S. Coppola

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.

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.

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.

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.

Random amplified polymorphic DNA and amplified ribosomal DNA spacer polymorphism : powerful methods to differentiate Streptococcus thermophilus strains

Streptococci from different collections and dairy materials were characterized by conventional and molecular methods. After amplification of the 16S–23S rDNA spacer region, all the strains referable to the genus Streptococcus exhibited a single polymerase chain reaction (PCR) product, allowing their differentiation from enterococci. Cleaving this PCR product with Hae III, two different restriction patterns could be observed, allowing Streptococcus salivarius DSM 20560T, Strep. thermophilus NCDO 822 and two strains of Streptococcus spp. to be gathered in one group and all the other strains in another. In order to achieve strain typing, all the cultures were investigated by random amplified polymorphic DNA (RAPD)‐PCR analysis employing two selected primers. The results were treated by cluster analysis, appearing significantly consistent with both the taxonomic position and the origin of the strains. Pulsed‐field gel electrophoresis (PFGE) of Sma I digests of the genomic DNA from 11 representative strains with decreasing levels of RAPD similarity allowed their diversity to be confirmed, even though RAPD‐PCR proved to be less discriminating than PFGE analysis. The results are discussed with reference to the capability of the analytical procedures used to aid both identification and strain typing of streptococci, as well as the taxonomic structure of the species Strep. thermophilus.

Microbial diversity in Natural Whey Cultures used for the production of Caciocavallo Silano PDO cheese

The microbial diversity of sixty-three Natural Whey Cultures (NWCs) for the manufacture of Caciocavallo Silano cheese PDO was studied. The NWCs were collected from different dairies covering the whole territory of PDO production including five different regions of southern Italy. The microbial species diversity was determined by direct DNA extraction from NWCs and Polymerase Chain Reaction (PCR) amplification of variable regions of the 16S rRNA gene followed by denaturing gradient gel electrophoresis (DGGE) and denaturing high performance liquid chromatography (DHPLC). DGGE and DHPLC fingerprinting yielded the same results in terms of number of bands/peaks and specific migration/retention time of the amplicons. The DHPLC technique was used in this study for the first time to assess a food-related mixed microbial community by a culture-independent approach and proved to be at least as effective as DGGE in profiling species diversity in NWCs. Cluster analysis of DGGE and DHPLC data revealed that the species-related groups of similarity were not dependent on the geographical origin of the NWCs. The presence of three groups of 10-14 NWCs at 100% of species similarity indicated that some species associations are very commonly occurring in the NWCs for Caciocavallo Silano cheese PDO. A RAPD-PCR analysis of the NWCs was also performed for the members of the above groups and it showed that, though characterized by the same species diversity, most of the identical NWCs included different biotypes. The sequences of DGGE bands and DHPLC peaks revealed the occurrence of mainly thermophilic lactic acid bacteria such as Lactobacillus delbrueckii, L. helveticus and Streptococcus thermophilus even though the mesophilic Lactococcus lactis also occurred in some NWCs. In conclusion, the results of this study indicate that the microbial diversity of NWCs used for the Caciocavallo Silano PDO cheese is not high, it is not dependent on the geographical origin and the same microbial species occur within the territory examined. The microbiota of fermented PDO products and its possible link with territory should be studied case by case in order to have useful evidences for the assessment of product quality and authenticity.

Characterization of strains of Leuconostoc mesenteroides by analysis of soluble whole-cell protein pattern, DNA fingerprinting and restriction of ribosomal DNA

Of 215 leuconostocs isolated from field grass, natural whey cultures and water‐buffalo milk, 178 were identified as Leuconostoc mesenteroides ssp. mesenteroides while 37 strains could not be identified Biochemical characterization allowed seven groups to be defined. Representative strains of each group and different habitat and nine reference strains were selected for further analyses. Protein profiles appeared suitable for species discrimination, but did not differentiate between the three subspecies of Leuc. mesenteroides. The technique also showed some differences among equivocal strains. DNA fingerprinting for most strains of Leuc. mesenteroides ssp. mesenteroides examined showed a different restriction pattern from that of the type strain. Ribotyping was not useful for discriminating species and subspecies of the genus Leuconostoc: Leuc. mesenteroides ssp. mesenteroides and ssp. dextranicum showed the same ribopattern as Leuc. lactis while Leuc. mesenteroides ssp. cremoris exhibited a pattern distinct from all the other species examined. On the basis of ARDRA‐PCR, two main groups could be distinguished: the larger group included Leuc. mesenteroides, Leuc. lactis, Leuc. pseudomesenteroides and some unidentifiable strains; the second one included Leuc. citreum, Leuc. fallax, Weissella paramesenteroides and some unidentified strains.

Antilisterial activity of thermophilin 347, a bacteriocin produced by Streptococcus thermophilus

Streptococcus thermophilus 347 isolated from yogurt produces a bacteriocin referred as thermophilin 347. The bacteriocin was evidenced in the neutralized, filtered and catalase treated culture supernatant fluid of the producer strain. After partial purification, thermophilin 347 exhibited a bactericidal effect against Listeria monocytogenes and several closely related lactic acid bacteria. The activity of thermophilin 347 was lost after protease treatment but was maintained after heating at 100 degrees C for 1 h; after autoclaving at 121 degrees C for 15 min the activity was reduced by 50%. SDS-PAGE of partially purified thermophilin 347 was used to detect bacteriocin activity corresponding to an apparent molecular mass between 2.5 and 6.2 KDa.

Phenotypic and genotypic characterization of Oenococcus oeni strains isolated from Italian wines

A phenotypic and genotypic characterization of 84 Oenococcus oeni isolates from Italian wines of different oenological areas was carried out. Numerical analysis of fatty acid profiles grouped the isolates into two clusters at low level of similarity (63%), the minor cluster containing seven isolates besides the type and the reference strains. Forthy-eight O. oeni isolates, representative of the two clusters, showed no differences in their metabolic properties (heterolactic fermentation pattern, citrate degradation capability and formation of some secondary metabolites). Moreover, the analysis of species-specific randomly amplified polymorphic DNA and 16S-23S rDNA intergenic spacer region polymorphism as well as the sequence-specific separation of V3 region from 16S rDNA by denaturing gradient gel electrophoresis demonstrated a substantial homogeneity among the isolates. On the basis of ApaI Pulsed Field Gel Electrophoresis (PFGE) restriction patterns, the 84 isolates were grouped into five different clusters at 70% similarity, but no correlation with the phenotypic groups could be demonstrated. However, by combining phenotypic and genotypic data, the 84 O. oeni isolates grouped into eight phenotypic-genotypic combined profiles and a relationship between the origin of the isolates and their combined profile became evident, so that a sort of strain specificity can be envisaged for each wine-producing area.

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.