Method for the preparation of lower graft degree glucosylated steviol glycosides

Abstract

The present invention discloses a method for preparing lower graft degree GSGs, and belongs to the technical field of biosynthesis of sweeteners. The method uses amylase to catalyze hydrolysis of GSGs with a high graft degree, thereby obtaining GSGs with low graft degree mainly containing GSGs with a low grafting number. The content of mono- and di-glucosyl substituents in the SGs in the product was 60% or more of the total glycosides, and the mass percent of the GSGs with a glucosyl grafting number of 3 or less was higher than 70% of the total glycosides. The mono- and di-substituted GSGs obtained by enzyme catalysis by the present invention were structurally similar to those, belong to a mixture of the isomers thereof, and have good sweetness and a flavoring function.

Claims

1. A method for preparing a lower graft degree glucosylated steviol glycosides (GSGs) composition, wherein said method comprises: hydrolyzing a starting material composition comprising higher graft degree GSGs at from 55° C. to 60° C. for 3 to 24 hours, wherein hydrolyzing is catalyzed by 1000 to 4000 Units of α-amylase per gram of starting material, wherein the lower graft degree GSGs are GSGs with a grafting number of one to three, and the higher graft degree GSGs are GSGs with a grafting number of four or more, wherein total glycosides of the lower graft degree GSGs comprises both GSGs and steviol glycosides (SGs), wherein the lower graft GSGs comprise 60% or higher of the total glycosides and have a mass percent of more than 85.1% of the total glycosides, wherein the α-amylase derived from Bacillus subtilis, and wherein the starting material composition comprises 60 g/L to 240 g/L of the higher graft degree GSGs.

2. The method according to claim 1, wherein: a total mass percent of the higher graft degree GSGs in the starting material composition is higher than 40% of a total mass of total glycosides; or, a total mass percent of monoglucosyl grafted SGs and diglucosyl grafted SGs in the starting material composition is lower than 50% of the total mass of the total glycosides.

3. The method according to claim 1, further comprising one or more of: (a) recrystallizing the crude GSG product; and (b) adsorbing a by-product reducing sugar in the lower graft degree GSGs composition, produced by recrystallization of the lower graft degree GSGs composition, by macroporous resin and then eluting with water.

4. The method according to claim 1, wherein hydrolyzing is performed for 3 hours to 5 hours.

Description

BRIEF DESCRIPTION OF FIGURES

(1) FIG. 1 is the HPLC proof of hydrolysis of GSG catalyzed by Bacillus subtilis α-amylase (4000 U/g glycoside).

(2) FIG. 2 is the HPLC proof of hydrolysis of GSG catalyzed by Aspergillus niger sp. glucoamylase (50 U/g glycoside).

(3) FIG. 3 is the HPLC proof of hydrolysis of GSG catalyzed by Aspergillus niger sp. glucoamylase (100 U/g glycoside).

(4) FIG. 4 is the HPLC proof of hydrolysis of GSG catalyzed by Aspergillus niger sp. glucoamylase (300 U/g glycoside).

(5) FIG. 5 is the HPLC proof of hydrolysis of GSG catalyzed by Bacillus amyloliquefaciens α-amylase (800 U/g glycoside).

(6) FIG. 6 is the HPLC proof of hydrolysis of GSG catalyzed by Bacillus licheniformis β-amylase (300 U/g glycoside).

(7) FIG. 7 is the HPLC proof of hydrolysis of GSG catalyzed by Bacillus licheniformis α-amylase (300 U/g glycoside).

(8) FIG. 8 is the HPLC proof of hydrolysis of GSG catalyzed by Aspergillus niger sp. glucoamylase (50 U/g glycoside, 60° C.).

(9) FIG. 9A to 9C each is the effect of substrate concentration on preparation of lower graft degree GSGs. FIG. 9A: the concentration of substrate was 100 mg/mL; FIG. 9B: the concentration of substrate was 150 mg/mL; FIG. 9C: the concentration of substrate was 200 mg/mL; Time(h): .square-solid.: 1; .circle-solid.: 3; .diamond-solid.: 7; X: 11; .box-tangle-solidup.: 24.

(10) FIG. 10A to 10C each is the effect of pH on preparation of lower graft degree GSGs. FIG. 10A: preparation of GSG with a low grafting number under pH 5.0; FIG. 10B: preparation of GSG with a low grafting number under pH 6.0; FIG. 10C: preparation of GSG with a low grafting number under pH 6.5; Time(h): □: 0, .square-solid.: 1, .circle-solid.: 3, .diamond-solid.7; x: 11, .box-tangle-solidup.: 24.

(11) FIG. 11A to 11C each is the effect of the enzyme amount on preparation of lower graft degree GSGs. FIG. 11A: preparation of GSG with a low grafting number by 40 U/g glycoside; FIG. 11B: preparation of GSG with a low grafting number by 300 U/g glycoside; FIG. 11C: preparation of GSG with a low grafting number by 400 U/g glycoside; Time(h): □: 0; .circle-solid.: 3; X: 12; .box-tangle-solidup.: 24.

DETAILED DESCRIPTION

(12) Analytical Calculation Method:

(13) (1) The qualitative analysis of GSG adopted qualitative determination of a transglycosylation product using liquid chromatography tandem quadrupole time-of-flight mass spectrometer, and the detection conditions were as follow: an ACQUITY UPLC BEH HILIC amino chromatographic column was used, the column temperature was 30° C., gradient elution was carried out under the condition that acetonitrile:water=80:20, (2 min)−50:50 (30 min)(v/v), the injection volume was 1 μL, the injection concentration was 5 mg/mL, and the flow rate was 0.3 mL/min; the mass spectrometry condition: the collision voltage was 6 eV; the ionization mode adopts electrospray ionization (ESI) and a negative ion detection mode were applied, and the molecular weight range detected was 200 to 2000.

(14) (2) The quantitative analysis of GSG was based on an analytical method for determination of GSG in document No. 8 of the GB 2760-2014 Supplementary Document of the National Health Commission.

(15) (3) The glucoamylase activity and α-amylase activity were determined by an enzyme activity measurement method as in GB1886.174-2016.

EXAMPLE 1

Synthesis of Lower Graft Degree GSG Using α-Amylase Derived from Bacillus Subtilis as Catalyst

(16) Lower graft degree GSG was prepared using GSG derived from rebaudioside A as the starting material. The starting material is a commodity obtained from rebaudioside A and dextrin by transglycosylation catalyzed by a cyclodextrin glucose transferase. The mass percent of rebaudioside A in the starting material is 8.3%, and the total mass percent of mono- and di-substituents (monoglucosyl grafted SGs and diglucosyl grafted stevioside) is 23.3%.

(17) 20 g of water was added to a jacketed reactor, and after heating to 60° C., 1.2 g of starting material glycoside was added. After dissolving by stirring, an aqueous solution of α-amylase derived from Bacillus subtilis (produced by Wuxi Xuemei Enzyme Company) was added to the jacketed reactor, with a dosage of 4000 U/g of starting material during stirring. The reaction was carried out at 60° C. for 5 h and then the reaction was terminated. The content of total glycosides in the product was 98.7%, and the contents of mono- and di-substituted glucosyl SGs were 36.8% and 34.4% respectively, totally 71.2%. The total content of mono-, di-, and tri-substituted glucosyl SGs was 86.1%, and the sweetness and flavoring function were good. The high pressure liquid chromatography diagram of the product after hydrolysis was shown in FIG. 1 of the specification.

EXAMPLE 2

Synthesis of Lower Graft Degree GSG Using Glucoamylase Derived from Aspergillus sp. as Catalyst

(18) Lower graft degree GSGs was prepared using glucosyl SGs derived from rebaudioside A as the starting material. The mass percent of rebaudioside A in the starting material was 9.8%, and the total mass percent of mono- and di-substituents was 30.5%.

(19) 20 g of water was added to a jacketed reactor, and after heating to 55° C., 4.8 g of starting material glycoside was added. After dissolving by stirring, an aqueous solution of glucoamylase derived from Aspergillus sp. (produced by Shandong Xiya Chemical Industry Co., Ltd.) was added to the jacketed reactor, with a dosage of 50 U/g of starting material glycoside during stirring. The reaction was carried out at 55° C. for 7.5 h and then the reaction was terminated. The content of total glycosides in the product was 99.4%, and the contents of mono- and di-substituted GSGs were 43.9% and 25.9% respectively, totally 69.8%. The total content of mono-, di-, and tri-substituted GSGs was 76.3%, and the sweetness and flavoring function were good. The high pressure liquid chromatography diagram of the product after hydrolysis was shown in FIG. 2 of the specification.

EXAMPLE 3

Synthesis of Lower Graft Degree GSGs Using Glucoamylase Derived from Aspergillus sp. as Catalyst

(20) Lower graft degree GSGs was prepared using GSGs derived from rebaudioside A as the starting material. The mass percent of rebaudioside A in the starting material was 9.8%, and the total mass percent of mono- and di-substituents was 30.5%.

(21) 20 g of water was added to a jacketed reactor, and after heating to 55° C., 1.2 g of starting material glycoside was added. After dissolving by stirring, an aqueous solution of glucoamylase derived from Aspergillus sp. was added to the jacketed reactor, with a dosage of 100 U/g of starting material glycoside during stirring. The reaction was carried out at 55° C. for 2.5 h and then the reaction was terminated. The content of total glycosides in the product was 98.0%, and the contents of mono- and di-substituted GSGs were 40.2% and 25.3% respectively, totally 65.5%. The total content of mono-, di-, and tri-substituted GSGs was 75.6%, and the sweetness and flavoring function were good. The high pressure liquid chromatography diagram of the product after hydrolysis was shown in FIG. 3 of the specification.

EXAMPLE 4

Synthesis of Lower Graft Degree GSGs Using Glucoamylase Derived from Aspergillus sp. as Catalyst

(22) Lower graft degree GSGs was prepared using GSGs derived from rebaudioside A as the starting material. The mass percent of rebaudioside A in the starting material was 6.8%, and the total mass percent of mono- and di-substituents was 21.2%.

(23) 20 g of water was added to a jacketed reactor, and after heating to 55° C., 1.2 g of starting material glycoside was added. After dissolving by stirring, an aqueous solution of glucoamylase derived from Aspergillus sp. was added to the jacketed reactor, with a dosage of 300 U/g of starting material glycoside during stirring. The reaction was carried out at 55° C. for 0.5 h and then the reaction was terminated. The content of total glycosides in the product was 99.0%, and the contents of mono- and di-substituted GSGs were 36.5% and 28.9% respectively, totally 65.4%. The total content of mono-, di-, and tri-substituted glucosyl SGs was 80.2%, and the sweetness and flavoring function were good. The high pressure liquid chromatography diagram of the product after hydrolysis was shown in FIG. 4 of the specification.

EXAMPLE 5

Synthesis of Lower Graft Degree GSGs Using α-Amylase Derived from Bacillus Amyloliquefaciens as Catalyst

(24) Lower graft degree GSGs was prepared using GSGs in which the mass percent of rebaudioside A was 8.3% and the total mass percent of mono- and di-substituents was 23.3% as the starting material.

(25) 20 g of water was added to a jacketed reactor, and after heating to 60° C., 1.2 g of glycoside was added. After dissolving by stirring, an aqueous solution of α-amylase derived from Bacillus amyloliquefaciens was added to the jacketed reactor, with a dosage of 800 U/g of starting material glycoside during stirring. The reaction was carried out at 60° C. for 24 h and then the reaction was terminated. The content of total glycosides in the product was 99.0%, and the contents of mono- and di-substituted GSGs were 33.7% and 34.0% respectively, totally 67.7%. The total content of mono-, di-, and tri-substituted GSGs was 85.1%, and the sweetness and flavoring function were good. The high pressure liquid chromatography diagram of the product after hydrolysis was shown in FIG. 5 of the specification.

EXAMPLE 6

Synthesis of Lower Graft Degree GSGs Using β-Amylase Derived from Bacillus Licheniformis as Catalyst

(26) Lower graft degree GSGs was prepared using GSGs in which the content of rebaudioside A was 9.1% and the total content of mono- and di-substituents was 23.7% as the starting material.

(27) 20 g of water was added to a jacketed reactor, and after heating to 60° C., 4 g of glycoside was added. After dissolving by stirring, an aqueous solution of β-amylase derived from Bacillus licheniformis was added to the jacketed reactor, with a dosage of 300 U/g of starting material glycoside during stirring. The reaction was carried out at 60° C. for 3 h and then the reaction was terminated. The content of total glycosides in the product was 92.4%, the contents of mono- and di-substituted GSGs were 33.3% and 27.2% respectively, totally 60.5%, and the sweetness and flavoring function were good. The total content of mono-, di-, and tri-substituted GSGs was 71.7%. The high pressure liquid chromatography diagram of the product after hydrolysis was shown in FIG. 6 of the specification.

EXAMPLE 7

Synthesis of Lower Graft Degree GSGs Using α-Amylase Derived from Bacillus Licheniformis as Catalyst

(28) Lower graft degree GSGs was prepared using GSGs in which the content of rebaudioside A was 8.3% and the total content of mono- and di-substituents was 23.3% as the starting material.

(29) 20 g of water was added to a jacketed reactor, and after heating to 85° C., 6 g of glycoside was added. After dissolving by stirring, an aqueous solution of α-amylase derived from Bacillus licheniformis was added to the jacketed reactor, with a dosage of 300 U/g of starting material glycoside during stirring. The reaction was carried out at 85° C. for 3 h and then the reaction was terminated. The content of total glycosides in the product was 95.6%, the contents of mono- and di-substituted GSG were 37.7% and 25.9% respectively, totally 63.6%. The total content of mono-, di-, and tri-substituted GSGs was 71.6%. The high pressure liquid chromatography diagram of the product after hydrolysis was shown in FIG. 7 of the specification.

EXAMPLE 8

Synthesis of Lower Graft Degree GSGs Using Glucoamylase Derived from Aspergillus sp. as Catalyst

(30) Lower graft degree GSGs was prepared using GSGs in which the content of rebaudioside A was 9.8% and the total content of mono- and di-substituents was 30.5% as the starting material.

(31) 20 g of water was added to a jacketed reactor, and after heating to 60° C., 5 g of glycoside was added. After dissolving by stirring, an aqueous solution of glucoamylase derived from Aspergillus sp. was added to the jacketed reactor, with a dosage of 50 U/g of starting material glycoside during stirring. The reaction was carried out at 60° C. for 7 h and then the reaction was terminated. The content of total glycosides in the product was 96.1%, the contents of mono- and di-substituted GSGs were 47.6% and 15.3% respectively, totally 62.9%. The total content of mono-, di-, and tri-substituted GSGs was 71.9%. The high pressure liquid chromatography diagram of the product after hydrolysis was shown in FIG. 8 of the specification.

EXAMPLE 9

Effect of Starting Material Glycoside Concentration on Preparation of Lower Graft Degree GSGs

(32) The starting material glycoside was prepared at 60° C. into solutions with the concentration of 100 g/L, 150 g/L and 200 g/L respectively for reaction. An aqueous solution of α-amylase derived from Bacillus subtilis was added to the above solutions, with a dosage of 300 U/g of starting material glycoside during stirring. The contents of various SGs in the products were determined after reacting at 60° C. for different times. The results were shown in FIG. 9 of the specification.

(33) It can be seen from FIG. 9 that the increase in substrate concentration within the scope of experiment has no significant effect on the preparation of the lower graft degree GSGs. Certainly, the degree of influence may vary depending on the type of amylase. As the reaction time prolongs, within a certain range, the concentration of products with a high graft degree rapidly decreases, while the concentration of products with a low graft degree rapidly increases.

EXAMPLE 10

Effect of pH on Preparation of Lower Graft Degree GSGs

(34) The starting material glycoside was prepared into solutions with a concentration of 100 g/L at 60° C. for reaction with sodium dihydrogen phosphate-disodium hydrogen phosphate buffers (10 mmol/L) with different pH values. An aqueous solution of α-amylase derived from Bacillus subtilis was added to the above solutions, with a dosage of 300 U/g of starting material glycoside during stirring. The contents of various SGs in the products were determined after reacting at 60° C. for different times. The results were shown in FIG. 10 of the specification.

EXAMPLE 11

Effect of Enzyme Amount on Preparation of Lower Graft Degree GSGs

(35) The starting material glycoside was prepared into solutions with a concentration of 100 g/L with water for reaction at 70° C. respectively. An aqueous solution of α-amylase derived from Bacillus subtilis was added to the above solutions, with a dosage of 300 U/g of starting material glycoside during stirring. The contents of various SGs in the products were determined after reacting at 70° C. for different times. The results were shown in FIG. 11 of the specification.