JPH0660B2 - Novel microorganism capable of degrading chitin - Google Patents

Description

TECHNICAL FIELD The present invention relates to a novel microorganism capable of degrading chitin, and particularly effective chitosan obtained by subjecting chitin provided in the shells of crustaceans such as crabgrass to microbial culture. The present invention relates to an antibacterial composition containing a culture as a component or a culture obtained by culturing such a microorganism or a microorganism giving an anti-nematode agent.

(Background Art) In recent years, the shells of crustaceans such as crab crab are chemically treated to collect chitosan as a useful substance. In this chemical treatment method, first, the calcium-free shell obtained by decalcifying the shells of crustaceans such as crabs
% Alkaline or protease treatment and deproteinization to remove chitin, then remove it by 40%
The desired chitosan is obtained by treating with an alkaline aqueous 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 searched from the natural world for microorganisms that decompose chitin well, particularly have excellent ability to decompose chitin existing in the shells of crustaceans, and have high production ability of chitosan and various chitin-degrading enzymes. Among them, it was found that chitosan obtained by degrading chitin using Enterobacter G-1 strain has excellent antibacterial action and anti-nematode action, and further, such a microorganism was treated with chitin medium. It was also found that the culture (liquid) obtained by culturing in (1) also has an antibacterial action and an anti-nematode action.

(Problem to be solved) Here, the present invention has been completed based on such findings, and the problem to be solved is that an antibacterial composition or an anti-nematode having a microorganism-forming chitosan component as an active ingredient is used. It is an attempt to provide a novel microorganism that gives an agent.

(Solution) In order to solve the above problems, the present invention has the ability to decompose chitin and is a strain (microorganism) consisting of Enterobacter G-1 deposited as Microtechnical Research Institute No. 3140. Is its characteristic.

(Effects of the Invention) Such a specific strain (Enterobacter G-1) according to the present invention decomposes chitin present in the shells of crustaceans such as crabgrass, and has an effective antibacterial action or anti-nematode action. Isolated as a microorganism that gives a toxane having, and using such a microorganism, chitosan obtained by degrading chitin is prepared as an active ingredient into an antibacterial composition or an anti-nematode agent. By exhibiting an excellent antibacterial action against various pathogenic bacteria, such as grape root head cancer, rose root head cancer, rice Mongale disease, wasabi inking disease, and nematodes such as pine wood nematode. It has an effective insecticidal or anti-nematode effect against

(Specific Structure) By the way, the microorganism according to the present invention is a strain belonging to the genus Enterobacter, and is an Enterobacter G-
It is called 1. This strain was obtained from water collected at Gekkoji Temple in Matsue City, Shimane Prefecture, and was used in a medium containing only decalcified crab powder and 0.2% K ₂ HPO ₄ in order to induce chitin-degrading activity. One month
It was isolated from the one that was subjected to continuous cultivation 0 times,
On January 14th, "Mikoken Kenjoyori No. 3140 (FERM B
P-3140) ”, and its mycological properties are as follows.

(I) Morphological Properties This strain is a Gram-negative monobacillus (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) Days until a clear zone is created 16 ℃ ・ ・ ・ 2 days later 30 ℃ ・ ・ ・ 1 day after 37 ℃ ・ ・ ・ 1 day after 42 ℃ ・ ・ ・ 4 days after (2) Growth pH range ・ ・ ・ 4-9 (3) Reduction of nitrate ・ ・ ・ Positive ( 4) Generation of hydrogen sulfide: Positive (5) Formation of indole: 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 generation test from sugar Acid generation and gas generation: D-fructose , D-mannose, glucose, succharose acid, no gas is generated: maltose, D-galacto Scan, D- sorbitol, without generating a mannitol acid, gas does not occur: arabinose, lactose, D- xylose, rhamnose (IV) identified "Bajizu Manual of Systematic
Biotechnology (Bergey's manual of systematic
biotechnology) ”, Vol. 1 and“ Classification and identification of microorganisms <below> ”(edited by Takeharu Hasegawa, Academic Publishing Center), it is considered to belong to the genus Enterobacter. However, when searching from known strains of worms described in the Vergiz 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) Cultivation of microorganism For culturing this strain, a general method for culturing a radiant bacterium 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 the culturing method, culturing on a solid medium is also possible, but like the general yeast production method, it is preferable to adopt liquid culturing, in which case, for example, a liquid having the following composition: A medium is used.

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 performed at a culture temperature of about 20 to 40 ° C.

In the present invention, the above Enterobacter G
-1 is used to decompose chitin existing in the shells of crustaceans such as crabs to provide effective chitosan, but for this microbial treatment, the shells of crustaceans are preferably used. By treatment with a suitable acid such as hydrochloric acid, the calcium decalcified, in other words, the CaCO ₃ in the shell eluted and removed by the acid, is provided in the powder state. In particular, by carrying out such a calcium removal treatment, the volume of the entire shell can be reduced, which facilitates the handling thereof and has the advantage that the calcium treatment in the microorganism treatment tank is not necessary. From the advantage that sterilization of metamorphic microorganisms can be performed at the same time,
It will be advantageously used for the treatment of crab.

Further, such shells of crustaceans are dispersed in an appropriate dispersion medium such as water to obtain a dispersion liquid, which is then housed in an appropriate reaction container (bioreactor), and the specific microorganism according to the present invention is used. The microbial treatment is carried out. In addition, in this microbial treatment, the same components as in the above-mentioned culture solution composition are appropriately added, and the mixture is maintained at a temperature of 20 to 40 ° C. and cultivated for about 10 to 15 days under stirring to obtain the target shellfish. The shells of the kind are processed.

The ratio of crustacean shells and the amount of microorganisms added to the dispersion contained in the reaction vessel, the culturing temperature, the culturing period, etc., are such that the production amount of the desired chitosan in the culturing liquid is the maximum. It will be determined as appropriate.

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 is stopped at the time when the production amount of the target product reaches the maximum, and the target product is isolated and purified. As one of the methods, there is a method by centrifugal 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. In addition,
From the supernatant, useful degrading enzymes such as chitinase and chitosanase can be collected by ultrafiltration membrane fractionation, chitin affinity chromatography, isoelectric focusing and the like.

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 performing the microbial treatment in the culture tank. That is, for a predetermined period of time, the culture is taken out of 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,
Taking out microbial cells, chitin, chitosan, and separating chitosan from them, while returning the microbial cells and chitin to the culture tank again, and supplying the necessary raw materials such as decalcified crab shell to the culture tank, It is possible to continue the microbial treatment in such a culture tank.

The thus-obtained chitosan has an excellent antibacterial action or anti-radiation (insecticidal) action, and therefore, it is used as an active ingredient in accordance with a known formulation in accordance with a known formulation. It can be prepared or used as a mid-line agent. In addition, chitosan formed by such microbial treatment is 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 same antibacterial action or antifibrinolytic action is present in the culture solution (culture medium) obtained by culturing the same, and therefore It is also possible to prepare an antibacterial composition or an antifibrinolytic agent using various culture solutions.

Incidentally, the mechanism of chitosan obtained by the treatment with a microorganism according to the present invention inhibits the growth of pathogenic microorganisms and simultaneously activates plant cells, but at present, its mechanism is not fully understood. It is thought that the experimental results can be well explained by considering as follows. That is, in contaminated soil, plant cells are infected by pathogenic organisms, invaded in the line, or eaten by insects, and become physiologically functional fixed price, nutritional disorder, poor growth, and eventually die. 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 the coexisting microorganisms into a low-molecular-weight, water-soluble chitosan oligosaccharide, which enters the plant cells and
Promotes transcription of RNA from NA. Second, as a result,
In plant cells, protein synthesis becomes active, in which biosynthesis of chitinase, chitosanase sugar enzymes, and antibacterial substances such as phytoalexin are promoted. Thirdly, these enzymes decompose the cell wall of pathogenic microorganisms and enter phytoalexin, chitosan oligosaccharide, etc.
Suppresses A transcription and inhibits proliferation. Fourthly, new chitosan oligos and the like generated by this cell wall decomposition promote the activation of plant cells. Further, it is considered that the chitosan oligosaccharides and various enzymes thus produced attack the line and inhibit the growth. Then, these series of reactions start by being stimulated by chitosan, and the leading 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.

(Examples) Hereinafter, several examples of the present invention will be shown to clarify the present invention more specifically, but the present invention does not impose any restrictions due to the description of such examples. Needless to say, it is not something to receive.

In addition to the following embodiments, the present invention further includes various changes and modifications based on the knowledge of those skilled in the art, in addition to the above specific description, without departing from the spirit of the present invention. It should be understood that improvements and the like can be added.

The percentages in the following examples are indicated by gravimetric techniques unless otherwise specified.

Preparation of Microbial Treated Chitosan First, dehydrated calcium crab powder treated with dehydrated calcium in an aqueous solution of 1% HCl: 400 g, containing 0.025% peptone and 0.2% K ₂ HPO ₄ , and further enterobacter G-. A culture solution 30:30 having a pH of 7.0 containing 700 ml of the seed culture of Example 1 was prepared, and shake culture was carried out for 14 days while maintaining this at a temperature of 30 ° C. The precipitate obtained by centrifuging from the culture solution after such culturing was about 200 g in dry weight, and as a result of infrared absorption spectrum analysis, crude chitin that had been deacetylated at a maximum of about 50 to 60% was obtained. , A mixture of chitosan and the like. Also, when this is treated with acetic acid, it is about 90g of 48%.
Was obtained as acetic acid insoluble crude chitosan, and about 105 g of the remaining 52% 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 centrifugation,
About 200 g of Ca-free crab is present in a solubilized state.
It is considered that acetylglucosamine oligosaccharides, glucosamine oligosaccharides and partially acetylated glucosamine oligosaccharides are produced.

In addition, the activity of each enzyme produced by the above culture treatment was measured for chitinase: 0.5 ml of 0.5% colloidal chitin aqueous solution, 0.1 M citric acid-0.2 M disodium hydrogen phosphate buffer (pH 7.0) 1 ml And 0.5 ml of the above-mentioned culture filtrate (crude enzyme solution) were incubated at 30 ° C. for 30 minutes and boiled at 100 ° C. for 5 minutes to inactivate the enzyme.
HALES) modified method. 1 μmol of N-
The amount of enzyme that produces a reducing sugar corresponding to acetylglucosamine was defined as 1 unit. And again, the measurement of chitosanase activity was carried out with a 1% colloidal chitosan aqueous solution (pH 6.0).
Add 0.5 ml of the above culture filtrate to 0.5 ml and
Incubation for 30 minutes at
After boiling at 0 ° C for 5 minutes to inactivate the enzyme reaction,
The released reducing sugar was quantified and determined by a modified Scarless method.
The amount of enzyme that produces 1 μmol of glucosamine per minute is 1 unit. Furthermore, chitin deacetylase activity is 0.5% colloidal chitin aqueous solution 0.5 ml, 0.1M citric acid-0.2M disodium hydrogen phosphate buffer (pH 7.0)
1 ml and 0.5 ml of the above culture filtrate, 2 ml in total,
Incubate for 30 minutes at a temperature of ° C, then
The enzyme was deactivated by heating at a temperature of 100 ° C. for 5 minutes and the NH ₂ groups formed were determined by colloid titration.

As a result, 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 acetic acid-soluble components including chitosan from chitin is about 5
2%. At this time, 180 to 300 mg of chitinase and chitosanase enzymes are produced as enzyme proteins.
If the amount of the enzyme that cleaves 1 μmol of β-1,4-glucoside bond in 1 minute is 1 unit, it will be 360 to 600 units. Similarly, chitin deacetylase is produced as an enzyme protein in an amount of 120 to 180 mg and an enzyme amount of 210 mg.
It was found to be ~ 300 units.

Antibacterial test of microbial-treated chitosan and microbial culture, using the microbial-treated chitosan obtained as described above,
Agar medium added at each concentration such as 0.1%, 0.5% or 1% and control agar medium without addition, as well as various types of colloidal chitosan, powdered chitosan and colloidal chitin obtained by chemical treatment for comparison. A concentration of agar medium was 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, first of all, the pathogenic bacterium of the root canal carcinoma of the grape (Agroba
Regarding the antibacterial property against cterium tumefaciens, first, as the medium, an ordinary agar medium (meat extract 5 g, sodium chloride 5 g, peptone 10 g, agar 15 g / water 100 m) was used.
1, pH 7.0) was used as a control, and colloidal chitosan, powdered chitosan, microbially treated chitosan, and colloidal chitotin were added at various concentrations to prepare a pathogenic bacterium that had been grown in a liquid medium in advance. Was smeared on the medium and cultured at 30 ° C. for 3 days, and the number of bacteria was measured. The results are shown in Table 1 below.

In addition, the same results were obtained when the same antibacterial test was performed on rose root carcinoma.

In addition, the pathogen of the horseradish inking disease (Phoma wasabiae Yok
Regarding the antibacterial activity against ogi), the same agar medium as above was used as a control, and colloidal chitosan, powdered chitosan, microbially treated chitosan, and colloidal chitin were added to the medium at various concentrations, and 9 pathogenic bacteria were planted at 30 ° C. After culturing for 8 days, the antibacterial properties of each colony were examined by the average value of the colony diameter. The results are shown in Table 2 below.

Furthermore, regarding the antibacterial activity against the pathogenic bacterium of Mongale disease of rice, by controlling the same agar medium as above, colloidal chitosan, powdered chitosan, microbial treated chitosan, colloidal chitin was added to the medium at various concentrations in advance, The pathogenic bacteria that had been grown in the liquid medium were smeared and cultured at 30 ° C. for 1 day, after which the growth of colonies was observed. As a result, control of powdered chitosan
0.5%, 1% supplemented medium, 0.5% of colloidal chitin, 1%
It was confirmed that the addition medium and the 0.5% and 1% addition medium of colloidal chitosan all grew on the entire surface. On the other hand, the medium (0.5% added with microbially treated chitosan)
%, 1%, 5%), the growth of pathogenic bacteria was remarkably suppressed, and the growth of pathogenic bacteria was not observed at all in the medium containing 5% microbial-treated chitosan. .

As is clear from the results of the above 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, since it is considered that the microbially treated chitosan has 40 to 50% of acetyl groups, the low molecular weight chitosan gradually progresses, so that it is expected to have a rapid effect as well as a delayed effect. .

On the other hand, when the effect of pine, which is considered to be the main cause of pine beetle death with pine beetle, was investigated, it was found that, as shown in FIG. It was found to kill 90% of the nematodes at 25 ° C for a time. 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. It was also 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 effects indicate that the culture solution of microbially 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 activity of microbially treated chitosan against pine wood nematodes obtained in Examples.

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Claims (1)

[Claims] 1. An enterobacter G-, which has the ability to decompose chitin and has been deposited as Micromachine Research Institute No. 3140.
1.