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Method for generating resveratrol by converting glucose by using intracellular enzyme of alternaria microorganism

A technology of Alternaria genus and resveratrol, which is applied in the field of converting glucose into resveratrol by intracellular enzymes of Alternaria genus microorganisms, which can solve the problems of long production cycle, large influence on production status, and unstable yield. , to achieve the effects of short production time, low production cost and high production efficiency

Active Publication Date: 2014-12-24
西安天一生物技术股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The production of resveratrol by biosynthesis is mainly limited to the application of plant cells and microbial culture methods, but there are problems such as long production cycle, greatly affected by the production state of biological cells, and unstable yield

Method used

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  • Method for generating resveratrol by converting glucose by using intracellular enzyme of alternaria microorganism
  • Method for generating resveratrol by converting glucose by using intracellular enzyme of alternaria microorganism
  • Method for generating resveratrol by converting glucose by using intracellular enzyme of alternaria microorganism

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Step 1: Preparation of enzyme solution:

[0049] The Alternaria CCTCC No: M2011348 strain was inoculated into potato juice glucose liquid medium (1000 mL of 20% potato juice, 20 g glucose, natural pH), and cultured at 28°C and 120 rpm for 4 days. Take the culture to collect the mycelium by filtration, and rinse it with distilled water. The obtained mycelium was ground and broken in liquid nitrogen, and the enzyme extract (50mmol / L pH=7 phosphate buffer solution, 0.1g / L magnesium sulfate was added at the ratio of 8 ml crude enzyme extract per 4.0 g of bacteria , 0.2g / L calcium sulfate, 0.6mmol / L DTT). After mixing uniformly, centrifuge at 4°C and 1000 rpm for 10 min, and collect the supernatant. Add solid ammonium sulfate to the supernatant to its saturation concentration of 75%, let it stand overnight at 4°C and centrifuge at 1000rpm for 10 minutes, collect the precipitate and dissolve it with the enzyme extract, put it into a dialysis bag, and dialyze at 4°C until nothi...

Embodiment 2

[0057] Step 1: Preparation of enzyme solution:

[0058] The same as in Example 1.

[0059] Step 2: Generate resveratrol:

[0060] Take 100 mL of 50 mmol / L pH 7.0 phosphate buffer solution containing 0.1 g / L magnesium sulfate, 0.2 g / L calcium sulfate and 1 mmol / L glucose, add 5 mL of crude enzyme solution, and place it at 37°C and 120 rpm After reacting for 0.5 to 4 hours, mass spectrometry and high performance liquid chromatography were used to qualitatively and quantitatively analyze the resveratrol production in the reaction solution at different times.

[0061] Mass spectrometry and high performance liquid chromatography were used to qualitatively analyze the yield of resveratrol: the same as in Example 1.

[0062] Experimental results:

[0063] The content of resveratrol at 0.5 h and 4.0 h was much higher than other reaction times, indicating that it only takes 0.5 h to obtain higher resveratrol yield. The reaction efficiency is greatly improved.

Embodiment 3

[0065] Step 1: Preparation of enzyme solution:

[0066] The same as in Example 1.

[0067] Step 2: Generate resveratrol:

[0068] Take 100 mL of 50 mmol / L pH 7.0 phosphate buffer solution containing 0.1 g / L magnesium sulfate, 0.2 g / L calcium sulfate and different concentrations of glucose (0.5, 1.0, 1.5, 2.0, 2.5, 5.0, 10.0 mmol / L), Add 5 mL of crude enzyme solution, react for 0.5 h at 37°C and 120 rpm, and then use mass spectrometry and high performance liquid chromatography to qualitatively analyze the resveratrol output in the reaction solution.

[0069] Mass spectrometry and high performance liquid chromatography were used to qualitatively analyze the yield of resveratrol: the same as in Example 1.

[0070] Experimental results:

[0071] When the glucose concentration was 2.0 mmol / L, resveratrol produced the highest.

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Abstract

The invention relates to a method for generating resveratrol by converting glucose by using an intracellular enzyme of an alternaria microorganism. The production of the resveratrol mainly depends on the direct extraction from a plant, and the main plant which can be widely applied to the production is the root of the traditional Chinese medicine of wild polygonum cuspidatum, so that the development of the industry is severely limited. The method comprises the following steps: inoculating the alternaria strain under the CCTCC No:M2011348 (China Center For Type Culture Collection Number) into a liquid culture medium for culturing, filtering the cultured product, collecting mycelia, cleaning, grinding and crushing the mycelia, adding a crude enzyme extracting solution into the crushed mycelia, centrifuging, collecting the supernatant, adding solid ammonium sulfate into the supernatant, standing overnight, centrifuging the solution, collecting the sediment to obtain the crude enzyme solution, and adding the crude enzyme solution into a buffer solution and reacting to obtain the resveratrol. According to the method provided by the invention, the intracellular enzyme of the alternaria microorganism is used as the catalyst, the glucose is used as the substrate, and the resveratrol can be synthesized by conversion in a mild reaction system, and therefore, the method is safe, environment-friendly, high in production efficiency, strong in controllability, low in production cost, and suitable for large-scale industrial production.

Description

technical field [0001] The invention belongs to the technical field of production of stilbene compounds, and in particular relates to a method for converting glucose into resveratrol by intracellular enzymes of Alternaria microorganisms. Background technique [0002] Resveratrol is a plant stilbene compound with a variety of physiological activities. It has the functions of preventing and slowing down various diseases such as cancer, cardiovascular disease and ischemic injury, and has the ability to enhance stress resistance and prolong life from yeast to vertebrate The efficacy of a variety of biological life. At the same time, resveratrol is an important precursor for the production of acylated resveratrol, oxidized resveratrol and other functional active substances. It is widely used in functional food processing and pharmaceutical production, and has a large market demand. [0003] Currently, the production of resveratrol mainly relies on direct extraction from plants....

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P7/22C12R1/645
Inventor 师俊玲车金鑫
Owner 西安天一生物技术股份有限公司
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