Study On Bioactive Peptides From Milk

Casein glycomacropeptide(CGMP) is a kind of biological action of bioactive peptidesbearing the carbohydrate chain with sialic acid. Because CGMP has many physiologicalfunctions and unique nutritional attributes, it can widely be used in the function food andmedicine. This paper explored the technological process, enzymolysis, isolation andpurification of casein glycomacropeptide from casein. This thesis is enzyme experiment ofthis item, the main works are as follows:(1) Choosing the degree of glycosylated modification(sialic acid/protein, μg/mg)as indexto screen out the optimal enzyme from pepsin, trypsinase,chymosin, papain, neutral protease,flavor enzyme and alkaline protease, which enzymolysised casein to get caseinglycomacropeptide.(2) Choosing the sialic acid of non-dialyzable fraction of sample as the index,enzymolysis conditions of pepsin technology is optimized by single factor experiment. Theoptimal conditions for degradation of casein by pepsin enzymolysised are determined asfollows:The optimum temperature is37℃, the optimum pH is2.5, the optimum ratio ofenzyme and casein is800U/g protein, the optimum concentration of casein is4%, hydrolysistime is2h. Under such condition, sialic acid of sample is the highest,49.53μg/mL,containing CGMP0.61mg/mL.(3) Non-dialyzable fraction of sample was purified with DEAE-cellulose DE-52(2×20cm) preliminarily. The optimal separation conditions were as follows: equilibrium liquid was0.01mol/L NaAc-HAc buffer(pH=6.8), flow rate was0.5mL/min,3mL per tube, the gradientelution was conducted with0~0.4mol/L NaCl. The results showed that the peptides wereseparated into4chromatogram peaks by DE-52. Sialic acid was only found in the samplerecovered from the peak I eluted by0.1mol/L NaCl, but almost no sialic acid was found inother elution peaks.(4) The sample recovered from the peak I was further purified with Sephadex G-100column choromatography. The optimal separation conditions were as follows: equilibrium liquid was0.1mol/L NaAc-HAc buffer(pH=6.8), flow rate was0.25mL/min,1.5mL per tube.The results showed that the peptides were separated into3chromatogram peaks by SephadexG-100. Sialic acid was only found in the sample recovered from the peak I-1eluted byequilibrium liquid.(5) The purified product had a clear band on a SDS polyacrylamide gel electrophoresis,and its molecular mass was estimated to be about30kDa. The purity of caseinglycomacropeptide was determinated by UV spectrophotometric method, which can be up to92.1%.(6) Enzymatic preparation and separation technology of CGMP from casein weredetermined.