【作者】 邓克权；

【导师】 华欲飞；

【作者基本信息】 江南大学 ， 粮食、油脂及植物蛋白工程， 2013， 博士

【摘要】 大豆蛋白主要由大豆球蛋白和p-伴球蛋白,即11S和7S两个主要组分构成,分别占蛋白总含量的40%与30%左右。大豆蛋白的功能性质也主要是由大豆球蛋白与β-伴球蛋白决定,分级分离11S和7S组分有助于我们更好地理解其各组分在功能性质中所发挥的作用,评价其对营养及结构的影响,为新产品的研发奠定理论基础。有关大豆球蛋白和β-伴球蛋白分离纯化方面的工作已有较多的文献报道,但大多直接以低温脱脂豆粕为原料浸提提取。迄今为止,鲜有以低残余脂质豆粕(LRLSF)为原料分级分离大豆蛋白的报道。本论文重点探讨大豆球蛋白和β-伴球蛋白的制备及氧化对大豆蛋白结构及功能性质的影响。单因素试验以低残余脂质豆粕为原料,探讨了大豆β-伴球蛋白(7S)分级分离过程中提取条件如还原剂、冷沉时间和pH对蛋白得率及纯度的影响。结果表明,与亚硫酸氢钠(SBS)或二硫苏糖醇(DTT)相比,在浸提液中添加p-巯基乙醇(ME),大豆p-伴球蛋白(7S)的得率与纯度均最高。在蛋白提取液的冷沉阶段,随着冷沉时间的延长,后续分离所得7S组分的得率持续降低,而纯度逐渐提高,当冷沉时间超过12h以后,7S纯度增速放缓,此时在P>0.05水平上,巯基与二硫键总量并无明显差异。在7S组分酸沉阶段,采用pH4.70酸沉时7S组分的得率与纯度均最佳。在单因素试验基础上,通过响应面分析确定了7S组分提取的最佳工艺条件为：巯基乙醇浓度9.5mmol/L,冷沉时间12h,酸沉pH4.86。与传统蛋白制取相比,新工艺制取7S蛋白的得率增加了23.86%,纯度提高了8.64%。在分级分离7S组分的同时,以低残余脂质豆粕为原料,探讨了大豆球蛋白(11S)分级分离过程中pH、还原剂和冷沉时间等提取条件对蛋白得率及纯度的影响。试验表明,将浸提液pH由6.2调至6.6时,大豆球蛋白的得率降低了16.7%,而纯度提高了4.60%。随着浸提pH的提高,蛋白质的巯基与二硫键含量增加。与二硫苏糖醇(DTT)或β-巯基乙醇(ME)相比,在pH6.4的浸提液中添加亚硫酸氢钠(SBS),大豆球蛋白的得率最高。上述三种还原剂中,使用DTT时的11S组分得率与纯度最低。在蛋白沉淀阶段,当冷沉时间为10h时,蛋白的得率与纯度最佳,此时巯基与二硫键总量并无显著差异(P>0.05)。随着浸提时间的延长,部分巯基发生氧化形成二硫键,大豆球蛋白游离巯基含量降低。在单因素试验基础上,通过响应面分析确定了11S组分提取的最佳工艺条件为：SBS浓度8.89mmol/L,冷沉时间10h,pH6.32。与传统蛋白制取相比,新工艺制取11S蛋白的得率增加了28.48%,纯度提高了5.58%。在优化制备大豆p-伴球蛋白(7S)与大豆球蛋白(11S)工艺条件的基础上,分别以低氧化程度脱脂豆粕即低残余脂质豆粕、低温脱脂豆粕及高氧化程度脱脂豆粕为原料,研究大豆蛋白分级分离过程中脂质氧化与蛋白的相互作用对大豆p-伴球蛋白和大豆球蛋白结构特征、氧化程度以及聚集形态的影响。结果表明：(1)氧化使得β-大豆伴球蛋白和大豆球蛋白羰基值上升,游离巯基、总巯基含量和α-螺旋含量下降。在同等氧化条件下,p-大豆伴球蛋白更易于发生氧化。(2)氧化使得p-大豆伴球蛋白和大豆球蛋白表面疏水性下降,内源荧光强度下降并且λmax发生蓝移,说明蛋白质氧化使得p-大豆伴球蛋白和大豆球蛋白结构发生了变化。在同等氧化条件下,p-大豆伴球蛋白更易于发生氧化,β-大豆伴球蛋白易于发生氧化主要原因可能在于p-大豆伴球蛋白含有较多的脂肪族氨基酸残基侧链基团。(3)氧化使得p-大豆伴球蛋白和大豆球蛋白形成可溶性氧化聚集体,并且在同等氧化应激条件下,氧化β-大豆伴球蛋白聚集程度更高。此外,氧化还使得p-大豆伴球蛋白肽链断裂,使得大豆球蛋白分子解离。在结构表征的基础上,进一步研究了蛋白质氧化对大豆p-伴球蛋白和大豆球蛋白溶解性、持水性、吸油性、乳化性、起泡性和凝胶性等功能性质的影响,结果表明：(1)氧化使得p-大豆伴球蛋白和大豆球蛋白溶解性下降,在相同氧化条件下,p·大豆伴球蛋白溶解度下降幅度较大。(2)p-大豆伴球蛋白和大豆球蛋白持水性和吸油性随着蛋白质氧化程度的增大而减小,p-大豆伴球蛋白持水性和吸油性下降幅度较大。(3)氧化使得β-大豆伴球蛋白和大豆球蛋白乳化性、乳化稳定性、起泡性、泡沫稳定性、凝胶硬度以及凝胶强度下降,相比大豆球蛋白,p-大豆伴球蛋白功能特性下降幅度较大。(4)在凝胶形成过程中,蛋白质氧化使得p-大豆伴球蛋白和大豆球蛋白在升温阶段具有较高的初始G.,而在保温和降温阶段,氧化p-大豆伴球蛋白和大豆球蛋白的G’值随着蛋白质氧化程度的增加而减小。β-大豆伴球蛋白和大豆球蛋白凝胶频率扫描图中G’和G"的间距随着蛋白质氧化程度的增大而减小,表明蛋白质氧化使得大豆β-伴球蛋白和大豆球蛋白凝胶强度下降。在相同氧化条件下,氧化对大豆β-伴球蛋白流变性质影响幅度大于大豆球蛋白。基于以上实验结果,可以明确的是,原料残余脂质的不同氧化程度将影响终产品7S、11S蛋白的结构和功能性质,而低残余脂质的低变性脱脂豆粕可制备优质的7S、11S蛋白。更多还原

【Abstract】 Soybean proteins are composed of two major components, glycinin and β-conglycinin, which account for around40%and30%of total proteins, respectively. The glycinin and β-conglycinin, or11S and7S fractions, are primarily responsible for the functional properties of soybean proteins. Fractionation of these proteins is beneficial to better understand their separate contribution to functionality, to evaluate their impact on nutrition and structure, and to identify opportunities for new product development.Many methods have been reported in the literature for purifying the glycinin and β-conglycinin fraction. However, few data have been reported on the fractionation of soybean glycinin and β-conglycinin isolated from lower Residual lipids soybean flour (LRLSF). This dissertation focuses on the preparation of soybean glycinin and β-conglycinin, and the effect of oxidation on the structure and functionality of soybean proteins during the fractionation.Processing conditions such as reducing agent, store time and pH on yields and purity of β-conglycinin (7S) were evaluated in the fractionation of soybean β-conglycinin with LRLSF. Compared with sodium bisulfite (SBS) or dithiothreitol (DTT) reducing agent, the yield and purity of β-conglycinin was the highest when P-mercaptoethanol (ME) was added to the protein extract. Prolonging store time in the precipitate stage (protein extract), the yield of β-conglycinin decreased continuously, but the purity of the protein increased by degrees in the experiment. When store time exceeded12hours, the velocity increment of7S purity slowed, while there was no significant difference at P≥0.05for total sulfhydryl and disulfide content. The yield and purity of β-conglycinin was the best when the pH of3#protein extract was adjusted to4.7. Based on the single-factor experiments, the optimized extraction conditions of7S by response surface methoclology (RSM) were determined as ME concentration of9.5mmol/L, cryoprecipitation time of12h, and pH4.86. The yield and purity of β-conglycinin were23.86%and8.64%higher than traditional preparation, respectively.Processing conditions such as pH, reducing agent and store time on yields and purity of glycinin (11S) were also evaluated in the fractionation of soybean glycinin with LRLSF.. Adjusting the pH of protein extract from6.2to6.6, the yield of glycinin decreased by16.71%, while the purity of the protein increased by4.60%. Sulfhydryl and disulfide content of proteins increased by degrees with intensifying pH. Compared with dithiothreitol (DTT) or β-mercaptoethanol (ME) reducing agent, the yield of glycinin was the highest when sodium bisulfite (SBS) was added to the protein extract at pH6.4. The effect of DTT on yields of glycinin was the lowest of three kinds of reducing agent. It was similar to the purity of glycinin when three kinds of reducing agent were used. Prolonging store time in the precipitate stage,10h was the best for yields and purity of glycinin in the experiment, while there was no significant difference at P>0.05for total sulfhydryl and disulfide content. The decreased free sulfhydryl content of glycinin indicated that the oxidation of free sulfhydryl and the formation of disulfide bond while extract time prolonged. Based on the single-factor experiments, the optimized extraction conditions of11S by response surface methoclology (RSM) were determined as SBS concentration of8.89mmol/L, cryoprecipitation time of10h, and pH6.32. The yield and purity of glycinin were28.48%and5.58%higher than traditional preparation, respectively.Based the optimum condition, soybean glycinin and P-conglycinin were prepared from thee kinds of material, namely, lowly oxidative soybean flour, fresh low temperature defatted soybean flour and low temperature defatted soybean flour treated with high temperature. And the effects of interaction between lipid oxidation and protein on oxidation extent, structural characteristics, and aggregation morphology of P-conglycinin and glycinin were investigated during the fractionation of the protein. The results showed that oxidation resulted in an increase of protein carbonyl content of β-conglycinin and glycinin, a decrease of free sulfhydryl, total sulfhydryl, and the α-helix content. Compared to soybean glycinin, β-conglycinin was more susceptible to oxidation in the same oxidation condition. Oxidation led to a decrease of surface hydrophobicity of β-conglycinin and glycinin as well as maximum tryptophan fluorescence intensity, and blue-shifted wavelength of the maximum emission, indicating that protein oxidation resulted in aggregation of β-conglycinin and glycinin. β-conglycinin was more sensitive to oxidation than glycinin because β-conglycinin contains more amino acid residues with aliphatic side chain groups which could be easily oxidative attacked. Oxidation also caused formation of soluble oxidative aggregates, aggregation extent of β-conglycinin was higher than glycinin in the same oxidative stress environment. In addition, oxidation resulted in backbone fragmentation of β-conglycinin molecule as well as dissociation of glycinin.Effect of protein oxidation on solubility, water holding capacity, oil-absorbing capacity, emulsifying properties, foaming properties, and gelation was also investigated in the paper. Oxidation resulted in a decrease of β-conglycinin and glycinin solubility. Decline of β-conglycinin solubility was more significant than glycinin in the same oxidation conditions. Water holding capacity and oil-absorbing capacity of β-conglycinin and glycinin decreased as oxidation extent of protein increased, and decrease extent of β-conglycinin water holding capacity and oil-absorbing capacity was more significant. Oxidation led to a decrease of emulsifying properties, emulsifying stability, foaming properties, foaming stability, gel hardness, and gel strength of β-conglycinin and glycinin. Compared to glycinin, decrease extent of β-conglycinin functional properties was more obvious. Protein oxidation resulted in an increase of initial of G’ value in heating period in the process of gel formation. In the holding temperature and cooling period, G’of β-conglycinin and glycinin decreased as protein oxidation extent increased. The gap between curves of G’ and G" gradually decreased as extent of oxidation of β-conglycinin and glycinin increased, indicating that protein oxidation led to a decrease of gel strength of β-conglycinin and glycinin. In the same oxidation conditions, effect of oxidation on rheological properties of β-conglycinin was much more significant than glycinin.Based on the obtained facts, we can conclude that structure and functionality of7S and11S will be affected by different degree oxidation of the lipid remained in material, and high quality7S and11S can be prepared with lower lipid-remaied and lowly denaturalized soybean flour.更多还原