Study On The Preparation Of ACE Inhibitory Peptides From Wheat Gluten Based On Ultrasonic Pretreatment And In-situ Monitoring Of The Process

Wheat gluten(WG) is a potential source of plant protein which could be used to produce antihypertensive peptides with strong angiotensin I-converting enzyme(ACE) inhibitory activity without side effects. However, the enzymolysis efficiency of WG is low due to its poor dispersion. Meanwhile, the WG complex consists of gliadin and glutenins and they were easy to cross-linking in the water. The enzymolysis efficiency of protein could be improved by ultrasound pretreatment though changing the protein structure. However, the dynamic anylysis of protein structure in ultrasound pretreatment process and the relationship between protein structure and enzymolysis efficiency were not clear. Meanwhile, the in-situ monitoring of the protein structure in ultrasound pretreatment process, and the parameters during enzymatic process need to be established.The purpose of the present paper was to improve the enzymolysis efficiency by ultrasound pretreatment, and establish the quantitative analysis model of WG protein structure and ACE inhibitory activity of WG hydrolysate during ultrasonic pretreatment. Though in-situ and real-time spectra monitoring the change of sulfhydryl(-SH) and disulfide bond(S-S) content in wheat gluten by ultrasonic pretreatment, monitoring the changes of the degree of the hydrolysis(DH), peptide concentration and ACE inhibitory activity of WG hydrolysate during the enzymatic hydrolysis reaction. The main creative conclusions drawn by the research are as following:(1) Taking the DH and the ACE inhibitory activity as indexes study the effects of multimode ultrasound pretreatment on the WG enzymolysis characteristics and obtain the best ultrasonic pretreatment working mode and conditions. The results showed that synchronous dual-frequency mode was better than single frequency mode, while the alternate dual-frequency mode was better than the synchronous dual-frequency mode. In the best alternative dual-frequency operation mode, the optimal ultrasonic pretreatment parameters were: frequency combination 20-35 k Hz, alternating time 5 s, power density 200 W/L, pretreatment initial temperature 30 oC, material concentration 30 g/L, and pretreatment time 10 min. At this condition, the DH of WG protein was 12.51%, the ACE inhibitory activity was 80.03%, the IC50 was 0.696 mg/m L, and the conversion rate of protein was 75.4%. Compared to control(without ultrasound pretreatment), the alternate dual-frequency ultrasound pretreatment improved the DH by 2.37%, improved the ACE inhibitory activity by 19.30%, and decreased the IC50 value by 6.5%, respectively. There was no significant difference in the rate of protein conversion. The effects of alternate dual-frequency ultrasound(ADFU) pretreatment on the enzymatic hydrolysis process showed that the ACE inhibition rate of the enzymatic hydrolysate was 10 min in advance to reach its maximum value. The reason for this phenomenon is that the ultrasonic pretreatment improved the molecular weight distribution of 200-1000 Da of peptide in the early stage of the enzymatic hydrolysis reaction.(2) The effects of ultrasonic frequency, power and treatment time on the microstructure, nano-mechanical properties and molecular structure were studied by atomic force microscopy and fourier transform infrared spectroscopy. The results showed that the reticulation structures of WG were destroyed by ultrasound pretreatment. Appropriate ultrasonic pretreatments led to even distribution of surface molecules of the protein and the surface roughness became larger. However, excessive pretreatment(pretreatment duration>10 min, power density >150 W/L) caused aggregation of protein molecules and reduction in the surface roughness of WG. The nano-mechanical properties of WG were redistributed by ultrasound pretreatment. The average values of Young’s modulus(stiffness) and the average adhesion force decreased. The-SH and S-S content, H0 and the secondary structure of the WG protein was significantly changed by ADFU pretreatment. The changes were mainly in the increase of-SH and S-S content and H0 and decrease of α-helix.(3) The relationship between WG protein structure affected by ultrasound and ACE inhibitory activity of WG hydrolysate was developed by Pearson correlation analysis, principal component analysis and stepwise multiple linear regression(Stepwise-MLR). Pearson’s correlation analysis showed that the-SH content, Ra, Rq and H0 correlated with ACE inhibitory activity of WG hydrolysate linearly and significantly(P < 0.05); The average values of Young’s modulus, The average values of adhesion values and the relative percentage of α-helix negatively correlated with ACE inhibitory activity of the hydrolysate linearly and significantly(P<0.05); the relative percentage of β-sheet correlated linearly. Principal component analysis selected 3 principal components and represented 79.628% of the original structure content in the sample. The first principal component contained the-SH content, the average adhesion force, Ra, Rq and H0; the second principal component contained mainly β-sheet and random coil; the third principal component contained the S-S content of WG. The young’s modulus, adhesion and α-helix correlated closely. The decrease of α-helix might possibly led to decrease of adhesion and Young’s modulus. The results of Stepwise-MLR showed that the-SH contents, α-helix, S-S content were significantly correlated to ACE Inhibitory activity of WG hydrolysate, with the standard partial regression coefficients of 6.8410,-0.6870, and-0.1620, respectively. The R2 of this model was 0.9380.(4) In-situ and real-time spectrum monitoring system and spectrum analysis methods were established. It could collect the NIR spectrum of WG during ultrasound pretreatment process stable. Four different spectra data preprocessing method, standard normal variate transformation(SNV), multiplicative scatter correction(MSC), first and second derivative were used comparatively in this study. Consider the correlation coefficient R of calibration and prediction models and the Root mean squared error of prediction(RMSEP), the optimal spectra intervals were selected. Based on the optimal spectral intervals, the synergy interval least squares, Si-PLS and error back propagation neural network, BP-ANN of-SH, S-S contents were established. The results showed that the SNV was the best preprocessed method; the optimal spectral intervals of-SH content were 869-947, 1207-1284, 1458-1536 and 2205-2274 nm, the optimal spectral intervals of S-S content were 933-992, 1388-1446, 2091-2148 and 2217-2274 nm; the BP-ANN model was better than Si-PLS model. Finally, for the-SH content, the correlation coefficient of the prediction set is 0.9113, and the RMSEP is 0.38 μmol/g. For the SS content, the correlation coefficient of the prediction set is 0.7523, and the RMSEP was 6.56 μmol/g.(5) In-situ and real-time spectrum systerm was developed and used for monitoring the parameters(DH, peptide content and ACE inhibitory activity of hydrolysate) during enzymatic process of WG. Four different spectra data preprocessing method(SNV), MSC, first and second derivative were used comparatively. Si-PLS algorithm was performed to calibrate regression model. Experimental results showed that the SNV was the best preprocessed method. The correlations coefficients in the prediction set(Rp) were achieved as follows: Rp = 0.9270 for DH of WG, Rp = 0.9673 for peptide content of WG hydrolysates, Rp = 0.9507 for ACE inhibitory of WG hydrolysates. The RMSEP were 1.73%, 0.79 mg/m L and 5.12%, respectively. The miniature fiber optical spectrometer combined Si-PLS model, can be used as an alternative method for real-time monitoring the enzymatic process of WG in the enzymasis reactor which was the in-situ reaction set of enzymasis.In summary, alternating dual-frequency ultrasound pretreatment could significantly improve the ACE inhibitory activity of WG hydrolysate. The reason were ADFU pretreatment increased the-SH content, reduced of the relative percentage content of α-helix, the average young’s modulus and the average adhesion force by ultrasound pretreatment. The-SH content made the largest contribution. The in-situ real-time spectra system combined with Si-BP-ANN model could be well applicable for monitoring the-SH and S-S contents of WG in ultrasonic pretreatment process. Meanwhile, the in-situ real-time spectra system combined with Si-PLS model could be well applicable for monitoring the DH of WG protein and the peptide content of WG hydralysates in enzymatic process. The results have great significance in the in-situ and real-time monitoring the preparation of ACE inhibitory peptides by ultrasonic pretreatment, and can provide theoretical foundation and methodological reference for developing in-situ and real-time equipment for food processing process.