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1 7 10 Vol. 7 No Journal of Food Safety and Quality Oct., , 陈文平 2, 朱照武 3, 樊世杰 (1., ; 2., ; 3., ) 1, 2* 摘要 : 目的, 方法, ; ph, 结果 20 mmol/l,, U : 1%, 60, ph 5 结论,, 关键词 : ; ; Process optimization of ginger protease extraction by citric acid precipitation CHEN Wen-Ping 1, 2, ZHU Zhao-Wu 3 1, 2*, FAN Shi-Jie (1. Egg Quality Improvement And Safety Technical Engineering Laboratory, Beijing , China; 2. Beijing Huadu Yukou Poultry Industry Co. Ltd., Beijing , China; 3. Hubei University for Nationalities, Enshi , China) ABSTRACT: Objective To optimize the extraction technology of ginger protease by citric acid precipitation, and determine the optimal reaction conditions of enzymatic reaction. Methods Citric acid precipitation method was used to extract ginger protease from ginger juice. The enzyme activities after adding different concentrations of citric acid were compared. The specific activities of the enzyme were assayed to determine the optimum conditions for enzymatic reaction in different conditions of buffer ph, substrate concentrations and temperatures. Results The highest enzyme activity was U when the citric acid concentration was 20 mmol/l. The optimal enzymatic reaction conditions were as follows: concentration of substrate was 1%, reaction temperature was 60, and the ph of buffer was 5. Conclusion The citric acid precipitation method can extract ginger protease simply and efficiently, and the activity of ginger protease can be rapidly detected by means of the condition optimization of the ginger protease enzymatic reaction. KEY WORDS: ginger protease; citric acid precipitation method; enzyme activity assay 1 引言 (ginger protease), P2, [1-3],, [4-7], [8], * 通讯作者 :,,, fansj23@126.com *Corresponding author: FAN Shi-Jie, Master, Livestock Engineers, Egg Quality Improvement and Safety Technical Engineering Laboratory, Beijing , China. fansj23@126.com

2 10, : 4217 [9] ph ; [10,11], ; [12],,,, 2 材料与方法 2.1 仪器与试剂 主要仪器 ( ); Cary 300 ( ); AUX-20A ( ); ( ); Alpha 2-4LSC ( CHRIST ) 主要试剂 (, ); G-250(, ); (, Sigma ) 2.2 实验方法 生姜蛋白酶提取工艺路线 [13],, 1 1 [13] Fig. 1 Extraction flow chart of ginger protease 姜汁透光率的测定, 10 ( mmol/l) [13], 3, -, A=log1/T, T=10^(-A), T, A 生姜蛋白酶含量的测定 G-250 [14] G-250 G-250, NH 3+ G-250,, G-250,,, 生姜蛋白酶活力测定, (40 ), min 1 μg , 1 ml 1 ml 1%, min, 0.4 mol/l 2.0 ml, 10 min 1 ml, 2.2.2, 0, GB/T23527 U [11] 探索最佳酶反应条件 ph, ph : -, ph , 1%, 40, 10 min,, : 0.5% 1% 1.5% 2%, 40, - (ph=5) 10 min,, : 1%, , - (ph=5) 10 min,, 3 结果与分析 3.1 姜汁透光率的测定 2, 2, 600 nm,, 625 nm, 625 nm

3 4218 7,, [14] 20 mmol/l, U 2 (n=3) Fig.2 Transmittance changes of ginger juice under different wavelengths (n=3) 625 nm, 1 1, 8 mmol/l,, ; 8 mmol/l 40 mmol/l,, 40 mmol/l 93.36% 3.2 生姜蛋白质含量的测定 G-250,,, 3 4 4,,, 3.3 生姜蛋白酶活力的测定 Fig. 3 3 Correlation between absorbance and ginger protease content ph 5 5,,, ph, 2~20 mmol/l, ; 20~100 mmol/l,,,, 4 (n=3) Fig. 4 The effect of citric acid addition on the protein content in ginger juice (n=3) Table 1 表 1 不同柠檬酸浓度下的姜汁透光率 (n=3) Transmittances of ginger juice under different citric acid concentrations (n=3) (mmol/l) % 1.10± ± ± ± ± ± ± ± ± ±0.77

4 10, : 4219,, [16] 7 7, 1%,, 1%, 1%,, 5 ph (n=3) Fig. 5 Correlation between citrate acid addition and ph of ginger juice and protease activity (n=3) 3.4 最佳酶反应条件的研究 缓冲液 ph 值对生姜蛋白酶的影响,,, ph, [15], ph ph 6 6, ph 5,, ph 5,, ph=5, ph 7 (n=3) Fig. 7 Effects of substrate concentration on protease specific activity (n=3) 反应温度对生姜蛋白酶的影响 [11],,,,,,, 8 8, 30~60,, 60 60,, U/mg, 60 6 ph (n=3) Fig. 6 Effect of ph value of buffer on protease specific activity (n=3) 底物浓度对生姜蛋白酶的影响,,, ;,, Fig. 8 8 (n=3) Effects of temperature on protease specific activity (n=3)

5 结论,, U( 20 mmol/l), : ph 5, 1%, 60,, 参考文献 [1],,,. [J]., 2014, 35(14): Fan JB, Hou Y, Huang XW, et al. Effect of different process parameters on the rheological properties of ginger protease-induced milk gels [J]. Sci Technol Food Ind, 2014, 35(14): [2],,,. [J]., 2015, 6: Li TY, Wu YK, Zhao XY, et al. Optimization of extraction technique of ginger protease by orthogonal ultrasonic method and its application in the functional yogurt [J]. Food Sci Technol, 2015, 6: [3] Ohtsuki, Kozo, et al. Purification of ginger protease by DEAE-sepharose and isoelectric focusing [J]. Biochem Biophys Acta, 1995, 1423(2): [4],,,. [J]., 2002, (2): 17 18, 21. Tang XZ, Huang XS, Ma M, et al. Study of zingibain on tenderization of pork [J]. Meat Res, 2002, (2): 17 18, 21. [5],,. [J]., 2007, (11): Li YQ, Yu CQ, Cui JJ, et al. Study on tenderization of beef by ginger juice [J]. Meat Ind, 2007, (11): [6] Vasudeb G, Mendiratta SK. Studies on tenderization and preparation of enrobed pork chunks in microwave oven [J]. Meat Sci, 2006, 74: [7]. [D]. :, Sun GL. Studies on the extracting and usage in beef tenderization of ginger protease [D]. Taian: Shandong Agricultural University, [8],,,. [J]., 2009, 54(12): Huang DX, Li XH, Li L, et al. Research progress in ginger protease and its application in food processing [J]. Sci Technol Food Ind, 2009, 54(12): [9],. [J]., 2005, 26(3): Hao GX, Yang QJ. The influence of factors on the activity of ginger rhizome protease [J]. J Hanshan Teachers Coll, 2005, 26(3): [10],. [J]., 2003, 24(2): Dai JQ, Huang XS. Purification of ginger protease [J]. Food Sci, 2003, 24(2): [11],. [J]., 2010, 35(1): Song Q, Xu GR. A study on the method for evaluating of ginger protease activity [J]. Food Sci Technol, 2010, 35 (1): [12],. [J]., 2004, 16(6): , 551. Cai HY, Zhou XH. Prinary research of preparation and Anti-micelle extracting about Giner Protase(GP- Ⅱ) [J]. Nat Prod Res Dev, 2004, 16(6): , 551. [13],. [J]., 2010, 26(8): Ji QZ, Zhou T. Processing technology of the clarified ginger juice [J]. Mod Food Sci Technol, 2010, 26(8): [14]. [D]. :, Zhang YX. Extraction of ginger protease [D]. Jinan: Shandong University, [15] Adulyatham, Richard OA. Stabilization and partial purification of a protease from ginger rhizome (zingiber offinale roscoe) [J]. Food Sci, 2005, 70(3): [16],,. [J]., 2005, 36(2): Song GY, Wang M, Wang XY. Inhibition kinetics of substrate, product and its analogues on phytase [J]. J Shandong Agric Univ, 2005, 36(2): 作者简介 ( 责任编辑 : 姚菲 ) 陈文平, 硕士, 主要研究方向为食品科学 chenwenping_sp@163.com 樊世杰, 硕士, 畜牧师, 主要研究方向为蛋鸡品质改良与食品安全 fansj23@126.com