Fermentation production process of L-alanine

Abstract

The invention discloses a fermentation production process of L-alanine, and relates to the technical field of biology. The fermentation production process comprises the following steps: carrying out seed culture on L-alanine producing bacteria to obtain a seed solution; inoculating the seed solution into a fermentation culture medium, and carrying out fermentation culture to prepare L-alanine; in the fermentation culture process, the pH range is controlled to be 6.9 + / -0.1. By regulating the pH value of the fermentation environment, the situation that excessive ammonium ions force cells to consume more ATP for nitrogen assimilation (such as a glutamine synthetase pathway) is effectively avoided, so that energy waste is reduced, and energy supply for synthesis of L-alanine is increased. The process not only improves the yield of the L-alanine, but also optimizes the energy utilization efficiency in the fermentation process, and provides reliable technical support for efficient and economic industrial production of the L-alanine.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a fermentation production process of L-alanine. Background Art

[0002] L-alanine, an important non-essential amino acid, is widely used in food, pharmaceuticals, chemicals, and biomaterials. In the pharmaceutical industry, L-alanine is a key intermediate in the synthesis of vitamin B6, the Parkinson's disease medication levodopa (L-DOPA), and immunomodulators. In the food industry, demand for its use as a sweetener and preservative continues to grow. With the rapid development of downstream industries, market demand for L-alanine has steadily increased, and traditional production methods are no longer able to meet the high-efficiency, environmentally friendly, and cost-effective industrial requirements.

[0003] At present, the production methods of L-alanine mainly include chemical synthesis, enzymatic method and microbial fermentation. The chemical synthesis method usually uses acetaldehyde, hydrocyanic acid and ammonia as raw materials and prepares it through the Strecker synthesis reaction. However, the reaction conditions of this process are harsh (high temperature and high pressure), the raw materials are highly toxic and the environmental pollution is large, which is not in line with the development trend of green chemistry; the enzymatic method relies on immobilized enzymes or whole cells to catalyze the conversion of substrates (such as L-aspartic acid or fumaric acid). Although the conditions are mild, the enzyme cost is high, the substrate conversion rate is low, and the product separation and purification is difficult, which restricts large-scale application. In contrast, the microbial fermentation method uses renewable carbon sources (such as glucose) as raw materials and metabolically engineered strains to directly synthesize L-alanine. It has the advantages of a wide source of raw materials, mild reaction conditions and environmental friendliness, and has become a key research direction in the industry.

[0004] However, the existing microbial fermentation method still has a technical bottleneck of low yield. Based on this, the present invention intends to develop a new fermentation production process for L-alanine to achieve efficient fermentation production of L-alanine. Summary of the Invention

[0005] The present invention aims to provide a fermentation process for producing L-alanine to address the problems of the prior art. This fermentation process achieves precise control of the ammonium ion concentration in the fermentation system by optimizing the pH control strategy, thereby effectively increasing the yield of L-alanine.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a fermentation production process for L-alanine, comprising the following steps:

[0008] Performing seed culture on L-alanine producing bacteria to obtain seed liquid;

[0009] Inoculate the seed liquid into a fermentation medium to perform fermentation culture, so as to prepare L-alanine.

[0010] The pH range in the fermentation culture process is 6.9±0.1.

[0011] Further, when the ammonium ion concentration is less than 5 g / L, ammonia water is used to control pH; when the ammonium ion concentration is greater than 5 g / L, NaOH solution is used to control pH.

[0012] Further, the mass-volume concentration of the ammonia water is 25%; the mass-volume concentration of the NaOH solution is 40%.

[0013] Further, the fermentation medium comprises the following components: glucose 9-11 g / L, corn syrup dry powder 4-6 g / L, protein peptone 4-6 g / L, magnesium sulfate 1.5-2.5 g / L, betaine 0.4-0.6 g / L, potassium dihydrogen phosphate 2.5-3.5 g / L, ferrous sulfate 0.01-0.03 g / L, manganese sulfate 0.04-0.08 g / L, ammonium molybdate tetrahydrate 0.005-0.02 g / L, copper sulfate pentahydrate 0.01-0.03 g / L, cobalt chloride hexahydrate 0.04-0.08 g / L, and boric acid 0.1-0.3 g / L.

[0014] Preferably, the fermentation medium comprises the following components: glucose 10 g / L, corn syrup dry powder 5 g / L, protein peptone 5 g / L, magnesium sulfate 2 g / L, betaine 0.5 g / L, potassium dihydrogen phosphate 3 g / L, ferrous sulfate 0.02 g / L, manganese sulfate 0.06 g / L, ammonium molybdate tetrahydrate 0.01 g / L, copper sulfate pentahydrate 0.02 g / L, cobalt chloride hexahydrate 0.06 g / L, and boric acid 0.2 g / L.

[0015] Further, the glucose solution is started to be added at the 3rd hour of the fermentation culture, and the residual sugar concentration is controlled to be 15-20 g / L until the fermentation is completed.

[0016] Further, the concentration of the glucose solution is 700-800 g / L.

[0017] Further, the temperature of the fermentation culture is 35℃, and the fermentation period is 48 h.

[0018] Further, the medium used in the seed culture comprises the following components: yeast powder 5 g / L, protein peptone 10 g / L, and sodium chloride 10 g / L.

[0019] Further, the temperature of the seed culture is 35℃, the pH is 6.9±0.1, and the culture time is 4 h.

[0020] The present application discloses the following technical effects:

[0021] The present application develops a fermentation production process of L-alanine, the core of which is to realize the precise control of the concentration of ammonium ions (NH4 + ) in the fermentation system by optimizing the pH regulation strategy, thereby effectively improving the yield of L-alanine. The present application research finds that ammonium ions, as an important nitrogen source and metabolic regulator in microbial fermentation, have a significant impact on the synthesis of L-alanine. Excessive ammonium ion concentration may interfere with the biosynthesis pathway of L-alanine through mechanisms such as feedback inhibition of metabolic pathways, interference of enzyme activity, disruption of cell metabolic balance, and competition of byproduct accumulation and metabolic flow, thereby inhibiting the generation of products or increasing the metabolic burden. The present application effectively avoids the excessive ammonium ions forcing the cells to consume more ATP for nitrogen assimilation (such as glutamine synthetase pathway), thereby reducing energy waste and increasing energy supply for L-alanine synthesis. This process not only improves the yield of L-alanine, but also optimizes the energy utilization efficiency of the fermentation process, providing reliable technical support for efficient and economical industrial production of L-alanine. BRIEF DESCRIPTION OF DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0023] Figure 1 Time-ammonium ion concentration curve diagram of the fermentation process of Example 1 and Comparative Example 1;

[0024] Figure 2 Time-ammonium ion concentration curve diagram of the fermentation process of Example 1 and Comparative Example 1; DETAILED DESCRIPTION

[0025] The various exemplary embodiments of the present application will now be described in detail, which should not be considered as limiting the present application, but should be understood as a more detailed description of certain aspects, characteristics and embodiments of the present application.

[0026] It should be understood that the terms described in the present application are only for describing the specific embodiments, and are not intended to limit the present application. In addition, for the numerical range in the present application, it should be understood that each intermediate value between the upper limit and the lower limit 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 application. The upper limit and the lower limit of these smaller ranges can be independently included or excluded from the range.

[0027] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0028] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0029] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0030] The L-alanine-producing bacteria used in the present invention are Escherichia coli BA-15 (Escherichia coli), which was deposited in the General Microbiology Center of the China Microorganism Collection Administration on May 23, 2025, with the deposit address being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number being CGMCC No. 34653.

[0031] Example 1

[0032] A fermentation production process for L-alanine, comprising the following steps:

[0033] 1. After activating the L-alanine glycerol tube strain Escherichia coli BA-15, it was inoculated into a 5 L seed tank containing 3 L seed culture medium for seed culture for 4 hours to obtain seed liquid. The culture temperature was 35°C, the pH was 6.9±0.1, the ventilation volume was 0.5 vvm, the rotation speed was 300 rpm, and the tank pressure was 0.04 MPa.

[0034] The seed culture medium used for seed culture was: 5 g / L yeast powder, 10 g / L peptone and 10 g / L sodium chloride.

[0035] 2. The seed liquid was inoculated into a 50L fermenter (containing 25L of fermentation medium) at an inoculation amount of 5% (v / v) for fermentation culture, at a rotation speed of 400 rpm, a temperature of 35°C, a pH of 6.9±0.1, an ammonium ion concentration of less than 5g / L, and a tank pressure of 0.01Mpa. The pH was controlled by using 25% (w / v) ammonia water when the ammonium ion concentration was less than 5g / L, and by using 40% (w / v) NaOH when the ammonium ion concentration was greater than 5g / L. Anaerobic fermentation was used, and the fermentation period was 48h. The ammonium ion concentration was determined by using the indigo phenol blue reaction method.

[0036] The fermentation medium was as follows: glucose 10g / L, corn syrup dry powder 5g / L, peptone 5g / L, magnesium sulfate 2g / L, betaine 0.5g / L, potassium dihydrogen phosphate 3g / L, ferrous sulfate 0.02g / L, manganese sulfate 0.06g / L, ammonium molybdate tetrahydrate 0.01g / L, copper sulfate pentahydrate 0.02g / L, cobalt chloride hexahydrate 0.06g / L, and boric acid 0.2g / L.

[0037] The feeding process during the fermentation culture was as follows:

[0038] Based on 25L of initial fermentation medium, the following feeding process was used during the fermentation culture: at the 3rd hour of fermentation culture, glucose solution (concentration of 750g / L) was started to be fed to control the residual sugar concentration at 15-20g / L until the end of fermentation.

[0039] 3. During the fermentation culture, samples were taken every 6h, centrifuged to obtain supernatant, and the ammonium ion concentration and L-alanine content of the supernatant were detected, with the results shown in Figure 1 and Figure 2 The results showed that the yield of L-alanine was 150g / L, and the conversion rate reached 94.8%.

[0040] Example 2

[0041] An L-alanine fermentation production process, and the specific steps were as follows:

[0042] 1. After the L-alanine glycerol tube fungus Escherichia coli BA-15 was activated, it was inoculated into a 5L seed tank containing 3L of seed culture medium for seed culture for 4h to obtain seed liquid, at a culture temperature of 35°C, a pH of 6.9±0.1, a ventilation amount of 0.5vvm, a rotation speed of 300 rpm, and a tank pressure of 0.04Mpa.

[0043] The seed culture medium used for seed culture was as follows: yeast powder 5g / L, peptone 10g / L, and sodium chloride 10g / L.

[0044] 2. The seed liquid is inoculated into a 50L fermenter (containing 25L of fermentation medium) at an inoculation amount of 4% (v / v) for fermentation culture, at a rotation speed of 400 rpm, a temperature of 35°C, a pH of 6.9±0.1, an ammonium ion concentration of less than 5g / L, and the pH is controlled by 25% (w / v) ammonia water; the ammonium ion concentration is greater than 5g / L, and the pH is controlled by 40% (w / v) NaOH, anaerobic fermentation is adopted, the tank pressure is 0.01Mpa, and the fermentation period is 48h. The ammonium ion concentration is determined by indigo phenol blue reaction method.

[0045] The fermentation medium is: glucose 9g / L, corn syrup dry powder 6g / L, proteose peptone 4g / L, magnesium sulfate 2.5g / L, betaine 0.4g / L, potassium dihydrogen phosphate 3.5g / L, ferrous sulfate 0.01g / L, manganese sulfate 0.08g / L, ammonium molybdate tetrahydrate 0.005g / L, copper sulfate pentahydrate 0.03g / L, cobalt chloride hexahydrate 0.04g / L and boric acid 0.3g / L.

[0046] The feeding process of the fermentation culture process is as follows:

[0047] Based on 25L of initial fermentation medium, the following feeding process is carried out during the fermentation culture process: at the 3th hour of fermentation culture, start adding glucose solution (concentration of 700g / L), control the residual sugar concentration at 15-20g / L, until the end of fermentation.

[0048] 3. After the fermentation culture, the fermentation liquid is taken, centrifuged to obtain the supernatant, and the L-alanine content of the supernatant is detected, and the results show that the yield of L-alanine is 147g / L.

[0049] Example 3

[0050] An L-alanine fermentation production process, the specific steps are as follows:

[0051] 1. After the L-alanine glycerol tube fungus Escherichia coli BA-15 is activated, it is inoculated into a 5L seed tank containing 3L of seed culture medium for seed culture for 4h to obtain seed liquid, the culture temperature is 35°C, the pH is 6.9±0.1, the aeration amount is 0.5vvm, the rotation speed is 300rpm, and the tank pressure is 0.04Mpa.

[0052] The seed culture medium used for seed culture is: yeast powder 5g / L, proteose peptone 10g / L and sodium chloride 10g / L.

[0053] 2. The seed liquid was inoculated into a 50L fermenter (containing 25L of fermentation medium) at an inoculation amount of 4% (v / v) for fermentation culture, at a rotation speed of 400 rpm, a temperature of 35°C, a pH of 6.9±0.1, an ammonium ion concentration of less than 5g / L, and the pH was controlled by 25% (w / v) ammonia water; an ammonium ion concentration of more than 5g / L was controlled by 40% (w / v) NaOH, anaerobic fermentation was adopted, the tank pressure was 0.01Mpa, and the fermentation period was 48h. The ammonium ion concentration was determined by indigo phenol blue reaction method.

[0054] The fermentation medium was as follows: glucose 11g / L, corn syrup dry powder 4g / L, proteose peptone 6g / L, magnesium sulfate 1.5g / L, betaine 0.6g / L, potassium dihydrogen phosphate 2.5g / L, ferrous sulfate 0.03g / L, manganese sulfate 0.04g / L, ammonium molybdate tetrahydrate 0.02g / L, copper sulfate pentahydrate 0.01g / L, cobalt chloride hexahydrate 0.08g / L, and boric acid 0.1g / L.

[0055] The feeding process during the fermentation culture was as follows:

[0056] Based on 25L of initial fermentation medium, the following feeding process was carried out during the fermentation culture: at the 3th hour of fermentation culture, glucose solution (concentration of 800g / L) was started to be fed to control the residual sugar concentration at 15-20g / L until the end of fermentation.

[0057] 3. After the end of fermentation culture, the fermentation liquid was taken, centrifuged to obtain supernatant, and the L-alanine content of the supernatant was detected, and the results showed that the yield of L-alanine was 149g / L.

[0058] Comparative Example 1

[0059] The same as Example 1, except that during the fermentation culture, the pH was controlled by 25% (w / v) ammonia water throughout the whole process.

[0060] During the fermentation culture, samples were taken every 6h, centrifuged to obtain supernatant, and the ammonium ion concentration and L-alanine content of the supernatant were detected, and the results are shown in Figure 1 and Figure 2 The results showed that the yield of L-alanine was 128g / L, and the conversion rate was 89.6%.

[0061] Comparative Example 2

[0062] The same as Example 1, except that the fermentation medium was as follows: glucose 10g / L, corn syrup dry powder 5g / L, proteose peptone 5g / L, magnesium sulfate 2g / L, potassium dihydrogen phosphate 3g / L, ferrous sulfate 0.02g / L, manganese sulfate 0.06g / L, ammonium molybdate tetrahydrate 0.01g / L, copper sulfate pentahydrate 0.02g / L, cobalt chloride hexahydrate 0.06g / L, and boric acid 0.2g / L.

[0063] After the end of the fermentation culture, the fermentation liquid was taken, centrifuged to take the supernatant, and the L-alanine content of the supernatant was detected, and the results showed that the yield of L-alanine was 117 g / L.

[0064] The above-described embodiments are merely preferred modes of the present application and are not intended to limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by those of ordinary skill in the art shall fall within the protection scope of the present application as defined by the claims.

Claims

1. A fermentation production process for L-alanine, characterized in that: The following steps are involved: Performing seed culture on L-alanine producing bacteria to obtain seed liquid; inoculating the seed liquid into a fermentation medium, performing fermentation culture, and preparing L-alanine; During the fermentation process, the pH is controlled within the range of 6.9±0.

1.

2. The fermentation production process according to claim 1, characterized in that When the ammonium ion concentration is less than 5 g / L, ammonia water is used to control the pH; when the ammonium ion concentration is greater than 5 g / L, NaOH solution is used to control the pH.

3. The fermentation production process according to claim 2, characterized in that: The mass volume concentration of the ammonia water is 25%; the mass volume concentration of the NaOH solution is 40%.

4. The fermentation production process according to claim 1, characterized in that The fermentation medium comprises the following components: 9-11 g / L of glucose, 4-6 g / L of corn steep liquor powder, 4-6 g / L of peptone, 1.5-2.5 g / L of magnesium sulfate, 0.4-0.6 g / L of betaine, 2.5-3.5 g / L of potassium dihydrogen phosphate, 0.01-0.03 g / L of ferrous sulfate, 0.04-0.08 g / L of manganese sulfate, 0.005-0.02 g / L of ammonium molybdate tetrahydrate, 0.01-0.03 g / L of copper sulfate pentahydrate, 0.04-0.08 g / L of cobalt chloride hexahydrate, and 0.1-0.3 g / L of boric acid.

5. The fermentation production process according to claim 4, characterized in that: The fermentation medium includes the following components: 10 g / L glucose, 5 g / L corn steep liquor powder, 5 g / L peptone, 2 g / L magnesium sulfate, 0.5 g / L betaine, 3 g / L potassium dihydrogen phosphate, 0.02 g / L ferrous sulfate, 0.06 g / L manganese sulfate, 0.01 g / L ammonium molybdate tetrahydrate, 0.02 g / L copper sulfate pentahydrate, 0.06 g / L cobalt chloride hexahydrate, and 0.2 g / L boric acid.

6. The fermentation production process according to claim 1, characterized in that: At the 3rd hour of fermentation, glucose solution was added to control the residual sugar concentration at 15-20 g / L until the end of fermentation.

7. The fermentation production process according to claim 6, characterized in that: The concentration of the glucose solution is 700-800 g / L.

8. The fermentation production process according to claim 1, characterized in that: The fermentation temperature is 35° C. and the fermentation period is 48 hours.

9. The fermentation production process according to claim 1, characterized in that: The culture medium used for the seed culture comprises the following components: 5 g / L yeast powder, 10 g / L peptone and 10 g / L sodium chloride.

10. The fermentation production process according to claim 1, characterized in that: The seed culture temperature is 35° C., the pH is 6.9±0.1, and the culture time is 4 hours.

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