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Aspergillus is a widespread fungal genus contaminating and degrading food and feeds mostly during storage. Some species can also produce carcinogenic mycotoxins harmful for human and animal health. In this study, Bacillus amyloliquefaciens AG1 (BaAG1), active against fungal pathogens was evaluated by in vitro tests against seven Aspergillus species belonging to six Aspergillus sections. After 7 days of incubation at 25°C, mycelial growth of all fungal species was reduced from 46.53% to 60.92%, compared to the controls. The inhibitory effect of BaAG1 cell-free culture filtrate was tested by turbidimetric measurements by using ScanReady P-800 that allow the automatic and continuous monitoring of the changes in the O.D. of fungal growth over time. Two culture mediums for growth curve optimization were tested, and PDB (Potato Dextrose Broth) was selected. The activity of two concentration of BaAG1 cell-free culture filtrate against three strains (Aspergillus niger SAAF 7, A. flavus SAAF 43 and A. fumigatus SAAF 47) was assessed over 7 d periods with automated measurements every 60 min. Significant differences in AUC (area under growth curve) were recorded between the treatments and the controls. Furthermore, macro and microscopic observation on the fungal colonies showed that both the concentrations of BaAG1 culture filtrates induced morphological changes, such as absence of conidiophores and conidia, presence of cleistothecia and hyphal alterations.

The present research aimed at determining the optimal conditions for the lactic acid fermentation of green Spanish-style table olives. The work is a follow-up, and focuses on the performance of the commercial starter strain Lactobacillus pentosus OM13 by applying an acclimatization step and the addition of nutrients, and concentrations of lactic acid that were previously investigated. The acclimatization of the cells warranted the dominance of the starter culture even at an inoculation level of 2 Log cycles lower than that commonly used in standard fermentation. A significant effect was found in terms of acidification kinetics within the first week of fermentation, with the highest decrease in pH, at ~2.5 units, which occurred in the trial and after inoculation with 106 CFU/mL of starter after acclimatation (EO3) that showed values similar to control C obtained with Lactobacillus pentosus OM13 at a concentration of 107 CFU/mL. After day 3, further decreases in pH of up to 4.30 were observed until day 30, and then these values remained almost constant until the end of process (day 190) when lower pH values were reached for trial EO3 and control C. The results of microbiological dynamics, the changes in VOCs and finally the effect of the processes on the sensory analysis of the fermented product were investigated by multivariate analysis. The acclimatization process and the initial inoculation level influenced the bacterial microflora, aromatic compounds and organoleptic characteristics of the final product. Finally, the trials C, EO2 and EO3 showed higher values (60-80%) of preferences and satisfaction compared to other experimental productions. La presente investigación tuvo como objetivo establecer las condiciones óptimas para la fermentación láctica de aceitunas verdes de mesa estilo español. El trabajo se centra en el seguimiento del rendimiento de la cepa iniciadora comercial Lactobacillus pentosus OM13 aplicando una etapa de aclimatación, adición de nutrientes y concentraciones de ácido láctico que fueron previamente investigadas. La aclimatación de las células garantizó la dominancia del cultivo iniciador, incluso a un nivel de inoculación 2 ciclos Log menor que el utilizado habitualmente en una fermentación estándar. Se encontró un efecto significativo en términos de cinéticas de acidificación en la primera semana de fermentación, donde el mayor descenso de pH de ~2,5 unidades ocurrió en el ensayo inoculado con 106 CFU/mL del iniciador después de la aclimatación (EO3), que mostró valores similares al control C obtenido con Lactobacillus pentosus OM13 a la concentración de 107 CFU/mL. Después del día 3, hasta el 30 se observaron descensos adicionales de pH hasta 4,30, manteniéndose casi constantes hasta el final del proceso (día 190) donde se alcanzaron los menores valores en el ensayo EO3 y el control. Los resultados de dinámicas microbiológicas, cambios en COVs y el efecto de los procesos en el análisis sensorial del producto fermentado se estudiaron mediante análisis multivariante. El proceso de aclimatación y el nivel de inoculación inicial influenciaron la microflora bacteriana, los compuestos aromáticos y las características organolépticas del producto final. Por último, los ensayos C, EO2 y EO3 mostraron un valor mayor (60-80%) de preferencias y satisfacción comparada con otras producciones experimentales.

The aim of this study was to evaluate substance P (SP) levels and the effect of a non-steroidal anti-inflammatory drug (NSAID), ketoprofen, on SP in the pericoronal gingival tissue after extraction of upper third molars. A sample of 20 young non-smoking systemically healthy adults of both sexes, with a healthy upper third molar to extract for orthodontic purposes, was selected. After extraction, a sample of the gingival tissue of the pericoronal region was collected with a sterile scalpel, placed into test tubes and kept frozen at -20°C until the SP determination. SP levels were determined by using a commercially available enzyme immunoassay (ELISA) kit. The subjects were randomly divided into two groups: group 1 received a single dose of ketoprofen 30 minutes prior to the experimental procedure. The subjects of group 2 did not receive any kind of drug administration before extraction. The patients were asked to complete a diary on the postoperative pain. A relevant amount of SP was measured in all the gingival samples. No statistically significant difference could be detected in SP expression between the two groups. In group 1 pain appearance was significantly delayed (6.2±0.13 hours) in comparison with group 2 (3.95±0.2 hours). In this small selected group of subjects and limited study design, preventive administration of ketoprofen did not significantly affect the gingival levels of SP, the clinical recommendation emerging is that of NSAID administration postoperatively but before pain appearance in order to optimize the management of pain of the patient.

Obiettivo di questa ricerca . stato validare le attitudini tecnologiche alla trasformazione di olive da mensa cv. “Nocellara del Belice” da parte del ceppo Lactobacillus pentosus OM13. Lo starter Lb. pentosus OM13 ha dimostrato di dominare l’intero processo fermentativo e ha determinato un sensibile abbassamento del pH, inibendo cos. lo sviluppo di microrganismi potenzialmente patogeni e/o alterativi. All’inizio della sperimentazione e fino al 16¢X giorno di fermentazione, i lieviti hanno mostrato un incremento in entrambe le lavorazioni, con starter e di controllo, ma con un livello di carica microbica maggiore nella tesi di controllo. Nella fase di sperimentazione, successiva al 16¢X giorno, i lieviti hanno raggiunto concentrazioni pressoché costanti. I composti organici volatili hanno presentato una maggiore concentrazione nelle olive del controllo rispetto a quelle sperimentali. In definitiva, l’uso dello starter ha garantito un’adeguata stabilità microbiologica e chimico-fisica e ha potenzialmente incrementato le proprietà salutistiche del prodotto finito.

This chapter presents the results of studies conducted to (i) detect and localize endophytic bacteria in symptomless and symptomatic leaf tissues of grapevine (Vitis vinifera) using fluorescence in situ hybridization (FISH) in combination with confocal laser scanning microscopy (CLSM); and (ii) test the antagonistic activity of Bacillus amyloliquefaciens AG1, previously isolated from grape wood affected by the esca syndrome, against grapevine fungal pathogens: Alternaria alternata, obtained from grapevine leaves showing necrotic concentric spots; Aspergillus carbonarius, A. ochraceus and Penicillium verrucosum, isolated from grapes affected by secondary rot; Botrytis cinerea, isolated from grapes with grey mould; Cladosporium viticola, agent of grapevine leaf spots; Fomitiporia mediterranea, obtained from grapevines with white rot symptoms; Fusarium oxysporum, isolated from the grapevine rhizosphere; Lasiodiplodia theobromae, isolated from vine wood with cortical cankers; Phaeoacremonium aleophilum and Phaeomoniella chlamydospora, both isolated from grapevine showing esca symptoms; Phoma glomerata from grape leaves showing necrotic spots; and Verticillium dahliae, obtained from decaying vine. The FISH/CLSM approach was successfully applied to visualize endophytic bacteria in grapevine leaves, including also those that it is not possible to cultivate in sterilized medium. Also, B. amyloliquefaciens AG1 showed a potential role as a biological control agent against trunk fungal pathogens as well as other fungal phytopathogens.

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.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.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.The potential of the findings is discussed with reference to the development of flavouring adjuncts for the dairy industry.

Enterococcus faecalis 226 NWC, isolated from natural whey cultures utilized as starter in water-buffalo Mozzarella cheese manufacture, produces a bacteriocin, designated Enterocin 226 NWC, which is inhibitory to Listeria monocytogenes. Plasmid analysis of E. faecalis 226 NWC showed a single 5.2-kb plasmid, pEF226. In conjugation experiments, pEF226 was transferred into a plasmid-free strain of E. faecalis JH2-2. The transfer required direct cell-to-cell contact and was not inhibited by DNase. The identity of conjugation was confirmed by digestion with SmaI restriction endonuclease and subsequent pulsed-field gel electrophoresis (PFGE) of the genomic DNA of E. faecalis 226, E. faecalis JH2-2 and of the isolates after the mating. The data indicate that the ability of E. faecalis 226 NWC to produce the bacteriocin is linked to the 5.2-kb conjugative plasmid pEF226.

The area-intensity law of geometrical optics is derived from Maxwell’s equations in general-relativistic refractive media.

The Einstein field equations in the presence of a polytropic fluid performing self‐similar motion are reduced to a dynamical system. Qualitative properties of the dynamical system are investigated in the case when the fluid motion is with shock waves.

New application of self‐similar generalizations of homogeneous cosmological models is shown in the problem of nonstationary nonspherically‐symmetric accretion of self‐gravitating gas on the center. For self‐similar Bianchi types II and III, solutions of the system of Einstein equations are reduced to some dynamical system of small order. A number of exact solutions in empty space and for the stiff equation of state of matter is found.

The present work presents a successful attempt to achieve an enhanced and more predictable fermentation process in Spanish-style green olive technology by selection and use of autochthonous starter cultures. During the first phase of this work, two Spanish-like fermentations of green table olives of cultivar (cv) “Nocellara del Belice”, coming from irrigated and not irrigated fields, were monitored, in order to highlight the best agricultural conditions for drupe production and to isolate lactic acid bacteria strains with relevant technological properties. Among 88 identified isolates, one Lactobacillus pentosus strain showed remarkable biochemical features and high acidification rate in synthetic brine. In the second phase, the selected strain was used as starter culture in three different trials to establish the best conditions for its use. Microbial counting, as well as starter tracking by M13 RAPD-PCR, reflected the optimal adaptation of the strain to the environment. Spontaneous fermentation needed a 14-day long lag phase to reach the same population as the inoculated trials. Moreover, sensory traits of table olives obtained with adjunct culture showed better characteristics compared to those processed in the other trials, in particular concerning the presence of off-odours.

Grapes of the “Grillo” variety, used to produce Marsala wine, were harvested from five vineyards with different climatic and agronomic parameters, in order to obtain a first mapping of lactic acid bacteria (LAB) inhabiting the production area. Marsala base wine production was followed at a large-scale, and also two experimental vinifications, with different lysozyme and SO2 concentrations and in combination, were carried out at pilot-plant scale. LAB communities and conventional chemical parameters were periodically analysed. LAB were found on grapes at an average concentration of about 102 CFU g−1 which decreased during the transformation process. A total of 146 colonies were collected, but only 35 were recognized as presumptive LAB. On the basis of phenotypic differences and isolation source, 16 isolates were then subjected to genotypic identification and assembled into the following species: Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris, Enterococcus faecium, Leuconostoc fallax and Sporolactobacillus nakayamae subsp. nakayamae. Lactococcus lactis subsp. lactis strains were the most frequently isolated during winemaking which showed the highest resistance to SO2 and lysozyme.

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 degrees C. This strain could be selectively counted in the presence of a normal background microflora and it was shown that it could survive for 1 week at 4 degrees C in the raw food studied.

The microbial composition of artisan and industrial animal rennet pastes was studied by using both culture-dependent and -independent approaches. Pyrosequencing targeting the 16S rRNA gene allowed to identify 361 operational taxonomic units (OTUs) to the genus/species level. Among lactic acid bacteria (LAB), Streptococcus thermophilus and some lactobacilli, mainly Lactobacillus crispatus and Lactobacillus reuteri , were the most abundant species, with differences among the samples. Twelve groups of microorganisms were targeted by viable plate counts revealing a dominance of mesophilic cocci. All rennets were able to acidify ultrahigh-temperature-processed (UHT) milk as shown by pH and total titratable acidity (TTA). Presumptive LAB isolated at the highest dilutions of acidified milks were phenotypically characterized, grouped, differentiated at the strain level by randomly amplified polymorphic DNA (RAPD)-PCR analysis, and subjected to 16S rRNA gene sequencing. Only 18 strains were clearly identified at the species level, as Enterococcus casseliflavus , Enterococcus faecium , Enterococcus faecalis , Enterococcus lactis , Lactobacillus delbrueckii , and Streptococcus thermophilus , while the other strains, all belonging to the genus Enterococcus , could not be allotted into any previously described species. The phylogenetic analysis showed that these strains might represent different unknown species. All strains were evaluated for their dairy technological performances. All isolates produced diacetyl, and 10 of them produced a rapid pH drop in milk, but only 3 isolates were also autolytic. This work showed that animal rennet pastes can be sources of LAB, mainly enterococci, that might contribute to the microbial diversity associated with dairy productions.

Aims: The positive influence of two selected extremely halophilic archaea strains in the production of salted anchovies (Engraulis encrasicolus, L., 1758) was highlighted. Methods and Results: Anchovies produced with salt artificially contaminated with halophiles exhibited lower loads of staphylococci, Enterobacteriaceae and lactic acid bacteria, and a reduced content of histamine as well as an improved organoleptic acceptance. Conclusions: The findings of this survey are expected to enhance the safety of salted anchovies, with regard to the histamine formation during ripening, and to improve the sensory attributes of this product. Significance and Impact of the Study: This study represents the first report on the positive influence of halophilic archaea in traditional salted anchovies production, thus suggesting new perspectives about a conscious employment of properly selected haloarchaea strains in this traditional manufacture

Soil solarization (SS) seems one of the most promising techniques to control soilborne pathogens, but an important limitation to the diffusion of this technique is due to the drawback regarding the disposal of the used plastic materials. A possible solution to this problem is the use of biodegradable plastics. The aim of this study was compare the impact of SS with biodegradable materials with SS with plastic film and others pest management techniques (i.e. organic matter amendment, calcium cyanamide and Dazomet fungicide application) on crop productivity, soilborne disease incidence, weed suppression, and soil chemical (total N, ammonium, nitrate, available phosphorus, organic matter, hydrolysis of the fluorescein diacetate) and microbial parameters (cultivable Pseudomonas, DGGE fingerprinting of ribosomal DNA of soil bacteria and fungi). We carried out field experiments on two types of soil with different textures (clay and sandy) artificially inoculated with Fusarium oxysporum f.sp. lycopersici (vs. tomato) and Sclerotinia minor (vs. lettuce). Temperature of soils covered with solarizing materials was always higher than that of bare soils, but plastic cover was more effective and consistent in rising soil temperature compared to biodegradable materials. Plant growth promotion by SS was limited, especially compared to Dazomet and organic matter application, and a positive effect was observed only for lettuce in the clay soil. Differently, both plastic and biodegradable solarizing materials were effective in reducing lettuce drop caused by Sclerotinia minor. Weed development was significantly suppressed by Dazomet application and SS with plastic film, while weed control with biodegradable materials was limited. SS has a variable and limited effect on chemical and microbial parameters, with the exception of a general tendency of reduction of bacterial and fungal richness. Dazomet fungicide has the strongest effect on the soil microbial community, with the most pronounced diversity reduction in both soil types and a significant stimulation of the fluorescent Pseudomonas group. Organic amendment significantly enhanced organic matter content, the hydrolysis of the fluorescein diacetate and the Pseudomonas population. Among all measured soil parameters, fluorescent Pseudomonas population emerged as the most important parameter in relation to crop productivity. In conclusion, the results of this experimentation show the potential of using the biodegradable solarizing materials in place of plastic films, but also, indicate the need for improving their properties to obtain performances comparable to those of other pest management techniques.

A total of 98 rhizobial strains, isolated during the winter of the years 2003 (35 isolates), 2004 (33 isolates), and 2005 (30 isolates) were analyzed to determine the genetic diversity of the natural population nodulating Vicia faba plants and to identify dominant genotypes. All isolates were identified as Rhizobium leguminosarum bv. viciae by biovar-specific polymerase chain reaction amplification of the nodC gene. Intraspecific DNA polymorphism was evaluated through the restriction endonucleases analysis combined with pulsed-field gel electrophoresis. Four genotypes characterized 53% of the isolates, showing a high occurrence; moreover, they were recovered over the 3 years, thus showing a lasting persistence in the soil, which could mean a high degree of saprophytic competitiveness. The richness, diversity, and dominance indexes of genotypes were calculated to monitor the evolution of the rhizobial population during the 3 years. The genetic diversity of the analyzed strains decreased along the 3 years. In fact, the biodiversity index H' decreased from 2.6 in the first and second year to 1.9 in the third year; probably, as a result of bean monocropping, specific genotypes of Rh. leguminosarum bv. viciae were naturally selected.

Microbial diversity of the raw milk for the production of Fior di Latte di Agerola and its changes during cheesemaking were studied. Viable counts showed that at the end of curd ripening, loads of lactic acid bacteria, both mesophilic and thermophilic rods and cocci, higher than those commonly evidenced in similar cheeses produced by using natural or commercial starters, were detected. Identification of 272 isolates, supported by molecular diagnostic aids, evidenced representative cultures of a high number of bacterial taxa of interest as participating in the process, although most of the isolates belonged to Lactococcus lactis and Lactobacillus helveticus species. RAPD-PCR and REA-PFGE biotyping were performed for the isolates of the above species and it was shown that most of the strains isolated from the raw milk occurred during the whole cheesemaking process, and an active role of these strains in the fermentation was supposed. The results offer further proof of the importance of the raw milk as source of technologically interesting strains of lactic acid bacteria capable of driving the fermentation of traditional cheeses.

Twenty-seven new Rhizobium isolates were obtained from root nodules of wild and crop legumes belonging to the genera Vicia, Lathyrus and Pisum from different agroecological areas in central and southern Italy. A polyphasic approach including phenotypic and genotypic techniques was used to study their diversity and their relationships with other biovars and species of rhizobia. Analysis of symbiotic properties and stress tolerance tests revealed that wild isolates, showed a wide spectrum of nodulation and a marked variation in stress tolerance compared with reference strains tested in this study. All rhizobial isolates (except for the isolate CG4 from Galega officinalis) were presumptively identified as Rhizobium leguminosarum biovar viciae both by their symbiotic properties and the specific amplification of the nodC gene. In particular, we found that the nodC gene could be used as a diagnostic molecular marker for strains belonging to the bv. viciae. RFLP-PCR 16S rDNA analysis confirms these results, with the exception of two strains that showed different RFLP-genotypes from those of the reference strains of R. leguminosarum bv. viciae. Analysis of intraspecies relationship among strains by using the RAPD-PCR technique showed a high level of genetic polymorphism, grouping our isolates and reference strains into six different major clusters with a similarity level of 20%. Data from seven parameters of phenotypic and genotypic analyses were evaluated by using principal component analysis which indicated the differences among strains and allowed them to be divided into seven different groups. (c) 2005 Elsevier GmbH. All rights reserved.

D . E R C O L I N I , G . M A U R I E L L O , G . B L A I O T T A , G . M O S C H E T T I A N D S . C O P P O L A . 2003. 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� 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.

Separation of amplified V3 region from 16S rDNA by denaturing gradient gel electrophoresis (PCR-DGGE) and 16S-23S rDNA intergenic spacer region polymorphism (ISR-PCR) analyses were tested as tool for differentiation of staphylococcal strains commonly isolated from fermented sausages. Variable V3 regions of 25 staphylococcal reference strains and 96 wild strains of species belonging to the genera Staphylococcus, Micrococcus and Kocuria were analyzed. PCR-DGGE profiles obtained were species-specific for S. sciuri, S. haemolyticus, S. hominis, S. auricularis, S. condimenti, S. kloosi, S. vitulus, S. succinus, S. pasteuri, S. capitis and S. (Macrococcus) caseolyticus. Moreover, 7 groups could be distinguished gathering the remaining species as result of the separation of the V3 rDNA amplicons in DGGE. Furthermore, the combination of the results obtained by PCR-DGGE and ISR-PCR analyses allowed a clear differentiation of all the staphylococcal species analysed, with exception of the pairs S. equorum-S. cohnii and S. carnosus-S. schleiferi. The suitability of both molecular techniques and of the combination their results for the identification of staphylococci was validated analysing partial nucleotide sequence of the 16S rDNA of a representative number of wild strains.

Molecular typing of several cheeses was achieved by PCR-DGGE analysis of microbial DNA directly extracted from the dairy samples. Cheese-specific fingerprints were obtained analyzing the variable V3 region of 16S rDNA, the profiles showed to be repeatable and reliable after repeated analysis of the same sample. Statistical analysis of molecular data showed relevant differences among cheeses highlighting the potential of the PCR-DGGE fingerprinting for the discrimination of dairy products. The variability of the fingerprints obtained by different samples of the same type of cheese definitely compromises the use of the technique to develop class-specific profiles to be used for cheeses classification, allowing just to detect the degree of complexity of the microbiota.

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. Conclusions: 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.

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.

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.