Studies On Characteristic, Composition And Application Of The Collagen From Anglerfish (Lophius Litulon) Skin

Anglerfish(Lophius litulon) lived in the cold bottom layer of west of northern Pacific Ocean,and northern Donghai sea,Huanghai sea and Bohai sea of China.In Anglerfish processing industry,skins were dumped as waste without further utilization.Collagen is an important structural protein,which plays roles in supporting organs and protecting bodies.It is widely distributed in connective tissue,skin and bone,and also is the principle factor in deciding the tensile of connective tissues. Due to its low toxicity,low immunogenicity,biodegradability,nutrient and moisture retentivity,collagen was widely used in the fields of food, cosmetic and biomedical materials.In this paper,extraction, characterization,composition and application of collagen from skin of Anglerfish was investigated,in order to provide theoretical basis for utilization and exploration of Anglerfish skin collagen.Extract collagen from Anglerfish skin by acid and enzyme. Collagens from Crucian carp skin,Snakehead skin,Promfret skin and porcine skin were studied as control.Anglerfish skin was extracted with 7.5%(w/v) NaCl to remove non-collagenous proteins and pigments,and with 10%(v/v) butyl alcohol to remove fat.Acid soluble collagen(ASC) was then extracted using 0.5M acetic acid solution with a yield of 35.5%. Undissolved residue,obtained after acid extraction,was thoroughly rinsed with distilled water,then extracted with 2.5%(w/v) papain, defined as papain soluble collagen(PSC) with a yield of 15.5%.ASC and PSC was further purified by precipitation and re-dissolvation with NaCl and 0.5M acetic acid solution respectively,after then dialysed against acetic acid solution and distilled water.Study on physiochemical properties,including solubility,viscosity and denaturation temperature(T_d),of Anglerfish ASC and other collagens.The solubility of Anglerfish collagen and other skin collagens reached maxima at pH 3 and minimum at pH 7.The effect of NaCl on the solubility of collagens from Anglerfish and other skins didn't observed until up to 2%(w/v),a sharp decrease of solubility was occurred of all collagens with increasing NaCl concentration.Viscosities of Anglerfish collagen and other skin collagens were decreased with an increasing temperature,and decrease rate speed up when the temperature up to some specific point,while the initial temperatures of speeding up were different.T_d of Anglerfish ASC was 22.09℃determined by differential scanning calorimeter(DSC),which is close to Promfret ASC (21.91℃).T_d results showed that,ASC from marine fish was lower than those from freshwater fish(e.g.Crucian carp ASC:28.38℃,Snakehead ASC:28.73℃),besides,ASC from fish skin was lower than those from mammalian skins(e.g.bovine tendon collagen:38.10℃,porcine ASC: 38.91℃).The results suggested that thermal stability of collagens were highly depending on which animal species it from.Subunit compositions and types of Anglerfish collagen and other collagens were identified by SDS-PAGE,results showed that collagens obtained in this work were electrophoretic purity.These collagens were comprised of twoα₁ chains and oneα₂ chain,and classified as typeâ… collagen.The molecular weight(MW) ofαandβcomponents of PSC was lower than ASC from Anglerfish skin.Compared MW ofαchain of all collagens,it found that mammalian was higher than fish,and freshwater fish higher than marine fish,showed a distinguished resource variations.Amino acid compositions of collagens from Anglerfish and other fish skins were analyzed by pre-column derivation of HPLC.Anglerfish skin ASC results showed,it was rich in glycine(40%),the contents of proline and hydroxyl proline,lysine and hydroxyl lysine were 14.69% and 2.66%respectively,in total amino acids,besides,hydroxylation degree of proline and lysine was 28.43%.The contents of amino acids which related to collagen's thermal stability were behaved consistently with T_d.The contents of proline and lysine from mammalian were higher than freshwater fish,and freshwater fish higher than marine fish.Peptide mixture hydrolyzed by trypsin fromα₁ andα₂ chain of Anglerfish ASC and Bovine tendon collagen were detected by HPLC-MS/MS.Amino acid sequences of signature peptides were concluded from mass spectrum results.Mass spectrum results showed partial signature peptides could be detected in allαchains,while the mass spectrum break pattern and relative abundance of same peptide was differed withαchains.Moreover,eachαchain have its specific peptide,it need further validation to use it to identifyαchain.In this paper,physiochemical properties,haemostatic effects and biocompatibilities of Anglerfish collagen-chondroitin sulfate(CS) sponge and Porcine collagen-CS sponge which as control were studied. Physiochemical properties results of Anglerfish and Porcine collagen sponge met all requirements of medical materials.Haemostatic ability was evaluated by haemostatic time and blood quantity of collagen sponge on rabbit cut ears,results showed haemostatic ability of Anglerfish collagen sponge was similar to Porcine collagen sponge,but much better than surgical cotton.Biocompatibility of Anglerfish collagen sponge was characterized by cellular compatibility, sensitization response in vivo and biodegradability.Anglerfish collagen sponge's cellular compatibility indicated that it was affinity to cell,could stimulate cell to proliferation and differentiation,and have optimum pore size to provide enough inner areas for cell attachment and extra-cellular matrix accumulation.Degradability in vivo results showed,Anglerfish collagen sponge was much better than Porcine collagen sponge.In conclusion,Anglerfish collagen sponge,compared with Porcine collagen, was a better choice in application on haemostatic sponge,artificial skin and tissue engineering materials.