Luca Tipaldi

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.

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.

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.

Biodiversity of Lactobacillus plantarum from traditional Italian wines

In this study, 23 samples of traditional wines produced in Southern Italy were subjected to microbiological analyses with the aim to identify and biotype the predominant species of lactic acid bacilli. For this purpose, a multiple approach, consisting in the application of both phenotypic (API 50CHL test) and biomolecular methods (polymerase chain reaction-denaturing gradient gel electrophoresis and 16S rRNA gene sequencing) was used. The results showed that Lactobacillus plantarum was the predominant species, whereas Lb. brevis was detected in lower amount. In detail, out of 80 isolates 58 were ascribable to Lb. plantarum and 22 to Lb. brevis. Randomly amplified polymorphic DNA-polymerase chain reaction was used to highlight intraspecific variability among Lb. plantarum strains. Interestingly, the cluster analysis evidenced a relationship between different biotypes of Lb. plantarum and their origin, in terms of wine variety. Data acquired in this work show the possibility to obtain several malolactic fermentation starter cultures, composed by different Lb. plantarum biotypes, for their proper use in winemaking processes which are distinctive for each wine.

Lactobacillus plantarum 29 Inhibits Penicillium spp. Involved in the Spoilage of Black Truffles (Tuber aestivum)

The effect of an antifungal culture of Lactobacillus plantarum to be used in the storage at refrigeration temperature of fresh black truffles was examined. The strain was selected among 29 lactobacilli isolated from foods and evaluated for their viability and acidification activity at 4 °C, as well as for their inhibitory activity against 11 Penicillium strains isolated from truffles stored at refrigeration temperature. Lb. plantarum 29 showed the ability to hold not only the growth of Penicillium isolated from truffles, but also that of P. digitatum DSM 2750, a green mold involved in the spoilage of truffles. The antifungal activity was observed in vitro and in situ, and the sensory characteristics of truffles were preserved during the cold storage.

Sub-optimal pH Preadaptation Improves the Survival of Lactobacillus plantarum Strains and the Malic Acid Consumption in Wine-Like Medium

Forty-two oenological strains of Lb. plantarum were assessed for their response to ethanol and pH values generally encountered in wines. Strains showed a higher variability in the survival when exposed to low pH (3.5 or 3.0) than when exposed to ethanol (10 or 14%). The study allowed to individuate the highest ethanol concentration (8%) and the lowest pH value (4.0) for the growth of strains, even if the maximum specific growth rate (μmax) resulted significantly reduced by these conditions. Two strains (GT1 and LT11) preadapted to 2% ethanol and cultured up to 14% of ethanol showed a higher growth than those non-preadapted when they were cultivated at 8% of ethanol. The evaluation of the same strains preadapted to low pH values (5.0 and 4.0) and then grown at pH 3.5 or 3.0 showed only for GT1 a sensitive μmax increment when it was cultivated in MRS at pH 3 after a preadaptation to pH 5.0. The survival of GT1 and LT11 was evaluated in Ringers solution at 14% ethanol after a long-term adaptation in MRS with 2% ethanol or in MRS with 2% ethanol acidified at pH 5.0 (both conditions, BC). Analogously, the survival was evaluated at pH 3.5 after a long-term adaptation in MRS at pH 5.0 or in MRS BC. The impact of the physiologic state (exponential phase vs stationary phase) on the survival was also evaluated. Preadapted cells showed the same behavior of non-preadapted cells only when cultures were recovered in the stationary phase. Mathematical functions were individuated for the description of the survival of GT1 and LT11 in MRS at 14% ethanol or at pH 3.5. Finally, a synthetic wine (SW) was used to assess the behavior of Lb. plantarum GT1 and LT11 preadapted in MRS at 2% ethanol or at pH 5.0 or in BC. Only GT1 preadapted to pH 5.0 and collected in the stationary phase showed constant values of microbial counts after incubation for 15 days at 20°C. In addition, after 15 days the L-malic acid resulted completely degraded and the pH value increased of about 0.3 units.

Antimicrobial effect of Malpighia punicifolia and extension of water buffalo steak shelf-life

Abstract In the present study, a multiple approach was used to characterize Malpighia punicifolia extract and to evaluate its inhibitory activity against several meat spoilage bacteria. First, volatile fraction, vitamins and phenolic compounds of the extract obtained by supercritical fluid extraction were determined by GC‐MS and HPLC. Then, the antimicrobial action of the extract was in vitro evaluated against Pseudomonas putida DSMZ 291T, Pseudomonas fluorescens DSMZ 50009T, Pseudomonas fragi DSMZ 3456T, and Brochothrix thermosphacta DSMZ 20171T by the agar well diffusion assay and by the agar dilution test. Based on the results of the minimum inhibitory concentration (MIC) against the assayed bacteria, 4 different concentrations of the extract were used in a challenge test on water buffalo steaks stored for 21 d at 4 °C. Results of chemical analyses showed that M. punicifolia extract is characterized by the presence of several compounds, already described for their antimicrobial (phenolic acids, flavonones, and furanes) and antioxidant (ascorbic acid) properties. The in vitro detection of antimicrobial activities highlighted that the extract, used at 8% concentration, was able to inhibit all the target bacteria. Moreover, very low MIC values (up to 0.025%) were detected. In situ tests, performed on water buffalo steaks treated with the extract in the concentration range 0.025% to 0.05%, showed a strong inhibition of both intentionally inoculated bacteria and naturally occurring microorganisms. Positive results, in terms of color and odor, were also observed during the entire storage of steaks preserved with the extract.

Lactic Acid Bacteria in Pharmaceutical Formulations: Presence and Viability of "Healthy Microorganisms"

Many probiotic formulations are available in the market and are advertised for several preventive or curative roles. The aim of this study was the identification of microorganisms composing different lactic acid bacteria based pharmaceutical formulations and the ascertainment of their ability to survive gastro-intestinal (GI) stresses, the main requisite to produce beneficial effects. For this purpose, viable bacteria were enumerated by plate counts in different media. Denaturing Gradient Gel Electrophoresis-Polymerase Chain Reaction (PCR-DGGE) analysis was applied on pure isolates and on crude formulations to confirm the composition in species. Also, crude formulations were subjected to stresses characteristic of the GI tract (GIT) to assess cell survival. Results highlighted concentrations lower than those reported in the labels in almost all the formulations. Moreover, some discrepancies were observed between reported species and those ascertained through the identification, and the use of an erroneous nomenclature was highlighted. The GI stress test revealed that bacteria are strongly injured, and this fact was evidenced by a marked reduction in viable counts after the stress. In conclusion, a widespread number of lactic acid bacteria based formulations are sold as probiotics, but their probiotic requisites are not adequately observed.