Method for identifying gallnut honey by utilizing characteristic markers

Abstract

The invention provides a method for identifying gallnut honey by utilizing a characteristic marker, and belongs to the technical field of honey detection. According to the method, kynurenic acid is taken as a characteristic marker, quantitative detection is carried out on kynurenic acid through a high performance liquid chromatography-diode array detector method, and the content of kynurenic acid in gallnut honey is not lower than 14 mg / kg. The kynurenic acid is used as the characteristic marker of the gallnut honey for the first time, the developed identification method also can successfully discriminate the gallnut honey, and the method has the advantages of simple pretreatment, common instruments and mobile phases, low cost, short time, accuracy and the like, and is suitable for industrial production. The method has application potential in gallnut honey identification and product grading.

Description

Technical Field

[0001] The invention relates to the technical field of honey detection, and in particular to a method for identifying gallnut honey by utilizing characteristic markers. Background Art

[0002] The flavor and biological activity of honey are the main factors affecting the price of honey, and the flavor and biological activity mainly depend on the variety of honey, that is, the plant source of honey, so the price difference of different varieties of honey is obvious. In order to avoid selling inferior products as good ones and to maintain the order of consumers and the honey market, it is necessary to identify the authenticity of various honeys. At present, the most common means of authenticating honey is to screen the characteristic markers of honey by chromatography or chromatography-mass spectrometry.

[0003] Characteristic markers have been a hot topic in the honey research field in recent years, and they have important applications in the honey industry. On the one hand, characteristic markers are an effective method for identifying the authenticity of honey. By identifying the characteristic markers of honey, it is possible to effectively avoid the counterfeiting of cheap honey varieties as high-value honey varieties; on the other hand, biologically active characteristic markers can also be used as indicators for the grading of honey products. Honey can be divided into different grades according to the content level of characteristic markers, and pricing and sales are carried out separately, which is conducive to improving the economic value of honey. Therefore, characteristic markers are crucial for the authentication and quality control of honey.

[0004] Existing studies have reported the characteristic markers of some honey varieties. However, due to the diversity of nectar plants, the characteristic markers of many honey varieties have not been fully explored, including gallnut honey. Gallnut honey is a high-value medicinal honey formed by bees collecting nectar from Rhus chinensis plants and fully brewing. The annual output of gallnut honey exceeds 700 tons, generating huge economic benefits. Although a study published in 2023 (Characteristic components and authenticity evaluation of Chinese honeys from three different botanical sources. J. Agric. Food Chem. 71(45), 17284-17294.) showed that benzoic acid, phaseolic acid, leucoflavin, leucochrome, trans-trans-abscisic acid, cis-trans-abscisic acid, gallin, etc. are present in gallnut honey, the study only analyzed the components of gallnut honey due to the lack of control honey, and did not fully prove that the above substances can be used as characteristic markers of gallnut honey. Therefore, it is still necessary to explore the characteristic markers of gallnut honey to ensure the authentication and quality control of gallnut honey. At the same time, a simple, universal and time-saving quantitative method should be developed for the discovered characteristic markers of gallnut honey to lay the foundation for its practical application in the gallnut honey industry. Summary of the invention

[0005] In view of the technical problems existing in the background technology, the present invention aims to explore the characteristic markers in gallic honey and realize accurate identification of gallic honey based on the characteristic markers, aiming to solve the problem of lack of using characteristic markers for gallic honey identification and product grading.

[0006] The technical solution of the present invention is as follows: A method for identifying gallnut honey using a characteristic marker, wherein the characteristic marker is kynurenic acid, and the content of kynurenic acid in the gallnut honey is not less than 14 mg / kg.

[0007] By comparing the gallnut honey extract with other honey extracts through high performance liquid chromatography, it was found that kynurenic acid existed in the gallnut honey and the content was significantly higher; in other plant-based honeys, kynurenic acid was basically not detected or the content was significantly lower than that in the gallnut honey. Therefore, based on this characteristic discovery, the present invention has developed a method for identifying the gallnut honey using kynurenic acid as a characteristic marker.

[0008] Preferably, in the above method, the content of kynurenic acid in the gallnut honey is 14-300 mg / kg.

[0009] Preferably, in the above method, the following steps are specifically included: S1, pre-treating honey to obtain a honey solution; S2. Quantitative analysis of kynurenic acid in honey solution was performed using high performance liquid chromatography-diode array detector.

[0010] Compared with the high performance liquid chromatography tandem mass spectrometry method and the high performance liquid chromatography-fluorescence detector method, the high performance liquid chromatography-diode array detector method for quantitative analysis of kynurenic acid in the present invention has the advantages of low instrument cost, high convenience and simple operation.

[0011] More preferably, in the above method, step S1 is specifically: dissolving honey in methanol aqueous solution, and filtering to obtain a honey solution. For example, in some embodiments of the present invention, the methanol aqueous solution is a 35% methanol aqueous solution, and the filtration is performed using a 0.22 μm filter membrane.

[0012] In view of the fact that the kynurenic acid content in gallnut honey is relatively high, and other honey varieties do not contain kynurenic acid or have a low content of kynurenic acid, the scheme of the present invention directly uses a methanol aqueous solution to prepare the honey into a liquid phase honey solution to be tested when preparing the honey solution. On the one hand, operations such as extraction and concentration are avoided, which greatly simplifies the detection process. On the other hand, it is more conducive to distinguishing gallnut honey based on kynurenic acid.

[0013] More preferably, in the above method, the detection conditions of step S2 include: C18 chromatographic column; phase A in the mobile phase is 1% acetic acid aqueous solution, phase B is methanol, and isocratic elution; flow rate is 1 mL / min; column temperature is 30°C; injection volume is 10 μL; and detection wavelength is 260 nm.

[0014] More preferably, in the above method, step S2 is to quantitatively calculate the kynurenic acid in the honey solution to be tested by an external standard method based on the liquid phase detection result.

[0015] It is understandable that the above method provided by the present invention can be used to distinguish gallnut honey from other honey varieties. For example, in one embodiment of the present invention, gallnut honey is successfully distinguished from acacia honey, vitex honey, rape honey, vetch honey, and litchi honey. Similarly, the above method provided by the present invention can also be used for quantitative detection of kynurenic acid in honey samples.

[0016] Compared with the prior art, the present invention has the following beneficial effects: By comparing the composition differences between gallnut honey extract and other honey extracts, it was found that kynurenic acid existed in gallnut honey and its content was significantly different from that of other honeys; based on this discovery, the present invention proposed and successfully established a method for identifying gallnut honey using kynurenic acid as a characteristic marker for the first time. In the method provided by the present invention, the pretreatment of the honey sample is extremely simple, and only dissolution and filtration are required without extraction. At the same time, conditions for quantifying kynurenic acid using high performance liquid chromatography-diode array detector method are developed, which effectively shortens the detection time and reduces the instrument cost, and the verification results show that the method of the present invention is accurate and reliable. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solution of the present invention, the following is a brief introduction to the drawings used in the present invention. Obviously, the drawings described below are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained based on these drawings without creative work.

[0018] Figure 1 It is a flow chart of the method for identifying gallnut honey using kynurenic acid in an embodiment of the present invention; Figure 2 : are component analysis results of the gallnut honey extract and other honey extracts in the embodiment of the present invention, A is a liquid chromatogram of the gallnut honey extract and other honey extracts at a wavelength of 340 nm, B is an absorption spectrum of compound a, and C is an absorption spectrum of compound b; Figure 3 is the mass spectrum of compound a in the embodiment of the present invention, A is the primary mass spectrum in positive ion mode, B is the primary mass spectrum in negative ion mode, and C is the secondary mass spectrum in positive ion mode; Figure 4 : is the mass spectrum of compound b in the embodiment of the present invention, A is the primary mass spectrum in positive ion mode, B is the primary mass spectrum in negative ion mode, and C is the secondary mass spectrum in positive ion mode; Figure 5 1 is the liquid chromatography test result of the honey sample in the embodiment of the present invention, A is the liquid chromatogram of kynurenic acid and 6 varieties of honey solutions, B is the absorption spectrum of kynurenic acid, and C is the absorption spectrum of the compound corresponding to chromatographic peak 1. DETAILED DESCRIPTION

[0019] The following embodiments of the technical solution of the present invention are described in detail in conjunction with the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and are therefore only used as examples, and cannot be used to limit the protection scope of the present invention.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by technicians in the technical field to which the present invention belongs; the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention; the terms "including" and "having" in the description and claims of the present invention and any variations thereof are intended to cover non-exclusive inclusions.

[0021] The characteristic markers of honey are crucial for the authentication and quality control of honey, but the characteristic markers of gallnut honey have not been fully explored, and there is currently a lack of characteristic markers that can be effectively used for the grading of gallnut honey products. Existing research, such as patent application CN 117589749 A, discloses a scheme for judging whether gallnut honey is adulterated with sugar using laser induced breakdown spectroscopy and a strategy for identifying gallnut honey and its quality by analyzing gallin, flavin, benzoic acid, and tannic acid content by LC-MS and GC-MS. However, the scheme has many compounds to be tested and the operation is relatively complicated. In addition, although LC-MS and GC-MS have high sensitivity, the equipment cost is also high, and the instrument penetration rate is limited, which is not conducive to practical application.

[0022] In order to solve the technical problem of low-cost identification of gallnut honey and its quality, the present invention provides a new method for identifying gallnut honey using characteristic markers by exploring new characteristic markers of gallnut honey. The method has obvious advantages in terms of instrument cost, sample pretreatment process, reagent cost, detection time cost, etc., is more convenient, and is conducive to promotion in enterprises.

[0023] In the first aspect, the present invention screened out a compound that can be used as a characteristic marker of gallnut honey, namely kynurenic acid, by comparing gallnut honey extracts with extracts of other honey varieties. Compared with other varieties of honey that do not contain or contain very low amounts of kynurenic acid, a large amount of validation data shows that kynurenic acid in gallnut honey is significantly higher, specifically in the range of 14 to 300 mg / kg.

[0024] It is understandable that the characteristic markers of honey do not necessarily mean that they are unique to that type of honey, as long as they can be combined with the detection method and other factors to specifically distinguish that type of honey from different varieties of honey. Taking photochrome as an example, a 2010 study (Lumichrome and Phenyllactic Acid as Chemical Markers of Thistle ( Galactites tomentosaMoench) Honey) suggested that photochrome is a chemical marker for thistle honey; two studies in 2012 (Riboflavin and lumichrome in Dalmatian sage honey and other unifloral honeys determined by LC–DAD technique; Floral markers of cornflower ( Centaurea cyanus ) honey and its peroxide antibacterial activity for an alternative treatment of digital dermatitis) suggested that photopigments could also be used as markers in sage honey and cornflower honey, respectively; a 2017 study (Polish yellow sweet clover( Melilotus officinalis L.) honey, chromatographic fingerprints, and chemical markers) believes that photopigments are suitable for distinguishing rock-grown curved fruit purpurogenous purpurogenous honey from other honeys; the 2023 study (Characteristic components and authenticity evaluation of Chinese honeys from three different botanical sources) once again reiterated that photopigments can be used as characteristic phytochemical components of gallnut honey, safflower honey and dragon beard honey. In addition, patent application CN 111398497 A believes that bee species can also cause slight changes in the content of kynurenic acid in some honeys, and uses the content of kynurenic acid as a standard for distinguishing Italian honey from Chinese honey; however, it can be noted that the content of kynurenic acid in Chinese honey is between 0.05 and 23.70 μg / kg, and the content of kynurenic acid in Italian honey is between 18.61 and 375.04 μg / kg, that is, different bee species have little effect on the content of kynurenic acid; and the kynurenic acid in gallnut honey is hundreds and thousands of times that of the above scheme, which shows that there is a significant difference. Therefore, based on kynurenic acid, it is expected that reliable identification of gallnut honey can be achieved by developing a supporting quantitative method.

[0025] In view of the fact that there are very few reports on kynurenic acid being used as a characteristic marker of a certain honey variety, and kynurenic acid has a variety of good biological activities, which is beneficial to human health, and the kynurenic acid content in gallnut honey is significantly higher than that of other honey varieties reported and detected by the present invention, kynurenic acid has the potential to be used as a grading indicator for gallnut honey products. For example, Manuka honey uses its characteristic component UMF as a grading indicator. The higher the UMF content, the higher the grade and the higher the selling price.

[0026] Please refer to Figure 1 In a second aspect, an embodiment of the present invention provides a method for identifying gallnut honey using kynurenic acid, the method comprising the following steps: S1. Dissolve the honey solution in methanol aqueous solution and filter to obtain the honey solution; S2. Use high performance liquid chromatography-diode array detector method to quantitatively analyze kynurenic acid in honey solution, and honey with kynurenic acid content not less than 14 mg / kg can be identified as gallnut honey.

[0027] Preferably, in the above method, the kynurenic acid content in the gallnut honey is within 14-300 mg / kg.

[0028] Since the content of plant-derived ingredients in honey is relatively low, it is usually necessary to extract and concentrate the honey before testing, and a more sensitive detection method must be used for the detection, such as the use of high performance liquid chromatography tandem mass spectrometry in patent application CN 111398497 A to detect kynurenic acid at a lower content. However, the equipment cost of high performance liquid chromatography tandem mass spectrometry is much higher than that of high performance liquid chromatography, and the cost of use and maintenance is high, which is not conducive to the actual promotion and application of the identification method. In addition, when detecting kynurenic acid as a compound based on liquid chromatography, the high performance liquid chromatography-fluorescence detector method (such as Optimization of Zn 2+ -containing mobile phase forsimultaneous determination of kynurenine, kynurenic acid and tryptophan inhuman plasma by high performance liquid chromatography. J. Chromatogr. B 878(5-6), 603-608). Its mobile phase contains salt ions such as zinc ions or ammonium ions. However, fluorescence detectors are relatively rare, and the most widely used detectors on the market are ultraviolet detectors or diode array detectors. Moreover, salt ions in the mobile phase increase the risk of chromatographic column blockage.

[0029] Based on the discovery that the content of kynurenic acid in gallnut honey is significantly higher, the present invention optimizes and simplifies the pretreatment of honey, and based on the high performance liquid chromatography-diode array detector, re-explores the liquid chromatography conditions suitable for the quantitative detection of kynurenic acid in honey, and successfully establishes a method for quantitative analysis of kynurenic acid in honey samples using high performance liquid chromatography. Compared with the existing kynurenic acid quantitative method, the method of the present invention has the characteristics of low instrument cost, short detection method time (only 10 minutes is needed to complete the determination of a sample), not easy to cause chromatographic column blockage in the mobile phase, simple honey pretreatment, etc., which is very convenient for practical application in enterprises and grassroots testing units.

[0030] Some specific examples are listed below. It should be noted that the examples described below are exemplary and are only used to explain the present invention, and cannot be understood as limiting the present invention. If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used; if the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be obtained commercially.

[0031] Example 1 In this example, the characteristic markers of gallnut honey were obtained by comparing gallnut honey with other varieties of honey, including the following operations: (1) Preparation of honey extract.

[0032] 5 g of honey samples of each variety were weighed and dissolved in 20 mL of acidified water (pH adjusted to 2 with hydrochloric acid) to obtain honey solutions. In addition, the Waters Oasis HLB solid phase extraction column was preactivated with 5 mL of methanol and 5 mL of acidified water with pH=2 in sequence. The honey solution was loaded into the solid phase extraction column and dripped at a rate of 1 drop / s. After all the honey solution flowed through the solid phase extraction column, 5 mL of acidified water with pH=2 was added to the solid phase extraction column to remove the compounds not adsorbed by the filler. Subsequently, 5 mL of methanol solution was added to elute the adsorbed compounds, and the eluate was collected. The eluate was blown to dryness with a nitrogen blower and stored to prepare honey extracts of different varieties. The dried honey extract needs to be reconstituted with 50% methanol solution before use.

[0033] (2) Screening of characteristic chromatographic peaks of gallnut honey.

[0034] Thermo Fisher's ultimate3000 high performance liquid chromatography system was used to analyze the components of honey extracts of different varieties. Specifically, the liquid phase analysis procedure is as follows: Chromatographic column: Thermo Fisher ODS2 (250 mm ×4.6 mm, 5 μm); Mobile phase: Phase A was 1% acetic acid in water, and phase B was methanol solution; gradient elution program was: 0 min, 5% B; 10 min, 28% B; 12 min, 31% B; 15 min, 31% B; 18 min, 34% B; 20 min, 35% B; 23 min, 36% B; 33 min, 37% B; 47 min, 40% B; 50 min, 50% B; 55 min, 56% B; 75 min, 57% B; 80 min, 5% B; 85 min, 5% B; Flow rate: 0.5 mL / min; column temperature: 30°C; injection volume: 10 μL; detection wavelength: 340 nm.

[0035] The HPLC results of gallnut honey extract and other honey extracts are as follows Figure 2 As shown in Figure A, it can be found that at retention times of 17.0 min and 43.2 min, compound a and compound b are present in the gallnut honey extract, and the contents of these two compounds are relatively high, while these two compounds are not found in the five control honeys, namely, acacia honey, vitex honey, rapeseed honey, litchi honey and vetch honey. The absorption wavelengths of compound a and compound b were analyzed, and the characteristic absorption wavelengths of compound a are 242 nm and 333 nm ( Figure 2 B), the characteristic absorption wavelengths of compound b are 261 nm and 354 nm ( Figure 2 C).

[0036] (3) UPLC-QTOF-MS identification of characteristic chromatographic peaks of gallnut honey.

[0037] The compounds of the characteristic chromatographic peaks of gallnut honey were identified using the Agilent Accurate-Mass Q-TOF LC / MS 6520 system. Specifically, the UPLC-QTOF-MS liquid chromatography procedure is as follows: Chromatographic column: Thermo Fisher ODS2 (250×4.6 mm, 5 μm); Mobile phase: Phase A was 1% acetic acid in water, and phase B was methanol solution; gradient elution program was: 0 min, 5% B; 10 min, 28% B; 12 min, 31% B; 15 min, 31% B; 18 min, 34% B; 20 min, 35% B; 23 min, 36% B; 33 min, 37% B; 47 min, 40% B; 50 min, 50% B; 55 min, 56% B; 75 min, 57% B; 80 min, 5% B; 85 min, 5% B; Flow rate: 0.5 mL / min; column temperature: 30°C; injection volume: 10 μL; detection wavelength: 340 nm.

[0038] UPLC-QTOF-MS mass spectrometry parameters are: Ion source temperature, 350 °C; ion spray voltage, 4000 V (positive ion mode) / -400 0 V (negative ion mode); drying gas (nitrogen) flow rate: 10 L / min; nebulizer pressure: 35 psi; mass-to-charge ratio scanning range: 50-1200.

[0039] For compound a, in positive ion mode, its quasi-molecular ion [M+H] + is 190 ( Figure 3 A); in negative ion mode, its quasi-molecular ion [MH] - is 188 ( Figure 3 B), therefore, combined with the mass-to-charge ratio of the quasi-molecular ion in the positive and negative ion modes, the relative molecular mass of compound a was determined to be 189. In the secondary mass spectrometry in the positive ion mode ( Figure 3 C), the fragment ions produced by compound a include m / z 144, which indicates that compound a contains a carboxyl group; in addition, existing studies (Kynurenine and kynurenic acid: Two human neuromodulators found in Cannabis sativa LJPharmaceut. Biomed. 211, 114636) showed that the fragment ion m / z 116 and m / z 89 is the characteristic fragment ion of kynurenic acid. At the same time, the maximum absorption wavelength of kynurenic acid is 333 nm, which is basically consistent with compound a. In summary, combining the spectral information and mass spectrometry information, compound a was identified as kynurenic acid.

[0040] For compound b, in positive ion mode, its quasi-molecular ion [M+H] + is 243 ( Figure 4 A); in negative ion mode, its quasi-molecular ion [MH] - is 241 ( Figure 4 B), therefore, combined with the mass-to-charge ratio of the quasi-molecular ion in the positive and negative ion modes, the relative molecular mass of compound a was determined to be 242. In the secondary mass spectrometry in the positive ion mode ( Figure 4 C), fragment ions produced by compound b m / z 198. m / z 172 and m / z 145, have been reported in the literature (e.g. Lumichrome and phenyllactic acid as chemical markers of thistle ( Galactites tomentosa Moench) honey. J. Agric. Food Chem. 59(1), 364-369) showed that it was a characteristic fragment ion of photopigment. At the same time, the maximum absorption wavelength of photopigment was 353 nm, which was basically consistent with compound b. In summary, combined with spectral information and mass spectrometry information, compound b was identified as photopigment.

[0041] Example 2 This example provides a method for identifying gallnut honey based on kynurenic acid, comprising the following steps: (1) Pretreatment of honey samples.

[0042] Take 0.3 g of honey and dissolve it in 35% methanol aqueous solution. Stir to make it completely dissolved, and make up the volume to 3 mL with 35% methanol aqueous solution. Filter with a 0.22 μm filter membrane.

[0043] (2) Liquid chromatography detection.

[0044] The quantification of kynurenic acid in honey extract was carried out by HPLC-diode array detection, and the detection conditions were as follows: Chromatographic column: Thermo Fisher Hypersil Gold C18 (250×4.6 mm, 5 μm); Mobile phase: Phase A is 1% acetic acid in water, phase B is methanol; isocratic elution at a ratio of 35% for 10 minutes; Flow rate: 1 mL / min; column temperature: 30°C; injection volume: 10 μL; detection wavelength: 260 nm.

[0045] Under the above conditions, the retention time of kynurenic acid was about 5.7 min.

[0046] (3) Quantitative calculation of kynurenic acid was performed by external standard method.

[0047] Example 3 In this example, under the detection conditions provided in Example 2, the kynurenic acid standard was dissolved in 35% methanol, and the methodological validation was performed by measuring the detection limit, quantitation limit, linear range, intra-day precision, inter-day precision and spike recovery according to the "Guidelines for Validation of Analytical Methods in Chinese Pharmacopoeia 9101". Specifically, the following steps were included: The detection limit and quantification limit were determined using the signal-to-noise ratio (S / N) method, with the concentrations of the kynurenic acid solution corresponding to S / N=3 and S / N=10 as the detection limit and quantification limit, respectively. Specifically, the liquid phase diagram of 35% methanol was used as a blank control, a low concentration of kynurenic acid solution was prepared and adjusted according to the signal-to-noise ratio. When the S / N was 3 and 10, respectively, the corresponding concentrations were the detection limit and quantification limit.

[0048] The linear range and linear equation were determined based on eight concentrations of kynurenic acid solution: 0.2, 0.5, 1, 2, 5, 10, 20, and 50 μg / mL.

[0049] Intra-day precision was determined using three concentrations of kynurenic acid solution: 5, 10, and 20 μg / mL, and the results are expressed as relative standard deviation (RSD%). Inter-day precision was also determined using three concentrations of kynurenic acid solution: 5, 10, and 20 μg / mL, and the results were also expressed as RSD%.

[0050] The spike recovery was determined by adding 5, 10, and 20 μg / mL of kynurenic acid solution to blank honey (i.e., honey samples that were determined to contain no kynurenic acid), and the results were expressed as recovery (%).

[0051] The results of the method validation are shown in Table 1: the detection limit was 0.08 μg / mL, the quantification limit was 0.28 μg / mL, the linear range was 0.2~50 μg / mL, the linear equation was y=0.1937x-0.1296, and R 2 The RSD of intra-day precision was 0.17-0.52%, the RSD of inter-day precision was 1.08-3.85%, and the recovery rate of spiked was 95.71-103.06%. The above results show that the method of the present invention meets the requirements of the "Guidelines for Validation of Analytical Methods in Chinese Pharmacopoeia 9101" and is reliable.

[0052] Table 1 Methodology validation results

[0053] Example 4 In this case, kynurenic acid in the actual honey sample was analyzed and the gallnut honey was successfully identified as follows: In this example, 55 gallnut honey samples and 32 non-galnut honey samples were collected as test samples. Among them, the 32 non-galnut honey samples were specifically 10 acacia honeys, 10 vitex honeys, 6 rape honeys, 4 sweet potato honeys, and 2 litchi honeys. The above test samples were pre-treated according to the method in step (1) of Example 2.

[0054] According to the conditions in step (2) of Example 2, the honey solutions obtained by pretreatment were tested using a Thermo Fisher Ultimate 3000 HPLC system. Figure 5 A is the liquid chromatogram of various honey solutions. Figure 5 A shows that the retention time of kynurenic acid is around 5.7 min, and near the retention time of kynurenic acid, there is a chromatographic peak 1 in gallnut honey, while no obvious chromatographic peak is observed in other honeys. Figure 5 B) compared to chromatographic peak 1 ( Figure 5 C) is consistent with the characteristic wavelength of kynurenic acid, indicating that chromatographic peak 1 is kynurenic acid, that is, kynurenic acid exists in gallnut honey.

[0055] Based on the liquid chromatography results, kynurenic acid in each sample was quantified by the external standard method, and the quantitative results are shown in Tables 2 and 3.

[0056] Table 2 Content of kynurenic acid in gallnut honey samples

[0057] As shown in Table 2, the content of kynurenic acid in 55 gallnut honeys ranged from 14.78 to 250.09 mg / kg, with a median of 80.92 mg / kg and an average of 79.00 mg / kg.

[0058] Table 3 Contents of kynurenic acid in non-gallic honey samples

[0059] In Table 3, samples 1-10 are acacia honey, samples 11-20 are vitex honey, samples 21-26 are rape honey, samples 27-30 are vetch honey, and samples 31-32 are litchi honey. As shown in Table 3, kynurenic acid was not detected in acacia honey, vitex honey, rape honey, litchi honey, and vetch honey, and it may be that its content is below the detection limit.

[0060] In summary, kynurenic acid can be used as a characteristic marker in gallnut honey, and the method developed by the present invention can be used to identify gallnut honey, and the method has the advantages of saving materials and time in the extraction process, the instruments and mobile phases used are common, the time is short, the method is simple and accurate, etc.

[0061] It should be noted that the present invention is not limited to the above-mentioned embodiments. The above-mentioned embodiments are merely examples, and embodiments having substantially the same structure as the technical idea and exerting the same effects within the scope of the technical solution of the present invention are all included in the technical scope of the present invention. In addition, within the scope of the main purpose of the present invention, various modifications that can be thought of by those skilled in the art to the embodiments, and other methods of combining some of the constituent elements in the embodiments to construct are also included in the scope of the present invention.

Claims

1. A method for identifying Chinese nut honey using characteristic markers, characterized in that: The characteristic marker is kynurenic acid, and its content in gallnut honey is not less than 14 mg / kg.

2. The method according to claim 1, characterized in that The following steps are involved: S1, pre-treating honey to obtain a honey solution; S2. Quantitative analysis of kynurenic acid in honey solution was performed using high performance liquid chromatography-diode array detector.

3. The method according to claim 2, characterized in that Step S1 specifically comprises: dissolving honey in a methanol aqueous solution, and filtering to obtain a honey solution.

4. The method according to claim 3, characterized in that The filtration was performed using a 0.22 μm filter membrane.

5. The method according to claim 2, characterized in that The detection conditions of step S2 include: C18 chromatographic column; phase A in the mobile phase is 1% acetic acid aqueous solution, and phase B is methanol; the flow rate is 1 mL / min; the column temperature is 30°C; the injection volume is 10 μL; and the detection wavelength is 260 nm.

6. The method according to claim 5, characterized in that Isocratic elution was performed using the mobile phase.

7. The method according to claim 2, characterized in that The quantitative analysis was calculated using the external standard method.

8. Use of any one of the methods of claims 1-7 in distinguishing Chinese gall honey from other honey varieties.

9. The use according to claim 8, characterized in that: The other honey varieties include acacia honey, vitex honey, rapeseed honey, vetch honey and litchi honey.

10. Use of the method according to any one of claims 2 to 7 in the quantitative detection of kynurenic acid in honey.

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