Study On The Preparation Of Ace Inhibitors From Cordyceps Militaris Medium Residues Based On Ultrasonic Pretreatment And In-situ Monitoring Of The Process

Cordycepsmilitaris,compared with Cordycepssinensis,contains a variety of bioactive substances such as cordycepin.The culturing scale of Cordyceps has gradually been expanded in recent years.Cordycepsmilitaris medium residue?CMMR?,by-products of the sporocarp after the harvest of Cordyceps,is lack of effectively high-value utilization method.Additionally,CMMR is rich in protein and has the potential to develop angiotensin I-converting enzyme?ACE?inhibitors containing peptides.In this study,based on the determination of the basic components and ACE inhibition activity of CMMR,ultrasounic-assisted enzymolysis was applied for preparation of the ACE inhibitors from CMMR.Near-infrared spectroscopy technique was also used to monitor the change of the ACE inhibition activity of CMMR hydrolysate during the enzymatic hydrolysis.This laid a foundation for developing the preparation method of bioactive substances from CMMR high-valuably,high-efficiently and intelligently.The main work and conclusions were presented as follows:1.Study on the technical pathway of preparation of ACE inhibitors using CMMR.The basic components such as the amino acid composition of CMMR were determined to explore the feasibility of preparing ACE inhibitors from CMMR.According to the ACE inhibition activity,the optimal way of preparation of ACE inhibitors was screened.Results were presented as follows:CMMR contained 2.97%of non-starch polysaccharides and 15.83%of proteins?including macromolecular proteins and small molecule polypeptides?,in which hydrophobic amino acids and tyrosines accounted for 31.20%of total proteins.Additionally,both the direct extraction hydrolysates and the enzymatic hydrolysates of CMMR had the ACE inhibition activity.Also,the optimal way for preparing ACE inhibitor was showed as follows:the ACE inhibitors with high activity were obtained through ultrasonic pretreatment,followed by the enzymatic hydrolysis with amylase and subsequently with protease.2.Study on preparation of ACE inhibitors from CMMR using ultrasonic pretreatment.The effects of ultrasonic mode,ultrasonic power density and pretreatment time of multi-frequency S-type ultrasonic equipment,concentration,pH,initial temperature of the treatment liquid and pretreatment time on the ACE inhibitory activity and dry matter content of the enzymatic hydrolysates were studied.The optimal ultrasonic mode was selected as 20/28 kHz?sequential dual-frequency countercurrent ultrasound?.The optimal ultrasonic pretreatment parameters were as follows:pretreatment time of 30 min,power density of 128 W/L,material concentration of 40g/L,pH of 10 and initial temperature of 50°C.Under the optimal pretreatment conditions,the ACE inhibition rate was used to optimize the enzymatic hydrolysis parameters of amylase and protease.The optimal enzymatic hydrolysis conditions were determined as:enzymatic hydrolysis of amylase for 15 min at pH of 7.5 and then of protease for the next 40 min at pH of 9.5.Under the optimal ultrasonic pretreatment and enzymatic hydrolysis conditions,the IC₅₀ value,yield,total polysaccharide,protein and peptide contents for inhibiting ACE activity of the enzymatic hydrolysate was 4.62 mg/mL,64.62%,64.06%,21.13%,and 19.73%,respectively.3.Study on in-situ and real-time monitoring technology for the preparation of ACE inhibitors during amylase and protease enzymatic processes of CMMR.By combination of near-infrared spectroscopy and chemometrics,an on-line monitoring system was established.The near-infrared spectroscopy equipment with an immersion micro-optical fiber was used to collect the spectra of enzymatic hydrolysis processes using the amylase and protease.The spectra pretreated by standard normal variate transformation?SNV?were combined with three different variable screening methods?Synergy interval?Si?,Random Frog?RF?,Competitive Adaptive Reweighted Sampling?CARS?coupled with Partial Least Squares?PLS??to establish quantitative prediction models for ACE inhibition rate during enzymatic hydrolysis.Results showed that the predictive effect and stability of the CARS-PLS model was best.During the enzymatic hydrolysis process using amylase,the Rp and RMSEP of ACE inhibition rate in the prediction modelwere 0.9693 and 0.01,respectively.During the enzymatic hydrolysis process using protease,Rp and RMSEP were 0.9454 and 0.03,respectively.In summary,it is feasible to establish an in-situ and real-time monitoring system for preparation of ACE inhibitors during the enzymatic processes using amylase and protease of CMMR.