Eleftherios H. Drosinos

Characterization of the microbial flora from a traditional Greek fermented sausage

The microbial flora of naturally fermented sausages was studied. Lactic acid bacteria were the dominant species at the end of fermentation in all 3 batches (ca. 10(8) cfu g(-1)). Enterobacteria, Pseudomonas, yeasts and aerobic spore-formers decreased during fermentation and the ripening process and were below the detection limit in the end product. Enterococci exceeded 10(4)-10(5) cfu g(-1) during fermentation and remained constant at this level during ripening. Gram-positive, catalase-positive cocci exceeded 10(5) cfu g(-1), except for batch 1, during the first days of fermentation and then decreased until the end of ripening (10(2)-10(4) cfu g(-1)). No pathogenic staphylococci, sulfite reducing clostridia or Salmonella spp. were detected. Listeria spp. occurred in the first days of fermentation but were eliminated by the end of whole process in all batches. Identification showed that the majority of lactobacilli isolated from MRS agar strains were assigned to the species of Lactobacillus plantarum and Lb. plantarum/pentosus. All the isolated strains from the mannitol salt agar belonged to the genus of Staphylococcus. The predominant species were Staphylococcus saprophyticus, Staphylococcus xylosus and Staphylococcus simulans. The tests used to characterize the lactic acid bacteria and staphylococci as well as their distribution on the three batches were also discussed.

Applicability of an Arrhenius Model for the Combined Effect of Temperature and CO2 Packaging on the Spoilage Microflora of Fish

ABSTRACT The temperature behavior of the natural microflora on the Mediterranean fish red mullet (Mullus barbatus) was examined as a case study. The growth of the spoilage bacteriaPseudomonas spp., Shewanella putrefaciens,Brochothrix thermosphacta, and lactic acid bacteria was modeled as a function of temperature and the concentration of carbon dioxide in modified atmosphere packaging. Combined models were developed and comparatively assessed based on polynomial, Belehradek, and Arrhenius equations. The activation energy parameter of the Arrhenius model, EA, was independent of the packaging atmosphere and ranged from 75 to 85 kJ/mol for the different bacteria, whereas the preexponential constant decreased exponentially with the packaging CO2 concentration. We evaluated the applicability of the models developed by using experimental bacterial growth rates obtained from 42 independent experiments performed with three Mediterranean fish species and growth rates predicted from the models under the same temperature and packaging conditions. The accuracy factor and bias factor were used as statistical tools for evaluation, and the developed Arrhenius model and the Belehradek model were judged satisfactory overall.

Inhibition of resident microbial flora and pathogen inocula on cold fresh fish fillets in olive oil, oregano, and lemon juice under modified atmosphere or air

The development of a microbial population was studied in Mediterranean gilt-head seabream ( Sparus aurata , tsipoura in Hellenic) dressed with olive oil, lemon juice, and oregano, inoculated with Staphylococcus aureus and Salmonella enteritidis , and stored under a modified atmosphere (MA) of 40% CO2, 30% O2, and 30% N2 or air at 0 ± 1°C. The treatment had bacteriostatic and bactericidal effects on both inoculated pathogens as well as on the autochthonous flora. Brochothrix thermosphacta and pseudomonads dominated the spoilage flora under MA and under air respectively. Shewanella putrefaciens was clearly inhibited.

Traditional dry fermented sausages produced in small-scale processing units in Mediterranean countries and Slovakia. 1: Microbial ecosystems of processing environments

Microbial ecosystems were surveyed in 314 environmental samples from 54 Southern and Eastern European small-scale processing units (PUs) manufacturing traditional dry fermented sausages. The residual microflora contaminating the surfaces and the equipment were analysed after cleaning and disinfection procedures. All the PU environments were colonised at various levels by spoilage and technological microflora with excessive contamination levels in some of the PUs. Sporadic contamination by pathogenic microflora was recorded. Salmonella and Listeria monocytogenes were detected in 4.8% and 6.7% of the samples, respectively, and Staphylococcus aureus was enumerated in 6.1% of the samples. Several critical points were identified, such as the machines for S. aureus and the tables and the knives for L. monocytogenes; this knowledge is crucial for the improvement of hygiene control systems in small and traditional meat processing industries. The variability of the residual contamination emphasized the different cleaning, disinfecting and manufacturing practices routinely followed by these small-scale processing units.

Control of spoilage microorganisms in minced pork by a self-developed modified atmosphere induced by the respiratory activity of meat microflora.

The changes in microbial flora of minced pork during aerobic storage at 0, 5, 10 and 15 degrees C were studied. Minced pork samples (100g) were packed using two types of packaging films: (a) a common food film with high permeability (HPF) and (b) a film with low permeability (LPF). The respiratory activity of meat microflora and the use of a LPF resulted in a modified atmosphere in the package headspace developed during storage. Oxygen concentration decreased from 18.7% (after packaging) to 7% (after 15 days of storage) in packages with LPF, stored at 0 degrees C, while CO(2) increased from 3% to 10.5%, respectively. On the contrary, no significant atmosphere changes were observed during storage of HPF packages. The self-developed modified atmosphere in LPF packages resulted in a significant inhibition of pseudomonad growth which was more pronounced at low storage temperatures. For example, during storage at 0 degrees C, the growth rate of pseudomonads in meat packed with LPF was reduced by 48.7% compared to HPF. At 10 degrees C the latter reduction decreased to 13.7%. LPF packaging was also found to inhibit the growth of Brochothrix thermosphacta but this inhibition was weaker compared to pseudomonads. The effect of storage temperature on the growth rate of pseudomonads and B. thermosphacta in minced pork packed with the different films was modeled using an Arrhenius equation. For both bacteria, the activation energy was higher for LPF packaging. This can be attributed to the increased inhibitory effect of the modified atmosphere at lower storage temperature. The Arrhenius model was further used to evaluate the effect of temperature on the time required by the two bacteria to reach a spoilage level of 10(7)CFU/g. The results showed that when LPF packaging is combined with effective temperature control the time-to-spoilage can be significantly extended compared to HPF packaging.

Screening of bacterial strains capable of converting biodiesel-derived raw glycerol into 1,3-propanediol, 2,3-butanediol and ethanol

The ability of bacterial strains to assimilate glycerol derived from biodiesel facilities to produce metabolic compounds of importance for the food, textile and chemical industry, such as 1,3‐propanediol (PD), 2,3‐butanediol (BD) and ethanol (EtOH), was assessed. The screening of 84 bacterial strains was performed using glycerol as carbon source. After initial trials, 12 strains were identified capable of consuming raw glycerol under anaerobic conditions, whereas 5 strains consumed glycerol under aerobiosis. A plethora of metabolic compounds was synthesized; in anaerobic batch‐bioreactor cultures PD in quantities up to 11.3 g/L was produced by Clostridium butyricum NRRL B‐23495, while the respective value was 10.1 g/L for a newly isolated Citrobacter freundii. Adaptation of Cl. butyricum at higher initial glycerol concentration resulted in a PDmax concentration of ∼32 g/L. BD was produced by a new Enterobacter aerogenes isolate in shake‐flask experiments, under fully aerobic conditions, with a maximum concentration of ∼22 g/L which was achieved at an initial glycerol quantity of 55 g/L. A new Klebsiella oxytoca isolate converted waste glycerol into mixtures of PD, BD and EtOH at various ratios. Finally, another new C. freundii isolate converted waste glycerol into EtOH in anaerobic batch‐bioreactor cultures with constant pH, achieving a final EtOH concentration of 14.5 g/L, a conversion yield of 0.45 g/g and a volumetric productivity of ∼0.7 g/L/h. As a conclusion, the current study confirmed the utilization of biodiesel‐derived raw glycerol as an appropriate substrate for the production of PD, BD and EtOH by several newly isolated bacterial strains under different experimental conditions.

Study of the effect of lethal and sublethal pH and aw stresses on the inactivation or growth of Listeria monocytogenes and Salmonella Typhimurium

During food processing, microorganisms are commonly exposed to multiple sublethal or lethal stresses (commonly a(w), pH) sequentially or simultaneously. The objectives of the present study were: (i) to comparatively evaluate the survival of Listeria monocytogenes and Salmonella Typhimurium in lethal acid (pH 4.0 and 4.5 with lactic acid) or osmotic conditions (15 and 20% NaCl), applied singly, sequentially (pH then NaCl or NaCl then pH), or simultaneously at 5 and 10 degrees C; and, (ii) to quantify the effect of osmotic shifts at pH 7.0, 6.0 or 5.0 (adjusted with lactic acid) on the lag phase and growth rate of L. monocytogenes at 10 degrees C. In sequential lethal stress applications, the second stress was applied 2 or 3 days after the first for Salmonella and L. monocytogenes, respectively. Acid tolerance of L. monocytogenes was higher than osmotic tolerance and the opposite was observed for Salmonella. Higher inactivation was observed after exposure to pH 4.0 compared to pH 4.5 as well as after exposure to 20% NaCl compared to 15% NaCl. Exposure to stresses sequentially resulted in faster (P<0.05) reductions than the exposure to single or double stresses applied simultaneously. The pH then NaCl sequence was more detrimental for pathogens than the reverse sequence. Incubation temperature (5 and 10 degrees C) did not show any profound (P<0.05) effect on microbial inactivation. When L. monocytogenes was incubated at a(w) 0.930 or 0.995 at 30 degrees C, then the lag phase increased both in subsequent osmotic downshift and upshift, respectively, at 10 degrees C. Shorter lag phase and higher ability to initiate growth at lower a(w) was observed after pre-adaptation at pH 6.0 or 5.0 compared to neutral pH. The results may contribute to the review of critical limits in low pH (with lactic acid) and water activity products, considering the risk of L. monocytogenes and Salmonella survival. In addition, the present indications may address the points in processing where stricter sanitation procedures should be applied in order to minimize the risk of survivors.

Quantifying nonthermal inactivation of Listeria monocytogenes in European fermented sausages using bacteriocinogenic lactic acid bacteria or their bacteriocins: a case study for risk assessment.

Listeria monocytogenes NCTC10527 was examined with respect to its nonthermal inactivation kinetics in fermented sausages from four European countries: Serbia-Montenegro, Hungary, Croatia, and Bosnia-Herzegovina. The goal was to quantify the effect of fermentation and ripening conditions on L. monocytogenes with the simultaneous presence or absence of bacteriocin-producing lactic acid bacteria (i.e., Lactobacillus sakei). Different models were used to fit the experimental data and to calculate the kinetic parameters. The best model was chosen based on statistical comparisons. The Baranyi model was selected because it fitted the data better in most (73%) of the cases. The results from the challenge experiments and the subsequent statistical analysis indicated that relative to the control condition the addition of L. sakei strains reduced the time required for a 4-log reduction of L. monocytogenes (t(4D)). In contrast, the addition of the bacteriocins mesenterocin Y and sakacin P decreased the t(4D) values for only the Serbian product. A case study for risk assessment also was conducted. The data of initial population and t(4D) collected from all countries were described by a single distribution function. Storage temperature, packaging method, pH, and water activity of the final products were used to calculate the inactivation of L. monocytogenes that might occur during storage of the final product (U.S. Department of Agriculture Pathogen Modeling Program version 7.0). Simulation results indicated that the addition of L. sakei strains significantly decreased the simulated L. monocytogenes concentration of ready-to-eat fermented sausages at the time of consumption.

Characterization of two bacteriocins produced by Leuconostoc mesenteroides L124 and Lactobacillus curvatus L442, isolated from dry fermented sausages

Leuconostoc mesenteroides L124 and Lactobacillus curvatus L442, isolated from dry fermented sausages, produce bacteriocins antagonistic towards closely related species and pathogens, such as Listeria monocytogenes. The bacteriocins were inactivated by proteolytic enzymes and lipase but not by catalase and lysozyme. They were also heat stable, retaining activity after heating at 100 °C for 60 min. The bacteriocins were stable at pH values ranging from 2.0 to 8.0. Bacteriocin production was observed at low temperatures (10 and 4 °C) and in meat juice. The maximum bacteriocin activity was observed at the end of the exponential growth phase. The bacteriocins were produced in media with initial pH values ranging from 5.0 to 7.5, but not in media with a pH lower than 5.0 (weak bacteriocin activity of the antibacterial compound produced by Ln. mesenteroides L124 was observed at pH 4.5). Both bacteriocins exhibited strong bactericidal activity following cell/bacteriocin contact.

Purification and characterization of a bacteriocin produced by Leuconostoc mesenteroides E131.

Leuconostoc mesenteroides E131, isolated from Greek traditional fermented sausage, prepared without the addition of starters, produces a bacteriocin which is active against the pathogen Listeria monocytogenes. The bacteriocin was purified by 50% ammonium sulphate precipitation, cation exchange, and reverse-phase chromatography. Bacteriocin is active at pH values between 4.0 and 9.0 and retains activity after incubation for 1h at 100°C. Proteolytic enzymes inactivated the bacteriocin after 1h of incubation, while renin resulted in full inactivation only after 24h. Lipase resulted in full inactivation after 4h. Applying molecular methods, it was determined that the bacteriocin produced, named as mesenterocin E131, was identical to mesenterocin Y105 and was expressed during the exponential growth phase.