A method for extracting active monomer compounds from Luzhou-flavor liquor vinasse

Abstract

The present application belongs to the technical field of detection and analysis, and relates to a method for extracting active monomer compounds from sauce-flavor liquor vinasse. The method comprises the following steps: (1) primary extraction; (2) secondary extraction; (3) qualitative confirmation: qualitatively confirming the components in the secondary extraction extract obtained after the secondary extraction; (4) separation and purification: based on the components possibly existing in the secondary extraction extract qualitatively confirmed in step (3), the active components are separated and purified in sequence by using macroporous adsorption column chromatography, ODS column chromatography and Pre-HPLC chromatography method, so that the monomer compounds are obtained. The method provided by the present application can simultaneously obtain difficult volatile active components such as large-polarity phenolic acids, amino acids, dipeptides and flavonoids from sauce-flavor liquor vinasse, further verifies the existence of active substances in vinasse, deepens the understanding of the polar active components in sauce-flavor liquor vinasse, and provides theoretical support for the utilization and development of vinasse.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of detection analysis, and relates to a method for extracting active monomer compounds from sauce-flavor liquor lees. BACKGROUND

[0002] Liquor lees are solid mixtures left after liquor brewing, and the amount of liquor lees produced by liquor brewing in China is huge every year. At present, the most common way for the development and utilization of liquor lees in China is to use them as raw materials or bases in the feed industry. This traditional way of preparing products from liquor lees has low added value and poor economic benefits of comprehensive development and recycling. Some studies have reported that liquor lees are rich in various bioactive substances such as flavonoids, tocopherols and phytosterols, and have great development potential. In recent years, scholars at home and abroad have successfully applied the bioactive substances extracted from liquor lees to the fields of cosmetics and medicine by extracting bioactive substances from liquor lees, greatly improving the added value of liquor lees.

[0003] Chinese Patent CN104745431A reports a method for extracting organic acid substances from liquor lees, but this method is limited to extracting organic acid substances from liquor lees, and the final extraction state is a mixture of acid substances, and monomer substances cannot be extracted. Chinese Patent CN102746703A reports a production method for extracting active substances from beer lees, but this method can only extract pigments, proteins and dietary fibers from beer lees, and cannot extract other active ingredients or separate monomer compounds. The methods reported in other patents and documents can only extract one or several compounds from liquor lees, or only identify and analyze small and medium polar components in liquor lees, and do not study difficult-to-volatile large-polarity active substances in liquor lees.

[0004] Therefore, the present application aims to provide a method for extracting difficult-to-volatile large-polarity active monomer compounds from sauce-flavor liquor lees. SUMMARY

[0005] The present application aims to provide a method for extracting active monomer compounds from sauce-flavor liquor lees.

[0006] In one aspect, the present application provides a method for extracting active monomer compounds from sauce-flavor liquor lees, which comprises:

[0007] (1) primary extraction;

[0008] (2) secondary extraction;

[0009] (3) qualitative confirmation: qualitatively confirming the components in the secondary extraction extract obtained after the secondary extraction;

[0010] (4) Separation and purification: based on the components possibly existing in the secondary extraction extract qualitatively confirmed in step (3), the active components are separated and purified by using macroporous adsorption column chromatography, ODS column chromatography and Pre-HPLC chromatography in turn to obtain the monomer compound.

[0011] In some embodiments, the primary extraction comprises the following steps: adding an extraction solvent to a distiller's grains sample, performing ultrasonic extraction, and then performing vacuum concentration to obtain a primary extraction extract, thereby completing the primary extraction.

[0012] In some embodiments, the extraction solvent used is 60%-80% ethanol aqueous solution; preferably, the extraction solvent is 70%-80% ethanol aqueous solution; preferably, the extraction solvent is 74%-76% ethanol aqueous solution; preferably, the extraction solvent is 75% ethanol aqueous solution.

[0013] In some embodiments, the vacuum concentration conditions are as follows: water bath temperature is 60-70°C, rotation speed is 70-90 rpm, vacuum degree is 0.08-0.1 MPa, and circulating refrigerator temperature is -8--12°C.

[0014] In some embodiments, the addition ratio of the extraction solvent to the distiller's grains sample is 1-5:1 by v / v.

[0015] In some embodiments, the addition ratio of the extraction solvent to the distiller's grains sample is 1-2:1; preferably, the number of ultrasonic extractions is 2-4 times.

[0016] In some embodiments, the secondary extraction comprises the following steps: adding a secondary extraction solvent to the primary extraction extract obtained after the primary extraction, performing ultrasonic extraction, and then performing vacuum concentration on the extract to obtain a secondary extraction extract.

[0017] In some embodiments, the secondary extraction solvent is n-butanol.

[0018] In some embodiments, the addition ratio of the secondary extraction solvent to the primary extraction extract is 1:1-3 by v / v; preferably, the addition ratio of the secondary extraction solvent to the primary extraction extract is 1:2.

[0019] In some embodiments, the number of ultrasonic extractions is 1-4 times; preferably, the number of ultrasonic extractions is 3 times.

[0020] In some embodiments, the vacuum concentration conditions are as follows: water bath temperature is 40-60°C, rotation speed is 70-90 rpm, vacuum degree is 0.08-0.1 MPa, and circulating refrigerator temperature is -8--12°C.

[0021] In some embodiments, in step (3), the qualitative confirmation comprises: using HPLC-Q-TOF-MS technology to qualitatively confirm the active components in the secondary extraction extract obtained after the secondary extraction.

[0022] In some embodiments, the qualitative confirmation comprises the following steps:

[0023] Based on the HPLC-Q-TOF-MS analysis results, combined with the component and mass spectrum information in the PubChem, MassBank, and ChemicalBook databases, a chemical component information database is established.

[0024] The accurate relative molecular mass of the main chromatographic peaks obtained by HPLC-Q-TOF-MS is compared with the established chemical component information database, the molecular formula of the active components in the secondary extraction extract is speculated, and each chromatographic peak is identified, attributed, and qualitatively confirmed combined with the mass spectrum fragment information.

[0025] In some embodiments, the chromatographic conditions of HPLC-Q-TOF-MS are as follows: the chromatographic column is SHIMADZU Shim-pack VP-ODS column (250 mm x 4.6 mm x 5 μm).

[0026] In some embodiments, the mass spectrometry conditions of HPLC-Q-TOF-MS are as follows: the split ratio is set to 1:4, the positive ion is full scanned (ESI), the scan mass range is m / z 100-1000; the volume flow rate of dry gas is 6 L / min, the dry gas temperature is 180°C, the atomization gas pressure is 80 kPa. In the positive ion mode, the capillary voltage is 4500V.

[0027] In some embodiments, in step (4), the separation and purification comprises the following steps: based on the active components possibly present in the secondary extraction extract qualitatively confirmed in step (3), according to the peak time and peak position of the active components, the proportion of the mobile phase and the elution time of each mobile phase used in the separation and purification step are evaluated.

[0028] In some embodiments, the separation and purification comprises the following steps:

[0029] 1) Using macroporous adsorption column chromatography to separate the components in the secondary extraction extract;

[0030] 2) Using ODS column chromatography to separate the secondary sub-components obtained after macroporous adsorption column chromatography separation;

[0031] 3) Using Pre-HPLC chromatography to purify the tertiary sub-components obtained after ODS column chromatography separation to obtain the monomeric compound.

[0032] In some embodiments, the macroporous adsorption column chromatography is a D-101 type.

[0033] In some embodiments, the gradient elution of the components in the secondary extract infusion extract by the macroporous adsorption column chromatography comprises gradient elution of the components by elution reagents.

[0034] In some embodiments, the elution solvent in the gradient elution is 10%-100% ethanol aqueous solution.

[0035] In some embodiments, the gradient elution is performed by increasing gradient of elution solvent 8-12%; preferably, the gradient elution is performed by increasing gradient of elution solvent 10%.

[0036] In some embodiments, the concentration gradient of the elution solvent in the gradient elution is respectively 10% ethanol aqueous solution, 20% ethanol aqueous solution, 30% ethanol aqueous solution, 40% ethanol aqueous solution, 50% ethanol aqueous solution, 60% ethanol aqueous solution, 70% ethanol aqueous solution, 80% ethanol aqueous solution, 90% ethanol aqueous solution, and 100% ethanol aqueous solution.

[0037] In some embodiments, the ODS column chromatography is an ODS-C18 type.

[0038] In some embodiments, the gradient elution of the secondary sub-components obtained after the macroporous adsorption column chromatography by the ODS column chromatography comprises gradient elution of the components by elution reagents.

[0039] In some embodiments, the elution reagent in the gradient elution is 30%-60% methanol aqueous solution.

[0040] In some embodiments, the concentration gradient of the elution reagent in the gradient elution is respectively 30% methanol aqueous solution, 50% methanol aqueous solution, and 60% methanol aqueous solution.

[0041] In some embodiments, the purification of the tertiary sub-components obtained after the ODS column chromatography by the Pre-HPLC chromatography comprises gradient elution of the components by elution reagents.

[0042] In some embodiments, the elution reagent in the gradient elution is 30%-60% methanol aqueous solution.

[0043] In some embodiments, the concentration of the elution reagent in the gradient elution is respectively 35% methanol aqueous solution and 53% methanol aqueous solution.

[0044] In some embodiments, the method further comprises: using nuclear magnetic resonance, high-resolution mass spectrometry to identify the structure of the separated monomer compound, and finally determining the monomer compound.

[0045] In summary, the present application includes at least one of the following beneficial technical effects:

[0046] (1) The present application provides a method for obtaining simultaneously large-polarity phenolic acids, amino acids, dipeptides and flavonoids and other difficult-to-volatile active components from Jiangxiang Baijiu (liquor) vinasse.

[0047] (2) Using the extraction method provided by the present application, a plurality of large-polarity active monomer compounds are separated, purified and identified from Jiangxiang Baijiu vinasse, which further verifies the existence of active substances in vinasse and deepens the understanding of the polar active components in Jiangxiang Baijiu vinasse, thereby providing a theoretical support for the utilization and development of vinasse.

[0048] (3) In the prior art disclosed methods, most of them are directed to medium and small polarity substances in vinasse, and the obtained are a type of active substances or a few active substances, and most of the extraction states are mixed liquid; while through the method provided by the present application, the extraction of large-polarity substances in vinasse is realized, and through the extraction method provided by the present application, an extraction liquid containing at least 3 types of large-polarity active substances and more than 15 compounds can be obtained, and finally 10 different types of monomer compounds can be obtained. BRIEF DESCRIPTION OF DRAWINGS

[0049] Figure 1 It is the separation and extraction diagram of macroporous adsorption column chromatography in the embodiment of the present application;

[0050] Figure 2 It is the separation and purification flow chart of ODS column chromatography and Pre-HPLC chromatography in the embodiment of the present application;

[0051] Figure 3 It is the strong-polarity component compound in Jiangxiang Baijiu vinasse extracted in the embodiment of the present application;

[0052] Figure 4 It is the structure of cyclo(proline-leucine) in the embodiment of the present application 1 HNMR analysis schematic diagram;

[0053] Figure 5 It is the structure of cyclo(proline-leucine) in the embodiment of the present application 13 C NMR analysis schematic diagram;

[0054] Figure 6 It is the chromatographic comparison diagram of primary extraction of vinasse samples using different solvents in the comparative example 1 of the present application;

[0055] Figure 7 Chromatogram comparison chart of primary extraction of the distiller's grains sample in the present application comparative example 1 using different solvents;

[0056] Figure 8 Chromatogram comparison chart of secondary extraction of the distiller's grains sample in the present application comparative example 2 using different solvents. DETAILED DESCRIPTION

[0057] The technical solutions of the present application are further illustrated below by specific examples, which do not represent a limitation on the scope of protection of the present application. Some non-essential modifications and adjustments made by others according to the concept of the present application still fall within the scope of protection of the present application.

[0058] Example 1 A method for extracting active monomer compounds from Jiangxiang Baijiu distiller's grains

[0059] (1) Primary extraction: 100 g of Jiangxiang Baijiu distiller's grains sample was taken, 75% ethanol was added in an amount of 2 times the volume of the distiller's grains sample, and ultrasonic extraction was performed for 3 times. After the extraction liquid was combined, the obtained extraction liquid was concentrated under reduced pressure using a rotary evaporator, with a water bath temperature of 60℃, a rotation speed of 80 rpm, a vacuum degree of 0.09 MPa, and a circulating chiller temperature of -10℃. The rotary evaporation was performed until there was basically no extraction solvent, and a paste was obtained, which was the primary extraction extract of Jiangxiang Baijiu distiller's grains extracted by 75% ethanol. Subsequently, it was dispersed in water;

[0060] (2) Secondary extraction: n-butanol was used as the secondary extraction solvent to perform secondary extraction on the primary extraction extract of Jiangxiang Baijiu distiller's grains extracted by 75% ethanol obtained in step (1), ultrasonic extraction was performed for 3 times, the extraction liquid was combined, and the obtained extraction liquid was concentrated under reduced pressure using a rotary evaporator, with a water bath temperature of 50℃, a rotation speed of 80 rpm, a vacuum degree of 0.09 MPa, and a circulating chiller temperature of -10℃. The rotary evaporation was performed until there was basically no extraction solvent, and a paste was obtained, which was the secondary extraction extract of Jiangxiang Baijiu distiller's grains. The components in the secondary extraction extract were polar and difficult to volatilize, which were active components;

[0061] (3) The components in the secondary extraction extract obtained in step (2) were preliminarily confirmed by high performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (HPLC-Q-TOF-MS), as follows:

[0062] Based on the results of high performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry analysis, combined with the component and mass spectrometry information in the PubChem, MassBank, and ChemicalBook databases, a chemical component information database was established, and MassHunter B.06.00 software was used to collect and process data;

[0063] The accurate relative molecular mass of the main chromatographic peaks obtained by high performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry is compared with the self-built chemical component information library to deduce the molecular formula of each compound, and the mass spectrometry fragment information is combined to identify, attribute and qualify each chromatographic peak;

[0064] The chromatographic conditions of high performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry are as follows: the chromatographic column is SHIMADZU Shim-pack VP-ODS column (250 mm*4.6 mm*5 μm); the mass spectrometry conditions are as follows: the split ratio is set to 1:4, the positive ion is full scanned (ESI), the scanning mass range is m / z 100-1000; the volume flow rate of dry gas is 6 L / min, the dry gas temperature is 180°C, and the atomization gas pressure is 80 kPa. In the positive ion mode, the capillary voltage is 4500 V.

[0065] By the above-mentioned manner, the results in Table 1 are obtained. As shown in Table 1, by the above-mentioned manner, it is deduced that the strong polar active components in the secondary extraction dreg extract of Jiangxiang Baijiu obtained in step (2) mainly include 15 kinds, covering phenolic acids, amino acids, dipeptides and flavonoids, specifically lactic acid, p-hydroxyphenethyl alcohol, 4-octyloxybenzoic acid, tartaric acid, 4-methyl-2,6-di-tert-butylphenol, N-(tert-butoxycarbonyl)-D-valine, fructosyl valine, isoleucine, L-phenylalanine, proline, cyclo(proline-leucine), methyl gallate, rutin, 3,6-dimethylpiperazine-2,5-dione and cyclo(leucine-leucine). Phenolic acid compounds have biological activities such as anti-inflammatory, antibacterial, anticancer and antioxidant; amino acids are the basic units of proteins and can be metabolized in the body to synthesize hormones; flavonoids have wide beneficial effects in clinical fields such as antioxidant, cancer, diabetes, vascular disease and neurology.

[0066] Table 1 Deduction of chemical components in the secondary extraction mixed solution

[0067]

[0068]

[0069] (4) Based on the active components possibly existing in the secondary extraction extract deduced in step (3), macroporous adsorption resin column chromatography, ODS column chromatography and Pre-HPLC are used to separate and purify the active components in the secondary extraction extract of Jiangxiang Baijiu obtained in step (2), and finally monomer compounds are obtained, which are as follows:

[0070] In this step, the proportion of the mobile phase and the elution time of each mobile phase used in this step are evaluated according to the peak time and peak position of the corresponding active components.

[0071] First, the macroporous adsorption resin is used to mix the sample with twice the amount of n-butanol extract, and the components in the secondary extract of the n-butanol extract are separated by D-101 macroporous adsorption column chromatography. Gradient elution is performed with 10% as the elution solvent, and the concentration gradient of the elution solvent is 10% ethanol, 20% ethanol, 30% ethanol, 40% ethanol, 50% ethanol, 60% ethanol, 70% ethanol, 80% ethanol, 90% ethanol, and 100% ethanol. As shown in Figure 1 , 10 secondary sub-components Fr1, Fr2, Fr3, Fr4, Fr5, Fr6, Fr7, Fr8, Fr9, and Fr10 are obtained;

[0072] The secondary sub-components obtained are further separated and purified by ODS-C18 column chromatography, and gradient elution is performed with 30% methanol, 50% methanol, and 60% methanol as the elution solvent to obtain tertiary sub-components;

[0073] The tertiary sub-components obtained are purified by Pre-HPLC, and gradient elution is performed with 35% methanol and 53% methanol as the elution solvent to obtain monomeric compounds,

[0074] (5) The monomeric compounds obtained in step (4) are identified by nuclear magnetic resonance (NMR) and high-resolution mass spectrometry (MS), and the monomeric compounds obtained are finally determined, as follows:

[0075] As shown in Figure 2 , taking the secondary sub-component Fr4 as an example, after separation and purification by ODS-C18 column chromatography and Pre-HPLC, three compounds are finally obtained, and their structures are identified by nuclear magnetic resonance and high-resolution mass spectrometry. Taking Fr4-1-2 as an example, the structure is analyzed as follows:

[0076] This compound is a white powder, and ESI-MS shows m / z 211.14 [M+H] + . According to 1 D NMR information, the molecular formula is C 11 H 18 N2O2. As shown in Figure 4 , 1H-NMR (600MHz, CDCl3) showed two azomethine protons [δH 4.14 (1H, t, J = 8.2 Hz, H-8a), 4.04 (1H, t, J = 6.0 Hz, H-3)], one aliphatic methine proton [δH 1.55 (1H, m, H-10)], four methylene protons [δH 3.41-3.51 (2H, m, H-6), 2.09-2.34 (2H, m, H-8), 1.92-2.02 (2H, m, H-7), 1.66 (2H, m, H-9)], and two methyl groups [δH 1.05 (3H, d, J = 6.4 Hz, H-11), 0.98 (3H, t, J = 6.4 Hz, H-12)]; as Figure 5 shown, 13 C-NMR (150MHz, CDCl3) showed 11 carbon signals, including two amide carbons (δC 170.1, 166.1), two azomethine carbons (δC 59.0, 53.4), one azomethine carbon signal (δC 45.5), three aliphatic methylene carbon signals (δC 38.6, 35.8, 28.1, 24.7), one aliphatic methine (δC 23.3), two methyl groups (δC 22.8, 21.2). According to the molecular formula, it is speculated that the compound is formed by the dehydration condensation of two amino acids. The two doublet methyl proton signals indicate that one of the compounds contains an isopropyl structure. Among the 20 common amino acids, only valine and isoleucine contain an isopropyl structure. According to the remaining unsaturation and the number of carbon and hydrogen, it is speculated that the compound is formed by the dehydration cyclization of proline and isoleucine. The nuclear magnetic resonance data of the compound is consistent with the reference, and it is determined that Fr4-1-2 is cyclo(proline-leucine).

[0077] The structures of other compounds separated by the above similar method were identified, and finally it was determined that compound Fr4-1-1 was 3,6-dimethylpiperazine-2,5-dione, and Fr4-2-1 was rutin.

[0078] As Figure 3 shown, using the above implementation method of the application, 10 monomer compounds were finally separated and extracted from the Jiangxiang Baijiu (Luzhou-flavor liquor) vinasse sample, specifically isoleucine, tartaric acid, methyl gallate, 4-hydroxyphenethyl alcohol, 2-phenylalanine, 4-(octyloxy)benzoic acid, cyclo(leucine-leucine), 3,6-dimethylpiperazine-2,5-dione, cyclo(proline-leucine), and rutin.

[0079] Comparative Example 1: Exploring the effect of different solvents on the initial extraction of vinasse samples

[0080] 100g of lees from a soy sauce-flavored baijiu (Chinese liquor) was extracted three times with ultrasonic extraction. The extracts were combined and concentrated under reduced pressure to obtain a lees extract. Appropriate amounts of the extracts were dissolved in methanol, filtered through a 0.45μm filter membrane, and analyzed using analytical high-performance liquid chromatography (HPLC). The results are shown below. Figure 6 , Figure 7 As shown.

[0081] Using the number of peaks and resolution in the liquid chromatogram of the extract as evaluation indicators, the extraction effect of each solvent was evaluated. The comparison showed that the liquid chromatogram of the extract with 75% ethanol could separate various components well and had more peaks than other solvents. It could take into account chemical components in various polarity ranges, indicating that 75% ethanol can extract chemical components from the lees more completely.

[0082] Comparative Example 2 investigated the effect of different solvents on the secondary extraction effect of the primary extract of Maotai-flavor liquor extracted with 75% ethanol.

[0083] Polar solvents such as n-pentanol, isopentanol, and n-butanol were used as secondary extraction solvents to perform secondary extraction on the same mass of 75% ethanol extract of Maotai-flavor liquor lees, obtaining a mixed solution of polar active components in the Maotai-flavor liquor lees. The solution was analyzed using analytical high-performance liquid chromatography (HPLC), and the results are shown below. Figure 8 As shown.

[0084] The number and resolution of peaks in the liquid chromatogram of the secondary extract were used as evaluation indicators to assess the secondary extraction effect of each solvent on the primary extract. Figure 8 The results showed that in the extraction of highly polar components from the lees of Maotai-flavor liquor using n-pentanol, isoamyl alcohol, and n-butanol, the peak response values ​​of the compounds in the liquid chromatograms of n-pentanol and isoamyl alcohol were lower than those of the n-butanol extract. n-Butanol showed two main peaks with higher response values, making it more suitable for the extraction of highly polar chemical components. Moreover, n-butanol is inexpensive and readily available. In other words, even if similar solvents are selected for secondary extraction, the extraction effects of different solvents will be different.

[0085] Comparative Example 3

[0086] This comparative study investigated the effects of different types of macroporous resins on the separation and purification of components in the secondary extraction mixture of Maotai-flavor liquor lees samples, as detailed below:

[0087] Firstly, the present application mainly extracts and separates large polar active components in Jiangxiang Baijiu (liquor) vinasse samples, while in the prior art, methods such as normal pressure silica gel chromatographic column are mainly used to extract and separate active components in vinasse samples. Experimental research shows that the method of using a normal pressure silica gel chromatographic column cannot realize the separation and purification of large polar active components in the vinasse sample of the present application. Based on this, the present application further explores the separation effect of macroporous adsorption resin on the components in the secondary extraction extractives obtained in step (2) of Example 1. The results show that not all types of macroporous adsorption resins have the same separation effect on the active components in the secondary extraction extractives of vinasse. Specifically as follows:

[0088] By taking the adsorption rate and elution rate of water-soluble components such as organic acids in Jiangxiang Baijiu vinasse as indicators, the static adsorption test is used to compare the adsorption capacity of different types of macroporous resins for water-soluble components such as organic acids, and further explore the influence of different types of macroporous resins on the separation and purification effect of components in the secondary extraction extractives of Jiangxiang Baijiu vinasse samples. The results are shown in Table 2. Compared with AB-8, HPD-100 and S-8, the adsorption rate and elution rate of D-101 type macroporous resin are the best.

[0089] Table 2 Separation effect of different types of macroporous adsorption resins on secondary extraction extractives of vinasse

[0090]

[0091] Comparative Example 4

[0092] This comparative example explores the influence of gradient elution concentration with different increasing gradients as elution solvents on the separation effect of secondary sub-components after separating the components in the secondary extractives extracted by n-butanol through a D-101 type macroporous adsorption column. The elution of ethanol with different increasing gradients under the same mass is compared, and the results are shown in Table 3.

[0093] Table 3 Elution separation effect of different increasing gradients

[0094]

[0095] The results in Table 3 show that using 5% ethanol gradient elution, the polarity of the elution solvent is small, which increases the elution time and the amount of elution solvent, the elution time is as long as 90h, the amount of elution solvent needs 5L, and the collected elution solvent is concentrated under reduced pressure to obtain 8 sub-components. Although the elution time is shortened and the amount of elution solvent is reduced after using 20% and 30% ethanol gradient elution, the number of sub-components obtained by concentrating the collected elution solvent under reduced pressure is small, and the sub-components contain multiple compounds, which increases the workload of subsequent separation and purification, and is not conducive to the separation and extraction of monomer compounds. When 10% ethanol gradient elution is used, the elution time is 72h, the amount of elution solvent needs 4L, and the sub-components obtained by concentrating the collected elution solvent under reduced pressure are the most, and 10 sub-components can be obtained. The more sub-components obtained at this stage, the more conducive to the subsequent separation and purification of monomer compounds. That is, the above results show that after using D-101 macroporous adsorption column chromatography to separate the components in the secondary extract of n-butanol, different ethanol gradients have a great influence on the sub-components that can be separated.

[0096] Comparative Example 5

[0097] This comparative example further explores the method of further separating and purifying the secondary sub-components separated in Example 1, as follows:

[0098] Firstly, by taking the adsorption rate and elution rate of water-soluble components such as organic acids in Jiangxiang Baijiu (Liquor) vinasse as indicators, the static adsorption test is used to compare the adsorption capacity of different types of chromatographic columns for water-soluble components such as organic acids, and further explore the influence of different types of chromatographic columns on the separation results, and the results are shown in Table 4.

[0099] Table 4 Influence of different types of chromatographic columns on separation results

[0100]

[0101] The results in Table 4 show that the ODS-C18 chromatographic column has the best separation effect on the n-butanol sub-component of vinasse.

[0102] After determining the ODS-C18 chromatographic column, the influence of methanol elution ratio on the separation effect is further explored, and the influence of different proportions of methanol elution solvent on the elution of the same sub-component is compared, and the results are shown in Table 5.

[0103] The results in Table 5 show that when the proportion of methanol is too high, the separation effect of the compounds is poor, the compounds cannot be effectively separated, and the effect of fine separation cannot be achieved. When the proportion of methanol is too low, the compounds cannot be eluted, and the elution time is long.

[0104] Table 5 Separation effect of different gradient methanol elution

[0105]

[0106] It is to be understood that the present application is described by way of example only, and that modifications or alterations can be made to the features and embodiments described without departing from the spirit and scope of the application. In addition, modifications can be made to the features and embodiments described to accommodate specific situations and materials without departing from the spirit and scope of the application. Accordingly, the application is not limited to the specific embodiments disclosed herein, but rather, the scope of the application includes all embodiments falling within the scope of the claims.

Claims

1. A method for extracting active monomer compounds from Zao-flavor liquor lees, characterized in that, The active monomer compounds include isoleucine, tartaric acid, methyl gallate, 4-hydroxyphenethyl alcohol, 2-phenylalanine, 4-(octyloxy)benzoic acid, cyclo(Leu-Leu), 3,6-dimethylpiperazine-2,5-dione, cyclo(Pro-Leu), and rutin; and the method includes: (1) primary extraction: the primary extraction includes the following steps: adding an extraction solvent to a distiller's grains sample, performing ultrasonic extraction, and then performing vacuum concentration to obtain a primary extraction extract, thereby completing the primary extraction; The extraction solvent used is 60%-80% ethanol aqueous solution; the addition ratio of the extraction solvent to the distiller's grains sample is 1-5:1 in terms of v / v; (2) secondary extraction: the secondary extraction includes the following steps: adding a secondary extraction solvent to the primary extraction extract obtained after the primary extraction, performing ultrasonic extraction, and then performing vacuum concentration on the extraction liquid to obtain a secondary extraction extract; The secondary extraction solvent is n-butanol; The addition ratio of the secondary extraction solvent to the primary extraction extract is 1:1-3 in terms of v / v; (3) qualitative confirmation: the components in the secondary extraction extract obtained after the secondary extraction are qualitatively confirmed; (4) separation and purification: based on the active components possibly existing in the secondary extraction extract qualitatively confirmed in step (3), the active components are sequentially separated and purified by using macroporous adsorption column chromatography, ODS column chromatography, and Pre-HPLC chromatography methods, thereby obtaining the monomer compounds; The separation and purification includes the following steps: based on the active components possibly existing in the secondary extraction extract qualitatively confirmed in step (3), the proportion of the mobile phase and the elution time length of each mobile phase used in the separation and purification step are evaluated according to the peak time and peak position of the active components; based on the evaluation result, the proportion of the mobile phase and the elution time length of each mobile phase used in the separation and purification step are determined; The separation and purification includes the following steps: 1) the components in the secondary extraction extract are separated by using macroporous adsorption column chromatography; The separation of the components in the secondary extraction extract by using macroporous adsorption column chromatography includes gradient elution of the components by using an elution reagent; The concentration gradient of the elution solvent of the gradient elution is respectively 10% ethanol aqueous solution, 20% ethanol aqueous solution, 30% ethanol aqueous solution, 40% ethanol aqueous solution, 50% ethanol aqueous solution, 60% ethanol aqueous solution, 70% ethanol aqueous solution, 80% ethanol aqueous solution, 90% ethanol aqueous solution, and 100% ethanol aqueous solution; 2) the secondary sub-components obtained after the separation by using macroporous adsorption column chromatography are separated by using ODS column chromatography; The ODS column chromatography is of the ODS-C18 type; the separation of the secondary sub-components obtained after the separation by using macroporous adsorption column chromatography by using ODS column chromatography includes gradient elution of the components by using an elution reagent; The concentration gradient of the elution solvent of the gradient elution is respectively 30% methanol aqueous solution and 50% methanol aqueous solution; and 60% methanol aqueous solution. 3) purifying the tertiary sub-fraction obtained after the ODS column chromatography by using Pre-HPLC chromatography to obtain the monomeric compound; The D-101 type is used in the macroporous adsorption column chromatography. The purifying the tertiary sub-fraction obtained after the ODS column chromatography by using Pre-HPLC chromatography includes gradient elution of the sub-fraction by using elution reagents. The elution reagent concentration of the gradient elution is 35% methanol and 53% methanol respectively.

2. The method of claim 1, wherein, The extraction solvent is 70%-80% ethanol aqueous solution.

3. The method of claim 1, wherein, The extraction solvent is 74%-76% ethanol aqueous solution.

4. The method of claim 1, wherein, The addition ratio of the extraction solvent to the vinasse sample is 1-2:

1.

5. The method of claim 1, wherein, In the step (3), the qualitative confirmation includes the following steps: using HPLC-Q-TOF-MS technology to qualitatively confirm the active components in the secondary extraction extract obtained after the secondary extraction.

6. The method of claim 5, wherein, The qualitative confirmation includes the following steps: Based on the HPLC-Q-TOF-MS analysis results, the component and mass spectrum information in the PubChem, MassBank and ChemicalBook databases are combined to establish a chemical component information database. The accurate relative molecular mass of the main chromatographic peak obtained by HPLC-Q-TOF-MS is compared with the established chemical component information database to deduce the molecular formula of the active components in the secondary extraction extract, and the mass spectrum fragment information is combined to identify, attribute, and qualitatively confirm each chromatographic peak.

7. The method of claim 6, wherein, The chromatographic conditions of the HPLC-Q-TOF-MS are as follows: the chromatographic column is SHIMADZU Shim-pack VP-ODS column with specifications of 250mm×4.6mm×5μm.

8. The method of claim 7, wherein, The mass spectrum conditions of the HPLC-Q-TOF-MS are as follows: the split ratio is 1:4, the positive ion is full scanned with a scanning mass range of m / z 100-1000; the volume flow rate of the drying gas is 6L / min, the drying gas temperature is 180℃, and the atomization gas pressure is 80kPa; in the positive ion mode, the capillary voltage is 4500V.

9. The method of claim 1, wherein, The method further includes using nuclear magnetic resonance and high-resolution mass spectrometry to identify the structure of the monomeric compound obtained by separation, and finally determining the monomeric compound.

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