Method for detecting pectin in honey based on chemical derivatization-surface enhanced Raman technology

Abstract

A method for detecting pectin in honey based on chemical derivatization-surface enhanced Raman technology, first preparing galacturonic acid working solutions of different concentrations, and then performing derivatization treatment to obtain galacturonic acid standard derivative solutions with different concentrations; then combining the SERS substrate with the galacturonic acid standard derivative solution for signal enhancement, and then performing Raman spectroscopy test, and then drawing the Raman spectroscopy standard curve of the galacturonic acid standard derivative solution; finally verifying the Raman spectroscopy standard curve of the galacturonic acid standard derivative solution. The present invention does not need to pre-treat honey, and directly performs surface enhanced Raman test on the derivatized solution, so as to realize the qualitative and quantitative analysis of pectin in honey. The pectin content is measured by galacturonic acid content, which effectively simplifies the operation steps, and can simply, quickly and accurately realize the qualitative and quantitative analysis of the pectin content in honey.

Description

Technical Field

[0001] The invention relates to the technical field of pectin detection, in particular to a method for detecting pectin in honey based on chemical derivatization-surface enhanced Raman technology. Background Art

[0002] Pectin exists in the cell walls and inner layers of plants and is an important component of plant cells. It is mainly a chain compound of galacturonic acid connected by α-1,4-glycosidic bonds. It can exist stably in acidic media and is insoluble in ethanol. Honey, as a nutritious and complex natural food, has high edible and medicinal value and is a traditional natural nutritional health product that is loved by people. With the improvement of people's living standards, people's requirements are getting higher and higher. Improving the quality of honey has become one of the important issues of food safety. Honey is a food that is very easy to be adulterated. In order to make huge profits, some manufacturers add pectin to inferior honey to change the sensory properties of honey and achieve the purpose of passing inferior products off as good ones. What's more, they directly use fructose syrup or corn syrup to compound with pectin to prepare fake honey that is difficult to distinguish between true and false with the naked eye. Therefore, it is of great significance to accurately determine the pectin content in honey.

[0003] At present, there are many research methods for pectin detection, including gravimetric method, colorimetric method, high performance liquid chromatography (HPLC), gas chromatography (GC) and enzymatic hydrolysis-flow analysis method. Among them, the gravimetric method has complicated operation steps and poor experimental repeatability. Both colorimetric method and high performance liquid chromatography hydrolyze pectin into galacturonic acid, but the colorimetric method has poor selectivity. Although high performance liquid chromatography solves the selectivity problem, its instrument configuration is expensive and the analysis time is long, which is not suitable for universal promotion; gas chromatography requires derivatization and is complicated to operate; and the flow analysis method has poor instrument stability and wastes reagents.

[0004] Like infrared spectroscopy, Raman spectroscopy is a type of molecular vibration spectroscopy, which has the advantages of simple operation, high sensitivity, short test time, and no need for pretreatment, and can be used for rapid on-site detection and identification. When the test sample is well combined with the "hot spot" of the enhanced substrate, the intensity of the Raman signal will increase sharply; however, some organic matter has poor ability to bind to the substrate, and the Raman spectral signal is weak and cannot be used for detection. Studies have found that chemical derivatization can enhance the Raman spectral signal of organic matter, so how to apply this technology to the detection of pectin content in honey has become an important issue that needs to be solved in this field. Summary of the invention

[0005] The technical problem solved by the present invention is to provide a method for detecting pectin in honey based on chemical derivatization-surface enhanced Raman technology to solve the problems in the above-mentioned background technology.

[0006] The technical problem solved by the present invention is achieved by adopting the following technical solutions:

[0007] The method for detecting pectin in honey based on chemical derivatization-surface enhanced Raman technology, the specific steps are as follows:

[0008] (1) Preparation of galacturonic acid working solution

[0009] Preparation of 1000 mg / L galacturonic acid stock solution: dissolve 0.1 g galacturonic acid in 100 mL ultrapure water to obtain a galacturonic acid stock solution with a galacturonic acid mass concentration of 1000 mg / L, and then dilute the prepared galacturonic acid stock solution with ultrapure water to obtain galacturonic acid working solutions with concentrations of 0.01 mg / L, 0.1 mg / L, 1 mg / L, 5 mg / L, 10 mg / L, and 100 mg / L;

[0010] (2) Derivatization of galacturonic acid standards

[0011] Take equal volumes of the following galacturonic acid working solutions of different concentrations: 0.01 mg / L, 0.1 mg / L, 1 mg / L, 5 mg / L, 10 mg / L, 100 mg / L, and then add equal volumes of hydrochloric acid solution to the galacturonic acid working solutions of different concentrations in turn, mix and shake well, place in a 50°C to 90°C water bath and heat for 5 to 12 minutes, and cool naturally to room temperature to obtain a galacturonic acid standard derivative solution;

[0012] (3) Raman spectroscopy test of galacturonic acid standard derivative solution

[0013] The SERS substrate (made of gold or silver nanoparticles) was combined with a galacturonic acid standard derivative solution to enhance the signal, and then the Raman spectrum test was performed;

[0014] (4) Draw the Raman spectrum standard curve and UV-visible spectrum standard curve of the galacturonic acid standard derivative solution

[0015] The concentration of galacturonic acid standard derivative solution is used as the horizontal axis, 1083cm -1 The Raman intensity of is taken as the ordinate, and the Raman spectrum standard curve of the galacturonic acid standard derivative solution is drawn. The galacturonic acid working curve equation and correlation coefficient can be obtained from the measured Raman intensity and the concentration of the galacturonic acid working solution;

[0016] The pectin content is calculated as the amount of galacturonic acid X, as follows:

[0017]

[0018] In formula (1), the mass concentration of galacturonic acid in the c-galacturonic acid working solution is in milligrams per liter (mg / L); V is the volume of the solution, in milliliters (mL); the mass of the m-galacturonic acid working solution is in grams (g);

[0019] The average of three parallel determinations is taken as the determination result of the galacturonic acid working solution. The relative average deviation between the three parallel determinations should not be greater than 5%, and the result is retained to two decimal places;

[0020] Take a certain volume of galacturonic acid standard derivative solution with different concentrations: 1 mg / L, 10 mg / L, 30 mg / L, 50 mg / L, 70 mg / L, 100 mg / L, and then add equal volumes of carbazole sulfuric acid test solution to the above galacturonic acid standard derivative solutions with different concentrations in turn, mix and shake well, heat in a boiling water bath, cool to room temperature, and perform UV-visible spectrum test, and finally draw a UV-visible spectrum standard curve of the galacturonic acid standard derivative solution according to the UV spectra of the galacturonic acid standard derivative solution in different concentrations with the concentration of the galacturonic acid standard derivative solution as the horizontal axis and the absorbance at 525 nm as the vertical axis;

[0021] (5) Verification of the aforementioned detection method

[0022] The honey sample was subjected to Raman spectroscopy detection. The pectin content was measured as galacturonic acid content. Three honey samples were accurately weighed, each weighing 0.5 g (accurate to 0.0001 g). A certain amount of pure pectin standard was added to each of the honey samples to prepare a honey sample derivative solution. Then, according to the above step (3), the SERS substrate and the honey sample derivative solution were subjected to Raman spectroscopy signal enhancement, and then a Raman spectrometer was used to perform Raman spectroscopy testing. The prepared honey sample derivative solution was then subjected to UV-visible spectroscopy testing. The galacturonic acid concentration of the honey sample derivative solution was calculated using the obtained UV-visible spectroscopy standard curve of the honey sample derivative solution to verify the accuracy of the galacturonic acid concentration calculated from the Raman detection data in step 1. The results showed that the recovery rate of galacturonic acid was between 92.0% and 105.2%, indicating that the determination method had good accuracy and high recovery rate.

[0023] Beneficial effects: The present invention uses hydrochloric acid to carry out decarboxylation reaction on the galacturonic acid working solution, and then directly performs surface enhanced Raman detection and analysis on the derivatized solution. Compared with the prior art, the operation steps are effectively simplified, and the qualitative and quantitative analysis of pectin in honey can be simply and quickly realized; the surface enhanced Raman technology is used to determine the pectin content, and its detection limit is 0.01 mg / L, the spiked recovery rate is 92.0% to 105.2%, and the RSD is 3.19% to 5.17%. The method is simple to operate, highly sensitive, rapid and accurate, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of surface enhanced Raman spectrum of honey-derived liquid in a preferred embodiment of the present invention.

[0025] Figure 2 Schematic diagram of Raman spectra of galacturonic acid standard derivative solutions with different concentrations in a preferred embodiment of the present invention.

[0026] Figure 3 Schematic diagram of the Raman spectrum standard curve of the galacturonic acid standard derivative solution in a preferred embodiment of the present invention.

[0027] Figure 4 Schematic diagram of the UV-visible spectrum standard curve of the galacturonic acid standard derivative solution in a preferred embodiment of the present invention. DETAILED DESCRIPTION

[0028] In order to make the technical means, creative features, objectives and effects achieved by the present invention easy to understand, the present invention is further explained below with reference to specific diagrams.

[0029] The method for detecting pectin in honey based on chemical derivatization-surface enhanced Raman technology, the specific steps are as follows:

[0030] (1) Preparation of galacturonic acid working solution

[0031] Preparation of 1000 mg / L galacturonic acid stock solution: dissolve 0.1 g galacturonic acid in 100 mL ultrapure water to obtain a galacturonic acid stock solution with a galacturonic acid mass concentration of 1000 mg / L, and then dilute the prepared galacturonic acid stock solution with ultrapure water to obtain galacturonic acid working solutions with concentrations of 0.01 mg / L, 0.1 mg / L, 1 mg / L, 5 mg / L, 10 mg / L, and 100 mg / L;

[0032] (2) Derivatization of galacturonic acid standards

[0033] Take equal volumes of the following galacturonic acid working solutions of different concentrations: 0.01 mg / L, 0.1 mg / L, 1 mg / L, 5 mg / L, 10 mg / L, 100 mg / L, and then add equal volumes of hydrochloric acid solution to the galacturonic acid working solutions of different concentrations in turn, mix and shake well, place in a 50°C to 90°C water bath and heat for 5 to 12 minutes, cool naturally to room temperature, and obtain galacturonic acid standard derivative solution for standby use;

[0034] (3) Raman spectroscopy test of galacturonic acid standard derivative solution

[0035] The SERS substrate (made of gold or silver nanoparticles) was combined with the galacturonic acid standard derivative solution to enhance the signal, and then the Raman spectrum test was performed. The obtained Raman spectrum is as follows: Figure 1 As shown;

[0036] The test parameters of the Raman spectrum are shown in Table 1:

[0037] Table 1 Raman spectroscopy test parameters

[0038] Exposure time 1.0s Sample exposure 2 times Slit 25μm Grating 400 lines / mm Laser wavelength 785nm Estimated resolution <![CDATA[2.4-4.3cm -1 ]]>

[0039] Under the above Raman spectroscopy test parameters, the galacturonic acid standard derivative solution and honey samples were analyzed and tested;

[0040] (4) Raman spectroscopy standard curve and detection and quantification limits of galacturonic acid standard derivative solution

[0041] A. Standard curve: Take a certain volume of galacturonic acid working solution of different concentrations: 0.01 mg / L, 0.1 mg / L, 1 mg / L, 5 mg / L, 10 mg / L, and then add equal volumes of hydrochloric acid test solution to the above galacturonic acid working solution of different concentrations in turn, mix and shake well, heat in a 50-90°C water bath for 5-12 minutes, cool naturally, and test with the test parameters of Raman spectrum in Table 1 in step (3), with the concentration of galacturonic acid standard derivative solution as the horizontal axis, 1083 cm -1 The Raman intensity of is taken as the ordinate, and the Raman spectrum standard curve of the galacturonic acid standard derivative solution is drawn. The galacturonic acid working curve equation and correlation coefficient can be obtained from the measured Raman intensity and the concentration of the galacturonic acid working solution, as shown in Table 2;

[0042] B. Limit of detection and limit of quantification

[0043] The lowest concentration of galacturonic acid working solution was taken and tested 15 times repeatedly. The standard deviation was calculated based on the test results. Three times of the standard deviation was used as the detection limit of the instrument, and 10 times was used as the quantification limit, as shown in Table 2:

[0044] Table 2 Linear range, linear equation, correlation coefficient, detection limit and quantification limit of galacturonic acid

[0045]

[0046] (5) Recovery rate

[0047] The recovery rate of the honey sample was analyzed and tested. Three honey samples were accurately weighed, each weighing 0.5 g (accurate to 0.0001 g). A certain amount of pure pectin standard was added to each of them to prepare a honey sample derivative solution. Then, according to the above step (3), the SERS substrate and the honey sample derivative solution were subjected to Raman spectral signal enhancement, and then Raman spectroscopy was tested using a Raman spectrometer. The results are shown in Table 3. The pectin content was calculated as galacturonic acid content. The pectin content in the honey sample was obtained by comparing the Raman spectral standard curve in step (4). The recovery rate of the honey sample was calculated by the spiked amount, the unspiked amount and the measured amount after spiked. The recovery rate of galacturonic acid was between 92.0% and 105.2%, indicating that the determination method had good accuracy and high recovery rate.

[0048] The pectin content is calculated as the amount of galacturonic acid X, as follows:

[0049]

[0050] In formula (1), c is the mass concentration of galacturonic acid in the honey sample, in milligrams per liter (mg / L); V is the volume of the solution, in milliliters (mL); m is the mass of the honey sample, in grams (g);

[0051] The average of three parallel determinations is taken as the determination result of the galacturonic acid working solution. The relative average deviation between the three parallel determinations should not be greater than 5%, and the result is retained to two decimal places;

[0052] Table 3 Detection results of pectin in honey samples

[0053] serial number Theoretical value Calculated value Relative deviation (n=5) 1 10 9.79 -2.10％ 2 1 0.92 -4.31％ 3 0.1 0.09 -6.22％

[0054] (6) Verification of Raman spectroscopy standard curve of galacturonic acid standard derivative solution

[0055] Take a certain volume of galacturonic acid standard derivative solution with different concentrations: 1 mg / L, 10 mg / L, 30 mg / L, 50 mg / L, 70 mg / L, 100 mg / L, then add equal volume of carbazole sulfuric acid test solution to the above galacturonic acid standard derivative solution with different concentrations, mix and shake well, heat in a boiling water bath, cool to room temperature, and perform UV-visible spectrum test;

[0056] The test parameters of the UV-visible spectrum are shown in Table 4:

[0057] Table 4 UV-visible spectrum test parameters

[0058] Wavelength range 200~800nm Vertical axis range 0.00~4.00Abs Scan interval 1.00nm Slit 5.0nm Scan speed fast

[0059] (7) Drawing of UV-visible spectrum standard curve of galacturonic acid standard derivative solution

[0060] With the concentration in the galacturonic acid standard derivative solution as the horizontal axis and the absorbance at 525nm as the vertical axis, a UV-visible spectrum standard curve of the galacturonic acid standard derivative solution is drawn according to the UV spectra of the galacturonic acid standard derivative solution in different concentrations, such as Figure 4 As shown;

[0061] (8) Verification of Raman Detection Results of Honey Sample Derivative Liquid

[0062] The honey sample derivative liquid prepared in step (5) is subjected to UV-visible spectroscopy test under the test conditions of Table 4. The galacturonic acid concentration of the honey sample derivative liquid is calculated by using the obtained UV-visible spectroscopy standard curve of the honey sample derivative liquid to verify the accuracy of the galacturonic acid concentration calculated from the Raman detection data.

[0063] Practical application shows that the chemical derivatization-surface enhanced Raman spectroscopy analysis method for determining the pectin content in honey is simple to operate, fast, highly sensitive, has a high recovery rate, and requires few consumables. It is suitable for the determination of pectin in complex food samples.

Claims

1. A method for detecting pectin in honey based on chemical derivatization-surface enhanced Raman technology, characterized in that: The specific steps are as follows: (1) preparing galacturonic acid working solutions of different concentrations; (2) performing a derivatization treatment on the galacturonic acid working solutions of different concentrations prepared in step (1), specifically: first taking equal volumes of galacturonic acid working solutions of different concentrations, then adding equal volumes of hydrochloric acid solution to the taken galacturonic acid working solutions of different concentrations, mixing and shaking, heating in a water bath not higher than 100° C. for several minutes, and naturally cooling to room temperature to obtain galacturonic acid standard derivative solutions of different concentrations; (3) Raman spectroscopy test of galacturonic acid standard derivative solution The SERS substrate is combined with the galacturonic acid standard derivative solution obtained in step (2) to enhance the signal, and then a Raman spectrum test is performed; (4) Draw the Raman spectrum standard curve and UV-visible spectrum standard curve of the galacturonic acid standard derivative solution The concentration of galacturonic acid standard derivative solution is used as the horizontal axis, 1083cm -1 The Raman intensity of is taken as the ordinate, and the Raman spectrum standard curve of the galacturonic acid standard derivative solution is drawn; With the concentration of the galacturonic acid standard derivative solution as the abscissa and the absorbance at 525 nm as the ordinate, a UV-visible spectrum standard curve of the galacturonic acid standard derivative solution is drawn according to the UV spectra of the galacturonic acid standard derivative solution in different concentrations; (5) Verification of the Raman spectroscopy standard curve of the galacturonic acid standard derivative solution The honey sample derivative liquid obtained by derivatization treatment of the honey sample is subjected to Raman spectroscopy detection, and then the prepared honey sample derivative liquid is subjected to UV-visible spectroscopy test. The galacturonic acid concentration in the honey sample derivative liquid is calculated by the obtained UV-visible spectroscopy standard curve to verify the accuracy of the galacturonic acid concentration calculated by the Raman spectroscopy standard curve of the galacturonic acid standard derivative liquid; the pectin content in the honey sample is calculated as the galacturonic acid content.

2. The method for detecting pectin in honey based on chemical derivatization-surface enhanced Raman technology according to claim 1, characterized in that: In step (1), the preparation method of the galacturonic acid working solution is: first prepare a 1000 mg / L galacturonic acid stock solution, dissolve 0.1 g galacturonic acid in 100 mL ultrapure water to obtain a galacturonic acid stock solution with a galacturonic acid mass concentration of 1000 mg / L, and then dilute the prepared galacturonic acid stock solution with ultrapure water to obtain galacturonic acid working solutions with concentrations of 0.01 mg / L, 0.1 mg / L, 1 mg / L, 5 mg / L, 10 mg / L, and 100 mg / L.

3. The method for detecting pectin in honey based on chemical derivatization-surface enhanced Raman technology according to claim 1, characterized in that: The water bath temperature is 50° C. to 90° C., and the heating time is 5 to 12 minutes.

4. The method for detecting pectin in honey based on chemical derivatization-surface enhanced Raman technology according to claim 1, characterized in that: In step (3), the SERS substrate is made of nanomaterials.

5. The method for detecting pectin in honey based on chemical derivatization-surface enhanced Raman technology according to claim 1, characterized in that: In step (5), the Raman spectroscopy detection of the honey sample derivative liquid is specifically as follows: Accurately weigh several portions of honey samples, 0.5 g each, and add a certain amount of pure pectin standard to each honey sample to prepare a bee sample derivative solution. Then, according to step (3), the SERS substrate and the bee sample derivative solution are subjected to Raman spectral signal enhancement, and then a Raman spectrometer is used to perform Raman spectral testing.

6. The method for detecting pectin in honey based on chemical derivatization-surface enhanced Raman technology according to claim 1, characterized in that: In step (5), the pectin content of the honey sample is calculated as galacturonic acid content, as shown in the following formula: In formula (1), X-galacturonic acid content, c-galacturonic acid mass concentration in honey sample, unit is mg / L; V-solution volume, unit is mL; m-mass of honey sample, in g; The average value of three parallel determinations is taken as the determination result of the honey sample. The relative average deviation between the three parallel determinations should not be greater than 5%, and the result is retained to two decimal places.

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