JPH0651651B2 - 2,7,11-Cembratriene-4,6,20-triol, process for producing the same, and flavor enhancer for tobacco comprising the compound - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to 2,7,11-sembratriene-4,6-
Novel compound 2,7,11-sembratriene-4,6,2 obtained by allowing microorganisms to act on diol
It relates to 0-triol. This compound is a novel substance effective for improving the flavor and taste of tobacco.

[Prior art]

Heretofore, a large number of compounds have been already known that are effective in imparting a more favorable taste and flavor or improving the flavor and taste of a smoking material material by adding it to a smoking article in the manufacturing process of tobacco. However, the compounds of the present invention are also novel as chemical substances, and thus have never been used in the manufacture of cigarettes.

[Problems to be solved by the invention]

The present invention has been made for the purpose of developing and providing a new flavoring agent that can meet various consumer needs for the recent flavor and taste of tobacco products.

[Means for solving problems]

The present inventors have found that 2,7,11-sembratriene-4,6-diol represented by the formula (II) [hereinafter referred to as compound (II)] has Bacillus megaterium N
Excellent scent as a conversion substance by the action of H5 (Microtechnology Research Institute, No. 4448) It was found that a novel compound 2,7,11-sembratriene-4,6,20-triol represented by the formula (I) having a taste improving effect can be obtained, and the present invention forms the present invention. Came to.

Below, the analytical data of this compound are shown.

Molecular formula: C ₂₀ H ₃₄ O ₃ Molecular weight: 322 Infrared absorption spectrum: 3344 cm ⁻¹ Nuclear magnetic resonance spectrum: (Measuring instrument Bruker AM 500 spectrometer) ¹ H-NMR ((CD ₃ ) ₂ CO, TMS) δ (ppm) : 5.47 d, J = 15.7 5.23 dd 5.22 d J = 7.2 5.18 dd J = 15.6,9.3 4.78 m 4.16 dd J = 6.2,12.2 3.96 dd J = 4.6,12.2 2.28 m 2.23 m 2.11 m 2.05 m 2.01 dd J = 14.0 , 1.1 1.91 m 1.75 dd J = 13.8,9.1 1.67 s 1.50 m 1.35 s 0.83 d J = 6.7 0.80 d J = 6.8 ¹³ C-NMR (δ in CDCl ₃ , TMS) (Measuring instrument Bruker AM500 spectrometer) 16.0 (-CH _{3), 19.2 (-CH 3)} , 20.7 (-CH 3) 22.5 (-CH 2), 27.9 (-CH 2), 28.9 (-CH 3) 32.0 (-CH 2), 32.8 (-CH), 38.9 (-CH ₂ ) 46.5 (-CH), 52.6 (-CH ₂ ), 59.9 (-CH ₂ ) 64.4 (-CH), 71.5 (-CR ₃ ), 127.9 (-CH) 130.2 (-CH), 131.7 ( -CH), 136.1 (-CR ₃ ) 136.3 (-CH), mass spectrum GC-MS 257 (2) 159 (9) 145 (8) 133 (12) 107 (18) 91 (21) 81 (38) 71 (31) 55 (35) 43 (100) Specific rotation ▲ (α) ²⁵ _D ▼ + 140.3 (Ethanol, C = 0.32) Next, a method for producing compound (I) by microbial conversion of compound (II) will be described step by step.

First, Bacillus megaterium NH5 is cultured as the inoculum by the following method. That is, a solid medium or a liquid medium is inoculated with the bacterium and cultured at 30 to 43 ° C for 1 to 2 days. These inoculums were further inoculated into a liquid medium at 37 ° C.
Shake or aeration stirring culture for 2 to 6 hours. Nutrient sources used for the liquid medium and the solid and liquid medium for inoculum culture used here include glucose, sucrose, corn steep liquor, peptone, meat extract, yeast extract, urea, sodium nitrate, ammonium nitrate, ammonium sulfate, and phosphoric acid. Potassium, potassium chloride, iron sulfate,
Appropriate ones can be selected and used from carbon sources such as zinc sulfate, calcium chloride and calcium carbonate, nitrogen sources, inorganic salts, growth factors and the like. These nutrients should be in the form of an aqueous solution.
Add 2.0% agar and sterilize by heating or filtration before use.

Next, the compound (II) is added to the bacterial cell culture solution precultured as described above, and then shaken or aerated and stirred. By this operation, the compound (II) is gradually converted to the compound (I). The amount of compound (II) to be added is usually 0.1 to 1 g per 1 cell culture solution.

Whether or not the compound (II) is converted in the culture medium can be quickly determined by, for example, the following operation. That is, withdrawing a culture solution containing 3 to 10 ml of bacterial cells from the flask or tank in which the conversion is in progress,
Extract with an organic solvent such as ethyl acetate without adjusting the pH, and immediately analyze by gas chromatography or high performance liquid chromatography. The time required is less than 1 hour including the sampling time.

Fig. 1-a shows a typical gas chromatogram of the converted product containing the compound (II) and Fig. 1-b the compound (II) 120 hours after the start of the conversion operation. The gas chromatogram in the figure was obtained under the following conditions. That is, a Tsushima GC-4CM type gas chromatograph is equipped with silicon O
Inner diameter 0.28 with V-101 coated on the inner wall of the column
Equipped with a glass capillary column having a length of 30 mm and a length of 30 m, at a column temperature of 250 ° C., 1 u of an ethyl acetate extract of the above-mentioned culture solution was flown with 0.96 ml / min of helium as a carrier gas.
It was measured by injecting 1 as a sample.

Next, in the culture of the converted product, the microbial cells were filtered off, and then the fragrance component was extracted from the liquid using an organic solvent. In order to purify and isolate the compound (I), which is a flavor component, first, the converted culture is filtered and separated into cells and liquid. An organic solvent such as ethyl acetate is added to the liquid, and the organic solvent obtained by washing the bacterial cells is combined to form an organic phase. The solvent is distilled off under reduced pressure to obtain a conversion product. The obtained converted product is applied to a column using silica gel as an adsorbent, eluted with a mixed solution of n-hexane: ethyl acetate, and fractionated by a fraction collector. Further, if necessary, by high performance liquid chromatography, the compound (I)
Is isolated.

When the compound (I) of the present invention is added to tobacco and smoking is performed,
Both of them have a good harmony with the original scent of tobacco, suppress irritation, and have a long-lasting effect, so that they have many excellent effects such as less dissipation during the manufacturing process and product storage of tobacco.

That is, this compound can be diluted with a solvent such as ethanol to an appropriate concentration and used as a flavor and taste improving agent for tobacco. This compound can be used alone or in combination with other flavors and additives for tobacco as appropriate. The added amount is 0.1 to 50 ppm (W / W), preferably 1 to 30 ppm (W / W) with respect to the cut tobacco product, and the above-mentioned effects are exhibited. The type of tobacco to which the compound of the present invention can be effectively applied is not particularly limited, and is not limited to tobacco obtained by cultivation, and regenerated tobacco produced from scrap tobacco as a raw material and aroma flavor of pipe tobacco and the like. It is also effective for improvement.

The bacterium used in the present invention has been deposited at the Ministry of International Trade and Industry, Institute of Industrial Technology, Institute of Microbial Technology, and the deposit number is Microtechnology Research Institute
It is No. 8.

Below, the mycological properties of this bacterium are described in the Manual of Microbiological Method.
The results of examination according to the method described in (ical Method) are shown.

I Morphological properties 1. Microscopic findings Bacillus, 1.0-1.5μ × 3.5-5.0μ, wavy chain, sometimes lumpy, motile, oval endospore formation, 1.0-1.2μ ×
1.5-2.0μ, 1 per cell, central position, spore-like swelling, gram positive, acid-negative.

2. Culture findings (1) Plate culture Circular, slightly hill-like ridge, smooth, full edge, opaque, white with slight yellow color, slightly glossy.

(2) Slope culture Growing, smooth, slightly shiny, pale yellowish white, opaque, no odor, no change in medium.

(3) Liquid culture Medium growth, faint surface growth, slightly cloudy, medium precipitation.

II Physiological properties Growth temperature: 15 to 42 ℃, optimum 30 to 37 ℃ Growth pH: pH 3.5 to 10.0, optimum 6.5 to 7.5 Oxygen requirement: aerobic Gelatin liquefaction: changes into a donut shape Litmus milk: acidic, peptone Indole formation: Negative Nitrate reduction: Negative Starch hydrolysis: Positive Methyl red reaction: Positive Forgesproscauer reaction: Negative Catalase formation: Positive Urease formation: Positive Salt concentration and growth: Grow up to 7% Growth inhibition by sodium azide: Does not grow at 0.02% Growth inhibition by lysozyme: Does not grow at 0.001%.

Growth on Sabouraud agar: grows Growth on Sabouraud liquid: grows OF test: fermentative oxidase activity: positive Citric acid utilization: positive lecithinase activity: negative lepan formation: positive Casein hydrolysis: positive Deamination of phenylalanine: Positive III Utilization of sugars No acid, no gas: glucose, arabinose, mannitol, no acid, no gas: xylose Based on the above bacteriological properties, Verjay's Manual of Detergent Native Bacteriology 8
Edition (Bergey's Mannual of Determinative Bacteriology
This strain belongs to the genus Bacillus when searched according to the 8th Edition), and Bacillus megaterium
Was identified.

Next, the present invention will be specifically described with reference to examples.

Example 1. A nutrient agar (manufactured by Difco) medium was prepared in a test tube, and 1 platinum loop of Bacillus megaterium NH5 was ingested into the medium and allowed to stand for 2 days to form spores. Next, Nutrient Broth (Difco) 0.8g, tap water 10
One platinum loop of the bacterium was inoculated into 0 ml of a sterilized liquid medium, the mixture was placed on a rotary shaker at 200 rpm, and cultured at 37 ° C. for 24 hours to serve as a seed mother. 2 ml of this seed matrix was inoculated into an Erlenmeyer flask with 3 Cobbs containing 1 medium prepared by the same method and proportion as described above, and the mixture was subjected to rotary shaking culture at 37 ° C. and 200 rpm for 3 hours, and the absorbance at 610 nm was 0.3. A microbial cell culture solution was obtained. To this, 0.1 g of the compound (II) was added and subsequently shaken. Five days after the conversion, the culture was taken out and separated into cells and liquid by centrifugation. The compound (I) in the liquid was extracted with ethyl acetate, and this ethyl acetate layer was combined with the ethyl acetate obtained by washing the cells. The ethyl acetate solution was concentrated under reduced pressure to obtain 0.05 g of a concentrate. The same operation was repeated 6 times to obtain a total of 0.3 g of concentrate. The conversion product, compound (I), was then isolated from this concentrate as follows. 0.3 g of concentrate 5
It was added to the upper part of a glass column with a diameter of 30 mm filled with 0 g of silica gel G, and then a mixture of hexane-ethyl acetate (V / V), 85:15, 80:20, 60:40, 50:50 was added to 1250, 1500, 900, and 2300 ml were added respectively and eluted sequentially.

Next, for the 50:50 fraction, the mobile phase was methyl alcohol: acetonitrile: water = 1: 4: 9 (V / V), and the Lic
hrosorb RP-18 column (diameter 4 mm, length 25 cm,
High performance liquid chromatography was performed using Merck. The main component was separated and concentrated under reduced pressure at 35 ° C. or lower to remove the organic solvent. The aqueous phase was transferred to a separating funnel and liquid-liquid partition was performed using diethyl ether to extract the converted product. After dehydration with Glauber's salt, it was concentrated under reduced pressure. The concentrate was a colorless oil and the yield was 0.004 g.

Example 2 Waste tobacco was extracted with hot water at 100 ° C. to separate it into a water-soluble part and a water-insoluble part (extraction residue), and then the water-insoluble part was beaten, to which 15% of the dry matter weight of a softwood tree was cut. A mixture of kraft pulp was molded into a thin paper, and 100 g of sheet-like regenerated cigarette made by returning the water-soluble part to the thin paper,
0.5 mg of the compound (I) obtained in Example 1 was dissolved in 3 ml of ethanol, sprayed and added thereto, and then paper was engraved by a conventional method, and 0.5 mg of the compound (II) was treated in the same manner as above. Using the refined product as a control, the flavor and taste were compared by the two-point identification method for odor, taste and irritation. The evaluation of 20 specially trained professional panels was as shown in Table 1. The majority of the panel members evaluated that the stimulus was suppressed and the cigarette was given a comment.

(Effects of the Invention) The compound of the present invention is a product tobacco product, pipe tobacco, by addition of ppm order to regenerated tobacco and leaf tobacco, imparts tobacco-likeness, the effect of suppressing irritation and improving taste, that is, It has the effect of improving the flavor and taste of tobacco, which improves the quality of tobacco products.

[Brief description of drawings]

FIG. 1-a shows the compound (II), and FIG. 1-b shows the culture time when the compound (II) was converted into microorganisms by the method of the present invention.
The respective gas chromatograms of the compound (II) conversion product after 20 hours are shown. In the figure, peak (1) is a solvent, peak (2) is a compound (II), peak (4) is a compound (II), peak
(3) and (5) are by-products. The vertical axis represents peak height, and the horizontal axis represents retention time.

Claims (3)

[Claims] 1. A 2,7,11-sembratriene-4,6,20-triol represented by the following formula (I). 2. A flavor and taste improving agent for tobacco comprising 2,7,11-sembratriene 4,6,20-triol represented by the following formula (I). 3. A bacterium of the genus Bacillus (Bacill) is added to 2,7,11-sembratriene 4,6-diol represented by the following formula (II).
us sp.) is applied to collect 2,7,11-sembratriene-4,6,20-triol represented by the following formula (I), which is represented by the following formula (I). Method of manufacturing compound.