Study On Enzymatic Preparation Of Cholesterol-lowering Macromolecular Peptides By Ultrasound Coupled With Sodium Bisulfite Pretreatment And Its On-line Monitoring In Process

The traditional method to prepare cholesterol-lowering peptides is to directly convert proteins into small molecular peptides with high cholesterol-lowering activity by enzymatic hydrolysis in vitro.However,this method does not consider the use of normal human gastrointestinal digestive function,which not only increases the production cost,but also greatly reduces the activity of small molecular peptides by further gastrointestinal digestion.In order to overcome the above shortcomings,this study constructed a technical scheme for the preparation of macromolecular cholesterol-lowering peptides by in vitro restriction enzymolysis based on in vivo gastrointestinal digestion,that is,using restriction enzymolysis in vitro,soybean protein was transformed into macromolecular peptides with high cholesterol-lowering activity after simulated gastrointestinal digestion.Using animal experiments,the cholesterol-lowering activity of macromolecular peptides degraded into small peptides in vivo was detected,and the rationality of the preparation scheme of "in vitro enzymatic hydrolysis + in vivo digestion" macromolecular peptides was evaluated.In order to improve the efficiency of enzymatic hydrolysis and the intelligent level of the production process,the synergistic effect of ultrasound coupled with sodium bisulfite pretreatment on enzymatic hydrolysis and its mechanism were studied,and the near infrared spectroscopy in situ real-time online monitoring model of enzymatic hydrolysis process was established.The main research work and conclusions are as follows:(1)Study on the basic conditions of enzymatic preparation of soybean cholesterol-lowering macromolecular peptides: taking the cholesterol-lowering activity and peptide content of soybean cholesterol-lowering macromolecular peptides after simulated gastrointestinal digestion as the evaluation index,the protease that can maximize the conversion of soybean protein into cholesterol-lowering macromolecular peptides was screened from six common proteases,and the optimum degree of hydrolysis for cholesterol-lowering macromolecular peptides was determined.The results showed that when soybean protein was hydrolyzed to 9%degree of hydrolysis by Alcalase,the cholesterol-lowering activity of the hydrolysate was the highest after simulated gastrointestinal digestion.Compared with the traditional small molecular peptides,the degree of hydrolysis decreased by 57.14%and the activity increased by 2.36 times,which means that the production cost is greatly reduced and the product quality is significantly improved.(2)Study on the promoting effect of ultrasound coupled with sodium bisulfite pretreatment on soybean enzymatic hydrolysis: the technology of ultrasound coupled with sodium bisulfite pretreatment on soybean protein was used to improve the cholesterol-lowering activity of enzymatic hydrolysate after gastrointestinal digestion.Under the optimum conditions,it was found that when the concentration of sodium bisulfite was 1.5 g / L,the cholesterol-lowering activity of the hydrolysate was the highest;Based on the optimal concentration of sodium bisulfite,it was found that the effect of 28 k Hz single frequency ultrasound made the enzymatic hydrolysate with the highest cholesterol-lowering activity after gastrointestinal digestion;Based on the optimal ultrasonic working mode,the optimal ultrasonic treatment conditions are as follows: ultrasonic power density of 100 W / L,ultrasonic time of 30 min.Under the above conditions,the cholesterol-lowering activity of the hydrolysate after gastrointestinal digestion was as high as 55.18%,which was 81.51% higher than that without ultrasound coupled with sodium bisulfite pretreatment and 28.59% higher than that with simple sodium bisulfite pretreatment.It was also found that ultrasound coupled with sodium bisulfite pretreatment had no significant effect on the peptide content of soy protein hydrolysate after gastrointestinal digestion.(3)Molecular mechanism of ultrasound coupled with sodium bisulfite pretreatment improving cholesterol-lowering activity of soybean protein hydrolysates:in order to explore the molecular mechanism of ultrasound coupled with sodium bisulfite pretreatment,the effects of ultrasound coupled with sodium bisulfite pretreatment on secondary structure,surface hydrophobicity,sulfhydryl group,disulfide bond,microstructure and molecular weight of soybean protein were studied.The results showed that ultrasound coupled with sodium bisulfite pretreatment increased the content of ?-sheet and random coil,the surface hydrophobicity and sulfhydryl content of soybean protein,and decreased the content of ?-helix,?-turn and disulfide bond.Pearson correlated coefficient analysis showed that there was a significant negative correlation between cholesterol-lowering activity and disulfide bond content,and a significant positive correlation between cholesterol-lowering activity and surface hydrophobicity.Therefore,it is suggested that the increase of surface hydrophobicity and the decrease of disulfide bond content of soybean protein induced by ultrasound coupled with sodium bisulfite pretreatment is the molecular mechanism of improving the cholesterol-lowering activity of macromolecular peptides.This study once again proved that ultrasound can promote the enhancement of biological activity related to the increase of surface hydrophobicity even if it cannot increase the ratio of protein to peptides.(4)Establishment of near infrared spectroscopy in situ real-time monitoring model for soybean proteolysis process: in order to realize the intelligent control of the preparation process of soybean cholesterol-lowering macromolecular peptides,near infrared spectroscopy technology was used to build a model of spectral information,degree of hydrolysis in the enzymatic hydrolysis process and cholesterol-lowering activity of enzymatic hydrolysate after gastrointestinal digestion.From the standard normal transformation,polynomial convolution smoothing,first derivative and second derivative,the optimal preprocessing method is polynomial convolution smoothing.From the three modeling methods of partial least squares(PLS),interval partial least squares(i PLS)and joint interval partial least squares(Si-PLS),the best one was selected as Si-PLS.In the optimum spectral range,R value of the calibration model for hydrolysis degree was 0.9340,the error was 0.0143,and R value of the prediction model was 0.9522,the error was 0.0126.In the best spectral range,R value of the calibration model for cholesterol-lowering activity of the hydrolysate after gastrointestinal digestion was 0.9522,the error value was 0.0189,R value of the prediction model was 0.9661,the error value was 0.0169.The results show that pretreatment method of the polynomial convolution smoothing spectral and modeling method of Si-PLS can be used to establish the in situ real-time monitoring model of soybean proteolysis process and support the development of intelligent control system.(5)Animal experimental study on lowering cholesterol of soybean cholesterollowering macromolecular peptides: in order to explore the cholesterol-lowering effect of soybean cholesterol-lowering macromolecular peptides in mice and its mechanism of action in vivo,the preventive animal experiment was carried out.The results showed that the soybean macromolecular peptides could significantly reduce the content of total cholesterol(T-CHO),triglyceride(TG)and low-density lipoprotein cholesterol(LDL-C)cholesterol in serum and liver,and increase the content of high-density lipoprotein cholesterol(HDL-C)in serum.Compared with the traditional soybean small molecule peptide high-dose group,the content of T-CHO,TG and LDL-C in the high-dose soybean macromolecular peptides group decreased by16.13%,9.70% and 18.57%,respectively.The high dose group of soybean macromolecular peptides was better than the traditional soybean small molecule peptide high-dose group on the improvement of liver cells.The results showed that the mechanism of cholesterol-lowering activity of soybean peptides was to promote cholesterol conversion into bile acid and apolipoprotein to increase its catabolism,and reduce its absorption and metabolism by inhibiting the formation of esterifying cholesterol.The mechanism of simvastatin in lowering cholesterol was to inhibit the synthesis of cholesterol by regulating the activity of HMG-Co AR,and to promote cholesterol conversion into bile acid and apolipoprotein to increase its catabolism.The difference of cholesterol-lowering mechanism between simvastatin and soybean peptides in vivo is that simvastatin can inhibit cholesterol synthesis by regulating activity of HMG-Co AR,while soybean peptides can reduce its absorption and metabolism by inhibiting the production of esterified cholesterol.