壳寡糖酶法糖基化大豆蛋白提高乳液冻融稳定性及机制研究_张智慧.pdf

第 14 卷 第 2 期 食 品 安 全 质 量 检 测 学 报 Vol.14 No.2 2023 年 1 月 Journal of Food Safety and Quality Jan.,2023 基金项目:东北农业大学“青年才俊”项目(19QC28)、黑龙江省普通本科高等学校青年创新人才培养计划项目(UNPYSCT-2020106)Fund:Supported by the“Young Talents”Project in Northeast Agricultural University(19QC28),and the Youth Innovation Talent Training Plan in Undergraduate College of Heilongjiang Province(UNPYSCT-2020106)#张智慧、马 楠为共同第一作者#ZHANG Zhi-Hui and MA Nan are Co-first Authors*通信作者:王喜波,博士,教授,主要研究方向为粮食、油脂及植物蛋白工程。E-mail: 张晓南,博士,副教授,主要研究方向为食品科学。E-mail:*Corresponding author:WANG Xi-Bo,Ph.D,Professor,College of Food Science,Northeast Agricultural University,No.600,Changjiang Street,Xiangfang District,Harbin 150030,China.E-mail: ZHANG Xiao-Nan,Ph.D,Associate Professor,College of Life Science,Jiaying University,No.100,Meisong Road,Miejiang District,Meizhou 514015,China.E-mail: 壳寡糖酶法糖基化大豆蛋白提高乳液 冻融稳定性及机制研究 张智慧1#,马 楠1#,谷眉宇1,王 旭1,刘媛媛2,王喜波1*,张晓南3*(1.东北农业大学食品学院,哈尔滨 150030;2.夏庄畜牧兽医站,荣成 264300;3.嘉应学院生命科学学院,梅州 514015)摘摘 要要:目的目的 通过谷氨酰胺转氨(transglutaminase glycosylation,TG)酶催化技术制备壳寡糖(chitosan oligosaccharide,COS)糖基化大豆分离蛋白(soy protein isolate,SPI),并研究改性产物结构变化及其功能性改进机制。方法方法 以 COS 与 SPI 为原料,采用 TG 酶催化技术制备大豆分离蛋白-壳寡糖共价改性产物(SPI-COS)。采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate-polyacrylamide gel electrophoresis,SDS-PAGE)、傅里叶变换红外光谱(Fourier transform-infrared spectroscopy,FT-IR)、荧光光谱研究 SPI-COS 的结构特征变化,采用乳析指数(creaming index,CI)、出油率、絮凝程度(flocculation degree,FD)、聚结程度(coalescence degree,CD)、电子显微镜观察等方法表征不同 SPI 产物乳液的冻融稳定性。结果结果 蛋白乳液在经历 1、2、3 次冻融循环处理后,与对照 SPI 相比,SPI-COS 乳析指数分别降低了 58.66%、25.51%和 13.63%,出油率分别降低了 49%、69%和 48%,冻融稳定性显著提高。SDS-PAGE 和 FT-IR 分析表明,SPI 与 COS 形成共价键,SPI-COS 二级结构中-螺旋和-转角的含量显著减少(P0.05),而-折叠和无规则卷曲的含量显著增加(P0.05)。微观状态分析表明,与对照 SPI 冻融乳液相比,SPI-COS 乳液不规则,聚集体更小,状态更稳定。结论结论 酶法糖基化蛋白可以通过改变大豆蛋白理化结构特性,进而提高其乳液的冻融稳定性,本研究为制备高冻融稳定性 SPI 提供理论指导及技术支持,可以扩大大豆蛋白乳液在冷冻食品中的应用。关键词关键词:大豆分离蛋白;壳寡糖;谷氨酰胺转氨酶;冻融稳定性 Investigation on freeze-thaw stability and mechanism of glycosylated soy protein isolate-chitosan oligosaccharide emulsion by transglutaminase ZHANG Zhi-Hui1#,MA Nan1#,GU Mei-Yu1,WANG Xu1,LIU Yuan-Yuan2,WANG Xi-Bo1*,ZHANG Xiao-Nan3*(1.College of Food Science,Northeast Agricultural University,Harbin 150030,China;2.Xiazhuang Animal Husbandry&Veterinary Medicine,Rongcheng 264300,China;3.Department of Biology,Jiaying University,Meizhou 514015,China)DOI:10.19812/ki.jfsq11-5956/ts.2023.02.021第 2 期 张智慧,等:壳寡糖酶法糖基化大豆蛋白提高乳液冻融稳定性及机制研究 131 ABSTRACT:Objective To prepare soy protein isolate(SPI)and chitosan oligosaccharide(COS)by transglutaminase glycosylation(TG)enzyme catalytic technology,and study the structural changes and functional improvement mechanism of the SPI modified product.Methods In this experiment,the covalent modification of soy protein isolate-chitosan oligosaccharide(SPI-COS)was prepared by TG enzyme catalytic technology using COS and SPI as raw materials.The structural change of the modified SPI-COS was evaluated by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE),Fourier transform-infrared spectroscopy(FT-IR),and fluorescence spectra.Changes in the creaming index(CI),oiling off,flocculation degree(FD),coalescence degree(CD),and electron microscopic observation were used to characterize the freeze-thaw stability of different SPI product emulsions.Results Compared with the control SPI,after the protein emulsions were treated with 1,2,and 3 freeze-thaw cycles,the SPI-COS emulsion creaming index was reduced by 58.66%,25.51%,and 13.63%;the oiling off was reduced by 49%,69%,and 48%,and the freeze-thaw stability was significantly improved.SDS-PAGE and FT-IR spectroscopic analysis showed that SPI formed covalent bonds with COS,and the content of-helix and-turn in the SPI-COS secondary structure was significantly decreased(P0.05),while the content of-folding and random coil was significantly increased(P0.05).The microstate analysis showed that the SPI-COS emulsion was changed to irregular morphology,and the aggregates were made smaller and more stable than the control SPI freeze-thaw emulsion.Conclusion Enzymatic glycosylation of proteins improves the freeze-thaw stability of soy protein emulsions by modifying their physicochemical structural properties.This study provides theoretical guidance and technical support for the preparation of highly freeze-thaw stable SPI.It expands the application of soy protein emulsions in frozen foods.KEY WORDS:soy protein isolate;chitosan oligosaccharide;transglutaminase glycosylation;freeze-thaw stability 0 引 言 大豆分离蛋白(soy protein isolate,SPI)在油水界面处具有高效乳化性,常被用作乳化剂1。其乳液的冻融性质也被广泛关注。冻融循环过程中,由于水相在冷冻时会形成冰晶导致体积膨胀,挤压乳液的界面膜,产生一定的机械作用,对食物的质地、外观、风味等都会造成不可逆的损害,乳液发生聚集2。融化时乳剂会出现分层、絮凝、聚结、奥氏熟化、相分离等不稳定现象,使乳剂稳定性下降3,从而影响其在食品乳化体系中的应用。为了进一步改善蛋白质乳液的冻融稳定性,可以使用物理、化学和酶促方法对 SPI 进行修饰,从而改善其功能特性4。其中,糖基化修饰作为一种反应简单、作用明显的化学改性方法备受关注。糖基化改性方法主要有两种:美拉德反应和谷氨酰胺转氨(transglutaminase glycosylation,TG)酶途径5,但过度美拉德反应会导致营养物质的损失,并产生有毒中间产物(如丙烯酰胺)6。近年来,TG 酶途径由于其反应控制较简单、条件温和,生成物性质稳定,在蛋白质改性修饰中的应用非常广泛7。TG 酶可以催化谷氨酰胺残基和赖氨酸残基在蛋白质中的交联聚合,也可以催化酰基转移及脱酰胺反应8。使用 TG 酶改性修饰的 SPI 在乳液中能够更加均匀地包裹在油滴表面,有效防止液滴聚集