JP3071233B2 - Method for producing 4-hydroxy-4- (3-keto-1-butenyl) -3,5,5-trimethyl-2-cyclohexen-1-one - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to 4-hydroxy-
4- (3-keto-1-butenyl) -3, 5, a method for manufacturing a 5-trimethyl-2-cyclohexen-1-one.

[0002]

2. Description of the Related Art Hitherto, in the manufacture of tobacco, it has been generally practiced to add a carotenoid decomposition product, which is a component of leaf tobacco, in order to improve the flavor and taste of inferior leaf tobacco.

[0003] However, the conventionally known carotenoid decomposed products have a drawback that the effect of improving the taste of tobacco is weak, and when used in large amounts, there is an odor generated from the carotenoid decomposed products themselves.

[0004] The present invention has been made in view of the above points, and an object of the present invention is to provide a method for producing a compound capable of imparting or enhancing flavor to cigarettes in a very small amount.

[0005]

Means for Solving the Problems In order to solve the above-mentioned problems, the present inventor has conducted a wide search for substances effective in improving the taste of tobacco, and as a result, has found that 4-hydroxy-4- (3-keto-1-butenyl) -3, 5, onsets seen to have an improved effect of tobacco flavor and taste having excellent 5-trimethyl-2-cyclohexen-1-one, the compound, 4-hydro-oxy - 4- (3-hydroxy-
1-butenyl) -3, 5, we have found that it is possible to readily prepared by conversion by the action of microorganisms 5-trimethyl-2-cyclohexen-1-one.

Namely, the present invention is represented by the general formula (II) shown in Chemical formula 3 4 Hydro-4- (3-hydro-1-butenyl) -3, 5, 5-trimethyl-2-cyclohexen 1-one (II) 4-hydro-4-formula shown bacteria are allowed to formula 4 act belonging to the genus Aspergillus (I) to (3-keto-1-butenyl) -3, 5, 4-hydroxy-4- (3-keto-1-butenyl) -3, which is converted to 5-trimethyl-2-cyclohexen-1-one (I).
5, to provide a production method of 5-trimethyl-2-cyclohexen-1-one.

[0007]

[0008]

Embedded image

[0009]

Embedded image Hereinafter, the present invention will be described in detail.

[0010] The 4-hydroxy-4- (3-
Keto-1-butenyl) -3, 5, 5-trimethyl-2
Cyclohexen-1-one (I) [hereinafter, compound (I)
Is a compound isolated / extracted from Podocacarus blumei by chromatography.
- Hydro-4- (3-hydro-1-butenyl) -3, 5, 5-trimethyl-2-cyclohexen-1-one (II) [Burumenoru A, below, compound (II) and referred] derivatives (JCS Che)
m. Com. , 3,9 FEB, P. 113-114,
(1972)). It is known that compound (I) can be obtained by oxidizing α-ionone.

The compound (I) of the present invention can be produced, for example, by the following steps:
It is performed as follows. The microorganism used in the production of the compound (I) is a bacterium belonging to the genus Aspergillus. As a specific example, Aspergillus niger (A
spergillus niger) JTS-191 strain [Microtechnical Laboratory No. 5087 (FERP P-508)
7)], Aspergillus awamori (Aspergil)
rus awamori IFO4314, Aspergillus soja
e) IFO5241, Aspergillus hoenesis (A
spergillus phoenicis) IFO8
874, Aspergillus Itaconix (Asperg)
illus itaconicus) IFO4419 and the like, but Aspergillus niger JTS-19.
One strain is preferred. The IFO is a fermentation research institute.

First, spores or hypha of the cells of the genus Aspergillus niger as described above are cultured to obtain a seed. (1) When spores are used as inoculum; static culture is carried out at 24 to 37 ° C. in a solid medium or liquid medium to form sufficient spores. Usually, spores are formed on the entire surface of the bacterium from about 3 days after inoculation.

(2) When a mycelium is used as a seed fungus; shaking or aeration and stirring in a liquid medium at 24 to 37 ° C. for 24 to 60 hours to obtain a porridge mycelium suspension, which is used. Alternatively, the cells obtained by static culture are ground with a Waring-brader or the like to obtain a porridge mycelium suspension, which is used.

The inoculum thus obtained is further inoculated into a liquid medium, and cultured at 24 to 37 ° C. for 24 to 48 hours with shaking or aeration and stirring. At this time, as the inoculation form of the microorganism, in addition to the spore or mycelium suspension as described above, a pre-cultured culture solution or the like can be appropriately selected.

The nutrients used in the liquid medium and the solid and liquid medium for culturing the inoculum include potato, wort, corn, glucose, sucrose, starch, corn steep liquor, peptone, and the like.
Meat extract, yeast extract, urea, sodium nitrate, ammonium nitrate, ammonium sulfate, potassium phosphate, potassium chloride, iron sulfate, zinc sulfate, calcium chloride, calcium carbonate, and other carbon sources, nitrogen sources, inorganic salts, growth factors, etc. It can be appropriately selected and used. These nutrient sources are used as an aqueous solution or a solid medium obtained by adding 1.5 to 2.0% agar to the aqueous solution, and then sterilized by heating or filtration before use.

Next, as described above, the compound (II) is added to the cell culture solution prepared in advance, followed by culturing by shaking or stirring with aeration. Thereby, the compound (II) is gradually converted to the compound (I). The amount of the compound (II) to be added to the cell culture solution was 0.5 to 1 liter of the cell culture solution.
~ 1.0 g is appropriate.

Next, after confirming that the compound (II) has been sufficiently converted to the compound (I), the culture is extracted with ethyl acetate. The extract is washed successively with an alkaline solution such as a 5% aqueous sodium hydrogen carbonate solution and water, and the organic layer is dried over anhydrous sodium sulfate and the like, and the solvent is distilled off under reduced pressure.

The thus obtained neutral fraction is subjected to column chromatography using silica gel 30 times or more the neutral fraction and eluted sequentially with a mixed solution of hexane and ethyl acetate to give compound (I). Is isolated and purified.
For example, elution was first performed with 200 ml of a hexane / ethyl acetate mixed solution in which hexane and ethyl acetate were mixed at a ratio of 2: 1, and then with 200 ml of a hexane / ethyl acetate mixed solution having a 1: 1 mixing ratio. In this case, 30 fractions were obtained, among which fractions 16 to 19 contained compound (I).
Is obtained.

When the compound (I) is used as a tobacco flavor enhancer, its addition amount is, for example, from 0.01 ppm to 100 pp based on the weight of the tobacco cut.
The range of m is preferred.

[0020]

The following is a description of the present invention.
(3-keto-1-butenyl) -3, 5, will be described in detail an example of a manufacturing method of the 5-trimethyl-2-cyclohexen-1-one (I).

First, a modified Tzapek Dox medium (sucrose 30) contained in a 3-liter triangular flask with a bump was used.
g, 2 g of sodium nitrate, 1 g of dicalcium phosphate, 1000 ml of distilled water) and 1 ml of spores of Aspergillus niger JTS-191 in 6 ml of sterilized water so that the spore concentration is 2 to 4 × 10 ⁴ per ml of medium. The suspension was inoculated, and shaking culture was performed at 28 ° C. and 200 rpm for 72 hours. Here, the spores of Aspergillus niger used were those grown sufficiently on a slant of potato glucose agar medium.

[0022] When mycelia culturing the spores as described above were grown sufficiently, the medium per liter of 1 g 4-hydro-4- (3-hydro-1-butenyl) -3, 5, 5 Trimethyl-2-cyclohexene-
1-one (II) (trade name Blumenol A, manufactured by Soda Corporation) was added, followed by shaking culture under the same conditions. At this time, in order to confirm the progress of conversion of Blumenol A (II), cultures were collected every 2, 4, 6, and 13 days, and confirmed by thin-layer chromatography. On the 13th day, disappearance of the spot of Blumenol A (II) was confirmed, and the culture was stopped.

Next, this culture was filtered to separate the cells into filtrate and filtrate, and the filtrate was extracted with ethyl acetate. This extract was washed twice with an alkaline solution such as a 5% aqueous sodium hydrogen carbonate solution, and then once with distilled water. Then, anhydrous sodium sulfate was immediately added to remove water in the extract, and the solvent was distilled off under reduced pressure to obtain 0.7 g of a converted product.

The total amount of the thus obtained converted product was dissolved in a small amount of ethyl acetate and subjected to silica gel column chromatography. As the column, a column tube of column diameter × height = 40 mm × 250 filled with Wakogel C300 (manufactured by Wako Pure Chemical Industries, Ltd.) was used, and hexane and ethyl acetate were mixed at a ratio of 2: 1. Eluted sequentially with 200 ml of the hexane / ethyl acetate mixed solution and 200 ml of the hexane / ethyl acetate mixed solution having a mixing ratio of 1: 1.
30 fractions (fraction 1-fraction 10) of 13 ml each were obtained. Of these, 0.3 g of the conversion product was obtained in fractions 16 to 19.

MS spectrum of the obtained conversion product, I
R spectrum, ¹ H-NMR spectrum and ¹³ C-NM
The R spectrum was as follows.

MS: m / e, 223 (M +), 180,
166, 124, 43 IR: cm ⁻¹ , 3500 (OH), 1650 (C = 0) ¹ H-NMR (CDCl ₃ ): 1.03 (3H, s),
1.11 (3H, s), 1.91 (3H, s), 2.3
1 (3H, s), 2.41 (2H, q, J = 17 Hz),
3.10 (1H, s, OH), 6.49 (1H, d, J
= 6HZ), 6.86 (1H, d, J = 6HZ) 13 C-NMR (CDCl 3): 197.81,197.
46, 161.22, 145.50, 130.30, 1
27.44, 79.07, 49.48, 41.44, 2
8.15, 24.28, 22.90, 18.77.

The above ¹³ C-NMR spectrum shows that the number of carbon atoms is 13, that the chemical shift of carbon at position 9 shifts from 68.73 ppm before conversion to a lower magnetic field side, and that the CH-carbon before conversion is Indicates that the carbon at the 9-position has changed to C-.

Further, the ¹ H-NMR spectrum is as follows.
401 ppm of protons have disappeared, and the chemical shift of methyl at position 10 has shifted to the low magnetic field side of 2.31 ppm, indicating a singlet, and the carbon at position 9 has been changed to C-. You can see that it is doing.

The above measurement results and the fact that the conversion products are produced only from Burumenoru A (II), the conversion product 4-hydro-4- (3-keto-1-butenyl) -3, 5 , 5-Trimethyl-2-cyclohexene-
It was determined as 1-one (I).

Example 2 4-Hydroxy-4- (3-keto-obtained in Example 1)
1-butenyl) -3, 5, 5-trimethyl-2-amount of cyclohexene-1-one (I) is suitably dissolved in ethanol so as to 0.01ppm and 100ppm per unit weight of cut tobacco, cigarettes Spray was added uniformly to the tobacco cut of the leaf set. Thereafter, the mixture was allowed to stand at room temperature (25 ° C.) for 2 days to allow the compound (I) to sufficiently adapt to cigarettes, and then rolled up in a cigarette form to obtain a flavored product.

The obtained flavored product was subjected to a two-point discrimination test with a non-scented product to which no compound (I) was added, and a panel specializing in the taste of cigarettes of 20 persons for flavor, smoke volume, and softness was obtained. Was used to perform a sensory test. The results are shown in Table 1 below.

Table 1 Category Flavor Smell of smoke and softness Scented product 1 0.01 ppm 19 19 2 １９ 19 193 １９ 19 19 1 100 ppm 19 19 2 １８ 18 193 〃 18 19 Unscented product 1 1 * Indicate the number of panelists who are good.

As is apparent from Table 1 above, in the case of the fragranced product, both the flavor, the amount of smoke and the crispness were increased at both the addition amounts of 0.01 ppm and 100 ppm, and excellent tobacco aroma was obtained. It was confirmed that it had an effect of improving the taste.

On the other hand, in the non-scented test sample, no improvement was found in any of the flavor, the feeling of smoke amount, and the crispness.

[0035]

As described above, as described above, the 4-hydroxy-4- (3-keto-1-butenyl) -3,
5, 5-trimethyl-2-cyclohexen-1-according to the production method of the on, can be readily applied to enhance the excellent flavor and taste to the tobacco by adding a small amount to the cut tobacco or the like, the tobacco product quality Can be easily produced by allowing a microorganism to act on a starting material.

──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Shizuo Suhara 6-2 Umegaoka, Midori-ku, Yokohama-shi, Kanagawa Prefecture Inside the Tobacco Central Research Laboratories of Tobacco Inc. (72) Inventor Yasuhiro Shinozaki 6 Umegaoka, Midori-ku, Yokohama-shi, Kanagawa Prefecture Address 2 Tobacco Central Research Laboratory, Japan Tobacco Inc. (72) Inventor Jin Shigematsu 6-2 Umedaoka, Midori-ku, Yokohama-shi, Kanagawa Prefecture Tobacco Central Research Laboratory, Japan Tobacco Inc. (56) -9038 (JP, B2) (58) Fields investigated (Int. Cl. ⁷ , DB name) C12P 7/26 CA (STN) REGISTRY (STN)

Claims (1)

(57) [Claims] 1. A 4-hydro-4- (3-hydro-1-butenyl) -3, 5, 5-trimethyl -
4-Hydroxy-4- (3-keto-1-butenyl) represented by the general formula (I) shown in Chemical Formula 1 by allowing a microorganism belonging to the genus Aspergillus to act on 2-cyclohexen-1-one.
-3, 5, 5-trimethyl-2-cyclohexen-1-
And converting the on (I) 4-hydro-4- (3-keto-1-butenyl) -3, 5, 5
A method for producing trimethyl-2-cyclohexen-1-one. Embedded image