JPH10101704A - Preparation of degradation product of polysaccharide having biological activity against plant utilizing radiation irradiation - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an antimicrobial activity inducing material having a high elicitor activity in a large amt. with high efficiency by irradiating a polysaccharide with an ionizing radiation. SOLUTION: A naturally occurring polysaccharide, such as pectin or chitosan, in a dry or aq. soln. form is irradiated with an ionizing radiation at a dose of 0.01 to 2000KGy to obtain a degradation product comprising an oligosaccharide having biological activity against plants comprising glycerin or bisatin.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for producing a degradation product having high biological activity, which is obtained by irradiating a polysaccharide with ionizing radiation to decompose the polysaccharide. In particular, the present invention relates to a method for efficiently producing a large amount of a novel oligosaccharide which is an antibacterial active substance (phytoalexin) having an activity as a signal transmitting substance (elicitor) that mediates a resistance reaction to plant pests and pathogenic bacteria. How to do it.

[0002]

2. Description of the Related Art Hitherto, as a method for producing a bioactive substance obtained from a polysaccharide, a method for producing a biologically active substance by enzymatically or acid-decomposing a cell wall of a plant or a pathogenic bacterium has been used.

[0003] However, in the enzymatic decomposition treatment, there is a problem that the location of cleavage of the polysaccharide main chain is limited, and the type of decomposition product obtained is limited. Furthermore, among the enzymes known so far, only a few enzymes can be used for large-scale treatment, and in the enzymatic digestion treatment, the reaction is performed in an aqueous solution. There was a problem that it was necessary.

On the other hand, in the acid decomposition treatment, it is necessary to hydrolyze polysaccharides under severe conditions using concentrated hydrochloric acid or the like. As a result, a large amount of monosaccharides is produced and conversion efficiency is poor. It is necessary to use an acid-resistant reaction vessel, and there are also problems such as difficulty in recovering the reagent.

[0005]

In view of the above problems,
The present invention provides a method for efficiently producing a substance having high elicitor activity from a polysaccharide in a large amount,
Another object of the present invention is to produce a novel active substance which cannot be obtained by a conventional method.

[0006]

In order to solve the above problems, the present inventors irradiate polysaccharides with ionizing radiation, thereby quickly, simply and effectively treating plant pests or plants. It has been found that various oligosaccharides having high elicitor (antibacterial activity inducer) activity against pathogenic bacteria can be produced, and that novel substances having high elicitor activity can be produced.

That is, the method of the present invention for producing an elicitor active substance by irradiation can treat a large amount of a polysaccharide continuously and in a short time, and can be carried out in the form of a dry powder or a solution of the polysaccharide. Is also possible. In addition, since the degradation of polysaccharides by radiation occurs randomly, it has become possible to generate a novel oligosaccharide having a structure not obtained by the conventional method.

[0008]

DETAILED DESCRIPTION OF THE INVENTION The polysaccharide used in the present invention is used in the broadest sense and includes all natural products,
Also included are products from microorganisms and their processed products. As natural polysaccharides, for example, pectin, chitin, chitosan, xylan, cellulose, starch, inulin, alginic acid, carrageenan contained in seaweed,
There are polygalactosamine, mannan, pullulan, etc., which are polysaccharides produced by microorganisms, and lentinan, schizophyllan, etc. produced by basidiomycetes. These polysaccharides may be used alone or as a mixture. In the present invention, it is particularly preferable to use pectin, chitin, and chitosan as the polysaccharide.

In the present invention, α-rays, β-rays, γ-rays, electron beams and X-rays are used as the ionizing radiation for irradiating the polysaccharide. Practically, it is convenient to use γ-rays, electron beams and X-rays. The dose of ionizing radiation is
It is desirable to be in the range of 0.01 to 2,000 kGy. If the dose is too low (0.01 kGy or less), no effect is obtained, and if the dose is too high (2,000 kGy or more), the decomposition proceeds too much and the activity is lost.

When the polysaccharide is irradiated with radiation, the polysaccharide is adjusted to a dried or aqueous solution. When the polysaccharide is in a dry state, the dose is desirably as high as about 100 to 2,000 kGy. Radiation treatment in a dry state is advantageous from the viewpoint that mass treatment is easy. When the polysaccharide is in an aqueous solution, the dose is 0.01 to 10 k.
There is an advantage that the dose can be as low as Gy and the required dose can be reduced. As described above, when the polysaccharide is in a dry state, high-dose irradiation is necessary.In such a case, the irradiation process can be completed in a short time under a high dose rate using an electron accelerator. it can.

[0011]

The present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not limited to these examples.

[0012]

Example 1 Pectin in a dry powder state is irradiated with various doses of radiation to decompose pectin,
The inducing activity of the antibacterial active substance glyceon in soybean was investigated. For comparison, the effect of inducing the pectin degradation product by the enzyme dolyserase was also examined. FIG.
As shown in FIG.
It was found that increasing the amount of y to 1,000 kGy significantly increased the amount of glyceone produced.
At a high dose of 2,000 kGy, 1,000
Although the production amount was smaller than in the case of kGy, the production amount at 1,000 kGy was the highest in the irradiation dose range used. On the other hand, when the enzyme doriserase was used, the production of glyceone was observed, but 1,000 kG
Irradiation of y and 2,000 kGy produced higher amounts of glyceolin.

Next, the irradiated pectin degradation product was subjected to molecular weight fractionation by HPLC (High Performance Liquid Chromatography). FIG. 2 shows the H of the pectin-degraded product at an irradiation dose of 1,000 kGy at which the amount of produced glyceone was the highest.
3 shows a PLC pattern. Table 1 shows that a (elution time: 9.
7-10.7 min. ), B (elution time: 10.7-1)
1.7 min. ) And c (elution time: 11.7-14.
0 min. 3) indicates the amount of glyceone induced and produced. As a result, irradiation dose of 1,000 kGy
In the case of the pectin degradation in, a very high glyceolin-inducing activity was observed in the fraction c. By the way, in the case of pectin degradation at an irradiation dose of 2,000 kGy, the glyceolin-inducing activity was highest in the fraction a, but only about 1/6 of that in the c fraction of 1,000 kGy was obtained.

[0014]

[Table 1] Table 1. Induction and production of glyceolin by pectin degradation / fractionation components Sample Glyceolin (nmol / g) Untreated 1.0 500 kGy-a 5.4 500 kGy-b 7.0 500 kGy-c 5.4 1000 kGy-a 9.4 1000 kGy-b 10.2 1000 kGy-c 61.9 2000 kGy-a 11.7 2000 kGy-b 5.5 2000 kGy-c 5.8

Therefore, the most active of 1,000 kGy
The glycein-inducing activity was examined for the c fraction of the pectin that had been irradiated. As a result of examining the required amount of pectin to be added to the cotyledon (cotyledone), it was found that the addition concentration of 30 μg was optimal as shown in FIG.

[0016]

Example 2 Induction of peasin, a pea phytoalexin, was investigated. Pea is 50 after the start of cultivation
Young pods after a day were harvested and used. A solution containing elicitor was dropped on the inner membrane of the pea pod and cultured, and the produced pisatin was isolated and analyzed by HPLC.

As shown in FIG. 4, the pectin-decomposed product irradiated with 1,000 kGy showed an activity of inducing pisatin, and it was found that it functions as an elicitor. The yield of pisatin in pectin degradation products by the enzyme endopolygalacturonase was lower.

On the other hand, when the induced amount of pisatin in the degradation product obtained by irradiating chitosan, which is a polymer of glucosamine, was examined, it was found that the amount of pisatin produced was extremely high in chitosan irradiated with 1,000 kGy. Obtained. In addition, in chitosan irradiated with 2,000 kGy, the amount of generated pisatin was significantly reduced as compared with the case of irradiation with 1,000 kGy.

Thus, from these results, the induced production of pisatin can be observed in irradiated pectin,
A 1,000 kGy irradiation of chitosan was found to be much more effective at producing pisatin.

[0020]

Example 3 The anti-auxin effect in pea was examined using a radiolysis product of pectin. The hypocotyl of the third node of pea on day 7 was used as a specimen. Its fragment 1c
m, and after weighing each of the 20 fragments, they were combined with 1 mM indoleacetic acid (IAA) and 0.3 mM, respectively.
It was left still for 3 hours in a solution containing g / ml pectin degradation product. After recovering each fragment, it was weighed again, and the anti-auxin effect of the oligomer as a degradation product was calculated from the following equation. Anti-auxin effect = (CT) / C × 100 (%) C: Weight increase in untreated case T: Weight increase in treated batch

As shown in FIG. 5, the inhibitory effect of the auxin effect on the promotion of pea elongation was 800 kGy.
Were hardly observed in the pectin-decomposed product irradiated with the above-mentioned radiation, but a remarkable inhibitory effect was observed in the pectin-decomposed product irradiated with the 1,000 kGy and 2,000 kGy radiations.

In addition, endopolygalacturonase (End)
An inhibitory effect was also observed with a pectin degradation product treated with an enzyme such as oPG) or driselase, but the inhibitory effect of a pectin degradation product irradiated with 2,000 kGy radiation was the highest.

The result of using an oligomer mixture which is a pectin degradation product by the enzyme endopolygalacturonase is as follows:
In contrast to a 20% inhibitory effect, the result of using a pectin degradation product irradiated with 2,000 kGy of radiation has a higher inhibition efficiency than using a degradation product of the enzyme. Therefore, it was clarified that the pectin-degraded product by radiation has a high inhibitory activity against the promotion of elongation by auxin in plants.

[0024]

The present invention provides a method for irradiating a polysaccharide such as pectin or chitosan with ionizing radiation.
The effect is that a novel oligosaccharide having a high biological activity as an antibacterial activity inducer against plant pests and pathogenic bacteria can be produced in large quantities and efficiently.

[Brief description of the drawings]

FIG. 1 is a diagram showing glyceone-induced production using a pectin degradation product.

FIG. 2 is a view showing a molecular weight fractionation of a pectin degradation product by HPLC.

FIG. 3 is a graph showing the amount of glyceolin induced production relative to the amount of pectin added to cotyledons in the 1,000 kGy-c fraction.

FIG. 4 is a graph showing the amount of pisatin produced by a decomposition product obtained by radiation treatment of a polysaccharide.

FIG. 5 is a graph showing the effect of inhibiting degradation of pea hypocotyls by pectin degradation products.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code FI C08B 37/08 C08B 37/08 A

Claims (3)

[Claims] 1. A method for producing a decomposed product of a polysaccharide having a biological activity on a plant, which comprises irradiating the polysaccharide with ionizing radiation to produce a decomposed product composed of an oligosaccharide. 2. The polysaccharide is pectin or chitosan,
The method according to claim 1, wherein the degradation product is glyceolin or pisatin. 3. A radiation dose of 0.01 to 2,000 k.
2. The method according to claim 1, which is Gy.