Conversion extraction process of high-concentration gamma-aminobutyric acid

Abstract

The invention discloses a conversion and extraction process of high-concentration gamma-aminobutyric acid, and relates to the technical field of gamma-aminobutyric acid preparation, and the conversion and extraction process comprises the following steps: thallus culture and collection, conversion, impurity removal, concentration and crystallization, crystal washing and crystal drying; activated carbon used in the traditional impurity removal process is replaced with a certain amount of absolute ethyl alcohol, after most impurities are removed through filter paper, part of soluble impurities are separated out by adjusting the pH and heating, then soluble macromolecular substances are separated out by adding absolute ethyl alcohol, and the impurity removal effect is improved. According to the method, gamma-aminobutyric acid is taken as a raw material, and then a biological flocculant is added for flocculation and impurity precipitation, so that the impurity removal effect is ensured, and the loss of gamma-aminobutyric acid is greatly reduced; and pigments and impurities can be removed by using a solvent in a coexistence state of a small amount of water and ethanol, so that the purity of the gamma-aminobutyric acid crystal is improved.

Description

Technical Field

[0001] The invention relates to the technical field of gamma-aminobutyric acid preparation, and in particular to a conversion and extraction process of high-concentration gamma-aminobutyric acid. Background Art

[0002] γ-Aminobutyric acid (GABA) is a non-protein natural amino acid widely distributed in animals, plants and microorganisms. It is the most important inhibitory neurotransmitter in the central nervous system of mammals. γ-Aminobutyric acid not only has analgesic, anti-anxiety, anticonvulsant, blood pressure lowering, anti-epileptic, hormone secretion regulating and nerve cell nutrition effects on humans, but also has the effects of promoting animal feeding, regulating endocrine, regulating hormone secretion and its reproductive physiological effects, and improving animal immunity.

[0003] Corynebacterium glutamicum is used as the chassis bacteria to construct a high-yield strain of GABA whole cell catalysis, and the whole cell is used as a catalyst to catalyze the conversion of sodium glutamate into GABA. This production technology has the advantages of high yield, few by-products, high conversion rate, and controllable reaction. However, in the existing γ-aminobutyric acid conversion and extraction process, the impurity removal step usually uses the method of adding activated carbon to the reaction solution for decolorization and impurity removal. Due to the presence of a large number of holes inside the activated carbon, a large amount of γ-aminobutyric acid will also be adsorbed, resulting in a significant reduction in the finished product yield of γ-aminobutyric acid, and the activated carbon after adsorption of pigments belongs to hazardous waste, which will cause greater environmental pressure; in addition, in the existing process, high-concentration ethanol is usually directly used for washing in the crystal washing step. Directly using high-concentration ethanol can only promote the precipitation of γ-aminobutyric acid crystals, and at the same time, a part of impurities will be precipitated and mixed in the γ-aminobutyric acid crystals, resulting in a decrease in the purity of the γ-aminobutyric acid crystals. Therefore, a high-concentration γ-aminobutyric acid conversion and extraction process is needed to solve the above problems. Summary of the invention

[0004] The purpose of the present invention is to provide a high-concentration γ-aminobutyric acid conversion and extraction process to solve the problems existing in the prior art mentioned in the above background technology.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] A conversion and extraction process for high-concentration γ-aminobutyric acid comprises the following steps:

[0007] S1: Bacterial culture and collection: First, the bacteria are cultured to induce the production of glutamate decarboxylase, the bacteria are filtered and enriched, and the bacteria are washed to remove damaged and senescent cells;

[0008] S2: Conversion: Add 500-700 g / L of glutamic acid or glutamate to the enzyme catalytic system, adjust the pH value to 7.0, and resuspend the bacteria therein for conversion reaction;

[0009] S3: Impurity removal: After the conversion is completed, the conversion liquid is filtered with filter paper to remove cells and other impurities, dilute hydrochloric acid is added to adjust the pH to 5-6, the conversion filtrate is heated at 70-90°C for 15-25 minutes, 5-15% volume of anhydrous ethanol is added to the filtrate and stirred continuously, and then a flocculant is added to the conversion liquid, and stirred continuously until the flocculant is completely dissolved, and the mixture is allowed to stand for 25-40 minutes, and then the impurities are removed by filter pressing;

[0010] S4: Concentration and crystallization: The supernatant after impurity removal is concentrated by low-temperature vacuum freezing, and is taken out when there is no obvious liquid solution;

[0011] S5: Crystal washing: Add ethanol solution to the crystals obtained in S4 for washing, so that the crystals are completely dispersed in the ethanol solution, let stand for 50-70 minutes, and remove the ethanol solution by suction; add ultrapure water 3 times the weight of the obtained crystals to stir and dissolve, use low-temperature vacuum freezing to concentrate, and take out when there is no obvious liquid solution; add ethanol solution to wash again, so that the crystals are completely dispersed in the ethanol solution, let stand for 50-70 minutes, and remove the ethanol solution by suction;

[0012] S6: Crystal drying: Dry the crystals and collect them to obtain γ-aminobutyric acid crystals.

[0013] Preferably, in S1, the bacteria are one of lactic acid bacteria and Corynebacterium glutamicum, centrifugation or membrane filtration is selected according to the volume of the reaction solution, and the filtration membrane is one of ceramic filter membrane and fiber bundle membrane.

[0014] Preferably, in S1, the bacterial culture method is: under sterile conditions, the microorganisms are inoculated into a seed culture medium, cultured at 35-37°C for 8-14 hours, and then the liquid is inoculated into a fermentation culture medium at an inoculation rate of 2.5-5% for 4 hours, and 10 mg / L of an inducer is added and cultured at a temperature of 28-32°C for 6-8 hours to obtain a microbial fermentation liquid.

[0015] Preferably, in S2, the resuspension is performed to control the OD600 to be 3-4, glutamate or glutamate salt is added to the substrate in two batches, the conversion temperature is 37° C., and the conversion time is 24 h.

[0016] Preferably, in S3, the flocculant is one of chitin, chitosan and cellulose, and the concentration is 0.5 g / L.

[0017] Preferably, in S4, the concentration temperature is set to -80°C, and the concentration is stopped when the material is dried to a solid content of ≥90%.

[0018] Preferably, in S5, an ethanol solution having a concentration of 70-80% and a volume three times that of the crystals is added for the first time, and an ethanol solution having a concentration of 85-95% and a volume four times that of the crystals is added for the second time.

[0019] Preferably, in S6, the specific step of drying the crystals is: dispersing the obtained crystals in a wide-mouth container, and drying them in an oven at 60° C. for 6-12 hours.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] 1. The present invention replaces the activated carbon used in the impurity removal process of the traditional process with a certain amount of anhydrous ethanol. First, filter paper is used to remove most of the impurities, and then some soluble impurities are precipitated by adjusting the pH and heating. Then, anhydrous ethanol is added to precipitate soluble macromolecular substances, and then a biological flocculant is added to flocculate and precipitate impurities, thereby ensuring the impurity removal effect and greatly reducing the loss of γ-aminobutyric acid.

[0022] 2. The present invention adopts a gradient concentration of ethanol for washing in the crystal washing step, which can promote the precipitation of γ-aminobutyric acid crystals. At the same time, a solvent in which a small amount of water and ethanol coexist can be used to remove pigments and impurities, thereby improving the purity of the γ-aminobutyric acid crystals. DETAILED DESCRIPTION

[0023] In order to make the technical means, creative features, objectives and effects achieved by the present invention easy to understand, the present invention is further explained below in conjunction with specific implementation methods.

[0024] The present invention provides the following technical solutions:

[0025] Example 1

[0026] A conversion and extraction process for high-concentration γ-aminobutyric acid comprises the following steps:

[0027] S1: Bacterial culture and collection: First, culture Corynebacterium glutamicum to induce the production of glutamate decarboxylase, filter and enrich the bacteria, wash the bacteria to remove damaged and senescent cells, and wash the bacteria twice with a buffer solvent;

[0028] The bacterial culture method is as follows: under sterile conditions, the microorganisms are inoculated into a seed culture medium and cultured at 36°C for 10 hours, then the liquid is inoculated into a fermentation culture medium at a 3% inoculation rate and cultured for 4 hours, 10 mg / L of an inducer is added and cultured at 30°C for 7 hours to obtain a microbial fermentation liquid.

[0029] S2: Conversion: 600 g / L sodium glutamate was added to the enzyme catalytic system in two batches, the pH value was adjusted to 7.0, the bacteria were resuspended in it for conversion reaction, the OD600 was controlled to 3 after resuspension, the conversion temperature was 37°C, and the conversion time was 24 h.

[0030] S3: Impurity removal: After the conversion is completed, the conversion liquid is filtered with filter paper to remove cells and other impurities, and dilute hydrochloric acid is added to adjust the pH to 5.5. The conversion filtrate is heated at 80°C for 20 minutes, and 10% of the volume of the filtrate is added with anhydrous ethanol and stirred continuously. Then, a chitosan flocculant with a concentration of 0.5 g / L is added to the conversion liquid, and the flocculant is stirred continuously until it is completely dissolved. The mixture is allowed to stand for 30 minutes and filtered to remove impurities.

[0031] S4: Concentration and crystallization: The supernatant after impurities removal is concentrated by low-temperature vacuum freezing. When there is no obvious liquid solution, it is taken out. The concentration temperature is set to -80°C. When the material is dried to a solid content ≥90%, the concentration is stopped.

[0032] S5: Crystal washing: Add 70% ethanol solution with a volume 3 times the volume of the crystal to the crystals obtained in S4 for washing, so that the crystals are completely dispersed in the ethanol solution, let it stand for 60 minutes, and remove the ethanol solution by suction; add 3 times the weight of ultrapure water to the obtained crystals, stir and dissolve, use low-temperature vacuum freezing to concentrate, and take out when there is no obvious liquid solution; then add 85% ethanol solution with a volume 4 times the volume of the crystal to wash, so that the crystals are completely dispersed in the ethanol solution, let it stand for 60 minutes, and remove the ethanol solution by suction.

[0033] S6: Crystal drying: The obtained crystals are dispersed in a wide-mouth container, and dried in an oven at 60° C. for 6-12 h to collect the γ-aminobutyric acid crystals.

[0034] Liquid phase detection was performed on the γ-aminobutyric acid crystals obtained in Example 1, and the purity was 98.2%, and the finished product yield was 98.1%.

[0035] Example 2

[0036] The difference between Example 2 and Example 1 is that the concentration of the ethanol solution added for the first time in S5 is 80%, the concentration of the ethanol solution added for the second time is 95%, and the other steps and parameters are the same as those in Example 1; the γ-aminobutyric acid crystals obtained in Example 2 were subjected to liquid phase detection, and the purity was 98.6%, and the finished product yield was 98.5%.

[0037] Comparative Example 1

[0038] The difference between Comparative Example 1 and Example 1 is that in S3, anhydrous ethanol is replaced with activated carbon commonly used in the existing process, and the solution is decolorized and impurized by activated carbon, and the other steps and parameters are the same as those in Example 1; the γ-aminobutyric acid crystals obtained in Comparative Example 1 are subjected to liquid phase detection, and the purity is 98.4%, and the finished product yield is 90.6%.

[0039] Comparative Example 2

[0040] The difference between Comparative Example 2 and Example 1 is that the concentration of the ethanol solution added for the first time and the concentration of the ethanol solution added for the second time in S5 are both 95%, and the other steps and parameters are the same as those in Example 1; the γ-aminobutyric acid crystals obtained in Comparative Example 2 were subjected to liquid phase detection, and the purity was 91.3%, and the finished product yield was 97.8%.

[0041] It can be analyzed from the above examples 1-2 and comparative examples 1-2 that the present invention replaces the traditional activated carbon with anhydrous ethanol in the impurity removal step, uses filter paper to remove most of the impurities, adjusts the pH and heats to precipitate some soluble impurities, and then uses ethanol to precipitate macromolecular substances (cell polysaccharides, proteins, etc.) insoluble in ethanol, and then adds a bioflocculant to flocculate and precipitate impurities, which not only ensures the impurity removal effect but also greatly reduces the loss of γ-aminobutyric acid, thereby improving the yield of finished products; in addition, the present invention uses an ethanol gradient concentration addition method to wash in the crystal washing step, and the purpose is to: directly use high-concentration ethanol can only promote the precipitation of γ-aminobutyric acid crystals, and the purpose of setting the concentration gradient is to promote the precipitation of γ-aminobutyric acid crystals, and at the same time, a solvent in a state where a small amount of water and ethanol coexist is used to remove pigments and impurities, so that the purity of γ-aminobutyric acid crystals is improved. The method of the present invention is used to prepare γ-aminobutyric acid, which can effectively improve the purity and finished product yield of γ-aminobutyric acid, and can effectively reduce production costs and operating cycles.

[0042] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A conversion and extraction process for high-concentration γ-aminobutyric acid, characterized in that: The following steps are involved: S1: Bacterial culture and collection: First, the bacteria are cultured to induce the production of glutamate decarboxylase, the bacteria are filtered and enriched, and the bacteria are washed to remove damaged and senescent cells; S2: Conversion: Add 500-700 g / L of glutamic acid or glutamate to the enzyme catalytic system, adjust the pH value to 7.0, and resuspend the bacteria therein for conversion reaction; S3: Impurity removal: After the conversion is completed, the conversion liquid is filtered with filter paper to remove cells and other impurities, dilute hydrochloric acid is added to adjust the pH to 5-6, the conversion filtrate is heated at 70-90°C for 15-25 minutes, 5-15% volume of anhydrous ethanol is added to the filtrate and stirred continuously, and then a flocculant is added to the conversion liquid, and stirred continuously until the flocculant is completely dissolved, and the mixture is allowed to stand for 25-40 minutes, and then the impurities are removed by filter pressing; S4: Concentration and crystallization: The supernatant after impurity removal is concentrated by low-temperature vacuum freezing, and is taken out when there is no obvious liquid solution; S5: Crystal washing: Add ethanol solution to the crystals obtained in S4 for washing, so that the crystals are completely dispersed in the ethanol solution, let stand for 50-70 minutes, and remove the ethanol solution by suction; add ultrapure water 3 times the weight of the obtained crystals to stir and dissolve, use low-temperature vacuum freezing to concentrate, and take out when there is no obvious liquid solution; add ethanol solution to wash again, so that the crystals are completely dispersed in the ethanol solution, let stand for 50-70 minutes, and remove the ethanol solution by suction; S6: Crystal drying: Dry the crystals and collect them to obtain γ-aminobutyric acid crystals.

2. The conversion and extraction process of high-concentration γ-aminobutyric acid according to claim 1, characterized in that: In the S1, the bacteria are one of lactic acid bacteria and Corynebacterium glutamicum, centrifugation or membrane filtration is selected according to the volume of the reaction solution, and the filtration membrane is one of ceramic filter membrane and fiber bundle membrane.

3. The conversion and extraction process of high-concentration γ-aminobutyric acid according to claim 1, characterized in that: In S1, the bacterial culture method is: under sterile conditions, the microorganisms are inoculated into a seed culture medium, cultured at 35-37°C for 8-14 hours, and then the liquid is inoculated into a fermentation culture medium at an inoculation rate of 2.5-5% for 4 hours, and 10 mg / L of an inducer is added and cultured at a temperature of 28-32°C for 6-8 hours to obtain a microbial fermentation liquid.

4. The conversion and extraction process of high-concentration γ-aminobutyric acid according to claim 1, characterized in that: In S2, the OD600 is controlled to be 3-4 by resuspending, and glutamate or glutamate salt is added to the substrate in two batches, with a conversion temperature of 37° C. and a conversion time of 24 h.

5. The conversion and extraction process of high-concentration γ-aminobutyric acid according to claim 1, characterized in that: In the S3, the flocculant is one of chitin, chitosan and cellulose, and the concentration is 0.5 g / L.

6. The conversion and extraction process of high-concentration γ-aminobutyric acid according to claim 1, characterized in that: In S4, the concentration temperature is set to -80°C, and the concentration is stopped when the material is dried to a solid content of ≥90%.

7. The conversion and extraction process of high-concentration γ-aminobutyric acid according to claim 1, characterized in that: In S5, an ethanol solution having a concentration of 70-80% and a volume three times that of the crystals is added for the first time, and an ethanol solution having a concentration of 85-95% and a volume four times that of the crystals is added for the second time.

8. The conversion and extraction process of high-concentration γ-aminobutyric acid according to claim 1, characterized in that: In S6, the specific steps of drying the crystals are: dispersing the obtained crystals in a wide-mouth container, and drying them in an oven at 60° C. for 6-12 hours.