A kind of separation and purification method of pyrroloquinoline quinone in methylotrophic bacteria fermentation broth and its application

A technology for separation and purification of pyrroloquinoline quinone is applied in the field of separation and purification of pyrroloquinoline quinone, which can solve the problems of environmental pollution and high application cost, and achieve the effects of simple process operation, high degree of connection and single crystal form

Active Publication Date: 2019-05-14
FUJIAN NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The above method needs to use a large amount of organic solvents such as n-hexane, methanol or ethanol, and the industrial application cost is high and pollutes the environment

Method used

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  • A kind of separation and purification method of pyrroloquinoline quinone in methylotrophic bacteria fermentation broth and its application
  • A kind of separation and purification method of pyrroloquinoline quinone in methylotrophic bacteria fermentation broth and its application
  • A kind of separation and purification method of pyrroloquinoline quinone in methylotrophic bacteria fermentation broth and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1, pyrroloquinoline quinone produced by denitrification hyphae microbacteria FJNU-R8

[0040] 1. Fermentation and supernatant preparation of denitrifying hyphaetic bacteria FJNU-R8

[0041] (1) Pick a single colony of FJNU-R8 into a Erlenmeyer flask filled with medium, and culture it at 32°C and 220rpm for 24-36 hours (OD 650 Between 2.5-3.0, adjust to zero with the seed medium) to obtain the seed solution.

[0042] The seed liquid is transferred to the fermentation medium with an inoculum amount of 10% (volume fraction), and fermented and cultivated at 30°C for 5-6 days to obtain a fermentation liquid; fermentation conditions: liquid loading (volume ratio of the fermentation medium to the fermenter) During the fermentation process, by feeding methanol and ammonia water, the concentration of methanol in the fermentation broth is controlled at 1-2g / L, and the pH is controlled at 6.8-7.0; stirring is associated with dissolved oxygen, and the dissolved oxygen (...

Embodiment 2

[0067] Embodiment 2, pyrroloquinoline quinone produced by denitrifying hyphaetic bacteria DSM 1869

[0068] 1. Fermentation and supernatant preparation of denitrifying hyphaetic bacteria DSM 1869

[0069] (1) Pick a single colony of DSM 1869 into a Erlenmeyer flask equipped with seed medium, and cultivate it for 24-36 hours at 32°C and 220rpm (OD 650 Between 2.5-3.0, adjust to zero with the seed medium) to obtain the seed liquid.

[0070] Transfer the seed liquid into the fermentation medium with an inoculation amount of 10% (volume fraction), and ferment and cultivate at 30°C for 7-8 days to obtain a fermented liquid; fermentation and culture conditions: the liquid loading is 70%, and methanol is added during the fermentation process and ammonia water, so that the concentration of methanol in the fermentation broth is controlled at 1-2g / L, and the pH is controlled at 6.8-7.0; stirring is associated with dissolved oxygen, and the dissolved oxygen amount (DO) is controlled at ...

Embodiment 3

[0095] Embodiment 3, the production of pyrroloquinoline quinone by Methylobacter spirochete CGMCC 1.6987

[0096] 1. Fermentation and supernatant preparation of Methylobacter spirochete CGMCC 1.6987

[0097] (1) Pick a single colony of CGMCC 1.6987 into a Erlenmeyer flask equipped with seed medium, and cultivate it for 24-36 hours at 32°C and 220rpm (OD 650 Between 2.5-3.0, adjust to zero with the seed medium) to obtain the seed liquid.

[0098] The seed liquid is transferred to the fermentation medium with an inoculum amount of 10% (volume fraction), and fermented and cultivated at 30°C for 4-5 days to obtain a fermented liquid; fermented culture conditions: the liquid load is 70%, and methanol is added during the fermentation process and ammonia water, so that the concentration of methanol in the fermentation broth is controlled at 1-2g / L, and the pH is controlled at 6.8-7.0; stirring is associated with dissolved oxygen, and the dissolved oxygen amount (DO) is controlled at...

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Abstract

The invention discloses a method for separating and purifying pyrroloquinoline quinone from a methylotrophic bacteria fermentation broth and an application thereof. The method of the present invention comprises the following steps: (1) enriching the fermented liquid through a macroporous resin, and then successively eluting through a buffer solution and water, collecting the eluent, and obtaining an enriched solution rich in pyrroloquinoline quinone; (2) purifying the enriched solution rich in pyrroloquinoline quinone through a hydrophilic silica gel filler to obtain a pyrroloquinoline quinone mother liquor; (3) separating out the pyrroloquinoline quinone crystallization in the pyrroloquinoline quinone mother liquor , to obtain pyrroloquinoline quinone crude crystals; (4) recrystallize the pyrroloquinoline quinone crude crystals by alkali-dissolving acid precipitation method to obtain pyrroloquinoline quinone. It is proved by experiments that the method of the present invention has the characteristics of simple process operation, high coherence, high product recovery rate and high purity, and can be used for large-scale industrial production to prepare pyrroloquinoline quinone.

Description

technical field [0001] The invention belongs to the field of biochemical industry, and in particular relates to a method for separating and purifying pyrroloquinoline quinone in a fermentation liquid of methylotrophic bacteria and an application thereof. Background technique [0002] Pyrroloquinoline quinone (PQQ) belongs to the group of aromatic tricyclic orthoquinones and acts as the third oxidoreductase cofactor found after riboflavin and pyridine nucleotides. PQQ participates in the oxidation-reduction process of dehydrogenase, cooperates with COQ to complete the electron transfer of the respiratory chain, exists widely in organisms, and has the functions of promoting the synthesis and repair of nerve growth factors, preventing nerve cell fibrosis, scavenging free radicals, and maintaining mitochondrial energy metabolism, etc. Physiological functions have broad application prospects in the fields of medicine, food and cosmetics. Due to many isomers and by-products, low ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07D471/04C12P17/18C12N1/20C12R1/01
CPCC07B2200/13C07D471/04C12P17/182C12N1/205C12R2001/01
Inventor 柯崇榕黄建忠杨欣伟任洋魏杰廖灿
Owner FUJIAN NORMAL UNIV
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