A method for identifying the authenticity of honey

Abstract

The present invention proposes a method for identifying the authenticity of honey, which belongs to the technical field of food detection. The method comprises the following steps: 1) adding an organic reagent to a raw honey sample to obtain a raw honey sample solution, determining the stable oxygen isotope ratio of water in the raw honey sample solution, and constructing a database of the stable oxygen isotope ratio of water in the raw honey sample; 2) adding an organic reagent to a honey sample to be tested to obtain a honey sample solution to be tested, and determining the stable oxygen isotope ratio of water in the honey sample to be tested; 3) comparing the determination result of the stable oxygen isotope ratio of water in the honey sample to be tested obtained in step 2) with the database of the stable oxygen isotope ratio of water in the raw honey sample obtained in step 1), if the determination result of the honey sample to be tested is less than the minimum value of the database, it is determined that the honey to be tested is not real honey. The present invention effectively solves the application technology problem of the lack of a simple and rapid method for identifying the authenticity of honey in the field of honey adulteration detection / authenticity assurance.

Description

Technical Field

[0001] The invention belongs to the field of food detection and relates to a method for identifying the authenticity of honey. Background Art

[0002] Honey is a popular health food. After decades of development, my country's honey industry has made great achievements: At present, the total number of bee colonies in my country has reached 9.2 million, accounting for 13% of the world's total bee colonies. my country's honey production has increased from 411,800 tons in 2011 to 542,500 tons in 2018, ranking first in the world, and the export volume is 129,300 tons, a year-on-year increase of 0.74%, accounting for 1 / 4 of the world's honey trade volume.

[0003] At present, my country's bee industry has entered a fast-growing track. More than 1,300 bee product processing enterprises of different sizes have obtained national certification, including seven national leading enterprises, 35 provincial leading enterprises, and 65 industry leading enterprises. Since honey is an industry that depends on the weather, honey fraud incidents have occurred frequently in recent years. In order to reduce costs and make profits, some honey production enterprises mix cheap rapeseed honey with higher-priced honey. Some enterprises even add cheap raw materials such as sucrose and syrup to honey in order to increase production.

[0004] In order to regulate the development of the honey industry, combat counterfeit and substandard products, and protect the rights and interests of consumers, my country's national standards draw on the advanced experience of the American Association of Analytical Chemists (AOAC), add inspection methods for determining the authenticity of honey, and clarify the use of carbon isotope technology to detect carbon-4 plant sugars in honey. However, this method is limited to detecting carbon-4 plant sugars such as cane sugar and corn syrup. Substances such as beets belong to the same carbon-3 plants as nectar plants, so carbon isotope technology loses its effect. Hydrogen isotope technology can be used to detect carbon-3 beet sugars in honey, but it is difficult to detect other carbon-3 plant sugars such as rice syrup and wheat syrup.

[0005] Stable isotope technology has important application value in food adulteration detection and authenticity identification. It is different from other technologies such as chromatography and mass spectrometry in that it studies the characteristics and differences of constant substances rather than trace substances, just like the carbon and hydrogen isotope technology mentioned above. However, the constant substances in honey are not only organic matter containing carbon and hydrogen elements, but also water at a constant level. Although the water content is less than that of sugar organic matter, there is no difference in order of magnitude. Summary of the invention

[0006] The present invention proposes a method for identifying the authenticity of honey. Starting from the index characteristics of macro-compounds, the stable isotope technology is used to accurately measure the stable oxygen isotope characteristics of water in honey, and a database of the characteristics is constructed, and the authenticity of the product is identified based on this.

[0007] The present invention provides a method for identifying the authenticity of honey, comprising the following steps:

[0008] 1) adding an organic reagent to a raw honey sample to obtain a raw honey sample solution, determining the stable oxygen isotope ratio of water in the raw honey sample solution, and constructing a database of stable oxygen isotope ratios of water in the raw honey sample;

[0009] 2) adding an organic reagent to the honey sample to be tested to obtain a honey sample solution to be tested, and determining the stable oxygen isotope ratio of water in the honey sample to be tested;

[0010] 3) Compare the stable oxygen isotope ratio of water in the honey sample to be tested obtained in step 2) with the stable oxygen isotope ratio database of water in the raw honey sample obtained in step 1). If the measurement result of the honey sample to be tested is less than the minimum value of the database, it is determined that the honey sample to be tested is not real honey.

[0011] Furthermore, in step 1) and step 2), the organic reagent is at least one of esters, alcohols, nitriles, ethers, and ketones;

[0012] Preferably, the organic reagent is an alcohol;

[0013] More preferably, the organic agent is ethanol.

[0014] Furthermore, in step 1) and step 2), the organic reagent is subjected to anhydrous treatment.

[0015] Further, in step 1), the mass ratio of the raw honey sample to the organic reagent is 1:0.6-1;

[0016] Preferably, the mass ratio of the raw honey sample to the organic reagent is 1:1;

[0017] In step 2), the mass ratio of the honey sample to be tested to the organic reagent is 1:0.6-1;

[0018] Preferably, the mass ratio of the honey sample to be tested to the organic reagent is 1:1.

[0019] Furthermore, in step 3), the data in the database are obtained from raw honey samples of the same type of honey across the country every year, and the number is at least 30.

[0020] Furthermore, in step 1) and step 2), a gas chromatography-isotope ratio mass spectrometer is used to measure the stable oxygen isotope ratio of water.

[0021] Furthermore, the raw honey or the honey to be tested is plant nectar; the plant nectar includes woody plant nectar and herbaceous plant nectar;

[0022] Preferably, woody plant nectar includes linden honey, locust tree honey, jujube honey; herbaceous plant nectar includes multiflora honey

[0023] The present invention has the following advantages:

[0024] The present invention is based on physical chemistry, stable isotope thermodynamic fractionation principle and stable isotope ratio analysis technology, and measures the stable oxygen isotope ratio of water in honey, thereby identifying the authenticity of honey. Due to the low water content in honey, an organic solvent is used to dissolve and extract the water in honey. Under the action of an organic reagent, water is fully contacted with carbon dioxide, and a gas chromatography-stable isotope ratio mass spectrometry system is used to measure the stable oxygen isotope ratio of carbon dioxide. Finally, the stable oxygen isotope ratio of water in honey is obtained by calibration with a standard product, thereby achieving the determination of the oxygen isotope ratio in water in a viscous state in honey. By analyzing the oxygen isotope characteristics of water in real honey and establishing a database, and then comparing the honey sample to be tested with the data in the database, the authenticity of the honey sample to be tested can be determined. The method is simple and fast, has low cost, and can accurately identify the authenticity of honey. DETAILED DESCRIPTION

[0025] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0026] The inventor of the present application has found that no matter what kind of cheap sugar (sugar or syrup) is mixed in the counterfeiting of honey, in order to meet the physical and chemical indicators of honey, water will inevitably be introduced. Therefore, the stable oxygen isotope difference of water in honey can be used to identify the authenticity of honey, but due to the low water content in honey, it is difficult to use existing analytical techniques for sample analysis and characteristic research. The inventor of the present application uses an organic reagent to dissolve the water in honey so that water can be fully contacted with carbon dioxide, and then uses a gas chromatography-stable isotope ratio mass spectrometry system to measure the stable oxygen isotope ratio of carbon dioxide, and finally calibrates with a standard product to obtain the stable oxygen isotope ratio of water in the honey sample to be tested. By analyzing the oxygen isotope characteristics of water in real honey and establishing a database, and then comparing the honey sample to be tested with the data in the database, the authenticity of the honey sample to be tested can be determined.

[0027] An embodiment of the present invention provides a method for identifying the authenticity of honey, comprising the following steps:

[0028] 1) adding an organic reagent to a raw honey sample to obtain a raw honey sample solution, determining the stable oxygen isotope ratio of water in the raw honey sample solution, and constructing a database of stable oxygen isotope ratios of water in the raw honey sample;

[0029] 2) adding an organic reagent to the honey sample to be tested to obtain a honey sample solution to be tested, and determining the stable oxygen isotope ratio of water in the honey sample to be tested;

[0030] 3) Compare the stable oxygen isotope ratio of water in the honey sample to be tested obtained in step 2) with the stable oxygen isotope ratio database of water in the raw honey sample obtained in step 1). If the measurement result of the honey sample to be tested is less than the minimum value of the database, it is determined that the honey sample to be tested is not real honey.

[0031] In other words, if the measurement result of the honey sample to be tested is within the distribution range of the database, the honey to be tested is determined to be real honey.

[0032] In one embodiment of the present invention, in step 1) and step 2), the organic reagent is at least one of esters, alcohols, nitriles, ethers, and ketones. Preferably, the organic reagent is alcohol. More preferably, the organic reagent is ethanol.

[0033] In one embodiment of the present invention, in step 1), a raw honey sample is selected and dissolved with an organic reagent. After the water is released from the viscous state, the water-organic reagent mixture is used as the main research object, the stable oxygen isotope ratio of water is determined, and a database of stable oxygen isotope ratios of water in raw honey is constructed.

[0034] In one embodiment of the present invention, in step 2), the honey sample to be tested is dissolved with an organic reagent, and after the water is released from the viscous state, subsequent analysis is performed based on the water-organic reagent mixture to determine the stable oxygen isotope ratio of water in the honey sample to be tested.

[0035] Furthermore, in step 1) and step 2), the organic reagent is subjected to anhydrous treatment. Preferably, the organic reagent is subjected to anhydrous treatment through a molecular sieve.

[0036] In one embodiment of the present invention, in step 1), the mass ratio of the raw honey sample to the organic reagent is 1:0.6-1. Preferably, the mass ratio of the raw honey sample to the organic reagent is 1:1.

[0037] In step 2), the mass ratio of the honey sample to be tested to the organic reagent is 1:0.6-1. Preferably, the mass ratio of the honey sample to be tested to the organic reagent is 1:1.

[0038] In one embodiment of the present invention, in step 1) and step 2), a gas chromatography-isotope ratio mass spectrometer (GC-IRMS) is used to measure the stable oxygen isotope ratio in water. The measurement methods of step 1) and step 2) are the same.

[0039] Specifically, taking step 2) as an example, step 2) specifically includes:

[0040] A) adding an organic reagent to the honey sample to be tested, mixing and letting stand, taking the upper layer of liquid to obtain a solution to be tested;

[0041] B) placing the test solution obtained in step A) in a bottle, sealing it, replacing the air in the bottle with high-purity carbon dioxide gas, and then placing the bottle containing the test solution and high-purity carbon dioxide under constant temperature conditions to react to obtain a reaction solution;

[0042] C) Using a gas chromatography-stable isotope ratio mass spectrometry system, after determining the stable oxygen isotope ratio of carbon dioxide in the reaction solution, the stable oxygen isotope ratio of water in the honey sample to be measured is obtained by calibration with a standard substance.

[0043] Wherein, in step C), standard calibration specifically includes: selecting standard water (stable oxygen isotope ratio is known), adding organic reagent to water, and obtaining a standard solution to be tested; placing the standard solution to be tested in a bottle, sealing it, replacing the air in the bottle with high-purity carbon dioxide gas, reacting it under constant temperature, using a gas chromatography-stable isotope ratio mass spectrometry system, determining the stable oxygen isotope ratio of carbon dioxide in the standard reaction solution, and establishing an oxygen isotope standard curve between water and carbon dioxide; combining the above standard curve, according to the stable oxygen isotope ratio of carbon dioxide in the reaction solution, the stable oxygen isotope ratio of water in the reaction solution can be calculated. Wherein, the determination conditions of the standard solution to be tested are the same as those of the reaction solution.

[0044] In step B), the replacement time is 1 to 20 minutes, the reaction temperature is 10 to 100° C. under constant temperature conditions, and the reaction time is 4 to 48 hours under constant temperature conditions.

[0045] In one embodiment of the present invention, in step 1) and step 2), the raw honey or the honey to be tested is plant nectar; the plant nectar includes woody plant nectar and herbaceous plant nectar. Preferably, the woody plant nectar includes linden honey, locust tree honey, jujube honey; the herbaceous plant nectar includes multi-flower honey. In the embodiment of the present invention, due to the stable oxygen isotope ratio (δ 18 O) is significantly higher than the δ 18 O, so it can be detected by this method.

[0046] In one embodiment of the present invention, in step 3), the data in the database are obtained from raw honey samples of the same type of honey across the country every year, and the number is at least 30.

[0047] The present invention will be described in detail below with reference to embodiments.

[0048] Example 1 Authenticity identification of acacia honey to be tested

[0049] a) Collect control samples: Collect pure, additive-free acacia honey samples (31) from beehives and record the sampling time and location;

[0050] b) Obtaining the samples to be tested: registering the detailed information of the acacia honey to be tested, which is divided into sample 1 and sample 2;

[0051] c) Take 3g of acacia honey from each control sample and the sample to be tested, and add 3g of anhydrous ethanol (10g of activated Molecular sieves were dissolved in anhydrous ethanol and mixed evenly, and then allowed to stand, and the liquids were taken for testing respectively;

[0052] d) Pour the solution obtained in c) into a crimp-top headspace injection bottle, seal it with a crimp cap, insert a gas guide needle, fill it with high-purity CO2 and exhaust all the air, then remove the gas guide needle. The whole process lasts for 10 minutes;

[0053] e) Place the headspace bottle in d) at a constant temperature of 25°C and shake for more than 8 hours.

[0054] f) taking the headspace injection bottle after oscillation for 8 hours in e), collecting the gas in the headspace of the headspace injection bottle, injecting it into the GC-IRMS system, and determining the stable oxygen isotope ratio of carbon dioxide;

[0055] g) According to the PIT (identical treatment) principle of stable isotope analysis and testing [Werner RA, Brand WA. Referencing strategies and techniques in stable isotope ratio analysis. Rapid Commun Mass Spectrom. 2001; 15(7): 501-19. doi: 10.1002 / rcm.258. PMID: 11268135.], the stable isotope ratio of carbon dioxide is calibrated with a standard substance to obtain the stable oxygen isotope ratio of water in honey.

[0056] The stable oxygen isotope ratio (δ 18 O) value distribution range is 0.74‰~3.11‰;

[0057] The sample 1 was measured to be δ 18 O value is -1.19‰, and the δ 18 O value is 1.58‰;

[0058] h) Compare the stable oxygen isotope ratios (δ 18 O) It can be seen that:

[0059] δ of sample 1 18 If the O value is less than the minimum value in the database, the product can be judged to be fake honey;

[0060] δ of sample 2 18The O value is consistent with the distribution range of the database, and the product can be determined to be real honey.

[0061] Test Example 1 Verify the accuracy of the detection method of the present invention

[0062] a) A raw honey sample (No. 1#) was selected as the research object, and its water content was measured to be 19.6%. Beet sugar and tap water were selected as the research objects, and a syrup with a water content of 19.6% was prepared (No. 2#);

[0063] b) adding syrup to honey in a mass ratio of 2:8, 4:6, 6:4 and 8:2, thereby obtaining simulated samples with syrup mass ratios of 20%, 40%, 60% and 80%, which are numbered 3#, 4#, 5# and 6# respectively;

[0064] c) Take 3g of each of the above 6 samples, add 3g of ethanol and mix thoroughly, let stand and take the upper layer solution for later use;

[0065] d) Pour the solution obtained in c) into a crimp-top headspace injection bottle, seal it with a crimp cap, insert a gas guide needle, fill it with high-purity CO2 and exhaust all the air, and then remove the gas guide needle;

[0066] e) Place the headspace bottle in d) at a constant temperature of 25°C and shake for more than 8 hours.

[0067] f) taking the headspace injection bottle after oscillation for 8 hours in e), collecting the gas in the headspace of the headspace injection bottle, injecting it into the GC-IRMS system, and determining the stable oxygen isotope ratio of carbon dioxide;

[0068] g) According to the PIT (treat as if nothing is wrong) principle of stable isotope analysis and testing, the stable isotope ratio of carbon dioxide was corrected with a standard substance to obtain the stable oxygen isotope ratio of water in each sample. The results are shown in Table 1.

[0069] Table 1 δ in water of each sample 18 O value (‰)

[0070] Sample No. 1# 2# 3# 4# 5# 6# Measured value 1.24 -8.11 -0.79 -2.23 -4.62 -6.51 Predicted value - - -0.63 -2.50 -4.37 -6.24 difference - - 0.16 -0.27 0.25 0.27

[0071] As can be seen from Table 1, the absolute value of the difference between the predicted value and the measured value is <0.3‰, which meets the standard deviation requirements of the stable isotope ratio mass spectrometer oxygen isotope analysis test in water. The t-test analysis between the measured value and the predicted value shows that there is no significant difference between the two, and the measured value is linearly negatively correlated with the syrup incorporation ratio. Therefore, it can be shown that the method of the present invention can accurately analyze the stable oxygen isotope ratio of water in honey.

[0072] δ of water in honey in Example 1 18 O database comparison, in this verification example, when the proportion of honey mixed with syrup is higher than 20%, the δ18 If the O value is less than the minimum value in the database, it can be determined to be fake honey.

[0073] Example 2 Authenticity identification of the tested honey

[0074] a) Collect control samples: Collect pure, additive-free raw honey samples (34) from beehives and record the sampling time and location;

[0075] b) Obtaining the samples to be tested: registering the detailed information of the honey to be tested, which is divided into sample 1 and sample 2;

[0076] c) Take 3g of honey from each control sample and the sample to be tested, and add 3g of anhydrous ethanol (10g of activated Molecular sieves were dissolved in anhydrous ethanol and mixed evenly, and then allowed to stand, and the liquids were taken for testing respectively;

[0077] d) Pour the solution obtained in c) into a crimp-top headspace injection bottle, seal it with a crimp cap, insert a gas guide needle, fill it with high-purity CO2 and exhaust all the air, then remove the gas guide needle. The whole process lasts for 10 minutes;

[0078] e) Place the headspace bottle in d) at a constant temperature of 25°C and shake for more than 8 hours.

[0079] f) taking the headspace injection bottle after oscillation for 8 hours in e), collecting the gas in the headspace of the headspace injection bottle, injecting it into the GC-IRMS system, and determining the stable oxygen isotope ratio of carbon dioxide;

[0080] g) According to the PIT (identical treatment) principle of stable isotope analysis and testing, the stable isotope ratio of carbon dioxide is calibrated with a standard substance to obtain the stable oxygen isotope ratio of water in honey.

[0081] h) Measure the stable oxygen isotope ratio of water in the raw honey database (δ 18 O) value distribution range is -0.38‰~3.76‰;

[0082] The sample 1 was measured to be δ 18 O value is -0.02‰, and the δ 18 O value is 0.99‰;

[0083] i) Compare the stable oxygen isotope ratios (δ 18 O) It can be seen that:

[0084] δ of sample 1 and sample 2 18 The O values ​​all conform to the distribution range of the database, and the product can be determined to be real honey.

[0085] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for identifying the authenticity of honey, characterized in that: The steps include: 1) adding an organic reagent to a raw honey sample to obtain a raw honey sample solution, determining the stable oxygen isotope ratio of water in the raw honey sample solution, and constructing a database of stable oxygen isotope ratios of water in the raw honey sample; wherein the mass ratio of the raw honey sample to the organic reagent is 1:1; 2) adding an organic reagent to the honey sample to be tested to obtain a honey sample solution to be tested, and determining the stable oxygen isotope ratio of water in the honey sample to be tested; wherein the mass ratio of the honey sample to be tested to the organic reagent is 1:1; 3) comparing the stable oxygen isotope ratio of water in the honey sample to be tested obtained in step 2) with the database of stable oxygen isotope ratios of water in the raw honey sample obtained in step 1), and if the measurement result of the honey sample to be tested is less than the minimum value of the database, it is determined that the honey sample to be tested is not real honey; In step 1) and step 2), the organic reagent is ethanol; the stable oxygen isotope ratio of water is determined using a gas chromatography-isotope ratio mass spectrometer; wherein step 2) specifically comprises: A) adding an organic reagent to the honey sample to be tested, mixing and letting stand, taking the upper layer of liquid to obtain a solution to be tested; B) placing the test solution obtained in step A) in a bottle, sealing it, replacing the air in the bottle with high-purity carbon dioxide gas, and then placing the bottle containing the test solution and high-purity carbon dioxide under constant temperature conditions to react to obtain a reaction solution; C) Using a gas chromatography-stable isotope ratio mass spectrometry system, after determining the stable oxygen isotope ratio of carbon dioxide in the reaction solution, the stable oxygen isotope ratio of water in the honey sample to be measured is obtained by calibration with a standard substance.

2. The method according to claim 1, characterized in that In step 1) and step 2), the organic reagent is subjected to anhydrous treatment.

3. The method according to claim 1 or 2, characterized in that: In step 3), the data in the database are obtained from raw honey samples of the same type of honey across the country every year, with a number of at least 30.

4. The method according to claim 1, characterized in that The raw honey or the honey to be tested is the nectar of plants, and the nectar of plants includes the nectar of woody plants and the nectar of herbaceous plants.