JPS5835680B2 - Method for producing S-nornicotine - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing S-nornicotine by a culture method.

S-nornicotine is a type of alkaloid that is contained in leaf tobacco along with S-nicotine, and its content varies depending on the type of leaf tobacco, but usually cultivated leaf tobacco contains o, i to 2.5 weight scale, There are 3 in wild leaf tobacco.
~4 weight φ is contained.

As is well known, S-nicotine is considered to be an extremely important factor in providing satisfaction from smoking cigarettes, but recent research has shown that S-nornicotine also contributes to the satisfaction of smoking cigarettes, just like S-nicotine. It is effective in imparting a sense of
- It has been found that adding a small amount of nornicotine to various tobacco raw materials has a quality-improving effect, such as suppressing the bad habit of tobacco smoke and making the smoking taste mellow.

Furthermore, since the physiological effects of S-nornicotine are weaker than S-nicotine, efforts have been made in recent years to provide a sense of smoking satisfaction and improve the flavor of artificial cigarettes or regenerated cigarettes that have undergone nicotine removal treatment. Therefore, the use of S-nornicotine is considered to be extremely effective.

Furthermore, heat condensates of S-nornicotine and various sugars are known to be excellent tobacco flavor improvers (Special Publication No. 6639/1982), and the uses of S-nornicotine are expected to expand in the future. It is expected.

Conventionally, the following two methods can be considered as methods for producing nornicotine.

That is, the first method is a method of extracting and purifying S-nornicotine from leaf tobacco grown and harvested,
The second method is to chemically synthesize myosmin and reduce it to obtain nornicotine.

The first method has the advantage of being able to directly extract S-nornicotine, but since the S-nornicotine content in leaf tobacco is extremely small, the wild type of leaf tobacco that has a relatively high S-nornicotine content is Cotiana glutinosa. etc.), the yield per field area of wild species is about 0% higher than that of cultivated species, and the alkaloids of wild species are a mixture of S-nornicotine and S-nicotine, so they cannot be extracted. , separation and purification processes become complicated.

In the second method, racemic nornicotine is produced, and since optical resolution of this is not known, it is currently impossible to obtain only S-type nornicotine.

The present inventors have conducted intensive research on a method for producing S-nornicotine by a culture method without using the above-mentioned method, and as a result, they have discovered a strain that can decompose S-nicotine to produce S-nornicotine. I came up with this.

That is, the present invention is a method for extracting and collecting S-nornicotine from a culture obtained by inoculating and cultivating nicotine-degrading bacteria belonging to Pelicularia filamentosa in a medium containing S-nicotine.

Until now, the only bacteria that can decompose S-nicotine and produce S-nornicotine are the incomplete bacteria Cunninghamella.
Echinulata (Cunninghamellaech)
inulata) has been reported (Applied
andEnvironmental Microbi
ology, 38.836 (1979)).

However, the amount of S-nornicotine produced by this bacterium is extremely low and can only be detected using analytical equipment.

However, the S-nicotine degrading bacteria discovered by the present inventors efficiently produces S-nornicotine from S-nicotine, and is extremely useful.

This strain is Pelicularia filamentosa (Pelcularia filamentosa), which was isolated in 1978 in the field of Japan Monopoly Corporation Okayama Experimental Station.
licularia filamentosa )J
TS-208 (hereinafter also referred to as this strain) is a well-known bacterium that has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology under contract number 5955, and is conventionally known as the tobacco buckthorn bacterium. be.

Its mycological properties are written by Hisao Nakamura, Tobacco Plant Pathology P, 78-
81, (1955) published by Breakfast Shoten.

Next, the present invention will be explained in detail.

First, the MY medium having the composition shown in Table 1 is sterilized by heating and pressure using an autoclave or the like in a conventional manner.

The medium composition shown in Table 1 is known as MY medium for preserving mold and yeast.

Next, a potato glucose agar medium (hereinafter referred to as PDA medium) was placed in a test tube having an outer diameter of 18 mm.

), add one test tube worth of bacterial cells of this strain that have been cultured in advance at 20 to 30°C for 15 to 10 days, preferably one week.
The medium 11 shown in Table 1 was added to a 31-volume Erlenmeyer flask, sterilized, inoculated aseptically, and cultured aerobically with shaking at a temperature of 20 to 30°C.

Cultivate for 3 to 5 days, preferably 4 days, and when the number of bacterial cells reaches almost the maximum amount, add an aqueous S-nicotine solution (adjusted to pH 5.5 with hydrochloric acid) that has been over-sterilized in advance to approximately 0.1 weight φ or less. Addition and culturing are continued until the concentration is as follows.

When the S-nicotine concentration exceeds 0.1 weight of the medium,
This is undesirable because it inhibits the growth of bacteria.

The culture period is preferably in the range of 7 to 20 days after addition of S-nicotine.

If the culture is carried out for less than 7 days, the amount of S-nornicotine produced is insufficient, and even if the culture is continued for 20 days or more, the amount of S-nornicotine produced is only slightly increased.

By this culture operation, about 10% of S-nicotine as a substrate can be converted to S-nornicotine.

Furthermore, the conversion of S-nicotine to S-nornicotine using this strain can also be carried out in a Jar Fermentor by a conventional method with aeration and stirring.

Next, S-nornicotine produced in this culture solution is extracted.

That is, after the culture is completed, an alkali such as potassium carbonate or caustic soda is added to the solution to adjust the pH to about 10 or higher, and then an organic solvent is added for extraction.

A hydrochloric acid aqueous solution having a pH of 3 or less is added to this organic solvent layer for further extraction to obtain an S-nornicotine dihydrochloric acid aqueous solution.

Ether, chloroform, etc. can be used as the organic solvent for extraction.

In addition, by concentrating the liquid after completion of culture under reduced pressure prior to extraction and then performing solvent extraction, it is possible to save on organic solvents and the like.

This aqueous hydrochloric acid solution is concentrated under reduced pressure using an evaporator or the like.

However, in this method, S-nicotine is also extracted along with S-nornicotine.

Therefore, S-nornicotine and residual S-nicotine are separated by conventional methods such as thin layer chromatography, high performance liquid chromatography, or distillation.

Since the method for producing S-nornicotine according to the present invention is a culture method, the operation can be carried out at room temperature and normal pressure, and has the advantage that it can be produced easily with high yield compared to the method of extracting it from leaf tobacco. .

Next, the present invention will be explained by examples.

Example 1 In an Erlenmeyer flask containing MY medium 51 having the composition shown in Table 1, which had been sterilized by pressure and heat at 121°C using an autoclave,
Pre-incubate 5 test tubes of sterile PDA slants at 28°C.
Add 10 ml of sterile water to this strain, which had been cultured for 7 days at The cells were cultured with shaking for 4 days.

Then, sterilized 10% S whose pH was adjusted to 5.5 with hydrochloric acid.
-Nicotine aqueous solution is respectively 0.5 as S-nicotine.
The cells were added in an amount of 1.5 g each and cultured for 14 days under the same conditions.

The average concentration of S-nornicotine in the total volume 51 of this cultured solution was 0.055 m97ml (12
(equivalent to φ (molar ratio)).

This cultured solution 51 was filtered through gauze to remove bacterial cells, and then adjusted to pH 1 using a 10% potassium carbonate aqueous solution.
The mixture was extracted with chloroform in a total amount of about 21 g in three portions, and the combined chloroform was extracted with 700 ml of a 0.5N aqueous hydrochloric acid solution.

This aqueous hydrochloric acid solution was concentrated under reduced pressure using an evaporator made by Tokyo Rikagaku Co., Ltd., and then applied to a thin layer plate (thickness 5 mff1) of silica gel 60 made by Merck Co., Ltd. as a developing agent at a mixing ratio of chloroform:methanol:58 dammonia water: 60:10: 1
After the development was completed, 0.2% isatin (dissolved in 50% acetic acid water) was sprayed on one corner of the air-dried product using a spray bottle. Heated at ℃ for 15 minutes, scraped off the part with the same Rf value as the position where the blue color developed, extracted with 50 ml of 1N hydrochloric acid aqueous solution, concentrated under reduced pressure in an evaporator, and obtained S-nornicotine dihydrochloride 0.3759 [ 11 pieces (molar ratio) of S-nicotine were obtained.

Example 2 MY medium 71 shown in Table 1 was prepared, placed in a 101 volume MD 500 fermenter manufactured by Marubishi Rika Seki Kenkyusho, and sterilized under pressure and heat in an autoclave at 121°C for 15 minutes.

This strain was previously inoculated into 7 test tubes of sterilized PDA slant medium, and after culturing at 25°C for 17 days, 10 ml of sterile water was added to each test tube to scrape off the bacterial bodies and the entire amount was transferred to a sterilized fermenter. It was added to the culture medium and cultured with stirring and aeration at 25°C for 13 days.

The rotational speed of the stirring propeller was 30C)rl)In, and the ventilation amount was 0.5VVM.

Add 3. sterile S-nicotine to this medium. ! Ill was added, and culture was continued for 7 days under the same conditions.

After completion of this culture, the amount of S-nornicotine was 0.415g (
13 molar ratio of the substrate s-nicotine].

This cultured solution 71 was added to Toyosawa Paper Arashi 1 using Nutsche.
The bacterial cells were removed by passing it through a piece of paper.

After concentrating under reduced pressure using an evaporator until the pH was about 11, the pH was adjusted to 10 with a 10% potassium carbonate aqueous solution, extracted in three portions with ether 31, and further extracted with 0.5
Extraction was performed with a specified aqueous hydrochloric acid solution.

Hydrochloric acid aqueous solution was concentrated to about 10 ml using an evaporator, and a 1=1 mixture of methanol and 0.05 molar ammonium bicarbonate aqueous solution was added as a solvent to a Microbondapak CI8 column using a high-performance liquid chromatograph manufactured by Waters Inc. for 1 minute. The solvent at the peak corresponding to S-nornicotine was collected in a 0.5 N hydrochloric acid aqueous solution and concentrated under reduced pressure to a volume of about 20 ml.

This concentrated solution was adjusted to pf-111 again with 2N caustic soda, and then mixed with 30 ml of chloroform and 1 ml of 0.5N hydrochloric acid aqueous solution.
Extracted with 0 Tnl and concentrated under reduced pressure to obtain 0.55 g of S-nornicotine dihydrochloride, 11.5% of the C substrate S-nicotine (
equivalent to the molar ratio) was obtained.

Since S-nornicotine is a highly reactive and unstable substance, it is stored in the form of S-nornicotine dihydrochloride and used as S-nornicotine using an alkaline solvent immediately before use.

In the present invention, S-nornicotine was determined by the following method.

That is, add an equal amount of methanol to the culture solution and use a centrifuge for s, o o every minute.

The supernatant from which bacterial cells and precipitates were removed by rotation was transferred to a Waters high-performance liquid chromatograph, Microbondapak CI.
8 columns, methanol and 0.05 molar ammonium bicarbonate aqueous solution 1:l as solvent, wavelength 254 as detector
Using a spectrophotometer adjusted to r/L11, the amount of S-nornicotine was determined from the peak area on the chart.

Claims (1)

[Scope of Claims] 1 S-Nornicotine is extracted and collected from the culture obtained by inoculating and culturing nicotine-degrading bacteria belonging to Pelicularia filamentosa in a medium to which S-Nicotine has been added. Method for producing nornicotine. 2. The method for producing S-nornicotine according to claim 1, wherein the nicotine-degrading bacteria is Pelicularia filamentosa JTS-208 strain.