Chemical Modification Of Soy Protein Isolate (SPI)

Soy protein isolate (SPI) is the most important component of soybean. Owing to its sustainability, abundance, low cost and functionality, SPI has attracted great interest for the development of environmentally friendly materials. There have been several soy protein-based materials, such as plastics, gels, films, additives or coatings, and biomedical materials reported in the recent literature. However, it well known that soy protein is a globular protein, the interaction between the molecules in the unmodified SPI may be only the friction force from the individual globular proteins. Therefore, the mechanical properties as materials in real application are very poor. As far as we are aware, there is no report on preparation and application of pure soy protein film. If we want to make best use of this renewable natural polymer, we should modify their fundamental properties though physical and chemical methods. At present, a variety of modification methods, including bulk and surface modifications, have been developed to improve the drawbacks of SPI-based materials. However, most of these were made by simply crosslinking with aldehydes or blending with other synthetic/natural polymers to overcome their fundamental limitations, such as water sensitivity, poor processibility, and in particular, low mechanical strength. Although those methods can improve the mechanical properties of SPI at certain level, they also change the natural properties to a lager extent. What’s more, the mechanical properties of final modified soy protein materials mainly depend on the properties of added materials. As for these modification methods, there still exist some drawbacks, for example, low interfacial adhesion between SPI and hydrophobic plasticizers or polymers and toxicity of chemical cross-linkers containing aldehyde groups, etc. Therefore, novel approaches still need to be developed with respect to good performance of resultant SPI plastic materials as well as high feasibility of modification.In this project, we try to adopt a chemical modification method which is rarely used in previous researchers to improve the mechanical properties. This method is not simply crosslinking with aldehydes, nor blending with other synthetic/natural polymers, but modifying the polar amino acid residues. There are large amount of polar amino acid residues in SPI, and the results of the amino acid analysis of our SPI samples indicated that the percentage of five polar amino acid residues (Arg, Lys, His, Asp, and Glu) was 47.1%, almost held the half number of the total amino acid residues. Among them, the percentage of Asp and Glu containing the carboxyl group is the biggest, which is almost twice than the percentage of Arg and Lys containing the amino group. The aim to modify SPI is that though such a modification reduce the electrostatic interactions between the acidic and basic amino acid residues, and increase the steric hindrance within the polypeptide chains of soy protein, finally, the globular structure of soy protein was destroyed and the polypeptide chains became more freely to interconnect each other, which lay a solid foundation for preparing excellent mechanical properties of soy protein film material.In the first part of my work, diethoxy phosphoryl group was successfully grafted onto the soy protein chains via a specific Atherton-Todd reaction with different grafting ratio (0.15±1.18%). The phosphoryl rates for homologous nucleophiles are inversely related to their pKa values, so normally primary amino group of Arg and Lys residues in SPI with a low pKa has the high reactivity. After modification, the isoelectric point, the apparent viscosity, the storage and loss modulus (G’and G"), and the conformation of protein were changed correspondingly compared to those of pure SPI. We suggested such a modification reduced the electrostatic interactions between the acidic and basic amino acid residues, and increased the steric hindrance within the polypeptide chains of soy protein. As a noticeable application, a robust soy protein film without any crosslink agent and plasticizer can be obtained through such a modification. The mechanical properties of the soy protein film in both dry and wet state were good enough for the potential application as a biomaterial because only a few amino acid residues (for instance, less than 0.5%) have been modified. In conclusion, the phosphoryl modification of soy protein provides a practical route to improve the mechanical properties of soy protein materials and broaden the application area.Although a robust soy protein film in dry state was successfully obtained by Atherton-Todd reaction in the first part of my work, which can also meet the requirement of the real application. However, in the real dry environment, theelongation at break of this films is still low (about 2.5%), which is not satisfied the requirement of processibility and restrict their further application. Therefore, in the second part of my work, we want to find a chemical agent which not only can improve the tensile strength of soy protein film, but also can bear large mechanical deformation. Finally, we choose tetrakis (hydroxymethyl) phosphonium chloride (THPC) to react with amino group of Arg and Lys residues though Mannich-type reaction. After modification, we obtained a more flexible and ductile soy protein film than begin in dry state as expected, and we did not use any crosslinking agent and plasticizer that were almost unavoidable in the previous works reported in the literature. The tensile strength and the elongation at break of our soy protein films were 10±5 MPa,25±0.5% in dry state, and 3.8±1.5 MPa,125±5% in wet state, respectively. Although the tensile strength of this modified soy protein film is one third of previous one, the elongation at break is ten times than that of previous one. Furthermore, the elongation at break can reach about 200%.In previous two parts of my work, we successfully modified the amino group of Arg and Lys residues of soy protein and obtain two different modified soy protein films which have different mechanical properties in dry and wet state. According to the results of the amino acid analysis of our SPI samples, the percentage of Asp and Glu containing the carboxyl group (about 28.6 wt%) is almost twice than the percentage of Arg and Lys containing the amino group (about 14.0 wt%). Therefore, in the third part of my work, we try to modify the carboxyl group of Asp and Glu residues by glucosamine as EDC/HOBT is coupling agent. In this reaction process, there is not only the reaction between carboxyl group of soy protein and amino group of glucosamine, but also the reaction between carboxyl group of soy protein and amino group of soy protein itself. We expected that such a modification reduced the electrostatic interactions between the acidic and basic amino acid residues, and increased the steric hindrance within the polypeptide chains of soy protein. Meanwhile, the hydroxyl group of glucosamine which have been grafted on soy protein probably can be served as a plasticizer, which will make a great contribution to improve flexibility of modified soy protein. Finally, we obtained a modified soy protein film without any crosslinking agent and plasticizer, which have excellent mechanical properties. In particular, the elongation at break of this film is 350±30%, suggesting that it will have more obvious improvement in aspect of processibility than two previous modified soy proteins. The result of cytotoxicity of this modified soy protein film shown that it has excellent biocompatibility.In this paper, we successfully modified the amino group and carboxyl group soy protein and obtain three different modified soy protein films which all have different and more excellent mechanical properties in dry and wet state than pure soy protein. We believe that those methods we developed provide a practical approach to improve the mechanical properties and broaden the applications of natural soy protein based material.