Laccases (LCs) are multicopper oxidases that find application as versatile biocatalystsfor the green bioremediation of environmental pollutants and xenobiotics. In this study weelucidate the degrading activity of Lac2 pure enzyme form Pleurotus pulmonarius towards aflatoxinB1 (AFB1) and M1 (AFM1). LC enzyme was purified using three chromatographic steps andidentified as Lac2 through zymogram and LC-MS/MS. The degradation assays were performedin vitro at 25 °C for 72 h in buffer solution. AFB1 degradation by Lac2 direct oxidationwas 23%. Toxin degradation was also investigated in the presence of three redox mediators,(2,20-azino-bis-[3-ethylbenzothiazoline-6-sulfonic acid]) (ABTS) and two naturally-occurring phenols,acetosyringone (AS) and syringaldehyde (SA). The direct effect of the enzyme and the mediatedaction of Lac2 with redox mediators univocally proved the correlation between Lac2 activity andaflatoxins degradation. The degradation of AFB1 was enhanced by the addition of all mediators at10 mM, with AS being the most effective (90% of degradation). AFM1 was completely degraded byLac2 with all mediators at 10 mM. The novelty of this study relies on the identification of a pureenzyme as capable of degrading AFB1 and, for the first time, AFM1, and on the evidence that themechanism of an effective degradation occurs via the mediation of natural phenolic compounds.These results opened new perspective for Lac2 application in the food and feed supply chains as abiotransforming agent of AFB1 and AFM1

The aim of this study was to test the antibacterial activity of Bacillus subtilis TR50 isolated from a traditional cured sausage against ten food-borne pathogenic bacteria. Most of the pathogens were sensitive to cell-free concentrated supernatant from TR50. The inhibitory activity was retained at pH values between 4 and 8, up to80 _C for 30 min and after treatment with pepsin and trypsin, whereas it disappeared after chymotrypsin hydrolysis; lipase treatment abolished inhibitory activity only against one target strain. The ammonium sulphate precipitation of TR50 concentrated cell-free culture retained the most residual antibacterial activity unlike the ethyl acetate and acid fractions. All these results suggest that the antimicrobial compound(s) produced by TR50 could be of proteinaceous nature and require a lipid moiety for activity against the pathogens tested. This is the first report on the antimicrobial activity against food-borne pathogens of a B. subtilis strain occurring in a traditional sausage potentially useful for food safety improvement.

Citrus pomace is a huge agro-food industrial waste mostly composed of peels and traditionally used as compost or animal feed. Owing to its high content of compounds beneficial to humans (e.g., flavonoids, phenol-like acids, and terpenoids), citrus waste is increasingly used to produce valuable supplements, fragrance, or antimicrobials. However, such processes require sustainable and efficient extraction strategies by solvent-free techniques for environmentally-friendly good practices. In this work, we evaluated the antimicrobial and antibiofilm activity of water extracts of three citrus peels (orange, lemon, and citron) against ten different sanitary relevant bacteria. Both conventional extraction methods using hot water (HWE) and microwave-assisted extraction (MAE) were used. Even though no extract fully inhibited the growth of the target bacteria, these latter (mostly pseudomonads) showed a significant reduction in biofilm biomass. The most active extracts were obtained from orange and lemon peel by using MAE at 100 °C for 8 min. These results showed that citrus peel water infusions by MAE may reduce biofilm formation possibly enhancing the susceptibility of sanitary-related bacteria to disinfection procedures.

Several spices are used to process 'Nduja, a traditional Calabrian sausage, well knownfor its typical flavor and piquancy. Despite wide use of spices for meat curing andcontrolling undesired microorganisms, no studies reported antimicrobial effectivenessof the spices in the 'Nduja processing. The aim of this work was to evaluate theantimicrobial activities against 10 foodborne pathogens of processed spices used inthe 'Nduja industrial manufacturing. Spice extracts (using ethanol, methanol andwater as solvents) were investigated by disk diffusion assay for their antimicrobialactivity. Methanol extracts showed the best results, mainly against Bacillus cereusstrains. Thus, they were further evaluated, through microdilution automated spectrofluorimetricabsorbance readings. Many spice methanol extracts showed low antimicrobialactivities, although B. cereus strains were still more sensitive. In addition, twoout of the three spice mixtures showed medium and high inhibition activity againstEscherichia coli ATCC 35401 and Yersinia enterocolitica DSM 4780, respectively.

The aim of this work was to check the efficacy of bovine lactoferrin (BLF) and its pepsin-digested hydrolysate (LFH) to control spoilage bacteria contaminating the governing liquid of high moisture (HM) Mozzarella cheese during cold storage. These natural substances resulted effective when tested in vitro against five potential spoilage bacteria contaminating cold-stored HM Mozzarella cheese. Among six LFH fractions, only the fraction containing lactoferricins, mainly represented by LfcinB?????, resulted effective against Escherichia coli K12 at the same extent of the whole pepsin-digested hydrolysate. LFH tested throughout seven days for its antimicrobial activity against the main bacterial groups growing in cold-stored commercial HM Mozzarella cheese samples delayed significantly the growth of pseudomonads and coliforms in comparison with the un-treated samples. This is the first report providing a direct evidence of the ability of LFH to inhibit the growth of cheese spoilage bacteria.

Milk protein crosslink through the action of enzymes represents a feasible strategy to impart new functionalities to cheese. In this work we reported the effects of a laccase mediator system (LMS) on protein crosslink and antioxidant property of curd. The crosslinking activity of a purified recombinant laccase Ery4 and a commercial enzyme preparation (cLC), with three mediators was firstly evaluated in milk and then applied before curd manufacture. Only Ery4-LMS significantly increased curd weight compared to that of the control sample. SDS-PAGE revealed that similar high molecular weight bands produced by both LMSs in milk were also retained in curds. The antioxidant activity recorded in curds with Ery4-LMS was the highest among all samples both before and after gastro-pancreatic digestion. This is the first time that a CGA-based LMS is used in manufacture of curd with improved antioxidant properties. These results open new perspectives for dairy applications.

Psychrotrophic Pseudomonas spp. were proved to cause dramatic quality losses in fresh foods like leafy greens and dairy products resulting in the shortening of their shelf-life. These spoilage microorganisms are resistant to most disinfection treatments. This work was addressed to assay the antimicrobial efficacy of the food-grade bovine lactoferrin (BLF) and its hydrolysates (LFH), obtained after digestion with pepsin or the enzymatic cardoon extract. The inhibition assays were carried out in vitro on selected Pseudomonas spp. strains involved in off-color development in ready-to-eat vegetables (RTE) and in mozzarella cheese. A higher antimicrobial activity against most of the tested strains was found for pepsin digested BLF in comparison with BLF digested with the enzymatic cardoon extract. A solution of the antimicrobial peptide lactoferricin B (LFcinB), purified from the pepsin-LFH, was applied to both cold stored RTE lettuce leaves and mozzarella cheeses, inoculated with the spoilage strains. A significant reduced tissue browning of RTE lettuce leaves, as well as absence of mozzarella cheese discoloration applying LFcinB. The results obtained show the possibility to use milk-derived antimicrobial peptides in the control of food spoilage caused by Pseudomonas strains.

Le pigmentazioni anomale di mozzarelle sono state associate a ceppi psicrotrofi di Pseudomonas fluorescens presenti a basse concentrazioni nel liquido di governo già nelle prime fasi di frigoconservazione di tale formaggio fresco. Il mancato controllo di questi batteri alterativi ne causa la crescita determinando il ritiro dal mercato dei prodotti alterati. Scopo di questo lavoro è stato valutare l'efficacia antimicrobica in vitro ed in mozzarella dell'idrolizzato peptico di lattoferrina bovina (HLF) contro P. fluorescens 84095 isolato da mozzarelle blu. I risultati hanno mostrato che la crescita in vitro a 4?C del ceppo è stata inibita da 50 mg/mL di HLF. La stessa concentrazione, applicata a mozzarelle inoculate, ha determinato una significativa riduzione delle conte di 84095 rispetto a quelle registrate in campioni senza HLF fino al 14 giorno di frigoconservazione. Il trattamento ha inoltre bloccato lo sviluppo della colorazione blu. Le analisi LC-MS hanno confermato la presenza della leucoindigoidina, forma ridotta ed incolore del pigmento blu indigoidina solo nei campioni di mozzarella inoculati non trattati con HLF. Questo lavoro, pertanto, suggerisce l'applicazione di un idrolizzato antimicrobico di una proteina del latte, efficace per prevenire la colorazione blu, senza modificare la composizione della mozzarella. Parole chiave: peptidi antimicrobici, shelf-life, controllo alterazioni

La laccasi (LC) è un enzima ampiamente studiato per modulare le proprietà reologiche e tecnologiche degli alimenti. L'applicazione in latte e prodotti lattiero caseari è tuttavia limitata. Recentemente è stato studiato il suo ruolo nella degradazione in latte dell'aflatossina M1, oltre che nella creazione di crosslink proteici per migliorare la texture e la reologia dei prodotti lattiero caseari. Quest' ultima sua proprietà potrebbe essere sfruttata per favorire il recupero di proteine dal siero durante la caseificazione aumentando la resa casearia. D'altra parte l'applicazione della LC richiede la combinazione con l'acido clorogenico (CGA), un polifenolo naturale che potrebbe conferire al formaggio prodotto nuove caratteristiche qualitative, come un'aumentata attività antiossidante.Pertanto, l'obiettivo di questo lavoro è stato valutare l'aumento in resa casearia e attività antiossidante della cagliata ottenuta pretratrattando il latte per 45' a 38°C con concentrazioni note di LC + CGA prima dell'utilizzo del caglio. La cagliata ottenuta è stata comparata a quella controllo non trattata valutando anche il profilo elettroforetico su SDS-PAGE, e l'attività antiossidante dopo digestione gastrico-pancreatica simulata.Il trattamento del latte con il sistema laccasi-acido clorogenico ha causato la formazione di oligomeri ad alto e medio peso molecolare (>200 e 50 kDa, rispettivamente) rilevabili in SDS-PAGE. Parallelamente, le bande associate alle ?-, ?- e ?-caseine, hanno subito una lieve ma significativa riduzione di intensità, suggerendo un loro coinvolgimento nella formazione dei suddetti oligomeri, confermato successivamente anche dall'analisi in cromatografia liquida associata a spettrometria di massa. Questo pattern elettroforetico, osservato in latte prima dell'aggiunta del caglio, è stato osservato invariato anche nelle cagliate prodotte, indice che tali oligomeri sono incorporati nella rete caseinica al momento della coagulazione. Contrariamente della transglutaminasi, che modifica il sito di taglio della ?-caseina, le modalità d'impiego del sistema LC + CGA non hanno invece influenzato l'azione del caglio, suggerendo pertanto una sua facile implementazione nel processo di caseificazione industriale. La cagliata ottenute hanno inoltre mostrato una resa casearia significativamente superiore, circa 25%, a quella della cagliata controllo sia in peso fresco (2.89g vs 2.14g) che in peso secco (1.66g vs 1.28g), presumibilmente ascrivibile all'aumento totali del contenuto proteico totale.La cagliata trattata con il sistema LC + CGA, sottoposta a digestione gastrico pancreatica simulata, ha mostrato un aumento dell'attività antiossidante di quasi 4 volte rispetto a quella del controllo non trattato (2273.3 vs 586 µmol TEAC/L). I dati presentati dimostrano come un unico trattamento enzimatico, facilmente implementabile nel processo produttivo, potrebbe apportare un significativo contributo in campo lattiero caseario su più fronti. L'azione degrad

In questo lavoro, mais triturato combinato alternativamente con frumento, frumento parboiled,riso e patata è stato bollito in eccesso di acqua e sottoposto a fermentazione lattica per circa16 ore al fine di realizzare 4 tipologie di bevande (B1, B2, B3 e B4) ad elevato contenuto di amidiresistenti (RS), una frazione dell'amido che sfugge alla digestione del primo tratto intestinaleed è fermentata nel colon. Le bevande, dal contenuto medio di acqua (in peso) del 76,8%,hanno mostrato valori medi di pH di 4,06 e di acidità totale titolabile di 0,53% (w/w) dopofrigoconservazione e viscosità apparente variabile in funzione della loro composizione (da 19,4a 37,9 P.s). Larga parte della conta batterica totale, prossima in tutti i casi ai 9 log ufc/g, risultavacostituita da batteri lattici. A seguito di determinazioni enzimatico-spettrofotometriche lebevande hanno mostrato percentuali di RS, sulla sostanza secca, superiori al 5,5%. Quantitàcrescenti di amilosio, registrate nelle miscele di mais e frumento, hanno determinato incrementisignificativi di RS nelle rispettive bevande fino a raggiungere in media il 48% del contenutototale di amido. Il presente lavoro pone le basi per lo sviluppo di un nuovo tipo di bevandapotenzialmente utile per il controllo della risposta glicemica post-prandiale, come disciplinatodal Regolamento Europeo n. 432/2012 in materia di indicazioni nutrizionali-salutistiche.

La qualità e la shelf-life della mozzarella è strettamente correlata ai cambiamenti della composizione chimico-fisica e microbiologica del liquido di governo, nel quale si riversano zuccheri, peptidi e sali minerali provenienti dalla mozzarella che favoriscono la crescita microbica anche in condizioni di frigo conservazione. Nel presente lavoro, il liquido di governo di bocconcini di mozzarella è stato sostituito con biopolimeri ottenuti da soluzioni di siero neutralizzati, pastorizzati e arricchiti con differenti concentrazioni di alginato di sodio (E401). Le mozzarelle sono state, quindi, conservate a 4°C per 30 giorni, monitorando i parametri reologici, chimici e microbiologici.A seguito di valutazioni preliminari, solo sul prodotto conservato con il biopolimero al 2% è proseguito il monitoraggio, valutando che le mozzarelle conservate in alginato mostravano percentuali maggiori di deformazioni, rispetto a quelle riscontrate sulle mozzarelle di riferimento. Parallelamente, la capacità di recupero elastico di queste ultime risultava significativamente maggiore di quella dei campioni inclusi in alginato, suggerendo un possibile indebolimento della struttura del formaggio trattato.A differenza del normale liquido di governo che tende ad acidificarsi significativamente a partire dal settimo giorno di frigoconservazione, il siero alginato mostrava maggiore stabililtà raggiungendo i medesimi valori di pH del liquido di governo con un ritardo di circa 14 giorni. Il trend di acidificazione è stato registrato anche in mozzarella, sebbene le differenze tra i due metodi di conservazione risultino inferiori. Come atteso, in condizioni di stress fisico controllato, è stato osservato che le mozzarelle in liquido di governo rilasciavano molto più siero interno, soprattutto entro sette giorni di frigoconservazione, rispetto alle mozzarelle conservate con il biopolimero di siero alginato. I dati microbiologici non rivelano significative differenze tra i campioni in liquido di governo e quelli in siero alginato, sia nelle cinetiche che nei valori della carica microbica delle popolazioni lattiche e non. I risultati ottenuti in questo lavoro, sebbene preliminari, dimostrano che il siero alginato può essere utilizzato come metodo di conservazione alternativo al liquido di governo per mantenere i parametri strutturali e chimici della mozzarella nel corso della conservazione. Tale metodo inoltre può essere ulteriormente migliorato al fine di favorire il parziale recupero del siero, attualmente considerato un sottoprodotto delle lavorazioni lattiero casearie dagli onerosi costi di smaltimento.

Bovine lactoferrin and lactoferricin B, well-known for their antimicrobial properties, were individually immobilized on two different coatings functionalized with - COOH groups deposited in the inner part of polyethylene micro tubes by means of a plasma deposition (PE-CVD) process fed with ethylene and acrylic acid vapors. The resulting functionalized tubes were tested for antimicrobial activity against three Pseudomonas strains responsible for casein hydrolysis and cheese pigmentation. The cell counts of these spoilage bacteria, incubated for 30 h under their optimal growth conditions, were found to be significantly reduced after 24 h in micro tubes functionalized with lactoferricin B, whereas a very low antimicrobial activity against the same strains, often undistinguishable from that of control samples, was observed in tubes functionalized with lactoferrin.This is the first work in which a plasma coating functionalized by lactoferricin B was studied to make an active packaging useful to control cheese spoilage by Pseudomonas.Industrial relevance: The current study describes a new method to immobilize two food grade proteinaceous natural compounds. The resulting plasma-functionalized lactoferricin B-immobilized coating is a promising tool for the control of spoilage microorganisms and shelf-life extension of cheeses.

The aim of this work was to analyze changes occurring in cereal protein and starch composition throughout the production process of Boza for its potential exploitation as functional beverage. A sharp decrease in the extractability of albumins, globulins, glutenins and prolamins was found in the processed samples after the cooking step. This phenomenon was confirmed analyzing the extracted cereal proteins on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which also revealed an apparent decrease in the number of wheat albumin/globulin, glutenin and prolamin bands in the gruel samples in comparison with that shown by the related flours. The application of an improved extraction procedure and reverse-phase high-performance liquid chromatography analysis confirmed that the prolamin content in Boza decreased markedly in comparison with that of the flour samples. Under the experimental conditions applied, the resistant starch content of Boza resulted significantly higher than that of flours; thus, this beverage can be regarded as a "high fiber" food in accordance with the recent European regulations. This is the first report that throws light on changes occurring in protein and starch components throughout the production process of Boza and provides direct evidence on the low prolamin and high resistant starch contents of this traditional cereal-based beverage. (IF 1.566)

TR50 CFCS showed antimicrobial activities against several food-borne pathogens and seven strains belonging to B. pumilus, B. licheniformis and B. thuringensis. The EA fraction retained inhibitory activity against six food-borne pathogenic strains. An inhibition halo against B. cereus DSM 4312 was produced by one peptide band with an apparent molecular weight of about 2.5 kDa, resolved on Tricine SDS-PAGE. The loss of inhibitory activities of TR50 CFCS against some tested strains and the comparison between CFCS and EA RP-HPLC profiles sustains the hypothesis that TR50 produces various active compounds responsible for its antimicrobial activity. Inhibition halos produced by bands scraped from TLC indicates the presence of lipopeptides. These preliminary results highlight that the antibacterial potential of B. subtilis TR50 could be exploited in several food sectors and in the sausage curing process. Further analyses are in progress to identify the active compounds.

Citrus pomace is a huge agro-food industrial waste mostly composed of peels. Owing to its high content of compounds beneficial to humans (e.g., flavonoids, phenol-like acids, and terpenoids), citrus waste is increasingly used to produce valuable supplements, fragrance, or antimicrobials. Sustainable and efficient extraction strategies by solvent-free techniques for environmentally-friendly good practices should be desirable. In this work, both conventional extraction methods using hot water (HWE) and microwave-assisted extraction (MAE) were used to obtain water extracts. The antimicrobial and antibiofilm activity of water extracts of three citrus peels (orange, lemon, and citron) against ten different sanitary relevant bacteria were evaluated.Even though no extract fully inhibited the growth of the target bacteria, these latter (mostly pseudomonads) showed a significant reduction in biofilm biomass. The most active extracts were obtained from orange and lemon peel by using MAE at 100 °C for 8 min. These results showed that citrus peel water infusions by MAE may reduce biofilm formation possibly enhancing the susceptibility of sanitary-related bacteria to disinfection procedures

A wide range of biopeptides potentially able to lower blood pressure through inhibition of the angiotensin-I converting enzyme (ACE) is produced in fermented foods by proteolytic starter cultures. This work applies a procedure based on recombinant DNA technologies for the synthesis and expression of three ACE-inhibitory peptides using a probiotic cell factory. ACE-inhibitory genes and their pro-active precursors were designed, synthesized by PCR, and cloned in Escherichia coli; after which, they were cloned into the pAM1 E. coli-bifidobacteria shuttle vector. After E. coli transformation, constructs carrying the six recombinant clones were electrotransferred into the Bifidobacterium pseudocatenulatum M115 probiotic strain. Interestingly, five of the six constructs proved to be stable. Their expression was confirmed by reverse transcription PCR. Furthermore, transformed strains displayed ACE-inhibitory activity linearly correlated to increasing amounts of cell-free cellular lysates. In particular, 50 ?g of lysates from constructs pAM1-Pro-BP3 and pAM1-BP2 showed a 50% higher ACE-inhibitory activity than that of the controls. As a comparison, addition of 50 ng of Pro-BP1 and Pro-BP3 synthetic peptides to 50 ?g of cell-free extracts of B. pseudocatenulatum M115 wild-type strain showed an average of 67% of ACE inhibition; this allowed estimating the amount of the peptides produced by the transformants. Engineering of bifidobacteria for the production of biopeptides is envisioned as a promising cell factory model system.

Mozzarella is one of the most known Italian traditional fresh cheeses, usually stored into a cold governing liquid in order to maintain its typical organoleptic characteristics. These storage conditions favour the growth of psychrotrophic bacteria among which some are responsible for cheese spoilage. In this work, bovine lactoferrin (BLF) and its pepsin-digested hydrolysate (LFH) were tested in vitro to evaluate their efficacy as potential control agents of Mozzarella cheese spoilage bacteria growing during refrigeration period. At first, five bacterial strains (Pseudomonas fragi 23A, P. gessardi 2A, Serratia proteamaculans 19A, Aeromonas salmonicida 22DB and Rahnella aquatilis 19B), previously isolated from different cold-stored Mozzarella cheese samples, were tested for their ability to grow at 4 °C in the governing liquid-based medium, in which Mozzarella releases debris, sugars and salts, and to promote proteolytic activities against casein fractions. In order to check the efficacy of BLF or LFH to control spoilage bacteria, target strains were inoculated in the governing liquid-based medium at two levels of initial inoculum (3 and 6 log cfu/mL), and incubated at 4 °C or 30 °C for two weeks. At the initial inoculum of 3 log cfu/mL all strains grew well in the governing liquid at 4 °C, reaching 7.1 log cfu/mL, on average, after four days of cold storage. The most proteolytic strains were P. gessardi 2A and P fragi 23A that were able to digest completely all casein fractions, flowed into the governing liquid, after five and eight days, respectively. The remaining strains caused the disappearance of some casein bands on SDS-PAGE patterns, after twelve days of incubation. Viable cells of P. gessardi 2A and R. aquatilis 19B were undetectable when 3 log cfu/ml of each microorganism were inoculated in the governing liquid supplemented with 10 mg/mL of LFH. S. proteomaculans 19A, A. salmonicida 22DB and P. fragi 23A resulted partially inhibited as their bacterial loads resulted 2.3 log cfu/mL, on average, lower than those of the un-supplemented control up to day 5 of cold storage. After additional two days at 4 °C, this difference decreased by 1 log cfu/mL. The inhibitory activity of LFH resulted higher than BLF against all strains grown in the governing liquid-based medium. No antimicrobial activity was observed when the governing liquid, amended with BLF or LFH, was inoculated with target strains at 6 log cfu/mL. Also the increase in temperature of incubation advantaged microbial cells, reducing the inhibitory efficacy of BLF and LFH: after one day of incubation at 30 °C, a decrease of bacterial growth was registered only for P. fragi 23A and R. aquatilis 19B and only at 3 log cfu/ml of initial inoculum level. As expected, casein band intensities in SDS-PAGE patterns were comparable with those of the uninoculated control sample only in experiments showing the lowest microbial viable cell concentration. In conclusion, we demonstrat

In this work the development of table grape sour rot, a disease sustained by a consortium of non-Saccharomyces yeasts (NSY) and acetic acid bacteria (AAB), was observed under postharvest conditions inoculating wounded berries with 8 NSY strains, or 7 AAB, or their combinations (56). Candida zemplinina CBS 9494 (Cz) and Acetobacter syzygii LMG 21419 (As) produced the highest percentage of rotten berries (100%) after 6 days at 25 °C. Subsequently, the efficacy of low doses of gaseous ozone (2.14 mg m-3) was evaluated in wounded berries inoculated with this simplified As-Cz microbial consortium after ten days at 4 °C. At the end of cold storage, no sample showed sour rot symptoms, although ozonation mainly affected the As viability. In order to simulate sale conditions, all samples were additionally stored at 25°C for 12 days. After this period, the sour rot index of inoculated As-Cz berries previously cold-stored under ozone or air accounted for 22.6 ± 3.7% and 66.7 ± 4.5%, respectively. Inoculated rotten berries showed a slight reduction in monosaccharide content and the different increases in concentration of acetic acid, gluconic acid, ethanol and glycerol. Microbiological analyses of sound and rotten berries revealed, in addition to As and Cz, the occurrence of 15 new strains potentially responsible for sour rot development and mainly belonging to Gluconobacter albidus and Hanseniaspora uvarum. In this work we demonstrated that cold ozonation was useful to extend the shelf-life of cold stored table grapes even when, at the end of treatment, they were further stored at room temperature.

The objective of this study was to develop a new synbiotic beverage evaluating the ability of some bifidobacteria strains to grow in this beverage which was fortified with whey proteins up to 20 g L-1, and enriched with 10 g L-1 of prebiotic inulin or resistant starch. The ability of Bifidobacterium strains to survive for 30 days at 4°C was evaluated in two synbiotic whey protein fortified beverages formulated with 2% of whey proteins and 1% of inulin or resistant starch. Microbial growth was significantly affected by the whey protein amount as well as by the kind of prebiotic fibre. Resistant starch promoted the growth of the Bifidobacterium pseudocatenulatum strain and its viability under cold storage, also conferring higher sensory scores. The development of this new functional beverage will allow to carry out in vivo trials in order to validate its pre- and probiotic effects.

Bio-composite coatings, consisting of an organic matrix embedding a bioactive molecule, have been deposited by means of atomizer-assisted atmospheric pressure plasma. Ethylene was chosen as the precursor of the matrix, while the atomizer was fed with a water solution of lysozyme. Coatings chemical composition was investigated by XPS, FTIR and MALDI-TOF spectroscopies, and it has been proved that the one-step inclusion of protein domains in the composite coatings is successful and lysozyme chemical structure is only slightly altered. The amount of embedded lysozyme is as high as 14?g/cm² as evaluated from water release test. Finally, the activity of the plasma-embedded protein is close to that of pure lysozyme as verified against Micrococcus lysodeikticus ATCC 4698 through an agar plate diffusion test.

Pseudomonas fluorescens is a genetically and phenotypically heterogeneous species that is often reported as a spoiler of fresh foods, but it has recently been implicated in clinical infection. In this study, we sequenced the genome of P. fluorescens strain ITEM 17298, isolated from mozzarella cheese and able to cause several alterations under cold storage.

The aim of this work was to check the efficacy of an annular ultraviolet reactor for stabilizing the growth of lactic acid bacteria (LAB), the main components of the microbiota of three raw fermented beverages, Boza, Ayran and Matsoni during cold storage. The ultraviolet (UV) dose of 260 mJ cm-2 caused a reduction of 2.6 and 1 log CFU mL-1 in the LAB loads of Boza and Ayran samples, respectively, whereas multiple UV treatments were required for Matsoni; they caused the decrease of 3 log CFU mL-1 in LAB counts. Twenty-two bacterial strains, belonging to 11 species, were identified in the natural microflora of the three beverages before and after the UV treatments. Only a minimal post-acidification process was registered up to day 30 in all the three beverages. This ultraviolet device is at disposal of beverage manufacturers, who could apply it for producing microbially-stable drinks with an extended shelf-life

The aim of this work was to check the efficacy of an annular ultraviolet reactor for stabilizing the growth of lactic acid bacteria (LAB), the main components of the microbiota of three raw fermented beverages, Boza, Ayran and Matsoni during cold storage. The ultraviolet (UV) dose of 260 mJ cm-2 caused a reduction of 2.6 and 1 log CFU mL-1 in the LAB loads of Boza and Ayran samples, respectively, whereas multiple UV treatments were required for Matsoni; they caused the decrease of 3 log CFU mL-1 in LAB counts. Twenty-two bacterial strains, belonging to 11 species, were identified in the natural microflora of the three beverages before and after the UV treatments. Only a minimal post-acidification process was registered up to day 30 in all the three beverages. This ultraviolet device is at disposal of beverage manufacturers, who could apply it for producing microbially-stable drinks with an extended shelf-life.

The microbial content of plant tissues has been reported to cause the spoilage of ca. 30% of chlorine-disinfectedfresh vegetables during cold storage. The aim of this work was to evaluate the efficacy of antimicrobial peptidesin controlling microbial vegetable spoilage under cold storage conditions. A total of 48 bacterial isolateswere collectedfrom ready-to-eat (RTE) vegetables and identified as belonging to Acinetobacter calcoaceticus, Aeromonasmedia, Pseudomonas cichorii, Pseudomonas fluorescens, Pseudomonas jessenii, Pseudomonas koreensis, Pseudomonasputida, Pseudomonas simiae and Pseudomonas viridiflava species. Reddish or brownish pigmentation was foundwhen Pseudomonas strains were inoculated in wounds on leaves of Iceberg and Trocadero lettuce and escarolechicory throughout cold storage. Bovine lactoferrin (BLF) and its hydrolysates (LFHs) produced by pepsin, papainand rennin, were assayed in vitro against four Pseudomonas spp. strains selected for their heavy spoiling ability.As the pepsin-LFH showed the strongest antimicrobial effect, subsequent experiments were carried out using thepeptide lactoferricin B (LfcinB), well known to be responsible for its antimicrobial activity. LfcinB significantly reduced(P <= 0.05) spoilage by a mean of 36% caused by three out of four inoculated spoiler pseudomonads on RTElettuce leaves after six days of cold storage. The reduction in the extent of spoilage was unrelated to viable celldensity in the inoculated wounds. This is the first paper providing direct evidence regarding the application ofan antimicrobial peptide to control microbial spoilage affecting RTE leafy vegetables during cold storage.

This work aims at studying the efficacy of low doses of gaseous ozone in postharvest control of the table grape sour rot, a disease generally attributed to a consortium of non-Saccharomyces yeasts (NSY) and acetic acid bacteria (MB). Sour rot incidence of wounded berries, inoculated with 8 NSYstrains, or 7 AAB, or 56 yeast-bacterium associations, was monitored at 25 degrees C up to six days. Sour rot incidence in wounded berries inoculated with yeast-bacterium associations resulted higher than in berries inoculated with one single NSY or AAB strain. Among all NSY-AAB associations, the yeast-bacterium association composed of Candida zemplinina CBS 9494 (Cz) and Acetobacter syzygii LMG 21419 (As) showed the highest prevalence of sour rot; thus, after preliminary in vitro assays, this simplified As-Cz microbial consortium was inoculated in wounded berries that were stored at 4 degrees C for ten days under ozone (2.14 mg m(-3)) or in air. At the end of cold storage, no berries showed sour-rot symptoms although ozonation mainly affected As viable cell count. After additional 12 days at 25 degrees C, the sour rot index of inoculated As-Cz berries previously cold-stored under ozone or in air accounted for 22.6 +/- 3.7% and 66.7 +/- 4.5%, respectively. Molecular analyses of dominant AAB and NSY populations of both sound and rotten berries during post refrigeration period revealed the appearance of new strains mainly belonging to Gluconobacter albidus and Hanseniaspora uvarum species, respectively. Cold ozonation resulted an effective approach to extend the shelf-life of table grapes also after cold storage. (C) 2017 Elsevier Ltd. All rights reserved.

Nduja is a traditional Calabrian (Italy) sausage made from pork trimmings (underbelly, shoulder and head) and manufactured with high amounts of different spices (ca. 5%), mostly represented by the red hot chili pepper that confers its typical flavour and piquancy. In addition, spices are well known for their antimicrobial activity against several microorganisms; in fact, no yeast and mould growth was found on this sausage throughout ripening and storage. In this study, methanolic, ethanolic and aqueous extracts of processed sweet, hot chili pepper powder (SP and HCP), and three spice mixtures (SM1, SM2, SM3), used in industrial manufacturing of 'Nduja, were assayed for their antimicrobial activity against ten food-borne bacteria (Figura 1) by using agar disc diffusion assay.

Allo scopo di incrementare la resa casearia della caciotta sono state condotte prove di caseificazioni con latte vaccino arricchito con differenti quantità (2,5 - 10%) di siero concentrato in polvere (WPC) proveniente da reflui caseari.I risultati preliminari hanno evidenziato che ad elevate concentrazioni (dal 5 al 10%) l'aggiunta di WPC aumentava significativamente i tempi di coagulazione e diminuiva la consistenza del formaggio rispetto a quella del campione controllo. Al contrario l'aggiunta di WPC al 2,5% ha prodotto un aumento della resa casearia, nonostante la maggior parte delle sieroproteine (SP) aggiunte fossero perse durante il processo di coagulazione. Durante la stagionatura la riduzione dell'umidità dei formaggi, inferiore nelle caciotte con WPC rispetto a quelle controllo, ha consentito un sensibile aumento del livello di SP unicamente nei formaggi arricchiti con WPC. E' stato possibile infatti integrare nella caciotta stagionata oltre il 10% della quantità di SP aggiunta nel latte, ottenendo rese casearie superiori per circa l'1,5%. La produzione di ricotta proveniente dal siero delle caciotte arricchite in WPC ha inoltre permesso un recupero di SP pari a circa il 50% in più di quanto ottenuto dalle ricotte controllo.A termine della stagionatura (1 mese), le caciotte con e senza WPC sono state confezionate sottovuoto e conservate per tre mesi a 4°C per la valutazione della loro shelf life. Le popolazioni batteriche mesofile (CBT), i batteri lattici e i coliformi hanno mostrato una modesta riduzione delle cariche vitali senza particolari differenze tra le due tipologie di formaggio. Le prove di assaggio hanno evidenziato una maggiore cremosità della caciotta arricchita in WPC rispetto a quella controllo. Tuttavia a fine del periodo di shelf-life i formaggi con WPC presentavano un lieve retrogusto amaro che era assente nelle caciotte controllo.Le valutazioni organolettiche delle ricotte realizzate tra le 24 e 48 ore dalla loro produzione non hanno mostrato sostanziali differenze.I risultati ottenuti mostrano per la prima volta che, applicando un processo di lavorazione integrato, è possibile recuperare le SP presenti nel siero per ottenere formaggi con aumentata resa casearia ed un migliorato profilo proteico. Il controllo dei fenomeni di proteolisi legati alla produzione di peptidi amari necessita di ulteriore sperimentazione.

Le malattie cardiovascolari, cancro, malattie non infettive dell'apparato respiratorio e diabete sono malattie croniche, anche dette "noncommunicable diseases" (NCDs) legate a disordini metabolici come l'incremento della pressione sanguigna, sovrappeso ed obesità, iperglicemia e iperlipidemia. I dati raccolti dalla Word Health Organization indicano come nei Paesi altamente sviluppati l'87% della mortalità è attribuita alle NCDs il cui più importante fattore di rischio è rappresentato dall'elevata pressione sanguigna. E' stato ampiamente dimostrato che gli alti livelli di sodio nella dieta sono legati all'incremento della pressione sanguigna e, per tale motivo, la riduzione della quantità di sodio assunta con la dieta è un obiettivo portato avanti a livello nazionale e comunitario. Dal momento che circa il 70-75% del sodio assunto proviene da alimenti processati, ridurre l'uso del sale nei vari processi di trasformazione alimentare potrebbe portare ad un ridotto consumo giornaliero di sodio. Dal momento che l'aggiunta in etichetta di indicazioni nutrizionali come "a basso (o bassissimo) contenuto di sodio/sale" è favorevolmente accolta dai consumatori, numerose ricerche sono in atto per superare gli ostacoli tecnici legati alle modifiche di processo necessarie per l'ottenimento di formaggi sicuri e di buona qualità a basso contenuto di sodio. Il Cacioricotta è un formaggio a pasta semidura che può raggiungere un contenuto di sale superiore al 6% a fine stagionatura. La salatura è cruciale per l'ottenimento delle caratteristiche chimiche fisiche, sensoriali e microbiologiche ottimali di questo formaggio, per cui, la riduzione del sale potrebbe determinare la riduzione dei livelli di sicurezza e la shelf-life di tale formaggio. In questo studio la salatura del Cacioricotta è stata realizzata sostituendo il sale con quantità crescenti di un concentrato minerale contenente circa il 10% di sodio. Le caratteristiche chimico-fisiche, microbiologiche, le concentrazioni di sodio e di altri metalli e i profili di consistenza strumentale (TPA) sono stati confrontate con quelli dei campioni di riferimento (0 e 100% cloruro di sodio) e monitorate durante 90 giorni di frigoconservazione sotto vuoto.Durante i 90 giorni di frigoconservazione i valori di pH e acidità totale sono risultati simili in tutti i campioni analizzati mentre il valore dell'attività dell'acqua è risultato inversamente proporzionale alla concentrazione di sodio aggiunta con valori medi di 0,901 ± 0,03 al termine della frigoconservazione. Le differenti concentrazioni di sodio, monitorate mediante ICP-OES, non hanno influenzato le cariche vitali di enterococchi, stafilococchi, funghi e lieviti e batteri lattici, mentre le popolazioni di coliformi ed Escherichia coli risultavano controllate in maniera più efficace all'aumentare della quantità di cloruro di sodio usato. I formaggi con parziale e totale sostituzione del sale mostravano valori di durezza in media più elevati (86,26 N) di qu

Ayran is a traditional Turkish milk drink which is fermented and salted. Inadequate production andstorage conditions contribute to its variable organoleptic quality and stability during shelf-life. A thoroughphysico-chemical, nutritional and microbial characterization of artisanal Ayran was carried out inorder to standardize its overall quality without altering its original traits. Ayran microbial ecosystem waslargely dominated by Streptococcus thermophilus (ST) and Lactobacillus delbrueckii subsp. bulgaricus(LDB). High counts of other lactic acid bacteria species, including Lactobacillus helveticus (LH), Lactobacillusfermentum (LF), and Lactobacillus paracasei (LP), were also found. Selected LDB, LP and LH strainsgrew well in milk displaying fast acidification and high proteolysis, differently from ST and LF strains thatdid not cause noticeable changes. A selected autochthonous three-strain culture (TSC), composed of onestrain of LDB, LP and ST, was applied for the pilot-scale production of traditional Ayran. The Ayranproduced with this TSC resulted in the most extensive shelf-life (one month) and in the best terms of itsnutritional and sensory quality nevertheless altering its typical pleasant yogurt and cottage cheese notes.This TSC is at disposal of SMEs who need to standardize the overall quality of this traditional fermentedmilk, preserving its typical traits

Fifty-one yeasts were randomly isolated from four different preparations of Boza, a Turkish cereal-based beverage in order to throw light upon their biotechnological potential. Sequence analysis of 26S rDNA D1/D2 domain revealed that the isolated yeasts belonged to Candida, Clavispora, Cryptococcus, Cystofilobasidium, Geotrichum, Coniochaeta, Pichia, Rhodotorula, Saccharomyces, Torulaspora and Trichosporon genera. Interestingly, Pichia fermentans resulted the dominant species with 21 isolates; a high polymorphism among these yeasts resulted by RAPD-PCR analysis which allowed to distinguish 15 strains that were characterized for their biochemical and technological features. In addition, all P. fermentans strains did not assimilate d-maltose and d-sucrose and expressed a very low invertase activity, suggesting their possible coexistence with maltose- and sucrose-fermenting lactic acid bacteria in Boza microbial ecosystem. Three yeasts produced a high amount of the rose-like fragrance 2-phenylethanol that might be involved in flavor development of this traditional beverage. Moreover, these yeasts also displayed an antagonistic activity against Trichosporon cutaneum, a pathogen occurring in Boza. These selected yeasts are at disposal of Boza manufacturers who could apply them, in combination with LAB, for producing a high quality beverage in accordance with the Turkish quality standards.(IF 3.150)

The aim of this study was to analyse non-lactic acid bacteria populations (NLABPs) and evaluate their role in proteolysis of cold-stored high moisture (HM) Mozzarella cheese. NLABPs reached values close to 8log cfumL -1 after seven days of cold storage. Sequencing of 16 rDNA and rpoB genes and molecular biotyping allowed to identify 66 bacterial strains belonging to 25 species from 15 genera, mainly represented by Pseudomonas, Acinetobacter, and Rahnella. Fifteen strains showed proteolytic activity values higher than 1000.00?g GlymL -1 after 24h of growth in skimmed milk. Moreover, as shown by Urea-PAGE, 11 proteolytic strains caused partial or total disappearance of at least one of the caseins. Their proteolytic behaviour was assessed even when they grew inside the governing liquid together with HM Mozzarella cheese at 4°C for 12 days. This is the first report that throws light on the complexity of NLABPs in HM Mozzarella cheese, demonstrating that some strains caused the partial hydrolysis of ?, ?, and ? caseins on its outer surface where a concomitant wrinkling and successive exfoliation became visible without significant changes in texture characteristics. (IF 3.283)

Bacillus genus is a widespread heterogeneous group of Gram-positive, facultative anaerobic, endospore-forming bacteria occurring in many types of foods. Bacillus has been reported to produce more than 45 antimicrobial molecules: some of them are of clinical value, whereas others have been assayed in vitro against food-borne microorganisms. In this work, the antibacterial activity of the cell-free concentrate supernatant of Bacillus subtilis TR50, a strain isolated from the natural microflora of an Italian traditional cured sausage, was tested by the disc diffusion assay against B. cereus DSM4312, B. cereus DSM4313, Enterococcus faecalis V583, Listeria monocytogenes DSM20600, Staphylococcus aureus NCTC8325, Aeromonas hydrophila DSM30187, Escherichia coli ATCC35401, Helicobacter pylori ATCC43504, Salmonella enterica ATCC13311, Yersinia enterocolitica DSM4780 and 17 Bacillus spp. strains isolated from traditional and industrial cured sausages. All pathogens, except E. faecalis, resulted inhibited by the cell-free concentrated TR50 supernatant. The average size of the inhibition halos on the bacterial lawns of the Bacillus species assayed decreased in the following order: B. pumilus > B. licheniformis > B. thuringensis, whereas no antimicrobial activity was detected against B. amyloliquefaciens and the other B. subtilis strains. The cell-free concentrate TR50 supernatant was extracted with ethyl acetate and the resultant organic fraction proved to retain antimicrobial activity. The same fraction was both spotted on silica thin layer chromatography (TLC) plates and loaded on Tricine SDS-PAGE gel; both TLC plates and the Tricine SDS-PAGE gel were overlaid with the Isosensitest soft-agar inoculated with Bacillus cereus DSM 4312 or B. cereus 4313. The halos on the bacterial lawn agar resulted correlated with the retention factor (Rf) of the spots, that were subsequently scraped from TLC and extracted again with methanol; among the spots (Rf 0.50-0.88), only the compounds with Rf ranging from 0.49 to 0.58 and from 0.69 to 0.77 retained antimicrobial activity. Among bands revolved by Tricine SDS-PAGE, one with an apparent molecular weight of about 2.5 kDa, displayed inhibitory activity against the same indicator strains. Finally, the organic fraction further purified by reverse phase (RP)-HPLC showed that only 20% of the TR50 specific peaks were retained; some of them fell in the chromatographic region common to commercial iturins. These preliminary results highlight the antibacterial potential of B. subtilis TR50 against widespread meat-borne pathogens that could be exploited in the sausage curing process. Further analyses are in progress to identify active compounds even though results of analytical techniques suggest the presence of lipopeptides responsible antimicrobial activity.

The aim of this work was to check the efficacy of bovine lactoferrin hydrolyzed by pepsin (LFH) to prevent blue discoloration of Mozzarella cheese delaying the growth of the related spoilage bacteria. Among 64 Pseudomonas fluorescens strains, isolated from 105 Mozzarella samples, only ten developed blue discoloration in cold-stored Mozzarella cheese slices. When Mozzarella cheese samples from dairy were treated with LFH and inoculated with a selected P.fluorescens strain, no pigmentation and changes in casein profiles were found up to 14 days of cold storage. In addition, starting from day 5, the count of P.fluorescens spoiling strain was steadily ca. one log cycle lower than that of LFH-free samples. ESI-Orbitrap-based mass spectrometry analyses allowed to reveal the pigment leucoindigoidine only in the blue LFH-free cheese samples indicating that this compound could be considered a chemical marker of this alteration. For the first time, an innovative mild approach, based on the antimicrobial activity of milk protein hydrolysates, for counteracting blue Mozzarella event and controlling psychrotrophic pigmenting pseudomonads, is here reported. © 2014 Elsevier Ltd.

8-Hydroxyquinolines show a wide range of pharmacological activities, and some are marketed as therapeutic agents. Despite the significant number of biologically active hydroxyquinolines proposed, there is a continued interest in the development of new active derivatives to overcome the drawbacks associated with their use. Herein, we report the synthesis and characterization of a set of positional isomers of hydroxyquinoline-mannose conjugates. Since in many cases the complexation ability of 8-hydroxyquinolines seems to be responsible for their pharmacological activities, we investigated the capacity of these systems to complex copper(ii) ions. We also examined diverse biological activities (antiproliferative, antimicrobial and antioxidant) of the new derivatives and their copper(ii) complex and compared them to those of their parent compounds and an analogous glucose-quinoline conjugate. All compounds show antioxidant activity that depends on the regioisomer. Moreover, specific isomers show significant antibacterial activity against P. aeruginosa and S. aureus. Furthermore only a regioisomer shows a pharmacologically relevant antiproliferative activity against human tumor cells, in the presence of copper(ii) ions.

In questo lavoro, quattro tipologie di bevande (B1, B2, B3 e B4) a differentecomposizione (mais, frumento, riso e patata) e realizzate mediante cottura esuccessiva fermentazione lattica, sono state analizzate per il loro contenutodi amido resistenti (RS). Le bevande, conservate a 4°C, hanno mostrato elevateconcentrazioni di batteri lattici (in media 8,65 log ufc/g) e ridotti valori di pH(3,71 in media). Nonostante un contenuto in peso di acqua di circa il 76,8%, lebevande hanno presentato differenti valori di viscosità apparente (da 19,4 a37,9 Pa·s) e comportamento simile a quello di un fluido pseudoplastico. Inoltrele bevande hanno mostrato percentuali in peso di RS in media superiori al5,89%, in funzione di quantità crescenti di amilosio presenti negli ingredientiutilizzati. Le analisi hanno mostrato che, in media, il 48% del contenuto totaledi amido era costituito da RS. Il presente lavoro pone le basi per lo sviluppodi bevande a base di cereali potenzialmente utili al controllo della rispostaglicemica post-prandiale, come disciplinato dal Regolamento Europeo n.432/2012 in materia di indicazioni nutrizionali-salutistiche.

The viability and competitiveness of Staphylococcus xylosus in meat mostly depend on the ability to adapt itself to rapid oxygen and nutrients depletion during meat fermentation. The utilization of nitrite instead of oxygen becomes a successful strategy for this strain to improve its performance in anaerobiosis; however, metabolic pathways of this strain underlying this adaptation, are partially known. The aim of this study was to provide an overview on proteomic changes of S. xylosus DSM 20266T cultured under anaerobiosis and nitrite exposure. Thus, two different cultures of this strain, supplemented or not with nitrite, were in vitro incubated in aerobiosis and anaerobiosis monitoring cell viability, pH, oxidation reduction potential and nitrite content. Protein extracts, obtained from cells, collected as nitrite content was depleted, were analyzed by 2DE/MALDI-TOF/TOF-MS. Results showed that DSM 20266T growth was significantly sustained by nitrite in anaerobiosis, whereas no differences were found in aerobiosis. Accordingly, nitrite content was depleted after 13 h only in anaerobiosis. At this time of sampling, a comparative proteomic analysis showed 45 differentially expressed proteins. Most differences were found between aerobic and anaerobic cultures without nitrite; the induction of glycolytic enzymes and glyoxylate cycle, the reduction of TCA enzymes, and acetate fermentation were found in anaerobiosis to produce ATP and maintain the cell redox balance. In anaerobic cultures the nitrite supplementation partially restored TCA cycle, and reduced the amount of glycolytic enzymes. These results were confirmed by phenotypic microarray that, for the first time, was carried out on cell previously adapted at the different growth conditions. Overall, metabolic changes were similar between aerobiosis and anaerobiosis NO2-adapted cells, whilst cells grown under anaerobiosis showed different assimilation profiles by confirming proteomic data; indeed, these latter extensively assimilated substrates addressed at both supplying glucose for glycolysis or fueling alternative pathways to TCA cycle. In conclusion, metabolic pathways underlying the ability of S. xylosus to adapt itself to oxygen starvation were revealed; the addition of nitrite allowed S. xylosus to take advantage of nitrite to this condition, restoring some metabolic pathway underlying aerobic behavior of the strain.

Bacillus subtilis TR50, a gram-positive endospore-forming bacteria isolated from a cured sausage, proved to be source of several antimicrobial compounds against food-borne pathogenic bacteria (Caputo et al., 2011). The wide chemical variability shown by these metabolites and their related antimicrobial efficacy depend on both the specific strain producer and on nutritional/environmental growth conditions (Baruzzi et al., 2011;Dhouha and Ellouze-Chaabouni, 2011).The difficulty for reliably identifying the antimicrobial compounds released by strains hampers their application in many fields such as controlling food microbial spoilage or fighting the emergence of antibiotic resistance to human pathogens. In general, identification of these antimicrobial compounds such as bacteriocins and lipopeptides in culture filtrates is accomplished by MALDI-TOF analysis or filtrate fractionation followed by HPLC separation and ESI -MS/MS analysis (Pabel et al., 2003; Kim et al., 2004; Caputo et al., 2011).In this work we describe a LC/MS method for the rapid screening of antimicrobial compounds produced by Bacillus strain TR50. By using HPLC separation and High Resolution MS analysis on a benchtop Orbitrap(TM)-based mass spectrometer equipped with a collision chamber we identified sixteen compounds belonging to three family of lipopeptides in the cell-free supernatant directly.This approach is herein described and proposed as a high throughput tool for the rapid identification of antimicrobial and antifungal compounds in microbial cultures.

Several Bacillus strains, typically isolated from different food sources, represent renowned producers of a multitude of low and high molecular weight compounds, including lipopeptides and macrolactones, with an importance for their antimicrobial activity. The high homology shared by many of these compounds also occurring as closely related isoforms poses a challenge in their prompt detection.Identification and structural elucidation is generally achieved by matrix-assisted laser desorption/ionization (MALDI) or liquid chromatography (LC) coupled to mass spectrometry (MS) after a pre-fractionation and/or purification step of the extract. In this paper we report the application of a method based on LC separation and high-resolution Orbitrap(TM)-based MS for the rapid screening of raw filtrate of the strain Bacillus subtilis TR50 endowed with antimicrobial activity, without requiring any sample pre-treatment.Upon direct analysis of the cell-free filtrate of Bacillus subtilis TR50 by high-resolution mass spectrometry (HRMS), different compounds families, that proved to exert a remarked antimicrobial activity against several foodborne pathogens, can be readily displayed along the chromatographic run. Among them, three different classes were identified and characterized belonging to the iturin, fengycin and surfactin groups. The high resolving power and accurate mass accuracy provided by the HRMS system in use ensured an enhanced selectivity compared to other mass spectrometers. In addition, after activation of the HCD cell, the HR-MS/MS spectra can provide insights in the structural elucidation of several compounds.The acquisition of HRMS spectra of raw filtrates of subtilis strains allows untargeted analysis of the major classes of compounds produced to be performed, thus facilitating identification of other unknown bioactive molecules after retrospective analysis. These features make this approach a fast tool applicable to the rapid screening and further identification of antimicrobial compounds released by Bacillus strains in raw filtrates

The global interest in saving food resources is leading to recycle wasted-food materials to extract useful nutrients. In dairy industry, the recycling of whey proteins determines their utilization in the healthy-addressed foods, which, however, can cause immunological responses in allergic subjects. In this work, a whey protein concentrate (WPC) was alternatively hydrolyzed with pepsin, papain, trypsin and rennin in order to attenuate or abolish the ?-lactoglobulin (BLG) antigenicity. The electrophoretic profiles of both pepsin and papain WPC hydrolysates proved the disappearance of the BLG band, even though a slight antigenicity was still found by ELISA. Pepsin hydrolysates, filtered through a 10-kDa cut-off membrane, did not produce immunological response. A deeper investigation carried out on pepsin digested and ultrafiltered samples by LC-MS/MS showed the disappearance of the immunoreactive BLG-fragment IVTQMKGLDIQKVAGTW. The remaining peptides, partially overlapped to major IgE binding epitopes, were not able to give immunoreactivity response. The combined WPC pepsin digestion with ultrafiltration confirmed to be a user-friendly strategy to reduce markedly the WPC antigenicity. The improvement of this two-steps process could be used to produce novel hypoallergenic infant food formulas.

L'aflatossina M1 (AFM1) è il principale metabolita derivante dall'idrossilazionedell'aflatossina B1 (AFB1) presente nel latte di animali alimentati con mangimi contaminati da AFB1 ed è classificato nel gruppo 2B, potenzialmente cancerogeno per l'uomo, dall'AgenziaInternazionale per la Ricerca sul Cancro (IARC). Il livello limite della AFM1 nel latte crudo,trattato termicamente e destinato alla produzione di prodotti a base di latte è fissato a50ng/kg dal regolamento europeo numero 1881 del 2006. Essendo resistente ai comunitrattamenti dell'industria alimentare, la presenza di AFM1 è documentata in tutti i prodottidella filiera lattiero casearia, inclusi yogurt e formaggi, e rappresenta un serio pericolo perla salute. Lo sviluppo di metodi per la riduzione della contaminazione di aflatossine è untema cruciale e attuale, ed è complicato dalla necessità di preservare le qualità organolettichee nutrizionali della matrice trattata. In questo lavoro è stata valutata la capacità degradativadi una laccasi da Pleurotus eryngii verso l'AFM1, sia in buffer che in latte, ed il suo effettosulla componente proteica di questa matrice al fine di verificarne l'applicazione per ilmiglioramento della sicurezza di prodotti lattiero caseari. La riduzione di AFM1 in bufferdi sodio acetato pH 6.5 1mM, a 25°C, è di ca 50% dopo 1h di incubazione e risulta completadopo 72h. Simili risultati sono stati ottenuti in latte, sebbene la cinetica di degradazioneabbia registrato un rallentamento nelle prime tre ore di trattamento. L'analisi dei patternproteici in SDS-PAGE ha evidenziato una riduzione nell'intensità delle bande di ? e ?caseine, di ?-lattoglobulina e sieroalbumina bovina, contemporaneamente alla comparsadi aggregati proteici di peso molecolare superiore ai 200kDa. I dati presentati dimostranoil potenziale applicativo della laccasi per lo sviluppo di metodologie green di degradazionedi AFM1 in prodotti a base di latte e per applicazioni tecnologiche volte al miglioramentodella reologia e alla riduzione della componente allergenica in prodotti lattierocaseari.Parole chiave: sicurezza, latte, laccasi, aflatossina M1, cross-link di proteine, reologia dellatte, allergeni

La qualità e la shelf-life della mozzarella tradizionale è correlata ai cambiamentidella composizione chimico-fisica e microbiologica del liquido di governo, nel quale siriversano zuccheri, peptidi e sali minerali provenienti dalla mozzarella. Il liquido di governo,normalmente costituito da acqua e sale, si trasforma così in un substrato di crescita in cuiproliferano differenti popolazioni microbiche, incluse quelle alterative e patogene. Ne derivache il controllo microbiologico del liquido di governo rappresenta uno dei fattori principaliper la salubrità e la shelf-life della mozzarella durante la fase di conservazione e distribuzione.Una delle possibilità per contrastare l'alterazione della mozzarella è rappresentata dall'utilizzodi un siero a composizione definita e costante. Nel presente lavoro il liquido di governo dibocconcini di mozzarella è stato sostituito con soluzioni di siero neutralizzate, pastorizzatee gelificate differenti concentrazioni di alginato di sodio (siero-alginato). Le mozzarelle,conservate a 4°C per 25 giorni, sono state valutate per i parametri reologici, chimici emicrobiologici. I risultati non hanno evidenziato significative variazioni di pH del sieroalginatofino a 7 giorni di frigoconservazione. A differenza del liquido di governo che tendead acidificarsi raggiungendo valori di pH pari a 5,75 già al settimo giorno di frigoconservazione,il siero-alginato ha mostrato maggiore stabilità mostrando simili valori di pH dopo 17giorni (5,96); trend di acidificazione simili sono stati registrati anche nelle rispettivemozzarelle. Le mozzarelle in siero-alginato hanno registrato una minore deformazione epiù elevate percentuali di recupero quando sottoposte a sforzo meccanico, rispetto ai valoriregistrati per le mozzarelle controllo. Inoltre, le mozzarelle controllo rilasciavano circa il5% in più del liquido al settimo giorno di frigoconservazione, rispetto alle mozzarelleconservate in siero alginato probabilmente a causa dell'assorbimento di liquido di governoall'interno della matrice caseinica. I dati microbiologici non hanno rivelato significativedifferenze tra i campioni in liquido di governo e quelli in siero alginato, sia nelle cineticheche nei valori della popolazioni microbiche analizzate. I risultati ottenuti in questo lavoro,sebbene preliminari, dimostrano che il siero-alginato può essere utilizzato come metodo diconservazione alternativo al liquido di governo per stabilizzare almeno i parametri strutturalie chimici della mozzarella. Inoltre, l'implementazione in caseificio di tale metodo diconservazione, favorirebbe il parziale riutilizzo del siero riducendo gli onerosi costi del suosmaltimento.

The Institute of Sciences of Food Production (ISPA) is a centre of excellence acting in the fields of scientific research, innovation and technology transfer aimed to improve safety and quality of agro-food products. The Institute policy targets continuous innovation and socio-economic growth of territories by building and implementing strategic cooperative partnerships with world leader Institutions in the area of agro-food (FAO, EFSA, FSA, USDA, etc.) and with SMEs and large enterprises as well as local universities and Consortia of P.D.O. products within funding programs promoted by local, national and international bodies.

Whey is largely discarded by cheese manufacturing plants and in the least transformed to ricotta cheese and whey concentrates. Whey-derived yeasts have recently been considered for both spoilage activity and biotechnological potential of these products. Nevertheless, their ability to hydrolyze lactose and whey proteins is less studied. In this work yeast isolates (106), randomly collected from different samples of whey, ricotta, de-proteinized whey (scotta) and fermented ricotta cheese were identified and screened for glucose, galactose and lactose assimilation and lipase and proteolytic activities. Most of isolates belonged to Kazachstania servazzii (45%) and Kluyveromyces spp. (28%), whereas the remaining genus isolates were in descending order represented by Candida (11%), Trichosporon (7%), Pichia (6%), Yarrowia (2%) and Debaryomyces (1%). Interestingly, Kazachstania servazzii predominated in all whey-based products, in contrast to microbial whey composition that varied more. Most of yeast isolates highly metabolized glucose and galactose, whereas few (17 out of 73) fermented lactose. In particular, lactose was mostly metabolized by Kluyveromyces marxianus and K. lactis but not by all K. servazzii isolates. However, three lactose negative isolates of K. marxianus (RFY9, RFY30 and RFY34) were found for the first time. Lipolytic activity were registered only for Candida spp. Yarrowia lipolytica and Trichosporon spp. isolates and for K. marxianus RFY95. In addition, few K. marxianus and K. servazzii showed proteolytic activity towards milk total proteins, but only K. marxianus RF15 was able to hydrolyze whey proteins. These results suggest that the dominant yeast species of whey and whey by-products encompass isolates with different sugar preferences. In fact, unlike the most represented K. servazzii, most of K. marxianus isolates was well adapted to whey environment because of their biochemical features. On the other hand, the dominance of K. servazzii could be supported by lactose-fermenting species such as K. marxianus or lactic acid bacteria.