Method for optimizing thalli by using alpha-ketoglutaric acid and producing inositol by fermentation

Abstract

The invention belongs to the technical field of microorganisms, and discloses a method for optimizing thalli by using alpha-ketoglutaric acid and producing inositol by fermentation, which comprises the following steps: carrying out gradient dilution on the thalli containing inositol monophosphate by using sterile water, then inoculating to a selective culture medium containing alpha-ketoglutaric acid, and culturing to obtain the inositol by using the alpha-ketoglutaric acid. The method comprises the following steps of: selecting uniform and single bacterial colonies with raised bacterial colonies and bacterial colony diameter of 2-3mm, transferring the bacterial colonies to a plate culture medium, shaking a flask and carrying out fermentation culture, regulating fermentation conditions by adding arabinose and sodium carbonate, and finally detecting the content of inositol by using HPLC (High Performance Liquid Chromatography) to screen. The method is simple, the high-yield strain screening efficiency is high, the obtained strain is high in stability, and the yield of inositol produced through fermentation is high.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a method for optimizing bacteria by utilizing alpha-ketoglutaric acid and fermenting to produce inositol. Background Art

[0002] Inositol (myo-inositol), also known as cyclohexanhexol, has a molecular formula of C6H12O6 and a molecular weight of 180. Almost all organisms contain inositol in free or bound form. Inositol itself does not directly promote the growth of culture, but it can help the active substances to play their role, improve the effect of vitamin B1, participate in carbohydrate metabolism, phospholipid metabolism and ion balance, thereby promoting the growth of culture and the formation of embryoids and buds.

[0003] The production method of inositol has hydrolysis, microbiological method, chemical synthesis and ion exchange method at present.Hydrolysis is the most important method of producing inositol, mainly with rice bran, corn bran etc. as raw material extraction, its production process is complicated, low in output, and large in environmental harm.Along with the development of biotechnology, utilizing biological method to produce inositol is more and more taken seriously, but the output of biological method is also relatively low at present, and disclosed patent CN116286923B provides a method for producing inositol and its special engineering bacteria, and bacterial strain preservation number is M20221796, and classification and naming is Escherichia coli (Escherichiacoli) BW25113, and its output reaches 53g / L after 96h fed-batch fermentation.

[0004] In the process of producing inositol by microbial fermentation, the screening and optimization of bacteria are key steps to improve the yield and stability of strains. At present, the screening methods commonly used in the industry are often inefficient, and the growth of bacteria and the yield of inositol during fermentation are not always ideal. Therefore, there is an urgent need for a method that can efficiently screen bacteria and optimize their fermentation performance to improve the production efficiency and quality of inositol. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a method for optimizing bacteria using α-ketoglutaric acid and producing inositol by fermentation, wherein an α-ketoglutaric acid-containing culture medium is used to screen and optimize Escherichia coli containing inositol monophosphatase, and the fermentation process is optimized, the efficiency of screening high-yield strains is high, the obtained strains have strong stability, and the yield of inositol produced by fermentation is high.

[0006] In order to solve the above technical problems, the technical solution of the present invention is:

[0007] The method of optimizing bacteria by using α-ketoglutarate and fermenting to produce inositol comprises the following steps:

[0008] a. The bacteria containing inositol monophosphatase are gradiently diluted with sterile water to obtain a dilution;

[0009] b. The dilution in step a was inoculated onto a selective medium, wherein the selective medium formula was 5 g / L yeast powder, 10 g / L peptone, 10 g / L sodium chloride, 20 g / L agar powder, 2 g / L inositol, 1 g / L α-ketoglutaric acid, and the rest was deionized water, with a natural pH value. When the temperature dropped to 40 to 50 ° C, chloramphenicol at a final concentration of 50 mg / L and ampicillin at a final concentration of 25 mg / L were added; and cultured at 37 ° C for 20 to 30 h;

[0010] c. In step b, the colonies cultured on the medium with darker and uniform color, raised colonies, and a colony diameter of 2 to 3 mm are selected and inoculated on a plate culture medium, wherein the culture medium formula is 5 g / L yeast powder, 10 g / L peptone, 10 g / L sodium chloride, 20 g / L agar powder, and the rest is deionized water, and the pH value is natural; culture at a constant temperature of 37°C for 10 to 18 hours;

[0011] d. A portion of the colony obtained by culture in step c was used for preservation, and a portion was inoculated into a shake flask containing 100 ml of liquid culture medium, wherein the liquid culture medium was formulated as 5 g / L yeast powder, 10 g / L peptone, 10 g / L sodium chloride, and the rest was deionized water, and the pH value was natural; the culture was cultured at 37 ° C and 220 to 260 rpm until the OD600 value of the bacterial solution was 1.6 to 2.0;

[0012] e. The single colony cultured in the shake flask in step d was inoculated into 1L fermentation medium at an inoculum size of 5%, and cultured at 37°C. After culturing for 10 hours, arabinose with a final concentration of 2g / L was added. After culturing for 20 hours, α-ketoglutaric acid with a final concentration of 2.0mM was added. After culturing for 36 hours, 1g / L arabinose was added again. The fermentation was terminated after 72 hours. During this period, the ventilation volume was maintained at 1vvm, the rotation speed was set to 600rpm, the dissolved oxygen was linked to the feed, and the feed was started when the DO was greater than 30%. A 25% mass concentration sodium carbonate solution was used to adjust the pH value and maintain it at 6.8-7.2; HPLC was used to detect the inositol content, and the strain with high yield was selected.

[0013] Preferably, the gradient dilution in step a is from 10 -1 to 10 -8 .

[0014] Preferably, the step b is cultured at 37° C. for 24 hours.

[0015] Preferably, in step c, the colony morphology is blue, the colonies are raised, and the colony diameter is 2 to 3 mm; and the culture is carried out at a constant temperature of 37° C. for 16 hours.

[0016] Preferably, in step d, the colony morphology is blue, the colonies are raised, and the colony diameter is 2 to 3 mm; the culture is carried out at a constant temperature of 240 rpm until the OD600 value of the bacterial solution is 1.8.

[0017] Preferably, the components of the feeding solution used for feeding in step e are 500 g / L glucose, 100 g / L glycerol, and the rest is water.

[0018] Preferably, the components of the fermentation medium in step e are glucose 5g / L; glycerol 10g / L; potassium dihydrogen phosphate 7g / L; magnesium sulfate heptahydrate 2g / L; ammonium sulfate 2g / L; yeast powder 5g / L; citric acid 2g / L; defoamer PPE 0.5mL / L; trace element mother solution 1mL / L; add water to make up to 1L; wherein the components of the trace element mother solution are: weigh FeCl3 5g, CoCl·6H2O2.5g, MnCl2·4H2O 0.15g, CuCl2·2H2O1.5g, H3BO3 3g, NaMnO4·2H2O 2.5g, Zn(CH3COO)2·2H2O 13g, and add water to make up to 1L.

[0019] Due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0020] The present invention: 1. By introducing alpha-ketoglutaric acid as a screening factor in a selection medium, a bacterial cell with high fermentation performance is successfully screened out; 2. During the fermentation process, by accurately controlling the addition timing and concentration of arabinose and alpha-ketoglutaric acid, alpha-ketoglutaric acid is used to accelerate the circulation and metabolic process of the bacterial cell, while continuously enhancing the stimulation of arabinose on bacterial inositol monophosphatase related genes, promoting the expression of the genes, increasing the amount of inositol monophosphatase, and significantly improving the growth rate of the bacterial cell and the yield of inositol; 3. Using a sodium carbonate solution with a mass concentration of 25% as a pH value regulator not only solves the problem of high nitrogen element in wastewater and difficult wastewater treatment caused by the use of ammonia water to adjust the pH value during conversion due to the lack of nitrogen consumption in the process, but also can significantly improve the yield of inositol; 4. The strain yield obtained by the method reaches more than 70g / L, which is significantly improved compared with 53g / L in the prior art.

[0021] In conclusion, the method of the present invention is simple, has high efficiency in screening high-yield strains, has strong stability of the obtained strains, and has high yield of inositol produced by fermentation. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 HPLC chart of the fermentation product of strain A113 in Example 1 of the present invention;

[0023] Figure 2 This is the HPLC chart of inositol standard. DETAILED DESCRIPTION

[0024] The technical solution of the present invention is further described below in conjunction with embodiments:

[0025] Embodiment 1:

[0026] a. Dilute the cells containing inositol monophosphatase with sterile water in a gradient dilution from 10 -1 to 10 -8 Get the dilution.

[0027] b. Inoculate the dilution in step a onto the selection medium, the selection medium formula is 5g / L yeast powder, 10g / L peptone, 10g / L sodium chloride, 20g / L agar powder, 2g / L inositol, 1g / L α-ketoglutaric acid, and the rest is deionized water, the pH value is natural, and when the temperature drops to 40-50°C, add chloramphenicol at a final concentration of 50mg / L and ampicillin at a final concentration of 25mg / L; and culture at a constant temperature of 37°C for 24h.

[0028] c. Select the colonies cultured on the culture medium in step b, which are blue in color, have raised colonies, and have a colony diameter of 2 to 3 mm, and inoculate them on a plate culture medium. The culture medium formula is 5 g / L yeast powder, 10 g / L peptone, 10 g / L sodium chloride, 20 g / L agar powder, and the rest is deionized water, with a natural pH value; culture at a constant temperature of 37°C for 16 hours.

[0029] d. A portion of the blue colonies with raised colonies and a colony diameter of 2 to 3 mm obtained by culturing in step c was used for preservation, and a portion was inoculated into a shake flask containing 100 ml of liquid culture medium, wherein the liquid culture medium formula was 5 g / L yeast powder, 10 g / L peptone, 10 g / L sodium chloride, and the rest was deionized water, and the pH value was natural; the culture was constant temperature cultured at 37°C and 240 rpm until the OD600 value of the bacterial solution was 1.8; the preserved colonies were named A101, A102, A103........A115 in sequence.

[0030] e. Prepare 1L of feeding solution, the feeding solution components of which are 500g / L glucose, 100g / L glycerol, and the rest is water; inoculate the single colonies cultured in the shake flask in step d into 1L of fermentation medium at an inoculum size of 5% and culture at 37°C; the components of the fermentation medium are 5g / L glucose; 10g / L glycerol; 7g / L potassium dihydrogen phosphate; 2g / L magnesium sulfate heptahydrate; 2g / L ammonium sulfate; 5g / L yeast powder; 2g / L citric acid; 0.5mL / L defoamer PPE; 1mL / L trace element mother solution; add water to make up to 1L. The components of the trace element mother solution are: weigh 5g FeCl3, 2.5g CoCl·6H2O, 0.15g MnCl2·4H2O, 1.5g CuCl2·2H2O, 33g H3BO3, 2.5g NaMnO4·2H2O, and 13g Zn(CH3COO)2·2H2O, and add water to make up to 1L.

[0031] After 10 hours of cultivation, arabinose was added at a final concentration of 2 g / L. After 20 hours of cultivation, α-ketoglutarate was added at a final concentration of 2.0 mM. After 36 hours of cultivation, 1 g / L arabinose was added again. The fermentation was terminated after 72 hours. During this period, the ventilation volume was maintained at 1 vvm and the rotation speed was set to 600 rpm. The dissolved oxygen was linked to the feeding. Feeding was started when DO was greater than 30%. During the fermentation process, 25% sodium carbonate solution was used to adjust the pH value and maintain it at 6.8-7.2.

[0032] After the fermentation, the inositol content was detected by HPLC (chromatographic column: Alltech amino column (4.6 mm×250 mm), mobile phase: acetonitrile: water = 75:25, flow rate: 1 mL / min, column constant temperature: 30°C, detector: differential detector, injection volume: 10 μL). The yield of each strain is shown in Table 1.

[0033] Embodiment 2:

[0034] α-Ketoglutarate was not added during the screening culture medium and fermentation process. The remaining components and operations were the same as in Example 1. Ten single colonies were screened out by this screening method and named A201 to A210. The yield of each strain after fermentation is shown in Table 1.

[0035] Embodiment 3:

[0036] The screening process was the same as that in Example 1, and 10 single colonies were screened out by the screening method and named A301 to A310; during the fermentation process, arabinose was not added before and after the addition of α-ketoglutaric acid, or 2 g / L arabinose was added only once, wherein A301 before, A302 before, A303 before, A304 before, and A305 before were experimental examples of adding arabinose with a final concentration of 2 g / L after 10 h of culture; A306 after, A307 after, A308 after, A309 after, and A310 after were experimental examples of adding arabinose with a final concentration of 2 g / L after 36 h of culture; A301 without, A302 without, A303 without, A304 without, and A305 without were experimental examples of not adding arabinose with a final concentration of 2 g / L after 10 h and 36 h of culture. The yield of each strain after fermentation is shown in Table 1.

[0037] The fermentation yields of each strain in Examples 1-3 are shown in Table 1:

[0038] Table 1 Fermentation yield of each strain in Examples 1-3

[0039]

[0040]

[0041] From the above results, it can be seen that strains A304, A102, A103, A104, A106, A107, A111, A113, A114, and A115 have higher yields.

[0042] Example 4: Stability test

[0043] The A304, A102, A103, A104, A106, A107, A111, A113, A114, and A115 strains were subjected to genetic stability experiments, and the results showed that the yield of A304, A113, and A103 remained unchanged after 50 generations, proving that they can be used for industrial production.

[0044] Embodiment 5:

[0045] The inositol fermentation was carried out using the genetically stable strain A113 in Example 4, and the pH of the fermentation liquid was regulated using 25% mass concentration of sodium carbonate solution, 20% mass concentration of sodium bicarbonate solution, ammonia water, 10% mass concentration of potassium hydroxide solution, and 10% mass concentration of sodium hydroxide solution, respectively, and the remaining operations were the same as in Example 1. The final inositol yield is shown in Table 2.

[0046] Table 2 Effect of different pH regulators on inositol production

[0047] category Inositol yield g / L Alkali consumption g / L 25% Sodium Carbonate 72.4 3.2 20% Sodium Bicarbonate 70.3 4.8 ammonia 70.1 3.0 10% Potassium Hydroxide 65.3 5.7 10% Sodium Hydroxide 67.6 4.6

[0048] From the above results, it can be seen that 25% sodium carbonate solution has the best effect as a pH regulator; although sodium bicarbonate and ammonia water are similar to sodium carbonate in effect as pH regulators, the addition of ammonia water will cause the ammonia nitrogen content in the fermentation broth to increase, which has certain adverse effects on sewage treatment and the environment, and ammonia water is easy to volatilize and has high requirements for storage and transportation; although the use of sodium bicarbonate can reduce the ammonia nitrogen content in sewage and has low requirements for storage and transportation, its effect on promoting production is not significant, so the best choice is 25% sodium carbonate solution.

[0049] Result analysis:

[0050] The following conclusions were drawn from the analysis of the inositol yield of each strain in the example: 1. α-ketoglutaric acid can significantly affect the screening of high-yield strains and the yield of inositol. Not using α-ketoglutaric acid will reduce the final inositol yield, and the number of excellent strains that can be screened is relatively small; 2. The addition of arabinose also has a significant effect on the yield of inositol. If the addition of arabinose is not on time or is reduced, it will have a certain impact on the final inositol yield; therefore, accurately controlling the timing and concentration of adding arabinose and α-ketoglutaric acid is of great help in increasing the inositol yield; 3. The selection of pH regulator can also significantly affect the yield of inositol. The use of 25% sodium carbonate solution not only overcomes the defects of the prior art, but also significantly increases the yield of inositol.

[0051] It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope limited by the appended claims of the application.

Claims

1. A method for optimizing bacteria using α-ketoglutarate and fermenting to produce inositol, characterized in that: The following steps are involved: a. The bacteria containing inositol monophosphatase are gradiently diluted with sterile water to obtain a dilution; b. The dilution in step a was inoculated onto a selective medium, wherein the selective medium formula was 5 g / L yeast powder, 10 g / L peptone, 10 g / L sodium chloride, 20 g / L agar powder, 2 g / L inositol, 1 g / L α-ketoglutaric acid, and the rest was deionized water, with a natural pH value. When the temperature dropped to 40 to 50 ° C, chloramphenicol at a final concentration of 50 mg / L and ampicillin at a final concentration of 25 mg / L were added; and cultured at 37 ° C for 20 to 30 h; c. In step b, the colonies cultured on the medium with darker and uniform color, raised colonies, and a colony diameter of 2 to 3 mm are selected and inoculated on a plate culture medium, wherein the culture medium formula is 5 g / L yeast powder, 10 g / L peptone, 10 g / L sodium chloride, 20 g / L agar powder, and the rest is deionized water, and the pH value is natural; culture at a constant temperature of 37°C for 10 to 18 hours; d. A portion of the colony obtained by culture in step c was used for preservation, and a portion was inoculated into a shake flask containing 100 ml of liquid culture medium, wherein the liquid culture medium was formulated as 5 g / L yeast powder, 10 g / L peptone, 10 g / L sodium chloride, and the rest was deionized water, and the pH value was natural; the culture was cultured at 37 ° C and 220 to 260 rpm until the OD600 value of the bacterial solution was 1.6 to 2.0; e. The single colony cultured in the shake flask in step d was inoculated into 1L fermentation medium at an inoculum size of 5%, and cultured at 37°C. After culturing for 10 hours, arabinose with a final concentration of 2g / L was added. After culturing for 20 hours, α-ketoglutaric acid with a final concentration of 2.0mM was added. After culturing for 36 hours, 1g / L arabinose was added again. The fermentation was terminated after 72 hours. During this period, the ventilation volume was maintained at 1vvm, the rotation speed was set to 600rpm, the dissolved oxygen was linked to the feed, and the feed was started when the DO was greater than 30%. A 25% mass concentration sodium carbonate solution was used to adjust the pH value and maintain it at 6.8-7.2; HPLC was used to detect the inositol content, and the strain with high yield was selected.

2. The method for optimizing bacteria using α-ketoglutarate and fermenting to produce inositol as claimed in claim 1, characterized in that: The gradient dilution in step a is from 10 -1 to 10 -8 .

3. The method for optimizing bacteria using α-ketoglutarate and fermenting to produce inositol as claimed in claim 1, characterized in that: In step b, the mixture is cultured at a constant temperature of 37° C. for 24 hours.

4. The method for optimizing bacteria using α-ketoglutarate and fermenting to produce inositol as claimed in claim 1, characterized in that: In the step c, the colonies are blue in morphology, convex in colonies, and have a colony diameter of 2 to 3 mm; and the colonies are cultured at a constant temperature of 37° C. for 16 hours.

5. The method for producing inositol by fermentation using α-ketoglutarate to optimize bacteria as claimed in claim 1, characterized in that: In the step d, the colony morphology is blue, the colony is raised, and the colony diameter is 2 to 3 mm; the culture is carried out at a constant temperature of 240 rpm until the OD600 value of the bacterial solution is 1.

8.

6. The method for optimizing bacteria using α-ketoglutarate and fermenting to produce inositol as claimed in claim 1, characterized in that: The components of the feeding solution used for feeding in step e are 500 g / L of glucose, 100 g / L of glycerol, and the rest is water.

7. The method for producing inositol by fermentation using α-ketoglutarate to optimize bacteria as claimed in claim 1, characterized in that: The components of the fermentation medium in step e are glucose 5g / L; glycerol 10g / L; potassium dihydrogen phosphate 7g / L; magnesium sulfate heptahydrate 2g / L; ammonium sulfate 2g / L; yeast powder 5g / L; citric acid 2g / L; defoamer PPE 0.5mL / L; trace element mother solution 1mL / L; add water to make up to 1L; wherein the components of the trace element mother solution are: weigh FeCl3 5g, CoCl·6H2O 2.5g, MnCl2·4H2O0.15g, CuCl2·2H2O1.5g, H3BO3 3g, NaMnO4·2H2O 2.5g, Zn(CH3COO)2·2H2O 13g, add water to make up to 1L.

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