John Samelis

Characterisation of lactic acid bacteria isolated from naturally fermented Greek dry salami.

A total of 348 lactic acid bacteria isolated from five batches of naturally fermented dry salami at various stages of ripening were characterised. The majority of the strains were assigned to two main phylogenetic groups of species: (i) the psychrotrophic, formerly called atypical, meat streptobacteria (169 strains) and (ii) a new genus Weissella (120), which was recently proposed (Collins et al., 1993) to include Leuconostoc paramesenteroides and some other closely related species. Meat streptobacteria were identified as Lactobacillus curvatus (88 strains) and L. sake (76), whereas 5 strains were indistinguishable and, thus designated L. sake/curvatus. Non-psychrotrophic streptobacteria were also isolated and identified as L. plantarum (34 strains), L. farciminis (10), L. coryniformis (1) and L. casei subsp. pseudoplantarum (1). The majority of the Weissella strains (86) were leuconostoc-like bacteria; four of them were identified as W. viridescens, 11 belonged to the newly described W. hellenica (Collins et al., 1993), another 11 resembled W. paramesenteroides, whereas 60 isolates were not classified to any species. The latter group comprised strains that produced D(L)-lactate. The remaining Weissella were gas-forming, arginine-positive rods assigned to W. minor (31) and W. halotolerans (3). Other species identified were Enterococcus faecium (10), Leuconostoc mesenteroides (1), L. brevis (1) and Pediococcus sp. (1). The main criteria used to distinguish between above species as well as their distribution on the five salami batches in relation to their succession with time and suitability as starters were discussed.

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.

Lipolytic and microbial changes during the natural fermentation and ripening of Greek dry sausages.

The hydrolytic changes in the lipid fraction and the changes in microbial growth during the natural fermentation and ripening of Greek dry sausages were studied. Two batches were manufactured under industrial conditions, without the addition of starter cultures. The results of the microbiological analyses indicated that, among the micro-organisms well known for their lipolytic activity, micrococci were predominant during the whole fermentation-ripening process. It was shown that the fatty acid composition of the hydrolysed lipid fraction did not remain constant with time. Analysis of the experimental results indicated that, given the length of the aliphatic chain (16 or 18 carbon atoms), the more unsaturated fatty acids were hydrolysed in preference to the saturated homologues. On the other hand, fatty acids having 16 carbon atoms were hydrolysed in preference to the iso-unsaturated ones having 18 carbon atoms.

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.

The microbial association of Greek taverna sausage stored at 4 and 10 °C in air, vacuum or 100% carbon dioxide, and its spoilage potential

J. SAMELIS and K.G. GEORGIADOU.2000.Strains of the Lactobacillus sakei/curvatus group, mainly non‐slime‐producing Lact. sakei, dominated the microbial flora of industrially manufactured taverna sausage, a traditional Greek cooked meat, stored at 4 °C and 10 °C in air, vacuum and 100% CO2. Atypical, arginine‐positive and melibiose‐negative strains of this group were isolated. The isolation frequency of Lact. sakei/curvatus from sausages stored anaerobically was as high as 92–96%, while other meat spoilage organisms were practically absent. Conversely, in air‐stored sausages, leuconostocs, mainly Leuconostoc mesenteroides ssp. mesenteroides, had a considerable presence (14–21%), whereas Brochothrix thermosphacta, pseudomonads and Micrococcaceae grew, but failed to increase above 105 cfu g−1 in all samples during storage. Only yeasts were able to compete against LAB and reached almost 107 cfu g−1 after 30 d of aerobic storage at 10 °C. The great dominance (> 108 cfu g−1) of LAB caused a progressive decrease of pH and an increase of the concentration of l‐lactate, d‐lactate and acetate in all sausage packs. The growth of LAB and its associated chemical changes were more pronounced at 10 °C than 4 °C. At both storage temperatures, l‐lactate and acetate increased more rapidly and to a higher concentration aerobically, unlike d‐lactate, which formed in higher amounts anaerobically. Storage in air was the worst packaging method, resulting in greening and unpleasant off‐odours associated with the high acetate content of the sausages. Carbon dioxide had no significant effect on extending shelf‐life. The factors affecting the natural selection of Lact. sakei/curvatus in taverna sausage are discussed. Moreover, it was attempted to correlate the metabolic activity of this group with the physicochemical changes and the spoilage phenomena occurring in taverna sausage under the different storage conditions.

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.