Method for efficiently producing 2-O-glucose-based ascorbic acid

An ascorbic acid and glucose-based technology, applied in the field of high-efficiency production of 2-O-glucosyl ascorbic acid by biological methods, can solve problems such as low yield and affect the large-scale production of 2-O-glucosyl ascorbic acid, and achieves reduction of production costs and optimization. The effect of the production process

Inactive Publication Date: 2010-12-15
JIANGNAN UNIV
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Lower Yield Affects Large-Scale Prod

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0015] Example 1 Preparation of Paenibacillus macerans cyclodextrin glucosidase

[0016] Peanibacillus macerans (Peanibacillus macerans JFB05-01), the deposit number is CCTCC NO: M 208063, can be purchased at the China Type Culture Collection, and the strain is submitted for this laboratory. Take a ring of slant bacteria to inoculate 500ml with 80mL seed culture medium. Cultivate in a triangular flask with a shaker speed of 200r / min and culture at 37°C for 18h. Inoculate the cultured seed culture solution into a 500mL Erlenmeyer flask containing 80mL fermentation medium according to an inoculum of 10% (volume ratio), and ferment for 24h at 37°C. The HYG II type rotary constant temperature and speed regulating shaker cabinet The speed is 200r / min. After the fermentation is stopped, the fermentation broth is centrifuged at 12000r / min for 20min, and the supernatant is taken as the crude enzyme.

[0017] Seed medium (g*L -1 ) Composition: soluble starch 40, peptone 25, corn steep li...

Example Embodiment

[0019] Example 2 Preparation of Bacillus licheniformis cyclodextrin glucosidase transferase

[0020] Take a ring of slant bacteria to inoculate 250mL with 40mL seed culture medium. Cultivate in a triangular flask with a shaker speed of 200r / min and culture for 12 hours at 38°C. The cultured seed culture solution was inoculated into a flask (250 mL) containing 40 mL of enzyme-producing medium according to an inoculum of 10% (volume ratio). After the inoculation, it was incubated at 38°C with constant temperature shaking for 96 hours, and then centrifuged at 4000 r / min for 20 minutes. The supernatant is the crude enzyme solution.

[0021] Seed medium (g*L -1 ) Composition: soluble starch 10, peptone 5, yeast extract 5, MgSO 4 ·7H 2 O0.2, K 2 HPO 4 ·3H 2 O 1, NaH 2 PO 4 ·2H 2 O 1, NaCO 3 5. The pH value is 7.0, and sterilized at 121°C for 15 minutes.

[0022] Fermentation medium (g*L -1 ) Composition: soluble starch 10, peptone 5, yeast extract 5, MgSO 4 ·7H 2 O0.2, K 2 HPO 4 ·3H 2 O ...

Example Embodiment

[0023] Example 3 Synthesis of 2-O-glucosyl ascorbic acid

[0024] Dissolve ascorbic acid and β-cyclodextrin in 100 mM acetate buffer to prepare a mixed solution, add the crude cyclodextrin glucosidyl transferase enzyme solution to make the ascorbic acid concentration 50g / L and β-cyclodextrin concentration 50g / L L, the concentration of cyclodextrin glucosidase transferase is 160 U / mL.

[0025] Adjust pH to 5.5, temperature to 37°C, avoid light and oxygen, and shake at 100 revolutions per minute for 24 hours. Sampling was taken during the reaction, and the production of 2-O-glucosyl ascorbic acid was detected by HPLC. When the reaction reached 24 h, the yield of 2-O-glucosyl reached 7 g / L.

[0026] 2-O-glucosyl ascorbic acid determination method (HPLC method):

[0027] Liquid instrument: Agilent 1200 liquid instrument

[0028] Column: Agilent SB-Aq

[0029] Flow rate: 0.5mL / min

[0030] Detector: DAD

[0031] Column temperature: 25℃

[0032] Detection wavelength: 238nm

[0033] Mobile phase:...

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Abstract

The invention discloses a method for efficiently producing 2-O-glucose-based ascorbic acid. The 2-O-glucose-based ascorbic acid is synthesized by using L-ascorbic acid and beta-cyclodextrin as substrates through transformation, wherein the synthesizing conditions are as follows: the temperature is 35-45 DEG C, the pH value is 4.5-5.5, the concentration of the beta-cyclodextrin is 25-100g/L, the concentration of the ascorbic acid is 25-100g/L, and the amount of the glycosyltransferase is 50-200U/mL. The method of the invention has the advantages of simple operation, easy control, low requirements on transformation condition and high output of the 2-O-glucose-based ascorbic acid of 8.5g/L, and provides possibility for massively producing the 2-O-glucose-based ascorbic acid by using a biotransformation method.

Description

technical field [0001] The invention relates to a method for producing 2-O-glucosyl ascorbic acid, in particular to a method for efficiently producing 2-O-glucosyl ascorbic acid through a biological method. Background technique [0002] L-ascorbic acid (abbreviated as VC) plays an important physiological role in the body, but its reducing property is strong and extremely unstable, which limits its application. The derivatives of VC greatly improved its stability without affecting its physiological function. This patent concerns the biosynthesis of one of its derivatives, 2-O-glucosyl ascorbic acid, under the action of glycosyltransferase. [0003] 2-O-glucosyl ascorbic acid is an important derivative of L-ascorbic acid, which has wider application value than L-ascorbic acid. In 1990, the Hayashibara Institute of Biochemistry in Japan and the Department of Pharmacy of Okayama University jointly discovered 2-O-glucosyl ascorbic acid AA-2G, and have determined a large number ...

Claims

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Application Information

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IPC IPC(8): C12P19/60
Inventor 陈坚堵国成李江华刘龙张子臣
Owner JIANGNAN UNIV
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