Effects of essential oil from mint (Mentha piperita) on Salmonella enteritidis and Listeria monocytogenes in model food systems at 4° and 10°C

The effect of mint ( Mentha piperita ) essential oil (0·5, 1·0, 1·5 and 2·0%, v/w) on Salmonella enteritidis and Listeria monocytogenes in a culture medium and three model foods; tzatziki (pH 4·5), taramosalata (pH 5·0) and pâté (pH 6·8), inoculated at 10⁷ cfu g^-1, at 4° and 10°C for ca 1 week was studied. In the culture medium supplemented with the essential oil, no growth was observed over 2 d at 30°C determined by a conductance method with a Malthus 2000 growth analyser. Salmonella enteritidis died in tzatziki in all treatments and declined in the other foods except for pâté at 10°C as judged with viable counts. Listeria monocytogenes populations showed a declining trend towards the end of the storage period but was increased in pâté. Mint essential oil antibacterial action depended mainly on its concentration, food pH, composition, storage temperature and the nature of the micro-organism.     

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.     

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 PD_max 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.     

Modeling the Effect of Abrupt Acid and Osmotic Shifts within the Growth Region and across Growth Boundaries on Adaptation and Growth of Listeria monocytogenes

This study aims to model the effects of acid and osmotic shifts on the intermediate lag time of Listeria monocytogenes at 10°C in a growth medium. The model was developed from data from a previous study (C. I. A. Belessi, Y. Le Marc, S. I. Merkouri, A. S. Gounadaki, S. Schvartzman, K. Jordan, E. H. Drosinos, and P. N. Skandamis, submitted for publication) on the effects of osmotic and pH shifts on the kinetics of L. monocytogenes. The predictive ability of the model was assessed on new data in milk. The effects of shifts were modeled through the dependence of the parameter h₀ (“work to be done” prior to growth) induced on the magnitude of the shift and/or the stringency of the new environmental conditions. For shifts across the boundary, the lag time was found to be affected by the length of time for which the microorganisms were kept at growth-inhibiting conditions. The predicted concentrations of L. monocytogenes in milk were overestimated when the effects of this shift were not taken into account. The model proved to be suitable to describe the effects of osmotic and acid shifts observed both within the growth domain and across the growth boundaries of L. monocytogenes.The lag phase of a microorganism is usually seen as a period of transition from an initial physiological state to the state of balanced growth. The duration of the lag phase, denoted by lag in what follows, depends on the amount of work to be carried out by the cells prior to exponential growth and the rate at which this work is undertaken (6, 13). According to Baranyi and Roberts (2), the “work to be done” is proportional to h₀, the product of the lag time and the rate at which the work is carried out. Robinson et al. (13) pointed out that there is no direct way to measure this rate, and it is often assumed that it is equal to the specific growth rate characteristic of the growth conditions (2). Some authors use the relative lag time (RLT) (7, 8) as a replacement for the “work to be done” h₀ parameter. In fact the two concepts appear to be very similar, RLT and h₀ being proportional to each other.The transient phase following inoculation is commonly called the initial lag phase. Many authors (6, 7, 14, 17) observed that subsequent abrupt changes in the environmental conditions (temperature, pH, and water activity [a_w]) during the growth phase were able to induce a so-called “intermediate” lag phase. In other words, abrupt changes cause extra “work to be done” that cells have to perform before reinitiating their growth. Most of the studies on intermediate lag times have focused on abrupt thermal changes. For example, some authors have proposed models for the effects of temperature shifts on the lag time of Escherichia coli (14) and Lactobacillus plantarum (17). Using the data of Whiting and Bagi (15), for Listeria monocytogenes, Delignette-Muller et al. (3) highlighted a linear relationship between the “work to be done” and the magnitude and direction of the temperature shifts. Less attention has been given to the effects of acid and osmotic shifts, although such shifts pose a higher energetic burden to the cells than temperature shifts, especially around the growth boundaries (C. I. A. Belessi, Y. Le Marc, S. I. Merkouri, A. S. Gounadaki, S. Schvartzman, K. Jordan, E. H. Drosinos, and P. N. Skandamis, submitted for publication). Generally, the “work to be done” increases with the magnitude of the shifts applied (3, 7) and the cells in exponential phase are more sensitive to abrupt shifts than those in stationary phase (7). Muñoz-Cuevas et al. (9) proposed a model for the lag time of L. monocytogenes induced by temperature and water activity downshifts within the growth region. For osmotic shifts, the authors found that the “work to be done” was related not only to the magnitude of the shift and but also to the level of the environmental factors (temperature and water activity) after the shift.In most of the available modeling packages, predictions in dynamic environments are based on the assumption that when the environmental conditions change, the specific growth rate changes instantaneously relative to the new conditions. The intermediate lag times caused by abrupt changes in the environmental conditions are commonly neglected in the models. Besides, the models usually ignore the effects of shifts across the growth boundary and the duration of the period the cells spend above the growth/no-growth boundary on the physiological state of the cells. However, such abrupt shifts are important, as they may occur for example during fermentation and ripening of dairy products (10, 11) or during cross-contamination (e.g., when L. monocytogenes is accidently transferred to a different environment). The aim of this work was to develop a model to describe the effects of such abrupt shifts (within the growth range or across the growth boundary) on the possibly induced intermediate lag time of L. monocytogenes. The analysis here is based on the data from a previous study (Belessi et al., submitted) on the effects of acid and osmotic shifts on the kinetics of L. monocytogenes at 10°C. We also used new data in milk to explore the possibility of integrating the proposed approach in a generic growth model.     

Biotechnological valorization of low-cost sugar-based media for bacteriocin production by Leuconostoc mesenteroides E131

The aim of the present study was to evaluate the suitability of low-cost carbon sources for bacteriocin production by Leuconostoc mesenteroides strain E131. For this purpose, inexpensive sugars derived from a sugar refinery plant (glucose, fructose and sucrose) as well as waste molasses were utilized as carbon sources in submerged shake-flask experiments and the kinetic response of the microorganism was evaluated. Interestingly, in the case of molasses, non-negligible decolorization-detoxification (up to ～27%) of the residue was performed together with the production of bacteriocin. In all instances the initial concentration of sugars employed was adjusted at 20 and 30 g/L, therefore the effect of both the nature and the initial quantity of sugar upon the growth of the microorganism was assessed. All media proved to be suitable for both biomass and bacteriocin production by L. mesenteroides, whereas variable quantities of lactate, acetate and ethanol were detected into the medium. Employment of fructose, sucrose or molasses as carbon sources resulted in the accumulation of mannitol (in some cases in significant quantities) into the medium; remarkable portion thus of the available or released fructose acted as electron acceptor instead of carbon source by the microorganism. The highest bacteriocin production achieved (=640 AU/mL) was obtained when initial glucose at 30 g/L was used as substrate. Finally, utilization of waste molasses as carbon source by L. mesenteroides resulted in satisfactory bacteriocin production (up to 320 AU/mL) besides the decolorization of the residue.     

Microbial interaction in cooked cured meat products under vacuum or modified atmosphere at 4 degrees C

AIMS: To investigate the antagonistic activity of two lactic acid strains against the spoilage microflora in cooked cured meat products, vacuum or modified atmosphere packed at 4 degrees C and to determine the inhibitory capacity of their bacteriocins. METHODS AND RESULTS: Frankfurter-type sausages and sliced cooked cured pork shoulder were inoculated with Leuconostoc mesenteroides L124 and Lactobacillus curvatus L442 or with their bacteriocins. The microbial, physico-chemical (pH, L- and D-lactate, acetate and ammonia) and colour changes were studied. Results under vacuum packaging showed that in the uninoculated samples of the pork product the spoilage microflora grew but in the inoculated ones the spoilage microorganisms (e.g. Brochothrix thermosphacta and enterococci) reduced during the storage. This observation was more pronounced in the samples with the addition of bacteriocins. In the frankfurter-type sausages the spoilage microflora did not grow in the uninoculated and inoculated samples. In the modified atmosphere enriched in CO2 the population of spoilage microflora remained at low levels in both products, indicating that CO2 has an effect on the spoilage microorganisms' growth. In the pork product the concentrations of acetate and d-lactate increased while L-lactate decreased, but in the frankfurter-type sausages increase of acetate and D-lactate was not observed. CONCLUSIONS: Lactic acid strains had an effect on the spoilage microflora growth but did not affect, negatively, the organoleptic properties of the products. These strains may be used as biopreservative cultures or their bacteriocins could be an important contribution to microbiological quality of meat products. SIGNIFICANCE AND IMPACT OF STUDY: Establishment of biopreservation as a method for extension of shelf life of meat products.     

Metabolic activities of pseudomonads in batch cultures in extract of minced lamb

E.H. DROSINOS AND R.G. BOARD. 1994. Pseudomonas fragi, Ps. lundensis and Ps. fluorescens were studied in axenic batch cultures growing in a lamb juice (pH 6.0) aerobically or in an atmosphere ( Ps. fragi only) enriched with carbon dioxide at 4C. With all but a glucose dehydrogenase-deficient strain of Ps. fluorescens there was a sequential catabolism of glucose and lactate. Diauxic growth was observed in a nutrient-deficient meat juice supplemented with glucose and lactate. A transient peak in the concentration of gluconate and pyruvate was associated with the catabolism of glucose and lactate respectively. With Ps. fluorescens deficient in glucose dehydrogenase there was simultaneous catabolism of glucose and lactate. The stereoisomers of lactate were catabolized simultaneously in a laboratory medium. Glucose-6-phosphate was oxidized to 6-phosphogluconate by Ps. fragi and Ps. lundensis under aerobic conditions only. Creatine and creatinine were catabolized by Ps. fragi under aerobic conditions only. There was a slight decrease in the concentration of total amino acids (ninhydrin-reactive material) during the exponential phase of growth. The results suggest that the dominance of Ps. fragi in the climax populations in meat is due to catabolism of amino acid related substrates, creatine and creatinine.     

Purification and characterization of curvaticin L442, a bacteriocin produced by Lactobacillus curvatus L442

Lactobacillus curvatus L442, isolated from Greek traditional fermented sausage prepared without the addition of starters, produces a bacteriocin, curvaticin L442, which is active against the pathogen Listeria monocytogenes. The bacteriocin was purified by 50% ammonium sulphate precipitation, cation exchange, reverse phase and gel filtration chromatography. Partial N-terminal sequence analysis using Edman degradation revealed 30 amino acid residues, revealing high homology with the amino acid sequence of sakacin P. Curvaticin L442 is active at pH values between 4.0 and 9.0 and it retains activity even after incubation for 5 min at 121 °C with 1 atm of overpressure. Proteolytic enzymes and α-amylase inactivated this curvaticin, while the effect of lipase was not severe.     

Performance of Pseudomonas CFC-selective medium in the fish storage ecosystems

Pseudomonas agar base supplemented with cephaloridine, fucidin, and cetrimide (CFC) was used to count Pseudomonas populations on fish. Both Enterobacteriaceae and Shewanella putrefaciens were able to grow on the CFC medium. Evaluation of the performance of CFC-selective for pseudomonads medium, on fish samples stored aerobically and under a modified atmosphere at 0, 10 and 20 degrees C was tested.The selectivity of the medium was affected by storage temperatures and the type of packaging of the fish samples. The selectivity of the medium diminished as the population increased and for samples stored at high temperature (20 degrees C) or under modified atmospheres.When designing adequate selectivity of a medium, interfering organisms should be taken into account, especially when the background flora tends to be more robust than the organisms to be counted or detected.     

Applicability of an Arrhenius model for the combined effect of temperature and CO(2) packaging on the spoilage microflora of fish

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 bacteria Pseudomonas 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, E(A), 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 CO(2) 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.