WO2012071766A1

WO2012071766A1 - Preparation method of l-arginine-α-ketoglutarate by direct crystallization from zymotic fluid

Info

Publication number: WO2012071766A1
Application number: PCT/CN2011/000629
Authority: WO
Inventors: 李少平; 方佳茂; 陈伟滨; 许正宏; 史劲松; 孙志浩
Original assignee: 广东环西生物科技股份有限公司
Priority date: 2010-11-29
Filing date: 2011-04-11
Publication date: 2012-06-07
Also published as: CN102020593A; CN102020593B

Abstract

A preparation method of L-arginine-α-ketoglutarate by direct crystallization from zymotic fluid is provided. The method includes: respectively pretreating L-arginine zymotic fluid and α-ketoglutaric acid zymotic fluid by flocculation, filtration or centrifugation, and then mixing them for complexing; then concentrating in vacuo, crystallizing, decolorizing with activated carbon, recrystallizing and drying to obtain the product with a high purity.

Description

Method for preparing L-arginine-α-ketoglutarate by direct crystallization from fermentation broth

The invention belongs to the field of biotechnology and relates to a preparation technology of arginine-ketoglutarate. Background technique

L-arginine-α-ketoglutarate, English name is L-Arginine-alpha-Ketoglutarate, referred to as AAKG, white or light yellow crystal, soluble in water, HC1 and NaOH solution, slightly soluble in ethanol, insoluble In acetone, ether, etc.

L-arginine-α-ketoglutarate is an amino acid salt health supplement that is increasing in sales on the international market. It is used as a functional nutritional supplement and liver protection drug, mainly for physical reinforcement and promotion. The rapid growth and recovery of muscles promotes the absorption of nutrients and energy by liver cells and maintains normal liver function. Its main physiological mechanism is that AAKG can promote the level of nitric oxide in a short period of time, promote and expand muscle movement, thereby promoting the rapid growth and recovery of muscles, enhancing strength and energy.

AAKG has 1: 1 and 2: 1 (L-arginine: α-ketoglutaric acid) two kinds of salt, namely monoarginine ketoglutarate (MAAKG) and diarginine ketone Diacid salt (DAAKG), the physical and chemical properties of the two AAKG are close, but there are also some differences in function and application. MAAKG's main role is to increase blood nitric oxide levels, stimulate vasodilation, and promote muscle growth after training. It is favored as a health care product exercise agent in the US market (http://www.mims.com/).

DAAKG is an organic acid salt with a variety of physiological effects. High-dose DAAKG is given to patients with cirrhosis, and its liver detoxification function is enhanced. Plasma ammonia and serum phenol concentrations are significantly reduced, and its oxidative decomposition is enhanced. This effect was also demonstrated in animal model trials for the treatment of ammonia poisoning or for enhancing liver detoxification, with a higher survival rate in the treated group relative to the control group and less coma. The DAAKG liver-protecting drug Euligen (trade name Youlikang) of Ruifang Pharmaceutical Co., Ltd. has carried out a series of clinical research on the basis of pharmacological analysis, toxicological identification, animal liver tissue regeneration, animal detoxification and other experiments. It has been proved that DAAKG has significant effects in liver diseases and its complications, alcoholism and its complications.

As early as 1977, studies have reported that high doses of arginine alpha-ketoglutaric acid in patients with cirrhosis can enhance their liver detoxification ability (Muting et al. Munch Med Wochenschr, 1977). At present, research on the clinical application and metabolic mechanism of AAKG is in-depth. U.S. Patent No. 2005/0085498 A1, animal experiments show that supplementation with AAKG can increase the levels of arginine and glutamine in skeletal muscle, while also stimulating the function of the immune system. A study of AAKG (15g/d) supplementation in children also showed that AAKG can increase the level of synthetic auxin and promote the metabolism of amino acids including insulin-like growth factor (IGF1), glutamine and glutamate. After a month, the growth rate increased significantly. Similarly, AAKG can promote the secretion of auxin and insulin in postoperative patients, and the metabolites glutamine and proline can also promote wound healing. In a study of AAKG anti-catabolism, 14 patients with multiple traumatic lesions with high catabolism and metabolic enhancement participated in the experiment. The test group received 20 g of AAKG per day, showing protein conversion, insulin, auxin and valley. The blood levels of free amino acids such as aminoamide, proline, and arginine are also significantly higher. In addition, studies on healthy men showed that the daily dose of 10 g of AAKG increased insulin levels by 20 to 30%, whereas this increase was not observed in the study group supplemented with arginine or alpha-ketoglutarate alone. . In vitro cell culture showed that AAKG can significantly induce the growth of human fibroblasts, which are similar to myofibroblasts in that their effects have a significant dose-effect relationship, whereas the corresponding arginine or α-ketoglutarate does not. (USP 2005/ 0085498 Al).

In addition, AAKG can be dissociated in aqueous solution to form arginine and α-ketoglutaric acid, and thus can also be used as a donor of α-ketoglutaric acid or arginine, in some cases, as arginine. Or an alternative to alpha-ketoglutaric acid. Arginine is an essential essential amino acid and synthetic protein and creatine indispensable in the human body. It is an important intermediate metabolite of the urea cycle of the organism and a precursor of nitric oxide. And α-ketoglutaric acid has a strong regulation effect on protein metabolism, and is an important substance and productivity carrier for promoting the tricarboxylic acid cycle. (US Patent USP 7645742; USP 6905707; USP 7, 645, 742)

In the production of AAKG, there are mainly three ways: First, the direct mixing method, that is, L-arginine and α-ketoglutaric acid are weighed according to the molar ratio, and mixed at 1:1 or 2:1. This product is a simple mixture of arginine and ketoglutaric acid. There is no salt bond between the two. The quality of the product is greatly affected by the quality of the raw materials and the uniformity of mixing. This method is not used in general pharmaceutical or food production; The second is the dissolution-lyophilization method, respectively preparing a certain concentration of L-arginine, α-ketoglutaric acid solution, mixing according to a certain molar ratio, and then freeze-drying the solution to become AAKG, two substances in the method Forms a stable organic acid salt, but the lyophilization process consumes a large amount of energy and is generally only suitable for AAKG injections. Production; Chinese patents 200710063477.4, 200710063478.9, 200710063479.3 all involve the above two methods. The third method is crystallization. After the solution is mixed, the solution is concentrated or organic solvent is added to reduce the solubility of AAKG, and the AAKG crystal is precipitated. After washing and drying, the AAKG product is obtained. The purity of the product prepared by the method is high, and the product quality can be guaranteed. However, the process is complicated and the cost is high (CN 101591271A:).

The L-arginine and α-ketoglutaric acid required for the preparation of AAKG by the above three methods must meet the requirements of the raw material drug, especially the first two methods, and the preparation process cannot remove impurities introduced by the raw material. Put forward higher requirements for quality control of products. The production process of the crystallization process is the preferred method for preparing AAKG.

CN101591271A describes a preparation method for crystallizing L-arginine α-ketoglutarate, which is prepared by mixing and crystallizing a commercially available L-arginine and α-ketoglutaric acid raw material, and is known as L-arginine currently marketed. Acid is extracted from the hair (usually pig hair, human hair) by acid hydrolysis. (X-ketoglutaric acid is a petroleum-based raw material chemical synthesis product. Both sources of raw materials have biosafety concerns. L- Arginine and α-ketoglutaric acid, as described in CN101011372A, a commercially available chemical synthetic product, an α-ketoglutarate-arginine salt preparation derived from a hair extract raw material, but a pharmaceutical raw material Not necessarily suitable for health foods.

Summary of the invention

The object of the present invention is to provide a method for preparing AAKG from a fermentation source of L-arginine or α-ketoglutaric acid raw material, and to directly use the fermentation liquid, in order to overcome the disadvantages of the above AAKG product and its preparation process. L-arginine and α-ketoglutaric acid in the fermentation broth are used as raw materials for the preparation of AAKG, instead of extracting the refined crystalline product.

In the present invention, the desired raw materials L-arginine and α-ketoglutaric acid are fermentation products obtained by metabolic fermentation of non-transgenic wild type microorganisms using plant-derived starch glucose as a carbon source, referred to as fermentation liquid, The α-ketoglutaric acid in the fermentation broth is mixed with L-arginine in the fermentation broth in a certain ratio, and the salt is chelated. The L-arginine-α- is directly crystallized by a common chemical separation technology unit and equipment. Ketoglutarate, the preparation process includes fermentation unit pretreatment, chelation, concentration, crystallization, dissolution and decolorization, recrystallization and crystal drying, etc., L-arginine fermentation broth pretreatment, removal of bacteria and large Molecular impurities, adding a certain amount of α-ketoglutarate fermentation filtrate which is simultaneously purified for mixing, ie, chelation, concentration by vacuum evaporation, and then obtaining L-sperm ammonia by means of cooling crystallization The crude product of acid α-ketoglutarate, the crude product is decolorized by dissolution, and recrystallized from an organic solvent, and finally dried to obtain high-purity L-arginine α-ketoglutarate.

In the present invention, the fermentation liquid pretreatment technology unit is characterized in that the L-arginine fermentation liquid or the α-ketoglutaric acid fermentation liquid is flocculated, filtered or centrifuged according to a known method, and the bacteria are removed and larger. Solid particles, the process of obtaining a clear filtrate. The flocculation operation uses a flocculant such as aluminum sulfate, CaCl ₂ , polyacrylamide or chitosan or other inorganic aluminum salts, iron salts, calcium salts and high-molecular flocculants. The filtering operation is carried out by using equipment such as plate and frame filtration, vacuum filtration or belt filtration, and is particularly suitable for large-scale preparation process; the centrifugal operation is carried out by centrifugal separation equipment for solid-liquid separation, and is particularly suitable for a small-scale preparation process. .

In the present invention, the chelation technology unit is a process in which a certain amount of α-ketoglutaric acid fermentation broth or α-ketoglutaric acid solution is added according to the content of L-arginine in the filtrate. The molar content of α-ketoglutaric acid added is 0.3 to 1.5 times the molar amount of L-arginine in the system.

In the present invention, the concentration technology unit is a process of concentrating a solution containing L-arginine and α-ketoglutaric acid to bring the AAKG into a supersaturated state. The preferred concentration apparatus is a vacuum concentration, two-effect concentration or multi-effect concentration apparatus, and the solution is concentrated to 15% to 50% of the volume of the L-arginine original solution.

In the crystallization technology unit of the present invention, the concentrated liquid is placed at a temperature of -5 ° C to 15 ° C for crystallization, or a crystallization pot is used, and the solution is cooled by a cooling liquid, and the crystallization process is controlled at 4 to 50 ho. The crystals are then collected by filtration or centrifugation.

In the present invention, the dissolving and decolorizing technique unit is characterized in that the crystal is redissolved in 2 to 5 parts by weight of deionized water, stirred and sufficiently dissolved, and then deactivated by adding granular activated carbon. The amount of activated carbon added is equivalent to 0.5% to 3.0% of the mass of the AAKG crystal. The mixture is stirred and heated to 40 to 90 ° C for 20 to 60 minutes, and then filtered through a plate frame to collect the AAKG filtrate.

In the present invention, the recrystallization technology unit is a hydrophilic organic solvent which is slowly added to the solution volume by 1 to 4 times in the AAKG filtrate, and is recrystallized by standing at room temperature or in a refrigerator for 8 to 50 hours, and collected by filtration. Crystals were washed 2 to 3 times with a small amount of cold fresh solvent. The hydrophilic organic solvent includes ethanol, methanol, acetone, isopropanol, glycerin, acetonitrile and the like, and the preferred solvent is ethanol or acetone.

In the present invention, the crystal drying technology unit performs crystal drying by using a device such as vacuum drying, hot air circulation oven or air flow drying, and the drying temperature is 50° (: 150 150 ° C). In the present invention, the technical unit of the fermentation liquid pretreatment, chelation, concentration, crystallization, dissolution decolorization, recrystallization, crystal drying, etc. may be appropriately added, reduced, adjusted or combined.

The raw materials for preparing AAKG, L-arginine and α-ketoglutaric acid, are all fermentation source products, using plant-derived starch glucose as a carbon source, and using non-transgenic microorganisms (self-screening modified wild bacteria, CN 03112896.3; CN 200310106298.6) Metabolic fermentation is carried out, and the obtained fermentation product L-arginine or α-ketoglutaric acid has high purity and stable quality. More importantly, AAKG prepared from α-ketoglutaric acid, a source of non-hair-derived amino acids and a non-petroleum-based chemical synthesis source, is used for health foods, and there are no concerns about biosafety and food safety.

The present invention directly uses L-arginine in the fermentation broth and α-ketoglutaric acid in the fermentation broth as the raw material of AAKG, L-arginine, α-ketoglutaric acid, instead of using the crystalline product thereof. This avoids more repeated operations with the post-extraction of the refined arginine or α-ketoglutaric acid by fermentation. The extraction of L-arginine or α-ketoglutaric acid from the fermentation broth generally requires flocculation, filtration, resin adsorption, elution, concentration, crystallization, drying, and the like. The invention is directly prepared by mixing (chelating) and concentrating crystallization of the fermentation broth, and can link and improve the production process of L-arginine or a-ketoglutaric acid and the preparation process of AAKG, and can be shortened. Good results in production, lower production costs, and improved quality control.

The method for preparing AAKG crystal directly from the fermentation liquid provided by the invention has the following significant advantages as compared with the prior art: (1) directly from the L-arginine fermentation broth and the a-ketoglutaric acid fermentation broth Preparation, organic integration of the production of AAKG into the post-extraction process of the fermentation product, so the overall process is shortened, the production cost is significantly reduced; (2) the purification step in the preparation process of the fermentation arginine and the fermentation a-ketoglutaric acid is removed, Reduce energy consumption and waste water production; (3) Process has greater flexibility, through the appropriate adjustment of parameters, can achieve two or more ratios of AAKG production; (4) product quality is easy to control, using low temperature Crystallization, organic solvent recrystallization process, and decolorization with activated carbon can effectively ensure the purity of AAKG.

DRAWINGS

Figure 1 is an HPLC analysis of the example L-arginine a-ketoglutarate, column Waters Atantis dCl 8, mobile phase 20 mmol / L NaH ₂ P0 ₄ -Na ₂ HP0 ₄ , pH 2.7, column temperature 30 "C, UV 210 nm.

detailed description

The invention provides a plant-derived starch glucose as a carbon source, and uses a non-transgenic strain to enter Microbial metabolic fermentation (refer to the patent CN 03112896.3 method and patent CN 200310106298.6, respectively) to obtain a fermentation broth containing L-arginine or a metabolite containing α-ketoglutaric acid, and directly preparing L- from its fermentation broth The crystallization process of arginine-α-ketoglutarate is mainly composed of technical units such as pretreatment, chelation, concentration, crystallization, dissolution and decolorization, recrystallization, and crystal drying of the fermentation broth. The technical parameters proposed in the invention are explained from the perspectives of technology optimization, product quality improvement and technology cost reduction.

The purpose of the fermentation liquid pretreatment unit is to remove the bacteria and larger solid particles, and by adding the flocculant, the content of the macromolecular substance in the fermentation liquid can be further reduced, and the flocculation effect of CaCl ₂ can generally achieve a good flocculation effect. In the large-scale preparation process, the filtration operation can select equipment such as plate and frame filtration, vacuum filtration or belt filtration. In the small test process, the centrifuge can be used for solid-liquid separation.

Before acidification, the contents of L-arginine and α-ketoglutaric acid in the two fermentation filtrates should be determined separately. Usually, the L-arginine content in the fermentation broth is between 4% and 6% (mass volume percent, After the same), the content of α-ketoglutaric acid is 3% to 4%. It should be optimized according to the preparation target product. When preparing MAAKG, the molar amount of α-ketoglutaric acid added should be 0.8~1.5 of the molar amount of L-arginine, and the optimized range is 0.95~1.05. When preparing DAAKG, the molar amount of α-ketoglutaric acid added should be 0.2 to 0.8 of the molar amount of L-arginine, and the optimum range is 0.35 to 0.55.

The mixed solution containing L-arginine and α-ketoglutaric acid is concentrated to make the AAKG supersaturated. The ideal concentration equipment should be vacuum concentrated. The multi-effect vacuum concentration device is more suitable for large-scale preparation. Process, small test preparation is suitable for film concentration or rotary evaporator. The volume of L-arginine and α-ketoglutarate solution before mixing should be concentrated to 15%~50% of the volume, and the suitable concentration volume is 20%~25%, and the concentration is larger. The crystallization recovery rate is higher, but the purity is lower; when the concentration is small, the crystal precipitation amount is small, and the crystallization operation time needs to be moderately extended, but the crystal purity of the precipitated crystal is generally high.

Further, the chelating operation can be carried out after the concentration operation, but it is avoided that the volume of the system is increased by the addition of the α-ketoglutaric acid solution after the concentration is completed.

The suitable temperature for crystallization is -5 ° (~ 15 ° C, preferably the temperature is 5 ° C ~ 10 ° C. In the large production process using a crystallization pot, when the system is cooled by the cooling liquid, the cooling rate should be controlled, the preferred temperature drop The rate is between 2 ° C and 5 ° C / h. The crystallization time is controlled at 4 to 50 h, and the preferred time is 20 to 30 h.

The crystal is redissolved in 2 to 5 times the mass of deionized water, preferably 3 times the volume of water; The amount of granular activated carbon to be added should be moderately adjusted according to the purity of the crystal. The preferred addition amount is 0.5% to 1.5%, and the suitable decolorization temperature is 60 ° C to 90 ° C. It is suitable to maintain 30 to 60 minutes, and then filtered through the plate frame. The filtrate is collected. In the small batch production, the AAKG recrystallization can be selected by centrifugation or vacuum filtration. The preferred solvent is ethanol or acetone. The preferred addition volume is 2 times, and the recrystallization time is 8~30 h. After the crystals are precipitated, the crystals are obtained by filtration and washed with a small amount of cold fresh solvent for 2 to 3 times, usually 50% or more of ethanol or acetone. The washing solvent should be pre-cooled to the crystallization temperature (5 to 10 ° C) to Reduce the solubility of AAKG.

The recrystallization operation can also be carried out directly in the aqueous phase without using an organic solvent, and the purity of AAKG can be appropriately increased, and can be determined according to the quality index of the product. Recrystallization is carried out using an aqueous phase, and the crystal is dissolved using 2 times by volume of water, which can be heated appropriately to increase the dissolution rate.

Generally, the crystal is dried by vacuum drying at a temperature of 60 ° C to 90 ° C, preferably 70 ° C to 75 ° C, and a large-scale production can be carried out by using a gas flow drying device.

The contents of the present invention are not specified for mother liquor treatment and solvent recovery, and specific implementation or improvement measures can be employed in the specific implementation stage to increase the yield and reduce emissions.

In order to further illustrate the process idea of the present invention, a specific implementation case will now be described. [Example 1] Preparation of monoarginine ketoglutarate MAAKG

Add 25g of CaCl ₂ powder to the 5 L arginine fermentation broth, stir it to dissolve fully, let stand for 2 h, and then centrifuge in a large-capacity centrifuge (8000 r/min, 10 min) to collect 4.6 L of the filtrate. . It was determined that the L-arginine content was 45.0 g/L, and it was calculated that the solution contained 1.19 mol of L-arginine. In a similar manner to α- ketoglutarate fermentation centrate pretreatment, and calculates the amount of _β mol

Calculate the molar ratio of L-arginine: α-ketoglutaric acid = 1 : 1 , and slowly add α-ketoglutarate fermentation filtrate to the L-arginine fermentation filtrate while stirring. Completely mixed. The mixture was concentrated by a rotary evaporator at a water bath temperature of 65 ° C to obtain a concentrate of 1200 mL, which was 26.1% of the pre-mixed L-arginine and α-ketoglutarate fermentation filtrate. Put it into a 5 Ό freezer and let it stand for 20 hours. A large amount of crystals are precipitated in the solution.

The crude crystals were collected by vacuum filtration to obtain 272.3 g (dry weight), and the recovery was calculated to be 71.5%. The purity of the crude product was determined by HPLC. The purity of the crude product was 92.0% (excluding crystal water, the same below), L-arginine: α- The molar ratio of ketoglutaric acid was 1: 1.11. The crude product 270 g was dissolved in 500 mL of deionized water (2.5 times water volume of the crude product volume), powdered activated carbon (100 mesh:) was added at 0.5% of the crude product mass, and the mixture was decolorized by stirring, and the temperature was raised to 70 ° C for 40 min. Then, vacuum filtration was performed to remove activated carbon.

After the filtrate was cooled to room temperature, 3 volumes of ethanol were slowly added, and the chamber was allowed to stand for 50 h for recrystallization. The crystals were collected by vacuum filtration and rinsed with 85% (by volume) ethanol 10 to 15 mL for 3 times, and then drained 20 mir! ~ 30 min. The crystals were transferred to a watch glass, placed in a vacuum oven, and dried at 70 ° C for 48 h.

After drying, the product quality of recrystallization was 239.6 g, and the purity of the product determined by HPLC was 99.2%, wherein the molar ratio of L-arginine: α-ketoglutaric acid was 1:1.06. The recrystallization yield reached 88.0%, and the total yield of AAKG was 62.9%.

[Example 2] Preparation of double arginine ketoglutaic acid DAAKG

Add 5 mL of 1.0% chitosan hydrochloride (1.0%) solution to 5 L arginine fermentation broth, stir and let stand for 1.0 h, vacuum filter, and collect 4.2 L of filtrate. The L-arginine content was determined to be 52.0 g/L, and the solution was found to contain L-arginine 1.25 moL α-ketoglutaric acid fermentation. The fermentation filtrate was obtained in the same manner.

Take 91.6 g of a-ketoglutaric acid fermentation filtrate (according to L-arginine: α-ketoglutaric acid = 1: 0.5 molar ratio), slowly add to the L-arginine fermentation broth while stirring In, make it completely mixed. The mixture was concentrated using a rotary evaporator at a water bath temperature of 65 ° C to obtain a concentrate of 1680 mL, which was 40% relative to the L-arginine and α-ketoglutarate fermentation filtrate before mixing. Put in a 0 ° C refrigerator, let stand for 48 h, crystals precipitated in the solution.

The crude crystals were collected by vacuum filtration, and the dry weight was 190.3 g (dry weight). The calculated recovery was 61.4%. The purity of the crude product was 88.6% by HPLC. L-arginine: α-ketoglutarate molar ratio Is 1.92: 1.

The crude product 185 g was dissolved in 370 mL of deionized water (2.0 times water volume of the crude product), and powdered activated carbon (100 mesh) was added at 0.5% of the crude mass, stirred and decolorized, and warmed to 70 ° C for 40 min. Then, the mixture was vacuum filtered, and the activated carbon was removed, and the filtrate was 350 mL.

After the filtrate was cooled to room temperature, 1400 mL of acetone (4 times the volume of the filtrate) was slowly added, and placed under a refrigerator at 0 ° C for 8 h. After crystallizing, the crystals were collected by vacuum filtration, and cold acetone at 0 ° C was used. The crystal was rinsed 3 times with mL and continued to drain for 20 min to 30 min. The crystals were transferred to a watch glass, placed in a vacuum oven, and dried at 70 ° C for 24 h to be a DAAKG product. The product quality of recrystallization was 165.1 g, and the purity of the product determined by HPLC was 98.7%, wherein the molar ratio of L-arginine: α-ketoglutarate was 1.99:1. The recrystallization yield reached 86.7%, and the total yield of AAKG was 53.3%.

[Example 3] AAKG preparation technology under pilot scale

The L-arginine fermentation broth was 5 Μ ³ and the arginine content was 48 g/L. 25 kg of CaCl ₂ powder was added, stirred to dissolve fully, and allowed to stand for 2 h, filtered through a plate and frame filter device (filter area of about 25.0 m ² ), and the filter residue was washed. A clear filtrate of 4350 L was collected.

The α-ketoglutaric acid used for the salt reaction with arginine is also used as a fermentation broth. The pretreatment process is similar to that of L-arginine, and the content of α-ketoglutarate in the clarified filtrate is 39.2 g/L.

Taking the production of MAAKG as an example, the α-ketoglutaric acid fermentation filtrate is added to the L-arginine filtrate according to the L-arginine: α-ketoglutaric acid = 1 : 1 molar ratio, Stir well and mix the fermentation broth with a total of 8.82 Μ ³ .

Using a two-effect vacuum concentrating device, it was concentrated to 1500 L, and the amount of the concentrate was 34.5% of the volume of the original L-arginine filtrate and the α-ketoglutarate filtrate mixture.

The concentrate is crystallized by using a crystallizing pot, and cooling water is introduced. When the temperature of the filtrate is cooled to 30 ° C, the cooling rate is controlled, and the temperature is slowly lowered to 5 ° C at a rate of 2 ° C per hour, and the stirring speed of the crystal pot is 10 to 15 r/min, crystal growth time 10 h. The plate crystal was filtered to separate the coarse crystals. The crude crystal was reduced to a dry weight of 342.7 kg, and the crude product was 88.6%, and the recovery was 79.1%.

The crude product was redissolved in 600 L of deionized water, added with powdered activated carbon (100 mesh) 1500 g, stirred and heated to 70 ° C for 60 min. The activated carbon was removed by plate frame filtration, and the filtrate was charged into a crystallization tank, and cooled to room temperature with condensed water. Slowly add 1800 L of 96% (v/v) ethanol. The cooling water was cooled to 15 ° C, and allowed to stand for 12 h to be crystallized. The mixture was vacuum filtered, washed with 96% (v/v) ethanol, dried under vacuum and dried at 50 °C. Recrystallization AAKG 222.4 kg was obtained.

The above process is a preparation process of MAAKG, and the total yield of the product is about 57.3%, and the product purity is >99.0%. If the mother liquor is treated and reused, the actual yield is above 70%.

[Example 4] Effect of the amount of α-ketoglutaric acid on the purity and yield of crystalline products

In order to study the effect of the amount of a-ketoglutaric acid added on the purity and yield of the crystalline product, 100 mL of L-arginine filtrate was added, and different amounts of α-ketoglutaric acid were added, and concentrated in "Example 1". The crystallization method was operated, and the purity and yield of each batch of MAAKG were determined by HPLC.

Table 1 Effect of α-ketoglutaric acid addition ratio on crystallization yield and purity Amount of L-arginine (Arg) Amount of α-ketoglutaric acid (KG) MAAKG yield MAAKG purity / mol / mol /% /%

0.025 0.020 0.8 55.1 92.7

0.025 0.025 1.0 71.5 92.0

0.025 0.030 1.2 67.5 91.0

0.025 0.035 1.4 60.0 89.0

0.025 0.040 1.6 57.7 87.0 The ratio of a-ketoglutaric acid can be adjusted within a certain range, but for MAAKG, the quantitative addition of 1:1 is the optimal solution.

[Example 5] Effect of crystallization temperature and cooling rate on product yield and purity

The cooling rate has a certain influence on the crystallization yield and product purity. According to the solution system (Arg: KG = 1 : 1) in "Example 4", each group was pre-cooled to 30 °C, and different cooling rates and crystallization temperatures were used to investigate the crystallization yield and product purity of MAAKG. See the table below for the results.

Effect of crystal temperature and temperature drop rate on yield and purity

[Example 6] HPLC determination of AAKG

The test sample MAAKG 0.100 g was weighed and dissolved in 100 mL of distilled water as the sample to be tested. The HPLC was analyzed using a Waters Atantis dC18 column with a mobile phase of 20 mmol/L NaH ₂ P0 ₄ -Na ₂ HP0 ₄ , pH 2.7, a column temperature of 30 ° C, and a UV detector for a wavelength of 210 nm. The injection volume is 10μ.

MAAKG dissociates into two separate compounds, L-arginine and alpha-ketoglutaric acid, in aqueous solution, thus presenting two peaks in the HPLC profile (see figure). The peaks of these two components were well obtained by the above chromatographic conditions, and the retention times were 3.35 min and 5.79 min, respectively. use For the standards of L-arginine and α-ketoglutaric acid, it is determined that these two peaks are L-arginine and α-ketoglutaric acid, respectively. The content of the sample can be calculated according to the standard, and the molar ratio of the composition of L-arginine and α-ketoglutaric acid in MAAKG can also be calculated.

Claims

Rights request

1. A process for preparing L-arginine-α-ketoglutarate by direct crystallization from a fermentation broth, characterized in that the desired raw materials L-arginine and α-ketoglutaric acid are plant sources The starch glucose is a carbon source, and the fermentation product obtained by metabolic fermentation of a non-transgenic wild-type microorganism, referred to as a fermentation broth, includes a fermentation broth pretreatment, chelation, concentration, crystallization, dissolution and decolorization, recrystallization, and The crystal is dried, the L-arginine fermentation broth is pretreated, the bacteria and macromolecular impurities are removed, and a certain amount of α-ketoglutarate fermentation filtrate which is simultaneously purified and purified is mixed, that is, chelated, and concentrated by vacuum evaporation. The crude product of L-arginine α-ketoglutarate is obtained by cooling crystallization. The crude product is decolorized by dissolving and recrystallization from organic solvent, and finally dried to obtain high purity L-arginine α-ketopentane Acid salt.

2. The process for preparing L-arginine-α-ketoglutarate by direct crystallization from a fermentation broth according to claim 1, wherein: the fermentation liquid pretreatment process is L-arginine The process of flocculation, filtration or centrifugation of the acid fermentation liquid and the α-ketoglutaric acid fermentation liquid to remove the bacteria and the larger solid particles to obtain the clarified fermentation filtrate of L-arginine and α-ketoglutaric acid, The flocculation operation is a flocculant such as aluminum sulfate, calcium chloride, polyacrylamide or chitosan or other commercially available inorganic aluminum salts, iron salts, calcium salts and high-molecular flocculants; It is especially suitable for large-scale preparation process by equipment such as plate and frame filtration, vacuum filtration or belt filtration. The centrifugal operation is carried out by centrifugal separation equipment for solid-liquid separation, and is especially suitable for small-scale preparation.

3. The process for preparing L-arginine-α-ketoglutarate by direct crystallization from a fermentation broth according to claim 1, wherein: the chelation process is based on L-fine in the filtrate. The content of the amino acid, adding a certain amount of α-ketoglutaric acid fermentation filtrate, or α-ketoglutaric acid solution for the chelation reaction process, the molar content of α-ketoglutaric acid added, is the L-fine in the system The molar amount of the acid is 0.3 to 1.5 times.

4. The process for preparing L-arginine-α-ketoglutarate by direct crystallization from a fermentation broth according to claim 1, wherein: the concentration process comprises L-arginine The process of concentrating the chelate solution of α-ketoglutaric acid to super-saturate the AAKG. The preferred concentration equipment is vacuum concentration, two-concentration or three-effect concentration equipment, and the solution is concentrated to L-arginine and The volume of the α-ketoglutarate solution mixture is 15% to 50%.

5. The process for preparing L-arginine-α-ketoglutarate by direct crystallization from a fermentation broth according to claim 1, wherein: the crystallization process is to place the concentrate at a temperature - Crystallization is carried out at 5 ° C to 15 ° C, or the solution is cooled by a cooling liquid using a crystallizing pot, and the crystallization process is controlled at 4 to 50 h, and the crystals are collected by filtration or centrifugation after completion.

6. The process for preparing L-arginine-α-ketoglutarate by direct crystallization from a fermentation broth according to claim 1, wherein: the dissolving and decoloring process re-dissolves the crystal in the 2 to 5 times by weight of deionized water, stir to fully dissolve, then add granular activated carbon for decolorization, the amount of activated carbon added is equivalent to 0.5%~3.0% of the mass of AAKG crystal, stir and heat the solution to 40~90 °C, and maintained for 20 to 60 minutes, and then filtered through the plate frame, collecting AAKG filtrate.

7. The process for preparing L-arginine-α-ketoglutarate by direct crystallization from a fermentation broth according to claim 1, wherein the recrystallization process is in the AAKG filtrate. Slowly add 1~4 times of the hydrophilic organic solvent in the volume of the solution, recrystallize at room temperature or cool down for 8~50 h, collect the crystal by filtration, and wash it with a small amount of solvent for 2~3 times, the hydrophilic organic solvent, These include ethanol, methanol, acetone, isopropanol, glycerol and acetonitrile, with the preferred solvent being ethanol or acetone.

8. The process for preparing L-arginine-α-ketoglutarate by direct crystallization from a fermentation broth according to claim 1, wherein: the crystal drying process is vacuum drying and hot air circulation. The oven or the air drying apparatus performs crystal drying, and the drying temperature is 50 ° C to 150 ° C.