Fermented or unfermented milk using Bifidobacterium animalis subsp. lactis HN019: Technological approach determines the probiotic modulation of mucosal cellular immunity C.S.B. Bogsan a , L. Ferreira b , C. Maldonado c , G. Perdigon c , S.R. Almeida b , M.N. Oliveira a, ⁎ a Department of Biochemical Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, University of São Paulo, Brazil b Department of Clinical Analysis, Faculty of Pharmaceutical Sciences, University of São Paulo, Brazil c CERELA-CONICET, Chacabuco 145, Tucuman, Argentina abstract article info Article history: Received 10 February 2014 Received in revised form 13 May 2014 Accepted 28 May 2014 Available online 11 June 2014 Keywords: Fermented milk Immunomodulation B1 cells Matrix–probiotic–mucosa interaction Bifidobacterium animalis subsp. lactis HN019 Functional foods are important sources of probiotic delivery, mainly by fermented milk products. The physiological benefits attributed to bifido bacteria are their abilities to interfere with the adhesion of pathogenic species to surfaces of intestinal cells, and to enhance the host's immune function through their metabolic activities. However, the effects of a technological approach — fermentation or addition of probiotic in milk, and its efficacy in health are rarely taken into consideration. Hence, fermented or unfermented milk using Bifidobacterium animalis subsp. lactis HN019 were administered to BALB/c mice for 14 days. After that, the architecture of the gut was histologically investigated, and the related immune cells were examined by flow cytometry and immunofluorescence. Increase in mucus and cellularity production, changes in immune pattern and preservation of mucosal epithelia in health BALB/c mice were observed only in the fermented milk group. This suggested that changes in functionality of bifidobacteria and/or the metabolites produced by the fermentation process are the keys to improving beneficial effects in the host of the gut mucosa. © 2014 Elsevier Ltd. All rights reserved. 1. Introduction The intestinal homeostasis is maintained by immunomodulation of the intestinal mucosa. This modulation is promoted by the interaction of indigenous microbioma with medicines, food and a large variety of orally taken substances, including probiotics (Yan & Polk, 2011). Since 2002, probiotics have been defined as “live microorganisms which, when administered in adequate amounts, confer unspecified health benefits on the host” (FAO/WHO, 2002) without attention to their interactions with the matrix where they were delivered, or the changes undergone by the technology applied to the development of a probiotic product, as fermentation. The probiotic health benefits are strain-specific, and should confer at least, one of the most significant health effects. Metabolic effects of fermentation of indigestible dietary fiber (Nilsson, Stman, Holst, & Björck, 2008), positive influence on transit of luminal contents by peristalsis (Matsumoto et al., 2012), competition with pathogenic microbes for nutrients and binding sites on mucosal epithelial cells (Candela, Biagi, Turroni, Vitali, & Brigidi, 2008) and modulation of the host's immune response (Mazmanian, Liu, Tzianabos, & Kasper, 2005) were described. The probiotics of human consumption are preferentially of human origin, they are non- pathogenic, and they survive gastrointestinal transit (Hardy, Harris, Lyon, Beal, & Foey, 2013; Ouwehand et al., 2008). On the other hand, the exact mechanism of action is not fully understood (Collado, Isolauri, Salminen, & Sanz, 2009). The probiotics of genera Bifidobacterium are believed to be beneficial to human health (Collado et al., 2009) because of their abilities to inter- fere with the adhesion of pathogenic species to surfaces of intestinal cells, even though their abilities to enhance the host's immune function are attributed as the major physiological benefits to host (Gopal, Prasad, & Gil, 2003). The hallmark of mucosal immunity is the B-lymphocyte IgA + , where the clonal expansion of this B-lymphocyte is associated to the modula- tion of the mucosal immune response which may also be promoted by probiotic microorganisms, without enhancement of the inflammatory immune response. The probiotics may regulate the innate immune response upwardly or downwardly, in order to maintain the gut homeostasis (Galdeano, de Le Blanc, Vinderola, Bonet, & Perdigon, 2007; Macpherson, Geuking, & McCoy, 2011). Functional foods are the main delivery forms of probiotics; although the differences between fermented (FBM) or unfermented (UFBM) milk benefits to health-related subjects are not often taken into consider- ation. The probiotic activity is changed not only by the strain specificity, Food Research International 64 (2014) 283–288 Abbreviations: CFU, colony forming units; CM, control milk; CW, control water; FBM, fermented bifido milk; FBMHT, fermented bifido milk heat treated; PP, Peyer's patches; UFBM, unfermented bifido milk. ⁎ Corresponding author at: Department of Biochemical Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, University of São Paulo, Av. Prof. Lineu Prestes, 580, B16, 05508-000 SP, Brazil. Tel.: +55 11 30913690; fax: +55 11 38156386. E-mail address: monolive@usp.br (M.N. Oliveira). http://dx.doi.org/10.1016/j.foodres.2014.05.036 0963-9969/© 2014 Elsevier Ltd. All rights reserved. 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