A high-yield ethyl acetate yeast strain under low temperature conditions and its application
A low-temperature condition and ethyl acetate technology, applied in the field of bioengineering, can solve the problems of many types of metabolites, high fermentation temperature, and high production cost, so as to reduce fermentation temperature, increase ethyl acetate content, and reduce production energy consumption and cost Effect
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Embodiment 1
[0019] The screening of embodiment 1 ester-producing yeast
[0020] Grind the koji medicine and fermented unstrained spirits with a mortar, weigh 10g to 100ml of sterile normal saline, and vibrate at 150r / min for 30min to obtain the bacterial suspension, perform ten-fold serial dilution, and spread the dilution on the primary screening culture Base, select the ratio of the diameter of the transparent circle and the diameter of the colony to be a large colony, that is, a strain with significant ester production. Then transfer the strain, carry out shake flask fermentation and re-screening, use HS-SPME-GC-MS method to determine the type of ester in the fermentation liquid, use the method for determining the total ester of liquor stipulated in GB / T 10345-2007 to determine the content of ethyl acetate in the fermentation liquid , and screened to obtain strains with strong ability to produce ethyl acetate.
[0021] The initial screening culture conditions for ester production are:...
Embodiment 2
[0027] Example 2 Ethyl acetate-producing yeast and its molecular biology identification
[0028] The obtained ethyl acetate-producing yeast was identified by molecular biology, and the 26SrDNA fragments of the strains were amplified with yeast-specific classification and identification primers, and detected by gel electrophoresis. Sequencing comparison was then carried out to determine the species of the screened yeast. The strain was deposited in the General Microorganism Center of China Committee for Culture Collection of Microorganisms, and the yeast species and preservation number were: Wickerhamomycesanomalus (Wickerhamomycesanomalus) CGMCC NO: 10370.
[0029] The process of confirming the species of yeast is as follows:
[0030] Identification of 26SrDNA sequence homology of isolated strains: Extraction of yeast DNA: inoculate the yeast in liquid YPD medium after activation, and culture it on a shaker at 28°C for 12 hours, then take a small amount of bacteria into a ste...
Embodiment 3
[0035] The acid resistance test of embodiment 3 yeast strains
[0036] Adjust the pH of the YPD liquid medium to 1.0-7.0, increase the gradient with pH 0.5, do three parallels for each gradient, inoculate 10% (V / V) yeast suspension, at 30°C, 150r / min, after culturing for 24h, Use a microplate reader to measure the turbidity of the culture medium at a wavelength of 600nm to determine the growth of the bacteria. The acid resistance results of the strains are shown in Table 1 below.
[0037] Table 1 strain acid resistance (OD 600 value)
[0038] pH
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
OD 600
0.002
1.085
1.291
1.341
1.335
1.403
1.403
1.425
1.421
1.417
1.433
[0039] Table 1 shows the OD of the strains at pH 2.5 600 reached 1.085, indicating that the strain can grow normally under the environment of pH2.5,
[0040] And with the increase of pH, the strain can still grow well in acidi...
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