Ethanol-assisted Extraction Of Related Bioactive Components Of Stevia Leaves And Evaluation Of Their Function

Stevia leaf is a natural,green,high-quality resource with dual functions of sweetness and health.The content of glycosides in the leaf is the highest,and it also contains various functional substances such as protein,cellulose,fat,flavonoids and trace elements.Therefore,research on the comprehensive processing and utilization of stevia leaves are very important.Based on ethanol-assisted extraction to prepare steviol glycosides,the paper further studied the enzymatic hydrolysis of proteins and cellulose in stevia leaves and the functional activities of related extracts,as well as carried out experimentation related to enzymes in a feasible way in the laboratory in order to provide a guide for green and healthy approaches to the extensive development and utilization of stevia leaves.First,a mixed solvent of ethanol and water was used to extract and prepare stevioside.Then,the response surface software was used to optimize the extraction and hydrolysis of protein and cellulose in the extract powder of stevia leaves,and the related activities of the extracts of stevia leaves were evaluated.Finally,the technology and properties of enzymes produced by fermentation of ethanol-extracted powder were studied.The test results were summarized as the following:1.In the process of preparing stevioside,the content of the main chemical components of stevia leaves were first determined in accordance with national standards and other methods.The res ults were:13.83%�0.36%of stevioside,11.15%�0.05%of moisture,7.95%�0.39%of ash,12.11%�0.21%of protein and 10.37%�0.18%of crude fiber.Then,the stevia leaves were pretreated.In the process of extraction,the stability of separation and purification of stevioside in a mixed solvent of ethanol and water were tested.According to the single factor test was performed,the effects of the number of milled meshes,concentration of ethanol,time of extraction,temperature of extraction and the ratio of material to liquid on the extraction rate of stevioside were investigated.Then,by using IBM SPSS Statistics 25 orthogonal processing software to obtain the optimal the number of milled meshes was300,the optimal concentration of ethanol was 60%,the optimal time of extraction was 60 min,the optimal temperature of extraction was 55?and the optimal material-liquid ratio was 1:12.Finally,under the optimal technological conditions,three parallel purification tests of stevioside were conducted,and the purity of stevioside obtained was 90.48%�0.65%.2.In the process of hydrolysis of stevia leaf ethanol extract powder by neutral protease,the protein extraction rate was determined by factors such as the pH of enzymolysis,the ratio of material to liquid,the temperature of enzymolysis,the concentration of enzymolysis,the time of enzymolysis and the number of milled meshes.After conducting process research and analysis,according to Box-Behnken test design and using Design-Expert.V8.0.6.1 software to perform response surface tests and perform data fitting of related factors.The optimized process extrac tion conditions were:the pH of enzymolysis was 7.97,the ratio of material to liquid was 1:8.64,the temperature of enzymolysis was53.12?and the concentration of enzymolysis was 3.73%.The software predicted the protein extraction rate through the corresponding regression equation was 86.72%.For the convenience of subsequent experiments,the optimization process was modified:the pH of enzymolysis was 8.0,the ratio of material to liquid was 1:9,the temperature of enzymolysis was 53.1?,the concentration of enzymolysis was 4%and the protein extraction rate of 87.05%,which was almost the same as that of theoretical predictions,show ing that the model was reliable.In addition,under the same optimized conditions,by adding stevioside and chlorogenic acid to the prepared gelatin solution,the components that inhibit protease activity in stevia leaves were investigated analogously,and the results showed that the protein extraction rate was not affected by stevioside,stevioside had no inhibitory effect on the activity of protease.The presence of chlorogenic acid would obviously inhibit the enzymatic hydrolysis of protein.This had a guiding significance for further improving the efficiency of proteolytic hydrolysis,increasing the enzymatic hydrolysis rate of stevia leaf protein.In the process of cellulase hydrolyzing of ethanol extraction powder of stevia leaf,after single factor analysis and Design-Expert.V8.0.6.1 software processing,the extraction conditions were optimized as follows:the ratio of material to liquid was 1:13.73,the concentration of enzymolysis was 1.58%,the time of enzymolysis was 2.48 h and the number of milled meshes was 283.48.After correcting this properly,the optimal process conditions were modified to be as follows:the ratio of material to liquid was 1:14,the concentration of enzymolysis was 1.6%,the time of enzymolysis was 2.5 h and the number of milled meshes was 300.The cellulose hydrolysis rate was measured to 74.88%and the error was negligible compared to the value of 73.32%.3.In the comparative study of the ACE inhibitory activity of the protein hydrolysate of stevia leaf obtained by protease hydrolysis,ethanol-extracted stevioside solution and pure stevioside sample solution,it was found that all three samples showed a significant inhibitory effect,and the protein hydrolysate had a highest inhibitory activity.In the determination of the functional properties of the concentrated stevia leaf proteolytic sample,the results showed that the water holding capacity of 2.921g/g,the oil holding capacity of 2.262 g/g,the foaming property of 115.640%,the foaming stability of65.333%,the emulsifying property of 51.956%and the emulsifying stability of 83.013%.In the determination of the antioxidant activity of the protein hydrolyzed of stevia leaf by enzymes,the results showed that the scavenging activity of the hydroxyl radicals was significantly stronger than that of the superoxide anion radicals.And when the sample concentration of the stevia leaf proteolysate was increased to 0.5 mg/ml,the scavenging activities of the two radicals also reached to the maximum,respectively 68.25%and 44.24%.It indicated that the sample of the protein hydrolysate of stevia leaf had certain in vitro antioxidant properties.4.The stevia leaf ethanol extract powder was subjected to the natural fermentation culture and the preparation of crude enzyme solution,the strongest enzyme activity was determined,at 168 h,the strongest enzyme activity of the protease was 1.811 U/ml;at 144 h,the strongest enzyme activity of the cellulase determined by the CMC method was 0.287 U/ml;at 192 h,the strongest cellulase activity determined by the FPA method was 0.447 U/ml.After simple purification by dialysis desalting and freeze drying,it was found that the final specific activity of the protease was 0.867 U/mg and the purification factor was 1.292 times;the final specific activity of the cellulase measured by CMC-Na was 0.152 U/mg and the purification factor was 1.288 times;the final specific activity of the cellulase measured by FPA method was 0.219 U/mg and the purification factor was 1.273 times.In addition,in the determination of some properties of the enzyme,the optimum pH and the optimum temperature for the protease reaction were 6.0 and 35�C,respectively.The optimum pH and optimum temperature of the cellulase obtained by the two determination methods were the same,which were 4.5 and 55?,respectively.Moreover,both enzymes showed good pH stability and thermal stability in the reaction.The results of the analysis of the effects of several metal ions on the two enzymes showed that Na⁺,Cu²⁺,Zn²⁺,and Al³⁺had an activating effect on protease enzyme activity,and K⁺had an inhibitory effect on enzyme activity.Ba²⁺had an inhibitory effect on cellulase enzyme activity,and the effects of other ions were not obvious.