Fermentation method for producing L-alanine and application thereof

Abstract

The invention relates to a fermentation method for producing L-alanine and application of the L-alanine, and the fermentation method comprises the following steps: inoculating a strain seed solution into a culture medium for fermentation to obtain the L-alanine, the concentration of the iron element in the culture medium is 5-30 [mu] mol / L, and the concentration of the magnesium element in the culture medium is 1-3 mmol / L. According to the fermentation method for producing L-alanine provided by the invention, the content of iron element and magnesium element in the fermentation culture medium is controlled, and the iron element and the magnesium element have a synergistic effect, so that the production efficiency of fermentation production of L-alanine is improved; furthermore, the dissolved oxygen level in the fermentation liquid is adjusted, so that the yield of the L-alanine is effectively improved, the fermentation period is shortened, the production cost is reduced, and the method has a good large-scale application prospect.

Description

Technical Field

[0001] This invention relates to the field of biochemical technology, and in particular to a fermentation method for producing L-alanine and its application. Background Technology

[0002] L-Alanine, also known as L-α-alanine, or L-aminopropionic acid, is one of the smallest chiral compounds. Its molecular formula is CH3CH(NH2)COOH. It appears as a white crystalline powder, is non-toxic and odorless, and is readily soluble in water (16.72 g / 100 ml) and ethanol. It has stable physicochemical properties and is widely used in the food, pharmaceutical, and daily chemical industries. L-alanine has a distinctive sweetness, 1.6 times sweeter than glycine, and can be used as a food additive to improve food flavor. Furthermore, L-alanine promotes sugar and amino acid metabolism, making it a valuable nutritional fortifier to enhance immunity and glycemic capacity. Additionally, L-alanine can be used as a raw material to synthesize vitamin B6, aminopropanol, the antibacterial drug ofloxacin, the hypertension drug enalapril, and the novel hepatitis C treatment drug sofosbuvir. In the daily chemical industry, L-alanine is primarily used to synthesize the green, phosphate-free detergent methylglycine diacetic acid (MGDA). MGDA is a novel, environmentally friendly chelating agent that has gradually become the highest-quality green chelating agent since BASF established its first production facility in 2010. With the continuous exploration of the application potential of L-alanine, global demand is rapidly increasing, posing significant challenges to production technology and costs.

[0003] The traditional production method of L-alanine mainly uses L-aspartic acid as a raw material, which undergoes a decarboxylation reaction catalyzed by aspartic acid decarboxylase. Currently, most domestic manufacturers use this method to produce L-alanine. However, the raw material aspartic acid in this method comes from maleic anhydride, which in turn comes from benzene. Therefore, L-alanine production is limited by petroleum. With the increasing scarcity and rising price of petroleum resources, maleic anhydride resources are becoming scarce and prices are rising, leading to significant fluctuations in the supply and price of aspartic acid, thus greatly impacting L-alanine production. Furthermore, with increasing emphasis on environmental protection and reducing dependence on petroleum fossil resources, there is an urgent need to develop a green, environmentally friendly, energy-efficient, and high-performance L-alanine production technology.

[0004] In industrial production, microbial fermentation for L-alanine production has advantages such as low substrate cost and mild conditions, and has gradually replaced the original enzyme catalysis method using L-aspartic acid as raw material, becoming the mainstream method for large-scale L-alanine production. Efficient L-alanine fermentation technology is a key process to improve production intensity and reduce production costs, and is also an essential path to industrialization. Currently, methods to improve L-alanine production efficiency include: (1) adding high concentrations of organic nitrogen sources, such as yeast extract, corn steep liquor, and soybean peptone, to the culture medium to improve the production performance of the strain, thereby improving L-alanine production efficiency; (2) using two-stage fermentation to decouple strain growth from L-alanine synthesis, with the aerobic stage used for strain growth and the anaerobic stage used for L-alanine synthesis, to improve L-alanine production efficiency. However, adding high concentrations of organic nitrogen sources will increase raw material costs, and two-stage fermentation will cause the strain to decline too quickly during the anaerobic period.

[0005] In conclusion, developing a fermentation method for producing L-alanine with high yield, high conversion rate, and short cycle based on metabolic engineering principles and fermentation engineering technology is of great significance for the industrial production of L-alanine. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a fermentation method for producing L-alanine and its application. By controlling the concentrations of iron and magnesium in the culture medium, the yield of L-alanine can be effectively increased, the fermentation cycle shortened, and the production cost reduced, showing promising prospects for large-scale application.

[0007] To achieve this objective, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a fermentation method for producing L-alanine, the fermentation method comprising the following steps:

[0009] The bacterial seed culture was inoculated into a culture medium for fermentation to obtain the L-alanine;

[0010] The concentration of iron in the culture medium is 5-30 μmol / L and the concentration of magnesium is 1-3 mmol / L.

[0011] The fermentation method for producing L-alanine provided by this invention uses a culture medium with specific iron and magnesium concentrations. The two elements work synergistically to enhance the L-alanine biosynthetic pathway, achieving high yield and short cycle production of L-alanine with high carbon source conversion rate.

[0012] The concentration of iron in the culture medium is 5-30 μmol / L, for example, it can be 8 μmol / L, 10 μmol / L, 12 μmol / L, 15 μmol / L, 18 μmol / L, 20 μmol / L, 22 μmol / L, 25 μmol / L or 28 μmol / L, as well as specific values ​​between the above values. Due to space limitations and for the sake of brevity, this invention will not exhaustively list the specific values ​​included in the range.

[0013] The concentration of magnesium in the culture medium is 1-3 mmol / L, for example, it can be 1.2 mmol / L, 1.5 mmol / L, 1.8 mmol / L, 2 mmol / L, 2.2 mmol / L, 2.5 mmol / L or 2.8 mmol / L, as well as specific values ​​between the above values. Due to space limitations and for the sake of brevity, the present invention will not exhaustively list the specific values ​​included in the range.

[0014] Preferably, the strain includes Escherichia coli.

[0015] Preferably, the Escherichia coli is an engineered Escherichia coli.

[0016] Preferably, the inoculation amount of the strain seed into the culture medium is 1-10%, for example, it can be 2%, 3%, 4%, 5%, 6%, 7%, 8% or 9%, and specific values ​​between the above points. Due to space limitations and for the sake of brevity, the present invention will not exhaustively list the specific values ​​included in the range.

[0017] Preferably, the culture medium includes a carbon source, a nitrogen source, a phosphorus source, iron, and magnesium.

[0018] Preferably, the iron element includes ferric ions and / or ferrous ions.

[0019] Preferably, the magnesium element includes magnesium ions.

[0020] Preferably, the carbon source includes glucose.

[0021] Preferably, the concentration of glucose in the culture medium is 10-100 g / L, for example, it can be 15 g / L, 20 g / L, 30 g / L, 40 g / L, 50 g / L, 60 g / L, 70 g / L, 80 g / L or 90 g / L, as well as specific values ​​between the above values. Due to space limitations and for the sake of brevity, the present invention will not exhaustively list the specific values ​​included in the range. More preferably, it is 30-60 g / L.

[0022] Preferably, the nitrogen source includes ammonia and / or ammonium salts.

[0023] Preferably, the ammonium salt comprises ammonium sulfate.

[0024] Preferably, the concentration of ammonium sulfate in the culture medium is 1-5 g / L, for example, it can be 1.5 g / L, 2 g / L, 2.5 g / L, 3 g / L, 3.5 g / L, 4 g / L or 4.5 g / L, as well as specific values ​​between the above values. Due to space limitations and for the sake of brevity, the present invention will not exhaustively list the specific values ​​included in the range. More preferably, it is 2-3 g / L.

[0025] Preferably, the phosphorus source includes phosphate.

[0026] Preferably, the phosphate comprises disodium hydrogen phosphate and / or potassium dihydrogen phosphate.

[0027] Preferably, the concentration of disodium hydrogen phosphate in the culture medium is 1-10 g / L, for example, it can be 1.5 g / L, 2 g / L, 2.5 g / L, 3 g / L, 3.5 g / L, 4 g / L, 4.5 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L or 9 g / L, as well as specific values ​​between the above values. Due to space limitations and for the sake of brevity, the present invention will not exhaustively list the specific values ​​included in the range, but it is more preferably 1-5 g / L.

[0028] Preferably, the concentration of potassium dihydrogen phosphate in the culture medium is 1-5 g / L, for example, it can be 1.5 g / L, 2 g / L, 2.5 g / L, 3 g / L, 3.5 g / L, 4 g / L or 4.5 g / L, as well as specific values ​​between the above values. Due to space limitations and for the sake of brevity, the present invention will not exhaustively list the specific values ​​included in the range, but it is more preferably 1-3 g / L.

[0029] Preferably, the fermentation temperature of the fermentation method is 36-38℃, for example, it can be 36.2℃, 36.5℃, 36.8℃, 37℃, 37.2℃, 37.5℃ or 37.8℃, as well as specific values ​​between the above points. Due to space limitations and for the sake of brevity, the present invention will not exhaustively list the specific values ​​included in the range.

[0030] Preferably, the pH value of the fermentation method is 6.0-7.5, for example, it can be 6.2, 6.5, 6.8, 7 or 7.2, as well as specific values ​​between the above values. Due to space limitations and for the sake of brevity, the present invention will not exhaustively list the specific values ​​included in the range.

[0031] Preferably, the dissolved oxygen content of the fermentation broth during the fermentation process is 0-1.5 mg / L, for example, it can be 0.1 mg / L, 0.2 mg / L, 0.3 mg / L, 0.4 mg / L, 0.5 mg / L, 0.6 mg / L, 0.8 mg / L, 1 mg / L, 1.2 mg / L or 1.4 mg / L, as well as specific values ​​between the above values. Due to space limitations and for the sake of brevity, the present invention will not exhaustively list the specific values ​​included in the range.

[0032] Preferably, the fermentation includes a first fermentation stage with a fermentation time ≤ 5h (e.g., 0.5h, 1h, 2h, 3h or 4h, etc.), a third fermentation stage with a fermentation time ≥ 30h (e.g., 32h, 34h, 36h, 38h, 40h, 42h, 45h or 48h, etc.), and a second fermentation stage between the first and third fermentation stages.

[0033] Preferably, the dissolved oxygen content in the fermentation broth of the first fermentation stage is greater than or equal to the dissolved oxygen content in the fermentation broth of the third fermentation stage, which in turn is greater than or equal to the dissolved oxygen content in the fermentation broth of the second fermentation stage.

[0034] Preferably, the dissolved oxygen content in the fermentation broth of the first fermentation stage is greater than the dissolved oxygen content in the fermentation broth of the third fermentation stage, which in turn is greater than the dissolved oxygen content in the fermentation broth of the second fermentation stage.

[0035] Preferably, the dissolved oxygen content in the fermentation broth during the first fermentation stage is 0-1.5 mg / L, for example, it can be 0.1 mg / L, 0.2 mg / L, 0.3 mg / L, 0.4 mg / L, 0.5 mg / L, 0.6 mg / L, 0.8 mg / L, 1 mg / L, 1.2 mg / L or 1.4 mg / L, as well as specific values ​​between the above values. Due to space limitations and for the sake of brevity, the present invention will not exhaustively list the specific values ​​included in the range.

[0036] Preferably, the dissolved oxygen content in the fermentation broth during the second fermentation stage is 0-0.5 mg / L, for example, it can be 0.05 mg / L, 0.1 mg / L, 0.15 mg / L, 0.2 mg / L, 0.25 mg / L, 0.3 mg / L, 0.35 mg / L, 0.4 mg / L or 0.45 mg / L, as well as specific values ​​between the above values. Due to space limitations and for the sake of brevity, the present invention will not exhaustively list the specific values ​​included in the range.

[0037] Preferably, the dissolved oxygen content in the fermentation broth during the third fermentation stage is controlled to be 0-0.75 mg / L, for example, it can be 0.05 mg / L, 0.1 mg / L, 0.15 mg / L, 0.2 mg / L, 0.25 mg / L, 0.3 mg / L, 0.4 mg / L, 0.5 mg / L, 0.55 mg / L, 0.6 mg / L or 0.7 mg / L, as well as specific values ​​between the above values. Due to space limitations and for the sake of brevity, this invention will not exhaustively list the specific values ​​included in the range.

[0038] As a preferred technical solution of the present invention, the dissolved oxygen content in the fermentation broth is controlled at different stages of fermentation. In the early stage, higher dissolved oxygen conditions are controlled to promote the rapid growth of the strain; in the middle stage, lower dissolved oxygen conditions are controlled to promote the efficient synthesis of L-alanine; and in the later stage, moderate dissolved oxygen conditions are controlled to balance the growth of the strain and the synthesis of L-alanine. This ensures that the strain has strong biological activity while allowing most of the metabolic flux to be used for the synthesis of L-alanine. By controlling the different dissolved oxygen levels in the fermentation broth, it is beneficial to increase the yield of L-alanine and shorten the production cycle.

[0039] Preferably, the fermentation method includes the following steps:

[0040] (1) Inoculate the culture medium with the strain seed solution at an inoculation rate of 1-10%;

[0041] The culture medium contains 5-30 μmol / L iron, 1-3 mmol / L magnesium, 10-100 g / L glucose, 1-5 g / L ammonium sulfate, 1-10 g / L disodium hydrogen phosphate, and 1-5 g / L potassium dihydrogen phosphate.

[0042] (2) Fermentation was carried out at 36-38℃ with pH controlled at 6.0-7.5. In the first fermentation stage, the dissolved oxygen content in the fermentation broth was controlled at 0-1.5 mg / L. In the second fermentation stage, the dissolved oxygen content in the fermentation broth was controlled at 0-0.5 mg / L. In the third fermentation stage, the dissolved oxygen content in the fermentation broth was controlled at 0-0.75 mg / L to obtain the L-alanine.

[0043] In this invention, there are no special restrictions on the purification method for L-alanine; any conventional method that can be used for L-alanine purification is applicable to this invention.

[0044] Secondly, the present invention provides an application of the fermentation method according to the first aspect in the industrial production of L-alanine.

[0045] Compared with the prior art, the present invention has at least the following beneficial effects:

[0046] The fermentation method for producing L-alanine provided by this invention improves the production efficiency of L-alanine fermentation by controlling the iron and magnesium content in the fermentation medium, which synergistically enhances the effect of the two elements. Furthermore, by adjusting the dissolved oxygen level in the fermentation broth, the L-alanine yield is ≥29.7g / L. This method is characterized by high yield, short processing time, and low cost, and has good prospects for large-scale application. Detailed Implementation

[0047] The technical solution of the present invention will be further illustrated below through specific embodiments. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention is determined by the claims.

[0048] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0049] 1. Strains and seed culture preparation methods

[0050] The strains used in the following examples are all engineered Escherichia coli B21-p28 / 33 / 4 / Δ, which were obtained from CN117143792A.

[0051] In this embodiment of the invention, the engineered Escherichia coli seed culture was prepared according to the following method:

[0052] Engineered Escherichia coli B21-p28 / 33 / 4 / Δ was inoculated from cryopreservation tubes onto LB agar plates, inverted in a 37°C incubator, and cultured for 16 h to complete strain activation. A small number of activated single colonies were picked and transferred to liquid LB agar, and cultured at 37°C and 200 rpm for 12 h to complete seed culture preparation.

[0053] 2. L-Alanine Detection Method

[0054] L-alanine was detected using the method reported in the literature “Production of L-alanine by metabolically engineered Escherichia coli, Xueli Zhang et al., Appl Microbiol Biotechnol, Vol. 77, pp. 355-366”.

[0055] Example 1

[0056] This embodiment provides a fermentation method for producing L-alanine, the fermentation method comprising the following steps:

[0057] Using a 5L fermenter, the engineered Escherichia coli seed culture was inoculated into the culture medium that had been sterilized by high-temperature steam (121℃, 20min) at an inoculation rate of 5%. The initial volume of the fermentation broth after inoculation was 2.5L.

[0058] The culture medium contained iron ions at a concentration of 5 μmol / L, magnesium ions at a concentration of 1 mmol / L, glucose at a concentration of 50 g / L, ammonium sulfate at a concentration of 2 g / L, disodium hydrogen phosphate at a concentration of 1.5 g / L, and potassium dihydrogen phosphate at a concentration of 1.5 g / L.

[0059] During fermentation, the pH was controlled at 6.8±0.2 using 28wt% ammonia solution, and the temperature was controlled at 37±0.5℃. Dissolved oxygen in the fermentation broth was controlled at 1.5 mg / L for 0-5 hours, 0.5 mg / L for 5-30 hours, and 0.75 mg / L after 30 hours. 1 mL of fermentation broth was collected every 6 hours to measure cell density and L-alanine concentration.

[0060] The experimental results are shown in Table 1. After 48 hours of fermentation, the cell density OD... 600 The value was 15.2, and the L-alanine yield was 37.5 g / L.

[0061] Example 2

[0062] This embodiment provides a fermentation method for producing L-alanine, the fermentation method comprising the following steps:

[0063] Using a 5L fermenter, the engineered Escherichia coli seed culture was inoculated into the culture medium that had been sterilized by high-temperature steam (121℃, 20min) at an inoculation rate of 5%. The initial volume of the fermentation broth after inoculation was 2.5L.

[0064] The culture medium contained iron ions at a concentration of 30 μmol / L, magnesium ions at a concentration of 3 mmol / L, glucose at a concentration of 50 g / L, ammonium sulfate at a concentration of 2 g / L, disodium hydrogen phosphate at a concentration of 3.5 g / L, and potassium dihydrogen phosphate at a concentration of 1.5 g / L.

[0065] During fermentation, the pH was controlled at 6.8±0.2 using 28wt% ammonia solution, and the temperature was controlled at 37±0.5℃. Dissolved oxygen in the fermentation broth was controlled at 0.75 mg / L for 0-5 hours, 0.25 mg / L for 5-30 hours, and 0.5 mg / L after 30 hours. 1 mL of fermentation broth was collected every 6 hours to measure cell density and L-alanine concentration.

[0066] The experimental results are shown in Table 1. After 48 hours of fermentation, the cell density OD600 was 17.1 and the L-alanine yield was 40.9 g / L.

[0067] Example 3

[0068] This embodiment provides a fermentation method for producing L-alanine, the fermentation method comprising the following steps:

[0069] Using a 15L fermenter, the engineered Escherichia coli seed culture was inoculated into the culture medium that had been sterilized by high-temperature steam (121℃, 20min) at an inoculation rate of 5% (0.375L). The initial volume of the fermentation broth after inoculation was 7.5L.

[0070] The culture medium contained iron ions at a concentration of 15 μmol / L, magnesium ions at a concentration of 2 mmol / L, glucose at a concentration of 50 g / L, ammonium sulfate at a concentration of 2 g / L, disodium hydrogen phosphate at a concentration of 4.5 g / L, and potassium dihydrogen phosphate at a concentration of 1.5 g / L.

[0071] During fermentation, the pH was controlled at 6.8±0.2 using 28wt% ammonia water, and the temperature was controlled at 37±0.5℃. Dissolved oxygen in the fermentation broth was controlled at 0.75 mg / L for 0-5 hours, 0 mg / L for 5-30 hours, and 0.25 mg / L after 30 hours. Cell density and L-alanine concentration were measured every 6 hours (1 mL of fermentation broth).

[0072] The experimental results are shown in Table 1. After 48 hours of fermentation, the cell density OD600 was 16.2 and the L-alanine yield was 45.1 g / L.

[0073] Example 4

[0074] This embodiment provides a fermentation method for producing L-alanine, the fermentation method comprising the following steps:

[0075] Using a 100L fermenter, the engineered Escherichia coli seed culture was inoculated into the culture medium that had been sterilized by high-temperature steam (121℃, 20min) at an inoculation rate of 5%. The initial volume of the fermentation broth after inoculation was 50L.

[0076] The culture medium contained iron ions at a concentration of 15 μmol / L, magnesium ions at a concentration of 2 mmol / L, glucose at a concentration of 50 g / L, ammonium sulfate at a concentration of 2 g / L, disodium hydrogen phosphate at a concentration of 1.5 g / L, and potassium dihydrogen phosphate at a concentration of 1.5 g / L.

[0077] During fermentation, the pH was controlled at 6.8±0.2 using 28wt% ammonia water, and the temperature was controlled at 37±0.5℃. Dissolved oxygen in the fermentation broth was controlled at 0.75 mg / L for 0-5 hours, 0 mg / L for 5-30 hours, and 0.25 mg / L after 30 hours. Cell density and L-alanine concentration were measured every 6 hours (1 mL of fermentation broth).

[0078] The experimental results are shown in Table 1. After 48 hours of fermentation, the cell density OD600 was 16.5 and the L-alanine yield was 42.8 g / L.

[0079] Example 5

[0080] This embodiment provides a fermentation method for producing L-alanine, the fermentation method comprising the following steps:

[0081] Using a 5L fermenter, the engineered Escherichia coli seed culture was inoculated into the culture medium that had been sterilized by high-temperature steam (121℃, 20min) at an inoculation rate of 5%. The initial volume of the fermentation broth after inoculation was 2.5L.

[0082] The culture medium contained iron ions at a concentration of 30 μmol / L, magnesium ions at a concentration of 3 mmol / L, glucose at a concentration of 50 g / L, ammonium sulfate at a concentration of 2 g / L, disodium hydrogen phosphate at a concentration of 1.5 g / L, and potassium dihydrogen phosphate at a concentration of 1.5 g / L.

[0083] During fermentation, the pH was controlled at 6.8±0.2 using 28wt% ammonia solution, and the temperature was controlled at 37±0.5℃. Dissolved oxygen in the fermentation broth was controlled at 1.5 mg / L for 0-5 hours, 0.5 mg / L for 5-30 hours, and 0.5 mg / L after 30 hours. Cell density and L-alanine concentration were measured every 6 hours (1 mL of fermentation broth).

[0084] The experimental results are shown in Table 1. After 48 hours of fermentation, the cell density OD600 was 15.7 and the L-alanine yield was 29.7 g / L.

[0085] Comparative Example 1

[0086] This comparative example provides a fermentation method for producing L-alanine, which differs from Example 1 only in that the iron ion concentration in the culture medium is 0 μmol / L.

[0087] Comparative Example 2

[0088] This comparative example provides a fermentation method for producing L-alanine, which differs from Example 1 only in that the magnesium ion concentration is 0 mmol / L.

[0089] Comparative Example 3

[0090] This comparative example provides a fermentation method for producing L-alanine, which differs from Example 1 only in that the iron ion concentration in the culture medium is 40 μmol / L.

[0091] Comparative Example 4

[0092] This comparative example provides a fermentation method for producing L-alanine, which differs from Example 1 only in that the iron ion concentration in the culture medium is 3 μmol / L.

[0093] Comparative Example 5

[0094] This comparative example provides a fermentation method for producing L-alanine, which differs from Example 1 only in that the magnesium ion concentration in the culture medium is 5 mmol / L.

[0095] Comparative Example 6

[0096] This comparative example provides a fermentation method for producing L-alanine, which differs from Example 1 only in that the magnesium ion concentration in the culture medium is 0.5 mmol / L.

[0097] The cell density and L-alanine concentration in the fermentation broth after 48 hours of fermentation were measured, and the results are shown in Table 1.

[0098] Table 1

[0099]

[0100]

[0101] As can be seen from Examples 1-5, this invention, by controlling the iron concentration in the culture medium to 5-30 μmol / L and the magnesium concentration to 1-3 mmol / L, and further by controlling the dissolved oxygen in the fermentation broth, achieves an L-alanine yield ≥29.7 g / L. This fermentation production method is high-yield, time-efficient, and low-cost, showing good prospects for large-scale application. Examples 2 and 5 demonstrate that this invention, by regulating the dissolved oxygen level in the fermentation broth, further improves the L-alanine production efficiency, achieving an L-alanine yield as high as 40.9 g / L.

[0102] As can be seen from Example 1 and Comparative Examples 1-6, by adding iron and magnesium to the culture medium and further controlling the concentration of iron in the culture medium to 5-30 μmol / L and the concentration of magnesium to 1-3 mmol / L, the present invention can increase the yield of L-alanine to more than 29.7 g / L. However, when iron or magnesium ions are lacking, or when the content of the corresponding ions is not within the range of the present invention, the yield of L-alanine will be greatly reduced.

[0103] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A fermentation method for producing L-alanine, characterized in that, The fermentation method includes the following steps: The bacterial seed culture was inoculated into a culture medium for fermentation to obtain the L-alanine; The concentration of iron in the culture medium is 5-30 μmol / L and the concentration of magnesium is 1-3 mmol / L.

2. The fermentation method according to claim 1, characterized in that, The strains include Escherichia coli; Preferably, the Escherichia coli is engineered Escherichia coli; Preferably, the inoculation amount of the strain seed liquid into the culture medium is 1-10%.

3. The fermentation method according to claim 1 or 2, characterized in that, The culture medium includes a carbon source, a nitrogen source, a phosphorus source, iron, and magnesium. Preferably, the iron element includes ferric ions and / or ferrous ions; Preferably, the magnesium element includes magnesium ions.

4. The fermentation method according to claim 3, characterized in that, The carbon source includes glucose; Preferably, the concentration of glucose in the culture medium is 10-100 g / L, more preferably 30-60 g / L.

5. The fermentation method according to claim 3, characterized in that, The nitrogen source includes ammonia water and / or ammonium salts; Preferably, the ammonium salt comprises ammonium sulfate; Preferably, the concentration of ammonium sulfate in the culture medium is 1-5 g / L, more preferably 2-3 g / L.

6. The fermentation method according to claim 3, characterized in that, The phosphorus source includes phosphate; Preferably, the phosphate comprises disodium hydrogen phosphate and / or potassium dihydrogen phosphate; Preferably, the concentration of disodium hydrogen phosphate in the culture medium is 1-10 g / L, more preferably 1-5 g / L; Preferably, the concentration of potassium dihydrogen phosphate in the culture medium is 1-5 g / L, more preferably 1-3 g / L.

7. The fermentation method according to any one of claims 1-6, characterized in that, The fermentation temperature is 36-38℃; Preferably, the pH value of the fermentation is 6.0-7.

5.

8. The fermentation method according to any one of claims 1-7, characterized in that, The dissolved oxygen content of the fermentation broth during the fermentation process is 0-1.5 mg / L; Preferably, the fermentation includes a first fermentation stage with a fermentation time of ≤5h, a third fermentation stage with a fermentation time of ≥30h, and a second fermentation stage between the first and third fermentation stages; Preferably, the dissolved oxygen content in the fermentation broth of the first fermentation stage is greater than or equal to the dissolved oxygen content in the fermentation broth of the third fermentation stage, which in turn is greater than or equal to the dissolved oxygen content in the fermentation broth of the second fermentation stage. Preferably, the dissolved oxygen content in the fermentation broth during the first fermentation stage is 0-1.5 mg / L; Preferably, the dissolved oxygen content in the fermentation broth during the second fermentation stage is 0-0.5 mg / L; Preferably, the dissolved oxygen content in the fermentation broth during the third fermentation stage is 0-0.75 mg / L.

9. The fermentation method according to any one of claims 1-8, characterized in that, The fermentation method includes the following steps: (1) Inoculate the culture medium with the strain seed solution at an inoculation rate of 1-10%; The culture medium contains 5-30 μmol / L iron, 1-3 mmol / L magnesium, 10-100 g / L glucose, 1-5 g / L ammonium sulfate, 1-10 g / L disodium hydrogen phosphate, and 1-5 g / L potassium dihydrogen phosphate. (2) Fermentation was carried out at 36-38℃ with pH controlled at 6.0-7.

5. In the first fermentation stage, the dissolved oxygen content in the fermentation broth was controlled at 0-1.5 mg / L. In the second fermentation stage, the dissolved oxygen content in the fermentation broth was controlled at 0-0.5 mg / L. In the third fermentation stage, the dissolved oxygen content in the fermentation broth was controlled at 0-0.75 mg / L to obtain the L-alanine.

10. The application of a fermentation method according to any one of claims 1-9 in the industrial production of L-alanine.

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