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[EN] The antimicrobial activity of eight essential oils (EOs) extracted from the fruit peel of Citrus genotypes (orange, mandarin and lemon) was evaluated against 76 strains of Listeria monocytogenes, previously isolated from different food matrices. EOs showing the most (EO L2 and EO L8) and least (EO O3 and EO M7) effective inhibition activities were chemically characterized by gas chromatography coupled with mass spectrometry (GC/MS) to compare their composition. EO L2 and EO L8 were chosen to determine the MIC and to evaluate the cell viability of the most sensitive strains (L. monocytogenes LM35 and LM69) after 1, 2, 4 and 6 h of exposure. The effectiveness of chitosan (CH) and methylcellulose (MC) edible films, alone and in combination with EO L2 and EO L8, was determined against LM35 and LM69 at 37 C for 0, 8 and 24 h and at 8 C for 0, 1, 3 and 7 days. In addition, the analysis of the microstructure of the films were performed by scanning electron microscope (SEM) to evidence the interactions between the polymers and EOs. Thirty-five and twenty-nine strains were clearly inhibited by EO L2 and EO L8, respectively, while the other Citrus EOs showed poor (EO M1, O4, O5, O6) or minimal (EO O3 and M7) antimicrobial activity. A total of 36 chemical volatile substances was identified by GC/MS to detect the compounds that might play an important role in the characterization of the EOs. The chemical characterization points to oxygenated monoterpenes as relevant compounds in inhibiting Listeria strains, since they have been detected in lemon EOs in concentrations four/five folds higher than orange EOs. Generally, CH- and MCbased films containing EO L2 and EO L8 showed antilisterial activities, even though, the best performances were observed in case of CH-films at 8 C, with a major reduction up to 3 log (CFU/cm2) in case of EO L2 incorporation. The microstructures observed by SEM suggested a better incorporation of the EOs in CH matrix, where a higher amount of oil droplets was distinguished. Therefore, lemon EOs incorporated into chitosan films could be an efficient tool to control Listeria monocytogenes, especially in refrigerated applied conditions., WR was supported by the "Student Mobility for Placement - SMP" grant of the EU Life Learning Program. The authors thank the "Azienda Sperimentale Palazzelli C.R.A. - Centro di ricerca per l'agrumicoltura e le colture mediterranee Contrada Palazzelli Scordia" (CT, Italy) for providing some of the fruits used for EOs extractions.

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.

Four obligate heterofermentative lactic acid bacteria (LAB) strains (Weissella cibaria PON10030 and PON10032 and Leuconostoc citreum PON 10079 and PON10080) were tested as single strain starters, mono-species dual strain starters, and multiple strain starter for the preparation and propagation of sourdoughs for the production of a typical bread at industrial level. The kinetics of pH and TTA during the daily sourdough refreshments indicated a correct acidification process for all trials. The concentration of lactic and acetic acid increased consistently during fermentation. The resulting molar ratios between these two organic acids in the experimental trials were lower than those observed in the control trial. The microbiological investigation showed levels of approximately 109 CFU/mL in almost all sourdoughs and the comparison of the genetic polymorphisms of the dominating LAB with those of the pure cultures evidenced the persistence of the added strains over time. The resulting breads were evaluated for several quality parameters. The breads with the greatest height were obtained with the quadruple combination of leuconostocs and weissellas. The highest softness was registered for the breads obtained from fermentations performed by W. cibaria PON10032 alone and in combination. The different inocula influenced also the color, the void fraction, the cell density and the mean cell area of the breads. Different levels of acids, alcohols, aldehydes, esters, hydrocarbons, ketones, terpenes, furans and phenol were emitted by the breads. The sensory tests indicated the breads from the sourdoughs fermented with the seven LAB inocula as sweeter and less acidic than control breads and the breads from the trials with the highest complexity of LAB inoculums were those more appreciated by tasters. A multivariate approach found strong differences among the trials. In particular, control breads and the breads obtained with different starter LAB were quite distant and a more strict relation was found among the productions carried out by W. cibaria strains. This study proved the suitability of the selected strains of L. citreum and W. cibaria for industrial-scale level applications in sourdough bread production.

Proper management of soil fertility requires specific tools for predicting N availability for crops as a consequence of different fertilization strategies. More information is required, especially for organic fertilizers, depending on their mineralization rate, composition, and processing (i.e., fresh or composted manure), as well as their effects on soil properties. Laboratory soil incubations were used as a proxy for understanding plant–soil N dynamics under field conditions. Chemical and microbiological measurements as contents of mineral N, potentially mineralizable N and the abundance of key genes regulating the overall N cycle were used as predictors of mineral N availability to maize in two contrasting pedoclimatic conditions. Our results showed that there was a good correlation between chemical and microbiological measurements from laboratory soil incubation experiments and soil–plant N dynamics of maize cropping systems. Mineralization patterns from soil incubation proved to be useful for optimizing fertilization management of maize under field conditions as long as incubation time is normalized over maize growth cycle, according to the simplified model of growth degree days. Average cumulative soil mineral N values calculated over a short incubation period (42 days) showed a significant correlation (R2 = 0.72, p

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.

During a survey of yeasts vectored by migratory birds in the Mediterranean basin, isolations from the cloacae of members of the order Passeriformes collected in Ustica (Italy) were performed. Based on phylogenetic analysis of the D1/D2 domain of the 26S rRNA gene and the internal transcribed spacer ITS1-5.8S rRNA gene-ITS2 region, five yeast isolates clustered in a new lineage within the Microstromatales clade. The DNA sequences of these isolates differed from those of their closest relatives, Jaminaea angkorensis and Jaminaea lanaiensis, by 20 and 25 nt substitutions in the D1/D2 domain and 119 and 131 nt substitutions in the complete ITS region, respectively. In addition, the five isolates showed phenotypic characteristics not observed in their closest relatives, such as the ability to grow at 44 °C and at pH 2.5, which suggests a possible adaptation to the bird gastrointestinal tract. On the basis of the isolation source, phenotypic features and molecular strain typing carried out with randomly amplified polymorphic DNA (RAPD)-PCR and mini-satellite-primed (MSP)-PCR analysis, the five isolates were characterized as five distinct strains of a novel species formally described as Jaminaea phylloscopi sp. nov., with 551B6T ( = PYCC 6783T = CBS 14087T) as the type strain. The Mycobank accession number is MB811984.

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.

Twenty Apulian and Sicilian cheeses were analysed for their concentrations of eight biogenic amines (BAs), free amino acids, pH, water activity, and subjected to microbiological characterisation. In addition, lactic acid bacteria isolated from cheeses were assayed for their capacity to generate BAs. Principal component analysis was performed to find the effect of different parameters on the distribution of the cheeses. Although short-ripened (≤30 d) cheeses did not show significant BA concentrations, the only BA showing high positive correlation with time of ripening was histamine. Concentration of histidine and, especially, percentage of histidine-decarboxylase bacteria presumably affected histamine concentration. High pH values were negatively correlated to the concentration of tyramine, putrescine, and cadaverine. Fifty percent of the cheeses contained at least one BA at potentially toxic concentrations. Unambiguous and ever-valid relations among parameters and BAs are difficult to determine, because BAs are the result of combined and varied factors.

Fifteen sourdoughs produced in western Sicily (southern Italy) were analysed by classical methods for their chemico-physical characteristics and the levels of lactic acid bacteria (LAB). pH and total titratable acidity (TTA) were mostly in the range commonly reported for similar products produced in Italy, but the fermentation quotient (FQ) of the majority of samples was above 4.0, due to the low concentration of acetic acid estimated by high performance liquid chromatography (HPLC). Specific counts of LAB showed levels higher than 10(8) CFU g(-1) for many samples. The colonies representing various morphologies were isolated and, after the differentiation based on phenotypic characteristics, divided into 10 groups. The most numerous group was composed of facultative heterofermentative isolates, indicating a relevance of this bacterial group during fermentation. The genetic analysis by randomly amplified polymorphic DNA (RAPD)-PCR, 16S rRNA gene sequencing and species-specific PCRs identified 33 strains as Lactobacillus plantarum, Lactobacillus curvatus and Lactobacillus graminis. Due to the consistent presence of L. plantarum, it was concluded that this species codominates with obligate heterofermentative LAB in sourdough production in this geographical area. In order to evaluate the performances at the basis of their fitness, the 29 L. plantarum strains were investigated for several technological traits. Twelve cultures showed good acidifying abilities in vitro and L. plantarum PON100148 produced the highest concentrations of organic acids. Eleven strains were positive for extracellular protease activity. Bacteriocin-like inhibitory substances (BLIS) production and antifungal activity was scored positive for several strains, included L. plantarum PON100148 which was selected as starter for experimental sourdough production. The characteristics of the sourdoughs and the resulting breads indicated that the best productions were obtained in presence of L. plantarum PON100148.

The technology of “pied de cuve” (PdC) is applied in food process only to produce wines with an enriched community of pro-technological yeasts. PdC promotes the growth of the desirable microbial strains in a small volume of grape must acting as a starter inoculums for higher volumes. The aim of the present work was to investigate the use of partially fermented brines, a technology known as PdC, developed with lactic acid bacteria (LAB) on the microbiological, chemical and sensory characteristics of green fermented table olives during two consecutive campaigns. The experimental plan included two trials based on different PdCs: trial A, PdC obtained with Lactobacillus pentosus OM13; trial B, PdC obtained through a spontaneous fermentation. Two control additional trials without PdC were included for comparison: trial C, spontaneous fermentation; trial D, direct inoculation of L. pentosus OM13. The use of PdCs favoured the rapid increase of LAB concentrations in both trials A and B. These trials showed levels of LAB higher than trial C and almost superimposable to that of trial D. Trial B was characterized by a certain diversity of L. pentosus strains and some of them dominated the manufacturing process. These results indicated PdC as a valuable method to favour the growth of autochthonous L. pentosus strains. Hierarchical cluster analysis (HCA) and principal component analysis (PCA) visibly discriminated olive processes fermented with the two experimental PdCs. Interestingly, on the basis of microbial and pH variables, both approaches showed that the olives produced with PdC technology are closely related to those of trial D, with the advantages of reducing the amount of starter to inoculate (trial A) and a higher LAB biodiversity (trial B). Volatile organic compound (VOC) composition and sensory analysis showed trials A and B different from the trials with no PdC added, in both years. Furthermore, the trial B showed the highest scores of green olive aroma and taste complexity. Spoilage microorganisms were estimated at very low levels in all trials. Undesired off-odours and off-flavours were not revealed at the end of the process.

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.

This study was conducted to characterize the Bacillus populations associated with dead Rhynchophorus ferrugineus, to develop a biological control for the red palm weevil. Dead adult beetles, collected throughout Sicily, were used for isolating internal and external spore forming bacteria (SFB) microbiota. The isolates, preliminarily allotted to the Bacillaceae family, were tested at 4 concentrations (103 to 106 CFU/mL) for their ability to inhibit hatching of eggs of R. ferrugineus and were used at 106 CFU/mL to monitor their insecticidal activity against 10 day-old larvae. Total amounts of SFB measured outside the skeleton and in the inners part of the beetles were 5.59-6.94 and 5.17-7.05 Log CFU/g, respectively. Hatching was inhibited markedly by 9 isolates, representing 9 distinct strains of 7 species (Bacillus amyloliquefaciens, Bacillus cereus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus subtilis, and Lysinibacillus sphaericus), especially by the strains B. pumilus GC43 and GC51, which exhibited lethal concentrations 50 (LC50) values of 1.60 × 103 and 9.84 × 103 CFU/mL, respectively. Among all the strains tested, only B. licheniformis CG62 exhibited significant insecticidal activity against red palm weevil larvae. The Bacillus isolates characterized and tested in this study inhibited the hatching of red palm weevils in a contact-dependent manner. Thus, these isolates can be used as a preventive rather than as a curative treatment. Keywords Bacillus, Rhynchophorus ferrugineus, hatching assays, larvae, Palm

The present work was undertaken to evaluate the effect of natural wine making on the microbial and chemical composition of Marsala base wine. To this purpose, a large-scale vinification process of Grillo grape cultivar was monitored from the grape harvest to the final product. Total yeasts (TY) showed a rapid increase after must pressings and reached values that were almost identical to those registered during conventional wine makings. Lactic acid bacteria (LAB) were registered at the highest levels simultaneous to those of yeast growth at the beginning of the process. Saccharomyces cerevisiae was the species found at the highest concentrations in all samples analysed. Several strains (n = 16) was registered at high levels during the alcoholic fermentation (AF) and/or aging of the wine; only two of them were detected on the grape surface. Lactobacillus plantarum was the LAB species most frequently isolated during the entire vinification process. The ethanol content was approximately 14 % (v/v) at the end of vinification. The pH value did not greatly vary during the process, and the volatile acidity (VA) was detected at low concentrations during the entire transformation. The concentration of malic acid rapidly decreased during the AF; on the other hand, lactic acid showed an irregular trend during the entire process. Trans-caffeoyl tartaric acid was the most abundant hydroxycinnamoyl tartaric acid, and volatile organic compounds (VOC) were mainly represented by isoamylic alcohol and isobutanol.

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.

Obiettivo di questa ricerca . stato validare le attitudini tecnologiche alla trasformazione di olive da mensa cv. “Nocellara del Belice” da parte del ceppo Lactobacillus pentosus OM13. Lo starter Lb. pentosus OM13 ha dimostrato di dominare l’intero processo fermentativo e ha determinato un sensibile abbassamento del pH, inibendo cos. lo sviluppo di microrganismi potenzialmente patogeni e/o alterativi. All’inizio della sperimentazione e fino al 16¢X giorno di fermentazione, i lieviti hanno mostrato un incremento in entrambe le lavorazioni, con starter e di controllo, ma con un livello di carica microbica maggiore nella tesi di controllo. Nella fase di sperimentazione, successiva al 16¢X giorno, i lieviti hanno raggiunto concentrazioni pressoché costanti. I composti organici volatili hanno presentato una maggiore concentrazione nelle olive del controllo rispetto a quelle sperimentali. In definitiva, l’uso dello starter ha garantito un’adeguata stabilità microbiologica e chimico-fisica e ha potenzialmente incrementato le proprietà salutistiche del prodotto finito.

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”.

Risulta comprovata nell’industria agroalimentare l’efficacia di oli essenziali di agrumi quale tecnologia per esaltare la sapidità di cibi e bevande. Ad oggi, il rinnovato interesse verso gli oli essenziali deriva dalle loro proprietà antibatteriche ed antifungine, che possono trovare ampia applicazione in sistemi hurdle-technology ai fini di prolungare la shelf-life di agroprodotti, specie in cibi minimally-processed e di IV gamma. Infatti, la necessità di ricercare nuove sostanze non di sintesi con proprietà antimicrobiche deriva dai cambiamenti legislativi in campo alimentare, dalla tendenza dei consumatori ad acquistare cibi processati naturalmente e dalla crescente resistenza dei patogeni alimentari ai comuni composti antimicrobici usati nel comparto alimentare. Gli EOs sono sostanze “generalmente riconosciute come sicure” (“generally recognized as safe”, GRAS) dalla U.S. FDA, per cui il loro potenziale utilizzo negli alimenti soddisfa non solo i requisiti legali, le esigenze dell’industria di trasformazione ma anche la richiesta dei consumatori. L’applicazione di tali biotecnologie, però, non può prescindere dalla caratterizzazione preliminare delle proprietà chimiche ed antimicrobiche degli oli essenziali. A tal fine, sono stati estratti gli oli essenziali (EOs) presenti nelle bucce di agrumi appartenenti a tre specie differenti del genere Citrus. Gli EOs sono stati caratterizzati mediante Gas-Cromatografia accoppiata a Spettrometria di Massa (GC-MS) ed identificati per confronto con libreria NIST, nel caso in cui mostravano una similarità maggiore del 90%. Per valutare l’attività antimicrobica, gli EOs sono stati provati in-vitro tramite un saggio di diffusione in pozzetto. A tal fine, gli EOs estratti sono stati testati contro 47 ceppi fungini alterativi e/o patogeni, precedentemente isolati da matrici alimentari. Gli EOs di limone hanno dimostrato possedere in vitro un’attività inibente della crescita fungina nettamente maggiore confrontata con gli oli estratti da mandarino ed arancia. La differente composizione chimica, evidenziata tramite GC/MS, è responsabile della diversa attività antifungina. In particolare, la presenza di idrocarburi monoterpenici ossigenati e sequiterpeni negli EOs con maggiore attività inibente (EO L1 e EO L2), suggerisce che siano proprio queste classi di composti a possedere le proprietà antifungine evidenziate nei test microbiologici.

In order to investigate the seasonal variations of antimicrobial properties and chemical composition of essential oils (EOs), three different cultivars of Citrus limon L. Burm. spp. (Femminello Santa Teresa, Monachello and Femminello Continella) were collected at 6-week intervals, from December 2012 to April 2013, for a total of four harvests. The EOs were extracted from lemon peel by hydro-distillation. The antimicrobial activity, tested by paper disc diffusion method, was evaluated against common food-related pathogenic bacteria (Listeria monocytogenes, Staphylococcus aureus, Salmonella enterica and Enterobacter spp.). EOs were more effective against Gram-positive than Gram-negative bacteria at each collection time, but a strong strain dependence was evidenced. Monachello EOs showed the highest inhibition power. The chemical characterisation of the EOs performed by gas chromatography/mass spectrometry identified from 36 to 42 molecules. The chemical difference registered among samples and seasons may explain the different antimicrobial efficacies recorded.

The microbial composition of artisan and industrial animal rennet pastes was studied by using both culture-dependent and -independent approaches. Pyrosequencing targeting the 16S rRNA gene allowed to identify 361 operational taxonomic units (OTUs) to the genus/species level. Among lactic acid bacteria (LAB), Streptococcus thermophilus and some lactobacilli, mainly Lactobacillus crispatus and Lactobacillus reuteri , were the most abundant species, with differences among the samples. Twelve groups of microorganisms were targeted by viable plate counts revealing a dominance of mesophilic cocci. All rennets were able to acidify ultrahigh-temperature-processed (UHT) milk as shown by pH and total titratable acidity (TTA). Presumptive LAB isolated at the highest dilutions of acidified milks were phenotypically characterized, grouped, differentiated at the strain level by randomly amplified polymorphic DNA (RAPD)-PCR analysis, and subjected to 16S rRNA gene sequencing. Only 18 strains were clearly identified at the species level, as Enterococcus casseliflavus , Enterococcus faecium , Enterococcus faecalis , Enterococcus lactis , Lactobacillus delbrueckii , and Streptococcus thermophilus , while the other strains, all belonging to the genus Enterococcus , could not be allotted into any previously described species. The phylogenetic analysis showed that these strains might represent different unknown species. All strains were evaluated for their dairy technological performances. All isolates produced diacetyl, and 10 of them produced a rapid pH drop in milk, but only 3 isolates were also autolytic. This work showed that animal rennet pastes can be sources of LAB, mainly enterococci, that might contribute to the microbial diversity associated with dairy productions.

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.

Cereals represent the main crop in Africa (45% of arable land) and make an important contribution to the human diet in this continent. Cereals for humans are not consumed raw. Fermentation technology is widely used in Africa to transform cereals into edible products as well as to preserve and enhance the nutritional and safety aspects of cereals. In general, in Africa, this process is not controlled and is devoid of good manufacturing practices (GMPs). Spoilage and/or pathogenic microorganisms may compromise the final quality when the fermentation is not controlled. This article focuses on the role that the fermentation process could play in creating safe food conditions in Africa. Taking into account the increasing demand for cereals, in particular maize and rice, which contain undesired microorganisms, fermentation can play an important role in creating food security and safety. Here, the authors report a detailed analysis of the cereal market, the main transformation processes used to obtain a variety of products from cereals, the most current information on the microbial ecology of the most important traditional fermented cereals, and the safety aspects of and the technological parameters for the selection of the strains to be used as starters for African cereal-based fermented products.

In this study, the composition of the cultivable microbial populations of 38 nectar honey and honeydew honey samples of different botanical and geographical origin were assessed. After growth in specific media, various colonies with different appearance were isolated and purified before phenotypic (morphological, physiological and biochemical traits) and genotypic [randomly amplified polymorphic DNA (RAPD), repetitive DNA elements-PCR (rep-PCR) and restriction fragment length polymorphism (RFLP)] differentiation. The identification was carried out by 16S rRNA gene sequencing for bacteria and, in addition to RFLP, by sequencing the D1/D2 region of the 26S rRNA gene for yeasts and the 5.8S-ITS rRNA region for filamentous fungi. The results showed the presence of 13 species of bacteria, 5 of yeasts and 17 of filamentous fungi; the species most frequently isolated were Bacillus amyloliquefaciens, Zygosaccharomyces mellis and Aspergillus niger for the three microbial groups, respectively. The highest microbial diversity was found in multifloral honeys. No correlation among the microbial species and the botanical/geographical origin was found, but some strains were highly adapted to these matrices since they were found in several samples of different origin.

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.

Nine yeast species belonging to genera Candida , Cryptococcus , Phaffomyces , Rhodotorula and Wickerhamomyces , and one species of Aureobasidium genus were isolated from the cloaca of migratory birds. Candida glabrata and C. inconspicua were the species most frequently isolated and Wickerhamomyces sylviae , which has recently been described as a new species isolated from bird cloaca, was again found. The majority of isolates showed the ability to grow up to 40 °C and/or at pH 3.0, two environmental conditions typical of the digestive tract of birds. The phylogenetic analysis of the D1/D2 domain of 26S rRNA gene placed the cultures of Phaffomyces in a new lineage that differed from the closest species, P. opuntiae , by 13 nucleotide substitutions. The new species was able to grow at 40 °C and at pH 2.5, which suggests a possible adaptation to the bird cloaca. Moreover, the ability to grow in the presence of digitonin at pH 3.7 and the assimilation of ethyl acetate indicates a potential cactophilic origin. For the first time, the presence of yeasts belonging to the Phaffomyces clade in Europe and also in non-cactus environments is reported. The new species is formally described as P. usticensis sp. nov. (PYCC 6346T = CBS 12958T).

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.

In the present work, we investigated the phylogenetic position and phenotypic characteristics of eight yeast isolates collected from migratory birds on the island of Ustica, Italy. A phylogenetic analysis based on the D1/D2 region of the large-subunit rRNA gene showed that all isolates clustered as a single separate lineage within the Wickerhamomyces clade. They exhibited distinct morphological and physiological characteristics and were clearly separated from their closest relatives, Wickerhamomyces lynferdii, Wickerhamomyces anomalus and Wickerhamomyces subpelliculosus, in blastn searches. On the basis of the isolation source, physiological features and molecular strain typing carried out with randomly amplified polymorphic DNA (RAPD)-PCR and minisatellite-primed (MSP)-PCR analysis, the isolates were identified as strains of the same species. The name Wickerhamomyces sylviae f.a., sp. nov. is proposed to accommodate these novel strains; the type strain is U88A2T ( = PYCC 6345T = CBS 12888T). The MycoBank number is MB 804762.

The aim of the present work was to investigate the microbiological, chemical, and sensory characteristics of red wine subjected to post-fermentation maceration that was extended to 90 days. For this purpose, the ‘Aglianico di Taurasi’ grape was used as a case study. The total yeast concentration increased until day 40 of maceration and decreased thereafter, whereas the concentration of lactic acid bacteria slightly increased. Dekkera/Brettanomyces spp. and acetic acid bacteria were not detected. The yeast community was composed of Saccharomyces cerevisiae, Zygosaccharomyces bisporus, Metschnikowia pulcherrima, Hanseniaspora guilliermondii, Hanseniaspora uvarum, Pichia guilliermondii, Aureobasidium pullulans and Debaryomyces carsonii. Nine S. cerevisiae strains were detected at high levels at different times of maceration. The results of all the conventional chemical analyses of the wines were in agreement with the regulations of commercial production and, interestingly, the changes in terms of concentration demonstrated the presence of yeast and LAB populations that were not only alive but also in a metabolically active state until day 90 of maceration. The alcohol and glycerol contents slightly increased until day 90. The concentrations of malic acid decreased, whereas those of lactic acid slightly increased throughout the maceration process. Furthermore, different durations of maceration resulted in significant differences in the total polyphenol content, which was higher at 40–50 days. The main phenolic compounds were benzoic and cinnamic acids and catechins. Interestingly, the highest ratio between (+)-catechin and (−)-epicatechin was found on day 40. In addition, the highest antioxidant activity was observed between days 40 and 50. The concentration of volatile organic compounds, which were mainly represented by alcohols, increased until the end of the maceration process. Sensory analysis revealed that samples that were subjected to maceration for a long period of time showed the highest odour and taste complexity and no off-odours and/or off-flavours were detected. These data confirmed that extending post-fermentation maceration to 90 days has no negative impact on the microbiological, chemical and sensory composition of wines, but affects the polyphenol content and potential health benefits of the resulting wine.

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.

Il presente lavoro ha previsto inizialmente l’isolamento, la tipizzazione e l’identificazione dei batteri lattici (BL) presenti in campioni di farine utilizzate per prodotti da forno a lievitazione naturale prelevati presso diversi panifici siciliani. Le conte in piastra dei BL sono state ottenute su tre differenti terreni colturali, generici e specifici. I risultati hanno evidenziato una concentrazione massima di BL di 4.75 Log UFC/g. Tutte le colonie diverse sono state isolate e purificate. Le colture sono state differenziate sulla base delle caratteristiche fenotipiche e dei profili generati mediante la tecnica randomly amplified polymorphic DNA (RAPD)-PCR che ha permesso il riconoscimento di 50 diversi ceppi. L’identificazione genotipica, condotta mediante il sequenziamento del gene della sub-unità ribosomiale 16S, ha individuato 11 specie di BL appartenenti a sei generi. Le specie più frequentemente riscontrate sono state Weissella cibaria, Lactobacillus plantarum, Leuconostoc citreum e Leuconostoc pseudomesenteroides. La tecnica denaturing gradient gel electrophoresis (DGGE) ha confermato la presenza delle specie di BL isolati precedentemente in uno stato vitale e coltivabile. Tutti i 50 ceppi di BL sono stati testati in vitro per la capacità di acidificare un terreno sintetico ottenuto con estratto di cereali e 11 ceppi sono risultati tecnologicamente rilevanti per le applicazioni in situ. Tali ceppi sono stati testati individualmente in impasti (DY = 160) ottenuti con la farina sottoposta a trattamento ai raggi γ e, successivamente, in farina non trattata. I dati ottenuti dagli impasti prima [conta in piastra, pH, acidi organici, perdita di peso, e componente organica volatile (COV)] e dopo (colore, altezza, volume, consistenza, analisi dell’immagine, acidi organici e COV) della cottura hanno determinato l’idoneità dei ceppi W. cibaria PON10030, PON10032 e PON100337, Ln. citreum PON10079 e PON10080 per l’applicazione su scala industriale.

Two traditional sausage products (“salsiccia” and “salame”) processed from the raw meat of the Black Sicilian swine “Suino Nero dei Nebrodi” were microbiologically investigated during the manufacturing and ripening stages. Both products were dominated by lactic acid bacteria (LAB), especially rod-shaped types. The concentration of enterococci was consistent in salame. Coagulase-negative cocci increased slower than LAB. Yeasts showed an increasing trend during the ripening of both products. Enterobacteriaceae were counted at a constant level of about 105 CFU/g in both products, while pseudomonads diminished during ripening. Coagulase-positive staphylococci, Listeria monocytogenes and Salmonella spp. were not detected at the end of the ripening process. Characterisation of LAB at the strain and species level revealed that Lactococcus lactis was found only in the meat mixture, while Lactobacillus sakei and various enterococci persisted during the monitoring period. Some LAB strains isolated from sausages were also identified on the surface of the factory equipment. Two strains (Lactobacillus sakei SS106A and Enterococcus faecalis SS91) were characterised by their anti-Listeria properties due to bacteriocin-like inhibitory substance production. A multiple strain starter composed of Lactobacillus sakei and enterococci has been proposed to maintain the typical characteristics of the two fermented meat products microbiologically investigated in this study.

I microrganismi caratteristici di un prodotto tipico fermentato come il vino, dai quali dipendono molte delle proprietà organolettiche, riflettono, in molti casi, le caratteristiche dell’ambiente e dei sistemi di produzione. Infatti, i prodotti alimentari di nicchia legano la loro storia alla zona di produzione non solo per le tradizioni che si tramandano nel tempo, ma anche e soprattutto per la presenza di specie e ceppi di microrganismi che colonizzano la materia prima, nonché gli ambienti e le attrezzature di lavorazione, contribuendo in maniera decisiva a caratterizzare la tipicità del prodotto finale. Nello spirito delle “Denominazioni d’Origine”, particolare importanza deve essere ancora attribuita a tutti quegli elementi che creano il “legame” tra un determinato prodotto e una particolare zona geografica, la cultura della popolazione e tutti quegli aspetti che possono essere sintetizzati nell’insieme dei fattori storico-sociali nei quali risiedono i motivi dello sviluppo di un prodotto in una data area antropizzata. I microrganismi rientrano sicuramente tra questi fattori e costituiscono, anzi, uno dei “segreti” dei produttori artigianali che, nel tempo, sono stati svelati e convertiti in moderna tecnologia di trasformazione. Anche l’impiego di ceppi “autoctoni”, selezionati durante la trasformazione di prodotti tradizionali e legati allo sviluppo di componenti aromatiche desiderate, al prolungamento della shelf-life, agli aspetti salutistici dei prodotti finiti e cosìvia, può richiedere l’adattamento di protocolli produttivi già esistenti per migliorare le performance del(i) microrganismo(i). Tuttavia, tale innovazione non può essere “selvaggia”, bensì dovrebbe essere rispettosa degli aspetti essenziali e peculiari della tradizione di un prodotto. In questo lavoro svolto nell'areale del Fiano di Avellino DOCG si sono selezionati dall'ambiente vigneto 2 ceppi di Saccharomyces cerevisiae da utilizzare come starter nella produzione di qualità del vino Fiano

Il mandarino denominato “Tardivo di Ciaculli” ha avuto origine e viene prodotto in provincia di Palermo (Sicilia) nei pressi della contrada Ciaculli. Ha un’epoca di maturazione tardiva che si concentra nei mesi da febbraio ad aprile. I frutti sono piccoli e con buccia di medio spessore [1]. In generale gli oli essenziali di mandarino sono particolarmente apprezzati per le loro proprietà chimico-fisiche costituendo un fondamentale ingrediente nell’industria cosmetica e alimentare come esaltatori di sapore in cibi e bevande. Inoltre trovano un impiego sempre maggiore nelle biotecnologie applicate al settore agroalimentare per ottimizzare le proprietà nutrizionali degli alimenti e prolungarne la shelf life [2]. Nel presente lavoro è stata caratterizzata, mediante Gas Cromatografia accoppiata alla Spettrometria di Massa (GC-MS), la componente volatile dell’olio essenziale estratto dalle bucce dei frutti di Mandarino Tardivo di Ciaculli. Il pattern aromatico è stato poi confrontato con quello di un olio di mandarino commerciale. Sono state effettuate inoltre delle prove preliminari dell’attività antimicrobica sulla crescita di ceppi batterici Gram-positivi (Staphylococcus aureus e Listeria monocytogenes) e Gram-negativi (Salmonella enterica e Enterobacter hormaechei), mediante il metodo della “diffusione su disco di carta”. Per quanto riguarda le molecole volatili identificate, i risultati ottenuti presentano una significativa differenza nella classe dei monoterpeni ossigenati, con risaputa attività antimicrobica [4]. L’inibizione è risultata essere maggiore per i batteri Gram-positivi, in particolare per Listeria monocytogenes, rispetto ai Gram-negativi. L’olio essenziale di Mandarino Tardivo di Ciaculli potrebbe quindi rappresentare una buona alternativa all’utilizzo di additivi sintetici in campo alimentare.

The application of heat treatment is especially important in maintaining the organoleptic characteristics of fermented agro-food products, which potential consumers perceive as reflecting the quality of these foodstuffs. The aim of this study was to evaluate the influence of pasteurization time and temperature on the flesh firmness of table olives cv. Nocellara del Belice, packaged in glass jars, and to study their effect on the microbial community of table olives. Four different treatments were applied, one for each of the pasteurization temperatures adopted: 65 °C, 75 °C, 85 °C and 95 °C. A control test was also carried out (test Tc, not pasteurized). Each test was repeated with treatments lasting 4, 8 and 12 minutes. In order to determine the mechanical properties of the olives, all of the batches were subsequently subjected to a laboratory compression test using a dynamometer (Imada DPS 5R - USA) connected to a stand and a PC for data download. The compression force was recorded continuously along the entire pulp sample. Microbiological analyses were performed on olives and brines immediately after the heat treatment (t0) and after 2 months (t1). The results obtained here show that it is appropriate to employ pasteurization temperatures at least equal to 75 °C for a time not lower than 8 minutes from the microbiological point of view. Regarding the olive fruits mechanical properties, it is advisable to use a pasteurization temperature of 75 °C even for a time of 4 minutes in order to maintain a good flesh firmness thus satisfying the consumers' taste.

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.

Gli oli essenziali, estratti dalle piante, sono composti lipofili contenenti una ampia varietà di elementi volatili. Vengono normalmente ottenuti per distillazione o estrazione con solventi. Trovano impiego nella preparazione di cosmetici, per aggiungere sapore ai cibi e alle bevande e per la formulazione di profumi sia personali che per l’ambiente. Gli oli essenziali hanno recentemente trovato impiego nelle biotecnologie applicate al settore agroalimentare. Sono stati, infatti, utilizzati per ottimizzare le proprietà nutrizionali degli alimenti e per prolungarne la shelf life [1,2]. Gli oli essenziali ottenuti da mandarino sono tradizionalmente usati come antidolorifici, sedativi e curativi per patologie dell’apparato circolatorio [3,4]. Le qualità antisettiche, già individuate agli inizi del secolo scorso, stanno oggi conquistando l’interesse dei ricercatori [5]. Lo scopo del presente lavoro è stato quello di testare l’attività antimicrobica di un olio essenziale estratto da mandarino prodotto in Sicilia (zona di Ciaculli, Palermo) come inibitore della crescita di ceppi batterici Gram-positivi (Staphylococcus aureus e Listeria monocytogenes) e Gram-negativi (Salmonella enterica e Enterobacter hormaechei). L’attività antimicrobica, testata con il metodo “paper disc diffusion” [7], è risultata maggiore contro i batteri Gram-positivi; l’attività più evidente, in termini di diametro della zona di inibizione, è stata registrata nei confronti dei ceppi appartenenti alla specie Listeria monocytogenes, in particolare contro gli isolati di origine alimentare L. monocytogenes 180 e 182. Interessante è anche l’inibizione osservata contro alcuni ceppi di Enterobacter hormaechei, sebbene inferiore a quella mostrata contro i batteri Gram-positivi. Analisi di Gas Cromatografia accoppiata alla Spettrometria di Massa (GC-MS) sono state, inoltre, effettuate per caratterizzare e identificare le molecole volatili dell’intero aroma. Molecole quali D-limonene , gamma.-terpinene, alpha.-terpineol e beta.-myrcene, sono risultate essere i maggiori costituenti dell’aroma. A tali molecole è già stata attribuita bibliograficamente attività antibatterica [6]. Gli oli essenziali di mandarino, pertanto, potrebbero rappresentare un’alternativa all’utilizzo di additivi sintetici in campo alimentare.

The composition of yeast and lactic acid bacteria (LAB) communities and the chemical evolution of the large-scale commercial vinification of Catarratto IGT Sicilia, carried out under the biological regime, was followed from grape harvest until bottling. Simultaneously to the maximum growth of yeasts, LAB counts reached high level of concentration (6-7 log CFU mL(-1)) during the first steps of the alcoholic fermentation. Yeast identification was determined applying different molecular methods. The highest species biodiversity was observed on grape and must samples taken soon after pressing. Saccharomyces cerevisiae was detected at dominant concentrations during the entire winemaking process. LAB cultures were grouped and identified by a combined phenotypic and genotypic approach. Leuconostoc mesenteroides, Lactobacillus hilgardii and Lactobacillus plantarum species were identified; the last was the main LAB recognized during vinification. The winemaking process was also chemically monitored. The alcoholic content was approximately 12.67% (v v(-1)) at bottling; pH, volatile acidity and total acidity showed a moderate increase during vinification. Tartaric, citric and malic acids decreased until bottling, while lactic acid showed a rapid increase at the end of maceration and bottling. Trans-caffeil tartaric acid was the most abundant phenolic compound and volatile organic compounds (VOC) were mainly represented by isoamylic alcohol, isobutanol, ethyl acetate and octanoic acid.

The interest towards the application of essential oils (EOs) is increasing due to the antimicrobial potential to be used as “natural” alternative to the chemical preservation of foods [1]. EOs are aromatic oily liquids produced as secondary metabolites in various plants exerting different biological properties, such as antibacterial, antiviral, antimycotic and insecticidal. EOs are complex mixtures of lipophilic substances and the chemical composition, thereby the biological activities, is influenced by the raw plant material (genotype, part of the plant, harvest time, geographical, ecological conditions and cultural techniques) and extraction method [2].

article i nfo Several lactic acid bacteria (LAB) were evaluated in situ for their potential in sourdough fermentation. The strains belonged to Lactobacillus plantarum, Lactobacillus sakei, Lactobacillus sanfranciscensis, Leuconostoc citreum, Leuconostoc mesenteroides, Leuconostoc pseudomesenteroides and Weissella cibaria. LAB were used, in individual inocula, to carry out the fermentation of γ-ray treated (sterile) flour and untreated commercial flour, in order to evaluate their performances both in the absence and presence of the native microbiota of flour. The pH and total titratable acidity (TTA) showed a strong and fast acidification of the experimental sourdough determined by W. cibaria and Ln. citreum strains. All strains were followed during fermentation by plate count. Randomly amplified polymorphic DNA (RAPD)-PCR analysis applied on the colonies isolated from the highest dilution of samples confirmed the dominance of the added strains in all sourdoughs prepared with sterile and non-sterile flour. The analysis of organic acids, performed by high-performance liquid chromatography (HPLC), confirmed that some W. cibaria and Ln. citreum strains showed an optimal fermentation quotient. The volatile organic compound (VOC) composition resulting from the gas chromatography coupled with mass spectrometry (GC/MS) analysis of sourdough headspace recognised 51 chemical compounds including acids, alcohols, aldehydes, esters, ketones, lactones, acetate, alkane, and phenol, most of which are of LAB origin and are relevant for the final bread. After baking, the breads were evaluated for the height of the central slices, colour of crust and crumb, hardness and number and distribution of alveolus. The combination of these results indicated that strains Ln. citreum PON10079 and PON10080 and W. cibaria PON10030 and PON10032 are suitable cultures to use in industrial production.

The potential for the transport and diffusion of some pathogenic microorganisms by migratory birds is of concern. Migratory birds may be involved in the dispersal of microorganisms and may play a role of mechanical and biological vectors. The efficiency of dispersal of pathogenic microorganisms depends on a wide range of biotic and abiotic factors that influence the survival or disappearance of a given agent in a geographical area. In the present study, 349 migratory birds were captured in four sites (Mazara del Vallo, Lampedusa, Ustica and Linosa), representing the main stop-over points during spring and autumnal migration, and analyzed for the presence of filamentous fungi. A total of 2,337 filamentous fungi were isolated from 216 birds and identified by a combined phenotypic-genotypic approach to species level. Twelve species were identified in the study, with Cladosporium cladosporioides, Alternaria alternata, and Aspergillus niger as the most abundant. The transport of these fungal species isolated in this study is of considerable importance because some of these species can create dangers to human health.

In this work, the yeast ecology associated with the spontaneous fermentation of Grillo cultivar grapes from 10 vineyards was analyzed from grape harvest till complete consumption of must sugars. The microbiological investigation started with the plate count onto two culture media to distinguish total yeasts (TY) and presumptive Saccharomyces (PS). Yeasts were randomly isolated and identified by a combined genotypic approach consisting of restriction fragment length polymorphism (RFLP) of 5.8S rRNA gene and 26S rRNA and sequencing of D1/D2 domain of the 26S rRNA gene, which resulted in the recognition of 14 species belonging to 10 genera. The distribution of the yeasts within the vineyards showed some differences in species composition and concentration levels among 2008 and 2009 vintages. Due to the enological relevance, all Saccharomyces cerevisiae isolates were differentiated applying two genotypic tools (interdelta analysis and microsatellite multiplex PCR of polymorphic microsatellite loci) that recognized 51 strains. Based on the low production of H(2)S, acetic acid and foam, ethanol resistance, growth in presence of high concentrations of potassium metabisulphite (KMBS) and CuSO(4) and at low temperatures, 14 strains were selected and used as starter to ferment grape must at 13 °C and 17 °C in presence of 100 mg/L of KMBS. Three strains (CS160, CS165 and CS182) showed optimal technological aptitudes.

We investigated the effect of residual salts from the previous summer’s irrigation on two non-irrigated cover crops—broad bean and common vetch—and on their rhizobial symbiontics. Before sowing, seeds were inoculated with a salt-tolerant strain and a salt-sensitive strain of Rhizobium leguminosarum biovar viciae. An increase in the electrical conductivity of the saturated-soil extract from 2.0 dS m−1 to 6.0 dS m−1 caused a severe reduction of broad bean biomass, while growth of common vetch was almost unaffected by the salinity level. Our results clearly indicate that common vetch as a cover crop may increase the availability of nitrogen in soil more than broad bean also in saline environments. A high ability of both inoculated strains to nodulate Vicia plants was observed under controlled conditions. In contrast, inoculated strains were not able to nodulate plants in salinized soils, showing lower competitiveness compared to natural rhizobial population. Indeed, the new isolates from nodules were salt-sensitive when tested in vitro. The complex interaction among bacteria, plant and soil determined the survival of endogenous salt sensitive rhizobial strains and limited the success of inoculation with exogenous salt resistant rhizobia.

Antifungal metabolites produced by Bacillus amyloliquefaciens AG1, previously isolated from wood of grapevine with “esca syndrome”, were studied. The crude protein extract (CPE) obtained from culture supernatant fluid by precipitation with ammonium sulfate was assayed against many grapevine fungal pathogens. B. amyloliquefaciens strain AG1 showed a broad spectrum of antifungal activity, inhibiting mycelial growth in vitro of all tested fungal microorganisms. The metabolites contained in CPE were heat stable and remained active over a wide pH range (2–10). Their activity was not affected by proteolytic or glycolytic enzymes. Tricine- SDS-polyacrylamide gel electrophoresis revealed a single band within the range of 2,510–3,480 Da, that showed inhibitory activity when used in the antifungal assay. Mass spectrometry analysis of this band allowed the substances involved in antibiosis to be identified as two tryptic peptides that correspond to the N-terminal sequence of subtilisin BPN’. These results suggest a potential role of B. amyloliquefaciens AG1 as a biocontrol agent.

The genomic grapevine (Vitis vinifera L.) DNA extraction is difficult because of secondary metabolites that interfere with DNA isolation procedures and subsequent applications. We developed a simple, rapid and efficient method for the extraction of genomic DNA from asymptomatic and pathogen-infected grape leaves. The protocol reported, based on a modified cetyl trimethylammonium bromide (CTAB) extraction procedure, allowed the rapid DNA extraction from little amounts of leaf material without employment of liquid nitrogen for initial tissue grinding. The protocol included polyvinylpyrrolidone (PVP) to bind phenolic compounds, β-mercaptoethanol to inhibit the oxidation of polyphenols, and a high concentration of NaCl (2.5 M) to increase the solubility of polysaccharides, thus reducing their co-precipitation with DNA. Final DNA solution did not contain polysaccharides, polyphenols and other major contaminants. The purity of genomic DNA was confirmed by A260/280 and A260/230 ratios calculated from the spectrophotometric readings. In addition, the quality of the DNA extracted from asymptomatic, Oidium tuckeri- and Plasmopara viticola-infected leaves of V. vinifera L. was evaluated in polymerase chain reaction (PCR) analyses by using different set of primers to be able to amplify vegetal, fungal and bacterial DNA. Key words: Vitis vinifera L., DNA extraction, PCR, fungi, bacteria.

The present work was undertaken to evaluate the contribution of migratory birds in the environmental dissemination of yeasts. Four sites (Mazara del Vallo, Lampedusa, Ustica and Linosa), representing the main stop-over points in Sicily, were analysed during spring and autumnal bird migration and 349 birds (belonging to 10 families) were ringed and analysed for yeast presence. A total of 125 yeasts were isolated and identified by a multiple genotypic approach, consisting of restriction fragment length polymorphism (RFLP) of 5.8S rRNA gene and 26S rRNA and sequencing of D1/D2 domain of the 26S rRNA gene, which resulted in the recognition of 18 species, including the technological relevant Saccharomyces cerevisiae which were characterized at strain level applying three techniques (interdelta analysis, minisatellite analysis based on the separate amplification of three genes and microsatellite multiplex PCR of polymorphic microsatellite loci). The evaluation of the persistence of living S. cerevisiae in birds for about 12 h from ingestion of inoculated feed allowed the conclusion that yeasts with technological potential are disseminated during migration.

The antagonistic activity of the essential oils (EOs) extracted by hydrodistillation from the fruit peel of several citrus genotypes (pummelo, grapefruit, orange, kumquat, mandarin and lemon) was evaluated against foodborne pathogen bacteria (43 strains of Listeria monocytogenes , 35 strains of Staphylococcus aureus and 14 strains of Salmonella enterica ). Five commercial EOs were used for comparison. Most of the EOs were more effective against the Gram-positive bacteria rather than Salmonella . EOs of lemon genotypes 14 and 15 showed the best results in terms of number of strains inhibited and width of the inhibition zone. The most susceptible strain of each species ( L. monocytogenes 133, St. aureus 473 and Salmonella Newport 50404) was used as indicator for the evaluation of the minimum inhibition concentration (MIC) of the EOs 14 and 15. The last two EOs, EO 16 (showing the weakest inhibition power among lemon EOs), and the commercial EO L (lemon) were analysed for their chemical composition. A total of 30 major compounds were identified by gas chromatography/mass spectrometry (GC/MS) and the oxygenated monoterpenes were suggested to be implicated in the process of bacterial inhibition by citrus EOs.

The present work was carried out to evaluate the effect of two salting technologies [dry salting (DS) and the combined dry-brine salting (DBS)] on the chemicophysical and microbiological characteristics of PDO Pecorino Siciliano cheeses of different final weight (6 and 12 kg). Dry matter was significantly influenced by both salting process and final size. Twelve kilogram cheeses treated by DBS showed higher protein content with higher soluble nitrogen per cent than 6 kg cheeses. Salt content was in the range 3.1–4.0% on dry matter. The colour did not show significant differences for any of the factors, but 12 kg cheeses subjected to DS showed higher yellow index than the other cheeses. The resistance at 30% of strain was influenced by cheese size, with 6 kg cheeses showing higher resistances than 12 kg cheeses. All cheeses were dominated by coccus LAB, but pseudomonads and Enterobacteriaceae showed comparable levels of about 105 cfu/g. Significant microbiological differences were evidenced only for enterococci and yeasts concerning the final cheese size. Thirteen species of LAB, belonging to five genera (Enterococcus, Lactobacillus, Lactococcus, Pediococcus and Streptococcus), were identified, but several spoilage/ pathogenic species were also identified, especially Pseudomonas putida, Citrobacter freundii and Stenotrophomonas maltophilia. LAB isolates were preliminary evaluated for their physiological characteristics in view of developing autochthonous starters to improve the microbiological quality of PDO Pecorino Siciliano cheese.