Bacterial contamination of foods reduces their shelf-life due to microbial metabolic activity. The presence of Bacillus spp. spores in cereal products plays a key role in the development of "bread rope" and represents a real concern for industrial production. To quantitatively predict the contamination risk, it is essential to determine kinetic data of the microorganism contaminating a specific food. Enterprises find difficult to perform experimental trials, and predictive tools can assist product and/or process development reducing laboratory experiments. Within the WP6 "Model food applicability", studies were performed to obtain information on food safety and quality when the WP2-5 models were challenged by pathogens and spoilers. The objective of this study was to obtain experimental data taking into account all the food/microorganism characteristics affecting bacterial growth. Particularly, we determined the growth/no growth boundaries and the behaviour of a selected Bacillus amyloliquefaciens strain in the bread model.

The raw materials used in the production of bakery products are the main source of sporeforming bacteria associated with the alteration of "bread rope" which occurs predominantly in humid hot weather and which results in significant economic losses in the bakery product sector (Valerio et al., 2012 , Int. J. Food Microbiol, 156, 278-285). The spores, surviving the cooking process, germinate and the vegetative cells start a degrading process of starch and bread proteins leading to the formation of slime. In the present study a screening test was carried out on 176 isolates from raw materials to evaluate which species could cause alteration in bread. Bacillus amyloliquefaciens, together with B. subtilis and B. pumilus, were the main species capable of causing the spoilage. Moreover, among the identified species, strains belonging to the B. cereus group account for 17%, of which 38% were found to be able to cause bread rope. The B. cereus group strains belong to the phylogenetic groups III and IV associated with high risk of intoxication, particularly those of group III, for which a high thermal resistance of the spores has been demonstrated during a test that simulates the thermal profile of the baking process (De Bellis et al., 2015, Int. J. Food Microbiol., 197, 30-39). To estimate the risk of bread spoilage during shelf-life, Symp Previus tool was used: cardinal parameters and growth / no growth boundaries of three strains were determined for three B. amyloliquefaciens strains isolated from bread with clear alteration symptoms, wheat and semolina. In addition, challenge tests were conducted by inoculating the spores of one strain in the mixing phase: various bread formulations were prepared to evaluate their effect on bacterial growth and the experimental results were compared with silica simulations. Finally, to estimate the probability of product contamination during shelf-life, two storage temperatures and a threshold of 5 log cfu / g were considered, a value which could cause alteration and / or represent a risk to consumer health. The characterization of the growth behavior of the three strains showed a difference in the contamination probability and subsequent alteration of the product, demonstrating the need to consider biological variability in predictive microbiological studies in order to obtain a realistic estimate of the risk of contamination. In conclusion, this study demonstrated the applicability of predictive microbiological instruments, known for assessing the risk of food contamination from pathogenic microorganisms, to the study of the behavior of alterative microorganisms to control the quality of food (Valerio et al. 2015 Food Microbiol., 45, 2-9).

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 manuscript describes antifungal, structural, thermal and morphological properties of anovel, eco-friendly bioplastic film, based on poly(butylenesuccinate) and cavoxin, a chalconephytopathogenic metabolite, isolated from cultures by the fungus Phoma cava and included insidethe biodegradable polymeric matrix. The antagonistic activity of the film explored against twofungal food contaminants Penicilliumroqueforti and Aspergillusniger, highlighted that cavoxin wasfungicide at MIC concentrations. The chemico-physical analysis were performed on films beforeand after antifungal activity. Thermal analysis evidenced both the higher thermal stability of poly(butylenesuccinate) and cavoxin blend and the sharp nucleating action of cavoxin on thepolymer. Infrared spectroscopy highlighted the presence of physical interaction between thepolar groups of the blend components, whereas gel permeation chromatography highlighted thatthe hydrolytic effect of cavoxin on poly(butylenesuccinate) was responsible for the diffusion andreleasing of the biomolecule from the matrix and UV-Vis spectroscopy provided quantitativeinformation of release kinetics. Finally, morphological analysis confirmed both the polymericsurface hydrolysis exerted by cavoxin, and the adhesion of fungal spores on films after contactoccurred during the antifungal assay.

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.

Raw materials used in bread-making process may be a rich source of spore-forming bacteria whose presence after cooking may represent a spoilage concern for bakery industries and a risk to consumer health. The aim of this study was to investigate the toxigenic potential of 54 spore-forming bacterial strains isolated from bread ingredients and bread, mainly of the Bacillus genus, and their resistance to a thermal treatment reproducing the bread cooking process to ascertain if they could represent a health concern for consumers.The potential toxigenicity of the strains was evaluated by screening the cytotoxic activity on HT-29 cells using bacterial culture filtrates after growing bacterial cells in BHI and in the bread-based medium BEB. The results showed a high cytotoxic activity of B. cereus strains, although it was lower in BEB medium. PCR analyses detected the presence of genes involved in the production of NHE, HBL or CytK toxins in B. cereus strains, while none of the tested strains contained the gene for cereulide production. Production of NHE and HBL toxins was also confirmed by specific immunoassays only for B. cereus strains. Cytotoxic activity of 13 strains belonging to B. amyloliquefaciens (7 strains), Paenibacillus spp. (3) B. mojavensis (1), B. simplex (1) and B. pumilus (1) was also detected.Interestingly, B. cereus strains assigned to phylogenetic group IV exhibited a thermal resistance markedly lower than B. cereus group III; furthermore, B. amyloliquefaciens strains almost completely survived the heat treatment, but showed a low cytotoxic activity. It is also relevant that single strains belonging to B. mojavensis and B. simplex showed a cytotoxic activity higher after growth in BEB than in BHI and a spore resistance enough to survive the bread cooking process. In conclusion, our study indicates that spore-forming bacteria could represent a risk to consumer health related to strains able to produce toxic substances and to survive bread cooking conditions.

Different spore-forming bacterial species contaminate durum wheat semolina which may affect final bread quality.

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.

Bread is one of the principal components of the human diet, but it generally undergoes staling process and microbial contamination within few days from its production. The aim of the study was to investigate on the effects of wheat bran and of a Lactobacillus brevis based-bioingredient (LbBio), obtained after 18 h growth in flour-based medium, on microbiological, physico-chemical and textural quality of yeast leavened bread (WWB) during storage.

The effects of wheat bran and of a Lactobacillus brevis based-bioingredient (LbBio), obtained after growth in flour-based medium, on quality of yeast leavened wheat bread (WWB) were investigated. Bran was used in bread formulation by substituting a part (20 g/100 g) of white wheat flour (WBB), while LbBio was used instead of the water content (WWB + LbBio and WBB + LbBio). The use of LbBio in WWB resulted in the biological acidification of the dough due to lactic, phenyllactic and OH-phenyllactic acid contents determining a high fermentation quotient value and an improved bread texture and microbiological quality. Conversely, wheat bran reduced the specific volume and crumb hardness during storage at 25 °C, and affected the antibacterial ability of LbBio during 30 °C storage. Our findings demonstrated that LbBio counteracted the negative effects of bran and allowed to obtain an enriched fibre bread with specific volume and soft crumb comparable to bread without bran.

In this study, chitosan based microbeads containing Ungeremine, an antimicrobial alkaloid particularly active against Penicillium roqueforti, a filamentous fungus responsible of the bakery products deterioration, were prepared by external gelation by using sodium tripolyphosphate (TPP) as crosslinking agent. The stability of the beads, as well as the loading efficiency of the bioactive molecule, were assessed at different pH and TPP concentrations resulting particularly enhanced at low pH. All the microbeads evidenced antimicrobial activity against Penicillium roqueforti. The release kinetics of Ungeremine was tailored by opportunely modulating pH and TPP concentrations. Morphological analysis evidenced the improvement of the structural crosslinking density of microbeads including Ungeremine and spectroscopic analysis emphasized the active participation of Ungeremine to the crosslinking process occurring between chitosan and TPP. Finally, thermogravimetric analysis confirmed the increasing of free volume in three-dimensional networks and their liability to thermal degradation.

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.

A Lactobacillus plantarum fermentation product (Bio21B), obtained after strain growth (14 h) in a wheat flour-based medium, was applied in the bread-making process as taste enhancer, in order to obtain a yeast-leavened bread with reduced salt content (20% and 50%) with respect to a reference bread (REF) not containing the fermentation product. Sensory analysis indicated that the Bio21B bread with salt reduced by 50% had a pleasant taste similar to the salt-containing bread (REF). L32 Glutamate and total free amino acid content did not differ between REF and Bio21B breads, while the acids lactic, acetic, phenyllactic, 4-OH-phenyllactic and indole-3-lactic were present only in Bio21B breads. Moreover, the presence of several umami (uridine monophosphate, inosine monophosphate, adenosine, and guanosine) and kokumi (gamma-L-glutamyl-L-valine) taste-related molecules was ascertained both in REF and in Bio21B breads. Therefore, a possible role of the acidic molecules in compensating the negative perception of salt reduction can be hypothesized.

Spore-forming bacteria are frequently isolated from raw materials of vegetable origin and they may cause spoilage and toxicity problems when these materials are used for food production. The rope spoilage of bread is associated to the presence of spores of Bacillus species in raw materials and may represent a microbiological concern relevant for bakery industries mainly under warm and humid conditions that may be frequent in Mediterranean countries. Moreover, ropy bread could also be a risk to consumer health; in fact, Bacillus species are known to cause food poisoning through the production of toxins. A relevant outbreak of rope spoilage in Southern Italy prompted this study aimed to identify spore-forming bacterial species more frequently contaminating semolina and associated to rope in bread, by using appropriate techniques for their molecular characterization and identification. A total of 176 spore-forming bacterial isolates, from 93 samples of durum wheat semolina, durum wheat grain, bread improvers, brewer's yeast and bread, were analysed by rep-PCR. Isolates representative of each of the 54 different rep-PCR profiles were identified by 16S rRNA gene sequencing and also by sequencing the gyrA or gyrB genes. Strains of the B. cereus group were further characterized by partial sequencing of the panC gene. Moreover, in order to assess the ability of all strains to cause ropy, a screening test was performed in bread slices. Our results indicated that more than 102 spores/g - amount that may cause microbial alteration in the final product - were detected in 23% of durum wheat semolina samples and in 42 % of other raw material samples. Molecular identification of isolates revealed a high bacterial diversity at the species level leading to the identification of the following species: Bacillus amyloliquefaciens, B. subtilis, B. licheniformis, B. mojavensis, B. safensis, B. oleronius, B. simplex, B. megaterium, B. pumilus, Paenibacillus peoriae, P. lautus, Paenibacillus spp., Lysinibacillus spp. and species of the B. cereus group. On the basis of partial sequencing of the panC gene, strains of the B. cereus group were included in phylogenetic groups which also include potentially dangerous strains with a cytotoxic activity and involved in food poisoning. It is also noteworthy that B. amyloliquefaciens was the species more frequently isolated and its importance for rope spoilage was recognized in this work for the first time, probably due to its previous misidentification as B. subtilis. Our results also indicated that strains of other species, mainly B. subtilis and those of the toxigenic B. cereus group, could cause rope spoilage, although different strains of the same species may behave differently. In conclusion, our results indicate that raw materials used to produce bread may represent a source of spore-forming bacteria potentially responsible for spoilage and toxicity problems, therefore more attention should be devoted to this aspect.

The aim of the current study was to improve the antifungal activity of eight lactic acid bacterial (LAB) strains by the addition of phenylpyruvic acid (PPA), a precursor of the antifungal compound phenyllactic acid (PLA), to a defined growth medium (DM). The effect of PPA addition on the LAB's antifungal activity related to the production of organic acids (PLA, D-lactic, L-lactic, acetic, citric, formic and 4-hydroxy-phenyllactic acids) and of other phenylpyruvic-derived molecules, was investigated. In the presence of PPA the inhibitory activity (expressed as growth inhibition percentage) against fungal bread contaminants Aspergillus niger and Penicillium roqueforti significantly increased and was, even if not completely, associated to PLA increase (from a mean value of 0.44 to 0.93 mM). While the inhibitory activity against Endomyces fibuliger was mainly correlated to the low pH and to lactic, acetic and p-OH-PLA acids. When the PCA analysis based on data of growth inhibition percentage and organic acid concentrations was performed, strains grown in DM+PPA separated from those grown in DM and the most active strains Lactobacillus plantarum 21B, Lactobacillus fermentum 18B and Lactobacillus brevis 18F grouped together. The antifungal activity resulted to be strain-related, based on a different mechanism of action for filamentous fungi and the yeast and was not exclusively associated to the increase of PLA. Therefore, a further investigation on the unique unidentified peak in HPLC-UV chromatograms, was performed by LC-MS/MS analysis. Actually, full scan mass spectra (negative ion mode) recorded at the retention time of the unknown compound, showed a main peak of m/z 291.0 which was consistent with the nominal mass of the molecular ion [M-H]- of polyporic acid, a PPA derivative whose antifungal activity has been previously reported (Brewer et al., 1977). In conclusion, the addition of PPA to the growth medium contributed to improve the antifungal activity of LAB strains and resulted in the production of the polyporic acid, here ascertained in LAB strains.

Fungal growth is the most frequent cause of spoilage in baked goods determining great economic losses. Lactic acid bacteria (LAB) are applied in the process of several bakery products since they significantly contribute to technological and nutritional properties, influence the food flavour and prevent spoilage by producing organic acids, hydrogen peroxide, cyclic dipeptides, bacteriocins, fatty acids, carbon dioxide, ethanol and diacetyl. Among organic acids, lactic, acetic, phenyllactic (PLA) and p-OH-phenyllactic acids (OH-PLA) play a role in inhibiting fungal and bacterial growth [1]. In particular, the inhibitory properties of PLA have been demonstrated against several fungal species. Recently, the research has been moved towards the application of biotechnology aimed to improve PLA production by microorganisms (bacteria and yeast). PLA is a by-product of phenylalanine (Phe) metabolism and its production can be significantly improved by the addition of precursors such as Phe, phenylpyruvic acid (PPA) and ?-ketoglutaric acid to the growth medium. The aim of the current study was to improve the antifungal activity of eight lactic acid bacterial (LAB) strains by the addition of PPA to a defined growth medium (DM) [2]. The effect of PPA addition on the LABs antifungal activity related to the production of organic acids (PLA, D-lactic, L-lactic, acetic, citric, formic and 4-hydroxy-phenyllactic acids) and of other phenylpyruvic-derived molecules, was investigated. In the presence of PPA the inhibitory activity (expressed as growth inhibition percentage) against fungal bread contaminants Aspergillus niger and Penicillium roqueforti significantly increased and was, even if not completely, associated to PLA increase (from a mean value of 0.44 to 0.93 mM).While the inhibitory activity against the yeast Endomyces fibuliger was mainly correlated to the low pH and to lactic, acetic and p-OH-PLA acids. When the Principal Component Analysis (PCA) analysis based on data of growth inhibition percentage and organic acid concentrations was performed, strains grown in DM + PPA separated from those grown in DM and the most active strains Lactobacillus plantarum 21B, Lactobacillus fermentum 18B and Lactobacillus brevis 18F grouped together. The antifungal activity resulted to be strain-related, based on a different mechanism of action for filamentous fungi and the yeast and was not exclusively associated to the increase of PLA. Therefore, a further investigation on the unique unidentified peak in HPLC-UV chromatograms was performed by LC- MS/MS analysis. Full scan mass spectra (negative ion mode) recorded at the retention time of the unknown compound, showed a main peak of m/z 291.0 which was consistent with the nominal mass of the molecular ion [M-H]- of polyporic acid, a PPA derivative whose antifungal activity has been previously reported against 12 species of fungi [3]. Actually, in microorganisms different from LAB, as fungi and lichens, PPA is a precursor

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.

The study aimed at improving the nutritional profile of yeast leavened salt reduced sliced bread and puccia type bread fortified with a wheat-based Lactobacillus plantarum ITM21B fermentation product (Bio21B). The protein content of bread made under laboratory conditions was increased by using: (i) chickpea flour (CF) (15% wt/wt flour) and Bio21B or (ii) the Bio21B containing a fungal protease to favour the gluten hydrolysis. Products showed increased protein and total amino acid content and improved protein digestibility. Moreover, the formula significantly affected the protein pattern of breads which, according to the results of the microfluidic two-dimensional electrophoresis (?2DE) protein pattern, were discriminated as observed by the PCA plot. The use of CF was validated at industrial pilot plant producing salt reduced sliced bread and puccia type bread. The resulting products showed improved nutritional profile and a sensory quality comparable to the company's products containing salt.

Meat is subjected to chemical and microbiological deterioration, caused by oxidative degradation of meat fat and by the presence of microbial populations, particularly Pseudomonas spp.. Therefore spoilage can be monitored through their growth and changes in TBARS, thiols and carbonyls. The aim of the current study is to evaluate the correlation between chemical and microbiological data by PCA analysis and predict meat spoilage by pseudomonads contamination under isothermal and dynamic conditions or by chemical parameters in high and low fat ground meat by multivariate statistical analysis.Beef ground meat containing a total fat amount of about 5% or 15% of fat was divided into 150 g portions, stored under isothermal conditions (0, 5, 10 and 15°C) and checked for the presence of Pseudomonas spp., Brochothrix spp., total viable count (TVC). Water loss, pH, thiols, carbonyls, TBARS, metmyoglobin, deoxymioglobin, oxymioglobin and colour changes were determined on all samples. All data were subjected to PCA analysis to evaluate the correlation between microbiological and chemical variables. Subsequently, a square root model was fitted to the growth rates of pseudomonads and used to predict spoilage of ground meat under isothermal and dynamic conditions. Additionally, multivariate statistical analysis was applied to fit a PLS (Partial Least Square Regression) model, aiming at predicting microbial counts of minced meat, based on chemical data, regardless of time-temperature storage profile. Growth models allow to predict the evolution of food spoilage during storage as a function of extrinsic conditions food characteristics. Results by the PCA analysis showed that, among microbiological and chemical indexes monitored during refrigerated storage, a positive correlation was found between increase in TBARS (0.70) and levels of pseudomonads, which were inversely correlated with thiols (-0.71) and oxymioglobin (-0.81). Moreover, results demonstrated that the fat level of ground meat did not influence the growth rate of pseudomonads allowing the application of a single model for predicting the growth of pseudomonads and time to spoilage of ground meat given dynamic temperature conditions, regardless of fat content. PLS analysis was performed considering all the microbiological parameters as Y-variables, while the chemical data were used as X-variables. The model (R2CV 0.81, RMSECV of 0.68 for pseudomonads prediction) can allow to use chemical data to predict the microbial load of ground meat during refrigerated shelf life, regardless of fat content and time-temperature conditions. Moreover, the PLS model can allow to discriminate acceptable (TVC<7 log cfu/g; TBARS 0.28-3.37 mg/kg; thiols 29.89-69.39 nmol/mg protein) from unacceptable (TVC> 7 log cfu/g; TBARS 0.13-1.33 mg/kg; thiols 52.94-73.06 nmol/mg protein) samples. Knowledge of perishability of food matrices associated to the storage history provide important information on the quality and edibility o

Alternaria species are ubiquitous and includes both pathogens and saprophytes that may damage crops and cause postharvest decay. Alternaria spp. are able to grow at low temperature and may responsible for spoilage during refrigerated transport and storage. Certain species are also capable of producing mycotoxins which can contaminate plant products. Due to their thin skin, tomatoes are particularly at risk of being infected by Alternaria, and rapid growth may occur in soft tissues, causing the well-known black mold. In that context, predictive models of mycotoxin synthesis may be helpful in determining the levels of these mycotoxins for conditions supporting growth of Alternaria spp. The objective of this study was to evaluate the effects of temperature and pH on the toxin production of an A. alternata strain in a tomato-based mediumA full factorial design of six pH levels (ranging from 2 to 7) and 11 incubation temperatures (ranging from 3.2 to 37°C) was undertaken. Tomato-based agar plates at aw 0.99 were centrally inoculated with a standardized inoculum of 40 spores/5µL. Each experimental condition was tested in triplicate. For each of the 35 combinations supporting fungal development, mycotoxin accumulation was measured. Measurements of TeA, AOH and AME concentrations were performed on plates fully covered by the fungus, using HPLC. Quantification was performed by measuring peak areas at toxins retention time and comparing them with the relevant calibration curves. The experimental observations suggest that increasing pH induces a linear increase in the square root of mycotoxin synthesis, followed by a logistic decrease. The equation used to describe this relationship is based on the model of Bréand et al. (1997) and is defined by the following parameters: pHminTOX (minimum pH for toxin production), pHoptTOX (optimum pH for toxin production), TQoptTOX (toxin quantity at pHoptTOX) and TQminTOX (the toxin quantity at high pH values). Secondary models were developed to describe the relationship between temperature and the parameters (pHminTOX, pHoptTOX and TQoptTOX) while TQminTOX was assumed to be independent of temperatureTeA has been described as the major mycotoxin produced by Alternaria spp. on tomatoes. Perhaps not surprisingly, only TeA could be measured in this work while AOH and AME have not been detected. TeA measurements show an important contribution of acidic pH to mycotoxin synthesis, in particular near pH 4. At similar pH levels, incubation at low temperatures (from 6.5°C to 12°C) resulted in higher TeA production than at temperatures above 20°C. The developed model for TeA synthesis describes accurately the experimental measurements (R2=0.98). The predicted maximum toxin quantity TQoptTOX increases from of 180 µg/g at 6.5°C to 290 µg/g at 12°C. It then decreases until reaching 70 µg/g at 30°C. The parameters pHminTOX and pHoptTOX were also found to be temperature dependentThe model for toxin production can be used togethe

In this paper, a novel and sustainable process for the fruit dehydration was described. Specifically, ediblecoatings based on pectin and honey were prepared and used as dehydrating and antimicrobial agents of cut fruitsamples, in this way promoting the fruit preservation from irreversible deteriorative processes.Pectin-honey coating was tested on apple, cantaloupe melon, mango and pineapple. The analysis were performedalso on uncoated dehydrated fruits (control). The coated fruit evidenced enhanced dehydration percentage,enriched polyphenol and vitamin C contents, improved antioxidant activity and volatile moleculesprofile. Moreover, the antimicrobial activity against Pseudomonas and Escherichia coli was assessed. Finally,morphological analysis performed on fruit fractured surface, highlighted the formation of a non-sticky andhomogeneous thin layer. These outcomes suggested that the novel fruit dehydration process, performed by usingpectin-honey coating, was able to both preserve the safety and quality of dehydrated fruits, and enhance theirauthenticity and naturalness.

Baby lettuce leaves are generally produced for the unwashed ready-to-eat market. The aim of this research was to predict sensory and microbiological aspects of this product based on physicochemical properties during storage at 4 and 10°C. Products were analysed at sampling times chosen on the basis of five sensory visual quality (VQ) levels. Samples scoring VQ5 and VQ4 were considered acceptable (Ac), whereas the remaining scores were defined as unacceptable (UAc). Each VQ level was then characterized for physico-chemical (colour, ammonium, antioxidant activity, electrolytic leakage, phenols, chlorophyll, respiratory activity) and microbiological (total viable count, Pseudomonas spp., Enterobacteriaceae, lactic acid bacteria, yeast and moulds) parameters. UAc samples also proved unsatisfactory from a microbiological point of view (total viable count >=107 CFU g-1). Partial least squares (PLS) regression analysis allowed us to identify colour change (?E*) and total chlorophyll (TC) as suitable variables to predict the microbial load (TVC) associated with each sensory VQ level. The model obtained showed R2CV=0.94, RMSECV=0.41 and a relative error of 5.61%. In conclusion, the use of these parameters as quality indicators could be a new strategy for discriminating green leafy vegetables into acceptable or unacceptable products.

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.

Table olives represent a new patented and marketable vegetable food to transport probiotics in the human intestine. The vegetable surface hosts a variety of micro-organisms which can interact with a probiotic strain with a consequent impact on the product quality. The aim of this study was to assess the dynamics of microbial populations associated with olive surface in industrial fermentation sets inoculated with the probiotic strain L. paracasei LMGP22043. The results indicated that the probiotic strain successfully colonized the olive surface and persisted in high numbers until the end of fermentation. The dynamics of microbial populations associated with olive surface and belonging to different groups indicated that olives inoculated with the probiotic, and held at room temperature, did not host Enterobacteriaceae at the end of fermentation. Yeast populations were present in a low number throughout the process. The results indicated that strain L. paracasei LMGP22043 can be used in the dual role of starter and probiotic culture making it possible to control fermentation processes, even in adverse conditions, and to obtain a functional product.

Lactobacillus plantarum and Lactobacillus paracasei are species generally used as starters in food fermentation and/or as probiotics. The growth cardinal values of these strains, characteristic parameters independent from the food matrix, can be exploited in predictive microbiology to set the appropriate food processing/manufacturing conditions. In particular, the decision making tool Sym'Previus (http://www.symprevius.org) is used to describe the behaviour of bacteria on a food matrix using these parameters. In the current study, the growth kinetics of four L. plantarum strains (from sourdough and table olives) and four L. paracasei strains (from table olives and probiotic human isolates) were investigated in order to identify the optimal temperature conditions for growth. Strains were grown in liquid medium and incubated at nine temperature levels (5.5, 11, 18, 20, 22, 27, 35, 39, 40°C). The growth was automatically monitored by a Bioscreen C using the turbidimetry method or determined manually after static incubation. Maximum growth rates (?max) for each temperature were obtained fitting data by the Rosso model. To estimate the cardinal growth values, the ?max values relevant to each temperature were fitted to the growth cardinal model. The following average of cardinal values were identified: for Lactobacillus plantarum strains Tmin 2.05±0.54°C, Topt 33.74±0.63°C and Tmax 39.79±0.50°C. In the case of L. paracasei strains: Topt 34.90±1.70 °C and Tmax 38.99±1.72°C. The Tmin values were lower than 0°C for three out of four strains highlighting a strain variability in growth abilities at low temperatures. The ?opt values ranged from 0.778 and 0.90 h-1 for L. plantarum strains and from 0.553 and 0.654 h-1 for L. paracasei strains. This preliminary study demonstrates a bacterial variability in growth abilities, mainly for L. paracasei strains, which should be taken into account in predictive microbiology to obtain a reliable prediction of growth in food conditions.

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

Fifty-four spore-forming bacterial strains isolated from bread ingredients and bread, mainly belonging to the genus Bacillus (including Bacillus cereus), together with 11 reference strains were investigated to evaluate their cytotoxic potential and heat survival in order to ascertain if they could represent a risk for consumer health. Therefore, we performed a screening test of cytotoxic activity on HT-29 cells using bacterial culture filtrates after growing bacterial cells in Brain Heart Infusion medium and in the bread-based medium Bread Extract Broth (BEB). Moreover, immunoassays and PCR analyses, specifically targeting already known toxins and related genes of B. cereus, as well as a heat spore inactivation assay were carried out. Despite of strain variability, the results clearly demonstrated a high cytotoxic activity of B. cereus strains, even if for most of them it was significantly lower in BEB medium. Cytotoxic activity was also detected in 30% of strains belonging to species different from B. cereus, although, with a few exceptions (e.g. Bacillus simplex N58.2), it was low or very low. PCR analyses detected the presence of genes involved in the production of NHE, HBL or CytK toxins in B. cereus strains, while genes responsible for cereulide production were not detected. Production of NHE and HBL toxins was also confirmed by specific immunoassays only for B. cereus strains even if PCR analyses revealed the presence of related toxin genes also in some strains of other species. Viable spore count was ascertained after a heat treatment simulating the bread cooking process. Results indicated that B. amyloliquefaciens strains almost completely survived the heat treatment showing less than 2 log-cycle reductions similarly to two strains of B. cereus group III and single strains belonging to Bacillus subtilis, Bacillus mojavensis and Paenibacillus spp. Importantly, spores from strains of the B. cereus group IV exhibited a thermal resistance markedly lower than B. cereus group III with high values of log-cycle reductions. In conclusion, our results indicate that spore-forming bacteria contaminating bread ingredients and bread could represent a source of concern for consumer health related to the presence of strains, such as strains of B. cereus group III and single strains of other species, showing the ability to produce toxic substances associated to a thermal resistance enough to survive the bread cooking conditions.

IntroductionLactobacillus plantarum and Lactobacillus paracasei are species generally used as starters in food fermentation and/or as probiotics. The growth cardinal values of these strains, characteristic parameters independent from the food matrix, can be exploited in predictive microbiology to set the appropriate food processing/manufacturing conditions. The aim of this study was to transfer available and validated mathematical models used to ensure food safety to technological microflora in order to assess the impact of environmental conditions on bacterial growth for further industrial applications.Material and methodsA total of 4 strains of L. plantarum isolated from sourdough and table olives as well as 4 strains of L. paracasei strains isolated from table olives and probiotic human isolates were investigated to determine growth cardinal values. For that purpose strains were grown in liquid medium and incubated at nine temperature levels from 5.5 to 40°C. The growth was automatically monitored by a Bioscreen C using the turbidimetry method or determined manually after static incubation. Maximum growth rates (?max) for each temperature were obtained fitting data by the Rosso model. To estimate the cardinal growth values, the ?max values relevant to each temperature were fitted to the growth cardinal model using Sym'Previus decision making tool (www.symprevius.eu).ResultsCardinal values were determined for Lactobacillus plantarum strains with the following average values for Tmin 2.05±0.54°C, Topt 33.74±0.63 °C and Tmax 39.79±0.50°C. In the case of L. paracasei strains, a wider diversity was obtained with average values for Topt 34.90±1.70 °C and Tmax 38.99±1.72°C. The Tmin values were lower than 0°C for three out of four strains. The ?opt values ranged from 0.778 and 0.90 h-1 for L. plantarum strains and from 0.553 and 0.654 h-1 for L. paracasei strains.DiscussionThe determination of growth cardinal values is useful to assess variability in bacterial growth abilities. This preliminary study also demonstrates the transfer of already available mathematical models to technological and probiotic strains. Indeed several user friendly tools are available to facilitate the practical use recognized mathematical models for growth prediction. The generic approach used in Sym'Previus is applicable to pathogenic, spoilage and technological microflora to further predict the impact of environmental conditions on bacterial growth. Besides the importance of using real life strains, this study further underlines the importance of characterized collection for the selection of the bacterial strain to be used in challenge test to optimize process/shelf-life and ensure food safety and quality.

Dehydrated raw materials used to produce bread are known to be highly contaminated with spore-forming bacteria. Once incremented in the bread, spores will germinate and exponential growth of amylase producing species such as Bacillus amyloliquefaciens may cause ropy bread spoilage in Mediterranean countries and huge economical costs. The aim of this study was to perform challenge test studies to compare growth experimental counts in bread and in silico growth predictions for various bread recipes.B. amyloliquefaciens ISPA-S109.3 was selected after previous characterization underlining its resistance to the baking process and high spoilage potential. Challenge tests were performed according to standardized methods with ISPA-S109.3 spore inoculation (4log spore/g) in the ingredient mixture. Home bread-making machine was used to bake the 4 bread recipes, i.e. wheat bran bread (WBB) and white wheat bread +/- bioingredient yielding various intrinsic parameters (aw: 0.93-0.96 and pH: 4.67-5.87). Bread incubation was performed at 20, 25 and 30°C +/- 0.01. Artificially contaminated pan bread and controls were prepared in three replicates. Distinction between ISPA-S109.3 and naturally contaminated Bacillus was performed using Rep PCR fingerprinting. The enumeration of ISPA-S109.3 was performed to determine growth kinetics and mathematical modeling using Sym'Previus.Based on fitted experimental growth kinetics, growth rate in WBB at 30°C was determined (µmax= 0.415+/-0.013h-1) and optimal growth rate calculated (µopt=1.61+/-0.048h-1) to further predict growth for the 5 tested conditions. Comparisons between experimental kinetics and growth predictions underlined that challenge test data were comprised into the 90% confidence interval of the predictions. These results highlight that major impact on growth was due to pH, aw and temperature while the impact of food matrix quantified by the µopt was sufficient, even in the case of sourdough. This study received funding from FP7-222-654-2 DREAM project and data related to strain and behavior diversity were incremented in Sym'Previus.

Le materie prime impiegate nella produzione di prodotti panari rappresentano la principale fonte di batteri sporigeni associati all'alterazione "pane filante" che si manifesta prevalentemente in condizioni di clima caldo umido e che determina rilevanti perdite economiche nel settore panario (Valerio et al., 2012, Int. J. Food Microbiol, 156, 278-285). Le spore, sopravvivendo al processo di cottura germinano e le cellule vegetative avviano un processo degradativo dell'amido e delle proteine del pane che porta alla formazione di esopolisaccaridi (slime). Nell'ambito del presente studio è stato condotto un test di screening su 176 isolati da materie prime per valutare quali fossero le specie in grado di causare l'alterazione nel pane. Bacillus amyloliquefaciens, insieme con B. subtilis e B. pumilus, sono risultate le principali specie in grado di causare il fenomeno alterativo. Inoltre, tra le specie identificate, i ceppi appartenenti al gruppo B. cereus rappresentano il 17%, e tra questi il 38% è risultato in grado di provocare l'alterazione. I ceppi del gruppo B. cereus appartengono ai gruppi filogenetici III e IV, associati ad elevato rischio di intossicazione, in particolare quelli del gruppo III, per i quali è stata evidenziata un'elevata resistenza termica delle spore durante un test che simula il profilo termico del processo di cottura del pane (De Bellis et al. 2015, Int. J. Food Microbiol., 197, 30-39). Per stimare il rischio di insorgenza dell'alterazione del pane durante la shelf-life, è stata impiegata la microbiologia predittiva (Sym'Previus tool): sono stati determinati i parametri cardinali e i limiti di crescita (growth/no growth boundaries) di tre ceppi di B. amyloliquefaciens isolati rispettivamente da pane con chiari sintomi di alterazione, da grano e da semola. Inoltre sono stati condotti challenge test inoculando le spore di uno dei ceppi nella fase di impasto: sono state preparate varie formulazioni del pane per valutarne l'effetto sulla crescita batterica e i risultati sperimentali sono stati confrontati con le simulazioni in silico. Infine, per stimare la probabilità di contaminazione del prodotto durante la shelf-life, sono state considerate due temperature di conservazione e la soglia di 5 log cfu/g che può causare l'alterazione e/o rappresentare un rischio per la salute del consumatore. La caratterizzazione del comportamento di crescita dei tre ceppi ha evidenziato una differenza nella probabilità di contaminazione e successiva alterazione del prodotto, dimostrando la necessità di considerare la variabilità biologica negli studi di microbiologia predittiva al fine di ottenere una stima realistica del rischio di contaminazione. In conclusione, questo studio ha dimostrato l'applicabilità degli strumenti di microbiologia predittiva, noti per la valutazione del rischio di contaminazioni alimentari da microrganismi patogeni, allo studio del comportamento di microrganismi alterativi per il controllo della qualità dei prodotti a