Study On The Technology Of Increasing Mogroside V Content Of Momordica Grosvenory By Physical And Enzymatic Methods And Its Structure Verification Of Transformation Products

There are 12 kinds of cucurbitane triterpenoid glycosides from the fruits of the Momordica grosvenory.Among them,mogroside V is the main component of sweet taste.Mogroside V is cucurbitane type tetracyclic triterpenoid glycosides,which is 300 times sweeter than sucrose,low in calories,has the effect of moistening lung,relieving cough and sliding bowels,has protective effect on caries teeth,such as obesity and diabetes.It accounts for 30?40%of total glycosides,and the rest of the other 60?70%is other low sweet glycosides and even no sweet glycosides,these parts of glycosides are not used,resulting in a waste of resources.This paper studies the postprocessing conditions of the fruits of the Momordica grosvenory,the extraction and purification of glycosides from Momordica grosvenory,study on the conditions of enzymatic conversion of non glycoside V substances in Momordica grosvenory extracts by biochemical method.Finally,the structure of the enzymatic conversion products were determined by nuclear magnetic resonance spectroscopy,in order to make full use of the resources and to improve the yield of mogroside V for the theoretical basis.The main research contents and results are as follows:1.Study on postprocessing conditions of the unripe Momordica grosvenoryAccording to the rule of physiological accumulation,the mogroside V was transformed from the low glycosides of Momordica grosvenory with the increase of maturity.Therefore,Study on process conditions of green Momordica grosvenory after-ripening to promote the transformation of sweet glycosides.Postharvest unripe Momordica grosvenory as the object of study,in this study,the effects of light,illumination time,constant temperature and temperature change on the transformation of mogroside V were studied,each treatment was randomly divided 6 into a group as the parallel group,and set at room temperature under natural conditions,which was always shading treatment as the control group with the content of mogroside V as the index and the content of mogroside V was detected by UV spectrophotometry.According to the results and cost saving considerations,choosing the appropriate process conditions for purchasing back green Momordica grosvenory is put into 20?,daily light 3?6h 9D,light intensity of 1060 lux(Lx),then mogroside V content of sweet glycosides can reach the peak value of 0.112g/100mL.Compared with the initial value,the increase was about 4.71 times.2.Study on extraction and purification of glycosides from Momordica grosvenoryIn this study,dry fruit of Momordica grosvenory as raw materials,according to the solid-liquid ratio was 1:10,the use of ultrasonic assisted water extraction of mogroside V,the extractions of glycosides pH was transferred to 9,then separated by AB-8 macroporous adsorption resin,and then eluted with deionized water,40%ethanol,60%ethanol and 80%ethanol in turn.The flow rate was 1mL/min,and the elution amount of each eluent was 3BV,which was completed for 3 times.The eluate was collected and detected by UV Spectrophotometry after concentration,and was confirmed by thin-layer chromatography(Thin Layer Chromatography,TLC).The results showed that mogroside V could be eluted completely from the AB-8 macroporous adsorption resin by 60%ethanol.Thus,mogroside V and non glycoside V components were separated.3.Study on the conditions of enzymatic conversionStudy on the conditions of enzymatic conversion of non glycoside V substances from Momordica grosvenory extraction(The initial content of mogroside V was 0.016g/100mL).The cyclodextrin glycosyltransferase(CGTase)method was used to study the enzymatic conversion conditions of non glycoside V substances.Through the single factor experiment to study the influence effects of pH value in enzyme reaction,enzyme reaction type,temperature,reaction time,enzyme and substrate ratio,solid-liquid ratio to improve the contents of Mogroside V,and the 4 selected factors which had a great influence on the results are used to the orthogonal test.The optimum conditions of enzymatic conversion were obtained.The results showed that pH 5.5,glucose as the substrate of the enzyme reaction,the reaction time was 10min at 45 0C,the ratio of enzyme to extract was 1:1(v/v,mL),the ratio of material to liquid was 1:14(material refers to the substrate glucose,liquid refers to the enzyme solution and the extraction of Momordica grosvenory glycosides(m/v,g/mL),namely 1g:(7mL+7mL)).The optimum conditions were used to verify the experiment and repeated 3 times.Under these conditions,the content of mogroside V was 0.051g/100mL,compared with initial value before conversion,the maximum increase was about 2.25 times.4.Study on the separation and purification conditions of transformed products and the choice of coloration methods of conversion productsThe optimum enzymatic conversion process was used to obtain the product.The content of mogroside V was determined by UV Spectrophotometry.By using thin layer chromatography(TLC)detected results before and after enzyme addition.The study was carried out to investigate the separation effect of n-butanol:ethanol:water=5:1:1,chloroform:methanol:water=60:10:1 and N-butanol:acetic acid:water=4:1:1,all these 3 kinds of developing agents on the conversion products after enzyme addition.The results showed that the developing agent was n-butanol:glacial acetic acid:water=4:1:1,which was suitable for the separation of the conversion products,causing the overall expansion speed of the silica gel plate was moderate and the separation degree of the enzyme conversion product was better.Finally,4 separate products were obtained,named as L1,L2,L3,L4.Using n-butanol:glacial acetic acid:water=4:1:1 as the developing agent for the separation of the conversion products.Three kinds of methods of coloration separation for TLC were compared respectively,such as UV coloration,iodine staining coloration and 10%sulfuric acid ethanol coloration.According to the results of coloration circumstances,simple operation and experimental safty were used to select the appropriate coloration method.The results showed that the iodine staining method was the best for the subsequent purification of the conversion products,which was easy to be recognized.5.Purification and purity identification of transformed productsUsing 10×10(cm)silica gel G plate,10?L sample into 1cm spots on the thin layer plate,then expanded with the best developing agent of n-butanol:glacial acetic acid:water=4:1:1,scraped out the 4 spots,and dissolved by ethanol,centrifuged to extract the supernatant,then removed ethanol to obtain the 4 pure conversion products.High performance liquid chromatography(HPLC)was used to identify the 4 products.The chromatographic conditions:Japan's SHIMADZU Iner Sustain C18 column(4.6mm X 250mm,5?m);column temperature:40?;the mobile phase was acetonitrile water,gradient elution of acetonitrile water(0-20min,80:20),20min later,acetonitrile water(76:24);detection wavelength:210nm;volume flow:0.75mL/min;sample size:20?L.Time for 30min.Liquid chromatography analysis was used by software Lab Solution version 5.82.The results showed that there was a significant peak of mogroside V at the retention time of 12.419min,which illustrated the transformed products contained mogroside V.6.Structure verification of enzymatic conversion productsThe enzymatic conversion products were initially identified by TLC.L1,L 2,L3,L4 four kinds of samples were isolated,and then analyse through high performance liquid chromatography(HPLC).It was further identificated that th-e addition of CGTase could increase the content of mogroside V.At last,usi-ng nuclear magnetic resonance(NMR)spectroscopy for structural verification,and finally inferred the chemical structure of the convertion products L1,L2,L3 and L4.L1:mogrol-3-O-?-D-glucopyranosyl(6?1)-24-O-?-D-glucopyranosy-1(2?1),namely mogroside ? E;L2 and L3:mogrol-3-O-?-D-glucopyranosyl(6?1)-24-O-?-D-glucopyranosyl(6?1)-?-D-glucopyranosyl(2?1),namely Mogro-side V;L4:non glycosides.The results showed that L1 was mogroside ? E,L2 and L3 were identified as mogroside V,indicated that non glycoside V s-ubstances could be converted into glycoside V by CGTase method.To sum up,relying on the postharvest ripening and processing of green Momordica grosvenory and enzymatic conversion method could improve the content of Mogroside V so that the Momordica grosvenory resources could be fully used and also lay a theoretical foundation for improving the yield of Mogroside V.