A kind of bacterial strain and application thereof for promoting alcoholic fermentation of high-yield sugarcane molasses
A technology of sugarcane molasses and alcohol fermentation, applied in the biological field, can solve the problems of low alcohol fermentation level, lack of high-performance industrial strains, environmental pollution, etc., and achieve the effects of improving utilization rate, significant economic benefits, and reducing production costs
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Embodiment 1
[0014] Example 1: Cloning of the PH04 gene of low-yielding yeast MC15.
[0015] 1. Extraction of genomic DNA of low-yielding yeast MC15.
[0016] Medium YPD: 2% glucose, 2% peptone, 1% yeast powder.
[0017] Yeast activation and culture overnight, collect 2mL bacterial precipitate, wash twice with water, add 250 microliters of lysate, 100 microliters of 1XTE, and sterile quartz sand about one-third of the volume of the liquid, shake for 3 minutes, add an equal volume of phenol Chloroform (1:1), slowly invert and shake several times, centrifuge at 12000rpm for 10 minutes, absorb 100 microliters of supernatant, wash twice with appropriate amount of 70% ethanol, air dry, add appropriate amount of ddH 2 O, stored in -20°C refrigerator.
[0018] 2. Cloning of the PH04 gene, electrophoresis inspection, and sequencing verification.
[0019] PCR reaction system: 5 microliters of 10×ExTaq PCR Buffer, 2 microliters of PH04-F / R primers, 2 microliters of dNTPMixture (2.5mM each), 1 mic...
Embodiment 2
[0026] Example 2: Obtaining of recombinant high-yielding yeast MF01-PH04.
[0027] 1. Sporulation culture of original high-yielding yeast.
[0028] McClay sporulation medium: glucose 0.1%, potassium chloride 0.18%, sodium acetate 0.82%, yeast powder 0.25%.
[0029] After the yeast strains were activated, the seed liquid was collected, the bacterial precipitate was washed twice with sterile water, inoculated into McClay sporulation medium, cultured at 28°C for about 5 days, and the sporulation effect was detected by sampling microscope.
[0030] 2. Preparation of original high-yielding yeast haploid protoplasts.
[0031] Collect the spore-forming culture solution in the above 1, kill the vegetative cells in a 58°C water bath for 15 minutes, wash twice with sterile water, remove the supernatant, and resuspend the precipitate with a mixed enzyme solution (4% helicase, 2% cellulase), 30 Enzymolysis at ℃ 200rpm for 8h (after 4h, microscopic examination every 1h), observe the cond...
Embodiment 3
[0041] Example 3: Alcoholic fermentation test of sugarcane molasses by recombinant high-yield yeast MF01-PH04.
[0042] 1. Differences in gas production between the recombinant high-yielding strain MF01-PH04 and the original high-yielding strain MF01.
[0043] Activate the strains to the logarithmic phase, count microscopically, and control the number of bacteria to be consistent. The initial number of bacteria is 1×10 6 The final concentration of each milliliter was inoculated into a test tube equipped with an inverted Duchenne tubule (full of YPD liquid medium) and 6 mL of YPD, placed in an incubator, cultured at 30°C, and the gas production was observed every 1 hour starting from 4 hours. MF01-PH04 produces gas faster than the original high-yielding strain MF01 (see figure 2 ).
[0044] 2. Comparison of sugarcane molasses alcoholic fermentation experiments among low-yielding strains, original high-yielding strains and recombinant high-yielding strains.
[0045] Molasses...
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