Method for imparting resistance to yeast using polysaccharide

Abstract

To provide novel means capable of easily imparting alkali resistance and high-temperature resistance to yeast without the use of genetic engineering.SOLUTION: A means for imparting alkali resistance and / or high-temperature resistance to yeast includes the culturing of yeast in an aqueous solution containing polysaccharides with an anionic functional group.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for imparting resistance to yeast using a polysaccharide. [Background technology]

[0002] Microorganisms are a general term for tiny organisms that can generally only be observed under a microscope. Microorganisms include mold, yeast, bacteria, and algae. Microorganisms live everywhere on Earth, including in soil, the deep sea, hot springs, food, the human body, and the air. It is said that there are approximately 12 million species of organisms on Earth, of which approximately 3 million species (25%) are microorganisms.

[0003] Humans have used many microorganisms in industry since ancient times. Yeasts in particular are a type of extremely useful industrial microorganism that are used in a wide range of fields, including the production of food such as alcohol and bread, the production of useful substances such as pharmaceuticals and biofuels, and environmental remediation, such as for environmentally friendly detergents and the decomposition of environmental pollutants.

[0004] One of the limitations of yeast use is that it must be used under conditions in which the yeast can survive. For example, in terms of managing microorganisms contained in wastewater from food factories, disinfection using alkali is a simple method, but when disinfecting wastewater using alkali, all contained microorganisms are killed, which causes the problem of killing useful microorganisms that are sprayed into the wastewater for the purpose of decomposing environmental pollutants. In addition, since many species of yeast are not alkaline resistant, there is also the problem that in the production of bread and alcohol, the raw materials must be neutralized before adding yeast to them.

[0005] As a method for imparting resistance to a specific stress (e.g., alkaline conditions, etc.) to a microorganism, a technique of growing the microorganism in an environment where it is continuously exposed to the stress (i.e., "acclimation") is known. However, acclimation generally takes time, and it is known that it is difficult to predict whether a microorganism that has acquired the desired resistance will be obtained. In addition, the useful characteristics originally possessed by the microorganism may change through acclimation, and there are many challenges in imparting resistance to microorganisms through acclimation.

[0006] In addition, as another method for imparting specific resistance to microorganisms, a resistance imparting method by genetic recombination has been established. For example, Patent Document 1 teaches a method for imparting alkaline resistance to yeast by genetic recombination. However, since microorganisms produced by a method involving genetic recombination are prohibited by law from being released into the natural environment, the situations in which they can actually be used in the industrial world are extremely limited. Therefore, even if such a method can technically impart resistance to microorganisms, there remain major problems in terms of industrial use.

[0007] In addition to imparting alkali resistance to microorganisms, there is also a strong need to impart high-temperature resistance to microorganisms, but there have been few reports on a simple method for imparting high-temperature resistance to microorganisms that has few restrictions on use.

[0008] Polysaccharides such as deacylated gellan gum (DAG) form a three-dimensional network (irregular structure) in a solution by aggregating through metal cations (e.g., divalent metal cations such as calcium ions). When cells are cultured in a liquid medium containing this three-dimensional network, the cells are trapped in the three-dimensional network and do not settle, so a technique has been reported in which the cells are cultured in a state where they are uniformly dispersed in a suspended state in the medium without the need for shaking or rotation (Patent Document 2). [Prior art documents] [Patent documents]

[0009] [Patent Document 1] JP 2007-61025 A [Patent Document 2] International Publication No. 2014 / 017513 [Non-patent literature]

[0010] [Patent Document 1] Antonio Casamayor et al., Biochem J. 2012 May 15;444(1):39-49. Summary of the Invention [Problem to be solved by the invention]

[0011] In view of the above, an objective of the present invention is to provide a novel means for imparting alkaline resistance and / or thermotolerance to yeast simply and without genetic manipulation, and a method for producing yeast having the desired resistance, which can be safely and legally applied to industry. [Means for solving the problem]

[0012] As a result of intensive efforts to solve the above problems, the present inventors discovered that it is possible to impart resistance to alkali and high temperatures by culturing yeast in suspension in an aqueous solution containing a specific polysaccharide, and further research based on this finding led to the completion of the present invention.

[0013] [1] A method for imparting alkaline resistance and / or high temperature resistance to yeast, comprising culturing the yeast in an aqueous solution containing a polysaccharide having an anionic functional group. [2] The method according to [1], wherein the polysaccharide having an anionic functional group is at least one selected from the group consisting of deacylated gellan gum, alginic acid, gellan gum, hyaluronic acid, rhamsan gum, diutan gum, hexuronic acid, fucoidan, pectin, pectic acid, pectinic acid, heparan sulfate, heparin, heparitin sulfate, keratosulfate, chondroitin sulfate, dermatan sulfate, and rhamnan sulfate. [3] The method according to [2], wherein the polysaccharide having an anionic functional group is deacylated gellan gum, or deacylated gellan gum and alginic acid. [4] The method according to any one of [1] to [3], wherein the aqueous solution containing the polysaccharide having an anionic functional group is a plastic fluid. [5] The method according to any one of [1] to [4], wherein the culture is a suspension culture. [6] A method for producing an alkaline-tolerant and / or high-temperature-tolerant yeast, comprising culturing the yeast in an aqueous solution containing a polysaccharide having an anionic functional group. [7] The method according to [6], wherein the polysaccharide having an anionic functional group is at least one selected from the group consisting of deacylated gellan gum, alginic acid, gellan gum, hyaluronic acid, rhamsan gum, diutan gum, hexuronic acid, fucoidan, pectin, pectic acid, pectinic acid, heparan sulfate, heparin, heparitin sulfate, keratosulfate, chondroitin sulfate, dermatan sulfate, and rhamnan sulfate. [8] The method according to [7], wherein the polysaccharide having an anionic functional group is deacylated gellan gum, or deacylated gellan gum and alginic acid. [9] The method according to any one of [6] to [8], wherein the aqueous solution containing the polysaccharide having an anionic functional group is a plastic fluid.

[10] The method according to any one of [6] to [9], wherein the culture is a suspension culture.

[11] An agent for imparting alkaline resistance and / or high temperature resistance to yeast, comprising a polysaccharide having an anionic functional group.

[12] The agent according to

[11] , wherein the polysaccharide having an anionic functional group is at least one selected from the group consisting of deacylated gellan gum, alginic acid, gellan gum, hyaluronic acid, rhamsan gum, diutan gum, hexuronic acid, fucoidan, pectin, pectic acid, pectinic acid, heparan sulfate, heparin, heparitin sulfate, keratosulfate, chondroitin sulfate, dermatan sulfate, and rhamnan sulfate.

[13] The agent according to

[12] , wherein the polysaccharide having an anionic functional group is deacylated gellan gum, or deacylated gellan gum and alginic acid. Effect of the Invention

[0014] According to the present invention, alkali tolerance and / or high temperature tolerance can be imparted to yeast simply and without genetic manipulation. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] The present invention will be described in detail below.

[0016] 1. Method for imparting alkaline tolerance and / or high temperature tolerance to yeast The present invention provides a method for imparting alkaline resistance and / or high temperature resistance to yeast, which comprises culturing yeast in an aqueous solution containing a polysaccharide having an anionic functional group (hereinafter, sometimes referred to as the "method of the present invention").

[0017] The polysaccharide having an anionic functional group used in the method of the present invention is not particularly limited as long as it can obtain the desired effect of the present invention. In one embodiment, the polysaccharide having an anionic functional group can be an acidic polysaccharide having an anionic functional group. The acidic polysaccharide having an anionic functional group is not particularly limited, but examples thereof include polysaccharides having uronic acid in the structure (e.g., glucuronic acid, iduronic acid, galacturonic acid, mannuronic acid, etc.); polysaccharides having sulfate or phosphoric acid in the structure, or polysaccharides having both structures. More specifically, at least one selected from the group consisting of deacylated gellan gum (hereinafter sometimes referred to as "DAG" in this specification), alginic acid, gellan gum, hyaluronic acid, rhamsan gum, diutan gum, hexuronic acid, fucoidan, pectin, pectic acid, pectinic acid, heparan sulfate, heparin, heparitin sulfate, keratosulfate, chondroitin sulfate, dermatan sulfate, rhamnan sulfate, and salts thereof can be exemplified. Examples of salts include, but are not limited to, salts of alkali metals such as lithium, sodium, and potassium; salts of alkaline earth metals such as calcium, barium, and magnesium; and salts of aluminum, zinc, copper, iron, ammonium, organic bases, and amino acids.

[0018] The weight-average molecular weight of these polysaccharides is preferably 10,000 to 50,000,000, more preferably 100,000 to 20,000,000, and even more preferably 1,000,000 to 10,000,000. For example, the molecular weight can be measured in terms of pullulan by gel permeation chromatography (GPC). Furthermore, phosphorylated DAG can also be used. The phosphorylation can be carried out by a known method.

[0019] Polysaccharides may be used in combination of multiple types (e.g., two types). The type of polysaccharide combination is not particularly limited, but preferably, the combination includes DAG or a salt thereof. That is, suitable polysaccharide combinations include DAG or a salt thereof, and polysaccharides other than DAG or a salt thereof (e.g., alginic acid, xanthan gum, alginic acid, locust bean gum, methylcellulose, diutan gum, or salts thereof). Specific polysaccharide combinations include, but are not limited to, DAG and alginic acid, DAG and xanthan gum, DAG and sodium alginate, DAG and locust bean gum, DAG and methylcellulose, DAG and diutan gum, etc.

[0020] In a preferred embodiment of the method of the present invention, the polysaccharide having anionic functional groups may be DAG or a mixture of DAG and alginic acid.

[0021] In one embodiment of the method of the present invention, the aqueous solution containing the polysaccharide having anionic functional groups may be a plastic fluid.

[0022] As used herein, a "plastic fluid" refers to a fluid that requires a yield stress in order to flow, i.e., has a yield value. A plastic fluid may be a Bingham or non-Bingham fluid.

[0023] The plastic fluid used in the method of the present invention is not particularly limited as long as it can hold yeast without sedimentation in a stationary state, but the yield value of the plastic fluid is preferably 8 mPa or more from the viewpoint of dispersing and holding yeast, and is preferably 500 mPa or less from the viewpoint of operability such as filling into a storage container. The yield value can be measured, for example, by the method described in the Examples below. Specifically, it can be derived by measurement using a rheometer (manufactured by Anton Paar, model: MCR301, cone rotor: CP75-1).

[0024] From the viewpoint of operability, the viscosity of the plastic fluid used in the method of the present invention is preferably 1.5 to 200 mPa·s at 1 to 25° C. The viscosity can be measured, for example, by the method described in the Examples below. Specifically, the viscosity can be measured using an E-type viscometer (manufactured by Toki Sangyo Co., Ltd., TV-22 type viscometer, model: TVE-22L, cone rotor: standard rotor 1°34′×R24, rotation speed 10 to 100 rpm).

[0025] By adding an appropriate amount of polysaccharide to a liquid that is not a plastic fluid, the liquid can be made into a plastic fluid.

[0026] The concentration of polysaccharides required to turn a liquid that is not a plastic fluid into a plastic fluid depends on the type of polysaccharide, but is usually 0.0005% to 1.0% (weight / volume), preferably 0.001% to 0.4% (weight / volume), more preferably 0.005% to 0.1% (weight / volume), and even more preferably 0.005% to 0.05% (weight / volume).

[0027] For example, in the case of DAG, it may be 0.001% to 1.0% (weight / volume), preferably 0.003% to 0.5% (weight / volume), more preferably 0.005% to 0.1% (weight / volume), even more preferably 0.01% to 0.05% (weight / volume), and most preferably 0.01% to 0.03% (weight / volume).In the case of native gellan gum, it may be 0.05% to 1.0% (weight / volume), preferably 0.05% to 0.1% (weight / volume).

[0028] When a combination of DAG or a salt thereof with a polysaccharide other than DAG or a salt thereof is used, the concentration of DAG or a salt thereof is, for example, 0.005 to 0.02% (weight / volume), preferably 0.01 to 0.02% (weight / volume), and the concentration of the polysaccharide other than DAG or a salt thereof is, for example, 0.005 to 0.4% (weight / volume), preferably 0.1 to 0.4% (weight / volume). Specific examples of the combination of concentration ranges are as follows. DAG or a salt thereof: 0.005 to 0.08% (preferably 0.01 to 0.04%) (weight / volume) Polysaccharides other than DAG Xanthan gum: 0.01-0.2% (weight / volume) Sodium alginate: 0.01-0.2% (weight / volume) Locust bean gum: 0.01-0.2% (weight / volume) Methylcellulose: 0.01 to 0.2% (weight / volume) (preferably 0.2 to 0.4% (weight / volume)) Diutan gum: 0.01 to 0.2% (weight / volume) Carboxymethylcellulose: 0.01 to 0.2% (weight / volume)

[0029] In addition, when preparing a plastic fluid by adding polysaccharides, it may be preferable to add metal cations, such as divalent metal cations (calcium ions, magnesium ions, zinc ions, iron ions, copper ions, etc.), preferably calcium ions, to an aqueous solution that is not a plastic fluid. For example, water-soluble polysaccharides such as DAG make a liquid that is not a plastic fluid into a plastic fluid by forming a three-dimensional network through metal cations. Therefore, if the aqueous solution for culturing yeast does not contain a sufficient amount of metal cations for polysaccharides to form a three-dimensional network and make a liquid that is not a plastic fluid into a plastic fluid, it is preferable to add metal cations.

[0030] In the method of the present invention, the aqueous solution (not a plastic fluid) for culturing yeast is not particularly limited as long as it does not adversely affect the survival of yeast. Examples of the aqueous solution used in the method of the present invention include, but are not limited to, water, a buffer solution, and a liquid medium for yeast culture. In a preferred embodiment, the aqueous solution may be a buffer solution such as a phosphate buffer or a liquid medium for yeast culture. Liquid media for yeast culture are known (e.g., YM medium, YPD medium, etc.), and commercially available ones may be used.

[0031] The yeast to which resistance is imparted by the method of the present invention is not particularly limited, but is preferably a yeast known to be industrially applicable. Examples include yeasts of the genus Yarrowia, Aspergillus, Candida, Pichia, Hansenura, Saccharomyces, Kluyveromyces, Trichosporon, Lipomyces, Rhodotorula, Schizosaccharomyces, Lactobacillus, Enterococcus, etc. In a preferred embodiment, the yeast may be a yeast of the genus Yarrowia, Aspergillus, or Saccharomyces, more preferably a yeast of the genus Yarrowia, and particularly preferably Yarrowia lipolytica.

[0032] The method of the present invention is characterized in that yeast is cultured in an aqueous solution containing polysaccharides. In one embodiment of the present invention, the yeast may be cultured as a suspension culture. As used herein, "suspension culture" refers to culture in which the yeast is supported by a three-dimensional network formed by polysaccharides present in the aqueous solution, and is not concentrated on the bottom surface of the culture vessel, but is dispersed with a three-dimensional spread. The suspension culture of yeast may or may not involve stirring or shaking. In a preferred embodiment, the method of the present invention may be carried out by suspension culture in which the aqueous solution is a plastic fluid and does not involve stirring or shaking (i.e., "static suspension culture"). In this specification, the term "yeast culture" refers to any state in which yeast survives. In this specification, yeast culture may or may not result in yeast proliferation. In other words, a state in which yeast survival is maintained may also be included in the term "yeast culture" in this specification.

[0033] In the method of the present invention, the conditions for culturing yeast are not particularly limited as long as the yeast survives and the desired resistance is imparted. For example, the culture period is usually 1 minute or more, preferably 30 minutes or more (1 hour or more, 2 hours or more, 3 hours or more, 4 hours or more, 5 hours or more, 6 hours or more, 7 hours or more, 8 hours or more, 9 hours or more, 10 hours or more, 11 hours or more, or 12 hours or more), and more preferably 1 day or more (2 days or more, or 3 days or more). The upper limit of the culture period is also not particularly limited as long as the desired effect is obtained, but it may be usually 1 year or less, preferably 6 months or less, and more preferably 1 month or less (2 weeks or less, or 1 week or less). The culture temperature may usually be 2 to 50°C, and preferably 3 to 40°C (e.g., 4 to 37°C, 4 to 36°C, 4 to 35°C, 4 to 34°C, 4 to 33°C, 4 to 32°C, 4 to 31°C, 4 to 30°C, 4 to 29°C, 4 to 28°C, 4 to 27°C, 4 to 26°C, 4 to 25°C, 4 to 24°C, 4 to 23°C, 4 to 22°C, 4 to 21°C, 4 to 20°C, 4 to 19°C, 4 to 18°C, 4 to 17°C, 4 to 16°C, 4 to 15°C, 4 to 14°C, 4 to 13°C, 4 to 12°C, 4 to 11°C, 4 to 10°C, 4 to 9°C, 4 to 8°C, 4 to 7°C, 4 to 6°C, 4 to 5°C). Moreover, since yeast is generally preferably cultured under weakly acidic conditions, the pH may usually be 5.0 to 7.0, and preferably 5.5 to 6.5.

[0034] In one embodiment, the aqueous solution containing the polysaccharide having an anionic functional group in the method of the present invention may contain components other than the polysaccharide, such as, for example, a substance that serves as a nutrient source for yeast or a substance that can enhance the biological function or viability of yeast.

[0035] The substance that serves as a nutrient source for yeast is not particularly limited, but examples thereof include a carbon source, a nitrogen source, and inorganic salts.

[0036] Examples of carbon sources include sugars such as glucose, fructose, cellobiose, raffinose, xylose, maltose, galactose, sorbose, glucosamine, ribose, arabinose, rhamnose, sucrose, trehalose, α-methyl-D-glucoside, salicin, melibiose, lactose, melezitose, inulin, erythritol, glucitol, mannitol, galactitol, N-acetyl-D-glucosamine, starch, starch hydrolysates, molasses, blackstrap molasses, natural products such as wheat and rice, alcohols such as glycerol, methanol, ethanol, organic acids such as acetic acid, lactic acid, succinic acid, gluconic acid, pyruvic acid, citric acid, hydrocarbons such as hexadecane, etc. One or more of the above carbon sources may be contained.

[0037] Examples of nitrogen sources include organic nitrogen sources such as meat extract, fish extract, peptone, polypeptone, yeast extract, malt extract, soybean hydrolysate, soybean powder, casein, milk casein, casamino acid, various amino acids such as glycine, glutamic acid, aspartic acid, corn steep liquor, other animal, plant, and microbial hydrolysates, and inorganic nitrogen sources such as ammonia, ammonium salts such as ammonium nitrate, ammonium sulfate, and ammonium chloride, nitrates such as sodium nitrate, nitrites such as sodium nitrite, and urea. In addition, one or more of the above nitrogen sources may be contained.

[0038] Examples of inorganic salts include salts of magnesium, manganese, calcium, sodium, potassium, copper, iron, zinc, etc. (e.g., phosphates, hydrochlorides, sulfates, acetates, carbonates, bicarbonates, chlorides, etc.) In addition, one or more of the above inorganic salts may be contained.

[0039] The substance capable of enhancing the biological function or viability of yeast is not particularly limited as long as the desired purpose is achieved, and may be appropriately selected depending on the type of yeast. For example, when the yeast is capable of decomposing fats and oils, an oil may be added to maintain or enhance the biological function.

[0040] In the present specification, the term "oil" refers to edible or industrial fats and oils that are rich in glycerides such as triglycerides, diglycerides, and monoglycerides, as well as fatty acids. Examples of the oils and oils that can be used in the method of the present invention include edible fats and oils such as olive oil, canola oil, coconut oil, sesame oil, rice oil, rice bran oil, safflower oil, soybean oil, corn oil, rapeseed oil, palm oil, palm kernel oil, sunflower oil, cottonseed oil, coconut oil, peanut oil, beef tallow, lard, chicken oil, fish oil, whale oil, butter, margarine, fat spread, and shortening; industrial fats and oils such as linseed oil, jatropha oil, tall oil, seaweed oil, castor oil, and jojoba oil; and butyric acid, hexanoic acid, heptanoic acid, octanoic acid, decanoic acid, lauric acid, tridecanoic acid, myristic acid, pentadecanoic acid, palmitic acid, heptane acid, octane ... Fatty acids such as tadecanoic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, decenoic acid, myristoleic acid, pentadecenoic acid, palmitoleic acid, heptadecenoic acid, oleic acid, icosenoic acid, docosenoic acid, tetracosenoic acid, hexadecadienoic acid, hexadecatrienoic acid, hexadecatetraenoic acid, linoleic acid, α-linolenic acid, γ-linolenic acid, octadecatetraenoic acid, icosadienoic acid, icosatrienoic acid, icosatetraenoic acid, arachidonic acid, icosapentaenoic acid, henicosapentaenoic acid, docosadienoic acid, docosatetraenoic acid, docosapentaenoic acid, and docosahexaenoic acid are preferred.

[0041] The amount of oil to be added is not particularly limited, and may be appropriately selected according to the type of microorganism. As an example, the amount of oil to be added may be 1 to 30 g, more preferably 5 to 15 g, per 1 L of the composition of the present invention, but is not limited thereto. The oil may be added alone or in combination of two or more kinds.

[0042] Metal salts may also be added to the aqueous solution containing the polysaccharides having an anionic functional group. Examples of metal salts include, but are not limited to, sulfates, sulfites, hyposulfites, persulfates, thiosulfates, carbonates, phosphates, pyrophosphates, hydrochlorides, nitrates, nitrites, acetates, propionates, butyrates, citrates, oxalates, and halides (e.g., fluorides, chlorides, bromides, and iodides) of metal elements such as sodium, potassium, magnesium, calcium, manganese, iron, nickel, zinc, and copper. More specifically, examples of metal salts include sodium sulfate, sodium sulfite, sodium hyposulfite, sodium thiosulfate, sodium carbonate, sodium persulfate, monosodium phosphate, disodium phosphate, trisodium phosphate, sodium acetate, sodium nitrate, sodium nitrite, sodium citrate, sodium oxalate, sodium chloride, potassium sulfate, potassium sulfite, potassium hyposulfite, potassium thiosulfate, potassium carbonate, potassium persulfate, monopotassium phosphate, dipotassium phosphate, tripotassium phosphate, potassium acetate, potassium nitrate, potassium nitrite, potassium citrate, potassium oxalate, potassium chloride, magnesium sulfate, magnesium sulfite, magnesium thiosulfate, magnesium carbonate, magnesium monophosphate, magnesium diphosphate, magnesium triphosphate, magnesium pyrophosphate, magnesium nitrate, magnesium nitrite, magnesium acetate, magnesium citrate, magnesium oxalate, magnesium chloride, calcium sulfate, calcium sulfite, calcium thiosulfate, calcium carbonate, calcium nitrate, calcium nitrite, calcium acetate, calcium citrate, calcium oxalate, calcium chloride, etc. Metal salts may be used alone or in combination of two or more.

[0043] In one embodiment, from the viewpoint of good survival rate of yeast, the metal salt is preferably a sulfate, carbonate, phosphate, or a combination thereof of an element selected from the group consisting of sodium, potassium, magnesium, and calcium, and more preferably a sulfate of magnesium and / or calcium. Note that the "metal salt" in this specification also includes hydrates and solvates of the metal salt.

[0044] In another embodiment, the aqueous solution containing a polysaccharide having an anionic functional group may contain monosaccharides such as glucose and fructose; disaccharides other than trehalose, such as sucrose, lactose, and maltose; polysaccharides such as cyclodextrin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, carboxymethyl cellulose, crystalline cellulose, and corn starch; proteins such as soy protein; protein hydrolysates and peptides such as soy peptides, gelatin, peptone, and tryptone; oils and fats such as soybean oil, rapeseed oil, palm oil, sesame oil, and olive oil; vitamins such as ascorbic acid or a salt thereof, and tocopherol; surfactants such as polyglycerin fatty acid esters, sucrose fatty acid esters, and sorbitan fatty acid esters; polyethylene glycol, glycerin, and the like.

[0045] The aqueous solution containing the polysaccharide having an anionic functional group may also contain a pH adjuster or the like.

[0046] 2. Method for producing yeast having alkali resistance and / or high temperature resistance The present invention also provides a method for producing an alkaline-tolerant and / or high-temperature-tolerant yeast, which comprises culturing the yeast in an aqueous solution containing a polysaccharide having an anionic functional group (hereinafter sometimes referred to as the "production method of the present invention").

[0047] The specific embodiment of the production method of the present invention is the same as the above-mentioned method of the present invention.

[0048] 3. Agents for imparting alkaline and / or high temperature resistance to yeast The present invention also provides an agent for imparting alkaline tolerance and / or high temperature tolerance to yeast, which comprises a polysaccharide having an anionic functional group (hereinafter, sometimes referred to as "the agent of the present invention").

[0049] The polysaccharide having an anionic functional group contained in the agent of the present invention is the same as that described in the method of the present invention. In one embodiment, the polysaccharide having an anionic functional group contained in the agent of the present invention can be DAG or a mixture of DAG and alginic acid.

[0050] The amount of the polysaccharide having an anionic functional group to be incorporated in the agent of the present invention is not particularly limited, but is usually 0.001 to 100% by weight, preferably 1 to 100% by weight, and more preferably 10 to 100% by weight, based on the total weight of the agent, but is not limited thereto.

[0051] The agent of the present invention may contain components other than the polysaccharide having an anionic functional group. Such components include, for example, substances that serve as nutrient sources for yeast and substances that can enhance the biological function or viability of yeast, and these components are the same as those described in the method of the present invention.

[0052] The dosage form of the agent of the present invention is also not particularly limited, but may generally be any dosage form such as a solid, granular, powder, liquid, or slurry.

[0053] The present invention will be described in more detail in the following examples, but the present invention is not limited to these examples in any way. EXAMPLES

[0054] [Test Example 1] Preparation of deacylated gellan gum (DAG) aqueous solution A 0.8% (weight / volume) DAG aqueous solution was prepared by adding 100 parts by volume of tap water and 0.8 parts by weight of DAG (manufactured by Sansho Co., Ltd.) to a glass medium bottle and sterilizing the bottle in an autoclave (121° C., 20 minutes).

[0055] [Test Example 2] Preparation of a liquid composition for imparting resistance to yeast An aqueous solution (hereinafter sometimes referred to as "basic aqueous solution") was prepared by mixing urea and various metal salts with tap water. The basal aqueous solution was mixed with the 0.8% (weight / volume) DAG aqueous solution prepared in Test Example 1, or a DAG aqueous solution diluted with tap water to 0.08% (weight / volume), to prepare a liquid composition containing 0.04% (weight / volume) DAG (hereinafter sometimes referred to as "liquid composition for imparting resistance"). The basal aqueous solution and the DAG aqueous solution were mixed using a three-dimensional medium preparation kit (Nissan Chemical FCeM (registered trademark)-series Preparation Kit) according to the following procedure.

[0056] <Preparation of a liquid composition for imparting resistance using a 3D medium preparation kit> In preparing the liquid composition for conferring resistance using the 3D medium preparation kit, 47.5 mL of 1x basal aqueous solution was dispensed into a 50 mL conical tube (manufactured by Sumitomo Bakelite Co., Ltd.), and an adapter cap, which is a component of the kit, was attached. Next, the tip of a disposable syringe filled with 2.5 mL of 0.8% (weight / volume) DAG aqueous solution was fitted into the cylindrical part of the adapter cap to connect it, and the plunger of the syringe was manually pressed, and the DAG aqueous solution in the syringe was vigorously ejected into the container and mixed with the basal aqueous solution to prepare a liquid composition for conferring resistance containing 0.04% DAG.

[0057] [Test Example 3] Imparting alkaline resistance and high temperature resistance using a resistance-imparting liquid composition 1. Preparation of Microorganisms 0.1 L of a standard suspension containing Y. lipolytica, a type of yeast with the ability to decompose fats and oils, was dispensed into a centrifuge tube and centrifuged (6000 x g, 10 minutes, 15°C). After centrifugation, the supernatant was discarded, and the entire yeast pellet obtained was suspended in the base aqueous solution to prepare a 40-fold concentrated standard suspension of Y. lipolytica. In addition, the same procedure was performed for Burkholderia arboris (B. arboris), a type of bacterium with the ability to decompose fats and oils, as a control bacterium, to prepare a 40-fold concentrated suspension of the standard suspension of B. arboris.

[0058] Static culture using the liquid composition for conferring resistance was carried out as follows: 9.5 mL of the liquid composition for conferring resistance containing 0.04% (weight / volume) DAG or 9.5 mL of a basal aqueous solution containing no DAG was added to a 15 mL tube, to which 0.5 mL of a 40-fold concentrated solution of Y. lipolytica or B. arboris was added. Two of the above samples were prepared. The prepared samples were left to stand for 14 days under refrigeration.

[0059] After the samples were left to stand for 14 days under refrigeration, 3.2 mL was taken from each sample and re-cultured in an aqueous solution containing nutrients necessary for the survival and proliferation of Y. lipolytica and B. arboris (urea: 45 mg / L, KH2PO4: 5.25 mg / L, Na2HPO4: 1.41 mg / L, canola oil: 500 ppm / L, Triton-X: 50 ppm / L) (hereinafter referred to as "oil-containing aqueous solution").

[0060] In order to confirm whether the microorganisms were resistant to the static culture, the culture in the oil-containing aqueous solution was carried out under the following two conditions: [High temperature conditions] 40℃, pH 7.3, constant stirring (120 rpm) [Alkaline conditions] 30℃, pH 10, constant stirring (120 rpm)

[0061] The microorganisms were cultured using the oil-containing aqueous solution for 6 hours. Sampling for counting the number of bacteria was performed at the start of the culture and 6 hours after the start of the culture.

[0062] The number of bacteria of the cultured microorganisms was counted according to the following procedure. The samples after culture were thoroughly suspended. Each sample was appropriately diluted to a concentration suitable for counting, 0.1 mL of the diluted sample was taken and dropped onto two plate media, and the bacterial liquid was spread on the medium using a cone-larger stick. An oil agar medium was used for colony formation of B.arboris, and an LB agar medium supplemented with an antibiotic was used for colony formation of Y.lipolytica. The plate media on which the microorganisms were spread were cultured at 30°C for more than three days, and then the number of colonies formed on the plate media was counted and used as the viable bacterial count. The number of colonies was the average of the two plate media. The results are shown in Tables 1 to 4. Table 1 shows the results of B.arboris cultured under high temperature conditions, Table 2 shows the results of Y.lipolytica cultured under high temperature conditions, Table 3 shows the results of B.arboris cultured under alkaline conditions, and Table 4 shows the results of Y.lipolytica cultured under alkaline conditions.

[0063] [Table 1]

[0064] [Table 2]

[0065] [Table 3]

[0066] [Table 4]

[0067] As shown in Tables 1 to 4, when cultured for 6 hours under high temperature conditions (40°C) or alkaline conditions (pH 10), the survival rates of both microorganisms decreased in samples that were left in the basal aqueous solution for 14 days. On the other hand, it was confirmed that 8% of Y. lipolytica that was left for 14 days in the liquid composition for imparting resistance survived when cultured under high temperature conditions. Moreover, surprisingly, the survival rate of Y. lipolytica that was left for 14 days in the liquid composition for imparting resistance increased to 152% when cultured under alkaline conditions.

[0068] The above results indicate that culturing Y. lipolytica in a liquid composition for imparting resistance to high temperatures and alkalies can be imparted to the bacteria. On the other hand, no such resistance-imparting effect was observed in the case of B. arboris, an oil-decomposing bacterium.

[0069] Without wishing to be bound by theory, it is expected that in the present invention, resistance is conferred to yeast via the following mechanism: (1) When yeast is cultured in a liquid composition containing a polysaccharide, the yeast becomes attached to the polysaccharide; (2) adhesion to polysaccharides alters yeast gene expression profiles; (3) As a result of the change in gene expression profile, the protein expression profile of the yeast changes, and the composition of the yeast cell wall and other components is modified, resulting in the acquisition of resistance. [Industrial Applicability]

[0070] According to the present invention, it is possible to impart alkaline resistance and thermotolerance to yeast simply and without genetic manipulation, and therefore the present invention is extremely useful in the fields of food, pharmaceutical, biofuel manufacturing, and environmental restoration.

Claims

1. A method for imparting alkaline resistance and / or high temperature resistance to yeast, comprising culturing yeast in an aqueous solution containing a polysaccharide having an anionic functional group.

2. 2. The method of claim 1, wherein the polysaccharide having an anionic functional group is at least one selected from the group consisting of deacylated gellan gum, alginic acid, gellan gum, hyaluronic acid, rhamsan gum, diutan gum, hexuronic acid, fucoidan, pectin, pectic acid, pectinic acid, heparan sulfate, heparin, heparitin sulfate, keratosulfate, chondroitin sulfate, dermatan sulfate, and rhamnan sulfate.

3. The method according to claim 2, wherein the polysaccharide having an anionic functional group is deacylated gellan gum or a combination of deacylated gellan gum and alginic acid.

4. 4. The method according to claim 1, wherein the aqueous solution containing the polysaccharide having an anionic functional group is a plastic fluid.

5. The method according to any one of claims 1 to 3, wherein the culture is a suspension culture.

6. A method for producing an alkaline-tolerant and / or thermo-tolerant yeast, comprising culturing the yeast in an aqueous solution containing a polysaccharide having an anionic functional group.

7. The method according to claim 6, wherein the polysaccharide having an anionic functional group is at least one selected from the group consisting of deacylated gellan gum, alginic acid, gellan gum, hyaluronic acid, rhamsan gum, diutan gum, hexuronic acid, fucoidan, pectin, pectic acid, pectinic acid, heparan sulfate, heparin, heparitin sulfate, keratosulfate, chondroitin sulfate, dermatan sulfate, and rhamnan sulfate.

8. 8. The method according to claim 7, wherein the polysaccharide having an anionic functional group is deacylated gellan gum or deacylated gellan gum and alginic acid.

9. The method according to any one of claims 6 to 8, wherein the aqueous solution containing the polysaccharide having an anionic functional group is a plastic fluid.

10. The method according to any one of claims 6 to 8, wherein the culture is a suspension culture.

11. An agent for imparting alkaline resistance and / or high temperature resistance to yeast, comprising a polysaccharide having an anionic functional group.

12. The agent according to claim 11, wherein the polysaccharide having an anionic functional group is at least one selected from the group consisting of deacylated gellan gum, alginic acid, gellan gum, hyaluronic acid, rhamsan gum, diutan gum, hexuronic acid, fucoidan, pectin, pectic acid, pectinic acid, heparan sulfate, heparin, heparitin sulfate, keratosulfate, chondroitin sulfate, dermatan sulfate, and rhamnan sulfate.

13. The agent according to claim 12, wherein the polysaccharide having an anionic functional group is deacylated gellan gum or a combination of deacylated gellan gum and alginic acid.