鸡蛋壳膜角蛋白不同提取工艺的效果比较

Comparison of eggshell membrane protein extracted effects by different methods

为探索不同方法提取的鸡蛋壳膜角蛋白的得率及其结构的区别，为鸡蛋壳膜角蛋白工业化生产方法的选择提供理论依据，分别采用氢氧化钠碱法、过氧化氢氧化法、碱性蛋白酶法从鸡蛋壳膜中提取角蛋白，以其反应速率及产品角蛋白的得率、分子量、结构完整性等为指标，比较3种不同提取方法对鸡蛋壳膜角蛋白性质的影响。试验比较结果表明，角蛋白得率：氧化法（24.33%）＞碱法（19.31%）＞酶法（15.19%）；反应速率：氢氧化钠碱法最快，过氧化氢氧化法次之，碱性蛋白酶法最慢；角蛋白分子量：碱性蛋白酶法（31～43 kDa）＞氢氧化钠碱法（14.4～20.1 kDa）＞过氧化氢氧化法（＜14.4 kDa）；角蛋白结构：碱性蛋白酶法和氢氧化钠碱法提取的角蛋白，二级结构保存完整，过氧化氢氧化法提取的角蛋白二级结构受到了破坏。氢氧化钠碱法和碱性蛋白酶法可用于生产分子量高、二级结构完整的角蛋白产品，而过氧化氢氧化法是生产小分子肽类角蛋白产品较好的方法。

Recently, the exploitation and utilization of eggshell membrane keratin has aroused wide interest. Eggshell membrane contains about thirty percent of keratin, is easy to obtain, and is an abundant raw material for keratin production and application. In this paper, the author uses the sodium hydroxide alkali method, hydrogen peroxide oxidation method, and alkali protease method to extract keratin from eggshell membrane. By comparing the three methods’ reaction rate and keratin products’ extraction rate, formula weight, and structure integrity, study the influence of three extraction methods on eggshell membrane keratin’s characteristic, find the difference on the results of each method. The ratio of pure keratin weight to eggshell membrane weight represents the extraction rate of keratin. According to the sodium dodecyl sulfate polyacrylamide gel electrophoresis method, identify the molecular weight of the three kinds of keratin. The structural integrity of keratin is identified by ultraviolet absorption spectra and using a Fourier infrared spectrometer. The test results showed that there are differences between hydrolytic processes and properties of keratin extracted by different methods. The extraction rate of keratin by hydrogen peroxide oxidation method is 24.33 percent, higher than the sodium hydroxide alkali method （19.31 percent）, and the alkali protease method （15.19 percent）. The reaction rate of sodium hydroxide alkali method is fastest;the egg membrane completely dissolvesin about twenty-five minutes. The reaction rate of hydrogen peroxide oxidation method comes second; the egg membrane completely dissolves in about two hours. The hydrolysis rate by alkali protease method is the lowest;the eggshell membrane completely dissolves in up to two and a half hours. The sodium dodecyl sulfate polyacrylamide gel electrophoresis result shows that keratin product’s purity and quality was better. The formula weight of eggshell membrane keratin is 31 kDa to 43 kDa in the alkaline protease method, 14.4 kDa to 20.1 kDa in the sodium hydroxide alkali method, and less than 14.4 kDa in the hydrogen peroxide oxidation method. Ultraviolet absorption spectra shows that the maximum absorption peak of the eggshell membrane keratin extracted by sodium hydroxide alkali method, hydrogen peroxide oxidation method, and alkali protease method are in the 290 nm place, the 296 nm place, and the 287 nm place, respectively, and the ultraviolet absorption peak moves to longer wavelengths. Ultraviolet absorption band occurred red shift may be associated with the destruction of extraction solvent on the structure of keratin. The ultraviolet absorption peak red shift degree of eggshell membrane keratin extracted by alkali protease method is lowest, indicating that the destruction of enzymatic method on the structure of eggshell membrane keratin is minimal. The result of Fourier infrared spectra shows that, Eggshell membrane keratin extracted by sodium hydroxide alkali method and alkaline protease method has a characteristic absorption peak in the infrared spectral region amideⅠ, amide Ⅱ and amide Ⅲ belt, indicating that keratin extracted by the alkali method and the alkaline protease method have kept the keratin’s secondary structure integrity. There is no characteristic absorption peak in the infrared spectral region amide Ⅰ belt and amide Ⅲ belt of the eggshell membrane keratin extracted by the hydrogen peroxide oxidation method, but in the amide Ⅱ belt appeared two characteristic absorption peaks, in the 1 489 cm-1 place and the 1478 cm-1 place. There are three characteristic absorption peaks near the 1 000 cm-1 place, which are produced by the N-H bending vibration and the S-O stretching vibration, thus indicating that the disulfide bond in the eggshell membrane keratin is opened by hydrogen peroxide, and the keratin’s secondary structure is destroyed.