Enzymatic Synthesis of Galactooligosaccharides From Whey Permeate
Galactooligosaccharides (GOS) are prebiotics that have a beneficial effect on human health by promoting the growth of probiotic bacteria in the gut. GOS are commonly produced from lactose in a reaction catalysed by β-galactosidase, termed transglycosylation. In the present work the synthesis of GOS from Whey Permeate (WP) using commercially available β-galactosidases was studied. The enzymes used were from Kluyveromyces lactis (Maxilact® L2000) and Escherichia coli. Initially, a novel
... , a novel quantitative TLC-based assay to monitor GOS synthesis was developed. This method was employed for kinetic analysis but precision and bias problems in quantification were observed. An HPLC assay was subsequently developed and used to quantitate the kinetics of GOS synthesis. The influence of substrate concentrations of WP and enzyme concentrations were examined. The reaction kinetics showed an exponential consumption of lactose, while the GOS reached a maximum level and decreased thereafter. The data showed that the enzyme and WP concentrations influenced the maximum level of GOS synthesis. The maximum yield of GOS from WP was found to be 24%. Modelling of GOS synthesis profiles using a full reaction mechanism (Kim et al., 2004) fitted the experimental data. However, high correlation between kinetic parameters and high standard errors in parameter estimates were found. Therefore, a simplified GOS synthesis mechanism based on simplifying assumptions previously identified in literature was devised. This reduced model fitted data appropriately and parameter estimation and associated uncertainty was improved. The influence of low amounts of organic solvents on GOS synthesis was examined. The progress curve in the presence of solvents was probed using the reduced reaction mechanism model. To examine the influence of the source of enzyme on GOS synthesis, two βgalactosidases were compared. Data showed that when reaction conditions were identical there was no significant difference in GOS synthesis observed. These studies show Whey Permeate is a useful material for GOS synthesis. They confirm the literature observations that enzyme and substrate concentrations strongly influence GOS yields. The use of organic solvents was found to modify the reaction kinetics, with promising applications to increase GOS yield. However, the source of enzyme may not influence GOS synthesis to the extent believed in the literature. DECLARATION I certify that this thesis which I now submit for examination for the award of MPhil, is entirely my own work and has not been taken from the work of others, save and to the extent that such work has been cited and acknowledged within the text of my work. This thesis was prepared according to the regulations for postgraduate study by research of the Dublin Institute of Technology and has not been submitted in whole or in part for another award in any Institute. The work reported on in this thesis conforms to the principles and requirements of the Institute's guidelines for ethics in research. The Institute has permission to keep, lend or copy this thesis in whole or in part, on condition that any such use of the material of the thesis be duly acknowledged. Signature __________________________________ Date _______________ iv ACKNOWLEDGEMENTS My foremost thank goes to my supervisors, Prof. Gary T. M. Henehan and Dr. Jesús María Frías Celayeta, for their support, guidance and encouragements throughout the project. I appreciate their contributions of knowledge, insights and suggestions that helped to shape my research skills. This work was conducted with the financial support from Technological Sector Research grant (2007)(2008)(2009)(2010). I would like to acknowledge them for giving me the opportunity to carry out my research. I would also like to convey thanks to DIT administrative staff for providing the financial means and laboratory facilities.