Method to Elevate the Amount of Steviol Glycosides Remained in the Recrystallization Mother Liquor of Stevia Extract

Abstract

The disclosure discloses a method to elevate the amount of steviol glycosides remained in the recrystallization mother liquor of Stevia extract, and belongs to the field of modification and extraction of natural compounds. The disclosure adopts lactase for highly specific catalytic hydrolysis of stevioside to convert stevioside into rubusoside which usually can only be obtained from leaves of Rubus suavissimus S. Lee; and meanwhile, after stevioside in the mother liquor glycosides is converted, since the polarity difference between rubusoside and rebaudioside A is far larger than the polarity difference between stevioside and rebaudioside A, rebaudioside A is more easily separated out of the mother liquor glycosides. A technology provided by the disclosure is also applicable to value increment of mixed steviol glycosides containing stevioside and is beneficial for separating out rebaudioside A from the mixed steviol glycosides.

Claims

1. A method of elevating amount of steviol glycosides remained in recrystallization mother liquor of Stevia extract for preparing rubusoside, comprising: with Stevia mother liquor glycosides as a substrate, using lactase from Kluyveromyces fragilis to assist a reaction for 1-3 h at a temperature of 25-45 C. for specific catalytic hydrolysis of stevioside in the Stevia mother liquor glycosides, so that stevioside is hydrolyzed into rubusoside, wherein rebaudioside A does not participate in the reaction but continues to stay in the Stevia mother liquor glycosides; and after the reaction ends, separating out rubusoside and rebaudioside A; wherein an enzyme concentration of the lactase is 10-100 U/g stevioside, and mass of the added lactase does not exceed 1% of mass of the Stevia mother liquor glycosides used.

2. The method according to claim 1, wherein the Stevia mother liquor glycosides are mother liquor or a dry product of the mother liquor generated after the steviol glycosides are extracted or purified in a process of extracting the steviol glycosides from Stevia rebaudiana leaves.

3. The method according to claim 2, wherein the enzyme concentration of the lactase is 10-100 U/g stevioside.

4. The method according to claim 3, wherein during the catalytic reaction with the lactase, a pH of the substrate is adjusted to 4-6.

5. The method according to claim 1, wherein after the reaction ends, other steviol glycosides including rebaudioside A are separated excluding rubusoside through exclusion chromatography or recrystallization.

6. The method according to claim 1, wherein the Stevia mother liquor glycosides is replaced with mixed steviol glycosides containing stevioside.

7. The method according to claim 1, wherein a solution with a predetermined concentration is prepared first by adding water to the Stevia mother liquor glycosides, and then the lactase is added to catalyze stevioside to produce rubusoside; and a mass concentration of steviol glycosides in an aqueous solution of the Stevia mother liquor glycosides is 100-500 g/L.

8. A method of use of stevioside to synthesize rubusoside, comprising: using lactase from Kluyveromyces fragilis to perform a reaction for specific catalytic hydrolysis of stevioside to hydrolyze stevioside into rubusoside through efficient catalytic conversion.

9. The method according to claim 8, wherein the lactase comes from Kluyveromyces fragilis, the lactase exists in a form of a concentrated solution, freeze-dried powder or an immobilized enzyme, and an enzyme concentration does not exceed 1% of mass of Stevia mother liquor glycosides used.

10. The method according to claim 8, wherein the reaction is performed for 1-3 h at a temperature of 25-45 C. for specific catalytic hydrolysis of stevioside in the Stevia mother liquor glycosides to hydrolyze stevioside into rubusoside.

11. The method according to claim 9, wherein the reaction is performed for 1-3 h at a temperature of 25-45 C. for specific catalytic hydrolysis of stevioside in the Stevia mother liquor glycosides to hydrolyze stevioside into rubusoside.

12. The method according to claim 10, wherein the reaction is performed for 2-3 h at a temperature of 30 C. for specific catalytic hydrolysis of stevioside in the Stevia mother liquor glycosides to hydrolyze stevioside into rubusoside.

Description

BRIEF DESCRIPTION OF FIGURES

[0027] FIG. 1A and FIG. 1B are HPLC of Stevia after enzymatic reaction; FIG. 1A shows that lactase with an enzyme concentration of 60 U/g stevioside is added to a solution of Stevia for reaction for 2 h; and FIG. 1B shows that lactase with an enzyme concentration of 150 U/g stevioside is added to a solution of Stevia for reaction for 2 h.

[0028] FIG. 2 is an HPLC of reaction mixture obtained after lactase with an enzyme concentration of 30 U/g stevioside is added to a solution of mother liquor glycosides for reaction for 2 h.

[0029] FIG. 3 is an HPLC of reaction mixture obtained after stevioside is catalyzed by lactase to react for 2 h.

[0030] FIG. 4A-FIG. 4C are an HPLC after rebaudioside A itself or a mixture of rebaudioside A and stevioside is catalyzed by lactase to react; FIG. 4A shows an HPLC after 50 wt % of rebaudioside A and 50 wt % of stevioside are catalyzed by lactase to react for 2 h at an enzyme concentration of 200 U/g stevioside, where rebaudioside A does not react; FIG. 4B shows an HPLC of rebaudioside A; and FIG. 4C shows an HPLC of 50 wt % of rebaudioside A and 50 wt % of stevioside.

[0031] FIG. 5 is an HPLC after rebaudioside A and lactase coexist for 2 h.

DETAILED DESCRIPTION

[0032] A Measurement Method

[0033] 1. Definition of enzyme activity of lactase: with ONPG as a substrate, the lactase performs enzymolysis on the ONPG at a constant temperature of 35 C. and releases an amount of enzyme required by 1 mol/L o-nitrophenol every minute, which is defined as one enzyme activity unit.

[0034] Measurement of enzyme activity of lactase: 500 L of a pH6.5 phosphate buffer solution and 200 L of a 0.25% ONPG solution are added into a colorimetric tube for warm bath for 5 min at 35 C., 500 L of properly-diluted enzyme liquid is added for reaction for 10 min, 800 L of a 5% Na.sub.2CO.sub.3 solution is added to stop reaction, a volume is fixed to 10 mL with distilled water, the absorbance of the solution at 420 nm is measured through an ultraviolet spectrophotometer, and the enzyme activity is calculated according to a standard curve of nitrophenol.

[0035] 2. Qualitative Analysis of Steviol Glycosides

[0036] Qualitative analysis is performed on hydrolysis and transglucosyl products through a liquid chromatography tandem quadrupole rod time-of-flight mass spectrometer, and the detection conditions include: a Kromasil 100-5-C18 chromatographic column (4.6*250 mm), a column temperature of 40 C., acetonitrile to water=32:68 (v/v) for isocratic elusion, a sample size of 10 L, a sample injection concentration of 10 mg/mL, and a flowing velocity of 1 mL/min; a mass spectrometry condition: a fragmentor voltage of 6 eV; an ionization mode: electrospray ionization (ESI), an anion detection mode, and a molecular weight of 200-2000.

[0037] 3. Quantitative Analysis of Steviol Glycosides

[0038] A standard curve is drawn with rebaudioside A (HPLC 99%) as a standard sample, concentrations of rebaudioside A, stevioside and rubusoside are figured out through the standard curve of rebaudioside A by means of an analysis and detection method of steviol glycosides in JECFA2016.

[0039] A conversion rate calculation formula of stevioside:

=[(C.sub.0C.sub.t)/C.sub.0]100%(1)

[0040] where represents a conversion rate of stevioside, C.sub.0 represents a concentration of stevioside when a solution does not react, and C.sub.t represents a concentration of stevioside in reaction mixture at time t.

[0041] A yield calculation formula of rubusoside:

=(C.sub.m/C.sub.n)100%(2)

[0042] where represents a yield of rubusoside, C.sub.m represents a concentration of rubusoside when reaction ends, and C.sub.n represents a concentration of rubusoside that can be produced theoretically. C.sub.m and C.sub.n are both figured out by means of a method disclosed in JECFA2016.

rubusoside yield=(mass of rubusoside obtained after separation and purification of each gram of raw material/mass of rubusoside that can be produced from each gram of raw material theoretically)*100%

Example 1 A Preparation Method of Lactase from Kluyveromyces fragilis (ATCC 8554)

[0043] A strain of Kluyveromyces fragilis (ATCC 8554) is inoculated into a YPD agar slant culture medium for shake cultivation at 30 C. Then, 10% of a cultivated seed culture solution is inoculated into a liquid fermentation culture medium (containing lactose and yeast powder) for shake cultivation at 28 C. at 180 rpm for 36 h. The fermentation culture is then centrifuged for 15 min at 4000 r/min. After discasted the supernatant, phosphate buffer (pH7.0) is added to the deposited yeast cells, stirred and then processed by a ball mill at 180 r/min for 60 min. Triple volume of phosphate buffer solution (pH7.0) is then added, stirred to even, flowed by centrifuge to remove the yeast cell fragments; and then the crude enzyme can be obtained. A ultrafiltration membrane with a molecular weight cut-off of 30 kDa is used, the crude enzyme is subjected to ultrafiltration under pressure of 70 KPa to obtain a concentrated solution. The membrane is washed with a buffer solution. The concentrated solution and the washing solution are combined to obtain a concentrated enzyme solution, and the concentrated enzyme solution is freeze-dried to obtain freeze-dried powder with a specific enzyme activity of 40000 U/g.

Example 2 Preparation of Rubusoside from Stevia Mother Liquor Glycosides and Further Separation of Rebaudioside A

[0044] A Stevia mother liquor glycoside solution (19% of rebaudioside A, 66% of stevioside and 9% of rebaudioside C) obtained as the residue from recrystallization of rebaudioside A, is then concentrated to reach a solid content of 300 g/L. The lactase obtained from Example 1 is added to reach an enzyme concentration of 100 U/g stevioside for reaction at 30 C. for 2 h. The reaction mixture is dried and then recrystallized with 95% ethanol to separate out rubusoside with a purity of 97.1% and a yield of 88.9%, and rebaudioside A is separated out with a purity of 95.8% and a yield of 86%.

Comparative Example 1

[0045] Stevia mother liquor glycoside solid (19% of rebaudioside A, 66% of stevioside and 9% of rebaudioside C) obtained as the residue from recrystallization of rebaudioside A is recrystallized with 95% ethanol to separate out rebaudioside A, the purity is 84.2% and the yield is 54.9%.

[0046] Compared with Example 2, it can be seen that by converting the mother liquor glycosides through an enzyme method recorded in Example 2, the purity of rebaudioside A can be improved, an absolute yield of rebaudioside A is improved by 31.1%, and a relative yield is improved by 56.6%.

Example 3 Preparation of Rubusoside from Stevia Mother Liquor Glycosides and Further Separation of Rebaudioside A

[0047] A Stevia mother liquor glycoside solution (33% of rebaudioside A, 41% of stevioside and 11% of rebaudioside C), as a by-product during refining of rebaudioside A, is concentrated to reach a solid content of 30%. The lactase obtained in Example 1 is added to reach an enzyme concentration of 80 U/g stevioside for reaction at 30 C. for 1 h. The reaction mixture is spray-dried and then recrystallized with 95% ethanol to separate out rubusoside. The obtained rubusoside possess a purity of 96.7% and a yield of 91.7%, and rebaudioside A is separated out with a purity of 95.7% and a yield of 87.2%.

Comparative Example 2

[0048] Stevia mother liquor glycosides (33% of rebaudioside A, 41% of stevioside and 11% of rebaudioside C), as by-products of refining of rebaudioside A, are spray-dried and then recrystallized with 95% ethanol to separate out rebaudioside A, the obtained RA possess a purity of 86.2% and a yield of 61.8%

[0049] Compared with Example 3, it can be seen that by converting mother liquor glycosides through an enzyme method recorded in Example 3, the purity of rebaudioside A can be improved, an absolute yield of rebaudioside A is improved by 25.4%, and a relative yield is improved by 41.1%.

Example 4 Preparation of Rubusoside from Stevia Sugar

[0050] 250 g/L glycoside liquid is prepared with deionized water from Stevia sugar (containing 22% of rebaudioside A, 68% of stevioside and 3% of rebaudioside C in percentages by mass). The lactase freeze-dried powder obtained in Example 1 is added to reach an enzyme concentration of 60 U/g stevioside for reaction at 35 C. for 2 h. The reaction mixture is analyzed through a high performance liquid chromatograph, the HPLC profile of the product is shown in FIG. 1A, a conversion rate of stevioside reaches 98.3%, and a yield of rubusoside is 97.7%.

Comparative Example 3

[0051] 250 g/L glycoside liquid is prepared with deionized water from Stevia sugar (containing 22% of rebaudioside A, 68% of stevioside and 3% of rebaudioside C in percentages by mass). The lactase freeze-dried powder obtained in Example 1 is added to reach an enzyme concentration of 150 U/g stevioside for reaction at 35 C. for 24 h. The reaction mixture is analyzed through the high performance liquid chromatograph, the HPLC profile of the product is shown in FIG. 1 (b), a conversion rate of stevioside reaches 99.5%, and a yield of rubusoside is 89.1%. It can be seen in FIG. 1B that transglucosyl products are generated when an enzyme concentration of the lactase is too high and an LC-MS analysis result shows that a molecular weight of the transglucosyl products conforms to that of St-Glc2 or RA-Glc1.

Example 5 Preparation of Rubusoside from Stevia Mother Liquor Glycosides

[0052] 100 g/L glycoside liquid is prepared from the Stevia mother liquor glycosides (containing 47% of rebaudioside A, 19% of stevioside and 7% of rebaudioside C in percentages by mass). The lactase freeze-dried powder obtained in Example 1 is added to reach an enzyme concentration of 30 U/g stevioside for reaction at 25 C. for 0.5 h. The reaction mixture is analyzed through the high performance liquid chromatograph, the HPLC profile of the reaction mixture is shown in FIG. 2; a conversion rate of stevioside reaches 72.7%, and a yield of rubusoside is 70.5%. It can be seen that a conversion rate of stevioside will drop at a low temperature, a low enzyme concentration and a short reaction time.

Example 6 Preparation of Rubusoside from Stevioside

[0053] A 200 g/L stevioside solution is prepared by dissolving 20 g of stevioside (97%, HPLC) in 100 mL of deionized water into a reactor. The lactase freeze-dried powder obtained in Example 1 is added to reach an enzyme concentration of 90 U/g stevioside for reaction at 35 C. for 2 h. The reaction mixture is analyzed through the high performance liquid chromatograph, the HPLC profile of the product is shown in FIG. 3, a conversion rate of stevioside reaches 98.9%, and a yield of rubusoside is 97.9%.

Comparative Example 4

[0054] The lactase freeze-dried powder obtained in Example 1 is added to a mixed glycoside solution (50% of stevioside and 50% of rebaudioside A) with a solid content of 200 g/L to reach an enzyme concentration of 200 U/g stevioside, stirred at 35 C. for 2 h. The reaction mixture is analyzed through the high performance liquid chromatograph, a conversion rate of stevioside reaches 99.5%, a yield of rubusoside is 83%, the HPLC profile of the product is shown in FIG. 4A, transglucosyl products are produced, and a molecular weight of the transglucosyl products is 1128 as measured by LC-MS analysis.

[0055] A 20 mg/mL solution of stevioside (97%, HPLC) and a 20 mg/mL 50 wt % rebaudioside A+50 wt % stevioside are prepared respectively, the two types of glycoside liquid is analyzed through the high performance liquid chromatograph respectively, and HPLC profiles of the two glycoside liquids are shown in FIG. 4B and FIG. 4C, respectively.

Comparative Example 5

[0056] A 10 g/L rebaudioside A solution is prepared by dissolving 1 g of rebaudioside A (99%, HPLC) in 100 mL of deionized water and preheated for 30 min at 30 C. The lactase freeze-dried powder obtained in Example 1 is added to reach an enzyme concentration of 100 U/g rebaudioside A for reaction for 24 h at 30 C. The reaction mixture is analyzed through the high performance liquid chromatograph, the HPLC profile of the product is shown in FIG. 5, and rebaudioside A did not react.

Comparative Example 6

[0057] 450 g/L glycoside liquid is prepared with a pH5 phosphate-potassium phosphate buffer solution (50 mmol/L) from Stevia sugar (containing 22% of rebaudioside A, 68% of stevioside and 3% of rebaudioside C in percentages by mass). The lactase (Lactozym Pure 7500L, purchased from Novozymes company in China) is added to reach an enzyme concentration of 100 U/g stevioside for reaction at 30 C. for 2 h. Then, the reaction mixture is analyzed through the high performance liquid chromatograph, and the results show that there is no rubusoside produced in the reaction mixture and stevioside was not converted either.

Comparative Example 7

[0058] 450 g/L glycoside liquid is prepared with a pH5 phosphate-potassium phosphate buffer solution (50 mmol/L) from Stevia sugar (containing 22% of rebaudioside A, 68% of stevioside and 3% of rebaudioside C in percentages by mass). The lactase (trade name: Lactozym 3000L HP-G, purchased from Novozymes company in China) from Kluyveromyces lactis is added to reach an enzyme concentration of 100 U/g stevioside for reaction at 30 C. for 2 h. Then, the reaction mixture is analyzed through the high performance liquid chromatograph, and the results show that the content of stevioside in the reaction mixture is not reduced, stevioside is not converted and there is no rubusoside produced.

Comparative Example 8

[0059] 450 g/L glycoside liquid is prepared with a pH5 phosphate-potassium phosphate buffer solution (50 mmol/L) from Stevia sugar (containing 22% of rebaudioside A, 68% of stevioside and 3% of rebaudioside C in percentages by mass). The lactase (trade name: Maxilact LG 2000, purchased from Royal DSM company in N.V.) from Kluyveromyces lactis is added to reach an enzyme concentration of 100 U/g stevioside for reaction at 30 C. for 2 h. Then, the reaction mixture is analyzed through the high performance liquid chromatograph; and the results show that the content of stevioside in the reaction mixture has no change and there is no rubusoside produced, indicating that stevioside is not converted. However, if reaction time is regulated from 2 h to 20 h, there is still no rubusoside produced, but steviolbioside is produced.

Comparative Example 9

[0060] 450 g/L glycoside liquid is prepared with a pH5 phosphate-potassium phosphate buffer solution (50 mmol/L) from Stevia sugar (containing 22% of rebaudioside A, 68% of stevioside and 3% of rebaudioside C in percentages by mass). The lactase from Aspergillus niger (ATCC 9029) is added to reach an enzyme concentration of 100 U/g stevioside for reaction at 30 C. for 2 h. Then, the reaction mixture is analyzed through the high performance liquid chromatograph, and the results show that the content of stevioside in the reaction mixture has no change and there is no rubusoside produced.