The Study Of Chinese Soft-Shelled Turtle Calipash Collagen Extraction And The Mechanism Of Collagen Peptide On Rat Wound Healing

Chinese soft-shelled turtle is a nutritious aquatic product with delicious taste and high edible and medicinal value.Calipash is the protective skin tissue around the shell of Chinese soft-shelled turtle,which contains abundant collagen.At present,the global production of Chinese soft-shelled turtle is rising with the gradually developed cultivation technology.However,there are few work majoring inthe deep processing of calipash and its related products.Therefore,this research focused on the calipash resources of the soft-shelled turtle,it extracted the calipash collagen and further developed the collagen peptide products,studied the properties and functions of products,which could provide a theoretical basis for industrial production.Firstly,the calipash of soft-shelled turtle was extracted by acid method and enzymatic method.The acid-soluble collagen?ASC?and pepsin-soluble collagen?PSC?were obtained.The structure and physichemical properties of the two collagens were analyzed.The results showed that the yields of ASC and PSC under the optimal extraction conditions were 35.69%and 65.50%,respectively.The SDS-PAGE gel electrophoresis and matrix-assisted laser desorption/ionization time of flight mass spectrometry showed that both ASC and PSC were I type collagen.The scanning electron microscopy observed that the structure of ASC was arranged regularly and compactly between layers,while the PSC was arranged orderly and loosely between layers.The circular dichroism spectrum further indicated that PSC has a more complete three-stage helical structure.The thermal denaturation temperatures?T_d?of acid-soluble collagen and enzymatic collagenwere measured by differential scaning calorimetry scanner,the T_d were 38.39°C and 38.11°C,respectively.The amino acid analysis showed the amount of flavor amino acid and essential amino acid in ASC and PSC were similar,and the amount of sub-amino acid and non-essential amino acid were slightly different.Among them,the sub-amino acid in ASC and PSC were 20.46%and 21.53%,respectively;the amount of non-essential amino acid were 12.74%and 11.62%,respectively.Secondly,the optimization of preparation process of collagen peptide from calipash was studied.Four enzymes?pepsin,alkaline protease,neutral protease,papain?were used to hydrolyze calipash collagen.Based on the results of hydrolysis degree,the optimal enzymatic conditions were determined.It was found that the pepsinase exhibited the best enzymatic effect and the optimum enzymatic hydrolysis conditions were:pH 2.5,substrate concentration3%,enzyme dosage 8000 U/g,hydrolysis time 5 h,hydrolysis degree was 24.13%.Besides,two drying methods?spray drying and vacuum freeze drying?were applied to dry collagen peptide hydrolysate.The color,structure,water absorption,oil absorption,water holding capacity,solubility,emulsification,foam stability were studied to determine the optimum drying process.The results showed that the vacuum freeze dried sample has lower brightness?L^*?with a loosely arranged lamellar structure,and it has good water absorption,oil absorption andwater holding capacities and solubility.The spray dried samplehad higher brightness?L^*?with a smooth spherical structure,and it exhibited a good emulsion stability and foam stability.High performance liquid chromatography?HPLC?showed that the collagen peptides that has molecular weight below 2000 Da was 66.23%and the molecular weight below 1000Da was 41.96%.Thirdly,the antioxidant properties of the STCCP?Soft-shelled turtle calipash collagen peptides?with different molecular weight were compared.The collagen peptides were divided into three groups with different molecular weight grades by ultrafiltration.The DPPH·scavenging rate,ABTS·⁺scavenging rate,OH·free radical scavenging rate,lipid oxidation inhibition rate and the reducing power of three groups were measured.The results showed that the STCCP-III?<1 kDa?has the best antioxidant activity,followed by STCCP-II?1 kDa³ kDa?and STCCP-I?3 kDa⁵ kDa?.Apart from these,the effects and mechanisms of soft-shelled turtle calipash collagen peptide?STCCP?from the calipash of Chinese soft-shelled turtle on wound healing in rats were studied.The study used SD rats as experimental objects to establish an excisional skin wound model.Low(0.20 g·kg^-1·bw^-1),medium(0.66 g·kg^-1·bw^-1)and high(2.00 g·kg^-1·bw^-1)doses of collagen peptide solutions was given to the SD rats.The wound healing and its biological mechanism were analyzed by histopathology and immune-histochemistry.The results showed that the wound healing rate of the STCCP group was higher than control from the 4th day after modeling.The histopathological observation showed that STCCP-M and STCCP-H have higher collagen and hydroxyproline amount in the wound skin than control group after 10 days and 15 days.The healing rate of the medium and high doses groups of STCCP were significantly?P<0.05?higher than that control after 15 days of modeling.The immunohistochemical analysis indicated that the expression of vascular endothelial growth factor?VEGF?in STCCP group was significantly?P<0.01?higher than control after 15 days of modeling.STCCP could increase the expression of vascular endothelial growth factor and CD31 in rat wound skin tissue,promoted neovascularization and enhanced collagen synthesis.This might be one of the mechanisms of STCCP promoting wound healing in rats.