Method for measuring concentration of brazzein

Abstract

Provided in the present invention is a method for measuring the concentration of Brazzein. The method comprises the following steps: (a) performing liquid chromatography detection by using Brazzein solution standards having different concentrations, making a standard curve on the basis of the chromatographic peak areas and the concentrations of Brazzein of the standards at the maximum absorption wavelength, and performing fitting to obtain a regression equation of the standard curve; and (b) performing liquid chromatography detection on a sample, and calculating the concentration of Brazzein in the sample on the basis of the chromatographic peak area of the sample at the maximum absorption wavelength and the regression equation of the standard curve.

Description

Method for detecting the concentration of Brazilian sweet

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent application number CN202311104566.4 and filing date August 29, 2023, which is incorporated herein by reference in its entirety. Technical Field

[0003] The present invention relates to a sweet protein detection technology, in particular to a method for detecting the concentration of brassica rapa in a sample. Background Art

[0004] Brazzein is a sweet protein isolated and purified from the fruit of the wild plant Pentadiplandra Brazzeana in West Africa. Together with other plant-derived proteins with similar properties, such as thaumatin, monellin, and miraculin, it is collectively referred to as sweet proteins. Brazzein is a single-chain protein composed of 54 amino acid residues. It has the smallest molecular weight of the plant sweet protein family, with a relative molecular mass of 6.5 kDa and an isoelectric point of 5. Its sweetness is approximately 2000 times that of an equal mass of sucrose. Brazzein exhibits excellent water solubility, thermodynamic stability, and pH stability. Compared to traditional carbohydrate sugars or chemically synthesized sweeteners, these natural sweet proteins generally offer advantages such as high sweetness, low calories, a pleasant taste, and safety and non-toxicity. They also improve food flavor, can be degraded into essential amino acids, and do not cause blood sugar spikes or tooth decay. They are considered highly promising food additives.

[0005] Among the sweet proteins, only thaumatin has a reported detection method. The national standard for thaumatin can only detect pure products with a purity greater than 95%, and cannot accurately detect fermentation broth samples with complex components. Patent CN109682894A reports a method for detecting thaumatin in beverages, using ultra-high performance liquid chromatography and SDS-PAGE for qualitative and quantitative analysis.

[0006] However, there is no report on the relevant detection method for brazilian tartrate. At present, the brazilian tartrate protein produced by microorganisms urgently needs a relevant detection method to measure the concentration to meet the needs of fermentation production.

[0007] Summary of the Invention

[0008] The present invention provides a method for detecting the concentration of brazilian tea in a sample. The method can accurately detect the concentration of brazilian tea prepared by microbial fermentation by high performance liquid chromatography, thereby meeting the needs of microbial fermentation production.

[0009] The method for detecting the concentration of brazilian sweetener in a sample of the present invention comprises:

[0010] (a) establishing a standard curve, comprising performing liquid chromatography detection using standard solutions of different concentrations of brazilian sweetener, preparing a standard curve based on the chromatographic peak area of ​​the standard solution at the maximum absorption wavelength and the brazilian sweetener concentration, and fitting to obtain a regression equation for the standard curve;

[0011] (b) Sample testing, including performing liquid chromatography on the sample, and calculating the concentration of brassica oleracea in the sample based on the chromatographic peak area at the maximum absorption wavelength of the sample and the regression equation of the standard curve.

[0012] In some embodiments, the maximum absorption wavelength range is 270-285 nm, preferably 275-280 nm, and most preferably 279 nm.

[0013] In some embodiments, in step (b), the sample is pretreated before being subjected to liquid chromatography detection. The pretreatment comprises diluting the sample with a 0.2M-0.5M sodium chloride solution and then filtering to remove impurities.

[0014] In some embodiments, the filler material for liquid chromatography detection is a cation exchange resin.

[0015] In some embodiments, liquid chromatography detection uses phosphate buffer as the equilibration solution and phosphate buffer with sodium chloride added as the eluent, wherein the pH of the phosphate buffer is 6.8-7.2, preferably 7.0.

[0016] In some embodiments, the concentration of sodium chloride in the eluent is 0.5M to 1.0M, preferably 0.8M.

[0017] In some embodiments, the brazilian tamarind solution standard has a brazilian tamarind concentration between 50 μg / mL and 1000 μg / mL, preferably between 80 μg / mL and 500 μg / mL.

[0018] In some embodiments, the standard curve regression equation is Y=1.5512X-29.394, where X is the concentration of brazilian salsa and Y is the chromatographic peak area.

[0019] The detection method of the present invention has the following advantages:

[0020] 1. Filled the gap in the detection method of Brazilian sweet;

[0021] 2. It can not only detect pure products with a purity greater than 95%, but also fermentation tank samples with complex environmental components and post-processing samples to meet the testing needs of fermentation production;

[0022] 3. Environmentally friendly, the mobile phase and reagents used only involve buffer salt reagents;

[0023] 4. High accuracy. According to the recommendation of GB / T 22554-2010 "Linear calibration based on standard samples", a standard curve is prepared. The linear performance of the curve reaches above 0.999. After the standard curve is prepared, the content of the substance to be tested can be found according to the standard curve. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] FIG1 is a full wavelength scan of the test solution containing brazilin in Example 1.

[0025] FIG2 is a standard curve of braziliana concentration-peak area established in Example 1. DETAILED DESCRIPTION

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. All publications, patent applications, patents and other references mentioned herein are incorporated herein by reference in their entirety. In the event of a conflict, the present specification (including definitions) shall prevail. In addition, the materials, methods and examples described herein are illustrative only and are not intended to be limiting.

[0027] The terms "about" and "approximately," when used in connection with a numerical variable, generally mean that the value of the variable and all values ​​of the variable are within measurement or experimental error (e.g., a 95% confidence interval for the mean) or within the broader range of the specified value (e.g., ±5% or ±10%).

[0028] The term "comprise" or its variations such as "contains," "has," and "includes" means including the stated steps or elements but not excluding any other steps or elements. "Consisting of" means excluding unrecited steps or elements. "Consisting essentially of" means not excluding steps or elements that do not materially affect the basic and novel characteristics of the claimed invention. The term "comprising" specific steps or elements and its variations also include "consisting of" and "consisting essentially of" specific steps or elements.

[0029] When a numerical range is mentioned, it should be considered that the specific values ​​of its upper and lower limits are specifically disclosed, as well as all intermediate ranges included therein, such as intermediate ranges between its upper or lower limit and any intermediate value, or intermediate ranges between any two intermediate values. In addition, any intermediate ranges, subranges and all individual values ​​described in the numerical range can be excluded from the numerical range.

[0030] The term "and / or" used herein should be understood as including any one element or any combination of several elements among the multiple elements connected by the term.

[0031] In the present invention, when referring to the concentration (or content) of a substance in a mixture, unless otherwise specified, when the mixture is a solution and the substance is a solid substance before being prepared into the mixture, the concentration refers to the concentration (or content) of mass / volume; when the mixture is a solution and the substance is a liquid substance before being prepared into the mixture, the concentration refers to the concentration (or content) of volume / volume; when the mixture is a solid substance and the substance is a solid substance before being prepared into the mixture, the concentration refers to the concentration (or content) of mass / mass.

[0032] The invention provides a method for detecting the concentration of sweet protein-brazil in a sample.

[0033] Brazzein is a 54-amino acid sweet protein isolated and purified from the fruit of the wild plant Pentadiplandra brazzeana in West Africa (Ming D, Hellekant G. Brazzein, a new high-potency thermostable sweet protein from Pentadiplandra brazzeana B. FEBS Lett. 1994; 355:106–108). The detection target "brazzein" in the detection method of the present invention can include wild-type brazzein protein and various forms thereof, such as various naturally occurring forms of the protein, various mutants (including natural or artificial variants), truncated forms of the protein, and chemically modified proteins. The brazzein detected in the present invention can be purified from natural sources or prepared through genetic engineering methods.

[0034] The method for detecting the concentration of brasiliensis of the present invention comprises the following steps:

[0035] (a) establishing a standard curve, comprising performing liquid chromatography detection using standard solutions of different concentrations of brazilian sweetener, preparing a standard curve based on the chromatographic peak area of ​​the standard solution at the maximum absorption wavelength and the brazilian sweetener concentration, and fitting to obtain a regression equation for the standard curve;

[0036] (b) Sample testing, including performing liquid chromatography on the sample, and calculating the concentration of brassica oleracea in the sample based on the chromatographic peak area of ​​the sample at the maximum absorption wavelength and the regression equation of the standard curve.

[0037] In some embodiments, the maximum absorption wavelength range is 270-285nm, for example 270nm, 271nm, 272nm, 273nm, 274nm, 275nm, 276nm, 277nm, 278nm, 279nm, 280nm, 281nm, 282nm, 283nm, 284nm, 285nm. Those skilled in the art can reasonably select the maximum absorption wavelength according to the structure of brazilian sweet. As is well known to those skilled in the art, "maximum absorption wavelength" refers to the wavelength corresponding to the peak of the maximum absorption peak on the absorption spectrum. An absorption spectrum is a graph of absorbance versus wavelength. The maximum absorption wavelength used in the above-mentioned detection method can be obtained by full wavelength scanning, for example, by carrying out a full wavelength scan on the brazilian sweet protein to be detected.

[0038] In some embodiments, the brazilian sweet solution standard is a series of brazilian sweet solutions of different concentrations prepared using purified brazilian sweet. In some embodiments, the concentration of the brazilian sweet solution standard is between 50 μg / mL and 1000 μg / mL, for example, between 50 μg / mL and 800 μg / mL, between 80 μg / mL and 800 μg / mL, or between 80 μg / mL and 500 μg / mL. In some embodiments, purified brazilian sweet refers to brazilian sweet with a purity of ≥95% or higher. The number of brazilian sweet solutions of different concentrations contained in the brazilian sweet solution standard can be 5, 6, 7, 8 or more. The brazilian sweet solution standard can be prepared from purified brazilian sweet and water or buffer.

[0039] The method of the present invention measures the concentration of brazilian sweet by liquid chromatography. The term "liquid chromatography (LC)" may refer to the process of selective retardation of one or more components of a fluid solution when the fluid passes through a column of finely divided material or is filtered uniformly through a capillary channel. Retardation is caused by the distribution of the components of a mixture between one or more stationary phases and a large amount of fluid (i.e., mobile phase) as the fluid moves relative to the stationary phase (or multiple stationary phases). Examples of "liquid chromatography" include reverse phase liquid chromatography (RPLC), high performance liquid chromatography (HPLC), and turbulent flow liquid chromatography (TFLC) (sometimes referred to as high turbulence liquid chromatography (HTLC) or high throughput liquid chromatography). In some embodiments, the concentration of brazilian sweet is measured by high performance liquid chromatography. The term "high performance liquid chromatography (HPLC)" as described herein is also known as high pressure liquid chromatography, which refers to liquid chromatography in which a mobile phase is forced under pressure through a stationary phase (usually a tightly packed column), thereby increasing the degree of separation.

[0040] In some embodiments, the filler of the liquid chromatography is a cation exchange resin. In some embodiments, the ion exchange group of the liquid chromatography filler is a sulfopropyl group. In some embodiments, the liquid chromatography uses IEC-SP-8-5000 (sodium ion analysis column) as a filler. In some embodiments, the liquid chromatography uses sodium chloride elution, preferably using 0.5M to 1.0M, such as 0.5M, 0.6M, 0.7M, 0.8M, 0.9M or 1.0M sodium chloride for elution. In some embodiments, the elution step of the liquid chromatography includes: first, allowing the equilibrium solution without sodium chloride to flow through the chromatographic column, and then allowing the mobile phase (eluent) containing sodium chloride to flow through the chromatographic column for elution. In some embodiments, the elution step of the liquid chromatography includes: 0-5min, allowing the equilibrium solution without sodium chloride to flow through the chromatographic column; 5-15min, allowing the mobile phase (eluent) containing sodium chloride to flow through the chromatographic column for elution. In some embodiments, the liquid chromatography uses a phosphate buffer as an equilibrium solution, and the pH of the phosphate buffer is 6.8-7.2, preferably 7.0. In some embodiments, the liquid chromatography uses a phosphate buffer to which sodium chloride is added as an eluent, and the pH of the phosphate buffer is 6.8-7.2, preferably 7.0. In some embodiments, the concentration of sodium chloride in the eluent is 0.5M to 1.0M, such as 0.5M, 0.6M, 0.7M, 0.8M, 0.9M or 1.0M. The phosphate buffer can be a sodium phosphate buffer or a potassium phosphate buffer. In some embodiments, the phosphate buffer is a sodium phosphate buffer, which contains disodium hydrogen phosphate and sodium dihydrogen phosphate. In some embodiments, the phosphate buffer is composed of disodium hydrogen phosphate, sodium dihydrogen phosphate and water.

[0041] In some embodiments, the column temperature of the liquid chromatography detection is 32° C.-38° C., preferably 35° C. In some embodiments, the equilibration and / or elution flow rate of the liquid chromatography detection is 0.6-1.0 mL / min, preferably 0.8 mL / min.

[0042] In some embodiments, the sample and the standard of the brazilian sweet solution are subjected to the same liquid chromatography test procedure. Specifically, the reagents, test procedures, operating parameters, etc. used in the liquid chromatography test are the same.

[0043] In some embodiments, the standard curve regression equation is a linear equation. The fitting method of the standard curve regression equation is well known to those skilled in the art, such as the least squares method. In some embodiments, the R of the standard curve regression equation is 2 ≥0.99, or R 2≥0.999. In some embodiments, when preparing a standard curve, the protein (i.e., brazilian sweet) concentration is used as the abscissa (X) and the chromatographic peak area is used as the ordinate (Y), and a standard curve regression equation is fitted. In some embodiments, the standard curve regression equation is Y=1.5512X-29.394.

[0044] In the present invention, the sample can be any sample to be tested containing brazilian sweet, for example, it can be purified brazilian sweet, or it can be a food, beverage, fermentation broth (such as a fermentation product prepared by biological fermentation, such as microbial fermentation) containing brazilian sweet, and a sample of the fermentation broth after subsequent treatment. In some embodiments, the sample is liquid, or the substance to be tested can be dissolved in water or a buffer solution and tested as a sample. In some embodiments, the fermentation broth is a fermentation broth for producing brazilian sweet, for example, a fermentation broth produced by fermenting cells (such as microorganisms) that contain a nucleotide sequence encoding brazilian sweet and can express brazilian sweet.

[0045] In some embodiments, before the sample is subjected to liquid chromatography detection, the sample is pretreated, and the pretreatment may include filtration to remove impurities in the sample. The filtration may be, for example, filtration using a filter membrane. The filter membrane may be a microfiltration membrane or an ultrafiltration membrane. In some embodiments, the filter membrane may be an aqueous filter membrane (also referred to as an aqueous filter membrane or water membrane in the present invention). The term "aqueous filter membrane" refers to a filter membrane for filtering an aqueous solution. In some embodiments, the filter membrane may be a cellulose acetate filter membrane or a mixed cellulose ester filter membrane (MCEM). In some embodiments, the pore size of the filter membrane is about 0.2 μm to 0.22 μm.

[0046] In some embodiments, the pretreatment further comprises diluting the sample. The sample is diluted before detection so that the concentration of brazilian tartrate in the sample is within a range suitable for detection, for example, between the highest concentration and the lowest concentration of the standard, to obtain more accurate results. Those skilled in the art know how to determine an appropriate dilution factor. For example, a dilution factor can be preset first and then detected. The appropriateness of the preset dilution factor can be determined based on whether the detection concentration of the diluted sample is between the highest concentration and the lowest concentration of the standard, and the dilution factor can be adjusted as appropriate. Those skilled in the art should understand that when calculating the concentration of brazilian tartrate in the sample, the dilution factor needs to be considered. Specifically, when the protein concentration value corresponding to the diluted sample is obtained according to the standard curve regression equation, the concentration value is multiplied by the dilution factor to obtain the brazilian tartrate concentration value of the sample before dilution.

[0047] In some embodiments, the sample is diluted before filtering. In some embodiments, the sample is diluted after filtering. In some embodiments, the sample is diluted before filtering and further diluted after filtering.

[0048] In some embodiments, the sample is diluted with sodium chloride solution before filtering. The concentration of the sodium chloride solution for diluting the sample can be 0.2M-0.5M, for example 0.2M, 0.3M, 0.4M or 0.5M. Compared with filtering after diluting the sample with equilibration solution A, filtering after diluting the sample with sodium chloride can reduce the adsorption loss of brazilin on the filter membrane during the filtration process. In some embodiments, the sample is detected after diluting with equilibration solution after filtering. In some embodiments, the sample is diluted with sodium chloride solution before filtering and detects after diluting with equilibration solution after filtering.

[0049] In some embodiments, the pretreatment may further include other processing steps, such as centrifugation to obtain the supernatant.

[0050] Those skilled in the art will appreciate that pretreatment is not a necessary step. When the sample contains relatively few impurities, the impurity removal step may not be necessary. When the concentration of the parathalam in the sample is expected to be within a range suitable for detection, the dilution step may not be necessary.

[0051] The technical solutions of the present invention are further described in detail below through examples and in conjunction with the accompanying drawings, but the present invention is not limited to the following examples.

[0052] Example 1 Establishment of a Method for Detecting Brazilian Sweetener Concentration

[0053] (1) Determine the detection wavelength of brasilien:

[0054] (1-1) diluting the fermented brasilien sample with water to an appropriate multiple;

[0055] (1-2) Baseline zeroing was performed with water, and the dilution obtained in (1-1) was scanned at the full wavelength of 210 nm to 740 nm. The highest peak appeared at 279 nm, so the wavelength was selected for detection at 279 nm, as shown in Figure 1;

[0056] (2) Chromatographic conditions:

[0057] Agilent 1220 ultra-high performance liquid chromatograph; chromatographic column: IEC-SP-8-5000 sodium ion analysis column; balance solution A (equilibrium solution): disodium hydrogen phosphate dodecahydrate / sodium dihydrogen phosphate dihydrate, pH 7.0; mobile phase B (eluent): disodium hydrogen phosphate dodecahydrate / sodium dihydrogen phosphate dihydrate / 0.8 M sodium chloride, pH 7.0; elution conditions: 0-5 min, balance solution A, i.e., 100% disodium hydrogen phosphate dodecahydrate / sodium dihydrogen phosphate dihydrate; 5-15 min, mobile phase B, i.e., 100% disodium hydrogen phosphate dodecahydrate / sodium dihydrogen phosphate dihydrate / 0.8 M sodium chloride; column temperature: 35°C; flow rate: 0.8 mL / min; sample volume: 20 μL.

[0058] Reagent preparation:

[0059] Balanced solution A: disodium hydrogen phosphate dodecahydrate / sodium dihydrogen phosphate dihydrate at pH 7.0.

[0060] Weigh 2.18 g of disodium hydrogen phosphate dodecahydrate and 0.61 g of sodium dihydrogen phosphate dihydrate respectively and dissolve them in ultrapure water. After dissolution, dilute to 1 L with ultrapure water, adjust the pH to 7.0 with 5 mol / L glacial acetic acid, and store at room temperature before use.

[0061] Mobile phase B: sodium hydrogen phosphate dodecahydrate / sodium dihydrogen phosphate dihydrate / 0.8 M sodium chloride at pH 7.0.

[0062] Weigh 2.18 g of disodium hydrogen phosphate dodecahydrate, 0.61 g of sodium dihydrogen phosphate dihydrate, and 46.75 g of sodium chloride respectively and dissolve them in ultrapure water. After dissolution, dilute to 1 L with ultrapure water, adjust the pH to 7.0 with 5 mol / L sodium hydroxide solution, and store at room temperature before use.

[0063] (3) Establish a standard curve:

[0064] 30 mg of brazilian sweet (purified sample, purity ≥95%) was weighed and added to 30 mL of H2O to prepare a 1 mg / mL brazilian sweet solution standard stock solution, which was then further prepared into brazilian sweet samples with concentrations of 500 μg / mL, 400 μg / mL, 200 μg / mL, 160 μg / mL, and 80 μg / mL. High performance liquid chromatography was performed and a standard curve was plotted based on the chromatographic peak area at 279 nm and the corresponding concentration. The standard curve regression equation was obtained: Y = 1.5512X - 29.394, R 2 =0.9998, as shown below:

[0065] Table 1

[0066] (4) Sample pretreatment

[0067] Take 1 mL of the sample supernatant, add 1 mL of 0.2 M sodium chloride, 0.4 M sodium chloride, 0.5 M sodium chloride and balance solution A respectively, and filter with a 0.22 μm water membrane filter to obtain a pretreated sample. The purpose of filtering the sample with a water membrane filter is to prevent the pigment and other impurities in the sample from entering the liquid phase instrument and chromatographic column, causing damage to the instrument and chromatographic column. Filtering with a water membrane filter will cause the adsorption of brassina. Diluting and filtering with sodium chloride will reduce the adsorption loss of brassina during the filtration process. Compared with filtering with balance solution A, diluting with sodium chloride significantly reduces the adsorption loss of brassina, and the yield before and after filtration reaches more than 95%. The specific data are shown in Table 2 below:

[0068] Table 2

[0069] Repeatability (parallelism) of the method established in Example 2

[0070] A 1 mg / mL mother liquor of a brazilian tartrate standard (purified sample, purity ≥95%) was diluted 2-fold with equilibration solution A for liquid phase detection. High performance liquid chromatography (HPLC) detection was performed under the chromatographic conditions described in Example 1, with eight repetitions. The concentration of the mother liquor brazilian tartrate was calculated using the standard curve established in Example 1. The relative deviation was calculated as (average value - actual measured value) / average value * 100%, which was within 5%, meeting the requirements of fermentation production. The test results are as follows:

[0071] Table 3

[0072] The fermentation tank supernatant sample was diluted 2 times with 0.4M NaCl, filtered through a water membrane filter, and then diluted 3 times with equilibrium solution A for liquid phase detection. HPLC detection was performed under the chromatographic conditions described in Example 1, and repeated detection was performed 3 times. The concentration of the fermentation tank supernatant sample brazilin was calculated according to the standard curve established in Example 1. The relative deviation was within 5%, which met the requirements of fermentation production. The test results are as follows:

[0073] Table 4

[0074] Stability of the method established in Example 3

[0075] The concentration of brazilian tamarind protein in samples from different shake flask fermentations was tested at different time periods to verify the stability of this method.

[0076] The shake flask sample was diluted 2-fold with 0.4 M NaCl, filtered with a water membrane filter, and then diluted with equilibration solution A to a standard curve range of 80-500 μg / mL. HPLC detection was performed under the chromatographic conditions described in Example 1, and the concentration of brazilian tamarind in the fermentation sample was calculated based on the standard curve established in Example 1. Each sample was repeated three times, and the relative deviation = (average value - actual detection value) / average value * 100% was within 5%. The test results are as follows:

[0077] Table 5

[0078] Example 4 Determination of recovery of the method

[0079] Sample Preparation: Sample 1: Dilute the standard (1 g / L) 2-fold with water; Sample 2: Dilute the supernatant from the shake flask sample 2-fold with 0.4 M sodium chloride and filter through a water filter; Sample 3: Take equal volumes of the mixture of Sample 1 and Sample 2. Each of these three samples was subjected to HPLC analysis using the chromatographic conditions described in Example 1. The concentration of brazilin was calculated using the standard curve established in Example 1. The calculated results are as follows: The recovery rate is 100%. The test results are as follows:

[0080] Table 6

[0081] The embodiments of the present invention are not limited to the above embodiments. Without departing from the spirit and scope of the present invention, ordinary technicians in this field can make various changes and improvements to the present invention in form and details, and these are all considered to fall within the scope of protection of the present invention.

Claims

1. The method for detecting the concentration of Brazilian sweetener in the sample includes: (a) performing liquid chromatography detection using standard solutions of different concentrations of brazilian sweetener, preparing a standard curve according to the chromatographic peak area of ​​the standard solution at the maximum absorption wavelength and the brazilian sweetener concentration, and obtaining a regression equation of the standard curve by fitting; (b) Performing liquid chromatography on the sample, and calculating the concentration of brazilian sweetener in the sample based on the chromatographic peak area at the maximum absorption wavelength of the sample and the regression equation of the standard curve.

2. The detection method according to claim 1, wherein the maximum absorption wavelength range is 270-285 nm, preferably 275-280 nm, and most preferably 279 nm.

3. The detection method according to claim 1 or 2, wherein in step (b), before the sample is subjected to liquid chromatography detection, the sample is pretreated, and the pretreatment comprises diluting the sample with a 0.2M-0.5M sodium chloride solution and then filtering to remove impurities.

4. The detection method according to any one of claims 1 to 3, wherein the filler for liquid chromatography detection is a cation exchange resin.

5. The detection method according to any one of claims 1 to 4, wherein the liquid chromatography detection uses a phosphate buffer as a balancing solution, and a phosphate buffer to which sodium chloride is added as an eluent, and the pH of the phosphate buffer is 6.8-7.2, preferably 7.

0.

6. The detection method according to claim 5, wherein the concentration of sodium chloride in the eluent is 0.5M to 1.0M, preferably 0.8M.

7. The detection method according to any one of claims 1 to 6, wherein the concentration of the Brazilian sweet solution standard is between 50 μg / mL and 1000 μg / mL, preferably between 80 μg / mL and 500 μg / mL.

8. The detection method according to any one of claims 1 to 7, wherein the standard curve regression equation is Y=1.5512X-29.394, wherein X is the concentration of brazilian sweetener and Y is the chromatographic peak area.

9. The detection method according to any one of claims 1 to 8, wherein the brazilian sweetener is a purified sample or a fermentation product prepared by biological fermentation.

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