Ultrasound Assisted Preparation Of High Bioavailability Corn Protein And Near Infrared Spectroscopy In Situ Monitoring Of Processing

Corn gluten meal?CGM?is considered as an excellent plant protein source.Nonetheless,CGM has not been fully used in the food industry because of its poor solubility,dense structure,and rich hydrophobic amino acids content,which causes decrease in protein bioavailability.To overcome these restrictions,the protein structure can be modified by physical,chemical,and biological methods to improve its bioavailability.In this work,the enzymatic modification was carried out to enhance the bioavailability of CGM.Further,the effect of sonochemical action on enzymatic hydrolysis efficiency and protein dissolution rate was examined.To realize the intelligent enzymatic hydrolysis process,in situ real-time monitoring technology was used in the ultrasonic pretreatment process,and the enzymatic hydrolysis process,respectively.The main research contents and observations in this work were as follows:?1?Enzymatic modification had a direct effect on the bioavailability of CGM by SD rat in the growth and metabolism test and SD rat in vivo small intestine perfusion experimental model.At degree of hydrolysis of 15%,the bioavailability of moderate enzymolysis solution was better.By increasing the degree of hydrolysis,the content of components with molecular weights less than200 Da increased,but the content of components with molecular weights of 200-500 Da did not change significantly.The amino acid composition showed an enhancement in the content of Asp,Ser,and Glu in the free hydrophilic amino acid increases,whilst the content of the total amino acid and hydrophobic amino acid increased first and then decreased.When the degree of hydrolysis was 15%,it reached the maximum value.Furthermore,the digestibility of hydrophobic amino acids Pro,Thr,and Ala decreased,and the digestibility of Trp and Met increased.The degree of hydrolysis had no statistical significance on the digestibility of hydrophilic and hydrophobic amino acids.The in vivo one-way small intestine perfusion test of SD rats showed that the protein absorption rate of hydrolyzed CGM reached its maximum value at 15%degree of hydrolysis,and was easily absorbed by the body.?2?The effect of ultrasound pretreatment on the enzymatic hydrolysis efficiency and protein dissolution rate of CGM was studied.The cup-shaped dual-frequency countercurrent ultrasound equipment,cylindrical five-frequency countercurrent ultrasound equipment,and slit-type six-frequency ultrasound equipment were used to pretreat CGM,with the aim of improving the enzymatic hydrolysis efficiency and dissolution rate.The results indicated that the slit-type six-frequency ultrasound equipment had the best effect on the enzymolysis efficiency.Additionally,the dual-frequency working model was significantly better than the mono-frequency,tri-frequency,quad-frequency,and five-frequency working modes,and the sequential working mode was better than the synchronous working mode.The optimum conditions were observed at sequential working mode?20/40 kHz?,ultrasonic power density of 80 W/L,ultrasonic treatment time of 30min,ultrasonic temperature of 30?,ultrasonic intermittent ratio 5:2?s/s?,substrate concentration50 g/L,initial pH 8.5.After slit dual-frequency ultrasonic pretreatment,the enzymatic hydrolysis efficiency and dissolution rate of CGM increased by 23.63%and 35.07%,respectively.Compared to control,the molecular weight distribution of the sonicated samples was mainly concentrated in200-500 Da,the total amino acid content decreased,and the ratio of hydrophobic amino acid content to total amino acid content increased.?3?The results of thiol and disulfide bonds showed that ultrasound pretreatment changed the content of thiol and disulfide bonds of CGM by increasing the thiol content and decreasing the disulfide bonds content.Also,the surface hydrophobicity increased,resulting in a more stretched protein structure.Fourier transform infrared spectroscopy indicated that ultrasound pretreatment altered the secondary structure of the protein,which was mainly reflected in the decrease of?-helix and?-turn content and the increase of?-sheet and random coil content.The ultrasonic power density has a greater impact on protein secondary structure.Protein particle size,polydispersity coefficient,and atomic force microscopy results indicated that after ultrasound pretreatment,the particle size of CGM decreased,the number of small protein particles increased,the distribution of protein particles was more concentrated and uniform,the surface roughness increased.As the surface area increased,these changes were conducive to the proteolysis reaction.?4?The relationship between the structural characteristics of CGM and the enzymatic hydrolysis efficiency during the ultrasonic pretreatment was studied through the Pearson correlation analysis,principal component analysis,stepwise multiple regression analysis.The results indicated that the increase of sulfhydryl content,surface hydrophobicity,and Zeta potential value,and the decrease of PDI value and particle size were the main determinant of improvement in the CGM proteolysis efficiency.The stepwise regression equation of the structure characterization and relative enzymolysis efficiency of CGM with ultrasound pretreatment was established as Y=16.008+1.722X₁+0.383X₁₀(Y?X₁?X₁₀ stand for relative enzymolysis efficiency,mercapto group content,Zeta potential,R²=0.9480.).In the process of ultrasound pretreatment,thiol content and Zeta potential value could be used to predict the relative enzymatic hydrolysis efficiency of CGM,so as to judge the best limits of ultrasound pretreatment.?5?An in situ and real-time spectroscopic monitoring system for the content of sulfhydryl groups during the ultrasonic pretreatment of CGM was established.Near infrared spectroscopy data of the changes in the content of sulfhydryl groups during the ultrasonically assisted alkali pretreatment could be dynamically collected.The MSC pretreatment had the best effect on the model established by the collected near infrared spectrum data.According to the model prediction established by Si-PLS,the best spectral bands for sulfhydryl content screening were1265.06¹323.19 nm,1432.76¹490.65 nm,2001.72²052.51 nm,2109.58²166.59 nm.Under the best spectral bands,the Si-PLS quantitative analysis model of sulfhydryl content during the ultrasonic pretreatment process was established.The correlation coefficient R_c of the corrected model of sulfhydryl content was 0.9350,and RMSECV was 0.984?mol/g.The correlation coefficient R_p of the prediction model was 0.8468,and RMSEP was 0.937?mol/g.This model could realize in situ and real-time monitoring of sulfhydryl content of CGM in the process of ultrasound pretreatment.?6?In situ and real-time monitoring technology of CGM proteolysis process was developed.The methods of multivariate scattering correction?MSC?,polynomial convolution smoothing?SG?,first-order derivation(1^st Der),second-order derivation(2^nd Der),standard normal transformation?SNV?,and mean centralization?MC?were used for near-infrared original spectrum.The Si-PLS modeling method was used to develop a model between spectrum data proteolysis degree and protein dissolution rate.The model constructed after treatment showed strong predictive power and good robustness.The best spectral bands of the degree of hydrolysis of CGM were1316.73¹387.68 nm,1387.68¹464.93 nm,1612.58¹689.40 nm,1989.01²058.85 nm.The best spectral bands of protein dissolution rate were 1297.36¹387.68 nm,1387.68¹471.361 nm,1644.61¹734.16 nm,1906.34¹989.01 nm.The Si-PLS quantitative analysis model for the hydrolysis degree and protein dissolution rate of CGM proteolysis was established.The correlation coefficient R_p value of the prediction model of hydrolysis degree of CGM was 0.9503,and RMSEP was 1.79%.In the prediction model of dissolution rate of CGM,the correlation coefficient R_p value was 0.9518,and RMSEP was 3.74%.This model could realize in situ and real-time monitoring of the degree of hydrolysis and protein dissolution rate during the proteolysis of CGM.