Extraction Of Fish Scale Collagen And Preparation Of Collagen Hemostatic Sponge

Collagen is a structural protein of the extracellular matrix. The special triplehelical structure endows collagen with many excellent biological activities such asgood biocompatibility, biodegradability, low antigenicity, hemostatic properties and soon. Collagen has been widely used in biomedical materials, tissue engineering, healthproducts, food, cosmetics and other fields.Fish scale is one of the main byproduct of the aquatic product processing, andmost is treatmented as waste, not only cause a serious waste of resources, but alsobring great pollution to environment. The paper optimized the decalcificationtechnique of fish scale by the method of response surface with Tilapia mossambicafish scale as raw material, and discussed the optimum process condition of extractingfish scale collagen by low temperature biological enzymolysis technology. To get thehemostatic sponge, the fish scale typeⅠcollagen and chitosan were mixed together,and the preparation process of collagen-chitosan hemostatic sponge was optimized byorthogonal experiment. At the same time, through the animal experiments of mousemodel, hemostatic properties, allergenicity and biocompatibility of collagen-chitosanhemostatic sponge were further explored. The main research contents were as follows:1、Though analysied the component of fish scale, it found that ash content was of34.2%and calcium content was up to15.8%. These impacted on the extractionefficiency of fish scale collagen directly, so acid method was chosen for thedecalcification of fish scale in the test. Hydrochloric acid concentration, substrateconcentration, reaction temperature and reaction time were chosen as the impactfactors based on decalcification rate of fish scale. The appropriate levels and factorswere selected by previous single factor experiments. Depending on the dynamicequation model which was established according to the Box-Behnken responsesurface method, significance of each factor and interaction were analyzed. Theoptimum condition of decalcification technique of Tilapia mossambica fish scale was obtained as follows: hydrochloric acid concentration of2.9%, substrate concentrationof6.3%, reaction time of1.9h, and the decalcification rate of fish scale could reach99.55%under this condition.2、TypeⅠcollagen was extracted by low temperature biological enzymolysistechnology from fish scale after decalcification. Substrate concentration, enzymecontent, pH and enzyme extraction time were chosen as the impact factors based onextraction rate of fish scale. The appropriate levels and factors were selected byprevious single factor experiments. Depending on the dynamic equation model whichwas established according to the Box-Behnken response surface method, significanceof each factor and interaction were analyzed. The optimum condition of collagenextraction technique of Tilapia mossambica fish scale was obtained as follows:substrate concentration of7.8%, enzyme content of43.4U/g, pH of1.8, and thecollagen extraction rate of fish scale could reach17.41%under this condition. Inaccordance with optimum condition, fish scale collagen was extracted by enzymolysistechnology three consecutive times. The total extraction rate was46.96%. Afterfreeze-drying, the total yield of fish scale collagen was of12.21%. The collagen offish scale was identified as typacal typeⅠcollagen by technical means such asSDS-PAGE gel electrophoresis, amino acid composition, UV spectrum scanning,infrared spectrum scanning.3、The fish scale typeⅠcollagen and chitosan were mixed together, and thecollagen-chitosan hemostatic sponge was prepared. The main factors affecting thecharacteristics of the hemostatic sponge were researched by single factor experimentsand orthogonal test. The optimal preparation process of collagen-chitosan hemostaticsponge was obtained as follows: proportion of collagen and chitosan of7∶3,preparative liquid concentration of1.4%, glutaraldehyde concentration of0.015%.On this condition, collagen-chitosan hemostatic sponge had excellent hemostaticproperties and honeycomb porous three-dimensional structure was observed by scanning electron microscopy.4、Through the femoral artery hemostasis test of mouse, the hemostatic time ofcollagen-chitosan hemostatic sponge was found that（68±9）s, faster than absorbablegelatin sponge and medical absorbent cotton. Hemostatic effect of collagen-chitosanhemostatic sponge is ideal. Through the single exposure experiment of back healthyskin and back damaged skin of guinea pig, the characteristics of no skin sensitization,non-irritating, high security of the hemostatic sponge were found. It confirmed thatthe hemostatic sponge was completely adsorption by liver of mouse within8weeksthrough the compatibility test of liver of mouse. The result showed hemostatic spongehas good biocompatibility.