Method for producing indigo pigment with bacillus subtilis

A technology of Bacillus subtilis and indigo pigment, which is applied in the field of producing natural indigo pigment, can solve the problems of enzyme instability, low pigment secretion ability, and long strain culture period, and achieve the same synthetic ability, simple operation, and high production efficiency. Effect

Inactive Publication Date: 2013-06-12
XUZHOU UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the reported microbial catalysis also has many problems in the production and application of indigo pigment: such as the long culture period of the strain, low pigment secretion ability, unstable enzyme in the reaction medium, etc. strains are significant

Method used

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  • Method for producing indigo pigment with bacillus subtilis
  • Method for producing indigo pigment with bacillus subtilis
  • Method for producing indigo pigment with bacillus subtilis

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] The screening identification of embodiment 1 producing indigo pigment strain

[0030] 1. Sample collection

[0031] Soil samples were collected from the sludge near the sewage treatment plant in Xuzhou City, Jiangsu Province.

[0032] 2. Isolation and screening of bacterial strains On September 12, 2011, soil samples were collected from the sludge of Kui River, Xuzhou City, Jiangsu Province, and the soil samples were collected by Gao Zhaojian.

[0033] Prepare solid primary screening medium (unit g / 100ml): yeast extract 0.2, NaCl 0.2, (NH 4 ) 2 SO 4 0.2, NH 4 NO 3 0.1,K 2 HPO 4 0.01, MgSO 4 ·7H 2 O0.01, FeSO 4 ·7H 2 O0.004, agar powder 1.5, pH7.5. The prepared culture is based on 121°C, sterilized for 15 minutes, and poured onto the plate. Indole was made into an indole solution with a final concentration of 0.5g / 100ml with ethanol, and the sterilized medium was cooled to about 50°C, and 3ml of indole solution was added per 100ml of medium, mixed well and t...

Embodiment 2

[0039] The fermentation culture of embodiment 2 bacterial strains and the extraction of indigo pigment crude product

[0040] 1. Culture of strains

[0041] Prepare seed liquid medium (unit g / 100ml): peptone 1, glucose 0.5, yeast powder 0.5, NaCl 0.2, (NH 4 ) 2 5O 4 0.2, NH 4 NO 3 0.1,K 2 HPO 4 0.01, MgSO 4 ·7H 2 O0.01, pH7.5. The prepared culture is based on 121°C and sterilized for 15 minutes. Inoculate a single colony of the strain JWDL into the above medium, fill a 250ml Erlenmeyer bottle with 40ml of liquid, and culture with shaking at 32°C and 180r / min for 20h.

[0042] Preparation of fermentation medium (g / L): peptone 0.5, glucose 0.5, yeast powder 0.5, (NH 4 ) 2 5O 4 0.25, MgSO 4 ·7H 2 O0.01, FeSO 4 ·7H 2 O0.04, NH 4 NO 3 0.1, pH7.5. The prepared culture is based on 121°C and sterilized for 15 minutes. Inoculate the cultured seed liquid into the sterilized fermentation medium according to the inoculum amount of 4%, fill the volume of liquid in a 10...

Embodiment 3

[0045] The structural identification of embodiment 3 pigment

[0046] 1. Thin-layer chromatography (TLC) analysis: the prepared crude indigo sample was dissolved in N,N-dimethylformamide (DMF). The sample and the standard sample were respectively spotted on the thin electrode of TLC. The developing solvent is methanol-acetone / 1:1 (v / v) solvent system. After fully unfolding, blow dry with a hair dryer. The result is as figure 2 As shown, a blue spot appears at the same position on the chromatographic plate.

[0047] 2. Ultraviolet-visible broad-spectrum scanning analysis: the indigo sample dissolved in N, N-dimethylformamide (DMF) is the same as the standard indigo sample, and the ultraviolet-visible (250nm-700nm) broad-spectrum scanning is performed. The result is as image 3 As shown, the prepared sample is basically consistent with the scanning broad-spectrum absorption curve of the standard sample, and both show absorption peaks at 285nm and 600nm. This proves that t...

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Abstract

The invention relates to a method for producing natural indigo pigment and particularly discloses a method for producing indigo pigment with the bacillus subtilis. The method includes the following steps: (1) bacterial strain fermental cultivation: inoculating seed solution of China General Microbiological Culture Collection Center (CGMCC) 5698 of the bacillus subtilis producing the indigo pigment into a sterilized liquid fermentation medium, and cultivating the bacillus subtilis at 26-34 DEG C for 46-60h, wherein the rotation speed of a swing bed is 200r/min; and (2) extracting indigo pigment from the bacillus subtilis. The method for producing the indigo pigment with the bacillus subtilis has the advantages that bacterial strains of the bacillus subtilis can synthesize indigo pigment in the liquid fermentation medium efficiently, and the production efficiency is high. The requirements for a carbon source and a nitrogen source by the bacterial strains are low, the genetic stability is good and the operation is simple.

Description

Technical field: [0001] The present invention relates to a method of producing natural indigo pigment. Background technique: [0002] Indigo is a bright and stable blue pigment. Structurally, indigo is an aromatic compound mainly used for dyeing of fabrics, such as jeans are obtained by dyeing textiles with indigo. In addition to being used as dyes, indigo and indirubin (a kind of indigo pigment) also have important applications in the pharmaceutical industry, and can have important therapeutic effects on some diseases, including cancer-related diseases, Alzheimer's disease, and delayed hypersensitivity. In the food industry, indigo can be added to food as a kind of food coloring additive. The traditional indigo production is mainly extracted from plants, and the leaves of some plants such as woad, Polygonum indigo, horse blue, and wild green tree are rich in indigo pigment. Indigo extracted from plants has high safety and less pollution, but its yield and purity are low,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P17/16C12R1/125
Inventor 高兆建刘全德纪伟陈尚龙巫永华吴如波
Owner XUZHOU UNIV OF TECH
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