Characterization of exopolysaccharide and ropy capsular polysaccharide formation by Weissella
Introduction
Bacterial exopolysaccharides (EPS) produced by lactic acid bacteria (LAB) are extensively investigated biomolecules that exhibit heterogeneous composition, structure and broad range of physicochemical properties and applications (Ruas-Madiedo et al., 2009). EPS are reported to function as health promotors and to improve rheological properties, mouthfeel and storability of foods, for e.g. fermented milk and breads (Galle et al., 2012a, Hassan, 2008, Ruas-Madiedo et al., 2009). EPS and capsular polysaccharide (CPS) producers are frequently identified among Streptococcus, Lactococcus, Lactobacillus, Leuconostoc and Weissella species (Bounaix et al., 2009, Monsan et al., 2001, Mozzi et al., 2006).
LAB EPS comprise a heterogenous group of polymers which are mostly classified based on composition and cellular attachment. Homo- and heteropolysaccharides (HoPS and HePS) consist of one or several monomer components, respectively. EPS are loosely attached to the cell or secreted to the environment while CPS are covalently bound to the cell surface (Sutherland, 1972). The formation of HePS, CPS and β-glucan (HoPS) requires sugar nucleotide intermediates as precursors and is directly linked to the growth and the central carbon metabolism of the producer organism (De Vuyst and Degeest, 1999). HePS and CPS formation is controlled by complex eps or cps gene clusters (Jolly and Stingele, 2001, Yasuda et al., 2008) while β-glucans are polymerized from UDP-glucose by a single transmembrane glycosyltransferase (Werning et al., 2006). The most abundant monomer building blocks of HePS are glucose, galactose and rhamnose while N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, fucose, mannose, ribose as well as inorganic substitutions might be present with lower frequency (De Vuyst and Degeest, 1999). CPS can structurally be of the HoPS or HePS type (Mozzi et al., 2006).
In contrast, β-fructan and α-glucan HoPS synthesis involves single extracellular or cell-wall associated fructansucrases (FS) and glucansucrases (GS), respectively. FS and GS are glycoside hydrolases (GH) and classified into families GH68 and GH70, respectively (www.cazy.org). Hydrolysis of the glycosidic bond of sucrose provides these glycansucrases with the energy required to catalyse the transfer reaction of the glycosyl moiety to the growing polymer-chain, this reaction is thus uncoupled from growth (Monsan et al., 2001). The α-glucan polymers are classified based on types of glycosidic linkages into dextrans (>50% α-1,6), mutans (>50% α-1,3), alternans (alternating α-1, 3 and α-1,6-linkages) and reuterans (α-1,4 with some α-1,6 branches) (Monsan et al., 2001). Beta-fructans are separated into levans consisting of mainly (2,6)-linked-β-D-fructofuranosyl units, and inulins with (2,1)-linked-β-D-fructofuranosyl residues (Monsan et al., 2001). Glycansucrases can also produce oligosaccharides (OS) in the presence of acceptor molecules (Tieking et al., 2005).
Cereals, vegetables and other plant materials are natural habitats of Weissella cibaria and Weissella confusa, which are associated with mixed LAB populations and are frequently isolated from traditionally fermented foods such as sourdough (Robert et al., 2009), fermented vegetables (Park et al., 2013, Shukla and Goyal, 2011), raw milk and fermented dairy (Ayeni et al., 2011, Van der Meulen et al., 2007). Dextran production from sucrose is regarded as phenotypic identification characteristic of the closely related W. confusa and W. cibaria (Bjorkroth et al., 2002). W. cibaria and W. confusa strains have been used for in-situ dextran and gluco-oligosaccharide formation to improve wheat and gluten-free breads (Galle et al., 2012a, Katina et al., 2009, Schwab et al., 2008). Furthermore, strains of W. cibaria were suggested as probiotic bacteria for oral health, inhibiting Streptococcus mutans glucan biofilm formation and proliferation by their produced dextran (Kang et al., 2006). Most studies on EPS-producing Weissella strains focused on dextran produced by strains isolated from European sourdoughs. This study aimed to investigate EPS production of W. confusa and W. cibaria strains previously isolated from African fermented milk and cassava products (Kastner, 2008, Wullschleger et al., 2013). Isolates with different EPS phenotypes were characterized to elucidate the EPS structures and identify genes and enzymes involved in EPS biosynthesis.
Section snippets
Materials, strains and growth conditions
Strains were grown in standard MRS media (De Man et al., 1960) containing 20 g/l glucose (Biolife, Labo-Life Sàrl, Pully, Switzerland) or modified MRS in which glucose was replaced with sucrose (MRSsuc), maltose (MRSmal) or raffinose (MRSraf) (Table 1). To promote growth, raffinose media were supplemented with 5 g/l maltose and/or glucose as indicated (Table 1). Solid plates were prepared by addition of 1.5% (w/v) agar. Liquid media were filter-sterilized (0.2 μm PES, bottle-top vacuum
Prevalence of EPS and CPS production in W. cibaria and W. confusa
A phenotypic screening using agar plates supplemented with sucrose, raffinose or glucose was used for identification and differentiation of EPS phenotypes within 123 strains of the genus Weissella. All W. confusa (110) and W. cibaria (13) strains produced EPS from sucrose. W. cibaria strains formed EPS only from sucrose but no polymer was formed on raffinose indicating glucan but no fructan biosynthesis. In contrast, 18 W. confusa strains (16.4%) synthesized fructan from raffinose additionally
Discussion
LAB produce compositionally and structurally diverse EPS which find applications in food industry and could have therapeutic potential (Ruas-Madiedo et al., 2009). Weissella sp. have been mainly investigated for dextran formation (Bounaix et al., 2009, Shukla et al., 2014), very few information is available on other EPS types. In this study we evaluated the diversity of 123 EPS-producing phenotypes of W. confusa and W. cibaria isolated from spontaneous African cassava and milk fermentations.
Conclusion
We showed that Weissella strains can synthesize an outstanding pool of oligosaccharides and polymers, including high MM, low branched dextran, levan and inulin, in addition to gluco- and fructooligosaccharides, and cell associated ropy polymers. The present study offers novel insight in the diversity of EPS types produced by W. confusa and W. cibaria thereby broadening general knowledge on this species and opening numerous possibilities for future applications. Strains producing more than one
Acknowledgements
This study was supported by a grant from the Swiss Confederation Innovation Promotion Agency (CTI, project number 10276). The Academy of Finland (project number 255755, H.N.M, T.M) is also acknowledged for the financial support. We thank Franziska Baeriswyl for technical support and Alfonso Díe for assistance in HPLC analysis.
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