WO2021066145A1 - Oil/fat accumulation induction medium for oil/fat producing yeast - Google Patents
Oil/fat accumulation induction medium for oil/fat producing yeast Download PDFInfo
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- WO2021066145A1 WO2021066145A1 PCT/JP2020/037550 JP2020037550W WO2021066145A1 WO 2021066145 A1 WO2021066145 A1 WO 2021066145A1 JP 2020037550 W JP2020037550 W JP 2020037550W WO 2021066145 A1 WO2021066145 A1 WO 2021066145A1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
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- the present invention relates to a fat accumulation-inducing medium and a fat accumulation-inducing method for fat-producing yeast.
- Non-Patent Document 1 an Ls medium having the composition shown in Table 1 in Example 1 described later (Non-Patent Document 1) is known.
- An object of the present invention is to provide a fat accumulation induction medium and a fat accumulation induction method capable of reducing the cost of the medium itself and increasing the amount of fat accumulation in cells.
- a fat accumulation-inducing medium for fat-producing yeast containing sugars and ammonium sulfate [1] A method for inducing fat accumulation of fat-producing yeast, which comprises the step of culturing in the medium of [1]. Can be solved by.
- the present invention it is possible to provide a fat accumulation induction medium and a fat accumulation induction method capable of reducing the cost of the medium itself and increasing the amount of fat accumulation in cells.
- lipomyces were cultured using various media having different presence or absence of biotin, sucrose, and ammonium sulfate, and 25 hours after culturing. It is a photograph instead of a drawing showing the state of lipomyces. In order to examine the effect of ammonium sulfate concentration on fat accumulation, lipomyces are cultured using media having different ammonium sulfate concentrations, and the state of lipomyces after 24, 48, and 70 hours of culture is shown in a photograph instead of a drawing.
- the fat accumulation-inducing medium or the fat accumulation-inducing method of the present invention can be used to induce intracellular fat-accumulation in fat-producing yeast.
- the fat-producing yeast that can be used in the present invention is not particularly limited as long as it is a yeast that can accumulate fats and oils in cells. Yeasts belonging to the genus Endomyces, Candida, Rhodotorula, Cryptococcus, or Rhodosporidium can be mentioned, with yeast belonging to the genus Lipomyces being preferred. ..
- fat and oil means triglyceride (neutral fat) which is an ester of fatty acid and glycerin.
- the fat-producing yeast produces triglyceride and accumulates in the cells.
- the oil / fat accumulation induction medium of the present invention contains saccharides and does not contain ammonium sulfate.
- the oil / fat accumulation induction medium of the present invention does not contain ammonium sulfate.
- the present inventor has been searching for a fat accumulation-inducing medium having a higher fat-accumulation efficiency than the Ls medium, which is one of the conventionally known fat-accumulation-inducing media.
- the initial composition was set, the reason why the oil and fat accumulation efficiency was lower when the Ls medium was used as compared with the medium of the present invention was further investigated. As shown in Example 5, ammonium sulfate had an effect of suppressing oil and fat accumulation. Found to show.
- ammonium sulfate (5.3 g / L) is the most abundant inorganic salt among the inorganic salts contained in the Ls medium, and it is highly expected that ammonium sulfate will exhibit an action of suppressing fat accumulation. It was an outside finding.
- ammonium sulfate-free means that it does not contain an amount of ammonium sulfate exhibiting an oil / fat accumulation inhibitory effect, and more specifically, in an embodiment having an ammonium sulfate content, it is 5.3 g / L or less. 1 g / L or less in some embodiments, 0.5 g / L or less in some embodiments, 0.1 g / L or less in some embodiments, 50 mg / L or less in some embodiments, 10 mg / L or less in some embodiments, 10 mg / L or less in some embodiments. It means that it is 5 mg / L or less.
- the oil / fat accumulation induction medium of the present invention contains saccharides as a carbon source.
- saccharide include glucose, sucrose, sorbitol, galactose, raffinose, fructose, xylose, trehalose, maltose, lactose, and the like, and one kind selected from these saccharides or two or more kinds are combined. It can be used together.
- the values are 15% in some embodiments, 10% in some embodiments, 5% in some embodiments, 3% in some embodiments, 1% in some embodiments, and 0.5% in some embodiments.
- the lower limit and the upper limit can be arbitrarily combined as desired.
- the pH of the fat accumulation-inducing medium of the present invention is not particularly limited as long as it can accumulate fats and oils in cells, and is pH 3 to 8 in some embodiments and pH 4 to 7 in some embodiments. In some embodiments, the pH is 4.5-6.5.
- the lower limit and the upper limit can be arbitrarily combined as desired.
- the oil / fat accumulation induction medium of the present invention may contain at least one inorganic salt, if desired.
- the inorganic salt include potassium dihydrogen phosphate, magnesium sulfate, iron chloride, copper chloride, zinc sulfate, sodium molybdate, and potassium iodide (hereinafter, these may be collectively referred to as a specific inorganic salt).
- a specific inorganic salt potassium dihydrogen phosphate, magnesium sulfate, iron chloride, copper chloride, zinc sulfate, sodium molybdate, and potassium iodide.
- the oil / fat accumulation induction medium of the present invention may contain an inorganic salt other than the specific inorganic salt exemplified above, if desired.
- the inorganic salt other than the specific inorganic salt include potassium hydroxide or sodium hydroxide that can be used to adjust the pH, or calcium chloride or sodium chloride contained in a conventionally known fat accumulation induction medium. it can.
- One or more of these inorganic salts may be used together with the specific inorganic salt, if desired, but from the viewpoint of cost, it is preferable that no inorganic salt other than the specific inorganic salt is contained. For example, as compared with the conventionally known Ls medium, the types of inorganic salts can be reduced, and the cost applied to the medium can be reduced.
- oil / fat accumulation induction medium of the present invention can contain vitamins such as biotin, thiamine, ascorbic acid, pantothenic acid, folic acid, niacin and the like, if desired.
- the amount of each inorganic salt and each vitamin (particularly biotin) contained in the oil / fat accumulation induction medium of the present invention is not particularly limited as long as it can accumulate oil / fat in cells, and is not particularly limited. It can be appropriately determined based on the specific description of the examples to be carried out. For example, based on each content shown in Table 1 described later, in some embodiments, the amount is 1/2 to 2 times the reference amount, and in some embodiments, 1/5 to 5 times the reference amount. In some embodiments, 1/10 to 10 times the reference amount, in some embodiments 1/20 to 20 times the reference amount, in some embodiments 1/50 to 50 times the reference amount, in some embodiments. An amount of 1/100 to 100 times the reference amount can be used. The lower limit and the upper limit can be arbitrarily combined as desired.
- the oil / fat accumulation induction method of the present invention can be carried out according to a conventionally known oil / fat accumulation induction method, except that culturing is performed using the oil / fat accumulation induction medium of the present invention.
- the main culture can be carried out by pre-culturing in advance and inoculating the culture into the oil / fat accumulation induction medium of the present invention.
- the culture temperature can be, for example, 20 to 35 ° C., and the culture is shaken for about 1 to 7 days.
- the fat accumulation in the fat accumulation yeast can be confirmed and quantified by a conventionally known method.
- a method of separating and quantifying the oil and fat components present in yeast can be mentioned.
- Example 1 Confirmation of increase in fat accumulation amount by the medium of the present invention
- Table 1 shows the initial composition of the medium of the present invention initially set by the present inventor and the composition of the conventionally known Ls medium (Non-Patent Document 1).
- Non-Patent Document 1 the difference in intracellular fat and oil accumulation in Lipomyces was confirmed using the medium of the present invention having the composition shown in Table 1 and the Ls medium shown in Table 1 as a comparative example.
- Lipomyces starkeyi was used as the lipomyces. Lipomyces starkeyi was cultured in YPD medium at 25 ° C. for 1 to 2 days, the cells were washed with sterile water, implanted in Ls medium or the medium of the present invention, and cultured at 25 ° C. A part of the culture solution was separated and fixed with 2.5% glutaraldehyde. The fixed cells were washed and then stained with 0.5% Oil red stain for 30 minutes to 1 hour. After washing the cells, microscopic observation was performed. In addition, a cell extract was prepared by lysing or physically destroying cells, and the fat and oil components existing in the extract were separated, and the amount of intracellular fat and oil accumulated was calculated.
- FIG. 1 The state after 24 hours of culturing is shown in FIG.
- LS medium shown in FIG. 1 Intracellular fat and oil accumulation was about 3% per single cell, whereas the medium of the present invention having the composition shown in Table 1 was used.
- New LS medium shown in FIG. 1 oil and fat accumulation of 30% or more per single cell was confirmed.
- Example 2 Examination of sugar concentration in the medium of the present invention >> In this example, the effect of sugar concentration on fat accumulation was examined.
- the medium of the present invention in the initial composition shown in Table 1, 3 g of sucrose was replaced with 30 g of sucrose (3% sucrose concentration), 3 g (0.3% sucrose), and 0.3 g (0.03% sucrose). 3 types of sucrose-containing medium and 3 types of glucose in which 3 g of sucrose was replaced with 30 g of glucose (3% glucose concentration), 3 g (0.3% of the same), and 0.3 g (0.03% of the same). A medium was prepared.
- the state before culturing (0 hr) and the state after 24 hours of culturing (24 hr) are shown in FIG.
- the medium of the present invention was used (New Ls medium shown in FIG. 2)
- fats and oils were accumulated at sugar concentrations of 3% and 0.3% for both sucrose and glucose sugars, but cells were accumulated at sugar concentrations of 0.03%.
- the morphology was distorted and the efficiency of fat accumulation was not good.
- Ls medium having a sugar concentration of 0.3% Ls medium shown in FIG. 2
- the cells proliferated almost no fats and oils were accumulated.
- Example 3 Examination of the type of sugar in the medium of the present invention >> In this example, the effect of the type of sugar on the accumulation of fats and oils was examined.
- the medium in the initial composition shown in Table 1, 3 g of sucrose, 3 g of glucose (glucose concentration 0.3%), 3 g of sucrose (sucrose concentration 0.3%), 3 g of sorbitol (sorbitol concentration 0.3%), Six types of media were prepared in place of galactose 3 g (galactose concentration 0.3%), raffinose 3 g (raffinose concentration 0.3%), and glycerol 3 g (glycerol concentration 0.3%). The culture was carried out in the same manner as in Example 1.
- Fats and oils were accumulated in each medium containing sugars other than glycerol, that is, glucose, sucrose, sorbitol, galactose, and raffinose, but no fats and oils were accumulated in the glycerol-containing medium.
- Example 4 Examination of pH in the medium of the present invention >>
- the effect of pH in the medium of the present invention on fat accumulation was investigated.
- a medium having a pH of 6.5 having the initial composition shown in Table 1 and a medium having a pH of 4.5 obtained by removing potassium hydroxide from the initial composition shown in Table 1 were prepared.
- the pH of the medium was based on the presence or absence of potassium hydroxide, and the pH was not separately adjusted with an acid or an alkali.
- Example 5 Examination of types of inorganic salts in the medium of the present invention (1) >>
- inorganic salts ammonium sulfate, calcium chloride, sodium chloride
- the medium of pH 4.5 prepared in Example 4 (New Ls pH 4.5 shown in FIG. 4) was used as the basic medium, and the media (1) to (8) to which various inorganic salts shown in Table 2 were added.
- the amount added was the same as the content of ammonium sulfate, calcium chloride, and sodium chloride in the Ls medium shown in Table 1.
- Example 6 Examination of types of inorganic salts in the medium of the present invention (2) >>
- the inorganic salts commonly contained in the medium of the present invention and the Ls medium potassium dihydrogen phosphate, magnesium sulfate, iron chloride, copper chloride, zinc sulfate, sodium molybdate, or potassium iodide; hereinafter, these are used.
- the effect of (sometimes collectively referred to as specific inorganic salt) on fat accumulation was investigated.
- potassium dihydrogen phosphate and magnesium sulfate it was confirmed in another experiment that fats and oils accumulated even if they were not added, so this was not carried out.
- a medium obtained by removing potassium dihydrogen phosphate and magnesium sulfate from the medium having a pH of 4.5 (New Ls pH 4.5 shown in FIG. 4) prepared in Example 4 as a basic medium is further described in Table 3.
- (1) to (6) [however, the medium (6) is the basal medium in Example 6] were prepared.
- the composition of the basal medium in this example is shown in Table 4.
- Example 7 Determination of essential components in the medium of the present invention >>
- the accumulation of fats and oils was confirmed even when one of the specific inorganic salts was removed. Therefore, in this Example, all 7 components of the specific inorganic salt were not added, and the presence or absence of biotin and sucrose and the presence or absence of sucrose and The effect of suppressing the accumulation of fats and oils by adding ammonium sulfate was investigated.
- Example 8 Examination of the effect of ammonium sulfate on suppressing the accumulation of fats and oils >> Regarding the medium (2) used in Example 5, four types of media in which the ammonium sulfate concentration was replaced with 5.3 g / L, 530 mg / L, 53 mg / L, and 5.3 mg / L were prepared, and the ammonium sulfate concentration was accumulated in fats and oils. The effect on the culture medium was examined.
- FIG. 7 shows a state after 24 hours of culturing (24h), a state after 48 hours of culturing (48h), and a state after 70 hours of culturing (70h).
- Ammonium sulfate concentration of 5.3 g / L, 530 mg / L showed an effect of suppressing fat accumulation, but 5.3 mg / L did not suppress fat accumulation.
- the oil and fat accumulation induction medium and the oil and fat accumulation induction method of the present invention can be used in the field of oil and fat production using yeast.
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Abstract
The present invention provides an oil/fat accumulation induction medium and an oil/fat accumulation induction method, which enable cost reduction of the medium itself and enable the oil/fat accumulation amount in cells to be increased. The oil/fat accumulation induction medium contains a sugar but does not contain ammonium sulfate. The oil/fat accumulation induction method includes a step for performing culturing in said oil/fat accumulation induction medium.
Description
本発明は、油脂生産酵母の油脂蓄積誘導培地および油脂蓄積誘導方法に関する。
The present invention relates to a fat accumulation-inducing medium and a fat accumulation-inducing method for fat-producing yeast.
バイオ燃料への期待が高まっているが、トウモロコシなどを原料にするために食糧自給とのバランスがあり、なかなか進まない。近年、ユーグレナのような微細藻類の研究が進められており、単細胞あたり３０％ほどの油脂を蓄積する。一方、一部の微生物でも油脂を蓄積するものもあり、リポミセス（Ｌｉｐｏｍｙｃｅｓ）は単細胞あたり７０％もの油脂を蓄積することが知られており、急速に研究が進められている。
リポミセスの油脂蓄積誘導培地として、後述の実施例１における表１に示す組成からなるＬｓ培地（非特許文献１）が知られている。 Expectations for biofuels are rising, but it is difficult to make progress due to the balance with food self-sufficiency because corn is used as a raw material. In recent years, research on microalgae such as Euglena has been advanced, and about 30% of fats and oils are accumulated per single cell. On the other hand, some microorganisms also accumulate fats and oils, and Lipomyces is known to accumulate as much as 70% of fats and oils per single cell, and research is being rapidly advanced.
As an oil / fat accumulation-inducing medium for Lipomyces, an Ls medium having the composition shown in Table 1 in Example 1 described later (Non-Patent Document 1) is known.
リポミセスの油脂蓄積誘導培地として、後述の実施例１における表１に示す組成からなるＬｓ培地（非特許文献１）が知られている。 Expectations for biofuels are rising, but it is difficult to make progress due to the balance with food self-sufficiency because corn is used as a raw material. In recent years, research on microalgae such as Euglena has been advanced, and about 30% of fats and oils are accumulated per single cell. On the other hand, some microorganisms also accumulate fats and oils, and Lipomyces is known to accumulate as much as 70% of fats and oils per single cell, and research is being rapidly advanced.
As an oil / fat accumulation-inducing medium for Lipomyces, an Ls medium having the composition shown in Table 1 in Example 1 described later (Non-Patent Document 1) is known.
本発明の課題は、培地自体のコストを下げることができ、且つ、細胞内の油脂蓄積量を増加させることのできる油脂蓄積誘導培地および油脂蓄積誘導方法を提供することにある。
An object of the present invention is to provide a fat accumulation induction medium and a fat accumulation induction method capable of reducing the cost of the medium itself and increasing the amount of fat accumulation in cells.
前記課題は、本発明による、
［１］糖類を含み、硫酸アンモニウムを含まない、油脂生産酵母の油脂蓄積誘導培地、
［２］［１］の培地中で培養する工程を含む、油脂生産酵母の油脂蓄積誘導方法、
により解決することができる。 The subject is according to the present invention.
[1] A fat accumulation-inducing medium for fat-producing yeast containing sugars and ammonium sulfate.
[2] A method for inducing fat accumulation of fat-producing yeast, which comprises the step of culturing in the medium of [1].
Can be solved by.
［１］糖類を含み、硫酸アンモニウムを含まない、油脂生産酵母の油脂蓄積誘導培地、
［２］［１］の培地中で培養する工程を含む、油脂生産酵母の油脂蓄積誘導方法、
により解決することができる。 The subject is according to the present invention.
[1] A fat accumulation-inducing medium for fat-producing yeast containing sugars and ammonium sulfate.
[2] A method for inducing fat accumulation of fat-producing yeast, which comprises the step of culturing in the medium of [1].
Can be solved by.
本発明によれば、培地自体のコストを下げることができ、且つ、細胞内の油脂蓄積量を増加させることのできる油脂蓄積誘導培地および油脂蓄積誘導方法を提供することができる。
According to the present invention, it is possible to provide a fat accumulation induction medium and a fat accumulation induction method capable of reducing the cost of the medium itself and increasing the amount of fat accumulation in cells.
（定義）
本発明の油脂蓄積誘導培地または油脂蓄積誘導方法は、油脂生産酵母において細胞内に油脂蓄積を誘導するために用いることができる。
本発明で用いることのできる油脂生産酵母としては、細胞内に油脂を蓄積できる酵母である限り、特に限定されるものではなく、例えば、リポミセス（Lipomyces）属、オイディウム（Oidium）属、エンドミセス（Endomyces）属、カンジダ（Candida）属、ロドトルラ（Rhodotorula）属、クリプトコッカス（Cryptococcus）属、又はロドスポリディウム（Rhodosporidium）属に属する酵母を挙げることができ、リポミセス（Lipomyces）属に属する酵母が好ましい。 (Definition)
The fat accumulation-inducing medium or the fat accumulation-inducing method of the present invention can be used to induce intracellular fat-accumulation in fat-producing yeast.
The fat-producing yeast that can be used in the present invention is not particularly limited as long as it is a yeast that can accumulate fats and oils in cells. Yeasts belonging to the genus Endomyces, Candida, Rhodotorula, Cryptococcus, or Rhodosporidium can be mentioned, with yeast belonging to the genus Lipomyces being preferred. ..
本発明の油脂蓄積誘導培地または油脂蓄積誘導方法は、油脂生産酵母において細胞内に油脂蓄積を誘導するために用いることができる。
本発明で用いることのできる油脂生産酵母としては、細胞内に油脂を蓄積できる酵母である限り、特に限定されるものではなく、例えば、リポミセス（Lipomyces）属、オイディウム（Oidium）属、エンドミセス（Endomyces）属、カンジダ（Candida）属、ロドトルラ（Rhodotorula）属、クリプトコッカス（Cryptococcus）属、又はロドスポリディウム（Rhodosporidium）属に属する酵母を挙げることができ、リポミセス（Lipomyces）属に属する酵母が好ましい。 (Definition)
The fat accumulation-inducing medium or the fat accumulation-inducing method of the present invention can be used to induce intracellular fat-accumulation in fat-producing yeast.
The fat-producing yeast that can be used in the present invention is not particularly limited as long as it is a yeast that can accumulate fats and oils in cells. Yeasts belonging to the genus Endomyces, Candida, Rhodotorula, Cryptococcus, or Rhodosporidium can be mentioned, with yeast belonging to the genus Lipomyces being preferred. ..
本明細書において「油脂」とは、脂肪酸とグリセリンとのエステルであるトリグリセリド（中性脂肪）を意味する。前記の油脂生産酵母は、トリグリセリドを生産し、菌体内に蓄積する。
In the present specification, "fat and oil" means triglyceride (neutral fat) which is an ester of fatty acid and glycerin. The fat-producing yeast produces triglyceride and accumulates in the cells.
（本発明の油脂蓄積誘導培地）
本発明の油脂蓄積誘導培地は、糖類を含み、硫酸アンモニウムを含まない。 (Medium for inducing fat accumulation of the present invention)
The oil / fat accumulation induction medium of the present invention contains saccharides and does not contain ammonium sulfate.
本発明の油脂蓄積誘導培地は、糖類を含み、硫酸アンモニウムを含まない。 (Medium for inducing fat accumulation of the present invention)
The oil / fat accumulation induction medium of the present invention contains saccharides and does not contain ammonium sulfate.
本発明の油脂蓄積誘導培地は、硫酸アンモニウムを含まない。本発明者は、従来公知の油脂蓄積誘導培地の１つであるＬｓ培地に対して、より油脂蓄積効率の高い油脂蓄積誘導培地を探索する中で、後述の表１に記載の本発明培地の初期組成を設定したが、前記の本発明培地と比べて前記Ｌｓ培地を用いた場合に油脂蓄積効率が低い理由を更に検討したところ、実施例５に示すように、硫酸アンモニウムが油脂蓄積抑制作用を示すことを見出した。後述の表１に示すように、Ｌｓ培地に含まれる無機塩の中で硫酸アンモニウム（５．３ｇ／Ｌ）は最も多量に含まれる無機塩であり、硫酸アンモニウムが油脂蓄積抑制作用を示すことは極めて予想外の知見であった。
The oil / fat accumulation induction medium of the present invention does not contain ammonium sulfate. The present inventor has been searching for a fat accumulation-inducing medium having a higher fat-accumulation efficiency than the Ls medium, which is one of the conventionally known fat-accumulation-inducing media. Although the initial composition was set, the reason why the oil and fat accumulation efficiency was lower when the Ls medium was used as compared with the medium of the present invention was further investigated. As shown in Example 5, ammonium sulfate had an effect of suppressing oil and fat accumulation. Found to show. As shown in Table 1 below, ammonium sulfate (5.3 g / L) is the most abundant inorganic salt among the inorganic salts contained in the Ls medium, and it is highly expected that ammonium sulfate will exhibit an action of suppressing fat accumulation. It was an outside finding.
本明細書において「硫酸アンモニウムを含まない」とは、油脂蓄積抑制作用を示す量の硫酸アンモニウムを含まないことを意味し、より具体的には、硫酸アンモニウム含有量がある態様では５．３ｇ／Ｌ以下、ある態様では１ｇ／Ｌ以下、ある態様では０．５ｇ／Ｌ以下、ある態様では０．１ｇ／Ｌ以下、或る態様では５０ｍｇ／Ｌ以下、或る態様では１０ｍｇ／Ｌ以下、或る態様では５ｍｇ／Ｌ以下であることを意味する。
In the present specification, "ammonium sulfate-free" means that it does not contain an amount of ammonium sulfate exhibiting an oil / fat accumulation inhibitory effect, and more specifically, in an embodiment having an ammonium sulfate content, it is 5.3 g / L or less. 1 g / L or less in some embodiments, 0.5 g / L or less in some embodiments, 0.1 g / L or less in some embodiments, 50 mg / L or less in some embodiments, 10 mg / L or less in some embodiments, 10 mg / L or less in some embodiments. It means that it is 5 mg / L or less.
本発明の油脂蓄積誘導培地は、炭素源となる糖類を含む。前記糖類としては、例えば、グルコース、スクロース、ソルビトール、ガラクトース、ラフィノース、フルクトース、キシロース、トレハロース、マルトース、ラクトースなどを挙げることができ、これらの糖類から選んだ１種を、あるいは、２種以上を組合わせて、用いることができる。本発明の油脂蓄積誘導培地に含まれる糖類の含有量（２種以上を用いる場合はそれらの総量）は、下限値として、或る態様では０．０３％（＝０．３ｇ／Ｌ）、或る態様では０．０５％、或る態様では０．１％、或る態様では０．３％、或る態様では０．５％、或る態様では１％、或る態様では３％、上限値として、或る態様として１５％、或る態様として１０％、或る態様として５％、或る態様では３％、或る態様では１％、或る態様では０．５％である。なお、前記の下限と上限は、所望により、任意に組み合わせることができる。
The oil / fat accumulation induction medium of the present invention contains saccharides as a carbon source. Examples of the saccharide include glucose, sucrose, sorbitol, galactose, raffinose, fructose, xylose, trehalose, maltose, lactose, and the like, and one kind selected from these saccharides or two or more kinds are combined. It can be used together. The content of saccharides contained in the oil / fat accumulation induction medium of the present invention (the total amount thereof when two or more kinds are used) is 0.03% (= 0.3 g / L) in some embodiments as a lower limit value, or 0.05% in some embodiments, 0.1% in some embodiments, 0.3% in some embodiments, 0.5% in some embodiments, 1% in some embodiments, 3% in some embodiments, upper limit. The values are 15% in some embodiments, 10% in some embodiments, 5% in some embodiments, 3% in some embodiments, 1% in some embodiments, and 0.5% in some embodiments. The lower limit and the upper limit can be arbitrarily combined as desired.
本発明の油脂蓄積誘導培地のｐＨは、細胞内に油脂を蓄積することのできるｐＨであれば、特に限定されるものではなく、或る態様ではｐＨ３～８、或る態様ではｐＨ４～７、或る態様ではｐＨ４．５～６．５である。なお、前記の下限と上限は、所望により、任意に組み合わせることができる。
The pH of the fat accumulation-inducing medium of the present invention is not particularly limited as long as it can accumulate fats and oils in cells, and is pH 3 to 8 in some embodiments and pH 4 to 7 in some embodiments. In some embodiments, the pH is 4.5-6.5. The lower limit and the upper limit can be arbitrarily combined as desired.
本発明の油脂蓄積誘導培地は、所望により、少なくとも１つの無機塩を含むことができる。前記無機塩としては、例えば、リン酸二水素カリウム、硫酸マグネシウム、塩化鉄、塩化銅、硫酸亜鉛、モリブデン酸ナトリウム、又はヨウ化カリウム（以下、これらを総称して特定無機塩と称することがある）を挙げることができる。これらの特定無機塩から選んだ１種を、あるいは、２種以上を組合わせて、用いることができ、特定無機塩の全て、すなわち、リン酸二水素カリウム、硫酸マグネシウム、塩化鉄、塩化銅、硫酸亜鉛、モリブデン酸ナトリウム、及びヨウ化カリウムを全て含むこともできる。
The oil / fat accumulation induction medium of the present invention may contain at least one inorganic salt, if desired. Examples of the inorganic salt include potassium dihydrogen phosphate, magnesium sulfate, iron chloride, copper chloride, zinc sulfate, sodium molybdate, and potassium iodide (hereinafter, these may be collectively referred to as a specific inorganic salt). ) Can be mentioned. One selected from these specific inorganic salts, or a combination of two or more, can be used, and all of the specific inorganic salts, that is, potassium dihydrogen phosphate, magnesium sulfate, iron chloride, copper chloride, can be used. It can also contain all of zinc sulfate, sodium molybdate, and potassium iodide.
また、本発明の油脂蓄積誘導培地は、所望により、先に例示した特定無機塩以外の無機塩を含むこともできる。特定無機塩以外の無機塩としては、例えば、ｐＨを調整するのに用いることができる水酸化カリウム若しくは水酸化ナトリウム、又は従来公知の油脂蓄積誘導培地に含まれる塩化カルシウム若しくは塩化ナトリウムを挙げることができる。これらの無機塩１種または２種以上を、所望により、特定無機塩と一緒に用いることもできるが、コストの面から、特定無機塩以外の無機塩は含まないことが好ましい。例えば、従来公知のＬｓ培地と比較すると、無機塩の種類を少なくすることができ、培地にかけるコストを下げることができる。
Further, the oil / fat accumulation induction medium of the present invention may contain an inorganic salt other than the specific inorganic salt exemplified above, if desired. Examples of the inorganic salt other than the specific inorganic salt include potassium hydroxide or sodium hydroxide that can be used to adjust the pH, or calcium chloride or sodium chloride contained in a conventionally known fat accumulation induction medium. it can. One or more of these inorganic salts may be used together with the specific inorganic salt, if desired, but from the viewpoint of cost, it is preferable that no inorganic salt other than the specific inorganic salt is contained. For example, as compared with the conventionally known Ls medium, the types of inorganic salts can be reduced, and the cost applied to the medium can be reduced.
また、本発明の油脂蓄積誘導培地は、所望により、ビタミン、例えば、ビオチン、チアミン、アスコルビン酸、パントテン酸、葉酸、ナイアシンなどを含むことができる。
Further, the oil / fat accumulation induction medium of the present invention can contain vitamins such as biotin, thiamine, ascorbic acid, pantothenic acid, folic acid, niacin and the like, if desired.
本発明の油脂蓄積誘導培地に含まれる各無機塩及び各ビタミン（特にビオチン）の量は、細胞内に油脂を蓄積することのできる量であれば、特に限定されるものではなく、例えば、後述する実施例の具体的記載に基づいて適宜決定することができる。例えば、後述の表１に記載の各含有量を基準として、或る態様では基準量の１／２量～２倍量、或る態様では基準量の１／５量～５倍量、或る態様では基準量の１／１０量～１０倍量、或る態様では基準量の１／２０量～２０倍量、或る態様では基準量の１／５０量～５０倍量、或る態様では基準量の１／１００量～１００倍量を用いることができる。なお、前記の下限と上限は、所望により、任意に組み合わせることができる。
The amount of each inorganic salt and each vitamin (particularly biotin) contained in the oil / fat accumulation induction medium of the present invention is not particularly limited as long as it can accumulate oil / fat in cells, and is not particularly limited. It can be appropriately determined based on the specific description of the examples to be carried out. For example, based on each content shown in Table 1 described later, in some embodiments, the amount is 1/2 to 2 times the reference amount, and in some embodiments, 1/5 to 5 times the reference amount. In some embodiments, 1/10 to 10 times the reference amount, in some embodiments 1/20 to 20 times the reference amount, in some embodiments 1/50 to 50 times the reference amount, in some embodiments. An amount of 1/100 to 100 times the reference amount can be used. The lower limit and the upper limit can be arbitrarily combined as desired.
（本発明の油脂蓄積誘導方法）
本発明の油脂蓄積誘導方法は、本発明の油脂蓄積誘導培地を使用して培養を行うこと以外は、従来公知の油脂蓄積誘導方法に従って実施することができる。
例えば、本発明の油脂蓄積誘導方法では、予め予備培養を行っておき、その培養物を本発明の油脂蓄積誘導培地に植菌することにより本培養を実施することができる。培養温度は、例えば、２０～３５℃で実施することができ、１～７日間程度振とう培養する。 (Method for Inducing Fat Accumulation of the Present Invention)
The oil / fat accumulation induction method of the present invention can be carried out according to a conventionally known oil / fat accumulation induction method, except that culturing is performed using the oil / fat accumulation induction medium of the present invention.
For example, in the oil / fat accumulation induction method of the present invention, the main culture can be carried out by pre-culturing in advance and inoculating the culture into the oil / fat accumulation induction medium of the present invention. The culture temperature can be, for example, 20 to 35 ° C., and the culture is shaken for about 1 to 7 days.
本発明の油脂蓄積誘導方法は、本発明の油脂蓄積誘導培地を使用して培養を行うこと以外は、従来公知の油脂蓄積誘導方法に従って実施することができる。
例えば、本発明の油脂蓄積誘導方法では、予め予備培養を行っておき、その培養物を本発明の油脂蓄積誘導培地に植菌することにより本培養を実施することができる。培養温度は、例えば、２０～３５℃で実施することができ、１～７日間程度振とう培養する。 (Method for Inducing Fat Accumulation of the Present Invention)
The oil / fat accumulation induction method of the present invention can be carried out according to a conventionally known oil / fat accumulation induction method, except that culturing is performed using the oil / fat accumulation induction medium of the present invention.
For example, in the oil / fat accumulation induction method of the present invention, the main culture can be carried out by pre-culturing in advance and inoculating the culture into the oil / fat accumulation induction medium of the present invention. The culture temperature can be, for example, 20 to 35 ° C., and the culture is shaken for about 1 to 7 days.
本発明においては、油脂蓄積酵母内の油脂蓄積は、従来公知の方法により確認・定量することができる。例えば、油脂をオイルレッドのような染色液で染色後、顕微鏡下で酵母写真を撮影して目視により確認する方法や、細胞壁を溶解または物理的破壊により細胞抽出液を作成し、その抽出液内に存在する油脂成分を分離・定量する方法を挙げることができる。
In the present invention, the fat accumulation in the fat accumulation yeast can be confirmed and quantified by a conventionally known method. For example, a method in which fats and oils are stained with a staining solution such as oil red and then a yeast photograph is taken under a microscope to visually confirm the fats and oils, or a cell extract is prepared by lysing or physically destroying the cell wall and inside the extract. A method of separating and quantifying the oil and fat components present in yeast can be mentioned.
以下、実施例によって本発明を具体的に説明するが、これらは本発明の範囲を限定するものではない。
Hereinafter, the present invention will be specifically described with reference to Examples, but these do not limit the scope of the present invention.
《実施例１：本発明培地による油脂蓄積量増加の確認》
本発明者が最初に設定した本発明培地の初期組成と、従来公知のＬｓ培地の組成（非特許文献１）を表１に示す。本実施例では、表１に示す組成の本発明培地と、比較例として表１のＬｓ培地を用いて、リポミセス（Ｌｉｐｏｍｙｃｅｓ）における細胞内油脂蓄積の違いを確認した。 << Example 1: Confirmation of increase in fat accumulation amount by the medium of the present invention >>
Table 1 shows the initial composition of the medium of the present invention initially set by the present inventor and the composition of the conventionally known Ls medium (Non-Patent Document 1). In this example, the difference in intracellular fat and oil accumulation in Lipomyces was confirmed using the medium of the present invention having the composition shown in Table 1 and the Ls medium shown in Table 1 as a comparative example.
本発明者が最初に設定した本発明培地の初期組成と、従来公知のＬｓ培地の組成（非特許文献１）を表１に示す。本実施例では、表１に示す組成の本発明培地と、比較例として表１のＬｓ培地を用いて、リポミセス（Ｌｉｐｏｍｙｃｅｓ）における細胞内油脂蓄積の違いを確認した。 << Example 1: Confirmation of increase in fat accumulation amount by the medium of the present invention >>
Table 1 shows the initial composition of the medium of the present invention initially set by the present inventor and the composition of the conventionally known Ls medium (Non-Patent Document 1). In this example, the difference in intracellular fat and oil accumulation in Lipomyces was confirmed using the medium of the present invention having the composition shown in Table 1 and the Ls medium shown in Table 1 as a comparative example.
リポミセスとしては、Lipomyces starkeyiを使用した。Lipomyces starkeyiをＹＰＤ培地で１～２日間２５℃で培養後、細胞を滅菌水で洗浄、Ｌｓ培地または本発明培地に植え込み、２５℃で培養した。培養液の一部を分取し、２．５％グルタルアルデヒド固定を行った。固定した細胞を洗浄後、０．５％オイルレッド（Oil red）染色液で３０分～１時間染色した。細胞を洗浄後、顕微鏡観察を行った。また、細胞を溶解または物理的破壊により細胞抽出液を作成し、その抽出液内に存在する油脂成分を分離し、細胞内油脂蓄積量を算出した。
Lipomyces starkeyi was used as the lipomyces. Lipomyces starkeyi was cultured in YPD medium at 25 ° C. for 1 to 2 days, the cells were washed with sterile water, implanted in Ls medium or the medium of the present invention, and cultured at 25 ° C. A part of the culture solution was separated and fixed with 2.5% glutaraldehyde. The fixed cells were washed and then stained with 0.5% Oil red stain for 30 minutes to 1 hour. After washing the cells, microscopic observation was performed. In addition, a cell extract was prepared by lysing or physically destroying cells, and the fat and oil components existing in the extract were separated, and the amount of intracellular fat and oil accumulated was calculated.
培養２４時間後の状態を図１に示す。
従来公知のＬｓ培地を用いた場合には（図１に示すＬＳ ｍｅｄｉｕｍ）、細胞内油脂蓄積が単細胞当たり約３％であったのに対して、表１に示す組成の本発明培地を用いた場合には（図１に示すＮｅｗ ＬＳ ｍｅｄｉｕｍ）、単細胞当たり３０％以上の油脂蓄積が確認された。 The state after 24 hours of culturing is shown in FIG.
When a conventionally known Ls medium was used (LS medium shown in FIG. 1), intracellular fat and oil accumulation was about 3% per single cell, whereas the medium of the present invention having the composition shown in Table 1 was used. In some cases (New LS medium shown in FIG. 1), oil and fat accumulation of 30% or more per single cell was confirmed.
従来公知のＬｓ培地を用いた場合には（図１に示すＬＳ ｍｅｄｉｕｍ）、細胞内油脂蓄積が単細胞当たり約３％であったのに対して、表１に示す組成の本発明培地を用いた場合には（図１に示すＮｅｗ ＬＳ ｍｅｄｉｕｍ）、単細胞当たり３０％以上の油脂蓄積が確認された。 The state after 24 hours of culturing is shown in FIG.
When a conventionally known Ls medium was used (LS medium shown in FIG. 1), intracellular fat and oil accumulation was about 3% per single cell, whereas the medium of the present invention having the composition shown in Table 1 was used. In some cases (New LS medium shown in FIG. 1), oil and fat accumulation of 30% or more per single cell was confirmed.
《実施例２：本発明培地における糖濃度の検討》
本実施例では、糖濃度が油脂蓄積に与える影響を検討した。本発明培地としては、表１に記載の初期組成において、スクロース３ｇを、スクロース３０ｇ（スクロース濃度として３％）、３ｇ（同０．３％）、０．３ｇ（同０．０３％）に代えた３種類のスクロース含有培地と、スクロース３ｇを、グルコース３０ｇ（グルコース濃度として３％）、３ｇ（同０．３％）、０．３ｇ（同０．０３％）に代えた３種類のグルコース含有培地とを準備した。比較例として、表１のＬｓ培地において、グルコース３０ｇを、スクロース３ｇ（スクロース濃度として０．３％）、グルコース３ｇ（グルコース濃度として０．３％）に代えた２種類の培地を準備した。培養は、実施例１と同様にして実施した。 << Example 2: Examination of sugar concentration in the medium of the present invention >>
In this example, the effect of sugar concentration on fat accumulation was examined. As the medium of the present invention, in the initial composition shown in Table 1, 3 g of sucrose was replaced with 30 g of sucrose (3% sucrose concentration), 3 g (0.3% sucrose), and 0.3 g (0.03% sucrose). 3 types of sucrose-containing medium and 3 types of glucose in which 3 g of sucrose was replaced with 30 g of glucose (3% glucose concentration), 3 g (0.3% of the same), and 0.3 g (0.03% of the same). A medium was prepared. As a comparative example, in the Ls medium shown in Table 1, two types of media were prepared in which 30 g of glucose was replaced with 3 g of sucrose (0.3% as sucrose concentration) and 3 g of glucose (0.3% as glucose concentration). The culture was carried out in the same manner as in Example 1.
本実施例では、糖濃度が油脂蓄積に与える影響を検討した。本発明培地としては、表１に記載の初期組成において、スクロース３ｇを、スクロース３０ｇ（スクロース濃度として３％）、３ｇ（同０．３％）、０．３ｇ（同０．０３％）に代えた３種類のスクロース含有培地と、スクロース３ｇを、グルコース３０ｇ（グルコース濃度として３％）、３ｇ（同０．３％）、０．３ｇ（同０．０３％）に代えた３種類のグルコース含有培地とを準備した。比較例として、表１のＬｓ培地において、グルコース３０ｇを、スクロース３ｇ（スクロース濃度として０．３％）、グルコース３ｇ（グルコース濃度として０．３％）に代えた２種類の培地を準備した。培養は、実施例１と同様にして実施した。 << Example 2: Examination of sugar concentration in the medium of the present invention >>
In this example, the effect of sugar concentration on fat accumulation was examined. As the medium of the present invention, in the initial composition shown in Table 1, 3 g of sucrose was replaced with 30 g of sucrose (3% sucrose concentration), 3 g (0.3% sucrose), and 0.3 g (0.03% sucrose). 3 types of sucrose-containing medium and 3 types of glucose in which 3 g of sucrose was replaced with 30 g of glucose (3% glucose concentration), 3 g (0.3% of the same), and 0.3 g (0.03% of the same). A medium was prepared. As a comparative example, in the Ls medium shown in Table 1, two types of media were prepared in which 30 g of glucose was replaced with 3 g of sucrose (0.3% as sucrose concentration) and 3 g of glucose (0.3% as glucose concentration). The culture was carried out in the same manner as in Example 1.
培養前の状態（０ｈｒ）と、培養２４時間後の状態（２４ｈｒ）を図２に示す。
本発明培地を用いた場合（図２に示すＮｅｗ Ｌｓ ｍｅｄｉｕｍ）には、スクロース、グルコースいずれの糖でも、糖濃度３％、０．３％で油脂蓄積したが、糖濃度０．０３％では細胞形態がいびつで油脂蓄積の効率もよくなかった。糖濃度０．３％のＬｓ培地（図２に示すＬｓ ｍｅｄｉｕｍ）では、細胞増殖するものの、ほとんど油脂蓄積しなかった。 The state before culturing (0 hr) and the state after 24 hours of culturing (24 hr) are shown in FIG.
When the medium of the present invention was used (New Ls medium shown in FIG. 2), fats and oils were accumulated at sugar concentrations of 3% and 0.3% for both sucrose and glucose sugars, but cells were accumulated at sugar concentrations of 0.03%. The morphology was distorted and the efficiency of fat accumulation was not good. In the Ls medium having a sugar concentration of 0.3% (Ls medium shown in FIG. 2), although the cells proliferated, almost no fats and oils were accumulated.
本発明培地を用いた場合（図２に示すＮｅｗ Ｌｓ ｍｅｄｉｕｍ）には、スクロース、グルコースいずれの糖でも、糖濃度３％、０．３％で油脂蓄積したが、糖濃度０．０３％では細胞形態がいびつで油脂蓄積の効率もよくなかった。糖濃度０．３％のＬｓ培地（図２に示すＬｓ ｍｅｄｉｕｍ）では、細胞増殖するものの、ほとんど油脂蓄積しなかった。 The state before culturing (0 hr) and the state after 24 hours of culturing (24 hr) are shown in FIG.
When the medium of the present invention was used (New Ls medium shown in FIG. 2), fats and oils were accumulated at sugar concentrations of 3% and 0.3% for both sucrose and glucose sugars, but cells were accumulated at sugar concentrations of 0.03%. The morphology was distorted and the efficiency of fat accumulation was not good. In the Ls medium having a sugar concentration of 0.3% (Ls medium shown in FIG. 2), although the cells proliferated, almost no fats and oils were accumulated.
《実施例３：本発明培地における糖の種類の検討》
本実施例では、糖の種類が油脂蓄積に与える影響を検討した。培地としては、表１に記載の初期組成において、スクロース３ｇを、グルコース３ｇ（グルコース濃度０．３％）、スクロース３ｇ（スクロース濃度０．３％）、ソルビトール３ｇ（ソルビトール濃度０．３％）、ガラクトース３ｇ（ガラクトース濃度０．３％）、ラフィノース３ｇ（ラフィノース濃度０．３％）、グリセロール３ｇ（グリセロール濃度０．３％）に代えた６種類の培地を準備した。培養は、実施例１と同様にして実施した。 << Example 3: Examination of the type of sugar in the medium of the present invention >>
In this example, the effect of the type of sugar on the accumulation of fats and oils was examined. As the medium, in the initial composition shown in Table 1, 3 g of sucrose, 3 g of glucose (glucose concentration 0.3%), 3 g of sucrose (sucrose concentration 0.3%), 3 g of sorbitol (sorbitol concentration 0.3%), Six types of media were prepared in place of galactose 3 g (galactose concentration 0.3%), raffinose 3 g (raffinose concentration 0.3%), and glycerol 3 g (glycerol concentration 0.3%). The culture was carried out in the same manner as in Example 1.
本実施例では、糖の種類が油脂蓄積に与える影響を検討した。培地としては、表１に記載の初期組成において、スクロース３ｇを、グルコース３ｇ（グルコース濃度０．３％）、スクロース３ｇ（スクロース濃度０．３％）、ソルビトール３ｇ（ソルビトール濃度０．３％）、ガラクトース３ｇ（ガラクトース濃度０．３％）、ラフィノース３ｇ（ラフィノース濃度０．３％）、グリセロール３ｇ（グリセロール濃度０．３％）に代えた６種類の培地を準備した。培養は、実施例１と同様にして実施した。 << Example 3: Examination of the type of sugar in the medium of the present invention >>
In this example, the effect of the type of sugar on the accumulation of fats and oils was examined. As the medium, in the initial composition shown in Table 1, 3 g of sucrose, 3 g of glucose (glucose concentration 0.3%), 3 g of sucrose (sucrose concentration 0.3%), 3 g of sorbitol (sorbitol concentration 0.3%), Six types of media were prepared in place of galactose 3 g (galactose concentration 0.3%), raffinose 3 g (raffinose concentration 0.3%), and glycerol 3 g (glycerol concentration 0.3%). The culture was carried out in the same manner as in Example 1.
培養前の状態（０ｈｒ）と、培養２５時間後の状態（２５ｈｒ）を図３に示す。
グリセロール以外の糖、すなわち、グルコース、スクロース、ソルビトール、ガラクトース、ラフィノースを含有する各培地では油脂を蓄積したが、グリセロール含有培地では油脂蓄積が見られなかった。 The state before culturing (0 hr) and the state after 25 hours of culturing (25 hr) are shown in FIG.
Fats and oils were accumulated in each medium containing sugars other than glycerol, that is, glucose, sucrose, sorbitol, galactose, and raffinose, but no fats and oils were accumulated in the glycerol-containing medium.
グリセロール以外の糖、すなわち、グルコース、スクロース、ソルビトール、ガラクトース、ラフィノースを含有する各培地では油脂を蓄積したが、グリセロール含有培地では油脂蓄積が見られなかった。 The state before culturing (0 hr) and the state after 25 hours of culturing (25 hr) are shown in FIG.
Fats and oils were accumulated in each medium containing sugars other than glycerol, that is, glucose, sucrose, sorbitol, galactose, and raffinose, but no fats and oils were accumulated in the glycerol-containing medium.
《実施例４：本発明培地におけるｐＨの検討》
本実施例では、本発明培地におけるｐＨが油脂蓄積に与える影響を検討した。本発明培地として、表１に記載の初期組成からなるｐＨ６．５の培地と、表１に記載の初期組成から水酸化カリウムを除いたｐＨ４．５の培地とを準備した。なお、培地のｐＨは、水酸化カリウムの有無によるものであり、別途、酸又はアルカリによるｐＨ調整は行わなかった。実施例１と同様に培養を実施したところ、培養２４時間後において、両培地における油脂蓄積に差異は認められなかった。 << Example 4: Examination of pH in the medium of the present invention >>
In this example, the effect of pH in the medium of the present invention on fat accumulation was investigated. As the medium of the present invention, a medium having a pH of 6.5 having the initial composition shown in Table 1 and a medium having a pH of 4.5 obtained by removing potassium hydroxide from the initial composition shown in Table 1 were prepared. The pH of the medium was based on the presence or absence of potassium hydroxide, and the pH was not separately adjusted with an acid or an alkali. When the culture was carried out in the same manner as in Example 1, no difference was observed in the accumulation of fats and oils in bothmedia 24 hours after the culture.
本実施例では、本発明培地におけるｐＨが油脂蓄積に与える影響を検討した。本発明培地として、表１に記載の初期組成からなるｐＨ６．５の培地と、表１に記載の初期組成から水酸化カリウムを除いたｐＨ４．５の培地とを準備した。なお、培地のｐＨは、水酸化カリウムの有無によるものであり、別途、酸又はアルカリによるｐＨ調整は行わなかった。実施例１と同様に培養を実施したところ、培養２４時間後において、両培地における油脂蓄積に差異は認められなかった。 << Example 4: Examination of pH in the medium of the present invention >>
In this example, the effect of pH in the medium of the present invention on fat accumulation was investigated. As the medium of the present invention, a medium having a pH of 6.5 having the initial composition shown in Table 1 and a medium having a pH of 4.5 obtained by removing potassium hydroxide from the initial composition shown in Table 1 were prepared. The pH of the medium was based on the presence or absence of potassium hydroxide, and the pH was not separately adjusted with an acid or an alkali. When the culture was carried out in the same manner as in Example 1, no difference was observed in the accumulation of fats and oils in both
《実施例５：本発明培地における無機塩の種類の検討（１）》
本実施例では、Ｌｓ培地にのみ含まれる無機塩（硫酸アンモニウム、塩化カルシウム、塩化ナトリウム）が油脂蓄積に与える影響を検討した。培地としては、実施例４で調製したｐＨ４．５の培地（図４に示すＮｅｗ Ｌｓ ｐＨ４．５）を基本培地として、表２に記載の各種無機塩を添加した培地（１）～（８）を準備した。なお、添加量は、表１に示すＬｓ培地における硫酸アンモニウム、塩化カルシウム、塩化ナトリウムの各含有量と同じになるように添加した。 << Example 5: Examination of types of inorganic salts in the medium of the present invention (1) >>
In this example, the effect of inorganic salts (ammonium sulfate, calcium chloride, sodium chloride) contained only in the Ls medium on fat and oil accumulation was examined. As the medium, the medium of pH 4.5 prepared in Example 4 (New Ls pH 4.5 shown in FIG. 4) was used as the basic medium, and the media (1) to (8) to which various inorganic salts shown in Table 2 were added. Prepared. The amount added was the same as the content of ammonium sulfate, calcium chloride, and sodium chloride in the Ls medium shown in Table 1.
本実施例では、Ｌｓ培地にのみ含まれる無機塩（硫酸アンモニウム、塩化カルシウム、塩化ナトリウム）が油脂蓄積に与える影響を検討した。培地としては、実施例４で調製したｐＨ４．５の培地（図４に示すＮｅｗ Ｌｓ ｐＨ４．５）を基本培地として、表２に記載の各種無機塩を添加した培地（１）～（８）を準備した。なお、添加量は、表１に示すＬｓ培地における硫酸アンモニウム、塩化カルシウム、塩化ナトリウムの各含有量と同じになるように添加した。 << Example 5: Examination of types of inorganic salts in the medium of the present invention (1) >>
In this example, the effect of inorganic salts (ammonium sulfate, calcium chloride, sodium chloride) contained only in the Ls medium on fat and oil accumulation was examined. As the medium, the medium of pH 4.5 prepared in Example 4 (New Ls pH 4.5 shown in FIG. 4) was used as the basic medium, and the media (1) to (8) to which various inorganic salts shown in Table 2 were added. Prepared. The amount added was the same as the content of ammonium sulfate, calcium chloride, and sodium chloride in the Ls medium shown in Table 1.
培養前の状態（０ｈｒ）と、培養４８時間後の状態（４８ｈｒ）を図４に示す。
硫酸アンモニウムが含まれている培地〔培地（２）、（５）、（６）、（８）〕を用いた場合にのみ、油脂蓄積が抑制されており、硫酸アンモニウムが油脂蓄積抑制作用を示すことが判明した。 The state before culturing (0 hr) and the state after 48 hours of culturing (48 hr) are shown in FIG.
Only when a medium containing ammonium sulfate [medium (2), (5), (6), (8)] is used, fat accumulation is suppressed, and ammonium sulfate exhibits an action of suppressing fat accumulation. found.
硫酸アンモニウムが含まれている培地〔培地（２）、（５）、（６）、（８）〕を用いた場合にのみ、油脂蓄積が抑制されており、硫酸アンモニウムが油脂蓄積抑制作用を示すことが判明した。 The state before culturing (0 hr) and the state after 48 hours of culturing (48 hr) are shown in FIG.
Only when a medium containing ammonium sulfate [medium (2), (5), (6), (8)] is used, fat accumulation is suppressed, and ammonium sulfate exhibits an action of suppressing fat accumulation. found.
《実施例６：本発明培地における無機塩の種類の検討（２）》
本実施例では、本発明培地とＬｓ培地に共通に含まれる無機塩（リン酸二水素カリウム、硫酸マグネシウム、塩化鉄、塩化銅、硫酸亜鉛、モリブデン酸ナトリウム、又はヨウ化カリウム；以下、これらを総称して特定無機塩と称することがある）が油脂蓄積に与える影響を検討した。なお、リン酸二水素カリウム、硫酸マグネシウムについては、別の実験で、添加しなくても油脂蓄積することを確認していたため、今回は実施しなかった。
培地としては、実施例４で調製したｐＨ４．５の培地（図４に示すＮｅｗ Ｌｓ ｐＨ４．５）からリン酸二水素カリウム及び硫酸マグネシウムを除いた培地を基本培地として、更に、表３に記載の各種無機塩を除いた培地（１）～（６）〔但し、培地（６）は実施例６における基本培地〕を準備した。本実施例における基本培地の組成を表４に示す。 << Example 6: Examination of types of inorganic salts in the medium of the present invention (2) >>
In this example, the inorganic salts commonly contained in the medium of the present invention and the Ls medium (potassium dihydrogen phosphate, magnesium sulfate, iron chloride, copper chloride, zinc sulfate, sodium molybdate, or potassium iodide; hereinafter, these are used. The effect of (sometimes collectively referred to as specific inorganic salt) on fat accumulation was investigated. Regarding potassium dihydrogen phosphate and magnesium sulfate, it was confirmed in another experiment that fats and oils accumulated even if they were not added, so this was not carried out.
As the medium, a medium obtained by removing potassium dihydrogen phosphate and magnesium sulfate from the medium having a pH of 4.5 (New Ls pH 4.5 shown in FIG. 4) prepared in Example 4 as a basic medium is further described in Table 3. (1) to (6) [however, the medium (6) is the basal medium in Example 6] were prepared. The composition of the basal medium in this example is shown in Table 4.
本実施例では、本発明培地とＬｓ培地に共通に含まれる無機塩（リン酸二水素カリウム、硫酸マグネシウム、塩化鉄、塩化銅、硫酸亜鉛、モリブデン酸ナトリウム、又はヨウ化カリウム；以下、これらを総称して特定無機塩と称することがある）が油脂蓄積に与える影響を検討した。なお、リン酸二水素カリウム、硫酸マグネシウムについては、別の実験で、添加しなくても油脂蓄積することを確認していたため、今回は実施しなかった。
培地としては、実施例４で調製したｐＨ４．５の培地（図４に示すＮｅｗ Ｌｓ ｐＨ４．５）からリン酸二水素カリウム及び硫酸マグネシウムを除いた培地を基本培地として、更に、表３に記載の各種無機塩を除いた培地（１）～（６）〔但し、培地（６）は実施例６における基本培地〕を準備した。本実施例における基本培地の組成を表４に示す。 << Example 6: Examination of types of inorganic salts in the medium of the present invention (2) >>
In this example, the inorganic salts commonly contained in the medium of the present invention and the Ls medium (potassium dihydrogen phosphate, magnesium sulfate, iron chloride, copper chloride, zinc sulfate, sodium molybdate, or potassium iodide; hereinafter, these are used. The effect of (sometimes collectively referred to as specific inorganic salt) on fat accumulation was investigated. Regarding potassium dihydrogen phosphate and magnesium sulfate, it was confirmed in another experiment that fats and oils accumulated even if they were not added, so this was not carried out.
As the medium, a medium obtained by removing potassium dihydrogen phosphate and magnesium sulfate from the medium having a pH of 4.5 (New Ls pH 4.5 shown in FIG. 4) prepared in Example 4 as a basic medium is further described in Table 3. (1) to (6) [however, the medium (6) is the basal medium in Example 6] were prepared. The composition of the basal medium in this example is shown in Table 4.
培養４８時間後の状態を図５に示す。
前記特定無機塩のいずれを除いても、油脂蓄積を確認することができた。 The state after 48 hours of culturing is shown in FIG.
Accumulation of fats and oils could be confirmed by removing any of the specific inorganic salts.
前記特定無機塩のいずれを除いても、油脂蓄積を確認することができた。 The state after 48 hours of culturing is shown in FIG.
Accumulation of fats and oils could be confirmed by removing any of the specific inorganic salts.
《実施例７：本発明培地における必須成分の確定》
前記実施例６において、特定無機塩の１つを除いてもいずれも油脂蓄積が確認できたため、本実施例では、特定無機塩７成分の全てを非添加とし、更に、ビオチン、スクロースの有無および硫酸アンモニウムの添加による油脂蓄積抑制効果を検討した。 << Example 7: Determination of essential components in the medium of the present invention >>
In Example 6, the accumulation of fats and oils was confirmed even when one of the specific inorganic salts was removed. Therefore, in this Example, all 7 components of the specific inorganic salt were not added, and the presence or absence of biotin and sucrose and the presence or absence of sucrose and The effect of suppressing the accumulation of fats and oils by adding ammonium sulfate was investigated.
前記実施例６において、特定無機塩の１つを除いてもいずれも油脂蓄積が確認できたため、本実施例では、特定無機塩７成分の全てを非添加とし、更に、ビオチン、スクロースの有無および硫酸アンモニウムの添加による油脂蓄積抑制効果を検討した。 << Example 7: Determination of essential components in the medium of the present invention >>
In Example 6, the accumulation of fats and oils was confirmed even when one of the specific inorganic salts was removed. Therefore, in this Example, all 7 components of the specific inorganic salt were not added, and the presence or absence of biotin and sucrose and the presence or absence of sucrose and The effect of suppressing the accumulation of fats and oils by adding ammonium sulfate was investigated.
培養２５時間後の状態を図６に示す。
硫酸アンモニウム（硫安）の添加により油脂蓄積は抑制された（図６におけるＮｏ．１、Ｎｏ．３、Ｎｏ．５）。
ビオチンの有無で油脂蓄積に影響はなかった（図６におけるＮｏ．２（ビオチン有り）とＮｏ．４（ビオチン無し）の比較）。
スクロースが含まれていないと、油脂蓄積できなかった（図６におけるＮｏ．４（スクロース有り）とＮｏ．６（スクロース無し）の比較）。 The state after 25 hours of culturing is shown in FIG.
The addition of ammonium sulfate (ammonium sulfate) suppressed the accumulation of fats and oils (No. 1, No. 3, No. 5 in FIG. 6).
The presence or absence of biotin did not affect the accumulation of fats and oils (comparison between No. 2 (with biotin) and No. 4 (without biotin) in FIG. 6).
If sucrose was not contained, fats and oils could not be accumulated (comparison between No. 4 (with sucrose) and No. 6 (without sucrose) in FIG. 6).
硫酸アンモニウム（硫安）の添加により油脂蓄積は抑制された（図６におけるＮｏ．１、Ｎｏ．３、Ｎｏ．５）。
ビオチンの有無で油脂蓄積に影響はなかった（図６におけるＮｏ．２（ビオチン有り）とＮｏ．４（ビオチン無し）の比較）。
スクロースが含まれていないと、油脂蓄積できなかった（図６におけるＮｏ．４（スクロース有り）とＮｏ．６（スクロース無し）の比較）。 The state after 25 hours of culturing is shown in FIG.
The addition of ammonium sulfate (ammonium sulfate) suppressed the accumulation of fats and oils (No. 1, No. 3, No. 5 in FIG. 6).
The presence or absence of biotin did not affect the accumulation of fats and oils (comparison between No. 2 (with biotin) and No. 4 (without biotin) in FIG. 6).
If sucrose was not contained, fats and oils could not be accumulated (comparison between No. 4 (with sucrose) and No. 6 (without sucrose) in FIG. 6).
《実施例８：硫酸アンモニウムの油脂蓄積抑制効果の検討》
実施例５で用いた培地（２）に関して、硫酸アンモニウム濃度を５．３ｇ／Ｌ、５３０ｍｇ／Ｌ、５３ｍｇ／Ｌ、５．３ｍｇ／Ｌに代えた４種類の培地を用意し、硫酸アンモニウム濃度が油脂蓄積に与える影響を検討した。
培養２４時間後の状態（２４ｈ）、培養４８時間後の状態（４８ｈ）、培養７０時間後の状態（７０ｈ）を図７に示す。硫酸アンモニウム濃度５．３ｇ／Ｌ、５３０ｍｇ／Ｌでは油脂蓄積抑制作用を示したが、５．３ｍｇ／Ｌでは油脂蓄積を抑制しなかった。 << Example 8: Examination of the effect of ammonium sulfate on suppressing the accumulation of fats and oils >>
Regarding the medium (2) used in Example 5, four types of media in which the ammonium sulfate concentration was replaced with 5.3 g / L, 530 mg / L, 53 mg / L, and 5.3 mg / L were prepared, and the ammonium sulfate concentration was accumulated in fats and oils. The effect on the culture medium was examined.
FIG. 7 shows a state after 24 hours of culturing (24h), a state after 48 hours of culturing (48h), and a state after 70 hours of culturing (70h). Ammonium sulfate concentration of 5.3 g / L, 530 mg / L showed an effect of suppressing fat accumulation, but 5.3 mg / L did not suppress fat accumulation.
実施例５で用いた培地（２）に関して、硫酸アンモニウム濃度を５．３ｇ／Ｌ、５３０ｍｇ／Ｌ、５３ｍｇ／Ｌ、５．３ｍｇ／Ｌに代えた４種類の培地を用意し、硫酸アンモニウム濃度が油脂蓄積に与える影響を検討した。
培養２４時間後の状態（２４ｈ）、培養４８時間後の状態（４８ｈ）、培養７０時間後の状態（７０ｈ）を図７に示す。硫酸アンモニウム濃度５．３ｇ／Ｌ、５３０ｍｇ／Ｌでは油脂蓄積抑制作用を示したが、５．３ｍｇ／Ｌでは油脂蓄積を抑制しなかった。 << Example 8: Examination of the effect of ammonium sulfate on suppressing the accumulation of fats and oils >>
Regarding the medium (2) used in Example 5, four types of media in which the ammonium sulfate concentration was replaced with 5.3 g / L, 530 mg / L, 53 mg / L, and 5.3 mg / L were prepared, and the ammonium sulfate concentration was accumulated in fats and oils. The effect on the culture medium was examined.
FIG. 7 shows a state after 24 hours of culturing (24h), a state after 48 hours of culturing (48h), and a state after 70 hours of culturing (70h). Ammonium sulfate concentration of 5.3 g / L, 530 mg / L showed an effect of suppressing fat accumulation, but 5.3 mg / L did not suppress fat accumulation.
本発明の油脂蓄積誘導培地および油脂蓄積誘導方法は、酵母を用いる油脂生産の分野で利用することができる。
The oil and fat accumulation induction medium and the oil and fat accumulation induction method of the present invention can be used in the field of oil and fat production using yeast.
Claims (2)
- 糖類を含み、硫酸アンモニウムを含まない、油脂生産酵母の油脂蓄積誘導培地。 A fat accumulation induction medium for fat-producing yeast that contains sugars and does not contain ammonium sulfate.
- 請求項１に記載の培地中で培養する工程を含む、油脂生産酵母の油脂蓄積誘導方法。 A method for inducing fat accumulation of fat-producing yeast, which comprises the step of culturing in the medium according to claim 1.
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| JP2010158219A (en) * | 2009-01-09 | 2010-07-22 | Univ Of Yamanashi | Method for culturing oil- and fat-producing microorganism |
| JP2012040006A (en) * | 2010-08-13 | 2012-03-01 | Fu Jen Catholic Univ | Method for producing oil by yeast of genus cystofilobasidium |
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2019
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2020
- 2020-10-02 WO PCT/JP2020/037550 patent/WO2021066145A1/en active Application Filing
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| JP2010158219A (en) * | 2009-01-09 | 2010-07-22 | Univ Of Yamanashi | Method for culturing oil- and fat-producing microorganism |
| JP2012040006A (en) * | 2010-08-13 | 2012-03-01 | Fu Jen Catholic Univ | Method for producing oil by yeast of genus cystofilobasidium |
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