JPH0316116B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing a resin that can be used as a chewing gum base material by tissue culturing Achras sapota L, a plant belonging to the Acetaceae family.

Traditionally, the sap (latex) of trees found in tropical regions has been solidified and used as the raw material for chewing gum. Among them, resin obtained from sapodilla, a plant belonging to the family Acateaceae (hereinafter referred to as chicle resin), is considered to be the most suitable raw material for gum base. The main component of chicle resin is fatty acid ester of triterpene alcohol (Journal of Japan Food Industry Association, Vol. 27, No. 9, 1980, pp. 419-425).

However, mature sapodilla trees from which resin can be harvested are scattered throughout tropical forests, and the reality is that their collection relies on manual labor. Furthermore, with the development of forest belts, the amount that can be mined is decreasing, and it is predicted that it will not be possible to meet the demand of the chewing gum manufacturing industry in the future. Therefore, it would be extremely meaningful if such a useful resin could be obtained, for example, by culturing plant cells, without relying on collection from natural tropical plants.

As a result of research on tissue culture using Sapodilla, the present inventors succeeded in culturing the tissue and found that resin (fatty acid ester of triterpene alcohol equivalent to or similar to chicle resin) could be obtained from the culture. .

Therefore, the general object of the present invention is to provide an industrial method for producing triterpene alcohol fatty acid esters similar to triterpene alcohol fatty acid esters, which are the main component of chicle resin, by tissue culture of chicle.

In order to achieve this purpose, the present invention uses Achras sapota (achras sapota).
L) The cell mass separated from L) is tissue cultured, and the resin is collected from the culture.

The medium used in the present invention contains inorganic salts, sugars,
Any medium containing vitamins, amino acids, auxin agents, cytokinin agents, and other plant hormones can be used.

To tissue culture sapodilla according to this invention,
For example, small pieces of sapodilla leaves, roots, stems, seeds, and other organs or tissues are surface sterilized, then planted in a tissue culture medium and cultured at 25-30°C and pH 5.0-7.5. The medium may be solidified by adding agar or the like, or liquid without the addition of agar. Culture start 2~
After 4 weeks, when the induced callus is obtained, it is transplanted into a new medium, and the callus cells are grown by static culture, shaking culture, or aeration culture, and this culture is repeated to obtain stable callus. Sapodilla plant cells prepared to be suitable for tissue culture are inoculated into a resin production medium and cultured.

Resin is usually produced and accumulated in greater amounts in callus. Therefore, as a method for collecting the resin, a method commonly used for collecting fat-soluble substances from natural products is used. That is, callus is separated from the medium, dried, and extracted with an organic solvent. Extracting solvent at normal pressure,
Alternatively, the target resin can be obtained as a residue by distilling it off under reduced pressure. Gas chromatography (Model: Shimadzu GC-6A Column: Diasolid ZT φ2mm x 30cm: Column temperature 100→360℃, 5
C/min), three main peaks were observed, and each peak was identified as fatty acid ester of triterpene alcohol (acetate, palmitate, stearate).

These are close to the components of chicle resin and are sufficient substitutes for it.

According to this invention, triterpene alcohol fatty acid ester, which is the main component of chicle resin, can be obtained from sapodilla tissue culture, which can be used as chewing gum base raw material.

Next, examples of the present invention will be specifically described, but these examples are not intended to limit the present invention in any way.

Example 1 A small piece of Sapodilla (Achras sapota L) leaf was washed with deionized water and placed in 70% ethanol for 1 minute.
Then, after immersing it in a 1% aqueous sodium hypochlorite solution for 15 minutes, it was thoroughly washed with sterile water.

Add this small piece to 150ml of coconut milk/0.5% of 2-4 dichlorophenoxyacetic acid (2-4D).
The cells were aseptically transplanted into Linsmeyer-Skoog medium (LS medium) (Table 1) containing 0.2 mg/mg/kinetin and 0.2 mg/kinetin, and cultured at 26.5°C to derive callus.

A portion of this callus is cut out and subcultured in a new medium having the same composition as above.

<Table-1> KNO ₃ 1900mg/ NH ₄ NO ₃ 1650〃 CaCl ₂・2H ₂ O 440〃 MgSO ₄・7H ₂ O 370〃 KH ₂ PO ₄ 170〃 Na ₂ −EDTA 37.3〃 FeSO ₄・7H ₂ O 27.8 〃 MnSO ₄・4H ₂ O 22.3〃 ZnSO ₄・4H ₂ O 8.6〃 H ₃ BO ₃ 6.2〃 KI 0.83〃 Na ₂ MoO ₄・2H ₂ O 0.25〃 CuSO ₄・5H ₂ O 0.025〃 CoCl ₂・6H ₂ O 0.025〃 Myo-inositol 100〃 Thiamine/HCl 1〃 Sucrose 30×10 ^3〃 Agar 9000〃 The pH was adjusted to 6.1, and the product was autoclaved at 2 atm at 120°C for 15 minutes before use.Example 2 Implementation The callus obtained in Example 1 was placed in a 500ml culture medium containing 100ml of a liquid medium with the same medium composition as described in Example 1, except for the agar.
ml shake flask and culture with shaking at 26.5°C. Callus grows as dispersed cells in culture. This is subcultured in a new medium with the same composition as above to obtain a homogeneous cell solution.

Example 3 The callus obtained in Example 1 was mixed with 150 ml of coconut milk having the medium composition described in Example 1 and malt extract 4.
g/, and kinetin 0.2 mg/
It was inoculated onto a solid medium at a concentration of 2.0 mg/ml and cultured at 26.5°C.

Example 4 The homogeneous cell suspension obtained in Example 2 was inoculated into a 3-volume Erlenmeyer flask containing liquid medium 1 used in Example 2, and cultured with shaking at 26.5°C.

Example 5 After dividing the tissue culture obtained in Examples 3 and 4 into callus and medium, the callus is freeze-dried. Dry callus was extracted with n-hexane for 30 minutes using a Soxhlet extractor.
Extract time. The extracted fractions were subjected to thin layer chromatography (developing solvent: n-hexane:benzene-3:
1, coloring reagent, iodine vapor), spots presumed to be fatty acid esters of triterpene alcohols were obtained.

The fatty acid ester components of triterpene alcohols were separated and purified using preparative thin layer chromatography, and gas chromatography (model: Shimadzu GC-
6A, Column: Diasolid ZT φ2 mm x 30 cm: Column temperature 100→360°C 5°C/min) As a result, it was identified as triterpene alcohol acetate, palmitate, and stearate.

[Brief explanation of the drawing]

Figure 1 is an infrared absorption spectrum diagram of the resin (KBr method), with the vertical axis representing transmittance (%) and the horizontal axis representing wave number (cm ^-1 ). Figure 2 is a gas chromatogram diagram of the resin, with the horizontal axis representing the wave number (cm -1 ). Time (minutes) and the vertical axis represent the sensitivity of sample components by the FID method.

Claims (1)

[Scope of Claims] 1. Achras sapota (achras sapota)
L) A method for producing resin, which comprises tissue culturing the cell mass separated from L) and collecting resin from the culture. 2. The method for producing a resin according to claim 1, wherein the tissue culture medium is a normal tissue culture medium mainly containing inorganic salts, saccharides, vitamins, and amino acids to which plant hormones are added. 3. The method for producing a resin according to claim 2, wherein the ordinary tissue culture medium is a Linsmeyer-Skoog medium. 4. The method for producing a resin according to claim 2, wherein the plant hormone is auxin and/or cytokinin. 5. The method for producing a resin according to any one of claims 1 to 4, wherein tissue culture is carried out in a solid medium using agar. 6. The method for producing a resin according to any one of claims 1 to 4, wherein tissue culture is carried out in a liquid medium. 7. The method for producing a resin according to claim 1, wherein the resin is obtained as a residue obtained by distilling off the solvent from an extract extracted from a dried tissue culture product with a non-polar solvent. 8. The method for producing a resin according to claim 1 or 7, wherein the resin is a mixture of acetate, palmitate, and stearate of triterpene alcohol compounds.