A method for fermentative production of 2-keto-L-gulonic acid

Abstract

The present invention provides a method for fermentatively producing 2-keto-L-gulonic acid, which includes using a culture medium substantially free of sodium source during the fermentation process, adjusting the total nitrogen content of the fermentation medium used at the start of fermentation, using an ammonia-containing basic substance to adjust the pH value during the fermentation process, adjusting the dosage ratio of ammonia in sorbose and the ammonia-containing basic substance, etc. The method of the present invention can reduce the generation of sodium chloride waste salt, shorten the fermentation cycle, reduce the production cost, improve the synthesis efficiency of 2-keto-L-gulonic acid, etc.

Description

Technical Field

[0001] The present invention provides a method for fermentatively producing 2-keto-L-gulonic acid, which includes using a culture medium substantially free of sodium source during the fermentation process, and using ammonia-containing alkaline substances to adjust the pH value during the fermentation process, etc., so as to achieve the production purpose of reducing the generation of sodium chloride waste salt, being environmentally friendly, and reducing production costs. Background Art

[0002] Gulonic acid is an important precursor for the synthesis of vitamin C. Currently, the two-step fermentation method (Patent Document 1) is mainly used for the fermentative production of 2-keto-L-gulonic acid (2-KGA for short). In this two-step fermentation method, microorganisms are first used to ferment to produce sodium gulonate, and then the fermentation broth is subjected to solid-liquid separation by ultrafiltration to remove the thalli to obtain a sodium gulonate ultrafiltrate. Then, gulonic acid is obtained through ion exchange, and a large amount of sodium chloride is generated at the same time. Finally, 2-KGA crystals are obtained through crystallization (Patent Document 2).

[0003] In the above-mentioned gulonic acid production process, in order to maintain the pH environment for efficient acid production by the two-step bacteria, it is usually necessary to add an alkali for neutralization to convert the generated gulonic acid into gulonate. Currently, the alkali used in gulonic acid fermentation is usually sodium hydroxide. The sodium chloride waste salt comes from two parts. One part is the sodium hydroxide introduced during the two-step fermentation process, which is converted into sodium chloride in the ion exchange process of post-extraction. The other part is the sodium hydroxide used for alkali regeneration of the resin in the ion exchange process. This part of sodium hydroxide generates sodium chloride waste salt after being neutralized with hydrochloric acid.

[0004] Currently, there are two methods for treating sodium chloride waste salt. One is to landfill it as solid waste after environmental protection treatment. This method is simple but causes relatively large environmental pollution, and this method is currently adopted in the industry. The other is to extract sodium chloride as a by-product through crystallization. The equipment investment and subsequent operating costs of this method are both relatively large, significantly increasing the production cost of 2-KGA.

[0005] Prior Art Documents:

[0006] Patent Document 1: CN106011043B

[0007] Patent Document 2: CN112028766A Summary of the Invention

[0008] Problems to be Solved by the Invention

[0009] In the existing process for fermentatively producing 2-KGA, there are problems such as a large amount of sodium chloride waste salt generation, relatively large environmental pollution, and high post-treatment costs.

[0010] Solutions for Solving the Problems

[0011] The present invention solves the above problems by using a substance substantially free of sodium in the gluconic acid fermentation process, adjusting the total nitrogen content of the initial medium, adjusting the pH value during the fermentation process with an ammonia-containing basic substance, and controlling the dosage ratio of ammonia in the fed sorbose and the ammonia-containing basic substance.

[0012] Specifically, in order to control the source of sodium in the fermentation process and reduce the generation of sodium chloride waste salt in the process, the present invention preferably uses a medium "substantially free of sodium source".

[0013] "Substantially free of sodium source" means that the medium does not use substances from sodium sources, such as sodium salts like sodium sulfate, sodium chloride, sodium bicarbonate, sodium hydrogen carbonate, etc.

[0014] Previous fermentation processes also used ammonia-containing basic substances to replace sodium-containing basic substances to adjust the pH value during the fermentation process. Although it can reduce the generation of sodium chloride waste salt in the fermentation and separation and extraction processes and avoid the post-treatment process of sodium chloride waste salt, there are the following problems:

[0015] 1) Since the ammonia-containing basic substance is used to replace the sodium-containing basic substance, the keto group in sorbose is converted into an amino group in gluconate, and the amino group will inhibit the reaction, resulting in an extended fermentation cycle.

[0016] 2) Excessive nitrogen source leads to a longer fermentation cycle, and simply reducing the initial nitrogen source content cannot completely eliminate the influence, and the fermentation cycle is still extended.

[0017] The inventor of the present invention found through research that by adjusting the total nitrogen content of the initial medium during the fermentation process and controlling the dosage ratio of ammonia in the fed sorbose and the ammonia-containing basic substance during the fermentation process, the extension of the fermentation cycle caused by the ammonia-containing basic substance can be inhibited.

[0018] The nitrogen in the initial medium mainly comes from organic nitrogen sources, and the total nitrogen content is in the range of 0.35 g / L to 1.05 g / L. More preferably, the total nitrogen content is 0.50 g / L to 0.80 g / L to promote the growth of the bacterial cells, obtain a higher yield of 2-KGA, and shorten the fermentation cycle.

[0019] As the organic nitrogen source, nitrogen-containing organic substances commonly used in the art can be used. Preferably, it can be one or more of corn steep liquor, peptone, yeast powder, yeast extract, and urea. From the perspective of reducing costs and convenient access, corn steep liquor is more preferably used. If cost is not considered, from the perspective of increasing the yield of 2-KGA, yeast powder is preferably used as the organic nitrogen source. The content of corn steep liquor is preferably 10.0 to 30.0 g / L, and from the perspective of promoting the growth of the bacterial cells and obtaining a higher yield of 2-KGA, the content of corn steep liquor is more preferably 15.0 to 25.0 g / L.

[0020] The fermentation medium used in the fermentation process further contains a carbon source and inorganic salts.

[0021] The method for producing 2-KGA in this application belongs to the sorbose pathway, so sorbose is used as the carbon source.

[0022] The inorganic salts can be the inorganic salts commonly used in the art. For example, phosphates, sulfates, etc. The phosphate can be the phosphate commonly used in the art, preferably the potassium salt of phosphoric acid, more preferably potassium dihydrogen phosphate. The sulfate can be the sulfate commonly used in the art, preferably magnesium sulfate.

[0023] Preferably, the fermentation medium used in the fermentation process of this application contains (g / L): L-sorbose 20.0 - 60.0, corn steep liquor 10.0 - 30.0, potassium dihydrogen phosphate 1.0 - 3.0, anhydrous magnesium sulfate 0.1 - 0.5, pH 6.0 - 8.0.

[0024] Preferably, in the initial stage after the start of fermentation, ammonia water is added dropwise to control the pH to 6.8 - 7.2. When the sorbose concentration drops to 10 - 20 g / L, ammonia water is added dropwise to control the pH to 7.0 - 7.6. At the same time, sorbose is added dropwise to control the molar ratio of sorbose to ammonia in ammonia water to 1 - 1.2:1. When the content of 2-KGA in the fermentation broth is greater than 90 mg / g, adding sugar is stopped, and when the residual sugar concentration is lower than 0.05%, the fermentation ends.

[0025] Preferably, during the fermentation process, the temperature is controlled at 27 - 33 °C, the tank pressure is 0.02 - 0.06 MPa, the ventilation rate is 0.8 - 1.2 vvm, and the stirring speed is 300 - 500 rpm. Regarding the stirring speed, an air-lift fermenter with the same stirring effect can also be used instead.

[0026] The ammonia-containing basic substance can be selected from one or more of ammonia water, liquid ammonia, ammonium bicarbonate, and its concentration and dosage can be adjusted according to the pH value and residual sugar concentration required during the fermentation process. For example, the concentration of ammonia water used to adjust the pH value can be 15 - 35%. Preferably, from the perspective of facilitating the prevention of bacterial contamination, the concentration of ammonia water is 20% - 30%. If ammonium bicarbonate is used, its concentration is preferably 1 - 8%, more preferably 2.5 - 6.5%. The concentration of the ammonia-containing basic substance within the preferred range can quickly and efficiently adjust the pH value during the fermentation process to the range of 6.5 - 7.5, reducing the inhibitory reaction caused by the generation of amines. Generally, without adjusting the pH of the fermentation broth, being slightly alkaline is more conducive to the production of 2-KGA.

[0027] Further, to inhibit the prolongation of the fermentation cycle caused by ammonia-containing basic substances, the molar ratio of the amount of sorbose added during the fermentation process to the amount of ammonia in the ammonia-containing basic substance is preferably between 1:1 and 1.2:1. If this molar ratio is too large or too small, it will affect the reaction cycle and make the cycle longer. Excessive ammonia will cause the reaction to stagnate and make fermentation difficult to proceed.

[0028] The inventors of the present invention also found that when adding sorbose and ammonia-containing basic substances during the fermentation process, it is preferably to end the fermentation when the content of 2-KGA in the fermentation broth is greater than 90 mg / g and the residual sugar concentration is lower than 0.05%, so as to greatly improve the yield of 2-KGA within a relatively short fermentation cycle. When the residual sugar concentration is greater than 0.05%, ending the fermentation prematurely will reduce the yield of 2-KGA.

[0029] The present invention uses a seed solution of a mixed bacterial system including small bacteria and associated bacteria, and inoculates it into a culture medium for fermentative production of 2-KGA. The mixed bacterial system including small bacteria and associated bacteria is not limited, as long as 2-KGA can be obtained through this mixed bacterial system when using the culture medium of the present invention. The small bacteria include strains commonly used in the art, preferably Ketogulonigenium vulgare, that is, ketogulonic acid bacteria. The associated bacteria include strains commonly used in the art, preferably Bacillus megaterium, Bacillus cereus, Bacillus subtilis, or Bacillus thuringiensis, etc.

[0030] Effects of the Invention

[0031] The method of the present application can significantly reduce the alkali cost, reduce the generation of sodium chloride waste salt, and produce 2-KGA with high yield and in a green and environmentally friendly manner within a relatively short fermentation cycle. Detailed Embodiments

[0032] The method of the present application will be described in detail below.

[0033] The strains, culture medium, mixed bacterial system, fermentation culture conditions, separation and purification conditions, and product determination methods used in the method of the present application can be obtained or implemented in the following ways.

[0034] <Source of Strains>

[0035] The minor bacterium and the associated bacterium of the mixed bacterial system used in the following examples are as follows: the minor bacterium is Ketogulonigenium vulgare (formerly named Gluconobacter oxydans, the preservation number of this Gluconobacter oxydans is CGMCC NO.1.110, purchased from China General Microbiological Culture Collection Center, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, postal code 100101); the associated bacterium is Bacillus megaterium (preservation number is CGMCC NO.1.432, purchased from China General Microbiological Culture Collection Center, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, postal code 100101).

[0036] <Preparation of culture medium>

[0037] The components of the culture medium of the present invention can use commonly used materials in the art and are prepared by methods well known in the art.

[0038] The composition of each culture medium is as follows:

[0039] 1). Solid culture medium (g / L): L-sorbose 60.0 - 80.0, corn steep liquor 4.0 - 6.0, urea 11.0 - 13.0, potassium dihydrogen phosphate 0.5 - 1.5, anhydrous magnesium sulfate 0.1 - 0.2, calcium carbonate 4.0 - 6.0, agar 15 - 20, pH 6.0 - 8.0.

[0040] 2). Liquid culture medium (g / L): L-sorbose 80.0 - 120.0, corn steep liquor 4.0 - 6.0, urea 11.0 - 13.0, potassium dihydrogen phosphate 0.5 - 1.5, anhydrous magnesium sulfate 0.1 - 0.2, calcium carbonate 4.0 - 6.0, pH 6.0 - 8.0.

[0041] 3). Fermentation culture medium (g / L): L-sorbose 20.0 - 60.0, corn steep liquor 10.0 - 30.0, potassium dihydrogen phosphate 1.0 - 3.0, anhydrous magnesium sulfate 0.1 - 0.5, pH 6.0 - 8.0.

[0042] 4). Sterilization conditions: Use conventional sterilization equipment, sterilize at 115°C for 25 min, and make the solid culture medium into a plate after sterilization.

[0043] Among them, L-sorbose can be replaced by sorbose mash solution produced by one-step fermentation and having the same quality of L-sorbose.

[0044] <Preparation of mixed bacterial system>

[0045] Inoculate the minor bacterium and the associated bacterium separately on a solid medium and culture them at 28 °C for 24 to 36 h. Then, prepare the suspensions of the minor bacterium and the associated bacterium on the solid medium respectively, and inoculate them into a liquid seed medium at an inoculation amount of 2%. Culture at 28 °C to 30 °C, with a stirring speed of 280 to 320 rpm and an aeration ratio of 0.8 to 1.2 vvm for 18 to 20 h to obtain a seed liquid of the mixed bacterial system. During the fermentation process, detect the residual sugar, 2-KGA content, pH value, and microscopically examine the cell morphology to obtain a seed liquid with better cell growth and a higher 2-KGA yield.

[0046] <Fermentation culture conditions>

[0047] Inoculate the seed liquid of the mixed bacterial system into a fermenter at an inoculation amount of 10% to 20% and perform fermentation using a 10 to 15 L fermenter. After the start of fermentation, add ammonia water to control the pH to 6.8 to 7.2, and the concentration of ammonia water is 15% to 35%. When the concentration of sorbose drops to 10 to 20 g / L, add ammonia water to control the pH to 7.0 to 7.6. At the same time, add sorbose and control the molar ratio of the amount of ammonia in sorbose and ammonia water to be 1:1 to 1.2:1. Stop adding sugar when the content of 2-KGA in the fermentation broth is greater than 90 mg / g, and end the fermentation when the residual sugar concentration is lower than 0.05%.

[0048] <Feed addition of sorbose>

[0049] The added sorbose can be pure sorbose or sorbose mash obtained by fermentation.

[0050] <Detection method>

[0051] 1) Determination of total nitrogen content

[0052] Use the Kjeldahl method to determine the total nitrogen content in the sample.

[0053] Digest the sample together with sulfuric acid and a catalyst by heating to decompose the protein. The decomposed ammonia combines with sulfuric acid to form ammonium sulfate, then alkalize and distill to separate ammonia, absorb it with boric acid, and then titrate with a standard solution of sulfuric acid or hydrochloric acid. The total nitrogen content is obtained by multiplying the acid consumption by the conversion factor.

[0054] Calculation formula:

[0055] Nitrogen content in the sample (%) = 0.014 × (A - B) × 100%

[0056] A ---- Milliliters of HCl solution consumed by the sample

[0057] B ---- Milliliters of HCl solution consumed by the blank

[0058] 2) Determination of 2-KGA content

[0059] Determination principle:

[0060] In a strong acid medium, 2-KGA is converted to VC through lactone enolization reaction. After measuring the content of VC by using the redox reaction between VC and iodine, the content of 2-KGA is calculated by conversion.

[0061] Determination method: Use a pipette to aspirate 2 mL of the fermentation broth as a sample into a test tube, add 2 mL of 7 mol / L sulfuric acid, shake well and heat in a boiling water bath for 25 min (2-KGA is lactonized to form VC). After taking it out and cooling, rinse it 3 - 4 times with purified water and collect it into a 250 mL Erlenmeyer flask. Add 3 mL of 0.5% starch solution, and titrate with 0.1 mol / L iodine solution until the blue color does not fade for 30 s (iodine can oxidize VC, and when VC is consumed completely, iodine reacts with starch to turn blue), which is the titration end point.

[0062] Calculation method:

[0063]

[0064] Where:

[0065] C---Concentration of iodine titrant (mol / L)

[0066] V---Volume of iodine titrant consumed by the sample (mL)

[0067] 0.9072--Molecular weight of VC / Molecular weight of 2-KGA

[0068] 8.806---Each 1 mL of 0.05 mol / L iodine standard solution is equivalent to 8.806 mg of VC

[0069] A---Molar conversion rate of 2-KGA converted to VC (calculated as 63.10%)

[0070] B---Volume of fermentation broth aspirated (mL)

[0071] 0.05---Theoretical concentration of iodine standard solution

[0072] 3) Determination of residual sugar concentration

[0073] The anthrone method is used to determine the residual sugar in the fermentation broth. Specifically: Take 1 mL of the fermentation broth sample in a clean volumetric flask, add purified water to make up the volume and dilute to a certain multiple. Then aspirate 1 mL of the diluted solution into a clean and dry test tube, add 6 mL of anthrone solution, shake well with a vortex oscillator, and let it stand at room temperature for 20 min. The blank control is operated as above with purified water instead of the fermentation broth sample. Use a UV-visible spectrophotometer to measure the absorbance value at a wavelength of 620 nm, and then convert it to the residual sugar content. When the residual sugar content in the fermentation broth ≤ 0.05%, it is the fermentation end point.

[0074] To better describe the present invention, the following embodiments are provided for reference. It should be noted that the following embodiments are only used to describe the present invention, and the present invention is not limited thereto.

[0075] Example 1

[0076] 1) Prepare the fermentation medium (g / L): L-sorbose 40.0, corn steep liquor 20.0, potassium dihydrogen phosphate 2.0, anhydrous magnesium sulfate 0.2, adjust the pH to 7.0, sterilize at 115 °C for 25 min, and sterilize sorbose alone at 115 °C for 25 min. The total nitrogen content in the medium is 0.70 g / L. The total nitrogen content of the medium in the example is derived from corn steep liquor.

[0077] 2) Inoculate the seed liquid of the mixed bacterial system into a 15 L fermenter at an inoculation amount of 20%.

[0078] 3) After the start of fermentation, feed 25 wt% ammonia water to control the pH at 7.0. Control the temperature at 30 °C, the tank pressure at 0.04 Mpa, the ventilation rate at 1 vvm, and the stirring speed at 400 rpm. Sorbose is not fed in this stage.

[0079] 4) When the sorbose concentration drops to 15 g / L, feed 25 wt% ammonia water to control the pH at 7.3. At the same time, feed sorbose, control the molar ratio of sorbose to ammonia in ammonia water at 1.1:1, stop feeding sugar when the content of 2-KGA in the fermentation broth is greater than 90 mg / g, and end the fermentation when the residual sugar concentration is lower than 0.05%.

[0080] Example 2

[0081] Produce 2-KGA according to the method of Example 1, except that the content of corn steep liquor in the initial fermentation medium is adjusted to 10 g / L, and the total nitrogen content in the medium is 0.35 g / L.

[0082] Example 3

[0083] Produce 2-KGA according to the method of Example 1, except that the content of corn steep liquor in the initial fermentation medium is adjusted to 30 g / L, and the total nitrogen content in the medium is 1.05 g / L.

[0084] Example 4

[0085] Produce 2-KGA according to the method of Example 1, except that the concentration of ammonia water fed during fermentation is adjusted to 20 wt%, and the pH of the fermentation broth is controlled at 6.5.

[0086] Example 5

[0087] Produce 2-KGA according to the method of Example 1, except that the concentration of ammonia water fed during fermentation is adjusted to 35 wt%, and the pH of the fermentation broth is controlled at 7.5.

[0088] Example 6

[0089] 2-KGA was produced according to the method of Example 1, except that the molar ratio of sorbose to ammonia in ammonia water added during the fermentation process was adjusted to 1:1.

[0090] Example 7

[0091] 2-KGA was produced according to the method of Example 1, except that the molar ratio of sorbose to ammonia in ammonia water added during the fermentation process was adjusted to 1.2:1.

[0092] Example 8

[0093] 2-KGA was produced according to the method of Example 1, except that 20 g / L of corn steep liquor in the initial fermentation medium was adjusted to 10 g / L of yeast powder, and the total nitrogen content was the same.

[0094] Comparative Example 1

[0095] 2-KGA was produced according to the method of Example 1, except that the content of corn steep liquor in the initial fermentation medium was adjusted to 40 g / L, and the total nitrogen content in the medium was 1.40 g / L.

[0096] Comparative Example 2

[0097] 2-KGA was produced according to the method of Example 1, except that 25 wt% ammonia water added during the fermentation process was adjusted to 25 wt% sodium hydroxide.

[0098] Comparative Example 3

[0099] 2-KGA was produced according to the method of Example 1, except that when the sorbose concentration dropped to 15 g / L, sorbose was added to maintain the residual sugar concentration at 2.5%.

[0100] Comparative Example 4

[0101] 2-KGA was produced according to the method of Example 1, except that the concentration of ammonia water added during the fermentation process was adjusted to 10 wt%.

[0102] Comparative Example 5

[0103] 2-KGA was produced according to the method of Example 1, except that the molar ratio of sorbose to ammonia in ammonia water added during the fermentation process was adjusted to 0.8:1.

[0104] Comparative Example 6

[0105] 2-KGA was produced according to the method of Example 1, except that the molar ratio of sorbose to ammonia in ammonia water added during the fermentation process was adjusted to 1.4:1.

[0106] The fermentation cycles and 2-KGA synthesis efficiencies of 2-KGA obtained by the methods of Examples 1-8 and Comparative Examples 1-6 are listed below.

[0107] Table 1

[0108]

[0109] As can be seen from the data in Table 1, the fermentation cycles of Examples 1-8 are in the range of 35 h to 38 h, and no sodium source medium is used. The fermentation cycles of Comparative Examples 1-6 are longer, in the range of 42 h to 62 h.

[0110] In Comparative Example 1, the amount of corn steep liquor was increased and the total nitrogen content was very high. In Comparative Example 2, sodium hydroxide, a sodium source component, was used to adjust the pH value. In Comparative Example 3, fermentation was terminated when the residual sugar concentration was higher than 0.05%. In Comparative Example 4, the concentration of ammonia water used was low. The molar ratio of sorbose to ammonia in Comparative Examples 5 and 6 was not within the range of 1:1 to 1.2:1. Therefore, Comparative Examples 1-6 have problems such as long fermentation cycles or lower 2-KGA synthesis efficiency than Examples 1-8.

[0111] From this, it can be seen that even without using a sodium source medium, by controlling the nitrogen content in the initial fermentation medium, the pH value during the fermentation process, the molar ratio of sorbose to ammonia in ammonia water, etc., the yield of 2-KGA can be increased.

[0112] Industrial applicability

[0113] In this application, by using a substance substantially free of sodium in the fermentation process, controlling the total nitrogen content in the initial fermentation medium, the pH value during the fermentation process, and the molar ratio of ammonia in the added sorbose and ammonia-containing basic substance, the purpose of reducing the generation of sodium chloride waste salt, shortening the fermentation cycle, reducing production costs, and improving the synthesis efficiency of 2-keto-L-gulonic acid is achieved.

Claims

1. A method for fermentatively producing 2-keto-L-gulonic acid, characterized in that, The method includes using a culture medium substantially free of a sodium source during the fermentation process, and using an ammonia-containing alkaline substance to adjust the pH value during the fermentation process. The total nitrogen content of the fermentation medium used at the start of the fermentation is 0.35 g / L to 1.05 g / L. During the fermentation process, an ammonia-containing alkaline substance is added to control the pH of the fermentation broth to 6.5 to 7.

5. Sorbitol is added while adding the ammonia-containing alkaline substance, and the molar ratio of the amount of sorbitol to the ammonia in the ammonia-containing alkaline substance is between 1:1 and 1.2:

1. The fact that it is substantially free of a sodium source means that the culture medium does not use substances from a sodium source. The method includes inoculating a seed solution into the culture medium for fermentation culture to produce 2-keto-L-gulonic acid. The seed solution is a mixed bacterial system of a small bacterium and an associated bacterium. The small bacterium is Ketogulonigenium vulgare, and the associated bacterium is selected from the group consisting of Bacillus megaterium, Bacillus cereus, Bacillus subtilis, and Bacillus thuringiensis.

2. The method according to claim 1, characterized in that, The ammonia-containing alkaline substance is one or more of ammonia water, liquid ammonia, and ammonium bicarbonate.

3. The method according to claim 1 or 2, characterized in that, The fermentation medium contains, in g / L: L-sorbose 20.0 to 60.0, corn steep liquor 10.0 to 30.0, potassium dihydrogen phosphate 1.0 to 3.0, anhydrous magnesium sulfate 0.1 to 0.5, pH 6.0 to 8.

0.

4. The method according to claim 1 or 2, characterized in that, After the start of the fermentation, the pH of the fermentation broth is adjusted to 6.5 to 7.5 using the ammonia-containing alkaline substance, the temperature is 27 to 33 °C, the tank pressure is 0.02 to 0.06 MPa, the aeration rate is 0.8 to 1.2 vvm, and the stirring speed is 300 to 500 rpm.

5. The method according to claim 4, characterized in that, After the start of the fermentation, ammonia water is fed to control the pH of the fermentation broth to 6.8 to 7.

2. When the sorbitol concentration drops to 10 to 20 g / L, the ammonia water is continuously fed to control the pH of the fermentation broth to 7.0 to 7.

6. Sorbitol is fed until the content of 2-keto-L-gulonic acid in the fermentation broth is greater than 90 mg / g, and the fermentation ends when the residual sugar concentration is lower than 0.05%.

Images