A way to follow the viability of encapsulated Bifidobacterium bifidum subjected to a freeze-drying process in order to target the colon: Interest of flow cytometry F. Martin-Dejardin a,b , B. Ebel a,b , G. Lemetais a,b , H. Nguyen Thi Minh a,b , P. Gervais a,b , R. Cachon a,b , O. Chambin a,c,⇑ a Unité Procédés Alimentaires et Microbiologiques (UMR A 02.102), AgroSup Dijon-Université de Bourgogne, 1 esplanade Erasme, 21000 Dijon, France b Equipe Procédés Microbiologiques et Biotechnologiques, AgroSup Dijon, 1 esplanade Erasme, 21000 Dijon, France c Equipe Procédés Alimentaires et Physico-Chimie, Faculté de Pharmacie, 7 boulevard Jeanne d’Arc, 21079 Dijon Cedex, France article info Article history: Received 10 August 2012 Received in revised form 12 February 2013 Accepted 20 February 2013 Available online 28 February 2013 Keywords: Alginate–pectinate beads Encapsulation Probiotics Freeze-drying Viability Flow cytometry abstract The aim of this work was to apply flow cytometry in order to assess and compare the viability of freeze- dried entrapped bacteria with an usual technique by quantification by plate count techniques. It also aimed at studying the effect of various cryoprotectants on the viability of an entrapped Bifidobacterium bifidum subjected to freeze-drying to check their ability to be delivered all along the gastro-intestinal tract. The alginate–pectinate beads were chosen as the encapsulation matrix added with different protec- tants. The beads were characterized by scanning electron microscopy and the viability was checked by both methods. The best combination to improve viability of entrapped bacteria subjected to freeze-dry- ing is made of glycerol 20% (one cryoprotectant) and sodium ascorbate 10% (one anti-oxidative com- pound). This study also demonstrates that flow cytometry allows assessment of entrapped bacteria viability. Indeed we showed that viability evaluated by plate method is correlated to that obtained by flow cytometry. So, flow cytometry is a rapid method to determine cell viability after encapsulation and freeze-drying. Finally, these beads seem to be a promising probiotic delivery system to target the colon. Ó 2013 Elsevier B.V. All rights reserved. 1. Introduction Nowadays consumers expect their food to be healthy and to prevent illness. Their interest increased about the role of probiotic bacteria in human health (Sultana et al., 2000). Probiotics have been defined as ‘live microorganisms, which when administered in adequate amounts confer a health benefit on the host’ (FAO/ WHO, 2001). Therefore, providing living bacteria with a physical barrier to resist adverse environmental conditions (large pH varia- tions, enzymes, biliary salts, peristaltisis) is a challenge currently receiving considerable interest (Kailasapathy, 2009). Encapsulation is exactly a technology which has been developed for use in the food industry (functional foods and probiotic bacteria) not only to protect bacteria cells against an adverse environment but also to control release site (Burgain et al., 2011). Natural polymer-based materials represent promising matrices as controlled delivery sys- tems due to their biodegradability, compatibility, food-grade nat- ure and wide availability (Doherty et al., 2011). One of the most common methods of encapsulation involves cell entrapment within calcium alginate gel beads. Alginate is a polysaccharide obtained from brown algae nontoxic, biodegrad- able and biocompatible (Morales et al., 2008; Santagapita et al., 2011). But to ensure specified delivery of the probiotic in the colon site, the dosage form must be formulated by taking into consider- ation the gastrointestinal tract. Low methoxy pectin, a polysaccha- ride nontoxic, biodegradable and biocompatible, has the ability to form gels in presence of some divalent cations (calcium or zinc) by ionotropic gelation (Assifaoui et al., 2011). In this state, it delays probiotic release in the upper gastrointestinal tract because of its insolubility (Sriamornsak and Nunthanid, 1998). The combination of these two polymers provides a consolidated matrix and thus ob- tain better protection against bile salts (Sandoval-Castilla et al., 2010). The strain used in this study must precisely be released in the colon. Bifidobacterium have been accorded the generally recognized as safe (GRAS) status (Salminen et al., 1998) due to their long his- tory of safe use in food; thus, probiotics have been highlighted as suitable candidates for the development of functional foods. Bifido- bacterium are one of the most important bacterial groups found in the intestinal microbiota of infants (Turroni et al., 2009). Bifidobac- terium bifidum is a probiotic which reduces the incidence of 0928-0987/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejps.2013.02.015 ⇑ Corresponding author at: Unité Procédés Alimentaires et Microbiologiques (UMR A 02.102), AgroSup Dijon-Université de Bourgogne, 1 esplanade Erasme, 21000 Dijon, France. Tel.: +33 3 80 39 32 15; fax: +33 3 80 39 33 00. E-mail address: odile.chambin@u-bourgogne.fr (O. Chambin). European Journal of Pharmaceutical Sciences 49 (2013) 166–174 Contents lists available at SciVerse ScienceDirect European Journal of Pharmaceutical Sciences journal homepage: www.elsevier.com/locate/ejps