Method for rapid detection of cyclamate in alcoholic beverages

Abstract

The application discloses a method for rapidly detecting cyclamate in wine, and the method comprises the following steps: preparing a natural low eutectic solvent fluorescent probe by using two raw materials of xylitol and acetamide, and determining the content of cyclamate in the wine to be measured by utilizing the linear relationship between the cyclamate concentration and the fluorescence intensity; the whole detection process has good stability, consumes less time, is accurate in result, and has obvious detection effect, and the method shows obvious fluorescence enhancement under a lower cyclamate concentration, the fluorescence intensity is increased by ten times, and stable determination can be realized in a wider pH range.

Description

Technical Field

[0001] This invention belongs to the field of food additive detection technology, and relates to a method for rapid detection of cyclamate in alcoholic beverages. Background Technology

[0002] Cyclamate (sodium cyclohexylsulfamate) is a commonly used food additive. Its sweetness is 30-40 times that of sucrose, and it can be used as a sweetener in formulated wines to enhance their quality and taste. The Food and Agriculture Organization of the United Nations and the World Health Organization stipulate that the acceptable daily intake is 0-11 mg / kg, so its usage needs to be strictly controlled to ensure food safety. my country's National Food Safety Standard GB 2760-2014 clearly stipulates that the maximum amount of cyclamate added to formulated wines is 0.65 g / kg, and the addition of cyclamate to baijiu (Chinese liquor) and wine is prohibited. Cyclamate can provide a sweeter taste to formulated wines, and the reasonable addition of cyclamate content ensures the quality of the formulated wines. How to efficiently detect the cyclamate content in food is an important prerequisite for ensuring the normal operation of my country's food export trade, a major way to ensure that its content meets national standards, and a key to improving food safety and public health. Therefore, it is essential to establish a rapid and green method for detecting cyclamate in alcoholic beverages.

[0003] Currently, the main methods for detecting cyclamate in alcoholic beverages are liquid chromatography-mass spectrometry (HPLC-MS / MS), which serves as the third method for cyclamate detection and is also used for detecting cyclamate in imported and exported foods in my country. In recent years, HPLC-MS has made significant progress in the application of biology, medicine, chemical engineering, and environmental fields, and it also occupies an important position in cyclamate detection. Recently, headspace gas chromatography (HGC) has been greatly optimized for rapid quantitative detection of cyclamate in alcoholic beverages. Besides chromatography, atomic absorption spectrometry can also indirectly calculate the cyclamate content. With the continuous development of detection technology, new detection methods tend towards online, rapid, and highly sensitive detection. Fluorescence detection has the advantages of short detection time and low detection limit. This invention represents a new advancement in the development of fluorescent probe materials for cyclamate detection, realizing a rapid and quantitative fluorescence analysis method for the detection of cyclamate content in alcoholic beverages. Summary of the Invention

[0004] The present invention aims to provide a rapid method for detecting cyclamate in alcoholic beverages. The entire detection process is stable, time-saving, accurate, and has obvious detection effects. It shows significant fluorescence enhancement at low concentrations of cyclamate, with fluorescence intensity increasing by up to ten times, and can achieve stable determination over a wide pH range.

[0005] The technical solution of the present invention is as follows:

[0006] A rapid method for detecting cyclamate in alcoholic beverages comprises the following steps in sequence:

[0007] S1. A natural eutectic solvent fluorescent probe was prepared using xylitol and acetamide as raw materials, and the probe was prepared into a solution as a reaction solution, denoted as DES solution.

[0008] S2. Prepare standard solutions of cyclamate with different concentrations. Mix DES solution and cyclamate standard solution at a volume ratio of 1:1 and react at room temperature for 5 min. Measure the fluorescence intensity of the solution after reaction using a fluorescence spectrophotometer under the conditions of excitation wavelength of 330 nm and emission wavelength of 400 nm. There is a linear relationship between cyclamate concentration and fluorescence intensity.

[0009] S3. Mix the DES solution with the wine to be tested at a volume ratio of 1:1 and react at room temperature for 5 minutes. Measure the fluorescence intensity of the solution after the reaction using a fluorescence spectrophotometer under the conditions of excitation wavelength 330 nm and emission wavelength 400 nm. Based on the linear relationship obtained in S2, determine the content of cyclamate in the wine to be tested.

[0010] As a first limitation of the present invention, in step S1, both xylitol and acetamide are solid particles, and their molar ratio is 4:1.

[0011] As a second limitation of the present invention, in step S1, the fluorescent probe is prepared according to the following procedure:

[0012] S11. Dissolve acetamide and xylitol in n-heptane and heat the mixture at 60–100 °C for 25–35 min, then transfer it to a rotary evaporator for vacuum evaporation.

[0013] S12. After evaporation, cool at room temperature for 20 minutes to obtain a fluorescent probe with good flowability in an acetamide-xylitol eutectic solvent.

[0014] As a third limitation of the present invention, in step S11, the temperature of vacuum evaporation is 60°C and the time is 1.5 to 2 hours.

[0015] As a fourth limitation of the present invention, in step S11, the volume ratio of n-heptane to the total weight ratio of acetamide and xylitol is (2-3) mL: 1 g.

[0016] As a fifth limitation of the present invention, in step S2, the linear relationship is expressed as y = 0.3815x + 0.8036, R 2 =0.9985.

[0017] As a sixth limitation of the present invention, the natural eutectic solvent fluorescent probe solution is prepared by dissolving the fluorescent probe in ultrapure water to form a solution with a concentration of 1 g / mL.

[0018] The above-described technical solution, as a whole, involves interconnected steps that collectively influence the accuracy and detection limit. In this invention, the fluorescent probe formed from xylitol and acetamide exhibits a specific response to cyclamate in alcoholic beverages. This is primarily due to the fluorescent probe forming a more stable supramolecular structure with the cyclamate surface groups through intermolecular hydrogen bonds and van der Waals forces. Intermolecular hydrogen bonds, a weak chemical bond, provide structural stability, while van der Waals forces are attractive forces arising from the uneven distribution of electrons at a given moment. The synergistic effect of these two forces significantly contributes to the stability of the supramolecular structure. This supramolecular structure formation can further enhance the performance of the fluorescent probe and offers potential advantages for the detection of cyclamate and related applications.

[0019] The beneficial effects achieved by the present invention after adopting the above technical solution are as follows:

[0020] ① The probe material used in the detection method of this invention is easy to prepare and has stable properties;

[0021] ② The present invention has obvious detection effect, showing obvious fluorescence enhancement at low concentrations of cyclamate, with fluorescence intensity increasing by up to ten times;

[0022] ③ The detection method in this invention can achieve stable measurement over a wide pH range, making it more widely applicable;

[0023] ④ The detection method of the present invention has good results when applied to real samples and the spiked recovery rate is good, ranging from 92% to 106%;

[0024] ⑤ This invention has good selectivity for common additives such as fruit acids, sugars, and pigments in formulated wines.

[0025] This invention is applicable to the rapid detection of cyclamate in alcoholic beverages.

[0026] The specific embodiments of the present invention will now be described in further detail with reference to the accompanying drawings. Attached Figure Description

[0027] Figure 1 This is a graph showing the fluorescence intensity variation of the acetamide-xylitol eutectic solvent fluorescent probe under different Ex values ​​in Example 1 of the present invention.

[0028] Figure 2 The fluorescent probe NMR of this invention 1 H spectrum;

[0029] Figure 3 The fluorescent probe NMR of this invention 13 C spectrum;

[0030] Figure 4 This is the infrared spectrum of the fluorescent probe of the present invention;

[0031] Figure 5 The fluorescence response test diagram of the present invention is shown in the figure below. From left to right, the diagrams are: acetamide-xylitol eutectic solvent under visible light, eutectic solvent and cyclamate mixed solution under visible light, acetamide-xylitol eutectic solvent under ultraviolet light, and eutectic solvent and cyclamate mixed solution under ultraviolet light.

[0032] Figure 6 This is a fluorescence enhancement diagram of the present invention under different cyclamate concentrations;

[0033] Figure 7 This is a graph showing the linear relationship between cyclamate concentration and fluorescence intensity in this invention.

[0034] Figure 8 This is a graph showing the changes in fluorescence intensity of the reaction system at different pH values ​​according to the present invention.

[0035] Figure 9 This is a comparison chart of the time stability of the fluorescent probes of this invention;

[0036] Figure 10 The fluorescence curve of the fluorescent probe of this invention changes over time.

[0037] Figure 11 The fluorescence response diagrams of the preferred acetamide-xylitol eutectic solvent fluorescent probe of this invention to different additives are shown.

[0038] Figure 12 The fluorescence response diagrams of the cyclamate of this invention with fluorescent probes of different comparative proportions are shown.

[0039] Figure 13 The fluorescence response test diagram is a comparative example selected for this invention. Detailed Implementation

[0040] Unless otherwise specified, all reagents used in the following embodiments are commercially available reagents, and all experimental and detection methods used in the following embodiments are existing experimental and detection methods.

[0041] Example 1: A method for rapid detection of cyclamate in alcoholic beverages

[0042] 1. The preparation method of the eutectic solvent fluorescent probe is carried out according to the following procedure:

[0043] Under the protection of 30 mL of n-heptane, solid particles of acetamide and xylitol were heated at 60 °C for 30 min in a molar ratio of 1:4 (total mass 10 g), followed by vacuum evaporation at 60 °C for 1.5 h in a rotary evaporator. After cooling to room temperature for 20 min, a free-flowing acetamide-xylitol eutectic solvent fluorescent probe was obtained. The optimal excitation wavelength of this fluorescent probe was determined using a fluorescence spectrophotometer. Figure 1As shown, the optimal Ex is 330nm.

[0044] The NMR spectra of the above fluorescent probes were characterized using the deuterated solvent DMSO, see [link to relevant documentation]. Figure 2 , Figure 3 As shown, the results are as follows:

[0045] 1 H NMR (500MHz, DMSO-d6) δ: 6.98 (d, J = 301.1Hz, 2H), 4.45 (t, J = 5.6Hz, 0H), 4.43 (s, 2H), 4.42 (s, 2H), 4.09 (s, 0H) ,4.07(s,0H),3.59–3.47(m,1H),3.47–3.40(m,1H),3.39(s,0H),3.39–3.30(m,1H),2.50(s,0H),1.76(s,3H).

[0046] 13C NMR (126MHz, DMSO-d6) δ: 172.19, 72.96, 70.65, 63.17, 39.93 (dp, J = 42.0, 21.0Hz), 22.97.

[0047] The FT-IR spectra of the above fluorescent probes were characterized using a coating method. For example... Figure 4 As shown, the characterization results are as follows:

[0048] OH(3306.94cm -1 (stretching vibration peak); CH,CH2 (2933.55cm) -1 (stretching vibration peak); C=O(1661.98cm) -1 (bending vibration peak); NH2 (1610.92cm) -1 (in-plane bending vibration peak); CH3 (1398.90cm) -1 (bending vibration peak); CC (1211.29cm) -1 (stretching vibration peak); CH (1001.01cm) -1 (bending vibration peak); CH2 (874.84cm) -1 (Planar rocking vibration peak); see Figure 4 As shown.

[0049] 2. Construct a standard curve for cyclamate detection:

[0050] Prepare standard solutions of cyclamate at different concentrations of 1, 5, 10, 15, 20, and 25 μmol / L. Mix the cyclamate standard solutions with a eutectic solvent solution (concentration 1 g / mL) at a 1:1 volume ratio and react at room temperature for 5 min. The resulting fluorescence response diagrams are shown below. Figure 5 As shown, the fluorescence intensity of the solution system at different cyclamate concentrations was measured using a fluorescence spectrophotometer under excitation wavelength of 330 nm and emission wavelength of 400 nm. Specifically... Figure 6 As shown, the results indicate that the fluorescence intensity of the eutectic solvent increases with increasing cyclamate concentration, and exhibits a good linear relationship between fluorescence intensity and cyclamate concentration in the range of 0–25 μmol / L. Figure 7 As shown, the obtained linear equation is y = 0.3815x + 0.8036(R). 2 =0.9985), according to the limit of detection (LOD) calculation principle (i.e. LOD = 3S / K, where S represents the standard deviation of the blank signal and K represents the slope of the standard curve), the detection limit is calculated to be 0.95 μmol / L. Table 1 compares the method of this application with existing methods for detecting cyclamate, showing that the method of this application has a lower detection limit.

[0051] Table 1. Comparison of linear range and detection limit of different methods for detecting cyclamate content in alcoholic beverages

[0052]

[0053] Example 2: Investigation of pH stability of fluorescent probes

[0054] 1. The pH stability of the fluorescent probe prepared in Example 1 was tested: solutions with pH values ​​of 2–9 were prepared using concentrated hydrochloric acid and sodium hydroxide. Different pH solutions were mixed with DES at a ratio of 5:1, and changes in fluorescence intensity were observed. The results are as follows: Figure 8 As shown, the detection method of the present invention can achieve stable measurement over a wide pH range.

[0055] 2. Testing the time stability of the fluorescent probe prepared in Example 1: The changes in fluorescence intensity were measured after 1 day, 5 days, 15 days, and 30 days in the eutectic solvent. Figure 9 As shown, the fluorescent probe prepared by this invention has relatively stable fluorescence intensity, and the fluorescence attenuation fitting curve is as follows. Figure 10 As shown, the formula is y = -3.0624x + 1805.54. According to the formula, it takes about 60 days for the fluorescence intensity of the probe to decrease to 90%, which shows that the probe is relatively stable.

[0056] The fluorescent probe provided by this invention can achieve stable measurement over a wide pH range, has a wider range of applications, and has good practical value.

[0057] Example 3: Specificity and responsiveness analysis of fluorescent probes.

[0058] The specificity and responsiveness of the fluorescent probe prepared in Example 1 were analyzed. 15 μmol / L solutions of cyclamate, carmine, gallic acid, fructose, sucrose, citric acid, and malic acid were prepared and mixed with the eutectic solvent solution provided in Example 1 at a 1:1 volume ratio. Fluorescence was then measured, and the fluorescence enhancement effects of different additives and the blank sample were compared as follows: Figure 11 As shown. From Figure 11 It was found that the acetamide-xylitol eutectic solvent only responded to cyclamate, with a fluorescence enhancement rate as high as 653.5%.

[0059] Examples 4-6: Methods for Rapid Detection of Cyclamate in Alcoholic Beverages

[0060] This embodiment is a method for rapid detection of cyclamate in alcoholic beverages. The specific process is similar to that in Embodiment 1, except that the technical parameters in the detection process are different, as shown in the table below.

[0061] Table 2 Technical Parameters

[0062]

[0063]

[0064] The fluorescence intensity of the eutectic solvents prepared in Examples 4-6 increased with the increase of cyclamate concentration. Experimental results showed that the fluorescence intensity and cyclamate concentration exhibited a good linear relationship in the range of 0-25 μmol / L.

[0065] Comparative Example 1: Choline chloride-sorbitol as a eutectic solvent for the detection of cyclamate.

[0066] Under the protection of 30 mL of n-heptane, choline chloride and sorbitol were heated at 60 °C for 30 min in a 1:1 molar ratio and then evaporated under vacuum in a rotary evaporator (the evaporation process was the same as in Example 1). After cooling at room temperature for 20 min, a free-flowing choline chloride-sorbitol eutectic solvent fluorescent probe was obtained.

[0067] A standard curve between the fluorescent probe and the cyclamate solution was constructed using the method described in Example 1. The results showed that the choline chloride-sorbitol eutectic solvent had a relatively small response to cyclamate. Figure 12 As shown.

[0068] Comparative Example 2: Citric acid-xylitol as a eutectic solvent for the detection of cyclamate.

[0069] Under the protection of 30 mL of n-heptane, solid particles of citric acid and xylitol were heated at 80 °C for 60 min in a molar ratio of 1:4, and then evaporated under vacuum in a rotary evaporator (the evaporation process was the same as in Example 1). After cooling at room temperature for 20 min, a citric acid-xylitol eutectic solvent fluorescent probe with good flowability was obtained.

[0070] A standard curve between the fluorescent probe and the cyclamate solution was constructed using the method described in Example 1. The results showed that the citric acid-xylitol eutectic solvent had a relatively small response to cyclamate. Figure 12 As shown.

[0071] Comparative Example 3: Detection of Cyclamate in Citric Acid-Proline Eutectic Solvent

[0072] Under the protection of 30 mL of n-heptane, solid particles of citric acid and proline were heated at 100 °C for 40 min at a molar ratio of 3:1, and then evaporated under vacuum in a rotary evaporator (the evaporation process was the same as in Example 1). After cooling at room temperature for 20 min, a highly mobile citric acid-proline eutectic solvent fluorescent probe was obtained.

[0073] The fluorescence response curves between the fluorescent probe and the cyclamate solution were constructed using the method described in Example 1. The results showed that the citrate-proline eutectic solvent had a relatively small response to cyclamate. Figure 12 As shown.

[0074] As shown in Comparative Examples 1-3, only the acetamide-xylitol eutectic solvent fluorescent probe provided by this invention can be used to detect cyclamate, and it exhibits good specificity. The fluorescence response test graphs for the actual comparative experiments are shown below. Figure 13 .

[0075] Application Example 1:

[0076] The detection method described in Example 1 of this invention was applied to verify the detection effect of cyclamate in simulated wine samples. The specific steps included:

[0077] 1. Preparation of simulated wine sample: Prepare a 12% ethanol solution by volume, add tartaric acid at a ratio of 5 g / L, and adjust the pH to 3.5.

[0078] 2. Detection of cyclamate in alcohol:

[0079] The concentrations prepared were 2×10 -6 mol / L, 1×10 -5 mol / L, 2×10 -5 Prepare a 1 mol / L cyclamate solution; take 1 mL of the simulated wine sample to be tested, add 1 mL of cyclamate solution and 1 mL of eutectic solvent, mix well, add to a cuvette, and detect using a fluorophotometer.

[0080] The results are shown in Table 3 below. The recovery rate in the simulated wine sample was between 92.40% and 100.50%, indicating a good recovery effect.

[0081] Table 3. Recovery of cyclamate in simulated wine samples

[0082]

[0083] Application Example 2:

[0084] The detection method described in Example 1 of this invention was applied to verify the detection effect of cyclamate in a white wine sample. The white wine sample was from the commercially available Chardonnay dry white wine Concha y Toro Reserve Chardonnay series, with an alcohol content of 12.5% ​​vol, and the origin was the Central Valley of Chile. The specific detection steps are as follows:

[0085] The concentrations prepared were 2×10 -6 mol / L, 1×10 -5 mol / L, 2×10 -5 Prepare a 1 mol / L cyclamate solution; take 1 mL of the white wine sample to be tested, add 1 mL of cyclamate solution and 1 mL of eutectic solvent, mix well, add to a cuvette, and detect using a fluorophotometer.

[0086] The results are shown in Table 4 below. The recovery rate in the white wine samples ranged from 101.00% to 106.80%, indicating a good recovery effect.

[0087] Table 4. Recovery of cyclamate in white wine

[0088]

[0089] In summary, the method for detecting cyclamate in alcoholic beverages provided by this invention is simple and rapid, with advantages such as high fluorescence intensity, high selectivity, and high sensitivity. It can achieve accurate detection of cyclamate and has good application value and promotion significance.

[0090] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A method for rapid detection of cyclamate in alcoholic beverages, characterized in that, Follow these steps in sequence: S1. A natural eutectic solvent fluorescent probe was prepared using xylitol and acetamide as raw materials, and the probe was prepared into a solution as a reaction solution, denoted as DES solution. The fluorescent probe was prepared according to the following procedure: S11. Dissolve acetamide and xylitol in n-heptane and heat the mixture at 60–100°C for 25–35 min. Then transfer the mixture to a rotary evaporator for vacuum evaporation at 60°C for 1.5–2 h. S12. After evaporation, cool at room temperature for 20 min to obtain a fluorescent probe with good flowability in an acetamide-xylitol eutectic solvent. S2. Prepare standard solutions of cyclamate with different concentrations. Mix DES solution and cyclamate standard solution at a volume ratio of 1:1 and react at room temperature for 5 min. Measure the fluorescence intensity of the solution after reaction using a fluorescence spectrophotometer under the conditions of excitation wavelength of 330 nm and emission wavelength of 400 nm. There is a linear relationship between cyclamate concentration and fluorescence intensity. The linear relationship is expressed as y = 0.3815x + 0.8036, R 2 =0.9985; S3. Mix the DES solution with the wine to be tested at a volume ratio of 1:1 and react at room temperature for 5 minutes. Measure the fluorescence intensity of the solution after the reaction using a fluorescence spectrophotometer under the conditions of excitation wavelength 330 nm and emission wavelength 400 nm. Based on the linear relationship obtained in S2, determine the content of cyclamate in the wine to be tested.

2. The method for rapid detection of cyclamate in alcoholic beverages according to claim 1, characterized in that, In step S1, both xylitol and acetamide are solid particles, and their molar ratio is 4:

1.

3. The method for rapid detection of cyclamate in alcoholic beverages according to claim 1, characterized in that, In step S11, the volume ratio of n-heptane to the total weight ratio of acetamide and xylitol is (2-3) mL: 1 g.

4. A method for rapid detection of cyclamate in alcoholic beverages according to any one of claims 1-3, characterized in that, The natural eutectic solvent fluorescent probe solution is prepared by dissolving the fluorescent probe in ultrapure water to form a solution with a concentration of 1 g / mL.

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