Patrizio Tremonte

Homology-based modeling of Universal Stress Protein from Listeria innocua up-regulated under acid stress conditions

An Universal Stress Protein (USP) expressed under acid stress condition by Listeria innocua ATCC 33090 was investigated. The USP was up-regulated not only in the stationary phase but also during the exponential growth phase. The three dimensional (3D) structure of USP was predicted using a combined proteomic and bioinformatics approach. Phylogenetic analysis showed that the USP from Listeria detected in our study was distant from the USPs of other bacteria (such as Pseudomonas spp., Escherichia coli, Salmonella spp.) and clustered in a separate and heterogeneous class including several USPs from Listeria spp. and Lactobacillus spp. An important information on the studied USP was obtained from the 3D-structure established through the homology modeling procedure. In detail, the Model_USP-691 suggested that the investigated USP had a homo-tetrameric quaternary structure. Each monomer presented an architecture analogous to the Rossmann-like α/β-fold with five parallel β-strands, and four α-helices. The analysis of monomer-monomer interfaces and quality of the structure alignments confirmed the model reliability. In fact, the structurally and sequentially conserved hydrophobic residues of the β-strand 5 (in particular the residues V146 and V148) were involved in the inter-chains contact. Moreover, the highly conserved residues I139 and H141 in the region α4 were involved in the dimer association and functioned as hot spots into monomer–monomer interface assembly. The hypothetical assembly of dimers was also supported by the large interface area and by the negative value of solvation free energy gain upon interface interaction. Finally, the structurally conserved ATP-binding motif G-2X-G-9X-G(S/T-N) suggested for a putative role of ATP in stabilizing the tetrameric assembly of the USP. Therefore, the results obtained from a multiple approach, consisting in the application of kinetic, proteomic, phylogenetic and modeling analyses, suggest that Listeria USP could be considered a new type of ATP-binding USP involved in the response to acid stress condition during the exponential growth phase.

Raw milk from vending machines: Effects of boiling, microwave treatment, and refrigeration on microbiological quality

In Italy, the sale of raw milk from vending machines has been allowed since 2004. Boiling treatment before its use is mandatory for the consumer, because the raw milk could be an important source of foodborne pathogens. This study fits into this context with the aim to evaluate the microbiological quality of 30 raw milk samples periodically collected (March 2013 to July 2013) from 3 vending machines located in Molise, a region of southern Italy. Milk samples were stored for 72 h at 4 °C and then subjected to different treatments, such as boiling and microwaving, to simulate domestic handling. The results show that all the raw milk samples examined immediately after their collection were affected by high microbial loads, with values very close to or even greater than those acceptable by Italian law. The microbial populations increased during refrigeration, reaching after 72 h values of about 8.0 log cfu/mL for Pseudomonas spp., 6.5 log cfu/mL for yeasts, and up to 4.0 log cfu/mL for Enterobacteriaceae. Boiling treatment, applied after 72 h to refrigerated milk samples, caused complete decontamination, but negatively affected the nutritional quality of the milk, as demonstrated by a drastic reduction of whey proteins. The microwave treatment at 900 W for 75 s produced microbiological decontamination similar to that of boiling, preserving the content in whey proteins of milk. The microbiological characteristics of raw milk observed in this study fully justify the obligation to boil the raw milk from vending machines before consumption. However, this study also showed that domestic boiling causes a drastic reduction in the nutritional value of milk. Microwave treatment could represent a good alternative to boiling, on the condition that the process variables are standardized for safe domestic application.

Interactions between Lactobacillus sakei and CNC (Staphylococcus xylosus and Kocuria varians) and their influence on proteolytic activity

Aims:  To evaluate interactions between Lactobacillus sakei and coagulase negative cocci (CNC) (Staphylococcus xylosus and Kocuria varians) and to investigate the influence of these interactions on their own proteolytic activity.

Interactions between strains of Staphylococcus xylosus and Kocuria varians isolated from fermented meats

Aims:  To evaluate the interactions of Staphylococcus xylosus on Kocuria varians strains isolated from fermented meat products.

Irradiation Treatments to Improve the Shelf Life of Fresh Black Truffles (Truffles Preservation by Gamma-Rays)

The aim of this study was to individuate a method to improve the shelf life of fresh truffles, hypogeous fungi highly prized but very perishable as well. Microbial ecology of fresh products was investigated and the effect of irradiation at different doses on microbial and sensorial parameters was assayed. Data showed that fresh truffles are strongly contaminated from different groups of microorganisms. Irradiation was able to influence their growth, determining an impressive reduction. Treatments at 5 and 10 kGy doses produced the highest microbial decontamination but negatively influenced sensorial parameters. Irradiation at a 1.5 kGy dose was the best solution to improve the shelf life of truffles.

Microbiological and fermentative properties of baker's yeast starter used in breadmaking.

This study assessed the levels of microbial contaminants in liquid, compressed and dry commercial bakers yeasts used as starters in breadmaking. Eumycetes, Enterobacteriaceae, total and fecal coliforms, Bacillus spp., and lactic acid bacteria (LAB), in particular enterococci, were quantified. Results obtained in this study highlighted that bakers yeast could represent a potential vehicle of spoilage and undesirable microorganisms into the baking environment, even if these do not influence the leavening activity in the dough, as ascertained by rheofermentometer analysis. Different microbial groups, such as spore-forming bacteria and moulds, were found in bakers yeast starters. Moreover, different species of LAB, which are considered the main contaminants in large-scale yeast fermentations, were isolated and identified by Denaturing Gradient Gel Electrophoresis (DGGE) and 16S rDNA sequencing. The most recurrent species were Lactobacillus plantarum, Enterococcus faecalis, and Enterococcus durans, isolated from both compressed and dry starters, whereas strains belonging to Leuconostoc and Pediococcus genera were found only in dry ones. Nested-Polymerase Chain Reaction (Nested-PCR) and Randomly Amplified Polymorphic DNA-PCR (RAPD-PCR) were also used to highlight the biodiversity of the different commercial yeast strains, and to ascertain the culture purity.

Shelf Life of Fresh Sausages Stored under Modified Atmospheres

The aim of this study was to investigate differences in modified atmosphere packaging (MAP) for the improvement of the shelf life of fresh meat products. Three different conditions for preserving fresh sausages were tested: MAP1 (20% CO2, 70% O2, and 10% N2), MAP2 (40% CO2 and 60% O2), and MAP3 (40% CO2, 30% O2, and 30% N2). Samples from the MAP2 group had fewer spoilage bacteria, stable red color (no change of a* value), and good physical attributes (high water-holding capacity, little loss from cooking, and low shear force needed for cutting) compared with samples from other treatment groups. Thus, high concentrations of CO2 (40%) and O2 (60%) resulted in a longer shelf life for fresh sausages.

Innovative Caciocavallo cheeses made from a mixture of cow milk with ewe or goat milk

This study assessed and compared the physicochemical, microbiological, and sensorial characteristics of Caciocavallo cheeses, made from cow milk and a mixture of cow with ewe or goat milk, during ripening. Different cheese-making trials were carried out on an industrial scale following the standard procedure of pasta filata cheeses, with some modifications. The percentage of the different added milk to cow milk influenced compositional and nutritional characteristics of the innovative products, leading to a satisfactory microbiological and sensorial quality.

Preservation by freezing of potentially probiotic strains ofLactobacillus rhamnosus

The aim of this work was to detect the best conditions to preserve by freezing potentially probiotic strains ofLactobacillus rhamnosus isolated from food. Four strains isolated from Parmigiano Reggiano cheese, the commercial strainLactobacillus GG and the type strain ATCC 7469T were used in the present study. Two different pre-incubation times (5 and 24 h), three protective media (Skim milk, Skim milk plus glucose and MRS plus glycerol) and two storage temperatures (−20 and −80 °C) were used for a preservation period of 90 days. A sensible loss of survival of the strains was detected and the acidifying activity decreased depending on the different factors analysed. Moreover, plate counts performed in MRS plus bile salts evidenced that a considerable percentage of cells suffers damages deriving from cold. This study showed that the growth phase of the cells plays an important role for the resistance to the storage by freezing. Finally, Skim milk had the best protective action, showing the highest activity at −80 °C.

Detection of different microenvironments and Lactobacillus sakei biotypes in Ventricina, a traditional fermented sausage from central Italy

The present study evaluated the physico-chemical and microbiological features of Ventricina, considering for the first time the presence of different compartments deriving from the technology of production. In fact meat pieces (pork muscle and fat cut into cubes of about 10-20cm3), mixed with other ingredients and then stuffed into pig bladder, are still distinguishable at the end of the ripening. They appear delimited on the outside by the casing and inside by thin layers consisting of spices (mainly red pepper powder), salt and meat juices. Our results showed that the exterior (portion of the product in contact with the casing), the interstice (area between the different cubes of meat or fat) and the heart (the inner portion of meat cubes) had distinctive values of pH and aw, and a typical microbial progression, so that they can be considered as different ecological niches, here called microenvironments. The study of lactic acid bacteria population, performed with PCR-DGGE and sequence analysis targeting the V1-V3 region of the 16S rRNA gene (rDNA), highlighted the presence of a few species, including Lactobacillus sakei, Lb. plantarum, Weissella hellenica and Leuconostoc mesenteroides. The RAPD-PCR analysis performed on Lb. sakei, recognised as the predominant species, allowed the differentiation into three biotypes, with that characterised by the highest acidifying and proteolytic activities and the highest ability to grow in the presence of sodium chloride prevailing. This leading biotype, detectable in the interstice during the entire ripening period, was isolated in the microenvironments exterior and heart starting from the 30th d of ripening, and it was the sole biotype present at the end of the ripening. The analysis of microenvironments through the scanning electron microscopy (SEM) evidenced the presence of micro-channels, which could favour the microbial flow from the interstice to the exterior and the heart. Moreover, the SEM analysis allowed the detection of biofilms, recognised as responsible for the correct colonisation of the different meat niches.