Study On Preparation Of High Bioavailability Corn Protein Based On Computer Simulation And Ultrasound Assisted Proteolysis

Corn gluten meal (CGM), a byproduct of the corn wet-milling process in the production of corn starch, contains 70% of protein. The CGM has limited uses in the human food industry due to its water insolubility and deficient in lysine and tryptophan. The purpose of the present paper was to improve the bioavailability of CGM by enzymolysis, and to promote the enzymolysis efficiency and increase the substrate conversion rates by adopting the dual frequencies power ultrasound to assist enzymolysis of CGM. Firstly, the bioinformatics platform for computer simulation of proteolysis was established and the best protease for producing corn peptide with higher bioavailability was screened out with the aid of this software. Secondly, the effects of degree of hydrolysis on the bioavailability of CGM and its molecular mechanism were studied. Because of the drawback in molecular structure of the CGM, the effects of dual frequencies power ultrasound (sweeping frequency and pulsed ultrasound (SFPU), sequential dual frequency ultrasound (SDFU)) pretreatment on the enzymolysis of CGM were invesitigated. Furthermore, the molecular mechanism of dual frequencies ultrasound accelerated enzymolysis of CGM was explored with the aid of UV-Vis, fluorescent spectra, FT-IR, circular dichroism (CD) spectra, scanning electron microscope (SEM) and atomic force microscope (AFM). Finally, the effects of SFPU pretreatment on the kinetics and thermodynamics of enzymolysis of CGM were investigated by using Michaelis-Menten equation, the first-order kinetic model, Arrhenius law, and transition state theory. The main creative conclusions drawn by the research are as following:(1) The platform which can meet the requirements of basic computer simulation of proteolysis and results analysis was developed. The software has the functions of simulating enzymolysis of protein by single and dual enzyme, and screening out the protease to obtain hydrolysate with a certain degree of hydrolysis (DH), and performing the statistic service of DH, molecular weight distribution (MWD) and the hydrophobicity of terminal amino acids.(2) The method of enzymolysis of CGM improved its bioavailability significantly, which was highly correlated with DH. The in vitro digestion showed that the bioavailability of the enzymatic hydrolysates was influenced by their molecular weight distribution and the composition of amino acids (AA). The content of fraction 200-500 Da almost did not change under the action of pepsin and trypsin. With increasing the DH of the hydrolysates, the free hydrophobic AA content especially the Ala, Met, Ile, Val and Phe increased, the content of Thr and Leu decreased. The content of total AA and the hydrophobic AA especially the Leu, Pro and Ala content increased when increasing the DH of the hydrolysates. The analysis of digestibility of amino acids showed that the digestibility of the hydrophobic AA Met, Ile, Pro, Ala decreased while that of Thr increased significantly, and the DH did not have significant influence on the average digestibility of the hydrophilic and hydrophobic AA.(3) Taking the DH and conversion rate (CR) of CGM as indexes, the best ultrasonic pretreatment conditions for both kind of dual frequencies power ultrasound (SDFU and SFPU) were obtained. As compared to the traditional enzymolysis, the results showed that SDFU pretreatment improved the DH by 45.3% and improved the CR by 59.3%, while the SFPU pretreatment improved the DH and the CR by 38.8% and 54.7%, respectively. Moreover, the MWD range of hydrolysates resulted from ultrasound assisted enzymolysis was narrowed when compared with that of the control The results also indicated that the ultrasonic pretreatment especially the SDFU pretreatment had a positive effect on the formation of peptides with a MW of 200-1000 Da.(4) The results of UV-Vis spectra, fluorescent emission and excitation spectra, and surface hydrophobicity indicated that ultrasound pretreatment made the protein molecules unfolded, which was beneficial for the enzymolysis. The FT-IR spectra and CD analysis showed that ultrasound pretreatment altered the relative content of secondary structure (a-helix, (3-sheet, P-turn and random coil) of zein and glutelin, meanwhile the ultrasound had stronger impact on glutelin than on zein. The results of SEM showed that ultrasound pretreatment destroyed the microstructures of protein, reduced the particles size, which resulted in an increase in the contact area between enzyme and protein, in spite of the aggregation of zein induced by SDFU was observed. Furthermore, the results of AFM indicated that ultrasound pretreatment reduced the height and the surface roughness of glutelin while this pretreatment decreased the Young’s modulus and stiffness as well as increased the adhesion of zein. Additionally, the ptesent research also found that the SFPU was superior to SDFU provided that the hydrophobicity of the protein was extremly strong, otherwise the SDFU was more suitable.(5) The results of kinetics study showed that, compared with traditional enzymolysis, the SFPU pretreatment decreased the apparent constant (KM) value by 26.1% and increased the apparent break-down rate constant (KA) value by 7.3%, indicating that the SFPU pretreatment increased the affinity between enzyme and CGM. In addition, the thermodynamic analysis showed that the values of reaction rate constant k for ultrasound pretreatment were increased by 84.8%,41.9%,28.9%, and 18.8% at the temperature of 293,303,313 and 323 K, respectively. The thermodynamics parameters Ea, △H and △S of enzymolysis were reduced by 23.0%, 24.3% and 25.3% respectively, which means that the SFPU pretreatment could make the enzymolysis proceeding more easily.