GB2485862A - Preparation process of L-arginine-alpha-ketoglutarate by direct crystallisation from fermentation broth - Google Patents

Abstract

A method of preparing L-arginine-alpha-ketoglutarate (AAKG) is disclosed comprising adding L-arginine and α-ketoglutarate which are obtained as fermentation products from microbes, the method further comprising concentration in a vacuum, crystallisation, dissolution discolouration, re-crystallisation and crystal drying.

Description

PREPARATION PROCESS OF L-ARGININE-a-KETOGLUTARATE BY

DIRECT CRYSTALLIZATION FROM FERMENTATION BROTH

Field of the Invention

The present invention belongs to biotechnology field and involves a preparation technology of Arginine-Alpha-ketoglutarate.

Background of the Invention

L-arginine-alpha-ketoglutarate, Called AAKG, is white or yellow crystal, soluble in water, HC1 and NaOH solution, slightly soluble in ethanol and insoluble in acetone and ether, etc. L-arginine-ct-ketoglutarate is an amino acid salt health product with increasing sales in the international market. As the functional nutrition intensifying agent and hepatinica, it is mainly used as the tonica to improve physique, accelerate and resume rapid muscle growth, promoting the nutrition and energy absorption of hepatic cells, and maintaining normal liver function and etc. The main physiological mechanism is that AAKG can promote nitrogen monoxide level and muscular movement, and further accelerate rapid growth and rejuvenation of muscle, enhancing strength and energy.

There are two AAKG salts: I:1 and 2:1 mixture ratio (L-arginine: a-ketoglutarate), namely Mono arginine ketoglutarate (MAAKG) and Di-arginine ketoglutarate (DAAKG). Two AAKG salts have similar physicochemical property, but are different in function and application fields. The primary function of MAAKG is to increase the nitric oxide level in blood, stimulating vasorelaxation, and accelerating the muscle growth after exercise. As the exercise invigorator

I

health product, it is very popular in American market (http://www.rnirns.com/) DAAKG is a organic acid salt with multiple physiological functions. Applying high doses of DAAKG to hepatoeirrhosis patients can enhance detoxication function of liver, which can significantly reduce pEasma ammonia and phenol. The function is also confirmed in the treatment of ammonia poisoning or intensification liver detoxification in animal experiments. Comparing with the control group, the treatment group has higher survival rate and less coma symptom. Based on pharmacological analysis, toxicological evaluation, regeneration of animal hepatic tissue, in vivo detoxification and other animal experiments, a series of clinical studies on DAAKG hepatinica Euligen was performed by France Refarland Pharmaceutical Factory, in which significant curative effect on liver disease, alcoholism and their complications was observed.

As early as 1977, it was reported that liver detoxification of hepatocirrhosis patients could be improved when high dose arginine-LI -ketoglutarate was applied (Muting et al. Munch Mcd Wochensehr, 1977). In recent years, there has been increasing studies on the AAKG clinical application and metabolic mechanism. It is described in United States Patent USP 2005/0085498 Al that in animal experiment, arginine and glutamine level in skeletal muscles can be improved and immune system can be stimulated by supplementing AAKG. It is also shown in a research aiming at supplement of AAKG (15 g/d) to children that AAKG can improve growth hormone level, promote metabolism of insulin-like growth factor (IGF1), glutamine, glutamate and other aminoacid. The growth rate is significantly increased after 5-month supplement. Similarly, AAKG can promote growth hormone and insulin secretion of patients after surgery, the intermediary metabolism substance glutamine and proline can also promote wound recovery. In a research on the anti-catabolism ftinction of AAKG, 14 multiple-traumatism patients with high catabolic and enhanced metabolism participated in the experiment. 20g AAKG is supplied to patients in the test group. It is shown that protein transformation and blood level of insulin, growth substance, glutamine, praline, arginine and other free amino acids were also significantly improved. In addition, study on healthy male with a daily supplement of lOg AAKG, the insulin level was enhanced by 2030%. However, no improvement is observed in group with separated arginine or a-ketoglutarate supplement. It is indicated from in vitro cell culture that AAKG can significantly induce the growth of human fibroblasts, a cell similar to muscle fiber cell. There is a remarkable dose-effect, whereas arginine or a-ketoglutarate has no such characteristic (USP 2005/008 5498 Al).

Furthermore, in water solution, AAKG can be dissociated to form arginine and ct-ketoglutarate. Therefore, it can be utilized as donor for ct-ketoglutarate or arginine. Under certain condition, it can be applied as substitute of arginine or a-ketoglutarate. Arginine is an essential amino acid as well as material for the synthesis of protein and creatine in vivo, it is also an important metabolic intermidiate for urea cycle and a precursor of nitrogen monoxide, whereas ct-kctoglutarate has strong regulation effect on protein metabolism, also an important substance to promote tricarboxylic acid cycle. (United States Patent USP 7645742; USP 6905707; USP 7,645,742) There are three methods for the production of AAKG. The first method is direct mixing process, in which L-arginine and a-ketoglutarate are mixed according to molar ratio of 1:1 and 2:1. The product is simple mixture of arginine and ketoglutarate, in which no salt bond exists, and the product quality is greatly affected by raw material quality and mixture homogeneity. Therefore, this method is not used in medicine or food production. The second method is dissolution-lyophilization. L-arginine and a-ketoglutarate solution in certain concentration are mixed in certain molar ratio, and the solution is lyophilized into AAKG, in which two substances can form stable organic acid salt. However, due to its high energy eomsumption during the lyophilization process, this method is only applicable in the production of AAKG injection. Above two methods are described in Chinese patent 200710063477.4, 200710063478.9 and 200710063479.3. The third method is crystallization process. After the solution is mixed, the solution is concentrated or treated with organic solvent to reduce solubility of AAKG and obtain AAKG crystals, which shall be washed and dried to obtain AAKG product.

Product prepared by this method has high purity, and the product quality can be guaranteed. However, the process is complicated arid the cost is high (CN 101591271 A).

L-arginine and a-ketoglutarate required in preparation of AAKG in above three methods must meet the requirement of raw pharmaceutical material. Especially in previous two methods, the impurity can not be eliminated during preparation process, and, higher requirement on product quality control is therefore required.

The crystallization method becomes optimization method in preparation of AAKG.

Preparation method of L-arginine-ct-ketoglutarate crystallization is described in CN1O1591271A. L-arginine and a-ketoglutarate raw material purchased from the market were used to prepare solution mixture crystallization preparation. It is well known that present L-arginine on the market is extracted from hair (usually pig or human hair) acid hydrolysis. a-ketoglutarate is chemically synthesized from petroleum material. There are biosafety concerns in source of these two raw materials. Just as described in CNlOlOll372A, ct-ketoglutarate-arginine salt prepared from chemosynthcsis products and hair extracts raw material might not be suitable for health food.

SUMMARY OF THE INVENTION

The present invention is to overcome above problems in AAKG product and its preparation technology, provide a method to prepare AAKG from ferrnentative L-arginine, a-ketoglutarate raw material. L-arginine and ct-ketoglutarate in the fermentation broth are directly used as AAKG preparation raw material, instead of refined crystallization products.

In present invention, raw material L-arginine and a-ketoglutarate were fermentation products obtained from non-transgenic wild-type microbe metabolism and fermentation using starch glucose from plant as the carbon source, which is called fermentation broth. a-ketoglutarate and L-arginine in fermentation broth are mixed and chelated to form salt, then L-arginine-cz-ketoglutarate salt is prepared using general chemical isolation technique and equipment for direct crystallization.

The preparation processes comprise fermentation broth pretreatment, chelation, concentration, crystallization, dissolution discoloration, recrystallization, crystal dryness and other technical units. In pretreatment process of L-arginine fermentation liquid, microbial cells and macromolecule impurities arc eliminated.

Certain amount of same purified a -ketoglutarate ferment broth is added and mixed, which is called chelation. Evaporate and concentrate the mixture in vacuum, L-argininecz-ketoglutarate crude product could be obtained under cooling and crystallization condition. The crude products is dissolved and decolorized, and then recrystallized in organic solvent. Finally, L-arginine a-ketoglutarate in high purity can be obtained after drying process.

In the present invention, the mentioned fermentation liquid pretreatment technique unit is a process to respectively flocculate, filtrate or centrifuge L-arginine fermentation broth or a-ketoglutarate fermentation broth in accordance with common methods to remove cells and large solid particles and obtain clarified filter liquor. In flocculation operation, aluminum sulfate, CaC12, polyaerylamide, chitosan and other flocculants or other inorganic aluminium salt, molysitc, calcium salt and polymeric flocculant are adopt. In the mentioned filter operation, plate-and-frame filtering, vacuum filtering or band type filter devices, etc. are especially suitable for large scale preparation process. In the mentioned centrifugation operation, a centriftigation device for solid-liquid separation is especially suitable for small sample preparation process.

In the present invention, the mentioned chelation technology unit is the process to add certain ct-Icetoglutarate fermentation liquid or ct-ketoglutarate solution based on the L-argininc content in filter liquor. The molar content of ct-ketoglutarate is 0.3-A.5 times of L-arginine in the system.

In the present invention, the mentioned concentration technique unit is a process to concentrate solution with L-arginine and a-kctoglutarate so that AAKG can reach to a hyper-saturated state. The preferred concentration device is vacuum concentration, double-effect concentration or multiple-effect concentration equipment. The solution is concentrated to I 5%-'50% of original L-arginine solution volume.

In the present invention, the mentioned crystallization technology unit is to incubate the concentrated solution under S DC -15 DC for crystallization or use a crystallizer tank to cool solution with coolant. The crystallization process is controlled within 4-50 hours. In the end, adopt filtration or centrifugation to collect crystals.

In the present invention, the mentioned dissolution decolorization technology unit is to dissolve above crystals again in 2-5 times (by weight) de-ionized water, stir the mixture so that it can be dissolved completely. Add particle activated carbon into the mixture for discoloration. The amount of activated carbon amount added is equivalent to 0.5%3.0% of AAKG crystal quality. Stir and then heat the solution to 4O-90 ° for 20--'60 minutes. In the end, collect AAKG filter liquor through plate-and-frame filtering.

In the present invention, the mentioned recrystallization technology unit is to slowly add hydrophilicity organic solvent with l4 times of solution volume into above AAKG filter liquor. Perform recrystallization after incubation under room temperature or in refrigerator statically for 850h. Collect crystals through filtration and which the crystal for 23 times with small amount of cold and fresh solvent, The mentioned hydrophilicity organic solvents include ethanol, methanol, acetone, isopropanol, glycerol, acetonitrile etc, where the preferred solvent is ethanol or acetone.

In the present invention, the mentioned crystal dryness technology unit adopts vacuum drying, heated air circulation oven, flash drying or other devices for crystal dryness. The drying temperature is 50 °C 150 °C.

In the present invention, the mentioned fermentation liquid pretreatment, 1 5 chelation, concentration, crystallization, dissolution discoloration, recrystallization, crystal dryness and other technical units can be added eliminated, adjusted or combined properly.

In the present invention, raw materials L-arginine and a-ketoglutarate for preparing AAKG are fermentation products using starch glucose with plant origin as the carbon source and non-transgenie microbe (independently isolated wild-type microorganism, CN 03112896.3; CN 200310106298.6). The obtained fermented product L-arginine or ct-ketoglutarate are in high in purity and stable in quality.

Importantly, AAKG is prepared from non-hair source arginine and non-petroleum based a-ketoglutarate raw material. It can be applied in health food without biosafety and food safety issues.

In the present invention, L-arginine in fermentation liquid and a-ketoglutarate in fermentation liquid are taken as raw materials of AAKG, instead of crystallization products. Tn this way, repetitive operation in purifying arginine or a-ketoglutarate from fermentation broth can be avoided. Flocculation, filtration, resin adsorption, elution, concentration, crystallization, dryness and other steps shall be adopted to extraction L-arginine or ct-ketoglutarate from fermentation liquid. In the present invention, fermentation liquid is directly used to perform mixing (chelation), concentration, crystallization and preparation in certain proportion. In this way, production process of L-arginine or ct-ketoglutarate as well as preparation process of AAKG can be connected and improved, the production gap can be shortened, the production cost can be reduced and quality control level can be improved.

Compared with existing technique, the method (prepare AAKG crystal directly from fermentation liquid) in the present invention has following remarkable advantages: (1) the crystal is directly prepared from L-arginine fermentation liquid and cz-ketoglutarate fermentation liquid, AAKG production is integrated to post-extraction process of fermentation products. Therefore, the entire production flow is shortened and the manufacturing cost is reduced remarkably; (2) purification step in arginine fermentation and a-ketoglutarate preparation fermentation process are eliminated. Therefore, energy and substance consumption, waste water generation are reduced; (3) the technology possesses great flexibility.

By proper parameter adjustment, AAKG in two mixture ratios or several mixture ratios can be produced; (4) product quality can be controlled easily. Purity of AAKG can effectively guarantee by low temperature crystallization, organic solvent recrystallization technology and discoloration with activated carbon.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is an execution example; L-arginine ct-ketoglutarate HPLC ehromatograph, column Waters Atantis dC 18, mobile phase 20 mmol/L NaH2PO4 -Na2HPO4, pH 2.7, column temperature 30 °C,UY 210 mm

DESCRIPTION OF THE PREFERRED EMBODIMENTS

In the present invention, starch glucose of plant origin is used as carbon source and non-transgenic microorganism is used as fermentation strain (refer to methods in patent CN 03112896.3 and patent CN 200310106298.6) to obtain fermentation broth containing metabolic products of L-arginine or a-ketoglutarate. L-arginine -a -ketoglutarate crystal is directly prepared from the fermentation broth. The process involves fermentation liquid pretreatment, chelation, concentration, crystallization, dissolution discoloration, recrystallization, crystal dryness and other technique.

Here, technical parameters presented in the invention are described based on technique optimization, improvement of product quality, and reduction of technique cost.

Fermentation liquid pretreatment unit is to eliminate cells and large solid particles. Macrornolecular substance content in the fermentation broth can be further reduced by adding flocculant. A good flocculation effect can be obtained through CaCl2 flocculation. In scaled-up preparation process, plate-and-frame filtering, vacuum filtering or band type filtering device can be adopted. In small sample process, a centrifuge can be adopted for solid-liquid separation.

Before acidification, L-arginine and a-ketoglutarate content in two fermentation filter liquor are measured respectively. Generally, L-arginine content in fermentation broth is 4%-6% (mass and volume percent, same in following contents), a-ketoglutarate content is 3%4%. Optimization should be performed according to target products. While preparing MAAKG, molar weight of a-ketoglutarate added shall be 0.8-4.5 of L-arginine molar weight. The optimization range is within 0.95--i.05. However, molar weight of added ct-lcetoglutarate shall be 0.2-0.8 of L-arginine molar weight while preparing DAAKG. The optimization range is within 0.35-0.55.

Mixture solution of L-arginine and a-ketoglutarate is concentrated so that AAKG can reach hypersaturated state. An ideal concentration device shall be in vacuum concentration mode. Multiple-effect vacuum concentration device is more feasible for large-scale preparation process, while membrane concentration or rotary distillatory is suitable for in small-scale preparation. Take original volume of L-arginine and ct-ketoglutarate solution before mixture as the standard, concentrate the solution to 1 5%-50% of the original volume. The proper concentrated volume is 20%-25%. In this method, the larger the concentration volume, the higher crystallization recovery and lower purity; the less the concentration volume, the less crystallization recovery, the higher purity and prolonged crystallization time.

is Furthermore, chelation operation can be performed after concentration operation. However, after concentration is completed, system volume may be increased due to the addition of ct-ketoglutarate solution. This situation should be avoided.

The proper crystallization temperature is within -5 ° C 15 ° C, and the preferred temperature is within 5 ° C 10 ° C. In large-scale production, crystallizer tank is used. System cooling is performed with coolant and the cooling rate should be controlled. The preferred cooling rate is within 2 ° C 5 °C/h, the crystallization time is control within 450h, and the preferred time is 2030h.

Crystals are dissolved in 2-5 times of deionized water, the preferred volume is 3 times of water. The amount of particle activated carbon shall be adjusted properly according to crystal purity, and the preferred adding amount is 0.5%-1.5%. Proper decolorization temperature is 60 ° C -90 °C, and maintain the temperature for 30'-óomin. Filter liquor is collected after plate-frame filter. In small-scale production, AAKG filter liquor can be obtained through centrifugation or vacuum filtration.

Re-crystallization is performed in organic solvent system, and the preferred solvents are ethanol or acetone; preferred adding volume is 2 times. The re-crystallization time is 830h. After separation, crystals are obtained through filtration and washed with small amount of cold and fresh solvent for twice or three times, usually ethanol or acetone of over 50%. The wash solvent should be pre-cooled to crystallization temperature (5 ° C -10 ° C) to reduce the solubility to AAKG.

Re-crystallization can be directly operated in a water phase instead of organic solvent in. perform recrystallization. AAKG purity can also be improved, which can be determined according to quality standard of product. When recrystallization is performed in a water phase, 2 times water volume should be used to dissolve the crystal, and the crystal can be heated to accelerate dissolving rate.

Vacuum drying is generally used for crystal dryness within 60 °C 90 ° C. The preferred temperature is 70 ° C 75 °C. A pneumatic conveying dryer can be adopted in large-scale production.

The present invention content does not specify mother solution processing and solvent recovery. In implementation phase, the general or improved measure in the area can be adopted to improve the production yield and reduce waste release.

In order to elaborate on process of the present invention, specific execution cases are adopted to describe the content.

EXAMPLES

Example 1

Preparation of Mono arginine ketoglutarate MAAKG Add 25g CaC12 powder in 5L arginine fermentation liquid. Mix them sufficiently and put the solution statically for 2h. Centrifuge the solution in batch through high-capacity centrifuge (8000 r/min, 10 mm). Collect filter liquor in 4.6 L. L-arginine concentration is calculated to be 45.Og/L. The solution contains 1.1 9mol L-arginine in total. et-Ketoglutarate fermentation filtration liquid is pretreated according to similar method and the molar amount is calculated.

It is calculated that the L-arginine:ct-ketoglutarate molar ratio is equal to 1:1.

Stir the solution while slowly adding a-ketoglutarate fermentation filtration liquid into L-arginine fermentation filtration liquid so that they are mixed completely.

Adopt a rotary distillator to concentrate mixed liquor under 65 °C water bath temperature and l200mL concentrated solution is obtained. The solution volume is 26.1% of L-arginine and a-ketoglutarate fermentation filtration liquid before mixing. Leave the solution in a S °C refrigerator still for 20h, and large crystals are precipitated from the solution.

Through vacuum filtration, 272.3g (dry weight) crude product crystal is collected and the recovery ratio calculated is 71.5%. Through HPLC measurement, the crude product purity is 92.0% (without crystal water, same hereinafter), L-arginine:ct-ketoglutarate molar ratio is 1:1.11.

Take 270g crude product and dissolve in 500mL deionized water (2.5 times water volume of crude product). Active carbon powder (100 meshes) is added to 0.5% of crude product quality. Stir and decolorize the solution. Heat the solution to °C and maintain the condition for 40 mm. Perform vacuum filtration and then eliminate active carbon.

After the filter liquor is cooled to room temperature. Slowly add ethanol in 3 times volume. Leave the solution under room temperature for 50h, and then perform re-crystallization. Perform vacuum filtration and collect crystals. Wash crystals with 10-i 5mL of 85% (volume percent) ethanol for 3 times. Further dry crystals for 20 min'-30 mm. Transfer crystals to watch glass, which shall be put into vacuum drying oven under 70 °C and dried for 48 hours.

After dryness, the recrystalliscd product quality is 239.6g. Product purity is 99.2% by HPLC measurement. Where L-arginine:a-ketoglutaratc molar ratio is 1:1.06. Re-crystallised yield is 88.0%. Total yield of AAKG is 62.9%.

Example 2

Preparation of Di-arginine ketoglutaratc DAAKG Add 2SmL 1.0% chitosan hydrochloric acid (1.0%) in 5L arginine fermentation liquid. Stir and put it statically for 1.Oh. Perforrrt vacuum filtration and collect 4.2L filter liquor. It is measured that L-arginine content is 52.Og/L. It is calculated that there is totally 1.25mol L-arginine in the solution. fermentation filter liquor from a-ketoglutarate fermentation is obtained according to same method.

Take fermentation filter liquor with 91.6g a-ketoglutaratc (add filter liquor according to L-arginine: a-kctoglutaratc = 1:0.5 molar ratio). Slowly add the filter liquor into L-arginine fermentation liquid while stirring the mixture at the same time so that they can mix completely. Adopt a rotary distillatory to concentrate mixed liquor under 65 °C water bath and obtain 1680 mL concentrated solution, which is 40% volume of L-arginine and a -ketoglutarate fermentation filtration liquid before mixing. Put the mixture into 0 ° C refrigerator and leave statically for 48h. Crystals are precipitated from the solution.

Perform vacuum filtration to collect crude product crystal. The converted dry weight is 190.3g (dry weight). The recovery ratio is calculated to be 61.4%. It is measured through HPLC that the crude product purity is 88.6%, L-arginine: a-ketoglutarate mol ratio is 1.92:1.

Take 1 85g crude product and dissolve it in 370mL deionized water (2.0 times of the volume of crude product). Add active carbon powder (100 meshes) in 0.5% of crude product quality. Stir and decolorize the solution. Heat the solution to 70 C and maintain for 40 mm. Perform vacuum filtration and then eliminate active carbon to obtain 350mL filter liquor.

After the filter liquor is cooled to room temperature. Slowly add l400mL acetone (4 times filter liquor volume). Leave the solution into a 0 °C refrigerator for 8h. After crystallization, collect crystals by vacuum filtration, wash crystals with 1 0-4 SmL 0 °C acetone for 3 times, further vacuum dry crystals for 20 min-30 mm. Transfer crystals to watch glass, which shall be put into vacuum drying oven under 70 °C for 24 h to obtain DAAKG product.

Recrystal product is 165.1g. Product purity is measured to be 98.7% by HPLC, where L-arginine: ct-ketoglutarate molar ratio is 1.99:1. Re-crystal yield is 86.7%, and total yield of AAKG is 53.3%.

Example 3

AAKG preparation technology under pilot plant test scale Take S m3 L-arginine fermentation liquid containing 48g/L arginine. Add 25kg CaCl2 powder, stir the mixture so that they can dissolve completely. Put the solution statically for 2h. Adopt plate-frame filter plant (about 25.0m2 filtration area for filtration. Wash the filtration residue. Collect 4350L clarification filter liquor.

The pretreatment process for fermentation broth of ct-ketoglutarate is similar to L-arginine. The ct-ketoglutarate content in clarification filter liquor is 39.2g/L.

Taking MAAICG production as the example, add ct-ketoglutarate fennentation filtration liquid into L-arginine filtration liquid according to L-arginine: a-ketoglutarate =1:1 molar ratio. Stir the mixture evenly; the mixed fermentation liquid is 8.82 m3 in total.

Adopt double-effect vacuum concentration device to concentrate the solution to l 500L. Amount of concentrated solution is 3 4.5% of original volume of L-arginine filter liquor and ct-ketoglutarate filter liquor.

Crystallize concentrated solution mentioned above with a crystallizer tank connected with cooling water. When the filter liquor temperature is cooled to 30 C, control the cooling rate at 2 ° per hour to 5 °C. Stirring speed of crystallizer tank is l-45 r/min.. Incubate crystals for I Oh. Adopt plate-frame filtration to separate raw crystal. The raw crystal is 342.7Kg in dry weight afler conversion.

Crude product purity is 88.6% and the recovery ratio is 79.1%.

Dissolve crude product in 600L deionized water. Add 1 SOOg active carbon powder (100 meshes). Stir and heat the solution to 70 ° C, decolorize the solution for 60 mm. Eliminate active carbon with plate-frame filtration method. Fill the filter liquor into a crystallizing tank connected with cooling water and cool the filter liquor to room temperature. Add 1 800L 96% (v/v) ethanol slowly, cool the solution to 15 ° C, leave the solution still for 1 2h. Perform vacuum filtration and wash crystals with 96% (v/v) ethanol, dry crystals under 50 ° C in vacuum condition, then 222.4kg re-crystal AAKG can be obtained.

MAAKG preparation process is as described above. Total yield of the product is about 57.3% with more than 99.0% product purity. If the mother solution is recycled after treatment, the actual yield is more than 70%.

Example 4

Influence of a-ketoglutarate addition on purity and yield of crystallization product In order to study the effect of ct-ketoglutarate addition amount on purity and productivity of crystallization products, various amount of ix-ketoglutarate is added into 1 OOmL L-arginine filter liquor. The process is performed according to concentration and crystallization method in "the first implementation ease". It is measured by HPLC that purity and productivity under different batches are as follows.

Table I Influence of a-ketoglutarate addition on crystallization productivity and purity cc-ketoglutarate KG:Arg L-arginine MAAKG MAAKG (1(G) /mol:mol (Arg)/mol yield/% purity 1% /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 amount of a-ketoglutarate added can be adjusted within certain range.

However, compared to MAAKG, the optimal ratio is 1:1.

Example 5

Effect of crystallization temperature and cooling rate on product yield and purity Cooling rate has certain effect on crystallization yield and product purity.

According to solution system in "the fourth ease implemented" (Arg:KG = 1:1), each group is pre-eooled to 30 ° C and then adopt different cooling rate and crystallization temperature. MAAKG crystal yield and product purity is shown in

table below.

Table 2 Affect of crystallization temperature and cooling rate on productivity and purity Crystallization MAAKG MAAKG Cooling rate -__temperature yield/% purity / % °C 24 °C/h 20.7 98.8 "C Rapid cooling, 0.5 lh 22.2 97.7 "C 24 "C/h 60.6 96.4 "C Rapid cooling, 0.5 lh 67.5 95.0 "C 2-4 "C/h 71.5 92.0 "C Rapid cooling, 0.5 -lb 74.7 87.0

Example 6

HPLC measurement of AAKG Weigh 0.1 OOg MAAKG product to be measured, and dissolve in 1 OOmL distilled water as the sample to be measured. HPLC analysis conditions are: Waters Atantis dCl8 column, 2Ommol/L NaH2PO4 -Na2HPO4, pH 2.7 mobile phase. Set the column temperature as 30 C. Use a UV-detector with 21 Onm detection wavelength. The sample injection volume is lOj.tL.

Two compounds L-arginine and a-ketoglutarate are dissociated from MAAKG in water solution, and therefore two peaks are observed in HPLC chromatograph (see Figure attached). These two peaks can be obtained by using above HPLC condition with retention time of 3.35 mm and 5.79 mm respectively. Standard samples of L-arginine, ci-ketoglutarate are used to confirm that these two peaks are L-arginine and ix-ketoglutarate respectively. Sample content can be calculated according to the standard sample, and molar ratio of L-arginine, ct-ketoglutarate can be calculated at the same time.

Claims (8)

1. CLAIMS: 1. A method or process for the preparation of L-arginine-ct-ketoglutarate from fermentation broth, characterized in that the required raw materials L-arginine and ct-ketoglutarate are obtained from a fermentation product, which is called fermentation broth for short, that starch glucose of plant origin is used as a carbon source and that a non-transgenic wild-type microbe is used as a fermentation strain, the preparation processes comprises: fermentation broth pretreatment, chelation, concentration, crystallization, dissolution discoloration, recrystallization and crystal drying, L-arginine fermentation broth pretreatment, elimination of cells and macromolecule impurities; wherein purified ct-ketoglutarate fermentation filter liquor is added, this process being called chelation, the mixture is evaporated and concentrated in vacuum to obtain L-argininc-a-ketoglutarate crude product with cooling and crystallization; crude products arc further treated by dissolving decolorization and organic solvent recrystallization; finally, L-arginine-ct-ketoglutarate in high purity can be obtained by drying.
2. 2. The method of claim 1, wherein the process of fermentation broth pretreatment is that to flocculate, filtrate or centrifuge L-arginine fennentation liquid or ct-ketoglutarate fermentation liquid respectively to remove cells and large solid particles to obtain clarified L-arginine and a-ketoglutarate fermentation filter liquor; wherein in the flocculation operation aluminum sulfate, calcium chloride, polyacrylamide, chitosan and other flocculants or other commercial inorganic aluminium salt, molysite, calcium salt and polymeric flocculant was adopted; filtration operations including plate-and-frame filtering, vacuum filtering or band type filter devices, etc. are adopted for filtration; the process being especially suitable for large-scale preparation technology; and a centrifugation device is suitable for solid-liquid separation in small-scale preparation process.
3. 3. The method of claim 1, wherein the process of chelation is that to add certain amount of a-ketoglutarate fermentation filter liquor or a-ketoglutarate solution in accordance with L-arginine content in filter liquor for chelation reaction; the molar weight of a-ketoglutarate added being 0.3-4.5 times the L-arginine molar weight in the system.
4. 4. The method of claim 1, wherein the concentration process is that to concentrate chelate solution containing L-arginine and a-ketoglutarate so that AAKG can reach to hypersaturated state; the preferred concentration devices being vacuum concentration, double-effect concentration or triple-effect concentration device; the solution being concentrated to 15% 50% of original volume of L-arginine and a -ketoglutarate solution mixture.
5. 5. The method of claim 1, wherein the process of crystallization is that to leave the concentrated solution under -5 15 for crystallization or use a crystallizer tank to cool solution with coolant. The crystallization process is controlled with 4'-SO hours. In the end, adopt filtration or centrifügation to collect crystals.
6. 6. The method of claim 1, wherein the process of dissolving and decolorization is that to dissolve above crystals again in 2'-' 5 times of deionized water, stir the mixture so that it can be dissolved completely. Add activated carbon particle into the mixture for decolorization. The amount of activated carbon added is equivalent to 0.5%'-3.0% of AAKG crystal quality. Stir and then heat the solution to 40'-90D for 20'-60 minutes. In the end, collect AAKG filter liquor through plate-and-frame filtering.
7. 7. The method of claim 1, wherein the process of recrystallization is that to slowly add hydrophilieity organic solvent of 1 -4 times of solution volume into above AAKG filter liquor. Perform recrystallization after it is incubated under room temperature or in refrigerator statically for 8 SOb. Collect crystals through filtration and wash the crystal for 2'--3 times with small amount of cold solvent.The mentioned hydrophilieity organic solvents include ethanol, methanol, acetone, isopropanol, glycerol, acetonitrile etc., where the preferred solvent is ethanol or acetone.
8. 8. The method of claim 1, wherein the process of crystal drying is that to adopt vacuum drying, heated air circulation oven or pneumatic conveying, the drying temperature is between SOLI-JSOLJ.