CN111154662B - High-concentration beer strain and screening method thereof - Google Patents
High-concentration beer strain and screening method thereof Download PDFInfo
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- CN111154662B CN111154662B CN202010014507.8A CN202010014507A CN111154662B CN 111154662 B CN111154662 B CN 111154662B CN 202010014507 A CN202010014507 A CN 202010014507A CN 111154662 B CN111154662 B CN 111154662B
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12C—BEER; PREPARATION OF BEER BY FERMENTATION; PREPARATION OF MALT FOR MAKING BEER; PREPARATION OF HOPS FOR MAKING BEER
- C12C12/00—Processes specially adapted for making special kinds of beer
- C12C12/002—Processes specially adapted for making special kinds of beer using special microorganisms
- C12C12/006—Yeasts
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/02—Separating microorganisms from their culture media
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/36—Adaptation or attenuation of cells
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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
- Y02E50/10—Biofuels, e.g. bio-diesel
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- Food Science & Technology (AREA)
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Abstract
The invention relates to a high-concentration beer strain and a screening method thereof, belonging to the technical field of beer brewing. The screening method comprises the following steps: domestication: inoculating the starting strain into a composite culture medium containing predetermined amino acid for domestication for 3-7 times, wherein the sugar degree of the composite culture medium is 25-30 DEG P; separating: fermenting and culturing the culture solution obtained by the final domestication, and selecting yeast meeting the preset standard as preset separated yeast; coating the selected colony of the predetermined separation yeast in a composite culture medium flat plate with the sugar degree of 25-30 DEG P, carrying out streak culture separation, activating, and separating by adopting a gradient dilution method to obtain a single colony; screening: inoculating the single colony obtained by separation into 18°P wort, culturing at 12 ℃, adding 8% alcohol when the sugar degree of wort is reduced to 12°P, culturing at 16 ℃, and selecting strains with high alcohol concentration resistance. The obtained yeast can resist 30 DEG P ultra-high concentration wort to ferment and produce beer with 13.8% alcohol content.
Description
Technical Field
The invention relates to the technical field of beer brewing, in particular to a high-concentration beer strain and a screening method thereof.
Background
Along with the development of the world-wide refined beer, various kinds of beer with diversified flavors and distinct characteristics are emerging. In the aspect of high-alcoholicity beer, the alcohol separated is generally added back to improve the alcoholicity by beer distillation, but the method has high energy consumption and complex process, is inapplicable at present for advocating energy conservation and consumption reduction, is environment-friendly and efficient, and achieves the aim of high alcoholicity by screening ultra-high concentration yeast to be matched with a high-concentration brewing method.
For the brewing technology with high alcohol content, although the conventional technology is about 20 resistance ° Fermentation research of P wort high-concentration yeast, which is limited to laboratory stage, is not applied to mass production. The high-concentration brewing of the beer products is 16-18 ° P wort brewing rarely exceeds 18 ° P wort brewing to produce beer. The main reason is that the stability of the fermentation performance and quality of the high-concentration yeast is difficult to control, the higher the concentration of wort is, the worse the living environment of the yeast is, and the higher the osmotic pressure and the alcoholic strength required to be born by the yeast are, so that the common beer yeast is difficult to be qualified, and the yeast which is resistant to the high-concentration wort and has good fermentation performance and genetic stability must be bred.
Disclosure of Invention
In view of the above, it is necessary to provide a high-concentration beer strain and a method for screening the same, by which the strain screened can withstand fermentation with high concentration wort and alcoholicity.
A screening method of a high-concentration beer strain comprises the following steps:
domestication: inoculating the starting strain into a composite culture medium containing predetermined amino acid for domestication for 3-7 times, wherein the sugar degree of the composite culture medium is 25-30 DEG P;
separating: fermenting and culturing the culture solution obtained by the final domestication, detecting the death rate and the alcohol degree of the yeast, and selecting the yeast meeting the preset standard as the preset separation yeast; coating the selected colony of the predetermined separation yeast in a composite culture medium flat plate with the sugar degree of 25-30 DEG P for streak culture separation, finding out a single colony for single colony separation, picking up a colony with good growth, inoculating the colony into wort liquid for activation, and separating the single colony by adopting a gradient dilution method;
screening: inoculating the single colony obtained by separation into 18°P wort, culturing at 12 ℃, adding 8% alcohol when the sugar degree is reduced to 12°P, culturing at 15 ℃, and selecting strains with high alcohol concentration resistance.
The inventor finds that free amino acid is one of the most important factors for yeast growth in earlier research, so that the content of amino acid in wort directly affects each biological metabolism synthesis path, and the screening method of the high-concentration beer strain is that the inventor effectively shortens the number of experiments through ingenious experimental design (such as DOE design and the like) and calculates the experimental steps, and simultaneously grasps the type and concentration of main amino acid affecting yeast biological metabolism in fermenting the ultrahigh-concentration wort after considering the most effective parameter information affecting the ultrahigh-concentration fermentation performance of the yeast, and utilizes the influenced amino acid as a screening preparation culture medium to screen out the yeast capable of resisting the fermentation of the ultrahigh-concentration wort.
In the screening step, the colony is inoculated in an environment with the wort concentration lower than that in the domestication step, and is cultivated at a temperature lower than that in the domestication step, after the wort concentration is reduced to a preset concentration, alcohol is added, and the temperature is increased for cultivation, so that the yeast with high-concentration wort resistance and alcohol concentration can be screened.
In one implementation, in the acclimatizing step, the predetermined amino acid is: 40-120mg/L aspartic acid, 25-75mg/L glutamic acid, 55-165mg/L asparagine, 40-120mg/L serine, 5-15mg/L glutamine, 30-90mg/L histidine, 20-60mg/L glycine, 35-105mg/L threonine, 60-180mg/L alanine, 75-225mg/L arginine, 55-165mg/L tyrosine, 70-210mg/L valine, 20-60mg/L methionine, 40-120mg/L isoleucine, 35-105mg/L tryptophan, 65-195mg/L phenylalanine, 85-255mg/L leucine, 55-165mg/L lysine, 5-15mg/L cysteine, and 180-540mg/L proline. The strain domestication is performed by adopting the predetermined amino acid, so that the strain domestication method has a good effect.
In one implementation, the predetermined amino acid is: 80mg/L aspartic acid, 50mg/L glutamic acid, 110mg/L asparagine, 80mg/L serine, 10mg/L glutamine, 90mg/L histidine, 60mg/L glycine, 70mg/L threonine, 120mg/L alanine, 150mg/L arginine, 110mg/L tyrosine, 140mg/L valine, 40mg/L methionine, 80mg/L isoleucine, 70mg/L tryptophan, 195mg/L phenylalanine, 170mg/L leucine, 110mg/L lysine, 10mg/L cysteine, 360mg/L proline. The strain domestication is carried out by adopting the predetermined amino acid, so that the strain domestication effect is optimal.
In one implementation, in the acclimating step, the complex medium further includes complex sugar, where the complex sugar is: 21.5-25.6g/L glucose, 2.9-3.8g/L fructose, 12.1-14.5g/L sucrose, 172.7-180.5g/L maltose, 41.8-46.9g/L maltotriose, 52.3-56.7g/L maltotetraose. The composite sugar is a carbon source formula required by yeast in a 30 DEG P composite culture medium for domestication.
In one implementation, the temperature is maintained at 15 ℃ during the acclimation step.
In one embodiment, the acclimatizing step, the starting bacterial species is selected from the group consisting of: lager brewing yeast. The strain is used as a starting strain, and a high-concentration beer strain with a good effect can be obtained.
In one embodiment, the isolation culture, the fermentation culture is specifically: inoculating the domesticated culture solution into 18 DEG P wort, culturing at 25 ℃ for 24 hours for activation, and then streaking the activated bacterial solution on a composite culture medium without alcohol addition, and fermenting and culturing at 15 ℃. Individual strains were isolated by streaking.
In one implementation, the acclimatization step is preceded by an activation step in which the hairline strain is picked up and inoculated into 18°p wort for cultivation at 25 ℃ for 24 hours.
In one embodiment, the screening step is followed by an evaluation step in which individual strains isolated by streaking are subjected to a pilot test to evaluate their production suitability.
Specifically, the above-mentioned pilot test may employ the following method: selecting a ring of the screened strain; inoculating into 10mL wort, and culturing at 28deg.C for 24 hr; and then all bacterial liquid in the activated test tube is filled into a 30 DEG P wort triangle bottle added with 30mg/L histidine, 20mg/L glycine and 65mg/L phenylalanine, the mixture is sealed by a fermentation plug, and is cultured and fermented for 8 days at the temperature of 12 ℃, the mixture is weighed every day, the daily weight loss is recorded, and when the single daily weight loss is within 0.2 g, the fermentation is finished. By this evaluation step, strains capable of propagating and rapidly fermenting with 30°p wort were selected.
The invention also discloses the high-concentration beer strain obtained by screening by the screening method of the high-concentration beer strain.
Compared with the prior art, the invention has the following beneficial effects:
according to the screening method of the high-concentration beer strain, through ingenious experimental design, the experimental quantity is effectively shortened through calculation and test steps, and after the most effective parameter information affecting the ultrahigh-concentration fermentation performance of the yeast is considered, the main amino acid types and the concentration affecting the biological metabolism of the yeast in fermenting the ultrahigh-concentration wort are mastered, and the influenced amino acid is used as a screen to prepare a culture medium, so that the yeast capable of resisting the fermentation of the ultrahigh-concentration wort is screened. The obtained yeast can resist 30 DEG P ultra-high concentration wort to ferment and produce beer with 13.8% alcohol content.
Detailed Description
In order that the invention may be readily understood, a more complete description of the invention will be rendered by reference to the embodiments that are illustrated below. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.
The starting strains used in the examples below were all conventional commercial lager brewing yeasts.
Example 1
A screening method of high-concentration beer strain, the original strain is lager brewing yeast, comprising the following steps:
1. the amino acid concentration of the culture medium is designed.
1) The amino acid content of 18℃P wort and 30℃P wort were examined, and the results are shown in the following table.
TABLE 1 amino acid content of wort
The amino acid concentration was calculated according to the amino acid concentration shown in the above table by designing the DOE method, and the specific results are shown in the following table.
TABLE 2 amino acid concentration
2) The compound sugar is prepared according to sugar components required by yeast, and specifically comprises the following components: 21.5-25.6g/L glucose, 2.9-3.8g/L fructose, 12.1-14.5g/L sucrose, 172.7-180.5g/L maltose, 41.8-46.9g/L maltotriose, 52.3-56.7g/L maltotetraose.
3) A complex medium was prepared according to the amino acid concentration and the complex carbohydrate content shown in Table 2 above and 25g/L agar.
2. Domestication.
Inoculating the starting strain into the composite culture medium for domestication for 5 times, wherein the sugar degree of the composite culture medium is 30 DEG P; the method comprises the following specific steps:
all bacterial liquid in the activated test tube is poured into 500mL triangular flasks, 300mL of the composite culture medium is added into each flask, and domestication is carried out at 15 ℃ for 5 times. And (3) fermenting and culturing the culture solution obtained by the final domestication, and detecting the death rate and the alcohol degree of the yeast.
3. And (5) separating.
The method comprises the following steps: the strain is picked out and inoculated into 10mL18 DEG P wort for 24h culture at 25 ℃ for activation. And then streaking the activated bacterial liquid on the composite culture medium without alcohol addition, and culturing at 15 ℃. Individual strains were isolated by streaking and stored.
4. And (5) screening.
Selecting one ring of each separated single strain, inoculating into 10mL of 18 DEG P wort, culturing at 25 ℃ for 24 hours for activation, pouring all bacterial liquid in activated test tubes into 500mL triangular bottles, adding 300mL of the composite culture medium without adding alcohol into each bottle, and fermenting at 15 ℃ until the diacetyl content is detected to be lower than 0.25 ppm. After the fermentation is completed, the fermentation conditions of the fermentation liquid yeast, including the yeast death rate and the ethanol yield, are detected, and the results are shown in the following table.
TABLE 3 fermentation assay results
| Round of | Yeast mortality (Single) | Alcohol degree (Single) |
| 1 | 21.9 | 8.5 |
| 2 | 23.5 | 9.2 |
| 3 | 26.7 | 9.7 |
| 4 | 19.4 | 7.6 |
| 5 | 18.8 | 9.9 |
| 6 | 20.1 | 7.3 |
| 7 | 25.6 | 8.1 |
| 8 | 22.0 | 9.4 |
| 9 | 21.3 | 8.7 |
| 10 | 19.9 | 10.8 |
| 11 | 15.3 | 13.8 |
| 12 | 18.4 | 9.2 |
| 13 | 17.6 | 10.9 |
| 14 | 24.6 | 11.2 |
| 15 | 22.8 | 8.4 |
| 16 | 16.2 | 9.2 |
| 17 | 23.3 | 8.3 |
| 18 | 26.1 | 10.6 |
| 19 | 21.5 | 7.9 |
| 20 | 20.5 | 9.6 |
| 21 | 27.3 | 8.8 |
| 22 | 19.6 | 9.4 |
From the above test results, the 11 th round of amino acid comparisons enabled the yeast to tolerate 30℃P sugar degree and to produce higher alcohol degree.
Taking the yeast of the 11 th round as a scheduled separation yeast; the selected colony of the predetermined separation yeast is coated in a compound culture medium flat plate with the 11 th round of amino acid ratio and the sugar degree of 30 DEG P for streak culture separation, single colony is found out for single colony separation, the colony with good growth is picked up and inoculated into 10mL wort liquid for activation, and the single colony is obtained by adopting a gradient dilution method for separation.
Inoculating the single colony obtained by separation into 18 DEG P wort, culturing at 12 ℃, adding 8% alcohol when the wort degree is reduced to 12 DEG P, culturing at 15 ℃, and selecting strains capable of resisting high alcohol concentration according to the death rate of yeast after the sugar degree is reduced to 5 DEG P.
5. And (5) evaluating.
Fermenting and culturing the newly obtained strain: selecting a ring of the screened strain; inoculating into 10mL wort, and culturing at 28deg.C for 24 hr; and then all bacterial liquid in the activated test tube is filled into a 30 DEG P wort triangle bottle added with 30mg/L histidine, 20mg/L glycine and 65mg/L phenylalanine, the mixture is sealed by a fermentation plug, and is cultured and fermented for 8 days at the temperature of 12 ℃, the mixture is weighed every day, the daily weight loss is recorded, and when the single daily weight loss is within 0.2 g, the fermentation is finished. After the fermentation is completed, the fermentation index is detected, and the results are shown in the following table.
TABLE 4 fermentation index
The indexes show that the strain bred by the culture medium of the invention by using the screening method is greatly improved compared with the fermentation performance indexes of the original strain.
Example 2
The selected strain obtained by selection using example 1 above was fermented by the following fermentation steps:
1) Saccharifying, preparing raw wort with concentration of 16 deg.P by using 65-74% malt and 25-36% rice, adding syrup into boiling pot, adjusting wort concentration to 30 deg.P, and adding 30mg/L histidine, 20mg/L glycine and 65mg/L phenylalanine when cooling;
2) Fermenting, cooling wort, adding oxygen 10-12ppm, inoculating strain, and controlling the inoculum size at 17-21×10 6 The fermentation temperature is 15 ℃, and the pressure boosting sugar degree is controlled to be 11-13 DEG P;
3) The performance index was measured after fermentation, and the results are shown in the following table.
TABLE 5 fermentation Performance index
From the results, the breeding method can obtain the high-concentration beer strain which can be fermented at 30 DEG P ultra-high-concentration wort and 14% alcohol.
Example 3
A screening method of a high-concentration beer strain, wherein a starting strain is lager brewing yeast, screening is carried out according to the method of the embodiment 1 to obtain a selective breeding strain, and fermentation culture is carried out on the selective breeding strain:
selecting a ring of the screened strain; inoculating into 10mL wort, and culturing at 28deg.C for 24 hr; and then all bacterial liquid in the activated test tube is filled into a 300mL30 DEG P wort triangular flask added with 30mg/L histidine, 20mg/L glycine and 65mg/L phenylalanine, the flask is sealed by a fermentation plug, the fermentation is carried out at 12 ℃ for 8 days, the weight is weighed every day, the weight loss every day is recorded, and when the weight loss per day is within 0.2 g, the fermentation is finished. After the fermentation is completed, the fermentation index is detected, and the results are shown in the following table.
TABLE 6 fermentation index
The indexes show that the strain bred by the culture medium of the invention by using the screening method is greatly improved compared with the fermentation performance indexes of the original strain.
Example 4
A screening method of a high-concentration beer strain, wherein a starting strain is lager brewing yeast, screening is carried out according to the method of the embodiment 1 to obtain a selective breeding strain, and fermentation culture is carried out on the selective breeding strain:
selecting a ring of the screened strain; inoculating into 10mL wort, and culturing at 28deg.C for 24 hr; and then all bacterial liquid in the activated test tube is filled into a 30 DEG P wort triangle bottle added with 30mg/L histidine, 20mg/L glycine and 65mg/L phenylalanine, the mixture is sealed by a fermentation plug, and is cultured and fermented for 8 days at the temperature of 12 ℃, the mixture is weighed every day, the daily weight loss is recorded, and when the single daily weight loss is within 0.2 g, the fermentation is finished. After the fermentation is completed, the fermentation index is detected, and the results are shown in the following table.
TABLE 7 fermentation index
The indexes show that the strain bred by the culture medium of the invention by using the screening method is greatly improved compared with the fermentation performance indexes of the original strain.
The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (2)
1. The screening method of the high-concentration beer strain is characterized by comprising the following steps of:
activating: picking out the strain, inoculating into 18 DEG P wort, and culturing at 25 ℃ for 24 hours;
domestication: inoculating the lager brewing yeast serving as a starting strain into a composite culture medium containing predetermined amino acid for domestication, keeping the temperature at 15 ℃, and performing domestication for 5 times, wherein the sugar degree of the composite culture medium is 30 DEG P; the predetermined amino acids are: 80mg/L aspartic acid, 50mg/L glutamic acid, 110mg/L asparagine, 80mg/L serine, 10mg/L glutamine, 90mg/L histidine, 60mg/L glycine, 70mg/L threonine, 120mg/L alanine, 150mg/L arginine, 110mg/L tyrosine, 140mg/L valine, 40mg/L methionine, 80mg/L isoleucine, 70mg/L tryptophan, 195mg/L phenylalanine, 170mg/L leucine, 110mg/L lysine, 10mg/L cysteine, 360mg/L proline; the compound culture medium also comprises compound sugar, wherein the compound sugar is as follows: 21.5-25.6g/L glucose, 2.9-3.8g/L fructose, 12.1-14.5g/L sucrose, 172.7-180.5g/L maltose, 41.8-46.9g/L maltotriose, 52.3-56.7g/L maltotetraose;
separating: inoculating the culture solution obtained by the final domestication into 18 DEG P wort, culturing for 24 hours at 25 ℃, activating, pouring the activated bacterial solution into a composite culture medium without alcohol addition, fermenting and culturing at 15 ℃, detecting the death rate and the alcohol degree of yeast, and selecting the yeast meeting the preset standard as the preset separation yeast; coating the selected colony of the predetermined separation yeast in a composite culture medium flat plate with the sugar degree of 25-30 DEG P for streak culture separation, finding out a single colony for single colony separation, picking up a colony with good growth, inoculating the colony into wort liquid for activation, and separating by a gradient dilution method to obtain the single colony;
screening: inoculating the single colony obtained by separation into 18°P wort, culturing at 12 ℃, adding 8% alcohol when the sugar degree of wort is reduced to 12°P, culturing at 15 ℃, and selecting strains with high alcohol concentration resistance.
2. The method according to claim 1, wherein the step of screening further comprises an evaluation step of performing a pilot test on the strain resistant to high alcohol concentration obtained by screening to evaluate its production adaptability.
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