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
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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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