CN110205257A - The saccharomyces cerevisiae of one plant of yield of higher alcohol and its application in Xiaoqu rice wine brewing - Google Patents
The saccharomyces cerevisiae of one plant of yield of higher alcohol and its application in Xiaoqu rice wine brewing Download PDFInfo
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- CN110205257A CN110205257A CN201910561987.7A CN201910561987A CN110205257A CN 110205257 A CN110205257 A CN 110205257A CN 201910561987 A CN201910561987 A CN 201910561987A CN 110205257 A CN110205257 A CN 110205257A
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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
- C12G3/00—Preparation of other alcoholic beverages
- C12G3/02—Preparation of other alcoholic beverages by fermentation
- C12G3/021—Preparation of other alcoholic beverages by fermentation of botanical family Poaceae, e.g. wheat, millet, sorghum, barley, rye, or corn
- 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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- 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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Abstract
The invention discloses the saccharomyces cerevisiae of one plant of yield of higher alcohol and its applications in Xiaoqu rice wine brewing, belong to wine brewing engineering field.The present invention from the Traditional bulk Chinese yeast of Guizhou separation screening to one plant can yield of higher alcohol and the excellent saccharomyces cerevisiae of alcoholization ability, which is preserved in China typical culture collection center on May 29th, 2019, and deposit number is CCTCC NO:M 2019406.The bacterial strain shows excellent fermenting property, preferable alcoholization ability and lower higher alcohol yield in rice wine brewing, during wine brewing using the bacterial strain can improve Xiaoqu rice wine because advanced alcohol content it is high caused by the symptoms such as " top ".
Description
Technical field
The invention discloses the saccharomyces cerevisiae of one plant of yield of higher alcohol and its applications in Xiaoqu rice wine brewing, belong to wine
Wine engineering field.
Background technique
Higher alcohol refers to that carbon atom number is more than 2 monohydric alcohol, mainly includes normal propyl alcohol, isoamyl alcohol, isobutanol, activity penta
Alcohol, benzyl carbinol, tyrosol, tryptophol and methyl mercaptan etc..Higher alcohol is that (benzyl carbinol of such as low concentration is in flower for flavor substance important in wine
Flavor), but its content can excessively damage nervous system.Guizhou traditional rice wine is since higher alcohol, methanol content are high, easily
Cause occur phenomena such as " taking the lead " after drinking.Higher alcohol mainly passes through the cometabolism of yeast during the fermentation and generates, therefore
The screening and application of yield of higher alcohol yeast are of great significance to Guizhou Xiaoqu rice wine quality is improved.In addition, saccharomycete
Alcoholization ability directly affect vinosity and fermentation efficiency, therefore be also one of the principal concern of yeast screening assay.
Correlative study mainly obtains low yield height by monoploid preparation, induction mutation of bacterium, from modes such as nature separation screenings
Grade alcohol yeast, all saccharomyces cerevisiaes of yield of higher alcohol yeast of acquisition.Research report saccharomycete can have low yield height simultaneously
The functional character of the pure and mild high yield wine of grade.Chinese yeast is one of big distiller's yeast in China four, is widely used, but at present for Chinese yeast and wherein function
The research report of energy microorganism is less, there is no the separation screening of yield of higher alcohol yeast and high liquor-making yeast in Chinese yeast at present and answers
With report.
The invention discloses one plant of separation screenings from the saccharomyces cerevisiae of Guizhou Traditional bulk Chinese yeast, and the saccharomyces cerevisiae is in Chinese yeast
The good characteristic of the fast and high alcoholization power of yield of higher alcohol, fermenting speed is presented in rice wine brewing, which is for the first time in Guizhou
The excellent saccharomyces cerevisiae screened in Xiaoqu, is provided simultaneously with the function of yield of higher alcohol and high yield wine.
Summary of the invention
First technical problem that the present invention solves is to provide the saccharomyces cerevisiae of one plant of yield of higher alcohol
(Saccharomyces cerevisiae), the Isolation and screening of bacterial strain was from the Traditional bulk Chinese yeast of Guizhou, on May 29th, 2019
It is preserved in China typical culture collection center, deposit number is CCTCC NO:M 2019406.
A method of separation obtains saccharomyces cerevisiae described in claim 1, comprising the following steps:
(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 are chosen single colonie method of scoring and are purified 3-4 times in WL culture medium culture, record 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 saccharomyces cerevisiae.
The specific features of screening acquisition saccharomyces cerevisiae include: that WL culture medium pure culture is milky spheroidal protrusion, cream
Shape and edge are complete；Microexamination cell is rounded or oval, budding are obvious；5.8S-ITS-RFLP analysis obtains PCR product
For 840bp,HaeIII digestion product is 300+220+175+125bp；26S D1/D2 area's sequencing analysis obtains the effective sequence of 577bp
Column are compared by the Blast of GenBank database, determine its with saccharomyces cerevisiae (Saccharomyces cerevisiae) mould
The similitude of formula bacterial strain is 99%, this strain sequence has been filed on to GenBank database, sequence accession number MK722474.
Second technical problem that the present invention solves is to provide application of the bacterial strain in Xiaoqu rice wine brewing, is in rice
The bacterial strain is inoculated in liquor brewing to reduce the content of rice wine Higher Alcohols.
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
Inoculation rhizopus saccharification preparation after pressure sterilizing, cooling.
The saccharomyces cerevisiae, which is added, in the form of liquid inoculation during rice wine brewing can be improved fermentation rate, improve wine
Change ability reduces the content of rice wine Higher Alcohols (propyl alcohol, isobutanol, butanol, isoamyl alcohol, hexanol and bata-phenethyl alcohol), to improvement
The quality of Chinese tradition Xiaoqu rice wine is of great significance.
Detailed description of the invention
Fig. 1 bacterial strain microexamination cell characteristic
Fig. 2 fermentation diagram
Fig. 3 bacterial strain brewed rice wine fermentation liquid higher alcohols content gas chromatographic detection figure.
Specific embodiment
A kind of method that separation obtains the saccharomyces cerevisiae of embodiment 1, comprising the following steps:
(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 are chosen single colonie method of scoring and are purified 3-4 times in WL culture medium culture, record 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 saccharomyces cerevisiae.
The specific features of screening acquisition saccharomyces cerevisiae include: that WL culture medium pure culture is milky spheroidal protrusion, cream
Shape and edge are complete；Microexamination cell is rounded or oval, budding are obvious；5.8S-ITS-RFLP analysis obtains PCR product
For 840bp,HaeIII digestion product is 300+220+175+125bp；26S D1/D2 area's sequencing analysis obtains the effective sequence of 577bp
Column are compared by the Blast of GenBank database, determine its with saccharomyces cerevisiae (Saccharomyces cerevisiae) mould
The similitude of formula bacterial strain is 99%, this strain sequence has been filed on to GenBank database, sequence accession number MK722474.
Fermentation situation of the 2 saccharomyces cerevisiae CTCC NO:M 2019406 of embodiment in laboratory simulation rice wine brewing and
Higher alcohol yield research
Laboratory simulation rice wine brewing process is inoculated with the Wine brewing yeast strain in glutinous rice fermentation medium, is monitored by weighing
It is as follows to detect ethyl alcohol and advanced alcohol content, concrete scheme in final fermentation liquid by gas chromatograph for fermenting speed:
1. the preparation of glutinous rice fermentation medium: accurately weighing 100.0 g glutinous rice respectively, add distilled water immersion to stay overnight, put after draining
Enter 500 mL triangular flasks, 121 DEG C of 20 min of sterilizing add 150mL sterile water after cooling, and activation root is added with 0.3% ratio
Mould song Q303(activation method: add 28 DEG C of 10 min of a small amount of water) it is spare afterwards.The pH of culture medium is 6.5 or so.
2. simulation rice wine brewing experiment: the saccharomyces cerevisiae is inoculated with the malt extract medium into 10 mL, 30 DEG C of standings
Overnight incubation.According to inoculum concentration 1 × 10 after being counted with blood counting chamber6Cells/mL is seeded to fermentation medium.Sealing weighing
It is placed on 30 DEG C to be cultivated, every 24 h weighs and records, when weightlessness regards as fermentation ends less than 0.2 g/d.
3. ethyl alcohol and higher alcohol detection method of content in fermentation liquid: 100 mL of fermentating liquid filtrate being taken to be transferred to 500 mL band glass
In the cucurbit of glass pearl, start to distill after adding 100 mL distilled water, stops distillation after collecting 100 mL distillates, distillate is close
Envelope is preserved in 4 DEG C.2 mL rice wine fermenting distillate samples are drawn, are added in sample bottle with after 0.22 μm of membrane filtration, with first
Alcohol is external standard test sample higher alcohols content.GC conditions: chromatographic column is the mm of DB-FFAP(30 m × 0.25,
0.25 μm), 260 DEG C of injector temperature, program be 45 DEG C of holding 3 min of heating, 16 DEG C/min to 120 DEG C of heating rate
3 min are kept, then 220 DEG C of 5 min of holding, fid detector detection temperature are warming up to as 260 DEG C, hydrogen with 50 DEG C/min rate
30 mL/min of gas velocity, 300 mL/min of air velocity, input mode is split sampling: 1 μ L of sample volume, split ratio 40:
1。
The strain fermentation period is 17 days, and final to generate 11.7% ethyl alcohol, higher alcohol total amount is 277.75mg/L, wherein third
Alcohol 34.99mg/L, isobutanol 66.6mg/L, butanol 0.89mg/L, isoamyl alcohol 120.57mg/L, hexanol 0.94mg/L, β-benzene second
Alcohol 53.77mg/L.
Embodiment 3 compare fermentation situation of the strain Saccharomyces cerevisiae FBKL2.8003 in laboratory simulation rice wine brewing and
Higher alcohol yield research
Laboratory simulation rice wine brewing process is inoculated with the Wine brewing yeast strain in glutinous rice fermentation medium, is monitored by weighing
Fermenting speed detects ethyl alcohol and advanced alcohol content in final fermentation liquid, concrete scheme such as 2 institute of embodiment by gas chromatograph
It states.
Comparing strain Saccharomyces cerevisiae FBKL2.8003 fermentation period is 39 days, final to generate 9.96% ethyl alcohol, higher alcohol total amount
For 277.82mg/L, wherein propyl alcohol 46.48mg/L, isobutanol 46.61mg/L, butanol 1.12mg/L, isoamyl alcohol 90.57mg/L,
Hexanol 9.10mg/L, bata-phenethyl alcohol 83.95mg/L.Compared to saccharomyces cerevisiae CTCC NO:M 2019406, though the comparison bacterial strain
Higher alcohol yield is not much different, but fermentation rate is low, and alcoholization ability is weak.
Claims (7)
1. one plant of yield of higher alcohol saccharomyces cerevisiae (Saccharomyces cerevisiae), it is characterised in that: its preservation is compiled
Number be CCTCC NO:M 2019406.
2. a kind of method that separation obtains saccharomyces cerevisiae described in claim 1, which comprises the following steps:
(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 are chosen single colonie method of scoring and are purified 3-4 times in WL culture medium culture, record 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 saccharomyces cerevisiae.
3. application of the saccharomyces cerevisiae of yield of higher alcohol as described in claim 1 in Xiaoqu rice wine brewing.
4. application according to claim 3, which is characterized in that the higher alcohol includes propyl alcohol, isobutanol, butanol, isoamyl
Alcohol, hexanol and bata-phenethyl alcohol.
5. application according to claim 3, which is characterized in that be inoculated with the wine brewing ferment during rice wine brewing
Mother, the inoculation is inoculated liquid microbial inoculum.
6. application according to claim 5, which is characterized 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.
7. application according to claim 5, which is characterized in that the rice wine brewing uses glutinous rice fermentation medium, described
Glutinous rice culture medium be glutinous rice is impregnated, is drained, high pressure sterilization, it is cooling after inoculation rhizopus saccharification preparation.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113528361A (en) * | 2021-07-12 | 2021-10-22 | 江南大学 | Saccharomyces cerevisiae suitable for brewing rice wine by liquefaction method and application thereof |
| CN115558611A (en) * | 2022-10-24 | 2023-01-03 | 贵州大学 | Schizosaccharomyces japonicus strain with high flocculation property and application thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113528361A (en) * | 2021-07-12 | 2021-10-22 | 江南大学 | Saccharomyces cerevisiae suitable for brewing rice wine by liquefaction method and application thereof |
| CN115558611A (en) * | 2022-10-24 | 2023-01-03 | 贵州大学 | Schizosaccharomyces japonicus strain with high flocculation property and application thereof |
| CN115558611B (en) * | 2022-10-24 | 2024-02-23 | 贵州大学 | High flocculation property schizosaccharomyces japonica strain and application thereof |
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