| Silver carp (Hypophthalmichthys molitrix) is one of the main freshwater species in China because of its fast growth, easy to farming, high nutritional value. Large byproducts (eg, head, skin, scales, bones and internal organs, etc.) generated during processing silver carp that are usually treated as waste and discarded. The total weight of protein in the scale about50%-70%, and mainly collagen and keratin, which is a very good fish scale collagen raw materials. Use scale production collagen, which can protect environment, but also significantly improve the added value of aquatic products processing. In this paper, silver carp scale as the raw material and extracted collagen. Free radical scavenging rate as the main indicators, selected the best enzyme which prepared the antioxidant activity peptides, and optimized preparation conditions. Using In vitro and in vivo anti-oxidation test, systematic research of the silver carp scale collagen peptide (SCSCP) antioxidant activity, physicochemical properties and stability. Silver carp scale collagen antioxidant activity peptide were purified and identification by the modern biochemical separation techniques. Research results for the development and utilization of the silver carp scale collagen proteolysis antioxidant peptides provide a theoretical basis. The main results are as follows:1. In order to obtain a higher extraction rate of collagen from silver carp scale, first of all decalcification for scales. Explored by single factor experiment and orthogonal experiment design optimization to obtain the optimal process conditions of decalcification:liquid ratio1:30, EDTA concentration0.17mol/L, soaking time30h. In optimal conditions, the silver carp scales decalcification was94.11%. Optimization of pepsin extracts collagen conditions from silver carp scale by using response surface experiment design. Optimal extraction conditions:liquid ratio (v/w)18.0, the amount of pepsin added25.51U·mg-1, extraction time61.9h. In optimal conditions, collagen extraction rate was4.02±0.11%.Using SDS-PAGE gel electrophoresis to analysis the silver carp scale collagen, And compared with the stanurd type I collagen to determine the silver carp scale collagen is type Ⅰ collagen. UV scan shows silver carp scale collagen characteristic absorption at the wavelength of235nm. Amino acid composition analysis showed that glycine is the most abununt amino acids in silver carp scale collagen, followed by glutamic, alanine, proline and hydroxyproline. FTIR scan results show that the collagen better keep a helical structure.Silver carp scale collagen isoelectric point was about7.0, denaturation temperature24.1 ℃. Its solubility highest and lowest pH values were3.0and7.0(P<0.05), respectively, sodium chloride concentration greater impact on collagen solubility, water absorption and water retention are better than glycerin, emulsifying and emulsion stability, foaming and foam stability are better.2. The degree of hydrolysis (DH), DPPH·,O2-·and·OH free radical scavenging rate as the indicators. Compare the effects and the antioxidant activity of product of alkaline protease, neutral protease and papain hydrolysis on the silver carp scale collagen. The results showed strongest antioxidant activity that alkaline protease hydrolyzate. Its DPPH·,O2-·and·OH free radical scavenging rate were76.93±0.64%,43.11±0.48%and58.21±0.80%, respectively.Optimization the preparation antioxidant activity peptides conditions of alkaline protease hydrolysis silver carp scale collagen by using response surface experimental design. Optimal preparing conditions:the substrate concentration5.47%, hydrolysis time4.24h, enzyme dosage4200U·g-1. In optimal conditions, Antioxidant peptides on DPPH·,O2-·and·OH and·OH free radical scavenging rate was79.62±0.1.04%,46.28±0.96%and63.47±0.88%, respectively. Compared with before optimization, antioxidant peptide on the free radical scavenging rate has been significantly increased (P<0.05).3. Silver carp scale collagen peptide (SCSCP) was separation grading by using MWCO of ultrafiltration membrane were5kU,3kU and1kU, respectively. Get four different ultrafiltration components:SCSCP-Ⅰ (>5kU), SCSCP-Ⅱ (3-5kU), SCSCP-Ⅲ (1-3kU), SCSCP-Ⅳ(<1kU). Evaluate the antioxidant activity strength of ultrafiltration components by six indicators of the in vitro antioxidant. The results showed that the molecular weight less than1kU (SCSCP-Ⅳ) have strongest antioxidant activity in vitro(P<0.05). Amino acid composition analysis showed. SCSCP-Ⅳ total content of hydrophobic amino acids and arginine were higher, presumably with a higher antioxidant activity are closely related. Animal experiments show that SCSCP-Ⅳ can significantly increase serum antioxidant enzymes SOD and GSH-Px activity (P<0.05), decreased MU content (P<0.05).4. Physicochemical properties for SCSCP-Ⅳ findings suggest that, compared with the silver carp scale collagen, SCSCP-Ⅳ solubility less affected by pH changes, its solubility more than90%. SCSCP-Ⅳ emulsifying and emulsion stability are better than collagen (P <0.05). SCSCP-Ⅳ water absorption and water holding capacity superior to collagen and glycerol (P<0.05).The results of research on stability of SCSCP-Ⅳ showed that urk, sunlight and natural light had some umaging effects on SCSCP-Ⅳ antioxidant activity. Compared with sunlight and natural light, urk stored less impact. SCSCP-Ⅳ has good heat resistance. SCSCP-Ⅳ antioxidant activity in an acidic environment to maintain good activity, but in an alkaline environment lost quickly. Sodium chloride, sucrose and glucose although for SCSCP-Ⅳ radical scavenging activity had some umaging effects, but SCSCP-Ⅳ on free radical scavenging activity remained at95%. Metal ions in varying degrees reduce the SCSCP-Ⅳ radical scavenging activity. Sodium benzoate and potassium sorbate at different levels reduce the activity SCSCP-Ⅳ and potassium sorbate more negative effect than sodium benzoate. Compared with the spray-drying, the product has urker color, larger particles and dissolves faster by freeze-drying. In addition, it’s for O2-·and·OH free radical scavenging activity better than spray-dried product (P<0.05).5. The relative molecular weight less than1kU antioxidant peptide (SCSCP-Ⅳ) were isolated and purified by continuous use of DEAE-52anion exchange chromatography, Sephadex G-15Sephadex chromatography and RP-HPLC. One pair of free radicals scavenging activity stronger component (E3-4) was collected. E3-4on DPPH·,O2-·and·OH free radical scavenging rate was91.80±1.08%,79.23±4.10%and82.17±2.35%, respectively. Component E3-4was identified as a single component of high purity by the analytical RP-HPLC. Amino acid composition analysis showed that the component E3-4was composed by Glu, His, Pro, and Tyr, and the four amino acids in a molar ratio was n(Glu): n(His):n(Pro):n(Tyr)=1:1:1:1. Component E3-4was analyzed by TOF-MS/MS and MALDI-TOF/TOF MS, the relative molecular weight was576.9, the amino acid sequence HPEY (His-Pro-Glu-Tyr). Through the retrieval, it’s novel antioxidant peptides. Preliminary analysis of the antioxidant tetrapeptide HPEY (His-Pro-Glu-Tyr) antioxidant activity is provided by the proton ability Tyr, His ability to chelate metal ions, strong hydrophobic properties and structural stability of its own. |
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