| In food industry, most pigskin gelatin was used as stabilizer and emulsifier to improve food’s original form, flavor, etc. Because of its single uses, lower added value limitation in food industry, the antioxidant activity of the petides were evaluated through vitro chemical model, ultrafiltration was applied to the separation of petides. Moreover, the antifatigue function was also studied combined with animal model so as to provide the theoretical basis for the development of antifatigue foods. The main research contents and conclusions were as follows:(1) The hydrolysis process of pigskin gelatin were studied: the clearance rate of superoxide anion radical(O2-?), degree of hydrolysis(DH) were selected as main and secondary indexes. The effect of substrate concentration, reaction temperature, time, p H, the amount of enzyme on hydrolysis were investigated respectively, and the optimum hydrolysis combination of alkaline protease, ZHM protease and PAM protease were obtained by single factor and orthogonal tests.The optimum hydrolysis conditions of alkaline protease were determined: the concentration of substrate was 9%(w/v), p H was 8.0, hydrolysis time was 4h, reaction temperature was 58℃, ratio of alkaline protease was 6%(w/v), under these conditions the hydrolysis degree was 16.49%, while the scavenging capacities of O2-? could reach 44.53%. The optimum hydrolysis conditions of ZHM protease were determined: concentration of gelatin was 9%, p H was 6.5, hydrolysis time was 4h, reaction temperature was 40℃, ratio of ZHM protease was 4%, under these conditions the scavenging capacities of O2-? could reach 59.02%, while the hydrolysis degree of gelatin was 9.18%. The optimum hydrolysis conditions of PAM protease were determined: the concentration of gelatin was 7%, p H was 6.0, hydrolysis time was 4h, reaction temperature was 65℃, ratio of alkaline protease was 4%, under these conditions the hydrolysis degree of gelatin was 9.74%, while the scavenging capacities of O2-? could reach 58.17%.Through comprehensive comparison, the results showed: the ZHM protease hydrolyzate presented the strongest scavenging capacities of O2-?, followed by PAM protease, the difference was not significant(p>0.05), the clearance rate of alkaline protease hydrolysates for O2-? was relatively lower, the difference was significant(p<0.05).(2) Four different ultrafiltration microporous membrane having a range of molecular weight cut-offs of 30, 10, 1 and 5k Da were used to separate the enzymatic hydrolysis. Measure the scavenging capacities for O2-? of each molecular weight range for each peptide, results showed that when the concentration was 10mg/m L, the clearance rates for O2-? of less than 5k Da molecular weight peptides could reach 38.89% and 40.21%, significantly higher than the enzyme dope and other molecular weight peptides(p<0.05).The antioxidant properties for different concentrations of hydrolysates were determined in vitro. The results showed that at the same concentration, two protease enzyme mixture of DPPH? scavenging activity was significantly higher than the molecular weight of less than 5k Da hydrolysates(p<0.01), when the concentration reached 60 mg/m L, the clearance rate for DPPH? of PAM protease hydrolysis up to 94.38%, increased by 13.21% when compared with ZHM protease hydrolyzate. When the concentration reached 48 mg/m L, four hydrolysates of ?OH scavenging ability were higher than 94%, a molecular weight of less than 5k Da PAM protease hydrolysates of ?OH scavenging ability was significantly higher than the enzyme mixture(p<0.05), at the same concentration, PAM protease hydrolysates of O2-? scavenging activity was higher than ZHM protease hydrolyzate(p<0.05). Overall, four different pigskin gelatin hydrolysates exhibited antioxidant effect.(3) The anti-fatigue effect for four hydrolysates were studied combined with animal model. The screened hydrolysates which has strong antioxidant capacity after isolation and purification, water bath concentrate, frozen, vacuum freeze-dried into powder. After the last administration, the mice were measured swimming time, physical and chemical indicators of blood urea nitrogen(BUN), blood lactate, muscle lactate, glycogen, muscle glycogen. Results showed that amylase conditions were as follows: four kinds of hydrolysates can significantly prolong mice swimming time, the mice swimming time could reach 3668.5±308.12(s) for hydrolyzates of PAM protease for molecular weight less than 5k Da group. Compared with the control group, the content of BUN and muscle lactate decreased by 16.74% and 18.52%, the difference was significant(p<0.05), while the content of liver glycogen increased by 29.57% and 32.19%, which proved pigskin gelatin hydrolyzates having function of alleviating physical fatigue. |
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