JP2018121532A - Farnesyl acid, method for producing farnesyl acid production yeast, novel yeast, farnesyl acid production composition and use thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing farnesyl acid capable of stably producing farnesyl acid inexpensively, efficiently, a composition for producing farnesyl acid, a novel yeast, a method for producing yeast for producing farnesyl acid, and a composition for producing farnesyl acid. And a method for producing sake lees using the same, and a sake lees and yeast culture containing farnesyl acid. A method for producing farnesylate using a yeast for producing farnesylate, which is Saccharomyces cerevisiae, in which guanine at position 714 of the base sequence of the ERG9 gene encoding squalene synthase is replaced with adenine. [Selection diagram] None

Description

  The present invention relates to a method for producing farnesic acid, a composition for producing farnesic acid, a novel yeast having the ability to produce farnesic acid, a method for producing yeast for producing farnesic acid, and production of sake lees using the composition for producing farnesic acid. The present invention relates to a method, and sake lees and yeast cultures containing farnesic acid.

  In general, the first step in terpenoid synthesis is the generation of isoprene units, where acetyl CoA is activated as malonyl CoA, reacts with the other two molecules of acetyl CoA, and is reduced to produce mevalonic acid. Further, this is decarboxylated into isopentenyl diphosphate (C5) and dimethylallyl diphosphate (C5). As the isopentenyl diphosphate is sequentially bonded to dimethylallyl diphosphate, the number of carbons increases by 5 to synthesize geranyl diphosphate (C10), farnesyl diphosphate (C15), and the like.

  Among these, farnesyl diphosphate is an important biosynthetic intermediate, and includes steroids such as ergosterol, side chains of quinones, sesquiterpenes, squalene, anchor molecules of farnesylated proteins, dolichol, bactoprenol, natural rubber Although it is known as an aromatic substance in essential oils used as a fragrance, it is also an important substance as a starting material for the synthesis of compounds including the above-mentioned vitamins useful as pharmacological agents.

Farnesol (FOH) produced by dephosphorylation of farnesyl diphosphate has been reported to function in the transcriptional activation of target genes of peroxisome proliferator-activated receptors (PPARs) (non-) Patent Document 1). Such farnesol becomes a ligand of PPARs and increases PPARs activity, and thus anti-obesity and anti-hyperlipidemic effects are expected (see Non-Patent Document 2).
However, farnesol has a problem that stability at high temperatures is low and application to foods is difficult (see Patent Document 1).

  On the other hand, farnesic acid (FA), which is a metabolite of farnesol, is known to have an activation effect of PPARα (peroxisome proliferator-activated receptor α) superior to farnesol (see Non-Patent Document 3). Furthermore, farnesyl acid has the advantage of high temperature stability compared to farnesol.

However, since farnesol, a precursor of farnesyl acid, is extracted from essential oils of plants, its availability and price fluctuate in relation to the availability of plants and the stability of the country of origin and supply it stably. There is a problem that it is difficult (see Patent Documents 2 and 3). In addition, farnesyl acid marketed as a standard product has a problem that it is very expensive even in a small amount.
Therefore, at present, it is strongly desired to provide a new method for producing farnesic acid that can be supplied inexpensively and stably.

JP 2010-229051 A Japanese translation of PCT publication No. 2006-500902 Special table 2008-546384 gazette

Takahashi. N, et al. , FEBS Letters, Volume 514, Issue 2-3, March 13, 2002, pp. 315-322 Takahashi. H et al. , J Lipid Res, Volume 56, Issue 2, Feb, 2015, pp. 254-265 Joe JH et al. , Cancer Lett, Volume 287, Issue 2, Jan 28, 2010, pp. 123-135

  An object of the present invention is to solve the conventional problems and achieve the following objects. That is, the present invention provides a method for producing farnesic acid that can produce farnesic acid stably at low cost, efficiently, a composition for producing farnesic acid, a novel yeast, a method for producing a yeast for producing farnesic acid, the farnesic acid It aims at providing the manufacturing method of the sake lees using the composition for production, and the liquor and yeast culture containing farnesic acid.

Means for solving the problems are as follows. That is,
<1> Yeast for farnesic acid production which is Saccharomyces cerevisiae mutated to the gene represented by SEQ ID NO: 1 in which guanine at position 714 of the base sequence of the ERG9 gene encoding squalene synthase is replaced with adenine Is a method for producing farnesic acid.
<2> Yeast for farnesic acid production which is Saccharomyces cerevisiae mutated to the gene represented by SEQ ID NO: 1 in which the guanine at position 714 of the base sequence of the ERG9 gene encoding squalene synthase is replaced with adenine Is a composition for producing farnesic acid.
<3> Saccharomyces cerevisiae HL305 strain (Accession number: NITE P-02406), which is a yeast characterized by having the ability to produce farnesic acid.
<4> Farnesyl, characterized in that in Saccharomyces cerevisiae , guanine at the base sequence position 714 of the ERG9 gene encoding squalene synthase is substituted with adenine and mutated to the gene represented by SEQ ID NO: 1. It is the manufacturing method of the yeast for acid production.
<5> A method for producing a sake lees characterized in that the composition for producing farnesic acid according to <2> is used.
<6> A sake lees characterized by containing farnesic acid.
<7> A yeast culture characterized by containing farnesic acid.

  According to the present invention, a method for producing farnesic acid, a composition for producing farnesic acid, which can solve the above-mentioned problems and can achieve the above-mentioned object, can produce farnesic acid at low cost, efficiently and stably. Product, a novel yeast, a method for producing farnesyl acid-producing yeast, a method for producing sake mash using the farnesic acid-producing composition, and a mash and yeast culture containing farnesic acid.

1 is a diagram showing a liquid chromatogram of an extract of HL305 strain in Test Example 1. FIG. FIG. 2 is a diagram showing a liquid chromatogram of an extract of sake yeast in Test Example 1. FIG. 3 is a diagram showing a liquid chromatogram of a farnesyl acid standard product in Test Example 1. FIG. 4 is a diagram showing a liquid chromatogram of an extract of HL305 strain before high-temperature storage in Test Example 3. Peak 2 is the farnesic acid peak, and peak 3 is the farnesol peak. FIG. 5 is a diagram showing a liquid chromatogram of an extract of HL305 strain after high-temperature storage in Test Example 3. Peak 2 is the farnesic acid peak, and peak 3 is the farnesol peak.

(yeast)
The yeast of the present invention is Saccharomyces cerevisiae HL305 strain (Accession No .: NITE P-02406) (hereinafter sometimes referred to as “HL305 strain”), which is a yeast having the ability to produce farnesic acid. is there.
The HL305 strain is a strain isolated by the present inventors.
The HL305 strain was filed with the National Institute of Technology and Evaluation Biotechnology Center Patent Microorganisms Deposit Center (Room 2-5-8 Kazusa Kamashi, Kisarazu City, 292-0818, Japan). Deposited as NITE P-02406 on March 17.

  Since the yeast has the ability to produce farnesic acid, it can be suitably used in the method for producing farnesic acid, the composition for producing farnesic acid, and the method for producing sake lees described below.

(Composition for farnesyl acid production)
The composition for producing farnesyl acid of the present invention is a gene represented by SEQ ID NO: 1 in which guanine at position 714 in the base sequence of the ERG9 gene encoding squalene synthase is substituted with adenine (hereinafter referred to as “erg9-2 gene”). A yeast for producing farnesic acid, which is mutated in Saccharomyces cerevisiae (sometimes referred to as “ Saccharomyces cerevisiae” ) and, if necessary, further containing other components.

<Farnesyl acid production yeast>
The yeast for producing farnesic acid is a yeast having the ability to produce farnesic acid. The farnesyl acid-producing yeast may be haploid or diploid as long as it has the ability to produce farnesic acid.
The yeast for producing farnesyl acid may be a Saccharomyces cerevisiae mutated to the erg9-2 gene represented by SEQ ID NO: 1 in which the guanine at position 714 of the base sequence of the ERG9 gene encoding the squalene synthase is replaced with adenine. There is no particular limitation, and it can be appropriately selected according to the purpose, but preferably has the following bacteriological properties, and Saccharomyces cerevisiae having the strain number of NITE P-02406 is attached. It is particularly preferred that it is a cervicier.

<< Mycological properties >>
(1) Bacterial morphology when grown in YPD liquid medium Vegetative cell size: 4 μm to 8 μm, width: 4 μm to 6 μm
2. 2. Vegetative cell shape: egg shape Form of growth: budding (2) Physiological properties a. Sugar assimilation glucose: +
Galactose: +
Sucrose: +
Maltose: +
Lactose:-
b. Fermentation of sugar Glucose: +
Galactose: +
Sucrose: +
Maltose: +
Lactose:-
Melibiose:-
Raffinose: +
(3) Bondability The haploid has bondability, but the diploid has no bondability.
(4) Sporulation A haploid does not sporulate. The diploid forms a spore when cultured in a spore-forming medium at 30 ° C. for 4 days.

  As seen in other yeasts, the farnesyl acid-producing yeast may change in properties, but for example, mutant strains derived from the farnesyl acid-producing yeast (for example, natural mutants and Artificial mutant strains that can be obtained by mutation treatment of ultraviolet rays, X-rays, radiation, drugs, etc.), zygotes, recombinants, etc., which have the ability to produce farnesic acid are included in the microorganism of the present invention It is.

  The farnesic acid-producing yeast does not require a special medium to produce farnesic acid, and does not need to add ergosterol. That is, it is advantageous in that farnesyl acid can be produced only by aerobic culture in a medium generally used for yeast culture, for example, YPD medium.

  The yeast for producing farnesic acid can be suitably produced by the method for producing yeast for producing farnesic acid of the present invention described later.

  There is no restriction | limiting in particular as content of the said yeast for farnesic acid production in the said composition for farnesic acid production, According to the objective, it can select suitably. Further, the farnesic acid production composition may be the farnesyl acid production yeast itself.

<Other ingredients>
The other components are not particularly limited and may be appropriately selected depending on the purpose. For example, among pharmacologically acceptable carriers, auxiliary raw materials or additives used for foods and drinks, and the like. Can be appropriately selected according to the purpose. These may be used alone or in combination of two or more.

  Here, the food and drink are those which are less likely to harm human health and are taken by oral or gastrointestinal administration in normal social life. For example, general foods taken orally; health foods; special purpose foods such as functional health foods (nutrient functional foods and foods for specified health use) and sick foods; Quasi-drug; means a wide variety of drugs.

-Pharmacologically acceptable carrier-
Examples of the pharmacologically acceptable carrier include additives, adjuvants, and water. The additive or the adjuvant is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include bactericides, preservatives, binders, thickeners, fixing agents, binders, and coloring agents. , Stabilizers, pH adjusters, buffers, isotonic agents, solvents, antioxidants, UV inhibitors, crystal precipitation inhibitors, antifoaming agents, physical property improvers, preservatives, and the like.

  There is no restriction | limiting in particular as said disinfectant, According to the objective, it can select suitably, For example, cationic surfactants, such as benzalkonium chloride, benzethonium chloride, and cetylpyridinium chloride, etc. are mentioned.

  There is no restriction | limiting in particular as said preservative, According to the objective, it can select suitably, For example, paraoxybenzoic acid esters, chlorobutanol, cresol, etc. are mentioned.

  The binder, thickener, or fixing agent is not particularly limited and may be appropriately selected depending on the intended purpose. For example, starch, dextrin, cellulose, methylcellulose, ethylcellulose, carboxymethylcellulose, hydroxyethylcellulose, hydroxy Propylcellulose, hydroxypropylmethylcellulose, carboxymethyl starch, pullulan, sodium alginate, ammonium alginate, propylene glycol ester alginate, guar gum, locust bean gum, gum arabic, xanthan gum, gelatin, casein, polyvinyl alcohol, polyethylene oxide, polyethylene glycol, ethylene Propylene block polymer, sodium polyacrylate, polyvinylpyrrolidone, etc. That.

  The binder is not particularly limited and may be appropriately selected depending on the intended purpose. For example, water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, hydroxypropylcellulose, hydroxy Examples include propyl starch, methyl cellulose, ethyl cellulose, shellac, calcium phosphate, and polyvinyl pyrrolidone.

  The colorant is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include titanium oxide and iron oxide.

  The stabilizer is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include tragacanth, gum arabic, gelatin, sodium pyrosulfite, ethylenediaminetetraacetic acid (EDTA), thioglycolic acid, and thiolactic acid. Is mentioned.

  There is no restriction | limiting in particular as said pH adjuster or said buffering agent, According to the objective, it can select suitably, For example, sodium citrate, sodium acetate, sodium phosphate, sodium hydroxide, sodium hydrogencarbonate, sodium carbonate Etc.

  The isotonizing agent is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include sodium chloride and glucose.

-Auxiliary raw materials or additives-
The auxiliary raw material or additive is not particularly limited and may be appropriately selected depending on the intended purpose. For example, glucose, fructose, sucrose, maltose, sorbitol, stevioside, rubusoside, corn syrup, lactose, citric acid , Tartaric acid, malic acid, succinic acid, lactic acid, L-ascorbic acid, dl-α-tocopherol, sodium erythorbate, glycerin, propylene glycol, glycerin fatty acid ester, polyglycerin fatty acid ester, sucrose fatty acid ester, sorbitan fatty acid ester, Arabia Examples include gum, carrageenan, casein, gelatin, pectin, agar, vitamin Bs, nicotinic acid amide, calcium pantothenate, amino acids, calcium salts, pigments, fragrances, preservatives and the like.

  There is no restriction | limiting in particular as content of the said other component in the said composition for a farnesic acid production, unless the effect of this invention is impaired, It can select suitably according to the objective.

  The farnesic acid production composition may be used as it is, may be a dried product of the farnesic acid production composition, and is in the form of an extract obtained by extracting the farnesic acid production composition with a solvent or the like. There may be.

  There is no restriction | limiting in particular as said solvent, According to the objective, it can select suitably, For example, diethyl ether, ethanol, methanol, acetone, butanol, acetonitrile, dimethylsulfoxide (DMSO) etc. are mentioned. These may be used alone or in combination of two or more.

  The composition for producing farnesic acid can be suitably used in the method for producing sake lees of the present invention described later.

(Method for producing farnesyl acid)
The method for producing farnesic acid according to the present invention comprises a Saccharomyces cerevisiae mutated to the erg9-2 gene represented by SEQ ID NO: 1 in which the guanine at position 714 of the base sequence of the ERG9 gene encoding squalene synthase is substituted with adenine. Saccharomyces cerevisiae ) is a method for producing farnesic acid using a yeast for producing farnesic acid.
As the farnesic acid-producing yeast, those described in the section <Farnesic acid-producing yeast> in the “farnesic acid-producing composition” are preferably used.
The farnesyl acid production method is not particularly limited as long as the farnesyl acid-producing yeast is used, and can be appropriately selected according to the purpose, but preferably includes a culture step, and further includes a collection step. More preferably, it may further include other steps as necessary.

<Culture process>
The culturing step is a step of culturing the farnesyl acid-producing yeast. The method for culturing is not particularly limited and may be appropriately selected depending on the intended purpose. For example, the yeast for producing farnesic acid is inoculated in a nutrient medium (hereinafter simply referred to as “medium”). And a method of culturing at a temperature suitable for production of the farnesic acid.

There is no restriction | limiting in particular as said nutrient medium, According to the objective, it can select suitably, For example, the well-known thing conventionally utilized for culture | cultivation of yeast can be used, A liquid medium may be sufficient. It may be a solid (agar) medium.
The nutrient source added to the nutrient medium is not particularly limited and can be appropriately selected according to the purpose.For example, commercially available soybean flour, wheat germ, pressed wheat, peptone, cottonseed meal, yeast extract, Nitrogen sources such as meat extract, corn steep liquor, ammonium sulfate, sodium nitrate, urea, etc .; carbohydrate sources such as tomato paste, glycerin, starch, glucose, galactose, dextrin, bacto-soyton; carbon sources such as fat;
Furthermore, inorganic salts such as sodium chloride and calcium carbonate can be added to the medium for use, and in addition, a trace amount of metal salt can be added to the medium for use.
Any of these materials may be used as long as they are used by the farnesic acid-producing yeast and are useful for producing the farnesic acid, and all known culture materials can be used.

  The seed culture solution for producing the farnesic acid is not particularly limited and may be appropriately selected according to the purpose. The farnesic acid may be selected on a medium such as a liquid medium, a plate medium, a slant medium, a half slant medium, or the like. A grown product obtained by culturing the production yeast can be used.

The culture method is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include shaking culture, stationary culture, and tank culture.
The temperature of the culture is not particularly limited as long as it is within a range that can produce the farnesic acid without substantially inhibiting the growth of the farnesic acid-producing yeast, and is appropriately selected according to the producing bacteria to be used. However, 10 ° C. to 40 ° C. is preferable, and 35 ° C. to 40 ° C. is particularly preferable in terms of good production efficiency of farnesic acid.
The pH of the culture is not particularly limited as long as it does not substantially inhibit the growth of the farnesic acid-producing yeast and can produce the farnesic acid, depending on the farnesic acid-producing yeast to be used. For example, pH 4.0-7.5 etc. are mentioned.
There is no restriction | limiting in particular as the period of the said culture | cultivation, According to accumulation | storage of the said farnesic acid, it can select suitably.

<Collection process>
The collecting step is a step of collecting farnesic acid from the culture obtained in the culturing step.

The culture is not particularly limited as long as it is obtained in the culture step and contains the farnesic acid, and can be appropriately selected according to the purpose. The cells, the culture supernatant after liquid culture, Examples thereof include a solid medium after solid culture and a mixture thereof.
When the cells are used as the culture, the farnesyl acid is extracted from the cells by an extraction method using an appropriate organic solvent, an elution method by disrupting the cells, etc. Or you may use for refinement | purification.

  The collection method is not particularly limited, and a method used for collecting a metabolite produced by a microorganism can be appropriately selected. For example, a solvent extraction method, a method using a difference in adsorption affinity for various adsorbents, a chromatographic method, and the like can be mentioned. These farnesyl acids separated and / or purified can be collected by using these methods alone or in combination as appropriate, and repeatedly using them in some cases.

  There is no restriction | limiting in particular as a solvent used for the said solvent extraction method, According to the objective, it can select suitably, For example, diethyl ether, ethanol, methanol, acetone, butanol, acetonitrile, dimethyl sulfoxide (DMSO) etc. are mentioned. . These may be used alone or in combination of two or more.

  There is no restriction | limiting in particular as said adsorbent, According to the objective, it can select suitably from well-known adsorbents, For example, a polystyrene type adsorption resin etc. are mentioned.

The chromatographic method is not particularly limited and may be appropriately selected depending on the intended purpose. For example, gas chromatogram method, thin layer chromatographic method, high performance liquid chromatograph (HPLC) using normal phase or reverse phase column ) Method.
There is no restriction | limiting in particular as a support | carrier used for the said chromatographic method, According to the objective, it can select suitably, For example, ion exchange resin, gel filtration, a silica gel, an alumina, activated carbon etc. are mentioned.

  The method for eluting the farnesic acid from the adsorbent or the carrier in the chromatographic method is not particularly limited and may be appropriately selected depending on the kind and properties of the adsorbent and the carrier.

The farnesic acid can be produced as described above.
In addition, there is no restriction | limiting in particular as the grade of the refinement | purification of the said farnesic acid in the said collection | recovery process, A crudely purified product may be sufficient and the crudely purified product may be refined | purified.

<Other processes>
There is no restriction | limiting in particular as said other process, According to the objective, it can select suitably, A washing | cleaning process, a refinement | purification process, etc. are mentioned.
The washing step is a step of washing the culture obtained in the culturing step or the farnesic acid obtained in the collecting step. There is no restriction | limiting in particular as the said washing | cleaning method, A well-known method can be selected suitably and can be performed.
The purification step is a step of purifying the farnesic acid obtained in the collecting step. There is no restriction | limiting in particular as the said purification method, A well-known method can be selected suitably and can be performed, for example, the method similar to the method described in the said collection process is mentioned.

  The method for producing farnesic acid is advantageous in that farnesyl acid can be produced efficiently and stably by an inexpensive and simple method.

(Method for producing farnesyl acid-producing yeast)
The method for producing a farnesic acid-producing yeast according to the present invention comprises substituting guanine at position 714 of the ERG9 gene encoding squalene synthase in Saccharomyces cerevisiae with adenine. This is a method for producing a yeast for producing farnesic acid by mutating the erg9-2 gene.
In Saccharomyces cerevisiae , guanine at position 714 in the base sequence of the ERG9 gene encoding squalene synthase is replaced with adenine, so that tryptophan at amino acid sequence 238 in squalene synthase is replaced with a start codon. As a result, farnesyl acid productivity can be imparted to the yeast.

  There is no restriction | limiting in particular as said Saccharomyces cerevisiae, According to the objective, it can select suitably, For example, sake yeast, shochu yeast, wine yeast, beer yeast, baker's yeast, etc. are mentioned. These may be used alone or in combination of two or more. Among these, sake yeast is preferable.

The method for mutating the ERG9 gene in Saccharomyces cerevisiae to the erg9-2 gene is not particularly limited and may be appropriately selected depending on the intended purpose. Examples include mutation, trait conjugation, and gene recombination.
The mutation may be a natural mutation or a method of artificially performing a mutation process. The method for artificially performing the mutation treatment is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include ultraviolet irradiation, X-ray irradiation, radiation irradiation, and chemical treatment.
Among these, the method of artificially performing the mutation treatment is preferable because mutation is easily induced, and ultraviolet irradiation is particularly preferable.

  The method for selecting a strain in which the ERG9 gene in Saccharomyces cerevisiae is mutated to the erg9-2 gene is not particularly limited and can be appropriately selected according to the purpose. For example, the productivity of farnesyl acid is used as an indicator. The method of selection is mentioned.

Examples of the method for confirming the productivity of the farnesic acid include the following methods.
After the strain to be selected is cultured in a YPD medium liquid medium at 35 ° C. for 3 days, the cultured cells are freeze-dried, and farnesyl acid in the freeze-dried cells is usually used in a solvent. By extracting by a method and analyzing farnesic acid using high performance liquid chromatography (HPLC), a strain in which farnesyl acid is detected can be selected as a yeast having farnesyl acid productivity.
There is no restriction | limiting in particular as said solvent, According to the objective, it can select suitably, For example, diethyl ether, ethanol, methanol, acetone, butanol, acetonitrile, dimethylsulfoxide (DMSO) etc. are mentioned. These may be used alone or in combination of two or more.

  The yeast for producing farnesic acid is further produced by backcrossing the yeast having farnesyl acid productivity with a yeast haploid to form spores, and then dissecting the ascomb, and then producing the farnesic acid with high yield. Crossing between body strains to produce a diploid farnesyl acid-producing yeast is preferred in that a farnesyl acid-producing yeast that produces farnesyl acid at a higher concentration can be obtained.

  There is no restriction | limiting in particular as said yeast haploid, According to the objective, it can select suitably, For example, sake yeast etc. are mentioned. These may be used alone or in combination of two or more.

  The method for confirming that the ERG9 gene is mutated to the erg9-2 gene in the yeast having farnesyl acid productivity is not particularly limited and can be appropriately selected depending on the purpose. And a DNA sequence analysis method.

  The farnesic acid-producing yeast produced by the above-described method for producing farnesic acid-producing yeast is a yeast having the mycological properties described in the section <Farnesic acid-producing yeast> of the “Farnesic acid-producing yeast”. Preferably, it is Saccharomyces cerevisiae to which the strain number of NITE P-02406 is attached.

(Sake lees and its manufacturing method)
The method for producing sake lees according to the present invention is a method for producing sake lees using the farnesyl acid producing composition.
Moreover, the sake lees of the present invention contain at least farnesic acid, and further contain other components as necessary. The sake lees can be suitably manufactured by the method for producing the sake lees.
Hereinafter, the sake lees will be described in detail together with the description of the method for producing the sake lees.

<Manufacturing method of sake lees>
The method for producing the sake lees is not particularly limited as long as the composition for producing farnesyl acid is used, and can be appropriately selected according to the purpose.The initial addition treatment, the intermediate treatment, and the distillation treatment are performed. It is preferable to include the koji making process and the upper tank process, and may further include other processes as necessary.

<< Sashimi making process >>
In sake brewing, the koji making process is basically performed in a three-stage process of initial addition processing, intermediate addition processing, and distillation processing. In the three-stage preparation, rice bran, steamed rice (hereinafter sometimes referred to as “kake rice”), and water (hereinafter sometimes referred to as “pumped water”) are added at each stage.

-Initial treatment-
The initial addition process is a process of adding rice cake, rice bran, scooped water, and the farnesic acid producing composition to a container charged with a liquor mother. In the initial addition treatment, lactic acid may be further added, or a liquor may be previously charged in the container.

  There is no restriction | limiting in particular as a manufacturing method of the said liquor mother, A well-known method can be suitably selected according to the objective.

  There is no restriction | limiting in particular as the usage-amount of the said composition for farnesyl acid production and lactic acid, According to the objective, it can select suitably.

  There is no restriction | limiting in particular as hook rice in the said initial addition process, According to the objective, it can select suitably, For example, the rice etc. which polished rice to 75% of refinement | purification rates are mentioned.

There is no restriction | limiting in particular as a koji in the said initial addition process, According to the objective, it can select suitably, For example, what made the koji mold grow to what steamed polished rice of refinement | purification ratio 75% etc. is mentioned. There is no restriction | limiting in particular as said koji mold, A well-known koji mold can be suitably selected according to the objective.
There is no restriction | limiting in particular as the addition amount of the soot in the said initial addition process, According to the objective, it can select suitably.

  Moreover, there is no restriction | limiting in particular also as the addition amount of a rice cake and rice bran (henceforth "total rice") in the said initial addition process, According to the objective, it can select suitably.

-Mediation processing-
The initial addition process is a process of adding rice cake, rice bran, and pumped water to the initial addition process.

  There is no restriction | limiting in particular as the time which performs the said intermediate addition process, According to the objective, it can select suitably, For example, the 2nd day after the said initial addition process etc. are mentioned.

  There is no restriction | limiting in particular as the rice and rice bran in the said intermediate addition process, According to the objective, it can select suitably, For example, the thing similar to the said initial addition process is mentioned.

  There is no restriction | limiting in particular as addition amount of the rice cake, rice bran, and total rice in the said intermediate | middle process, According to the objective, it can select suitably.

-Deposition treatment-
The distillation process is a process of adding rice cake, rice bran, and pumped water to the intermediated one. In the distillation treatment, an enzyme agent may be added as necessary.

  There is no restriction | limiting in particular as the time which performs the said addition treatment, According to the objective, it can select suitably, For example, the 1st day after the said intermediate treatment etc. are mentioned.

  There is no restriction | limiting in particular as the rice and rice bran in the said addition process, According to the objective, it can select suitably, For example, the thing similar to the said initial addition process is mentioned.

  There is no restriction | limiting in particular as the addition amount of rice cake, rice bran, and total rice in the said distillation process, According to the objective, it can select suitably.

There is no restriction | limiting in particular as said enzyme agent, A well-known enzyme agent can be selected suitably. The said enzyme agent may be used individually by 1 type, and may use 2 or more types together.
There is no restriction | limiting in particular as addition amount of the said enzyme agent, According to the objective, it can select suitably.

There is no restriction | limiting in particular as temperature of the said koji making process, According to the objective, it can select suitably, For example, 15 degreeC constant etc. are mentioned.
Moreover, there is no restriction | limiting in particular as a timing which carries out the tank obtained from the said koji making process, According to the objective, it can select suitably.

<< Upper tank process >>
The upper tank process is a process of separating the koji obtained in the koji making process into sake and sake lees.
There is no restriction | limiting in particular as a method of isolate | separating the said koji into refined sake and sake lees, According to the objective, it can select suitably, For example, the method etc. which put in a pressing machine, etc. are mentioned.

<< Other processes >>
As said other process, unless the effect of this invention is impaired, there is no restriction | limiting in particular, The process performed with the manufacturing method of a well-known liquor can be performed suitably.

<Sake lees>
The form of the sake lees can be appropriately selected according to the use and the like, and may be the sake lees themselves, the dried liquor form, the solid form (such as those obtained by removing water from the liquor) In addition, it may be in the form of a liquid (such as those obtained by removing solid content from the sake lees), or in the form of a liquor extract obtained by extracting the liquor with a solvent or the like.

  There is no restriction | limiting in particular as said solvent, According to the objective, it can select suitably, For example, diethyl ether, ethanol, methanol, acetone, butanol, acetonitrile, dimethylsulfoxide (DMSO) etc. are mentioned. These may be used alone or in combination of two or more.

  Since the sake lees contain farnesic acid, it is advantageous in that it has anti-obesity and anti-hyperlipidemic effects.

(Yeast culture)
The yeast culture of the present invention contains at least farnesic acid, and further contains other components as necessary.

  The yeast culture is not particularly limited as long as it is a yeast culture containing farnesic acid, and can be appropriately selected according to the purpose. However, the yeast for producing farnesic acid in the “composition for producing farnesic acid” Preferably, the culture is

The culture is not particularly limited as long as it contains farnesyl acid, and can be appropriately selected according to the purpose. Yeast cells, yeast cell disruptions, Examples thereof include a solid medium after solid culture of yeast and yeast, and a mixture thereof.
In addition, the crushed material of the said yeast microbial cell means the state from which the crushed microbial cell was lost under microscope observation.

<Farnesic acid>
There is no restriction | limiting in particular as content of the said farnesic acid in the said yeast culture, According to the objective, it can select suitably.

<Other ingredients>
There is no restriction | limiting in particular as said other component, According to the objective, it can select suitably, For example, the culture medium component at the time of culture | cultivating the said yeast etc. are mentioned.

  The medium component is not particularly limited and may be appropriately selected depending on the intended purpose. Examples of the medium component include nutrient medium components described in the section <Culture process> in the above-mentioned “farnesyl acid production method”.

  There is no restriction | limiting in particular as content of the said other component in the said yeast culture, According to the objective, it can select suitably.

  The form of the yeast culture can be appropriately selected according to the use and the like, and may be a culture itself, or a dry form of the culture, a solid form (from a culture to a culture solution, etc. Etc.), liquid forms (such as those obtained by removing solids from the culture), and extracts obtained by extracting the culture with a solvent or the like.

  There is no restriction | limiting in particular as said solvent, According to the objective, it can select suitably, For example, diethyl ether, ethanol, methanol, acetone, butanol, acetonitrile, dimethylsulfoxide (DMSO) etc. are mentioned. These may be used alone or in combination of two or more.

  Since the yeast culture contains farnesic acid, it is advantageous in that it has anti-obesity and anti-hyperlipidemic effects.

<Method for producing yeast culture>
The present invention also relates to a method for producing the yeast culture.
The method for producing the yeast culture is not particularly limited as long as the yeast culture containing at least the farnesic acid can be produced, and can be appropriately selected according to the purpose. Is preferable, and other steps may be included as necessary.

<< Culture process >>
The culturing step is a step of culturing yeast having the ability to produce farnesic acid.
As the yeast having the ability to produce farnesic acid, it is preferable to use the yeast for producing farnesic acid in the “composition for producing farnesic acid”.
The method for culturing is not particularly limited and may be appropriately selected depending on the intended purpose. However, the method described in <Culture Step> in the above-mentioned “Method for producing farnesic acid” is preferably used.

<< Other processes >>
There is no restriction | limiting in particular as said other process, According to the objective, it can select suitably, A crushing process, a washing | cleaning process, etc. are mentioned.

  The crushing step is a step of crushing the cells obtained in the culturing step. The method for disrupting the cells is not particularly limited as long as it is a method that eliminates unbroken cells under microscopic observation, and can be appropriately selected according to the purpose. For example, physical disruption, chemical Crushing treatment method. These treatment methods can be performed according to a known method using a known device or the like.

  The washing step is a step of washing the yeast culture obtained in the culturing step. There is no restriction | limiting in particular as the said washing | cleaning method, A well-known method can be selected suitably and can be performed.

  The yeast culture is a pharmaceutical composition including pharmaceuticals and quasi-drugs; cosmetics; special foods such as general foods, general foods, health foods, health functional foods (nutrient functional foods and foods for specified health), and foods for the sick. It can be suitably used for foods such as foods for use.

  The yeast culture may be used alone or in combination with a pharmaceutical composition, cosmetic or food containing other ingredients as active ingredients. Moreover, the said composition may be used in the state mix | blended in the pharmaceutical composition which uses another component as an active ingredient, cosmetics, or a foodstuff.

  The present invention will be specifically described below with reference to production examples and test examples, but the present invention is not limited to these production examples and test examples.

(Production Example 1)
<Manufacture of yeast for producing farnesyl acid>
A wild-type laboratory yeast strain X2180-1A strain (ATCC (registered trademark) 204504, MATa) was induced by irradiating with ultraviolet rays, and then selected using the productivity of farnesic acid confirmed by the following method as an index, and sequenced. As a result, it was confirmed that guanine at position 714 in the base sequence of the ERG9 gene was replaced with adenine and mutated to the erg9-2 gene.
Next, the strain mutated to the erg9-2 gene and the haploid of sake yeast are crossed, sporulated, then dissected ascending, and then the farnesyl acid productivity is indicated by the same method as described above. The haploid strains that produced a high yield of farnesic acid selected as described above were crossed to obtain diploid farnesic acid-producing yeast. This farnesyl acid-producing yeast was designated as “HL305 strain”.

<< Confirmation of farnesyl acid productivity >>
The strain to be selected is cultured at 35 ° C. for 3 days in a YPD liquid medium (yeast extract 10 g, peptone 20 g, glucose 20 g / water 1,000 mL, the same applies hereinafter), and the cultured cells are lyophilized. did. To 60 mg of the lyophilized cells, 400 μL of dimethyl sulfoxide (DMSO) is added and suspended sufficiently by vortexing, then centrifuged at 10,000 rpm for 5 minutes, and the supernatant is filtered (product name: Minisart RC4, pore size). : 0.45 μm, manufactured by Satorius). Farnesyl acid in the supernatant was analyzed by high performance liquid chromatography (HPLC) under the following conditions.
-HPLC conditions-
-Device: LC-2000 Plus series (manufactured by JASCO Corporation)
(Intelligent HPLC pump PU-2080, Degasser DG-2080-53 (triple), Dynamic mixer MX-280-32, Intelligent autosampler AS-2057, Intelligent column oven CO-2067, UV / VIS Wavelength detector MD-2010 type)
Column: Xbridge BEH C18 column, 130 mm, particle size: 3.5 μm, inner diameter: 2.1 mm, length: 150 mm (manufactured by Waters)
Mobile phase: [A] distilled water / [B] acetonitrile (gradient conditions are shown in Table 1 below)
・ Column temperature: 50 ℃
・ Injection volume: 10 μL
・ Flow rate: 0.3 mL / min

The mycological properties of the HL305 strain are shown below.
(1) Bacterial morphology when grown in YPD liquid medium Vegetative cell size: 4 μm to 8 μm, width: 4 μm to 6 μm
2. 2. Vegetative cell shape: egg shape Form of growth: budding (2) Physiological properties a. Sugar assimilation glucose: +
Galactose: +
Sucrose: +
Maltose: +
Lactose:-
b. Fermentation of sugar Glucose: +
Galactose: +
Sucrose: +
Maltose: +
Lactose:-
Melibiose:-
Raffinose: +
(3) Bondability Since it is a diploid, there is no bondability.
(4) Spore formation As a result of culturing HL305 strain in a spore formation medium at 30 ° C. for 4 days, spore formation was performed.

From the above bacteriological properties, it is shown that the HL305 strain is a yeast belonging to Saccharomyces cerevisiae ( Saccharomyces cerevisiae ).

(Test Example 1)
The HL305 strain and sake yeast were each cultured in a YPD liquid medium at 35 ° C. for 3 days, and the cultured cells were lyophilized. To 60 mg of the lyophilized cells, 400 μL of dimethyl sulfoxide (DMSO) is added and suspended sufficiently by vortexing, then centrifuged at 10,000 rpm for 5 minutes, and the supernatant is filtered (product name: Minisart RC4, pore size). : 0.45 μm, manufactured by Satorius Co., Ltd.) to obtain an extract of each culture solution. Each obtained extract and a standard product of farnesyl acid (manufactured by Echelon Biosciences) were analyzed by high performance liquid chromatography (HPLC) under the same conditions as in Production Example 1.

  FIG. 1 shows a liquid chromatogram of an extract of the HL305 strain, FIG. 2 shows a liquid chromatogram of an extract of sake yeast, and FIG. 3 shows a liquid chromatogram of a farnesyl acid standard product. The results of quantifying farnesic acid production are shown in Table 2 below.

  From the result of the standard product (FIG. 3), the retention time of farnesic acid was 36.5 minutes, and farnesic acid was detected in the extract of HL305 strain (FIG. 1 and Table 2). On the other hand, farnesyl acid was not detected in the extract of sake yeast (FIG. 2 and Table 2).

(Test Example 2)
<Small preparation test>
A small preparation test was performed by the following method using HL305 strain and sake yeast.
The HL305 strain and sake yeast were each cultured overnight at 30 ° C. in 20 mL of YPD liquid medium. After incubation, the mixture was centrifuged at 4,000 rpm for 10 minutes to collect HL305 strain and sake yeast, washed with distilled water, suspended in 5 mL of distilled water, and the whole amount was used for a small preparation test. The small preparation test was conducted in a three-stage preparation using the preparation composition shown in Table 3 below. The koji was fermented at a constant temperature of 15 ° C. Once a day, the fermentation progress was observed by measuring the amount of CO ₂ weight reduction of the koji, and the upper tank was observed on the 12th day of fermentation, and separated into sake lees and sake.

<Farnesic acid content in sake lees>
The resulting HL305 strain and sake yeast sake lees were freeze-dried. To 60 mg of the lyophilized sake cake, 400 μL of dimethyl sulfoxide (DMSO) is added and suspended sufficiently by vortexing, then centrifuged at 10,000 rpm for 5 minutes, and the supernatant is filtered (product name: Minisart RC4, pore size: Each extract of sake lees was prepared by filtration through 0.45 μm, manufactured by Satorius. The content of farnesic acid in this extract of sake lees was quantified under the same conditions as the HPLC conditions described in Production Example 1. The results are shown in Table 4 below.

  Farnesic acid was detected in the sake extract of HL305 strain. On the other hand, farnesic acid was not detected in the sake yeast extract of sake yeast.

<General analysis value>
Moreover, the general analysis value of the obtained sake is shown in Table 5 below.

  The HL305 strain showed a sufficient amount of alcohol productivity.

(Test Example 3)
<High-temperature stability of farnesic acid and farnesol>
The HL305 strain was cultured in a YPD liquid medium at 35 ° C. for 3 days, and the cultured cells were lyophilized. The freeze-dried cells were placed in a vial and allowed to stand for 1 month in a constant temperature room at 55 ° C. to obtain a high-temperature storage sample. To this high-temperature storage sample and 60 mg of lyophilized cells before high-temperature storage, 400 μL of dimethyl sulfoxide (DMSO) is added and suspended sufficiently by vortexing, then centrifuged at 10,000 rpm for 5 minutes, and the supernatant is filtered. The extract of each microbial cell was obtained by filtering with (Product name: Minisart RC4, the hole diameter: 0.45 micrometer, the product made by Satorius). Each obtained extract was analyzed by high performance liquid chromatography (HPLC) under the same conditions as in Production Example 1 to detect farnesic acid and farnesol. The retention time of farnesol is 38.0 minutes.

FIG. 4 shows a liquid chromatogram of the extract of the HL305 strain before high-temperature storage, and FIG. 5 shows a liquid chromatogram of the extract of the HL305 strain as a high-temperature storage sample. 4 and 5, peak 2 represents a farnesic acid peak, and peak 3 represents a farnesol peak.
Based on the peak areas of FIGS. 4 and 5, for each of farnesic acid (peak 2) and farnesol (peak 3), the ratio of the peak area after high temperature storage to the peak area before high temperature storage, The residual ratio of the compound was calculated.
Table 6 below shows the peak areas of farnesic acid and farnesol in the extract of HL305 strain before and after high-temperature storage, and the residual ratio of each compound after high-temperature storage.

  From the results in Table 6, farnesyl acid had a higher residual rate after high-temperature storage. This indicates that farnesyl acid is more stable under high temperature conditions than farnesol.

Examples of the aspect of the present invention include the following.
<1> Yeast for farnesic acid production which is Saccharomyces cerevisiae mutated to the gene represented by SEQ ID NO: 1 in which guanine at position 714 of the base sequence of the ERG9 gene encoding squalene synthase is replaced with adenine Is a method for producing farnesic acid.
<2> The method for producing farnesic acid according to <1>, wherein the yeast for producing farnesic acid is Saccharomyces cerevisiae HL305 strain (accession number: NITE P-02406).
<3> Yeast for farnesic acid production which is Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) mutated to the gene represented by SEQ ID NO: 1 in which the guanine at position 714 of the base sequence of the ERG9 gene encoding squalene synthase is replaced with adenine Is a composition for producing farnesic acid.
<4> The composition for producing farnesic acid according to the above <3>, wherein the yeast for producing farnesic acid is Saccharomyces cerevisiae HL305 strain (accession number: NITE P-02406).
<5> Saccharomyces cerevisiae HL305 strain (Accession number: NITE P-02406), which is a yeast characterized by having the ability to produce farnesic acid.
<6> Farnesyl, characterized in that in Saccharomyces cerevisiae , guanine at the base sequence position 714 of the ERG9 gene encoding squalene synthase is substituted with adenine and mutated to the gene represented by SEQ ID NO: 1. It is the manufacturing method of the yeast for acid production.
<7> The method for producing a farnesic acid-producing yeast according to the above <6>, wherein the farnesyl acid-producing yeast is Saccharomyces cerevisiae HL305 strain (accession number: NITE P-02406).
<8> A method for producing a sake lees characterized by using the farnesic acid producing composition according to any one of <3> to <4>.
<9> A sake lees characterized by containing farnesic acid.
<10> A yeast culture characterized by containing farnesic acid.

  NITE P-02406

Claims (10)

Use of a yeast for producing farnesic acid which is Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) mutated to the gene represented by SEQ ID NO: 1 in which the guanine at position 714 of the base sequence of the ERG9 gene encoding squalene synthase is replaced with adenine A process for producing farnesic acid characterized by the above. The method for producing farnesic acid according to claim 1, wherein the yeast for producing farnesic acid is Saccharomyces cerevisiae HL305 strain (accession number: NITE P-02406). Containing a yeast for producing farnesic acid, which is Saccharomyces cerevisiae mutated to the gene represented by SEQ ID NO: 1 in which the guanine at position 714 of the base sequence of the ERG9 gene encoding squalene synthase is replaced with adenine A composition for producing farnesic acid. The composition for producing farnesic acid according to claim 3, wherein the yeast for producing farnesic acid is Saccharomyces cerevisiae HL305 strain (accession number: NITE P-02406). Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) HL305 strain (Accession number: NITE P-02406), which has the ability to produce farnesic acid. For farnesic acid production, characterized in that in Saccharomyces cerevisiae , guanine at the base sequence position 714 of the ERG9 gene encoding squalene synthase is substituted with adenine and mutated to the gene represented by SEQ ID NO: 1. Yeast production method. The method for producing a yeast for producing farnesic acid according to claim 6, wherein the yeast for producing farnesic acid is Saccharomyces cerevisiae HL305 strain (accession number: NITE P-02406).   A method for producing a sake lees characterized by using the farnesic acid-producing composition according to claim 3.   A sake lees characterized by containing farnesyl acid. A yeast culture characterized by containing farnesic acid.