CN110218662A - A kind of thermophilic low temperature produces fragrant characteristic U.S. pole plum surprise yeast outstanding and its application - Google Patents
A kind of thermophilic low temperature produces fragrant characteristic U.S. pole plum surprise yeast outstanding and its application 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
- C12G1/00—Preparation of wine or sparkling wine
- C12G1/02—Preparation of must from grapes; Must treatment and fermentation
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Abstract
A kind of thermophilic low temperature produces fragrant characteristic U.S.A pole plum surprise yeast outstanding and its application in ice-wine is made, and belongs to technical field of microbe application.The saccharomycete is stored in Guangdong Province's Culture Collection on April 12nd, 2019, deposit number is GDMCC No:60631, classification naming is Metschnikowia pulcherrima Y6, and the nucleotide sequence of bacterial strain 26S rDNA gene is as shown in SEQ ID No.1.The strain isolation of the saccharomycete is from the surface of Jilin Province's Ji'an City frost Vidal grape, and the bacterium colony at 15 DEG C after YPD solid medium 48h culture is circle, light red, glossy, and neat in edge, thallus is sticky, easily provokes.Cell is in oval, monopole budding.The saccharomycete is fast with fermenting speed at low temperature, and produces the prominent feature of fragrant characteristic, can be applied to the brew of ice-wine and other grape wine.
Description
Technical field
The invention belongs to technical field of microbe application, and in particular to a kind of thermophilic fragrant characteristic U.S. pole plum outstanding of low temperature production is odd
Yeast and its application in ice-wine brewing.
Background technique
Fragrance is the key factor for determining grape wine quality, is the result of multiple compounds interaction.In grape wine
There are three fragrance main sources: the kind fragrance of grape fruit, the fermentation fragrance that fermentative microorganism metabolism generates, and in ageing
Ageing fragrance caused by period.Wine Aroma ingredient is affected by many factors, such as grape variety, raw material maturity, brewing
Technique, ageing condition etc..
Saccharomycete plays critical effect in wine production.By taking ice liquor brewing as an example, yeast be responsible in high sugar and
Sugar is converted to alcohol in hypertonic environment, finally obtain it is a give off a strong fragrance, dense sweet wine unique in taste.And saccharomyces cerevisiae
Quality be determine wine quality key factor, most sugar in grape juice are converted alcohol and titanium dioxide by it
Carbon, while the metabolites such as glycerol, higher alcohol, ester, aldehyde are generated, directly affect the color, fragrance and mouthfeel of grape wine.Therefore
Saccharomyces cerevisiae is the soul for controlling wine quality, and different saccharomyces cerevisiaes can assign grape wine different sense organ flavors.
For ice wine compared with common claret, fermentation has the high sugar of raw material, and high acid environment and fermentation process require low
The particular surroundings such as temperature, cold fermentation can promote the synthesis of the aromatic substances such as vinegar class, alcohols, ketone, mushroom alkenes, improve grape wine
Quality, this just needs to filter out low temperature saccharomyces cerevisiae preferable to the tolerance of low temperature, that fermentation is fast.
China is still in infancy in terms of the research and utilization of wine yeast, makes the yeast of ice wine mostly from foreign countries
It introduces.In order to meet the fast development of wine brewing industry, filters out the yeast with unique fermentation character and have become current grape wine
The trend of industry.
Summary of the invention
The object of the present invention is to provide a kind of thermophilic low temperature to produce fragrant characteristic U.S. pole plum surprise saccharomycete Y6 outstanding
(Metschnikowia pulcherrima Y6) and its application in ice-wine brewing.The saccharomycete is in April, 2019
It being stored within 12nd Guangdong Province's Culture Collection (GDMCC), address is Guangzhou, Guangdong Province, China city martyr road 100,
Guangdong Microbes Inst (510075), deposit number are GDMCC No:60631, classification naming Metschnikowia
pulcherrima.The strain isolation of the saccharomycete freezes Vidal from Jilin Province's Ji'an City (Vidal is a kind of grape variety)
The surface of grape.
U.S. pole plum surprise saccharomycete Y6 (Metschnikowia pulcherrima Y6) of the present invention, passes through at 15 DEG C
Bacterium colony after crossing YPD solid medium 48h culture is circle, light red, glossy, and neat in edge, thallus is sticky, easily chooses
It rises.Cell is in oval, monopole budding.
The present invention also provides application of the saccharomycete in ice-wine brewing.Saccharomycete provided by the invention has
Fermenting speed is fast under low temperature, and produces the prominent feature of fragrant characteristic, can be applied to the brew of ice-wine and other grape wine.
Detailed description of the invention
Fig. 1 is aspect graph of the U.S. pole plum surprise saccharomycete Y6 under YPD plate (a) and microscope (b)；
The 26S rDNA sequential system that Fig. 2 is the U.S. pole plum surprise saccharomycete Y6 develops tree；
Fig. 3 is the optimum growth temperature curve of the U.S. pole plum surprise saccharomycete Y6；
The chromatography of ions figure of the fragrant analysis of the production that Fig. 4 is the U.S. pole plum surprise saccharomycete Y6；
The chromatography of ions figure of the fragrant analysis of the production that Fig. 5 is commercial References yeast strain R2.
Specific embodiment
Method in embodiment is unless otherwise instructed conventional method.
YPD Liquid Culture based formulas is as follows in embodiment: glucose (20g/l000mL), peptone (20g/1000mL),
Yeast extract (10g/1000mL), deionized water 1000mL, pH value is naturally, 121 DEG C of sterilizing 15min.YPD solid medium is
It is obtained after adding agar (20g/1000mL) in aforesaid liquid culture medium.
Embodiment 1: the acquisition and identification of the U.S. pole plum surprise saccharomycete Y6
The strain isolation of U.S. pole plum surprise saccharomycete of the present invention freezes the surface of Vidal grape from Jilin Province's Ji'an City.
Separation process are as follows: the sundries such as acquisition Vidal grape removal grape pedicel are impregnated 10 minutes at 15 DEG C with physiological saline, take 5mL
In soak addition 100mL enriched medium (enriched medium manner of formulation: 1 gram of yeast powder, 2 grams of peptone, 5 grams of glucose,
Chloramphenicol and each 10mg of streptomysin is added after cooling in 100mL, pH=5.0,121 DEG C of sterilizing 20min of deionized water), 15 DEG C, 150
Turn/min culture 2 days, obtains enrichment culture liquid.Enrichment culture liquid is again with liquid YPD medium dilution 106Times, it is solid to be coated on YPD
On body culture medium, 15 DEG C are cultivated 3 days.Choose growth it is fast, colonial morphology and color it is different bacterium colony it is 22 total, cross
Purifying, colony lift after purification are cultivated into YPD fluid nutrient medium to logarithmic phase.It is inoculated with respectively with the inoculum concentration of 2% (V/V)
In the Vidal grape juice of frost squeezing, ferment 60 days under the conditions of 15 DEG C.By sensory judgments, (above-mentioned 22 bacterium colonies are both needed to
Ice is prepared after drinking, according to the strong degree of fragrance using sensory judgments), the saccharomycete for finally selecting label Y6 (is denoted as Y6 bacterium
Strain), the ice wine aroma prepared by the saccharomycetes to make fermentation is the strongest.By Y6 bacterial strain in 15 DEG C of conditions of YPD solid culture primary surface
Lower culture 48 hours, colony colour is creamy white, colony shape is round, surface is smooth, central protuberance, regular edges, viscosity
Greatly and it is easy to provoke shown in (such as Fig. 1 a).Y6 bacterial strain observes (such as Fig. 1 b) at microscope (10x40), and thallus is ellipse, nothing
Branch can be observed often to have two cells of size to be connected, thus it is speculated that the bacterial strain is saccharomycete, and the reproduction in a manner of sprouting.
Will culture to logarithmic growth phase Y6 strain cultured solution (use YPD fluid nutrient medium) by centrifugation (8000 turns/
Point) collect thallus.Yeast genome is extracted using pastoris genomic dna extracts kit (Bio Teke company).To be extracted
Genome be template, with saccharomycete 26S rDNA gene D1/D2 region sequence amplimer NL1 and NL417, (forward primer is
5'-GCATATCAATAAGCGGAGGAAAAG-3', reverse primer are as follows: 5'-GGTCCGTGTTTCAAGACGG-3') it is primer, lead to
Cross the method (PCR amplification system are as follows: 10xPCR buffer 5uL, dNTPs 4uL, primers F 1 and each 1uL of R1, Taq enzyme of PCR
0.5uL, template 1uL, ultrapure water are supplemented to 50uL；Amplification condition are as follows: 94 DEG C of initial denaturation 3min, 94 DEG C of denaturation 3min, 54 DEG C are moved back
Fiery 1min, 72 DEG C of extension 1min, 30 circulations, 72 DEG C of ends extend 10min) the 26S rDNA gene order of amplification bacterial strain.PCR
The measurement of Product Sequence is completed by Jilin provincial treasury U.S. Biotechnology Co., Ltd.Sequencing result carries out in ncbi database
BLAST carries out homologous comparison, chooses the high bacterial strain of homology and carrys out phylogenetic tree construction.The nucleosides of bacterial strain 26S rDNA gene
Acid sequence as shown in SEQ ID No.1, systematic evolution tree as shown in Figure 2 (Fig. 2 illustrates this sequence and KY108496.1, i.e., with
U.S. pole plum surprise yeast is most like, so naming this bacterial strain is U.S. pole plum surprise saccharomycete Y6).The bacterium and GenBank:
KY108496.1 (Metschnikowia pulcherrima- U.S. pole plum surprise yeast) similarity highest, homology reach 99%.
It can primarily determine that the bacterial strain belongs to U.S. pole plum surprise yeast in conjunction with the morphological observations of bacterial strain, the preliminary designation yeast strain is
U.S. pole plum surprise saccharomycete Y6 (Metschnikowia pulcherrima Y6).
Embodiment 2: the thermophilic Low-temperature yeast bacterial strain optimum growth temperature measurement
U.S. pole plum surprise saccharomycete Y6 (Metschnikowia pulcherrima Y6) that embodiment 1 obtains is inoculated in
In YPD fluid nutrient medium, cultivated 2 days under the conditions of 15 DEG C.Bacterial strain after above-mentioned activation is inoculated in 2% (V/V) inoculum concentration
It in YPD fluid nutrient medium, dispenses into 5 groups of test tubes, being respectively put into culture in 5,10,15,20,25 DEG C of shaking table, (three groups flat
Row), the absorbance of culture is measured, at 600nm with ultraviolet-visible photometer two days later to probe into the most suitable growth of bacterial strain
Temperature.As shown in figure 3, the U.S. pole plum surprise saccharomycete Y6 (Metschnikowia pulcherrima Y6) is under the conditions of 5 DEG C
It can grow, as the temperature increases, the speed of growth is faster, and optimum growth temperature is 15 DEG C, more than 15 DEG C, growth speed
Degree eases up, and illustrates that the bacterial strain is thermophilic low temperature yeast.
Embodiment 3: the comparative experiments of the thermophilic Low-temperature yeast bacterial strain and commercial yeast brewing ice-wine.
The U.S. pole plum surprise saccharomycete Y6 (Metschnikowia pulcherrima Y6) and business that embodiment 1 is obtained make
Brewer yeast R2 (being given by Hua Xin grape wine factory, Jilin Province) is passed on YPD solid and fluid nutrient medium respectively, by l05CFU/mL
It is inoculated in the Vidal grape juice that 3L is squeezed in ice conditions, stationary culture 67 days under the conditions of 15 DEG C.During the fermentation
The U.S. pole plum surprise saccharomycete Y6 (Metschnikowia pulcherrima Y6), which can be observed, can quickly reach growth
Animated period, thallus number rapidly increase, and quantity is more than business saccharomyces cerevisiae R2, and stablize in a very long time inner cell sum
Higher level has embodied very strong adaptive capacity to environment and higher fermentative activity.
The measurement of the Volatile infochemicals content such as esters, higher alcohol, organic acid: Agilent6890 gas-chromatography is used
(GC) and Agilent5975 mass spectrum (MS) combined instrument detects various Volatile infochemicals kinds in the making grape wine of above-mentioned acquisition
Class and content.
It is as shown in Figure 4 and Figure 5 that Volatile infochemicals analyze result.Fermentation fragrance complexity in terms of and content side
Face, the U.S. pole plum surprise saccharomycete Y6 (Metschnikowia pulcherrima Y6) have been above business saccharomyces cerevisiae.
Compare from volatile species, U.S. pole plum surprise saccharomycete Y6 (the Metschnikowia pulcherrima
Y6 esters, alcohols, acids, aldehydes, ketone, alkanes, substance in terpenes and phenols 8) are produced, and business saccharomyces cerevisiae is not
Generate phenolic substances.
From the type of main flavor, (Fig. 4, Fig. 5 and table 1 are 100% calculating, table 1 to detect the total amount of volatile substance
In numerical value be volatile substance percentage contents) on compare, the U.S. pole plum surprise saccharomycete Y6 (Metschnikowia
Pulcherrima Y6) produce ethyl palmitate, isobutanol and ethyl myristate that business saccharomyces cerevisiae does not generate；And
It is more unexistent that U.S. pole plum surprise saccharomycete Y6 (Metschnikowia pulcherrima Y6) produces business saccharomyces cerevisiae
Trace aroma substance, as heptyl acetate, methoxyacetic acid pentyl ester, caprylic acid isobutyl ester, the non-keto acid ethyl ester of 8-, isobutyl acetate,
Decyl methyl esters, cis- -4- octene acetoacetic ester, third heptalactone, pentadecanoic acid ethyl ester, methyl hexadecanoate, ethyl stearte, 3- are pungent
Alcohol, 1- amino -2- propyl alcohol, n-butanol, 1 nonyl alcohol, 2- undecyl alcohol, guaiacol, o-cresol, metacresol etc., these ingredients are to wine
Fragrance equally have contribution outstanding.
Table 1: flavor substance detects comparing result in ice wine
Compare from main flavor content, the U.S. pole plum surprise saccharomycete Y6 (Metschnikowia
Pulcherrima Y6) generate main flavor, such as isoamyl acetate, ethyl palmitate, ethyl myristate, acetic acid benzene second
Ester, elaidic acid ethyl ester, isoamyl hexanoate (fruity, fresh banana flavor), methyl laurate (dry fruit taste), benzyl alcohol (fragrance of a flower, rose
Color smell), be 2.5 times of business saccharomyces cerevisiae yield respectively, 2.5 times, 1.9 times, 2.75 times, 3.48 times, 3.1 times, 2 times,
3.15 again.Furthermore ethyl laurate, ethyl acetate (fruity, ester fragrant), ethyl butyrate (fruity, strawberry are fragrant), hexyl acetate etc.
Content, which also has, significantly to be improved.
It can be seen that the thermophilic Low-temperature Ice fermented glutinous rice brewer yeast that this experiment preferably obtains can not only quickly complete entire ice wine
Brewing process complies with national standard, while can effectively improve ice wine aroma, increases the giving off a strong fragrance property of ice wine and answers
Polygamy.
<110>Jilin University
<120>a kind of thermophilic low temperature produces fragrant characteristic U.S. pole plum surprise yeast outstanding and its application
<130> nm:
<160> 1
<170> PatentIn version
<210>SEQ ID NO:1
<211> 490
<212> DNA
<213>U.S. pole plum surprise yeast Y6
<220>
<221> rDNA
<400> 1
TGCCTCAGTA ACGGCGAGTG AAGCGGCAAA AGCTCAAATT TGAAATCCCC CGGGAATTGT 61
AATTTGAAGA GATTTGGGTC CGGCCGGCAG GGGTTAAGTC CACTGGAAAG TGGCGCCACA 121
GAGGGTGACA GCCCCGTGAA CCCCTTCAAC GCCTTCATCC CAGATCTCCA AGAGTCGAGT 181
TGTTTGGGAA TGCAGCTCTA AGTGGGTGGT AAATTCCATC TAAAGCTAAA TACCGGCGAG 241
AGACCGATAG CGAACAAGTA CAGTGATGGA AAGATGAAAA GCACTTTGAA AAGAGAGTGA 301
AAAAGTACGT GAAATTGTTG AAAGGGAAGG GCTTGCAAGC AGACACTTAA CTGGGCCAGC 361
ATCGGGGCGG CGGGGAGCAA AACCACCGGG GAATGTACCT TTCGAGGATT ATAACCCCGG 421
TCCTTACTCC CACACCATCC CGAGGCCTGC AATCTAAGGA TGCTGGCGTA ATGGTTGCAA 481
GTCGCCCGT 490
Claims (2)
1. a kind of thermophilic low temperature produces perfume characteristic U.S. pole plum surprise saccharomycete Y6 outstanding (Metschnikowia pulcherrima Y6),
It is characterized by: the saccharomycete is stored in Guangdong Province's Culture Collection on April 12nd, 2019, deposit number is
GDMCC No:60631, classification naming are Metschnikowia pulcherrima, the nucleotide of bacterial strain 26S rDNA gene
Sequence is as shown in SEQ ID No.1.
2. a kind of thermophilic low temperature described in claim 1 produces fragrant characteristic U.S. pole plum surprise saccharomycete Y6 (Metschnikowia outstanding
Pulcherrima Y6) ice-wine brewing in application.
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| CN112725203A (en) * | 2021-01-22 | 2021-04-30 | 宁夏农产品质量标准与检测技术研究所(宁夏农产品质量监测中心) | Meiji yeast strain YC15 for high yield of aroma substances and application thereof |
| CN113502233A (en) * | 2021-07-14 | 2021-10-15 | 河北科技师范学院 | Meiji meiqi yeast and application thereof in wine brewing |
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