Method for extracting coronatine from fermentation liquor by using membrane separation technique

A technology of fermented liquid and coronatin, applied in the direction of membrane technology, semi-permeable membrane separation, chemical instruments and methods, etc., to achieve the effect of less separation steps, simple process and mild conditions

Inactive Publication Date: 2012-09-05
JIANGXI AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report on the use of membrane separation technology to separate and extract coronatine from fermentation broth.

Method used

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  • Method for extracting coronatine from fermentation liquor by using membrane separation technique
  • Method for extracting coronatine from fermentation liquor by using membrane separation technique

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Determination of coronatin content

[0029] High-performance liquid chromatography was used for detection, and the chromatographic working conditions were: chromatographic column Symmetry C18 column (Waters, 4.6×150mm, 5 μm); the mobile phase was 60% methanol solution containing 0.05% phosphoric acid; the detection wavelength was 230nm; the flow rate was 1.0mL / min ; Column temperature 35°C. Standards were provided by the Laboratory of Organic Analytical Chemistry, Hokkaido University, Japan.

Embodiment 2

[0030] Example 2 Preparation of coronatin fermentation broth

[0031] 1. Strains

[0032] Coronatine-producing bacteria Burkholderia cepacia (Burkholderia cepacia).

[0033] 2. Medium

[0034] Incline medium (g / L): glucose 10, beef extract 3, peptone 5, yeast extract 1, agar 15, pH 6.8-7.0.

[0035] Seed medium (g / L): glucose 20, beef extract 20, KH 2 PO 4 4.1, K 2 HPO 4 1.8, MgSO 4 ·7H 2 O0.2, FeCl 3 (1.5μmol / L), pH6.8-7.0.

[0036] Basic fermentation medium (g / L): glucose 10, soybean cake powder 20, urea 10, molasses 10, KH 2 PO 4 4.1, K 2 HPO 4 3.6, MgSO 4 ·7H 2 O 0.2, FeCl 3 (1.5μmol / L), pH6.8-7.0.

[0037] 3. Preparation of coronatin fermentation broth

[0038] The strain is cultured on a slant for 1 day, inserted into a seed medium and cultured for 16 hours, then transferred to a fermentation medium, fermented for 3 days, and placed in a tank to obtain a fermented liquid containing coronatin.

Embodiment 3

[0040] 10L of coronatin fermented liquid is passed through a polyvinyl chloride microfiltration membrane with a pore size of 1 μm to remove impurity particles and some bacterial cells. The operating pressure is 0.2MPa, the temperature is 30°C, the concentration is 20 times, and the dialyzed water volume is 2L; the crude extract after filtration flows through Polyolefin ultrafiltration membrane with a pore size of 0.05 μm removes remaining protein, nucleic acid, and colloidal particle macromolecular impurities in the fermentation broth. The operating pressure is 0.15 MPa, the temperature is 30°C, the concentration factor is 15 times, and the dialysis water volume is 4 L; the same as in Example 1 As determined by the method, the total yield of coronatine was 90%. The permeate was further processed by column chromatography, concentrated and crystallized, and the finished product of coronatin with a purity of 97% was obtained.

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Abstract

The invention discloses a method for extracting coronatine from fermentation liquor by using a membrane separation technique, comprising micro-filtration and hyper-filtration steps: filtering a fermentation liquor containing coronatine by a micro filtering membrane with the aperture of 0.1-10 Mum so as to remove impurity particles and thalli; then filtering the liquor by a hyper-filtration membrane with the aperture of 0.001-0.1 Mum so as to remove macromolecule impurities of proteins, nucleic acids and colloid granules to obtain clear filtrate; and then carrying out common resin exchange or column chromatography, concentrating and crystallizing to obtain the coronatine. In order to improve the effect of separating purification, pre-treatment can be added before the micro-filtration, namely, the coronatine fermentation liquor is filtered though the filtration membrane with the aperture of 1-10 Mum; nano-filtration can be added after the hyper-filtration, namely, the effluence liquor is filtered by a nano-membrane system with cut-off molecular weight of 400-800. The method in the invention has simple operation and high yield which can be over 90%; in addition, the method has no influence on the structure and activity of the coronatine; and has low energy consumption and low pollution, thus completely satisfying the requirements of industrialized production.

Description

technical field [0001] The invention belongs to the field of biochemical industry, and in particular relates to a method for separating and extracting active components from fermented liquid by using membrane separation technology. Background technique [0002] Coronatine (I), also known as coronatine (COR for short), is a physiologically active substance produced by microorganisms discovered in the late 1970s. It consists of two special structural parts, namely bicyclic carboxylic acids, called crowns. Acid (II) (coronafacic acid, referred to as CFA), and cyclopropyl amino acid, known as crown amino acid (III) (coronamicacid, referred to as CMA). [0003] Coronatins exhibit multiple functions in plant growth regulation, such as promoting tuber formation and expansion, inhibiting seed germination and root growth, and promoting fruit abscission, etc. (Weiler, 1994; Vignutelli, 1998; Koda, 1994). A number of patents have been applied for the application of coronatin. For exa...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07C235/82C07C231/24B01D61/58B01D61/14
Inventor 吴晓玉
Owner JIANGXI AGRICULTURAL UNIVERSITY
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