CN102816706B - Screening and application of yeast CGMCC 4745 capable of high yield of ethanol and low yield of fusel oil in production of Chinese Maotai-flavor liquor - Google Patents
Screening and application of yeast CGMCC 4745 capable of high yield of ethanol and low yield of fusel oil in production of Chinese Maotai-flavor liquor Download PDFInfo
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Abstract
The invention provides screening and application of the yeast CGMCC 4745 capable of high yield of ethanol and low yield of fusel oil in production of Chinese Maotai-flavor liquor, which belongs to the technical field of bioengineering. A bacterial strain used in the invention is Zygosaccharomyces bailii CGMCC 4745. The bacterial strain is separated from a variety of Daqu, fermented grains, brewage raw materials and brewing environment materials in a plurality of famous distilleries in China. The screening method for the bacterial strain comprises the following steps: taking and diluting a Daqu sample, fermented grains, brewage raw materials or brewing environment materials, coating diluted substances on a TTC primary screening plate, choosing a substantially red bacterium colony, inoculating the substantially red bacterium colony into a shake flask for culture, carrying out secondary screening, determining the content of ethanol and fusel oil in obtained fermentation broth by respectively using a distillation specific gravity method and HS-SPME-GC-MS and screening out a bacterial strain with high concentration of ethanol and low concentration of fusel oil. The bacterial strain can be used in the industries of food and wine making.
Description
Technical field
The present invention relates to the screening method of the yeast of high-yield ethanol low yield potato spirit, and yeast and the application in food technology, brewing wine industry thereof of this method of application screening high-yield ethanol low yield potato spirit in Chinese Maotai-flavor liquor 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 and help the essential substance of pastil in the liquor, 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 as the potato spirit too high levels, not only can cause product peppery, bitter, puckery, influence the mouthfeel of wine, and to the toxic effect of human body.Potato spirit oxidation rate in human body is slow than ethanol, and the residence time is long, can cause headache, symptoms such as neural system hyperemia.Potato spirit also is to cause liquor one of reason of white opacity to occur simultaneously, thus its content must control certain model with in, the content of potato spirit is≤0.20g/100 mL (in isopropylcarbinol, the primary isoamyl alcohol of content maximum in the liquor) in the GB regulation 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, make its control in suitable scope, 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 problems such as reverse mutation, have risk in the production.Therefore have only 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 name Maotai-flavor liquor factory of the many families of China 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 effectively be applied to the traditional fermentation liquor industry, optimizes the brewing wine production technique, reduces the brewing wine production cost, improves the drinks quality.
Summary of the invention
(1) technical problem that 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 called after Bayer zygosaccharomyces (
Zygosaccharomyces bailii) BJY-6, deposit number CGMCC No. 4745, 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 Chinese Maotai-flavor liquor production with Daqu, wine unstrained spirits, brewing materials or after brewageing surrounding material and sterilized water mixing, through dilution coating TTC primary dcreening operation flat board, select red significant bacterium colony, transfer again in liquid fermentation medium, shake-flask culture carries out multiple sieve, fermented liquid adopts distillation hydrometer method and HS-SPME-GC-MS to measure ethanol and potato spirit content respectively, 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.Coating TTC primary dcreening operation is cultivated 24h for dull and stereotyped back 30 ℃, grow visible bacterium colony after, pour about 12mL TTC upper strata substratum into and cover original bacterium colony, at 30 ℃ of following lucifuges insulation 2～3h, judge yeast product alcohol ability by the colour generation of bacterium colony afterwards.
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 cooling back adds the saccharifying enzyme of 10～50U/g, 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 wine industry and the Application in Food Industry of various brewing wines, liquor, assembled alcoholic drinks.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 effectively improve the content of 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 the loss of weight method 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 is interior mark with 4-methyl-2-amylalcohol, 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 effectively be applied to 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 Chinese Maotai-flavor liquor is produced, its called after of classifying: 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 Bayer 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 flat board, select red significant bacterium colony, transfer again and carry out multiple sieve in shake-flask culture, fermented liquid adopts distillation hydrometer method and HS-SPME-GC-MS to measure ethanol and potato spirit content respectively, 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, at 30 ℃ of following lucifuges insulation 2～3h, judge yeast product alcohol ability by the colour generation of bacterium colony afterwards.
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 cooling back adds the saccharifying enzyme of 10～50 units/g, 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 comparison of checking order subsequently, determine sieve the kind of yeast.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
Embodiment 3: strain fermentation producing and ethanol and potato spirit are measured
The 2 strain liquor-making yeasts used with certain brewery are contrast, the yeast of the different high-yield ethanol low yield potato spirit that this patent is provided, and 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 called after of classifying:
The Bayer zygosaccharomyces (
Zygosaccharomyces bailii) BJY-6, deposit number CGMCC No. 4745, 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 the described high-yield ethanol low yield of claim 1 potato spirit is characterized in that the application in the brewery industry of various brewing wines, liquor, assembled alcoholic drinks.
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| CN 201210248862 CN102816706B (en) | 2011-05-12 | 2011-05-12 | Screening and application of yeast CGMCC 4745 capable of high yield of ethanol and low yield of fusel oil in production of Chinese Maotai-flavor liquor |
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| CN 201210248862 CN102816706B (en) | 2011-05-12 | 2011-05-12 | Screening and application of yeast CGMCC 4745 capable of high yield of ethanol and low yield of fusel oil in production of Chinese Maotai-flavor liquor |
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| CN105238706A (en) * | 2015-10-29 | 2016-01-13 | 杭州胡庆余堂药业有限公司 | Environmental microorganism zygosaccharomyces bailii |
| CN117165458A (en) * | 2022-03-15 | 2023-12-05 | 重庆江记酒庄有限公司 | Boehmeria bayer joint yeast and method for brewing original flavor of white spirit |
| CN115093981B (en) * | 2022-06-10 | 2024-02-13 | 贵州茅台酒股份有限公司 | Method for improving wine producing performance of wine producing yeast under acid stress environment |
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| CA2990939A1 (en) * | 2007-12-23 | 2009-07-09 | Gevo, Inc. | Yeast organism producing isobutanol at a high yield |
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