CN110205254A - The abnormal Brunswick Durham yeast of one plant of yield of higher alcohol and its application in low rice wine brewing - Google Patents
The abnormal Brunswick Durham yeast of one plant of yield of higher alcohol and its application in low rice wine brewing Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12G—WINE; PREPARATION THEREOF; ALCOHOLIC BEVERAGES; PREPARATION OF ALCOHOLIC BEVERAGES NOT PROVIDED FOR IN SUBCLASSES C12C OR C12H
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- C12G3/022—Preparation of other alcoholic beverages by fermentation of botanical family Poaceae, e.g. wheat, millet, sorghum, barley, rye, or corn of botanical genus Oryza, e.g. rice
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Abstract
The invention discloses the abnormal Brunswick Durham yeast of one plant of yield of higher alcohol and its applications in low rice wine brewing, belong to wine brewing engineering field.Present invention separation screening from the Traditional bulk Chinese yeast of Guizhou obtains one plant of abnormal Brunswick Durham yeast, and the characteristic that yield of higher alcohol, fermenting speed are fast, liquor-producing ability is medium is shown in rice wine brewing, is suitably applied raising rice wine quality in low rice wine brewing.The bacterial strain is preserved in China typical culture collection center on May 31st, 2019, and deposit number is CCTCC NO:M2019412.
Description
Technical field
The invention discloses the abnormal Brunswick Durham yeast of one plant of yield of higher alcohol and its answering in low rice wine brewing
With belonging to wine brewing engineering field.
Background technique
Higher alcohol is flavor substance important in wine, mainly includes normal propyl alcohol, isoamyl alcohol, isobutanol, active-amyl alcohol, benzene second
Alcohol, tyrosol, tryptophol and methyl mercaptan etc., but its content can excessively damage nervous system, and isoamyl alcohol and isobutanol are to human body
Toxicity is maximum, and alcoholic beverage Higher Alcohols total amount should be less than 10g/L.Higher alcohol is mainly generated by yeast metabolism, therefore low yield
The screening and application of advanced alcohol yeast are of great significance to rice wine quality is improved.
Correlative study obtains yield of higher alcohol yeast by modes such as Natural Selection and genetic mutagenesis, the low yield reported at present
The advanced all saccharomyces cerevisiaes of alcohol yeast.There is stronger ethyl alcohol to generate ability, be suitable for as main alcoholization yeast for saccharomyces cerevisiae
The brewing of height rice wine.Brewing for high-quality minuent rice wine needs to develop corresponding yeast.Guizhou traditional rice wine is mostly used small
Qu Zuowei fermenting agent is made, thus need to from Chinese yeast separation screening other types in addition to saccharomyces cerevisiae saccharomycete, lead to
Excessive phase separation answers the higher alcohol yield and alcoholization ability of saccharomycete, function yeast needed for obtaining brewing high-quality minuent rice wine
Bacterium.
Summary of the invention
First technical problem that the present invention solves is to provide the abnormal Brunswick Durham yeast of one plant of yield of higher alcohol
(Wickerhamomyces anomalus), the Isolation and screening of bacterial strain was from the Traditional bulk Chinese yeast of Guizhou, on May 31st, 2019
It is preserved in China typical culture collection center, deposit number is CCTCC NO:M2019412.
A method of separation obtains the abnormal Brunswick Durham yeast of the yield of higher alcohol comprising the steps of:
(1) aseptically by the complete Chinese yeast grind into powder of several piece, sterile water the separation of WL culture medium and microexamination: is added
Oscillation, dilution spread is in WL culture medium culture；Choose single colonie method of scoring to purify 3-4 times, records colony characteristics, while picking bacterium
It falls and slide is made, 400 × microscopically observation cell morphological characteristic；
(2) 5.8S-ITS-RFLP analysis and 26S D1/D2 area's sequencing analysis identify separated bacterial strain: extracting separated pure culture
Bacterial strain DNA, carry out 5.8S-ITS-RFLP analysis and the area 26S D1/D2 sequencing analysis, pass through restriction enzyme mapping compare and sequence
It compares, determines the characterization of molecules of exception Brunswick Durham yeast.
The specific features that screening obtains abnormal Brunswick Durham yeast include: that WL culture medium pure culture is convex for milky spheroidal
Rise, butyrous and edge it is complete；Microexamination cell is in oval；It is 650bp that 5.8S-ITS-RFLP analysis, which obtains PCR product,HaeIII digestion product is 650bp；26S D1/D2 area's sequencing analysis obtains 564bp ordered sequence, passes through GenBank database
Blast compare, determine its with abnormal Brunswick Durham yeast (Wickerhamomyces anomalus) type strain it is similar
Property is 99%, this strain sequence has been filed on to GenBank database, sequence accession number MK722475.
Second technical problem that the present invention solves is to provide application of the bacterial strain in low rice wine brewing, is in rice
The bacterial strain is inoculated in liquor brewing to reduce the content of rice wine Higher Alcohols, institute's brewed rice wine wine degree is lower (8% vol or so).
The inoculation is inoculated liquid microbial inoculum, and the liquid microbial inoculum is that seed liquor is inoculated into aseptic liquid nutrient medium,
28-30 DEG C of culture 1-2d；The rice wine brewing uses glutinous rice fermentation medium, and glutinous rice culture medium is to impregnate glutinous rice, drain, is high
Rhizopus preparation after pressure sterilizing, cooling after inoculation activation.
Beneficial effects of the present invention: the abnormal Brunswick Durham is added in the form of liquid inoculation during rice wine brewing
Fermentation rate can be improved in yeast, reduces rice wine Higher Alcohols (propyl alcohol, isobutanol, butanol, isoamyl alcohol, hexanol and bata-phenethyl alcohol)
Content, especially reduction isoamyl alcohol and isobutyl alcohol content, are of great significance to the quality for improving Chinese tradition minuent rice wine.
Detailed description of the invention
Fig. 1 bacterial strain WL cultivates colony characteristics (a) and microexamination cell characteristic (b)；
Fig. 2 bacterial strain brewed rice wine fermentation liquid higher alcohols content gas chromatographic detection figure.
Specific embodiment
The separation method of 1 exception Brunswick Durham yeast CTCC NO:M2019412 of embodiment
(1) aseptically by the complete Chinese yeast grind into powder of several piece, sterile water the separation of WL culture medium and microexamination: is added
150r/min vibrates 20min, and dilution spread is in WL culture medium, 28-30 DEG C of culture 5d.Choose single colonie method of scoring to purify 3-4 times,
Colony characteristics are recorded, while slide, 400 × microscopically observation cell morphological characteristic is made in picking colony.
(2) 5.8S-ITS-RFLP analysis and 26S D1/D2 area's sequencing analysis identify separated bacterial strain: extracting separated pure
The DNA of the bacterial strain of culture, carry out 5.8S-ITS-RFLP analysis and the area 26S D1/D2 sequencing analysis, by restriction enzyme mapping compare and
Sequence alignment determines the characterization of molecules of exception Brunswick Durham yeast.
WL culture medium pure culture is that milky spheroidal protrusion, butyrous and edge are complete；Microexamination cell is in oval
Shape；It is 650bp that 5.8S-ITS-RFLP analysis, which obtains PCR product,HaeIII digestion product is 650bp；The sequencing of the area 26S D1/D2
Analysis obtains 564bp ordered sequence, is compared by the Blast of GenBank database, determines itself and abnormal Brunswick Durham yeast
(Wickerhamomyces anomalus) type strain similitude be 99%, this strain sequence has been filed on to GenBank data
Library, sequence accession number MK722475.
The abnormal Brunswick Durham yeast CTCC NO:M2019412 of embodiment 2 is advanced in laboratory room small-sized rice wine brewing
Alcohol yield research
When laboratory room small-sized rice wine brewing, the exception Brunswick Durham yeast strain is inoculated in glutinous rice fermentation medium, passes through title
Fermentation termination is monitored again, and it is as follows to detect ethyl alcohol and advanced alcohol content, concrete scheme in final fermentation liquid using gas chromatograph:
1. the preparation of glutinous rice fermentation medium: accurately weighing 100.0 g glutinous rice, add distilled water immersion to stay overnight, be put into 500 after draining
ML triangular flask, 121 DEG C of 20 min of sterilizing add 150mL sterile water after cooling, and activation rhizopus is added with 0.3% ratio
Q303(activation method: add 28 DEG C of 10 min of a small amount of water) it is stand-by afterwards, the pH of prepared fermentation medium is 6.5 or so.
2. laboratory room small-sized rice wine brewing is tested: the abnormal Brunswick Durham yeast is accessed to the brewer's wort culture of 10 mL
Base, 30 DEG C of stationary cultures are stayed overnight.According to inoculum concentration 1 × 10 after being counted with blood counting chamber6Cells/mL is seeded to fermentation training
Support base.Sealing weighing is placed on 30 DEG C and is cultivated, and every 24 h weighs and records, when weightlessness thinks fermentation knot less than 0.2 g/d
Beam.
3. ethyl alcohol and higher alcohol detection method of content in fermentation liquid: taking 100 mL of fermentating liquid filtrate that 100 mL distilled water is added to turn
It moves in 500 cucurbits of the mL with bead and distills, collect 100 mL distillates and sealing preservation in 4 DEG C.Draw 2 mL meters
Wine fermentation distillate sample, with after 0.22 μm of membrane filtration using methanol as external standard test sample higher alcohols content.Gas phase color
Spectral condition: chromatographic column be the mm of DB-FFAP(30 m × 0.25,0.25 μm), 260 DEG C of injector temperature, temperature program(me) 45
DEG C keep 3 min, 120 DEG C of 3 min of holding are warming up to 16 DEG C/min rate, then be warming up to 220 with 50 DEG C/min rate
DEG C keep 5 min, fid detector detection temperature be 260 DEG C, 30 mL/min of hydrogen flow rate, 300 mL/min of air velocity, into
Sample loading mode is split sampling: sample volume 1 μ L, split ratio 40:1.
The strain fermentation period is 17 days, and final fermentation liquid wine degree is 7.1%vol, and higher alcohol total amount is 169.66mg/L,
Wherein propyl alcohol 30.29mg/L, isobutanol 27.14mg/L, butanol 1.36mg/L, isoamyl alcohol 61.02mg/L, hexanol 0.9mg/L, β-
Benzyl carbinol 48.94mg/L.
3 control strain Clavisporalusitaniae of embodiment (Clavispora lusitaniae) FBKL2.8013 testing
Higher alcohol yield research in the brewing of room small rice-wine
When laboratory room small-sized rice wine brewing, the Clavisporalusitaniae bacterial strain is inoculated in glutinous rice fermentation medium, passes through weighing
Fermentation termination is monitored, detects ethyl alcohol and advanced alcohol content in final fermentation liquid, concrete scheme such as embodiment 2 using gas chromatograph
It is described.
The Clavisporalusitaniae strain fermentation period is 38 days, and final fermentation liquid wine degree is 9.86%vol, and higher alcohol is total
Amount is 275.02mg/L, wherein propyl alcohol 51.87mg/L, isobutanol 51.49mg/L, butanol 0.97mg/L, isoamyl alcohol 105.21mg/
L, hexanol 4.34mg/L, bata-phenethyl alcohol 61.12mg/L.Compared to the control strain, abnormal Brunswick Durham yeast CTCC NO:
Faster, higher alcohol yield is lower for M2019412 fermentation rate, is more suitable for low rice wine brewing.
Claims (7)
1. one plant of yield of higher alcohol abnormal Brunswick Durham yeast (Wickerhamomyces anomalus), it is characterised in that:
Its deposit number is CCTCC NO:M2019412.
2. a kind of method that separation obtains the abnormal Brunswick Durham yeast of yield of higher alcohol described in claim 1, it is characterised in that:
(1) aseptically by the complete Chinese yeast grind into powder of several piece, sterile water the separation of WL culture medium and microexamination: is added
Oscillation, dilution spread is in WL culture medium culture；Choose single colonie method of scoring to purify 3-4 times, records colony characteristics, while picking bacterium
It falls and slide is made, observe cell morphological characteristic；
(2) 5.8S-ITS-RFLP analysis and 26S D1/D2 area's sequencing analysis identify separated bacterial strain: extracting separated pure culture
Bacterial strain DNA, carry out 5.8S-ITS-RFLP analysis and the area 26S D1/D2 sequencing analysis, pass through restriction enzyme mapping compare and sequence
It compares, determines the characterization of molecules of exception Brunswick Durham yeast.
3. application of the abnormal Brunswick Durham yeast of yield of higher alcohol as described in claim 1 in low rice wine brewing.
4. application according to claim 3, it is characterised in that: the higher alcohol includes propyl alcohol, isobutanol, butanol, isoamyl
Alcohol, hexanol and bata-phenethyl alcohol.
5. application according to claim 3, it is characterised in that: further include being inoculated with the abnormal prestige during rice wine brewing
Gram Durham yeast, is inoculated with as inoculated liquid microbial inoculum.
6. application according to claim 5, it is characterised in that: the liquid microbial inoculum is that seed liquor is inoculated into sterile liquid
In culture medium, 28-30 DEG C of culture 1-2d；The rice wine brewing uses glutinous rice fermentation medium.
7. application according to claim 6, it is characterised in that: the glutinous rice culture medium be glutinous rice is impregnated, is drained, high pressure
Rhizopus preparation after sterilizing, cooling after inoculation activation.
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Cited By (5)
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| CN111019789A (en) * | 2019-12-30 | 2020-04-17 | 贵州大学 | Method for brewing Japanese sake by using saccharomyces cerevisiae |
| CN113528359A (en) * | 2021-05-25 | 2021-10-22 | 黄鹤楼酒业有限公司 | Candida planiformis and isolated culture method and application thereof |
| CN114134055A (en) * | 2021-10-22 | 2022-03-04 | 南昌大学 | Abnormal yeast Weikehan for high yield of phenethyl alcohol and application thereof |
| CN114752510A (en) * | 2022-03-28 | 2022-07-15 | 贵州大学 | Abnormal hanm kawakamii yeast and application thereof in rice wine |
| CN114854514A (en) * | 2022-03-04 | 2022-08-05 | 重庆江记酒庄有限公司 | New application of abnormal yeast Wiekeem and application of abnormal yeast Wiekeem in production of white spirit |
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| CN114854514A (en) * | 2022-03-04 | 2022-08-05 | 重庆江记酒庄有限公司 | New application of abnormal yeast Wiekeem and application of abnormal yeast Wiekeem in production of white spirit |
| CN114752510A (en) * | 2022-03-28 | 2022-07-15 | 贵州大学 | Abnormal hanm kawakamii yeast and application thereof in rice wine |
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