JPS60188082A - Preparation of (2s)-2-hydroxy-2,6,6-trimethyl-1,4- cyclohexanedione and agent for improving taste and flavor of tobacco composed of said compound - Google Patents

Abstract

PURPOSE:To prepare a cyclohexane derivative having excellent effect as an agent for improving the taste and flavor of tobacco, by the hydroxylation reaction using microorganisms. CONSTITUTION:A microbial strain belonging to Aspergillus genus, e.g. Aspergillus niger JTS191, Aspergillus awamori IFO4314, etc. is cultured. The obtained culture liquid containing the microbial cells is added with (6R)-2,2,6-trimethyl-1,4- cyclohexanedione, the cultivation is continued, and the objective (2S)-2-hydroxy- 2,2,6-trimethyl-1,4-cyclohexanedione is separated from the culture product. The compound is added to the cut tobacco in an amount of about 0.01-30ppm.

Description

[Detailed description of the invention] The present invention is based on =#Kikikairen=≠4(2S) -2-,',
The present invention relates to a method for producing riftyl-1,4-cyclohexanedio/, and a flavor improver for tobacco using the compound.

In recent years, cigarette preferences are rapidly shifting to so-called light-tasting cigarettes with low nicotine and low tar, and at the same time, demands for flavor and flavor are becoming increasingly diverse. Therefore, in order to respond to this trend, research into various additives including flavoring agents is an important issue, especially the development of new flavoring agents that are effective in diversifying tobacco flavors.

From this perspective, the present invention was made with the purpose of providing a compound effective for improving the aroma and taste of tobacco.
The present inventors discovered that certain filamentous fungi (6LL)
It has been discovered that -2,2,6-drimethyl-1,4-cyclohexanedione can be converted to exhibit excellent effects as a converted flavor improver, and the present invention has been completed. That is, the present invention provides (28)-2- represented by structural formula (1)
A method for producing hydroxy-2,6,6-drimethyl-1゜4-cyclohexanedione and a tobacco flavor improver comprising the compound's proboscis 0(1) (28)-2-hydroxy-2,6, 6-Trimethyl-1,4-cyclohexanedione (hereinafter referred to as the present compound) is a crystalline substance and is accompanied by a bitter odor.

The spectral data and physical and chemical properties of this compound are shown below.

Mass spectrometry; m/z (%) 170 (M, 3), 152 (M, 18.3), 142
(M-28, 30).

127 (15), Zoo (8), 96. (4), 85(
12).

70 (15), 69 (15), 58 (100), 57 (
15).

56 (42), 43 (60), 41 (32)”H-NM
几″1 Yellow 1 Deuteroxane-Deuterochloroform 7:3°MS +2. H in the right column indicates that it corresponds to the signal in the left column.

■Disappears with the addition of 3° heavy water Molecular formula: C, H1403 Physical and chemical properties: White crystal m, p, 73-74°C [α)” + 13.6° (C = 0.28.CHCl3
) Has an oily, faded odor Conventionally, the optical rotation of the enantiomer of this compound represented by the structural formula (If) (a)"-' -13: 1° (C = 1.06
3.

CHCl3) compound (2R)-2-hydroxy-2,
6,6-)dimethyl-1,4-cyclohexanedione (
Hereinafter, it will be referred to as compound ■. ) has been reported. (Wad
a.
(] 965)) (IT) Compound 0) is Dlgitalis purpurea
Dlgiprola, a component of L.
This is a compound obtained in the process of determining the structure of cton (digiprolactone).

However, nothing is known about the method for producing this compound or its use as a flavor improver for tobacco.

-Craw゛.

The present compound exhibiting the above-mentioned spectral data and physicochemical properties is represented by the following structural formula (1) ((,R)-2,2,6
-) riftyl-1,4-cyclohexanedione (hereinafter referred to as
Compound I, which is obtained in one step by microbial oxidation from compound I, is preferably as pure as possible.

In addition, the microorganisms used in the present invention belong to the genus Aspergillus (order Aspergillus), and specific examples include the following: Aspergillus niger (order Aspergillus 1us', ni
ger) JTS 191 (Kikoken Bacteria No. 5087), Aspergillus awamori (Aspergi Il.
uaawamorl) IFO4314, Aspergillus Salk+ (Aspergillus 5oJa
e) IFO5241% Aspergillus sufoenizos (
Aspergillus 1 us pboenicis) I
FO8874゜UFO is a microorganism preservation institution of the Japan Federation of Microbial Strains Preservation Institutions, and is a foundation of Fermentation Research Institute. Cultivate and use as seed bacteria using methods such as 01
) When using spores as seed: Cultivate statically on a solid or liquid medium at 24-37°C and allow sufficient spore formation.Usually, spores will be formed on the top surface of the bacterial flora around 3 days after inoculation. Yes, but generally it is preferable that it is from 7th to JO day02)
When using mycelium as a seed; incubate the liquid medium at 24-37℃ for 24 hours.
Either perform shaking or aeration stirring culture for ~60 hours to obtain a porridge-like mycelial suspension and use this, or grind the bacterial cells obtained by static culture using a Waring grinder or the like. , a porridge-like mycelial suspension is obtained and used. These inoculum are further inoculated into a liquid medium and cultured with shaking or aeration with stirring at 24 to 37°C for 24 to 48 hours. At this time, as the inoculation form of the microorganism, in addition to the above-mentioned spores and hyphae, pre-cultured culture solution and the like can be appropriately selected.

In addition, the nutrient sources used in the liquid medium used here and the solid and liquid medium for culturing the inoculum include potatoes,
Wort, corn, g# j -7,, sucrose, f
Carbon flour, corn steep liquor, peptone, meat extract, yeast extract, urea, sodium nitrate, ammonium nitrate, ammonium sulfate, potassium phosphate, potassium chloride, magnesium sulfate, iron sulfate, zinc sulfate, calcium chloride, calcium carbonate, etc. source of nitrogen, unattached salt,
Appropriate growth factors can be selected and used. These nutrient sources are used in the form of an aqueous solution, and when used as a solid medium, 15 to 2.0 g of agar is added and sterilized by heating or p-filtration.

Next, a compound solution is added to the bacterial cell culture solution previously cultured as described above, followed by shaking or aeration.

By this operation, compound 1 is gradually converted to the present compound. The amount of compound 1 added is usually 11 parts of the bacterial culture solution and 0.
5-3g is appropriate.

The physicochemical properties and spectrometry results are as described above. If the molecular formula has one more oxygen than the starting compound, the signal corresponding to the proton of 20 (sweet) CLI disappears in the IH-NMR spectrum, and instead >C(Or() Since a signal corresponding to the structure of CH3 appears, the planar structure of this compound is 2-hydroxy-2,6,6-)dimethyl-1,4
- Determined to be fuchlorohexanedione. In addition, literature (C
hem, Pharm, Bull, 13.43-49,
(1965)) (+)-2-hydroxy-2,6
,6-trimethyl-1,4-cyclohexanedione optical rotation [α]2←5-13.10 (C=1-063.CH
CJ6) From the ratio of 111, the absolute structure of this compound is (
28)-2-hydroxy-2,6,6-)dimethyl-1
, 4-Fuclohexanedione≠
The present compound can be diluted with a solvent such as ethyl alcohol, water, or ethylene glycol to an appropriate concentration and used as a tobacco flavoring agent. The present compound may be used alone, or may be used in combination with other tobacco flavorings, additives, etc.The present compound may be used in an amount of 0.01 to 30 ppm (W/W) based on the shredded tobacco product. degree,
Preferably, when added in an amount of 0.1 to 5 ppm (w/w), it has the effect of imparting a sweet taste and a stinky odor and alleviating irritation. The types of tobacco to which the present compound can be effectively applied are not particularly limited, but it is also effective for improving the flavor and aroma of tobacco such as tangerine tobacco.

Example 1. (Production example) Prepare a potato glucose agar slant medium in a test tube, and add Aspergillus nikah to it.
1us njger) JTS 191 strain (Kikoken Bacteria No. 51157) was inoculated and cultured at 28°C for 7 days to form spores. Next, 30 g of sucrose, 2 g of sodium nitrate, 111 dipotassium phosphate, 05 potassium chloride
g, magnesium sulfate heptahydrate 0.5 g, f ('f) A liquid medium consisting of 1 g of mother extract and 1000 m6 of distilled water was
Pour into a 1 volume Erlenmeyer flask and sterilize at 121°C for 15 minutes.
The spores of the above strain were inoculated at a concentration of approximately 20,000 spores per tmg of culture medium, and then subjected to a rotary shaker and a mill at 210 rpm.
ms 1 yen for 48 hours of culture at 28°C. The culture obtained at this time has a pH of about 3.8 and contains about 5 g of bacterial cells as a dry weight.

Immediately, 11 g of the compound was added thereto, and after culturing at 28° C. for 7 days, a converted culture with a pH of 5.5 and a dry weight of 8 g of bacterial cells was obtained.

From this converted culture, the present compound is purified and isolated. In other words, ethyl acetate is added once to the culture, and 250 ml of
Add 1' each and stir and extract twice. Combine both extracts and add 5% hydrogen carbonate) IJum aqueous solution - 50nl each for 2
Wash twice and then once with distilled water. After dehydration with anhydrous sodium sulfate, the solvent was distilled off under reduced pressure to obtain a neutral brown color with a sweet aroma of 0.90. get il. Next, a column was made using a wet method using 30 g of solica gel and hexane9, and the neutral section was prepared at a mixing ratio of 90:10, 80:
20,70:30 hexane-ethyl acetate mixture ((
Elute each 1 hour with 1 (l Om 1) and separate into 1 (l). and aspergillus awamori (Aspergillus awamori).
IFO4314 strain (Ius awamori) was inoculated and cultured at 28°C for 7 days to form spores.

Next, 30 g of sucrose, 211 sodium nitrate, 11 dipotassium phosphate, and 0.0 g of potassium chloride were added. ．． 5', 9. Magnesium sulfate heptahydrate 0.5.9. Yeast extract 1g, distilled water 1
A liquid medium containing 0100O was placed in a 31-volume Erlenmeyer flask, and after sterilization at 121'C for 15 minutes, the spores of the above-mentioned bacteria were added to a concentration of about 1 ml of the medium.The resulting culture had a pH of about 4.0 and a dry matter. Contains about 6g of bacterial cells. Immediately, compounds 1f and 9 were added thereto, and after 7 days of continuous culture at 28°C, a converted culture with a p1 of 5.0 and a bacterial dry weight of 9 g was obtained.

From this converted culture, the present compound is purified and isolated. That is, 250 mJ of ethyl acetate was added to the culture once.
Add one by one and stir and extract twice.

Combine both extracts and add 5 parts each of sodium bicarbonate aqueous solution.
Wash twice with 0ml and then once with distilled water. After dehydration with anhydrous sulfuric acid (IJ), the solvent was distilled off under reduced pressure to obtain a neutral brown color with a sweet aroma of 0.879. Next, a column was prepared using a wet method using 30 g of silica gel and hexane, and the neutral section was prepared at a mixing ratio of 90:10.
The mixture was sequentially eluted with 100 rJ' of a hexane-ethyl acetate mixture having a ratio of 80:20° and 70:30, and fractionated in log portions. This compound was eluted in the 16th to 18th fractions, the yield was 0.15 g, the yield was 15%, and the purity was over 99%.
It was two.

Example 3. (Production example) Prepare a potato glucose agar slant medium in a test tube, and add Aspergillus soja to this.
lus 5ojae) IFO5241 strain,
Culture at 28°C for 7 days to form spores. Tsuitesoyo [3J/, sodium nitrate 2g, potassium chloride 0.59, magnesium sulfate heptahydrate 0.59, yeast extract 1g, k≧distilled water 10100
Pour a liquid medium consisting of O into a 31-volume Erlenmeyer flask, and add 12
After sterilizing at 1'C for 15 minutes, the spores of the above bacteria were inoculated at a concentration of about 20,000 per ml of medium, and placed in a rotary shaker at 21'C (rpm, 48℃ at 28℃). The culture obtained at this time has a pH of 3.9 and a dry weight of about 5.
Contains 59 bacterial cells. Immediately add compound (1) 1. ! 7
After shaking culture at 28°C for 7 days, pH 4.8,
A converted culture with a bacterial dry weight of 7.51 was obtained.

Purification and single riF of this compound are performed from this converted culture. That is, 250 ml of ethyl acetate was added to the culture once.
Add 1ml each and stir and extract twice.

Combine the exudate from both sleeves and add 5% aqueous sodium bicarbonate solution, 5 t each.
Wash twice with 0 m4 and then once with distilled water. After dehydration with IJum, the solvent was distilled off under reduced pressure to obtain a neutral brown color with a sweet aroma of 0.70. li+
get. Next, a column was prepared using a wet method using 30 g of silica gel and hexane, and the neutral section was prepared at a mixing ratio of 90:
Elute with 100 mJ each of hexane-ethyl acetate mixtures of 10, 80:20, and 70:30, and collect 10 g each.

The present compound was eluted in the 16th to 18th fractions at a concentration of 1 or more. Example 4: 0.5 # of the present compound was added to 100.!9 of the J Debagumi carved product (product name: JASS) from Japan Monopoly Corporation 1r5. It was dissolved in 3 ml of ethanol, sprayed and added, then rolled into paper, and compared with the roll of the above shredded tobacco without the addition of this compound as a control, using a two-point discrimination method for odor, taste, and irritation when smoked. The evaluations of the 20 expert panels were as shown below.

(Note) Number of people who said number C1 was good ■ indicates that there is a significant difference at a risk rate of 1%.

Procedural amendment (voluntary) April 10, 1980 Commissioner of the Japan Patent Office Kazuo Wakasugi 1 Indication of the case 1982 Patent Application No. 43841 2 Name of the invention (2S)-2-hydroxy-2,6,6-drimethyl- 1,4 - Process for producing nclohexanedione and tobacco flavor improver comprising said compound 3 Relationship with the case of the person making the amendment Patent applicant address 2-2-1 Toranomon, Minato-ku, Tokyo Name (456)
) Representative of Japan Monopoly Public Corporation President Minoru Nagaoka 1 Address of designated agent 2-2-2 Toranomon, Minato-ku, Tokyo ■ No. 5 Target of amendment ``Designation-1!1'' and ``Claims 4 in the specification section
('Id J 6 Contents of amendment (1) As per Attachment 1 (2) As per Attachment 2 (Attachment 2) 2. Claims (6R)-2,2,6-)limethyl-1.4 in the culture medium −
A compound (2'5)-2-hydroxy-2,616-
Method for producing a compound represented by 4 (゛4 formula ()) characterized by methyl-1,4-cyclohene

Claims (1)

[Claims] 1. Kuumakiiba (6R)-2,2,6-) Rimethi/l/
The compound (2S)-2-hydroxy-2,6,
6-) Generate remethyl-1,4-cyclohexanedione. A method for producing a compound represented by structural formula (1), characterized in that it has a taste. (1) 2. A flavor improver for tobacco, which is a compound represented by the structural formula (1). (1)