Study On The Extraction And In Vitro Self-assembly Properties Of Freshwater Fish Collagen

Collagen is one of the most abundant and complex structural proteins of the extracellular matrix,which is of great significance to the organism's structure and life activities.In vitro self-assembly is one of the characteristics of collagen,which refers to the collagen relies on non-covalent bonds between collagen molecules and molecular force to form a stable structure of supramolecular structure or molecular aggregation process under certain conditions.The mechanical properties,enzymatic degradation and thermal stability of collagen fibers formed by self-assembly are of great advantages.There are many self-assembly studies on the source of collagen from terrestrial sources,but the self-assembly research on the aquatic source collagen is still in its infancy.In recent years with frequent Revelations of mad cow disease,the disease such as massive security incidents at home and abroad,as well as the traditional religious and ethical factors limit,lead to terrestrial sources of collagen in security applications widely in people's lives,so more and more researchers began to turn to the source of aquatic collagen.Freshwater fish production in China is huge,its processing still belongs to extensive use of basic rough machining,machining to produce by-products such as bones,most of the scales,fish innards are discarded,the waste resources,also caused great pollution to the environment.In freshwater fish scales as the research object,this paper,the acid method and enzymatic extraction of collagen,the self-assembly properties of freshwater fish collagen,for its application in the hemostatic sponge scaffold materials to provide the corresponding theoretical basis.Will first scales after cleaning,removing impurity,decalcified,in turn by acid method and enzymatic extraction of collagen protein,on the basis of single factor experiment,using the response surface method to optimize the extraction technology of fish scale collagen,get scales acid method to extract the best process is: material liquid than 1:10,60 h extraction time,extraction temperature 20 ?,the concentration of acetic acid 0.5 mol/L.The extraction rate was 13.02%;The best technology of enzymatic extraction is: pepsin 50 Unit/mg,48 h extraction time,extraction temperature 25 ?,acetic acid concentration of 0.59 mol/L.The extraction rate was 33.82%.The effect of the external environment on self-assembly of collagen was studied by the solution turbidity.The effects of ultrasonic treatment,buffer system,temperature,pH value,ion type,ion intensity and collagen concentration on the self-assembly of collagen were discussed.Results show that the temperature at 25 ? and 30 ? was more fish collagen self-assembly,PBS buffer system is more advantageous to collagen self-assembly,fish collagen concentration of 3 mg/mL in a greater degree of collagen assemble,potassium ions on collagen self assembly has a promoting effect,ionic strength under the condition of the tendency for 200 / L self-assembly rate faster.The collagen self-assembly system has a pH of 7.3 which is more conducive to self-assembly of collagen.Then using polyacrylamide gel electrophoresis(SDS-PAGE),automatic amino acid analyzer,scanning electron microscope(SEM)and circular dichroism(CD)on collagen structure and morphological analysis before and after assembly.The results of sds-page showed that the collagen protein extracted by acetic acid and gastric protein was the type I collagen with complete tertiary structure.CD tests known fish collagen and pigskin collagen samples at 15 ? to keep a full set of three spiral structure.Through SEM observation,all the self-assembled collagen samples were composed of either thick or thin fibers with obvious spatial network structure.Finally pass the test before and after the self-assembly of collagen swelling,water retention and in vitro degradation,and found that the swelling of self-assembly can increase collagen,water retention,prolong the time of degradation,thus increase collagen play a role of time,as biomedical scaffold materials for its laid a good foundation.