Carbohydrate Polymers 92 (2013) 241–248 Contents lists available at SciVerse ScienceDirect Carbohydrate Polymers jo urnal homepage: www.elsevier.com/locate/carbpol Soluble ␤-1,3/1,6-glucan in seaweed from the southern hemisphere and its immunomodulatory effect a, b b a Francisca Bobadilla ∗, Carolina Rodriguez-Tirado , Mónica Imarai , María José Galotto , c Roger Andersson a Facultad Tecnológica, Universidad de Santiago de Chile, Av. Libertador Bernardo, O’Higgins, 3363 Estación Central, Santiago, Chile b Facultad de Química y Biología, Universidad de Santiago de Chile, Av. Libertador Bernardo, O’Higgins, 3363 Estación Central, Santiago, Chile c Swedish University of Agricultural Sciences, Department of Food Science, P.O. Box 7051, SE-750 07 Uppsala, Sweden a r t i c l e i n f o a b s t r a c t Article history: Five types of macroalgae from the southern hemisphere were analysed for the presence of ␤-1,3/1,6- Received 8 June 2012 glucan and its immunostimulant properties. We were able to extract soluble ␤-1,3/1,6-d-glucan from Received in revised form Durvillaea antarctica (Chamisso) Hariot (DA). The morphology of the brown algae influenced extraction, 25 September 2012 and the highest percentage of ␤-glucan was found in the fronds. The content of ␤-glucan in the stipes Accepted 26 September 2012 and holdfast was on average 33% and <5%, respectively, of that in the fronds. A simple laboratory extrac- Available online 4 October 2012 tion process was developed. A highly pure water-soluble polysaccharide, mainly composed of glucose residues, was obtained with a dominant average molecular weight of 6.9 kDa. NMR spectroscopy con- Keywords: firmed the polysaccharide structure to be of ␤-1,3/1,6-glucan type, comprising a ␤-1,3-glucan backbone Durvillaea antarctica (Chamisso) Hariot Polysaccharide and 21% degree of branching of ␤-1,6-glucan side chains. Mouse cells were exposed to four DA extract Polymer structure concentrations in water (50, 100, 250 and 500 ␮g/mL) and no adverse effects on survival were noted. ␤-1,3/1,6-Glucan Remarkably, the ␤-glucan induced a 16.9% increase in activated CD19+ B lymphocytes compared with Immunological action the control sample. The optimal concentration for maximum activity was 100 ␮g DA extract/mL. © 2012 Elsevier Ltd. All rights reserved. 1. Introduction 5401727, US Patent 6476003, WO 2004030613, CN 101117357, etc.). Soluble ␤-1,3/1,4-glucan from barley (US Patent 7462607) and Naturally occurring non-specific immunostimulants, such as soluble ␤-1,3/1,6-glucan from fungi (KR 2007 1037035) have been ␤-glucan, have been proved to have immunomodulatory effects developed for use in food and medicinal applications (US Patent in aquaculture (Bohn & BeMiller, 1995; Kumari & Sahoo, 2006; 7462607). These include use as quasi drugs for cancer therapy, Robertsen, Rorstad, Engstad, & Raa, 1990; Sahoo & Mukherjee, developed for the purpose of enhancing the efficacy of antibod- 2001). Prophylactic immunomodulators might be of use as a pro- ies (Modak, Koehne, Vickers, O’Reilly, & Cheung, 2009). To a large phylactic treatment to prevent seasonal outbreaks and also as a extent, the applications are orientated towards supplementing suppressive treatment for latent and sublethal pathogens (Gannam diets to generally improve resistance against several diseases of & Schrock, 2001, chap. 12). Compared with antibiotics, the advan- bacterial, fungal and parasitic origin (Mantovani et al., 2008), but tage of naturally occurring non-specific immunostimulants is that they also have great potential as an adjuvant complementing fish they do not cause safety concerns in terms of residual contaminant, vaccination to increase antibacterial response (Shoemaker, Klesius, and particularly water quality damage, when used as prophylactics. & Lim, 2001, chap. 7; Siwicki et al., 2004). However, the main draw- ␤-Glucans are long-chain polysaccharides found in the cell wall back limiting the development of many of these polysaccharides is of fungi, yeasts and certain bacteria and plants (Gannam & Schrock, the lack of efficient processes for their extraction and purification 2001, chap. 12; Rice et al., 2005). Yeast glucans are the most com- and their cost (Laroche & Michaud, 2007). monly used immunomodulators (US Patent 6444448, US Patent To a large extent, commercial applications have been devel- oped to incorporate immunomodulators as a dietary supplement in feed and food production. However, Gannam and Schrock (2001, chap. 12) noted that the use of glucans in a bath treatment for fish ∗ Corresponding author. Permanent Postal Address: Kristinebergs Strand 33, has not been examined in depth. Furthermore, Soltanian, Stuyven, Stockholm 11252, Sweden. Tel.: +56 25709642. Cox, Sorgeloos, and Bossier (2009) indicated that oral administra- E-mail addresses: [email protected] (F. Bobadilla), tion of water-soluble ␤-glucan preparations has several advantages [email protected] (C. Rodriguez-Tirado), [email protected] compared with other routes of administration (i.e. intravenous, (M. Imarai), [email protected] (M.J. Galotto), [email protected] (R. Andersson). intraperitoneal, and peroral). Sandula, Kogan, Kacurakova, and 0144-8617/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.carbpol.2012.09.071 242 F. Bobadilla et al. / Carbohydrate Polymers 92 (2013) 241–248 Machova (1999) reported problems with insoluble, or sparingly 2. Experimental soluble, ␤-glucan causing significant adverse effects (e.g. granu- loma formation, microembolisation, inflammation, and pain) when 2.1. Materials administered by parenteral routes and indicated that oral admin- istration of the water-soluble glucan preparations, when tested in Five species within the red (Gracilariaceae) and brown (Phaeo- mice, had several advantages. phyceae) algae were studied. These were Gracilaria chilensis, The use of natural immunostimulants in decapods and bivalves Durvillaea antarctica (DA), Macrocystis pyrifera, Lessonia trabeculata to protect them, especially from infectious diseases, is promising, and Lessonia nigrescens, all of which originated from the intertidal since these animals do not have in their system the capacity for zone of the Chilean coast. The samples were washed, freeze-dried, specific immunity against infectious agents (Klesius, Shoemaker, milled and stored frozen at 20 C inside vacuum-sealed bags until − ◦ Evans, & Lim, 2001, chap. 17). In addition, the innate immune sys- analysis. All chemicals and reagents used were of analytical grade. tem is of primary importance in combating infections in fish and Furthermore, samples of DA algae were collected from two loca- the reason is basically the intrinsic inefficiency of the acquired tions: A (34◦34′S, 72◦4′W and B (34◦46′S, 72◦6′W), and tissues of system due to its evolutionary status (Magnadóttir, 2006). Further- frond, stipe and holdfast were analysed separately. more, it is well known that most larval marine fish will not readily accept formulated feeds and thus when rearing marine species it is 2.2. Extraction often necessary to supply nutrient and health supplements by other methods (Weirich & Reigh, 2001, chap. 15). One of these meth- Extraction was carried out for the five types of algae in order to ods is the absorption of prophylactics dissolved in bath treatments determine the content of ␤-1,3/1,6-glucan and its behaviour dur- through the skin (Strand and Dalmo, 1997). Therefore, fish in the ing the extraction process. Milled algal material (1 g) was placed early stages of development and inland cultures of crustaceans are in a 80 mL beaker with 20 mL 85% ethanol solution and soaked in urgent need of a soluble ␤-glucan bath treatment application overnight at room temperature (Fig. 1). On the following day, and it has been suggested that soluble ␤-glucan from algae may the sample was mixed in an Ika ultraturrax T-25 homogeniser at have the potential to enhance immunity in early life stages before 8000 g (Janke and Kunkel) for 1 min, transferred to a 15 mL capped × the development of acquired immunity (Gannam & Schrock, 2001, tube and placed in a water bath at 70 ◦C for approximately 3 h. chap. 12). The unextractable residue was collected by centrifugation Research on ␤-glucan bioactivity has shown increased at 3000 g (Heraeus Multifuge 3S) for 10 min. The pellet was × macrophage and neutrophil numbers, enhanced macrophage extracted with 20 mL CaCl2, homogenised by ultra-turrax (Kine- phagocytic activity, elevated serum lysozyme and up-regulated matica Polytrom PT 3000) at 20,000 g for 1 min and incubated at × macrophage respiratory burst activity (Guselle, Markham, & 70 ◦C with continuous mixing for 1 h. The sample was centrifuged Speare, 2006). Furthermore, it is possible that the treatment of fish at 3000 g (Heraeus Multifuge 3S) for 10 min and the supernatant × with ␤-glucan may enhance adaptive immunity, as demonstrated collected. The extraction cycle was repeated twice with 10 ml of in Labeo rohita by Misra, Das, Mukherjee, and Pradhan (2006). CaCl2 each. This study sought to characterise the properties of a soluble Pooled supernatant was concentrated in a conical flask with ␤-1,3/1,6-glucan extracted from brown algae samples from the a Büchi evaporator at 40 ◦C. The sample was defatted with 2 mL southern hemisphere. Percentage recovery was measured and the chloroform, mixed and centrifuged. The supernatant was dial- content monitored throughout the process by sugar and ␤-1,3/1,6- ysed in 29 mm diameter regenerated cellulose tubing membrane