CN112540072A - Preparation method of surface-enhanced Raman spectrum substrate material for detecting xanthoxylin in hotpot condiment - Google Patents
Preparation method of surface-enhanced Raman spectrum substrate material for detecting xanthoxylin in hotpot condiment Download PDFInfo
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Abstract
The invention belongs to the technical field of rapid detection of foods, and relates to a preparation method of a surface-enhanced Raman spectrum substrate material for detecting xanthoxylin in hotpot condiment; firstly, preparing silicon-coated metal nanoparticles, adding the silicon-coated metal nanoparticles into a silane coupling agent solution, reacting in a constant-temperature water bath, centrifuging after reaction to obtain silicon amide-coated metal nanoparticles, mixing with deionized water or a buffer solution and a glutaraldehyde aqueous solution, stirring, centrifuging, drying to obtain a nano material, mixing with a polyphenol oxidase solution, incubating, centrifuging, and drying to obtain an SERS substrate material with ligase; the SERS substrate material with the ligase can be further used for detecting sanshoamides in the hotpot condiment; the substrate material disclosed by the invention is simple to prepare, low in cost, high in detection speed, accurate in result, portable, suitable for accurate detection of large-scale zanthoxylum tingle essence on site, and provides technical support for intelligent analysis and detection of zanthoxylum tingle essence.
Description
Technical Field
The invention belongs to the technical field of rapid detection of foods, and particularly relates to a preparation method of a surface-enhanced Raman spectrum substrate material for detecting xanthoxylin in hotpot condiment.
Background
The chafing dish is a traditional special food in China and is favored by more and more consumers. With the development of the catering industry, the chafing dish culture is developing vigorously as the traditional culture of China, and the chafing dish bottom material plays a great promoting role in the development of the chafing dish, thereby not only determining the quality of the chafing dish, but also having important influence on the taste of the chafing dish. The chafing dish bottom flavoring is a compound condiment, which is prepared by frying vegetable oil or animal oil and various ingredients. The pepper is used as a spicy flavor developing raw material in the spicy hot pot bottom material, and the content of spicy flavor substances is directly related to the strength and the taste of spicy flavor in the bottom material. The main numb-taste substances of the pepper are chain unsaturated fatty acid amides represented by sanshoamides, and mainly comprise hydroxy-alpha-sanshoamides, hydroxy-beta-sanshoamides, hydroxy-gamma-sanshoamides, hydroxy-epsilon-sanshoamides and the like.
The content of sanshoamides is one of the important indexes for evaluating the quality of the hotpot condiment. At present, methods for detecting sanshoamides mainly comprise an ultraviolet spectrophotometry, a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS), a thin layer chromatography, an electrochemical analysis method and the like. Wherein, the ultraviolet spectrophotometry and the thin layer chromatography are simple and convenient to operate and high in detection speed, but the result accuracy is low, so that the method is not suitable for complex food samples such as hotpot condiment; the HPLC-MS method can accurately qualitatively and quantitatively analyze the zanthoxylum bungeanum hemsl component, has high precision and good repeatability, but has the defects of expensive and large volume of instruments, long detection time, complicated operation procedure, need of professional operators and the like, so that the method is difficult to realize large-scale application in the production of the hotpot condiment. Although the electrochemical analysis method is simple to operate and high in accuracy, specific molecular structure information of the sanshoamides cannot be provided. Therefore, the established detection method which can qualitatively identify the zanthoxylum bungeanum hemsl ingredient and can quickly and accurately quantify the zanthoxylum bungeanum hemsl ingredient has important practical significance for quality monitoring and quality evaluation of the hotpot condiment.
The Surface Enhanced Raman Scattering (SERS) technology is an effective product of the combination of nano materials and the SERS technology, is a very effective tool for representing molecular structure information, has the advantages of high analysis speed, simplicity in operation, simplicity in sample pretreatment, high sensitivity and the like, and is widely applied to rapid detection in the field of food and agricultural products.
Noble metal nanomaterials are widely used in SERS substrate materials due to their unique Localized Surface Plasmon Resonance (LSPR) and surface enhanced raman scattering properties. However, the precious metal nanoparticles alone, as the SERS substrate material, are susceptible to interference and the accuracy and sensitivity of detection are not sufficient. The ingredients of the hot pot seasoning matrix are complex, and the interfering substances are more. Therefore, the SERS substrate material with strong anti-interference performance, high detection signal intensity, good sensitivity and accurate detection result is prepared, so that the interference of other substances such as capsaicin, polyphenol and the like in the hotpot condiment can be effectively avoided in the detection of complex food systems such as the hotpot condiment, the structural information of the xanthoxylin can be quickly reflected, and the content of the xanthoxylin can be accurately measured.
Disclosure of Invention
The invention aims to overcome the defects in the existing technology for detecting the xanthoma in the hotpot condiment, construct an SERS substrate material with strong anti-interference performance, high detection signal intensity, good sensitivity and accurate detection result, and realize the purposes of quickly, qualitatively and accurately and quantitatively detecting the xanthoma in the hotpot condiment by utilizing the surface-enhanced Raman spectroscopy technology.
In order to achieve the purpose, the invention adopts the following technical means:
the invention provides a surface-enhanced Raman spectrum substrate material for detecting xanthoxylin in hotpot condiment, which specifically comprises the following steps:
(1) preparing metal nanoparticle sol, adjusting the pH value to be alkaline by using an alkaline solution, adding a silicon source solution into the metal nanoparticle sol in several times, uniformly mixing, placing in a constant-temperature water bath for reaction, and centrifuging after the reaction to obtain silicon-coated metal nanoparticles which are marked as a nano material A;
(2) adding the nano material A prepared in the step (1) into a silane coupling agent solution, uniformly mixing, reacting in a constant-temperature water bath, and centrifuging after reaction to obtain silicon amide coated metal nano particles, wherein the nano particles are marked as a nano material B;
(3) adding the nano material B prepared in the step (2) into deionized water or buffer solution and glutaraldehyde water solution for mixing to obtain mixed solution, carrying out magnetic stirring, then carrying out centrifugation, and drying the centrifuged product in flowing nitrogen to obtain a nano material C; and finally, mixing the nano material C with a polyphenol oxidase solution, incubating at a certain temperature T, centrifuging after incubation, and drying a centrifuged product in flowing nitrogen to obtain the final SERS substrate material with the ligase, wherein the final SERS substrate material is marked as a nano material D.
Further, in the step (1), the metal nanoparticles are selected from at least one of gold, silver, copper, platinum and their alloys; the particle size of the metal nanoparticles is 10-100 nm; the temperature of the constant-temperature water bath is 20-100 ℃, and the time of the constant-temperature water bath reaction is 12-48 h.
Further, in the step (1), the alkaline solution is any one or more of potassium hydroxide, sodium carbonate, potassium carbonate and ammonia water;
further, in the step (1), the volume fraction of the silicon source solution is 5-50%; wherein the silicon source is at least one of tetraethoxysilane, hexamethyldisiloxane and tetramethyltetravinylcyclotetrasiloxane; the solvent of the silicon source solution is at least one of absolute ethyl alcohol, methanol, ethyl acetate or cyclohexane,
further, in the step (1), the silicon source solution is added with the metal nanoparticle sol for 2-6 times.
Further, in the step (2), the volume fraction of the silane coupling agent solution is 5-50%; the silane coupling agent is any one of 3-aminopropyltrimethoxysilane, 3-aminopropyltriethoxysilane, phenylaminomethyltriethoxysilane or phenylaminomethyltrimethoxysilane; the solvent of the silane coupling agent is any one of absolute ethyl alcohol, toluene, 2-propanol and cyclohexane.
Further, in the step (2), the temperature of the constant-temperature water bath is 20-100 ℃, and the time of the constant-temperature water bath reaction is 5-48 hours.
Further, in the step (3), the buffer solution is any one of a phosphate buffer solution, a citric acid buffer solution and an acetic acid buffer solution, and the concentration is 0.02-0.2M.
Further, in the step (3), the dosage ratio of the nano material B, the deionized water or the buffer solution and the glutaraldehyde aqueous solution is 1 g: (20-100) mL: (0.1-2) mL; the magnetic stirring time is 2-24 h.
Further, in the step (3), the concentration of the polyphenol oxidase solution is 0.1-10 g/L, and the solvent is phosphate buffer solution, citric acid buffer solution or acetic acid buffer solution.
Further, in the step (3), the dosage ratio of the nano material C to the polyphenol oxidase solution is 1 g: (0.1-20) L; the temperature T is 0-60 ℃; the incubation time is 5-48 h.
Further, in the steps (2) to (3), the centrifugation conditions are: 2000-9000 rpm, 3-30 min;
the application of the SERS substrate material with ligase in detecting xanthoxylin in hotpot condiment comprises the following specific steps:
(1) the SERS substrate material with the ligase obtained by the method is used for detecting the sanshoamides standard solution with different concentrations, then the characteristic Raman peak of the sanshoamides standard solution is analyzed, and a standard curve is drawn according to the concentration of the sanshoamides standard solution and the corresponding characteristic Raman peak intensity value;
(2) firstly, preparing a sample solution to be detected, detecting the sanshoamides in the sample solution to be detected by using the SERS substrate material with the ligase obtained by the method, qualitatively judging the detection result and the characteristic Raman peak contrast of the sanshoamides standard solution in the step (1), and judging whether the hotpot condiment contains the sanshoamides; and (3) substituting the detected characteristic Raman peak into the standard curve in the step (1) to obtain the concentration of the sanshoamides in the sample solution to be detected, and further calculating to obtain the content of the sanshoamides in the sample to be detected.
Further, the specific operation of the step (1) is as follows: firstly, preparing a series of zanthoxylum bungeanum Maxim standard solutions with the concentration range of 0-20mg/mL, then respectively taking the zanthoxylum bungeanum Maxim standard solutions with the same volume and different concentrations, and mixing the zanthoxylum bungeanum Maxim standard solutions with SERS substrate material suspension with ligase according to the ratio of 1: (0.5-5) uniformly mixing in a volume ratio, and standing for 0-1 h at the temperature of 20-60 ℃; then detecting a Raman spectrogram of a mixed solution of the sanshoamides standard solution and the SERS substrate material suspension with the ligase by using a Raman spectrometer, recording a characteristic Raman signal peak value, and drawing a standard curve by using the concentration of the sanshoamides standard solution and a corresponding characteristic Raman peak intensity value thereof; the SERS substrate material suspension with the ligase is a mixed solution of the SERS substrate material with the ligase and ultrapure water or buffer solution, and the mass volume fraction of the mixed solution is 10% -60% (w/v).
Further, in the step (1), the characteristic Raman peak of the zanthoxylum bungeanum standard solution is 1140-1190 cm-1，1250～1270cm-1，1440～1470cm-1，1620～1650cm-1The peak is the characteristic Raman peak of sanshoamides.
Further, in the step (2), the qualitative judgment specifically includes: 1140-1190 cm in the spectrogram-1，1250～1270cm-1，1440～1470cm-1，1620～1650cm-1When the characteristic Raman peak exists, the to-be-detected sample solution contains sanshoamides.
Further, the specific operation of the step (2) is as follows: firstly, preparing a sample solution to be detected, then respectively taking the sample solution to be detected with the same volume and different concentrations, and mixing the sample solution to be detected with SERS substrate material suspension with ligase according to the ratio of 1: (0.5-5) uniformly mixing in a volume ratio, and standing for 0-1 h at the temperature of 20-60 ℃; and (2) detecting a Raman spectrogram of a mixed solution of the sample to be detected and the SERS substrate material suspension with the ligase by using a Raman spectrometer, recording a characteristic Raman signal peak value, substituting the detected Raman signal peak value into the standard curve drawn in the step (1) for comparison, obtaining the concentration of the sanshoamides in the solution of the sample to be detected, and further calculating to obtain the content of the sanshoamides in the sample to be detected.
Further, in the step (2), the operation of preparing the sample solution to be tested is specifically: weighing hotpot condiment, adding absolute ethyl alcohol A into the hotpot condiment w by mass, shaking up the hotpot condiment by shaking, and performing ultrasonic extraction for 5-40 min at normal temperature to obtain a crude extract; centrifuging the crude extract, and collecting supernatant; collecting the remaining filter residue, adding absolute ethyl alcohol B for dissolving, centrifuging again after shaking, and collecting supernatant; repeating the steps, mixing the supernatant liquid collected for multiple times to obtain a sample solution to be detected, and recording the total volume V; the whole process is operated under the condition of keeping out of the sun; the using amount ratio of the hotpot condiment to the absolute ethyl alcohol A is 1 g: (3-50) mL; the centrifugation conditions were: the rotating speed is 1500-4000 r/min, and the time is 3-20 min.
Has the advantages that:
the invention provides a surface-enhanced Raman spectrum substrate material for detecting xanthoxylin in hotpot condiment, which mainly solves the defects that the existing main technical means can not accurately, qualitatively and quantitatively analyze and detect the xanthoxylin in the hotpot condiment, the operation procedure is complicated, the analysis time is long and the like, and on the basis of the successful preparation of the SERS substrate material, the surface-enhanced Raman spectrum technology can be used for rapidly identifying the xanthoxylin in the hotpot condiment to obtain molecular structure information and accurately and quantitatively detect the content of the xanthoxylin substances in the hotpot condiment, and has the following specific beneficial effects:
(1) the surface-enhanced Raman spectrum substrate material is simple to prepare, low in cost and accurate in detection result, and enhances the original Raman spectrum signal intensity of sanshoamides in the hotpot seasoning; the SERS substrate material can be used for obtaining definite molecular group identification information of the xanthoxylin, can be used for quickly and qualitatively judging whether the hotpot condiment contains the xanthoxylin, and can be used for accurately and quantitatively detecting the xanthoxylin.
(2) By means of the SERS substrate material, the SERS technology is applied to detection of the xanthoxylin in the hotpot condiment for the first time, the detection speed is high (detection of each sample is completed within 1 min), the result is accurate (the error rate with the HPLC-MS result is less than 10%), and a new thought is provided for the hotpot condiment industry for quickly judging the existence of the xanthoxylin and accurately and quantitatively detecting the xanthoxylin.
(3) The hotpot condiment contains a large amount of capsaicin, polyphenol, pigment and other substances, the substances are easy to generate fluorescence, and have certain interaction with the sanshoamides, so that the influence on the Raman spectrum of the sanshoamides is very large; according to the surface-enhanced Raman substrate material prepared by the invention, polyphenol, capsaicin and other substances in a hotpot condiment solution to be detected are greatly removed by utilizing polyphenol oxidase, so that the interference of the substances on the detection of the xanthoxylin is effectively avoided, the detection precision of the surface-enhanced Raman spectrum for the xanthoxylin in the hotpot condiment is greatly improved, and the detection requirement of the hotpot condiment on the market is basically met by the method.
(4) The method has the advantages of simple operation, high detection speed, low detection cost, no need of strong professional background of operators, small required sample amount, simple pretreatment and portability, and provides technical support for intelligent analysis and detection of the spicy substances in the hotpot condiment.
Drawings
FIG. 1 shows Raman characteristic peaks of Zanthoxylum bungeanum Maxim standard substance.
FIG. 2 is a Raman spectrum of the zanthoxylum bungeanum hemsl standard substance detection.
FIG. 3 is an example of a Raman spectrum for detecting xanthoxylin in the hotpot seasoning solution to be detected.
Detailed Description
In order to more clearly illustrate the content of the present invention, the present invention will be further described with reference to specific examples, and it is apparent that the described examples are only a part of the examples of the present invention and should not be construed as all the examples of the present invention.
1. Experimental samples, Primary reagents and instruments
Samples were purchased from the Zhenjiang European supermarket: the method adopts 5 spicy hotpot seasonings with different brands as detection objects, which are respectively as follows: taking a No. 1 sample, and fishing out from the seabed; sample No. 2, dezhuang; sample No. 3, small dragon ridge; sample No. 4, bridgehead; sample No. 5, famous.
Reagent: the zanthoxylum bungeanum hemsl standard substance is purchased from Chengdu Mai Desheng science and technology limited company, and the purity is more than 98 percent. The water used in the experimental process is deionized water, and the experimental reagents are analytically pure.
The instrument comprises the following steps: portable Raman spectrometer (SR-510Pro, Shanghai Uli optical instruments Co.)
Example 1:
the specific preparation process of the gold nanorod/aminated silicon dioxide/polyphenol oxidase nanocomposite material is as follows:
1) preparation of gold nanorods (AuNRs): to prepare the gold seed solution, 5mL of cetyltrimethylammonium bromide CTAB solution (0.2M) was mixed with HAuCl chloroauric acid4Solution (0.5mM) was mixed at 1: 1, then rapidly adding 0.6mL sodium borohydride NaBH4(0.01M, made up in ice water) and stirred vigorously for 2 min. The solution turned dark brown at this point, indicating the formation of a gold seed solution. The gold seed solution was incubated for 2 hours at 30 ℃ in a constant temperature water bath. To prepare the growth solution, 50mL of HAuCl will be included at 30 deg.C4(1mM) and 1.0mL silver nitrate AgNO3CTAB (50mL, 0.2M) was added to the solution mixture of solution (4 mM). Subsequently, a further 0.7mL of ascorbic acid solution (0.0788M) was added with gentle mixing, whereupon the solution turned from dark yellow to colorless. Finally, 0.12mL of gold seed solution is rapidly added, the preparation of the growth solution is completed, and the growth solution is placed in a constant-temperature water bath at 30 ℃ for a whole night (14 h); centrifuge at 8500rpm for 15min to remove excess CTAB, remove supernatant and finally transfer to 100mL deionized water for further use.
2) Preparation of gold nanorods/silica (AuNRs @ SiO)2): adding 1mL of NaOH solution (0.1M) into the AuNRs prepared in the step 1) under stirring, wherein the pH is 10.0, adding 1mL of 20% volume fraction ethyl orthosilicate (TEOS) ethanol solution, dropwise adding the solution once every 30min for three times, and reacting for 2 days in a constant-temperature water bath at the temperature of 30 ℃; centrifuging at 8500rpm 15min, remove supernatant for further use.
3) Preparation of gold nanorods/aminated silica (AuNRs @ SiO)2-NH2): AuNRs @ SiO prepared in the step 2)2Soaking in 2-propanol solution of 2% 3-aminopropyltriethoxysilane APTES in the presence of 0.1% acetic acid for 12h, centrifuging at 4500rpm for 15min, removing the supernatant, redissolving with 2-propanol, centrifuging again, removing the supernatant, redissolving with phosphate, centrifuging again, repeating this step 3 times, and drying under flowing nitrogen before use.
4) Preparation of gold nanorod/aminated silica/polyphenol oxidase nanocomposite (AuNRs @ SiO)2-NH2@ PPO): 200mg of AuNRs @ SiO2-NH2Nanoparticles, 10mL phosphate buffer (0.1M, pH 7) and 0.25mL glutaraldehyde (50% aqueous solution) were mixed and the reaction mixture was kept for 7h under magnetic stirring; glutaraldehyde-activated AuNRs @ SiO prepared using the same buffer2-NH2The nanoparticles were washed three times with centrifugation/redissolution cycles (4500rpm, 10min) and dried in flowing nitrogen to give glutaraldehyde-activated AuNRs @ SiO2-NH2Nanoparticles. 200mg of glutaraldehyde activated AuNRs @ SiO2-NH2The nanoparticles were mixed with 200mL polyphenol oxidase solution (5g/L, prepared with the same buffer) and incubated at 25 ℃ for 12h, finally rinsed with the same buffer and dried in flowing nitrogen to give AuNRs @ SiO2-NH2@ PPO nanoparticle powder. Before use, AuNRs @ SiO2-NH2The @ PPO nano particles are added with a proper amount of buffer solution to be dissolved, ultrasonic dispersion is carried out to obtain suspension, and AuNRs @ SiO is added into the suspension2-NH2The mass ratio w/v of the @ PPO substrate material is 40%
Accurately weighing 20mg of sanshoamides standard substance, dissolving with 1mL of anhydrous ethanol to obtain standard mother liquor, diluting the standard mother liquor with anhydrous ethanol respectively, preparing a series of hydroxy-alpha-sanshoamides standard solutions with different concentrations, and storing at 4 deg.C in dark place; 50 mu L of AuNRs @ SiO is taken2-NH2@ PPO nano particles are respectively added with 50 mu L of standard liquid with different concentrations and evenly mixed by vortex to obtain mixed liquidDripping 5 mu L of the solution on a silicon wafer, and drying the solution at 400-2500 cm-1And performing spectrum scanning by using the portable Raman spectrometer in the range to obtain a Raman spectrogram, and smoothing the spectrogram and optimizing a base line. FIG. 1 shows Raman characteristic peaks of Zanthoxylum bungeanum Maxim standard substance. As can be seen from the figure, the characteristic peak shift of sanshoamides occurs at 1154cm-1、1178cm-1、1260cm-1、1452cm-1、1630cm-1Respectively belonging to C-C stretching vibration, C-H deformation vibration, CH3Deformation vibration, C ═ O stretching vibration. FIG. 2 is a Raman spectrum of zanthoxylum oil standard solution at 785nm excitation wavelength, with Raman shift on the abscissa and Raman signal on the ordinate, wherein a-e represent AuNRs @ SiO2-NH2And the @ PPO nano particles can measure Raman signals of the zanthoxylum bungeanum maxim standard solutions with different concentrations of 0.5, 4.0, 6.0, 9.0, 12.0 and 20 mg/mL. In the Raman characteristic peak of sanshoamides, 1630cm-1The characteristic peak is selected as a quantitative analysis spectrum peak because the characteristic peak is attributed to C ═ O stretching vibration and has no influence of overlapping peaks. Measurement 1630cm-1±2cm-1And drawing a standard curve by using the Raman peak intensity value I to the concentration of the sanshoamides, and obtaining a function relation of the standard curve with a linear range of 0.5-20mg/mL as follows: 81.34x +4318, coefficient of correlation R2The linear relationship was good at 0.9949. The detection working conditions of the portable Raman spectrometer are as follows: laser power: 100 mW; integration time: 10 s; average times: 6; smoothing parameters: 2.
(2) preparing a sample solution to be tested:
weighing hotpot condiment, wherein the weight of the hotpot condiment is 10g, adding 50mL of absolute ethyl alcohol, shaking up, and performing ultrasonic extraction for 20min at normal temperature to obtain a crude extract; centrifuging the crude extractive solution (2500r/min, 10min), and collecting supernatant; collecting filter residues, adding 10mL of absolute ethyl alcohol, shaking, centrifuging again, and collecting supernate; repeating the steps, mixing the supernatants collected for multiple times, fixing the volume to 100mL by using absolute ethyl alcohol to obtain a sample solution to be detected, recording the total volume as V, and refrigerating the sample solution for later use; the whole step is operated under the condition of keeping out of the sun;
(3) raman detection of a sample solution to be detected:
and (3) adding 50 mu L of the sample solution to be detected prepared in the step (2) into a suspension containing 50 mu L of SERS substrate material, mixing uniformly in a vortex manner, reacting for 5min at 45 ℃, then dropping 5 mu L of the sample solution on a silicon wafer, drying, performing spectral scanning by using a Raman spectrometer to obtain a Raman spectrogram, and smoothing the spectrogram and optimizing a baseline. FIG. 3 is a Raman spectrum of a solution of a sample to be measured at 785nm excitation wavelength, with Raman shift on the abscissa and Raman peak intensity on the ordinate. Wherein, a represents a Raman spectrogram of a sample solution to be detected obtained by adopting a gold nanorod substrate without any modification, and b represents the Raman spectrogram of the sample solution to be detected. In addition, as can be seen from fig. 3, the sample solution to be tested does not generate raman signal on the gold nanorod substrate without any modification, because of interference from other components in the sample solution to be tested; while passing through AuNRs @ SiO2-NH2The @ PPO nano particle substrate shows a strong Raman signal.
(4) And (3) qualitative judgment:
preprocessing the Raman spectrogram obtained in the step (3), and then obtaining 1140-1190 cm in the spectrogram-1，1250～1270cm-1，1440～1470cm-1，1620～1650cm-1When the characteristic Raman peak exists, the to-be-detected sample solution contains sanshoamides;
(5) quantitative determination:
based on the detection result of the step (4), the Raman spectrogram of the sample solution to be detected obtained in the step (3) is 1630cm-1±2cm-1And (2) taking the characteristic Raman peak as a quantitative reference peak, measuring the Raman peak intensity value at the displacement position of the quantitative reference peak, obtaining the concentration of the sanshoamides in the solution of the sample to be detected according to the hydroxyl-alpha-sanshoamides standard curve obtained in the step (1), marking the concentration as X, and further calculating the content Y of the sanshoamides in the hotpot condiment sample according to a formula.
Y: the content of sanshoamides in the hotpot seasoning sample is mg/g;
x: obtaining the concentration of the sanshoamides in the to-be-detected sample solution according to the standard curve, namely mg/L;
v: the volume of the sample solution to be measured, L;
w: sample mass of hotpot condiment, g.
(6) Verification experiment
And respectively measuring the five sample solutions to be measured by adopting an HPLC-MS method and calculating the content of sanshoamides in the hotpot condiment samples so as to verify the accuracy of the measurement result of the method. In the HPLC-MS detection, a zanthoxylum tingle standard substance is used as a standard sample, absolute ethyl alcohol is used for dissolving zanthoxylum tingle to prepare a series of standard solutions with different concentrations for liquid chromatography detection, and the zanthoxylum tingle concentration is used as a horizontal coordinate, and a peak area is used as a vertical coordinate to draw a standard curve. Respectively sucking 1.5mL of sample solution to be detected, filtering the sample solution by a 0.22 mu m filter membrane, and then carrying out liquid chromatography detection; taking a peak with retention time of 32-34min as a target peak, calculating according to the sum of the calculated peak areas and a linear regression equation corresponding to a standard curve to obtain the concentration of sanshoamides in the to-be-detected sample solution, and then converting according to the formula in the step (5) to obtain the content of sanshoamides in the hotpot condiment sample; the content of sanshoamides measured by the Raman detection method and HPLC-MS is shown in Table 1. The relative error range between the HPLC-MS measurement value and the Raman spectroscopy of different hotpot condiment samples is 2.59-8.33%, and is lower than 10%. The results show that: the zanthoxylum bungeanum content in the hotpot condiment sample can be well obtained by utilizing a Raman spectroscopy method, and the result is highly similar to that of HPLC-MS.
TABLE 1 comparison of HPLC-MS and Raman Spectroscopy measurements
Description of the drawings: the above embodiments are only used to illustrate the present invention and do not limit the technical solutions described in the present invention; thus, while the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted; all such modifications and variations are intended to be included herein within the scope of this disclosure and the present invention and protected by the following claims.
Claims (10)
1. A preparation method of a surface-enhanced Raman spectrum substrate material for detecting xanthoxylin in hotpot condiment is characterized by comprising the following specific steps:
(1) firstly, preparing silicon-coated metal nanoparticles, and marking as a nano material A;
(2) adding the nano material A prepared in the step (1) into a silane coupling agent solution, uniformly mixing, reacting in a constant-temperature water bath, and centrifuging after reaction to obtain silicon amide coated metal nano particles, wherein the nano particles are marked as a nano material B;
(3) adding the nano material B prepared in the step (2) into deionized water or buffer solution and glutaraldehyde water solution for mixing to obtain mixed solution, carrying out magnetic stirring, then carrying out centrifugation, and drying the centrifuged product in flowing nitrogen to obtain a nano material C; and finally, mixing the nano material C with a polyphenol oxidase solution, incubating at a certain temperature T, centrifuging after incubation, and drying a centrifuged product in flowing nitrogen to obtain the final SERS substrate material with the ligase, wherein the final SERS substrate material is marked as a nano material D.
2. The preparation method of the surface-enhanced Raman spectroscopy substrate material for detecting xanthoxylin in hotpot condiment according to claim 1, wherein in the step (2), the volume fraction of the silane coupling agent solution is 5-50%; the silane coupling agent is any one of 3-aminopropyltrimethoxysilane, 3-aminopropyltriethoxysilane, phenylaminomethyltriethoxysilane or phenylaminomethyltrimethoxysilane; the solvent of the silane coupling agent is any one of absolute ethyl alcohol, toluene, 2-propanol or cyclohexane.
3. The preparation method of the surface-enhanced Raman spectrum substrate material for detecting xanthoxylin in the hotpot condiment according to claim 1, wherein in the step (2), the temperature of the constant-temperature water bath is 20-100 ℃, and the reaction time of the constant-temperature water bath is 5-48 hours.
4. The preparation method of the surface-enhanced Raman spectroscopy substrate material for detecting xanthoxylin in hotpot condiment according to claim 1, wherein in the step (3), the dosage ratio of the nano material B, deionized water or buffer solution and glutaraldehyde aqueous solution is 1 g: (20-100) mL: (0.1-2) mL; the magnetic stirring time is 2-24 h.
5. The preparation method of the surface-enhanced Raman spectroscopy substrate material for detecting xanthoxylin in the hotpot condiment according to claim 1, wherein in the step (3), the concentration of the polyphenol oxidase solution is 0.1-10 g/L, and the solvent is a phosphate buffer solution, a citric acid buffer solution or an acetic acid buffer solution; the dosage ratio of the nano material C to the polyphenol oxidase solution is 1 g: (0.1-20) L; the temperature T is 0-60 ℃; the incubation time is 5-48 h.
6. The preparation method of the surface-enhanced Raman spectroscopy substrate material for detecting xanthoxylin in hotpot condiment according to claim 1, wherein in the steps (2) to (3), the centrifugation conditions are as follows: 2000-9000 rpm, 3-30 min.
7. The use of the substrate material prepared by the method according to claims 1-6 for the detection of sanshoamides in hotpot seasonings is characterized by comprising the following specific steps:
(1) detecting the zanthoxylum bungeanum Maxim standard liquid with different concentrations by utilizing an SERS substrate material with ligase, then analyzing the characteristic Raman peak of the zanthoxylum bungeanum Maxim standard liquid, and drawing a standard curve by using the concentration of the zanthoxylum bungeanum Maxim standard liquid and the corresponding characteristic Raman peak intensity value thereof;
(2) firstly, preparing a sample solution to be detected, detecting the sanshoamides in the sample solution to be detected by utilizing an SERS substrate material with ligase, qualitatively judging the detection result and the characteristic Raman peak contrast of the sanshoamides standard solution in the step (1), and judging whether the hotpot condiment contains the sanshoamides; and (3) substituting the detected characteristic Raman peak into the standard curve in the step (1) to obtain the concentration of the sanshoamides in the sample solution to be detected, and further calculating to obtain the content of the sanshoamides in the sample to be detected.
8. The use according to claim 7, characterized in that the specific operations of step (1) are: firstly, preparing a series of zanthoxylum bungeanum Maxim standard solutions with the concentration range of 0-20mg/mL, then respectively taking the zanthoxylum bungeanum Maxim standard solutions with the same volume and different concentrations, and mixing the zanthoxylum bungeanum Maxim standard solutions with SERS substrate material suspension with ligase according to the ratio of 1: (0.5-5) uniformly mixing in a volume ratio, and standing for 0-1 h at the temperature of 20-60 ℃; then detecting a Raman spectrogram of a mixed solution of the sanshoamides standard solution and the SERS substrate material suspension with the ligase by using a Raman spectrometer, recording a characteristic Raman signal peak value, and drawing a standard curve by using the concentration of the sanshoamides standard solution and a corresponding characteristic Raman peak intensity value thereof;
the SERS substrate material suspension with the ligase is a mixed solution of the SERS substrate material with the ligase and ultrapure water or buffer solution, and the mass volume fraction of the mixed solution is 10% -60%;
the characteristic Raman peak of the zanthoxylum bungeanum Maxim standard solution ranges from 1140 cm to 1190cm-1，1250～1270cm-1，1440～1470cm-1，1620～1650cm-1The peak is the characteristic Raman peak of sanshoamides.
9. The use according to claim 7, wherein in step (2), the qualitative judgment is specifically: 1140-1190 cm in the spectrogram-1，1250～1270cm-1，1440～1470cm-1，1620～1650cm-1When the characteristic Raman peak exists, the to-be-detected sample solution contains sanshoamides.
10. The use according to claim 7, characterized in that the specific operations of step (2) are: firstly, preparing a sample solution to be detected, then respectively taking the sample solution to be detected with the same volume and different concentrations, and mixing the sample solution to be detected with SERS substrate material suspension with ligase according to the ratio of 1: (0.5-5) uniformly mixing in a volume ratio, and standing for 0-1 h at the temperature of 20-60 ℃; detecting a Raman spectrogram of a mixed solution of a sample solution to be detected and an SERS substrate material suspension with ligase by using a Raman spectrometer, recording a characteristic Raman signal peak value, substituting the detected Raman signal peak value into the standard curve drawn in the step (1) for comparison to obtain the concentration of the sanshoamides in the sample solution to be detected, and further calculating to obtain the content of the sanshoamides in the sample to be detected;
the specific operation of preparing the sample solution to be detected is as follows: weighing hotpot condiment, adding absolute ethyl alcohol A into the hotpot condiment w by mass, shaking up the hotpot condiment by shaking, and performing ultrasonic extraction for 5-40 min at normal temperature to obtain a crude extract; centrifuging the crude extract, and collecting supernatant; collecting the remaining filter residue, adding absolute ethyl alcohol B for dissolving, centrifuging again after shaking, and collecting supernatant; repeating the steps, mixing the supernatant liquid collected for multiple times to obtain a sample solution to be detected, and recording the total volume V; the whole process is operated under the condition of keeping out of the sun; the using amount ratio of the hotpot condiment to the absolute ethyl alcohol A is 1 g: (3-50) mL; the centrifugation conditions were: the rotating speed is 1500-4000 r/min, and the time is 3-20 min.
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