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P S Cocconcelli

Publications and source records attributed to P S Cocconcelli.

15 recordsLinked to original sources

Indigenous raw milk microbiota influences the bacterial development in traditional cheese from an alpine natural park.

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.

Animals↗

Microbial community dynamics during the Scamorza Altamurana cheese natural 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.

Animals↗

Identification of fungi from dairy products by means of 18S rRNA analysis.

The role of fungi as cause of spoilage of dairy products, such as cheese and yoghurt, has been clearly demonstrated. Despite of this, there is still a lack in rapid methods for the identification of food-associated fungi. In the course of the present work, molecular taxonomical techniques were developed and used to identify yeasts involved in the spoilage of yoghurt and moulds responsible for spoilage of vacuum-packaged hard cheese. Three methods for DNA extraction and purification were evaluated and the fungus-specific primers TR1 and TR2 were used to amplify a 581-bp fragment within the gene, coding for the small ribosomal subunit (18S rRNA) of fungi. The 18S rRNA sequence analysis of fungi isolated from yoghurt and packaged cheese allowed to identify yeast belonging to Zygosaccharomyces microellipsoides and moulds belonging to Penicillium chrysogenum and Cladosporium cladosporoides.

Animals↗

Changes in the Lactobacillus community during Ricotta forte cheese natural fermentation.

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.

Animals↗

Molecular characterization of the Lactobacillus community in traditional processing of Mozzarella cheese.

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.

Carbohydrate Metabolism↗

Physiological and molecular techniques for the study of bacterial community development in sausage fermentation.

The development of the microbial community involved in the production process of Italian dry sausage was investigated using physiological analysis and molecular techniques for strain typing and taxonomical identification. A cycle of sausage production was followed collecting samples during the 2 months of ripening process. Microbiological analysis allowed the identification of the main bacterial groups responsible for the fermentation process as lactobacilli and coagulase-negative staphylococci. The use of a polymerase chain reaction-based technique of strain typing, RAPD fingerprinting, demonstrated that the environmental parameters interact to select a limited number of strains that dominate the fermentation process. The staphylococcal populations were characterized for their physiological properties and the two dominant strains were identified as Staphylococcus xylosus and Staph. sciuri. The use of 16S rDNA sequencing allowed the definition of the taxonomical position of the two dominant strains of lactic acid bacteria, as belonging to Lactobacillus sake and Lact. plantarum.

Bacterial Typing Techniques↗

Use of RAPD and 16S rDNA sequencing for the study of Lactobacillus population dynamics in natural whey culture.

The development of communities of the thermophilic microflora of natural whey culture for Parmigiano Reggiano cheese production was studied by means of molecular techniques. RAPD analysis facilitates the identification of the Lactobacillus strains involved in this microbial association and permitted the study of population dynamics during two cycles of whey fermentation. Analysis of RAPD fingerprints revealed the presence of four biotypes that dominate the whey fermentation process. Sequence analysis of 16S rDNA demonstrated that the strains isolated from whey belong to Lact. helveticus and Lact. delbrueckii ssp. lactis species.

Base Sequence↗

Genetic analysis of the replication region of the Lactobacillus plasmid vector pPSC22.

The sequence and genetic organization of the 1,600-bp replication region of the Lactobacillus vector pPSC22, a plasmid derived from a 7-kb cryptic plasmid of L. plantarum used for the cloning of heterologous genes in several lactobacilli, were determined. Sequence analysis revealed the presence of a plus origin of replication containing the two functional elements nic and bind, required for initiation of the leading strands typical of the rolling circle (RC)-replicating plasmids belonging to the pLS1 family. Two open reading frames (copA and repA) were located within the Lactobacillus portion of pPSC22. The repA gene product, a 234-amino acid protein, showed homologies with the Rep protein of the streptococcal plasmid pLS1 and contained the three conserved domains detected in most Rep proteins of RC-replicating plasmids and ss-coliphages. The genetic organization of the replication region of pPSC22 shared relevant homologies with the lactococcal plasmids pWVO1 and pFX2.

Base Sequence↗

In vitro permeability evaluation and colonization of membranes for periodontal regeneration by Porphyromonas gingivalis.

The use of membranes for periodontal regeneration is well established. In clinical use, the exposure of membranes to the oral microflora may result in a pathway for periodontal infections. An important role in this process is played by Porphyromonas gingivalis. The purpose of the present study was to examine the colonization of 6 different bioresorbable and nonresorbable membranes for periodontal regeneration by the strain DSM 20709 of P. gingivalis and to determine the time needed by this microorganism to pass through the membranes. A device consisting of a tube sealed with the membranes and filled with a medium suitable for the growth of P. gingivalis was incubated in a bigger tube containing the same medium to study the process of colonization and the crossing of membranes. The outer tube was inoculated with 10(4) cells of P. gingivalis DSM 20709. The passage of bacteria through the membranes was monitored at 6, 24, and 48 hours by counting the number of cells in the inner tube. The colonized membranes were observed using a scanning electron microscope. Differences in the behavior of the 6 membranes analyzed were demonstrated.

Bacterial Adhesion↗

Development of RAPD protocol for typing of strains of lactic acid bacteria and enterococci.

A protocol for typing strains of lactic acid bacteria and enterococci based on randomly amplified polymorphic DNA (RAPD) fragments has been developed. Using a single 10-mer primer, fingerprints were achieved without the need to isolate genomic DNA. Different conditions of DNA release and amplification were investigated in order to obtain reproducible results and high discrimination among strains. This RAPD protocol was successfully applied for the typing of strains belonging to the species Lactobacillus acidophilus, Lact. helveticus, Lact. casei, Lact. reuteri, Lact. plantarum, Enterococcus faecalis, Ent. faecium and Streptococcus thermophilus.

Bacterial Typing Techniques↗

Plasmid transformation of Ruminococcus albus by means of high-voltage electroporation.

To apply recombinant DNA techniques to the genetic manipulation of cellulolytic ruminal bacteria, a plasmid vector transformation system must be available. The objective of this work was to develop a system for plasmid transformation of Ruminococcus albus. Using high voltage electrotransformation, pSC22 and pCK17 plasmid vectors, derived from lactic acid bacteria plasmids and replicating via single-stranded DNA intermediate, were successfully introduced into three freshly isolated R. albus strains and into R. albus type strain ATCC 27210. The optimization of the electrotransformation condition raised the electroporation efficiency up to 3 x 10(5) transformants per microgram of pSC22 plasmid.

DNA, Bacterial↗

Use of DNA probes in the study of silage colonization by Lactobacillus and Pediococcus strains.

A technique to monitor lactic acid bacteria inoculants in silage, based on specific DNA probes, was developed and used to evaluate the colonization properties of two strains of Lactobacillus plantarum and one strain of Pediococcus pentosaceus which were used as maize silage inoculants in farm conditions. The results indicated that these three strains were able to dominate the natural microflora of the silage, representing more than the 95% of the bacterial biomass of the maize silage. These studies indicate that the colony hybridization with specific DNA probes may be an effective method for monitoring bacteria and evaluating the colonization properties of inoculants in maize silage.

Animals↗

Lactobacillus protoplast transformation.

A method for the transformation of Lactobacillus protoplasts by plasmid DNA is reported. The procedure involves polyethylene glycol treatment of protoplasts to induce DNA uptake. A transformation efficiency ranging from 5 to 1000 transformants per microgram of DNA is achieved; the efficiency of protoplast regeneration ranged from 10 to 20%.

DNA, Bacterial↗

Fast and slow milk-coagulating variants of Lactobacillus helveticus HLM 1.

Slow milk-coagulating variants were isolated from Lactobacillus helveticus HLM 1, a fast strain which coagulates milk in 16 h at 42 degrees C. Variants were isolated after subculturing in reconstituted skim milk or complex broth media. Analysis of plasmid content revealed that in slow variants a 3.5-megadalton plasmid was missing.

Animals↗

Single-stranded DNA plasmid, vector construction and cloning of Bacillus stearothermophilus alpha-amylase in Lactobacillus.

Vector plasmids were constructed by ligating chloramphenicol and erythromycin resistance genes to TaqI-digested DNA of a cryptic plasmid from Lactobacillus plantarum. The minimal region of Lactobacillus plasmid DNA that was required for DNA replication was defined and a single-stranded DNA intermediate replication system was observed. Homologies with other origins of replication of plasmids from Gram-positive bacteria, replicating via rolling circle mechanism, were found. It was shown that the constructed vectors, named pPSC20 and pPSC22, were transformable into L. plantarum, Lactobacillus acidophilus, Lactobacillus reuteri, Lactobacillus fermentum, Lactobacillus helveticus, Lactococcus lactis subsp. lactis, Bacillus subtilis, and Escherichia coli. Using plasmid pPSC22, the alpha-amylase gene of Bacillus stearothermophilus was cloned and expressed in several Lactobacillus species.

DNA, Single-Stranded↗