CN103627646B - Wine yeast with low yield of higher alcohol - Google Patents
Wine yeast with low yield of higher alcohol Download PDFInfo
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- CN103627646B CN103627646B CN201310633733.4A CN201310633733A CN103627646B CN 103627646 B CN103627646 B CN 103627646B CN 201310633733 A CN201310633733 A CN 201310633733A CN 103627646 B CN103627646 B CN 103627646B
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- higher alcohol
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Abstract
The invention relates to wine yeast with low yield of higher alcohol. The wine yeast is characterized in that a certain amount of isoamyl chloroacetate is added in a flat plate for cultivating yeast, the isoamyl chloroacetate generates chloroacetic acid under the catalysis of saccharomycetes isoamyl acetate hydrolase; the chloroacetic acid has a strong restraining effect on the growth of saccharomycetes, if the activity of the ester decomposition enzyme of a mutant strain is high (the higher the activity of the ester decomposition enzyme is, the more higher alcohol produced by the strain is), more chloroacetic acid is produced by decomposition, so that a bacterial colony grows slowly under the inhibition effect of the chloroacetic acid. The wine yeast can be utilized to accurately, conveniently and quickly screen out saccharomycetes with low yield of the higher alcohol. When the saccharomycetes with low yield of the higher alcohol, which is bred through the wine yeast, is used for producing grape wine, the content of the higher alcohol in the grape wine can be lowered by about 10% to 15%; when the saccharomycetes with low yield of the higher alcohol, which is bred through the wine yeast, is used for alcohol fermentation, the content of the higher alcohol in grape wine can be lowered effectively, and the health-care values of the grape wine can be improved.
Description
(1) technical field
The present invention relates to the wine yeast of a strain yield of higher alcohol, belong to wine making field.
(2) background technology
Higher alcohols is yeast secondary metabolite the most common in grape wine, as too high levels in fruit wine can cause human consumer to drink rear top, headache and drunk, has a strong impact on health-care effect vinous.
The higher alcohols that wine yeast metabolism produces mainly contains primary isoamyl alcohol, isopropylcarbinol, n-propyl alcohol and isohexyl alcohol, and wherein primary isoamyl alcohol accounts for 40% ~ 50% in grape wine higher alcohols composition.Existing research reduces the content of grape wine higher alcohols mainly through diazonium dyeing flat band method screening yield of higher alcohol yeast strain.Diazonium dyeing dull and stereotyped colour developing principle: the 1-naphthyl acetate on yeast Isoamyl Acetate FCC Hydrolases catalyze flat board generates naphthols, and the latter and diazo salt (fast blue salts B) react, and generates azopigment and takes on a red color.When the timing of substrate (1-acetic naphthalene) concentration one, esterase activity is higher, and the amount of product naphthols is larger, and the azopigment of generation is more, and yeast colony color is darker, and higher alcohols growing amount is larger.
The ester lytic enzyme expression activitiy of current commercial yeast is low, and is through repeatedly domestication ability for industrial production, if transformed these bacterial classifications further, ester lytic enzyme activity change amplitude is not very large again.If screened it by diazonium dyeing flat band method, the colour development difference between starting strain and object bacterial strain is not obvious, is unfavorable for selecting of mutant strain.Institute thinks brewages low higher alcohols grape wine, and it is necessary that searching one screens the saccharomycetic method of yield of higher alcohol fast, accurately, easily.
(3) summary of the invention
The object of this invention is to provide the wine yeast of a strain yield of higher alcohol, and provide one fast, accurately, the saccharomycetic method of seed selection yield of higher alcohol easily, utilize this yeast production grape wine can solve higher alcohols in grape wine too high levels and make human consumer drink the problem of rear top, headache.
The approach that patent of the present invention addresses this problem selects isoamyl chloroacetate plate screening model.Its principle is: in the flat board of culturing yeast, add a certain amount of isoamyl chloroacetate, isoamyl chloroacetate generates Mono Chloro Acetic Acid under yeast Isoamyl Acetate FCC Hydrolases catalyze, Mono Chloro Acetic Acid has strong restraining effect to saccharomycetic growth, if the ester lytic enzyme of mutant strain is active greatly, (esterase lytic enzyme is active large, the higher alcohols that bacterial strain generates is many), and then it is just many to decompose the Mono Chloro Acetic Acid produced, will be very little so bacterium colony is long under chloroacetic restraining effect, utilize the present invention can filter out the barms of yield of higher alcohol accurately, easily and fast.
Utilize the yield of higher alcohol barms of seed selection of the present invention to produce grape wine, the content of higher alcohols in grape wine can be made to reduce about 10% ~ 15%.
The present invention utilizes common yeast saccharomyces cerevisiae to be separated the wine yeast CP1118(Saccharomyces cerevisiae cultivating a strain yield of higher alcohol), the experiment proved that it has the ability reducing higher alcohols in grape wine.
The wine yeast CP1118(Saccharomyces cerevisiae of one strain yield of higher alcohol) be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) on June 28th, 2013; Deposit number: CGMCCNO.7828.
Depositary institution address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City institute of microbiology of the Chinese Academy of Sciences
Classification And Nomenclature: yeast saccharomyces cerevisiae (Saccharomyces cerevisiae).
The wine yeast of one strain yield of higher alcohol, prepares by the following method:
A. use transfering loop picking one ring business wine yeast thalline as starting strain, be inoculated in 20mL YPD liquid nutrient medium, 30 DEG C of shaking culture 16 ~ 18h;
B. then get the centrifugal 15s of YPD liquid nutrient medium 12000r/min after 1mL step a cultivation, abandon supernatant collection yeast cell; The cell collected is washed 1 time again with sterilized water;
C. be the 7.0 Sterile phosphate sodium damping fluid (mixed solutions of SODIUM PHOSPHATE, MONOBASIC, Sodium phosphate dibasic and sterilized water by 1mL pH value; The concentration 0.1mol/L of damping fluid), suspension cell; In the cell after suspending, add 30 μ L ethylmethane sulfonate (EMS) stostes, vibrating dispersion cell, be placed in 30 DEG C of shaking tables and cultivate the centrifugal 15s of 12000r/min after 1h;
D. the aseptic Na that 1mL concentration is 5% is added in the cell after centrifugal to step c
2s
2o
3solution (Na
2s
2o
3with the mixed solution of sterilized water) washed cell twice, with the mutation effect of inactivation EMS.
E. collect the cell after step d) washing, then with aseptic washing twice, after cell is coated with isoamyl chloroacetate flat board (CL is dull and stereotyped), cultivate choosing colony the maximum after 3 days for 30 DEG C and be higher alcohols low yield bacterial strain (object bacterial strain).Be CP1118(Saccharomyces cerevisiae by object Strain Designation).
To bacterial strain CP1118(Saccharomyces cerevisiae) carry out genetic stability test and carry out the detection of higher alcohols growing amount, after proving that this strain passage is cultivated, still there is the effect reducing higher alcohols growing amount, its stabilization characteristics of genetics is described.
Analyzed by higher alcohols growing amount, determine that the growing amount of object bacterial strain higher alcohols obviously reduces than the higher alcohols growing amount of the bacterium that sets out.
The main biological property of this bacterial strain is: in oval or circle.Bacterium colony is creamy white, surface wettability, thickness, opaque, and neat in edge is easily provoked.The optimum growth conditions of this bacterial strain is pH value 7.0, temperature 30 DEG C.
Beneficial effect of the present invention:
Utilize the higher alcohols low yield bacterial strain of seed selection of the present invention to carry out the content that zymamsis effectively can reduce higher alcohols in grape wine, improve health value vinous.
(3) accompanying drawing explanation
Fig. 1 starting strain grape wine higher alcohols measures gas chromatogram
The growing amount of starting strain higher alcohols is gone out as benchmark;
Fig. 2 object bacterial strain grape wine higher alcohols measures gas chromatogram
The growing amount of object bacterial strain CP1118 higher alcohols is gone out as benchmark;
(4) embodiment
Below in conjunction with concrete case study on implementation, the present invention is described further, but protection scope of the present invention is not limited in this:
The present invention can use common commercial wine yeast as starting strain.
YPD liquid nutrient medium: 1% yeast extract, 2% peptone, 2% glucose, to dissolve with distilled water; Sterilizing 15min at 121 DEG C; PH nature;
YPD solid medium: 1% yeast extract, 2% agar powder, 2% peptone, 2% glucose, dissolve with distilled water;
Isoamyl chloroacetate flat board (CL is dull and stereotyped) substratum: the yeast nitrogen basis of 0.67%, glucose, 2% agar, the 90mg/L isoamyl chloroacetate of 2%; Dissolve with distilled water and be adjusted to pH7.0.
Embodiment 1: the seed selection of yield of higher alcohol wine yeast
1) mutagenesis of starting strain:
A. from the wine yeast EC1118 inclined-plane a small amount of yeast thalline of inoculating needle picking as starting strain, be inoculated into and fill in 20mL YPD liquid nutrient medium, shaking culture 16 ~ 18h at 30 DEG C, obtain starting strain bacterium liquid.
B. then get 1mL starting strain bacterium liquid 12000r/min in centrifuge tube, centrifugal 15s collecting cell, washs 1 time with sterilized water.
C. be the 7.0 Sterile phosphate sodium damping fluid (mixed solutions of SODIUM PHOSPHATE, MONOBASIC, Sodium phosphate dibasic and sterilized water by 1mL pH value; The concentration 0.1mol/L of damping fluid) suspension cell, add 30 μ L ethylmethane sulfonate (EMS) stostes, vibrating dispersion cell, centrifugal after being placed in 30 DEG C of shaking tables cultivation 1h.
D. then add in centrifuge tube with step c centrifuge tube in centrifugal after the isopyknic concentration of liquid be centrifugally after 5% aseptic Na2S2O3 solution remove supernatant liquor, repetitive operation 2 times, with the mutation effect of inactivation EMS.
2) screening of object bacterial strain:
A. the cell of collected by centrifugation in step 1) d is got, and then with aseptic washing secondary and to adjust to barm cell concentration in diluting soln be 10
3individual/mL time, namely obtain bacteria suspension; Draw 30 microlitre bacteria suspension coating isoamyl chloroacetates flat board (CL is dull and stereotyped), cultivate 3 days for 30 DEG C, choosing colony the maximum is higher alcohols low yield bacterial strain (object bacterial strain).This object Strain Designation is CP1118 and carries out genetic stability test.
3) the genetic stability test of object bacterial strain
A. CP1118 transfering loop is inoculated a ring in YPD solid medium, continue cultivation 2 days, obtain generation CP1118; Inoculate a ring generation CP1118 in new YPD solid medium with transfering loop again, continue cultivation 2 days, obtain two generation CP1118; By that analogy, continuous passage cultivates 10 times;
B. by Secondary Culture to the CP1118 in the 10th generation in YPD liquid nutrient medium, 28 DEG C of shaking tables cultivate 2 days, obtain yeast CP1118 bacterium liquid.
C. with sterilized water, yeast CP1118 bacterium liquid being diluted to concentration is 10
6individual/mL.Then the yeast CP1118 bacterium liquid drawing 1mL dilution ferments in the 50mL Sucus Vitis viniferae of sterilizing, gets 1mL starting strain bacterium liquid simultaneously and adds and ferment as a control group in the same Sucus Vitis viniferae of 50mL; Two groups of fermentation conditions are identical; After fermentation ends, (to residual sugar content lower than 4g/L) carries out the detection of higher alcohols to two groups of Sucus Vitis viniferaes, detected result shows to use the content of the higher alcohols in grape wine of bacterial strain CP1118 fermentation significantly to lower compared with the grape wine using starting strain to ferment, and this bacterial strain higher alcohols growing amount is stablized, still there is after bacterial strain CP1118 Secondary Culture 10 generation of this genetic stability test card improving eyesight the effect reducing higher alcohols growing amount, its stabilization characteristics of genetics is described.
Embodiment 2: starting strain and object bacterial strain higher alcohols growing amount are analyzed
The extraction of higher alcohols: the grape wine that accurate measuring 8mL bacterial strain CP1118 ferments, put into the ml headspace bottle of 15mL, add 1g NaCl, promote the volatilization of higher alcohols, add mark (4-methyl-2-amylalcohol) in 30 μ L again, compress with the sealing of PTFE/ silicon rubber dottle pin at once.When grape wine sample temperature arrives 45 DEG C, with the extracting head extraction 50min of DVB/CAR/PDMS, sample introduction carries out gas chromatography-mass spectrometry analysis.
GC/MS condition: chromatographic column, Stabilwax-DA capillary column ((30m × 0.32mm × 0.25 μm)); Heating schedule: 30 DEG C keep 1min, rise to 100 DEG C, be warming up to 200 DEG C with 3 DEG C/min, be warming up to 210 DEG C with 10 DEG C/min with 6 DEG C/m in, keep 3min; Sampler temperature 250 DEG C; Detector temperature 250 DEG C, Splitless injecting-Sample.Electronics bombardment (EI) ion source; Electron energy 70eV; Ion source temperature 200 DEG C; Full scan pattern, mass scan range: 30 ~ 400u.The standard mass spectrum provided by computer search and NIST08 and WILEY7 mass spectral database is contrasted and confirms.
The quantification and qualification of higher alcohols: higher alcohols composition is separated through gas-chromatography, different components forms its respective chromatographic peak, carries out Analysis and Identification with gas chromatography-mass spectromet.Analytical results uses computer spectrum storehouse (NIST08 and WILEY7) to carry out preliminary search and data analysis, then carries out artificial spectrum elucidation in conjunction with document, the content of this yeast-leavened higher alcohols in grape wine of qualitative and quantitative analysis.Result shows that the total higher alcohols growing amount of bacterial strain CP1118 is 237.43mg/L, and comparatively bacterial strain EC1118 total higher alcohols growing amount (276.39mg/L) reduces nearly 14%.
Claims (2)
1. a strain preserving number is yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) CP1118 of CGMCC NO.7828, is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on June 28th, 2013.
2. yeast saccharomyces cerevisiae as claimed in claim 1 is reducing the application in grape wine higher alcohols.
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| CL2015003744A1 (en) | 2015-12-28 | 2016-07-29 | Neobiotec S A | Methodology for the elaboration of fermented beverages reduced in ethanol |
| CN117757650B (en) * | 2024-01-03 | 2024-06-04 | 泰山学院 | Saccharomyces cerevisiae and application thereof in production of low-higher alcohol and/or high-ethyl acetate wine products |
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| RU2299902C1 (en) * | 2006-08-17 | 2007-05-27 | Артак Симони Погосян | Alcohol-containing beer-like drink, composition for preparing beer wort and method for preparing special beer |
| CN101792719B (en) * | 2009-11-16 | 2011-09-14 | 中国农业大学 | Saccharomyces cerevisiae and application thereof in wine brewing |
| CN101914461B (en) * | 2010-07-16 | 2012-11-14 | 天津科技大学 | Low-yield higher-alcohol saccharomyces cerevisiae engineering bacterium and construction method thereof |
| CN103305431A (en) * | 2012-03-16 | 2013-09-18 | 湖北智敏农业有限公司 | Saccharomyces cerevisiae and application thereof in brewing of plum wine |
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Inventor after: Qin Weishuai Inventor after: Di Heng Inventor after: Zhang Na Inventor after: Du Yuanpeng Inventor before: Di Heng Inventor before: Qin Weishuai Inventor before: Zhang Na Inventor before: Du Yuanpeng |
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Granted publication date: 20150513 Termination date: 20191202 |