Process for producing threonine through large-scale fermentation

Abstract

The invention belongs to the technical field of biological fermention.The invention discloses a process for producing threonine through large-scale fermention.The process comprises the following steps of 1, strain recovery, 2, shake-flask culture, 3, seed amplification culture and 4, fermentation tank culture.A strain attachment carrier is added in the fermentation tank culture.The process can be applied to large-scale production of threonine by optimizing fermentation conditions.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological fermentation, and relates to a process for large-scale fermentation production of threonine. BACKGROUND

[0002] It is a research hotspot in recent years to optimize the strain performance by adjusting the medium components so as to improve the fermentation yield of threonine. The previous patent of the applicant “CN2025114129035, a method for improving threonine fermentation efficiency by optimizing medium components” proves that the addition of guar gum or EGCG in the culture medium can promote the formation of biofilm of Brevibacterium flavum on the surface of six-hole plate culture containers, and the two can synergistically improve the yield of L-threonine. The above-mentioned achievement is in the research and development pilot stage, and further exploration of the strain characteristics and process parameters is needed to provide basic data for subsequent scale-up. The pilot data involved in the above-mentioned patent technology only reflect the basic characteristics of the strain in the pilot stage, and there may be differences in the product yield with actual production (fermenter), which needs to be verified through subsequent scale-up. The fermenter culture maintains the optimal state of bacterial metabolism through real-time control of dissolved oxygen, pH and feeding, supports high-density culture, and can break through the limitations of shake flasks or culture dishes in environmental control and productivity, which is the core guarantee for yield improvement in industrial production. SUMMARY

[0003] The application provides a process for large-scale fermentation production of threonine based on the laboratory pilot development of threonine, which is intended to be applied to industrial production.

[0004] The application is realized by the following technical solutions.

[0005] A process for large-scale fermentation production of threonine, comprising the following steps: 1) resuscitation, 2) shake flask culture, 3) seed expansion culture, 4) fermenter culture; a strain adhesion carrier is added in the fermenter culture.

[0006] Further, the strain adhesion carrier is a porous ceramic particle.

[0007] Further, the fermenter culture medium used in the fermenter culture contains guar gum or / and EGCG.

[0008] Preferably, the components of the fermenter culture medium are: glucose 50 g / L, yeast powder 20 g / L, potassium dihydrogen phosphate 1 g / L, dipotassium hydrogen phosphate 1 g / L, magnesium sulfate heptahydrate 100 mg / L, ferrous sulfate heptahydrate 10 mg / L, manganese sulfate monohydrate 10 mg / L, VB1 0.5 mg / L, VB2 0.5 mg / L, biotin 0.1 mg / L, guar gum 0.8 g / L.

[0009] Preferably, the components of the fermentation tank medium are: Glucose 50 g / L, yeast powder 20 g / L, potassium dihydrogen phosphate 1 g / L, dipotassium hydrogen phosphate 1 g / L, magnesium sulfate heptahydrate 100 mg / L, ferrous sulfate heptahydrate 10 mg / L, manganese sulfate monohydrate 10 mg / L, VB1 0.5 mg / L, VB2 0.5 mg / L, biotin 0.1 mg / L, EGCG 0.4 mg / L.

[0010] More preferably, the components of the fermentation tank medium are: Glucose 50 g / L, yeast powder 20 g / L, potassium dihydrogen phosphate 1 g / L, dipotassium hydrogen phosphate 1 g / L, magnesium sulfate heptahydrate 100 mg / L, ferrous sulfate heptahydrate 10 mg / L, manganese sulfate monohydrate 10 mg / L, VB1 0.5 mg / L, VB2 0.5 mg / L, biotin 0.1 mg / L, guar gum 0.8 g / L, EGCG 0.4 mg / L.

[0011] Specifically, the amount of the porous ceramic particles added is 5-20 g per liter of the fermentation tank medium.

[0012] Further, the components of the seed culture medium used in the seed expansion culture are: glucose 40 g / L, yeast powder 10 g / L, ammonium sulfate 8 g / L, potassium dihydrogen phosphate 0.5 g / L, dipotassium hydrogen phosphate 0.5 g / L, magnesium sulfate heptahydrate 50 mg / L, ferrous sulfate heptahydrate 10 mg / L, manganese sulfate monohydrate 10 mg / L.

[0013] Specifically, the process comprises the following steps: 5) Revival: the threonine-producing Brevibacterium flavum is taken out and revived; 6) Shaking culture: inoculated into LB liquid medium, 37°C, 100 rpm shaking culture for 24 hours, coated on LB solid medium, 37°C culture for 24 hours, then picked up the vigorous colonies, inoculated into a shaking flask containing LB liquid medium, 37°C, 100 rpm shaking culture until the concentration is 1 x 10 7 CFU / mL; 7) Seed expansion culture: inoculated into a 10 L seed tank containing 4 L seed culture medium at a 3% inoculation amount for seed expansion culture, cultured for 48 hours to obtain a seed liquid; 8) Fermentation tank culture: porous ceramic particles are added to the fermentation tank medium at a ratio of 10 g: 1 L, then the seed liquid is inoculated into the fermentation tank medium at a 5% inoculation amount for fermentation culture, the temperature is controlled at 36°C, the stirring speed is 300 rpm, the aeration ratio is 0.8-1.0 VVm, and the antifoam agent is bubble enemy, the fermentation time is 48 h, the fermentation is stopped, and the fermentation broth is collected.

[0014] Further, in the fermentation tank culture process, the sugar content is controlled to be not less than 1% by adding 50% glucose solution, and the pH is controlled to be 7.0 by adding ammonia water.

[0015] The technical effects obtained by the present application mainly include, but are not limited to, the following aspects: adding guar gum and epigallocatechin gallate in the fermentation tank medium can promote the adhesion of the strain and the formation of the biofilm, and synergistically improve the yield of L-threonine, thereby providing a basis for large-scale production of threonine; adding porous ceramic particle carriers in the fermentation tank is beneficial to the formation of the biofilm of the strain on the surface of the carrier, and the yield of L-threonine cannot be effectively improved by adding guar gum or / and epigallocatechin gallate under the culture condition without adding the adhesion carrier. DETAILED DESCRIPTION

[0016] Those skilled in the art can improve the process parameters according to the content herein. It should be particularly pointed out that all similar replacements and changes are obvious to those skilled in the art, and they are all regarded as included in the present application. The products and methods of the present application have been described by the preferred embodiments, and the relevant personnel can obviously modify or appropriately change and combine the products and methods described herein to realize and apply the present application technology without departing from the content, spirit and scope of the present application. In order to further understand the present application, the present application will be described in detail below in combination with the embodiments.

[0017] Embodiment 1

[0018] The formula of the LB liquid culture medium is as follows: 0.5 g of yeast powder, 1 g of peptone, 1 g of sodium chloride, and distilled water is added to make up to 100 mL, and sterilized at 121℃ for 20 minutes.

[0019] The LB solid culture medium is obtained by adding 1.5% agar to the liquid LB and sterilizing it to be ready for use.

[0020] The seed culture medium is as follows: 40 g / L of glucose, 10 g / L of yeast powder, 8 g / L of ammonium sulfate, 0.5 g / L of potassium dihydrogen phosphate, 0.5 g / L of potassium hydrogen phosphate , 50 mg / L of magnesium sulfate heptahydrate, 10 mg / L of ferrous sulfate heptahydrate, and 10 mg / L of manganese sulfate monohydrate.

[0021] Fermentation tank medium: glucose 50 g / L, yeast powder 20 g / L, potassium dihydrogen phosphate 1 g / L, dipotassium hydrogen phosphate 1 g / L, magnesium sulfate heptahydrate 100 mg / L, ferrous sulfate heptahydrate 10 mg / L, manganese sulfate monohydrate 10 mg / L, VB1 0.5 mg / L, VB2 0.5 mg / L, biotin 0.1 mg / L; set the final concentration of guar gum added to be 0, 0.1, 0.2, 0.4, 0.8, 1.6 (unit g / L) respectively. Take each raw material to dissolve in water to configure the above final concentration, and autoclave.

[0022] Attached carrier: porous ceramic particles (Ceram Tec, Germany), diameter 1.5-2.0 mm, pore volume 0.2 ml / g, particle density 1.26 g / cm3.

[0023] Take out the Flavobacterium ATCC14067, recover, inoculate into LB liquid medium, shake culture at 37℃, 100 rpm for 24 hours, spread on LB solid medium, culture at 37℃ for 24 hours, then pick up the vigorous colonies, inoculate into a flask containing LB liquid medium, shake culture at 37℃, 100 rpm until the concentration is 1×10 7 CFU / mL, then inoculate into a 10L seed tank containing 4L seed medium at an inoculation amount of 3% for seed expansion culture, culture for 48 hours to obtain seed liquid. Add porous ceramic particles to the fermentation tank medium at a ratio of 10g:1L, then inoculate the seed liquid into the fermentation tank medium at an inoculation amount of 5% for fermentation culture, control the temperature at 36℃, the stirring speed at 300 rpm, and the aeration ratio at 0.8-1.0 VVm, use antifoam to foam, and the fermentation time is 48h. Stop fermentation and collect the fermentation broth; during the fermentation culture, control the sugar content to be not less than 1% by feeding 50% glucose solution, and control the pH to be 7.0 by feeding ammonia water until the end of fermentation.

[0024] Detect the yield of L-threonine in the fermentation broth to verify the correlation between threonine yield and guar gum.

[0025] Table 1

[0026] Set three groups of parallel tests, take the control as the benchmark, use SPSS 16.0 software for single factor analysis of variance, P<0.05 indicates significant difference, P>0.05 indicates no significant difference, mark P<0.05 as *.

[0027] As can be seen from Table 1, similar to the results of the small-scale study, guar gum can improve the production of threonine by promoting the formation of B. flavum biofilm. The difference is that in the small-scale culture study, the optimal concentration of guar gum is 0.2 g / L, while in the fermenter culture, the optimal concentration in the culture medium is 0.8 g / L.

[0028] Example 2

[0029] The formula of LB liquid medium is: 0.5 g of yeast powder, 1 g of peptone, 1 g of sodium chloride, and distilled water to 100 mL, 121 ℃ high pressure sterilization for 20 minutes.

[0030] LB solid medium: add 1.5% agar to the liquid LB, pour flat after sterilization.

[0031] Seed medium: glucose 40 g / L, yeast powder 10 g / L, ammonium sulfate 8 g / L, potassium dihydrogen phosphate 0.5 g / L, dipotassium hydrogen phosphate 0.5 g / L, magnesium sulfate heptahydrate 50 mg / L, ferrous sulfate heptahydrate 10 mg / L, manganese sulfate monohydrate 10 mg / L.

[0032] Fermenter medium: glucose 50 g / L, yeast powder 20 g / L, potassium dihydrogen phosphate 1 g / L, dipotassium hydrogen phosphate 1 g / L, magnesium sulfate heptahydrate 100 mg / L, ferrous sulfate heptahydrate 10 mg / L, manganese sulfate monohydrate 10 mg / L, VB1 0.5 mg / L, VB2 0.5 mg / L, biotin 0.1 mg / L; set the final concentration of epigallocatechin gallate (EGCG) to be added to 0, 0.1, 0.2, 0.4, 0.8, 1.6 (unit mg / L) respectively. Take each raw material to dissolve in water to configure the above final concentration, high pressure sterilization.

[0033] Attached carrier: porous ceramic particles (Ceram Tec, Germany), diameter 1.5-2.0 mm, pore volume 0.2 ml / g, particle density 1.26 g / cm3.

[0034] Take out B. flavum ATCC14067, recover, inoculate into LB liquid medium, 37℃, 100 rpm shaking incubator, cultivate for 24 hours, spread on LB solid medium, cultivate at 37℃ for 24 hours, pick up vigorous colonies, inoculate into a flask containing LB liquid medium, cultivate at 37℃, 100 rpm shaking incubator until the concentration is 1×10 7CFU / mL, and then inoculated into a 10 L seed tank containing 4 L of seed medium at an inoculation amount of 3% for seed expansion culture, and cultured for 48 h to obtain a seed liquid. Porous ceramic particles were added to the fermentation tank medium at a ratio of 10 g: 1 L, and the seed liquid was inoculated into the fermentation tank medium at an inoculation amount of 5% for fermentation culture, with the temperature controlled at 36°C, the stirring speed at 300 rpm, the aeration ratio at 0.8-1.0 VVm, and the antifoam agent at 0.1 mL / L. The fermentation time was 48 h, and the fermentation was stopped, and the fermentation liquid was collected. During the fermentation culture, the sugar content was controlled to be no less than 1% by feeding 50% glucose solution, and the pH was controlled to be 7.0 by feeding ammonia water until the end of the fermentation.

[0035] The L-threonine yield in the fermentation liquid was detected to verify the correlation between the threonine yield and EGCG.

[0036] Table 2

[0037] Three groups of parallel tests were set up, and the control was used as a benchmark. Single factor variance analysis was performed using SPSS 16.0 software, and P<0.05 indicated significant difference, and P>0.05 indicated no significant difference. P<0.05 was marked as *.

[0038] As can be seen from Table 2, similar to the results of the small-scale study, EGCG can improve the yield of threonine by promoting the formation of Brevibacterium flavum biofilm.

[0039] Example 3

[0040] The combination of the two was used to promote the synthesis efficiency of threonine in large-scale production. In combination with Example 1 and Example 2, the addition concentration of guar gum was selected as 0.8 g / L, and the addition concentration of EGCG was selected as 0.4 mg / L for subsequent experiments. Both were not added as a control group. The groups of guar gum and EGCG were set up for single use and combination, and the carrier control group without adding porous ceramic particles was set up. The experimental procedure was the same as Example 1. The L-threonine yield detection results are shown in Table 3.

[0041] Table 3

[0042] P<0.05 indicates significant difference, P>0.05 indicates no significant difference. Compared with the control group, P<0.05 marked a, compared with guar gum group, P<0.05 marked b, compared with EGCG group, P<0.05 marked c. As can be seen from table 3, the addition of guar gum and epigallocatechin gallate in the fermentor medium can synergistically improve the yield of L-threonine; It should be noted that the adhesion carrier is a necessary condition, if the porous ceramic particle carrier is not added, guar gum or / and epigallocatechin gallate cannot effectively promote the improvement of L-threonine yield.

[0043] The above description is only the preferred embodiment of the present application, not any form of limitation on the present application, although the present application has been disclosed as above with the preferred embodiment, however, any skilled person in the art, without departing from the technical solution range of the present application, of course, will use the disclosed technical content to make some changes or modifications, become equivalent embodiments of equivalent changes, but as long as it does not deviate from the technical solution of the present application, according to the technical essence of the present application, any simple modification, equivalent change and modification of the above embodiments, all belong to the scope of the technical solution of the present application.

Claims

1. A process for the production of threonine by fermentation on a large scale, characterized in that, The process comprises the following steps: 1) strain recovery, 2) shake flask culture, 3) seed expansion culture, 4) fermenter culture; the strain adhesion carrier is added in the fermenter culture.

2. The process according to claim 1, characterized in that, The strain adhesion carrier is porous ceramic particles.

3. Process according to claim 1 or 2, characterized in that, The fermenter culture medium used in the fermenter culture contains guar gum or / and EGCG.

4. The process of claim 3, wherein, The components of the fermenter culture medium are: glucose 50 g / L, yeast powder 20 g / L, potassium dihydrogen phosphate 1 g / L, dipotassium hydrogen phosphate 1 g / L, magnesium sulfate heptahydrate 100 mg / L, ferrous sulfate heptahydrate 10 mg / L, manganese sulfate monohydrate 10 mg / L, VB1 0.5 mg / L, VB2 0.5 mg / L, biotin 0.1 mg / L, guar gum 0.8 g / L.

5. The process of claim 3, wherein, The components of the fermenter culture medium are: glucose 50 g / L, yeast powder 20 g / L, potassium dihydrogen phosphate 1 g / L, dipotassium hydrogen phosphate 1 g / L, magnesium sulfate heptahydrate 100 mg / L, ferrous sulfate heptahydrate 10 mg / L, manganese sulfate monohydrate 10 mg / L, VB1 0.5 mg / L, VB2 0.5 mg / L, biotin 0.1 mg / L, EGCG 0.4 mg / L.

6. The process of claim 3, wherein, The components of the fermenter culture medium are: glucose 50 g / L, yeast powder 20 g / L, potassium dihydrogen phosphate 1 g / L, dipotassium hydrogen phosphate 1 g / L, magnesium sulfate heptahydrate 100 mg / L, ferrous sulfate heptahydrate 10 mg / L, manganese sulfate monohydrate 10 mg / L, VB1 0.5 mg / L, VB2 0.5 mg / L, biotin 0.1 mg / L, guar gum 0.8 g / L, EGCG 0.4 mg / L.

7. The process of claim 3, wherein, The addition amount of the porous ceramic particles is 5-20 g per liter of the fermenter culture medium.

8. Process according to any one of claims 1 to 7, characterized in that, The components of the seed culture medium used in the seed expansion culture are: glucose 40 g / L, yeast powder 10 g / L, ammonium sulfate 8 g / L, potassium dihydrogen phosphate 0.5 g / L, dipotassium hydrogen phosphate 0.5 g / L, magnesium sulfate heptahydrate 50 mg / L, ferrous sulfate heptahydrate 10 mg / L, manganese sulfate monohydrate 10 mg / L.

9. Process according to any one of claims 1-7, characterized in that, The process comprises the following steps: 1) strain recovery: taking out threonine-producing Brevibacterium flavum for recovery; 2) Shaking culture: inoculate into LB liquid medium, 37°C, 100 rpm shaking culture for 24 hours, then spread on LB solid medium, 37°C culture for 24 hours, then pick up the vigorous colonies, inoculate into the flask containing LB liquid medium, 37°C, 100 rpm shaking culture until the concentration is 1 x 10 7 CFU / mL; 3) seed expansion culture: inoculating the shake flask culture liquid obtained in step 2) into a seed tank containing seed culture medium at a 3% inoculation amount for seed expansion culture, culturing for 48 hours to obtain seed liquid; 4) fermenter culture: adding porous ceramic particles into the fermenter culture medium at a ratio of 10 g: 1 L, then inoculating the seed liquid into the fermenter culture medium at a 5% inoculation amount for fermentation culture, controlling the temperature at 36℃, the stirring speed at 300 rpm, the aeration ratio at 0.8-1.0 VVm, using foam control agent to control the foam, and the fermentation time at 48 h, stopping the fermentation, and collecting the fermentation liquid.

10. The process of claim 9, wherein, In the fermenter culture process, 50% glucose solution is added to control the sugar content to be no less than 1%, and ammonia water is added to control the pH at 7.0.

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