| Silkworm (Bombyx mori) pupae are considered main msilk industry waste. Because of shorting at the technical support, silkworm pupae mostly consumed as animal feed and fertilizer. The protein of silkworm pupae (PSP) alone consists of about40%-60%of the total dry pupae weight, containing18known amino acids and is considered as a good nutritional source of protein. In this thesis, in accordance with the deep processing application and mechanism of protein hydrolysis about PSP, extraction condition was optimized by one-factor for PSP from silkworm pupae. Then the hydrolyzates of PSP catalyzed by neutrase and alacase exerted the higher antioxidant and angiotensin I-converting enzyme (ACE) inhibitory activity. The hydrolyzing conditions were optimized by response surface methodology (RSM). At last, the macro dynamic equation and lumped dynamic equation are deduced about neutrase and alacase reaction system, the main results are as follow.Amino acid composition and of PSP was analyzed with HPLC. Experiment results show that8essential amino acids (EAA) and hydrophobic amino acid was48.03%and46.69%of total amino, respectively. It is worthwhile to do the further research on antioxidative activity and angiotemin-I-converting enzyme (ACE) inhibitory activity because of its enormous potentials. Molecular weight distribution was obtained by HPSEC. The results indicated that the PSP included a variety of small proteins and peptide with the molecular weight range from500to100000Da.The hydrolyzates of silkworm pupae protein were catalyzed by neutrase, alcalase, bromelain, trypsin and papain. The ACE inhibitory activity and antioxidative activity were identified by HPLC and spectrophotometry. The hydrolyzates catalyzed by neutrase and alcalase exerted the higher antioxidative activity and inhibitory activity. The hydrolyzing conditions were optimized by one-factor and Box-Behnken design methods, and response surface methodology (RSM). Statistical analyses showed that regression of the second-order model equation is suitable to describe degree of hydrolysis, and the average regressive error was less than5%. The neutrase optimum technology conditions were:hydrolysis temperture at49℃, pH7.00, enzyme concentration0.38%(w/w) and reaction time180min. The alcalase optimum technology conditions were:hydrolysis temperature at51℃, pH9.05, enzyme concentration0.70%(w/w) and reaction time180min. The reliability of the model was verified by RSM, and the average regression error was less than5%.Taking into account the reaction mechanism, a kinetic model was established to characterize the enzymatic hydrolysis curves about neutrase and alcalase. The reaction constants in neutrase were determined as michaelis constant4.02g·L-1, product formation rate constant130.9min-1, substrate inhibition constant110.4g·L-1and enzyme inactivation rate constant24.6min. The reaction constants in alcalase were determined as michaelis constant3.5g·L-1, product formation rate constant251.7min-1,substrate inhibition constant94.8g·L-1and enzyme inactivation rate constant41.4min-1. It proved that the substrate inhibition existed in neutrase and alcalase system, and its optimum substrate concentration were determined as32.7g·L-1and23.4g·L-1. The experimental data were substituted this kinetic model, the regressive results agree well with the experimental data, i. e. the average relative error was only3.80%and4.60%. The kinetics of hydrolysis and solubilization for all experiments could be represented, and the empirical model was defined and the relationship between hydrolysis and solubilization was power found for all experimental data. Taking into account the reaction mechanism, this paper applied lumping kinetics to the PSP-neutrase and PSP-alcalase systems. The principle of lumping kinetic was built base on solubility of PSP and molecular weight distribution of hydrolysates. The lumped components of the complex reaction system were defined and a three-lumping reaction network was proposed in two systems. Reaction of the lumps was described with intrinsic kinetic equations, which contained product inhibition; substrate inhibition, enzyme inactivation and the variation of different lumped components in different time, and system of differential equations for this reaction of lumps were established, and its reaction activation energy were determined as22.22kJ-mol-1and9.52kJ-mol-1. The reliability of the model was verified by comparing the computed values with the experimental values, and the kinetic equations could be used to model the hydrolysis process. The functional test of different lumped components showed that it is holds better ACE inhibitory activity and antioxidative activity in lump C. By means of the combination of macro dynamic equation and lumped dynamic equation, system of equations in two systems were deduced about degree of hydrolysis, different lumped components and reaction time. These equations offer the theory support for the technological process of bioactive peptides... |
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