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Abstracts From the Meeting

Journal of Clinical Gastroenterology: November/December 2014 - Volume 48 - Issue - p S113-S120
doi: 10.1097/MCG.0000000000000245
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Lactobacillus paracaseiF19 Modulates the Inflammatory Response in an Ex Vivo/In Vitro Model of Biopsies Samples From Diarrhea-predominat IBS Patients

Alessandra Cianflone*, Debora Compare*, Marco Sanduzzi Zamparelli*, Emilia Russo*, Maria Timone*, Alberto Martino*, Gerardo Nardone*. *Department of Clinical Medicine and Surgery, University of Naples Federico II, Naples, Italy.

Objective: Mounting evidence suggests that the gut microbiota-related low-grade mucosal inflammation and local immune activation may play a key role in the pathogenesis of irritable bowel syndrome (IBS). This hypothesis opens the way to new treatment strategies that target the gut microbiota, including probiotics. Aim of this study was to evaluate the effect of Lactobacillus paracasei F19 in modulating the TLRs-mediated inflammatory immune response in diarrheapredominant IBS.

Methods: We enrolled 4 outpatients diagnosed as IBS, according to Rome III criteria. Patients underwent lower endoscopy with mucosal biopsies that were placed in organ culture dishes and treated with 4×106 probiotic bacteria (Lactobacillus paracasei F19) for 6 and 24 hours. Semiquantitative RT-PCR was performed for the evaluation of the transcriptional levels of TLR4, TLR2, Myd-88, p-65, IL-10, IL-4, TGF-β baseline and after 6 hours of probiotic treatment. Protein levels of p-65 were evaluated by Western blot baseline and after 24 hours of probiotic treatment.

Results: Lactobacillus paracasei F19 treatment reduced transcriptional levels of TLR4, Myd-88, and p-65 and increased TGF-β and IL-4 with respect to no-treated biopsies. However, only the variation of IL-4 and p-65 reached a significant P-value (P=0.01 and P=0.05, respectively). At protein levels, probiotic treatment reduced p-65 expression with respect to no-treated samples. Finally, Lactobacillus paracasei F19 treatment did not induce differences of TLR2 and IL-10 expression in respect to basal condition.

Conclusions: Our preliminary results demonstrated that in an ex vivo organ culture model of IBS the anti-inflammatory Lactobacillus paracasei F19 may modulate the inflammatory response. Further analyses are needed to confirm this data.

The Probiotic StrainB. animalisSubsp.lactisBI07 Differently Modulates the Inflammation-dependent Unbalances of the Mucosa-associated Intestinal Microbiota of Breastfed Infants and Adults

Clarissa Consolandi*, Manuela Centanni†, Silvia Turroni†, Simone Rampelli†, Clelia Peano*, Marco Severgnin*, Elena Biagi†, Gianluca De Bellis*, Patrizia Brigidi†, Marco Candela†. *Institute of Biomedical Technologies, Italian National Research Council, Segrate, Milan; †Department of Pharmacy and Biotechnology, University of Bologna, Bologna, Italy.

Objective: This study aims to highlight the inflammation-dependent unbalances of the mucosal-adherent fraction of the gut microbiota (GM) in healthy breastfed infants and adults, evaluating the potential of the probiotic strain Bifidobacterium animalis subsp. lactis BI07 to recover a mutualistic profile.

Methods: Fecal stools from 30-year-old and breastfed infants donors were allowed to interact in adhesion assays with nonstimulated and TNF-α-stimulated HT29 cells, the latter in the presence or absence of B. animalis subsp. lactis BI07. The GM of breastfed infants and adults was analyzed by pyrosequencing. Slurries and adherent microbial communities were characterized by qPCR and HTF-Microbi.Array.

Results: Breastfed infants and adults possess phylogenetically and functionally different GM mucosa-associated fraction. While in adults the mucosal fraction is distorted by the inflammatory stimulus, in breastfed infants it remains unchanged. B. animalis subsp. lactis BI07 was effective in partially readdressing the inflammation-dependent dysbiosis of the GM mucosa-associated fraction in adults.

Conclusions: Our ex vivo system can be used in noninvasive GM case-control screenings to assess the potential of probiotics to modulate different disease-associated deviations from a healthy mucosa-associated GM profile, either in adults or infants.

Intestinal Mucus Alterations Induced by a Chronic Stress are Linked to a Shift in O-Glycosylation Rather Than to Mucin Expression Changes: Prevention by a Probiotic Treatment

Stéphanie Da Silva*, Catherine Robbe-Masselot†, Alessandro Mancuso†, Myriam Mercade-Loubière*, Christel Cartier‡, Marion Gillet‡, Afifa Ait-Belgnaoui§, Pascal Loubière*, Etienne Dague∥, Vassilia Théodorou‡, Muriel Mercier-Bonin*. *LISBP-CNRS/INRA, INSA; ‡UMR 1331-INRA TOXALIM, EI Purpan; §UMR 1331-INRA TOXALIM/Lallemand, EI Purpan; ∥LAAS CNRS-ITAV-UMS3039, Université de Toulouse, Toulouse; †Unité de Glycobiologie Structurale et Fonctionnelle, CNRS, Université de Lille 1, Villeneuve d’Ascq, France.

Objective: In contrast to well-known disruption of intestinal epithelial barrier in irritable bowel syndrome (IBS), changes in structure and physical properties of the mucus layer remain unknown. Despite efficacy of probiotics on IBS symptoms, their influence on the mucus barrier integrity has not been yet investigated.

Methods: Thus, we aimed at evaluating in rats whether (i) a chronic stress (Water Avoidance Stress, WAS) modifies the number of intestinal goblet cells, Muc2 expression, the biochemical structure of mucin O-glycans, as well as the mucus layer morphology; and (ii) a probiotic treatment with Lactobacillus farciminis prevents these alterations.

Results: WAS did not modify neither the number of goblet cells nor Muc2 expression in the colon and ileum. In contrast, WAS strongly affected O-glycosylation of ileal and colonic mucins, as reflected by the appearance of more elongated polylactosaminic O-glycan structures. Mucin sialylation and sulfation remained unchanged. Scrapped mucus of stressed rats was flattened and less cohesive versus controls, as shown by Atomic Force Microscopy. Such structural and morphologic modifications influence physicochemical interactions within the mucin fiber network, negatively impacting the mucus barrier integrity. The probiotic treatment prevented the WAS-induced changes in O-glycan structure and partially restored the mucus gel morphology.

Conclusions: In conclusion, we have shown for the first time that a chronic stress in rats induces changes in O-glycan structure and mucus layer morphology without affecting mucin expression. The probiotic treatment with L. farciminis, by preventing these changes, contributes to the enhancement of the mucus barrier function.

Influence of the Microbial Community in Kefir Grains on the Survival and Growth ofStaphylococcus aureusin Milk

Sabina Fijan*, Sonja Sostar Turk*. *Faculty of Health Sciences, University of Maribor, Maribor, Slovenia.

Objective: According to literature, Kefir contains live active cultures of strong strains of microorganisms that help to overtake pathogenic organisms. Regular Kefir consumption can help relieve all intestinal disorders, create a healthier digestive system, and possesses antibacterial activity in vitro against gram-positive and gram-negative bacteria and against some fungi. Staphylococcus aureus is an important potential pathogen that can cause severe hospital-acquired infections. The objective of our study was to investigate the influence of the microbiota in Kefir grains on the survival and growth of S. aureus.

Methods: Different concentrations of S. aureus cultures were inoculated into 20 mL of milk with 1 g of Kefir grains. The samples were incubated at 20°C for 48 hours. Serial 10-fold dilutions were plated on different nutrient and selective media. The results were expressed as colony-forming units (CFU).

Results: Milk with 1 g of Kefir grains had a total count of aerobic microbes of 5.0×105 CFU/mL after 48-hour incubation. The initial concentrations of S. aureus inoculated in milk and milk with Kefir grains were 1.0×1016 to 6.88×1012 CFU/mL. Concentrations of S. aureus in milk and milk with Kefir grains after 48-hour incubation were 2.3×1010 to 6.5×105 CFU/mL and 3.7×105 to 4.0×100 CFU/mL, respectively.

Conclusions: Although the concentration of S. aureus in milk was decreased after 48-hour incubation for 6 log steps, the decrease in milk with Kefir grains was much higher at 11 log steps. Therefore, the microbiota in Kefir grains successfully inhibited the growth of S. aureus in milk.

Probiotic Supplementation Modulates Intestinal Microflora and Improve NK Cytotoxic Activity in Pediatric Patients After Hematopoietic Stem Cell Transplantation (HSCT)

Sara Carlotta Tagliacarne*, Chiara Valsecchi*, Lorenza Montagna*, Maria Luisa Callegari†, Patrizia Comoli‡, Anna Maria Castellazzi*. *Department of Clinical, Surgical, Diagnostic and Pediatric Sciences, University of Pavia; ‡Oncohaematological Unit, Foundation IRCCS Policlinico San Matteo, Pavia; †Biotechnology Research Centre, Università Cattolica del Sacro Cuore, Piacenza, Italy.

Conclusions: Combined prescription of multiprobiotic Symbiter with broad-spectrum antibiotics, for example, β-lactam antibiotics, is effective to prevent development of hepatic and colonic dysfunctions.

Objective: In this study we evaluated, in pediatric HSCT recipients, the role of probiotic supplementation in preventing infections and intestinal complications and in improving microbiological and immune reconstitution. We characterized intestinal microflora and analyzed the probiotic modulation of innate immunity in recipients and controls without probiotic supplementation.

Methods: Two patients were supplemented with L. paracasei I 1688, L. salivarius I 1794, and 1 patients was supplemented with B. bifidum W23, B. lactis W52, Lactococcus lactis W58, since 1 to 2 months after HSCT. Gut microflora was evaluated through gel electrophoresis in denaturing gradient (DGGE), following RT-PCR and innate antiviral response was detected through NK cytotoxicity assay. NK cells population was characterized in flow cytometry.

Results: Conditioning therapy for HSCT induced intestinal dysbiosis, with an increasing of Enterococcus faecium, while probiotic supplementation restore the normal heterogeneity of microbiological pattern, with an improvement of Ruminococcus species. Probiotic supplementation in patients improve NK activity, not detected in the control group. No patients developed bacterial infections during follow-up. One patient, presenting an IV degree acute intestinal GVHD unresponsive to therapy, showed resolution of inflammatory lesions, and reduction to II degree GVHD after probiotic supplementation.

Conclusions: These data may suggest that probiotics can be useful to stimulate motility through the restoration of normal intestinal microflora. Probiotics stimulate proliferation and activation of NK cells, underlying a positive effect on antiviral response. Data on GvHD complications show an improvement in 2 patients’ prognosis, suggesting a possible role of probiotics in the improvement of immunologic reconstitution and patients’ follow-up.

Effect ofLactobacillus salivariusStrain LPLM-O1 in a Murine Model ofSalmonella typhimuriumInfection

Erica Castro*†‡§, Juan P. Mellado*, Pamela Contreras*, Maria J. Aguayo*, Karen Pardo*, Elizabeth Monsalves*, Fernando Cárcamo*, Rodrigo Vera*, Jaime Cofré*, Hernán Montecinos†, Margarita González‡. *Laboratory of Lactic Bacteria; †Departments of Biological Sciences; ‡Pharmacy, University de Concepción; §Department of Medicine, St. Sebastian University. Concepción, Chile.

The prevention of bacterial infections with probiotics is an interesting study field. The objective of this study was to research the preventive effect of Lactobacillus salivarius LPLM-O1, isolated from breast milk, in a murine model of infection with Salmonella typhimurium ATCC 14028. Balb/c mice (n=76), 6 to 8 weeks old, were grouped on: G1 (base diet), G2 (109 CFU of LPLM-O1, once daily for 7 d), G3 (108 CFU of S. typhimurium), and G4 (daily oral dose of 109 CFU of LPLM-O1 for 7 d, and then 108 CFU of S. typhimurium). Survival rate, splenic index (SE), specific growth rate (SGR), differential leukocyte count, and bacterial count were quantified. The survival rate in groups G1 and G2 was 100%. In G3 and G4, there was a 40% mortality rate at days 7 and 10, with 100% and 86% rates at day 16, respectively. SGR was for G1=1.6, G2=1.3, G3=0.1, and G4=0.6, with no diarrhea or bleeding. Differential leukocyte count was indicative of acute bacterial infection. The highest Salmonella counts were on infected mice that had not been previously treated with the strain LPLM-O1. S. typhimurium count in feces was significantly lower in G4 (105 CFU/g) than in G3 (107 CFU/g) (P≤0.05). Once Salmonella had colonized organs like the liver, spleen, and intestines, LPLM-O1 administration did not have an impact on the count. Taking into account the high infection rate, preventive administration of LPLM-O1 slightly raised the SGR and the survival rate, while lowering the pathogenic microbial load in the intestines.

This work was financed with founds of the INNOVA CHILE 09CAVC-6955 grant.

Protective Effects ofLactobacillus acidophilusCRL 1014 and Yacon (Smallanthus sonchifolius) on DMH-induced Colon Carcinogenesis in Wistar Rats

Camilla Martins Avi*, Ana Paula Silva Almeida*, Maria Angela Tallarico Adorno†, Elizeu Antõnio Rossi*, Katia Sivieri*. *Department of Food & Nutrition, Faculty of Pharmaceutical Sciences, São Paulo State University, Araraquara; †Department of Hydraulic and Sanitation, University of São Paulo, São Carlos, Brazil.

Objective: In this study, the Lactobacillus acidophilus CRL 1014 and yacon (Smallanthus sonchifolius) were used to compare their protective potential against 1,2 dimethylhydrazine dihydrochloride (DMH)-induced chemical colon carcinogenesis in Wistar rats.

Methods: The rats (n=25) were divided into 5 groups. Colon cancer was induced by the application of DMH twice a week in a dose of 40 mg/kg SC in groups G2-G5. Before 2 weeks of DMH-initiation, groups were fed basal diet (G1 and G2) or basal diet+L. acidophilus CRL 1014 at 108 CFU/mL (G3), basal diet+yacon (G4), basal diet+L. acidophilus CRL 1014 at 108 CFU/mL, and yacon (G5) for 8 weeks. Stool samples were collected, every 15 days during the experimental protocol for the pH, ammonium, short chain fatty acids (SCFA) analysis, and intestinal microbiota composition. For count of Aberrant Crypts Foci (ACF), the proximal, medial, and distal colons were stained in Leishman (Merck) for 2 minutes.

Results: The pH was significantly lower in the group fed with probiotics. All groups showed increased in ammonium ion. A significantly increase in SCFA contents was observed in the group 5. The group 5 showed the significantly increase in Lactobacillus spp. and Bifidobacterium spp., and Clostridium spp. was not a significant difference. A significant reduction in number of ACF was observed in groups 3, 4, and 5; however, the greater reduction was observed in group 5.

Conclusions: This study indicates that yacon plus L. acidophilus CRL 1014 intake may reduce the development of chemically induced colon cancer.

Effects of Multistrain Probiotic Preparation on the Cytokine Profile in Rat Liver and Pancreas Upon Long-term Gastric Hypochlorhydria

Lyudmila Ostapchenko*, Kateryna Dvorshchenko*, Oksana Bernyk*, Sergii Vakal*. *ESC “Institute of Biology,” Taras Shevchenko National University of Kyiv, Kyiv, Ukraine.

Objective: Long-term gastric hypochlorhydria (LTGH) can lead to digestive disorders, dysbiosis, and inflammation development in gastrointestinal tract, including liver and pancreas. Probiotics are widely used for restoration of gastrointestinal homeostasis by virtue of a broad spectrum of biological activities. Cytokine profile is a useful indicator of inflammation upon different diseases. So, the aim of study was to determine the cytokine profile in rat liver and pancreas upon LTGH and administration of multistrain probiotic (MSP).

Methods: Experiments were performed on white nonstrain rats. LTGH was induced by 28-day-long abdominal omeprazole injection (14 mg/kg/d). The second animal group was simultaneously orally treated with multiprobiotic preparation containing 18 strains. The levels of interleukins (IL) and TNF-α were determined in liver and pancreatic homogenates by immunochemiluminometry.

Results: The increased levels of IL-6, IL-8, and TNF-α (in 8, 1,2 and 1,3 times, respectively) and decreased concentration of IL-10 (in 1,3 times) in rat liver in comparison with control upon LTGH were observed. Moreover, levels of IL-6, IL-8, and TNF-α were higher than control in 1,5, 4,5 and 3 times, respectively, whereas the concentration of IL-10 was 1,8 times lower. Joint administration of MSP with omeprazole was associated with normalization of studied parameters in liver and pancreatic tissues.

Conclusions: The increase in proinflammatory cytokines (IL-6, IL-8, TNF-α) and decrease in anti-inflammatory cytokine IL-10 indicate the inflammation development in rat liver and pancreas upon LTGH. MSP ameliorates endogenous intoxication and promotes immunity correction of hypochlorhydria-related systemic consequences, probably through tolerated microbiota restoration.

Probiotic Cheese With Cardioprotective Effect

Epp Songisepp*, Pirje Hütt*, Merle Rätsep*, Mihkel Zilmer†. *Bio-Competence Centre of Healthy Dairy Products LLC, Bio-Competence Centre of Healthy Dairy Products LLC; †Department of Biochemistry, University of Tartu, Tartu, Estonia.

Cardiovascular disease is the most frequent cause of mortality and morbidity in elderly. Long-term exposure to elevated blood pressure (BP) in younger subjects also leads to partly irreversible risk situation, shortening otherwise longer life expectancy. Previously (6th Probiotics, Prebiotics, New Food, OC2.11) we reported that 3-week consumption of L. plantarum TENSIA (DSM 21380) containing cheese helps to maintain normal BP.

Objective: To demonstrate sustained effect of continuous consumption of the probiotic over longer period (8 wk) as suggested by EFSA (2011).

Methods: A randomized, blind, controlled, parallel-designed 2-armed intervention (ISRCTN29105501) was carried out. Clinically healthy adults (n=206; 43.6±11.1 y) consumed 50 g of control or probiotic cheese for 4+4 weeks (daily dose of TENSIA 10.0 logCFU). Primary outcome: significant (P<0.05) SBP reduction. Secondary outcome measure: significant DBP reduction. Additional markers: changes in oxidized-LDL, total peroxide count (TPX), total antioxidant capacity (TAC), oxidative stress index (OSI).

Results: Median change during 4- and 8-week probiotic consumption: SBP (−5.28 and −7.97 mm Hg, P=0.0002, P≤0.0001, respectively), DPB (−5.65 and −7.92 mm Hg, P=0.0001, P≤0.0001, respectively), OSI (−0.4 and −3.5, P=0.050, P≤0.001, respectively), TPX (−5.0 and −32.0 mol/L, P=0.193, P≤0.001, respectively), TAC (0.02 and 0.08 μmol Trolox equivalent/L, P=0.135, P=0.046, respectively), Ox-LDL (−2.1 and −4.2 U/L, P=0.050, P≤0.001, respectively).

Conclusions: The probiotic cheese containing L. plantarum TENSIA has cardioprotective effect through lowering blood pressure and reduction of some subclinical oxidative stress markers.

Production of New Oat Grains Probiotic Yoghurt

Hoda Mahrous*, Amal Abd El Razek†, Wedad M. El-Kholy‡. *Industrial Biotechnology Department, Genetic Engineering and Biotechnology Research Institute, Menofiya University, Al-Minufya; †Food Science and Technology Department, Faculty of Agriculture, El-Shatby, Alexandria University; ‡Dairy Research Department, Food Technology Research Institute, A.R.C., Alexandria, Egypt., alex, Egypt.

Abstract: Food industry companies place rather high expectations in food products made to meet the consumers’ demand for a healthy life style. In this context, “Functional Foods” play a special role. In many of the studies that have involved examination of the use of probiotics and prebiotics in infants, the primary health outcomes have been microbiological in nature. This study aimed to develop probiotic yoghurt inclusion of oat which attends to the human’s energy and nutritional demands, as well as functional because of its high content of soluble fiber, which are highly beneficial to humans’ health. The results indicated that: the effect of probiotic lactic acid bacteria strain tested with and without oat for their ability to assimilation of Cholesterol In Vivo and In Vitro and their effect on feeding mice on general health indicators, hematological parameters and their ability to reduce the cholesterol level in blood serum; the strain with oat effect on reduce the level of cholesterol in the blood serum. The formulae had presented excellent microbiological quality and the sensory evaluation through multiple comparisons had pointed out that the samples did not differ between themselves, and that they had satisfactory acceptance by the consumers.

Epidemiological Factors and Food: Which is the Role inHelicobacter pyloriReinfection in Pediatric Age?

Tamara Sabbi*. *Asl rmg, Asl rmg, Palestrina, Italy.

Background:Helicobacter pylori (Hp) infection has been recognized as a cause of chronic gastritis, peptic ulcer, atrophic gastritis, and gastric cancer. Its acquisition is related with poor socioeconomic conditions while the relationship of nutrition and Hp is still a question.

Aim: To analyze if socioeconomic factors and dietary contribute to Hp reinfection in pediatric age.

Patients and Methods: A total of 150 patients (92 males; age range, 5 to 16 y) with Hp infection treated and eradicated in the past. Fifty-five patients with Hp reinfection and 95 patients not reinfected. We interviewed the children with questionnaire about socioeconomics factors, hygiene, living conditions, and their dietary habits.

Results: A lower frequency of fermented dairy food, fruits, and vegetable consumption was registered among children with Hp reinfection as compared with not been reinfected. Among persons with Hp reinfection were noted low socioeconomic markers such as crowded living conditions, a large number of siblings, and unclean water.

Conclusions: The risk of Hp reinfection might decrease with the use of probiotics, vitamin C, and antioxidants contained in fruit and vegetables. Risk factors for Hp reinfection are low socioeconomics factors, hygiene, and living conditions.

Selection of a Probiotic Strain Potentiating the Immunomodulatory Properties of an Elderberry Extract (EE)

Christine Libon*, Marie-Francoise Aries*, Laila Haddioui†, Christian Latge*.*Pierre Fabre Research Institute, Pierre Fabre R&D Center; †Fonderephar, Paul Sabatier University, Toulouse, France.

Objective: There is growing evidence for associating plant extracts with probiotics to enhance immune responses. We previously identified an immunomodulating EE. As bacterial strains have disparate effects in stimulating the immune system, the aim of this work was to select a probiotic strain able to potentiate the properties of EE.

Methods: Peripheral blood mononuclear cell (PBMC) from healthy subjects were stimulated 24 hours with live bacterial cells at different ratios bacteria:PBMC. Cytokines in supernatant were quantified using the Luminex technology.

Results: Seven strains were screened for their IL-6 production. The 3 Bifidobacterium spp. tested were the strongest inducers of IL-6. We choose to conduct further studies with EE with the 4 Lactobacillus spp. strains. Upon addition of EE, only L. rhamnosus GG was found to significantly potentiate IL-6 secretion. Cytokine profiles showed L. rhamnosus GG did not stimulate the production of IL-2, IL-4, IL-5, IL-9, IL-13, IL-15, IL-17A, typically produced by T cells. In contrast, substantial amounts of inflammatory (IL-1β, IL-12p70, TNF-α, IFN-γ, and GM-CSF) and anti-inflammatory (IL-1RA and IL-10) cytokines and chemokines (CCL2, CCL3, CCL4, CCL22, CXCL8, and CXCL10) were induced. In the presence of EE, the production of 6 cytokines was significantly potentiated, whereas the production of CXCL10 was antagonized.

Conclusions:L. rhamnosus GG did not stimulate T cells. In contrast, it upregulated the production of cytokines involved in the recruitment and activation of immune cells, such as monocytes/macrophages, neutrophils, and lymphocytes. Based on these properties and its strain-specific interactions with EE, L. rhamnosus GG was selected.

Fermented Milk Dairy Product ContainingBifidobacterium lactisCNCM I2494 Enhances Gut Immune Barrier Function Via a REG-TH17 Immune Pathway

Simona Agostini*, Monique Goubern†, Mathilde Leveque*, Vassilia Theodorou*, Raphael Moriez†, Sophie Legrain-Raspaud†, Helene Eutamene*. *UMR 1331 Toxalim, INRA/INP/UPS, Neurogastroenterology & Nutrition group, Toulouse; †DANONE RESEARCH, Center Daniel Carasso, Palaiseau, France.

Objective: Irritable bowel syndrome (IBS) is characterized by visceral hypersensitivity, increased intestinal permeability and gut immune cells activation. A fermented milk (FM) product containing Bifidobacterium lactis CNCM-I2494 suppresses visceral hypersensitivity by normalizing intestinal epithelial barrier dysfunction induced by acute stress in rats. Recent outcomes showed that balance between Tregs and Th17 cells is critical for maintaining gut barrier integrity. Our aim was to investigate the impact of FM product on gut immune barrier in basal and acute stress conditions.

Methods: Wistar rats were orally given (15 d) FM product or saline. At day 15, paracellular and transcellular permeability were measured in Ussing chamber on ileal Peyer’s patches, ileum, and colonic segments in basal and after 2 hours of acute stress. IL-10, IL-12, TGF-β, and IL-17A levels were measured in ileal Peyer’s patches and CD4+CD25+Foxp3+ cells were quantified by flow cytometry both in ileal Peyer’s patches and in mesenteric lymph nodes (MLN).

Results: FM product counteracted the increase of paracellular and transcellular permeability induced by acute stress at all the gut segments tested. In Peyer’s patches, the ratio IL-10/IL-12 reduced by stress was restored to control values after FM consumption. In basal condition, FM product increased IL-10, IL-17A, and TGF-β levels and significantly decreased Treg cells (CD4+CD25+Foxp3+) population in Peyer’s patches without affecting their population in MLN.

Conclusions: A chronic consumption of a marketed FM product containing Bifidobacterium lactis CNCM-I2494 seems to upregulate gut local reg-TH17 immune pathway contributing in turn on restoring epithelial gut permeability integrity altered by acute stress.

Could Probiotics Stimulate Immune Response in Human In Vitro Model?

Stephanie Beauchemin*, Francois A. Leblond*, Marie-Eve Boyte†, Chad MacPherson†, Pierre Burguiere†, Thomas A. Tompkins†, Vincent Pichette*. *Centre de recherche de l’Hopital Maisonneuve-Rosemont, Universite de Montreal; †Lallemand Health Solutions, R&D, Montreal, QC, Canada.

Objective: We evaluated the capacity of probiotic strains to stimulate colon and immune system cells in an in vitro human model searching for an efficient strategy to compare probiotic strains immunomodulatory potential.

Methods: In the coculture model, T84 human cell line is used to represent the intestinal barrier and freshly isolated human Peripheral Blood Mononuclear Cells (PBMC) are used as immune cells. These 2 cell types are separated by a semipermeable membrane. Probiotics are cocultured (107 CFU of Lactobacillus or Bifidobacterium) with T84 cells for 6 and 48 hours and cells and media were screened during 48 hours. Cytokine secretion is evaluated by immunoassays, shift in PBMC’s proportion by FACS, and gene modulation by PCR.

Results: Among the 12 inflammatory cytokines evaluated, few were not stimulated by the tested probiotics (IL-2, IL-4, IL-5, IL-12, and IFNγ). Others were induced but did not allow strain discrimination (eg, IL-1β peaking at 200 to 300 pg/mL after 12 h for both strains). Finally, TNF secretion is more stimulated (peaks at 100 ng/mL) and longer sustained (>15 ng/mL after 48 h) when in contact with Lactobacillus in comparison with Bifidobacterium (35 ng/mL and <2.5 ng/mL after 24 h). Among PBMC, only Treg subpopulation was found significantly modulated by both tested probiotic strains (up to 3-fold increase; P<0.05).

Conclusions: Our model proved that a measurable immune response could be elicited by probiotic strains. This model could discriminate probiotic strains by induced inflammatory response. This model could be used as a tool to predict the potential of a probiotic strain to induce an immune response.

Effects of Probiotics on Intestinal Colonization and Infiammatory Bowel Disease in Preterm Infants With BW<1500 g

Morena De Angelis*, Maria Grazia Capretti*. *NICU, University and General Hospital, Bologna, Italy.

Objective: To assess the effectiveness of oral administration of Lactobacillus reuteri to reduce gut colonization with pathogenic bacteria in very low birth weight (BW) infants (BW<1500 g).

Methods: A prospective, randomized, blind study was performed in the NICU of Sant’Orsola- Malpighi Hospital, Bologna. VLBW infants were started on Lactobacillus reuteri (5 drops/day=108 CFU) or placebo (5 drops/day) within the seventh day from birth. Culture for aerobic and anaerobic gram-negative bacteria, and measurement of fecal calprotectin, were perfomed once a week, for 4 weeks, on rectal swab.

Results: Seventy-seven newborns (33 male) were evaluated: 37 received L. reuteri and 40 placebo. Treated and control infants did not differ in terms of gestational age (mean±SD; 28±2.27 vs. 29±2.18 wk, respectively), birth weight (1003±294.44 vs. 1076.45±260 g, respectively), and rate of cesarean section (35/37 vs. 36/40). Gut colonization by pathogenic species was different in the 2 groups: treated infants were colonized later than controls by E. coli, Stenotrophomonas spp., and Pseudomonas spp. (P=0.015). Average values of fecal calprotectin were higher in controls (177 vs. 140 μg/g, respectively). Fecal calprotectin values of >280 μg/g were detected in 8/150 (5.3%) samples from treated infants and in 21/148 (14.2%) samples from controls (P=0.031).

Conclusions: Probiotic supplementation influences gut colonization by pathogenic bacteria; further studies are needed to clarify its potential role in preventing intestinal inflammation in VLBW infants.

Phagebiotics in Prophylaxis Against Food-borne Infections

Svetlana Bochkareva*. *Bphage LLC, R&D Company, Moscow, Russian Federation.

Objective: The risk of infection caused by the food contaminated with food-borne pathogens is very high. Peroral form of phagebiotics will allow to reduce the risks of outbreaks and sporadic cases of these infections.

Methods: Using the clinical material a collection of strains of enterobacteria and other medically significant microorganisms was created. Bacteriophages, active against these pathogens, were detected and isolated, focusing close attention to their lytic activity, productivity, ability to retain their characteristics in storage as well as safety for laboratory animals and humans.

Results: The phage cocktail (PhC) against S. aureus, E. coli, S. enterica, and L. monocytogenes with titer 106 pfu/mL was created. None of the phages carry any known undesirable genes. The PhC was purified from endotoxins. We did not detect any intoxication symptoms during estimation of acute and chronic toxicity in outbred mice and guinea pigs. Specific efficiency of PhC was studied during experimental salmonellosis infection of 20 outbred white mice. When the PhC was used 70% of them survived. Complex safety and efficiency assessments of PhC were carried out within the rehabilitation program of 46 men with IBS. Thirty patients who took the PhC stopped complaining sooner, dyspepsia and meteorism ceased, their stool normalized, and tong plaque and bowel sounds disappeared; the number of patients with dysbacteriosis stages 2 and 3 decreased by 33%, whereas 37% of the patients showed complete normalization of microbiocenosis.

Conclusions: The introduction to the world market of the novel class of probiotics: phagebiotics, will reduce the risk of sporadic cases and outbreaks of food-borne infections.

Effect of a Probiotic Soy Beverage and Sulfassalazine on Fecal Microbiota of Animals With Colitis Induced by Dextran-Sodium Sulfate

Larissa Sbaglia Celiberto*, Mariana Nougalli Roselino*, Raquel Bedani†, Elizeu Antonio Rossi*, Roseli Aparecida Pinto*, Nadiége Dourado Pauly-Silveira*, Graciela Font de Valdez‡, Daniela Cardoso Umbelino Cavallini*. *Department of Food and Nutrition, School of Pharmaceutical Sciences, UNESP, Univ Estadual Paulista, Araraquara; †School of Pharmaceutical Sciences, USP, University of São Paulo, São Paulo, Brazil; ‡Centro de Referência para Lactobacilos, CERELA, Tucumã, Argentina.

Objective: The aim of this work was to evaluate the effect of a probiotic fermented soy beverage (fermented with Enterococcus faecium CRL183 and Lactobacillus helveticus 416 with addition of Bifidobacterium longum ATTC15707) and sulfassalazine on the fecal microbiota of rats with induced ulcerative colitis.

Methods: Four experimental groups (n=10) of rats receive water (C: control); DSS 4% in the drinking water for 7 days (CL); DSS plus probiotic beverage (CLP: 108 CFU, starting 1 wk before colitis induction); and DSS plus sulfassalazine (CLS: 100 mg/kg/d, starting after colitis induction) for 30 days with basal diet. Fecal samples were collected weekly during the study and fecal microbiota was characterized by enumerating the Lactobacillus spp., Bifidobacterium spp., Enterococcus spp., Enterobacterium spp., Clostridium spp., and Bacteroides population.

Results: At the end of the protocol, the group that received the probiotic fermented product (CLF) exhibited the greatest increase in population of Lactobacillus spp. (0.84 log10 CFU/g) and Bifidobacterium spp. (1.35 log10 CFU). The animals treated with sulfassalazine presented reduction on Bifidobacterium spp. population in the same period (0.93 log10 CFU/g). The other groups of microorganism showed no significant alteration during the experimental protocol.

Conclusions: The results revealed that regular ingestion of the probiotic product resulted in an increase of Lactobacillus spp. and Bifidobacterium spp. population, microorganisms considered important for maintaining the integrity of the epithelial cells of the colon. The positive modulation of microbiota may be related to the protective effect of probiotc product on colitis development, verified in others studies.

Interaction ofLactobacillus fermentumBGHI14 With Rat Colonic Mucosa—Implications for Colitis Induction

Jovanka Lukic*, Ivana Strahinic*, Marina Milenkovic†, Natasa Golic*, Milan Kojic*, Ljubisa Topisirovic*, Jelena Begovic*. *Institute of Molecular Genetics and Genetic Engineering; †Faculty of Pharmacy, University of Belgrade, Belgrade, Serbia.

Objective: The research aimed to test the response of colon mucosa of healthy and colitic rats to oral application of Lactobacillus fermentum BGHI14. Considering potential antigenic nature of administered bacteria, we tested mucosal reaction after different BGHI14-treatment durations and impact of treatment in defined time point of colitis induction.

Methods: Healthy rats were orally gavaged with BGHI14 for 16 or 28 days. Rats from colitic group were pretreated for 16 days with BGHI14 before intrarectal TNBS administration and killed after 48 hours. Control vehicle-treated groups were set for each BGHI14-treated group. Treatment effects were assessed by weight measurements during the experiment and by H&E staining and qPCR analysis of colonic IL-1β, TNF-α, IL-17F, Hsp70, and Tjp1 mRNA expression. DGGE was employed for intestinal microflora profile analysis.

Results: Treatment with BGHI14 for 16 days stimulated healthy mucosa which was apparent from histologic scores, elevated TNF-α mRNA synthesis and transient body mass decrease. Twenty-eight-day BGHI14-treated rats did not show immune reaction histologically although TNF-α and IL-1β mRNA levels were increased. BGHI4 pretreatment elevated Hsp70 mRNA synthesis without affecting neither other genes’ expression nor histologic scores of colitic rats. No microflora perturbation was detected by DGGE after BGHI14 treatment.

ProbioticLactobacillusStrains Activity AgainstCandidaspp.: In Vitro Assays

Maria Magdalena Coman*, Maria Cristina Verdenelli†, Stefania Silvi‡, Cinzia Cecchini†, Carla Orpianesi†, Alberto Cresci‡. *School of Advanced Studies, University of Camerino; †Synbiotec S.r.l., Spin-off of UNICAM; ‡School of Biosciences and Biotechnologies, University of Camerino, Camerino, Italy.

Objective: To analyze Lactobacillus strains for properties related to vaginal mucosa colonization and antagonism towards Candida species. The aim of the study was to confirm the efficacy of these strains and to use them for the design of a product for local application to the vaginal tract.

Methods: Several probiotic lactobacilli strains were screened for their antipathogenic potential against Candida spp. by 4 different methods of assay. In addition, we investigated the in vitro adherence blockage of 8 Candida pathogens by 8 Lactobacillus strains to HeLa cells, considering 3 possible mechanisms: exclusion by adhered lactobacilli, displacement of adhered pathogens, and competition for receptor sites (inhibition test). Vaginal ovules and douches were produced using lyophilized/microgranulated SYNBIO, a 1:1 combination of Lactobacillus rhamnosus IMC501 and Lactobacillus paracasei IMC502 as preliminary evaluation of different matrix and cell viability during 6 months.

Results: All strains showed an antipathogenic activity against the Candida strains tested. Six probiotic strains demonstrate a high adhesion capacity to HeLa cells. The inhibition results highlight a significant (P<0.05) competition of L. plantarum 319, L. rhamnosus IMC501, L. paracasei IMC502, and SYNBIO against all the Candida strains. During the storage of vaginal ovules at 25°C for 6 months, Witepsol was the matrix that showed the highest suitability to preserve viable microorganisms thus increasing the shelf-life of the product respect to PEG formulation.

Conclusions: The probiotic strains analyzed demonstrated an inhibitory action against Candida spp. growth and a high adhesion capacity to HeLa cells. The results suggest that the probiotic strains used in the present study could prevent colonization of the urogenital tract by relevant pathogens such as Candida strains through barrier and interference mechanisms (mainly displacement and competition). An advantage for women is that they can self-administer the probiotics.

Clostridium botulinumandListeria monocytogenes: Single Species or Consortia of Probiotics to Limit the Pathogens Risk?

Concetta Scalfaro*, Angelo Iacobino*, Giovanna Franciosa*. *Istituto Superiore di Sanità, Government Institution, Rome, Italy.

Objective:Clostridium botulinum and Listeria monocytogenes are 2 of the current microbiological hazards associated with foods, as both have been implicated in intestinal infection diseases. Probiotics used in some foods and supplements have a potential role for microbial risk control. The purpose of this study was to determine whether some associations of different probiotic species (consortia) may be more effective in the inactivation of the above pathogens, compared with the same probiotic species used individually.

Methods: The study had therefore 2 steps: (1) analysis of reciprocal inhibition of different probiotic species commonly used in the consortia and selection of the combinations with less reciprocal inhibition; (2) evaluation of the antimicrobial activity of the different selected probiotic consortia versus C. botulinum and L. monocytogenes, compared with the activity of the single probiotic species. Specifically, we tested the reciprocal antibacterial activity of 18 single probiotic species belonging to 5 different genera. On the bases of the obtained results, we selected 3 probiotic consortia to be tested. The inhibitory activity was evaluated using the agar spot test by measurement of the inhibition area around spots.

Results: Most of the single probiotic strains used in our consortia showed a higher antimicrobial activity versus the 2 pathogens, compared with the selected probiotic consortia.

Conclusions: In conclusion, we have identified some strains of probiotics that have a low reciprocal inhibition, but a considerable inhibitory activity toward C. botulinum and L. monocytogenes. These strains, preferably used individually, still retain a significant activity also in consortia.

Probiotic-enriched Foods and Dietary Supplements Containing Synbio Positively Affect Bowel Habits of Healthy Adults

Stefania Silvi*, Maria Cristina Verdenelli†, Cinzia Cecchini†, Maria Magdalena Coman‡, Maria Simonetta Bernabei§, Jessica Rosati§, Renato De Leone§, Carla Orpianesi†, Alberto Cresci*. *Scuola di Bioscenze e Biotecnologie; ‡School of Advances Studies; §Scuola di Scienze e Tecnologie, Unversità di Camerino; †Synbiotec Srl, Spin-off di UNICAM, Camerino, Italy.

Objective: The present study was a randomized, double-blind, placebo-controlled, parallel group study assessing the effect of daily consumption of the combination of 2 probiotic bacterial strains, Lactobacillus rhamnosus IMC 501 and Lactobacillus paracasei IMC 502, named SYNBIO, by probiotic-enriched foods (dairy and not-dairy foods) or by dietary supplement (capsules) on the bowel habits of healthy adults.

Methods: The study was performed with a 4-week run-in period followed by a 12-week intervention period. Primary and secondary outcomes assessed by a questionnaire gave the overall assessment of bowel well-being, a self-administration of Psychological General Well-Being Index (PGWBI) estimated the health-related quality of life, and gastrointestinal tolerance was determined with Gastrointestinal Symptom Rating Scale (GSRS). Support Vector Machine models for the classification problem have been used to validate the total outcomes of the bowel well-being.

Results: SYNBIO consumption positively affects bowel habits of volunteers consuming either probiotic-enriched foods or probiotic capsules. The intestinal regularity, the stool volume, and the PGWBI global score were significantly improved in the supplemented groups respect to controls. Support Vector Machine models validated the accuracy of these features. The persistence of strains in the gastrointestinal tract of people was confirmed by an high percentage of recovery of probiotic bacteria from the feces of subjects for each supplemented group.

Conclusions: The results indicate that the probiotic combination SYNBIO is effective in colonizing the gut by either functional foods or capsules. The persistence in the intestinal tract favored the intestinal regularity and contributed to maintain and improve intestinal well-being of consumers.

Encapsulation of Probiotic Microorganisms in Aqueous Dispersions of Cellulose Derivatives

Milos Beran*, Marian Urban*, Frantisek Toman*, Josef Drahorad*, Lubomir Adamek*. *Food Research Institute Prague, Radiova 7, Prague.

Objective: The objective of this work was development of a new technology of enteric probiotic encapsulation and verification of the ability of the polymeric coating to protect the probiotic microorganisms against the deleterious effects in gastrointestinal tract and to prolong their shelf-life and durability in different food matrices.

Methods: Two different methods of encapsulation of Lactobacillus acidophilus and Bifidobacterium lactis bacterial strains with commercial enteric FMC’s Aquacoat ECD (ethyl cellulose) and Aquacoat CPD (cellulose acetate phthalate) pseudolatex preparations were tested and compared—conventional spray drying and nebulisation by pressured carbon dioxide. Prebiotic Frutafit HP inulin was added to the pseudolatex preparations as a bacterially degradable polysaccharide to facilitate release of the probiotic microorganisms from the coating in the colon.

Results: Spray drying provided larger particle aggregates and decreased bacterial vitality by 1 or 2 orders in comparison with the nebulisation method. The survivability of the encapsulated probiotics in vitro in bile salt solution and simulated gastric juice were improved significantly in comparison with control bacterial sample by several orders.

Conclusions: The composite of inulin and enteric cellulosic derivatives combines the enzymatic susceptibility of inulin and the protective properties of ethyl cellulose. Kinetics of the probiotic bacteria release by the time-dependent erosion of the coating and by enzymatic activities of the colon microflora can be adjusted in a wide range by changing Aquacoat ECD, Aquacoat CPD and insulin ratios.

This work was supported by the Research Grant QJ1210093 of the National Agency for Agriculture Research, Czech Republic.

Glycaemic Response to 4 Different Sweeteners in Type II Diabetes (Sucrose in 3 Different Preparation Versus Acaloric Sweetener)

Patrizia Marrino*, Dazzi Davide†, Giulio Marchesini‡. *Sos Diabetologia, asl Reggio Emilia, Reggio Emilia; †ambulatorio di diabetologia, ospedale “Di Vaio”, Parma; ‡SSD Malattie del metabolismo e Dietetica Clinica, Azienda Ospedaliero-Universitaria S.Orsola-Malpighi, Bologna, Bologna, Italy.

Objective: The underlying hypothesis was that sucrose in a different preparation with respect to the purified one could remain inside in the gut in order not to augment glycemia and arrive to the place of GLP1 production and stimulate it.

Methods: In 64 patients (age, 70.76±8.05 y; M=44, F=20, HbA1c 7.05±0.72, BMI=28.9±4.7) with type 2 diabetes mellitus we compared the glycaemic response to the intake of a brioche prepared with different sweeteners. Sucrose was used in different preparations: purified sucrose, molasses of beet, or pulp of beet, tested versus acaloric erythritol plus sucralose. The 4 brioches (≈50 g) were consumed in double-blind and randomized order; after 30, 60, 90, 120 minutes capillary glucose sample was taken using a Bayer glucometer.

Results: A systematic glucose response to brioche intake was observed in all cases, with blood glucose increasing from 130.29±24.04 mg/dL at time 0’ to 157.03±28.83; 170.19±28.32; 161.61±29.13; 144.49±26.63 during the observation period; with no significant differences according to the type of sweetener (3.5 to 4.0 g of sucrose inside in each preparation).

Conclusions: We conclude that glucose response is mainly driven by carbohydrates (15 g) in the flour (mainly manitoba flour), not by added sweetener.

Melanzana Rossa Di Rotonda: Phytochemical Analysis and Biological Activity Towards its Validation for Nutraceuticals Market

Marcello Nicoletti*†, Chiara Toniolo*†, Gabriela Mazzanti*‡, Antonella Di Sotto*‡, Lorena Abete*‡. *University Sapienza of Rome; †Dipartimento di Biologica Ambientale; ‡Dipartimento di Fisiologia e Farmacologia “V. Erspamer,” Università Sapienza di Roma, Roma, Italia.

Variability is the main character of life and is highly expressed in plants. Among the human mission, we must consider the capacity of expanding natural variability to the possible limits. This peculiar attitude is evident in the agriculture cultivars. Selection and development of these cultivars is the result of talent and application of generations of farmers, which have domesticated wild types, generating, with patient work, completely different productive plants. This enormous work and indispensable treasure is now in danger. Concentration of production and market control on food acts against the survival of many cultivars, despite their potential economic value. This potentiality could reach new interest and possibility in the recent nutraceuticals and botanicals market. However, these products need urgently scientific validation and technology.

We report the case of an ecotype of Solanum aethiopicum L., cultivated in the Basilicata region, where it is known as “melanzana rossa di Rotonda.” It received the DOP character, being essentially present in this part of Italy. It belongs to the aubergine category, but differs for many characters, like the shape and color resembling those of tomato. The taste is practically unique and it is enhanced by the cooking in different ways.

We report the results of the composition analysis of this species, as whole and of different parts, mainly based on HPTLC devices and showing a high content in polyphenols. This account is in accordance with the pharmacological tests, showing a strong antioxidant activity. Data were compared with the aubergines cultivars actually utilized in the Italian market, that is, Solanum melongena var. bianca di Senise and S. melongena var. purpose, utilizing certified ALSIA products. We were able to show the different composition of melanzana rossa, in particular the content in chlorogenic acid, using the HPTLC analysis and comparing the fingerprints. The same method was also experimented for quantitative determinations. A remarkable antioxidant activity was detected, in different amounts for the different parts of the fruits.

These results encourage further study to use “melanzana rossa di Rotonda” for developing nutraceuticals or functional foods.

Chloride Secretion Induced by Rotavirus NSP4 is Oxidative Stress-dependent and is Inhibited bySaccharomyces boulardiiin Human Enterocytes

Vittoria Buccigrossi*, Carla Russo*, Serena Orlando*, Alfredo Guarino*. *Department Translational Medical Science, Section of Pediatrics, University of Naples “Federico II,” Naples, Italy.

Objective: Rotavirus (RV) infection leads to watery diarrhea through multiple mechanisms, the main being the chloride secretions which may depend on NSP4 enterotoxic activity in human enterocyte but its mechanisms are largely unknown. Redox unbalance is a common event in cells infected by viruses, but the role of oxidative stress in RV infection is still unknown. The aim of this study was to investigate the link between the oxidative stress and chloride secretion induced by RV. In addition, because Saccharomyces boulardii (Sb) has been effectively used in RV diarrhea, we tested its effects in RV-infected cells.

Methods: Intestinal ion transport was evaluated in Caco-2 cells infected with RV or exposed to its enterotoxin NSP4 in Ussing chambers. Reactive oxygen species (ROS) and reduced (GSH)/oxidized (GSSG) glutathione ratio were assessed using dichlorofluorescein and a colorimetric assay, respectively. S. boulardii culture supernatant (SbS) was added to Caco-2 cells before and after RV infection.

Results: Chloride secretion was associated with ROS increase in Caco-2 cells infected with RV SA11 strain. Caco-2 cells treated with NSP4 produced a dose-dependent chloride secretion and this was associated with an increase in reactive oxygen species (ROS). RV increased ROS levels and reduced reduced (GSH)/oxidized (GSSG) glutathione ratio inducing oxidative stress. N-acetylcysteine (NAC), a potent antioxidant, strongly inhibited ROS increase and GSH unbalance induced by the virus. The inhibition of NSP4-induced chloride secretion by NAC preincubation suggests that there is a link between oxidative stress and RV-induced diarrhea. SbS prevented RV-induced oxidative stress and chloride secretion in Caco-2 cells. And these results were confirmed in human intestinal biopsies.

Conclusions: These results demonstrate that chloride secretion induced by NSP4 is oxidative stress dependent and inhibited by S. boulardii acting on oxidative stress through its soluble metabolites.

Structure-Function Relationship of Antioxidant Peptides Isolated From Wheat Sprouts. Functional Binding With Phospholipids

Gian Luigi Gianfranceschi*, Isabella Calzuola*, Valeria Marsili†. *NPP Nutraceutical & Phytochemical Products, 3A Parco Tecnologico Agroalimentare dell’Umbria, Pantalla di Todi (PG); †Dipartimento di Biologia Cellulare e Ambientale, Università degli Studi di Perugia, Via Pascoli, Perugia, Italy.

Wheat sprouts’ extract was studied to characterize peptide and phospholipid fractions. The aim of this research was prompted by evidences we reported that the peptide molecules present in the extract are responsible of specific biological activities including the control of cell proliferation in cancer cells in vitro. Recently, we observed that the strong antioxidant activity exerted by wheat sprout extract is for 70% due to antioxidant peptides. Consequently, the structure of the peptides extracted from wheat sprouts and fractionated by HPLC was analyzed by mass spectrometry and mass spectrometry of second order (MS/MS). The mass/mass spectrometry results were analyzed by a recently reported (J Pep Sci. 2005, 11: 225-234) automatic combinatorial method that carries out the computation of all amino acid sequences compatible with a given molecular ion. The possible sequences are automatically obtained by considering all the sequences compatible with the molecular weights of the peptide compound (MH+) and of its potential breakdown products present in the MS/MS spectrum. The designed sequences were scanned to recognize potential antioxidant sequences. The evidence of peptides with antioxidant activity is quite recent, but the number of papers demonstrating a strong antioxidant activity by peptides is quickly increasing. In this last decade >800 papers have been reported in the international literature. From the analysis of reported data, it is difficult to recognize a peptide sequence specific towards an antioxidant function. It is probable that the specific antioxidant activity is linked both to sequence and structure of the peptide. We can see that the antioxidant peptides some time do not contain antioxidant amino acids (such as cystein, methionin, taurine); in this case the presence of aromatic amino acids appears necessary.

Some of the obtained results obtained are reported:

MH+: 541.0; sequence: AcPhe-Cys-Ala-Gly-CysNH2

MH+: 663.5; sequence: AcAsn-Val-Ala-Leu-Cys-CysNH2

MH+: 785.5; sequence: NH2Tyr-Met-Thr-Val-Val-Ala-CysNH2

It is noteworthy that the C-terminal of many sequences is necessarily constituted by amidated cysteine. Moreover the results show another peculiarity: the presence of domains with 2 residues of cysteine, some time together with aromatic amino acids. A question associated to biologically active peptides is their poor bioavailability. It has been reported that the complex with phospholipids can significantly increase this bioavailability. In this context the phospholipids molecular species extracted from wheat sprouts were characterized. The peptide sequences were scanned also to recognize binding sites for phospholipids following the vector machine prediction method. The results show several sequences suitable for the binding with phospholipids, according to previously reported results (J Pep Sci. 2011, 17: 741-760). Peptide sequences containing both potential antioxidant domains and interaction sites for phospholipids are selected. Some of the above reported sequences are synthesized. The synthetic peptides shows a strong antioxidant activity “in vitro” evaluated as radical scavenging of the superoxide ion and capacity to inhibit the production of thiobarbituric acid reactive substances (TBARS) (J Clin Pathol. 2001, 54: 356-361).

Probiotics for the Treatment of Irritable Bowel Syndrome: A Systematic Review and Meta-Analysis of Evidence in Children and Adults

Lo Vecchio Andrea*, Bruzzese Eugenia*, Viscovo Sara*, Chiatto Fabrizia*, Fedele Cristina*, Guarino Alfredo*, Giannattasio Antonietta†, Sarnelli Giovanni‡. *Department of Translational Medical Science, Section of Pediatrics; ‡Gastroenterology Unit, Department of Clinical and Experimental Medicine, University of Naples, “Federico II”; †Medicine and Health Sciences Department, University of Molise, Campobasso, Italy.

Objective: Probiotics may have a role in reducing the severity of symptoms in IBS. The objective of the study was to systematically review the evidence on the treatment with probiotics in children and adults with IBS, and to perform a meta-analysis of the major clinical outcomes comparing the effect of intervention in different age ranges.

Design: Medline and CENTRAL databases were searched for randomized controlled trials on the use of probiotics in children and adults with IBS (defined according to age-specific Rome criteria). Trials scoring ≥3 according to the Linde Internal Validity Scale (LIVS) were included in meta-analysis.

Results: Thirty-one trials (4 on children and 27 on adults) were included in the qualitative analysis and 23 in the meta-analysis. Differently from adults, children treated with probiotics had a significant improvement in severity (SMD=−0.25; 95% CI, −0.45 to −0.05; P<0.001) and frequency (SMD=−1.05; 95% CI, −1.44 to −0.66; P<0.001) of abdominal pain. A significant reduction of abdominal distention was demonstrated both in adults (SMD=−0.21; 95% CI, −0.35 to −0.08; P=0.002) and children (SMD=−2.15; 95% CI, −2.80 to −1.50). However, standardized IBS symptoms scores (SMD=−0.15; 95% CI, −0.92 to 0.62; P=0.70) and quality of life scores (SMD=−0.10; 95% CI, −0.28 to 0.08; P=0.26) were not significantly affected by probiotics in the adult population.

Conclusions: Probiotics may have a slight efficacy in reducing IBS-related symptoms, mainly abdominal pain and distention. This effect seems to be more evident in children rather than adults. However, the high heterogeneity among studies may partially affect interpretation of results.

© 2014 by Lippincott Williams & Wilkins