CN116813679A - Stevioside RN crystal form H1 and preparation method and application thereof - Google Patents

Abstract

The invention discloses a steviol glycoside RN crystal form H1 and its preparation method and use. It relates to the field of sweeteners. The steviol glycoside RN crystal form H1 uses X-ray powder diffraction analysis measured with Cu-Kα rays, and is expressed in degrees. The 2θ is 3.7±0.2, 5.6±0.2, 7.6±0.2, 8.6±0.2, 9.0±0.2, 11.4±0.2, 12.0±0.2, 12.6±0.2 and 18.3±0.2. There are obvious characteristic diffraction peaks at stevia. Glycoside RN crystal form H1 has good crystallinity, low hygroscopicity and high chemical stability.

Description

A kind of steviol glycoside RN crystal form H1 and its preparation method and use

Technical field

The present invention relates to the field of sweeteners, and in particular to a steviol glycoside RN crystal form H1 and its preparation method and use.

Background technique

Stevia is native to the Amambai Mountains on the border between Paraguay and Brazil in South America. It is a plant with a high sweetness. White powdered steviol glycoside extracted from stevia is a pure natural, high-intensity, zero-calorie sweetener and natural sugar substitute. Stevia is the third best-selling product in the international high-intensity sweetener market. The first generation of stevia was mainly mixed sugar, but it was often accompanied by a bitter taste. The second-generation stevia sugar is mainly composed of highly pure steviol glycoside A and has been widely used in food, health products and medicines. With the continuous popularity of steviol glycoside A, other glycosides in stevia are gradually receiving attention, such as steviol glycoside B, steviol glycoside C, stevia glycoside D, stevia glycoside M and stevia glycoside N. The combination of different steviol glycosides is expected to provide similar flavor characteristics to sucrose.

Steviol glycoside RN (also known as rebaudioside N) has a molecular formula of C ₅₆ H ₉₀ O ₃₂ and a structure of:

Chinese patent CN109890973A discloses an enzymatic preparation method of Rebaudioside N, which uses Rebaudioside A or Rebaudioside J as the substrate, and makes the substrate react with UDP sugar in the presence of a glycosyl donor. The reaction is catalyzed by UDP glycosyltransferase and/or recombinant cells containing UDP glycosyltransferase to generate rebaudioside N.

Chinese patent CN111868252A discloses the biosynthetic production of the steviol glycosides rebaudioside J and rebaudioside N, which uses rebaudioside A as a substrate and involves the biosynthesis of various 1,2RhaT-rhamnosyltransferases Pathway to produce the steviol glycoside Rebaudioside N.

As we all know, different crystal forms may lead to differences in color, morphology, stability, hygroscopicity and solubility, thereby affecting the storage conditions, appearance and taste of food. Different crystal forms of stevia sugar compounds have a great impact on their taste, stability, hygroscopicity and solubility. Chinese patents CN103739639A and CN103739640A report two crystal forms of steviol glycoside A. Among them, 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 a stevia D glycoside crystal form A, its preparation method and its application. Stevia D glycoside crystal form A has the advantages of high crystallinity, good water solubility and high chemical stability. At present, studies on the crystal forms of steviol glycoside A, steviol glycoside B, steviol glycoside C, and stevia glycoside D have been reported, but the crystal form of steviol glycoside N has never been reported.

There is an urgent need in this field to provide a crystal form of Steviol Glycoside N with better properties, such as a new crystal form with good crystallinity, low hygroscopicity and high stability. At the same time, there is an urgent need to provide preparation methods and uses of the above crystal forms.

Contents of the invention

The first technical problem to be solved by the present invention is to provide a steviol glycoside RN crystal form H1 with good crystallinity, low hygroscopicity and high chemical stability in view of the shortcomings of the existing technology.

In order to solve the above-mentioned first technical problem, the technical solution of the present invention is:

A kind of steviol glycoside RN crystal form H1, the crystal form H1 uses X-ray powder diffraction analysis measured by Cu-Kα ray, and the 2θ expressed in degrees is 3.7±0.2, 5.6±0.2, 7.6±0.2, 8.6± There are obvious characteristic diffraction peaks at 0.2, 9.0±0.2, 11.4±0.2, 12.0±0.2, 12.6±0.2 and 18.3±0.2.

Preferably, the thermogravimetric analysis chart of the crystal form H1 shows a weight loss of 5.2% at 30-100°C and begins to decompose at 250°C.

Preferably, the differential scanning calorimetry spectrum of the crystal form H1 has a characteristic endothermic peak in the range of 30 to 180°C.

Preferably, the dynamic moisture adsorption pattern of the crystalline form H1 is in the range of 0-35% relative humidity, and the mass percentage of water absorbed is 0-3.4%; in the range of relative humidity 35-40%, the mass percentage of water absorbed is 0-3.4%. The mass percentage of water is 3.4-6.1%; in the relative humidity range of 40-80%, the mass percentage of moisture absorbed is 6.1-9.2%.

The infrared spectrum of the crystalline form HT is at 3300cm ^-1 , 2920cm ^-1 , 1731cm -1 , 1635cm ^-1 , 1639cm ^-1 , 1388cm ^-1 , 1333cm ^-1 , 1226cm ^-1 , 1069cm ^{-1 ,} 1017cm ^-1 , There are characteristic peaks at 989cm ^-1 and 895cm ^-1 , and the error range is ±2cm ^-1 .

The second technical problem to be solved by the present invention is to provide a method for preparing the steviol glycoside RN crystal form H1 in view of the shortcomings of the existing technology. The prepared steviol glycoside RN crystal form H1 has good crystallinity, low hygroscopicity, High chemical stability.

In order to solve the above second technical problem, the technical solution of the present invention is:

A method for preparing steviol glycoside RN crystal form H1. The preparation method is one or more mixed crystallization methods selected from the suspension method, solution evaporation method, cooling method or anti-solvent method, and includes the following steps:

(1) Suspension: Mix steviol glycoside RN and solvent 1 in the temperature range of 0 to 100°C for 1 to 48 hours to obtain a suspension solution;

(2) Cooling: Dissolve steviol glycoside RN in one or more solvents such as methanol, ethanol, and water in the temperature range of 30 to 100°C to form a saturated solution, filter while it is hot, and cool the filtrate to 0 to 30°C. temperature until a large amount of white solid precipitates to obtain a suspension solution;

(3) Volatilization: Dissolve steviol glycoside RN in solvent 2 and volatilize at room temperature with a vacuum pressure less than or equal to 0.1MPa until a large amount of white solid precipitates to obtain a suspension solution;

(4) Antisolvent: Dissolve stevia RN in methanol or water to prepare a saturated solution of RN, and add the poor solvent dropwise until a large amount of white solid precipitates to obtain a suspension solution;

(5) Filtration: Filter or centrifuge the suspension solution in steps (1), (2), (3) or (4) in the temperature range of 0 to 100°C to obtain a white solid, which is dried to obtain steviol glycoside RN. Crystal form H1.

Preferably, the dry matter purity of steviol glycoside RN described in step (1) is between 80% and 100%.

Preferably, the solvent 1 described in step (1) is methanol, ethanol, isopropyl alcohol, acetonitrile, acetone, methyl ethyl ketone, tetrahydrofuran, ethyl acetate, isopropyl acetate, n-hexane, n-heptane, dichloromethane, One or more than two kinds of chloroform, or a mixed solvent of the above solvents and water;

Solvent 2 in step (3) is a mixed solvent of methanol, ethanol, isopropyl alcohol, acetonitrile, acetone, methyl ethyl ketone, tetrahydrofuran, ethyl acetate, isopropyl acetate, dichloromethane, chloroform and water.

The third technical problem to be solved by the present invention is to provide a food composition containing the stevioside RN crystal form H1.

The fourth technical problem to be solved by the present invention is to provide the application of the stevioside RN crystal form H1 and its preparation method in the preparation of food, health products and medicines.

Due to the adoption of the above technical solutions, the beneficial effects of the present invention are:

The preparation method of the steviol glycoside RN crystal form H1 provided by the present invention has a simple process and is easy to operate, and the prepared product has high crystallinity, low hygroscopicity and good stability.

Description of the drawings

Figure 1 is an X-ray powder diffraction (XRPD) pattern of the steviol glycoside RN crystal form H1 provided by the present invention;

Figure 2 is a differential scanning calorimetry (DSC) diagram of the steviol glycoside RN crystal form H1 provided by the present invention;

Figure 3 is a thermogravimetric analysis (TG) diagram of the steviol glycoside RN crystal form H1 provided by the present invention;

Figure 4 is a hygroscopicity analysis (DVS) diagram of the steviol glycoside RN crystal form H1 provided by the present invention;

Figure 5 is an infrared (IR) diagram of the steviol glycoside RN crystal form H1 provided by the present invention;

Figure 6 is a comparison chart of the hygroscopicity (DVS) of the steviol glycoside RN crystal form H1 and the amorphous form provided by the present invention;

Figure 7 is an X-ray powder diffraction (XRPD) comparison chart of the stevioside RN crystal form H1 provided by the present invention when stored for two weeks at °C and a relative humidity of 75%.

Detailed ways

The present invention will be further described below in conjunction with the examples.

Example 1

At room temperature, add 0.2g steviol glycoside RN to 10.0 mL methanol, heat to dissolve, filter and bring to room temperature. Leave at room temperature for 24 hours and a large amount of solid will precipitate. Filter to obtain a white solid. The white solid is vacuum dried at 25°C to obtain Steviol glycoside RN crystal form H1.

Example 2

At room temperature, add 0.2g of steviol glycoside RN to 10.0 mL of a mixed solution of ethanol: water = 1:1 (v/v), and volatilize at room temperature and vacuum pressure of 0.1MPa until a large amount of white solid precipitates to obtain The suspension solution was centrifuged and dried to obtain Steviol glycoside RN crystal form H1.

Example 3

At room temperature, add 0.2g of steviol glycoside RN to 10.0 mL of acetone, stir for 36 hours at room temperature, filter to obtain a white solid, and dry the white solid under vacuum at 25°C to obtain the crystal form H1 of steviol glycoside RN.

Example 4

At room temperature, add 0.2g of steviol glycoside RN to 10.0 mL of ethyl acetate, stir for 48 hours at room temperature, filter to obtain a white solid, and dry the white solid under vacuum at 25°C to obtain the crystal form H1 of steviol glycoside RN.

Example 5

At room temperature, 0.2g of steviol glycoside RN was added to 10.0 mL of n-hexane, stirred at room temperature for 16 hours, filtered to obtain a white solid, and the white solid was vacuum dried at 25°C to obtain steviol glycoside RN crystal form H1.

Example 6

At room temperature, add 0.2g of steviol glycoside RN to 10.0 mL of ethanol, stir for 24 hours at room temperature, filter to obtain a white solid, and dry the white solid under vacuum at 25°C to obtain the crystal form H1 of steviol glycoside RN.

Example 7

At room temperature, add 0.2g steviol glycoside RN to 10.0mL anhydrous methanol, filter to obtain a saturated solution, add 50mL of poor solvent acetone to the saturated solution while stirring, a large amount of solid precipitates, filter to obtain a white solid, and the white solid is obtained at 25 After vacuum drying at ℃, the steviol glycoside RN crystal form H1 was obtained.

Example 8

At room temperature, add 0.2g steviol glycoside RN to 10.0mL anhydrous methanol, filter to obtain a saturated solution, add 50mL of poor solvent ethyl acetate to the saturated solution while stirring, a large amount of solid precipitates, filter to obtain a white solid, white solid After vacuum drying at 25°C, the steviol glycoside RN crystal form H1 was obtained.

Example 9

At 70°C, add 0.2g of steviol glycoside RN to 10.0 mL of a mixed solution of n-hexane: n-heptane = 1:1 (v/v), stir at 70°C for 8 hours, and filter to obtain a white solid. After vacuum drying at 25°C, the steviol glycoside RN crystal form H1 was obtained.

Example 10

At 100°C, add 0.4g of steviol glycoside RN to 10.0 mL of water, stir at room temperature for 3 hours, filter to obtain a white solid, and dry the white solid under vacuum at 25°C to obtain steviol glycoside RN crystal form H1.

Example 11

At 50°C, add 0.2g steviol glycoside RN to 10.0mL methanol, mix and stir for 1 hour, filter while hot to obtain a filtrate, cool the filtrate to 0°C until a white solid precipitates, filter to obtain a white solid, and dry the white solid under vacuum at 25°C , to obtain the steviol glycoside RN crystal form H1.

Example 12

At 90°C, add 0.4g steviol glycoside RN to 10.0mL water, mix and stir for 1 hour, filter while hot to obtain a filtrate, cool the filtrate to 30°C until a white solid precipitates, filter to obtain a white solid, and dry the white solid in a vacuum at 25°C. Steviol glycoside RN crystal form H1 was obtained.

Example 13

At room temperature, add 0.2g of steviol glycoside RN to 10.0 mL of a mixed solution of isopropyl acetate: water = 1:1 (v/v), and volatilize at room temperature and vacuum pressure of 0.05MPa until a large amount of white color precipitates. The solid was filtered, and the resulting suspension solution was dried at room temperature to obtain Steviol glycoside RN crystal form H1.

X-ray powder diffraction analysis (XRPD), differential scanning calorimetry (DSC), thermogravimetric analysis (TG), dynamic moisture adsorption analysis (DVS), etc. were performed on the steviol glycoside RN crystal form H1 prepared in the above example.

XRPD analysis: It uses a Bruker D8 advance diffractometer from Bruker Instruments Co., Ltd. in Germany for detection at room temperature, using Cu–Kα rays. The 2θ angle scan is from 3 degrees to 40 degrees, and the scanning speed is 0.1 degrees/second. The analysis results are shown in Figure 1. The XRPD spectrum shows that the steviol glycoside RN crystal form H1 prepared in the above example has good crystallinity.

In the X-ray powder diffraction pattern of sample powder, the diffraction pattern obtained from a specific crystal form is often characteristic. Because of differences in crystallization conditions, particle size, relative content of the mixture, and other testing conditions, the diffraction spectrum may produce preferential orientation effects, resulting in changes in the relative intensity of certain bands in the spectrum (especially at low angles). Therefore, the relative intensity of the diffraction peak is not characteristic of the target crystal. When judging whether it is the same as a 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, attention should be paid to maintaining the overall concept, because it is not a diffraction line that represents a phase, but a specific set of "d-I/I1" data that represents a certain phase. It should also be pointed out that in the identification of mixtures, some diffraction lines will be missing due to factors such as content reduction. In this case, there is no need to rely on all the bands observed in the high-purity sample. Even one band may be correct for the given sample. Certain crystals are characteristic.

DSC analysis: All DSC spectra of this patent were detected by the DSC Q2000 differential scanning calorimeter of the American TA Company. The atmosphere was nitrogen and the heating rate was 10°C/min.

TG analysis: All TGA spectra of this patent were detected by the TGA 55 thermogravimetric analyzer of the American TA Company. Temperature range: 30-400°C, scan rate: 10°C/min, purge gas: 40mL/min.

DVS analysis: All DVS spectra of this patent were measured by the British SMS company's DVS advantage adsorption instrument. Relative humidity range: 0~95%, temperature: 25°C. The analysis results are shown in Figure 4. The hygroscopicity of the steviol glycoside RN crystal form H1 prepared in the above embodiment is significantly lower than that of the amorphous steviol glycoside RN under the condition of 40%-80% relative humidity. The amorphous form absorbs about 5.9-14.2% of water, and H1 absorbs 6.1-9.2% of water. Comparatively The results are shown in Figure 6.

The steviol glycoside RN crystal form H1 prepared in the above example was stored for two weeks at 40°C and 75% relative humidity. The analysis results are shown in Figure 7. It can be seen from Figure 7 that its crystal form remains unchanged, indicating that this crystal form has good physical stability under normal storage conditions.

It should be understood that these examples are only used to illustrate the invention and are not intended to limit the scope of the invention. In addition, it should be understood that after reading the teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims (10)

1. A steviol glycoside RN crystalline form H1, characterized in that: the crystal form H1 has obvious characteristic diffraction peaks at the positions of 3.7+/-0.2, 5.6+/-0.2, 7.6+/-0.2, 8.6+/-0.2, 9.0+/-0.2, 11.4+/-0.2, 12.0+/-0.2, 12.6+/-0.2 and 18.3+/-0.2 of 2 theta measured by using Cu-K alpha rays through X-ray powder diffraction analysis.

2. A steviol glycoside RN form H1 according to claim 1, wherein: the thermal weight loss analysis spectrum of the crystal form H1 loses weight by 5.2% at 30-100 ℃ and starts to decompose at 250 ℃.

3. A steviol glycoside RN form H1 according to claim 1, wherein: the differential scanning calorimetric spectrum of the crystal form H1 has characteristic endothermic peaks in the range of 30-180 ℃.

4. A steviol glycoside RN form H1 according to claim 1, wherein: the dynamic moisture adsorption spectrum of the crystal form H1 is in the range of 0-35% of relative humidity, and the mass percentage of the absorbed moisture is 0-3.4%; the mass percentage of the absorbed water is 3.4 to 6.1 percent within the range of 35 to 40 percent of relative humidity; the mass percentage of the absorbed moisture is 6.1-9.2% within the relative humidity range of 40-80%.

5. A steviol glycoside RN form H1 according to claim 1, wherein: the infrared spectrum of the crystal form HT is 3300cm ^-1 、2920cm ^-1 、1731cm ^-1 、1635cm ^-1 、1639cm ^-1 、1388cm ^-1 、1333cm ^-1 、1226cm ^-1 、1069cm ^-1 、1017cm ^-1 、989cm ^-1 And 895cm ^-1 The position has characteristic peak with error range of + -2 cm ^-1 。

6. The method for preparing steviol glycoside RN crystalline form H1 according to any one of claims 1 to 5, wherein the preparation method is a mixed crystallization method of one or more of a suspension method, a solution evaporation method, a cooling method or an antisolvent method, comprising the steps of:

(1) Suspending: mixing stevioside RN with a solvent 1 for 1-48 h at the temperature of 0-100 ℃ to obtain a suspension solution;

(2) And (3) cooling: dissolving stevioside RN in one or more solvents of methanol, ethanol and water at the temperature of 30-100 ℃ to prepare a saturated solution, filtering while the saturated solution is hot, and cooling the filtrate to the temperature of 0-30 ℃ until a large amount of white solids are separated out to obtain a suspension solution;

(3) Volatilizing: dissolving stevioside RN in a solvent 2, volatilizing at room temperature, and keeping the vacuum pressure less than or equal to 0.1MPa until a large amount of white solid is separated out to obtain a suspension solution;

(4) Antisolvent: dissolving stevioside RN in methanol or water to prepare a saturated solution of RN, and dripping poor solvent until a large amount of white solid is separated out to obtain a suspension solution;

(5) And (3) filtering: filtering or centrifuging the suspension solution in the step (1), (2), (3) or (4) at the temperature of 0-100 ℃ to obtain white solid, and drying to obtain the stevioside RN crystal form H1.

7. The method for preparing stevioside RN crystal form H1 according to claim 1, wherein the method comprises the following steps: the purity of the stevioside RN dry matter in the step (1) is 80-100%.

8. The method for preparing stevioside RN form H1 according to claim 1, wherein; the solvent 1 in the step (1) is one or more of methanol, ethanol, isopropanol, acetonitrile, acetone, methyl ethyl ketone, tetrahydrofuran, ethyl acetate, isopropyl acetate, n-hexane, n-heptane, dichloromethane and chloroform, or a mixed solvent of the solvent and water;

the solvent 2 in the step (3) is a mixed solvent of methanol, ethanol, isopropanol, acetonitrile, acetone, methyl ethyl ketone, tetrahydrofuran, ethyl acetate, isopropyl acetate, dichloromethane, chloroform and water.

9. A food composition characterized by: the food composition comprising steviol glycoside RN form H1 according to any one of claims 1 to 5.

10. Use of stevioside RN form H1 according to any one of claims 1 to 5 and the process for the preparation of stevioside RN form H1 according to any one of claims 6 to 8 in the preparation of food, health products and pharmaceuticals.