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The genus Vibrio includes bacteria with different morphological and metabolic characteristics responsible for different human and animal diseases. An accurate identification is essential to assess the risks in regard to aquatic organisms and consequently to public health. The Multilocus Sequence Analysis (MLSA) scheme developed on the basis of 4 housekeeping genes (gyrB, pyrH, recA and atpA) was applied to identify 92 Vibrio strains isolated from crustaceans in 2011. Concatenated sequences were used for the phylogenetic and population analyses and the results were compared with those from biochemical identification tests. From the phylogenetic analysis, 10 clusters and 4 singletons emerged, whereas the population analysis highlighted 12 subpopulations that were well supported by phylogeny with few exceptions. The retrospective analysis allowed correct re-attribution of isolated species, indicating how, for some pathogens, there may be an overestimation of phenotypic identification (e.g. V. parahaemolyticus). Use of the PubMLST Vibrio database highlighted a possible genetic link between Sequence Type (ST) 529 and ST195 (V. alginolyticus) isolated from a human case in Norway during 2018. In addition to the identification of major risk groups of V. cholerae, V. vulnificus and V. parahaemolyticus, MLSA could be a valid support for species considered a minor risk, such as V. alginolyticus, V. mimicus and V. fluvialis. Due to the increased incidence of vibriosis in Europe, the application of different tools will also have to be considered to investigate the possible epidemiological links of the various species in the perspective of Open Science to protect the consumer.

Processing of alpine milk in malga farms is carried out under conditions that can favor contamination by coliforms, coagulase-positive staphylococci, or pathogens such as Listeria monocytogenes. With the aim to improve the hygienic characteristics and safety of cheese produced in four malga farms the use of lyophilized Natural Milk Culture prepared with selected strains was tested. Two cheesemaking tests were carried out in the same day always starting from the same milk: in the first case following the malga recipe that uses either Natural Whey Culture or without the addition of a starter, in the second one using a Natural Milk Culture. Cheesemaking were carried out in four malga farms located in the west area of Trentino region within the same week. For hygienic and safety evaluation, aerobic colony count, coagulase-positive staphylococci, Escherichia coli, staphylococcal toxins, Listeria monocytogenes , and Salmonella spp, pH and aw were determined in raw milk from evening and morning milking, curd in vat, curd after extraction and two months-ripened cheese. Pathogens or toxins, high values of coagulase- positive staphylococci and E. coli were not found in cheese samples. However, in the curd coagulase-positive staphylococci reached values almost of 5 Log CFU/g in the two malga without starter cultures. The use of Natural Milk Culture reduced E. coli counts. In addition, DNA was extracted from cheese samples and from Natural Milk Culture and the composition of the microbial community determined by Next Generation Sequencing method. The determination of cheese microbial communities demonstrated that the use of Natural Milk Culture exerted different effects in the different malga, in any case preserving bacterial biodiversity.

Near infrared spectroscopy (NIRs) and ultraviolet visible spectroscopy (UV-vis) have been investigated as rapid techniques to characterize foodborne bacteria through the analysis of the spectra of whole cells or microbial suspensions. The use of spectra collected from broth cultures could be used as a fingerprint for strain classification using a combined polyphasic approach. The aim of this study was to evaluate the feasibility of NIRs and UV-vis for the characterization of blue strains belonging to the Pseudomonas fluorescens group. The bacteria were isolated from different food matrices, including some spoiled samples (blue discoloration). Eightyone strains previously identified at the species level were grown in Minimal Bacterial Medium broth under standard conditions at 22°C. Two biological replicates were centrifuged in order to separate the bacterial cells from the extracellular products. Six aliquots per strain were analyzed on a small ring cup in transflectance mode (680-2500 nm, gap 2 nm). A subset of 39 strains was evaluated by UV-vis to determine changes in the spectral characteristics at 48 and 72 hours. Several chemometric approaches were tested to assess the performance of NIRs and UVvis. According to the variable importance in projection (VIP), the 1892-2020 nm spectral region showed the highest level of discrimination between blue strains and others. Additional information was provided in the 680-886 and 1454-1768 nm regions (aromatic C-H bonds) and in the 2036-2134 nm region (fatty acids). Changes in UV-vis spectral data (at 48 and 72 hours) appear to indicate the presence of phenazine and catecholic compounds in extracellular products.

Challenge tests are a clear opportunity for manufacturers interested in the evaluation of their management system with the aim to reduce the spread of foodborne pathogens. This is a main concern especially in ready-to-eat food in relation to the risk associated with Listeria monocytogenes. For small and medium-scale food industry the manufacturing practices and products formulation are characterised by a wider variability and poor repeatability. The use of ad hoc challenge test and the comparison among different processing systems are strongly required. This paper reports a preliminary comparison among different challenge tests (n=12) commissioned by three manufacturers of raw-fermented salami during a period of three years (2013-2016). The challenge tests were designed to evaluate the growth potential (δ) of L. monocytogenes during the whole processing period of the salami. The doughs were prepared according to different formulations: the simplest formulation was represented by the use of salt, potassium nitrate, black pepper and starter cultures, while the most composited formulations also included the use of sugars and ascorbic acid in addition to nitrite salt. All the processing steps were conducted within an experimental laboratory dedicated for the processing of meat. After stuffing, the salami were dried and ripened under temperature and relative humidity control. The sugar inclusion can be considered as a protective factor, while the drying step at high temperature (above 20°C) was associated with higher δ values (δ>0.5 log10 cfu/g). The addition of starter cultures, and the subsequent acidification highlighted the importance of pH as the parameter able to affect the L. monocytogenes growth.

Bacterial proteases are involved in food spoilage and shelf-life reduction. Among the bacterial proteases, a predominant role in spoilage of dairy products seems to be played by the thermostable metallo-protease AprX, which is produced by various strains of Pseudomonas fluorescens. Differences in AprX enzyme activity among different strains were highlighted, but the most proteolytic strains were not identified. In this study, the presence of the aprX gene was evaluated in 69 strains isolated from food matrices and 18 reference strains belonging to the P. fluorescens group, which had been previously typed by the multi locus sequence typing method. Subsequently, a subset of reference strains was inoculated in ultra-high temperature milk, and the expression of the aprX gene was evaluated at 22 and 6°C. On the same milk samples, the proteolytic activity was then evaluated through Azocasein and trinitrobenzenesulfonic acid solution assays. Finally, to assess the applicability of the former assay directly on dairy products the proteolityc activity was tested on industrial ricotta samples using the Azocasein assay. These results demonstrate the spread of aprX gene in most strains tested and the applicability of Azocasein assay to monitor the proteolytic activity in dairy products.

Phenols are plant metabolites characterised by several interesting bioactive properties such as antioxidant and bactericidal activities. In this study the application of a phenols concentrate (PC) from olive vegetation water to two different fresh products – gilt-head seabream (Sparus aurata) and chicken breast – was described. Products were treated in a bath of PC (22 g/L; chicken breast) or sprayed with two different solutions (L1:0.75 and L2:1.5 mg/mL; seabream) and then stored under refrigeration conditions. The shelf life was monitored through microbiological analyses – quality index method for seabream and a specific sensory index for raw breast. The secondary products of lipid-peroxidation of the chicken breast were determined using the thiobarbituric acid reactive substances (TBARs) test on cooked samples. Multivariate statistical techniques were adopted to investigate the impact of phenols and microbiological data were fitted by DMfit software. In seabream, the levels of PC did not highlight any significant difference on microbiological and sensory features. DMfit models suggested an effect only on H2S producing bacteria with an increased lag phase compared to the control samples (C: 87 h vs L2: 136 h). The results on chicken breast showed that the PC bath clearly modified the growth of Pseudomonas and Enterobacteriaceae. The phenol dipping was effective in limiting lipid-peroxidation (TBARs) after cooking. Treated samples disclosed an increase of shelf life of 2 days. These could be considered as preliminary findings suggesting the use of this concentrate as preservative in some fresh products.

The aim of the study was to assess the in vitro effect of phenols extracted from oil vegetation water (PEOW) on several food-borne strains. Antibacterial activity of PEOW was based on the minimum bactericidal concentration (MBC) on microtitre assay. The taxa tested were: Staphylococcus (n. 5), Listeria (n. 4), Escherichia (n. 2), Salmonella (n. 1), Pseudomonas (n. 3), Lactobacillus (n. 2) and Pediococcus (n. 1). S. aureus and L. monocytogens showed the lowest level of resistance to PEOW (MBC=1.5-3 mg/mL). In contrast, the Gram negative strains (e.g. S. Typhimurium and Pseudomonas spp.) were in some cases unaffected by the tested doses and the MBCs ranged between 6 to 12 mg/mL. Starter cultures were dramatically reduced on growth (e.g. Staphylococcus xylosus; 0.75 mg/mL MBC). The thresholds for pathogenic strains could be considered for further applications of PEOW in food models (e.g. shelf life or challenge test studies).

A purified extract of phenols compounds (65% of phenolic content of which decarboxymethyl oleuropein aglycone represented 45% of the wet mass) obtained from vegetation water (a by-product of oil mill) was added to a ground meat dough intended for salami manufacture in two concentration levels: 75 and 150 mg/100 g of dough (F1 and F2, respectively). The control batch was composed of lean and fat cuts of pork in 70:30 ratio, 2.7% salt and a mixed starter culture of staphylococci and pediococci. After stuffing into natural casings, salamis were aged until they reached a total weight loss of 30%. The product was then sliced and packaged in a protective atmosphere (nitrogen:carbon dioxide 80:20) and placed in a refrigerator thermostat (2-4°C) with alternating 12 h of artificial light and darkness. The samples were analysed for the measurement of pH, water activity, organic acidity, peroxide number and secondary products of lipid peroxidation at the time of slicing and after 10, 20 and 30 days of storage into the refrigerated thermostat. The pH and water activity were not substantially different between the control and the two enriched batches. The peroxide number and secondary products of lipid peroxidation values in the two batches with phenols were at least substantially lower than the control sample. In conclusion, the phenol compounds obtained from vegetation water have shown no interference with the ripening process while protecting the dough from oxidation.