Method for judging transparency condition of stevioside and application thereof

Abstract

The invention relates to the technical field of plant extracts, and particularly discloses a method for judging the transparency condition of stevioside and application of the method. The invention provides a method for judging the transparency condition of stevioside. According to the method, the transparency condition can be judged according to the content of myricetin and trihydroxy-kaurane diterpenoid glycoside in the stevioside. The higher the content of the myricetin and the trihydroxy-kaurane diterpenoid glycoside is, the poorer the transparency of the stevioside is, and the lower the content of the myricetin and the trihydroxy-kaurane diterpenoid glycoside is, the better the transparency of the stevioside is. The method is simple and easy to implement, and the transparency condition of stevioside can be judged in time in the production process, so that the production method can be adjusted in good time, and a high-light-transmittance product can be obtained.

Description

Technical Field

[0001] The invention relates to the technical field of plant extracts, in particular to a method for judging the transparency of stevioside and an application thereof. Background Art

[0002] As a natural, zero-calorie, high-intensity sweetener, stevioside is widely used in many fields such as food and beverage, pharmaceutical products, and daily chemical products due to its wide source, high safety, and taste similar to sucrose. With the improvement of consumers' awareness of healthy lifestyles and the growth of low-sugar and sugar-free market demand, the quality requirements for stevioside products are also constantly increasing.

[0003] High transmittance helps to identify product adulteration, and high-transmittance stevioside is also easy to use in products, reducing the impact on the overall color of the product. However, the transmittance of high-purity products is not necessarily ideal, so it is not possible to accurately judge its transmittance based solely on the test results of product purity. At present, the transmittance detection of stevioside is to extract stevioside, dry it, and then configure it into a solution of a specific concentration, and then detect it by optical methods. This method requires adjustment of the concentration of the test solution, which is generally performed after the product is dried, which is not conducive to monitoring the transmittance during the product production process, and is not conducive to timely and corresponding adjustment of the production method according to the actual production situation, and is not conducive to industrial processing and production of high-transmittance stevioside products. Therefore, it is necessary to further study the method for judging the transparency of stevioside. Summary of the invention

[0004] One of the purposes of the present invention is to provide a new method for judging the transparency of stevioside.

[0005] The invention provides a method for judging the transparency of stevioside, which judges the transparency according to the contents of salicylic acid and trihydroxy-kaurane type diterpene glycosides in stevioside; the higher the contents of salicylic acid and trihydroxy-kaurane type diterpene glycosides, the worse the transparency of stevioside, and the lower the contents of salicylic acid and trihydroxy-kaurane type diterpene glycosides, the better the transparency of stevioside.

[0006] At present, there are still few studies on substances that affect transmittance in stevioside. In the process of developing high-transmittance stevioside, the present invention unexpectedly found that the transmittance of stevioside can be judged according to the content of trihydroxy-kaurane diterpene glycoside and the content of salicin. The method directly detects the content of the above two substances to judge the transparency, which can be directly applied to the intermediate products (such as crude extract) in the production process of stevioside, and can also be tested and judged on the finished product. It is not necessary to dry / adjust the concentration of the stevioside to be tested according to the conventional transparency test method, so that the transparency of the product can be known more timely, which is conducive to timely adjustment of the production process (such as adding the step of adjusting the content of salicin and trihydroxy-kaurane diterpene glycoside to change the transparency), avoiding the disadvantage of not being able to adjust the transparency of the product in time after the product is dried and prepared.

[0007] The method of the present invention was discovered through a large sample size study during the long-term research and development and production process of stevioside and has good universality.

[0008] In the present invention, the structural formula of salicin is shown in formula (I), the English name is Gein, and the molecular formula is C 21 H 30 O 11 , molecular weight is 458.17, CAS number is 585-90-0.

[0009] Formula (I).

[0010] The structural formula of trihydroxy-kaurane-type diterpene glycoside is shown in formula (II), the English name is Cofaryloside, and the molecular formula is C 26 H 42 O 10 , molecular weight is 514.27, CAS number is 63558-43-0.

[0011] Formula (II).

[0012] In the method of the present invention, when the content of aconitum in the stevioside is ≤0.1% and the content of trihydroxy-kaurane-type diterpene glycoside is ≤1.5%, the transparency of the stevioside is judged to be high.

[0013] In the method of the present invention, the high transparency refers to a light transmittance of ≥85%.

[0014] The present invention also provides application of the method in producing steviol glycosides, in monitoring the transparency of steviol glycoside products, or in screening steviol glycoside products with high transparency.

[0015] In the application of the present invention, the high transparency refers to a light transmittance of ≥85%.

[0016] The present invention also provides a method for producing steviol glycosides. During the process of producing steviol glycosides, the transparency of the steviol glycosides is monitored by the above method.

[0017] In the method of the present invention, during the process of producing stevioside, the transparency of the stevioside solution or crude product obtained by extraction is monitored.

[0018] In the method of the present invention, when it is found that the content of salicin in the stevioside solution or crude product is greater than 0.1% and the content of trihydroxy-kaurane type diterpene glycosides is greater than 1.5%, a step of removing salicin and trihydroxy-kaurane type diterpene glycosides is added.

[0019] The beneficial effects of the present invention are at least: The present invention provides a new method for judging the transparency of stevioside. The method is simple and easy to implement, and can timely judge the transparency of products in the intermediate link of stevioside extraction during the production process, thereby facilitating timely adjustment of the production method and obtaining products with high light transmittance. DETAILED DESCRIPTION

[0020] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0021] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are commercially available or prepared according to conventional methods in the art unless otherwise specified.

[0022] In the examples and comparative examples of the present invention, the conventional steps for determining the light transmittance (transmittance) of steviol glycosides are as follows: Weigh about 1.00 g (accurate to 0.01 g) of the sample into a beaker, add pure water to about 7.86 g, and stir with a magnetic stirrer (29 gears, about 700 rpm) to dissolve. Use a PAL refractometer to measure the concentration of the solution and adjust the concentration of the solution to 14.0%. Use an ultraviolet spectrophotometer to detect the transmittance of the adjusted solution at 420 nm; the value obtained is the transmittance of 14%. The test results are rounded to 1 decimal place.

[0023] The content of steviol glycosides was determined according to GB 1886.355-2022.

[0024] Salicylin, trihydroxy-kaurane-type diterpene glycosides, dihydroxy-kaurane-type diterpene diglycosides, and citrus glycoside B were detected by ultra-high performance liquid chromatography-tandem time-of-flight mass spectrometry as follows: Take the sample to be tested, dissolve it with 50% methanol-water solution and dilute it to a solid concentration of about 1.0 mg / mL, then filter it through a 0.22 um filter membrane, transfer it to a sample injection vial, and wait for testing.

[0025] The detection conditions were as follows: mobile phase: phase A was 1% formic acid-water solution, phase B was acetonitrile, and gradient elution was used for analysis (gradient elution conditions are shown in Table 1); flow rate was controlled during elution: 0.2 mL / min; column temperature: 40°C; chromatographic column: BEHC18 column (2.1 mm × 100 mm, particle size 1.7 μm); injection volume: 1.0 μL. The retention time of citrus glycoside B was about 5.2 min, the retention time of salicylic acid was about 6.9 min, the retention time of dihydroxy-kaurane type diterpene diglycoside was about 7.7 min, and the retention time of trihydroxy-kaurane type diterpene glycoside was about 9.2 min.

[0026] Table 1 Gradient elution conditions The Xinjiang stevia raw materials involved in the following embodiments and comparative examples were provided by the Shuanglonggou Stevia Planting Professional Cooperative of the 65th Regiment of Huocheng County; the Gansu stevia raw materials were provided by Zhangye Xinheng Agricultural Development Co., Ltd.; and the Jiangsu Dongtai City stevia raw materials were provided by Dongtai Gaohua Stevia Professional Cooperative.

[0027] Example 1 This embodiment provides a new method for determining the transparency of stevioside during the preparation of stevioside, as follows: 1. Preparation of steviol glycosides from stevia: The method provided in Example 1 of Chinese Patent Publication No. CN102295668B is used for operation, and the process is as follows: After crushing the leaves of Xinjiang Stevia, pass through a 40-mesh sieve, weigh 100 g of the sample, add 1500 mL of pure water and extract at 60℃ for 2 h, and filter to obtain the extract. Then add 5 g of FeSO4·H2O to the extract, adjust the pH to 8 ~ 9 with Ca (OH)2, stir for 10 min, let stand for 20 min, and filter to obtain a light yellow impurity-free extract. The impurity-free extract is concentrated under reduced pressure to a total concentration of stevioside of 20 mg / mL~40 mg / mL before loading, and the flow rate during adsorption of ADS-7 resin is 3BV / h. The adsorption-saturated resin is first rinsed with pure water at a flow rate of 4BV / h, and the amount is 2BV for impurity removal. Then 4BV of 60% ethanol is used for elution at a rate of 4BV / h. The eluate of 60% ethanol is collected.

[0028] 2. Determine the transparency: The dry basis content of salicylic acid glycoside and trihydroxy-kaurane diterpene glycoside in the eluent was tested. The content of salicylic acid glycoside was 2.153%, and the content of trihydroxy-kaurane diterpene glycoside was 3.173%. The content of salicylic acid glycoside was greater than 0.1%, and the content of trihydroxy-kaurane diterpene glycoside was greater than 1.5%. Therefore, it was judged that its transmittance was low.

[0029] The eluate obtained in step 1 is dried to obtain crude stevioside, which is then tested according to a conventional method for determining the transmittance of stevioside. The transmittance is 47.2%, which is consistent with the conclusion obtained by the new method for determining the transparency of stevioside of the present invention.

[0030] In order to further obtain a product with high transparency, the crude stevioside obtained after drying was further dissolved in a mobile phase and then separated by a medium-pressure silica gel chromatography column by a wet method, and developed with a mobile phase (n-butanol: ethanol: water = 5:1:0.5), a mobile phase velocity of 4 BV / h, a constant flow pump pressure of 9.5 kgf / cm, and an ultraviolet detector was used for online detection at 210 nm, and the effluent corresponding to the chromatographic peak was collected. Stevioside can be eluted using a mobile phase of 5 BV and completely separated from impurities.

[0031] Determine the transparency: The dry content of salicin and trihydroxy-kaurane diterpene glycosides in the effluent was tested. The salicin content was less than 0.01%, and the trihydroxy-kaurane diterpene glycosides were 0.304%. The salicin content was less than 0.1%, and the trihydroxy-kaurane diterpene glycosides content was less than 1.5%. Therefore, it was judged that its light transmittance was high.

[0032] The effluent was dried under reduced pressure to obtain stevioside, which was then tested according to a conventional method for determining the transmittance of stevioside. The transmittance was 95.9%, which was consistent with the conclusion obtained by the new method for determining the transparency of stevioside of the present invention.

[0033] This indicates that the method of the present invention can effectively judge the transparency of the actual product after drying through the process liquid in the preparation process.

[0034] In order to prove the applicability of the new method for judging the transparency of stevioside of the present invention, the present invention also applies it to the preparation / purification process of stevioside using raw materials from different methods or origins, as shown in Examples 2-9.

[0035] Example 2 This embodiment provides a new method for determining the transparency of stevioside during the preparation process of stevioside, as follows: 1. Preparation of steviol glycosides from stevia: The method provided in Example 1 of Chinese Patent Publication No. CN109293712B is used for operation, and the process is as follows: (1) After crushing the leaves of Xinjiang stevia, pass them through a 40-mesh sieve, weigh 1 kg of the sample, use 85% ethanol aqueous solution as the extracting solution, and extract the stevia powder three times at 50° C. The solid-liquid ratios during the three extractions were 1:5 / 3.5 / 3.5, respectively. The first extraction time was 1.5 h, and the second and third extraction times were both 1 h. The filtrates were combined as the extracting solution; (2) Concentrating the extract to 1 / 10 of the original volume in a water bath at 60° C. and under vacuum conditions of -0.08 MPa, adding an equal volume of n-hexane to the concentrate for liquid-liquid extraction three times, taking the water layer, and obtaining an extract free of low-polarity impurities; (3) The pH of the extract after removing low-polar impurities was adjusted to 10.0 and the solid content was adjusted to 10%, and then it was adsorbed by 1.5L T28 resin, and the flow rate of the liquid during the adsorption process was adjusted to 0.2BV / h; after the adsorption was completed, the T28 resin was eluted with 2BV of water, and the flow rate of the water was controlled to 0.2BV / h in the first BV, and the flow rate of the water was controlled to 1BV / h in the second BV. After the elution was completed, the resin was analyzed with 2BV of 70% ethanol aqueous solution, and the flow rate of the analysis liquid was controlled to 1BV / h during the analysis process. Collect the analysis liquid.

[0036] 2. Determine the transparency: The dry basis content of salicin and trihydroxy-kaurane diterpene glycosides in the analytical solution was tested. The salicin content was 3.154%, and the trihydroxy-kaurane diterpene glycosides were 4.231%. The content of salicin was greater than 0.1%, and the content of trihydroxy-kaurane diterpene glycosides was greater than 1.5%. Therefore, it was judged that its transmittance was low.

[0037] The analytical solution obtained in step 1 was concentrated into powder at 60° C. and vacuum -0.08 MPa. Then, the transmittance of the stevioside was tested according to the conventional method for determining the transmittance of stevioside, and the transmittance was 22.4%, which was consistent with the conclusion obtained by the new method for determining the transparency of stevioside of the present invention.

[0038] In order to further obtain a product with high transparency, the analytical solution was further concentrated in this embodiment and then desalted (001×14.5 resin purchased from Nankai Hecheng Technology Co., Ltd.), decolorized (001×14.5 resin purchased from Nankai Hecheng Technology Co., Ltd.), and refined (LX-8 resin purchased from Lanxiao Technology New Materials Co., Ltd.) with resin, and the flow rate was 2~3 BV / h to obtain refined solution.

[0039] Determine the transparency: The dry content of salicylic acid and trihydroxy-kaurane diterpene glycosides in the semen extract was tested. The salicylic acid content was 0.049%, and the trihydroxy-kaurane diterpene glycosides were 0.769%. The content of salicylic acid was less than 0.1%, and the content of trihydroxy-kaurane diterpene glycosides was less than 1.5%. Therefore, it is judged that its light transmittance is high.

[0040] The above-mentioned refined liquid was dried to obtain stevioside, which was then tested according to a conventional stevioside transmittance determination method. The transmittance was 90.6%, which was consistent with the conclusion obtained by the new stevioside transparency determination method of the present invention.

[0041] Example 3 This embodiment provides a new method for judging the transparency of stevioside during the preparation process of stevioside. The specific method is basically the same as that of Example 2, except that the origin of the raw materials is changed, specifically, Xinjiang stevia is replaced by Gansu stevia.

[0042] The dry basis content of salicylic acid in the analytical solution obtained in the preparation is 3.232%, and the dry basis content of trihydroxy-kaurane type diterpene glycosides is 4.188%. The content of salicylic acid is greater than 0.1%, and the content of trihydroxy-kaurane type diterpene glycosides is greater than 1.5%. Therefore, it is judged that its transmittance is low.

[0043] The above analytical solution was concentrated into powder at 60°C and vacuum -0.08MPa. Then, the transmittance of stevioside was tested according to the conventional method for determining the transmittance of stevioside, and the transmittance was 24.4%, which was consistent with the conclusion obtained by the new method for determining the transparency of stevioside of the present invention.

[0044] After further refining the analytical solution, the dry basis content of salicin and trihydroxy-kaurane diterpene glycosides in the refined solution was tested, and the salicin content was 0.042%, and the trihydroxy-kaurane diterpene glycosides were 0.729%. The content of salicin was less than 0.1%, and the content of trihydroxy-kaurane diterpene glycosides was less than 1.5%. Therefore, it was judged that its transmittance was high.

[0045] The above-mentioned refined liquid was dried to obtain stevioside, and then tested according to the conventional stevioside transmittance determination method. The transmittance was 91%, which was consistent with the conclusion obtained by the new stevioside transparency determination method of the present invention.

[0046] Example 4 This embodiment provides a new method for judging the transparency of stevioside during the preparation process of stevioside. The specific method is basically the same as that of Example 2, except that the origin of the raw materials is changed, specifically, Xinjiang stevia is replaced by stevia from Dongtai City, Jiangsu Province.

[0047] The dry basis content of salicin in the analytical solution obtained in the preparation is 3.226%, and the dry basis content of trihydroxy-kaurane type diterpene glycosides is 4.190%. The content of salicin is greater than 0.1%, and the content of trihydroxy-kaurane type diterpene glycosides is greater than 1.5%. Therefore, it is judged that its transmittance is low.

[0048] The above analytical solution was concentrated into powder at 60°C and vacuum -0.08MPa. Then, the transmittance of stevioside was tested according to the conventional method for determining the transmittance of stevioside, and the transmittance was 22.1%, which was consistent with the conclusion obtained by the new method for determining the transparency of stevioside of the present invention.

[0049] After further refining the analytical solution, the dry basis content of salicin and trihydroxy-kaurane diterpene glycosides in the refined solution was tested, and the salicin content was 0.066%, and the trihydroxy-kaurane diterpene glycosides were 0.653%. The content of salicin was less than 0.1%, and the content of trihydroxy-kaurane diterpene glycosides was less than 1.5%. Therefore, it was judged that its transmittance was high.

[0050] The above-mentioned refined liquid was dried to obtain stevioside, which was then tested according to a conventional stevioside transmittance determination method. The transmittance was 90.5%, which was consistent with the conclusion obtained by the new stevioside transparency determination method of the present invention.

[0051] Example 5 This embodiment provides a new method for determining the transparency of stevioside during the preparation process of stevioside, as follows: 1. The method described in 1.1.3 of Chinese Patent Publication No. CN 106236808 B is used to prepare stevioside, and the process is as follows: Take Xinjiang stevia leaves and crush them, then pass them through a 40-mesh sieve. Weigh 100 kg of the sample and add 1.5 m 3 Water, 65℃ extraction, repeated extraction three times, combined extracts, passed through 5 nm ceramic membrane. The permeate was washed with 201-H adsorption resin, 1BV water, water washing flow rate 1BV / h, 1.5BV 0.4% NaOH aqueous solution, flow rate 1BV / h, ethanol analysis, the analysis liquid was concentrated and ethanol was recovered, and then desalted with resin (001×14.5 resin purchased from Nankai Hecheng Technology Co., Ltd.), decolorized (001×14.5 resin purchased from Nankai Hecheng Technology Co., Ltd.), and refined (LX-8 resin purchased from Lanxiao Technology New Materials Co., Ltd.), the flow rate was 2~3BV / h, and refined solution was obtained.

[0052] 2. Determine the transparency: The dry basis content of salicin and trihydroxy-kaurane diterpene glycosides in the semen desorption liquid was tested. The salicin content was 0.334%, and the trihydroxy-kaurane diterpene glycosides were 1.831%. The content of salicin was greater than 0.1%, and the content of trihydroxy-kaurane diterpene glycosides was greater than 1.5%. Therefore, it was determined that its light transmittance still needs to be improved.

[0053] The refined liquid obtained in step 1 is dried to obtain crude stevioside, which is then tested according to a conventional method for determining the transmittance of stevioside. The transmittance is 81.3%, which is consistent with the conclusion obtained by the new method for determining the transparency of stevioside of the present invention.

[0054] In order to further obtain a product with high transparency, this example further takes 100 g of the above crude stevioside and adds 400 mL of 90% methanol solution to dissolve it, cools it to 10° C., stands for 4 h, collects the crystals by filtration, and vacuum-dries it to obtain a stevioside product.

[0055] The dry basis content and light transmittance of the obtained stevioside product of salicin and trihydroxy-kaurane diterpene glycosides were tested, and the light transmittance was 95.7%, salicin was 0.014%, and trihydroxy-kaurane diterpene glycosides was 0.258%. The content of salicin was less than 0.1%, and the content of trihydroxy-kaurane diterpene glycosides was less than 1.5%. It can also be judged that its light transmittance is high, which is consistent with the actual test results.

[0056] Example 6 This embodiment provides a new method for determining the transparency of stevioside during the preparation of stevioside, as follows: 1. Preparation of steviol glycosides from stevia: The method provided in Example 1 of Chinese Patent Publication CN 105001281 B is used for operation, and the process is as follows: Take the leaves of Xinjiang stevia, grind them and pass them through a 40-mesh sieve, weigh 100 g of the sample, add 15 times of water, reflux and extract at 100°C for 2.5 h, filter to obtain the extract, repeat 3 times, combine the extracts, add 1% flocculant ferrous sulfate under insulation conditions, add CaO under stirring conditions to adjust the pH to 11.0, let it stand at 65°C for 60 min, filter to obtain filter mud and filtrate, and the filtrate is adsorbed by macroporous adsorption resin ADS-4 at a flow rate of 1.5BV / h, analyzed with 50% ethanol aqueous solution, and purified by desalting resin 001×6 and decolorizing resin 330 to obtain a purified solution.

[0057] 2. Determine the transparency: The dry basis content of salicin and trihydroxy-kaurane diterpene glycosides in the purified solution was tested. The salicin content was 0.151%, and the trihydroxy-kaurane diterpene glycosides were 1.614%. The content of salicin was greater than 0.1%, and the content of trihydroxy-kaurane diterpene glycosides was greater than 1.5%. Therefore, it was determined that its light transmittance still needs to be improved.

[0058] The purified solution obtained in step 1 is dried to obtain crude stevioside, and then tested according to the conventional stevioside transmittance determination method. The transmittance is 82.8%, which is consistent with the conclusion obtained by the new stevioside transparency determination method of the present invention. At the same time, it can be seen that the content of amaranthin and trihydroxy-kaurane-type diterpene glycosides in the purified solution is already low, so the transmittance of the dried product is relatively high.

[0059] In order to further obtain a product with high transparency, in this example, 1 g of the crude stevioside obtained after drying was further added with 10 mL of water for ultrasonic dissolution, and the dissolved solution was subjected to chromatographic separation to remove the components of amaranthin and trihydroxy-kaurane-type diterpene glycosides, and the stevioside components were collected, as follows: The stevioside solution was separated by using a waters BEH C18 (10 mm×250 mm, 5 um) column under the following conditions: injection volume 500 uL; column temperature 30°C; flow rate 3 mL / min; detection wavelength 210 nm; mobile phase A was 0.1% formic acid aqueous solution, mobile phase B was acetonitrile; elution gradient: 25% B, 0-16.4 min; 25%-32% B, 16.4-19.0min; 32% B, 19.0-50.5 min; 32-25% B, 50.5-53.0 min; 25% B, 53.0-57.0 min. According to the above elution gradient and method, the stevioside components were collected, and the components collected from 10 to 40 minutes were specifically the target solution.

[0060] Determine the transparency: The dry basis content of salicin and trihydroxy-kaurane diterpene glycosides in the target solution was tested. The content of salicin was 0.013%, and the content of trihydroxy-kaurane diterpene glycosides was 0.223%. The content of salicin and trihydroxy-kaurane diterpene glycosides further decreased, so it was judged that the transmittance was further increased. The content of salicin was less than 0.1%, and the content of trihydroxy-kaurane diterpene glycosides was less than 1.5%, indicating that the transmittance was high.

[0061] The concentrated target solution is dried to obtain a stevioside product, which is then tested according to a conventional stevioside transmittance determination method. The transmittance is 96.6%, which is consistent with the conclusion obtained by the new stevioside transparency determination method of the present invention.

[0062] Example 7 This embodiment provides a new method for determining the transparency of stevioside during the preparation process of stevioside, as follows: 1. Take Xinjiang stevia leaves and crush them, then pass them through a 40-mesh sieve, weigh 1 kg of the sample, use an aqueous solution as the extract, and extract the stevia powder 3 times at 50° C. The solid-liquid ratios in the 3 extraction processes are 1:5 / 3.5 / 3.5, respectively. The first extraction time is 1.5 h, and the second and third extraction times are both 1 h. The filtrates are combined as the extract; 2. Adjust the solid content of the extract to 10%, and then adsorb it through 1.5L T28 resin, and adjust the flow rate of the liquid during the adsorption process to 0.2BV / h; after the adsorption is completed, use 2BV of 20% ethanol aqueous solution to elute the T28 resin, and control the flow rate of the first BV to 0.2BV / h, and the second BV to control the flow rate of 1BV / h. After the elution is completed, use 2BV 70% ethanol aqueous solution to analyze the resin, and control the flow rate of the analytical solution to 1BV / h during the analytical process. Collect the analytical solution, concentrate it, and then use the resin for desalting (001×14.5 resin purchased from Nankai Hecheng Technology Co., Ltd.) and decolorization (001×14.5 resin purchased from Nankai Hecheng Technology Co., Ltd.), with a flow rate of 2~3 BV / h to obtain a desalted and decolorized solution.

[0063] 3. The dry basis content of salicin and trihydroxy-kaurane diterpene glycosides in the desalted and decolorized liquid was tested. The salicin content was 0.1%, and the trihydroxy-kaurane diterpene glycosides content was 1.486%. The salicin content was equal to 0.1%, and the trihydroxy-kaurane diterpene glycosides content was slightly less than 1.5%. Therefore, it was judged that its transmittance was high.

[0064] The desalted and decolorized liquid was dried to obtain stevioside, which was then tested according to a conventional method for determining the transmittance of stevioside. The transmittance was 85.1%, which was consistent with the conclusion obtained by the new method for determining the transparency of stevioside of the present invention.

[0065] Example 8 This embodiment provides a new method for determining the transparency of stevioside during the preparation process of stevioside, as follows: 1. Take the Xinjiang stevia leaves and crush them, then pass them through a 40-mesh sieve, weigh 1 kg of the sample, use 50% ethanol as the extract, repeat the extraction 3 times at 60°C, the solid-liquid ratio is 1:5, the extraction time is 1h, and the filtrate is combined as the extract; 2. The extract is first filtered through a ceramic membrane with a pore size of 30 mm, and then filtered through an ultrafiltration membrane with a throttling molecular weight of 1000 Da. After the filtration is completed, a stevioside filtrate is obtained.

[0066] 3. The filtrate in step 2 is adsorbed by 1.5L of D101 resin, and the flow rate of the liquid during the adsorption process is adjusted to 0.3BV / h; after the adsorption is completed, the D101 resin is eluted with 2BV of water, the flow rate of the water is controlled to 0.2BV / h in the first BV, and the flow rate of the water is controlled to 1BV / h in the second BV. After the elution is completed, the resin is analyzed with 2BV of 80% ethanol aqueous solution, and the flow rate of the analytical solution is controlled to 1BV / h during the analysis process. The analytical solution is collected, concentrated, and then desalted with resin (001×14.5 resin purchased from Nankai Hecheng Technology Co., Ltd.), decolorized (001×14.5 resin purchased from Nankai Hecheng Technology Co., Ltd.), and refined (LX-8 resin purchased from Lanxiao Technology New Materials Co., Ltd.), with a flow rate of 2~3 BV / h to obtain refined desalted liquid.

[0067] Determine the transparency: The dry content of salicin and trihydroxy-kaurane diterpene glycosides in the semen extract was tested, and the salicin content was 1.148%, and the trihydroxy-kaurane diterpene glycosides were 2.126%. The content of salicin was greater than 0.1%, and the content of trihydroxy-kaurane diterpene glycosides was greater than 1.5%. Therefore, it was judged that its light transmittance was low.

[0068] The refined liquid obtained in step 3 is dried to obtain a stevioside product. Then, the stevioside transmittance is tested according to a conventional stevioside transmittance determination method, and the transmittance is 72.7%, which is consistent with the conclusion obtained by the new stevioside transparency determination method of the present invention.

[0069] Example 9 This embodiment provides a new method for determining the transparency of stevioside during the preparation process of stevioside, as follows: 1. Take Xinjiang stevia leaves and crush them, then pass them through a 40-mesh sieve, weigh 1 kg of the sample, use n-hexane as the extracting liquid, extract the stevia powder 3 times at 50° C., the solid-liquid ratio is 1:5, the extraction time is 1 h, and the residue is collected; 2. Using aqueous solution as the extracting solution, the filter residue in (1) was repeatedly extracted three times at 50°C. The solid-liquid ratios during the three extractions were 1:5 / 3.5 / 3.5, respectively. The extraction time was 1 h. The filtrates were combined as the extracting solution. 3. The extract was adsorbed by 1.5L of S-8 resin, and the flow rate of the liquid during the adsorption process was 0.2BV / h. Afterwards, the resin was eluted with 3BV of 20% ethanol aqueous solution to remove polar impurities. During the elution process, the flow rate was controlled to be 1BV / h, and the water washing amount was 5 times. Then, stevioside was analyzed with 85% ethanol aqueous solution. During the analysis process, the flow rate of the analysis liquid was controlled to be 1BV / h, and the water washing amount was 7 times to obtain stevioside analysis liquid; 4. Concentrate the analytical solution to a solid content of 20%, and adjust the volume fraction of ethanol in the analytical solution to 90%, cool to 4°C, let stand for 24 hours, filter and collect the crystals, and vacuum dry to obtain stevioside products.

[0070] The dry basis content and light transmittance of the obtained stevioside product of salicin and trihydroxy-kaurane diterpene glycosides were tested, and the light transmittance was 96.2%, salicin was 0.014%, and trihydroxy-kaurane diterpene glycosides was 0.227%. The content of salicin was less than 0.1%, and the content of trihydroxy-kaurane diterpene glycosides was less than 1.5%. It can also be judged that its light transmittance is high, which is consistent with the actual test results.

[0071] Comparative Experiment 1 The present invention detects the dry basis contents of stevioside, citrus glycoside B, a substance similar to citrus glycoside, and dihydroxy-kaurane-type diterpene diglycoside, a substance similar to trihydroxy-kaurane-type diterpene glycoside, respectively, while detecting the citrus glycoside and trihydroxy-kaurane-type diterpene glycoside in the eluate obtained in step 1 of Example 1, and detecting the citrus glycoside and trihydroxy-kaurane-type diterpene diglycoside in the effluent after purification in Example 1.

[0072] In the present invention, the structural formula of the dihydroxy-kaurane type diterpene diglycoside is shown in formula (III), the English name is Tricalysioside Z, and the molecular formula is C 32 H 52 O 15 , molecular weight is 722.32. Methods Determination of content.

[0073] Formula (III) The structural formula of Citrusin B is shown in formula (IV), and its English name is Citrusin B, cas: 105279-10-5, and its molecular formula is C 27 H 36 O 13 , molecular weight is 568.57. Methods Determination of content.

[0074] Formula (IV) Results The dry basis content of stevioside in the eluate of Example 1 was 75.34%, dihydroxy-kaurane diterpene diglycoside was 0.454%, and citrus glycoside B was 0.054%. The content of stevioside in the refined effluent was 97.07%, dihydroxy-kaurane diterpene diglycoside was 0.222%, and citrus glycoside B was 0.013%.

[0075] The present invention detects the dry basis contents of stevioside, citrus glycoside B, a substance similar to citrus glycoside, and dihydroxy-kaurane-type diterpene diglycoside, while detecting the citrus glycoside and trihydroxy-kaurane-type diterpene glycoside in the analytical solution obtained in step 1 of Example 2-4, and detecting the citrus glycoside and trihydroxy-kaurane-type diterpene glycoside in the refined extract after purification in Example 2-4.

[0076] Results The content of stevioside in the analytical solution of Example 2 was 35.05%, dihydroxy-kaurane diterpene diglycoside was 1.388%, and citrus glycoside B was 0.172%. The content of stevioside in the refined deionized solution after purification was 94.22%, dihydroxy-kaurane diterpene diglycoside was 1.227%, and citrus glycoside B was 0.164%.

[0077] The content of steviol glycoside in the analytical solution of Example 3 was 33.14%, the content of dihydroxy-kaurane type diterpene diglycoside was 1.311%, and the content of citrus glycoside B was 0.224%. The dry content of steviol glycoside in the refined deionized liquid after purification was 93.92%, the content of dihydroxy-kaurane type diterpene diglycoside was 1.194%, and the content of citrus glycoside B was 0.171%.

[0078] The dry basis content of steviol glycosides in the analytical solution of Example 4 was 31.06%, dihydroxy-kaurane diterpene diglycosides were 1.314%, and citrus glycoside B was 0.201%. The content of steviol glycosides in the refined extract after purification was 91.87%, dihydroxy-kaurane diterpene diglycosides were 1.211%, and citrus glycoside B was 0.151%.

[0079] The present invention detects the dry basis contents of stevioside, citrus glycoside B, a substance similar to citrus glycoside, and dihydroxy-kaurane-type diterpene diglycoside, while detecting the citrus glycoside and trihydroxy-kaurane-type diterpene glycoside in the refined solution obtained in step 1 of Example 5, and detecting the citrus glycoside and trihydroxy-kaurane-type diterpene glycoside in the stevioside product obtained after refining in Example 5.

[0080] Results The content of steviol glycoside in the refined solution of Example 5 was 91.54%, dihydroxy-kaurane diterpene diglycoside was 0.866%, and citrus glycoside B was 0.114%. The content of steviol glycoside in the refined steviol glycoside product was 95.39%, dihydroxy-kaurane diterpene diglycoside was 0.259%, and citrus glycoside B was 0.149%.

[0081] The present invention detects the dry basis contents of stevioside, citrus glycoside B, a substance similar to citrus glycoside, and dihydroxy-kaurane-type diterpene diglycoside, a substance similar to trihydroxy-kaurane-type diterpene glycoside, respectively, while detecting the citrus glycoside and trihydroxy-kaurane-type diterpene glycoside in the purified solution obtained in step 1 of Example 6, and detecting the citrus glycoside and trihydroxy-kaurane-type diterpene glycoside in the target solution obtained after chromatographic separation in Example 6.

[0082] Results The dry basis content of stevioside in the purified solution of Example 6 was 90.33%, dihydroxy-kaurane diterpene diglycoside was 0.546%, and citrus glycoside B was 0.065%. The dry basis content of stevioside in the target solution was 94.74%, dihydroxy-kaurane diterpene diglycoside was 1.517%, and citrus glycoside B was 0.369%.

[0083] The present invention not only detects the dry basis content of stevioside, a substance similar to stevioside, namely, citrus glycoside B, and a substance similar to trihydroxy-kaurane-type diterpene glycoside, namely, dihydroxy-kaurane-type diterpene diglycoside, while detecting the stevioside and trihydroxy-kaurane-type diterpene glycoside in the desalted and decolorized liquid obtained in step 2 of Example 7.

[0084] Results The contents of steviol glycoside in the desalted and decolorized liquid of Example 7 were 90.69%, dihydroxy-kaurane-type diterpene diglycosides were 0.549%, and citrus glycoside B were 0.222%.

[0085] While detecting the aconitum glycoside and trihydroxy-kaurane-type diterpene glycosides in the seminal fluid obtained in step 3 of Example 8, the present invention also detects the dry basis content of stevioside, a substance similar to aconitum glycoside - citrus glycoside B, and a substance similar to trihydroxy-kaurane-type diterpene glycoside - dihydroxy-kaurane-type diterpene diglycoside.

[0086] Results The dry basis content of stevioside in the seminal extract of Example 8 was 94.65%, that of dihydroxy-kaurane-type diterpene diglycoside was 0.333%, and that of citrus glycoside B was 0%.

[0087] While detecting the aconitum glycoside and trihydroxy-kaurane-type diterpene glycosides in the steviol glycoside product obtained in step 4 of Example 9, the present invention also detects the dry basis content of stevioside, a substance similar to aconitum glycoside - citrus glycoside B, and a substance similar to trihydroxy-kaurane-type diterpene glycoside - dihydroxy-kaurane-type diterpene diglycoside.

[0088] Results: The content of steviol glycoside in the steviol glycoside product of Example 9 was 93.88%, the content of dihydroxy-kaurane-type diterpene diglycoside was 1.024%, and the content of citrus glycoside B was 0.153%.

[0089] By comparing the above results, it can be found that the samples with high content of steviol glycosides are not necessarily high in transparency. For example, the content of steviol glycosides in the refined effluent of Example 1 is 97.07%, and the transparency of the dried product is 95.9%. The content of steviol glycosides in the target solution of Example 6 is 94.74%, which is lower than that of Example 1, and the transparency of the dried product is 96.6%, which is higher than that of Example 1. The content of steviol glycosides in the refined effluent of Example 2 is 94.22%, and the transparency of the dried product is 90.6%. The content of steviol glycosides in the refined effluent of Example 3 is 93.92%, which is lower than that of Example 2, and the transparency of the dried product is 91%, which is higher than that of Example 2. The content of steviol glycosides in the refined effluent of Example 8 is 94.65%, and the transparency of the dried product is 72.7%. The content of steviol glycosides in the steviol glycoside product of Example 9 is 93.88%, which is lower than that of Example 8, and the transmittance is 96.2%, which is higher than that of Example 8.

[0090] When the dry content of salicylic acid glycosides in the sample is less than 0.1% and the dry content of trihydroxy-kaurane-type diterpene glycosides is less than 1.5%, the transmittance of the sample is high (≥85%). And the lower the content of salicylic acid glycosides and trihydroxy-kaurane-type diterpene glycosides in the sample, the higher the transmittance of the sample. The accuracy of judging the transmittance of the sample based on the content of salicylic acid glycosides and trihydroxy-kaurane-type diterpene glycosides is better, and there is no need to dry the sample and / or adjust the concentration, which is conducive to real-time detection of the transmittance of the product in industrial production, so as to make timely adaptive adjustments to the production method (for example, adding a step to remove the content of salicylic acid glycosides and trihydroxy-kaurane-type diterpene glycosides to improve the transmittance).

[0091] However, the changes in the transparency of the samples of substances similar to amaranthin and trihydroxy-kaurane diterpene glycosides, citrus glycoside B and dihydroxy-kaurane diterpene diglycosides, cannot be determined based on the changes in their contents. For example, in Example 1, as the transparency of the sample before and after refining increases, the dry basis contents of dihydroxy-kaurane diterpene diglycosides and citrus glycoside B both decrease. In Example 6, the transmittance of the crude stevioside obtained after drying the purified solution is 82.8%, dihydroxy-kaurane diterpene diglycosides is 0.546%, and citrus glycoside B is 0.065%. The transparency of the target solution obtained by further chromatographic separation after drying is increased to 96.6%, dihydroxy-kaurane diterpene diglycosides is increased to 1.517%, and citrus glycoside B is increased to 0.369%. That is, as the transparency of the sample before and after refining increases, the dry basis contents of dihydroxy-kaurane diterpene diglycosides and citrus glycoside B are also increased, and not decreased.

[0092] In order to further verify the accuracy of the method for judging the transparency of stevioside provided by the present invention, the present invention further conducted a reverse addition experiment of related substances, as shown in Comparative Experiment 2.

[0093] Comparative experiment 2 1. Take 1g of the stevioside powder finally prepared in Example 2 (the content of stevioside is 94.22%, the transmittance is 90.6%, the water salicin is 0.049%, the trihydroxy-kaurane diterpene glycoside is 0.769%, the dihydroxy-kaurane diterpene diglycoside is 1.227%, and the citrus glycoside B is 0.164%), add 0.03g of water salicin in reverse, mix well, and then test the dry content. The content of stevioside in the sample is measured to be 91.44%, the transmittance is 75.5%, the water salicin is 2.934%, the trihydroxy-kaurane diterpene glycoside is 0.745%, the dihydroxy-kaurane diterpene diglycoside is 1.218%, and the citrus glycoside B is 0.16%. It can be seen that the content of water salicin is increased and the transmittance of stevioside is significantly reduced.

[0094] Then, 1 g of the stevioside powder prepared in Example 2 was taken, and 0.05 g of trihydroxy-kaurane-type diterpene glycoside was added in reverse, and the dry content was detected after mixing evenly. The content of stevioside in the sample was measured to be 90.07%, the transmittance was 77.3%, the content of salicin was 0.047%, the content of trihydroxy-kaurane-type diterpene glycoside was 5.225%, the content of dihydroxy-kaurane-type diterpene diglycoside was 1.148%, and the content of citrus glycoside B was 0.152%. It can be seen that the content of trihydroxy-kaurane-type diterpene glycoside increased, and the transmittance of stevioside decreased significantly.

[0095] Continue to take 1g of the stevioside powder prepared in Example 2 above, add 0.05g of dihydroxy-kaurane type diterpene diglycoside in reverse, mix well and then test the dry content. The content of stevioside in the sample is 90.28%, the transmittance is 90.4%, the content of salicin is 0.037%, the content of trihydroxy-kaurane type diterpene glycoside is 0.706%, the content of dihydroxy-kaurane type diterpene diglycoside is 5.833%, and the content of citrus glycoside B is 0.119%. It can be seen that the content of dihydroxy-kaurane type diterpene diglycoside is increased, and the transmittance of stevioside is not significantly reduced.

[0096] Next, 1 g of the stevioside powder prepared in Example 2 was taken, and 0.01 g of citrus glycoside B was added in reverse, and the mixture was mixed evenly, and the dry content was detected. The content of stevioside in the sample was measured to be 93.54%, the transmittance was 91.2%, the content of salicin was 0.045%, the content of trihydroxy-kaurane type diterpene glycoside was 0.765%, the content of dihydroxy-kaurane type diterpene diglycoside was 1.212%, and the content of citrus glycoside B was 1.106%. It can be seen that as the content of citrus glycoside B increases, the transmittance of stevioside increases instead.

[0097] 2. Take 1 g of the stevioside powder prepared in Example 5 (the content of stevioside is 95.39%, the transmittance is 95.7%, the content of salicin is 0.014%, the content of trihydroxy-kaurane diterpene glycoside is 0.258%, the content of dihydroxy-kaurane diterpene diglycoside is 0.259%, and the content of citrus glycoside B is 0.149%), add 0.01 g of salicin in reverse, mix well, and then test the dry content. The content of stevioside in the sample is measured to be 94.27%, the transmittance is 81.8%, the content of salicin is 1.031%, the content of trihydroxy-kaurane diterpene glycoside is 0.25%, the content of dihydroxy-kaurane diterpene diglycoside is 0.24%, and the content of citrus glycoside B is 0.131%.

[0098] Take 1 g of the stevioside powder prepared in Example 5, add 0.03 g of trihydroxy-kaurane diterpene glycosides in reverse, mix well, and then test the dry content. The stevioside content in the sample is 92.49%, the transmittance is 82.6%, the aconitum glycoside is less than 0.01%, the trihydroxy-kaurane diterpene glycoside is 3.128%, the dihydroxy-kaurane diterpene diglycoside is 0.236%, and the citrus glycoside B is 0.127%.

[0099] Take 1 g of the stevioside powder prepared in Example 5, add 0.03 g of dihydroxy-kaurane diterpene diglycoside in reverse, mix well, and then test the dry content. The stevioside content in the sample is 93.14%, the transmittance is 95.5%, the aconitum glycoside is less than 0.01%, the trihydroxy-kaurane diterpene glycoside is 0.231%, the dihydroxy-kaurane diterpene diglycoside is 3.215%, and the citrus glycoside B is 0.135%.

[0100] Take 1 g of the stevioside powder prepared in Example 5, add 0.01 g of citrus glycoside B in reverse, mix well, and then test the dry content. The stevioside content in the sample is 94.52%, the transmittance is 96.0%, the aconitum glycoside is less than 0.01%, the trihydroxy-kaurane type diterpene glycoside is 0.251%, the dihydroxy-kaurane type diterpene diglycoside is 0.248%, and the citrus glycoside B is 1.104%.

[0101] 3. Take 1 g of the stevioside powder prepared in Example 6 (the content of stevioside is 94.74%, the transmittance is 96.6%, the content of salicin is 0.013%, the content of trihydroxy-kaurane diterpene glycoside is 0.223%, the content of dihydroxy-kaurane diterpene diglycoside is 1.517%, and the content of citrus glycoside B is 0.369%), add 0.01 g of salicin in reverse, mix well, and then test the dry content. The content of stevioside in the sample is measured to be 94.13%, the transmittance is 80.3%, the content of salicin is 1.004%, the content of trihydroxy-kaurane diterpene glycoside is 0.219%, the content of dihydroxy-kaurane diterpene diglycoside is 1.514%, and the content of citrus glycoside B is 0.366%.

[0102] Take 1 g of the stevioside powder prepared in Example 6, add 0.03 g of trihydroxy-kaurane diterpene glycoside in reverse, mix well, and then test the dry content. The stevioside content in the sample is 91.49%, the transmittance is 82.1%, the aconitum glycoside is 0.012%, the trihydroxy-kaurane diterpene glycoside is 3.213%, the dihydroxy-kaurane diterpene diglycoside is 1.455%, and the citrus glycoside B is 0.357%.

[0103] Take 1 g of the stevioside powder prepared in Example 6, add 0.05 g of dihydroxy-kaurane diterpene diglycoside in reverse, mix well, and then test the dry content. The stevioside content in the sample is 90.47%, the transmittance is 96.4%, the aconitum glycoside is less than 0.01%, the trihydroxy-kaurane diterpene glycoside is 0.204%, the dihydroxy-kaurane diterpene diglycoside is 6.113%, and the citrus glycoside B is 0.353%.

[0104] Take 1 g of the stevioside powder prepared in Example 6, add 0.03 g of citrus glycoside B in reverse, mix well, and then test the dry content. The stevioside content in the sample is 91.72%, the transmittance is 96.7%, the aconitum glycoside is less than 0.01%, the trihydroxy-kaurane type diterpene glycoside is 0.211%, the dihydroxy-kaurane type diterpene diglycoside is 1.454%, and the citrus glycoside B is 3.165%.

[0105] In summary, after reverse addition of salicylic acid glycoside and trihydroxy-kaurane-type diterpene glycosides, the transparency of the product decreased significantly, while reverse addition of substances similar to salicylic acid glycosides and trihydroxy-kaurane-type diterpene glycosides, such as citrus glycoside B and dihydroxy-kaurane-type diterpene diglycosides, failed to show consistent effects on transparency.

[0106] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A method for determining the transparency of steviol glycosides, characterized in that: The transparency is judged according to the content of salicylic acid glycoside and trihydroxy-kaurane type diterpene glycoside in stevioside; the higher the content of salicylic acid glycoside and trihydroxy-kaurane type diterpene glycoside, the worse the transparency of stevioside, and the lower the content of salicylic acid glycoside and trihydroxy-kaurane type diterpene glycoside, the better the transparency of stevioside.

2. The method according to claim 1, characterized in that: When the content of amaranthin in the stevioside is ≤0.1% and the content of trihydroxy-kaurane-type diterpene glycoside is ≤1.5%, it is judged that the transparency of the stevioside is high.

3. The method according to claim 2, characterized in that The high transparency refers to a light transmittance of ≥85%.

4. Use of the method according to any one of claims 1 to 3 in the production of steviol glycosides.

5. Application of the method according to any one of claims 1 to 3 in monitoring the transparency of steviol glycoside products.

6. Use of the method according to any one of claims 1 to 3 in screening steviol glycoside products with high transparency.

7. The use according to claim 6, characterized in that: The high transparency refers to a light transmittance of ≥85%.

8. A method for producing steviol glycosides, characterized in that: In the process of producing steviol glycoside, the transparency of steviol glycoside is monitored by the method described in any one of claims 1 to 3.

9. The method according to claim 8, characterized in that During the production of stevioside, the transparency of the extracted stevioside solution or crude product is monitored.

10. The method according to claim 9, characterized in that When it is found that the content of salicin in the stevioside solution or crude product is greater than 0.1% and the content of trihydroxy-kaurane type diterpene glycosides is greater than 1.5%, a step of removing salicin and trihydroxy-kaurane type diterpene glycosides is added.

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