A B S T R A C TExopolysaccharides (EPSs) produced by Bifidobacteria have received considerable attention due to their ability to modify the rheological and physicochemical properties of dairy products. However, the quantification and characterisation of Bifidobacterial EPS is hampered by the presence of EPSequivalent (EPS-E) substances in complex media such as Reinforced Clostridial Medium (RCM).This study has developed a media based on RCM which both supports the growth of Bifidobacterium animalis ssp. lactis AD011 and does not interfere with the quantification and characterisation of the EPS generated. Media development involved the identification of EPE-E containing components via NMR analysis followed by their removal, substitution or pre-treatment. Both beef extract and casein acid hydrolysate required chemical pre-treatment to remove polysaccharide components before the media was free of EPS-E materials. Once EPS-E free components had been identified, lactose, glucose and galactose were evaluated as potential carbon sources. Glucose was found to be the optimum carbon source. The final media composition supported growth to the same extent as RCM providing significant EPS yields and no interferences during analysis.

Three strains of Bifidobacterium breve (JCM 7017, JCM 7019 and JCM 2258) and two strains of Bifidobacterium animalis subsp. lactis (AD011 and A1dOxR) were grown in broth cultures or on plates, and a standard exopolysaccharide extraction method was used in an attempt to recover exocellular polysaccharides. When the extracted materials were analysed by NMR it was clear that mixtures of polysaccharides were being isolated including exopolysaccharides (EPS) cell wall polysaccharides and intracellular polysaccharides. Treatment of the cell biomass from the B. breve strains, or the B. animalis subsp. lactis AD011 strain, with aqueous sodium hydroxide provided a very similar mixture of polysaccharides but without the EPS. The different polysaccharides were partially fractionated by selective precipitation from an aqueous solution upon the addition of increasing percentages of ethanol. The polysaccharides extracted from B. breve JCM 7017 grown in HBM media supplemented with glucose (or isotopically labelled D-glucose-1-C) were characterised using 1D and 2D-NMR spectroscopy. Addition of one volume of ethanol generated a medium molecular weight glycogen (Mw=1×10 Da, yield 200 mg/l). The addition of two volumes of ethanol precipitated an intimate mixture of a low molecular weight β-(1→6)-glucan and a low molecular weight β-(1→6)-galactofuranan which could not be separated (combined yield 46 mg/l). When labelled D-glucose-1-C was used as a carbon supplement, the label was incorporated into >95% of the anomeric carbons of each polysaccharide confirming they were being synthesised in situ. Similar H NMR profiles were obtained for polysaccharides recovered from the cells of B. animalis subsp. lactis AD011and A1dOxR (in combination with an EPS), B. breve JCM 7017, B. breve JCM 7019, B. breve JCM 2258 and from an EPS (-ve) mutant of B. breve 7017 (a non-EPS producer).