| In order to use the resource of bovine bone, improve the deep processing and the added value, we did this research. The bovine bone were used as material, first we extracted the collagen. The physical and chemical properties of collagen were also researched. The pepsin soluble collagen which was purified was used to prepare collagen sponge, the structure and the physico-chemical test of the sponge were also studied. The main research contents and results are as follows:To improve the extraction rate of bovine bone collagen, used yield as an indicator to study the collagen extraction and purification process, and defatted, decalcified, salting, enzymes dissolve and other various conditions to be optimized. Low temperature reflow4-5h for defatted at60℃, the decalcified rate was97.74%. Then with0.5mol/L HCl to decalcified, ratio of material to liquid was1:10, every2hours to change dialysate acid (changed5times), the ash content of bone meal decreased from64.23%to3.18%. Collagen salting conditions:pH5.6, NaCl concentration was0.9mol/L. Extract conditions of enzyme soluble collagen (PSC) by orthogonal optimization were:enzyme amount was250u/g, time was10h, temperatue was25℃, pH2.1, ratio of material to liquid was1:10. The yield were2.34g acid soluble collagen (ASC) and8.20g enzyme soluble collagen (PSC) per100g of defatted bone meal, respectively.To identify collagen type of bovine bone, this paper identified the physicochemical characterization of acid soluble and enzyme soluble collagen after purification. UV spectrum analysis show that the maximum absorptions of the acid soluble and enzyme soluble collagen extracted from bovine bone were at near230nm, there were no absorption peak at280nm, which showed collagen did not contain tryptophan, and phenylalanine, tyrosine content was extremely low. Analysis of the amino acid composition showed that glycine accounted for about1/3of the amino acids, was typical characteristics of type I collagen. In the IR spectrum, absorption peak of acid soluble collagen was at1240.79, and absorption peak of enzyme soluble collagen was at1241.15, showed bovine bone collagen had triple helix structure. In the SDS-PAGE gel electrophoresis of ASC and PSC, there was a band near250KDa, on behalf of the (3subunit of collagen, and two electrophoretic bands near130KDa, on behalf of al and a2subunit respectively, which reflected the triple helix structure of type I collagen. In Circular dichroism figure of ASC and PSC, there was a negative absorption peak in the vicinity of197nm, and a positive absorption peak at220nm, that showed triple helix structure of type I collagen was complete. By differential scanning calorimetry analysis of acid soluble collagen and enzyme soluble collagen of bovine bone, it was found that thermal denaturation temperature of acid soluble collagen was73.93℃and thermal denaturation temperature of enzyme soluble collagen was71.94℃. These results indicated that extracted collagen still had complete triple helix structure.In order to improve the performance of collagen sponge, the key technical steps and preparation were studied in this study. The preparation processes such as compositing, crosslinking, freeze-dring of the composite sponge were studied and optimized, sensory evaluation, absorbent multiples, tensile strength, rate of elongation at break as indicator. The sponge preparation condition ultimately determined were:the perfusion thickness was6nm, the total concentration of the collagen/chitosan was1.2%, collagen/chitosan rate was2:8, the concentration of glutaraldehyde was1.5%, the temperature of pre-freezing was-40℃. In this condition, the weight of collagen absorbent.To test the physical, chemical and structural analysis of the homemade collagen composite sponge. Their physical properties were porosity of70.23±1.98%, apparent density36.03±1.46mg/cm3, the permeability of28.57±1.40%, the water absorbent38.67±0.47times, the absorption rate of10.17±0.65%, moisture3.68±0.16g/g; chemical properties as water-soluble weight loss2.57±0.12%, pH6.92±0.03, residue on ignition1.03±0.21%; in the saline tolerance test, they remained intact in25d period and no serious damage, dissolution, dispersion phenomena, the structure was more complete and close; electron microscopy showed the structure of the sponge was three-dimensional lamellar structure formated by accumulation of porous layers, they had good air permeability and high porosity. |
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