CN111944708B - Yeast for high yield of isoamyl acetate and application thereof - Google Patents
Yeast for high yield of isoamyl acetate and application thereof Download PDFInfo
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- CN111944708B CN111944708B CN202010879643.3A CN202010879643A CN111944708B CN 111944708 B CN111944708 B CN 111944708B CN 202010879643 A CN202010879643 A CN 202010879643A CN 111944708 B CN111944708 B CN 111944708B
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- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
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- 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
- C12N1/14—Fungi; Culture media therefor
- C12N1/145—Fungal isolates
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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
Abstract
The invention belongs to the technical field of development and utilization of non-saccharomyces cerevisiae, and particularly relates to a yeast for high yield of isoamyl acetate and application thereof. Aiming at the problem that a pure culture strain of yeast of Kazachstania turiensis is not adopted for solid state fermentation at present, the invention provides yeast for producing isoamyl acetate in high yield by solid state fermentation, and the preservation number is CGMCC 19812. The yeast has good adaptation to the solid-state fermentation environment of grains and vigorous metabolism, and the main volatile flavor substances of the yeast comprise 700mg/kg of isoamyl acetate, 19.8mg/kg of acetic acid, 307mg/kg of beta-phenethyl alcohol, 8mg/kg of n-propanol, 27mg/kg of isoamyl alcohol, 3mg/kg of n-butyl alcohol and only 2mg/kg of methanol. The fermented grains of the yeast show obvious flavor characteristics of flower and fruit. The invention provides a new fermentation yeast for producing the white spirit with richer flavor by solid state fermentation, widens the means for regulating and controlling the flavor of the white spirit, and has important practical significance.
Description
Technical Field
The invention belongs to the technical field of development of non-saccharomyces cerevisiae, and particularly relates to a yeast for high yield of isoamyl acetate and application thereof.
Background
The strong aromatic Chinese spirits adopt the traditional mixed bacteria natural solid state fermentation process, and after many rounds of continuous vinasse fermentation for hundreds of years, relatively stable functional microbial populations are gradually formed in the fermented grains, the functional microbes are important characteristic microbial resources in China, particularly yeast resources therein, which not only take the task of converting reducing sugar to generate ethanol, but also have more important value compared with yeast in other fermentation systems in that the quality of the Chinese spirits is influenced by producing various flavor substances such as alcohol, ester, acid, ester and the like, the microbial populations are involved in co-metabolism by intermediate metabolites, and the ecological environment in the fermentation systems is influenced by producing active substances. Compared with the existing commercial yeast with the ethanol yield of more than 11%, the ethanol content in the fermented grains of the Luzhou-flavor liquor after fermentation is usually not more than 6-7%, and the main characteristics of the yeast in the fermented grains, except for ethanol production, are different from other environmental yeasts, so that the yeast is most closely related to the quality of the Luzhou-flavor liquor, and the improvement of the liquor quality by breeding functional yeast strains is always a research field of important attention of the industry.
The yeast is a kind of microorganism which is safer to use, has important application value in the process of brewing white spirit, and various functional yeasts which comprise ester-producing yeast and can be used for brewing white spirit are selected and bred at present, but pure culture yeasts of other species except saccharomyces cerevisiae are not used in production so far, and the saccharomyces cerevisiae is only used for producing vinasse, on the one hand, the solid state fermentation characteristic of aroma-producing yeast is not exact or unstable; on the other hand, considering that the microbial ecological environment of natural fermentation of white spirit may be affected by the addition of exogenous yeast, it is preferable to separate the yeast originally present in the brewing environment and use the yeast for solid-state fermentation of white spirit on the basis of clarifying the solid-state fermentation characteristics.
Research shows that yeasts of various genera such as Pichia anomala, P.kudriavzevii, Zygosaccharomyces bailii and the like in fermented grains or Daqu of white spirit are related to generation of flavor substances in the white spirit with various flavors, and part of the species are separated into pure culture strains with special brewing functions. Researches in recent two years find that the yeast of the genus Kazachstania is the dominant yeast in the fermented grains of the strong aromatic Chinese spirits and may have a special brewing function.
However, at present, the brewing function of K.turicensis yeast is not deeply researched in the industry, and no report on the application of the pure culture strain of the yeast to solid state fermentation or related industries is found.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: there is no problem of pure culture strains of K.turiensis genus yeast for solid state fermentation or related industries.
The technical scheme for solving the technical problems comprises the following steps: provides a yeast with high yield of isoamyl acetate, and the preservation number is CGMCC 19812. The preservation time is 2020, 05 and 13 days, the preservation center is the common microorganism center of China Committee for culture Collection of microorganisms, and the address is the institute of microorganisms of China academy of sciences No. 3, West Lu No. 1, North Cheng, the Korean district, Beijing.
Wherein, the 26S rDNA sequence of the yeast for highly producing isoamyl acetate is shown as SEQ ID NO. 1.
26S rDNA sequence of yeast with high yield of isoamyl acetate of SEQ ID NO. 1
gctcaaatttgaaatctggtaccttcggtgcccgagttgtaatttgtagagggcgactttggggcggctccttgtctatgttccttggaacaggacgtcatagagggtgagaatcccgtgtggcgaggagtgcggttccgtgtaaagcgctctcgaagagtcgagttgtttgggaatgcagctctaagtgggtggtaaattccatctaaagctaaatattggcgagagaccgatagcgaacaagtacagtgatggaaagatgaaaagaactttgaaaagagagtgaaaaagtacgtgaaattgttgaaagggaagggcatttgatcagacatggtgttttgcgcccctcgctccttgtgggtgggggactctcgcagctcactgggccagcatcagttttggcggtcggataaaaccaggggaacgtagcttgcttcgggaagtgttatagcccttgggaatacggccagccgggactgaggaacgcgattcgtcaaggatgctggcataatggttatatgccgcccgtcttgaa。
Wherein the yield of isoamyl acetate produced by the yeast with high yield of isoamyl acetate reaches 700mg/kg fermented grains.
Wherein the yield of the isoamyl acetate after the yeast for highly producing the isoamyl acetate is subjected to solid state fermentation is 700mg/kg of fermented grains, the yield of acetic acid is 19.8mg/kg of fermented grains, the yield of beta-phenethyl alcohol is 307mg/kg of fermented grains, the yield of n-propyl alcohol is 8mg/kg of fermented grains, the yield of isoamyl alcohol is 27mg/kg of fermented grains, the yield of n-butyl alcohol is 3mg/kg of fermented grains, and the yield of methanol is 2mg/kg of fermented grains.
Wherein the yeast for producing isoamyl acetate with high yield is obtained by separating from fermented grains of wuliangye.
Wherein the growth conditions of the yeast for producing the isoamyl acetate with high yield are that the temperature is 24-32 ℃, and the pH value is 3.0-6.0.
Preferably, the growth conditions of the yeast for producing isoamyl acetate with high yield are that the temperature is 28 ℃ and the pH value is 5.0.
Wherein the ethanol tolerance of the yeast with high yield of isoamyl acetate is 12 percent Vol.
Wherein, the yeast with high yield of isoamyl acetate is characterized in that: after 24 hours of culture on WL culture medium at 28 ℃, a round and yellow-green flat colony is formed, the culture time is prolonged, the colony is darker in green, is easy to pick, has fragrance of flowers and fruits, is nearly round in cells, and germinates.
Further, the composition of the WL medium comprises: 0.5 percent of yeast extract powder, 0.5 percent of tryptone, 5 percent of glucose, 2 percent of agar, 0.055 percent of monopotassium phosphate, 00425 percent of potassium chloride, 0.0125 percent of calcium chloride, 0.00025 percent of ferric chloride, 0.0125 percent of magnesium sulfate, 0.00025 percent of manganese sulfate, 0.0022 percent of bromocresol green, 6.5 percent of pH value and 20 minutes of sterilization at 121 ℃.
Wherein, the yeast with high yield of isoamyl acetate is preserved by adopting a lactic acid YPD culture medium, and the components of the yeast comprise: 10g of yeast extract powder, 20g of peptone, 20g of glucose, 1000mL of distilled water, adjusting the pH value to 5.0 by lactic acid, and sterilizing at 121 ℃ for 20 min.
Wherein, the yeast preservation method for high yield of isoamyl acetate is a slant subculture method or a freeze-drying method.
The invention also provides application of the yeast for producing isoamyl acetate with high yield in brewing of strong-flavor, Maotai-flavor, fen-flavor or rice-flavor liquor.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides a yeast strain for high yield of isoamyl acetate, which mainly produces 15 volatile flavor substances by solid fermentation of grains, mainly comprises isoamyl acetate, C6 and more than a plurality of fatty acid ethyl esters, beta-phenethyl alcohol, diethyl succinate, phenethyl acetate, ethyl phenylacetate and the like, particularly has the yield of isoamyl acetate reaching 700mg/kg fermented grains, and also produces other flavor substances at high yield, such as: 19.8mg/kg of acetic acid, 307mg/kg of beta-phenethyl alcohol, 8mg/kg of n-propyl alcohol, 27mg/kg of isoamyl alcohol, 3mg/kg of n-butyl alcohol and only 2mg/kg of methanol.
The yeast of the invention has special significance to the ecological environment in the cellar: the weight loss caused by solid-state fermentation inoculated into grains for the same time is 145 percent of that of the saccharomyces cerevisiae, the residual sugar after fermentation is only 1.7 percent, but the yield of the ethanol is only 3.72mL/100g, which shows that the fermented grains have vigorous respiratory metabolism characteristics and are richer in metabolites, the moisture of the fermented grains can be increased to 64.70 percent by 24 days of fermentation, the moisture is increased by 3.8 times of that of the saccharomyces cerevisiae, and the acidity is increased by 2.3 times of that of the saccharomyces cerevisiae.
The yeast of the invention is highly suitable for the solid fermentation environment of white spirit, the lowest growth pH is 3.0, the temperature range for normal growth is 24-32 ℃, the yeast can be inoculated into the mixed bacteria natural fermentation grains for 1 day to proliferate in large quantity, the cell number reaches the highest value when the yeast is inoculated for 10-12 days, and the yeast can be continuously fermented for 24 days.
The yeast with high yield of isoamyl acetate is preserved in China general microbiological culture Collection center (CGMCC) at 13.05.2020, with the preservation number of CGMCC 19812, and the China general microbiological culture Collection center of China Committee for culture Collection in the preservation place has the following addresses: the microbial research institute of China academy of sciences, No. 3, Xilu No. 1, Beijing, Chao Yang district, is named Kazachstania turiensis by classification.
Drawings
FIG. 1 shows the colony morphology of the yeast with high isoamyl acetate yield of the present invention after culturing at 28 ℃ for 48 hours on WL agar medium.
FIG. 2 shows the cell morphology of the present invention after culturing at 28 ℃ for 48 hours on WL agar medium for high yield of isoamyl acetate.
FIG. 3 shows the total ion diagram of the main volatile substances produced by fermenting 5 kinds of grains with high isoamyl acetate yield according to the present invention.
Detailed Description
The invention provides a yeast for producing isoamyl acetate with high yield, and the preservation number is CGMCC 19812. The preservation time is 2020, 05 and 13 days, the preservation center is the common microorganism center of China Committee for culture Collection of microorganisms, and the address is the institute of microorganisms of China academy of sciences No. 3, West Lu No. 1, North Cheng, the Korean district, Beijing.
Wherein, the 26S rDNA sequence of the yeast for highly producing isoamyl acetate is shown as SEQ ID NO. 1.
Currently, there has been no study on the use of pure culture strains of K.turiensis yeast for solid state fermentation. The inventor of the invention separates K.turicensis yeast from fermented grains fermented by five grain liquids, and finds that the quantity of the K.turicensis yeast in the fermented grains naturally fermented by mixed bacteria reaches 106The yeast is the main dominant yeast species in the strong fermentation period of the strong aromatic Chinese spirits. Separation according to the inventionThis pure K.turiciensis yeast was cultured and named WLY 54. Researches show that the yeast is adopted to carry out solid state fermentation to mainly produce 15 volatile flavor substances, mainly comprising isoamyl acetate, C6 and above multiple fatty acid ethyl esters, beta-phenethyl alcohol, diethyl succinate, phenethyl acetate, ethyl phenylacetate and the like, particularly the yield of the isoamyl acetate reaches 700mg/kg fermented grains, and other flavor substances are also produced at high yield, such as: 19.8mg/kg of acetic acid, 307mg/kg of beta-phenethyl alcohol, 8mg/kg of n-propyl alcohol, 27mg/kg of isoamyl alcohol, 3mg/kg of n-butyl alcohol and only 2mg/kg of methanol.
The following examples are intended to illustrate specific embodiments of the present invention without limiting the scope of the invention to the examples.
EXAMPLE 1 isolation and characterization of high-yielding isoamyl acetate yeast strains
Separation: the sampling pit is a pit which is continuously fermented for more than 5 years in a production workshop of Yibin wuliangye Co. Sampling at 12d of fermentation, inserting a special sampler (hollow double-layer stainless steel sleeve) into a pit, filling fermented grains with 5 sampling points at the center and the periphery of the pit, which are 0.5-1.5 m from the surface of the pit, into a sterile sampling bag, transporting an ice box back to a laboratory, and separating yeast strains within 1 hour after sampling according to the following method: the fermented grain sample is diluted with sterile water 1000, 10000, 100000 times, and spread on YPD medium (1% yeast extract, 2% peptone, 2% xylose, 2% agar, and sterilized at 121 deg.C for 20min) with xylose instead of glucose and lactic acid to adjust pH to 5.0. After culturing at 28 ℃ for 36 hours, colonies which were wet, white, fine villous on the surface and edges were picked and purified 3 times on the same medium for use.
And (3) identification: DNA was extracted according to the procedure of E.Z.N.A. Yeast DNA kit (Shanghai Weijin Biotech Co., Ltd.), and the Yeast strain 26S rDNA sequence was amplified using NL1 and NL4 as primers (the nucleotide sequence of NL1 primer is shown in SEQ ID NO: 2: 5'-GCATATCAATAAGCGGAGGAAAAG-3' (SEQ ID NO:2) and the nucleotide sequence of NL4 primer is shown in SEQ ID NO: 3: 5'-GGTCCGTGTTTCAAGACGG-3' (SEQ ID NO: 3)). Detecting the amplification product by agarose gel electrophoresis, sending to Shanghai Meiji sequencing company for sequencing, carrying out BLASTN on the obtained sequence and the sequence of the model strain in GenBank, identifying the sequence with the sequence similarity of more than 99 percent as the same species, further comparing the morphological characteristics and physiological and biochemical characteristics of the separated yeast and the yeast species in literature records, and identifying the species when the morphological characteristics and physiological and biochemical characteristics are completely matched.
Example 2 physiological and biochemical characteristics of the yeast for producing isoamyl acetate in high yield by solid state fermentation of the invention
Culturing in L-YPD agar medium at 28 deg.C for 2d, and observing colony and cell morphology; culturing yeast by taking glucose, maltose, trehalose, erythrose, ribulose, xylose, arabinose, sucrose, lactose, galactose, melibiose, cellobiose, lactose and melezitose as the only carbon source in YNB culture medium, observing the growth condition of the yeast, and evaluating the sugar assimilation capability of the yeast; inoculating yeast to urea culture medium, culturing for 24 hr, and evaluating the urea utilization capacity of yeast according to whether the culture medium turns red. 5mL of the bacterial suspension was inoculated with a lactic acid YPD medium (pH5.0), and cultured with shaking at 20, 24, 28, 32, 36, and 40 ℃ at 100r/min for 36 hours, to detect the temperature tolerance, 5mL of the bacterial suspension was inoculated into a lactic acid YPD medium (pH5.0) containing 1, 2, 3, 4, 5, 6, 7, and 8% ethanol, respectively, and cultured with shaking at 28 ℃ and 100r/min for 36 hours, to detect the ethanol tolerance, and 5mL of the bacterial suspension was inoculated into a lactic acid YPD medium having pH values of 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, and 6.0, respectively, and cultured with shaking at 28 ℃ and 100r/min for 36 hours, to detect the pH adaptation, and the increase in OD560 was regarded as a growth exceeding 50%. The physiological and biochemical characteristics of the strain with the number of CGMCC 19812 are shown in Table 1.
TABLE 1 major physiological characteristics of Kazachstania turiensis CGMCC 19812
Example 3 application of the Yeast of the present invention in brewing Luzhou-flavor liquor
Firstly, inoculating 1 ring of yeast strain preserved on a slant into a lactic acid YPD liquid culture medium by using an inoculating needle, culturing for 1 day at 28 ℃, centrifuging the obtained yeast suspension for 5min at 25 ℃ and 3000rpm, pouring out supernatant, adding sterile water, uniformly mixing, centrifuging again under the same condition, washing thalli, and preparing the yeast suspension with the concentration of about 0.5OD for later use.
The yeast was inoculated into cellar grain stillage at an inoculum size of 0.1% for 70 days, and the major flavor substances produced by the yeast compared to the naturally fermented grain stillage (control) without yeast inoculation are shown in table 2. The content of acetic acid isoprene in the grains is increased from 0.007g/kg to 0.341g/kg, the content of beta-phenethyl alcohol is increased from 0.014g/kg to 0.207g/kg, the total content of fatty acid ethyl ester with the carbon number of more than 6 is increased from 1.014g/kg to 3.516g/kg, and sensory evaluation shows that fermented grains fermented by inoculating the yeast of the invention have strong fragrance, are remarkably superior to a control and have typical flower and fruit fragrance characteristics.
TABLE 2 major volatile flavor substances produced by solid fermentation of grains with Kazachstania turiensis CGMCC 19812 Strain
Example 4 production of Luzhou-flavor liquor flavoring liquor by using the yeast of the invention
Yeast strain was prepared in the same manner as in example 2.
The yeast is inoculated into cellar grain tank for fermentation for 70 days in an inoculation amount of 0.5%, compared with natural fermented grain tank (control) without yeast, n-amyl acetate is used as an internal standard, semi-quantification is carried out on the produced flavor substances, and the response of beta-phenylethyl alcohol under the detection condition is far higher than that of the internal standard, so that the external standard method is adopted for quantifying the beta-phenylethyl alcohol alone, and the result is shown in table 3.
TABLE 3 amount of flavor substances produced from fermented grains g/kg by Kazachstania turicensis CGMCC 19812
The yeast increases the total amount of flavor substances in the grains from 7.48g/kg to 18.07g/kg by 1.42 times, wherein the content of isoamyl acetate reaches 0.980g/kg, the content of beta-phenethyl alcohol reaches 0.312g/kg, and the yield of ethanol is reduced by 0.7 percent Vol.
Sequence listing
<110> Yibin wuliangye GmbH
<120> yeast for high yield of isoamyl acetate and application thereof
<130> A200687K (preface)
<141> 2020-08-27
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 535
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
gctcaaattt gaaatctggt accttcggtg cccgagttgt aatttgtaga gggcgacttt 60
ggggcggctc cttgtctatg ttccttggaa caggacgtca tagagggtga gaatcccgtg 120
tggcgaggag tgcggttccg tgtaaagcgc tctcgaagag tcgagttgtt tgggaatgca 180
gctctaagtg ggtggtaaat tccatctaaa gctaaatatt ggcgagagac cgatagcgaa 240
caagtacagt gatggaaaga tgaaaagaac tttgaaaaga gagtgaaaaa gtacgtgaaa 300
ttgttgaaag ggaagggcat ttgatcagac atggtgtttt gcgcccctcg ctccttgtgg 360
gtgggggact ctcgcagctc actgggccag catcagtttt ggcggtcgga taaaaccagg 420
ggaacgtagc ttgcttcggg aagtgttata gcccttggga atacggccag ccgggactga 480
ggaacgcgat tcgtcaagga tgctggcata atggttatat gccgcccgtc ttgaa 535
<210> 2
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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gcatatcaat aagcggagga aaag 24
<210> 3
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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ggtccgtgtt tcaagacgg 19
Claims (2)
1. A yeast for producing isoamyl acetate is characterized in that: the taxonomic name of the yeast isKazachstania turicensisThe preservation number is CGMCC 19812.
2. Use of the isoamyl acetate producing yeast of claim 1 in the brewing of Luzhou, Maotai, fen or rice white spirits.
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