CN114107077A - Ester-producing yeast strain and application thereof - Google Patents
Ester-producing yeast strain and application thereof Download PDFInfo
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- CN114107077A CN114107077A CN202111509846.4A CN202111509846A CN114107077A CN 114107077 A CN114107077 A CN 114107077A CN 202111509846 A CN202111509846 A CN 202111509846A CN 114107077 A CN114107077 A CN 114107077A
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- strain
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- pichia
- pichia kudriavzevii
- culturing
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- 239000006228 supernatant Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 229960000344 thiamine hydrochloride Drugs 0.000 description 1
- 235000019190 thiamine hydrochloride Nutrition 0.000 description 1
- 239000011747 thiamine hydrochloride Substances 0.000 description 1
- DPJRMOMPQZCRJU-UHFFFAOYSA-M thiamine hydrochloride Chemical compound Cl.[Cl-].CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N DPJRMOMPQZCRJU-UHFFFAOYSA-M 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
- 239000007222 ypd medium Substances 0.000 description 1
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 description 1
- 229910000368 zinc sulfate Inorganic materials 0.000 description 1
- 229960001763 zinc sulfate Drugs 0.000 description 1
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Abstract
The invention provides an ester-producing yeast strain and application thereof. The invention provides a Pichia kudriavzevii strain, which is prepared from the following components in parts by weight: the strain of Pichia kudriavzeii C4.12 (Pichia kudriavzeii C4.12) is deposited in the chinese typical culture collection (CCTCC) with the deposit number of CCTCC NO: m2021124. The yeast strain provided by the invention has the characteristics of multi-environment tolerance, broad-spectrum applicability of a substrate and ester production, and can improve the sensory flavor of a traditional brewed product, improve the quality of the product and enrich the taste of the product.
Description
Technical Field
The invention belongs to the field of microorganisms, and particularly relates to an ester-producing yeast strain and application thereof.
Background
The raw materials, the process, the microorganisms and the environmental conditions in the liquor brewing process can cause the difference of the types and the contents of flavor substances in the liquor, wherein the microorganisms are the most important factors, the yeast is the main microorganism contributing to the flavor of the liquor, and the yeast is divided into saccharomyces cerevisiae and non-saccharomyces cerevisiae, wherein ester-producing yeast in the non-saccharomyces cerevisiae has different degrees of esterification capacity on acid and alcohol, can produce ester substances such as ethyl acetate, ethyl lactate, phenethylacetate and the like, is a key compound for improving the sensory flavor of the liquor and promoting the flavor of the liquor to be fuller, and the difference of the components of various grain raw materials can react under the action of the microorganisms in the fermentation process to generate different flavor substances, so that the liquor can show different flavor characteristics.
Disclosure of Invention
During fermentation, yeast is subjected to various environmental stresses, mainly including osmotic pressure, temperature, ethanol, low pH and the like, which can destroy the cell structure of yeast to influence the growth, metabolism and physiological functions of yeast, resulting in low growth rate and survival rate of yeast, and finally influencing the fermentation efficiency.
Therefore, the yeast strain needs to have the characteristics of multiple environmental tolerance, broad-spectrum substrate adaptability, ester production and the like in the brewing process, so that the fermentation efficiency can be improved, the production cost can be reduced, and the product quality can be improved in industrial production.
Aiming at the problems of poor environmental tolerance, poor substrate adaptability and low ester production of yeast strains in the prior art, the invention provides the yeast strain with multi-environmental tolerance, broad-spectrum applicability to substrates and ester production.
The invention provides a Pichia kudriavzevii strain, which is prepared from the following components in parts by weight: the strain of Pichia kudriavzeii C4.12 (Pichia kudriavzeii C4.12) was deposited at the chinese typical culture collection center (CCTCC) at 2021-21 days in 2021, with the deposit number of CCTCC NO: m2021124.
Preferably, the 26S rDNA gene sequence of the Pichia kudriavzevii strain C4.12 is shown in SEQ ID NO. 1.
The invention also provides a fermentation preparation method of the Pichia kudriavzevii microbial inoculum, which comprises the following steps: and culturing the Pichia kudriavzevii strain.
Preferably, the preparation method comprises the following steps:
(1) amplifying and culturing the Pichia kudriavzevii strain;
(2) adding the product obtained in the step (1) into a liquid culture medium, and fermenting and culturing at the temperature of 26-40 ℃.
The invention also provides a microbial inoculum which is obtained by the fermentation preparation method.
The invention also provides the Pichia kudriavzevii strain or the application of the microbial inoculum in brewing.
The invention also provides a fermentation product which is obtained by culturing the Pichia kudriavzevii strain or the microbial inoculum.
Preferably, the fermentation product contains phenethyl alcohol, phenethyl acetate, isoamyl acetate, ethyl phenylacetate, ethyl caprylate and allo-albizim alcohol;
preferably, the fermentation product contains phenethyl alcohol with the concentration of 1000-1300ng/g, phenethyl acetate with the concentration of 50-100ng/g, isoamyl acetate with the concentration of 5-20ng/g, ethyl phenylacetate with the concentration of 1-15ng/g, ethyl octanoate with the concentration of 0.5-5ng/g and albizium alcohol with the concentration of 0.1-1 ng/g;
more preferably, the fermentation product contains phenethyl alcohol with the concentration of 1160-1170ng/g, phenethyl acetate with the concentration of 70-80ng/g, isoamyl acetate with the concentration of 10-15ng/g, ethyl phenylacetate with the concentration of 5-10ng/g, ethyl octanoate with the concentration of 1-2ng/g and albizium alcohol with the concentration of 0.3-0.6 ng/g.
The invention also provides a method for using the Pichia kudriavzevii strain or the microbial inoculum for brewing wine, which comprises the following steps: and culturing the Pichia kudriavzevii strain or the microbial inoculum.
The invention also provides wine which is obtained by adding the Pichia kudriavzevii strain or the microbial inoculum into a brewing raw material and brewing.
The yeast strain provided by the invention has the characteristics of multi-environment tolerance, broad-spectrum applicability of a substrate and ester production, and can improve the sensory flavor of a traditional brewed product, improve the quality of the product and enrich the taste of the product.
The yeast strain provided by the invention can efficiently utilize glucose and raffinose, can take kernels of vegetables, fruits, rice and some oil crops, such as beet, wheat, rice, cottonseed, soybean and sunflower seeds, as fermentation substrates, and has broad-spectrum substrate applicability.
The yeast strain provided by the invention can normally grow and propagate under the conditions that the pH is 3.0, the temperature is 40 ℃, the ethanol content (V/V) is 14 percent and the lactic acid content (V/V) is 6 percent, and has multi-environment tolerance.
The yeast strain provided by the invention can generate 61 flavor substances, and the total content reaches 1959.9327 ng/g.
Information on strain preservation
The Pichia kudriavzeii C4.12 strain (Pichia kudriavzeii C4.12) provided by the invention is preserved in China Center for Type Culture Collection (CCTCC) at 1 month and 21 days 2021, and the preservation number is CCTCC NO: m2021124.
Drawings
FIG. 1 shows the morphology of Pichia kudriavzevii C4.12 colonies;
FIG. 2 shows a microscopic observation image of Pichia kudriavzevii C4.12;
FIG. 3 shows growth curves of Pichia kudriavzevii strain C4.12 under different carbon source conditions;
FIG. 4 shows growth curves of Pichia kudriavzevii strain C4.12 under different pH conditions;
FIG. 5 shows growth curves of Pichia kudriavzevii strain C4.12 at different temperatures;
FIG. 6 shows the growth curves of Pichia kudriavzevii strain C4.12 under different ethanol concentrations;
FIG. 7 shows growth curves of Pichia kudriavzevii strain C4.12 under different lactic acid concentrations;
FIG. 8 shows a gas chromatography peak profile.
Detailed Description
The invention provides a Pichia kudriavzevii strain, which is prepared from the following components in parts by weight: the strain of Pichia kudriavzeii C4.12 (Pichia kudriavzeii C4.12) was deposited at the chinese typical culture collection center (CCTCC) at 2021-21 days in 2021, with the deposit number of CCTCC NO: m2021124.
The Pichia kudriavzeii C4.12 strain (Pichia kudriavzeii C4.12) provided by the invention is obtained by separating from cellar mud of a farmhouse corn wine house, and the colony is cheese-shaped, milk-white in color and smooth in surface; microscopic morphology was oval, 3.0X 7.5 μm in size, and microscopic observation was indicative of budding. The 26S rDNA gene sequence of the Pichia kudriavzevii C4.12 strain is shown in SEQ ID NO. 1.
The Pichia kudriavzeii C4.12 strain (Pichia kudriavzeii C4.12) provided by the invention has the characteristics of multi-environment tolerance, wide-spectrum applicability of a substrate and ester production, and has special contribution to the sensory flavor of a product, and the flavor of a traditional brewed product can be improved.
The medium components referred to in the examples are as follows:
YNB medium: 5000mg of ammonium sulfate, 2mg of inositol, 0.4mg of nicotinic acid, 0.4mg of thiamine hydrochloride, 0.04mg of copper sulfate, 1000mg of monopotassium phosphate, 0.5mg of boric acid, 0.4mg of pyridoxine hydrochloride, 0.4mg of calcium pantothenate, 0.2mg of p-aminobenzoic acid, 500mg of magnesium sulfate, 0.4mg of manganese sulfate, 0.4mg of zinc sulfate, 0.2mg of ferric chloride, 0.2mg of riboflavin, 100mg of calcium chloride, 0.1mg of potassium iodide, 0.2mg of sodium molybdate, 0.002mg of biotin, 0.002mg of folic acid, 100mg of sodium chloride and 1000mL of water, and then filtering and sterilizing.
YPD medium: 10g of yeast extract powder, 20g of glucose, 20g of peptone, 20g of agar and 1000mL of water, and sterilizing at 115 ℃ for 30 min;
carbon source culture medium: 0.67g YNB, 2g carbon source (glucose, xylose, arabinose, galactose, lactose, maltose, melibiose, sucrose, trehalose, cellobiose, melezitose, raffinose, glycerol, sorbitol), 1000mL water, filtration sterilized.
The reagents and instrument sources used in the examples of the invention are shown in table 1 below.
TABLE 1 list of reagents and instrument source information used in the examples
Example 1 isolation and identification of Yeast strains in the present invention
Dissolving the Hubei Enshi farmhouse corn wine house cellar mud sample in sterile water, mixing, absorbing the bacterial suspension, and preparing 10 times of the bacterial suspension by serial dilution-5、10-6Coating the bacterial suspension on YPD culture medium, culturing at 30 deg.C for 24-48h, making sheet, observing yeast morphology under microscope, observing single colony characteristic on plate, separating strain with typical yeast colony characteristic, streaking, purifying, inoculating on YPD slant culture medium, and storing at 4 deg.C. Obtaining a strain of bacteria, wherein the colony texture of the bacteria is cheese-shaped, the color of the bacteria is milky white, and the surface of the bacteria is smooth; microscopic morphology was oval, 3.0X 7.5 μm in size, and microscopic observation was indicative of budding. Extracting the yeast strain genome, taking NL1 (5'-GCATATCAATAAGCGGAGGAAAAG-3') and NL4 (5'-GGTCCGTGTTTCAAGACGG-3') as primers, carrying out PCR (polymerase chain reaction) program of 94 ℃ pre-denaturation for 5min, 94 ℃ denaturation for 30S, 55 ℃ annealing for 45S, 72 ℃ extension for 90S, 30 cycles, and finally 72 ℃ extension for 10min, wherein the PCR program is used for amplifying a yeast 26S rDNA sequence, and detecting and sequencing by 1% gel electrophoresis to obtain the 26S rDNA sequence SEQ ID NO.1 of the strain as shown in the specification:
combining morphological analysis and molecular identification, the strain is Pichia kudriavzeii (Pichia kudriavzeii) and is named as Pichia kudriavzeii C4.12 strain (Pichia kudriavzeii C4.12). The strain is preserved in China Center for Type Culture Collection (CCTCC) at 21/1/2021, with the preservation number being CCTCC NO: m2021124. FIG. 1 shows the colony morphology of the Pichia kudriavzeii strain C4.12 (Pichia kudriavzeii C4.12). FIG. 2 is a micrograph of Pichia kudriavzeiiii C4.12 strain (Pichia kudriavzeiiic4.12) of yeast.
Example 2 substrate utilization characterization of Pichia kudriavzevii strain C4.12
Inoculating the Pichia kudriavzevii C4.12 strain into a test tube filled with 5mLYPD liquid culture medium, culturing at 30 ℃ and 180rpm for 20h, inoculating the strain into a 100-hole culture plate filled with 300uL of carbon source liquid culture medium according to the inoculation amount of 3%, preparing a Bioscreen instrument for determination, and setting parameters: measuring data at 30 deg.C for 24 hr and wavelength of 600nm every 30min, and determining growth curve with OD600nmGrowth rates exceeding 50% are considered as growth.
OD 1-initial OD600nm value;
OD 2-OD 600nm value at end;
as shown in figure 3, the results show that the Pichia kudriavzevii C4.12 strain can utilize the 15 carbon sources, and when glucose and raffinose are utilized, the logarithmic phase and the stable phase are reached preferentially, the glucose and raffinose can be utilized efficiently, and the raffinose is widely existed in nature and comprises seeds and kernels of vegetables, fruits, rice and some oil crops, wherein beet, wheat, rice, cotton seeds, soybeans and sunflower seeds are common fermentation substrates, namely the strain has broad-spectrum substrate applicability and can be used as a grain substrate and a strain special for soybean meal fermentation.
Example 3 tolerance analysis of Pichia kudriavzevii strain C4.12
(1) pH tolerance test: inoculating the Pichia kudriavzevii C4.12 strain into a test tube filled with 5mL of YPD liquid culture medium, culturing at 30 ℃ and 180rpm for 20h, then inoculating into a 100-hole culture plate filled with 300uL of YPD liquid culture medium according to 3% inoculation amount, adjusting the pH of the YPD liquid culture medium to 2.0, 3.0, 4.0, 5.0 and 6.0 respectively, preparing a Bioscreen instrument for on-machine determination, and setting parameters: measuring the data at 30 deg.C for 24h and wavelength of 600nm every 30min, and determining the growth curve.
As shown in fig. 4, the growth curves of the pichia kudriavzevii strain C4.12 at pH 3.0, 4.0, 5.0, and 6.0 respectively show a typical sigmoid curve model, and the growth curves of the strain at pH 2.0 are approximately a straight line, i.e., the strain can normally grow at pH 3.0, 4.0, 5.0, and 6.0, but the strain cannot normally grow at pH 2.0, so that the pH of the strain is maximally tolerated at 3.0.
(2) Temperature resistance test: inoculating the Pichia kudriavzevii C4.12 strain into a test tube filled with 5mL YPD liquid culture medium, culturing at 30 ℃ and 180rpm for 20h, then inoculating the strain into a 100-hole culture plate filled with 300uL YPD liquid culture medium according to the inoculation amount of 3%, preparing a Bioscreen instrument for determination, and setting parameters: measuring data at 30 deg.C for 24h and wavelength of 600nm every 30min, and measuring growth curve; the temperature resistance tests at 35 ℃, 45 ℃ and 50 ℃ were in accordance with the procedure described above.
As shown in FIG. 5, the growth curves of the Pichia kudriavzevii C4.12 strain at 30 ℃, 35 ℃ and 40 ℃ are typical S-shaped curve models, the growth curve of the strain at 45 ℃ is approximately a straight line, namely the strain can normally grow at 30 ℃, 35 ℃ and 40 ℃, but the strain at 45 ℃ can not normally grow, so the temperature tolerance of the strain is 40 ℃ at most.
(3) Ethanol tolerance test: inoculating a Pichia kudriavzevii C4.12 strain into a test tube filled with 5mL of YPD liquid culture medium, culturing at 30 ℃ and 180rpm for 20h, then inoculating the strain into a 100-hole culture plate filled with 300uL of YPD liquid culture medium according to the inoculation amount of 3 percent to ensure that the ethanol concentration in the YPD liquid culture medium is respectively 2 percent, 4 percent, 6 percent, 8 percent, 10 percent, 12 percent, 14 percent, 16 percent, 18 percent and 20 percent, preparing a Bioscreen instrument for on-machine determination, and setting parameters: measuring the data at 30 deg.C for 24h and wavelength of 600nm every 30min, and determining the growth curve.
As shown in FIG. 6, the Pichia kudriavzevii C4.12 strain can grow normally under the conditions of ethanol concentration of 2%, 4%, 6%, 8%, 10%, 12% and 14%, respectively, while the strain can not grow normally under the conditions of 16%, 18% and 20%, so that the maximum ethanol tolerance (V/V) of the strain is 14%.
(4) Lactic acid tolerance: inoculating a Pichia kudriavzevii C4.12 strain into a test tube filled with 5mL of YPD liquid culture medium, culturing at 30 ℃ and 180rpm for 20h, then inoculating the strain into a 100-hole culture plate filled with 300uL of YPD liquid culture medium according to the inoculation amount of 3 percent to ensure that the volume concentration (v/v) of lactic acid in the YPD liquid culture medium is respectively 2 percent, 4 percent, 6 percent, 8 percent, 10 percent and 12 percent, preparing a Bioscreen instrument for on-machine determination, and setting parameters: measuring the data at 30 deg.C for 24h and wavelength of 600nm every 30min, and determining the growth curve.
As shown in FIG. 7, the Pichia kudriavzevii C4.12 strain can grow normally under the conditions of 2%, 4% and 6% of lactic acid concentration, respectively, while the strain can not grow normally under the conditions of 8% and 10%, so that the lactic acid tolerance (V/V) of the strain is 6% at maximum.
Example 4 analysis of volatile Components in fermentation broth
Inoculating the Pichia kudriavzevii C4.12 strain into a test tube filled with 5mL of YPD liquid culture medium, culturing at 30 ℃ and 180rpm for 20h, then inoculating into a triangular flask filled with 300mL of YPD liquid culture medium according to the inoculation amount of 0.6%, culturing at 30 ℃ and 180rpm for 20h, centrifuging, collecting supernatant, filtering by 0.22 mu m, taking 10mL of filtrate, adding 20 mu L of internal standard solution (n-butyl acetate), mixing uniformly according to the chromatographic conditions: the split ratio is 40:1, the injection port temperature is 250 ℃, the detector temperature is 250 ℃, the chromatographic column is an AT.LZP-930 white spirit column, the initial column temperature is kept at 50 ℃ for 2min, the temperature is increased to 110 ℃ at the speed of 5 ℃/min, and the holding time is 0 min; heating to 130 deg.C at a rate of 3 deg.C/min, and maintaining for 0 min; the volatile component analysis was carried out by raising the temperature to 230 ℃ at a rate of 15 ℃/min for 2min, the gas chromatography peak was as shown in FIG. 8, and the information on the main volatile components is shown in Table 2.
TABLE 2 information on the major volatile constituents in the fermentation broths
The result shows that 61 flavor substances are detected in the fermentation liquor, the total content is 1959.9327ng/g, the main volatile substances are phenethyl alcohol, phenethyl acetate, isoamyl acetate, phenethyl acetate, ethyl caprylate and albizzia alcohol, wherein the phenethyl alcohol content is the highest, the characteristic flavor is rose fragrance, and the estimated concentration is 1169 ng/g.
The above embodiments are only for further illustration and understanding of the technical solutions of the present invention, and are not intended to limit the present invention, and any modifications that do not make a prominent substantive feature or significant progress on the basis of the above embodiments should fall within the scope of the present invention.
Sequence listing
<110> Angel Yeast Co Ltd
<120> ester-producing yeast strain and application thereof
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ggaacagggc gcccaggagg gtgagagccc cgtgggatgc cggcggaagc agtgaggccc 180
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gctaaatact ggcgagagac cgatagcgaa caagtactgt gaaggaaaga tgaaaagcac 300
tttgaaaaga gagtgaaaca gcacgtgaaa ttgttgaaag ggaagggtat tgcgcccgac 360
atggggattg cgcaccgctg cctctcgtgg gcggcgctct gggctttccc tgggccagca 420
tcggttcttg ctgcaggaga aggggttctg gaacgtggct cttcggagtg ttatagccag 480
ggccagatgc tgcgtgcggg gaccgaggac tgcggccgtg taggtcacgg atgctggcag 540
aacggcgcaa caccgcccgt cttgaacccg gaccaaa 577
Claims (10)
1. A Pichia kudriavzevii strain is characterized in that the Pichia kudriavzevii strain is:
the Pichia kudriavzeii strain C4.12 (Pichia kudriavzeii C4.12) is deposited in the chinese typical culture collection (CCTCC) with the collection number of CCTCC NO: m2021124.
2. The Pichia kudriavzevii strain of claim 1, wherein the 26S rDNA gene sequence of the Pichia kudriavzevii strain C4.12 is shown in SEQ ID No. 1.
3. A fermentation preparation method of a Pichia kudriavzevii microbial inoculum is characterized by comprising the following steps: culturing the Pichia kudriavzevii strain of claim 1 or 2.
4. The method of claim 3, comprising the steps of:
(1) amplifying and culturing the Pichia kudriavzevii strain of claim 1 or 2;
(2) adding the product obtained in the step (1) into a liquid culture medium, and fermenting and culturing at the temperature of 26-40 ℃.
5. A microbial inoculum obtained by the fermentation production method according to claim 3 or 4.
6. Use of a pichia kudriavzevii strain according to claim 1 or 2 or a microbial inoculum according to claim 5 in brewing.
7. A fermented product obtained by culturing the Pichia kudriavzevii strain of claim 1 or 2 or the microbial agent of claim 5.
8. Fermentation according to claim 7,
the fermentation product contains phenethyl alcohol, phenethyl acetate, isoamyl acetate, ethyl phenylacetate, ethyl caprylate and alloy albizzia alcohol;
preferably, the fermentation product contains phenethyl alcohol with the concentration of 1000-1300ng/g, phenethyl acetate with the concentration of 50-100ng/g, isoamyl acetate with the concentration of 5-20ng/g, ethyl phenylacetate with the concentration of 1-15ng/g, ethyl octanoate with the concentration of 0.5-5ng/g and albizium alcohol with the concentration of 0.1-1 ng/g;
preferably, the fermentation product contains phenethyl alcohol with the concentration of 1160-1170ng/g, phenethyl acetate with the concentration of 70-80ng/g, isoamyl acetate with the concentration of 10-15ng/g, ethyl phenylacetate with the concentration of 5-10ng/g, ethyl octanoate with the concentration of 1-2ng/g and albizium alcohol with the concentration of 0.3-0.6 ng/g.
9. The method for brewing wine using the pichia kudriavzevii strain of claim 1 or 2 or the microbial agent of claim 5, comprising the steps of: culturing the Pichia kudriavzevii strain of claim 1 or 2 or the microbial agent of claim 5.
10. A wine brewed by adding the Pichia kudriavzevii strain of claim 1 or 2 or the microbial agent of claim 5 to a raw material for brewing wine.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114149932A (en) * | 2021-11-08 | 2022-03-08 | 泸州老窖股份有限公司 | Saccharomyces cerevisiae LJ-2 and application thereof |
| CN114149933A (en) * | 2021-11-08 | 2022-03-08 | 泸州老窖股份有限公司 | Saccharomyces cerevisiae LJ-1 and application thereof |
| CN114517157A (en) * | 2022-03-08 | 2022-05-20 | 北京工商大学 | Screening and identification of pichia kudriavzevii X-8 for producing phenethyl acetate and application of pichia kudriavzevii X-8 in white spirit brewing |
| CN116083263A (en) * | 2022-07-06 | 2023-05-09 | 贵州大学 | Ester-producing type Pichia kudriavzevii strain and application thereof |
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| WO2023104005A1 (en) | 2023-06-15 |
| CN114107077B (en) | 2023-10-27 |
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