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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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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Lower Yield Affects Large-Scale Production of 2-O-Glucosyl Ascorbic Acid

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] The preparation of embodiment 1 Paenibacillus macerans cyclodextrin glucosidyl transferase

[0016] Peanibacillus macerans JFB05-01, the preservation number is CCTCC NO: M 208063, can be purchased in China Center for Type Culture Collection, and the strain submitted by this laboratory. Take a ring of slant bacteria and inoculate to 500ml containing 80mL seed medium. Cultivate in a Erlenmeyer flask with a shaker speed of 200r / min, and cultivate at 37°C for 18h. The cultivated seed culture solution was inoculated into a 500mL Erlenmeyer flask containing 80mL of fermentation medium according to the inoculum amount of 10% (volume ratio), and fermented at 37°C for 24 hours. The rotating speed is 200r / min. After stopping the fermentation, the fermentation liquid was centrifuged at 12000r / min for 20min, and the supernatant was taken as the crude enzyme.

[0017] Seed medium (g*L -1 ) consists of: soluble starch 40, peptone 25, corn steep liquor 5, MgSO 4 ·7H 2 O 0.5, CaC...

Embodiment 2B

[0019] The preparation of embodiment 2 Bacillus licheniformis cyclodextrin glucosidyl transferase

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

[0021] Seed medium (g*L -1 ) consists of: 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. pH value 7.0, sterilized at 121°C for 15 minutes.

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

Embodiment 3

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

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

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

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

[0027] Liquid phase instrument: Agilent 1200 liquid phase instrument

[0028] Column: Agilent SB-Aq

[0029] Flow rate: 0.5mL / min

[0030] Detector: DAD

[0031] Column temperature: 25°C

[0032] Detectio...

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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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