Studies On The Enzymatic Synthesis Of Galacto-oligosaccharide And Its Functional Properties

Galacto-oligosaccharide(GOS)is a mixed heteroglycan formed by connecting 1 to 7galactosyl groups in the form of?-glycosidic bonds on the galactosyl side of the lactose molecule.As a new type of functional prebiotic,GOS has the functions of promoting the proliferation of bifidobacteria in the intestine,improving the absorption of mineral elements,improving lipid metabolism,preventing and treating constipation.The use of safe and efficient?-D-galactosidase and the enzymatic synthesis of GOS have received extensive attention in recent years.In this study,a high-yielding?-D-galactosidase strain Kluyveromyces marxianus ZX-5 was isolated and screened from Tibetan mushroom.The enzyme production conditions of the strain were optimized,and the product protease was purified to obtain high-purity?-D-galactosidase.Using this enzyme,the technology of enzymatic synthesis of galactooligosaccharides was studied.Afterwards,membrane separation technology was used to study the separation and purification process of galactooligosaccharide.Finally,the functional characteristics of galactooligosaccharides on Lactobacillus plantarum YW11 and its application in the development of functional probiotic fermentation ice cream products were studied.The main findings are as follows:(1)Using Kluyveromyces marxianus ZX-5 as the experimental strain,the medium composition and fermentation conditions were optimized.The optimization of the medium composition is mainly from the two single factors of carbon source and nitrogen source,and the optimization of the fermentation conditions is mainly from the three single factors of temperature,shaker speed,and initial p H.The final result is:the optimal medium:galactose2%as carbon Source,tryptone 1%as nitrogen source.Optimal fermentation conditions:fermentation temperature 30?,initial p H 6.5,liquid volume 30%,rotation speed 100 r/min,inoculation volume 2%,fermentation under this condition for 36 h,the crude enzyme liquid enzyme activity was 2.60 U/mg.The?-D-galactosidase produced by Kluyveromyces marxianus ZX-5 was separated and purified by ammonium sulfate fractionation precipitation and DEAE-Sepharose fast flow ion exchange chromatography.The purified enzyme had a specific enzyme activity of 157.35 U/mg and was purified.The multiple is 60.5 times,and the recovery rate is 58.6%.(2)The purified enzyme was used to study the process of enzymatic synthesis of galactooligosaccharides,and compared with the results of GOS production from two different sources of commercial?-D-galactosidase.?-D-galactosidase derived from Kluyveromyces marxianus ZX-5,at 40?,p H 6.0,initial lactose concentration 50%(m/v),plus enzyme amount 16 U/m L,K~+added at 0.01 mol/L for 10 h,GOS synthesis rate was34.51%,concentration was 162.53 g/L,and lactose hydrolysis rate was 66.25%;?-D-galactosidase from commercial yeast,at 40?,Ph 6.0,initial lactose concentration 50%(m/v),added enzyme amount 16 U/m L,K~+added amount 0.01 mol/L for 12 h,GOS synthesis rate is 33.28%,concentration is 232.95 g/L,and the lactose hydrolysis rate is65.63%;?-D-galactosidase derived from commercial Aspergillus oryzae at 50?,p H 6.0,initial lactose concentration of 50%(m/v),enzyme addition 8 U/m L,Na~+addition amount0.01 mol/L for 12 h,the GOS synthesis rate was 30.89%,the concentration was 185.33 g/L,and the lactose hydrolysis rate was 55.48%.(3)The effect of ultrafiltration and nanofiltration on the separation and purification of galactooligosaccharides was investigated.The roll-type ultrafiltration membrane module with a molecular weight cut-off of 3 k Da was selected to obtain the optimal operating parameters of the ultrafiltration process operation:operating pressure 0.2 MPa,operating temperature 35?,feed liquid concentration 10%,feed liquid p H 4.0.After ultrafiltration,the impurity protein content dropped to 0.12%,the galactooligosaccharide content increased to 43.04%,and the product yield after ultrafiltration was 83.58%.The roll-type nanofiltration membrane module with a molecular weight cut-off of 400 Da was selected to obtain the optimal operating parameters of the nanofiltration process operation:operating pressure 0.55MPa,operating temperature 35?,feed liquid p H 4.0,nanofiltration water addition amount is 4 times of raw Liquid volume.After nanofiltration,the free glucose content decreased to1.31%,the impurity protein content decreased to 0.10%,the galactooligosaccharide content increased to 68.12%,and the final yield of the product after nanofiltration was 72.86%.(4)The addition of 4%(m/m)galactooligosaccharide can significantly promote the growth of the number of viable bacteria of Lactobacillus plantarum YW11,the protein density of probiotic bacteria increases by 40%,and GOS-probiotic metabolic products have good bacteriostatic effect to Shiga Bacteria,Salmonella and E.coli.The rheological experiment of ice cream slurry showed that galactooligosaccharides accelerated the fermentation process of the slurry and increased the viscoelasticity of the fermentation end of the slurry.Adding 4%(m/m)galactooligosaccharides,the viable count of probiotic ice cream reached 5.25×10~8CFU/g.The acidity,melting rate and swelling rate of the GOS added group were not significantly different from the fermented ice cream of the control group(p>0.05),and the viscosity of the ice cream slurry and the hardness of the product increased significantly(p<0.05).The highest survival rate of probiotic bacteria in artificial simulated gastric juice experiment is 87.10%,and the highest survival rate of artificial simulated pancreatic juice experiment is 85.11%,indicating that galactooligosaccharides have a significant protective effect on Lactobacillus plantarum YW11 under strong acid and bile environment.