| Hypertension is the leading risk factor for cardiovascular diseases (CVDs). Traditionally, it has been controlled and prevented using synthetic ACE inhibitory drugs aside maintaining a healthy lifestyle and modification of diet. In recent times however, there has been an increased interest among researchers to identify food-protein derived peptides which have antihypertensive properties, due to the potential side effects of the synthetic ACE inhibitory drugs. Another focus of investigators is to optimize process factors in the production of natural ACE inhibitors with modern technologies. One such technology is the use of ultrasound which has been identified by some experimenters as an efficient way of improving the ACE inhibitory activity of some bioactive peptides. In this study, wheat gluten (WG) was studied as a source of ACE inhibitory peptides.Specifically, the objectives of the study were to assess the effect of (1) enzymatic hydrolysis on the ACE inhibitory activity of wheat gluten (WG) protein isolates,(2) process factors (substrate concentration, enzyme-substrate ratio and hydrolysis time) on the degree of hydrolysis (DH) and ACE inhibitory activity of WG hydrolysate,(3) counter-flow (CF) ultrasonic pretreatment on the DH and ACE inhibitory activity of WG hydrolysates and (4) counter-flow (CF) ultrasound pretreatment on the hydrolysis kinetics of wheat gluten.Firstly, after the isolation of different proteins from the WG and enzymatic hydrolysis, the results (according to the amount of hydrophobic amino acids and tyrosine) showed that gliadin’s contribution to ACE inhibitory activity was:theoretical value42.91%, and the actual value59.79%while glutenin’s contribution was:theoretical value57.09%, and the actual value40.21%. The theoretical contribution of glutenin was33.06%higher than gliadin while the actual contribution of gliadin was48.69%higher than glutenin. Albumin and globulin had relatively low contribution to the ACE inhibitory activity.Secondly, a Box-Benhnken response surface methodology (RSM) design was used to study the effect of process conditions on the degree of hydrolysis (DH) and ACE inhibitory activity of WG hydrolyzed with alcalase, and the mean responses were fitted to a second order polynomial to obtain regression equations. The study revealed that enzyme-substrate ratio and time increased DH significantly (p<0.05) while substrate concentration increased ACE inhibitory activity significantly (p<0.05). The optimized conditions obtained were5.04%substrate concentration,5.94%enzyme-substrate ratio, and30.79min hydrolysis time. The best condition gave a point prediction of a12.74%DH and82.28%ACE inhibitory activity.Thirdly, to further improve the ACE inhibitory activity of the peptides produced, CF ultrasound pretreatment of WG samples were carried out before being hydrolyzed with alcalase. CF ultrasound pretreatment parameters of pulse on-time and off-time, initial reaction temperature, pretreatment time and the speed of circulation pump were studied. Results of single factor experiment showed that ultrasonic pretreatment had significant effect on DH and ACE inhibitory activity (p<0.05) within the range of values tested. However, the relationship between the DH and ACE inhibitory activity was quite unclear. Therefore, based on the results obtained, a central composite response surface methodology study was designed with only ACE inhibitory activity as response. This study gave optimized conditions of2.58s ultrasonic on-time,4.24s ultrasonic off-time,30.14℃initial reaction temperature,20.06min pretreatment time and198.54rpm speed of circulation pump. Under these conditions, the ACE inhibitory activity of WGH measured as IC50(mg/mL) was0.41mg/mL土0.01while that without ultrasound pretreatment was0.65mg/mL土0.002. IC50of the ultrasound pretreated WGH was significantly decreased by36.92%while increasing ACE inhibitory activity by29.97%over the control.Finally, in order to understand the effect of the CF ultrasound pretreatment on the hydrolysis process of WG, the hydrolysis kinetics of ultrasound pretreated WG was studied and compared with that of traditional hydrolysis. It was observed that the initial rate of reaction of CF ultrasound-assisted hydrolysis was generally lower than the traditional hydrolysis and the highest decrease in the initial rate of reaction (22.98%) was obtained at20.0g/L substrate concentration. The apparent breakdown rate constant (KA) was decreased by41.12%and interestingly the apparent constant (KM) was also decreased by43.52%, the reasons for this phenomenon needs further study, but may be related to increasing protein surface hydrophobicity caused with ultrasound. Overall, although because ultrasonic induced increase in hydrophobic surface protein, resulting in molecular aggregates and solubility decreases, it did not improve the efficiency of gluten protein hydrolysis, but the rich hydrophobic amino acids in terminal of molecules is characteristic of ACE inhibitory peptides, so the ultrasonic pretreatment significantly increased the ACE inhibitory activity of the hydrolysates.This study has provided optimized enzymatic hydrolysis conditions and counter-flow ultrasonic pretreatment conditions for the production and enhancement of ACE inhibitory peptides from WG which can be useful to the functional food and nutraceutical industries. |
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