WO2019114067A1 - Method for rapid industrial production and preparation of glucose-based stevioside mixture - Google Patents

Abstract

A method for rapid industrial production and preparation of a glucose-based stevioside mixture, the method comprising the following steps: step 1: enzymatic reaction: dissolving oxidised starch and stevioside and adding a CGT enzyme to implement an enzymatic reaction, the temperature of the enzymatic reaction being 40℃ to 50℃, and the enzymatic time being 7-12h; deactivating the enzyme activity to end the reaction; step 2: decolouring and deodorising: and step 3: filtering, concentrating, and drying to obtain the product. The oxidised starch is used as a glycosyl donor, reducing the processing steps and improving the transglycosylation effect, the process being simple and easy to operate. In addition, the demands of industrialisation on equipment and energy consumption are reduced, work time is shortened, costs are reduced, and unit production capacity is increased.

Description

Method for industrially rapidly producing and preparing glucose-based stevioside mixture Technical field

The invention belongs to the technical field of stevioside production process, and more particularly to a method for industrially rapidly producing a mixture of glucosylstearosides.

Background technique

With the promotion of stevioside, it has been found that it has a bitter aftertaste. Although stevia has many advantages, its economic benefits have been severely affected in some areas under the influence of people's taste habits formed over the years. Shock. In recent years, research on the improvement of stevia taste has become another hot issue after the research on the safety of stevioside; it is foreseeable that the establishment of a highly efficient and specific method to improve the quality of stevia will inevitably lead to the whole stevia. The sugar industry has reached a new height.

In the prior art, the enzymatic modification of stevioside using glucosyltransferase, fructofuranosidase or galactosidase has a low overall yield and a low unit productivity; at the same time, more than two grape-based products are grafted. Low, the product's sweetness improvement effect is not ideal. For example, "The study on the enzymatic synthesis of α-glucosyl-stevioside" published in the 11th issue of the Journal of Chemistry of Higher Education, 1996, using glucosyltransferase to enzymatically modify stevioside and starch solutions. The rate reached 56.1%; "Food and Fermentation Industry", No. 3, 2009, "The Production of β-Fructofuranosidase and Enzymatic Modification of Stevioside and Rebaudioside A" Using Fructofuranosidase The stevia and sucrose solutions were enzymatically modified, and the conversion rate of stevioside was 65%. The article published in the Proceedings of the Chinese Resource Biotechnology and Sugar Engineering Symposium, 2006, "β-galactosidase-modified stevia The study of sugar" enzymatically modified stevioside and lactose solutions by galactosidase method, the total yield reached 48.1%.

U.S. Patent No. 8,257,948 B2, Spectrotech Co., Ltd. uses enzymatic modification of stevioside by using α-amylase, CGTase and β-amylase in combination, and the yield is improved as follows: α-glucosyl stevioside Preparation method: adding starch to water to form a starch suspension; adding a mixture of α-amylase and CGT enzyme to the starch suspension and incubating at about 75-80 ° C for about 0.5 to 2 hours to form a liquefied starch suspension The liquid is inactivated by low-pH heat treatment, and the stevioside is added to the liquefied starch suspension to form a reaction mixture to adjust the pH to 5.5-7; the stevioside and the hatched β-amylase are added at 35-55. °C enzymatic 12-24 hours; wherein, β-amylase incubation conditions: 12 to 48 hours, 55-75 ° C; heat treatment to eliminate enzyme activity after enzymatic termination; and decolorization, macroporous adsorption resin adsorption; ethanol elution After passing through an ion exchange resin, it is concentrated and dried to obtain a finished product.

However, this technical solution has certain limitations and is not suitable for industrial production: it requires three different enzyme treatments, the steps are cumbersome, and three different enzymes are used, the enzyme dosage and cost are too high; High temperature treatment, the first time need to boil the enzyme for 5min under the condition of pH 2.8 acidity, which is more damage to the iron-containing equipment, or requires higher quality and more expensive acid-proof equipment, and the temperature control system There is also a higher requirement. The high temperature inactivation of the enzyme requires more energy and increases carbon emissions; the third enzyme treatment consumes a longer total working time, and the three enzyme treatment times exceed 37 hours in total, and the efficiency is low. Due to the three enzyme treatments, more impurities are introduced, the grafting effect is not ideal, and more starch-enzyme products remain. The macroporous resin is removed, the ion exchange resin is desalted, and the working hours are further extended. The cost.

Summary of the invention

In order to overcome the deficiencies of the prior art, the present invention provides a method for industrially rapid production of a mixture of glucosylstearosides. The method does not use starch as a donor of glucose residues, uses oxidized starch as a donor of glucose residues; reduces the type of enzyme and the number of treatments, and uses only CGT enzyme for one enzyme transglycosylation treatment; optimizes the reaction of enzyme treatment Conditions, shortened working hours, reduced equipment requirements, reduced costs, and increased unit capacity.

The present inventors have found that starch has poor solubility in an aqueous solution, insufficiently dissolved starch, and the starch chain is not sufficiently opened by amylase and CGTase, resulting in a limited amount of hydrolyzed glucose residues, thereby transglycosylation effect. Poor, low conversion rate. Without being treated with amylase, it has been shown to have good water solubility with a slight heating and high solubility. Oxidized starch is added to the α-amylase after starch gelatinization, and the α-1,4 glucosidic bond in the starch is randomly cut to obtain dextrin and oligosaccharide, thereby reducing the viscosity of the solution and increasing the solubility of the starch. Monosaccharide disaccharides such as glucose and maltose are not suitable as glyco donors, and oxidized starch is a superior glycosyl donor, and cost-effective, and inexpensive oxidized starch is selected as the glycosyl donor of the present invention.

The object of the present invention can be achieved by the following technical solutions:

The invention provides a method for industrially and rapidly producing a mixture of glucosylstevioside, which comprises the following steps:

Step one, enzymatic reaction: dissolving oxidized starch and stevioside, adding CGT enzyme for enzymatic reaction, the enzymatic reaction temperature is 40 ° C ~ 55 ° C, the enzymatic time is 7 ~ 12 h, the end of the enzyme activity;

Step two, bleaching and deodorizing;

Step three, filtering, concentrating, and drying to obtain a product.

When the CGT enzyme is used to catalyze the modification of industrial stevioside with different glycosyl donors, the conversion rate of oxidized starch as a glycosyl donor stevioside is as high as 93%, and the conversion rate of other starches as a glycosyl donor It is poor, only about 40% to 60% conversion rate; dextrin products as a glycosyl donor is better than starch, the conversion rate of stevioside is significantly improved to a grade of 70% to 85% The cyclodextrin effect is the best in dextrin-based glycosyl donors, but there is a significant difference compared to oxidized starch.

Considering thermodynamic factors, the temperature is too low, the CGT enzyme activity is low, more CGT enzyme needs to be added, and the cost is too high. In the optimization of the process of the present invention, it is found that the CGT enzyme is used in the stevia application when the temperature is above 40 ° C. Will be activated; while considering thermal stability, when the temperature is too high, CGT enzyme will be inactivated due to protein denaturation, although the CGT enzyme can withstand high temperatures, the supplier Novozymes recommended the reaction temperature is 85 ° C, but this The inventors found in the process of process optimization: CGT enzyme in the field of stevia application, the lower temperature is beneficial to the graft glycosylation of stevioside, when the temperature exceeds 55 °C, the glycosylation reaction of stevioside This is another important finding of the present invention.

Preferably, the dissolved solvent of the first step is tap water or purified water. The inventors have found that when the purified water is selected as the reaction medium, less impurity ions are introduced, which is favorable for the subsequent reaction; the amount is 5~ of the total mass of the oxidized starch and the stevioside 20 times; by controlling the amount of oxidized starch, the oxidized starch is fully dissolved. If the oxidized starch is not sufficiently dissolved, the starch chain is not opened by the CGT enzyme, resulting in a limited amount of hydrolyzed glucose residues, resulting in poor transglycosylation and conversion. low.

Preferably, the dissolution temperature of the first step is 55 ° C to 100 ° C; the inventors have found that the microorganisms are easily fermented in summer, affecting the reaction, and the microorganisms are more likely to multiply in the summer, causing the fermentation of the raw materials and the solution, affecting the enzymatic reaction, so the summer and autumn seasons , boiled to 100 ° C after dissolution, can better kill microorganisms.

Preferably, the mass ratio of the oxidized starch to the stevioside in the first step is from 1 to 1.5:1; when the substrate mass ratio is increased, that is, as the amount of the glycosyl donor is increased, the conversion rate is increased, and the yield is also increased. Considering the raw material utilization rate and subsequent separation, the mass ratio of oxidized starch to stevioside is 1 to 1.5:1, which is the optimal substrate ratio range. In this range, the conversion rate and the initial reaction rate increase with the increase of the enzyme amount. A significant increase. In order to fully dissolve it, the oxidized starch can be added at a constant rate under stirring conditions, and after being sufficiently dissolved, the stevioside is uniformly added to the dissolved oxidized starch solution under stirring to obtain a uniformly dissolved oxidized starch and stevioside mixture. .

Preferably, the amount of the CGT enzyme is 5% to 15% of the mass of the stevioside; when the amount of the enzyme is more than 15%, the yield is not increased significantly, taking into account the cost of the enzyme, and selecting 5% to 15% of stevioside The mass is the optimum amount of enzyme added.

Preferably, the step-enzymatic reaction has a pH of 3.5 to 5.

Preferably, the step 1 inactivation temperature is from 95 ° C to 100 ° C, and the time is from 10 to 20 min;

Preferably, the step two is decolorized and deodorized using activated carbon;

Preferably, the amount of the activated carbon in the step 2 is 0.03 to 0.15 times the weight of the stevioside.

Preferably, the temperature of the decolorization and deodorization in the second step is 85 to 100 ° C, and the time is 20 to 40 min.

Preferably, the third step is concentrated to concentration under reduced pressure, the temperature is 70-80 ° C, and the degree of vacuum is -0.07--0.09 MPa.

Preferably, the third step of drying is vacuum drying, the temperature is 70-80 ° C, and the degree of vacuum is -0.07--0.09 MPa.

Compared with the prior art, the method of the invention has the following advantages:

1. Commercialized oxidized starch as a glycosyl donor can reduce process steps compared to using starch as a glycosyl donor, which is more time-saving, time-saving, high-efficiency, good glycosylation effect, and lower cost.

2. Only a single CGT enzyme is used for the transglycosylation reaction, which eliminates a series of steps such as amylase treatment of starch, saves the cost of the enzyme, and also eliminates the process steps of high temperature inactivation of amylase at low pH, and the process is simple. Easy to operate.

3. The optimized enzymatic reaction conditions are mild, the transglycosylation of stevioside reaches 85%-95%, the conversion rate is high, and the time is shortened to less than 12 hours.

4. Since the steps are simple, no other impurities such as amylase are introduced, and the dextrin which is not fully utilized after hydrolyzing the starch is omitted, and the macroporous resin and the ion exchange resin are omitted. Process steps such as impurities and ionic ash. Thereby further reducing production costs and improving efficiency.

Detailed ways

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

Example 1

(1) 1000 ml of tap water is heated to 55 ° C, and 100 g of oxidized starch is uniformly added under stirring to make it dissolve completely in a transparent liquid; and then 100 g of 90% stevioside is uniformly added to the transparent liquid under stirring to form A homogeneous clear mixture of oxidized starch and stevioside.

(2) The above uniformly dissolved oxidized starch and stevioside mixture were cooled to 40 °C.

(3) To the above-mentioned uniformly mixed oxidized starch and stevioside mixture, 5 g of CGTase (manufactured by Novozymes) was added, and the pH was adjusted to 3.5 with hydrochloric acid, and the glycosylation reaction was carried out for 7 hours under stirring.

(4) The above glycosylation reaction product was heated to 95 ° C for 10 minutes to extinguish the CGT enzyme activity, and the glycosylation reaction was terminated.

(5) Adding 3 g of activated carbon powder, decolorizing and deodorizing at 85 ° C for 20 minutes; removing activated carbon powder and CGT enzyme by filtration.

(6) The filtrate is concentrated under reduced pressure to obtain a concentrated liquid. The concentration under reduced pressure is 70 ° C, and the degree of vacuum is -0.07 MPa; the concentrated liquid is vacuum dried, the temperature is 70 ° C, and the degree of vacuum is -0.07 MPa; finally, the finished product is finally obtained. The product was 184.24 g, the stevioside content was 6.16%, and the stevioside conversion rate was 1-(184.24 x 6.16%) / (100 x 90%) = 87.4%.

Example 2

(1) heating 2000 ml of tap water to 100 ° C, adding 150 g of oxidized starch at a constant rate under stirring to fully dissolve the transparent liquid; and then adding 100 g of 95% stevioside at a constant rate to the transparent liquid to form A homogeneous clear mixture of oxidized starch and stevioside.

(2) The above uniformly dissolved oxidized starch and stevioside mixture were cooled to 55 °C.

(3) To the above-mentioned uniformly mixed oxidized starch and stevioside mixture, 15 g of CGTase (manufactured by Novozymes) was added, and the pH was adjusted to 5 with hydrochloric acid, and the glycosylation reaction was carried out for 12 hours while stirring.

(4) The above glycosylation reaction product was heated to 100 ° C for 20 minutes to extinguish the CGT enzyme activity, and the glycosylation reaction was terminated.

(5) 15 g of activated carbon powder was added, and the mixture was dehydrated and deodorized by being kept at 100 ° C for 40 minutes; the activated carbon powder and the CGT enzyme were removed by filtration.

(6) The filtrate is concentrated under reduced pressure to obtain a concentrated liquid, and the concentration under reduced pressure is a temperature of 80 ° C, a vacuum degree of -0.09 MPa; the concentrated liquid is vacuum dried, the temperature is 80 ° C, the degree of vacuum is -0.09 MPa; the final product is 227.36 g. The stevioside content was 6.18%, and the stevioside conversion rate was 1-(227.36×6.18%)/(100×95%)=85.2%.

Example 3

(1) 1600 ml of purified water is heated to 60 ° C, and 130 g of oxidized starch is added at a constant rate under stirring to fully dissolve the transparent liquid; and then 100 g of 95% stevioside is uniformly added to the transparent liquid under stirring. A uniform clear mixture of oxidized starch and stevioside is formed.

(2) The above uniformly dissolved oxidized starch and stevioside mixture were cooled to 45 °C.

(3) To the above-mentioned uniformly mixed oxidized starch and stevioside mixture, 10 g of CGTase (manufactured by Novozymes) was added, and the pH was adjusted to 4 with hydrochloric acid, and the glycosylation reaction was carried out for 11 hours while stirring.

(4) The above glycosylation reaction product was heated to 98 ° C for 13 minutes to extinguish the CGT enzyme activity, and the glycosylation reaction was terminated.

(5) Adding 5 g of activated carbon powder, decolorizing and deodorizing at 90 ° C for 30 minutes; removing activated carbon powder and CGT enzyme by filtration.

(6) The filtrate is concentrated under reduced pressure to obtain a concentrated liquid, and the concentration under reduced pressure is a temperature of 75 ° C, a vacuum degree of -0.08 MPa; the concentrated liquid is vacuum dried, the temperature is 75 ° C, the degree of vacuum is -0.08 MPa; the final product is 210.11 g. The stevioside content was 4.25%, and the stevioside conversion rate was 1-(210.11×4.25%)/(100×95%)=90.6%.

Example 4

(1) heating 2000 ml of purified water to 65 ° C, adding 120 g of oxidized starch at a constant rate under stirring to fully dissolve the transparent liquid; and then uniformly adding 100 g of 95% stevioside to the transparent liquid under stirring. A uniform clear mixture of oxidized starch and stevioside is formed.

(2) The above uniformly dissolved oxidized starch and stevioside mixture were cooled to 50 °C.

(3) To the above-mentioned uniformly mixed oxidized starch and stevioside mixture, 12 g of CGTase (manufactured by Novozymes) was added, and the pH was adjusted to 4.5 with hydrochloric acid, and the glycosylation reaction was carried out for 9 hours under stirring.

(4) The above glycosylation reaction product was heated to 96 ° C for 15 minutes to extinguish the CGT enzyme activity, and the glycosylation reaction was terminated.

(5) Adding 8 g of activated carbon powder, decolorizing and deodorizing at 90 ° C for 35 minutes; removing activated carbon powder and CGT enzyme by filtration.

(6) The filtrate is concentrated under reduced pressure to obtain a concentrated liquid, and the concentration under reduced pressure is a temperature of 72 ° C, a vacuum degree of -0.08 MPa; the concentrated liquid is vacuum dried, the temperature is 72 ° C, the degree of vacuum is -0.08 MPa; finally, the finished product is 201.49 g. , the content of stevioside was 3.12%, and the conversion rate of stevioside was 1-(201.49×3.12%)/(100×95%)=93.4%

Example 5

(1) 1500 ml of purified water is heated to 78 ° C, and 110 g of oxidized starch is uniformly added under stirring to fully dissolve the transparent liquid; and then 100 g of 95% stevioside is uniformly added to the transparent liquid under stirring. A uniform clear mixture of oxidized starch and stevioside is formed.

(2) The above uniformly dissolved oxidized starch and stevioside mixture were cooled to 48 °C.

(3) 6 g of CGTase (manufactured by Novozymes) was added to the above-mentioned mixed oxidized starch and stevioside mixture, and the pH was adjusted to 4.3 with hydrochloric acid, and the glycosylation reaction was carried out for 10 hours under stirring.

(4) The above glycosylation reaction product was heated to 100 ° C for 18 minutes to extinguish the CGT enzyme activity, and the glycosylation reaction was terminated.

(5) Add 8 g of activated carbon powder, decolorize and deodorize at 98 ° C for 25 minutes; remove activated carbon powder and CGT enzyme by filtration.

(6) The filtrate is concentrated under reduced pressure to obtain a concentrated liquid, and the concentration under reduced pressure is a temperature of 76 ° C, a vacuum degree of -0.09 MPa; the concentrated liquid is vacuum dried, the temperature is 72 ° C, the degree of vacuum is -0.07 MPa; finally, the finished product is 192.86 g. The stevioside content was 4.83%, and the stevioside conversion rate was 1-(192.86×4.83%)/(100×95%)=90.2%.

Example 6

(1) 1400 ml of purified water is heated to 95 ° C, and 145 g of oxidized starch is added at a constant rate under stirring to fully dissolve the transparent liquid; and then 100 g of 95% stevioside is uniformly added to the transparent liquid under stirring. A uniform mixture of oxidized starch and stevioside is formed.

(2) The above uniformly dissolved oxidized starch and stevioside mixture were cooled to 52 °C.

(3) To the above-mentioned mixed oxidized starch and stevioside mixture, 8 g of CGTase (manufactured by Novozymes) was added, and the pH was adjusted to 4.8 with hydrochloric acid, and the glycosylation reaction was carried out for 11 hours while stirring.

(4) The above glycosylation reaction product was heated to 96 ° C for 12 minutes to extinguish the CGT enzyme activity, and the glycosylation reaction was terminated.

(5) 13 g of activated carbon powder was added, and the mixture was dehydrated and deodorized at 88 ° C for 36 minutes; the activated carbon powder and the CGT enzyme were removed by filtration.

(6) The filtrate is concentrated under reduced pressure to obtain a concentrated liquid. The concentration under reduced pressure is 78 ° C, and the degree of vacuum is -0.08 MPa; the concentrated liquid is vacuum dried, the temperature is 73 ° C, the degree of vacuum is -0.09 MPa; the final product is 223.05 g. The stevioside content was 5.41%, and the stevioside conversion rate was 1-(223.05×5.41%)/(100×95%)=87.3%.

Comparative Example 1-2 was used to evaluate the difference in technical effect obtained by the technical solution of the present invention from the technical solution of the use of starch as a donor of glucose residues in US Pat. No. 8,257,948 B2.

Comparative example 1

(1) Suspending 100g of tapioca starch in 300mL of water, pH 6.5

(2) adding 2 g of α-amylase and 5 g of CGTase (produced by Novozymes), and performing starch liquefaction at 80 ° C for about one hour;

(3) The pH of the reaction mixture was adjusted to pH 2.8 by hydrochloric acid, and the mixture was boiled at 100 ° C over 5 minutes to inactivate the enzyme.

(4) After cooling to 65 ° C, the pH was adjusted to pH 6.0 with a sodium hydroxide solution, and 100 g of 95% stevioside passed was added to the liquefied starch and stirred until a homogeneous solution was obtained.

(5) 6 g of CGTase was added to the solution, and the mixture was kept at a temperature of 65 ° C for 24 hours with continuous stirring.

(6) The temperature was then lowered to 45 ° C and 8 g of β-amylase was added to the reaction mixture. The reaction was continued for another 12 hours.

(7) The obtained reaction mixture was heated at 95 ° C for 15 minutes to inactivate the enzyme.

(8) 20 g of activated carbon was added, and the mixture was heated to 75 ° C for 30 minutes.

(9) The mixture was filtered and the filtrate was diluted with water to a 5% solid content, and passed through a column each filled with 4000 mL of Amberlite XAD 7HP macroporous adsorption resin. The column was washed with 5 volumes of water and 2 volumes of 20% (v/v) ethanol, and the adsorbed glycoside was eluted with 50% ethanol.

(10) The obtained eluate was passed through a column packed with Amberlite FPC23 (H ⁺ ) and Amberlite FPA51 (OH ^- ) ion exchange resin.

(11) Ethanol was evaporated, and the desalted and decolored aqueous solution was concentrated in vacuo at 72 ° C, and then dried into a powder form using a laboratory spray drier to obtain 151 g of a product having a stevioside content of 9.69% and a stevioside conversion ratio of: 1-(151 x 9.69%) / (100 x 95%) = 84.6%.

Comparative example 2

(1) 100 g of tapioca starch was suspended in 300 mL of water (pH 6.5).

(2) 2 g of α-amylase and 6 g of CGTase (manufactured by Novozymes) were added, and starch liquefaction was carried out at 80 ° C for about 1.5 hours.

(3) The pH of the reaction mixture was adjusted to pH 2.8 by hydrochloric acid, and the mixture was boiled at 100 ° C over 5 minutes to inactivate the enzyme.

(4) After cooling to 65 ° C, the pH was adjusted to pH 6.0 with a sodium hydroxide solution. 100 g of 95% stevioside extract was added to the liquefied starch and stirred until a homogeneous solution was obtained.

(5) 7 g of CGTase was added to the solution, and the mixture was kept at a temperature of 65 ° C for 24 hours with continuous stirring.

(6) The temperature was then lowered to 45 ° C and 12 g of β-amylase was added to the reaction mixture. The reaction was continued for another 12 hours.

(7) The obtained reaction mixture was heated at 95 ° C for 15 minutes to inactivate the enzyme.

(8) 20 g of activated carbon was added, and the mixture was heated to 75 ° C for 30 minutes.

(9) The mixture was filtered and the filtrate was diluted with water to a 5% solid content, and passed through a column packed with Amberlite FPC23 (H ⁺ ) and Amberlite FPA51 (OH ^- ) ion exchange resin.

(12) The desalted solution was concentrated in vacuo at 72 ° C and dried to a powder form using a laboratory spray drier to obtain 166 g of product having a stevioside content of 7.55% and a stevioside conversion rate of 1- (166 × 9.69). %) / (100 x 95%) = 86.8%.

Table 1 Comparison of various process requirements and technical effects

Comparative examples 1, 2 need to be treated with 3 different enzymes, the steps are cumbersome, and 3 different enzymes are used, the amount of enzyme is large and the cost is too high; in which two enzymes are inactivated, high temperature treatment is needed, the first time needs to be Under the condition of pH 2.8 acidity, the enzyme is burned for 5 minutes at high temperature, which is harmful to the iron-containing equipment, or requires higher quality and higher price acid-resistant equipment, and has higher requirements for the temperature control system. Enzyme activity requires more energy and increases carbon emissions; 3 times of enzyme treatment, the total working time is longer, and the 3 times of enzyme treatment time exceeds 37 hours in total, and the efficiency is low. Due to the three enzyme treatments, more impurities are introduced, the grafting effect is not ideal, and more starch-enzyme products remain. The macroporous resin is removed, the ion exchange resin is desalted, and the working hours are further extended. The cost. In Comparative Examples 1 and 2, there was also a low conversion rate, and the conversion yield did not exceed 90%, and the unit productivity was low.

Effects of different glycosyl donors on enzyme-modified stevioside products

Comparative example 3-8

Comparative Examples 3 to 8 were used to evaluate the effect of non-oxidized starch as a glycosyl donor on the enzyme-modified stevioside product, using corn starch, sweet potato starch, tapioca starch, maltodextrin, dextrin, cyclodextrin and other 6 non- The technical solution of oxidized starch as a donor of glucose residues differs from the technical effect obtained by the technical solution of the present invention. Comparative Examples 3 to 8 respectively replaced corn starch as a glycosyl donor with corn starch, sweet potato starch, tapioca starch, maltodextrin, dextrin and cyclodextrin. The other steps were the same as those in Example 4. The contents of the obtained products were as follows:

Table 2 Effect of different glycosyl donors on the grafting conversion rate of stevioside

Glycosyl donor	Conversion rate(%)	Total glycosides (%)	RD (%)	RA (%)	STV (%)	RF (%)	RC (%)	DA (%)	Sweet glycosides (%)	RB (%)	SB (%)
corn starch	55.53	23.29	0.44	10.26	9.35	0.39	1.76	0.15	0.42	0.32	0.20
Sweet potato starch	51.86	25.21	0.45	11.08	10.12	0.45	1.89	0.18	0.39	0.33	0.32
Cassava starch	46.36	28.09	0.50	12.75	10.95	0.45	1.92	0.15	0.62	0.38	0.37
Maltodextrin	69.75	15.84	0.32	6.81	5.72	0.34	1.51	0.11	0.51	0.23	0.29
dextrin	74.32	13.45	0.19	6.22	4.86	0.25	1.13	0.10	0.31	0.18	0.21
Cyclodextrin	84.53	8.10	0.11	3.46	2.89	0.18	0.97	0.08	0.14	0.11	0.16
Oxidized starch	93.39	3.46	-	1.56	1.50	-	0.40	-	-	-	-

From the above table, the following conclusions can be drawn: the difference in graft saccharification of stevioside by each glycosyl donor is very obvious, and the effect of oxidized starch is the best.

From the index of conversion rate, when oxidized starch is used as a glycosyl donor, the conversion rate of stevioside is as high as 93% or more, while the conversion rate of other starches as a glycosyl donor is poor, only about 40%~ 60% conversion rate; the effect of dextrin products as a glycosyl donor is better than that of starch. The conversion rate of stevioside is obviously increased to a level of 70% to 85%, and the effect of cyclodextrin is amylin. Sugar-based donors work best, but there is a significant difference compared to oxidized starch.

From the content of total glucosides and steviosides in the glycosylation products that are not involved in the reaction, the total glycosides remaining in the starch as a glycosyl donor are higher, and the total glycosides remaining in the product are about 20% to 30%. It indicates that the starch substances are not fully involved in the reaction, which may be related to their own molecular size and solubility in water. Because of the large molecular weight of starchy substances, the solubility in water is not good enough, and it is not fully developed for CGT enzyme hydrolysis and transglycosylation. Grafting affects the working efficiency of CGT enzyme; dextrin products are processed by further processing of starch products, the molecular weight is smaller than starch, and the water solubility is also greatly improved, so when it is used as a glycosyl donor CGT enzyme can hydrolyze more glucose residues more easily and faster, and carry out graft glycosylation reaction, thereby increasing the conversion rate of stevioside, and the residual stevioside total glycosides not involved in the reaction are greatly reduced. Total glycosides remain 8% to 16%, especially when cyclodextrin is used as a glycosyl donor, but the overall effect is not as good as oxidized starch, oxidized starch as sugar. The total glycoside residue in the product can be less than 4%, and only a small amount of RA, RC and STV are the three steviosides. The bitterness and astringency are completely removed, the taste is relatively pure, close to sucrose, unlike other glycosyl groups. The product obtained from the body has more ingredients and is more heterozygous, with bitter and grassy taste.

Compared with corn starch, sweet potato starch, tapioca starch, maltodextrin, dextrin, cyclodextrin and other six non-oxidized starches as donors of glucose residues, the conversion rate of oxidized starch is high, and the residual unreacted total glycosides are low. The residual unreacted excipient has low content, good taste and thorough removal of bitterness.

Comparative Example 9 to 12

Comparative Examples 9 to 12 were used to evaluate the difference in technical effects between the technical solutions using the other four modified starches as donors of glucose residues and the technical solution of the present invention. In Comparative Examples 9 to 12, the oxidized starch was replaced by acid-decomposed starch, esterified starch, cross-linked starch, and etherified starch, respectively, and the other procedures were the same as in Example 4.

[Correct according to Rule 26 27.02.2018]

[Correct according to Rule 26 27.02.2018]
As shown in the above table, four of the modified starches were able to detect the formation of a transglucoside product. Acid-decomposed starch, esterified starch, cross-linked starch, and etherified starch were used as glycosyl donors for subsequent experiments. Compared with other modified starches as donors of glucose residues (Comparative Examples 9, 10, 11, 12), the conversion rate of stevioside was higher in the enzymatic reaction system of oxidized starch and stevioside, about 9- Twice of 12, indicating that the technical effect of the technical solution of the present invention using oxidized starch as a donor of glucose residues is superior to that of four modified starches such as other acid-decomposed starches.

In the present invention, the oxidized starch is used as the glycosyl donor, and the conversion rate of St reaches 93.4%, and the mono-, di-, and poly-substituted transglycosylation products are obtained; and the CGTase is used to catalyze the reaction of stevioside and oxidized starch to obtain a single substitution. There are nine kinds of di- and tri-substituted transglycosyl products, and the taste of the mono- and di-substituted transglycosylation products is greatly improved, while the other four modified starch transglycosylation products are relatively more. less. At the same time, in the catalytic reaction of St-RA mixture with 4 different starches, it was found that when oxidized starch was used as glycosyl donor, CGTase had higher transglucosidic activity (higher for both St and RA), while high substitution products were also significantly higher. For other modified starches. When using acid-decomposed starch as a glycosyl donor, it was found that the disproportionation activity of CGTase was stronger than that of oxidized starch, but the conversion rate of stevioside modification was about 60%, which was lower than that of oxidized starch. The reason may be the difference in the length and length of the sugar-based donor, the difference in molecular weight, the balance of hydrolysis and disproportionation after substrate binding, and finally the difference in the conversion rate of stevioside.

Effects of different enzymes on enzyme-modified stevioside products

Comparative Examples 13 to 15 were used to evaluate the difference in technical effects obtained by the technical solution of enzymatic glycosylation using other enzymes and the technical solution of the present invention. In Comparative Example 7-9, GGTase was replaced by glucosyltransferase, fructofuranosidase and galactosidase. The temperature and pH of each reaction system were designed according to the optimum temperature and pH value of each enzyme. Example 4.

Comparative example 13

(1) heating 2000 ml of purified water to 58 ° C, adding 120 g of oxidized starch at a constant rate under stirring to fully dissolve the transparent liquid; and then uniformly adding 100 g of 95% stevioside to the transparent liquid under stirring to form A homogeneous clear mixture of oxidized starch and stevioside.

(2) The above uniformly dissolved oxidized starch and stevioside mixture were cooled to 56 °C.

(3) To the above-mentioned homogeneously mixed oxidized starch and stevioside mixture, 15 g of glucosidase (manufactured by Novozymes) was added, and the pH was adjusted to 6.5 with a buffer, and the glycosylation reaction was carried out for 12 hours under stirring.

(4) The above glycosylation reaction product was heated to 100 ° C for 10 minutes to kill the glucosidase enzyme activity, and the glycosylation reaction was terminated.

(5) 40 mL of ethanol was added, and after shaking for 5 minutes, it was allowed to stand for 2 hours, and the insoluble residual starch, dextrin, and the like were removed by filtration.

(6) Add 8 g of activated carbon powder, decolorize and deodorize at 90 ° C for 35 minutes; remove activated carbon powder and glucosidase by filtration.

(7) The filtrate is concentrated under reduced pressure to obtain a concentrated liquid, and the concentration under reduced pressure is a temperature of 72 ° C, a vacuum degree of -0.08 MPa; the concentrated liquid is vacuum dried, the temperature is 72 ° C, and the degree of vacuum is -0.08 MPa; The obtained dried product was dried to 200.69 g, the stevioside content was 31.62%, and the stevioside conversion rate was 1- (200.69 × 31.62%) / (100 × 95%) = 33.2%.

Comparative example 14

(1) heating 2000 ml of purified water to 58 ° C, adding 120 g of oxidized starch at a constant rate under stirring to fully dissolve the transparent liquid; and then uniformly adding 100 g of 95% stevioside to the transparent liquid under stirring to form A homogeneous clear mixture of oxidized starch and stevioside.

(2) The above uniformly dissolved oxidized starch and stevioside mixture were cooled to 40 °C.

(3) Adding 15 g of fructofuranosidase (produced by Novozymes) to the above uniformly dissolved oxidized starch and stevioside mixture, adjusting the pH value of 7 with a phosphate buffer solution, and performing glycosylation reaction under stirring. hour.

(4) The above glycosylation reaction product was heated to 100 ° C for 10 minutes, and the fructofuranosidase enzyme activity was completed to terminate the glycosylation reaction.

(5) Add 8 g of activated carbon powder, decolorize and deodorize after being kept at 90 ° C for 35 minutes; remove the activated carbon powder and CGT enzyme after passing through the microporous membrane.

(6) The filtrate is concentrated under reduced pressure to obtain a concentrated liquid, and the concentration under reduced pressure is a temperature of 72 ° C, a vacuum degree of -0.08 MPa; the concentrated liquid is vacuum dried, the temperature is 72 ° C, and the degree of vacuum is -0.08 MPa; The dried product obtained by drying was 199.52 g, the stevioside content was 17.38%, and the stevioside conversion rate was 1-(199.52×17.38%)/(100×95%)=63.5%.

Comparative example 15

(1) heating 2000 ml of purified water to 58 ° C, adding 120 g of oxidized starch at a constant rate under stirring to fully dissolve the transparent liquid; and then uniformly adding 100 g of 95% stevioside to the transparent liquid under stirring to form A homogeneous clear mixture of oxidized starch and stevioside.

(2) The above uniformly dissolved oxidized starch and stevioside mixture were cooled to 50 °C.

(3) 20 g of galactosidase (produced by Novozymes) was added to the above-mentioned mixed oxidized starch and stevioside mixture, and the pH was adjusted to 6.4 with hydrochloric acid, and the glycosylation reaction was carried out for 48 hours under stirring.

(4) The above glycosylation reaction product was heated to 100 ° C for 10 minutes, and the galactosidase enzyme activity was terminated to terminate the glycosylation reaction.

(5) 8 g of activated carbon powder was added, and the mixture was dehydrated and deodorized at 90 ° C for 35 minutes; the activated carbon powder and galactosidase were removed by filtration.

(6) The filtrate is concentrated under reduced pressure to obtain a concentrated liquid, and the concentration under reduced pressure is a temperature of 72 ° C, a vacuum degree of -0.08 MPa; the concentrated liquid is vacuum dried, the temperature is 72 ° C, and the degree of vacuum is -0.08 MPa; finally, the finished product is finally obtained. The dried product obtained by concentration drying was 200.43 g, the stevioside content was 35.79%, and the stevioside conversion rate was 1- (200.43 × 35.79%) / (100 × 95%) = 24.5%.

[Correct according to Rule 26 27.02.2018]

[Correct according to Rule 26 27.02.2018]
As can be seen from the above table, in comparison with the other enzymes: glucosyltransferase, fructofuranosidase, galactosidase, enzymatic glycosylation, the technical solution of the present invention using GGT enzyme is in the glycosylation reaction. GGT enzyme has the highest transglucosidic activity in the glycosyltransferase used in the experiment, the content of untransformed stevioside is 3.12%, and the content of untransformed stevioside in galactosidase is 35.79%, so the stevioside of GGTase acts. The conversion rate was significantly higher than that of the comparative examples 7 to 9; since the enzyme reaction system in the comparative example is the optimal reaction condition of each enzyme, the interference of other factors has been excluded, indicating that the technical scheme of the present invention adopts GGT enzyme glycosylation/ligation The branching effect is superior to the glycosylation/grafting effect of other enzymes such as glucosyltransferase.

The technical solution of the present invention is superior to the prior art such as Comparative Document 1-4 in improving the conversion effect of stevioside. At the same time, the requirements for industrialization on equipment and energy consumption are reduced, working hours are reduced, costs are reduced, and unit capacity is increased.

Although the present invention has been described in detail above with the aid of the general description, the specific embodiments and the examples of the invention, it may be obvious to those skilled in the art . Therefore, such modifications or improvements made without departing from the spirit of the invention are intended to be within the scope of the invention.

Claims (12)

1. A method for industrially rapidly producing a mixture of glucosylsteviosides, comprising the steps of:

   Step one, enzymatic reaction: dissolving oxidized starch and stevioside, adding CGT enzyme for enzymatic reaction, the enzymatic reaction temperature is 40 ° C ~ 55 ° C, the enzymatic time is 7 ~ 12 h, the end of the enzyme activity;

   Step two, bleaching and deodorizing;

   Step three, filtering, concentrating, and drying to obtain a product.
2. The method for industrially rapidly producing a mixture of glucosylstevioside according to claim 1, wherein the solvent of the first step is tap water or purified water.
3. The method for industrially rapidly producing a mixture of glucosylstevioside according to claim 1, wherein the first step has a dissolution temperature of 55 ° C to 100 ° C.
4. The method according to claim 1, wherein the mass ratio of the oxidized starch to the stevioside in the first step is from 1 to 1.5:1.
5. The method for industrially rapidly producing a mixture of glucosylstevioside according to claim 1, wherein the step C-C enzyme is used in an amount of 5% to 15% by mass of the stevioside.
6. A method for industrially rapidly producing a mixture of glucosylsteviosides according to claim 1, wherein the step-enzymatic reaction has a pH of 3.5 to 5.
7. The method for industrially rapidly producing a mixture of glucose-based stevioside according to claim 1, wherein the step 1 is inactivated at a temperature of from 95 ° C to 100 ° C for 10 to 20 minutes.
8. A method of industrially rapidly producing a mixture of glucosylsteviosides according to claim 1, characterized in that the step two is subjected to decolorization and deodorization using activated carbon.
9. A method for industrially rapidly producing a mixture of glucosylstevioside according to claim 8, wherein the amount of activated carbon in the second step is from 0.03 to 0.15 times the weight of stevioside.
10. The method for industrially rapidly producing a mixture of glucosylstevioside according to claim 8, wherein the temperature of the decolorization and deodorization in the step 2 is 85 to 100 ° C and the time is 20 to 40 min.
11. The method for industrially rapidly producing a mixture of glucose-based stevioside according to claim 1, wherein the step three is concentrated to a concentration under reduced pressure, a temperature of 70 to 80 ° C, and a degree of vacuum of -0.07 to -0.09 MPa.
12. The method for industrially rapidly producing and preparing a mixture of glucose-based stevioside according to claim 1, wherein the step three is vacuum drying, the temperature is 70-80 ° C, and the degree of vacuum is -0.07 - -0.09 MPa. .