Method for producing 2-KGA through one-step fermentation of ketogulonigenium vulgare

Abstract

The invention relates to a method for producing 2-KGA through one-step fermentation of ketogulonigenium vulgare, and belongs to the technical field of fermentation engineering. In earlier-stage research, an inventor obtains a strain of ketogulonigenium sp. WTF0114 through mutagenesis screening, and accidentally finds that the strain can be directly converted into 2-keto-L-gulonic acid (2-KGA) by using D-sorbitol in one step, so that D-sorbitol is directly converted into 2-keto-L-gulonic acid by using the ketogulonigenium sp. WTF0114 and adopting a single-bacterium one-step fermentation method. Compared with a mainstream three-bacterium two-step fermentation method, the production process can be greatly simplified, the fermentation cost is reduced, the production period is shortened, and good economic benefits are achieved.

Description

TECHNICAL FIELD

[0001] The application relates to a method for one-step fermentation of Ketogulonigenium vulgare to produce 2-KGA, and belongs to the technical field of fermentation engineering. BACKGROUND

[0002] Vitamin C is one of the essential vitamins for the human body, has physiological functions such as antioxidant and free radical scavenging, and is widely used in the fields of medicine, food and cosmetics. At present, industrial production of vitamin C is mainly through fermentation method to first synthesize the precursor 2-keto-L-gulonic acid (2-KGA), and then obtain vitamin C through chemical conversion.

[0003] At present, the mainstream industrial production method of 2-keto-L-gulonic acid is three-bacteria two-step fermentation method, that is, first, through the oxidation of Gluconobacter oxydans, D-sorbitol is converted into L-sorbose, and then through the mixed fermentation of Ketogulonigenium vulgare (K. vulgare) and Bacillus megaterium, L-sorbose is converted into 2-keto-L-gulonic acid. Although this method has high conversion rate, due to the need for two fermentations and two sterilizations, the production energy consumption is high; in addition, the double-bacterial synergistic mechanism in the second step of mixed fermentation is not clear, which also leads to complex fermentation process and poor control stability. In the three-bacteria two-step fermentation method, the second step of mixed fermentation uses L-sorbose generated in the first step of fermentation as the substrate, and uses the mixed fermentation of K. vulgare and Bacillus to generate 2-KGA; for example, Chinese patent document CN102586381A (application number 201110338281.8) provides a production process for improving the fermentation intensity of 2-keto-L-gulonic acid, the fermentation substrate used is L-sorbose, and the physiological relationship between the two bacteria in the fermentation of 2-keto-L-gulonic acid is adjusted by means of staged and fed-batch culture, which effectively improves the biosynthesis rate of the mixed bacteria. This production process directly uses L-sorbose as the production substrate, and uses two bacteria one-step method to produce 2-keto-L-gulonic acid. The production technology of D-sorbitol is mature, the production scale is large, and the market supply is sufficient. Compared with D-sorbitol, the production scale of L-sorbose is small, and the output is limited, so the price is expensive. Therefore, the production process has high cost, which is not conducive to industrial production. At present, the single-bacterium one-step fermentation method is mostly through protoplast fusion or preparation of recombinant bacteria to integrate key enzymes into one strain to one-step ferment 2-KGA from D-sorbitol; for example, Chinese patent document CN119061081A (application number 202411197796.4) provides a method for producing 2-keto-L-gulonic acid through protoplast fusion technology, and the method fuses Gluconobacter oxydans WTF0512 and Ketogulonigenium vulgare WTF0114 through protoplast fusion technology to construct a fusant that can one-step ferment 2-keto-L-gulonic acid. Ketogulonicigenium vulgare

[0004] ​At present, there is no report about using the ketogulonigenus bacterium as the fermentation strain to directly ferment D-sorbitol to produce 2-keto-L-gulonic acid. SUMMARY

[0005] In view of the deficiencies in the prior art, the present application provides a method for producing 2-KGA by one-step fermentation of ketogulonigenus bacterium, which has good industrial application value.

[0006] The technical scheme of the present application is as follows: The method for producing 2-KGA by one-step fermentation of ketogulonigenus bacterium is to use ketogulonigenus bacterium as the fermentation strain and D-sorbitol as the substrate to synthesize 2-keto-L-gulonic acid by one-step fermentation.

[0007] Preferably, the ketogulonigenus bacterium is ketogulonigenus bacterium WTF0114, which is preserved in the China Center for Type Culture Collection and has the preservation number CCTCC NO: M2024406.

[0008] Preferably, the method for producing 2-KGA by one-step fermentation of ketogulonigenus bacterium has the following specific steps: (1) inoculate the ketogulonigenus bacterium into a solid culture medium and cultivate for 42-48 h to grow single colonies; (2) pick the single colonies and inoculate into a seed culture medium to cultivate for 28-32 h to obtain a seed liquid; and (3) inoculate the seed liquid into a fermentation culture medium at a volume ratio of 20%-30% to carry out fermentation cultivation, so as to obtain a fermentation liquid containing 2-KGA.

[0009] Further preferably, the cultivation conditions in step (1) are as follows: 25-35℃ inverted cultivation.

[0010] Further preferably, the cultivation conditions in step (2) are as follows: 25-35℃, 150-220 rpm oscillation cultivation.

[0011] Further preferably, the components of the fermentation culture medium in step (3) are as follows: D-sorbitol 60-80 g / L, yeast extract 2-4 g / L, corn syrup 8-12 g / L, urea 8-14 g / L, potassium dihydrogen phosphate 0.5-1 g / L, magnesium sulfate 0.1-0.2 g / L, and calcium carbonate 0.5-1 g / L.

[0012] Further preferably, the fermentation cultivation conditions in step (3) are as follows: pH 6.0-7.0, temperature 28-37℃, aeration intensity 1.5-2.0 vvm, the dissolved oxygen concentration is maintained at 20-30%, the rotation speed is associated with the dissolved oxygen, and the fermentation period is 48-72 h.

[0013] Further preferably, the components of the seed culture medium in step (2) are: L-sorbose 16-24 g / L, peptone 10 g / L, yeast extract 3 g / L, beef extract 3 g / L, corn steep liquor 3 g / L, urea 1 g / L, potassium dihydrogen phosphate 1 g / L, magnesium sulfate 0.2 g / L, calcium carbonate 1 g / L, and water as solvent, and the pH is 6.4-6.7; and the solid culture medium in step (1) is 20 g / L agar added to the seed culture medium.

[0014] Advantages: In the previous study, the inventors screened a ketogulonic acid-producing bacterium WTF0114 by mutagenesis, and unexpectedly found that the strain can directly utilize D-sorbitol to generate 2-keto-L-gulonic acid in one step. Based on this, the present application uses the ketogulonic acid-producing bacterium WTF0114 to directly convert D-sorbitol to 2-keto-L-gulonic acid in one step by single-bacterium fermentation. Compared with the mainstream three-bacterium two-step fermentation method, the production process can be greatly simplified, the fermentation cost can be reduced, the production cycle can be shortened, and good economic benefits can be achieved. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 is a standard curve graph of D-sorbitol, L-sorbose and 2-KGA; Figure 2 is a high performance liquid chromatogram of a mixed sample; Figure 3 is a high performance liquid chromatogram of the fermentation broth obtained in Example 1; Figure 4 is a high performance liquid chromatogram of the fermentation broth obtained in Example 2; Figure 5 is a key component concentration and bacterial liquid OD value line graph of the fermentation broth obtained at different fermentation times in Example 1; Figure 6 is a key component concentration and bacterial liquid OD value line graph of the fermentation broth obtained at different fermentation times in Example 2; Figure 7 is a key component concentration and bacterial liquid OD value line graph of the fermentation broth obtained at different fermentation times of a commercially available ketogulonic acid-producing bacterium. DETAILED DESCRIPTION

[0016] The technical solutions of the present application will be further described below in combination with examples, but the scope of protection of the present application is not limited thereto.

[0017] The drugs and reagents used in the examples are ordinary products on the market if not otherwise specified, and the contents not specifically described in the examples are all according to the existing technology in the art.

[0018] The ketogulonigenus bacteria WTF0114 is deposited in China Center for Type Culture Collection, and the deposit number is CCTCC NO: M2024406. The ketogulonigenus bacteria WTF0114 is obtained by ARTP mutagenesis from the starting strain ketogulonigenus bacteria WTF. The strain has been disclosed in the Chinese patent document CN 119061081 A (application number 202411197796.4), and the preservation of the patent strain is not involved in the present application.

[0019] The culture medium and detection method involved in the following examples are as follows: Seed culture medium: L-sorbose 20 g / L, peptone 10 g / L, yeast extract 3 g / L, beef extract 3 g / L, corn syrup 3 g / L, urea 1 g / L, potassium dihydrogen phosphate 1 g / L, magnesium sulfate 0.2 g / L, calcium carbonate 1 g / L, solvent is water, pH 6.4-6.7; solid culture medium is 20 g / L agar added to the seed culture medium.

[0020] Detection method of key components in fermentation broth: The contents of D-sorbitol, L-sorbose and 2-KGA in the fermentation broth are detected by high performance liquid chromatography (HPLC). Chromatographic column: Aminex HPX-87H; mobile phase: 5 mM sulfuric acid; column temperature: 65°C; flow rate: 0.6 mL / min; sample size: 20 μL.

[0021] D-sorbitol, L-sorbose and 2-KGA concentration gradient samples of 0.1 g / L, 0.5 g / L, 1 g / L, 2 g / L, 4 g / L, 6 g / L, 8 g / L and 10 g / L and a single standard sample of 10 g / L are prepared with the mobile phase as diluent. The standard curve is established according to the peak time of the D-sorbitol, L-sorbose and 2-KGA single standard sample and the detection results of the mixed standard sample gradient. The R 2 ≥0.999, that is, it meets the requirements. The standard curve is shown in Figure 1 , and the high performance liquid chromatogram of the D-sorbitol, L-sorbose and 2-KGA mixed standard sample is shown in Figure 2 .

[0022] The fermentation broth sample is stored in a-20°C refrigerator. Before detection, the sample is taken out and thawed at room temperature. The bacteria are removed by centrifugation at 12000 rpm for 10 min. The sample is diluted with the mobile phase to the concentration of the detection substance within the detection range of the standard curve. The concentrations of D-sorbitol, L-sorbose and 2-keto-L-gulonic acid are detected by HPLC.

[0023] Example 1 One-step fermentation of ketogulonigenus bacteria WTF0114 to produce 2-keto-L-gulonic acid (1) Seed culture: take the glycerol strain preserved at -20°C, melt at room temperature, and use a sterile inoculation loop to take a small amount of bacterial liquid, streak inoculate on a solid culture medium plate, and invert culture in a 30°C incubator for 2 days. The colony of Ketogulonicum bacteria WTF0114 is slightly protruding on the surface, with neat edges, light yellow or colorless transparent, smooth and moist surface, and about 1-2 mm in size. After confirming that the plate is free of mixed bacteria, a single colony is inoculated into the seed culture medium, and a seed liquid is obtained by incubating at 30°C, 200 rpm constant temperature and oscillation for 28-32 h.

[0024] (2) Fermentation culture: inoculate the seed liquid into the fermentation medium at a volume ratio of 30%, maintain pH at 6.4, temperature at 28°C, aeration at 1.5 vvm, maintain DO (dissolved oxygen concentration) at 30%, and the rotation speed is associated with DO. The fermentation period is 72 h, and a fermentation liquid is obtained.

[0025] The components of the above fermentation medium are: D-sorbitol 60 g / L, yeast extract 2 g / L, corn syrup 8 g / L, urea 8 g / L, potassium dihydrogen phosphate 1 g / L, magnesium sulfate 0.2 g / L, calcium carbonate 1 g / L, and water as the solvent.

[0026] Example 2 One-step fermentation of Ketogulonicum bacteria to produce 2-keto-L-gulonic acid (1) Seed culture: the seed liquid is obtained according to step (1) of Example 1.

[0027] (2) Fermentation culture: inoculate the seed liquid into the fermentation medium at a volume ratio of 30%, maintain pH at 6.7, temperature at 30°C, aeration at 2.0 vvm, maintain DO at 20%, and the rotation speed is associated with DO. The fermentation period is 72 h, and a fermentation liquid is obtained.

[0028] The components of the above fermentation medium are: D-sorbitol 80 g / L, yeast extract 2 g / L, corn syrup 10 g / L, urea 10 g / L, potassium dihydrogen phosphate 1 g / L, magnesium sulfate 0.2 g / L, calcium carbonate 1 g / L, and water as the solvent.

[0029] Comparative Example A method for one-step fermentation of Ketogulonicum bacteria to produce 2-keto-L-gulonic acid, the specific steps are the same as Example 2, and the difference from Example 2 is that a commercially available Ketogulonicum bacteria (Ketogulonicum bacteria WTF0114, purchased from Puruiting Biotechnology (Beijing) Co., Ltd.) is used for fermentation. Ketogulonicigenium vulgare

[0030] Experimental Example ​The fermentation broth obtained in the fermentation process of Example 1, Example 2, Comparative Example 0, 12, 24, 36, 48, 60, 72 h was determined by HPLC to determine the content of D-sorbitol, L-sorbose and 2-KGA, and the OD value of the bacterial solution 600 , Figure 3 , Figure 4 are the high performance liquid chromatograms of the fermentation broth of Example 1, Example 2, respectively, 72 h fermentation broth, Figure 5 , Figure 6 are the key component concentration and bacterial solution OD value line graphs of the fermentation broth obtained at different times in Example 1, Example 2, respectively, from the figure, the concentration of D-sorbitol gradually decreases with the fermentation time, the concentration of 2-KGA gradually increases with the fermentation time, and the concentration of L-sorbose remains at a low level, which shows that the ketogulonic acid-producing bacteria WTF0114 can utilize D-sorbitol and directly convert it to 2-KGA, and the final yield of 2-KGA can be as high as 14.94 g / L. Figure 7 are the key component concentration and bacterial solution OD value line graphs of the fermentation broth obtained at different times in Example 1, Example 2, respectively, from the figure, the concentration of D-sorbitol gradually decreases with the fermentation time, the concentration of 2-KGA gradually increases with the fermentation time, and the concentration of L-sorbose remains at a low level, which shows that the ketogulonic acid-producing bacteria WTF0114 can utilize D-sorbitol and directly convert it to 2-KGA, and the final yield of 2-KGA can be as high as 14.94 g / L.

[0031] In the prior art, ketogulonic acid-producing bacteria are all used to generate 2-KGA with L-sorbose as the substrate, but the ketogulonic acid-producing bacteria WTF0114 of the present application is a mutant strain, and the starting strain is improved after mutagenesis to improve its ability to directly generate 2-KGA with D-sorbitol as the substrate. The fermentation method for preparing 2-ketogulonic acid by single bacteria of the ketogulonic acid-producing bacteria established in the present application can greatly simplify the production process, reduce equipment investment and production energy consumption cost, and has good economic and social benefits and broad application prospect.

Claims

1. A method for one-step fermentation of 2-KGA by Ketogulonigenium vulgare, characterized in that, The method is to ferment D-sorbitol as a substrate by using Ketogulonigenium vulgare as a fermentation strain to synthesize 2-keto-L-gulonic acid.

2. The method of claim 1, wherein, The Ketogulonigenium vulgare is Ketogulonigenium vulgare WTF0114, which is preserved in China Center for Type Culture Collection with a preservation number of CCTCC NO: M2024406.

3. The method of claim 1, wherein, The specific steps are as follows: (1) inoculate the Ketogulonigenium vulgare into a solid culture medium and cultivate for 42-48 h to grow single colonies; (2) pick the single colonies and inoculate into a seed culture medium to cultivate for 28-32 h to obtain a seed liquid; and (3) inoculate the seed liquid into a fermentation culture medium at a volume ratio of 20%-30% to perform fermentation cultivation to obtain a fermentation liquid containing 2-KGA.

4. The method of claim 3, wherein, The culture conditions in step (1) are as follows: 25-35℃ inverted cultivation.

5. The method of claim 3, wherein, The culture conditions in step (2) are as follows: 25-35℃, 150-220 rpm oscillation cultivation.

6. The method of claim 3, wherein, The components of the fermentation culture medium in step (3) are as follows: D-sorbitol 60-80 g / L, yeast extract 2-4 g / L, corn syrup 8-12 g / L, urea 8-14 g / L, potassium dihydrogen phosphate 0.5-1 g / L, magnesium sulfate 0.1-0.2 g / L, and calcium carbonate 0.5-1 g / L.

7. The method of claim 3, wherein, The fermentation cultivation conditions in step (3) are as follows: pH 6.0-7.0, temperature 28-37℃, aeration intensity 1.5-2.0 vvm, the dissolved oxygen concentration is maintained at 20-30%, the rotation speed is associated with the dissolved oxygen, and the fermentation period is 48-72 h.

8. The method of claim 3, wherein, The components of the seed culture medium in step (2) are as follows: L-sorbitol 16-24 g / L, peptone 10 g / L, yeast extract 3 g / L, beef extract 3 g / L, corn syrup 3 g / L, urea 1 g / L, potassium dihydrogen phosphate 1 g / L, magnesium sulfate 0.2 g / L, calcium carbonate 1 g / L, and water as a solvent, and the pH is 6.4-6.7; and the solid culture medium in step (1) is a solid culture medium with 20 g / L agar added to the seed culture medium.

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