Extraction method for converting Reb A into Reb M through enzyme catalysis

Abstract

The invention discloses an extraction method for converting Reb A into Reb M through enzyme catalysis, belongs to the technical field of separation and purification of stevioside M subjected to enzyme conversion, and aims to solve the problems that a stevioside M conversion solution subjected to enzyme conversion contains analogues such as stevioside A, stevioside B and stevioside D, and also contains a substrate, inorganic salt and the like, the solubility of stevioside substances in water is only 0.1-1%, and the stevioside M conversion solution subjected to enzyme conversion is low in cost. The method comprises the following steps: S1, adding absolute ethyl alcohol into the conversion liquid, and uniformly stirring the absolute ethyl alcohol and the conversion liquid, so that the absolute ethyl alcohol and the conversion liquid are uniformly stirred; the external standard content of the stevioside M subjected to concentration and crystallization treatment is not lower than 95%, the yield reaches 86%, meanwhile, the purification method of the conversion liquid of the stevioside M subjected to enzymatic conversion is simple in process, only pure water, ethyl alcohol and other basic industrial raw materials are needed, and the method has the advantages of being low in cost, high in purification yield, free of complex secondary treatment and suitable for industrial production. The high-purity and high-content stevioside M can be obtained, and the method has extremely high industrial popularization and application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of separation and purification of enzymatically converted steviol glycoside M, and specifically relates to an extraction method for enzymatically catalyzing the conversion of Reb A to Reb M. Background Art

[0002] The contents of St and Reb A in stevia leaves are the highest, accounting for 5%-10% and 2%-4% of the dry weight of the leaves respectively. Therefore, the main components of the extraction products in the existing research technologies are St and Reb A. However, the contents of Reb D and Reb M in stevia are extremely low, only accounting for 0.4%-0.5% of the dry weight of the leaves, indicating that the main differences among different types of steviol glycosides lie in the number and position of glycosyl groups, thus presenting different sweetness and tastes. The sweetness of trisaccharide-based St and tetrasaccharide-based Reb A is 250-300 times that of sucrose, with a slight bitter aftertaste. With the increase in the number of glycosyl groups, the sweetness of pentasaccharide-based Reb D and hexasaccharide-based Reb M can reach 350 times that of sucrose, with almost no bitter aftertaste and better taste.

[0003] The common production processes of steviol glycosides mainly involve extracting with stevia leaves, and gradually developing to fermentation by fermentation methods. There are also enterprises using enzymatic methods to convert steviol glycosides into high-end products.

[0004] Patent Publication No. CN110229201B discloses a method for extracting steviol glycoside M from plants, which does not involve enzymatically converted steviol glycoside M. No other methods for extracting steviol glycoside M from the enzymatic conversion solution have been found yet. The enzymatic conversion solution of steviol glycoside M contains analogues such as steviol glycoside A, steviol glycoside B, steviol glycoside D, etc., and also contains substrates, inorganic salts, etc. The solubility of steviol glycoside substances in water is only 0.1%-1%. A large amount of steviol glycosides in the conversion solution exist in the form of syrup, seriously affecting the extraction process. Therefore, we propose an extraction method for enzymatically catalyzing the conversion of Reb A to Reb M. Summary of the Invention

[0005] The purpose of the present invention is to provide an extraction method for enzymatically catalyzing the conversion of Reb A to Reb M to solve the problems raised in the above background art.

[0006] To achieve the above purpose, the present invention provides the following technical solution: An extraction method for enzymatically catalyzing the conversion of Reb A to Reb M, comprising the following steps:

[0007] S1: Add anhydrous ethanol to the conversion solution, stir evenly, keep warm, and remove the bacteria.

[0008] S2: Perform vacuum distillation on the filtrate obtained in step S1, and then add pure water until it dissolves.

[0009] S3: Adsorb the solution obtained in step S2 onto an AB-8 column and wash it with pure water until it becomes colorless.

[0010] S4: Analyze the solution in S3 to obtain an analytical solution.

[0011] S5: Add activated carbon to the analytical solution in S4, then perform heat preservation, vacuum filtration to remove the activated carbon, and negative pressure concentration until crystals precipitate, and finally perform cooling crystallization.

[0012] S6: Filter the crystals obtained in S5 overnight, wash them clean with pure water, and repeat step S3 for the mother liquor.

[0013] S7: Dry, crush, bag, and test the crystals in S6 in a vacuum.

[0014] It should be noted in the solution that the heat preservation temperature in step S1 is 80°C and the heat preservation time is 1 hour.

[0015] Furthermore, it is worth noting that the absolute ethanol in step S1 is 4 - 6 times that of the conversion solution.

[0016] Even further, it should be noted that 40 - 70% ethanol is used in step S4.

[0017] As a preferred implementation manner, 1 - 3% activated carbon is used in step S5.

[0018] As a preferred implementation manner, the heat preservation temperature in step S5 is 80°C and the heat preservation time is 1 hour.

[0019] As a preferred implementation manner, the crystallization cooling temperature in step S5 is 5°C.

[0020] As a preferred implementation manner, the crystal drying temperature in step S7 is 85°C and the drying time is 3 - 6 hours.

[0021] Compared with the prior art, the extraction method for the enzymatic conversion of Reb A to Reb M provided by the present invention has at least the following beneficial effects:

[0022] By setting resin adsorption and analysis to remove most other sugar impurities, the external standard content of stevioside M after concentration and crystallization treatment is not less than 95%, and the yield reaches 86%. At the same time, the purification method for the conversion solution of enzymatically converted stevioside M has a simple process, only requiring basic industrial raw materials such as pure water and ethanol, with low cost, high purification yield, and no need for complex secondary treatment to obtain high-purity and high-content stevioside M, which has great industrial promotion and application value. Description of the Drawings

[0023] Figure 1 It is the detection spectrum of the conversion liquid in step S1 of the present invention;

[0024] Figure 2 It is the detection spectrum of the absorption tail liquid in step S3 of the present invention;

[0025] Figure 3 It is the detection spectrum of the mother liquid in step S6 of the present invention;

[0026] Figure 4 It is the detection spectrum of the crystal in step S7 of the present invention. Detailed implementation manners

[0027] The following describes in detail an extraction method for converting Reb A into Reb M by enzymatic catalysis provided by the present invention in combination with the accompanying drawings and specific embodiments. At the same time, it should be noted here that in order to make the embodiments more detailed, the following embodiments are the best and preferred embodiments. For some well-known technologies, those skilled in the art can also adopt other alternative methods for implementation; moreover, the accompanying drawings are only for more specifically describing the embodiments and are not intended to specifically limit the present invention.

[0028] It should be noted that in the specification, when referring to "an embodiment", "embodiment", "exemplary embodiment", "some embodiments", etc., it indicates that the described embodiment may include specific features, structures or characteristics, but not necessarily every embodiment includes such specific features, structures or characteristics. Additionally, when combining embodiments to describe specific features, structures or characteristics, implementing such features, structures or characteristics in combination with other embodiments (whether explicitly described or not) should be within the knowledge of those skilled in the relevant art.

[0029] Generally, the terms can be understood at least in part from their use in the context. For example, at least in part depending on the context, the term "one or more" used herein can be used to describe any feature, structure or characteristic in a singular sense, or can be used to describe a combination of features, structures or characteristics in a plural sense. Additionally, the term "based on" can be understood as not necessarily aiming to convey a set of exclusive factors, but rather, at least in part depending on the context, allowing for the existence of other factors that may not be explicitly described.

[0030] It can be understood that the meanings of "on...", "above...", and "over..." in the present invention should be interpreted in the broadest manner, such that "on..." not only means "directly on" something, but also includes the meaning of being "on" something with intermediate features or layers therebetween, and "above..." or "over..." not only means "above" or "over" something, but also can include the meaning of being "above" or "over" something with no intermediate features or layers therebetween.

[0031] In addition, spatial relative terms such as "under", "below", "lower part", "above", "upper part", etc. may be used in this text for convenience of description to describe the relationship between one element or feature and another or more elements or features, as shown in the drawings. The spatial relative terms are intended to cover different orientations in the use or operation of the device in addition to the orientation depicted in the drawings. The device may be oriented in other ways, and the spatial relative descriptive terms used in this text may be similarly interpreted accordingly.

[0032] The following embodiments are used to illustrate the present invention, but cannot be used to limit the protection scope of the present invention. The conditions in the embodiments can be further adjusted according to specific conditions. Any simple improvement of the method of the present invention under the premise of the concept of the present invention belongs to the scope of protection required by the present invention.

[0033] Please refer to Figures 1-4 , the present invention provides an extraction method for enzymatically catalyzing the conversion of Reb A to Reb M, comprising the following steps:

[0034] S1: Add 4 - 6 times absolute ethanol to the conversion solution, stir evenly, heat to 80 °C and keep warm for 1 hour, and remove the thallus through a ceramic membrane;

[0035] S2: When the ethanol in the filtrate obtained in step S1 is distilled under negative pressure until there is no ethanol, add pure water until it is dissolved;

[0036] S3: Adsorb the solution obtained in step S2 into an AB - 8 column at a rate of one volume per unit time. After the feeding is completed, wash it with pure water until it is colorless;

[0037] S4: Elute the solution in S3 with 40 - 70% ethanol at a rate of one volume per unit time to obtain an eluate

[0038] S5: Add 1 - 3% activated carbon to the eluate in S4, heat to 80 °C and keep warm for 1 hour, filter out the activated carbon by vacuum filtration, concentrate under negative pressure until crystals precipitate, and then cool down to 5 °C for crystallization;

[0039] S6: Filter the crystals in S5 overnight by suction filtration, wash them clean with pure water, and repeat step S3 for the mother liquor;

[0040] S7: Dry the crystals in S6 in a vacuum at 85 °C for 3 - 6 hours, then pulverize, bag and detect them.

[0041] Unless otherwise defined, the technical terms or scientific terms used in the present invention shall have the ordinary meanings as understood by those of ordinary skill in the field to which the present invention pertains. The words such as "comprising" or "including" used in the present invention mean that the elements or items appearing before this word cover the elements or items listed after this word and their equivalents, without excluding other elements or items. The words such as "connected" or "coupled" do not limit to physical or mechanical connections, and may also include electrical connections, whether direct or indirect. The terms such as "upper", "lower", "left", and "right" are only used to represent relative positional relationships. When the absolute position of the object being described changes, the relative positional relationship may also change accordingly.

[0042] Although the embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. An extraction method for enzymatically converting Reb A to Reb M, comprising the following steps: S1: adding anhydrous ethanol to the transformation solution and stirring evenly to keep it warm and remove the bacteria; S2: subjecting the filtrate obtained in step S1 to negative pressure distillation, and then adding pure water until dissolved; S3: The solution obtained in step S2 is adsorbed on an AB-8 column and washed with pure water until colorless; S4: Analyzing the solution in S3 to obtain an analytical solution; S5: Add activated carbon to the analytical solution in S4, then keep warm, vacuum filter to remove the activated carbon, and concentrate under negative pressure until crystals precipitate, and finally cool and crystallize; S6: Filter the crystals in S5 overnight, wash them with pure water, and repeat step S3 for the mother liquor; S7: Dry, crush, bag and inspect the crystals in S6 in a vacuum.

2. The method of claim 1 for enzymatically converting Reb A into Reb M, characterized in that: In step S1, the insulation temperature is 80° C. and the insulation time is 1 hour.

3. The method of claim 1 for enzymatically converting Reb A into Reb M, characterized in that: In step S1, the amount of anhydrous ethanol is 4-6 times that of the conversion solution.

4. The method of claim 1 for enzymatically converting Reb A into Reb M, characterized in that: In step S4, 40-70% ethanol is used.

5. The method of claim 1 for enzymatically converting Reb A into Reb M, characterized in that: In the step S5, 1-3% activated carbon is used.

6. The method of claim 1 for enzymatically converting Reb A into Reb M, characterized in that: In step S5, the insulation temperature is 80° C. and the insulation time is 1 hour.

7. The method of claim 1 for enzymatically converting Reb A into Reb M, characterized in that: The crystallization cooling temperature in step S5 is 5°C.

8. The method of claim 1 for enzymatically converting Reb A into Reb M, characterized in that: In step S7, the crystal drying temperature is 85° C. and the drying time is 3-6 hours.

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