ACCEL and pediocin NV5 from P. acidilactici LAB5 were purified by this method (Mandal et al., 2008). Bacteriocins can also be purified by organic solvent extraction method, in which bacteriocin is extracted using an organic solvent, butanol. Pediocin A from P. pentosaceus FBB61 was purified using this method (Piva and Headson, 1994).
Fig. 1.8 Purification strategies of bacteriocin from Lactic acid bacteria.
A. Homopolysaccharides: polymers composed of glucose or fructose units, such as glucans which contain repetitive glucose units joined by α-(1→6) glycosidic linkages.
B. Heteropolysaccharides: polymers composed of a variety of sugar residues, mainly glucose, galactose, fructose and rhamnose, such as kefiran that contains equal proportion of galactose and glucose. In some Heteropolysaccharides charged groups such as acetate, phosphate or glycerol phosphate are also present (De Vuyst and Degeest, 1999).
Fig. 1.9 Classification of microbial exopolysaccharides.
The function of exopolysaccharides in the microbial host is to provide protection against dehydration by retaining water (De Vuyst and Degeest, 1999;
Tallon et al., 2003). The slimy and sticky nature of some EPS helps in adhering to surfaces, impeding the diffusion of toxic compounds to the host (De Vuyst and Degeest, 1999). The exopolysaccharides derived from LAB play crucial role in improving rheology, texture, mouth feel of fermented food formulations and conferring beneficial physiological effects on human health, such as antitumour
activity, immunomodulating bioactivity and anticarcinogenicity (Robyt, 1986; Talon et al., 2003; Puruma and Goyal, 2005; Patel et al., 2012; Shukla and Goyal., 2013)
1.8.1 Glucans from LAB
Four different genera of lactic acid bacteria, Streptococcus, Leuconostoc, Pediococcus and Lactobacillus are known to produce glucan. Although the glucan synthesis from Leuconostoc spp. has been extensively studied (Monsan et al., 2001;
Majumder et al., 2009; Purama et al., 2009). The culture conditions of glucan and glucansucrase from Leuconostoc spp. has been optimized by statistical approach method. Leuconostoc mesenteroides NRRL B-640 is shown to produce glucansucrase that gives highly linear and soluble glucan (Uzochukwu et al., 2002, Puruma et al., 2008). A novel dextran produced by Leuconostoc dextranicum NRRL-B-18242 having a slushy, applesauce like appearance with a particulate gel-like structure is used in foods and other applications where texture is important (Pucci and Kunka, 1990).
1.8.2 Structure and function of glucan 1.8.2.1 α-D-Glucan
α-D-Glucan synthesized by glucansucrase in presence of sucrose is categorized mainly in four groups;
i) Dextran with α-(1→6) linkages, or with a majority of α-(1→6) linkages and α- (1→2), α-(1→3), and/or α-(1→4) branched linkages are mainly found in Leuconostoc species. The schematic representation of dextran is shown in Fig.
1.10.
ii) Mutan with a majority of α-(1→3) linkages generally found in Streptococcus species.
iii) Alternan with alternating α-(1→3) and α-(1→6) linkages, only reported in L.
mesenteroides.
iv) Reuteran being a highly branched structure with mainly α-(1→4) linkages mainly found in Lactobacillus reuteri. (Monchois et al., 1999; van Leeuwen et al., 2009).
Other than these four distinct group of α-D-glucan, another group with containing large amounts of α-(1→2) linkages (predominantly α-(1→2,6) branching points), produced by Leuconostoc mesenteroides strain NRRL-B1299 and Leuconostoc mesenteroides NRRL B-1355 (mutant) was also reported (Smith et al., 1998; Bozonnet et al., 2002). The solubility of glucans depends upon the percentage of branched linkages. Presence of 95% linear linkages makes glucan water-soluble, and suitable for various applications (Leathers, 2002).
Fig. 1.10 Structure of α-D-glucan showing α(1→6) glycosidic bonds in main chain and possible branches of smaller chains with α(1→2), α(1→3) or α(1→4) links.
1.8.2.2 β-D-glucans
β-D-Glucan consists of linear un-branched polysaccharides of linked β-(1→3) and β-(1→4)-D-glucopyranose units, and it is a natural water-soluble polymer that cannot be digested by human enzymes, but is degraded by probiotic bacteria in the colon into short-chain fatty acids (SCFAs) in anaerobic condition (Dols-Lafargue et al., 2008). LAB strains belonging to the genera Pediococcus and Lactobacillus isolated from cider (Garai-Ibabe et al., 2010; Elizaquivel et al., 2011) and Oenococcus isolated from wine (Dols-Lafargue et al., 2008) synthesize β-glucan with the same primary structure: a trisaccharide repeating unit, with two (1,3)-β linked residues in the main chain, one of which is branched at position 2 by a terminal glucose residue (Dols-Lafargue et al., 2008; Garai-Ibabe et al., 2010; Elizaquivel et al., 2011).
1.8.3 Applications of glucan in foods and pharmaceuticals
Lactic acid bacteria produce a wide variety of food grade exopolysaccharides (EPS) with the help of glucosyltransferases that have nutritional and health applications. Certain exopolysaccharides are also potential therapeutic agents (Korakli and Vogel, 2006). They are also used as viscosifying, stabilizing, emulsifying, sweetening, flocculating, gelling, texuturing or water-binding agents in the food as well as in the non-food industries owing to their non-ionic character and good stability under normal operating conditions (Sutherland, 1998; Welman and Maddox, 2000; Tallon et al., 2003; Dols-Lafargue et al., 2008; Bhavnani and Nisha, 2010).
Glucan also show,
A. Cholesterol lowering effect: it absorbs the cholesterol, helps in lowering the blood cholesterol level, thus reduces the risk of cardiovascular disease.
B. Lowering of the glycaemic index: Glucans are very viscous in nature which makes the gastric content thicker and help in slowing down the absorption rate of glucose. β-glucan spreads glucose absorption time (i.e. reduction of glycaemic index) and helps the body to fight against diabetes.
β-glucans are also used as edible film and as stabilizers in the manufacture of low-fat products such as salad dressings (Kontogiorgos et al., 2004), ice creams and yoghurts and cheese (Brennan et al., 2002). Two strains Pediococccus parvulus CUPV226 and Lactobacillus suebicus CUPV221 isolated from cider producing a 2- branched (1,3)-β-D-glucan have been reported, which decrease the serum cholesterol levels and affects the activation of human macrophages (Elizaquivel et al., 2011). The prebiotic properties of the 2-branched (1,3)-β-D-glucan produced by Pediococcus parvulus 2.6 has also been reported. This branched β-D-glucan also can resist the hydrolysis by the enzymes present in gastrointestinal tract and can induce the production of inflammation-related cytokines by polarized macrophages (Fernàndez de Palencia et al., 2007).
It has been reported that the α-D-glucan produced by Leuconostoc dextranicum NRRL B-1146, having α-(1→4) and α-(1→6) linkages, showed non- Newtonian pseudoplastic behaviour indicating its branched nature and also have unique rheological properties because of its potential of forming very viscous solution at low concentration and can be used as thickening or gelling agent in food
(Majumder and Goyal, 2009). Dextran from Pediococcus pentosaceus holds potential for usage as gelling agent in food formulations and as drug delivery carriers (Patel et al., 2010). The cytotoxicity test of dextran from Pediococcus pentosaceus on human cervical cancer (HeLa) cell line showed that there is no effect on the viability of HeLa cells for 72 h even at high concentration of 1000 mg/ml showing that it is non-toxic and biocompatible, rendering it safe for drug delivery, tissue engineering and various other biomedical applications (Patel et al., 2010). Recently, in vitro cytotoxicity analysis of novel dextran (2.93 x 105 Da) from Pediococcus pentosaceus CRAG3 displaying anti-cancer activity against cervical cancer (HeLa) and colon cancer (HT29) cell lines has been reported (Shukla and Goyal, 2013). The prebiotic effect of low molecular weight dextran with branched α-(1→2) linkages has also been reported (Sarbini et al., 2013). This particular type of dextran induces the growth of beneficiary bacteria such as Bifidobacterium sp. and Lactobacillus sp. (Sarbini et al., 2013). Major food and pharmaceutical applications of glucan are shown in Fig. 1.11.
Fig. 1.11 Applications of glucan in foods and pharmaceuticals.