CN102796790A - Method for conversing steviolbioside to rebaudiodside B - Google Patents
Method for conversing steviolbioside to rebaudiodside B Download PDFInfo
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- CN102796790A CN102796790A CN201210288781XA CN201210288781A CN102796790A CN 102796790 A CN102796790 A CN 102796790A CN 201210288781X A CN201210288781X A CN 201210288781XA CN 201210288781 A CN201210288781 A CN 201210288781A CN 102796790 A CN102796790 A CN 102796790A
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- glycosides
- stevia rebaudianum
- rebaudiodside
- lai baodi
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Abstract
The invention discloses a method for conversing steviolbioside to rebaudiodside B, which belongs to the food chemical field, steviolbioside is taken as a raw material, cane sugar is taken as a carbohydrate source, so that the rebaudiodside B can be conversed under the catalysis of aspergillns niger III. According to the invention, the conversion rate of the rebaudiodside B which is obtained by taking steviolbioside as the raw material is greater than or equal to 70%, the rebaudiodside B content in a solid content can reach at least 60%, and can reach 80% after further separation and purification, and even can reach greater than 90%, the rebaudiodside B tastes odorless, and can be individually used as a sweetener, the rebaudiodside B produced by the method has the advantages of low cost and easy amplification, and is suitable for industrial production.
Description
Technical field
The invention belongs to the food chemical field, be specifically related to the method that the two glycosides of a kind of stevia rebaudianum are converted into Lai Baodi glycosides B.
Background technology
Steviol glycoside (Steviol glycosides) is a general name to qualities of stevia extract; Its sweet ingredient comprises stevioside (Stevioside); Lai Baodi glycosides A, B, C, D, E (Rebaudi oside A/B/C/D/E), the single glycosides (Steviolmonoside) of stevia rebaudianum, the two glycosides (Steviolbioside) of stevia rebaudianum; Du Ke glycosides A (Dulcoside A) etc., the structural formula of steviol glycoside composition is following:
Lai Baodi glycosides B (Rebaudioside B is hereinafter to be referred as RB) is one of sweet ingredient in the steviol glycoside, discovers it except that stronger sugariness is arranged, and also has the physiological function of reducing blood-fat, is functional sweetener of new generation.But the content of RB in steviol glycoside is less than 1%, if produce RB through the purifying steviol glycoside, its cost is very high, is difficult to mass production and promotes the use of.
Present research for the steviol glycoside bio-transformation focuses mostly on and on existing molecule, adds long sugar chain.
Patented claim CN 201110379733.7 discloses a kind of method that improves steviol glycoside flavor matter with beta-cyclodextrin glucanotransferase (CGTase); It is characterized in that: water is mixed with the reaction solution of the starch of the steviol glycoside that contains 2-5% weight and 2-5% weight; Enzyme work is even for thorough mixing in the beta-cyclodextrin glucanotransferase adding reaction solution of 60-70U/ml; The add-on of transferring enzyme is 300-500U/ steviol glycoside weight g, is 37-50 ℃ in temperature and carries out conversion reaction 48-72 hour.
Patented claim 201010131126.4 discloses the method with microwave-assisted cyclomaltodextrin glucanotransferase (CGTase) catalyzing and synthetizing modified steviol glycoside of planting.This method is a raw material with stevioside sweet solution and starch hydrolyzate, in the microwave reaction device via microwave radiation and the substituted steviol glycoside of the catalytic acting in conjunction synthesis of glucose of CGTase base.In processing condition provided by the invention, not only can significantly accelerate the carrying out of enzymic catalytic reaction, and can not produce under the usual conditions phenomenon that microwave in the aqueous systems makes enzyme deactivation.
More than these methods all be with cyclomaltodextrin glucanotransferase (CGTase) catalyzing and synthetizing modified steviol glycoside; But CGTase can not be added to a single glucosyl residue on the steviol glycoside molecule, and the position that its catalysis sugar chain is transferred on the steviol glycoside is not single-minded.Therefore, the catalysate of CGTase is complicated mixture, is difficult to meet the standard of U.S. FDA and the JECFA of European Union.
Up to now, be not raw material also with the two glycosides of stevia rebaudianum, through microbial fermentation, be that glycosyl donor carries out the report that Lai Baodi glycosides B is produced in bio-transformation with sucrose.
Summary of the invention
It is raw material with the two glycosides of stevia rebaudianum that the technical problem that the present invention solved provides a kind of, is sugared source with sucrose, produces Lai Baodi glycosides B through enzymatic conversion method, thereby improves the method for its mouthfeel.
Technical scheme of the present invention is: the two glycosides of stevia rebaudianum are converted into the method for Lai Baodi glycosides B, and it is a raw material with the two glycosides of stevia rebaudianum, is sugared source with sucrose, under the catalysis of black mold III, is converted into Lai Baodi glycosides B.
Further, the method for conversion may further comprise the steps:
(1) by weight, with being mixed with the aqueous solution in the two glycosides of 1 portion of stevia rebaudianum and 4～8 parts of sucrose dissolved entry.
(2) in the aqueous solution that step (1) obtains, add the black mold III, addition is by weight: stevia rebaudianum Shuan Gan ︰ black mold III=1200～1800 ︰ 1 is preferably 1600 ︰ 1, then reaction under certain condition.
(3) reaction is accomplished, and filtering reacting liquid is drying to obtain the solid substance that contains Lai Baodi glycosides B.
Further, amount of water is 20～40 weight parts in the step (1).
Further, the condition of reaction is in the step (2): temperature 30～50 degree, and pH6～8, the reaction times is 3～7 hours.
The crucial part of method for transformation of the present invention is: sucrose is converted into Lai Baodi glycosides B as catalyzer with the two glycosides of stevia rebaudianum by the black mold III as saccharide donor.
Sucrose is as one of raw material, because its physicochemical property differ greatly with the stevia rebaudianum monoglycosides, so can also adopt modes such as column chromatography and crystallization to remove unreacted sucrose and reacted fructose after reacting.After having removed sucrose, fructose, black mold III in the solid substance, Lai Baodi glycosides B content can be greater than 80% in the solid substance, even can be greater than 90%.
As the key factor black mold III in the method for transformation of the present invention, it is from black-koji mould CICC 40417 (Chinese industrial microbial strains preservation administrative center deposit number: 40417) the specific specificity glycosyltransferase that obtains of fermentation inducement.Concrete, it utilizes following method to obtain:
(1) picking black-koji mould CICC 40417 mono-clonals are seeded to the 250ml that contains 25ml BMGY nutrient solution and shake in the bottle, and 26-30 degree, 200-300rpm are cultured to OD600=2-6 (about 16-18 hour).The 25ml culture medium inoculated is shaken in the bottle to the 2-4L that contains 1L BMGY nutrient solution, and 26-30 degree thermal agitation (200-300rpm) is to logarithmic phase (OD600=2-6).
(2) with the sterilization centrifuge tube, the centrifugal 5min collecting cell of room temperature 2000-3000g is removed supernatant; With BMMY solution re-suspended cell to OD600=1.0 (2-6L); Packing substratum to several 3-4L dividing plates shake bottle, cover with 2 layers of sterile gauze or cheese cloth, put into shaking table 26-30 degree and continue to cultivate.
(3) per 24 hours, add methyl alcohol to 0.4-0.6% concentration, until arriving best induction time.Under the room temperature condition, the centrifugal 5min collecting cell of 2000-3000g.Clear enzyme solution in the reservation places 4 degree precoolings to concentrate.Use polydextran gel press molecular weight to thick enzyme segmentation, wherein molecular weight is the black mold III at the enzyme of 68-73kD, be stored in-80 spend subsequent use.
Preferably, the preparation method of black mold III is following:
(1) picking black-koji mould CICC 40417 mono-clonals are seeded to the 250ml that contains 25ml BMGY nutrient solution and shake in the bottle, 28 degree, and 250rpm is cultured to OD600=4 (about 16-18 hour); The 25ml culture medium inoculated is shaken in the bottle to the 3L that contains 1L BMGY nutrient solution, and 28 degree thermal agitations (250rpm) are to logarithmic phase (OD600=4).
(2) with the sterilization centrifuge tube, the centrifugal 5min collecting cell of room temperature 2500g is removed supernatant; With BMMY solution re-suspended cell to OD600=1.0 (2-6L); Packing substratum to several 3-4L dividing plates shake bottle, cover with 2 layers of sterile gauze or cheese cloth, put into shaking table 28 degree and continue to cultivate.
(3) per 24 hours, add methyl alcohol to 0.5% concentration, until arriving best induction time; Under the room temperature condition, the centrifugal 5min collecting cell of 2500g.Clear enzyme solution in the reservation places 4 ℃ to concentrate.Use polydextran gel to press molecular weight to thick enzyme segmentation, wherein molecular weight is the black mold III at the enzyme of 68-73kD, be stored in-80 ℃ subsequent use.
The present invention is transformation efficiency >=70% that raw material obtains Lai Baodi glycosides B with the two glycosides of stevia rebaudianum, and Lai Baodi glycosides B content can reach 60% at least in the solid substance, further can reach 80% after the separation and purification; Even can＞90%; RB mouthfeel free from extraneous odour can use as sweeting agent separately, and it is low to produce the RB cost with this method; Be prone to amplify, be applicable to industrial production.
Embodiment
In embodiment, the two glycosides (Steviolbioside) of stevia rebaudianum abbreviate SB as, and Lai Baodi glycosides B (Rebaudioside B) abbreviates RB as, and sucrose (Sucrose) abbreviates SU as.
It below is the shaker test of carrying out to key parameter in the method for transformation of the present invention.
Test the influence that 1 reaction substrate condition transforms RB
Embodiment 1～9 is for investigating the influence of reaction substrate condition to the RB bio-transformation; Experimentize with orthogonal test method, (1:20～1:40m/v, 10～40 ℃, churning time 30～60min) carry out reaction substrate and prepare by table 1 condition with 9 parts of 20g SB (95%) respectively.After the preparation, according to the ratio 1:6 of SB dry powder and SU, 1200:1-1600:1 (m/m, SB dry powder: make a gesture of measuring and add the black mold III, under 7,40 ℃ of conditions of pH value, is incubated the enzymatic bio-transformation of carrying out of 5h by the weight black mold III) again.
Table 1 reaction substrate condition test scheme and interpretation of result
The invention is not restricted to the specified conditions of given conversion, purity and the dissolving SB dry powder of RB among the embodiment.
Test the influence that 2 bio-transformation conditions transform RB
Embodiment 10～18 carries out orthogonal experiment for investigating the influence of bio-transformation condition to the RB transformation efficiency with the bio-transformation condition element.At first, 9 parts of 20g SB (90%) are not pressed table 3 condition, with the ratio 1:30 of SB dry powder and water, churning time 45min, 25 ℃ of solvent temperatures, stirring velocity 90rpm carries out reaction substrate and prepares.Press SB then: black mold III=1400:1 (m/m) adds the black mold III, again according to the table 2 condition (orthogonal test that SB:SU=1:4～1:8m/m, 30～50 ℃ of temperature, acidity-basicity ph 6～8, reaction times 3～7h) are carried out bio-transformation.Behind the bioconversion reaction, adopt the HPLC detection method among the GB GB8270-1999, detect the growing amount of RB, and calculate the RB transformation efficiency, the result sees table 2.
Table 2 bio-transformation condition is to the influence of RB transformation efficiency
The invention is not restricted to the specified conditions of given conversion, purity and the enzyme treatment S B of RB among the embodiment.
Experiment 3 investigate to be filtered, method of enrichment is to the influence of solid substance RB content
After obtaining reaction solution according to the method for transformation of embodiment 1-18, if adopt filter paper filtering, drying obtains solid substance A; If adopt crystallization can obtain solid substance B; If solid substance B recrystallize after drying is obtained solid substance C, adopt the HPLC detection method among the GB GB8270-1999 to detect RB, the result sees table 3.
Table 3 investigate to filter, method of enrichment is to the influence of solid substance RB content
Visible from table 3, RB content can reach 60% at least in the employing solid substance, can reach 80% after further making with extra care, even can＞90%.
Claims (5)
1. the two glycosides of stevia rebaudianum are converted into the method for Lai Baodi glycosides B, it is characterized in that: it is a raw material with the two glycosides of stevia rebaudianum, is sugared source with sucrose, under the catalysis of black mold III, is converted into Lai Baodi glycosides B.
2. the two glycosides of stevia rebaudianum according to claim 1 are converted into the method for Lai Baodi glycosides B, and it is characterized in that: the method for conversion may further comprise the steps:
(1) by weight, with being mixed with the aqueous solution in the two glycosides of 1 portion of stevia rebaudianum and 4～8 parts of sucrose dissolved entry;
(2) in the aqueous solution that step (1) obtains, add the black mold III, addition is by weight: stevia rebaudianum Shuan Gan ︰ black mold III=1200～1800 ︰ 1, reaction under certain condition then;
(3) reaction is accomplished, and filtering reacting liquid is drying to obtain the solid substance that contains Lai Baodi glycosides B.
3. the two glycosides of stevia rebaudianum according to claim 1 are converted into the method for Lai Baodi glycosides B, and it is characterized in that: amount of water is 20～40 weight parts in the step (1).
4. the two glycosides of stevia rebaudianum according to claim 2 are converted into the method for Lai Baodi glycosides B, it is characterized in that: in the step (2), the addition of black mold III is the two glycosides ︰ black mold III of stevia rebaudianum=1600 ︰ 1 by weight.
5. the two glycosides of stevia rebaudianum according to claim 2 are converted into the method for Lai Baodi glycosides B, and it is characterized in that: the condition of reaction is in the step (2): temperature 30～50 degree, and pH6～8, the reaction times is 3～7 hours.
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| CN 201210288781 CN102796790B (en) | 2012-08-14 | 2012-08-14 | Method for conversing steviolbioside to rebaudiodside B |
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| CN 201210288781 CN102796790B (en) | 2012-08-14 | 2012-08-14 | Method for conversing steviolbioside to rebaudiodside B |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105899088A (en) * | 2013-12-16 | 2016-08-24 | 嘉吉公司 | Stabilized steviol glycoside in concentrated syrup |
| CN107164435A (en) * | 2017-05-27 | 2017-09-15 | 中国药科大学 | A kind of Rebaudiodside A KA preparation method |
| CN113583064A (en) * | 2021-08-28 | 2021-11-02 | 诸城市浩天药业有限公司 | Process method for producing rebaudioside B by high-temperature cracking method |
| CN114805456A (en) * | 2022-03-21 | 2022-07-29 | 翁源广业清怡食品科技有限公司 | Method for preparing high-purity rebaudioside b by adopting rebaudioside a alkaline hydrolysis |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101775425A (en) * | 2010-03-24 | 2010-07-14 | 江南大学 | Method for catalyzing and synthetizing modified stevioside by using microwave auxiliary cyclodextrin glucosyltransferase |
| CN102492757A (en) * | 2011-11-25 | 2012-06-13 | 中国农业大学 | Method for improving taste quality of stevioside by using beta-cyclodextrin glucosyltransferase |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101775425A (en) * | 2010-03-24 | 2010-07-14 | 江南大学 | Method for catalyzing and synthetizing modified stevioside by using microwave auxiliary cyclodextrin glucosyltransferase |
| CN102492757A (en) * | 2011-11-25 | 2012-06-13 | 中国农业大学 | Method for improving taste quality of stevioside by using beta-cyclodextrin glucosyltransferase |
Non-Patent Citations (1)
| Title |
|---|
| A. ERKUCUK, 等: "Supercritical CO2 extraction of glycosides from stevia rebaudiana leaves: Identification and optimization", 《THE JOURNAL OF SUPERCRITICAL FLUIDS》, vol. 51, no. 1, 30 November 2009 (2009-11-30), XP026587020, DOI: doi:10.1016/j.supflu.2009.07.002 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105899088A (en) * | 2013-12-16 | 2016-08-24 | 嘉吉公司 | Stabilized steviol glycoside in concentrated syrup |
| CN107164435A (en) * | 2017-05-27 | 2017-09-15 | 中国药科大学 | A kind of Rebaudiodside A KA preparation method |
| CN107164435B (en) * | 2017-05-27 | 2020-03-31 | 中国药科大学 | Preparation method of rebaudioside KA |
| CN113583064A (en) * | 2021-08-28 | 2021-11-02 | 诸城市浩天药业有限公司 | Process method for producing rebaudioside B by high-temperature cracking method |
| CN113583064B (en) * | 2021-08-28 | 2024-03-12 | 诸城市浩天药业有限公司 | Process method for producing rebaudioside B by high-temperature pyrolysis method |
| CN114805456A (en) * | 2022-03-21 | 2022-07-29 | 翁源广业清怡食品科技有限公司 | Method for preparing high-purity rebaudioside b by adopting rebaudioside a alkaline hydrolysis |
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Inventor after: Li Qintong Inventor after: Ou Yangjin Inventor after: Li Cuan Inventor before: Li Qintong Inventor before: Ou Yangjin |
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