Structural Characterization And Physicochemical Properties Of Kefiran And Their Interactions With Milk Protein

Kefir is a kind of complex fermented milk with acid and low alcohol content,which is formed by Kefir fermentation.Because Kefir exopolysaccharides have many biological activities,such as anti-microbial and anti-tumor,hypoglycemic,immune cell balance,protection of liver injury,wound healing and so on,potential development prospects and application value,it has gradually become one of the hot spots in the field of exopolysaccharide research.In this thesis,Kefir granules and Kefir fermented milk were used as raw materials.Response Surface Methodology(RSM)was used to optimize the extraction process of Kefir exopolysaccharides,the physical and chemical properties,primary structure and anti-oxidation properties of the crude polysaccharide were studied by means of SEM,UV,IR,emulsification,flocculation and anti-oxidation,secondly,the exopolysaccharides were separated and purified by DEAE Sepharose Fast Flow anion exchange column and BRT-GS system,and the structure was characterized,finally,the interaction between Kefiran and milk protein was studied by rheometer and Zeta potential analyzer.The findings are as follows:1.Single factor test and response surface methodology were used to optimize the extraction process of Kefiran from Kefir fermentation broth and Kefiran-G from Kefir granules.The optimum conditions of Kefiran extraction were as follows:The volume of ethanol 1.5 times,the concentration of ethanol 95%,the settling time of ethanol 23 h,the extraction rate of Kefiran 2.75%,the ratio of material to liquid 1:9,the extraction time 1h,the extraction temperature 90°C,the extraction rate of Kefiran-G was 5.37%.2.The structure,physicochemical properties and antioxidant activity of crude exopolysaccharides Kefiran and Kefiran-G from different sources were determined.The results of SEM showed that the microstructure of Kefiran-G was characterized by many spherical structures with similar size and shape adhered to the edge dendritic structure,and the structure of Kefiran was loose and reticulate.Both Kefiran and Kefiran-G contained a small amount of protein,and Kefiran-G had better emulsifying activity and flocculating activity than Kefiran.Kefiran showed better antioxidant activity than Kefiran-G.3.Kefiran-III with molecular weight of 1.98×10~4Da and Kefiran-G-I neutral polysaccharides with molecular weight of 1.41×10~6Da were isolated and purified by Ion Exchange and GEL column chromatography.Kefiran-III monosaccharides are composed of fucose,aminogalactose hydrochloride,rhamnose,Arabinose,Glucosamine,Galactose,glucose,n-acetyl-d Glucosamine,xylose and mannose,its molar ratio is0.016:0.014:0.103:0.047:0.052:0.126:0.503:0.027:0.048:0.062,containing a small amount of protein,Kefiran-G-I is composed of two monosaccharides,galactose and glucose.The ratio of substance is 0.588:0.412,pyran-type polysaccharides containingβ-glycoside andα-glycoside bonds and having carboxyl groups.4.After the Skim Milk was fermented,the particle size of the solution increased,the Zeta-potential decreased,the fluorescence intensity decreased,and the microstructure changed from spherical structure with different size and smooth surface to flaky structure with smooth surface,when Kefiran was added to the fermented Skim Milk,the particle size increased,the Zeta-potential decreased,the fluorescence intensity was the lowest,the surface of the sheet structure formed part of the network structure,the apparent viscosity and the viscoelasticity increased significantly,the frequency dependence is small,stability is high.In Kefiran/WP system,compared with WP solution,the particle size of Kefiran/WP mixed solution increases,the Zeta-potential decreases,the intensity of maximum absorption peak decreases,and the fluorescence intensity also decreases.The structure of Kefian/WP mixed solution is fragmented and its surface is compact and smooth,apparent viscosity increase.