Method for separating mogroside by combining pulsed electric field ultrasonic extraction with preparative chromatography technology

Abstract

The invention relates to a method for separating mogroside by combining pulsed electric field ultrasonic extraction with preparative chromatography, which comprises the following steps: (1) pulsed electric field ultrasonic extraction: taking momordica grosvenori, crushing, adding alkaline water, continuously passing through pulsed electric field ultrasonic extraction equipment, coarsely filtering and centrifuging to obtain a momordica grosvenori extracting solution; the alkaline water contains a reducing agent-cyclodextrin inclusion compound; (2) membrane filtration; (3) enriching the siraitia grosvenorii total glycosides through macroporous adsorption resin; (4) enabling the macroporous adsorption resin desorption solution to pass through an aluminum oxide resin column, and concentrating and drying effluent; and (5) carrying out gradient elution by preparative chromatography, collecting eluent in sections, and respectively concentrating and drying the eluent to obtain different types of mogroside components. According to the method, oxidation and loss of active ingredients, especially siamenoside I, are inhibited through an electric breaking and sound extraction technology, and the finally obtained product has the advantages of high content and high yield.

Description

Technical Field

[0001] The invention relates to a separation technology of natural active ingredients of plants, and in particular to a method for separating mogroside by combining pulse electric field ultrasonic extraction with preparative chromatography technology. Background Art

[0002] Monk fruit, the fruit of a perennial vine of the Cucurbitaceae family, is produced in mountainous areas such as Guilin, Guangxi, Shaoyang, Huaihua, Yongzhou, Hunan, and Qiandongnan, Guizhou. It is a precious local specialty in my country and one of the first batch of "dual-purpose medicine and food" materials approved by the state. Monk fruit has high nutritional value and is rich in vitamin C, mogroside, fructose, glucose, protein, pectin and lipids. According to the "Chinese Materia Medica", Monk fruit is sweet and cool in nature, belongs to the lung and large intestine meridians, has the effects of moistening the lungs and relieving coughs, promoting fluid production and quenching thirst, and is suitable for coughs caused by lung heat or dryness, whooping cough, and thirst caused by heat damage to body fluids, etc. In addition, it has the effect of moistening the intestines and promoting bowel movements. Modern medical research has confirmed that the mogroside contained in Monk fruit is a natural sweetener that is 200-600 times sweeter than sucrose, but it does not produce calories, so it is a sweet substitute for people who are not suitable for eating sugar, such as those with diabetes and obesity. As a food, mogroside is safe and non-toxic. The national mandatory standard "GB2760 Food Additive Usage Standard" stipulates that mogroside can be used in all kinds of food without limit. Due to its many excellent properties, mogroside can replace sucrose in whole or in part, so it is widely used in the food industry. In addition, mogroside also has very broad application prospects and market potential in the fields of pharmaceuticals, cosmetics and health foods.

[0003] Mogrosides are a class of natural active ingredients unique to monk fruit, including mogroside V, 11-oxy-mogroside V, mogroside III, mogroside IV, simenoside I, mogroside VI, etc. So far, researchers have found more than ten types of mogrosides in monk fruit. The types and contents of mogrosides usually vary depending on factors such as monk fruit variety, planting conditions and maturity. Generally, the highest content and most important glycoside in monk fruit is mogroside V (the content in fresh fruit is about 0.3%-0.5%; the proportion of total glycosides is as high as 80%; the sweetness multiple is about 400 times that of sucrose).

[0004] The sweetest glycoside in monk fruit is simonoside I, which is about 560 times sweeter than sucrose. Since simonoside I itself has a very low content and unstable properties, there is relatively little research on simonoside I in the academic community, and even fewer literature reports on the separation and purification of simonoside I.

[0005] CN111018933A discloses a monk fruit extract product and its preparation method and use. The product is made of monk fruit as raw material, and different types of monk fruit extracts are obtained through the steps of crushing, water reflux extraction, concentration, centrifugation, pH adjustment, macroporous resin adsorption, ethanol analysis, MCI column adsorption, ethanol gradient elution, chromatography detection, concentration, drying, etc. This patent can produce simenoside I extract, mogroside V extract, and 11-O-mogroside V extract, but all the steps and process parameter ranges are exactly the same, but it is claimed that different mogroside monomers with high content and high yield can be obtained. There are errors in principle, and the authenticity is questionable.

[0006] CN110628861A discloses a method for increasing the overall sweetness of mogrosides based on increasing the content of simanoside, comprising the following steps: 1) dissolving: dissolving mogrosides in a fermentation culture medium of the same concentration, wherein the mogroside concentration is 45-50 mg / ml, and obtaining a stock solution after sterilization; 2) screening dominant strains: 2-1) different strains are separated from the skin of monk fruit, the soil in which monk fruit grows, and the monk fruit residues, and each strain is inoculated in a culture medium with cellulose and starch as substrates. After culturing for 24 hours, the dominant strain that can produce exogenous glycosidase is confirmed by detecting the concentration of glucose in the substrate, and a total of five dominant strains are screened, numbered Sc001, VSc01, CN01, CN01-A, and T-CN00, and the five dominant strains are all Saccharomyces cerevisiae strains; 2-2) Saccharomyces cerevisiae strains Sc001, VSc01, CN01, CN01-A, and T-CN00 that can produce exogenous glycosidase are inoculated respectively. In a culture solution with mogroside V as a substrate, ferment at room temperature for 168 hours, compare the sweetness of the fermented solution with that of the stock solution every 24 hours, and screen out the strain corresponding to the highest sweetness of the fermented solution as the specific sake yeast CN01; 3) Fermentation: After the specific sake yeast CN01 is rejuvenated with a potato slant culture medium, it is inoculated with an MS culture medium for expansion, and after two-stage expansion, it is used as a fermentation seed solution, and then the stock solution obtained in step 1) is inoculated with the fermentation seed solution, and the inoculation amount is 1 / 1 of the stock solution 0-12%, control the fermentation temperature to 30-32°C, naturally ferment for 72-80h, and obtain fermentation liquid; 4) centrifugal filtration: centrifuge and filter the fermentation liquid, and collect the supernatant; 5) chromatography: separate the above supernatant through the transformed ion chromatography resin, with a column flow rate of 2BV / h, column flow for 2h, and collect the effluent within 45min-1.2h; 6) concentration and freeze drying: vacuum decompression and concentrate the above effluent, and freeze-dry to obtain mogroside with improved sweetness. This patent converts mogroside into highly sweet simone glycoside through specific fermentation, but does not specifically prepare simone glycoside I.

[0007] CN102942611A discloses a method for preparing high-purity simonoside I, comprising the following steps: 1) crushing Momordica grosvenori into coarse powder, boiling with water, centrifuging and collecting supernatant; 2) adsorbing the supernatant obtained in step 1) with a macroporous resin, washing impurities with 40% (v / v) alcohol, eluting with 60-80% (v / v) alcohol, and finally recovering ethanol from the eluate to obtain an extract; 3) dissolving the extract with 60% (v / v) alcohol, centrifuging, taking the supernatant and applying it to an ODS column, first washing impurities with 60% (v / v) alcohol, and then eluting with 70-90% (v / v) alcohol; finally recovering ethanol from the eluate and performing HPLC detection, combining the fractions containing simonoside I to obtain a crude product of simonoside I; 4) subjecting the crude product of simonoside I to Sephadex LH-20 column chromatography, ethanol gradient elution, HPLC detection and merging of the fractions containing simenoside I, and finally recovery of the solvent to obtain the refined simenoside I. The patented monk fruit raw material undergoes high-temperature decoction, and simenoside will be oxidized and lost, resulting in a low simenoside yield; and the HPLC technology is used multiple times, which is not convenient for large-scale industrial production. Summary of the invention

[0008] The technical problem to be solved by the present invention is to overcome the defects of the prior art and provide a technology for separating mogrosides by pulsed electric field ultrasonic extraction fused with preparative chromatography technology, which can simultaneously obtain multiple high-value mogrosides, has high product content and high yield, has strong operability in the production process, has low production cost, and is suitable for large-scale industrial production.

[0009] The technical solution adopted by the present invention to solve the technical problem is as follows: a method for separating mogroside by pulsed electric field ultrasonic extraction fused with preparative chromatography technology, comprising the following steps:

[0010] (1) Pulsed electric field ultrasonic extraction: Take the monk fruit, crush it, add alkaline water, continuously pass it through a pulsed electric field ultrasonic extraction device, coarsely filter it, and centrifuge it to obtain a monk fruit extract; the alkaline water contains a reducing agent @ cyclodextrin inclusion complex;

[0011] (2) membrane filtration: filtering the monk fruit extract with a ceramic membrane to obtain a ceramic membrane filtrate;

[0012] (3) Enrichment of total glycosides from Momordica grosvenori: The filtrate from the ceramic membrane was passed through a macroporous adsorption resin column until the column was completely loaded, first washed with pure water, then analyzed with an alcohol-water solution, and the ethanol analysis solution was collected for later use;

[0013] (4) Purification of total glucosides of Momordica grosvenori: the alcohol aqueous solution analytical solution is passed through an alumina resin column, the effluent is concentrated to obtain a concentrated solution, and the concentrated solution is dried to obtain a total glucosides extract of Momordica grosvenori; the alumina resin column is impregnated with acid water and washed;

[0014] (5) Preparative chromatography separation: Take a portion of the total glycosides extract of Momordica grosvenori, dissolve it in pure water, filter it through a microporous filter membrane, pass the filtrate through an MCI preparative chromatography column, perform gradient elution, and collect the eluate in sections for later use;

[0015] (6) Concentration and drying: The eluates collected in the segments are concentrated and dried respectively to obtain different types of mogroside components.

[0016] Preparative chromatography refers to the use of chromatographic techniques to prepare pure substances, that is, to separate and collect one or more chromatographically pure substances. The concept of "preparation" in preparative chromatography refers to obtaining sufficient amounts of a single compound to meet research and other purposes.

[0017] Preferably, in step (1), the reducing agent @ cyclodextrin inclusion complex is obtained by embedding the reducing agent in a cyclodextrin substance, the reducing agent is selected from at least one of astaxanthin and β-carotene, and the cyclodextrin substance is selected from at least one of β-cyclodextrin, hydroxypropyl-β-cyclodextrin, and sulfobutyl ether-β-cyclodextrin; the reducing agent content in the reducing agent @ cyclodextrin inclusion complex is 15-20wt%, and the reducing agent @ cyclodextrin inclusion complex in alkaline water is 1-5wt%.

[0018] The preparation of the reducing agent @ cyclodextrin inclusion complex is well known in the art. The reducing agent and cyclodextrin substances are added in a molar ratio of 1:1-3, and an alcohol aqueous solution is added. The mixture is stirred under an inert atmosphere until the embedding reaches equilibrium, and the mixture is spin-dried and then freeze-dried to obtain the reducing agent @ cyclodextrin inclusion complex. The alcohol aqueous solution is an ethanol solution with a volume fraction of 70-95%, and the volume ratio of the alcohol aqueous solution to the reducing agent is 3-5L:1g.

[0019] Preferably, in step (1), the alkali is one or more of sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate or sodium bicarbonate, the mass percentage concentration of the alkaline water is 0.1-2wt%, preferably 0.4-0.7wt%, and the amount of the alkaline water is 3-10 times the weight of the monk fruit raw material, preferably 4-5 times. The purpose of using alkaline water as the extraction solvent in the present invention is, first, to prevent the material from becoming rancid and the degradation of mogrosides due to the growth of microorganisms during extraction and subsequent processes; second, under an alkaline pH environment, the conductivity and ionic strength of the solution are greatly increased, which can maximize the advantages of the pulsed electric field "electrical breaking and acoustic extraction", and quickly and efficiently leach mogrosides.

[0020] Preferably, in step (1), the electric field strength of the pulsed electric field is 0.5-10 kV / cm, the number of times is 10 to 30 times, the power of the ultrasound is 20-1000 w, the ultrasound frequency is 60-150 kHz, the pulsed electric field ultrasonic extraction time is 10-100 min, and the extraction temperature is 5-30 ° C, preferably 10-25 ° C. The purpose of the pulsed electric field assisted extraction of the present invention is to overcome the two disadvantages of partial oxidation of mogrosides during high-temperature extraction and low extraction rate of mogrosides during low-temperature extraction in the conventional method, and to achieve the purpose of preventing oxidation of mogrosides at low temperature and efficiently extracting mogrosides. Simenoside I is the mogroside with the highest sweetness multiple. The inventors found that Simenoside I can be oxidized to 11-oxy-simenoside I (the sweetness multiple is only one tenth of Simenoside I) under high temperature conditions. The use of low-temperature alkaline water + pulsed electric field ultrasonic assisted extraction can greatly reduce the oxidation of Simenoside I, thereby improving the yield of Simenoside I.

[0021] In step (1), the centrifugation method includes but is not limited to horizontal screw centrifugation, butterfly centrifugation, tubular centrifugation, and a combination of the above multiple centrifugation methods.

[0022] Preferably, in step (2), the material of the ceramic membrane is zirconium oxide, aluminum oxide, or titanium oxide; the pore size of the ceramic membrane is 0.1-1.2 μm; and the filtration pressure of the ceramic membrane is 0.6-1.0 MPa.

[0023] Preferably, in step (3), the macroporous adsorption resin used in the macroporous adsorption resin column is a non-polar macroporous adsorption resin, a medium-polar macroporous adsorption resin or a weakly polar macroporous adsorption resin, or a combination of two or more of the above resins. For example, the macroporous adsorption resin is selected from at least one of D101, LX-T28, and AB-8, the resin volume dosage and the fresh fruit mass ratio of monk fruit is 10-30L:100kg, the height-to-diameter ratio of the resin column is 3-8:1, and the column flow rate is 0.5-5Bv / h. The pure water washing amount is 2-4BV, and the alcohol aqueous solution analysis amount is 2-3BV, and the alcohol aqueous solution is a 50-70% solution of methanol and / or ethanol.

[0024] Preferably, in step (4), the types of alumina include neutral alumina, acidic alumina and basic alumina, and the amount of alumina used is 1-10wt% of the weight of the monk fruit raw material. The purpose of using alumina is that alumina can specifically adsorb impurities such as flavonoids and pigments in the ethanol analysis solution, thereby improving the purity of total glycosides in the material and reducing the interference of impurities on the subsequent preparative chromatographic separation step.

[0025] Preferably, in step (4), the alumina resin column is immersed in acid water with a pH of 3-4 for 10-60 minutes, and then washed with water until the effluent is close to neutral (pH = 7 ± 0.5), and the acid water is at least one of dilute hydrochloric acid and dilute sulfuric acid. The inventors found that various commercially available aluminas have irreversible dead adsorption to simonoside I due to the aluminum hydroxide usually attached to their surfaces, which will lead to the loss of simonoside I. The content of simonoside I in Momordica grosvenori itself is relatively low, and the loss during the extraction and purification process is the main reason why there has been no simonoside I product. The inventors found that after the alumina resin column was immersed in acid water, it did not affect the adsorption of other impurities, but greatly reduced the adsorption of simonoside I.

[0026] Preferably, in step (5), the filler types used in the MCI preparative chromatography include polystyrene / divinylbenzene type (PS / DVB) and acrylate type, and the particle size of the filler is 4-300 μm.

[0027] Preferably, in step (5), the gradient elution is performed by using an alcohol aqueous solution with an increasing volume fraction of alcohol, wherein the alcohol is selected from methanol and / or ethanol, and the volume fraction of alcohol increases from 0% (i.e., pure water) to 100% (i.e., alcohol). Using alcohol solutions with different volume fractions, mogroside is eluted in sequence according to the difference in polarity, thereby obtaining a variety of high-content mogroside monomers.

[0028] More preferably, in step (5), the gradient elution is controlled at a flow rate of 0.5-2BV / h, and eluted in sequence with 3-5BV pure water, 3-5BV 10-20% alcohol aqueous solution by volume, 3-5BV 20-30% alcohol aqueous solution, 3-5BV 30-40% alcohol aqueous solution, 3-5BV 40-60% alcohol aqueous solution, 1-2BV 60-70% alcohol aqueous solution, and 1-2BV>70% alcohol aqueous solution.

[0029] The pulse electric field will have a regular effect on the permeability of plant tissue structure; ultrasonic vibration can generate powerful energy, causing the solvent and plant tissue to oscillate continuously, forming a cavitation effect, which helps solute diffusion and accelerates the leaching of effective active ingredients in plants. When the two technologies are coupled, pulse electric field instant wall breaking and ultrasonic enhanced mass transfer extraction (i.e.: "electric breaking and acoustic extraction" technology) can be achieved simultaneously, and plant tissues can be instantly and accurately broken to form micropores of different pore sizes, realizing short-time and efficient extraction of active ingredients at low temperature to room temperature. Thanks to pulse electric field wall breaking and ultrasonic enhanced mass transfer, the active ingredients are fully and efficiently leached. At the same time, due to the lower extraction temperature and shortened extraction time, the probability of thermal decomposition and oxidative denaturation of active ingredients (especially heat-sensitive, photosensitivity, and easily oxidized active ingredients) is greatly reduced, thereby reducing the loss of active ingredients and increasing the yield.

[0030] MCI (full name: MCI GEL) preparative chromatography is a medium-pressure preparative chromatography that uses chromatographic separation gel (also known as small-particle chromatographic filler based on polymer matrix) as filler. Due to the advantages of high adsorption capacity, uniform particles, good mechanical strength, not easy to break, less residue, and convenient pretreatment, this filler is widely used in the separation and purification of natural products, fermentation products and small organic molecules.

[0031] The beneficial effects of the method of the present invention are as follows:

[0032] (1) With the implementation of the "electrical extraction and acoustic extraction" technology, the extraction of active ingredients no longer relies on the high temperature and long-term boiling required in traditional processes, and the energy consumption of steam, water, and electricity has been greatly reduced.

[0033] (2) Compared with C-18 bonded silica gel filler, MCI filler has the advantages of more stable chemical properties and longer service life, and is more suitable for reversed-phase chromatography separation.

[0034] (3) The method of the present invention provides a new method for separating mogroside monomers, which is combined with the "electrolysis and sonication" extraction technology to fully extract and inhibit the oxidation of active ingredients. The final product has the advantages of high content and high yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is the HPLC spectrum of fraction A in Example 1.

[0036] Figure 2 This is the HPLC spectrum of fraction B in Example 1.

[0037] Figure 3 This is the HPLC spectrum of fraction C in Example 1. DETAILED DESCRIPTION

[0038] The present invention will be further described below in conjunction with the embodiments.

[0039] The Momordica grosvenori raw material used in the embodiment of the present invention was purchased from Shaoyang, Hunan, wherein the mass percentage content of mogroside V was 0.452%, the mass percentage content of 11-oxy-mogroside V was 0.053%, and the mass percentage content of simenoside I was 0.015%; the macroporous adsorption resin used in the embodiment of the present invention was purchased from Xi'an Lanxiao Technology New Materials Co., Ltd.; the preparative chromatographic filler MCI GEL CHP used in the embodiment of the present invention was purchased from Mitsubishi Chemical of Japan; the raw materials or chemical reagents used in the embodiment of the present invention, unless otherwise specified, were obtained through conventional commercial channels.

[0040] In the examples of the present invention, the contents of various mogrosides were detected by high performance liquid chromatography external standard method.

[0041] Preparation Example 1

[0042] 10g astaxanthin and hydroxypropyl-β-astaxanthin were added in a molar ratio of 1:2, and 40L 95% ethanol was added. The mixture was stirred for 24 hours under nitrogen atmosphere to reach embedding equilibrium. The mixture was first rotary evaporated to a paste, and then freeze-dried to obtain a powder, which was the astaxanthin@hydroxypropyl-β-cyclodextrin inclusion complex. After testing, the astaxanthin content in the astaxanthin@hydroxypropyl-β-cyclodextrin inclusion complex was 15.4wt%.

[0043] Example 1

[0044] (1) Pulse electric field ultrasonic extraction: Take 100 kg of fresh Momordica grosvenori fruit, crush it, add 400 L of alkaline aqueous solution containing 0.4 wt % NaOH and 1 wt % of the inclusion compound prepared in Preparation Example 1, and continuously pass through a pulse electric field ultrasonic extraction device (electric field strength of 30 kV / cm, ultrasonic frequency of 120 kHz, power of 100 W) at room temperature, coarsely filter, and centrifuge to obtain a Momordica grosvenori extract;

[0045] (2) Membrane filtration: The monk fruit extract was filtered through an alumina ceramic membrane with a pore size of 0.4 μm and a filtration pressure of 0.8 MPa to obtain a ceramic membrane filtrate;

[0046] (3) Enrichment of total glycosides of Momordica grosvenori: The ceramic membrane filtrate was passed through a macroporous adsorption resin column (wherein the macroporous adsorption resin was labeled D101, the resin dosage was 15 L, and the height-to-diameter ratio of the resin column was 4:1) at a flow rate of 1 BV / hour until the column was completed. The effluent was first washed with 3 BV of pure water until it was neutral, and then analyzed with 2 BV of 70% ethanol by volume, and the ethanol analysis solution was collected for later use;

[0047] (4) Purification of total glycosides of Momordica grosvenori: The ethanol analytical solution was passed through an alumina resin column (wherein the type of alumina was neutral chromatography alumina, 100-200 mesh, 5 kg in amount, the height-to-diameter ratio of the resin column was 5:1, the alumina resin was first immersed in dilute sulfuric acid at pH 3 for 30 min, and then washed with pure water until the effluent was neutral), the flow rate of the column was 0.5 BV / hour, the effluent of the alumina resin column was collected, concentrated to be alcohol-free, and a refined total glycoside concentrate was obtained, which was dried to obtain 0.741 kg of total glycosides extract of Momordica grosvenori;

[0048] (5) Preparative chromatographic separation: Take 200 g of total glycosides extract of Momordica grosvenori, dissolve it in pure water to a solid concentration of about 10%, and then filter it with a microporous filter membrane. Pass the filtrate through an MCI preparative chromatographic column (wherein the filler model is MCI GEL CHP20SS, the filler amount is 1 L, and the column pressure is 2 bar). Control the flow rate at 2 BV / h, and use 5 BV of pure water, 3 BV of methanol with a volume fraction of 10%, 3 BV of methanol with a volume fraction of 20%, 5 BV of methanol with a volume fraction of 35%, 3 BV of methanol with a volume fraction of 50%, 1 BV of methanol with a volume fraction of 62%, 1 BV of methanol with a volume fraction of 70%, and 5 BV of methanol with a volume fraction of 100% to elute the chromatographic column, and collect the eluents of each section for standby use.

[0049] (6) Concentration and drying: Select the eluate eluted with 20% methanol, concentrate and dry to obtain fraction A, weighing 13.58 g; select the eluate eluted with 35% methanol, concentrate and dry to obtain fraction B, weighing 115.72 g; select the eluate eluted with 62% methanol, concentrate and dry to obtain fraction C, weighing 3.75 g.

[0050] Determined by high performance liquid chromatography external standard method, in fraction A, the mass percentage content of 11-oxo-mogroside V was 97.10%, and the yield of 11-oxo-mogroside was 92.18%; the mass percentage content of mogroside V in fraction B was 97.71%, and the yield of mogroside V was 92.68%; the mass percentage content of simonoside I in fraction C was 96.23%, and the yield of simonoside I was 89.14%.

[0051] Figure 1 is the HPLC spectrum of fraction A, Figure 2 is the HPLC spectrum of fraction B, Figure 3 This is the HPLC spectrum of fraction C.

[0052] Example 2

[0053] (1) Pulse electric field ultrasonic extraction: Take 100 kg of fresh Momordica grosvenori fruit, crush it, add 0.6 wt% KOH, 1.5 wt% alkaline aqueous solution of the inclusion compound prepared in Preparation Example 1, and continuously pass it through a pulse electric field ultrasonic extraction device (electric field strength of 50 kV / cm, ultrasonic frequency of 60 kHz and power of 100 W) at room temperature, coarsely filter and centrifuge to obtain Momordica grosvenori extract;

[0054] (2) membrane filtration: filtering the monk fruit extract with a ceramic membrane to obtain a ceramic membrane filtrate;

[0055] (3) Enrichment of total glycosides of Momordica grosvenori: The ceramic membrane filtrate was passed through a macroporous adsorption resin column (wherein the macroporous adsorption resin was labeled LX-T28, the resin dosage was 15 L, and the height-to-diameter ratio of the resin column was 4:1) at a flow rate of 1 BV / hour until the column was completed. The effluent was first washed with 3 BV of pure water until it was neutral, and then 3 BV of 65% ethanol was used for analysis. The ethanol analysis solution was collected for later use;

[0056] (4) Purification of total glycosides of Momordica grosvenori: The ethanol analytical solution was passed through an alumina resin column (wherein the type of alumina was alkaline chromatography alumina, 100-200 mesh, 4 kg in amount, the height-to-diameter ratio of the resin column was 4:1, the alumina resin was first immersed in dilute hydrochloric acid at pH 4 for 10 min, and then washed with pure water until the effluent was neutral), the flow rate of the column was 0.5 BV / hour, the effluent of the alumina resin column was collected, concentrated to be alcohol-free, and a refined total glycoside concentrate was obtained, which was dried to obtain 0.78 kg of total glycosides extract of Momordica grosvenori;

[0057] (5) Preparative chromatographic separation: Take 200 g of total glycosides extract of Momordica grosvenori, dissolve it in pure water to a solid concentration of about 10%, and then filter it with a microporous filter membrane. Pass the filtrate through an MCI preparative chromatographic column (wherein the filler model is MCI GEL CHP20P, the filler amount is 1 L, and the column pressure is 2.5 bar). Control the flow rate to 2 BV / h, and use 5 BV of pure water, 3 BV of 15% ethanol by volume, 3 BV of 25% ethanol by volume, 5 BV of 40% ethanol by volume, 3 BV of 55% ethanol by volume, 1 BV of 69% ethanol by volume, 1 BV of 80% ethanol by volume, and 5 BV of 95% ethanol to elute the chromatographic column in sequence, and collect the eluents of each section for standby use.

[0058] (6) Concentration and drying: Select the eluate eluted with 25% ethanol, concentrate and dry to obtain fraction A, weighing 12.83 g; select the eluate eluted with 40% ethanol, concentrate and dry to obtain fraction B, weighing 109.19 g; select the eluate eluted with 69% ethanol, concentrate and dry to obtain fraction C, weighing 3.61 g.

[0059] Determined by high performance liquid chromatography external standard method, the content of 11-oxo-mogroside V in fraction A was 97.32wt%, and the yield of 11-oxo-mogroside V was 91.89%; the content of mogroside V in fraction B was 98.28wt%, and the yield of mogroside V was 92.64%; the content of simanoside I in fraction C was 95.09wt%, and the yield of simanoside I was 89.26%.

[0060] Comparative Example 1

[0061] Step (1) adopts traditional hot water countercurrent extraction: 100 kg of fresh Momordica grosvenori fruit is taken, crushed, countercurrently extracted with 500 L of hot water at a temperature of 95° C., roughly filtered, and centrifuged to obtain a Momordica grosvenori extract. The other steps are consistent with Example 1.

[0062] Determined by high performance liquid chromatography external standard method, the content of 11-oxo-mogroside V in fraction A was 97.28wt%, and the yield of 11-oxo-mogroside V was 84.90%; the content of mogroside V in fraction B was 98.35wt%, and the yield of mogroside V was 86.17%; the content of simanoside I in fraction C was 92.48%, and the yield of simanoside I was 53.13%.

[0063] Comparative Example 2

[0064] The other conditions are the same as those in Example 1, except that in step (1), the alkaline aqueous solution contains 0.4wt% NaOH and 1wt% astaxanthin, i.e. astaxanthin is used to replace the inclusion complex prepared in Preparation Example 1. Final product: In fraction A, the content of 11-oxo-mogroside V is 97.14wt%, and the yield of 11-oxo-mogroside V is 90.06%; in fraction B, the content of mogroside V is 98.30wt%, and the yield of mogroside V is 91.84%; in fraction C, the content of simenoside I is 94.32wt%, and the yield of simenoside I is 73.26%.

[0065] Comparative Example 3

[0066] The other conditions are the same as those in Example 1, except that in step (1), the alkaline aqueous solution contains 0.4wt% NaOH and 1wt% vitamin C, that is, vitamin C is used to replace the inclusion compound prepared in Preparation Example 1. Final product: In fraction A, the mass percentage content of 11-oxo-mogroside V is 97.21%, and the yield of 11-oxo-mogroside is 92.35%; the mass percentage content of mogroside V in fraction B is 97.64%, and the yield of mogroside V is 92.70%; in fraction C, the mass percentage content of simenoside I is 96.41%, and the yield of simenoside I is 78.37%.

[0067] Comparative Example 4

[0068] The other conditions are the same as those in Example 1, except that in step (4), the alumina resin column is not pre-treated by acid water impregnation and washing. Final product: In fraction A, the mass percentage content of 11-oxo-mogroside V is 97.14%, and the yield of 11-oxo-mogroside is 92.19%; the mass percentage content of mogroside V in fraction B is 97.65%, and the yield of mogroside V is 92.57%; the mass percentage content of simenoside I in fraction C is 93.31%, and the yield of simenoside I is 75.49%.

Claims

1. A method for separating mogrosides by pulsed electric field ultrasonic extraction and preparative chromatography, characterized in that: The following steps are involved: (1) Pulsed electric field ultrasonic extraction: Take the monk fruit, crush it, add alkaline water, continuously pass it through a pulsed electric field ultrasonic extraction device, coarsely filter it, and centrifuge it to obtain a monk fruit extract; the alkaline water contains a reducing agent @ cyclodextrin inclusion complex; (2) membrane filtration: filtering the monk fruit extract with a ceramic membrane to obtain a ceramic membrane filtrate; (3) Enrichment of total glycosides from Momordica grosvenori: The filtrate from the ceramic membrane was passed through a macroporous adsorption resin column until the column was completely loaded, first washed with pure water, then analyzed with an alcohol-water solution, and the ethanol analysis solution was collected for later use; (4) Purification of total glucosides of Momordica grosvenori: the alcohol aqueous solution analytical solution is passed through an alumina resin column, the effluent is concentrated to obtain a concentrated solution, and the concentrated solution is dried to obtain a total glucosides extract of Momordica grosvenori; the alumina resin column is impregnated with acid water and washed; (5) Preparative chromatography separation: Take a portion of the total glycosides extract of Momordica grosvenori, dissolve it in pure water, filter it through a microporous filter membrane, pass the filtrate through an MCI preparative chromatography column, perform gradient elution, and collect the eluate in sections for later use; (6) Concentration and drying: The eluates collected in the segments are concentrated and dried respectively to obtain different types of mogroside components.

2. The preparation method according to claim 1, characterized in that: In step (1), the reducing agent @ cyclodextrin inclusion complex is obtained by embedding the reducing agent in a cyclodextrin substance, the reducing agent is selected from at least one of astaxanthin and β-carotene, and the cyclodextrin substance is selected from at least one of β-cyclodextrin, hydroxypropyl-β-cyclodextrin, and sulfobutyl ether-β-cyclodextrin; the reducing agent content in the reducing agent @ cyclodextrin inclusion complex is 15-20wt%, and the reducing agent @ cyclodextrin inclusion complex in alkaline water is 1-5wt%.

3. The preparation method according to claim 1, characterized in that: In step (1), the preparation method of the reducing agent @ cyclodextrin inclusion complex comprises the following steps: adding the reducing agent and the cyclodextrin substance in a molar ratio of 1:1-3, adding an alcohol aqueous solution, stirring under an inert atmosphere until the embedding reaches equilibrium, spinning and then freeze-drying to obtain the reducing agent @ cyclodextrin inclusion complex; Preferably, the alcohol aqueous solution is an ethanol solution with a volume fraction of 70-95%, and the volume ratio of the alcohol aqueous solution to the reducing agent is 3-5L:1g.

4. The preparation method according to claim 1, characterized in that: In step (1), the alkali is one or more of sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate or sodium bicarbonate, the mass percentage concentration of the alkaline water is 0.1-2wt%, preferably 0.4-0.7wt%, and the amount of the alkaline water is 3-10 times the weight of the monk fruit raw material, preferably 4-5 times.

5. The preparation method according to claim 1, characterized in that: In step (1), the electric field strength of the pulse electric field is 0.5-10 kV / cm, the number of times is 10 to 30 times, the power of the ultrasound is 20-1000 w, the ultrasound frequency is 60-150 kHz, the pulse electric field ultrasonic extraction time is 10-100 min, and the extraction temperature is 5-30° C., preferably 10-25° C.

6. The preparation method according to claim 1, characterized in that: In step (2), the material of the ceramic membrane is zirconium oxide, aluminum oxide, or titanium oxide; the pore size of the ceramic membrane is 0.1-1.2 μm; and the filtration pressure of the ceramic membrane is 0.6-1.0 MPa.

7. The preparation method according to claim 1, characterized in that: In step (3), the macroporous adsorption resin used in the macroporous adsorption resin column is a non-polar macroporous adsorption resin, a medium-polar macroporous adsorption resin or a weak-polar macroporous adsorption resin, or a combination of two or more of the above resins; Preferably, the macroporous adsorption resin is selected from at least one of D101, LX-T28, and AB-8, the volume ratio of the resin to the mass of the fresh monk fruit is 10-30L:100kg, the height-to-diameter ratio of the resin column is 3-8:1, and the column flow rate is 0.5-5Bv / h; the pure water washing amount is 2-4BV, and the alcohol-water solution analysis amount is 2-3BV, and the alcohol-water solution is a 50-70% solution of methanol and / or ethanol.

8. The preparation method according to claim 1, characterized in that: In step (4), the types of alumina include neutral alumina, acidic alumina and basic alumina, and the amount of alumina used is 1-10wt% of the weight of the monk fruit raw material.

9. The preparation method according to claim 1, characterized in that: In step (4), the alumina resin column is immersed in acid water with a pH of 3-4 for 10-60 minutes, and then washed with water until the effluent is close to neutral; preferably, the acid water is at least one of dilute hydrochloric acid and dilute sulfuric acid.

10. The preparation method according to claim 1, characterized in that: In step (5), the filler types used in the MCI preparative chromatography include polystyrene / divinylbenzene type (PS / DVB) and acrylate type, and the particle size of the filler is 4-300 μm; the gradient elution is performed by using an alcohol aqueous solution according to the increasing volume fraction of the alcohol, and the alcohol is selected from methanol and / or ethanol, and the volume fraction of the alcohol increases from 0% to 100%; Preferably, in step (5), the gradient elution is controlled at a flow rate of 0.5-2BV / h, and elution is performed in sequence using 3-5BV pure water, 3-5BV 10-20% alcohol aqueous solution by volume, 3-5BV 20-30% alcohol aqueous solution, 3-5BV 30-40% alcohol aqueous solution, 3-5BV 40-60% alcohol aqueous solution, 1-2BV 60-70% alcohol aqueous solution, and 1-2BV>70% alcohol aqueous solution.

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