Francesca Clementi

Twelve-hour PCR-based method for detection of Salmonella spp. in food.

ABSTRACT A PCR-based method for the detection of Salmonella spp. in food was developed. The method, set up on typical salami from the Italian region of Marche, is sensitive and specific and shows excellent correlation with the conventional method of reference when naturally contaminated foods are analyzed. Moreover, it can be easily performed within a maximum of 12 h from food sampling, thus allowing prompt detection of Salmonella spp. in the food stocks analyzed.

Contribution of winery-resident Saccharomyces cerevisiae strains to spontaneous grape must fermentation.

The origin of the Saccharomyces cerevisiae strains that are responsible for spontaneous grape must fermentation was investigated in a long-established industrial winery by means of two different approaches. First, seven selected components of the analytical profiles of the wines produced by 58 strains of S. cerevisiae isolated from different sites and phases of the production cycle of a Grechetto wine were subjected to Principal Components Analysis. Secondly, the same S. cerevisiae isolates underwent PCR fingerprinting by means of δ primers. The results obtained by both methods demonstrate unequivocally that under real vinification conditions, the S. cerevisiae strains colonising the winery surfaces are the ones that carry out the natural must fermentation.

The combined effect of nisin, leucocin F10, pH, NaCl and EDTA on the survival of Listeria monocytogenes in broth.

The combined effect of the bacteriocins nisin (1-2100 IU/ml) and leucocin F10 (1-2100 AU/ml), pH (4.7-6.5), NaCl (0.7-4.5% w/l), ethylene diaminetetraacetic acid disodium salt (EDTA, 0.08-4.72 mmol/l) and inoculum level (10(3)-10(8) cfu/ml) on the survival of a pool of three strains of Listeria monocytogenes in broth was evaluated in three factorial experiments. Several factor combinations were found to prevent growth. Logistic regression analysis of the categorical data (survival/no survival) was used to generate predictive models for the probability of survival in 0.01 ml (P0.01) or 1 ml (P1). Predicted and observed probabilities of survival were not significantly different in 72% and 68.9% of treatments for P0.01 and P1, respectively. Unsafe predictions were obtained in 9.4% and 14.8% of treatments for P0.01 and P1, respectively. Nisin had a major effect on the probability of survival but the addition of leucocin F10 was necessary to prevent the survival of L. monocytogenes. Lower pH values significantly decreased the probability of survival, while NaCl and EDTA had only a minor effect. Doses of bacteriocins > 250 AU/ml, pH < 5.6 and EDTA > 0.2 mmol/l (0.074 g/l) were needed to reliably prevent survival of Listeria monocytogenes.

Bacteria and yeast microbiota in milk kefir grains from different Italian regions

Kefir grains are a unique symbiotic association of different microrganisms, mainly lactic acid bacteria, yeasts and occasionally acetic acid bacteria, cohabiting in a natural polysaccharide and a protein matrix. The microbial composition of kefir grains can be considered as extremely variable since it is strongly influenced by the geographical origin of the grains and by the sub-culturing method used. The aim of this study was to elucidate the bacteria and yeast species occurring in milk kefir grains collected in some Italian regions by combining the results of scanning electron microscopy analysis, viable counts on selective culture media, PCR-DGGE and pyrosequencing. The main bacterial species found was Lactobacillus kefiranofaciens while Dekkera anomala was the predominant yeast. The presence of sub-dominant species ascribed to Streptococcus thermophilus, Lactococcus lactis and Acetobacter genera was also highlighted. In addition, Lc. lactis, Enterococcus sp., Bacillus sp., Acetobacter fabarum, Acetobacter lovaniensis and Acetobacter orientalis were identified as part of the cultivable community. This work further confirms both the importance of combining culture-independent and culture-dependent approaches to study microbial diversity in food and how the combination of multiple 16S rRNA gene targets strengthens taxonomic identification using sequence-based identification approaches.

PCR‐DGGE analysis of lactic acid bacteria and yeast dynamics during the production processes of three varieties of Panettone

Aims:  To study lactic acid bacteria (LAB) and yeast dynamics during the production processes of sweet‐leavened goods manufactured with type I sourdoughs.

Interactions between Saccharomyces cerevisiae and malolactic bacteria: preliminary characterization of a yeast proteinaceous compound(s) active against Oenococcus oeni

Aims:  To investigate the occurrence and extent of Saccharomyces cerevisiae and Oenococcus oeni interactions.

Characterization of natural starter cultures used in the manufacture of Pasta Filata cheese in Basilicata (Southern Italy)

Microbiological, chemical and technological analyses were used to characterize nine natural starter cultures used for the manufacture of Pasta Filata cheese in Basilicata (Southern Italy). The cultures were either dominated by thermophilic rods or mesophilic and/or thermophilic cocci. Principal component and cluster analysis discriminated between rods and coccus cultures and helped to estabilish relationships between the cultures and to evaluate the variability of cultures obtained from the same plant. A total of 156 isolates of lactic acid bacteria were obtained from the cultures and tentatively classified using biochemical and physiological tests and cluster analysis. Most of the lactobacilli were identified as Lactobacillus helveticus. Most cocci were classified in the genera Lactococcus or Enterococcus but identification at the species level was often impossible. The acid production and proteolytic activity of the isolates in skim milk were evaluated. Cluster analysis was used to group the isolates according to technological properties. Isolates belonging to some phenotypic clusters were consistently associated with some technological clusters.

Investigation of the microbial ecology of Ciauscolo, a traditional Italian salami, by culture-dependent techniques and PCR-DGGE

The microbial ecology of 22 samples of commercially available Ciauscolo salami were investigated using a polyphasic approach, based on culture-dependent and -independent techniques. The viable counts of pathogen and hygiene indicator microorganisms highlighted the adequate application of good manufacturing practices, while the viable counts of the lactic acid bacteria, coagulase negative cocci, and yeasts showed dominance of the first of these microbial groups. Bacterial and fungal DNA were extracted directly from the salami and amplified by PCR, using two primer sets targeting the 16S and 28S rRNA genes, respectively. Denaturing gradient gel electrophoresis (DGGE) and sequencing of selected bands were used to investigate the microbial ecology of these Ciauscolo salami. The most frequently found bacterial species were Lactobacillus sakei and Lb. curvatus, while Debaryomyces hansenii was the prevalent yeast species detected. Cluster analysis of the DGGE profiles and calculation of biodiversity indices allowed the degree of microbial similarity across these salami to be determined.

Resident lactic acid bacteria in raw milk Canestrato Pugliese cheese

Aims:  Investigation of the autochthonous lactic acid bacteria (LAB) population of the raw milk Protected Designation of Origin Canestrato Pugliese cheese using phenotypic and genotypic methodologies.

Minisatellites in Saccharomyces cerevisiae genes encoding cell wall proteins: a new way towards wine strain characterisation

With the aim of developing new tools for the characterisation of wine yeasts, by means of databases available on-line we scanned the genome of Saccharomyces cerevisiae in search of potentially polymorphic targets. As we have previously observed for SED1, we found that other genes coding for cell wall proteins contain minisatellite-like sequences. A polymerase chain reaction (PCR) survey of SED1 and three of these others, namely AGA1, DAN4 and HSP150, in a population of wild S. cerevisiae demonstrated that these genes are highly polymorphic in length and represent a sink of unexplored genetic variability. The primer pairs designed on the gene open reading frames yield stable and repeatable amplification profiles that show a level of resolution that allows the clear discriminate between different strains. These can therefore be utilised for PCR-based typing of S. cerevisiae.