Gut production of GABA by a probiotic formula: an in vitro study.

The objective of this study was to evaluate the influence of a probiotic formula on the production of neuroactive compounds in different parts of the colon in batch culture and in vitro gut simulator experiments. Thirteen lactic acid bacterial strains, belonging to the species Levilactobacillus brevis, Lactiplantibacillus plantarum, Lacticaseibacillus paracasei, Ligilactobacillus salivarius, Streptococcus thermophilus, were characterised for their in vitro ability to produce neurotransmitters. L. brevis P30021 and L. plantarum P30025 were selected based on their capability to produce γ-aminobutyric acid (GABA) and acetylcholine in vitro. A probiotic formulation with potential psychobiotic activity was prepared and tested in a batch culture of human microbiota monitoring the formation of GABA and acetylcholine. Samples of the three colonic tracts were taken from the Simulator of the Human Intestinal Microbiota (SHIME®) evaluating the production of GABA and other neurotransmitters by LC-MS. Short chain fatty acids (SCFA) by GC and microbiota composition by 16S rRNA gene sequencing were also determined. Probiotic supplementation led to the formation of GABA and acetylcholine with a decrease in glutamate concentrations in the in vitro batch fermentation. Production of GABA after the treatment with probiotics was confirmed in the SHIME® short-term experiment. No differences in short-chain fatty acids were observed up to 72 h of fermentation. Different microbiota composition was found in the three different parts of the colon, with a higher abundance of Veillonellaceae in the ascending colon vessels. The probiotic-exposed microbiota showed higher levels of Bacteroides, a gut microbe associated with anti-inflammatory activities and a potential GABA producer. Results demonstrate the impact of the tested probiotic formula on gut microbiota structure and GABA production. In conclusion, the probiotic treatment changed the microbiota composition and increased neuroactive metabolites production, indicating promising potential as psychobiotics, even if further clinical evidence is needed to confirm the effectiveness of these probiotics in improving mental health.

Antibiotic-induced microbiota perturbation causes gut endocannabinoidome changes, hippocampal neuroglial reorganization and depression in mice.

The microbiota-gut-brain axis (MGBA) regulates the reciprocal interaction between chronic inflammatory bowel and psychiatric disorders. This interaction involves multiple pathways that are highly debated. We examined the behavioural, biochemical and electrophysiological alterations, as well as gut microbiota composition in a model of antibiotic-induced experimental dysbiosis. Inflammation of the small intestine was also assessed. Mice were exposed to a mixture of antimicrobials for 2weeks. Afterwards, they received Lactobacillus casei DG (LCDG) or a vehicle for up to 7days via oral gavage. Perturbation of microbiota was accompanied by a general inflammatory state and alteration of some endocannabinoidome members in the gut. Behavioural changes, including increased immobility in the tail suspension test and reduced social recognition were observed, and were associated with altered BDNF/TrkB signalling, TRPV1 phosphorylation and neuronal firing in the hippocampus. Moreover, morphological rearrangements of non-neuronal cells in brain areas controlling emotional behaviour were detected. Subsequent probiotic administration, compared with vehicle, counteracted most of these gut inflammatory, behavioural, biochemical and functional alterations. Interestingly, levels of Lachnospiraceae were found to significantly correlate with the behavioural changes observed in dysbiotic mice. Our findings clarify some of the biomolecular and functional modifications leading to the development of affective disorders associated with gut microbiota alterations.

Spoilage-related microbiota associated with chilled beef stored in air or vacuum pack.

In order to study the spoilage-related microbiota of beef at species level, a combination of culture-independent and culture-dependent methods was used to analyse nine different beef samples stored at 4°C in air or in vacuum pack. Plate counts on selective agars after 0, 7 and 20 days of storage showed that vacuum packaging reduced the viable counts of Brochothrix thermosphacta, Pseudomonas spp. and Enterobacteriaceae, whereas the growth of lactic acid bacteria (LAB) was unaffected. Storage in vacuum pack mainly affected viable counts and not necessarily the species diversity of microbial populations on meat. Such populations were studied by PCR-DGGE of DNA directly extracted from meat and from bulk cells from culture media, followed by sequencing of DGGE fragments. Pseudomonas spp., Carnobacterium divergens, B. thermosphacta, Rahnella spp. and Serratia grimesii, or close relatives were detected in the meat at time zero. The use of the culture-independent method highlighted the occurrence of species that were not detected by plating. Photobacterium spp. occurred in most meat samples stored in air or in vacuum pack, which indicates this organism probably has a role in spoilage. In contrast, culture-dependent analysis allowed detection of bacterial species that were not found in DNA extracted directly from meat. This was the case for several species of Serratia or Rhanella among the enterobacteria, and Leuconostoc spp. among the LAB. Besides advancing our knowledge of the species involved in the spoilage of vacuum-packaged meat, this study shows the benefits of combining culture-based and direct approaches to enhance understanding of populations of spoilage bacteria.

Characterization of Streptococcus thermophilus lytic bacteriophages from mozzarella cheese plants.

Phage infection still represents the main cause of fermentation failure during the mozzarella cheese manufacturing, where Streptococcus thermophilus is widely employed as starter culture. Thereby, the success of commercial lactic starter cultures is closely related to the use of strains with low susceptibility to phage attack. The characterization of lytic S. thermophilus bacteriophages is an important step for the selection and use of starter cultures. The aim of this study was to characterize 26 bacteriophages isolated from mozzarella cheese plants in terms of their host range, DNA restriction profile, DNA packaging mechanism, and the variable region VR2 of the antireceptor gene. The DNA restriction analysis was carried out by using the restriction enzymes EcoRV, PstI, and HindIII. The bacteriophages were distinguished into two main groups of S. thermophilus phages (cos- and pac-type) using a multiplex PCR method based on the amplification of conserved regions in the genes coding for the major structural proteins. All the phages belonged to the cos-type group except one, phage 1042, which gave a PCR fragment distinctive of pac-type group. Furthermore, DNA sequencing of the variable region VR2 of the antireceptor gene allowed to classify the phages and examine the correlation between typing profile and host range. Finally, bacterial strains used in this study were investigated for the presence of temperate phages by induction with mitomycin C and only S. thermophilus CHCC2070 was shown to be lysogenic.

Characterization of bacteriocin-coated antimicrobial polyethylene films by atomic force microscopy.

Antimicrobial agents can be used to give antimicrobial properties to polymeric materials used to envelope foods for packaging purposes. In this study, we exploited an atomic force microscopy (AFM) analysis for the characterization of plastic films activated with antimicrobial agents. The aim was to acquire information on the distribution of the antimicrobials on plastic materials with the ultimate scope of understanding the mechanisms of interaction between antimicrobials and materials to be used for food packaging. Four polyethylene films differing in linear, EVA, and erucamide content were activated by 3 different bacteriocins as antimicrobials, namely, nisin and bacteriocins Bac162W from Lactobacillus curvatus and BacAM09 from Lactobacillus plantarum. The spectrum of activity of the bacteriocins was assayed and shown to include several strains of Listeria monocytogenes. The plastic films were activated by a previously developed coating procedure and the surfaces of the active films were examined by AFM. In addition, roughness parameters related to the single surfaces were investigated by an appropriate software. Significant differences were found between the bacteriocin activated and control (nonactivated) films and the activated surfaces showed lower values of average roughness and surface area ratio. It was not always possible to obtain a homogeneous distribution of the bacteriocin preparation following the coating procedure. This result was dependent on the bacteriocin used and its distribution on the different plastic films. Overall, the bacteriocin Bac162W showed the most homogeneous distribution while surfaces treated with nisin, showing a sort of microtexturing, always gave the highest roughness values. Although the issue needs further investigation, the connection between AFM imaging, roughness, and antimicrobial distribution on active packaging showed the potential to improve the understanding of the interactions between plastic films and antimicrobial preparations that can be important for the innovation in food packaging and science.

Behaviour of Brochothrix thermosphacta in presence of other meat spoilage microbial groups.

The microbial flora of fresh meat stored aerobically at 5 degrees C up to spoilage was enumerated and collected in order to have mixed spoilage bacterial groups to be used in competition tests against Brochothrix thermosphacta. The bacterial groups collected as bulk colonies were identified by PCR-DGGE followed by partial 16S rDNA sequencing. The predominant bacteria associated with the spoilage of the refrigerated beef were B. thermosphacta, Pseudomonas spp, Enterobacteriaceae and lactic acid bacteria (LAB). The interactions between B. thermosphacta and the other spoilage microbial groups were studied in vitro at 5 degrees C. The results showed that a decrease of the growth of B. thermosphacta was evidenced in presence of LAB at 5 degrees C while the bacterium is the dominant organism when inoculated with mixtures of Pseudomonas spp., LAB and Enterobacteriaceae. A better understanding of bacterial meat spoilage interactions may lead to improved quality of fresh meat stored in refrigerated conditions.

Effect of a bacteriocin-activated polythene film on Listeria monocytogenes as evaluated by viable staining and epifluorescence microscopy.

AIMS: To evaluate the effect of a bacteriocin-activated polythene film on resting and growing populations of Listeria monocytogenes. METHODS AND RESULTS: The active polythene films were industrially obtained by coating a solution of bacteriocin 32Y from Lactobacillus curvatus upon the surface of the film to be in contact with the packaged material. The behaviour of live Listeria populations was examined in liquid suspensions directly in contact with the bacteriocin-activated film, packed in antimicrobial film, and in a challenge test of storage of frankfurters superficially contaminated by L. monocytogenes and packed in antimicrobial film. In all the experiments, live and dead cells of L. monocytogenes were counted in epifluorescence microscopy after viable staining, which proved to be a suitable method to evaluate the action of bacteriocins on populations of L. monocytogenes. The results showed that the direct contact between active film surface and L. monocytogenes cells is effective for a fast and irreversible inactivation of the population by determining a direct cell disruption. This was confirmed by the results of the challenge test indicating that the antimicrobial package was effective in inhibiting the growth and survival of the pathogen on the surface of frankfurters during storage. CONCLUSIONS: The use of the antimicrobial film is encouraged especially for solid food products where the superficial contaminants come immediately in contact with the antimicrobial film. SIGNIFICANCE AND IMPACT OF THE STUDY: A fast inactivation of the bacterial population, coupled with appropriate conditions of storage, can improve the quality and safety and prolong the shelf-life of the food products packed in antimicrobial films.

Antimicrobial activity of a nisin-activated plastic film for food packaging.

AIMS: To determine the effectiveness of a packaging film coated with nisin to inhibit Micrococcus luteus ATCC 10240 in tryptone soya broth (TSB) and the microbiota of raw milk during storage. A further aim was to examine the release of nisin from the activated film. METHODS AND RESULTS: An active package, obtained from nisin-treated film, was filled with 1 l of M. luteus ATCC 10240 (ML) suspension in TSB and stored at 4 and 25 degrees C for 2 days. After 24 h at 25 degrees C there was a remarkable reduction of M. luteus ATCC 10240 compared with the control, while at 4 degrees C a slight reduction was observed. Moreover, microbial growth was controlled when 1 l of three different kinds of milk was poured into the active package and stored at 4 degrees C for 7 days. The most significant results were observed in raw milk and pasteurized milk with a reduction of 0.9 log and 1.3 log, respectively. The release experiments showed that nisin release from the film was unpredictable, but it was favoured by low pH and high temperature. CONCLUSIONS: It appears that nisin-coated films were effective in inhibiting M. luteus ATCC 10240 in TSB and the bacterial flora in milk, and the release of nisin was pH and temperature dependent. SIGNIFICANCE AND IMPACT OF THE STUDY: Nisin-activated film may control bacterial growth, maintaining food quality, safety and extending the shelf-life of food products.

Response of Escherichia coli O157:H7, Listeria monocytogenes, Salmonella typhimurium, and Staphylococcus aureus to the thermal stress occurring in model manufactures of Grana Padano cheese.

The purpose of this research was to investigate the effect of temperature in the technology of production of Grana cheese against Escherichia coli O157:H7, Listeria monocytogenes, Salmonella Typhimurium, and Staphylococcus aureus. According to the technology of production, the cheese curds are cooked at 55 degrees C and then cooled at room temperature (25 degrees C). A curd-cooling model was developed to estimate the temperature variation across the curd during cooling, and the thermal stress was applied to the pathogens according to the model in model-scale productions of Grana cheese artificially contaminated with approximately 10(4) cfu/mL of the selected pathogens. According to the numerical results, the initial temperature inside the cheese is kept at almost the initial value (above 50 degrees C) for at least 4 h during cooling, whereas the crust of the curd cools rapidly to 30 degrees C in the first hour. The best case was that of the core of the cheese where the high temperature was able to efficiently eliminate the contaminating pathogens. Moreover, the worst case was where the external ring of the curd in which a more rapid cooling allowed bacterial survival. Therefore, the thermal stress in the technology of production of Grana cheese can be only partially effective in the control of the selected pathogens. However, the whole technology of production includes other hurdles that can affect the survival of the pathogens and that need to be taken into account as a whole to evaluate the safety of Grana Padano cheese.

Presence and characterisation of verotoxin producing E. coli in fresh Italian pork sausages, and preparation and use of an antibiotic-resistant strain for challenge studies.

One hundred and twenty six samples of fresh pork sausages were analysed for the presence of verocytotoxigenic Escherichia coli (VTEC). Selective enrichment followed by DNA extraction and PCR amplification of the stx1 and stx2 genes highlighted the occurrence of the above mentioned genes in 20 out of 126 samples screened. From the stx positive enriched cultures, isolation was performed on CT-SMAC agar plates after immuno-magnetic separation of E. coli O157. Fifty three non-sorbitol fermenting isolates were obtained and further characterised, along with the reference strain E. coli ATCC 35150(T). All the isolates were characterised by PCR assays, assessing the presence of stx1, stx2, rfbE(O157:H7), eae and hlyA genes. The overall prevalence of VTEC was found to be 16%. VTEC strains were also characterised by plasmid profiling and REA-PFGE analysis, which allowed strain clustering into 5 and 8 groups, respectively. In addition, an antibiotic resistant E. coli O157:H7 strain was selected and used in challenge tests of raw pork at 4°C. This strain could be selectively counted in the presence of a normal background microflora and it was shown that it could survive for 1week at 4°C in the raw food studied.

PCR detection of staphylococcal enterotoxin genes in Staphylococcus spp. strains isolated from meat and dairy products. Evidence for new variants of seG and seI in S. aureus AB-8802.

AIMS: Evaluation of the occurrence of most known staphylococcal enterotoxin (SE) genes, egc (enterotoxin gene cluster) and TSST1 (toxic shock syndrome toxin 1) gene in both coagulase-positive (CPS) and coagulase-negative (CNS) staphylococcal strains isolated from meat and dairy products. METHODS AND RESULTS: Specificity and reliability of the PCR detection methods used were ascertained by using nine reference strains of Staphylococcus (S. aureus) harbouring SE genes (seA to seE; seG, seH, seI, seM, seJ, seN and seO) and egc (containing the following sequence of genes: seO, seM, seI, phient1, phient2, seN and seG). Of 109 wild Staphylococcus spp. strains analysed, only 11 S. aureus strains were SE and/or TSST1 PCR-positive. The last 11 strains also appeared to harbour the egc. Restriction endonuclease analysis of part of the egc of both reference and wild strains showed that different variants of the egc exist. Moreover, nucleotide sequences of seG and seI indicate that the egc of the strain AB-8802 is characterized by the presence of variants of these enterotoxins (seGv and seIv). CONCLUSIONS: The occurrence of SE genes in CNS and other non-S. aureus species isolated from Napoli-type salami, raw water buffalo milk and natural whey cultures used for mozzarella cheese manufacturing is very rare. SIGNIFICANCE AND IMPACT OF THE STUDY: During this study it was shown that at least five different egc may exist in S. aureus. A thorough study of egc polymorphism should provide further insight into the phylogenetics of the egc.

Development of polythene films for food packaging activated with an antilisterial bacteriocin from Lactobacillus curvatus 32Y.

AIMS: The aims of this work were to (i) use a bacteriocin produced by Lactobacillus curvatus 32Y active against Listeria monocytogenes to activate polythene films by different methods, (ii) implement a large-scale process for antilisterial polythene films production and (iii) verify the efficacy of the developed films in inhibiting the growth of L. monocytogenes during the storage of meat products. METHODS AND RESULTS: The film was made active by using the antilisterial bacteriocin 32Y by Lact. curvatus with three different procedures: soaking, spraying and coating. The antimicrobial activity of the activated films was tested in plate assays against the indicator strain L. monocytogenes V7. All the used procedures yielded active polythene films although the quality of the inhibition was different. The coating was therefore employed to develop active polythene films in an industrial plant. The antimicrobial activity of the industrially produced films was tested in experiments of food packaging involving pork steak and ground beef contaminated by L. monocytogenes V7 at roughly 10(3) CFU cm(-2) and gram respectively. The results of the challenge tests showed the highest antimicrobial activity after 24 h at 4 degrees C, with a decrease of about 1 log of the L. monocytogenes population. CONCLUSIONS: Antimicrobial packaging can play an important role in reducing the risk of pathogen development, as well as extending the shelf life of foods. SIGNIFICANCE AND IMPACT OF THE STUDY: Studies of new food-grade bacteriocins as preservatives and development of suitable systems of bacteriocin treatment of plastic films for food packaging are important issues in applied microbiology and biotechnology, both for implementing and improving effective hurdle technologies for a better preservation of food products.

PCR-based detection of enterotoxigenic Staphylococcus aureus in the early stages of raw milk cheese making.

AIMS: To define PCR-based detectability of Staphylococcus aureus in raw milk and intermediate products of raw milk cheese making in the presence of a complex background microflora by targetting different specific genes harboured by a single strain. METHODS AND RESULTS: The strain Staph. aureus FRI 137 harbouring nuc, sec, seg, seh and sei genes was used in this study. Raw milk artificially contaminated by different concentrations of Staph. aureus FRI 137 was employed in dairy processing resembling traditional raw milk cheese making. Samples of milk and curds were PCR-analysed after DNA extraction by targetting all the above genes. The pathogen was detected when the initial contamination was 10(4) CFU ml(-1) by amplification of nuc and seh genes. 10(5) and 10(7) CFU ml(-1) were needed when seg or sei and sec genes were targetted, respectively. Enrichment cultures from raw milk and curd samples proved to increase the detection limit of 1 log on average. CONCLUSIONS: The direct detection of the pathogen in the raw material and dairy intermediates of production can provide rapid results and highlight the presence of loads of Staph. aureus potentially representing the risk of intoxication. However, every target gene to be used in the analysis has to be studied in advance in a system similar to the real case in order to determine the level of contamination potentially predictable. SIGNIFICANCE AND IMPACT OF THE STUDY: The detection in real dairy systems of significant loads of Staph. aureus by multiple targets PCR can be more accurate.

PCR-DGGE fingerprints of microbial succession during a manufacture of traditional water buffalo mozzarella cheese.

AIMS: To monitor the process and the starter effectiveness recording a series of fingerprints of the microbial diversity occurring at different steps of mozzarella cheese manufacture and to investigate the involvement of the natural starter to the achievement of the final product. METHODS AND RESULTS: Samples of raw milk, natural whey culture (NWC) used as starter, curd after ripening and final product were collected during a mozzarella cheese manufacture. Total microbial DNA was directly extracted from the dairy samples as well as bulk colonies collected from the plates of appropriate culture media generally used for viable counts of mesophilic and thermophilic lactic acid bacteria (LAB) and used in polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) experiments. The analysis of the DGGE profiles showed a strong influence of the microflora of the NWC on the whole process because after the starter addition, the profile of all the dairy samples was identical to the one shown by the NWC. Simple indexes were calculated for the DGGE profiles to have an objective estimation of biodiversity and of technological importance of specific groups of organisms. LAB grown on Man Rogosa Sharp (MRS) and Rogosa agar at 30 degrees C showed high viable counts and the highest diversity in species indicating their importance in the cheese making, which had not been considered so far. Moreover, the NWC profiles were shown to be the most similar to the curd profile suggesting to be effective in manufacture. CONCLUSIONS: The PCR-DGGE analysis showed that in premium quality manufacture the NWC used as starter had a strong influence on the microflora responsible for process development. SIGNIFICANCE AND IMPACT OF THE STUDY: The molecular approach appeared to be valid as a tool to control process development, starter effectiveness and product identity as well as to rank cheese quality.

Selection of Lactobacillus strains from fermented sausages for their potential use as probiotics.

A rapid screening method was used to isolate potentially probiotic Lactobacillus strains from fermented sausages after enrichment in MRS broth at pH 2.5 followed by bile salt stressing (1% bile salts w/v). One hundred and fifty acid- and bile-resistant strains were selected, avoiding preliminary and time-consuming isolation steps. Strains were further characterized for survival at pH 2.5 for 3 h in phosphate-buffered saline and for growth in the presence of 0.3% bile salts with and without pre-exposure at low pH. Twenty-eight strains showed a survival >80% at pH 2.5 for 3 h; moreover, most of the strains were able to grow in the presence of 0.3% bile salts. Low pH and bile resistance was shown to be dependent on both the species, identified by phenotypic and molecular methods, and the strain tested. This is the first report on the direct selection of potentially probiotic lactobacilli from dry fermented sausages. Technologically interesting strains may be used in the future as probiotic starter cultures for novel fermented sausage manufacture.

Relationships between flavoring capabilities, bacterial composition, and geographical origin of natural whey cultures used for traditional water-buffalo mozzarella cheese manufacture.

Natural whey cultures (NWC) (n = 29) used for traditional water-buffalo Mozzarella cheese manufacture and arising from different geographical areas of production were characterized and grouped on the basis of their capability to develop neutral volatile compounds and according to their microbial diversity as revealed by molecular analysis. The flavoring properties of NWC were studied in dairy microcosms resembling the specific technological procedure used in the traditional water-buffalo Mozzarella cheese-making. Neutral volatile compounds were identified by high-resolution gas chromatography (HRGC)-mass spectrometry analysis while information on the microbial diversity occurring in the NWC was retrieved by PCR-denaturing gradient gel electrophoresis (DGGE) analysis of 16S rDNA after direct DNA extraction. Neoformation volatile substances (n = 27) were found; 23 were identified and some of them recognized as odor-conferring molecules. Eight different bands, referable to eight microbial species, were obtained by PCR-DGGE analysis of the NWC. Statistical analyses were applied to PCR-DGGE and HRGC data. Interestingly, the flavoring capabilities and the microbial diversity of the NWC proved to be closely linked and both related to the geographical origin of the NWC. These results suggested a possible use of the molecular characterization of the dairy products to support the traceability criteria of typical dairy products like water-buffalo Mozzarella cheese.

Molecular evaluation of microbial diversity occurring in different types of Mozzarella cheese.

AIMS: The microbial community of different types of unripened Pasta Filata cheese was investigated by culture-independent methods with the aim of rapidly achieving knowledge about cheese microbiota and discriminating traditional and industrial cheeses. METHODS AND RESULTS: The microbial DNA extracted directly from the samples was used as a template in PCR experiments to amplify the 16S-23S rDNA spacer region and the V3 region of the 16S rDNA. Conventional electrophoresis of the amplified spacers allowed known classes of these DNA fragments belonging to genera and species of lactic acid bacteria to be distinguished. Denaturing gradient gel electrophoresis analysis of V3 amplicons was supported by reference cultures of LAB used as markers. CONCLUSION: Both molecular approaches furnished the expected information about microbial diversity and were quite valid for discriminating industrial, semi-artisanal or traditional cheeses, characterized by increasingly complex DNA profiles. SIGNIFICANCE AND IMPACT OF THE STUDY: Both methods could be used for legal purposes when products obtained through prescribed manufacturing regulations are to be analysed.

Behavior of variable V3 region from 16S rDNA of lactic acid bacteria in denaturing gradient gel electrophoresis.

Separation of amplified V3 region from 16S rDNA by denaturing gradient gel electrophoresis (DGGE) was tested as a tool for differentiation of lactic acid bacteria commonly isolated from food. Variable V3 regions of 21 reference strains and 34 wild strains referred to species belonging to the genera Pediococcus, Enterococcus, Lactococcus, Lactobacillus, Leuconostoc, Weissella, and Streptococcus were analyzed. DGGE profiles obtained were species-specific for most of the cultures tested. Moreover, it was possible to group the remaining LAB reference strains according to the migration of their 16S V3 region in the denaturing gel. The results are discussed with reference to their potential in the analysis of LAB communities in food, besides shedding light on taxonomic aspects.

The potential of a polyphasic PCR-dGGE approach in evaluating microbial diversity of natural whey cultures for water-buffalo Mozzarella cheese production: bias of culture-dependent and culture-independent analyses.

A polyphasic PCR-DGGE approach was used to describe the microbial population occurring in natural whey cultures (NWCs) for water-buffalo Mozzarella cheese production. Total microbial community was assessed without cultivation by analyzing DNA directly extracted from the original samples of NWC. In addition, DNA extracted from bulks of cells formed by harvesting colonies from the serial dilution agar plates of a variety of culture media was used to profile the "cultivable" community. The 16S rDNA V3 region was amplified using DNA from NWC as well as DNA from bulks as templates and the amplicons were separated by DGGE. The microbial entities occurring in NWCs were identified by partial 16S rDNA sequencing of DGGE bands: four lactic acid bacteria (LAB) closest relative of Streptococcus thermophilus, Lactococcus lactis, Lactobacillus delbrueckii and Lactobacillus crispatus were revealed by the analysis of DNA directly extracted from NWC while two other LAB, Lactobacillus fermentum and Enterococcus faecalis, were identified by analyzing DNA from the cultivable community. The developed PCR-DGGE analysis of the "cultivable" community showed good potential in evaluating microbial diversity of a dairy environment: it usefully highlighted the bias introduced by selective amplification when compared to the analysis of the total community from NWC and allowed suitability of media and growth conditions to be evaluated. Moreover, it could be used to complete the culture independent study of microbial diversity to give information on concentration ratios among species occurring in a particular environment and can be proposed for rapid identification of dominant microorganisms in alternative to traditional tools.

Conditions for conjugative transposon transfer in Lactococcus lactis.

Three different techniques for bacterial mating were applied to wild type and culture collection strains of Lactococcus lactis harbouring transposons: direct plate conjugation, filter mating and mating on milk agar. Efficiencies and frequencies of transfer were compared. Transconjugants were characterized by marker properties and molecular assays. Transposon-coded Suc+ Nis+ phenotype as well as Suc+ Bac+ Nis- phenotype were transferred with frequencies ranging between 10-9 and 10-6. Milk agar plate mating was the best technique for obtaining gene transfer events involving wild type lactococci.