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Method utilizing mixed bacteria evolution subculturing to improve 2-keto-L-gulonic acid yield

A subculture and gulonic acid technology, which is applied in the field of mixed bacteria evolution subculture to increase the output of 2-keto-L-gulonic acid, can solve the problems of low acid production efficiency and slow growth, and achieve improved transformation efficiency and growth speed , the effect of improving utilization efficiency

Active Publication Date: 2012-02-15
TIANJIN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Bacillus megaterium (Bacillus megaterium) and Gluconobacter oxydans (Gluconobacter oxydans) mixed bacteria fermentation used in mixed fermentation, wherein, Gluconobacter oxydans (Gluconobacter oxydans) is an acid producing bacterium, Bacillus megaterium (Bacillus megaterium) is an associated bacterium , if Gluconobacter oxidans is used alone without adding Bacillus megaterium, the growth is extremely slow and the acid production efficiency is low. When Bacillus megaterium is added, the growth rate of Gluconobacter oxidans can be increased, and the amount of acid production can be increased. However, Bacillus megateria There is still room for improvement in the conversion rate of 2-keto-L-gulonic acid in the mixed fermentation of Bacillus megaterium and Gluconobacter oxydans

Method used

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  • Method utilizing mixed bacteria evolution subculturing to improve 2-keto-L-gulonic acid yield
  • Method utilizing mixed bacteria evolution subculturing to improve 2-keto-L-gulonic acid yield

Examples

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Embodiment 1

[0027] A method for improving the yield of 2-keto-L-gulonic acid by subculture of mixed bacteria evolution, comprising the steps of:

[0028] (1) Solid culture:

[0029] The preparation of the solid medium is as follows: Weigh 20g of L-sorbose, 3g of corn steep liquor, 3g of beef extract, 3g of yeast extract powder, 1g of urea, 10g of peptone, 20g of agar, KH 2 PO 4 1g, MgSO 4 0.2g, CaCO 3 Add 1 g of water to 1 L, adjust the pH to 6.8, and sterilize at 121°C for 20 minutes to make a solid medium;

[0030] Inoculate 150 μL of Gluconobacter oxydans stored in 20% aqueous glycerol and 150 μL of Bacillus megaterium stored in 20% aqueous glycerol in liquid nitrogen, respectively. Incubate on solid medium at 30°C for 48 hours;

[0031] (2) Seed cultivation:

[0032] The preparation of the seed medium is as follows: weigh 20g of L-sorbose, 3g of corn steep liquor, 3g of beef extract, 3g of yeast extract powder, 1g of urea, 10g of peptone, KH 2 PO 4 1g, MgSO 4 0.2g, CaCO 3 Ad...

Embodiment 2

[0049] A method for improving the yield of 2-keto-L-gulonic acid by subculture of mixed bacteria evolution, comprising the steps of:

[0050] (1) Solid culture:

[0051] Take 10 μL of Gluconobacter oxydans stored in 30% aqueous glycerol solution and 10 μL of Bacillus megaterium stored in 30% aqueous glycerol solution and inoculate them respectively Incubate on solid medium at 35°C for 24 hours;

[0052] (2) Seed cultivation:

[0053] Transfer the Bacillus megaterium and Gluconobacter oxidans cultivated in step (1) into the seed culture medium respectively, and cultivate them on a shaking table at 35°C and 200r / min for 24 hours to obtain the Bacillus megaterium seed liquid and the Gluconobacter oxidans seed liquid;

[0054] Inoculate the new seed medium with Bacillus megaterium and Gluconobacter oxydans so that the density of Bacillus megaterium is 2×10 7 cfu / ml, so that the density of Gluconobacter oxydans is 2×10 8 cfu / ml, at 35°C, shake culture at 200r / min on a shaker, t...

Embodiment 3

[0064] A method for improving the yield of 2-keto-L-gulonic acid by subculture of mixed bacteria evolution, comprising the steps of:

[0065] (1) Solid culture:

[0066] Take 200 μL of Gluconobacter oxydans (Gluconobacter oxydans) preserved in 15% aqueous glycerol solution and 200 μL of Bacillus megaterium (Bacillus megaterium) preserved in 15% aqueous glycerol solution stored in liquid nitrogen and inoculate them respectively Incubate on solid medium at 30°C for 36 hours;

[0067] (2) Seed cultivation:

[0068] Transferring the Bacillus megaterium and Gluconobacter oxidans cultured in step (1) into the seed culture medium respectively, and cultured on a shaking table at 30° C. at 240 r / min for 36 hours to obtain the Bacillus megaterium seed liquid and the Gluconobacter oxidans seed liquid;

[0069] Inoculate the new seed medium with Bacillus megaterium and Gluconobacter oxydans so that the density of Bacillus megaterium is 2×10 8 cfu / ml, so that the density of Gluconobacte...

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Abstract

The invention discloses a method utilizing mixed bacteria evolution subculturing to improve the 2-keto-L-gulonic acid yield, which comprises the following steps of: (1) solid culture; (2) seed culturing: the bacillus megaterium and gluconobacter oxydans are inoculated into a new seed culture medium, are shake cultured in a shaker of 200-280r / min at 28-35 DEG C, 24h-48h is taken as the passage period, the volume ratio of 1%-10% is taken as the passage ratio to inoculate into the new seed culture medium and passages for 50-80 days; (3) purification; and (4) fermentation. The method utilizes themixed bacteria evolution subculturing to culture for tens of generations, the growth rate of the bacillus megaterium and gluconobacter oxydans and the efficiency of utilizing the gluconobacter oxydans to produce 2-keto-L-gulonic acid can be obviously improved, accordingly, the utilization rate of the culture medium can be improved, and the L-sorbose transformation efficiency is improved about 10%.

Description

technical field [0001] The invention belongs to the field of industrial microbes, and relates to a method for improving the yield of 2-keto-L-gulonic acid through evolution and subculture of mixed bacteria. Background technique [0002] Vitamin C is a trace water-soluble vitamin necessary for the nutrition and growth of the body, and plays an important role in anti-oxidation and maintaining metabolic balance. Vitamin C can be used as medicine, health products, food additives and cosmetic nutrients, its application scope is expanding and the market is stable. [0003] At present, the method of producing vitamin C in my country is a "two-step fermentation method". The first step of fermentation uses Acetobacter black to convert sorbitol into L-sorbose. Precursor 2-keto-L-gulonic acid. Bacillus megaterium (Bacillus megaterium) and Gluconobacter oxydans (Gluconobacter oxydans) mixed bacteria fermentation used in mixed fermentation, wherein, Gluconobacter oxydans (Gluconobacter ...

Claims

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Application Information

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IPC IPC(8): C12P39/00C12P7/60C12R1/01C12R1/11
Inventor 元英进邹旸胡梦龙吕亚金汪洋
Owner TIANJIN UNIV
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