Quick throughput screening method of high temperature and high acid resistant citric acid high-yield strain

A high-yield strain, citric acid technology, applied in the field of microorganisms, can solve the problems of low efficiency and directional

Inactive Publication Date: 2018-10-30
TTCA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0018] The technical problem to be solved in the present invention is to overcome the defects of low efficiency and poor orientation of mutagenesis means in the prior art, and provide a high-throughp

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Prepare medium

[0076] Prepare the medium required for strain screening and fermentation, and the composition and proportion of each medium are as follows:

[0077] PDA solid medium (g / L) consists of: 20 grams of glucose, 200 grams of boiled potatoes, 20 grams of agar, MgSO 4 7H2O 1 g, K 2 HPO 4 2 grams, 0.5 grams of ammonium sulfate; sterilize at 0.1 MPa, 121°C for 20 minutes, and cool.

[0078] Bromocresol green selective medium: PDA solid medium supplemented with 0.04% bromocresol green.

[0079] High-acid type selection medium: add 80g citric acid on the basis of bromocresol green selection medium.

[0080] Mycelia growth medium: on the basis of PDA liquid medium without agar, add yeast extract 5g / L and peptone 5g / L.

[0081] Plate hyperosmotic medium: PDA solid medium supplemented with 0.6mol / L NaCl.

[0082] Shake flasks and fermentation medium were prepared through the following preparation steps:

[0083] Take 100 grams of corn flour, add 300 ml of wate...

Embodiment 2

[0085] Preparation of Aspergillus niger fusion strain:

[0086] The preparation of Aspergillus niger fusion strain comprises selection of Aspergillus niger parent strain and carrying out the cultivation step and the fusion step, specifically as follows:

[0087] Select the two parent Aspergillus niger strains as the starting strains, which are high-temperature-resistant and high-acid-resistant mutagenic strains respectively, and are marked as Aspergillus niger A (Aspergillus niger, the preservation number is CCTCC NO: M 2011359) and Aspergillus niger B (the preservation number is CGMCCNo. .5751). Under the condition of aseptic operation, it is diluted and separated, spread on the medium plate, and cultured at 36-37° C. for 48 hours to prepare the fusion strain.

[0088] The preparation of Aspergillus niger fusion strain comprises the preparation and inactivation of protoplast, the steps of protoplast fusion, and concrete steps are:

[0089] S1: Preparation of protoplasts:

...

Embodiment 3

[0098] Spot the fused strains on each group of hyperosmotic plates onto selective medium plates, select three places in each group of plates to plant three selective medium plates, and culture them at 42°C for 3 days, and select at least A group of colonies with a large color change circle obtained a total of 90 fused Aspergillus niger strains that were initially screened for high temperature resistance.

[0099] Since the citric acid produced during the growth of Aspergillus niger can change the pH of the medium around the colony, the bromocresol green will change color, and a yellow color circle will appear. Observe and compare the ratio of the diameter of the color change circle to the diameter of the colony, and choose the color change circle first. Strains with a larger ratio of diameter to colony diameter are ready for screening.

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Abstract

The invention provides a quick screening method of a high temperature and high acid resistant citric acid high-yield strain. The quick screening method includes: preparing a culture medium; selectinga parent strain; preparing a fusion strain, preliminarily screening the fusion strain, screening the high temperature and high acid resistant citric acid high-yield strain, performing shake-flask fermentation re-screening and strain genetic stability testing and performing optional re-mutation breeding. The high-yield high temperature and high suspension resistant strain is obtained by applying the high-throughput quick screening method combining preliminary screening, re-screening and re-mutation, size of a selective culture medium indicating ring cultured at high temperature is taken as a preliminary screening basis for high-throughput large-scale elimination, and a HPLC measuring method is combined to accurately select the high-yield strain, so that screening efficiency is improved. Thehigh-throughput quick screening method is simple and efficient in operation, high in mutant strain directionality and important in production value.

Description

technical field [0001] The invention relates to the technical field of microbes, in particular to a rapid screening method for high-temperature resistant high-acid citric acid production strains. Background technique [0002] The chemical name of citric acid is 2-hydroxypropane-1,2,3-tricarboxylic acid, which is widely used in food, medicine and chemical industries, and is the most biochemically produced organic acid in the world. In recent years, the demand for citric acid is increasing. At present, the main methods of producing citric acid are chemical synthesis and biological fermentation. The chemical synthesis method uses a lot of chemical reagents, and the synthesis steps are cumbersome and the raw materials are expensive, so it is difficult to apply to food production. The biological fermentation method has mature technology, low cost and large output, and it is widely supplied to the food industry. More than 90% of citric acid currently on the market is produced by...

Claims

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Application Information

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IPC IPC(8): C12Q1/04C12N15/04C12R1/685
CPCC12Q1/04C12N15/04G01N2333/38
Inventor 刘海清于清闫锡升王泽宇王江涛赵培红张光祥
Owner TTCA
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