L-malic acid high-yield strain and application thereof
A high-yielding strain, malic acid technology, applied in the field of bioengineering, can solve problems such as low-price competition
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[0026] The preparation method of the fermentation medium is:
[0027] Mix corn flour and water at a mass-to-volume ratio of 1:4 (g / mL), and heat to 70°C to obtain dry starchy raw materials;
[0028] Add high-temperature resistant a-amylase according to the added amount of 800U / g dry starch raw material, heat it up to 90°C and keep it warm until liquefaction is complete to obtain a corn meal liquefied liquid. After the liquefied liquid is hot, filtered with a filter cloth to obtain a liquefied clear liquid;
[0029] The liquefied liquid and the liquefied supernatant are mixed and adjusted with water to a total sugar concentration of 9-10%, 0.1-0.2 g / L urea is added, and an appropriate amount of calcium carbonate is added to adjust the pH to 6-7.
[0030] HPLC method for the determination of L-malic acid: quantitative detection of fermentation components by HPLC method, using DIONEXHPLC P680 workstation, Alltech organic acid chromatography column No. 88645 column 250 mm×4.6 mm, UV detect...
Embodiment 1
[0031] Example 1: ARTP mutagenesis treatment of Aspergillus niger
[0032] Take a spore-rich and black starting strain (Control) slant (the slant medium is the PDA solid medium) that has been cultivated for 5 to 6 days, rinse the cultivated fresh slant spores with 5 mL sterile water, and filter through sterile gauze Afterwards, place it in a 250 mL Erlenmeyer flask containing glass beads and Cha's liquid medium (the glass beads should be roughly covered with the bottom of the bottle), and shake well for 30 minutes at 30°C and 200 rpm to disperse the spores evenly. Single spore suspension, take 0.5mL and appropriately dilute with sterile normal saline, then measure the concentration of the bacterial suspension by hemocytometer, adjust the concentration of spore solution to 10 7 Pcs / mL, as the mutagenic stock solution. The normal temperature and pressure (ARTP) plasma mutagenizer was used for mutagenesis of Aspergillus niger. The working gas during mutagenesis is helium. Accordin...
Embodiment 2
[0033] Example 2: Screening of strains
[0034] Preliminary screening: Take 100 μL of the spore suspension obtained after ARTP mutagenesis for 180 s and apply it to an acid-producing indicator plate and incubate at 30-35°C for 48-120 hours. Observe whether a color circle is produced to determine whether the strain produces acid. . Since nystatin is added to the acid production indicator plate, the growth rate of mold is inhibited to a certain extent, and a single colony is easily obtained. Join CaCO 3 It can make the solid medium turbid, and can form calcium salt after acid production, improve the transparency of the medium around the colony, and make the edge of the bromocresol green color circle become clear. By spreading the mutagenized strain on the plate, selecting a single colony with a larger ratio of the discolored transparent circle to the colony diameter, and preliminarily determining that it is a strain with higher acid production, and transferring it to the PDA solid...
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