| In order to solve the problems of long hydrolysis time and high production costs,we proposes a new design concept of?preparation of macromolecules and absorption of small molecules?instead of the classical criteria of?preparation of small molecules and absorption of small molecules?for the production of functional peptide at home and abroad.Basing on this concept,an overall design scheme for the preparation of macromolecular rice peptides by?in vitro enzymatic digestion and in vivo digestion?was constructed.Under the guidance of this program,a series of key innovative technologies such as ultrasound-assist enzymatic extraction of rice protein,screening of hydrolysis conditions and establishment of system of macromolecular rice peptide,ultrasonic enhancement of enzymatic hydrolysis reaction of rice protein,and in situ real-time monitoring of enzymatic hydrolysis reaction processing for the preparation of macromolecular rice peptides were created.Finally,the goal of preparation of macromolecular rice hypotensive peptides was finally achieved.In this paper,a set of new technical methods for the preparation of macromolecular peptides were proposed in terms of theory and method.The following main conclusions were drawn in this paper:?1?Ultrasound-assisted extraction of rice protein:To increase the extraction rate of rice protein,ultrasound-assisted extraction of rice protein using?-amylase under low alkaline condition was established.Ultrasound-assisted starch degradation method increased the extraction rate of rice protein significantly with the extraction rate of rice protein of 89.58%under 20/35 kHz sequential dual-frequency ultrasound.The results of protein and starch content,fourier transform infrared spectroscopy,amino acid analysis and SDS-PAGE electrophoresis showed that the ultrasound-assisted starch degradation method promoted the protein extraction rate by destroying the starch-protein agglomerates structure.The solubilization,emulsification and foaming ability of rice protein extracted by ultrasound-assisted starch degradation method were increased significantly.These changes in properties was benefit for the proteolytic effect of subsequent experiments.?2?The preparation system of macromolecular rice hypotensive peptide:To take full advantage of gastrointestinal digestive function and prevent excessive enzymatic hydrolysis of peptides,the?enzymolysis in vitro and digestion in vivo?method was used to achieve the maximum hypotensive activity of the rice peptide after digestion in vivo.It was found that considering the gastrointestinal simulated digestion,the ACE inhibitory activity?detection index of hypotensive effect in vitro?of the neutrase-hydrolysate of rice protein achieved the highest with the degree of hydrolysis of 8%.Different enzymatic hydrolysis parameters did not significantly alter the ACE inhibitory activity of the proteolysis product and its gastrointestinal simulated digestion products?p>0.05?,but they had significant effect?p<0.05?on the enzymolysis time at the degree of hydrolysis of 8%of rice protein.Therefore,basing on the enzymolysis time as response value,a four-factor three-level response surface test was designed at the hydrolysis degree of rice protein reaching 8%.The shortest enzymolysis time was predicted by software with the material concentration of 46.24g/L,the enzyme amount of 7.94%,pH of 7.11 and enzymolysis temperature of53.46°C with the enzymolysis time was 37.10 min.Under this condition,the enzymolysis time was verified to be 36.82 min,indicating that the regression model for software analysis had good-fit results.Under these conditions,the ACE inhibitory activity of hydrolysate after gastrointestinal simulated digestion was 61.74%.The peptide content,ACE inhibitory activity and relative molecular weight distribution of macromolecular peptides?degree of hydrolysis 8%?and their gastrointestinal simulated digestion products were comparable to that of small molecule peptides?degree of hydrolysis 16%?.The ACE inhibitory activity and peptide content of macromolecular peptides after gastrointestinal simulated digestion was significantly higher than that of small molecule peptides.The content of peptides can be absorbed directly after digestion?molecular weight between 200-500 Da?increased significantly,and the amino acid content decreased.All the results above showed that the preparation system of macromolecular rice peptides shortened the enzymolysis time and increased the ACE inhibitory activity of the hydrolysate after gastrointestinal simulated digestion.This showed the feasibility of establishing a system basing on gastrointestinal simulated digestion of macromolecular rice peptides.At the same time,rice protein hydrolysate of the molecular weight more than 1000 Da over 90%or the molecular weight more than 3000 Da over 80%is defined as macromolecular rice peptide with ACE inhibitory activity.?3?Enhancment of proteolytic effect by multi-mode ultrasound for the preparation of macromolecular rice hypotensive peptides:To overcome the current problems of the use of relatively backward and single working mode of ultrasonic equipments in ultrasound-assisted enzymatic reactions,five new ultrasonic devices developed by our research group were used to improve the proteolysis effect of rice protein for the preparation of macromolecular rice peptides.The results showed that all ultrasonic pretreatments had significant effect on ACE inhibitory activity of rice hydrolysates and their gastrointestinal simulated digestion products,of which the20/40 kHz dual-frequency S-mode ultrasound increased the most.Pretreatment of rice protein with different ultrasonic power density and time did not significantly reduce the enzymolysis time as the degree of hydrolysis of rice protein reached 8%,but they significantly increased the ACE inhibitory activity of proteolysis product and its gastrointestinal simulated digestion product.Basing on the structural analysis of rice protein,ultrasound pretreatment decreased the content of?-helix and?-turn of rice protein and increased the content of?-sheet and random coil.It also increased the hydrophobicity of protein and decreased the content of disulfide bond.All of these affected the enzymatic hydrolysis effect of rice protein,thereby improving the ACE inhibitory activity of the hydrolyzed product after gastrointestinal simulated digestion.According to the definition of macromolecular rice peptide with ACE inhibitory activity,the hydrolysate of rice protein which was pretreated by ultrasound was still macromolecular rice peptide with ACE inhibitory activity.?4?In-situ real-time monitoring of the preparation process of macromolecular rice hypotensive peptides:The key to intellectualization of the peptide preparation process could be achieved lies in the real-time acquisition of target process parameters.For this purpose,probe-type miniature raman spectroscopy was used to construct a mathematical model that could accurately predict the target product.According to the establishment of the Si-PLS model,the optimal joint range of the degree of hydrolysis of rice protein were 418.427-600.406,1276.204-1426.07,1854.47-1984.029 and2464.368-2571.622 cm-1.The best combination interval of ACE inhibitory activity of hydrolysate after gastrointestinal simulated digestion were 214.423-398.846,742.877-903.002,1063.515-1212.354 and 1503.061-1636.945 cm-1.Under the optimal sub-interval conditions for screening,a quantitative Si-PLS model was established for detection of the hydrolysis degree of rice protein and the ACE inhibitory rate of hydrolysate after gastrointestinal digestion.The calibration model for the degree of rice protein hydrolysis was 0.9048 with RMSECV of 1.09%and the predictive model r value was 0.8389 with RMSEP of 1.45%.The Rc of the ACE inhibitory rate of the enzymatic hydrolyzate after gastrointestinal digestion was0.9733 with RMSECV of 3.42%,the Rp of the prediction model was 0.9381 with the RMSEP of 5.57%.Through the establishment of mathematical model between Raman spectroscopy and key parameters of the enzymatic hydrolysis process,the in situ real-time monitoring of the preparation of macromolecular rice peptide was realized.?5?Safety of macromolecular peptides and their effects on digestion and absorption and hypotensive effect:To evaluate the safety and hypotensive effect of macromolecular rice peptides,acute oral toxicity test in mice,digestion and absorption test in SD rats and gastric pressure test in primary hypertensive rats were performed.According to the results of acute toxicity tests,the maximum oral toxicity test for macromolecular peptides was more than 15000 mg/kg,indicating that the macromolecular peptides were non-toxic.Compared with small molecular peptides,intragastric macromolecular rice peptides significantly increased the peptide content amplification in blood of SD rats.Through the ACE activity of the organ and the determination of serum biochemical indicators results,macromolecular rice peptides inhibited ACE inhibitory activity in the lungs and aorta,which also influenced nitric oxide,nitric oxide synthase,angiotensin II and renin in signaling factors to reduce blood pressure.Unlike captopril,peptides have no effect on the activity of creatine kinase isoenzymes and lactate dehydrogenase isoenzymes. |
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