CN113215007B - Schizosaccharomyces pombe for high acid resistance millet wine and application of schizosaccharomyces pombe in Maotai-flavor liquor brewing - Google Patents
Schizosaccharomyces pombe for high acid resistance millet wine and application of schizosaccharomyces pombe in Maotai-flavor liquor brewing Download PDFInfo
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- WIIZWVCIJKGZOK-IUCAKERBSA-N 2,2-dichloro-n-[(1s,2s)-1,3-dihydroxy-1-(4-nitrophenyl)propan-2-yl]acetamide Chemical compound ClC(Cl)C(=O)N[C@@H](CO)[C@@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-IUCAKERBSA-N 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12G—WINE; PREPARATION THEREOF; ALCOHOLIC BEVERAGES; PREPARATION OF ALCOHOLIC BEVERAGES NOT PROVIDED FOR IN SUBCLASSES C12C OR C12H
- C12G3/00—Preparation of other alcoholic beverages
- C12G3/02—Preparation of other alcoholic beverages by fermentation
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12G—WINE; PREPARATION THEREOF; ALCOHOLIC BEVERAGES; PREPARATION OF ALCOHOLIC BEVERAGES NOT PROVIDED FOR IN SUBCLASSES C12C OR C12H
- C12G3/00—Preparation of other alcoholic beverages
- C12G3/02—Preparation of other alcoholic beverages by fermentation
- C12G3/021—Preparation of other alcoholic beverages by fermentation of botanical family Poaceae, e.g. wheat, millet, sorghum, barley, rye, or corn
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
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Abstract
The invention belongs to the technical field of microbial fermentation, and particularly relates to schizosaccharomyces pombe with strong acid resistance and strong ethanol production capacity and application thereof. In particular to Schizosaccharomyces pombe (Schizosaccharomyces pombe) Sujiu.065, which is obtained by separating and screening soy sauce flavor fermented grains, and the preservation number is CGMCC No.21792. The strain has excellent acid resistance and the wine production capacity of the strain is equivalent to that of industrial saccharomyces cerevisiae under the non-stress condition. Under the high-acid environment of a simulated Maotai-flavor liquor liquid fermentation medium and a solid fermentation medium, the method has outstanding liquor production advantages and is beneficial to improving the production of tetramethylpyrazine. The method can help solve the problems of low utilization rate of raw materials, production waste, high production cost and the like caused by large acidity of fermented grains and insufficient yeast fermentation capacity in the production of the Maotai-flavor liquor.
Description
The technical field is as follows:
the invention belongs to the technical field of microbial fermentation, and particularly relates to schizosaccharomyces pombe with strong acid resistance and strong ethanol production capacity and application thereof.
Background art:
the yeast is a core microorganism in the fermentation process of the ethanol beverage, and the growth and metabolism abilities of the yeast are highly related to the alcohol yield.
The solid fermentation of the white spirit is a multi-micro co-fermentation process, and researches show that the initial temperature is one of the key factors for driving microbial community succession in the white spirit fermentation process. The high environmental temperature can promote the rapid propagation of lactic acid bacteria and metabolize to generate a large amount of acetic acid and lactic acid, and the large accumulation of substances such as acetic acid and lactic acid obviously increases the acidity of the fermented grains, so that the ecological environment in the cellar gradually becomes a relatively extreme environment. Particularly in the production of Maotai-flavor liquor, the content of acetic acid can reach 4-6g/kg of fermented grains, the content of lactic acid is 30 g/kg, and the lowest pH value of the fermented grains is 2.
The increase of acidity seriously affects the growth and metabolic capability of yeast, causes 'discharge' in production, and causes huge economic loss for enterprises. It was reported that during the fermentation, the increase in acidity of fermented grains by 1 degree, with a loss of 4.5kg per 100kg of starch, corresponds to a decrease in the yield by 3.68% (by 60% vol/vol).
The existing 'temperature-reducing and acid-controlling' process has a certain effect, but consumes a large amount of manpower and material resources, and increases the production cost. The breeding of the yeast strains with high acid resistance and strong wine production capability is one of the effective ways to solve the problem.
The invention content is as follows:
in order to solve the technical problems, the invention provides fission yeast schizosaccharomyces pombe with strong acid resistance and good wine production capacity and application thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
the Schizosaccharomyces pombe is obtained by separating and screening soy sauce flavor fermented grains, is preserved in the common microorganism center of China Committee for culture Collection of microorganisms in 2.1.2021, and has the address: beijing, chaoyang district, beicheng Xilu No.1, microbial institute of Chinese academy of sciences, zip code 100101, preservation number CGMCC No.21792, classification name: schizosaccharomyces pombe.
The 26SrDNA of the Schizosaccharomyces pombe Sujiu.065 has a nucleotide sequence shown in a sequence table SEQ ID NO. 1;
SEQ ID NO.1：
AGCGGAGGAAAAGAAAATAACCATGATTCCCTCARKWACGGCGAGTGAAGCGGGA AAAGCTCAAATTTGAAATCTGTCAACATTTCTTTTGTTGTCCGAGTTGTAATTTCAAGAA GCTGCTTTGAGTGTAGACGATCGGTCTAAGTTCCTTGGAACAGGACGTCAGAGAGGGTG AGAACCCCGTCTTTGGTCGATTGGATATGCCATATAAAGCGCTTTCGAAGAGTCGAGTTG TTTGGGAATGCAGCTCTAAATGGGTGGTAAATTTCATCTAAAGCTAAATATTGGCGAGAG ACCGATAGCGAACAAGTAGAGTGATCGAAAGATGAAAAGAACTTTGAAAAGAGAGTTA AATAGTACGTGAAATTGCTGAAAGGGAAGCATTGGAAATCAGTCTTACCTGGGTGAGAT CAGTAGTCTCTTCGCGAGACTATGCACTCTGAACCTGTGGTAGGTCAGCATCAGTTTTCG GGGGCGGAAAAAGAATAAGGGAAGGTGGCTTTCCGGGTTCTGCCTGGGGAGTGTTTAT AGCCCTTGTTGTAATACGTCCACTGGGGACTGAGGACTGCGGCTTCGTGCCAAGGA；
the Schizosaccharomyces pombe Sujiu.065 has OD after culturing in YPD medium containing 20g/L acetic acid for 34 hr 600 A value above 0.7; OD of the cells cultured in YPD medium containing 60g/L of lactic acid for 22 hours 600 The value is 1.2 or more.
The invention also provides application of the schizosaccharomyces pombe Sujiu.065 in liquor brewing.
Further, schizosaccharomyces pombe Sujiu.065 is adopted to carry out liquid fermentation of Maotai-flavor liquor.
Further, fission yeast schizosaccharomyces pombe Sujiu.065 is adopted to carry out solid state fermentation on the Maotai-flavor liquor.
The alcohol content of said Schizosaccharomyces pombe Sujiu.065 in fermentation in liquid sorghum juice medium in which 6g/L acetic acid and 30g/L lactic acid coexist reaches 7.24 + -0.23 vol%, the alcohol content of the control strain Saccharomyces cerevisiae AY12 is 1.53 + -0.13 vol%, the alcohol content of the commercial Schizosaccharomyces pombe ATCC 169979 is 1.59 + -0.16 vol%;
the ethanol content of the schizosaccharomyces pombe Sujiu.065 in the solid-state fermentation of the simulated maotai-flavor liquor is 47.41 +/-2.72 g/kg of fermented grains, the ethanol content of a reference strain Saccharomyces cerevisiae AY12 is 41.53 +/-1.24 g/kg, the ethanol content of a commercial schizosaccharomyces pombe ATCC 169979 is 41.8 +/-1.41 g/kg, and the ethanol content of a non-inoculated reference group is 41.5 +/-0.6 g/kg.
The fission yeast schizosaccharomyces pombe Sujiu.065 is beneficial to improving the yield of tetramethylpyrazine in the solid state fermentation of the simulated Maotai-flavor liquor, and is 31.2 +/-3.19 mg/kg fermented grains.
Has the advantages that:
the fission yeast schizosaccharomyces pombe with excellent acid resistance is separated from the soy sauce flavor fermented grains, and the production capacity of the fission yeast schizosaccharomyces pombe is equivalent to that of industrial saccharomyces cerevisiae under the non-stress condition. Under the high-acid environment of simulating the liquid fermentation and solid fermentation culture medium of the Maotai-flavor liquor, the method has outstanding liquor production advantages and is beneficial to improving the production of the tetramethylpyrazine. The invention can provide help for solving the problems of low utilization rate of raw materials, production waste, high production cost and the like caused by large acidity of fermented grains and insufficient fermentation force of yeast in the production of Maotai-flavor liquor. The strain has strong application value, is beneficial to improving the traditional white spirit production, promotes the improvement of the yield and the output value of the white spirit production, and generates great economic benefit.
Description of the drawings:
FIG. 1 shows a simulated white spirit liquid state fermentation strain CO without stress 2 And ethanol yield plot;
a-alcohol content; B-CO 2 And (4) yield.
FIG. 2 shows that 6g/L acetic acid and 30g/L lactic acid simultaneously stimulate acid to simulate liquor liquid state fermentation strain CO 2 And ethanol yield plot;
a-alcohol content; B-CO 2 And (4) yield.
FIG. 3 is a graph of ethanol yield of a simulated white spirit solid state fermentation strain under acid stress.
FIG. 4 is a graph of starch residue of a simulated white spirit solid state fermentation strain under acid stress.
FIG. 5 is a graph of the yield of tetramethylpyrazine in a simulated white spirit solid state fermentation strain under acid stress.
FIG. 6 is a phylogenetic tree of Schizosaccharomyces pombe Sujiu.065.
FIG. 7 shows the growth of Schizosaccharomyces pombe under no stress and different concentrations of acetic acid.
FIG. 8 shows the growth of Schizosaccharomyces pombe at different concentrations of lactic acid.
In the above-mentioned figure SP.65 is Sujiu.065 of Schizosaccharomyces pombe (Schizosaccharomyces pombe) of the present invention.
The specific implementation mode is as follows:
in order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and do not limit the invention.
EXAMPLE 1 screening of acid-tolerant, high-yield ethanol Yeast from fermented grains
(1) The strain source is as follows: the sixth and seventh round of fermented grains of Maotai-flavor liquor.
(2) Culture medium:
enrichment culture medium: taking a wort culture medium as an enrichment culture medium, crushing malt, sieving by a 120-mesh sieve, and mixing the malt with the culture medium according to the weight ratio of the malt: water =1, 4, liquefying at 65 ℃, and saccharifying for 1h. Filtering to remove residue, boiling for 1 hr, adding flos Lupuli at a ratio of 0.4 ‰ for 40min, centrifuging after boiling, collecting supernatant, sterilizing, and adding 100mg/L chloramphenicol;
WL solid medium: weighing 78.25g of WL solid culture medium, heating and dissolving in 1000mL of distilled water, subpackaging in triangular flasks, autoclaving at 121 ℃ for 15min, and adding chloramphenicol to reach a final concentration of 100mg/L for later use.
YPD liquid medium: 20g/L glucose, 20g/L peptone and 10g/L yeast extract powder, sterilizing at 115 deg.C for 20min, and using.
(3) The screening method comprises the following steps:
strain enrichment and coating:
weighing 10g of fermented grains, adding the fermented grains into 100mL of enrichment medium, standing and culturing for 48h at 30 ℃, taking 100 mu L of enrichment medium, coating the enrichment medium on a WL solid culture medium by adopting a gradient dilution method, culturing for 4-5 days at 30 ℃, selecting colonies with good growth vigor, purifying each colony twice by adopting three-zone dividing lines, finally obtaining 158 purified strains in total, and storing in a glycerol tube for later use.
Primary screening:
the initial analysis of the fermentation and ethanol production capacity of the obtained strain is carried out by adopting a Duchenne tubule fermentation method, which comprises the following specific operations: respectively inoculating the obtained strain and industrial saccharomyces cerevisiae AY12 in a liquid YPD culture medium, culturing at 30 ℃ for 24h at 180r/min, and transferring the bacterial liquid into 5mL of fresh YPD liquid culture medium to activate to logarithmic metaphase. Adjusting OD value of logarithmic phase bacterial liquid of each strain to be 1.0-1.2, taking 100 mu L of each bacterial liquid, respectively inoculating each bacterial liquid into YPD culture medium with Duchen tubules, standing and culturing at 30 ℃, monitoring gas production condition, and specifically judging standard is shown in Table 1. And detecting the glucose consumption and ethanol production of each strain by liquid chromatography. Finally obtaining 34 strains which can produce gas and ethanol.
TABLE 1 evaluation criteria for gassing power
Re-screening:
selecting 34 strains which produce gas and can produce ethanol on the basis of the primary screening result, performing a Duchenne tubule fermentation experiment under the condition of YPD liquid culture medium containing 60g/L of lactic acid, continuously observing the gas production and ethanol production performance, and setting 3 strains in parallel. The 27 strains which still produce ethanol under 60g/L of lactic acid are obtained by co-screening, wherein the ethanol yield of the 65-encoding strain is relatively high, the fermentation speed is higher, and 18 hours can fill the Duchen tubule with gas, as shown in Table 2.
TABLE 2 Du's tubule fermentation-60 g/L lactic acid (observation period 36 h)
Example 2 molecular biological identification
The isolated 65-encoding strain was subjected to 26SrDNA identification. The universal primers selected were:
NL1(5'-GCATATCAATAAGCGGAGGAAAAG-3')；
NL4(5'-GGTCCGTGTTTCAAGACGG-3')。
the length of the amplified fragment is about 600bp. The target fragment is subjected to intellectual sequencing, and the sequencing result is compared with 26S rDNA sequences (MK 749861.1, MK749858.1, MK749856.1, KY296084.1 and the like) registered in GenBank for analysis, so that the homology reaches 99.49 percent (see figure 6). Through morphological characteristics and 26S rDNA sequence analysis, the strain of the strain code 65 is determined to be Schizosaccharomyces pombe, namely the Schizosaccharomyces pombe (Schizosaccharomyces pombe) Sujiu.065 of the invention with the preservation number of CGMCC No.21792.
Example 3 simulation of liquid fermentation of Baijiu
Schizosaccharomyces pombe Sujiu.065, saccharomyces cerevisiae AY12 and commercial Schizosaccharomyces pombe ATCC 169979 are respectively used as fermentation strains.
(1) Preparing sorghum hydrolysate:
high-quality sorghum is taken as a raw material, the sorghum powder is mixed with water according to a ratio of 1:3 (w/v) by using a 20-mesh sieve, high-temperature-resistant alpha-amylase (10U/g of raw material) is added for acting in a water bath at 90 ℃ for 1h, then the mixture is continuously heated and boiled for 30min, and then water is supplemented to the original volume, and the temperature is immediately reduced to 60 ℃. Saccharification: saccharifying enzyme is added at a ratio of 250U/g raw material, and saccharification is carried out at 60 deg.C for 4h. After saccharification is finished, the temperature is reduced to 40 ℃, and acid protease (30U/g raw material) is added for water bath action for 4 hours. Cooling to room temperature and filtering with 4 layers of gauze to obtain clear solution, and adjusting sugar degrees to 8 ° Bx, 12 ° Bx and 16 ° Bx respectively for later use.
(2) Preparing a seed culture medium and a liquid fermentation culture medium:
preparing a seed culture medium: adding 0.5% yeast extract powder into the hydrolysis liquid of 8 ° Bx and 12 ° Bx respectively as primary seed culture medium and secondary seed culture medium, and sterilizing at 121 deg.C for 20 min.
Non-stressed simulated liquid medium: 6g/L of ammonium sulfate, 1.2g/L of magnesium sulfate and 2.4g/L of dipotassium phosphate are added into 16-degree Bx hydrolysis liquid to serve as an unstressed simulated culture medium, and the mixture is sterilized at the high temperature of 121 ℃ for 20min for later use.
Acid stress simulated liquid medium: acetic acid and lactic acid are simultaneously added into the non-stress simulated liquid culture medium, and the final concentrations are respectively 6g/L and 30g/L (the concentrations are set according to the contents of the acetic acid and the lactic acid in the fermented grains of the Maotai-flavor liquor reported by the literature).
(3) Fermentation process
First-order seed culture: scraping a ring yeast from the solid culture medium into a test tube filled with a primary seed culture medium, loading the liquid in the test tube with the volume of 10mL/30mL, and performing static culture at 30 ℃ for 24 hours;
secondary seed culture: inoculating the primary seed liquid into a secondary seed culture medium according to the proportion of 10%, filling the liquid in the secondary seed culture medium in a volume of 100mL/250 mL, and performing static culture at 30 ℃ for 16h to obtain the yeast secondary seed liquid.
Fermentation and inoculation: yeast secondary seed liquid according to 1 × 10 7 Inoculating the CFU/mL inoculum size to a liquid fermentation culture medium, loading the liquid in the liquid fermentation culture medium in a volume of 150mL/250mL, and performing static culture at 30 ℃ for 1-6d. Weighing for 1 time every 12h, and finishing fermentation when the weight loss of the two times is less than 0.3 g. And (3) taking 100mL of mash after the fermentation is finished, adding 100mL of water, distilling out 100mL of wine sample, and measuring the alcoholic strength.
Under the stress-free condition, the results are shown in figure 1, the two schizosaccharomyces pombe strains complete fermentation within 48 hours, the fermentation speed of the saccharomyces cerevisiae AY12 is slightly superior, the fermentation can be completed within 36 hours, and the final alcohol yield is between 6.7 and 7 without significant difference. Under acid stress, the results are shown in FIG. 2, after fermentation for 108h, the Schizosaccharomyces pombe Sujiu.065 provided by the invention has the alcohol content of 7.24 + -0.23% vol, the control strain Saccharomyces cerevisiae AY12 has the alcohol content of 1.53 + -0.13% vol, the commercial Schizosaccharomyces pombe ATCC 169979 has the alcohol content of 1.59 + -0.16% vol, and the Schizosaccharomyces pombe Sujiu.065 shows significant advantages.
Example 4 simulation of solid fermentation of Maotai-flavor liquor
Schizosaccharomyces pombe Sujiu.065, saccharomyces cerevisiae AY12, and commercial Schizosaccharomyces pombe ATCC 169979 were used as fermentation strains, respectively, and no inoculation was used as a control.
(1) Material steaming: high-quality sorghum is used as a raw material, and the grinding degree of the sorghum accounts for 70-75% when passing through a 20-mesh sieve. The sorghum flour is evenly mixed with water with the temperature of 60-70 ℃ which is equivalent to 60 percent of the raw material, and the materials are piled up and moistened for 18-20 hours. And (3) mixing the moistened material with rice hulls (which are steamed to be clear) with the sorghum flour content being 20%, uniformly stirring, steaming the material, and steaming for 1 hour after circular steaming.
(2) And (3) ingredient fermentation:
basic ingredients: cooling the steamed material to about 30 ℃, and adding saccharifying enzyme according to the proportion of 250U/g sorghum flour for later use;
adding high temperature Daqu into distiller's grains according to proportion of 7% of distiller's grains, inoculating yeast seed liquid (1 × 10) according to proportion of 2% of distiller's grains 7 CFU/mL), ready for use.
According to the following steps: 5, mixing the vinasse containing the yeast seed liquid into the basic ingredients, filling the mixture into a fermentation jar, sealing the fermentation jar with water, and fermenting for 30 days at the temperature of 30 ℃. After the fermentation is finished, 100g of fermented grains are taken, 300mL of water is added, and 100mL of wine samples are steamed.
The results of ethanol content measurement and residual starch measurement are shown in FIGS. 3 and 4. In the solid state fermentation of the simulated Maotai-flavor liquor, the ethanol content of each kg of fermented grains of the Schizosaccharomyces pombe Sujiu.065 can reach 47.41 +/-2.72 g, the ethanol content of the fermented grains fermented by the reference strain Saccharomyces cerevisiae AY12 is 41.53 +/-1.24 g/kg, the ethanol content of the commercial Schizosaccharomyces pombe ATCC 169979 is 41.8 +/-1.41 g/kg, the ethanol content of the non-inoculated Sujiu.065 is higher than that of the fermented grains inoculated with the industrial Saccharomyces cerevisiae AY12, the commercial Schizosaccharomyces pombe ATCC 169979 and the non-inoculated yeasts by about 6 percent. The ethanol yield and residual starch content of industrial saccharomyces cerevisiae AY12 and commercial schizosaccharomyces pombe ATCC 169979 are similar to those of non-inoculated yeasts, which indicates that the two strains are inhibited in the solid state fermentation of the simulated Maotai-flavor liquor and cannot play a role in improving the alcohol content.
The content of tetramethylpyrazine (distilled 100mL of wine sample) in the product is determined by the following method: gas chromatograph: agilent 7890C; and (3) chromatographic column: a column special for white spirit, AT.LZP-930, 230 ℃,50m multiplied by 320 mu m multiplied by 1 mu m; a detector: FID detector, detector temperature: 200 ℃; carrier gas: high-purity nitrogen with the flow rate of 5mL/min; detection conditions are as follows: temperature programming, keeping the temperature at 50 ℃ for 8min, heating the temperature to 120 ℃ at 5 ℃/min, and keeping the temperature for 8min; sample inlet temperature: 200 ℃; sample introduction amount: 1.0 μ L; a flow splitting mode: splitting, wherein the splitting ratio is 10:1.
as shown in FIG. 5, it was found that the Schizosaccharomyces pombe provided by the present invention also contributes to the production of tetramethylpyrazine, and the content of tetramethylpyrazine in the Schizosaccharomyces pombe is 31.2 + -1.24 mg/kg fermented grains.
Example 5 evaluation of acetic acid resistance
YPD liquid medium: 20g/L glucose, 20g/L peptone and 10g/L yeast extract powder, sterilizing at 115 deg.C for 20min, and using.
Acetic acid stress medium YPD liquid medium was supplemented with acetic acid at final concentrations of 4g/L,8g/L,12g/L,16g/L, 18g/L, and 20g/L, respectively.
Stress experiments were performed with Sujiu.065 as the subject and Schizosaccharomyces pombe commercial strain ATCC 169979 as the control strain. The growth of both strains under stress-free treatment, i.e.in YPD liquid medium, was first investigated before the stress experiments.
Inoculating Schizosaccharomyces pombe in solid culture medium to liquid culture medium containing 5mLYPD, and shake culturing at 30 deg.C and 180r/min to stationary phase. Inoculating 10% of the strain into fresh YPD liquid culture medium (30 deg.C, 180 r/min), and culturing to middle and late logarithmic phase (viable count is about 1X 10) 7 CFU/mL) were further inoculated to a non-stressed medium and an acetic acid-stressed medium at an inoculum size of 3% and the growth tendency of the strain was measured using a fully automated growth profiler (Bioscreen C, finland) (30 ℃, static culture).
The results are shown in FIG. 7, and the culture without stressWhen the two strains enter the stationary phase within 12h, the strains grow OD 600 The value is about 1.6, and the difference of growth states is small. Under the stress of acetic acid, the fission yeast schizosaccharomyces pombe provided by the invention is cultured in a YPD culture medium with 20g/L of acetic acid for 34h, and the growth OD value is more than 0.72. The control strain ATCC 169979 stopped growing upon treatment with 4g/L acetic acid.
Example 6 evaluation of lactic acid resistance
Lactic acid stress medium adding lactic acid into YPD liquid medium at final concentrations of 20g/L,40g/L,60g/L and 80g/L, respectively.
Sujiu.065 was used as the subject, and Schizosaccharomyces pombe commercial strain ATCC 169979 was used as the control strain. Inoculating Schizosaccharomyces pombe in a solid culture medium into a liquid culture medium containing 5mLYPD, and performing shake culture at 30 ℃ and 180r/min to a stationary phase. Inoculating 10% of the culture medium into fresh YPD liquid medium (30 deg.C, 180 r/min), and culturing to middle and late logarithmic phase (viable count is about 1X 10) 7 CFU/mL) was further transferred to a lactic acid stress medium (30 ℃, static culture) at an inoculation amount of 3% and the growth tendency of the strain was determined using a full-automatic growth curve machine (Bioscreen C, finland). As shown in FIG. 8, the Schizosaccharomyces pombe of the present invention was cultured in YPD medium in the presence of 60g/L lactic acid for 24 hours to grow OD 600 The value is around 1.24. Control strain ATCC 169979 was cultured at this concentration for 24h and the OD was grown 600 The value was 0.65.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
SEQUENCE LISTING
<110> Guizhou national platform wine industry Co., ltd
Tianjin University of Science and Technology
<120> high acid resistance Schizosaccharomyces pombe and application thereof in maotai-flavor liquor brewing
<130> 1
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 585
<212> DNA
<213> Schizosaccharomyces pombe (Schizosaccharomyces pombe) Sujiu.065
<400> 1
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taatacgtcc actggggact gaggactgcg gcttcgtgcc aagga 585
Claims (3)
1. Schizosaccharomyces pombe (A)Schizosaccharomyces pombe ) It is characterized in that the yeast is schizosaccharomyces pombe (Schizosaccharomyces pombe)Schizosaccharomyces pombe) Sujiu.065, preservation number CGMCC No.21792.
2. Use of the Schizosaccharomyces pombe Sujiu.065 of claim 1 in the brewing of white spirit.
3. Use of the Schizosaccharomyces pombe Sujiu.065 of claim 1 for the production of ethanol.
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