Antimicrobial activity of gallic acid against food-related Pseudomonas strains and its use as biocontrol tool to improve the shelf life of fresh black truffles.

Refrigeration alone or in combination with other technologies represents the main tool used in the last decades to preserve the freshness of black truffles. This is principally due to the delicateness and vulnerability of this edible hypogeous fungus, so that other invasive preservation practices cannot be adopted. However, the proliferation of some microbial species during the cold storage still represents an unsolved problem. Pseudomonads are among the main spoiler bacteria responsible for the deterioration of refrigerated black truffles. Their growth ability at low temperatures requires the use of additional hurdles to prolong the shelf-life of truffles without altering their major features. The use of natural compounds may represent an alternative system for the biocontrol of this kind of product. Specifically, gallic acid (GA) is a phenolic acid naturally present in different foods, whose effectiveness was in vitro demonstrated against Pseudomonas spp. In our study, we reported the antimicrobial activity expressed by GA not only in vitro, using as target bacteria Pseudomonas putida DSMZ 291T, P. fluorescens DSMZ 50090T, P. fragi DSMZ 3456T and Pseudomonas spp. P30-4, previously isolated from black truffles, but also in situ on fresh black truffles stored at 4°C for 28days. Our results showed Minimum Inhibitory Concentrations (MIC) of 2.5mg/mL GA for all tested strains, except for P. fluorescens DSMZ 50090T, having a MIC corresponding to 5mg/mL GA. The Minimum Bactericidal Concentration (MBC) was 10mg/mL for all strains. The analysis of kinetic parameters showed that the survival declined passing from 2.5 to 10mg/mL GA concentrations, with P. fluorescens confirmed to be the most resistant strain. Moreover, images obtained from Scanning Electron Microscopy revealed that Pseudomonas cells were strongly injured by the treatment with GA at 2.5mg/mL concentration, displaying visible pores on the cellular surfaces, absence of flagella and lysis with loss of cytoplasmic material. The storage test performed on fresh black truffles confirmed in situ the GA antimicrobial activity observed in vitro, with a drastic reduction not only of Pseudomonas spp., but also of the other assessed microbial groups, including Enterobacteriaceae and Eumycetes. Finally, sensory analysis established the absence of off-flavours and the preservation of positive features in black truffles treated with 2.5mg/mL GA and stored for 28 d at 4°C. The results obtained in this study suggest that GA is a potential biocontrol tool to decontaminate and preserve fresh black truffles during refrigerated storage.

Pre-cultivation with Selected Prebiotics Enhances the Survival and the Stress Response of Lactobacillus rhamnosus Strains in Simulated Gastrointestinal Transit.

In our study, we dwelled upon combinations of lactobacilli/prebiotics, considering four different strains belonging to the Lactobacillus rhamnosus species, including Lactobacillus rhamnosus GG (LGG), and different prebiotics often found in commercial synbiotic products, such as inulin, lactulose and polyols mannitol and sorbitol. In the first step of the research, the survival, the growth kinetic parameters and the protein expression of Lb. rhamnosus strains cultivated in presence of the different prebiotics as a unique carbon source were evaluated. In the second step, the influence of pre-cultivation in medium added of metabolizable prebiotics on the strains survival to simulated gastrointestinal (GI) transit, assayed without prebiotics addition, was estimated. Our results showed that the presence in the medium of certain low fermented prebiotics, specific for each strain, represents a stress factor that significantly affects the growth of Lb. rhamnosus strains, inducing the up-regulation of several proteins. In detail, all added prebiotics used as unique carbon source caused a growth retard compared with glucose, as testified by increased values of the lag phase and decreased values of the µmax. Mannitol evidenced intermediate µmax values between those registered with glucose and those detected with the other assayed prebiotics. Moreover, the cultivation with prebiotics induced the over expression of 7 protein bands. Interestingly, we found a correlation between the up-regulation of two specific stress proteins, called P4 (ATP-binding subunit Clpx) and P7 (GrpE), and the death kinetic parameters (resistance and cells viability) registered during the simulated GI transit of strains pre-cultivated with specific, low fermented prebiotics. Specifically, the highest resistance and gastric-vitality scores were highlighted for the strain AT195 when pre-cultivated in presence of sorbitol. Conversely, the lowest values were found in the case of DSM20021 pre-cultivated with mannitol. Among the up-regulated stress proteins, P7 resulted involved in the response to the starvation. Finally, it is possible to conclude that the pre-cultivation with certain prebiotics as a unique carbon source represents a strain-specific, sub-lethal stress able to enhance the resistance of Lb. rhamnosus strains and consequently their viability under simulated GI transit.

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 (µ max ) 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 µ max 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 Ringer's 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.

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.

Variability in chemical and microbiological profiles of long-ripened Caciocavallo cheeses.

Five protected designation of origin (PDO) Caciocavallo Silano and 6 non-PDO Caciocavallo cheeses, ripened for 6mo and collected in the 5 Italian regions of the PDO area (Apulia, Basilicata, Calabria, Campania, and Molise, Italy), were studied to assess their physico-chemical (pH, acidity, moisture, fat, ash, protein, and free amino acid composition) and microbiological profiles. Analyses evidenced a certain fluctuation of previous parameters among samples regardless of the kind of cheese evaluated (PDO and non-PDO). The PCR-denaturing gradient gel electrophoresis analysis performed on the DNA directly extracted from cheeses gave different results, but a low number of bands was always observed. Only one band, corresponding to the species Streptococcus thermophilus, was detectable in 1 PDO and in 2 non-PDO cheese samples, whose free amino acid content was the lowest. Analyses were repeated on experimental Caciocavallo cheeses. Specifically, 2 productions were made, one mimicking the industrial technology (pasteurized milk and selected starter culture) and one the artisanal technology (raw milk and natural whey starter). Results obtained on experimental cheeses at 6mo of ripening showed that industrial samples had lower amounts of total free amino acids then the artisanal ones (1,188.2 vs. 7,523.67mg/100 g of dry matter). Moreover, the PCR-denaturing gradient gel electrophoresis analysis evidenced the sole presence of S. thermophilus in the case of the industrial technology. These data sustain the hypothesis that, out of 11 cheeses analyzed previously, 1 PDO Caciocavallo Silano and 2 non-PDO Caciocavallo cheeses were obtained with the industrial technology. These results could be of help in the discrimination of PDO products, taking into account that the PDO production regulation does not allow the milk pasteurization, nor the use of selected starters.

Antimicrobial Effect of Malpighia Punicifolia and Extension of Water Buffalo Steak Shelf-Life.

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 291(T), Pseudomonas fluorescens DSMZ 50009(T), Pseudomonas fragi DSMZ 3456(T), and Brochothrix thermosphacta DSMZ 20171(T) 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.

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/ß-fold with five parallel ß-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 ß-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.

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.

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.

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.

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.