Safety and Immunomodulatory Effects of Three Probiotic Strains Isolated from the Feces of Breast-Fed Infants in Healthy Adults: SETOPROB Study

et al. (2013) Safety and Immunomodulatory Effects of Three Probiotic Strains Isolated from the Feces of Breast-Fed Infants in Healthy Adults: SETOPROB Study. PLoS ONE 8(10): e78111. doi:10.1371/journal.pone.0078111 Safety and Immunomodulatory Effects of Three Probiotic Strains Isolated from the Feces of Breast-Fed Infants in Healthy Adults: SETOPROB Study Julio Plaza-Diaz 0 Carolina Gomez-Llorente 0 Laura Campaa-Martin 0 Esther Matencio 0 Inmaculada 0 Ortuo 0 Rosario Martnez-Silla 0 Carlos Gomez-Gallego 0 Maria Jess Periago 0 Gaspar Ros 0 Empar 0 Chenoll 0 Salvador Genovs 0 Beatriz Casinos 0 ngela Silva 0 Dolores Corella 0 Olga Portols 0 Fernando Romero 0 Daniel Ramn 0 Antonio Perez de la Cruz 0 Angel Gil 0 Luis Fontana 0 Ernesto T. A. Marques, University of Pittsburgh, United States of America 0 1 Department of Biochemistry & Molecular Biology II, School of Pharmacy, University of Granada , Granada , Spain , 2 Institute of Nutrition & Food Technology Jose Mataix, Biomedical Research Center, University of Granada , Granada , Spain , 3 Hero Global Technology Center , Hero Spain, S.A., Alcantarilla, Murcia , Spain , 4 Department of Human Nutrition and Food Science, Faculty of Veterinary Sciences, University of Murcia , Murcia , Spain , 5 Department of Food Biotechnology, Biopolis s.l., Parc Cientific Universitat de Valencia , Paterna, Valencia , Spain , 6 Department of Preventive Medicine and Public Health, School of Medicine, University of Valencia , Valencia, Spain, 7 CIBER Fisiopatologia de la Obesidad y Nutricion , Instituto de Salud Carlos III , Madrid , Spain , 8 Unit of Nutrition and Dietetics, Virgen de las Nieves Hospital , Granada , Spain We previously described the isolation and characterization of three probiotic strains from the feces of exclusively breast-fed newborn infants: Lactobacillus paracasei CNCM I-4034, Bifidobacterium breve CNCM I-4035 and Lactobacillus rhamnosus CNCM I-4036. These strains were shown to adhere to intestinal mucus in vitro, to be sensitive to antibiotics and to resist biliary salts and low pH. In the present study, a multicenter, randomized, doubleblind, placebo-controlled trial with 100 healthy volunteers in three Spanish cities was carried out to evaluate the tolerance, safety, gut colonization and immunomodulatory effects of these three probiotics. Volunteers underwent a 15-day washout period, after which they were randomly divided into 5 groups that received daily a placebo, a capsule containing one of the 3 strains or a capsule containing a mixture of two strains for 30 days. The intervention was followed by another 15-day washout period. Patients did not consume fermented milk for the entire duration of the study. Gastrointestinal symptoms, defecation frequency and stool consistency were not altered by probiotic intake. No relevant changes in blood and serum, as well as no adverse events occurred during or after treatment. Probiotic administration slightly modified bacterial populations in the volunteers' feces. Intestinal persistence occurred in volunteers who received L. rhamnosus CNCM I-4036. Administration of B. breve CNCM I-4035 resulted in a significant increase in fecal secretory IgA content. IL-4 and IL-10 increased, whereas IL-12 decreased in the serum of volunteers treated with any of the three strains. These results demonstrate that the consumption of these three bacterial strains was safe and exerted varying degrees of immunomodulatory effects. Trial Registration: ClinicalTrials.gov NCT01479543 - Funding: Part of the research currently in progress in the authors' laboratory is funded by the company Hero Spain, S. A. through the grant #3582 managed by the Fundacion General Empresa-Universidad de Granada. No additional external funding received for this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: Part of the research currently in progress in the authors' laboratory is funded by the company Hero Spain, S. A. through the grant #3582 managed by the Fundacion General Empresa-Universidad de Granada. EM, IO, RMS and FR are employed by Hero Global Technology Center, Hero Spain, S.A. This center is part of the food company HERO, headquartered in Switzerland. EC, SG, BC, AS and DR are employed by Biopolis S.L., a spin-off of the Cover Letter National Spanish Research Council (Consejo Superior de Investigaciones Cientficas), Ministry of Education, Spain. There are no patents, products in development or marketed products to declare. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors. The Food and Agriculture Organization (FAO) and the World Health Organization (WHO) define probiotics as live microorganisms that confer a health benefit to the host when administered in adequate amounts [1]. Strains belonging to Bifidobacterium and Lactobacillus, the predominant and subdominant groups of the gastrointestinal microbiota, respectively [2,3], are the most widely used probiotic bacteria and are included in many functional foods and dietary supplements [4-6]. The FAO/WHO [1] and the European Union (EU)-funded Product Safety Enforcement Forum of Europe (EU-PROSAFE) project [7] have attempted to create consensus guidelines for probiotic safety evaluation. These groups have recommended that i) the genus and species of the microorganism must first be definitively determined by phenotypic and genotypic techniques, ii) the strains must be deposited in an internationally recognized culture collection, and iii) the safety of the bacterial strain must be evaluated through acute ingestion studies in murine models and the estimation of potential side effects in human studies. For probiotics to be successful, they must possess certain characteristics. The criteria for the selection of probiotics include tolerance to gastrointestinal conditions (gastric acid and bile), ability to adhere to the gastrointestinal mucosa and competitive exclusion of pathogens [8,9]. We have previously described the isolation of three lactic acid bacteria (LAB) strains from the feces of exclusively breastfed newborn infants. These strains were selected based on their probiotic properties, such as adhesion to intestinal mucus, sensitivity to antibiotics and resistance to biliary salts and low pH. We identified these strains as Lactobacillus paracasei CNCM I-4034, Bifidobacterium breve CNCM I-4035 and Lactobacillus rhamnosus CNCM I-4036 [10]. In addition, their safety has been assessed by acute ingestion in immunocompetent and immunosuppressed BALB/c mouse models. The three strains inhibited Listeria monocytogenes, the etiological agent of meningitis, and human rotavirus infections in vitro [10]. The immunomodulatory effects of probiotics have been demonstrated in experimental models of allergy, autoimmunity and inflammatory bowel disease [2]. In the presen (...truncated)