The main aim of this work was the identification of genetic determinants involved in bacteriocin production by strain ITM317 of Pseudomonas savastanoi pv. savastanoi, besides bacteriocin characterization. The bacteriocin was observed to be a heat-sensitive, high molecular weight proteinaceous compound. We identified a transposon (Tn5)-induced mutant which had lost its ability to produce the bacteriocin. The Tn5 insertions responsibility for the above mutated phenotype was demonstrated by marker-exchange mutagenesis. An EcoRI DNA fragment, corresponding to the EcoRI Tn5-containing fragment of the mutant, was also cloned from the wild-type strain, and its introduction into the mutant complemented the mutation. Moreover, that fragment enabled bacteriocin production by P. s. pv. savastanoi ITM302, a strain not previously capable of doing so. DNA sequence analysis revealed that Tn5 insertion occurred in the mutant within a large ORF encoding a protein which showed similarity with proteins from the Rhs family. The DNA region including that ORF showed features which have been considered typical of the Rhs genetic elements previously identified in other bacteria but whose function is as yet unclear. The results of this study for the first time identify an Rhs-like element in P. s. pv. savastanoi, and for the first time indicate that an Rhs element is involved in bacteriocin production, also suggesting this possible function for Rhs genetic elements previously characterized in other bacteria.

The survival of 3 pathogens Listeria monocytogenes ATCC19115, Salmonella enterica subsp. enterica ATCC13311, and Escherichia coli ATCC8739 was evaluated over time in ready-to-eat (RTE) artichoke products processed or not with the probiotic strain Lactobacillus paracasei LMGP22043. Both probiotic and standard products (final pH about 4.0; aw=0.98) dressed with oil and packaged in modified atmosphere were inoculated with pathogens at a level of about 3 log CFU/g and stored at 4 °C for 45 d. Pathogens decreased in the probiotic product in 2 descent phases, without shoulder and/or tailing as observed by fitting the models available in the GInaFit software to the experimental data. S. enterica subsp. enterica was completely inactivated after 14 and 28 d in probiotic and standard products, respectively; E. coli was inhibited in the probiotic food at day 4 (count <detection limit (DL) 1 log CFU/g), while in the standard product, it survived until the end of experiment. L. monocytogenes decreased in the probiotic product at day 1 reaching values below the DL after 14 d, while 21 d were needed in the standard product, and survived in both samples until the end of the experimental period. Therefore, the probiotic strain, representing always more than the 93% of lactic acid bacteria (about 7 log CFU/g) during the entire experimental period, combines the efficacy of a protective culture, which can control the development of pathogens during storage with probiotic benefits.

Probiotic strains used as starter cultures may combine the positive image of fermented foods with a functional appeal. Moreover, these bacteria can be used as bioprotective cultures to extend the shelf-life of ready-to-eat (RTE) foods and to control harmful microorganisms Indeed, new probiotic vegetable products - table olives, artichokes and cabbage - have been obtained after a mild fermentation process supported by the probiotic strain Lactobacillus paracasei LMGP22043 (Valerio et al. 2013; Lavermicocca et al. 2016). In this study the probiotic strain L. paracasei LMGP22043, was applied as bioprotective culture (7 log CFU/g) to preserve RTE artichokes and as a starter to ferment blanched white cabbage thus preserving its nutritional quality. The probiotic strain was used to pilot the fermentation of cabbage leading, after 71 h fermentation, to a final product containing about log 8 CFU/g live cells and the 35% of the total glucosinolates (GSs) detected before fermentation otherwise completely lost during conventional fermentation of sauerkraut (Sarvan et al. 2013). After 30 days of refrigerated vacuum packed storage, GSs and the probiotic concentration still persisted. Moreover, the bioprotective features of the probiotic strain were ascertained in RTE artichokes challenged (3 log CFU/g) with pathogens Listeria monocytogenes ATCC19115, Salmonella enterica subsp. enterica ATCC13311, or Escherichia coli ATCC8739 and stored at 4°C for 45 days. All pathogens decreased in the probiotic RTE product, whereas a longer survival was observed in the standard product (control). At the same time, the probiotic RTE product still contained an adequate amount (more than 7 log CFU/g) of live probiotic cells (Valerio et al. 2013). Therefore, studies demonstrated that vegetables can be processed to obtain functional products, using a probiotic strain which plays the double role of starter and bioprotective culture.

This study aims at the characterisation of growth behaviour of three strains of Bacillus amyloliquefaciens,isolated from ropy bread (ATCC8473), wheat grain (ISPA-S109.3) and semolina (ISPA-N9.1) to estimaterope spoilage risk in pan bread during shelf-life using the Sym'Previus tool. Cardinal values and growth/no growth boundaries were determined in broth, while artificial spore inoculations were performed indough for various pan bread recipes to compare experimental counts with in silico growth simulations.Finally, two storage scenarios were tested to determine the probability to reach a spoilage thresholdduring bread shelf-life. Similarly to the safety criteria fixed for Listeria monocytogenes contamination infoodstuff complying with EC regulation, a potential rope spoilage threshold was arbitrary fixed at 5 logCFU/g for B. amyloliquefaciens. This study further underlines a higher rope spoilage potential of the ISPAstrains as compared to the ATCC strain, thus emphasizing the interest to characterise both wild strainsand reference strain to account for biological variability. In conclusion, this study showed that availabledecision making tools which are largely recognized to predict behaviour of pathogenic strains, shall alsobe used with spoilage strains to help maintain food quality and extend shelf-life.

This study examines the diversity of spore-forming bacteria isolated from raw materials/bread using molecular methods along with a rapid and innovative technology, the FT-NIR spectroscopy. Microbiological analysis showed that 23% of semolina and 42% of other raw materials (including grain, brewer yeast, improvers) contained more than 100 spores/g and more than 50% of each kind of sample was contaminated at a level ranging from 1 to 100 spores/g. A high bacterial diversity characterized rawmaterials. In total 176 isolates were collected and characterized: 13 bacterial species belonging to Bacillus (10) and Paenibacillus (3) generawere identified by sequencing of 16S rRNA, gyrA or gyrB genes. The two closely related species Bacillus amyloliquefaciens (strain N45.1) and Bacillus subtilis (strain S63) were also analyzed by the spectroscopic technique FT-NIR. This analysis gave clear discrimination between the strains in the score plot obtained by the PCA and allowed to identify the spectral region 5600-4000 cm-1 as the information-rich region for discrimination. B. amyloliquefaciens, possibly misidentified as B. subtilis in previous studies, was recognized as the most frequent species, found also in ropy bread. Moreover, the screening test for rope production indicated that mainly B. amyloliquefaciens, together with B. subtilis and Bacillus pumilus, could cause spoilage in bread, even if the last two species were represented by a low number of isolates. The Bacillus cereus group and Bacillus megaterium showed a lower percentage (30-70%) of isolates potentially able to cause the rope, but considering the high number of B. cereus group isolates detected in this study, this bacterial group should also be considered important in rope spoilage. In conclusion, results demonstrate that raw materials used to produce bread represent a rich source of sporeforming bacteria, therefore their microbiological quality should be monitored before use. Moreover, this study highlights for the first time the importance of the species B. amyloliquefaciens in rope spoilage and indicates that other species may also cause this alteration although strains of the same species may behave differently.

Goals: To determine whether the consumption of artichokes enriched with a probiotic Lactobacillus paracasei strain affects faecal microbiota composition, faecal enzyme activity and short chain fatty acids (SCFAs) production as well as symptom profile in patients suffering from constipation. Background: Constipation is a common gastrointestinal disorder often related to the food diet. The beneficial effects of probiotics and prebiotics on human health are under investigation. Moreover, recent studies assessed the suitability of some vegetables, particularly olives and artichokes, to vehicle probiotic strains into the gastrointestinal tract. Study: For 15 days, 8 volunteers (3M/5F age 40±14 yr) integrated their normal diet with artichokes (180 gr) enriched with 20 billions of Lactobacillus paracasei LMGP22043. Faecal samples were subjected to microbiological and biochemical analyses. Besides, investigations on symptom profile of subjects and stool consistency were performed by using a validated questionnaire (GSRS) and the Bristol stool form chart.Results: The gut of all subjects resulted to be colonized by the probiotic strain after 15 days feeding. No significant differences in the microbial counts throughout the experimental period were registered, while a significant increase of butyric and valeric acids with a concomitant decrease of lactic acid was registered. At the same time the faecal ²-glucuronidase activity was significantly reduced. Finally, the analysis of symptom profile indicated a marked reduction in abdominal distension and feeling of incomplete evacuation.Conclusions: These preliminary data suggest that novel approaches for treating constipation can come through ingestion of probiotic vegetable products, that, acting as symbiotics, can ameliorate this common disorder.

In virtù della relazione tra salute e batteri benefici, il mercato degli alimenti funzionali probiotici ha subito negli ultimi anni un forte impulso orientandosi sull'individuazione di alimenti della dieta quotidiana in grado di agire da carrier biologici per il trasporto di cellule vive e attive nell'intestino. In quest'ambito è stato realizzato un filetto di pesce spada pronto da mangiare (Ready-To-Eat) e in grado di trasportare il Lactobacillus paracasei IMPC2.1 (LMG P-22043), noto per le sue proprietà probiotiche e tecnologiche (Valerio et. al 2015; Riezzo et al. 2012), nell'intestino umano. È stato condotto un trial nutrizionale su 8 soggetti sani che hanno inserito nella loro dieta a giorni alterni porzioni di filetto probiotico (100 g contenenti 9 log CFU di L. paracasei) per un totale di 20 giorni. Dopo il consumo di sole 5 porzioni di pesce (10 giorni), l'intestino di cinque soggetti risultava già colonizzato dal microorganismo (6.30-7.74 log CFU/g feci), fornendo quindi gli stessi benefici di un'assunzione quotidiana che è generalmente suggerita per gli alimenti probiotici. Dopo 20 giorni (10 porzioni) il ceppo probiotico era presente in tutti soggetti a concentrazioni comprese tra 6.15-7.47 log CFU/g di feci. Questo studio ha dimostrato che 5 porzioni di filetti di pesce probiotico Ready-To-Eat consumate a giorni alterni consentono la colonizzazione transiente dell'intestino da parte di L. paracasei IMPC 2.1. Inoltre, il pesce probiotico amplia l'offerta di alimenti funzionali e rappresenta un modo per raggiungere l'obiettivo di "dieta funzionale" anche per consumatori/pazienti sottoposti ad un regime dietetico modificato, come ad esempio diete a basso contenuto di colesterolo o senza lattosio. Inoltre lo studio ha dimostrato che il ceppo probiotico selezionato sopravvive nel prodotto nel corso della conservazione in una marinatura a ridotto contenuto di sale e contribuisce a preservare le proprietà nutrizionali del pesce, mantenendo inalterato il contenuto di amminoacidi ed il profilo proteico.

Conventional fermentation of cabbage like in sauerkraut production leads to a complete elimination of glucosinolates (GSs). In order to retain GSs in fermented cabbage, the effect of a thermal treatment (blanching) followed by fermentation (4% brine at 25 °C) by the probiotic strain Lactobacillus paracasei LMG P22043, was investigated. After 71 h fermentation the probiotic blanched cabbage still contained 27.2 ± 2.3 ?mol/100 g GSs, corresponding to the 35% of the total GSs before fermentation. A final count of L. paracasei of 8.26 ± 1.2 log10 CFU/g, and a final pH of 4.12 ± 0.1 were reached. After 30 days of refrigerated vacuum packed storage, 23.7 ± 1.5 ?mol/100 g of GSs still persisted. In the control cabbage (blanched and not inoculated with L. paracasei) no fermentation occurred and as a result final pH was 6.10 ± 0.21, leading to a product not suitable for storage and consumption. Compared to traditional sauerkraut the final product has the advantage of containing a high content of phytochemicals in combination with a high count of live probiotic bacterial cells.

La produzione delle olive da mensa avviene mediante un processo spontaneo di fermentazione ad opera dei batteri lattici naturalmente presenti sull'oliva o provenienti dal processo produttivo. Lo studio delle specie microbiche coinvolte nella fermentazione è essenziale per evitare processi alterativi e ottenere un prodotto con migliori caratteristiche organolettiche e maggiore conservabilità. L'analisi delle proteine di parete è un valido metodo di discriminazione di popolazioni provenienti da differenti nicchie ecologiche ma nessuna caratterizzazione ha riguardato i batteri lattici provenienti dall'ecosistema olivo (filloplano e salamoie). Lo studio, condotto mediante elettroforesi automatizzata, ha consentito una accurata discriminazione fra 102 isolati - appartenenti alle specie Lactobacillus plantarum, Leuconostoc mesenteroides e Enterococcus faecium - e la distinzione di ceppi strettamente correlati all'interno della stessa specie. L'analisi delle sequenze del gene 16S rRNA di ceppi selezionati ha confermato l'identificazione fenotipica. L'utilizzo dell'elettroforesi automatizzata che sfrutta i principi della tecnologia microfluidica può rappresentare una valida alternativa alla tradizionale metodica SDS-PAGE per la discriminazione di grandi popolazioni microbiche di interesse agro-alimentare.

Bread shelf-life is generally compromised by fungi mainly belonging to Aspergillus and Penicillium genera which colonize the surface of the product within few days from the production. The aim of this study was to select a Lactobacillus brevis-based bioingredient (LbBio) able to inhibit the growth of Aspergillus niger ITEM5132 on pan bread in order to prolong its shelf-life. Four LbBio formulations, obtained by growing a selected L. brevis strain in a flour-based medium containing different carbon sources or acid precursor (fructose, LbBio1; fructose and maltose, LbBio2; ?-chetoglutaric acid, LbBio3; short-chain fructooligosaccharides, LbBio4), were evaluated for their content of organic acids (lactic, acetic, propionic, phenyllactic, 4-hydroxyl-phenyllactic, valeric, isovaleric acids). The LbBio formulations were applied in yeast-leavened bread during bread-making trials and the resulting products were inoculated after baking with A. niger spore suspension and the fungal growth was monitored during storage (25°C for 6 days). The formulation showing the highest inhibitory activity was separated by ultra-filtration method, and whole and fractions obtained were evaluated for their in vitro activity. The fraction showing the highest activity was further separated by gel-filtration and the resulting products were investigated for their protein content and in vitro inhibition. The results from the bread-making trials performed using different formulations of LbBio showed a delay in fungal growth (1 day) respect to the bread not containing the bioingredient (control) or including calcium propionate (0.3% w/w). The formulation LbBio2, prepared with fructose and maltose 1% (w/vol), contained the highest amount of total organic acids, including phenyllactic and hydroxyl-phenyllactic acids, and reduced the visual spoilage of bread. This formulation was separated by ultra-filtration and fractions containing metabolites with molecular weight higher than 30 kDa showed high inhibitory effect in the in vitro assay. In particular, the microfluidic analysis highlighted the presence of a protein with a molecular weight of 56 kDa only in the active fraction. Further studies have to be done in order to identify the protein involved in the antifungal activity.

This paper proposes bio-preservation as a tool to assure quality and safety of Spanishstyletable olives cv. Bella di Cerignola. Lactobacillus plantarum 5BG was inoculatedin ready to sell olives packaged in an industrial plant by using a half-volume brine (4%NaCl; 2% sucrose). The samples were stored at 4C. The survival of the inoculatedstrain, the microbiological quality, the sensory scores and the survival of a strain ofListeria monocytogenes inoculated in brines were evaluated. The persistence of theLb. plantarum bio-preserving culture was confirmed on olives (6.5 Log CFU/g) andin brine (7 Log CFU/ml). Bio-preserved olives (SET1) showed a better sensory profilethan chemically acidified control olives (SET2) and the texture was the real discriminativeparameter among samples. Bio-preserved olives recorded better scores during storagebecause of their ability to retain good hardness, crunchiness, and fibrousness withoutcracks. The inoculation of Lb. plantarum positively acted on the safety of olives, asthe D-value of L. monocytogenes was reduced from 40 (SET2) to 5 days (SET1).In conclusion, Lb. plantarum 5BG and the physico-chemical conditions achieved inthe settled procedure are suitable for the industrial packaging of Bella di Cerignolatable olives, improving the process by halving brining volumes and avoiding chemicalstabilizers, and significantly reducing the salt concentration. The final product is alsosafely preserved for almost 5 months as suggested by the reduction of the survival rateof L. monocytogenes.

Ready-to-eat (RTE) swordfish fillets were used in a human feeding study involving 8 volunteersto assess the suitability of the fish matrix in delivering viable cells of the probioticLactobacillus paracasei IMPC 2.1. The strain did not affect the protein profile and total freeamino acid content along the fillet shelf life. Volunteers consumed 100 g of probiotic RTEportion delivering 9 log CFU of bacterial cells, on alternate days over 20 days. Five subjectsresulted to be colonized by the probiotic strain, at levels ranging from 6.30 to 7.74 log CFU/gof faeces, after eating 5 portions of the product (T1, 10 days). After 20 days (T2, 10 portions)the probiotic strain was recovered in the faeces of all subjects at concentrations rangingfrom 6.15 to 7.47 log CFU/g. This study demonstrated that 5 portions of probiotic RTE fishfillets consumed on alternate days allowed the transient colonization of gut by L. paracaseiIMPC 2.1.

This study reports the dynamics of microbial populations adhering on the surface of debittered green olives cv. Bella di Cerignola in fermentation sets inoculated with the probiotic strain Lactobacillus paracasei IMPC2.1 in different brining conditions (4% and 8% (w/v) NaCl) at room temperature and 4oC. The probiotic strain successfully colonized the olive surface dominating the natural LAB population and decreasing the pH of brines to 5.0 after 30 days until the end of fermentation. The dynamics of microbial populations associated with olive surface and belonging to the different groups indicated that inoculated olives held at room temperature did not host Enterobacteriaceae at the end of fermentation. Yeast populations were present in a low number ( log10 5.7CFU/g) throughout the process. A considerable genetic diversity of LAB species colonizing the olive surface was found mainly in inoculated set brined in 8% NaCl, as indicated by the Shannon diversity index calculated for each set. Generally, strains of Lactobacillus coryniformis, L. paracasei, L. plantarum, L. pentosus, L. rhamnosus, L. brevis, L. mali, L. vaccinostercus, L.casei, Leuconostoc mesenteroides, Leuc. pseudomesenteroides, Lactococcus lactis, Weissella paramesenteroides, W. cibaria, Enterococcus casseliflavus group and E. italicus were identified during the whole process. In particular, L. pentosus was the most frequently isolated species and it showed a high strain diversity throughout fermentation in all processes except for the one held at 4oC. Also a notable incidence of Leuc. mesenteroides on olives was highlighted in this study during all fermentation. Results indicated that the human strain L. paracasei IMPC2.1 can be considered an example of a strain used in the dual role of starter and probiotic culture which allowed the control of fermentation processes and the realization of a final probiotic product with functional appeal.

Quality of marinated swordfish ready-to-eat (RTE) fillets, with or withoutinoculation of probiotic strain Lactobacillus paracasei IMPC 2.1, was assessed over 3 months of refrigerated storage at 4°C. Fish fillets from probiotic-inoculated and control RTE samples were sampled at 7, 14, 30, 60, and 90 days of storage. Microbiological tests, fatty acid (FA) profiles and malondialdehyde content were examined. Microbiological counts, including total viable count, lactic acid bacteria (LAB), yeasts and moulds, Enterobacteriaceae and Pseudomonadaceae were determined.RESULTS: Inoculation successfully ensured the growth of the probiotic strain, preventing thegrowth of other LAB types. The two RTE products showed significant differences in lipid profile and lipid oxidation along storage. In particular, inoculation with L. paracasei IMPC2.1 increased the amount of polyunsaturated FAs, and limited the amount of monounsaturated FAs and oleic acid, as well as lipid oxidation, thus representing an interesting strategy to preserve fish fillets chemical quality and to alternatively deliver probiotics.CONCLUSION: Probiotic inoculation seemed to delay the fish flesh lipid oxidation and increased the retention of polyunsaturated FAs suggesting a potential application of this probiotic strain in the seafood industry.

The beneficial relationship between the host health and its gut microbiota has led in the last decades to the enormous increase of research and commercial interest in the development of probiotic preparations to manipulate microbiome in helping host physiology or preventing diseases [1]. Producers of functional foods are exploring technological solutions for developing foods supplemented with additional functional benefits but with high level of consumer satisfactions (health-orientated as well as taste-orientated). Many strains belonging to lactic acid bacterial (LAB) species are widely used as probiotics in commercial products since, additionally to functional properties conferred to foods, they may also act as starters establishing a mild fermentation that protects products from deterioration. Particularly, a L. paracasei strain (IMPC 2.1) has been studied and used for the development of innovative patented functional foods based on the association of that probiotic strain with vegetables (olives, artichokes, cabbage etc.) whose commercialization has been authorized by the Italian Ministry of Health [2]. Clinical trials performed on subjects suffering from constipation and healthy subjects demonstrated the efficacy of L. paracasei IMPC 2.1 carried by ready-to-eat artichokes in transiently colonize the human gut thus modulating potentially harmful bacteria, faecal enzyme activity and short chain fatty acid production as well as symptom profile [3]. Currently among probiotic foods, fish products have been rarely investigated as vehicles for probiotic strains in humans, even if they are suitable to sustain viable LAB populations [4]. Therefore our investigation aimed to evaluate the ability of the probiotic L. paracasei strain IMPC 2.1 to survive in marinated ready-to-eat swordfish fillets and to reach, viable, the human gut in an alternate day- based study. Probiotic ready-to-eat swordfish fillets (PR-RTE) were prepared by brining (3% NaCl) fillets for 2 days with live cells of the probiotic strain at 7 log CFU/g; then fillets were drained, seasoned with sunflower seed oil, spiced with parsley and stored under vacuum packing at 4°C in polyethylene trays (shelf life 4 months). The final product, containing more than 7 log CFU/g of the probiotic strain, showed physicochemical characteristics (protein profile and total free amino acid content, pH, aw) similar to those of control-RTE fillets (traditionally processed with an acidic marinade) along shelf life. The probiotic fillets were used in a human feeding study involving 8 volunteers to assess the suitability of the fish matrix in delivering viable cells of the probiotic strain [5]. Participants integrated their dietary intakes throughout the study period with a portion (100 g) of PR-RTE fillets containing about log 9 CFU of probiotic cells. The protocol was approved by a local Scientific and Ethics Committee. The dietary intervention study lasted 27 days and subjects consumed PR-RTE fillets on

To evaluate the positive influence of the probiotic strain Lactobacillusparacasei LMGP22043 carried by artichokes into the human gut with specialreference to faecal bacterial balance, short-chain fatty acid concentrations andenzyme activities in a randomized, double-blind human trial in comparisonwith probiotic-free artichokes (control).Methods: Twenty subjects were randomized into two groups, which consumeddaily 180 g of the artichoke product (probiotic or control) during two 15-daystudy periods (periods 1 and 2) separated by a 15-day washout in a crossovermanner. Faecal samples were subjected to microbiological and biochemicalanalyses, and a strain-specific PCR was performed to monitor the probioticstrain.Results: The probiotic strain, transported by the vegetable matrix, transientlycolonized the gut of 17 D 20 subjects (median 6Æ87 log CFU g)1 faeces), antagonizedEscherichia coli and Clostridium spp. and increased the genetic diversityof lactic population based on REP-PCR profiles, mainly after period 1.Conclusions: The probiotic L. paracasei LMGP22043 successfully colonizedthe human gut and positively influenced faecal bacteria and biochemicalparameters.Significance and Impact of the Study: The association of the probiotic L. paracaseiwith a food carrier rich in fibre can represent a new strategy for favouringa daily supply of probiotics and attracting more consumers to vegetable food fortified with probiotic strains.

The use of protective microbial strains in combination with modified atmospherepackaging (MAP) and refrigerated storage on the shelf life of tuna burgers wasinvestigated. Preliminary, the protective ability of three lactic acid bacteria (LAB)cultures (Lactobacillus casei, Lactobacillus paracasei and Lactobacillus plantarum)have been assessed on ready-to-cook tuna fish burgers. Among them, L. paracaseishowed the best preserving performance and significantly controlled both aerobicmesophilic bacteria and Pseudomonas spp. growth. Subsequently, the efficacy of theselected LAB culture under MAP conditions (5% O2 and 95% CO2) was assessedevaluating microbial and sensory quality, as well as volatile aldehydes content. Resultsindicated that the shelf life of burgers containing L. paracasei and packaged underMAP was 4 days longer than the control (shelf life about 6 days) and that the appliedprocedure represents an effective approach for the mild preservation of fish products.