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The environment of hard cheese encourages bacterial synergies and competitions along the ripening process, which might lead in defects such as clostridial blowing. In this study, Denaturing Gradient Gel Electrophoresis (DGGE), a quantitative Clostridium tyrobutyricum PCR and next-generation Illumina-based sequencing of 16S rRNA gene were applied to study 83 Grana Padano spoiled samples. The aim was to investigate the community of clostridia involved in spoilage, the ecological relationships with the other members of the cheese microbiota, and the effect of lysozyme. Three main genera were dominant in the analysed cheeses, Lactobacillus, Streptococcus and Clostridium, and the assignment at the species level was of 94.3% of 4,477,326 high quality sequences. C. tyrobutyricum and C. butyricum were the most prevalent clostridia. Hierarchical clustering based on the abundance of bacterial genera, revealed three main clusters: one characterized by the highest proportion of Clostridium, a second where Lactobacillus was predominant and the last, dominated by Streptococcus thermophilus. Ecological relationships among species were found: cheeses characterized by an high abundance of S. thermophilus and L. rhamnosus were spoiled by C. tyrobutyricum while, when L. delbrueckii was the most abundant Lactobacillus, C. butyricum was the dominant spoiling species. Lysozyme also shaped the bacterial community, reducing C. tyrobutyricum in favour of C. butyricum. Moreover, this preservative increased the proportion of L. delbrueckii and obligate heterofermentative lactobacilli and lowered L. helveticus and non-starter species, such as L. rhamnosus and L. casei., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Coagulase-negative staphylococci (CoNS) belong to saprophytic microbiota on the skin and mucous membranes of warm-blooded animals and humans, but are also isolated from foodstuffs such as meat, cheese, and milk. In other circumstances, some CoNS can act as pathogens. Thus the presence of CoNS may not be an immediate danger to public health, but can become a risk factor. In particular antibiotic-resistant genes could be transferred to other potentially pathogenic microorganisms. Furthermore, CoNS are known to be strong biofilm producers and this is also a risk factor for public health. The aim of the present work was to determine the genotypic and phenotypic profiles of 106 CoNS belonging to four different species isolated from five different Italian cheeses for the presence of some adhesion and virulence features. In order to verify a possible correlation between the formation of biofilm and staphylococcal virulence factors, we checked the presence of adhesin genes by PCR and we investigated the ability of these strains to make biofilm at different temperatures. Furthermore, in some conditions, we analyzed surface proteins and autolytic pattern of selected strains. In conclusion, we checked the presence of norA and mecA genes responsible for fluoroquinolones and methicillin resistance, respectively. We found resistant genes in a proportion of the food isolates in amounts of 9.4% (mecA) and 5.7% (norA). These data support the importance to continuously examine the microbiota not only for the creation of a database but also to safeguard public health., (© The Author(s) 2015.)

Six strains of non-starter lactic acid bacteria (NSLAB) were used to extend the shelf-life of the fresh cheese Tosèla manufactured with pasteurised cows' milk. The acidification kinetics of three Lactobacillus paracasei, one Lactobacillus rhamnosus and two Streptococcus macedonicus were studied in synthetic milk medium. Lb. paracasei NdP78 and NdP88 and S. macedonicus NdP1 and PB14-1 showed an interesting acidifying capacity and were further characterised for growth in UHT milk and production of antimicrobial compounds. Lb. paracasei NdP78 and S. macedonicus NdP1 grew more than 2 log cycles in 6 h. Lb. paracasei NdP78 was also found to produce a bacteriocin-like inhibitory substance (BLIS) active against Listeria monocytogenes. The four NSLAB strains (singly or in combination) were used to produce experimental pilot-scale cheeses which were compared by a panel. The cheese manufactured with the mixed culture Lb. paracasei NdP78 - S. macedonicus NdP1 was the most appreciated for its sensory properties. The cheeses produced at factory-scale showed higher concentrations of lactobacilli (7.90 log CFU/g) and streptococci (6.10 log CFU/g), but a lower development of coliforms (3.10 log CFU/g) and staphylococci (2.78 log CFU/g) than control cheese (4.86, 4.89, 4.93 and 5.00 log CFU/g of lactobacilli, streptococci, coliforms and staphylococci, respectively) processed without NSLAB addition. The food pathogens Salmonella spp. and Listeria monocytogenes were never detected. The dominance of the species inoculated was demonstrated by denaturing gradient gel electrophoresis (DGGE), whereas strain recognition was evaluated by randomly amplified polymorphic DNA (RAPD)-PCR. From the results obtained, Lb. paracasei NdP78 and S. macedonicus NdP1 were able to persist during the storage of Tosèla cheese and their combination influenced positively the sensory characteristics and shelf-life of the final product., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The composition of the bacterial consortia of the smear Italian cheeses and their role on quality and safety is still poorly understood. The objective of this study was to identify and characterize the bacterial communities present on the surface of five traditional Italian cheeses, Casera Valtellina, Scimudin, Formaggio di Fossa, Gorgonzola and Taleggio. DGGE analysis performed using total DNA obtained from cheese surfaces enabled us to identify the dominant bacterial populations. Bands showing different intensity and identified as Staphylococcus, Micrococcus, Psychrobacter, Enterococcus and Brevibacterium species were detected on the surface of cheeses. The cluster analysis showed that Gorgonzola, Taleggio and Formaggio di Fossa cheeses present high similarity in their surface bacterial composition while major differences in the DGGE profiles were observed in Scimudin and Casera. The molecular taxonomical identification among the Gram positive isolates, reveals the presence of the following bacterial genera: Staphylococcus, Micrococcus, Macrococcus, Enterococcus, Lactobacillus, Carnobacterium, Leuconostoc, Brevibacterium, Corynebacterium, Brochothrix, Bacillus. The combination of culture dependent and independent techniques allowed us to obtain information about the bacterial species covering the surface of five different traditional Italian cheeses., ((c) 2010 Elsevier B.V. All rights reserved.)

Nostrano di Primiero is a 6-month ripened cheese produced from raw milk collected in the Paneveggio-Pale di San Martino Natural Park area in the Italian Dolomites. In summer, this cheese is made using milk collected from two different areas, Passo Rolle and Vanoi, in the Paneveggio Natural Park. During the experiment, the milk from the two areas was separately processed, and cheeses were made in the same cheese factory using the same technological process. The microbiota of raw milk and cheeses of the two areas was isolated and the dominant population was monitored by RAPD analysis and identified by 16S rRNA sequence. The milk of the Passo Rolle area was mainly composed of mesophilic strains, thermophilic Streptococcus thermophilus, and low amounts of enterococci were also found; the milk of the Vanoi area was dominated by mesophilic microbiota mostly Lactococcus lactis ssp. cremoris and ssp. lactis and Lactobacillus paracasei ssp. paracasei. The plating of the natural starter culture revealed the presence of a relevant community of thermophilic cocci and lower amounts of enterococci. The dynamic population analysis showed the importance of the natural starter culture in the first 2 days of cheese ripening in both cheeses. Moreover, the large biodiversity observed in the raw milks was also detected in the cheeses during ripening. The Vanoi cheese was dominated by Enterococcus faecium and Streptococcus macedonicus in the first two days and mesophilic 21 Lb. paracasei ssp. paracasei became the most represented population after 15 days of ripening. In the first few days, the Rolle cheese was characterized by being mainly composed of thermophilic S. macedonicus and S. thermophilus and secondarily by mesophilic cocci. During ripening, the microbiota composition changed, and at 15 days, mesophilic lactobacilli were the dominant population, but later, this was mainly composed of mesophilic cocci and lactobacilli. The taxonomical identification by 16S rRNA sequence confirmed a large biodiversity related to raw milk microbiota and only five strains of S. macedonicus, Lactobacillus plantarum, 21 Lb. paracasei ssp. paracasei, Lactobacillus fermentum and E. faecium were detected in both cheeses.

This study assessed the frequency of transfer of two mobile genetic elements coding for virulence determinants and antibiotic resistance factors, into food associated enterococci during fermentation processes. First, the transfer of the pheromone-inducible pCF10 plasmid, carrying tetracycline resistance and aggregation substance (AS) as virulence factor, between clinical and food strains of Enterococcus faecalis, was investigated in models of cheese and fermented sausage. The experiments demonstrated that even in the absence of selective tetracycline pressure, plasmid pCF10 was transferred from E. faecalis OG1rf cells to food strain E. faecalis BF3098c and that the plasmid subsequently persisted in these environments. Very high frequency of transfer was observed in sausage (10(-3)/recipient) if compared to cheese (10(-6)) and plate mating (10(-4)). Transconjugants were subsequently verified by PCR. The second transmissible element was the plasmid harbouring the vancomycin resistance (VanA phenotype) from E. faecalis A256. The transfer of this antibiotic resistance to a food strain of E. faecalis was studied in vitro and in food models. Although the transfer of vancomycin resistance was achieved in all the environments, the highest conjugation frequencies were observed during the ripening of fermented sausages, reaching 10(-3) transconjugants/recipient cell. PCR confirmed the transfer of the VanA genotype into a food associated Enterococcus strain. This study showed that even in the absence of selective pressure, mobile genetic elements carrying antibiotic resistance and virulence determinants can be transferred at high frequency to food associated enterococci during cheese and sausage fermentation.

The growth dynamics of the natural microbial community responsible for the fermentation of Scamorza Altamurana, a typical Southern Italian cheese made using backslopping, was investigated applying a polyphasic approach combining 1) microbial enumeration with culture media, 2) randomly amplified polymorphic DNA (RAPD) fingerprinting of microbial communities, 3) sequencing of partial 16S ribosomal DNA (rDNA) genes, and 4) physiological tests. Viable cell counts on different culture media showed that the cocci community prevailed during the 18 h of curd fermentation and the 6 d of cheese ripening. RAPD fingerprinting made it possible to isolate 25 different strains identified by 16S rDNA sequencing as belonging to five species of Lactobacillus, three species of Streptococcus, one species of Weissella, and one species of Enterococcus. The physiological analyses of all lactic acid bacteria strains revealed that the isolates belonging to Streptococcus genus were the most acidifying, whereas lactobacilli were most proteolytic. Streptococcus thermophilus C48W and Lactobacillus delbrueckii subsp. bulgaricus B15Z dominated all through the fermentation process. Furthermore, they seemed to be stable in a subsequent whey sample analyzed after 7 mo. The recovery of strains endowed with interesting technological features, such as acidifying and proteolytic activities, and surviving in natural whey could allow the upscaling of cheese processing safeguarding the organoleptic characteristics of Scamorza Altamurana and could possibly improve other fermented dairy products.

The loss of microbial biodiversity due to the increase in large-scale industrial processes led to the study of the natural microflora present in a typical little known dairy product. The community of lactobacilli was studied in order to understand the natural fermentation of Ricotta forte cheese. The combined use of RAPD analysis, 16S rDNA sequencing and physiological tests allowed 33 different strains belonging to 10 species of Lactobacillus to be characterized. RAPD analysis revealed the heterogeneity of both the Lact. kefiri and Lact. paracasei species. The sequence analysis of the large 16S/23S rRNA spacer region enabled Lact. plantarum to be distinguished from Lact. paraplantarum, two closely related species belonging to the Lact. plantarum group. The recovery of strains endowed with interesting physiological characteristics, such as strong stress resistance, could improve technological and/or organoleptic characteristics of Ricotta forte cheese and other fermented foods.

The natural Lactobacillus community involved in traditional Mozzarella cheese production has been investigated. The bacterial associations of whey, curd before stretching and Mozzarella were analyzed using randomly amplified polymorphic DNA (RAPD) to follow growth kinetics, and 16S rDNA sequencing to identify the taxonomical position of isolated strains. Analysis of RAPD fingerprints revealed that the Lactobacillus community was composed of 13 different biotypes and the sequence analysis of 16S rDNA demonstrated that the isolated strains belong to L. plantarum, L. fermentum, L. helveticus and L. casei subsp. casei. In addition, two strains of Weissella hellenica were isolated on selective media for lactobacilli. The four L. casei subsp. casei strains and W. hellenica contained sequences related to the prtP gene coding for proteinase, and the highest proteolytic activity in milk was found in one strain of L. casei subsp.casei.