Studies On The Dissolution And Separation Of Silkworm Pupa Protein Based On Ionic Liquids

Silkworm pupa protein (SPP) is a kind of natural protein extracted from waste silkworm pupa. Its reasonable amino acid ratio, good bioactivity and biocompatibility make it suitable for bio-pharmaceutical, textiles, food and cosmetics. The current methods for the the extraction of SPP suffer from the defects of the use of acid and alkali, strong corrosivity, serious pollution to the environment, low yield and high fat content. In order to optimally utilize the pupae material and obtain purified protein, a clean and efficient technology is urgently needed.In this study, a process for the extraction of SPP was established based on ionic liquids (ILs) by using silkworm pupa as the raw material, which was cheap and easy to get. It provided some scientific basis for developmenting new technologies to fabricate SPP. The main research contents and the results are as follows.(1) Firstly, the analysis methods of products were deternaibed. Experiments were conducted to verify the traditional sepatation technology and primary improving suggestions were proposed. The results illustrated that the yield of SPP extracted from traditional methods was only about 45%, but its fat content was as high as 7%。(2) The extraction of SPP from silkworm pupa was explored based on ILs. ILs were found to be efficient solvents for silkworm pupa. The effects of dissolution temperature, dissolution time, coagulation solvent and water content were investigated. FT-IR, XRD and TGA were used to examine the structures and properties of regenerated SPP. Taking the yield, purity, protein content and fat content into consideration, dissolution temperature at 90℃, dissolution time for 24 h and coagulation solvent of ethanol were chosen as optimal conditions. When the experiements were conducted at optimal conditions, the regenerated SPP with a yield of 59.6% and an extremely low fat content of less than 0.5% could be obtained by using [Amim]Cl, whose water content was less than 0.5%.(3) A series of ILs with different onion or anion compositions were used to investigate the effect of the structure of IL on the dissolution and regeneration of SPP. Both the cation and anion were found to play important roles in the dissolution and regeneration of SPP. For identical chloride-containing ILs, the extraction of protein decreased in the following order:[Emim]Cl> [Bmim]Cl> [Amim]Cl> [Hmim]Cl> [Omim]Cl. With the increase in the length of alkyl chain on the methylimidazolium cation, there was a decrease in dissolution rate of silkworm pupa and yield of SPP. [Amim]Cl also exhibited a good efficiency because of its short chain and C=C bond. A series of ILs, containing the [Emim]+ cation and a variety of anions, were selected to investigate the effect of anion. [Emim]OAc was demonstrated to have the best ability to dissolve the pupa power. However, a big dissolution rate did not mean a good yield. The yield of regenerated SPP increased in the following order:[NTf2]"< [DCA]-< Br-< OAc-< DMP-< DEP"< Cl". The extraction efficiency obtained from [Emim]Cl was the highest, which can up to 62.63%, and the fat content of the regenerated SPP was less than 0.5%。(4) FI-IR, XRD,13C NMR and TGA were used in order to examine the structural changes and prpoerities of regenerated SPP. It was observed that the dissolution process was physical dissolution, which did not make major chemical changes on the polypeptide chain. However, the crystalline domains of SPP were disrupted by the dissolving process and some a-helices and β-sheets were not restored during the process of regeneration, causing a decrease in the degree of crystallinity. Meanwhile, the regenerated SPP displayed a decrease in fraction of the a-helix and an increase in fraction of the β-sheet+ coil random. TGA data showed that the regenerated proteins had slightly better thermal stabilities than raw material.(5) The dissolution and regeneration mechanisms of SPP were proposed based on the experimental results. The anions and cations associated with the H atoms in-NH- and 0 atoms in C=O, respectively. The interaction disrupted hrdrogen bonding between protein macromolecules and led to the dissolution of silkworm pupa. Then coguation solvents were added into SPP/IL solution. Since the coguation solvents would dissolve IL other than SPP, the hydrogen bonds between protein macromolecules would be rebuilt, resulting in the regeneration of SPP.