WO2025016411A1 - 一种利用母液糖制备葡萄糖基甜菊糖苷、分离纯化葡萄糖基甜菊糖苷并回收葡萄糖的方法 - Google Patents

一种利用母液糖制备葡萄糖基甜菊糖苷、分离纯化葡萄糖基甜菊糖苷并回收葡萄糖的方法 Download PDF

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WO2025016411A1
WO2025016411A1 PCT/CN2024/106037 CN2024106037W WO2025016411A1 WO 2025016411 A1 WO2025016411 A1 WO 2025016411A1 CN 2024106037 W CN2024106037 W CN 2024106037W WO 2025016411 A1 WO2025016411 A1 WO 2025016411A1
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solution
glucosyl
glucose
purifying
stevioside
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English (en)
French (fr)
Inventor
朱理平
何冬生
郭晓杰
刘浩
曹欣欣
鞠敏
臧鹏飞
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Dongtai Haorui Biotechnology Co Ltd
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Dongtai Haorui Biotechnology Co Ltd
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Priority claimed from CN202310879842.8A external-priority patent/CN116987132A/zh
Priority claimed from CN202311239776.4A external-priority patent/CN117327131A/zh
Priority claimed from CN202311848635.2A external-priority patent/CN117844886A/zh
Application filed by Dongtai Haorui Biotechnology Co Ltd filed Critical Dongtai Haorui Biotechnology Co Ltd
Priority to KR1020257026064A priority Critical patent/KR20250130672A/ko
Priority to EP24842400.4A priority patent/EP4640840A1/en
Priority to JP2025541591A priority patent/JP2026502602A/ja
Publication of WO2025016411A1 publication Critical patent/WO2025016411A1/zh
Priority to MX2025009075A priority patent/MX2025009075A/es
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/20—Preparation of compounds containing saccharide radicals produced by the action of an exo-1,4 alpha-glucosidase, e.g. dextrose
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C07H1/06—Separation; Purification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H3/00—Compounds containing only hydrogen atoms and saccharide radicals having only carbon, hydrogen, and oxygen atoms
    • C07H3/02—Monosaccharides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/22—Preparation of compounds containing saccharide radicals produced by the action of a beta-amylase, e.g. maltose
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
    • C12P19/56—Preparation of O-glycosides, e.g. glucosides having an oxygen atom of the saccharide radical directly bound to a condensed ring system having three or more carbocyclic rings, e.g. daunomycin, adriamycin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/20—Carbocyclic rings
    • C07H15/24—Condensed ring systems having three or more rings
    • C07H15/256—Polyterpene radicals

Definitions

  • the invention relates to the technical field of food additives, and in particular to a method for preparing glucose-based steviol glycoside by utilizing mother liquor sugar, separating and purifying glucose-based steviol glycoside and recovering glucose.
  • Stevioside is a natural sweetener extracted from the herbaceous plant Stevia of the Asteraceae family. It has the functional characteristics of high sweetness and low calories. Its sweetness is 100 to 300 times that of sucrose, and its calories are only 1/300 of sucrose. Stevioside has both sweetness and bitterness, and the taste of both flavors is related to the number of sugar groups connected to its main part and the type of glycosidic bonds between sugar groups.
  • Stevioside mother liquor sugar is a powder solid made by spray drying the remaining crystallized mother liquor after recovering high-purity rebaudioside A (RA) and stevioside (ST) by solution extraction.
  • RA rebaudioside A
  • ST stevioside
  • stevioside mother liquor sugar is sold as a low-priced sweetener; another idea is to separate the steviol glycosides (such as RA, STV, RC, etc.) in the mother liquor sugar by resin method (such as mixed resin chromatography, modified resin chromatography, etc.), and then further utilize the separated steviol glycosides separately.
  • the total glycoside content in the mother liquor sugar is low, which does not belong to the qualified stevioside product, and contains residual polyphenols and flavonoids impurities, which not only increases the bitterness of the mother liquor sugar, but also affects the recovery of glycosides.
  • Glucosyl steviol glycoside is a mixture of steviol glycoside and dextrin and catalyzed by enzymes. Glucosylation is the process of attaching one or more glucose groups to stevioside through covalent bonds, and then evaporating, concentrating, and spray drying to obtain glucosyl stevioside, which is a modified product with improved taste and reduced bitterness, and has been approved for use in the United States, China and other countries.
  • the DE value of dextrin is related to its degree of hydrolysis, and the more dextrin and macromolecular polysaccharides there are, the greater the viscosity of the product, resulting in low conversion efficiency of glucose groups.
  • Glucosyl stevioside is a flavor and fragrance widely used in the food and beverage industry. In the industrial production process, glucose stevioside needs to be purified to achieve high purity and purity requirements.
  • the commonly used purification method for glucosyl stevioside is through ion exchange chromatography technology.
  • Ion exchange chromatography is a separation method based on the interaction between ions in the sample and the stationary phase.
  • the stationary phase carries ionic functional groups, which can selectively adsorb and release the target components in the sample.
  • the commonly used ion exchange resin is a strong cation exchange resin. The concentrated solution is added to the strong cation exchange resin column. After appropriate elution conditions, the glucosyl stevioside can be adsorbed on the resin, and other irrelevant components are eluted. Finally, by adjusting the elution conditions, the glucosyl stevioside adsorbed on the resin can be eluted to obtain a purer product.
  • Patent application number 202010447914.8 provides a method for separation and purification of glucose-syl steviol glycosides The method is specifically as follows: the material generated in the previous stage is subjected to ion exchange resin to remove cations in the material; the material is pumped into a simulated moving bed at a speed of 0.5-1m 3 /h, the temperature is controlled at 25-40°C, and the time is 1-2.5h; the total time is calculated according to the feeding speed, and the corresponding material is intercepted in sections according to the time period of 1:3:1, the feeding speed is controlled at 0.3-2m 3 /h, the temperature is 20-35°C, and the feeding time is 0.5-3h; the material obtained after 30-90 minutes is pumped into a single-effect concentrator, the pressure is controlled at -0.6 ⁇ -0.9Mpa, the temperature is 45°C-60°C, and the sugar concentration is more than 50%.
  • the concentrated material is filtered and sent to a concentrated sugar liquid tank for spray drying.
  • the material is decolorized by resin and then separated by a simulated moving bed.
  • the technical problem to be solved by the present invention is: in view of the deficiencies in the prior art, a method for preparing glucose-based stevioside by using mother liquor sugar is provided.
  • the present invention enriches the ways of utilizing mother liquor sugar and fills the technical gap of preparing glucose-based stevioside by using mother liquor sugar.
  • the glucose-based stevioside prepared from mother liquor sugar has good taste, high yield and high total glycoside content.
  • the method optimizes and improves the purification process of glucosyl steviol glycosides and effectively recycles auxiliary material dextrin, thereby not only reducing the discharge of waste liquid, greatly reducing the energy consumption and cost of production, but also improving the yield of high-purity glucosyl steviol glycosides.
  • the method comprises the following steps: dissolving the glucose-based stevioside, and then performing concentration, nanofiltration, flocculation sedimentation, and cooling crystallization to obtain high-purity glucose-based stevioside, and the recovery of glucose is achieved with low cost.
  • the technical solution of the present invention is:
  • a method for preparing glucosyl steviol glycoside by using mother liquor sugar comprises the following steps:
  • step (2) Adding activated carbon to the enzymatic hydrolyzate obtained in step (1) to remove impurities, obtaining a de-impurity liquid, performing enzyme inactivation treatment on the de-impurity liquid, and then filtering the de-impurity liquid, and the obtained filtrate enters into a macroporous adsorption resin for adsorption deal with;
  • step (1) the mass ratio of the mother liquor sugar, maltodextrin and water is 1:(1-3):(5-10).
  • the enzyme is a mixture of glucosyltransferase and amylase
  • the glucosyltransferase is ⁇ -cyclodextrin glucosyltransferase
  • the glucosyltransferase is added in the form of a glucosyltransferase solution, and the concentration of the glucosyltransferase solution is 0.8 to 1.2 wt %.
  • the amylase is added in the form of a solid amylase, and the concentration of the solid amylase is 0.4 to 0.6 wt %.
  • the amount of glucosyltransferase solution added to 100 g of mother liquid sugar is 0.5 to 2 ml
  • the amount of solid amylase added to 100 g of mother liquid sugar is 0.1 to 1 g.
  • step (1) the enzymatic hydrolysis conditions are: enzymatic hydrolysis temperature is 60-80°C, pH is 5.5-6.0, and enzymatic hydrolysis time is 36-48h.
  • step (2) the amount of activated carbon added is 1 to 3% of the mass of the enzymatic hydrolyzate, and the impurity removal time is 1 to 6 hours.
  • step (2) when macroporous adsorption resin is used for adsorption, the feed volume of the filtrate is 30-60% of the volume of the macroporous adsorption resin, and the feed rate is 0.5-2 BV/h.
  • the adsorbed macroporous adsorption resin before analyzing the adsorbed macroporous adsorption resin, it also includes: washing the macroporous adsorption resin with pure water, the amount of pure water used during washing is 1 to 3 BV of the volume of the macroporous adsorption resin, and the flow rate is 1 to 3 BV/h.
  • the concentration of the ethanol solution is 40-60 v/v%
  • the concentration of the hydrochloric acid solution is 0.3-0.5 wt%
  • the volume of the acid-alcohol solution is It is 2 to 4 BV of the volume of the macroporous adsorption resin
  • the flow rate is 1 to 3 BV/h.
  • step (4) the molecular weight cutoff of the nanofiltration membrane is 400 to 500 Da.
  • the taste modifier is a composition of erythritol, sodium alginate and valine, and the mass ratio of the three is 2: (2-4): (5-8); the added amount of the taste modifier is 0.1-0.5% of the weight of the product in the concentrate.
  • Stevioside mother liquor sugar is characterized by low total glycoside content and obvious bitterness due to the presence of residual polyphenols and flavonoid impurities.
  • the present invention pioneered the use of mother liquor sugar as raw material, supplemented with maltodextrin as a glucose substrate, and obtained glucose-based stevioside by dual catalysis using glucosyltransferase and amylase, thereby reducing the bitterness of the mother liquor sugar.
  • Activated carbon was then used to remove impurities and deodorize, thereby further improving the taste of glucose-based stevioside and removing impurities.
  • Glucoside was then enriched and separated by adsorption by macroporous adsorption resin, and the adsorbed resin was analyzed by acid-alcohol solution to adjust the solution pH to 5-6, thereby ensuring the stability of glycoside substances in the subsequent concentration process and improving the yield and quality. Subsequently, the glucose-based stevioside was further modified by adding a taste modifier to adjust its taste, and finally, after spray drying, a glucose-based stevioside product with good taste and high total glycoside content that can be directly sold on the market was obtained.
  • the present invention adds amylase to the existing glucosyltransferase to solve the problem of low glucosyl conversion rate caused by low hydrolysis degree of dextrin, large amount of dextrin and macromolecular polysaccharides, and high product viscosity; by using a combination of glucosyltransferase and amylase, the hydrolysis of dextrin and the transglycosylation of glucosyl are accelerated, thereby improving the glucosyl conversion rate.
  • the present invention uses a mixed solution of hydrochloric acid solution and ethanol solution for analysis, which has a good analysis effect and will not cause partial product loss, thereby greatly improving the product yield.
  • the present invention also uses pure water to wash the resin before analysis, which not only allows the residual unadsorbed liquid in the resin to re-enter the resin for adsorption, but also can effectively remove the monosaccharides and oligosaccharides produced during the transglycoside process, further improving the analysis effect and product yield.
  • the present invention directly converts stevioside mother liquor sugar into commercially available glucose-based stevioside products through a reasonable process. Compared with the prior art, the present invention makes more complete use of mother liquor sugar, enriches the ways of utilizing mother liquor sugar, and fills the technical gap in preparing glucose-based stevioside from mother liquor sugar.
  • the technical solution of the present invention is:
  • a method for purifying glucosyl steviol glycoside comprises the following steps:
  • step (2) concentrating the mixture of the effluent obtained in step (1) and the acid water analysis solution, adding ⁇ -1,4-glucose hydrolase to the concentrate, heating the solution for hydrolysis, and adjusting the pH of the reaction system to 3 after the hydrolysis is completed to inactivate the enzyme to obtain a hydrolyzate;
  • the high alcohol analysis solution is concentrated to a solid content of 40-50 wt %, and then the concentrated solution is spray-dried to obtain high-purity glucosyl stevioside B.
  • the concentration of the ethanol solution is 5-15wt%; the concentration of glucosyl steviol glycoside in the glucosyl steviol glycoside ethanol solution is 30-50g/L.
  • step (1) during the adsorption treatment, the flow rate of the glucose-syl stevioside ethanol solution is 0.25BV/h-1BV/h.
  • the acid aqueous solution is a hydrochloric acid solution with a concentration of 0.04-0.06wt%; and the concentration of the high-purity ethanol solution is 70-80wt%.
  • step (1) during analysis, the volume of the acid aqueous solution is 2-3BV, and the flow rate is 2-3BV/h; the volume of the high-purity ethanol solution is 2-3BV, and the flow rate is 2-3BV/h.
  • the volume of the effluent is 1-2 BV of the resin volume
  • the volume of the acid water analysis solution is 2-3 BV of the resin volume.
  • step (2) the concentration multiple during the concentration treatment is 3-5 times.
  • step (2) 0.03-0.1 ml of ⁇ -1,4-glucose hydrolase is used for every 100 g of crude glucosyl steviol glycoside.
  • step (2) the temperature of the hydrolysis reaction is 40-50°C, and the time of the hydrolysis reaction is 1-3h.
  • the steviol glycoside is steviol glycoside STV; and 0.3-0.5 ml of glucosyltransferase is used for every 100 g of crude glucosyl steviol glycoside.
  • step (3) the temperature of the temperature-raising reaction is 70-80°C, and the time is 6-10h; and the temperature of the temperature-raising inactivation is 95°C.
  • the present invention first dissolves the crude glucosyl stevioside in an ethanol solution, then performs adsorption treatment with a macroporous adsorption resin, and then sequentially performs analytical treatment with an acid aqueous solution and a high-purity ethanol solution;
  • the macroporous adsorption resin is a highly efficient adsorption material, has a large specific surface area and a strong adsorption capacity, can effectively adsorb the target substance, and has good selectivity for other impurities; through the action of the macroporous adsorption resin, the glucosyl stevioside can be separated from the impurities therein to improve the purity.
  • the acid aqueous solution can analyze the dextrin mixed and adsorbed in the macroporous adsorption resin, and the high-purity ethanol solution can desorb the glucosyl stevioside from the adsorption resin, thereby improving the purity of the glucosyl stevioside and removing the residual impurities.
  • the present invention also mixes the effluent after adsorption by the macroporous adsorption resin and the acid water analysis liquid, adds ⁇ -1,4-glucose hydrolase to carry out hydrolysis reaction, and then mixes with steviol glycoside to react, so as to obtain high-purity glucose steviol glycoside; the dextrin recovered by the present invention is reused to prepare high-purity glucose-based steviol glycoside, which not only reduces waste liquid discharge, reduces the production energy consumption and cost of glucose-based steviol glycoside, but also improves the yield of high-purity glucose-based steviol glycoside.
  • the method provided by the present invention is relatively simple to operate and does not require complex equipment and conditions to achieve the adsorption separation of glucosyl steviol glycosides.
  • the macroporous adsorption resin has a high adsorption capacity and regeneration capacity and can be recycled. This can not only improve the efficiency of the purification process, but also save costs.
  • the technical solution of the present invention is:
  • a method for separating and purifying glucosyl steviol glycosides and recovering glucose comprises the following steps:
  • the solid content of the feed liquid is 1wt%-5wt%.
  • step (2) when nanofiltration concentration is performed, the molecular weight cutoff of the nanofiltration membrane is 500Da-800Da, and the operating pressure during nanofiltration concentration is 3.5-5bar.
  • step (3) the solvent in the membrane permeate and the membrane interception liquid is removed at a temperature of 80° C. and a pressure of ⁇ 0.1 MPa.
  • step (4) the concentration of methanol is 95wt%-99wt%, and the mass ratio of the intermediate product A to methanol is 1:(5-10).
  • step (4) the mixing and settling time is 2h-8h.
  • step (5) the added amount of the ⁇ -1,4-glucose hydrolase is 0.2%-0.3% of the total weight of the intermediate product B and the filter cake.
  • step (5) the temperature of the enzymatic hydrolysis treatment is 35°C-45°C, the pH is 4.0-4.5, and the time is 5h-7h; the feed liquid is heated to 90°C to inactivate the enzyme.
  • step (6) the temperature of the high-temperature concentration treatment is 80°C-90°C, and the solid content of the extract-like intermediate product C is 70wt%-75wt%.
  • step (6) the slow cooling rate is 10°C/h-15°C/h, and the temperature is lowered to 20°C and kept for crystallization for 5h-7h.
  • the invention adds water to dissolve the crude glucosyl stevioside again, and then uses a nanofiltration membrane to concentrate and perform preliminary purification treatment.
  • the relative molecular masses of unreacted rebaudioside A and glucose in the feed liquid and the product glucosyl stevioside are greatly different.
  • the glucosyl stevioside can be separated to a large extent by using a 500Da-800Da nanofiltration membrane. Then, the membrane interception liquid is concentrated to remove the solvent, and methanol is added to purify again to obtain high-purity glucosyl stevioside.
  • the present invention removes the solvent from the membrane permeate collected in the nanofiltration process, mixes it with the filter cake in the flocculation precipitation process, adds ⁇ -1,4-glucose hydrolase to carry out enzymatic hydrolysis, and then concentrates and cools down the solution to crystallize it under appropriate conditions to obtain high-purity glucose.
  • the process of the present invention is simple and easy to operate, and can not only purify glucosyl stevioside to obtain a high-purity glucosyl stevioside product, but also recover the unreacted substrate to a large extent, which can be reused, thereby reducing the production cost.
  • the present invention provides a method for preparing glucosyl steviol glycoside by using mother liquid sugar, comprising the following steps:
  • step (2) adding activated carbon to the enzymatic hydrolyzate obtained in step (1) to remove impurities, thereby obtaining a de-impurity liquid, performing an enzyme deactivation treatment on the de-impurity liquid, and then filtering the de-impurity liquid, and then passing the obtained filtrate into a macroporous adsorption resin for adsorption treatment;
  • the mother liquor sugar in the present invention comes from the crystallization mother liquor obtained after stevia leaves are extracted with solution.
  • the total glycoside content thereof is 60-80%, including RA 20%-40%, STV 10%-25%, RC 10%-20%, and other glycosides 5-20%.
  • the invention firstly mixes and dissolves mother liquid sugar, maltodextrin and water, and then adds enzyme to carry out enzymolysis reaction to obtain enzymolysis solution.
  • the mass ratio of the mother liquor sugar, maltodextrin and water is preferably 1: (1-3): (5-10).
  • the present invention has no special requirements on the source of maltodextrin, and commercially available products can be used.
  • the DE value of maltodextrin in the present invention is preferably 5-10; the present invention has no special requirements on water, and purified water is preferably used.
  • the present invention also includes stirring, and the stirring rate is preferably 10-100rpm.
  • the enzymatic hydrolysis reaction is preferably started after the mother liquor sugar and maltodextrin are completely dissolved; further, the enzyme is preferably a mixture of glucosyltransferase and amylase, and the glucosyltransferase is preferably ⁇ -cyclodextrin glucosyltransferase.
  • the DE value of maltodextrin the lower the degree of hydrolysis of dextrin, the more substances such as dextrin and macromolecular polysaccharides, and the greater the viscosity of the product.
  • the combined addition of glucosyltransferase and amylase in the present invention can accelerate the hydrolysis of dextrin and the transglycosylation of glucosyl groups, thereby improving the conversion rate.
  • the glucosyltransferase in the present invention is preferably added in the form of a glucosyltransferase solution, the concentration of the glucosyltransferase solution is preferably 0.8-1.2wt%, further preferably 1wt%, and the activity of the glucosyltransferase is preferably 100U/g;
  • the amylase is preferably added in the form of a solid amylase, the concentration of the solid amylase is preferably 0.4-0.6wt%, further preferably 0.5wt%, and the activity of the amylase is preferably 1000-3000U/g.
  • the addition amount of the glucosyltransferase solution is preferably 0.5 to 2 ml per 100 g of mother liquid sugar, and the addition amount of the solid amylase is preferably 0.1 to 1 g per 100 g of mother liquid sugar.
  • the conditions for the enzymatic hydrolysis reaction of the present invention are preferably: a reaction temperature of 60 to 80° C., a pH of 5.5 to 6.0, and a reaction time of 36 to 48 hours.
  • the present invention has no special requirements for the reagent for adjusting the pH, and preferably uses a potassium hydroxide solution or a sodium hydroxide solution; the present invention has no special restrictions on the concentrations of the potassium hydroxide solution and the sodium hydroxide solution, for example, the concentration of the potassium hydroxide solution can be 1 to 10 v/v%, and the concentration of the sodium hydroxide solution can be 1 to 10 v/v%.
  • the present invention adds activated carbon to the enzymolysis solution for impurity removal.
  • activated carbon By utilizing the adsorption of activated carbon, impurities such as flavonoids, saponins, alkaloids, etc. in the enzymolysis solution can be effectively adsorbed; in addition, the activated carbon also has a certain deodorization effect, removing part of the bitterness of glucosyl stevioside.
  • the addition amount of the activated carbon is preferably 1-3% of the mass of the enzymolysis solution, and the impurity removal time is preferably 1-6h; the present invention has no special requirements for the pore size of the activated carbon, for example, it can be 2-50nm; the present invention has no special requirements for the source of the activated carbon, and commercially available products can be used.
  • the present invention performs enzyme inactivation treatment on the impurity removal liquid and filters to obtain a filtrate.
  • high temperature inactivation is preferably used for enzyme inactivation treatment, and the inactivation temperature is preferably 100-120°C; the present invention has no special limitation on the inactivation time.
  • the enzyme is inactivated when the temperature reaches 100-120°C.
  • the filtration is preferably plate-and-frame filtration, the filter cloth pore size is preferably 30-50 ⁇ m, and the operating pressure is preferably 0.5-2.5 MPa.
  • the purpose of the present invention to deactivate the enzyme in the impurity removal liquid is to stop the reaction, prevent the enzyme from affecting the final product, and ensure the product quality.
  • the purpose of the present invention to use the plate frame for filtering is to filter out the activated carbon after adsorption and to filter out the inactivated enzyme.
  • the filtrate obtained is adsorbed by a macroporous adsorption resin.
  • the present invention has no special limitation on the type of the macroporous adsorption resin, and is preferably one of SD-9, AB-8, LX-T81, LX-T83, and LX-T28; the present invention has no special requirements on the source of the resin, and commercially available products can be used; the present invention has no special limitation on the feeding method, such as pumping feeding; the present invention has no special requirements on the carrier of the macroporous adsorption resin, such as a resin column.
  • the macroporous adsorption resin is washed with pure water; wherein, preferably, the amount of the pure water is 1 to 3 BV of the volume of the macroporous adsorption resin, and the flow rate is preferably 1 to 3 BV/h.
  • the purpose of using pure water for washing in the present invention is to adsorb the filtrate remaining in the resin column by the macroporous adsorption resin, thereby achieving the full adsorption of the filtrate as much as possible and improving the adsorption rate.
  • the present invention analyzes the adsorbed macroporous adsorption resin and adjusts the pH of the effluent to 5-6 to obtain an analytical solution.
  • the analytical agent used in the analysis is an acid-alcohol solution
  • the preparation method of the acid-alcohol solution includes: adding hydrochloric acid with a concentration of 0.3-0.5wt% to an ethanol solution with a concentration of 40-60v/v%, and adjusting the pH to 1-3, so as to obtain an acid-alcohol solution.
  • the amount of the acid-alcohol solution is preferably 2-4BV of the volume of the macroporous adsorption resin, and the flow rate is preferably 1-3BV/h.
  • the present invention does not specifically limit the pH adjusting agent, and the pH adjusting agent commonly used in the art can be used; the purpose of adjusting the pH of the effluent to 5-6 after analysis in the present invention is to avoid the destruction of glycosides due to excessive acidity during the subsequent concentration process.
  • the analytical solution is concentrated through a nanofiltration membrane to obtain a concentrated solution; the molecular weight cutoff of the nanofiltration membrane is preferably 400-500 Da; and the solid content of the concentrated solution is 30-50 wt%.
  • S7 Add 0.14 g of taste modifier (0.028 g of erythritol, 0.042 g of sodium alginate, and 0.07 g of valine) to 350 ml of the concentrated solution for modification, and obtain 113 g of glucose-based stevioside product by spray drying.
  • the inlet air temperature during spray drying is 190° C. and the outlet air temperature is 90° C.
  • S7 Add 0.45 g of taste modifier (0.09 g of erythritol, 0.135 g of sodium alginate, and 0.225 g of valine) to 430 ml of the concentrated solution for modification, and obtain 126 g of glucose-based stevioside product by spray drying.
  • the inlet air temperature during spray drying is 200° C. and the outlet air temperature is 80° C.
  • 3L of the adsorbed resin is analyzed with an acid-alcohol solution, the amount of the acid-alcohol solution is 2BV of the volume of the macroporous adsorption resin, the flow rate is 3BV/h, and then the pH of the effluent is adjusted to 6 to obtain 6L of analytical solution; wherein the acid-alcohol solution is a solution with a pH of 2 composed of an ethanol solution with a concentration of 60v/v% and a hydrochloric acid solution with a concentration of 0.5wt%;
  • S7 Add 0.75 g of taste modifier (0.15 g of erythritol, 0.225 g of sodium alginate, and 0.375 g of valine) to 300 ml of the concentrated solution for modification, and obtain 131 g of glucose-based stevioside product by spray drying.
  • the inlet air temperature during spray drying is 180° C. and the outlet air temperature is 100° C.
  • step S2 of this comparative example 2 L of macroporous adsorption resin SD-300 was used to replace the activated carbon, and other conditions were the same as those in Example 1.
  • Example 2 Compared with Example 2, the enzyme inactivation treatment in step S3 was omitted in this comparative example, and other conditions were the same as those in Example 2.
  • Example 3 Compared with Example 3, in this comparative example, the macroporous adsorption resin (T81) in step S4 is replaced by an equal amount of T-28 resin, and other conditions are the same as those in Example 3.
  • step S7 of this comparative example is replaced by an equal amount of sodium citrate, and other conditions are the same as those of Example 4.
  • the samples were diluted to 500ppm, and the test conditions were: temperature 29°C, humidity 50RH%.
  • 10 professional sensory evaluators were selected according to the provisions of GB/T 16291.2-2010 to form a sensory evaluation team.
  • the sensory evaluators restricted their diet within 1 hour before the evaluation experiment, especially restricting the consumption of foods that can seriously affect the taste.
  • the sensory evaluators scored the bitterness of the samples on a 10-point scale, and the final score was averaged.
  • the evaluation standard was 5wt% sucrose solution. The test results are shown in Table 2.
  • the present invention sets up 4 groups of comparative experiments to test the taste difference between the glucose-syl steviol glycoside prepared by the present invention and the steviol glycoside in the original mother solution sugar, and the samples taken are diluted to 500ppm respectively. This is mainly because the sweetness multiple of steviol glycoside products is high, and dilution is required for distinction and evaluation, as follows:
  • the evaluation standard was a 5 wt% sucrose solution.
  • Test conditions temperature 29°C, humidity 50RH%;
  • Sensory evaluators gave comprehensive scores for sweeteners based on sweetness, sweetening speed, aftersweetness, bitterness, astringency, miscellaneous flavors, and overall preference. Among them, sweetening speed represents the speed of feeling sweetness, and aftersweetness represents the duration of sweetness. Except for overall preference, which is a percentage system (5wt% sucrose solution has a full score of 100 points), all other scores are based on a 10-point system, and the final score is the average.
  • Table 3 The test results of taste evaluation are shown in Table 3 below, where the larger the value of each sensory organ, the more obvious the taste characteristics.
  • glucose-syl stevioside prepared by the present invention has improved taste in all aspects compared with the original mother liquid sugar, especially in sweetness and sweetening speed, while significantly reducing the bitterness and astringency in the raw mother liquid sugar.
  • the content of total stevioside (TSG) in 100 g of crude glucosyl steviol glycoside is 71 wt %
  • the content of glucosyl steviol glycoside (GSG) is 59 wt %
  • the content of dextrin is 28 wt %.
  • Liquid chromatography-tandem quadrupole time-of-flight mass spectrometry was used to qualitatively analyze the transglycoside products.
  • ESI electrospray ionization
  • Quantitative analysis method of the product Based on the analytical detection method for glucosyl stevioside in the supplementary document No. 8 of the National Health and Family Planning Commission GB2760-2014.
  • S1 Dissolve 100g of crude glucosyl stevioside in 2L of 10wt% ethanol solution, then use macroporous adsorption resin for adsorption, control the liquid flow rate to 0.5BV/h during adsorption, collect the effluent, and then use 0.05wt% hydrochloric acid aqueous solution for analysis, control the flow rate of the hydrochloric acid aqueous solution to 2BV/h, the volume to 2BV, and collect the acid water analysis liquid; preferably, use 70wt% ethanol solution for analysis, control the flow rate of the ethanol solution to 2BV/h, the volume to 2BV, and collect the high alcohol analysis liquid;
  • the content of total stevioside (TSG) in high-purity glucose syl steviol glycoside A was 93wt%
  • the content of glucose syl steviol glycoside (GSG) was 77wt%
  • the content of dextrin was 5.8wt%
  • the content of total stevioside (TSG) in high-purity glucose syl steviol glycoside B was 99wt%
  • the content of glucose syl steviol glycoside (GSG) was 92wt%
  • the content of dextrin was 0.2wt%.
  • S1 Dissolve 100g of crude glucosyl stevioside in 3L of 10wt% ethanol solution, then use macroporous adsorption resin for adsorption, control the liquid flow rate to 0.75BV/h during adsorption, collect the effluent, and then use 0.05wt% hydrochloric acid aqueous solution for analysis, control the flow rate of the hydrochloric acid aqueous solution to 2BV/h, the volume to 2BV, and collect the acid water analysis liquid; preferably use 80wt% ethanol solution for analysis, control the flow rate of the ethanol solution to 3BV/h, the volume to 3BV, and collect the high alcohol analysis liquid;

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Abstract

一种利用母液糖制备葡萄糖基甜菊糖苷、分离纯化葡萄糖基甜菊糖苷并回收葡萄糖的方法，属于食品添加剂技术领域。提供一种利用母液糖制备葡萄糖基甜菊糖苷的制备方法，以母液糖为原料制备葡萄糖基甜菊糖苷，丰富了母液糖的利用途径，填补了以母液糖为原料制备葡萄糖基甜菊糖苷的技术空白，同时制备得到的产品还具有口感良好、总苷含量高的优点；提供一种葡萄糖基甜菊糖苷的纯化方法，将糊精进行回收利用，减少了废液的排放，极大降低了生产的能耗与成本，提高了高纯度葡萄糖基甜菊糖苷的收率；还提供一种分离纯化葡萄糖基甜菊糖苷并回收葡萄糖的方法，该方法工艺简单，易操作，不仅得到了高纯葡萄糖基甜菊糖苷，且对未反应底物进行了回收利用，降低成本。

Description

一种利用母液糖制备葡萄糖基甜菊糖苷、分离纯化葡萄糖基甜菊糖苷并回收葡萄糖的方法
本申请要求于2023年12月29日提交中国专利局、申请号为202311848635.2、发明名称为“一种利用母液糖制备葡萄糖基甜菊糖苷的制备方法”的中国专利优先权、2023年7月18日提交中国专利局、申请号为202310879842.8、发明名称为“一种葡萄糖基甜菊糖苷的纯化方法”的中国专利优先权以及2023年9月25日提交中国专利局、申请号为202311239776.4、发明名称为“一种分离纯化葡萄糖基甜菊糖苷并回收葡萄糖的方法”的中国专利优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及食品添加剂技术领域，特别涉及一种利用母液糖制备葡萄糖基甜菊糖苷、分离纯化葡萄糖基甜菊糖苷并回收葡萄糖的方法。
背景技术
甜菊糖苷是从菊科草本植物甜叶菊中提取的天然甜味剂，它具有高甜度、低热量的功能特点，其甜度是蔗糖的100～300倍，热量仅为蔗糖的1/300。甜菊糖苷同时具有甜味和苦味，且两种味道的味质均与其主体部分连接的糖基数量以及糖基之间的糖苷键类型有关。
甜菊糖苷母液糖(MLS)是通过溶液浸提法回收掉高纯度的莱鲍迪苷A(RA)和甜菊苷(ST)之后，剩余得到的结晶母液经喷雾干燥制成的粉末固体。一般来讲，甜菊糖苷母液糖用作低价甜味剂进行销售；还有一种思路是通过树脂法(例如混合树脂层析法、改性树脂层析法等)将母液糖中的甜菊糖苷(例如RA、STV、RC等)进行分离，然后将分离得到的甜菊糖苷分别进行进一步利用。但是母液糖中的总苷含量较低，不属于合格的甜菊糖产品，且含有残余的多酚、黄酮类杂质，不仅加重了母液糖的苦味，更加影响了糖苷的回收。
葡萄糖基甜菊糖苷是将甜菊糖苷和糊精混合并以酶催化法对甜菊糖苷进 行葡萄糖基化，即在甜菊糖苷上通过共价键接上一个至数个葡萄糖基，然后经蒸发浓缩、喷雾干燥而得的葡萄糖基甜菊糖苷，其属于一种味质改善、苦味下降的改性产品，并且已经在美国、中国等国家获得批准使用。在实际生产过程中，由于糊精的DE值与其水解程度相关，而且糊精和大分子多糖等物质越多，产品粘度大，导致葡萄糖基的转化效率低下。
基于上述问题，如何利用母液糖来高效率制备葡萄糖基甜菊糖苷成为亟需解决的问题。
葡萄糖基甜菊糖苷是一种香精香料，广泛应用于食品和饮料工业中。在工业生产过程中，葡萄糖基甜菊糖苷需要进行纯化以达到高纯度和纯净度的要求。
目前葡萄糖基甜菊糖苷常用的纯化方法是通过离子交换色谱技术。离子交换色谱是一种基于样品中离子与固定相之间相互作用的分离方法。在离子交换色谱柱中，固定相上带有离子功能基团，可以选择性地吸附和释放样品中的目标组分。在葡萄糖基甜菊糖苷纯化过程中，常用的离子交换树脂是强阳离子交换树脂。将浓缩溶液加入强阳离子交换树脂柱，经过适当的洗脱条件，可以使葡萄糖基甜菊糖苷吸附到树脂上，其他无关组分则被洗脱。最后，通过调整洗脱条件，可以将吸附在树脂上的葡萄糖基甜菊糖苷洗脱下来，得到较纯的产品。
现有技术中在葡萄糖基甜菊糖苷的制备和纯化工艺中，有大量的辅料糊精生成，后续废液需要进行处理才能进行排放，提高了生产能耗和成本。
葡萄糖基甜菊糖苷的转化过程，并不是单纯的化合反应(A+B＝C)，由于所用酶的性质，会呈现出并不单一的糖链与糖基配体的组合形式，反应体系中出现不同接枝数葡萄糖基甜菊糖苷，且反应转化过程不易控制，反应程度并不能到达100％，导致制得的葡萄糖基甜菊糖苷的纯度较低，因此需要对其进行分离纯化。
申请号为202010447914.8的专利提供了一种葡萄糖基甜菊糖苷分离纯化 的方法，具体为：上一工段生成的物料，通过离子交换树脂去除物料中阳离子；物料以0.5-1m3/h的速度泵入模拟移动床，控制温度25-40℃,时间1-2.5h；根据入料速度计算总时间，按照1:3:1的时间段进分段截取相应物料，控制入料速度0.3-2m3/h，温度20-35℃,进料时间0.5-3h；将30-90分钟所得物料泵入单效浓缩器，控制压力-0.6～-0.9Mpa，温度为45℃-60℃，浓缩至糖浓度50％以上，浓缩后的物料过滤后送入浓缩糖液槽，进行喷雾干燥。上述申请中对物料进行树脂脱色后采用模拟移动床进行分离，虽然能得到高纯葡萄糖基甜菊糖苷，但是对设备要求高、成本高。
发明内容
本发明所要解决的技术问题是：针对现有技术存在的不足，提供一种利用母液糖制备葡萄糖基甜菊糖苷的制备方法，本发明丰富了母液糖利用的途径，填补了以母液糖制备葡萄糖基甜菊糖苷的技术空白，由母液糖制得的葡萄糖基甜菊糖苷口感好，产量高，总苷含量高。
提供一种葡萄糖基甜菊糖苷的纯化方法，该方法通过优化和改进葡萄糖基甜菊糖苷的纯化工艺，将辅料糊精进行有效回收利用，不仅减少了废液的排放，极大降低了生产的能耗与成本，而且提高了高纯度葡萄糖基甜菊糖苷的收率。
提供一种分离纯化葡萄糖基甜菊糖苷并回收葡萄糖的方法，该方法对葡萄糖基甜菊糖苷溶解后，经浓缩、纳滤、絮凝沉降、冷却结晶，得到了高纯葡萄糖基甜菊糖苷，且实现了葡萄糖的回收，成本低。
为解决上述技术问题，本发明的技术方案是：
一种利用母液糖制备葡萄糖基甜菊糖苷的制备方法，包括以下步骤：
(1)将母液糖、麦芽糊精和水混合搅拌至固体溶解，加入酶进行酶解得到酶解液；
(2)向步骤(1)得到的酶解液中加入活性炭进行除杂，得除杂液，将除杂液进行酶灭活处理，然后过滤，得到的滤液进入到大孔吸附树脂内进行吸附 处理；
(3)采用乙醇溶液与盐酸溶液组成的pH为1～3的酸醇溶液进行解析，得解析液；
(4)调节上述解析液的pH至5～6，然后采用纳滤膜进行浓缩，得浓缩液；
(5)向浓缩液中加入口感修饰剂，然后采用喷雾干燥得到葡萄糖基甜菊糖苷产品。
作为一种改进的技术方案，步骤(1)中，所述母液糖、麦芽糊精和水的质量比为1：(1～3)：(5～10)。
作为一种改进的技术方案，步骤(1)中，所述酶为葡萄糖基转移酶和淀粉酶的混合物，所述葡萄糖基转移酶为α-环糊精葡萄糖基转移酶，具体添加时，所述葡萄糖基转移酶的添加形式是以葡萄糖基转移酶溶液的形式添加，葡萄糖基转移酶溶液的浓度为0.8～1.2wt％，所述淀粉酶的添加形式是以固体淀粉酶的形式添加，所述固体淀粉酶的浓度为0.4～0.6wt％。
进一步的，每100g母液糖中葡萄糖基转移酶溶液的添加量为0.5～2ml，每100g母液糖中固体淀粉酶的添加量为0.1～1g。
作为一种改进的技术方案，步骤(1)中，所述酶解的条件为：酶解温度为60～80℃，pH为5.5～6.0，酶解时间为36～48h。
作为一种改进的技术方案，步骤(2)中，所述活性炭的添加量为所述酶解液质量的1～3％，除杂时间为1～6h。
作为一种改进的技术方案，步骤(2)中，采用大孔吸附树脂吸附时滤液的进料体积为大孔吸附树脂体积的30～60％，进料速度为0.5～2BV/h。
作为一种改进的技术方案，将吸附后的大孔吸附树脂进行解析前，还包括：用纯水洗涤所述大孔吸附树脂，洗涤时所述纯水的用量为所述大孔吸附树脂体积的1～3BV，流速为1～3BV/h。
作为一种改进的技术方案，步骤(3)中，所述乙醇溶液的浓度为40～60v/v％，所述盐酸溶液的浓度为0.3～0.5wt％，解析时，酸醇溶液的体积 为大孔吸附树脂体积的2～4BV，流速为1～3BV/h。
作为一种改进的技术方案，步骤(4)中，所述纳滤膜的截留分子量为400～500Da。
作为一种改进的技术方案，步骤(5)中，所述口感修饰剂为赤藓糖醇、海藻酸钠和缬氨酸的组合物，三者质量比为2：(2～4)：(5～8)；所述口感修饰剂的添加量为浓缩液中产品重量的0.1～0.5％。
由于采用了上述技术方案，本发明的有益效果是：
(1)甜菊糖苷母液糖的特点是总苷含量低，且由于残余的多酚、黄酮类杂质的存在，存在明显的苦味，本发明开创性的以母液糖为原料，辅之麦芽糊精为葡萄糖基底物，经采用葡萄糖基转移酶和淀粉酶进行双催化得到了葡萄糖基甜菊糖苷，降低了母液糖的苦味，再经活性炭除杂、脱味，进一步提升了葡萄糖基甜菊糖苷的口感并去除杂质；再经大孔吸附树脂吸附对葡萄糖基甜菊糖苷进行富集和分离，吸附的树脂经酸醇溶液解析后调节溶液pH至5～6，保证了糖苷类物质在后续浓缩过程中的稳定性，提升了产量和品质；后续通过添加口感修饰剂进一步对葡萄糖基甜菊糖苷进行修饰，调整其口感，最后经喷雾干燥后得到了口感良好、总苷含量高的可直接市售的葡萄糖基甜菊糖苷产品。
(2)本发明在现有葡萄糖基转移酶的基础上添加了淀粉酶辅助，解决了由于糊精的水解程度低、糊精和大分子多糖等物质多、产品粘度大导致的葡萄糖基转化率低的问题；通过葡萄糖基转移酶和淀粉酶的复配使用，加速了糊精的水解和葡萄糖基的转苷，提高葡萄糖基转化率。
(3)本发明采用盐酸溶液和乙醇溶液的混合液进行解析处理，解析效果好，不会造成部分产品流失，大大提高了产品的收率，而且本发明在解析前，还采用纯水对树脂进行洗涤，不仅使得树脂中残余的未被吸附的料液重新进入到树脂中吸附，而且还可以有效去掉转苷过程中产生的单糖和低聚糖，进一步提高解析效果和产品收率。
(4)本发明通过合理的工艺将甜菊糖苷母液糖直接转化成了可市售的葡萄糖基甜菊糖苷产品，相较于现有技术，本发明对母液糖的利用更为充分，丰富了母液糖利用的途径，填补了以母液糖制备葡萄糖基甜菊糖苷的技术空白。
为解决上述技术问题，本发明的技术方案是：
一种葡萄糖基甜菊糖苷的纯化方法，包括以下步骤：
(1)将葡萄糖基甜菊糖苷粗品溶于乙醇溶液中，然后将得到的葡萄糖基甜菊糖苷乙醇溶液经大孔吸附树脂吸附处理，收集流出液；之后依次采用酸水溶液以及高纯度乙醇溶液进行解析，收集酸水解析液和高醇解析液；
(2)将步骤(1)得到的流出液和酸水解析液的混合液浓缩处理，之后向浓缩液中加入α-1,4葡萄糖水解酶，升温水解反应，水解结束后调节反应体系pH至3，对酶进行灭活，得到水解液；
(3)向水解液中加入甜菊糖苷，然后调节溶液pH至6.0，加入葡萄糖基转移酶，升温反应，反应结束后继续升温灭活，得到反应液，将反应液浓缩至固含量为40-50wt％，然后将浓缩液进行喷雾干燥，得到高纯葡萄糖基甜菊糖苷A；
(4)将高醇解析液浓缩至固含量为40-50wt％，然后将浓缩液进行喷雾干燥，得到高纯葡萄糖基甜菊糖苷B。
作为上述技术方案的优选，步骤(1)中，所述乙醇溶液的浓度为5-15wt％；所述葡萄糖基甜菊糖苷乙醇溶液中葡萄糖基甜菊糖苷的浓度为30-50g/L。
作为上述技术方案的优选，步骤(1)中，吸附处理时，葡萄糖基甜菊糖苷乙醇溶液的流速为0.25BV/h-1BV/h。
作为上述技术方案的优选，步骤(1)中，所述酸水溶液为浓度为0.04-0.06wt％的盐酸溶液；所述高纯度乙醇溶液的浓度为70-80wt％。
作为上述技术方案的优选，步骤(1)中，解析时，酸水溶液的体积为2-3BV，流速为2-3BV/h；高纯度乙醇溶液的体积为2-3BV，流速为2-3BV/h。
作为上述技术方案的优选，步骤(1)中，流出液的体积为树脂体积的1-2BV，酸水解析液的体积为树脂体积的2-3BV。
作为上述技术方案的优选，步骤(2)中，所述浓缩处理时的浓缩倍数为3-5倍。
作为上述技术方案的优选，步骤(2)中，每100g葡萄糖基甜菊糖苷粗品使用0.03-0.1ml的α-1，4葡萄糖水解酶。
作为上述技术方案的优选，步骤(2)中，所述水解反应的温度为40-50℃，水解反应的时间为1-3h。
作为上述技术方案的优选，步骤(3)中，所述甜菊糖苷为甜菊糖苷STV；每100g葡萄糖基甜菊糖苷粗品使用0.3-0.5ml的葡萄糖基转移酶。
作为上述技术方案的优选，步骤(3)中，所述升温反应的温度为70-80℃，时间为6-10h；所述升温灭活的温度为95℃。
由于采用了上述技术方案，本发明的有益效果是：
本发明首先将葡萄糖基甜菊糖苷粗品溶于乙醇溶液，然后经大孔吸附树脂进行吸附处理，之后依次采用酸水溶液、高纯度乙醇溶液进行解析处理；大孔吸附树脂是一种高效的吸附材料，具有较大的比表面积和较强的吸附能力，能够有效吸附目标物质，对其他杂质具有较好的选择性；通过大孔吸附树脂的作用，可以实现将葡萄糖基甜菊糖苷与其中的杂质分离，提高纯度。在大孔吸附树脂吸附处理后，酸水溶液可以将夹杂吸附于大孔吸附树脂内的糊精解析下来，而高纯度乙醇溶液可以将葡萄糖基甜菊糖苷从吸附树脂上脱附下来，进而提高葡萄糖基甜菊糖苷的纯度，去除残留的杂质。
此外，本发明还将大孔吸附树脂吸附后的流出液以及酸水解析液混合加入α-1，4葡萄糖水解酶进行水解反应，之后与甜菊糖苷混合反应，得到高纯葡萄糖甜菊糖苷；本发明回收的糊精重新利用来制备高纯葡萄糖基甜菊糖苷，不仅减少了废液排放，降低葡萄糖基甜菊糖苷的生产能耗和成本，且提高了高纯葡萄糖基甜菊糖苷的收率。
本发明提供的方法操作相对简单，不需要复杂的设备和条件支持，就可以实现对葡萄糖基甜菊糖苷的吸附分离。大孔吸附树脂具有较高的吸附容量和再生能力，可以循环使用。这样不仅可以提高纯化过程的效率，还能节约成本。
为解决上述技术问题，本发明的技术方案是：
一种分离纯化葡萄糖基甜菊糖苷并回收葡萄糖的方法，包括以下步骤:
(1)将葡萄糖基甜菊糖苷粗品加纯化水重新进行溶解，制得料液；
(2)将料液进行纳滤浓缩处理，收集膜截流液和膜透过液；
(3)分别将膜截流液和膜透过液中的溶剂去除，制得中间产品A和中间产品B；
(4)将中间产品A和甲醇混合沉降，然后真空抽滤，收集滤饼和滤液，滤液干燥后得到高纯葡萄糖基甜菊糖苷；
(5)将中间产品B和滤饼混合并加入纯化水，搅拌至固体溶解，加入α-1,4-葡萄糖水解酶进行酶解处理，酶解结束后加热料液使酶失活，制得含葡萄糖溶液；
(6)将含葡萄糖溶液高温浓缩处理，得到浸膏状中间产品C，将浸膏状中间产品C进行缓慢降温结晶，最后进行真空抽滤，滤液回收留作后续生产套用，滤饼为高纯葡萄糖。
作为一种优选的技术方案，步骤(1)中，所述料液的固含量为1wt％-5wt％。
作为一种优选的技术方案，步骤(2)中，采用纳滤浓缩时纳滤膜的截留分子量为500Da-800Da，纳滤浓缩时的操作压力在3.5-5bar。
作为一种优选的技术方案，步骤(3)中，在温度为80℃，压力为-0.1MPa的条件下去除膜透过液和膜截流液中的溶剂。
作为一种优选的技术方案，步骤(4)中，所述甲醇的浓度为95wt％-99wt％，所述中间产品A与甲醇的质量比为1：(5-10)。
作为一种优选的技术方案，步骤(4)中，所述混合沉降的时间为2h-8h。
作为一种优选的技术方案，步骤(5)中，所述α-1,4-葡萄糖水解酶的添加量为中间产品B和滤饼总重量的0.2％-0.3％。
作为一种优选的技术方案，步骤(5)中，所述酶解处理的温度为35℃-45℃，pH为4.0-4.5，时间为5h-7h；加热料液至90℃使酶失活。
作为一种优选的技术方案，步骤(6)中，高温浓缩处理的温度为80℃-90℃，所述浸膏状中间产品C的固含量为70wt％-75wt％。
作为一种优选的技术方案，步骤(6)中，所述缓慢降温的速率为10℃/h-15℃/h，降温至20℃保温结晶5h-7h。
由于采用了上述技术方案，本发明的有益效果是：
本发明将葡萄糖基甜菊糖苷粗品重新加水溶解后，采用纳滤膜浓缩，进行初步纯化处理，料液中未反应完全的莱鲍迪苷A、葡萄糖与产物葡萄糖基甜菊糖苷的相对分子质量有很大区别，采用500Da-800Da纳滤膜可以很大程度上将葡萄糖基甜菊糖苷分离出来；然后将膜截流液进行浓缩除去溶剂后加入甲醇进行再次纯化，得到了高纯葡萄糖基甜菊糖苷。
本发明将纳滤过程中收集的膜透过液去除溶剂后与絮凝沉淀过程中的滤饼混合加入α-1,4-葡萄糖水解酶进行酶解反应，然后在合适的条件下进行浓缩、降温结晶，得到的高纯葡萄糖。
综上所述，本发明工艺简单，易操作，既实现了葡萄糖基甜菊糖苷的提纯，得到高纯度的葡萄糖基甜菊糖苷产品，又很大程度上回收了未反应底物，可以再次利用，降低生产成本。
具体实施方式
下面结合实施例进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。
本发明提供了一种利用母液糖利用母液糖制备葡萄糖基甜菊糖苷的制备方法，包括以下步骤：
(1)将母液糖、麦芽糊精和水混合搅拌至固体溶解，加入酶进行酶解得到酶解液；
(2)向步骤(1)得到的酶解液中加入活性炭进行除杂，得除杂液，将除杂液进行酶灭活处理，然后过滤，得到的滤液进入到大孔吸附树脂内进行吸附处理；
(3)采用乙醇溶液与盐酸溶液组成的pH为1～3的酸醇溶液进行解析，得解析液；
(4)调节上述解析液的pH至5～6，然后采用纳滤膜进行浓缩，得浓缩液；
(5)向浓缩液中加入口感修饰剂，然后采用喷雾干燥得到葡萄糖基甜菊糖苷产品。
本发明中的母液糖来源为甜叶菊叶片经溶液浸提后得到的结晶母液，一般来讲，其总苷含量为60～80％，其中，RA 20％～40％，STV 10％～25％，RC 10％～20％，其他苷5～20％。
本发明首先将母液糖、麦芽糊精和水混合溶解，加入酶进行酶解反应得到酶解液。
在本发明中，所述母液糖、麦芽糊精和水的质量比优选为1：(1～3)：(5～10)。本发明对麦芽糊精的来源并无特殊要求，采用市售产品即可，本发明中麦芽糊精的DE值优选为5～10；本发明对水并无特殊要求，优选采用纯净水。在混合溶解过程中，本发明还包括进行搅拌，所述搅拌的速率优选为10～100rpm。
在本发明中，所述酶解反应优选在母液糖、麦芽糊精完全溶解后开始；进一步的，所述酶优选为葡萄糖基转移酶和淀粉酶的混合物，所述葡萄糖基转移酶优选为α-环糊精葡萄糖基转移酶。麦芽糊精的DE值越低，糊精的水解程度越低，糊精和大分子多糖等物质越多，产品粘度大，而本发明采用葡萄糖基转移酶和淀粉酶联合添加可以加速糊精的水解和葡萄糖基的转苷，提高转化率。
在具体添加时，本发明中所述葡萄糖基转移酶的添加形式优选为葡萄糖基转移酶溶液，葡萄糖基转移酶溶液的浓度优选为0.8～1.2wt％，进一步的优选为1wt％，葡萄糖基转移酶的活力优选为100U/g；所述淀粉酶的添加形式优选为固体淀粉酶，固体淀粉酶的浓度优选为0.4～0.6wt％，进一步的优选为0.5wt％，淀粉酶的活力优选为1000～3000U/g。
在本发明中，每100g母液糖中，葡萄糖基转移酶溶液的添加量优选为0.5～2ml，每100g母液糖中，所述固体淀粉酶的添加量优选为0.1～1g。
进一步的，本发明在进行酶解反应中的条件优选为：反应温度为60～80℃，pH为5.5～6.0，反应时间为36～48h，此外，本发明对调节pH的试剂并无特殊要求，优选采用氢氧化钾溶液或氢氧化钠溶液；本发明对所述氢氧化钾溶液和氢氧化钠溶液的浓度并无特殊限定，例如氢氧化钾溶液的浓度可以为1～10v/v％，氢氧化钠溶液的浓度可以为1～10v/v％。
本发明中，酶解结束后，本发明向酶解液中加入活性炭进行除杂，利用活性炭的吸附性，可以对酶解液中存在的黄酮、皂苷、生物碱类等杂质进行有效吸附；另外，活性炭还具备一定的脱味效果，去除葡萄糖基甜菊糖苷的部分苦味。进一步的，所述活性炭的添加量优选为所述酶解液质量的1～3％，除杂时间优选为1～6h；本发明对所述活性炭的孔径并无特殊要求，比如可以为2～50nm；本发明对所述活性炭的来源并无特殊要求，采用市售产品即可。
本发明中，在经活性炭除杂得到除杂液后，本发明将所述除杂液进行酶灭活处理，过滤得到滤液。进一步的，酶灭活处理时优选采用高温灭活，灭活温度优选为100～120℃；本发明对灭活时间并无特殊限定，一般来讲，温度达到100～120℃过程中，酶已经失活。进一步的，所述过滤优选采用板框过滤，滤布孔径优选为30～50μm，操作压力优选为0.5～2.5MPa。
本发明对除杂液进行酶灭活的目的是为了终止反应进行，防止酶对最终的产品产生影响，保证产品品质。本发明采用板框进行过滤的目的一是为了滤除吸附后的活性炭，二是为了滤除灭活后的酶。
在本发明中，本发明将得到的滤液通过大孔吸附树脂进行吸附。本发明对所述大孔吸附树脂的种类并无特殊限定，优选为SD-9、AB-8、LX-T81、LX-T83、LX-T28中的一种；本发明对所述树脂的来源并无特殊要求，采用市售产品即可；本发明对所述进料的方式并无特殊限定，比如可以采用泵送进料；本发明对所述大孔吸附树脂的载体并无特殊要求，比如可以采用树脂柱。具体的，在进液时，所述滤液的进料量优选为所述大孔吸附树脂体积的30～60％，进料速度优选为0.5～2BV/h。本发明经大孔吸附树脂吸附后的流出液含有一定量的糊精和葡萄糖，可以进一步回收利用制备其他有价值的副产品。
进一步地，在将所述滤液通过大孔吸附树脂进行吸附后，采用纯水洗涤所述大孔吸附树脂；其中，优选的，所述纯水的用量为所述大孔吸附树脂体积的1～3BV，流速优选为1～3BV/h。本发明采用纯水进行洗涤的目的是将树脂柱中残留的滤液通过大孔吸附树脂进行吸附，从而尽可能的实现滤液的全部吸附，提高吸附率。
在本发明中，经大孔吸附树脂吸附后，本发明将吸附后的大孔吸附树脂进行解析，并调节流出液的pH至5～6，得解析液。具体的，解析时采用的解析剂为酸醇溶液，所述酸醇溶液的配制方法包括：向浓度为40～60v/v％的乙醇溶液中加入浓度0.3～0.5wt％的盐酸，调节pH为1～3，即得酸醇溶液。在解析时，所述酸醇溶液的用量优选为所述大孔吸附树脂体积的2～4BV，流速优选为1～3BV/h。本发明对所述调节pH的调节剂并无特殊限定，采用本领域常用的pH调节剂即可；本发明中在解析后调节流出液的pH至5～6的作用是避免后续浓缩过程中酸度过高导致糖苷的破坏。
在本发明中，将所述解析液通过纳滤膜进行浓缩，得到浓缩液；所述纳滤膜的截留分子量优选为400～500Da；所述浓缩液的固含量为30～50wt％。
在得到浓缩液后，本发明向所述浓缩液中加入口感修饰剂，并经喷雾干燥得到葡萄糖基甜菊糖苷产品。具体的，所述口感修饰剂为赤藓糖醇、海藻酸钠和缬氨酸的组合物，三者质量比优选为2：(2～4)：(5～8)，进一步的优 选为2:3:5；所述口感修饰剂的添加量优选为所述浓缩液中产品质量的0.1～0.5％。本发明中所使用的口感修饰剂优选以固态形式添加；本发明添加口感修饰剂的目的是调节产品的口感，降低产品的苦涩味。在本发明中，所述喷雾干燥的进风温度为180～200℃，出风温度为80～100℃。
为了进一步说明本发明，下面结合实施例对本发明提供的一种利用母液糖制备葡萄糖基甜菊糖苷的制备方法进行详细地描述，但不能将它们理解为对本发明保护范围的限定。特别说明，以下实施例以及对比例中所用原料如无特殊说明均为市售原料。下述实施例中的甜菊糖苷母液糖的主要成分如表1所示，该批甜菊糖苷母液糖均由诸城市浩天药业有限公司提供。
表1 1～4批次母液糖甜菊糖苷成分
实施例1
S1：取批次1的母液糖100g，DE值为8的麦芽糊精150g，加入700mL水中，在50rpm的转速下搅拌至固体完全溶解，然后加入0.5mlα-环糊精葡萄糖基转移酶(活力为100U/g)以及0.1g淀粉酶(活力1000～3000U/g)，在温度为70℃，pH为5.8的条件下酶解反应42h，得到850mL酶解液；
S2：向850mL酶解液中加入10g孔径为30nm的活性炭，除杂3h后得到830mL除杂液；
S3：将除杂液升温至110℃进行酶灭活处理，再由滤布孔径为40μm的板框过滤机在操作压力为2.0MPa的条件下进行过滤，得815mL滤液；
S4：将815mL滤液进料至装填有2L大孔吸附树脂(SD-9)的树脂柱中进行吸附处理，进料速度控制为1BV/h；
S5：将吸附后的2L树脂用酸醇溶液进行解析，酸醇溶液的用量为大孔吸附树脂体积的4BV，流速为2BV/h，然后调节流出液的pH至5得到7.8L解析液；其中，所述酸醇溶液是由40v/v％乙醇溶液与浓度为0.5wt％的盐酸溶液混合组成的pH为2的溶液；
S6：将7.8L解析液经截留分子量为400Da的纳滤膜浓缩，收集纳滤截留液，得到350ml固含量为40wt％的浓缩液；
S7：向350ml浓缩液中添加0.14g口感修饰剂(赤藓糖醇0.028g、海藻酸钠0.042g、缬氨酸0.07g)进行修饰，并经喷雾干燥得到113g葡萄糖基甜菊糖苷产品，喷雾干燥时的进风温度为190℃，出风温度为90℃。
实施例2
S1：取批次2的母液糖100g，DE值为5的麦芽糊精100g，加入500mL水中，在10rpm的转速下搅拌至固体完全溶解，然后加入1mlα-环糊精葡萄糖基转移酶(活力为100U/g)以及0.3g淀粉酶(活力1000～3000U/g)，在温度为80℃，pH为5.5的条件下反应48h，得到750mL酶解液；
S2：向750mL酶解液中加入15g孔径为2nm的活性炭，除杂6h后得到730mL除杂液；
S3：将除杂液升温至120℃进行酶灭活处理，再由滤布孔径为50μm的板框过滤机在操作压力为0.5MPa的条件下过滤，得到720mL滤液；
S4：将720mL滤液进料至装填有2.4L大孔吸附树脂(AB-8)的树脂柱中进行吸附处理，进料速度控制为0.5BV/h；
S5：将吸附后的2.4L树脂用酸醇溶液进行解析，酸醇溶液的用量为大孔吸附树脂体积的3BV，流速为1BV/h，然后调节流出液的pH至5.5，得到7L解析液；其中，所述酸醇溶液是由浓度为50v/v％的乙醇溶液与浓度为0.5wt％的乙醇溶液组成的pH为2的溶液；
S6：将7L解析液经截留分子量为450Da的纳滤膜浓缩，收集纳滤截留液，得到430ml固含量为35wt％的浓缩液；
S7：向430ml浓缩液中添加0.45g口感修饰剂(赤藓糖醇0.09g、海藻酸钠0.135g、缬氨酸0.225g)进行修饰，并经喷雾干燥得到126g葡萄糖基甜菊糖苷产品，喷雾干燥时的进风温度为200℃，出风温度为80℃。
实施例3
S1：取批次3的母液糖100g，DE值为10的麦芽糊精200g，加入800mL水中，在100rpm的转速下搅拌至固体完全溶解，然后加入1.5mlα-环糊精葡萄糖基转移酶(活力为100U/g)和0.7g淀粉酶(活力1000～3000U/g)，在温度为60℃，pH为6.0的条件下反应36h，得到1200mL酶解液；
S2：向1200mL酶解液中加入20g孔径为50nm的活性炭，除杂1h后得到1900mL除杂液；
S3：将除杂液升温至100℃进行酶灭活处理，再由滤布孔径为30μm的板框过滤机在操作压力为2.5MPa的条件下过滤，得到1800mL滤液；
S4：将1800mL滤液进料至装填有3L大孔吸附树脂(T81)的树脂柱中进行吸附处理，进料流速控制为2BV/h；
S5：将吸附后的3L树脂用酸醇溶液进行解析，酸醇溶液的用量为大孔吸附树脂体积的2BV，流速为3BV/h，然后调节流出液的pH至6，得到6L解析液；其中，所述酸醇溶液是由浓度为60v/v％的乙醇溶液与浓度为0.5wt％的盐酸溶液组成的pH为2的溶液；
S6：将6L解析液经截留分子量为500Da的纳滤膜浓缩，收集纳滤截流液，得到300ml固含量为50wt％的浓缩液；
S7：向300ml浓缩液中添加0.75g口感修饰剂(赤藓糖醇0.15g、海藻酸钠0.225g、缬氨酸0.375g)进行修饰，并经喷雾干燥得到131g葡萄糖基甜菊糖苷产品，喷雾干燥时的进风温度为180℃，出风温度为100℃。
实施例4
S1：取批次4的母液糖100g，DE值为6的麦芽糊精250g，加入1000mL水中，在80rpm的转速下搅拌至固体溶解完全，然后加入2mlα-环糊精葡萄 糖基转移酶(酶浓度0.15％，活力为100U/g)和1g淀粉酶(活力1000～3000U/g)，在温度为70℃，pH为5.6的条件下反应40h，得到850mL酶解液；
S2：向850mL酶解液中加入25g孔径为20nm的活性炭，除杂2h后得到830mL除杂液；
S3：将除杂液升温至100℃进行酶灭活处理，再由滤布孔径为40μm的板框过滤机在操作压力为1.0MPa的条件下过滤，得到815mL滤液；
S4：将815mL滤液进料至装填有1.5L大孔吸附树脂(T83)的树脂柱中吸附处理，流速1.5BV/h；再用2BV纯水对吸附后的大孔吸附树脂进行洗涤，流速为2BV/h；
S5：将洗涤后的1.5L树脂用酸醇溶液进行解析，酸醇溶液的用量为大孔吸附树脂体积的4BV，流速为1.5BV/h，然后调节流出液的pH至5.5，得到6L解析液；其中，所述酸醇溶液是由浓度为60v/v％的乙醇溶液与浓度为0.5wt％的盐酸溶液组成的pH为2的溶液；
S6：将6L解析液经截留分子量为500Da的纳滤膜浓缩，收集纳滤截流液，得到400ml固含量为40wt％的浓缩液；
S7：向400ml浓缩液中添加0.8g口感修饰剂(赤藓糖醇0.16g、海藻酸钠0.24g、缬氨酸0.4g)进行修饰，并经喷雾干燥得到145g葡萄糖基甜菊糖苷产品，喷雾干燥时的进风温度为180℃，出风温度为90℃。
对比例1
相对于实施例1，本对比例步骤S2中，采用2L大孔吸附树脂SD-300来替换活性炭，其他条件和实施例1相同。
对比例2
相对于实施例1，本对比例先采用活性炭进行除杂，再进行酶解反应，具体的除杂条件以及酶解反应条件及其他条件和实施例1相同。
对比例3
相对于实施例1，本对比例S5中，先采用pH为2的盐酸溶液进行解析， 再采用浓度为40v/v％的乙醇溶液进行解析，并合并两次的流出液，其他条件和实施例1相同。
对比例4
相对于实施例1，本对比例S5中，先采用浓度为40v/v％的乙醇溶液进行解析，再采用pH为2的盐酸溶液进行解析，并合并两次的流出液，其他条件和实施例1相同。
对比例5
相对于实施例1，本对比例S5中调节流出液的pH至9，得解析液，其他条件和实施例1相同。
对比例6
相对于实施例2，本对比例取消步骤S3中的酶灭活处理，其他条件和实施例2相同。
对比例7
相对于实施例3，本对比例将步骤S4中的大孔吸附树脂(T81)等量替换为T-28树脂，其他条件和实施例3相同。
对比例8
相对于实施例4，本对比例将步骤S7中的口感修饰剂替换为等量的柠檬酸钠，其他条件和实施例4相同。
一、产品质量、纯度以及口感测试
本发明实施例1-4以及对比例1-8中葡萄糖基甜菊糖苷含量的测定方法参考国家标准GB 2760-2014执行；
口感测试时，分别将所取样品稀释至500ppm，测试条件：温度29℃，湿度50RH％。具体的，根据GB/T 16291.2-2010的规定选择10名专业感官评价人员，组成感官评定小组，评价时感官评价人员在评定实验开始前1h内限制饮食，尤其是限制食用能严重影响味觉的食物。感官评价人员针对样品的苦味进行打分，10分制，最终分数取平均值。评价标准品为5wt％的蔗糖溶 液。测试结果如表2所示。
表2
从表2测试结果可以看出，相对于对比例，本发明以母液糖为原料，通过优化条件，制得的葡萄糖基甜菊糖苷不仅产量高，且总苷含量高，产品口味很好。
二、甜味剂感官评定实验
本发明设置了4组对比实验来测试本发明制备得到的葡萄糖基甜菊糖苷与原先母液糖中甜菊糖苷的口感差异，分别将所取样品稀释至500ppm，这主要是因为甜菊糖苷类产品的甜度倍数较高，需要进行稀释才能进行区别和评价，具体如下：
实验1：500ppm的母液糖(总苷68％)；
实验2：500ppm的实施例1制得的葡萄糖基甜菊糖苷；
实验3：500ppm的母液糖(总苷80％)；
实验4：500ppm的实施例4制得的葡萄糖基甜菊糖苷；
评价标准品为5wt％的蔗糖溶液。
测试条件：温度29℃，湿度50RH％；
测试时间：2023年05月16日。
根据GB/T 16291.2-2010的规定选择10名专业感官评价人员，组成感官评定小组。
具体评价时，感官评价人员在评定实验开始前1h内限制饮食，尤其是限制食用能严重影响味觉的食物。
感官评价人员分别针对甜味剂的甜度、起甜速度、后甜、苦味、涩味、杂味、整体喜好等指标进行综合打分；其中，起甜速度表征的是感受到甜味时的快慢，后甜表征的是甜味的持续时间；除整体喜好为百分制外(5wt％的蔗糖溶液为满分100分)，其他均为10分制，最终分数取平均值。口感评价的测试结果见下表3，其中，各感官数值越大，代表口感特征越明显。
表3
从表3测试结果可以看出：本发明制备得到的葡萄糖基甜菊糖苷相较于原先的母液糖在口感的各方面都有了提升，尤其在甜度、起甜速度上改善明显，同时显著降低了原料母液糖中的苦味和涩味。
下述实施例中100g葡萄糖基甜菊糖苷粗品中甜菊糖总苷(TSG)含量为71wt％，葡萄糖基甜菊糖苷(GSG)的含量为59wt％，糊精的含量为28wt％。
产品的定性分析方法：采用液相色谱串联四极杆飞行时间质谱仪定性转苷产物，检测条件如下：ACQUITY UPLC BEH HILIC氨基色谱柱，柱温为30℃， 乙腈：水＝80：20,(2min)-50：50(30min)(v/v)下梯度洗脱，进样量1μL，进样浓度为5mg/mL，流速为0.3mL/min；质谱条件为碰撞电压为6eV；离子化方式电喷雾电离(ESI)，负离子检测模式，分子量范围：200-2000。
产品的定量分析方法：依据GB2760-2014卫计委增补文件第8号文件中葡萄糖基甜菊糖苷的分析检测方法。
实施例5
S1：将100g葡萄糖基甜菊糖苷粗品溶于2L的10wt％乙醇溶液中，然后采用大孔吸附树脂进行吸附，吸附时控制液体流速为0.5BV/h，收集流出液，之后用0.05wt％浓度的盐酸水溶液进行解析，控制盐酸水溶液的流速为2BV/h，体积为2BV，收集酸水解析液；最好采用70wt％浓度乙醇溶液进行解析，控制乙醇溶液的流速为2BV/h，体积为2BV，收集高醇解析液；
S2：将流出液和酸水解析液混合浓缩得到500ml浓缩液，向浓缩液中加入0.05mlα-1，4葡萄糖水解酶，升温至45℃，水解2h，然后调节反应体系pH至3，对酶进行灭活，得到水解液；
S3：向500ml水解液中加入30g甜菊糖苷STV，调节溶液pH至6.0，升温至75℃，加入0.3ml葡萄糖基转移酶反应8小时，反应结束后升温至95℃灭活，得到反应液；将反应液浓缩至固含量为50wt％，然后进行喷雾干燥，得到60g高纯葡萄糖基甜菊糖苷A；
S4：将高醇解析液浓缩至固含量为50wt％，然后进行喷雾干燥，得到69g高纯葡萄糖基甜菊糖苷B。
经检测，高纯葡萄糖基甜菊糖苷A中甜菊糖总苷(TSG)含量为93wt％，葡萄糖基甜菊糖苷(GSG)含量为77wt％，糊精含量为5.8wt％；高纯葡萄糖基甜菊糖苷B中甜菊糖总苷(TSG)含量为99wt％，葡萄糖基甜菊糖苷(GSG)含量为92wt％，糊精含量为0.2wt％。
实施例6
S1：将100g葡萄糖基甜菊糖苷粗品溶于3L的10wt％乙醇溶液中，然后采用大孔吸附树脂进行吸附，吸附时控制液体流速为0.75BV/h，收集流出液，之后用0.05wt％浓度的盐酸水溶液进行解析，控制盐酸水溶液的流速为2BV/h，体积为2BV，收集酸水解析液；最好采用80wt％浓度乙醇溶液进行解析，控制乙醇溶液的流速为3BV/h，体积为3BV，收集高醇解析液；
S2：将流出液和酸水解析液混合浓缩得到500ml浓缩液，向浓缩液中加入0.05mlα-1，4葡萄糖水解酶，升温至50℃，水解2h，然后调节反应体系pH至3，对酶进行灭活，得到水解液；
S3：向500ml水解液中加入30g甜菊糖苷STV，调节溶液pH至6.0，升温至75℃，加入0.4ml葡萄糖基转移酶反应7.5小时，反应结束后升温至95℃灭活，得到反应液；将反应液浓缩至固含量为40wt％，然后进行喷雾干燥，得到61g高纯葡萄糖基甜菊糖苷A；
S4：将高醇解析液浓缩至固含量为40wt％，然后进行喷雾干燥，得到68g高纯葡萄糖基甜菊糖苷B。
经检测，高纯葡萄糖基甜菊糖苷A中甜菊糖总苷(TSG)含量为93.5wt％，葡萄糖基甜菊糖苷(GSG)含量为79.5wt％，糊精含量为5.7wt％；高纯葡萄糖基甜菊糖苷B中甜菊糖总苷(TSG)含量为99wt％，葡萄糖基甜菊糖苷(GSG)含量为93wt％，糊精含量为0.2wt％。
实施例7
S1：将100g葡萄糖基甜菊糖苷粗品溶于2.5L的10wt％乙醇溶液中，然后采用大孔吸附树脂进行吸附，吸附时控制液体流速为0.8BV/h，收集流出液，之后用0.05wt％浓度的盐酸水溶液进行解析，控制盐酸水溶液的流速为3BV/h，体积为3BV，收集酸水解析液；最好采用75wt％浓度乙醇溶液进行解析，控制乙醇溶液的流速为2BV/h，体积为2BV，收集高醇解析液；
S2：将流出液和酸水解析液混合浓缩得到500ml浓缩液，向浓缩液中加 入0.05mlα-1，4葡萄糖水解酶，升温至45℃，水解2h，然后调节反应体系pH至3，对酶进行灭活，得到水解液；
S3：向500ml水解液中加入30g甜菊糖苷STV，调节溶液pH至6.0，升温至75℃，加入0.5ml葡萄糖基转移酶反应8小时，反应结束后升温至95℃灭活，得到反应液；将反应液浓缩至固含量为45wt％，然后进行喷雾干燥，得到61g高纯葡萄糖基甜菊糖苷A；
S4：将高醇解析液浓缩至固含量为45wt％，然后进行喷雾干燥，得到68.5g高纯葡萄糖基甜菊糖苷B。
经检测，高纯葡萄糖基甜菊糖苷A中甜菊糖总苷(TSG)含量为93wt％，葡萄糖基甜菊糖苷(GSG)含量为78wt％，糊精含量为5.6wt％；高纯葡萄糖基甜菊糖苷B中甜菊糖总苷(TSG)含量为99.5wt％，葡萄糖基甜菊糖苷(GSG)含量为93wt％，糊精含量为0.15wt％。
下述实施例中葡萄糖基甜菊糖苷的收率计算方法如下：
收率(％)＝(目的产物实际生成量/目的产物的理论生成量)×100％。
下述实施例中GSG为葡萄糖基甜菊糖苷，TSG为所有甜菊糖苷总称。
实施例8
S1：取100g反应所得葡萄糖基甜菊糖苷粗品(TSG85wt％，GSG79wt％，糊精14.5wt％)，加入5L纯化水常温常压下搅拌至固体溶解，制得固含量为2wt％的料液；
S2：将上述制得的料液经截留分子量为800Da的纳滤膜操作压力在4bar范围内进行浓缩处理，收集膜截流液和膜透过液；
S3：采用旋转蒸发器将步骤S2得到的膜截流液以及膜透过液中的溶剂在80℃，压力为-0.1MPa的条件下分别去除，制得85g中间产品A、15g中间产品B；
S4：取85g中间产品A，在烧杯中加入850ml浓度为99wt％的甲醇并至 于磁力搅拌器上，缓慢加入中间产品A并持续搅拌，待完全溶解后开始计时，沉降5h，反应结束；之后真空抽滤，滤饼回收待用，滤液去除溶剂得83.3g高纯度葡萄糖基甜菊糖苷产品，该产品中葡萄糖基甜菊糖苷的含量为92.3wt％(即产品纯度)，总苷含量为98wt％，糊精含量＜0.5wt％，葡萄糖基甜菊糖苷收率为97.3％；
S5：将中间产品B和S4中得到的滤饼合并得16.7g固体样品，将固体样品和纯化水以质量比为1:10的比例混合溶解，加入固体样品重量0.2％的α-1,4-葡萄糖水解酶，在40℃、pH4.5的条件下酶解6h，酶解结束后将反应液加热至90℃使酶灭活得到含葡萄糖溶液；
S6：将含葡萄糖溶液在80℃条件下浓缩至固含量为75wt％的浸膏状中间产品C；将浸膏状中间产品C降温至20℃保温，此时有晶体缓慢析出，静置6h，真空抽滤，滤液回收，滤饼为高纯葡萄糖，葡萄糖的纯度为80％。
实施例9
S1：取100g反应所得葡萄糖基甜菊糖苷粗品(TSG90wt％，GSG83.6wt％，糊精10wt％)，加入10L纯化水常温常压下搅拌至固体溶解，制得固含量为1wt％的料液；
S2：将上述制得的料液经截留分子量为800Da的纳滤膜操作压力在4bar范围内进行浓缩处理，收集膜截流液和膜透过液；
S3：采用旋转蒸发器将步骤S2得到的膜截流液以及膜透过液中的溶剂在80℃，压力为-0.1MPa的条件下分别去除，制得92g中间产品A、8g中间产品B；
S4：取92g中间产品A，在烧杯中加入850ml浓度为99wt％的甲醇并至于磁力搅拌器上，缓慢加入中间产品A并持续搅拌，待完全溶解后开始计时，沉降8h，反应结束；之后真空抽滤，滤饼回收待用，滤液去除溶剂得86g高纯度葡萄糖基甜菊糖苷产品，该产品中总苷含量为98.4wt％,葡萄糖基甜菊 糖苷含量为92.9wt％；糊精含量＜0.6wt％，葡萄糖基甜菊糖苷收率为95.6％；
S5：将中间产品B和S4中得到的滤饼合并得12.5g固体样品，将固体样品和纯化水以质量比为1:10的比例混合溶解，加入固体样品重量0.3％的α-1,4-葡萄糖水解酶，在40℃、pH为4.5的条件下酶解7h，酶解结束后将反应液加热至90℃使酶灭活得到含葡萄糖溶液；
S6：将含葡萄糖溶液在90℃条件下浓缩至固含量为70wt％的浸膏状中间产品C；将浸膏状中间产品C降温至20℃保温，此时有晶体缓慢析出，静置7h，真空抽滤，滤液回收，滤饼为高纯葡萄糖，纯度为83％。
实施例10
S1：取100g反应所得葡萄糖基甜菊糖苷粗品(TSG85wt％，GSG79wt％，糊精14.5wt％)，加入5L纯化水常温常压下搅拌至固体溶解，制得固含量为2wt％的料液；
S2：将上述制得的料液经截留分子量为700Da的纳滤膜操作压力在4bar范围内进行浓缩处理，收集膜截流液和膜透过液；
S3：采用旋转蒸发器将步骤S2得到的膜截流液以及膜透过液中的溶剂在80℃，压力为-0.1MPa的条件下分别去除，制得83g中间产品A、16.3g中间产品B；
S4：取83g中间产品A，在烧杯中加入850ml浓度为99wt％的甲醇并至于磁力搅拌器上，缓慢加入中间产品A并持续搅拌，待完全溶解后开始计时，沉降5h，反应结束；之后真空抽滤，滤饼回收待用，滤液去除溶剂得82.9g高纯度葡萄糖基甜菊糖苷产品，产品中总苷含量为98wt％，葡萄糖基甜菊糖苷含量为93.1wt％，糊精含量＜0.5wt％，葡萄糖基甜菊糖苷收率为97.7％；
S5：将中间产品B和S4得到的滤饼合并得17g固体样品，将固体样品和纯化水以质量比为1:10的比例混合溶解，加入固体样品重量0.2％的α-1,4- 葡萄糖水解酶，在40℃、pH4.5的条件下酶解6h，酶解结束后将反应液加热至90℃使酶灭活得到含葡萄糖溶液；
S6：将含葡萄糖溶液在80℃条件下浓缩至固含量为75wt％的浸膏状中间产品C；将浸膏状中间产品C降温至20℃保温，此时有晶体缓慢析出，静置6h，真空抽滤，滤液回收，滤饼为高纯葡萄糖，葡萄糖纯度为83％。
实施例11
S1：取100g反应所得葡萄糖基甜菊糖苷粗品(TSG85wt％，GSG79wt％，糊精14.5wt％)，加入5L纯化水常温常压下搅拌至固体溶解，制得固含量为2wt％的料液；
S2：将上述制得的料液经截留分子量为600Da的纳滤膜操作压力在4bar范围内进行浓缩处理，收集膜截流液和膜透过液；
S3：采用旋转蒸发器将步骤S2得到的膜截流液以及膜透过液中的溶剂在80℃，压力为-0.1MPa的条件下分别去除，制得83.9g中间产品A、15.5g中间产品B；
S4：取83.9g中间产品A，在烧杯中加入850ml浓度为99wt％的甲醇并至于磁力搅拌器上，缓慢加入中间产品A并持续搅拌，待完全溶解后开始计时，沉降5h，反应结束；之后真空抽滤，滤饼回收待用，滤液去除溶剂得80g高纯度葡萄糖基甜菊糖苷产品，产品中总苷含量为99wt％，葡萄糖基甜菊糖苷含量为94.2wt％，糊精含量＜0.3wt％，葡萄糖基甜菊糖苷收率为95.4wt％；
S5：将中间产品B和S4得到的滤饼合并得16g固体样品，将固体样品和纯化水以质量比为1:10的比例混合溶解，加入固体样品重量0.2％的α-1,4-葡萄糖水解酶，在40℃、pH4.5的条件下酶解6h，酶解结束后将反应液加热至90℃使酶灭活得到含葡萄糖溶液；
S6：将含葡萄糖溶液在80℃条件下浓缩至固含量为75wt％的浸膏状中间 产品C；将浸膏状中间产品C降温至20℃保温，此时有晶体缓慢析出，静置6h，真空抽滤，滤液回收，滤饼为高纯葡萄糖，葡萄糖纯度为82％。
实施例12
S1：取100g反应所得葡萄糖基甜菊糖苷粗品(TSG85wt％，GSG79wt％，糊精14.5wt％)，加入5L纯化水常温常压下搅拌至固体溶解，制得固含量为2wt％的料液；
S2：将上述制得的料液经截留分子量为500Da的纳滤膜操作压力在4bar范围内进行浓缩处理，收集膜截流液和膜透过液；
S3：采用旋转蒸发器将步骤S2得到的膜截流液以及膜透过液中的溶剂在80℃，压力为-0.1MPa的条件下分别去除，制得88.6g中间产品A、11g中间产品B；
S4：取88g中间产品A，在烧杯中加入850ml浓度为99wt％的甲醇并至于磁力搅拌器上，缓慢加入中间产品A并持续搅拌，待完全溶解后开始计时，沉降5h，反应结束；之后真空抽滤，滤饼回收待用，滤液去除溶剂得81.9g高纯度葡萄糖基甜菊糖苷产品，该产品中总苷含量为97.3wt％，葡萄糖基甜菊糖苷含量为91.2wt％，糊精含量＜1.0wt％，葡萄糖基甜菊糖苷收率为94.5％；
S5：将中间产品B和S4得到的滤饼合并得17g固体样品，将固体样品和纯化水以质量比为1:10的比例混合溶解，加入固体样品重量0.2％的α-1,4-葡萄糖水解酶，在40℃、pH4.5的条件下酶解6h，酶解结束后将反应液加热至90℃使酶灭活得到含葡萄糖溶液；
S6：将含葡萄糖溶液在80℃条件下浓缩至固含量为75wt％的浸膏状中间产品C；将浸膏状中间产品C降温至20℃保温，此时有晶体缓慢析出，静置6h，真空抽滤，滤液回收，滤饼为高纯葡萄糖，葡萄糖的纯度为85％。
实施例13
S1：取100g反应所得葡萄糖基甜菊糖苷粗品(TSG85wt％，GSG79wt％，糊精14.5wt％)，加入5L纯化水常温常压下搅拌至固体溶解，制得固含量为2wt％的料液；
S2：将上述制得的料液经截留分子量为800Da的纳滤膜操作压力在4bar范围内进行浓缩处理，收集膜截流液和膜透过液；
S3：采用旋转蒸发器将步骤S2得到的膜截流液以及膜透过液中的溶剂在80℃，压力为-0.1MPa的条件下分别去除，制得84.6g中间产品A、15g中间产品B；
S4：取84.6g中间产品A，在烧杯中加入850ml浓度为99wt％的甲醇并至于磁力搅拌器上，缓慢加入中间产品A并持续搅拌，待完全溶解后开始计时，沉降5h，反应结束；之后真空抽滤，滤饼回收待用，滤液去除溶剂得83.5g高纯度葡萄糖基甜菊糖苷产品，该产品中总苷含量为97.8wt％，葡萄糖基甜菊糖苷含量为92.9wt％，糊精含量＜0.5wt％，葡萄糖基甜菊糖苷收率为98.2％；
S5：将中间产品B和S4得到的滤饼合并得16.5g固体样品，将固体样品和纯化水以质量比为1:10的比例混合溶解，加入固体样品重量0.2％的α-1,4-葡萄糖水解酶，在45℃、pH4.5的条件下酶解8h，酶解结束后将反应液加热至90℃使酶灭活得到含葡萄糖溶液；
S6：将含葡萄糖溶液在80℃条件下浓缩至固含量为75wt％的浸膏状中间产品C；将浸膏状中间产品C降温至20℃保温，此时有晶体缓慢析出，静置6h，真空抽滤，滤液回收，滤饼为高纯葡萄糖，葡萄糖的纯度为84.3％。
实施例14
S1：取100g反应所得葡萄糖基甜菊糖苷粗品(TSG85wt％，GSG79wt％，糊精14.5wt％)，加入5L纯化水常温常压下搅拌至固体溶解，制得固含量为2％的料液；
S2：将上述制得的料液经截留分子量为800Da的纳滤膜操作压力在4bar范围内进行浓缩处理，收集膜截流液和膜透过液；
S3：采用旋转蒸发器将步骤S2得到的膜截流液以及膜透过液中的溶剂在80℃，压力为-0.1MPa的条件下分别去除，制得84.8g中间产品A、14.9g中间产品B；
S4：取84g中间产品A，在烧杯中加入850ml浓度为99wt％的甲醇并至于磁力搅拌器上，缓慢加入中间产品A并持续搅拌，待完全溶解后开始计时，沉降5h，反应结束；之后真空抽滤，滤饼回收待用，滤液去除溶剂得82.5g高纯度葡萄糖基甜菊糖苷产品，该产品中总苷含量为98.1wt％，葡萄糖基甜菊糖苷含量为92.9wt％，糊精含量＜0.7wt％，葡萄糖基甜菊糖苷收率为97％；
S5：将中间产品B和S4得到的滤饼合并得17g固体样品，将固体样品和纯化水以质量比为1:10的比例混合溶解，加入固体样品重量0.2％的α-1,4-葡萄糖水解酶，在35℃、pH4.5的条件下酶解12h，酶解结束后将反应液加热至90℃使酶灭活得到含葡萄糖溶液；
S6：将含葡萄糖溶液在80℃条件下浓缩至固含量为75wt％的浸膏状中间产品C；将浸膏状中间产品C降温至20℃保温，此时有晶体缓慢析出，静置8h，真空抽滤，滤液回收，滤饼为高纯葡萄糖，葡萄糖的纯度为83.6％。
实施例15
S1：取100g反应所得葡萄糖基甜菊糖苷粗品(TSG85wt％，GSG79wt％，糊精14.5wt％)，加入5L纯化水常温常压下搅拌至固体溶解，制得固含量为2wt％的料液；
S2：将上述制得的料液经截留分子量为800Da的纳滤膜操作压力在4bar范围内进行浓缩处理，收集膜截流液和膜透过液；
S3：采用旋转蒸发器将步骤S2得到的膜截流液以及膜透过液中的溶剂在 80℃，压力为-0.1MPa的条件下分别去除，制得86.1g中间产品A、13.8g中间产品B；
S4：取86g中间产品A，在烧杯中加入850ml浓度为99wt％的甲醇并至于磁力搅拌器上，缓慢加入中间产品A并持续搅拌，待完全溶解后开始计时，沉降5h，反应结束；之后真空抽滤，滤饼回收待用，滤液去除溶剂得84g高纯度葡萄糖基甜菊糖苷产品，产品中总苷含量为98.5wt％，葡萄糖基甜菊糖苷含量为93.1wt％，糊精含量＜0.6wt％，葡萄糖基甜菊糖苷的收率为99％；
S5：将中间产品B和S4得到的滤饼合并得15g固体样品，将固体样品和纯化水以质量比为1:10的比例混合溶解，加入固体样品重量0.2％的α-1,4-葡萄糖水解酶，在30℃、pH4的条件下酶解6h，酶解结束后将反应液加热至90℃使酶灭活得到含葡萄糖溶液；
S6：将含葡萄糖溶液在80℃条件下浓缩至固含量为75wt％的浸膏状中间产品C；将浸膏状中间产品C降温至20℃保温，此时有晶体缓慢析出，静置6h，真空抽滤，滤液回收，滤饼为高纯葡萄糖，葡萄糖的纯度为80％。
综上所述，本申请实施例中制得的葡萄糖的纯度高达85％，制得的产品总苷含量高达99wt％，葡萄糖基甜菊糖苷含量高达94.2wt％，葡萄糖基甜菊糖苷收率高达99％。
此外应理解，在阅读了本发明讲授的内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (30)

  1. 一种利用母液糖制备葡萄糖基甜菊糖苷的制备方法，其特征在于，包括以下步骤：
    (1)将母液糖、麦芽糊精和水混合搅拌至固体溶解，加入酶进行酶解得到酶解液；
    (2)向步骤(1)得到的酶解液中加入活性炭进行除杂，得除杂液，将除杂液进行酶灭活处理，然后过滤，得到的滤液进入到大孔吸附树脂内进行吸附处理；
    (3)采用乙醇溶液与盐酸溶液组成的pH为1～3的酸醇溶液进行解析，得解析液；
    (4)调节上述解析液的pH至5～6，然后采用纳滤膜进行浓缩，得浓缩液；
    (5)向浓缩液中加入口感修饰剂，然后采用喷雾干燥得到葡萄糖基甜菊糖苷产品。
  2. 根据权利要求1所述的一种利用母液糖制备葡萄糖基甜菊糖苷的制备方法，其特征在于：步骤(1)中，所述母液糖、麦芽糊精和水的质量比为1：(1～3)：(5～10)。
  3. 根据权利要求1所述的一种利用母液糖制备葡萄糖基甜菊糖苷的制备方法，其特征在于：步骤(1)中，所述酶为葡萄糖基转移酶和淀粉酶的混合物，所述葡萄糖基转移酶为α-环糊精葡萄糖基转移酶，添加时所述葡萄糖基转移酶的添加形式为葡萄糖基转移酶溶液，所述葡萄糖基转移酶溶液的浓度为0.8～1.2wt％，所述淀粉酶的添加形式为固体淀粉酶，所述固体淀粉酶的浓度为0.4～0.6wt％；
    和/或每100g母液糖中葡萄糖基转移酶溶液的添加量为0.5～2ml，每100g母液糖中固体淀粉酶的添加量为0.1～1g。
  4. 根据权利要求1所述的一种利用母液糖制备葡萄糖基甜菊糖苷的制备方法，其特征在于：步骤(1)中，所述酶解的条件为：酶解温度为60～80℃，pH为5.5～6.0，酶解时间为36～48h。
  5. 根据权利要求1所述的一种利用母液糖制备葡萄糖基甜菊糖苷的制备方法，其特征在于：步骤(2)中，所述活性炭的添加量为所述酶解液质量的1～3％，除杂时间为1～6h。
  6. 根据权利要求1所述的一种利用母液糖制备葡萄糖基甜菊糖苷的制备方法，其特征在于：步骤(2)中，采用大孔吸附树脂吸附时滤液的进料体积为大孔吸附树脂体积的30～60％，进料速度为0.5～2BV/h。
  7. 根据权利要求6所述的一种利用母液糖制备葡萄糖基甜菊糖苷的制备方法，其特征在于：将吸附后的大孔吸附树脂进行解析前，还包括：用纯水洗涤所述大孔吸附树脂，洗涤时所述纯水的用量为所述大孔吸附树脂体积的1～3BV，流速为1～3BV/h。
  8. 根据权利要求1所述的一种利用母液糖制备葡萄糖基甜菊糖苷的制备方法，其特征在于：步骤(3)中，所述乙醇溶液的浓度为40～60v/v％，所述盐酸溶液的浓度为0.3～0.5wt％，解析时，酸醇溶液的体积为大孔吸附树脂体积的2～4BV，流速为1～3BV/h。
  9. 根据权利要求1所述的一种利用母液糖制备葡萄糖基甜菊糖苷的制备方法，其特征在于：步骤(4)中，所述纳滤膜的截留分子量为400～500Da。
  10. 根据权利要求1所述的一种利用母液糖制备葡萄糖基甜菊糖苷的制备方法，其特征在于：步骤(5)中，所述口感修饰剂为赤藓糖醇、海藻酸钠和缬氨酸的组合物，三者质量比为2：(2～4)：(5～8)；所述口感修饰剂的添加量为所述浓缩液中产品质量的0.1～0.5％。
  11. 一种葡萄糖基甜菊糖苷的纯化方法，其特征在于，包括以下步骤：
    (1)将葡萄糖基甜菊糖苷粗品溶于乙醇溶液中，然后将得到的葡萄糖基甜菊糖苷乙醇溶液经大孔吸附树脂吸附处理，收集流出液；之后依次采用酸水溶液以及高纯度乙醇溶液进行解析，收集酸水解析液和高醇解析液；
    (2)将步骤(1)得到的流出液和酸水解析液的混合液浓缩处理，之后向浓缩液中加入α-1,4葡萄糖水解酶，升温水解反应，水解结束后调节反应体系pH 至3，对酶进行灭活，得到水解液；
    (3)向水解液中加入甜菊糖苷，然后调节溶液pH至6.0，加入葡萄糖基转移酶，升温反应，反应结束后继续升温灭活，得到反应液，将反应液浓缩至固含量为40-50wt％，然后将浓缩液进行喷雾干燥，得到高纯葡萄糖基甜菊糖苷A；
    (4)将高醇解析液浓缩至固含量为40-50wt％，然后将浓缩液进行喷雾干燥，得到高纯葡萄糖基甜菊糖苷B。
  12. 根据权利要求11所述的一种葡萄糖基甜菊糖苷的纯化方法，其特征在于，步骤(1)中，所述乙醇溶液的浓度为5-15wt％；所述葡萄糖基甜菊糖苷乙醇溶液中葡萄糖基甜菊糖苷的浓度为30-50g/L。
  13. 根据权利要求11所述的一种葡萄糖基甜菊糖苷的纯化方法，其特征在于，步骤(1)中，吸附处理时，葡萄糖基甜菊糖苷乙醇溶液的流速为0.25BV/h-1BV/h。
  14. 根据权利要求11所述的一种葡萄糖基甜菊糖苷的纯化方法，其特征在于，步骤(1)中，所述酸水溶液为浓度为0.04-0.06wt％的盐酸溶液，所述高纯度乙醇溶液的浓度为70-80wt％；解析时，酸水溶液的体积为2-3BV，流速为2-3BV/h，高纯度乙醇溶液的体积为2-3BV，流速为2-3BV/h。
  15. 根据权利要求11所述的一种葡萄糖基甜菊糖苷的纯化方法，其特征在于，步骤(1)中，流出液的体积为树脂体积的1-2BV，酸水解析液的体积为树脂体积的2-3BV。
  16. 根据权利要求11所述的一种葡萄糖基甜菊糖苷的纯化方法，其特征在于，步骤(2)中，所述浓缩处理时的浓缩倍数为3-5倍。
  17. 根据权利要求11所述的一种葡萄糖基甜菊糖苷的纯化方法，其特征在于，步骤(2)中，每100g葡萄糖基甜菊糖苷粗品使用0.03-0.1ml的α-1，4葡萄糖水解酶。
  18. 根据权利要求11所述的一种葡萄糖基甜菊糖苷的纯化方法，其特征在 于，步骤(2)中，所述水解反应的温度为40-50℃，水解反应的时间为1-3h。
  19. 根据权利要求11所述的一种葡萄糖基甜菊糖苷的纯化方法，其特征在于，步骤(3)中，所述甜菊糖苷为甜菊糖苷STV；每100g葡萄糖基甜菊糖苷粗品使用0.3-0.5ml的葡萄糖基转移酶。
  20. 根据权利要求11所述的一种葡萄糖基甜菊糖苷的纯化方法，其特征在于，步骤(3)中，所述升温反应的温度为70-80℃，时间为6-10h；所述升温灭活的温度为95℃。
  21. 一种分离纯化葡萄糖基甜菊糖苷并回收葡萄糖的方法，其特征在于，包括以下步骤:
    (1)将葡萄糖基甜菊糖苷粗品加纯化水重新进行溶解，制得料液；
    (2)将料液进行纳滤浓缩处理，收集膜截流液和膜透过液；
    (3)分别将膜截流液和膜透过液中的溶剂去除，制得中间产品A和中间产品B；
    (4)将中间产品A和甲醇混合沉降，然后真空抽滤，收集滤饼和滤液，滤液干燥后得到高纯葡萄糖基甜菊糖苷；
    (5)将中间产品B和滤饼混合并加入纯化水，搅拌至固体溶解，加入α-1,4-葡萄糖水解酶进行酶解处理，酶解结束后加热料液使酶失活，制得含葡萄糖溶液；
    (6)将含葡萄糖溶液高温浓缩处理，得到浸膏状中间产品C，将浸膏状中间产品C进行缓慢降温结晶，最后进行真空抽滤，滤液回收留作后续生产套用，滤饼为高纯葡萄糖。
  22. 根据权利要求21所述的一种分离纯化葡萄糖基甜菊糖苷并回收葡萄糖的方法，其特征在于：步骤(1)中，所述料液的固含量为1wt％-5wt％。
  23. 根据权利要求21所述的一种分离纯化葡萄糖基甜菊糖苷并回收葡萄糖的方法，其特征在于：步骤(2)中，采用纳滤浓缩时纳滤膜的截留分子量为500Da-800Da，纳滤浓缩时的操作压力在3.5-5bar。
  24. 根据权利要求21所述的一种分离纯化葡萄糖基甜菊糖苷并回收葡萄糖的方法，其特征在于：步骤(3)中，在温度为80℃，压力为-0.1MPa的条件下去除膜透过液和膜截流液中的溶剂。
  25. 根据权利要求21所述的一种分离纯化葡萄糖基甜菊糖苷并回收葡萄糖的方法，其特征在于：步骤(4)中，所述甲醇的浓度为95wt％-99wt％，所述中间产品A与甲醇的质量比为1：(5-10)。
  26. 根据权利要求21所述的一种分离纯化葡萄糖基甜菊糖苷并回收葡萄糖的方法，其特征在于：步骤(4)中，所述混合沉降的时间为2h-8h。
  27. 根据权利要求21所述的一种分离纯化葡萄糖基甜菊糖苷并回收葡萄糖的方法，其特征在于：步骤(5)中，所述α-1,4-葡萄糖水解酶的添加量为中间产品B和滤饼总重量的0.2％-0.3％。
  28. 根据权利要求21所述的一种分离纯化葡萄糖基甜菊糖苷并回收葡萄糖的方法，其特征在于：步骤(5)中，所述酶解处理的温度为35℃-45℃，pH为4.0-4.5，时间为5h-7h；加热料液至90℃使酶失活。
  29. 根据权利要求21所述的一种分离纯化葡萄糖基甜菊糖苷并回收葡萄糖的方法，其特征在于：步骤(6)中，高温浓缩处理的温度为80℃-90℃，所述浸膏状中间产品C的固含量为70wt％-75wt％。
  30. 根据权利要求21所述的一种分离纯化葡萄糖基甜菊糖苷并回收葡萄糖的方法，其特征在于：步骤(6)中，所述缓慢降温的速率为10℃/h-15℃/h，降温至20℃保温结晶5h-7h。
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