WO2021012424A1 - 一种高含量罗汉果皂苷单体产品的制备方法及其应用 - Google Patents

一种高含量罗汉果皂苷单体产品的制备方法及其应用 Download PDF

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WO2021012424A1
WO2021012424A1 PCT/CN2019/111851 CN2019111851W WO2021012424A1 WO 2021012424 A1 WO2021012424 A1 WO 2021012424A1 CN 2019111851 W CN2019111851 W CN 2019111851W WO 2021012424 A1 WO2021012424 A1 WO 2021012424A1
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mogroside
content
extract
eluent
ethanol
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PCT/CN2019/111851
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English (en)
French (fr)
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李伟
黄华学
赵冠宇
刘永胜
叶桂芳
陈江林
黄�俊
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湖南艾达伦科技有限公司
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
    • A23L27/30—Artificial sweetening agents
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
    • A23L27/30—Artificial sweetening agents
    • A23L27/33—Artificial sweetening agents containing sugars or derivatives
    • A23L27/34—Sugar alcohols
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J17/00—Normal steroids containing carbon, hydrogen, halogen or oxygen, having an oxygen-containing hetero ring not condensed with the cyclopenta(a)hydrophenanthrene skeleton
    • C07J17/005—Glycosides
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

  • the invention relates to a preparation method of mogroside saponins, in particular to a preparation method and application of a high-content mogroside saponins monomer product.
  • Momordica grosvenori is a unique plant in my country, mainly produced in Lingui County and Yongfu County of Guilin City. It is known as a fairy fruit and is the first batch of precious Chinese medicinal materials for medicine and food announced by the Ministry of Health.
  • the fruits and leaves of Momordica grosvenori contain the triterpene saponins of Momordica grosvenori, as well as a large amount of fructose, more than ten kinds of essential amino acids, fatty acids, flavonoids, vitamin C, and trace elements.
  • As the main ingredient of the sweetener of Momordica grosvenori it is a kind of triterpene glucoside with sweet taste. The sweetness is up to 300 times sweeter than sucrose, but it does not generate heat.
  • Luo Han Guo is not only a traditional Chinese medicine for relieving cough and reducing phlegm, but also a sweet tea. Its specific effects include: clearing away heat and moisturizing lungs, relieving throat and sound, moistening the intestines, improving immune function, protecting liver, inhibiting bacteria, Lower blood sugar, anti-tumor, etc.
  • Mogroside is a low-calorie, high-sweetness, non-nutritive, non-fermented sweetener. As a food, it is safe and non-toxic. According to the national compulsory standard "GB2760 Food Additives Use Standards", it is stipulated that Mogroside can be used in unlimited quantities For all kinds of food.
  • mogroside V The types and content specifications of the common Momordica grosvenori extract on the market are usually mogroside V with a content of ⁇ 50%. Mogroside V products with a content of more than 50%, and other mogroside products other than mogroside V are very rare. This is mainly due to the limitation of production technology. Although there are public reports in the literature on the preparation of high-content mogroside V, these technologies only remain in the laboratory stage and do not have the feasibility of batch industrialization. In addition, the separation and purification of other mogrosides other than mogroside V (ie: 11-O-mogroside V, mogroside VI, siamenoside I, mogroside IV, mogroside III), especially for industrial preparation methods Report, not yet seen.
  • CN107936079A discloses a method for preparing high-purity mogroside V, which uses fresh mogroside V as raw material through pretreatment, countercurrent extraction, multi-stage filtration, strong alkaline anion exchange resin decolorization, concentration, and drying to obtain a content of 65% -68% Mogroside V products.
  • this method has an extraction process, it does not have a process of enriching mogroside V.
  • the subsequent processing completely relies on the removal of impurities to increase the content of mogroside V.
  • mogroside V also has the risk of being removed together with impurities. Therefore, the yield of this method and the content of mogroside V in the product are both low.
  • CNCN104892717A discloses an industrial-grade preparative liquid chromatographic separation method for mogroside V. It uses crude mogroside products as raw materials and passes through the steps of dissolution filtration, centrifugal ultrafiltration, preparative liquid chromatography separation, concentration, and drying to obtain a purity of ⁇ 95% Mogroside V. This method can only prepare a high-content mogroside monomer.
  • CN106967142A discloses a method for simultaneously extracting mogroside V, VI and 11-O-based glycoside V. It uses the refined mother liquor of mogroside V as a raw material, through n-butanol extraction, concentration and crystallization, polyamide chromatography, gradient After elution, concentration, drying and other steps, three high-content monomers of mogroside V, mogroside VI and 11-O-based glycoside V are obtained. This method can only prepare three high-content mogroside monomers, and cannot obtain the remaining three mogroside monomers-siamenoside I, mogroside IV and mogroside III.
  • the technical problem to be solved by the present invention is to overcome the above-mentioned defects in the prior art, and provide a simple process, strong operability, no pollution, no toxic and harmful chemical solvents, green and environmental protection, and each monomer of mogroside in the product With high content and high yield, it is suitable for the preparation method of industrialized production of high-content mogroside saponins monomer products.
  • a compound sweetener prepared with high-content mogroside saponins monomer is provided.
  • a preparation method of high-content mogrosides includes the following steps:
  • Dispersion and dissolution take the crude extract of Luo Han Guo, add ethanol and stir to disperse, to obtain the crude extract of Luo Han Guo ethanol mixed solution;
  • step (2) Precipitation and precision filtration: the ethanol mixed solution of the crude extract of Momordica grosvenori obtained in step (1) is allowed to stand for precipitation, and the insoluble matter is precipitated, and the ceramic membrane is used for precision filtration to collect the ceramic membrane filtrate;
  • step (3) Dilution and nanofiltration: dilute the ceramic membrane filtrate obtained in step (2) with water, and then filter with a nanofiltration membrane to collect the nanofiltration membrane retentate;
  • the mass percentage content of the total saponins of mogroside in the crude extract of momordica grosvenori is 20%-95%, and the mass percentage content of mogroside V is 25%-70%.
  • the volume percentage concentration of the ethanol is 60%-90%, the ratio of the volume of the ethanol to the weight of the crude extract of Luo Han Guo is 5-10, and the volume consumption unit of the ethanol is L, The weight of the crude extract of Luo Han Guo kg.
  • concentration of ethanol used is high, the effect of dispersing and dissolving the crude extract of Luo Han Guo is better: first, the total saponins in the crude extract of Luo Han Guo can be dissolved in ethanol to the greatest extent, and the second is high Concentration ethanol cannot dissolve macromolecular substances such as pectin and protein.
  • the macromolecular substances such as pectin and protein in the crude extract of Luo Han Guo are suspended in a high-concentration ethanol solution to reduce the dissolved impurities in the solution to reduce subsequent preparations. Difficulty of chromatographic separation. If the volume percentage concentration of ethanol is too low, the macromolecular substances such as pectin and protein will be partially dissolved in ethanol, and the purpose of complete suspension and free pectin, protein and other impurities cannot be achieved; if the volume percentage concentration of ethanol is too high, it will As a result, the total saponins of Luo Han Guo cannot be fully dissolved. If the amount of high-concentration ethanol is too small, the total saponins of Momordica grosvenori will not be fully dissolved; if the amount of high-concentration ethanol is too much, it will cause waste of materials and energy.
  • the time for the standing precipitation is 0.5 to 2.0 hours.
  • the purpose of the static precipitation is to fully analyze and settle macromolecular substances such as pectin and protein that are insoluble in high ethanol. If the time of standing for precipitation is too short, the precipitation of macromolecular substances such as pectin and protein will not be complete; if the time of standing for precipitation is too long, the production cycle will be prolonged.
  • the material of the ceramic membrane is zirconia, alumina or titania
  • the pore size of the ceramic membrane is 0.1-1.0 ⁇ m
  • the pressure of the precision filtration is 0.1-0.3Mpa.
  • the purpose of precision filtration with ceramic membranes is to remove macromolecular substances such as pectin and protein that precipitate and settle in the ethanol solution.
  • the amount of water added is 0.5 to 4 times (more preferably 1 to 3 times) the volume of the ceramic membrane filtrate; the volume fraction of ethanol in the diluted ceramic membrane filtrate is 10% to 50 %.
  • the purpose of diluting the ceramic membrane filtrate with water is to reduce the volume fraction of ethanol in the ceramic membrane filtrate so that the volume fraction of ethanol in the diluted low ceramic membrane filtrate is the same as the volume fraction of ethanol in the eluent used in the subsequent step (5) Consistent.
  • the molecular weight cut-off of the nanofiltration membrane is 300-1000 Da (more preferably 500-800 Da), and the pressure of the nanofiltration is 0.4-0.6Mpa.
  • the purpose of nanofiltration is to remove glucose, fructose and inorganic salts in the low-ceramic membrane filtrate (from the crude extract of Luo Han Guo).
  • the small molecular substances such as glucose, fructose and inorganic salts will not pass through the nanofiltration membrane and be removed; Excessive filtration pressure will cause the total saponins of momordica grosvenori to pass through the nanofiltration membrane, resulting in the loss of total saponins of momordica grosvenori.
  • the preparative chromatographic column is a low pressure, medium pressure or high pressure compression column, and the column pressure is 0.1Mpa-50Mpa (further preferably, the column pressure is 0.5-35Mpa, and even more preferably the column pressure is 35Mpa).
  • the type of chromatographic filler is C4, C6, C8, C12 or C18, and the particle size of the chromatographic filler is 5-30 ⁇ m.
  • the amount of the chromatographic filler is 5-20 times the weight of the crude extract of Luo Han Guo.
  • the purpose of loading the nanofiltration membrane retentate to a preparative chromatographic column equipped with chromatographic packing is to adsorb and concentrate the total saponins of momordica grosvenori in the nanofiltration membrane retentate in the preparative chromatographic column.
  • the height-to-diameter ratio of the chromatographic packing packing column is 1-20:1 (preferably 2.5-11:1).
  • the eluent is ethanol-water solution, wherein the volume fraction of ethanol is 10%-50%.
  • the purpose of eluting the preparative chromatographic column with ethanol-water solution is to sequentially elute the mogroside monomers adsorbed in the preparative chromatographic column and collect them in sections to obtain a single high-content mogroside monomer.
  • volume fraction of ethanol is too small, multiple mogroside monomers adsorbed on the preparative chromatographic column will not be able to desorb; if the volume fraction of ethanol is too large, multiple mogroside monomers adsorbed on the preparative chromatographic column will quickly After being desorbed, different mogroside monomers may be desorbed at the same time, or the time interval between successive outflows is too short, which makes it impossible to collect in sections, and thus cannot obtain high-content mogroside monomers.
  • the elution is isocratic.
  • the purpose of isocratic elution is to simplify the difficulty of operation and facilitate the reuse of solvent after recovery.
  • the elution flow rate is 5-20 BV/h (further preferably 13-18 BV/h). If the elution flow rate is too slow, the total collection time will be too long and the production cycle will be too long; if the elution flow rate is too fast, it will not be conducive to accurate segmented collection, resulting in low content of mogroside monomers.
  • the segmented collection refers to the timing from the time the eluent enters the preparative chromatographic column, 15 to 23 minutes is eluent A section, and 20 to 35 minutes is eluent B section. 32 ⁇ 40min is eluent C section, 38 ⁇ 45min is eluent D section, 45 ⁇ 65min is eluent E section, and 65 ⁇ 85min is eluent F section.
  • the concentration is vacuum concentration under reduced pressure.
  • the drying is microwave drying.
  • step (6) in the stepwise concentration and drying, after the eluent A is concentrated and dried, the high-content mogroside monomer obtained is 11-O-mogroside V.
  • step (6) in the stepwise concentration and drying, after the eluent B is concentrated and dried, the high-content mogroside monomer obtained is mogroside V.
  • step (6) in the stepwise concentration and drying, after the eluate C is concentrated and dried, the high-content mogroside monomer obtained is mogroside VI.
  • step (6) in the stepwise concentration and drying, after the eluent is concentrated and dried in step D, the high-content mogroside monomer obtained is siamenoside I.
  • step (6) in the stepwise concentration and drying, after the eluent E is concentrated and dried, the high-content mogroside monomer obtained is mogroside IV.
  • step (6) in the stepwise concentration and drying, after the eluent F is concentrated and dried, the high-content mogroside monomer obtained is mogroside III.
  • the different types of high-content mogroside monomers obtained after drying are all white solids, and their content ranges are all 75.0% to 99.9%.
  • the mechanism of the method of the present invention In addition to the total saponins of Luo Han Guo, the components in the crude extract of Luo Han Guo also contain macromolecular impurities such as protein and pectin, as well as small molecular impurities such as fructose, glucose, and inorganic salts.
  • the invention first uses the principle that high-concentration ethanol does not dissolve protein, pectin and other macromolecular substances, and removes this type of macromolecular impurities through precision filtration; and then uses nanofiltration membranes to permeate small molecules such as fructose, glucose, and inorganic salts.
  • the principle of the substance is that the relatively large molecular weight cutoff of mogrosides is to remove small molecular impurities such as fructose, glucose, and inorganic salts; after both macromolecular impurities and small molecular impurities are removed, the remaining components in the ethanol solution are mogrosides.
  • the total saponins of Momordica grosvenori are adsorbed and enriched in a preparative chromatographic column, and the preparative chromatographic column is eluted with an eluent. Since the polarity of each mogroside is different, the order of elution is different.
  • the saponin with the most polarity is always eluted first and flows out of the preparative column, and the saponin with the least polarity is eluted last and flows out of the preparative column. Therefore, by collecting the eluate at different time periods, different mogroside monomers can be obtained.
  • the high-content mogroside monomer prepared in the present invention is used to prepare a compound sweetener, and the compound sweetener refers to one or more high-content mogroside monomers, and one or more natural high sweetness Mixtures (liquid or solid) containing sweeteners, synthetic sweeteners, sugar alcohols, monosaccharides, disaccharides, polysaccharides or plant/microbial extracts in any ratio.
  • the compound sweetener refers to one or more high-content mogroside monomers, and one or more natural high sweetness Mixtures (liquid or solid) containing sweeteners, synthetic sweeteners, sugar alcohols, monosaccharides, disaccharides, polysaccharides or plant/microbial extracts in any ratio.
  • the high-content mogroside monomer is one or more of 11-O-mogroside V, mogroside V, mogroside VI, simenoside I, mogroside IV, and mogroside III, and its content The range is 75.0% to 99.9%.
  • the natural high-intensity sweetener is stevioside, rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, and rebaudioside F , Duke glycoside A, sweet tea glycosides, steviol diglycoside, glycyrrhizin, thaumatin, dimethoate protein, capersin, curculoside A, betadine, blazine or one of Several kinds.
  • the synthetic sweetener is sucralose, aspartame, alitame, sodium saccharin, neotame, acesulfame K, cyclamate, neohesperidin dihydrochalcone.
  • the sugar alcohol is sorbitol, maltitol, xylitol, isomalt, mannitol, lactitol, erythritol.
  • the monosaccharide is one or more of glucose, fructose, psicose, galactose, ribose, and deoxyribose.
  • the disaccharide is one or more of sucrose, lactose, maltose and trehalose.
  • the polysaccharide is starch, dextrin, glycogen, cellulose, hemicellulose, chitin, polyfructose (inulin), fructo-oligosaccharide, polygalactose, galacto-oligosaccharide, glycosamine One or more of glycans.
  • the plant/microbial extract is Momordica grosvenori extract (Mogroside V content ⁇ 25%), Boluohui extract, Andrographis paniculata extract, onion extract, guarana extract, ivy extract, Tribulus Terrestris Extract, Rutin Extract, Ginseng Extract, Green Tea Extract (Theanine, Tea Polyphenols), Grape Seed Extract, Valerian Extract, Capsicum Red Pigment, Uncaria Extract, Artichoke Extract , Silybum marianum extract, liangmianzi extract, hijiki extract, rhodiola extract, pine bark extract, pomegranate peel extract, persimmon leaf extract, astragalus extract, Ganoderma lucidum extract, green coffee beans Extract, Acanthopanax senticosus extract, Magnolia bark extract, Schisandra extract, Haematococcus pluvialis extract, Yeast extract, Chlorella extract, Ginkgo biloba extract, Garcinia cambogia extract, Pupil water
  • the compound sweetener of the present invention can be used in foods, medicines, condiments, health products, cosmetics, flavors and fragrances.
  • the beneficial effects of the present invention are as follows: (1) The maximum content of mogroside monomers obtained can reach 99.1%, and the highest yield is nearly 95%; (2) six kinds of high-content mogroside monomers can be obtained simultaneously, which fills up the industry’s Blank, far-reaching meaning; (3) Simple process, strong operability, no pollution, no toxic and harmful chemical solvents, environmental protection, and suitable for industrial production.
  • Figure 1 is the HPLC chart of the crude extract of Luo Han Guo in Example 1;
  • FIG. 1 is an HPLC chart of 11-O-mogroside V prepared in Example 1;
  • Figure 3 is an HPLC chart of mogroside V prepared in Example 1;
  • Figure 4 is an HPLC chart of mogroside VI prepared in Example 1;
  • Figure 5 is an HPLC chart of siamenoside I prepared in Example 1;
  • Fig. 6 is an HPLC chart of mogroside IV prepared in Example 1;
  • Fig. 7 is an HPLC chart of mogroside III prepared in Example 1.
  • HPLC high performance liquid chromatography
  • step (2) Precipitation and precision filtration: The ethanol solution of the crude extract of Momordica grosvenori obtained in step (1) is allowed to stand for 1 hour to precipitate a large amount of insoluble matter, and the ceramic membrane is used for precision filtration; the ceramic membrane used is made of alumina and ceramic membrane The pore size is 0.5 ⁇ m, the pressure of ceramic membrane filtration is 0.2Mpa; the ceramic membrane filtrate is collected;
  • step (3) Dilution and nanofiltration: add the ceramic membrane filtrate obtained in step (2) to 1.67 times the volume of pure water, dilute the volume fraction of ethanol to 30%, and then filter with a nanofiltration membrane; the molecular weight cut-off of the nanofiltration membrane used 500Da, the pressure of nanofiltration is 0.5Mpa; collect the retentate of the nanofiltration membrane;
  • mogroside V (white solid) was obtained, with a weight of 315.58 g and a content of 98.21%.
  • mogroside VI (white solid) was obtained, with a weight of 18.73 g and a content of 96.76%.
  • siamenoside I (white solid) was obtained, with a weight of 10.45 g and a content of 97.11%.
  • mogroside IV (white solid) was obtained with a weight of 8.74 g and a content of 95.56%.
  • mogroside III (white solid) is obtained, weighing 7.13 g, and content 96.03%.
  • step (2) Precipitation and precision filtration: The ethanol solution of the crude extract of Momordica grosvenori obtained in step (1) is allowed to stand for 1.5 hours to precipitate a large amount of insoluble matter, which is precision filtered with a ceramic membrane; the material of the ceramic membrane is zirconia, ceramic membrane The pore size is 0.2 ⁇ m, and the pressure of ceramic membrane filtration is 0.3Mpa. Collect ceramic membrane filtrate;
  • step (3) Dilution and nanofiltration: add the ceramic membrane filtrate obtained in step (2) to 1 volume of pure water, dilute the volume fraction of ethanol to 35%, and then filter with nanofiltration membrane; the molecular weight cut-off of the nanofiltration membrane used It is 800Da, and the pressure of nanofiltration is 0.4Mpa. Collect the retentate of the nanofiltration membrane;
  • the elution and collection of preparative chromatography use eluent to elute the preparative chromatography column in step (4), and collect the eluent in different time periods; the eluent used is 35 in volume fraction % Ethanol-water solution, the elution flow rate is 17BV/hour; from the time the eluent enters the preparative chromatographic column, 17 ⁇ 22min is eluent A section, 23 ⁇ 34min is eluent B section, and 35 ⁇ 39min is Eluent C segment, 40 ⁇ 44min is eluent D segment, 45 ⁇ 63min is eluent E segment, and 65 ⁇ 83min is eluent F segment;
  • mogroside V (white solid) was obtained, with a weight of 312.29 g and a content of 99.10%.
  • mogroside VI (white solid) was obtained, weighing 17.52 g, and content 97.67%.
  • siamenoside I (white solid) was obtained, with a weight of 9.83 g and a content of 97.28%.
  • mogroside IV (white solid) was obtained, with a weight of 8.60 g and a content of 96.19%.
  • mogroside III (white homologous) is obtained, with a weight of 6.92 g and a content of 97.33%.
  • step (2) Precipitation and precision filtration: The ethanol solution of the crude extract of Momordica grosvenori obtained in step (1) is allowed to stand for 2 hours to precipitate a large amount of insoluble matter, which is then precision filtered with a ceramic membrane; the ceramic membrane used is made of titanium oxide and ceramic membrane The pore size is 0.8 ⁇ m, the pressure of ceramic membrane filtration is 0.1Mpa; the ceramic membrane filtrate is collected;
  • step (3) Dilution and nanofiltration: add 1 volume of pure water to the ceramic membrane filtrate obtained in step (2), dilute the volume fraction of ethanol to 32.5%, and then filter with nanofiltration membrane; the molecular weight cut-off of the nanofiltration membrane used It is 300Da, the pressure of nanofiltration is 0.6Mpa; collect the retentate of nanofiltration membrane;
  • the elution and collection of preparative chromatography eluting the preparative chromatography column in step (4) with an eluent, and collect the eluent in different time periods; the eluent used is 32.5 in volume fraction % Ethanol-water solution, the elution flow rate is 18 BV/hour.
  • the timing starts when the eluent enters the preparative chromatographic column, 18 ⁇ 23min is eluent A section, 24 ⁇ 35min is eluent B section, 36 ⁇ 40min is eluent C section, and 42 ⁇ 45min is eluent D section , 46 ⁇ 65min is eluent E section, 67 ⁇ 85min is eluent F section;
  • mogroside V (white solid) was obtained, with a weight of 313.37 g and a content of 98.59%.
  • mogroside VI (white solid) was obtained, with a weight of 18.10 g and a content of 97.67%.
  • siamenoside I (white solid) was obtained, with a weight of 10.07 g and a content of 96.91%.
  • mogroside IV (white solid) was obtained with a weight of 8.55 g and a content of 96.87%.
  • mogroside III (white solid) is obtained, with a weight of 6.99 g and a content of 97.24%.
  • step (2) Precipitation and precision filtration: The ethanol solution of the crude extract of Momordica grosvenori obtained in step (1) is allowed to stand for 1 hour to precipitate a large amount of insoluble matter, and the ceramic membrane is used for precision filtration; the ceramic membrane used is made of alumina and ceramic membrane The pore size is 0.5 ⁇ m, the pressure of ceramic membrane filtration is 0.2Mpa; the ceramic membrane filtrate is collected;
  • step (3) Dilution and nanofiltration: add the ceramic membrane filtrate obtained in step (2) to 1.43 times the volume of pure water, dilute the volume fraction of ethanol to 35%, and then filter with nanofiltration membrane; the molecular weight cut-off of the nanofiltration membrane used It is 500Da, and the pressure of nanofiltration is 0.45Mpa. Collect the retentate of the nanofiltration membrane;
  • the elution and collection of preparative chromatography use eluent to elute the preparative chromatography column in step (4), and collect the eluent in different time periods; the eluent used is 35 in volume fraction % Ethanol-water solution, the elution flow rate is 13 BV/hour.
  • the timing starts when the eluent enters the preparative chromatographic column. 17-22 min is eluent A section, 23-33 min is eluent B section, 34-38 min is eluent C section, and 39-44 min is eluent D section , 45 ⁇ 64min is eluent E section, 65 ⁇ 83min is eluent F section.
  • mogroside V (white solid) was obtained, with a weight of 490.93 g and a content of 98.07%.
  • mogroside VI (white solid) was obtained, with a weight of 29.52 g and a content of 96.60%.
  • siamenoside I (white solid) was obtained, weighing 15.79 g and content 97.36%.
  • mogroside IV (white solid) was obtained with a weight of 12.93 g and a content of 96.69%.
  • mogroside III (white solid) was obtained, with a weight of 10.63 g and a content of 96.52%.
  • step (2) Precipitation and precision filtration: The ethanol solution of the crude extract of Momordica grosvenori obtained in step (1) is allowed to stand for 2 hours to precipitate a large amount of insoluble matter, which is then precision filtered with a ceramic membrane; the ceramic membrane used is made of zirconia, ceramic membrane The pore size is 0.2 ⁇ m, the pressure of ceramic membrane filtration is 0.3Mpa; the ceramic membrane filtrate is collected;
  • step (3) Dilution and nanofiltration: add the ceramic membrane filtrate obtained in step (2) to 1.27 times the volume of pure water, dilute the volume fraction of ethanol to 33%, and then filter with nanofiltration membrane; the molecular weight cut-off of the nanofiltration membrane used It is 800Da, and the pressure of nanofiltration is 0.4Mpa. Collect the retentate of the nanofiltration membrane;
  • mogroside V (white solid) was obtained, with a weight of 478.79 g and a content of 98.83%.
  • mogroside VI (white solid) was obtained, with a weight of 28.38 g and a content of 97.49%.
  • siamenoside I (white solid) was obtained, weighing 15.36 g and content 97.07%.
  • mogroside IV (white solid) was obtained with a weight of 12.31 g and a content of 96.41%.
  • mogroside III (white solid) is obtained, with a weight of 10.25 g and a content of 96.24%.
  • step (2) Precipitation and precision filtration: The ethanol solution of the crude extract of Momordica grosvenori obtained in step (1) is allowed to stand for 1.5 hours to precipitate a large amount of insoluble matter, which is precision filtered with a ceramic membrane; the material of the ceramic membrane is titanium oxide, ceramic membrane The pore size is 0.8 ⁇ m, the pressure of ceramic membrane filtration is 0.1Mpa; the ceramic membrane filtrate is collected;
  • step (3) Dilution and nanofiltration: add the ceramic membrane filtrate obtained in step (2) to 1 volume of pure water, dilute the volume fraction of ethanol to 30%, and then filter with nanofiltration membrane; the molecular weight cut-off of the nanofiltration membrane used 300Da, the pressure of nanofiltration is 0.55Mpa; collect the retentate of nanofiltration membrane;
  • mogroside V (white solid) was obtained, with a weight of 489.64 g and a content of 98.27%.
  • mogroside VI (white solid) was obtained, with a weight of 28.69 g and a content of 96.42%.
  • siamenoside I (white solid) was obtained with a weight of 15.17 g and a content of 96.71%.
  • mogroside IV (white solid) was obtained, with a weight of 12.24 g and a content of 97.35%.
  • mogroside III (white solid) is obtained, with a weight of 9.98 g and a content of 96.97%.
  • step (3) Dilution and nanofiltration: add the ceramic membrane filtrate obtained in step (2) to 1.67 times the volume of pure water, dilute the volume fraction of ethanol to 30%, and then filter with a nanofiltration membrane; the molecular weight cut-off of the nanofiltration membrane used 500Da, the pressure of nanofiltration is 0.5Mpa. Collect the retentate of the nanofiltration membrane;
  • mogroside V (white solid) was obtained, with a weight of 503.34 g and a content of 80.50%.
  • mogroside VI (white solid) was obtained, with a weight of 33.21 g and a content of 77.56%.
  • siamenoside I (white solid) was obtained, weighing 18.31 g and content 79.35%.
  • mogroside III (white solid) was obtained, with a weight of 11.75 g and a content of 80.67%.
  • step (2) Precipitation and precision filtration: The ethanol solution of the crude extract of Momordica grosvenori obtained in step (1) is allowed to stand for 1 hour to precipitate a large amount of insoluble matter, which is then precision filtered with a ceramic membrane; the ceramic membrane used is made of zirconia, ceramic membrane The pore size is 0.2 ⁇ m, the pressure of ceramic membrane filtration is 0.3Mpa; the ceramic membrane filtrate is collected;
  • step (3) Dilution and nanofiltration: add the ceramic membrane filtrate obtained in step (2) to 1 volume of pure water, dilute the volume fraction of ethanol to 35%, and then filter with nanofiltration membrane; the molecular weight cut-off of the nanofiltration membrane used 800Da, the pressure of nanofiltration is 0.4Mpa; collect the retentate of the nanofiltration membrane;
  • the elution and collection of preparative chromatography use eluent to elute the preparative chromatography column in step (4), and collect the eluent in different time periods; the eluent used is 35 in volume fraction % Ethanol-water solution, the elution flow rate is 16BV/hour; from the time the eluent enters the preparative chromatographic column, 17 ⁇ 22min is eluent A section, 23 ⁇ 34min is eluent B section, and 35 ⁇ 39min is Eluent C segment, 40 ⁇ 44min is eluent D segment, 45 ⁇ 63min is eluent E segment, and 65 ⁇ 83min is eluent F segment;
  • mogroside V (white solid) was obtained, with a weight of 501.57 g and a content of 82.72%.
  • mogroside VI (white solid) was obtained, with a weight of 32.87 g and a content of 76.91%.
  • siamenoside I (white solid) was obtained, weighing 17.58 g and content 80.65%.
  • mogroside IV (white solid) was obtained with a weight of 14.96 g and a content of 80.69%.
  • mogroside III (white solid) was obtained, with a weight of 11.07 g and a content of 81.43%.
  • step (2) Precipitation and precision filtration: The ethanol solution of the crude extract of Momordica grosvenori obtained in step (1) is allowed to stand for 1.5 hours to precipitate a large amount of insoluble matter, which is precision filtered with a ceramic membrane; the material of the ceramic membrane is titanium oxide, ceramic membrane The pore size is 0.8 ⁇ m, the pressure of ceramic membrane filtration is 0.1Mpa; the ceramic membrane filtrate is collected;
  • step (3) Dilution and nanofiltration: add 1 volume of pure water to the ceramic membrane filtrate obtained in step (2), dilute the volume fraction of ethanol to 32.5%, and then filter with nanofiltration membrane; the molecular weight cut-off of the nanofiltration membrane used It is 300Da, the pressure of nanofiltration is 0.6Mpa; collect the retentate of nanofiltration membrane;
  • mogroside V (white solid) was obtained, with a weight of 502.07 g and a content of 82.69%.
  • mogroside VI (white solid) was obtained, with a weight of 32.98 g and a content of 78.78%.
  • siamenoside I (white solid) was obtained, with a weight of 17.06 g and a content of 80.75%.
  • mogroside IV (white solid) was obtained, with a weight of 13.89 g and a content of 82.61%.
  • mogroside III (white solid) was obtained, with a weight of 10.23 g and a content of 82.36%.
  • This implementation provides a compound sweetener containing high-content mogroside monomers, which includes the following components in weight percentage:
  • Mogroside V weight percentage content: 98.21%): 11.5%
  • Rebaudioside A (weight percentage content: 98.06%): 32.0%;
  • This implementation provides a compound sweetener containing high-content mogroside monomers, which includes the following components in weight percentage:
  • Neohesperidin dihydrochalcone weight percentage content: 98.06%): 5.0%;
  • This implementation provides a compound sweetener containing high-content mogroside monomers, which includes the following components in weight percentage:
  • Mogroside V weight percentage content: 98.21%): 15.0%;
  • Rebaudioside A (weight percentage content: 98.06%): 82.5%.
  • This implementation provides a compound sweetener containing high-content mogroside monomers, which includes the following components in weight percentage:
  • Mogroside V weight percentage content: 98.21%): 10.0%
  • Rebaudioside A (weight percentage content: 98.06%): 38.5%.
  • This implementation provides a compound sweetener containing high-content mogroside monomers, which includes the following components in weight percentage:
  • Mogroside V weight percentage content: 98.21%): 15.0%;
  • Glycyrrhizin (weight percentage content: 98.06%): 22.0%;
  • This implementation provides a compound sweetener containing high-content mogroside monomers, which includes the following components in weight percentage:
  • Mogroside V weight percentage content: 98.21%): 15.0%;
  • Neohesperidin dihydrochalcone weight percentage content: 98.06%): 5.0%;
  • Polyfructose 40%;
  • Green tea extract (theanine): 40%.
  • This implementation provides a compound sweetener containing high-content mogroside monomers, which includes the following components in weight percentage:
  • Yeast extract 15%.
  • This implementation provides a compound sweetener containing high-content mogroside monomers, which includes the following components in weight percentage:
  • Simenoside I weight percentage content: 97.11%): 8%;
  • Rebaudioside D (weight percentage content: 96.67%): 15%;
  • Chrysanthemum extract 27.0%.
  • This implementation provides a compound sweetener containing high-content mogroside monomers, which includes the following components in weight percentage:
  • Mogroside V weight percentage content: 80.50%): 50.0%
  • This implementation provides a compound sweetener containing high-content mogroside monomers, which includes the following components in weight percentage:
  • Mogroside V (weight percentage content: 98.21%): 5.0%
  • Rebaudioside A (weight percentage content: 98.06%): 90.0%
  • This implementation provides a compound sweetener containing high-content mogroside monomers, which includes the following components in weight percentage:
  • Mogroside V weight percentage content: 98.21%): 10.0%
  • Rebaudioside A (weight percentage content: 98.06%): 60.0%

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Abstract

一种高含量罗汉果皂苷单体产品的制备方法，包括以下步骤：(1)分散与溶解；(2)沉淀与精密过滤；(3)稀释与纳滤：将陶瓷膜滤液加水稀释，再用纳滤膜过滤，收集纳滤膜截留液；(4)制备色谱的上样；(5)制备色谱的洗脱与收集；(6)浓缩与干燥。本发明还包括所述高含量罗汉果皂苷单体产品制备复配甜昧剂的应用。本发明工艺简单，绿色环保，产品中罗汉果皂苷各单体含量高、收率高，适宜于工业化生产。

Description

一种高含量罗汉果皂苷单体产品的制备方法及其应用 技术领域
本发明涉及一种罗汉果皂苷的制备方法，具体涉及一种高含量罗汉果皂苷单体产品的制备方法及其应用。
背景技术
罗汉果是我国特有的一种植物，主要产于桂林市的临桂县和永福县，被人们誉为神仙果，是卫生部首批公布的药食两用名贵的中药材。罗汉果的果实和和叶均含有罗汉果三萜皂苷，还有大量的果糖、十多种人体必需氨基酸、脂肪酸、黄酮类化合物、维生素C、微量元素等。罗汉果皂苷作为罗汉果甜味剂的主要成分，是一种具有甜味的三萜烯葡萄糖苷，甜度最高比蔗糖甜300倍，但是不产生热量，是一种饮料、糖果行业的名贵原料，是蔗糖的最佳替代品。常饮罗汉果茶可以防多种疾病，适用于痰多咳嗽以及百日咳患者饮用。罗汉果不仅是一种止咳化痰的中药，更是一种口味甘甜的茶饮，具体的功效包括：清热润肺、利咽开音、润肠通便、提高免疫功能、保护肝脏、抑菌、降血糖、抗肿瘤等。
罗汉果皂苷作为属低热量、高甜度、非营养、非发酵型的甜味剂，作为食品，是安全无毒的，在国家强制标准《GB2760食品添加剂使用标准》中规定，罗汉果甜可不限量用于各类食品。
目前市场上常见的罗汉果提取物的种类及含量规格，通常是罗汉果苷V、含量≤50％。含量超过50％的罗汉果苷V产品，以及罗汉果苷V以外的其他罗汉果皂苷产品很少。这主要是因为生产技术的制约，尽管有文献公开报道高含量罗汉果苷V的制备方法，但是，这些技术仅仅停留在实验室阶段，并不具备批量工业化的可行性。此外，对罗汉果苷V以外的其他罗汉果皂苷(即：11-O-罗汉果苷V、罗汉果苷VI、赛门苷I、罗汉果苷IV、罗汉果苷III)的分离及纯化，尤其是工业化制备方法的报道，尚未见到。
CN107936079A公开了一种制备高纯度罗汉果甜苷V的方法，是以鲜罗汉果为原料，通过预处理、逆流提取、多级过滤、强碱性阴离子交换树脂脱色、浓缩、干燥，得到含量在65％-68％罗汉果甜苷V产品。该方法虽然有提取过程，但是没有富集罗汉果甜苷V的过程，后续处理完全是依靠杂质的除去而提高罗汉果甜苷V的含量。在除去杂质的过程，罗汉果甜苷V同样有跟杂质一起被除去的风险，因此，该方法的收率和产品中的罗汉果甜苷V的含量都偏低。
CNCN104892717A公开了一种罗汉果甜苷V的工业级制备液相色谱分离方法，是以罗汉果粗制品为原料，通过溶解过滤、离心超滤、制备液相色谱分离、浓缩、干燥等步骤， 得到纯度≥95％的罗汉果甜苷V。该方法只能制备一种高含量的罗汉果皂苷单体。
CN106967142A公开了一种同时提取罗汉果甜苷V、VI和11-O基苷V的方法，是以罗汉果甜苷V精制母液为原料，通过正丁醇萃取、浓缩析晶、聚酰胺层析、梯度洗脱、浓缩、干燥等步骤，得到罗汉果苷V、罗汉果苷VI和11-O基苷V三种高含量的单体。该方法只能制备三种高含量的罗汉果皂苷单体，无法得到其余的三种罗汉果皂苷单体——赛门苷I、罗汉果苷IV和罗汉果苷III。
目前尚未见到有关同时提取制备高含量罗汉果皂苷各单体(即：11-O-罗汉果苷V、罗汉果苷V、罗汉果苷VI、赛门苷I、罗汉果苷IV、罗汉果苷III)方法及复配甜味剂的报道。
发明内容
本发明所要解决的技术问题是，克服现有技术存在的上述缺陷，提供一种工艺简单，可操作性强，无污染，不使用有毒有害的化学溶剂，绿色环保，产品中罗汉果皂苷各单体含量高、收率高，适宜于工业化生产的高含量罗汉果皂苷单体产品的制备方法。同时，提供一种用高含量罗汉果皂苷单体制备的复配甜味剂。
本发明解决其技术问题所采用的技术方案如下：一种高含量罗汉果皂苷的制备方法，包括以下步骤：
(1)分散与溶解：取罗汉果粗提物，加入乙醇搅拌分散，得罗汉果粗提物乙醇混合溶液；
(2)沉淀与精密过滤：将步骤(1)所得的罗汉果粗提物乙醇混合溶液静置，沉淀，析出不溶物，用陶瓷膜精密过滤，收集陶瓷膜滤液；
(3)稀释与纳滤：将步骤(2)所得的陶瓷膜滤液加水稀释，再用纳滤膜过滤，收集纳滤膜截留液；
(4)制备色谱的上样：将步骤(3)所得的纳滤膜截留液上样至装有色谱填料的制备色谱柱；
(5)制备色谱的洗脱与收集：用洗脱剂对步骤(4)中的制备色谱柱进行洗脱，分段收集不同时间段的洗脱液；
(6)浓缩与干燥：将步骤(5)所得的不同时间段的洗脱液分别浓缩，干燥，得到不同种类的高含量罗汉果皂苷单体。
优选地，步骤(1)中，所述罗汉果粗提物中罗汉果总皂苷的质量百分比含量为20％～95％，其中罗汉果苷V的质量百分比含量为25％～70％。
优选地，步骤(1)中，所述乙醇的体积百分比浓度为60％～90％，所述乙醇的体积与罗汉果粗提物的重量之比为5～10，乙醇的体积用量单位为L，罗汉果粗提物的重量kg。 研究表明，所用乙醇的浓度较高时，对罗汉果粗提物进行分散与溶解的效果较好：第一是，可将罗汉果粗提物中的总皂苷最大限度溶解于乙醇，第二是，高浓度乙醇无法溶解果胶、蛋白质等大分子物质，据此，使罗汉果粗提物中的果胶、蛋白质等大分子物质悬浮于高浓度乙醇溶液中，减少溶液中溶解的杂质，以降低后续制备色谱分离的难度。若乙醇的体积百分比浓度过低，将导致果胶、蛋白质等大分子物质部分溶解于乙醇，无法达到彻底悬浮和游离果胶、蛋白质等杂质的目的；若乙醇的体积百分比浓度过高，又会导致罗汉果总皂苷无法充分溶解。若高浓度乙醇的用量过少，同样会导致罗汉果总皂苷无法充分溶解；若高浓度乙醇的用量过多，将造成物料和能源的浪费。
优选地，步骤(2)中，所述静置沉淀的时间为0.5～2.0小时。静置沉淀的目的是使高度乙醇不溶解的果胶、蛋白质等大分子物质充分析出并沉降。若静置沉淀的时间过短，果胶、蛋白质等大分子物质的析出将不彻底；若静置沉淀的时间过长，将延长生产的周期。
优选地，步骤(2)中，所述陶瓷膜的材质为氧化锆、氧化铝或氧化钛，陶瓷膜的孔径为0.1～1.0μm，精密过滤的压力为0.1～0.3Mpa。使用陶瓷膜精密过滤的目的，是除去乙醇溶液中析出和沉降的果胶、蛋白质等大分子物质。
优选地，步骤(3)中，所述加水的量为陶瓷膜滤液体积的0.5～4倍(进一步优选1～3倍)；稀释后的陶瓷膜滤液中，乙醇的体积分数为10％～50％。用水稀释陶瓷膜滤液的目的，是降低陶瓷膜滤液中乙醇的体积分数，使稀释后的低陶瓷膜滤液中的乙醇的体积分数，与后续步骤(5)所用洗脱剂中的乙醇的体积分数一致。
优选地，步骤(3)中，所述纳滤膜的截留分子量为300～1000Da(进一步优选500～800Da)，纳滤的压力为0.4～0.6Mpa。纳滤的目的是除去低陶瓷膜滤液中的葡萄糖、果糖和无机盐等小分子物质(来自于罗汉果粗提物)。若纳滤膜的截留分子量过小或纳滤的压力过小，都将导致葡萄糖、果糖和无机盐等小分子物质无法透过纳滤膜而除去；若纳滤膜的截留分子量过大或纳滤的压力过大，都将导致罗汉果总皂苷透过纳滤膜，造成罗汉果总皂苷的损失。
优选地，步骤(4)中，所述制备色谱柱为低压、中压或高压压缩柱，柱压为0.1Mpa～50Mpa(进一步优选柱压为0.5～35Mpa，更进一步优选柱压为35Mpa)。
优选地，步骤(4)中，所述的色谱填料类型为C4、C6、C8、C12或C18，色谱填料的粒度为5～30μm。
优选地，步骤(4)中，所述色谱填料的用量为罗汉果粗提物重量的5～20倍。将纳滤膜截留液上样至装有色谱填料的制备色谱柱的目的，是将纳滤膜截留液中的罗汉果总皂苷吸附并富集在制备色谱柱中。若色谱填料的用量过少，将导致罗汉果总皂苷无法完全被吸 附，造成各个单一罗汉果皂苷成分的洗脱收率偏低；若色谱填料的用量过多，不但会造成资源的浪费，还将增大洗脱的难度，延长洗脱的时间，造成浪费。
优选地，步骤(4)中，所述色谱填料装柱的高径比为1～20∶1(优选2.5～11∶1)。
优选地，步骤(4)中，所述上样的流速为5～20BV/h(进一步优选8～13BV/h)；1BV＝色谱填料装柱体积。
优选地，步骤(5)中，所述的洗脱剂为乙醇-水溶液，其中乙醇的体积分数为10％～50％。使用乙醇-水溶液对制备色谱柱洗脱的目的是，将吸附在制备色谱柱中的罗汉果皂苷单体按顺序依次洗脱，并分段收集，从而得到单一的高含量罗汉果皂苷单体。若乙醇的体积分数过小，将无法对吸附在制备色谱柱中的多个罗汉果皂苷单体解吸；若乙醇的体积分数过大，吸附在制备色谱柱中的多个罗汉果皂苷单体将快速的被解吸，不同的罗汉果皂苷单体可能会同时被解吸，或者先后流出的时间间隙过短，导致无法分段收集，从而无法得到高含量的罗汉果皂苷单体。
优选地，步骤(5)中，所述的洗脱为等度洗脱。等度洗脱的目的是，简化操作的难度，有利于溶剂回收后的重复利用。
优选地，步骤(5)中，所述洗脱流速为5～20BV/h(进一步优选13～18BV/h)。若洗脱的流速过慢，将导致收集的总时间过长，造成生产周期过长；若洗脱的流速过快，将不利于精准的分段收集，造成罗汉果皂苷单体的含量偏低。
优选地，步骤(5)中，所述的分段收集指的是，从洗脱剂进入制备色谱柱开始计时，15～23min为洗脱液A段，20～35min为洗脱液B段，32～40min为洗脱液C段，38～45min为洗脱液D段，45～65min为洗脱液E段，65～85min为洗脱液F段。
优选地，步骤(6)中，所述的浓缩为真空减压浓缩。
优选地，步骤(6)中，所述的干燥为微波干燥。
优选地，步骤(6)中，所述的分段浓缩与干燥中，洗脱液A段浓缩、干燥后，得到的高含量罗汉果皂苷单体为11-O-罗汉果苷V。
优选地，步骤(6)中，所述的分段浓缩与干燥中，洗脱液B段浓缩、干燥后，得到的高含量罗汉果皂苷单体为罗汉果苷V。
优选地，步骤(6)中，所述的分段浓缩与干燥中，洗脱液C段浓缩、干燥后，得到的高含量罗汉果皂苷单体为罗汉果苷VI。
优选地，步骤(6)中，所述的分段浓缩与干燥中，洗脱液D段浓缩、干燥后，得到的高含量罗汉果皂苷单体为赛门苷I。
优选地，步骤(6)中，所述的分段浓缩与干燥中，洗脱液E段浓缩、干燥后，得到 的高含量罗汉果皂苷单体为罗汉果苷IV。
优选地，步骤(6)中，所述的分段浓缩与干燥中，洗脱液F段浓缩、干燥后，得到的高含量罗汉果皂苷单体为罗汉果苷III。
优选地，步骤(6)中，所述干燥后得到的不同种类的高含量罗汉果皂苷单体均为白色固体，其含量范围均为75.0％～99.9％。
本发明方法的机理：罗汉果粗提取物中的成分，除了罗汉果总皂苷，还含有蛋白质、果胶等大分子杂质，以及果糖、葡萄糖、无机盐等小分子杂质。本发明先利用适当的高浓度乙醇不溶解蛋白质、果胶等大分子物质的原理，通过精密过滤除去这一类大分子杂质；再利用纳滤膜可以透过果糖、葡萄糖、无机盐等小分子物质的原理，截留分子量相对比较大的罗汉果皂苷，由此将果糖、葡萄糖、无机盐等小分子杂质除去；大分子杂质和小分子杂质都除去之后，乙醇溶液中剩余的成分为罗汉果总皂苷。将罗汉果总皂苷吸附并富集在制备色谱柱中，用洗脱剂对制备色谱柱进行洗脱。由于各个罗汉果皂苷的极性大小各有不同，被洗脱的顺序各不相同。在洗脱的过程中，总是极性最大的皂苷最先被洗脱而流出制备色谱柱，极性最小的皂苷最后被洗脱而流出制备色谱柱。因此，在不同的时间段收集洗脱液，可以得到不同的罗汉果皂苷单体。
本发明制备的高含量罗汉果皂苷单体用于制备复配甜味剂，所述复配甜味剂是指一种或多种高含量罗汉果皂苷单体，与一种或多种天然高倍甜味剂、合成甜味剂、糖醇、单糖、双糖、多糖或植物/微生物提取物，以任意比例组合成的含有甜味剂的混合物(液体或固体)。
优选的，所述高含量罗汉果皂苷单体是11-O-罗汉果苷V、罗汉果苷V、罗汉果苷VI、赛门苷I、罗汉果苷IV、罗汉果苷III中的一种或多种，其含量范围均为75.0％～99.9％。
优选的，所述的天然高倍甜味剂是甜菊苷、瑞鲍迪苷A、瑞鲍迪苷B、瑞鲍迪苷C、瑞鲍迪苷D、瑞鲍迪苷E、瑞鲍迪苷F、杜克苷A、甜茶苷、甜菊双糖苷、甘草甜素、奇甜蛋白、应乐果甜蛋白、马槟榔甜蛋白、仙茅甜蛋白、倍他丁、布拉齐因中的一种或几种。
优选的，所述的合成甜味剂是三氯蔗糖、阿斯巴甜、阿力甜、糖精钠、纽甜、安赛蜜、甜蜜素、新橙皮苷二氢查耳酮。
优选的，所述的糖醇是山梨糖醇、麦芽糖醇、木糖醇、异麦芽糖醇、甘露糖醇、乳糖醇、赤藓糖醇。
优选的，所述的单糖是葡糖糖、果糖、阿洛酮糖、半乳糖、核糖、脱氧核糖中的一种或几种。
优选的，所述的双糖是蔗糖、乳糖、麦芽糖、海藻糖中的一种或几种。
优选的，所述的多糖是淀粉、糊精、糖元、纤维素、半纤维素、壳多糖、多聚果糖(菊 粉)、低聚果糖、多聚半乳糖、低聚半乳糖、糖胺聚糖中的一种或几种。
优选的，所述的植物/微生物提取物是罗汉果提取物(罗汉果苷V含量≤25％)、博落回提取物、穿心莲提取物、洋葱提取物、瓜拉纳提取物、常春藤提取物、刺蒺藜提取物、芦丁提取物、人参提取物、绿茶提取物(茶氨酸、茶多酚)、葡萄籽提取物、缬草提取物、辣椒红色素、钩藤提取物、朝鲜蓟提取物、水飞蓟提取物、两面针提取物、羊栖菜提取物、红景天提取物、松树皮提取物、石榴皮提取物、柿叶提取物、黄芪提取物、灵芝提取物、绿咖啡豆提取物、刺五加提取物、厚朴提取物、五味子提取物、雨生红球藻提取物、酵母浸出物、小球藻提取物、银杏叶提取物、藤黄果提取物、柔毛水杨梅提取物、亚麻籽提取物、千层塔提取物、燕麦提取物、桑葚提取物、麦芽提取物、莴苣提取物、薄荷提取物、菊花提取物、虎杖提取物、生姜提取物、白桦树皮提取物、皱叶酸模提取物、山药提取物、枸杞提取物、白柳皮提取物、育亨宾提取物、丝兰提取物、葫芦巴提取物、淫羊藿提取物、杜仲提取物、灯盏花提取物、月见草提取物、吴茱萸提取物、满山红提取物、钩果草提取物、蔓越橘提取物、玉米须提取物、可可提取物、枳实提取物、蛇床子提取物、接骨木提取物、菊苣提取物、积雪草提取物、白屈菜提取物、水皂角提取物、卡图巴提取物、西红柿提取物、辣椒提取物、大麦草提取物、杨梅树皮提取物、白桦树提取物、越橘提取物、苦橙提取物、黑加仑提取物、黑苦薄荷提取物、黑芝麻提取物、红茶提取物、黑莓提取物、墨角藻提取物、赐福蓟草提取物、蓝旗提取物、蓝莓提取物、玻璃苣籽提取物、乳香提取物、蓝升麻提取物、苎麻根提取物、地瓜儿苗提取物、牛蒡根提取物、金雀花提取物、艳紫铆提取物、蜂斗菜提取物、紫花苜蓿提取物、知母提取物、泽泻提取物、药蜀葵提取物、西印度樱桃提取物、西洋参提取物、天门冬提取物、山金车提取物、芹菜提取物、苹果提取物、芦荟提取物、茴香籽提取物、葛根提取物、多香果提取物、当归提取物、菜蓟叶提取物、白术提取物、巴西莓提取物、艾叶提取物、南非醉茄提取物、巴拿巴提取物、罗勒提取物、苦瓜提取物、东革阿里提取物、红景天提取物、加纳籽提取物、仙人掌提取物、射干提取物、竹茹提取物、巴戟天提取物、车前草提取物、黄柏提取物、地黄提取物、花生衣提取物、芙蓉花提取物、木槿提取物、山楂提取物、圣罗勒提取物、栀子提取物、龙胆根提取物、老鹤草提取物、高良姜提取物、大蒜提取物、浙贝母提取物、芒果籽提取物、小麦草提取物、白芸豆提取物、艾草提取物、葫芦巴提取物、何首乌提取物、紫锥菊提取物、土木香提取物、桉树叶提取物、小米草提取物、蒲公英提取物、达米阿拉提取物、菟丝子提取物、番红花提取物、延胡索提取物、痉挛树皮提取物、山茱萸提取物、香菜提取物、冬虫夏草提取物、黄连提取物、款冬提取物、党参提取物、丁香提取物、猪殃殃提取物、肉桂提取物、猴面包提取物、繁缕提取物、圣洁莓提取物、洋甘菊提取物、矢车菊提 取物、猫爪草提取物、决明子提取物、胡萝卜提取物、甘蓝提取物、假马齿笕提取物、竹子提取物、伏牛花提取物、大麦芽提取物、蜂花粉、甜菜根提取物、槟榔子提取物、黑升麻提取物、黑萝卜提取物、黑胡桃提取物、天竺葵提取物、波尔多叶提取物、芥兰提取物、糙米提取物、西印度醋粟提取物、西洋龙芽草提取物、甜杏仁提取物、木通提取物、轮叶沙参提取物、菠萝提取物、槟榔提取物、熊果叶提取物中的一种或若干种。
本发明的复配甜味剂，可运用于食品、药品、调味品、保健品、化妆品、香精香料中。
本发明的有益效果如下：(1)所得罗汉果皂苷单体的最高含量可达到99.1％，最高收率将近95％；(2)可同时获得六种高含量的罗汉果皂苷单体，填补了行业的空白，意义深远；(3)工艺简单，可操作性强，无污染，不使用有毒有害的化学溶剂，绿色环保，适宜于工业化生产。
附图说明
图1为实施例1罗汉果粗提物的HPLC图谱；
图2为实施例1制备的11-O-罗汉果苷V的HPLC图谱；
图3为实施例1制备的罗汉果苷V的HPLC图谱；
图4为实施例1制备的罗汉果苷VI的HPLC图谱；
图5为实施例1制备的赛门苷I的HPLC图谱；
图6为实施例1制备的罗汉果苷IV的HPLC图谱；
图7为实施例1制备的罗汉果苷III的HPLC图谱。
具体实施方式
下面结合实施例对本发明作进一步说明。
本发明实施例所使用的化学试剂，如无特殊说明，均通过常规商业途径获得。
实施例中，采用高效液相色谱(HPLC)外标法测定罗汉果总皂苷和各个罗汉果皂苷单体的含量。
实施例1
本实施例包括以下步骤：
(1)分散与溶解：取罗汉果粗提物0.5kg，罗汉果总皂苷含量为82.26％(其中，11-O-罗汉果苷V的含量为7.71％，罗汉果苷V的含量为65.25％，罗汉果苷VI的含量为3.90％，赛门苷I的含量为2.21％，罗汉果苷IV的含量为1.76％，罗汉果苷III的含量为1.43％)，加入体积分数为80％的乙醇5L，搅拌分散，得罗汉果粗提物高浓度乙醇溶液；
(2)沉淀与精密过滤：将步骤(1)所得的罗汉果粗提物乙醇溶液静置沉淀1小时，析出大量不溶物，用陶瓷膜精密过滤；所用陶瓷膜的材质为氧化铝，陶瓷膜的孔径为0.5 μm，陶瓷膜过滤的压力为0.2Mpa；收集陶瓷膜滤液；
(3)稀释与纳滤：将步骤(2)所得的陶瓷膜滤液加入1.67倍体积的纯水，将乙醇的体积分数稀释至30％，再用纳滤膜过滤；所用纳滤膜的截留分子量为500Da，纳滤的压力为0.5Mpa；收集纳滤膜截留液；
(4)制备色谱的上样：将步骤(3)所得的纳滤膜截留液上样至装有色谱填料的制备色谱柱；所用的色谱填料类型为C18，填料的粒径为10μm，填料的用量为4kg，色谱填料装柱的高径比为2.5∶1；所用的制备色谱柱为高压压缩柱，柱压35Mpa；上样的流速为12BV/小时(1BV＝3.75L)；
(5)制备色谱的洗脱与收集：用洗脱剂对步骤(4)中的制备色谱柱进行洗脱，分段收集不同时间段的洗脱液；所用的洗脱剂为体积分数为30％的乙醇-水溶液，洗脱的流速为15BV/小时。从洗脱剂进入制备色谱柱开始计时，15～22min为洗脱液A段，23～34min为洗脱液B段，35～40min为洗脱液C段，41～45min为洗脱液D段，46～63min为洗脱液E段，67～80min为洗脱液F段。
(6)浓缩与干燥：将步骤(5)所得的不同时间段的洗脱液(A段、B段、C段、D段、E段、F段)分别真空减压浓缩，微波干燥，得到不同种类的高含量罗汉果皂苷单体。
洗脱液A段浓缩、干燥后，得到11-O-罗汉果苷V(白色固体)，重量36.25g，含量97.55％。
洗脱液B段浓缩、干燥后，得到罗汉果苷V(白色固体)，重量315.58g，含量98.21％。
洗脱液C段浓缩、干燥后，得到罗汉果苷VI(白色固体)，重量18.73g，含量96.76％。
洗脱液D段浓缩、干燥后，得到赛门苷I(白色固体)，重量10.45g，含量97.11％。
洗脱液E段浓缩、干燥后，得到罗汉果苷IV(白色固体)，重量8.74g，含量95.56％。
洗脱液F段浓缩、干燥后，得到罗汉果苷III(白色固体)，重量7.13g，含量96.03％。
实施例2
本实施例包括以下步骤：
(1)分散与溶解：取罗汉果粗提物0.5kg，罗汉果总皂苷含量为82.26％(其中，11-O-罗汉果苷V的含量为7.71％，罗汉果苷V的含量为65.25％，罗汉果苷VI的含量为3.90％，赛门苷I的含量为2.21％，罗汉果苷IV的含量为1.76％，罗汉果苷III的含量为1.43％)。加入体积分数为70％的高浓度乙醇4.5L，搅拌分散，得罗汉果粗提物高浓度乙醇溶液；
(2)沉淀与精密过滤：将步骤(1)所得的罗汉果粗提物乙醇溶液静置沉淀1.5小时，析出大量不溶物，用陶瓷膜精密过滤；所用陶瓷膜的材质为氧化锆，陶瓷膜的孔径为0.2μm，陶瓷膜过滤的压力为0.3Mpa。收集陶瓷膜滤液；
(3)稀释与纳滤：将步骤(2)所得的陶瓷膜滤液加入1倍体积的纯水，将乙醇的体积分数稀释至35％，再用纳滤膜过滤；所用纳滤膜的截留分子量为800Da，纳滤的压力为0.4Mpa。收集纳滤膜截留液；
(4)制备色谱的上样：将步骤(3)所得的纳滤膜截留液上样至装有色谱填料的制备色谱柱；所用的色谱填料类型为C12，填料的粒径为20μm，填料的用量为4.5kg，色谱填料装柱的高径比为3∶1；所用的制备色谱柱为中压压缩柱，柱压10Mpa。上样的流速为10BV/小时(1BV＝4.4L)；
(5)制备色谱的洗脱与收集：用洗脱剂对步骤(4)中的制备色谱柱进行洗脱，分段收集不同时间段的洗脱液；所用的洗脱剂为体积分数为35％的乙醇-水溶液，洗脱的流速为17BV/小时；从洗脱剂进入制备色谱柱开始计时，17～22min为洗脱液A段，23～34min为洗脱液B段，35～39min为洗脱液C段，40～44min为洗脱液D段，45～63min为洗脱液E段，65～83min为洗脱液F段；
(6)浓缩与干燥：将步骤(5)所得的不同时间段的洗脱液(A段、B段、C段、D段、E段、F段)分别真空减压浓缩，微波干燥，得到不同种类的高含量罗汉果皂苷单体。
洗脱液A段浓缩、干燥后，得到11-O-罗汉果苷V(白色固体)，重量35.75g，含量97.52％。
洗脱液B段浓缩、干燥后，得到罗汉果苷V(白色固体)，重量312.29g，含量99.10％。
洗脱液C段浓缩、干燥后，得到罗汉果苷VI(白色固体)，重量17.52g，含量97.67％。
洗脱液D段浓缩、干燥后，得到赛门苷I(白色固体)，重量9.83g，含量97.28％。
洗脱液E段浓缩、干燥后，得到罗汉果苷IV(白色固体)，重量8.60g，含量96.19％。
洗脱液F段浓缩、干燥后，得到罗汉果苷III(白色同体)，重量6.92g，含量97.33％。
实施例3
本实施例包括以下步骤：
(1)分散与溶解：取罗汉果粗提物0.5kg，罗汉果总皂苷含量为82.26％(其中，11-O-罗汉果苷V的含量为7.71％，罗汉果苷V的含量为65.25％，罗汉果苷VI的含量为3.90％，赛门苷I的含量为2.21％，罗汉果苷IV的含量为1.76％，罗汉果苷III的含量为1.43％)；加入体积分数为65％的高浓度乙醇4L，搅拌分散，得罗汉果粗提物高浓度乙醇溶液；
(2)沉淀与精密过滤：将步骤(1)所得的罗汉果粗提物乙醇溶液静置沉淀2小时，析出大量不溶物，用陶瓷膜精密过滤；所用陶瓷膜的材质为氧化钛，陶瓷膜的孔径为0.8μm，陶瓷膜过滤的压力为0.1Mpa；收集陶瓷膜滤液；
(3)稀释与纳滤：将步骤(2)所得的陶瓷膜滤液加入1倍体积的纯水，将乙醇的体 积分数稀释至32.5％，再用纳滤膜过滤；所用纳滤膜的截留分子量为300Da，纳滤的压力为0.6Mpa；收集纳滤膜截留液；
(4)制备色谱的上样：将步骤(3)所得的纳滤膜截留液上样至装有色谱填料的制备色谱柱；所用的色谱填料类型为C8，填料的粒径为30μm，填料的用量为5kg，色谱填料装柱的高径比为3.3∶1；所用的制备色谱柱为低压压缩柱，柱压0.5Mpa。上样的流速为8BV/小时(1BV＝4.8L)；
(5)制备色谱的洗脱与收集：用洗脱剂对步骤(4)中的制备色谱柱进行洗脱，分段收集不同时间段的洗脱液；所用的洗脱剂为体积分数为32.5％的乙醇-水溶液，洗脱的流速为18BV/小时。从洗脱剂进入制备色谱柱开始计时，18～23min为洗脱液A段，24～35min为洗脱液B段，36～40min为洗脱液C段，42～45min为洗脱液D段，46～65min为洗脱液E段，67～85min为洗脱液F段；
(6)浓缩与干燥：将步骤(5)所得的不同时间段的洗脱液(A段、B段、C段、D段、E段、F段)分别真空减压浓缩，微波干燥，得到不同种类的高含量罗汉果皂苷单体。
洗脱液A段浓缩、干燥后，得到11-O-罗汉果苷V(白色固体)，重量35.07g，含量97.87％。
洗脱液B段浓缩、干燥后，得到罗汉果苷V(白色固体)，重量313.37g，含量98.59％。
洗脱液C段浓缩、干燥后，得到罗汉果苷VI(白色固体)，重量18.10g，含量97.67％。
洗脱液D段浓缩、干燥后，得到赛门苷I(白色固体)，重量10.07g，含量96.91％。
洗脱液E段浓缩、干燥后，得到罗汉果苷IV(白色固体)，重量8.55g，含量96.87％。
洗脱液F段浓缩、干燥后，得到罗汉果苷III(白色固体)，重量6.99g，含量97.24％。
实施例4
本实施例包括以下步骤：
(1)分散与溶解：取罗汉果粗提物1kg，罗汉果总皂苷含量为65.08％(其中，11-O-罗汉果苷V的含量为6.11％，罗汉果苷V的含量为51.77％，罗汉果苷VI的含量为3.10％，赛门苷I的含量为1.69％，罗汉果苷IV的含量为1.33％，罗汉果苷III的含量为1.08％)；加入体积分数为85％的高浓度乙醇8L，搅拌分散，得罗汉果粗提物高浓度乙醇溶液；
(2)沉淀与精密过滤：将步骤(1)所得的罗汉果粗提物乙醇溶液静置沉淀1小时，析出大量不溶物，用陶瓷膜精密过滤；所用陶瓷膜的材质为氧化铝，陶瓷膜的孔径为0.5μm，陶瓷膜过滤的压力为0.2Mpa；收集陶瓷膜滤液；
(3)稀释与纳滤：将步骤(2)所得的陶瓷膜滤液加入1.43倍体积的纯水，将乙醇的体积分数稀释至35％，再用纳滤膜过滤；所用纳滤膜的截留分子量为500Da，纳滤的压力 为0.45Mpa。收集纳滤膜截留液；
(4)制备色谱的上样：将步骤(3)所得的纳滤膜截留液上样至装有色谱填料的制备色谱柱；所用的色谱填料类型为C8，填料的粒径为10μm，填料的用量为12kg，色谱填料装柱的高径比为5.5∶1；所用的制备色谱柱为高压压缩柱，柱压35Mpa。上样的流速为11BV/小时(1BV＝11.5L)；
(5)制备色谱的洗脱与收集：用洗脱剂对步骤(4)中的制备色谱柱进行洗脱，分段收集不同时间段的洗脱液；所用的洗脱剂为体积分数为35％的乙醇-水溶液，洗脱的流速为13BV/小时。从洗脱剂进入制备色谱柱开始计时，17～22min为洗脱液A段，23～33min为洗脱液B段，34～38min为洗脱液C段，39～44min为洗脱液D段，45～64min为洗脱液E段，65～83min为洗脱液F段。
洗脱液A段浓缩、干燥后，得到11-O-罗汉果苷V(白色固体)，重量57.03g，含量97.50％。
洗脱液B段浓缩、干燥后，得到罗汉果苷V(白色固体)，重量490.93g，含量98.07％。
洗脱液C段浓缩、干燥后，得到罗汉果苷VI(白色固体)，重量29.52g，含量96.60％。
洗脱液D段浓缩、干燥后，得到赛门苷I(白色固体)，重量15.79g，含量97.36％。
洗脱液E段浓缩、干燥后，得到罗汉果苷IV(白色固体)，重量12.93g，含量96.69％。
洗脱液F段浓缩、干燥后，得到罗汉果苷III(白色固体)，重量10.63g，含量96.52％。
实施例5
本实施例包括以下步骤：
(1)分散与溶解：取罗汉果粗提物1kg，罗汉果总皂苷含量为65.08％(其中，11-O-罗汉果苷V的含量为6.11％，罗汉果苷V的含量为51.77％，罗汉果苷VI的含量为3.10％，赛门苷I的含量为1.69％，罗汉果苷IV的含量为1.33％，罗汉果苷III的含量为1.08％)；加入体积分数为75％的高浓度乙醇7L，搅拌分散，得罗汉果粗提物高浓度乙醇溶液；
(2)沉淀与精密过滤：将步骤(1)所得的罗汉果粗提物乙醇溶液静置沉淀2小时，析出大量不溶物，用陶瓷膜精密过滤；所用陶瓷膜的材质为氧化锆，陶瓷膜的孔径为0.2μm，陶瓷膜过滤的压力为0.3Mpa；收集陶瓷膜滤液；
(3)稀释与纳滤：将步骤(2)所得的陶瓷膜滤液加入1.27倍体积的纯水，将乙醇的体积分数稀释至33％，再用纳滤膜过滤；所用纳滤膜的截留分子量为800Da，纳滤的压力为0.4Mpa。收集纳滤膜截留液；
(4)制备色谱的上样：将步骤(3)所得的纳滤膜截留液上样至装有色谱填料的制备色谱柱；所用的色谱填料类型为C6，填料的粒径为20μm，填料的用量为15kg，色谱填 料装柱的高径比为5.8∶1；所用的制备色谱柱为中压压缩柱，柱压10Mpa；上样的流速为12BV/小时(1BV＝14.7L)；
(5)制备色谱的洗脱与收集：用洗脱剂对步骤(4)中的制备色谱柱进行洗脱，分段收集不同时间段的洗脱液；所用的洗脱剂为体积分数为33％的乙醇-水溶液，洗脱的流速为14BV/小时。从洗脱剂进入制备色谱柱开始计时，18～23min为洗脱液A段，25～34min为洗脱液B段，35～39min为洗脱液C段，40～44min为洗脱液D段，45～64min为洗脱液E段，65～82min为洗脱液F段；
(6)浓缩与干燥：将步骤(5)所得的不同时间段的洗脱液(A段、B段、C段、D段、E段、F段)分别真空减压浓缩，微波干燥，得到不同种类的高含量罗汉果皂苷单体。
洗脱液A段浓缩、干燥后，得到11-O-罗汉果苷V(白色固体)，重量56.17g，含量98.15％。
洗脱液B段浓缩、干燥后，得到罗汉果苷V(白色固体)，重量478.79g，含量98.83％。
洗脱液C段浓缩、干燥后，得到罗汉果苷VI(白色固体)，重量28.38g，含量97.49％。
洗脱液D段浓缩、干燥后，得到赛门苷I(白色固体)，重量15.36g，含量97.07％。
洗脱液E段浓缩、干燥后，得到罗汉果苷IV(白色固体)，重量12.31g，含量96.41％。洗脱液F段浓缩、干燥后，得到罗汉果苷III(白色固体)，重量10.25g，含量96.24％。
实施例6
本实施例包括以下步骤：
(1)分散与溶解：取罗汉果粗提物1kg，罗汉果总皂苷含量为65.08％(其中，11-O-罗汉果苷V的含量为6.11％，罗汉果苷V的含量为51.77％，罗汉果苷VI的含量为3.10％，赛门苷I的含量为1.69％，罗汉果苷IV的含量为1.33％，罗汉果苷III的含量为1.08％)；加入体积分数为60％的高浓度乙醇7.5L，搅拌分散，得罗汉果粗提物高浓度乙醇溶液；
(2)沉淀与精密过滤：将步骤(1)所得的罗汉果粗提物乙醇溶液静置沉淀1.5小时，析出大量不溶物，用陶瓷膜精密过滤；所用陶瓷膜的材质为氧化钛，陶瓷膜的孔径为0.8μm，陶瓷膜过滤的压力为0.1Mpa；收集陶瓷膜滤液；
(3)稀释与纳滤：将步骤(2)所得的陶瓷膜滤液加入1倍体积的纯水，将乙醇的体积分数稀释至30％，再用纳滤膜过滤；所用纳滤膜的截留分子量为300Da，纳滤的压力为0.55Mpa；收集纳滤膜截留液；
(4)制备色谱的上样：将步骤(3)所得的纳滤膜截留液上样至装有色谱填料的制备色谱柱；所用的色谱填料类型为C18，填料的粒径为30μm，填料的用量为13kg，色谱填料装柱的高径比为6.4∶1；所用的制备色谱柱为低压压缩柱，柱压0.5Mpa；上样的流速为 11BV/小时(1BV＝12.8L)；
(5)制备色谱的洗脱与收集：用洗脱剂对步骤(4)中的制备色谱柱进行洗脱，分段收集不同时间段的洗脱液；所用的洗脱剂为体积分数为30％的乙醇-水溶液，洗脱的流速为15BV/小时；从洗脱剂进入制备色谱柱开始计时，16～21min为洗脱液A段，22～33min为洗脱液B段，34～39min为洗脱液C段，40～44min为洗脱液D段，45～65min为洗脱液E段，66～85min为洗脱液F段；
(6)浓缩与干燥：将步骤(5)所得的不同时间段的洗脱液(A段、B段、C段、D段、E段、F段)分别真空减压浓缩，微波干燥，得到不同种类的高含量罗汉果皂苷单体。
洗脱液A段浓缩、干燥后，得到11-O-罗汉果苷V(白色固体)，重量56.25g，含量97.68％。
洗脱液B段浓缩、干燥后，得到罗汉果苷V(白色固体)，重量489.64g，含量98.27％。
洗脱液C段浓缩、干燥后，得到罗汉果苷VI(白色固体)，重量28.69g，含量96.42％。
洗脱液D段浓缩、干燥后，得到赛门苷I(白色固体)，重量15.17g，含量96.71％。
洗脱液E段浓缩、干燥后，得到罗汉果苷IV(白色固体)，重量12.24g，含量97.35％。
洗脱液F段浓缩、干燥后，得到罗汉果苷III(白色固体)，重量9.98g，含量96.97％。
实施例7
本实施例包括以下步骤：
(1)分散与溶解：取罗汉果粗提物2kg，罗汉果总皂苷含量为32.76％(其中，11-O-罗汉果苷V的含量为3.21％，罗汉果苷V的含量为25.65％，罗汉果苷VI的含量为1.63％，赛门苷I的含量为0.89％，罗汉果苷IV的含量为0.78％，罗汉果苷III的含量为0.60％)；加入体积分数为80％的高浓度乙醇14L，搅拌分散，得罗汉果粗提物高浓度乙醇溶液；
(2)沉淀与精密过滤：将步骤(1)所得的罗汉果粗提物乙醇溶液静置沉淀2小时，析出大量不溶物，用陶瓷膜精密过滤；所用陶瓷膜的材质为氧化铝，陶瓷膜的孔径为0.5μm，陶瓷膜过滤的压力为0.2Mpa；收集陶瓷膜滤液；
(3)稀释与纳滤：将步骤(2)所得的陶瓷膜滤液加入1.67倍体积的纯水，将乙醇的体积分数稀释至30％，再用纳滤膜过滤；所用纳滤膜的截留分子量为500Da，纳滤的压力为0.5Mpa。收集纳滤膜截留液；
(4)制备色谱的上样：将步骤(3)所得的纳滤膜截留液上样至装有色谱填料的制备色谱柱；所用的色谱填料类型为C18，填料的粒径为10μm，填料的用量为10kg，色谱填料装柱的高径比为9.7∶1；所用的制备色谱柱为高压压缩柱，柱压35Mpa；上样的流速为13BV/小时(1BV＝9.5L)；
(5)制备色谱的洗脱与收集：用洗脱剂对步骤(4)中的制备色谱柱进行洗脱，分段收集不同时间段的洗脱液；所用的洗脱剂为体积分数为30％的乙醇-水溶液，洗脱的流速为14BV/小时；从洗脱剂进入制备色谱柱开始计时，15～22min为洗脱液A段，23～34min为洗脱液B段，35～40min为洗脱液C段，41～45min为洗脱液D段，46～63min为洗脱液E段，67～80min为洗脱液F段；
(6)浓缩与干燥：将步骤(5)所得的不同时间段的洗脱液(A段、B段、C段、D段、E段、F段)分别真空减压浓缩，微波干燥，得到不同种类的高含量罗汉果皂苷单体。
洗脱液A段浓缩、干燥后，得到11-O-罗汉果苷V(白色固体)，重量65.72g，含量76.20％。
洗脱液B段浓缩、干燥后，得到罗汉果苷V(白色固体)，重量503.34g，含量80.50％。
洗脱液C段浓缩、干燥后，得到罗汉果苷VI(白色固体)，重量33.21g，含量77.56％。
洗脱液D段浓缩、干燥后，得到赛门苷I(白色固体)，重量18.31g，含量79.35％。
洗脱液E段浓缩、干燥后，得到罗汉果苷IV(白色固体)，重量15.26g，含量81.74％。
洗脱液F段浓缩、干燥后，得到罗汉果苷III(白色固体)，重量11.75g，含量80.67％。
实施例8
本实施例包括以下步骤：
(1)分散与溶解：取罗汉果粗提物2kg，罗汉果总皂苷含量为32.76％(其中，11-O-罗汉果苷V的含量为3.21％，罗汉果苷V的含量为25.65％，罗汉果苷VI的含量为1.63％，赛门苷I的含量为0.89％，罗汉果苷IV的含量为0.78％，罗汉果苷III的含量为0.60％)；加入体积分数为70％的高浓度乙醇12L，搅拌分散，得罗汉果粗提物高浓度乙醇溶液；
(2)沉淀与精密过滤：将步骤(1)所得的罗汉果粗提物乙醇溶液静置沉淀1小时，析出大量不溶物，用陶瓷膜精密过滤；所用陶瓷膜的材质为氧化锆，陶瓷膜的孔径为0.2μm，陶瓷膜过滤的压力为0.3Mpa；收集陶瓷膜滤液；
(3)稀释与纳滤：将步骤(2)所得的陶瓷膜滤液加入1倍体积的纯水，将乙醇的体积分数稀释至35％，再用纳滤膜过滤；所用纳滤膜的截留分子量为800Da，纳滤的压力为0.4Mpa；收集纳滤膜截留液；
(4)制备色谱的上样：将步骤(3)所得的纳滤膜截留液上样至装有色谱填料的制备色谱柱；所用的色谱填料类型为C12，填料的粒径为20μm，填料的用量为14kg，色谱填料装柱的高径比为11∶1；所用的制备色谱柱为中压压缩柱，柱压10Mpa；上样的流速为11BV/小时(1BV＝13.6L)；
(5)制备色谱的洗脱与收集：用洗脱剂对步骤(4)中的制备色谱柱进行洗脱，分段 收集不同时间段的洗脱液；所用的洗脱剂为体积分数为35％的乙醇-水溶液，洗脱的流速为16BV/小时；从洗脱剂进入制备色谱柱开始计时，17～22min为洗脱液A段，23～34min为洗脱液B段，35～39min为洗脱液C段，40～44min为洗脱液D段，45～63min为洗脱液E段，65～83min为洗脱液F段；
(6)浓缩与干燥：将步骤(5)所得的不同时间段的洗脱液(A段、B段、C段、D段、E段、F段)分别真空减压浓缩，微波干燥，得到不同种类的高含量罗汉果皂苷单体。
洗脱液A段浓缩、干燥后，得到11-O-罗汉果苷V(白色固体)，重量63.65g，含量77.39％。
洗脱液B段浓缩、干燥后，得到罗汉果苷V(白色固体)，重量501.57g，含量82.72％。
洗脱液C段浓缩、干燥后，得到罗汉果苷VI(白色固体)，重量32.87g，含量76.91％。
洗脱液D段浓缩、干燥后，得到赛门苷I(白色固体)，重量17.58g，含量80.65％。
洗脱液E段浓缩、干燥后，得到罗汉果苷IV(白色固体)，重量14.96g，含量80.69％。
洗脱液F段浓缩、干燥后，得到罗汉果苷III(白色固体)，重量11.07g，含量81.43％。
实施例9
本实施例包括以下步骤：
(1)分散与溶解：取罗汉果粗提物2kg，罗汉果总皂苷含量为32.76％(其中，11-O-罗汉果苷V的含量为3.21％，罗汉果苷V的含量为25.65％，罗汉果苷VI的含量为1.63％，赛门苷I的含量为0.89％，罗汉果苷IV的含量为0.78％，罗汉果苷III的含量为0.60％)；加入体积分数为65％的高浓度乙醇13L，搅拌分散，得罗汉果粗提物高浓度乙醇溶液；
(2)沉淀与精密过滤：将步骤(1)所得的罗汉果粗提物乙醇溶液静置沉淀1.5小时，析出大量不溶物，用陶瓷膜精密过滤；所用陶瓷膜的材质为氧化钛，陶瓷膜的孔径为0.8μm，陶瓷膜过滤的压力为0.1Mpa；收集陶瓷膜滤液；
(3)稀释与纳滤：将步骤(2)所得的陶瓷膜滤液加入1倍体积的纯水，将乙醇的体积分数稀释至32.5％，再用纳滤膜过滤；所用纳滤膜的截留分子量为300Da，纳滤的压力为0.6Mpa；收集纳滤膜截留液；
(4)制备色谱的上样：将步骤(3)所得的纳滤膜截留液上样至装有色谱填料的制备色谱柱；所用的色谱填料类型为C8，填料的粒径为30μm，填料的用量为12kg，色谱填料装柱的高径比为10.6∶1；所用的制备色谱柱为低压压缩柱，柱压0.5Mpa；上样的流速为9BV/小时(1BV＝11.7L)；
(5)制备色谱的洗脱与收集：用洗脱剂对步骤(4)中的制备色谱柱进行洗脱，分段收集不同时间段的洗脱液；所用的洗脱剂为体积分数为32.5％的乙醇-水溶液，洗脱的流速 为17BV/小时；从洗脱剂进入制备色谱柱开始计时，18～23min为洗脱液A段，24～35min为洗脱液B段，36～40min为洗脱液C段，42～45min为洗脱液D段，46～65min为洗脱液E段，67～85min为洗脱液F段；
(6)浓缩与干燥：将步骤(5)所得的不同时间段的洗脱液(A段、B段、C段、D段、E段、F段)分别真空减压浓缩，微波干燥，得到不同种类的高含量罗汉果皂苷单体。
洗脱液A段浓缩、干燥后，得到11-O-罗汉果苷V(白色固体)，重量63.75g，含量77.65％。
洗脱液B段浓缩、干燥后，得到罗汉果苷V(白色固体)，重量502.07g，含量82.69％。
洗脱液C段浓缩、干燥后，得到罗汉果苷VI(白色固体)，重量32.98g，含量78.78％。
洗脱液D段浓缩、干燥后，得到赛门苷I(白色固体)，重量17.06g，含量80.75％。
洗脱液E段浓缩、干燥后，得到罗汉果苷IV(白色固体)，重量13.89g，含量82.61％。
洗脱液F段浓缩、干燥后，得到罗汉果苷III(白色固体)，重量10.23g，含量82.36％。
实施例10
本实施提供一种含有高含量罗汉果皂苷单体的复配甜味剂，其包括以下重量百分比的各组分：
罗汉果苷V(重量百分比含量：98.21％)：11.5％；
瑞鲍迪苷A(重量百分比含量：98.06％)：32.0％；
赤藓糖醇：50.0％；
阿洛酮糖：6.5％。
实施例11
本实施提供一种含有高含量罗汉果皂苷单体的复配甜味剂，其包括以下重量百分比的各组分：
11-O-罗汉果苷V(重量百分比含量：97.55％)∶23.0％；
新橙皮苷二氢查尔酮(重量百分比含量：98.06％)∶5.0％；
低聚果糖：72.0％。
实施例12
本实施提供一种含有高含量罗汉果皂苷单体的复配甜味剂，其包括以下重量百分比的各组分：
罗汉果苷V(重量百分比含量：98.21％)∶15.0％；
11-O-罗汉果苷V(重量百分比含量：97.55％)∶2.5％；
瑞鲍迪苷A(重量百分比含量：98.06％)∶82.5％。
实施例13
本实施提供一种含有高含量罗汉果皂苷单体的复配甜味剂，其包括以下重量百分比的各组分：
罗汉果苷V(重量百分比含量：98.21％)∶10.0％；
11-O-罗汉果苷V(重量百分比含量：97.55％)∶1.5％；
瑞鲍迪苷A(重量百分比含量：98.06％)∶38.5％。
赤藓糖醇：50％
实施例14
本实施提供一种含有高含量罗汉果皂苷单体的复配甜味剂，其包括以下重量百分比的各组分：
罗汉果苷V(重量百分比含量：98.21％)∶15.0％；
甘草甜素(重量百分比含量：98.06％)∶22.0％；
赤藓糖醇：63.0％。
实施例15
本实施提供一种含有高含量罗汉果皂苷单体的复配甜味剂，其包括以下重量百分比的各组分：
罗汉果苷V(重量百分比含量：98.21％)∶15.0％；
新橙皮苷二氢查尔酮(重量百分比含量：98.06％)∶5.0％；
多聚果糖：40％；
绿茶提取物(茶氨酸)：40％。
实施例16
本实施提供一种含有高含量罗汉果皂苷单体的复配甜味剂，其包括以下重量百分比的各组分：
11-O-罗汉果苷V(重量百分比含量：97.55％)∶12.0％；
三氯蔗糖：35.0％；
海藻糖：38％；
酵母浸出物：15％。
实施例17
本实施提供一种含有高含量罗汉果皂苷单体的复配甜味剂，其包括以下重量百分比的各组分：
赛门苷I(重量百分比含量：97.11％)∶8％；
瑞鲍迪苷D(重量百分比含量：96.67％)∶15％；
赤藓糖醇：50.0％；
菊花提取物：27.0％。
实施例18
本实施提供一种含有高含量罗汉果皂苷单体的复配甜味剂，其包括以下重量百分比的各组分：
罗汉果苷V(重量百分比含量：80.50％)∶50.0％
罗汉果提取物(罗汉果苷V含量＝20.3％)∶50.0％
实施例19
本实施提供一种含有高含量罗汉果皂苷单体的复配甜味剂，其包括以下重量百分比的各组分：
罗汉果苷V(重量百分比含量：98.21％)∶5.0％
罗汉果提取物(罗汉果苷V含量＝20.3％)∶5.0％
瑞鲍迪苷A(重量百分比含量：98.06％)∶90.0％
实施例20
本实施提供一种含有高含量罗汉果皂苷单体的复配甜味剂，其包括以下重量百分比的各组分：
罗汉果苷V(重量百分比含量：98.21％)∶10.0％
11-O-罗汉果苷V(重量百分比含量：97.55％)∶2.0％
罗汉果提取物(罗汉果苷V含量＝15.7％)∶10.0％
瑞鲍迪苷A(重量百分比含量：98.06％)∶60.0％
赤藓糖醇：18.0％

Claims (10)

  1. 一种高含量罗汉果皂苷单体产品的制备方法，其特征在于，包括以下步骤：
    (1)分散与溶解：取罗汉果粗提物，加入乙醇搅拌分散，得罗汉果粗提物乙醇混合溶液；
    (2)沉淀与精密过滤：将步骤(1)所得的罗汉果粗提物乙醇混合溶液静置，沉淀，析出不溶物，用陶瓷膜精密过滤，收集陶瓷膜滤液；
    (3)稀释与纳滤：将步骤(2)所得的陶瓷膜滤液加水稀释，再用纳滤膜过滤，收集纳滤膜截留液；
    (4)制备色谱的上样：将步骤(3)所得的纳滤膜截留液上样至装有色谱填料的制备色谱柱；
    (5)制备色谱的洗脱与收集：用洗脱剂对步骤(4)中的制备色谱柱进行洗脱，分段收集不同时间段的洗脱液；
    (6)浓缩与干燥：将步骤(5)所得的不同时间段的洗脱液分别浓缩，干燥，得不同种类的高含量罗汉果皂苷单体。
  2. 根据权利要求1所述的高含量罗汉果皂苷产品的制备方法，其特征在于，步骤(1)中，所述罗汉果粗提物中罗汉果总皂苷的质量百分比含量为20％～95％，其中罗汉果苷V的质量百分比含量为25％～70％；所述乙醇的体积百分比浓度为60％～90％，所述乙醇的体积与罗汉果粗提物的重量之比为5～10，乙醇的体积单位为L，罗汉果粗提物的重量单位为kg。
  3. 根据权利要求1或2所述的高含量罗汉果皂苷产品的制备方法，其特征在于，步骤(2)中，所述静置沉淀的时间为0.5～2.0小时；所述陶瓷膜的材质为氧化锆、氧化铝或氧化钛，陶瓷膜的孔径为0.1～1.0μm，精密过滤的压力为0.1～0.3Mpa。
  4. 根据权利要求1～3之一所述的高含量罗汉果皂苷产品的制备方法，其特征在于，步骤(3)中，所述加入水的量为陶瓷膜滤液体积的0.5～4倍，稀释后的乙醇溶液中，乙醇的体积分数为10％～50％；所述纳滤膜的截留分子量为300～1000Da，纳滤的压力为0.4～0.6Mpa。
  5. 根据权利要求1～4之一所述的高含量罗汉果皂苷产品的制备方法，其特征在于，步骤(4)中，所述制备色谱柱为低压、中压或高压压缩柱，柱压为0.1Mpa～50Mpa；所述的色谱填料类型为C4、C6、C8、C12、C18，色谱填料的粒度为5～30μm；所述色谱填料的用量为罗汉果粗提物重量的5～20倍；所述色谱填料装柱的高径比为1～20∶1；所述上样的流速为5～20BV/h，所述1BV＝色谱填料装柱体积。
  6. 根据权利要求1～5之一所述的高含量罗汉果皂苷产品的制备方法，其特征在于，步骤(5)中，所述的洗脱剂为乙醇-水溶液，其中乙醇的体积分数为10％～50％；所述的洗脱为等度洗脱；所述洗脱流速为5～20BV/小时；所述的分段收集指的是，从洗脱剂进入制备色谱柱开始计时，15～23min为洗脱液A段，20～35min为洗脱液B段，32～40min为洗脱液C段，38～45min为洗脱液D段，45～65min为洗脱液E段，65～85min为洗脱液F段。
  7. 根据权利要求1～6之一所述的高含量罗汉果皂苷产品的制备方法，其特征在于，步骤(6)中，所述的浓缩为真空减压浓缩；所述的干燥为微波干燥。
  8. 根据权利要求1～7之一所述的高含量罗汉果皂苷产品的制备方法，其特征在于，步骤(6)中，所述的分段浓缩与干燥中，洗脱液A段浓缩、干燥后，得到的高含量罗汉果皂苷单体为11-O-罗汉果苷V；洗脱液B段浓缩、干燥后，得到的高含量罗汉果皂苷单体为罗汉果苷V；洗脱液C段浓缩、干燥后，得到的高含量罗汉果皂苷单体为罗汉果苷VI；洗脱液D段浓缩、干燥后，得到的高含量罗汉果皂苷单体为赛门苷I；洗脱液E段浓缩、干燥后，得到的高含量罗汉果皂苷单体为罗汉果苷IV；洗脱液F段浓缩、干燥后，得到的高含量罗汉果皂苷单体为罗汉果苷III。
  9. 根据权利要求1～8之一所述的高含量罗汉果皂苷产品的制备方法，其特征在于，步骤(6)中，所述干燥后得到的不同种类的高含量罗汉果皂苷单体均为白色固体，其含量范围均为75.0％～99.9％。
  10. 一种如权利要求1～9之一所述制备方法制得的高含量罗汉果皂苷单产品在复配甜味剂制备中的应用。
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