Angiolella Lombardi

Biochemical properties of enterococci relevant to their technological performance

A total of 129 E. faecium, E. faecalis and E. durans strains of food, veterinary and human origin were screened for biochemical properties relevant to their technological performance. Strains exhibited low milk acidifying ability and low extracellular proteolytic activity, with food origin and E. faecalis strains being generally more active. Their peptidase activities were low and mainly specific against glycine-proline- and glutamate-4-nitroanilide, while only food origin and E. durans strains showed broader substrate specificity. In contrast, their lipolytic activities were relatively higher; food and veterinary origin and E. faecalis strains were the most lipolytic. The post-electrophoretic detection of esterase activities showed that the esterolytic system of enterococci was rather complex. All species showed strain-to-strain variation in their ability to metabolise citrate and pyruvate, with E. faecalis strains being generally more active. The main volatile compounds produced in milk were acetaldehyde, ethanol and acetoin; generally, E. faecalis strains produced the highest concentrations. None of the strains decarboxylated histidine, lysine and ornithine, but the majority produced tyramine from tyrosine, independently of origin and species. In respect of most biochemical properties considered in this study, E. faecalis strains were generally more active compared to E. faecium and E. durans. This was also the case for the isolates of food origin compared to those of veterinary and human origin. Results obtained allow the selection of enterococci strains to be used as adjunct starters in food fermentations. However, a final selection should take into account the potential virulence factors of enterococci.

Some probiotic properties of yeast isolates from infant faeces and Feta cheese

Yeast isolates from infant faeces and Feta cheese were characterized to species level by phenotypic criteria, Randomly Amplified Polymorphic DNA (RAPD)-PCR and mitochondrial DNA (mt-DNA) restriction analysis. Results suggested that there is a good agreement between phenotypic characterization of yeasts and RAPD-PCR at species level; in addition, RAPD-PCR as well as mt-DNA restriction analysis provided good discrimination at strain level. Some technological and probiotic properties of selected strains were also investigated. The test strains exhibited lipolytic and proteolytic activities. They also tolerated low pH and survived satisfactory in gastric juice in vitro as well as in the presence of bile. In general, the isolates from faeces were more resistant to low pH and bile than those from Feta cheese. Selected strains could be used as starter supplements for industrial fermentations.

Proteolytic, lipolytic and molecular characterisation of Yarrowia lipolytica isolated from cheese.

This work studied the qualitative and quantitative proteolytic and lipolytic activities of Yarrowia lipolytica strains isolated from two cheese types. Randomly amplified polymorphic DNA-PCR (RAPD-PCR) analysis was used to compare the cheese strains of Y. lipolytica with strains isolated from other food products and with the type strain of the species in order to investigate the genetic diversity and occurrence of specific environmental groups. Diversity of proteolytic and especially lipolytic activity within Y. lipolytica strains isolated from dairy products was observed. In particular, the degree of specificity for saturated or unsaturated fatty acids as well as for even- or odd-numbered carbon free fatty acids (FFAs) varied among the strains. The RAPD-PCR profiles showed low genetic relatedness between many of the food isolates and the type strain of the species. Such genetic variability needs to be further evaluated. Most of the Y. lipolytica strains appeared to be specific to the particular environment from which they were isolated. However, phenotypic characteristics having technological importance in dairy products and, particularly, lipolytic activities did not correspond to the genetic differences observed by RAPD-PCR analysis.

Yeast population dynamics during pilot-scale storage of grape marcs for the production of Grappa, a traditional Italian alcoholic beverage

The composition and population dynamics of the yeast microflora of grape marcs were investigated during a pilot scale fermentation study using two white grape varieties, namely Moscato and Prosecco, from two distinct areas of the Veneto Region. Yeast counts were made at the beginning, after 4 and after 15 days of marc storage under anaerobic conditions. Seventy isolates from each sampling time were identified to species by RAPD-PCR analysis and subsequent ITS region sequencing. A good biodiversity of yeasts occurred in both marcs at the beginning of fermentation, with high presence of Hanseniaspora opuntiae, but without detectable presence of Saccharomyces strains, which instead became the dominant yeast after just 4 days of fermentation, remaining at that level until the end of fermentation. Colonization of Moscato marc by S. cerevisiae resulted better, in relation to its higher sugar content. Characterization of S. cerevisiae isolates by mitochondrial DNA restriction analysis revealed the presence of 66 different strains in the marc from the Moscato grapes, without the occurrence of a clearly dominant strain, while in the marc from the Prosecco grapes only 23 different profiles were scored, with a dominant strain that accounted for 62.7% of the Saccharomyces population after 4 days of fermentation.

Phenotypic and Genotypic Characterization of Staphylococcus aureus Strains from Italian Dairy Products

Staphylococcus aureus is a known major cause of foodborne illnesses, and milk and dairy products are often contaminated by enterotoxigenic strains of this bacterium. In the present study, 122 S. aureus isolates collected from different dairy products were characterised by phenotypic properties, by the distribution of genes encoding staphylococcal enterotoxins (sea, sec, sed, seg, seh, sei, sej, and sel) and by randomly amplified polymorphic DNA PCR (RAPD-PCR). Moreover, strain resistance to vancomycin and methicillin (oxacillin) was studied. The differences in the RAPD-PCR profiles obtained with the primers M13 and AP4 revealed the presence of a great genetic heterogeneity among the different S. aureus strains. Using the primer AP4 and M13, eight groups were distinguished by RAPD-PCR cluster analysis, although, except in few cases, it was not possible to correlate the isolates of different animal species (cow or ovine) with the presence of se genes. None of the isolates showed resistance to vancomycin or methicillin.

Biodiversity and characterization of indigenous coagulase-negative staphylococci isolated from raw milk and cheese of North Italy

The aim of the current study was to detect coagulase-negative staphylococci (CNS) in raw milk and cheeses produced in North Italy, and to analyze isolates for their biodiversity, safety aspects and technological properties. Molecular identification methods revealed a high biodiversity among isolates and assigned them to 17 species. The most recovered species were Staphylococcus equorum (12%), Staphylococcus lentus (12%), Staphylococcus simulans (12%), Staphylococcus sciuri (10%), and Staphylococcus xylosus (9%). The presence of ten transferable antibiotic resistance (AR) genes was verified by PCR and 19% of isolates were positive, with tet(K) being the most frequent gene (10%); interestingly, no strain carried multiple AR genes. Twenty-four isolates displayed hemolytic activity; tyrosine decarboxylase gene (tdcA) was found in two isolates, while histidine decarboxylase gene (hdcA) and enterotoxin genes (se) were not detected. Isolates were further characterized for the presence of some relevant technological properties; 16% of isolates displayed proteolytic activity and 39% lipolytic activity, while no one of the isolates was found to exhibit antimicrobial activity against Staphylococcus aureus. This study provided evidence of a low occurrence of safety hazards in CNS isolated from dairy products.

Identification of Microbiota Present on the Surface of Taleggio Cheese Using PCR–DGGE and RAPD–PCR

UNLABELLED Microbial DNA from 9 batches of Taleggio PDO cheese sampled at various times during ripening, brines, swabs of wooden shelves used for cheese dry-salting, and 13 commercial cheeses were analyzed by denaturing gradient gel electrophoresis (PCR-DGGE) and/or random amplification of polymorphic DNA (RAPD-PCR). Sequencing allowed the detection of 12 genera, 27 species, and 2 unclassified bacteria. Molecular analysis allowed for the detection of microorganisms not previously associated with Taleggio such as Lactobacillus paracasei, Carnobacterium maltaromaticum, Bacillus licheniformis, Corynebacterium variabile, Psychrobacter cibarius, and Staphylococcus carnosus. For the first time Massilia spp. was detected in a dairy ecosystem. PRACTICAL APPLICATION Indigenous species and strains of bacteria identified by this study could be used for the selection of dairy cultures to be employed routinely by manufacturers to control the Taleggio cheese production. The new cultures may give the bases for driving dairy processes and, consequently, control the typical flavor resulting from metabolic actions of environmental microorganisms.

Molecular characterization and genotypic antimicrobial resistance analysis of Campylobacter jejuni and Campylobacter coli isolated from broiler flocks in northern Italy

Genetic variability and genotypic antimicrobial resistance (AMR) of Campylobacter jejuni and Campylobacter coli from commercial broiler farms were investigated in this study. Campylobacter isolates were genetically characterized by random amplified polymorphic DNA (RAPD)-polymerase chain reaction (PCR) and flaA-SVR and flaB-SVR sequence-based typing. Eight RAPD types were identified in C. jejuni and three in C. coli, while 16 fla profiles were detected among all isolates. Further, 13 flaA-SVR and 13 flaB-SVR alleles were identified. Both typing methods detected a high level of genetic diversity, but fla-SVR typing showed a higher discriminatory power. Indeed, Simpsons index of fla typing (D=0.920) was higher than that of RAPD typing (D=0.814). Moreover, the association of flaA-SVR and flaB-SVR sequence analysis showed a higher discriminatory power compared with the sequence analysis of single loci. Isolates were also analysed by the mismatch amplification mutation assay PCR test and the detection of cmeB gene to determine the occurrence of genetic determinants of AMR to macrolides and fluoroquinolones and multidrug resistance. The A2074C and A2075G mutations in the 23S rRNA gene, the C257T mutation in the gyrA gene, and the cmeB gene were higher in C. coli (19.0%, 67.0%, 100.0% and 100.0%, respectively) than in C. jejuni (0.0%, 3.1%, 48.3% and 48.3%, respectively). This study showed a high degree of genetic diversity and a high prevalence of genetic determinants of macrolide resistance, fluoroquinolone resistance and multidrug resistance among C. jejuni and C. coli isolates from Italian commercial broiler farms.

A Longitudinal Study on Thermophilic Campylobacter spp. in Commercial Turkey Flocks in Northern Italy: Occurrence and Genetic Diversity

SUMMARY. Poultry are recognized as a main reservoir of thermophilic campylobacters, but few studies have been carried out on commercial meat turkeys. This study was aimed at assessing the occurrence of thermophilic Campylobacter spp., their genetic diversity, and the trend of the infection during the whole production cycle of three turkey flocks from different farms in Northern Italy. Flocks were monitored from the time of housing 1-day-old poults to slaughter time by collecting samples (meconium and cloacal swabs) at weekly intervals up to the recovery of Campylobacter spp. and then twice a month. A conventional culture method and a multiplex PCR assay were used for Campylobacter detection and identification. A subset of isolates was genetically characterized by random amplified polymorphic DNA-PCR (RAPD-PCR) and flagellin gene A short variable region (flaA-SVR) sequencing. Although at different times, all flocks became colonized by Campylobacter jejuni or Campylobacter coli (or both) that persisted throughout the entire production cycle. Overall, nine RAPD types and 14 flaA-SVR types were detected with differences in their distribution among flocks and sampling times. Moreover, changes in the Campylobacter genotypes colonizing turkeys were observed over time within each flock. These findings suggest that Italian commercial turkeys might be widely colonized by different genotypes of C. jejuni and C. coli and also suggest that differences in the distribution and epidemiologic dynamics of these microorganisms might occur among flocks.

Outlining a selection procedure for Saccharomyces cerevisiae isolated from grape marc to improve fermentation process and distillate quality.

Nowadays grape marc represents one of the main by-product of winemaking. Many South Europe countries valorize this ligno-cellulosic waste through fermentation and distillation for industrial alcoholic beverage production. The storage of marcs is a crucial phase in the distillation process, due to the physicochemical transformations ascribed to microbial activity. Among the methods adopted by distillers to improve the quality of spirits, the use of selected yeasts has not been explored so far, therefore in this work we evaluated the selection criteria of Saccharomyces cerevisiae strains for grape marc fermentation. The proposed selection procedure included three steps: characterization of phenotypical traits, evaluation of selected strains on pasteurised grape marc at lab-scale (100 g) and pilot-scale fermentation (350 kg). This selection process was applied on 104 strains isolated from grape marcs of different origins and technological treatment. Among physiological traits, β-glucosidase activity level as quality trait seems to be only partially involved in increasing varietal flavour. More effective in describing yeast impact on distillate quality is the ratio higher alcohols/esters that indicates strain ability to increase positive flavours. Finally, evaluating grape marc as source of selected yeasts, industrial treatment rather than varietal origin seems to shape strain technological and quality traits.