A method for detecting steviol glycosides in sugar-free beverages based on surface-enhanced raman spectroscopy

Abstract

The application discloses a method for detecting steviol glycoside in sugar-free beverage based on surface-enhanced Raman spectroscopy, and belongs to the field of analysis and detection. The method comprises the following steps: (1) mixing silver nitrate solution and sodium citrate solution uniformly to perform hydrothermal reaction, and then performing centrifugal re-dispersion and concentration to prepare AgNPs substrate solution; then, 4-MPBA aqueous solution is added to modify the AgNPs to obtain 4-MPBA@AgNPs substrate solution; (2) a series of steviol glycoside sugar-free beverage solutions with different concentrations of steviol glycoside are prepared; then, the obtained 4-MPBA@AgNPs substrate solution is added; and after mixing, the surface-enhanced Raman spectrum of the mixed sample solution is determined; (3) a linear model is constructed by using the characteristic peak at 1572 cm ‑1 ±5 cm ‑1 in the Raman spectrum and the concentration of the steviol glycoside sugar-free beverage solution. In the method, the 4-MPBA@AgNPs substrate can be used as a Raman enhancement substrate to detect steviol glycoside in sugar-free beverage, the detection time is within 5 minutes, and the detection limit is as low as 0.169 mg / L.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for detecting steviol glycosides in sugar-free beverages based on surface-enhanced Raman spectroscopy, belonging to the field of analysis and detection. BACKGROUND

[0002] Stevioside is a rich component of Stevia rebaudiana leaves, which is well known for its strong sweetness (250-300 times sweeter than sucrose) and is used as a non-caloric sweetener in some countries. Many studies have shown that in addition to sweetness, steviol glycosides and related compounds have anti-hyperglycemic, anti-hypertensive, anti-inflammatory, anti-tumor, anti-diarrhea, diuretic and immunomodulatory effects. However, some articles have pointed out that steviol glycosides may be converted to steviol in the body and absorbed through the gastrointestinal wall, and metabolically activated steviol may have mutagenic activity and thus significant toxicity to the human body. Therefore, many countries in the world have set standards for the addition of steviol glycosides in food. Therefore, there is a need for a sensitive and reliable method for detecting steviol glycosides in the food industry.

[0003] Currently, the main methods for detecting steviol glycosides are high-performance liquid chromatography, electrophoresis, etc. These methods have high precision and reliability, but the detection steps are mostly complex and time-consuming, requiring sample pretreatment and professional instruments and manpower. Therefore, there is an urgent need to find a simpler, faster and more sensitive method for detecting steviol glycosides.

[0004] Raman spectroscopy is a commonly used vibrational spectroscopy for identifying chemical and biological molecules. Raman spectroscopy can provide valuable information related to chemical bonds and has great potential in food detection and pharmacological analysis. In addition, it is a non-destructive detection technique that does not require any pretreatment of food samples. Surface-enhanced Raman spectroscopy (SERS) technology combines the fingerprint recognition ability of Raman spectroscopy and the high sensitivity of plasmonic enhancement, making it effective for ultra-sensitive detection. SERS technology has been widely used in environmental monitoring, chemistry and biomedical fields. It has the advantages of non-contact, low detection limit and short detection time, making it more suitable for the detection of small molecules such as steviol glycosides. Moreover, there are few reports on using SERS technology to detect steviol glycosides in aqueous solutions in the field of steviol glycoside detection, so this method is the first to be proposed for this type of substance. SUMMARY

[0005] [Technical problem]

[0006] Currently, the main methods for detecting steviol glycosides are high-performance liquid chromatography, electrophoresis, etc. These detection steps are mostly complex and time-consuming, requiring sample pretreatment and professional instruments and manpower. At the same time, there are few reports on using SERS technology to detect steviol glycosides in sugar-free beverages, and the sensitivity and detection limit have certain advantages compared to traditional methods.

[0007] [Technical solution]

[0008] To solve the above at least one problem, the application applies the Raman enhanced substrate of 4-MPBA@AgNPs to detect steviol glycoside in sugar-free beverage, and realizes the effects of short detection time, high detection precision and low detection limit.

[0009] The first object of the application is to provide a method for detecting steviol glycoside in sugar-free beverage based on surface enhanced Raman spectrum, comprising the following steps:

[0010] (1) mixing silver nitrate solution and sodium citrate solution uniformly to perform hydrothermal reaction, collecting the solid by centrifugation after the reaction is finished, and then concentrating after re-dispersing, to prepare AgNPs substrate solution; then adding 4-MPBA aqueous solution to functionalize and modify the AgNPs, to obtain 4-MPBA@AgNPs substrate solution;

[0011] (2) mixing different concentrations of steviol standard solution with sugar-free beverage to obtain a series of steviol glycoside sugar-free beverage solutions with different concentrations of steviol; then adding the obtained 4-MPBA@AgNPs substrate solution; mixing uniformly, and determining the surface enhanced Raman spectrum of the mixed sample solution;

[0012] (3) using the characteristic peak at 1572cm -1 ±5cm -1 in the Raman spectrum to construct a linear model with the concentration of steviol glycoside sugar-free beverage solution;

[0013] (4) mixing the test object with 4-MPBA@AgNPs substrate solution uniformly to obtain the test sample, determining the surface enhanced Raman spectrum of the test sample, and obtaining the concentration of steviol glycoside in the test sample according to the linear model in step (3).

[0014] In an embodiment of the application, the concentration of the silver nitrate solution in step (1) is 0.1-0.5mg / mL; specifically, 0.18mg / mL can be selected.

[0015] In an embodiment of the application, the concentration of the sodium citrate solution in step (1) is 0.5-2wt%; specifically, 1wt% can be selected.

[0016] In an embodiment of the application, the volume ratio of the silver nitrate solution to the sodium citrate solution in step (1) is 50:1.

[0017] In an embodiment of the application, the synthetic material of the AgNPs in step (1) is specifically: adding 0.036g of AgNO3 powder to 200mL of deionized water, and then adding 4mL of 1wt% concentration sodium citrate solution.

[0018] In one embodiment of the present invention, the hydrothermal reaction conditions in step (1) are: continuous addition and stirring for 1 hour under boiling conditions.

[0019] In one embodiment of the present invention, the amount of 4-MPBA material used in step (1) is: 200 μL of 10-MPBA material is added to every 10 mL of AgNPs substrate solution. -3 M in 4-MPBA aqueous solution.

[0020] In one embodiment of the present invention, the 10mLAgNPs substrate solution used for modification in step (1) is a product that has been concentrated by one-fold after centrifugation and redispersion following the hydrothermal reaction.

[0021] In one embodiment of the present invention, the conditions for modifying the 4-MPBA material in step (1) are as follows: after adding the 4-MPBA aqueous solution, stir slowly for 10 minutes and then incubate for about 20 minutes.

[0022] In one embodiment of the present invention, in step (2), the volume ratio of the steviol glycoside sugar-free beverage solution to the 4-MPBA@AgNPs base solution is 1:(0.4-2.0); specifically, 1:1 is optional.

[0023] In one embodiment of the present invention, the method for preparing the steviol glycoside standard solution in step (2) is as follows: prepare a 1 mM steviol glycoside stock solution, and dilute the stock solution with sugar-free beverages to 1, 3, 5, 7, 9, 10, 15, 20, 30, 50, 100 μM respectively.

[0024] In one embodiment of the present invention, the sample solution or the sample to be tested and the substrate solution are thoroughly mixed in step (2) or (4) before the spectrum is detected.

[0025] In one embodiment of the present invention, the centrifugation parameters in step (1) are: centrifugation speed of 10000 rpm and centrifugation time of 5 min.

[0026] In one embodiment of the present invention, the conditions for Raman detection in step (2) or (4) are as follows: Raman detection is performed using a Raman spectrometer, the excitation source wavelength of the Raman spectrometer is 532 nm, the integration time is 10 s, and the laser power is 5%.

[0027] In one embodiment of the present invention, the method for preparing the standard curve model in step (3) includes the following steps:

[0028] A series of steviol glycoside sugar-free beverage solutions with varying concentrations were prepared. These solutions were then mixed with a 4-MPBA@AgNPs substrate at a 1:1 volume ratio to obtain the sample solution. Raman spectroscopy was then performed to obtain Raman spectra. Finally, the characteristic peak at 1572 cm⁻¹ in the Raman spectrum was utilized. -1±5cm -1 A linear model, i.e., a standard curve model, was constructed by comparing the peak intensity at a given point with the concentration of steviol glycosides.

[0029] In one embodiment of the present invention, during the construction of the standard curve, the sample solution and the 4-MPBA@AgNPs substrate are mixed by vortexing for 1 min.

[0030] In one embodiment of the present invention, the standard curve model in step (3) is: I SERS =36199.113 + 627.023x, correlation coefficient R 2 The value is 0.981, where x is the concentration of steviol glycosides in μmol / L; I SERS Raman, 1572cm -1 ±5cm -1 Relative intensity of characteristic peaks.

[0031] The second objective of this invention is the application of the method described herein in the field of food testing.

[0032] [Beneficial Effects]

[0033] (1) In this invention, a Raman-enhanced substrate of functionalized 4-MPBA@AgNPs is used for detection. The chemical structure of the substrate enhances the substrate’s ability to grasp the target molecules and improves the detection level.

[0034] (2) The nano-silver reduced by sodium citrate in this invention has surface plasmon resonance properties, which enhance the Raman signal.

[0035] (3) The 4-MPBA@AgNPs substrate in this invention can be used as a Raman-enhanced substrate to detect steviol glycosides in sugar-free beverages. The detection time is short and the detection limit is as low as 0.169 mg / L, which is of great significance for supervising the addition of steviol glycosides in food. Attached Figure Description

[0036] Figure 1 This is a flowchart for detecting steviol glycosides based on surface-enhanced Raman spectroscopy.

[0037] Figure 2 This illustrates the effect of reaction time in Example 2.

[0038] Figure 3 This illustrates the effect of different ratios of substrate and test solution on the intensity of Raman characteristic peaks in Example 3.

[0039] Figure 4 The concentration of steviol glycosides in the reaction system of Example 1 is related to 1572 cm⁻¹. -1 The gradient concentration relationship of Raman intensity at a given location.

[0040] Figure 5 The concentration of steviol glycosides in the reaction system of Example 1 is related to 1572 cm⁻¹. -1 The relationship curve between Raman intensities at the given location.

[0041] Figure 6 The concentration of steviol glycosides in the reaction system of Example 1 is related to 1572 cm⁻¹. -1 The linear model is obtained by fitting the relationship curve of Raman intensity at a given location.

[0042] Figure 7 Photos of a concentrated AgNPs aqueous solution (left) and a 4-MPBA@AgNPs solution (right). Detailed Implementation

[0043] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0044] Example 1: Preparation of 4-MPBA@AgNPs substrate

[0045] The preparation method of 4-MPBA@AgNPs substrate includes the following steps:

[0046] Add 200 mL of deionized water to a 250 mL Erlenmeyer flask, add 0.036 g of AgNO3 powder, stir thoroughly, and heat to boiling. While continuing to heat, add 4 mL of 1 wt% sodium citrate solution, stir and heat for 1 h, then allow to cool naturally to obtain a gray-green AgNPs substrate solution.

[0047] The AgNPs substrate solution was concentrated by centrifugation at 10,000 rpm for 5 min and then redispersed in 100 mL of deionized water. 200 μL of 10% HCl was then added to 10 mL of the concentrated solution. -3 The 4-MPBA solution of M was slowly stirred for 10 minutes, then stirred and allowed to stand for about 20 minutes. The final product was a yellowish-brown 4-MPBA@AgNPs substrate (solution).

[0048] Figure 7 Images show a concentrated AgNPs aqueous solution (left) and a 4-MPBA@AgNPs solution (right). The 4-MPBA@AgNPs solution is a darker, yellowish-brown color than the concentrated AgNPs aqueous solution.

[0049] Example 2: Construction of the Standard Curve

[0050] Constructing a linear measurement model:

[0051] (1) Preparation of sample solution: 4-MPBA@AgNPs base solution obtained in Example 1, and steviol glycoside sugar-free beverage solution with concentrations of 1, 3, 5, 7, 9, 10, 15, 20, 30, 50, and 100 μM; the two solutions were mixed in a 1:1 ratio, vortexed for 1 minute, and their Raman spectra were measured.

[0052] (2) Raman spectrum determination: Scanning conditions: laser source wavelength is 532nm, integration time is 20s, laser power is 5%, and the corresponding Raman spectrum is determined.

[0053] (3) Constructing a linear determination model: A model was constructed to correlate different steviol glycoside concentrations with the concentration at 1572 cm⁻¹ in Raman spectra. 1 The relationship curve between the relative intensities of the characteristic peaks is as follows: Figure 5 and Figure 6 As shown, the relative intensity values ​​of steviol glycosides at concentrations of 1-20 μM showed a linear relationship with the steviol glycoside concentration, and the linear regression equation was I. SERS =36199.113+627.023x (μmol / L), correlation coefficient R 2 The value was 0.981, and the calculated detection limit was 0.169 mg / L (0.210 μM).

[0054] Example 3: Investigating the effect of reaction time on detection in the system

[0055] Referring to Example 1, after stirring during modification, the signal peak of 4-MPBA@AgNPs changed significantly over time. Adding 200 μL of 10-MPBA@AgNPs to 10 mL of AgNPs solution... 3 After adding 4-MPBA aqueous solution of M, stir slowly for 10 min. The incubation time is set within 0-30 min, and Raman detection of the substrate is performed every 5 min. Figure 2 A comparison diagram showing the changes in the Raman spectrum of this reaction system over time; as time progresses, the 1584 cm⁻¹ spectrum attributed to 4-MPBA... 1 The Raman peak begins to weaken, while the 1572cm- 1 The characteristic peaks gradually appeared. The left image shows the detection using 10% laser intensity because the overall intensity was not high during the 5-15 minute period when the modification was not yet complete. The right image shows the detection using 5% laser intensity because the overall signal intensity was greatly improved after 20 minutes when the modification was initially completed.

[0056] Example 4: Investigating the effect of different ratios of substrate and test solution in the system on the Raman intensity of steviol glycoside characteristic peaks.

[0057] Referring to Example 1, the mixing volume ratio of the steviol glycoside sugar-free beverage solution and the 4-MPBA@AgNPs substrate solution was varied (1:0.4, 1:0.6, 1:0.8, 1:1, 1:1.2, 1:1.4, 1:1.6, 1:1.8, 1:2) to investigate the effect of different proportions of substrate and test solution in the system on the characteristic peak (1572 cm⁻¹). 1 The influence of Raman intensity. When the volume ratio is greater than 1, the Raman signal intensity gradually increases. However, when the volume ratio is less than 1, the Raman signal intensity begins to weaken. Finally, the Raman peak intensity attributable to the substrate begins to affect the characteristic Raman peak intensity of the sample. A large part of the signal intensity at the characteristic peak is the intensity of the substrate itself, and the addition of the analyte may even further promote substrate aggregation.

[0058] The results are as follows Figure 3 As shown. In this invention, the mixing ratio of the substrate and the test solution is selected as 1:1.

[0059] Experimental Example 5: Detection of Sugar-Free Beverages in the Environment

[0060] Detection in the environment of sugar-free beverages

[0061] Referring to Example 1, under the above optimal conditions, the concentration of steviol glycosides at 6 and 20 μM was measured, and the detection results are shown in Table 1. The detection results are satisfactory, indicating that this method is accurate and feasible.

[0062] Table 1. Detection of steviol glycoside concentration in sugar-free beverages.

[0063] Spiked concentration / mM Detection concentration / mM Recovery 6 6.565 109.42％ 20 19.686 98.43％

[0064] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method for detecting steviol glycosides in sugar-free beverages based on surface-enhanced Raman spectroscopy, characterized in that, Includes the following steps: (1) Silver nitrate solution and sodium citrate solution were mixed and subjected to hydrothermal reaction. After the reaction was completed, the solid was collected by centrifugation, redispersed and concentrated to prepare AgNPs substrate solution. Then, 4-MPBA aqueous solution was added to functionalize AgNPs to obtain 4-MPBA@AgNPs substrate solution. (2) Mix stevia standard solutions of different concentrations with sugar-free beverages to obtain a series of stevia glycoside sugar-free beverage solutions with different stevia concentrations; Then add the 4-MPBA@AgNPs substrate solution obtained in step (1); mix well, and measure the surface-enhanced Raman spectrum of the mixed sample solution; (3) Using the 1572cm in the Raman spectrum -1 ±5cm -1 A linear model was constructed between the characteristic peaks and the concentrations of steviol glycosides in the sugar-free beverage solution. (4) Mix the analyte with the 4-MPBA@AgNPs substrate solution to obtain the sample to be tested. Measure the surface-enhanced Raman spectrum of the sample to be tested. According to the linear model in step (3), obtain the concentration of steviol glycosides in the sample to be tested.

2. The method according to claim 1, characterized in that, The concentration of silver nitrate solution in step (1) is 0.1-0.5 mg / mL.

3. The method according to claim 1, characterized in that, The concentration of sodium citrate solution in step (1) is 0.5-2 wt%.

4. The method according to claim 1, characterized in that, In step (1), the volume ratio of silver nitrate solution to sodium citrate solution is 50:

1.

5. The method according to claim 1, characterized in that, The conditions for the hydrothermal reaction in step (1) are: continuous addition and stirring for 1 hour under boiling conditions.

6. The method according to claim 1, characterized in that, In step (1), the amount of 4-MPBA material used is: 200 μL of 10-MPBA solution is added to every 10 mL of AgNPs substrate solution. -3 M in 4-MPBA aqueous solution.

7. The method according to claim 1, characterized in that, The conditions for modifying the 4-MPBA material in step (1) are as follows: after adding the 4-MPBA aqueous solution, stir slowly for 10 min and then incubate for 20 min.

8. The method according to claim 1, characterized in that, In step (2), the volume ratio of the stevioside sugar-free beverage solution to the 4-MPBA@AgNPs base solution is 1:(0.4-2.0).

9. The method according to any one of claims 1-8, characterized in that, The conditions for Raman detection in step (2) or (4) are as follows: Raman detection is performed using a Raman spectrometer, the excitation source wavelength of the Raman spectrometer is 532nm, the integration time is 10s, and the laser power is 5%.

10. The application of the method according to any one of claims 1-9 in the field of food testing.

Images