Distribution of aminogenic activity among potential autochthonous starter cultures for dry fermented sausages.

Any bacterial strain to be used as starter culture should have suitable characteristics, including a lack of amino acid decarboxylase activity. In this study, the decarboxylase activity of 76 bacterial strains, including lactic acid bacteria and gram-positive, catalase-positive cocci, was investigated. These strains were previously isolated from European traditional fermented sausages to develop autochthonous starter cultures. Of all the strains tested, 48% of the lactic acid bacteria strains and 13% of gram-positive, catalase-positive cocci decarboxylated one or more amino acids. Aminogenic potential was strain dependent, although some species had a higher proportion of aminogenic strains than did others. Thus, all Lactobacillus curvatus strains and 70% of Lactobacillus brevis strains had the capacity to produce tyramine and beta-phenylethylamine. Some strains also produced other aromatic amines, such as tryptamine and the diamines putrescine and cadaverine. All the enterococcal strains tested were decarboxylase positive, producing high amounts of tyramine and considerable amounts of beta-phenylethylamine. None of the staphylococcal strains had tyrosine-decarboxylase activity, but some produced other amines. From the aminogenic point of view, Lactobacillus plantarum, Lactobacillus sakei, and Staphylococcus xylosus strains would be the most suitable for use as autochthonous starter cultures for traditional fermented sausages.

Lactobacillus role during conditioning of refrigerated and vacuum-packaged Argentinean meat.

The role of Lactobacillus strains with bioprotective and technological potential on raw beef during 15days of storage under vacuum at 7°C was investigated. The assayed strains were able to grow on the meat, Lactobacillus curvatus CRL705 and Lactobacillus sakei 23K showing the highest competitiveness. A net increase of amino acids was determined in inoculated samples when compared to the control, this being maximal for Lactobacillus plantarum CRL681. Although an important endogenous activity of meat sarcoplasmic proteins was observed, the disappearance of protein bands and the generation of a new one were detected as a consequence of Lactobacillus growth. A synergistic effect of Lactobacillus in combination with the muscle proteolytic enzyme complex can be suggested. From the studied strains, the bacteriocin producer L. curvatus CRL705 may be considered as a good candidate to contribute to meat ageing by means of small peptides and free amino acids generation while improving shelf life.

Microbial ecosystems of traditional fermented meat products: The importance of indigenous starters.

This paper reviews the diversity of microbiota, both in the environment and in traditional fermented European sausages. The environments of processing units were colonised at variable levels by resident spoilage and technological microbiota, with sporadic contamination by pathogenic microbiota. Several critical points were identified such as the machines, the tables and the knives - knowledge crucial for the improvement of cleaning and disinfecting practices. Traditionally fermented sausages generally did not present a sanitary risk. The great diversity of lactic acid bacteria and staphylococci was linked to manufacturing practices. Development of indigenous starters is very promising because it enables sausages to be produced with both high sanitary and sensory qualities. Our increasing knowledge of the genomes of technological bacteria will allow a better understanding of their physiology in sausages.

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.

Diversity of microorganisms in the environment and dry fermented sausages of small traditional French processing units.

Naturally fermented sausages produced in nine traditional French processing units and their environmental surfaces were characterised by microbial and physico-chemical analyses. Salmonella and Staphylococcus aureus were not detected in the environment whereas Listeria monocytogenes was detected in four samples. Staphylococcus/Kocuria, lactic acid bacteria (LAB), Enterobacteriaceae, Pseudomonas, yeasts/moulds and enterococci contaminated the surfaces of two processing units, indicating insufficient cleaning and disinfection procedures. The final sausages did not present any health risk in seven of the processing units. In two of the processing units, the final sausages were contaminated with S. aureus and L. monocytogenes, respectively, at levels exceeding the maximum tolerable limit. Staphylococcus/Kocuria and LAB grew well in the products. Biogenic amines were found in the majority of the final products. Their occurrence was associated with high numbers of lactic acid bacteria and enterococci. The study outlined the processing and microbial diversities of French naturally fermented sausages.

Formation of biofilm by Staphylococcus xylosus.

The ability of 12 Staphylococcus xylosus strains to form biofilm was determined through the study of different criteria. Eleven out of the 12 strains were able to form biofilm, 10 preferentially on hydrophilic support (glass) and one, S. xylosus C2a, on both hydrophilic and hydrophobic (polystyrene) supports. The determination of bacterial surface properties showed that all strains were negatively charged with five strains moderately hydrophobic and seven hydrophilic. The bap and icaA genes, important for biofilm formation of some staphylococci, were searched. All strains were bap positive but icaA negative. Furthermore, S. xylosus strain C2a was studied on two supports widely used in the food industry, polytetrafluoroethylene (PTFE, hydrophobic) and stainless steel (hydrophilic) and appeared to adhere preferentially on stainless steel. Addition of 20 g/l of NaCl to Tryptic Soy Broth medium (TSB) did not improve significantly its adhesion but enhanced both bacterial growth and cell survival, which were optimum in this medium. Environmental scanning electron microscopy showed that S. xylosus C2a colonized the surface of stainless steel chips with intercellular spaces. The strain formed cell aggregates embedded in an amorphous polysaccharidic matrix. Indeed, synthesis of polysaccharides increased during growth on stainless steel chips in TSB.

Identification of beta-oxidation and thioesterase activities in Staphylococcus carnosus 833 strain.

Staphylococcus carnosus 833, inoculated into sausage meat, increased the level of methyl ketones, which contributed to the cured aroma. These ketones can arise from incomplete beta-oxidation followed by two enzymatic activities: a thioesterase and a decarboxylase. In this study we identified the beta-oxidative pathway (through the measure of 3-hydroxyacyl-CoA dehydrogenase activity) and the thioesterase activity in extracts of S. carnosus cells grown in the presence of different methyl esters. The beta-oxidative system was induced by methyl esters and highest induction was found with a 12-carbon substrate. It was specific for medium chain length fatty acyl CoA substrates. Its maximal activity was observed at the end of stationary growth phase. HPLC analyses of acyl-CoA after incubation of cell extracts with palmitoyl-CoA showed that the beta-oxidation system released preferentially long chain hydroxyacyl-CoAs, enoyl-CoAs, and acyl-CoAs. The time-course of intermediate formation indicated a precursor product relationship indicative of a model of free intermediates which could be further deacylated by a thioesterase. The thioesterase activity was enhanced when S. carnosus was grown in the presence of methyl esters with at least 12 carbons and this enzyme was specific for short chain acyl-CoAs. The maximal activity was reached at the stationary growth phase.

Decarboxylase activity involved in methyl ketone production by Staphylococcus carnosus 833, a strain used in sausage fermentation.

Staphylococcus carnosus strain 833, inoculated into sausage, increased the levels of methyl ketones which contributed to the cured aroma. These ketones were predicted to arise from incomplete beta-oxidation followed by a decarboxylation. To check this hypothesis, we measured the beta-decarboxylase activity in resting cells of S. carnosus grown in complex or in synthetic medium, using as substrate a beta-ketoacid, which can be an intermediate of the beta-oxidation pathway. This activity was present throughout the growth period. The enzyme appeared to be constitutive because no induction was observed. High aeration, a pH of 5 and the presence of nitrate promoted the production of methyl ketones.

Roles of superoxide dismutase and catalase of Staphylococcus xylosus in the inhibition of linoleic acid oxidation.

Staphylococcus xylosus used as starter culture in sausages decreases the level of volatile organic compounds arising from lipid oxidation and so contributes to the aroma by avoiding rancidity. The aim of this study was to characterize the roles of catalase and superoxide dismutase (SOD) in the inhibition of free fatty acid oxidation by comparing antioxidant capacity of the S. xylosus wild-type strain with those of the katA mutant and the sod mutant. Antioxidant capacity was determined by measuring the volatile organic compounds and the conjugated diene hydroperoxides arising from linoleic acid oxidation. The three strains inhibited the oxidation of linoleic acid. However, the katA mutant, and especially the sod mutant, had less antioxidant capacity than the S. xylosus wild-type strain. Thus both catalase and SOD of S. xylosus contributed to the inhibition of lipid oxidation.

Prediction of Streptococcus salivarius subsp. thermophilus and Lactobacillus delbrueckii subsp. bulgaricus populations in yoghurt by Curie point pyrolysis-mass spectrometry.

We evaluated the potential of pyrolysis-mass spectrometry (PyMS) for quantifying the binary mixed population of Streptococcus salivarius subsp. thermophilus and Lactobacillus delbrueckii subsp. bulgaricus in yoghurt. For this purpose, a new analytical approach was developed. The yoghurt was transparised and its total bacterial population was recovered by centrifugation and estimated by turbidimetric measurement. The quantity of each population (L. bulgaricus, S. thermophilus) was then estimated in the pellet by PyMS, and the data were analysed by artificial neural networks (ANNs). In parallel, streptococci and lactobacilli were numerated on SYL agar and these data were used as reference values to predict the bacterial counts of each population by PyMS. A close correlation was established between the streptococci and the lactobacilli counts on SYL agar and PyMS measurements (r(2)=0.98 for S. thermophilus and r(2)=0.96 for L. bulgaricus). Combined turbidimetric measurement and PyMS/ANNs seemed to be a powerful method for obtaining rapid counts of binary mixtures of bacteria in yoghurt.

Hydrolysis of esters by staphylococci.

The objective of this work was to characterize the hydrolysis of esters by staphylococci in order to understand if they could contribute to the release of free fatty acids in sausage. Cell-free extracts and extracellular concentrates of staphylococci were examined for esterase activities against p-nitrophenyl esters and for lipolytic activities against triolein. Staphylococci showed intracellular and extracellular esterase activities with different esterase electrophoretic patterns. Cell-free extracts of S. xylosus, S. warneri and S. saprophyticus preferentially hydrolysed p-nitrophenyl butyrate whereas their extracellular concentrates were mainly active against p-nitrophenyl butyrate, p-nitrophenyl caproate and p-nitrophenyl caprylate. In addition their extracellular concentrates hydrolysed triolein. The two strains of S. carnosus differed as they did not show pronounced p-nitrophenyl substrate specificity and did not hydrolyse triolein. Staphylococci hydrolysed esters at a high rate between 15 and 25 degrees C; acidic conditions inhibited the hydrolysis. The hydrolysis was also reduced when the water activity was decreased by addition of polyethylene glycol or glycerol.

Proteolytic and lipolytic activities of Micrococcus roseus (65), Halomonas elongata (16) and Vibrio sp. (168) isolated from Danish bacon curing brines.

Viable cells, cell free extracts and extracellular concentrates of Micrococcus roseus (65), Halomonas elongata (16) and Vibrio sp. (168) isolated from Danish bacon curing brines were examined for lipase, esterase, proteinase and aminopeptidase activities on natural and synthetic substrates. Micrococcus roseus (65) produced one intracellular esterase with affinity for short chain esters, and two intracellular aminopeptidases with affinity for nonpolar amino acids and L-arginine, respectively. One extracellular aminopeptidase with affinity for L-proline was also observed. Three intracellular esterases with affinity for short chain esters and a membrane bound esterase with affinity for butyric to capric esters were found in Halomonas elongata (16), but almost no aminopeptidase activity was found. Vibrio sp. (168) had four intracellular esterases with affinity for short chain esters and one esterase with affinity for all tested esters. Furthermore, an enzyme, which did not migrate on electrophoretic gels, had activity on all examined esters and tributyrin. Small intracellular activity on L-alanine was observed for this bacterial strain. There was no proteinase activities in the tested bacteria.

Histamine and tyramine production by bacteria from meat products.

A series of 94 strains of lactic acid bacteria and Micrococcaceae were tested for their ability to decarboxylate histidine and tyrosine in a laboratory medium. Histamine and tyramine were quantified by using a fluorimetric and a HPLC method. There was no significant difference between the results obtained with either method. Among the strains tested, only three released histamine. On the other hand, all the strains of Carnobacterium produced high concentrations of tyramine (2193 micrograms/ml). Some strains of Lactobacillus curvatus and also Lactobacillus plantarum showed tyramine production. Micrococcaceae and Lactobacillus sake did not produce tyramine.

Effect of nitrate and incubation conditions on the production of catalase and nitrate reductase by staphylococci.

The objective of this work was to study the production of catalase and nitrate reductase by staphylococci in order to understand their role in lipid oxidation during sausage manufacturing. Catalase and nitrate reductase were measured in resting cells and supernatants of staphylococci grown in different conditions. All staphylococci (except S. warneri) synthetized nitrate reductase. In static condition, the synthesis was maximal during exponential growth phase, whereas in shaking condition, the synthesis was maximal at the beginning of stationary phase. The production of nitrate reductase was increased in presence of nitrate, this effect was particularly important for the two S. carnosus strains which exhibited the highest activity. For all staphylococci, the production of catalase was maximal at the end of the exponential growth phase. The lowest amount of catalase was produced by S. warneri and the highest by S. carnosus. Only S. xylosus 873 and S. saprophyticus 852 released high amounts of catalase in the supernatant growth. Staphylococci produced higher amounts of catalase in shaking conditions. Addition of nitrate in the growth media favoured the synthesis of catalase, with a pronounced effect for S. carnosus. Nitrate also favoured the release of catalase.

Factors influencing leucine catabolism by a strain of Staphylococcus carnosus.

The metabolism of leucine by resting cells of Staphylococcus carnosus 833 was studied according to three physicochemical factors: preculture condition (defined medium; complex medium), nitrate concentration (0% and 0.03%) and stirring condition (static or shaking). A factorial design was set up to test the effects of these factors, each at two levels. The results showed that resting cells of S. carnosus 833 produced 3-methyl butanal, 3-methyl butanol and 3-methyl butanoic acid from leucine. Whatever the incubation conditions, there was greater quantity of 3-methyl butanoic acid than 3-methyl butanal and 3-methyl butanol. The preculture and incubation conditions influenced the level of production of the 3 metabolites. The highest overall production of the 3 metabolites was observed when cells were incubated without nitrate in the reaction mixture. 3-methylbutanoic acid production was enhanced when S. carnosus 833 was precultivated in complex medium. 3-methylbutanal was only detected when cells were precultivated in defined medium. Stirring condition had no effect on leucine catabolism of S. carnosus 833.

Production of esters by Staphylococci.

The ability of resting cells and extracellular concentrates of Staphylococci to synthesize ethyl esters was studied in the presence of ethanol and short chain acids considered individually. All the strains synthesized ethyl esters, S. warneri was the highest producer and S. carnosus the lowest. Resting cells esterified preferentially butanoic acid, extracellular concentrates esterified butanoic, valeric and hexanoic acids. Acetic, decanoic and branched acids were poorly esterified. The activity of the extracellular concentrates with ethanol and butanoic acid was not modified by the pH (pH 5.5 or 7.0); but it was decreased at a temperature of 14 degrees C compared to 24 degrees C. For the resting cells it was the opposite, the activity was inhibited by acid pH and was not influenced by the temperatures. So the Staphylococci could produce esters during sausage manufacture.

Growth and effect of staphylococci and lactic acid bacteria on unsaturated free fatty acids.

The growth and the effects of four species of staphylococci and six lactic acid bacteria (LAB) of the genus Carnobacterium, Lactobacillus and Pediococcus on unsaturated free fatty acids were studied. The strains were grown in complex medium supplemented either with oleic, linoleic or linolenic acids. Growth was followed and oxidation of the substrates measured by TBARS. The strains of Staphylococcus xylosus 873, 16, Staphylococcus warneri 863 and Staphylococcus saprophyticus grew well on all the substrates. Whereas, the growth of the two strains of Staphylococcus carnosus and Staphylococcus xylosus 831 was inhibited in the media with linolenic acid. The addition of manganese to this media allowed good growth of these strains. All the LAB did not grow well in the media with linoleic acid, but their growth was favoured by addition of manganese to the media. Under our conditions, only linoleic and linolenic acids were oxidised. All the strains had no prooxidant activity. All the staphylococci limited oxidation of linoleic acid and had a small effect on linolenic acid. LAB did not limit oxidation of linoleic acid. With manganese in the media: the oxidation of the sterile controls was delayed for 2 days and then increased; strains of S. carnosus and S. xylosus inhibited oxidation of linolenic acid; and Lactobacillus plantarum and Pediococcus pentosaceus limited oxidation of linoleic acid. The two Carnobacterium, whatever the conditions, had no antioxidant properties.

Bacterial role in flavour development.

The role of bacteria in the production of non volatile and volatile compounds involved in the fermented meat flavour is discussed. Lactic acid bacteria produce D-lactic and acetic acids which may give a sour note. By reducing the pH, they also modulate the other bio-chemical bacterial activities. In muscle tissue proteins are degraded into peptides and lipids into fatty acids mainly by endogenous enzymes. In fermented meat products with a high pH lipases from very lipolytic species of Staphylococcus could increase lipolysis. Bacteria could also play a role in the production and degradation of free amino acids. Staphylococcus and to a lesser extent, lactic acid bacteria could participate in the production of methyl-branched aldehydes and their corresponding alcohols and acids from branchedchain amino acids. By their nitrate reductase and catalase Staphylococcus species limit fatty acid oxidation and aldehyde production. Staphylococcus could contribute to the ester content as they can produce or hydrolyse esters in vitro.

Effects of starter cultures on the biochemical characteristics of French dry sausages.

Bacterial starter cultures, consisting of lactic acid bacteria (Lactobacillus, Pediococcus) and staphylococci (Staphlylococcus carnosus. S. saprophyticus, S. warneri), have an important effect on the pH, lactate, acetate and free fatty acid contents of sausages. Sausages made with L. sake had the lowest pH whereas no change of pH was noticed in the controls and in the sausages inoculated with P. acidilactici. Inoculation of S. saprophyticus led to sausages with a high acetate content. Lipolysis occurred not only in inoculated samples but also in the controls, but it was the highest in the sausages inoculated with S. warneri.

Effects of starter cultures on the formation of flavour compounds in dry sausage.

The purpose of this work was to study the impact of starter cultures on the production of flavour compounds in dry sausages. The effect of six starter cultures corresponding to different combinations of lactic acid bacteria (Lactobacillus sake L110, Pediococcus acidilactici 725, P. pentosaceus 716) and different Staphylococcus species (S. carnosus 833, S. warneri 863, S. saprophyticus M31) strains were tested in a total of 30 dry sausages without spices. The analysis of flavour compounds using a dynamic headspace apparatus coupled to a gas chromatograph and a mass spectrometer enabled us to identify about 80 volatile compounds. They were of various origins-lipids oxidation, fermentations, amino acid catabolism and animal feedstuffs. The influence of the starters and especially the flavouring strains proved to have a major effect on the level of volatile compounds in dry sausages. The flavour tests led to more accurate determination of the sensory characteristics of important molecules in the flavour of dry sausages. The sensory analyses showed that the butter odour of dry sausages largely depends on the catabolism of carbohydrates and that curing and rancid odours were correlated with some typical compounds of lipid oxidation.