Method for producing pyrroloquinoline quinine through microbial fermentation and fermentation medium used in same

A pyrroloquinoline quinone and microbial fermentation technology, applied in the field of microbial fermentation, can solve the problems of long production cycle of strains, low yield, restriction of synthesis regulation and the like

Active Publication Date: 2013-07-31
INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The main factors affecting the large-scale application of microbial fermentation to produce PQQ are: (1) There are not many microorganisms suitable for industrial production; (2) The PQQ production strains reported in the literature have a long production cycle and low yield; (3) PQQ as a Coenzymes are not secondary metabolites of microorganisms, and their synthesis regulation is strictly restricted by culture conditions and metabolic regulation

Method used

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  • Method for producing pyrroloquinoline quinine through microbial fermentation and fermentation medium used in same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] Embodiment 1 The activation of methylotrophic bacteria Methylovorus sp.MP688

[0088] Methylovorus sp.MP688 is preserved in CGMCC4096 of China General Microorganism Culture Collection Center, and it is a strain whose PQQ production reaches the production level. Use the original strain (glycerol preservation or freeze-dried state) to streak on a solid medium plate, and culture it at 30°C for 2 days; pick a single colony, inoculate it into 5ml liquid medium, culture it at 30°C, shaker at 200 rpm, and cultivate it for 48 hours as activated seeds. The solid medium is to add agar powder and carbon source methanol on the basis of the basic medium, and the concentration of the agar powder in the fixed medium is 1g / L; Carbon source methanol is added on the basis; wherein the concentration of the methanol in the fixed medium or liquid medium is 10g / L; the components and proportioning of the basic medium are as follows:

[0089] Basic culture medium (in 1L)

[0090] MgSO 4 ·7...

Embodiment 2

[0104] The preparation of embodiment 2 seed liquid

[0105] Get 2ml of the bacterial solution obtained in Example 1, and inoculate it into a conical flask containing 100ml of liquid medium, at 30°C, on a shaker at 200 rpm, and cultivate for 36 hours, and activate it 1-2 times under the same conditions to make the final fine density ( OD 600 ) is 1.2 to 1.5, made into seed solution. The liquid medium used is the same as in Example 1.

Embodiment 3

[0106] Embodiment 3 carries out PQQ production example A in 5L automatic fermenter

[0107] With the seed liquid of embodiment 2, inoculate in the 7.5L automatic fermenter according to the ratio of 1:20. After inoculation, the initial ventilation volume of the fermentation was controlled at 1vvm, the stirring speed was controlled at 300rpm, the temperature was controlled at 0°C, and the initial pH value was set at 7.0; the initial concentration of methanol as a carbon source was 7.5g / L, and the initial concentration of ammonium sulfate as a nitrogen source was 15g / L. L; described fermentation medium, concrete composition and proportioning are as follows:

[0108] Fermentation medium (according to 1L)

[0109] MgSO 4 ·7H 2 O 0.4g,

[0110] (NH 4 ) 2 SO 4 3g,

[0111] K H 2 PO 4 2.8g,

[0112] Na 2 HPO 4 12H 2 O 6g,

[0113] Ferric Citrate 60mg,

[0114] CaCl 2 0.05mg,

[0115] MnCl 2 4H 2 O 5mg,

[0116] ZnSO 4 ·7H 2 O 5...

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Abstract

The invention discloses a method for producing pyrroloquinoline quinine through microbial fermentation and a fermentation medium used in the method for producing the pyrroloquinoline quinine through the microbial fermentation. An oxygen control fermentation culture method is used for culturing methylotrophic bacteria, a carbon source contained in the fermentation medium is methyl alcohol, and a nitrogen source contained in the fermentation medium is ammonium sulfate, a dissolved oxygen level is controlled by stages to be 30%-50% of saturated dissolved oxygen in the fermentation process, pH is controlled to be 5.5-6.0 through fed-batch, the fed-batch is achieved through a pH-nitrogen source joint control technology, the methylotrophic bacteria are cultured for six days in a 7.5L fermentation tank, and eventually the cell density (OD 600) exceeds 10, yield of PQQ reaches 2g/L, and production intensity of the PQQ is 333.33mg/L/day. The method for producing the pyrroloquinoline quinine through the microbial fermentation is simple in operation, automatic control and continuous fermentation can be achieved easily, and a solid foundation for industrially producing the pyrroloquinoline quinine through the methylotrophic bacteria is laid.

Description

technical field [0001] The invention relates to the research field of microbial fermentation technology, in particular to a method for efficiently producing pyrroloquinoline quinone (PQQ) by microbial fermentation and a fermentation medium used. Background technique [0002] Pyrroloquinoline quinone (PQQ) is a coenzyme of the third type of oxidoreductase, and is also known as a new vitamin. It has important medical value in regulating the level of free radicals, resisting radiation damage, cardiovascular disease and diabetes. [0003] At present, PQQ is mainly a chemical synthesis method. Microbial synthesis has the advantages of clean and low-carbon, mild and controllable, and low cost. It is a production method with great potential. The main factors affecting the large-scale application of PQQ produced by microbial fermentation are: (1) There are not many microorganisms suitable for industrial production; (2) The PQQ production strains reported in the literature have a lo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P17/18C12R1/01
Inventor 葛欣张惟材熊向华汪建华
Owner INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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