JPH02152904A - Antibacterial and nematocidial composition and microorganism therefor - Google Patents

Abstract

PURPOSE:To obtain the subject antibacterial and nematocidial composition containing chitosan prepared by decomposing chitin using G-1 strain belonging to Enterobacter genus as the active component and having effects against various pathogenic fungi, pinewood nematode, etc. CONSTITUTION:The subject antibacterial and nematocidial agent contains chitosan prepared by decomposing chitin using G-1 strain belonging to Enterobacter genus which is Enterobacter G-1 capable of decomposing chitin and entrusted as bikokenki no. 10392 and the chitin is obtained from shells of a crustacean such as crab shells subjected to decalcium treatment. A cultured material obtained by culturing the G-1 strain belonging to Enterobactor genus on a chitin medium can be used as the antibacterial and nematocidial agent without treatment. The subject agent is effective against grape crown gall, rose crown gall, rice sheath blight, phama wasabiae Yokogi, pinewood nematode, etc.

Description

Detailed Description of the Invention (Technical Field) The present invention relates to antibacterial and antibacterial compositions and microorganisms therefor, and in particular to chitin obtained by microbial culture treatment of chitin provided in the shells of crustaceans such as crab shells. The present invention relates to an antibacterial composition or an antinematode agent containing chitosan as an active ingredient, or a culture obtained by culturing such a microorganism, and a microorganism that provides the same.

(Background Art) In recent years, chitosan, which is a useful substance, has been collected by chemically treating the shells of crustaceans such as crab shells. In addition, in this chemical treatment method, first, the decalcified shell obtained by decalcifying the shell of a crustacean such as crab shell is treated with 1
% alkali or protease treatment to remove protein, then remove chitin by 40%
The desired chitosan is obtained by treatment with an aqueous alkali solution and deacetylation. The chitosan obtained in this way is one of the few natural basic polythiols, and is mainly used as a flocculant for wastewater treatment, and is also useful as surgical thread, artificial skin, fertilizer, etc. It is recognized that

Under these circumstances, the present inventors have developed microorganisms that can effectively decompose chitin, particularly the chitin present in the shells of crustaceans, and that have a high ability to produce chitosan and various chitin-degrading enzymes (from the natural world). During our search, we discovered that chitosan, which is obtained by decomposing chitin using the Enterobacter strain G-1, has excellent antibacterial and antinematode effects, and we have also discovered that chitosan, which is obtained by decomposing chitin using Enterobacter strain G-1, has excellent antibacterial and antinematode effects. It was discovered that the culture (liquid) obtained by culturing in a chitin medium similarly has antibacterial and anti-nematode effects.

(Problem to be solved) The present invention has been completed based on this knowledge, and the problem to be solved is a novel antibacterial composition or an antibacterial composition containing a microorganism-formed chitosan component as an active ingredient. The aim is to provide a nematode agent.

(Solution Means) In order to solve this problem, the present invention provides an antibacterial composition or an anti-cottonworm agent containing chitosan obtained by decomposing chitin using Enterobacter strain G-1. The gist thereof is as follows, but it also provides an antibacterial composition or an anti-nematode composition using a culture (liquid) obtained by culturing the G-1 strain of the Enterobacter genus in a chitin medium. The agent also
This is the summary.

Furthermore, the present invention is also characterized by a strain (microorganism) consisting of Enterobacter G-1, which has the ability to decompose chitin and was deposited as Fiber Science and Technology Research Institute No. 10392.

(Effects of the Invention) The specific strain (Enterobacter G-1) according to the present invention decomposes chitin present in the shells of crustaceans such as crabgrass, and exhibits effective antibacterial and antinematode effects. It is isolated as a microorganism that gives chitosan having the following properties, and the chitosan obtained by decomposing chitin using such a microorganism is used as an active ingredient to prepare an antibacterial composition or an antinematode agent. As a result, it exhibits excellent antibacterial activity against various pathogenic bacteria, such as grape root carcinoma, rose root carcinoma, rice Sengare disease, and wasabi inking disease.
It also shows insecticidal or anti-nematode effects against nematodes such as nematodes.

(Specific configuration) By the way, the microorganism according to the present invention is a strain belonging to the genus Enterobacter, and is a strain of Enterobacter G-
1. This strain was obtained from water collected at Gesshoji Temple in Matsue City, Shimane Prefecture, and was cultured in a medium containing only decalcified crabgrass powder and 0.2% HPO to induce chitinolytic activity. It was isolated from 10 times of continuous culture for 5 months, and was submitted to the Institute of Microbiology, Agency of Industrial Science and Technology on November 14, 1988 as FERM
P-10392) J, and its mycological properties are as follows.

(1) Morphological properties This strain is a Durham-negative monobacillus (0.7-1.2 μm
1.0-1.5 μm) and does not form spores.

(II) Properties on various media (1) Broth medium Colonies are cream-colored and spread widely. The surface of the colony is smooth and the surroundings are circular. Also, the colony is slightly raised.

(2) Colonies on colloidal chitin agar medium are white in color, and after culturing at 30″C for 1 day,
A transparent rear zone is created around the colony. The surface of the colony has small irregularities. The surface of the colony is wavy.

Colonies are also spreading to the lower layer of the agar.

(I Number of days until production: 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 production...Negative (6) V-P test...Positive (7) O-
F test...Fermentation (8) Methyl red test
... Negative (9) Catalase ... Positive 00) Oxidase ... Negative (I +) Urease ... Negative 02) Test for acid and gas production from sugars Acid production and gas production: D-fructose, Produces D-mannose, glucose, saccharose acid and no gas: Does not produce maltose, D-galactose, D-sorbitol, mannitol acid and does not produce gas: Arabinose, lactose, D-xylose, rhamnose (IV) Identification “Birshi’s Manual of Systematic
Biotechnology (Vergey'so+annual
of systemattc biotechnol
ogy)J.

Vol. 1 and [Classification and Identification of Microorganisms (Part 2)] (edited by Takeharu Hase, Gakkai Publishing Center), it is considered to belong to the genus Enterobacter. However, when searching among the known strains listed in the Bersey's Manual, there was no complete match in the sugar-to-acid production test. In addition, this strain is positive for the generation of hydrogen sulfide, but none of the known strains are positive, and this strain is recognized as a new strain.
terobacter) G-1.

(V) Cultivation of microorganisms For culturing this strain, a normal method for culturing actinomycetes is used. The carbon source for the culture medium should be mainly chitin, such as colloidal chitin, in order to prevent the loss of chitinolytic activity induced by bacteria, and should be used in combination with known appropriate carbon sources as necessary. Become.

Further, as the nitrogen source, ammonium salt, nitrate, yeast extract, peptone, etc. are used alone or in combination, and as the P source, phosphate, etc. are used. Furthermore, inorganic salts such as alkali metal salts, magnesium sulfate, iron sulfate, zinc sulfate, manganese chloride, etc. may be added as appropriate.

Although cultivation on a solid medium is possible as a culture method, it is preferable to use liquid culture as in the general yeast production method. A medium is used.

Colloidal chitin: 4g K, HPO,; 0.7 g KH, HPO,: 0.3g M g S Oa ・5 H2O: 0.5 gF
e SO= ・7 HzO: 0.01 gZnSO
n: 0.001 g MnClt: 0.001 g Yeast extract: 0.25 g Peptone: 0.25 g Agar: 15 g Distilled water: 100100 OH: 7.0 In addition, such culture is generally carried out at about 20 to 40°C. The culture is carried out at a temperature of

The present invention uses Enterobacter G-1 described above to decompose chitin present in the shells of crustaceans such as crab shells to obtain chitosan. The shell of a crustacean is decalcified by treating it with an appropriate acid such as hydrochloric acid, in other words, the CaC0j in the shell is eluted and removed by the acid, and the product is provided in powder form. becomes. In particular, by performing such decalcification treatment, the volume of the entire shell can be reduced, which makes it easier to handle.
It has the advantage of not requiring calcium treatment in the microbial treatment tank, and also has the advantage that blackening microorganisms can be sterilized at the same time, so it is advantageously employed in crab shell treatment.

In addition, the shells of such crustaceans are dispersed in a suitable dispersion medium such as water to form a dispersion liquid, and then placed in a suitable reaction vessel (bioreactor) to use the specific microorganism according to the present invention. Microbial treatment is then carried out. In addition, during this microbial treatment, components similar to the above-mentioned culture solution composition are added as appropriate, and the target is cultured at a temperature of 20 to 40°C with stirring for about 10 to 15 days. Crustacean shells are processed.

Note that the ratio of crustacean shells and the amount of microorganisms added in the dispersion liquid stored in the reaction vessel, as well as the culture temperature and culture period, etc., should be determined so that the desired amount of chitosan produced in the culture liquid can be maximized. It will be decided as appropriate to ensure that.

Moreover, by such microbial treatment, the reaction proceeds as follows. In other words, crustacean shells, especially decalcified shells, undergo protein removal through microbial treatment and become chitin.
By microbial treatment of chitin, chitinase, chitin deacetylase, chitosanase, etc. are produced, resulting in the production of chitosan, its lower molecular weight, and chitin's lower molecular weight. Then, when the culture produced by the reaction by such microbial treatment reaches the maximum production amount of the target product, the culture is stopped and the target product is isolated and purified. One such method is a method using centrifugal fractionation. That is, by this centrifugal fractionation, the culture is transformed into a supernatant (
The culture filtrate) and precipitate are separated, and chitosan powder is collected from the precipitate. Note that useful degrading enzymes such as chitinase and chitosanase can be collected from the supernatant by ultrafiltration membrane fractionation, chitin affinity chromatography, isoelectric focusing, etc.

Furthermore, in the present invention, it is also possible to employ a method in which the culture is taken out and the products are successively separated from it while continuously performing microbial treatment in the culture tank. That is, for a predetermined period of time, the culture is removed from a culture tank that has been treated with microorganisms and separated using a filter (membrane) that cuts out substances with a molecular weight of 200,000 or more, thereby removing microbial cells, chitin, and chitosan. While removing the chitosan and separating the chitosan from it, the microbial cells and chitin are returned to the culture tank, and necessary raw materials such as decalcified crab shells are supplied into the culture tank, and microbial treatment is carried out in the culture tank. You can force it to continue.

The chitosan thus obtained has excellent antibacterial or nematicidal (insecticidal) effects, and therefore it can be used as an active ingredient in antibacterial compositions or antibacterial compositions according to known formulations. It can be prepared or used as a nematode agent. Furthermore, the chitosan formed by such microbial treatment can be used as a nutrient medium for culturing the microorganism (Enterobacter G-1) according to the present invention.
Since a chitin medium, such as a colloidal chitin agar medium, is used, the culture fluid (culture) obtained by culturing it also has similar antibacterial or antinematode effects, and therefore, such It is also possible to prepare an antibacterial composition or an antinematode agent using the culture solution.

The mechanism by which chitosan obtained by the microbial treatment according to the present invention inhibits the growth of pathogenic microorganisms and simultaneously activates plant cells has not yet been fully elucidated, but at present, the mechanism is as follows. It is thought that the experimental results can be well explained by thinking like this. That is, in contaminated soil, plant cells are infected by pathogenic microorganisms, invaded by nematodes, or damaged by insects, resulting in decreased physiological function, malnutrition, poor growth, and eventually wither. On the other hand, in activated soil to which chitosan has been added, the following four functions are induced. First, the added chitosan is hydrolyzed by coexisting microorganisms to form low-molecular, water-soluble chitosan oligosaccharides, which enter plant cells and promote transcription of RNA from DNA. Second, as a result, protein synthesis becomes active in plant cells, which promotes the biosynthesis of enzymes such as chitinase and chitosanase and antibacterial substances such as phytoalexins. Third, these enzymes decompose the cell walls of pathogenic microorganisms, and phytoalechin, chitosan oligosaccharides, etc. enter the cells, suppressing RNA transcription and inhibiting proliferation. Fourth, new chitosan oligosaccharides produced by this cell wall decomposition promote the activation of plant cells. Furthermore, the chitosan oligosaccharides and various enzymes produced in this way are thought to attack nematodes and inhibit their growth. These series of reactions are stimulated and started by chitosan, but the main character is
It can be said to be a group of microorganisms in the soil that decompose chitosan into chitosan oligosaccharides. This can be said to demonstrate the superiority of chitosan produced using the functions of microorganisms.

(Examples) Below, some examples of the present invention will be shown to clarify the present invention more specifically, but the present invention is not limited in any way by the description of such examples. Needless to say, it is not something that can be accepted.

In addition to the following examples and the above-mentioned specific description, the present invention includes various changes, modifications, and changes based on the knowledge of those skilled in the art, as long as they do not depart from the spirit of the present invention. It should be understood that improvements and the like may be made.

Note that the percentages in the following examples are expressed on a weight basis unless otherwise specified.

' 几 Kisan's i,' First, 400 g of decalcified crabgrass powder treated with a 1% aqueous solution of former 1, containing 0.025% peptone and 0.2% HPO, and further A pH 7.0 culture solution: 302 containing 700 m2 of a seed culture of Bacterium G-1 was prepared and cultured with shaking for 14 days while maintaining this at a temperature of 30°C. After such cultivation, the precipitate obtained by centrifugation from the culture solution weighs about 200 g on a dry basis, and as a result of infrared absorption spectrum analysis, crude chitin containing up to about 50 to 60% deacetylation was obtained. , chitosan, etc. In addition, when this was treated with acetic acid, about 90 g of 48% was obtained as acetic acid-insoluble crude chitosan, and about 1 of the remaining 52% was obtained as acetic acid-insoluble crude chitosan.
05 g was obtained as an acetic acid soluble component. This acetic acid soluble component contains chitosan and an acetic acid soluble protein obtained from crabgrass binding protein.

On the other hand, in the culture filtrate obtained by the above centrifugation,
200g of Ca-free crabgrass is present in solubilized form, and this is composed of soluble low-molecular components such as chitin and chitosan, that is, crude N-
It is thought that acetylglucosamine oligosaccharides, glucosamine oligosaccharides, partially acetylated glucosamine oligosaccharides, etc. are produced.

In addition, for chitinase, the activity of each enzyme produced by the above culture treatment was measured using 0.5 ml of 0.55% colloidal chitin aqueous solution, 0.1 M citric acid-0,
2M disodium hydrogen phosphate buffer (pH 7,0) 1m
j2 and the above culture filtrate (crude enzyme solution) 0.5 m 1.
Incubate a total of 2 ml at 30°C for 30 minutes, then boil at 100°C for 5 minutes to inactivate the enzyme, and quantify the resulting reducing end using a modified SC IALES method. I asked. Note that the amount of enzyme that produces reducing sugar equivalent to 1 μmol of N-acetylglucosamine was defined as 1 unit. Furthermore, chitosanase activity was measured using a 1% colloidal chitosan aqueous solution (pH 6.0).
Add 0.5 ml of the above culture filtrate to 0.5 mf and incubate for 30 minutes at a temperature of 30 °C.
Thereafter, the enzyme reaction was inactivated by boiling at a temperature of 100°C for 5 minutes, and the liberated reducing sugars were determined by a modified Scarless method. Note that the amount of enzyme that produces 1 μmol of glucosamine per minute is defined as 1 unit. Furthermore, chitin deacetylase activity was determined by 0.5% colloidal chitin aqueous solution.
．． A total of 2 mff of 5 ml of 0.1 M citric acid-0.2 M sodium hydrogen phosphate buffer pH 7.0) and 0.5 ml of the above culture filtrate was incubated at a temperature of 30°C for 30 minutes. After incubation, the enzyme was inactivated by heating at a temperature of 100'C for 5 minutes and the resulting NHt groups were determined by colloid titration.

As a result, it was found that an acetic acid soluble component containing up to about 210 g of chitosan was produced, and several g of N-acetylglucosamine and glucosamine were obtained. The production rate of acetic acid-soluble components including chitosan from chitin is approximately 5
It becomes 2%. At this time, 180 to 300 mg of chitinase and chitosanase enzymes are produced as enzyme proteins.

If the amount of enzyme that cleaves 1 μmol of β-1,4-glucoside bond per minute is 1 unit, it is 360 to 600 units. Similarly, chitin deacetylase is produced as an enzyme protein in an amount of 120 to 180 mg, and the amount of enzyme is 21
It was found that it was 0 to 300 units.

Using the microbial-treated chitosan obtained as described above, an agar medium to which chitosan was added at various concentrations such as 091%, 0.5%, or 1%, and a control agar medium without additives; prepared agar media containing various concentrations of colloidal chitosan, powdered chitosan, and colloidal chitin obtained by chemical treatment for comparison. On the other hand, we obtain infected plants or pathogenic microorganisms of grape root carcinoma, rice Sengare disease, and wasabi inking disease, and transplant them onto the above-mentioned microorganism-treated chitosan-added agar medium or chemically-treated chitosan-added agar medium, etc. I looked into gender.

More specifically, first, the pathogenic bacterium Agr.
Regarding the antibacterial properties against (obacterium tumefaciens), we first used ordinary agar medium (5 g of meat extract, 5 g of sodium chloride, 10 g of peptone) as a medium.
, agar 15g/water 10100O, pH 7.0) was used as a control, and colloidal chitosan, powdered chitosan, microbial-treated chitosan, and colloidal chitin were added to this at various concentrations, and the samples were grown in a liquid medium in advance. The prepared pathogenic bacteria were smeared onto the medium, and after culturing at 30°C for 3 days, the number of bacteria was measured. The results are shown in Table 1 below.

Furthermore, when the same antibacterial test as above was carried out for Hara radicular carcinoma, similar results were obtained.

Table 1 also shows the pathogenic bacteria of wasabi inking disease (Phoma wasabi).
As for the antibacterial properties against Abiae Yokogi), a normal agar medium similar to the above was used as a control, and pathogenic cavities were inoculated at 9 locations on a medium containing various concentrations of colloidal chitosan, powdered chitosan, microbe-treated chitosan, and colloidal chitin. 30. After culturing at ℃ for 8 days, the antibacterial properties of each strain were examined based on the average colony diameter. The results are shown in Table 2 below.

Table 2 Furthermore, regarding the antibacterial properties against the pathogenic bacteria of Sengare disease of rice, the same ordinary agar medium as above was used as a control, and the culture medium to which colloidal chitosan, powdered chitosan, microbe-treated chitosan, and colloidal chitin were added at various concentrations was tested. After smearing pathogenic bacteria that had been grown in a liquid medium in advance and culturing at 30°C for 1 day, the growth status of colonies was observed. As a result, control, 0.5% of powdered chitosan, 1% supplemented medium, 0.5% of colloidal chitin
%, 1% supplemented medium, 0.5% of colloidal chitosan, 1
% supplemented medium, growth was observed on the entire surface. On the other hand, it was observed that the growth of pathogenic bacteria was significantly suppressed in the culture medium to which microbial-treated chitosan was added (0.5%, 1%, 5%). No growth of pathogenic bacteria was observed in the supplemented medium.

As is clear from the results of the above antibacterial tests, it was recognized that the microorganism-treated chitosan was excellent in effect in all cases. This is thought to be because, compared to chemically treated chitosan, microbe-treated chitosan has a relatively low molecular weight and is easily dispersed and solubilized in water, so that its antibacterial properties are quickly exhibited. In addition, microbial-treated chitosan is thought to have 40 to 50% acetyl groups, so it is expected that as the chitosan is gradually converted into a low-molecular-weight chitosan, it is expected to be both quick and slow-acting. .

On the other hand, pine beetles cause death of pine trees! As shown in Figure 1, when applying 5% microorganism-treated chitosan, 90% Approved to kill nematodes. On the other hand, when chemically treated chitosan was applied, it was found that there was almost no effect under the same conditions.

This seems to be due to the difference in the dispersion and solubility of both chitosans in water. In addition, Enterobacter
The culture solution of G-1 concave cultured for 2 weeks was at a concentration of 25%,
It was found that 95% of nematodes were killed in 5 hours at 25°C.

The effect is thought to be due to the coexistence of low-molecular-weight chitosan and various chitinases in the culture solution of the microorganism.

These results also indicate that microbial-treated chitosan and Enterobacter G-1 culture solution may be used as natural antibacterial and antinematode agents in practical applications and in agriculture. .

[Brief explanation of the drawing]

FIG. 1 is a graph showing the insecticidal properties of microbial-treated chitosan against pine nematode obtained in Examples. Applicant Sanin Construction Industry Co., Ltd.

Claims (6)

[Claims] (1) An antibacterial composition containing chitosan obtained by decomposing chitin using Enterobacter strain G-1. (2) The antibacterial composition according to claim 1, wherein the chitin is provided by decalcified crab shell. (3) An anti-nematode agent containing chitosan obtained by decomposing chitin using Enterobacter strain G-1. (4) An antibacterial composition containing a culture obtained by culturing Enterobacter strain G-1 in a chitin medium. (5) An anti-nematode agent containing a culture obtained by culturing Enterobacter strain G-1 in a chitin medium. (6) Has the ability to decompose chitin, and has the ability to decompose chitin.
Enterobacter G-1 deposited as No. 392.