CN112322686A - Method for producing rebaudioside B by enzyme method - Google Patents
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- CN112322686A CN112322686A CN202011355519.3A CN202011355519A CN112322686A CN 112322686 A CN112322686 A CN 112322686A CN 202011355519 A CN202011355519 A CN 202011355519A CN 112322686 A CN112322686 A CN 112322686A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C—CHEMISTRY; METALLURGY
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- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
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- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
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Abstract
The invention relates to a method for producing rebaudioside B by an enzymatic method, belonging to the technical field of biosynthesis. The technical problem to be solved by the invention is to provide a novel method for producing rebaudioside B by an enzyme method. The method comprises the following steps: the method comprises the steps of catalyzing substrate reaction in an aqueous solution by taking beta-galactosidase as a catalyst to obtain rebaudioside B, wherein the substrate is at least one of rebaudioside A, rebaudioside D, rebaudioside I and rebaudioside M. The method provided by the invention adopts beta-galactosidase as a catalyst for the first time, catalyzes a substrate reaction to obtain RB, and is simple, easy to operate, high in substrate conversion rate and low in cost.
Description
Technical Field
The invention relates to a method for producing rebaudioside B by an enzymatic method, belonging to the technical field of biosynthesis.
Background
Among the stevioside compounds, rebaudioside a (ra) has been widely used as a sweetener in food and beverage products. But has the disadvantage of having a natural aftertaste or grassy smell, which also limits its further sugar substitution.
Rebaudioside b (rb) is also one of the sweet components in steviol glycosides. The sweetness is equivalent to that of RA, the bitterness is weaker than that of RA, therefore, the mouthfeel of RB is better than that of RA, besides being used as a sweetening agent, the RB has the effect of reducing blood sugar, and is a stevioside compound with larger application potential. However, the content of RB in steviol glycosides is small, less than 1%. If the RB is obtained by a plant extraction mode, the RB is limited by the yield and the cost of raw materials, and is difficult to produce and popularize.
Because the content of the RB in stevia rebaudiana leaves is low, the RB is mainly prepared by hydrolyzing stevia rebaudiana sugar with similar structures such as RA, RD and RI by sodium hydroxide, for example, Chinese patent CN 104725437A. The chemical hydrolysis method has the disadvantages of extreme reaction conditions, generation of a large amount of hazardous waste reagents, great environmental protection pressure and the like, and the development of the technology is accompanied by great tendency that the biological enzyme method is used for replacing the chemical method for production.
The Chinese patent with publication number CN102796790A discloses a method for converting steviolbioside into rebaudioside B, which takes steviolbioside as a raw material and sucrose as a sugar source and converts the steviolbioside into rebaudioside B under the catalysis of aspergillus niger III. According to the method, specific aspergillus niger III is required to be adopted as a catalyst, steviolbioside is used as a raw material, an additional sugar source is required to be added, the cost of the raw material is high, the conversion rate of rebaudioside B obtained by the method is 70-75%, and the conversion rate is required to be further improved.
Disclosure of Invention
Aiming at the defects, the technical problem solved by the invention is to provide a novel method for producing rebaudioside B by an enzyme method.
The method for producing rebaudioside B by using the enzymatic method comprises the following steps: catalyzing substrate reaction in aqueous solution by taking beta-galactosidase as a catalyst to obtain rebaudioside B, wherein the substrate is at least one of Rebaudioside A (RA), Rebaudioside D (RD), Rebaudioside I (RI) and Rebaudioside M (RM).
In one embodiment of the invention, the beta-galactosidase is derived from sulfolobus, aspergillus niger or kluyveromyces.
In one embodiment of the invention, the beta-galactosidase is derived from kluyveromyces lactis.
In one embodiment of the invention, the amount of beta-galactosidase added is 1000 to 20000U/g substrate.
In one embodiment of the invention, in the catalytic reaction system, the temperature is controlled to be 25-60 ℃, the pH value is 6.0-10.0, the substrate concentration is 20-80 g/L, and the reaction time is more than or equal to 6 h.
In one embodiment of the present invention, glycerol and β -mercaptoethanol are also added to the catalytic reaction system of the present invention.
In some embodiments of the invention, glycerol is added in an amount of 50% (v/v) or less and beta-mercaptoethanol is added in an amount of 10% (v/v) or less.
In one embodiment of the present invention, after the catalytic reaction, the rebaudioside B product is obtained by performing separation and purification by the following method:
a. heating and coarsely filtering the reaction solution after reaction, and then performing ultrafiltration to remove insoluble impurities and denatured proteins to obtain ultrafiltration permeate;
b. separating rebaudioside B from the ultrafiltration permeate in a nanofiltration mode to obtain nanofiltration retentate;
c. concentrating, crystallizing and drying the nanofiltration trapped fluid to obtain a rebaudioside-B product; or concentrating the trapped fluid and then spray-drying to obtain the rebaudioside-B product.
Compared with the prior art, the invention has the following beneficial effects:
the method provided by the invention adopts beta-galactosidase as a catalyst for the first time, catalyzes a substrate reaction to obtain RB, and is simple, easy to operate, high in substrate conversion rate and low in cost.
Detailed Description
The method for producing rebaudioside B by using the enzymatic method comprises the following steps: catalyzing substrate reaction in aqueous solution by taking beta-galactosidase as a catalyst to obtain rebaudioside B, wherein the substrate is at least one of Rebaudioside A (RA), Rebaudioside D (RD), Rebaudioside I (RI) and Rebaudioside M (RM).
According to the method, the RB is obtained by catalyzing substrate reaction by using beta-galactosidase as a catalyst. Beta-galactosidase (EC3.2.1.23) is called lactase for short, can hydrolyze lactose into galactose and is initially applied to the food industry, hydrolyze lactose in dairy products and prepare functional oligosaccharide, thereby meeting the requirements of lactose intolerance people on dairy products. Beta-galactosidase also has transglycosylation activity, for example, beta-galactosidase from Aspergillus can hydrolyze a glucose residue at C13 to produce suspensoid with stevioside as substrate. The research of the invention finds that the beta-galactosidase can hydrolyze glucosyl in RA, RD, RI and RM to generate RB. The method is simple, the operation is easy, and the conversion rate of the substrate is high.
The general structural formulas of RA, RB, RD, RI and RM are shown in Table 1.
TABLE 1
In one embodiment of the invention, the beta-galactosidase is derived from sulfolobus, aspergillus niger or kluyveromyces. The beta-galactosidase from the sources has better substrate specificity and can well convert the substrate to generate RB. In one embodiment of the invention, the beta-galactosidase is derived from kluyveromyces lactis.
The amount of beta-galactosidase added correlates with the amount of substrate. In order to better promote the reaction and simultaneously take account of the reaction rate, in one embodiment of the invention, the addition amount of the beta-galactosidase is 1000-20000U/g substrate.
In addition to the amount of enzyme and substrate, other factors may also affect the enzymatic reaction. In one embodiment of the invention, in the catalytic reaction system, the temperature is controlled to be 25-60 ℃, the pH value is 6.0-10.0, the substrate concentration is 20-80 g/L, and the reaction time is more than or equal to 6 h.
Temperature affects the activity of the enzyme and thus the progress of the catalytic reaction. In the embodiment of the invention, the reaction temperature is controlled to be 25-60 ℃. In one embodiment, the reaction temperature is controlled to be 25-40 ℃. In some embodiments of the invention, the temperature may be controlled to 25 deg.C, 26 deg.C, 28 deg.C, 30 deg.C, 32 deg.C, 34 deg.C, 35 deg.C, 36 deg.C, 37 deg.C, 38 deg.C, or 40 deg.C, etc.
The pH may be adjusted by a buffer, in one embodiment of the invention, a phosphate buffer, e.g., Na2HPO4/NaH2PO4To adjust the pH. In some embodiments of the present invention, the pH of the reaction system is 6.5 to 8. Specifically, the pH may be 6.5, 6.8, 7.0, 7.2, 7.5, 7.8, 8.0, or the like.
With the progress of the catalytic reaction, the amount of the substrate in the system is lower and lower, and in some embodiments of the invention, the concentration of the substrate is controlled to be 20-80 g/L. Specifically, the substrate concentration may be 20g/L, 25g/L, 30g/L, 35g/L, 40g/L, 45g/L, 50g/L, 55g/L, 60g/L, 65g/L, 70g/L, 75g/L, or 80g/L, etc.
In some embodiments of the invention, the reaction time is ≧ 6 h. After the catalytic reaction is carried out for a period of time, the substrate reacts completely, and at the moment, the yield cannot be increased by prolonging the reaction time, so that the reaction efficiency is influenced. In one embodiment of the invention, the reaction time is 6 h.
In the catalytic reaction system of the invention, glycerol and beta-mercaptoethanol can also be added. Glycerol can act as a protectant of the enzyme, while beta-mercaptoethanol can act as an activator of the enzyme. After adding both, the conversion rate of the substrate can be improved.
Both glycerol and beta-mercaptoethanol are useful in the present invention in amounts commonly used in the art. In some embodiments of the present invention, glycerol is added in an amount of 50% (v/v) or less, and β -mercaptoethanol is added in an amount of 10% (v/v) or less, i.e., glycerol is added in an amount of 50% or less by volume of the reaction system, and β -mercaptoethanol is added in an amount of 10% or less by volume of the reaction system.
After the completion of the catalysis, RB can be produced, but in this case, RB is in a solution containing impurities such as β -galactosidase, unreacted substrate, and the like in addition to RB, and thus it is necessary to separate and purify the solution to obtain a product. Separation and purification methods commonly used in the art are suitable for use in the present invention.
In one embodiment of the invention, the following method is adopted for separation and purification to obtain the RB product:
a. heating and coarsely filtering the reaction solution after reaction, and then performing ultrafiltration to remove insoluble impurities and denatured proteins to obtain ultrafiltration permeate;
b. separating rebaudioside B from the ultrafiltration permeate in a nanofiltration mode to obtain nanofiltration retentate;
c. concentrating, crystallizing and drying the nanofiltration trapped fluid to obtain a rebaudioside-B product; or concentrating the trapped fluid and then spray-drying to obtain the rebaudioside-B product.
In one embodiment of the invention, the ultrafiltration membrane used in step a has a specification of 10kD and a transmembrane pressure of 1.0-1.5 MPa, and at this time, the water-soluble protein is in the trapped fluid, and the reaction substrate and the product exist in the permeated fluid.
In one embodiment of the invention, the nanofiltration membrane used in the step b has a specification of 0.5kD, the transmembrane pressure is 1.5-2.0 MPa, the substrate/product is in the trapped liquid, and the rest of the small molecular impurities are in the permeate liquid.
Preferably, the crystallization method adopted in the step c is that the nanofiltration concentrated solution is concentrated to a solid content of 10-30%, ethanol is added to adjust the ethanol concentration of the crystallization system to 10-80%, the mixture is heated to boiling, cooled to 0-40 ℃, and crystallized for 1-60 hours under the crystallization conditions.
Preferably, the spray drying conditions used in step d are: concentrating the nanofiltration trapped fluid to a solid content of 10-60%, and drying by using a spray drying method, wherein the temperature of an air inlet is 80 ℃, and the temperature of an air outlet of the spray drying method is 120 ℃.
The following examples are provided to further illustrate the embodiments of the present invention and are not intended to limit the scope of the present invention. The beta-galactosidase in the examples was derived from Kluyveromyces lactis, available from Novoxil (China) Biotechnology Inc.
Example 1
Taking RA as a substrate, weighing RA, buffer salt and glycerol with proper concentrations, adding beta-galactosidase after complete dissolution, carrying out water bath reaction for a certain time, sampling, measuring the concentrations of RA and RB in a reaction solution by HPLC, and calculating the conversion rate of RA. Specific reaction parameters and conversion results are shown in table 2.
The calculation method of the conversion rate in the reaction system comprises the following steps:
RA molar conversion RB molar concentration/(RA molar concentration + RB molar concentration). times.100%
TABLE 2 investigation of the catalytic System
Example 2
The effect on transformation was examined by adding different concentrations of protective/activating agents to RA as substrate in group 4 of example 1, and the results are shown in Table 3.
TABLE 3 Effect of protectant/activator on transformation
Example 3
The conversion effect of RB produced by using RA, RD, RI, and RM as substrates was examined, and the results are shown in Table 4.
TABLE 4 comparison of different substrate conversions RB
Example 4
The RB in group 1 of example 1 is separated and purified, and the specific steps are as follows:
1. separation with ultrafiltration membrane
Diluting the converted mixed solution by 2 times with purified water, and heating to 60-80 ℃ to obtain a clear solution. The clear solution is filtered by an ultrafiltration membrane with the specification of 10kD, the transmembrane pressure is respectively controlled at 0.5MPa, 1.0MPa and 1.5MPa, the solid content of the clear solution is detected by respectively taking trapped fluid and permeate, the influence of the ultrafiltration transmembrane pressure on the RB permeability is examined, and the result is shown in table 5.
When the pressure of the ultrafiltration transmembrane is more than or equal to 1.0MPa, RB can permeate the ultrafiltration membrane with the 10kD specification, and the effect of separating products from macromolecular impurities is achieved.
TABLE 5 Effect of ultrafiltration transmembrane pressure on RB recovery
2. Separation and enrichment of nanofiltration membrane
The clear liquid passes through an ultrafiltration membrane with the specification of 10kD, the transmembrane pressure is controlled to be 1.5MPa, then the ultrafiltration permeate passes through a nanofiltration membrane with the specification of 0.5kD, the transmembrane pressure is respectively controlled to be 1.0MPa, 1.5MPa and 2.0MPa, the content of solid matters in the trapped liquid and the permeate are respectively detected, the influence of the nanofiltration transmembrane pressure on the elimination of small molecular impurities is examined, and the result is shown in table 6.
TABLE 6 influence of nanofiltration transmembrane pressure on the elimination of small molecular impurities
When the nanofiltration transmembrane pressure is more than or equal to 1.5MPa, the small molecular impurities can permeate the nanofiltration membrane with the specification of 0.5kD, and the effect of separating the small molecular impurities from RB is achieved.
3. Crystallization of
And heating the nanofiltration trapped fluid to concentrate the solid content to be 20%, stirring for 12h at normal temperature, separating out RB crystals, leaching with a small amount of purified water after filtering, and drying to obtain a RB crude product. And (3) concentrating the crystallization mother liquor to the original volume of 1/10, stirring at 4 ℃ for 12h for crystallization, collecting crystals, leaching with a small amount of purified water, and drying to obtain a RB crude product.
4. Refining
Adding 50% ethanol solution with 4 times volume of the RB crude product, heating and dissolving to prepare supersaturated solution, adding 1% active carbon, stirring for 30 minutes while the solution is hot, filtering, and stirring for 14 hours at 4 ℃ for crystallization. Filtering and collecting crystals, and drying at 80 ℃ to obtain RB crystals with the purity of more than 95%.
Claims (7)
1. A method for producing rebaudioside B by an enzymatic method, comprising the following steps: the method comprises the steps of catalyzing substrate reaction in an aqueous solution by taking beta-galactosidase as a catalyst to obtain rebaudioside B, wherein the substrate is at least one of rebaudioside A, rebaudioside D, rebaudioside I and rebaudioside M.
2. The method for enzymatically producing rebaudioside B according to claim 1, characterized in that: the beta-galactosidase is derived from sulfolobus, aspergillus niger or kluyveromyces lactis; preferably, the beta-galactosidase is derived from Kluyveromyces lactis.
3. The method for enzymatically producing rebaudioside B according to claim 1 or 2, characterized in that: the addition amount of the beta-galactosidase is 1000-20000U/g substrate.
4. The method for enzymatically producing rebaudioside B according to any one of claims 1 to 3, characterized in that: in a catalytic reaction system, the temperature is controlled to be 25-60 ℃, the pH value is 6.0-10.0, the substrate concentration is 20-80 g/L, and the reaction time is more than or equal to 6 hours.
5. The method for enzymatically producing rebaudioside B according to any one of claims 1 to 4, characterized in that: in the catalytic reaction system, glycerin and beta-mercaptoethanol are also added.
6. The method for enzymatically producing rebaudioside B according to claim 5, characterized in that: in the reaction system, the addition amount of the glycerol is less than or equal to 50 percent (v/v), and the addition amount of the beta-mercaptoethanol is less than or equal to 10 percent (v/v).
7. The method for enzymatically producing rebaudioside B according to any one of claims 1 to 6, characterized in that: after the catalytic reaction, the rebaudioside B product is obtained by adopting the following method for separation and purification:
a. heating and coarsely filtering the reaction liquid after reaction, and then performing ultrafiltration to obtain ultrafiltration permeate;
b. separating rebaudioside B from the ultrafiltration permeate in a nanofiltration mode to obtain nanofiltration retentate;
c. concentrating, crystallizing and drying the nanofiltration trapped fluid to obtain a rebaudioside-B product; or concentrating the trapped fluid and then spray-drying to obtain the rebaudioside-B product.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114366754A (en) * | 2021-09-13 | 2022-04-19 | 兰州大学第二医院 | Application of Reb B in preparation of medicine for treating lung ischemia-reperfusion injury |
| 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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| CN104725437A (en) * | 2013-12-19 | 2015-06-24 | 江南大学 | Method for preparation of laminaribiose and rebaudiodside B by basic hydrolysis of rebaudiodside I |
| CN108103127A (en) * | 2017-12-13 | 2018-06-01 | 桂林莱茵生物科技股份有限公司 | A kind of method for industrializing quick production and preparing glucosyl group steviol glycoside mixture |
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Patent Citations (2)
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| CN104725437A (en) * | 2013-12-19 | 2015-06-24 | 江南大学 | Method for preparation of laminaribiose and rebaudiodside B by basic hydrolysis of rebaudiodside I |
| CN108103127A (en) * | 2017-12-13 | 2018-06-01 | 桂林莱茵生物科技股份有限公司 | A kind of method for industrializing quick production and preparing glucosyl group steviol glycoside mixture |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114366754A (en) * | 2021-09-13 | 2022-04-19 | 兰州大学第二医院 | Application of Reb B in preparation of medicine for treating lung ischemia-reperfusion injury |
| 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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