CN114836332B - Pichia kudriavzevii with high tolerance and low isoamyl alcohol yield and application thereof - Google Patents
Pichia kudriavzevii with high tolerance and low isoamyl alcohol yield and application thereof Download PDFInfo
- Publication number
- CN114836332B CN114836332B CN202210590776.8A CN202210590776A CN114836332B CN 114836332 B CN114836332 B CN 114836332B CN 202210590776 A CN202210590776 A CN 202210590776A CN 114836332 B CN114836332 B CN 114836332B
- Authority
- CN
- China
- Prior art keywords
- pichia
- pichia pastoris
- wine
- brewing
- kudriavzevii
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 241000235645 Pichia kudriavzevii Species 0.000 title claims abstract description 24
- PHTQWCKDNZKARW-UHFFFAOYSA-N isoamylol Chemical compound CC(C)CCO PHTQWCKDNZKARW-UHFFFAOYSA-N 0.000 title claims description 12
- 235000014101 wine Nutrition 0.000 claims abstract description 35
- 241000235058 Komagataella pastoris Species 0.000 claims abstract description 24
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims abstract description 17
- 238000002360 preparation method Methods 0.000 claims abstract description 3
- 238000002156 mixing Methods 0.000 claims description 16
- 238000000855 fermentation Methods 0.000 claims description 12
- 230000004151 fermentation Effects 0.000 claims description 12
- 238000012258 culturing Methods 0.000 claims description 11
- 238000000034 method Methods 0.000 claims description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 7
- 235000013339 cereals Nutrition 0.000 claims description 5
- 239000000463 material Substances 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 4
- 239000007858 starting material Substances 0.000 claims description 4
- 235000015099 wheat brans Nutrition 0.000 claims description 4
- 238000013124 brewing process Methods 0.000 claims description 3
- 238000001035 drying Methods 0.000 claims description 3
- 239000002994 raw material Substances 0.000 claims description 3
- 235000020097 white wine Nutrition 0.000 claims description 3
- 241000235648 Pichia Species 0.000 claims 2
- 241000196324 Embryophyta Species 0.000 claims 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 abstract description 41
- 239000001760 fusel oil Substances 0.000 abstract description 12
- 230000000877 morphologic effect Effects 0.000 abstract description 3
- 235000009508 confectionery Nutrition 0.000 abstract description 2
- 239000001963 growth medium Substances 0.000 description 16
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 14
- 239000001888 Peptone Substances 0.000 description 13
- 108010080698 Peptones Proteins 0.000 description 13
- 235000019319 peptone Nutrition 0.000 description 13
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 11
- 229940041514 candida albicans extract Drugs 0.000 description 11
- 239000012138 yeast extract Substances 0.000 description 11
- 150000001298 alcohols Chemical class 0.000 description 10
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 9
- 239000008103 glucose Substances 0.000 description 9
- 240000006394 Sorghum bicolor Species 0.000 description 7
- 235000011684 Sorghum saccharatum Nutrition 0.000 description 7
- 239000000796 flavoring agent Substances 0.000 description 7
- 238000012216 screening Methods 0.000 description 5
- 239000002253 acid Substances 0.000 description 4
- 239000006285 cell suspension Substances 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 101100522111 Oryza sativa subsp. japonica PHT1-11 gene Proteins 0.000 description 3
- 150000002148 esters Chemical class 0.000 description 3
- 235000019634 flavors Nutrition 0.000 description 3
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 238000004321 preservation Methods 0.000 description 3
- 238000007790 scraping Methods 0.000 description 3
- 238000002791 soaking Methods 0.000 description 3
- 238000010025 steaming Methods 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 235000019640 taste Nutrition 0.000 description 3
- 239000004382 Amylase Substances 0.000 description 2
- 102000013142 Amylases Human genes 0.000 description 2
- 108010065511 Amylases Proteins 0.000 description 2
- 238000007476 Maximum Likelihood Methods 0.000 description 2
- 235000019418 amylase Nutrition 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 239000008121 dextrose Substances 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 description 2
- 239000006481 glucose medium Substances 0.000 description 2
- FUZZWVXGSFPDMH-UHFFFAOYSA-N hexanoic acid Chemical compound CCCCCC(O)=O FUZZWVXGSFPDMH-UHFFFAOYSA-N 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- ZXEKIIBDNHEJCQ-UHFFFAOYSA-N isobutanol Chemical compound CC(C)CO ZXEKIIBDNHEJCQ-UHFFFAOYSA-N 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 238000002386 leaching Methods 0.000 description 2
- 238000009630 liquid culture Methods 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 238000002470 solid-phase micro-extraction Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- 108020004463 18S ribosomal RNA Proteins 0.000 description 1
- 108091005508 Acid proteases Proteins 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 229920004934 Dacron® Polymers 0.000 description 1
- 230000005526 G1 to G0 transition Effects 0.000 description 1
- 206010028813 Nausea Diseases 0.000 description 1
- 206010033557 Palpitations Diseases 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 235000019658 bitter taste Nutrition 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 235000010633 broth Nutrition 0.000 description 1
- 125000004432 carbon atom Chemical group C* 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000004821 distillation Methods 0.000 description 1
- 230000035622 drinking Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- -1 ester compounds Chemical class 0.000 description 1
- 235000013312 flour Nutrition 0.000 description 1
- 238000004817 gas chromatography Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000001319 headspace solid-phase micro-extraction Methods 0.000 description 1
- 239000010903 husk Substances 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 230000002906 microbiologic effect Effects 0.000 description 1
- 230000008693 nausea Effects 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 239000005020 polyethylene terephthalate Substances 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 229930010796 primary metabolite Natural products 0.000 description 1
- 238000004886 process control Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000010563 solid-state fermentation Methods 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 235000013599 spices Nutrition 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000005728 strengthening Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 230000004580 weight loss Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12G—WINE; PREPARATION THEREOF; ALCOHOLIC BEVERAGES; PREPARATION OF ALCOHOLIC BEVERAGES NOT PROVIDED FOR IN SUBCLASSES C12C OR C12H
- C12G3/00—Preparation of other alcoholic beverages
- C12G3/02—Preparation of other alcoholic beverages by fermentation
- C12G3/021—Preparation of other alcoholic beverages by fermentation of botanical family Poaceae, e.g. wheat, millet, sorghum, barley, rye, or corn
-
- 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
- C12G3/04—Preparation of other alcoholic beverages by mixing, e.g. for preparation of liqueurs
-
- 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
-
- 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
- C12N1/165—Yeast isolates
-
- C—CHEMISTRY; METALLURGY
- 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
- C12R2001/84—Pichia
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
Abstract
The invention provides a high-tolerance and low-isoamyl alcohol-producing Pichia kudriavzevii and application thereof, wherein the strain is the Pichia kudriavzevii @ kudriavzeviiPichia kudriavzevii) M3, deposit number is: cctccc NO: m2022254. The yeast has high tolerance, high ethanol yield and low fusel oil yield, and the colony is circular with sawtooth edge and large shape. The micro-bulge is milky white in appearance, dried, and observed under a 400-time microscope, the cell morphological characteristics of the strain are elliptic or prolate, and the propagation mode is bud. The invention also relates to application of the pichia pastoris in the field of brewing and/or in yeast preparations; the wine is blended with the base wine, so that the purposes of improving the purity and the high-quality rate of the wine can be achieved, and the high-quality wine which is pure, sweet and free of top-quality can be obtained.
Description
Technical Field
The invention relates to the field of brewing microorganisms, in particular to a pichia pastoris strain with good tolerance, high ethanol yield and low fusel oil yield and application thereof.
Background
Higher alcohols are the generic names of monohydric alcohols containing more than three carbon atoms, and include n-propanol, isobutanol, isoamyl alcohol, etc.; higher alcohols are also one of the three major aromatic substances, and yeast is the main microorganism producing higher alcohols in the brewing process, and the substrate is sufficient in the early stage of fermentation, and the yeast proliferates in a large amount while the higher alcohols are primary metabolites, so that the higher alcohols are mainly produced in this stage; and a part of the higher alcohols and acid substances mainly including acetic acid generate corresponding ester compounds under the catalysis of enzymes in the later fermentation stage, and a part of the higher alcohols and acid substances mainly including acetic acid are continuously present in the wine body. In the wine body, a proper amount of higher alcohol has the function of balancing taste and aroma, but researches show that the higher alcohol in the wine body can not only bring bitter taste and unpleasant 'fusel oil taste', but also cause serious post-wine stress effects such as head rising, nausea, palpitation and the like after drinking. Because of the problems of excessive higher alcohol, the method for reducing the content of higher alcohol in wine body is an important means for controlling and improving the quality of wine, and the method for removing the higher alcohol mainly comprises the steps of process control, rectification separation, material adsorption, breeding of microorganisms with low yield of higher alcohol and the like.
Disclosure of Invention
The invention provides a pichia kudriavzevii with high tolerance and low yield of isoamyl alcohol and application thereof, and the strain has the characteristics of sugar resistance, ethanol resistance, acid resistance, high yield of ethanol and low yield of fusel oil.
The technical scheme of the invention is that the pichia kudriavzevii with high tolerance and low isoamyl alcohol yield is pichia kudriavzevii (Pichia kudriavzevii) M3, and the strain is preserved in China center for type culture collection, and the preservation number is: cctccc NO: m2022254.
Further, the DNA splice sequence of the strain is shown as SEQ ID NO. 1.
Further, the bacterial colony of the strain is circular, the edge is serrated, the appearance is milky white, and the bacterial colony is dried; the cell buds are in oval or prolate form under a microscope, the propagation mode is bud, and the cell buds in propagation are clearly visible.
The invention also relates to application of the pichia pastoris in the field of brewing and/or in yeast preparations.
Further, the brewing is white wine brewing or yellow wine brewing.
Further, in the field of brewing, pichia pastoris is used in any step of the brewing process, including but not limited to mixing with starter propagation material during starter propagation; or mixing with saccharified and gelatinized brewing materials; or mixing with fermented grains during middle and late stages of fermentation.
The invention also relates to a reinforced bran koji which contains the pichia pastoris.
Further, wheat bran is used as raw material, water is added, the mixture is uniformly mixed and sterilized, and then pichia pastoris is scattered until the cell number is 10 7 -10 8 And then inoculating the pichia pastoris to bran according to the proportion of 6-8wt% for culturing, and drying to obtain the pichia pastoris reinforced bran koji.
The invention also relates to a blending wine, and in the process of preparing the wine, the pichia pastoris or the reinforced bran koji is adopted for solid state fermentation. Further, the wine for blending is mixed with the base wine, so that the fen-flavor white wine can be blended.
The invention has the following beneficial effects
The Pichia pastoris (Pichia kudriavzevii) strain M3 provided by the invention has excellent physiological tolerance and unique capability of high ethanol yield and low fusel oil yield. Can continuously adapt to the change of physicochemical environment in the fermentation process. Can be widely applied to the brewing field, in particular to the white spirit field and the yellow wine field. Meanwhile, the reinforced bran koji prepared from the pichia pastoris is mixed with common distiller's yeast, so that the quality of the wine can be obviously improved, and the content of fusel oil in the raw wine can be reduced.
The invention also provides a wine prepared by pure fermentation of the Pichia pastoris (Pichia kudriavzevii) strain M3, which is used for blending in the fen-flavor liquor, and the wine stored for a proper time is blended with the fen-flavor liquor base wine, so that the ester flavor of the base wine can be obviously improved to coordinate the ester flavor and the grain flavor; reduce the sense of coarse and hot and bitter. So as to achieve the aim of improving the purity and the high-quality rate of the wine and obtain the high-quality wine which is pure, sweet and free from top-quality.
Drawings
FIG. 1 is a graph showing the tolerability analysis of a plurality of low-fusogenic yeast strains.
FIG. 2, colony morphology of Pichia kudriavzevii M3 (Pichia kudriavzevii) on yeast extract peptone glucose medium (Yeast Extract Peptone Dextrose Medium, YEPD).
FIG. 3, pichia kudriavzevii M3 (Pichia kudriavzevii) strain pattern (blue stain) under 400-fold microscope.
FIG. 4, genetic development tree diagram of Pichia kudriavzevii M3 (Pichia kudriavzevii).
FIG. 5, GC-MS total ion flow chromatogram of volatile components in Pichia kudriavzevii M3 (Pichia kudriavzevii) fermentation broths.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only for illustrating 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 in high-quality fermented grains, which comprises the following steps:
firstly, screening more than 60 yeast strains with excellent performance from high-quality fermented grains, directionally screening for ethanol production and low-yield fusel oil, equally dividing a prepared yeast extract peptone glucose culture medium (YEPD) plate into six areas, preparing a plurality of plates in advance, then, dibbling all the strains on the plates, culturing for two days at 30 ℃, pouring TTC culture medium cooled to 45 ℃ into the upper layer of the yeast extract peptone glucose culture medium (YEPD) plate, preserving for two hours in the dark, observing color change, and selecting the strains with darker red color as alternative strains (specific numbers are Shao, fen II, PD1, PT11, PT17, Y-3, JY26, PF4, M3, M4, 4-16, 2300, 1312 and 10#). Then placing the strain in yeast extract peptone glucose culture medium (YEPD) liquid culture medium at 30deg.C for 180r/min for 18 hr as seed solution, inoculating to sorghum juice culture medium with sugar degree of 12Brix according to 5% inoculum size, culturing at 30deg.C for 180r/min for 12 hr, standing for culturing, weighing every 12 hr, and recording CO 2 And when the weight loss is less than 0.2g, the fermentation is finished, the fusel oil is detected by adopting a gas chromatography after the fermentation is finished, the ethanol is detected by adopting a distillation method, the strain with high ethanol yield and low fusel oil yield is selected and compared with a production strain SC (Saccharomyces cerevisiae (commercially available) of a certain company), and compared with the production strain SC of a certain factory, the total amount of three higher alcohols of strains FenII, PT11, JY26, 2300, 1312 and M3 is respectively reduced by 22.32%, 22.05%, 23.29%, 10.41%, 11.13% and 15.93%, and meanwhile, the strains have the capacity of high ethanol yield.
The composition of the yeast extract peptone glucose medium (YEPD) in this example was: yeast extract 10g, glucose 25g, peptone 25g, water 1200g and natural pH value; 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 selected strains and production strains
Example 2: physiological characteristics of pichia pastoris with high ethanol yield and low fusel oil yield:
scraping Fen II, PT11, JY26, 2300, 1312 and M3 in a ring of inclined planes to 30 ℃ in a liquid yeast leaching powder peptone glucose culture medium (YEPD), culturing for 16h at 180r/min, inoculating the liquid yeast leaching powder peptone glucose culture medium into the YEPD liquid culture medium at 30 ℃ according to 2% of inoculation amount, culturing for 24h at 180r/min, and detecting the concentration of the strain in ethanol at 1%, 3%, 5%, 7%, 9%, 11% and 13% by adopting an ultraviolet spectrophotometer with OD600 as an index; the pH value is 2, 3, 4, 5, 6, 7 and 8; the sugar degree value is 5Brix, 10Brix, 15Brix, 20Brix, 25Brix, 30Brix, 35Brix; the culture temperature was 18 ℃, 22 ℃, 26 ℃,30 ℃, 34 ℃, 38 ℃, 42 ℃; the lactic acid concentration is: 0%, 1%, 2%, 3%, 4%, 5%, caproic acid concentration: 0.2%, 0.4%, 0.6%, 0.8%, 1.0%; the acetic acid concentration is: the tolerance conditions under the conditions of 0%o, 1%o, 2%o, 3%o, 4%o and 5%o are shown in figure 1. The M3 strain was found to have optimal physiological tolerance.
The screened M3 strain is preserved in China center for type culture Collection, and the preservation address is that: the preservation number of the Chinese medicine is CCTCC NO: m2022254, date of deposit 2022-03-14. The strain is identified as Pichia kudriavzevii (Pichia kudriavzevii) M3.
(1) Microbiological morphological characteristics:
taking the cultured inclined plane, scraping a ring by an inoculating loop, scattering in sterile normal saline to prepare a cell suspension, diluting the cell suspension according to a 10-time gradient, taking 100 mu L of the cell suspension, coating the cell suspension in a yeast extract peptone glucose culture medium (Yeast Extract Peptone Dextrose Medium, YEPD) culture medium, inverting a flat plate, placing the flat plate into a 30 ℃ incubator for culturing for 48 hours, and observing the morphological characteristics of a colony: in the YEPD plates, the colonies were round, with serrated edges, large morphology, micro-ridges, milky white in appearance, and dried, see in particular FIG. 2.
The cell morphology of the strain was observed under a 400-fold microscope and characterized by: in oval or prolate form, the propagation mode is bud, and the buds of the cells in propagation are clearly visible, and are particularly shown in FIG. 3.
Example 3: molecular biological identification of M3 Strain
The strain obtained by screening is sent to Hua Dacron gene technology Co., ltd for 18S rRNA gene sequencing, the sequencing result is placed in NCBI (National Center for Biotechnology Information) database for comparison, and the strain sequence with higher similarity is selected to construct phylogenetic tree by using the maximum likelihood method (Maximum Likelihood Estimation, MLE) in MEGA 11.0 software, which is shown in figure 4.
Example 4 application of Strain M3 to white spirit brewing and blending
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 M3 strain seed solution cultured in liquid yeast extract peptone glucose culture medium (YEPD), washing, precipitating, adding physiological saline, and scattering to obtain cell number of 10 7 -10 8 Then inoculating 8wt% of the mixture into bran, uniformly stirring, and culturing in a 30 ℃ incubator for 3d. During the culture period, bottle buckling is carried out every 12 hours. After the culture is completed, the bran koji is placed in a constant temperature drying oven at 38-40 ℃ and dried until the moisture is less than 10%, and the reinforced bran koji is obtained.
Brewing white spirit: soaking 3000g of high-quality sorghum in hot water at 70-80 ℃ for 20cm, soaking for 24h, washing for seven to eight times, steaming at high temperature and high pressure of 0.25MPa for 30min, soaking the first steamed sorghum in hot water at 80 ℃ for 30min, steaming at high temperature and high pressure of 0.16MPa for 20min until the husks are broken, adding mixed yeast with the mixing ratio of 0.7wt% of mixed reinforced bran yeast to brewing yeast being 1:1, stirring uniformly, saccharifying and accumulating for 24h, respectively loading into small jars, sealing and placing into room temperature for fermentation for 7d. After fermentation, distilling, and collecting distillate by pinching head and removing tail with two sides slightly higher than the middle by 1-2 cm. Or adding 0.1% amylase, 0.2% saccharifying enzyme and 0.01% acid protease after sorghum is steamed to bloom and break skin, saccharifying for 24h in a stacking saccharifying box, adding 2-5% pure bran koji prepared from pichia pastoris M3, sealing and fermenting for 7-14d, steaming and distilling, and collecting fraction to obtain the wine for blending.
Blending white spirit: the wine for blending can be blended with other wine samples of raw wine to replace essence and spice, so that the quality of the wine can be improved to meet the pursuit of consumers on taste, and the characteristics before and after blending are shown in the following table 2.
Table 2 post-ticking scoring table
Example 5 analysis of Strain M3 Metabolic products
Scraping Pichia pastoris in a ring of inclined plane, culturing at 30deg.C in liquid yeast extract peptone glucose culture medium (YEPD) at 180r/min for 16h, and inoculating into sorghum juice culture medium to ensure that the number of cells after inoculation is 10 6 -10 7 Culturing at 30deg.C and 180r/min for 18 hr to stationary phase, standing at the same temperature for 4d, and extracting flavor compound by headspace-solid phase microextraction method in combination with GC-MS detection.
TABLE 3 determination of volatile component content (fraction) using HS-SPME method
Note that: the extraction rate and other problems should be considered when the HS-SPME method is used for extraction, and all volatile products of the M3 strain are not contained.
From table 3, it can be seen that the metabolites of strain M3 contain esters, acids and alcohols which are common in wine, and are suitable for pure brewing of wine to achieve the aim of blending and improving the quality of wine, or pure yeast is prepared for fermenting and improving the quality of wine with traditional yeast.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. 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 (7)
1. A high tolerance, low isoamyl alcohol producing pichia kudriavzevii, characterized in that: the Pichia is Pichia kudriavzeviiPichia kudriavzevii) M3, deposited in China center for type culture Collection, with a deposit number of: cctccc NO: m2022254.
2. The pichia kudriavzevii according to claim 1, wherein: the pichia pastoris colony is round, has sawtooth-shaped edge and milky white appearance, and is dried; the plant is in an elliptic or prolate form under a microscope, and the propagation mode is bud.
3. The use of pichia pastoris according to any one of claims 1 to 2 in the field of wine brewing and/or in yeast preparations.
4. A use according to claim 3, characterized in that: the brewing is white wine brewing or yellow wine brewing.
5. A use according to claim 3, characterized in that: when the pichia pastoris is specifically applied in the brewing field, the pichia pastoris is applied to any step in the brewing process and comprises mixing with starter propagation raw materials in the starter propagation process; or mixing with saccharified and gelatinized brewing materials; or mixing with fermented grains during middle and late stages of fermentation.
6. A fortified bran koji characterized in that: the bran koji contains the pichia pastoris according to any one of claims 1 to 2.
7. A fortified bran koji according to claim 6, wherein: the wheat bran is taken as raw material, water is added, the mixture is uniformly mixed and sterilized, and then the pichia pastoris is scattered until the cell number is 10 7 -10 8 And then inoculating the pichia pastoris to bran according to the proportion of 6-8wt% for culturing, and drying to obtain the pichia pastoris reinforced bran koji.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210590776.8A CN114836332B (en) | 2022-05-27 | 2022-05-27 | Pichia kudriavzevii with high tolerance and low isoamyl alcohol yield and application thereof |
| CN202310951335.0A CN116925871A (en) | 2022-05-27 | 2022-05-27 | Application of high-tolerance low-isoamyl alcohol yield pichia kudriavzevii in brewing and blending white spirit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210590776.8A CN114836332B (en) | 2022-05-27 | 2022-05-27 | Pichia kudriavzevii with high tolerance and low isoamyl alcohol yield and application thereof |
Related Child Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310951335.0A Division CN116925871A (en) | 2022-05-27 | 2022-05-27 | Application of high-tolerance low-isoamyl alcohol yield pichia kudriavzevii in brewing and blending white spirit |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114836332A CN114836332A (en) | 2022-08-02 |
| CN114836332B true CN114836332B (en) | 2023-10-20 |
Family
ID=82572529
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210590776.8A Active CN114836332B (en) | 2022-05-27 | 2022-05-27 | Pichia kudriavzevii with high tolerance and low isoamyl alcohol yield and application thereof |
| CN202310951335.0A Pending CN116925871A (en) | 2022-05-27 | 2022-05-27 | Application of high-tolerance low-isoamyl alcohol yield pichia kudriavzevii in brewing and blending white spirit |
Family Applications After (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310951335.0A Pending CN116925871A (en) | 2022-05-27 | 2022-05-27 | Application of high-tolerance low-isoamyl alcohol yield pichia kudriavzevii in brewing and blending white spirit |
Country Status (1)
| Country | Link |
|---|---|
| CN (2) | CN114836332B (en) |
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 |
|---|
| Distribution and function of dominant yeast species in the fermentation of strong‐flavor baijiu;You Ling等;World Journal of Microbiology and Biotechnology;第37卷;文献号26，第1-12页 * |
| Isolation, identification, and kinetic and thermodynamic characterization of a Pichia kudriavzevii yeast strain capable of fermentation;Leonel Nieto-Sarabia等;Food and Bioproducts Processing;第131卷;第109-124页 * |
| 酱香型白酒酒糟中生香酵母的筛选及鉴定;蔡雪梅等;中国酿造;第36卷(第7期);第42-47页 * |
| 酵母对浓香型白酒糟醅中高级醇生成的影响;游玲等;食品与发酵工业;第42卷(第2期);第23-28页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN116925871A (en) | 2023-10-24 |
| CN114836332A (en) | 2022-08-02 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP3214168B1 (en) | Saccharomyces cerevisiae capable of being co-fermented by multiple carbon sources and application thereof | |
| CN108118002B (en) | Acremonium ramosum and application thereof | |
| CN112662575B (en) | Saccharomycetes capulae with high protease activity and high liquor yield, and composition and application thereof | |
| CN102102084A (en) | Issatchenkia orientalis and composition and application thereof | |
| CN108018218A (en) | One plant height produces ethyl acetate yeast strain and its cultural method and application | |
| CN102586125A (en) | Highly ester producing issatchenkia orientalis yeast and composition as well as application thereof | |
| CN109439556B (en) | Mulberry fruit wine saccharomyces cerevisiae and application thereof | |
| CN111621428B (en) | Salt-tolerant rhodotorula mucilaginosa strain for high yield of phenethyl alcohol and application thereof | |
| CN106591160A (en) | Compound Xiaoqu and Xiaoqu Baijiu production method | |
| CN114574375B (en) | Saccharomyces cerevisiae, starter and application thereof in preparation of fermented food | |
| CN110317734B (en) | Monascus with high yields of saccharifying enzyme, esterifying enzyme and protease and separation culture method and application thereof | |
| CN111925951A (en) | Saccharomyces cerevisiae, microbial inoculum and application thereof, white spirit and yellow wine and brewing method thereof | |
| CN111484941A (en) | Saccharomyces cerevisiae with low fusel oil yield and application thereof | |
| CN112322509B (en) | Candida parapsilosis with low temperature resistance and high alcohol yield, and composition and application thereof | |
| CN114606152B (en) | Bacillus bailii, microbial agent and application thereof | |
| CN108165501B (en) | Saccharomyces cerevisiae strain and culture method and application thereof | |
| CN103789219B (en) | Saccharomyces cerevisiae for brewing white spirit | |
| CN114836332B (en) | Pichia kudriavzevii with high tolerance and low isoamyl alcohol yield and application thereof | |
| CN113528361B (en) | Saccharomyces cerevisiae suitable for brewing rice wine by liquefaction method and application thereof | |
| CN114621880A (en) | Abnormal yeast Weikehan for producing ester and application of abnormal yeast Weikehan in white spirit Daqu | |
| CN109666594B (en) | Method for intensively producing Shanxi mature vinegar by utilizing excellent native high-alcohol-yield saccharomyces cerevisiae and Artemisia annua Candida | |
| CN113773977A (en) | Yeast strain with low ethanol yield and high fragrance yield and application thereof | |
| CN113502233A (en) | Meiji meiqi yeast and application thereof in wine brewing | |
| CN109198578A (en) | A method of high concentration esters blending liquor is prepared using soybean-flavor liquor by-product | |
| CN114763517B (en) | High-temperature resistant saccharomyces cerevisiae and high-temperature fermentation process development of saccharomyces cerevisiae in fermented food |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |