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

Stevioside RN crystal form H1 and preparation method and application thereof Download PDF

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Publication number
CN116813679A
CN116813679A CN202310748737.0A CN202310748737A CN116813679A CN 116813679 A CN116813679 A CN 116813679A CN 202310748737 A CN202310748737 A CN 202310748737A CN 116813679 A CN116813679 A CN 116813679A
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stevioside
crystal form
solvent
steviol glycoside
white solid
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Inventor
朱理平
梅雪峰
宋维才
朱冰清
何冬生
李胜强
池磊
刘浩
鞠敏
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Dongtai Haorui Biological Technology Co ltd
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Dongtai Haorui Biological Technology Co ltd
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Abstract

The application discloses a stevioside RN crystal form H1 and a preparation method and application thereof, and relates to the field of sweeteners, wherein the stevioside RN crystal form H1 has obvious characteristic diffraction peaks at 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 in terms of degrees through X-ray powder diffraction analysis measured by Cu-K alpha rays, and the prepared stevioside RN crystal form H1 has good crystallinity, small hygroscopicity and high chemical stability.

Description

Stevioside RN crystal form H1 and preparation method and application thereof
Technical Field
The application relates to the field of sweeteners, in particular to a stevioside RN crystal form H1 and a preparation method and application thereof.
Background
Stevia rebaudiana is native to the Arman Bayer mountain bordering Paraguay and Brazil in south America and is a plant with high sweetness. The white powdery stevioside extracted from stevia rebaudiana is a natural sweetener with high sweetness and zero calories and natural sugar substitute. Stevioside is the third most popular product in the international high-intensity sweetener market. First generation steviol sugar is based on mixed sugar, but it is often accompanied by a bitter taste. The second generation stevioside, mainly high purity stevioside A, has been widely used in foods, health products and medicines. With the increasing popularity of stevioside A, other glycoside compounds in stevia rebaudiana have also received increasing attention, such as stevioside B, stevioside C, stevioside D, stevioside M and stevioside N. The compounding of different steviol glycosides is expected to provide flavor profiles similar to sucrose.
Stevioside RN (also referred to as rebaudioside N) of formula C 56 H 90 O 32 And the structure is as follows:
chinese patent CN109890973a discloses a method for preparing rebaudioside N by an enzymatic method, in which rebaudioside a or rebaudioside J is used as a substrate, and the substrate is reacted under the catalysis of UDP glycosyltransferase and/or recombinant cells containing UDP glycosyltransferase in the presence of a glycosyl donor to produce rebaudioside N.
Chinese patent CN111868252a discloses the biosynthetic production of steviol glycoside rebaudioside J and rebaudioside N using rebaudioside a as a substrate and a biosynthetic pathway involving various 1,2 rhat-rhamnosyltransferases to produce steviol glycoside rebaudioside N.
It is well known that the difference in crystal forms may result in differences in color, morphology, stability, hygroscopicity and solubility, which in turn affect the storage conditions, appearance and mouthfeel of the food product. The different crystal forms of stevioside compounds have great influence on the taste, stability, hygroscopicity and solubility. Chinese patents CN103739639a and CN103739640a report two crystal forms of stevioside a glycoside, of which 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 stevioside D glycoside crystal form A, a preparation method and application thereof, wherein the stevioside D glycoside crystal form A has the advantages of high crystallinity, good water solubility and high chemical stability. At present, crystal form researches on stevioside A, stevioside B, stevioside C and stevioside D are reported, but crystal forms of stevioside N are never reported.
There is a strong need in the art to provide a crystalline form of steviol glycoside N with better properties, such as good crystallinity, low hygroscopicity and stability Gao Xin. Meanwhile, there is an urgent need to provide a preparation method and use of the above crystal forms.
Disclosure of Invention
The first technical problem to be solved by the application is as follows: aiming at the defects existing in the prior art, the stevioside RN crystal form H1 is provided, and has the advantages of good crystallinity, small hygroscopicity and high chemical stability.
In order to solve the first technical problem, the technical scheme of the application is as follows:
a steviol glycoside RN crystalline form H1, which crystalline form H1 has distinct characteristic diffraction peaks at 2Θ in degrees 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, as determined by X-ray powder diffraction analysis using Cu-ka radiation.
Preferably, 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 ℃.
Preferably, the differential scanning calorimetric spectrum of the crystal form H1 has characteristic endothermic peaks in the range of 30-180 ℃.
Preferably, 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%.
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 。
The second technical problem to be solved by the application is as follows: aiming at the defects existing in the prior art, the preparation method of the stevioside RN crystal form H1 is provided, and the prepared stevioside RN crystal form H1 has the advantages of good crystallinity, small hygroscopicity and high chemical stability.
In order to solve the second technical problem, the technical scheme of the application is as follows:
a preparation method of stevioside RN crystal form H1 is one or more than two mixed crystallization methods selected from a suspension method, a solution volatilization method, a cooling method and an antisolvent method, and comprises the following steps:
(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.
Preferably, the steviol glycoside RN dry matter purity in step (1) is between 80 and 100%.
Preferably, 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 above solvents 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.
The third technical problem to be solved by the application is: a food composition containing the steviol glycoside RN form H1 is provided.
The fourth technical problem to be solved by the application is that: provides the stevioside RN crystal form H1 and the application of the preparation method thereof in the preparation of foods, health products and medicines.
Due to the adoption of the technical scheme, the application has the beneficial effects that:
the preparation method of the stevioside RN crystal form H1 provided by the application has the advantages of simple process, easiness in operation, high crystallinity, low hygroscopicity and good stability.
Drawings
FIG. 1 is an X-ray powder diffraction (XRPD) pattern of stevioside RN form H1 provided by the present application;
FIG. 2 is a Differential Scanning Calorimetric (DSC) diagram of stevioside RN form H1 provided by the present application;
fig. 3 is a thermogravimetric analysis (TG) of stevioside RN form H1 provided by the present application;
FIG. 4 is a graph of the moisture absorption analysis (DVS) of stevioside RN form H1 provided by the present application;
FIG. 5 is an Infrared (IR) diagram of stevioside RN crystal form H1 provided by the present application;
FIG. 6 is a graph comparing steviol glycoside RN form H1 provided by the present application with amorphous hygroscopicity (DVS);
FIG. 7 is a graph comparing X-ray powder diffraction (XRPD) of stevioside RN form H1 provided by the present application stored at 75% relative humidity for two weeks at C.
Detailed Description
The application is further illustrated by the following examples.
Example 1
Under the condition of room temperature, 0.2g of stevioside RN is added into 10.0mL of methanol, heated for dissolution, filtered and then placed to room temperature, and a large amount of solids are separated out after standing for 24 hours at room temperature, the white solids are obtained after filtration, and the white solids are dried in vacuum at 25 ℃ to obtain stevioside RN crystal form H1.
Example 2
0.2g steviol glycoside RN was added to 10.0mL ethanol at room temperature: water = 1: and volatilizing in the mixed solution of 1 (v/v) under the conditions of normal temperature and vacuum pressure of 0.1MPa until a large amount of white solids are separated out, centrifuging the obtained suspension, and drying to obtain the stevioside RN crystal form H1.
Example 3
0.2g stevioside RN is added into 10.0mL acetone at room temperature, stirred for 36H at room temperature, filtered to obtain white solid, and the white solid is dried in vacuum at 25 ℃ to obtain stevioside RN crystal form H1.
Example 4
0.2g stevioside RN is added into 10.0mL ethyl acetate at room temperature, stirred for 48H at room temperature, filtered to obtain white solid, and the white solid is dried in vacuum at 25 ℃ to obtain stevioside RN crystal form H1.
Example 5
0.2g stevioside RN is added into 10.0mL of n-hexane at room temperature, stirred for 16H at room temperature, filtered to obtain a white solid, and the white solid is dried in vacuum at 25 ℃ to obtain stevioside RN crystal form H1.
Example 6
0.2g stevioside RN is added into 10.0mL of ethanol at room temperature, stirred for 24 hours at room temperature, filtered to obtain white solid, and the white solid is dried in vacuum at 25 ℃ to obtain stevioside RN crystal form H1.
Example 7
Under the condition of room temperature, 0.2g of stevioside RN is added into 10.0mL of absolute methanol, a saturated solution is obtained by filtering, 50mL of poor solvent acetone is added into the saturated solution while stirring, a large amount of solids are separated out, a white solid is obtained by filtering, and the white solid is dried in vacuum at 25 ℃ to obtain stevioside RN crystal form H1.
Example 8
Under the condition of room temperature, 0.2g of stevioside RN is added into 10.0mL of absolute methanol, a saturated solution is obtained by filtering, 50mL of poor solvent ethyl acetate is added into the saturated solution while stirring, a large amount of solids are separated out, a white solid is obtained by filtering, and the white solid is dried in vacuum at 25 ℃ to obtain stevioside RN crystal form H1.
Example 9
0.2g steviol glycoside RN was added to 10.0mL of n-hexane at 70 ℃): n-heptane=1: 1 (v/v), stirring for 8 hours at 70 ℃, filtering to obtain a white solid, and vacuum drying the white solid at 25 ℃ to obtain the stevioside RN crystal form H1.
Example 10
0.4g stevioside RN is added into 10.0mL of water at the temperature of 100 ℃, stirred for 3 hours at room temperature, filtered to obtain white solid, and the white solid is dried in vacuum at the temperature of 25 ℃ to obtain stevioside RN crystal form H1.
Example 11
Adding 0.2g stevioside RN into 10.0mL of methanol at 50 ℃, mixing and stirring for 1H, filtering while the mixture is hot to obtain a filtrate, cooling the filtrate to 0 ℃ until white solid is separated out, filtering to obtain white solid, and drying the white solid in vacuum at 25 ℃ to obtain stevioside RN crystal form H1.
Example 12
Adding 0.4g stevioside RN into 10.0mL of water at 90 ℃ for mixing and stirring for 1H, filtering while the mixture is hot to obtain a filtrate, cooling the filtrate to 30 ℃ until white solid is separated out, filtering to obtain white solid, and drying the white solid in vacuum at 25 ℃ to obtain stevioside RN crystal form H1.
Example 13
0.2g stevioside RN was added to 10.0mL isopropyl acetate at room temperature: water = 1: and volatilizing in the mixed solution of 1 (v/v) under the conditions of normal temperature and vacuum pressure of 0.05MPa until a large amount of white solid is separated out, filtering the obtained suspension, and drying filter residues at room temperature to obtain the stevioside RN crystal form H1.
X-ray powder diffraction analysis (XRPD), differential scanning calorimetric analysis (DSC), thermogravimetric analysis (TG), dynamic moisture adsorption analysis (DVS) and the like are performed on the stevioside RN crystal form H1 prepared in the above examples.
XRPD analysis: it adopts a Bruker D8 advanced diffractometer of Bruker instruments, germany, to detect at room temperature, adopts Cu-K alpha raysThe 2 theta angle scan is from 3 degrees to 40 degrees with a scan speed of 0.1 degrees/second. The analysis results are shown in FIG. 1. The XRPD spectrum shows that stevioside RN crystal form H1 prepared in the embodiment has good crystallinity.
In the sample powder X-ray powder diffraction patterns, the diffraction patterns resulting from a particular crystal form are often characteristic. Because of differences in crystallization conditions, particle size, relative amounts of the mixture, and other testing conditions, the diffraction patterns may produce preferential orientation effects, resulting in changes in the relative intensities of certain bands (especially at low angles) in the patterns. Thus, the relative intensities of the diffraction peaks are not characteristic for the crystals to which they are directed, and it is more important to note the positions of the peaks than their relative intensities when judging whether they are the same as the known crystal forms. In addition, care should be taken to maintain the general concept of whether the crystalline forms are identical, because instead of a diffraction line representing a phase, a specific set of "d-I/I1" data represents a phase. It should also be noted that in the identification of mixtures, the loss of part of the diffraction lines may be caused by content reduction, etc., and that, in this case, it is not necessary to rely on all bands observed in the high purity sample, even one band may be characteristic for a given crystal.
DSC analysis: all DSC spectra of this patent were detected by DSC Q2000 type differential scanning calorimeter from the company TA of America, the atmosphere was nitrogen, and the heating rate was 10deg.C/min.
TG analysis: all TGA spectra of this patent were measured by TGA 55 thermogravimetric analyzer from TA company in the united states, temperature range: scanning rate at 30-400 deg.c: 10 ℃/min, purge gas: 40mL/min.
DVS analysis: all DVS spectra of this patent were measured by DVS advantage type adsorber from SMS company, uk, relative humidity range: 0-95%, temperature: 25 ℃. The analysis results are shown in FIG. 4. The hygroscopicity of the stevioside RN crystal form H1 prepared in the embodiment is obviously lower than that of the stevioside RN amorphous form under the condition of 40% -80% relative humidity, the amorphous form absorbs water by about 5.9% -14.2%, and the H1 absorbs water by 6.1% -9.2%, and the comparison result is shown in figure 6.
The stevioside RN form H1 prepared in the above example was stored at 40℃and 75% relative humidity for two weeks, and the analysis results are shown in FIG. 7. From fig. 7, it can be seen that the crystal form is unchanged, which indicates that the crystal form has good physical stability under normal storage conditions.
It is to be understood that these examples are illustrative of the present application and are not intended to limit the scope of the present application. Furthermore, it should be understood that various changes and modifications can be made by one skilled in the art after reading the teachings of the present application, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.

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.
CN202310748737.0A 2023-06-25 2023-06-25 Stevioside RN crystal form H1 and preparation method and application thereof Pending CN116813679A (en)

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