Steviol glycoside ra1g crystal form h4, methods of making and uses

Abstract

The application discloses a steviol glycoside RA1G crystal form H4, a preparation method and application thereof, relates to the technical field of sweeteners, and the X-ray powder diffraction analysis of the crystal form H4 measured by using Cu-K alpha rays has obvious characteristic diffraction peaks at 3.86, 7.89, 9.67, 14.12, 17.49, 20.60, 22.45 and 23.73 in degrees of 2 theta angle, and the obtained crystal form H4 has the advantages of good crystallinity, small hygroscopicity and high chemical stability.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of sweeteners, in particular to a steviol glycoside RA1G crystal form H4, a preparation method and use thereof. BACKGROUND

[0002] Stevia rebaudiana, a plant with high sweet taste, is originally from the Amambai Mountains on the border between Paraguay and Brazil. The white powder steviol glycoside extracted from Stevia rebaudiana is a natural, high-intensity, zero-calorie sweetener. Steviol glycoside is the third best-selling product in the international high-intensity sweetener market. The first generation of steviol glycoside is mainly mixed sugar, but it often has a bitter taste. The second generation of steviol glycoside is mainly high-purity steviol A glycoside, which has been widely used in food, health products and drugs. With the continuous popularity of steviol A glycoside, other glycosides in Stevia rebaudiana have also gradually attracted attention. At present, steviol glycosides used on the market include steviol B glycoside, steviol C glycoside, steviol D glycoside, steviol M glycoside and steviol A glycoside derivative steviol RA1G. Steviol RA1G significantly reduces the bitterness of steviol A glycoside and improves the taste.

[0003] Steviol RA1G (also known as rebaudioside A1G) has a molecular formula of C 50 H 80 O 28 and a structure of:

[0004]

[0005] Chinese patent CN108753871A discloses a double-enzyme method for preparing steviol glycoside derivative rebaudioside AlG and its application. The double-enzyme method uses steviol glycoside rebaudioside A (RA) as the acceptor substrate and glucose as the donor substrate to obtain a new steviol glycoside derivative RA1G. Chinese patent CN108727443A discloses a crystallization method for increasing the content of rebaudioside AlG, the product and its use.

[0006] It is well known that different crystal forms can lead to differences in color, morphology, stability, hygroscopicity and solubility, which in turn affect the storage conditions, appearance and taste of food. Different crystal forms of steviol glycosides have a great influence on their taste, stability, hygroscopicity and solubility. Chinese patents CN103739639A and CN103739640A report two crystal forms of steviolbioside, wherein crystal form 7 has the advantages of good taste and low hygroscopicity, and crystal form 9 has the advantages of high stability and high water solubility. Chinese patent CN105037458A discloses that crystal form A of steviolbioside has the advantages of high crystallinity, good water solubility and high chemical stability. At present, the crystal forms of steviolbioside A, steviolbioside B, steviolbioside C and steviolbioside D have been reported, but the crystal form of steviol glycoside RA1G has never been reported.

[0007] There is an urgent need in the art to provide a crystal form of steviol glycoside RA1G with better performance, such as a new crystal form with good crystallinity, low hygroscopicity and high chemical stability. At the same time, there is an urgent need to provide a preparation method and use of the above crystal form. SUMMARY

[0008] The first technical problem to be solved by the present application is to provide a steviol glycoside RA1G crystal form H4 with good crystallinity, low hygroscopicity and high chemical stability to overcome the shortcomings of the prior art.

[0009] To solve the above-mentioned first technical problem, the technical solution of the present application is:

[0010] A steviol glycoside RA1G crystal form H4, wherein the crystal form H4 has obvious characteristic diffraction peaks at 2θ angles of 3.86, 7.89, 9.67, 14.12, 17.49, 20.60, 22.45 and 23.73, measured by X-ray powder diffraction analysis using Cu-Kα rays.

[0011] Preferably, the crystal form G has the following characteristics: the 2θ values measured by X-ray powder diffraction analysis using Cu-Kα rays, the error range is ±0.2°, the interplanar spacing d expressed in

[0012]

[0013]

[0014] Preferably, the differential scanning calorimetry pattern of the crystal form H4 has characteristic endothermic peaks in the range of about 30-100℃ and 200-225℃.

[0015] Preferably, the thermal gravimetric analysis of the crystal form H4 starts to decompose at 210±20℃.​

[0016] Preferably, the dynamic water adsorption spectrum of the crystal form H4 has a moisture absorption percentage of 0-0.8% at a relative humidity of 0-40%; and a moisture absorption percentage of 0.8-1.1% at a relative humidity of 40-80%.

[0017] Preferably, the infrared spectrum of the crystal form H4 has characteristic peaks at at least 3343cm-1, 2943cm-1, 1727cm-1, 1744cm-1, 1341cm-1, 1222cm-1, 1035cm-1, 910cm-1and 568cm-1, with an error range of ±2cm-1. -1 -1 -1 -1 -1 -1 -1 -1 -1 . -1 .

[0018] The second technical problem to be solved by the present application is to provide a preparation method of steviol glycoside RA1G crystal form H4 to address the deficiencies in the prior art, so that the prepared steviol glycoside RA1G crystal form H4 has good crystallinity, low hygroscopicity and high chemical stability.

[0019] To solve the above-mentioned second technical problem, the technical scheme of the present application is:

[0020] A preparation method of steviol glycoside RA1G crystal form H4, the preparation method being one or a combination of two or more of suspension method, solution evaporation method or cooling method, comprising the following steps:

[0021] (1) Suspension: mixing steviol glycoside RA1G with a solvent at a temperature of 0-100℃ for 1-48h to obtain a suspension solution;

[0022] (2) Cooling: filtering the suspension solution in step (1) while hot, and cooling the filtrate to a temperature of 0-30℃ until a large amount of white solid is precipitated to obtain a suspension solution;

[0023] (3) Evaporation: evaporating the suspension solution in step (1) after filtration at room temperature under a vacuum pressure of less than or equal to 0.1MPa until a large amount of white solid is precipitated to obtain a suspension solution;

[0024] (4) Filtration: filtering or centrifuging the suspension solution in step (1), (2) or (3) at a temperature of 0-100℃ to obtain white solid, which is dried to obtain steviol glycoside RA1G crystal form H4.

[0025] ​​​​​​​​Further, the dry substance purity of the steviol glycoside RA1G in step (1) is in the range of 80-100%;

[0026] The solvent in step (1) is one or more of methanol, ethanol, acetonitrile, acetone, tetrahydrofuran, isopropanol, or a mixture of the above-mentioned solvents and water or water.

[0027] A third technical problem to be solved by the present application is to provide a food composition containing the steviol glycoside RA1G crystal form H4.

[0028] A fourth technical problem to be solved by the present application is to provide the application of the steviol glycoside RA1G crystal form H4 and the preparation method thereof in the preparation of food, health products and medicines.

[0029] Due to the adoption of the above technical solutions, the present application has the following beneficial effects:

[0030] The preparation method of the steviol glycoside RA1G crystal form H4 provided by the present application has the advantages of simple process, easy operation, and the ability to prepare the steviol glycoside RA1G crystal form H4 through various methods. The prepared product has high crystallinity, low hygroscopicity, and high stability. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 is the X-ray powder diffraction (XRPD) pattern of the steviol glycoside RA1G crystal form H4 provided by the present application;

[0032] Figure 2 is the differential scanning calorimetry (DSC) pattern of the steviol glycoside RA1G crystal form H4 provided by the present application;

[0033] Figure 3 is the thermogravimetric analysis (TG) pattern of the steviol glycoside RA1G crystal form H4 provided by the present application;

[0034] Figure 4 is the hygroscopicity analysis (DVS) pattern of the steviol glycoside RA1G crystal form H4 provided by the present application;

[0035] Figure 5 is the infrared (IR) pattern of the steviol glycoside RA1G crystal form H4 provided by the present application;

[0036] Figure 6 is the hygroscopicity (DVS) comparison pattern of the steviol glycoside RA1G crystal form H4, crystal form H1 and crystal form H2 provided by the present application;

[0037] Figure 7 is the X-ray powder diffraction (XRPD) comparison pattern of the steviol glycoside RA1G crystal form H4 stored at 40℃ and 75% relative humidity for two weeks provided by the present application. DETAILED DESCRIPTION

[0038] The application is further described below in conjunction with examples.

[0039] Example 1

[0040] At room temperature, 0.2 g of steviol glycoside (crystal form H1) RA1G with a purity of 99% was added into 1 mL of pure water, and a large amount of solid was precipitated after standing for 12 h. The white solid was obtained by filtration, and the white solid was dried at 25 °C under vacuum to obtain steviol glycoside RA1G crystal form H4.

[0041] Example 2

[0042] At 50 °C, 2.0 g of steviol glycoside (crystal form H1) RA1G with a purity of 99% was added into 1 mL of acetonitrile, and a large amount of solid was precipitated after mixing for 2 h. The white solid was obtained by filtration, and the white solid was dried at 15 °C under vacuum to obtain steviol glycoside RA1G crystal form H4.

[0043] Example 3

[0044] At room temperature, 0.2 g of steviol glycoside (crystal form H1) RA1G with a purity of 99% was added into 1 mL of mixed solvent (methanol and water in a volume ratio of 1:3), and a large amount of solid was precipitated after standing for 36 h at room temperature. The white solid was obtained by filtration, and the white solid was dried at 25 °C under vacuum to obtain steviol glycoside RA1G crystal form H4.

[0045] Example 4

[0046] At 90 °C, 2.0 g of steviol glycoside (crystal form H1) RA1G with a purity of 99% was added into 1 mL of mixed solvent (methanol and acetone in a volume ratio of 1:1), and a large amount of solid was precipitated after standing for 42 h after being placed at room temperature. The white solid was obtained by centrifugation, and the white solid was dried at 25 °C under vacuum to obtain steviol glycoside RA1G crystal form H4.

[0047] Example 5

[0048] At 0 °C, 0.2 g of steviol glycoside (crystal form H1) RA1G with a purity of 99% was added into 1 mL of mixed solvent (tetrahydrofuran and water in a volume ratio of 1:2), and a large amount of solid was precipitated after standing for 1 h at 0 °C. The white solid was obtained by centrifugation, and the white solid was dried at 25 °C under vacuum to obtain steviol glycoside RA1G crystal form H4.

[0049] Example 6

[0050] 70℃, 2.0 g of steviol glycoside (crystalline form H1) RA1G with a purity of 99% was added into 1 mL of mixed solvent (acetonitrile and water with a volume ratio of 1:3), and a large amount of solid was precipitated after being placed at 50℃ for 48 h. White solid was obtained by filtration, and the white solid was dried at 25℃ under vacuum to obtain steviol glycoside RA1G crystalline form H4.

[0051] Example 7

[0052] 30℃, 20 g of steviol glycoside (crystalline form H1) RA1G with a purity of 99% was added into 10 mL of pure water, and a large amount of solid was precipitated after being placed at room temperature for 24 h. White solid was obtained by filtration, and the white solid was dried at 25℃ under vacuum to obtain steviol glycoside RA1G crystalline form H4.

[0053] Example 8

[0054] 50℃, 20 g of steviol glycoside (crystalline form H1) RA1G with a purity of 99% was added into 10 mL of pure water, and the filtrate and residue were obtained by filtration while hot after being placed at 50℃ for 24 h. The filtrate was cooled to 25℃ to obtain a suspension solution, and white solid was obtained by centrifugation of the suspension solution. The white solid and residue were dried at 25℃ under vacuum to obtain steviol glycoside RA1G crystalline form H4.

[0055] Example 9

[0056] 50℃, 20 g of steviol glycoside (crystalline form H1) RA1G with a purity of 99% was added into 10 mL of pure water, and the filtrate and residue were obtained by filtration while hot after being placed at 50℃ for 24 h. The filtrate was evaporated at room temperature under a vacuum pressure of 0.1 MPa until a large amount of solid was precipitated to obtain a suspension solution. White solid was obtained by filtration of the suspension solution, and the white solid and residue were dried at 25℃ under vacuum to obtain steviol glycoside RA1G crystalline form H4.

[0057] Example 10

[0058] 25℃, 10 g of steviol glycoside RA1G (amorphous) with a purity of 99% was added into 200 mL of ethanol (with a purity of 95%), and white solid was obtained by filtration after stirring for 24 h. The white solid was dried at 25℃ under vacuum to obtain steviol glycoside RA1G crystalline form H1.

[0059] Example 11

[0060] 80%±5% relative humidity, 25℃, 5 g of crystalline form H1 of Example 10 was placed flat for 2 days to obtain steviol glycoside RA1G crystalline form H2.

[0061] The steviol glycoside RA1G crystal form H4 prepared in the above examples was subjected to X-ray powder diffraction analysis (XRPD), differential scanning calorimetry analysis (DSC), thermogravimetric analysis (TG), dynamic vapor sorption analysis (DVS), etc.

[0062] XRPD analysis: It was detected by a diffractometer of Bruker D8 advance of Bruker Corporation in Germany at room temperature, using Cu-Ka ray 2θ angle scanning from 3 degrees to 40 degrees, scanning speed was 0.1 degree / s. The analysis results are shown in Figure 1 . The XRPD spectrum showed that the steviol glycoside RA1G crystal form H4 prepared in the above examples had good crystallinity.

[0063] In the X-ray powder diffraction spectrum of the sample powder, the diffraction spectrum obtained from a specific crystal form is often characteristic. Because of the differences in crystallization conditions, particle size, relative content of the mixture and other test conditions, the diffraction spectrum may have a preferred orientation effect, resulting in changes in the relative intensity of some bands in the spectrum (especially at low angles). Therefore, the relative intensity of the diffraction peak is not characteristic of the crystal, and when judging whether it is the same as the known crystal form, more attention should be paid to the position of the peak rather than their relative intensity. In addition, when judging whether the crystal forms are the same, the overall concept should be paid attention to, because not a diffraction line represents a phase, but a specific "d-I / I1" data represents a phase. It should also be pointed out that in the identification of mixtures, the decrease in content and other factors may cause the absence of some diffraction lines, at this time, it is not necessary to rely on all the bands observed in high-purity samples, even one band may be characteristic of the given crystal.

[0064] DSC analysis: All DSC spectra of the present patent were detected by a differential scanning calorimeter of DSC Q2000 of TA Corporation in the United States, the atmosphere was nitrogen, and the heating speed was 10 ℃ / min.

[0065] TG analysis: All TGA spectra of the present patent were detected by a thermogravimetric analyzer of TGA 55 of TA Corporation in the United States, temperature range: 30-400 ℃, scanning rate: 10 ℃ / min, purge gas: 40 mL / min.

[0066] DVS analysis: All DVS spectra of the present patent were measured by an adsorber of DVS advantage of SMS Corporation in the United Kingdom, relative humidity range: 0-95%, temperature: 25 ℃. The analysis results are shown in Figure 4 . The hygroscopicity of the steviol glycoside RA1G crystal form H4 prepared in the above examples was significantly lower than that of the steviol glycoside RA1G crystal form H1 and crystal form H2 at 25 ℃, 40% RH, and the comparison results are shown in Figure 6See Table 1. Meanwhile, the hygroscopicity of steviol glycoside RA1G crystal form H4 is lower than that of crystal forms H1 and H2 under normal storage conditions (40%-80% RH).

[0067] Table 1

[0068]

[0069] The steviol glycoside RA1G crystal form H4 obtained in the above examples was stored at 40°C and 75% RH for two weeks. The analytical results are shown in [Figure number missing]. Figure 7 .from Figure 7 The results show that its crystal form remains unchanged, indicating that the crystal form has good physical stability under normal storage conditions.

[0070] The steviol glycoside RA1G raw material (crystal form H1) used in the above embodiments was provided by Shandong Zhucheng Haotian Pharmaceutical Co., Ltd.

[0071] It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A steviol glycoside RA1G crystalline Form H4 characterized by: The X-ray powder diffraction analysis of the crystal form H4 has obvious characteristic diffraction peaks at 2θ angles of 3.86, 7.89, 9.67, 14.12, 17.49, 20.60, 22.45 and 23.73, measured using Cu-Kα rays.

2. A steviol glycoside RA1G crystalline Form H4 according to claim 1, characterized in that, The X-ray powder diffraction analysis of the crystal form H4 has the following characteristics: 2θ values in degrees, error range of ±0.2°, interplanar spacing d in Å, and relative intensity of diffraction peaks in percentage: 。 3. The steviol glycoside RA1G crystalline Form H4 of claim 1, characterized by: The differential scanning calorimetry analysis of the crystal form H4 has characteristic endothermic peaks in the temperature ranges of 30-100℃ and 200-225℃.

4. The steviol glycoside RA1G crystalline Form H4 of claim 1, characterized by: The thermal gravimetric analysis of the crystal form H4 starts to decompose at 210±20℃.

5. A steviol glycoside RA1G crystalline Form H4 according to claim 1, characterized by: The dynamic moisture adsorption analysis of the crystal form H4 has a moisture absorption percentage of 0-0.8% at a relative humidity of 0-40%, and a moisture absorption percentage of 0.8-1.1% at a relative humidity of 40-80%.

6. A steviol glycoside RA1G crystalline Form H4 according to claim 1, characterized by: The infrared spectrum of said crystalline Form H4 has characteristic peaks at 3343 cm -1 -1, 2943 cm -1 -1, 1727 cm -1 -1, 1744 cm -1 -1, 1341 cm -1 -1, 1222 cm -1 -1, 1035 cm -1 -1, 910 cm -1 -1, and 568 cm -1 -1, with an error range of ± 2 cm -1 -1.

7. The method of preparing steviol glycoside RA1G crystalline Form H4 according to any one of claims 1-6, wherein, The preparation method is one or a combination of more than two of suspension method, solution evaporation method or cooling method, comprising the following steps: (1) Suspension: mixing steviol glycoside RA1G with a solvent at a temperature of 0-100℃ for 1-48h to obtain a suspension solution, wherein the solvent is one or a combination of more than two of methanol, ethanol, acetonitrile, acetone, tetrahydrofuran, isopropanol, or a mixture of the above-mentioned solvents and water, or water; (2) Cooling: filtering the suspension solution of step (1) while hot, and cooling the filtrate to a temperature of 0-30℃ until a large amount of white solid is precipitated to obtain a suspension solution; (3) Evaporation: filtering the suspension solution of step (1), and evaporating at room temperature under a vacuum pressure of less than or equal to 0.1 MPa until a large amount of white solid is precipitated to obtain a suspension solution; (4) Filtration: filtering or centrifuging the suspension solution of step (1), (2) or (3) at a temperature of 0-100℃ to obtain white solid, and drying to obtain steviol glycoside RA1G crystal form H4.

8. The method of preparing steviol glycoside RA1G crystalline Form H4 of claim 7, wherein: The steviol glycoside RA1G in step (1) has a dry matter purity of 80-100%.

9. A food composition characterized in that: The food composition contains the steviol glycoside RA1G crystal form H4 according to any one of claims 1-5.

10. Application of the steviol glycoside RA1G crystal form H4 obtained by the preparation method of the steviol glycoside RA1G crystal form H4 according to any one of claims 1-6 and 7-8 in the preparation of food and health products.

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