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Exopolysaccharides (EPS) are known for their positive contribute to the technological properties of many foods, including bakery products. These molecules can be obtained performing piloted fermentation with lactic acid bacteria (LAB). In order to select strains able to produce EPS, a screening test in agar medium containing sucrose, fructose or glucose as carbohydrate source was performed on 21 LAB strains. Results allowed to select 8 Weissella cibaria, 2 Weissella confusa and 2 Leuconostoc spp. strains as EPS producers only in the presence of sucrose. A further screening in liquid medium enriched with sucrose (10%) (mMRS_S) indicated the W. cibaria strain C43-11 as the higher EPS producer. The selected strain was used to develop liquid sourdoughs (LSs) with dough yield (DY) 500, fermented for 15 h and based on wheat flour and wheat gluten or pseudocereals (quinoa or amaranth) in ratio 1:1, in the presence or not of sucrose at 3% (w/w, LS weight), in comparison to Lactobacillus plantarum ITM21B, a strain not producing EPS in mMRS_S. Results indicated that the use of pseudocereals favored the EPS production. Formulations were optimized by modifying DY (500 or 250), sucrose concentration (3 or 6%) and flour ratio. LSs were characterized for the content of organic acids (lactic, acetic, phenyllactic, OH-phenyllactic), pH, TTA, EPS, viscosity, total protein degradation and protein pattern. The highest EPS production (20.79 g/Kg) and viscosity (1168 mPa s) were obtained in LS (DY 250, sucrose 6%) based on quinoa flour and started with C43-11 strain. The LS was characterized by the presence of phenyllactic and OH-phenyllactic acids, protein degradation by 51.7% and proteins in the range 14-80 kDa. In these conditions, also strain ITM21B was able to produce EPS at level of 4.61 g/Kg and to degrade proteins by 53.8% in LS based on wheat and quinoa flours (1:1) (DY250 and sucrose 3%). Therefore, results demonstrated that the use of selected conditions (flour type, DY, sucrose) can stimulate specific attributes of strains making them suitable for production of short fermented (15h) liquid sourdoughs which can be used as bread improvers.

Production of the traditional Italian Focaccia bread (FcBread) requires reformulation to meet the new rules for healthier foods. We tested the applicability of a short-fermented (15 h) liquid sourdough (LS) (dough yield, DY, 250), obtained using quinoa (Q) or amaranth (Am) flour fermented with a Weissella cibaria strain (C43-11) producing exopolysaccharides (EPS) as fat replacer in yeast-leavened FcBread. The LSs were applied during the FcBread-making process at industrial pilot plant, reducing the added fat amount by 20% (FcBread-QLS and FcBread-AmLS) and products were compared with reference samples not containing EPS. All the products were analysed for physico-chemical properties (pH, TTA, organic acids, protein content and profile, instrumental colour), textural properties, sensory quality and glycemic index. The application of both pseudo-cereal-based W. cibaria LSs in FcBread allowed obtaining products with a reduced amount of fat, increased protein amount, mainly due to the albumin and glutenin/glutelin fractions, reduced glycemic index, improved texture and preserved in the traditional sensory profile of 'focaccia'.

A pectin coated dehydrated apple snack containing ≥9 log colony forming units/20 g portion of the probiotic Lactobacillus paracasei IMPC2.1, was developed. The strain was incorporated into the apple slices using vacuum impregnation (IP1) or soaking/stirring (IP2); apple slices were then coated with citrus pectin and dehydrated at 4 °C for 14 days. Microbial application led to colonization of the apple surface, as seen using scanning electron microscope images, and all probiotic products showed comparable radical scavenging activity and total phenol content. IP2 led to better bacterial survival during 30 days storage at 4 °C and color parameters comparable to the fresh apple. The simulated gastro-intestinal (GI) digestion suggested the ability of the strain to survive the digestive process. This inclusion/coating/dehydration process led to a probiotic snack, which could successfully deliver bacterial cells in a viable form to the GI tract.

The use of protective microbial strains in combination with modified atmosphere packaging (MAP) and refrigerated storage on the shelf life of tuna burgers was investigated. 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 burgers. Among them, L. paracasei showed the best preserving performance and significantly controlled both aerobic mesophilic bacteria and Pseudomonas spp. growth. Subsequently, the efficacy of the selected LAB culture under MAP conditions (5% O2 and 95% CO2) was assessed evaluating microbial and sensory quality, as well as volatile aldehyde content. Results indicated that the shelf life of burgers containing L. paracasei and packaged under MAP was 4 days longer than the control (shelf life about 6 days) and that the applied procedure represents an effective approach for the mild preservation of fish products.

The aim of this study was the characterization and selection of bacterial strains suitable for the production of a &ldquo, yeast-free&rdquo, bread. The strains Leuconostoc citreum C2.27 and Weissella confusa C5.7 were selected for their leavening and acidification capabilities and individually used as starters in bread-making tests. Liquid type-II sourdoughs, singly inoculated with the two selected strains, were characterized and employed for bread-making, through the set-up of a biotechnological protocol without the use of baker&rsquo, s yeast as a leavening agent. Aiming to verify the ability of the selected strains to dominate the fermentation process, bacteria and yeasts were isolated from liquid sourdoughs and doughs, genetically characterized and identified. Both the selected strains were suitable for the production of bread, even if L. citreum C2.27 showed the highest leavening capacity and was able to dominate the dough microbiota. The effects of different salt concentrations on the selected strain performances were also investigated. The applicability of the developed protocol, adapted for the production of the typical Apulian bread, &ldquo, puccia&rdquo, and the suitability of the strain L. citreum C2.27 were confirmed at pilot scale in an industrial bakery. The puccia bread, which was produced with the liquid sourdough fermented with L. citreum C2.27, without baker&rsquo, s yeast and salt, was similar in appearance to the conventional product containing baker&rsquo, s yeast and was judged positively by a sensory analysis.

This paper proposes bio-preservation as a tool to assure quality and safety of Spanish-style table olives cv. Bella di Cerignola. Lactobacillus plantarum 5BG was inoculated in ready to sell olives packaged in an industrial plant by using a half-volume brine (4% NaCl; 2% sucrose). The samples were stored at 4°C. The survival of the inoculated strain, the microbiological quality, the sensory scores and the survival of a strain of Listeria monocytogenes inoculated in brines were evaluated. The persistence of the Lb. plantarum bio-preserving culture was confirmed on olives (≥6.5 Log CFU/g) and in brine (≥7 Log CFU/ml). Bio-preserved olives (SET1) showed a better sensory profile than chemically acidified control olives (SET2) and the texture was the real discriminative parameter among samples. Bio-preserved olives recorded better scores during storage because of their ability to retain good hardness, crunchiness, and fibrousness without cracks. The inoculation of Lb. plantarum positively acted on the safety of olives, as the 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 in the settled procedure are suitable for the industrial packaging of Bella di Cerignola table olives, improving the process by halving brining volumes and avoiding chemical stabilizers, and significantly reducing the salt concentration. The final product is also safely preserved for almost 5 months as suggested by the reduction of the survival rate of L. monocytogenes.

Background The quality of marinated ready-to-eat (RTE) swordfish fillets, with or without inoculation with the probiotic strain Lactobacillus paracasei IMPC 2.1, was assessed over 3 months of refrigerated storage at 4 °C. RTE probiotic and control fish fillets were sampled after 7, 14, 30, 60, and 90 days of storage. Microbiological tests were performed, and fatty acid (FA) profiles and malondialdehyde content were examined. Microbiological counts, including total viable count, lactic acid bacteria (LAB), yeasts, moulds, Enterobacteriaceae, and Pseudomonadaceae were determined. Results Inoculation successfully ensured the growth of the probiotic strain and prevented the growth of other LAB. The two RTE products showed significant differences in lipid profile and lipid oxidation during storage. In particular, inoculation with L. paracasei IMPC 2.1 increased the amount of polyunsaturated FAs and limited the amount of monounsaturated FAs and oleic acid, as well as lipid oxidation. It thus represents an interesting strategy for preserving the chemical quality of fish fillets and an alternative means of delivering probiotics. Conclusion Probiotic inoculation with Lactobacillus paracasei seemed to delay lipid oxidation of the fish flesh and increase the retention of polyunsaturated FAs, suggesting a potential application for this strain in the seafood industry. © 2018 Society of Chemical Industry.

Il pane è tra gli alimenti più consumati al mondo quotidianamente e, sebbene di semplice composizione, è interessato da una costante e crescente ricerca di innovazione. Al fine di realizzare prodotti da forno senza aggiunta di lievito di birra commerciale, è stato definito un protocollo biotecnologico che prevede lo sviluppo di prodotti fermentati da batteri lattici da aggiungere direttamente, nella formulazione dei prodotti panari, agli ingredienti nella fase di impasto. La selezione di ceppi batterici adatti alla preparazione di impasti, dotati di buona capacità lievitante in assenza di lievito di birra, è stata effettuata mediante caratterizzazione tecnologica e molecolare di 12 ceppi appartenenti a 8 specie di batteri lattici. I risultati ottenuti hanno permesso di individuare due ceppi più promettenti, Leuconostoc citreum C2.27 e Weissella confusa C5.7, con i quali sono state effettuate prove di panificazione in laboratorio. In azienda è stata confermata l'applicabilità del protocollo definito e l'idoneità in particolare del ceppo L. citreum C2.27.

Ready-to-eat (RTE) swordfish 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 Lactobacillus paracasei IMPC 2.1. The strain did not affect the protein profile and total free amino acid content along the fillet shelf life. Volunteers consumed 100 g of probiotic RTE portion delivering 9 log CFU of bacterial cells, on alternate days over 20 days. Five subjects resulted to be colonized by the probiotic strain, at levels ranging from 6.30 to 7.74 log CFU/g of 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 ranging from 6.15 to 7.47 log CFU/g. This study demonstrated that 5 portions of probiotic RTE fish fillets consumed on alternate days allowed the transient colonization of gut by L. paracasei IMPC 2.1.

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 estimate rope 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 in dough 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 threshold during bread shelf-life. Similarly to the safety criteria fixed for Listeria monocytogenes contamination in foodstuff complying with EC regulation, a potential rope spoilage threshold was arbitrary fixed at 5 log CFU/g for B. amyloliquefaciens. This study further underlines a higher rope spoilage potential of the ISPA strains as compared to the ATCC strain, thus emphasizing the interest to characterise both wild strains and reference strain to account for biological variability. In conclusion, this study showed that available decision making tools which are largely recognized to predict behaviour of pathogenic strains, shall also be used with spoilage strains to help maintain food quality and extend shelf-life.

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 oC 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

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 raw materials. In total 176 isolates were collected and characterized: 13 bacterial species belonging to Bacillus (10) and Paenibacillus (3) genera were 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 spore-forming 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.

Aims: To evaluate the positive influence of the probiotic strain Lactobacillus paracasei LMGP22043 carried by artichokes into the human gut with special reference to faecal bacterial balance, short-chain fatty acid concentrations and enzyme activities in a randomized, double-blind human trial in comparison with probiotic-free artichokes (control). Methods: Twenty subjects were randomized into two groups, which consumed daily 180 g of the artichoke product (probiotic or control) during two 15-day study periods (periods 1 and 2) separated by a 15-day washout in a crossover manner. Faecal samples were subjected to microbiological and biochemical analyses, and a strain-specific PCR was performed to monitor the probiotic strain. Results: The probiotic strain, transported by the vegetable matrix, transiently colonized the gut of 17 ⁄20 subjects (median 6AE87 log CFU g )1 faeces), antagonized Escherichia coli and Clostridium spp. and increased the genetic diversity of lactic population based on REP-PCR profiles, mainly after period 1. Conclusions: The probiotic L. paracasei LMGP22043 successfully colonized the human gut and positively influenced faecal bacteria and biochemical parameters. Significance and Impact of the Study: The association of the probiotic L. paracasei with a food carrier rich in fibre can represent a new strategy for favouring a daily supply of probiotics and attracting more consumers to vegetable food fortified with probiotic strains.

Goals: To determine whether the consumption of artichokes enriched with a probiotic Lactobacillus paracasei strain affects fecal microbiota composition, fecal enzyme activity, and short-chain fatty acids production and 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 y) integrated their normal diet with artichokes (180 gr) enriched with 20 billions of L. paracasei LMGP22043. Faecal samples were subjected to microbiologic and biochemical analyses. Besides, investigations on symptom profile of the volunteers and stool consistency were carried out by using a validated questionnaire (Gastrointestinal Symptom Rating Scale) and the Bristol stool form chart. Results: The gut of all volunteers resulted to be colonized by the probiotic strain after 15 days feeding. No significant differences in the microbiological counts throughout the experimental period were registered, whereas a significant increase of butyric and valeric acids with a concomitant decrease of lactic acid was registered. At the same time, the fecal β-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.

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 4 degrees C. The probiotic strain successfully colonized the olive surface dominating the natural LAB population and decreasing the pH of brines to

The ability of potentially probiotic strains of Lactobacillus plantarum and Lactobacillus paracasei to survive on artichokes for at least 90 days was shown. The anchorage of bacterial strains to artichokes improved their survival in simulated gastrointestinal digestion. L. paracasei IMPC2.1 was further used in an artichoke human feeding study involving four volunteers, and it was shown that the organism could be recovered from stools.

With the aim of developing new functional foods, a traditional product, the table olive, was used as a vehicle for incorporating probiotic bacterial species. Survival on table olives of Lactobacillus rhamnosus (three strains), Lactobacillus paracasei (two strains), Bifidobacterium bifidum (one strain), and Bifidobacterium longum (one strain) at room temperature was investigated. The results obtained using a selected olive sample demonstrated that bifidobacteria and one strain of L. rhamnosus ( Lactobacillus GG) showed a good survival rate, with a recovery of about 10 6 CFU g −1 after 30 days. The Lactobacillus GG population remained unvaried until the end of the experiment, while a slight decline (to about 10 5 CFU g −1 ) was observed for bifidobacteria. High viability, with more than 10 7 CFU g −1 , was observed throughout the 3-month experiment for L. paracasei IMPC2.1. This strain, selected for its potential probiotic characteristics and for its lengthy survival on olives, was used to validate table olives as a carrier for transporting bacterial cells into the human gastrointestinal tract. L. paracasei IMPC2.1 was recovered from fecal samples in four out of five volunteers fed 10 to 15 olives per day carrying about 10 9 to 10 10 viable cells for 10 days.

Pseudomonas syringae pv. ciccaronei strain NCPPB2355 was found to produce a bacteriocin inhibitory against strains of Ps. syringae subsp. savastanoi, the causal agent of olive knot disease. Treatments with mitomycin C did not substantially increase the bacteriocin titre in culture. The purification of the bacteriocin obtained by ammonium sulphate precipitation of culture supernatant fluid, membrane ultrafiltration, gel filtration and preparative PAGE, led to the isolation of a high molecular weight proteinaceous substance. The bacteriocin analysed by SDS-PAGE revealed three protein bands with molecular weights of '76, 63 and 45 kDa, respectively. The bacteriocin was sensitive to heat and proteolytic enzymes, nias resistant to non-polar organic solvents and was active between pH 5.0-7.0. Plasmid-DNA analysis of Ps. syringae ciccaronei revealed the presence of 18 plasmids; bacteriocin-negative variants could not be obtained by cure experiments.

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 ºC 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 (countdetection 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.

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 insertion’s 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.

A fully automated electrophoretic method for separating cell-wall proteins was applied to 102 isolates from olive phylloplane and brine previously phenotypically identified as Lactobacillus plantarum (10), Leuconostoc mesenteroides subsp. mesenteroides (60), Leuconostoc spp. (12) and Enterococcus faecium (19). The high sensitivity of this technique allowed to discriminate isolates and to distinguish closely related strains within the same species. L. plantarum was characterized by proteins at 70.4 and 163.9 kDa, while 3 proteins (20.4, 52.9, 68. kDa) were typical for E. faecium. No characteristic proteins were found for Leuconostoc isolates except for that at 31.8 kDa mainly related to the ecological origin since only isolates from brine showed this protein. However, Leuc. mesenteroides subsp. mesenteroides and Leuconostoc spp. were differentiated by additional proteins. Sequence analysis of the 16S rRNA gene of selected strains was in agreement with the phenotypic identification and allowed to identify all Leuconostoc spp. isolates as Leuc. mesenteroides. Cell-wall protein fingerprinting performed by microfluidic technology completed the phenotypic characterization of olive-related lactic acid bacteria allowing their strain typing.

Four fermentation products (FPs) of the lactic acid bacterium Lactobacillus plantarum ITM21B were screened for their anti-Bacillus activity in vitro and in bread-making trials. Results of the storage tests performed with loaves prepared with an FP or calcium propionate demonstrated that after 3 days at 30 degrees C, gross spoilage was evident in only the control loaves, which contained Bacillus subtilis at numbers of about 10(9) cfu/g. The highest inhibitory activity was shown by DM-FP obtained by growing L. plantarum in a defined medium (DM). Significantly, this medium contained an amino acceptor of the aminoacid transamination, namely alpha-ketoglutaric acid, and an aminoacid pool. With loaves prepared using the DM-acid mixture which simulated the DM-FP composition, the same reduction of ropy spoilage as with DM-FP was obtained after 3 days, while the efficacy of the mixture decreased after 7 days. This result suggests the potential involvement of some unknown metabolites in the inhibitory activity of DM-FP. In baked products made with flour based media (M1-FP, M2-FP, M3-FP), no ropy symptoms were noticeable after 3 days storage although a considerable Bacillus count was detected. DM-FP was as effective as calcium propionate (0.3% w/w, based on flour mass) in prolonging the Bacillus free-shelf life of yeast-leavened bread for 7 days.

Strains of Lactobacillus spp., isolated from sourdough and olive brines (seven strains), and three human isolates were screened for their antagonistic activity in coculture against the ureolytic pathogen Yersinia enterocolitica. A general reduction in the pathogen population was observed after 6 h when each Lactobacillus strain was cocultured with the pathogen at a ratio of 100:1 cfu ml(-1), causing an almost complete inhibition of urease activity. Strains were also screened for their performances in in vitro tests such as adhesion ability to Caco-2 cells, tolerance to low pH, bile salts, and simulated digestion, which enabled the differences between strains to be highlighted. Three strains, L. paracasei IMPC2.1 and L. plantarum ITM21B and ITM5BG, met the main criteria for selecting effective probiotics: the ability to inhibit the pathogen Y. enterocolitica and, consequently, its urease activity (ITM21B); survival of simulated digestion (ITM21B and IMPC2.1); strong adhesion ability to enterocytes and good survival at low pH and in the presence of bile salts (ITM5BG and IMPC2.1).

A bacteriocin produced by Pseudomonas syringae pv. ciccaronei, used at three purification levels and several concentrations, inhibited the multiplication in olive tissues of P. s. subsp. savastanoi, the causal agent of olive knot disease and affected its epiphytic survival on leaves and twigs of olive plants. We determined that 6,000 Active Units ml−1 of crude bacteriocin, obtained by a simple purification step, is a concentration suitable to obtain an effective reduction of olive knot formation. Moreover, treatments of olive phylloplane with 6,000 AU m1−1 of crude bacteriocin were able to prevent bacterial multiplication by controlling the epiphytic survival of the pathogen. The inhibitory effect lasted until the end of observation (30 days). P. s. pv. ciccaronei bacteriocin, purified by ammonium sulphate precipitation, membrane ultrafiltration, gel filtration and gel electrophoresis when analysed by SDS-PAGE revealed three protein bands with molecular weights of 76, 63 and 45 kDa, respectively.

Strain ITM317 of Pseudomonas syringae subsp. savastanoi, which causes hyperplastic symptoms (knots) on olive plants, shows antagonistic activity against other strains of this plant pathogenic bacterium because of the production of a bacteriocin-like substance (BUIS). BLIS was sensitive to heat and proteolytic enzymes, slightly affected by lipolytic and glycolytic enzymes and active between pH 5.5 and 7.5, and showed a molecular mass, based on ultrafiltration, ranging between 100 and 300 kDa. In order to identify the genetic determinants involved in the antagonistic activity, a collection of Tn5-induced mutants from strain ITM317 was screened and prototrophic mutant ITM317–636, which had lost its ability to inhibit the growth of other P. s. subsp. savastanoi strains, was identified. Southern blot analysis demonstrated that the genome of mutant ITM317–636 contained a single copy of the Tn5 element and that Tn5 insertion occurred on a plasmid. The responsibility of Tn5 insertion for the above-mentioned mutated phenotype (BLIS phenotype) was demonstrated by marker-exchange mutagenesis carried out in parental strain ITM317. The EcoRI Tn5-containing fragment from mutant ITM317–636 was cloned and the DNA regions flanking Tn5 insertion were in part sequenced. Preliminary results on sequence similarity searches revealed that Tn5 insertion occurred within a DNA region encoding a predicted product 37% identical and 52% similar to a putative RHS-related protein of Xanthomonas campestris pv. campestris.

A bacteriocin produced by Pseudomonas syringae pv. ciccaronei, used at different purification levels and concentrations in culture and in planta, inhibited the multiplication of P. syringae subsp. savastanoi , the causal agent of olive knot disease, and affected the epiphytic survival of the pathogen on the leaves and twigs of treated olive plants. Treatments with bacteriocin from P. syringae pv. ciccaronei inhibited the formation of overgrowths on olive plants caused by P. syringae subsp. savastanoi strains PVBa229 and PVBa304 inoculated on V-shaped slits and on leaf scars at concentrations of 10 5 and 10 8 CFU ml −1 , respectively. In particular, the application of 6,000 arbitrary units (AU) of crude bacteriocin (dialyzed ammonium sulfate precipitate of culture supernatant) ml −1 at the inoculated V-shaped slits and leaf scars resulted in the formation of knots with weight values reduced by 81 and 51%, respectively, compared to the control, depending on the strains and inoculation method used. Crude bacteriocin (6,000 AU ml −1 ) was also effective in controlling the multiplication of epiphytic populations of the pathogen. In particular, the bacterial populations recovered after 30 days were at least 350 and 20 times lower than the control populations on twigs and on leaves, respectively. These results suggest that bacteriocin from P. syringae pv. ciccaronei can be used effectively to control the survival of the causal agent of olive knot disease and to prevent its multiplication at inoculation sites.