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1 响应面法优化香港巨牡蛎蛋白酶解工艺的研究徐艳 1, 孙雪萍 1, 张秀国 2, 杨家林 1, 黄国强 1 1, 张琴 (1., ; 2., ) 摘要 : 以水解度为指标, 比较了 6 种蛋白酶对香港巨牡蛎 (Crassostrea hongkongensis) 的水解效果在单因素考察实验的基础上, 采用 Box-Behnken 模型响应面设计, 建立香港巨牡蛎蛋白酶解的二次回归模型, 确定最佳的水解条件实验结果表明, 海产品专用蛋白酶对香港巨牡蛎蛋白的水解效果优于其他蛋白酶, 该酶的最佳水解条件为 : 酶与底物比为 5.93 %, 酶解时间为 4.07 h, 酶解温度为 54.6 在此条件下, 海产品专用蛋白酶酶解香港巨牡蛎蛋白的水解度达到 % ± 2.39 % 本研究为香港巨牡蛎的开发和利用提供了理论依据关键词 : 香港巨牡蛎 (Crassostrea hongkongensis); 水解度 ; 响应面分析中图分类号 : TS254.1 Q516 文献标识码 : A 文章编号 : (2015) doi: /hykx (Oxtrea rivularis Gould),,, [1],,, Sea Milk [2], [3], [4],,, [5],,,, [6] [2], [7, 8], [9] [10],,,,, [2],,, 1 实验部分 1.1 材料与试剂,,, 80 ; (TNBS), Sigma ; ; 0.1 mol/l HCl ; 1 % ; mol/l ph 8.2 ; 20 g/l ; 40 % NaOH( ); Alcalase (Protamex) (Flavourzyme) ; 1.2 仪器与设备 DK-S24 ( : ; : : (13YJ22HYS15); ( ) : (1981-),,,,,, : ; : , @qq.com 42 / 2015 / 39 / 3

2 ); SPECTRA MR (DYNEX); ( ); FA1004N ( ); ( ) 1.3 实验方法 [11] 63.5 % ± 6.44 % g, 30 ml, 1 6 (,, Alcalase,, ), 5 %, 4 h, 10 min, g 10 min, 表 1 6 种蛋白水解酶最适宜的反应条件 Tab.1 The optimal experimental conditions of six enzymes ph ( ) Alcalase [12] 0.5 ml, 1 % SDS 2.5 ml 15 μl, 1% SDS 35 μl, ph μl, 0.1 % (TNBS)500 μl 50 1 h 0.1 % HCl 2 ml, 30 min 340 nm (DH, Degree of Hydrolysis) = 水解后的游离氨基浓度水解前的游离氨基浓度 100% 总肽键浓度 = h tot (, 9.6 mmol/g) 1.3.4, (E/S, ), 1.5 g, 30 ml, (E/S, 3 %, 4 %, 5 %, 6 %, 7 %), 55 4 h, 10 min, 4000 g 10 min, 1.5 g, 30 ml, E/S 6 %, 55 (2, 3, 4, 5, 6 h), 10 min, g 10 min, 1.5 g, 30 ml, E/S 6 %, (30, 40, 50, 60, 70 ) 5 h, 10 min, g 10 min, Design Expert 8.05, Box- Behnken,,,, (E/S),, 2 表 2 响应面实验因素水平表 Tab. 2 Factors and levels of RSM experiment X 1 (E/S, %) X 2 (, ) X 3 (, h) , Design Expert 8.05, 2 结果与讨论 2.1 水解用酶的筛选 1, 6 Alcalase, % ± 3.16 % 2.2 单因素考察 2, E/S, Marine Sciences / Vol. 39, No. 3 /

3 1 6 Fig. 1 Comparison of C. hongkongensis protein hydrolysis by six proteases 4 Fig. 4 Effect of temperature on hydrolytic degree, 响应面实验设计分析及结果 2 E/S Fig. 2 Effect of E/S on hydrolytic degree E/S 6 %,,, E/S 5 % 7 % 3,, 2~5 h,, 5~6 h 5 h, 4 6 h 3 Fig. 3 Effect of time on hydrolytic degree 4,, 30 50, 50 60,,,, Design-expert 2, (Y) (X 1 ), (X 2 ) (X 3 ) : Y = X X X X 1 X X 1 X X 2 X X X X 2 3 3, X 1 X 2 F,, 3,,, P , P ,, (R 2 = ),,, 2.3.2, 5A 5a,,,,, 5B 5b,,,,,, 5C 5c,, Design-expert, : 44 / 2015 / 39 / 3

4 表 3 Box-Behnken 试验设计及结果 Tab.3 Experimental design and results of Box-Behnken X 1 (E/S, %) X 2 (, ) X 3 (, h) Y (DH, %) 表 4 响应面回归方程方差分析 Tab.4 ANOVA for response surface quadratic model F P F X X X X 1 X X 1 X X 2 X X 1 2 X 2 2 X %, 4.07 h, 54.6, %, : 5.9 %, 4 h, 54 (n=3), 25.73% ± 2.39 %,, 3 结论,, Box-Behnken,, 5.9 %, 4 h, 54, % ± 2.39 %,,,, [13-15],,,,,,,,,,,,, Marine Sciences / Vol. 39, No. 3 /

5 Fig. 5 Three-dimensional chart and corresponding contour plot : [1],. [J]., 2005, 26(6): 39-44 [2],,,. [J]., 2003, 22(3): 97-100 [3],,. [J]., 1997, l8(1): 112-116 [4],,,. [J]., 2009, 25(3): 262-265 [5],,,.

5 5 Fig. 5 Three-dimensional chart and corresponding contour plot : [1],. [J]., 2005, 26(6): [2],,,. [J]., 2003, 22(3): [3],,. [J]., 1997, l8(1): [4],,,. [J]., 2009, 25(3): [5],,,. [J]., 2008, 7: [6]. [D]. :, [7] Jung K S, Min J L, Hye J G, et a1 Purification and antimicrobial function of ubiquitin isolated from the gill of Pacific oyster, Crassostrea gigas[j]. Molecular Immunology, 2013, 53(1/2): [8] Liu Z Y, Zeng M Y, Dong S Y, et a1. Effect of an antifungal peptide from oyster enzymatic hydrolysates for control of gray mold (Botrytis cinerea) on harvested strawberries[j]. Postharvest Biology and Technology, 2007, 46(1): [9] Wang J P, Hu J N, Cui J Z, et a1 Purification and identification of a ACE inhibitory peptide from oyster proteins hydrolysate and the antihypertensive effect of hydrolysate in spontaneously hypertensive rats[j]. Food Chemistry, 2008, 111(2): [10] Qian Z J, Jung W K, Byun H G, et a1 Protective effect of an antioxidative peptide purified from gastrointestinal digests of oyster, Crassostrea gigas against free radical induced DNA damage[j]. Bioresource Technology, 2008, 99(9): [11],,,. [J]., 2014, 39(3): [12] Cian R E, Martínez-Augustin O, Drago S R Bioactive properties of peptides obtained by enzymatic hydrolysis from protein byproducts of Porphyra columbina [J]. Food Research International, 2012, 49(1): [13] Je J Y, Park P J, Byun H G, et a1 Angiotensin I convening enzyme (ACE) inhibitory peptide derived from the sauce of fermented blue mussel, Mytilus edtdis [J]. Bioresouree Technology, 2005, 96 (14): [14],,,. [J]., 2008, 29(11): [15],,,. [J]., 2007, 28(5): / 2015 / 39 / 3

6 Improvement of Crassostrea hongkongensis hydrolysis by Response Surface Methodology XU Yan 1, SUN Xue-ping 1, ZHANG Xiu-guo 2, YANG Jia-lin 1, HUANG Guo- qiang 1, ZHANG Qin 1 (1. Guangxi Institute of Oceanology, Guangxi Key Laboratory of Marine Biotechnology, Beihai , China; 2. Beihai College of Beihang University, Beihai , China) Received: Jun., 9, 2014 Key words: Crassostrea hongkongensis; Degress of hydrolysis; Response surface methodology (RSM) Abstract: Using DH (the degree of hydrolysis) as the main index, the hydrolysis of Crassostrea hongkongensis using six proteases was compared. On the basis of single factor experiment, response surface methodology and Box-Behnken model were employed to establish the quadratic model for optimization of the hydrolysis conditions. Results showed that the DH of marine products protein-specific protease was higher than that of others. The optimum hydrolysis condition was as follows: the ratio of enzyme to substrate was 5.93%, hydrolysis time was 4.07 h and the hydrolysis temperature was Under this condition, DH reached 25.73%±2.39 %. This study aims to provide theoretical basis for the development and utilization of C. hongkongensis. ( 本文编辑 : 康亦兼 ) Marine Sciences / Vol. 39, No. 3 /

H 2 SO ml ml 1. 0 ml C 4. 0 ml - 30 min 490 nm 0 ~ 100 μg /ml Zhao = VρN 100% 1 m V ml ρ g

H 2 SO ml ml 1. 0 ml C 4. 0 ml - 30 min 490 nm 0 ~ 100 μg /ml Zhao = VρN 100% 1 m V ml ρ g 16 6 2018 11 Chinese Journal of Bioprocess Engineering Vol. 16 No. 6 Nov. 2018 doi 10. 3969 /j. issn. 1672-3678. 2018. 06. 004 1 2 1 1 1. 330200 2. 330006 4 Box-Behnken 1 25 g /ml 121 92 min 2 6. 284%