| Due to the high biological value, hypoallergenic, amino acid balance and other certain important advantages of rice protein (RP), an increasing number of researchers have studied the RP largely to develop it as functional food. However, disadvantages such as insolubility limit its application in food industry. Since the RP was rich in alkali soluble proteins, the purpose of this paper was to enhance the efficiency of enzymatic hydrolysis using ultrasound and ultrasound synergy alkali as pretreatments to modify the structure of substrate. Meanwhile, processes of the pretreatment and enzymatic hydrolysis were also simulated via models in this study. The main conclusions are as follows.(1) Taking the amount of protein eluate and degree of hydrolysis (DH) of RP as indexes, the optimum performance of ultrasound pretreatment was obtained. Based on this, the pH of RP solution was adjusted to 8.5 for ultrasonic synergy alkali pretreatment, which was the optimum pH value of the alkaline protease used in this experiment. In comparison with the traditional methods of enzymolysis, using ultrasound pretreatment improved the DH and the angiotensin-I converting enzyme(ACE) inhibitory activity by 8.59% and 5.68%, respectively; while the ultrasound synergy alkali pretreatment enzymolysis improved the DH by 13.02% and the ACE inhibitory activity by 52.04%, respectively. Furthermore, the molecular weight distribution (MWD) ranges of hydrolysates resulted from the ultrasound synergy alkali pretreatment were narrowed when compared to that of the control. This kind of MWD range was mainly distributed in less than 1000 Da.(2) After pretreated by ultrasound and ultrasound synergy alkali, the surface hydrophobicity, disulfide bond content, fluorescent spectra and ultraviolet-visible(UV) spectroscopy of RP were all modified. The results indicated that ultrasound pretreatment could unfold the protein molecules and the ultrasound synergy alkali pretreatment could destroy parts of the compact protein structure. The circular dichroism (CD) and Fourier transform infrared (FT-IR) spectra analysis showed that different ultrasound pretreatments altered the relative content of secondary structure of RP. The results of scanning electron microscope (SEM) indicated that ultrasound and ultrasound synergy alkali pretreatments could destroy the structure and reduce the particle size of the RP, which led to an increase of the contact probability between enzyme and protein. The results of atomic force microscope (AFM) showed that ultrasound pretreatment reduced the height and increased the surface roughness of RP while the ultrasound synergy alkali pretreatment decreased the size of RP. Moreover,the composition of amino acids (AA) pretreated via different ultrasound methods has changed significantly. As far as the ratio of hydrophobic amino acid (HAA) to total AA content was concerned, it increased compared to the unpretreated RP.(3) The dynamic equation y=[y∞-/(1+kd*t)]*[1-exp(-k*t)]+[y0/(1+kd*t)]*exp(-k*t)was used to simulate the process of ultrasound and ultrasound synergy alkali pretreatments on RP. The aggregation coefficient was zero when the ultrasound synergy alkali method was used to pretreat RP. Rate constant k of the ultrasound synergy alkali pretreatment on RP increased with the pH value of solution from 7.0 to 8.5. Using ultrasound pretreatment, the kinetic equation was established and the result indicated that the rate constant k and aggregation coefficient kd were related to the temperature, time, as well as the power density of ultrasound. Then the kd rose with the increase of temperature and ultrasound power density. Furthermore, the dynamic equation could be better to fit the experimental data at different temperatures and ultrasound power density.(4) According to the results of the hydrolysis curve at different ultrasound parameters, the diffusion - reaction kinetic equation was constructed to describe the ultrasound- assisted enzymatic hydrolysis. The results showed that RP hydrolyzed by alkaline protease fite for the no substrate inhibition when the substrate concentration was less than 10 g/L; but the excessive amount of RP (10～40 g/L) might cause the enzyme activity inhibition. The most suitable concentration of substrate in traditional enzymatic hydrolysis calculated using model was 10.05 g/L, which was very close to the experimental result of 10 g/L. So the kinetic model of diffusion-reaction was more fitful to simulate the enzymatic hydrolysis. Additionally, the optimum substrate concentration of ultrasound-assisted enzymatic hydrolysis calculated by the model was 13.35 g/L, which was higher than result obtained from the traditional method.Therefore, the ultrasonic pretreatment was able to delay the derepressed concentration of substrate to some extent.(5) The pK values of AA side chain groups at different pH values were analyzed and the values were calculated based on the model. The results showed that the catalytic activity groups of alkaline protease might contain serine amino acid. The secondary structure of alkaline protease was analyzed under different pH values. The result indicated that the total content of alpha helix and random curl increased with the growth of the pH value, while the content decreased when the pH reached 8.5.Therefore, the maximum reaction rate was obtained at pH value of 8.5. Based on the analysis results of RP enzymatic hydrolysis at different pH values, the kinetic equation was built. The calculated values by kinetics anodel showed a good fit with experiment data. In addition, the optimum pH value calculated by model was close to the experimental result.(6) The secondary structure analysis results of alkaline protease at different reaction temperatures showed that the amount of random curl and beta turn rose with the increase of the temperature, while the amount of alpha helix and beta sheet decreased, indicating that the increased temperature induced the initially ordered structure of alkaline protease turning to the disordered statement. Based on the analysis results of enzymatic hydrolysis of RP at different temperature, the reaction rate equation was established and the thermal inactivation model of alkaline protease was built. Then, the apparent inactivation rate constant Ai and half-life T1/2 of alkaline protease at different temperatures were obtained from the nonlinear regression. The value of A; was proved using the model that well-agreed with the experimental results(the average relative error was 4.23%). |
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