JPS61167637A - 2-hydroxy-alpha-irone and composed of said compound for improving taste and flavor of tobacco - Google Patents

Abstract

NEW MATERIAL:2-Hydroxy-alpha-irone of formula I. USE:An agent for improving taste and flavor of tabacco. Effective to impart the character of tobacco, improve the taste, suppress the stimulant taste, and improve the quality of tobacco product by adding 0.05-30ppm (W/W), preferably 0.1-5ppm (W/W) of the compound to shredded tobacco, cut tobacco, regenerated tobacco and leaf tobacco. PREPARATION:Aspergillus niger JIS191 strain (FERM P-5087) is inoculated in a liquid medium and cultured at 24-37 deg.C for 24-48hr under shaking or under aeration and agitation. The alpha-irone of formula II is added to the culture medium, and the cultivation is continued under shaking or under aeration and agitation to obtain the objective compound of formula I.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a novel 2-hydroxy-α-ylon obtained by allowing microorganisms to act on α-ylon. This compound is a new substance effective in improving tobacco flavor.

[Prior art]

In the production of cigarettes, a large number of compounds are already known that are effective when added to smoking materials to impart more desirable taste and flavor, or to improve the aroma and taste of smoking materials. However, the compound of the present invention is also a new chemical substance, and therefore has never been used in the production of cigarettes.

[Problem that the invention seeks to solve]

This was done with the aim of developing and providing a new flavoring agent that can meet the diverse tastes of consumers regarding the aroma and taste of recent tobacco products.

[Means for solving problems]

The present inventors have investigated the α-iron represented by the formula: Aspergillus niger (A
spergillus n1g5r) JTS 1
It has been discovered that 2-hydroxy-α-ylon, which has an excellent effect on improving the taste of smoked tobacco as a converting substance, can be obtained by reacting with the 91 strain (Feikoken Bacterial Serial No. 5087) represented by the formula:

The spectral data and gas chromatography (GC) retention ratio (Rr) of this compound are shown below. The retention ratio is 1.0 for the retention time of dimethyl phthalate (DMP).
It is shown as a relative value when it is set to 0.

Molecular formula: C,, H, e O.

M8 m/! :207,204,161,147,1
37.133,119,109,105,95,93,
91,86.77.71.43 (Jl quasi-peak)'
H-N M R(CD CLs * TMS) tδ(
ppm): 0.84 (3Hs8), 0.91 (3Hs8)
,8)tl,21(3Hs8)*1.59(31(,8
), 1.97 (IH, dd, J=6.5Hz.

19Hz), 2.28 (3H, 8), 2.32 (IH,
dd, J=5.1Hz, 19Hz), 2.87(IH
,d,J=10.8)h)5.45(IH,8,bro
ad), 6.18 (IH, d, J=1&8Hz), 6.
65 (IH, dd, J==10.8Hz, 16.8Hri) C-N M R (CD C1s s T M 8
) *' (ppm): 20.0(q),
20.9((1), 22..5(q), 23.5(
(1), 27.1(q), 38.1(t), 39.9
(s), 51.3(d), 72.7(S), 120.1
(d), 132.9(s) 1345(d).

148.3(d), 197.7(s) (a)'+147. s°(C=g0g4.MeOH)G
C: Rr 1.69 (DMP=1.00) Next, the above 2-
The method for producing hydroxy-α-yron will be explained step by step.

The spores or hyphae of the trout, Aspergillus niger J'r8191 strain, are cultured to 8M, for example, by the following method. 1) When using spores as a seed; statically culture on a solid medium or liquid medium at 24 to 37°C to form sufficient spores.

Usually, spore formation is observed on the upper surface of the omega from about 3 days after inoculation, but spore formation is generally preferable from 7 to 10 days.

2) When using mycelium as a seed; liquid medium 4C 24-37℃
Shake or stir culture for 24 to 60 hours to obtain a porridge-like mycelial suspension and use this, or grind the bacterial cells obtained by static culture using a waring blender, etc. 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 inoculum, a pre-cultured culture solution 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, gluten, sucrose, starch,
Corn steep liquor, peptone, meat extract, yeast extract, urea, sodium nitrate, ammonium nitrate,
An appropriate carbon source, nitrogen source, inorganic salt, growth factor, etc., such as ammonium sulfate, potassium phosphate, potassium chloride, iron sulfate, zinc sulfate, calcium chloride, and calcium carbonate, can be selected and used. These nutrient sources are in the form of an aqueous solution, and in the case of a solid medium, 1.5~
Add 2.0% agar and sterilize by heating or filtering before use.

Next, α-
Add iron and continue to shake or aerate.

By this operation, σ-ylon is gradually converted to 2-hydroxy-α-ylon. The appropriate amount of α-iron to be added is usually 0.5 to 3 t per lt of the bacterial culture solution. It is desirable that the α-ion added here be of high purity, but ordinary industrially synthesized products may also be used. It has been found that, for example, by the following method, continuous reuse is possible almost permanently.

That is, in the method described above, the bacterial cells that have already been used to convert α-iron and have already had the conversion ability are filtered out,
This can be reused as it is, or it can be washed with an organic solvent such as ethyl acetate or hexane to remove the adhering α-iron conversion product, and then the solvent can be removed by ventilation or the like and then reused.

When using such reused bacterial cells, α-iron converting enzyme is already sufficiently induced in the bacterial cells, so α-iron is added to a separately sterilized or sterilized medium. Even if the bacterial cells to be reused are added to this, the conversion time is shorter than in the case of the above-mentioned method because α-Iron starts converting as soon as it comes into contact with the bacterial cells.

In addition, the α-iron conversion capacity per unit cell weight is greater in reused bacterial cells, and they can be used repeatedly any number of times.

Whether or not α-iron has been converted in a culture solution containing bacterial cells can be quickly determined, for example, by the following procedure.

That is, a culture solution containing 3 to 10 cnt of bacterial cells is extracted from a flask or tank in which conversion is in progress, and the pI (
3.0 or less, extract with a barbed solution such as ethyl acetate, and immediately analyze by gas chromatography or high performance liquid chromatography. The required time is within 30 minutes including sampling time.

Changes in the chromatogram pattern over time depend on the culture conditions (
If the temperature, aeration-stirring conditions, bacterial cell mass-a-ratio of added amount of iron, etc.) were kept constant, the process would be almost the same, so 1. The converted product at each culture stage was used as a tobacco flavoring agent in advance. A standard chromatogram pattern is created by associating the sensory test evaluation of the case with its chromatogram pattern.When actually performing conversion, the chromatogram obtained by the above method and the above standard chromatogram pattern are created. By comparing the patterns, it can be determined that a conversion stage has been reached where the conversion exhibits the desired composition.

In addition, FIG. 1 shows a typical gas chromatogram of α-iron, and FIG. 2 shows a typical gas chromatogram of the α-iron converted product obtained at 48 hours according to the present invention. As is clear from the figure, α-iron is completely converted into other substances according to the present invention.

The gas chromatogram shown in the figure was obtained under the following conditions. That is, a glass capillary column (length 30 m) with an inner diameter of 0.28 fi coated with polyethylene glycol 6000 on the inner wall of the column was attached to a Hitachi 663 model gas chromatograph, and helium was pumped at 1 m/min as a carrier gas at a column temperature of 160°C.
While flowing, 1 μt of the ethyl acetate extract of the above culture solution was injected as a sample.

The culture containing flavor components obtained as described above can be used as a cigarette flavor after filtering out the bacterial cells and concentrating as is, or the culture can be divided into organic solvent-soluble neutral sections by a conventional method. , fractionated into organic solvent soluble acidic category, water soluble category, etc.

It is possible to create tobacco flavorings with different aroma tones. However, among these, organic solvent-soluble fragrances have the most significant impact.

In order to fractionate the σ-iron converted product, the converted culture is filtered and separated into bacterial cells and filtrate. The σ-iron conversion product is extracted from the filtrate with an organic solvent such as ethyl acetate, and the bacterial cells are combined with the organic solvent in which the cells were washed to form an organic phase. This is divided into a neutral section and an acidic section using a saturated sodium bicarbonate solution in a conventional manner, and the solvent is distilled off under reduced pressure to obtain a neutral section. The neutral fraction obtained here was applied to a column using silica gel as an adsorbent, and n-hexane:
Elute with a mixture of ethyl acetate and fractionate with a fraction collector. Further, if necessary, high performance liquid chromatography is used to isolate the 2-hydroxy-α-ylon represented by formula (1).

When these compounds are added to cigarettes and smoked, they harmonize well with the natural aroma of tobacco, suppress irritation, and have long-lasting effects, so they are less likely to escape during the cigarette manufacturing process and product storage. It has been found that it has many excellent effects, including less.

The compound of the present invention can be diluted with a solvent such as ethanol, water, ethylene glycol, etc. to an appropriate concentration and used as a tobacco flavor improver. The tobacco compounds may be used alone or in combination, and other tobacco flavorings, additives, etc. may be appropriately blended and used.

All of the compounds of the present invention are applied to the peripheral side of the product tobacco at 0.
05-30 ppm (w/w), preferably 0.1
By adding ~5 ppm (w/w), the above-mentioned effect is exhibited. The types of tobacco to which the compounds of the present invention can be effectively applied are not particularly limited, and include not only tobacco obtained through cultivation, but also improved flavor and flavor of regenerated tobacco and pipe tobacco produced from tobacco waste. It is also effective for

(Example) Example 1゜A potato juice agar medium was prepared in a test tube, one platinum loopful of Aspergillus niger JT8191 was inoculated thereto, and the culture was incubated at 28°C.
The mixture was allowed to stand for 7 to 10 days to form spores. Next, add 30 f of sucrose, dipotassium phosphate IF, and 21.
Magnesium sulfate (heptahydrate) 0, 5t, potassium chloride 0
．． 5F, yeast extract lf, pH 7 consisting of 1 t of tap water,
0 sterilized liquid medium (modified Czapek-Dox medium)
The spores of strain No. 9 were inoculated at a concentration of about 20,000 spores per tmt of the medium, and cultured at 27-28°C for 48 hours under rotational shaking at 20 rpm.

A bacterial culture solution with a bacterial dry weight of 4.5F% pHa and a bacterial cell culture solution of 5 was obtained.

α-Iron iF was immediately added to this, and the mixture was continuously shaken. 48 hours after the addition of α-iron, a converted culture was obtained with a pH of 3.5, a residual glue of 20%, and a bacterial cell wet state of 6Of. This culture was fractionated according to the procedure described above. That is, the culture was filtered and separated into bacterial cells and filtrate. The α-irone conversion product was extracted from the filtrate with ethyl acetate, and the bacterial cells were combined with the washed ethyl acetate. This ethyl acetate solution was fractionated by a conventional method into a neutral section and an acidic section, and the neutral section was concentrated at 0.6f under reduced pressure.
I got it. This neutral solution was added to the top of a column with a diameter of 395 W filled with 302 silica gel G, and then a mixture of hexane and ethyl acetate (ma/ma) 99:1, 95:5 was added.
．． 90:10.85:15, Kazumi = 200 each
mt was eluted into the stone.

The 85:15 fraction was then converted to 8I60 with the mobile phase being hexane-ethyl acetate 85:15 (v/v).
Silica isolated. The yield was 0.05f.

Example λ Waste tobacco is extracted with hot water at 10°C, separated into a water-soluble part and a water-insoluble part (extraction residue), the water-insoluble part is beaten, and 15% of its dry weight is added to softwood kraft. For 100 tons of recycled tobacco sheets made by molding the mixture with pulp into thin paper and returning the water-soluble portion to this thin paper,
2-Hydroxy-α-ylon (formula (I) obtained by the method described above)
)) 0.5q was dissolved in 3 mt of ethanol, sprayed and added to the latter half, and rolled into paper. Using the cut and rolled pieces of the above-mentioned sheet-shaped regenerated tobacco without the addition of the compound of the present invention as a control, the aroma and flavor were compared using a two-point discrimination method in terms of K odor, taste, and irritation.

The evaluations of the 20 panelists were as shown in Table 1. In addition, many members of the panel found it to be more tobacco-like, sweet, and
We received comments that it gave off a musty odor.

Table 1 ((1) The number indicates the number of people who said it was good. The mark in the middle indicates a significant difference between samples with a risk rate of 1%.

Example 3゜Example 1. The neutral category described in is an alcohol solution,
The additive amount was 5 ppm sprayed on chopped low-grade barley type tobacco, then rolled up in paper, and the above-mentioned rolled tobacco was sprayed with only alcohol as a control product. A comparison was made using the two-point discrimination method. The evaluations of the 20 panelists were as shown in Table 2.

Table 2 ((1) The number is the number of people who said it was good.

The umbrella mark indicates a significant difference between samples at 1%.

〔Effect of the invention〕

The compound of the present invention can be added to tobacco products, pipe tobacco, regenerated tobacco, and leaf tobacco in ppm order to impart tobacco-like characteristics, improve taste, and suppress irritation, i.e., to improve tobacco flavor and flavor. It has an improving effect, which improves the quality of tobacco products.

[Brief explanation of the drawing]

Figure 1 shows α-ilon, and Figure 2 shows the constant temperature after 48 hours of culture when α-ilon was converted by microorganisms according to the present invention.
Gas chromatograms of the converted products of iron are shown. In the figure, peak (1) is the solvent, peak (2) is α
-ylon, peak (3) is 2-hydroxyα-ylon, and peaks (4) e (5) and (6) are by-products. The vertical axis shows the peak height and the horizontal axis shows the retention time.

Claims (2)

[Claims] (1) General formula ▲There are mathematical formulas, chemical formulas, tables, etc.▼(I) 2-hydroxy-α-ylon represented by (2) A tobacco flavor improver consisting of 2-hydroxy-α-ylon represented by the general formula ▲ Numerical formula, chemical formula, table, etc. ▼ (I).