CN105294790A - Method for extracting high-purity steviol glycosides from stevia rebaudiana - Google Patents

Method for extracting high-purity steviol glycosides from stevia rebaudiana Download PDF

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CN105294790A
CN105294790A CN201510882914.XA CN201510882914A CN105294790A CN 105294790 A CN105294790 A CN 105294790A CN 201510882914 A CN201510882914 A CN 201510882914A CN 105294790 A CN105294790 A CN 105294790A
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sweet stevia
stevia
stevioside
stevia rebaudiana
high purity
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CN105294790B (en
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龙伟岸
黄华学
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Hunan Huacheng Biotech Inc
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Hunan Huacheng Biotech Inc
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Abstract

The invention relates to a method for extracting high-purity steviol glycosides from stevia rebaudiana. The method comprises the following steps: (1) soaking a ground stevia rebaudiana raw material into water, and adding a cellulase preparation for enzymolysis; (2) carrying out heating extraction and centrifugation, and filtering, so as to obtain stevia rebaudiana enzyme extraction liquid; (3) carrying out water-bath heating on the stevia rebaudiana enzyme extraction liquid, adding an inorganic salt flocculating agent, stirring, flocculating, carrying out centrifugation, and filtering, so as to obtain stevia rebaudiana edulcorated liquid; (4) carrying out decolorization adsorption on the stevia rebaudiana edulcorated liquid by virtue of a macroporous adsorption resin column, carrying out elution through eluent, carrying out nanofiltration, and concentrating, so as to obtain a stevia rebaudiana concentrated liquid; and (5) carrying out silica-gel column chromatography and elution on the stevia rebaudiana concentrated liquid, collecting each fraction, concentrating, and drying, so as to obtain multiple high-purity steviol glycosides products. The purity of the products obtained by virtue of the method can reach up to 98.4%, and the final yield is more than 90%; the residual amounts of ethanol and ethyl ester are low, so that the method is environmentally friendly and safe; the method is simple in process, high in operability, low in energy consumption and cost and applicable to industrial production.

Description

A kind of method extracting high purity stevioside from sweet Stevia
Technical field
The present invention relates to a kind of method extracting high purity stevioside, be specifically related to a kind ofly be applicable to the industrial method extracting high purity stevioside from sweet Stevia.
Background technology
Sweet Stevia originates in Amambay and the Mbaxacayu mountain range of Paraguay, belongs to sugar plant, and whole vegetative period, temperature was all more than 20 DEG C, had thermophily.Within 1970, Japan introduces sweet Stevia from Brazil, starts domestication, cultivation, glycosides processed, carries out the test such as toxicity, food test simultaneously, and first develop sweet Stevia product---stevioside.China introduced a fine variety in 1976 by from Japan, planted experimentally, and succeeded, and the beginning of the eighties is to popularizing planting in all parts of the country.
Stevioside is the tetracylic diterpene derivatives that a class is mainly present in sweet Stevia blade, mainly comprise stevioside (Stevioside), rebaudioside A (RebaudiosideA), RB (RebaudiosdeB), 8 kinds of glucosides such as dulcoside B (RebaudiosideC), its sugariness is approximately 300 times of sucrose.There are some researches show, stevioside has high sugariness, low in calories, without the feature of obvious toxic-side effects.It can suppress hyperglycemia, hypertension have anti-inflammatory, antitumor, antidiarrheal diuresis and assist immunoregulatory effect.At present, South America, Japan and China are widely used in stevioside in food and drink as a kind of natural sweeting agent.
The extracting method of existing stevioside mainly contains cooking process, cold water soak method, fermentation method, electrolytic process, ultrasonic extraction etc.Cooking process, cold water soak method, ultrasonic extraction are industrial a kind of most widely used methods, but it is while extraction stevioside, is also extracted out by a large amount of impurity; And biological fermentation process, electrolytic process can introduce tramp material in extracting solution, bring difficulty to the purifies and separates of subsequent handling.The purification process of existing stevioside mainly contains preparative chromatography, organic solvent extraction recrystallization method etc.Preparing chromatograph in industry has high input, and operation easier is large; And there is the defects such as solvent usage quantity is large, product yield is low in organic solvent extraction recrystallization method.
CN101062077B discloses a kind of method simultaneously preparing the total stevioside of sweet Stevia and sweet Stevia total flavones, and be by sweet Stevia cured leaf organic solvent extraction, rear supercritical fluid extraction, column chromatography obtain high purity stevioside product.But because its extraction have employed organic solvent method, cost is high, and safety coefficient is low, later-period purification is separated and adopts the defects such as overcritical, column chromatography existence investment is large, operation easier is large.
CN101805768B discloses a kind of biological enzymolysis and purification method of high-quality stevioside, is to adopt enzyme to carry, and macroporous resin purification, solvent extraction, recrystallization obtains high purity product, and uses dehydrated alcohol in a large number in crystallisation process.Its defect is not suitable for industrial scale to produce, and yield is low, cost is high.
CN102199177A discloses a kind of production method of pure natural high-purity stevioside, be that after adopting enzymolysis, ultrasonic continuous countercurrent extraction technology is extracted, then clarify material with membrane filtration, then adsorb by Multi-functional analog moving-bed separation system, ethanol elution, obtains product.But the purity of products obtained therefrom is the highest can only reach 96%, and finished product is single, and product recovery rate is low, is not suitable for suitability for industrialized production.
Summary of the invention
Technical problem to be solved by this invention is, overcomes the above-mentioned defect that prior art exists, provides a kind of technique simple, energy consumption is low, environment friendly and pollution-free, suitability for industrialized is produced, the method extracting high purity stevioside from sweet Stevia that product yield is high, purity is high.
The technical solution adopted for the present invention to solve the technical problems is as follows: a kind of method extracting high purity stevioside from sweet Stevia, comprises the following steps:
(1) enzyme-added immersion: be soaked in water after Stevia rebaudiana starting material is pulverized, add cellulase preparation and carry out enzymolysis, obtain sweet Stevia enzymolysis solution;
(2) lixiviate is heated: by step (1) gained sweet Stevia enzymolysis solution heating lixiviate, be cooled to room temperature, centrifugal, filter, obtain the enzyme-added vat liquor of sweet Stevia;
(3) flocculation purification: the enzyme-added vat liquor of step (2) gained sweet Stevia is carried out heating in water bath, adds inorganic salt flocculation agent, stirs, after carrying out flocculation treatment, centrifugal, filters, obtains sweet Stevia removal of impurities liquid;
(4) macroporous adsorbent resin decolouring purifying: step (3) gained sweet Stevia removal of impurities liquid is crossed macroporous adsorptive resins and carries out decolorization adsorption, then use elution, elutriant carries out nanofiltration, concentrated, obtains sweet Stevia concentrated solution;
(5) silica gel column chromatography is separated: by silica gel column chromatography on step (4) gained sweet Stevia concentrated solution, then carry out wash-out with the mixed solvent of ethanol and ethyl acetate, collect each cut respectively, concentrated, dry, obtains multiple highly purified stevioside glycoside product.
In step (1), in described Stevia rebaudiana starting material, the mass content of stevioside is: stevioside 4 ~ 5%, rebaudioside A 5 ~ 6%, dulcoside B 0.5 ~ 1.0%, and total glucosides is 10 ~ 12%.
Further, in step (1), particle diameter after described Stevia rebaudiana starting material pulverizing is 10 ~ 30 orders, the feed liquid mass ratio of described Stevia rebaudiana starting material and water is 1:10 ~ 12, interpolation quality (g) of the described cellulase preparation 0.1 ~ 0.3w/v%(preferably 0.15 ~ 0.20w/v% of volume of water (mL) for adding).If soak water consumption lower than 10 times, then glucosides yield can sharply decline, and water consumption higher than glucosides yield after 12 times almost without increasing, then it is meaningless to continue increase water consumption; Adding of described cellulase preparation, be conducive to the fragmentation of Stevia rebaudiana starting material cell walls, thus be beneficial to the stripping of all kinds of glucosides, if but addition is less than 0.1w/v% that hydrolysis result can be caused sharply to decline, and addition also promotes without positive effect higher than 0.3w/v%, continue to increase enzyme dosage and only understand loss zymin.
Further, in step (2), the temperature of described lixiviate is 40 ~ 50 DEG C (preferably 43 ~ 46 DEG C), and the pH value of lixiviate is 5.0 ~ 6.0, and the time of lixiviate is 60 ~ 70min.Need the activity and the extracting efficiency that ensure enzyme reagent due to during heating lixiviate simultaneously, contriver studies discovery, under described Extracting temperature, pH value, extracting effect is best, and upon extracting between be greater than 60min after, extracting efficiency tends to be steady state, yield increases hardly, continues to extend extraction time meaningless.
Further, in step (3), the temperature of described heating in water bath is 60 ~ 80 DEG C (preferably 68 ~ 72 DEG C), and the pH value of described flocculation treatment is 8 ~ 9, and the time of flocculation treatment is 30 ~ 40min.Contriver studies discovery, when water temperature 50 ~ 80 DEG C, pH value be 8 ~ 10 time, flocculating effect substantially in the same horizontal line, but flocculation rate of loss have larger difference, rate of loss of flocculating at described temperature and pH is minimum; Flocculation time is longer, and rate of loss is larger, and for ensureing the balance of yield, flocculating effect, flocculation time is that 30 ~ 40min is best.
Further, in step (3), the addition of described inorganic salt flocculation agent is 3.0 ~ 4.0g/L.Flocculation agent adds more, and can lose larger undoubtedly, when addition reaches 4.0g/L, flex point appears in rate of loss, and rate of loss sharply rises, and flocculating effect promotes without obvious after 4.0g/L, when addition can not reach flocculation object lower than flocculating effect during 3.0g/L.
Further, in step (3), described inorganic salt flocculation agent is chitosan, or is the mixture of iron(ic) chloride and calcium oxide; In the mixture of described iron(ic) chloride and calcium oxide, the mass ratio of preferred iron(ic) chloride and calcium oxide is 1:0.2 ~ 1.5(more preferably 1:0.25 ~ 1.20).The mixture flocculating effect of described iron(ic) chloride and calcium oxide is good, cheap, and addition is few, meet industrial needs, and the requirement producing shearing force in flow process can be met, under described mass ratio after iron(ic) chloride flocculation, flocculation solution shows slightly alkalescence, is beneficial to flocculation.
In step (2), described centrifugal preferred tripodia sedimentation is centrifugal, and its rotating speed is 1000 ~ 1500 turns/min; In step (3), described centrifugal preferably sleeping spiral shell is centrifugal+disk centrifugal series connection, and sleeping spiral shell centrifugal rotational speed is 2500 ~ 3500 turns/min, and disk centrifugal rotating speed is 5000 ~ 7000 turns/min.
Further, in step (4), the adsorption flow rate of described decolorization adsorption is 2 ~ 3BV/h, and the pH value of decolorization adsorption is 7.5 ~ 8.5, and the aspect ratio of macroporous adsorptive resins is 5 ~ 9:1.Adsorption flow rate starts pleasantly sweet more than upper prop effluent liquid during 3.5BV/h, shows that flow velocity there occurs leakage soon, if but flow velocity can cause adsorption efficiency too low too slowly.In decolorization adsorption process, the effluent liquid after adopting high performance liquid chromatograph continuous detecting sweet Stevia removal of impurities liquid to cross post, when detecting stevioside, changes post absorption.
Further, in step (4), described elutriant is the ethanolic soln of volume fraction 60 ~ 65%, and the flow velocity of wash-out is 0.5 ~ 2BV/h, and the consumption of elutriant is 1 ~ 2BV.The temperature of described wash-out is room temperature.Contriver studies discovery, and the product be adsorbed on resin can all elute by the ethanolic soln of volume fraction 60%, the industrial difficulty for reducing ethanolic soln preparation, the ethanolic soln of preferred volume mark 60 ~ 65%.
In step (4), described macroporous adsorbent resin is AB-8, DC-7, D101, D301 or KB-9.Preferably be purchased from the D101 type macroporous adsorbent resin of Xi'an Sunresin New Materials Co., Ltd. and be purchased from the anti-AB-8 type macroporous adsorbent resin in Shandong, Shandong.Described macroporous adsorbent resin preferably first carries out activating pre-treatment: be first fill post after the alcohol solution dipping of 95% by resin volume fraction, with 4 ~ 8BV column volume, volume fraction be 95% ethanolic soln clean with the flow velocity of 0.2 ~ 0.3BV/h, until flow out ethanol add water not muddy till, to clean out residual vinylbenzene, divinylbenzene, toluene, the material of the resins synthesis such as dimethylbenzene, linking agent, perforating agent etc., wash ethanol in most post with water, use 2 ~ 3BV again, the dilute hydrochloric acid solution of massfraction 5% cleans with the flow velocity of 0.2 ~ 0.3BV/h, remain with the metal ion removed wherein, then wash with water to pH=6 ~ 7, use 2 ~ 3BV instead again, the diluted sodium hydroxide solution of massfraction 4% cleans with the flow velocity of 0.2 ~ 0.3BV/h, to remove wherein residual organism, finally wash with water to pH=7 ~ 8, for subsequent use.
Further, in step (4), the molecular weight cut-off of described nanofiltration nanofiltration membrane used is 300 ~ 600Da, and working pressure is 1.0 ~ 1.5Mpa; Described simmer down to vacuum concentration, vacuum pressure is-0.08 ~-0.04Mpa, and thickening temperature is 50 ~ 80 DEG C, and being concentrated into degree Beaume is till 35 ~ 40.Described nanofiltration membrane preferred Tao Shi DOWNF90-400 desalination type nanofiltration membrane, the effect of nanofiltration is recovery ethanol, concentrates, decolours, except inorganic salt etc.
In step (5), describedly collect each cut respectively and refer to: according to the change of chromatogram layer, determine the wash-out elution time that various stevioside glycoside product is corresponding, to determine the time period of collecting.
Further, in step (5), the volume ratio of the mixed solvent of described ethanol and ethyl acetate is preferred 1:6 ~ 9 of 1:1 ~ 10().Described ethanol and ethyl acetate are the food grade materials of volume fraction 95%.
In step (5), the preferred vacuum-drying of described drying.
The inventive method tool has the following advantages:
(1) the multiple high purity stevioside glycoside product extracted according to the inventive method is all in white or beige white powder shape, detect through high performance liquid chromatography, the purity of gained stevioside can up to 98.4%, rebaudioside A purity can up to 97.6%, foreign matter content is few, the ultimate yield > 90% of stevioside;
(2) method that the present invention adopts enzyme-added immersion, heating lixiviate, flocculation purification to combine improves the maximum leaching content of stevioside greatly, makes the impurity stripping quantity in sweet Stevia minimize simultaneously, facilitates subsequent purification;
(3) in the inventive method, a large amount of solvents is mainly sloughed in nanofiltration, as ethanol, water, inorganic salt etc., reaches certain concentrated object, while employing nanofiltration mode carries out purifying, also to a certain degree reduces the energy consumption of follow-up vacuum concentration, reduces production cost;
(4) the inventive method technique is simple, and cost is low, strong operability, and be applicable to plant produced, organic solvent residual quantity in the final product controls at below 100ppm, relative to the harmful organic solvent Environmental Safety more used in prior art.
Embodiment
Below in conjunction with embodiment, the invention will be further described.
The sweet Stevia cured leaf that the embodiment of the present invention uses is commercially available; The cellulase preparation model used: letter Cellucast1.5LFG(is active in Novi: 150,000 U/g), is purchased from lucky precious (Qingdao) bio tech ltd; The AB-8 type macroporous resin used is purchased from Shandong, Shandong and resists, and D101 type macroporous adsorbent resin is purchased from Xi'an Sunresin New Materials Co., Ltd.; The nanofiltration membrane used is Tao Shi DOWNF90-400 desalination type nanofiltration membrane, is purchased from Guangzhou film water treatment equipment company limited of China; The ethanol that wash-out uses and ethyl acetate are the food grade materials of volume fraction 95%; Other chemical reagent used, if no special instructions, is all obtained by routine business approach.
The activation pre-treatment of macroporous adsorbent resin: be first fill post after the alcohol solution dipping of 95% by 40mL resin volume fraction, by 320mL volume fraction be 95% ethanolic soln clean with the flow velocity of 0.25BV/h, until flow out ethanol add water not muddy till, ethanol in most post is washed with pure water, clean with the flow velocity of 0.25BV/h with the dilute hydrochloric acid solution of 120mL massfraction 5% again, pH=7 is washed till with pure water, clean with the flow velocity of 0.25BV/h with the diluted sodium hydroxide solution of 120mL massfraction 4% again, finally wash with water to pH=7, for subsequent use.
embodiment 1
(1) enzyme-added immersion: by 50g sweet Stevia cured leaf (containing stevioside 4.27%, rebaudioside A 5.52%, dulcoside B 0.58%, total sweet glycosides is 10.37%) be crushed to 10 ~ 30 orders after, be soaked in 510mL water, add 1g cellulase preparation and carry out enzymolysis, obtain sweet Stevia enzymolysis solution;
(2) heat lixiviate: by step (1) gained sweet Stevia enzymolysis solution at 45 DEG C, pH value is 5.5 times, and lixiviate 60min, is cooled to room temperature, carries out tripodia sedimentation centrifugal under 1200r/min speed, filter, obtain the enzyme-added vat liquor of 350mL sweet Stevia;
(3) flocculation purification: the enzyme-added vat liquor of 350mL step (2) gained sweet Stevia is placed in 70 DEG C of water-baths and heats, add 1.05g inorganic salt flocculation agent (wherein, iron(ic) chloride 0.70g, calcium oxide 0.35g), stir, pH value 8.5 times, after flocculation treatment 40min, it is centrifugal first flocculation solution to be carried out under 3000r/min speed sleeping spiral shell, then carries out disk centrifugal under 6000r/min speed, filter, obtain 340mL sweet Stevia removal of impurities liquid;
(4) macroporous adsorbent resin decolouring purifying: 340mL step (3) gained sweet Stevia removal of impurities liquid is crossed the resin column (aspect ratio is 6:1) that 20mLAB-8 type macroporous adsorbent resin is housed, be 2BV/h at adsorption flow rate, pH value 8 times, carry out decolorization adsorption (when post completes excessively, effluent liquid does not detect stevioside), use the ethanolic soln of volume fraction 60% as elutriant again, with elution flow rate 0.5BV/h, the consumption of elutriant is 1BV, at room temperature carry out wash-out, elutriant nanofiltration membrane (molecular weight cut-off 400Da) carries out nanofiltration, working pressure is 1.2Mpa, then at vacuum pressure-0.05MPa, under temperature 60 C, till vacuum concentration to 38 degree Beaume, obtain sweet Stevia concentrated solution,
(5) silica gel column chromatography is separated: by silica gel column chromatography on step (4) gained sweet Stevia concentrated solution, then be that the ethanol of 12:88 and the mixed solvent of ethyl acetate carry out wash-out by volume ratio, collect 2 cuts respectively according to the change of chromatogram layer and (carry out collection first cut when wash-out carries out 40 ~ 53min, collection second cut is carried out) when wash-out carries out 65 ~ 78min, carry out vacuum concentration respectively again, vacuum-drying, obtains 1.96g stevioside and 2.6g rebaudioside A.
Products obtained therefrom is all in white or beige white powder, and detect through high performance liquid chromatography, wherein, the purity of stevioside is 98.3%, and the purity of rebaudioside A is 97.2%; The ultimate yield of sweet Stevia main products stevioside is 90.24%, and the ultimate yield of rebaudioside A is 91.56%.
embodiment 2
(1) enzyme-added immersion: by 50g sweet Stevia cured leaf (containing stevioside 4.27%, rebaudioside A 5.52%, dulcoside B 0.58%, total sweet glycosides is 10.37%) be crushed to 10 ~ 30 orders, be soaked in 500mL water, add 1g cellulase preparation and carry out enzymolysis, obtain sweet Stevia enzymolysis solution;
(2) heat lixiviate: by step (1) gained sweet Stevia enzymolysis solution at 43 DEG C, pH value is 5.0 times, and lixiviate 65min, is cooled to room temperature, carries out tripodia sedimentation centrifugal under 1000r/min speed, filter, obtain the enzyme-added vat liquor of 351mL sweet Stevia;
(3) flocculation purification: the enzyme-added vat liquor of 351mL step (2) gained sweet Stevia is placed in 68 DEG C of water-baths and heats, add 1.2285g inorganic salt flocculation agent (wherein, iron(ic) chloride 0.6825g, calcium oxide 0.546g), stir, pH value 8.0 times, after flocculation treatment 30min, it is centrifugal first flocculation solution to be carried out under 2500r/min speed sleeping spiral shell, then carries out disk centrifugal under 5000r/min speed, filter, obtain 332mL sweet Stevia removal of impurities liquid;
(4) macroporous adsorbent resin decolouring purifying: 332mL step (3) gained sweet Stevia removal of impurities liquid is crossed the resin column (aspect ratio is 5:1) that 20mLD101 type macroporous adsorbent resin is housed, be 2.5BV/h at adsorption flow rate, pH value 7.5 times, carry out decolorization adsorption (when post completes excessively, effluent liquid does not detect stevioside), use the ethanolic soln of volume fraction 65% as elutriant again, with elution flow rate 0.7BV/h, the consumption of elutriant is 1.5BV, at room temperature carry out wash-out, elutriant nanofiltration membrane (molecular weight cut-off 300Da) carries out nanofiltration, working pressure is 1.0Mpa, then at vacuum pressure-0.04MPa, under temperature 50 C, till vacuum concentration to 37 degree Beaume, obtain sweet Stevia concentrated solution,
(5) silica gel column chromatography is separated: by silica gel column chromatography on step (4) gained sweet Stevia concentrated solution, then be that the ethanol of 12:72 and the mixed solvent of ethyl acetate carry out wash-out by volume ratio, collect 2 cuts respectively according to the change of chromatogram layer and (carry out collection first cut when wash-out carries out 39 ~ 53min, collection second cut is carried out) when wash-out carries out 63 ~ 75min, carry out vacuum concentration respectively again, vacuum-drying, obtains 1.97g stevioside and 2.56g rebaudioside A.
Products obtained therefrom is all in white or beige white powder, and detect through high performance liquid chromatography, wherein, the purity of stevioside is 97.9%, and the purity of rebaudioside A is 97.4%; The ultimate yield of sweet Stevia main products stevioside is 90.33%, and the ultimate yield of rebaudioside A is 90.34%.
embodiment 3
(1) enzyme-added immersion: by 100g sweet Stevia cured leaf (containing stevioside 4.27%, rebaudioside A 5.52%, dulcoside B 0.58%, total sweet glycosides is 10.37%) be crushed to 10 ~ 30 orders, be soaked in 1050mL water, add 2g cellulase preparation and carry out enzymolysis, obtain sweet Stevia enzymolysis solution;
(2) heat lixiviate: by step (1) gained sweet Stevia enzymolysis solution at 46 DEG C, pH value is 6.0 times, and lixiviate 70min, is cooled to room temperature, carries out tripodia sedimentation centrifugal under 1500r/min speed, filter, obtain the enzyme-added vat liquor of 746mL sweet Stevia;
(3) flocculation purification: the enzyme-added vat liquor of 746mL step (2) gained sweet Stevia is placed in 72 DEG C of water-baths and heats, add 2.984g inorganic salt flocculation agent (wherein, iron(ic) chloride 1.492g, calcium oxide 1.492g), stir, pH value 9.0 times, after flocculation treatment 35min, it is centrifugal first flocculation solution to be carried out under 3500r/min speed sleeping spiral shell, then carries out disk centrifugal under 7000r/min speed, filter, obtain 710mL sweet Stevia removal of impurities liquid;
(4) macroporous adsorbent resin decolouring purifying: 710mL step (3) gained sweet Stevia removal of impurities liquid is crossed the resin column (aspect ratio is 7:1) that 40mLAB-8 type macroporous adsorbent resin is housed, be 3BV/h at adsorption flow rate, pH value 8.5 times, carry out decolorization adsorption (when post completes excessively, effluent liquid does not detect stevioside), use the ethanolic soln of volume fraction 65% as elutriant again, with elution flow rate 2.0BV/h, the consumption of elutriant is 2.0BV, at room temperature carry out wash-out, elutriant nanofiltration membrane (molecular weight cut-off 600Da) carries out nanofiltration, working pressure is 1.5Mpa, then at vacuum pressure-0.08MPa, at temperature 80 DEG C, till vacuum concentration to 40 degree Beaume, obtain sweet Stevia concentrated solution,
(5) silica gel column chromatography is separated: by silica gel column chromatography on step (4) gained sweet Stevia concentrated solution, then be that the ethanol of 12:108 and the mixed solvent of ethyl acetate carry out wash-out by volume ratio, collect 2 cuts respectively according to the change of chromatogram layer and (carry out collection first cut when wash-out carries out 41 ~ 54min, collection second cut is carried out) when wash-out carries out 65 ~ 77min, carry out vacuum concentration respectively again, vacuum-drying, obtains 3.93g stevioside and 5.20g rebaudioside A.
Products obtained therefrom is all in white or beige white powder, and detect through high performance liquid chromatography, wherein, the purity of stevioside is 98.1%, and the purity of rebaudioside A is 97.2%; The ultimate yield of sweet Stevia main products stevioside is 90.29%, and the ultimate yield of rebaudioside A is 91.56%.
embodiment 4
(1) enzyme-added immersion: by 100g sweet Stevia cured leaf (containing stevioside 4.27%, rebaudioside A 5.52%, dulcoside B 0.58%, total sweet glycosides is 10.37%) be crushed to 10 ~ 30 orders, be soaked in 1100mL water, add 2g cellulase preparation and carry out enzymolysis, obtain sweet Stevia enzymolysis solution;
(2) heat lixiviate: by step (1) gained sweet Stevia enzymolysis solution at 45 DEG C, pH value is 5.5 times, and lixiviate 60min, is cooled to room temperature, carries out tripodia sedimentation centrifugal under 1200r/min speed, filter, obtain the enzyme-added vat liquor of 746mL sweet Stevia;
(3) flocculation purification: the enzyme-added vat liquor of 746mL step (2) gained sweet Stevia is placed in 70 DEG C of water-baths and heats, add 2.25g inorganic salt flocculation agent (wherein, iron(ic) chloride 1.50g, calcium oxide 0.75g), stir, pH value 8.5 times, after flocculation treatment 40min, it is centrifugal first flocculation solution to be carried out under 3000r/min speed sleeping spiral shell, then carries out disk centrifugal under 6000r/min speed, filter, obtain 722mL sweet Stevia removal of impurities liquid;
(4) macroporous adsorbent resin decolouring purifying: 722mL step (3) gained sweet Stevia removal of impurities liquid is crossed 40mL AB-8 type macroporous adsorbent resin resin column (aspect ratio is 8:1) is housed, be 2BV/h at adsorption flow rate, pH value 8 times, carry out decolorization adsorption (when post completes excessively, effluent liquid does not detect stevioside), use the ethanolic soln of volume fraction 60% as elutriant again, with elution flow rate 1.0BV/h, the consumption of elutriant is 2.0BV, at room temperature carry out wash-out, elutriant nanofiltration membrane (molecular weight cut-off 500Da) carries out nanofiltration, working pressure is 1.4Mpa, then at vacuum pressure-0.06MPa, under temperature 70 C, till vacuum concentration to 39 degree Beaume, obtain sweet Stevia concentrated solution,
(5) silica gel column chromatography is separated: by silica gel column chromatography on step (4) gained sweet Stevia concentrated solution, then be that the ethanol of 12:88 and the mixed solvent of ethyl acetate carry out wash-out by volume ratio, collect 2 cuts respectively according to the change of chromatogram layer and (carry out collection first cut when wash-out carries out 43 ~ 53min, collection second cut is carried out) when wash-out carries out 63 ~ 74min, carry out vacuum concentration respectively again, vacuum-drying, obtains 3.91g stevioside and 5.16g rebaudioside A.
Products obtained therefrom is all in white or beige white powder, and detect through high performance liquid chromatography, wherein, the purity of stevioside is 98.4%, and the purity of rebaudioside A is 97.6%; The ultimate yield of sweet Stevia main products stevioside is 90.10%, and the ultimate yield of rebaudioside A is 91.23%.

Claims (10)

1. from sweet Stevia, extract a method for high purity stevioside, it is characterized in that, comprise the following steps:
(1) enzyme-added immersion: be soaked in water after Stevia rebaudiana starting material is pulverized, add cellulase preparation and carry out enzymolysis, obtain sweet Stevia enzymolysis solution;
(2) lixiviate is heated: by step (1) gained sweet Stevia enzymolysis solution heating lixiviate, be cooled to room temperature, centrifugal, filter, obtain the enzyme-added vat liquor of sweet Stevia;
(3) flocculation purification: the enzyme-added vat liquor of step (2) gained sweet Stevia is carried out heating in water bath, adds inorganic salt flocculation agent, stirs, after carrying out flocculation treatment, centrifugal, filters, obtains sweet Stevia removal of impurities liquid;
(4) macroporous adsorbent resin decolouring purifying: step (3) gained sweet Stevia removal of impurities liquid is crossed macroporous adsorptive resins and carries out decolorization adsorption, then use elution, elutriant carries out nanofiltration, concentrated, obtains sweet Stevia concentrated solution;
(5) silica gel column chromatography is separated: by silica gel column chromatography on step (4) gained sweet Stevia concentrated solution, then carry out wash-out with the mixed solvent of ethanol and ethyl acetate, collect each cut respectively, concentrated, dry, obtains multiple highly purified stevioside glycoside product.
2. from sweet Stevia, extract the method for high purity stevioside according to claim 1, it is characterized in that: in step (1), particle diameter after described Stevia rebaudiana starting material pulverizing is 10 ~ 30 orders, the feed liquid mass ratio of described Stevia rebaudiana starting material and water is 1:10 ~ 12, and the interpolation quality of described cellulase preparation is 0.1 ~ 0.3w/v% of the volume of water added.
3. according to claim 1 or 2, extract the method for high purity stevioside from sweet Stevia, it is characterized in that: in step (2), the temperature of described lixiviate is 40 ~ 50 DEG C, and the pH value of lixiviate is 5.0 ~ 6.0, and the time of lixiviate is 60 ~ 70min.
4. according to the method extracting high purity stevioside one of claims 1 to 3 Suo Shu from sweet Stevia, it is characterized in that: in step (3), the temperature of described heating in water bath is 60 ~ 80 DEG C, and the pH value of described flocculation treatment is 8 ~ 9, and the time of flocculation treatment is 30 ~ 40min.
5. according to the method extracting high purity stevioside one of Claims 1 to 4 Suo Shu from sweet Stevia, it is characterized in that: in step (3), the addition of described inorganic salt flocculation agent is 3.0 ~ 4.0g/L.
6. according to the method extracting high purity stevioside one of Claims 1 to 5 Suo Shu from sweet Stevia, it is characterized in that: in step (3), described inorganic salt flocculation agent is chitosan, or is the mixture of iron(ic) chloride and calcium oxide; In the mixture of described iron(ic) chloride and calcium oxide, the mass ratio of iron(ic) chloride and calcium oxide is 1:0.2 ~ 1.5.
7. according to the method extracting high purity stevioside one of claim 1 ~ 6 Suo Shu from sweet Stevia, it is characterized in that: in step (4), the adsorption flow rate of described decolorization adsorption is 2 ~ 3BV/h, and the pH value of decolorization adsorption is 7.5 ~ 8.5, and the aspect ratio of macroporous adsorptive resins is 5 ~ 9:1.
8. according to the method extracting high purity stevioside one of claim 1 ~ 7 Suo Shu from sweet Stevia, it is characterized in that: in step (4), described elutriant is the ethanolic soln of volume fraction 60 ~ 65%, and the flow velocity of wash-out is 0.5 ~ 2BV/h, and the consumption of elutriant is 1 ~ 2BV.
9. according to the method extracting high purity stevioside one of claim 1 ~ 8 Suo Shu from sweet Stevia, it is characterized in that: in step (4), the molecular weight cut-off of described nanofiltration nanofiltration membrane used is 300 ~ 600Da, and working pressure is 1.0 ~ 1.5Mpa; Described simmer down to vacuum concentration, vacuum pressure is-0.08 ~-0.04Mpa, and thickening temperature is 50 ~ 80 DEG C, and being concentrated into degree Beaume is till 35 ~ 40.
10. according to the method extracting high purity stevioside one of claim 1 ~ 9 Suo Shu from sweet Stevia, it is characterized in that: in step (5), the volume ratio of the mixed solvent of described ethanol and ethyl acetate is 1:1 ~ 10.
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