| Collagen is the main component of animal connective tissue,with good biocompatibility,biodegradability and bioactivity.It is widely used in food,medicine,tissue engineering and other fields.At present,the main source of collagen is animal tissues from livestock and poultry,but the use of collagen in terrestrial mammals is restricted due to animal diseases and some religious beliefs.Now it is being developed in aquatic life.As a by-product of aquatic processing,fish skin is rich in collagen and has potential development prospects.Based on the analysis of the physical and chemical properties of the collagen from Carassius auratus skin,the effect of ultrasonic wave and silk fibroin peptide on the self-assembly behavior of the collagen from Carassius auratus skin was studied in this paper,so as to provide theoretical and technical support for the further application of the collagen from Carassius auratus skin.Carassius auratus skin was riched in acid soluble(ASC)and pepsin-soluble collagen(PSC),and easy to extract,and its total extraction rate was as high as 50%.The amino acid composition of ASC and PSC was basically similar,and the content of glycine was about 33%.The ASC and PSC from Carassius auratus skin were characterized as a type?collagen,made up of two different?-chains and one?-chain,maximum absorption wavelength of 218 nm,and its secondary structures contain the highest?-folds and the lowest?-helices.Collagen concentration,pH,NaCl concentration and temperature had significant effects on the self-assembly behavior of the ASC and PSC from Carassius auratus skin.At the collagen concentration of 2.1 mg/mL,pH7.2,NaCl concentration of 200-600mmol/L and temperature of 20?,the self-assembly degree of the ASC from Carassius auratus skin was better;At the collagen concentration of 1.3 mg/mL,pH 7.2,NaCl concentration of200-400 mmol/L and temperature of 20°C,the self-assembly degree of the PSC from Carassius auratus skin was better.Ultrasonic treatment had a significant effect on the self-assembly of collagen in crucian carp.When the ultrasonic pretreatment power was below 200W,it could significantly promote the self-assembly of ASC.When the ultrasonic pretreatment power was below200W for 1min,the highest self-assembly degree of ASC could be achieved.The results of circular dispectral analysis showed that the triple helix structure of ASC was destroyed when the ultrasonic power was greater than 200W.However,200W,short time treatment(less than 5min)had no effect on the triple helix structure of collagen.Atomic force microscopy(AFM)further showed that the suitable ultrasonic pretreatment made the fiber density of the self-assembled products from ASC greater,and the cross-linking between the fiber micelles more complex and more aggregation.The texture analysis showed that the hardness of self-assembled products from ASC was not significantly affected by 200W and short time treatment(less than 5min)or<200W long time treatment.Cell proliferation experiments showed that the self-assembly product of skin collagen of crucian carp treated with ultrasound could significantly improve the proliferation capacity of NIH3T3 cells.Among them,the self-assembly product from ASC treated with 200W for 5min had the best proliferation effect on NIH3T3 cells,which was 10.11%higher than that of untreated.Silkworm fibroin peptide had a significant effect on the self-assembly of ASC from Carassius auratus skin.At the condition of the<5kDa fibroin peptide component with the concentration of 0.5 mg/mL,ASC from Carassius auratus skin had the highest degree of self-assembly.SDS-PAGE analysis showed that the adding of fibroin peptide collagen self-assembly product still retain the characteristics of typical type?collagen structure.The content of helix in collagen was significantly increased and the molecular order was enhanced.Atomic force microscopy(AFM)and scanning electron microscopy(SEM)analysis showed that the adding<5kDa fibroin peptide collagen self-assembly of products,components of collagen fiber network more intense,more regularity,formed by the three helical structure of collagen is more stable,in addition,fibroin peptide had no significant effect of collagen fiber D period,where<5kDa fibroin peptide component in the process of self-assembly from ASC could promote collagen fiber strand crosslinking.The results of texture analysis showed that the self-assembly product of silk peptide cross-linked ASC had the characteristics of macromolecular gel.At the<5kDa fibroin peptide concentration of 0.5mg/mL,the hardness of the self-assembled product reached the maximum value of0.15N/cm~2.The study also found that the cohesion of self-assembly products of the fibroin peptide group was lower than that of the collagen group,which indicated that the fibroin peptide and collagen fiber formed by self-assembly together had higher strength.In addition,fibroin peptide had no obvious effect on the adhesion of crucian carp skin collagen.Cell proliferation experiments showed that the self-assembly product of crucian carp skin collagen with<5kDa silk fibroin peptide could significantly improve the value-added capacity of NIH3T3 cells,which was 9.3%higher than that of the control group(without addition). |
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