KIND OF METHOD FOR PREPARING L-CITRULLINE BY USING AEROMONAS SP.

Abstract

The present invention discloses a kind of method for preparing L-citrulline by using Aeromonas sp., which is related to the technical field of bioengineering. The said method consists of following steps: fermenting Aeromonas sp. in a culture medium to obtain seed liquid, and the liquid is inoculated into the fermenter at an inoculum size of 1-2% by volume. The initial pH is 3.5, and the pH in the medium is controlled below 4.0 all the time. Then, conducts shaking culture for 15-20 hours to obtain the fermentation broth. The fermentation broth is centrifuged to obtain precipitation solution and supernatant; Use the said supernatant to prepare the substrate solution, and the pH of the substrate solution is controlled at 3-5, and then, add the precipitation solution to react at the temperature of 30-50° C., the reaction time is 5-8 hours. After reaction, the L-citrulline is obtained by the method of concentration and crystallization. The present invention provides a method for preparing L-citrulline with low cost, high L-citrulline content and high purity.

Claims

1. A kind of method for preparing L-citrulline by using Aeromonas sp., characterized in that it consists of following steps: (1) Cultivate strains: Ferment Aeromonas sp. in the culture medium to obtain seed liquid, and inoculate the seed liquid in the fermenter at an inoculum size of 1-2% by volume. The initial pH is 3.5, and the pH in the medium is controlled below 4.0 all the time, the loaded liquid volume of the fermentation tank is ⅓ of the total volume, the volume of aeration is 12-15 m.sup.3/h, the tank pressure is 0.03-0.05 MPa, the rotation speed is 80-90 r/min, and the culture temperature is at 35-37° C. Add L-arginine solution during the process of fermentation, then conducts shaking culture for 15-20 hours, and thus obtains fermentation broth. After that, the fermentation broth is centrifuged, and thus obtains precipitation solution and supernatant; (2) Prepare L-citrulline: prepare a substrate solution with the said supernatant, and the pH of the substrate solution is controlled at 3-5, add the precipitation solution obtained in step (1), react at the temperature of 30-50° C., and the reaction time is 5-8 hours. After reaction, the L-citrulline feed liquid is obtained, and thus obtains L-citrulline by the method of concentration and crystallization.

2. The said method for preparing L-citrulline by using Aeromonas sp. according to claim 1, characterized in that the said culture medium consists of following components: glucose 0.5-1.0 g/mL, (NH4)2HPO4 0.02-0.3 g/mL, arginine 0.01-0.03 g/mL, (NH4)2HPO4 0.01-0.08 g/mL, manganese sulfate 0.02-0.07 g/mL, starch hydrolysate 2-5 g/mL, glutamic acid 0.001-0.005 g/mL, aspartic acid 0.01-0.03 g/mL, NaCl0.3-0.6 g/mL.

3. The said method for preparing L-citrulline by using Aeromonas sp. according to claim 1, characterized in that adding L-arginine solution in step (1) to make the concentration of L-arginine in the fermentation broth be 5-15 g/L.

4. The said method for preparing L-citrulline by using Aeromonas sp. according to claim 1, characterized in that the concentration of L-arginine in the substrate solution in step (2) is 100-130 g/L.

5. The said method for preparing L-citrulline using Aeromonas sp. according to claim 1, characterized in that the said method of obtaining L-citrulline by concentration and crystallization described in step (2) specifically consists of following steps: a. decolor and concentrate the prepared L-citrulline feed liquid, and thus obtains the first batch of L-citrulline concentrates; b. crystallize the first batch of the L-citrulline concentrates obtained in step a, and thus obtains the first batch of crude L-citrulline and the feed liquid after the crude L-citrulline is separated; c. concentrate the feed liquid after the crude L-citrulline is separated in step b, and thus obtains the second batch of L-citrulline concentrates; d. crystallize the second batch of L-citrulline concentrates obtained in step c, and thus obtains the second batch of crude L-citrulline and the feed liquid after the crude L-citrulline is separated; the said crystallization conditions are the same as those in step b; e. repeat steps c and d, and thus prepares the third batch of L-citrulline, which is the finished L-citrulline.

6. The said method for preparing L-citrulline by using Aeromonas sp. according to claim 5, characterized in that the said decolorization described in step a refers to adding activated carbon to the L-citrulline feed liquid, and thus obtains decolorized L-citrulline feed liquid; the amount of activated carbon added is 0.5-0.6 wt % of the mass of the L-citrulline feed liquid.

7. The said method for preparing L-citrulline by using Aeromonas sp. according to claim 5, characterized in that, the said concentration described in step a refers to stirring the said decolorized L-citrulline feed liquid at the temperature of 70-80° C. and under the vacuum condition of −0.08-0.07 MPa, when the Baume degree of the said decolorized L-citrulline feed liquid reaches 1.085-1.095, stop stirring, and thus obtains the L-citrulline concentrates; the said speed of stirring is 70-90 r/min.

8. The said method for preparing L-citrulline by using Aeromonas sp. according to claim 5, characterized in that the said crystallization described in step b refers to lowering the temperature of the first batch of L-citrulline concentrates to 0-5° C., and stirring the concentrates at a speed of 30-40 r/min in the meantime of cooling down, and thus obtains the crude L-citrulline.

9. The said method for preparing L-citrulline by using Aeromonas sp. according to claim 5, characterized in that the said concentration described in step c refers to stirring the said L-citrulline concentrates at the temperature of 70-80° C. and under the vacuum condition of −0.08-0.07 MPa, when the Baume degree of the L-citrulline concentrates reaches 1.250-1.325, stop stirring, and thus obtains the second batch of L-citrulline concentrates; the said speed of stirring is 70-90 r/min.

10. A kind of L-citrulline, characterized in that is prepared by any of the preparation methods described in claims 1-9, and the optical rotation of the said L-citrulline is 24.8°-25.75°, the light transmittance is >99.99%.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0032] Various exemplary embodiments of the present invention will be described in detail herein. The detailed description should not be constructed as a limitation to the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and embodiments of the present invention.

[0033] It should be interpreted that the said terms described in the present invention are only used to describe specific embodiments rather than limiting the present invention. In addition, as for the numerical range described in the present invention, it should be interpreted that each intermediate value between the upper and the lower limits of the range is also specifically disclosed. Each smaller range between any stated value or intermediate value within the stated range and any other stated value or intermediate value within the stated range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0034] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the said art of the present invention. Although the present invention only describes preferred methods and materials, any method and material similar or equivalent to those described herein can also be used in the embodiment or testing of the present invention. All documents mentioned in the present specification are incorporated by reference, and used to disclose and describe methods and/or materials related to the said documents. In the event of conflict with any incorporated document, the content of the present specification shall prevail.

[0035] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the present specification, which is obvious to those skilled in the art. Other embodiments derived from the specification of the present invention will be obvious to the skilled person. The specification and embodiments of the present invention are only exemplary.

[0036] Such terms as “consist of”, “include”, “have”, “contain”, etc., used herein are all open words, which mean including but not limited to.

[0037] The said Aeromonas sp. of the present invention is Aeromonas sp. YQ, which is preserved in the Chinese Type Culture Collection, the preservation number: CCTCC No: M 2015271, the preservation date: May 3, 2015, and the address: Wuhan University, Wuhan, China, 430072.

[0038] The said bacterial strain of the present invention is obtained from: the soil samples collected from the watermelon greenhouse in Gouzhuang, Hangzhou City, Zhejiang Province, which is subjected to primary screening and secondary screening, and thus obtains a pure culture capable of converting the substrate L-arginine to produce L-citrulline. The Morphological, physiological, and biochemical identification of the pure culture was carried out, and it was identified as Aeromonas sp., named as Aeromonas sp. YQ.

Embodiment 1

[0039] The said method for preparing L-citrulline by using Aeromonas sp. in this embodiment consists of following steps:

[0040] (1) Cultivate strains: Ferment Aeromonas sp. in the culture medium to obtain seed liquid, and inoculate the seed liquid in a 300 L fermenter at an inoculum size of 1% by volume. The initial pH is 3.5, and the pH in the medium is controlled below 4.0 all the time, the loaded liquid volume of the fermentation tank is ⅓ of the total volume, the volume of aeration is 12 m.sup.3/h, the tank pressure is 0.03 MPa, the rotation speed is 80 r/min, and the culture temperature is at 35° C. Add L-arginine solution during the process of fermentation to make the concentration of L-arginine in the fermentation broth is 5 g/L, then conducts shaking culture for 15 hours, and thus obtains fermentation broth. After that, the fermentation broth is centrifuged, and thus obtains precipitation solution and supernatant;

[0041] The said culture medium consists of following components: glucose 0.5 g/mL, (NH4)2HPO4 0.3 g/mL, arginine 0.03 g/mL, (NH4)2HPO4 0.01 g/mL, manganese sulfate 0.07 g/mL, starch hydrolysate 2 g/mL, glutamic acid 0.001 g/mL, aspartic acid 0.03 g/mL, NaCl 0.6 g/mL;

[0042] (2) Prepare L-citrulline: prepare a substrate solution with the said supernatant, and the concentration of L-arginine in the substrate solution is 100 g/L, the pH of the substrate solution is controlled at 3, add the precipitation solution obtained in step (1), react at the temperature of 30° C., and the reaction time is 8 hours. After reaction, the L-citrulline feed liquid is obtained, and thus obtains L-citrulline by the method of concentration and crystallization.

[0043] The said method of obtaining L-citrulline by concentration and crystallization described in step (2) specifically consists of following steps:

[0044] a. add activated carbon to the L-citrulline feed liquid for decolorization and concentration, and thus obtains the first batch of L-citrulline concentrates. The amount of activated carbon added is 0.5 wt % of the mass of the L-citrulline feed liquid, the said concentration refers to stirring the said decolorized L-citrulline feed liquid at the temperature of 70° C. and under the vacuum condition of −0.08 MPa, when the Baume degree of the said decolorized L-citrulline feed liquid reaches 1.085-1.095, stop stirring, and thus obtains the L-citrulline concentrates; the said speed of stirring is 70 r/min;

[0045] b. crystallize the first batch of the L-citrulline concentrates obtained in step a, and thus obtains the first batch of crude L-citrulline and the feed liquid after the crude L-citrulline is separated, the said crystallization refers to lowering the temperature of the L-citrulline concentrates to 0° C., and stirring the concentrates at a speed of 30 r/min in the meantime of cooling down, and thus obtains the crude L-citrulline;

[0046] c. concentrate the feed liquid after the crude L-citrulline is separated in step b, and thus obtains the second batch of L-citrulline concentrates, the said concentration refers to stirring the said L-citrulline concentrates at the temperature of 70° C. and under the vacuum condition of −0.08 MPa, when the Baume degree of the L-citrulline concentrates reaches 1.250-1.325, stop stirring, and thus obtains the second batch of L-citrulline concentrates; the said speed of stirring is 90 r/min;

[0047] d. crystallize the second batch of L-citrulline concentrates obtained in step c, and thus obtains the second batch of crude L-citrulline and the feed liquid after the crude L-citrulline is separated; the said crystallization conditions are the same as those in step b;

[0048] e. repeat steps c and d, and thus prepares the third batch of L-citrulline.

[0049] The optical rotation of the finished L-citrulline prepared in the present embodiment is 24.8°, the light transmittance is 99.8%, the purity of L-citrulline is 70%, and the total recovery is 80%.

Embodiment 2

[0050] The said method for preparing L-citrulline by using Aeromonas sp. in this embodiment consists of following steps:

[0051] (1) Cultivate strains: Ferment Aeromonas sp. in the culture medium to obtain seed liquid, and inoculate the seed liquid in a 200 L fermenter at an inoculum size of 1% by volume. The initial pH is 3.5, and the pH in the medium is controlled below 4.0 all the time, the loaded liquid volume of the fermentation tank is ⅓ of the total volume, the volume of aeration is 15 m.sup.3/h, the tank pressure is 0.05 MPa, the rotation speed is 90 r/min, and the culture temperature is at 37° C. Add L-arginine solution during the process of fermentation, and the concentration of L-arginine added in the fermentation broth is 15 g/L, then conducts shaking culture for 20 hours, and thus obtains fermentation broth. After that, the fermentation broth is centrifuged, and thus obtains precipitation solution and supernatant;

[0052] The said culture medium consists of following components: glucose 1.0 g/mL, (NH4)2HPO4 0.08 g/mL, arginine 0.01 g/mL, (NH4)2HPO4 0.08 g/mL, manganese sulfate 0.02 g/mL, starch hydrolysate 3 g/mL, glutamic acid 0.003 g /mL, aspartic acid 0.02 g/mL, NaCl 0.5 g/mL;

[0053] (2) Prepare L-citrulline: prepare a substrate solution with the said supernatant, and the concentration of L-arginine in the substrate solution is 120 g/L, the pH of the substrate solution is controlled at 4, add the precipitation solution obtained in step (1), react at the temperature of 50° C., and the reaction time is 5 hours. After reaction, the L-citrulline is obtained by the method of concentration and crystallization.

[0054] The said method of obtaining L-citrulline by concentration and crystallization described in step (2) specifically consists of following steps:

[0055] a. add activated carbon to the L-citrulline feed liquid for decolorization and concentration, and thus obtains the first batch of L-citrulline concentrates. The amount of activated carbon added is 0.6 wt % of the mass of the L-citrulline feed liquid, the said concentration refers to stirring the said decolorized L-citrulline feed liquid at the temperature of 80° C. and under the vacuum condition of 0.07 MPa, when the Baume degree of the said decolorized L-citrulline feed liquid reaches 1.085-1.095, stop stirring, and thus obtains the L-citrulline concentrates; the said speed of stirring is 80 r/min;

[0056] b. crystallize the first batch of the L-citrulline concentrates obtained in step a, and thus obtains the first batch of crude L-citrulline and the feed liquid after the crude L-citrulline is separated, the said crystallization refers to lowering the temperature of the L-citrulline concentrates to 3° C., and stirring the concentrates at a speed of 35 r/min in the meantime of cooling down, and thus obtains the crude L-citrulline;

[0057] c. concentrate the feed liquid after the crude L-citrulline is separated in step b, and thus obtains the second batch of L-citrulline concentrates, the said concentration refers to stirring the said L-citrulline concentrates at the temperature of 75° C. and under the vacuum condition of 0.07 MPa, when the Baume degree of the L-citrulline concentrates reaches 1.250-1.325, stop stirring, and thus obtains the second batch of L-citrulline concentrates; the said speed of stirring is 80 r/min;

[0058] d. crystallize the second batch of L-citrulline concentrates obtained in step c, and thus obtains the second batch of crude L-citrulline and the feed liquid after the crude L-citrulline is separated; the said crystallization conditions are the same as those in step b;

[0059] e. repeat steps c and d, and thus prepares the third batch of L-citrulline.

[0060] The optical rotation of the finished L-citrulline prepared in the present embodiment is 25.45°, the light transmittance is 99.8%, the purity of L-citrulline is 75%, and the total recovery is 83%.

Embodiment 3

[0061] The said method for preparing L-citrulline by using Aeromonas sp. in this embodiment consists of following steps:

[0062] (1) Cultivate strains: Ferment Aeromonas sp. in the culture medium to obtain seed liquid, and inoculate the seed liquid in a 300 L fermenter at an inoculum size of 2% by volume. The initial pH is 3.5, and the pH in the medium is controlled below 4.0 all the time, the loaded liquid volume of the fermentation tank is ⅓ of the total volume, the volume of aeration is 15 m.sup.3/h, the tank pressure is 0.04 MPa, the rotation speed is 85 r/min, and the culture temperature is at 36° C. Add L-arginine solution during the process of fermentation, and the concentration of L-arginine added in the fermentation broth is 8 g/L, then conducts shaking culture for 20 hours, and thus obtains fermentation broth. After that, the fermentation broth is centrifuged, and thus obtains precipitation solution and supernatant;

[0063] The said culture medium consists of following components: glucose 0.8 g/mL, (NH4)2HPO4 0.08 g/mL, arginine 0.02 g/mL, (NH4)2HPO4 0.05 g/mL, manganese sulfate 0.03 g/mL, starch hydrolysate 3 g/mL, glutamic acid 0.003 g /mL, aspartic acid 0.02 g/mL, NaCl 0.5 g/mL;

[0064] (2) Prepare L-citrulline: prepare a substrate solution with the said supernatant, and the concentration of L-arginine in the substrate solution is 130 g/L, the pH of the substrate solution is controlled at 5, add the precipitation solution obtained in step (1), react at the temperature of 30° C., and the reaction time is 8 hours. After reaction, the L-citrulline is obtained by the method of concentration and crystallization.

[0065] The said method of obtaining L-citrulline by concentration and crystallization described in step (2) specifically consists of following steps:

[0066] a. add activated carbon to the L-citrulline feed liquid for decolorization and concentration, and thus obtains the first batch of L-citrulline concentrates. The amount of activated carbon added is 0.6 wt % of the mass of the L-citrulline feed liquid, the said concentration refers to stirring the said decolorized L-citrulline feed liquid at the temperature of 75° C. and under the vacuum condition of 0.07 MPa, when the Baume degree of the said decolorized L-citrulline feed liquid reaches 1.085-1.095, stop stirring, and thus obtains the L-citrulline concentrates; the said speed of stirring is 90 r/min;

[0067] b. crystallize the first batch of the L-citrulline concentrates obtained in step a, and thus obtains the first batch of crude L-citrulline and the feed liquid after the crude L-citrulline is separated, the said crystallization refers to lowering the temperature of the L-citrulline concentrates to 5° C., and stirring the concentrates at a speed of 40 r/min in the meantime of cooling down, and thus obtains the crude L-citrulline;

[0068] c. concentrate the feed liquid after the crude L-citrulline is separated in step b, and thus obtains the second batch of L-citrulline concentrates, the said concentration refers to stirring the said L-citrulline concentrates at the temperature of 80° C. and under the vacuum condition of 0.05 MPa, when the Baume degree of the L-citrulline concentrates reaches 1.250-1.325, stop stirring, and thus obtains the second batch of L-citrulline concentrates; the said speed of stirring is 80 r/min;

[0069] d. crystallize the second batch of L-citrulline concentrates obtained in step c, and thus obtains the second batch of crude L-citrulline and the feed liquid after the crude L-citrulline is separated; the said crystallization conditions are the same as those in step b;

[0070] e. repeat steps c and d, and thus prepares the third batch of L-citrulline.

[0071] The optical rotation of the finished L-citrulline prepared in the present embodiment is 25.75°, the light transmittance is 99.9%, the purity of L-citrulline is 73%, and the total recovery is 85%.

Comparative Example 1

[0072] Same as the Embodiment 3, the only difference is that the initial pH in step (1) is 6.8, and the pH is maintained at about 7.2 during the fermentation process.

[0073] The optical rotation of the finished L-citrulline prepared in the present comparative example is 23.6°, the light transmittance is 99.1%, the purity of L-citrulline is 33%, and the total recovery is 35%.

Comparative Example 2

[0074] Same as the Embodiment 3, the only difference is that the pH of the substrate solution in step (2) is controlled at 6.

[0075] The optical rotation of the finished L-citrulline prepared in the present comparative example is 23.1°, the light transmittance is 99.2%, the purity of L-citrulline is 31%, and the total recovery is 33%.

[0076] The above-mentioned embodiments only describe the preferred mode of the present invention, rather than limiting the scope of the present invention. Without departing from the design and the spirit of the present invention, any variation and improvement to the technical schemes of the present invention made by those skilled in the art shall fall within the protection scope determined by the claims of the present invention.