阅读说明：本技术 一种l-瓜氨酸的制造方法 (Method for preparing L-citrulline ) 是由 张礼 秦振华 于 2020-12-29 设计创作，主要内容包括：本发明公开了一种L-瓜氨酸的制造方法,步骤1：将含有L-瓜氨酸的转化液经超滤膜过滤,得到含有L-瓜氨酸的超滤清液；步骤2：将超滤清液脱盐,获得透析清液；步骤3：将透析清液的pH调节至4.2-4.6,在透析清液中加入活性炭脱色；步骤4：将脱色液在真空度进行浓缩,浓缩至L-瓜氨酸的含量为60-70%的浓缩液；步骤5：将浓缩液冷却至10℃条件下,加入晶种,冷却结晶3-4h,抽滤得L-瓜氨酸的晶体；步骤6：将L-瓜氨酸的晶体进行真空干燥,得纯化的L-瓜氨酸产品。本发明采用的制备L-瓜氨酸的方法与传统化学合成法、酶法和微生物发酵法相比,过程控制较为简单,生产过程中不会产生大量的污染物质,对环境友好。(The invention discloses a method for preparing L-citrulline, which comprises the following steps: filtering the conversion solution containing the L-citrulline by an ultrafiltration membrane to obtain ultrafiltration clear solution containing the L-citrulline; step 2: desalting the ultrafiltration clear solution to obtain a dialysis clear solution; and step 3: adjusting pH of the dialyzed clear solution to 4.2-4.6, and adding activated carbon into the dialyzed clear solution for decolorization; and 4, step 4: concentrating the decolorized solution at vacuum degree to obtain a concentrated solution containing L-citrulline 60-70%; and 5: cooling the concentrated solution to 10 ℃, adding seed crystals, cooling and crystallizing for 3-4h, and performing suction filtration to obtain crystals of L-citrulline; step 6: and (3) carrying out vacuum drying on the crystals of the L-citrulline to obtain a purified L-citrulline product. Compared with the traditional chemical synthesis method, the enzyme method and the microbial fermentation method, the method for preparing the L-citrulline has the advantages of simpler process control, no generation of a large amount of pollutants in the production process and environmental friendliness.)

1. A method for producing L-citrulline, which is characterized by comprising the following steps:

step 1: and (3) ultrafiltration: filtering the conversion solution containing L-citrulline by an ultrafiltration membrane, removing the conversion solution, recovering and utilizing arginine deiminase to obtain ultrafiltration clear solution containing L-citrulline, and cleaning the ultrafiltration clear solution by using a small amount of pure water;

step 2: electrodialysis: carrying out electrodialysis treatment on the washed ultrafiltration clear liquid of the L-citrulline to further desalt and residue arginine, and obtaining dialysis clear liquid;

and step 3: activated carbon decolorization: adjusting pH of the dialyzed clear solution to 4.2-4.6, adding activated carbon into the dialyzed clear solution, decolorizing at 50-60 deg.C for 25-35min, and filtering with filter membrane to obtain decolorized solution containing L-citrulline;

and 4, step 4: concentration: concentrating decolorized solution at 50-55 deg.C under vacuum degree of 0.095Mpa or more to obtain concentrated solution containing L-citrulline 60-70%;

and 5: and (3) crystallization: cooling the concentrated solution to 10 ℃, adding seed crystals, cooling and crystallizing for 3-4h, and performing suction filtration to obtain crystals of L-citrulline;

step 6: and (3) drying: and (3) carrying out vacuum drying on the crystals of the L-citrulline to obtain a purified L-citrulline product.

2. The method for producing L-citrulline according to claim 1, characterized in that: in the step 1, when the ultrafiltration membrane is used for filtration, the filtration temperature is less than 50 ℃, and the filter membrane aperture of the ultrafiltration membrane is 3000-10000D.

3. The method for producing L-citrulline according to claim 1, characterized in that: in step 2, the voltage during electrodialysis is 5V, the current of electrodialysis is changed under the condition of constant voltage, the change range of the current is 0.1-1A, the temperature is 20-30 ℃, the pH value of a fresh water chamber is maintained at 5.8-6.0 during desalination, and the final fresh water conductivity is 1-5 ms/cm.

4. The method for producing L-citrulline according to claim 1, characterized in that: in step 3, the volume ratio of the dialyzed clear solution to the activated carbon is 1: 0.5-1%.

5. The method for producing L-citrulline according to claim 1, characterized in that: in step 3, a 0.22 μm filter was used for filtration.

6. The method for producing L-citrulline according to claim 1, characterized in that: in step 5, the volume ratio of the concentrated solution to the seed crystal is 1: 0.1-0.5%.

7. The method for producing L-citrulline according to claim 1, characterized in that: in step 6, the temperature of vacuum drying is 50-60 ℃.

Technical Field

The invention relates to the technical field of bioengineering, in particular to a method for preparing L-citrulline.

Background

L-Citrulline (L-Citrulline) with the chemical name of L- (+) -2-amino-5-urea valeric acid and the molecular formula of CAS No 372-75-8C₆H₁₃N₃O₃Molecular weight of 175.19, melting point of 222 ℃, isoelectric point of 5.92, and specific optical rotation [ alpha ]]D25= +24.5 to +26.8 degrees, white crystal or crystalline powder, sour, soluble in water, insoluble in ethanol and ether. L-citrulline is an alpha amino acid named from the watermelon from which citrulline was first extracted.

L-citrulline is an important non-essential amino acid, is an important intermediate in the urea cycle process, and has a plurality of important physiological functions such as free radical removal, foreign body rejection effect indicator, vasodilation, blood pressure stabilization, rheumatoid arthritis diagnosis, antioxidation and the like; in addition, it has very obvious functions of restoring energy and raising immunity of human body. In recent years, L-citrulline is more and more emphasized in the fields of food, cosmetics, medicines and the like, and has wide development and application prospects.

At present, the main production methods of L-citrulline include chemical synthesis methods, enzymatic methods and microbial fermentation methods.

(1) The chemical method means that L-arginine is hydrolyzed under alkaline condition to obtain L-citrulline, the process is difficult to control, the product contains optical antipode D-citrulline, the product quality is influenced, a large amount of waste water is generated in the production process, and the environment is greatly polluted.

(2) The difficulty of the fermentation production is that the yield of the L-citrulline in unit volume is low due to the feedback inhibition in the citrulline metabolic pathway, so the cost for extracting the L-citrulline from the fermentation liquor is high, and the sewage quantity is large. The fermentation method has the advantages of low cost, high yield, high product purity, no toxic substances in the production process of the product, and convenience for post-processing of the product. At present, the country most studied for the production of citrulline by fermentation is japan, and a certain level of fermentation has been achieved.

(3) An enzyme method comprises the following steps: under the action of arginine deiminase, L-arginine is converted into L-citrulline, the production condition is mild, no toxic substance is generated, impurities in a conversion system are less, the extraction process is simple, and the pollution is less. The method takes microbial somatic cells with strong specificity and high conversion rate as a catalyst to catalyze the deimination of arginine to generate L-citrulline. The enzymatic method is used for synthesizing the citrulline, and a one-step enzymatic reaction is mostly adopted, so that the complex feedback regulation effect in the complete synthesis path of the citrulline can be avoided, and the citrulline can be accumulated to a higher concentration. The immobilized enzyme or immobilized cell is adopted for catalysis, so that the difficulty of subsequent separation and purification is further reduced.

However, in the process of separating and purifying the L-citrulline from the enzyme conversion solution, arginine cannot be completely converted, and a large amount of ammonium salt byproducts are generated in the conversion solution, which has a certain influence on the product purity. The existing process has complex extraction process and multiple steps, some of the processes need to use ion exchange resin for product separation, the sewage production amount is large, and the environmental pressure maintaining capacity is high. The citrulline has high solubility in water phase, so the crystallization temperature is low, about 4 ℃, the production has strict requirements on temperature conditions, and the crystallization yield is low.

Disclosure of Invention

The present invention is directed to a method for producing L-citrulline, which solves the problems set forth in the background art.

In order to achieve the purpose, the invention provides the following technical scheme: a method for producing L-citrulline, comprising the following steps:

step 1: and (3) ultrafiltration: filtering the conversion solution containing L-citrulline by an ultrafiltration membrane, removing the conversion solution, recovering and utilizing arginine deiminase to obtain ultrafiltration clear solution containing L-citrulline, and cleaning the ultrafiltration clear solution by using a small amount of pure water;

step 2: electrodialysis: carrying out electrodialysis treatment on the washed ultrafiltration clear liquid of the L-citrulline to further desalt and residue arginine, and obtaining dialysis clear liquid;

and step 3: activated carbon decolorization: adjusting pH of the dialyzed clear solution to 4.2-4.6, adding activated carbon into the dialyzed clear solution, decolorizing at 50-60 deg.C for 25-35min, and filtering with filter membrane to obtain decolorized solution containing L-citrulline;

and 4, step 4: concentration: concentrating decolorized solution at 50-55 deg.C under vacuum degree of 0.095Mpa or more to obtain concentrated solution containing L-citrulline 60-70%;

and 5: and (3) crystallization: cooling the concentrated solution to 10 ℃, adding seed crystals, cooling and crystallizing for 3-4h, and performing suction filtration to obtain crystals of L-citrulline;

step 6: and (3) drying: and (3) carrying out vacuum drying on the crystals of the L-citrulline to obtain a purified L-citrulline product.

Preferably, in the step 1, when the ultrafiltration membrane is used for filtration, the filtration temperature is less than 50 ℃, and the membrane aperture of the ultrafiltration membrane is 3000-10000D.

Preferably, in step 2, the voltage during electrodialysis is 5V, the current during electrodialysis is changed under the condition of constant voltage, the current is changed within the range of 0.1-1A, the temperature is 20-30 ℃, the pH value of the fresh water chamber is maintained at 5.8-6.0 during desalination, and the final fresh water conductivity is 1-5 ms/cm.

Preferably, in step 3, the volume ratio of the dialyzed clear solution to the activated carbon is 1: 0.5-1%.

Preferably, in step 3, a 0.22 μm filter is used for filtration.

Preferably, in step 5, the volume ratio of the concentrated solution to the seed crystal is 1: 0.1-0.5%.

Preferably, in step 6, the temperature for vacuum drying is 50-60 ℃.

Compared with the prior art, the invention has the beneficial effects that:

compared with the traditional chemical synthesis method, the enzyme method and the microbial fermentation method, the method for preparing the L-citrulline has the advantages that the process control is simpler, a large amount of pollutants cannot be generated in the production process, and the method is environment-friendly; meanwhile, ethanol is not used in the production process, and the concentrated crystal is directly crystallized under certain process conditions, so that the method has the advantages of mild production conditions, short concentration time, high product purity, high crystallization yield, high yield, low production cost and the like.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The invention provides a technical scheme that: a method for producing L-citrulline, comprising the following steps:

example 1:

the novel extraction process of the L-citrulline comprises the following steps:

1) and (3) ultrafiltration: filtering the conversion solution containing L-citrulline by an ultrafiltration membrane, removing the conversion solution, namely arginine deiminase (purchased) and recycling to obtain ultrafiltration clear solution containing L-citrulline, and washing a small amount of pure water in the system. And (3) ultrafiltration membrane filtration conditions: the temperature is 10 ℃, and the pore diameter is 3000D;

2) electrodialysis: performing electrodialysis treatment on the microfiltration clear liquid of the L-citrulline for one-step desalination to obtain dialysis clear liquid; electrodialysis conditions: the voltage is 5V, the current of the electrodialysis equipment is a changing process but not constant under the condition of constant voltage, the changing range of the current is 0.1-1A, the temperature is 20 ℃, the pH value of the fresh water chamber is maintained at 5.8, and the final fresh water conductivity is 1 ms/cm;

3) activated carbon decolorization: adjusting pH of the nanofiltration clear liquid to 4.2, adding active carbon according to 0.5% of volume ratio, decolorizing at 50 deg.C for 25 min; filtering with 0.22 μm filter membrane to obtain decolorized solution containing L-citrulline;

4) concentration and crystallization: concentrating the pre-concentrated solution at a vacuum degree of not less than 0.095Mpa and a concentration temperature of 50 ℃ to obtain a concentrated solution with the L-citrulline content of 60-70%; cooling the concentrated solution to 10 ℃, adding 0.1% of seed crystal, cooling and crystallizing for 3h, and performing suction filtration to obtain a crystal of L-citrulline; the crystallization mother liquor returns to electrodialysis circulation for recovery;

5) and (3) drying: and (3) carrying out vacuum drying on the crystals of the L-citrulline at 50 ℃ to obtain a purified L-citrulline product.

Example 2:

the novel extraction process of the L-citrulline comprises the following steps:

1) and (3) ultrafiltration: filtering the conversion solution containing L-citrulline by an ultrafiltration membrane, removing the conversion solution, namely arginine deiminase (purchased) and recycling to obtain ultrafiltration clear solution containing L-citrulline, and washing a small amount of pure water in the system. And (3) ultrafiltration membrane filtration conditions: temperature 20 ℃, aperture 5000D;

2) electrodialysis: performing electrodialysis treatment on the microfiltration clear liquid of the L-citrulline for one-step desalination to obtain dialysis clear liquid; electrodialysis conditions: the voltage is 5V, the current of the electrodialysis equipment is a changing process but not constant under the condition of constant voltage, the changing range of the current is 0.1-1A, the temperature is 20 ℃, the pH value of the fresh water chamber is maintained at 5.8, and the final fresh water conductivity is 1 ms/cm;

3) activated carbon decolorization: adjusting pH of the nanofiltration clear liquid to 4.5, adding active carbon according to 0.7% of volume ratio, decolorizing at 55 deg.C for 30 min; filtering with 0.22 μm filter membrane to obtain decolorized solution containing L-citrulline;

4) concentration and crystallization: concentrating the pre-concentrated solution at a vacuum degree of not less than 0.096Mpa and a concentration temperature of 50 ℃ to obtain a concentrated solution with the L-citrulline content of 60-70%; cooling the concentrated solution to 10 ℃, adding 0.3% of seed crystal, cooling and crystallizing for 3.5h, and performing suction filtration to obtain a crystal of L-citrulline; the crystallization mother liquor returns to electrodialysis circulation for recovery;

5) and (3) drying: and drying the crystals of the L-citrulline at 55 ℃ in vacuum to obtain a purified L-citrulline product.

Example 3:

the novel extraction process of the L-citrulline comprises the following steps:

1) and (3) ultrafiltration: filtering the conversion solution containing L-citrulline by an ultrafiltration membrane, removing the conversion solution, namely arginine deiminase (purchased) and recycling to obtain ultrafiltration clear solution containing L-citrulline, and washing a small amount of pure water in the system. And (3) ultrafiltration membrane filtration conditions: the temperature is 40 ℃, and the pore diameter is 10000D;

2) electrodialysis: performing electrodialysis treatment on the microfiltration clear liquid of the L-citrulline for one-step desalination to obtain dialysis clear liquid; electrodialysis conditions: the voltage is 5V, the current of the electrodialysis equipment is a changing process but not constant under the condition of constant voltage, the changing range of the current is 0.1-1A, the temperature is 20 ℃, the pH value of the fresh water chamber is maintained at 5.8, and the final fresh water conductivity is 1 ms/cm;

3) activated carbon decolorization: adjusting the pH value of the nanofiltration clear liquid to 4.6, adding activated carbon according to 1% of the volume ratio, and decoloring at 60 ℃ for about 35 min; filtering with 0.22 μm filter membrane to obtain decolorized solution containing L-citrulline;

4) concentration and crystallization: concentrating the pre-concentrated solution at a vacuum degree of more than or equal to 0.098Mpa and a concentration temperature of 55 ℃ to obtain a concentrated solution with the L-citrulline content of 60-70%; cooling the concentrated solution to 10 ℃, adding 0.5% of seed crystal, cooling and crystallizing for 4h, performing suction filtration to obtain crystals of L-citrulline, and returning the crystallization mother liquor to electrodialysis for recycling;

5) and (3) drying: and (3) carrying out vacuum drying on the crystals of the L-citrulline at 60 ℃ to obtain a purified L-citrulline product.

In the three embodiments, the yield of ultrafiltration and electrodialysis reaches 99%, the comprehensive yield of the concentrated and crystallized product is more than 90%, and the method has the advantages of mild production conditions, short concentration time, high product purity, high crystallization yield, high yield, low production cost and the like.

Application method

A method for producing L-citrulline, comprising the following steps:

and (3) ultrafiltration: filtering the conversion solution containing L-citrulline by an ultrafiltration membrane, removing the conversion solution, recovering and utilizing arginine deiminase to obtain ultrafiltration clear solution containing L-citrulline, and cleaning the ultrafiltration clear solution by using a small amount of pure water;

step 2: electrodialysis: carrying out electrodialysis treatment on the washed ultrafiltration clear liquid of the L-citrulline to further desalt and residue arginine, and obtaining dialysis clear liquid;

and step 3: activated carbon decolorization: adjusting pH of the dialyzed clear solution to 4.2-4.6, adding activated carbon into the dialyzed clear solution, decolorizing at 50-60 deg.C for 25-35min, and filtering with filter membrane to obtain decolorized solution containing L-citrulline;

and 4, step 4: concentration: concentrating decolorized solution at 50-55 deg.C under vacuum degree of 0.095Mpa or more to obtain concentrated solution containing L-citrulline 60-70%;

and 5: and (3) crystallization: cooling the concentrated solution to 10 ℃, adding seed crystals, cooling and crystallizing for 3-4h, and performing suction filtration to obtain crystals of L-citrulline;

step 6: and (3) drying: and (3) carrying out vacuum drying on the crystals of the L-citrulline to obtain a purified L-citrulline product.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.