超声波联合酶解处理对β-伴大豆球蛋白抗原性的影响
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摘要
试验以大豆分离蛋白为原料,选择超声波和酶解联合处理大豆蛋白,采用间接竞争ELISA法和免疫印迹法测定大豆主要抗原蛋白β-伴大豆球蛋白的抗原性和免疫活性,并分析超声波和酶解处理后大豆蛋白的结构变化。结果表明,超声波联合酶解处理能够显著降低β-伴大豆球蛋白的抗原性和免疫活性, 400 W、15 min超声波预处理的大豆蛋白质再经酶解处理后,β-伴大豆球蛋白的抗原抑制率降低了61.15%;电泳结果显示,超声波联合酶解处理能够使蛋白质肽键断裂,分解为小分子肽段;表面疏水性结果表明,蛋白质的空间结构发生解聚和重新聚合,三四级结构被破坏,构象型表位可能被改变。β-伴大豆球蛋白的抗原性的变化主要是因为超声波与酶解联合引起大豆蛋白氨基酸序列及空间结构的变化。
In this experiment, soy protein isolate was used as raw material, ultrasonic and enzymatic hydrolysis were combined to treat soy protein, and the antigenicity and immunological activity of soybean main antigen protein β-conglycinin were determined by indirect competitive ELISA and immunoblotting. Structural changes in soybean after ultrasonic and enzymatic treatment were detected. The results showed that the combination of ultrasonic and enzymatic treatment could significantly reduce the antigenicity and immunological activity of β-conglycinin. The antigenic inhibition rate of-conglycinin decreased by 61.15% after 400 W and 15 min ultrasonic pretreatment of soybean protein by enzymatic treatment. SDS-PAGE results showed that ultrasonic combined with enzymatic treatment could break the protein peptide bond and change the linear epitope. Surface hydrophobicity results showed that the spatial structure of the protein was depolymerized and repolymerized, the third and fourth structures were destroyed, and conformational epitope was changed. The antigenic change of β-conglycinin is mainly due to the change of amino acid sequence and spatial structure of soybean protein caused by ultrasonic combined enzymatic hydrolysis.
In this experiment, soy protein isolate was used as raw material, ultrasonic and enzymatic hydrolysis were combined to treat soy protein, and the antigenicity and immunological activity of soybean main antigen protein β-conglycinin were determined by indirect competitive ELISA and immunoblotting. Structural changes in soybean after ultrasonic and enzymatic treatment were detected. The results showed that the combination of ultrasonic and enzymatic treatment could significantly reduce the antigenicity and immunological activity of β-conglycinin. The antigenic inhibition rate of-conglycinin decreased by 61.15% after 400 W and 15 min ultrasonic pretreatment of soybean protein by enzymatic treatment. SDS-PAGE results showed that ultrasonic combined with enzymatic treatment could break the protein peptide bond and change the linear epitope. Surface hydrophobicity results showed that the spatial structure of the protein was depolymerized and repolymerized, the third and fourth structures were destroyed, and conformational epitope was changed. The antigenic change of β-conglycinin is mainly due to the change of amino acid sequence and spatial structure of soybean protein caused by ultrasonic combined enzymatic hydrolysis.
引文
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[22] O’SULLIVAN J, PARK M, BEEVERS J. The effect of ultrasound upon the physicochemical and emulsifying properties of wheat and soy protein isolates[J]. Journal of Cereal Science,2016(69):77-84.
[23] FRACZEK R J, KOSTYRA E, KOSTYRA H. Immunogenic potential of antigens isolated from trypsin pea protein hydrolysates[J]. Polish Journal of Food&Nutrition Sciences,2008, 58(4):491-496.
[2]刘宾.大豆主要致敏原的免疫检测研究[D].北京:中国农业科学院, 2011.
[3]杨慧,陈红兵,程伟,等.大豆主要过敏原及其脱敏方法的研究进展[J].食品科学, 2011, 32(21):273-277.
[4] LI H, ZHU K, ZHOU H, et al. Effects of high hydrostatic pressure treatment on allergenicity and structural properties of soybean protein isolate for infant formula[J]. Food Chemistry,2012, 132(2):808-814.
[5]王锦欣,秦贵信,龙国徽,等.β-伴大豆球蛋白酶解物的抗消化性及免疫活性变化的研究[J].中国畜牧杂志, 2014,50(9):45-49.
[6] GOVINDARAJU K, SRINIVAS H. Controlled enzymatic hydrolysis of glycinin:Susceptibility of acidic and basic subunits to proteolytic enzymes[J]. LWT-Food Science and Technology, 2007, 40(6):1056-1065.
[7] ZHOU M, LIU J, ZHOU Y, et al. Effect of high intensity ultrasound on physicochemical and functional properties of soybean glycinin at different ionic strengths[J]. Innovative Food Science&Emerging Technologies, 2016(34):205-213.
[8] HU H, LI-CHAN E C Y, LI W, et al. The effect of high intensity ultrasonic pre-treatment on the properties of soybean protein isolate gel induced by calcium sulfate[J]. Food Hydrocolloids, 2013, 32(2):303-311.
[9]孙英杰.超声波处理对大豆分离蛋白结构和功能性质影响研究[D].哈尔滨:东北农业大学, 2014.
[10] HUANG L, DING X, DAI C, et al. Changes in the structure and dissociation of soybean protein isolate induced by ultrasound-assisted acid pretreatment[J]. Food Chemistry,2017, 232:727-732.
[11] CHEN L, CHEN J, REN J, et al. Effects of ultrasound pretreatment on the enzymatic hydrolysis of soy protein isolates and on the emulsifying properties of hydrolysates[J].Journal of Agricultural&Food Chemistry, 2011, 59(6):2600-2609.
[12] YANG X, LI Y, LI S, et al. Effects of multi-frequency ultrasound pretreatment under low power density on the enzymolysis and the structure characterization of defatted wheat germ protein[J]. Ultrasonics Sonochemistry, 2017(38):410-420.
[13]邓涵,祖琴琴,朱杰瑞,等.超声处理对大豆7S蛋白潜在致敏性的影响[J].食品科学, 2017, 38(5):32-37.
[14]郭兴凤.蛋白质水解度的测定[J].中国油脂, 2000, 25(6):176-177.
[15] BU G, ZHANG N, CHEN F. The influence of glycosylation on the antigenicity, allergenicity, and structural properties of11S-lactose conjugates[J]. Food Research International, 2015,76(3):511-517.
[16]黄婷,布冠好,陈复生.酶解改性对大豆蛋白抗原性的影响研究[J].河南工业大学学报(自然科学版), 2016, 37(4):11-17.
[17] DHAKAL S, LIU C, ZHANG Y, et al. Effect of high pressure processing on the immunoreactivity of almond milk[J]. Food Research International, 2014, 62(1):215-222.
[18] LAEMMLI U K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4[J]. Nature, 1970,227(5259):680-685.
[19]余晶梅,林东强,童红飞,等.荧光探针法研究牛血清白蛋白和免疫球蛋白的表面疏水特性[J].高校化学工程学报, 2014(4):771-776.
[20] ZHANG Y H, WANG Q A. Peanut protein hydrolyzing by Alcalase to prepare peanut oligopeptides[J]. Transactions of the Chinese Society of Agricultural Engineering, 2007, 23(4):258-263.
[21]李素云.超声和超声协同碱预处理对大米蛋白酶解效果影响及过程模拟研究[D].镇江:江苏大学, 2017.
[22] O’SULLIVAN J, PARK M, BEEVERS J. The effect of ultrasound upon the physicochemical and emulsifying properties of wheat and soy protein isolates[J]. Journal of Cereal Science,2016(69):77-84.
[23] FRACZEK R J, KOSTYRA E, KOSTYRA H. Immunogenic potential of antigens isolated from trypsin pea protein hydrolysates[J]. Polish Journal of Food&Nutrition Sciences,2008, 58(4):491-496.