CN109439557A - High acid, low yield fusel oil S. cervisiae and combinations thereof and application - Google Patents
High acid, low yield fusel oil S. cervisiae and combinations thereof and application Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
- C12N1/18—Baker's yeast; Brewer's yeast
- C12N1/185—Saccharomyces isolates
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/85—Saccharomyces
- C12R2001/865—Saccharomyces cerevisiae
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- 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
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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
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
- C12N1/18—Baker's yeast; Brewer's yeast
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
- C12N1/20—Bacteria; Culture media therefor
Abstract
The invention discloses one plant of new S. cervisiaes, the wine brewing microbial inoculum containing the bacterium and its application in brewed spirit.It is strong that S. cervisiae disclosed by the invention has both production acid, ester producing capacity, advanced alcohol content is low, high temperature resistant, it is acidproof, producing and ethanol ability with higher, the advantages such as the white wine of fermenting and producing has total acid, total ester content high, and fusel oil content is low, wine brewing antimicrobial composition and wine brewing reinforcing song can apply to white wine solid state fermentation and quality of white spirit and yield can be improved.
Description
Technical field
The invention belongs to wine brewing fields, specifically, wine brewing microbial inoculum the present invention relates to S. cervisiae, containing the bacterium and its
Application in brewed spirit.
Background technique
Organic acid is the Main Fragrance to form white wine taste, and generates the precursor of esters.In white wine
Lactic acid and acetic acid content highest, suitable acetic acid can make white wine have frank sense (liquor production technology pandect, p769, the side Shen Yi master
It compiles, China Light Industry Press 2013).Acid can eliminate the bitter taste of white wine, and acid amount is insufficient, in fact it could happen that wine is bitter, and wine is not clean, single
It adjusts uncoordinated etc..Acid is best flavor agent, and organic acid can make wine become multi-flavor, and sweet tea sense occurs back in rich in taste, is increased pure and mild
It spends, the water taste (great-grandfather's instruction, wine brewing, 2014,41 (2): 3-5) of low wine in reduction.Acetic acid also has sterilizing and anti-virus, expansion blood
Manage, delay the function (Zhuan Mingyang, wine brewing, 2006,33 (6): 109-110) of vascular sclerosis.Butyric acid is the feature of mixed-flavouring liquor
Ingredient (Shen Yifang, liquor production technology pandect, p772).Top grade wine in China's white wine national standard (GB1078.1-1078.3)
Generally than other the more lower grades white wine of total acid content total acid content it is high.Organic acid in white wine mainly by making wine during
The microorganisms such as bacterium, yeast and the mould of fermentation are participated in generate.Screening in relation to high acid unartificial yeast bacterium at present and improvement wine
Flavor research it is less.Marvin's is auspicious equal (food and fermentation industries, 2017,43 (7): 135-139)) from 3, Xinjiang producing region
39 saccharomycetes are filtered out in grape and soil altogether, by primary dcreening operation and secondary screening, 1 plant is obtained and produces the high saccharomyces cerevisiae Y6 of acid content,
Acid amount is produced in small-size wine fermentation production run improves 1.84g/L.Lv Huiwei etc. (Food Science, 2010,31 (11):
3 saccharomycete strains 197-201) are separated from strawberry natural fermentation broth, and are control with saccharomyces cerevisiae, pass through hypoglycemic speed
Rate produces the fermentation character that alcohol ability, production acid amount and sensory quality assessment compare different strains.
Fusel oil be make fermentation process in yeast eubolism by-product, including aliphatic acids, aliphatic alcohols,
Higher alcohols.In white wine as fusel oil too high levels will affect white wine mouthfeel and to human health generate adverse effect (HofiH,
Equal .Alcoholism:Clinical and Experimental Research, 2003,27 (S1): 37S-41S).In tradition
Brewed wine in, the universal higher problem of fusel oil content has caused the extensive concern of industry.Breeding low yield fusel oil and
The yeast of high-yield ethanol is the most fundamental solution.Tang fetches etc. (brewing science and technology, 2016,5:35-37) and sends out in Xiaoqu
It is inoculated with the high ester yield low-yield higher-alcohol saccharomyces cerevisiae of a certain amount of purebred culture during ferment, containing for ethyl acetate can be significantly improved
Amount, and significantly reduce advanced alcohol content.Zhang Cuiying (brewing science and technology, 2013,7:62-64) adds low-yield higher-alcohol saccharomyces cerevisiae AY
Δ BAT2 carries out Xiaoqu wine semi-solid ferment, on Xiaoqu wine wine degree, residual sugar, total acid and total ester group this without influence, but higher alcohol contains
Sharp fall is measured, isoamyl alcohol content reduces 50.8% compared with pure small koji fermentation.The reduction of advanced alcohol content exists
The flavor of Xiaoqu wine can be improved to a certain extent, improve Xiaoqu wine quality.
Saccharomycete is the main bacteria seed that alcohol and white wine correlation flavor substance are formed.Fermented grain is in height during white wine liquor brewing
Temperature accumulation and pit entry fermentation stage have the characteristics that high temperature, highly acidity and contain certain density alcohol, and therefore, breeding is excellent
Yeast strain with height endurability simultaneously is applied to be the technology for improving raw material availability and liquor output rate and vinosity in liquor production
Key has Important Economic meaning.The screening and application of the good quality yeast of heatproof, acid-fast ability can be improved liquor fermentation effect
Rate reduces production cost, improves former wine mouthfeel and improves white wine quality amount (the superfine wine brewing of Hu nation, 2017,44 (2): 13-18；
The such as Dong Yongsheng wine brewing, 2005,32 (5): 30-33；Tang Youhong wine brewing, 2011,38 (5): 45-47).Wang Yong (brewing science and technology,
2017,4:61-64) more outstanding wine brewing in terms of alcohol ability, alcohol-tolerant ability, fermenting power and tunning is produced in screening
2 plants of saccharomycete, it is fabricated to reinforcing wheat bran respectively and is added in wine brewing productive experiment, plays increase ethyl acetate, it is appropriate to control
Ethyl lactate processed reduces fusel oil content and improves the effect of distillation yield.Lu Xiaowei etc. (microbiology notification, 2015,42
(11): 2098-2107 the Wine brewing yeast strain MT1 of one plant of function admirable) is screened from paste flavor liquor brewing environment, which has
The ability of excellent tolerance high temperature, high concentration ethanol and low pH.
Presently commercially available saccharomyces cerevisiae less varieties, have a single function, and general production alcohol ability is stronger, but acid producing ability is poor,
Advanced content is higher, and heat-resisting ability is also poor, is mainly used for alcoholic fermentation industry.After these yeast are applied to liquor production
It frequently can lead to distillation yield to improve, but poor taste, the phenomenon that former wine quality decline, or since the heat-resisting quantity of bacterial strain is poor,
It inactivates and does not work after high temperature stacking fermentation.Therefore, screening tolerance is good, and acid producing ability is strong, can improve former wine quality
Saccharomyces cerevisiae has significant application value for liquor production.Chinese patent 201310587108.0 report a kind of production octanoic acid and
The yeast of capric acid and its corresponding ethyl ester.Chinese patent 201210059667.X reports thermotolerant ethanol fermentation yeast and its is separately cultured
Method.To saccharomyces cerevisiae, acid producing ability lacks research report during liquor fermentation at present.According to existing research and patent
Retrieval, does not have both high acid still simultaneously in brewed spirit industry at present, and yield of higher alcohol simultaneously has the characteristics such as high temperature resistant
The application report of Wine brewing yeast strain and its combination microbial inoculum being combined with bacillus subtilis.
Summary of the invention
It is an object of the present invention to provide a kind of new Wine brewing yeast strain, the bacterial strain high temperature resistants, acidproof, the energy of resistance to ethyl alcohol
Power is strong, the bacterial strain organic acids ability such as production acetic acid and caproic acid with higher, white wine of fermenting and producing under the conditions of liquor fermentation
Fusel oil content is low, and containing liquor flavors component substances such as higher 4- hydroxylphenylethyl alcohol and ethyl caprates, to improvement white wine
Mouthfeel, improving white wine quality measurer has important value.
It is a further object to provide a kind of wine brewing microbial inoculum containing saccharomyces cerevisiae.
The application that it is also another object of the present invention to provide saccharomyces cerevisiaes in liquor fermentation.
It is also another object of the present invention to provide the methods of preparation wine brewing microbial inoculum.
It is also another object of the present invention to provide application of the wine brewing microbial inoculum in brewed spirit.
The invention discloses one plant of new S. cervisiaes, which is characterized in that CY62 plants of Saccharomyces Cerevisiae in S
Saccharomyces cerevisiaeSCY62 is preserved in China typical culture collection center, deposit number CCTCC
NO:M2018799.Preservation date is on November 18th, 2018.CY62 plants of Saccharomyces Cerevisiae in S are divided from distillery, Hubei Province fermented grain
It is obtained from through screening.The bacterial strain has the following characteristics that
Saccharomyces Cerevisiae in S CY62 plant shape state is characterized in: on YPD 2~3d of cultured on solid medium, bacterium colony milky, surface
Smooth, relatively wet, neat in edge cultivates 12~14h in YPD fluid nutrient medium at 37 DEG C, and cell is rounded or ellipse, and one
Bring out bud.
CY62 plants of biochemical characters of Saccharomyces Cerevisiae in S are: can assimilate and utilize glucose, maltose, sucrose, galactolipin, maltose；
Xylose, lactose cannot be utilized.
CY62 plants of high temperature resistants of Saccharomyces Cerevisiae in S and growth characteristics are: being seeded on YPD solid medium, the bacterial strain is at 42 DEG C
Under remain to preferably grow, heat-resisting ability is strong.Compared with the increment that 30 DEG C are cultivated, SCY62 plants of relative growth yield at 42 DEG C
Up to 40.7%, and reference culture CICC1002 is not grown completely at this temperature.SCY62 bacterial strain heat-resisting ability compares reference culture
CICC1002 high.
CY62 plants of ability characteristics of resistance to ethyl alcohol of Saccharomyces Cerevisiae in S are: at 40 DEG C, in the YPD culture medium that ethanol content is 8%,
SCY62 is still grown very well, stronger than its reference culture growth ability of resistance to ethyl alcohol.
CY62 plants of acid-fast abilities of Saccharomyces Cerevisiae in S are characterized in: at 40 DEG C, Saccharomyces Cerevisiae in S CY62 is cultivated in the YPD that pH is 4.0
Still well-grown in base, and reference culture CICC1002 growth is poor, hence it is evident that it is suppressed.
To CY62 plants of Saccharomyces Cerevisiae in S carry out 26S rDNA PCR amplification and sequencing test, analysis shows that: bacterial strain SCY62 with
The homology of the 26S rDNA of Saccharomyces cerevisiaestrain CICC1002 has reached 99%, determines bacterial strain
SCY62 is a kind of saccharomyces cerevisiae.
The invention also discloses a kind of CY62 containing Saccharomyces Cerevisiae in S plants of wine brewing microbial inoculum, which is to make
SCY62 plants of brewer yeast and extracellular products.
Preferentially, the invention also discloses CY62 containing Saccharomyces Cerevisiae in S plants of wine brewing microbial inoculums of one kind, it is characterised in that under it contains
Column component (inoculum concentration):
Saccharomyces cerevisiae 10x108cfu/g
Bacillus subtilis 1x108cfu/g
Wherein saccharomyces cerevisiae is CCTCC NO:M2018799, and bacillus subtilis is CCTCC NO:M2017450.
Bacillus subtilis CCTCC NO:M2017450 is one plant of Patent Deposit bacterial strain, and strain characteristics are recorded in application
Number for 201710852757.7 Chinese patent in, bacterial strain was preserved in China typical culture collection on September 6th, 2017
The heart, scientific worker can ask for China typical culture collection center.Saccharomyces cerevisiae CICC1002 is common common micro-organisms
Strain is published in Chinese industrial Microbiological Culture Collection administrative center catalogue, and in open state, scientific worker can Xiang Zhongguo
Research for Industrial Microbial Germ preservation administrative center is asked for.
Wine brewing microbial inoculum disclosed by the invention is liquid or solid-state.Preferential, wine brewing microbial inoculum disclosed by the invention is solid-state.
The invention also discloses the methods for preparing solid brewing antimicrobial composition, and this method comprises the following steps:
(1) actication of culture: inoculation CY62 plants of slant strains of one ring Saccharomyces Cerevisiae in S are in the YPD liquid triangular flask culture of sterilizing
In base, 30 DEG C shaking table culture 18-24 hours；
(2) solid state rheology: wheat bran 100% fills plastic casing or polybag, 121 DEG C of sterilizings after adding water according to material-water ratio 1:1
30min, after being cooled to room temperature, by the Saccharomyces Cerevisiae in S CY62 bacterium solution of activation culture in the wheat bran culture of 10% ratio inoculation sterilizing
It in base, is uniformly mixed, is kept for 28 DEG C of room temperature or so, 35 DEG C of product temperature or so, culture finishes for 2 days, primary every stirring and evenly mixing for 24 hours；
(3) dry: forced air drying processing to be carried out at 35-42 DEG C to the yeast solids sample after fermentation, when sample is aqueous
Stop drying when rate is between 10%-25%, solid powdery wine brewing is prepared into after being crushed dry culture with pulverizer
Microbial inoculum.The viable count that sampling dilution plate measuring method measures yeast agent reaches 1,000,000,000/g or so.
(4) Saccharomyces Cerevisiae in S CY62 and bacillus subtilis CCTCC NO:M2017450 mixing and packaging: is pressed into viable count
Wine brewing antimicrobial composition is made than sealing after polybag after the mixing of=10:1 ratio.
Strengthen bent, preferential wine brewing the invention also discloses a kind of CY62 containing Saccharomyces Cerevisiae in S plants of wine brewing and strengthen song, contains
Following component:
Traditional song 95%
Wine brewing antimicrobial composition 5%
The wherein yeast or Chinese yeast for brewed spirit of tradition Qu Weiyong conventional method preparation, wine brewing antimicrobial composition are
Saccharomyces cerevisiae is CCTCC NO:M2018799 and bacillus subtilis is the mixture microbial inoculum of CCTCC NO:M2017450 composition.
The invention also discloses CY62 plants of the Saccharomyces Cerevisiae in S new applications in white wine solid state fermentation.
The invention also discloses application of the microbial inoculum in distillery's raising yeast of white wine and wine quality of making wine.
The invention also discloses wine brewing to strengthen the bent application that yeast of white wine and wine quality are improved in distillery.
Advantages of the present invention:
Saccharomyces Cerevisiae in S CY62 plants provided by the invention are one plant of new bacterial strains, and advantage is as follows:
1, the bacterial strain has very strong high temperature resistant growth ability.The bacterial strain remains to well-grown at 42 DEG C, trains with 30 DEG C
Feeding temperature is compared, and relative growth yield reaches 40.7%, and reference culture saccharomyces cerevisiae CICC1002 does not give birth to completely at 42 DEG C
It is long.
2, Saccharomyces Cerevisiae in S CY62 plants of acid production rates are high: the acetic acid of sorghum saccharified liquid fermented distilled liquor sample, butyric acid, valeric acid and oneself
Acid content higher than reference culture CICC1002 117.9%, 99.2%, 62.9% and 40.0% respectively.SCY62 plants of production total acid contents
It is higher than reference culture CICC1002 by 32.7%.
3, Saccharomyces Cerevisiae in S CY62 plant height grade alcohol content is low: normal propyl alcohol, the isobutanol of the Spirit sample of sorghum saccharified liquid fermentation
25.0%, 52,3% and 58.9% is reduced than reference culture CICC1002 respectively with the content of isoamyl alcohol.
4, it is strong that 4- hydroxylphenylethyl alcohol, the liquor flavors physical capacity such as certain herbaceous plants with big flowers acetoacetic ester are produced: SCY62 plant production 4- hydroxylphenylethyl alcohols with
The relative amount of certain herbaceous plants with big flowers acetoacetic ester is respectively up to 1.66% and 0.55%, and reference culture content is zero.
5, resistance to ethyl alcohol and acid-fast ability are good: the concentration of alcohol of SCY62 bacterial strain tolerable 8% or so, compared with the resistance to wine of reference culture
Smart ability is strong.The acidic growth environment that the tolerable pH of SCY62 bacterial strain is 4.0 is strong compared with reference culture acid-fast ability.
I.e. Saccharomyces Cerevisiae in S CY62 plants have high temperature resistant, resistance to ethyl alcohol, acidproof and high yield compared with reference culture CICC1002
The ability of the liquor flavors ingredient such as the organic acid substances such as acetic acid and caproic acid and 4- hydroxylphenylethyl alcohol, ethyl caprate, and have low
Produce the characteristic of fusel oil.
The present invention is CCTCC NO:M2017450 group by saccharomyces cerevisiae CCTCC NO:M2018799 and bacillus subtilis
At wine brewing antimicrobial composition the advantages of it is as follows:
1, SCY62 makes wine the antimicrobial composition saccharomyces cerevisiae microbial inoculum more single than its with higher distillation yield.
2, SCY62 make wine the antimicrobial composition saccharomyces cerevisiae microbial inoculum fermented wine more single than its vinegar drone content it is higher, just
Alcohol content is lower.
It is as follows that bent advantage is strengthened in the wine brewing of the strain of CY62 containing Saccharomyces Cerevisiae in S of the invention:
1, it is bent higher than the distillation yield of traditional yeast to add CY62 containing Saccharomyces Cerevisiae in S plants of wine brewing reinforcing.
2, it adds CY62 containing Saccharomyces Cerevisiae in S plants of wine brewing and strengthens total acid and total ester content that song is remarkably improved former wine, drop
The advanced alcohol content such as low normal propyl alcohol is obvious to the quality effect for improving former wine.
3, traditional yeast dosage 30% can be reduced by adding CY62 containing Saccharomyces Cerevisiae in S plants of wine brewing reinforcing song.
Detailed description of the invention
Fig. 1 is CY62 plants of 26S rDNA D1/D2 region sequence systematic growth tree graphs of Saccharomyces Cerevisiae in S
Specific embodiment
Below with reference to specific embodiment, the present invention is further explained.It should be appreciated that these embodiments are merely to illustrate
The present invention, and cannot limit the scope of the invention.
Embodiment
The screening and identification of 1 height endurability of embodiment, high acid rate Saccharomyces Cerevisiae in S CY62 bacterial strain
The separation of 1.1 height endurability yeast strains
It separates and obtains from Hubei distillery high temperature nuclear reactor buildup.Take the 10g high temperature nuclear reactor of different time sections and different parts
It more parts of buildup, is respectively added to equipped in acid bean sprout juice triangular flask enrichment culture liquid of the 90ml containing 5% (v/v) concentration of alcohol
(being adjusted to pH4.0 with lactic acid), 37 DEG C of enrichment culture 2-3d.Enrichment culture liquid of the 100 μ l after certain gradient dilution is respectively taken, point
It is not applied on Martin's rose bengal medium solid plate, after being placed in 37 DEG C of cultures 48 hours, picking products of typical yeast form
Single colonie, then scribing line purifying is repeated on same medium solid plate, after being placed in 40 DEG C of cultures 48 hours, 40 DEG C of cultures are obtained altogether
At a temperature of 50 plants of the well-grown yeast strain of single colonie.By isolated 50 plants of yeast with preferable high temperature resistance
Bacterium is inoculated with the YPD fluid nutrient medium containing 8% (v/v) concentration of alcohol respectively and adds the YPD Liquid Culture that lactic acid is adjusted to pH4.0
Base, 40 DEG C, 170rpm shaking table culture for 24 hours, observes the turbidity of more each yeast liquid growth, and it is high to pick out bacterium solution turbidity
Bacterial strain, turbidity is higher to show that its tolerance and growth result are better.Pass through high-temperature cultivation and resistance to ethyl alcohol, acidproof culture experiment ratio
Compared with, and compared with saccharomyces cerevisiae reference culture CICC1002,10 plants of the further screening tools from 50 plants of thermotolerant yeast bacterium
There is the bacterial strain (table 1) of preferable resistance to ethyl alcohol and acid-fast ability, 4 DEG C of preservations are carried out to it.Wherein YP10, SCY62, SCY57, SY18
The tolerance of four plants of yeast is most strong.
The measurement result of 1 10 plant height tolerance yeast of table
Note: +++ indicate that growth is fine；++ indicate that growth is preferable；+ indicate that growth is faint.
The screening of the high yeast strain SCY62 of 1.2 acid production rates
In order to obtain the quality yeast bacterial strain for having both height endurability and high acid rate, the present invention is to above-mentioned separation and filters out
10 plant height tolerance yeast strains carried out fermented glutinous rice test and acid production rate comparison, therefrom further preferably go out acid production rate
High bacterial strain.Method particularly includes: above-mentioned purebred each 1 ring of 10 plant height tolerance saccharomycete screened is taken with oese, is connect respectively
Enter in YPD liquid culture medium, 30 DEG C, 170rpm shaking table culture for 24 hours, is prepared into saccharomycete seed liquor, and with blood counting chamber meter
Saccharomycete sum in each bacterium solution of number.It then is 1 × 10 by inoculum concentration6A/mL is inoculated with each saccharomycete seed liquor in preparatory system respectively
The glutinous rice saccharified liquid got ready is (the preparation method comprises the following steps: by material after glutinous rice is crushed: after water=1:2.5 mixing, setting water-bath and be warming up to 90
Addition liquefaction enzyme solution 90min after DEG C, with lactic acid tune PH 4.5；It is cooled to 65 DEG C of addition saccharification enzymatic conversion 30min again, after cooling
It is collected by centrifugation saccharified liquid, fills triangular flask, 115 DEG C of sterilizing 30min) in, it 28 DEG C, after 170rpm shaking table culture 6h, then is left to ferment
7d.After fermentation, each fermentation broth sample 100ml+100ml distilled water is taken respectively, is carried out alcohol distillation, is respectively picked up 100ml's
Distillate (wine sample) is used for gas Chromatographic Determination alcoholic strength, and 50ml wine sample is taken to measure total acid by GB/T 10345-2007.Measurement
It the results are shown in Table 2.The result shows that the acid producing ability of SCY62 is most strong in 10 plant height resistant strains, while producing alcohol ability also most
Height, the total acid content of Spirit sample is than standard bacteria plant height 47.8%.
The different yeast strain ferments glutinous rice of table 2 produce alcohol, acid producing ability measurement result
The present inventor by one plant of separation new CY62 plants of Saccharomyces cerevisiaeSCY62 of Saccharomyces Cerevisiae in S,
It is preserved in China typical culture collection center, deposit number is CCTCC NO:M2018799, and preservation day is November 18 in 2018
Day.China typical culture collection center is located at Wuhan City, Hubei Province Wuchang District Wuhan University in the school, phone: 027-
68752319, EMAIL:cctcc@whu.edu.cnThe public can obtain the bacterial strain reproduction present invention in accordance with the law.
The identification of 1.3 CY62 plants of Saccharomyces Cerevisiae in S
Through observation of morphological characteristics, physiological and biochemical test and 26S rDNA D1/D2 regional gene the sequencing results pair
SCY62 is identified.In authentication, this experimental selection saccharomyces cerevisiae CICC1002 is micro- from Chinese industrial as control strain
Biological inoculum preservation administrative center, scientific worker can ask for.
1.3.1 Saccharomyces Cerevisiae in S CY62 plant shape state observation of characteristics
In YPD 2~3d of cultured on solid medium, bacterium colony milky, surface is smooth, relatively wet, neat in edge, in YPD
12~14h is cultivated in fluid nutrient medium at 37 DEG C, cell is rounded or ellipse, one end are sprouted.
1.3.2 Phylogenetic identification is carried out by the analysis of 26S rDNA D1/D2 regional sequence
Pcr amplification reaction is done by template of the genomic DNA of yeast strain, expands the area yeast strain 26S rDNA D1/D2
Domain, amplimer use universal primer NL1 (5'-GTAGTCATATGCTTGTCTC-3') and NL4 (5'-
CTTCCGTCAATTCCTTTAAG-3')。
PCR reaction system (50 μ L): Premix Taq (2 ×) 25 μ L, primer NL1/NL4 (20 μm of ol/L) each 1 μ L, template
DNA 1 μ L, sterile 22 μ L of tri-distilled water.
PCR amplification condition: 94 DEG C of initial denaturation 5min, 94 DEG C of denaturation 40s, 50 DEG C of annealing 40s, 72 DEG C of extension 60s, 30 are followed
After ring, 72 DEG C of extension 5min.Gained PCR product serves Hai Shenggong bioengineering and purifies and be sequenced, sequencing primer with above-mentioned NL1,
NL4。
After being sequenced by molecule, the 26S of bacterial strain SCY62 and Saccharomyces cerevisiae CICC1002 are found
The homology of rDNA has reached 99%, and difference is no more than between meeting the fixed interior different strains of the same race of Kuttzman&Robnett
1% standard determines that bacterial strain SCY62 is an Accharomyces cerevisiae.Fig. 1 is to be according to what 26S rDNA D1/D2 regional sequence was done
System development tree.
1.3.3 Saccharomyces Cerevisiae in S CY62 plants of bio-chemical characteristics measurement result
(1) biochemical character fermentation test
SCY62 bacterial strain the results are shown in Table 3 to the utilization of sugar.
The fermentation test result of 3 Saccharomyces Cerevisiae in S CY62 different carbon source of table
(2) Saccharomyces Cerevisiae in S CY62 high temperature resistant growth ability measures
Two Yeasts seed liquors of culture are seeded in YPD fluid nutrient medium by same ratio, respectively in 30 DEG C, 37
DEG C and 42 DEG C at 170rpm shaking table culture for 24 hours, measure OD600Value.Testing result is shown in Table 4, the results showed that SCY62 bacterial strain is at 42 DEG C
Under remain to normal growth, have very strong heat-resisting quantity, with 30 DEG C cultivate increment compared with, the relative growth yield at 42 DEG C reaches
40.7%, and reference culture CICC1002 is not grown completely at this temperature.
The high temperature resistant growth ability measurement result of 4 SCY62 of table and reference culture CICC1002
(3) SCY62 bacterial strain is compared with reference culture CICC1002 alcohol-tolerant ability
Containing concentration of alcohol (v/v) be respectively 4% by the access of the seed liquor of bacterial strain SCY62 and reference culture CICC1002,
6%, in 8% YPD fluid nutrient medium, 30 DEG C are placed in, after 170rpm shaking flask culture for 24 hours, survey OD600Value.As shown in table 5, adding
In the culture medium for having added 6% (v/v) ethyl alcohol, SCY62 and CICC1002 can be grown, but the tolerance of SCY62 is better than
CICC1002, the OD of bacterial strain SCY62600Value is the 131.1% of CICC1002；In the culture medium for being added to 8% ethyl alcohol, SCY62
Tolerance be still better than CICC1002, the OD of bacterial strain SCY62600It is the 120.3% of CICC1002.
CY62 plants of 5 Saccharomyces Cerevisiae in S of table is compared with reference culture CICC1002 alcohol-tolerant ability
(4) Saccharomyces Cerevisiae in S CY62 plants compared with reference culture CICC1002 acid-fast ability
The seed liquor of bacterial strain SCY62 and reference culture CICC1002 are transferred to respectively with acetic acid: lactic acid=1:1 adjusts pH
In respectively 4.0,4.5,5.0,5.5 YPD fluid nutrient medium, be placed in 30 DEG C, the culture of 170rpm shaking flask for 24 hours, dilution 10 times after
Survey OD600Value.As can be seen from Table 6, Saccharomyces Cerevisiae in S CY62 still well-grown in the YPD culture medium that pH is 4.0, and standard bacteria
Strain CICC1002 growth is poor, hence it is evident that is suppressed.
CY62 plants of 6 Saccharomyces Cerevisiae in S of table is compared with reference culture CICC1002 acid-fast ability
CY62 plants of fermentation sorghum saccharified liquids of 2. Saccharomyces Cerevisiae in S of embodiment, which produce alcohol, produce acid and produce higher alcohol ability, compares wine
SCY62 plants of brewer yeast have higher producing and ethanol ability.The results are shown in Table 7 for white wine liquid state fermentation, CY62 plants of Saccharomyces Cerevisiae in S fermentations
Ethanol production is high.Under the conditions of 28 DEG C of white wine liquid state fermentations, SCY62 ethanol production reaches 6.81% (v/v), compares reference culture
CICC1002 ethanol production is slightly higher.But the white wine after Saccharomyces Cerevisiae in S CY62 fermentation sorghum saccharified liquid distillation produces total acid and compares standard bacteria
The total acid of strain CICC1002 will be higher by 32.7%.Wherein, the acetic acid closely related with liquor flavor and caproic acid content are respectively than mark
Quasi- bacterial strain improves 117.8% and 40.0%；And main higher alcohol includes the content of normal propyl alcohol, isobutanol and isoamyl alcohol in white wine
25.0%, 52,3% and 58.9% is reduced than reference culture respectively.Show Saccharomyces Cerevisiae in S CY62 in liquor fermentation application more
It is advantageous.
The different yeast strain liquid state fermentation white liquor distilling wine sample gas chromatographic analysis results of table 7
CY62 plants of the above-mentioned Saccharomyces Cerevisiae in S for having separated acquisition and reference culture CICC1002 are prepared into seed respectively
Liquid, inoculation enters extraction volatile component progress GC-MS measurement after culture 72h in sorghum candy liquid culture medium, as a result such as the following table 8
It is shown:
The relative quantification result of other volatile ingredients after the fermentation of 8 saccharomycete single culture of table
Sour except common alcohol, other than ester, GC-MS analysis is it has also been found that Saccharomyces Cerevisiae in S CY62 can generate more aromatics
The liquor flavors substances such as object, 4- hydroxylphenylethyl alcohol, 2,3-butanediol and ethyl caprate are closed, and are not detected in reference culture
It arrives.
The preparation method of 3 SCY62 of embodiment wine brewing microbial inoculum and combinations thereof
5.1 actication of culture: inoculation CY62 plants of slant strains of one ring Saccharomyces Cerevisiae in S are in the YPD liquid triangular flask culture of sterilizing
In base, 30 DEG C shaking table culture 18-24 hours；
5.2 solid state rheologies: wheat bran 100% fills plastic casing or polybag, 121 DEG C of sterilizings after adding water according to material-water ratio 1:1
30min, after being cooled to room temperature, by the Saccharomyces Cerevisiae in S CY62 bacterium solution of activation culture in the wheat bran culture of 10% ratio inoculation sterilizing
It in base, is uniformly mixed, is kept for 28 DEG C of room temperature or so, 35 DEG C of product temperature or so, culture finishes for 2 days, primary every stirring and evenly mixing for 24 hours；
5.3 is dry: forced air drying processing is carried out at 35-42 DEG C to the yeast solids sample after fermentation, when sample is aqueous
Stop drying when rate is between 10%-25%, solid powdery wine brewing is prepared into after being crushed dry culture with pulverizer
Microbial inoculum.The viable count that sampling dilution plate measuring method measures yeast agent reaches 1,000,000,000/g or so.
5.4 mixing and packaging: the wine containing single culture is made after sealing after the solid fungicide dress polybag containing SCY62 plants
Wine microbial inoculum；Saccharomyces Cerevisiae in S CY62 and bacillus subtilis CCTCC NO:M2017450 is mixed in viable count ratio=10:1 ratio
Wine brewing antimicrobial composition is made in sealing after polybag afterwards.
Application of the 4 SCY62 microbial inoculum of embodiment and combinations thereof in Xiaoqu liquor by solid fermentation fermentation
Using distilled liquor process for solid state fermentation, to sorghum after bored fine strain of millet, steaming fine strain of millet processing, spreading for cooling is cooled to 25 DEG C of left sides
The right side, moisture are controlled 55% or so, and the sorghum after spreading for cooling is grouped 2500g/ group, saccharomyces cerevisiae microbial inoculum according to sorghum net weight
According to 1 × 106The inoculum concentration of cfu/g sorghum is inoculated with, and 39-41 DEG C of temperature is controlled after inoculation and carries out accumulation 30h or so, accumulation
After bottling 28 DEG C fermentation 15 days after sample distillation.By CY62 plants of Saccharomyces Cerevisiae in S and bacillus subtilis CCTCC NO:
M2017450 makees leavening than mixing with 3 kinds of different viable counts, and is compared with saccharomyces cerevisiae CICC1002, and each group is done three
A Duplicate Samples.50g fermented grain is taken after fermentation, and 150mL distilled water is added, 100mL distillate is steamed, in the sample of distillation
The component contents such as alcohols, acids, esters carry out gas chromatographic analysis.Experimental result is shown in Table 9.
As shown in Table 9, be inoculated with single SCY62 plants and its with bacillus subtilis CCTCC NO:M2017450 different proportion
The total acid and total ester of mixed composition fermented wine sample are higher than control group, and wherein SCY62 plants and bacillus subtilis CCTCC
NO:M2017450 is best by the composition ferment effect that viable count 10:1 is prepared, the alcoholic strength of fermented wine sample, ethyl acetate,
Total acid and total ester content are higher by 6.6%, 57.1%, 47.6% and 43.5% than being inoculated with the control group of CICC1002 respectively, and positive third
Alcohol content reduces 16.0%, while its vinegar drone content improves 61.1% than control group.Show to be inoculated with SCY62 microbial inoculum and its group
The distillation yield of white wine and the quality of white wine, and the S. cervisiae that SCY62 composition is more single than its can be significantly improved by closing object
Using effect is more preferably in terms of improving distillation yield and vinegar drone content, reducing the indexs such as normal propyl alcohol content for agent.
Main component gas chromatographic analysis result in the fermentation fermented grain Spirit sample of table 9
The bent application in raising " Daqu " white spirit distillation yield and wine quality is strengthened in 5 SCY62 of embodiment wine brewing
5.1 test method
By, according to 250kg/ group stacking, control group inoculation is traditional after the fermented grain spreading for cooling after the distillation of certain Liquor-making Enterprises & brew house
The ratio of yeast is the 14% of inventory, and experimental group is inoculated with the SCY62 wine brewing microbial inoculum prepared by traditional yeast and embodiment 3 respectively
Composition strengthens song by the wine brewing that different proportion mixes, and the ratio of addition is 9.8% (reduction yeast dosage of inventory
30%).It is carried out high temperature stacking fermentation 3 days after inoculation, heap temperature samples distillation up to after 45 DEG C after pit entry fermentation 30d.Each processing three
A Duplicate Samples.Gas-chromatography point is carried out to component contents such as alcohols, acids, esters in the former wine sample of distillation after fermentation
Analysis.Key instrument: gas chromatographicanalyzer.
5.2 vinosity chromatography results and sensory evaluation
As can be seen from Table 10: by traditional yeast: SCY62 wine brewing antimicrobial composition is mixed in 95%:5% ratio
The ferment effect that song is strengthened in wine brewing is best, and the total acid of the experimental group fermented wine and total ester are respectively than 100% tradition yeast of addition
Control group former wine improve 44.5% and 44.6%；Concentration of alcohol improves 3.6% than control group；Ethyl acetate content compares
27.5% is improved according to group；Vinegar drone content improves 95.2% than control group；And isoamyl alcohol, normal propyl alcohol and the isobutyl of the experimental group
Alcohol content reduces 53.3%, 51.5% and 57.9% than control group respectively.The result shows that: addition wine brewing containing SCY62 is strengthened bent
It is significant for improving former wine distillation yield and the equal function and effect of former wine quality, and high-temperature daqu dosage 30% can be reduced.
Table 10 adds SCY62 wine brewing and strengthens bent daqu fermentation former wine analysis result
Sensory evaluation comparison are as follows: control group wine sample score in 90-95, comment be fragrance compared with harmony, sweet mellow, wine body compared with
It is plentiful；Addition is by traditional yeast: SCY62 wine brewing antimicrobial composition strengthens bent reality in the wine brewing that 95%:5% ratio mixes
Group wine sample score is tested in 95-100, comment is fragrance harmony, taste alcohol is graceful, wine body is plentiful, quiet and tastefully laid out exquisiteness.Due to total acid, total ester
With the increase of vinegar drone the equal Main Fragrance content for white wine, the reduction of the advanced alcohol content such as normal propyl alcohol, isoamyl alcohol shows to pass
System yeast can be obviously improved vinosity with the use of SCY62 wine brewing microbial inoculum, coordinate the fragrance in wine more, mouthfeel is more preferably.
Claims (10)
1. one plant of high acid, low yield fusel oil S. cervisiae saccharomyces cerevisiae, can be used for brewed spirit, which is characterized in that the bacterium
Strain is CY62 plants of Saccharomyces cerevisiae SCY62 Saccharomyces Cerevisiae in S, is preserved in China typical culture collection
The heart, deposit number are CCTCC NO:M2018799.
2. a kind of CY62 plants of Saccharomyces Cerevisiae in S of wine brewing microbial inoculum containing described in claim 1, it is characterised in that its active constituent is
CY62 plants of thallus of Saccharomyces Cerevisiae in S and its metabolite.
3. a kind of wine brewing antimicrobial composition containing bacterial strain described in claim 1.
4. wine brewing antimicrobial composition according to claim 3, it is characterised in that it contains following component (inoculum concentration):
Saccharomyces cerevisiae 10x108cfu/g
Bacillus subtilis 1x108cfu/g
Wherein saccharomyces cerevisiae is CCTCC NO:M2018799, and bacillus subtilis is CCTCC NO:M2017450.
5. a kind of wine brewing containing bacterial strain described in claim 1 is strengthened bent.
6. wine brewing according to claim 5 is strengthened bent, it is characterised in that it contains following component:
Traditional song 95%
Wine brewing antimicrobial composition 5%
The wherein yeast or Chinese yeast for brewed spirit of tradition Qu Weiyong conventional method preparation, wine brewing antimicrobial composition are right
It is required that component described in 4.
7. wine brewing antimicrobial composition according to claim 3, it is characterised in that the wine brewing antimicrobial composition is solid
State microbial inoculum.
8. the method for preparing solid brewing antimicrobial composition described in claim 3, includes the following steps:
(1) actication of culture: being inoculated with CY62 plants of slant strains of a ring Saccharomyces Cerevisiae in S in the YPD liquid triangular flask culture medium of sterilizing,
30 DEG C shaking table culture 18-24 hours；
(2) solid state rheology: wheat bran 100% adds according to material-water ratio 1:1 and fills plastic casing or polybag after water, 121 DEG C of sterilizing 30min,
After being cooled to room temperature, by bran mass of the Saccharomyces Cerevisiae in S CY62 bacterium solution of activation culture in the inoculation sterilizing of 10% ratio, mix
It closes uniformly, is kept for 28 DEG C of room temperature or so, 35 DEG C of product temperature or so, culture finishes for 2 days, primary every stirring and evenly mixing for 24 hours；
(3) dry: forced air drying processing to be carried out at 35-42 DEG C to the yeast solids sample after fermentation, when sample moisture content exists
Stop drying when between 10%-25%, solid powdery wine brewing microbial inoculum be prepared into after being crushed dry culture with pulverizer,
The viable count that sampling dilution plate measuring method measures yeast agent reaches 1,000,000,000/g or so；
(4) mixing and packaging: by Saccharomyces Cerevisiae in S CY62 and bacillus subtilis CCTCC NO:M2017450 by viable count ratio=
Wine brewing antimicrobial composition is made in sealing after polybag after the mixing of 10:1 ratio.
9. the Saccharomyces Cerevisiae in S CY62 plants of application in brewed spirit described in claim 1.
10. wine brewing antimicrobial composition described in claim 3 is in the application in brewed spirit.
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Application publication date: 20190308 |