| In this study, experiment material was after supercritical CO2 fluid extraction corn germ meal. The separation of corn germ protein was by the traditional methods, and studied the characterization analysis of corn germ protein, including Sephadex G-100 chromatography separation, SDS-PAGE electrophoresis, amino acid analysis and circular dichroism spectrum analysis; In addition, the corn germ protein was used as the raw material for preparing corn germ peptide by enzymatic hydrolysis. The anti-fatigue model of animal was established, studied the anti-fatigue activity of corn germ peptides. The main results of investigation were as follows:(1) With corn germ protein extraction rate as evaluation indexes, using the single factor experiment and orthogonal test of corn germ protein extraction process was optimized. Results showed that p H value,solvent/solid ratio,extraction temperature and extraction time influenced the protein extracting rate significantly. And the optimum extraction condition was: p H value 9.5,solvent/solid ratio 10:1(m L/g),temperature 55℃ and extraction time 120 min. Under this condition,extraction rate of corn germ protein was(38.79±0.63)%.(2) The amino acid analysis of corn germ protein results showed that: The corn germ protein contains 17 kinds of amino acids, including seven kinds of essential amino acids for the human body. In these amino acids, the most abundant was glutamate, arginine and leucine was following. Branched chain amino acids(BCAA) was accounted for 17.68% of total amino acids and some academic research thinked BCAA can be reduce fatigue in a certain extent. After Sephadex G-100 chromatography separation, we can collected two protein components named peak-1, peak-2. Run SDS-PAGE electrophoresis with corn germ protein and collection of peak-1, peak-2, results map showed six contents, the molecular weight of each protein component was 69.857, 48.634, 34.588, 30.449, 25.594, 20.036 k Da; Peak-1 not appeared, peak-2 showed as a single strip, it showed that the molecular weight of peak-2 was 30.449 k Da. On-line analysis for protein circular dichroism spectrum results showed that the secondary structure of corn germ protein was β-fold type. The content of β-fold and α-helix was 51.9% and 18.3%.(3) This was chosen several proteases were used in its optimum conditions for enzymatic hydrolysis of corn germ protein by measuring the degree of hydrolysis of hydrolyzate comparison of several enzyme hydrolysis effects. Using the degree of hydrolysis(DH) of corn germ protein enzymolysis liquid as the evaluation index. And using the single factor experiment and orthogonal test of optimize the corn germ protein enzyme hydrolysis process, and the optimum enzyme hydrolysis condition was: enzyme hydrolysis p H value 9, the substrate concentration 8%, and E/S ratio of 5%, enzymolysis temperature 55℃. Under this condition the DH of enzymolysis was(24.1±0.55) %. Enzymatic liquid at room temperature ultrafiltration to obtain four different molecular weight component, named CGP-I(>10KDa), CGP-II(5-10KDa), CGP-II(1-5KDa), CGP-IV(<1KDa), the components were freeze-dried and measured antioxidant activity, measured CGP-IV was the highest rate, and the ability to clear the order of components is: CGP-IV> CGP-III> CGP-II> CGP-I.(4) Establish animal anti-fatigue model, with corn germ protein as the positive control group, corn germ peptide as the experimental group. After a month’s diet, swimming time,the contents of BUN, liver glycogen and blood lactic acid were observed. The result indicated that the swimming time of mice with corn germ peptide high dose group significantly compared with blank control group, the test result is positive; the liver glycogen contents of experimental groups were higher than blank control group after swimming, the test result is positive; the result of BUN content is different with liver glycogen, the test results were negative; blood lactic acid curve area of experimental groups were significantly lower than blank control group and the corn germ protein group, the test result is positive.According to the 《Health food inspection and evaluation of technical specifications》(2003) issued by the national ministry of health, the result determination of alleviate physical fatigue function test: swimming test result is positive, and two of the blood lactic acid, BUN and liver glycogen test results were positive, we can determined this test sample with the function of alleviate physical fatigue. In a word, the corn germ peptide(<1000Da) with alleviate physical fatigue function. |
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