Optimization Of Enzymatic Conditions And Isolation Of Antihypertensive Peptides From Pig Scapula

This experiment adopted the pig scapular as substrate material, preparing antihyp-ertensive peptides with protease by hydrolysis and use Angiotensin-converting enzyme(ACE) inhibition as a main index. The single-factor and response surface methodology were used as approaches to optimize the enzymatic conditions; On this basis, optimi-ze the ultrasonic pretreatment conditions with the single-factor and response surface method; Sephadex G-25 column chromatography, ultrafiltration were used to separate hydrolyzed pig scapula collagen.This paper investigated the stability of pig scapular antihypertensive peptide in human digestion process and learned the effects of physic-al and chemical properties to it.The most suitable enzyme was Flavourzyme, with the optimal hydrolysis conditi-ons of enzyme/substrate ratio of 6900 U/g, hydrolysis temperature of 53℃ and hydrol-ysis time of 4.5 h. The value of ACE inhibitory activity was 65.31%. The ACE inh-ibitory Half-inhibitory concentration(IC5o)was 1.34 mg/mL.The optimal ultrasonic conditions of pig scapula peptide were ultrasonic power of 717 W, ultrasonic time of 25 min, ultrasonic temperature of 40℃ and enzymolysis ti me after ultrasonic of 3 h. The value of ACE inhibitory activity was 75.58% and IC5 0 was 0.90 mg/mL.The ultrafiltration membranes were used to get three fractions, that is, less than 5 k Da, between 5 kDa and 10 kDa, more than 10 kDa. In addition, the concentration of each fraction was determined. The results showed that peptides less than 5 KDa pos sessed the highest inhibition activity, with the IC50 at 1.12 mg/mL. The molecules les s than 5 kDa was further separated through Sephadex G-25 column chromatography. The effect of concentration, elution flow rate and sample volume on separation were i nvestigated. Finally, the optimized condition was as follows:20 mg/mL of sample co ncentration, ultrapure water as eluent with 0.5 mL/min of eluent velocity,1.2 mL of s ample volume. Gel chromatography Sephadex G-25 column separated the molecules le ss than 5 kDa into 6 peaks and the second peak had the the highest ACE inhibit-ory activity with an IC50 of 0.56mg/mL.This experiment assayed the stability of pig scapular antihypertensive peptide in human digestion process and learned the effects of physical and chemical properties to it. The simulated gastrointestinal digestion process results showed that stability of anti-hypertensive peptides less than 5kDa was better than enzymatic hydrolysate.Gastric and gastrointestinal fluid digestion did not have a negative impact on the stability of antihypertensive peptide in ultrafiltrate. Antihypertensive peptide in-20℃ had the best stability among -20℃,4℃,25℃,37℃ four storage temperatures, while it in 37℃ had the worst after 30 days storage period. An hour’s boiling did nothing to antihyp-ertensive peptides. pH of the neutral environment brought minimal impact on the stab-ility of antihypertensive peptides.Soluble starch influenced the activity most, beta cy-clodextrin and glucose, citric acid followed it.