CN111662941A - Preparation method of glucosyl stevioside - Google Patents
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Abstract
The invention relates to a preparation method of glucosyl stevioside, which is characterized by comprising the following steps: (1) stevia extract: the cyclodextrin is mixed according to the mass ratio of 1: 1-2, preparing a 10% -60% mixed solution, and stirring and preheating for 1-3h at 40-80 ℃ to generate a suspension; (2) adding cyclodextrin glycosyltransferase, controlling the mass of cyclodextrin to be 2-10%, stirring at 40-80 ℃ and 300rpm/min for reaction for 1-96h, and heating to 105 ℃ for 30-60 min; (3) decolorizing the reaction solution with decolorizing resin, desalting the decolorized solution with anion and cation exchange resin, removing non-diterpene compounds from the desalted solution with macroporous adsorbent resin, washing the column with water, desorbing with 20-70% ethanol, and collecting ethanol desorption solution; (4) evaporating and concentrating the ethanol desorption solution, controlling the mass concentration of the stevioside to be more than 60%, and performing spray drying. The invention has the advantages that: the conversion rate of the invention is more than 90%, and the residual quantity of glycosyl donor is below 20%; the reaction temperature is lower, the stirring speed is low, the process is simple and the energy consumption is lower.
Description
Technical Field
The invention belongs to the field of biosynthesis and novel sweeteners, and particularly relates to a preparation method of glucosyl stevioside.
Background art:
recognizing that many diseases are associated with the consumption of high-sugar foods and beverages, alternatives to sucrose are receiving increasing attention. However, many artificial sweeteners, such as sodium cyclamate, cyclamate and saccharin, are banned or restricted in some countries due to safety concerns. As a result, non-caloric sweeteners of natural origin are becoming increasingly popular. Various diterpene glycosides are produced in stevia leaves, and have high-intensity sweetness and characteristics of being not easily absorbed by human bodies, so that the natural sweetener stevioside extracted from the dry leaves of stevia can be an excellent product for replacing cane sugar.
The natural sweeteners steviol glycosides are a class of extracts with a common aglycone (steviol) and differ by the number and type of carbohydrate residues at the C13 and C19 positions, the major glycosides found in stevia leaves being rebaudioside a (2-10%), stevioside (2-10%) and rebaudioside C (1-2%). Other glycosides, such as rebaudioside B, D, E and F, steviolbioside and rubusoside, are found at much lower levels (approximately 0-1%).
Rebaudioside a in the natural sweetener steviol glycoside principal has the least astringency, least bitterness and the least lingering aftertaste and therefore has the most favorable sensory attributes, however, even in a highly purified state, rebaudioside a still has undesirable taste attributes such as bitterness, sweet aftertaste, licorice flavor, and the like. These undesirable taste attributes in stevia sweeteners have been one of the major obstacles in commercialization, affecting the taste quality of stevia, and limiting its broader use.
The glucosyl stevioside is prepared by using the natural stevioside as a raw material and carrying out polymerization reaction on components such as stevioside, rebaudioside A and the like in the stevioside and glucosyl by methods such as enzyme catalysis and the like, the sweetness multiple of the stevioside is higher than that of sucrose by 100-250 times, bad mouthfeel such as bitter taste, sweet aftertaste, liquorice taste and the like of the natural stevioside is greatly improved, and the stevioside has excellent taste quality and is widely applied.
At present, a biological enzyme method is used for producing glucosyl stevioside, but because the stevioside raw material extracted from plants has low purity, and the influence of the variety, structure and the like of glycosyl donors causes the enzyme modified stevioside to generally have the defects of low conversion rate, more glycosyl donor residues, poor taste and color, complex operation steps, resource waste and the like.
Disclosure of Invention
The invention aims to overcome the defects of low conversion rate, more glycosyl donor residues, poor taste and color, complicated operation steps, resource waste and the like in the process of producing glucosyl stevioside by using the conventional biological enzyme method.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a preparation method of glucosyl stevioside is characterized by comprising the following steps:
(1) and (2) mixing the stevia extract: the cyclodextrin is mixed according to the mass ratio of 1: 1-2, adding water to prepare a mixed solution with the mass concentration of 10% -60%, then placing the mixed solution at 40-80 ℃, and preheating for 1-3 hours under the stirring condition to generate suspension;
(2) adding cyclodextrin glycosyltransferase (CGTase) into the suspension, controlling the mass of the cyclodextrin glycosyltransferase to be 2-10% of that of cyclodextrin, then placing the mixture at 40-80 ℃ and stirring the mixture at the rotating speed of 100-300rpm/min for reaction for 1-96h, continuously heating the mixture to more than 105 ℃ for 30-60min, and inactivating the cyclodextrin glycosyltransferase (CGTase) at a continuous high temperature to stop the reaction;
(3) after the reaction is finished, decoloring the reaction liquid by using decoloring resin (Weihua SHD-806), collecting the decolored solution, desalting the decolored solution by using anion-cation exchange resin (001 × 8 macroporous strong acid styrene cation exchange resin; 201 × 7 macroporous strong base styrene anion exchange resin) continuously, collecting the desalted solution, removing non-diterpene compounds (such as a plurality of sequentially connected columns filled with macroporous adsorption resin) by using macroporous adsorption resin, washing the columns by using distilled water or deionized water after the adsorption operation, desorbing by using 20-70% volume fraction ethanol, and collecting the ethanol hydrolysis imbibition containing diterpene compounds (stevioside);
(4) evaporating and concentrating the ethanol hydrolysis liquid containing diterpene compounds (stevioside), controlling the mass concentration of the stevioside in the concentrated solution to be more than 60%, continuously carrying out spray drying on the concentrated solution, controlling the inlet temperature (110-.
Further, the total glycoside content in the stevia extract in the step (1) is more than or equal to 90 wt%, wherein the stevioside accounts for 40-60 wt%, and the rebaudioside A accounts for 30-50 wt%.
Further, the cyclodextrin in the step (1) is one of alpha cyclodextrin, beta cyclodextrin and gamma cyclodextrin or any mixture thereof.
Further, the reaction temperature in the step (2) is preferably 55-65 ℃, the reaction time is preferably 24-50h, and the stirring speed is preferably 180-240 rpm/min.
Further, the macroporous adsorption resin in the step (3) is a resin product with higher selectivity for adsorbing diterpene compounds (stevioside) only or a resin product which is commercialized and can adsorb diterpene compounds (stevioside) only with higher selectivity.
The invention has the beneficial effects that:
1. the invention has higher conversion rate to the stevioside raw material, and the conversion rate of components such as stevioside, rebaudioside A and the like in the stevioside raw material can reach more than 90 percent through one-time reaction;
2. the residue of glycosyl donor in the system after the reaction is less and is below 20 percent of mass fraction;
3. the invention has the advantages of lower reaction temperature, low stirring speed, simple process and lower energy consumption, can realize concentration, recovery and cyclic utilization of the used solvent (ethanol and the like), realizes lower environmental pollution and higher economic benefit, and provides a reliable method for producing high-quality glucosyl stevioside.
Detailed Description
A preparation method of glucosyl stevioside comprises the following specific implementation steps:
example 1
(1) Accurately weighing 100g of commercial stevia extract as a raw material (preparing a first detection sample), 150g of commercial food-grade beta cyclodextrin as a glycosyl donor, adding 250g of pure water, stirring and dissolving, and then placing in a 70 ℃ water bath condition, and preheating at 200rpm/min for 1h to generate suspension;
(2) adding 7g of cyclodextrin glucoside transferase (produced by Novozymes) into the suspension, continuously reacting at 70 deg.C and stirring at 200rpm/min for 48h, continuously heating to 105 deg.C and maintaining for 60min, and inactivating and denaturing cyclodextrin glycosyl transferase (CGTase) by continuous high temperature to stop reaction;
(3) after the reaction is finished, naturally cooling the reaction solution to room temperature, carrying out decolorization treatment by using decolorizing resin (Weihua SHD-806), collecting decolorized solution, continuously desalting the decolorized solution by using anion and cation exchange resin (Dongda 001 × 8 macroporous strong acid styrene cation exchange resin; Dongda 201 × 7 macroporous strong basic styrene anion exchange resin), collecting desalted solution, removing non-diterpene compounds by using macroporous adsorption resin (Suqing DA 201-H), washing a separation column by using distilled water, desorbing the separation column by using 50% ethanol with volume fraction, and collecting ethanol desorption liquid containing stevioside;
(4) and (3) evaporating and concentrating ethanol desorption solution (12L) containing stevioside, concentrating the ethanol desorption solution to 400ml, carrying out spray drying on the concentrated solution by using a spray dryer (inlet temperature is 125 ℃), collecting 220g of dried powdery product (preparation detection sample II), and packaging to obtain the finished glucosyl glycoside product.
The liquid phase detection results of the first sample and the second sample are as follows:
by adopting the method of example 1, the RA conversion rate of the stevioside raw material is 92.18%; the stevioside conversion was 94.9%.
Example 2
(1) Accurately weighing 100g of commercial stevia extract as a raw material (preparing a first detection sample), 120g of commercial food-grade alpha cyclodextrin as a glycosyl donor, adding 250g of pure water, stirring and dissolving, and then placing in a water bath condition at 60 ℃ and preheating at 150rpm/min for 1h to generate suspension;
(2) adding 5g cyclodextrin glucoside transferase (Novozymes), reacting at 60 deg.C and stirring at 150rpm/min for 28 hr, heating to 110 deg.C and 60min, and inactivating cyclodextrin glycosyltransferase (CGTase) at high temperature to stop reaction;
(3) after the reaction is finished, naturally cooling the reaction solution to room temperature, carrying out decolorization treatment by using decolorizing resin (Weihua SHD-806), collecting decolorized solution, continuously desalting the decolorized solution by using anion and cation exchange resin (Dongda 001 × 8 macroporous strong acid styrene cation exchange resin; Dongda 201 × 7 macroporous strong basic styrene anion exchange resin), collecting desalted solution, removing non-diterpene compounds by using macroporous adsorption resin (Suqing DA 201-H), washing a separation column by using distilled water, desorbing the separation column by using 50% ethanol with volume fraction, and collecting ethanol desorption liquid containing stevioside;
(4) and (3) evaporating and concentrating ethanol desorption solution (11L) containing stevioside, concentrating the ethanol desorption solution to 350ml, carrying out spray drying on the concentrated solution by using a spray dryer (the inlet temperature is 120 ℃), collecting 200g of dried powdery product (preparation detection sample II), and packaging to obtain the finished glucosyl glycoside product.
The liquid phase detection results of the first sample and the second sample are as follows:
by adopting the method of the embodiment 2, the RA conversion rate of the stevioside raw material is 91.14%; the stevioside conversion was 95%.
Example 3
(1) Accurately weighing 100g of commercial stevia extract as a raw material (preparing a detection sample I), 75g of commercial food-grade alpha cyclodextrin and 75g of commercial food-grade beta cyclodextrin as glycosyl donors, adding 250g of pure water, stirring and dissolving, and then placing in a 70 ℃ water bath condition, preheating at 200rpm/min for 1h to generate suspension;
(2) adding 6g cyclodextrin glucoside transferase (Novozymes), placing at 70 deg.C and stirring at 150rpm/min for 48 hr, heating to 110 deg.C and 60min, and inactivating and denaturing cyclodextrin glycosyl transferase (CGTase) at high temperature to stop reaction;
(3) after the reaction is finished, naturally cooling the reaction solution to room temperature, carrying out decolorization treatment by using decolorizing resin (Weihua SHD-806), collecting decolorized solution, continuously desalting the decolorized solution by using anion and cation exchange resin (Dongda 001 × 8 macroporous strong acid styrene cation exchange resin; Dongda 201 × 7 macroporous strong basic styrene anion exchange resin), collecting desalted solution, removing non-diterpene compounds by using macroporous adsorption resin (Suqing DA 201-H), washing a separation column by using distilled water, desorbing the separation column by using 50% ethanol with volume fraction, and collecting ethanol desorption liquid containing stevioside;
(4) and (3) evaporating and concentrating ethanol desorption solution (9.2L) containing stevioside, concentrating the ethanol desorption solution to 400ml, carrying out spray drying on the concentrated solution by using a spray dryer (the inlet temperature is 120 ℃), collecting 230g of dried powdery product (a second preparation detection sample), and packaging to obtain the finished glucosyl glycoside product.
The liquid phase detection results of the first sample and the second sample are as follows:
by adopting the method of example 3, the RA conversion rate of the stevioside raw material is 93.25 percent; the stevioside conversion was 93.83%.
Example 4
(1) Accurately weighing 100g of commercial stevia extract as a raw material (preparing a first detection sample), taking 50g of commercial food-grade alpha cyclodextrin, 50g of commercial food-grade beta cyclodextrin and 50g of commercial food-grade gamma cyclodextrin as glycosyl donors, adding 250g of pure water, stirring and dissolving, and then placing in a water bath condition at 65 ℃ and preheating at 150rpm/min for 1h to generate suspension;
(2) adding 7g of cyclodextrin glucoside transferase (produced by Novozymes) into the suspension, continuously reacting at 65 deg.C and stirring at 150rpm/min for 48h, continuously heating to 110 deg.C and maintaining for 60min, and inactivating and denaturing cyclodextrin glycosyl transferase (CGTase) by continuous high temperature to stop reaction;
(3) after the reaction is finished, naturally cooling the reaction solution to room temperature, carrying out decolorization treatment by using decolorizing resin (Weihua SHD-806), collecting decolorized solution, continuously desalting the decolorized solution by using anion and cation exchange resin (Dongda 001 × 8 macroporous strong acid styrene cation exchange resin; Dongda 201 × 7 macroporous strong basic styrene anion exchange resin), collecting desalted solution, removing non-diterpene compounds by using macroporous adsorption resin (Suqing DA 201-H), washing a separation column by using distilled water, desorbing the separation column by using 50% ethanol with volume fraction, and collecting ethanol desorption liquid containing stevioside;
(4) and (3) evaporating and concentrating ethanol desorption solution (12L) containing stevioside, concentrating the ethanol desorption solution to 400ml, carrying out spray drying on the concentrated solution by using a spray dryer (the inlet temperature is 120 ℃), collecting 225g of dried powdery product (preparation detection sample II), and packaging to obtain the finished glucosyl glycoside product.
The liquid phase detection results of the first sample and the second sample are as follows:
by adopting the method of example 4, the RA conversion rate of the stevioside raw material is 93.14%; the stevioside conversion was 94.16%.
The method for measuring the total stevioside is as follows:
a detection step: detecting the content of each glycoside according to the following liquid chromatography conditions;
reagent: acetonitrile, with a detected transmittance of > 95% at 210 nm;
and (3) standard product specification:
stevioside standard substance: dry weight purity > 99%;
rebaudioside a standard: dry weight purity > 99%;
reference solution containing 9 steviol glycosides: comprising stevioside, rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside F, dulcoside A, rubusoside and steviolbioside, a standard solution of 9 Steviols was diluted with a 7:3 solution of water acetonitrile to determine the time-to-peak of each of the Steviols, which is commercially available from Wako of Japan and ChromaDex of USA.
Preparing a standard solution: accurately weighing 100 mg of stevioside standard substance and 100 mg of rebaudioside-A standard substance, respectively putting the stevioside standard substance and the 100 mg of rebaudioside-A standard substance into 100 ml volumetric flasks, and then adding a 7:3 aqueous acetonitrile solution to dissolve to 100 ml scales;
preparing a sample solution: sample one: accurately weighing 100 mg of stevioside serving as a raw material, putting the stevioside sample into a volumetric flask of 100 ml, and adding a water ethyl wax solution of 7:3 to dissolve to 100 ml of scales; sample two: accurately weighing 100 mg of reacted and processed glucosyl stevioside sample, putting the glucosyl stevioside sample into a volumetric flask of 100 ml, and adding a 7:3 aqueous ethyl wax solution to dissolve to 100 ml of scales;
a detection step:
HPLC detection of the injected sample solution was performed under the following conditions:
chromatographic column Acclaim 120C 18 chromatographic column of Agilent company, Luna5uC18 (2) 100A chromatographic column of Phenomenex company or chromatographic column with equivalent specification (length: 250 mm; inner diameter: 2.1 mm, filler granularity: 5 μm);
mobile phase: a mixture of acetonitrile and sodium dihydrogen phosphate buffer (specification: 10mmol/L, pH 2.6) in a ratio of 30: 70;
the preparation method of the sodium phosphate buffer solution comprises the following steps: dissolving 1.2 g of sodium dihydrogen phosphate, fixing the volume to 1L of water solution, and adding phosphoric acid to adjust the pH value to 2.6;
flow rate: 1 ml/min;
a detector: ultraviolet detection at 210 nm;
temperature of the column: at 40 ℃;
recording the detection profile for about 30 minutes;
peak identification and calculation:
comparing the peak output time of the sample solution with the peak output time of a reference solution containing 9 kinds of stevioside, and determining glucoside corresponding to each peak output in the sample solution; calculating peak areas corresponding to various glycosides in the sample solution, and calculating peak areas of the stevioside standard solution and the rebaudioside A standard solution;
the percentage of stevioside to rebaudioside a and the percentage of each of the 7 glycosides in addition to rebaudioside a in the sample were calculated according to the following formula:
X%= [WS/W]x [fxAx/AS]xlOO
calculating the percentage of rebaudioside a and stevioside in the sample according to the formula:
rebaudioside A or stevioside A% = [ WR/W ] x [ AX/AR ] xlOO
In the formula: x represents each glycoside;
ws is the dry weight (mg) of stevioside in a Steviol standard solution;
WR is the dry weight (mg) of rebaudioside a (stevioside) in rebaudioside a or stevioside standard solutions;
AR is the peak area of rebaudioside a (stevioside) in rebaudioside a or stevioside standard solutions;
w is the dry weight (mg) of the sample in the sample solution;
as is the peak area of the stevioside standard solution;
ax is the peak area of each glycoside of the sample solution;
fx is the ratio of molecular weight per one sugar to one stevia molecule: 1.00 (stevioside), 1.20 (rebaudioside a), 1.00 (rebaudioside B), 1.18 (rebaudioside C), 1.40 (rebaudioside D), 1.16 (rebaudioside F), 0.98 (dulcoside a abbreviated as DA), 0.80 (rubusoside abbreviated as DB), 0.80 (steviolbioside abbreviated as STV);
the contents of stevioside, RA, and total glycosides can be calculated as above.
The foregoing is merely a preferred embodiment of the invention and is not intended to limit the invention in any manner; those skilled in the art can make numerous possible variations and modifications to the present teachings, or modify equivalent embodiments to equivalent variations, without departing from the scope of the present teachings, using the methods and techniques disclosed above. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments according to the technical essence of the present invention are still within the scope of the protection of the technical solution of the present invention.
Claims (5)
1. A preparation method of glucosyl stevioside is characterized by comprising the following steps:
(1) and (2) mixing the stevia extract: the cyclodextrin is mixed according to the mass ratio of 1: 1-2, adding water to prepare a mixed solution with the mass concentration of 10% -60%, then placing the mixed solution at 40-80 ℃, and preheating for 1-3 hours under the stirring condition to generate suspension;
(2) adding cyclodextrin glycosyltransferase into the suspension, controlling the mass of the cyclodextrin glycosyltransferase to be 2-10% of that of cyclodextrin, then placing the mixture at 40-80 ℃, stirring and reacting the mixture at the rotating speed of 300rpm/min of 100-80 ℃ for 1-96h, continuously heating the mixture to more than 105 ℃ for 30-60min, and inactivating and denaturing the cyclodextrin glycosyltransferase through continuous high temperature to stop the reaction;
(3) after the reaction is finished, decoloring the reaction solution by using decoloring resin, collecting the decolored solution, desalting the decolored solution by using anion and cation exchange resin, collecting the desalted solution, removing non-diterpene compounds from the desalted solution by using macroporous adsorption resin, washing a column by using distilled water or deionized water after adsorption operation, desorbing by using ethanol with the volume fraction of 20-70%, and collecting the ethanol hydrolysis imbibition containing the diterpene compounds;
(4) evaporating and concentrating the ethanol hydrolysis liquid containing diterpene compounds, controlling the mass concentration of stevioside in the concentrated solution to be more than 60%, continuously carrying out spray drying on the concentrated solution, collecting the dried product in a powder shape, and packaging to obtain the finished glucosyl glycoside product.
2. The method of claim 1, wherein the method comprises the steps of: the total glycoside content in the stevia extract obtained in the step (1) is more than or equal to 90 wt%, wherein the stevioside accounts for 40-60 wt%, and the rebaudioside A accounts for 30-50 wt%.
3. The method of claim 1, wherein the method comprises the steps of: the cyclodextrin in the step (1) is one of alpha cyclodextrin, beta cyclodextrin and gamma cyclodextrin or any mixture thereof.
4. The method of claim 1, wherein the method comprises the steps of: the reaction temperature in the step (2) is preferably 55-65 ℃, the reaction time is preferably 24-50h, and the stirring speed is preferably 180-240 rpm/min.
5. A process according to any one of claims 1 to 4, wherein the reaction mixture comprises: the macroporous adsorption resin in the step (3) is a resin product which has higher selectivity and only adsorbs diterpene compounds or a commercialized resin product which can have higher selectivity and only adsorb diterpene compounds.
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| CN115433751A (en) * | 2022-08-18 | 2022-12-06 | 湖南绿蔓生物科技股份有限公司 | Method for improving taste quality of stevioside |
| CN115710295A (en) * | 2022-12-17 | 2023-02-24 | 安徽金禾实业股份有限公司 | Method for extracting rebaudioside-A from reaction liquid obtained by catalyzing stevioside through biological enzyme method |
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