A method for preparing 5-aminolevulinic acid by fermentation

Abstract

The invention provides a method for preparing 5-aminolevulinic acid by fermentation, and belongs to the technical field of fermentation of 5-aminolevulinic acid; the method comprises activation culture, primary seed culture, secondary seed culture and fermentation culture; the fermentation culture step comprises: when fermentation reaches 14h, adding a fermentation promoter, the flow acceleration is 0.04-0.06g / h / L, the flow addition time is 4.0-4.4h, and the fermentation cycle is controlled to be 34.8-35.1h, so as to obtain a fermentation culture liquid; the preparation method of the fermentation nutrient solution comprises: adding isoleucine and aspartic acid to deionized water, raising the temperature to 35-37°C and stirring, then adding sophorolipids and glycerol, and performing ultrasonic treatment to obtain a fermentation nutrient solution; the fermentation culture liquid prepared by the method of the invention has a high content of 5-aminolevulinic acid and a low content of by-products.

Description

Technical Field

[0001] The invention belongs to the technical field of 5-aminolevulinic acid fermentation, and particularly relates to a method for preparing 5-aminolevulinic acid by fermentation. Background Art

[0002] 5-Aminolevulinic acid (5-ALA) is a prefix compound of tetrahydropyrrole (tetrahydropyrrole is a component of heme, cytochrome, and vitamin B12), and is an indispensable substance for the biosynthesis of chlorophyll, heme, vitamin B12, etc.; as a substance with high environmental compatibility and selectivity, 5-ALA has important application value in the medical and agricultural fields; in medicine, 5-ALA is a new type of photodynamic drug, which is not only used for the treatment of local or systemic skin cancer, but also can be used for the diagnosis of bladder cancer, digestive tract cancer, lung cancer, etc.; in the agricultural field, it can be used as a plant growth regulator to improve photosynthetic efficiency, promote photosynthesis, and participate in the regulation process of plant growth and development; it can also be used as a green herbicide, pesticide, etc.

[0003] At present, the methods for producing 5-ALA mainly include chemical synthesis and microbial fermentation. Regarding the chemical synthesis method, researchers have successively synthesized 5-ALA using hippuric acid, succinic acid, tetrahydrofurfurylamine and levulinic acid as raw materials. However, it has the disadvantages of high reagent price, difficulty in obtaining, high toxicity, low yield and harsh reaction conditions.

[0004] Regarding the microbial fermentation method, CN118109527A discloses a process for producing 5-aminolevulinic acid by fermenting Aspergillus nidulans, comprising the following steps: constructing an engineered strain of Aspergillus nidulans, preparing Aspergillus nidulans spore liquid, fermenting to prepare 5-aminolevulinic acid, exogenously adding pyridoxal phosphate and iron ions, adjusting the rotation speed and pH, and on this basis, exogenously adding levulinic acid, and using a 5L fermenter for fed-batch fermentation.

[0005] This patent increases the production of 5-aminolevulinic acid to 4.134±0.256 g / L by adding exogenous substances to the strain and optimizing the fermentation conditions. However, the fermentation time is long, which reduces the yield. Summary of the invention

[0006] In order to solve the technical problems existing in the prior art, the present invention provides a method for preparing 5-aminolevulinic acid by fermentation, which shortens the fermentation time and improves the yield.

[0007] In view of the above technical problems, the present invention adopts the following technical solutions:

[0008] 1. Activation culture

[0009] Placing Corynebacterium glutamicum in an activation culture medium for activation culture, the culture time is 23-25 ​​hours, the culture temperature is 28-30° C., and an activation culture solution is obtained;

[0010] The Corynebacterium glutamicum is purchased from the market;

[0011] The activation culture medium comprises 10.2-10.6 g / L of tryptone, 0.7-0.9 g / L of magnesium sulfate, 1.8-2.2 g / L of agar, 3.4-4.0 g / L of yeast extract, 0.8-1.2 g / L of sodium chloride, and a pH of 6.5-6.7.

[0012] 2. Primary seed culture

[0013] The activated culture solution is inoculated on the primary seed culture medium at an inoculation rate of 0.25% to perform primary seed culture to obtain a primary seed culture solution; the primary seed culture conditions are as follows: a culture temperature of 28-30° C., a rotation speed of 195-206 rpm, and a culture time of 16-18 h;

[0014] The components of the primary seed culture medium are glucose 8.5-9.0 g / L, tryptone 7.2-7.6 g / L, yeast powder 12.1-12.5 g / L, dipotassium hydrogen phosphate 1.0-1.4 g / L, potassium dihydrogen phosphate 0.8-1.2 g / L, and growth agent 1.1-1.3 g / L;

[0015] The growth agents are thiamine and brassinolide, and the mass ratio of thiamine to brassinolide is 1.2-1.4:1.6-1.8.

[0016] 3. Secondary seed culture

[0017] The primary seed culture solution is inoculated on the secondary seed culture medium at an inoculation rate of 5% to perform secondary seed culture, thereby obtaining a secondary seed culture medium; the secondary seed culture conditions are as follows: a culture temperature of 30-33° C., a rotation speed of 215-225 rpm, and a culture time of 19-22 h;

[0018] The components of the secondary seed culture medium are glucose 6.7-7.2 g / L, tryptone 6.0-6.5 g / L, magnesium sulfate 0.6-1.0 g / L, FeSO4·7H2O 0.8-1.2 g / L, ammonium sulfate 0.7-1.1 g / L, and stabilizer 0.30-0.35 g / L;

[0019] The stabilizer is a mixture of cobalamin and mannitol, and the mass ratio of the cobalamin to mannitol is 1:0.8-1.2.

[0020] 4. Fermentation culture

[0021] The secondary seed culture liquid is inoculated on the fermentation medium at an inoculation rate of 10% for fermentation culture. During the fermentation process, the stirring speed is controlled to be 263-274rpm, the fermentation temperature is 35-37°C, the pressure is 0.05-0.07MPa, and the pH value is controlled to be 6.2-6.5 with a 24-26wt% ammonia solution. The dissolved oxygen content is controlled to be 28-32% at the 0th to 24th hour of fermentation. After 24 hours of fermentation until the end of fermentation, the dissolved oxygen content is controlled to be 9-11%. When the fermentation reaches the 2.5th hour, an IPTG inducer with a final concentration of 0.08-0.12mmol / L and a glycine solution with a final concentration of 3.8-4.2g / L are added. When the fermentation reaches the 14th hour, a fermentation promoter is added at a flow acceleration of 0.04-0.06g / h / L, and the flow addition time is 4.0-4.4h. The fermentation cycle is controlled to be 34.8-35.1h to obtain a fermentation culture liquid;

[0022] The fermentation medium comprises 16.4-17.0 g / L glucose, 9.6-10.0 g / L tryptone, 0.013-0.017 g / L manganese sulfate, 3.0-3.4 g / L ammonium chloride, and 1.4-1.6 g / L fermentation nutrient solution;

[0023] The preparation method of the fermentation nutrient solution comprises the following steps: adding isoleucine and aspartic acid to deionized water, raising the temperature to 35-37° C., stirring at 242-255 rpm for 27-33 min, adding sophorolipids and glycerol after the stirring, and performing ultrasonic treatment, wherein the ultrasonic time is 14-17 min, the ultrasonic frequency is 23-28 kHz, and the ultrasonic power is 225-234 W. After the ultrasonic treatment, a fermentation nutrient solution is obtained;

[0024] The mass ratio of deionized water, isoleucine, aspartic acid, sophorolipid and glycerol is 13.7-14.1:2.5-2.7:2.6-3.0:0.8-1.2:1.2-1.6;

[0025] The fermentation promoter is composed of oleic acid and glutathione, and the mass ratio of the oleic acid to the glutathione is 1.6-1.8:1.0-1.2.

[0026] Compared with the prior art, the present invention has achieved the following beneficial effects:

[0027] 1. The present invention performs two-stage seed culture on Corynebacterium glutamicum, and adds a growth agent in the first-stage seed culture process, which can promote cell division and growth of the bacteria, stimulate growth and reproduction, shorten the delay of the strain in the seed culture medium, and is conducive to improving the activity of the strain in the middle and late stages of fermentation, and promotes the decomposition and metabolism of carbohydrates, provides energy for the growth of microorganisms, and accelerates the growth and metabolism rate of the strain while providing nutrition, thereby increasing 5-ALA Yield: In the secondary seed culture, stabilizers are added, which can optimize the metabolic pathways of microorganisms, provide carbon sources and energy, promote the growth and metabolism of bacteria, provide sufficient inoculation volume and high-density microbial population for the subsequent fermentation process, and improve the consistency and efficiency of fermentation. In the fermentation step, the specific fermentation nutrient solution in the fermentation medium, the amino acids and glycerol components therein, mainly provide energy for microorganisms to use, promote microbial metabolism, and improve production efficiency, while sophorolipids can reduce surface tension, increase cell permeability, and improve the interaction between bacteria and culture medium, thereby improving the absorption and utilization rate of nutrients by bacteria, reducing energy consumption, improving acid production performance, and increasing the yield of 5-aminolevulinic acid. It is combined with fermentation promoters to regulate the fluidity of microbial cell membranes and protect the stability of strains during the fermentation process. Under the joint action of various additives, the fermentation process and the accumulation of 5-aminolevulinic acid are promoted, the content of by-products is reduced, and the yield of 5-ALA is further increased.

[0028] 2. The fermentation broth obtained by the method of the present invention has a 5-ALA content of 21.2-21.8 g / L, a lactic acid content of 1.09-1.14 g / L, and an acetic acid content of 0.64-0.68 g / L. DETAILED DESCRIPTION

[0029] In order to more clearly understand the technical features, purposes and effects of the present invention, specific embodiments of the present invention are now described.

[0030] Example 1

[0031] 1. Activation culture

[0032] Placing Corynebacterium glutamicum in an activation culture medium for activation culture, the culture time is 24 hours, the culture temperature is 29° C., and an activation culture solution is obtained;

[0033] The Corynebacterium glutamicum is purchased from the market;

[0034] The activation culture medium comprises 10.4 g / L tryptone, 0.8 g / L magnesium sulfate, 2.0 g / L agar, 3.7 g / L yeast extract, 1.0 g / L sodium chloride, and a pH of 6.6.

[0035] 2. Primary seed culture

[0036] The activated culture solution was inoculated on the primary seed culture medium at an inoculum amount of 0.25% to perform primary seed culture, thereby obtaining a primary seed culture solution; the primary seed culture conditions were as follows: a culture temperature of 29° C., a rotation speed of 200 rpm, and a culture time of 17 h;

[0037] The components of the primary seed culture medium are glucose 8.8 g / L, tryptone 7.4 g / L, yeast powder 12.3 g / L, dipotassium hydrogen phosphate 1.2 g / L, potassium dihydrogen phosphate 1.0 g / L, and growth agent 1.2 g / L;

[0038] The growth agents are thiamine and brassinolide, and the mass ratio of thiamine to brassinolide is 1.3:1.7.

[0039] 3. Secondary seed culture

[0040] The primary seed culture solution was inoculated into the secondary seed culture medium at an inoculation rate of 5% to perform secondary seed culture, thereby obtaining a secondary seed culture medium; the secondary seed culture conditions were as follows: a culture temperature of 31° C., a rotation speed of 220 rpm, and a culture time of 20 h;

[0041] The components of the secondary seed culture medium are glucose 7.0 g / L, tryptone 6.2 g / L, magnesium sulfate 0.8 g / L, FeSO4·7H2O 1.0 g / L, ammonium sulfate 1.0 g / L, and stabilizer 0.32 g / L;

[0042] The stabilizer is a mixture of cobalamin and mannitol, and the mass ratio of the cobalamin to mannitol is 1:1.

[0043] 4. Fermentation culture

[0044] The secondary seed culture liquid was inoculated on the fermentation medium at an inoculation rate of 10% for fermentation culture. During the fermentation process, the stirring speed was controlled to be 270 rpm, the fermentation temperature was 36°C, the pressure was 0.06 MPa, and the pH value was controlled to be 6.3 with a 25wt% ammonia solution. The dissolved oxygen content was controlled to be 30% during the 0-24h of fermentation. After 24h of fermentation until the end of fermentation, the dissolved oxygen content was controlled to be 10%. When the fermentation reached the 2.5h, an IPTG inducer with a final concentration of 0.1mmol / L and a glycine solution with a final concentration of 4.0g / L were added. When the fermentation reached the 14thh, a fermentation promoter was added at a flow acceleration of 0.05g / h / L, and the flow addition time was 4.2h. The fermentation cycle was controlled to be 34.8h to obtain a fermentation culture liquid.

[0045] The fermentation medium comprises 16.7 g / L glucose, 9.8 g / L tryptone, 0.015 g / L manganese sulfate, 3.2 g / L ammonium chloride, and 1.5 g / L fermentation nutrient solution;

[0046] The preparation method of the fermentation nutrient solution is as follows: adding isoleucine and aspartic acid to deionized water, raising the temperature to 36° C., stirring at 250 rpm for 30 min, adding sophorolipids and glycerol after the stirring, and performing ultrasonic treatment, wherein the ultrasonic time is 16 min, the ultrasonic frequency is 25 kHz, and the ultrasonic power is 230 W. After the ultrasonic treatment, a fermentation nutrient solution is obtained;

[0047] The mass ratio of deionized water, isoleucine, aspartic acid, sophorolipid and glycerol is 13.9:2.6:2.8:1.0:1.4;

[0048] The fermentation promoter is composed of oleic acid and glutathione, and the mass ratio of the oleic acid to the glutathione is 1.7:1.1.

[0049] The fermentation broth obtained by the method of Example 1 had a 5-ALA content of 21.8 g / L, a lactic acid content of 1.09 g / L, and an acetic acid content of 0.64 g / L.

[0050] Example 2

[0051] 1. Activation culture

[0052] Placing Corynebacterium glutamicum in an activation culture medium for activation culture, the culture time is 23 hours, the culture temperature is 28° C., and an activation culture solution is obtained;

[0053] The Corynebacterium glutamicum is purchased from the market;

[0054] The activation culture medium comprises 10.2 g / L tryptone, 0.7 g / L magnesium sulfate, 1.8 g / L agar, 3.4 g / L yeast extract, and 0.8 g / L sodium chloride, and the pH value is 6.5.

[0055] 2. Primary seed culture

[0056] The activated culture solution was inoculated on the primary seed culture medium at an inoculum amount of 0.25% to perform primary seed culture, thereby obtaining a primary seed culture solution; the primary seed culture conditions were as follows: a culture temperature of 28° C., a rotation speed of 195 rpm, and a culture time of 16 h;

[0057] The components of the primary seed culture medium are: 8.5 g / L glucose, 7.2 g / L tryptone, 12.1 g / L yeast powder, 1.0 g / L dipotassium hydrogen phosphate, 0.8 g / L potassium dihydrogen phosphate, and 1.1 g / L growth agent;

[0058] The growth agents are thiamine and brassinolide, and the mass ratio of thiamine to brassinolide is 1.2:1.6.

[0059] 3. Secondary seed culture

[0060] The primary seed culture solution was inoculated into the secondary seed culture medium at an inoculation rate of 5% to perform secondary seed culture, thereby obtaining a secondary seed culture medium; the secondary seed culture conditions were as follows: a culture temperature of 30° C., a rotation speed of 215 rpm, and a culture time of 19 h;

[0061] The components of the secondary seed culture medium are glucose 6.7 g / L, tryptone 6.0 g / L, magnesium sulfate 0.6 g / L, FeSO4·7H2O 0.8 g / L, ammonium sulfate 0.7 g / L, and stabilizer 0.30 g / L;

[0062] The stabilizer is a mixture of cobalamin and mannitol, and the mass ratio of the cobalamin to mannitol is 1:0.8.

[0063] 4. Fermentation culture

[0064] The secondary seed culture liquid was inoculated on the fermentation medium at an inoculation rate of 10% for fermentation culture. During the fermentation process, the stirring speed was controlled to be 263 rpm, the fermentation temperature was 35°C, the pressure was 0.05 MPa, and the pH value was controlled to be 6.2 with a 24wt% ammonia solution. The dissolved oxygen content was controlled to be 28% during the 0-24h of fermentation. After 24h of fermentation until the end of fermentation, the dissolved oxygen content was controlled to be 9%. When the fermentation reached the 2.5h, an IPTG inducer with a final concentration of 0.08mmol / L and a glycine solution with a final concentration of 3.8g / L were added. When the fermentation reached the 14thh, a fermentation promoter was added at a flow acceleration of 0.04g / h / L, and the flow addition time was 4.0h. The fermentation cycle was controlled to be 35.1h to obtain a fermentation culture liquid.

[0065] The fermentation medium comprises 16.4 g / L glucose, 9.6 g / L tryptone, 0.013 g / L manganese sulfate, 3.0 g / L ammonium chloride, and 1.4 g / L fermentation nutrient solution;

[0066] The preparation method of the fermentation nutrient solution is as follows: adding isoleucine and aspartic acid to deionized water, raising the temperature to 35° C., stirring at 242 rpm for 27 minutes, adding sophorolipids and glycerol after the stirring, and performing ultrasonic treatment, wherein the ultrasonic time is 14 minutes, the ultrasonic frequency is 23 kHz, and the ultrasonic power is 225 W. After the ultrasonic treatment, a fermentation nutrient solution is obtained;

[0067] The mass ratio of deionized water, isoleucine, aspartic acid, sophorolipid and glycerol is 13.7:2.5:2.6:0.8:1.2;

[0068] The fermentation promoter is composed of oleic acid and glutathione, and the mass ratio of the oleic acid to the glutathione is 1.6:1.0.

[0069] The fermentation broth obtained by the method of Example 2 had a 5-ALA content of 21.2 g / L, a lactic acid content of 1.14 g / L, and an acetic acid content of 0.68 g / L.

[0070] Example 3

[0071] 1. Activation culture

[0072] Placing Corynebacterium glutamicum in an activation culture medium for activation culture, the culture time is 25 hours, the culture temperature is 30° C., and an activation culture solution is obtained;

[0073] The Corynebacterium glutamicum is purchased from the market;

[0074] The activation culture medium comprises 10.6 g / L tryptone, 0.9 g / L magnesium sulfate, 2.2 g / L agar, 4.0 g / L yeast extract, and 1.2 g / L sodium chloride, and the pH value is 6.7.

[0075] 2. Primary seed culture

[0076] The activated culture solution was inoculated on the primary seed culture medium at an inoculum amount of 0.25% to perform primary seed culture, thereby obtaining a primary seed culture solution; the primary seed culture conditions were as follows: a culture temperature of 30° C., a rotation speed of 206 rpm, and a culture time of 18 h;

[0077] The components of the primary seed culture medium are: 9.0 g / L glucose, 7.6 g / L tryptone, 12.5 g / L yeast powder, 1.4 g / L dipotassium hydrogen phosphate, 1.2 g / L potassium dihydrogen phosphate, and 1.3 g / L growth agent;

[0078] The growth agents are thiamine and brassinolide, and the mass ratio of thiamine to brassinolide is 1.4:1.8.

[0079] 3. Secondary seed culture

[0080] The primary seed culture solution was inoculated into the secondary seed culture medium at an inoculation rate of 5% to perform secondary seed culture, thereby obtaining a secondary seed culture medium; the secondary seed culture conditions were as follows: a culture temperature of 33° C., a rotation speed of 225 rpm, and a culture time of 22 h;

[0081] The components of the secondary seed culture medium are glucose 7.2 g / L, tryptone 6.5 g / L, magnesium sulfate 1.0 g / L, FeSO4·7H2O 1.2 g / L, ammonium sulfate 1.1 g / L, and stabilizer 0.35 g / L;

[0082] The stabilizer is a mixture of cobalamin and mannitol, and the mass ratio of the cobalamin to mannitol is 1:1.2.

[0083] 4. Fermentation culture

[0084] The secondary seed culture liquid was inoculated on the fermentation medium at an inoculation rate of 10% for fermentation culture. During the fermentation process, the stirring speed was controlled to be 274 rpm, the fermentation temperature was 37°C, the pressure was 0.07 MPa, and the pH value was controlled to be 6.5 with a 26wt% ammonia solution. The dissolved oxygen content was controlled to be 32% during the 0-24h of fermentation. After 24h of fermentation until the end of fermentation, the dissolved oxygen content was controlled to be 11%. When the fermentation reached the 2.5h, an IPTG inducer with a final concentration of 0.12mmol / L and a glycine solution with a final concentration of 4.2g / L were added. When the fermentation reached the 14thh, a fermentation promoter was added at a flow acceleration of 0.06g / h / L, and the flow addition time was 4.4h. The fermentation cycle was controlled to be 35.0h to obtain a fermentation culture liquid.

[0085] The fermentation medium comprises 17.0 g / L glucose, 10.0 g / L tryptone, 0.017 g / L manganese sulfate, 3.4 g / L ammonium chloride, and 1.6 g / L fermentation nutrient solution;

[0086] The preparation method of the fermentation nutrient solution is as follows: adding isoleucine and aspartic acid to deionized water, raising the temperature to 37° C., stirring at 255 rpm for 33 min, adding sophorolipids and glycerol after the stirring, and performing ultrasonic treatment, wherein the ultrasonic time is 17 min, the ultrasonic frequency is 28 kHz, and the ultrasonic power is 234 W. After the ultrasonic treatment, a fermentation nutrient solution is obtained;

[0087] The mass ratio of deionized water, isoleucine, aspartic acid, sophorolipid and glycerol is 14.1:2.7:3.0:1.2:1.6;

[0088] The fermentation promoter is composed of oleic acid and glutathione, and the mass ratio of the oleic acid to the glutathione is 1.8:1.2.

[0089] The fermentation broth obtained by the method of Example 3 had a 5-ALA content of 21.6 g / L, a lactic acid content of 1.12 g / L, and an acetic acid content of 0.66 g / L.

[0090] The present invention performs two-stage seed culture on Corynebacterium glutamicum, and adds a growth agent in the first-stage seed culture process, which can promote cell division and growth of the bacteria, stimulate growth and reproduction, shorten the delay of the strain in the seed culture medium, and is conducive to improving the activity of the strain in the middle and late stages of fermentation, and promotes the decomposition and metabolism of carbohydrates, provides energy for the growth of microorganisms, and accelerates the growth and metabolism rate of the strain while providing nutrition, thereby increasing 5-ALA. Yield: In the secondary seed culture, a stabilizer is added, which can optimize the metabolic pathway of microorganisms, provide carbon source and energy, promote the growth and metabolism of bacteria, provide sufficient inoculation amount and high-density microbial population for the subsequent fermentation process, and improve the consistency and efficiency of fermentation; in the fermentation step, the specific fermentation nutrient solution in the fermentation medium, the amino acids and glycerol components therein, mainly provide energy for microorganisms to use, promote microbial metabolism, and improve production efficiency, while the sophorolipids can reduce surface tension, increase cell permeability, and improve the interaction between bacteria and culture medium, thereby improving the absorption and utilization rate of nutrients by bacteria, reducing energy consumption, improving acid production performance, and increasing the yield of 5-aminolevulinic acid; it is combined with a fermentation promoter to regulate the fluidity of the microbial cell membrane, protect the stability of the strain during the fermentation process, and under the joint action of various additives, promote the fermentation process and the accumulation of 5-aminolevulinic acid, reduce the content of by-products, and further increase the yield of 5-ALA.

[0091] Comparative Example 1

[0092] Based on Example 1, the changes are as follows:

[0093] In the primary seed culture step, the growth agent component in the primary seed culture medium is omitted;

[0094] In the fermentation culture step, the operation of "adding a fermentation promoter at the 14th hour of fermentation, with a flow rate of 0.05 g / h / L and a flow time of 4.2 h" was omitted, and the addition of the fermentation promoter was omitted in the entire fermentation culture step;

[0095] The rest of the operations are the same.

[0096] The fermentation culture liquid obtained by the method of Comparative Example 1 had a 5-ALA content of 16.4 g / L, a lactic acid content of 1.38 g / L, and an acetic acid content of 0.97 g / L.

[0097] Comparative Example 1 omits the growth agent component in the primary seed culture step, resulting in a large delay in the bacterial body in the seed culture, affecting the growth and reproduction of the bacterial body. Comparative Example 1 also omits the flow-added fermentation promoter during the fermentation culture process, thereby reducing the fluidity of the cell membrane, resulting in the inability to provide nutrients and metabolites to the bacterial body in a timely manner, and also resulting in uneven distribution of metabolites, ultimately affecting the synthesis of 5-ALA and increasing the content of by-products.

[0098] Comparative Example 2

[0099] Based on Example 1, the changes are as follows:

[0100] In the secondary seed culture step, the stabilizer component in the secondary seed culture medium is omitted;

[0101] In the fermentation culture step, the fermentation nutrient solution component in the fermentation medium is omitted;

[0102] The rest of the operations are the same.

[0103] The fermentation broth obtained by the method of Comparative Example 2 had a 5-ALA content of 14.2 g / L, a lactic acid content of 1.51 g / L, and an acetic acid content of 1.16 g / L.

[0104] In Comparative Example 2, in the secondary seed culture step, the stabilizer component was omitted, resulting in poor metabolism and low activity in the cells, and the fermentation nutrient solution component in the fermentation medium was also omitted, which reduced the nutrients in the fermentation process, reduced the permeability of the cell membrane, and hindered the absorption of nutrients, leading to a decrease in cell growth rate, weak metabolic activity, and reduced production vitality of the strain, ultimately resulting in a significant decrease in 5-ALA production efficiency and a high content of by-products.

[0105] Unless otherwise specified, all percentages used in the present invention are by mass.

[0106] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing 5-aminolevulinic acid by fermentation, characterized in that: It includes activation culture, primary seed culture, secondary seed culture and fermentation culture steps; The activation culture step comprises placing the Corynebacterium glutamicum in an activation culture medium for activation culture, the culture time is 23-25 ​​hours, the culture temperature is 28-30° C., and an activation culture solution is obtained; The activation medium comprises 10.2-10.6 g / L of tryptone, 0.7-0.9 g / L of magnesium sulfate, 1.8-2.2 g / L of agar, 3.4-4.0 g / L of yeast extract, 0.8-1.2 g / L of sodium chloride, and a pH of 6.5-6.7; The first-level seed culture step is to inoculate the activated culture solution on the first-level seed culture medium at an inoculation rate of 0.25% to perform first-level seed culture to obtain the first-level seed culture solution; The components of the primary seed culture medium are glucose 8.5-9.0 g / L, tryptone 7.2-7.6 g / L, yeast powder 12.1-12.5 g / L, dipotassium hydrogen phosphate 1.0-1.4 g / L, potassium dihydrogen phosphate 0.8-1.2 g / L, and growth agent 1.1-1.3 g / L; The growth agent is thiamine and brassinolide, and the mass ratio of thiamine to brassinolide is 1.2-1.4:1.6-1.8; The secondary seed culture step comprises inoculating the primary seed culture solution on the secondary seed culture medium at an inoculation rate of 5% to perform secondary seed culture, thereby obtaining the secondary seed culture medium; The components of the secondary seed culture medium are glucose 6.7-7.2 g / L, tryptone 6.0-6.5 g / L, magnesium sulfate 0.6-1.0 g / L, FeSO4·7H2O 0.8-1.2 g / L, ammonium sulfate 0.7-1.1 g / L, and stabilizer 0.30-0.35 g / L; The stabilizer is a mixture of cobalamin and mannitol, and the mass ratio of the cobalamin to mannitol is 1:0.8-1.2; The fermentation and culturing step comprises the following steps: inoculating the secondary seed culture solution on the fermentation medium at an inoculation rate of 10% for fermentation and culturing; during the fermentation process, controlling the stirring speed to 263-274 rpm, the fermentation temperature to 35-37° C., the pressure to 0.05-0.07 MPa, and using a 24-26 wt % ammonia solution to control the pH value to 6.2-6.5; controlling the dissolved oxygen content to 28-32% at the 0th to 24th hour of fermentation; controlling the dissolved oxygen content to 9-11% after 24 hours of fermentation until the end of fermentation; adding an IPTG inducer with a final concentration of 0.08-0.12 mmol / L and a glycine solution with a final concentration of 3.8-4.2 g / L at the 2nd to 2.5th hour of fermentation; and adding a fermentation promoter at the 14th hour of fermentation, with a flow acceleration of 0.04-0.06 g / h / L, a flow addition time of 4.0-4.4 hours, and controlling the fermentation cycle to 34.8-35.1 hours to obtain a fermentation culture solution; The fermentation medium comprises 16.4-17.0 g / L glucose, 9.6-10.0 g / L tryptone, 0.013-0.017 g / L manganese sulfate, 3.0-3.4 g / L ammonium chloride, and 1.4-1.6 g / L fermentation nutrient solution; The preparation method of the fermentation nutrient solution comprises the following steps: adding isoleucine and aspartic acid to deionized water, raising the temperature to 35-37° C., stirring at 242-255 rpm for 27-33 min, adding sophorolipids and glycerol after the stirring, and performing ultrasonic treatment, wherein the ultrasonic time is 14-17 min, the ultrasonic frequency is 23-28 kHz, and the ultrasonic power is 225-234 W. After the ultrasonic treatment, a fermentation nutrient solution is obtained; In the method for preparing the fermentation nutrient solution, the mass ratio of the deionized water, isoleucine, aspartic acid, sophorolipid and glycerol is 13.7-14.1:2.5-2.7:2.6-3.0:0.8-1.2:1.2-1.6; The fermentation promoter is composed of oleic acid and glutathione, and the mass ratio of the oleic acid to the glutathione is 1.6-1.8:1.0-1.

2.

2. The method for preparing 5-aminolevulinic acid by fermentation according to claim 1, characterized in that: In the primary seed culture step, the primary seed culture conditions are as follows: a culture temperature of 28-30° C., a rotation speed of 195-206 rpm, and a culture time of 16-18 h.

3. The method for preparing 5-aminolevulinic acid by fermentation according to claim 1, characterized in that: In the secondary seed culture step, the secondary seed culture conditions are as follows: a culture temperature of 30-33° C., a rotation speed of 215-225 rpm, and a culture time of 19-22 h.