CN105294790B - A method of extracting high-purity stevioside from STEVIA REBAUDIANA - Google Patents
A method of extracting high-purity stevioside from STEVIA REBAUDIANA Download PDFInfo
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Abstract
A method of it extracting high-purity stevioside from STEVIA REBAUDIANA, includes the following steps：（1）It is soaked in water after Stevia rebaudiana starting material is crushed, cellulase preparation is added and is digested；（2）Heating extraction, is centrifuged, and filtering obtains the enzyme leaching liquor of STEVIA REBAUDIANA；（3）By the enzyme leaching liquor heating water bath of STEVIA REBAUDIANA, inorganic salts flocculant, stirring is added, flocculation treatment is centrifuged, filtering, obtains STEVIA REBAUDIANA removal of impurities liquid；（4）STEVIA REBAUDIANA removal of impurities liquid is crossed into large pore resin absorption column and carries out decolorization adsorption, elution, nanofiltration is concentrated, obtains STEVIA REBAUDIANA concentrate；（5）By silica gel column chromatography on STEVIA REBAUDIANA concentrate, elution collects each fraction, concentration and drying, obtains the stevia glycoside product of a variety of high-purities.It may be up to 98.4% according to the method for the present invention products obtained therefrom purity, ultimate yield > 90%；Ethyl alcohol, ethyl ester residual quantity are low, Environmental Safety；The method of the present invention simple process, strong operability, energy consumption, at low cost, suitable plant produced.
Description
Technical field
The present invention relates to a kind of methods for extracting high-purity stevioside, and in particular to one kind is suitable for industrial
The method of high-purity stevioside is extracted from STEVIA REBAUDIANA.
Background technique
STEVIA REBAUDIANA originates in the mountain range Amambay and Mbaxacayu of Paraguay, belongs to sugar plant, entire growth period temperature
All at 20 DEG C or more, there is thermophily.Japan in 1970 introduces STEVIA REBAUDIANA from Brazil, starts to tame, cultivate, glycosides processed, while into
The test such as row toxicity, Food Inspection, and STEVIA REBAUDIANA product --- stevioside is developed and used first.China in 1976 by from
Japan introduces a fine variety, and plants experimentally, and succeeds, and the beginning of the eighties is to popularizing planting in all parts of the country.
Stevioside is a kind of tetracylic diterpene derivatives being primarily present in STEVIA REBAUDIANA blade, mainly includes stevioside
（Stevioside）, rebaudioside A（Rebaudioside A）, Rebaudioside B（Rebaudiosde B）, dulcoside B
（Rebaudioside C）Deng 8 kinds of glucosides, sugariness is about 300 times of sucrose.Some researches show that stevioside has height
Sugariness, it is low in calories, the characteristics of no obvious toxic-side effects.It can inhibit hyperglycemia, hypertension and has anti-inflammatory, antitumor, antidiarrheal benefit
Urinate and assist immunoregulatory effect.Currently, South America, Japan and China are using stevioside as a kind of natural sweet tea
Taste agent is widely used in food and beverage.
The extracting method of existing stevioside mainly has cooking process, cold water soak method, fermentation method, electrolysis method, ultrasound to mention
It follows the example of.Cooking process, cold water soak method, ultrasonic extraction are industrial a kind of most widely used methods, but it is extracting sweet tea
While leaf synanthrin glycosides, also a large amount of impurity is extracted out；And biological fermentation process, electrolysis method can introduce outside in extracting solution
Carry out impurity, brings difficulty to the purifies and separates of subsequent handling.The purification process of existing stevioside mainly has preparation chromatography, has
Solvent extraction and recrystallization method etc..Preparing chromatograph in industryization investment is big, and operation difficulty is big；And organic solvent extraction and recrystallization method is deposited
In the defects such as solvent usage amount is big, product yield is low.
CN101062077B discloses method that is a kind of while preparing STEVIA REBAUDIANA total stevioside and STEVIA REBAUDIANA general flavone, be by
STEVIA REBAUDIANA cured leaf is extracted with organic solvent, after high-purity stevia rebaudianum glycoside product is obtained with supercritical fluid extraction, column chromatography.
But organic solvent method is used since it is extracted, it is at high cost, and safety coefficient is low, later-period purification separation uses overcritical, column
There is the defects of investment is big, operation difficulty is big in chromatography.
CN101805768B discloses a kind of biological enzymolysis and purification method of high-quality stevioside, is mentioned using enzyme, macropore tree
Rouge purifying, solvent extraction are recrystallized to give high purity product, and dehydrated alcohol is largely used in crystallization process.Its defect is
Be not suitable for industrial scale production, yield is low, at high cost.
CN102199177A discloses a kind of production method of pure natural high-purity stevioside, is using ultrasonic wave after enzymatic hydrolysis
Continuous Countercurrent Extraction technology extracts, and then clarifies material with film filtering, then with Multi-functional analog moving bed separation system
It is adsorbed, ethanol elution obtains product.But the purity of products obtained therefrom only up to reach 96%, and finished product is single, produce
The product rate of recovery is low, is not suitable for industrialized production.
Summary of the invention
The technical problem to be solved by the present invention is to overcome drawbacks described above of the existing technology, provide a kind of technique letter
Single, low energy consumption, environment friendly and pollution-free, is suitable for industrialized production, extracts high-purity sweet tea in the slave STEVIA REBAUDIANA that product yield is high, with high purity
The method of leaf synanthrin glycosides.
The technical solution adopted by the present invention to solve the technical problems is as follows：One kind extracting high-purity sweetleaf from STEVIA REBAUDIANA
The method of synanthrin glycosides, includes the following steps：
（1）Enzyme immersion：It is soaked in water after Stevia rebaudiana starting material is crushed, cellulase preparation is added and is digested, obtains
STEVIA REBAUDIANA enzymolysis liquid；
（2）Heating extraction：By step（1）The heating extraction of gained STEVIA REBAUDIANA enzymolysis liquid, is cooled to room temperature, is centrifuged, and filtering obtains
The enzyme leaching liquor of STEVIA REBAUDIANA；
（3）Flocculation purification：By step（2）The enzyme leaching liquor of gained STEVIA REBAUDIANA carries out heating water bath, and inorganic salts flocculation is added
Agent, stirring after carrying out flocculation treatment, are centrifuged, filtering, obtain STEVIA REBAUDIANA removal of impurities liquid；
（4）Macroporous absorbent resin decoloration purifying：By step（3）Gained STEVIA REBAUDIANA removal of impurities liquid crosses large pore resin absorption column progress
Decolorization adsorption, then with elution, eluent carries out nanofiltration, concentration obtains STEVIA REBAUDIANA concentrate；
（5）Silica gel column chromatography post separation：By step（4）Silica gel column chromatography on gained STEVIA REBAUDIANA concentrate, then with ethyl alcohol and
The mixed solvent of ethyl acetate is eluted, and collects each fraction respectively, is concentrated, dry, obtains the stevioside of a variety of high-purities
Product.
Step（1）In, the mass content of stevioside is in the Stevia rebaudiana starting material：Stevioside 4~5%, Rebaudiodside A
A 5~6%, dulcoside B 0.5~1.0%, total glucosides are 10~12%.
Further, step（1）In, the smashed partial size of Stevia rebaudiana starting material is 10~30 mesh, the Stevia rebaudiana starting material
Feed liquid mass ratio with water is 1:10~12, the addition quality of the cellulase preparation（g）For the water volume of addition（mL）'s
0.1~0.3w/v%（It is preferred that 0.15~0.20w/v%）.If water consumption for immersion is lower than 10 times, glucosides yield can sharply decline, and
It is meaningless to be further continued for increase water consumption almost without increase for glucosides yield after water consumption is higher than 12 times；The cellulase preparation
Addition, be conducive to the broken of Stevia rebaudiana starting material cell wall, to be conducive to the dissolution of all kinds of glucosides, if but additive amount less than 0.1
W/v% will lead to hydrolysis result and sharply decline, and additive amount is higher than 0.3 w/v% and is also promoted without positive effect, continue growing enzyme use
Enzyme preparation can be only lost in amount.
Further, step（2）In, the temperature of the extraction is 40~50 DEG C（It is preferred that 43~46 DEG C）, the pH value of extraction is
5.0~6.0, the time of extraction is 60~70min.Since the activity and extraction effect of enzymatic reagent are needed while guaranteed when heating extraction
Rate, inventor the study found that extracting effect is best under the Extracting temperature, pH value, and upon extracting between be greater than 60min after,
Extracting efficiency tends to be steady state, and yield hardly increases, and continues to extend extraction time meaningless.
Further, step（3）In, the temperature of the heating water bath is 60~80 DEG C（It is preferred that 68~72 DEG C）, the flocculation
The pH value of processing is 8~9, and the time of flocculation treatment is 30~40min.Inventor is the study found that when water temperature is in 50~80 DEG C, pH
When value is 8~10, substantially in the same horizontal line, but loss late of flocculating has larger difference to flocculating effect, in the temperature and pH
Lower flocculation loss late is minimum；Flocculation time is longer, and loss late is bigger, and for the balance for guaranteeing yield, flocculating effect, flocculation time is
30~40min is best.
Further, step（3）In, the additive amount of the inorganic salts flocculant is 3.0~4.0g/L.Flocculant addition is got over
It is more, it can undoubtedly lose bigger, when additive amount reaches 4.0g/L, inflection point occurs in loss late, and loss late steeply rises, and effect of flocculating
Fruit is without being obviously improved after 4.0g/L, and when additive amount is lower than 3.0g/L, flocculating effect cannot reach flocculation purpose.
Further, step（3）In, the inorganic salts flocculant is chitosan, or the mixing for iron chloride and calcium oxide
Object；In the mixture of the iron chloride and calcium oxide, preferably the mass ratio of iron chloride and calcium oxide is 1:0.2~1.5（More preferably
1:0.25~1.20）.The mixture flocculating effect of the iron chloride and calcium oxide is good, and cheap, additive amount is few, meets industry
The needs of production, and can satisfy the requirement that shearing force is generated in flow process after iron chloride flocculation, under the mass ratio,
Flocculation solution shows slightly alkalinity, is conducive to flocculation.
Step（2）In, the preferred tripodia sedimentation centrifugation of centrifugation, revolving speed is 1000~1500 turns/min；Step（3）
In, the preferably sleeping spiral shell centrifugation of the centrifugation+disk centrifugal series connection, the spiral shell centrifugal rotational speed that crouches is 2500~3500 turns/min, disk centrifugal
Revolving speed is 5000~7000 turns/min.
Further, step（4）In, the adsorption flow rate of the decolorization adsorption is 2~3BV/h, and the pH value of decolorization adsorption is 7.5
~8.5, the ratio of height to diameter of large pore resin absorption column is 5~9:1.Upper prop efflux begins with sweet tea when adsorption flow rate is more than 3.5BV/h
Taste shows that flow velocity is leaked fastly, if but flow velocity to will cause very much adsorption efficiency slowly too low.During decolorization adsorption, use
High performance liquid chromatograph continuously detects STEVIA REBAUDIANA removal of impurities liquid and crosses the efflux after column, when detecting stevioside, changes column absorption.
Further, step（4）In, the eluent is the ethanol solution of volume fraction 60~65%, and the flow velocity of elution is
0.5~2BV/h, the dosage of eluent are 1~2BV.The temperature of the elution is room temperature.Inventor is the study found that volume fraction
60% ethanol solution can all elute the product being adsorbed on resin, and industrial is to reduce what ethanol solution was prepared
Difficulty, the ethanol solution of preferred volume score 60~65%.
Step（4）In, the macroporous absorbent resin is AB-8, DC-7, D101, D301 or KB-9.It is preferred that being purchased from Xi'an indigo plant
Know the D101 type macroporous absorbent resin of scientific and technological new material limited liability company and is purchased from the AB-8 type macroporous absorption tree of Shandong Shandong
Rouge.The macroporous absorbent resin preferably first carries out activation pre-treatment：The alcohol solution dipping for being first 95% by resin volume fraction
After fill column, with 4~8BV bed volume, the ethanol solution that volume fraction is 95% is cleaned with the flow velocity of 0.2~0.3BV/h, until
Until the ethyl alcohol of outflow adds water not muddy, to wash out the resins synthesis such as remaining styrene, divinyl benzene,toluene,xylene
Material, crosslinking agent, perforating agent etc., be washed with water ethyl alcohol in most column, then with 2~3BV, the dilute hydrochloric acid solution of mass fraction 5% with
The flow velocity of 0.2~0.3BV/h cleans, and to remove metal ion residual therein, is then washed with water to pH=6~7, then use 2 instead~
The diluted sodium hydroxide solution of 3BV, mass fraction 4% are cleaned with the flow velocity of 0.2~0.3BV/h, wherein remaining organic to remove
Object is finally washed with water to pH=7~8, spare.
Further, step（4）In, the molecular cut off of nanofiltration membrane used in the nanofiltration is 300~600Da, operating pressure
For 1.0~1.5Mpa；The concentration is vacuum concentration, and vacuum pressure is -0.08~-0.04Mpa, and thickening temperature is 50~80
DEG C, be concentrated into Baume degrees be 35~40 until.The preferred Tao Shi DOW NF90-400 desalination type nanofiltration membrane of nanofiltration membrane, nanofiltration
Effect be recycling ethyl alcohol, concentration, decoloration, except inorganic salts etc..
Step（5）In, it is described to collect each fraction respectively and refer to：Changed according to chromatography layer, determines that various steviosides produce
The product corresponding elution delivery time, to determine the period collected.
Further, step（5）In, the volume ratio of the mixed solvent of the ethyl alcohol and ethyl acetate is 1:1~10（It is preferred that 1:
6~9）.The ethyl alcohol and ethyl acetate are the food grade materials of volume fraction 95%.
Step（5）In, the drying is preferably dried in vacuo.
The method of the present invention has the following advantages that：
（1）According to the extracted a variety of high-purity stevia glycoside products of the method for the present invention are white or beige white powder
Shape is detected through high performance liquid chromatography, and the purity of gained stevioside may be up to 98.4%, and rebaudioside A purity may be up to 97.6%,
Impurity content is few, the ultimate yield > 90% of stevioside；
（2）The present invention greatly improves STEVIA REBAUDIANA using the method that enzyme immersion, heating extraction, flocculation purification combine
The maximum leaching content of glucosides, while the impurity the amount of dissolution in STEVIA REBAUDIANA is minimized, facilitate subsequent purification；
（3）Nanofiltration mainly sloughs a large amount of solvent, such as ethyl alcohol, water, inorganic salts in the method for the present invention, reaches certain
Purpose is concentrated, while being purified using nanofiltration mode, also reduces the energy consumption of subsequent vacuum concentration to a certain degree, reduces life
Produce cost；
（4）The method of the present invention simple process and low cost, strong operability are suitble to plant produced, and organic solvent is in final products
In residual quantity control in 100 ppm hereinafter, the organic solvent being harmful to the human body used in compared with the existing technology more ring
It ensures safety.
Specific embodiment
Below with reference to embodiment, the invention will be further described.
STEVIA REBAUDIANA cured leaf used in the embodiment of the present invention is commercially available；Used cellulase preparation model：Novi's letter
Cellucast 1.5 L FG（Activity：150000 U/g）, it is purchased from lucky treasured（Qingdao）Biotechnology Co., Ltd；Used AB-8
Type macroreticular resin is purchased from Shandong Shandong, and D101 type macroporous absorbent resin is purchased from Xi'an Sunresin New Materials Co., Ltd.
's；Used nanofiltration membrane is Tao Shi DOW NF90-400 desalination type nanofiltration membrane, is purchased from the limited public affairs of Guangzhou China film water treatment equipment
Department；Ethyl alcohol used in eluting and ethyl acetate are the food grade materials of volume fraction 95%；Chemistry examination used in other
Agent is obtained by routine business approach unless otherwise specified.
The activation pre-treatment of macroporous absorbent resin：After the alcohol solution dipping for being first 95% by 40mL resin volume fraction
Column is filled, is cleaned with the ethanol solution that 320mL volume fraction is 95% with the flow velocity of 0.25BV/h, until the ethyl alcohol of outflow adds water not
Until muddiness, ethyl alcohol in most column is washed with pure water, then clear with the flow velocity of 0.25BV/h with the dilute hydrochloric acid solution of 120mL mass fraction 5%
It washes, is washed till pH=7 with pure water, then cleaned with the flow velocity of 0.25BV/h with the diluted sodium hydroxide solution of 120mL mass fraction 4%, most
After be washed with water to pH=7, it is spare.
Embodiment 1
（1）Enzyme immersion：By 50g STEVIA REBAUDIANA cured leaf（Containing stevioside 4.27%, rebaudioside A 5.52%, dulcoside B
0.58%, total sweet tea glycosides is 10.37%）It after being crushed to 10~30 mesh, is soaked in 510mL water, 1g cellulase preparation is added and carries out
Enzymatic hydrolysis, obtains STEVIA REBAUDIANA enzymolysis liquid；
（2）Heating extraction：By step（1）Gained STEVIA REBAUDIANA enzymolysis liquid is at 45 DEG C, under pH value is 5.5, extracts 60min, cold
But to room temperature, tripodia sedimentation centrifugation is carried out under 1200r/min rate, filtering obtains the enzyme leaching liquor of 350mL STEVIA REBAUDIANA；
（3）Flocculation purification：By 350mL step（2）The enzyme leaching liquor of gained STEVIA REBAUDIANA is placed in 70 DEG C of water-baths and heats, and is added
1.05g inorganic salts flocculant（Wherein, iron chloride 0.70g, calcium oxide 0.35g）, stirring, under pH value 8.5, flocculation treatment
After 40min, flocculation solution is first subjected to sleeping spiral shell centrifugation under 3000r/min rate, then dish-style is carried out under 6000r/min rate
Centrifugation, filtering obtain 340mL STEVIA REBAUDIANA removal of impurities liquid；
（4）Macroporous absorbent resin decoloration purifying：By 340mL step（3）Gained STEVIA REBAUDIANA removal of impurities liquid is crossed equipped with 20mL AB-8
The resin column of type macroporous absorbent resin（Ratio of height to diameter is 6:1）, it is 2BV/h in adsorption flow rate, under pH value 8, carries out decolorization adsorption（It crosses
When column is completed, stevioside is not detected in efflux）, then use the ethanol solution of volume fraction 60% as eluent, to elute stream
The dosage of fast 0.5BV/h, eluent are 1BV, are eluted at room temperature, eluent nanofiltration membrane（Molecular cut off 400Da）
Nanofiltration is carried out, operating pressure 1.2Mpa is concentrated in vacuo to 38 Baumes then at vacuum pressure -0.05MPa, temperature 60 C
Until degree, STEVIA REBAUDIANA concentrate is obtained；
（5）Silica gel column chromatography post separation：By step（4）Silica gel column chromatography on gained STEVIA REBAUDIANA concentrate, then uses volume ratio
It is 12:88 ethyl alcohol and the mixed solvent of ethyl acetate are eluted, and collect 2 fractions respectively according to the variation of chromatography layer（It is washing
It is de- to carry out being collected first fraction when 40~53min, second fraction is collected when elution carries out 65~78min）,
It is concentrated in vacuo respectively again, is dried in vacuo, obtains 1.96g stevioside and 2.6g rebaudioside A.
Products obtained therefrom is white or beige white powder, detects through high performance liquid chromatography, wherein the purity of stevioside is
98.3%, the purity of rebaudioside A is 97.2%；The ultimate yield of STEVIA REBAUDIANA major product stevioside is 90.24%, rebaudioside A
Ultimate yield be 91.56%.
Embodiment 2
（1）Enzyme immersion：By 50g STEVIA REBAUDIANA cured leaf（Containing stevioside 4.27%, rebaudioside A 5.52%, dulcoside B
0.58%, total sweet tea glycosides is 10.37%）10~30 mesh are crushed to, are soaked in 500mL water, 1g cellulase preparation is added and carries out enzyme
Solution, obtains STEVIA REBAUDIANA enzymolysis liquid；
（2）Heating extraction：By step（1）Gained STEVIA REBAUDIANA enzymolysis liquid is at 43 DEG C, under pH value is 5.0, extracts 65min, cold
But to room temperature, tripodia sedimentation centrifugation is carried out under 1000r/min rate, filtering obtains the enzyme leaching liquor of 351mL STEVIA REBAUDIANA；
（3）Flocculation purification：By 351mL step（2）The enzyme leaching liquor of gained STEVIA REBAUDIANA is placed in 68 DEG C of water-baths and heats, and is added
1.2285g inorganic salts flocculant（Wherein, iron chloride 0.6825g, calcium oxide 0.546g）, stirring, under pH value 8.0, at flocculation
After managing 30min, flocculation solution is first subjected to sleeping spiral shell centrifugation under 2500r/min rate, then dish is carried out under 5000r/min rate
Formula centrifugation, filtering obtain 332mL STEVIA REBAUDIANA removal of impurities liquid；
（4）Macroporous absorbent resin decoloration purifying：By 332mL step（3）Gained STEVIA REBAUDIANA removal of impurities liquid is crossed equipped with 20mL D101
The resin column of type macroporous absorbent resin（Ratio of height to diameter is 5:1）, it is 2.5BV/h in adsorption flow rate, under pH value 7.5, carries out decoloration suction
It is attached（When crossing column completion, stevioside is not detected in efflux）, then use the ethanol solution of volume fraction 65% as eluent, with
The dosage of elution flow rate 0.7BV/h, eluent are 1.5BV, are eluted at room temperature, eluent nanofiltration membrane（Retain molecule
Measure 300Da）Nanofiltration is carried out, operating pressure 1.0Mpa is concentrated in vacuo then at vacuum pressure -0.04MPa, temperature 50 C
Until 37 Baume degrees, STEVIA REBAUDIANA concentrate is obtained；
（5）Silica gel column chromatography post separation：By step（4）Silica gel column chromatography on gained STEVIA REBAUDIANA concentrate, then uses volume ratio
It is 12:72 ethyl alcohol and the mixed solvent of ethyl acetate are eluted, and collect 2 fractions respectively according to the variation of chromatography layer（It is washing
It is de- to carry out being collected first fraction when 39~53min, second fraction is collected when elution carries out 63~75min）,
It is concentrated in vacuo respectively again, is dried in vacuo, obtains 1.97g stevioside and 2.56g rebaudioside A.
Products obtained therefrom is white or beige white powder, detects through high performance liquid chromatography, wherein the purity of stevioside is
97.9%, the purity of rebaudioside A is 97.4%；The ultimate yield of STEVIA REBAUDIANA major product stevioside is 90.33%, rebaudioside A
Ultimate yield be 90.34%.
Embodiment 3
（1）Enzyme immersion：By 100g STEVIA REBAUDIANA cured leaf（Containing stevioside 4.27%, rebaudioside A 5.52%, dulcoside B
0.58%, total sweet tea glycosides is 10.37%）10~30 mesh are crushed to, are soaked in 1050mL water, 2g cellulase preparation is added and carries out enzyme
Solution, obtains STEVIA REBAUDIANA enzymolysis liquid；
（2）Heating extraction：By step（1）Gained STEVIA REBAUDIANA enzymolysis liquid is at 46 DEG C, under pH value is 6.0, extracts 70min, cold
But to room temperature, tripodia sedimentation centrifugation is carried out under 1500r/min rate, filtering obtains the enzyme leaching liquor of 746mL STEVIA REBAUDIANA；
（3）Flocculation purification：By 746mL step（2）The enzyme leaching liquor of gained STEVIA REBAUDIANA is placed in 72 DEG C of water-baths and heats, and is added
2.984g inorganic salts flocculant（Wherein, iron chloride 1.492g, calcium oxide 1.492g）, stirring, under pH value 9.0, flocculation treatment
After 35min, flocculation solution is first subjected to sleeping spiral shell centrifugation under 3500r/min rate, then dish-style is carried out under 7000r/min rate
Centrifugation, filtering obtain 710mL STEVIA REBAUDIANA removal of impurities liquid；
（4）Macroporous absorbent resin decoloration purifying：By 710mL step（3）Gained STEVIA REBAUDIANA removal of impurities liquid is crossed equipped with 40mLAB-8
The resin column of type macroporous absorbent resin（Ratio of height to diameter is 7:1）, it is 3BV/h in adsorption flow rate, under pH value 8.5, carries out decolorization adsorption
（When crossing column completion, stevioside is not detected in efflux）, then use the ethanol solution of volume fraction 65% as eluent, to wash
The dosage of separation of flow speed 2.0BV/h, eluent are 2.0BV, are eluted at room temperature, eluent nanofiltration membrane（Molecular cut off
600Da）Nanofiltration is carried out, operating pressure 1.5Mpa is concentrated in vacuo to then at vacuum pressure -0.08MPa, 80 DEG C of temperature
Until 40 Baume degrees, STEVIA REBAUDIANA concentrate is obtained；
（5）Silica gel column chromatography post separation：By step（4）Silica gel column chromatography on gained STEVIA REBAUDIANA concentrate, then uses volume ratio
It is 12:108 ethyl alcohol and the mixed solvent of ethyl acetate are eluted, and collect 2 fractions respectively according to the variation of chromatography layer（It is washing
It is de- to carry out being collected first fraction when 41~54min, second fraction is collected when elution carries out 65~77min）,
It is concentrated in vacuo respectively again, is dried in vacuo, obtains 3.93g stevioside and 5.20g rebaudioside A.
Products obtained therefrom is white or beige white powder, detects through high performance liquid chromatography, wherein the purity of stevioside is
98.1%, the purity of rebaudioside A is 97.2%；The ultimate yield of STEVIA REBAUDIANA major product stevioside is 90.29%, rebaudioside A
Ultimate yield be 91.56%.
Embodiment 4
（1）Enzyme immersion：By 100g STEVIA REBAUDIANA cured leaf（Containing stevioside 4.27%, rebaudioside A 5.52%, dulcoside B
0.58%, total sweet tea glycosides is 10.37%）10~30 mesh are crushed to, are soaked in 1100mL water, 2g cellulase preparation is added and carries out enzyme
Solution, obtains STEVIA REBAUDIANA enzymolysis liquid；
（2）Heating extraction：By step（1）Gained STEVIA REBAUDIANA enzymolysis liquid is at 45 DEG C, under pH value is 5.5, extracts 60min, cold
But to room temperature, tripodia sedimentation centrifugation is carried out under 1200r/min rate, filtering obtains the enzyme leaching liquor of 746mL STEVIA REBAUDIANA；
（3）Flocculation purification：By 746mL step（2）The enzyme leaching liquor of gained STEVIA REBAUDIANA is placed in 70 DEG C of water-baths and heats, and is added
2.25g inorganic salts flocculant（Wherein, iron chloride 1.50g, calcium oxide 0.75g）, stirring, under pH value 8.5, flocculation treatment
After 40min, flocculation solution is first subjected to sleeping spiral shell centrifugation under 3000r/min rate, then dish-style is carried out under 6000r/min rate
Centrifugation, filtering obtain 722mL STEVIA REBAUDIANA removal of impurities liquid；
（4）Macroporous absorbent resin decoloration purifying：By 722mL step（3）Gained STEVIA REBAUDIANA removal of impurities liquid crosses 40mL equipped with AB-8
Type macroporous absorbent resin resin column（Ratio of height to diameter is 8:1）, it is 2BV/h in adsorption flow rate, under pH value 8, carries out decolorization adsorption（Cross column
When completion, stevioside is not detected in efflux）, then use the ethanol solution of volume fraction 60% as eluent, with elution flow rate
The dosage of 1.0BV/h, eluent are 2.0BV, are eluted at room temperature, eluent nanofiltration membrane（Molecular cut off 500Da）
Nanofiltration is carried out, operating pressure 1.4Mpa is concentrated in vacuo to 39 Baumes then at vacuum pressure -0.06MPa, temperature 70 C
Until degree, STEVIA REBAUDIANA concentrate is obtained；
（5）Silica gel column chromatography post separation：By step（4）Silica gel column chromatography on gained STEVIA REBAUDIANA concentrate, then uses volume ratio
It is 12:88 ethyl alcohol and the mixed solvent of ethyl acetate are eluted, and collect 2 fractions respectively according to the variation of chromatography layer（It is washing
It is de- to carry out being collected first fraction when 43~53min, second fraction is collected when elution carries out 63~74min）,
It is concentrated in vacuo respectively again, is dried in vacuo, obtains 3.91g stevioside and 5.16g rebaudioside A.
Products obtained therefrom is white or beige white powder, detects through high performance liquid chromatography, wherein the purity of stevioside is
98.4%, the purity of rebaudioside A is 97.6%；The ultimate yield of STEVIA REBAUDIANA major product stevioside is 90.10%, rebaudioside A
Ultimate yield be 91.23%.
Claims (23)
1. a kind of method for extracting high-purity stevioside from STEVIA REBAUDIANA, which is characterized in that include the following steps：
（1）Enzyme immersion：It is soaked in water after Stevia rebaudiana starting material is crushed, cellulase preparation is added and is digested, sweetleaf is obtained
Chrysanthemum enzymolysis liquid；
（2）Heating extraction：By step（1）The heating extraction of gained STEVIA REBAUDIANA enzymolysis liquid, is cooled to room temperature, is centrifuged, and filtering obtains sweetleaf
The enzyme leaching liquor of chrysanthemum；The temperature of the extraction is 40~50 DEG C, and the pH value of extraction is 5.0~6.0, the time of extraction is 60~
70min；
（3）Flocculation purification：By step（2）The enzyme leaching liquor of gained STEVIA REBAUDIANA carries out heating water bath, and inorganic salts flocculant is added, stirs
It mixes, after carrying out flocculation treatment, is centrifuged, filtering, obtain STEVIA REBAUDIANA removal of impurities liquid；
（4）Macroporous absorbent resin decoloration purifying：By step（3）Gained STEVIA REBAUDIANA removal of impurities liquid crosses large pore resin absorption column and decolourizes
Absorption, then use elution, eluent carry out nanofiltration, be concentrated, obtain STEVIA REBAUDIANA concentrate；
（5）Silica gel column chromatography post separation：By step（4）Then silica gel column chromatography on gained STEVIA REBAUDIANA concentrate is 1 with volume ratio:1
~10 ethyl alcohol and the mixed solvent of ethyl acetate are eluted, and collect each fraction respectively, are concentrated, dry, obtain a variety of high-purities
Stevia glycoside product.
2. the method for high-purity stevioside is extracted from STEVIA REBAUDIANA according to claim 1, it is characterised in that：Step
（1）In, the smashed partial size of Stevia rebaudiana starting material is 10~30 mesh, and the feed liquid mass ratio of the Stevia rebaudiana starting material and water is 1:
10~12, the addition quality of the cellulase preparation is 0.1~0.3w/v% of the water volume being added.
3. the method according to claim 1 or claim 2 for extracting high-purity stevioside from STEVIA REBAUDIANA, it is characterised in that：Step
Suddenly（3）In, the temperature of the heating water bath is 60~80 DEG C, and the pH value of the flocculation treatment is 8~9, the time of flocculation treatment
For 30~40min.
4. the method according to claim 1 or claim 2 for extracting high-purity stevioside from STEVIA REBAUDIANA, it is characterised in that：Step
Suddenly（3）In, the additive amount of the inorganic salts flocculant is 3.0~4.0g/L.
5. the method for high-purity stevioside is extracted from STEVIA REBAUDIANA according to claim 3, it is characterised in that：Step
（3）In, the additive amount of the inorganic salts flocculant is 3.0~4.0g/L.
6. the method according to claim 1 or claim 2 for extracting high-purity stevioside from STEVIA REBAUDIANA, it is characterised in that：Step
Suddenly（3）In, the inorganic salts flocculant is the mixture of iron chloride and calcium oxide；The mixture of the iron chloride and calcium oxide
In, the mass ratio of iron chloride and calcium oxide is 1:0.2~1.5.
7. the method for high-purity stevioside is extracted from STEVIA REBAUDIANA according to claim 3, it is characterised in that：Step
（3）In, the inorganic salts flocculant is the mixture of iron chloride and calcium oxide；In the mixture of the iron chloride and calcium oxide,
The mass ratio of iron chloride and calcium oxide is 1:0.2~1.5.
8. the method for high-purity stevioside is extracted from STEVIA REBAUDIANA according to claim 4, it is characterised in that：Step
（3）In, the inorganic salts flocculant is the mixture of iron chloride and calcium oxide；In the mixture of the iron chloride and calcium oxide,
The mass ratio of iron chloride and calcium oxide is 1:0.2~1.5.
9. the method according to claim 1 or claim 2 for extracting high-purity stevioside from STEVIA REBAUDIANA, it is characterised in that：Step
Suddenly（4）In, the adsorption flow rate of the decolorization adsorption is 2~3BV/h, and the pH value of decolorization adsorption is 7.5~8.5, macroporous absorption tree
The ratio of height to diameter of rouge column is 5~9:1.
10. the method for high-purity stevioside is extracted from STEVIA REBAUDIANA according to claim 3, it is characterised in that：Step
（4）In, the adsorption flow rate of the decolorization adsorption is 2~3BV/h, and the pH value of decolorization adsorption is 7.5~8.5, macroporous absorbent resin
The ratio of height to diameter of column is 5~9:1.
11. the method for high-purity stevioside is extracted from STEVIA REBAUDIANA according to claim 4, it is characterised in that：Step
（4）In, the adsorption flow rate of the decolorization adsorption is 2~3BV/h, and the pH value of decolorization adsorption is 7.5~8.5, macroporous absorbent resin
The ratio of height to diameter of column is 5~9:1.
12. the method for high-purity stevioside is extracted from STEVIA REBAUDIANA according to claim 6, it is characterised in that：Step
（4）In, the adsorption flow rate of the decolorization adsorption is 2~3BV/h, and the pH value of decolorization adsorption is 7.5~8.5, macroporous absorbent resin
The ratio of height to diameter of column is 5~9:1.
13. the method according to claim 1 or claim 2 for extracting high-purity stevioside from STEVIA REBAUDIANA, it is characterised in that：Step
Suddenly（4）In, the eluent is the ethanol solution of volume fraction 60~65%, and the flow velocity of elution is 0.5~2BV/h, eluent
Dosage is 1~2BV.
14. the method for high-purity stevioside is extracted from STEVIA REBAUDIANA according to claim 3, it is characterised in that：Step
（4）In, the eluent is the ethanol solution of volume fraction 60~65%, and the flow velocity of elution is 0.5~2BV/h, the use of eluent
Amount is 1~2BV.
15. the method for high-purity stevioside is extracted from STEVIA REBAUDIANA according to claim 4, it is characterised in that：Step
（4）In, the eluent is the ethanol solution of volume fraction 60~65%, and the flow velocity of elution is 0.5~2BV/h, the use of eluent
Amount is 1~2BV.
16. the method for high-purity stevioside is extracted from STEVIA REBAUDIANA according to claim 6, it is characterised in that：Step
（4）In, the eluent is the ethanol solution of volume fraction 60~65%, and the flow velocity of elution is 0.5~2BV/h, the use of eluent
Amount is 1~2BV.
17. the method for high-purity stevioside is extracted from STEVIA REBAUDIANA according to claim 9, it is characterised in that：Step
（4）In, the eluent is the ethanol solution of volume fraction 60~65%, and the flow velocity of elution is 0.5~2BV/h, the use of eluent
Amount is 1~2BV.
18. the method according to claim 1 or claim 2 for extracting high-purity stevioside from STEVIA REBAUDIANA, it is characterised in that：Step
Suddenly（4）In, the molecular cut off of nanofiltration membrane used in the nanofiltration is 300~600Da, and operating pressure is 1.0~1.5Mpa；It is described
Concentration is vacuum concentration, and vacuum pressure is -0.08~-0.04Mpa, and thickening temperature is 50~80 DEG C, and being concentrated into Baume degrees is 35
Until~40.
19. the method for high-purity stevioside is extracted from STEVIA REBAUDIANA according to claim 3, it is characterised in that：Step
（4）In, the molecular cut off of nanofiltration membrane used in the nanofiltration is 300~600Da, and operating pressure is 1.0~1.5Mpa；It is described dense
Be condensed to be concentrated in vacuo, vacuum pressure be -0.08~-0.04Mpa, thickening temperature be 50~80 DEG C, be concentrated into Baume degrees be 35~
Until 40.
20. the method for high-purity stevioside is extracted from STEVIA REBAUDIANA according to claim 4, it is characterised in that：Step
（4）In, the molecular cut off of nanofiltration membrane used in the nanofiltration is 300~600Da, and operating pressure is 1.0~1.5Mpa；It is described dense
Be condensed to be concentrated in vacuo, vacuum pressure be -0.08~-0.04Mpa, thickening temperature be 50~80 DEG C, be concentrated into Baume degrees be 35~
Until 40.
21. the method for high-purity stevioside is extracted from STEVIA REBAUDIANA according to claim 6, it is characterised in that：Step
（4）In, the molecular cut off of nanofiltration membrane used in the nanofiltration is 300~600Da, and operating pressure is 1.0~1.5Mpa；It is described dense
Be condensed to be concentrated in vacuo, vacuum pressure be -0.08~-0.04Mpa, thickening temperature be 50~80 DEG C, be concentrated into Baume degrees be 35~
Until 40.
22. the method for high-purity stevioside is extracted from STEVIA REBAUDIANA according to claim 9, it is characterised in that：Step
（4）In, the molecular cut off of nanofiltration membrane used in the nanofiltration is 300~600Da, and operating pressure is 1.0~1.5Mpa；It is described dense
Be condensed to be concentrated in vacuo, vacuum pressure be -0.08~-0.04Mpa, thickening temperature be 50~80 DEG C, be concentrated into Baume degrees be 35~
Until 40.
23. 3 method for extracting high-purity stevioside from STEVIA REBAUDIANA according to claim 1, it is characterised in that：Step
（4）In, the molecular cut off of nanofiltration membrane used in the nanofiltration is 300~600Da, and operating pressure is 1.0~1.5Mpa；It is described dense
Be condensed to be concentrated in vacuo, vacuum pressure be -0.08~-0.04Mpa, thickening temperature be 50~80 DEG C, be concentrated into Baume degrees be 35~
Until 40.
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| CN106674307A (en) * | 2016-11-03 | 2017-05-17 | 明光市大全甜叶菊专业合作社 | Method for extracting stevioside from stevia rebaudiana |
| CN106632539A (en) * | 2016-11-03 | 2017-05-10 | 明光市大全甜叶菊专业合作社 | A stevia rebaudiana glucoside extracting method |
| WO2018122579A1 (en) * | 2016-12-29 | 2018-07-05 | Villalba, Leandro | Industrial plant having an electroflocculating system, electroflocculating system and method for producing steviol glycosides for creating a noncaloric food additive |
| CN107371672A (en) * | 2017-06-14 | 2017-11-24 | 湖南祥民制药有限公司 | A kind of method for improving STEVIA REBAUDIANA REB D yield |
| CN107383128B (en) * | 2017-08-01 | 2019-06-18 | 江南大学 | A kind of discoloration method of steviol glycoside aqueous extract |
| CN107936071A (en) * | 2017-12-12 | 2018-04-20 | 蚌埠市华东生物科技有限公司 | A kind of method for extraction and purification of steviol glycoside |
| CN108047286A (en) * | 2017-12-12 | 2018-05-18 | 蚌埠市华东生物科技有限公司 | A kind of method that steviol glycoside is extracted from STEVIA REBAUDIANA |
| CN108676042B (en) * | 2018-07-09 | 2023-09-05 | 济宁市瑞信堂大禹药业有限公司 | Device for extracting stevioside from stevia rebaudiana and extraction method thereof |
| CN109757728A (en) * | 2019-03-15 | 2019-05-17 | 奎屯甜菊农业科技有限公司 | A kind of storage of STEVIA REBAUDIANA wet process and STEVIA REBAUDIANA extracting method |
| CN112047986B (en) * | 2019-06-06 | 2023-11-17 | 南京宸翔医药研究有限责任公司 | Preparation method of stevioside |
| CN110684813A (en) * | 2019-09-18 | 2020-01-14 | 江苏施宇甜生物科技有限公司 | Method for producing steviol glycosides in recombinant hosts |
| CN112940057B (en) * | 2021-02-04 | 2021-10-29 | 北京澳特舒尔保健品开发有限公司 | Preparation and purification method of stevioside and application of stevioside in anti-fatigue product |
| CN112851739B (en) * | 2021-03-30 | 2022-02-08 | 湖南华诚生物资源股份有限公司 | Method for recovering sweet glycosides from fructus Siraitiae Grosvenorii, folium Hydrangeae Strigosae or stevia rebaudiana flocculation residues |
| CN115669911A (en) * | 2022-09-22 | 2023-02-03 | 湖南绿蔓生物科技股份有限公司 | Composition containing stevia rebaudiana and application |
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