Method for increasing output of 2-keto-L-gulonic acid by strengthening mutual effect of two bacteria

A technology of gulonic acid and ketone group is applied in the field of strengthening the interaction between two bacteria and improving the production of 2-keto-L-gulonic acid, which can solve the problems of low acid production efficiency and slow growth, and achieves improvement of transformation efficiency and utilization efficiency. , the effect of improving efficiency

Inactive Publication Date: 2013-04-17
TIANJIN UNIV
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  • Summary
  • 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

Method used

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  • Method for increasing output of 2-keto-L-gulonic acid by strengthening mutual effect of two bacteria
  • Method for increasing output of 2-keto-L-gulonic acid by strengthening mutual effect of two bacteria
  • Method for increasing output of 2-keto-L-gulonic acid by strengthening mutual effect of two bacteria

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] The method for strengthening the interaction between two bacteria to improve the output of 2-keto-L-gulonic acid comprises the following steps:

[0029] (1) Solid culture:

[0030] 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;

[0031] Inoculate 150 μL of Gluconobacter oxydans 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;

[0032] (2) Seed cultivation:

[0033] 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 41g, MgSO 4 0.2g, CaCO 3 Add 1 g of water...

Embodiment 2

[0042] The method for strengthening the interaction between two bacteria to improve the output of 2-keto-L-gulonic acid comprises the following steps:

[0043] Step (1), (2) and (3) are the same as embodiment 1

[0044] (4) Fermentation:

[0045] The original Bacillus megaterium and the evolved Gluconobacter oxydans were inoculated into the fermentation medium so that the density of the original Bacillus megaterium was 2×10 7 cfu / ml, so that the density of the evolved Gluconobacter oxydans is 2 × 10 9 cfu / ml, culture at 30°C, 250r / min shaker for 96h to obtain 2-keto-L-gulonic acid.

[0046] Determination of 2-keto-L-gulonic acid and L-sorbose content:

[0047] High performance liquid chromatography (HPLC) was used.

[0048] Sample preparation: Take 1 mL of fermentation broth fermented for different times in a 1.5 mL centrifuge tube, centrifuge at 10,000 r / min for 3 min, take 100 μL of the supernatant in a 1.5 mL centrifuge tube, and add 900 μL of mobile phase (5 mM H 2 SO...

Embodiment 3

[0057] The method for strengthening the interaction between two bacteria to improve the output of 2-keto-L-gulonic acid comprises the following steps:

[0058] (1) Solid culture:

[0059] 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;

[0060] (2) Seed cultivation:

[0061] 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;

[0062] 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, 200r / min shaker cultur...

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Abstract

The invention discloses a method for increasing the output of 2-keto-L-gulonic acid by strengthening mutual effect of two bacteria, which comprises the following steps of (1) solid culture; (2) seed culture, namely, inoculating bacillus megatherium and gluconobacter oxydans into new seed medium, carrying out shaking culture in a table concentrator by 200-280r/min at 28-35 DEG C, and inoculating into new seed medium with 1%-10% of volume ratio as passage ratio and 24h-48h as passage period, and carrying out passage for 100-200 days; (3) separation and purification; and (4) fermentation. According to the method, after more than tens of generations of subculture by mixed bacteria evolution, the growing speed of bacillus megatherium and gluconobacter oxydans and the efficiency of the gluconobacter oxydans generating 2-keto-L-gulonic acid can be improved remarkably, so that the utilization efficiency of the culture medium can be improved, and the conversion efficiency of L-sorbose can be improved by 10%-15%.

Description

technical field [0001] The invention belongs to the field of industrial microorganisms, and relates to a method for strengthening the interaction between two bacteria and increasing the output of 2-keto-L-gulonic acid. 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 (2-KLG). Bacillus megaterium (Bacillus megaterium) and Gluconobacter oxydans (Gluconobacter oxydans) mixed bacteria fermentation used in mixed fermentation, wherein, Gluconobacter oxy...

Claims

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Application Information

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IPC IPC(8): C12P41/00C12P7/60C12R1/01C12R1/11
Inventor 元英进邹旸吕亚金胡梦龙汪洋
Owner TIANJIN UNIV
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