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IV International Symposium on Lactic Acid Bacteria: Food, Health and ApplicationsB3STUDY OF THE IMMUNOMODULATORY EFFECT OFKEFIRAN ON THP1 CELLS INFECTED WITH Bacillus cereusM. L. Larrouyet 1 , J Minnaard 1,2 , A. G. Abraham 1, 3 and P. F. Perez 1, 4 1Centro de Inves\gación y Desarrollo en Criotecnología de Alimentos. La Plata, Buenos Aires, Argen\na. CCT La Plata, CONICET. 2 Area Farmacia. Departamento de Ciencias Biológicas. Facultad de Ciencias Exactas. UNLP. 3 Area Bioquímica y Control de Alimentos. Facultad de Ciencias Exactas. UNLP. 4 Area Microbiología e Inmunología. Cátedra de Microbiología. Facultad de Ciencias Exactas. UNLP. 47 y 115. 1900. La Plata. E-­‐mail: pfp@biol.unlp.edu.ar Kefiran, an exopolysaccharide produce by kefir granules, is a neutral, ramified glucogalactane, non-­diges\ble, water-­‐soluble, with demonstrated ability to modulate immune response both in vitro and in vivo. The interac\on with phagocy\c cells could contribute to gain further insight on the immunomodulatory effect of kefiran. The aim of this work was to assess the effect of kefiran on the expression of HLA-­‐DR in a human monocy\c cell line (THP-­‐1 cells). THP-­‐1 cells were incubated with phorbol myristate acetate (PMA) 200 nM, kefiran 800 mg/L or vegeta\ve cells of Bacillus cereus B10502 (20 bacteria/THP-­‐1 cell – 100 µg/ml chloranphenicol). Expression of HLA-­‐DR was assessed by flow cytometry. Interac\on between bacteria and THP-­‐1 cells was studied by flow cytometry by labeling bacteria with CFDA-­‐SE. The presence of kefiran leads to modifica\on in the paIern of interac\on between bacteria and monocytes as evidenced by the presence of two sub-­popula\ons showing different levels of green fluorescence (FL1-­‐H). Cell ac\va\on was evidenced by morphological changes (increase of FSC and SSC values) as well as by the increase of the ra\o of cells expressing HLA-­‐DR. Indeed, whereas the ra\o of HLA-­‐DR (+) cells was 3.4 % aoer PMA s\mula\on these values increased to 6.4 % when cells were incubated with both PMA and kefiran. In another series of experiments where cells s\mulated with PMA were incubated with B. cereus; the presence of kefiran increases the percentage of HLA-­‐DR (+) cells from 15.3 to 21.8 %. Interes\ngly, kefiran was able to increase the ra\o of HLA-­‐DR (+) cells even when incuba\ons were conducted with B. cereus in the absence of PMA (values of 3.1 and 4.0 % for B. cereus and B. cereus + kefiran, respec\vely). To determine whether the effects are related to a general property of polysaccharides, the same experiments were performed with dextran of high molecular weight. Noteworthy, dextran did not modify the expression of HLA-­‐DR in THP1 cells s\mulated with B. cereus. Results of the present study demonstrate that kefiran is able to modify the interac\on between B. cereus and cultured human phagocy\c cells. These changes lead to the modula\on of the response of phagocy\c cells to chemical and bacterial s\muli. These findings provide further insight on the mechanisms responsible of the biological effects of kefiran as a func\onal ingredient of foods. San Miguel de Tucumán, Tucumán, ARGENTINA. October 16-18, 2013

IV International Symposium on Lactic Acid Bacteria: Food, Health and ApplicationsB4INTERACTION OF BIFIDOBACTERIA WITH THP1MONOCYTIC CELL LINEM. Diaz 1 , I. S. Rolny 1,4 , J. Minnaard 2,3 , P. F. Pérez 1,2 1Area Microbiología e Inmunología. Cátedra de Microbiología. Facultad de Ciencias Exactas. UNLP. 2 Centro de Inves\gación y Desarrollo en Criotecnología de Alimentos. CCT La Plata, CONICET. 3 Area Farmacia. Departamento de Ciencias Biológicas. Facultad de Ciencias Exactas. UNLP. 4 CCT La Plata, CONICET. 47 y 115. 1900 La Plata. E-­‐mail: pfp@biol.unlp.edu.ar Interac\on between bifidobacteria and host´s cells is relevant for the health promo\ng effects and studies with professional phagocy\c cells could contribute to the understanding of the mechanisms involved in the probio\c effect. The present work aimed to evaluated the interac\on of bifidobacteria with monocy\c cells from human origin (THP1 cells). Bifidobacterium bifidum 5310 and B. adolescen;s 5317 were cultured (48 hs; 37°C) in MRS broth in anaerobic condi\ons. Cultures were centrifuged, washed and suspended in DMEM medium. Bacteria were labeled with FITC. THP1 cells (1 x 106 cell/ml) were differen\ated with phorbol myristate acetate (PMA) 200 nm in DMEM 10% fetal bovine serum for 3 days at 37°C in 5% CO2 atmosphere. Coincuba\ons (THP1 cells-­‐bacteria) were performed for 1 h at 37°C at different mul\plici\es of infec\on (MOI). The effect of the presence of Bacillus cereus B10502 (MOI 25 or 50 for 30 min) was also studied. Associa\on and invasion were evaluated by flow cytometry (FL1+ cells), epifluorescence microscopy and plate counts in MRS agar. Necrosis was assessed by flow cytometry aoer propidium iodide staining (FL2+ cells). Internaliza\on was evaluated aoer quenching of exocellular bacteria with trypan blue. Bacteria were found as large clusters around the monocytes. This was par\cularly evident for strain CIDCA 5310. Flow cytometry analysis showed 92.3 ± 4.8 % of cells associated to strain CIDCA 5310 (FL1 + events) irrespec\vely of the MOI assayed. These findings suggest that monocyte-­‐bacteria interac\on is saturated at low MOI. In contrast, strain CIDCA 5317 showed a dose-­‐response behavior; i. e. 12.6 ± 3.8 %; 30.3 ± 6.0 % and 61.2 ± 17.6 % for MOI 25, 50 and 100 respec\vely. Aoer quenching with trypan blue, percentage of FL1 + cells ranged from 1 to 5 % thus sugges\ng that high number of bacteria remained exocellular. MOI of 50 and higher of strain CIDCA 5310 lead to around 70 % necrosis (FL2+ cells) whereas values remains similar to controls (20 %) for strain CIDCA 5317. When monocytes were incubated with bifidobacteria and strain B10502 of Bacillus cereus a 2 to 3 fold increase in the percentage of cells associated to bifidobacteria was found. Results demonstrated that bifidobacteria interact with phagocy\c cells in a strain-­‐dependent manner. These findings could be related to differences in surface proper\es already reported for the strains under study and could be relevant for the understanding of the immunomodula\ng effects of these poten\ally probio\c strains. San Miguel de Tucumán, Tucumán, ARGENTINA. October 16-18, 2013

IV International Symposium on Lactic Acid Bacteria: Food, Health and ApplicationsB5Lactobacillus paracasei SUBSP. Tolerans ISOLATED FROMJENYN`S SPRAT (Ramnogaster arcuata) AS PROBIOTIC FORJUVENILE RAINBOW TROUT (ONCORHYNCHUS MYKISS)(WALBAUM, 1792)M.G. Sica 1 , A. Lopez Cazorla 1,2 , L.I. Brugnoni 1,3 , P.L. Marucci 1 , M.A. CubiIo 1,4 1Departamento de Biología, Bioquímica y Farmacia -­‐ Universidad Nacional del Sur (UNS), San Juan 670 (8000) Bahía Blanca, Argen\na. 2 CONICET. 3 Planta Piloto de Ingeniería Química (UNS CONICET). 4 Centro de Recursos Naturales Renovables de la Zona Semiárida (UNS CONICET), Camino La Carrindanga Km 7, Bahía Blanca, Argen\na. Email: mgsica@uns.edu.ar The feed is the main cost of the commercial aquaculture, and therefore the feed quality and feeding strategy are of utmost importance. Slight improvements in feed efficiency can result in an important diminu\on of the produc\on costs and feed wastes that would otherwise alter the water quality. The use of probio\cs in aquaculture is an excellent choice not only for disease control, but also as growth promoters of the organisms. The beneficial effect on the feed efficiency and growth promo\on when probio\cs strains are given as dietary supplements, have aIracted increasing interest in recent years. The aim of this study was to evaluate the effects of a strain of Lactobacillus paracasei subsp tolerans-­‐F2 on growth parameters of juvenile rainbow trout, which is the predominant species reared in Argen\ne. The strain F2 was isolated from the gastrointes\nal tract of Ramnogaster arcuata (nv: Jenyn’s sprat), a fish that spends its whole life cycle in Bahía Blanca Estuary. The feeding experiment was conducted in 500 L fibreglass tanks, each provided with 98% re-­‐circulated aerated freshwater at a rate of 300 l h -­‐1 . The water temperature was maintained at 14 ± 2 ºC, oxygen levels above 80 % satura\on. Fish were held under natural photoperiod of la\tude 39 ºS. One system was employed for the probio\c treatment (commercial feed supplemented with the strain F2) and the other was used as control (commercial feed). The experiment was conducted in triplicate. During the 66 days of experiment there was no evidence of nega\ve effects of strain F2 on the fish, because no pathology was observed and behavior was normal. No significant differences in the survival in both systems were detected. The fish fed with the diet supplements with F2 showed a mean weight gain of 228% while it was of 211 % in the control diet fed fish. Significant differences in the specific growth rate and feed conversion ra\o in the treatment group, compared to the control one were detected. The improvement in growth performance and in feeding conversion when the fish were fed with F2-­‐suplemented diet indicated that the strain could produce a beIer u\liza\on of commercial feed. The enhancement in feed efficiency, results in a diminu\on of the amount of balanced food required to obtain the desired weight. These values might have a significant impact on the costs when the amount of food is scaled to the produc\on levels. The results of this study would indicate that the strain F2 could be used as a probio\c in juvenile rainbow trout culture. San Miguel de Tucumán, Tucumán, ARGENTINA. October 16-18, 2013

IV International Symposium on Lactic Acid Bacteria: Food, Health and ApplicationsB6DIVERSITY AND PROBIOTICS PROPERTIES OF LACTICACID BACTERIA ISOLATES FROM BRAZILIAN FOODPRODUCTSC. Lacerda Ramos 1,2 , L. Thorsen 2 , R. Freitas Schwan 1 , L. Jespersen 2 1Department of Biology, Federal University of Lavras, 37.200-­‐000, Lavras, MG, Brazil. 2 Food Microbiology, Department of Food Science, Faculty of Sciences, University of Copenhagen, Denmark. E-­‐mail: cin\alramos@yahoo.com.br Lac\c acid bacteria (LAB) have been used as food supplements, and are highly valued for their probio\c proper\es. A total of 234 LAB isolates from Brazilian food products were ini\ally screened for their ability to survive at pH 2.0. Fioy one of the isolates survived and were selected. They were characterized by phenotypic methods, rep-­‐PCR and iden\fied using 16S rRNA gene sequencing as Lactobacillus fermentum (34 isolates), Lactobacillus plantarum (10) and Lactobacillus brevis (7). Based on being either highly tolerant to bile, showing a ability for auto-­‐aggrega\on and/or hydrophobic proper\es, one L. fermentum (CH58), three L. plantarum (CH3, CH41 and SAU96) and two L. brevis (SAU105 and FFC199) were selected. The highest co-­‐aggrega\on ability with Escherichia coli was observed to L.plantarum CH41. L.brevis SAU105 and FFC199 and L. fermentum CH58 exhibited antagonis\c ac\vity towards the pathogens Listeria monocytogenes and Staphylococcus aureus. L. plantarum CH3 and CH41 and L. brevis FFC199 showed adhesion ability to Caco-­‐2 cells (1.6, 1.1 and 0.9 %, respec\vely) similar to the commercial probio\c, L. rhamnosus GG (1,5%) . They were able to increase the transepithelial electrical resistance (TEER) of Caco-­‐2 cells over 24 h (p

IV International Symposium on Lactic Acid Bacteria: Food, Health and ApplicationsB7EFFECTS OF MICROENCAPSULATION OF CLA-PRODUCING LACTIC ACID BACTERIAI. E. González 1 , G. R. Ross 1,2 , C. P. Van Nieuwenhove 3 , S. N. González 1,3 1Facultad de Bioquímica, Química y Farmacia, Universidad Nacional de Tucumán. Ayacucho 491 – 4000 – Tucumán, Argen\na. 2 Facultad de Ciencias de la Salud, Universidad del Norte Santo Tomás de Aquino, 9 de Julio 169-­‐ 4000-­‐ Tucumán, Argen\na. 3 Centro de Referencia para Lactobacilos CERELA-­‐CONICET. Chacabuco 145 – 4000 – Tucumán, Argen\na. E-­‐mail: romiross23@yahoo.com.ar Conjugated linoleic acid (CLA) is a mixture of posi\onal and geometric isomers of linoleic acid (LA) with conjugated double bonds. It is produced during the hydrogena\on of dietary LA in rumen, being dairy foods and ruminant meats the main natural sources of CLA. It has received great aIen\on for their beneficial health proper\es such as cancer and atherosclerosis preven\on, immunomodula\on, and body fat reduc\on. Natural CLA produc\on is far below physiological effec\ve level; so it is necessary to obtain CLA enriched food. Certain strains of lac\c acid bacteria (LAB) are able to conjugated LA in vivo. Therefore, microencapsula\on is an excellent alterna\ve to protect bacteria through gastrointes\nal tract. The aim of this work was to encapsulate CLA-­producing LAB, evalua\ng viability in simulated gastrointes\nal (SGI) condi\ons and CLA-­produc\on aoer encapsula\on process. CLA-­‐producing LAB (Lactobacillus plantarum CRL 353, Lactobacillus plantarum CRL 355 and Lactobacillus acidophilus CRL 44) were encapsulated by ionic gela\on method using: 1.8% (w/v) sodium alginate sterile solu\on, LAB suspension (10 9 CFU/mL) in 20% (w/v) non fat milk and 0.1M calcium chloride (hardening solu\on). Non fat milk (20%, w/v) bacteria suspension (10 9 CFU/mL) was used as Control. To evaluate SGI resistance, LAB alginate beads obtained were resuspended sequen\ally in gastric (NaCl 2 g/L, pepsin 3.2 g/L, and HCl 7 mL; pH 1.2) and intes\nal solu\ons (KH 2 PO 4 6.8 g/L, 0.2N NaOH 250 mL, and pancrea\n 10 g/L; pH 7.2). At different \me intervals, samples were withdrawn from the SGI medium. Viable cell counts were determinated by spreading on MRS agar and incuba\on at 37°C for 48 h. CLA-­‐bioproduc\on was evaluated before and aoer encapsula\on process. Capsules and Control suspension were inoculated in MRS broth containing LA (60 μg/mL) as substrate, and were anaerobically incubated at 37°C for 24 h. Lipids were extracted using chloroform/methanol (2:1, v/v) solu\on, they were saponificated with 0.9% (w/v) methanolic NaOH, deriva\zed to methyl esters and finally injected in a gas chromatograph equipped with flame ioniza\on detector and a HP-­‐88 capillary column (100 m × 0.32 mm interior diameter × 0.25 μm of thickness). FaIy acids were iden\fied by comparison with the reten\on \mes of methylated standards. Results were expressed as μg/mL of culture.Results show that encapsula\on significantly increased viability of L. plantarum CRL 353, L. plantarum CRL 355 and L. acidophilus CRL 44 under SGI condi\ons. It was also demonstrated that LA conjuga\on capacity of these strains was not affected by this process. Therefore, encapsula\on could be use as an efficient way to incorporate microorganisms in func\onal foods. San Miguel de Tucumán, Tucumán, ARGENTINA. October 16-18, 2013

IV International Symposium on Lactic Acid Bacteria: Food, Health and ApplicationsB8IN VITRO AND IN VIVO EFFECTS OF BENEFICIALVAGINAL LACTOBACILLI ON PATHOGENIC StreptococcusagalactiaeP. R. De Gregorio, M. S. Juárez Tomás, M. E. F. Nader-­‐Macías Centro de Referencia para Lactobacilos (CERELA-­‐CONICET). Chacabuco 145. San Miguel de Tucumán, Tucumán, CP: T4000ILC, Argen\na. E-­‐mail: fnader@cerela.org.ar Lactobacilli (Lb) are the dominant microorganisms of the vaginal microbiome of healthy women. An imbalance of the urogenital tract can produce a decrease of Lb popula\on and an increase of pathogens, meaning a higher suscep\bility to infec\ons. Streptococcus agalac;ae (Group B Streptococcus, GBS) is a commensal bacterium in the vaginal tract, but acts as pathogen in suscep\ble hosts. GBS can cause vagini\s in adolescent and young adult popula\ons and serious invasive infec\ons in suscep\ble adult popula\ons. In pregnant women, the presence of GBS is associated with the risk of neonatal infec\ons (bacteremia, sepsis, pneumonia, and/or meningi\s) and death. The intravaginal administra\on of beneficial LB could be a novel and alterna\ve strategy for the biological control of GBS infec\ons. The aim of this work was to evaluate the effects of beneficial human vaginal Lactobacillus on pathogenic S. agalac;ae, through in vitro and in vivo studies. In vitro associa\ve cultures of Lb (Lactobacillus gasseri CRL1509, Lactobacillus reuteri CRL1324 or Lactobacillus salivarius CRL1328) and S. agalac;ae NH17 were performed. Two Lb inoculum (10 6 -­‐10 8 CFU/ml) and one S. agalac;ae (10 5 CFU/ml) were assayed. Bacterial growth (by op\cal density and number of viable bacteria), pH, lac\c acid (by HPLC) and hydrogen peroxide (peroxidase chromogenic method) were quan\fied at different \mes during the culture at 37°C. In vivo studies were performed in female BALB/c mice in pseudo-­‐estrus state. Different groups of mice were intravaginally (i.v.) inoculated with L. gasseri CRL1509, L. reuteri CRL1324 or L. salivarius CRL1328 (10 7 -­‐10 8 CFU/dose) twice a day for two days, and then challenged i.v. with S. agalac;ae NH17 (5x10 5 CFU). Results from the in vitro and in vivo studies were analyzed by ANOVA (repeated measures and general linear models).The Lb-­‐S. agalac;ae co-­‐cultures showed a significant inhibi\on of the pathogen at 4h and 8h with the high and low Lb inocula, respec\vely. Parallel increases of lac\c acid and H 2 O 2 levels were evidenced. The lowest numbers of pathogen (4-­‐5 log units) were observed from 12 h of co-­‐culture with L. gasseri CRL1509 and L. salivarius CRL1328. L. reuteri CRL1324 inhibited the pathogen growth (4 log units) aoer 24 h only with the high Lb inoculum. L. gasseri CRL1509 showed a greater inhibi\on of S. agalac;ae being the highest lac\c acid producer. L. reuteri CRL1324, strains with higher H 2 O 2 produc\on, inhibited S. agalac;ae at lower levels, sugges\ng that the produc\on of organic acid could be responsible of the pathogen inhibi\on. However, in the experimental murine model, no significant differences were obtained in the number of streptococci recovered from the vaginal tract of control mice and those inoculated with L. gasseri CRL1509, L. reuteri CRL 1324 or L. salivarius CRL1328. In conclusion, different results were obtained through in vitro and in vivo assays: vaginal Lb exhibited in vitro an\microbial effects on S. agalac;ae, but these effects were not evidenced in the murine model used. Despite both types of assays are required to assess the beneficial characteris\cs of the different strains, the defini\ve characteriza\on and proper\es needs to be evaluated through phase I assays. San Miguel de Tucumán, Tucumán, ARGENTINA. October 16-18, 2013

IV International Symposium on Lactic Acid Bacteria: Food, Health and ApplicationsB9EFFECT OF ORAL ADMINISTRATION OF LACTIC ACIDBACTERIA ON INTESTINAL ESTERASE ACTIVITY ANDMETABOLIC PARAMETERS IN A CALORIC RESTRICTIONMURINE MODELM. Russo 1 , E. Fabersani 1 , M. C. Abeijón Mukdsi 1,2 , S. González 1,3 , P. Gauffin Cano 1,2 , R. B.Medina 1,2,3 1CERELA-­‐CONICET. Chacabuco 145. 4000. Tucumán, Argen\na. 2 Facultad Ciencias de la Salud-­‐UNSTA. 3 Universidad Nacional de Tucumán. E-­‐mail: rmedina@cerela.org.ar Caloric restric\on (CR) can be defined as undernutri\on without malnutri\on. CR regimen provides essen\al nutrients and vitamins, but limits the total calorie intake. CR represents an ideal model to study how changes in nutri\onal status influence on metabolic parameters. Human intes\nal bacteria with feruloyl esterase (FE) ac\vity have the ability to generate ferulic acid with an\oxidant proper\es in vivo. Administra\on of lac\c acid bacteria (LAB) with FE ac\vity can reverse metabolic markers present in animal models of metabolic syndrome. However, there is no informa\on about the effect of FE-­‐producing LAB administra\on in animal models of CR and its rela\onship with the host health. The aim of the present work was to evaluate the effect of LAB adminsitra\on on intes\nal FE ac\vity and metabolic parameters in mice fed a CR diet. Balb/c mice were daily fed a restricted amount of food during 45 days in order to reach a mild malnutri\on (CR group). Control mice received a standard diet (Control group). Two LAB supplemented CR groups were included: I) Lb group, administered with 10 8 cfu/day/mouse of Lact. fermentum CRL1446 (with FE ac\vity), and II) Lc group, administered with Lac. lac;s CRL1434 (without FE ac\vity) at the same dose. Animals were sacrified at day 1, 20, and 45. FE ac\vity was determined in gut content and mucose extract using methylferulate as substrate. Released ferulic acid was detected by HPLC. Animal metabolic status was evaluated by determina\on of plasma\c glucose concentra\on and lipid profile. Results showed a 1.3-­‐fold decrease in total intes\nal FE ac\vity in CR group, compared to control group at day 45. Lb group showed similar ac\vity at day 20 and 1.25-­‐fold higher ac\vity at day 45, compared to control group. In Lc group, ac\vity was similar to control group throughout the administra\on period. Both Lb and Lc groups showed significantly higher total intes\nal FE ac\vity than CR group. Plasma\c glucose, triglyceride and colesterol levels in animals from CR group were significantly lower than in control group. At day 45, Lb group showed lower glucose levels than CR group. Regarding total cholesterol, both treated groups showed lower levels than CR group, higher reduc\on being observed in mice from Lb group. No differences in triglycerides levels were observed among CR and treated groups throughout the trial. Results showed that oral administra\on of L. fermentum CRL 1446 to animals fed a caloric restric\on diet increases intes\nal FE ac\vity, reaching values similar or even higher than those from control group, favoring the bioavailability of an\oxidant free ferulic acid. Moreover, it showed the improvement of metabolic and oxida\ve status. San Miguel de Tucumán, Tucumán, ARGENTINA. October 16-18, 2013

IV International Symposium on Lactic Acid Bacteria: Food, Health and ApplicationsB10GROWTH AND BIOFILM FORMATION OF VAGINALLACTOBACILLI IN SIMULATING PHYSIOLOGICALUROGENITAL CONDITIONSM. C. Leccese Terraf 1 , M. S. Juárez Tomás 1 , E. Bru 1 , C. Silva 2 , M. E. F. Nader-­‐Macías 1 1Centro de Referencia para Lactobacilos (CERELA-­‐CONICET). Chacabuco 145. 4000 Tucumán, Argen\na. 2 Cátedra de Bacteriología. Facultad de Bioquímica, Química y Farmacia. Universidad Nacional de Tucumán. Ayacucho 471. 4000 Tucumán, Argen\na. E-­‐mail: fnader@cerela.org.ar Lactobacillus species are the dominant microorganisms in the vaginal environment, and inhibit pathogenic microorganisms by different mechanisms, as produc\on of organic acids, hydrogen peroxide and bacteriocins. Different syndromes, as bacterial vaginosis, aerobic vagini\s and urinary infec\on affect the balance and disrupt the vaginal ecosystem. The intravaginal administra\on of pharmaceu\cal formulas containing beneficial strains has been proposed to restore the healthy vaginal microbiome. The adhesion and biofilm forma\on (communi\es of bacteria, included in a self-­‐synthesized extracellular polymeric matrix) are strain-­‐proper\es that promote and help their permanence in the human vagina. The objec\ve of this study was to evaluate if beneficial vaginal lactobacilli (BVL) strains are able to grow and to form biofilm in laboratory experimental models in sterilized urines pools and in a culture medium simula\ng the vaginal content. Lactobacillus rhamnosus CRL1332, Lactobacillus reuteri CRL1324 and Lactobacillus gasseri CRL1263 were evaluated. L. rhamnosus CRL1332 and L.reuteri CRL1324 are biofilm-­‐forming strains in MRS broth without Tween 80 (MRS -­‐T ) and inhibits the growth of urogenital pathogens. L.gasseri CRL1263, a non-­‐biofilm forming strain in MRS -­‐T , was used as control. A 3x2x2 factorial design was applied, evalua\ng three BVL strains, two media (pooled human urine (PU) and medium simula\ng vaginal fluid (MSVF) and two Tween 80 concentra\ons (0 and 0.1%). Growth kine\c was determined by op\cal density (OD) at 540 nm, number of viable cells and pH. Organic acids (ace\c and lac\c) levels were quan\fied by HPLC. Biofilm forma\on under different condi\ons was carried out by crystal violet-­‐staining microplate assay. Results were analyzed by ANOVA (general linear mixed models). The BVL strains showed different behavior under the various condi\ons assayed. L. rhamnosus CRL1332 and L. reuteri CRL1324 were able to grow in MSVF and PU with and without Tween (PU -­‐T ). L. gasseri CRL 1263 has grown in MSVF with Tween, but not without Tween (MSVF -­‐T ). However, the growth of this strain in PU and in PU -­‐T was similar. The growth of L. gasseri was significantly higher in MSVF than in PU. When es\ma\ng the rela\onships between OD and pH, significant nega\ve correla\ons through \me were observed for the three strains. Among the three factors assayed, only the concentra\on of Tween 80 exerted sta\s\cally significant effects on biofilm forma\on. L. rhamnosus CRL1332 and L. reuteri CRL1324 were able to form biofilm in MSVF -­‐T and PU -­‐T . L. gasseri CRL 1263 formed biofilm only in MSVF -­‐T .The results demonstrate that BVL can grow, maintain their viability and form biofilm in condi\ons simula\ng the physiological environment of the tracts in which they will be applied. These beneficial and func\onal characteris\cs are basic selec\on criteria for inclusion of BVL in products to be used in the urogenital tract. San Miguel de Tucumán, Tucumán, ARGENTINA. October 16-18, 2013

IV International Symposium on Lactic Acid Bacteria: Food, Health and ApplicationsB11Lactobacillus reuteri CRL1098 SOLUBLE FACTORSATTENUATE INFLAMMATORY RESPONSE OF TOLL-LIKERECEPTOR 4-ACTIVATED MACROPHAGESM. Griet 1 , J. Villena 1,2 , Y. Tomosada 1 , S. Salva 1 , G. Font de Valdez 1 , H. Kitazawa 2 , V. Rodriguez 1, * 1Reference Centre for Lactobacilli (CERELA-­‐CONICET), CP 4000, Tucuman, Argen\na. 2 Food and Feed Immunology Group, Graduate School of Agricultural Science, Tohoku University, Sendai, Japan. E-­‐mail: anavirr@cerela.org.ar The immunomodulatory poten\al of probio\c bacteria is increasingly of interest for prophylac\c and therapeu\c op\ons in various complex disorders including inflammatory diseases. Previously, we demonstrated that Lactobacillus reuteri CRL1098 soluble factors (LrS) are able to reduce TNF-­‐α produc\on in peripheral blood mononuclear cells, indica\ng that LrS has a poten\al an\-­‐inflammatory effect. In the present work we aimed to confirm the immunoregulatory effect of LrS in mouse peritoneal macrophages under non-­‐inflammatory condi\ons and aoer the challenge with lipopolysaccharide (LPS). Peritoneal macrophages cultures were in vitro s\mulated with different concentra\ons LrS during 12, 24 or 48 hours and the produc\on of TNF-­‐α, IL-­‐1β, IL-­‐6, IL-­‐12 and IL-­‐10 was determined. We found that LrS is able to significantly decrease TNF-­‐α produc\on and increase IL-­‐10 levels in a dose-­‐ and \me-­‐dependent manner. S\mula\on with LrS diluted ½ during 24 hours was the op\mal dose/\me to achieve the immunoregulatory effect and was used for further experiments. We next evaluated whether LrS was able to modulate the response of macrophages to LPS challenge. Two series of experiments were design: a) peritoneal macrophages where s\mulated with LrS in the op\mal dose and then challenged with LPS (preven\ve treatment) or; b) macrophages were s\mulated with a coadministra\on of LPS plus LrS. Challenge of macrophages with LPS significantly increased the produc\on of the pro-­‐inflammatory cytokines TNF-­‐α, IL-­‐1β and IL-­‐6 (p

IV International Symposium on Lactic Acid Bacteria: Food, Health and ApplicationsB12POULTRY-FEED PRESERVATIVE POTENTIAL OF WHEYFERMENTED WITH KEFIR GRAINSA. Londero 1,* , A. León Peláez 2 , G. Diosma 3 , G. L. De Antoni 1,2 , A. G. Abraham 1 , G. L. Garrote 1,2 1Centro de Inves\gación y Desarrollo en Criotecnología de Alimentos (CIDCA, UNLP-­‐CONICET). Calle 47 y 116, CP 1900, La Plata, Argen\na. 2 Cátedra de Microbiología, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, Calle 47 y 115, CP 1900, La Plata, Argen\na. 3 Facultad de Ciencias Agrarias y Forestales, UNLP, 60 y 119, 1900 La Plata, Argen\na. *E-­‐mail: londeroalejandra@yahoo.com.ar Fungal contamina\on of poultry feed causes economic losses to industry and represents a poten\al risk to animal health. The aim of the present study was to analyze the applica\on of a by-­product of dairy industry fermented with kefir grains as an addi\ve to reduce fungal incidence on poultry feed. For this purpose, first the innocuousness and the effect on intes\nal microbiota of the fermented whey were studied in vivo. Twelve broiler chickens Ross PM3, 14-­‐day-­‐old, were divided in 2 equal groups: i) did not receive treatment and ii) received whey fermented with 10 % w/v of kefir grains (1 ml/chicken per day). The excreta moisture, the feed and water intake, along with the body weight were recorded daily for each chicken during 10 days. At the end of treatment the Eubacterial and Lactobacillus community of the ileum were analyzed by denaturing gradient gel electrophoresis. The an\fungal ac\vity of fermented whey against Aspergillus flavus, Aspergillus parasi;cus, Aspergillus terreus, Aspergillus fumigatus, Penicillium crustosum, Trichoderma longibrachiatum, and Rhizopus sp. was assessed by determining its capacity to inhibit conidial germina\on. Then, the fermented product was added to poultry feed (1g/m1) and dried in a convec\on oven at 50 °C. The survival of kefir microorganisms during storage for 0, 15, and 30 days was determined by viable counts on MRS and YGC agar. Finally, the resistance of the added feed to fungal contamina\on was analyzed. It was frac\onated on Petri plates (10 g feed/plate) and sprayed with 0.1 ml of a conidial (10 5 /ml) suspension. Plates were incubated at 20 °C and checked daily to determine the \me (in days) at which moulds become visible in the feed. Whey fermented with kefir grains was innocuous to chickens since there were not significant differences between control and treated groups for any of the parameters evaluated, whereas changes in Lactobacillus community of the ileum were detected. The fermented product showed high percentage of conidial germina\on inhibi\on (≥ 70%) on all fungal species evaluated. Furthermore, poultry feed added with the fermented whey was 2 to 4 \mes more resistant to fungal contamina\on than control feed depending on the fungal specie. It also contained 1 x10 8 colony forming units (CFU) of lac\c acid bacteria/kg and 6 x10 7 CFU of yeasts/kg even aoer 30 days of storage. Therefore whey fermented with kefir grains can be added to poultry feed as biopreserva\ve improving its resistance to fungi contamina\on and cons\tu\ng a source of safe microorganisms poten\ally beneficial to chicken health. San Miguel de Tucumán, Tucumán, ARGENTINA. October 16-18, 2013

IV International Symposium on Lactic Acid Bacteria: Food, Health and ApplicationsB13INMUNOMODULATORY PROPERTIES OF NONBACTERIAL FRACTION OF KEFIR:ROLE OF ORGANIC ACIDSC. Iraporda 1 , M. Rumbo 2 , G. L. Garrote 1 , A. G. Abraham 1,3 1Centro de Inves\gación y Desarrollo en Criotecnología de Alimentos (CIDCA, UNLP-­‐CONICET). Calle 47 y 116. (1900). La Plata, Argen\na. 2 Laboratorio de Inves\gaciones del Sistema Inmune (LISIN, UNLP). Calle 47 y 115. (1900). La Plata, Argen\na. 3 Área Bioquímica y Control de Alimentos, Facultad de Ciencias Exactas, UNLP. Calle 47 y 115. (1900). La Plata, Argen\na. E-­‐mail: carolinairaporda@gmail.com Kefir is obtained by milk fermenta\on with grains that are formed by a large number of lac\c acid bacteria, ace\c acid bacteria and yeast in a polysaccharide and protein matrix. It is considered a func\onal food since it contains bioac\ve ingredients that offer health beneficial effects and resistance to certain diseases. These beneficial proper\es could be aIributed to the presence of a complex microbiota as well as their metabolic products. The aim of this work is to study the bioac\ve proper\es of kefir fermented milk, focusing on the ability to modulate intes\nal epithelial innate response. Kefir grains CIDCA AGK1 were used to ferment milk at 20 °C, 24 h. Products were centrifuged, and the supernatants were neutralized and filtered. The effects of supernatants and solu\ons of lac\c and ace\c acid (in the same concentra\on found in the fermented milk) were studied on the reporter system Caco2ccl20:luc. This system consist of intes\nal Caco-­‐2 cells stably transfected with a luciferase reporter construc\on under the control of CCL20 promoter. In response to s\mula\on with different proinflamatory s\muli (flagellin 1µg/ml, IL-­‐1β 10ng/mL or TNF-­‐α 100ng/mL), the cells induce CCL20 expression. We found that a 30 min pre-­‐incuba\on of cells, with fermented milk supernatants and also with racemic lac\c acid solu\on (100 mM, pH 7.0) produced a very strong inhibi\on (>80%) of luciferase ac\vity, independently of the proinflammatory s\muli used, while a solu\on of ace\c acid, neutralized (

IV International Symposium on Lactic Acid Bacteria: Food, Health and ApplicationsB14THE TOLL-LIKE RECEPTOR FAMILY PROTEIN RP105/MD-1COMPLEX IS INVOLVED IN THE IMMUNOREGULATORYEFFECT OF Lactobacillus plantarum N14EXOPOLYSACCHARIDESY. Murofushi 1 , J. Villena 1,2 , K. Morie 1 , K. Hashiguchi 3 , M. Yoshida 3 , S. Alvarez 2 , T. Saito 1 , H. Kitazawa 1,* 1Food and Feed Immunology Group, Graduate School of Agricultural Science, Tohoku University, Sendai, Japan. 2Laboratory of Immunobiotechnology, Reference Centre for Lactobacilli (CERELA-­‐CONICET), Tucuman, Argen\na. 3Research Center, Momoya Co., Ltd, Saitama, Japan. E-­‐mail: jcvillena@cerela.org.ar The radioprotec\ve 105 (RP105)/MD-­‐1 complex is a member of the Toll-­‐like receptor (TLR) family of proteins. It was reported that this complex cooperates with the essen\al lipopolysaccharide (LPS) receptor TLR4/MD-­‐2 complex and plays a crucial role in LPS responses by immune cells. Previously, we cloned the cDNAs encoding porcine RP105 and porcine MD-­‐1 from Peyer's patches (PP) of adult swine and demonstrated in experiments with cells cotransfected with RP105 and MD-­‐1 (HEK RP105/MD-­‐1 cells) that Lactobacillus plantarum N14 (LP14) strongly ac\vate NF-­‐kB and induce the expression of various cytokines via RP105. In this work, we evaluated the immunoregulatory capaci\es of extracellular phosphopolysaccharides (EPS) from LP14 in intes\nal epithelial and immune cells from pigs. In addi\on, we aimed to gain insight in the knowledge of the mechanisms involved in the immunoregulatory effect of EPS by studying their capacity to s\mulate the RP105/MD-­‐1 complex, TLR4 and TLR2. EPS from LP14 were frac\onated into neutral (NPS) and acidic (APS) EPS by anion exchange chromatography. Our results showed that APS from LP14 is able to increase the mitogenic ac\vi\es of PP and mesenteric lymphoid node (MLN) immune cells in a dose dependent manner while NPS strongly s\mulated mitogenic ac\vity of MLN in low concentra\ons. In addi\on, APS upregulated the expression of IL-­‐6 and IL-­‐8 in immune cells from MLN and PP and in porcine intes\nal epithelial (PIE) cells while NPS significantly increased TGF-­‐β levels in both PP immune cells and PIE cells (p

IV International Symposium on Lactic Acid Bacteria: Food, Health and ApplicationsB15Lactobacillus jensenii TL2937 REGULATE THEINFLAMMATORY RESPONSE TRIGGERED BY TOLL-LIKERECEPTOR 4 AND IMPROVE PRODUCTIVITY IN POST-WEANING PIGJ. Villena 1,2 , Y. Suda 3 , Y. Takahashi 3 , S. Makino 4 , S. Ikegami 4 , S. Alvarez 2 , H. Kitazawa 1,* 1Food and Feed Immunology Group, Tohoku University, Sendai, Japan. 2 Laboratory of Immunobiotechnology, Reference Centre for Lactobacilli (CERELA-­‐CONICET), Tucuman, Argen\na. 3 Department of Food, Agriculture and Environment, Miyagi University, Sendai, Japan. 4 Division of Research and Development, Meiji Dairies Corpora\on, Kanagawa, Japan. E-­‐mail: jcvillena@cerela.org.ar The use of an\microbials in swine diets tends to be banned to avoid the risk of poten\al infec\ons with resistant pathogens, resul\ng in the reduc\on of produc\vity. As an alterna\ve, the use of immunoregulatory lac\c acid bacteria has been proposed to improve the immune system in piglets in order to avoid intes\nal infec\ons and reduce unproduc\ve inflammatory response aoer weaning. In this regard, we have demonstrated previously that Lactobacillus jensenii TL2937 (LJ) is able to aIenuate the inflammatory response triggered by ac\va\on of Toll-­‐like receptor 4 (TLR-­‐4) in porcine intes\nal epithelial (PIE) cells and an\gen presen\ng cells (APC) from Peyer’s patches (PP). In view of the cri\cal importance of intes\nal epithelial cells (IECs)-­‐APCs interac\ons in the regula\on of intes\nal immune responses, the aim of the present study was to examine the effect of LJ on ac\va\on paIerns of APCs from swine PPs in co-­‐cultures with PIE cells. Therefore, we evaluated the func\onal consequences of indirect exposure of APCs to LJ under non-­‐inflammatory and inflammatory condi\ons. In addi\on, this study aimed to inves\gate whether the previously reported in vitro effects of LJ and extended in this study, were able to beneficially modulate intes\nal immunity of piglets aoer weaning to improve immune-­‐health status. We observed that s\mula\on of PIE-­‐APCs co-­‐cultures with LJ increased the expression of MHC-­‐II, CD80/86 and IL-­‐10 in CD172a + CD11R1 -­‐ and CD172a + CD11R1 high APC cells (p

IV International Symposium on Lactic Acid Bacteria: Food, Health and ApplicationsB16Lactobacillus casei MODULATES TISSUE FACTOREXPRESSION, PARs AND PLATELET ACTIVATION IN ANEXPERIMENTAL INFECTION IN MALNOURISHED MICEH. Zelaya 1,2 , J. Laiño 1,2 , G. Agüero 1,* 1Ins\tuto de Bioquímica Aplicada. Facultad de Bioquímica, Química y Farmacia, UNT, Tucumán, Argen\na. 2 CERELA-­‐CONICET, Tucumán, Argen\na. *Email: gaguero@Žqf.unt.edu.ar Previously, it was demonstrated that Lactobacillus casei CRL431 (Lc), a well-­‐known immunomodulatory bacterium, regulates coagula\on ac\va\on, fibrin forma\on in lung, endothelial ac\va\on, and pro-­‐inflammatory state induced by malnourishment and infec\on. The objec\ve was to evaluate the effect of Lc on \ssue factor expression, Proteases Ac\vated Receptors (PARs) and platelet ac\va\on in an experimental respiratory infec\on model in malnourished mice. Malnourished Swiss Albino mice received during 7 days (d) balanced conven\onal diet (BCD) or BCD supplemented with Lc in the drink water (10 9 cells/d/mouse) during the last 2 d of the renutri\on (BCD+Lc). Once the feeding procedures were completed, BCD+Lc, BCD, malnourished control (MNC) and well-­‐nourished control (WNC) were intranasally challenged with Streptococcus pneumoniae. At different hours post-­‐infec\on (hpi) were determined in peripheral blood: a) platelet counts, b) PARs and P-­‐selec\n expression in platelets and c) \ssue factor (TF) in monocytes by flow cytometry; and in lung: a) P-­‐selec\n in platelet by immunofluorescence, b) TF by flow cytometry, and c) PAR1 and PAR4 by immunohistochemistry. Before the infec\on, malnourishment altered the most of the evaluated parameters and only renutri\on diet supplemented with Lc normalized them. In blood, the infec\on induced an increase in platelet counts, PARs ac\va\on and P-­‐selec\n expression in platelets, and TF in monocytes. In addi\on, an increase in TF expression, and PARs and platelet ac\va\on in lung were observed. The MNC group evidenced the highest altera\ons. The supplementa\on with Lc was effec\ve to normalize platelet counts in blood (WNC 12 hpi = 1492±5.7x10 9 /L; MNC= 1026±32.2; BCD= 1088±11.3; BCD+Lc= 1400±56.6; p

IV International Symposium on Lactic Acid Bacteria: Food, Health and ApplicationsB18IN VITRO STUDIES TO CHARACTERIZE BENEFICIALPROPERTIES OF LACTIC ACID BACTERIA ISOLATEDFROM BOVINE MILKM. Pellegrino 1 , I. Frola 1 , M. E. F. Nader Macias 2 , C. Bogni 1 1Dpto. Microbiología e Inmunología, Fac. Cs. Ex. Fco-­‐Qcas y Naturales, Univ. Nacional de Río Cuarto 2 CERELA-­‐CONICET, Tucumán. email: cbogni@exa.unrc.edu.ar Bovine Mas\\s is one of the most prevalent and expensive diseases of dairy cows, associated to distress to animal and a decreasement of milk produc\on. An interes\ng alterna\ve approach to prevent bovine mas\\s is the intramammary applica\on of microorganisms with beneficial proper\es, in order to restore the ecological balance of the udder and to protect the host from pathogens. In a previous report, 219 lac\c acid bacteria (LAB) strains were isolated from bovine milk of dairy herds from Córdoba and Tucumán. Nine LAB strains were selected as beneficial microorganisms by their high hydrophobicity, moderate auto aggrega\on, produc\on of lac\c acid, hydrogen peroxide and bacteriocins. The aim of the present study was to characterize the beneficial proper\es of the selected LAB for their possible inclusion in a product for the preven\on of bovine mas\\s in the dry period of the cow. The studies performed were: a) In vitro inhibitory capacity by the cross-­‐stria\ons technique in 1.2 % LAPTg agar against 20 microorganisms considered as major bovine mas\\s causing pathogen and 15 bacteria isolated from indigenous microbiota of the teat canal, b) LAB ability to co-­‐aggregate different mas\\s pathogens by co-­culture in MRS medium c) Adhesion of LAB to epithelial cells of the teat canal (ECTC). The results obtained have shown that LAB were able to inhibit, in different degrees (low, intermediate and high), all the bovine mas\\s pathogens assayed. Six LAB strains showed intermediate inhibi\on higher than 80% for Staphylococcus aureus, Streptococcus dysgalac;ae, Streptococcus agalac;ae and Streptococcus uberis. The rest of the strains showed low inhibi\on. Eight BAL strains evidenced a high inhibi\on percentage of pathogens in combina\on with a low inhibi\on of indigenous bacteria microbiota. Referred to co-­‐aggrega\on assays, all the BAL strains were able to co-­aggregate in 95% the bovine mas\\s pathogens, being S. aureus, S. agalac;ae and S. uberis those with a higher degree of co-­‐aggrega\on. Six of the nine LAB strains showed percentages of adhesion to ECTC higher than 85%. The results obtained have allowed toselect 3 LAB strains with beneficial characteris\cs: Lactobacillus perolens CRL1724, Lactococcus lac;s subsp lac;s CRL 1655 and Enterococcus hirae CRL1836. These strains showed some specific characteris\cs: high co-­aggrega\on with bovine mas\\s pathogens, high percentages of adherence to ECTC, moderate to high inhibi\on of bovine mas\\s pathogens and compa\bility with indigenous bacteria microbiota. The ability of L. lac;s subsp CRL 1655 and L. lac;s perolens CRL1724 to produce bacteriocins and high levels of lac\c acid respec\vely was also evidenced, as poten\al beneficial strains for the bovine mammary gland. The results obtained in this study indicate that the 3 LAB selected could be considered as poten\al strains for inclusion in a probio\c veterinary product for the preven\on of bovine mas\\s during the dry period. San Miguel de Tucumán, Tucumán, ARGENTINA. October 16-18, 2013

IV International Symposium on Lactic Acid Bacteria: Food, Health and ApplicationsB19SELECTION OF POTENTIAL PROBIOTIC LACTIC ACIDBACTERIA ISOLATED FROM Opuntia ficus-indica FRUITSTO OBTAIN A FERMENTED JUICE WITH FUNCTIONALPROPERTIESS. Torres 1,2 , D. Di Risio 2 , M. I. Isla 1,2 1Ins\tuto de Química del Noroeste (INQUINOA), CONICET-­‐UNT. 2 Fac. de Bioquímica, Qca. y Fcia., Universidad Nacional de Tucumán (UNT). Ayacucho 471. 4000 Tucumán, Argen\na. E-­‐mail: sebatk@hotmail.com Cactus fruits (Opun;a ficus-­‐indica) have aIracted great interest because of their nutri\onal and health improving proper\es. Scien\fic evidence has provided about the benefits from fruit inges\on with special considera\on about poten\ally ac\ve an\oxidant phytochemicals. In many places cactaceae fruits are consumed as juice. Unfortunately, its low acidity combined to high soluble solids content makes this fruit suscep\ble to microbial aIack limi\ng its storage life. Different procedures were developed to prevent spoilage. However, the final products do not resemble to original fresh juice due to changes in colour and flavour. The aim of this study was to isolate and select probio\c candidate strains for the prepara\on of a probio\c beverage from O. ficus-­‐indica fruit juice in order to preserve or improve its func\onal and sensorial proper\es. Mature fruits were collected in the north-­‐west of Tucuman (Colalao del Valle, Tucuman, Argen\na). Lac\c acid bacteria (LAB) were isolated from them, and also from commercial cow milk previously enriched in sterile juice. Isola\on of mesophilic LAB was made on MRS agar at 37 °C for 48-­‐72 h under anaerobiosis. Isolated strains were screened for probio\c traits such as acid and bile tolerance, acidifica\on of culture medium and hydrophobicity, and also for their ability to growth in pasteurized juice (64 °C, 30 min). The microbial viability, pH, pigments content (betanin and indicaxanthin) as well as the an\oxidant ac\vity (ABTS free radical scavenging ac\vity) were evaluated in fermented juices. 19 strains were isolated on MRS plates at 37 °C; 17 of them from cactus pear and 2 from milk. These isolates were Gram-­‐posi\ve, catalase-­‐nega\ve, non-­‐mo\le, homofermenta\ve and aerotolerant rods and coccobacilli, able to grow between 15 and 42 °C and were able to acidify MRS broth. Most isolates were resistant to pH 3 and all of them showed great resistance to bile salts (2 g/100 ml). The majority of the isolated strains showed low hydrophobicity values (

IV International Symposium on Lactic Acid Bacteria: Food, Health and ApplicationsB20MACROCAPSULES FOR MAINTENANCE OF PROBIOTICVIABILITY DURING STORAGE FOR YOUNG CALVESJ. A. Zimmermann 1 , D. M. Astesana 2 , A. P. Berisvil 1 , M. L. Fusari 1 , G. B. Con\ 1 , N. Gelo] 1 , L. E. Mar\ 1 , L. P. Soto 1,2 1Depto. de Salud Pública, Facultad de Ciencias Veterinarias, UNL. 2 Laboratorio de Análisis de Alimentos-­‐ICIVET-­‐CONICET. Kreder 2805 (S3080HOF) Esperanza, Santa Fe, Argen\na. zimmermann.jorge@hotmail.com Periodic administra\on of a probio\c inoculum of bovine origin may favor establishment of a stable and balanced intes\nal microbiota, which would improve the health of the calves. The viability and number of microorganisms inoculated is vital because the suggested minimum level (SML) of bacteria to produce beneficial effects is 10 6 CFU/ml. A technique that is currently being implemented to maintain the viability of probio\cs is encapsula\on. The aim of this study was to evaluate the viability of a co-­‐culture of Lactobacillus casei DSPV 318T and L. plantarum DSPV 354T macroencapsulated through \me in different store temperatures. Both strains were grown in pasteurized whey (PW) together over night. Then, this culture was mixed with alginate 1% w/v (final concentra\on). This mixture was disposed in 1 ml molds and frozen during 3 h. Thereaoer the capsules were suspended for 1 h in CaCl 2 solu\on (0.1 M) for polymeriza\on of alginate. Subsequently, half of the capsules were placed in a chitosan solu\on (0.4% w/v) for 40 min to create an outer coa\ng of the capsules. Then, both the coated capsules as uncoated were incubated in PW for 9 h at 37 °C to increase cell counts. Macrocapsules were stored at room temperature (28 °C) and refrigera\on (4 °C). For determina\on of viability, the capsules were dissolved in sodium citrate solu\on (1% w/v). Serial decimal dilu\ons were made and spread on MRS agar to determine the total counts and on Lactobacillus plantarum selec\ve medium (LPSM) for L. plantarum DSPV 354T counts. For determina\on of L. casei DSPV 318T counts, LPSM counts were subtracted from the total counts made in MRS. Cell viability was determined weekly for a period of 6 weeks Determina\ons were in triplicate. Results were analyzed using ANOVA and Tuckey’s test. Co-­‐culture (total counts) viability was higher (P < 0.001) for coated macrocapsules, in refrigera\on storage (P < 0.001), and the best (P = 0.032) interac\on between factors was coated capsule + refrigera\on, having 8.5 log CFU/g counts at the end of the experiment. L. plantarum DSPV 354T viability was higher (P < 0.001) for coated macrocapsules, in refrigera\on storage (P = 0.007), and the best (P < 0.001) interac\on between factors was: coated capsule + refrigera\on, obtaining counts of 7 log CFU/g at the end of the experiment. L. casei DSPV 318T viability was higher (P < 0.001) for coated macrocapsules, in refrigera\on storage (P

IV International Symposium on Lactic Acid Bacteria: Food, Health and ApplicationsB21PEPTIDOGLYCAN FROM Lactobacillus rhamnosus CRL1505BENEFICIALLY MODULATE THE RESPIRATORYINFLAMMATORY RESPONSE TRIGGERED BY TOLL-LIKERECEPTOR 3 ACTIVATIONSalva 1 , E. Chiba 2 , H. Zelaya 1 , Y. Kolling 1 , H. Kitazawa 2 , S. Alvarez 1 , J. Villena 1,* 1Laboratory of Immunobiotechnology, Reference Centre for Lactobacilli (CERELA-­‐CONICET), Tucuman, Argen\na. 2 Food and Feed Immunology Group, Graduate School of Agricultural Science, Tohoku University, Sendai, Japan. E-­‐mail: jcvillena@cerela.org.ar We have previously demonstrated that nasally administered Lactobacillus rhamnosus CRL1505 (Lr1505) beneficially modulate the inflammatory response triggered by the ac\va\on of Toll-­‐like receptor (TLR)-­‐3 and the outcome of Respiratory Syncy\al Virus (RSV) infec\on. In this work, we aimed to advance in the knowledge of the mechanism(s) involved in the immunoregulatory effect of Lr1505 by evalua\ng the capacity of its pep\doglycan (Pg1505) to regulate the respiratory an\viral response. To mimic the pro-­‐inflammatory and physiopathological consequences of RNA viral infec\ons in the lung, we used an experimental model of lung inflamma\on based on the administra\on of the ar\ficial viral pathogen-­‐associated molecular paIern poly(I:C). Six-­‐week old BALB/c mice were treated with Pg1505 (equivalent to 10 8 cells of Lr1505) by the nasal route during two consecu\ve days. Pg1505-­‐treated and untreated control mice were then nasally challenged with 250 μg poly(I:C) (equivalent to 10 mg/kg body weight). Mice received three doses of poly(I:C) with 24 hours rest period between each administra\on. Lung \ssue damage and respiratory and systemic immune responses were studied at several \me points aoer the last administra\on of poly(I:C). Nasal challenge with poly(I:C) induced a marked impairment of lung func\on that was accompanied by the produc\on of pro-­‐inflammatory mediators and inflammatory cell recruitment into the airways as we previously described. The preven\ve administra\on of Pg1505 reduced lung injuries and the produc\on of TNF-­‐α, IL-­‐6, IL-­‐8 and MCP-­‐1 in the respiratory tract aoer the challenge with poly(I:C) (p

IV International Symposium on Lactic Acid Bacteria: Food, Health and ApplicationsB23PRELIMINARY STUDY OF THE ANTAGONISTIC ACTIVITYOF Lactobacillus STRAINS ISOLATED FROM VAGINALFLUID OF HEALTHY WOMEN ON PATHOGENICBACTERIAE. Salas Osorio 1,2,4 , J. M. Jiménez 1,2,3 , R. Moreno 1,2 , K. Sanchez 1,2 , C. Mar\nez 4 , G. Medina Ramírez 1,3 1Laboratorio de Diagnos\co e Inves\gaciones Microbiológicas “Prof. Celina Araujo de Pérez”, 2 Laboratorio de Inmunología. Unidad de Probió\cos. Departamento de Microbiología y Parasitología, 3Sección de Biotecnología, Ins\tuto de inves\gaciones Facultad de Farmacia y Bioanálisis, 4 Grupo de Inves\gaciones Biopatológicas, Facultad de Odontología, Universidad de los Andes. Mérida -­‐ Venezuela. E-­‐mail: elaysalas72@yahoo.com Probio\cs are defined as live microorganisms that, when consumed in adequate amounts confer health benefits to the host, within the large group of genera belonging to the Lac\c Acid Bacteria, Lactobacillus is who has the largest number of species recognized as probio\c. The vaginal microbiota is cons\tuted mainly by lactobacilli. These bacteria possess healthy aIributes that can be assessed when they are used as probio\cs. Diverse healthy ac\ons are exerted by probio\cs through different mechanisms; growth inhibi\on of pathogen microorganisms is one of them. The aim of this study was to evaluate preliminarily the antagonis\c ac\vity of five (5) Lactobacillus strains isolated from vaginal fluid of healthy women in Mérida, Venezuela by agar diffusion assay. Antagonis\c ac\vity was evaluated on the lactobacilli strains of pathogenic bacteria, Listeria monocytogenes, Staphylococcus aureus, MRSA, Enterococcus faecalis, Salmonella typhi, Salmonella typhimurium, Salmonella enteri;dis, Shigella dysenteriae through an agar diffusion assay using wells standardized using different methodologies described in the literature. Pathogenic bacteria, with 24 hours of growth at 37 °C in Brain Heart Infusion broth BHI (HiMedia, India) were centrifuged at 4000 rpm × 10 min, the supernatant was discarded and the cell pellet was washed with physiological saline to 0,9%. Subsequently, they were resuspended to achieve equivalent cell suspension 0.5 tube PaIern Mac Farland (BioMérieux), approximately 1,5 x 10 8 bacteria / mL. Inoculated 160 uL of each suspension in 8 mL of BHI agar (Hi-­‐Media, India) tempered at +/-­‐ 45 °C to obtain agar inoculated with each pathogen. On Mueller Hinton agar plates (BBL, USA) were placed in sterile stainless steel cylinder of 8 mm diameter, then poured tempered BHI agar inoculated with the strain. Once solidified agar cylinders were removed with the forma\on of the wells. Finally, for each tested Lactobacillus strain was placed in a well 80 µL of a 24 hour culture in MRS broth (HiMedia, India) and in another well 80 µL of the supernatant obtained without neutralizing the centrifuga\on of a culture of 24 hours in MRS broth (HiMedia, India). The assay was performed in duplicate. All plates were incubated at 37 ° C aerobically for 48 hours, then observed and measured the inhibi\on halos. Is reported as posi\ve minimum inhibitory area around colonies of 0.5 mm. So any halo or greater diameter 9mmde present (including the 8 mm diameter of each well) was considered posi\ve for antagonis\c ac\vity. All tested strains showed antagonis\c ac\vity against pathogenic bacteria. 14V2 strain (L. acidophilus) inhibited all the indicator strains with the highest average diameter of the inhibi\on zones addi\onally 24V4 strain (L. paracasei ssp. Paracasei) and 12M (L. fermentum) were able to inhibit growth of almost all pathogens, which qualify as poten\ally probio\c microorganisms. San Miguel de Tucumán, Tucumán, ARGENTINA. October 16-18, 2013

IV International Symposium on Lactic Acid Bacteria: Food, Health and ApplicationsB24IMMUNOMODULATORY PROPERTIES OF POTENTIALPROBIOTIC Lactobacillus STRAINS FROM BREAST MILKAND NURSLINGS STOOLSE. Salas Osorio 1,2,4 , J. M. Jiménez 1,2,3 , R. Moreno 1,2 , K. Sanchez 1,2 , G. Medina Ramírez 1,3 1Laboratorio de Diagnos\co e Inves\gaciones Microbiológicas “Prof. Celina Araujo de Pérez”, 2 Laboratorio de Inmunología. Unidad de Probió\cos. Departamento de Microbiología y Parasitología, 3Sección de Biotecnología, Ins\tuto de inves\gaciones Facultad de Farmacia y Bioanálisis, 4 Grupo de Inves\gaciones Biopatológicas, Facultad de Odontología, Universidad de los Andes. Mérida -­‐ Venezuela. E-­‐mail: elaysalas72@yahoo.com At present, the developed socie\es face a progressive increase in health problems related to the immune system and intes\nal tract, such as allergies and autoimmune inflammatory diseases. Recent evidence suggests that nutri\onal strategies ups can contribute to the reduc\on of these diseases, by manipula\ng the microbiota through diet. Hence, the use of prebio\cs and probio\cs has become a priority area within the field of nutri\on and a promising tool to modulate the immune system in different popula\ons. Given its loca\on intes\nal and possibility of interac\ng with the mucosal epithelium of the probio\cs act on the intes\nal immunity, which is closely related to the beneficial effects on the host. However, it has been shown that some probio\c bacteria may also act as adjuvants of the systemic response. So not all probio\cs exert the same effects, as there is great variability immunological between species and even among strains belonging to the same species. Immunomodula\on produced by probio\c bacteria like BAL, depends on the interac\on of these microorganisms with the immune system. S\mula\on exerted by these bacteria to interact with epithelial cells and immune cause different paIerns of cytokine secre\on in the intes\ne. In this regard, the major cytokine profile observed in BAL probio\c is characterized by increases in tumor necrosis factor alpha (TNF-­‐α), interferon-­‐gamma (IFN-­‐γ) and interleukin regulatory IL10. This paIern is usually not associated with any increase in the inflammatory response. The aim of this study was to evaluate the immunomodulatory proper\es of two strains of L. paracasei (66 y 71) and one strain of L. rhamnosus (75); biochemically iden\fied by API 50CH galleries and characterized as poten\ally probio\c according to in vitro tests of resistance to gastric acidity, bile salts and antagonis\c ac\vity against intes\nal pathogens, isolated from samples of breast milk and nursling stools, obtained in Mérida, Venezuela. An inoculum of approximately 10 8 CFU / mL were administrated to BALB/c mice ad libitum in a drinking water for 7 days. Intes\nal fluid was collected from small intes\nes of mice in an Eppendorf tube by washes with phosphate buffered saline (PBS). To measure cytokines IFN-­‐γ and IL-­‐10 levels using ELISA assays (Biosciences). Histological slices of small intes\nes were stained with hematoxylin-­‐eosin to detect any inflammatory response. ANOVA one way test was used to assess the sta\s\cal significance of the differences between test and control groups. Significant differences between control and strains 71 and 75 groups, on the IFN-­‐γ e IL-­‐10 levels were observed. No inflammatory response was detected on the histological studies. These two strains were able to induce an immune response increasing IFN-­‐γ e IL-­‐10 cytokines. The magnitude of such s\mula\on did not enhance the inflammatory immune response in order to maintain the intes\nal homeostasis. Consequently they are probio\c bacteria with an adjuvant effect on the intes\nal immune system without the induc\on of collateral side effects. San Miguel de Tucumán, Tucumán, ARGENTINA. October 16-18, 2013

IV International Symposium on Lactic Acid Bacteria: Food, Health and ApplicationsB25USE OF FLUORESCEIN ISOTHIOCYANATE TO MONITORLactobacillus salivarius DSPV 003P DURING INTESTINALTRANSIT IN BROILERSJ. E. Blajman 2 , D. M. Astesana 2 , J. A. Zimmermann 1 , A. P. Berisvil 1 , C. R. Olivero 1 , G. J. Sequeira 1 , M. V. Zbrun 1, 2 , L. S. Frizzo 1, 2 1Depto. de Salud Pública, Facultad de Ciencias Veterinarias, Universidad Nacional del Litoral. 2 Laboratorio de Análisis de Alimentos, Ins\tuto de Ciencias Veterinarias del Litoral, Consejo Nacional del Inves\gaciones Cien_ficas y Técnicas (ICIVET-­‐CONICET). R.P. Kreder 2805 (S3080HOF) Esperanza, Santa Fe, Argen\na. E-­‐mail: jblajman@yahoo.com.ar There is liIle knowledge about the interac\on between probio\c bacteria and the gastrointes\nal tract in broilers. A good method to understand the mechanisms by which probio\cs mediate their effects is to mark probio\c bacteria and trace them. The objec\ve of this study was to label and monitor lac\c acid bacteria during intes\nal transit in broilers. Six broilers and a bacterial strain of avian origin and in vitro probio\c proper\es (Lactobacillus salivarius DSPV 003P) were used. Lactobacillus salivarius DSPV 003P was mul\plied in MRS broth for 24 h at 37°C. The fresh culture was centrifuged at 3500 rpm for 10 min and washed twice with phosphate-­‐buffered saline (PBS) solu\on (pH 7.2). Bacteria cells were resuspended in PBS solu\on with fluorescein isothiocyanate (FITC; Sigma) and incubated for 2 h at 37ºC in the dark. Labelled bacteria were washed six \mes with PBS solu\on to remove unincorporated FITC. The pellet was finally resuspended in PBS and administered orally (1 ml) to each broiler on a 7 Log colony forming-­‐units/ml dose. Thirty min, 1 h and 2 h aoer probio\c administra\on, two broilers were killed by cervical disloca\on. Histological slices of crop, duodenum, cecum and bursa of Fabricius were obtained following the Sainte-­‐Marie (1962) technique for paraffin inclusion. Tissues were observed using a fluorescence light microscope. Fluorescent bacteria were detected in all samples from the crop and the gut. No bacteria were found in the bursa of Fabricius. The number of bacterial aggregates observed was higher 30 min and 1 h aoer probio\c administra\on than the number observed 2 h aoer probio\c administra\on. In conclusion, use of FITC is a poten\al method to monitor the distribu\on and movement of lactobacilli in the gastrointes\nal tract of broilers. San Miguel de Tucumán, Tucumán, ARGENTINA. October 16-18, 2013

IV International Symposium on Lactic Acid Bacteria: Food, Health and ApplicationsB26GROWTH OF LACTIC ACID BACTERIA OF AVIAN ORIGINAT LOW pH AND BILEJ. E. Blajman 2 , M. L. Fusari 1 , G. B. Con\ 1 , A. Romero Scharpen 1 , G. P. Blanche 1 , E. Rossler 1 , M. L. Signorini 1 , M. R. Rosmini 1 1Departamento de Salud Pública, Facultad de Ciencias Veterinarias, Universidad Nacional del Litoral. 2 Laboratorio de Análisis de Alimentos, Ins\tuto de Ciencias Veterinarias del Litoral, Consejo Nacional del Inves\gaciones Cien_ficas y Técnicas (ICIVET-­‐CONICET). R.P. Kreder 2805 (S3080HOF) Esperanza, Santa Fe, Argen\na. E-­‐mail: jblajman@yahoo.com.ar A cri\cal aspect of the characteriza\on of probio\c strains is their capacity to avoid biological barriers during diges\on. Survival of probio\c bacteria through the gastrointes\nal tract is crucial to exert a posi\ve effect when administered in broilers. This study was performed to compare the growth of 11 lac\c acid bacteria strains of avian origin and in vitro probio\c proper\es at low pH and in the presence of bile. A 96 well plate was filled with 150 μl of MRS broth adjusted to pH 2.0 or 150 μl of MRS broth supplemented with 0,6 % p/v ox bile. One point five μl of an overnight grown LAB strain was inoculated. Bacterial op\cal density (OD), Vmax and Lag \me were measured at 630 nm for 24 h every 30 m at 37ºC using a Mul\ modal micro-­‐plate reader. The experiments were carried out in triplicate for each strain. Sta\s\cal analysis was performed using ANOVA followed by Duncan's test (P

IV International Symposium on Lactic Acid Bacteria: Food, Health and ApplicationsB27COMPARATIVE STUDIES OF THE ADMINISTRATION OFDAIRY PROPIONIBACTERIA AS DIETARY SUPPLEMENTIN POULTRY: EFFECT OF THE DOSESE. Argañaraz MarZnez 1 , J. D. Babot 2 , M. C. Apella 2,3 , A. Perez Chaia 1,2 1Univerdidad Nacional de Tucumán. Ayacucho 471. T4000INI Tucumán, Argen\na. 2 Centro de Referencia para Lactobacilos (CERELA)-­‐CCT Tucumán-­‐CONICET. Chacabuco 145. T4000ILC Tucumán, Argen\na. E-­‐mail: apchaia@cerela.org.ar Researchers engaged in the study of probio\cs for poultry have reported that the combined use of probio\c strains offers greater benefits during breeding by improving produc\on and preven\ng infec\ons. Propionibacteria are considered as poten\al probio\cs that exhibit physiological and func\onal features of great interest such as produc\on of short chain faIy acids (SCFA T ) in the intes\ne. They may influence the development of mucose and the establishment of beneficial microbiota popula\ons. Thus, the aim of this work was to evaluate the administra\on of Propionibacterium acidipropionici LET105 and LET107 combined (PAB) in BB chicks for 21 days. The doses of PAB tested were 10 6 and 10 8 CFU/mL supplemented in the drinking water. Feed conversion (FC) and products of cecal fermenta\on by HPLC were carried out. Furthermore, at the end of the trial, histomorphometric analysis by HE/PAS and cecal microbiota count by FISH with genus specific probes were done. FC was efficient in the group consuming the highest PAB dose. There were no differences in fermenta\on products between groups at day 7 except for the propionic acid detected only in the PAB group treated with 10 8 UFC/mL. Lac\c acid and ethanol showed lower concentra\ons in animals receiving PAB. The SCFA T at 14 days were significantly increased in the highest dose PAB group with 129.7 ± 18.7 µmol/g and a high amount of ace\c acid. This increase in organic acid coincided with the pH decrease observed in the intes\nal contents of these animals. Lac\c acid and ethanol levels decreased compared to day 7, indica\ng a change in the composi\on and / or ac\vity of microbiota. On day 21, the SCFA T decreased in all groups. Lactobacilli and enterococci popula\ons did not differ between groups and neither did clostridia and bacteroides. Bifidobacteria reached significant counts 7.95 ± 0.24 log cells/g in the group receiving the highest dose of PAB. Propionibacteria which joined the microbiota reach levels of 8.05 ± 0.41 log cells/g. These results are probably due to the produc\on of bifidogenic factors by PAB which also showed high counts. The length of the villi-­‐crypt unit from ileum was significantly greater for both PAB doses, being 569.82 ± 33.88 µm for the highest dose. This was accompanied by an increased epithelial cells number being 219 ± 11 cells/villi-­‐crypt unit. Animals who received the highest dose showed the largest number of goblet cells PAS (+), 50 ± 5 cells/villi-­‐crypt unit. We concluded that an equilibrium between the produc\on of SCFA T by the microbiota and the absorp\on of these acids as an energy source by the intes\ne was reached in the cecum. This allowed a prime development of the gut. According to the above findings, the combined administra\on of the strains P. acidipropionici LET105 and LET107 in dose 10 8 CFU/mL was the most efficient and effec\ve probio\c supplement during breeding poultry. San Miguel de Tucumán, Tucumán, ARGENTINA. October 16-18, 2013

IV International Symposium on Lactic Acid Bacteria: Food, Health and ApplicationsB28SAFETY OF A BACTERIAL MIXTURE INTENDED TO BEUSED INTO A PROBIOTIC PRODUCT FOR POULTRYJ. D. Babot 1 , E. Argañaraz Mar_nez 2 , L. Saavedra 1 , M. C. Apella 1,2 , A. Perez Chaia 1,2 1Centro de Referencia para Lactobacilos (CERELA)-­‐CCT Tucumán-­‐CONICET. Chacabuco 145. T4000ILC Tucumán, Argen\na. 2Universidad Nacional de Tucumán. Ayacucho 471. T4000INI Tucumán, Argen\na. E-­‐mail: apchaia@cerela.org.ar Laying hen and broiler diet components have secondary products from plant metabolism like lec\ns that act as defence mechanisms in vegetables. Their presence may impair intes\nal epithelia development and diges\ve enzyme ac\vity in baby chicks, diminishing their growth. Bacteria may decrease lec\n cytotoxic effects on intes\ne epithelial cells, capturing them by interac\ons with bacterial surface carbohydrates. Thus, the aim of this work was to assess the safety and compa\bility of a mixture of five strains to be administered to BB chicks as a probio\c supplement. To this effect, P. acidipropionici LET103, L. salivarius LET201, L. reuteri LET210, E. faecium LET301 and B. infan;s CRL1395, previously selected for their capacity to bind to deleterious dietary lec\ns were analyzed. Their compa\bility was evaluated through three different approaches: growth in co-­‐culture (with free and constant pH), well diffusion method and interac\on of different strain colonies in agarized media. Their safety was analyzed through the study of their an\bio\c sensibility paIern in LAPTg, LSM and MRS by the Kirby-­‐Bauer method, the presence of virulence determinants and the genes that encode vancomycin resistance by phenotypical tests and PCR using specific primers, respec\vely. None of the strains produced inhibitory substances against the others. LAB inhibited the development of P. acidipropionici LET103 and B. infan;s CRL1395 in MRS agar, but not in LAPTg agar. All the strains grew well in the co-­‐culture, except for P. acidipropionici LET103 because of the early decrease in pH. However, when the pH was fixed at 6.30, this bacterium could grow as expected. P. acidipropionici LET103 and B. infan;s CRL1395 showed sensi\vity to all the an\bio\cs tested in both media. E. faecium LET301 and L. reuteri LET210 presented resistance only to clindamycin and vancomycin, respec\vely, while L. salivarius LET201 was sensi\ve to all the an\bio\cs tested in both media with except for clindamycin in MRS. Virulence factors like gela\nase, hemolysin or hemagglu\nin were absent in all strains. E. faecium LET301 presented three out of the ten virulence determinants studied (efaAfm, efaAfs and gelE) and it lacked the genes encoding vancomycin resistance. In conclusion, the five strains showed acceptable compa\bility among them and proved to be highly safe for BB chicks, except for E. faecium LET301 whose resistance to clindamycin should be further studied. Nevertheless, in vivo trials should be performed in order to confirm the innocuous character of these strains. San Miguel de Tucumán, Tucumán, ARGENTINA. October 16-18, 2013

IV International Symposium on Lactic Acid Bacteria: Food, Health and ApplicationsB29ADMINISTRATION OF A BACTERIAL MIXTURE TO BBCHICKS: SAFETY AND EFFECTS ON GENERAL GROWTHPARAMETERSJ. D. Babot 1 , E. Argañaraz Mar_nez 2 , A. Perez Chaia 1,2 , M. C. Apella 1,2 1Centro de Referencia para Lactobacilos (CERELA)-­‐CCT Tucumán-­‐CONICET. Chacabuco 145. T4000ILC Tucumán, Argen\na. 2Universidad Nacional de Tucumán. Ayacucho 471. T4000INI Tucumán, Argen\na. E-­‐mail: mapella@cerela.org.ar In Argen\na, poultry feeds mainly contain soybean, corn, wheat, aminoacids, salts and growth factors. The vegetable components of these diets include highly resistant to heat and proteolysis proteins called lec\ns, which act as a defence mechanism in plants against the aIack of predator organisms. These proteins produce nega\ve effects on the animals that eat them: diminished diges\ve enzyme ac\vi\es and harmed intes\nal epithelial cells. This leads to an inefficient use of proteins, which is par\cularly relevant on the early life of chicks, when protein consump\on is crucial for weight gain. In previous studies, four strains (P. acidipropionici LET103, L. salivarius LET201, L. reuteri LET210 and B. infan;s CRL1395) were selected for their safety, in vitro probio\c proper\es and ability to bind to deleterious dietary lec\ns. E. faecium LET301 was included in the study due to its ability to bind to WGA. Thus, the aim of this work was to confirm the safety of a mixture of four strains administered to BB chicks as a probio\c supplement against lec\ns, and analyze its effects on general growth parameters along with E. faecium LET301. To this effect, the five strain mixture was daily administered to newly hatched BB chicks in their drinking water (10 6 -­‐10 7 CFU/mL of each strain), for three weeks (GT). A group without the bacterial mixture supplement was included as control (GC). Feed consump\on and mortality were daily noted. On the 3 rd , 6 th , 13 th and 20 th days of treatment, chicks were weighed and sacrificed, and transloca\on to liver and spleen, liver and spleen weights, and the ac\vity of diges\ve enzymes (alkaline phosphatase, amylase, leucine aminopep\dase, maltase, protease and sacarase) were analyzed. Feed conversion was evaluated weekly. The animals of group GT had significantly higher (p ≤ 0.05) weight than those of group GC from the 10 th day on. The feed conversion of the whole period was significantly (p ≤ 0.001) lower for the animals of group GT. No transloca\on to liver or spleen was observed. Amylase ac\vity was significantly higher (p ≤ 0.05) for the chicks of group GT on the 6 th day. Alkaline phosphatase ac\vity was significantly higher (p ≤ 0.01) on the 6 th and 13 th days for the group GT. Leucine aminopep\dasa presented significantly higher ac\vity (p ≤ 0.05) for the group GT on the 13 th day. Maltase ac\vity was significantly higher (p ≤ 0.05) for the animals in group GT on the 3 rd , 6 th and 13 th days. On the other hand, protease and sacarasa ac\vi\es showed no differences between the chicks of both experimental groups. In conclusion, the bacterial mixture increased the weight gain and the ac\vity of several diges\ve enzymes, and decreased food conversion, proving to be not only safe, but also to exert a posi\ve effect on the animals. Nevertheless, the intermediate resistance of E. faecium LET301 to clindamycin should be further studied, although the impact of this resistance on the poultry health is currently unknown. San Miguel de Tucumán, Tucumán, ARGENTINA. October 16-18, 2013

IV International Symposium on Lactic Acid Bacteria: Food, Health and ApplicationsB30PROBIOTIC PROPERTIES OF Lactobacillus mucosaeSTRAINS ISOLATED FROM BRAZILIAN GOAT MILKK. M. O. dos Santos 1 , L. R. de Abreu 1 , G. M. D. de Moraes 1,2 , C. R. Matos, L. M. F. da Silva 1 , B. D. G. de M. Franco 3 , S. D. Todorov 3 1Embrapa Caprinos e Ovinos, Caixa Postal 145, 62.010-­‐970, Sobral-­‐CE, Brazil. 2 Ins\tuto Federal de Educação, Ciência e Tecnologia do Ceará /RENORBIO. 3 Faculdade de Ciências Farmacêu\cas, Universidade de São Paulo. E-­‐mail: karina.dos-­‐santos@embrapa.br Firstly characterized in 2000, Lactobacillus mucosae is a species presen\ng proper\es related to probio\c poten\al, such as the ability to adhere to mucosal surfaces and ac\vity against pathogenic bacteria. Probio\c characteris\cs of three L. mucosae strains isolated from goat milk produced in Brazilian semiarid area were inves\gated. Resistance to simulated gastrointes\nal tract (GIT) condi\ons, bile salt hydrolase (BSH) ac\vity, and β-­‐galactosidase ac\vity of L. mucosae CNPC006, CNPC007, and CNPC009 were studied in vitro. To evaluate the strains tolerance to GIT, overnight cultures of each strain in MRS broth were centrifuged and the pellet was ressuspended in sodium chloride solu\on (0.85% w/v). Bacterial suspensions was added to simulated gastric juice containing pepsin (3.0 g/l) and lipase (0.9 mg/l), and the pH was adjusted to 2.3 with hydrochloric acid. Aoer 2h of incuba\on at 37 °C with agita\on (150 rpm), the enteric condi\ons were simulated with an ar\ficial intes\nal fluid containing pancrea\n (1.0 g/l) and bile salts (10.0 g/l) and adjusted to pH 5.0 to simulate the upper intes\nal condi\ons, and to pH 6.5 in the last 2 hours of incuba\on as above. Aliquots were taken for the enumera\on of viable cells at 0, 2, 4, and 6 h. The ability to perform bile salts deconjuga\on was screened by streaking 10 μl of overnight cultures on MRS agar plates containing either 0.5% (w/v) of the sodium salts of taurocholic acid (TC), taurodeoxycholic acid (TDC), glycocholic acid (GC), or glycodeoxycholic acid (GDC). Aoer anaerobic incuba\on at 37 °C for 72 h, the presence of precipitated bile acid around colonies (opaque halo) was considered a posi\ve result. β-­‐galactosidase ac\vity were assessed qualita\vely employing sterile filter paper disks impregnated with o-­‐nitrophenyl-­‐D-­‐galactopyranose. Overnight cultures were streaked on MRS agar plates, and incubated anaerobically at 37 °C for 48 h. A colony of each strain was emulsified with 0.1 ml of sterile 0.85% (w/v) sodium chloride solu\on in a tube containing an ONPG disk. The tubes were incubated at 35°C, and observed for up to 6 hours. The release of a yellow chromogenic compound, o-­‐nitrophenol, indicates a posi\ve result. All the tests was performed two \mes for each strain, in duplicates. The three L. mucosae strains showed good survival rates when exposed to the condi\ons simula\ng the GIT, higher than 50%. The cumula\ve survival rates obtained for strains CNPC006, CNPC007, and CNPC009 aoer 6 h were 58.25, 51.03 and 62.83 %, respec\vely. All the three L. mucosae strains showed β-­‐galactosidase ac\vity, were able to grow in presence of TDCA, TCA, GDCA or GCA, and to deconjugate GDC. L. mucosae CNPC007 was able to deconjugate all the tested bile salts. According to the results, the three L. mucosae strains are promising candidates to be further studied as probio\cs due to their tolerance to GIT condi\ons, and BSH and β-­‐galactosidase ac\vity, as well as for the development of novel dairy products. Acknowledgment: EMBRAPA and FAPESP for financial support. San Miguel de Tucumán, Tucumán, ARGENTINA. October 16-18, 2013

IV International Symposium on Lactic Acid Bacteria: Food, Health and ApplicationsB31PROBIOTIC STRAINS BB-­‐12 ® AND L. CASEI 431 ® INCREASE THE IMMUNE RESPONSE TO AN INFLUENZA VACCINE: A RANDOMIZED, DOUBLE-­‐BLIND, PLACEBO-­‐CONTROLLED STUDY L. Jespersen 1 , P. Calder 2 , M. Clerici 3 , G. Rizzardini 4 , A. Cape] 4 , D. Eskesen 1 1 Chr. Hansen A/S, 2970 Hørsholm, Denmark. E mail: dklij@chr-­‐hansen.com . 2 School of Medicine, University of Southampton, Southampton SO16 6YD, UK. 3 Department of Biomedical Science and Technology, University of Milan, 20090 Segrate-­‐Milano, Italy. 4 Department of Infec\ve Diseases, Luigi Sacco Hospital, 20157 Milan, Italy Many studies show that probio\cs may support the func\on of the immune system. The objec\ve of this study was to inves\gate the impact of the probio\c strains Bifidobacterium animalis ssp. lac;s, BB-­‐12 ® and Lactobacillus paracasei ssp. paracasei, L. casei 431 ® (Chr. Hansen A/S) on the func\onal capacity of the immune system in healthy humans using an influenza vaccina\on model. We conducted a randomized, double-­‐blind, placebo-­‐controlled, single-­‐center study in 211 healthy subjects (60/40 ra\o females/males, mean age (SD) 33.2 (13.1) years). Eligible subjects were randomized to BB-­‐12 ® or placebo capsule or L. casei 431 ® or placebo milk drink once daily for 6 weeks. Both probio\c products contained a minimum of 1x10 9 CFU/dose. Aoer two weeks subjects received the seasonal influenza vaccina\on. Plasma and saliva samples were collected at baseline and aoer 6 weeks of interven\on for analysis of vaccine-­‐specific and total an\bodies, cytokines IL-­‐2, IL-­‐10 and INF-­‐γ, and innate immune parameters. The vaccine-­‐specific an\body responses (IgG, IgG1, IgG3) were significantly increased in both probio\c groups compared to their corresponding placebo group (ANOVA p