Microsoft Word 王珊珊-二校

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1 6 10 Vol. 6 No Journal of Food Safety and Quality Oct., 2015 鲉王珊珊, 周德庆 *, 刘楠, 朱兰兰, 孙永, 赵峰 (, ) 摘要 : 目的鲉, 方法 (FTIR) - (SDS-PAGE) ; SCH 1(Mw>2000 U) SCH 2(Mw<2000 U), H 2 O 2 L929 结果 16.7%; FTIR ; SCH 1, SCH 2 SCH H 2 O 2,, 结论鲉 I,, H 2 O 2 关键词 : 鲉 ; ; ; Preparation and characterization of collagen and collagen peptides from the skin of rockfish Sebastes schlegeli WANG Shan-Shan, ZHOU De-Qing *, LIU Nan, ZHU Lan-Lan, SUN Yong, ZHAO Feng (Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao , China) ABSTRACT: Objective The collagen and collagen peptides were prepared from rockfish skin and the characterization was investigated. Methods The properties of collagen were analyzed by amino acid analysis, Fourier transform infrared spectroscopy(ftir), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and scanning electron microscope (SEM). SCH 1 (Mw>2000 U) and SCH 2 (Mw<2000 U) were prepared using ultrafiltration membranes. The water absorption capacity, moisture retention capacity, foaming expansion, foaming stability, and antioxidant activity in H 2 O 2 -stimulated L929 cells were analyzed. Results The amino acid composition showed the amino acid content of collagen was 16.7%; FTIR spectra showed collagen remained natural triple-helix structure. Collagen displayed a polyporous ultrastructure under SEM observation. SCH 1 possessed better foaming properties, SCH 2 showed a better water absorption capacity. SCH exhibited certain protective effect against cell damage induced by H 2 O 2. Conclusion The collagen extracted from the rockfish skin was type I collagen. SCH had good foaming properties and water absorption 基金项目 : (2015BAD17B01) (ZQ ) Fund: Supported by the National Science & Technology Support Plan Projects (2015BAD17B01) and Qingdao Postdoctoral Application Research Project (ZQ ). * 通讯作者 :,,, zhoudq@ysfri.ac.cn *Corresponding author: ZHOU De-Qing, Professor, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao , China. zhoudq@ysfri.ac.cn

2 10, : 鲉 3977 capabilities. Treatment of H 2 O 2 -induced L929 cells with SCH increased cell viability. KEY WORDS: rockfish; fish skin; collagen; collagen peptides 1 引言,,, [1],,,,,, [2], : I ( ) II XI ( ) V ( ) [3-4], Mendis [5], Wang [6],,, 鲉 Sebastes schlegeli),,, [7] 鲉,, 2 材料与方法 2.1 实验材料鲉 21.0~ 29.9 cm/ ; L929 ; (MTT) ; DMEM Gibco ; ; T 2.2 仪器与设备 Jouan BR4i, Jouan ; DYY-6C, ; , Hitachi ; Bruker Tensor 27, Bruker 2.3 实验方法基本组成分析 GB [8] ; GB [9] ; GB [10] ; GB/T [11] ; GB/T [12] 胶原蛋白的制备 (1) 4, [13] : 0.1 mol/l NaOH 1 d 0.5 mol/l (1:30, m:v) 3 d 9000 r/min 20 min, NaCl 0.9 mol/l 9000 r/min 20 min 0.5 mol/l 3 d 鲉 (2) T/%=(A/B) 100 : T,, %; A,, g; B,, g 胶原蛋白特性分析 (1) SDA-PAGE( - ) 0.5 mol/l, 2 mg/ml, ph 5% 7.5%, 15 μl, 100 V, 2~3 h, 20 min

3 (2) 10 mg, 6 mol/l h,, (Hitachi ) (3) 10 mg KBr,, Bruker Tensor ~500 cm -1, 2 cm -1 (4),, 20 kv, JEOL JSM 胶原肽的制备 10% ph 8.0, : 50, 90 min, 1.5% 5 min, 4000 r/min 15 min 2000 U,, (skin collagen hydrolysates, SCH)SCH 1: Mw>2000 U; SCH 2: Mw<2000 U 胶原肽特性分析 (1) (skin collagen hydrolysates, SCH) 5 mg/ml, ph ( ) V 0 SCH 1.5 min, V 1 3 min V 2 (foam expansion, FE) (foam stability, FS) : FE(%)=(V 1 V 0 )/V 0 100; FS(%)=(V 2 V 0 )/(V 1 V 0 ) 100; (2) : 0.5 g SCH,, (NH 4 ) 2 SO 4 (%)=(m n m 0 )/m 0 100; m 0 m n : 0.5 g SCH,, 10%, (NH 4 ) 2 SO 4, (%)=H n /H 0 100; H 0 H n (3) H 2 O 2 L929 MTT L929, 0.25%, DMEM /ml, h : 1) : SCH( mg/ml) 24 h, 1 mmol/l H 2 O 2 6 h; 2)H 2 O 2 : 1 mmol /LH 2 O 2 6 h, SCH( mg/ml) 24 h, (Control), (H 2 O 2 ) 1 mmol /LH 2 O 2 20 μl MTT 4 h, 150 μl (DMSO), 15 min, 570 nm A (%)=(A /A ) 100% 数据分析 x±s, SPSS 17.0, P< 结果与分析 3.1 基本组成 1, 鲉 (65.83%) (68.4~72.7%) [14], (29.41%) (21.6%) [14] (24.75%) [13], 11.1 [15] 鲉 (2.34%) (1.95%) [16] 25.97%, 88.32%, 鲉, 52.41% (0.42%), 3.2 SDS-PAGE 电泳 1, 鲉 α1 α2 β), β ; I [16],

10, : 鲉 3979 表 1 基本组成 (g/100 g) Table 1 Proximate analysis of samples (g/100 g) 65.83±0.45 29.41±1.22 4.01±0.69 1.05±0.38 2.34±0.02 5.12±0.38 93.68±1.56 NT 0.42±0.07 7.89±0.

SDS-PAGE patterns of collagen from rockfish (Sebastes schlegeli) I II III, β, 鲉 3.3 氨基酸组成 2, 鲉,, 鲉 + ) 167 /1000, I (215 /1000 ), [17] 3.4 傅里叶变换红外光谱 A N-H, 3400 3440 cm 1, NH 3300 cm 1 [18] 2, A 3306.

4 10, : 鲉 3979 表 1 基本组成 (g/100 g) Table 1 Proximate analysis of samples (g/100 g) 65.83± ± ± ± ± ± ±1.56 NT 0.42± ±0.08 表 2 Table 2 许氏平鲉胶原蛋白的氨基酸组成分析 Amino acid composition of collagen from rockfish (Sebastes schlegeli) /1000 (ASP) 46 (THR) 26 (SER) 56 (GLU) 71 : I: ; II: ( β ); III: ( β ); IV: I 1 鲉 SDS-PAGE Fig. SDS-PAGE patterns of collagen from rockfish (Sebastes schlegeli) I II III, β, 鲉 3.3 氨基酸组成 2, 鲉,, 鲉 + ) 167 /1000, I (215 /1000 ), [17] 3.4 傅里叶变换红外光谱 A N-H, cm 1, NH 3300 cm 1 [18] 2, A cm 1, I, C=O, (GLY) 329 (ALA) 126 (VAL) 35 (MET) 19 (ILE) 9 (LEU) 12 (TYR) 6 (PHE) 14 (LYS) 26 (HIS) 5 (ARG) 48 (PRO) 94 (HYP) cm 1[19] I : β-, cm 1 ; α-, cm 1 ; β-, cm 1[20] cm 1, I cm 1 III, 1454 cm 1 (0.85) 1; 1 [21] SDS-PAGE ( 1),, 3.5 扫描电镜鲉,

3980 6 ( 3),, [22] 3.6 起泡性质 4 ph SCH SCH,, SCH 1 SCH 2, Li [23], [24] SCH 1, ; SCH 2,, 3.7 水合性质 5 81% SCH, 0~4, SCH, 10 h,, SCH 20 h, SCH,, 2 鲉 Fig.

5 ( 3),, [22] 3.6 起泡性质 4 ph SCH SCH,, SCH 1 SCH 2, Li [23], [24] SCH 1, ; SCH 2,, 3.7 水合性质 5 81% SCH, 0~4, SCH, 10 h,, SCH 20 h, SCH,, 2 鲉 Fig. 2 FTIR of collagen from rockfish (Sebastes schlegeli) Fig. 3 3 鲉 SEM) Scanning electron microscope (SEM) images of collagen from rockfish (Sebastes schlegeli) A: 100; B: 1000

6 10, : 鲉 3981 SCH 2 SCH 1, SCH,,,, 3.8 胶原肽对 H 2 O 2 诱导 L929 细胞氧化损伤的保护作用 MTT SCH H 2 O 2 L929, 6 H 2 O 2, (0.2 mg/ml)sch, (0.6 mg/ml)sch H 2 O 2,, SCH 2, SCH 1 SCH %(P<0.01) 73.70%(P<0.01); H 2 O 2, SCH 1 SCH 2 : (P<0.05) 4 SCH (A) (B) Fig. 4 Foam expansion (A) and foam stability (B) of SCH Fig. 5 5 SCH (A) (B) Moisture-absorption (A) and moisture-retention (B) curve of SCH : A. ; B.H 2 O 2 6 SCH H 2 O 2 L929 Fig. 6 Effects of SCH on the cell viability of L929 cells oxidative damaged by H 2 O 2 # : P<0.05, ## : P<0.01 ; * : P<0.05, ** : P<0.01 H 2 O 2 # : P<0.05, ## : P<0.01 vs control group; * : P<0.05, ** : P<0.01 vs H 2 O 2 group

7 %(P<0.05) 63.86%(P<0.01) H 2 O 2, 鲉 H 2 O 2 L929, L929, H 2 O 2, 4 结论 SDS-PAGE 鲉 I,, SCH 1(Mw>2000 U) SCH 2(Mw<2000 U), SCH 1 SCH 2, SCH 2 SCH 1 SCH 1 SCH 2(0.6 mg/ml) H 2 O 2, 鲉参考文献 [1] Schuppan D, Schmid M, Somasundaram R, et al. Collagens in the liver extracellular matrix bind hepatocyte growth factor [J]. Gastroenterology, 1998, 114(1): [2] Badii F, Howell NK. Fish gelatin: structure, gelling properties and interaction with egg albumen proteins [J]. Food Hydrocolloid, 2006, 20(5): [3] Aidos I, Lie Ø, Espe M. Collagen content in farmed Atlantic salmon (Salmo salar L.) [J]. J Agr Food Chem, 1999, 47(4): [4],,,. [J]., 2009, 30(17): Jiao DL, Lu JF, Zhang WW, et al. Current research status and development of aquatic animal collagen [J]. Food Chem, 2009, 30(17): [5] Mendis E, Rajapakse N, Kim SK. Antioxidant properties of a radical-scavenging peptide purified from enzymatically prepared fish skin gelatin hydrolysate [J]. J Agr Food Chem, 2004, 53(3): [6] Wang J, Wang Y, Tang Q, et al. Antioxidation activities of low-molecular-weight gelatin hydrolysate isolated from the sea cucumber Stichopus japonicus [J].J Ocean U China, 2010, 9(1): [7] Lee SM, Jeon IG, Lee JY. Effects of digestible protein and lipid levels in practical diets on growth, protein utilization and body composition of juvenile rockfish (Sebastes schlegeli) [J]. Aquaculture, 2002, 211(1): [8] GB [S]. GB National food safety standards with the determination of moisture in food [S]. [9] GB [S]. GB National food safety standards with the determination of protein in food [S]. [10] GB [S]. GB National food safety standards with the determination of ash in food [S]. [11] GB/T [S]. GB/T National food safety standards with the determination of crude fat in foods [S]. [12] GB/T [S]. GB/T National food safety standards with the determination of hydroxyproline content in meat and meat products [S]. [13] Kittiphattanabawon P, Benjakul S, Visessanguan W, et al. Isolation and Characterisation of collagen from the skin of brownbanded bamboo shark (Chiloscyllium punctatum) [J]. Food Chem, 2010, 119: [14] Muyonga JH, Cole CGB, Duodu KG. Characterisation of acid soluble collagen from skins of young and adult Nile perch (Lates niloticus) [J]. Food Chem, 2004, 85(1): [15] Etherington DJ, Sims TJ. Detection and estimation of collagen [J]. J Sci Food Agr, 1981, 32(6): [16] Kittiphattanabawon P, Benjakul S, Visessanguan W, et al. Characterisation of acid-soluble collagen from skin and bone of bigeye snapper (Priacanthus tayenus) [J]. Food Chem, 2005, 89(3): [17] Karim AA, Bhat R. Fish gelatin: properties, challenges, and prospects as an alternative to mammalian gelatins [J]. Food Hydrocolloid, 2009, 23(3): [18] Doyle BB, Bendit EG, Blout ER. Infrared spectroscopy of collagen and collagen-like polypeptides [J]. Biopolymers, 1975, 14: [19] Wang L, An XX, Yang FM, et al. Isolation and characterization of collagens from the skin, scale and bone of deep-sea redfish (Sebastes mentella) [J]. Food Chem, 2008, 108:

[21] Plepis AMDG, Goissis G, Das-Gupta DK. Dielectric and pyroelectric characterization of anionic and native collagen [J]. Polym Eng Sci, 1996, 36(24): 2932 2938.

8 10, : 鲉 3983 [20] Ahmad M, Benjakul S, Nalinanon S. Compositional and physicochemical characteristics of acid solubilized collagen extracted from the skin of unicorn leatherjacket (Aluterus monoceros) [J]. Food hydrocolloid, 2010, 24: [21] Plepis AMDG, Goissis G, Das-Gupta DK. Dielectric and pyroelectric characterization of anionic and native collagen [J]. Polym Eng Sci, 1996, 36(24): [22] Ikoma T, Kobayashi H, Tanaka J, et al. Physical properties of type I collagen extracted from fish scales of Pagrus major and Oreochromis niloticas [J]. Int J Biol Macromol, 2003, 32: [23] Li Z, Wang B, Chi C, et al. Influence of average molecular weight on antioxidant and functional properties of cartilage collagen hydrolysates from Sphyrna lewini, Dasyatis akjei and Raja porosa [J]. Food Res Int, 2013, 51(1): [24] Liu J, Xu G, Yuan S et al. The effect of macromolecules on foam stability in sodium dodecyl sulfate/cetylpyridinium bromide mixtures [J]. J Disper Sci Technol, 2003, 24: 作者简介 ( 责任编辑 : 白洪健 ) 王珊珊, 博士, 主要研究方向为海洋功能食品 agate-w@163.com 周德庆, 博士, 研究员, 博士生导师 ; 主要研究方向为水产品加工与综合利用, 海洋功能食品 zhoudq@ysfri.ac.cn

Microsoft Word 陈伟婵-二校

Microsoft Word 陈伟婵-二校 6 10 Vol. 6 No. 10 2015 10 Journal of Food Safety and Quality Oct., 2015 陈伟婵 1 1,, 曾少葵 2*, 吴文龙 (1., 524088; 2. ( ), 524088) 1 摘要 : 目的 (Chlamys nobilis) 方法 -18, 结果 60 d,,, 81% 结论 -18 60 d,, 关键词 : ; ; Effect