JP2003250591A - Method for producing nootkatone - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing nootkatone having high purity and excellent flavor in very good yield and high selectivity. <P>SOLUTION: This method for producing nootkatone is to convert valencene to nootkatone by treating valencene with mycelia of the genus Mucor or their treated product. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing a nootkatone by treating valencene with a bacterial cell of a microorganism belonging to the genus Mucor or a treated product thereof to convert it into a nootkatone.

[0002]

2. Description of the Related Art In recent years, with the diversification of various food materials, food additives, foods and drinks, cosmetics, health and hygiene materials, new demands for the flavors used for them have been increased, and unique and highly palatable. There has been a demand for the development of fragrance substances having various odors. In particular, regarding citrus fruit-based flavors and food-based flavors, which are the most in demand among flavors, from the viewpoint of safety, natural compounds or substances derived from natural compounds are used as raw materials and obtained by biochemical conversion reaction by enzymes, cultured cells, etc. The development of fragrance materials was strongly desired.

The following equation

[Chemical 1] The Noot Katong represented by is one of the important fragrance materials, but in nature, the grapefruit oil typically contains less than about 1%, and the purified products obtained from natural products are very It is expensive. Therefore, in consideration of product safety and environmental protection in manufacturing such as the use of reagents by chemical synthesis and the disposal of large amounts of solvents, etc., we have utilized enzymatic reactions and conversion reactions by living organisms using natural products as raw materials. Development of the obtained Noot Katong has been eagerly awaited, and various manufacturing methods have been studied so far.

[0004] Further, the aroma intensity of the nootkatone is greatly different among the enantiomers, and the value of the nootkaton having the same absolute structure as that of natural one is very high. Under such circumstances, conventionally, a method for producing a nootkatone having the same absolute structure as a natural product by using a natural product of valencene as a raw material and utilizing a biochemical reaction has been reported.

[0005] For example, a method for producing Nootkatone using microbial of the genus Rhodococcus from valencene as a raw material (JP-A-6-303,967) or a bioconversion reaction using citrus cell suspension medium from valencene as a raw material. Method of manufacturing Noot Katong (Plant Cell Report,
Volume 3, p. 37, 1984) etc. have been reported. However, for example, in JP-A-6-303967, 500 mg in a 500 ml flask containing 50 ml cell suspension of Rhodococcus spp.
These methods have low conversion efficiency to nootkatone, as the amount of nootkaton obtained by culturing at 30 ° C. for 4 days with a small amount of 2.5 mg was small.
It is hard to say that it is industrially desirable.

[0006] Recently, a method has been reported in which valenecene is converted to valenecene hydroperoxide by a laccarase-catalyzed oxidation or enzymatic oxidation reaction, and then this hydroperoxide is decomposed to obtain nootkatone. No. 501052 and JP-A No. 2001-103989) are still not desirable in view of cost and yield problems.

[0007]

DISCLOSURE OF THE INVENTION The present invention aims to solve the above-mentioned various problems, has a high yield, and is suitable for industrial practical application from the viewpoint of optical activity selectivity. It is intended to provide a method for inexpensively producing Nootkatone from Valensen, which is a natural product, in consideration of safety and environmental protection in production.

[0008]

Means for Solving the Problems As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that valencene is converted by carrying out conversion using cells of a microorganism belonging to the genus Mucor or a treated product thereof. The inventors have found that it is possible to produce Nootkatone from the above, and have completed the present invention.

That is, the present invention provides the following (1)-
Provide (3). (1) A method for producing a nootkatone, which comprises producing valetene by treating valencene with a microbial cell belonging to the genus Mucor or a treated product thereof to convert into nootkaton.

(2) The microorganisms belonging to the genus Mucor are Mucor hiemalis, Mucor mucedo, and Muco javanicus.
r javanicus), Mucor fragili
s), Mucor circinelloi
des and Mucor genevensi
The production method according to (1), which is selected from the group consisting of s).

(3) The Mucor Hiemalis f. Mucor hiemalis f.
hiemalis) IFO8567, Mucor Hiemalis F. Hiemalis IFO8565, Mucor Hiemalis F. Hiemalis IFO8449, Mucor Hiemalis F. Hiemalis IFO5834, Mucor hiemalis F. Corticolus (Mucor hiemalis f. coryolus・ F Corticolas IFO9401 or Mucor hiemal
is f. luteus) IFO9410, Mucor Semdo is Mucor Mused IFO6750, Mucor Javanix is Mucor Javanics IFO5382, Mucor Fragilis is Mucor Fragilis IFO9402, Mucor Sircinerodes is Mucor Sircyneroides Ef Sircineroides circ f. circinelloides) IF
The production method according to (2), wherein O30470 and Mucor Genevensis are Mucor Genevensis IFO6415.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in more detail below. In the production method of the present invention, the valencene used as the substrate may be those isolated and purified from essential oils of citrus fruits such as grapefruit, orange and valencia orange, but are not particularly limited thereto. The purity of valencene is preferably 30% or more, and particularly preferably 50% or more.

In the method of the present invention, a microorganism belonging to the genus Mucor is used. Many species have been confirmed as the microorganism, and any microorganism can be used in the production method of the present invention as long as it has the ability to convert valencene into nootkatone. Such microorganisms of the genus Mucor include Mucor hiemali.
s), Mucor mucedo, Mucor
Examples thereof include Mucor javanicus, Mucor fragilis, Mucor circinelloides, Mucor genevensis, and the like. These microorganisms are widely distributed in the natural world, and it is possible to separate and obtain them from the natural world. For example, from the Fermentation Research Institute, 2-1785, 13-3 Honcho, Yodogawa-ku, Osaka, Japan The readily available Mucor hiemalis f. Hiemalis IFO8567, IFO8
565, IFO8449, IFO5834, Mucor hiemalis f. Corticolus IFO
9400, IFO9401, Mucor hiemalis f. Luteus IFO9410, Mucor
Mused IFO6750, Mucor Javanes IFO5382, Mucor Fragilis IFO9402, Mucor circinelloides
f. circinelloides) IFO30470 and Mucor Genevensis IFO6415 can be used. Also,
As long as it has the ability to be converted into Nootkatone, it may be a mutant strain obtained by artificially treating the cells of the above Mucor microorganism, for example, a mutation treatment, and further, a cell fusion method, recombination. It may be a fusion strain or a transformant strain obtained by applying a genetic engineering technique such as a DNA method.

The conversion in the method of the present invention is carried out using these microorganisms belonging to the genus Mucor. Basically, the conversion is not particularly limited as long as it is a method of converting microbial cells or a treated product thereof into valencene as a raw material in an aqueous medium to convert into nootkatone. Here, the treated product of the microbial cell means various treatments of the microbial cell such as a culture, freeze-dried product, ground product, extract of crude enzyme or purified enzyme, and immobilized microbial cell. Means what you have done. The treated cells are allowed to act in an aqueous medium as needed. As the aqueous medium, water,
A buffer solution, a culture solution, etc. may be mentioned, but the invention is not limited thereto. As a concrete method for performing the conversion of the present invention,
(1) A method of adding valencene to the culture of the microbial cells, (2) a method of culturing the microbial cells in a medium containing valencene, (3) a method of contacting valencene to the immobilized cells of the microorganism, There are (4) a method of bringing valencene into contact with the ground product of the microbial cells, (5) a method of bringing valencene into contact with an extracted enzyme solution of the microbial cells, and the like. For example, the method of adding valencene to the culture of the microbial cells can be carried out as follows.

The culture medium is not particularly limited as long as it can culture a microorganism of the genus Mucor, but for example, the Czapek-peptone medium described in Example 1 is used. The culture can be static culture, shaking culture, aeration-agitation culture, etc., and the culture temperature is 20 to 30 ° C., p
H is preferably 4 to 8. Of these, particularly preferred is 2
It is 7 ° C and pH 4-8. The number of culture days requires a period until reaching the number of cells required for conversion, and is usually about 5 to 20 days, but is not particularly limited to this.

The conversion reaction of the present invention can be achieved by adding valencene, which is a conversion substrate, to a culture of a microorganism of the genus Mucor grown as described above and further culturing. Valensene to be added varies depending on the purity and the type of the microorganism of the genus Mucor and the number of cells thereof, but the addition concentration is 0.5 mg / l to 50 g / l.
Is preferable, and for example, 500 mg or more of valencene can be added to 100 ml of a culture containing 1,000 mg / 100 ml of Mucor spp. Further, it is possible to increase the added amount by using a surfactant or the like. Valensene can be added in the form of solid or liquid, and the total amount thereof may be added in one step or in multiple steps of two or more steps, or continuously.

The culture for carrying out the conversion reaction can be carried out under any condition such as static culture, shaking culture, aeration and stirring culture, but shaking culture is preferable from the viewpoint of reaction rate,
Specifically, it is suitable to carry out at 0 to 150 rpm. The reaction temperature is preferably 20 to 30 ° C. and the pH is preferably 4 to 8. The conversion reaction is initiated with the addition of the substrate valencene, the reaction time being preferably 5-18 days.

In the above-mentioned method, the reaction product Nootkatone obtained by converting the bacterial cells of the microorganism belonging to the genus Mucor or a processed product thereof is a known method in the separation and purification of general organic compounds, for example, Filtration, extraction,
It can be recovered and purified from the culture broth by means such as distillation and column chromatography, or a combination thereof. The Nootkatone obtained by the present invention is useful as a perfume material such as various foods, food additives, foods and drinks, perfumes and cosmetics, and health and hygiene materials.

[0019]

The present invention will be described in more detail with reference to the following examples, which should not be construed as limiting the invention thereto. The devices used for measuring the physical properties in the examples are as follows. 1) Chemical purity gas chromatograph; HP-5890 (manufactured by Hewlett Packard) Column; DB-17 (0.25 mm x 30 m) (manufactured by J & W) Injection temperature 250 ° C, temperature rising program is initial temperature 50 ° C
To 250 ° C., 5 ° C./min., And the carrier gas was helium (1 ml / min.). 2) Nuclear magnetic resonance spectrum ¹ H-NMR; Mercury-300 type (300 MHz) (manufactured by Varian) ¹³ C-NMR; Mercury-300 type (75 MHz) (manufactured by Varian) 3) Infrared absorption spectrum (IR) FTIR -410 type (manufactured by JASCO Corporation) 4) Mass spectrum (MS): Gas chromatograph with mass selective detector; HP-5890 / HP5973
(Manufactured by Hewlett Packard) Column: DB-17 (0.25 mm x 30 m) (manufactured by J & W) 5) Optical rotation DIP-1000 (manufactured by JASCO Corporation)

[Example 1] Reaction of conversion of Valensen to Nootkatone by mucor hiemalis Mucor hiemaris F hyemaris IFO8567 obtained from Fermentation Research Institute MY medium 100 having the following composition
Each 500 ml flask containing 100 ml was inoculated and cultured with shaking for 6 days. (MY medium composition; per liter) Yeast extract 0.3 g Malt extract 0.3 g Polypeptone 0.5 g Glucose 1.5 g

Czapek-pep containing 5 ml of the culture medium shown below
It was inoculated into a 500 ml fold Sakaguchi flask containing 100 ml of tone medium. PH of (Czapek-peptone medium composition) Sucrose 15 g Glucose 15 g of polypeptone _{5 g K 2 HPO 4 1 g} MgSO 4 · 7H 2 O 0.5 mg KCl 0.5 mg FeSO 4 · 7H 2 O 0.01 g H 2 O 1.0 L ( medium Is adjusted to pH = 7.0 with HCl)

The cells were grown at 27 ° C. for 4 days with shaking at 150 rpm to grow the cells, and the substrate, valencene (([α] D
²⁰ + 96.5 ° (c = 0.99, CHCl ₃ ) was added with 50 mg in purity of 95% or more (from GC-MS), and further cultured at 27 ° C for 5 days with shaking at 150 rpm. Regarding the change with time of the reaction, a constant amount (1 ml) of the culture solution was collected every 24 hours in an adsorbent, Extrelute, and eluted with ether, and then the ether extract was subjected to GC-MS analysis (Hewlett-Packard). , capillary GC-quad
rupole MS system, Model HP-5890 (GC / MSD HP-5890 / HP
-5973); DB-17 column).

50 ml of ether was added to the culture solution for 15 hours.
After shaking at 0 rpm, suction filtration was performed, and the filtrate was separated into an aqueous layer and an ether layer. The ether layer was dried over magnesium sulfate, filtered, and concentrated under reduced pressure using a rotary evaporator at a low temperature of 30 ° C. to give a residue of 46.7.
to obtain mg. As a result of quantifying this residue by GC analysis by the internal vote method, the content of nootkatone was 26.5 mg (yield from valencene was 56.8%). Furthermore, when the residue obtained in this example was collected by GC and measured by NMR, it was in agreement with the standard product.

Example 2 Valensen's Mucor Musedo, Mucor Javanes, Mucor Fragilis,
Mucor Hiemalis conversion reaction to nootkatone Mucor Hiemaris ef Hiemaris IF of Example 1
Replace O8567 with Mucor Musedo IFO6750, Mucor Javanes IFO5382, Mucor Fragilis IFO9402, Mucor Hiemalis F Hiemalis IFO5834,
The same test as in Example 1 was performed. As a result, generation of the following Nootkatone was confirmed by GC analysis.

[0025]             Strains used Nootkatone production   Mucor Musede IFO6750 25.0 mg   Mucor Javanics IFO5382 24.6 mg   Mucor fragilis IFO9402 28.8 mg   Mucor Hiemalis F Hiemalis IFO5834 18.6 mg

The results of Examples 1 and 2 are shown in JP-A-6-30.
Compared with 3967, in JP-A-6-303967, 500 mg in a 500 ml flask containing 50 ml of cell suspension of Rhodococcus spp.
It was confirmed that the amount of produced Nootkatone of the present invention was remarkably high, while only 2.5 mg of Nootkaton was obtained when valensene was added and cultured at 30 ° C. for 4 days.

[Example 3] 5 ml of Czapek-peptone medium (200 ml)
The mixture was placed in a 00 ml Erlenmeyer flask, sterilized, and inoculated with Mucor sp. Collected from the moss Kunomono moss, followed by rotary culture (100 rpm) at 30 ° C. for 3 days. Valensene (([α] D ²⁰ + 96.5 ° (c = 0.99, C
About 300 mg of HCl ₃ ) with a purity of 95% or more (from GC-MS) was added. The change over time in the reaction is constant every 24 hours (1 m
l) The culture solution was collected in an adsorbent, Extrelute, and eluted with ether.
GC-MS analysis (Hewlett-Packard, capillary GC-quadrupole
MS system, Model HP-5890 (GC / MSDHP-5890 / HP-5973);
Confirmed by performing DB-17 column). Culture reaction 9
The reaction was stopped on the day, 50 ml of ether was added to the culture solution, and the mixture was shaken at 150 rpm for 1 hour and then suction-filtered to separate a filtrate into an aqueous layer and an ether layer. The ether layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure using a rotary evaporator at a low temperature of 10 ° C. to obtain a residue (243.2 mg). Furthermore, the residue was subjected to column chromatography using silica gel (20 g; Merck 70-230 mesh) with a hexane-ether solvent while increasing the content of ether, and a nootcatone (151.6 mg; valencene) having a purity of 95% or more was obtained. Yield of 47.2%) was obtained.

Specific rotation ([α] D ²⁰ +193.
5 ° (c = 1.10, CHCl ₃ )) coincided with the specific optical rotation ([α] D ²⁰ + 195.5 ° (c = 1.5, CHCl ₃ )) of the standard Nootkatone. In addition, MS spectrum, IR spectrum, ¹ H NMR spectrum,
^{The 13} C NMR spectrum was also in perfect agreement with the spectrum data of the standard product.

[Example 4] The amount of valencene added was 10
The conversion reaction was carried out using Mucor Spies collected from the moss Kumonosago moss under the same conditions as in Example 1 except that the reaction was stopped at 11 mg on the 11th day, and 99.5 mg of residue was obtained by concentration under reduced pressure. The residue is silica gel
(10 g; Merck 70-230 mesh) was subjected to column chromatography while increasing the content of ether with a hexane-ether solvent, and a purity of 95% or higher of Nootkatone (65.2 mg; yield from valencene was 61.0). %) Was obtained.

[0030]

INDUSTRIAL APPLICABILITY The method of the present invention is industrially suitable because nootkatone can be obtained in high yield and high selectivity as compared with the known prior art. In addition, the method of the present invention is a very efficient production method with very little generation of by-products. further,
The nootkatone obtained by the method of the present invention has a higher purity and is very excellent in aroma as compared with the nootkaton obtained by the conventional method. Therefore, various foods, food additives, foods and drinks, cosmetics, health and hygiene materials. It can be used in a wide range as a fragrance material such as.

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Chie Murakami             Tokushima Prefecture Tokushima City Yamashiro Town Higashihama Roadside 5-42 Petit             Maison Morimoto 203 (72) Inventor Mai Furusawa             3-71 Okihama East, Tokushima City, Tokushima Prefecture New DK Ha             Its 407 (72) Inventor Tsuneyoshi Kanizawa             Kanagawa Prefecture Hiratsuka City Nishihachiman 1-14-1 Taka             Sand Research Institute, Inc. (72) Inventor Makoto Emura             Kanagawa Prefecture Hiratsuka City Nishihachiman 1-14-1 Taka             Sand Research Institute, Inc. (72) Inventor Katsuhisa Mitsuhashi             Kanagawa Prefecture Hiratsuka City Nishihachiman 1-14-1 Taka             Sand Research Institute, Inc. F-term (reference) 4B064 AC40 CA05 CB11 CC03 DA16

Claims (3)

[Claims] 1. A process for producing a nootkatone, which comprises producing veneus caton by treating valencene with a bacterial cell of a microorganism belonging to the genus Mucor or a treated product thereof to convert into nootkaton. 2. The microorganisms belonging to the genus Mucor include Mucor hiemalis, Mucor mucedo, and Mucor javanics.
vanicus), Mucor fragili
s), Mucor circinelloi
des and Mucor genevensi
The method according to claim 1, which is selected from the group consisting of s). 3. A mucor hiemalis f. Hiemal is a mucor hiemalis f. Hiemal.
is) IFO8567, Mucor Hiemarith F Hiemaris IFO8565, Mucor Hiemaris F Hiemaris
IFO8449, Mucor Hiemarith F Hiemaris I
FO5834, Mucor hiemalis f. Corticolus IFO9400, Mucor hiemalis f. Corticolus IFO9401 or Mucor hiemalis f. Luteus (Mucor hiemalis f. Corticolus)
f. luteus) IFO9410, Mucor Semdo is Mucor Mused IFO6750, Mucor Javanix is Mucor Javanics IFO5382, Mucor Fragilis is Mucor Fragilis IFO9402, Mucor Sircineloides is Mucor Sircineroides ef Circineroides in circ. ) IFO304
70. The process according to claim 2, wherein 70 and Mucor Genevensis are Mucor Genevensis IFO6415.