WO2016065698A1 - 一株能以多碳源共发酵的酿酒酵母及其应用 - Google Patents

一株能以多碳源共发酵的酿酒酵母及其应用 Download PDF

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WO2016065698A1
WO2016065698A1 PCT/CN2014/093260 CN2014093260W WO2016065698A1 WO 2016065698 A1 WO2016065698 A1 WO 2016065698A1 CN 2014093260 W CN2014093260 W CN 2014093260W WO 2016065698 A1 WO2016065698 A1 WO 2016065698A1
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saccharomyces cerevisiae
ethyl
alcohol
acid
production
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French (fr)
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徐岩
吴群
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江南大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • C12N1/18—Baker's yeast; Brewer's yeast
    • C12N1/185—Saccharomyces isolates
    • 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
    • C12R2001/85—Saccharomyces
    • C12R2001/865—Saccharomyces cerevisiae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12C—BEER; PREPARATION OF BEER BY FERMENTATION; PREPARATION OF MALT FOR MAKING BEER; PREPARATION OF HOPS FOR MAKING BEER
    • C12C12/00—Processes specially adapted for making special kinds of beer
    • C12C12/002—Processes specially adapted for making special kinds of beer using special microorganisms
    • C12C12/006—Yeasts
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage
    • C12P7/08—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate
    • C12P7/10—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate substrate containing cellulosic material
    • 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

Definitions

  • the invention relates to a brewing yeast capable of co-fermenting with a multi-carbon source and an application thereof, and belongs to the technical field of microbes and the field of fermentation engineering technology.
  • Saccharomyces cerevisiae can use sugar to produce ethanol and has been used in industrial brewing for thousands of years, including the production of various types of beverages such as brandy, whiskey, vodka, gin, rum, Chinese white wine, wine. , rice wine, beer, etc.
  • beverages such as brandy, whiskey, vodka, gin, rum, Chinese white wine, wine. , rice wine, beer, etc.
  • humans began to use fuel yeast to produce fuel ethanol. Therefore, Saccharomyces cerevisiae is extremely important for the development of human society.
  • S. cerevisiae has the following three problems in production applications:
  • sugars such as lignocellulosic biomass (such as crop straw, chaff, bran, bagasse, forestry, forestry processing waste, grass, etc.), hydrolysis (using After acid or enzymatic method, glucose, xylose, arabinose, galactose and other oligosaccharides can be formed; after hydrolysis of starchy raw materials, glucose, maltose, maltotriose, isomaltose, panose and other oligosaccharides can be produced. And other ingredients.
  • This sugar diversity is not conducive to the fermentation of Saccharomyces cerevisiae.
  • Saccharomyces cerevisiae uses glucose to have a glucose effect, that is, when other sugars coexist with glucose, Saccharomyces cerevisiae can only use glucose. Only when the glucose is used up can other sugars be used. Secondly, Saccharomyces cerevisiae can only be used.
  • the use of glucose, maltose, fructose and other sugars, the above characteristics greatly limit the use of various sugars by Saccharomyces cerevisiae, not only reduce the wine production rate, but also reduce the utilization efficiency of carbon sources, and reduce the utilization of resources, resulting in Great waste of resources.
  • yeasts with multi-carbon source utilization capabilities have carried out a lot of work, including searching for yeasts with multi-carbon source utilization capabilities.
  • wild yeast does not have the ability to utilize multiple carbon sources. Therefore, genetic engineering methods are currently used to construct yeasts using multiple carbon sources. For example, protoplast fusion, exogenous gene introduction or key gene knockout. It has been reported that Saccharomyces cerevisiae recombined by genetic engineering technology can co-ferment glucose, xylose, mannose, arabinose and the like. However, due to the limited gene introduction of genetic engineering bacteria, the types of sugars used are still limited.
  • Saccharomyces cerevisiae is a single diploid coexisting strain
  • the unstable function of the strain is the biggest obstacle to the use of genetically engineered bacteria; and the method of genetic modification is in the beverage.
  • the use in wine production is not recognized.
  • Saccharomyces cerevisiae can only produce ethanol with higher tolerance of ethanol, and in the winemaking process, in addition to the brewing process, It is required that Saccharomyces cerevisiae can withstand high concentrations of ethanol, and it also needs to have the ability to withstand acidity and high temperature. Therefore, only Saccharomyces cerevisiae has the ability to withstand the above stress conditions in order to exert efficient fermentation capacity. However, currently used Saccharomyces cerevisiae can only tolerate 8% ethanol, 37 ° C temperature, pH 3.0.
  • Saccharomyces cerevisiae have high-efficiency fermentation ability, but also it has a good flavor metabolism ability, so that the beverage wine has more flavor composition.
  • the invention provides a brewing yeast capable of co-fermenting with a multi-carbon source and an application thereof, the yeast It is selected from the brewing environment of Chinese sauce-flavor liquor. It has the characteristics of multi-carbon source utilization, high temperature tolerance, high acid tolerance, high ethanol tolerance and rich flavor metabolism. It can be applied to beverage brewing and fuel ethanol. Production and other fields.
  • a first object of the present invention is to provide a strain of Saccharomyces cerevisiae , which is deposited on October 10, 2014 in the China Center for Type Culture Collection, and the deposit address is Wuchang, Wuhan, Hubei Province. Shan Wuhan University, the preservation number is CCTCC M 2014463.
  • Saccharomyces cerevisiae is screened from the brewing environment of the sauce-flavor liquor, and identified as Saccharomyces cerevisiae by 26srDNA sequence, 26s rDNA
  • the sequence is shown in SEQ ID NO. 1, and the sequence is 99% similar to the existing S. cerevisiae sequence after blast alignment on NCBI.
  • the Saccharomyces cerevisiae grows on YPD medium, the colonies are white, round, with neat edges and smooth surface.
  • Saccharomyces cerevisiae The morphology of the Saccharomyces cerevisiae under an electron microscope is shown in Figure 1, and it can be seen The cells of Saccharomyces cerevisiae are spherical or ellipsoidal, and the top buds indicate that the method of reproduction is budding reproduction.
  • Saccharomyces cerevisiae can utilize any of the following carbon sources: glucose, galactose, xylose, psicose, sucrose, maltose, melibiose, sucrose, trehalose, raffinose and other oligosaccharides.
  • Saccharomyces cerevisiae can also utilize any two or more of the following mixed carbon sources: glucose, galactose, xylose, psicose, sucrose, maltose, melibiose, pine disaccharide, trehalose, raffinose And other oligosaccharides.
  • the Saccharomyces cerevisiae can produce ethanol using one or more of the above mixed carbon sources.
  • Saccharomyces cerevisiae is resistant to high temperatures (44 °C), acid (pH 2.0) and alcohol resistant (16%).
  • the Saccharomyces cerevisiae is capable of metabolizing a variety of flavoring substances, including acid hexanoic acid, caprylic acid, citric acid, 9- Terpene acid, alcoholic alcohol, isobutanol, isoamyl alcohol, butanol, hexanol, -2-decene-1-ol, 3-methyl-1-hexanol, 1-octanol, decyl alcohol 3-methyl-1- Ethyl alcohol, phenylethyl alcohol, lauryl alcohol, trans-orange alcohol, farnesol, ethyl acetate of ethyl ester, isoamyl acetate, ethyl hexanoate, ethyl 4-hexenoate, ethyl octanoate, Ethyl citrate, acetic acid-2- Phenylethyl ester, phenylacetic acid-2-phenylethyl ester,
  • a second object of the present invention is to provide a method of application of the Saccharomyces cerevisiae.
  • the application method is to directly use Saccharomyces cerevisiae to produce beverage wine, and use a liquid or solid matrix composed of polysaccharides after saccharification of cereal raw materials as a substrate, and a fruit-based brewing raw material to ferment beverages.
  • the beverage includes: brandy, whiskey, vodka, gin, rum, Chinese liquor, tequila, wine, rice wine, beer, shochu, sake, and the like.
  • whisky, gin, vodka, tequila, Chinese liquor, rice wine, beer, shochu, sake these wines are mainly made from cereals (vegetable) raw materials, before brewing raw materials (wheat, corn, rye, barley, potato) , sorghum, pea, sweet potato, rice, glutinous rice) need to be saccharified
  • the Saccharomyces cerevisiae of the present invention can be used as a substrate by using a liquid or solid substrate containing a polysaccharide obtained by saccharification, or a fruit-based brewing raw material as a substrate, and fermenting a beverage.
  • Alcohol can increase carbon source utilization and alcohol production.
  • the application method may further be an enhanced application of the Saccharomyces cerevisiae in the production of beverage wine, and inoculation in a production system by using a liquid microbial agent or a solid microbial agent to improve the utilization rate of the carbon source and the rate of wine production.
  • the liquid microbial preparation method inoculating the activated seed liquid by 1 ⁇ 5% (w/w) (ie per 100g)
  • the liquid medium inoculated seed solution (1-5 g) was inoculated into a sterilized liquid medium and cultured at 25 to 35 ° C for 24 h to 72 h.
  • the liquid medium in one embodiment of the present invention, is a sorghum extract culture medium, and the preparation method is as follows: 20 to 200 g of the sorghum sample is pulverized, and 1 to 4 times of water is added in mL/g, and cooking is performed 1- 5h, paste, after cooling, add 10 ⁇ 50 units / g of glucoamylase, kept at 40 ⁇ 100 ° C for 2 ⁇ 10h, filtered, centrifuged filtrate, sugar content of 10 ⁇ 15 o Bx.
  • the solid microbial agent is prepared by inoculating the activated seed liquid (primary seed) in sorghum medium (inoculation amount is 1 to 5%, w/w) ), solid-state fermentation in the environment of 25 ⁇ 35 ° C for 32 ⁇ 72 h, into a solid bacteria preparation.
  • the application method may also be the use of Saccharomyces cerevisiae CCTCC M 2014463 for the production of fuel ethanol.
  • the Saccharomyces cerevisiae is inoculated into a liquid or solid matrix containing a polysaccharide composed of a starchy raw material and a cellulose raw material, and fermented to produce fuel ethanol to improve the utilization rate of carbon source and the yield of ethanol.
  • the starchy material comprises various cereal (plant) materials.
  • the cellulose raw material includes lignocellulosic biomass (such as crop straw, chaff, bran, bagasse, forestry, forestry processing waste, grass, etc.).
  • lignocellulosic biomass such as crop straw, chaff, bran, bagasse, forestry, forestry processing waste, grass, etc.
  • the brewing yeast of the invention is screened from the brewing environment of Chinese sauce-flavor liquor, and the brewing liquor of the sauce-flavor liquor belongs to the simultaneous saccharification and fermentation process, so that the system contains various carbon sources, and the system has high temperature and high acidity.
  • the characteristics of high ethanol concentration, etc. give the yeast a multi-carbon source utilization, and are resistant to high temperature, high acid and high ethanol concentration, and the Saccharomyces cerevisiae has rich flavor metabolism ability.
  • the yeast of the present invention is the first discovered Saccharomyces cerevisiae having the above-mentioned excellent brewing characteristics, so that the Saccharomyces cerevisiae has very important application potential and value for both beverage brewing and fuel ethanol brewing.
  • Saccharomyces cerevisiae CCTCC M 2014463 classified as Saccharomyces cerevisiae , was deposited with the China Center for Type Culture Collection (CCTCC) on October 10, 2014, and the deposit address is Wuhan University, and the accession number is CCTCC M 2014463.
  • FIG. 1 Electron micrograph of S. cerevisiae CCTCC M 2014463;
  • Figure 2 Saccharomyces Cerevisiae CCTCC M 2014463 and Saccharomyces Cerevisiae Model S288C Comparison of multi-carbon source utilization ability, * represents a significant difference (one-way ANOVA analysis P ⁇ 0.05);
  • FIG. 3 Saccharomyces Cerevisiae CCTCC M 2014463 and Saccharomyces Cerevisiae Model S288C Tolerance Temperature, pH Comparison of ethanol stress, * represents a significant difference (one-way ANOVA analysis P ⁇ 0.05);
  • Figure 4 Saccharomyces cerevisiae CCTCC M 2014463 Flavor metabolite GCMS spectrum.
  • Figure 5 Fermentation of strains in medium with glucose as a carbon source, where the solid icon represents S. cerevisiae CCTCC M 2014463, the hollow icon represents the control strain Saccharomyces cerevisiae S288c;
  • FIG. 6 Fermentation of strains in medium with glucose and maltose as mixed carbon sources, where the solid icon represents Saccharomyces Cerevisiae CCTCC M 2014463, the hollow icon represents the control strain Saccharomyces cerevisiae S288c;
  • Figure 7 Fermentation of strains in medium with glucose and galactose as mixed carbon sources, where the solid icon represents Saccharomyces Cerevisiae CCTCC M 2014463, the hollow icon represents the control strain Saccharomyces cerevisiae S288c.
  • Flavor substance detection volatile products were analyzed by headspace solid phase microextraction (HS-SPME) and gas chromatography-mass spectrometry (GC-MS). 8 mL samples were taken and placed in a headspace vial containing 3 g of NaCl. Among them, 10 ⁇ L of 4-methyl-2-pentanol having a concentration of 42.60 mg ⁇ L -1 was added as an internal standard. The headspace flask was extracted at 50 ° C for 45 min. GC-MS analysis was performed after extraction.
  • HS-SPME headspace solid phase microextraction
  • GC-MS gas chromatography-mass spectrometry
  • CCTCC M 2014463 can also utilize glucose, galactose, xylose and psicose, sucrose, maltose, Honey disaccharide, pine disaccharide, trehalose, raffinose and other oligosaccharides. Among them, the use of galactose and maltose has been verified by shake flask experiments.
  • the OD 600 was determined by inoculating 2% in the YPD liquid medium at 30 ° C, 37 ° C, and 44 ° C for 48 h.
  • Ethanol tolerance After the strain is activated, according to the inoculation amount of 2%, YPD liquid medium is used as the basic medium, adding 0% and 2% respectively. 4%, 6%, 8%, 10%, 12%, 14%, 16% (v/v) were determined by static culture at 30 °C for 48 h and OD600 was determined.
  • Acid tolerance After the strain is activated, according to the inoculum amount of 2%, the YPD liquid medium is used as the base medium, and the pH is adjusted to 5 with lactic acid. 4.5, 4, 3.5, 3, 2.5, 2 OD600 was determined after standing at 30 °C for 48 h.
  • Saccharomyces Cerevisiae CCTCC M 2014463 is capable of metabolizing multiple flavors. Inoculate it in sorghum juice medium at 30 ° C The fermented plug was prepared for static culture under the conditions, and the flavor metabolites in the fermentation broth were detected by GC-MS for 5 days.
  • Saccharomyces cerevisiae CCTCC M 2014463 is capable of metabolizing: acids (hexanoic acid, caprylic acid, citric acid, 9- Terpene acid), alcohol (ethanol, isobutanol, isoamyl alcohol, butanol, hexanol, -2-decene-1-ol, 3-methyl-1-hexanol, 1-octanol, hydrazine Alcohol, 3-methyl-1- Hexanol, phenylethyl alcohol, lauryl alcohol, trans- neroliol, farnesol, esters (ethyl acetate, isoamyl acetate, ethyl hexanoate, ethyl 4-hexenoate, ethyl octanoate) , ethyl citrate, acetic acid-2- Phenylethyl ester, phenylacetic acid-2-phenyleth
  • the sorghum juice medium can also be replaced by YNB medium.
  • S. cerevisiae under mixed carbon source medium containing 1% glucose and 1% galactose CCTCC M 2014463 has twice the biomass and ethanol production of S288c. From the sugar consumption curve, it can be seen that Saccharomyces Cerevisiae CCTCC M 2014463 Both glucose and maltose can be utilized.
  • Saccharomyces cerevisiae are not significantly different from those of S288c in a single carbon source medium containing only glucose; however, it is cultured in a mixed carbon source containing maltose and glucose or galactose and glucose.
  • the Saccharomyces cerevisiae strain has effectively utilized maltose and galactose in the early stage of its growth phase, and its growth curve has no secondary growth phenomenon.
  • the final ethanol production of Saccharomyces cerevisiae is twice that of Saccharomyces cerevisiae S288c, which also indicates that CCTCC M 2014463 in mixed carbon source medium has high carbon source utilization and high ethanol production.
  • a single colony of CCTCC M 2014463 and S288c was picked and inoculated into YPD liquid medium, and cultured at 200 rpm, 30 ° C for 16-20 h to obtain a seed liquid.
  • the flask was incubated at 200 rpm and 30 ° C for 48 h.

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Abstract

提供了一株能以多碳源共发酵的酿酒酵母菌株CCTCC M 2014463及其应用。该菌株可以利用葡萄糖、半乳糖、木糖和阿洛酮糖、蔗糖、麦芽糖、蜜二糖、松二糖、海藻糖、棉子糖，并且可以同时利用葡萄糖和以上的其它糖。同时，该酵母能够耐受高温、高乙醇和高酸，并产生酸类、醇类、酯类和苯乙醛、苯乙酮4-乙烯基愈疮木酚等多种风味物质。

Description

一株能以多碳源共发酵的酿酒酵母及其应用 说明书
技术领域
本发明涉及一株能以多碳源共发酵的酿酒酵母及其应用，属于微生物技术领域和发酵工程技术领域。
背景技术
酿酒酵母 Saccharomyces cerevisiae 可以利用糖来产生乙醇，被应用于工业酿造已有几千年的历史，包括各类饮料酒的生产，如白兰地、威士忌、伏特加、金酒、朗姆酒、中国白酒、葡萄酒、黄酒、啤酒等。同时，随着石油资源的枯竭，以及燃料乙醇生产资源的可再生性，人类开始利用酿酒酵母生产燃料乙醇。因此，酿酒酵母对人类社会的发展是极其重要的。然而，酿酒酵母在生产应用中存在以下三个方面的问题：
第一、不论是对于采用谷物类为原料的饮料酒酿造，还是以纤维素和淀粉为原料的燃料乙醇的生产，由于酿酒酵母不能直接利用纤维素和淀粉等聚合物原料，必须首先将上述多糖降解为单糖才能被酵母所使用。但是，由于纤维素和淀粉降解产生糖种类具有多样性，如木质纤维素类生物质（如农作物秸秆、谷壳、麸皮、蔗渣、林木、林业加工废弃物、草类等），水解（用酸法或酶法）后可生成葡萄糖、木糖、阿拉伯糖、半乳糖以及其它寡糖等成分；淀粉质原料水解后可生产葡萄糖、麦芽糖、麦芽三糖、异麦芽糖、潘糖及其它寡糖等成分。这种糖的多样性并不利于酿酒酵母的发酵。首先，酿酒酵母利用糖存在葡萄糖效应，也就是当其它糖与葡萄糖共存时，酿酒酵母只能利用葡萄糖，只有当葡萄糖利用完之后，才能开始利用其它可利用的糖；其次，酿酒酵母一般只能利用葡萄糖，麦芽糖、果糖等少数糖类，以上特点大大限制了酿酒酵母对各种糖类的使用，不仅使得产酒率降低，而且造成碳源利用效率的降低，以及资源利用率的降低，造成极大的资源浪费。
为解决以上问题，目前研究者开展大量工作，包括寻找具有多碳源利用能力的酵母等。但是，一般野生酵母都不具备多碳源利用的能力，因此，目前主要采用基因工程方法构建多碳源利用的酵母。例如原生质体融合、外源基因导入或关键基因敲除等方法。有研究报道，采用基因工程技术重组后的酿酒酵母可共发酵葡萄糖、木糖、甘露糖、阿拉伯糖等。但是由于基因工程菌导入基因有限，利用糖的种类仍然有限；同时，由于酿酒酵母为单双倍体共存的菌株，菌株功能不稳定是基因工程菌使用的最大障碍；并且基因改造的方法在饮料酒酿造生产中使用是不被认可的。
第二、在酿酒过程及燃料乙醇发酵过程中，酿造环境极其复杂，对于燃料乙醇的生产，酿酒酵母只有具有高浓度乙醇的耐受能力才能更有效的发酵生产乙醇；而在酿酒过程中，除了要求酿酒酵母能够耐受高浓度乙醇外，还需要其具有耐受酸性、以及高温的能力。因此，只有酿酒酵母具有耐受以上胁迫条件等能力，才能发挥高效的发酵能力。然而，目前常规使用的酿酒酵母只能耐受 8% 乙醇、 37°C 温度、 pH 3.0 。
第三、对于饮料酒的酿造，不仅要求酿酒酵母具有高效的发酵能力，还要求其具有良好的风味代谢能力，使得饮料酒具有更多样的风味组成。
以上特点对饮料酒与燃料乙醇等产业中酿酒酵母菌种的要求提出了 3 个方面的要求，但是由于目前技术的有限，以及野生酵母自身存在的缺陷，仍然要求在菌种方面需要重要突破。
发明内容
本发明提供一株能以多碳源共发酵的酿酒酵母及其应用，该酵母 是从中国酱香型白酒酿造环境中筛选获得的，具有多碳源利用、耐受高温、耐高酸、耐高乙醇浓度以及丰富的风味代谢能力等特征，能够应用于饮料酒酿造、燃料乙醇生产等领域。
本发明的第一个目的是提供一株酿酒酵母 Saccharomyces cerevisiae ，其特征在于，所述酿酒酵母于 2014 年 10 月 10 日保藏于 中国典型培养物保藏中心，保藏地址为湖北省武汉市武昌珞珈山武汉大学，保藏编号为 CCTCC M 2014463 。
所述酿酒酵母筛选自酱香型白酒酿造环境，经 26srDNA 序列鉴定为酿酒酵母，其 26s rDNA 序列如 SEQ ID NO.1 所示，该序列在 NCBI 上 blast 比对后，与现有酿酒酵母序列相似度是 99% 。
所述酿酒酵母在 YPD 培养基上生长，菌落为白色，圆形，边缘整齐，表面光滑。
所述酿酒酵母在电子显微镜下的形态如图 1 所示，可以看出 酿酒酵母的细胞为球形或者椭球形，顶端芽痕说明其繁殖 的方法为 出芽生殖 。
所述酿酒酵母能利用以下任意一种碳源：葡萄糖、半乳糖、木糖、阿洛酮糖、蔗糖、麦芽糖、蜜二糖、松二糖、海藻糖、棉子糖及其它寡糖。
所述酿酒酵母还能利用以下任意两种或者多种的混合碳源：葡萄糖、半乳糖、木糖、阿洛酮糖、蔗糖、麦芽糖、蜜二糖、松二糖、海藻糖、棉子糖及其它寡糖。
所述酿酒酵母能利用上述一种或者两种以上的混合碳源，来生产乙醇。
所述酿酒酵母能够耐受高温（ 44 ℃），耐酸（ pH 2.0 ），耐酒精（ 16% ）。
所述酿酒酵母能够代谢多种风味物质，包括酸类的己酸、辛酸、壬酸、 9- 癸烯酸，醇类的乙醇、异丁醇、异戊醇、丁醇、己醇、 -2- 壬烯 -1- 醇、 3- 甲基 -1- 己醇、 1- 辛醇、壬醇、 3- 甲基 -1- 己醇、苯乙醇、月桂醇、反式 - 橙花叔醇、法呢醇，酯类的乙酸乙酯、乙酸异戊酯、己酸乙酯、 4 - 己烯酸乙酯、辛酸乙酯、癸酸乙酯、乙酸 -2- 苯基乙酯、苯乙酸 -2- 苯基乙酯、异己基 -2- 苯乙酯、 2 ， 4- 己二烯酸乙酯，以及苯乙醛、苯乙酮 4- 乙烯基愈疮木酚。
本发明的第二个目的是提供所述酿酒酵母的应用方法。
所述应用方法，是将酿酒酵母直接用于生产饮料酒，以谷物类原料经过糖化后的含有多糖组成的液体或固体基质为底物，以及水果类酿造原料，发酵生产饮料酒。
所述饮料酒包括：白兰地、威士忌、伏特加、金酒、朗姆酒、中国白酒、特基拉、葡萄酒、黄酒、啤酒、烧酒、清酒等。其中威士忌、劲酒、伏特加、特基拉、中国白酒、黄酒、啤酒、烧酒、清酒，这些酒的酿造主要使用谷物（植物）类原料，在酿造之前原料（小麦、玉米、裸麦、大麦、马铃薯、高粱、豌豆、甘薯、大米、糯米）需要经过糖化，本发明的酿酒酵母能够以糖化后所得的含有多糖组成的液体或固体基质为底物，或者水果类酿造原料为底物，发酵生产饮料酒，能够提高碳源利用率和产酒率。
所述应用方法，还可以是所述酿酒酵母在饮料酒生产中的强化应用，采用液体菌剂或固体菌剂的方式接种于生产体系中，提高碳源利用率和产酒率。
所述液体菌剂制作方式：将活化的种子液按 1~5% （ w/w ）的接种量（即每 100g 液态培养基接种种子液 1-5g ）接种于灭菌的液态培养基中， 25~35℃ 培养 24 h~72 h 。
所述液态培养基，在本发明的一种实施方式中为高粱提取物培养基，制作方法为： 20 ～ 200g 高粱样品经粉碎后，加 mL/g 计 1 ～ 4 倍的水，蒸煮 1-5h ，呈糊状，冷却后加入 10 ～ 50 单位 /g 的糖化酶，于 40 ～ 100℃ 保持 2 ～ 10h ，过滤、离心所得滤液，糖度为 10~15oBx 。
所述固体菌剂的制作方式：将活化的种子液接种（一级种子）高粱培养基中（接种量为 1~5% ， w/w ），在 25~35℃ 环境中固态发酵 32~72 h ，制成固态菌制剂。
所述应用方法，还可以是将酿酒酵母 CCTCC M 2014463 用于燃料乙醇的生产， 是将所述酿酒酵母接种于以淀粉质原料和纤维素原料经过糖化后的含有多糖组成的液体或固体基质中，发酵生产燃料乙醇，提高碳源利用率和乙醇得率。
所述淀粉质原料，包括各种谷物（植物）原料。
所述纤维素原料，包括木质纤维素类生物质（如农作物秸秆、谷壳、麸皮、蔗渣、林木、林业加工废弃物、草类等）。
本发明的酿酒酵母是从中国酱香型白酒酿造环境中筛选获得的，由于酱香型白酒酿造属于同步糖化发酵过程，使得该体系中含有多样不同碳源组成，并且该系统具有高温、高酸、高乙醇浓度等特征，赋予了该酵母具有多碳源利用、以及耐受高温、高酸及高乙醇浓度的特征，并且该酿酒酵母具有丰富的风味代谢能力。本发明的酵母是首次发现的同时具有上述优良的酿造特征的酿酒酵母，使得该酿酒酵母不论对于饮料酒酿造还是对于燃料乙醇酿造都具有非常重要的应用潜力与价值。
生物材料保藏
酿酒酵母 Saccharomyces cerevisiae CCTCC M 2014463 ，分类学命名为 Saccharomyces cerevisiae ，于 2014 年 10 月 10 日保藏于 中国典型培养物保藏中心（ CCTCC ） ，保藏地址为武汉大学，保藏编号为 CCTCC M 2014463 。
附图说明
图 1 ：酿酒酵母 CCTCC M 2014463 的电镜照片；
图 2 ：酿酒酵母 CCTCC M 2014463 与酿酒酵母模式株 S288C 多碳源利用能力的比较，图中 * 代表显著性差异（ one-way ANOVA 分析 P<0.05 ）；
图 3 ：酿酒酵母 CCTCC M 2014463 与酿酒酵母模式株 S288C 耐受温度、 pH 、乙醇胁迫的比较，图中 * 代表显著性差异（ one-way ANOVA 分析 P<0.05 ）；
图 4 ：酿酒酵母 CCTCC M 2014463 风味代谢产物 GCMS 谱图。
图 5 ：菌株于以葡萄糖为碳源的培养基中的发酵情况，其中 实心图标代表酿酒酵母 CCTCC M 2014463 、空心图标代表对照菌株酿酒酵母 S288c ；
图 6 ：菌株于以葡萄糖、麦芽糖为混合碳源的培养基中的发酵情况，其中 实心图标代表酿酒酵母 CCTCC M 2014463 、空心图标代表对照菌株酿酒酵母 S288c ；
图 7 ： 菌株于以葡萄糖、半乳糖为混合碳源的培养基中的发酵情况，其中 实心图标代表酿酒酵母 CCTCC M 2014463 、空心图标代表对照菌株酿酒酵母 S288c 。
具体实施方式
风味物质检测 ：运用顶空固相微萃取技术 (HS-SPME) 和气相色谱 - 质谱 (GC-MS) 方法对挥发性产物进行分析，取 8mL 样品，放入装有 3gNaCl 的顶空进样瓶中，加入 10µL 浓度为 42.60 mg·L-1 的 4- 甲基 -2- 戊醇为内标。将顶空瓶于 50 ° C 恒温萃取 45min 。萃取完后进行 GC-MS 分析。
实施例 1 酿酒酵母 CCTCC M 2014463 碳源同化水平
采用 Biolog 碳源同化鉴定深孔板培养，挑取单菌落接种于 YPD 液体培养基， 200rpm ， 30°C 条件下培养 16-20h 后获得种子液。将种子液用生理盐水稀释至 OD600=0.05 后转接至 biolog YT 微孔鉴定版（购买于华粤行仪器公司），接种量 100μL ， 28°C 恒温培养 72h ，测定 OD590 和 OD730 。其中， OD590 、 OD730 大于 0.2 且小于 0.8 时，表示该碳源可以被利用但利用程度不高； OD590 、 OD730 大于 0.8 时，代表该碳源可以很好地利用。 biolog 做了 2 个平行，图上有 * 标记的含义表示经 one-way ANOVA 分析 P<0.05 。
结果如图 2 所示：相对于酿酒酵母模式株 Saccharomyces cerevisiae S288c 仅仅可以利用葡萄糖、蔗糖等常见碳源， CCTCC M 2014463 还可以利用葡萄糖、半乳糖、木糖和阿洛酮糖，蔗糖、麦芽糖、蜜二糖、松二糖、海藻糖，棉子糖及其它寡糖。其中，对于半乳糖和麦芽糖的利用已做摇瓶实验进行了验证。
实施例 2 酿酒酵母 CCTCC M 2014463 耐受胁迫条件能力
酿酒酵母 CCTCC M 2014463 与酿酒酵母模式菌株 Saccharomyces cerevisiae S288C 胁迫环境耐受能力的比较。
温度耐受性：菌株活化后，按接种量 2% ，以 YPD 液体培养基分别于 30 ℃、 37 ℃、 44 ℃静置培养 48h 后测定 OD600 。
乙醇耐受性：菌株活化后，按接种量 2% ，以 YPD 液体培养基为基础培养基，分别添加 0% 、 2% 、 4% 、 6% 、 8% 、 10% 、 12% 、 14% 、 16% （ v/v ）于 30 ℃静置培养 48h 后测定 OD600 。
酸耐受性：菌株活化后，按接种量 2% ，以 YPD 液体培养基为基础培养基，用乳酸调 pH 为 5 、 4.5 、 4 、 3.5 、 3 、 2.5 、 2 于 30 ℃静置培养 48h 后测定 OD600 。
结果如图 3 所示，由图 3 可以看出：酿酒酵母 CCTCC M 2014463 能够耐受能够耐受高温（ 44 ℃），耐酸（ pH 2.0 ），耐酒精（ 16 ％）。 CCTCC M 2014463 的耐酸能力强于对照菌株，在 pH 2 的条件下，对照菌株 OD 只能达到 0.2 ，几乎不生长，而本发明的酵母 OD 能达到 0.8 ，是 pH 5 条件下的 66.67% 。此外， CCTCC M 2014463 耐受乙醇的能力也强于对照菌株，其在 16% 酒精浓度下的最大生物量为对照菌株的 2 倍。
实施例 3 酿酒酵母 CCTCC M 2014463 风味代谢能力
酿酒酵母 CCTCC M 2014463 能够代谢多种风味物质。将其接种于高粱汁培养基，于 30°C 条件下配制发酵栓进行静置培养，发酵 5d 时间， 通过 GC-MS 检测发酵液中的风味代谢产物。
结果如图 4 所示，酿酒酵母 CCTCC M 2014463 能够代谢：酸类（己酸、辛酸、壬酸、 9- 癸烯酸）、醇类（乙醇、异丁醇、异戊醇、丁醇、己醇、 -2- 壬烯 -1- 醇、 3- 甲基 -1- 己醇、 1- 辛醇、壬醇、 3- 甲基 -1- 己醇、苯乙醇、月桂醇、反式 - 橙花叔醇、法呢醇）、酯类（乙酸乙酯、乙酸异戊酯、己酸乙酯、 4 - 己烯酸乙酯、辛酸乙酯、癸酸乙酯、乙酸 -2- 苯基乙酯、苯乙酸 -2- 苯基乙酯、异己基 -2- 苯乙酯、 2 ， 4- 己二烯酸乙酯）和苯乙醛、苯乙酮 4- 乙烯基愈疮木酚。
所述高粱汁培养基还可以被 YNB 培养基代替。
实施例 4 添加酿酒酵母 CCTCC M 2014463 提高碳源利用率和乙醇产量
挑取单菌落接种于 YPD 液体培养基， 200rpm ， 30°C 条件下培养 16-20h 后获得种子液。将种子液用生理盐水稀释至 OD600=1 转接至单一碳源或者混合碳源（总碳源含量为 2% ）培养基， 200rpm ， 30°C 条件下摇瓶培养 48h ，每隔 6h 取样一次， 测定 生物量及发酵液中乙醇产量和残糖，结果如图 5 至图 7 所示。
从图 6 可以看出：在含有 1% 葡萄糖和 1% 麦芽糖的混合碳源培养基条件下， 酿酒酵母 CCTCC M 2014463 的生物量和产乙醇量都达到对照菌株 S288c 的 2 倍。而从耗糖曲线可以看出酿酒酵母 CCTCC M 2014463 可以同时利用葡萄糖和半乳糖。
同理，从图 7 可以看出：在含有 1% 葡萄糖和 1% 半乳糖的混合碳源培养基条件下， 酿酒酵母 CCTCC M 2014463 的生物量和产乙醇量都达到 S288c 的 2 倍。而从耗糖曲线可以看出酿酒酵母 CCTCC M 2014463 可以同时利用葡萄糖和麦芽糖。
以上结果表明，在只含葡萄糖的单碳源培养基中，酿酒酵母的碳源利用、生长及产乙醇同 S288c 比较差异不明显；但是在含有麦芽糖和葡萄糖或者半乳糖和葡萄糖的混合碳源培养基中，该酿酒酵母菌株在其生长对数期初期已经有效利用麦芽糖以及半乳糖，其生长曲线没有二次生长现象，酿酒酵母的最终产乙醇量达到 Saccharomyces cerevisiae S288c 的 2 倍，这也说明在混合碳源培养基中 CCTCC M 2014463 的碳源利用率高、产乙醇量也高。
实施例 5 酿酒酵母 CCTCC M 2014463 的多碳源利用能力
挑取 CCTCC M 2014463 和 S288c 的单菌落接种于 YPD 液体培养基， 200rpm ， 30°C 条件下培养 16-20h 后获得种子液。将种子液用生理盐水稀释至 OD600=1 转接至碳源为 1% 蔗糖、 1% 木糖、 1% 蜜二糖的培养基中，于 200rpm ， 30°C 条件下摇瓶培养 48h ，每隔 6h 取样一次， 测定 生物量及发酵液中乙醇产量和残糖，结果发现 CCTCC M 2014463 在生长对数初期就能有效利用这三种糖，最终乙醇产量达到 Saccharomyces cerevisiae S288c 的 2.6 倍。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。

Claims (10)

  1. 一株酿酒酵母Saccharomyces cerevisiae，其特征在于，所述酿酒酵母于2014年10月10日保藏于中国 典型培养物保藏中心，保藏地址为湖北省武汉市武昌珞珈山武汉大学，保藏编号为CCTCC M 2014463。
  2. 根据权利要求1所述的酿酒酵母，其特征在于，所述酿酒酵母能利用以下任意一种、两种或者多种碳源 的混合：葡萄糖、半乳糖、木糖、阿洛酮糖、蔗糖、麦芽糖、蜜二糖、松二糖、海藻糖、棉子糖。
  3. 根据权利要求1所述的酿酒酵母，其特征在于，所述酿酒酵母能够耐受44℃温度，耐受pH 2.0的酸性环 境，耐受乙醇体积分数为16%的溶液。
  4. 根据权利要求1所述的酿酒酵母，其特征在于，所述酿酒酵母能够代谢多种风味物质，包括酸类的己酸 、辛酸、壬酸、9-癸烯酸，醇类的乙醇、异丁醇、异戊醇、丁醇、己醇、-2-壬烯-1-醇、3-甲基-1-己醇 、1-辛醇、壬醇、3-甲基-1-己醇、苯乙醇、月桂醇、反式-橙花叔醇、法呢醇，酯类的乙酸乙酯、乙酸异戊酯、己酸乙酯、4 -己烯酸乙酯、辛酸乙酯、癸酸乙酯、乙酸-2-苯基乙酯、苯乙酸-2-苯基乙酯、异己 基-2-苯乙酯、2，4-己二烯酸乙酯，以及苯乙醛、苯乙酮、4-乙烯基愈疮木酚。
  5. 权利要求1所述酿酒酵母在食品领域的应用。
  6. 权利要求1所述酿酒酵母在酿酒技术领域的应用。
  7. 根据权利要求6所述的应用，其特征在于，是将所述酿酒酵母直接用于生产饮料酒，以谷物类原料经过糖化后的含有多糖组成的液体或固体基质为底物，以及水果类酿造原料，发酵生产饮料酒，能够利用各种多糖组分，提高原料利用率和产物产率。
  8. 根据权利要求6所述的应用，其特征在于，是将所述酿酒酵母在饮料酒生产中的强化应用，采用液体菌 剂或固体菌剂的方式接种于生产体系中。
  9. 权利要求1所述酿酒酵母在燃料乙醇生产方面的应用。
  10. 根据权利要求9所述的应用，其特征在于，是将所述酿酒酵母接种于以淀粉质原料和纤维素原料经过 糖化后的含有多糖组成的液体或固体基质中，发酵生产燃料乙醇，能够利用各种多糖组分，提高原料利用 率和产物产率。
PCT/CN2014/093260 2014-10-30 2014-12-08 一株能以多碳源共发酵的酿酒酵母及其应用 WO2016065698A1 (zh)

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CN113186245A (zh) * 2020-01-14 2021-07-30 广东利世康低碳科技有限公司 一种耐受辣椒且同时利用葡萄糖和木糖发酵的酵母菌及其验证方法
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CN110628656A (zh) * 2019-09-03 2019-12-31 云南师范大学 一种酵母菌剂及其在酿酒中的应用
CN110628656B (zh) * 2019-09-03 2023-08-22 云南师范大学 一种酵母菌剂及其在酿酒中的应用
CN113186245A (zh) * 2020-01-14 2021-07-30 广东利世康低碳科技有限公司 一种耐受辣椒且同时利用葡萄糖和木糖发酵的酵母菌及其验证方法
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CN114149932A (zh) * 2021-11-08 2022-03-08 泸州老窖股份有限公司 酿酒酵母lj-2及其应用
WO2023140793A3 (en) * 2022-01-19 2023-10-26 National University Of Singapore Bioproduction of natural phenethyl acetate, phenylacetic acid, ethyl phenylacetate, and phenylethyl phenylacetate from renewable feedstock
CN117757650A (zh) * 2024-01-03 2024-03-26 泰山学院 一种酿酒酵母及其在生产低高级醇和/或高乙酸乙酯酒产品中的应用
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