Method for detecting and analyzing active substances in waxberry soaked wine

Abstract

The invention discloses a method for detecting and analyzing active substances in waxberry soaked wine. The method comprises the following steps: detecting higher alcohol substances by using a gas chromatography method, detecting esters by using a gas chromatography method and detecting polyphenols by using a liquid chromatography-tandem mass spectrometry method. The higher alcohol substance detection comprises the following steps: (1) taking an ethanol solution as a solvent and 4-methyl-2-amyl alcohol as an internal standard substance, and respectively preparing six higher alcohol series standard solutions with the same concentration of the internal standard substance, namely n-butyl alcohol, sec-butyl alcohol, isobutyl alcohol, n-propyl alcohol, active amyl alcohol and isoamyl alcohol; (2) taking 4-methyl-2-pentanol as an internal standard substance, and taking a to-be-detected wine sample to prepare a test solution; and (3) respectively detecting the six higher alcohol series standard solutions and the test solution by adopting a gas chromatographic method, and calculating the content of each alcohol in the wine sample to be detected. The method provided by the invention can provide a basis for index control of the waxberry wine and ensure the flavor and quality of the product.

Description

Technical Field

[0001] The present invention relates to the technical field of food detection, and more particularly to a method for detecting and analyzing active substances in bayberry-soaked wine. Background Art

[0002] Currently, there is no research on the active substances in bayberry wine or the characteristic substances in bayberry wines of different ages and types. The only research available is on optimizing the production process of bayberry wine, studying myricetin and other active substances in bayberry fruit, and analyzing the aromatic odor of bayberry. Therefore, it is necessary to establish a method for analyzing active substances in bayberry wine to gain a deeper understanding of the factors affecting the flavor and quality of bayberry wine, so as to improve the quality control level of bayberry wine. Summary of the Invention

[0003] The purpose of the present invention is to overcome the deficiencies of the above-mentioned prior art and provide a method for detecting and analyzing active substances in bayberry wine, so as to provide a basis for the index control of bayberry wine and ensure the flavor and quality of the product.

[0004] In order to achieve the above object, the present invention adopts the following technical solutions:

[0005] A method for detecting and analyzing active substances in bayberry-infused wine, comprising gas chromatography detection of higher alcohols, gas chromatography detection of esters, and liquid chromatography-tandem mass spectrometry detection of polyphenols;

[0006] The method for detecting higher alcohols comprises the following steps:

[0007] (1) Preparation of a series of standard solutions of higher alcohols: using ethanol solution as solvent and 4-methyl-2-pentanol as internal standard, prepare a series of n-butanol standard solutions, a series of sec-butanol standard solutions, a series of isobutanol standard solutions, a series of n-propanol standard solutions, a series of active amyl alcohol standard solutions, and a series of isoamyl alcohol standard solutions with the same internal standard concentration;

[0008] (2) Preparation of test solution: Using 4-methyl-2-pentanol as the internal standard, prepare a test solution with the same internal standard concentration as that of the standard solution in step (1) by taking the wine sample to be tested;

[0009] (3) Detection: Gas chromatography was used to detect the six higher alcohol series standard solutions and the test sample solutions, and the contents of n-propanol, sec-butanol, isobutanol, n-butanol, active amyl alcohol, and isoamyl alcohol in the test wine samples were calculated.

[0010] Furthermore, in step (3), the gas chromatography detection conditions are:

[0011] Chromatographic column: HP-INNOWax, 60m×0.25mm×0.25μm; carrier gas: high-purity nitrogen, 0.80 mL / min; split ratio: 2:1; makeup flow: 20mL / min to 30mL / min; hydrogen flow rate: maintained at 32mL / min; air flow rate: maintained at 400mL / min; detector temperature: 250°C; injection port temperature: 220°C; injection volume: 1μL;

[0012] Heating program: start at 40 °C and hold for 5 min, then increase to 75 °C at a rate of 3 °C / min and hold for 2 min, then increase to 80 °C at a rate of 3 °C / min and hold for 10 min.

[0013] Furthermore, in step (1), the internal standard concentrations of the n-butanol series standard solution, the sec-butanol series standard solution, the isobutanol series standard solution, the n-propanol series standard solution, the active amyl alcohol series standard solution, the isoamyl alcohol series standard solution, and the test solution are all 144.07 mg / L.

[0014] Furthermore, in step (1), the concentration gradient of the standard solution of n-propanol series is 34.430, 68.860, 137.72, 206.58, 275.44 mg / L; the concentration gradient of the standard solution of sec-butyl alcohol series is 31.193, 62.385, 124.77, 187.155, 249.54 mg / L; the concentration gradient of the standard solution of isobutyl alcohol series is 24.131, 48.26, 96.525, 144.79, 193.0 5mg / L; the concentration gradient of the n-butanol series standard solution is 23.754, 47.508, 95.016, 142.52, 190.03mg / L; the concentration gradient of the active amyl alcohol series standard solution is 29.703, 59.405, 118.81, 178.22, 237.62mg / L; the concentration gradient of the isoamyl alcohol series standard solution is 28.693, 57.385, 114.77, 172.16, 229.54mg / L.

[0015] Furthermore, the ester substance detection method comprises the following steps:

[0016] (a) Preparation of a mixed internal standard solution: Using ethanol as the solvent, prepare a tert-amyl alcohol-n-amyl acetate mixed internal standard solution containing 2.0 g of tert-amyl alcohol standard and 2.0 g of n-amyl acetate standard per 100 mL;

[0017] (b) Preparation of a standard mixed solution of ester substances: Using ethanol as the solvent, prepare a standard mixed stock solution of ester substances containing 2.5 g of ethyl acetate standard, 2.5 g of ethyl hexanoate standard, 2.5 g of ethyl butyrate standard, and 0.25 g of ethyl lactate standard per 100 mL; take 1 mL of the standard mixed stock solution of ester substances and place it in a 10 mL volumetric flask, add 0.1 mL of a mixed internal standard solution of tert-amyl alcohol and n-amyl acetate, and make up to the mark with ethanol solution. Shake well to obtain a standard mixed solution of ester substances;

[0018] (c) Preparation of test solution: Add tert-amyl alcohol-n-amyl acetate mixed internal standard solution to the wine sample to be tested to prepare a test solution containing 0.1 mL of tert-amyl alcohol-n-amyl acetate mixed internal standard solution per 10 mL;

[0019] (d) Detection: Take the standard mixed solution of ester substances and the test sample solution for gas chromatography detection respectively, and calculate the content of ethyl acetate, ethyl butyrate, ethyl lactate and ethyl caproate in the test wine sample.

[0020] Furthermore, in step (d), the gas chromatography detection conditions are:

[0021] Chromatographic column: polyethylene glycol capillary column; carrier gas: high-purity nitrogen, flow rate: 0.8 mL / min; tail gas: maintained at 25 mL / min; hydrogen flow rate: maintained at 30 mL / min; air flow rate: maintained at 400 mL / min; heating program: initial temperature: 35°C, maintained constant for 1 min, then increased to 70°C at 3°C / min, then increased to 180°C at 3.5°C / min, and finally increased to 210°C at 15°C / min, maintained constant for 6 min; detector temperature: 250°C;

[0022] Inlet temperature: 250°C; constant flow mode: 1 mL per minute; injection volume: 1 uL; split ratio 40:1.

[0023] Furthermore, the polyphenol detection method comprises the following steps:

[0024] (A) Preparation of a series of mixed standard working solutions: Using acetonitrile as the solvent, prepare mixed standard solutions with equal concentrations of gallic acid, catechin, vanillic acid, caffeic acid, syringic acid, p-coumaric acid, ferulic acid, quercetin, coumaric acid, protocatechuic acid, kaempferol, chlorogenic acid, vitexin, quercetin, hyperoside, rutin, epicatechin, ellagic acid, cinnamic acid, and sinapinic acid; then dilute the mixed standard solutions to prepare a series of mixed standard working solutions with different concentration gradients;

[0025] (B) Preparation of the test solution: dilute the alcohol sample to be tested several times with ultrapure water and adjust the pH to 2 with acid to obtain a pretreated alcohol sample; activate the HLB column with methanol and ultrapure water, load the pretreated alcohol sample onto the column, rinse the HLB column with ultrapure water after the sample is completely loaded, and elute the HLB column with a methanol-hydrochloric acid solution at pH 4. Collect the eluate, evaporate the eluate to dryness, dissolve the resulting solid with methanol, and filter to obtain the test solution;

[0026] (C) Detection: Liquid chromatography-tandem mass spectrometry was used to detect a series of mixed standard working solutions and test sample solutions, and the content of each polyphenol in the wine sample was measured using the standard curve method.

[0027] Furthermore, in step (A), the concentration of each polyphenol in the mixed standard solution is 500 μg / mL, and the concentration gradient of the series of mixed standard working solutions is 250, 100, 50, 10, and 5 μg / mL.

[0028] Furthermore, in step (C), the chromatographic conditions are:

[0029] Chromatographic column: Waters ACQUITY HSS T3; mobile phase A: 1 vol% acetic acid in water; mobile phase B: methanol; flow rate: maintained at 0.4 mL / min; injection volume: 1 μL; column temperature: 40°C.

[0030] Furthermore, in step (C), the mass spectrometry conditions are:

[0031] Electrospray ion source, ion source temperature: 550°C; electrospray voltage: -4500 V; detection mode: MRM mode; curtain gas pressure: 35.0 psi; nebulizer pressure: 50.0 psi; auxiliary heater pressure: 55.0 psi; collision gas flow rate: maintained at 9 mL per minute.

[0032] The beneficial effects of the present invention are:

[0033] The present invention establishes an efficient detection and analysis method for bayberry wine, which can analyze the influence of different years, different bayberry varieties and different preparation processes on the content of higher alcohols, esters and polyphenols in bayberry wine, determine the trend of active substances in bayberry wine changing over time and the active substances beneficial to the human body, provide a basis for the index control of bayberry wine, and ensure the flavor and quality of the product. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is the chromatogram of higher alcohol standard;

[0035] Figure 2 is the chromatogram of ester standards;

[0036] Figure 3This is the ion chromatogram of gallic acid standard;

[0037] Figure 4 This is the ion chromatogram of catechin standard;

[0038] Figure 5 This is the ion chromatogram of vanillic acid standard;

[0039] Figure 6 This is the ion chromatogram of the caffeic acid standard;

[0040] Figure 7 This is the ion chromatogram of syringic acid standard;

[0041] Figure 8 is the ion chromatogram of p-coumaric acid standard;

[0042] Figure 9 This is the ion chromatogram of ferulic acid standard;

[0043] Figure 10 is the ion chromatogram of quercetin standard;

[0044] Figure 11 This is the ion chromatogram of coumaric acid standard;

[0045] Figure 12 This is the ion chromatogram of the protocatechin standard;

[0046] Figure 13 This is the ion chromatogram of kaempferol standard;

[0047] Figure 14 This is the ion chromatogram of chlorogenic acid standard;

[0048] Figure 15 This is the ion chromatogram of vitexin standard;

[0049] Figure 16 This is the ion chromatogram of quercetin standard;

[0050] Figure 17 This is the ion chromatogram of hyperoside standard;

[0051] Figure 18 This is the ion chromatogram of rutin standard;

[0052] Figure 19 This is the ion chromatogram of epicatechin standard;

[0053] Figure 20 This is the ion chromatogram of ellagic acid standard;

[0054] Figure 21 This is the ion chromatogram of cinnamic acid standard;

[0055] Figure 22This is the ion chromatogram of sinapinic acid standard. DETAILED DESCRIPTION

[0056] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0057] Example 1 Detection and Analysis of Higher Alcohols

[0058] Using 4-methyl-2-pentanol as the internal standard, gas chromatography was used to analyze and detect n-propanol, sec-butanol (2-butanol), isobutanol, n-butanol, active amyl alcohol (2-methyl-1-butanol), and isoamyl alcohol (3-methyl-1-butanol) in wine samples. The specific steps are as follows:

[0059] (1) Preparation of internal standard solution

[0060] Take 1 mL of 4-methyl-2-pentanol in a 50 mL volumetric flask, dilute to the mark with 40 vol% ethanol solution, and shake well to obtain a 2 vol% 4-methyl-2-pentanol internal standard solution (4-methyl-2-pentanol concentration is 14407 mg / L).

[0061] (2) Preparation of standard solutions of higher alcohol series

[0062] Take ethanol and add water to prepare a 40 vol% ethanol solution; draw 1 mL of n-propanol into a 50 mL volumetric flask, dilute to the mark with 40 vol% ethanol solution, and shake well to obtain a 2 vol% n-propanol solution; according to the preparation method of n-propanol standard solution, prepare five solutions of sec-butanol, isobutanol, n-butanol, active amyl alcohol and isoamyl alcohol with a concentration of 2 vol%. 0.700 mL of n-propanol solution, 0.900 mL of sec-butanol solution, 0.500 mL of isobutanol solution, 0.600 mL of n-butanol solution, 0.600 mL of active amyl alcohol solution, and 0.600 mL of isoamyl alcohol solution were respectively placed into 6 10.0 mL volumetric flasks, and each was diluted to the scale with 40 vol% ethanol to prepare 1377.2 mg / L n-propanol intermediate solution, 1247.7 mg / L sec-butanol intermediate solution, 965.25 mg / L isobutanol intermediate solution, 950.16 mg / L n-butanol intermediate solution, 1188.1 mg / L active amyl alcohol intermediate solution, and 1147.7 mg / L isoamyl alcohol intermediate solution.

[0063] 0.25 mL of each of the six intermediate solutions was taken and placed in six 10 mL volumetric flasks, and 0.10 mL of 4-methyl-2-pentanol internal standard solution was added to each flask. The volume was adjusted to the mark with 40 vol% ethanol to prepare an n-propanol standard solution with an n-propanol concentration of 34.430 mg / L, a sec-butanol standard solution with a sec-butanol concentration of 31.193 mg / L, an isobutanol standard solution with an isobutanol concentration of 24.131 mg / L, an n-butanol standard solution with an n-butanol concentration of 23.754 mg / L, an active amyl alcohol standard solution of 29.703 mg / L, and an isoamyl alcohol standard solution of 28.693 mg / L. The concentration of 4-methyl-2-pentanol in each standard solution was 144.07 mg / L.

[0064] 0.50 mL of each of the six intermediate solutions was taken and placed in six 10 mL volumetric flasks, and 0.10 mL of 4-methyl-2-pentanol solution as internal standard was added to each flask. The volume was adjusted to the mark with 40 vol% ethanol to prepare an n-propanol standard solution with an n-propanol concentration of 68.860 mg / L, a sec-butanol standard solution with a sec-butanol concentration of 62.385 mg / L, an isobutanol standard solution with an isobutanol concentration of 48.263 mg / L, an n-butanol standard solution with an n-butanol concentration of 47.508 mg / L, an active amyl alcohol standard solution with an active amyl alcohol concentration of 59.405 mg / L, and an isoamyl alcohol standard solution with an isoamyl alcohol concentration of 57.385 mg / L. The concentration of 4-methyl-2-pentanol in each standard solution was 144.07 mg / L.

[0065] 1.00 mL of each of the six intermediate solutions was taken and placed in six 10 mL volumetric flasks, 0.10 mL of 4-methyl-2-pentanol internal standard solution was added to each flask, and each flask was diluted to the mark with 40 vol% ethanol to prepare an n-propanol standard solution with an n-propanol concentration of 137.72 mg / L, a sec-butanol standard solution with a sec-butanol concentration of 124.77 mg / L, an isobutanol standard solution with an isobutanol concentration of 96.525 mg / L, an n-butanol standard solution with an n-butanol concentration of 95.016 mg / L, an active amyl alcohol standard solution with an active amyl alcohol concentration of 118.81 mg / L, and an isoamyl alcohol standard solution with an isoamyl alcohol concentration of 114.77 mg / L. The concentration of 4-methyl-2-pentanol in each standard solution was 144.07 mg / L.

[0066] 1.50 mL of the above six intermediate solutions were respectively taken and placed in six 10 mL volumetric flasks, 0.10 mL of 4-methyl-2-pentanol internal standard solution was added to each flask, and each was diluted to the scale with 40 vol% ethanol to prepare an n-propanol standard solution with an n-propanol concentration of 206.58 mg / L, a sec-butanol standard solution with a sec-butanol concentration of 187.155 mg / L, an isobutanol standard solution with an isobutanol concentration of 144.79 mg / L, an n-butanol standard solution with an n-butanol concentration of 142.52 mg / L, an active amyl alcohol standard solution with an active amyl alcohol concentration of 178.22 mg / L, and an isoamyl alcohol standard solution with an isoamyl alcohol concentration of 172.16 mg / L. The concentration of 4-methyl-2-pentanol in each standard solution was 144.07 mg / L.

[0067] 2.00 mL of each of the six intermediate solutions was respectively pipetted into a 10 mL volumetric flask, and 0.10 mL of 4-methyl-2-pentanol internal standard solution was added to each of the flasks. The volumes of each of the six solutions were adjusted to the mark with 40 vol% ethanol to prepare an n-propanol standard solution with an n-propanol concentration of 275.44 mg / L, a sec-butanol standard solution with a sec-butanol concentration of 249.54 mg / L, an isobutanol standard solution with an isobutanol concentration of 193.05 mg / L, an n-butanol standard solution with an n-butanol concentration of 190.03 mg / L, an active amyl alcohol standard solution with an active amyl alcohol concentration of 237.62 mg / L, and an isoamyl alcohol standard solution with an isoamyl alcohol concentration of 229.54 mg / L. The concentration of 4-methyl-2-pentanol in each standard solution was 144.07 mg / L.

[0068] (3) Prepare the test solution

[0069] Use a pipette to transfer an appropriate amount of the wine sample to be tested into a 10mL volumetric flask, add 0.1mL of 4-methyl-2-pentanol internal standard solution, then use a pipette to draw up the volume of the wine sample to the mark, mix well, and the solution is obtained.

[0070] (4) Detection

[0071] Gas chromatography was used to detect six kinds of higher alcohol series standard solutions and test sample solutions, and the contents of n-propanol, sec-butanol, isobutanol, n-butanol, active amyl alcohol and isoamyl alcohol in the tested wine samples were calculated based on the obtained chromatograms.

[0072] Gas chromatography detection conditions are as follows:

[0073] Chromatographic column: HP-INNOWax, 60m×0.25mm×0.25μm; carrier gas: high-purity nitrogen, 0.80 mL / min; split ratio: 2:1; tail blow: 20mL / min to 30mL / min; hydrogen flow rate: maintain 32mL / min; air flow rate: maintain 400mL / min; detector temperature: 250℃; injection port temperature: 220℃; injection volume: 1μL; heating program: start temperature at 40℃ and keep constant for 5min, then increase to 75℃ at 3℃ / min and hold for 2min, then increase to 80℃ at 3℃ / min and hold for 10min.

[0074] The standard spectra of higher alcohols are shown in Figure 1 The analysis results of the standard curves of the various standards are shown in Table 1.

[0075] Table 1

[0076]

[0077] Example 2 Detection and Analysis of Esters in Bayberry Wine

[0078] Using a mixture of tert-amyl alcohol and n-amyl acetate as internal standards, gas chromatography was used to analyze and detect ethyl acetate, ethyl butyrate, ethyl lactate, and ethyl caproate in wine samples. The specific steps are as follows:

[0079] (1) Preparation of tert-amyl alcohol-n-amyl acetate mixed internal standard solution

[0080] Accurately weigh 2.0 g of tert-amyl alcohol standard and 2.0 g of n-amyl acetate standard, dissolve them in an appropriate amount of 50 vol% ethanol solution, transfer them to a 100 mL volumetric flask, make up to volume with 50 vol% ethanol solution, and mix well to prepare a tert-amyl alcohol-n-amyl acetate mixed internal standard solution.

[0081] (2) Preparation of standard mixed solution of ester substances

[0082] Accurately weigh 2.5 g of ethyl acetate standard, 2.5 g of ethyl hexanoate standard, 2.5 g of ethyl butyrate standard, and 0.25 g of ethyl lactate standard, respectively, add an appropriate amount of 50 vol% ethanol solution to dissolve, transfer to a 100 mL volumetric flask, make up to volume with 50 vol% ethanol solution, and shake thoroughly to obtain a standard mixed stock solution of ester substances.

[0083] Take 1 mL of the ester substance standard mixed stock solution in a 10 mL volumetric flask, add 0.1 mL of the tert-amyl alcohol-n-amyl acetate mixed internal standard solution, add 50 vol% ethanol solution to the scale, and shake well to obtain the ester substance standard mixed solution.

[0084] (3) Prepare the test solution

[0085] Use a pipette to transfer an appropriate amount of the wine sample to be tested into a 10mL volumetric flask, add 0.1mL of the mixed internal standard solution of tert-amyl alcohol and n-amyl acetate, then use a pipette to draw up the wine sample to the scale, and mix well.

[0086] (4) Detection

[0087] Take the ester standard mixed solution and the test solution for gas chromatography detection respectively, and calculate the content of ethyl acetate, ethyl butyrate, ethyl lactate and ethyl hexanoate in the wine sample according to the obtained chromatogram. Figure 2 shown.

[0088] Gas chromatography detection conditions are as follows:

[0089] Chromatographic column: polyethylene glycol capillary column; carrier gas: high-purity nitrogen, flow rate of 0.8 mL per minute; tail blow: maintain 25 mL per minute; hydrogen flow rate: maintain 30 mL per minute; air flow rate: maintain 400 mL per minute; heating program: initial temperature is 35°C, maintained constant for 1 min, then heated to 70°C at 3°C ​​per minute, then heated to 180°C at 3.5°C per minute, and finally heated to 210°C at 15°C per minute, maintained constant for 6 min; detector temperature: 250°C; injection port temperature: 250°C; constant flow mode: 1 mL per minute; injection volume: 1 uL; split ratio 40:1.

[0090] Example 3 Detection and Analysis of Polyphenols in Bayberry Wine

[0091] Solid phase extraction combined with liquid chromatography-tandem mass spectrometry was used to analyze and detect polyphenols in wine samples. The specific steps are as follows:

[0092] (1) Prepare a series of mixed standard working solutions

[0093] 5 mg of each of the 20 polyphenol standards (gallic acid, catechin, vanillic acid, caffeic acid, syringic acid, p-coumaric acid, ferulic acid, quercetin, coumaric acid, protocatechuic acid, kaempferol, chlorogenic acid, vitexin, quercetin, hyperoside, rutin, epicatechin, ellagic acid, cinnamic acid, and sinapic acid) were accurately weighed using an analytical balance. These were dissolved in 50 vol% acetonitrile by shaking to prepare 500 mg / mL single standard stock solutions, which were then stored at -18°C in the dark. The 20 single standard stock solutions were pipetted and mixed, and diluted with 50 vol% acetonitrile to obtain mixed standard solutions with a concentration of 500 μg / mL for each polyphenol. A certain amount of the mixed standard solution was then pipetted and serially diluted with 50 vol% acetonitrile to prepare a series of mixed standard working solutions with concentrations of 250, 100, 50, 10, and 5 μg / mL.

[0094] (2) Prepare the test solution

[0095] Use a pipette to accurately draw 2 mL of the wine sample to be tested and place it in a 50 mL centrifuge tube. Dilute the alcohol content of the wine sample to be tested five times with ultrapure water, and then adjust the pH value to 2 with 1 mol / L hydrochloric acid to obtain a pretreated wine sample; activate the HLB column with 6 mL of methanol, and after the liquid flows out, activate the HLB column with 6 mL of ultrapure water, set the high-throughput fully automatic solid phase extraction program, take the pretreated wine sample and load it, maintaining the flow rate at 1 mL / min; after the sample is completely loaded, rinse the HLB column with 6 mL of ultrapure water to remove interferences in the column, and then adjust the methanol to a solution pH of 4 with 1 mol / L hydrochloric acid, elute the HLB column, and collect the eluate; use a rotary evaporator, set the temperature to 35°C, rotary evaporate the eluate to dryness, then add 0.5 mL of methanol to dissolve it, and finally pass the collected sample through a 0.22 um organic filter membrane to obtain the test solution.

[0096] (3) Detection

[0097] Liquid chromatography-tandem mass spectrometry was used to detect a series of mixed standard working solutions and test solutions, and the content of each polyphenol in the wine samples was measured using the standard curve method.

[0098] The chromatographic conditions are:

[0099] Chromatographic column: Waters ACQUITY HSS T3 (2.1 mm × 100 mm, 1.8 μm);

[0100] Mobile phase A: 1 vol% acetic acid in water; mobile phase B: methanol; flow rate: maintain 0.4 mL / min;

[0101] Injection volume: 1 μL; column temperature: 40°C.

[0102] Mass spectrometry conditions are:

[0103] electrospray ionization source with a source temperature of 550°C;

[0104] Electrospray voltage: -4500 V;

[0105] Detection mode: MRM mode;

[0106] Air curtain pressure: 35.0psi;

[0107] Sprayer pressure: 50.0psi;

[0108] Auxiliary heater pressure: 55.0psi;

[0109] Collision gas flow rate: maintained at 9 mL per minute.

[0110] Ion chromatograms of 20 polyphenol standards are shown in Figure 2. Figure 3-Figure 22 The linear equations of the 20 polyphenol standards are shown in Table 2.

[0111] Table 2 Linear equations of 20 polyphenols

[0112]

[0113] Test results and analysis

[0114] (a) The higher alcohol content of various bayberry wines and millet wines was tested according to the method of Example 1. The results are shown in Table 3. (The following one-year-old bayberry wine, one-year-old Baisha bayberry wine, one- to five-year-old deep red bayberry wine, and five-year-old millet wine were purchased from the Jingmei Bayberry Professional Cooperative in Shangyu, Shaoxing.)

[0115] Table 3 Higher alcohol content in different wine samples (unit: μg / mL)

[0116]

[0117] An analysis and comparison of one-year-old bayberry-infused wine and one-year-old bayberry liquor (distilled liquor) showed that the content of sec-butanol in bayberry liquor was 1.1 times that of the deep red bayberry-infused wine, the content of isobutanol was 0.75 times that of the bayberry-infused wine, and the total content of active amyl alcohol and isopentanol was 1.1 times that of the bayberry-infused wine.

[0118] An analysis and comparison was conducted on one-year-old Baisha bayberry wine and one-year-old deep red bayberry wine: the content of sec-butanol in Baisha bayberry wine is 0.8 times that of deep red bayberry wine, the content of isobutanol is 0.6 times that of deep red bayberry wine, and the total content of active amyl alcohol and isopentanol is 0.8 times that of deep red bayberry wine.

[0119] Analysis and comparison of one-year, two-year, three-year, four-year, and five-year deep red bayberry wines: with the extension of storage time, the content of secondary butanol in the bayberry wine first decreased and then increased, reaching a minimum of 81.28 ug / mL in the third year; the content of isobutanol first decreased and then increased, decreasing by 2.8 times in the second year, and then slowly decreased, and increased in the fifth year; the content of active amyl alcohol and total isoamyl alcohol first decreased and then increased, decreasing by 2.2 times in the second year.

[0120] An analysis and comparison of five-year-old bayberry-infused wine and five-year-old millet wine showed that the content of sec-butanol in five-year-old millet wine was 3.4 times that of the bayberry-infused wine, the content of isobutanol was 2.5 times that of the bayberry-infused wine, and the total content of active amyl alcohol and isopentanol was 2.6 times that of the bayberry-infused wine.

[0121] (b) The ester content of different bayberry wine samples and millet wine was tested according to the method of Example 2. The results are shown in Table 4.

[0122] Table 4 Ester content in different wine samples (unit: μg / mL)

[0123]

[0124] An analysis and comparison of one-year-old Yangmei Shao (distilled liquor) and one-year-old Yangmei-infused liquor showed that the ethyl acetate content in Yangmei Shao was three times that in Yangmei-infused liquor, while no ethyl butyrate was detected in Yangmei Shao; the ethyl lactate content in Yangmei Shao was 2.3 times that in Yangmei-infused liquor.

[0125] An analysis and comparison of one-year-old Baisha bayberry soaked wine and one-year-old deep red bayberry soaked wine showed that the ethyl acetate content in Baisha bayberry soaked wine was 1.9 times that of the deep red bayberry soaked wine, the ethyl butyrate content was 1.1 times that of the deep red bayberry soaked wine, and the ethyl lactate content was 2 times that of the deep red bayberry soaked wine.

[0126] Analysis and comparison of one-year, two-year, three-year, four-year, and five-year old deep red bayberry soaked wines showed that with the extension of time, the ethyl acetate content first increased and then decreased, reaching a peak of 451.60ug / mL in the third year, and then began to decrease; the ethyl butyrate content first decreased and then increased, reaching a minimum of 142.98ug / mL in the third year, and then slowly increased; the ethyl lactate content first increased and then decreased, reaching a peak of 107.17ug / mL in the second year, and then slowly decreased.

[0127] An analysis and comparison of five-year-old millet liquor and five-year-old bayberry-infused wine showed that the ethyl acetate content in five-year-old millet liquor was 1.8 times that in bayberry-infused wine, while no ethyl butyrate content was detected in five-year-old millet liquor; the ethyl lactate content was 4.3 times that in bayberry-infused wine.

[0128] (c) The polyphenol content of different bayberry wine samples and millet wine was tested according to the method of Example 3. The results are shown in Table 5.

[0129] Table 5 Polyphenol content in different wine samples (unit: ng / mL)

[0130]

[0131] From Table 3 we can see that:

[0132] An analysis and comparison of one-year-old Yangmei Shao (distilled liquor) and one-year-old Yangmei wine showed that the total content of 20 polyphenols in Yangmei Shao was 118.19 ng / mL, which is negligible compared with the content of polyphenols in Yangmei wine.

[0133] An analysis and comparison of one-year-old Baisha bayberry soaked wine and one-year-old deep red bayberry soaked wine showed that the gallic acid content in the deep red bayberry soaked wine was 3.6 times that of the Baisha bayberry soaked wine, its vanillic acid content was 1.8 times that of the Baisha bayberry soaked wine, its syringic acid content was 2 times that of the Baisha bayberry soaked wine, its p-coumaric acid content was 5.2 times that of the Baisha bayberry soaked wine, its ferulic acid content was 4 times that of the Baisha bayberry soaked wine, its quercetin content was 6.3 times that of the Baisha bayberry soaked wine, its protocatechuic acid content was 4 times that of the Baisha bayberry soaked wine, its quercetin content was 1.3 times that of the Baisha bayberry soaked wine, its ellagic acid content was 1.6 times that of the Baisha bayberry soaked wine, and its cinnamic acid content was 2.8 times that of the Baisha bayberry soaked wine.

[0134] An analysis and comparison of wines soaked in deep red bayberries aged from one to five years showed that gallic acid, p-coumaric acid, quercetin, protocatechuic acid, quercetin, and ellagic acid all showed a trend of first decreasing and then increasing, vanillic acid gradually increased, ferulic acid gradually decreased, and cinnamic acid showed a trend of first decreasing, then increasing, and then decreasing, but the content remained above 520 ng / ml.

[0135] The polyphenol content detected in five-year-old millet wine was extremely low, and the difference was obvious compared with the five-year-old bayberry-soaked wine. Therefore, it can be determined that adding bayberry to millet wine can greatly increase the polyphenol content in the wine sample.

[0136] From the results analysis of Tables 3 to 5, it can be seen that the preparation of bayberry wine by soaking bayberries can reduce the content of alcohol substances in the wine samples, and can also reduce the content of ethyl acetate and ethyl lactate in the wine samples (among which, the content of higher alcohols in Baisha bayberry soaked wine is lower than that in deep red bayberry soaked wine, while the content of ester substances is higher than that in deep red bayberry soaked wine), but it will greatly increase the content of polyphenols in the wine samples. With increasing bayberry wine soaking time, alcohols in the wine samples showed a trend of first decreasing and then increasing. Esters, with the exception of ethyl butyrate, which gradually increased, showed a slow decrease. Polyphenols in the wine samples, including gallic acid, p-coumaric acid, quercetin, protocatechuic acid, quercetin, and ellagic acid, all showed a trend of first decreasing and then increasing (gallic acid and protocatechuic acid reached their lowest levels in the second year; p-coumaric acid, quercetin, quercetin, and ellagic acid reached their lowest levels in the third year, with gallic acid reaching a maximum of 9320.92 ng / mL and ellagic acid reaching a maximum of 15469.85 ng / mL). Vanillic acid gradually increased, while ferulic acid gradually decreased. Cinnamic acid showed a trend of first decreasing, then increasing, and then decreasing, but its content remained above 520 ng / mL. These results indicate that bayberry wine soaking significantly reduces harmful substances such as higher alcohols and enriches and increases polyphenols beneficial to the human body.

[0137] The above description is merely a preferred embodiment of the present invention. The scope of protection of the present invention is not limited to the above embodiment. All technical solutions based on the concept of the present invention are within the scope of protection of the present invention. It should be noted that for those skilled in the art, various improvements and modifications that do not depart from the principles of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. A method for detecting and analyzing active substances in bayberry-soaked wine, characterized in that: Including gas chromatography detection of higher alcohols, gas chromatography detection of esters and liquid chromatography-tandem mass spectrometry detection of polyphenols; The method for detecting higher alcohol substances comprises the following steps: (1) Preparation of a series of standard solutions of various higher alcohols: using ethanol solution as solvent and 4-methyl-2-pentanol as internal standard, prepare a series of n-butanol standard solutions, a series of sec-butanol standard solutions, a series of isobutanol standard solutions, a series of n-propanol standard solutions, a series of active amyl alcohol standard solutions and a series of isoamyl alcohol standard solutions with the same internal standard concentration; (2) Preparation of test solution: Using 4-methyl-2-pentanol as the internal standard, prepare a test solution with the same internal standard concentration as that of the standard solution in step (1) by taking the alcohol sample to be tested; (3) Detection: Gas chromatography was used to detect the six higher alcohol series standard solutions and the test sample solution, and the contents of n-propanol, sec-butanol, isobutanol, n-butanol, active amyl alcohol and isopentanol in the test wine sample were calculated.

2. The method for detecting and analyzing active substances in bayberry wine according to claim 1, characterized in that: In step (3), the gas chromatography detection conditions are: Chromatographic column: HP-INNOWax, 60m×0.25mm×0.25μm; Carrier gas: high purity nitrogen, 0.80 mL / min; Split ratio: 2:1; Tail blowing: 20mL per minute to 30mL per minute; Hydrogen flow rate: maintain 32 mL per minute; Air flow rate: maintain 400mL per minute; Detector temperature: 250°C; Inlet temperature: 220°C; Injection volume: 1 μL; Heating program: Initial temperature was 40°C and maintained for 5 min, then increased to 75°C at a rate of 3°C / min and maintained for 2 min, then increased to 80°C at a rate of 3°C / min and maintained for 10 min.

3. The method for detecting and analyzing active substances in bayberry-soaked wine according to claim 1, characterized in that: In step (1), the internal standard concentration of the n-butanol series standard solution, the sec-butanol series standard solution, the isobutanol series standard solution, the n-propanol series standard solution, the active amyl alcohol series standard solution, the isoamyl alcohol series standard solution, and the test solution is 144.07 mg / L.

4. The method for detecting and analyzing active substances in bayberry wine according to claim 1, characterized in that: In step (1), the concentration gradient of the standard solution of n-propanol series is 34.430, 68.860, 137.72, 206.58, 275.44 mg / L; the concentration gradient of the standard solution of sec-butyl alcohol series is 31.193, 62.385, 124.77, 187.155, 249.54 mg / L; the concentration gradient of the standard solution of isobutyl alcohol series is 24.131, 48.26, 96.525, 144.79, 193.05 mg / L / L; the concentration gradient of the n-butanol series standard solution is 23.754, 47.508, 95.016, 142.52, 190.03 mg / L; the concentration gradient of the active amyl alcohol series standard solution is 29.703, 59.405, 118.81, 178.22, 237.62 mg / L; the concentration gradient of the isoamyl alcohol series standard solution is 28.693, 57.385, 114.77, 172.16, 229.54 mg / L.

5. The method for detecting and analyzing active substances in bayberry-soaked wine according to claim 1, characterized in that: The ester substance detection method comprises the following steps: (a) Preparation of a mixed internal standard solution: using ethanol solution as solvent, prepare a tert-amyl alcohol-n-amyl acetate mixed internal standard solution containing 2.0 g of tert-amyl alcohol standard and 2.0 g of n-amyl acetate standard per 100 mL; (b) Preparation of ester standard mixed solution: using ethanol as solvent, prepare a standard mixed stock solution of esters containing 2.5 g of ethyl acetate standard, 2.5 g of ethyl hexanoate standard, 2.5 g of ethyl butyrate standard, and 0.25 g of ethyl lactate standard per 100 mL; take 1 mL of the standard mixed stock solution of esters into a 10 mL volumetric flask, add 0.1 mL of tert-amyl alcohol-n-amyl acetate mixed internal standard solution, add ethanol solution to the mark, shake well, and obtain a standard mixed solution of esters; (c) Preparation of test solution: adding tert-amyl alcohol-n-amyl acetate mixed internal standard solution to the wine sample to be tested to prepare a test solution containing 0.1 mL of tert-amyl alcohol-n-amyl acetate mixed internal standard solution per 10 mL; (d) Detection: Take the standard mixed solution of ester substances and the test sample solution for gas chromatography detection respectively, and calculate the content of ethyl acetate, ethyl butyrate, ethyl lactate and ethyl caproate in the test wine sample.

6. The method for detecting and analyzing active substances in bayberry-soaked wine according to claim 5, characterized in that: In step (d), the gas chromatography detection conditions are: Chromatographic column: polyethylene glycol capillary column; Carrier gas: high-purity nitrogen, gas flow rate 0.8 mL per minute; Tail blowing: maintain 25mL per minute; Hydrogen flow rate: maintain 30 mL per minute; Air flow rate: maintain 400mL per minute; Heating program: the initial temperature is 35°C, which is kept constant for 1 minute, then raised to 70°C at a rate of 3°C per minute, then raised to 180°C at a rate of 3.5°C per minute, and finally raised to 210°C at a rate of 15°C per minute, and kept constant for 6 minutes; Detector temperature: 250°C; Inlet temperature: 250°C; Constant flow mode: 1 mL per minute; Injection volume: 1uL; Split ratio 40:

1.

7. The method for detecting and analyzing active substances in bayberry-soaked wine according to claim 1, characterized in that: The polyphenol detection method comprises the following steps: (A) Preparation of a series of mixed standard working solutions: using acetonitrile solution as solvent, prepare mixed standard solutions with the same concentration of gallic acid, catechin, vanillic acid, caffeic acid, syringic acid, p-coumaric acid, ferulic acid, quercetin, coumaric acid, protocatechuic acid, kaempferol, chlorogenic acid, vitexin, quercetin, hyperoside, rutin, epicatechin, ellagic acid, cinnamic acid and sinapinic acid; then dilute the mixed standard solutions into a series of mixed standard working solutions with different concentration gradients; (B) Preparation of the test solution: dilute the wine sample to be tested with ultrapure water for several times, and then adjust the pH value to 2 with acid to obtain a pre-treated wine sample; activate the HLB column with methanol and ultrapure water, load the pre-treated wine sample, and after the sample is completely loaded, rinse the HLB column with ultrapure water, and then elute the HLB column with a methanol-hydrochloric acid solution with a pH value of 4, collect the eluate, evaporate the eluate to dryness, dissolve the obtained solid with methanol, and filter it to obtain the test solution; (C) Detection: Liquid chromatography-tandem mass spectrometry was used to detect a series of mixed standard working solutions and test sample solutions, and the content of each polyphenol in the wine sample was measured using the standard curve method.

8. The method for detecting and analyzing active substances in bayberry-soaked wine according to claim 7, characterized in that: In step (A), the concentration of each polyphenol in the mixed standard solution is 500 ug / mL, and the concentration gradient of the series of mixed standard working solutions is 250, 100, 50, 10, and 5 ug / mL.

9. The method for detecting and analyzing active substances in bayberry-soaked wine according to claim 7, characterized in that: In step (C), the chromatographic conditions are: Column: Waters ACQUITY HSS T3; Mobile phase A: 1 vol% acetic acid in water; Mobile phase B: methanol; Flow rate: maintain 0.4 mL per minute; Injection volume: 1 μL; Column temperature: 40℃.

10. The method for detecting and analyzing active substances in bayberry-soaked wine according to claim 7, characterized in that: In step (C), the mass spectrometry conditions are: Electrospray ion source, ion source temperature was 550°C; Electrospray voltage: -4500 V; Detection mode: MRM mode; Air curtain air pressure: 35.0psi; Sprayer pressure: 50.0psi; Auxiliary heater pressure: 55.0psi; Collision gas flow rate: maintained at 9 mL per minute.

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