Study On Preparation Of Collagen Peptides From Pseudosciaena Crocea Scales

Collagen is an important constructive protein of organism, while as thecaducity of human body, there becomes more crosslinking in collagen molecule, so itis necessary for body to take in new collagen. Generally, the lower molecular weightof the collagen peptides is, the easier for body to absorb them. Thus low molecularweight collagen products should be developed as a kind of functional component inhealth food and cosmetic. On the other side, some collagen peptides also havebiological activties such as antioxidative activity, so we should look for theantioxidative collagen peptides on the basis of low molecular weight to increase theeffect of cosmetology and health care. Pseudosciaena crocea is a kind of maincommercial fish of Zhejiang province. In the processing of pseudosciaena crocea,the scales of the fish are always wasted and bring about pollution to environment. Ifthe collagen in the scales could be utilized to make into peptides, the waste wouldbecome a new resource of collagen products, and the environment pollution wouldbe reduced as well. In this paper, pseudosciaena crocea scales were taken as rawmaterial, scale collagen was obtained after the pretreatment, proteases wereemployed to prepare the antioxidative hydrolysate, and the conditions of thehydrolysis was optimized. The hydrolysate was further isolated and purified toproduce antioxidative collagen peptides, and finally the peptides obtained wereidentified by MALDI-TOF/TOF. The main results are as follows:(1) In pretreatment, the time of decalcification with0.4M HCl should becontroled for1h, and the time of removing the protein with0.1M NaOH should becontroled for12h. It is benefit for hydrolysis to have high pressure processing of thecollagen before the hydrolyzing.(2) Gly, Pro, Ala, Glu, Hyp and Arg are the main amino acid of pseudosciaenacrocea scale collagen, while the contents of Phe and Tyr are very low.(3) Trpsin, neutral protease, protemax and alcalase were employedindependently to hydrolyze the scale collagen under their optimum pH andtemperature, and the degree of hydrolysate (DH), DPPH free radical scavenging rate(DSA), reducing power, ferrous ion binding ability, hydroxyl free radical scavengingrate (HSA) and molecular weight distribution of the four hydrolyzates were detected. Results showed that alcalase hydrolyzate had the highest DH and antioxidativeactivities, however the molecular weight distribution of the four hydrolyzates werenot so different.(4) According to the results of the single factor tests, response surfacemethodology (RSM) based on a central composite rotatable design (CCRD) wasapplied to optimise the hydrolysis of collagen with the aim to maximise DH andDSA. The optimum condition for maximum DH was: pH8.0, temperature54℃, E/S3.0%, time2.3h, under it the DH was46.63%; the optimum condition for maximumDSA was: pH7.8, temperature50.8℃, E/S2.9%, time2.1h, under it the DSA was14.97%.(5) The hydrolyzate was isolated and purified by the steps as follows: SephedaxLH-20, MCI-gel CHP20P, ODS-A. Six fractions of DPPH free radical scavengingactivity were obtained, and the DSAIC50values were: M1N1:168.16mg/mL; M1N2:112.36mg/mL; M1N3:55.65mg/mL; M1N4:29.43mg/mL; M2N1:72.41mg/mL;M2N2:31.62mg/mL.(6) With MALDI-TOF/TOF,12peptides were identified from five fractions, thesequence of the peptides in M1N2: EGRAAGPR, KDGLRGEP and GSPGEPGER;sequence of the peptides in M1N3: GLKGDRGQPR and QPGMPGAPGR; sequenceof the peptides in M1N4: QRPPEPR and EKVWKYCD; sequence of the peptides inM2N1: GPSGPVGER and QGSPGTPGQVG; sequence of the peptides in M2N2:GHQGPGGM*PGER, VGLPGLSGPVG and GDRGYEGPR.