Study On Enzymatic Hydrolysis Of Whey And Separation Purification Process Of Bovine Milk Oligosaccharides

Bovine milk oligosaccharides are a class of milk-derived oligosaccharides composing of3-10 specific monosaccharides linked by different glycosidic bonds.Although the contents and types of bovine milk oligosaccharides are not abundant compared to human milk oligosaccharides,some bovine milk oligosaccharides have the same structures as human milk oligosaccharides.They have a varity of physiological functions such as promoting the proliferation of beneficial bacteria in the gut,increasing the colonization of the anti-adhesion for intestinal pathogen,and enhancing intestinal barrier function for infants and young children.Besides,the monosaccharide of sialic acid can provide infant brain developmental and cognitive potential of nutrients.The production process of bovine milk oligosaccharides using milk whey as raw material can provide scientific basis for the development of industrial production of milk-derived oligosaccharides,infant milk formula and functional foods.In this study,a method of simultaneous determination of three sugars(glucose,galactose,lactose)was established by high performance liquid chromatography(HPLC)with anthranilamide(2-AB)derivatization.The limit of detection(LOD),limit of quantification(LOQ)were 0.5?g/L,0.5?g/L,1?g/L;1.8?g/L,1.8?g/L,and 3.5?g/L,respectively.The correlation coefficient of standard curve R~2 was 0.999.The recovery rate were from 100%to115%.This method has a high sensitivity,accuracy and repeatability,which can provide a measureent for galactooligosaccharide synthesis,and the determination of three sugars on membrane separation process or final product.The optimum process parameters for enzymolysis of milk whey by?-galactosidase were40°C,1‰for enzyme,200 g/L for feed solution,and 60 min.Under these conditions,the relative content of galacto-oligosaccharides was 26.63%and the residual lactose was 16.90%.Under the optimal enzymolysis conditions,the membrane flux,protein retention rate,and total sugar transmissibility were used as indicators to establish the optimal transmembrane pressure for the separation of bovine milk oligosaccharides by membrane separation technology to be 175 kPa.The protein retention rate was 84%,and most of the insoluble substances and proteins in the whey were retained;the optimal transmembrane pressure for ultrafiltration was275 kPa,and the total sugar transmission rate was 96%.Microfiltration and ultrafiltration processes can all achieve pilot scale production and provide important references for industrial production.The gel chromatography was used to further separate and purify the bovine milk oligosaccharides.The optimized injection volume was 40mL.By detecting content of the sialic acid,and galactooligosaccharide in each component,We determined 1st to 26th components both contained sialyllactose and galactooligosaccharide component.The final product of the separation was tested:the content of 3'-sialyllactose was3.29±0.096?g/mL,the content of 6?-sialyllactose was 0.28±0.068?g/mL,and the relative content of galacto-oligosaccharide was 56.62±0.48%;serine,glutamic acid,alanine and other 10amino acids were all less than 3.0ug/mL;calcium,magnesium,sodium and other minerals content were less than 70ug/mL;heavy metals such as arsenic,mercury,cadmium,and chromium were not detected.The content of lead(0.0143±0.0053?g/mL)conforms to the national pollutant limit standard in food.