A method for determining the stable oxygen isotope ratio of water in honey

Abstract

The present invention proposes a method for determining the stable oxygen isotope ratio of water in honey, which belongs to the technical field of stable isotope analysis of food. The method comprises the following steps: 1) adding an organic reagent to a honey sample to be tested, mixing and standing, taking the upper layer of liquid to obtain a solution to be tested; 2) taking the solution to be tested obtained in step 1) and placing it in a bottle, after sealing, replacing the air in the bottle with high-purity carbon dioxide gas, and then placing the bottle containing the solution to be tested and high-purity carbon dioxide under constant temperature conditions to react to obtain a reaction solution; 3) using a gas chromatography-stable isotope ratio mass spectrometry system, after determining the stable oxygen isotope ratio of carbon dioxide in the reaction solution, calibrating with a standard product, and obtaining the stable oxygen isotope ratio of water in the honey sample to be tested. The present invention can be used for simply and quickly determining the stable oxygen isotope ratio of water in honey, and can be used for research and application of honey authenticity or adulteration detection.

Description

Technical Field

[0001] The invention belongs to the field of stable isotope analysis and testing, and relates to a method for measuring the stable oxygen isotope ratio of water in honey. Background Art

[0002] According to the definition of GB 14963 "Honey", honey is "a natural sweet substance that bees collect from plants, secretions or honeydew, mix with their own secretions, and then fully brew." Because of its properties as a "natural sweet substance", it is quite popular among health care people and ordinary consumers. Statistics show that in 2018, the global honey production was 1.851 million tons, and my country's honey production of 542,500 tons ranked first in the world. The export volume was 129,300 tons, accounting for 1 / 4 of the world's honey trade volume. The export volume in 2021 was as high as 146,000 tons.

[0003] However, in recent years, honey fraud incidents have occurred frequently. In order to reduce costs and make profits, some honey production companies mix cheap rapeseed honey into higher-priced honey. Some companies even add cheap raw materials such as sucrose and syrup to honey in order to increase production.

[0004] Stable isotope technology has important applications in detecting food adulteration, especially stable oxygen isotope technology can be used to detect whether the product is adulterated with exogenous water. It is known that the water in honey comes from the nectar, secretions or honeydew collected in a sealed manner, and the remaining water after brewing. Due to the product properties and production process of sucrose and syrup, when they are used to make fake honey, water from non-honey sources will inevitably be introduced. Therefore, the stable oxygen isotope ratio in honey water can be studied to identify the authenticity of honey.

[0005] The current classic method for determining oxygen isotope ratios in water was developed by Epstein in 1953. Its principle is to use water and pure carbon dioxide to exchange oxygen atoms, extract carbon dioxide after the reaction reaches equilibrium, and measure its oxygen isotope composition. This technology can directly measure the oxygen isotope ratios in water samples, wine and juice, and has been adopted as an official method by international organizations such as the American Association of Analytical Chemists (AOAC), the European Commission (CEN), and the International Organization of Vine and Wine (OIV). Method standards for products such as wine and juice have also been formulated in my country. However, the reported methods such as dual-path injection-stable isotope ratio mass spectrometry, fully automatic equilibrium-stable isotope ratio mass spectrometry, and the existing operating methods disclosed in ZL201210473455.6 cannot directly measure honey samples because the water content of honey is very low (the water content of the first-grade honey is generally not more than 20%, and the water content of the second-grade honey is not more than 24%), and it is particularly viscous, and the water and carbon dioxide cannot effectively contact and cannot fully exchange oxygen isotopes. Therefore, there has been no report on the research and application of the oxygen isotope ratio of water in honey so far. Summary of the invention

[0006] The method for determining the stable oxygen isotope ratio of water in honey proposed by the present invention comprises the following steps: extracting water from honey by dissolving with an organic solvent, fully contacting water with carbon dioxide under the action of a high concentration organic reagent, determining the stable oxygen isotope ratio of carbon dioxide by a gas chromatography-stable isotope ratio mass spectrometry system, and finally calibrating with a standard substance to obtain the stable oxygen isotope ratio of water in honey.

[0007] The present invention provides a method for determining the stable oxygen isotope ratio of water in honey, comprising the following steps:

[0008] 1) adding an organic reagent to the honey sample to be tested, mixing and letting stand, and taking the upper layer of liquid to obtain a solution to be tested;

[0009] 2) Put the above-mentioned solution to be tested into a bottle, seal it, replace the air in the bottle with high-purity carbon dioxide gas, and then place the bottle containing the solution to be tested and high-purity carbon dioxide under constant temperature to react to obtain a reaction solution;

[0010] 3) 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.

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

[0012] Preferably, in step 1), the organic reagent is an alcohol;

[0013] More preferably, in step 1), the organic reagent is ethanol.

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

[0015] Preferably, in step 1), the organic reagent is anhydrous by molecular sieve.

[0016] Further, in step 1), the mass ratio of the honey sample to be tested to the organic reagent is 1:0.6-2;

[0017] Preferably, in step 1), the mass ratio of the honey sample to be tested to the organic reagent is 1:1.

[0018] Further, in step 2), the replacement time is 1 to 20 minutes;

[0019] Preferably, the replacement time is 10 min.

[0020] Further, in step 2), the reaction temperature is 10 to 100° C. under constant temperature conditions;

[0021] Preferably, in step 2), the reaction temperature is 25° C. under constant temperature conditions.

[0022] Furthermore, in step 2), the reaction time under constant temperature conditions is 4 to 48 hours.

[0023] Furthermore, the honey includes linden honey, locust tree honey, jujube honey or multi-flower honey.

[0024] The present invention has the following advantages:

[0025] The present invention realizes the determination of the oxygen isotope ratio of water in honey, and for the first time proposes to use an organic reagent to dissolve honey and then extract water therefrom, and with the assistance of the organic reagent, water and carbon dioxide are in a free state and fully contacted, and oxygen isotope exchange is completed continuously in the process of combining to form carbonic acid / carbonic acid hydrolysis, realizing the application of the "H2O-CO2 isotope exchange" principle, and finally successfully measuring the stable oxygen isotope ratio of water in honey. At the same time, the method of the present invention provides ideas for the stable oxygen isotope analysis of water in honey and its application in adulteration detection / authenticity assurance. DETAILED DESCRIPTION

[0026] 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.

[0027] The present invention provides a method for determining the stable oxygen isotope ratio of water in honey by way of an embodiment, comprising the following steps:

[0028] 1) adding an organic reagent to the honey sample to be tested, mixing and letting stand, and taking the upper layer of liquid to obtain a solution to be tested;

[0029] 2) placing the test solution obtained in step 1) 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;

[0030] 3) 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.

[0031] In one embodiment of the present invention, in step 1), the organic reagent is at least one of esters, alcohols, nitriles, ethers, and ketones. Preferably, in step 1), the organic reagent is an alcohol. More preferably, in step 1), the organic reagent is ethanol. In the embodiment of the present invention, the organic reagent is a reagent that is miscible with water. By utilizing the principle that organic reagents are similarly compatible with water, the organic reagent is used to dissolve the honey sample and make it in a solution state, and the water can be freed from the encapsulation of the sugar molecules in a non-viscous state.

[0032] In one embodiment of the present invention, in step 1), the organic reagent is subjected to anhydrous treatment. Preferably, in step 1), the organic reagent is subjected to anhydrous treatment using a molecular sieve.

[0033] In one embodiment of the present invention, in step 1), the mass ratio of the honey sample to be tested to the organic reagent is 1:0.6-2. Preferably, in step 1), the mass ratio of the honey sample to be tested to the organic reagent is 1:1.

[0034] In one embodiment of the present invention, in step 2), the bottle is a head space injection bottle. The head space injection bottle is a head space injection bottle of standard specifications, or a customized non-standard specification sample bottle, which is sealed with a clamp cap or a threaded plug.

[0035] In one embodiment of the present invention, in step 2), the replacement time is 1 to 20 minutes. Preferably, the replacement time is 10 minutes.

[0036] In one embodiment of the present invention, in step 2), the reaction temperature under constant temperature conditions is 10-100° C. Preferably, in step 2), the reaction temperature under constant temperature conditions is 25° C.

[0037] In one embodiment of the present invention, in step 2), the reaction time under constant temperature conditions is 4 to 48 hours.

[0038] In one embodiment of the present invention, in step 3), the standard calibration specifically includes: selecting standard water (stable oxygen isotope ratio is known), adding an organic reagent to the 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.

[0039] In one embodiment of the present invention, the honey includes linden honey, locust tree honey, jujube honey or multi-flower honey.

[0040] Furthermore, the determination method can also be used for oxygen isotope analysis in water of low-water-content foods, wherein low-water-content foods can also include concentrated fruit and vegetable juices and the like.

[0041] The present invention will be described in detail below with reference to embodiments.

[0042] Example 1 Determination of Stable Oxygen Isotope Ratios of Water in Linden Honey

[0043] a) Add 10 g of activated Molecular sieves to obtain anhydrous ethanol;

[0044] b) 3 g of honey was placed in a 10 mL centrifuge tube, 3 g of ethanol was added thereto, and the tube was shaken repeatedly for 10 min;

[0045] c) Transfer the upper layer of the solution obtained in b) to a 20 mL headspace injection bottle after it has been allowed to stand, cover it with a gasket and an aluminum gasket, clamp it tightly with a capping tool, and insert a gas guide needle;

[0046] d) Fill the headspace injection bottle with the lid tightly clamped in c) with high-purity carbon dioxide, exhaust the air in the bottle, and pull out the gas guide needle. The whole process lasts for 10 minutes;

[0047] e) Place the headspace injection bottle filled with high-purity carbon dioxide in d) in a constant temperature oscillating water tank, set the temperature of the water tank to 25°C, and allow the carbon dioxide and water to react for more than 8 hours under oscillating conditions;

[0048] f) When the test is about to begin, take out the headspace injection bottle that has completed the isotope exchange reaction in e), use a gas-tight injection needle to draw 2uL of gas from the headspace part of the bottle, and inject it into the GC-IRMS system to determine the stable oxygen isotope ratio of carbon dioxide (number 1#). The results are shown in Table 1;

[0049] g) For the linden honey sample, steps b) to f) were repeated three times (numbered 2# to 4#). The results are shown in Table 1.

[0050] Table 1 δ of carbon dioxide after isotope exchange of water in linden honey 18 O value (‰)

[0051] Sample No. 1# 2# 3# 4# <![CDATA[δ 18 The]]> -3.42 -3.46 -3.63 -3.57

[0052] It can be seen from the above table that the average value of the 4 measurement results is -3.52‰, the standard deviation is 0.086‰, and the standard deviation of every two adjacent samples is less than 0.30‰.

[0053] h) Select 3 known δ 18The water working standard with a value of 0 was used to establish the standard curve; the water content of the known honey was less than 24%, and the first grade was less than 20%. When 20% was used as the benchmark, the water content of the solution after the sample was treated in step b) was about 17%. According to the PIT principle of equal treatment for 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 anhydrous ethanol obtained in step a), three known δ 18 O value of water, prepared into an ethanol-water solution with a water content of about 17%, and the δ 18 O value, the results are shown in Table 2.

[0054] Table 2 δ of carbon dioxide after isotope exchange of water working standard 18 O value (‰)

[0055] Sample No. Water standard sample 1# Water Standard Sample 2# Water standard sample 3# <![CDATA[δ 18 O known value]]> -0.73 -6.42 -18.61 <![CDATA[δ 18 O measurement value in CO2]]> -5.44 -11.48 -23.30

[0056] Thus, a standard curve of the method can be established, and the average value of the determination results obtained in step f) and step g) is substituted into the standard curve equation to calculate that the stable oxygen isotope ratio of water in the linden honey sample is 1.35‰.

[0057] Example 2 Determination of Stable Oxygen Isotope Ratios of Water in Manuka Honey

[0058] a) Add 10 g of activated Molecular sieves to obtain anhydrous ethanol;

[0059] b) 3 g of Manuka honey was placed in a 10 mL centrifuge tube, 3 g of ethanol was added thereto, and the tube was repeatedly shaken for 10 min;

[0060] c) Transfer the upper layer of the solution obtained in a) to a 20 mL headspace injection bottle after it has been allowed to stand, cover it with a gasket and an aluminum gasket, clamp it tightly with a capping clamp, and insert a gas guide needle;

[0061] d) Fill the headspace injection bottle with the lid tightly clamped in b) with high-purity carbon dioxide, exhaust the air in the bottle and then pull out the gas guide needle. The whole process should last for more than 10 minutes;

[0062] e) Place the headspace injection bottle filled with high-purity carbon dioxide in c) in a constant temperature oscillating water tank, set the temperature of the water tank to 25°C, and allow the carbon dioxide and water to react for more than 8 hours under oscillating conditions;

[0063] f) When the test is about to begin, take out the headspace injection bottle that has completed the isotope exchange reaction in d), use a gas-tight injection needle to draw 2uL of gas from the headspace part of the bottle, and inject it into the GC-IRMS system to determine the stable oxygen isotope ratio (number 11#). The results are shown in Table 3;

[0064] g) For the linden honey sample, steps b) to f) were repeated three times (numbered 12# to 14#). The results are shown in Table 3.

[0065] Table 3 δ of carbon dioxide after isotope exchange in Manuka honey 18 O value (‰)

[0066] Sample No. 11# 12# 13# 14# <![CDATA[δ 18 The]]> -1.25 -1.47 -1.53 -1.39

[0067] As can be seen from the table above, the average value of the four measurement results is -1.41‰, the standard deviation is 0.121‰, and the standard deviation of each adjacent sample is less than 0.30‰. According to the standard curve obtained in Table 2 of Example 1, it can be seen that the δ 18 The O value is 3.47‰.

[0068] Example 3 Determination of Stable Oxygen Isotope Ratios of Water in Honey from Various Flowers

[0069] a) Add 10 g of activated Molecular sieves to obtain anhydrous ethanol;

[0070] b) Take 2g of honey and place it in a 10mL centrifuge tube, add 3g of ethanol and shake repeatedly for 10min;

[0071] c) Transfer the upper layer of the solution obtained in a) to a 20 mL headspace injection bottle after it has been allowed to stand, cover it with a gasket and an aluminum gasket, clamp it tightly with a capping clamp, and insert a gas guide needle;

[0072] d) Fill the headspace injection bottle with the lid tightly clamped in b) with high-purity carbon dioxide, exhaust the air in the bottle and then remove the gas guide needle. The whole process lasts for 10 minutes;

[0073] e) Place the headspace injection bottle filled with high-purity carbon dioxide in c) in a constant temperature oscillating water tank, set the temperature of the water tank to 25°C, and allow the carbon dioxide and water to react for more than 8 hours under oscillating conditions;

[0074] f) When the test is about to begin, take out the headspace injection bottle that has completed the isotope exchange reaction in d), use a gas-tight injection needle to draw 2uL of gas from the headspace part of the bottle, and inject it into the GC-IRMS system to determine the stable oxygen isotope ratio (No. 21#). The results are shown in Table 1;

[0075] g) For the multi-flower honey sample, steps b) to f) were repeated three times (numbered 22# to 24#). The results are shown in Table 4.

[0076] Table 4 δ of carbon dioxide after isotope exchange in honey of various flowers 18 O value (‰)

[0077] Sample No. 21# 22# 23# 24# <![CDATA[δ 18 The]]> -4.65 -4.82 -5.11 -4.97

[0078] As can be seen from the table above, the average value of the four determination results is -4.89‰, the standard deviation is 0.19‰, and the standard deviation of each adjacent sample is less than 0.30‰, which meets the repeatability requirements of water oxygen isotope determination. According to the standard curve obtained in Table 2 of Example 1, it can be seen that the δ 18 The O value is -0.02‰.

[0079] Verification Example 1 Determination of Stable Oxygen Isotope Ratios of Water in Different Ethanol-Water Solutions

[0080] The existing determination of the ratio of stable oxygen isotopes in water is based on water, fruit juice or low-alcohol beverages (such as wine, beer, with an alcohol content of less than 14% vol), while the embodiment of the present invention uses a large amount of organic reagents to dissolve and extract water. In order to verify the accuracy of the method, 3.5 g of each solution with an ethanol mass ratio of 0%, 30%, 50%, 70%, 80% and 90% was prepared with pure water, and then processed and measured according to steps c) to f) in Example 1. The measurement results are shown in Table 5.

[0081] Table 5 δ in water under different ethanol mass ratio conditions 18 O determination results

[0082] Ethanol ratio (%) 0 30 50 70 80 90 <![CDATA[δ 18 The(‰)]]> -11.98 -12.04 -12.15 -11.81 -11.86 -12.21

[0083] It can be seen from the above table that, relative to pure water samples, the differences between samples containing ethanol and pure water samples are less than 0.3‰. Therefore, when the mass ratio of ethanol is between 0% and 90% (water content of 10% to 100%), it does not affect the determination of the oxygen isotope ratio of water in the solution.

[0084] 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 determining the stable oxygen isotope ratio of water in honey, characterized in that: The steps include: 1) adding an organic reagent to a honey sample to be tested, mixing and standing, taking an upper layer of liquid to obtain a solution to be tested; wherein the mass ratio of the honey sample to be tested to the organic reagent is 1:1; wherein the organic reagent is ethanol; 2) placing the test solution 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; wherein the reaction temperature under constant temperature conditions is 10-100° C.; 3) 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), the organic reagent is subjected to anhydrous treatment.

3. The method according to claim 2, characterized in that In step 1), the organic reagent is anhydrous by molecular sieve.

4. The method according to claim 1, characterized in that: In step 2), the replacement time is 1 to 20 minutes.

5. The method according to claim 4, characterized in that In step 2), the replacement time is 10 min.

6. The method according to claim 1, characterized in that In step 2), the reaction temperature is 25° C. under constant temperature conditions.

7. The method according to claim 1, characterized in that In step 2), the reaction time is 4 to 48 h under constant temperature conditions.

8. The method according to claim 1, characterized in that The honey includes linden honey, locust tree honey, jujube honey or multi-flower honey.