| Collagen,the most abundant protein,which is biocompatible,has the advantages of biodegradability,low immunogenicity,and so on.Therefore,collagen can be used in biomedicine,food,cosmetics and many other areas.Its application is closely related with its molecular weight and other properties.In this paper,the raw materials of bovine hides were pretreated to remove impurities and depilate.The extraction of collagen from bovine hides with pepsin was investigated by single factor experiment.Five such variables as ratio of bovine hide weight and 0.5 mol/L acetic acid solution volume,enzyme dosage,temperature,pH and enzymolysis time,on the extraction of collagen were analyzed using collagen extraction rate as output.The structure and morphology of samples were observed by Fourier Transform Infrared Spectroscopy(FTIR)and Scanning Electron Microscopy(SEM).Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis(SDS-PAGE)was employed to determine the molecular weight distribution of collagen.Ultraviolet and Visible(UV)Spectrophotometer was used to study the fiber-forming ability in vitro of collagen at the wavelength of 313 nm.The denaturation temperature of collagen was characterized by Differential Scanning Calorimeter(DSC).The relationship between conditions of collagen extraction and main properties of collagen provided a theoretical basis for extraction and application of collagen.Little effect was found about the effect of pretreatment on solid content of bovine hides.When the pH of bovine hides is in the neutral range,the denaturation temperature is also consistent with that reported in the literature,which is 50.3°C.Single factor experiment showed that when other conditions were constant,with the increase of the volume of acetic acid solution,the collagen extraction rate increases gradually.When enzyme dosage increases,and the extraction rate of collagen increases.As the enzyme dosage increases from 1.5% to 2.5%,the extraction rate increases significantly.The change in temperature significantly affects the collagen extraction,which could lead to a significant decrease in extraction rate.Extraction rate is low at a low initial pH.As the pH is increased to above 2.0,however,the extraction rate is affected weakly in the range of pH gradient.The results showed that extraction rate increases with the prolongation of the enzymolysis time.The extraction rate cannot be improved by further extending the enzymolysis time after certain time.The results of FTIR,digital photo and SEM photograph showed that the sample was collagen with a lamellar structure,in pure white.Besides,the extraction conditions have little effect on the collagen structure.The molecular weight and distribution of collagen were less affected by ratio of bovine hide weight and 0.5 mol/L acetic acid solution volume,enzyme dosage,initial pH and enzymolysis time.All the electrophoretic patterns contained ?-bands and most of the bands were above 66.2 kDa.Extraction temperature could change the molecular weight of collagen.At 50°C,the ?-bands of collagen disappeared and the molecular weight distribution became wider.Therefore,temperature is a key factor in the extraction of collagen.Compared with the boiled collagen sample,the results of fiber-forming experiments in vitro declared that the collagen obtained under the different conditions of ratio of bovine hide weight and 0.5 mol/L acetic acid solution volume,enzyme dosage,initial pH and enzymolysis time all had fiber forming ability in vitro.Only the collagen extracted at 50°C lost the fiber forming ability.DSC results indicated that the denaturation temperature(Td)of collagen was all above 45°C.Enzyme dosage,initial pH and enzymolysis time have little effect on Td of collagen.Nevertheless,the Td of collagen decreases with the increase of the volume of acetic acid solution.When the temperature is lower than 37°C,extraction temperature affected it less.On the contrary,when temperature was higher than 37°C,the Td of collagen dropped to 50°C below. |
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