A method for extracting L-tyrosine from fermentation broth

Abstract

The invention provides a method for extracting L-tyrosine from a fermentation broth. The method comprises the following steps: low-speed centrifugation to obtain L-tyrosine crystals containing part of bacterial cells and a bacterial suspension containing a saturated L-tyrosine solution; high-speed centrifugation to separate the bacterial suspension to obtain bacterial cells and a supernatant, the supernatant is used for mixing and resuspending L-tyrosine crystals containing a small amount of bacterial cells obtained by low-speed centrifugation, and low-speed centrifugation to separate the crystals again to obtain L-tyrosine crystals containing a very small amount of bacterial cells; the crystals are slowly dissolved with an acid solution to obtain all L-tyrosine crystals in the obtained L-tyrosine crystal suspension to obtain an L-tyrosine solution containing a small amount of bacterial cells; the supernatant and the bacterial cells are separated by high-speed centrifugation, wherein the bacterial cells are recovered, the supernatant is decolorized by using activated carbon, and then is filtered by a plate and frame to remove waste activated carbon and a filtrate; ammonia water is slowly added to the filtrate to perform continuous crystallization, wet crystals are obtained by high-speed centrifugation, and high-yield and high-purity L-tyrosine crystals are obtained after drying.

Description

Technical Field

[0001] The invention relates to a method for extracting amino acids, in particular to a method for extracting L-tyrosine from fermentation liquid. Background Art

[0002] L-Tyrosine is one of the three major aromatic amino acids and an important conditionally essential amino acid. It plays an important role in the metabolism and growth and development of humans and animals, and has been widely used in the pharmaceutical, food, feed, chemical, agricultural and cosmetic industries.

[0003] The separation of products in the L-tyrosine industry not only requires that the final product meet relevant requirements, but also requires reducing L-tyrosine loss, improving product purity, saving water and electricity resources, and reducing extraction costs during the separation process.

[0004] The national pharmacopoeia requires that the purity of L-tyrosine should be greater than 99.0%, white crystals or crystalline powder, odorless, with a specific rotation of -11.3° to -12.1° and a light transmittance of ≥95.0%. The utility model patent (N209338523U) provides a device for extracting L-tyrosine from fermentation broth by centrifugation, but the crude L-tyrosine extracted according to the utility model still contains a large amount of fermentation broth, pigments, and bacteria, and the purity needs to be improved. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a method for extracting L-tyrosine from fermentation broth.

[0006] In order to solve the above technical problems, the technical solution of the present invention is:

[0007] A method for extracting L-tyrosine from a fermentation broth comprises the following steps: firstly, centrifuging at a low speed to obtain a bacterial suspension containing a part of bacterial L-tyrosine crystals and a saturated L-tyrosine solution; separating the bacterial suspension by high-speed centrifugation to obtain bacterial protein and a supernatant, wherein the supernatant is used for mixing and resuspending L-tyrosine crystals containing a small amount of bacterial cells obtained by low-speed centrifugation, and the resuspended liquid is centrifuged at a low speed again to obtain L-tyrosine crystals containing a very small amount of bacterial cells; the crystals are slowly dissolved with an acid solution to obtain all L-tyrosine crystals in the obtained L-tyrosine crystal suspension to obtain an L-tyrosine solution containing a small amount of bacterial cells; separating the supernatant and bacterial cells by high-speed centrifugation, wherein the bacterial cells are recovered, decolorizing the supernatant by using activated carbon, and then filtering the supernatant by plate and frame to remove the waste activated carbon and the filtrate; slowly adding ammonia water to the filtrate for continuous crystallization, and then separating by high-speed centrifugation to obtain wet L-tyrosine crystals, and drying to obtain L-tyrosine crystals.

[0008] Preferably, the above method for extracting L-tyrosine from a fermentation broth comprises the following specific steps:

[0009] (1) The fermentation liquid is subjected to low-speed centrifugation at a centrifugal speed of 2000-3000 r / min; and an upper bacterial liquid A and a crystal A are separated; the upper bacterial liquid A is composed of a solution containing saturated L-tyrosine and bacterial protein, and the crystal A is composed of bacterial protein and L-tyrosine crystals;

[0010] (2) subjecting the upper bacterial solution A obtained in step (1) to high-speed centrifugation at a centrifugal speed of 5000-6000 r / min to separate bacterial protein and a solution containing saturated L-tyrosine;

[0011] (3) stirring the saturated L-tyrosine solution obtained in step (2) and crystal A to obtain suspension A;

[0012] (4) subjecting the suspension A in step (3) to low-speed centrifugation at a speed of 2000-3000 r / min to separation to obtain an upper bacterial liquid B and crystals B; the upper bacterial liquid B is composed of a solution containing saturated L-tyrosine and bacterial protein, and the crystals B are L-tyrosine crystals still containing a small amount of bacterial cells;

[0013] (5) subjecting the upper bacterial solution B obtained in step (4) to high-speed centrifugation at a centrifugal speed of 5000-6000 r / min to separate bacterial protein and a solution containing saturated L-tyrosine;

[0014] (6) stirring the saturated L-tyrosine solution obtained in step (5) and crystal B to obtain suspension B;

[0015] (7) subjecting the suspension B in step (6) to low-speed centrifugation at a speed of 2000-3000 r / min to separation to obtain an upper bacterial liquid C and crystals C; the upper bacterial liquid C is composed of a solution containing saturated L-tyrosine and bacterial protein, and the crystals C are L-tyrosine crystals still containing a small amount of bacterial cells;

[0016] (8) subjecting the upper bacterial solution C obtained in step (7) to high-speed centrifugation at a centrifugal speed of 5000-6000 r / min to separate bacterial protein and a solution containing saturated L-tyrosine;

[0017] (9) stirring the saturated L-tyrosine solution obtained in step (8) and crystal C to obtain suspension C;

[0018] (11) slowly adding acid solution to the suspension C described in step (10) to dissolve all the L-tyrosine crystals to obtain a high-concentration L-tyrosine solution;

[0019] (12) subjecting the high-concentration L-tyrosine solution of step (11) to high-speed centrifugation to obtain supernatant A and a small amount of bacterial protein, and directly recovering the bacterial protein;

[0020] (13) decolorizing the supernatant A described in step (12) with activated carbon;

[0021] (14) subjecting the supernatant A mixed with activated carbon obtained in step (13) to plate-and-frame filtration to obtain a filtrate and waste activated carbon;

[0022] (15) adding aqueous ammonia to the filtrate stream described in step (14) to adjust the pH and perform continuous crystallization;

[0023] (16) performing high-speed centrifugation after the continuous crystallization in step (15) to separate wet L-tyrosine crystals and mother liquor;

[0024] (17) Drying the wet L-tyrosine crystals obtained in step (16) to obtain L-tyrosine crystals.

[0025] Preferably, in the above method for extracting L-tyrosine from a fermentation broth, the low-speed centrifugation speed in steps (1), (4) and (7) is 3000 r / min.

[0026] Preferably, in the above method for extracting L-tyrosine from a fermentation broth, the high-speed centrifugation speed in steps (2), (5), (8) and (16) is 5000 r / min.

[0027] Preferably, in the above method for extracting L-tyrosine from a fermentation broth, the acid solution in step (11) is dilute sulfuric acid, and the pH after adding the dilute sulfuric acid is controlled at 2-3.

[0028] Preferably, in the above method for extracting L-tyrosine from a fermentation broth, the activated carbon decolorization temperature in step (13) is room temperature, and the stirring speed of the decolorization tank is 200 r / min.

[0029] Preferably, in the above method for extracting L-tyrosine from fermentation broth, the ammonia water in step (15) is 20% ammonia water, and the pH is controlled at 5.6-5.7.

[0030] Preferably, in the above method for extracting L-tyrosine from a fermentation broth, the bacterial protein in step (12) is recovered and used as feed protein.

[0031] Preferably, in the above method for extracting L-tyrosine from a fermentation broth, the mother liquor in step (16) is used as a component of a later L-tyrosine fermentation medium.

[0032] Preferably, in the above method for extracting L-tyrosine from a fermentation broth, the temperature of the drying process in step (17) is 80°C.

[0033] Beneficial effects:

[0034] The method for extracting L-tyrosine from fermentation broth has the advantages of less L-tyrosine loss during the extraction process, high final yield and high product purity; no traditional membrane separation and ion exchange process is used in the extraction process, and no waste liquid is discharged; crystals are resuspended in the supernatant after centrifugation, and no fresh water is used, which has a significant water-saving effect; bacterial protein produced by fermentation is recovered and used as feed, reducing the discharge of waste residues; and the mother liquor finally obtained is rich in ammonium sulfate and used as a fermentation medium or organic fertilizer. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is a schematic diagram of the L-tyrosine extraction process. DETAILED DESCRIPTION

[0036] In order to enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention is further described in detail below in conjunction with specific implementation methods.

[0037] The fermentation broth used in the following examples was obtained by fermentation using the strain and method described in the invention patent, a L-tyrosine genetically engineered bacterium and a method for producing L-tyrosine (application number 201810963798.8).

[0038] Example 1

[0039] A method for extracting L-tyrosine from a fermentation broth, comprising the following steps:

[0040] a. Take 4L of L-tyrosine fermentation broth (L-tyrosine content is 45g / L), use a centrifuge to separate L-tyrosine crystals and most of the bacteria at a low speed of 3000r / min and a centrifugation time of 3min to obtain crystal A and the upper bacterial liquid A.

[0041] The upper bacterial liquid A obtained in step ba does not contain L-tyrosine crystals, and is subjected to high-speed centrifugation at a centrifugal speed of 5000 r / min for 5 min to separate the bacterial cells and the solution containing saturated L-tyrosine, and the bacterial cells are recovered and used as feed.

[0042] c. The suspension A is obtained by stirring and suspending the crystals A obtained in step a with the saturated L-tyrosine solution obtained in step b.

[0043] d. The suspension A was centrifuged at low speed to separate L-tyrosine crystals and most of the bacteria. The centrifugal speed was 3000 r / min and the centrifugal time was 3 min to obtain crystals B and the upper bacterial liquid B.

[0044] The upper bacterial liquid B obtained in step ed does not contain L-tyrosine crystals and is subjected to high-speed centrifugation at a speed of 5000 r / min for 10 min to separate the bacterial cells and the solution containing saturated L-tyrosine. The bacterial cells are recovered and used as feed.

[0045] f. The suspension B is obtained by stirring and suspending the crystals B obtained in step d using the saturated L-tyrosine solution obtained in step e.

[0046] g. Add dilute sulfuric acid to suspension B and adjust the pH to 2.0 to dissolve all L-tyrosine crystals.

[0047] h. Use the solution obtained in step g to perform high-speed centrifugation to obtain supernatant A.

[0048] i. The supernatant A was decolorized using activated carbon. The decolorization was performed at room temperature and the stirring speed of the decolorization tank was 200 r / min.

[0049] j. After decolorization, use a plate and frame filter to separate the filtrate and waste activated carbon.

[0050] k. Add 20% aqueous ammonia to the filtrate obtained in step j, adjust the pH to 5.6-5.7, and perform continuous crystallization.

[0051] 1. The L-tyrosine wet crystals and mother liquor were obtained by high-speed centrifugation at a speed of 5000 r / min and a centrifugation time of 10 min.

[0052] m. The wet L-tyrosine was dried at 80°C, and the L-tyrosine extraction yield was 92.3% and the purity was 99.1%.

[0053] Example 2

[0054] like Figure 1 As shown, compared with Example 1, the difference is that: the low-speed centrifugation and bacterial suspension are repeated 3 times, and after obtaining the suspension B, the low-speed centrifugation is performed to obtain the upper bacterial liquid C and crystal C, the upper bacterial liquid C is used for high-speed centrifugation to obtain bacterial protein and a solution containing saturated L-tyrosine, the solution containing saturated L-tyrosine is used to stir and suspend crystal C, and subsequent operations such as acid dissolution are continued. That is, the suspension B is subjected to low-speed centrifugation, the centrifugal speed is 3000r / min, the centrifugal time is 3min, and the upper bacterial liquid C and crystal C are separated; the upper bacterial liquid C is composed of a solution containing saturated L-tyrosine and bacterial protein, and the crystal C is an L-tyrosine crystal that still contains a small amount of bacterial cells; the obtained upper bacterial liquid C is subjected to high-speed centrifugation, the centrifugal speed is 5000r / min, and the centrifugal time is 10min, and the bacterial protein and the solution containing saturated L-tyrosine are separated; the obtained solution containing saturated L-tyrosine and crystal C are stirred evenly to obtain suspension C.

[0055] Compared with Example 1, the extraction yield of L-tyrosine in this example is 93.1% and the purity is 99.6%.

[0056] Example 3

[0057] Compared with Example 2, the difference is that the low-speed centrifugation and bacterial suspension are repeated 4 times, after obtaining the suspension C, low-speed centrifugation is performed to obtain the upper bacterial liquid D and crystal D, the upper bacterial liquid D is used for high-speed centrifugation to obtain bacterial protein and a solution containing saturated L-tyrosine, the solution containing saturated L-tyrosine is used to stir and suspend the crystal D, and subsequent operations such as acid addition and dissolution are continued. Compared with Example 2, the recovery rate of L-tyrosine in this example is 93.3%, and the purity is 99.6%.

[0058] Example 4

[0059] Compared with Example 2, the present embodiment is different in that dilute sulfuric acid is added to the suspension C to dissolve L-tyrosine, and the pH is controlled at 3.0. Compared with Example 2, the extraction yield of L-tyrosine in the present embodiment is 93.2% and the purity is 99.6%.

[0060] Example 5

[0061] Compared with Example 2, the difference between this example is that dilute sulfuric acid is added to the suspension C to dissolve L-tyrosine, and the pH is controlled at 4.0. Compared with Example 2, the extraction yield of L-tyrosine in this example is 92.7% and the purity is 99.5%.

[0062] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. The construction steps of the strain of the present invention are not in any particular order. Improvements and modifications such as strain transformation carried out by technicians in this technical field based on the method of the present invention or on the basis of the method are deemed to be within the scope of protection of the present invention.

Claims

1. A method for extracting L-tyrosine from a fermentation broth, characterized in that: The specific steps are as follows: (1) The fermentation liquid is centrifuged at a low speed of 2000-3000 r / min to separate the upper bacterial liquid A and crystal A; the upper bacterial liquid A is composed of a solution containing saturated L-tyrosine and bacterial protein, and the crystal A is composed of bacterial protein and L-tyrosine crystals; (2) subjecting the upper bacterial solution A obtained in step (1) to high-speed centrifugation at a centrifugal speed of 5000-6000 r / min to separate bacterial protein and a solution containing saturated L-tyrosine; (3) The saturated L-tyrosine solution obtained in step (2) and crystal A are stirred evenly to obtain suspension A; (4) subjecting the suspension A in step (3) to low-speed centrifugation at a speed of 2000-3000 r / min to separation to obtain an upper bacterial liquid B and crystals B; the upper bacterial liquid B is composed of a solution containing saturated L-tyrosine and bacterial protein, and the crystals B are L-tyrosine crystals still containing a small amount of bacterial cells; (5) subjecting the upper bacterial solution B obtained in step (4) to high-speed centrifugation at a speed of 5000-6000 r / min to separate bacterial protein and a solution containing saturated L-tyrosine; (6) The saturated L-tyrosine solution obtained in step (5) and crystal B are stirred evenly to obtain suspension B; (7) subjecting the suspension B in step (6) to low-speed centrifugation at a speed of 2000-3000 r / min to separation to obtain an upper bacterial liquid C and crystals C; the upper bacterial liquid C is composed of a solution containing saturated L-tyrosine and bacterial protein, and the crystals C are L-tyrosine crystals still containing a small amount of bacterial cells; (8) subjecting the upper bacterial solution C obtained in step (7) to high-speed centrifugation at a speed of 5000-6000 r / min to separate bacterial protein and a solution containing saturated L-tyrosine; (9) The saturated L-tyrosine solution obtained in step (8) and crystal C are stirred evenly to obtain a suspension C; (10) slowly adding dilute sulfuric acid to the suspension C described in step (9) to completely dissolve the L-tyrosine crystals, and controlling the pH at 2-3 to obtain a high-concentration L-tyrosine solution; (11) subjecting the high-concentration L-tyrosine solution of step (10) to high-speed centrifugation to obtain supernatant A and a small amount of bacterial protein, and directly recovering the bacterial protein; (12) decolorizing the supernatant A described in step (11) with activated carbon; (13) filtering the supernatant A mixed with activated carbon obtained in step (12) by plate and frame filtration to obtain a filtrate and waste activated carbon; (14) adding aqueous ammonia to the filtrate stream of step (13) to adjust the pH, and controlling the pH at 5.6-5.7 for continuous crystallization; (15) performing high-speed centrifugation after the continuous crystallization in step (14) to separate wet L-tyrosine crystals and mother liquor; (16) Drying the wet L-tyrosine crystals obtained in step (15) to obtain L-tyrosine crystals.

2. The method for extracting L-tyrosine from a fermentation broth according to claim 1, wherein: In the steps (1), (4) and (7), the low speed centrifugation speed is 3000 r / min.

3. The method for extracting L-tyrosine from a fermentation broth according to claim 1, wherein: In the steps (2), (5), (8) and (15), the high-speed centrifugal speed is 5000 r / min.

4. The method for extracting L-tyrosine from a fermentation broth according to claim 1, wherein: The pH after adding dilute sulfuric acid in step (10) is controlled at 2-3.

5. The method for extracting L-tyrosine from a fermentation broth according to claim 1, wherein: In the step (12), the activated carbon decolorization temperature is room temperature, and the stirring speed of the decolorization tank is 200 r / min.

6. The method for extracting L-tyrosine from a fermentation broth according to claim 1, wherein: In the step (11), the bacterial protein is recovered and used as feed protein.

7. The method for extracting L-tyrosine from a fermentation broth according to claim 1, wherein: The mother liquor in step (15) is used for preparing the L-tyrosine fermentation medium in the later stage.

8. The method for extracting L-tyrosine from a fermentation broth according to claim 1, wherein: The temperature of the drying process in step (16) is 80°C.

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