JPH0931071A - Production of (r)-(-)-massialactone, its production and perfume composition - Google Patents

Abstract

PROBLEM TO BE SOLVED: To simply obtain a large amount of the subject compound by culturing a specific fungus, chemically treating the culture product, by chemical reaction at least one stage, having a high optical purity, useful as a fruit juice, beverage, etc. SOLUTION: This compound of formula I is obtained by culturing a fungus and chemically treating its culture product. The compound is preferably a fungus capable of producing a compound of formula II ((n) is an integer of >=0; R1 to R5 are each arbitrary functional group), is preferably Exophiala pisciphila N1-10,102 (FERM-14,232). The fungus is preferably cultured under culture conditions at pH6-7 at 22-27 deg.C for 5-10 days. The chemical treatment, for example, is preferably acid treatment with sulfuric acid, phosphoric acid, etc.

Description

Detailed Description of the Invention

[0001]

The present invention has the following formula

[0002]

Embedded image (R)-(-)-massoy lactone (Ma)
The present invention relates to a method for producing sosialactone). The present inventors have developed a method for producing the present compound by a one-step chemical reaction by subjecting a culture of a microorganism to a simple chemical treatment. Specifically, the present invention relates to (R)-
(-)-A simple method for producing massoialactone, (R)-
The present invention relates to a perfume composition containing (-)-massoilactone and (R)-(-)-massoilactone as an active ingredient.

[0003]

2. Description of the Related Art (R)-(-)-Massoalactone, which is a known flavoring substance having a coconut milk-like flavor, is used as a food and drink or a cosmetic as a perfuming flavor imparting or modulating agent for various flavoring compositions. It is known to be useful in a wide range of fields such as chemicals, health care and pharmaceuticals, and is used in various fields. Up to now, (R)-(−)-massoy lactone has been produced by extraction from plants or chemical synthesis.
As an example of extraction from a plant, it has been reported that it can be obtained as a main component of bark oil of Cryptocaria massoia (Chemical Society of Japan 58, 246-251 (1937)). In addition to being isolated from sugar cane molasses, it is also obtained from coconut milk. On the other hand, most of the production methods by chemical synthesis have so far been reported on the synthesis of racemates (Japanese Patent Laid-Open Nos. 63-57583 and 63-21.
5676, JP-A-63-222164). In recent years, it has been reported that an optically active (R)-(-)-massoialactone synthesized from an optically active (+)-1,2-epoxyheptane in four steps is synthesized (Japanese Patent Laid-Open No. 2-59564). .

[0004]

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention It is known that Massoilactone has different odors between its (R)-(-) type optically active form and its racemic form. That is (R)-
The (-) type optically active substance has a scent of coconut milk, while the racemic substance has a buttery odor. Therefore, in the production of a perfume composition having a constant quality, (R)-(−)-massoilactone having high optical purity is indispensable according to the purpose, and development of a simple production method thereof is desired. It has been rare. The above-mentioned plant-derived manufacturing method requires cutting a large amount of plants and is not suitable for large-scale supply. In addition, seasonal environmental constraints cannot be escaped in order to obtain plants that produce (R)-(-)-massoilactone as needed. On the other hand, by chemical synthesis (R)
In the production method of-(-)-massoilactone, it is necessary to use a relatively expensive optically active substance having a high optical purity as a starting material in the starting material family in order to obtain a target substance having a high optical purity. In addition, the conversion of the starting material into the target product requires multiple steps. Furthermore, after the reaction in each step, it is necessary to separate the reaction reagent or by-product from the desired product and purify the intermediate or the desired product step by step. Therefore, in order to carry out industrial large-scale synthesis and rapidly and inexpensively supply optically pure (R)-(-)-massoy lactone, various restrictions peculiar to such chemical synthesis must be imposed.

[0005]

[Means for Solving the Problems]
As a result of various studies to solve the above problems, it was found that (R)-(-)-massoilactone can be obtained by culturing a microorganism and subjecting the culture to a simple chemical treatment. The present invention has been completed. The present invention is described in detail below.

According to the present invention, a culture of a microorganism is subjected to a simple chemical treatment to produce (R)-(-)-massoy lactone in the reaction solution, and then (R) is separated by a conventional separation method. -(-)-Massoy lactone is separated.

Any microorganism can be used as long as it can produce (R)-(-)-massoilactone by the treatment described in the present invention.

[0008]

Embedded image (However, n ≧ 0, R _{1 to} R ₅ are arbitrary functional groups)
A microorganism that produces the compound shown by can be used. For example,

[0009]

Embedded image Exophylline A (Exofilin
A) (however, R ₁ = H) or harimesin C (Hal
imecin C) where R ₁ = CH ₃ CO) or the following formula

[0010]

[Chemical 6] Halymecin A represented by
(However, R ₁ = CH ₃ CO, R ₂ = H) or halymecin B (however, R ₁ = CH ₃
A microorganism that produces CO, R ₂ = D-mannosyl) can be used.

The microorganism used in the present invention may be any microorganism as long as it produces (R)-(-)-massoialactone by the treatment described in the present invention, for example, Exophiala, Fusarium, Aureobasidi. Microorganisms belonging to either the genus Urum or the genus Trichoderma can be used.

An example of the microorganism belonging to the genus Exophiala is Exophiala picifila.

As an example of Exophiala pichiphila, Exophiala pichiphila NI10102 strain can be mentioned. The strain was deposited at the Institute of Biotechnology, Institute of Industrial Science, March 24, 1994, and the deposit number is Micromachine Research Institute No. 14232.

Cultivation of the microorganism is basically in accordance with the method for culturing general microorganisms, but deep culture in a liquid medium is usually suitable.

The medium used for culture may be a medium containing a culture source used by the microorganism, a synthetic medium, a semi-synthetic medium, or a natural medium. For the medium composition, for example, glucose is used as a carbon source, and L-asparagine is used as a nitrogen source. Other inorganic salts such as sodium chloride, potassium chloride and potassium dihydrogen phosphate are used as necessary.

The culture conditions are pH: 4-9, temperature: 10-3.
2 to 14 days at 5 ° C, preferably pH: 6 to 7, temperature:
Incubate at 22-27 ° C for 5-10 days.

The culture of the present invention is characterized in that it can be used without separating the cells and the medium. A culture, which is a mixture of cells and a medium, is used in the following chemical treatment in a mixed state. Of course, even if the cells or the medium alone are obtained by performing the separation operation on the culture, the extract of the culture, the extract of the cells, the extraction with the organic solvent such as the medium extract, etc. It goes without saying that any of the extracts obtained by adding an operation can be used.

The simple chemical treatment used in the present invention means that the chemical treatment on the culture product produces (R)-(-)-massoilactone, and a known reaction is used alone. Alternatively, these may be combined appropriately.

As the chemical treatment of the culture, acid treatment can be specifically shown as the most preferable example.
Alternatively, the treatment may be performed first with an alkali such as sodium hydroxide and then with an acid. After the acid treatment, dehydration treatment such as phosphorus oxychloride treatment in an organic solvent in the presence of pyridine may be performed. On the other hand, the chemical treatment of the culture extract can be achieved not only by the above example but also by allowing a dehydration condensing agent in an organic solvent to act. Specifically, it can be exemplified that dicyclohexylcarbodiimide is allowed to act in the presence of a catalytic amount of dimethylaminopyridine in an organic solvent. These chemical treatments can be used repeatedly or in appropriate combination.

The acid that can be used in the above acid treatment can be appropriately selected as needed. Specifically, sulfuric acid or phosphoric acid can be preferably exemplified.

The solvent used for the chemical treatment is not particularly required when a culture using a liquid medium is directly used, and is characterized in that the above-mentioned acid is added directly to the medium. When using the bacterial cells or various extracts or when culturing in a medium other than the liquid medium, they are used in combination with a solvent as appropriate. In any case, the addition amount of the acid is not limited, but for example, the concentration in the reaction solution is preferably 0.01 N to 6 N. More preferably, the range of 0.1 normal to 1 normal can be illustrated.

The type of solvent can be appropriately selected.
Specifically, water, methanol, ethanol, diethyl ether, tetrahydrofuran, benzene, toluene and the like can be exemplified.

The reaction temperature can be appropriately selected according to need, and a range from room temperature to reflux temperature can be preferably exemplified. More preferably, the reflux temperature can be exemplified. The reaction time may be, for example, in the range of about 1 hour to 6 hours.

After that, the desired substance is obtained by a usual separation means utilizing the physicochemical properties of (R)-(-)-massoilactone. For example, distillation, steam distillation, solvent extraction, ion exchange, adsorption or distribution column chromatography, gel filtration, dialysis method, precipitation method, etc. are used alone or in combination, and extracted and purified.

If necessary, the reaction mixture described in the preceding paragraph is extracted with a solvent and then concentrated under reduced pressure to obtain a residue. This residue is again dissolved or suspended in the solvent, and after the acid addition, heat treatment is performed again,
By performing the post-treatment as described above, the yield of (R)-(-)-massoilactone can be improved. The combination of the solvent and the acid may be appropriately selected, and sulfuric acid or phosphoric acid in water can be preferably exemplified. More preferably, p-toluenesulfonic acid in benzene or toluene can be exemplified. In addition, as a method other than the acid treatment, other known dehydration method, for example, by reacting phosphorus oxychloride in pyridine, similarly (R)-
(−)-Massoy lactone can be obtained.

The present inventors have added the following formula to the cells of the above-mentioned microbial culture.

[0027]

[Chemical 7] Exophylline A (Exofilin
It was confirmed that A) was included. The confirmation method is as follows. Cells were separated from the culture to obtain a chloroform-methanol extraction fraction. Next, this fraction was subjected to gel filtration and subsequently subjected to reverse phase HPLC to obtain exophylline A.
¹ H of exophylline A obtained by this confirmation test
NMR spectrum (solvent _{CD 3 OD: CDCl 3 = 3} :
1) is shown in FIG.

Therefore, the reaction of producing the (R)-(-)-massoilactone by treating the culture of the microorganism described in the present invention with an acid is carried out by the method described in 3, such as Exophylline A.
A multimer of 5-dihydroxydecanoic acid by an ester bond and 3,5-dihydroxydecanoic acid itself can be considered as an intermediate.

Massoia lactone obtained by the present invention has a specific optical rotation ([α] _D ) of -114 ° (c 0.14).
Met. Literature value ([α] _D −114.5 ° Tetr
ahedron: Assymetry 4,1017
-1026 (1993)), the massoia lactone obtained according to the invention has the absolute configuration of R, 99
It was confirmed that the optical purity was at least%. In addition, the (R)-(-)-massoialactone produced according to the present invention is as long-lasting and mildly sweet as coconut milk produced by known methods. It has a scent of

The massoia lactone produced according to the present invention has a scent similar to that of (R)-(-)-massoia lactone produced by the known method, and thus it can be produced by the conventional method (R). )-(-)-Massoy lactone containing the (R)-(-)-massoy lactone produced by the present invention as an active ingredient of aroma and flavor in the same manner as in the case of producing a fragrance composition containing the same. A perfume composition characterized by the above can be produced. Also, by adding this flavor composition, a fruit juice drink having the same flavor as before,
Beverages such as carbonated drinks, ice cream, Western confectionery, tobacco, shampoo, pomade, lipstick and other cosmetics,
We can provide health and pharmaceutical products such as indoor aroma deodorants, toothpaste, detergents for disinfection, and syrups.

Examples of the present invention will be shown below, but the present invention is not limited to the examples.

[0032]

【Example】

<Culture and collection> Glucose 1%, L- as medium
Asparagine 0.05%, potassium dihydrogen phosphate
A solution of which 0.05% was dissolved in seawater and adjusted to pH 7.0 was used. Exophiala picifila NI 10102 producing Exophylline A in 50 mL of the above liquid medium
1 platinum loop inoculation of the strain (Microtechnology Research Institute No. 14232)
Culture was carried out at 5 ° C. for 4 days with shaking (1). Further, (1) was added to 500 mL of a liquid medium having the same composition, and the mixture was cultured at 25 ° C. for 4 days with shaking (2). Finally, (2) was added to 15 L of medium having the same composition, and the mixture was cultured at 25 ° C for 10 days.

<Chemical Treatment and Purification> The above culture (10
Concentrated sulfuric acid (0.29 mL) was added to 0 mL) and the mixture was heated under reflux for 4 hours. The reaction solution is returned to room temperature, neutralized with 5N aqueous sodium hydroxide solution, and then diethyl ether (50 m
L) extracted 3 times. After concentrating the extract, the resulting residue is subjected to silica gel column chromatography (Merck K
ieselgel 60, Art. 7734 10g;
Purification with elution solvent hexane: ethyl acetate = 5: 1),
1.4 mg of (R)-(-)-massoy lactone was obtained as a colorless oil. (R) -obtained by this method
The physicochemical data of (-)-massoilactone are shown below.

¹ H NMR (90 MHz: CDCl ₃ )
Spectral data δ 0.90 (3H, t, J = 6Hz) 1.1-1.9 (m, 8H) 2.36 (2H, m) 4.45 (1H, m) 6.10 (1H, dt, J = 10,2 Hz) 7.00 (1H, dt, J = 10,5 Hz) Specific rotation [α] _D −114 ° (c 0.14, CHCl
₃

<Confirmation test for exophylline A> Cells were separated from the above culture (15 L), 5 volumes of chloroform-methanol (2: 1) were added, and the mixture was sonicated for 5 minutes (three times) with a disperser. Extracted for 5 minutes. After the extract was filtered, one-fourth the amount of water in the filtrate was added and the two layers were separated. The organic layer was dehydrated and then concentrated to dryness. Next, this fraction was purified by gel filtration (TOYOPEARL HW-40: mobile phase acetone). Fractions containing exophylline A were subsequently subjected to reverse phase HPLC (ODS Hibar RT250-10.
Lichrosorb RP-18: mobile phase acetonitrile: water: trifluoroacetic acid = 3: 1: 0.002
Flow rate of 2 mL / min) to obtain exophylline A having a peak at a retention time of 15 minutes.

[0036]

The effects of the present invention are listed below. (1) A microorganism culture is directly subjected to a chemical treatment without a separation operation, and (R)-(-) is carried out in at least one step chemical reaction.
-Since it is possible to obtain massoia lactone, it is convenient. (2) Since a culture of a microorganism is used, (R)-(-)-
Mass production of massoia lactone becomes possible when used. (3) The obtained (R)-(-)-massoy lactone has a high optical purity and can provide the same quality as conventional production methods. (4) Since no special reagent is used, the treatment of waste liquid is easy and safe.

[0037]

[Brief description of drawings]

FIG. 1 shows a ¹ H NMR spectrum of Exophylline A (solvent CD ₃ OD: CDCl ₃ = 3: 1).

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI technical display location // A24B 15/40 A24B 15/40 (C12P 7/62 C12R 1: 645) (C12P 7/62 (C12R 1:77) (C12P 7/62 C12R 1: 885) (72) Inventor Junko Doshida 559-6 Kitano-cho, Hachioji-shi, Tokyo Nihon Suisan Co., Ltd. Central Research Laboratory

Claims (8)

[Claims] 1. A method of culturing a microorganism, and subjecting the culture to a chemical treatment, the following formula: A method for producing (R)-(-)-massoilactone, which is characterized in that the compound (R)-(-)-massoalactone shown by is produced. 2. The microorganism has the following formula: (However, n ≧ 0, R _{1 to} R ₅ are arbitrary functional groups)
The method for producing (R)-(-)-massoalactone according to claim 1, which is a microorganism that produces a compound represented by: 3. The microorganism is an exophiala.
genus ala, Fusarium genus, Aureobasidium genus,
The microorganism (R)-(-)-according to claim 1, which is a microorganism belonging to any of the genus Trichoderma.
Massoy lactone manufacturing method. 4. A microorganism belonging to the genus Exophiala is Exophiala pis
(R)-(-) according to claim 3, which is C.
-A method for producing massoia lactone. 5. Exophiala pichiphila is Exophiala pichiphila NI 10102 (Microtechnology Research Institute
No. 4232), the method for producing (R)-(-)-massoilactone. 6. The method for producing (R)-(−)-massoialactone according to claim 1, wherein the chemical treatment is acid treatment. 7. (R)-(−)-Massoialactone produced by the method according to any one of claims 1 to 6. 8. A fragrance composition comprising the (R)-(−)-massoy lactone according to claim 7 as an active ingredient.