Separation and purification method of L-tyrosine and preparation method thereof

Abstract

The present invention relates to the field of biological fermentation technology, specifically, to a separation and purification method of L-tyrosine and a preparation method thereof. The separation and purification method includes: mixing and heating a fermentation broth and an acid so that L-tyrosine is completely dissolved; continuing to heat until the protein is completely denatured; adding activated carbon, maintaining the temperature and stirring, filtering to form a clarified liquid; maintaining the temperature of the clarified liquid, and adjusting the pH of the clarified liquid to 1-5.66 and then cooling and crystallizing; filtering to form L-tyrosine crystals and mother liquor; nanofiltration of the mother liquor to form a retained liquid and a passed liquid respectively; reverse osmosis concentration of the passed liquid to form a clear liquid circulated to the nanofiltration as a top water liquid and a reverse osmosis concentrated liquid circulated to the acid dissolution; the retained liquid is eluted and concentrated to form an eluted concentrated liquid after passing through a macroporous adsorption resin, and the eluted concentrated liquid is circulated to the fermentation end for use. The method can not only directly separate and extract L-tyrosine, but also the wastewater discharged from production realizes zero acid discharge and zero phenol discharge.

Description

Technical Field

[0001] The present invention relates to the field of biological fermentation technology, in particular to a separation and purification method of L-tyrosine and a preparation method thereof. Background Art

[0002] L-tyrosine, an aromatic polar α-amino acid containing phenolic hydroxyl group, has a molecular weight of 181.18900, CAS number: 60-18-4, white shiny fine needle-shaped crystals, odorless, bitter, insoluble in water (0.04%, 25℃), decomposes at 342-344℃, insoluble in anhydrous ethanol, ether and acetone, soluble in dilute acid or dilute alkali. Isoelectric point 5.66, specific rotation (°): [α] D22-10.6° (C=4, 1mol / L hydrochloric acid), [α] D18-13.2° (C=4, 3mol / L sodium hydroxide solution). Tyrosine is a non-essential amino acid and the raw material of many products of the body. Tyrosine can be converted into a variety of physiological substances in the body through different metabolic pathways. Tyrosine deficiency can lead to symptoms such as decreased intelligence, alkalosis, and albinism. Therefore, tyrosine is widely used in medicine, food, and chemical industries.

[0003] With the wide application of tyrosine, the production scale of tyrosine has gradually increased, and the defects in tyrosine production have also been magnified, which seriously restricts the sustainable development of tyrosine production. At present, the main production methods of tyrosine are: traditional protein hydrolysis extraction method, chemical synthesis method, microbial fermentation method and enzyme method. The extraction method uses natural protein resources such as casein, animal hair, hoof shell, horn, etc. as raw materials, acid hydrolysis alkali neutralization, alkali dissolution isoelectric point crystallization, which inevitably leads to high raw material cost, limited source, long production cycle and large environmental pollution. The chemical synthesis method cannot directly synthesize L-tyrosine, and DL-tyrosine needs to be further optically resolved, which inevitably leads to its harsh production conditions, large energy consumption, small production scale and low industrialization. The microbial fermentation method is to obtain L-tyrosine by fermenting cheap carbon sources such as molasses by excellent variants of Corynebacterium glutamicum. The screening and stability of excellent strains are limiting factors that restrict its development. The enzymatic method is a reaction in which β-tyrosinase converts substrates such as phenol-pyruvate-ammonia or phenol-L-serine into L-tyrosine. Although the development of this method is restricted by the highly toxic and corrosive phenol, the advantages of high conversion rate, high yield, easy process operation, and high feasibility of large-scale production make the enzymatic method of producing L-tyrosine still not to be underestimated.

[0004] In the prior art, the impurities in the fermentation broth of L-tyrosine produced by enzymatic method are mainly bacteria, protein, pigment and substrate. The purification focuses on repeated isoelectric point crystallization. Although the process is simple, the process is complicated, the amount of acid and alkali used is extremely large, the output wastewater is large, the substrate phenol is not treated, the environmental pollution problem is serious, the yield is low and the quality of L-tyrosine is not high. In view of this, the present invention is specially proposed. Summary of the invention

[0005] The object of the present invention is to provide a separation and purification method of L-tyrosine and a preparation method thereof. The separation and purification method provided by the embodiment of the present invention is efficient and low-pollution, and specifically, it can not only directly separate and extract L-tyrosine, but also achieve zero acid and zero phenol emissions in the wastewater discharged during production.

[0006] The present invention is achieved in that:

[0007] In a first aspect, the present invention provides a method for separating and purifying L-tyrosine, comprising: (1) mixing a fermentation broth containing L-tyrosine and an acid and heating the mixture so that the L-tyrosine is completely dissolved;

[0008] (2) Continue heating until the protein is completely denatured;

[0009] (3) adding activated carbon, stirring at maintained temperature, and filtering to form a clarified liquid;

[0010] (4) maintaining the temperature of the clarified liquid, adjusting the pH of the clarified liquid to 1-5.66, and then cooling and crystallizing;

[0011] (5) filtering to form L-tyrosine crystals and mother liquor;

[0012] (6) subjecting the mother liquor to nanofiltration to form a retentate and a pass liquid containing phenol and L-tyrosine, respectively;

[0013] The passed liquid is subjected to reverse osmosis concentration to form a clear liquid and an acid-containing reverse osmosis concentrate;

[0014] The clear liquid is circulated to the nanofiltration or reverse osmosis as a cleaning liquid (i.e., top water) and can also be circulated to the front-end process for crystallization cleaning, etc. The reverse osmosis concentrated liquid is circulated to step (1) for dissolving L-tyrosine;

[0015] (7) passing the retentate of step (6) through a macroporous adsorption resin to form an L-tyrosine-containing permeate, wherein the permeate is used to rinse a fermentation device for preparing L-tyrosine by fermentation;

[0016] After eluting the macroporous adsorption resin, the eluate is concentrated to form an eluate concentrate containing phenol, and the eluate concentrate is circulated to the fermentation end for preparing L-tyrosine for use.

[0017] In an optional embodiment, the heating temperature in step (1) is 60-80° C., the mass volume ratio of the fermentation broth to the acid is 1:1-1:1.4; and the acid is an inorganic acid, preferably hydrochloric acid.

[0018] In an optional embodiment, the heating time in step (2) is continued for 1-2 hours.

[0019] In an optional embodiment, the amount of activated carbon used in step (3) is 10-30 times the mass of the fermentation liquid, and the temperature and stirring time are maintained for 0.5-1 hour.

[0020] In an optional embodiment, the substance used to adjust the pH in step (4) is an alkali solution, preferably a hydroxide solution or ammonia water, preferably a sodium hydroxide solution;

[0021] Preferably, the concentration of the sodium hydroxide solution is 300-500 g / L, and the flow rate of the alkali solution is 200-500 ml / min;

[0022] Preferably, the temperature is lowered to 8-12°C for crystallization, and the cooling rate is 20-30°C / h.

[0023] In an optional embodiment, the L-tyrosine crystals formed by filtration in step (5) are sequentially washed, solid-liquid separated and dried.

[0024] Preferably, the filtration in step (5) and the above-mentioned solid-liquid separation are both carried out by plate-and-frame filtration, and the plate-and-frame pressure is 0.8-1.0 MPa;

[0025] Preferably, the water content of the L-tyrosine crystals after solid-liquid separation is ≤10%;

[0026] Preferably, the drying conditions are: temperature of 60-80°C and vacuum degree of -0.098Mpa.

[0027] In an optional embodiment, the pressure of the nanofiltration in step (6) is 0.1-0.2Mpa, and the flux is 200-450L / h;

[0028] The pressure of reverse osmosis concentration is 0.2-0.4Mpa, and the flux is 200-250L / h;

[0029] Preferably, the nanofiltration membrane used in step (6) nanofiltration is a rolled nanofiltration membrane with a molecular weight of 50-70D.

[0030] In an optional embodiment, the macroporous adsorption resin in step (7) is selected from any one of SD300, XDA8G and D101;

[0031] The solvent used for eluting the macroporous adsorption resin in step (7) is an alcohol solvent, preferably ethanol, preferably 20-25% ethanol.

[0032] In an optional embodiment, the fermentation broth used in step (1) is a fermentation product that has not been subjected to any treatment after fermentation.

[0033] In a second aspect, the present invention provides a method for preparing L-tyrosine, comprising the method for separating and purifying L-tyrosine according to any one of the aforementioned embodiments.

[0034] The present invention has the following beneficial effects: The embodiment of the present invention reduces the acid consumption by adopting the technology of single crystallization and membrane deacidification, reverse osmosis water removal, and resin adsorption of phenol-recovery. The acid-water separation and acid and water recycling in the above process achieve zero discharge of wastewater in the purification process, and the recycling of phenol achieves zero discharge of phenol. The L-tyrosine obtained by separation and purification has good quality and high yield, and can be widely used in the fields of medicine, food, chemical industry, daily chemicals, etc. Specifically, the separation and purification of the embodiment of the present invention has the following advantages:

[0035] (1) Heating during the acid dissolution process ensures that L-tyrosine is fully dissolved, while reducing acid consumption and also reducing alkali consumption for subsequent alkali adjustment and crystallization.

[0036] (2) Continuous high-temperature heating in an acidic environment ensures complete denaturation of the protein in the acid solution, reduces the viscosity of the solution, facilitates the filtration of activated carbon and improves the transmittance of the finished product, and also provides a material basis for achieving a single crystallization to obtain the finished product.

[0037] (3) The combination of nanofiltration-reverse osmosis-macroporous resin enables the effective separation of L-tyrosine and other components such as phenol, hydrochloric acid, and water, achieving zero emission of hydrochloric acid, water, and phenol. DETAILED DESCRIPTION

[0038] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0039] The embodiment of the present invention provides a method for separating and purifying L-tyrosine, comprising:

[0040] (1) The fermentation liquid containing L-tyrosine and an acid are mixed and heated to completely dissolve the L-tyrosine.

[0041] It should be noted that the fermentation liquid and the acid are mixed and heated by first mixing the two and then heating the mixture, or by heating the fermentation liquid and then adding the acid. In the embodiment of the present invention, the fermentation liquid is stirred and heated, and the acid is added until the L-tyrosine is completely dissolved, and then the addition of acid is stopped.

[0042] The fermentation broth can be a fermentation product that has not been treated in any way after fermentation, or a crude fermentation product that has been treated simply after fermentation. The fermentation broth used in the embodiment of the present invention is 48-60 kg of L-tyrosine fermentation product from Hangzhou Weibolai Biotechnology Co., Ltd., and the residual phenol content is about 600-1800 g and the L-tyrosine content is 40-60 kg. The fermentation broth used in the embodiment of the present invention is only an example and is not limited to the fermentation broth.

[0043] The acid used in step (1) is an inorganic acid, such as hydrochloric acid, sulfuric acid, etc. Any acid can be used as long as it can dissolve L-tyrosine and does not react with the solute in the fermentation broth to form impurities. The embodiment of the present invention uses hydrochloric acid, such as 37% dilute hydrochloric acid.

[0044] The heating temperature is 60-80° C., for example, 60° C., 61° C., 62° C., 63° C., 64° C., 65° C., 66° C., 67° C., 68° C., 69° C., 70° C., 71° C., 72° C., 73° C., 74° C., 75° C., 76° C., 77° C., 78° C., 79° C., and 80° C. Any value between 60-80° C. Heating and adding acid to dissolve L-tyrosine can reduce the amount of acid used.

[0045] The mass volume ratio of the fermentation broth (w / kg) to the acid (V / L) is 1:1-1:1.4, for example, any value between 1:1-1:4 such as 1:1, 1:2, 1:3 and 1:4.

[0046] (2) Continue heating until the protein is completely denatured;

[0047] Specifically, after L-tyrosine is completely dissolved, the addition of acid is stopped, and then heating is continued until the protein is completely denatured. The fermentation broth after acid addition can be analyzed by high-pressure liquid analysis to determine whether the protein is completely flocculated. Generally, the protein in the fermentation broth can be completely flocculated after continuous heating for 1-2 hours.

[0048] (3) adding activated carbon, stirring at maintained temperature, and filtering to form a clarified liquid;

[0049] Specifically, activated carbon is added and stirred to adsorb pigments and denatured proteins, and a high temperature is maintained during the stirring and adsorption process to prevent L-tyrosine from precipitating.

[0050] Among them, the mass ratio of activated carbon to fermentation liquid is 1:10-1:30, for example, 1:10, 1:15, 1:20, 1:25 and 1:30, etc., and the time for maintaining temperature stirring is 0.5-1 hour, for example, 30 minutes, 45 minutes and 1 hour, etc., any value between 0.5-1 hour.

[0051] The temperature of the fermentation liquid containing activated carbon is still maintained during filtration to prevent L-tyrosine from being dissolved. The filtration can adopt the existing solid-liquid separation method. The embodiment of the present invention adopts the method of passing the fermentation liquid containing activated carbon through a fluorine-lined plate frame pre-coated with a filter aid, and filtering to remove carbon to obtain a clarified liquid. The protein removal amount in the clarified liquid is ≥98%, and the transmittance T is ≥99.0%.

[0052] (4) maintaining the temperature of the clarified liquid, adjusting the pH of the clarified liquid to 1-5.66, and then cooling and crystallizing;

[0053] Specifically, the clarified liquid is introduced into a crystallization tank, and the temperature of the clarified liquid is always maintained, and then a substance for adjusting pH is slowly added to make the pH value be 1-5.66, for example, any value between 1-5.66 such as 1, 2, 3, 4, 5, 5.5 and 5.66.

[0054] Wherein, the substance used for adjusting pH is alkali solution, preferably hydroxide solution or ammonia water, preferably sodium hydroxide solution; the concentration of sodium hydroxide solution is 300-500g / L, for example, 350g / L, 400g / L, 450g / L and 500g / L, etc., any value between 300-500g / L; the flow rate of the alkali solution is 200-500ml / min, for example, 200ml / min, 250ml / min, 300ml / min, 350ml / min, 400ml / min, 450ml / min and 500ml / min, etc., any value between 200-500ml / min. And the stirring speed is 30-45rpm, for example, 30rpm, 32rpm, 35rpm, 38rpm, 40rpm and 45rpm, etc., any value between 30-45rpm.

[0055] After adjusting to the desired pH value, the temperature is lowered for crystallization, specifically, the temperature is lowered to 8-12°C, for example, 8°C, 9°C, 10°C, 11°C, 12°C, or any value between 8-12°C, wherein the cooling rate is 20-30°C / h, for example, 20°C / h, 21°C / h, 22°C / h, 23°C / h, 24°C / h, 25°C / h, 26°C / h, 27°C / h, 28°C / h, 29°C / h, 30°C / h, or any value between 20-30°C / h.

[0056] (5) filtering to form L-tyrosine crystals and mother liquor;

[0057] Specifically, after crystallization, filtration is performed, for example, plate-and-frame filtration is used for solid-liquid separation, and the formed L-tyrosine crystals are washed with 1-2 times of pure water, and plate-and-frame filtration is used for solid-liquid separation after each washing. The plate-and-frame pressure for solid-liquid separation is 0.8-1.0 MPa, for example, any value between 0.8-1.0 MPa, such as 0.8 MPa, 0.85 MPa, 0.9 MPa, 0.95 MPa, and 1.0 MPa.

[0058] The water content of the washed and separated crystals is ≤10%. The crystals are then dried, for example, vacuum dried, under the following drying conditions: temperature of 60-80°C, vacuum degree of -0.098Mpa. For example, the temperature is any value between 60-80°C, such as 60°C, 65°C, 70°C, 75°C and 80°C.

[0059] The embodiment of the present invention can separate and purify the finished product through only one crystallization, and the purity of the finished product is ≥99.9%, the transmittance T is ≥95.0%, and the yield is ≥95%.

[0060] The mother liquor formed by filtration is mixed with the crystal washing water obtained by solid-liquid separation after the crystal washing, and waits for the next step of treatment.

[0061] (6) subjecting the mother liquor to nanofiltration to form a retentate and a pass-through containing phenol and L-tyrosine, respectively; wherein the pass-through is subjected to reverse osmosis concentration to form a clear solution and a reverse osmosis concentrate containing acid; wherein the clear solution is circulated to the nanofiltration as a top water solution, and the reverse osmosis concentrate is circulated to step (1) to dissolve L-tyrosine.

[0062] Specifically, the mixed solution of the mother liquor and the crystal washing water is introduced into a rolled nanofiltration membrane with a molecular weight of 50D or 70D, and the liquid passing through the nanofiltration membrane is connected in series to the reverse osmosis membrane for further acid-water separation, thereby increasing the concentration and purity of the hydrochloric acid, so that the reverse osmosis concentrated liquid is circulated to step (1) for dissolving L-tyrosine. At the same time, the clear liquid passing through the reverse osmosis membrane is returned to the rolled nanofiltration membrane as the top water liquid for diluting the top water.

[0063] The pressure of the nanofiltration is 0.1-0.2 MPa, such as any value between 0.1-0.2 MPa, such as 0.1 MPa, 0.15 MPa, and 0.2 MPa. The flux is 200-450 L / h, such as any value between 200-450 L / h, such as 200 L / h, 250 L / h, 300 L / h, 350 L / h, 400 L / h, and 450 L / h.

[0064] The pressure of reverse osmosis concentration is 0.2-0.4 MPa, such as any value between 0.2-0.4 MPa, such as 0.2 MPa, 0.25 MPa, 0.3 MPa, 0.35 MPa and 0.4 MPa. The flux is 200-250 L / h; 200 L / h, 210 L / h, 220 L / h, 230 L / h, 240 L / h and 250 L / h, etc. Any value between 200-250 L / h.

[0065] (7) passing the retentate of step (6) through a macroporous adsorption resin to form an L-tyrosine-containing permeate, wherein the permeate is used to rinse a fermentation device for preparing L-tyrosine by fermentation;

[0066] Specifically, the retentate of the above step (6) is passed through a macroporous adsorption resin, and the phenol in the retentate is adsorbed by the macroporous adsorption resin, while L-tyrosine passes through the macroporous adsorption resin to form an effluent containing L-tyrosine, which is used for rinsing a fermentation device for fermenting and preparing L-tyrosine to increase the sample yield.

[0067] The macroporous adsorption resin is selected from any one of SD300, XDA8G and D101.

[0068] After eluting the macroporous adsorption resin, the eluate is concentrated to form an eluate concentrate containing phenol, and the eluate concentrate is circulated to the fermentation end for preparing L-tyrosine for use.

[0069] Specifically, an alcohol solvent, such as 20-25% ethanol, is used to elute the macroporous adsorption resin so that phenol is eluted into ethanol, and then the eluate is concentrated to remove the ethanol, and the ethanol is recovered to form an eluate concentrate containing phenol, which is circulated to the fermentation end for preparing L-tyrosine for secondary utilization.

[0070] The separation and purification method provided by the embodiment of the present invention improves the purification efficiency of enzymatic production of L-tyrosine and reduces the emission of acid water and phenol in the purification process. Specifically, the embodiment of the present invention can truly achieve zero emission of hydrochloric acid, water and phenol in the purification or production process only through primary crystallization and the combination of nanofiltration-reverse osmosis-macroporous adsorption resin, and the yield and purity of the finished product are high, with the purity of the finished product being ≥99.9% and the yield being ≥95%.

[0071] Although the purity of L-tyrosine obtained by the separation and purification method of L-tyrosine provided in the embodiment of the present invention is improved by about 1.5% relative to the purity of 98.2% of the existing separation and purification, the yield is improved by 5%, but it is well known to those skilled in the art that when the purity of the finished product reaches a certain degree, the purity of the finished product is extremely difficult to improve, so even if the purity is improved by 1.5%, it is also extremely difficult. Similarly, when the yield of the finished product reaches a certain degree, the yield of the finished product is extremely difficult to improve, so even if the yield is improved by 5%, it is also extremely difficult. For the improvement of purity and yield, since impurities (such as small molecule proteins, etc.) cannot be completely removed, some small molecule impurities can be filtered together with the desired product by corresponding filtration, so it will cause the product to be in the case of high purity, and its purity and yield are further improved. It is difficult, but the decolorization and protein removal in the present invention and subsequent multiple steps (such as high temperature, nanofiltration, resin adsorption, etc.) are all conducive to the further removal of impurities, and the goal of further improving purity and its yield is achieved in the case of high purity of the product.

[0072] The embodiment of the present invention further provides a method for preparing L-tyrosine, comprising the method for separating and purifying L-tyrosine according to any one of the aforementioned embodiments.

[0073] For the quality detection of L-tyrosine finished product, the embodiment of the present invention provides the following method:

[0074] 1.0000 g of L-tyrosine was accurately weighed, and the content of L-tyrosine purified in the embodiment of the present invention was determined by high performance liquid chromatography using an external standard method and 99% L-tyrosine purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. as a standard.

[0075] 0.5000 g of L-tyrosine was accurately weighed and dissolved in 1 mol / L hydrochloric acid solution. Its transmittance T was detected at a wavelength of 430 nm using a UV-visible spectrophotometer.

[0076] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0077] Example 1

[0078] (1) Acid dissolution: Take about 55 kg of the fermentation product, add 37% hydrochloric acid to the fermentation product, and heat the temperature to 70°C until the fermentation product is completely dissolved; wherein, the fermentation product (w / kg): hydrochloric acid (V / L) = 1:1.1.

[0079] (2) Protein denaturation: The acid-added solution in step (1) was heated at 70°C for 1 h.

[0080] (3) Decolorization and protein removal: The sample solution in step (2) is calculated based on the mass of the fermentation product, and activated carbon is added at a ratio of activated carbon (w): fermentation product (w) = 1:10. After adsorption at 70°C with stirring for 30 minutes, the sample solution is decarbonized in a decarbonizer.

[0081] (4) Crystallization: The clarified liquid after removing the activated carbon in step (3) was maintained at 70°C and entered into a crystallization kettle with a stirring speed of 40 rpm. NaOH solution was added to adjust the pH to 4. The flow acceleration was 300 ml / min. The temperature was cooled to 10°C and crystallization was completed. The cooling rate was 20°C / h.

[0082] (5) Solid-liquid separation: After crystallization, the material is filtered using a plate and frame filter at a pressure of 0.8 MPa. The filtered solid is washed with a small amount of pure water and filtered until the water content of the crystal is 10%. The crystal is then placed in a vacuum drying oven at a vacuum degree of -0.098 MPa and a temperature of 80°C.

[0083] (6) Acid-water separation: Take the mixed solution of crystal washing water and mother liquor obtained in step (5), enter it into a rolled nanofiltration membrane with a molecular weight of 50D, and the liquid passing through the nanofiltration membrane is connected in series to the reverse osmosis membrane. The pressure of the rolled nanofiltration membrane is 0.2Mpa, and the flux is 450L / h. The pressure of the reverse osmosis membrane is 0.25Mpa, and the flux is 250L / h.

[0084] (7) Phenol adsorption: The retentate of the nanofiltration membrane in step (6) was taken and the phenol concentration was 1.09 g / L and the L-tyrosine concentration was 8.36 g / L. The retentate was flowed into a 2L D101 macroporous resin column at a rate of 3 BV / h. When the retentate reached the column plane and the column loading was completed, the column was allowed to stand for 30 min. 2 BV of 25% ethanol was used to elute the eluate. The eluate was tested by liquid chromatography and its content was 0. The eluate was then concentrated at 60°C to remove alcohol. The sample after alcohol removal was returned to the catalytic end as water for secondary fermentation. The effluent was used to rinse the fermentation device for preparing L-tyrosine by fermentation.

[0085] Quality inspection of L-tyrosine finished product: 1.0153 g of L-tyrosine was accurately weighed, and the purity was 99.4% by liquid phase detection using external standard method. 0.5171 g of L-tyrosine was accurately weighed and dissolved in 10 ml of 1 mol / L hydrochloric acid solution, and its transmittance at 430 nm was measured by UV-visible spectrophotometer, and the transmittance was T = 95.4%, and the yield was 95.2%.

[0086] Example 2

[0087] (1) Acid dissolution: Take about 57 kg of the fermentation product, add 37% hydrochloric acid to the fermentation product, and heat the temperature to 80°C until the fermentation product is completely dissolved; wherein the fermentation product (w / kg): hydrochloric acid (V / L) = 1:1.

[0088] (2) Protein denaturation: The acid-added solution in step (1) was heated at 80°C for 1 h.

[0089] (3) Decolorization and protein removal: The sample solution in step (2) is calculated based on the mass of the fermentation product, and activated carbon is added in a ratio of activated carbon (w): fermentation product (w) = 1:10. After adsorption at 80°C with stirring for 30 minutes, the sample solution is decarbonized in a decarbonizer.

[0090] (4) Crystallization: The clarified liquid after removing the activated carbon in step (3) was maintained at 80°C and entered into a crystallization kettle with a stirring speed of 40 rpm. NaOH was added to adjust the pH to 4.21. The flow acceleration was 300 ml / min. The temperature was cooled to 10°C and crystallization was completed. The cooling rate was 20°C / h.

[0091] (5) Solid-liquid separation: After crystallization, the material is filtered using a plate and frame filter at a pressure of 1.0 MPa. The filtered solid is washed with a small amount of pure water and filtered until the water content of the crystal is 8.9%. The crystal is then placed in a vacuum drying oven at a vacuum degree of -0.098 MPa and a temperature of 80°C.

[0092] (6) Acid-water separation: The mixed solution of the crystal washing water and the mother liquor obtained in step (5) is introduced into a rolled nanofiltration membrane with a molecular weight of 50D. The liquid passing through the nanofiltration membrane is connected in series to the reverse osmosis membrane. The pressure of the rolled nanofiltration membrane is 0.15 MPa and the flux is 430 L / h. The pressure of the reverse osmosis membrane is 0.25 MPa and the flux is 250 L / h.

[0093] (7) Phenol adsorption: The retentate of the nanofiltration membrane in step (6) was taken and the phenol concentration was determined to be 0.92 g / L and the L-tyrosine concentration was 7.99 g / L. The retentate was flowed into a 0.9 L XDA8G macroporous resin column at a rate of 3 BV / h. When the retentate reached the column plane and the column loading was completed, the column was allowed to stand for 30 min and 3 BV of 20% ethanol was used to elute the eluted solution. The eluate was tested by liquid chromatography and its content was 0. The eluate was then concentrated at 60°C to remove alcohol. The sample after alcohol removal was returned to the catalytic end as water for secondary fermentation.

[0094] Quality inspection of L-tyrosine finished product: 1.0010 g of L-tyrosine was accurately weighed, and the purity was 103.21% by liquid phase detection using external standard method. 0.5037 g of L-tyrosine was accurately weighed and dissolved in 10 ml of 1 mol / L hydrochloric acid solution, and its transmittance at a wavelength of 430 nm was measured by UV-visible spectrophotometer, and the transmittance was T = 97.6%, and the yield was 95.4%.

[0095] Example 3

[0096] (1) Acid dissolution: Take about 55 kg of the fermentation product, add 37% hydrochloric acid to the fermentation product, and heat the temperature to 80°C until the fermentation product is completely dissolved; wherein, the fermentation product (w / kg): hydrochloric acid (V / L) = 1:1.

[0097] (2) Protein denaturation: The acid-added solution in step (1) was heated at 80°C for 1 h.

[0098] (3) Decolorization and protein removal: The sample solution in step (2) is calculated based on the mass of the fermentation product, and activated carbon is added in a ratio of activated carbon (w): fermentation product (w) = 1:20. After adsorption at 80°C with stirring for 30 minutes, the sample solution is decarbonized in a decarbonizer.

[0099] (4) Crystallization: The clarified liquid after removing the activated carbon in step (3) was maintained at 80°C and entered into a crystallization kettle with a stirring speed of 30 rpm. NaOH solution was added to adjust the pH to 3. The flow acceleration was 300 ml / min. The temperature was cooled to 10°C and crystallization was completed. The cooling rate was 20°C / h.

[0100] (5) Solid-liquid separation: After crystallization, the material is filtered using a plate and frame filter at a pressure of 1.0 MPa. The filtered solid is washed with a small amount of pure water and filtered until the water content of the crystal is 9.0%. The crystal is then placed in a vacuum drying oven at a vacuum degree of -0.098 MPa and a temperature of 80°C.

[0101] (6) Acid-water separation: The mixed solution of the crystal washing water and the mother liquor obtained in step (5) is introduced into a rolled nanofiltration membrane with a molecular weight of 50D. The liquid passing through the nanofiltration membrane is connected in series to the reverse osmosis membrane. The pressure of the rolled nanofiltration membrane is 0.2 MPa and the flux is 450 L / h. The pressure of the reverse osmosis membrane is 0.4 MPa and the flux is 250 L / h.

[0102] (7) Phenol adsorption: The retentate of the nanofiltration membrane in step (6) was taken and the phenol concentration was 1.21 g / L and the L-tyrosine concentration was 7.16 g / L. The retentate was flowed into a 1.00 L XDA8G macroporous resin column at a rate of 3 BV / h. When the retentate reached the column plane and the column loading was completed, the column was allowed to stand for 30 min and 3 BV of 20% ethanol was used to elute the eluted solution. The eluate was tested by liquid chromatography and its content was 0. The eluate was then concentrated at 60°C to remove alcohol. The sample after alcohol removal was returned to the catalytic end as water for secondary fermentation.

[0103] Quality inspection of L-tyrosine finished product: 0.9995 g of L-tyrosine was accurately weighed, and the purity was 102.98% by liquid phase detection using external standard method. 0.5001 g of L-tyrosine was accurately weighed and dissolved in 10 ml of 1 mol / L hydrochloric acid solution, and the transmittance T at 430 nm was measured by UV-visible spectrophotometer, and the yield was 96.3%.

[0104] Example 4

[0105] (1) Acid dissolution: Take about 56 kg of fermentation product, add 37% hydrochloric acid to the fermentation product, and heat the temperature to 80° C. until the fermentation product is completely dissolved; wherein, fermentation product (w / kg):

[0106] Hydrochloric acid (V / L) = 1:1.

[0107] (2) Protein denaturation: The acid-added solution in step (1) was heated at 80°C for 1 h.

[0108] (3) Decolorization and protein removal: In step (2), the sample solution is calculated based on the weight of L-tyrosine and the weight of activated carbon (w):

[0109] L-tyrosine (w) = activated carbon in a ratio of 1:30, adsorbed with stirring at 80°C for 60 min, and then decarbonized in a decarbonizer.

[0110] (4) Crystallization: The clarified liquid after removing the activated carbon in step (3) is maintained at 80°C and enters the crystallization kettle.

[0111] The stirring speed was 45 rpm, NaOH solution was added to adjust the pH to 1, and the flow acceleration was 200 ml / min.

[0112] The crystallization was completed when the temperature was lowered to 10°C, and the cooling rate was 20°C / h.

[0113] (5) Solid-liquid separation: After crystallization, the material is filtered using a plate and frame filter at a pressure of 1.0 MPa. The filtered 5% solid is washed with a small amount of pure water and filtered until the water content of the crystals is 8.8%. The crystals are then placed in a vacuum drying oven at a vacuum degree of -0.098 MPa and a temperature of 80°C.

[0114] (6) Acid-water separation: The mixed solution of the crystal washing water and the mother liquor obtained in step (5) is passed into a rolled nanofiltration membrane with a molecular weight of 50D. The liquid passing through the nanofiltration membrane is connected in series to the reverse osmosis membrane.

[0115] The membrane pressure is 0.2Mpa, the flux is 450L / h, the reverse osmosis membrane pressure is 0.4Mpa, the flux is 250L / h, the 0-roll nanofiltration membrane concentrate is 150L, ​​and the reverse osmosis concentrate is 60L.

[0116] (7) Phenol adsorption: The retentate of the nanofiltration membrane in step (6) was taken and the phenol concentration was determined to be 0.87 g / L and the L-tyrosine concentration was 4.79 g / L. The retentate was flowed into a 1.5 L XDA8G macroporous resin column at a rate of 3 BV / h. When the retentate reached the column plane and the column loading was completed, the column was allowed to stand for 30 min and 3 BV of 20% ethanol was used to elute the eluted solution. The eluate was tested by liquid chromatography and its content was 0. The eluate was then concentrated at 60°C to remove alcohol. The sample after alcohol removal was returned to the catalytic end as water for secondary fermentation.

[0117] Quality inspection of L-tyrosine finished product: 0.9867 g of L-tyrosine was accurately weighed, and the purity was 101.06% by liquid phase detection using external standard method. 0.5032 g of L-tyrosine was accurately weighed and dissolved in 10 ml of 1 mol / L hydrochloric acid solution, and its transmittance at a wavelength of 430 nm was measured by UV-visible spectrophotometer, and the transmittance was T = 96.0%, and the yield was 95.7%.

[0118] Comparative Example 1

[0119] (1) Acid dissolution: Take about 56 kg of the fermentation product, add 37% hydrochloric acid to the fermentation product, and heat the temperature to 80°C until the fermentation product is completely dissolved; wherein the fermentation product (w / kg): hydrochloric acid (V / L) = 1:1.

[0120] (2) Protein denaturation: The acid-added solution in step (1) was heated at 80°C for 1 h.

[0121] (3) Decolorization and protein removal: Activated carbon is added to the sample solution in step (2) at a ratio of activated carbon (w): L-tyrosine (w) = 1:30 based on the mass of L-tyrosine. After adsorption at 80°C for 60 min, the sample solution is decarbonized in a decarbonizer.

[0122] (4) Crystallization: The clarified liquid after removing the activated carbon in step (3) is maintained at 80°C and enters the crystallization kettle with a stirring speed of 45 rpm. NaOH solution is added to adjust the pH to 1. The flow acceleration is 200 ml / min. The temperature is cooled to 10°C and the crystallization is completed. The cooling rate is 20°C / h.

[0123] (5) Solid-liquid separation: After crystallization, the material is filtered using a plate and frame filter at a pressure of 1.0 MPa. The filtered solid is washed with a small amount of pure water and filtered until the water content of the crystal is 8.8%. The crystal is then placed in a vacuum drying oven at a vacuum degree of -0.098 MPa and a temperature of 80°C.

[0124] (6) Acid-water separation: Take the mixed solution of the crystal washing water and the mother liquor obtained in step (5), and enter it into a rolled nanofiltration membrane with a molecular weight of 50D. The liquid passing through the nanofiltration membrane is connected in series to the reverse osmosis membrane. The pressure of the rolled nanofiltration membrane is 0.2Mpa, and the flux is 450L / h. The pressure of the reverse osmosis membrane is 0.4Mpa, and the flux is 250L / h. The concentrated solution of the rolled nanofiltration membrane is 150L, ​​and the concentrated solution of the reverse osmosis is 60L.

[0125] (7) Phenol removal: The retentate of the nanofiltration membrane in step (6) was measured to have a phenol concentration of 0.67 g / L and an L-tyrosine concentration of 3.24 g / L. 60 g of NaOH was added to the concentrated solution to adjust the pH to a weakly alkaline state. After decomposing phenol and L-tyrosine, the waste liquid was discharged into a sewage treatment system and was no longer recycled.

[0126] Quality inspection of L-tyrosine finished product: 0.9456 g of L-tyrosine was accurately weighed, and the purity was 100.10% by liquid phase detection using external standard method. 0.5401 g of L-tyrosine was accurately weighed and dissolved in 10 ml of 1 mol / L hydrochloric acid solution, and its transmittance at a wavelength of 430 nm was measured by UV-visible spectrophotometer, and the yield was 95.0%.

[0127] Conclusion of Comparative Example 1: Considering the redox property of phenol, this method can only achieve zero emission temporarily, and the pollution of phenol to water bodies is not actually eliminated.

[0128] Comparative Example 2

[0129] (1) Acid dissolution: Take about 56 kg of the fermentation product, add 37% hydrochloric acid to the fermentation product, and heat the temperature to 80°C until the fermentation product is completely dissolved; wherein the fermentation product (w / kg): hydrochloric acid (V / L) = 1:1.

[0130] (2) Protein denaturation: The acid-added solution in step (1) was heated at 80°C for 1 h.

[0131] (3) Decolorization and protein removal: Activated carbon is added to the sample solution in step (2) at a ratio of activated carbon (w): L-tyrosine (w) = 1:30 based on the mass of L-tyrosine. After adsorption at 80°C for 60 min, the sample solution is decarbonized in a decarbonizer.

[0132] (4) Crystallization: The clarified liquid after removing the activated carbon in step (3) is maintained at 80°C and enters the crystallization kettle with a stirring speed of 45 rpm. NaOH solution is added to adjust the pH to 1. The flow acceleration is 200 ml / min. The temperature is cooled to 10°C and the crystallization is completed. The cooling rate is 20°C / h.

[0133] (5) Solid-liquid separation: After crystallization, the material is filtered using a plate and frame filter at a pressure of 1.0 MPa. The filtered solid is washed with a small amount of pure water and filtered until the water content of the crystal is 8.8%. The crystal is then placed in a vacuum drying oven at a vacuum degree of -0.098 MPa and a temperature of 80°C.

[0134] (6) Acid-water separation: The mixed solution of crystal washing water and mother liquor obtained in step (5) is fed into a multi-stage vacuum distillation system. The heating temperature of the first-stage distillation system is adjusted to 80°C and the cooling temperature is adjusted to 10°C; the heating temperature of the second-stage distillation system is adjusted to 60°C and the cooling temperature is adjusted to 4°C; the heating temperature of the third-stage distillation system is adjusted to 40°C and the cooling temperature is adjusted to -5°C. The recovered liquids of each stage are collected respectively. The first-stage recovered liquid and the second-stage recovered liquid are adjusted to a neutral pH and fed into a sewage treatment system. The third-stage recovered liquid is returned to the acid dissolution step. The concentrated liquids of each stage are collected and fed into the phenol adsorption to remove phenol.

[0135] (7) Phenol adsorption: The concentrated solutions from each stage of vacuum distillation in step (6) were measured to have a phenol concentration of 0.14 g / L and an L-tyrosine concentration of 4.67 g / L. The solutions were then flowed into a 0.4 L XDA8G macroporous resin column at a rate of 3 BV / h until they reached the column plane and were loaded onto the column. The solution was allowed to stand for 30 min and then eluted with 3 BV of 20% ethanol. The eluate was concentrated at 60°C to remove alcohol. The sample solution after removal of alcohol was returned to the catalytic end as water for secondary fermentation.

[0136] Quality inspection of L-tyrosine finished product: 1.0002 g of L-tyrosine was accurately weighed, and the purity was 99.98% by liquid phase detection using external standard method. 0.5321 g of L-tyrosine was accurately weighed and dissolved in 10 ml of 1 mol / L hydrochloric acid solution, and its transmittance at a wavelength of 430 nm was measured by UV-visible spectrophotometer, and the yield was 96.2%.

[0137] Conclusion of Comparative Example 2: This comparative example utilizes the traditional heating distillation method to recover hydrochloric acid and perform acid-water separation. The equipment investment cost is large, the separation is not thorough, and the residual hydrochloric acid in the concentrated liquid seriously affects the subsequent phenol adsorption efficiency. Due to the volatility of phenol, 0.26 g / L of phenol was detected in the tertiary recovered liquid. The introduction of phenol is bound to cause a decrease in the quality of the product after the hydrochloric acid is recovered and used.

[0138] Comparative Example 3

[0139] (1) Acid dissolution: Take about 56 kg of the fermentation product, add 37% hydrochloric acid to the fermentation product, and heat the temperature to 80°C until the fermentation product is completely dissolved; wherein the fermentation product (w / kg): hydrochloric acid (V / L) = 1:1.

[0140] (2) Protein denaturation: The solution after acid addition in step (1) was stirred at room temperature (25° C.) for 1 hour, and the protein was denatured only by the extremely high acid concentration.

[0141] (3) Decolorization and protein removal: Activated carbon is added to the sample solution in step (2) at a ratio of activated carbon (w): L-tyrosine (w) = 1:30 based on the mass of L-tyrosine. After adsorption at 80°C for 60 min, the sample solution is decarbonized in a decarbonizer.

[0142] (4) Crystallization: The clarified liquid after removing the activated carbon in step (3) is maintained at 80°C and enters the crystallization kettle with a stirring speed of 45 rpm. NaOH solution is added to adjust the pH to 1. The flow acceleration is 200 ml / min. The temperature is cooled to 10°C and the crystallization is completed. The cooling rate is 20°C / h.

[0143] (5) Solid-liquid separation: After crystallization, the material is filtered using a plate and frame filter at a pressure of 1.0 MPa. The filtered solid is washed with a small amount of pure water and filtered until the water content of the crystal is 8.8%. The crystal is then placed in a vacuum drying oven at a vacuum degree of -0.098 MPa and a temperature of 80°C.

[0144] (6) Acid-water separation: Take the mixed solution of the crystal washing water and the mother liquor obtained in step (5), and enter it into a rolled nanofiltration membrane with a molecular weight of 50D. The liquid passing through the nanofiltration membrane is connected in series to the reverse osmosis membrane. The pressure of the rolled nanofiltration membrane is 0.2Mpa, and the flux is 450L / h. The pressure of the reverse osmosis membrane is 0.4Mpa, and the flux is 250L / h. The concentrated solution of the rolled nanofiltration membrane is 150L, ​​and the concentrated solution of the reverse osmosis is 60L.

[0145] (7) Phenol adsorption: The retentate of the nanofiltration membrane in step (6) was taken and the phenol concentration was determined to be 0.84 g / L and the L-tyrosine concentration was 4.58 g / L. The retentate was flowed into a 1.5 L XDA8G macroporous resin column at a rate of 3 BV / h. When the retentate reached the column plane, the column loading was completed and the column was allowed to stand for 30 min. 3 BV of 20% ethanol was used for elution. The eluate was concentrated at 60°C to remove alcohol. The sample after removal of alcohol was returned to the catalytic end as water for secondary fermentation.

[0146] Quality inspection of L-tyrosine finished product: 0.9426 g of L-tyrosine was accurately weighed, and the purity was 88.6% by liquid phase detection using external standard method. 0.5001 g of L-tyrosine was accurately weighed and dissolved in 10 ml of 1 mol / L hydrochloric acid solution, and its transmittance at a wavelength of 430 nm was measured by UV-visible spectrophotometer, and the yield was 98.9%.

[0147] Comparative Example 4

[0148] (1) Acid dissolution: Take about 56 kg of the fermentation product, add 37% hydrochloric acid to the fermentation product, maintain the room temperature at 25°C, and stir until the fermentation product is completely dissolved; wherein, the fermentation product (w / kg): hydrochloric acid (V / L) = 1:1.

[0149] (2) Protein denaturation: The solution after acid addition in step (1) was stirred at room temperature (25° C.) for 1 hour, and the protein was denatured only by the extremely high acid concentration.

[0150] (3) Decolorization and protein removal: Activated carbon was added to the sample solution in step (2) at a ratio of activated carbon (w): L-tyrosine (w) = 1:30 based on the mass of L-tyrosine. After stirring and adsorbing at room temperature of 25°C for 60 minutes, the sample solution was decarbonized in a decarbonizer.

[0151] (4) Crystallization: The clarified liquid after removing the activated carbon in step (3) was maintained at room temperature of 25°C and entered into a crystallization kettle with a stirring speed of 45 rpm. NaOH solution was added to adjust the pH to 1. The flow acceleration was 200 ml / min. The temperature was cooled to 10°C and crystallization was completed. The cooling rate was 20°C / h.

[0152] (5) Solid-liquid separation: After crystallization, the material is filtered using a plate and frame filter at a pressure of 1.0 MPa. The filtered solid is washed with a small amount of pure water and filtered until the water content of the crystal is 8.8%. The crystal is then placed in a vacuum drying oven at a vacuum degree of -0.098 MPa and a temperature of 25°C, which is room temperature.

[0153] (6) Acid-water separation: Take the mixed solution of the crystal washing water and the mother liquor obtained in step (5), and enter it into a rolled nanofiltration membrane with a molecular weight of 50D. The liquid passing through the nanofiltration membrane is connected in series to the reverse osmosis membrane. The pressure of the rolled nanofiltration membrane is 0.2Mpa, and the flux is 450L / h. The pressure of the reverse osmosis membrane is 0.4Mpa, and the flux is 250L / h. The concentrated solution of the rolled nanofiltration membrane is 150L, ​​and the concentrated solution of the reverse osmosis is 60L.

[0154] (7) Phenol adsorption: The retentate of the nanofiltration membrane in step (6) was taken and the phenol concentration was determined to be 0.78 g / L and the L-tyrosine concentration was 4.66 g / L. The retentate was flowed into a 1.5 L XDA8G macroporous resin column at a rate of 3 BV / h. When the retentate reached the column plane, the column loading was completed and the column was allowed to stand for 30 min. 3 BV of 20% ethanol was used for elution. The eluate was concentrated at 60°C to remove alcohol. The sample after removal of alcohol was returned to the catalytic end as water for secondary fermentation.

[0155] Quality inspection of L-tyrosine finished product: 0.9852 g of L-tyrosine was accurately weighed, and the purity was 67.5% by liquid phase detection using external standard method. 0.5031 g of L-tyrosine was accurately weighed and dissolved in 10 ml of 1 mol / L hydrochloric acid solution, and its transmittance at a wavelength of 430 nm was measured by UV-visible spectrophotometer, and the transmittance was T = 8.3%, and the yield was 106.7%.

[0156] Comparative Example 5

[0157] (1) Acid dissolution: Take about 56 kg of the fermentation product, add 37% hydrochloric acid to the fermentation product, and heat the temperature to 80°C until the fermentation product is completely dissolved; wherein the fermentation product (w / kg): hydrochloric acid (V / L) = 1:1.

[0158] (2) Decolorization: Based on the mass of L-tyrosine in the sample solution in step (1), activated carbon is added at a ratio of activated carbon (w): L-tyrosine (w) = 1:30, and the mixture is stirred and adsorbed at 80°C for 60 min, and then decolorized in a decolorizer.

[0159] (3) Crystallization: The clarified liquid after decarbonization in step (2) is maintained at 80°C and enters the crystallization kettle with a stirring speed of 45 rpm. NaOH solution is added to adjust the pH to 1. The flow acceleration is 200 ml / min. The temperature is cooled to 10°C and crystallization is completed. The cooling rate is 20°C / h.

[0160] (4) Solid-liquid separation: After crystallization, the material is filtered using a plate and frame filter at a pressure of 1.0 MPa. The filtered solid is washed with a small amount of pure water and filtered until the water content of the crystal is 8.8%. The crystal is then placed in a vacuum drying oven at a vacuum degree of -0.098 MPa and a temperature of 80°C.

[0161] (5) Acid-water separation: Take the mixed solution of the crystal washing water and the mother liquor in step (4), and enter it into a rolled nanofiltration membrane with a molecular weight of 50D. The liquid passing through the nanofiltration membrane is connected in series to the reverse osmosis membrane. The pressure of the rolled nanofiltration membrane is 0.2Mpa, and the flux is 450L / h. The pressure of the reverse osmosis membrane is 0.4Mpa, and the flux is 250L / h. The concentrated solution of the rolled nanofiltration membrane is 150L, ​​and the concentrated solution of the reverse osmosis is 60L.

[0162] (6) Phenol adsorption: The retentate of the roll membrane in step (5) was taken and the phenol concentration was determined to be 0.66 g / L and the L-tyrosine concentration was 4.37 g / L. The retentate was flowed into a 1.5 L XDA8G macroporous resin column at a rate of 3 BV / h. When the retentate reached the column plane, the column loading was completed and the column was allowed to stand for 30 min. 3 BV of 20% ethanol was used for elution. The eluate was concentrated at 60°C to remove alcohol. The sample after removal of alcohol was returned to the catalytic end as water for secondary fermentation.

[0163] Quality inspection of L-tyrosine finished product: 0.9864 g of L-tyrosine was accurately weighed, and the purity was 86.55% by liquid phase detection using external standard method. 0.5041 g of L-tyrosine was accurately weighed and dissolved in 10 ml of 1 mol / L hydrochloric acid solution, and its transmittance at a wavelength of 430 nm was measured by UV-visible spectrophotometer, and the transmittance was T = 10.7%, and the yield was 99.2%.

[0164] Conclusion of Comparative Examples 3, 4 and 5: When the "protein denaturation" step or the high-temperature operation step therein is removed, a portion of impurities such as protein will remain. Therefore, when the quality of the final product is tested, the content of impurities such as residual protein is relatively high, resulting in a low L-tyrosine purity test result and a greatly reduced transmittance; the yield is slightly higher than that of the embodiment because some of the introduced impurities increase the weight of the product after purification, resulting in a higher calculated yield.

[0165] Comparative Example 6

[0166] This comparative example provides a method for separating and purifying L-tyrosine, comprising:

[0167] (1) Acid dissolution: Take about 48 kg of the fermentation product, add 37% hydrochloric acid to the fermentation product, and heat the temperature to 60°C until the fermentation product is completely dissolved; wherein, the fermentation product (w / kg): hydrochloric acid (V / L) = 1:1.4.

[0168] (2) Protein denaturation: The acid-added solution in step (1) was heated at 60°C for 1 h.

[0169] (3) Decolorization and protein removal: The sample solution in step (2) is calculated based on the mass of the fermentation product, and activated carbon is added in a ratio of activated carbon (w): fermentation product (w) = 1:10. After adsorption at 60°C with stirring for 30 minutes, the sample solution is decarbonized in a decarbonizer.

[0170] (4) Crystallization: The clarified liquid after removing the activated carbon in step (3) was maintained at 60°C and entered into a crystallization kettle with a stirring speed of 30 rpm. NaOH was added to adjust the pH to 5.66. The flow acceleration was 200 ml / min. The temperature was cooled to 10°C and crystallization was completed. The cooling rate was 30°C / h.

[0171] (5) Solid-liquid separation: After crystallization, the material is filtered using a plate and frame filter at a pressure of 0.6 MPa. The filtered solid is washed with a small amount of pure water and filtered until the water content of the crystal is 17%. The crystal is then placed in a vacuum drying oven at a vacuum degree of -0.098 MPa and a temperature of 60°C.

[0172] (6) Acid-water separation: The mixed solution of the crystal washing water and the mother liquor obtained in step (5) is taken and enters into a spiral nanofiltration membrane with a molecular weight of 70D. The liquid passing through the nanofiltration membrane is connected in series to the reverse osmosis membrane. The clear liquid of the reverse osmosis membrane is returned to the spiral nanofiltration membrane and used as the top water liquid to dilute the top water. The spiral nanofiltration membrane pressure is 0.15Mpa, the flux is 400L / h, and the reverse osmosis membrane pressure is 0.25Mpa, and the flux is 200L / h.

[0173] (7) Phenol adsorption: The retentate of the nanofiltration membrane in step (6) was measured to have a phenol concentration of 0.97 g / L and an L-tyrosine concentration of 8.49 g / L. The retentate was flowed into a 3.15 L SD300 macroporous resin column at a rate of 3 BV / h until it reached the column plane and the column loading was completed. The retentate was allowed to stand for 30 min. 5 BV of 20% ethanol was used for elution. The eluate was concentrated at 50°C to remove alcohol. The sample solution after alcohol removal was returned to the catalytic end as water for secondary fermentation. The permeate was used for rinsing the fermentation device for preparing L-tyrosine by fermentation.

[0174] Quality inspection of L-tyrosine finished product: 1.0003 g of L-tyrosine was accurately weighed, and the purity was 98.9% by liquid phase detection using external standard method. 0.5201 g of L-tyrosine was accurately weighed and dissolved in 10 ml of 1 mol / L hydrochloric acid solution, and its transmittance at a wavelength of 430 nm was measured by UV-visible spectrophotometer, and the yield was 96.2%.

[0175] Conclusion of Comparative Example 6: This comparative example changes the pressure of the plate and frame filtration, which not only causes the water content of the crystal to increase, but also significantly reduces the transparency.

[0176] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for separating and purifying L-tyrosine, characterized in that: include: (1) mixing a fermentation broth containing L-tyrosine and an acid and heating the mixture to completely dissolve the L-tyrosine; the heating temperature in step (1) is 60-80° C.; (2) continuing to heat until the protein is completely denatured; the heating time in step (2) is 1-2 hours; (3) adding activated carbon, stirring at maintained temperature, and filtering to form a clarified liquid; (4) maintaining the temperature of the clarified liquid, adjusting the pH of the clarified liquid to 1-4.21, and then cooling and crystallizing; (5) filtering to form L-tyrosine crystals and mother liquor; (6) subjecting the mother liquor to nanofiltration to form a retentate and a pass liquid containing phenol and L-tyrosine, respectively; The passed liquid is subjected to reverse osmosis concentration to form a clear liquid and an acid-containing reverse osmosis concentrate; wherein the reverse osmosis concentrated solution is circulated to step (1) for dissolving L-tyrosine; (7) passing the retentate of step (6) through a macroporous adsorption resin to form an L-tyrosine-containing permeate, wherein the permeate is used to rinse a fermentation device for preparing L-tyrosine by fermentation; After eluting the macroporous adsorption resin, the eluate is concentrated to form an eluate concentrate containing phenol, and the eluate concentrate is circulated to the fermentation end for preparing L-tyrosine for use.

2. The method for separation and purification of L-tyrosine according to claim 1, wherein The mass volume kg / L ratio of the fermentation liquid and the acid is: 1:1-1:1.4; the acid is an inorganic acid.

3. The method for separation and purification of L-tyrosine according to claim 1, wherein The acid is hydrochloric acid.

4. The method for separation and purification of L-tyrosine according to claim 1, wherein In step (3), the amount of activated carbon used is 10-30 times the mass of the fermentation liquid, and the temperature is maintained and stirred for 0.5-1 hour.

5. The method for separation and purification of L-tyrosine according to claim 1, characterized in that: The substance used to adjust the pH in step (4) is alkaline solution.

6. The method for separation and purification of L-tyrosine according to claim 5, characterized in that: The alkali solution is a hydroxide solution or ammonia water.

7. The method for separation and purification of L-tyrosine according to claim 5, wherein The alkali solution is sodium hydroxide solution.

8. The method for separation and purification of L-tyrosine according to claim 6 or 7, characterized in that: The concentration of the sodium hydroxide solution is 300-500 g / L, and the flow rate of the sodium hydroxide is 200-500 ml / min.

9. The method for separation and purification of L-tyrosine according to claim 1, wherein Cool down to 8-12℃ for crystallization, with a cooling rate of 20-30℃ / h.

10. The method for separation and purification of L-tyrosine according to claim 1, characterized in that: The L-tyrosine crystals formed by filtration in step (5) are sequentially subjected to crystal washing, solid-liquid separation and drying.

11. The method for separation and purification of L-tyrosine according to claim 10, characterized in that: The filtration in step (5) and the above-mentioned solid-liquid separation both adopt plate and frame filtration, and the plate and frame pressure is 0.8-1.0MPa.

12. The method for separation and purification of L-tyrosine according to claim 10, characterized in that: The water content of L-tyrosine crystals after solid-liquid separation is ≤10%; The drying conditions are: temperature 60-80°C, vacuum degree -0.098Mpa.

13. The method for separation and purification of L-tyrosine according to claim 1, characterized in that: In step (6), the nanofiltration pressure is 0.1-0.2Mpa and the flux is 200-450L / h; The pressure of reverse osmosis concentration is 0.2-0.4Mpa and the flux is 200-250L / h.

14. The method for separation and purification of L-tyrosine according to claim 1, characterized in that: The nanofiltration membrane used in step (6) nanofiltration is a rolled nanofiltration membrane with a molecular weight of 50-70D.

15. The method for separation and purification of L-tyrosine according to claim 1, characterized in that: In step (7), the macroporous adsorption resin is selected from any one of SD300, XDA8G and D101; The solvent used to elute the macroporous adsorption resin in step (7) is an alcohol solvent.

16. The method for separation and purification of L-tyrosine according to claim 15, characterized in that: The alcohol solvent is ethanol.

17. The method for separation and purification of L-tyrosine according to claim 15, characterized in that: The alcohol solvent is 20-25% ethanol.

18. The method for separation and purification of L-tyrosine according to claim 1, characterized in that: The fermentation liquid used in step (1) is a fermentation product that has not been subjected to any treatment after fermentation.

19. A method for preparing L-tyrosine, characterized in that: The invention relates to a method for separating and purifying L-tyrosine comprising the method according to any one of claims 1 to 18.