Abstract: Enzymatic synthesis of galacto-oligosaccharides targeting cholesterol reduction

Vasiliki Kachrimanidou(1), Oswaldo Hernández-Hernández(2), Sofia Kolida3, Katherine Stephens(1), Glenn R. Gibson(1), Robert A. Rastall(1)

(1) Department of Food and Nutritional Sciences, School of Chemistry, Food and Pharmacy, The

University of Reading, Whiteknights, P.O. Box 226, Reading RG6 6AP, Berkshire, United Kingdom

(2) Instituto de Investigación en Ciencias de la Alimentación (CIAL), Consejo Superior de Investigaciones

Científicas (CSIC), Calle Nicolás Cabrera, 9, 28049 Madrid, Spain

(3) Optibiotix, Health plc, Innovation Centre, Innovation Way, Heslington, York, United Kingdom

Introduction: The new definition of a prebiotic as “a substrate that is selectively utilised by host microorganisms conferring a health benefit” (Gibson et al., 2017), emphasises a prebiotic’s selective nature and impact on health. The aim of this work was to optimise the synthesis of galactooligosaccharides (GOS) using β-galactosidases from Lactobacillus plantarum LPLDL® and test the hypothesis that the GOS will selectively impact not only on the growth of the parent strain but also on its biological activity mediating a health benefit. Previous studies identified L. plantarum LPLDL® as a probiotic with high in vitro bile salt hydrolase (BSH) and cholesterol reduction activities and confirmed its impact on plasma cholesterol reduction in a human intervention study (Costabile et al, 2017). Methods: L. plantarum LPLDL® biomass was harvested at mid to late exponential growth phase and cells were mechanically lysed to release intra-cellular β-galactosidases. Following optimisation for temperature, pH, initial lactose concentration and enzymatic activity, GOS (LPGOS) was synthesized, purified and structurally characterised. The selective impact of LPGOS on the parent probiotic and its cholesterol reducing activity was evaluated in vitro pH-controlled faecal cultures and compared to commercially available GOS (BGOS). Samples were obtained for bacteriology (FISH, qPCR), short chain fatty acid (SCFA), BSH activity and cholesterol reduction determination.
Results: Lab scale synthesis optimisation gave yields of 39% for LPGOS, with initial lactose concentration, and temperature being the main factors affecting yield rather than the pH. Faecal culture evaluation showed a significant impact of LPGOS on Lactobacillus concentrations, cholesterol removal and BSH activity. The effect was further enhanced when used in combination with the parent strain. No significant impact on other numerically dominant and functionally significant bacterial groups was noted. SCFA profiles in the LPGOS and BGOS cultures were similar, however BGOS had no significant impact on cholesterol removal.
Discussion: Synthesis of LPGOS using β -galactosidases expressed by L. plantarum LPLDL® created a prebiotic which works in true synergy with the parent strain, not only increasing its populations in a complex culture environment, but also enhancing the biological activity the probiotic was selected for, cholesterol reduction. This approach may be used to rationally design targeted prebiotics for probiotics associated with impact on specific health biomarkers to further enhance their biological activity. This combination is termed an OptiBiotic®.

References

Gibson GR et al. (2017). Expert consensus document: The International Scientific Association for Probiotics and Prebiotics (ISAPP) consensus statement on the definition and scope of prebiotics. Nature Reviews Gastroenterology Hepatology 14, pages 491-502, doi:10.1038/nrgastro.2017.75

Costabile A. et al. (2017). An in vivo assessment of the cholesterol-lowering efficacy of Lactobacillus plantarum ECGC 13110402 in normal to mildly hypercholesterolaemic adults. PLoS One. 12 (12):e0187964. doi: 10.1371/journal.pone.0187964