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.

Composition and seasonal variation of soluble cuticular waxes in Actinidia deliciosa leaves.

This research examines the annual evolution and composition of soluble cuticular waxes of Actinidia deliciosa Chev. cv Hayward leaves. Soluble cuticular waxes of foliar blade were extracted in chloroform and analysed by GC-MS. The seasonal weighted mean of the wax coverage was about 24 microg cm(-2). The alkyl alkanoates were the main class of components (10 microg cm(-2)) followed by hydrocarbons (6 microg cm(-2)), terpenes (3 microg cm(-2)), alkanols (1 microg cm(-2)), ketones (1 microg cm(-2)), alkanoic acids (1 microg cm(-2)), alkanals (0.7 microg cm(-2)), and sterols (0.6 microg cm(-2)). The concentration of the soluble cuticular components reached a peak (43 microg cm(-2)) on the 83rd day after bud break. Different causes were proposed to explicate the seasonal evolution of the leaf waxes: biosynthesis of the waxes prevalently during rapid leaf growth; natural wax erosion and evaporation; progressive reduction in the extractability of the intracuticular free compounds due to the slow polymerization of the cutin matrix.

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.

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-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.

Isolation and technological properties of coagulase negative staphylococci from fermented sausages of Southern Italy.

The aims of this study were to characterize the population of Micrococcaceae in different types of fermented sausages of Southern Italy and to determine the technological properties of Staphylococcus strains in order to evaluate the suitability of selected strains as starter cultures in the processing of dry fermented pork sausages. Ninety-six strains were studied to evaluate nitrate reductase, proteolytic, lipolytic and antioxidant activities as well as growth ability at different temperatures, pH's and NaCl concentrations. All the strains were classified as Staphylococcus except for one isolate assigned to Kocuria spp. The species most often isolated were S. saprophyticus, S. xylosus and S. equorum, although they were not equally distributed within the different sausages. Other species isolated were, in descending order of abundance, S. succinus, S. warneri, S. lentus, S. vitulus, S. pasteuri, S. epidermidis, and S. haemolyticus. In general, the S. xylosus strains exhibited the best technological properties that would make them eligible as good starter cultures for fermented meat products. However, strains belonging to other species also showed good technological properties. Finally, all strains grew at 10, 15 and 20 °C, in the presence of 10% and 15% of NaCl and at pH 5.0 and 5.5. The results showed that it is possible to formulate a broad variety of staphylococcal starter cultures, adaptable to different technological conditions and sausage manufacture practices.

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.

Proteolytic activity of Staphylococcus xylosus strains on pork myofibrillar and sarcoplasmic proteins and use of selected strains in the production of "Naples type" salami.

AIMS: The aim of this study was to determine the proteolytic activities of Staphylococcus xylosus strains on sarcoplasmic and myofibrillar proteins in order to evaluate the suitability of selected strains as starter cultures in the processing of a dry fermented pork sausage. METHODS AND RESULTS: The proteolytic activity of 27 strains of Staphylococcus xylosus on sarcoplasmic and myofibrillar proteins was determined by agar plate method, o-phtaldialdehyde (OPA) spectrophotometric assay and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Four strains were selected for the formulation of six starter cultures to use in the production of "Naples type" salami. The proteolytic contribution of starters was determined by SDS-PAGE, comparing the protein profile of inoculated sausages with that of uninoculated sausages after 0, 15 and 33 days of ripening. The results showed that the proteolytic activity of some strains, determined by the agar plate method, were not confirmed by electrophoretic and spectrophotometric assays. In fact, of 24 strains of Staphylococcus xylosus able to hydrolyse muscle protein extracts on agar plate, only 12 strains were shown to change SDS-PAGE profile of pork proteins. The SDS-PAGE profile of sarcoplasmic proteins extracted from all sausages showed that the major changes were produced with starters S3, S4 and S5 after 15 days of ripening. Also myofibrillar proteins undergo major changes after 15 days of ripening and the protein profiles showed the same pattern in all samples, except for the sausages produced with starter S4. CONCLUSIONS: The results of this work showed that the muscle protein extracts hydrolysis test is suitable for preliminary screening of Staphylococcus xylosus strains on the basis of their proteolytic activity. However, evaluation of muscle protein hydrolysis in a food model system could then be more appropriate for selecting micro-organisms for use as starter cultures for fermented sausages. SIGNIFICANCE AND IMPACT OF THE STUDY: The potential of the findings is discussed with reference to the formulation of starter cultures for the dry fermented sausages production.

Characterization of lactic acid bacteria strains on the basis of neutral volatile compounds produced in whey.

AIMS: Seventy-eight strains of lactic acid bacteria belonging to five genera and showing six different phenotype combinations of Lac (lactose fermentation), Prt (proteolytic activity) and Cit (citrate degradation) characters were investigated for their main flavouring properties with the aim to detect variability among and within the groups. METHODS AND RESULTS: High resolution gas chromatography-mass spectrometry analysis of neutral volatile compounds produced in whey showed that, considering both neo-formation compounds and substances quantified in the whey cultures at different concentrations in comparison to the extract from sterile whey, the groups of lactococci, enterococci, thermophilic streptococci and mesophilic lactobacilli produced a higher number of volatiles than thermophilic lactobacilli and leuconostocs. Applying principal component analysis (PCA) to the results, enterococci, mesophilic lactobacilli and thermophilic streptococci showed a broad diversity, while lactococci included rather similar strains as well as strains with special flavouring properties. Applying PCA to thermophilic streptococci and enterococci, to lactococci and enterococci, to lactococci and thermophilic streptococci, or to mesophilic and thermophilic lactobacilli, the strains gathered consistently with their systematic position. CONCLUSION: The study evidenced strains producing some volatile compounds responsible for food flavouring. Flavouring properties were variable among the systematic groups and in some cases different within the same bacterial group. SIGNIFICANCE AND IMPACT OF THE STUDY: The potential of the findings is discussed with reference to the development of flavouring adjuncts for the dairy industry.

Detection and characterization of a bacteriocin, garviecin L1-5, produced by Lactococcus garvieae isolated from raw cow's milk.

AIMS: The identification of a bacteriocin-producing lactococcal strain isolated from raw cow's milk is reported, along with production conditions, physical and chemical properties, and mode of action of the bacteriocin. METHODS AND RESULTS: On the basis of resistance to clindamycin, species-specific PCR and amplification of the 16S-23S rDNA spacer region, the strain was identified as Lactococcus garvieae. Its bacteriocin, designated garviecin L1-5, was bactericidal against closely related species and strains of species from different genera, including Listeria monocytogenes and Clostridium spp. Garviecin L1-5 was shown to be proteinaceous by protease inactivation and was unaffected by heat treatments, also at low pH values. When amplifying known lactococcal bacteriocin genes using DNA from strain L1-5 as template, no amplification products were observed on the agarose gel. The molecular weight of garviecin L1-5 was about 2.5 kDa. As far as is known, no bacteriocins have been detected from Lactococcus garvieae. CONCLUSION: The general properties of garviecin L1-5 are characteristic of the low-molecular-weight bactericidal peptide group. SIGNIFICANCE AND IMPACT OF THE STUDY: The survey of micro-organisms for novel antimicrobial substances provided valuable information on their physiology, ecology and practical application.

Antibiotic resistance of coagulase-negative staphylococci isolated from artisanal Naples-type salami.

In the present paper 42 isolates from Italian salami were specified as Staphylococcus xylosus (30), Staph. capitis (1), Staph. saprophyticus (1), Staph. hominis (1), Staph. simulans (1), Staph. cohnii (1) and as Staph. spp. (7). These strains were coagulase-negative and were examined for resistance/sensitivity against 25 antibiotics including beta-lactams (7), macrolides (3), amynoglicosides (5), glycopeptides, lincosamides (4) and novobiocin, fusidic acid, chloramphenicol, rifampicin, tetracycline, minocycline. More than 64% of the strains were resistant to lincomycin, penicillin G, amoxicillin, fusidic acid and novobiocin. All the strains were multiresistant and displayed at least three resistances. Over 75% had a multiple antibiotic resistance (MAR) index between 0.2 and 0.5.

Microbial succession during ripening of Naples-type salami, a southern Italian fermented sausage.

Studies were carried out on the microbiological and physico-chemical changes which occurred during the ripening of five batches of Naples-type salami, manufactured without starter cultures. Salami were sampled internally and externally, and the following microbial groups were studied: lactic acid bacteria, Micrococcaceae and yeasts. The results obtained indicated that lactobacilli constituted the predominant flora, both on the surface and in the interior of the pieces throughout the ripening period. Micrococcaceae and yeasts were also found in considerable number in both locations. Characterisation of 191 lactic isolates indicated that the salami microflora was dominated by homofermentative lactobacilli; approximately 63% of them could be identified as Lactobacillus sake; 40% showing the traits of a racemase negative variant of this species, once referred to Lactobacillus bavaricus. Yeast population mainly comprised Debaryomyces strains. All the colonies grown on mannitol salt and Kranep agar were catalase-positive cocci; novobiocin-resistant staphylococci were the only Micrococcaceae found. The API Staph identification system did not prove to be reliable: 82% of the isolates remained unidentified. To achieve improved characterisation, cluster analysis was subsequently performed on this group, corroborating the existence of a fairly homogeneous group representing an intermediate variety between Staphylococcus xylosus and Staphylococcus saprophyticus that was isolated during the whole ripening process.

Singlet oxygen mediated degradation of Klason lignin.

After some results concerning photochemical generated singlet oxygen on lignins from steam explosion, the reactions of chemically generated singlet oxygen with Klason lignins from pine and beech are described. Singlet oxygen was produced through the reaction of hydrogen peroxide with sodium hypochlorite. The degradation of lignin was followed by uv spectroscopy and gel permeation chromatography. Extensive degradation of the lignins was observed when 20 mg of Klason lignin was treated with 1 ml of 30% hydrogen peroxide and 8.56 ml of 1.093 M sodium hypochlorite. In the uv spectra registered after the treatment with singlet oxygen the absorptions typical of lignin (210-220 nm and 250-280 nm) were completely absent. The gpc analysis of lignin after a treatment with 0.1 ml of hydrogen peroxide and 0.86 ml of sodium hypochlorite showed a clear reduction of signals due to the lignin and a shift to lower molecular weight. The potential use of this procedure in the bleaching procedure was tested by using recycled paper. A maximum reduction of 51% in the amount of lignin in this paper was observed.

Spray-drying of bacteriocin-producing lactic acid bacteria.

Cell survival, cellular damage, and antagonistic activity were investigated after spray-drying of four bacteriocin-producing strains of lactic acid bacteria: Lactococcus lactis subsp. lactis 140, isolated from natural whey culture and producing a narrow-inhibitory spectrum bacteriocin); L. lactis subsp. lactis G35, isolated from pizza dough and producing nisin; Lactobacillus curvatus 32Y and Lactobacillus sp. 8Z, isolated from dry sausages. Trials were performed with bacteria suspended in skimmed milk or directly grown in whey. Three air temperatures at the inlet of the drier (160, 180, and 200 degrees C) and three flow rates (10, 13, and 17 ml/min) were assayed. Cell viability and bacteriocin activity of the dried materials were determined immediately after the process and after 5, 15, 30, and 60 days of storage at 4 degrees C. There was no significant difference between the two feeding suspensions in cell survival, always decreasing with the increase of inlet-air temperature. No loss of bacteriocin activity was detected in reconstituted powders, nor was any loss of ability to produce bacteriocin found after drying. Investigations of sensitivity to NaCl revealed only temporary damage to dried bacteria. During storage for 2 months at 4 degrees C, all samples, but mainly the lactococcal strains, displayed a gradual decrease in cell survival. Bacteriocin activity remained at the same level, allowing powders to be considered as effective biopreservatives.

Effect of leavening microflora on pizza dough properties.

Fourteen different starter cultures containing one strain of Saccharomyces cerevisiae with and without individual or combinations of lactic acid bacteria (Lactobacillus plantarum, Lact. sanfrancisco, Enterococcus faecium, Leuconostoc mesenteroides) were employed to investigate the role of leavening microflora on the properties of pizza doughs. Microbiological, chemical and physical characteristics of doughs prepared with the same flour and under the same processing conditions were determined. Leavening times and acidification properties depended on the microbial association used. The proportions of lactic and acetic acid produced by lactic acid bacteria were consistent with the metabolic properties of the strains employed. The bacteria/yeast ratios arising from microbial counts at the end of the leavening process were always lower in comparison to sour- or bread-doughs. The size of the yeast population did not change much, while bacteria showed from one to four duplications. Rheologically, the fermented doughs could only be significantly distinguished from the control dough with regard to the elastic modulus. Principal Component Analysis was applied to the acidimetric data. The scattergram of the two principal components effectively discriminated 13 of the 14 pizza dough types.

Genotyping of Lactobacillus delbrueckii subsp. bulgaricus and determination of the number and forms of rrn operons in L. delbrueckii and its subspecies.

Three different approaches (whole-cell protein profiles, DNA fingerprinting combined with pulsed-field gel electrophoresis and analysis of rDNA genes) were used to characterize thirty-one strains of Lactobacillus delbrueckii subsp. bulgaricus from different dairy products, and three type strains belonging to L. delbrueckii subsp. delbrueckii, L. delbrueckii subsp. lactis and L. delbrueckii subsp. bulgaricus. Moreover, the number and different forms of rrn operons in L. delbrueckii and its subspecies were defined. At the strain typing level, Notl macrorestriction analysis permitted grouping of the 32 strains of L. delbrueckii subsp. bulgaricus into 23 restriction patterns, providing a high degree of discriminatory power. Among whole-cell protein profiles, PCR analysis of rDNA genes and ribotyping, the latter method seemed to be the most reliable approach to characterizing the subspecies belonging to L. delbrueckii. Ribotyping combined with enzymes such as HindIII and EcoRI showed that at least six rrn operons were present in L. delbrueckii and its subspecies; two forms of rrn operons were present in the subspecies lactis and bulgaricus and four forms were present in the subspecies delbrueckii.