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Method for preparing EGCG hydrolytic enzyme and producing EGC and gallic acid by aspergillus oryzae

A technology of gallic acid and Aspergillus oryzae, which is applied in the field of production of EGC and gallic acid, and the induction and preparation of EGCG hydrolase, can solve the problems of low hydrolysis rate, low enzyme yield, easy to be oxidized, etc., and achieves high conversion rate, simple operation, Controllable effect

Inactive Publication Date: 2013-03-06
DONGHUA UNIV
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AI Technical Summary

Problems solved by technology

However, conventional chemical methods such as acid or alkali hydrolysis have disadvantages such as extremely low hydrolysis rate, extremely unstable hydrolyzed products or easily oxidized during the reaction process.
In addition, according to literature reports, pig liver esterase cannot hydrolyze EGCG, and no other commercially available esterase or lipase can hydrolyze EGCG
Only Tannase (Tannase) can hydrolyze EGCG into non-ester type catechin EGC, but the production of this enzyme is small and the price is high

Method used

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  • Method for preparing EGCG hydrolytic enzyme and producing EGC and gallic acid by aspergillus oryzae
  • Method for preparing EGCG hydrolytic enzyme and producing EGC and gallic acid by aspergillus oryzae
  • Method for preparing EGCG hydrolytic enzyme and producing EGC and gallic acid by aspergillus oryzae

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] 1. Seed culture of Aspergillus oryzae ATCC20719

[0034] Aspergillus oryzae seed medium: 1% glucose, 0.1% peptone, 0.05% citric acid, 0.015% Tween80, 2% Vogel's medium N; use Aspergillus oryzae ATCC20719 as the production strain, at pH 5.0, 30°C, 200r / Min conditions for 36 hours.

[0035] 2. Fermentation of Aspergillus oryzae to produce enzymes

[0036] Aspergillus oryzae enzyme production medium: 1% glucose, 0.1% peptone, 0.05% citric acid, 0.015% Tween80, 2% Vogel's medium N, add EGCG to a final concentration of 2.5g / L, pH5.0; with 4% inoculum size, Cultivate at 30°C and 160r / min for 72h to induce enzyme production. The fermented supernatant liquid collected by filtration or centrifugation is the EGCG hydrolase liquid. In the experiment, the fermentation culture without adding EGCG was used as the control.

[0037] 3. Enzymatic hydrolysis of EGCG

[0038] Reaction system: 10g / L EGCG substrate 2mL, EGCG hydrolase solution 2mL, pH5.2 acetate buffer 2mL; EGCG hydro...

Embodiment 2

[0043] 1. Seed Culture of Aspergillus oryzae NRRL692

[0044]Aspergillus oryzae seed medium: 1% glucose, 0.1% peptone, 0.05% citric acid, 0.015% Tween80, 2% Vogel's mediumN; use Aspergillus oryzae NRRL692 as the production strain, pH 5.0, 25—30°C, 200rpm culture 36 hours.

[0045] 2. Fermentation of Aspergillus oryzae to produce enzymes

[0046] Aspergillus oryzae enzyme production medium: 1% glucose, 0.1% peptone, 0.05% citric acid, 0.015% Tween80, 2% Vogel's medium N, add EGCG to the final concentration of 5.0g / L, pH5.0; with 8% inoculum size, Cultivate at 30°C and 160r / min for 72h to induce enzyme production. The fermented supernatant liquid collected by filtration or centrifugation is the EGCG hydrolase liquid. In the experiment, the fermentation culture without adding EGCG was used as the control.

[0047] 3. Enzymatic hydrolysis of EGCG

[0048] Reaction system: 15g / L EGCG substrate 2mL, EGCG hydrolase solution 2mL, pH5.2 acetate buffer 2mL; EGCG hydrolase solution ...

Embodiment 3

[0051] 1. Seed culture of Aspergillus oryzae ATCC201873

[0052] Aspergillus oryzae seed medium: 1% glucose, 0.1% peptone, 0.05% citric acid, 0.015% Tween80, 2% Voge1's medium N; use Aspergillus oryzae ATCC201873 as the production strain, at pH5.0, 30°C, 200r / Min conditions for 36 hours.

[0053] 2. Fermentation of Aspergillus oryzae to produce enzymes

[0054] Aspergillus oryzae enzyme production medium: 1% glucose, 0.1% peptone, 0.05% citric acid, 0.015% Tween80, 2% Vogel's medium N, add EGCG to a final concentration of 10.0g / L, pH5.0; with 8% inoculum size, Cultivate at 30°C and 130r / min for 72h to induce enzyme production. The fermented supernatant liquid collected by filtration or centrifugation is the EGCG hydrolase liquid. In the experiment, the fermentation culture without adding EGCG was used as the control.

[0055] 3. Enzymatic hydrolysis of EGCG

[0056] Reaction system: 30g / L EGCG substrate 2mL, EGCG hydrolase solution 2mL, pH5.2 acetate buffer 2mL; EGCG hyd...

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Abstract

The process of inducing aspergillus oryzae with epigallocatechin gallate (EGCG) to produce EGCG hydrolase in high efficiency includes the following steps: 1. culturing aspergillus oryzae seed in the culture medium at 25-30 deg.c and 100-250 rpm for 36 hr; 2. inoculating to EGCG fermenting culture medium in the inoculated amount of 4-10 %, culturing at 25-30 deg.c and 100-250 rpm for 48-120 hr, filtering and centrifuging to obtain EGCG hydrolase. The process of utilizing EGCG hydrolase in hydrolyzing EGCG to produce epigallocatechin (EGC) and gallic acid includes the steps of constructing EGCG enzyme hydrolyzing reaction system, blank enzyme system and blank substrate system; reaction and substrate and product analysis. The present invention has EGC converting rate and yield, stable and easy-to-separate product, high safety, environment friendship and other advantages, and is suitable for industrial production.

Description

technical field [0001] The invention belongs to the field of enzyme preparation and catalytic transformation of enzyme, in particular relates to the induction preparation of EGCG hydrolase and the method for producing EGC and gallic acid by using the enzyme. Background technique [0002] Tea catechins are the main physiologically active components in tea. Tea catechins consist of epi-gallocatechin-3-gallate (EGCG), epicatechin-3-gallate (epicatechin-3- gallate, ECG), gallocatechin-3-gallate (gallocatechin-3-gallate, GCG), epicatechin (epicatechin, also known as cis-catechin, EC), epigallocatechin (epigallocatechin, also known as cis- gallocatechin, EGC) and other components. Catechin monomers are highly effective in anti-oxidation, scavenging free radicals, anti-tumor, anti-mutation, anti-aging, anti-virus, anti-allergy, anti-bacterial and anti-inflammatory, enhancing immune function, anti-radiation, lowering blood pressure, and lowering blood fat. It has a good effect on ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/14C12N9/14C12P7/42C12R1/69
Inventor 洪枫钟坤
Owner DONGHUA UNIV
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