A detection and analysis method for active substances in bayberry soaked wine

Abstract

The invention discloses a detection and analysis method for active substances in bayberry soaked wine, including gas chromatography detection of higher alcohol substances, gas chromatography detection of ester substances and liquid chromatography-tandem mass spectrometry detection of polyphenol substances; wherein the detection of higher alcohol substances includes: (1) using ethanol solution as solvent and 4-methyl-2-pentanol as internal standard, respectively preparing 6 higher alcohol series standard solutions of n-butanol, sec-butanol, isobutanol, n-propanol, active amyl alcohol and isoamyl alcohol with the same internal standard concentration; (2) using 4-methyl-2-pentanol as internal standard, taking a wine sample to be tested to prepare a test solution; (3) using gas chromatography to detect the 6 higher alcohol series standard solutions and the test solution, respectively, and calculating the content of each alcohol in the wine sample to be tested. The method of the invention can provide a basis for the index control of bayberry wine and ensure the flavor and quality of the product.

Description

Technical Field

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

[0002] At present, there is still a blank in the detection of active substances in bayberry wine and characteristic substances in bayberry wines of different years and types. Only the optimization research on the production process of bayberry wine, the research on myricetin and other active substances in bayberry fruit, and the analysis of the aromatic smell of bayberry can be found. Therefore, it is necessary to establish an analysis method for active substances in bayberry wine, to deeply understand the factors affecting the flavor and quality of bayberry wine, and 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 detection and analysis method for 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 solution:

[0005] A detection and analysis method for active substances in bayberry-infused wine, including gas chromatography detection of higher alcohol substances, gas chromatography detection of ester substances and liquid chromatography-tandem mass spectrometry detection of polyphenol substances;

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

[0007] (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;

[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 alcohol sample to be tested;

[0009] (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.

[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; tail gas: 20mL / min to 30mL / min; hydrogen flow rate: maintained at 32mL / min; air flow rate: maintained at 400mL / min; detector temperature: 250℃; injection port temperature: 220℃; injection volume: 1μL;

[0012] 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.

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

[0016] (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;

[0017] (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;

[0018] (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;

[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, gas flow rate of 0.8 mL per minute; tail gas: maintained at 25 mL per minute; hydrogen flow rate: maintained at 30 mL per minute; air flow rate: maintained at 400 mL per minute; heating program: initial temperature is 35°C, maintained constant for 1 min, then increased to 70°C at 3°C ​​per minute, then increased to 180°C at 3.5°C per minute, and finally increased to 210°C at 15°C per minute, 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] Further, the polyphenol detection method comprises the following steps:

[0024] (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;

[0025] (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;

[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 ug / mL, and the concentration gradient of the series of mixed standard working solutions is 250, 100, 50, 10, and 5 ug / mL.

[0028] Further, 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 per minute; 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 is 550°C; electrospray voltage: -4500V; detection mode: MRM mode; curtain gas pressure: 35.0psi; nebulizer pressure: 50.0psi; auxiliary heater pressure: 55.0psi; collision gas flow rate: maintained at 9mL per minute.

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

[0033] The 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 alcohol substances, ester substances and polyphenol substances in bayberry wine, determine the trend of active substances in bayberry wine changing over time and 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 sample;

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

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

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

[0038] Figure 5 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 the syringic acid standard;

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

[0042] Fig. 9 is the ion chromatogram of ferulic acid standard;

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

[0044] Fig.11 is the ion chromatogram of coumaric acid standard;

[0045] Fig.12 is the ion chromatogram of the protocatechin standard;

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

[0047] Fig.14 is the ion chromatogram of chlorogenic acid standard;

[0048] Fig.15 is the ion chromatogram of vitexin standard;

[0049] Fig.16 is the ion chromatogram of quercetin standard;

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

[0051] Fig.18 is the ion chromatogram of rutin standard;

[0052] Fig.19 is the ion chromatogram of epicatechin standard;

[0053] Fig. 20 is the ion chromatogram of ellagic acid standard;

[0054] Fig.21 is the ion chromatogram of cinnamic acid standard;

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

[0056] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. 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 creative work 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 isopentanol (3-methyl-1-butanol) in the wine sample. 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, 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 various 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, shake well to obtain a 2 vol% n-propanol solution; prepare five solutions of 2 vol% of sec-butanol, isobutanol, n-butanol, active amyl alcohol and isoamyl alcohol according to the preparation method of n-propanol standard solution. 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 in 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 the above six intermediate solutions were respectively taken and placed in 6 10 mL volumetric flasks, and 0.10 mL of 4-methyl-2-pentanol internal standard solution was added to each flask. Each flask was diluted to the mark with 40 vol% ethanol to prepare a 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, a 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 the above six intermediate solutions were respectively taken and placed in 6 10 mL volumetric flasks, 0.10 mL of 4-methyl-2-pentanol solution as internal standard was added to each flask, and each flask was made up to the scale 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 the above 6 intermediate solutions were respectively taken and placed in 6 10 mL volumetric flasks, and 0.10 mL of 4-methyl-2-pentanol internal standard solution was added to each flask. 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, and 0.10 mL of 4-methyl-2-pentanol internal standard solution was added to each flask. Each flask was diluted to the mark 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 the above six intermediate solutions were respectively taken into a 10 mL volumetric flask, and 0.10 mL of 4-methyl-2-pentanol internal standard solution was added to each of them. Each of them was made up to the scale 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) Preparation of 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 wine sample to the scale, mix well, and you have it.

[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 isopentanol in the tested wine samples were calculated based on the obtained chromatograms.

[0072] The 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℃ for 5min, then increase to 75℃ at 3℃ / min and maintain for 2min, then increase to 80℃ at 3℃ / min and maintain for 10min.

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

[0075] Table 1

[0076]

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

[0078] Using 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 the wine sample. 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, add 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, mix well, and prepare 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, add 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, shake well 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 tert-amyl alcohol-n-amyl acetate mixed internal standard solution, add 50 vol% ethanol solution to the scale, shake well, and obtain the ester substance standard mixed solution.

[0084] (3) Preparation of 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-n-amyl acetate, then use a pipette to draw up the wine sample to the scale, mix well and obtain the solution.

[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 caproate in the wine sample according to the obtained chromatogram. Figure 2 shown.

[0088] The gas chromatography detection conditions are as follows:

[0089] Chromatographic column: polyethylene glycol capillary column; carrier gas: high-purity nitrogen, gas 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 increased to 70°C at 3°C ​​per minute, then increased to 180°C at 3.5°C per minute, and finally increased 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: 1uL; 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 20 polyphenol standards were accurately weighed with an analytical balance (the 20 polyphenol standards were 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), and dissolved by shaking with 50 vol% acetonitrile to prepare a single standard stock solution of 500 mg / mL, which was stored at -18°C in the dark; the 20 single standard stock solutions were respectively drawn with a pipette to mix, and diluted with 50 vol% acetonitrile to obtain a mixed standard solution with a concentration of 500 ug / mL for each polyphenol substance, and then a certain amount of the mixed standard solution was drawn with a pipette, and diluted step by step with 50 vol% acetonitrile to form a series of mixed standard working solutions with concentrations of 250, 100, 50, 10, and 5 ug / mL.

[0094] (2) Preparation of test solution

[0095] Use a pipette to accurately draw 2 mL of the wine sample to be tested, put it into a 50 mL centrifuge test 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; use 6 mL of methanol to activate the HLB column, 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, and keep 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 use 1 mol / L hydrochloric acid to adjust the methanol to a solution pH of 4, 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, and finally pass the collected sample through a 0.22 um organic filter membrane to obtain a 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 by 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: maintained at 0.4 mL / min;

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

[0102] The mass spectrometry conditions were:

[0103] Electrospray ion source, ion source temperature was 550°C;

[0104] Electrospray voltage: -4500 V;

[0105] Detection mode: MRM mode;

[0106] Air curtain air 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 different bayberry wines and millet wines was tested according to the method of Example 1, and the results are shown in Table 3. (The following one-year-old bayberry wine, one-year-old Baisha bayberry soaked wine, one to five-year-old deep red bayberry soaked wine, and five-year-old millet wine were purchased from Shaoxing Shangyu Jingmei Bayberry Professional Cooperative)

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

[0116]

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

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

[0119] An analysis and comparison of one-year, two-year, three-year, four-year, and five-year deep red bayberry wines showed that 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 total content of active amyl alcohol and isopentanol first decreased and then increased, decreasing by 2.2 times in the second year.

[0120] An analysis and comparison of five-year-old bayberry wine and five-year-old millet wine showed that the content of secondary butanol in five-year-old millet wine was 3.4 times that of the bayberry wine, the content of isobutanol was 2.5 times that of the bayberry wine, and the total content of active amyl alcohol and isopentanol was 2.6 times that of the bayberry 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: ug / mL)

[0123]

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

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

[0126] An analysis and comparison of one-year, two-year, three-year, four-year, and five-year 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 wine and five-year-old bayberry-soaked wine showed that the ethyl acetate content in five-year-old millet wine was 1.8 times that in bayberry-soaked wine, and no ethyl butyrate content was detected in five-year-old millet wine; the ethyl lactate content was 4.3 times that in bayberry-soaked 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 Content of polyphenols 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 was conducted on the one-year-old Baisha bayberry wine and the one-year-old deep red bayberry wine: the gallic acid content in the deep red bayberry wine was 3.6 times that in the Baisha bayberry wine, its vanillic acid content was 1.8 times that in the Baisha bayberry wine, its syringic acid content was 2 times that in the Baisha bayberry wine, its p-coumaric acid content was 5.2 times that in the Baisha bayberry wine, its ferulic acid content was 4 times that in the Baisha bayberry wine, its quercetin content was 6.3 times that in the Baisha bayberry wine, its protocatechuic acid content was 4 times that in the Baisha bayberry wine, its quercetin content was 1.3 times that in the Baisha bayberry wine, its ellagic acid content was 1.6 times that in the Baisha bayberry wine, and its cinnamic acid content was 2.8 times that in the Baisha bayberry wine.

[0134] An analysis and comparison of wines soaked in deep red bayberry 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 520ng / ml.

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

[0136] From the results in 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 sample, and can also reduce the content of ethyl acetate and ethyl lactate in the wine sample (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 sample. And with the extension of the soaking time of bayberry wine, the alcohol substances in the wine samples showed a trend of first decreasing and then increasing; except for ethyl butyrate, the ester substances in the wine samples slowly decreased; the polyphenols in the wine samples, gallic acid, p-coumaric acid, quercetin, protocatechuic acid, quercetin, and ellagic acid all showed a trend of first decreasing and then increasing (among which gallic acid and protocatechuic acid dropped to the lowest in the second year; p-coumaric acid, quercetin, quercetin, and ellagic acid dropped to the lowest in the third year, the highest gallic acid content reached 9320.92 ng / mL, and the highest ellagic acid content reached 15469.85 ng / mL), vanillic acid gradually increased, ferulic acid gradually decreased, and cinnamic acid showed a trend of first decreasing, then increasing, and then decreasing, but its content remained above 520ng / mL. The results show that soaking bayberry wine can significantly reduce harmful substances such as higher alcohols and enrich and increase polyphenols that are beneficial to the human body.

[0137] The above is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiments. All technical solutions under the concept of the present invention belong to the protection scope of the present invention. It should be pointed out that for ordinary technicians in this technical field, some improvements and modifications without departing from the principle of the present invention should also be regarded as the protection scope 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: Use gas chromatography to detect the six higher alcohol series standard solutions and the test sample solution, and calculate the content of n-propanol, sec-butanol, isobutanol, n-butanol, active amyl alcohol, and isoamyl alcohol in the wine sample to be tested; 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 40℃ constant temperature for 5 min, then increase to 75℃ at a rate of 3℃ / min and hold for 2 min, then increase to 80℃ at a rate of 3℃ / min and hold for 10 min; 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 ester substance standard mixed solution 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 wine sample to be tested; 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; 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.

2. The method for detecting and analyzing active substances in bayberry 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.

3. The method for detecting and analyzing active substances in bayberry-soaked 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.

4. The method for detecting and analyzing active substances in bayberry wine according to claim 1, 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.

5. The method for detecting and analyzing active substances in bayberry-soaked wine according to claim 1, 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℃.

6. The method for detecting and analyzing active substances in bayberry-soaked wine according to claim 1, 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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