A method for extracting gamma-aminobutyric acid from liquor waste

Abstract

The present application provides a method for extracting γ-aminobutyric acid from waste liquor, comprising the following steps: S1, grinding the waste liquor after distillation, boiling it after adding water, filtering and centrifuging, collecting the supernatant, concentrating the supernatant and adjusting the pH to 7.0; S2, loading the supernatant onto a cation exchange resin, eluting it with an eluent after water balance, and collecting the eluted γ-aminobutyric acid solution; S3, loading the eluted γ-aminobutyric acid solution onto a macroporous resin for further purification; S4, crystallizing and freeze-drying the γ-aminobutyric acid solution after further purification by the macroporous resin to obtain pure γ-aminobutyric acid. The present application can obtain γ-aminobutyric acid products with high purity and high yield. The purity of γ-aminobutyric acid can reach 99.0%, and the yield can reach 93.3%. The extraction method of the present application has very simple operation steps and good reproducibility, is suitable for industrial promotion and use, and has very high application value.

Description

Technical Field

[0001] The present application belongs to the field of biotechnology, and more specifically, relates to a method for extracting gamma-aminobutyric acid from liquor waste. Background Art

[0002] γ-Aminobutyric acid (GABA) is a white or slightly yellowish crystalline powder with strong hygroscopicity. It is highly soluble in water, slightly soluble in hot ethanol, and insoluble in organic solvents such as benzene, cold ethanol, and ether. It has a slight odor and is optically inactive, making it a polar substance. In recent years, GABA has attracted increasing attention as a novel functional food ingredient. GABA has been shown to improve sleep quality, regulate cardiovascular function, treat epilepsy, promote hormone secretion, and improve liver and kidney function. Currently, GABA is primarily prepared through chemical synthesis, plant enrichment, and microbial fermentation.

[0003] Chemical synthesis methods have very high requirements for reaction conditions, poor safety, low production efficiency, and environmental pollution, which limits their application in the preparation of GABA. CN109535021B discloses a method for separating γ-aminobutyric acid from a chemical synthesis solution. Although the separation and purification of GABA prepared by chemical synthesis is relatively easy and high-purity GABA can be obtained, GABA purified by chemical synthesis cannot be used in the food industry.

[0004] Microbial synthesis of GABA offers low cost, high content, and good safety, making it suitable for use in the food industry. However, efficient microbial strains are often difficult to obtain, and the GABA purification process is often complex. It is difficult to obtain a high-purity GABA product in high yield after multiple purification steps. This means that both high purity and high yield cannot be achieved after purification. For example, CN106544372A discloses a method for purifying γ-aminobutyric acid from a fermentation broth. After multiple purification steps, the purity of γ-aminobutyric acid can reach over 98%, but the yield is only over 70%, which is relatively low.

[0005] Until now, the main limitation on the development of GABA in food applications has been the purifying of GABA with high yield and purity from complex, GABA-rich natural components. Distillers' grains contain a large number of yeast and lactic acid bacteria, both within their cells and in their metabolites, which are rich in GABA. Utilizing distillers' grains as a resource and increasing their added value has been a research hotspot. Current processes for extracting GABA from naturally occurring GABA-rich components generally suffer from multiple extraction steps, severe environmental pollution, and low product yield and purity. Summary of the Invention

[0006] The purpose of this application is to provide a method for extracting γ-aminobutyric acid from white wine waste, so as to solve the technical problem in the prior art that γ-aminobutyric acid cannot be purified with high yield and high purity from natural components with complex components and rich in GABA.

[0007] To achieve the above object, the present application provides a method for extracting γ-aminobutyric acid from liquor waste, comprising the following steps:

[0008] S1. Grind the waste lees after distillation, add water and boil, filter and centrifuge, collect the supernatant, concentrate the supernatant and adjust the pH to 7.0;

[0009] S2. Load the supernatant onto a cation exchange resin, elute with an eluent after water balance, and collect the eluted γ-aminobutyric acid solution;

[0010] S3, loading the eluted γ-aminobutyric acid solution onto a macroporous resin for further purification;

[0011] S4. Crystallizing and freeze-drying the γ-aminobutyric acid solution purified again by the macroporous resin to obtain pure γ-aminobutyric acid.

[0012] Furthermore, in step S1, the material-liquid ratio of the waste grains to water is 1:3-5.

[0013] Furthermore, the cation exchange resin is any one of C004 cation exchange resin, D152 cation exchange resin, LX1850-H resin, LX1850-Na resin, and D001 cation exchange resin.

[0014] Furthermore, the macroporous resin is any one of LX-68, LX-1600, D301, and DA201.

[0015] Furthermore, the eluent is any one of ammonia water, acetic acid, and acetic acid-sodium acetate buffer solution.

[0016] Furthermore, the concentration of the eluent is 0.2-0.5 mol / L.

[0017] Furthermore, the crystallization process in step S4 is as follows: adding ethanol to the γ-aminobutyric acid solution for thermal dissolution, and cooling at 4° C. for crystallization.

[0018] Furthermore, the concentration of ethanol added during the heat dissolution is 25-50%.

[0019] Furthermore, the eluent is 0.25-0.45 mol / L ammonia water or 0.35 mol / L acetic acid.

[0020] Furthermore, the cation exchange resin is LX1850-H resin or LX1850-Na resin, and the macroporous resin is LX-68 or LX-1600.

[0021] Compared with the existing technology, this application has the following technical effects:

[0022] The present invention discloses a method for extracting γ-aminobutyric acid from white wine spent grains. The method purifies the pretreated spent grains successively using a cation exchange resin and a macroporous resin, thereby obtaining a γ-aminobutyric acid product with high purity and high yield. The purity of the γ-aminobutyric acid can reach 99.0%, and the yield can reach 93.3%, which is significantly higher than the current level of γ-aminobutyric acid purified from natural components rich in GABA.

[0023] The present invention discloses a method for extracting γ-aminobutyric acid from white wine spent grains. By optimizing the selection of cation exchange resins and macroporous resins and the type and concentration of eluents used in the purification process, γ-aminobutyric acid can be purified from white wine spent grains with high yield and high purity. This method solves the problem of extracting GABA from the complex components of white wine spent grains, a by-product of winemaking, for the first time, thereby increasing the value of white wine spent grains and promoting the extension and development of the white wine industry chain to a certain extent.

[0024] The method for extracting γ-aminobutyric acid from white wine waste grains of the present application has very simple operation steps and good repeatability, is suitable for industrial promotion and use, and has very high application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following briefly introduces the drawings required for use in the embodiments or descriptions of the prior art. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0026] Figure 1 This is the liquid phase spectrum of the sample before purification provided in Example 5 of the present application;

[0027] Figure 2 This is a liquid phase spectrum of the sample after purification by cation exchange resin in step S2 of Example 5 of the present application;

[0028] Figure 3 This is the liquid phase spectrum of the sample obtained in step S4 after the purification treatment provided in Example 5 of the present application. DETAILED DESCRIPTION

[0029] In order to make the technical problems, technical solutions and beneficial effects to be solved by this application more clearly understood, the present application is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain this application and are not intended to limit this application.

[0030] The terms used in the embodiments of the present application are for the purpose of describing specific embodiments only and are not intended to limit the present application. The singular forms "a", "an", "the" and "the" used in the embodiments of the present application and the appended claims are also intended to include plural forms unless the context clearly indicates otherwise.

[0031] The weights of the relevant components mentioned in the examples of this application may not only refer to the specific content of each component, but also represent the weight ratio between the components. Therefore, as long as the content of the relevant components is proportionally enlarged or reduced according to the examples of this application, it is within the scope disclosed in the examples of this application. Specifically, the mass described in the examples of this application may be a mass unit known in the chemical industry, such as μg, mg, g, kg, etc.

[0032] Example 1

[0033] Example 1 of the present application provides a method for extracting γ-aminobutyric acid from white wine waste, comprising the following steps:

[0034] S1. Pretreatment of liquor spent grains: Collect the spent grains after distillation, grind and crush, add a certain amount of water, and prepare the solid-liquid ratio to 1:3. Boil for 1 hour, filter through gauze, centrifuge at 8000 rpm for 10 minutes, collect the supernatant, concentrate to a solid content of 10%, adjust the solution pH to 7.0, and store in a refrigerator.

[0035] S2. Load the supernatant from the S1 pretreatment onto LX1850-Na ion exchange resin at a rate of 2 BV / h. Equilibrate with water for 2 BV, collecting the supernatant every 0.2 BV. Elute with 0.35 M ammonia solution at a rate of 2 BV / h, collecting the supernatant every 0.2 BV. Measure the conductivity, pH, solids content, and GABA content of each tube. Combine the three tubes containing high-concentration GABA solutions.

[0036] S3. Add seed crystals to the γ-aminobutyric acid solution purified by S2, then heat-dissolve it with 40% ethanol solution, cool it at 4°C for crystallization, collect the crystals, crystallize them, and freeze-dry them to obtain a γ-aminobutyric acid sample with a purity of 83.7% and a yield of 90.3%.

[0037] Example 2

[0038] Example 2 of the present application provides a method for extracting γ-aminobutyric acid from liquor waste, comprising the following steps:

[0039] S1. Pretreatment of liquor spent grains: Collect the spent grains after distillation, grind and crush, add a certain amount of water, and prepare the solid-liquid ratio to 1:3. Boil for 1 hour, filter through gauze, centrifuge at 8000 rpm for 10 minutes, collect the supernatant, concentrate to a solid content of 10%, adjust the solution pH to 7.0, and store in a refrigerator.

[0040] S2. Load the supernatant from the S1 pretreatment onto D152 cation exchange resin at a rate of 2 BV / h. Equilibrate with water for 2 BV, collecting the supernatant every 0.2 BV. Elute with 0.35 M acetic acid at a rate of 2 BV / h, collecting the supernatant every 0.2 BV. Measure the conductivity, pH, solids content, and GABA content of each tube. Combine the three tubes containing high-concentration GABA solutions.

[0041] S3. The γ-aminobutyric acid solution eluted from S2 is loaded onto LX-1600 macroporous resin for further purification, the effluent is collected, and then vacuum concentrated to obtain a high-concentration colorless γ-aminobutyric acid solution.

[0042] S4. Add seed crystals to the γ-aminobutyric acid solution purified by S3, then heat-dissolve it with 40% ethanol solution, cool it at 4°C for crystallization, collect the crystals, crystallize them, and freeze-dry them to obtain a γ-aminobutyric acid sample with a purity of 87.3% and a yield of 89.1%.

[0043] Example 3

[0044] The difference from Example 2 is that: C004 cation exchange resin is used in step S2 and eluted with 0.35M acetic acid solution; D301 macroporous resin is used in step S3, and other process conditions are the same.

[0045] Step S4 obtains a γ-aminobutyric acid sample with a purity of 73.5% and a yield of 84.3%.

[0046] Example 4

[0047] The difference from Example 2 is that: LX1850-Na type ion exchange resin is used in step S2, and elution is performed with 0.45M ammonia solution; LX-1600 macroporous resin is used in step S3, and other process conditions are the same.

[0048] Step S4 obtains a γ-aminobutyric acid sample with a purity of 93.1% and a yield of 94.2%.

[0049] Example 5

[0050] The difference from Example 2 is that: LX1850-Na type ion exchange resin is used in step S2, and 0.35M ammonia solution is used for elution; LX-1600 macroporous resin is used in step S3, and other process conditions are the same.

[0051] Step S4 obtains a γ-aminobutyric acid sample with a purity of 99.0% and a yield of 93.3%.

[0052] Example 6

[0053] The difference from Example 2 is that: LX1850-Na type ion exchange resin is used in step S2, and 0.35M ammonia solution is used for elution; LX-68 macroporous resin is used in step S3, and other process conditions are the same.

[0054] Step S4 obtains a γ-aminobutyric acid sample with a purity of 93.7% and a yield of 92.5%.

[0055] Example 7

[0056] The difference from Example 2 is that: LX1850-Na type ion exchange resin is used in step S2, and 0.25M ammonia solution is used for elution; LX-1600 macroporous resin is used in step S3, and other process conditions are the same.

[0057] Step S4 obtains a γ-aminobutyric acid sample with a purity of 92.3% and a yield of 91.5%.

[0058] Example 8

[0059] The difference from Example 2 is that: LX1850-H ion exchange resin is used in step S2, and elution is performed with 0.35M ammonia solution; LX-1600 macroporous resin is used in step S3, and other process conditions are the same.

[0060] Step S4 obtains a γ-aminobutyric acid sample with a purity of 97.4% and a yield of 92.8%.

[0061] Example 9

[0062] The difference from Example 2 is that: LX1850-H ion exchange resin is used in step S2, and 0.35M acetic acid solution is used for elution; LX-1600 macroporous resin is used in step S3, and other process conditions are the same.

[0063] Step S4 obtains a γ-aminobutyric acid sample with a purity of 94.8% and a yield of 92.0%.

[0064] Example 10

[0065] The difference from Example 2 is that: D001 cation exchange resin is used in step S2, and 0.35M acetic acid-sodium acetate solution is used for elution; DA201 macroporous resin is used in step S3, and other process conditions are the same.

[0066] Step S4 obtains a γ-aminobutyric acid sample with a purity of 86.4% and a yield of 91.3%.

[0067] Example 11

[0068] The difference from Example 2 is that: C004 cation exchange resin is used in step S2, and 0.35M acetic acid-sodium acetate solution is used for elution; LX-1600 macroporous resin is used in step S3, and other process conditions are the same.

[0069] Step S4 obtains a γ-aminobutyric acid sample with a purity of 85.7% and a yield of 91.1%.

[0070] Example 12

[0071] The difference from Example 2 is that: D001 cation exchange resin is used in step S2, and 0.35M acetic acid-sodium acetate solution is used for elution; LX-18 macroporous resin is used in step S3, and other process conditions are the same.

[0072] Step S4 obtains a γ-aminobutyric acid sample with a purity of 83.4% and a yield of 93.5%.

[0073] Example 13

[0074] The difference from Example 2 is that step S2 is omitted, that is, no cation exchange resin is used for purification, and the supernatant of step S1 is directly loaded onto LX-1600 macroporous resin for purification. Other process conditions are the same.

[0075] A γ-aminobutyric acid sample was obtained with a purity of 22.7% and a yield of 87.5%.

[0076] The method for detecting γ-aminobutyric acid in step S4 of each embodiment of the present application is as follows: the sample obtained in step S4 is dissolved in a solution with an ethanol / water volume ratio of 4:1, and derivatized with 4-dimethylaminoazobenzene 4-sulfonyl chloride (DABS-Cl). The sample is determined by high performance liquid chromatography, with retention time used for qualitative analysis and external standard method used for quantitative analysis. The specific analysis steps are as follows:

[0077] (1) Sample extraction

[0078] Take 2.0 g of the sample obtained in step S4 and place it in a 50 mL centrifuge tube. Add ethanol / water solution to dissolve it to 10.0 g. Mix well and test.

[0079] (2) Derivatization

[0080] Accurately pipette 1 mL of the sample solution or standard solution from step (1) into a 2 mL Eppendorf tube, add 0.2 mL of sodium bicarbonate solution and 0.40 mL of 4-dimethylaminoazobenzene 4-sulfonyl chloride / acetonitrile derivatization reagent, mix well, and derivatize in a 70°C water bath for 20 minutes. Filter with a 0.45 μm microporous aqueous filter and wait for testing. Sodium bicarbonate solution: Dissolve 0.40 g of sodium bicarbonate in water and dilute to 10 mL. 4-Dimethylaminoazobenzene 4-sulfonyl chloride / acetonitrile derivatization reagent: Weigh 20.0 mg of 4-dimethylaminoazobenzene 4-sulfonyl chloride, dissolve in acetonitrile, and dilute to 10 mL.

[0081] (3) Chromatographic determination conditions

[0082] Chromatographic column: C18 column, 250 nm × 4.6 nm, 5 μm;

[0083] Detection wavelength: 436nm;

[0084] Column temperature: 30°C;

[0085] Injection volume: 10 μL;

[0086] Mobile phase: acetonitrile and sodium acetate trihydrate solution in a volume ratio of 35:65. Sodium acetate trihydrate solution: 3.40 g sodium acetate trihydrate was dissolved in water and diluted to 500 mL, and filtered through a 0.45 μm pore water filter.

[0087] Flow rate: 1.0 mL / min.

[0088] (4) Chromatographic analysis

[0089] The standard solution and sample solution were injected into the liquid chromatograph respectively, and the qualitative analysis was carried out by retention time, while the quantitative analysis was carried out by comparing the peak area of the sample solution with the peak area of the standard solution.

[0090] Figure 1 This is the liquid phase spectrum of the supernatant obtained in step S1 of Example 5 of the present application, that is, the sample before purification; Figure 2 This is a liquid phase spectrum of the sample after purification by cation exchange resin in step S2 of Example 5 of the present application; Figure 3 This is the liquid phase spectrum of the sample obtained in step S4 after the purification treatment provided in Example 5 of the present application. Figure 1 、 Figure 2 、 Figure 3 As can be seen in the figure, before the sample was purified, it contained many impurities. However, after purification by the cation exchange resin combined with the macroporous resin in the embodiment of the present application, almost no impurity peaks appeared, indicating that the purity of the sample after purification was very high, up to 99.0%.

[0091] Because only LX1850-Na ion exchange resin was used for treatment in Example 1 of the present application, without further treatment with a macroporous resin, the purity of the resulting sample reached only 83.7%, far lower than the purity of the samples obtained in Examples 2, 4, and 11 of the present application. Because Example 13 of the present application was purified only with a macroporous resin, without using a cation exchange resin, the purity of the resulting product was very low.

[0092] The purity of the samples obtained in Examples 2, 4, and 11 of the present application ranged from 85.7% to 99.0%, and the yields reached 89.1% to 94.2%. The purity of the samples obtained in Examples 5 and 8 both reached over 97%, and the yields reached over 91%.

[0093] The purification conditions and sample purification results of Examples 1 to 13 of the present application are shown in Table 1 below.

[0094] Table 1

[0095]

[0096] As can be seen from Table 1 above, when the cation exchange resin of the embodiment of the present application is LX-1850-Na or LX-1850-H, the macroporous resin is LX-1600 or LX-68, and the eluent is 0.25-0.45 mol / L ammonia water or 0.35 mol / L acetic acid, the purity and yield of the purified product can both reach more than 91.5%.

[0097] The above embodiments merely illustrate several implementation methods of the present application. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art could make various modifications and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. A kind of liquor discarded in the lees γ -A method for extracting aminobutyric acid, characterized in that The following steps are involved: S1. Grind the waste lees after distillation, add water and boil, filter and centrifuge, collect the supernatant, concentrate the supernatant and adjust the pH to 7.0; in step S1, the material-liquid ratio of the waste lees to water is 1:3-5; S2, the supernatant was loaded onto LX1850-Na type cation exchange resin, and after water balance, it was eluted with ammonia water, and the eluted γ - GABA solution; S3, the eluted γ - GABA solution was loaded onto LX-1600 macroporous resin for further purification; S4, the macroporous resin purified again γ -Aminobutyric acid solution was crystallized and freeze-dried to obtain γ -Pure aminobutyric acid.

2. The white wine waste as claimed in claim 1 γ -A method for extracting aminobutyric acid, characterized in that The concentration of the eluent is 0.25-0.45 mol / L.

3. A kind of liquor waste grains as claimed in claim 2 γ -A method for extracting aminobutyric acid, characterized in that The crystallization process in step S4 is: γ Add ethanol to the -aminobutyric acid solution for thermal dissolution and cool to 4°C for crystallization.

4. A kind of liquor waste grains as claimed in claim 3 γ -A method for extracting aminobutyric acid, characterized in that The concentration of ethanol added during the thermal dissolution is 25-50%.

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