CN102766582B - Screening and application of yeast CGMCC 4750 for high production of ethanol and low production of fusel oil in production of Chinese Maotai-flavor liquor - Google Patents
Screening and application of yeast CGMCC 4750 for high production of ethanol and low production of fusel oil in production of Chinese Maotai-flavor liquor Download PDFInfo
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Abstract
The invention relates to screening and application of yeast CGMCC 4750 for high production of ethanol and low production of fusel oil in production of Chinese Maotai-flavor liquor, belonging to the technical field of biological engineering. A strain is of abnormal fermentation Pichia pastoris CGMCC4750. The strain is obtained by separation from various Daqu, fermented grains, brewing raw materials and brewing environments of multiple famous liquor factories in China; and the screening method comprises the following steps of: taking substances of Daqu samples, fermented grains, brewing raw materials or brewing environments, diluting, coating a TTC (triphenyltetrazolium chloride) prescreening flat plate, selecting colonies with significant red color, further transferring into shaking flask culture for rescreening, determining the content of each of the ethanol and the fusel oil in a fermentation solution by adopting a distillation specific gravity method and HS-SPME-GC-MS respectively, and screening to obtain the strain with high concentration of the ethanol and low concentration of the fusel oil. The strain can be applied to food industry and brewed liquor industry.
Description
Technical field
The present invention relates to the screening method of the yeast of high-yield ethanol low yield potato spirit, and use yeast and the application in food technology, brewing wine industry thereof of the method screening high-yield ethanol low yield potato spirit in the Chinese Sauce aromatic white spirit is produced.Belong to technical field of bioengineering.
Background technology
Ethanol be most important in the brewing wine also be one of topmost composition, ethanol content is up to 40%～55%(v/v) in the China white wine; Be 14%～20% in the yellow rice wine; Be 2%～5% in the beer.Yeast is the major microorganisms that produces ethanol, also is indispensable microorganism in the brewing wine industry.Therefore, seek the yeast of high-yield ethanol and can optimize making method, reduce production costs, in liquor industry, have great importance.
Potato spirit is the secondary metabolite that yeast produces in brewing fermentation, comprises n-propyl alcohol, Virahol, propyl carbinol, isopropylcarbinol, Pentyl alcohol, sec.-amyl alcohol, hexanol and enanthol etc.It is pure and sweet in the liquor and helps the essential substance of pastil, to the local flavor that forms wine with impel that the wine body is plentiful, dense to play an important role.The soft coordination of mouthfeel, the wine body that suitable potato spirit content and the eurythmy between the various potato spirit can make wine is plentiful mellow and full and taste is unique, but in the liquor such as the potato spirit too high levels, not only can cause product peppery, bitter, puckery, affect the mouthfeel of wine, and to the toxic effect of human body.Potato spirit is slow than ethanol at the human body Internal oxidation rate, and the residence time is long, can cause headache, the symptoms such as neural system hyperemia.Potato spirit also is to cause liquor one of reason of white opacity to occur simultaneously, therefore its content must be controlled at certain model with in, the content of GB regulation Fusel Oil in Liquor is≤0.20g/100 mL (in isopropylcarbinol, the primary isoamyl alcohol of content maximum in the liquor).
At present, in traditional brewing wine, the general higher problem of potato spirit content has extensively caused the concern of industry.Therefore, for reducing its potato spirit content, it is controlled in the suitable scope, has taked all measures in the production, as adopting low temperature fermentation, optimize with Qu Liang etc.But because its complex process, influence factor is various, also is not well solved in the production.The most basic measure is to adopt the barms of low yield potato spirit to ferment, but, potato spirit is the inevitable by product of yeast in the ethanol fermentation process, and it is generally also stronger that the bacterial strain of high-yield ethanol ability produces the potato spirit ability, so high-yield ethanol and low yield potato spirit often are difficult to satisfactory to both parties.At present, the report by the distillery yeast bacterial strain of mutagenic and breeding low yield potato spirit is arranged also, but in brewery industry, have the problems such as reverse mutation, have risk in the production.Therefore only have the wild yeast of seed selection low yield potato spirit and high-yield ethanol to be only the most basic solution.
The yeast of acquisition that the present invention screens is to screen gained from the various Daqu of China many name Maotai-flavor liquors factory or brewing wine unstrained spirits, has the function of high-yield ethanol low yield potato spirit.This bacterial strain is the high-yield ethanol that therefrom obtains in state's brewed spirit process first and the yeast of low yield potato spirit.This bacterial strain is good brewing functional bacterial strain.This bacterial strain can effective application in the traditional fermentation liquor industry, optimize the brewing wine production technique, reduce the brewing wine production cost, improve the drinks quality.
Summary of the invention
The technical problem that (1) will solve
The technical problem that the present invention will solve provides the yeast strain of high-yield ethanol low yield potato spirit, another technical problem to be solved by this invention has provided the screening method of the yeast strain of high-yield ethanol low yield potato spirit, and the yeast of gained is applied in the brewing wine industry.
(2) technical scheme of the present invention
The yeast of high-yield ethanol low yield potato spirit, its Classification And Nomenclature for the fermentation pichia spp (
Pichia fermentans) BJY-11, deposit number CGMCC No. 4750, above-mentioned bacterial strains have been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center.
The screening of the yeast strain of high-yield ethanol low yield potato spirit:
The screening method of the yeast of described high-yield ethanol low yield potato spirit, in the production of Chinese Sauce aromatic white spirit with Daqu, wine unstrained spirits, brewing materials or after brewageing surrounding material and sterilized water mixing, dull and stereotyped through dilution spread TTC primary dcreening operation, select red significant bacterium colony, transfer again in liquid fermentation medium, shake-flask culture carries out multiple sieve, fermented liquid adopts respectively distillation hydrometer method and HS-SPME-GC-MS to measure ethanol and potato spirit content, and screening obtains producing and ethanol concentration height and produces the low bacterial strain of potato spirit concentration.
Get 0.5～5g Daqu, wine unstrained spirits, brewing materials or brewage surrounding material and be dissolved in 15～150mL stroke-physiological saline solution shaking table vibration, 30～150min, redilution, coating TTC primary dcreening operation flat board.
The TTC primary dcreening operation is cultivated: TTC upper strata substratum (g/L): glucose 5,15,121 ℃ of sterilizations of agar 20min when being cooled to 60 ℃ of left and right sides, behind the adding TTC solution (triphenyltetrazol triazole hydrochloride, final concentration 0.5), topples over bottom platform immediately.TTC lower floor substratum: YPD Agar.Dull and stereotyped rear 30 ℃ of coating TTC primary dcreening operation is cultivated 24h, grow visible bacterium colony after, pour about 12mL TTC upper strata substratum into and cover original bacterium colony, be incubated 2～3h 30 ℃ of lower lucifuges afterwards, judge producing Yeast alcohol ability by the colour generation of bacterium colony.
The TTC primary dcreening operation is selected red significant bacterium colony, transfers and carries out multiple sieve in shake-flask culture, and the shake flask fermentation culture condition is: inoculum size is 0.1 * 10
6～5 * 10
6Individual/mL, substratum is: adopt YPD, Chinese sorghum juice or malt juice liquid medium, Chinese sorghum juice or wort production method are: 20～200g Chinese sorghum or Fructus Hordei Germinatus sample are after crushed, the water that adds 1～4 times of mL/g meter, boiling 1-5h, in the pasty state, the saccharifying enzyme that adds 10～50U/g after the cooling, in 40～80 ℃ of maintenance 2～10h, filtration, centrifugal gained filtrate, pol is 10 ~ 15
oBx; Regulating pH is 2～5,25～35 ℃, 150rpm, shake-flask culture 24～120h.
The application of the yeast of described high-yield ethanol low yield potato spirit is in the wine industry of various brewing wines, liquor, assembled alcoholic drinks and the application in the foodstuffs industry.The application of yeast in brewing wine industry of high-yield ethanol low yield potato spirit: this bacterial strain is made the liquid or solid bacteria preparation, make an addition in the brewing wine brewing process, can effectively increase the biomass of this bacterial strain in brewageing, and the content of Effective Raise ethanol, reduce the generation of potato spirit, finally improve the quality of brewing wine.
Determining concentration of alcohol: the distillation hydrometer method is measured alcohol concn.Measure CO with weight reduction simultaneously
2Discharge quality.
The potato spirit concentration determination: adopt the HS-SPME-GC-MS method, add 5～10 mL clarified broths in the 15 mL head space bottles, 1～5 g NaCl take 4-methyl-2-amylalcohol as interior mark, carries out headspace solid-phase microextraction.The condition of headspace solid-phase microextraction is: three-phase (Car/DVB/PDMS) extracting head, 60 ℃ of preheating 5～10 min, extraction absorption 30～50 min, GC desorb 5～10 min.
(3) beneficial effect
The yeast of acquisition that the present invention screens is to screen gained from Chinese Maotai-flavor liquor brewing process, has the function of high-yield ethanol low yield potato spirit.This batch bacterial strain is the high-yield ethanol that therefrom obtains in state's brewed spirit process first and the yeast saccharomyces cerevisiae of low yield potato spirit.The every strain of this batch bacterial strain all is good brewing functional bacterial strain.These bacterial strains can effective application in the brewing wine industry, optimize the brewing wine production technique, reduce the brewing wine production cost, improve vinosity.
The biological material specimens preservation: the yeast of high-yield ethanol low yield potato spirit during the Chinese Sauce aromatic white spirit is produced, its Classification And Nomenclature is: unusual pichia spp (
Pichia anomala) BJY-1, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, address: No. 3 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, deposit number CGMCC No. 4740, preservation date on April 6th, 2011;
Schizosaccharomyces pombe (
Schizosaccharomyces pombe) BJY-5, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, deposit number CGMCC No. 4744, preservation date on April 6th, 2011;
The Baeyer zygosaccharomyces (
Zygosaccharomyces bailii) BJY-6, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, deposit number CGMCC No. 4745, preservation date on April 6th, 2011;
Trichosporon (
Trichosporon asahii) BJY-7, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, deposit number CGMCC No. 4746, preservation date on April 6th, 2011;
Yeast saccharomyces cerevisiae (
Saccharomyces cerevisiae) BJY-8, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, deposit number CGMCC No. 4747, preservation date on April 6th, 2011;
Saccharomyces exiguus (
Kazachstania exigua) BJY-9, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, deposit number CGMCC No. 4748, preservation date on April 6th, 2011;
The fermentation pichia spp (
Pichia fermentans) BJY-11, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, deposit number CGMCC No. 4750, preservation date on April 6th, 2011.
Description of drawings
Fig. 1. unusual pichia spp (
Pichia anomala) CGMCC No. 4740 cellular form electromicroscopic photographs.
Fig. 2. saccharomyces exiguus (
Kazachstania exigua) CGMCC No. 4748 cellular form electromicroscopic photographs.
Fig. 3. yeast saccharomyces cerevisiae (
Saccharomyces cerevisiae) CGMCC No. 4747 Chinese sorghum juice fermentation culture release CO
2Time curve.
Fig. 4. the fermentation pichia spp (
Pichia fermentans) CGMCC No. 4750 Chinese sorghum juice fermentation culture release CO
2Time curve.
Embodiment
Embodiment 1: the screening of the yeast strain of high-yield ethanol low yield potato spirit
Get 0.5g wine unstrained spirits and be dissolved in 15mL stroke-physiological saline solution shaking table vibration 30 minutes, redilution, coating TTC primary dcreening operation is dull and stereotyped, select red significant bacterium colony, transfer again and carry out multiple sieve in shake-flask culture, fermented liquid adopts respectively distillation hydrometer method and HS-SPME-GC-MS to measure ethanol and potato spirit content, and screening obtains alcohol concn height and the low bacterial strain of potato spirit concentration.
The TTC primary dcreening operation is cultivated: TTC upper strata substratum (g/L): glucose 5,15,121 ℃ of sterilizations of agar 20min when being cooled to 60 ℃ of left and right sides, behind the adding TTC solution (triphenyltetrazol triazole hydrochloride, final concentration 0.5), topples over bottom platform immediately.TTC lower floor: YPD Agar.Cultivate 24h for 30 ℃, grow visible bacterium colony after, pour about 12mL TTC upper strata substratum into and cover original bacterium colony, be incubated 2～3h 30 ℃ of lower lucifuges afterwards, judge producing Yeast alcohol ability by the colour generation of bacterium colony.
The shake-flask culture condition is: inoculum size is 0.1 * 10
6～5 * 10
6Individual/mL, culture medium condition is: adopt malt extract medium, production method is: 20g Fructus Hordei Germinatus sample after crushed, the water that adds 1～4 times of volume, boiling 1-5h, in the pasty state, the saccharifying enzyme that adds 10～50 units/g after the cooling, in 40～80 ℃ of maintenance 2～10h, filtration, centrifugal gained filtrate, pol is 10
oBx; Regulating pH is 4～5,25～35 ℃, 150rpm, shake-flask culture 24～120h.
Embodiment 2: yeast and the molecular biology identification thereof of high-yield ethanol low yield potato spirit
Yeast to the different high-yield ethanol low yield potato spirit that obtain carries out molecular biology identification, utilizes increase the respectively 26srDNA fragment of bacterial strain of the special classification primers designed of yeast, through detected through gel electrophoresis.The kind of yeast of sieving to get is determined in the comparison of checking order subsequently.Bacterial strain is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, and yeast kind and deposit number see Table 1.
The yeast of table 1 high-yield ethanol low yield potato spirit
| Numbering | Chinese name | English name | Deposit number |
| 1 | Unusual pichia spp | Pichia anomala | CGMCC No.4740 |
| 2 | Schizosaccharomyces pombe | Schizosaccharomyces pombe | CGMCC No.4744 |
| 3 | The Bayer zygosaccharomyces | Zygosaccharomyces bailii | CGMCC No.4745 |
| 4 | Trichosporon | Trichosporon asahi | CGMCC No.4746 |
| 5 | Yeast saccharomyces cerevisiae | Saccharomyces cerevisiae | CGMCC No.4747 |
| 6 | Saccharomyces exiguus | Kazachstania exigua | CGMCC No.4748 |
| 7 | The fermentation pichia spp | pichia fermentans | CGMCC No.4750 |
Embodiment 3: strain fermentation producing and ethanol and potato spirit are measured
Take the used 2 strain liquor-making yeasts of certain brewery as contrast, the yeast of the different high-yield ethanol low yield potato spirit that this patent is provided, adopting pH is 5, pol is 10
oThe Chinese sorghum juice substratum of Bx, 1 * 10
6The inoculum size of individual/mL, 30 ℃, 150rpm, shake-flask culture 72～96h, producing and ethanol and potato spirit concentration see Table 2.
Table 2 yeast Chinese sorghum juice substratum fermentation producing and ethanol result
Annotate: ethanol is 51.1% to the theoretical yield of sugar.
Claims (2)
1. the yeast of high-yield ethanol low yield potato spirit, its Classification And Nomenclature is
The fermentation pichia spp (
Pichia fermentans) BJY-11, deposit number CGMCC No. 4750, above bacterial strain have been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center.
2. the application of the yeast of high-yield ethanol low yield potato spirit claimed in claim 1 is characterized in that the application in the brewery industry of various brewing wines, liquor, assembled alcoholic drinks.
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| CN103525896B (en) * | 2013-09-27 | 2015-04-08 | 南京工业大学 | High-activity yeast cell quantitative screening method based on TTC staining method |
| CN106753994B (en) * | 2016-11-28 | 2020-05-19 | 山西农业大学 | Method for improving alcohol content of alcohol fermentation liquor and reducing isoamyl alcohol content by using high-ester-yield indigenous aroma-producing yeast enhanced yeast |
| CN107916198B (en) * | 2017-12-15 | 2021-01-29 | 江南大学 | Microbial agent for effectively inhibiting formation of monosodium glutamate in yeast for making hard liquor |
Citations (2)
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|---|---|---|---|---|
| WO1999050422A2 (en) * | 1998-03-27 | 1999-10-07 | Yissum Research Development Company Of The Hebrew University Of Jerusalem | Method for reduction of fusel oils in alcoholic beverages and food products |
| CN101845404A (en) * | 2010-01-08 | 2010-09-29 | 广西科学院 | Brewing yeast strain, breeding method thereof, and application of the strain in alcohol production |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999050422A2 (en) * | 1998-03-27 | 1999-10-07 | Yissum Research Development Company Of The Hebrew University Of Jerusalem | Method for reduction of fusel oils in alcoholic beverages and food products |
| CN101845404A (en) * | 2010-01-08 | 2010-09-29 | 广西科学院 | Brewing yeast strain, breeding method thereof, and application of the strain in alcohol production |
Non-Patent Citations (2)
| Title |
|---|
| "低产杂醇油啤酒酵母菌株的选育";肖东光 等;《酿酒科技》;20051231(第4期);第31-33页 * |
| 肖东光 等."低产杂醇油啤酒酵母菌株的选育".《酿酒科技》.2005,(第4期),第31-33页. |
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