JPS5867190A - Production of resin by tissue culture of achras sapota l - Google Patents

Abstract

PURPOSE:Achras sapota L in Sapotaceae is subjected to tissue culture to produce a resin that can be used as a chewing gum base. CONSTITUTION:Cell lumps are separated from Achras sapota L in Sapotaceae and subjected to tissue culture in a medium that is prepared by adding a plant hormone such as auxine or cytokinin to a customary tissue culture medium mainly containing inorganic salts, saccharides, vitamins and aminoacids, such as a Rinsmeyer-Scoog medium and the callus is separated from the medium, then dried. The dried callus is extracted with an organic solvent, the solvent is distilled off from the extract to collect the resin that is a mixture of acetates, palmitates and sterates of triterpene alcohols.

Description

DETAILED DESCRIPTION OF THE INVENTION This invention relates to a method for producing a resin that can be used as a chewing gum base material by tissue culturing Sapodilla (Achras 5apota L), a plant belonging to the family Acatellaceae.

Traditionally, the sap (latex) of trees found in tropical regions has been solidified and used as a raw material for chewing gum.

Among them, resin C (hereinafter referred to as chicle resin) obtained from Sabotilla, a plant belonging to the Acatellaceae family, is considered to be the most suitable raw material for a 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, the reality is that mature sabotzilla trees from which resin can be collected are scattered throughout the tropical forest zone, and their collection requires manual labor. Moreover, 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 useful resins could be obtained, for example, by culturing plant cells, without resorting to collecting natural tropical plants.

As a result of research on tissue culture using Sapodilla, the present inventors developed a tissue culture medium using Sapodilla, and found that resin (
It has been found that fatty acid esters of triterpene alcohols which are equivalent to or similar to chicle resins can be obtained.

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 objective, the present invention uses sapodilla (Achrasiapota L.
') The cell mass separated from the tissue culture [-5] is characterized by collecting resin from the culture.

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

K, who tissue culture sabotilla according to this method, for example, sterilizes the surface of sabotilla leaves, roots, graves, bulges, and other organs or small pieces of tissue, and then plants them in a tissue culture medium.
Culture at 5-SO°C, pH 5, Q-Z5. The culture medium may be solidified by adding agar or the like, or liquid without the addition of agar.After 2 to 4 weeks from the start of culture, once the induced callus has been grown, it is transplanted to a 4-cell culture medium and allowed to stand still. Callus cells are grown by standing culture, shaking culture, or aeration culture, and this culture is repeated to obtain stable callus. Sabotilla 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 resin, the means normally used for collecting fat-soluble substances from natural products are used. That is,
After separating the callus from the medium, it is dried and extracted with an organic solvent. The extraction solvent is distilled off under normal pressure or reduced pressure to obtain the desired product PivI as a residue. Gas chromatography C model: Shimadzu GC-6A Column Diasolid ZT 1252 rrm It was identified as palmitate, stearate).

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

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

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

Example 1 Sapodilla (Achras sapotm L) (D
The MtD pieces are washed with deionized water, immersed in 70% ethanol for 1 minute, then 1% hypochlorous acid (IJum) aqueous solution for 15 minutes, and then thoroughly washed with sterile water.

Add this small piece to 150m of coconut milk! , added 2-4 dichlorophenoxy vinegar 5i1 (2-4D) (15
■/1. Kinetin 0.2 Q/l Linsmeyer
The cells were randomly transplanted to Skoog's medium (L8 medium) (Table 1) and cultured at 265°C to derive callus.

This part of the callus is cut and subcultured in a new medium having the same composition as above.

<Table-1> KNo, 1900■/1NH4NO,
1650# CaCl2-2H20440# Mjl1804.7H20370# IG (2PUa 170 zNaz
-EDTA 37.3'k e80a,
7ti2o 27.8'-804 reference 4
)120 22.3 #Zn5Oa φ4
H20 &6' 11t, α21 Kl α83 1Na2 Mo
0a, 2H20α25 1CuSO4-5t(20α
025W/1CoCt2* 6H20α025 l Myo-inositol 100 I Thiamine・
HCI, 1 z sucrose
! , 0X10 1 cold weather 90
00 #9H1'i6. IK [[, 12 when used
Example 2: The callus obtained in Example 1 was placed in a 50 ONI shake flask containing 100 μl of a liquid medium with the same medium composition as described in Example 1, except for the agar. % 265° C. with shaking. Callus grows as dispersed cells in culture. This is subcultured in a new medium having the same composition as above to obtain a homogeneous cell solution.

Example 3 The callus obtained in Example 1 was treated with the medium composition described in Example 1, in which 150 d/l of coconut milk was replaced with 4 f/l of malt milk, and 0.2 my/l of kinetin was added to 2.0 d/l of malt milk. Hit/
The cells were inoculated onto a solid medium of 100 ml, and cultured at 26.5°C.

Example 4 The homogeneous cell suspension obtained in Example 2 was inoculated into an Erlenmeyer flask containing the liquid medium 1 used in Example 2.
Shaking culture was performed 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. The dried callus is extracted using a Soxhlet extractor with n-Φsan for 60 hours. When the extracted fraction was examined by thin layer chromatography (developing solvent: n-Φsan:benzene-3:1, coloring reagent, iodine vapor), a spot that was presumed to be a fatty acid ester of triterpene alcohol was obtained. .

Separate fatty acid ester components of triterpene alcohols using preparative thin layer chromatography! As a result of analysis using gas chromatography (machine: Island 5 (JC-6), column: Diasolid ZT (132 rrm x 30 cm: column size 100-360°C 5' c/m Tn), triterpene alcohol was detected. Acetate, palmitate and stearate were determined.

[Brief explanation of the drawing]

Figure 1 is an infrared absorption spectrum diagram of resin (K Br method)
The vertical axis represents transmittance (excellent), the horizontal axis represents wave number (cm),
FIG. 2 is a gas chromatogram of Fi resin, in which the horizontal axis represents time (minutes) and the vertical axis represents the sensitivity f of sample components by the FiFID method. Procedural amendment (August 19, 1981 Kazuo Wakasugi, Commissioner of the Patent Office1, Patent Application No. 164148 of 1982, Title of invention Method for producing resin by tissue culture of Sapodilla3, Amendment Relationship with the case of a person who does "Explanation" column. 6. i! Correct content ill 'A1 As per the judgment. Amendment 1, page 2, line 15 of the specification, "L," is amended to rLJ. 2. Specification Correct "■" in line 11 of page 8 to rmJJ.

Claims (1)

[Scope of Claims] (1) Achrassap (Achrassap), a plant belonging to the Acatellaceae family;
A method for producing a resin, which comprises culturing a cell mass separated from a cell mass (Ota L) and collecting the resin from the culture. Q) 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. 6) The method for producing a resin according to claim 2, wherein the normal tissue culture medium is a Linsmeyer-Skoog medium. (4) Claims that the plant hormones are auxin and/sumiretake cytokinin! A method for producing the resin described in Section 2. (2) 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. (2) A method for producing a resin according to any one of claims 1 to wX4, wherein tissue culture is carried out in a liquid medium. σ) The method for producing a resin according to claim 1, wherein the w fat 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. 7. The method for producing a resin according to claim 1, wherein the [F]l 41 resin is a mixture of triterpene alcohol compounds such as acetate, palmitate, and stearate.