Study On Preration Of Small Collagen Peptides By Bio-Enzymatic Hydrolysis And The Small Peptides Of Separation

Collagen peptides is the enzymatic hydrolysate of collagen and amixture peptides, because of its good functional properties, phydionlogicalactivities and extensive utilization prospect in the field of cosmetic &food, the molecular weight of hydrolyzed collagen is most importantfactors in producting collagen peptides to use as functional materials incosmetic.In the previous studies, there are some problems in the collagenhydrolyze, Hydrolysate' molecular weight is largo and the content of bigmolecular weight is high, so collagen peptides applications was astrictedin cosmetic, in the paper, adopted two- complex proteases to hydrolyzecollagen, so heightened the efficiency of hydrolyzation and solveddifficult hydrolyze.Using collagen as the substrate and the rate of acidity dissolutionpolypeptides as examination' index from the structure of collagen;Papain,Bromelin,Alkaline Protease,Neutral Protease and FlavorEnzyme principium chosen and used for the hydrolysis of collagen by Mono-Factor Experimentation. Complex Proteases was constituted ofPapain and Bromelin by the compare test of Complex Proteases. theresults showed that the optimal condition of hydrolysis by mono-factortest and orthogonal test: pH 6.5,time(t) 4h,temperature(T) 40℃,substance([S])12%,Pepsin and Bromelin E/S are 3000 and 2000U·g^-1respectively, the rate of acidity dissolution polypeptides and DH was 60%and 5.26% respectively. The SDS-polyacrylamide gel electrophores(SDS-PAGE) showed that the molecular weight of hydrolysis which thevestige big peptides concentrated on between 25.7 kDa and 40.3kDa.In order to tbtain small collagen peptides, the collagen hydrolysatesfurther hydrolyzed with Alkaline Protease and Neutral Protease ascomplex proteases, and optimized the conditions by mono-factorexperimentation: pH 9.5,time 3h,temperature 50℃,Alkaline Proteaseand Neutral Protease E/S 7000 and 4000U/g respectively, the result ofhydrolysate by Size Exclusion Chromatography-High PerformanceLiquid Chromatography(SEC-HPLC): The Average Molecular Weight(AMW) of hydrolysate was 386.3Da, the application request ofMolecular Weight satisfied to use in cosmetic.Activated carbon was chosed as decolouring agent of hydrolysate, itis an effective and simple decolourizer on the decolorization ofhydrolysate. The result showed that high degree of discolourization couldbe obtained under the following conditions: activated carbon concentr- ation 1.5%, pH 5.5, temperature 40℃, time 30min, decouring and the lossrate of peptides are 86.21%and 6.12% respectively.Molecular Weight Cut-Off (MWCO) 8kDa, 20kDa of ultrafiltrationmembrane were adopted to principium separate hydrolysate, The result ofremoval hydrolysate's SDS-PAGE, the Molecular Weight of hydrolysateare all 37.6 and 32.5kDa by ultrafiltration-membrane MWCO 8kDa and20kDa. We confirmed the MWCO 20kDa of ultrafiltration membrane bycombining the chromatogram map of peptides of hydrolysae, the optimalconditions: the comcentration of peptides 39.1 g/L, pressure 0.5MPa, pH5.0, the time of ultrafiltration 150min in rm. The permeate flux ofpeptides is 96.1%, the SEC-HPLC showed that the molecular weight ofhydrolysate less than 2kDa peak area is 99.79%.