Laura Franzetti

Characterisation ofPseudomonas spp. isolated from foods

PutativePseudomonas spp. (102 isolates) from different foods were first characterised by API 20NE and then tested for some enzymatic activities (lipase and lecithinase production, starch hydrolysis and proteolytic activity). However subsequent molecular tests did not always confirm the results obtained, thus highlighting the limits of API 20NE. Instead RFLP ITS1 and the sequencing of 16S rRNA gene grouped the isolates into 6 clusters:Pseudomonas fluorescens (cluster I),Pseudomonas fragi (duster II and V)Pseudomonas migulae (cluster III),Pseudomonas aeruginosa (cluster IV) andPseudomonas chicorii (cluster VI). The pectinolytic activity was typical of species isolated from vegetable products, especiallyPseudomonas fluorescens. InsteadPseudomonas fragi, predominantly isolated from meat was characterised by proteolytic and lipolytic activities.

Development of Genus/Species-Specific PCR Analysis for Identification of Carnobacterium Strains

Heterofermentative lactic acid bacteria belonging to the genus Carnobacterium are currently divided into seven different species, C. piscicola, C. mobile, C. gallinarum, C. inhibens, C. divergens, C. funditum, and C. alterfunditum. 16S rDNA-targeted PCR assay was carried out for the identification of the genus Carnobacterium. In addition, type strains of all Carnobacterium species were analyzed by 16S–23S rDNA intergenic spacer analysis in comparison with type strains of phylogenetically related lactic acid bacteria. These methods enabled the identification and the discrimination among Carnobacterium species and the other phylogenetically related lactic acid bacteria. Likewise, analogous results were obtained by restriction analysis of amplified 16S rDNA performed with HaeIII and HinfI as restriction enzymes.

Microbiological Quality and Shelf Life Modeling of Ready-to-Eat Cicorino

Growth of the microbial population resident in ready-to-use fresh cut cicorino, a variety of Chichorium intybus, was determined in the various preparation steps with the aim of defining the hazard critical control points. The investigation concerned cut cicorino from two producers. During the process a 1- to 1.5-fold increase of microbial counts was observed, and the retail product showed values of 10(5) to 10(6) CFU/g. The shelf life of the product was kinetically modeled in order to check the effects of storage temperature and assess the microbial indexes most relevant for hygiene and quality of the distributed product. A modified Gompertz function described the kinetics of microbial growth and allowed definition of a stability time and its dependence on temperature. Stability times were of 0.3, 3.7, and 4.7 days at storage temperatures of 20, 10, and 5 degrees C, respectively. Q10 (the fold decrease of stability time for an increase of 10 degrees C) was 3.85. The results from this study may be used to predict the effects of temperatures experienced in the distribution chain on bacterial levels in cicorino.

Phenotypic and genotypic characterization of Enterococcus spp. of different origins.

Sixty-four strains of Enterococcus spp. of different origin were identified using traditional phenotypic, biochemical and cultural tests, together with molecular tests. API 20 STREP tests identified the species at a preliminary level, only one strain remaining unidentified. Strains belonging to the genus Enterococcus were tested with genus-specific primers, while species-level identification was carried out with the 16S–23S rDNA intergenic region (ITS) and species-specific primers for E. faecium and E. faecalis. Those strains found to be negative with the species-specific primers were subjected to 16S rDNA partial sequencing.

Production of cyclopiazonic acid by molds isolated from Taleggio cheese.

Twenty-seven strains of Penicillium were isolated from the rind of Taleggio, a typical Italian cheese, so that we could test their capacity to produce cyclopiazonic acid (CPA); all strains produced CPA. The production was strongly influenced by the strain variety and growth conditions. Strains incubated at 25 degrees C for 7 days always produced CPA in mannitol broth, with concentrations ranging from 0.02 to 1 microg/ml, whereas only 33% of strains grown in yeast-extract broth produced CPA, with a maximum value of 0.1 microg/ml. In milk, maximum production (1.6 microg/ml) was observed after 14 days of incubation at 25 degrees C. In order to evaluate the presence of the toxin and its capacity for migrating into the cheeses, the rind, the cheese near the rind, and the cores from six Taleggio cheeses were analyzed. CPA was present in five cheeses, with a maximum concentration of 0.25 mg/kg in one rind, and in one cheese, the toxin migrated to the core. A positive correlation between CPA production and surface mold was found.

Shelf life of case-ready beef steaks (Semitendinosus muscle) stored in oxygen-depleted master bag system with oxygen scavengers and CO2/N2 modified atmosphere packaging

This study aims to evaluate the stability of beef from Semitendinosus muscle packaged in oxygen permeable wrapped-tray units and stored in a master bag system, with and without oxygen scavengers. Changes in the atmosphere composition, microbiological indexes, myoglobin forms and color parameters were monitored during the storage in master bag, blooming and display life. The presence of scavengers reduced rapidly the oxygen concentration and maintained it at values not detectable instrumentally. Within few days of storage in master bags, the resolution of the transient discoloration was completed and the meat quality was maintained over the anoxic storage. After the removal from master bags meat bloomed completely reaching OxyMb level and Chroma values higher than those on fresh meat at t(0). During 48 h of display life at 4 °C, quality attributes had a decay slower than samples stored traditionally in air. Without scavengers the oxygen caused the irreversible discoloration within 7 days, due to the formation of metmyoglobin on the surface.

Setup of a rapid method to distinguish among dead, alive, and viable but not cultivable cells of Pseudomonas spp. in mozzarella cheese

Pseudomonas spp. is the main psychrotrophic genus involved in the spoilage of raw milk and more in general of dairy products, such as mozzarella cheese. The members of this bacterial species are able to produce heat-resistant proteolytic enzymes, determining the casein hydrolysis, and as a consequence, a reduction of the shelf life and sensory quality of the products. Therefore, the spoilage activity could be attributed not only to viable, but also to viable but noncultivable (VBNC) cells. For this reason, the setup of a non-culture-based method is useful for a rapid detection of cells that are still alive, but no longer cultivable, such as VBNC cells. Here we propose a method based on DNA or RNA content (or both) to reveal the presence of dead, alive, and VBNC cells belonging to the genus Pseudomonas. The obtained results clearly indicate the limits of the classical plating count overcome by molecular detection of Pseudomonas spp. through DNA and RNA analysis, enabling us to establish the presence of different states of the cells.