A method for one-step fermentation of vitamin C precursor 2-keto-L-gulonic acid using two microorganisms

Abstract

This invention relates to a method for producing the vitamin C precursor 2-keto-L-gulonic acid via one-step fermentation using two bacteria, belonging to the field of bioengineering technology. The invention primarily involves the co-fermentation of *Acetobacter niger* and *Glucobacter oxygenase*, using D-sorbitol as a substrate. *Acetobacter niger*, as the sugar-producing bacterium, converts D-sorbitol to L-sorbose, while *Glucobacter oxygenase*, as the acid-producing bacterium, converts L-sorbose to 2-keto-L-gulonic acid. Sodium sulfide is added during the fermentation process. This one-step fermentation process, using a mixed-bacterial fermentation system, achieves the production of 2-keto-L-gulonic acid from D-sorbitol. This method requires only one fermentation and sterilization step, simplifying the production process, shortening the overall fermentation cycle, and reducing energy consumption.

Description

Technical Field

[0001] This invention relates to a method for producing vitamin C precursor 2-keto-L-gulonic acid by two-strain one-step fermentation, which belongs to the field of bioengineering technology. Background Technology

[0002] Vitamin C is a water-soluble vitamin essential for the human body, possessing strong reducing power and participating in many important biochemical reactions within organisms. In addition to its antioxidant function, vitamin C also participates in collagen synthesis, hormone synthesis, carnitine synthesis, gene transcription, and regulates translation through various mechanisms. Primates, guinea pigs, and fish lack the gulonolactone oxidase (GULO) required for vitamin C synthesis, therefore they must rely on exogenous vitamin C supplementation. Currently, the annual demand for vitamin C reaches 220,000 tons, and it is widely used in the pharmaceutical, food, health, cosmetic, and animal feed industries.

[0003] Currently, industrial production of vitamin C mainly utilizes the Rexroth process and the two-step fermentation process. The Rexroth process, first developed in 1933, converts D-glucose into vitamin C through six chemical reactions and a single fermentation step. However, this method is complex, difficult to scale up continuously, energy-intensive, environmentally challenging, and has a long production cycle. The three-strain two-step fermentation process, pioneered by Chinese scientists in 1975, is currently the only successfully applied microbial transformation method for industrial production and is widely used by Chinese vitamin C producers. Due to its significant cost advantages, this process has propelled my country to become the world's largest supplier of vitamin C. Currently, global demand for vitamin C exceeds 220,000 tons, with China supplying 70%, making it a crucial global vitamin C production base.

[0004] The first step of the two-step fermentation process involves *Glucosamine oxidans* converting sorbitol into L-sorbose. Currently, the conversion rate of the first step in industrial production can reach over 98%. The second step involves a mixed bacterial system composed of *Bacillus ketogenes* (commonly known as small bacteria) and its associated bacteria (commonly known as large bacteria) converting L-sorbose into 2-keto-L-gulonic acid (2-KGA). The conversion rate of the second step is over 90%. In the second step of the reaction, ketogenic gulonic acid bacteria use their own enzyme system to convert L-sorbose into 2-keto-L-gulonic acid. However, when cultured alone, this bacterium grows slowly and produces little acid, requiring co-culture with companion bacteria to promote its growth and acid production. Currently developed companion bacteria include Bacillus subtilis, Bacillus licheniformis, Bacillus megaterium, Bacillus thuringiensis, Bacillus cereus, Bacillus thuringiensis, Bacillus pumilus, Bacillus endophyticus, Pseudomonas streaksii, Stenotrophomonas maltophilia, and Saccharomyces cerevisiae, among which Bacillus megaterium is widely used in actual production.

[0005] Existing researchers have studied the relationship between two microorganisms in mixed fermentation, the control of fermentation conditions, the addition of exogenous substances, strain modification, selection of superior strains, and the construction of genomic metabolic network models. For example, Chinese patent document CN102321698A promotes the growth and acid production of ketgulonic acid bacteria by adding the exogenous substance lipoic acid. It improves the growth rate and acid production capacity of ketgulonic acid bacteria in the absence of large bacteria as companions. Lipoic acid is a B vitamin required for bacterial growth. As a component of coenzymes or prosthetic groups, it is an acyl carrier that performs coupling acyl transfer and electron transfer functions in α-keto acid oxidation and decarboxylation, playing an important role in metabolism. Chinese patent document CN 104152365A obtained a new strain of ketgulonic acid bacteria, SPUB-003, through the selection of superior strains, capable of independent growth and production of 2-keto-L-gulonic acid. Chinese patent document CN 109810975A describes the use of genetic engineering to modify ketgulonic acid bacteria, constructing a new sugar metabolism pathway and enabling single-strain production of 2-keto-L-gulonic acid.

[0006] Although the two-step fermentation method has achieved significant economic benefits in industry, it still suffers from problems such as secondary fermentation, secondary sterilization, long overall fermentation cycle, high energy consumption, and complex fermentation processes. There are also reports on one-step fermentation methods in existing technologies. For example, Chinese patent document CN 109234350 A achieves a one-pot conversion from D-sorbitol to 2-keto-L-gulonic acid by co-culturing Bacillus licheniformis overexpressing D-sorbitol dehydrogenase (SLDH) with the acid-producing bacterium *Bacillus ketogulonica* through strain modification. Chinese patent document CN 104404121 A directly mixes *Gluconobacterium oxysporum* and *Bacillus ketogulonica*, using sorbitol as a carbon source, to produce 2-keto-L-gulonic acid through one-step fermentation. However, due to the complexity of the growth and metabolic relationship between the two bacteria during mixed fermentation, there is still considerable room for improvement in conversion rate and production intensity. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a method for the one-step fermentation production of 2-keto-L-gulonic acid, a vitamin C precursor, using two microorganisms. This method utilizes *Acetobacter pungens* as a sugar-producing bacterium to convert D-sorbitol to L-sorbose, and *Glucobacter oxychloride* as an acid-producing bacterium to convert L-sorbose to 2-keto-L-gulonic acid. The one-step fermentation preparation of 2-keto-L-gulonic acid from D-sorbitol is achieved through a mixed-bacterial fermentation system composed of these two microorganisms. Compared to traditional two-step fermentation, this method simplifies the production process, reduces production costs, makes the production process easier to control, and reduces equipment investment, making it suitable for the industrial production of 2-keto-L-gulonic acid.

[0008] This invention utilizes *Acetobacter niger* and *Gluconobacter oxychloride* as the inoculants in a one-step fermentation process. The key to improving fermentation conversion rate and reducing the fermentation cycle lies in controlling the quantitative relationship between the two inoculants to maintain an appropriate ratio. Furthermore, consideration should also be given to improving their growth rate and conversion efficiency.

[0009] The technical solution of the present invention is as follows:

[0010] A method for producing vitamin C precursor 2-keto-L-gulonic acid by one-step fermentation using two bacteria involves co-fermenting *Acetobacter niger* and *Glucobacter oxygenase*. Using D-sorbitol as a substrate, *Acetobacter niger*, as the sugar-producing bacterium, converts D-sorbitol to L-sorbose, while *Glucobacter oxygenase*, as the acid-producing bacterium, converts L-sorbose to 2-keto-L-gulonic acid. Sodium sulfide is added during the fermentation process. This one-step fermentation process from D-sorbitol to 2-keto-L-gulonic acid is achieved through a mixed-bacterial fermentation system composed of the two bacteria.

[0011] According to a preferred embodiment of the present invention, the vinaigrette is Gluconbacter melanogenus H02, which was deposited on August 10, 2020 at the China Center for Type Culture Collection, address: No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, accession number CCTCC NO:M 2020409.

[0012] According to a preferred embodiment of the present invention, the Gluconobacter oxydans is Gluconobacter oxydans S19, which was deposited on August 17, 2020 at the China Center for Type Culture Collection, located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, with accession number CCTCC NO:M 2020427.

[0013] According to a preferred embodiment of the present invention, sodium sulfide is added at a constant flow rate during fermentation.

[0014] According to a preferred embodiment of the present invention, the sodium sulfide is added in a flow during the early stage of fermentation.

[0015] According to a preferred embodiment of the present invention, the sodium sulfide is added during the first 0-18 hours of the fermentation process.

[0016] According to a preferred embodiment of the present invention, the amount of sodium sulfide added is 0.005 to 0.01% of the mass of the mixed-culture fermentation system.

[0017] According to a preferred embodiment of the present invention, the sodium sulfide is added by preparing a sodium sulfide solution of 0.1-1 wt%, filtering and sterilizing it, and then adding it.

[0018] According to a preferred embodiment of the present invention, the seed liquid of Acetobacter purpureus and the seed liquid of Gluconobacter oxidase are mixed to obtain a mixed fermentation seed liquid, which is then inoculated into a fermentation medium for mixed fermentation.

[0019] Further preferably, the OD of the black vinegar seed liquid... 600 The OD value of the *Glucobacterium oxidans* seed culture was 7.5–8.0. 600 It ranges from 1.8 to 2.0.

[0020] Further preferably, the OD values ​​of *Acetobacter mellea* and *Glucobacter oxychloride* in the mixed-culture fermentation seed liquid are... 600 The ratio is (2~32):1.

[0021] More preferably, the inoculation amount of the mixed-culture fermentation seed liquid is 5-25% by volume.

[0022] More preferably, the fermentation medium consists of: 8-10% D-sorbitol, 0.2-0.5% yeast extract, 1.2-1.5% corn steep liquor powder, 0.2-0.5% urea, 0.1-0.2% potassium dihydrogen phosphate, 0.01-0.03% magnesium sulfate, and 0.1-0.3% calcium carbonate, all by mass percentage.

[0023] More preferably, the conditions for the mixed fermentation are: fermentation temperature 28-30℃, ventilation rate 3.0-3.5L / min, rotation speed 350-400rpm, and pH maintained at 7.2-7.3 during fermentation.

[0024] According to a preferred embodiment of the present invention, fermentation is terminated when the sum of the contents of D-sorbitol and L-sorbose in the mixed-culture fermentation system is ≤0.5 g / L.

[0025] A strain of glutenobacter melanogenus H02 was deposited on August 10, 2020, at the China Center for Type Culture Collection (CCTCC), located at No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, China, with accession number CCTCC NO: M2020409.

[0026] A strain of Gluconobacter oxydans S19 was deposited on August 17, 2020, at the China Center for Type Culture Collection (CCTCC), located at No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, China, with accession number CCTCC NO: M2020427.

[0027] In a preferred embodiment of the present invention, the method for producing vitamin C precursor 2-keto-L-gulonic acid by two-strain one-step fermentation comprises the following steps:

[0028] 1. Preparation of plate culture

[0029] Black vinegar bacteria solid culture medium, all by weight percentage: D-sorbitol 2%, yeast extract 0.6%, calcium carbonate 0.1%, agar powder 2%; pH 5.2–5.4.

[0030] The solid culture medium for *Glucobacter oxidans* consisted of the following percentages by weight: L-sorbose 2% (sterilized separately), peptone 1%, yeast extract 0.3%, beef extract 0.3%, corn steep liquor 0.3%, urea 0.1%, potassium dihydrogen phosphate 0.1%, magnesium sulfate 0.02%, calcium carbonate 0.1%, and agar powder 2%; pH 6.0–6.5.

[0031] Take glycerol tubes of *Acetobacter niger* and *Glucobacter oxygenase* respectively, streak them on the corresponding solid culture medium with an inoculation loop, and incubate them upside down at 30°C for 2-3 days. When a single colony grows, it is a plate culture.

[0032] 2. Seed liquid preparation:

[0033] Black vinegar bacteria liquid culture medium, all by weight percentage: D-sorbitol 2%, yeast extract 0.6%, calcium carbonate 0.1%; pH 5.2–5.4.

[0034] The liquid culture medium for *Glucobacterium oxysporum* consisted of the following weight percentages: L-sorbose 2% (sterilized separately), peptone 1%, yeast extract 0.3%, beef extract 0.3%, corn steep liquor 0.3%, urea 0.1%, potassium dihydrogen phosphate 0.1%, magnesium sulfate 0.02%, and calcium carbonate 0.1%; pH 6.0–6.5.

[0035] Single colonies of *Acetobacter oleracea* and *Glucobacter oxidans* were picked from agar plates using an inoculation loop and inoculated into their respective liquid culture media. The media were then incubated at 30°C with constant temperature shaking at 150–200 rpm until the *Acetobacter oleracea* culture reached its OD value. 600 The OD value was 7.5–8 for *Glucosamine oxidans* cultured to 0.05–0.05. 600 The seed solution is obtained when the concentration is 1.8–2; among which, OD 600 The determination involves aseptically sampling the cultured *Acetobacter purpureus* seed culture and *Glucobacter oxidans* seed culture, appropriately diluting them with 0.5–3% citric acid solution, and measuring the OD value at 600 nm. Then, the *Acetobacter purpureus* and *Glucobacter oxidans* seed cultures are mixed to obtain the mixed-culture fermentation seed culture. The OD values ​​of *Acetobacter purpureus* and *Glucobacter oxidans* in the mixed-culture fermentation seed culture are then measured. 600 The ratio is (2~32):1.

[0036] 3. Fermentation in fermentation tanks

[0037] Fermentation medium: D-sorbitol 8%, yeast extract 0.2-0.5%, corn steep liquor powder 1.2-1.5%, urea 0.2-0.5%, potassium dihydrogen phosphate 0.1-0.2%, magnesium sulfate 0.01-0.03%, calcium carbonate 0.1-0.3%.

[0038] All culture medium components except D-sorbitol were sterilized in a fermenter at 120°C for 20 min. D-sorbitol was prepared to a 40% concentration, sterilized at 115°C for 30 min, and then added to the fermenter. The prepared mixed-culture fermentation seed solution was inoculated into the fermentation medium at an inoculation rate of 5–25%. To improve the growth and acid production of *Glucobacterium oxysporum*, a 1 wt% filtered and sterilized sodium sulfide solution was added at a constant flow rate during the first 0–18 hours of fermentation. The amount of sodium sulfide added was 0.005–0.01% of the fermentation system mass. The fermentation temperature was set at 30°C, the aeration rate at 3.5 L / min, the rotation speed at 400 rpm, and the pH was maintained at 7.2 during fermentation. Fermentation was terminated when the sum of the contents of D-sorbitol and L-sorbose in the fermentation broth was ≤0.5 g / L.

[0039] The beneficial effects of this invention are as follows:

[0040] 1. This invention utilizes *Acetobacter pungens* as the sugar-producing bacteria to convert D-sorbitol to L-sorbose, and *Glucobacter oxychloride* as the acid-producing bacteria to convert L-sorbose to 2-keto-L-gulonic acid. A mixed-culture fermentation system composed of *Acetobacter pungens* and *Glucobacter oxychloride* achieves a one-step fermentation process from D-sorbitol to 2-keto-L-gulonic acid, ensuring high yield and conversion rate. The one-step fermentation of these two bacteria to produce the vitamin C precursor 2-keto-L-gulonic acid requires only one fermentation and one sterilization, simplifying the production process, shortening the overall fermentation cycle, and reducing energy consumption.

[0041] 2. This invention improves the yield of 2-keto-L-gulonic acid and shortens the fermentation cycle by controlling the ratio of the two strains, the inoculum size, and the addition of sodium sulfide. The yield reaches 76.9 g / L, the conversion rate reaches 90.2%, and the fermentation cycle is 42 hours. Sulfur is an essential macronutrient for organisms. Furthermore, the oxidative dehydrogenation reaction of vinaigrette bacteria in the early stages of fermentation increases the redox potential of the fermentation environment, thereby inhibiting the growth and metabolism of the strain. Sodium sulfide itself has strong reducing properties, which can lower the redox potential of the fermentation broth. Secondly, sodium sulfide is slowly oxidized in the fermentation system to sodium thiosulfate, sodium sulfite, sodium sulfate, and sodium polysulfide, serving as a slow-release sulfur donor. Attached Figure Description

[0042] Figure 1 This is a graph showing the fermentation process of experimental group 4 in Example 4. Detailed Implementation

[0043] The technical solution of the present invention will be further described below with reference to embodiments, but the scope of protection of the present invention is not limited thereto. Unless otherwise specified, the reagents and medicines involved in the embodiments are all commercially available products; unless otherwise specified, the experimental operations involved in the embodiments are performed in accordance with conventional operations in the art. Percentages not specifically mentioned in the embodiments are mass percentages.

[0044] Microbial sources involved in the examples:

[0045] The vinaigrette strain is Gluconbacter melanogenus H02, deposited on August 10, 2020 at the China Center for Type Culture Collection (CCTCC), located at No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, China, with accession number CCTCC NO: M2020409.

[0046] The Gluconobacter oxydans is S19, deposited on August 17, 2020 at the China Center for Type Culture Collection, located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, China, with accession number CCTCC NO:M 2020427.

[0047] Example 1: Preparation of seed culture of Acetobacter mellea and Gluconobacter oxidase

[0048] 1. Preparation of plate culture

[0049] Black vinegar bacteria solid culture medium: D-sorbitol 2%, yeast extract 0.6%, calcium carbonate 0.1%, agar powder 2%; pH 5.2-5.4.

[0050] Glucobacter oxidans solid culture medium: L-sorbose 2% (sterilized separately), peptone 1%, yeast extract 0.3%, beef extract 0.3%, corn steep liquor 0.3%, urea 0.1%, potassium dihydrogen phosphate 0.1%, magnesium sulfate 0.02%, calcium carbonate 0.1%, agar powder 2%; pH 6.0–6.5.

[0051] Take glycerol tubes of *Acetobacter niger* and *Glucobacter oxygenase* respectively, streak them on the corresponding solid culture medium with an inoculation loop, and incubate them upside down at 30°C for 2-3 days. When a single colony grows, it is a plate culture.

[0052] 2. Seed liquid preparation:

[0053] Black vinegar liquid culture medium: D-sorbitol 2%, yeast extract 0.6%, calcium carbonate 0.1%; pH 5.2-5.4.

[0054] Glucobacter oxidans liquid culture medium: L-sorbose 2% (sterilized separately), peptone 1%, yeast extract 0.3%, beef extract 0.3%, corn steep liquor powder 0.3%, urea 0.1%, potassium dihydrogen phosphate 0.1%, magnesium sulfate 0.02%, calcium carbonate 0.1%; pH 6.0-6.5.

[0055] Single colonies of *Acetobacter niger* and *Glucobacter oxygenase* were picked from agar plates using an inoculation loop and inoculated into their respective liquid culture media. The media were then incubated at 30°C and 200 rpm in a shaker. *Acetobacter niger* was cultured until its OD value reached 7.9, and *Glucobacter oxygenase* was cultured until its OD value reached 1.8 to obtain the seed culture.

[0056] Method for determining the OD value of seed culture: Aseptically sample the cultured seed culture, dilute it appropriately with 0.5-3% citric acid solution, and measure the OD value at 600 nm. Since the liquid culture medium of the strain contains insoluble calcium carbonate, citric acid solution is used to dilute the seed culture when measuring the OD value. The OD value of the diluted seed culture multiplied by the dilution factor is the original OD value of the seed culture.

[0057] Example 2: Optimization of the ratio of acetic acid bacteria and glucosamine oxidase seed culture

[0058] The vinaigrette seed solution prepared in Example 1 and the glucosamine oxidase seed solution were mixed to obtain a mixed fermentation seed solution. The ratio of the OD values ​​of the vinaigrette and glucosamine oxidase in the mixed fermentation seed solution was set to 1:1, 2:1, 8:1, 16:1, 32:1, and 64:1, respectively. That is, the original vinaigrette seed solution and glucosamine oxidase seed solution were mixed at volume ratios of 0.23:1, 0.46:1, 1.82:1, 3.65:1, 7.29:1, and 14.58:1, respectively.

[0059] 1. Fermentation in a fermentation tank:

[0060] Fermentation medium: D-sorbitol 8%, yeast extract 0.3%, corn steep liquor powder 1.5%, urea 0.5%, potassium dihydrogen phosphate 0.1%, magnesium sulfate 0.02%, calcium carbonate 0.3%.

[0061] In a 5L glass fermenter, all culture medium components except D-sorbitol were sterilized at 120°C for 20 minutes. D-sorbitol was prepared to a 40wt% concentration, sterilized at 115°C for 30 minutes, and then added to the fermenter. The prepared mixed-culture seed culture was inoculated into the fermentation medium at a rate of 15% by volume, resulting in a total fermentation volume of 3.5L. The fermentation temperature was set at 30°C, aeration rate of 3.5L / min, a rotation speed of 400rpm, and the pH was maintained at 7.2 during fermentation. Fermentation was terminated when the sum of D-sorbitol and L-sorbose in the fermentation broth was ≤0.5g / L.

[0062] 2. Determination of 2-keto-L-gulonic acid content

[0063] Sample preparation: Accurately pipette 0.1 mL of fermentation broth into 0.9 mL of sterile purified water, mix well, centrifuge at 12000 rpm for 10 min, and filter the supernatant through a 0.22 μm filter membrane to obtain the sample to be tested.

[0064] High Performance Liquid Chromatography (HPLC) Conditions:

[0065] Column: Aminex HPX-87H (300mm × 7.8mm)

[0066] Mobile phase: 5mM sulfuric acid solution

[0067] Flow rate: 0.6 mL / min

[0068] Column temperature: 65℃

[0069] Detector: Differential refractive index detector,

[0070] Injection volume: 20 μL.

[0071] The experimental results are shown in the table below:

[0072] Table 1: Production of 2-keto-L-gulonic acid under different strain ratios

[0073] 1 1:1 60.9 71.4 78 2 2:1 69.7 81.7 48 3 8:1 70.3 82.4 45 4 16:1 70.2 82.3 45 5 32:1 70.5 82.6 45 6 64:1 69.2 81.1 65

[0074] Table 1 shows that the yield of 2-keto-L-gulonic acid varies depending on the ratio of *Acetobacter niger* and *Gluconobacter oxygenase* during mixed fermentation. When the ratio of *Acetobacter niger* to *Gluconobacter oxygenase* in the mixed fermentation seed liquid is too low (i.e., the inoculation ratio of *Gluconobacter oxygenase* is too high), 2-keto-L-gulonic acid will be produced prematurely in the early stage of fermentation, thus inhibiting the growth of *Acetobacter niger* and the conversion of D-sorbitol to L-sorbose, resulting in a longer fermentation cycle and a lower conversion rate. Conversely, when the ratio of *Gluconobacter oxygenase* to *Gluconobacter oxygenase* in the mixed fermentation seed liquid is too high (i.e., the inoculation amount of *Gluconobacter oxygenase* is insufficient), acid production slows down in the later stage of fermentation, leading to a longer fermentation cycle and a lower conversion rate. The data in Table 1 show that the OD ratio of *Acetobacter niger* to *Gluconobacter oxygenase* in the mixed fermentation seed liquid of this invention is 2:1 to 32:1, which significantly shortens the fermentation cycle while ensuring a high conversion rate.

[0075] Example 3: Optimization of inoculum size for mixed-culture fermentation seed liquid

[0076] Seed cultures of Acetobacter niger and Gluconobacter oxychloride were prepared according to the method of Example 1, wherein the OD value of the Acetobacter niger seed culture was 7.5 and the OD value of the Gluconobacter oxychloride seed culture was 1.8. The prepared Acetobacter niger seed culture and Gluconobacter oxychloride seed culture were mixed to obtain a mixed fermentation seed culture, wherein the ratio of the OD values ​​of Acetobacter niger and Gluconobacter oxychloride in the mixed fermentation seed culture was 16:1.

[0077] 1. Fermentation in a fermentation tank:

[0078] Fermentation medium: D-sorbitol 8%, yeast extract 0.3%, corn steep liquor powder 1.5%, urea 0.5%, potassium dihydrogen phosphate 0.1%, magnesium sulfate 0.02%, calcium carbonate 0.3%.

[0079] In a 5L glass fermenter, all culture medium components except D-sorbitol were sterilized at 120°C for 20 minutes. D-sorbitol was prepared to a 40wt% concentration, sterilized at 115°C for 30 minutes, and then added to the fermenter. The prepared mixed-culture seed culture was inoculated into the fermentation medium at different volume percentages, with a total fermentation volume of 3.5L. The fermentation temperature was set at 30°C, aeration rate of 3.5L / min, a rotation speed of 400rpm, and the pH was maintained at 7.2 during fermentation. Fermentation was terminated when the sum of D-sorbitol and L-sorbose in the fermentation broth was ≤0.5g / L.

[0080] 2. Determination of 2-keto-L-gulonic acid content

[0081] The content of 2-keto-L-gulonic acid in the fermentation broth was determined according to the method in Example 2, and the results are shown in the table below:

[0082] Table 2: Production of 2-keto-L-gulonic acid under different inoculum amounts

[0083] 1 2％ 65.4 76.7 54 2 5％ 70.5 85.3 48 3 15％ 71.2 83.5 45 4 25％ 71.1 83.4 45 5 30％ 57.4 67.3 96

[0084] Table 2 shows that the yield of 2-keto-L-gulonic acid varies depending on the inoculum size of the mixed fermentation seed liquid when using *Acetobacter niger* and *Glucosamine oxyphylla* for co-fermentation. When the inoculum size is less than 5%, a certain amount of time and nutrients are needed for the reproduction and growth of the microorganisms in the early stage of fermentation, resulting in a longer fermentation cycle and a lower conversion rate. When the inoculum size reaches 30%, the separately sterilized D-sorbitol solution accounts for 20% of the fermentation liquid volume, and nutrients such as nitrogen sources and inorganic salts need to be prepared and sterilized in the remaining 50% volume. Excessive nutrient concentration during sterilization can lead to nutrient degradation, resulting in a decrease in fermentation yield and a longer fermentation cycle. The data in Table 2 indicate that the optimized inoculum size for the mixed fermentation seed liquid of this invention is 5–25%, which significantly shortens the fermentation cycle while ensuring a high conversion rate.

[0085] Example 4: Optimization of Sodium Sulfide Addition Amount and Method

[0086] Seed cultures of Acetobacter niger and Gluconobacter oxygenase were prepared according to the method of Example 1, wherein the OD value of the Acetobacter niger seed culture was 7.8 and the OD value of the Gluconobacter oxygenase seed culture was 1.9. The prepared Acetobacter niger seed culture and Gluconobacter oxygenase seed culture were mixed to obtain a mixed fermentation seed culture, wherein the ratio of the OD values ​​of Acetobacter niger and Gluconobacter oxygenase in the mixed fermentation seed culture was 16:1.

[0087] 1. Fermentation in a fermentation tank:

[0088] Fermentation medium: D-sorbitol 8%, yeast extract 0.3%, corn steep liquor powder 1.5%, urea 0.5%, potassium dihydrogen phosphate 0.1%, magnesium sulfate 0.02%, calcium carbonate 0.3%.

[0089] In a 5L glass fermenter, all culture medium components except D-sorbitol were sterilized at 120°C for 20 minutes. D-sorbitol was prepared to a 40wt% concentration, sterilized at 115°C for 30 minutes, and then added to the fermenter. The prepared mixed-culture seed culture was inoculated into the fermentation medium at a volume percentage of 15%, resulting in a total fermentation volume of 3.5L. To improve the growth and acid production of *Gluconobacterium oxysporum*, sodium sulfide was added during fermentation at a rate of 0.001–0.02% of the fermentation broth mass. The sodium sulfide was prepared to a 1wt% concentration, filtered for sterilization, and added at a constant flow rate during the first 0–18 hours of fermentation, or added all at once at the start of fermentation. The fermentation temperature was set at 30°C, the aeration rate at 3.5L / min, the rotation speed at 400rpm, and the pH maintained at 7.2 throughout fermentation. Fermentation is terminated when the sum of the contents of D-sorbitol and L-sorbose in the fermentation broth is ≤0.5 g / L.

[0090] 2. Determination of 2-keto-L-gulonic acid content

[0091] The content of 2-keto-L-gulonic acid in the fermentation broth was determined according to the method in Example 2, and the results are shown in the table below:

[0092] Table 3: Production of 2-keto-L-gulonic acid under different sodium sulfide addition amounts and methods

[0093] 1 0 / 71.8 84.2％ 45 2 0.001％ Constant flow addition 71.9 84.0％ 45 3 0.001％ One-off addition 73.3 86.0％ 42 4 0.005％ Constant flow addition 76.9 90.2％ 42 5 0.005％ One-off addition 73.2 85.9％ 45 6 0.01％ Constant flow addition 76.4 89.6％ 42 7 0.01％ One-off addition 69.3 81.3％ 45 8 0.02％ Constant flow addition 65.2 76.5％ 48 9 0.02％ One-off addition 60.4 70.8％ 48

[0094] Table 3 shows that the yield of 2-keto-L-gulonic acid varies depending on the amount and method of sodium sulfide addition during the co-fermentation of *Acetobacter niger* and *Gluconobacter oxynitrite*. Appropriate single-time or constant-rate addition of sodium sulfide allows it to act as a slow-release sulfur source and regulate the redox potential of fermentation, promoting the growth and acid production of *Gluconobacter oxynitrite*. Excessive sodium sulfide can lead to a low redox potential, inhibiting the growth and fermentation of both bacteria. The experimental group using constant-rate sodium sulfide addition avoided the inhibition of fermentation caused by excessive single-time addition of sodium sulfide, achieving a more ideal result. The data in Table 3 show that constant-rate addition of 0.005–0.01% sodium sulfide resulted in a higher conversion rate and shorter fermentation cycle compared to single-time addition. The changes in various components during the fermentation process in experimental group 4 are shown below. Figure 1 As shown.

[0095] Comparative Example 1: Comparison of different added compounds

[0096] The difference from Example 4 is that the compound added at a constant rate is different, while all other conditions are the same as in Example 4. The specific steps are as follows:

[0097] 1. Fermentation in a fermentation tank:

[0098] Fermentation medium: D-sorbitol 8%, yeast extract 0.3%, corn steep liquor powder 1.5%, urea 0.5%, potassium dihydrogen phosphate 0.1%, magnesium sulfate 0.02%, calcium carbonate 0.3%.

[0099] In a 5L glass fermenter, all culture medium components except D-sorbitol were sterilized at 120°C for 20 minutes. D-sorbitol was prepared to a 40wt% concentration, sterilized at 115°C for 30 minutes, and then added to the fermenter. The prepared mixed-culture seed culture was inoculated into the fermentation medium at a 15% volume percentage, resulting in a total fermentation volume of 3.5L. To enhance the growth and acid production of *Gluconobacterium oxidans*, different compounds were added during fermentation. These compounds were sodium sulfide, sodium sulfite, or lipoic acid, added at 0.005% of the fermentation medium mass. The added compounds were prepared to a 1wt% concentration, filtered for sterilization, and added at a constant flow rate during the first 0-18 hours of fermentation. The fermentation temperature was set at 30°C, the aeration rate at 3.5L / min, the rotation speed at 400rpm, and the pH maintained at 7.2 throughout fermentation. Fermentation is terminated when the sum of the contents of D-sorbitol and L-sorbose in the fermentation broth is ≤0.5 g / L.

[0100] 2. Determination of 2-keto-L-gulonic acid content

[0101] The content of 2-keto-L-gulonic acid in the fermentation broth was determined according to the method in Example 2, and the results are shown in the table below:

[0102] Table 4: Production of 2-keto-L-gulonic acid with the addition of different compounds

[0103] 1 Sodium sulfide 76.8 90.1％ 42 2 Sodium sulfite 75.2 88.2％ 45 3 Thioctic acid 75.7 88.8％ 42

[0104] Table 4 shows that the yield of 2-keto-L-gulonic acid varies depending on the compound added during the co-fermentation of *Acetobacter purpureus* and *Glucobacter oxidans*. Sodium sulfide, sodium sulfite, and lipoic acid all improve the one-step fermentation conversion rate of the two bacteria, but sodium sulfide is more effective than sodium sulfite and lipoic acid. Unlike sodium sulfite and lipoic acid, sodium sulfide can regulate the redox potential of the fermentation broth, thereby increasing the fermentation yield. In the early stage of fermentation, *Acetobacter purpureus* undergoes vigorous oxidative dehydrogenation to convert D-sorbitol to L-sorbose. The addition of sodium sulfide, which has strong reducing properties, at this stage is likely the main reason why the conversion rate and fermentation cycle are better than those of sodium sulfite and lipoic acid.

[0105] Comparative Example 2: Three-strain two-step fermentation method

[0106] 1. First step of fermentation

[0107] Black vinegar liquid culture medium: D-sorbitol 2%, yeast extract 0.6%, calcium carbonate 0.1%; pH 5.2-5.4.

[0108] Pick a single colony of vinaigrette using an inoculation loop, inoculate it into liquid culture medium, and incubate at 30°C and 200 rpm for 36 hours to obtain vinaigrette seed culture.

[0109] The first step fermentation medium consisted of 20% D-sorbitol (sterilized separately), 0.3% yeast extract, and 1% calcium carbonate.

[0110] In a 5L glass fermenter, all culture medium components except D-sorbitol were sterilized at 120°C for 20 minutes. D-sorbitol was prepared to a 40wt% concentration, sterilized at 115°C for 30 minutes, and then added to the fermenter. The prepared black vinegar bacteria seed culture was inoculated into the fermentation medium at a volume percentage of 15%, resulting in a total fermentation volume of 3.5L. The fermentation temperature was set at 30°C, aeration rate of 3.5L / min, a rotation speed of 400rpm, and the pH was maintained at 6.0 during fermentation.

[0111] Fermentation results: Fermentation cycle was 18 hours, fermentation yield was 78.6 g / L, and conversion rate was 98.3%.

[0112] After fermentation is complete, heat the fermentation liquid to 80°C and maintain the temperature for 30 minutes to obtain the first-step fermentation liquid.

[0113] 2. Second fermentation

[0114] Liquid culture medium for the strain: 2% L-sorbose (sterilized separately), 1% peptone, 0.3% yeast extract, 0.3% beef extract, 0.3% corn steep liquor powder, 0.1% urea, 0.1% potassium dihydrogen phosphate, 0.02% magnesium sulfate, 0.1% calcium carbonate; pH 6.0–6.5.

[0115] Using an inoculation loop, pick a single colony of epoxidobacterium cyclooxygenase and Bacillus megaterium, and inoculate them into the liquid culture medium of the above strains. Incubate at 30°C and 200 rpm for 24 hours to obtain the mixed seed culture.

[0116] Fermentation medium: 40% one-step fermentation broth (sterilized separately at low temperature), 0.3% yeast extract, 1.5% corn steep liquor powder, 0.5% urea, 0.1% potassium dihydrogen phosphate, 0.02% magnesium sulfate, and 0.3% calcium carbonate.

[0117] In a 5L glass fermenter, all culture medium components except the one-step fermentation broth were sterilized at 120°C for 20 minutes. Then, 40% of the low-temperature sterilized (70°C, 2 hours) one-step fermentation broth and 15% of a mixed seed culture of *Glucobacterium oxysporum* and *Bacillus megaterium* were added, bringing the total fermentation volume to 3.5L. The fermentation temperature was set at 30°C, the aeration rate at 3.5L / min, the rotation speed at 400 rpm, and the pH was maintained at 7.2 during fermentation.

[0118] Fermentation results: Fermentation cycle 30h, fermentation yield 76.5g / L, second-step fermentation conversion rate 91.3%.

[0119] Experimental results: The first fermentation step took 18 hours, the second fermentation step took 30 hours, and the total fermentation cycle was 48 hours; the yield was 76.5 g / L, and the molar conversion rate was 89.7%. Compared with the experimental results of Example 4, this invention successfully achieved the one-step fermentation production of vitamin C precursor 2-keto-L-gulonic acid. With similar yield and conversion rate, the fermentation cycle of this invention is shorter than the total fermentation cycle of the traditional three-strain two-step fermentation, and it simplifies the production process. Compared with the traditional three-strain two-step fermentation method, which requires secondary sterilization and secondary fermentation, it reduces the energy consumption of one sterilization and one fermentation. Furthermore, compared with the traditional three-strain two-step fermentation method, it reduces the equipment investment required for the fermentation tank in the first fermentation stage during the workshop construction phase, resulting in a significant cost advantage.

[0120] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method for producing vitamin C precursor 2-keto-L-gulonic acid by one-step fermentation using two microorganisms, characterized in that, The method involves mixed fermentation of vinaigrettes and glucosidobacterium oxidase, using D-sorbitol as a substrate. Vigna sorbitol is converted into L-sorbose by vinaigrettes as the sugar-producing bacteria, while glucosidobacterium oxidase is converted into 2-keto-L-gulonic acid by 2-keto-L-gulonic acid by glucosidobacterium oxidase as the acid-producing bacteria. Sodium sulfide is added at a constant rate in the early stage of fermentation. The one-step fermentation preparation of D-sorbitol to 2-keto-L-gulonic acid is achieved through a mixed fermentation system composed of two bacteria. The mixed fermentation involves mixing the seed culture of *Acetobacter purpureus* and *Glucobacter oxidans* to obtain a mixed fermentation seed culture, which is then inoculated into a fermentation medium for mixed fermentation. The OD values ​​of *Acetobacter purpureus* and *Glucobacter oxidans* in the mixed fermentation seed culture are... 600 The ratio is (2~32):1, and the inoculum amount of the mixed-culture fermentation seed liquid is 5%~25% by volume. Among them, the black vinegar bacteria is black vinegar bacteria ( Gluconbacter melanogenus H02, deposited on August 10, 2020 at the China Center for Type Culture Collection, address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, accession number CCTCCNO: M 2020409. The oxidizing glucosibib is oxidizing glucosibib ( Gluconobacter oxydans S19 was deposited on August 17, 2020 at the China Center for Type Culture Collection, address: No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, accession number CCTCC NO: M 2020427.

2. The method as described in claim 1, characterized in that, The sodium sulfide is added during the first 0-18 hours of the fermentation process.

3. The method as described in claim 1, characterized in that, The amount of sodium sulfide added is 0.005% to 0.01% of the mass of the mixed-culture fermentation system.

4. The method as described in claim 1, characterized in that, The sodium sulfide is added by preparing a sodium sulfide solution of 0.1wt% to 1wt%, filtering it for sterilization, and then adding it.

5. The method as described in claim 1, characterized in that, The OD of the black vinegar seed liquid 600 The OD value of the *Glucobacterium oxidans* seed culture was 7.5–8.

0. 600 It ranges from 1.8 to 2.

0.

6. The method as described in claim 1, characterized in that, The fermentation medium consists of the following components: D-sorbitol 8%~10%, yeast extract 0.2%~0.5%, corn steep liquor powder 1.2%~1.5%, urea 0.2%~0.5%, potassium dihydrogen phosphate 0.1%~0.2%, magnesium sulfate 0.01%~0.03%, and calcium carbonate 0.1%~0.3%, all by mass percentage.

7. The method as described in claim 1, characterized in that, The conditions for the mixed fermentation are: fermentation temperature 28℃~30℃, ventilation rate 3.0L / min~3.5L / min, rotation speed 350rpm~400rpm, and pH maintained at 7.2~7.3 during fermentation.

8. The method as described in claim 1, characterized in that, Fermentation is terminated when the sum of D-sorbitol and L-sorbose in the mixed-culture fermentation system is ≤0.5 g / L.

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