JPH0648904A - Antimicrobial and nematocidal composition - Google Patents

Abstract

PURPOSE:To provide an antimicrobial or nematocidal composition containing a microorganism-derived chitosan as the active component. CONSTITUTION:This antimicrobial or nematocidal composition contains, as the active component, chitosan obtained by decomposing chitin in the presence of Enterobactor G-1 strain.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an antibacterial / anti-nematode composition,
In particular, the present invention relates to an antibacterial / anti-nematode composition containing chitosan as an active ingredient, which is obtained by culturing microorganisms such as chitin provided in crustacean shells such as crab.

[0002]

BACKGROUND ART In recent years, shells of crustaceans such as crabs have been chemically treated to collect chitosan as a useful substance. In this chemical treatment method, first, the decalcified shell obtained by decalcining the shells of crustaceans such as crab shells is treated with 1% alkali or protease to deproteinize, and then the chitin is taken out. The desired chitosan is obtained by treating it with a 40% aqueous alkali solution and performing deacetylation. The thus-obtained chitosan is one of the few natural basic polysaccharides, is mainly used as a coagulant for wastewater treatment, etc., and is also useful as a surgical thread, artificial skin, fertilizer, etc. It is recognized that there is.

Under such circumstances, the present inventors have widely used microorganisms which are highly capable of degrading chitin and which are particularly excellent in degrading chitin existing in the shells of crustaceans and which are highly capable of producing chitosan and various chitin degrading enzymes. While searching from the natural world,
It was found that chitosan obtained by degrading chitin using Enterobacter G-1 strain has excellent antibacterial action and anti-nematode action, and culturing such a microorganism in a chitin medium. The obtained culture (liquid) is also
They found that they have antibacterial and anti-nematode effects.

[0004]

The present invention has been completed based on such findings, and the problem to be solved is to provide a novel antibacterial / anti-nematode composition containing a microorganism-forming chitosan component as an active ingredient. To try.

[0005]

In order to solve such a problem, the present invention provides an antibacterial / anti-nematode composition containing chitosan obtained by degrading chitin as an active ingredient using a G-1 strain of the genus Enterobacter. The thing is the thing.

The strain (microorganism) consisting of Enterobacter G-1 used in the present invention has been deposited as Micromachine Research Institute No. 10392.

[0007]

Action / Effect: The specific strain (Enterobacter G-1) according to the present invention decomposes chitin present in the shells of crustaceans such as crab, etc., and has an effective antibacterial action or anti-nematode action. And a chitosan obtained by decomposing chitin using such a microorganism to prepare an antibacterial or anti-nematode composition as an active ingredient. Therefore, it shows an excellent antibacterial action against various pathogenic bacteria, such as grape root head cancer, rose root head cancer, rice Mongare disease, wasabi inking disease, and nematodes such as pine wood nematode. It has an insecticidal or anti-nematode effect.

[0008]

SPECIFIC STRUCTURE By the way, the microorganism according to the present invention is a strain belonging to the genus Enterobacter and is called Enterobacter G-1. This strain was obtained from water collected at Gekkoji Temple in Matsue City, Shimane Prefecture, and it contains calcium-free crab powder and 0.2% K ₂ HP.
It was isolated from a culture medium containing only O _4, which was continuously cultured 10 times for about 5 months in order to induce chitin-degrading activity. On November 14, 1988, “Microtechnology Research Institute
No. 92 (FERM P-10392) ", and its mycological properties are as follows.

(I) Morphological Properties This strain is a gram-negative monococcus (0.7-1.2 μm ×
1.0-1.5 μm), no spores are formed.

(II) Properties on various media (1) Broth medium The color of colonies is cream and the colonies spread widely. The surface of the colony is smooth, and the surrounding condition is a circle.
Also, the colony is only slightly raised. (2) Colloidal chitin agar medium The color of the colony is white, and after culturing at 30 ° C for 1 day, a transparent clear zone is formed around the colony. The surface of the colony has small irregularities. The surface of the colony is wavy. In addition, colonies have spread to the lower layers of the agar.

(III) Physiological properties (1) Growth temperature range Liquid shaking culture; 15-33 ° C (optimum growth temperature 26 ° C) Colloidal chitin agar medium; 16-50 ° C (optimum growth temperature 30 ° C) Clear zone Number of days until making: 16 ° C ... 2 days later 30 ° C ... 1 day later 37 ° C ... 1 day later 42 ° C ... 4 days later (2) Growth pH range ... 4-9 (3) Nitrate Reduction: Positive (4) Hydrogen sulfide generation: Positive (5) Indole formation: Negative (6) VP test: Positive (7) OF test: Fermentation (8) ) Methyl red test ・ ・ ・ Negative (9) Catalase ・ ・ ・ Positive (10) Oxidase ・ ・ ・ Negative (11) Urease ・ ・ ・ Negative (12) Acid and gas production test from sugar: Acid production and gas Occurrence of D-fructose, D-mannose, glucose Produces no sucrose, no gas, no maltose, D-galactose, D-sorbitol, mannitol, no gas; arabinose, lactose, D-xylose, rhamnose

(IV) Identification "Bergie's Manual of Systematic
Biotechnology (Bergey's manual of systematic
biotechnology) ”, Vol. 1 and“ Classification and identification of microorganisms <bottom> ”(edited by Takeharu Hasegawa, Academic Publishing Center), it is considered to belong to the genus Enterobacter. However, when searching among known strains described in the Vergise Manual, there is no complete agreement in the sugar-to-acid production test. Also, regarding the generation of hydrogen sulfide, although this strain is positive, there is no positive one among known strains,
This strain was recognized as a new strain, and this strain was named Enterobacter G-1.

(V) Culturing of Microorganism For culturing this strain, a usual culturing method of actinomycetes is used. As the carbon source of the culture medium, in order not to lose the chitin-degrading activity induced by the bacterium, chitin such as colloidal chitin is mainly used, and if necessary, a known appropriate carbon source may be used in combination. Become. Further, as the nitrogen source, ammonium salts, nitrates, yeast extract,
Peptone or the like is used alone or in combination, and further, phosphate or the like is used as the P source. Furthermore, in addition, if necessary, an inorganic salt, for example, an alkali metal salt, magnesium sulfate, iron sulfate, zinc sulfate, manganese chloride, etc., will be appropriately added.

As a culturing method, it is possible to cultivate on a solid medium, but like the general yeast production method,
It is preferable to employ liquid culture, in which case a liquid medium having the following composition is used, for example. Colloidal _{chitin: 4g K 2 HPO 4: 0.7g} KH 2 HPO 4: 0.3g MgSO 4 · 5H 2 O: 0.5g FeSO 4 · 7H 2 O: 0.01g ZnSO 4: 0.001g MnCl 2: 0 0.001 g Yeast extract: 0.25 g Peptone: 0.25 g Agar: 15 g Distilled water: 1000 ml pH: 7.0

Further, such culture is generally carried out in the range of 20-40.
It is carried out at a culture temperature of about ° C.

The present invention is to obtain chitosan by degrading chitin existing in the shells of crustaceans such as Crabgrass using the above Enterobacter G-1. Is preferably decalcified by treating the shell of a crustacean with a suitable acid such as hydrochloric acid, in other words, CaCO ₃ in the shell is eluted and removed with an acid, and is in a powder state. Will be provided at. In particular, by performing such a calcium removal treatment,
The volume of the entire shell can be reduced, which makes it easier to handle, has the advantage that calcium treatment in the microbial treatment tank is unnecessary, and has the advantage that black disinfection microorganisms can be sterilized at the same time. Therefore, it can be advantageously used for the treatment of crab.

The shell of the crustacean is dispersed in a suitable dispersion medium such as water to obtain a dispersion, which is then housed in a suitable reaction vessel (bioreactor) to obtain the above-mentioned specific composition according to the present invention. Microbial treatment is performed using microorganisms. During the microbial treatment, the same components as in the above culture solution are appropriately added, and
The target crustacean shell is treated by maintaining the temperature at 40 ° C. and culturing for 10 to 15 days while stirring.

The proportion of crustacean shells in the dispersion liquid contained in the reaction vessel, the addition amount of microorganisms, the culture temperature, the culture period, etc. are determined by the production amount of the desired chitosan in the culture liquid. Will be appropriately determined so that

Further, the reaction proceeds as follows by such microbial treatment. That is, shells of crustaceans, in particular, decalcified shells, are deproteinized by their microbial treatment to become chitin, and by the microbial treatment of this chitin, chitinase, chitin deacetylase, chitosanase, etc. are produced to produce chitosan, The molecular weight reduction and chitin molecular weight reduction are carried out. Then, by the reaction by such microbial treatment, the resulting culture, at the time when the production amount of the target product reaches the maximum,
The culturing is stopped and the desired product is isolated and purified. One method is centrifugation fractionation. That is, by this centrifugal fractionation, the culture is fractionated into a supernatant (culture filtrate) and a precipitate, and chitosan powder is collected from the precipitate.
From the supernatant, ultrafiltration membrane fractionation, chitin affinity chromatography, isoelectric focusing, etc.
It is possible to collect useful degrading enzymes such as chitinase and chitosanase.

Further, in the present invention, it is also possible to adopt a system in which the culture is taken out and the products are sequentially separated from the culture while continuously carrying out microbial treatment in the culture tank. That is, for a predetermined period of time, the culture is taken out from the culture tank that has been treated with microorganisms, and separated by using a filter (membrane) that cuts, for example, those having a molecular weight of 200,000 or more, so that microbial cells, chitin, and chitosan are separated. take out,
While separating chitosan from it, microbial cells and chitin are returned to the culture tank again, and necessary raw materials such as decalcified crab shell are supplied to the culture tank to continue the microbial treatment in the culture tank. It can be done like this.

The thus-obtained chitosan has an excellent antibacterial action or anti-nematode (insecticidal) action, and therefore, it is used as an active ingredient in accordance with a known formulation to obtain antibacterial properties or It can be prepared or used as an anti-nematode composition. Further, the chitosan formed by such microbial treatment is one in which a chitin medium, for example, colloidal chitin agar medium is used as a nutrient medium for culturing the microorganism (Enterobacter G-1) according to the present invention. Therefore, the same antibacterial action or anti-nematode action also exists in the culture solution (cultured body) obtained by culturing it, and therefore, using such a culture solution, the antibacterial or anti-nematode composition is obtained. It is also possible to prepare things.

The chitosan obtained by the microbial treatment according to the present invention inhibits the growth of pathogenic microorganisms,
At the same time, the mechanism of activating plant cells has not yet been fully elucidated, but at the present time, it is considered that the mechanism can explain the experimental results well when the following is considered. That is, in contaminated soil, plant cells are infected by pathogenic microorganisms, invaded by nematodes, eaten by insects, and the like, resulting in decreased physiological functions, nutritional disorders, poor growth, and eventually death. On the other hand, in the active soil to which chitosan is added, the following four functions are induced. First, the added chitosan is hydrolyzed by coexisting microorganisms into a low-molecular-weight, water-soluble chitosan oligosaccharide, which enters plant cells and promotes transcription of RNA from DNA.
Second, as a result, plant cells become active in protein synthesis,
Among them, biosynthesis of enzymes such as chitinase and chitosanase and antibacterial substances such as phytoalexin is promoted. Thirdly, these enzymes decompose the cell wall of pathogenic microorganisms, and phytoalexin, chitosan oligosaccharide, etc. enter into the cells to suppress RNA transcription and inhibit proliferation.
Fourth, the new chitosan oligosaccharide produced by this cell wall degradation promotes the activation of plant cells. It is considered that the chitosan oligosaccharide and various enzymes thus produced attack nematodes and inhibit their growth. And
These series of reactions start by being stimulated by chitosan, and the main role can be said to be a group of microorganisms in the soil that decomposes chitosan into chitosan oligosaccharides. It can be said that this shows the excellent point of chitosan produced by utilizing the function of microorganisms.

[0023]

EXAMPLES Some examples of the present invention will be shown below to clarify the present invention in more detail. However, the present invention is not limited by the description of such examples. Needless to say, it is not something to receive. Further, in addition to the following specific examples, in addition to the following embodiments, the present invention includes various modifications based on the knowledge of those skilled in the art without departing from the spirit of the present invention.
It should be understood that modifications, improvements, etc. may be made. The percentages in the following examples are shown by weight unless otherwise specified.

Preparation of Chitosan Treated with Microorganisms First of all, Ca-depleted Crab Powder decalcified with a 1% HCl aqueous solution: 400 g, containing 0.025% peptone and 0.2% K ₂ HPO ₄ , and further enterobacter A culture solution (30 liters) having a pH of 7.0 containing 700 ml of the G-1 seed culture was prepared, and shake culture was carried out for 14 days while maintaining this at a temperature of 30 ° C. And, after such culture, the precipitate obtained by centrifugation from the culture solution,
The dry weight was about 200 g, and as a result of infrared absorption spectrum analysis, it was confirmed to be a mixture of crude chitin, chitosan and the like which were deacetylated at a maximum of about 50 to 60%. When this is treated with acetic acid, about 90 g of 48% is obtained as acetic acid-insoluble crude chitosan, and the remaining 52% of about 105 g is obtained.
g was obtained as an acetic acid soluble component. The acetic acid-soluble component contains an acetic acid-soluble protein obtained from chitosan and crabgrass-binding protein.

On the other hand, in the culture filtrate obtained by the above-mentioned centrifugation, 200 g of Ca-free crab is solubilized and present, which is a soluble low-molecular component of chitin and chitosan, that is, crude N-. It is considered that acetylglucosamine oligosaccharides, glucosamine oligosaccharides, and partially acetylated glucosamine oligosaccharides are produced.

The activity of each enzyme produced by the above culture treatment was determined by measuring 0.5 ml of a 0.5% colloidal chitin aqueous solution and 0.1 M citric acid-0.2 M disodium hydrogen phosphate buffer for chitinase. Liquid (pH 7.
0) 1 ml and 0.5 of the above culture filtrate (crude enzyme solution)
A total of 2 ml was incubated at 30 ° C. for 30 minutes, boiled at 100 ° C. for 5 minutes to inactivate the enzyme, and the resulting reducing end was quantified by a modified SCHALES method. The amount of the enzyme that produces a reducing sugar corresponding to 1 μmol of N-acetylglucosamine was defined as 1 unit. Also, the measurement of chitosanase activity was carried out in 0.5 ml of a 1% colloidal chitosan aqueous solution (pH 6.0),
0.5 ml of the above-mentioned culture filtrate was added and incubated at a temperature of 30 ° C. for 30 minutes and then boiled at a temperature of 100 ° C. for 5 minutes to inactivate the enzyme reaction. It was determined by the modified method. 1
One unit is the amount of enzyme that produces 1 μmol of glucosamine per minute. Furthermore, chitin deacetylase activity is 0.5
% Colloidal chitin aqueous solution 0.5 ml, 0.1 M citric acid-0.2 M disodium hydrogen phosphate buffer (pH 7.
0) 1 ml and 0.5 ml of the above culture filtrate, 2 ml in total
Is incubated at a temperature of 30 ° C. for 30 minutes,
The enzyme was then deactivated by heating at a temperature of 100 ° C. for 5 minutes and the NH ₂ groups formed were determined by colloid titration.

From the above results, it was found that acetic acid-soluble components containing up to about 210 g of chitosan were produced, and several g of N-acetylglucosamine and glucosamine were obtained. The production rate of the acetic acid-soluble component containing chitosan from chitin is about 52%. At this time, the chitinase and chitosanase enzymes are 180-300 mg as enzyme proteins.
Produced. If the amount of enzyme that cleaves 1 μmol of β-1,4-glucoside bond in 1 minute is 1 unit, it is 360 to 6
It becomes 00 units. Similarly, it was found that 120 to 180 mg of chitin deacetylase was produced as an enzyme protein and the amount of enzyme was 210 to 300 units.

Antibacterial test of microbial-treated chitosan and microbial culture solution Using the microbial-treated chitosan obtained as described above, 0.1%, 0.5% or 1% of each concentration was added to the agar medium and Control-free agar medium without addition, and for comparison, agar media with various concentrations of colloidal chitosan, powdered chitosan obtained by chemical treatment, and colloidal chitin were prepared. On the other hand, grape root head cancer, Mongare's disease of rice, obtaining the disease-causing microorganisms or pathogenic microorganisms of wasabi disease of wasabi, and substituting the above-mentioned microorganism-treated chitosan-containing agar medium or chemically treated chitosan-containing agar medium, etc., The antibacterial property was investigated.

More specifically, regarding the antibacterial activity against Agrobacterium tumefaciens, a first medium is an ordinary agar medium (meat extract 5 g,
Sodium chloride 5 g, peptone 10 g, agar 15 g / water 1000 ml, pH 7.0) was used as a control, and colloidal chitosan, powdered chitosan, microbially treated chitosan, and colloidal chitin were added at various concentrations, and prepared. Then, the pathogenic bacteria that have been grown in a liquid medium in advance are smeared on such a medium, and they are used for 30 days
After culturing at ℃, the number of bacteria was measured. The results are shown in Table 1 below.
As shown in.

The same results were obtained when the same antibacterial test was carried out for rose root carcinoma.

[0031]

[Table 1]

In addition, the pathogen of the horseradish inking disease (Phoma)
For antibacterial activity against wasabiae Yokogi), the same agar medium as above was used as a control, and colloidal chitosan, powdered chitosan, microbial treated chitosan,
The pathogenic bacteria were planted at 9 sites in a medium containing colloidal chitin and various concentrations, and cultured at a temperature of 30 ° C. for 8 days, and the antibacterial properties of each colony were examined. The results are shown in Table 2 below.

[0033]

[Table 2]

Further, regarding the antibacterial activity against the pathogenic bacterium of Mongarle's disease of rice, a medium in which colloidal chitosan, powdered chitosan, microbial treated chitosan, and colloidal chitin were added at various concentrations, using the same agar medium as above as a control. On the other hand, pathogenic bacteria that had been grown in a liquid medium in advance were smeared and cultured for 1 day at a temperature of 30 ° C., after which the growth of colonies was observed. As a result, the control, powdered chitosan 0.5%, 1% supplemented medium, colloidal chitin 0.5%, 1% supplemented medium, colloidal chitosan 0.5%, 1% supplemented medium were all spread over the entire surface. It was confirmed that it was growing. On the other hand, in the medium (0.5%, 1%, 5%) containing the microbial-treated chitosan, it was confirmed that the growth of pathogenic bacteria was remarkably suppressed. No growth of pathogenic bacteria was observed in the supplemented medium.

As is clear from the above results of the antibacterial test, it was confirmed that the effect of the microorganism-treated chitosan was excellent in all cases. This is considered to be because, compared with chemically treated chitosan, microbially treated chitosan has a relatively low molecular weight, is easily dispersed and solubilized in water, and its antibacterial property is rapidly exhibited. In addition, microbially treated chitosan is
Since it is considered to have 40 to 50% of acetyl groups, it is expected that the low molecular weight chitosan gradually progresses, and thus the rapid effect as well as the delayed effect is expected.

On the other hand, the effect of pine, which is considered to be the main cause of pine beetle mortality on pine wood nematodes, was examined. As shown in FIG. 5 hours, 25 ° C
Was found to kill 90% of the nematodes. On the other hand, when the chemically treated chitosan was applied, it was found that there was almost no effect under the same conditions. This seems to depend on the difference in the dispersion and solubility of both chitosans in water. Further, it was found that a culture solution of Enterobacter G-1 bacterium cultured for 2 weeks kills 95% of nematodes at 25 ° C. for 5 hours at a concentration of 25%. It is considered that the effect appeared because the low molecular weight chitosan and various chitinases coexisted in the culture medium of the microorganism. In addition, these results show that the culture solution of microbial-treated chitosan and Enterobacter G-1 bacterium can be practically used in agriculture as a natural antibacterial agent and anti-nematode agent. .

[Brief description of drawings]

FIG. 1 is a graph showing the insecticidal results of microbially treated chitosan against pine wood nematodes obtained in Examples.

Claims (3)

[Claims] 1. An antibacterial / anti-nematode composition comprising chitosan obtained by degrading chitin as an active ingredient using G-1 strain of the genus Enterobacter. 2. The antibacterial / anti-nematode composition according to claim 1, wherein the chitin is provided as decalcified crab mosquito. 3. The antibacterial / anti-nematode composition according to claim 1, wherein the chitin is provided in a chitin medium.