Fermentation production method of proline

Abstract

The invention relates to the technical field of fermentation production, and discloses a fermentation production method of proline, which comprises the following steps: inoculating a slant culture medium with a proline producing strain, and culturing until the strain grows fully; eluting the strain in the slant culture medium by using sterile water to form a bacterial solution, and inoculating the bacterial solution into a seed culture medium for enlarged culture to obtain a seed solution; inoculating the seed solution into a fermentation culture medium for fermentation culture, and starting to supplement an amino acid mixture adjusting solution until fermentation is finished when the seed solution is cultured to an initial logarithmic growth stage and the OD value of fermentation liquor reaches 20-30; when the reducing sugar content of the fermentation liquid is smaller than or equal to 1.5%, a glucose aqueous solution is supplemented in a fed-batch mode to maintain the reducing sugar content to range from 1.0% to 1.5%, the fed-batch amount of the glucose aqueous solution is 25%-40% of the initial volume of the fermentation culture medium, and fermentation is finished after the supplemented sugar is consumed. The amino acid mixture adjusting liquid is supplemented in the fermentation process until the fermentation is finished, so that the sugar-acid conversion rate and the yield of the target product proline are improved.

Description

Technical Field

[0001] The invention belongs to the technical field of fermentation production, and particularly relates to a fermentation production method of proline. Background Art

[0002] The chemical name of L-proline is (S)-pyrrolidine-2-carboxylic acid, and its molecular formula is C5H9NO2. L-proline contains a pyrroline ring and is the only non-essential amino acid without a complete amino group. This characteristic gives it a unique conformational role in protein structure. L-proline has physiological functions such as participating in protein synthesis, regulating the osmotic pressure of plant and animal cells, promoting collagen synthesis, and providing energy support for cells. In the food industry, it can be used as a nutritional supplement, flavor enhancer, and preservative for meat products. In the pharmaceutical, health food, and medical fields, it can be used in the formulation of amino acid injections, food additives, and nutritional supplements. In the synthetic industry, L-proline can also participate in inducing asymmetric reactions and act as a catalyst for hydrogenation, polymerization, and water-mediated reactions. The wide range of uses of L-proline gives it high commercial value.

[0003] Currently, proline production methods primarily include fermentation and chemical synthesis. Fermentation has become the primary method for industrial proline production due to its significant advantages in production costs and environmental friendliness. In fermentation production processes, sugar-acid conversion is a key cost-effectiveness indicator. Glucose, the primary carbon source in fermentation production, accounts for approximately 70% to 80% of the total raw material cost. Improving this conversion significantly reduces fermentation costs, which is crucial for manufacturers to enhance their market competitiveness. However, existing proline fermentation production methods achieve low conversion rates, necessitating the development of a method to improve the conversion rate of proline, the target product. Summary of the Invention

[0004] In order to address the deficiencies of the prior art, the present invention provides a fermentation production method for proline. By rationally adding an amino acid mixture regulating liquid during the fermentation process until the end of fermentation, the enzymatic activity of the bacterial metabolic process can be regulated, the metabolic flux of the synthetic pathway of the fermentation product can be enhanced, and the sugar-acid conversion rate of the target product proline can be effectively improved. At the same time, the yield of proline can also be correspondingly improved.

[0005] The technical effects to be achieved by the present invention are achieved through the following technical aspects: The present invention provides a fermentation production method of proline, comprising the following steps: S1. Inoculate the proline-producing bacteria into the slant culture medium and culture until the bacteria grow fully; S2. Elute the bacterial strain in the slant culture medium with sterile water to form a bacterial solution, inoculate the bacterial solution into a seed culture medium for expansion culture, and culture until the logarithmic growth phase to obtain a seed solution; S3, taking the seed liquid and inoculating it into a fermentation medium for fermentation culture, and culturing it until the OD value of the fermentation liquid reaches 20-30 in the early logarithmic growth stage, starting to feed the amino acid mixture regulating liquid until the fermentation is completed; when the reducing sugar content of the fermented liquid is ≤1.5%, feeding a glucose aqueous solution to maintain the reducing sugar content at 1.0%-1.5%, and feeding the glucose aqueous solution in an amount of 25%-40% of the initial volume of the fermentation medium, and terminating the fermentation after the sugar supplement is consumed; Wherein, each 100 mL of the amino acid mixture regulating solution comprises the following components: 8-12 g of threonine, 8-12 g of lysine, 6-10 g of valine, 3-5 g of isoleucine, and 1-3 g of leucine.

[0006] As a further description of the technical solution of the present invention, the amino acid mixture regulating solution is added by constant rate feeding, and the amount of the amino acid mixture regulating solution added per hour is 0.1% to 0.3% of the initial volume of the fermentation medium.

[0007] As a further description of the technical solution of the present invention, the amino acid mixture regulating solution is added in an intermittent quantitative manner, once every 4 hours, and the amount of the amino acid mixture regulating solution added each time is 0.7% to 0.9% of the initial volume of the fermentation medium.

[0008] As a further description of the technical solution of the present invention, the proline-producing bacteria is Corynebacterium aceticum.

[0009] As a further description of the technical solution of the present invention, in S3, the fermentation process controls the rotation speed of the fermentation equipment to be 300-600 rpm, the ventilation ratio to be 0.1-0.3, the temperature to be 30-34° C., the dissolved oxygen content of the fermentation liquid to be 10%-20%, the pH to be 6.8±0.1, and the fermentation cycle to be 72-80 h; The concentration of the glucose aqueous solution is 55% to 65%.

[0010] As a further description of the technical solution of the present invention, per 100 mL of the fermentation medium comprises the following components: 8-12 g of glucose, 8-10 g of corn steep liquor powder, 2-4 g of yeast extract powder, 1-2 g of ammonium sulfate, 0.1-0.2 g of KH2PO4, 0.04-0.06 g of MgSO4, 8-12 mg of MnSO4·H2O, and 8-12 mg of FeSO4.

[0011] As a further description of the technical solution of the present invention, in S2, the inoculation amount of the bacterial liquid is 1.0% to 1.4% of the initial volume of the seed culture medium; in S3, the inoculation amount of the seed liquid is 8% to 12% of the initial volume of the fermentation medium.

[0012] As a further description of the technical solution of the present invention, in S1, the culture temperature is 30±1°C, and the culture time is 20-24 hours; in S2, the temperature of the expanded culture is 30-34°C, and the culture time is 12-18 hours.

[0013] As a further description of the technical solution of the present invention, each 100 mL of the slant culture medium includes the following components: 1-3 g of glucose, 5-8 g of corn steep liquor powder, 3-5 g of yeast extract powder, 0.4-0.6 g of NaCl, and 2-4 g of agar.

[0014] As a further description of the technical solution of the present invention, each 100 mL of the seed culture medium includes the following components: 3-5 g of glucose, 8-12 g of corn steep liquor powder, 1-3 g of yeast extract powder, 0.1-0.3 g of KH2PO4, and 0.1-0.2 g of MgSO4.

[0015] In summary, the present invention has at least the following benefits: The fermentation production method of proline provided by the present invention, by starting to supplement the amino acid mixture regulating liquid when the OD value of the fermentation liquid reaches 20-30 until the fermentation is completed, can regulate the enzyme activity of the metabolic process of the bacterial body, reasonably intervene in the metabolic direction of glucose, strengthen the metabolic flux of glucose toward the synthesis direction of the target fermentation product proline, weaken bypass metabolism, reduce metabolic overflow, strengthen the metabolic flux of the synthetic pathway of the target fermentation product, thereby effectively improving the sugar-acid conversion rate of the target product proline, and at the same time, the yield of proline can also be correspondingly improved. By improving the sugar-acid conversion rate, the raw material cost of the fermentation production is effectively reduced, thereby reducing the comprehensive cost of the final product production, which is more conducive to improving the market competitiveness of the product. DETAILED DESCRIPTION

[0016] To facilitate understanding of the present invention, the present invention will be described more fully below in conjunction with specific embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to provide a more thorough and comprehensive understanding of the disclosure of the present invention.

[0017] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0018] The present invention provides a fermentation production method of proline, comprising the following steps: S1. Inoculate the proline-producing bacteria into the slant culture medium and culture until the bacteria grow fully; S2. Elute the bacteria from the slant culture medium with sterile water to form a bacterial solution, inoculate the bacterial solution into a seed culture medium for expansion and culture until the logarithmic growth phase to obtain a seed solution; S3, taking the seed liquid and inoculating it into the fermentation medium for fermentation culture, and culturing it until the early logarithmic growth stage, when the OD value of the fermentation liquid reaches 20-30, starting to feed the amino acid mixture regulating liquid until the fermentation is completed, and the total amount of the amino acid mixture regulating liquid fed is about 10%-12% of the initial volume of the fermentation medium; when the reducing sugar content of the fermented liquid is ≤1.5%, feeding the glucose aqueous solution to maintain the reducing sugar content at 1.0%-1.5%, and the feeding amount of the glucose aqueous solution is 25%-40% of the initial volume of the fermentation medium, and the fermentation is terminated after the supplemented sugar is consumed; Wherein, each 100 mL of the amino acid mixture adjustment solution comprises the following components: 8-12 g of threonine, 8-12 g of lysine, 6-10 g of valine, 3-5 g of isoleucine, and 1-3 g of leucine. Preferably, each 100 mL of the amino acid mixture adjustment solution comprises the following components: 10 g of threonine, 10 g of lysine, 8 g of valine, 4 g of isoleucine, and 2 g of leucine.

[0019] It is understood that proline-producing bacteria can induce the synthesis of corresponding enzymes or feedback inhibit corresponding enzymes based on the substrates provided by the fermentation environment, thereby enhancing their adaptability to the fermentation environment. When the end product of the metabolic pathway in the bacteria is excessive or already present in the fermentation broth, it will inhibit the further synthesis of key enzymes in the biosynthesis pathway, thereby reducing the production of the end product. The metabolic pathways of aspartic acid and branched-chain amino acids can be considered as branches of the proline production pathway. By weakening the metabolic flux of these two amino acid groups, more energy will be diverted to proline synthesis, thereby improving the sugar-acid conversion rate of the target fermentation product, proline. Threonine, lysine, and isoleucine in the amino acid mixture jointly inhibit aspartate kinase. Aspartate kinase phosphorylates aspartate to form aspartate-4-phosphate, a common precursor for the synthesis of essential amino acids such as threonine, lysine, and isoleucine. Aspartate kinase activity is subject to synergistic feedback inhibition by the end products of metabolism, threonine, lysine, and isoleucine. The presence of threonine, lysine, and isoleucine in the fermentation broth weakens the metabolic bypass for aspartate, increasing the flow of oxaloacetate and acetyl CoA into the tricarboxylic acid (TCA) cycle. Simultaneously, the enzyme system catalyzing the conversion of valine, isoleucine, and leucine is multivalently inhibited by valine and isoleucine, weakening the metabolic bypass for the conversion of pyruvate to valine and leucine. This allows more pyruvate to be converted into acetyl CoA, which then enters the TCA cycle and further produces proline.

[0020] By feeding the amino acid mixture regulator solution into the fermentation broth starting when the OD value reaches 20-30 and continuing until the end of fermentation, the enzyme activity of the bacterial metabolic process is regulated, strengthening the metabolic flux of glucose toward the synthesis of the target fermentation product, proline. This weakens the metabolic bypass of aspartic acid amino acids and branched-chain amino acids, reduces the energy consumption of the metabolic bypass, increases the flux of the TCA cycle, inhibits metabolic overflow, and strengthens the metabolic flux of the synthesis pathway of the target product, proline. This achieves the goal of improving the sugar-acid conversion rate of the target product, proline, and simultaneously increases the yield of proline. By improving the sugar-acid conversion rate, the raw material cost of fermentation production is effectively reduced, thereby reducing the overall cost of final product production and further improving the market competitiveness of the product.

[0021] In one embodiment, the amino acid mixture regulating liquid is fed at a constant rate, with the amount of the amino acid mixture regulating liquid fed per hour being 0.1% to 0.3%, preferably 0.2%, of the initial volume of the fermentation medium. Continuously feeding the amino acid mixture regulating liquid at a constant rate maintains the levels of threonine, lysine, valine, isoleucine, and leucine in the fermentation broth at stable levels, thereby stably inhibiting the metabolic bypass of aspartic acid family amino acids and branched-chain amino acids, allowing the bacteria to continuously and efficiently synthesize proline, thereby increasing the proline yield and sugar-acid conversion rate.

[0022] In one embodiment, the amino acid mixture regulating solution is added intermittently and quantitatively, with the amount added every 4 hours being 0.7% to 0.9%, preferably 0.8%, of the initial volume of the fermentation medium. This intermittent and quantitative addition method can reduce equipment energy consumption during the fermentation process, further reducing fermentation production costs, and thereby further improving the market competitiveness of the final product.

[0023] In some embodiments, the proline-producing bacteria may be Corynebacterium acidophilum. Corynebacterium acidophilum can directly utilize hydrolyzed starch sugars such as corn starch hydrolysate as its primary carbon source, thereby significantly reducing the amount of glucose used in the culture medium and effectively lowering the raw material costs of fermentation production. Furthermore, Corynebacterium acidophilum has a high fermentation acid production rate, which helps increase the yield of the target fermentation product, proline, shorten the fermentation cycle, reduce energy consumption, and further control production costs.

[0024] In S3, the fermentation process controlled the rotation speed of the fermentation equipment to be between 300 and 600 rpm, the ventilation ratio to be between 0.1 and 0.3, and the temperature to be between 30 and 34°C. The dissolved oxygen content of the fermentation liquid was controlled to be between 10% and 20%, and the pH was adjusted to 6.8±0.1 by adding 20% ​​ammonia water. The fermentation cycle was 72 to 80 h.

[0025] In some embodiments, the concentration of the glucose aqueous solution is 55% to 65%, preferably 60%.

[0026] In some embodiments, each 100 mL of fermentation medium includes the following components: 8-12 g glucose, 8-10 g corn steep liquor powder, 2-4 g yeast extract powder, 1-2 g ammonium sulfate, 0.1-0.2 g KH2PO4, 0.04-0.06 g MgSO4, 8-12 mg MnSO4·H2O, and 8-12 mg FeSO4.

[0027] In S2, the inoculum size of the bacterial solution is 1.0% to 1.4%, preferably about 1.3%, of the initial volume of the seed culture medium. In S3, the inoculum size of the seed solution is 8% to 12%, preferably 10%, of the initial volume of the fermentation culture medium.

[0028] In S1, the culture temperature is 30 ± 1°C for 20 to 24 hours. In S2, the expanded culture temperature is 30 to 34°C for 12 to 18 hours. The expanded culture process is carried out in a culture device such as a shaker or an automatic control tank, and the speed of the culture device is controlled at 300 rpm.

[0029] In some embodiments, each 100 mL of slant culture medium comprises the following components: 1-3 g glucose, 5-8 g corn steep liquor powder, 3-5 g yeast extract powder, 0.4-0.6 g NaCl, and 2-4 g agar.

[0030] In some embodiments, each 100 mL of seed culture medium includes the following components: 3-5 g glucose, 8-12 g corn steep liquor powder, 1-3 g yeast extract powder, 0.1-0.3 g KH2PO4, and 0.1-0.2 g MgSO4.

[0031] Example 1 This embodiment provides a fermentation production method of proline, comprising the following steps: Preliminary preparation: Preparation of slant medium: Each 100 mL of slant medium includes the following components: glucose 2 g, corn steep liquor 6 g, yeast extract 4 g, NaCl 0.5 g, agar 2 g, pH 6.8; Preparation of seed culture medium: Each 100 mL of seed culture medium includes the following components: glucose 4 g, corn steep liquor powder 10 g, yeast extract powder 2 g, KH2PO4 0.2 g, MgSO4 0.1 g, pH 6.8; Preparation of fermentation medium: Each 100 mL of fermentation medium includes the following components: glucose 10 g, corn steep liquor powder 9 g, yeast extract powder 3 g, ammonium sulfate 2 g, KH2PO4 0.15 g, MgSO4 0.05 g, MnSO4·H2O 10 mg, FeSO4 10 mg, pH 6.8; Preparation of amino acid mixture regulating solution: threonine 10g, lysine 10g, valine 8g, isoleucine 4g, leucine 2g; Preparation of glucose aqueous solution: The concentration of glucose aqueous solution is 60%.

[0032] S1. Apply 0.1 mL of glycerol stock of Corynebacterium acetophilum on a slant culture medium and incubate at 30°C for 20 h until the bacteria grow fully. S2. Elute the bacteria from the slant culture medium with 10 mL of sterile water to form a bacterial solution. Take 3 mL of the bacterial solution and inoculate it into a flask containing 300 mL of seed culture medium. Place the flask in a shaker for expansion culture at 300 rpm and 30°C for 14 h until the logarithmic growth phase to obtain the seed solution. S3, take 300mL seed liquid and inoculate it into 3L fermentation medium, and ferment and culture in a 5L fermentor. During the fermentation process, the speed of the fermentor is controlled at 300-600rpm, the ventilation ratio is at 0.1-0.3, and the temperature is 32°C. The dissolved oxygen content of the fermentation liquid is controlled at 10%-20%, and the pH is adjusted to 6.8 by adding 20% ​​ammonia water. After 24 hours of fermentation and culture, the OD value of the fermentation liquid reaches 20, and 24mL of amino acid mixture regulating solution is added. Thereafter, it is supplemented every 4 hours, and 24mL of amino acid mixture regulating solution is added each time until the fermentation is completed. When the reducing sugar content of the fermented liquid is ≤1.5%, glucose aqueous solution is started to be fed to maintain the reducing sugar content of the fermented liquid at 1.0%-1.5%. The fermentation is terminated after the sugar supplement is consumed. The final fermentation cycle is 72h. A total of 840mL of glucose aqueous solution and 312mL of amino acid mixture regulating solution are added during the fermentation process.

[0033] Example 2 This embodiment provides a fermentation production method of proline, comprising the following steps: Preliminary preparation: The preparation of slant culture medium, seed culture medium, fermentation culture medium, amino acid mixture regulating solution and glucose aqueous solution is the same as that in Example 1.

[0034] S1. Apply 0.4 mL of glycerol stock of Corynebacterium acetophilum on a slant culture medium and incubate at 30°C for 20 h until the bacteria grow fully. S2. Elute the bacteria from the slant culture medium with 40 mL of sterile water to form a bacterial solution. Inoculate 40 mL of the bacterial solution into 3000 mL of seed culture medium and culture in a 5 L automatic tank with an air volume of 5 L / min and a rotation speed of 300 rpm at 30°C for 16 h until the logarithmic growth phase to obtain a seed solution. S3. Take 3000 mL of seed liquid and inoculate it into 30 L of fermentation medium. Fermentation and culture are carried out in a 50 L fermentor. During the fermentation process, the speed of the fermentor is controlled at 300-600 rpm, the ventilation ratio is controlled at 0.1-0.3, and the temperature is 32 ° C. The dissolved oxygen content of the fermentation liquid is controlled at 10%-20%, and the pH is adjusted to 6.8 by adding 20% ​​ammonia water. After 22 hours of fermentation and culture, the OD value of the fermentation liquid reaches 25, and the amino acid mixture regulating liquid is continuously added at a constant flow rate of 60 mL / h until the fermentation is completed. When the reducing sugar content of the fermented liquid is ≤1.5%, glucose aqueous solution is started to be added to maintain the reducing sugar content of the fermentation liquid at 1.0%-1.5%. The fermentation is terminated after the added sugar is consumed. The final fermentation cycle is 78 hours. During the fermentation process, a total of 10.5 L of glucose aqueous solution and a total of 3.36 L of amino acid mixture regulating liquid are added.

[0035] Example 3 This embodiment provides a fermentation production method of proline, comprising the following steps: Preliminary preparation: The preparation of slant culture medium, seed culture medium, fermentation culture medium, amino acid mixture regulating solution and glucose aqueous solution is the same as that in Example 1.

[0036] S1. Apply 0.4 mL of glycerol stock of Corynebacterium acetophilum on a slant culture medium and incubate at 30°C for 20 h until the bacteria grow fully. S2. Elute the bacteria from the two slant cultures with 40 mL of sterile water to form 80 mL of bacterial solution. Inoculate 80 mL of bacterial solution into 6000 mL of seed culture medium and expand the culture in a 10 L automatic tank with an air volume of 15 L / min and a rotation speed of 300-500 rpm at 30°C for 17 h until the logarithmic growth phase to obtain the seed solution. S3. Take 6000 mL of seed liquid and inoculate it into 60 L of fermentation medium. Fermentation and culture are carried out in a 100 L fermentor. During the fermentation process, the speed of the fermentor is controlled at 300-600 rpm, the ventilation ratio is 0.1-0.3, the temperature in the early stage of fermentation is 32 ° C, and the temperature in the middle and late stages of fermentation is 34 ° C. The dissolved oxygen content of the fermentation liquid is controlled at 10%-20%, and the pH is adjusted to 6.8 by adding 20% ​​ammonia water. After 24 hours of fermentation, the OD value of the fermentation liquid reaches 25, and the amino acid mixture regulating liquid is continuously added at a constant flow rate of 120 mL / h until the fermentation is completed. When the reducing sugar content of the fermented liquid is ≤1.5%, glucose aqueous solution is added to maintain the reducing sugar content of the fermented liquid at 1.0%-1.5%. The fermentation is terminated after the added sugar is consumed. The final fermentation cycle is 80 hours. A total of 23.5 L of glucose aqueous solution and 6.72 L of amino acid mixture regulating liquid are added during the fermentation process.

[0037] Comparative Example 1 This comparative example provides a fermentation production method of proline, comprising the following steps: Preliminary preparation: The preparation of slant culture medium, seed culture medium, fermentation culture medium and glucose aqueous solution is the same as that in Example 1.

[0038] S1. Apply 0.4 mL of glycerol stock of Corynebacterium acetophilum on a slant culture medium and incubate at 30°C for 20 h until the bacteria grow fully. S2. Elute the bacteria from the slant culture medium with 40 mL of sterile water to form a bacterial solution. Inoculate 40 mL of the bacterial solution into 3000 mL of seed culture medium and culture in a 5 L automatic tank with an air volume of 5 L / min and a rotation speed of 300 rpm at 30°C for 16 h until the logarithmic growth phase to obtain a seed solution. S3. Take 3000 mL of seed liquid and inoculate it into 30 L of fermentation medium. Fermentation and culture are carried out in a 50 L fermentor. During the fermentation process, the rotation speed of the fermentor is controlled at 300-600 rpm, the ventilation ratio is controlled at 0.1-0.3, and the fermentation temperature is 32°C. The dissolved oxygen content of the fermentation liquid is controlled at 10%-20%, and the pH is adjusted to 6.8 by adding 20% ​​ammonia water. When the reducing sugar content of the fermentation liquid is ≤1.5%, glucose aqueous solution is started to be added to maintain the reducing sugar content of the fermentation liquid at 1.0%-1.5%. Fermentation is terminated after the added sugar is consumed. The final fermentation cycle is 80 h, and a total of 11.0 L of glucose aqueous solution is added during the fermentation process.

[0039] Test Example 1 In this test example, the fermentation broths from Examples 1-3 and Comparative Example 1 that had completed fermentation were centrifuged at 10,000 rpm for 10 minutes. The supernatant was obtained and diluted 6,000-fold with pure water. The absorbance at a characteristic wavelength of 515 nm was measured using a spectrophotometer. A standard curve was prepared and the absorbance values ​​were substituted into the standard curve equation to calculate the proline content. The sugar-acid conversion rate was calculated by calculating the ratio of the tank yield to the total sugar consumption for Examples 1-3 and Comparative Example 1. The results of the proline content and sugar-acid conversion rate are shown in Table 1.

[0040] Table 1 Proline content and sugar-acid conversion rate of fermentation broths of Examples 1 to 3 and Comparative Example 1

[0041] By comparing the results of Comparative Example 1 with Examples 1 to 3, it can be seen that by reasonably adding the amino acid mixture regulating liquid during the fermentation process, the sugar-acid conversion rate of the target product proline is significantly improved, and the proline content in the fermentation broth is also improved, which means that the yield of proline is also correspondingly improved, especially in Example 3. In Example 3, the sugar-acid conversion rate increased by 17% year-on-year compared with Comparative Example 1, and the improvement effect is most significant. By comparing the results of Example 1 with Examples 2 to 3, it can be seen that the sugar-acid conversion rate of the target product proline is higher when the amino acid mixture regulating liquid is added in a constant flow manner than when the amino acid mixture regulating liquid is added in an intermittent quantitative manner, and the proline content of the fermentation broth is also higher, indicating that the addition of the amino acid mixture regulating liquid in a constant flow manner can bring a more significant improvement effect on the sugar-acid conversion rate and the proline yield.

[0042] The above description is merely an example and illustration of the structure of the present invention. Although the description is relatively specific and detailed, it should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art may make various modifications and improvements without departing from the scope of the present invention, and such obvious alternatives are all within the scope of protection of the present invention.

Claims

1. A fermentation production method of proline, characterized in that: The following steps are involved: S1. Inoculate the proline-producing bacteria into the slant culture medium and culture until the bacteria grow fully; S2. Elute the bacterial strain in the slant culture medium with sterile water to form a bacterial solution, inoculate the bacterial solution into a seed culture medium for expansion culture, and culture until the logarithmic growth phase to obtain a seed solution; S3, taking the seed liquid and inoculating it into a fermentation medium for fermentation culture, and culturing it until the OD value of the fermentation liquid reaches 20-30 in the early logarithmic growth stage, starting to feed the amino acid mixture regulating liquid until the fermentation is completed; when the reducing sugar content of the fermented liquid is ≤1.5%, feeding a glucose aqueous solution to maintain the reducing sugar content at 1.0%-1.5%, and feeding the glucose aqueous solution in an amount of 25%-40% of the initial volume of the fermentation medium, and terminating the fermentation after the sugar supplement is consumed; Wherein, each 100 mL of the amino acid mixture regulating solution comprises the following components: 8-12 g of threonine, 8-12 g of lysine, 6-10 g of valine, 3-5 g of isoleucine, and 1-3 g of leucine.

2. The fermentation production method of proline according to claim 1, characterized in that The amino acid mixture regulating solution is added in a constant rate flow addition manner, and the flow addition amount of the amino acid mixture regulating solution per hour is 0.1% to 0.3% of the initial volume of the fermentation medium.

3. The fermentation production method of proline according to claim 1, characterized in that: The amino acid mixture regulating solution is added in an intermittent quantitative manner, once every 4 hours, and the amount of the amino acid mixture regulating solution added each time is 0.7% to 0.9% of the initial volume of the fermentation medium.

4. The fermentation production method of proline according to claim 1, characterized in that: The proline-producing bacteria is Corynebacterium acidophilum.

5. The fermentation production method of proline according to claim 1, characterized in that: In S3, the fermentation process controls the rotation speed of the fermentation equipment to be 300-600 rpm, the ventilation ratio to be 0.1-0.3, the temperature to be 30-34° C., the dissolved oxygen content of the fermentation liquid to be 10%-20%, the pH to be 6.8±0.1, and the fermentation cycle to be 72-80 h; The concentration of the glucose aqueous solution is 55% to 65%.

6. The fermentation production method of proline according to claim 1, characterized in that: The fermentation medium comprises the following components per 100 mL: glucose 8-12 g, corn steep liquor powder 8-10 g, yeast extract powder 2-4 g, ammonium sulfate 1-2 g, KH2PO4 0.1-0.2 g, MgSO4 0.04-0.06 g, MnSO4·H2O 8-12 mg, and FeSO4 8-12 mg.

7. The fermentation production method of proline according to claim 1, characterized in that: In S2, the inoculation amount of the bacterial liquid is 1.0% to 1.4% of the initial volume of the seed culture medium; in S3, the inoculation amount of the seed liquid is 8% to 12% of the initial volume of the fermentation culture medium.

8. The fermentation production method of proline according to claim 1, characterized in that: In the S1, the culture temperature is 30±1° C., and the culture time is 20-24 h; in the S2, the expanded culture temperature is 30-34° C., and the culture time is 12-18 h.

9. The fermentation production method of proline according to claim 1, characterized in that: Each 100 mL of the slant culture medium includes the following components: 1-3 g of glucose, 5-8 g of corn steep liquor powder, 3-5 g of yeast extract powder, 0.4-0.6 g of NaCl, and 2-4 g of agar.

10. The fermentation production method of proline according to claim 1, characterized in that: Each 100 mL of the seed culture medium includes the following components: 3-5 g of glucose, 8-12 g of corn steep liquor powder, 1-3 g of yeast extract powder, 0.1-0.3 g of KH2PO4, and 0.1-0.2 g of MgSO4.

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