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Listeria monocytogenes is a pathogen frequently found in dairy products, and its growth is difficult to control. Bacteriocinlike inhibitory substances (BLIS), produced by lactic acid bacteria (LAB), having proven in vitro anti-Listeria activity, could provide an innovative approach to control L. monocytogenes; however, this application needs to be evaluated in vivo. In this study, twenty LAB strains isolated from different Sicilian dairy environments were tested for control of growth of L. monocytogenes in three different experimental trials. First, raw and UHT milk were inoculated with LAB strains alone, and LAB strains mixed with L. monocytogenes. Second, mini-cheeses containing LAB and/or L. monocytogenes were produced. Third, two traditional Sicilian cheeses inoculated with a multi-strain LAB mixture combined with L. monocytogenes were produced. The addition of BLIS produced by LAB to milk and in mini-cheese production was unable to inhibit the growth of L. monocytogenes. However, an anti-Listeria effect was observed in the Pecorino Siciliano cheeses, where, after 15 days of ripening, the cheeses with added LAB had fewer L. monocytogenes compared to the control cheeses with no added LAB, while in the Vastedda della valle del Belìce cheeses, the multi-strain LAB mixture completely prevented the growth of L. monocytogenes.

This study aimed to evaluate the levels of enteric bacteria in ice cubes produced in different environments (home-made, prepared in bars and pubs with ice machines and produced in industrial plants) and to determine their survival in different alcoholic beverages and soft drinks. Members of the Enterobacteriaceae family were found in almost all samples analysed. All industrial and the majority of home-made samples did not contain coliforms. Enterococci were not identified in domestic samples while they were detected in two industrial and three bar/pub samples. The samples collected from bars and pubs were characterized by the highest levels of enteric bacteria. Fourteen strains representing 11 species of eight bacterial genera were identified, some of which are known agents of human infections. The most numerous groups included Enterococcus and Stenotrophomonas. The survival of Enterococcus faecium ICE41, Pantoea conspicua ICE80 and Stenotrophomonas maltophilia ICE272, that were detected at the highest levels (100–400 CFU/100 mL thawed ice) in the ice cubes, was tested in six drinks and beverages characterized by different levels of alcohol, CO2, pH and the presence of antibacterial ingredients. The results showed a species-specific behaviour and, in general, a reduction of the microbiological risks associated with ice after its transfer to alcoholic or carbonated beverages.

The irrigation of olive orchards is commonly applied to produce table olives with optimal size. No data have been published on the microbiological quality of drupes from irrigated olive groves during fermentation. The trials T100 and T50 (receiving a water amount equivalent to 100 % and 50 % of the required amount, respectively) and control T0 (rainfed trial) were monitored during two consecutive years. The results showed a significant increase of equatorial diameter and flesh:pit ratio of irrigated drupes. The decrease of pH and the numbers of lactic acid bacteria (LAB) registered for the irrigated trials during the fermentation were more consistent than those displayed by control T0. Lactobacillus pentosus, Lactobacillus plantarum, Lactobacillus coryniformis, and Pediococcus pentosaceus for LAB and Candida boidinii, Candida diddensiae, and Wickerhamomyces anomalus for yeasts were isolated at the highest concentrations. The global sensory acceptance was better for irrigated trials than control T0. The statistical multivariate analysis showed that the effect of irrigation was independent of the seasonal variability and it clearly distinguished the T100 and T50 trials from rainfed control. Irrigation improves significantly the quality of table olives. Interestingly, all explorative multivariate analyses showed low dissimilarity between irrigated trials; thus, the thesis T50 represents an effective approach to save water in olive orchards without compromising the quality of table olives.

Fresh vegetables are recommended for the daily diet because of their low caloric content, high amount of vitamins, minerals and fiber. Furthermore, these foods are source of various phytochemical compounds such as polyphenols, flavonoids and sterols exerting antioxidant activity. Some epidemiological and clinical studies clearly showed that a diet rich in fruits and vegetables might reduce the risk of some diseases, including some forms of cancer. Modern consumers pay a great attention to the “convenience foods”, foods characterized by ease of consumption. In particular, ready-to-eat products maintain almost all characteristics of fresh harvested products. These products are minimally processed and do not undergo further manipulation before consumption. However, ready to eat vegetables might represent a potential danger for the human health due to the high risk of growth of undesirable microorganisms. Bacteria belonging to the species Escherichia coli, Salmonella spp. and Listeria monocytogenes are the most common food pathogens that contaminate fresh vegetables and ready to eat vegetables. These microorganisms constitute one the most important causes of human infections. Besides the contamination deriving from the direct contact with soil, the manure from domestic or wild animals and the inappropriate packaging, the most frequent source of contamination by enteric bacteria is represented by the irrigation water. The application of inappropriate temperatures during storage determine the rapid increase of microbial levels in fresh cut vegetables.

The main aim of this research was to investigate the biopreservative effects of lemon essential oil (EO) micro-emulsions on salted sardines. The experimental design included two experimental trials, SR1 and SR2 carried out with 25 ml of lemon EO micro-emulsion at 0.3 and 1.0% (v/v), respectively, and a control trial performed without EO addition. Chemical analyses on salted sardines inoculated with the EOs clearly showed a substantial persistence of several volatile organic compounds (VOCs) belonging to groups of monoterpene hydrocarbons, oxygenated monoterpenes and sesquiterpene hydrocarbons derived from EOs during the entire period of ripening. In particular, the molecules mostly represented were limonene, p-cymene and (beta-pinene. Immediately after the addition of EOs, the concentrations of all microbial groups decreased. The presence of Enterobacteriaceae, staphylococci and rod lactic acid bacteria (LAB) observed in the trials SR1 and SR2 was significantly lower than that registered for the control. trial during the entire period of monitoring. Furthermore, the addition of EOs determined a lower accumulation of histamine in sardines compared to those of the control trial. The highest scores of sensory evaluation were registered for flavour and overall acceptability of the experimental trials in presence of EOs. On the basis of the increasing interest toward novel food preservatives, we conclude that the use of EOs to produce salted fishes represents a valid strategy to improve safety and sensory characteristics of salted sardines. This work has also economic implications, since the flavour improvement due to the addition of lemon EOs might increase the consumption of sardines by regular and new consumers. (C) 2016 Elsevier Ltd. All rights reserved.

The main objective was to set up a methodology to improve the high volume production of green table olives, cv. Nocellara del Belice. Lactobaccillus pentosus OM13 was applied during three different industrial processes of table olives as follows: trial one (IOP1) was subjected to an addition of lactic acid until a brine level of pH 7.0 was reached; trial two (IOP2) subjected to same addition of lactic acid as in trial one plus nutrient adjuvant; and trial three (IOP3) subjected to same addition of lactic acid as in trial one, but with the strain L. pentosus OM13 acclimatized in brine for 12 h before inoculation. These trials were compared against two untreated controls (spontaneously fermented and addition of L. pentosus OM13 only). Within the third day of fermentation, the pH of the brines decreased significantly, reaching pH 4.85 for trial three, pH 5.15 for trial two, and pH 5.92 for trial one. The pH of both controls decreased more slowly, and had values below pH 5.0 only after the fifteenth day of fermentation (control one) and the sixty-fifth day of fermtation (control two). Trial three reached the highest lactic acid bacteria (LAB) concentration on the third day of fermentation. After six days of fermentation, all trials showed similar values of LAB counts that were significantly higher compared to control number one. The result from genotypic identification showed that L. pentosus OM13 was the most frequently isolated in the inoculated trials. Lactobacillus plantarum, Lactobacillus coryniformis and Pediococcus pentosaceous were also detected at very low concentrations. Homoguaiacol, 2-butanol, 4-ethylphenol, phenylethyl alcohol and 4-ethylphenol were the volatile organic compounds detected at the highest levels in all experimental trials. Trial three showed a higher concentration of squalene that was not detected in other trials. The highest sensory scores of green olive aroma and overall satisfaction were found for all experimental olives, especially for those of trial one and trial two, that differed significantly from the untreated controls. This study provides evidence that the addition of lactic acid, nutrient adjuvants and, most importantly, the acclimatization of LAB cells significantly shortens the acidification process of olive brine, and improves safety and sensory quality. Shorter acidification processes result in a more rapid transformation of table olives, with reduced commodity loss and lower costs of production compared to conventional manufacturing protocols.

The aim of the research was to produce salami manufactured with meat of three different commercial categories of bovine breed: cow on retirement, beef and young bull. A total of six experimental productions, at small-scale plant, were carried out with and without starter culture inoculums. The evolution of physico-chemical parameters in all trials followed the trend already registered for other fermented meat products. Several LAB species were found during process with different levels of species diversity and frequency of isolation among inoculated (mainly Pediococcus pentosaceus and Staphylococcus xylosus) and uninoculated (mainly Enterococcus devriesei, Lactobacillus curvatus and Lactobacillus sakei) trials. Enterobacteriaceae were found at very low levels during the entire ripening period and no pathogenic bacteria were found in any samples. The multivariate analysis showed that starter inoculums and meat affected significantly the physico-chemical and the microbiological composition of salami. The sensory analysis evidenced the highest overall acceptability was displayed by salami produced with meat from cow on retirement.

Accumulation of biogenic amines (BAs) in cheese and other foods is a matter of public health concern. The aim of this study was to identify the enzyme activities responsible for BA degradation in lactic acid bacteria which were previously isolated from traditional Sicilian and Apulian cheeses. The selected strains would control the concentration of BAs during cheese manufacture. First, 431 isolates not showing genes encoding the decarboxylases responsible for BA formation were selected using PCR-based methods. Ninety-four out of the 431 isolates degraded BAs (2-phenylethylamine, cadaverine, histamine, putrescine, spermine, spermidine, tyramine, or tryptamine) during cultivation on chemically defined medium. As shown by random amplification of polymorphic DNA-PCR and partial sequencing of the 16S rRNA gene, 78 of the 94 strains were Lactobacillus species ( Lactobacillus casei , Lb. fermentum , Lb. parabuchneri , Lb. paracasei , Lb. paraplantarum , and Lb. rhamnosus ), Leuconostoc species ( Leuconostoc lactis and Ln. mesenteroides ), Pediococcus pentosaceus , Lactococcus lactis , Streptococcus species ( Streptococcus gallolyticus and S. thermophilus ), Enterococcus lactis , and Weissella paramesenteroides . A multicopper oxidase-hydrolyzing BA was purified from the most active strain, Lb. paracasei subsp. paracasei CB9CT. The gene encoding the multicopper oxidase was sequenced and was also detected in other amine-degrading strains of Lb. fermentum , Lb. paraplantarum , and P. pentosaceus . Lb. paracasei subsp. paracasei CB9CT and another strain (CACIO6CT) of the same species that was able to degrade all the BAs were singly used as adjunct starters for decreasing the concentration of histamine and tyramine in industrial Caciocavallo cheese. The results of this study disclose a feasible strategy for increasing the safety of traditional cheeses while maintaining their typical sensorial traits. IMPORTANCE Because high concentrations of the potentially toxic biogenic amines may be found in traditional/typical cheeses, the safety of these food items should be improved. Lactic acid bacteria selected for the ability to degrade biogenic amines may be used during cheese making to control the concentrations of biogenic amines.

Forty enterococci isolated along the production chains of three traditional cheeses (PDO Pecorino Siciliano, PDO Vastedda della Valle del Belìce, and Caciocavallo Palermitano) made in Sicily (southern Italy) were studied for the assessment of their antibiotic resistance and virulence by a combined phenotypic/genotypic approach. A total of 31 Enterococcus displayed resistance to at least one or more of the antimicrobials tested. The strains exhibited high percentages of resistance to erythromycin (52.5%), ciprofloxacin (35.0%), quinupristin–dalfopristin (20.0%), tetracycline (17.5%), and high-level streptomycin (5.0%). The presence of tet(M), cat(pC221), and aadE genes for resistance to tetracycline, chloramphenicol, and streptomycin, respectively, was registered in all strains with resistance phenotype. The erm(B) gene was not detected in any erythromycin-resistant strain. The Enterococcus strains were further tested by PCR for the presence of virulence genes, namely, gelE, asa1, efaA, ace, and esp. Twenty strains were positive for all virulence genes tested. Among the enterococci isolated from final cheeses, three strains (representing 33.3% of total cheese strains) were sensible to all antimicrobials tested and did not carry any virulence factor. Although this study confirmed that the majority of dairy enterococci are vectors for the dissemination of antimicrobial resistance and virulence genes, only two strains showed a high resistance to aminoglycosides, commonly administered to combat enterococci responsible for human infections. Furthermore, the presence of the strains E. casseliflavus FMAC163, E. durans FMAC134B, and E. faecium PON94 without risk determinants, found at dominating levels over the Enterococcus populations in the processed products, stimulates further investigations for their future applications in cheese making. All strains devoid of the undesired traits were isolated from stretched cheeses. Thus, this cheese typology represents an interesting environment to deepen the studies on the risk/benefit role of enterococci in fermented foods for their qualified presumption of safety (QPS) assessment.

The main hypothesis of this study was that the autochthonous lactic acid bacteria (LAB) selected for their dairy traits are able to stabilize the production of PDO (Protected Denomination of Origin) Pecorino Siciliano cheese, preserving its typicality. The experimental plan included the application of a multi-strain lactic acid bacteria (LAB) culture, composed of starter (Lactococcus lactis subsp. lactis CAG4 and CAG37) and non starter (Enterococcus faecalis PSL71, Lactococcus garviae PSL67 and Streptococcus macedonicus PSL72) strains, during the traditional production of cheese at large scale level in six factories located in different areas of Sicily. The cheese making processes were followed from milk to ripened cheeses and the effects of the added LAB were evaluated on the microbiological, chemico-physical and sensorial characteristics of the final products. Results highlighted a high variability for all investigated parameters and the dominance of LAB cocci in bulk milk samples. The experimental curds showed a faster pH drop than control curds and the levels of LAB estimated in 5-month ripened experimental cheeses (7.59 and 7.27 Log CFU/g for rods and cocci, respectively) were higher than those of control cheeses (7.02 and 6.61 Log CFU/g for rods and cocci, respectively). The comparison of the bacterial isolates by randomly amplified polymorphic DNA (RAPD)-PCR evidenced the dominance of the added starter lactococci over native milk and vat LAB, while the added non starter LAB were found at almost the same levels of the indigenous strains. The sensory evaluation showed that the mixed LAB culture did not influence the majority of the sensory attributes of the cheeses and that each factory produced cheeses with unique characteristics. Finally, the multivariate statistical analysis based on all parameters evaluated on the ripened cheeses showed the dissimilarities and the relationships among cheeses. Thus, the main hypothesis of the work was accepted since the quality parameters of the final cheeses were stabilized, but all cheeses maintained their local typicality.

Background and Aims: Some wineries, in order to promote the growth of yeasts able to ferment grape musts, traditionally produce wines using the ‘pied de cuve’ method. The aim of the present work was to study the performance of fortified pied de cuve (FPdC) prepared by addition of wine. Method and Results: Two FPdCs were prepared with the addition of wine at 1.5 and 3% (v/v) of ethanol to the musts and allowed to spontaneously ferment. The FPdCs were then added to fresh bulk musts in order to accelerate the spontaneous alcoholic fermentation (AF). Interestingly, several Saccharomyces cerevisiae strains isolated during the pied de cuve preparation were detected at the highest concentration throughout AF. The chemical composition of the wines conformed to commercial regulations. The volatile organic compounds (VOCs) were mainly represented by diethyl succinate and ethyl lactate, as well as by hexanol and isoamyl alcohol. Principal component analysis of the chemical parameters, VOCs and sensory data showed that the use of experimental pied de cuve influenced positively the composition of the final wines. Conclusion: The FPdC had no negative impact on the microbiological and chemical composition of wines, but it affected the diversity of the species S. cerevisiae present during AF and the sensory profile of the final wine. Significance of the Study: This study provided evidence that FPdC accelerates spontaneous AF and influences the sensory profile of red wines.

Wine is probably one of the main fermented beverages for which the recognition of the “territoriality” is fundamental for its appreciation. The sensory profile of wine is significantly affected by microbial activities, and indigenous microorganisms may significantly contribute to the expression of wine typicality. The microbial ecology of wines is complex and includes several species and strains of yeasts, bacteria and molds. Several works showed the positive effects of spontaneous fermentations on the quality of wine as a consequence of the growth of different species and/or strains together at high levels. Furthermore, a new style of “natural” winemaking is gaining importance, since the resulting wines are obtained thanks to the action of spontaneous autochthonous agents and the use of chemical addition is not allowed. In this contest, natural winemaking could provide enhanced opportunities for products with unique characters and popularly recognized as typical. The present work reports on microbial ecology and molecular profile characterizing natural large-scale vinifications, and an innovative procedure, named “fortified pied de cuve”, to accelerate the alcoholic fermentation performed spontaneously is also reported. Furthermore, this work reports on how the biotic factors, such as migratory birds, contribute in disseminating of wine-related yeasts over long distances, opening up new fields of research that will allow to unravel connection between wine and environmental factors.

Bacteriocins are antimicrobial proteins produced by bacteria that inhibit with a bactericidal or bacteriostatic mode of action, the growth of other bacteria. Many lactic acid bacteria (LAB) produce a high diversity of different bacteriocins. Bacteriocinogenic LAB are generally recognized as safe (GRAS) and useful to control the frequent development of pathogens and spoilage microorganisms; for this reason they are commonly used as starter cultures in food fermentations. In this study, the authors describe the results of a screening on 699 LAB isolated from wooden vat surfaces, raw milk and traditional Sicilian cheeses, for the production of bacteriocin-like inhibitory substances (BLIS), by comparing two alternative methods. The antagonistic activity of LAB and its proteinaceous nature were evaluated using the spot on the lawn and the well-diffusion assay and the sensitivity to proteolytic (proteinase K, protease B and trypsin), amylolytic (α-amylase) and lipolytic (lipase) enzymes. The indicator strains used were: Listeria monocytogenes, Staphylococcus aureus, E. coli, Salmonella enteritidis. A total of 223 strains (belonged to the species Enterococcus spp., Lactobacillus spp., Pediococcus spp., Streptococcus spp., Leuconostoc spp. and Lactococcus lactis) were found to inhibit the growth of Listeria monocytogenes by using the spot on the lawn method; only 37 of these were confirmed by using the well-diffusion assay. The direct addition of bacteriocin-producing cultures into dairy products can be a more practical and economic option for the improvement of the safety and quality of the final product.

In this work, an ad hoc method to identify and quantify polyphenols from caper berries was developed on high-performance liquid chromatography/electrospray ionisation source/mass spectrometry (HPLC-ESI-MS). The method was applied during fermentation carried out with Lactobacillus pentosus OM13 (Trial S) and without starter (Trial C). A total of five polyphenols were identified. All samples contained high concentrations of rutin. Epicatechin was found in untreated fruits, on the contrary quercetin was detected during fermentation. Trial S was characterised by a more rapid acidification and lower levels of spoilage microorganisms than Trial C. L. pentosus dominated among the microbial community of both trials and the highest biodiversity, in terms of strains, was displayed by Trial C. Aureobasidium pullulans was the only yeast species found. The analytical method proposed allowed a high polyphenolic compound recovery from untreated and processed caper berries in short time. The starter culture reduced the bitter taste of the final product.

Three Lactococcus lactis subsp. cremoris strains were used to develop ad hoc biofilms on the surfaces of virgin wooden vats used for cheese production. Two vats (TZ) were tested under controlled conditions (pilot plant), and two vats (TA) were tested under uncontrolled conditions (industrial plant). In each plant, one vat (TA1 and TZ1) was used for the control, traditional production of PDO Vastedda della Valle del Belìce (Vastedda) cheese, and one (TA2 and TZ2) was used for experimental production performed after lactococcal biofilm activation and the daily addition of a natural whey starter culture (NWSC). Microbiological and scanning electron microscopy analyses showed differences in terms of microbial levels and composition of the neoformed biofilms. The levels of the microbial groups investigated during cheese production showed significant differences between the control trials and between the control and experimental trials, but the differences were not particularly marked between the TA2 and TZ2 productions, which showed the largest numbers of mesophilic lactic acid bacterium (LAB) cocci. LAB populations were characterized phenotypically and genotypically, and 44 dominant strains belonging to 10 species were identified. Direct comparison of the polymorphic profiles of the LAB collected during cheese making showed that the addition of the NWSC reduced their biodiversity. Sensory evaluation showed that the microbial activation of the wooden vats with the multistrain Lactococcus culture generated cheeses with sensory attributes comparable to those of commercial cheese. Thus, neoformed biofilms enable a reduction of microbial variability and stabilize the sensorial attributes of Vastedda cheese.

The influence of the wooden equipment used for the traditional cheese manufacturing from raw milk was evaluated on the variations of chemico-physical characteristics and microbial populations during the ripening of Caciocavallo Palermitano cheese. Milk from two farms (A, extensive; B, intensive) was processed in traditional and standard conditions. Chemical and physical traits of cheeses were affected by the farming system and the cheese making technology, and changed during ripening. Content in NaCl and N soluble was lower, and paste consistency higher in cheese from the extensive farm and traditional technology, whereas ripening increased the N soluble and the paste yellow and consistency. The ripening time decreased the number of all lactic acid bacteria (LAB) groups, except enterococci detected at approximately constant levels (10 4 and 10 5 cfu g −1 for standard and traditional cheeses, respectively), till 120 d of ripening. In all productions, at each ripening time, the levels detected for enterococci were lower than those for the other LAB groups. The canonical discriminant analysis of chemical, physical and microbiological data was able to separate cheeses from different productions and ripening time. The dominant LAB were isolated, phenotypically characterised and grouped, genetically differentiated at strain level and identified. Ten species of LAB were found and the strains detected at the highest levels were Pediococcus acidilactici and Lactobacillus casei . Ten strains, mainly belonging to Lactobacillus rhamnosus and Lactobacillus fermentum showed an antibacterial activity. The comparison of the polymorphic profiles of the LAB strains isolated from the wooden vat with those of the strains collected during maturation, showed the persistence of three enterococci in traditional cheeses, with Enterococcus faecalis found at dominant levels over the Enterococcus population till 120 d; the absence of these strains in the standard productions evidenced the contribution of vat LAB during Caciocavallo Palermitano cheese ripening.

The biofilms of 12 wooden vats used for the production of the traditional stretched cheeses Caciocavallo Palermitano and PDO Vastedda della valle del Belìce were investigated. Salmonella spp. and Listeria monocytogenes were never detected. Total coliforms were at low numbers with Escherichia coli found only in three vats. Coagulase-positive staphylococci (CPS) were below the enumeration limit, whereas lactic acid bacteria (LAB) dominated the surfaces of all vats. In general, the dominance was showed by coccus LAB. Enterococci were estimated at high numbers, but usually between 1 and 2 Log cycles lower than other LAB. LAB populations were investigated at species and strain level and for their technological properties relevant in cheese production. Eighty-five strains were analysed by a polyphasic genetic approach and allotted into 16 species within the genera Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Pediococcus and Streptococcus. Enterococcus faecium was found in all wooden vats and the species most frequently isolated were Enterococcus faecalis, Lactococcus lactis, Leuconostoc mesenteroides, Pediococcus acidilactici and Streptococcus thermophilus. The study of the quantitative data on acidification rate, autolysis kinetics, diacetyl production, antibacterial compound generation and proteolysis by cluster and principal component analysis led to the identification of some strains with promising dairy characteristics. Interestingly, a consistent percentage of LAB was bacteriocin-like inhibitory substances (BLIS) producer. Thus, the microbial biofilms of the wooden vats analysed in this study might contribute actively to the stability of the final cheeses.

The aim of this study was to evaluate the effect of different packaging systems alternatives to vacuum that is commonly applied for PDO Vastedda della Valle del Belice (VdB) cheese, a tipical pasta filata cheese, made with raw ewes’ milk. After production following the traditional cheesemaking protocol, VdB cheeses were packaged as follows: vacuum, paraffin wax and two different Modified Atmosphere Packaging (MAP) consisting of a gas mixture of 70% CO2-30% N2 (MAP1) and 100% N2 (MAP2). Cheeses were stored at 4°C and analyzed (25 g) at time 0 and after 15, 30, 60, 90, 120 and 180 days. All the samples were subjected to microbiological analyses for the evaluation of total mesophilic aerobic bacteria, Enterobacteriaceae, total coliform, Escherichia coli, enterococci, lactic acid bacteria, positive coagulase staphylococci, sulphite reducing anaerobes, Pseudomonas spp., Listeria monocytogenes and Salmonella spp.). Moreover, the physicochemical parameters such as pH, water activity, dry matter, fat, protein, salt, total solids, total and soluble nitrogen and ash content were also determined. Chemical, physical and microbiological data were analyzed by a two factors ANOVA model (GLM procedure of SAS) which included the effect of packaging and time of storage. The results did not show significant differences among chemical and physical parameters of the cheeses subjected to the different packaging during storage. No pathogens and spoilage microbial species/group were detected in any of the samples examined. Lactic acid bacteria reached the highest levels for all samples. Total microbial counts detected for paraffin packaged cheeses were higher than those of the other cheeses, regardless the storage time. This observation was probably due to the higher percentage of oxygen allowed by paraffin respect to the other packaging strategies. For this reason, paraffin was excluded as a possible tool for long term storage of VdB cheese. It was also observed an increase of the total microbial load in all packaging systems after 60 days of storage. Sensory analyses are still ongoing.

Francesca et al (2010) studied the ecology of wine yeasts associated to birds caught in vineyards. The same authors were able to prove that migratory birds might carry living pro-technological yeasts for about 12 hours from the ingestion of inoculated feed (Francesca et al 2012). In subsequent studies, they tried to demonstrate that microorganisms are not only transported for a short period by birds, but microorganisms might be adapted to the specific conditions (body temperature about 42 °C and low pH) of the intestinal tract of birds. Hence, it was demonstrated that the majority of isolates carried by birds are thermotolerant. The most interesting results were the isolation of two new species of thermotolerant yeasts, isolated from birds (Francesca et al 2013, 2014). Presently, the main scope of this work is to investigate an additional number of seven new species of thermotolerant yeasts isolated from migratory birds. Bird’s cloacae were analyzed for the presence of yeasts (Francesca et al 2012). All isolates were subjected to phylogenetic and phenotypic analyses as reported by Francesca et al (2014). Twenty four cultures belonging to the genera Candida and Aureobasidium were isolated from birds. The phylogenetic analysis of D1/D2 domain of 26S and ITS region of 5.8S rRNA genes placed the cultures of Candida and Aureobasidium in new lineages that differed conspicuously from their closest relatives, C. verbasci and A. pullulans, respectively. For our Candida isolates the phenotypic analyses showed several discrepancies in assimilation tests between our cultures and C. verbasci, as well as represent a novel ecological niche of new species of thermotolerant yeasts gathered.

The essential oils (EOs) of green seeds from Daucus carota subsp. maximus growing wild in Pantelleria Island (Sicily, Italy) were characterized. EOs were extracted by steam distillation, examined for their inhibitory properties against food-borne Gram-positive and Gram-negative bacteria and analyzed for the chemical composition by gas chromatography (GC) and mass spectrometry (MS). Undiluted EOs showed a large inhibition spectrum against Gram-positive strains and also vs. Acinetobacter spp. and Stenotrophomonas maltophilia. The minimum inhibition concentration (MIC) was in the range 1.25 – 2.50 μl/ml for the most sensitive strains. The chemical analysis indicated that D. carota subsp. maximus EOs included 34 compounds (five monoterpene hydrocarbons, six oxygenated monoterpenes, 14 sesquiterpene hydrocarbons, four oxygenated sesquiterpenes, camphorene and four other compounds), accounting for 95.48% of the total oil, and that the major chemicals were carotol, β-bisabolene, and isoelemicin.

Three enterococci (WFE3, WFE20 and WFE31) selected as presumptive bacteriocin producers were found to be active against Listeria monocytogenes. In this study, due to their potential industrial/food applications, the three bacterial isolates were extensively characterized. Identification was performed by means of a combined 16S rRNA gene sequencing and multiplex PCR approach, and was confirmed with the sequencing of a partial region of a protein-encoding gene, namely pheS. The three isolates belonged unequivocally to the species Enterococcus mundtii. The randomly amplified polymorphic DNA (RAPD) analysis recognized three distinct strains. The supernatants were mainly active against Listeria spp., but some lactic acid bacteria were also inhibited. The proteinaceous nature of the three supernatants was detected after treatment with proteinase K, protease B and trypsin. The bacteriocins were found to be heat resistant, stable in a large pH range and in presence of ethanol. The bacteriocins were not adsorbed onto the surface of the producer cells and their effect was bactericidal. The production of bacteriocins was higher at neutral pHs and temperatures in the range 30-37. °C. The active supernatants did not show cytotoxicity against human erythrocytes and the three strains were susceptible to the action of common antibiotics. The genetic characterization of the bacteriocin genes showed that all three strains produced mundticin KS. They produced it in five food model systems, sterilized by thermal treatment or filtration, prepared from fresh vegetables, cereals, cheeses, meats and fishes. The in situ anti-listerial activities of the strains WFE3, WFE20 and WFE31 were quantitatively different. © 2013 Elsevier Ltd.

Traditional cheeses made without starter cultures can be characterised by the attribute of instability. The addition of autochthonous starter cultures can ensure stability without compromising the characteristics of the final product. This study aimed to characterise the autochthonous lactic acid bacteria (LAB) population in “Vastedda della valle del Belice” cheeses, which have a protected designation of origin (PDO) status, in order to develop an ad hoc starter culture to be used in its future production. Winter and spring productions were analysed to ensure isolation of specific LAB that had adapted to perform fermentation at low temperatures. Plate counts revealed total microbial numbers nearing 109 CFU.g−1. All of the cheese samples were dominated by coccus-shaped LAB. When enterobacteria were present, their concentrations were at similar levels (3.3–5.6 Log CFU.g−1) in both seasons. All of the colonies that differed in morphological appearance were isolated and differentiated on the basis of phenotypic characteristics and genetic polymorphisms, as analysed by random amplification of polymorphic DNA-polymerase chain reaction. A total of 74 strains were identified and further genotyped by sequencing the 16S rRNA gene, resulting in the identification of 16 LAB species belonging to five genera (Enterococcus, Lactobacillus, Lactococcus, Leuconostoc and Streptococcus). The species most frequently present were Streptococcus gallolyticus subsp. macedonicus, Streptococcus thermophilus, Lactococcus lactis and Leuconostoc mesenteroides. The 74 strains were also investigated in vitro for general dairy parameters such as acidification capacity, diacetyl generation and antibacterial activity. Several strains of the most frequently represented species displayed traits relevant to the production of PDO “Vastedda della valle del Belice”.

β-Lactoglobulin (β-lg) is the major whey milk protein and it represents the main allergen in cow and sheep milk (1). Microbial fermentation produces some proteolytic enzymes and leads to the degradation of milk protein allergens. In this study, the results of a screening on the ability of lactic acid bacteria (LAB) isolated from typical Sicilian cheeses to reduce the β-lg are shown. This screening was carried out through an indirect competitive ELISA. The lactic acid fermentation was found to be suitable for decreasing milk immunoreactivity. Twenty three strains of cheese LAB, belonging to six genera (Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Pediococcus and Streptococcus), were cultured for 48h in their optimal growth medium and, after washing and re-suspension in Ringer's solution, inoculated (1% vol/vol) in UHT milk. After 24h incubation at the optimal growth temperature, all samples were diluted (1:10) in distilled water and analysed by Ridascreen ß-Lactoglobulin kit (r-biopharm-Germany) according to the manufacture's instructions. Un-inoculated UHT milk was used as control. After milk fermentation by LAB, the amount of β-lg decreased in twelve samples with an inhibition rate ranging between 21% and 96%, as compared with unfermented milk. Eleven samples did not show a significant β-lg decrease. The strains that showed a higher capacity to hydrolyze the β-lg were represented by Leuconostoc mesenteroides, followed by Lactococcus lactis subsp. lactis (Figure 1). It is important to observe that two different strains of Leuconostoc mesenteroides give different results. Since β-lg is recognized as the major milk allergen and it is known that its hydrolysis may reduce milk allergenicity (2), this study can contribute to give some help in the development of hypoallergenic milk products. Future studies will be needed to evaluate the efficacy and suitability of the selected LAB useful in the processes of cheese-making.

Vastedda is a pasta filata cheese made from raw ewes’ milk without the addition of starter cultures. Cheese production is carried out in wooden vats characterized by microbial biofilms hosting lactic acid bacteria (LAB) responsible for the acidification of the curds. Vastedda cheese was traditionally produced during the summer season, but it is currently produced throughout the year. In order to minimize production variability, several LAB were isolated from Vastedda PDO cheeses, characterized for their technological potential (Gaglio et al., 2014a) and selected to obtain the best strain combination for the production (Gaglio et al., 2014b). In this study, the multiple strain culture composed of Lactococcus lactis subsp. cremoris PON36, PON153 and PON203 was applied during production carried out at large scale level in a dairy factory with bulk milk from different farms using new chestnut wooden vats [commercial production (CMP)]. The same trials were also carried out in controlled conditions at an experimental dairy factory using milk from only one farm [controlled production (CNP)]. In both CMP and CNP productions were used two new vats, one activated with a whey containing the lactococci selected and one traditionally activated with the whey. In the experimental trials, conducted in the vats activated with the lactococci, the same lactococci were also used as starter culture, while the control productions were performed without the addition of starter. Productions were performed every day for the first 5 d and at 5 d intervals for a month. Microbiological counts and strain recognition performed by randomly amplified polymorphic DNA (RAPD)-PCR demonstrated the ability of the triple Lactococcus combination to form a stable biofilm in the active vats. Cheeses analysed at T0 (soon after production) and after 15 days of refrigerated storage showed the persistence and dominance of the inoculated LAB.

article Twelve lactic acid bacteria (LAB), previously selected in vitro (Gaglio et al., 2014), were evaluated in situ for their potential to act as starter cultures for the continuous four-season production of Vastedda-like cheese, made with raw ewes' milk. The strains belonged to Lactobacillus delbrueckii, Lactococcus lactis subsp. cremoris, Leuconostoc mesenteroides subsp. mesenteroides and Streptococcus thermophilus .L AB werefirst inoculated in multiple-strain combinations on the basis of their optimal growth temperatures in three process conditions which differed for milk treatment and medium for strain development: process 1, growth of strains in the optimal synthetic media and pasteurised milk; process 2, growth of strains in whey based medium (WBM) and pasteurised milk; and process 3, growth of strains in WBM and raw milk. The strains that acidified the curds in short time, as shown by a pH drop, were all mesophilic and were then tested in a single inoculum through process 3. Ran- domly amplified polymorphic DNA (RAPD)-PCR analysis applied to the colonies isolated from the highest dilu- tions of samples confirmed the dominance of the added strains after curd acidification, stretching and storage. After 15 days of refrigerated storage, the decrease in pH values showed an activity of the mesophilic strains at low temperatures, but only Lc. lactis subsp. cremoris PON153, Ln. mesenteroides subsp. mesenteroides PON259 and PON559 increased their number during the 15 days at 7 °C. A sensory evaluation indicated that the cheeses obtained by applying protocol 3 and by inoculation with lactococci are the most similar to the protected denom- ination oforigin(PDO) cheese andreceivedthebest scores bythe judges. Thus,theexperimentalcheesesobtain- ed with raw milk and inoculated with single and multiple combinations of lactococci were subjected to the analysis of the volatile organic compounds (VOCs) carried out by a headspace solid phase microextraction (SPME) technique coupled with gas chromatography with mass spectrometric detection (GC/MS). The domi- nance of lactococci over thermophilic LAB of raw milk was verified during summer production and, based on the combination of VOC profiles and sensory evaluation of the final cheeses, the multi-strain Lactococcus culture resulted in the most suitable starter preparation for the full-year production of Vastedda-like cheese.

Lactic acid bacteria (LAB) were analysed from wheat flours used in traditional bread making throughout Sicily (southern Italy). Plate counts, carried out in three different media commonly used to detect food and sourdough LAB, revealed a maximal LAB concentration of approximately 4.75 Log CFU g−1. Colonies representing various morphological appearances were isolated and differentiated based on phenotypic characteristics and genetic analysis by randomly amplified polymorphic DNA (RAPD)-PCR. Fifty unique strains were identified. Analysis by 16S rRNA gene sequencing grouped the strains into 11 LAB species, which belonged to six genera: Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Pediococcus and Weissella. Weissella cibaria, Lactobacillus plantarum, Leuconostoc pseudomesenteroides and Leuconostoc citreum were the most prevalent species. The strains were not geographically related. Denaturing gradient gel electrophoresis (DGGE) analysis of total DNA of flour was used to provide a more complete understanding of the LAB population; it confirmed the presence of species identified with the culture-dependent approach, but did not reveal the presence of any additional LAB species. Finally, the technological characteristics (acidifying capacity, antimicrobial production, proteolytic activity, organic acid, and volatile organic compound generation) of the 50 LAB strains were investigated. Eleven strains were selected for future in situ applications.

This work was aimed to characterize the autochthonous lactic acid bacteria (LAB) of PDO Vastedda della valle del Belìce cheeses, produced in several dairy factories, for the development of an ad hoc starter culture preparation for the production of this cheese. To this purpose, winter and spring productions were analysed, in order to isolate LAB adapted to perform the fermentation at low temperatures. Plate counts showed the total microbial counts (TMC) till levels of 9 -1almost 10 CFU g and all cheese samples were dominated by coccus LAB. Not all samples were positive for the presence of enterobacteria, but when they were found their concentrations were at similar levels in both seasons. All colonies with different morphological appearance were isolated and differentiated on the basis of phenotypic characteristics and by randomly amplified polymorphic DNA (RAPD)- PCR analysis. A total of 65 strains were considered and subjected to the genotypic analysis by means of 16S rRNA gene sequencing, which identified 13 LAB species belonging to five genera (Enterococcus, Lactobacillus, Lactococcus, Leuconostoc and Streptococcus). The species most frequently found were Streptococcus macedonicus, Streptococcus thermophilus, Lactococcus lactis and Leuconostoc mesenteroides. The 65 strains were investigated in vitro for their general dairy aptitudes and some strains of the above four species showed technological traits relevant to act as starter strains for this cheese production. Among those strains, 12 LAB (Lactobacillus delbrueckii PON79, PON256 and PON405, Lactococcus lactis PON36, PON46 and PON203, Leuconostoc mesenteroides PON169, PON259 and PON559, Streptococcus thermophilus PON3, PON120 and PON261) were used in different combinations [all strains belonging to each species in triple combinations (Lb, lactobacilli; Lc, lactococci; Ln, leunostocs; St, streptococci), all thermophilic strains (Lb-St, lactobacilli and streptococci) and all mesophilic strains (Lc-Ln, lactococci and leuconostocs)] to produce experimental cheese by means of a dairy pilot plant. The different 7bacterial combinations (final concentration of approximately 10 CFU/mL) were tested in different conditions: 1, after growth in the optimal synthetic media, re- suspended in Ringer’s solution and inoculated in pasteurised ewes’ milk; 2, after growth in whey-based medium (WBM) and inoculated in pasteurised ewes’ milk; 3, after growth in WBM and inoculated in raw ewes’ milk. Plate counts and RAPD analysis were applied to monitor the bacterial evolution during the different trials, while pH was measured to follow their acidifying activities. All lactococci and leuconostocs were able to perform the rapid acidification of the curd in winter conditions; the experimentation was carried in February with 10°C as minimum ambient temperature registered in the room where the curd were left to acidify. A sensory evaluation of the resulting cheeses, obtained after stretching of the acidified curds, indicated the cheeses processed with lactococci in single and multiple combinations as those well appreciated by the judges. These cheeses were then subjected to the analysis of the volatile organic compounds carried out by gas chromatography coupled with mass spectrometry (GC/MS) which clearly showed substantial differences with the control cheese.

It is known that microbial interactions are important for the success of food fermentations, as well as for the establishment of the safety of the final products. In this study, we characterized extensively three enterococci (WFE3, WFE20 and WFE31) of flour origin, which were found to be active against Listeria monocytogenes, in order to investigate on their ability to produce bacteriocins in complex food model systems for their future industrial/food applications. The identification of the three bacterial isolates was performed by means of a combined 16S rRNA gene sequencing and multiplex PCR approach. Two isolates belonged unequivocally to the species E. mundtii, while one isolate could not be allotted into any described Enterococcus species. The randomly amplified polymorphic DNA (RAPD) analysis recognized three distinct strains. The supernatants were mainly active against Listeria spp., but some lactic acid bacteria were also inhibited. The proteinaceous nature of the three supernatants was detected after treatment with proteinase K, protease B and trypsin. The bacteriocins were found to be heat resistant, stable in a large pH range and in presence of ethanol. The bacteriocins were not adsorbed onto the surface of the producer cells and their mode of action was bactericidal. The production of bacteriocins was higher at neutral pHs and temperatures in the range 30-37°C. The active supernatants did not show cytotoxicity and the three strains were susceptible to the action of common antibiotics. The genetic characterization of the bacteriocin genes showed that all three strains produced mundticin KS. In order to evaluate the effect of different food components on the inhibitory activity of the E. mundtii strains, five food model systems were prepared from fresh vegetables, cereals, cheeses, meats and fishes. The broths were inoculated first singly with the strains WFE3, WFE20 and 6WFE31 at a final concentration of approximately 10 CFU ml-1. We observed that the three strains produced mundticin KS in all the five food model systems. Then the efficacy of the three E. mundtii strains was evaluated in situ against L. monocytogenes. The five model systems obtained from different food matrices were inoculated singly with one of the bacteriocin producing E. mundtii strains and 4 -1L. monocytogenes ATCC 19114 added at approximately 10 CFU mL . The in situ antilisterial activities of the strains WFE3, WFE20 and WFE31 were quantitatively different.

To evaluate the efficacy of lactic acid bacteria (LAB) to improve the hygienic safety of a traditional raw milk cheese, the raw ewes’ milk protected denomination of origin (PDO) Pecorino Siciliano cheese was used as a model system. Different Pecorino Siciliano curds and cheeses were used as sources of autochthonous LAB subsequently used as starter and non-starter LAB. These were screened for their acidification capacity and autolysis. Starter LAB showing the best performance were genotypically differentiated and identified: two strains of Lactococcus lactis subsp. lactis were selected. From the nonstarter LAB, Enterococcus faecalis, Lactococcus garvieae and Streptococcus macedonicus strains were selected. The five cultures were used in individual or dual inocula to produce experimental cheeses in a dairy factory for which production was characterised by high numbers of undesirable bacteria. At 5- month of ripening, the experimental cheeses produced with LAB were characterised by undetectable levels of enterobacteria and pseudomonads and the typical sensory attributes.