the ripened cheeses and their microbial consortia.

The microflora of four batches of traditional Greek Graviera cheese was studied at 5 weeks of ripening, and 200 lactic acid bacteria (LAB) isolates were phenotypically characterized and screened for antilisterial bacteriocins. The cheeses were also analyzed for organic acids by high-performance liquid chromatography and for the potential presence of 25 known LAB bacteriocin genes directly in cheese and their microbial consortia by PCR. All batches were safe according to the European Union regulatory criteria for Listeria monocytogenes, Salmonella, enterobacteria, and coagulase-positive staphylococci. The cheese flora was dominated by nonstarter Lactobacillus casei/paracasei (67.5%) and Lactobacillus plantarum (16.3%) strains, whereas few Streptococcus thermophilus (3.8%), Lactococcus lactis subsp. lactis (0.6%), and Leuconostoc (1.9%) organisms were present. Enterococcus faecium (9.4%) and Enterococcus durans (0.6%) were isolated among the dominant LAB from two batches; however, enterococci were present in all batches at 10- to 100-fold lower populations than mesophilic lactobacilli. Sixteen E. faecium isolates produced antilisterial enterocins. In accordance, enterocin B gene was detectable in all cheeses and enterocin P gene was present in one cheese, whereas the consortia of all cheeses contained at least two of the enterocin A, B, P, 31, L50A, and L50B genes. Plantaricin A gene was also amplified from all cheeses. Mean concentrations of lactic, acetic, citric, and propionic acids in the ripened cheeses exceeded 1.5% in total, of which approximately 0.9% was lactate. Thus, organic acid contents constitute an important hurdle factor for inhibiting growth of pathogens in traditional Graviera cheese products, with LAB bacteriocins, mainly enterocins, potentially contributing to increased cheese safety.

Effect of irradiation of frozen meat/fat trimmings on microbiological and physicochemical quality attributes of dry fermented sausages

Changes in microbiological and physicochemical quality attributes resulting from the use of irradiation in the production of Greek dry fermented sausage were investigated as a function of fermentation/ripening time. Results showed that irradiating meat/fat trimmings at 2 or 4kGy prior to sausage production eliminated natural contamination with Listeria spp., and reduced pseudomonads, enterococci and pathogenic staphylococci, and enterobacteria, to less than 2 and 1logcfug(-1), respectively. Pseudomonads were very sensitive (>3.4 log reduction) to either radiation dose. Yeasts were the most resistant followed by inherent lactic acid bacteria; their reductions on the trimmings were radiation dose-dependent. Residual effects of irradiation were noted against enterococci, but not against gram-negatives which died off fast during fermentation even in non-irradiated samples. Growth of the starter bacteria, Lactobacillus pentosus and Staphylococcus carnosus, inoculated in the sausage batters post-irradiation was unaffected by the 2 or 4kGy pre-treatment of the trimmings. Irradiation had little or no effect at the end of ripening period (28 days) on pH, moisture content and color (parameters L(∗), a(∗), and b(∗)). Changes in TBA values were small but statistically significant with irradiated samples having higher TBA values than control samples.

Characterization of a Wild, Novel Nisin A-Producing Lactococcus Strain with an L. lactis subsp. cremoris Genotype and an L. lactis subsp. lactis Phenotype, Isolated from Greek Raw Milk

ABSTRACT Several molecular taxonomic studies have revealed that many natural (wild) Lactococcus lactis strains of dairy origin which are phenotypically representative of the L. lactis subspecies lactis cluster genotypically within subspecies cremoris and vice versa. Recently, we isolated two wild nisin-producing (Nis+) L. lactis strains, M78 and M104, of the lactis phenotype from Greek raw milk (J. Samelis, A. Lianou, A. Kakouri, C. Delbès, I. Rogelj, B. B. Matijašic, and M. C. Montel, J. Food Prot. 72:783–790, 2009); strain M78 possess a novel nisin A sequence (GenBank accession number HM219853). In this study, the actual subspecies identity of M78 and M104 isolates was elucidated, using 16S rRNA and acmA (encoding lactococcal N-acetylmuramidase) gene and histidine biosynthesis operon polymorphisms and 16S rRNA and ldh (encoding lactate dehydrogenase) gene phylogenies. Except the acmA gene analysis, molecular tools revealed that isolates M78 and M104 clustered with strains of the cremoris genotype, including the LMG 6897T strain, while they were distant from strains of the lactis genotype, including the LMG 6890T strain. The two wild isolates had identical repetitive sequence-based PCR (rep-PCR), randomly amplified polymorphic DNA (RAPD), plasmid, and whole-cell protein profiles and shared high 16S rRNA (99.9%) and ldh (100%) gene sequence homologies. In contrast, they exhibited identical sugar fermentation and enzymatic patterns which were similar to those of the subspecies lactis LMG 6890T strain. To our knowledge, this is the first complete identification report on a wild L. lactis subsp. cremoris genotype of the lactis phenotype which is capable of nisin A production and, thus, has strong potential for use as a novel dairy starter and/or protective culture.

Leuconostoc carnosum associated with spoilage of refrigerated whole cooked hams in Greece.

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 degrees 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 Weissella spp. None of the isolates contained eicosenoic acid (n-C20:1), which is typically synthesized by W. viridescens, but all strains contained high amounts of C 19cycl acid, which is absent in W. hellenica and 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. carnosum type strain, NCFB 2776T. Based on the polyphasic taxonomic approach, the dominating lactic acid bacteria group was identified as L. carnosum.