Preparation Technology Of ACEI Peptides From Corn Gluten Meal By The Pulsed Ultrasonic-assisted Enzymolysis

Hypertension is one of the most common arterial diseases in our life, which fazes many people, so there are more and more studies on anti-hypertension medicine. Synthetic medicine such as Captopril has good effect in lowing blood pressure, but has some side effects such as cough, first-dose blood pressure response, tetter, et al. Therefore, the preparation of peptides, angiotensin-converting enzyme inhibitory （ACEI）, derived from food proteins, has attracted more and more researchers’ attention.In order to obtain safer ACEI peptide with good effect of anti-hypertension, corn gluten meal, a kind of corn starch byproduct, was used. Protein was ultrasound-assisted enzymatic hydrolyzed, hydrolysate was purified by ultra filtration and the effect of anti-hypertension was studied in vivo.The results as follows:A Micro Assay （MA） was developed for determination of angiotensin I-converting enzyme （ACE） activity in the presence of ACE inhibitors, using FA-Phe-Gly-Gly （FAPGG） as the ACE-specific substrate, 96-well plate and Microplate Photometer were instead of Colorimetric Cell and UV-Spectrometer, respectively. This method proposed here was shown to be direct, sensitive, accurate, reproducible, and less expensive, in comparison to the HPLC method. Briefly, the Micro analysis will facilitate the study of precursor proteins on a large scale and the specific release of bioactive peptides.Treated with Supercritical CO₂, the extract rate of CGM oil was about 90% （weight/weight）. And the effect of enzymolysis was improved. The chemical composition of the deflated corn gluten meal was measured, with protein content 60.45%, fat content 0.43%, moisture content 8.96%, fiber 1.62%, starch 14.23% and ash content 0.21%.To produce high activity, low bitterness and low salt antihypertensive peptides, seven kind of commercial proteolytic enzyme were employed to hydrolyze CGM, respectively. According to their ACE inhibitory activities (IC₅₀), two kind of enzyme （D and E） were chosen for further study. The influence of hydrolysis time, substrate concentration, amount of enzyme （[E]/[S]）, pH and reaction temperature was studied. By single factor and the response surface methodology （RSM） tests, enzyme E was finally selected by comparing their hydrolysates’ ACEI activities with two preliminary chosen enzymes under their optimum conditions, respectively. The optimum hydrolytic conditions of protease E were: reaction time, 40min, temperature, 50℃, pH, 7.0, substrate concentration, 1.75%, and amount of enzyme （[E]/[S]）, 3%. Under this condition, IC₅₀ was 0.259 mg/mL, the ratio of CGM peptides achieved 28.75%.Then the bioreaction mechanism and kinetic behavior of protein enzymatic hydrolysis were investigated. The kinetic model and relative parameter of controllable-enzymatic hydrolysis were obtained by use of mathematic deduction and experimental analysis. The result is as follows: kinetic model formula is1and DH = （?）;inetic parameter is k₂=9.2732 /min, k_d =3.8758/min, K_m=2.7222 g/L, K_s=72.7234 g/L, E_a=17.7936 kJ/mol, E_d=17.9691 kJ/mol. The relationship between kinetic constant and reaction temperature agreed well with the Arrhenius equation. The experiment tested that the theoretical DH of hydrolysis dynamics model controlled by enzyme E fit well with the experimental data. The results indicate that the kinetic model can be used to predict the bioreaction process of protein enzymatic hydrolysis, to calculate the thermodynamic and kinetic constants, and to optimize the operation parameters for bioreactor design.Three reaction models, which included the pulsed ultrasonic pretreatment of proteinase followed by enzymolysis （PUPPE）, the pulsed ultrasonic pretreatment of corn gluten meal solution followed by enzymolysis （PUPCE） and the pulsed ultrasonic-assisted enzymolysis processes （PUAE）, were investigated. The inhibitory activity of hydrolysate to ACE was the evaluated index. Traditional enzymatic hydrolysis （TE） without ultrasonic treatment was conducted as a control test. Results showed that three models all had significant effect, and PUPCE is the best. The optimum parameters of PUPCE obtained by orthogonal experiments were: solution volume 200mL, ultrasonic frequency 20 kHz, total ultrasonic treatment time 15min, ultrasonic power 2000 W, on-time of the pulse 2s and off-time 1s, initial temperature of solution 50℃. Compared with the traditional enzymatic hydrolysis without ultrasound pretreatment, the inhibitory activity of the hydrolysate to ACE, yield and protein conversion was increased by 74.08%, 55.11% and 55.06%, respectively, and Michaelis constant （K_m） was decreased by 33.30%. These results prove that the pulsed ultrasonic-assisted enzymatic hydrolysis is a high efficiency method of preparing ACEI peptides from CGM.Finally, the Mechanism of CGM hydrolysis reaction enhanced by ultrasound was investigated. The conformational changes of CGM protein and enzyme in different ultrasound power were measured by ultraviolet absorption spectrum, ultraviolet differential spectrum and fluorescence emission spectrum. The ultraviolet absorption peak and fluorescence emission peak of the protein and enzyme hadn’t shifted sharply. The ultraviolet absorption intensity and fluorescence emission peak intensity changed with increasing ultrasound power. Analysis of ultraviolet absorption spectrum, ultraviolet differential spectrum and fluorescence emission spectrum of the enzyme indicated that the conformation of enzyme and protein molecules had not been altered after the treatment of ultrasound. The activity of enzyme or degradability of protein was inhibited or incited and its emission maximum wavelength and fluorescence intensity were obviously changed by ultrasound treatment. The results showed that CGM protein and enzyme molecule hadn’t spread sufficiently, indicating the entire conformation of CGM protein and enzyme molecular was not changed obviously, however, the microenvironment of tyrosine and tryptophan of residues in enzyme and protein undergo some changes with ultrasound treatment. There was evidence that degradable behavior of protein or the activity of enzyme occurred more rapidly than its whole conformational change treated by ultrasound. It is suggested that the active site of the enzyme or protein is situated in a limited area of the enzyme molecule which is more flexible. The improve of enzymatic degradable behavior of protein or the activity of enzyme maybe origin from change of local conformation around the active sites.Moreover, several membranes with molecular weight cut-offs （MWCFs） of l-10kDa were used to filter the hydrolysate. The membranes of MW<3 kDa was chosed taking IC₅₀ value and the productivity as integrative index. Spray-drying was chosen to dry antihypertensive peptides with inlet air temperature 170℃and outlet air temperature 75℃. Results of stabilities test indicated that the CGM hydrolysates can keep their activities in different temperature, different pH and during proteolysis by gastrointestinal enzymes in vitro.Blood pressure of spontaneously hypertensive rats （SHR） decreased obviously after fed with antihypertensive peptides of MW <3k Da, in a single oral administration in 75, 150, and 300mkg/kg·bw dosage respectively. And the results of long term oral administration experiments （21-day continuous oral experiment） showed that the antihypertensive effect of CGM hydrolysates on SHR was steady. Result testified that the CGM hydrolysates had apparent effect on depressing blood pressure on SHR, but haven’t antihypertensive effect on normotensive rats.