Method for detecting concentration of Brazilian sweet

Abstract

The invention discloses a method for detecting concentration of Brazilian sweet. The invention provides a Brazilian sweet concentration detection method which comprises the following steps: (a) carrying out liquid chromatography detection by using Brazilian sweet solution standard substances with different concentrations, making a standard curve according to the chromatographic peak area of the standard substance at the maximum absorption wavelength and the Brazilian sweet concentration, and fitting to obtain a standard curve regression equation; and (b) carrying out liquid chromatography detection on the sample, and calculating the concentration of the Brazilian sweet in the sample according to the chromatographic peak area at the maximum absorption wavelength of the sample and the standard curve regression equation.

Description

[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 invention relates to a sweet protein detection technology, in particular to a method for detecting the concentration of basil in a sample. Background Art

[0004] Brazzein is a sweet protein isolated and purified from the fruit of the wild plant Pentadiplandra Brazzeana in western Africa. It is called sweet protein together with proteins with similar properties from other plant sources, such as Thaumatin, Monellin, and Miraculin. Brazzein is a single-chain protein composed of 54 amino acid residues. It is the smallest relative molecular weight in the plant sweet protein family, with a relative molecular mass of 6.5kDa and an isoelectric point of 5. Its sweetness is about 2000 times that of sucrose of the same mass. Brazzein has good water solubility, thermodynamic stability, and pH stability. Compared with traditional carbohydrate sugars or chemically synthesized sweeteners, these natural sweet proteins generally have the advantages of high sweetness, low calories, good taste, safety and non-toxicity. At the same time, they also have the characteristics of improving food flavor, being degradable into various amino acids required by the human body, and not causing blood sugar increase or tooth decay. They are considered to be food additives with great development prospects.

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

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

[0007] The invention provides a method for detecting the concentration of brazilian sweetener in a sample. The concentration of brazilian sweetener prepared by microbial fermentation can be accurately detected by high performance liquid chromatography, thereby meeting the demand for microbial fermentation production.

[0008] The method for detecting the concentration of brazilian sweetener in a sample of the present invention comprises:

[0009] (a) establishing a standard curve, comprising performing liquid chromatography detection with different concentrations of basil solution standard products, preparing a standard curve according to the chromatographic peak area of ​​the standard product at the maximum absorption wavelength and the basil concentration, and fitting to obtain a regression equation of the standard curve;

[0010] (b) Sample testing, including 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.

[0011] In some embodiments, the maximum absorption wavelength range is 270-285 nm, preferably 275-280 nm, and most preferably 279 nm.

[0012] 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.

[0013] In some embodiments, the filler material for liquid chromatography detection is a cation exchange resin.

[0014] In some embodiments, the liquid chromatography detection uses a phosphate buffer as the equilibration solution, and uses a phosphate buffer with sodium chloride added as the eluent, wherein the pH of the phosphate buffer is 6.8-7.2, preferably 7.0.

[0015] In some embodiments, the concentration of sodium chloride in the eluent is 0.5M to 1.0M, preferably 0.8M.

[0016] In some embodiments, 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.

[0017] In some embodiments, the standard curve regression equation is Y=1.5512X-29.394, where X is the concentration of brazilian sweetener and Y is the chromatographic peak area.

[0018] The detection method of the present invention has the following advantages:

[0019] 1. Filled the gap in the detection method of Brazilian sweet;

[0020] 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 detection needs of fermentation production;

[0021] 3. Environmentally friendly, the mobile phase and reagents used only involve buffer salt reagents;

[0022] 4. High accuracy. According to the recommendation of GB / T 22554-2010 "Linear calibration based on standard samples", the standard curve is made. The linear performance of the curve reaches above 0.999. After the standard curve is made, the content of the substance to be tested can be found out according to the standard curve. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a full wavelength scan of the test solution containing brazilian tamarind in Example 1.

[0024] Figure 2 This is the standard curve of Brazilian sweetener concentration-peak area established in Example 1. DETAILED DESCRIPTION

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally 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, this specification (including definitions) shall prevail. In addition, the materials, methods and examples described herein are illustrative only and are not intended to be limiting.

[0026] The terms "about" and "approximately" when used in conjunction 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., 95% confidence interval for the mean) or within a wider range of the specified value (e.g., ±5% or ±10%).

[0027] The term "comprise" or its variations such as "contains", "has", "includes" means including the stated steps or elements but not excluding any other steps or elements. "Consisting of" means not excluding steps or elements not listed. "Consisting essentially of" means not excluding steps or elements that do not substantially affect the basic and novel features of the invention being protected. The term "comprising" specific steps or elements and its variations also include "consisting of" and "consisting essentially of" specific steps or elements.

[0028] When a numerical range is mentioned, it should be deemed to specifically disclose the specific values ​​of its upper and lower limits, as well as all intermediate ranges included therein, such as the intermediate range between its upper or lower limit and any intermediate value, or the intermediate range between any two intermediate values ​​thereof. In addition, any intermediate range, subrange and all individual values ​​described in the numerical range can be excluded from the numerical range described.

[0029] The “and / or” described herein should be understood as any one element or any combination of several elements connected by this term.

[0030] 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 made 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 made 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 made into the mixture, the concentration refers to the concentration (or content) of mass / mass.

[0031] The invention provides a method for detecting the concentration of sweet protein-brazil in a sample.

[0032] Brazzein is a sweet protein with 54 amino acids isolated and purified from the fruit of the wild plant Pentadiplandra Brazzeana in western 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 object "Brazzein" in the detection method of the present invention may include wild-type protein of Brazzein and its various forms, such as various naturally occurring proteins, various mutants (including natural variants or artificial variants), truncated proteins and chemically modified proteins. The Brazzein detected in the present invention may be purified from natural sources or prepared by genetic engineering methods.

[0033] The method for detecting the concentration of Brazilian sweetener of the present invention comprises the following steps:

[0034] (a) establishing a standard curve, comprising performing liquid chromatography detection with different concentrations of basil solution standard products, preparing a standard curve according to the chromatographic peak area of ​​the standard product at the maximum absorption wavelength and the basil concentration, and fitting to obtain a regression equation of the standard curve;

[0035] (b) Sample testing, including performing liquid chromatography on the sample, and calculating the concentration of brassica 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.

[0036] 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. The absorption spectrum is a graph of absorbance and wavelength relationship. The maximum absorption wavelength used in the above detection method can be obtained by full wavelength scanning, for example, full wavelength scanning is performed on the Brazilian sweet protein to be detected.

[0037] 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 purity. 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.

[0038] The method of the present invention detects the concentration of brazilian sweet by liquid chromatography. The term "liquid chromatography (LC)" refers to the process of selective retardation of one or more components of a fluid solution when the fluid passes through a column of a subdivided substance or is uniformly filtered through a capillary channel. The retardation is caused by the distribution of the components of the mixture between one or more stationary phases and a large amount of fluid (i.e., mobile phase) when 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 detected by high performance liquid chromatography. The term "high performance liquid chromatography (HPLC)" described herein is also referred to as high pressure liquid chromatography, which refers to a liquid chromatography that forces the mobile phase to pass through a stationary phase (usually a tightly packed column) under pressure, thereby increasing the degree of separation.

[0039] 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 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, a balance solution without sodium chloride is passed through the chromatographic column, and then a mobile phase (eluent) containing sodium chloride is passed through the chromatographic column for elution. In some embodiments, the elution step of the liquid chromatography includes: 0-5min, a balance solution without sodium chloride is passed through the chromatographic column; 5-15min, a mobile phase (eluent) containing sodium chloride is passed through the chromatographic column for elution. In some embodiments, the liquid chromatography uses a phosphate buffer as a balancing 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.

[0040] In some embodiments, the column temperature of the liquid chromatography detection is 32° C.-38° C., preferably 35° C. In some embodiments, the flow rate of equilibrium and / or elution of the liquid chromatography detection is 0.6-1.0 mL / min, preferably 0.8 mL / min.

[0041] In some embodiments, the sample and the standard of the Brazilian sweet solution are subjected to liquid chromatography detection by the same procedure. Specifically, the reagents, detection procedures, operating parameters, etc. used in the liquid chromatography detection of the two are the same.

[0042] 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 the 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) to fit the standard curve regression equation. In some embodiments, the standard curve regression equation is Y=1.5512X-29.394.

[0043] In the present invention, the sample can be any sample to be tested containing brazilian sweet, for example, it can be purified brazilian sweet, and it can also be food, beverage, fermentation broth (such as fermentation products prepared by biological fermentation, such as microbial fermentation) containing brazilian sweet, and samples of fermentation broth after subsequent treatment, etc. In some embodiments, the sample is liquid, or the substance to be tested can be dissolved in water or buffer for detection 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 brazilian sweet with a cell (such as a microorganism) containing a nucleotide sequence encoding brazilian sweet and capable of expressing brazilian sweet.

[0044] 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 a water membrane in the present invention). The term "aqueous filter membrane" refers to a filter membrane used to filter 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.

[0045] In some embodiments, the pretreatment also includes diluting the sample. The sample is diluted before detection so that the concentration of brûlée in the sample is in 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 detection can be performed. According to whether the detection concentration of the diluted sample is between the highest concentration and the lowest concentration of the standard, it is judged whether the preset dilution factor is appropriate, and the dilution factor is adjusted as appropriate. Those skilled in the art should understand that when calculating the concentration of brûlée 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 brûlée concentration value of the sample before dilution.

[0046] 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.

[0047] 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, such as 0.2M, 0.3M, 0.4M or 0.5M. Compared with filtering after diluting the sample with balancing solution A, filtering after diluting the sample with sodium chloride can reduce the adsorption loss of brazilian sweet protein on the filter membrane during filtering. In some embodiments, the sample is detected after diluting with balancing solution after filtering. In some embodiments, the sample is diluted with sodium chloride solution before filtering and detected after diluting with balancing solution after filtering.

[0048] In some embodiments, the pretreatment may further include other processing steps, such as centrifugation to obtain supernatant, etc.

[0049] Those skilled in the art will appreciate that pretreatment is not a necessary step. When there are fewer impurities in the sample, there is no need to perform a removal step. When the concentration of brazilian sweetener in the sample is expected to be within a range suitable for detection, there is no need to perform a dilution step.

[0050] The technical solution of the present invention is further described in detail below through embodiments and in conjunction with the accompanying drawings, but the present invention is not limited to the following embodiments.

[0051] Example 1 Establishment of Brazilian sweetener concentration detection method

[0052] (1) Determine the detection wavelength of brasilien:

[0053] (1-1) diluting the tamarind sample obtained by fermentation with water to a suitable multiple;

[0054] (1-2) Use water to calibrate the baseline to zero, and scan the dilution obtained in (1-1) at the full wavelength of 210nm-740nm. The highest peak appears at 279nm, so the wavelength is selected for detection at 279nm. Figure 1 ;

[0055] (2) Chromatographic conditions:

[0056] Agilent 1220 ultra high performance liquid chromatograph; chromatographic column: IEC-SP-8-5000 sodium ion analysis column; balance solution A (balance solution): disodium hydrogen phosphate dodecahydrate / sodium dihydrogen phosphate dihydrate at pH 7.0; mobile phase B (eluent): disodium hydrogen phosphate dodecahydrate / sodium dihydrogen phosphate dihydrate / 0.8 M sodium chloride at 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 load: 20 μL.

[0057] Reagent preparation:

[0058] Balanced solution A: disodium hydrogen phosphate dodecahydrate / sodium dihydrogen phosphate dihydrate at pH 7.0.

[0059] 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, make up 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.

[0060] Mobile phase B: sodium phosphate dodecahydrate / sodium dihydrogen phosphate dihydrate / 0.8 M sodium chloride at pH 7.0.

[0061] 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, make up 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.

[0062] (3) Establishing a standard curve:

[0063] Weigh 30 mg of Brazilian sweet (purified sample, purity ≥ 95%), add 30 mL of H 2 O, a 1 mg / mL stock solution of the standard Brazilian sweet solution was prepared, and then Brazilian sweet samples with concentrations of 500 μg / mL, 400 μg / mL, 200 μg / mL, 160 μg / mL, and 80 μg / mL were prepared respectively, and high performance liquid chromatography was performed. A standard curve was drawn according to the chromatographic peak area at 279 nm and the corresponding concentration, and the standard curve regression equation was obtained: Y=1.5512X-29.394, R 2 =0.9998, as shown below:

[0064] Table 1

[0065] Concentration (μg / mL) Chromatographic peak area 80 95.726 160 216.152 200 284.035 400 586.131 500 749.536

[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 the chromatographic column, causing damage to the instrument and the chromatographic column. Filtering with a water membrane filter will cause the adsorption of Brazilian sweet protein. Diluting and filtering with sodium chloride will reduce the adsorption loss of Brazilian sweet protein during the filtration process. Compared with filtering with balance solution A, diluting with sodium chloride significantly reduces the adsorption loss of Brazilian sweet protein, and the yield before and after filtration reaches more than 95%. The specific data are shown in Table 2 below:

[0068] Table 2

[0069]

[0070] Repeatability (Parallelism) of the method established in Example 2

[0071] The 1 mg / mL mother liquor of the brazilian tartrate standard (purified sample, purity ≥ 95%) was diluted 2 times with the equilibrium solution A for liquid phase detection, and the HPLC detection was performed according to the chromatographic conditions described in Example 1, and the detection was repeated 8 times. The concentration of the mother liquor brazilian tartrate protein was calculated according to the standard curve established in Example 1, and the relative deviation = (average value-actual detection value) / average value*100%, and the relative deviation was within 5%, which met the requirements of fermentation production. The detection results are as follows:

[0072] Table 3

[0073]

[0074] The fermentation tank supernatant sample was diluted 2 times with 0.4M NaCl, filtered with a water membrane filter, and then diluted 3 times with equilibrium solution A for liquid phase detection. The HPLC detection was performed according to the chromatographic conditions described in Example 1, and the detection was repeated 3 times. The concentration of brazilian tamarind in the fermentation tank supernatant sample was calculated according to the standard curve established in Example 1, and the relative deviation was (average value-actual detection value) / average value*100%, and the relative deviation was within 5%, which met the requirements of fermentation production. The detection results are as follows:

[0075] Table 4

[0076]

[0077] Stability of the method established in Example 3

[0078] 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.

[0079] The shake flask sample was diluted 2 times with 0.4M NaCl, filtered with a water membrane filter, and then diluted with equilibrated solution A to a standard curve range of 80-500 μg / mL. High performance liquid chromatography was performed according to the chromatographic conditions described in Example 1, and the concentration of the fermented sample brazilian tamarind was calculated according to the standard curve established in Example 1; each sample was repeated three times, relative deviation = (average value - actual detection value) / average value * 100%, and the relative deviation was within 5%, and the test results were as follows:

[0080] Table 5

[0081]

[0082] Example 4 Determination of the recovery rate of the method

[0083] Sample treatment: Sample 1: dilute the standard (1 g / L) with water 2 times; Sample 2: dilute the supernatant of the shake flask sample with 0.4 M sodium chloride 2 times, and then filter with water membrane; Sample 3: take an equal volume of the mixed solution of Sample 1 and Sample 2; the above three samples are respectively subjected to high performance liquid chromatography detection according to the chromatographic conditions described in Example 1. The concentration of brazilian tamarind is calculated according to the standard curve established in Example 1. The calculation results are as follows, the recovery rate is 100%, and the detection results are as follows:

[0084] Table 6

[0085]

[0086] The implementation of the present invention is not limited to the above-mentioned 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 detail, and these are considered to fall within the protection scope 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.

Images