电子束辐照对带鱼鱼糜内源性蛋白酶活性及其构象单元的影响
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摘要
鱼类内源性蛋白酶是引起鱼糜凝胶劣化的重要因素之一。以不同剂量电子束辐照带鱼鱼糜,测定鱼糜内源性肌原纤维结合型丝氨酸蛋白酶(myofibril-bound serine proteinase,MBSP)和组织蛋白酶L(cathepsin L,Cat-L)活力及其最适反应温度,结合圆二色光谱分析其构象单元变化,探讨电子束辐照对鱼糜内源性MBSP和Cat-L的影响。结果表明:随着辐照剂量的增加,鱼糜MBSP和Cat-L活力降低,当辐照剂量为9 kGy时Cat-L活力下降更为明显;对照组和辐照组鱼糜MBSP的最适温度均为55℃,但3 kGy及9 kGy辐照对Cat-L的最适温度产生影响,使其由55℃降低至45℃;辐照引起鱼糜MBSP和Cat-L分子中的二级结构单元互相转化,导致其构象变化,削弱其降解肌原纤维蛋白的能力。结论:电子束辐照能够影响带鱼鱼糜MBSP和Cat-L的二级结构及活力,减轻二者对肌原纤维蛋白的降解作用,适宜剂量的电子束辐照能有效抑制鱼糜内源性蛋白酶活性,防止凝胶劣化,有利于鱼糜凝胶的形成。
Endogenous proteases in fish are one of the important causes of surimi gel degradation. In this study, to investigate the effect of electron beam(EB) irradiation on endogenous myofibril-bound serine proteinase(MBSP) and cathepsin L(Cat-L) in hairtail surimi, the activity and optimum temperature of both enzymes after being treated with different doses of EB irradiation were determined, and the secondary structure changes were studied by circular dichroism(CD) spectroscopy. The results indicated that the activity of endogenous MBSP and Cat-L decreased with the increase of irradiation dose; especially Cat-L activity decreased significantly at 9 kGy. The optimum temperature of both control and irradiated MBSP was 55 ℃, whereas the optimum temperature of Cat-L decreased from 55 ℃ to 45 ℃ after irradiation at 3 or 9 kGy. EB irradiation resulted in the transformation of secondary structure units in MBSP and Cat-L, thus leading to conformational change and weakening their ability to degrade myofibrillar protein. In conclusion, EB irradiation can affect the secondary structure and activity of endogenous MBSP and Cat-L from hairtail surimi. The appropriate dose of EB irradiation can effectively inhibit the activity of endogenous proteases in surimi, thereby weakening the degradation of myofibrillar protein and consequently facilitating the formation of surimi gel.
Endogenous proteases in fish are one of the important causes of surimi gel degradation. In this study, to investigate the effect of electron beam(EB) irradiation on endogenous myofibril-bound serine proteinase(MBSP) and cathepsin L(Cat-L) in hairtail surimi, the activity and optimum temperature of both enzymes after being treated with different doses of EB irradiation were determined, and the secondary structure changes were studied by circular dichroism(CD) spectroscopy. The results indicated that the activity of endogenous MBSP and Cat-L decreased with the increase of irradiation dose; especially Cat-L activity decreased significantly at 9 kGy. The optimum temperature of both control and irradiated MBSP was 55 ℃, whereas the optimum temperature of Cat-L decreased from 55 ℃ to 45 ℃ after irradiation at 3 or 9 kGy. EB irradiation resulted in the transformation of secondary structure units in MBSP and Cat-L, thus leading to conformational change and weakening their ability to degrade myofibrillar protein. In conclusion, EB irradiation can affect the secondary structure and activity of endogenous MBSP and Cat-L from hairtail surimi. The appropriate dose of EB irradiation can effectively inhibit the activity of endogenous proteases in surimi, thereby weakening the degradation of myofibrillar protein and consequently facilitating the formation of surimi gel.
引文
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[22]马艳萍,马惠玲,刘兴华,等.60Co-γ射线辐照对鲜食核桃采后膜脂过氧化作用的影响[J].农业机械学报,2011,42(12):171-176.DOI:10.3969/j.issn.1000-1298.
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[24]胡亚芹,姚燕佳,陈建初.南蓝鳕鱼糜中L型组织蛋白酶的研究[J].水产学报,2010,34(3):336-341.DOI:10.3724/SP.J.1231.2010.06619.
[25]ZHU B W,ZHAO L L,SUN L M,et al.Purification and characterization of a cathepsin L-like enzyme from the body wall of the sea cucumber Stichopus japonicus[J].Bioscience,Biotechnology and Biochemistry,2008,72(6):1430-1437.DOI:10.1271/bbb.70741.
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[27]MALIK M A,SHARMA H K,SAINI C S.Effect of gamma irradiation on structural,molecular,thermal and rheological properties of sunflower protein isolate[J].Food Hydrocolloids,2017,72(6):312-322.DOI:10.1016/j.foodhyd.2017.06.011.
[28]HERRERO A M,CARMONA P,ORDó?EZ J A,et al.Raman spectroscopic study of electron-beam irradiated cold-smoked salmon[J].Food Research International,2009,42(1):216-220.DOI:10.1016/j.foodres.2008.10.010.
[29]MOON S,SONG K B.Effect of γ-irradiation on the molecular properties of ovalbumin and ovomucoid and protection by ascorbic acid[J].Food Chemistry,2001,74(4):479-483.DOI:10.1016/S0308-8146(01)00166-2.
[30]GUAN Aiyan,MEI Kalin,LüMingchun,et al.The effect of electron beam irradiation on IgG binding capacity and conformation of tropomyosin in shrimp[J].Food Chemistry,2018,30:250-254.DOI:10.1016/j.foodchem.2018.05.051.
[2]纪蓉,顾伟钢,张进杰,等.鱼糜凝胶劣化现象相关蛋白水解酶[J].生物技术通讯,2011,22(1):143-148.DOI:10.3969/j.issn.1009-0002.2011.01.033.
[3]纪蓉.组织蛋白酶L对鱼糜凝胶劣化机理的研究[D].杭州:浙江大学,2012:34-35.
[4]JIANG S T,LEE B L,TSAO C Y,et al.Mackerel cathepsins B and Leffects on thermal degradation of surimi[J].Journal of Food Science,1997,62(2):310-315.DOI:10.1111/j.1365-2621.1997.tb03991.x.
[5]揭珍,徐大伦,胡小超,等.大豆胰蛋白酶抑制剂对带鱼鱼糜蛋白降解及其凝胶特性的影响[J].核农学报,2016,30(5):912-919.DOI:10.11869/j.issn.100-8551.2016.05.0912.
[6]SOMMERS C H,NIEMIRA B A.Inactivation of a virulent Yersinia pestis in beef bologna by gamma irradiation[J].Journal of Food Protection,2011,74(4):627-630.DOI:10.4315/0362-028X.JFP-10-421.
[7]YANG Z,WANG H Y,WANG W,et al.Effect of 10 MeV E-beam irradiation combined with vacuum-packaging on the shelf life of Atlantic salmon fillets during storage at 4℃[J].Food Chemistry,2014,145(2):535-541.DOI:10.1016/j.foodchem.2013.08.095.
[8]LIN Xianping,YANG Wenge,XU Dalun,et al.Improving gel properties of hairtail surimi by electron irradiation[J].Radiation Physics and Chemistry,2015,110(1):1-5.DOI:10.1016/j.radphyschem.2014.12.017.
[9]邓思瑶,杨文鸽,徐大伦,等.电子束辐照对梅鱼鱼糜肌原纤维蛋白结构及凝胶特性的影响[J].现代食品科技,2017,33(1):139-144;172.DOI:10.7506/spkx1002-6630-201618037.
[10]JACZYNSKI J,PARK J W.Physicochemical changes in Alaska Pollock surimi and surimi gel as affected by electron beam[J].Journal of Food Science,2004,69(1):53-57.DOI:10.1111/j.1365-2621.2004.tb17855.x.
[11]LIN Xianping,YANG Wenge,XU Dalun,et al.Effect of electron irradiation and heat on the structure of hairtail surimi[J].Radiation Physics and Chemistry,2015,114(5):50-54.DOI:10.1016/j.radphyschem.2015.05.028.
[12]DENG Siyao,LüLiangyu,YANG Wenge,et al.Effect of electron irradiation on the gel properties of Collichthys lucidus surimi[J].Radiation Physics and Chemistry,2017,130(1):316-320.DOI:10.1016/j.radphyschem.2016.08.022.
[13]CAO Minjie,OSATOMI K,HARA K,et al.Identification of a myofibril-bound serine proteinase(MBSP)in the skeletal muscle of lizard fish Saurida wanieso which specifically cleaves the arginine site[J].Comparative Biochemistry and Physiology. Part B: Biochemistry and Molecular Biology,2000,125(2):255-264.DOI:10.1016/S0305-0491(99)00176-5.
[14]HU Yaqin,MORIOKA K,ITOH Y.Distribution of cathepsins B,H,L,and trypsin-like proteases in natural actomyosin from washed meat of various fishes[J].Fisheries Science,2008,74(3):693-695.DOI:10.1111/j.1444-2906.2008.01577.x.
[15]XIONG Guangquan,CHENG Wei,YE Lixiu,et al.Effects of konjac glucomannan on physicochemical properties of myofibrillar protein and surimi gels from grass carp(Ctenopharyngodon idella)[J].Food Chemistry,2009,116(2):413-418.DOI:10.1016/j.foodchem.2009.02.056.
[16]LAEMMLI U K.Cleavage of structural proteins during the assembly of the head of bacteriophage T4[J].Nature,1970,227:680-685.DOI:10.1038/227680a0.
[17]CAO Minjie,OSATOMI K,PANGKEY H,et al.Cleavage specificity of a myofibril-bound serine proteinase from carp(Cyprinus carpio)muscle[J].Comparative Biochemistry and Physiology. Part B: Biochemistry and Molecular Biology,1999,123(4):399-405.DOI:10.1016/S0305-0491(99)00086-3.
[18]BARRETT A J.Fluorimetric assays for cathepsin B and cathepsin Hwith methylcoumarylamide substrates[J].Biochemical Journal,1980,187(3):909-912.DOI:10.1042/bj1870909.
[19]BARRETT A J,KIRSCHKE H.Cathepsin B,cathepsin H and cathepsin L[J].Methods in Enzymology,1981,80(5):535-561.DOI:10.1016/S0076-6879(81)80043-2.
[20]LEE Y,SONG K B.Effect of gamma-irradiation on the molecular properties of myoglobin[J].Journal of Biochemistry&Molecular Biology,2002,35(6):590-594.DOI:10.5483/BMBRep.2002.35.6.590.
[21]顾可飞.电子束辐照对多酚氧化酶活性及结构的影响[J].辐射研究与辐射工艺学报,2017,35(3):48-53.DOI:10.11889/j.1000-3436.2017.rrj.35.030401.
[22]马艳萍,马惠玲,刘兴华,等.60Co-γ射线辐照对鲜食核桃采后膜脂过氧化作用的影响[J].农业机械学报,2011,42(12):171-176.DOI:10.3969/j.issn.1000-1298.
[23]鲍晓瑾.引起鳙鱼鱼糜凝胶劣化的丝氨酸蛋白酶及其抑制研究[D].杭州:浙江工业大学,2007:2.
[24]胡亚芹,姚燕佳,陈建初.南蓝鳕鱼糜中L型组织蛋白酶的研究[J].水产学报,2010,34(3):336-341.DOI:10.3724/SP.J.1231.2010.06619.
[25]ZHU B W,ZHAO L L,SUN L M,et al.Purification and characterization of a cathepsin L-like enzyme from the body wall of the sea cucumber Stichopus japonicus[J].Bioscience,Biotechnology and Biochemistry,2008,72(6):1430-1437.DOI:10.1271/bbb.70741.
[26]吴烨.兔肌球蛋白热诱导凝胶化学作用力与凝胶结构功能关系的研究[D].南京:南京农业大学,2010:39-42.
[27]MALIK M A,SHARMA H K,SAINI C S.Effect of gamma irradiation on structural,molecular,thermal and rheological properties of sunflower protein isolate[J].Food Hydrocolloids,2017,72(6):312-322.DOI:10.1016/j.foodhyd.2017.06.011.
[28]HERRERO A M,CARMONA P,ORDó?EZ J A,et al.Raman spectroscopic study of electron-beam irradiated cold-smoked salmon[J].Food Research International,2009,42(1):216-220.DOI:10.1016/j.foodres.2008.10.010.
[29]MOON S,SONG K B.Effect of γ-irradiation on the molecular properties of ovalbumin and ovomucoid and protection by ascorbic acid[J].Food Chemistry,2001,74(4):479-483.DOI:10.1016/S0308-8146(01)00166-2.
[30]GUAN Aiyan,MEI Kalin,LüMingchun,et al.The effect of electron beam irradiation on IgG binding capacity and conformation of tropomyosin in shrimp[J].Food Chemistry,2018,30:250-254.DOI:10.1016/j.foodchem.2018.05.051.