| Silk is a natural polymer protein secreted by Bombyx mori,which is mainly composed of silk fibroin and sericin.Sericin is a layered colloidal protein wrapped around silk fibroin,which accounts for 25%to 30%of the total weight of silk.Sericin can bind silk fibroin together to form a firm structure of cocoon shell.And sericin is a multilayer protein.The sericin near outer layer is synthesized by the anterior cells of middle silk gland of the silkworm,while the sericin near inner layer is synthesized by the posterior cells of middle silk gland.In the silk industry,sericin not only affects the gloss and feel of silk fabrics,but also influences the dyeing processes.The process of removing sericin is called degumming,and sericin is often discharged with the degumming wastewater,causing serious environmental pollution and waste of biological resources.In recent years,with the rapid development of biomedical engineering,sericin has gradually attracted the attention of researchers due to its excellent biological properties.The silk culture has a thousands-years history.In the textile reeling process,hot water,alkaline solutions,acidic solutions,proteases,and surfactants are common degumming agent.In the process of recovering sericin,the degumming solution could be purified by modern processes such as dialysis or membrane ultrafiltration,but the cost is too high,which severely limits its practicability.Until now,there is still no standardized,practical,green silk degumming method available.Therefore,we need to develop low-cost,high-efficiency,greener sericin extraction methods.In the previous reports on new degumming methods,researchers mostly paid attention to the effects of degumming methods on silk fibroin,but ignored the effects on the recycle and properties of sericn.Therefore,we will extract three layers of sericin peptides by greener degumming methods,and analyze the physicochemical properties and biological activities in vitro of the three kinds of sericin peptides systematically.The severe degumming condition could improve the degumming efficiency,but the sericin would be degraded severely,and the mild degumming condition has a lower degumming efficiency.The sericin near inner layer is hard to remove completely.We found that the layered sericin could be extracted in two steps without introducing a degumming agent.Firstly,clean cocoon shells were degummed in boiling pure water for 1 h,and we could obtain the outer layered sericin with the degumming ratio was approximately 12%.Secondly,the cocoon shells,which have not been degummed completely,were degummed under 120℃ high temperature and high pressure water for 2 h to obtain the remaining inner layered sericin.The sericin obtained by the boiling water was named as outer layered sericin(OLS),and the sericin extracted by the high temperature and high pressure water was named as inner layered sericin(ILS).In addition,the calcium hydroxide[Ca(OH)2]aqueous solution could be used as a greener degumming agent,the alkaline degumming solution could be neutralized with sulfuric acid/phosphoric acid to cause calcium ion to form a calcium salt and precipitate.This degumming method could not only quickly isolate and purify sericin,but also recover the calcium salt precipitation,and then the sericin extracted by this method was named as whole sericin(WS).In the first part of this article,the physicochemical properties and biological activities of the three kinds of sericin peptides were analyzed and compared systematically.The degumming methods will affect the molecular weight range and amino acid composition of sericin peptides,which would affect its physicochemical properties and biological activities.The degumming ratios of OLS,ILS and WS are 11.4%,16.65%and 28.61%,respectively.The SDS-PAGE electrophoretic bands of WS were mainly concentrated at 10 kDa 一 30 kDa,the lowest molecular weight made WS having the best water solubility at room temperature.The solubility of the OLS and ILS which had large molecular weights would increase rapidly after the temperature rose to 70℃.Amino acid analysis showed that the content of polar amino acids such as glutamic acid,serine and lysine in OLS was significantly higher than that in ILS.OLS riched in hydrophilic groups showed a strong hygroscopicity and moisture retention capacity.And its moisture absorption ratio in 12 h exceeded 12.5%,and the moisture retention ratio in 8 h was about 85%.The thermal decomposition temperatures of OLS,ILS,and WS were approximately 308℃,322℃,and 311℃,respectively.The content of β-fold structure in ILS was higher than that in OLS,so the thermal decomposition temperature of ILS was higher approximately 14℃ than OLS,indicating that ILS had higher stability and could protect silk fibroin in natural environment.The three X-ray diffraction curves all showed crystallization peaks at 20.8°,indicating these degumming methods had no significant effects on the crystallinity of sericin peptides.In addition,all the three sericin peptides had obvious anti-ultraviolet activity,the small molecule WS which was more easily absorbed by the human body had the potential as a cosmetic raw material.The results of ABTS and FRAP tests showed that the order of the antioxidant capacity of the three sericins was:OLS>WS>ILS,and the inhibition ratio of 20 mg/mL OLS against E.coli could reach 50%.The IC50 values of tyrosinase inhibition curves of OLS and WS were 8.67 mg/mL and 3.49 mg/mL,respectively,and the inhibition ratios of 10 mg/mL OLS and WS against α-glucosidase reached 65.62%and 51.3%,respectively.These results showed that all the three kinds of layered sericin peptides had significant antioxidant,anti-ultraviolet,anti-bacterial,moisture absorption and moisturizing,whitening and hypoglycemic functions in vitro.Gelatin methacryloyl(GelMA)is a bioink commonly used in 3D bioprinting technology.In order to explore the possibility of using sericin in bioink,OLS,ILS,WS were mixed with GelMA to prepare SS/GelMA composite hydrogels.The results showed that the addition of OLS,ILS and WS would not affect the plasticity of pure GelMA hydrogel.The average pore size of the SS/GelMA composite hydrogel is approximate 150 μm,and its three-dimensional network structure could meet the needs of cell growth.Mechanical test results showed that ILS with β-sheet structure could increase the compressive strength of pure GelMA hydrogel from 0.21 MPa to 0.28 MPa,which improve the compressive performance by 25%.Since sericin did not change the molecular structure of GelMA,it has little effect on the swelling property of pure GelMA hydrogels.After 10 days of neutral protease treatment,except for WS/GelMA,the mass retention rate of the other SS/GelMA composite hydrogels were above 80%,and the drug release ratios of OLS/GelMA,ILS/GelMA,WS/GelMA composite hydrogels reached about 78%,62%,70%on the fifth day,respectively,which indicating that the three kinds of sericin peptides have the potential for application in the field of regenerative medicine.The results of cell culture in vitro showed that when the OLS concentration was 1%,excess OLS would make OLS/GelMA composite hydrogel to cause toxicity to L929 cells.After reducing the sericin concentration to 0.5%,0.25%,0.1%,all the SS/GelMA composite hydrogels showed higher cell viability.Compared with the cells on the pure GelMA hydrogel scaffold,the cells on the SS/GelMA composite hydrogel had longer pseudopods and grew in a spindle shape.It showed that SS/GelMA composite hydrogel could accelerate the process of L929 cells adhering to the hydrogel,and made the cells enter the rapid growth phase in advance,which showed excellent cell compatibility.Layered sericin peptides(OLS,ILS,WS)were prepared in greener degumming methods.A series of experimental results indicated that all the three sericin peptides had excellent physicochemical properties and biological activities in vitro.OLS with good biological activities and ILS with high thermal stability could be used in fields such as medical biomaterials and industrial surface modification materials,and small molecule WS had great potential for applications in biopharmaceuticals,functional health foods,and cosmetics.Meanwhile,these sericin peptides could also improve the mechanical property,cell adhesion property and biocompatibility of GelMA and had potential in the field of 3D bioprinting bioinks.These results provided important reference values for the sustainable development and high value-added products of the silk industry. |
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