| As a pretreatment method,ultrasound has a significant effect in promoting the preparation of functional peptides by protein hydrolysis.Micro optical fiber nearinfrared spectroscopy technology can be successfully applied to the in-situ real-time monitoring of key information in the enzymatic hydrolysis process.In recent years,domestic and foreign scholars including our research group have carried out a lot of research work around the above two aspects and made fruitful progress.However,the research work on the realization of ultrasonic frequency sweeping working mode and the construction of intelligent control system for protein hydrolysis process based on in-situ real-time monitoring of near-infrared spectroscopy is not deep enough,and the role of physical means in promoting and improving protein hydrolysis process is not fully played.Rice protein(RP)is a kind of plant protein with abundant resources and nutrients.Enzymatic hydrolysis of RP to prepare bioactive peptides has important commercial value.This project takes the enzymatic hydrolysis method preparation of rice peptides as the research object.Firstly,the design of ultrasonic power supply that can realize different sweeping frequency working modes and its application in the pretreatment of raw materials in rice enzymatic hydrolysis are carried out.Secondly,the structureactivity relationship of angiotensin I-converting enzyme(ACE)inhibitory activity of enzymatic hydrolysis products is studied,and the in-situ real-time monitoring technology of ACE inhibitory activity of enzymatic hydrolysis products in the enzymatic hydrolysis process based on micro optical fiber near-infrared spectroscopy is developed.Finally,the intelligent control system of enzyme hydrolysis endpoint discrimination and flexible switching of double enzyme hydrolysis is constructed.The main research contents and conclusions are as follows:(1)A random step sweeping frequency(the following is called random sweeping frequency)ultrasonic power supply is developed.The power rectifier circuit adopts bidirectional thyristor power regulation,which can realize multi-stage power regulation.The inverter circuit adopts a microcontroller unit(MCU)combined with a digital frequency synthesizer so that the power supply can quickly track the resonant frequency of the ultrasonic transducer,and can be adjusted to the fixed frequency(FF),cyclicsweeping frequency(CSF)or random-sweeping frequency(RSF)excitation mode with the center frequency of 28 kHz.The output terminal adopts series inductance for tuning and high-frequency transformer for impedance matching.The rated power of the designed ultrasonic power supply can reach 600 W.(2)The effect of sweeping frequency ultrasonic pretreatment on the strengthening effect of RP hydrolysis was studied.Taking the degree of hydrolysis(DH)and ACE inhibition rate of enzymatic hydrolysis products as the evaluation indexes,the RP was pretreated with the new sweeping frequency ultrasonic equipment developed above,and the enzymatic hydrolysis effects under three frequency modes of FF,CSF and RSF were compared.The comparative study found that the three modes improved DH and ACE inhibition rate of enzymatic hydrolysis products.Among them,RSF and FF ultrasonic mode were better than CSF,and RSF was the best.Under the RSF ultrasonic mode,the optimal ultrasonic pretreatment process parameters are:temperature 50℃,feed concentration 40 g/L,pretreatment time 20 min and pH 8.5.Compared with the control group without ultrasonic pretreatment,enzymatic hydrolysis for 60 min can increase the DH and ACE inhibition rate by 8.17%and 16.35%respectively.The molecular weight(MW)greater than 2000 Da content of the enzymatic hydrolysate products is 44.73%lower than that of the control,and the 200-1000 Da content is 22.57%higher than that of the control.(3)The structure-activity relationship between RP pretreated by RSF ultrasound and ACE inhibition rate of enzymatic hydrolysis products was studied.Moderate ultrasound can increase the sulfhydryl group(-SH)content,reduce the disulfide bond(S-S)content and increase the surface hydrophobicity.In the secondary structure,RSF ultrasound role reduces the α-helix content,increases the β-sheet and random coils,these indicate that ultrasound can stretch the protein structure,expose more hydrophobic groups and enzyme digestion sites.The above changes help to strengthen the enzymatic hydrolysis effect and improve the ACE inhibition rate of enzymatic hydrolysis products.Taking the protein structural characteristics as independent variable and ACE inhibition rate as variable,Pearson correlation analysis,principal component analysis and stepwise linear regression analysis were carried out.The analysis results show that the increase of-SH content,surface hydrophobicity and random coils,as well as the decrease of S-S and α-helix are the main factors for the ACE inhibition rate increase.There is a linear quantitative relationship between ACE inhibition rate and surface hydrophobicity in protein structural characteristics.The regression equation is:Y=6.200+0.113X3(Y is ACE inhibition rate,X3 is surface hydrophobicity,the correlation coefficient is 0.868).The equation fitting degree is high and can be used for quantitative judgment.(4)The in-situ monitoring method of near-infrared(NIR)spectroscopy in the RP hydrolysis process was studied.By comparing 14 spectral pretreatment methods,it was found that the ACE inhibition rate quantitative model established by SGF pretreatment was the best.The ACE inhibition rate quantitative model of RP hydrolysate was established by partial least squares regression(PLSR)analysis.The correlation coefficient of correction set was 0.9408,the root mean square error of cross validation was 3.25%.The correlation coefficient of the prediction set was 0.9323,and the root mean square error of prediction(RMSEP)was 3.27%.The evaluation index of the model is high and can be used to quantitatively predict the ACE inhibition rate of RP hydrolysates.(5)Using sensor detection,near-infrared spectroscopy in-situ monitoring and programmable logic conttroller(PLC)technology,an intelligent control system for RP hydrolysis process is established to detect and control the temperature and pH parameters in the reaction,and take the obtained real-time ACE inhibition rate as the index to determine the end point of enzymatic hydrolysis and the flexible switching application of double enzymatic hydrolysis.When the ultrasonic pretreatment RP solution with the feed solution concentration of 35,40 and 45 g/L reaches the set end point,the enzymatic hydrolysis reaction time is 84,75 and 99 min respectively.Compared with single enzymatic hydrolysis,using the same endpoint threshold,the double enzyme hydrolysis duration was shortened by 30,15 and 27 minutes respectively. |
