Preparation Of Collagen From The Skin Of Squid And Preliminary Study Of Collagen Sponge

Squid belong to Calamary Family，Cephalopoda，Phyllum Mollusca，are generallycalled calamari. Mainly distributed in the warm, high salt concentration sea，perched insurface and to1500m under the water. At present, the squid processing has become animportant part of the aquatic products processing industry. But the squid skin as squidprocessing by-product was processing simply into feed or buried for processing technologylimited, have not found good value-added way. The squid skin contains rich collagenprotein，collagen protein is an important structural protein, has important application value.Aquatic collagen has huge research and application value because of its low antigenicity，low allergic and easy to enzyme solution. The purpose of my experimental research was:extracted collagen from abandoned squid skin, researched its physical and chemicalproperties and the application in medical, In order to provide experiment data andtheoretical basis for further research or applicating collagen.The technology of extracted collagen from squid skin included: pretreatment (removemiscellaneous protein and fat), enzymatic extraction, salting-out, dissolved again to removesalt-soluble protein, dialyzed to remove small molecules protein, then freeze-drying. Toexplore the components of squid skin and the optimal pretreatment conditions for extractcollagen from squid skin by protease. Select four factors, three levels for each one, by L9(34)orthogonal experiment,the optimal conditions were determined. Results show, the optimalselection for the four factors Sodium hydroxide concentration, sodium hydroxide effecttime, isopropyl alcohol concentration, activated carbon concentration were0.15mol/L，6h，12%，0.8%, respectively. Enzymatic degradation was used to extract collagen from the skinof squid. Through single enzyme hydrolysis experiment, neutral protease was confirmed tobe the target enzyme. The condition of extraction was optimized by orthogonal design andresponse surface methodology. The optimum conditions for collagen extraction was:5%neutral protease was added to the skin of squid which had been through pretreatment,distilled water was added to the skin of squid in accordance with biomass-solvent ratio of1:14(g: mL), adjusted the pH to7.0, enzymolysis reaction for54min in51℃, collagenextraction yield is the highestThe physical and chemical properties of collagen and its composition characteristicswere analyzed.There was distinct absorbance peak obtained near220nm, which accordedwith the characteristic absorption peak at220-230nm. The Td of collagen was26.8℃, which was much lower than that of collagen from porcine skin (37℃) and calf skin(40.8℃). FTIR investigations showed the existence of triple-helical structure of Peru squidskin collagen, and the helical structure was not affected by pepsin digestion during collagenextraction. When pH was increased to7, marked decrease in solubility was observed andreached the minimum, on the contrary, when pH value passed over7, the solubility ofcollagen from squid skin increased. The increase of ionic strength causedhydrophobic-hydrophobic interaction between protein molecules, and then its solubilityreduced. It was found that the collagen was consisted of α chains (α1and α2chain), whichshowed two distinct species varying in their mobility, and its dimmer (β chain). Theexistence of at least two different subunits showed that a major collagen of squid skin mightbe type I collagen. The PSC isolated from squid skin was relatively high content of alanine,proline, hydroxylproline, glutamic acid and aspartic acid, and relatively low content ofcystine, methionine, threonine and isoleucine. The glycine content of the PSC was about33.5%, but tryptophan was not detected. The lyophilized collagen was loose and porous,and had uniform and regular alveolate pores. Uniform and regular network structures ofsponges are used as drug carriers are propitious, not only for well-proportioned distributionfor other drugs, but also for evaporation of fluid.This research manufactured collagen sponge with the collagen from squid skin.Through the determination of heavy metal content and potential of hydrogen of collagensponge, the results found that the pH of collagen sponge was6.12, heavy metal content was0.29μg/kg, accord with national standard of medical materials requirements. Through theresearch of rabbit ears hemostatic experiment, we found that the blood coagulation time ofcollagen sponge was141.24s, bleeding amount was0.519g. The index of that of gauzewas223.18s and0.843g. Not only blood coagulation time of the collagen sponge wasshorter, so far to blood coagulated, bleeding amount was less.