CN114836332A - Pichia kudriavzevii yeast with high tolerance and low isoamylol yield and application thereof - Google Patents
Pichia kudriavzevii yeast with high tolerance and low isoamylol yield and application thereof Download PDFInfo
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- 241000235645 Pichia kudriavzevii Species 0.000 title claims abstract description 29
- 240000004808 Saccharomyces cerevisiae Species 0.000 title claims abstract description 14
- PHTQWCKDNZKARW-UHFFFAOYSA-N isoamylol Chemical compound CC(C)CCO PHTQWCKDNZKARW-UHFFFAOYSA-N 0.000 title claims description 9
- 238000002156 mixing Methods 0.000 claims abstract description 22
- 241000235058 Komagataella pastoris Species 0.000 claims abstract description 21
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- 239000001760 fusel oil Substances 0.000 abstract description 11
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- 125000003118 aryl group Chemical group 0.000 description 1
- GONOPSZTUGRENK-UHFFFAOYSA-N benzyl(trichloro)silane Chemical compound Cl[Si](Cl)(Cl)CC1=CC=CC=C1 GONOPSZTUGRENK-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention provides a high-tolerance and low-isoamyl alcohol-producing Pichia kudriavzevii strain and application thereof, wherein the strain is Pichia kudriavzevii (Pichia kudriavzevii) ((R))Pichia kudriavzevii) M3, accession number: CCTCC NO: m2022254. The yeast has the capability of high tolerance, high ethanol yield and low fusel oil yield, and the bacterial colony is circular, has jagged edges and large shape. Slightly raised, milky white in appearance, dry, elliptical or prolate in cell morphology characteristic observed under 400 times microscope, and bud reproduction. The invention also relates to the application of the pichia pastoris in the field of brewing and/or yeast preparation; also provides a liquor for blending in fen-flavor liquor, which adoptsThe wine is blended with base wine, so that the purposes of improving the purity and the high-quality product rate of the wine can be achieved, and the high-quality wine which is pure and pure, sweet and not top-rated can be obtained.
Description
Technical Field
The invention relates to the field of brewing microorganisms, and particularly relates to a pichia pastoris strain with good tolerance, high ethanol yield and low fusel oil yield and application thereof.
Background
The higher alcohols are generic terms of monohydric alcohols containing three or more carbon atoms, and include n-propanol, isobutanol, isoamyl alcohol, etc.; the higher alcohol is also one of three aromatic substances, the yeast is the main microorganism for producing the higher alcohol in the brewing process, the substrate is sufficient in the early stage of fermentation, the yeast is proliferated in a large amount, and the higher alcohol belongs to a primary metabolite, so the higher alcohol is mainly generated in the stage; in the later stage of fermentation, part of the higher alcohols and acetic acid-based acid substances generate corresponding ester compounds under the catalytic action of enzyme, and part of the higher alcohols continuously exist in the wine. In the wine body, a proper amount of higher alcohol has the effect of balancing taste and aroma, but researches show that the higher alcohol in the wine body not only brings bitter taste and unpleasant fusel oil taste, but also causes serious after-drinking stress effects such as upper head, nausea, palpitation and the like after drinking. Because of the problems of excessive high alcohol, the method for removing the high alcohol mainly comprises process control, rectification separation, material adsorption, breeding of microorganisms with low high alcohol yield and the like.
Disclosure of Invention
The invention provides a Pichia kudriavzevii strain with high tolerance and low isoamylol yield and application thereof.
The technical scheme of the invention is that a strain of Pichia kudriavzevii with high tolerance and low isoamyl alcohol production is the Pichia kudriavzevii (Pichia kudriavzevii) M3, which is preserved in the China center for type culture collection with the preservation number: CCTCC NO: m2022254.
Furthermore, the DNA splicing sequence of the strain is shown as SEQ ID NO. 1.
Furthermore, the bacterial colony of the strain is round, the edge is saw-toothed, the appearance is milky white, and the bacterial colony is dry; the shape of the cell is oval or flat long under a microscope, the propagation mode is budding, and cell buds in propagation are clearly visible.
The invention also relates to application of the pichia pastoris in the field of brewing and/or yeast preparations.
Further, the brewing is white spirit brewing or yellow wine brewing.
Further, in specific applications in the field of brewing, pichia pastoris is applied at any step in the brewing process, including but not limited to mixing with a koji making raw material in the koji making process; or mixing with saccharified and gelatinized wine brewing raw material; or mixing with fermented grains in middle and late stage of fermentation.
The invention also relates to reinforced bran koji which contains the pichia pastoris.
Further, it is prepared from testa Tritici by mixing with water, sterilizing, and dispersing Pichia pastoris and normal saline until cell number is 10 7 -10 8 Then inoculating the mixture to bran according to the proportion of 6-8wt% for culture and drying to obtain the pichia pastoris fortified bran koji.
The invention also relates to wine for blending, and in the preparation process of the wine, the pichia pastoris or the reinforced bran koji are adopted for solid state fermentation. Further, the blending wine is mixed with base wine to prepare fen-flavor liquor.
The present invention has the following advantageous effects
The Pichia pastoris (Pichia kudriavzevii) strain M3 provided by the invention has excellent physiological tolerance and unique capacity of high yield of ethanol and low yield of fusel oil. Can continuously adapt to the change of physical and chemical environment in the fermentation process. Can be widely applied to the field of wine brewing, in particular to the field of white spirit and the field of yellow wine. Meanwhile, the strengthened bran koji made from the pichia pastoris is mixed with the common distiller's yeast for use, so that the wine quality can be obviously improved, and the content of fusel oil in the raw wine is reduced.
The invention also provides a wine for blending in fen-flavor liquor, which is prepared by pure fermentation of Pichia pastoris (Pichia kudriavzevii) strain M3, and the wine and the fen-flavor liquor base liquor are blended by storing the wine for a proper time, so that the ester fragrance of the base liquor can be obviously improved, and the ester fragrance and the grain fragrance are coordinated; reduce the rough and hot feeling and the bitter feeling. So as to achieve the purpose of improving the purity and the high-quality product rate of the wine and obtain the high-quality wine which is pure and pure, sweet and not top-end.
Drawings
FIG. 1 is a graph showing the analysis of the tolerance of a plurality of low-producing heteroalcoholic yeast strains.
FIG. 2, a colony morphology of Pichia kudriavzevii M3(Pichia kudriavzevii) on Yeast Extract Peptone glucose Medium (YEPD).
FIG. 3 shows a morphological diagram of Pichia kudriavzevii M3(Pichia kudriavzevii) under 400-fold microscope (Melanin staining).
FIG. 4 is a genetic developmental tree diagram of Pichia kudriavzevii (Pichia kudriavzevii) in Pichia kudriavzevii.
FIG. 5 GC-MS total ion chromatogram of volatile components in Pichia kudriavzevii M3(Pichia kudriavzevii) fermentation broth.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention.
Example 1:
screening of strains
The invention relates to a method for separating and screening pichia pastoris with high ethanol yield and low fusel oil yield from high-quality fermented grains, which comprises the following steps:
firstly, screening 60 strains of yeast with excellent performance from high-quality fermented grains, carrying out directional screening aiming at ethanol production and low-yield fusel oil, averagely dividing a prepared yeast extract peptone glucose medium (YEPD) plate into six areas, preparing a plurality of plates in advance, then dibbling all strains on the plates for two days at 30 ℃, pouring a TTC medium cooled to 45 ℃ into the upper layer of the yeast extract peptone glucose medium (YEPD) plate, preserving for two hours in the dark, observing color change, and selecting the strains with deeper red as alternative strains (specifically numbered as Shao, Fen II, PD1, PT11, PT17, Y-3, JY26, PF4, M3, M4, 4-16, 2300, 1312 and 10 #). Then placing the alternative strain in a liquid culture medium of yeast extract peptone glucose (YEPD) at 30 ℃, culturing for 18h at 180r/min to serve as a seed solution, inoculating the seed solution into a sorghum juice culture medium with the sugar degree of 12Brix according to the inoculation amount of 5 percent, culturing for 12h at 30 ℃, at 180r/min, transferring to static culture, weighing once every 12h, and recording CO 2 And (2) weight loss, ending fermentation when the weight loss is less than 0.2g, detecting fusel oil by using a gas chromatography after the fermentation is ended, selecting a strain with high ethanol yield and low fusel oil yield by using a distillation method, comparing the strain with a production strain SC (commercially available from a certain company), and respectively reducing the total amount of three higher alcohols of the strains Fen II, PT11, JY26, 2300, 1312 and M3 by 22.32%, 22.05%, 23.29%, 10.41%, 11.13% and 15.93% compared with the production strain SC of a certain plant, and simultaneously the strains have the high ethanol yield capability.
The yeast extract peptone glucose medium (YEPD) in this example consisted of: 10g of yeast extract powder, 25g of glucose, 25g of peptone and 1200g of water, and the pH value is natural; 15g of agar powder is additionally added into the solid culture medium. The sorghum juice culture medium comprises the following components: 2000g of sorghum flour, 3g of amylase, 75g of saccharifying enzyme, 8000g of water and natural pH value.
TABLE 1 comparison of fermentation parameters of the selected strains with the production strains
Example 2: the physiological characteristics of pichia pastoris for high ethanol yield and low fusel oil yield are as follows:
scraping FenII, PT11, JY26, 2300, 1312 and M3 in a ring inclined plane to a liquid yeast extract peptone glucose medium (YEPD) for culturing for 16h at 30 ℃ at 180r/min, then inoculating the strain into the YEPD liquid medium at 30 ℃ for culturing for 24h at 180r/min according to the inoculation amount of 2%, and detecting the concentration of the strain in ethanol by adopting an ultraviolet spectrophotometer with OD600 as an index to be 1%, 3%, 5%, 7%, 9%, 11% and 13%; the pH value is 2, 3, 4, 5, 6, 7 and 8; the sugar degree value is 5Brix, 10Brix, 15Brix, 20Brix, 25Brix, 30Brix, 35 Brix; the culture temperature is 18 deg.C, 22 deg.C, 26 deg.C, 30 deg.C, 34 deg.C, 38 deg.C, 42 deg.C; the concentration of lactic acid is: 0%, 1%, 2%, 3%, 4%, 5%, hexanoic acid concentration: 0 per mill, 0.2 per mill, 0.4 per mill, 0.6 per mill, 0.8 per mill, 1.0 per mill; the acetic acid concentration was: the tolerance conditions under the conditions of 0 per thousand, 1 per thousand, 2 per thousand, 3 per thousand, 4 per thousand and 5 per thousand are shown in the attached figure 1. It can be seen that the M3 strain has the best physiological tolerance.
The selected M3 strain is preserved in China center for type culture Collection with the preservation address: the preservation number of Wuhan university in Wuhan city of Hubei province is CCTCC NO: m2022254, preservation date 2022-03-14. Through identification, the strain is Pichia kudriavzevii (Pichia kudriavzevii) M3.
(1) Microbiological morphological characteristics:
taking a cultured inclined plane, scraping a ring by using an inoculating ring, scattering the ring in sterile normal saline to prepare a cell suspension, then diluting the cell suspension according to a gradient of 10 times, taking 100 mu L of the cell suspension, coating the cell suspension in a Yeast Extract Peptone Dextrose Medium (YEPD) culture Medium, inverting a flat plate, putting the flat plate into a 30 ℃ incubator for culturing for 48 hours, and observing the morphological characteristics of bacterial colonies: in the YEPD plate, the bacterial colony is circular, the edge is jagged, the shape is large, the bacterial colony is slightly raised, the appearance is milky white, and the bacterial colony is dried, which is specifically shown in figure 2.
The cell morphology of the strains was observed under 400-fold microscope and was characterized by: it is in oval or flat shape, the propagation mode is bud reproduction, and the cell bud in reproduction is clearly visible, as shown in FIG. 3.
Example 3: molecular biological identification of M3 strain
The strains obtained by screening are sent to Huada Gene science and technology Co., Ltd for 18S rRNA gene sequencing, the sequencing result is placed in an NCBI (national Center for Biotechnology information) database for comparison, and the strain sequences with higher similarity are selected and used for constructing phylogenetic trees by using the Maximum Likelihood Method (MLE) in MEGA 11.0 software, as shown in figure 4.
Example 4 application of the strain M3 in brewing and blending of white spirit
Strengthening wheat bran koji: weighing 20 parts of wheat bran into a 250mL triangular flask, adding 15 parts of water, uniformly stirring, sterilizing for 20min by high-pressure steam at 121 ℃, taking out, and cooling to room temperature; centrifuging seed liquid of M3 strain cultured in liquid yeast extract peptone glucose medium (YEPD), washing, precipitating, adding normal saline, and scattering to make cell number be 10 7 -10 8 Then inoculating 8wt% of the mixture into bran, uniformly stirring the mixture, and then placing the mixture into a 30 ℃ thermostat for culture for 3 d. During the culture period, flask buckling is carried out every 12 h. After the culture is finished, putting the wheat bran koji into a constant-temperature drying oven at 38-40 ℃, and drying until the moisture content is less than 10% to obtain the strengthened wheat bran koji.
Brewing white spirit: immersing 3000g of high-quality sorghum into hot water at 70-80 ℃ for 20cm, soaking for 24h, washing for seven to eight times, then cooking for 30min at high temperature and high pressure of 0.25MPa, soaking the first steamed sorghum in hot water at 80 ℃ for 30min, then cooking for 20min at high temperature and high pressure of 0.16MPa until the sorghum breaks and blooms, adding 0.7 wt% of mixed koji mixed with strengthened bran koji and brewing koji in a mixing ratio of 1:1, stirring uniformly, saccharifying and accumulating for 24h, then respectively loading into small jars, sealing and fermenting at room temperature for 7 d. After fermentation, distilling in a retort, slightly increasing the two sides to 1-2cm higher than the middle part when the fermentation is finished, and collecting fractions by 'pinching the ends and removing the tails'. Or steaming sorghum until the sorghum blooms and peels are broken, adding 0.1% of amylase, 0.2% of saccharifying enzyme and 0.01% of acid protease, saccharifying for 24 hours in an accumulation saccharifying box, adding 2-5% of pure bran koji made by pichia pastoris M3, fermenting for 7-14 days in a sealed manner, distilling in a retort, and collecting fractions to obtain the wine for blending.
Blending the white spirit: the wine for blending can be blended with other original wine samples to replace essence and spice so as to improve the wine quality and meet the pursuit of consumers on taste, and the characteristics before and after blending are shown in the following table 2.
TABLE 2 after-check scoring table
Example 5 metabolite analysis of Strain M3
Scraping Pichia pastoris from a ring of inclined planes into a liquid yeast extract peptone glucose medium (YEPD) at 30 ℃, culturing for 16h at 180r/min, and then inoculating into a sorghum juice liquid medium to ensure that the number of cells after inoculation is 10 6 -10 7 Culturing at 30 deg.C and 180r/min for 18h to stationary phase, standing at the same temperature for 4d, and extracting flavor compounds by headspace-solid phase microextraction method and detecting by GC-MS.
TABLE 3 determination of volatile component content by HS-SPME method (part)
Note: when the HS-SPME method is used for extraction, the problems of extraction rate and the like are considered, and the method does not contain all volatile products of the M3 strain.
As can be seen from Table 3, the metabolites of the strain M3 contain esters, acids and alcohols which are common in wine, and are suitable for pure-breed brewing of wine for blending and improving the high-quality product rate of wine, or making pure-breed koji for improving the quality of wine by matching with traditional koji for fermentation.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
SEQUENCE LISTING
SEQ ID NO.1
<110> Hubei Daohuaxiang wine industry Co Ltd
<120> Pichia kudriavzevii with low fusel oil yield and application thereof
<130> 1
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 501
<212> DNA
<213> Pichia kudriavzevii (Pichia kudriavzevii) M3
<400> 1
cttccgtagg gtgaacctgc ggaaggatca ttactgtgat ttactactac actgcgtgag 60
cggaacgaaa acaacaacac ctaaaatgtg gaatatagca tatagtcgac aagagaaatc 120
tacgaaaaaa caaacaaaac tttcaacaac ggatctcttg gttctcgcat cgatgaagag 180
cgcagcgaaa tgcgatacct agtgtgaatt gcagccatcg tgaatcatcg agttcttgaa 240
cgcacattgc gcccctcggc attccggggg gcatgcctgt ttgagcgtcg tttccatctt 300
gcgcgtgcgc agagttgggg gagcggagcg gacgacgtgt aaagagcgtc ggagctgcga 360
ctcgcctgaa agggagcgaa gctggccgag cgaactagac tttttttcag ggacgcttgg 420
cggccgagag cgagtgttgc gagacaacaa aaagctcgac ctcaaatcag gtaggaatac 480
ccgctgaact taagcatatc a 501
Claims (10)
1. The Pichia kudriavzevii yeast with high tolerance and low isoamyl alcohol production is characterized in that: the strain is Pichia kudriavzevii (A) (B)Pichia kudriavzevii) M3, deposited in China center for type culture Collection with the deposit number: CCTCC NO: m2022254.
2. The yeast according to claim 1, wherein: the DNA splicing sequence of the strain is shown as SEQ ID NO. 1.
3. The yeast according to claim 1, wherein: the bacterial colony of the strain is round, the edge is serrated, the appearance is milky white, and the strain is dry; the shape of the seed is oval or flat under a microscope, and the propagation mode is budding.
4. The use of Pichia pastoris according to any one of claims 1 to 3 in the field of brewing and/or yeast preparation.
5. Use according to claim 4, characterized in that: the brewing is white spirit brewing or yellow wine brewing.
6. Use according to claim 4, characterized in that: in particular application in the field of brewing, pichia pastoris is used at any step in the brewing process, including but not limited to mixing with the starter propagation raw material in the starter propagation process; or mixing with saccharified and gelatinized wine brewing raw material; or mixing with fermented grains in middle and late stage of fermentation.
7. The reinforced bran koji is characterized in that: the bran koji comprising the Pichia pastoris according to any one of claims 1 to 3.
8. The reinforced bran koji as claimed in claim 7, wherein: it is prepared from testa Tritici by mixing with water, sterilizing, and dispersing Pichia pastoris with normal saline until cell number is 10 7 -10 8 Then inoculating the mixture to bran according to the proportion of 6-8wt% for culture and drying to obtain the pichia pastoris fortified bran koji.
9. A wine for blending is characterized in that: in the preparation process of the wine, the pichia pastoris of any one of claims 1 to 3 or the reinforced bran koji of claim 7 is used for fermentation.
10. The use of the liquor of claim 9 in blending fen-flavor liquor by blending with a base liquor to prepare fen-flavor liquor.
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Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010151525A1 (en) * | 2009-06-22 | 2010-12-29 | Gevo, Inc. | Yeast organisms for the production of isobutanol |
| CN102226155A (en) * | 2011-05-12 | 2011-10-26 | 江南大学 | Screening and application of yeast with high ethanol yield and low fusel oil yield in Chinese Maotai-flavor liquor production |
| KR20140034644A (en) * | 2012-09-12 | 2014-03-20 | 안동대학교 산학협력단 | Issatchenkia orientalis ms-1 and its use |
| KR101618260B1 (en) * | 2014-12-18 | 2016-05-04 | 대한민국 | Pichia kudriavzevii n77-4 and its use |
| CN105861345A (en) * | 2016-06-14 | 2016-08-17 | 江南大学 | Pichia kudriavzevii yeast low in urea yield and capable of producing flavor and application thereof to food fermentation |
| CN110846235A (en) * | 2019-09-23 | 2020-02-28 | 广西科学院 | High-temperature-resistant stress-resistant Pichia kudriavzevii high-yield ethanol and application thereof |
| CN112831425A (en) * | 2021-02-04 | 2021-05-25 | 湖北稻花香酒业股份有限公司 | Pichia kudriavzevii and application thereof in XIN-flavor type white spirit fermentation |
| CN113717867A (en) * | 2021-09-16 | 2021-11-30 | 海南大学 | Pichia pastoris capable of producing no ethanol and application thereof |
-
2022
- 2022-05-27 CN CN202210590776.8A patent/CN114836332B/en active Active
- 2022-05-27 CN CN202310951335.0A patent/CN116925871A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010151525A1 (en) * | 2009-06-22 | 2010-12-29 | Gevo, Inc. | Yeast organisms for the production of isobutanol |
| CN102226155A (en) * | 2011-05-12 | 2011-10-26 | 江南大学 | Screening and application of yeast with high ethanol yield and low fusel oil yield in Chinese Maotai-flavor liquor production |
| KR20140034644A (en) * | 2012-09-12 | 2014-03-20 | 안동대학교 산학협력단 | Issatchenkia orientalis ms-1 and its use |
| KR101618260B1 (en) * | 2014-12-18 | 2016-05-04 | 대한민국 | Pichia kudriavzevii n77-4 and its use |
| CN105861345A (en) * | 2016-06-14 | 2016-08-17 | 江南大学 | Pichia kudriavzevii yeast low in urea yield and capable of producing flavor and application thereof to food fermentation |
| CN110846235A (en) * | 2019-09-23 | 2020-02-28 | 广西科学院 | High-temperature-resistant stress-resistant Pichia kudriavzevii high-yield ethanol and application thereof |
| CN112831425A (en) * | 2021-02-04 | 2021-05-25 | 湖北稻花香酒业股份有限公司 | Pichia kudriavzevii and application thereof in XIN-flavor type white spirit fermentation |
| CN113717867A (en) * | 2021-09-16 | 2021-11-30 | 海南大学 | Pichia pastoris capable of producing no ethanol and application thereof |
Non-Patent Citations (4)
| Title |
|---|
| LEONEL NIETO-SARABIA等: "Isolation, identification, and kinetic and thermodynamic characterization of a Pichia kudriavzevii yeast strain capable of fermentation", FOOD AND BIOPRODUCTS PROCESSING, vol. 131, pages 109 - 124, XP086913619, DOI: 10.1016/j.fbp.2021.10.013 * |
| YOU LING等: "Distribution and function of dominant yeast species in the fermentation of strong‐flavor baijiu", WORLD JOURNAL OF MICROBIOLOGY AND BIOTECHNOLOGY, vol. 37, pages 1 - 12 * |
| 游玲等: "酵母对浓香型白酒糟醅中高级醇生成的影响", 食品与发酵工业, vol. 42, no. 2, pages 23 - 28 * |
| 蔡雪梅等: "酱香型白酒酒糟中生香酵母的筛选及鉴定", 中国酿造, vol. 36, no. 7, pages 42 - 47 * |
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