Enzymatic Synthesis And Purification Of Galacto-oligosaccharides

Galacto-oligosaccharides are prebiotics and important components of human breast milk.They have physiological effects that include promoting the proliferation of Bifidobacteria,mineral absorption,and lipid metabolism.In this study,galacto-oligosaccharides were synthesized by a Lactobacillus plantarum-derived ?-galactosidase enzymatic method.The glycosidic linkage of galacto-oligosaccharide was identified.The galacto-oligosaccharide was then purified using Kluyveromyces lactis.The metabolic changes of yeast cells during purification were analyzed by related enzyme activities and transcriptomics.The main findings are as follows:1.Three strains with higher P-galactosidase activity were obtained from laboratory-preserved strains of lactic acid bacteria.The ?-galactosidase from L.plantarum was determined by measuring enzyme and transglycosylation activities for the production of galacto-oligosaccharides.The protein in the crude enzyme solution was separated by SDS-PAGE and identified by LC-MS.The results demonstrated that L.plantarum Go qi could produce ?-galactosidase with a molecular weight of 35 kDa.2.The optimal production process obtained by process optimization for the enzymatic synthesis of galacto-oligosaccharides included the following:temperature 35?,pH 7.0,lactose concentration 400 g/L,and reaction time of 6 h.The glycosidic bond structure of the galacto-oligosaccharide was identified by GC-MS.A total of eight disaccharide and four oligotrisaccharides were identified.Their main structural formula were ?-D-Galp-(1?6)-D-Lac,?-D-Galp-(1?3)-D-Lac,and ?-D-Galp,(1?6)-D-Glc?3.The galacto-oligosaccharide was purified using K.lactis.By determining sugar content changes of the purified solution during the purification process,it was proven that only lactose,glucose,and galactose were consumed,and galacto-oligosaccharide was not consumed.Analysis of K.lactis related enzyme activities and transcriptomics demonstrated that the synthesis of macromolecules such as proteins in yeast cells was blocked,but cell growth was resumed by increasing the processes of glucose metabolism and substance transport.K.lactis was successful in generating high-purity galacto-oligosaccharide.The optimal purification process conditions were soluble solid content of 10%and addition of 75 g/L yeast.The galacto-oligosaccharide content increased from 30%to 75%after 8 h of purification.In summary,galacto-oligosaccharide can be enzymatically synthesized by ?-galactosidase from L.plantarum Go qi.By combining the process of enzymatic synthesis with the fermentation of K.lactis,high purity galacto-oligosaccharides can be produced.