Enzymatic Hydrolysis And Molecular Weight Detection Method Of Fish Collagen Peptide

Fish collagen peptides have been widely applied to many fields, such as food, healthy food and cosmetics. Molecular weight is an important quality indicator, which is closely related to the digestion and absorption properties and the physiological. In this paper, the fish collagen protein hydrolysates were prepared by the skin and scale of tilapia with different degree of hydrolysis through controlled hydrolysis technique. The effect of enzymes and the degree of hydrolysis to the peptide molecular weight were analysis by gel filtration chromatography. In addition, the method validation towards gel chromatography was taken to determine the fitness of fish collagen peptides molecular weight detection. And ultrafiltration centrifugation combined with mass spectrometry was to verify the accuracy of gel chromatography conditions. The main conclusions are as follows:The degreased skin and the decalcified scale of tilapia were rich in collagen, the crude protein content was as high as 88% and 90%, which were suitable for preparing fish collagen peptides through enzyme hydrolysis technique. Collagen peptides molecular weight was related to enzyme species and reaction time, and papain, neutral protease, trypsin and alkaline protease enzyme were used to for fish skin and scale hydrolysis to analyze the degree of hydrolysis and molecular weight distribution. The results showed that the skin and scales hydrolysates with trypsin digestion for 8 h reached the highest degree of hydrolysis, which were 16.619±0.275% and 16.376±0.130%, respectively. The molecular weight distribution of each hydrolysate was determined by gel chromatography. The results showed that the higher the degree of hydrolysis of the hydrolysates, the smaller the molecular weight, and molecular weight distribution is concentrated substance. The skin trypsin digestion 8 h hydrolysates molecular weight mainly in 1,000 Da or less, having a number average molecular weight of 531.29 Da, a weight average molecular weight of 799.51 Da, the width of the distribution coefficient was 1.50.The method of experimental validation was used to compared the molecular weight distribution of fish collagen peptides by TSK GEL G2000 SWxl G2000 and Superdex Peptide HR 10/30 column in different mobile phase conditions. The results showed that bacitracin and Gly-Gly-Tyr-Arg were abnormal in the elution characteristics of phosphate buffer mobile phase. The accuracy and precision of Superdex Peptide HR 10/30 column were higher in the detection of small molecules collagen peptide distribution, the adding standard recovery was reached to 101.90%, and the value of RSD was below 1%. And the TSK GEL G2000 SWxl column was more suitable for large molecules detection, the adding standard recovery was reached to 106.66%, and the value of RSD was below 0.5%.The accuracy of gel chromatography collagen peptide molecular weight distribution was further verified by ultrafiltration centrifugation and mass spectrometry. The results showed that the sample concentration, speed and time of centrifugation have some effect on the sample separation of ultrafiltration centrifugal tube, but the protein ratio was closed in different separation condition. MALDI-TOF-MS spectrometry results indicated that collagen protein sample separation by ultrafiltration tubes was effective. Compare the ultrafiltration results with the results of gel permeation chromatography, showed that the results of Superdex Peptide HR 10/30 column detection and the ratio of centrifugal ultrafiltration method were closest in detecting the collagen peptide <1,000 Da and <3,000 Da components.