清酒乳杆菌细菌素研究的现状及展望

清酒乳杆菌不仅可作为发酵香肠的发酵剂赋予香肠良好的风味和品质,而且绝大多数清酒乳杆菌细菌素对食源性致病菌单核增生李斯特菌具有较强的抑制作用。清酒乳杆菌细菌素种类多,性质各异。本文分别从清酒乳杆菌细菌素的种类,肉制品环境对清酒乳杆菌和细菌素稳定性的影响以及清酒乳杆菌细菌素在食品中的应用研究进行了概述,为寻找新的具有良好性能的清酒乳杆菌细菌素提供了参考。     

Expression of the arginine deiminase pathway genes in Lactobacillus sakei is strain dependent and is affected by the environmental pH

The adaptation of Lactobacillus sakei to a meat environment is reflected in its metabolic potential. For instance, the ability to utilize arginine through the arginine deiminase (ADI) pathway, resulting in additional ATP, represents a competitive benefit. In L. sakei CTC 494, the arc operon (arcABCTDR) shows the same gene order and organization as that in L. sakei 23K, the genome sequence of which is known. However, differences in relative gene expression were found, and these seemed to be optimal in different growth phases, namely, the highest relative gene expression level was in the end exponential growth phase in the case of L. sakei CTC 494 and in the mid-exponential growth phase of L. sakei 23K. Also, the environmental pH influenced the relative expression level of the arc operon, as shown for L. sakei CTC 494, with the highest relative expression level occurring at the optimal pH for growth (pH 6.0). Deviations from this optimal pH (pH 5.0 and pH 7.0) resulted in an overall decline of the relative expression level of all genes of the arc operon. Furthermore, a differential relative expression of the individual genes of the arc operon was found, with the highest relative gene expression occurring for the first two genes of the arc operon (arcA and arcB). Finally, it was shown that some L. sakei strains were able to convert agmatine into putrescine, suggesting an operational agmatine deiminase pathway in these strains, a metabolic trait that is undesirable in meat fermentations. This study shows that this metabolic trait is most probably encoded by a previously erroneously annotated second putative arc operon.     

Protective effect of Lactobacillus sakei 2a against experimental challenge with Listeria monocytogenes in gnotobiotic mice

AIM: Lactobacillus sakei 2a isolated from sausage and presenting an in vitro antagonistic activity against Listeria monocytogenes Scott A was tested for a protective effect in mice experimentally challenged with the enterobacteria. METHODS AND RESULTS: In the experimental group, germ-free mice (n = 24) were inoculated intragastrically with 0.1 ml of a suspension containing 10(8) colony forming units (CFU) of Lact. sakei and 4 days later the animals were challenged intragastrically with 0.1 ml of a suspension containing 10(8) CFU of L. monocytogenes. Control group (n = 24) was only inoculated with the bacterial pathogen. Faecal counts showed that L. monocytogenes reached similar population levels (10(9) CFU g(-1) of contents) in both the groups. Animals in the control group showed lower (P = 0.0004) survival frequency (58.3%) when compared with the experimental one (100%). Anatomopathological examination confirmed the mortality data. CONCLUSIONS: Lactobacillus sakei 2a can survive in the mammal digestive tract where showed a protective effect against L. monocytogenes. This phenomenon was not due to an antagonistic activity. SIGNIFICANCE AND IMPACT OF THE STUDY: Use of Lact. sakei 2a as a meat starter could inhibit not only L. monocytogenes growth in the fermented product but also pathogen virulence in the gastrointestinal tract.     

Inhibition of Listeria monocytogenes in chicken cold cuts by addition of sakacin P and sakacin P-producing Lactobacillus sakei

AIMS: To evaluate the potential of sakacin P and sakacin P-producing Lactobacillus sakei for the inhibition of growth of Listeria monocytogenes in chicken cold cuts, by answering the following questions. (i) Is sakacin P actually produced in food? (ii) Is sakacin P produced in situ responsible for the inhibiting effect? (iii) How stable is sakacin P in food? METHODS AND RESULTS: Listeria monocytogenes, a Lact. sakei strain and/or the bacteriocin sakacin P were added to chicken cold cuts, vacuum packed and incubated at 4 or 10 degrees C for 4 weeks. Each of two isogenic Lact. sakei strains, one producing sakacin P and the other not, had an inhibiting effect on the growth of L. monocytogenes. The effect of these two isogenic strains on the growth of L. monocytogenes was indistinguishable, even though sakacin P was produced in the product by one of the two Lact. sakei strains. The addition of purified sakacin P had an inhibiting effect on the growth of L. monocytogenes. A high dosage of sakacin P (3.5 microg x g(-1)) had a bacteriostatic effect throughout the storage period of 4 weeks, while a low dosage (12 ng x g(-1)) permitted initial growth, but at a slow rate. After 4 weeks of storage, the number of L. monocytogenes in the samples with a low dosage of sakacin P was 2 logs below that in the untreated control. When using a high dosage of sakacin P, the bacteriocin was detected in samples stored for up to 6 weeks. CONCLUSIONS: (i) Sakacin P is produced by a Lact. sakei strain when growing on vacuum-packed chicken cold cuts. (ii) Inhibiting effects of Lact. sakei, other than sakacin P, are active in inhibiting the growth of L. monocytogenes growing on chicken cold cuts. (iii) Sakacin P is stable on chicken cold cuts over a period of 4 weeks. SIGNIFICANCE AND IMPACT OF THE STUDY: Both sakacin P and Lact. sakei were found to have potential for use in the control of L. monocytogenes in chicken cold cuts.     

The pentose moiety of adenosine and inosine is an important energy source for the fermented-meat starter culture Lactobacillus sakei CTC 494

The genome sequence of Lactobacillus sakei 23K has revealed that the species L. sakei harbors several genes involved in the catabolism of energy sources other than glucose in meat, such as glycerol, arginine, and nucleosides. In this study, a screening of 15 L. sakei strains revealed that arginine, inosine, and adenosine could be used as energy sources by all strains. However, no glycerol catabolism occurred in any of the L. sakei strains tested. A detailed kinetic analysis of inosine and adenosine catabolism in the presence of arginine by L. sakei CTC 494, a fermented-meat starter culture, was performed. It showed that nucleoside catabolism occurred as a mixed-acid fermentation in a pH range (pH 5.0 to 6.5) relevant for sausage fermentation. This resulted in the production of a mixture of acetic acid, formic acid, and ethanol from ribose, while the nucleobase (hypoxanthine and adenine in the case of fermentations with inosine and adenosine, respectively) was excreted into the medium stoichiometrically. This indicates that adenosine deaminase activity did not take place. The ratios of the different fermentation end products did not vary with environmental pH, except for the fermentation with inosine at pH 5.0, where lactic acid was produced too. In all cases, no other carbon-containing metabolites were found; carbon dioxide was derived only from arginine catabolism. Arginine was cometabolized in all cases and resulted in the production of both citrulline and ornithine. Based on these results, a pathway for inosine and adenosine catabolism in L. sakei CTC 494 was presented, whereby both nucleosides are directly converted into their nucleobase and ribose, the latter entering the heterolactate pathway. The present study revealed that the pentose moiety (ribose) of the nucleosides inosine and adenosine is an effective fermentable substrate for L. sakei. Thus, the ability to use these energy sources offers a competitive advantage for this species in a meat environment.     

Diversity of Lactobacillus sakei strains investigated by phenotypic and genotypic methods

The diversity of 10 strains of Lactobacillus sakei, a commercially important species of lactobacilli, was characterized by studying food isolates. Growth characteristics varied among the strains when examined after growth in a complex medium and a defined medium with either glucose or ribose. A commercial starter culture strain showed the fastest growth rates and high biomass formation on all media, while two of the strains hardly grew on ribose. Based on acidification properties in a meat model, some of the strains had the ability to compete with the indigenous microbiota of the meat batter in addition to being fast acid producers. Carbohydrate-fermentation abilities revealed a relatively wide variation, clustering the strains into two phenotypic groups. The isolates were analyzed using different genetic fingerprinting techniques, demonstrating a distinction between two genetic groups, a grouping consistent with previous studies dealing with L. sakei strains. Comparative genome hybridization (CGH) was introduced for clustering the strains and the same division into two genetic groups was observed. Chromosomal sizes of the strains were estimated by pulsed field gel electrophoresis (PFGE) and were found to vary from 1884 to 2175 kb. The genetic groups did not correlate with the clustering obtained with carbohydrate-fermenting abilities or with chromosomal sizes.     

Molecular characterization of tet(M) genes in Lactobacillus isolates from different types of fermented dry sausage

The likelihood that products prepared from raw meat and milk may act as vehicles for antibiotic-resistant bacteria is currently of great concern in food safety issues. In this study, a collection of 94 tetracycline-resistant (Tc(r)) lactic acid bacteria recovered from nine different fermented dry sausage types were subjected to a polyphasic molecular study with the aim of characterizing the host organisms and the tet genes, conferring tetracycline resistance, that they carry. With the (GTG)(5)-PCR DNA fingerprinting technique, the Tc(r) lactic acid bacterial isolates were identified as Lactobacillus plantarum, L. sakei subsp. carnosus, L. sakei subsp. sakei, L. curvatus, and L. alimentarius and typed to the intraspecies level. For a selection of 24 Tc(r) lactic acid bacterial isolates displaying unique (GTG)(5)-PCR fingerprints, tet genes were determined by means of PCR, and only tet(M) was detected. Restriction enzyme analysis with AccI and ScaI revealed two different tet(M) allele types. This grouping was confirmed by partial sequencing of the tet(M) open reading frame, which indicated that the two allele types displayed high sequence similarities (>99.6%) with tet(M) genes previously reported in Staphylococcus aureus MRSA 101 and in Neisseria meningitidis, respectively. Southern hybridization with plasmid profiles revealed that the isolates contained tet(M)-carrying plasmids. In addition to the tet(M) gene, one isolate also contained an erm(B) gene on a different plasmid from the one encoding the tetracycline resistance. Furthermore, it was also shown by PCR that the tet(M) genes were not located on transposons of the Tn916/Tn1545 family. To our knowledge, this is the first detailed molecular study demonstrating that taxonomically and genotypically diverse Lactobacillus strains from different types of fermented meat products can be a host for plasmid-borne tet genes.     

The effect of sodium acetate on the growth yield,the production of L- and D-lactic acid,and the activity of some enzymes of the glycolytic pathway of Lactobacillus sakei NRIC 1071(T) and Lactobacillus plantarum NRIC 1067(T)

The effect of sodium acetate was studied on the change of the growth yield, the production of L- and D-lactic acid, and the activity of lactate dehydrogenases (LDHs; L-lactate dehydrogenase [EC 1.1.1.27, L-LDH] plus D-lactate dehydrogenase [EC 1.1.1.28, D-LDH]), fructose-1, 6-bisphosphate aldolase [EC 4.1.2.13, FBP-aldolase], and phosphofructokinase [EC 2.7.1.11, PFK] of Lactobacillus sakei NRIC 1071(T) and Lactobacillus plantarum NRIC 1067(T). The growth yield of L. sakei NRIC 1071(T) was increased 1.6 times in the presence of sodium acetate compared with its absence. The activity of LDHs in L. sakei NRIC 1071(T) and L. plantarum NRIC 1067(T) was retained longer under the addition of sodium acetate in the reaction mixture. As a result, these strains produced much more lactic acid in the presence of sodium acetate compared with its absence. Furthermore, the activity of L-LDH in L. sakei NRIC 1071(T) cultivated in the presence of sodium acetate increased three times or more compared with the activity of the cells cultivated in its absence. Consequently, the type of stereoisomers of lactic acid produced by L. sakei shifted from the DL-type to the L-type because the ratio of L-lactic acid to D-lactic acid produced became larger with the addition of sodium acetate to culture media. This phenomenon was not observed in L. plantarum NRIC 1067(T). Further, the participation of lactate racemase is discussed from the viewpoint of the production of D-lactic acid by L. sakei.     

Identification by fluorescence spectroscopy of lactic acid bacteria isolated from a small-scale facility producing traditional dry sausages

Three different fluorescence spectra were recorded following excitation at 250 nm (aromatic amino acids+nucleic acids, AAA+NA), 316 nm (NADH) and 380 nm (FAD) for 20 type strain collections of lactic acid bacteria (LAB). Evaluation of the data using principal component analysis and factorial discriminant analysis showed a good discrimination of considered LAB at the genus, species and genus-species level. AAA+NA fluorophores showed the highest percentage of good classification. From AAA+NA spectra recorded on LAB isolated from a small-scale facility producing traditional dry sausages, we succeeded to identify 28 of 29 wild strains. This method allowed us to discriminate between Lactobacillus sakei subsp. carnosus and Lactobacillus sakei subsp. sakei. Thus, intrinsic fluorescence is an economical and powerful tool for the identification of wild LAB isolated from meat and meat products.     

Effect of Lactobacillus-protective cultures with bacteriocin-like inhibitory substances' producing ability on microbiological, chemical and sensory changes during storage of refrigerated vacuum-packaged sliced beef

AIMS: To investigate the effect of applying two different Lactobacillus-protective cultures, with bacteriocin-like inhibitory substances' (BLIS) producing ability, individually or in combination, on microbiological, chemical and sensory changes during storage of refrigerated vacuum-packaged sliced beef meat. METHODS AND RESULTS: Lactobacillus sakei CECT 4808 and Lactobacillus curvatus CECT 904(T), which were shown to be producers of BLIS, were inoculated individually or in combination on slices of beef M. semitendinosus. The samples were vacuum packaged and stored at 4 +/- 1 degrees C and were assessed during a 28-day storage period for microbiological [Enterobacteriaceae, Pseudomonas spp., lactic acid bacteria (LAB), Brochothrix thermosphacta and yeasts and moulds], chemical (pH, protein hydrolysis degree, lipid oxidation), sensory (abnormal odour) parameters and instrumental colour. Samples inoculated with the Lact. sakei strain and samples inoculated with the combination of the two strains had significantly (P < 0.05) lower spoilage microbial counts than those inoculated with the Lact. curvatus strain alone or the controls, while both chemical parameters (including lipid oxidation) and abnormal odour scores were also significantly (P < 0.05) improved by the former. Moreover, Lact. sakei alone showed a better preserving effect (P < 0.05) than the combination of both strains in the majority of the parameters tested. Instrumental colour measurements changed with storage time, but no treatment effects (P >or= 0.05) were observed during the whole 28-day storage period. CONCLUSIONS: The BLIS producer Lact. sakei CECT 4808 strain may be used for improving preservation of vacuum-packaged beef slices, as regards spoilage microbial counts and the chemical parameters tested in this study. SIGNIFICANCE AND IMPACT OF THE STUDY: Inoculation with the BLIS producer Lact. sakei CECT 4808 strain would provide an additional hurdle to improve storage life of refrigerated vacuum-packaged sliced beef. Furthermore, this strain demonstrated limited antioxidative ability, which could make a contribution to the prevention of lipid oxidation in meat and meat products.     

Development of a microbial time/temperature indicator prototype for monitoring the microbiological quality of chilled foods

A time/temperature indicator (TTI) system based on the growth and metabolic activity of a Lactobacillus sakei strain was developed for monitoring food quality throughout the chilled-food chain. In the designed system, an irreversible color change of a chemical chromatic indicator (from red to yellow) progressively occurs due to the pH decline that results from microbial growth and metabolism in a selected medium. The relation of the TTI response (color change) to the growth and metabolic activity (glucose consumption, lactic acid production, pH decrease) of L. sakei was studied. In addition, the temperature dependence of the TTI kinetics was investigated isothermally in the range of 0 to 16 degrees C and modeled with a system of differential equations. At all temperatures tested, the pH and color changes of the TTI system followed closely the growth of L. sakei, with the endpoint (the time at which a distinct visual color change to the final yellow was observed) of the TTI coinciding with a population level of 10(7) to 10(8) CFU/ml. The endpoint decreased from 27 days at 0 degrees C to 2.5 days at 16 degrees C, yielding an activation energy of 97.7 kJ/mol, which was very close to the activation energy of the L. sakei growth rate in the TTI substrate (103.2 kJ/mol). Furthermore, experiments conducted on the effect of the inoculum level showed a negative linear relationship between the level of L. sakei inoculated in the system medium and the endpoint of the TTI. For example, the endpoint at 8 degrees C ranged from 6 to 2 days for inoculum levels of 10(1) and 10(6) CFU/ml, respectively. This relationship allows the easy adjustment of the TTI endpoint at a certain temperature according to the shelf life of the food product of concern by using an appropriate inoculum level of L. sakei. The microbial TTI prototype developed in the present study could be used as an effective tool for monitoring shelf life during the distribution and storage of food products that are spoiled primarily by lactic acid bacteria or other bacteria exhibiting similar kinetic responses and spoilage potentials. Apart from the low cost, the main advantage of the proposed TTI is that its response closely matches the loss of the quality of a food product by simulating the microbial spoilage process in particular environments.     

Improvement of Raw Sausage Fermentation by Stress-Conditioning of the Starter Organism Lactobacillus sakei

Effective growth and high acidification activity during meat fermentation are key characteristics of starter lactobacilli to ensure hygienic safety and sensory quality of the product. In this study, we demonstrated that the performance of Lactobacillus sakei in sausage fermentation can be improved by preinoculation treatments with sublethal heat, cold, and salt stress. Sausages were produced and inoculated with stress-treated cells of L. sakei 23 K (pLPV111) and the isogenic mutant of the class III heat-shock repressor CtsR, which was previously shown to exhibit improved growth in fermenting sausages. The pH values of sausages fermented with stressed cells attained defined threshold values in a distinctly shorter time than those inoculated with unstressed cells. In particular, the cold-stressed cells (4 degrees C) reduced the pH to 5.0 within approximately 40 hours compared with approximately 70 hours for untreated cells. This enhanced acidification activity of the cold-stressed cells was consistent with an increased growth rate. Growth studies in culture medium showed that stress-treated cells with improved performance did not exhibit this advantage when exposed to curing salt, one of the major stressors at the beginning of sausage fermentation. Preinoculation stress treatment is a promising way to improve the effectiveness of meat starter lactobacilli.     

Effects of sodium acetate on the production of stereoisomers of lactic acid by Lactobacillus sakei and other lactic acid bacteria

Lactobacillus sakei and other lactic acid bacteria were studied on the change of the type of stereoisomers (the ratio of L-form to D-form) of lactic acid produced in the presence of sodium acetate and under other cultural conditions. Of 49 strains tested, only L. sakei NRIC 1071(T) and L. coryniformis subsp. coryniformis NRIC 1638(T) changed the type in the presence of 50 mm sodium acetate compared with the absence of sodium acetate. The type produced by L. sakei NRIC 1071(T) was shifted 30% or more from the DL-type to the L-type in the presence of 50 mm sodium acetate. L. sakei NRIC 1071(T) produced not only twice or more the amount of L-lactic acid but decreased the amount of D-lactic acid compared with the absence of sodium acetate. The shift of the DL-type to the L-type by L. sakei is due to the high production of L-lactic acid and the low production of D-lactic acid. The type of stereoisomers produced by 11 L. sakei strains was also shifted from the DL-type to the L-type in the presence of 50 mm sodium acetate. The shift of stereoisomers by the majority of L. sakei strains seems interesting from the viewpoint of the delineation of this species.     

The effect of sodium acetate on the activity of L- and D-lactate dehydrogenases in Lactobacillus sakei NRIC 1071(T) and other lactic acid bacteria

The effect of sodium acetate on the production of stereoisomers of lactic acid produced by Lactobacillus sakei NRIC 1071(T) and other lactic acid bacteria was studied. L. sakei NRIC 1071(T) started producing L-lactic acid at the early logarithmic phase and d-lactic acid at the late logarithmic phase. The activity of L-lactate dehydrogenase [EC 1.1.1.27, L-LDH] from the resting cells of L. sakei NRIC 1071(T) appeared at the early stage of the logarithmic phase during the growth, and the activity of D-lactate dehydrogenase [EC 1.1.1.28, D-LDH] at the late stage of the logarithmic phase. The resting cells and cell-free extracts of L. sakei NRIC 1071(T) did not produce DL-lactic acid from L- or D-lactic acid. Stained bands of L-LDH and D-LDH appeared in the cell-free extracts from the cells of L. sakei NRIC 1071(T). Consequently, L. sakei conclusively produced L- and D-lactic acid by the action of L-LDH and D-LDH. This finding leads to the conclusion that lactate racemase [EC 5.1.2.1] does not exist in this strain. When the specific activity of LDHs (the total activity of L-LDH plus D-LDH) from the cells cultivated in the presence of sodium acetate is compared with that cultivated in its absence, the ratio of the activity between the cells cultivated in the former condition and those in the latter fell from 1.7 on the cell-free extracts to 1.3 on the preparation of the QAE-Toyopearl 550c chromatography. This result indicates that the amount of LDHs in the cells of L. sake NRIC 1071(T) cultivated in the presence of 50 mM sodium acetate was much more than that in the cells cultivated in the absence of sodium acetate. The shift of the type of stereoisomers of lactic acid from the DL-type to the L-type is discussed in the case of L. sakei strains.     

Co-factor engineering in lactobacilli: effects of uncoupled ATPase activity on metabolic fluxes in Lactobacillus (L.) plantarum and L. sakei

The hydrolytic F(1)-part of the F(1)F(0)-ATPase was over-expressed in Lactobacillus (L.) plantarum NC8 and L. sakei Lb790x during fermentation of glucose or ribose, in order to study how changes in the intracellular levels of ATP and ADP affect the metabolic fluxes. The uncoupled ATPase activity resulted in a decrease in intracellular energy level (ATP/ADP ratio), biomass yield and growth rate. Interestingly, the glycolytic and ribolytic flux increased in L. plantarum with uncoupled ATPase activity compared to the reference strain by up to 20% and 50%, respectively. The ATP demand was estimated to have approximately 80% control on both the glycolytic and ribolytic flux in L. plantarum under these conditions. In contrast, the glycolytic and ribolytic flux decreased in L. sakei with uncoupled ATPase activity.     

Isolation and identification of tetracycline resistant lactic acid bacteria from pre-packed sliced meat products

In recent years, the food chain has been recognised as one of the main routes for transmission of antibiotic resistant bacteria between the animal and human population. In this regard, the current study aimed to investigate if tetracycline resistant (tetR) lactic acid bacteria (LAB) are present in ready-to-eat modified atmosphere packed (MAP) sliced meat products including fermented dry sausage, cooked chicken breast meat and cooked ham. From population graphs based on doubling tetracycline concentrations between 0 and 256 microg ml(-1), only fermented dry sausage was shown to contain a high-level retR LAB population (5.10(1) - 2,23.10(4) CFU/g), and this in four out of ten examined sausages. From these four positive sausages, a total of 100 strains were isolated on de Man, Rogosa and Sharpe-sorbic acid (MRS-S) agar without tetracycline (n = 45) and on MRS-S agar supplemented with a tetracycline breakpoint concentration of 64 microg ml(-1) (n = 55). Using resistance histograms derived from the disc diffusion method, all these strains were grouped as sensitive to rifampicin, erythromycin and ampicillin. All strains from the tetracycline-containing MRS-S plates were resistant to tetracycline. Identification with whole-cell protein profiling revealed that the total strain set represented four different species: Pediococcus pentosaceus, Lactobacillus plantarum, Lactobacillus sakei subsp. carnosus and Lactobacillus curvatus. All species are commonly associated with fermented dry sausage, either as starter culture or as natural contaminants. The latter three species were found to comprise all tetracycline resistant strains. To our knowledge, this is the first report providing evidence for the presence of tetR LAB in final ready-to-eat pre-packed fermented dry sausages.     

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

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 degrees C and 10 degrees 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 10(5) cfu g(-1) in all samples during storage. Only yeasts were able to compete against LAB and reached almost 10(7) cfu g(-1) after 30 d of aerobic storage at 10 degrees C. The great dominance (> 10(8) 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 degrees C than 4 degrees 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.