High-throughput enzymatic preparation of glucosylated steviol glycosides under programming temperatures

Abstract

The present invention discloses a method for preparing glucosyl steviol glycosides through enzymatic catalysis under programming temperatures in high throughput, belonging to the technical field of biosynthesis of sweeteners. By using cyclodextrin glucosyltransferase from Geobacillus sp. as a catalyst, steviol glycosides as the glycosyl receptor and dextrin or oligosaccharide as the glycosyl donor, taking a calcium/barium ion salt bridge as the main stabilizer and combining with glycerol to adjust the conformation and binding domain openness of the enzyme, and utilizing transglucosylation and hydrolytic activities of amylase at variable temperatures in different stages, thereby preparing the glucosyl steviol glycosides through enzymatic catalysis under programming temperatures in high throughput. The technology of the present invention can improve the utilization rate of the enzyme, and obtain glucosyl steviol glycosides with good sweetness and good taste.

Claims

1. A method for high throughput synthesis and production of glucosyl steviol glycosides which comprises: incubating cyclodextrin glucosyltransferase, steviol glycosides, dextrin or oligosaccharide, salt and polyol in an aqueous reaction mixture using the following steps: (a) dissolving the steviol glycosides and the dextrin or the oligosaccharide in water or a buffer solution at 80 to 85° C., (b) cooling to 60 to 65° C., (c) adding the salt and the polyol, and 10 to 100 U/g steviol glycoside of the cyclodextrin glucosyltransferase, (d) maintaining the temperature at 60 to 65° C. for 0.5 to 5 hours, (e) raising the temperature to 70 to 76° C. for 8 to 24 hours, and then (f) raising the temperature further to 75 to 85° C. for 8 to 24 hours until a change in the content of steviol glycosides in the reaction mixture is less than 0.1% per hour; wherein the steviol glycosides are rebaudioside A.

2. The method according to claim 1, wherein the cyclodextrin glucosyltransferase is a cyclodextrin glucosyltransferase or an immobilized enzyme thereof produced by a strain of Geobacillus sp.

3. The method according to claim 1, wherein a mass concentration of steviol glycoside is 10% to 25%, and a mass ratio of the steviol glycosides to the dextrin or oligosaccharide is 1:0.3 to 1:1.1.

4. The method according to claim 1, wherein the steviol glycosides are any one or a mixture of steviol glycosides extracted from stevia, or obtained by a fermentation method or by an enzymatic method.

5. The method according to claim 1, wherein the dextrin is any one or a mixture of maltodextrin, corn starch dextrin, cassava dextrin, and various cyclodextrins; and wherein the oligosaccharide is any one or a mixture of maltose, maltotriose, raffinose, and melibiose.

6. The method according to claim 1, wherein the salt is any one or a mixture of calcium chloride and barium chloride, present in an amount of 0.1% to 1% by mass of the cyclodextrin glucosyltransferase; and wherein the polyol is any one or a mixture of glycerol and sorbitol, present in amount of 0.1% to 5% by mass of the cylodextrin glucosyltransferase.

7. The method according to claim 1, wherein the water or the buffer solution is deionized water or a phosphoric acid-ammonium phosphate buffer solution with a pH in the range of 5 to 6.5.

8. The method according to claim 1, further comprising: spray drying, or concentrating and drying the glucosyl steviol glycosides produced to obtain a glucosyl steviol glycoside crude product, and removing residual dextrin or other reducing sugar by macroporous resin adsorption followed by elution with water.

Description

BRIEF DESCRIPTION OF FIGURES

(1) FIG. 1 HPLC chromatogram of the glucosyl rebaudioside A.

(2) FIG. 2 Effect of substrate concentration on conversion rate of steviol glycosides and yield of each product; .square-solid.: St, .diamond-solid.: St-Glc1, .box-tangle-solidup.: St-Glc2, □: St-Glc3, ⋄: St-Glc4, Δ: St-Glc5. St refers to stevioside; St-Glcx refers to stevioside grafted x mol of glucosyl residues.

(3) FIG. 3 Effect of ratio of steviol glycoside: glycosyl donor on conversion rate of steviol glycosides and yield of each product; .square-solid.: St, .diamond-solid.: St-Glc1, .box-tangle-solidup.: St-Glc2, □: St-Glc3, ⋄: St-Glc4, Δ: St-Glc5.

DETAILED DESCRIPTION

(4) Analytical Method and Calculation:

(5) (1) Quantitative analysis of unreacted steviol glycosides is based on the analysis and detection method of steviol glycosides presented in GB2760-2014 or JECFA2016.

(6) (2) Qualitative analysis of glucosyl steviol glycosides: the transglucosylation product is qualitatively determined by a liquid chromatography tandem quadrupole time-of-flight mass spectrometer. The detection conditions are as follows: ACQUITY UPLC BEH HILIC amino column, column temperature 30° C., gradient elution under acetonitrile:water=80:20 (2 min)-50:50 (30 min) (v/v), injection volume 1 μL, injection concentration 5 mg/mL, flow rate 0.3 mL/min; the mass spectrometry condition is a collision voltage of 6 eV; and the ionization manner: electrospray ionization (ESI), negative ion detection mode, molecular weight range: 200-2000.

(7) (3) Quantitative analysis of glucosyl steviol glycosides is based on the analysis and detection method of glucosyl steviol glycosides in the supplemental document of National Health and Family Planning Commission of the People's Republic of China in GB2760-2014.

(8) (4) The α-amylase activity is determined by the α-amylase activity determination method of α-amylase preparation in Chinese Standard GB/T 24401-2009.

(9) (5) The β-amylase activity is determined by a DNS (3,5-dinitrosalicylic acid) method, see P. Bernfeld, “Amylase, α and β,” in Methods in Enzymology, S. P. Colowick and N. O. Kaplan, Eds., pp. 149-158, Academic Press, New York, N.Y., USA, 1955.

(10) (6) Determination of cyclodextrin glucosyltransferase α- or β-cyclase activity:

(11) 0.1 mL of an appropriately diluted enzyme solution is added to a test tube containing 2 mL of a 1% (wt) soluble starch solution previously prepared with 10 mM phosphate buffer solution (pH 6.5). After reacting at 60° C. for 10 min, 1 mL of hydrochloric acid (1 mol/L) is added to stop the reaction, and the solution is rapidly cooled to room temperature.

(12) When determining the α-cyclase activity, 0.2 mL of a 0.5 mM methyl orange solution prepared with 10 mM phosphate buffer solution is added to the above solution, and allowed to stand at room temperature for 15 min, and the absorbance A.sub.505 at 505 nm is determined.

(13) When determining the β-cyclase activity, 3.5 mL of a NaOH aqueous solution (30 mM) is added to the above solution, and 0.5 mL of a 0.02% (wt) phenolphthalein solution prepared with a 5 mM Na.sub.2CO.sub.3 solution is added, and allowed to stand at room temperature for 15 min, and the absorbance A.sub.550 at 550 nm is determined.

(14) Then, respectively referring to the standard curves of A.sub.505 with respect to α-cyclodextrin concentration and A.sub.550 with respect to β-cyclodextrin concentration, the α- or β-cyclodextrin contents of the solution are respectively calculated. One α- or β-cyclase unit is defined as the number of μmol that produces α- or β-cyclodextrin per minute under the above conditions.

Example 1 Preparation of Cyclodextrin Glucosyltransferase (CGTase)

(15) A strain of Geobacillus sp. (Geobacillus thermoglucosidasius, ATCC 43742) was used as a production strain, inoculated at an inoculum size of 8% and followed by batch fermentation. A culture medium was composed of glucose 0.8%, lactose 0.05%, peptone 1.2%, yeast extract 2.4%, K.sub.2HPO.sub.4 0.3%, KH.sub.2PO.sub.4 0.98%, and CaCl.sub.2 0.28%. When the dissolved oxygen reached to 85%, the culture medium was supplemented, and the temperature was controlled at 33-37° C. and the dissolved oxygen was maintained at 25%-30% in the growth phase of the thalluses; when the OD.sub.600 of the thalluses reached 25, 1% glycine (mass volume fraction) was supplemented; when the OD.sub.600 of the thalluses reached 50, the temperature was lowered to 23-27° C. and 0.2-0.4 g.Math.l.sup.−1.Math.h.sup.−1 lactose was continuously added, and meanwhile, the culture medium was supplemented in a gradient descent manner; after 30 hours of fermentation and culture, the fermentation was terminated, and after removing the thalluses by centrifugation, the fermentation liquid was concentrated 10 times, wherein the α-amylase activity was 163.7 U/mL, the β-amylase activity was 42.3 U/mL, the α-cyclase activity of the cyclodextrin glucosyltransferase was 83.1 U/mL and the β-cyclase activity was 57.4 U/mL. When used as following cases, it is measured based on the α-cyclase activity of the cyclodextrin glucosyltransferase.

Example 2 Preparation of Glucosyl Steviol Glycosides with Cassava Dextrin and Rebaudioside A

(16) 100 kg of water was added to a jacketed reactor, and after heating to 85° C., 15 kg of cassava dextrin and 15 kg of rebaudioside A (HPLC content 98%) were sequentially added and stirred to dissolve. After being fully dissolved, the system was rapidly cooled to 65° C., and an aqueous solution containing 5 mL of 1% calcium chloride and barium chloride (mass ratio 0.2:1), 4 g of glycerin and 250 kU of a solution of the cyclodextrin glucosyltransferase obtained from Example 1 was added to the above jacketed reactor while stirring. The reaction was carried out at 65° C. for 3 h, then the temperature was raised to 75° C. to react for 24 h, then the temperature was raised to 80° C. to react for 10 h, and the reaction was terminated thereafter. The product was directly spray-dried to obtain the glucosyl steviol glycosides having a moisture content of 2.1%, and the product can be directly used without decolorization for a product which does not require a total glycoside content being not less than 95%. The content of rebaudioside A in the product was 2.9%.

Comparative Example 1 Preparation of Glucosyl Steviol Glycosides in the Absence of Stabilizer

(17) 100 kg of water was added to a jacketed reactor, and after heating to 85° C., 15 kg of cassava dextrin and 15 kg of rebaudioside A (HPLC content 98%) were sequentially added and stirred to dissolve. After being fully dissolved, the system was rapidly cooled to 65° C., and a solution containing 250 kU of a solution of the cyclodextrin glucosyltransferase obtained from Example 1 was added to the above jacketed reactor while stirring. The reaction was carried out at 65° C. for 3 h, then the temperature was raised to 75° C. to react for 24 h, then the temperature was raised to 80° C. to react for 10 h, and the reaction was terminated therafter. The product was directly spray-dried or slightly concentrated and dried to obtain the glucosyl steviol glycosides having a moisture content of 2.2%, wherein the content of rebaudioside A in the product was 5.2%.

Comparative Example 2 Preparation of Glucosyl Steviol Glycosides at Constant Temperature in the Absence of Stabilizer

(18) 100 kg of water was added to a jacketed reactor, and after heating to 75° C., 15 kg of cassava dextrin and 15 kg of rebaudioside A (HPLC content 98%) were sequentially added and stirred to dissolve. After being fully dissolved, 250 kU of a solution of the cyclodextrin glucosyltransferase obtained in Example 1 was added to the above jacketed reactor while stirring. The reaction was carried out at 75° C. for 48 h, and then terminated. The product was directly spray-dried to obtain the glucosyl steviol glycosides having a moisture content of 2.2%, wherein the content of rebaudioside A in the product was 6.1%.

Comparative Example 3 Effect of Steviol Glycoside Concentration on Preparation of Glucosyl Steviol Glycosides in the Absence of Stabilizer

(19) FIG. 2 shows the effect of concentration of steviol glycosides on preparation of glucosyl steviol glycosides in the absence of a stabilizer at constant temperature with stevioside as an example. Under the conditions of stevioside:β-cyclodextrin=1.06:1, 60° C. and enzyme amount of 10 U/g glucoside, after the reaction is carried out for 5 h at different concentrations, the substrate concentration is positively correlated with the reaction rate in the low concentration range, and when the substrate concentration exceeds the optimum substrate concentration (5%), the conversion rate of stevioside and the yield of glucosyl steviol glycosides are lowered because of the inhibiting action of the substrate. The conversion rate of stevioside and the yield of glucosyl steviol glycosides are calculated based on the concentration of the corresponding compound in the reactants and theoretical values.

Example 3 Preparation of Glucosyl Steviol Glycosides with β-Cyclodextrin and Rebaudioside A as Raw Materials

(20) 100 kg of water was added to a jacketed reactor, and after heating to 82° C., 10 kg of β-cyclodextrin and 25 kg of rebaudioside A (HPLC content 97%) were sequentially added and stirred to dissolve. After being fully dissolved, the system was rapidly cooled to 60° C., and an aqueous solution containing 10 mL of 1% calcium chloride and barium chloride (mass ratio 0.5:1), 2 g of glycerin, 2 g of sorbitol and 1000 kU of a solution of the cyclodextrin glucosyltransferase obtained in Example 1 was added to the above jacketed reactor while stirring. The reaction was carried out at 60° C. for 3 h, then the temperature was raised to 74° C. to react for 15 h, then the temperature was raised to 80° C. to react for 8 h, and the reaction was terminated. The product was directly spray-dried to obtain the glucosyl steviol glycosides having a moisture content of 2.0%, and the product can be directly used without decolorization for a product which does not require a total glycoside content being not less than 95%. The content of rebaudioside A in the product was 2.2%. The product was eluted with macroporous resin column until the total glycoside content was about 96%, wherein the content of rebaudioside A in the product was 3.97%.

Example 4 Preparation of Glucosyl Steviol Glycosides with Maltodextrin and Steviol Glycosides Obtained as Byproduct from Recrystallization of Rebaudioside A

(21) 100 kg of 0.01M phosphate buffer solution with the pH being 5.8 was added to a jacketed reactor, and after heating to 80° C., 10 kg of maltodextrin (DE value 16) and 20 kg of steviol glycosides obtained as byproduct from recrystallization of Rebaudioside A (HPLC content 97% of total steviol glycosides) were sequentially added and stirred to dissolve. After being fully dissolved, the system was rapidly cooled to 62° C., and an aqueous solution containing 20 mL of 0.8% barium chloride, 8 g of glycerin and 1250 kU of a solution of the cyclodextrin glucosyltransferase obtained in Example 1 was added to the above jacketed reactor while stirring. The reaction was carried out at 65° C. for 3 h, then the temperature was raised to 75° C. to react for 24 h, then the temperature was raised to 80° C. to react for 18 h, and the reaction was terminated. The product was concentrated and microwave-dried to obtain the glucosyl steviol glycosides having a moisture content of 2.8%, and the product can be directly used without decolorization for a product which does not require a total glycoside content being not less than 95%. The content of unconverted steviol glycosides in the product was 2.7%. The product was eluted with macroporous resin column until the total glycoside content was about 96%, wherein the content of unconverted steviol glycosides in the product was 5.4%.

Example 5 Preparation of Glucosyl Steviol Glycosides with Corn Starch and Stevioside as Raw Materials

(22) 100 kg of water was added to a jacketed reactor, and after heating to 80° C., 10 kg of corn starch and 12 kg of stevioside (HPLC content 95%) were sequentially added and stirred to dissolve. After being fully dissolved, the system was rapidly cooled to 65° C., and an aqueous solution containing 10 mL of 0.5% barium chloride, 3 g of glycerin and 250 kU of a solution of the cyclodextrin glucosyltransferase obtained in Example 1 was added to the above jacketed reactor while stirring. The reaction was carried out at 65° C. for 3 h, then the temperature was raised to 72° C. to react for 15 h, then the temperature was raised to 80° C. to react for 8 h, and the reaction was terminated. The product was directly spray-dried to obtain the glucosyl steviol glycosides having a moisture content of 2.3%, and the product can be directly used without decolorization for a product which does not require a total glycoside content being not less than 95%. The content of stevioside in the product was 2.7%.

Comparative Example 4 Effect of Steviol Glycoside:Glycosyl Donor Ratio on Preparation of Glucosyl Steviol Glycosides in the Absence of Stabilizer

(23) FIG. 3 shows the effect of the glucoside:glycosyl donor ratio on preparation of glucosyl steviol glycosides at constant temperature in the absence of a stabilizer with stevioside and β-cyclodextrin as an example: When the temperature is 60° C., the enzyme amount is 10 U/g and the β-cyclodextrin:stevioside ratio is 1.06:1, the conversion rate of stevioside is the highest. The conversion rate of stevioside is calculated based on the concentration of the corresponding compound in the reactants and theoretical values.

Example 6 Preparation of Glucosyl Steviol Glycosides with Maltotriose and Steviol Glycosides Obtained as Byproduct from Recrystallization of Rebaudioside A as Raw Materials

(24) 100 kg of water was added to a jacketed reactor, and after heating to 80° C., 18 kg of maltotriose and 20 kg of steviol glycosides obtained as byproduct from recrystallization of Rebaudioside A (HPLC content 97%) were sequentially added and stirred to dissolve. After being fully dissolved, the system was rapidly cooled to 65° C., and an aqueous solution containing 20 mL of 1% calcium chloride and barium chloride (mass ratio 1:0.5), 8 g of sorbitol and 1250 kU of a solution of the cyclodextrin glucosyltransferase obtained in Example 1 was added to the above jacketed reactor while stirring. The reaction was carried out at 65° C. for 5 h, then the temperature was raised to 75° C. to react for 14 h, then the temperature was raised to 80° C. to react for 6 h, and the reaction was terminated. The product was directly spray-dried to obtain the glucosyl steviol glycosides having a moisture content of 2.1%, and the product can be directly used without decolorization for a product which does not require a total glycoside content being not less than 95%. The content of unconverted steviol glycosides in the product was 3.7%. The product was eluted with macroporous resin column until the total glycoside content was about 96%, wherein the content of unconverted steviol glycosides in the product was 5.6%.