Extraction method and detection method of volatile substances in honey

Abstract

The invention belongs to the technical field of extraction and separation, and particularly relates to an extraction method and a detection method of volatile substances in honey. The extraction method comprises the following steps: mixing honey and a buffer solution to obtain a honey pretreatment solution; the honey pretreatment liquid is subjected to ultrasonic extraction by sequentially adopting a polar gradient deep eutectic solvent aqueous two-phase system (such as a deep eutectic solvent which is sequentially mixed with tert-butyl alcohol, isobutanol, n-butyl alcohol, n-propyl alcohol and isopropyl alcohol), and an obtained alcohol upper phase contains volatile substances. According to the method, the characteristics of active ingredients in the honey are combined, a polar gradient deep-eutectic solvent aqueous two-phase extraction system is constructed by utilizing the solvent polarity and similar dissolvability principle, simultaneous separation and enrichment of multiple types of active ingredients are realized, and the method is an efficient and green honey sample pretreatment method. According to the invention, a multi-stage gradient extraction technology is adopted, so that excellent extraction effect and sensitivity are shown; the invention provides a new solution for green extraction of natural products.

Description

Technical Field

[0001] This invention belongs to the field of extraction and separation technology, specifically relating to a method for extracting and detecting volatile substances in honey. Background Technology

[0002] Metabolomics enables in-depth molecular-level analysis of food components and biological functions. This systematic approach not only deepens our understanding of bioactive components in food but also significantly improves food quality control and traceability capabilities. Specifically, combining metabolomics with gas chromatography-mass spectrometry (GC-MS) provides a reliable framework for comprehensive analysis of the volatile metabolite profile of honey. This methodology lays the foundation for in-depth exploration of the aroma characteristics of honey. For example: Gao et al. (Gao YF, Zhou ZX, Wang MJ, Zhang YH. Volatile markers for unifloral safflowerhoney: An untargeted and targeted metabolomics analysis). Food Chem 471, 142734 (2025). Using a combination of non-targeted and targeted GC-MS metabolomics, three volatile biomarkers of safflower honey were identified; further GC-MS metabolomics studies revealed that hydroxy fatty acids can serve as novel biomarkers for determining the insect origin of honey.

[0003] However, the background matrices of foods such as honey are complex. Before conducting targeted or non-targeted metabolomics analysis using large instruments, a series of cumbersome pretreatment steps are required to separate interfering substances from the background matrix. Traditional separation methods, such as organic phase extraction (cyclohexane, diethyl ether, acetonitrile, and petroleum ether) and solid phase extraction, while achieving the separation of target components to some extent, suffer from problems such as cumbersome operation, high cost, and environmental pollution, limiting their widespread application. In contrast, aqueous two-phase extraction (ABS), as an emerging separation technology, has advantages such as mild extraction conditions, small overall volume, and high extraction rate, and has shown promising application prospects in the field of food science.

[0004] The quality characteristics and bioactivity of honey depend on the synergistic effects of multiple active ingredients. Therefore, the types and quantities of compounds extracted and separated directly affect the accuracy of food metabolomics research methods. Due to the complex background matrix of honey, the large number of bioactive ingredients in low concentrations, and the potential presence of unknown interfering substances, the application of current aqueous two-phase extraction (ABS) technology to the simultaneous extraction of multiple types of compounds from honey has not yielded ideal results and cannot achieve the separation and enrichment of a large number of bioactive ingredients in honey. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a method for extracting and detecting volatile substances in honey. The extraction method of this invention can fully extract volatile substances from honey, and yields a wide variety of volatile substances.

[0006] This invention provides a method for extracting volatile substances from honey, comprising the following steps: The honey and buffer solution were mixed to obtain a honey pretreatment solution; The honey pretreatment solution, eutectic solvent, and first alcohol are mixed and subjected to first ultrasonic extraction, followed by liquid-liquid separation to obtain the first alcohol upper phase and the first remaining eutectic solvent lower phase; the relative polarity of the first alcohol is 0.007~0.488; The lower phase of the first remaining eutectic solvent and the second alcohol are mixed and subjected to a second ultrasonic extraction, followed by liquid-liquid separation to obtain the upper phase of the second alcohol and the lower phase of the second remaining eutectic solvent; the relative polarity of the second alcohol is 0.489~0.568; The second remaining eutectic solvent lower phase and the third alcohol are mixed and subjected to a third ultrasonic extraction, followed by liquid-liquid separation to obtain the third alcohol upper phase and the third remaining eutectic solvent lower phase; the relative polarity of the third alcohol is 0.569~0.586; The lower phase of the third residual eutectic solvent and the fourth alcohol are mixed and subjected to fourth ultrasonic extraction, followed by liquid-liquid separation to obtain the upper phase of the fourth alcohol and the lower phase of the fourth residual eutectic solvent; the relative polarity of the fourth alcohol is 0.587~0.617; The lower phase of the fourth residual eutectic solvent and the fifth alcohol are mixed and subjected to fifth ultrasonic extraction, followed by liquid-liquid separation to obtain the upper phase of the fifth alcohol and the lower phase of the fifth residual eutectic solvent; the relative polarity of the fifth alcohol is 0.618~0.654; The first alcohol upper phase, the second alcohol upper phase, the third alcohol upper phase, the fourth alcohol upper phase, and the fifth alcohol upper phase contain volatile substances.

[0007] Preferably, the first alcohol is tert-butanol, the second alcohol is isobutanol, the third alcohol is n-butanol, the fourth alcohol is n-propanol, and the fifth alcohol is isopropanol.

[0008] Preferably, the eutectic solvent comprises a hydrogen bond acceptor and a hydrogen bond donor, wherein the hydrogen bond acceptor is betaine and the hydrogen bond donor is sucrose; the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1~2:1.

[0009] Preferably, the mass fraction of the first alcohol in the system after mixing the honey pretreatment solution, the eutectic solvent, and the first alcohol is 30-40%; the mass fraction of the small molecule alcohol in the system after mixing the remaining eutectic solvent lower phase and the small molecule alcohol is 30-40%, wherein the remaining eutectic solvent lower phase is a first remaining eutectic solvent lower phase, a second remaining eutectic solvent lower phase, a third remaining eutectic solvent lower phase, or a fourth remaining eutectic solvent lower phase, and the small molecule alcohol is a second alcohol, a third alcohol, a fourth alcohol, or a fifth alcohol.

[0010] Preferably, the mass fraction of the eutectic solvent in the system after mixing the honey pretreatment solution, the eutectic solvent and the first alcohol is 10-30%; the mass fraction of the eutectic solvent in the system after mixing the remaining eutectic solvent lower phase and the small molecule alcohol is 10-30%.

[0011] Preferably, the concentration of honey in the honey pretreatment solution is 0.2~0.4 g / mL.

[0012] Preferably, the temperature for the first, second, third, fourth, and fifth ultrasonic extractions is independently 40~50℃, and the time is independently 10~30min.

[0013] Preferably, the volatile substances include one or more of hydrocarbons, terpenes, esters, ethers, alcohols, aldehydes, ketones, phenols, acids, and amines.

[0014] This invention also provides a method for detecting volatile substances in honey, comprising the following steps: The extraction method described above yields an extract of volatile substances; the extract of volatile substances comprises five alcohol upper phases, namely, a first alcohol upper phase, a second alcohol upper phase, a third alcohol upper phase, a fourth alcohol upper phase, and a fifth alcohol upper phase; The five alcohol upper phases were diluted with ethyl acetate and then analyzed by gas chromatography-mass spectrometry to obtain the types or contents of volatile substances.

[0015] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a method for extracting volatile substances from honey. The method employs a polar gradient eutectic solvent aqueous two-phase system (eutectic solvent, sequentially mixed with a first alcohol with a relative polarity of 0.007~0.488, a second alcohol with a relative polarity of 0.489~0.568, a third alcohol with a relative polarity of 0.569~0.586, a fourth alcohol with a relative polarity of 0.587~0.617, and a fifth alcohol with a relative polarity of 0.618~0.654) to extract volatile substances from honey. The resulting alcohol phase contains volatile substances.

[0016] This invention, combining the characteristics of active ingredients in honey, utilizes solvent polarity and the principle of "like dissolves like" to construct a polar gradient eutectic solvent aqueous two-phase extraction system. This system achieves the simultaneous separation and enrichment of multiple types of active ingredients, providing a highly efficient and environmentally friendly honey sample pretreatment method. The multi-stage gradient extraction technology employed in this invention exhibits excellent extraction efficiency and sensitivity. Test results show that, compared with traditional single-polar liquid-liquid extraction, multi-stage polar gradient extraction technology has better extraction efficiency and higher sensitivity, yielding more characteristic components.

[0017] This invention uses a natural eutectic solvent (NADES) combined with small molecule alcohols for extraction, which has good designability, biodegradability and environmental friendliness, providing a new solution for the green extraction of natural products. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 This is a graph showing the binode curves formed by different NADES and n-propanol in Example 1; Figure 2 A graph showing the number of compounds extracted from the monopolar aqueous two-phase system of Comparative Example 1 and the polar gradient natural eutectic solvent aqueous two-phase system of Example 2; Figure 3 The results of polar gradient extraction of the various alcohol phases are shown in the figure. Figure 4 The graph shows the effect of different hydrogen bond donors (HBDs) on the effective peak area and total number of compounds in volatile substances. Figure 5 A graph showing the effect of the hydrogen bond donor (HBD) / hydrogen bond acceptor (HBA) molar ratio on the effective peak area and total number of compounds in volatile substances; Figure 6 This is a graph showing the effect of alcohol concentration on the effective peak area of ​​volatile substances and the total number of compounds. Figure 7 The graph shows the effect of natural eutectic solvent concentration on the effective peak area and total number of compounds of volatile substances. Figure 8 The graph shows the effect of extraction time on the effective peak area of ​​volatile substances and the total number of compounds. Figure 9 The graph shows the effect of extraction temperature on the effective peak area of ​​volatile substances and the total number of compounds. Figure 10The graph shows the effect of honey concentration on the effective peak area of ​​volatile substances and the total number of compounds. Figure 11 The graph shows the effect of pH value of honey pretreatment solution on the effective peak area of ​​volatile substances and the total number of compounds. Figure 12 A diagram showing the overlap of identified compounds in China's four major honey varieties; Figure 13 This is a map showing the relative abundance distribution of China's four major honey varieties. Figure 14 A graph showing the number of compounds detected in China's four major honey varieties; Figure 15 This is a diagram showing the electrostatic interaction energy between the characteristic compound and the solvent; Figure 16 The diagram shows the van der Waals interactions between the characteristic compounds and betaine and sucrose. Figure 17 This is a graph showing the greenness evaluation results of the extraction and detection methods of the present invention. Detailed Implementation

[0020] This invention provides a method for extracting volatile substances from honey, comprising the following steps: The honey and buffer solution were mixed to obtain a honey pretreatment solution; The honey pretreatment solution, eutectic solvent, and first alcohol are mixed and subjected to first ultrasonic extraction, followed by liquid-liquid separation to obtain the first alcohol upper phase and the first remaining eutectic solvent lower phase; the relative polarity of the first alcohol is 0.007~0.488; The lower phase of the first remaining eutectic solvent and the second alcohol are mixed and subjected to a second ultrasonic extraction, followed by liquid-liquid separation to obtain the upper phase of the second alcohol and the lower phase of the second remaining eutectic solvent; the relative polarity of the second alcohol is 0.489~0.568; The second remaining eutectic solvent lower phase and the third alcohol are mixed and subjected to a third ultrasonic extraction, followed by liquid-liquid separation to obtain the third alcohol upper phase and the third remaining eutectic solvent lower phase; the relative polarity of the third alcohol is 0.569~0.586; The lower phase of the third residual eutectic solvent and the fourth alcohol are mixed and subjected to fourth ultrasonic extraction, followed by liquid-liquid separation to obtain the upper phase of the fourth alcohol and the lower phase of the fourth residual eutectic solvent; the relative polarity of the fourth alcohol is 0.587~0.617; The lower phase of the fourth residual eutectic solvent and the fifth alcohol are mixed and subjected to fifth ultrasonic extraction, followed by liquid-liquid separation to obtain the upper phase of the fifth alcohol and the lower phase of the fifth residual eutectic solvent; the relative polarity of the fifth alcohol is 0.618~0.654; The first alcohol upper phase, the second alcohol upper phase, the third alcohol upper phase, the fourth alcohol upper phase, and the fifth alcohol upper phase contain volatile substances.

[0021] Unless otherwise specified, all materials and equipment used in this invention are commercially available products in the field.

[0022] In this invention, the volatile substances include one or more of hydrocarbons, terpenes, esters, ethers, alcohols, aldehydes, ketones, phenols, acids, and amines. Specifically, they may include 2,6,10,14-tetramethylhexadecane, 4-methylphenol, mesitylene, 1,3-divinylbenzene, furfural, dodecane, pentadecanoic acid, n-butyl ether, 4-carboxy-3-hexanone, or ethyl butyrate.

[0023] This invention mixes honey and a buffer solution to obtain a honey pretreated solution.

[0024] In this invention, the concentration of honey in the honey pretreatment solution is preferably 0.2~0.4 g / mL, specifically 0.29 g / mL or 0.3 g / mL, that is, the volume ratio of honey to buffer solution is preferably 0.2~0.4 g:1 mL, and the pH value of the buffer solution can be 8.

[0025] In this invention, the pH value of the honey pretreatment solution is preferably 7-8, specifically 7.67 or 8.

[0026] In this invention, the first alcohol is preferably tert-butanol, the second alcohol is preferably isobutanol, the third alcohol is preferably n-butanol, the fourth alcohol is preferably n-propanol, and the fifth alcohol is preferably isopropanol.

[0027] In this invention, the relative polarity is relative to the polarity of water (water is 1).

[0028] After obtaining the honey pretreatment solution, the present invention mixes the honey pretreatment solution, the eutectic solvent and tert-butanol and performs a first ultrasonic extraction, followed by liquid-liquid separation to obtain a first alcohol upper phase and a first remaining eutectic solvent lower phase.

[0029] In this invention, the eutectic solvent preferably includes a hydrogen bond acceptor and a hydrogen bond donor, wherein the hydrogen bond acceptor is preferably betaine, the hydrogen bond donor is preferably sucrose, and the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is preferably 1 to 2:1.

[0030] In this invention, the mass fraction of the first alcohol in the system after mixing the honey pretreatment solution, the eutectic solvent, and the first alcohol is preferably 30-40%, specifically 32% or 38%. The mass fraction of the eutectic solvent in the system after mixing the honey pretreatment solution, the eutectic solvent, and the first alcohol is preferably 10-30%, specifically 20% or 24%. In a specific embodiment of this invention, the ratio of the amount of honey pretreatment solution, the eutectic solvent, and the first alcohol (tert-butanol) can be 0.8 mL:0.5 g:1 mL.

[0031] In this invention, the temperature of the first ultrasonic extraction is preferably 40-50°C, specifically 45°C or 46.68°C, and the time is preferably 10-30 min, specifically 20 min. The liquid-liquid separation preferably includes standing and centrifugation, the standing time is preferably 30 min, the centrifugation speed is preferably 10000 rpm, and the time is preferably 5 min. After liquid-liquid separation, tert-butanol is the upper phase, and water and the eutectic solvent are the lower phase.

[0032] After obtaining the first remaining eutectic solvent lower phase, the present invention mixes the first remaining eutectic solvent lower phase and the second alcohol for a second ultrasonic extraction, followed by liquid-liquid separation to obtain the second alcohol upper phase and the second remaining eutectic solvent lower phase; the relative polarity of the second alcohol is 0.489~0.568; The second remaining eutectic solvent lower phase and the third alcohol are mixed and subjected to a third ultrasonic extraction, followed by liquid-liquid separation to obtain the third alcohol upper phase and the third remaining eutectic solvent lower phase; the relative polarity of the third alcohol is 0.569~0.586; The lower phase of the third residual eutectic solvent and the fourth alcohol are mixed and subjected to fourth ultrasonic extraction, followed by liquid-liquid separation to obtain the upper phase of the fourth alcohol and the lower phase of the fourth residual eutectic solvent; the relative polarity of the fourth alcohol is 0.587~0.617; The lower phase of the fourth residual eutectic solvent and the fifth alcohol are mixed and subjected to fifth ultrasonic extraction, followed by liquid-liquid separation to obtain the upper phase of the fifth alcohol and the lower phase of the fifth residual eutectic solvent; the relative polarity of the fifth alcohol is 0.618~0.654.

[0033] In this invention, the mass fraction of the small molecule alcohol in the system after mixing the remaining eutectic solvent lower phase and the small molecule alcohol is preferably 30-40%, specifically 32% or 38%. The remaining eutectic solvent lower phase is a first, second, third, or fourth remaining eutectic solvent lower phase, and the small molecule alcohol is a second, third, fourth, or fifth alcohol. The mass fraction of the eutectic solvent in the system after mixing the remaining eutectic solvent lower phase and the small molecule alcohol is preferably 10-30%, specifically 20% or 24%. In specific embodiments of this invention, the preferred ratio of the eutectic solvent to the second alcohol is 0.5 g:1 mL; the preferred ratio of the eutectic solvent to the third alcohol is 0.5 g:1 mL; the preferred ratio of the eutectic solvent to the fourth alcohol is 0.5 g:1 mL; and the preferred ratio of the eutectic solvent to the fifth alcohol is 0.5 g:1 mL.

[0034] In this invention, the conditions for the second, third, fourth, and fifth ultrasonic extractions are preferably the same as those for the first ultrasonic extraction, and will not be repeated here.

[0035] This invention also provides a method for detecting volatile substances in honey, comprising the following steps: The extraction method described above yields an extract of volatile substances; the extract of volatile substances comprises five alcohol upper phases, namely, a first alcohol upper phase, a second alcohol upper phase, a third alcohol upper phase, a fourth alcohol upper phase, and a fifth alcohol upper phase; The five alcohol upper phases were diluted with ethyl acetate and then analyzed by gas chromatography-mass spectrometry to obtain the types or contents of volatile substances.

[0036] In this invention, the chromatographic conditions for gas chromatography-mass spectrometry detection include: Capillary gas chromatography column: SH-Rxi-5MS, specifications: 30 m × 0.25 mm, 0.25 μm; high-purity helium was used as the carrier gas, with a chromatographic flow rate of 1.5 mL / min and a purge flow rate of 3.0 mL / min; splitless mode was used; the injection port temperature was 300℃, the injection time was 1 min, and the injection volume was 0.5 µL; the temperature program was: initial temperature held at 50℃ for 0 min, then increased to 230℃ at a rate of 3℃ / min. The purity of the high-purity helium was 99.999%.

[0037] Mass spectrometry conditions: ion source temperature 245℃; interface temperature 280℃; ionization voltage 70 eV; full scan mode; scan range 33~550 amu; solvent delay time 1 min.

[0038] To further illustrate the present invention, the extraction and detection methods for volatile substances in honey provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0039] Example 1: Construction and optimization of a natural eutectic solvent aqueous two-phase system (NADES-ABS) Sucrose (Suc), glucose (Glu), xylose (Xyl), maltose (Mal), and fructose (Fru) are mixed with betaine in a 1:1 molar ratio to form NADES.

[0040] To evaluate the phase formation behavior of aqueous two-phase systems (ABS) composed of natural eutectic solvents (NADES) and small molecule alcohols (such as n-propanol), phase diagrams were constructed using turbidity point titration. Measured binode line data for the five alcohol systems were fitted to the Merchuk equation (Equation (1)); subsequently, the junction length (TLL) of each system was calculated using Equation (2) according to the gravimetric method reported by Passos et al.

[0041] (1); (2); In the formula, , The mass fractions (wt%) of DES in the upper and lower phases are respectively. , , respectively, represent the mass fraction (wt%) of the small molecule alcohol in the upper and lower phases; a, b, and c represent fitting constants.

[0042] Figure 1 The graphs show binode lines formed by different NADES and n-propanol. It can be seen that the aqueous two-phase system formed by sucrose NADES and alcohol has better phase-forming ability (the closer the curve is to the origin, the better the phase-forming ability).

[0043] Example 2: Extraction Experiment of Polar Gradient Natural Eutectic Solvent Aqueous Two-Phase System (NADES-ABS) Five small molecule alcohol / betaine-sucrose (Bet-Suc) systems were used to sort the substances in order of increasing polarity, specifically: tert-butanol / Bet-Suc, isobutanol / Bet-Suc, n-butanol / Bet-Suc, n-propanol / Bet-Suc, and isopropanol / Bet-Suc; the molar ratio of betaine to sucrose in the natural eutectic solvent (NADES) was 1:1.

[0044] The extraction steps are as follows: Accurately weigh 0.5 g of NADES into a 10 mL centrifuge tube, and add 1.0 mL of tert-butanol and 0.8 mL of honey pretreatment solution (acacia honey mixed with a buffer solution at pH 8, honey concentration 0.3 g / mL). Extract the mixture using ultrasound-assisted extraction at 45°C for 20 min (40W), then allow it to stand at a constant temperature (45°C) for 30 min, followed by centrifugation at 10,000 rpm for 5 min. After phase separation, completely collect the alcohol-rich upper phase. Add isobutanol to the remaining NADES phase, maintaining a constant alcohol-to-NADES volume ratio (1.0 mL:0.5 g), and repeat the above aqueous two-phase (ABS) extraction process. This process was applied sequentially to the above five alcohol systems.

[0045] The alcohol phase obtained after each extraction was diluted twice with ethyl acetate, filtered through a 0.22 μm filter membrane, stored in a gas chromatography-mass spectrometry bottle, and then analyzed by GC-MS. The remaining raffinate was collected, freeze-dried, derivatized (see below for specific methods), and filtered before being analyzed by gas chromatography-mass spectrometry (GC-MS). Derivatization includes the following steps: N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) + 1 wt% trimethylchlorosilane (TMCS) was selected as the derivatization reagent. Since TMCS, when used as a catalyst, produces the byproduct HCl, pyridine was used as the acid removal solvent to remove the HCl before introducing it into the GC-MS system. 0.1 mL of the raffinate from the final step of the polar gradient extraction was accurately transferred to a 1.5 mL derivatization vial and freeze-dried to dryness under vacuum. 0.2 mL of a 30 mg / mL hydroxylamine hydrochloride anhydrous pyridine solution was added, and the mixture was heated at 70 °C for 30 min, then cooled to room temperature. 0.6 mL of BSTFA (containing 1 wt% TMCS) was quickly added, and the mixture was heated at 70 °C for 30 min, then cooled to room temperature. The sample was filtered through a 0.22 µm organic syringe filter before GC-MS analysis.

[0046] The conditions for gas chromatography-mass spectrometry include: Five derivatized samples of extracted alcohol phase and sucrose-based NADES phase, after drying (NADES phase freeze-dried, alcohol phase dried with anhydrous sodium sulfate), were analyzed by GC-MS. Separation was performed using an SH-Rxi-5MS (30 m × 0.25 mm, 0.25 μm) capillary gas chromatographic column. Chromatographic conditions: High-purity helium (99.999% purity) was used as the carrier gas, with a flow rate of 1.5 mL / min and a purge flow rate of 3.0 mL / min. Splitless mode was used. The injection port temperature was 300℃, and the injection time was 1 min. The injection volume for both the extracted alcohol phase and the sucrose-based NADES phase derivatized samples was 0.5 µL. The temperature program was set as follows: the initial temperature was held at 50℃ for 0 min, then increased to 230℃ at a rate of 3℃ / min. Mass spectrometry conditions: ion source temperature 245℃; interface temperature 280℃; ionization voltage 70 eV; full scan mode; scan range 33~550 amu; solvent delay time 1 min; program end time 60 min.

[0047] Qualitative analysis employed full-scan mode to acquire total ion current chromatograms and mass spectra of the analytes. Data preprocessing was performed using Shimadzu GC-MS reanalysis software. The mass spectrum fragmentation diagrams of each chromatographic peak were compared with the NIST17 standard library, and substances with a matching score (SI) ≥ 80 were selected as preliminary qualitative criteria. The peak areas of the components obtained from the five extracted alcohol phases were normalized before calculating their relative contents, while the peak areas of the derivatized samples in the raffinate aqueous phase were normalized separately before calculating their relative contents. GC-MS reanalysis software was used to perform peak correction, smoothing, background subtraction, and mass spectrum averaging on the original peak shapes, ultimately exporting TXT matrix data. Excel was then used for data merging and simplification preprocessing.

[0048] Comparative Example 1: Single-polar extraction The difference from Example 2 is that polar gradient extraction was not performed. Five honey pretreatment samples were prepared, and each was extracted using one of five small molecule alcohol / betaine-sucrose (Bet-Suc) systems. Taking tert-butanol as an example, the extraction steps are as follows: Accurately weigh 0.5 g NADES into a 10 mL centrifuge tube, add 1.0 mL tert-butanol and 0.8 mL honey pretreatment solution (acacia honey mixed with a buffer solution with a pH of 8, honey concentration 0.3 g / mL); perform ultrasonic-assisted extraction of the mixture at 45℃ for 20 min (40W), then allow it to stand at a constant temperature (45℃) for 30 min, and then centrifuge at 10000 rpm for 5 min; after phase separation, completely collect the alcohol-rich upper phase.

[0049] The extraction steps for other alcohols are the same.

[0050] After each extraction, 0.5 mL of the alcohol phase was taken, diluted twice with ethyl acetate, filtered through a 0.22 μm organic phase needle filter, and analyzed by gas chromatography-mass spectrometry (GC-MS).

[0051] Test Results Phase behavior and distribution of volatile substances in a polar gradient natural eutectic solvent aqueous two-phase system Figure 2 This is a comparison chart of the extraction effects (number of extracted compounds) of the single-polar aqueous two-phase system (ABS) and the polar gradient natural eutectic solvent aqueous two-phase system (NADES-ABS). Green represents the total number of extracted target compounds in the five alcohol phases of the polar gradient extraction method (multi-stage extraction) and the number of extracted target compounds in this alcohol phase in the polar gradient extraction method. Blue represents the sum of the number of extracted target compounds in the five alcohol phases of the single-polar extraction method (multi-stage extraction) and the number of extracted target compounds in this alcohol phase in the single-polar extraction method. Pink and magenta represent the unique components that were removed from the same component. Polar gradient aqueous two-phase system (ABS) shows a significant advantage over single-phase extraction in the extraction of volatile substances from acacia honey. Figure 2 The polar gradient extraction system identified a significantly larger number of compounds (158 substances in total, 133 after deduplication) than the single-polar method (116 substances, 96 after deduplication), highlighting its higher extraction efficiency and ability to capture rich chemical diversity. This performance stems from the fact that the polar gradient system can mitigate the saturation effect and analyte masking effect present in single-polar extraction. This invention utilizes ordered extraction based on the physicochemical properties of compounds, achieving highly efficient enrichment of a wider range of volatile substances with higher detection sensitivity.

[0052] Figure 3The results of polar gradient extraction of various alcohol phase substances are shown in the figure. The volatile components of acacia honey are mainly esters (41.55%) and hydrocarbons (16.37%), while the proportions of ethers, alcohols, amines, phenols and acids are relatively small.

[0053] Comparative Example 2 The difference from Example 2 is that sucrose is replaced with glucose, xylose, maltose and fructose, respectively.

[0054] Figure 4 The graph shows the effect of different hydrogen bond donors (HBDs) on the effective peak area (total peak area of ​​volatile substances) and the total number of compounds; it can be seen that sucrose-based NADES has the best extraction effect.

[0055] Example 3 1. Based on the total peak area and number of volatile compounds extracted from the polar gradient aqueous two-phase system (ABS), the optimal conditions were initially determined. Since a 1:3 hydrogen bond acceptor / hydrogen bond donor ratio presents synthetic difficulties, this ratio was excluded from further investigation. Only the effects of four feasible ratios (molar ratios of hydrogen bond acceptor and hydrogen bond donor of 1:2, 2:1, 1:1, and 3:1) were examined.

[0056] Figure 5 The graph shows the effect of the hydrogen bond acceptor (HBA) / hydrogen bond donor (HBD) molar ratio on the effective peak area (total peak area of ​​volatile substances) and the total number of compounds. It can be seen that the extraction effect is better at both 1:1 and 2:1 ratios. However, because NADES synthesized at a molar ratio of 1:1 is unstable at room temperature (easily crystallizes) and has a high viscosity, it is inconvenient to use. Therefore, a molar ratio of 2:1 was ultimately chosen to synthesize sucrose-based DES with an alcohol to form a polar gradient aqueous two-phase system for extraction experiments.

[0057] 2. Single-factor experimental design was used to systematically evaluate the following six parameters in sequence: alcohol concentration (16-48%, mass fraction), natural eutectic solvent (NADES) concentration (10-30%, mass fraction), extraction time (10-50 min), temperature (15-55℃), honey concentration (0.15-0.75 g / mL), and pH value (2-10). Optimization of subsequent factors was conducted using the optimal values ​​of the preceding factors. Alcohol concentration is defined as the percentage of alcohol in the total mass of (alcohol + honey pretreatment solution + eutectic solvent), and the concentration of natural eutectic solvent is defined similarly.

[0058] The results are as follows Figures 6-11 As shown, based on single-factor analysis, the optimal conditions are an alcohol concentration of 32 wt%, a NADES concentration of 20 wt%, an extraction time of 20 min, an ultrasonic temperature of 45 ℃, a honey concentrate concentration of 0.3 g / mL, and a pH value of 8.

[0059] This invention systematically optimizes the extraction process of a polar gradient aqueous two-phase system (ABS) using a combination of single-factor sequential experiments and response surface methodology (RSM). The effects of key parameters such as extraction time, temperature, type and concentration of natural eutectic solvent (NADES), alcohol concentration, pH, and honey concentration were comprehensively investigated. Statistical analysis using SPSS Statistics (version 27.0) identified four factors with significant influence: extraction temperature (A), pH (B), alcohol concentration (C), and honey concentration (D) (see Tables 1 and 2). These four factors were selected as independent variables, with total peak area (Y1) and the number of volatile compounds (Y2) as response values. A Box-Behnken design (BBD) model was then constructed to determine the optimal extraction parameters as follows: temperature 46.68℃, pH 7.67, alcohol concentration 32.32 wt%, honey concentrate concentration 0.29 g / mL, ultrasonic time 20 min, and NADES concentration 20 wt%.

[0060] Table 1. Results of ANOVA on the effect of each individual factor on the amount of volatile components.

[0061] Table 2. Analysis of variance (ANOVA) results of the influence of each individual factor on the effective peak area of ​​volatile components.

[0062] Data processing and multivariate statistical analysis All density functional theory (DFT) calculations were performed using Gaussian 16 (Revision C01). Geometric optimization was conducted at the B3LYP-D3 (BJ) / 6-31G (d,p) theoretical level, along with simple harmonic vibration frequency analysis at the same theoretical level to confirm that all optimized structures were local minima (without imaginary frequencies). Electrostatic potential (ESP) plots, dual descriptor and inter-interaction region indicator (IRI) analysis results were calculated and visualized using Multiwfn and VMD software. Molecular dynamics (MD) simulations were performed using the Forcite module of Materials Studio software. The initial structure was first optimized and energy minimized; simulations were conducted under controlled temperature (Andersen thermostat) and controlled pressure (Berendsen barostat) conditions. Non-bonded interactions were described using the COMPASS force field, with electrostatic and van der Waals interactions handled by the Ewald method and atomic-based summation, respectively, and a cutoff distance of 12.5 Å. Hierarchical clustering analysis (HCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) in SIMCA-P software (version 14.0, Umetrics) were used to perform chemical mapping analysis on the volatile components of four honey varieties. Characteristic markers were screened from the OPLS-DA model, and compounds with a variable projection importance (VIP) value greater than 1 were further validated for statistical significance using Duncan's test in SPSS software (p < 0.05). Using OmicStudio (https: / / www.omicstudio.cn / tool), heatmap clustering and Venn diagrams were used to visualize characteristic markers that simultaneously satisfied VIP > 1 and p < 0.05, highlighting the component differences among different honey varieties.

[0063] Example 4 The volatile substances in the four major types of honey in China (acacia honey, jujube honey, lychee honey and vitex honey) were extracted and detected using the optimal conditions selected by screening, and the volatile substance group spectrum of different honey varieties was obtained.

[0064] Figures 12-14 The graph shows the comparative analysis results of volatile substances in four types of honey. Figure 12 The overlap of identified compounds (Ven diagram); Figure 13 The distribution is based on relative abundance. Figure 14 The number of compounds detected.

[0065] This invention employs an integrated method combining polar gradient-type natural eutectic solvent aqueous two-phase system (NADES-ABS) extraction with gas chromatography-mass spectrometry (GC-MS) to characterize the volatile components of four major Chinese honey varieties. A total of 427 volatile compounds were identified, covering hydrocarbons, terpenes, esters, aldehydes, ketones, and acids. Venn diagram analysis was used (…). Figure 12 The data shows that different varieties of honey share some aroma components as well as have unique aroma components.

[0066] Acacia honey has the most unique compounds (147), followed by lychee honey (95), jujube honey (86), and vitex honey (81). Notably, acacia and lychee honey share 32 components, indicating a closer aroma correlation between the two; while acacia, lychee, and jujube honeys contain 9 shared compounds, which may be fundamental aroma characteristic substances of honey. Figure 13 As shown, there are significant differences in the relative abundance of various volatile substances in different varieties of honey. Acacia honey has the highest proportion of alkanes (20.81%), followed by esters (18.64%), consistent with its delicate and refined aroma. Jujube honey exhibits unique compositional characteristics, with abundant esters (23.64%), and alkanes (19.21%) and amines (16.82%) forming its sweet base. Lychee honey is characterized by a high content of amines (15.53%), which may be the core source of its fruity flavor. Vitex honey has a relatively simple composition, mainly composed of alkanes (25.49%) and esters (23.30%), with other compounds accounting for less than 8%, consistent with its mild aroma. The distribution of identified volatile compounds is summarized in [the table below]. Figure 14 Among the honeys tested, jujube honey (97 types), lychee honey (80 types), and vitex honey (92 types) showed the richest variety of alkane compounds. Aldehydes were also widely present, with acacia honey containing 50 types, jujube honey 42 types, lychee honey 46 types, and vitex honey 41 types. Alcohols and amines were also present in high amounts in all four types of honey. In summary, the volatile components of these honeys share common characteristics while also possessing unique attributes specific to each variety, providing a scientific basis for honey quality evaluation and plant origin traceability.

[0067] Mechanism of polar gradient NADES-ABS distribution process Figure 15 This is a diagram showing the electrostatic interaction energy between the characteristic compound and the solvent; Figure 16 This is a van der Waals interaction diagram of the characteristic compounds with betaine and sucrose.

[0068] Based on molecular dynamics (MD) simulations, this invention investigates the molecular interactions between five characteristic volatile compounds and different phases in a gradient-type natural eutectic solvent aqueous two-phase system (NADES-ABS). Figure 15 The electrostatic interaction energies between the target molecules and the solvent system were demonstrated. Except for 1,3,5-trimethylbenzene (trimethylbenzene), all other compounds exhibited more negative electrostatic interaction energies in the alcohol phase, indicating stronger attraction in these media. Notably, the trend of the electrostatic interaction energy is consistent with the solvent accessible surface area (SASA) results, further confirming the preferential affinity of these volatile compounds for alcohol-rich phases. Figure 16 The interaction strengths of characteristic compounds with hydrogen bond acceptors (HBA, betaine) and hydrogen bond donors (HBD, sucrose) in the natural eutectic solvent (NADES) were compared. The results showed that most analytes interacted more strongly with sucrose, indicating that the hydroxyl-rich sugar backbone plays a dominant role in molecular recognition and binding in the NADES system. In summary, these molecular dynamics (MD) simulations provide molecular-level evidence that, in polar gradient NADES-ABS extraction, most characteristic volatile compounds preferentially partition to the alcohol-rich phase, consistent with experimentally observed partitioning behavior and extraction efficiency.

[0069] The combination of efficient sample pretreatment technology and food metabolomics lays an important foundation for in-depth analysis of the quality characteristics and bioactivity of honey, and helps to re-understand the aroma mechanisms of different types of honey. This invention focuses on acacia honey, following the trend of solvent polarity progression, and designs and constructs a polar gradient natural eutectic solvent-aqueous two-phase system (NADES-ABS). Through phase diagram analysis, series length determination, and phase equilibrium mathematical modeling, its phase formation behavior is systematically investigated; and extraction conditions are optimized based on single-factor experiments (investigating DES type, DES concentration, alcohol concentration, gradient setting, pH value, extraction time, and temperature) and response surface methodology (RSM). Furthermore, density functional theory (DFT) and molecular dynamics simulations (MD) are used to deeply analyze the differentiated action mechanism of active molecules in the two-phase system, elucidating the extraction mechanism of gradient NADES-ABS. Based on this, the established polar gradient NADES-ABS was used for honey sample pretreatment. Non-targeted metabolomics analysis based on GC-MS, combined with a chemometrics pattern recognition algorithm, was employed. Through dynamic visualization using heatmaps, and by analyzing the algorithm's scores, loadings, and biplots, the volatile component characteristics of four representative Chinese honeys (acacia honey, jujube honey, lychee honey, and vitex honey) were systematically compared, and key aroma compounds in acacia honey were screened. Finally, the sustainability index of sample pretreatment (SPMS) and the AGREE (Aggregate Approval for Greenness) of analytical methods were used to comprehensively evaluate the greenness of the established extraction technology and analytical methods. Figure 17(See the results for AGREE and AGREEprep), ensuring compliance with green chemistry principles. This invention not only provides new research ideas for constructing novel green extraction systems but also lays the technical foundation for developing efficient separation and green enrichment methods for active ingredients in complex samples.

[0070] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments based on the present invention without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for extracting volatile substances from honey, characterized in that, Includes the following steps: The honey and buffer solution were mixed to obtain a honey pretreatment solution; The honey pretreatment solution, eutectic solvent, and first alcohol are mixed and subjected to first ultrasonic extraction, followed by liquid-liquid separation to obtain the first alcohol upper phase and the first remaining eutectic solvent lower phase; the relative polarity of the first alcohol is 0.007~0.488; The lower phase of the first remaining eutectic solvent and the second alcohol are mixed and subjected to a second ultrasonic extraction, followed by liquid-liquid separation to obtain the upper phase of the second alcohol and the lower phase of the second remaining eutectic solvent; the relative polarity of the second alcohol is 0.489~0.568; The second remaining eutectic solvent lower phase and the third alcohol are mixed and subjected to a third ultrasonic extraction, followed by liquid-liquid separation to obtain the third alcohol upper phase and the third remaining eutectic solvent lower phase; the relative polarity of the third alcohol is 0.569~0.586; The lower phase of the third residual eutectic solvent and the fourth alcohol are mixed and subjected to fourth ultrasonic extraction, followed by liquid-liquid separation to obtain the upper phase of the fourth alcohol and the lower phase of the fourth residual eutectic solvent; the relative polarity of the fourth alcohol is 0.587~0.617; The lower phase of the fourth residual eutectic solvent and the fifth alcohol are mixed and subjected to fifth ultrasonic extraction, followed by liquid-liquid separation to obtain the upper phase of the fifth alcohol and the lower phase of the fifth residual eutectic solvent; the relative polarity of the fifth alcohol is 0.618~0.654; The first alcohol upper phase, the second alcohol upper phase, the third alcohol upper phase, the fourth alcohol upper phase, and the fifth alcohol upper phase contain volatile substances.

2. The extraction method according to claim 1, characterized in that, The first alcohol is tert-butanol, the second alcohol is isobutanol, the third alcohol is n-butanol, the fourth alcohol is n-propanol, and the fifth alcohol is isopropanol.

3. The extraction method according to claim 1, characterized in that, The eutectic solvent includes a hydrogen bond acceptor and a hydrogen bond donor, wherein the hydrogen bond acceptor is betaine and the hydrogen bond donor is sucrose; the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1~2:

1.

4. The extraction method according to claim 1, characterized in that, The mass fraction of the first alcohol in the system after mixing the honey pretreatment solution, the eutectic solvent, and the first alcohol is 30-40%; the mass fraction of the small molecule alcohol in the system after mixing the remaining eutectic solvent lower phase and the small molecule alcohol is 30-40%, wherein the remaining eutectic solvent lower phase is a first remaining eutectic solvent lower phase, a second remaining eutectic solvent lower phase, a third remaining eutectic solvent lower phase, or a fourth remaining eutectic solvent lower phase, and the small molecule alcohol is a second alcohol, a third alcohol, a fourth alcohol, or a fifth alcohol.

5. The extraction method according to claim 4, characterized in that, The mass fraction of the eutectic solvent in the system after mixing the honey pretreatment solution, the eutectic solvent, and the first alcohol is 10-30%; the mass fraction of the eutectic solvent in the system after mixing the remaining eutectic solvent lower phase and the small molecule alcohol is 10-30%.

6. The extraction method according to claim 1, characterized in that, The concentration of honey in the honey pretreatment solution is 0.2~0.4 g / mL.

7. The extraction method according to claim 1 or 6, characterized in that, The pH value of the honey pretreatment solution is 7-8.

8. The extraction method according to claim 1, characterized in that, The temperature for the first, second, third, fourth, and fifth ultrasonic extractions is independently 40~50℃, and the time is independently 10~30min.

9. The extraction method according to claim 1, characterized in that, The volatile substances include one or more of the following: hydrocarbons, terpenes, esters, ethers, alcohols, aldehydes, ketones, phenols, acids, and amines.

10. A method for detecting volatile substances in honey, characterized in that, Includes the following steps: An extract of volatile substances is obtained by the extraction method according to any one of claims 1 to 9; the extract of volatile substances comprises five alcohol upper phases, namely a first alcohol upper phase, a second alcohol upper phase, a third alcohol upper phase, a fourth alcohol upper phase, and a fifth alcohol upper phase; The five alcohol upper phases were diluted with ethyl acetate and then analyzed by gas chromatography-mass spectrometry to obtain the types or contents of volatile substances.

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