CN114324633A

CN114324633A - Method for identifying authenticity of Chinese tallow tree honey

Info

Publication number: CN114324633A
Application number: CN202111528085.7A
Authority: CN
Inventors: 罗丽萍; 刘韬; 宁方建
Original assignee: Nanchang University
Current assignee: Nanchang University
Priority date: 2021-12-14
Filing date: 2021-12-14
Publication date: 2022-04-12
Anticipated expiration: 2041-12-14
Also published as: CN114324633B

Abstract

The invention discloses a method for identifying authenticity of sapium sebiferum honey, and belongs to the technical field of food inspection. The method takes the Sapium sebiferum element as a characteristic marker, if the content of a honey sample is 2.05-36.13mg/kg, the sample is judged to be the Sapium sebiferum honey, and otherwise, the honey sample is judged to be other honey or adulterated Sapium sebiferum honey. The method can quickly and accurately identify whether the honey to be detected is the Sapium sebiferum honey, and guarantees are provided for controlling the quality of the honey and maintaining the market order.

Description

Method for identifying authenticity of Chinese tallow tree honey

Technical Field

The invention belongs to the technical field of food inspection, and particularly relates to a method for identifying authenticity of sapium sebiferum honey.

Background

The Sapium sebiferum (Sapium discoor) is a plant of the genus Sapium of the family Euphorbiaceae, also called as Sapium sebiferum, mainly grows in Zhejiang, Jiangxi, Fujian, Taiwan, Guangdong, Guangxi, Yunnan, Guizhou and other places, often grows in hillside or mountain valley forest, is fond of deep and moist soil, and is one of important honey source plants in various regions in southern China.

The Chinese tallow tree usually begins to blossom in late 5 months, the blooming period from late 6 months to middle, and ends substantially in late 6 months. The florescence of the colony is long, is the main honey flow period in one year, is also the most vigorous period of the Chinese bee colony in one year, and the average annual specific yield can reach 10-20 kg/colony. In addition, the Chinese tallow tree honey is used as a main single nectar variety and has unique nutritional value and biological activity.

At present, no technology for effectively identifying the authenticity of the mountain tallow tree honey exists, and the national standard of the mountain tallow tree honey is not established yet. In view of the above, there is a need to develop a method for identifying sapium sebiferum honey conveniently, accurately, practically and effectively.

Disclosure of Invention

In order to solve the problems in the background art, the invention provides a method for identifying the authenticity of sapium sebiferum honey. The inventor finds that the Sapium sebiferum element (N1, N5, N10- (E) coumaroyl spermidine, CAS number 131086-78-7) is high in content and stable in the Sapium sebiferum honey, and the content of the Sapium sebiferum element in the Sapium sebiferum honey ranges from 2.05 mg/kg to 36.13mg/kg, so that the Sapium sebiferum element serving as a characteristic marker is applied to identification of the Sapium sebiferum honey.

The invention aims to provide a method for identifying the authenticity of sapium sebiferum honey, which takes sapium sebiferum element as a characteristic marker, judges that a honey sample to be detected is the sapium sebiferum honey if the content of the sapium sebiferum element in the honey sample to be detected is 2.05-36.13mg/kg, and judges that the honey sample is other honey or adulterated sapium sebiferum honey if the content of the sapium sebiferum element in the honey sample to be detected is not 2.05-36.13 mg/kg.

The invention is realized by the following technical scheme:

a method for identifying the authenticity of Chinese tallow tree honey comprises the following steps:

1) dissolving a honey sample to be detected in an acidified aqueous solution to obtain a honey sample aqueous solution;

2) extracting the honey sample water solution obtained in the step 1) by using an acetonitrile solution, centrifuging, and collecting an upper acetonitrile extracting solution;

3) diluting and filtering the acetonitrile extracting solution obtained in the step 2) to obtain a solution to be tested on a machine;

4) adding water into the acetonitrile extracting solution obtained in the step 2) for dilution to obtain a matrix sample solution, taking the matrix sample solution as a solvent, weighing different amounts of the Sapium sebiferum standard substance, diluting step by step to prepare different concentrations of the Sapium sebiferum standard substance working solution, performing data acquisition through a chromatography-mass spectrometer, and drawing an external standard curve;

5) and calculating the content of the Sapium sebiferum element in the sample according to the external standard curve and the target peak response area of the sample.

Further, the pH value of the acidified aqueous solution in the step 1) is 1-4, and the mixing proportion of the acidified aqueous solution and the honey sample to be detected is 1-4:1, mL: g.

Further, sodium chloride is added into the honey sample water solution in the step 1), and the concentration of the sodium chloride in the honey sample water solution is 5% -15%, w/v.

Further, the dilution in the step 3) adopts water with the volume 1-5 times that of the acetonitrile extracting solution.

Further, the filtration in the step 3) adopts a 0.22 μm hydrophilic PTFE filter membrane.

Further, the Sapium sebiferum element in the step 4) is N1, N5, N10- (E) cumaroyl spermidine.

Further, the concentration of the matrix working solution of the Sapium sebiferum standard substance in the step 4) is 1800, 1500, 1200, 1000, 500, 200, 100, 50 and 20ng/mL respectively.

The invention has the beneficial effects that:

the invention provides an identification method, which can quickly and accurately identify whether the honey to be detected is the Chinese tallow tree honey, and provides guarantee for controlling the quality of the honey and maintaining the market order.

Drawings

FIG. 1 is a chromatogram of Chinese tallow tree bark extract in standard solution and honey sample solution.

FIG. 2 is a standard curve of Sapium sebiferum matrix.

FIG. 3 is a total ion flow graph and an absorption spectrum graph of a quality control sample and a standard sample.

FIG. 4 is a second-order mass spectrum comparison of Sapium sebiferum extract with standard substance.

FIG. 5 is a comparison of the characteristic spectra of Sapium sebiferum extract and standard substance.

FIG. 6 shows the mass spectrum cracking characteristics of Sapium sebiferum.

Fig. 7 is a partially enlarged view of fig. 6.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below with reference to the embodiments. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

Examples

1. Dissolving 2g of honey to be tested in 4mL of acidified water with pH of 2, uniformly mixing by vortex, and adding sodium chloride into the aqueous solution to ensure that the concentration of the sodium chloride is 10% (w/v) to obtain an aqueous solution of the honey sample.

2. Adding 3mL of pure acetonitrile solution into the obtained honey sample water solution, mixing for 1min by vortex, centrifuging at the rotation speed of 5000rpm and the temperature of 4 ℃ for 4min, transferring the upper layer extracting solution, adding the equal volume of pure acetonitrile solution into the lower layer solution, repeatedly extracting once, combining the two extracting solutions and fixing the volume to 6 mL.

3. After vortex mixing 0.5mL of the combined acetonitrile extracts with 1.5mL of water, the mixture was filtered through a 0.22 μm hydrophilic PTFE membrane in a liquid phase vial and assayed.

4. After extraction according to the steps 1-2, taking 0.5mL of acetonitrile extract of a honey sample to be detected, carrying out vortex mixing to obtain a quality control sample (QC), and simultaneously taking part of the quality control sample and water to dilute according to a volume ratio of 1:3 to prepare a matrix sample solution. Weighing a certain amount of Sapium sebiferum extract standard, and dissolving with methanol to obtain stock solution. And (3) taking the matrix sample solution as a solvent, and preparing the Sapium sebiferum essence standard matrix working solution with different concentrations by stepwise dilution. The stock solution concentration of Sapium sebiferum extract standard is 400000ng/mL, and the working solution concentrations are 1800, 1500, 1200, 1000, 500, 200, 100, 50 and 20ng/mL respectively.

5. And measuring the honey extract by using a UPLC-Q-TOF system. The chromatographic conditions are as follows: the compound separation was carried out using a HST 3 (2.1X 100mm, 1.8 μm) column, the column temperature being 40 ℃. The mobile phase A is aqueous solution containing 0.02% formic acid, the mobile phase B is pure acetonitrile, the flow rate is 0.4mL/min, the sample injection volume is 5 muL, and the liquid phase separation of the extracting solution is carried out according to the procedure in the table 1; the mass spectrum conditions are as follows: double electric spray with LockSprayIon spray ion sources (ESI) that can simultaneously collect accurate mass numbers of analytes and calibration compounds and perform mass number corrections throughout the process. Acquiring mass spectrum information in a negative ion mode, and setting parameter information as follows: the capillary voltage is 2.5kV, the sample cone voltage is 40V, the desolvation gas temperature is 20 ℃, the ion source temperature is 120 ℃, atomization and taper hole gas in a mass spectrum system are nitrogen, the desolvation gas flow rate is 800L/h, the reverse taper hole gas flow rate is 50L/h, and after the instrument is started, the mass axis is checked and tuned by Leucine Enkephalin (LE) and sodium formate. The data acquisition software is MassLynx 4.1 and the data acquisition mode is set as MS^EThe mode can simultaneously acquire primary parent ions and secondary fragment ions by switching High and Low Collision Energy (CE), wherein the data structure is a profile map, the mass scanning range is 50-1200Da, the single scanning time is 0.2s, the High Energy Collision Energy (HCE) is 10-45V along with the increment of molecular weight, and the Low Energy Collision Energy (LCE) is 6V. The real-time calibration compound was LE (0.1. mu.g/mL) to set the flow rate at 10. mu.L/min and the voltage at 1.5 kV.

6. The Sapium sebiferum extract has an accurate molecular weight of 583.2680, and a fraction ion peak of M/z 582.2610([ M-H ]]^-) The secondary fragment ion peaks should contain one or more of m/z 462.2034, 316.1670, 119.0503, etc. with a deviation in ion m/z of less than 10 ppm. The retention time of chromatogram of Sapium sebiferum essence is 5.61min, and deviation is + -0.15 min. And (3) obtaining the peak area of the primary molecular ion peak of the Sapium sebiferum in the matrix standard solution, and fitting a standard curve with the corresponding concentration. And (3) acquiring the peak area of the primary molecular ion peak of the Sapium sebiferum in the sample solution, and substituting the peak area into a standard curve to calculate the content (mg/kg).

TABLE 1 liquid phase separation elution procedure

The above-described embodiments are only preferred embodiments of the present invention and are not intended to limit the present invention. Various changes and modifications can be made by one skilled in the art, and any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.

Claims

1. A method for identifying the authenticity of Chinese tallow tree honey is characterized by comprising the following steps:

2. The method for identifying the authenticity of the sapium sebiferum honey as claimed in claim 1, wherein the pH of the acidified aqueous solution in the step 1) is 1-4, and the mixing and dissolving ratio of the acidified aqueous solution to the honey sample to be detected is 1-4:1, mL: g.

3. The method for identifying the authenticity of the sapium sebiferum honey as claimed in claim 1, wherein sodium chloride is added into the honey sample aqueous solution in step 1), wherein the concentration of the sodium chloride in the honey sample aqueous solution is 5% -15%, and w/v.

4. The method for identifying the authenticity of the sapium sebiferum honey as claimed in claim 1, wherein the dilution in step 3) is performed with water in an amount of 1-5 times the volume of the acetonitrile extract.

5. The method for identifying the authenticity of the sapium sebiferum honey as claimed in claim 1, wherein the filtration in step 3) is performed with a 0.22 μm hydrophilic PTFE filter membrane.

6. The method of claim 1, wherein the Sapium sebiferum element of step 4) is N1, N5, N10- (E) Tricoumaroyl spermidine.

7. The method of claim 1, wherein the concentrations of the substrate working solution of the Sapium sebiferum standard substance in step 4) are 1800, 1500, 1200, 1000, 500, 200, 100, 50 and 20ng/mL respectively.