黑豆胰蛋白酶抑制剂性质分析及高温对其消化特性的影响
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摘要
胰蛋白酶抑制剂(trypsin inhibitor, TI)在食品科学和生物技术中具有重要的研究价值。为探讨黑豆胰蛋白酶抑制剂(black soybean trypsin inhibitor, BSTI)的性质及胃肠液消化特性,本研究通过脱脂、热处理、硫酸铵盐析以及阴离子柱层析等方法,从黑豆中纯化得到BSTI。SDS-PAGE显示BSTI呈单一条带,相对分子质量约为21 kDa。在60℃以下与pH2～11的范围内,BSTI呈现较高稳定性。当BSTI与胰蛋白酶的摩尔比达到1时,胰蛋白酶的活力被抑制在15%以下。抑制动力学结果表明,BSTI对胰蛋白酶的抑制属非竞争性抑制,其抑制常数K_i=0.24 nmol·L~(-1)。圆二色谱分析发现,在常温下BSTI的二级结构为:β-折叠43.4%,无规则卷曲29.0%,β-转角21.2%,α-螺旋8.1%。BSTI的变性温度为61±0.9℃。BSTI在模拟胃肠液消化过程中表现出很强的抗酶解作用,但经121℃热处理30 min后,其抗酶解作用显著降低,易被胃蛋白酶、胰蛋白酶和胰凝乳蛋白酶等主要消化酶消化。
Trypsin inhibitor(TI) has significant research value in food science and biotechnology. In order to investigate the properties of black soybean trypsin inhibitor(BSTI) and the digestive properties of gastrointestinal fluids, BSTI was purified from black soybeans by degreasing, heat treatment, ammonium sulfate salting out and anion column chromatography. SDS-PAGE showed a single band of BSTI with a relative molecular mass of approximately 21 kDa. BSTI was stable below 60℃ and in the range of pH2-11. When the molar ratio of BSTI to trypsin reached 1, the activity of trypsin was suppressed to 15% or less. Inhibition kinetics indicated that the inhibition of trypsin by BSTI was non-competitive type, and its inhibition constant K_i=0.24 nmol·L~(-1). The results of circular dichroism analysis showed that the secondary structure of BSTI at room temperature was β-sheet 43.4%, random coil 29.0%, β-turn 21.2%, α-helix 8.1% and its denaturation temperature was 61±0.9℃. BSTI was resistant to simulated gastrointestinal fluid digestion. However, after heat treatment at 121℃ for 30 min, the anti-enzymatic action of BSTI was significantly reduced, and was easily hydrolyzed by major digestive enzymes such as pepsin, trypsin and chymotrypsin.
Trypsin inhibitor(TI) has significant research value in food science and biotechnology. In order to investigate the properties of black soybean trypsin inhibitor(BSTI) and the digestive properties of gastrointestinal fluids, BSTI was purified from black soybeans by degreasing, heat treatment, ammonium sulfate salting out and anion column chromatography. SDS-PAGE showed a single band of BSTI with a relative molecular mass of approximately 21 kDa. BSTI was stable below 60℃ and in the range of pH2-11. When the molar ratio of BSTI to trypsin reached 1, the activity of trypsin was suppressed to 15% or less. Inhibition kinetics indicated that the inhibition of trypsin by BSTI was non-competitive type, and its inhibition constant K_i=0.24 nmol·L~(-1). The results of circular dichroism analysis showed that the secondary structure of BSTI at room temperature was β-sheet 43.4%, random coil 29.0%, β-turn 21.2%, α-helix 8.1% and its denaturation temperature was 61±0.9℃. BSTI was resistant to simulated gastrointestinal fluid digestion. However, after heat treatment at 121℃ for 30 min, the anti-enzymatic action of BSTI was significantly reduced, and was easily hydrolyzed by major digestive enzymes such as pepsin, trypsin and chymotrypsin.
引文
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[18] 宋欣欣,段小娟,徐颖,等.豆类种籽中胰蛋白酶抑制剂研究进展[J].粮食与油脂,2016,29(6):1-4.(Song X X,Duan X J,Xu Y,et al.Research progress of trypsin inhibitors of legume seeds[J].Cereals & Oils,2016,29(6):1-4.)
[19] Jiang T L,Cai Q F,Shen J D,et al.Establishment of immunological methods for the detection of soybean proteins in surimi products [J].LWT-Food Science and Technology,2015,64:344-349.
[20] Romero M P,Brandao C,Vivianne F A,et al.CgTI,a novel thermostable Kunitz trypsin-inhibitor purified from Cassia grandis seeds:Purification,characterization and termiticidal activity[J].International Journal of Biological Macromolecules,2018(118):2296-2306.
[21] Jiang X J,Zhang Z J,Cai H N,et al.The effect of soybean trypsin inhibitor on the degradation of myofibrillar proteins by an endogenous serine proteinase of crucian carp[J].Food Chemistry,2006,94:498-503.
[22] 程芬芬,刘春,杨晓泉.大豆胰蛋白酶抑制剂的制备及性质[J].食品科学,2017,38(3):37-44.(Cheng F F,Liu C,Yang X Q.Preparation and properties of soybean trypsin inhibitor[J].Food Science,2017,38(3):37-44.)
[23] Klomklao S,Benjakul S,Kishimura H,et al.A heat-stable trypsin inhibitor in adzuki bean (Vigna angularis):Effect of extraction media,purification and biochemical characteristics[J].International Journal of Food Science and Technology,2010,45:163-169.
[24] David U,Kathrin O,Kristin A,et al.Basic residues of β-sheet a contribute to heparin binding and activation of vaspin(Serpin A12)[J].Journal of Biological Chemistry,2016,292(3):994-1004.
[25] 赵伟伟.黑豆胰蛋白酶抑制剂的分离纯化及活性鉴定[D].太原:山西大学,2016.(Zhao W W.Purification and bioactivities of trypsin inhibitor from black soybean[D].Taiyuan:Shanxi University,2016.)
[26] 罗玉娇,李滨,舒衡平,等.Kunitz型胰蛋白酶抑制剂的研究进展[J].中国生化药物杂志,2012,(3)3:316-318.(Luo Y J,Li B,Shu H P,et al.Research advances in Kunitz trypsin inhibitor[J].Chinese Journal of Biochemical Pharmaceutics,2012,(3)3:316-318.)
[27] Guerra Y,Valiente P A,Pons T,et al.Structures of a bi-functional Kunitz-type STI family inhibitor of serine and aspartic protease:Could the aspartic protease inhibition have evolved from canonical serine protease-binding loop?[J].Journal of Structural Biology,2016,195:259-271.
[28] 袁燕,梅双喜.蒜头果蛋白的红外光谱及圆二色谱分析[J].安徽农业科学,2016,44(33):116-117.(Yuan Y,Mei S X.Infrared spectroscopy and circular dichroism spectrum of Malanin[J].Journal of Anhui Agricultural Sciences,2016,44(33):116-117.)
[29] Peng L,Tzi B N.A stable trypsin inhibitor from Chinese dull black soybeans with potentially exploitable activities[J].Process Biochemistry,2008,43:992-998.
[30] Cui Y,Hu Y H,Yu F,et al.Inhibition kinetics and molecular simulation of p-substituted cinnamic acid derivatives on tyrosinase[J].International Journal of Biological Macromolecules,2016,95:1289-1297.
[31] 杭虞杰,李学英,杨宪时,等.大豆胰蛋白酶抑制剂对南极磷虾类胰蛋白酶的抑制动力学[J].中国水产科学,2012,19(4):635-640.(Hang Y J,Li X Y,Yang X S,et al.Kinetics of inhibition of trypsin-like enzyme from Euphausia superba by STI[J].Journal of Fishery of Sciences of China,2012,19(4):635-640.)
[32] Ludovico M,Adeliana S d O,Elizeu A S,et al.Structural and mechanistic insights into a novel non-competitive Kunitz trypsin inhibitor from Adenanthera pavonina L.seeds with double activity toward serine- and cysteine-proteinases[J].Journal of Molecular Graphics and Modelling,2010,29:148-156.
[33] Guo J X,Peter T E,Alun R C,et al.Structure of Kunitz-type potato cathepsin D inhibitor[J].Journal of Structural Biology,2015,192:554-560.
[34] Bajpai S,Sharma A,Gupta M N.Removal and recovery of antinutritional factors from soybean flour[J].Food Chemistry,2005,89(4):497-501.
[35] Yu H L,Cao M J,Cai Q F,et al.Effects of different processing methods on digestibility of Scylla paramamosain allergen (tropomyosin)[J].Food & Chemical Toxicology,2011,49(4):791-798.
[2] Rebecca C,Alexander V K,Jane F,et al.An unusual helix-turn-helix protease inhibitory motif in a novel trypsin inhibitor from seeds of veronica (Veronica hederifolia L.) [J].Journal of Biological Chemistry,2007,282(38):27760-27768.
[3] Ma T C,Guo L,Zhou R H,et al.Soybean-derived Bowman-Birk inhibitor (BBI) blocks HIV entry into macrophages[J].Virology,2018,513:91-97.
[4] Lanza A,Tava A,Catalano M,et al.Effects of the Medicago scutellata trypsin inhibitor(MsTI) on cisplatin-induced cytotoxicity in human breast and cervical cancer cells[J].Anticancer Research:International Journal of Cancer Research and Treatment,2004,24(1):227-233.
[5] Li J G,Xiang Q S,Liu X F,et al.Inactivation of soybean trypsin inhibitor by dielectric-barrier discharge(DBD) plasma[J].Food Chemistry,2017,232:515-522.
[6] 江连洲,李佳妮,邹晓霜,等.花青素对大豆蛋白体外胃消化结构的影响[J].食品科学,2018,39(20):34-42.(Jiang L Z,Li J N,Zou X S,et al.Effects of anthocyanin on in vitro digestibility of soy protein isolate[J].Food Science,2018,39(20):34-42.)
[7] 俞红恩,康玉凡.豆类胰蛋白酶抑制剂研究进展[J].食品工业,2017,38(4):265-269.(Yu H E,Kang Y F.Research progress in legume trypsin inhibitor[J].Food Industry,2017,38(4):265-269.)
[8] 王荣春,孙建华,何述栋,等.胰蛋白酶抑制剂的结构与功能研究进展[J].食品科学,2013,34(9):364-368.(Wang R C,Sun J H,He S D,et al.Recent advance in research on the structure and function of trypsin inhibitor[J].Food Science,2013,34(9):364-368.)
[9] 俞红恩,康玉凡.豆类胰蛋白酶抑制剂亚基特性研究[J].食品工业科技,2017,38(9):133-138.(Yu H E,kang Y F.Subunit properties of legume trypsin inhibitor[J].Science and Technology of Food Industry,2017,38(9):133-138.)
[10] Astwood J D,Leach J N,Fuchs R L.Stability of food allergens to digestion in vitro[J].Nature Biotechnology,1996,14:1269-1273.
[11] Roychaudhuri R,Sarath G,Zeece M,et al.Stability of the allergenic soybean Kunitz trypsin inhibitor[J].Biochimica et Biophysica Acta:Proteins and Proteomics,2004,1699(1-2):207-212.
[12] 秦琦,张英蕾,张守文.黑豆的营养保健价值及研究进展[J].中国食品添加剂,2015(7):145-150.(Qin Q,Zhang Y L,Zhang S W.The nutritional value and research progress of black soybean[J].China Food Additives,2015(7):145-150.)
[13] 张瑞芬,池建伟,丘银清,等.黑大豆的营养及保健功能研究进展[J].广东农业科学,2006(11):13-16.(Zhang R F,Chi J W,Qiu Y Q,et al.Research progress of the nutrition and health-promoting functions of black soybean[J].Guangdong Agricultural Science,2006(11):13-16.)
[14] Lowry O H,Rosebrough N J,Farr A J.Protein measurement with the folinphenol reagent[J].Journal of Biological Chemistry,1951,193:265-275.
[15] Shen J D,Cai Q F,Liu G M,et al.A comparison study of the impact of boiling and high pressure steaming on the stability of soybean trypsin inhibitor[J].International Journal of Food Science and Technology,2013,48:1877-1883.
[16] United States Pharmacopeial Convention,Committee of Revison.The United States Pharmacopeia USP23:The national formulari NF 18;national formulary supplements 1-9[M].Netherlands:Springer,1987.
[17] 万楚君,游银川,翁凌,等.四种加工方式对皱纹盘鲍制品消化特性的影响[J].水产学报,2017,41(6):928-935.(Wan C J,You Y C,Weng L,et al.Comparison of the digestibility of abalone (Haliotis discus hannai) produced by four different processing treatments[J].Journal of Fisheries of China,2017,41(6):928-935.)
[18] 宋欣欣,段小娟,徐颖,等.豆类种籽中胰蛋白酶抑制剂研究进展[J].粮食与油脂,2016,29(6):1-4.(Song X X,Duan X J,Xu Y,et al.Research progress of trypsin inhibitors of legume seeds[J].Cereals & Oils,2016,29(6):1-4.)
[19] Jiang T L,Cai Q F,Shen J D,et al.Establishment of immunological methods for the detection of soybean proteins in surimi products [J].LWT-Food Science and Technology,2015,64:344-349.
[20] Romero M P,Brandao C,Vivianne F A,et al.CgTI,a novel thermostable Kunitz trypsin-inhibitor purified from Cassia grandis seeds:Purification,characterization and termiticidal activity[J].International Journal of Biological Macromolecules,2018(118):2296-2306.
[21] Jiang X J,Zhang Z J,Cai H N,et al.The effect of soybean trypsin inhibitor on the degradation of myofibrillar proteins by an endogenous serine proteinase of crucian carp[J].Food Chemistry,2006,94:498-503.
[22] 程芬芬,刘春,杨晓泉.大豆胰蛋白酶抑制剂的制备及性质[J].食品科学,2017,38(3):37-44.(Cheng F F,Liu C,Yang X Q.Preparation and properties of soybean trypsin inhibitor[J].Food Science,2017,38(3):37-44.)
[23] Klomklao S,Benjakul S,Kishimura H,et al.A heat-stable trypsin inhibitor in adzuki bean (Vigna angularis):Effect of extraction media,purification and biochemical characteristics[J].International Journal of Food Science and Technology,2010,45:163-169.
[24] David U,Kathrin O,Kristin A,et al.Basic residues of β-sheet a contribute to heparin binding and activation of vaspin(Serpin A12)[J].Journal of Biological Chemistry,2016,292(3):994-1004.
[25] 赵伟伟.黑豆胰蛋白酶抑制剂的分离纯化及活性鉴定[D].太原:山西大学,2016.(Zhao W W.Purification and bioactivities of trypsin inhibitor from black soybean[D].Taiyuan:Shanxi University,2016.)
[26] 罗玉娇,李滨,舒衡平,等.Kunitz型胰蛋白酶抑制剂的研究进展[J].中国生化药物杂志,2012,(3)3:316-318.(Luo Y J,Li B,Shu H P,et al.Research advances in Kunitz trypsin inhibitor[J].Chinese Journal of Biochemical Pharmaceutics,2012,(3)3:316-318.)
[27] Guerra Y,Valiente P A,Pons T,et al.Structures of a bi-functional Kunitz-type STI family inhibitor of serine and aspartic protease:Could the aspartic protease inhibition have evolved from canonical serine protease-binding loop?[J].Journal of Structural Biology,2016,195:259-271.
[28] 袁燕,梅双喜.蒜头果蛋白的红外光谱及圆二色谱分析[J].安徽农业科学,2016,44(33):116-117.(Yuan Y,Mei S X.Infrared spectroscopy and circular dichroism spectrum of Malanin[J].Journal of Anhui Agricultural Sciences,2016,44(33):116-117.)
[29] Peng L,Tzi B N.A stable trypsin inhibitor from Chinese dull black soybeans with potentially exploitable activities[J].Process Biochemistry,2008,43:992-998.
[30] Cui Y,Hu Y H,Yu F,et al.Inhibition kinetics and molecular simulation of p-substituted cinnamic acid derivatives on tyrosinase[J].International Journal of Biological Macromolecules,2016,95:1289-1297.
[31] 杭虞杰,李学英,杨宪时,等.大豆胰蛋白酶抑制剂对南极磷虾类胰蛋白酶的抑制动力学[J].中国水产科学,2012,19(4):635-640.(Hang Y J,Li X Y,Yang X S,et al.Kinetics of inhibition of trypsin-like enzyme from Euphausia superba by STI[J].Journal of Fishery of Sciences of China,2012,19(4):635-640.)
[32] Ludovico M,Adeliana S d O,Elizeu A S,et al.Structural and mechanistic insights into a novel non-competitive Kunitz trypsin inhibitor from Adenanthera pavonina L.seeds with double activity toward serine- and cysteine-proteinases[J].Journal of Molecular Graphics and Modelling,2010,29:148-156.
[33] Guo J X,Peter T E,Alun R C,et al.Structure of Kunitz-type potato cathepsin D inhibitor[J].Journal of Structural Biology,2015,192:554-560.
[34] Bajpai S,Sharma A,Gupta M N.Removal and recovery of antinutritional factors from soybean flour[J].Food Chemistry,2005,89(4):497-501.
[35] Yu H L,Cao M J,Cai Q F,et al.Effects of different processing methods on digestibility of Scylla paramamosain allergen (tropomyosin)[J].Food & Chemical Toxicology,2011,49(4):791-798.
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