| Preparation of collagen bioactive peptides derived from fish by-products has become one of the effective ways to achieve the comprehensive utilization of fish processing waste.Therefore,the acid soluble collagen was extracted from the skin of shark and tilapia,and the physico-chemical and enzymatic properties were investigated in this study.Then the relationship between peptide sequence and antioxidant activity was elucidated and the optimum decolorizing conditions of collagen peptides were determined.In addition,the anti-aging properties of collagen peptides were also investigated to provide reference for the using of collagen peptides in health food and cosmetics.Firstly,by the technology of amino acid analysis,SDS-PAGE,FTIR and HPLC the physico-chemical and enzymatic properties of the acid soluble collagens from the skin of shark(Prionace glanca)and tilapia(Tilapia zillii)were investigated.The amino acid composition showed that the isoleucine content of shark skin collagen was about 2-fold higher than that of tilapia skin collagen,but the proline content was lower.The molecular weight of ? chain and ? chain,and the thermal denaturation temperature of shark skin collagen were lower than those of tilapia skin collagen.However,no difference was observed in the FTIR and UV-Vis spectra.On the other hand,the data of SDS-PAGE and HPLC were indicated that both shark skin collagen and tilapia skin collagen could be hydrolyzed more easily by collagenase or trypsin than papain.When collagen was hydrolyzed with collagenase for 240 min,the collagen peptides content with molecular weight below 1000 Da from shark skin reached up to 23%,but only 17% was found in the tilapia skin collagen peptides.Nextly collagen peptides were prepared by multienzyme hydrolysis and the structure-function relationship was elucidated through the technology of size exclusion chromatography,HPLC,mass spectrum and peptide synthesis,et al.The results showed that the molecular weight of the hydrolysate mainly ranged from 150 to 2000 Da.After separated by size exclusion chromatography on Sephadex G-15 column,the fraction C which has the molecular weight of the smallest components presented the highest radical-scavenging activity among the three fractions.IC50 value for DPPH and hydroxyl radical was 0.57 mg/mL and 2.04 mg/mL for fraction C from shark skin collagen hydrolysate,but was 5.09 mg/mL and 0.76 mg/mL for tilapia,respectively.On the other hand,four sequences of collagen peptides and four kinds of amino acids were identified by MS/MS in fractions separated by RP-HPLC of fraction C from collagen hydrolysates.Their sequences were identified as Glu-Gly-Arg,Gly-Pro-Arg,Gly-Tyr and Gly-Phe,and their amino acids were Arg,Leu,Tyr and Phe,respectively.The peptide of Gly-Tyr displayed the highest DPPH and hydroxyl radical-scavenging activity among these peptides,and the IC50 value for DPPH and hydroxyl radical was 5.08 mg/mL and 0.62 mg/mL,but 5.08 mg/mL and 0.62 mg/mL for Tyr which displayed the highest radical-scavenging activity among these aminos.Then,we evaluated the anti-aging effect of collagen peptides on Hacat cells through determination of cell proliferation,protein content,SOD activity and MDA content.The dates showed that treatment of Hacat cells with collagen peptides enhanced cell proliferation,and being the highest cell counts with the dose of 0.20 mg/mL for shark and 0.10 mg/mL for tilapia.Under those dose of collagen peptides,the content of cell protein and the activity of SOD were significantly improved,and the content of MDA was significantly decreased.Howerer,the anti-aging effect was restrained when the dose of shark collagen peptides were more than 0.20 mg/mL,but not for tilapia collagen peptides.On the other hand,fraction C that separated from shark and tilapia collagen peptides by sephadex G-15 had better ability to promote cell proliferation compared to GY and tyrosine,and in improving cell SOD activity and inhibiting the production of MDA were even more superior than GSH.Lastly,the effects of activated carbon and macroporous resin on decolorizing of collagen peptides were investigated through the technology of amino acid analysis and HPLC.Based on the decolorizing rate and peptide loss,the optimum decolorizing conditions of collagen peptides were determined by orthogonal test.The results showed that under the optimum decolorizing conditions of activated carbon,the decolorizing rate and peptide loss of collagen peptides from shark skin were 95.47% and 10.60%,but 92.66% and 11.23% for tilapia.However,the decolorizing effect of macroporous resin was significantly inferior to activated carbon,which the decolorizing rate and peptide loss of collagen peptides from shark skin were 79.67% and 32.00%,but 83.02% and 30.77% for tilapia.Based on the results of the molecular weight distribution and amino acid composition,it was found that both component ratio of 500-1000 Da and amino acid conent of Tyr or Phe were significantly decreased after decolorizing whether with activated carbon or macroporous resin.In conclusion,many differences were found in the physico-chemical and enzymatic properties of the collagens from the skin of shark and tilapia,as well as the molecular weight and antioxidant activity of collagen peptides.However,GY and Tyr were the same main ingredients in the two types of collagen peptides.Therefore,the differences between seawater fish and freshwater fish should be considered when prepared the small molecule collagen peptides,but can be ignored the differences when prepared the antioxidant collagen peptides.On the other hand,the skin aging can inhibited significantly by the addition of collagen peptides,and the dose-effect relationship was more notable in shark.Therefore,the differences between seawater fish and freshwater fish should be considered in the use of collagen peptides.Furthermore,the fraction C was the active components in the collagen peptides,and through the synergistic effect to restrain the skin aging.Therefore,the results of this study showed that antioxidant and anti-aging properties of collagen peptides can be prepared from fish by-products,and achieved the efficient development of collagen from fish by-products. |
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