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.

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.

Ochratoxin A (OTA) is a mycotoxin with a main nephrotoxic activity contaminatingseveral foodstuffs. In the present report, five soil samples collected from OTA-contaminatedvineyards were screened to isolate microorganisms able to biodegrade OTA. When cultivated inOTA-supplemented medium, OTA was converted in OTalpha by 225 bacterial isolates. To revealclonal relationships between isolates, molecular typing by using an automated rep-PCR systemwas carried out, thus showing the presence of 27 different strains (rep-PCR profiles). The16S-rRNA gene sequence analysis of an isolate representative of each rep-PCR profiles indicatedthat they belonged to five bacterial genera, namely Pseudomonas, Leclercia, Pantoea, Enterobacter, andAcinetobacter. However, further evaluation of OTA-degrading activity by the 27 strains revealedthat only Acinetobacter calcoaceticus strain 396.1 and Acinetobacter sp. strain neg1, consistentlyconserved the above property; their further characterization showed that they were able to convert82% and 91% OTA into OTalpha in six days at 24 °C, respectively. The presence of OTalpha, asthe unique OTA-degradation product was confirmed by LC-HRMS. This is the first report onOTA biodegradation by bacterial strains isolated from agricultural soils and carried out underaerobic conditions and moderate temperatures. These microorganisms might be used to detoxifyOTA-contaminated feed and could be a new source of gene(s) for the development of a novelenzymatic detoxification system.

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.

Ochratoxin A (OTA) is a mycotoxin denoted by a nephrotoxic activity contaminating several foodstuffs. Nowadays, the biological systems for OTA degradation to the less toxic OT? aroused great interest by the scientific community. In the present study, bacteria able to biodegrade OTA were isolated from soil samples collected in OTA-contaminated vineyards. Soil samples were collected from five vineyards of Negroamaro and Primitivo grape cultivars in Salento (Southern Italy). They were cultured in appropriate media added with OTA, mycotoxin degradation was determined by HPLC/FLC analysis and bacterial colonies were isolated by plating. Clonal relationships between isolates was assessed by using an automated rep-PCR system and then each strain was identified by 16S rRNA gene sequencing. A total of 225 bacterial isolates were able to convert OTA in OT?. The molecular analysis of the above isolates showed the presence of 27 different strains (rep-PCR profiles). The sequence analysis of the 16S-rRNA gene indicated that they belonged to five bacterial genera: Pseudomonas, Leclercia, Pantoea, Enterobacter and Acinetobacter. Additional assessment of OTA-degrading capacity of the 27 strains indicated that only the Acinetobacter calcoaceticus strain 396.1 and the A. sp. strain neg1 conserved the above property: both strains were further studied thus showing that they were able to convert 82% and 91% OTA into OT? in 6 days at 24°C, respectively. The occurrence of OT?, as the sole OTA-degradation product was established by LC-MS/MS.This is the first description on OTA biodegradation under aerobic conditions and moderate temperature by bacterial strains from agricultural soils. These microorganisms might be used to detoxify OTA contaminated feed and could be a resource for the development of a new enzymatic detoxification system.

Many studies provided evidence that intestinal microbiota, including probiotic bacteria, can modulate the gut immune system(1). It is also recognized that different strains of the same bacterial species may differentially polarize the immune response. Considering the importance of the species Lactobacillus paracasei, also frequently isolated from different foods, we evaluated the ability of five genetically characterized L. paracasei strains to modulate the activity of mouse dendritic cells (DCs), which are potent antigen-presenting cells and can effectively induce primary immune responses. The results indicated that all the strains stimulated the phenotypic maturation of DCs, but cytokine profiles revealed a different secretion of interleukin IL-2, IL-12 and IL-10 by DCs. Specifically two strains, IMPC 4.1 and ATCC 334, stimulated the highest levels of IL-2 and IL-10. In this regard, IMPC 4.1 can be considered particularly efficient as an anti-inflammatory/regulatory strain. Other two strains, IMPC 2.1 and LMG P-17806, previously identified as probiotic strains, were characterized by an intermediate ability to induce cytokine secretion. While strain LMG 23554, characterized by a potential pathogenic behavior, was differentiated from the others by a very low ability to induce IL-10 and IL-12 secretions. In conclusions, our results indicate that the evaluation of types and/or levels of secreted cytokines by DCs could be relevant to define pro- or anti-inflammatory properties of a bacterial strain and its more appropriate clinical use. Moreover, a very low ability of a strain to induce cytokines secretion could suggest that further investigations are required to evaluate its possible use as a probiotic.

The modulation of the immune system is recognized as one of the main mechanisms by which probiotic bacteria exert health-promoting effects. To compare the immunomodulatory properties of L. paracasei strains, we studied their interaction with dendritic cells (DCs) which are important in the earliest bacterial recognition and in determination of the subsequent T-cell responses, playing a pivotal role in both innate and adaptive immunity. The results indicated that all the strains stimulated phenotypic maturation of DCs but they induced different cytokine secretion by DCs and, in particular, a different ratio IL-10/IL-12, which is considered indicative and predictive of in vivo pro- or anti-inflammatory properties. In fact, probiotic strains IMPC 2.1 and LMG P-17806 were characterized by a similar and intermediate ability to induce cytokine secretion, inducing a low pro-inflammatory response, while strain IMPC 4.1 was characterized by very interesting and peculiar anti-inflammatory properties. In contrast, the potentially unsafe strain LMG 23554 showed a very low ability to induce cytokine secretion. Further studies were carried out to ascertain if the immunomodulatory activity of L. paracasei strains on DCs was caused by bacterial metabolites released in the culture medium. Therefore, bacterial culture filtrates were obtained after the growth of the strains in two media generally used for the culture of DCs and their effects on the maturation of DCs and cytokine production were evaluated. An artichoke phenolic extract (APE) was also added to the media before the bacterial growth to reveal its potential activity on immunomodulation of DCs. The results revealed diverse immunomodulatory properties of the culture filtrates depending on the growth medium, on the strain and on the addition of APE; an interesting anti-inflammatory activity of a culture filtrate obtained after the growth of strain IMPC 2.1 in one of the media supplemented with APE was highlighted.

This study was performed to ascertain the immunomodulatory effect of Lactobacillus paracasei strains. These strains were also genetically characterized.Methods and Results: The strains were genetically differentiated by using the fluorescent-amplified fragment length polymorphism technique, which led to the identification of several molecular markers unique to each strain. To determine the immunomodulatory properties, we evaluated the effect of strains on dendritic cell maturation, dextran uptake, ability to induce proliferation of allogenic T cells and cytokine secretion. The results indicated that all the strains stimulated phenotypic maturation of dendritic cells (DCs), but they acted differently on DCs in relation to the other tested properties; notably, a different effect on cytokine secretion was detected.Conclusions: The results of this study revealed different immunomodulatory properties of strains of the species Lact. paracasei. Strain IMPC 4.1 showed an interesting anti-inflammatory ability. Probiotic strains IMPC 2.1 and LMG P-17806 were characterized by a similar and intermediate ability to induce cytokine secretion in contrast to the very low ability of strain LMG 23554.Significance and Impact of Study: Our results confirm that each single strain of a bacterial species appears to influence the immune system in a peculiar manner. The evaluation of the different types and D or levels of cytokines whose secretion is induced by each strain could be relevant to define its pro- or anti-inflammatory properties and its more appropriate clinical use.

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.

The most recent trend in research on probiotic bacteria aims at the exploitation of bioactive bacterial compounds that are responsible for health-promoting effects and suitable for medical applications. Therefore, the main purpose of this study was to ascertain if the immunomodulatory effects of L. paracasei strains on dendritic cells (DCs) were caused by bacterial metabolites released in the culture medium. For that reason, bacterial strains were grown in two media generally used for the culture of DCs, and the effects of culture filtrates on the maturation of DCs and cytokine production were evaluated. Moreover, to reveal potential synergistic effects on the immunomodulation of DCs, an artichoke phenolic extract (APE) was added to the media before bacterial growth. The experiments pointed out an interesting anti-inflammatory activity of a culture filtrate obtained after growing a probiotic L. paracasei strain in one of the media supplemented with APE. Therefore, this culture filtrate--which combines the anti-inflammatory activity and the other well-known health-promoting properties of artichoke phenolic compounds--could represent the basis for future particular exploitations.

Descrizione del Progetto "EZIOCONTROL": obiettivi, metodologie di ricerca, risultati

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.

Studi recenti indicano che diversi ceppi di Lactobacillus paracasei sono dotati di una interessante attività anti-infiammatoria dovuta alla produzione in coltura di una o più molecole attive. Tali sostanze (post-biotici) potrebbero essere utilizzate per la preparazione di nuovi alimenti funzionali privi di batteri vivi ed utili nel trattamento di patologie infiammatorie.

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.

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.

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.

Probiotic bacteria are generally available for consumers as concentrated preparations or incorporated in milk-based foods. Due to an increased interest of the market for probiotic foods as well as to meet a demand of industry for innovation, a new kind of probiotic food has been developed using table olives as a carrier. Green table olives, produced according to the Spanish-style, are obtained by a fermentation which can be carried out by spontaneous microflora, even if the use of starter cultures is desirable to obtain a more controlled process. In this regard, the selected strain Lactobacillus paracasei IMPC 2.1 of human origin was used in the dual role of starter and probiotic culture, and here we describe the different aspects which have been evaluated and solved to utilize that strain for the development of a new table olive-based probiotic food. These aspects include selection of the strain on the basis of its probiotic properties, molecular characterization, compatibility with the carrier food and efficacy as starter. The final product meets commercial and functional requirements throughout its shelf-life.

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.

Pseudomonas savastanoi pv. savastanoi is the causal agent of Olive knot disease, relying on the Type Three Secretion System (TTSS) for its pathogenicity. In this regard, nothing was known about the two other pathovars belonging to this species, pv. nerii and pv. fraxini, characterized by a different host range. Here we report on the organization of the entire TTSS cluster on the three pathovars, and a phylogenetic analysis including the TTSS of those bacteria belonging to the P. syringae complex sequenced so far, highlighting the evolution of each operon (hrpC, hrpJ, hrpRS, hrpU and hrpZ). Moreover, by Real-Time PCR we analyzed the in vitro expression of four main TTSS genes, revealing different activation patterns in the three pathovars, hypothetically related to their diverse virulence behaviors.

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