A method for purifying L-leucine using mono- and distearic acid glyceryl esters

Abstract

The present invention relates to the field of biotechnology, and more specifically, to a method for purifying L-leucine using mono- and distearic acid glycerol. After filtering and decolorizing the L-leucine fermentation broth, mono- and distearic acid glycerol is added to assist in crystallization, thereby obtaining high-purity, high-yield L-leucine. The method of the present invention features mild conditions, simple operation, a minimal number of separation steps, and clean production. It reduces the accumulation of material on the wall and aggregation during the evaporation and concentration process of L-leucine, increases the amount of L-leucine crystallization, and significantly improves the yield and purity of L-leucine.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a method for purifying L-leucine with the assistance of mono- and distearic acid glyceryl. Background Art

[0002] L-Leucine is an essential amino acid with the chemical formula C6H 13 NO2 occurs as white, shiny hexahedral crystals or a white crystalline powder at room temperature. It is odorless with a slightly bitter taste. It sublimes at 145-148°C. Its melting point is 293-295°C (decomposition). It is slightly soluble in ethanol (0.07%) and insoluble in ether. It is used for the diagnosis and treatment of idiopathic hyperglycemia in children. It is also indicated for disorders of glucose metabolism, liver disease with decreased bile secretion, anemia, poisoning, muscular dystrophy, post-poliomyelitis sequelae, neuritis, and mental illness.

[0003] Currently, the main method for producing L-leucine is fermentation. The fermentation broth of L-leucine produced by the fermentation method undergoes extraction, evaporation concentration, centrifugal drying, and other steps before it can be made into a finished product. Chinese Patent Publication CN109761833A discloses a method for separating and purifying L-leucine, which comprises: (1) adding a coagulant and a flocculant to the fermentation broth, allowing it to stand, and centrifuging to obtain a supernatant for later use; (2) adding sodium chloride and L-leucine seed crystals to the supernatant to separate the precipitate; (3) dissolving the precipitate in water, adding activated carbon for decolorization, centrifuging, and concentrating to precipitate crystals; (4) dissolving the crystals in water, passing them through ion exchange resin chromatography, and eluting with ammonia water to obtain L-leucine. Although this method is beneficial for improving the purity of leucine, the yield is low and the loss of leucine is large. In addition to the fermentation method, the hydrolysis method is also a common method for producing L-leucine. Chinese invention patent CN104926670A discloses a method for extracting L-leucine from plant protein by acid hydrolysis. This method uses plant protein - corn yellow powder, soybean paste, cottonseed protein or mulberry silk protein as raw materials. The production is not limited by animal-derived raw materials. In addition, the invention does not use a precipitant, but achieves the purpose of separating L-leucine by adjusting the pH, thereby solving the environmental hazard. Since a large amount of hydrochloric acid needs to be added to adjust the pH to the appropriate range during the hydrolysis process, and sodium metabisulfite oxidant is added, deacidification and desalination treatment are required, which increases production costs. The traditional production process for extracting L-leucine generally adopts an ion exchange process, which removes inorganic salts in the fermentation broth by ion exchange means. Chinese patent publication CN102757354A describes a method for extracting and isolating L-leucine from fermentation broth using a combination of membrane separation and electrodialysis to remove salts. This method involves directly desalting the L-leucine fermentation broth membrane filtrate through electrodialysis. However, because the L-leucine membrane filtrate still contains a large amount of impurities such as protein, the electrodialysis desalination rate is low, and the electrodialysis process has no decolorization effect on the membrane filtrate. Therefore, a simple combined extraction method is currently needed to improve the extraction yield and purity of L-leucine. Summary of the Invention

[0004] The present invention aims to provide a method for purifying L-leucine by using mono- and distearic acid glyceryl esters.

[0005] To achieve the above purpose, the present invention adopts the following technical solutions:

[0006] A method for purifying L-leucine using mono- and distearic acid glycerol assists, wherein the L-leucine fermentation broth is filtered and decolorized, and then mono- and distearic acid glycerol is added to assist crystallization, thereby obtaining high-purity and high-yield L-leucine.

[0007] Specifically:

[0008] S1. The L-leucine fermentation broth was filtered using a ceramic membrane, and then the ceramic filter liquid system was decolorized by adding activated carbon having a solid content of 0.3 to 3%, and the decolorized liquid was collected and set aside;

[0009] S2. Adjust the pH of the decolorized solution in S1 to 7±0.2, then add a crystallization aid and concentrate at 60-70°C to obtain a concentrate with precipitated crystals, and filter and collect the crystallization cake and filtrate;

[0010] S3. Add water to the crystallized filter cake in S2 for redissolution, collect the filtrate by filtration, concentrate and recrystallize to obtain high-yield and high-purity L-leucine.

[0011] The L-leucine fermentation broth in step S1 is prepared by culturing Brevibacterium flavum seed solution in a composite nutrient solution. Further reference is made to the method described in CN201811647345.0, "A method for increasing L-leucine fermentation yield."

[0012] The crystallization aid in step S2 is glyceryl mono- and distearate, and the amount of the added agent is 100 to 5000 ppm of the solid content in the decolorizing solution.

[0013] In the crystallization aid glyceryl mono- and distearate in step S2, the mass percentage of glyceryl mono-stearate is 30-70 wt%.

[0014] The concentration end point in step S2 is: the total mass of the concentrated solution from which the crystals are precipitated is twice the solid content of the decolorized solution.

[0015] The re-dissolution in step S3 is to add 40 to 50 times the mass of water to the crystallized filter cake to fully dissolve it.

[0016] In step S3, the re-dissolution temperature is 20-70° C., and after stirring for 30 minutes, the mixture is cooled to room temperature, and filtered or centrifuged after the mono- and distearic glyceryl esters are completely precipitated.

[0017] The end point of the recrystallization concentration in step S3 is: the total mass of the reconstituted concentrated solution is 3.5 times the solid content of the reconstituted solution.

[0018] The advantages of the present invention are:

[0019] The present invention introduces mono- and distearic acid glyceryl into the crystallization process after leucine decolorization, thereby promoting the nucleation and precipitation of leucine and forming hydrogen bonds with leucine. At the end of crystallization, the mono- and distearic acid glyceryl coat the periphery of the leucine crystals, thereby reducing the phenomenon of material wall adhesion and agglomeration during the evaporation and concentration process of L-leucine. The solvent content is reduced as much as possible at the end of evaporation and concentration, while maintaining fluidity, thereby improving the yield and output of leucine.

[0020] The method of the invention has the characteristics of mild conditions, simple operation, few separation steps and clean production. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a high performance liquid chromatogram of the decolorizing solution provided in an embodiment of the present invention.

[0022] Figure 2 This is a high performance liquid chromatogram of the crystalline filter cake of mono- and distearic acid glycerol with an addition amount of 2000ppm provided in an embodiment of the present invention.

[0023] Figure 3 The HPLC chromatogram of the recrystallized filter cake provided in the embodiment of the present invention. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0025] The present invention utilizes a method for extracting and purifying L-leucine using mono- and distearic acid glycerides, thereby promoting leucine crystallization and coating the exterior of leucine crystals through hydrogen bonding to enhance the degree of concentrated crystallization, thereby increasing the yield of L-leucine.

[0026] The raw material leucine fermentation broth in the following examples was prepared by culturing Brevibacterium flavum seed solution in a composite nutrient solution for 56 h; specifically, see CN201811647345.0 A method for increasing L-leucine fermentation yield for details.

[0027] Example 1: Inactivation, sterilization and decolorization of leucine fermentation broth

[0028] 475 kg of leucine fermentation broth was heated to 60°C and kept warm for 30 minutes under stirring for inactivation treatment. The material temperature was controlled below 40°C, the pressure was less than 0.65 MPa, and the flow rate was 200 L / h. The fermentation broth was filtered through a ceramic membrane to remove the bacterial residue and some impurities to obtain 450 kg of ceramic filtrate. Then, 369.9 g of activated carbon was added to the collected ceramic filtrate and stirred for decolorization for 1 hour. Ammonia water was gradually added dropwise to the decolorized liquid to adjust the pH of the system to 6.9, and finally 425 kg of decolorized liquid was obtained.

[0029] Table 1. Effects of filtration and decolorization steps on the purity and yield of L-leucine

[0030] (Note: Leucine yield in the filtration step = net mass of leucine in the filtration solution / net mass of leucine in the fermentation broth; leucine yield in the decolorization step = net mass of leucine in the decolorization solution / net mass of leucine in the filtration solution)

[0031] Table 1 shows that the filtration process removes large molecular bacterial enzymes, resulting in a higher leucine purity in the filtrate compared to the initial fermentation broth. However, the decolorization step has little effect on the leucine purity and only improves the color of the leucine fermentation broth.

[0032] Example 2: Purification Effects of Different Ratios of Mono- and Distearic Glyceryl Crystallization Aids

[0033] 5 kg of the collected decolorizing solution was divided into five equal parts, and mono- and distearic acid glyceryl having a solid content of 2000 ppm was added to each of the five parts, wherein the content of mono- and distearic acid glyceryl was 30 wt%, 40 wt%, 50 wt%, 60 wt%, and 70 wt% of the total mass of mono- and distearic acid glyceryl, respectively. The parts were then concentrated at 60° C. to twice the solid content of the decolorizing solution. The concentrated solution from each precipitated crystal was stirred for 30 min and then filtered to collect the crystallization filter cakes and filtrates.

[0034] Then, 40 times the mass of the crystallization filter cake was added for re-dissolution. After stirring at room temperature for 30 minutes, the leucine crystallization filter cake was completely dissolved. The water-insoluble mono- and distearic acid glycerides were filtered out, and the re-solution was concentrated to 3.5 times the solid content of the re-solution. The concentrated solution with precipitated crystals was stirred for 30 minutes and then filtered to collect the recrystallization filter cake and filtrate.

[0035] (Note: Leucine yield in the crystallization step = net mass of leucine in the crystallization filter cake / net mass of leucine in the decolorization solution; leucine yield in the recrystallization step after redissolution = net mass of leucine in the recrystallization filter cake / net mass of leucine in the crystallization filter cake)

[0036] Table 2 Effect of the ratio of mono- and distearic acid glycerol on the purity and yield of L-leucine

[0037] It can be seen from Table 2 that when monostearate (monoglyceride) accounts for 50% of the total mass of mono- and distearate glycerol, the purity and yield of L-leucine are optimal, but the overall difference is not large.

[0038] Example 3: Purification effect of different addition amounts of mono- and distearic glycerol crystallization aids

[0039] 5 kg of the collected decolorizing solution was divided into five equal parts, and mono- and distearic acid glyceryl (monoglyceride content was 50 wt%) with solid contents of 100 ppm, 1000 ppm, 3000 ppm, 4000 ppm, and 5000 ppm were added thereto, respectively. The mixture was then concentrated at 70° C. to twice the solid content in the decolorizing solution. The concentrate was stirred for 30 minutes and then filtered to collect the crystallized filter cakes and filtrate.

[0040] Then, 40 times the mass of the crystallization filter cake was added for re-dissolution, and the leucine crystallization filter cake was completely dissolved after stirring at 70°C for 30 minutes. After cooling to room temperature, the water-insoluble mono- and distearic acid glycerides were removed by filtration, and the reconstituted solution was concentrated to 3.5 times the solid content of the reconstituted solution. The concentrated solution with precipitated crystals was stirred for 30 minutes and then filtered to collect the recrystallization filter cake and filtrate.

[0041] Comparative Example 1: Blank Control Experiment

[0042] 1 kg of the decolorized liquid collected above was concentrated at 70° C. to 3 times the solid content in the decolorized liquid. The concentrated liquid was stirred for 30 minutes and then filtered to collect the crystallization cake and filtrate.

[0043] Then, 40 times the mass of the crystallization filter cake was added for re-dissolution, and the leucine crystallization filter cake was completely dissolved after stirring at 70°C for 30 minutes. The re-solution was concentrated to 3.5 times the solid content of the re-solution. The concentrated solution with precipitated crystals was stirred for 30 minutes and then filtered to collect the recrystallization filter cake and filtrate.

[0044] Comparative Example 2: Crystallization Aid Glycerol

[0045] 1 kg of the decolorized liquid collected above was added with glycerol having a solid content of 2000 ppm, and then concentrated at 70° C. to twice the solid content in the decolorized liquid. The concentrated liquid was stirred for 30 minutes and then filtered to collect the crystallized filter cake and filtrate.

[0046] Then, 40 times the mass of the crystallization filter cake was added to re-dissolve it, and the re-solution was concentrated to 3.5 times the solid content of the re-solution. The concentrated solution with precipitated crystals was stirred for 30 minutes and then filtered to collect the recrystallization filter cake and filtrate.

[0047] Comparative Example 3: Crystallization aid sucrose fatty acid diester

[0048] Take 1 kg of the decolorized liquid collected above, add sucrose fatty acid diester with a solid content of 2000 ppm, and then concentrate at 70°C to twice the solid content in the decolorized liquid. Stir the concentrate for 30 minutes and then filter to collect the crystallized filter cake and filtrate.

[0049] Then, 40 times the mass of the crystallization filter cake was added for re-dissolution, and the leucine crystallization filter cake was completely dissolved after stirring at 70°C for 30 minutes. After cooling to room temperature, the water-insoluble sucrose fatty acid diester was removed by filtration, and the re-solution was concentrated to 3.5 times the solid content of the re-solution. The concentrated solution with precipitated crystals was stirred for 30 minutes and then filtered to collect the recrystallization filter cake and filtrate.

[0050] Comparative Example 4: Crystallization aid sorbitan fatty acid ester S-60

[0051] Take 1 kg of the decolorized liquid collected above, add sorbitan fatty acid ester S-60 with a solid content of 2000 ppm, and then concentrate at 70°C to twice the solid content in the decolorized liquid. Stir the concentrate for 30 minutes and then filter to collect the crystallized filter cake and filtrate.

[0052] Then, 40 times the mass of the crystallized filter cake was added for re-dissolution, and the leucine crystallized filter cake was completely dissolved after stirring at 70°C for 30 minutes. After cooling to room temperature, the water-insoluble sorbitan fatty acid ester S-60 was removed by filtration, and the re-solution was concentrated to 3.5 times the solid content of the re-solution. The concentrated solution with precipitated crystals was stirred for 30 minutes and then filtered, and the recrystallization filter cake and filtrate were collected.

[0053] The purity and yield of L-leucine in Example 3 and the comparative examples are shown in the following table:

[0054] Table 3. Comparison of Purity and Yield of L-Leucine in Each Example and Comparative Example

[0055] As can be seen from the table, compared with the blank control group without crystallization aid, the purity of L-leucine decreased and the yield increased when glycerol, sucrose fatty acid ester, and sorbitan fatty acid ester were added as crystallization aids; when mono- and distearic acid glycerol were used as crystallization aids, the purity and yield of L-leucine were both improved, and when the mono- and distearic acid glycerol content was 50%, the optimal addition amount of mono- and distearic acid glycerol was 2000 ppm.

[0056] The reason for the above phenomenon is that after the crystallization aid is added, it forms hydrogen bonds with the leucine molecules outside the precipitated leucine crystals, thereby enhancing the fluidity of the precipitated leucine crystals, deepening the degree of their concentration, and thus improving the leucine extraction effect; however, if a water-soluble glycerol crystallization aid is used, it will remain in the leucine separation product. Therefore, although it increases the leucine purification yield, it will affect the quality of the leucine product. When other water-insoluble crystallization aids are used, some of them are still bonded to leucine after redissolution and cannot be completely dissociated and removed by filtration. Therefore, they cannot produce the same effect as mono- and distearic glycerol.

[0057] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the description and implementation methods. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to specific details.

Claims

1. A method for purifying L-leucine using mono- and distearic acid glyceryl esters, characterized in that: The L-leucine fermentation broth is filtered and decolorized, and then mono- and di-stearin is added to assist crystallization, thereby obtaining L-leucine; Specifically: S1. The L-leucine fermentation broth was filtered using a ceramic membrane, and then the ceramic filter liquid system was decolorized by adding activated carbon having a solid content of 0.3 to 3%, and the decolorized liquid was collected and set aside; S2. Adjust the pH of the decolorized solution in S1 to 7±0.2, then add a crystallization aid and concentrate at 60-70°C to obtain a concentrate containing precipitated crystals. Filter and collect the crystallization cake and filtrate. S3. The crystallized filter cake in S2 was redissolved in water, the filtrate was collected by filtration and concentrated for recrystallization to obtain L-leucine; In step S2, the crystallization aid is glyceryl mono- and distearate, and the amount added is 100-5000 ppm of the solid content of the decolorizing solution.

2. The method for purifying L-leucine using mono- and distearic acid glyceryl as claimed in claim 1, wherein: The L-leucine fermentation broth in step S1 is prepared by culturing Brevibacterium flavum seed solution in a composite nutrient solution.

3. The method for purifying L-leucine using mono- and distearic acid glyceryl as claimed in claim 1, wherein: In the crystallization aid glyceryl mono- and distearate in step S2, the mass percentage content of glyceryl mono-stearate is 30-70 wt %.

4. The method for purifying L-leucine using mono- and distearic acid glyceryl as claimed in claim 1, wherein: The concentration end point in step S2 is: the total mass of the concentrated solution from which the crystals are precipitated is twice the solid content of the decolorized solution.

5. The method for purifying L-leucine using mono- and distearic acid glyceryl as claimed in claim 1, wherein: The re-dissolution in step S3 is to add 40 to 50 times the mass of water to the crystallized filter cake to fully dissolve it.

6. The method for purifying L-leucine using mono- and distearic acid glyceryl as claimed in claim 1, wherein: In step S3, the re-dissolution temperature is 20-70° C., and after stirring for 30 minutes, the mixture is cooled to room temperature, and filtered or centrifuged after the mono- and distearic glyceryl esters are completely precipitated.

7. The method for purifying L-leucine using mono- and distearic acid glyceryl as an auxiliary agent according to claim 1, wherein: The end point of the recrystallization concentration in step S3 is: the total mass of the reconstituted concentrated solution is 3.5 times the solid content of the reconstituted solution.

Images