高乳化性大豆蛋白的制备及其界面流变性质的研究
发布时间：2019-05-16 04:44
【摘要】：大豆蛋白是一种产量大、价格低廉的植物蛋白资源。过去的几十年,针对大豆蛋白结构改造提高功能性质的研究很多,有大量研究集中在如何提高其加工功能性质,如乳化性、起泡性、凝胶性等等,也取得了很多进展。然而迄今为止,结构改造后大豆蛋白改性产品的加工功能性质例如乳化性等性质固然比大豆分离蛋白或者浓缩蛋白性质有显著提升,但尚未有具有很高商业价值的、可与酪蛋白酸钠等大分子乳化剂相媲美的高乳化性大豆蛋白产品。本论文利用酶水解和糖基化修饰等方法,对大豆蛋白进行结构改性,旨在研究和开发出一种高乳化性大豆蛋白。同时,基于对不同结构大豆蛋白界面流变性质的研究,进一步阐明具有良好乳化性的大分子乳化剂在油水两相界面的作用机制以及所需要的结构特征,为更好地开发和应用大豆蛋白作为表面活性物质和胶体稳定剂提供理论依据和指导。首先研究了限制性酶水解和糖基化法的组合应用对大豆蛋白乳化性质和界面性质的影响。研究发现,大豆蛋白水解产物中对乳化性质最有利的主要是大分子量部分(Mw30 k Da),小分子量的大豆肽乳化性质较差。肽的糖基化反应结果显示,随着糖分子质量的增加,糖基化大豆肽的乳化稳定性逐渐增加。糖的结合可以降低大豆肽的表面疏水性和净电荷,减少肽链间的排斥作用,增加界面吸附量。表面活性测定发现大豆肽-葡聚糖共聚物在界面上紧密排列,并且每个分子占据很小的面积。激光共聚焦显微镜观察发现大豆肽-葡聚糖共聚物可以在油滴表面形成厚的吸附层。界面流变测试说明大豆肽可以在界面形成具有高粘弹性和较宽线性粘弹区的界面膜,且大豆肽与葡聚糖共聚物可以在界面上形成多层吸附。研究结果表明了大豆肽-葡聚糖共聚物具有较好的表面性质,可以在界面上紧密排列并形成一定厚度的界面膜,包裹油滴,提高乳化体系的稳定性。在此基础上,基于保留大豆水解物中大分子量部分的目的,研究了选择性酶解方式(只水解大豆蛋白11S部分,保留7S部分)对大豆蛋白乳化性质和界面性质的影响。以SPI有限水解、选择性水解11S、7S以及SPI为原料,研究了不同结构的蛋白的界面性质和稳定乳状液的机制。研究发现,选择性酶解11S后大豆蛋白的乳化性质显著提高,可与7S蛋白以及酪蛋白酸钠相媲美。蛋白质稳定乳状液可以通过两种方式:一种是结构灵活,呈棒状结构的蛋白(如酪蛋白酸钠),尽管其界面模量低,但是蛋白可以沿轴向垂直于界面的方向进行吸附,界面吸附量大,排列紧密,通过形成厚的界面膜来稳定乳状液;另一种是分子量较小的球状蛋白(如7S),可以在界面上形成高粘弹性的界面膜,以此来保护油滴不发生絮凝和凝结现象。RG既具有球状结构(7S),又包含结构灵活的水解肽,因此形成的界面膜兼具模量高和界面吸附量大两个优点,可以形成稳定的乳状液。为了考察上述研究中具有良好乳化能力的7S和RG蛋白是否具有进一步提升乳化能力的可能,论文研究了以共价键和非共价键结合葡聚糖,对7S和RG蛋白乳化性质的影响。结果发现,不管是否与葡聚糖以共价键结合,RG的粒径均增大,表面净电荷量减少,制备的乳状液粒径较小,但是在酸性和高盐离子浓度条件下乳状液稳定性比酪蛋白酸钠稍差。可能因为RG蛋白中的小分子量肽部分的存在不利于乳状液的稳定性。由7S制备的乳状液,不管是否以共价键与葡聚糖结合,在酸性条件下,稳定性都显著优于酪蛋白酸钠;在高盐离子浓度的条件下,酪蛋白酸钠制备的乳状液具有较好的稳定性,7S与葡聚糖的糖基化产物制备的乳状液稳定性仅次于酪蛋白酸钠。为了进一步提高7S在等电点处的稳定性,选择了同样具有表面活性的两种阴离子多糖,可溶性大豆多糖(SSPS)和阿拉伯胶(GA),分别与7S进行静电复合,同时研究两种多糖对7S在气-水界面和油-水界面流变性质的影响。结果发现,两种多糖对7S在两种界面的吸附影响完全不同。对于7S-SSPS,在气-水界面,复合物的吸附行为由7S主导,吸附速率比单独的7S慢。相反的,在油-水界面,复合物的吸附行为一开始由SSPS主导,之后变为7S主导。乳状液稳定性测定表明7S-SSPS形成的乳状液完全不受p H影响,即使在7S等电点附近也具有很好的稳定性。可能是因为与7S结合的SSPS紧密缠绕在7S周围,通过空间位阻效应起到稳定乳状液的作用。GA的加入并没有改善7S乳状液在等电点附近的稳定性,主要是因为界面吸附行为由GA主导,而GA形成的乳状液同样受p H影响很大。令人意外的是,不管有无多糖的存在,7S在任意p H条件下均能形成具有高粘弹性模量的界面膜,说明等电点处乳状液的分层现象主要是液滴间的静电相互作用导致的,而与界面膜的机械强度无关。论文最终对具有高乳化能力的7S和RG在粉末油脂体系的应用效果进行了初探。以氢化椰子油为芯材,RG蛋白为乳化剂,在蛋白含量达到6%或者高于6%时,不论是喷雾干燥前形成的乳状液、喷雾干燥后制备的粉末油脂还是粉末油脂复水后形成的乳状液都具备粒径小、分布均匀的特点,且粉末油脂复水以后在室温下放置6小时也不析油、不分层,说明RG蛋白对氢化椰子油具有很好的包埋特性。单甘酯的加入会与RG蛋白发生竞争吸附,引起界面膜弹性模量的显著下降,不利于乳状液稳定性。7S与单甘酯可以形成300 nm左右的乳状液,但是喷雾干燥后制备的粉末油脂大多出现粘结现象,可能与7S在喷雾干燥过程中发生部分变性有关。本课题为高乳化性大豆蛋白产品的制备和应用提供了新思路。
[Abstract]:The soybean protein is a plant protein resource with large yield and low price. In the past few decades, many studies have been made on how to improve the functional properties of soybean protein structure, and there are a lot of research on how to improve its processing function, such as emulsibility, foamability, gel property and so on. however, to date, that nature of the processing function of the soy protein-modified product aft the structural modification, for example, the emulsifying property, is, for example, significantly higher than that of the soy protein isolate or the concentrate protein, but has not yet been of high commercial value, High-emulsifying soybean protein product comparable to that of sodium caseinate and the like. In this paper, the structure modification of soybean protein was carried out by means of enzyme hydrolysis and glycosylation modification, and the aim of this study was to study and develop a high-emulsifying soybean protein. At the same time, based on the research on the rheological property of the soybean protein interface with different structure, the action mechanism of the macromolecular emulsifier with good emulsifying property in the oil-water two-phase interface and the required structural characteristics are further clarified. In ord to better develop and apply soybean protein as a surface-active substance and a colloid stabilizer to provide that theoretical foundation and the guidance. The effect of the combination of restriction enzyme hydrolysis and glycosylation on the emulsifying properties and interface properties of soybean protein was studied. It has been found that the most advantageous effect of the soybean protein hydrolysate on the emulsifying properties is the large molecular weight fraction (Mw30 k Da), and the small molecular weight soybean peptide has poor emulsifying properties. The results of the glycosylation of the peptide show that, with the increase of the mass of the sugar molecules, the emulsion stability of the glycosylated soybean peptide is gradually increased. The combination of the sugar can reduce the surface hydrophobicity and the net charge of the soybean peptide, reduce the repulsion action between the peptide chains, and increase the adsorption amount of the interface. The surface activity assay found that the soybean peptide-dextran copolymer is closely aligned on the interface and each molecule occupies a very small area. The laser confocal microscope showed that the soybean peptide-glucan copolymer can form a thick adsorption layer on the surface of the oil droplet. The interface rheological test shows that the soybean peptide can form an interface film with high viscoelasticity and wide linear viscoelastic region at the interface, and the soybean peptide and the glucan copolymer can form multi-layer adsorption on the interface. The results show that the soybean peptide-glucan copolymer has good surface properties, can be closely arranged on the interface and form an interface film with a certain thickness, and the oil drops can be wrapped to improve the stability of the emulsion system. On this basis, based on the purpose of retaining the large molecular weight part of the soybean hydrolysate, the effect of selective enzymatic hydrolysis (hydrolysis of the part of the soybean protein 11S and the retention of the 7S part) on the emulsifying properties and the interface properties of the soy protein was studied. The interface properties of proteins with different structures and the mechanism of stable emulsion were studied by means of SPI limited hydrolysis, selective hydrolysis of 11S, 7S and SPI. The results showed that the emulsifying properties of the soybean protein after the selective enzymatic hydrolysis of the 11S were significantly higher, which could be compared with that of the 7S protein and the sodium caseinate. The protein-stable emulsion can be adopted in two ways: one is a protein (such as sodium caseinate) which is flexible in structure and has a rod-like structure, The emulsion is stabilized by forming a thick interfacial film, and the other is a spherical protein having a smaller molecular weight (e.g., 7S), which can form a highly viscoelastic interface film on the interface to protect the oil droplets from flocculation and coagulation. The RG has both a spherical structure (7S) and a structurally flexible hydrolyzed peptide, so that the formed interface film has two advantages of high modulus and large interface adsorption capacity, and can form a stable emulsion. In order to investigate whether the 7S and RG proteins with good emulsifying ability in the above-mentioned studies have the potential to further enhance the emulsifying ability, the effects of the covalent and non-covalent bonding of the dextran on the emulsifying properties of the 7S and RG proteins are studied. The results show that, whether or not the particle size of the RG is increased, the net charge amount of the surface decreases, and the prepared emulsion particle size is small, but the emulsion stability is slightly lower than that of the sodium caseinate under the condition of acid and high salt ion concentration. It is possible that the presence of small molecular weight peptide moieties in the RG protein is detrimental to the stability of the emulsion. The emulsion prepared from the 7S, whether in the combination of a covalent bond and a glucan, is obviously superior to the sodium caseinate under the acidic condition, and the emulsion prepared by the sodium caseinate has good stability under the condition of high salt ion concentration, The stability of the emulsion prepared with the glycosylation product of the 7S and the dextran is only after the sodium caseinate. In order to further improve the stability of 7S at the isoelectric point, two anionic polysaccharides, soluble soybean polysaccharides (SSPS) and gum arabic (GA), which have the same surface activity, were selected to be combined with 7S, respectively. The effects of two kinds of polysaccharides on the rheological properties of the gas-water interface and the oil-water interface were also studied. The results showed that the effect of two polysaccharides on the adsorption of 7S on the two interfaces was completely different. For 7S-SSPS, in the gas-water interface, the adsorption behavior of the complex is dominated by 7S, and the adsorption rate is slower than that of the individual 7S. On the contrary, in the oil-water interface, the adsorption behavior of the complex is dominated by the SSPS and then becomes 7S-dominated. The stability of the emulsion shows that the emulsion formed by the 7S-SSPS is completely unaffected by the p H, and has good stability even in the vicinity of the isoelectric point of the 7S. It is possible that the SSPS combined with the 7S is tightly wound around the 7S and acts as a stabilizing emulsion by the steric hindrance effect. The addition of GA does not improve the stability of the 7S emulsion near the isoelectric point, mainly because the adsorption behavior of the interface is dominated by GA, and the emulsion formed by GA is also affected by the p H. Surprisingly, no matter the presence of the polysaccharide, the 7S can form an interfacial film with a high viscosity elastic modulus under any of the p H conditions, indicating that the layered phenomenon of the emulsion at the isoelectric point is mainly due to the electrostatic interaction between the droplets, regardless of the mechanical strength of the interface film. Finally, the application effect of 7S and RG with high emulsifying ability in the powder and oil system was studied. When the hydrogenated coconut oil is a core material, the RG protein is an emulsifying agent, and when the protein content reaches 6% or higher, the emulsion formed before the spray drying, the powder oil prepared after the spray drying, or the emulsion formed by the powder grease and the water after the spray drying have a small particle size, The method has the characteristics of uniform distribution, and after the powder oil and grease are mixed with water, the oil is left at room temperature for 6 hours, and the oil is not separated, so that the RG protein has good embedding characteristics for hydrogenated coconut oil. The addition of the monoglyceride can compete with the RG protein to cause a significant decrease of the elastic modulus of the interface film, which is not conducive to the stability of the emulsion. May be related to the partial denaturalization of the 7s during the spray drying process. This subject provides a new thought for the preparation and application of high-emulsifying soybean protein products.
【学位授予单位】：江南大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：TS201.21
本文编号：2478027
[Abstract]:The soybean protein is a plant protein resource with large yield and low price. In the past few decades, many studies have been made on how to improve the functional properties of soybean protein structure, and there are a lot of research on how to improve its processing function, such as emulsibility, foamability, gel property and so on. however, to date, that nature of the processing function of the soy protein-modified product aft the structural modification, for example, the emulsifying property, is, for example, significantly higher than that of the soy protein isolate or the concentrate protein, but has not yet been of high commercial value, High-emulsifying soybean protein product comparable to that of sodium caseinate and the like. In this paper, the structure modification of soybean protein was carried out by means of enzyme hydrolysis and glycosylation modification, and the aim of this study was to study and develop a high-emulsifying soybean protein. At the same time, based on the research on the rheological property of the soybean protein interface with different structure, the action mechanism of the macromolecular emulsifier with good emulsifying property in the oil-water two-phase interface and the required structural characteristics are further clarified. In ord to better develop and apply soybean protein as a surface-active substance and a colloid stabilizer to provide that theoretical foundation and the guidance. The effect of the combination of restriction enzyme hydrolysis and glycosylation on the emulsifying properties and interface properties of soybean protein was studied. It has been found that the most advantageous effect of the soybean protein hydrolysate on the emulsifying properties is the large molecular weight fraction (Mw30 k Da), and the small molecular weight soybean peptide has poor emulsifying properties. The results of the glycosylation of the peptide show that, with the increase of the mass of the sugar molecules, the emulsion stability of the glycosylated soybean peptide is gradually increased. The combination of the sugar can reduce the surface hydrophobicity and the net charge of the soybean peptide, reduce the repulsion action between the peptide chains, and increase the adsorption amount of the interface. The surface activity assay found that the soybean peptide-dextran copolymer is closely aligned on the interface and each molecule occupies a very small area. The laser confocal microscope showed that the soybean peptide-glucan copolymer can form a thick adsorption layer on the surface of the oil droplet. The interface rheological test shows that the soybean peptide can form an interface film with high viscoelasticity and wide linear viscoelastic region at the interface, and the soybean peptide and the glucan copolymer can form multi-layer adsorption on the interface. The results show that the soybean peptide-glucan copolymer has good surface properties, can be closely arranged on the interface and form an interface film with a certain thickness, and the oil drops can be wrapped to improve the stability of the emulsion system. On this basis, based on the purpose of retaining the large molecular weight part of the soybean hydrolysate, the effect of selective enzymatic hydrolysis (hydrolysis of the part of the soybean protein 11S and the retention of the 7S part) on the emulsifying properties and the interface properties of the soy protein was studied. The interface properties of proteins with different structures and the mechanism of stable emulsion were studied by means of SPI limited hydrolysis, selective hydrolysis of 11S, 7S and SPI. The results showed that the emulsifying properties of the soybean protein after the selective enzymatic hydrolysis of the 11S were significantly higher, which could be compared with that of the 7S protein and the sodium caseinate. The protein-stable emulsion can be adopted in two ways: one is a protein (such as sodium caseinate) which is flexible in structure and has a rod-like structure, The emulsion is stabilized by forming a thick interfacial film, and the other is a spherical protein having a smaller molecular weight (e.g., 7S), which can form a highly viscoelastic interface film on the interface to protect the oil droplets from flocculation and coagulation. The RG has both a spherical structure (7S) and a structurally flexible hydrolyzed peptide, so that the formed interface film has two advantages of high modulus and large interface adsorption capacity, and can form a stable emulsion. In order to investigate whether the 7S and RG proteins with good emulsifying ability in the above-mentioned studies have the potential to further enhance the emulsifying ability, the effects of the covalent and non-covalent bonding of the dextran on the emulsifying properties of the 7S and RG proteins are studied. The results show that, whether or not the particle size of the RG is increased, the net charge amount of the surface decreases, and the prepared emulsion particle size is small, but the emulsion stability is slightly lower than that of the sodium caseinate under the condition of acid and high salt ion concentration. It is possible that the presence of small molecular weight peptide moieties in the RG protein is detrimental to the stability of the emulsion. The emulsion prepared from the 7S, whether in the combination of a covalent bond and a glucan, is obviously superior to the sodium caseinate under the acidic condition, and the emulsion prepared by the sodium caseinate has good stability under the condition of high salt ion concentration, The stability of the emulsion prepared with the glycosylation product of the 7S and the dextran is only after the sodium caseinate. In order to further improve the stability of 7S at the isoelectric point, two anionic polysaccharides, soluble soybean polysaccharides (SSPS) and gum arabic (GA), which have the same surface activity, were selected to be combined with 7S, respectively. The effects of two kinds of polysaccharides on the rheological properties of the gas-water interface and the oil-water interface were also studied. The results showed that the effect of two polysaccharides on the adsorption of 7S on the two interfaces was completely different. For 7S-SSPS, in the gas-water interface, the adsorption behavior of the complex is dominated by 7S, and the adsorption rate is slower than that of the individual 7S. On the contrary, in the oil-water interface, the adsorption behavior of the complex is dominated by the SSPS and then becomes 7S-dominated. The stability of the emulsion shows that the emulsion formed by the 7S-SSPS is completely unaffected by the p H, and has good stability even in the vicinity of the isoelectric point of the 7S. It is possible that the SSPS combined with the 7S is tightly wound around the 7S and acts as a stabilizing emulsion by the steric hindrance effect. The addition of GA does not improve the stability of the 7S emulsion near the isoelectric point, mainly because the adsorption behavior of the interface is dominated by GA, and the emulsion formed by GA is also affected by the p H. Surprisingly, no matter the presence of the polysaccharide, the 7S can form an interfacial film with a high viscosity elastic modulus under any of the p H conditions, indicating that the layered phenomenon of the emulsion at the isoelectric point is mainly due to the electrostatic interaction between the droplets, regardless of the mechanical strength of the interface film. Finally, the application effect of 7S and RG with high emulsifying ability in the powder and oil system was studied. When the hydrogenated coconut oil is a core material, the RG protein is an emulsifying agent, and when the protein content reaches 6% or higher, the emulsion formed before the spray drying, the powder oil prepared after the spray drying, or the emulsion formed by the powder grease and the water after the spray drying have a small particle size, The method has the characteristics of uniform distribution, and after the powder oil and grease are mixed with water, the oil is left at room temperature for 6 hours, and the oil is not separated, so that the RG protein has good embedding characteristics for hydrogenated coconut oil. The addition of the monoglyceride can compete with the RG protein to cause a significant decrease of the elastic modulus of the interface film, which is not conducive to the stability of the emulsion. May be related to the partial denaturalization of the 7s during the spray drying process. This subject provides a new thought for the preparation and application of high-emulsifying soybean protein products.
【学位授予单位】：江南大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：TS201.21
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