CN102796790B - Method for conversing steviolbioside to rebaudiodside B - Google Patents
Method for conversing steviolbioside to rebaudiodside B Download PDFInfo
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- CN102796790B CN102796790B CN 201210288781 CN201210288781A CN102796790B CN 102796790 B CN102796790 B CN 102796790B CN 201210288781 CN201210288781 CN 201210288781 CN 201210288781 A CN201210288781 A CN 201210288781A CN 102796790 B CN102796790 B CN 102796790B
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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 chemistry field, be specifically related to the method that the two glycosides of a kind of stevia rebaudianum are converted into RB.
Background technology
Steviol glycoside (Steviol glycosides) is a general name to qualities of stevia extract, its sweet ingredient comprises stevioside (Stevioside), rebaudioside A, B, C, D, E(Rebaudi oside A/B/C/D/E), SM (Steviolmonoside), the two glycosides (Steviolbioside) of stevia rebaudianum, Du Ke glycosides A(Dulcoside A) etc., the structural formula of steviol glycoside composition is as follows:
RB (Rebaudioside B is hereinafter to be referred as RB) is that in steviol glycoside, the research of one of sweet ingredient finds that it except stronger sugariness is arranged, 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 by the purifying steviol glycoside, its cost is very high, is difficult to produce in a large number and promote the use of.
Present research for the steviol glycoside bio-transformation focuses mostly on and add long sugar chain on existing molecule.
Patent application 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 is lived fully mix in reaction solution for the beta-cyclodextrin glucanotransferase of 60-70U/ml adds, 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.
Patent application 201010131126.4 discloses the method for planting with microwave-assisted cyclomaltodextrin glucanotransferase (CGTase) catalyzing and synthetizing modified steviol glycoside.The method is take stevioside sweet solution and starch hydrolyzate as raw material, the steviol glycoside that replaces via the acting in conjunction synthesis of glucose base of microwave radiation and CGTase catalysis in the microwave reaction device.In processing condition provided by the invention, not only can significantly accelerate the carrying out of enzymic catalytic reaction, and can not produce under usual conditions the phenomenon that microwave in aqueous systems makes enzyme deactivation.
Above these methods are all 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 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, also not take the two glycosides of stevia rebaudianum as raw material, by microbial fermentation, carry out the report that RB is produced in bio-transformation take sucrose as glycosyl donor.
Summary of the invention
Technical problem solved by the invention is to provide a kind of take the two glycosides of stevia rebaudianum as raw material, take sucrose as sugared source, produces RB by 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 RB, and it take sucrose as sugared source, is converted into RB take the two glycosides of stevia rebaudianum as raw material under the catalysis of black mold III.
Further, the method for conversion comprises the following steps:
(1) by weight, will be mixed with the aqueous solution in the two glycosides of 1 portion of stevia rebaudianum and 4～8 parts of sucrose dissolved entry.
(2) add the black mold III in the aqueous solution that obtains to step (1), addition is by weight: stevia rebaudianum Shuan Gan ︰ black mold III=1200～1800 ︰ 1 are preferably 1600 ︰ 1, then reaction under certain condition.
(3) reaction is completed, and filtering reacting liquid is drying to obtain the solid substance that contains RB.
Further, in step (1), amount of water is 20～40 weight parts.
Further, in step (2), the condition of reaction is: 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 RB 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 and stevia rebaudianum monoglycosides differ greatly, therefore can also adopt the modes such as column chromatography and crystallization to remove unreacted sucrose and reacted fructose after reaction.After having removed sucrose, fructose, black mold III in solid substance, in solid substance, RB content can be greater than 80%, even can be greater than 90%.
As the key factor black mold III in 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 species 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 in the 250ml shaking flask that contains 25ml BMGY nutrient solution, and 26-30 degree, 200-300rpm are cultured to OD600=2-6 (approximately 16-18 hour).To the 2-4L shaking flask that contains 1L BMGY nutrient solution, 26-30 degree thermal agitation (200-300rpm) is to logarithmic phase (OD600=2-6) with the 25ml culture medium inoculated.
(2) with the sterilization centrifuge tube, the centrifugal 5min collecting cell of room temperature 2000-3000g, remove supernatant, with BMMY solution re-suspended cell to OD600=1.0 (2-6L), the packing substratum is to several 3-4L dividing plate shaking flasks, cover with 2 layers of sterile gauze or cheese cloth, put into shaking table 26-30 degree and continue to cultivate.
(3) every 24 hours, add methyl alcohol to 0.4-0.6% concentration, until arrive best induction time.Under room temperature condition, the centrifugal 5min collecting cell of 2000-3000g.Clear enzyme solution in reservation is placed in 4 degree Preconcentrators.Use dextrane gel to press molecular weight to thick enzyme segmentation, wherein molecular weight is the black mold III at the enzyme of 68-73kD, is stored in-80 degree standby.
Preferably, the preparation method of black mold III is as follows:
(1) picking black-koji mould CICC 40417 mono-clonals are seeded in the 250ml shaking flask that contains 25ml BMGY nutrient solution, 28 degree, and 250rpm is cultured to OD600=4 (approximately 16-18 hour); To the 3L shaking flask that contains 1L BMGY nutrient solution, 28 degree thermal agitations (250rpm) are to logarithmic phase (OD600=4) with the 25ml culture medium inoculated.
(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), the packing substratum covers with 2 layers of sterile gauze or cheese cloth to several 3-4L dividing plate shaking flasks, puts into shaking table 28 degree and continues to cultivate.
(3) every 24 hours, add methyl alcohol to 0.5% concentration, until arrive best induction time; Under room temperature condition, the centrifugal 5min collecting cell of 2500g.Clear enzyme solution in reservation is placed in 4 ℃ and concentrates.Use dextrane 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 ℃ standby.
The present invention obtains the transformation efficiency of RB 〉=70% take the two glycosides of stevia rebaudianum as raw material, in solid substance, RB content can reach 60% at least, further can reach 80% after separation and purification, even can＞90%, RB mouthfeel free from extraneous odour can use as sweeting agent separately, produces the RB cost with this method low, easily amplify, be applicable to industrial production.
Embodiment
In embodiment, the two glycosides (Steviolbioside) of stevia rebaudianum are referred to as SB, and RB (Rebaudioside B) is referred to as RB, and sucrose (Sucrose) is referred to as SU.
It is below the shaker test of carrying out for key parameter in method for transformation of the present invention.
Test the impact that 1 reaction substrate condition transforms RB
Embodiment 1～9 is for investigating the reaction substrate condition to the impact of RB bio-transformation, test with orthogonal test method, respectively with 9 parts of 20g SB(95%) (1:20～1:40m/v, 10～40 ℃, churning time 30～60min) carry out reaction substrate and prepare by table 1 condition.After preparation, then according to the ratio 1:6 of SB dry powder and SU, and 1200:1-1600:1 (m/m, SB dry powder: the weight black mold III) is made a gesture of measuring and is added the black mold III, and insulation 5h's carries out enzymatic bio-transformation under pH value 7,40 ℃ of conditions.
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 in embodiment.
Test the impact that 2 biotransformation conditions transform RB
Embodiment 10～18 carries out orthogonal experiment for investigating biotransformation condition to the impact of RB transformation efficiency with the biotransformation condition factor.At first, not with 9 parts of 20g SB(90%) press 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.Then by SB: black mold III=1400:1 (m/m) adds the black mold III, then 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.After bioconversion reaction, adopt the HPLC detection method in GB GB8270-1999, detect the growing amount of RB, and calculate the RB transformation efficiency, the results are shown in Table 2.
The impact of table 2 biotransformation condition on the RB transformation efficiency
The invention is not restricted to the specified conditions of given conversion, purity and the enzyme treatment S B of RB in embodiment.
Experiment 3 investigate to be filtered, method of enrichment is on the impact 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 drying after solid substance B recrystallize is obtained solid substance C, adopt the HPLC detection method in GB GB8270-1999 to detect RB, the results are shown in Table 3.
Table 3 investigate to filter, method of enrichment is on the impact of solid substance RB content
As seen from Table 3, adopt that in solid substance, RB content can reach 60% at least, can reach 80% after further refining, even can＞90%.
Claims (4)
1. the two glycosides of stevia rebaudianum are converted into the method for RB, it is characterized in that: it take sucrose as sugared source, is converted into RB take the two glycosides of stevia rebaudianum as raw material under the catalysis of black mold III; Described black mold III is from black-koji mould (Aspergillus niger) CICC 40417(Chinese industrial microbial strains preservation administrative center deposit number: 40417) the species specificity glycosyltransferase that obtains of fermentation inducement, and it utilizes following method to obtain:
(1) picking black-koji mould CICC 40417 mono-clonals are seeded in the 250ml shaking flask that contains 25ml BMGY nutrient solution, and 26-30 ℃, 200-300rpm is cultured to OD600=2-6; To the 2-4L shaking flask that contains 1L BMGY nutrient solution, 26-30 ℃ of thermal agitation is to logarithmic phase with the 25ml culture medium inoculated;
(2) with the sterilization centrifuge tube, the centrifugal 5min collecting cell of room temperature 2000-3000g, remove supernatant, with BMMY solution re-suspended cell to OD600=1.0, the packing substratum is to several 3-4L dividing plate shaking flasks, cover with 2 layers of sterile gauze or cheese cloth, put into shaking table 26-30 ℃ and continue to cultivate;
(3) every 24 hours, add methyl alcohol to 0.4-0.6% concentration, until arrive best induction time; Under room temperature condition, the centrifugal 5min collecting cell of 2000-3000g; Clear enzyme solution in reservation is placed in 4 ℃ of Preconcentrators; Use dextrane gel to press molecular weight to thick enzyme segmentation, wherein molecular weight is black mold III at the enzyme of 68-73kD, be stored in-80 ℃ standby.
2. the two glycosides of stevia rebaudianum according to claim 1 are converted into the method for RB, it is characterized in that: it is take the two glycosides of stevia rebaudianum as raw material, and take sucrose as sugared source, the method that is converted into the conversion of RB under the catalysis of black mold III comprises the following steps:
(1) by weight, will be mixed with the aqueous solution in the two glycosides of 1 portion of stevia rebaudianum and 4～8 parts of sucrose dissolved entry;
(2) add black mold III in the aqueous solution that obtains to step (1), addition is by weight: stevia rebaudianum Shuan Gan ︰ black mold III=1200～1800 ︰ 1, and 30～50 ℃ of temperature, reacted 3～7 hours pH6～8;
(3) reaction is completed, and filtering reacting liquid is drying to obtain the solid substance that contains RB.
3. the two glycosides of stevia rebaudianum according to claim 2 are converted into the method for RB, and it is characterized in that: in method for transformation step (1), amount of water is 20～40 weight parts.
4. the two glycosides of stevia rebaudianum according to claim 2 are converted into the method for RB, and it is characterized in that: in method for transformation step (2), the addition of black mold III is stevia rebaudianum Shuan Gan ︰ black mold III=1600 ︰ 1 by weight.
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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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Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| US20160309761A1 (en) * | 2013-12-16 | 2016-10-27 | Cargill, Incorporated | Stabilized steviol glycoside in concentrated syrup |
| CN107164435B (en) * | 2017-05-27 | 2020-03-31 | 中国药科大学 | Preparation method of rebaudioside KA |
| 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 |
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 (2)
| Title |
|---|
| A. Erkucuk, 等.Supercritical CO2 extraction of glycosides from stevia rebaudiana leaves: Identification and optimization.《The Journal of Supercritical Fluids》.2009,第51卷(第1期), |
| Supercritical CO2 extraction of glycosides from stevia rebaudiana leaves: Identification and optimization;A. Erkucuk, 等;《The Journal of Supercritical Fluids》;20091130;第51卷(第1期);29-35 * |
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