JP2006129778A - Method for producing crude fraction of iturin produced by bacillus subtilis, crude fraction produced by the same, and use thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To efficiently provide an antimicrobially active substance such as iturin or surfactin which is difficult to commercialize because extraction and purification are conventionally difficult as a crude fraction with a high content and to readily provide the crude fraction having an antimicrobial activity against biological species such as molds or bacteria in the form of a mildewproof agent at a commercializable cost. <P>SOLUTION: A method for producing the crude fraction of the iturin is characterized as follows. The production efficiency of the crude fraction of the antimicrobially active substance consisting essentially of the iturin or surfactin which is an antibiotic effective against molds is increased by utilizing a Bacillus subtilis DB9011 strain that is a strain highly producing the iturin and naturally screening a cultured microorganism. The crude fraction containing the iturin or surfactin obtained from the Bacillus subtilis DB9011 strain is used as the mildewproof agent, a detergent having fungicidal or bactericidal/bacteriostatic or fungistatic and mildewproof effects and an edible detergent. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method for producing a crude fraction of Bacillus subtilis-produced Iturin. More specifically, the present invention extracts and purifies a crude fraction of Iturin produced from Bacillus subtilis DB9011, a type of Bacillus subtilis. The present invention relates to a production method, and further relates to, for example, antifungal, antibacterial / antibacterial, antifungal detergents and edible detergents which use the antibacterial activity function of the extracted and purified crude fraction.

  Conventionally, antifungal agents and fungicides have been sterilized, suppressed and removed with a solution of a strong alkali or a highly acidic cytotoxic chemical. However, solutions containing these chemicals may cause adverse effects on the human body and may be detrimental to health unless sufficient attention is given to their use. For example, if it adheres to the skin, it may become blind if the skin gets dripped or enters the eye. In addition, solutions containing chemicals always have dangers, such as accidental ingestion and health hazard if stored within the reach of infants. In addition, when these chemicals are used in household kitchens, toilets and bathrooms, or in food processing factories and other equipment that require anti-fungal and antibacterial properties, they are also used to drain water through drainage pipes such as household wastewater and factory wastewater. In its use, it leads to environmental damage such as inflow and water pollution, and it causes adverse effects not only on the human body but also on the environment.

  Against this background, research on biological drugs using microorganisms has been actively conducted in recent years, and the usefulness of microorganisms resistant to specific fungi, bacteria, and viruses has been recognized. Antifungal agents and bactericidal / antibacterial agents using microorganisms have been developed. In particular, iturin and surfactin, which are related to Bacillus natto and produced from Bacillus subtilis, which are highly safe, are substances produced outside Bacillus subtilis when they form spores, and are specific to fungi and bacteria. It is recognized as having an antibacterial action and is used as an antibacterial agent against plant pathogens (see, for example, Patent Documents 1 and 2). Further, for example, surfactin alone is used as an antifungal agent applied to agricultural products after harvest in the agricultural field, and as a throat disinfectant and a mouthwash in the medical field. Further, it has been clarified that when iturin is added to surfactin, the effect is much greater than that of surfactin alone (see Patent Documents 1 and 2).

  However, when iturin is produced artificially, the production process from its producing bacteria is very unstable, and it is impossible to produce it with a certain efficiency and costs much more than when it is collected naturally. There is.

Furthermore, as another problem of antifungal agents and disinfectants using conventional chemicals, antifungal agents using chemicals seem to have fallen on the surface when using bleach. In fact, the detergent is not as effective as fungi as it does it, so there is a problem that it is not effective in practice.
Japanese Patent No. 3132195 Japanese Patent No. 3237240

  Therefore, the present invention has been made in view of the above, and since it has been difficult to extract and purify in the past, it has been difficult to commercialize at a certain efficiency. The purpose is to provide a high rough fraction. Another object of the present invention is to provide a fungal inhibitor in the form of a fungicide or the like easily at a cost that allows commercial use of the crude fraction having antibacterial activity against biological species such as molds and bacteria.

  As a result of diligent studies to solve the above problems, production efficiency of crude fractions of antibacterial active substances mainly composed of iturin and surfactin, which are effective antibiotics against mold, using Bacillus subtilis DB9011 strain The present invention has been completed by finding that the above problem can be solved by raising

That is, the above purpose is to solidify or liquid culture Bacillus subtilis DB9011 strain,
This is achieved by a method for producing a crude fraction containing iturin, which comprises extracting and purifying a crude fraction having a high iturin content from the culture solution of the Bacillus subtilis DB9011 strain.

  According to the first aspect of the present invention, a crude fraction can be produced with a certain efficiency by a production method for extracting and purifying a crude fraction having a high iturin content from Bacillus subtilis DB9011 strain.

  The invention according to claim 2 is characterized in that, in the invention according to claim 1, a solid culture of the Bacillus subtilis DB9011 strain is extracted with water.

  According to invention of Claim 2, the novel extraction method which manufactures the crude fraction with a high content of iturin from Bacillus subtilis DB9011 strain can be provided.

  The invention according to claim 3 is the invention according to claim 1 or 2, wherein the crude fraction further contains surfactin.

  According to invention of Claim 3, it can have a more effective antimicrobial effect by including iturin and surfactin in a rough fraction.

  The invention according to claim 4 is achieved by a crude fraction containing Bacillus subtilis DB9011 strain-produced iturin produced by the method according to claims 1-3.

  According to the invention described in claim 4, it is possible to provide a crude fraction containing iturin produced by Bacillus subtilis DB9011, which has a specific antibacterial action against mold and can be produced with a certain efficiency.

  The invention according to claim 5 is characterized in that paraoxybenzoate is added to the invention according to claim 4.

  According to invention of Claim 5, it can have a more effective antimicrobial effect by adding paraoxybenzoic acid ester to the crude fraction containing iturin.

  The invention according to claim 6 is achieved by a fungal growth inhibitor using the antibacterial activity function of the crude fraction according to claim 4 or 5.

  According to the invention described in claim 6, by using a crude fraction having a high content of iturin having antibacterial activity specifically for fungi, a fungal growth inhibitor having an extremely high effect of combating fungi can be obtained.

  The invention according to claim 7 is the invention according to claim 6, wherein the fungal growth inhibitor is at least one of a fungicide, a bactericide, a detergent having an antifungal effect, or an edible detergent. Features.

  According to the seventh aspect of the present invention, as an antifungal agent that is neutral and harmless to the human body by at least one of an antifungal agent, a bactericidal agent, a detergent having an antifungal effect, or an edible detergent, It can be used as a mildewproofing agent that can provide mildewproofing at facilities, etc.

  According to the present invention, iturin having antibacterial activity specific to fungi using Bacillus subtilis DB9011 strain is produced, so that it has an extremely high effect of extinguishing mold and can stably produce iturin. . In addition, iturin is a cyclic peptide and has a weak surface activity, so even if it is accidentally swallowed, it is a side effect that causes the stomach to be damaged, and it is usually a safe substance that can be digested by humans, so it is edible An antifungal agent can be provided. Furthermore, even if it is disposed of in the environment, it is decomposed by the environmental microorganisms within approximately two weeks, so that it can be a fungicide or a bactericidal agent that does not burden the environment.

  Hereinafter, the best embodiment of the present invention will be described in detail.

  Iturin and surfactin are substances produced outside the cells when Bacillus subtilis forms spores, and are known as specific antibiotics against mold. As mentioned above, surfactin alone has antifungal and antibacterial action, but it has antifungal and antibacterial action even when the concentration of surfactin is low. Has sufficient antifungal and antibacterial activity.

  Conventional iturin has been extracted by culturing Bacillus sp. And treating the culture as an extracellular product, and has been purified by techniques such as chromatography. However, its production was unstable, the yield was very bad, and it was not suitable for commercialization.

  Therefore, the present invention is characterized in that production efficiency is increased by using a high iturin-producing strain, Bacillus subtilis DB9011, which has been naturally screened for bacteria to be cultured. In addition, most proteins including low molecular weight proteins such as iturin and surfactin are forcibly precipitated in a strong hydrochloric acid environment, and the same alcohol extraction as in the conventional method is used to efficiently produce a crude fraction with a high content of target iturin. Extract and purify. This crude fraction may contain low molecular weight proteins such as surfactin. In this process, filter sterilization is performed, and high molecular protein is removed by ultrafiltration to isolate a proteolytic enzyme that is considered to cause the titer decay of iturin and surfactin over time.

  The effectiveness of the crude antibacterial active substance fraction mainly composed of iturin and surfactin, which can be produced at a commercially available cost by efficiently carrying out the above operations, was confirmed on mold, bacteria, etc. In contrast to surfactin, which is effective at about 1000 ppm for tomato wilt, conventionally, it has an effective iturin content of about 12 ppm because it effectively suppresses mold growth and kills bacteria at about 70 ppm. A high crude fraction could be produced.

  In addition, iturin of the present invention is a natural substance of microorganisms produced outside the cells from Bacillus subtilis DB9011, and since it has little harm to the human body, it can be used as a detergent for sterilization of food according to the present invention. Become. In addition, it is possible to prevent molds at facilities in daily life as mildew-proofing agents that are neutral and harmless to the human body. Conventionally, in the countermeasure against pathogenic bacteria of a facility that uses Bacillus subtilis as a dominant bacterium, aseptic bacteria and fungi are sterilized / suppressed, and there is no host, so that viruses can also be used as a sterilizing agent for useful facilities. Conventionally, chemicals are strongly irritating and toxic and can be used as a mild fungicide that is safe to touch or drink, even if it is in direct contact with a fungicide that cannot be used in combination with other detergents.

Hereinafter, although the specific example implemented according to the manufacturing method of this invention by an Example is demonstrated in detail, this invention is not limited to these Examples.
Example 1
Instead of the conventional Bacillus sp., Bacillus subtilis DB9011 strain was used.

  According to the present invention, the high iturin-producing strain Bacillus subtilis DB9011 strain is cultured, and the culture solution is treated to extract and purify a crude fraction containing high iturin. As a specific treatment method of the culture solution, a liquid or solid medium of Bacillus subtilis DB9011 strain is prepared, and the culture solution is forcibly precipitated so that most proteins including low molecular weight proteins such as iturin and surfactin are precipitated. The crude fraction with a high content of iturin was extracted and purified efficiently by the same alcohol extraction as in the conventional method. The crude fraction obtained in this way may contain surfactin. In this process, filter sterilization was performed, and high molecular protein was removed by ultrafiltration to isolate a proteolytic enzyme that was considered to cause the titer decay of iturin and surfactin over time.

  In the following, the method for producing the crude fraction of iturin of the present invention will be described in more detail.

  First, a method for preparing Bacillus subtilis DB9011 strain as a pre-stage for producing a crude fraction of iturin will be described with reference to FIGS.

  FIG. 2 is a flowchart of a method for producing a Bacillus subtilis DB9011 strain. As can be seen from FIG. 2, the production method is roughly executed by each stage of culture consisting of seed culture and main culture, collection / drying, and production of the raw material for production. A more detailed description will be given below step by step.

First, a freeze-dried ampoule of the DB9011 strain, which is the inoculum for production (for example, ALS product of ATC Corporation, or the Institute of Microbial Engineering, Ministry of International Trade and Industry, Institute of Technology, Ministry of International Trade and Industry on May 21, 1991) (Institute of Industrial Science and Technology, Patent Biological Deposit Center) was opened as MICRO Kenjo No. 34185 (FERM BP-3418)), and sterilized water was added. ) And stir culture at 37 ° C. for 2 to 3 days. Sterile water is added to this flat plate medium, and 1 ml of the suspension prepared by scraping the bacteria is dispensed per screw tube and stored at -80 ° C. Characterization test (surviving under normal sterilization conditions of Bacillus bacteria (121 ° C., 1.2 atm, 20 minutes), producing iturin crude fraction at 1 × ^{10 10} cfu / g of 1 g / kg or more, 30 strains exhibiting the phenotype described in No. 3040234) and passing the strain are used as seeds for seed culture.

Next, a medium is prepared with the composition shown in FIG. 1, and seed culture is performed. Seed culture, that is, pre-culture, is performed in two stages: flask culture and 30 L fermenter (jar) culture. Specifically, the flask culture is performed by shaking culture of a 500 ml Erlenmeyer flask, and the culture conditions are described below.
Medium: 100 ml charge amount. As a defoaming agent, 0.1% of “Disform CC-118” manufactured by NOF Corporation is added.
Inoculation amount: Thaw frozen storage suspension and inoculate 100 μl into one flask.
Culture temperature: 37 ° C.
Number of revolutions: 200 rpm.
Incubation time: 16 to 24 hours.

This flask culture is followed by 30L jar culture, and the culture conditions are described below.
Medium: Charged amount 18L. As a defoaming agent, “Disform CC-118” manufactured by NOF Corporation is added 0.5% before sterilization of the medium, and 1% is dropped during the culture.
Inoculum: Inoculate two flasks cultured in the above flask.
Culture temperature: 37 ° C.
Number of revolutions: 500 rpm.
Internal pressure: 1 kg / cm ² -G.
Aeration rate: 0.1 to 0.4 vvm.
Culture time: 8 to 16 hours.

Following the previous seed culture, the main culture is cultured using a 3 cubic meter fermenter. The culture conditions are described below.
Medium: Charged amount 1800L. As a defoaming agent, “Disform CC-118” manufactured by NOF Corporation is added 0.5% before sterilization of the medium, and 1.5% is dropped during the culture.
Inoculum amount: Inoculate 18 L of culture solution cultured in the 30 L jar.
Culture temperature: 37 ° C.
Number of revolutions: 250 rpm.
Internal pressure: 1 kg / cm ² -G.
Aeration rate: 0.1 to 0.4 vvm.
Incubation time: 24 hours.

  After culturing is completed, the bacteria are collected and dried. The collection and drying stage consists of collection of bacteria, drying, a count method in lyophilized cells, and storage of lyophilized cells.

  Bacteria are collected at 8,000 rpm using a continuous centrifuge (Sharpless type) and transferred to the drying stage.

  Drying is performed by either freeze drying or spray drying.

  In the freeze-drying method, water is added to the collected wet cells (bacterial concentration of about 30%) to a bacterial concentration of 20%, suspended in a dispermill, and freeze-dried. The cell mass after drying is crushed with a mixer.

  On the other hand, in the spray drying method, water is added to the collected wet cells (bacterial concentration of about 30%) so that the bacterial concentration is 10 to 15%, and then suspended in a disper mill, and the outlet temperature is 80 to 80%. Apply to spray dryer at 100 ° C.

  After drying, a standard test method for Bacillus subtilis DB9011 strain (confirmed by colony hybridization with a PCR tag specific to DB9011 strain obtained by a pre-stored differential screening, and flow cytometry (only during pure culture) ) Or the number of bacteria in the lyophilized cells by the method of counting the number of bacteria according to the dilution plate method).

  The dried cells are then heat sealed in an aluminum laminate bag and stored at room temperature.

  This may then be used for further manufacturing process processing, for example as a manufacturing base for use in health supplements, pharmaceuticals and the like.

In this stage, so that the number of bacteria in the manufacture YoHara body becomes to any number of more than 10 ⁹ / g, lactose lyophilized cells, glucose, skimmed milk and the like is added, stirring and mixing. The number of bacteria in the production base conforms to the standard test method (by flow cytometry (only during pure culture) or dilution plate method) of the Bacillus subtilis DB9011 strain. The production base is placed in an aluminum laminate or polyethylene bag and stored at room temperature in a warehouse. The raw material for production thus prepared and stored may be appropriately modified and used in various applications utilizing the Bacillus subtilis DB9011 strain.

  In the present invention, at this stage, the Bacillus subtilis DB9011 strain is cultured in either a solid medium or a liquid medium, and then the culture solution is treated to extract and purify a crude fraction containing high iturin. (See FIGS. 3, 4 and 5).

In order to produce the crude fraction of iturin of the present invention, it is possible to culture in a liquid medium, but solid culture has higher iturin production efficiency of Bacillus subtilis DB9011, and Bacillus subtilis after culture. In general, culture in a solid medium is desirable in order to achieve favorable conditions for storage of DB9011 strain and subsequent treatment. “Fusuma” is generally used as a solid medium for solid culture, but “Okara” is used in the present invention. Solid culture using okara is a well-known technique and is described in detail in Makoto Masada (Professor, Institute of Resource Science, Tokyo Institute of Technology) "Production characteristics of antibacterial substances by Bacillus subtilis using liquid and solid culture" Therefore, only the outline is described here. The solid culture can be performed from a scale of 15 g to 3 kg, but sterilization of okara is required in any scale. As a specific sterilization treatment, KH ₂ PO ₄ and inorganic salts of MgSO ₄ and glucose are replenished, the water content is adjusted to approximately 80%, sterilization is performed at 120 ° C. for 20 minutes, and cooling at room temperature is performed. Again, sterilization at 120 ° C. for 20 minutes is necessary.

  In the case of 15 g scale solid culture, 15 g of okara is put in an Erlenmeyer flask, the above-mentioned Bacillus subtilis DB9011 strain is added, and culture at 25 ° C. in a thermostatic bath is preferable.

  In the case of 300-500 g scale solid culture, a glass cylindrical container is used. In this case, in order for the Bacillus subtilis DB9011 strain to increase the production amount of iturin, a pipe was inserted into the lower part of the container, oxygen was supplied from the outside, and the air flow rate was set to approximately 2 vvm.

  The solid culture of 3 kg scale can use a culture tank having an internal volume of 7 liters used for liquid culture, and air was blown from the bottom with a pipe as in the 300-500 g scale, and the speed was 2 vvm.

In liquid culture, the aforementioned Bacillus subtilis DB9011 strain was added to a medium having a composition of Polypeton 3%, glucose 1%, KH ₂ PO ₄ 0.1%, MgSO ₄ 7H ₂ O 0.05%, and shaken in a flask. Culture or stir culture in a culture tank.

  As shown in FIG. 3, in the case of solid culture, 3 to 5 times the volume of solid medium water is added and stirred, and squeezing extraction is repeated 2 to 3 times with a nell to obtain an extract. Thereafter, 0.45 micron filtration is performed with a cellulose membrane or the like to obtain a sterile original extract.

  As an alternative, when performing liquid culture, as shown in FIG. 4, perform centrifugation at 7000 g for 10 to 30 minutes, and then filter the supernatant by 0.45 micron with a cellulose membrane or the like. In the same manner as above, use a sterile raw extract.

  As shown in FIG. 5, the sterile raw extract obtained by any of the treatments is a strong hydrochloric acid having a pH of 2.5 to 3 so that most proteins including low molecular weight proteins such as iturin are forcibly precipitated. Precipitate in the environment and centrifuge at 10,000 rpm for 10 minutes. Of the two layers of precipitate, the opaque lower layer is extracted with the same alcohol as in the conventional method, for example, 3 to 5 volumes of ethanol or methanol. The mixture was centrifuged at 10,000 rpm for 10 minutes, and the resulting precipitate was extracted again with a few alcohol extraction treatments, and the crude fraction with high target iturin content was extracted and purified. . The crude fraction obtained in this way may contain surfactin. In this process, filter sterilization was performed, and high molecular protein was removed by ultrafiltration (molecular weight 50,000) using a cellulose membrane or the like to separate the proteolytic enzyme. The proteolytic enzyme removed here is considered to be the cause of the titer decay over time of iturin and surfactin.

The obtained crude fractions of iturin and surfactin with various iturin concentrations were applied to 8 types of molds. Detailed conditions such as mold medium including control experiments are described below.
Medium used: Potato dextrose agar (Eiken)
Culture conditions: 25 ° C., 7 days Test method:
1. Potato dextrose agar (PDA) was autoclaved at 121 ° C. for 15 minutes.
2. After high-pressure steam sterilization, a predetermined amount of iturin solution (0 ppm (control), 9 ppm, 17 ppm, 35 ppm, 68 ppm and 138 ppm) was added to each medium cooled to 50 ° C., mixed, and 20 ml was poured into the petri dish.
3. After the medium in Step 2 was solidified, the fungus grown on the agar medium was removed with a cork borer and placed in the center of the petri dish.
After culturing in the dark for 7 days at 4.25 ° C., the ratio of mold area to control at each Iturin concentration was measured in each petri dish.

  Table 1 is a table showing the antifungal action as an area ratio to the control, and FIG. 6 is a graph showing it.

この結果からわかるように、カビの種類に関わらず、イツリン濃度が１３８ｐｐｍの場合には、すべてのカビに対して、有効であることが分かる。また、カビの種類によっては、９ｐｐｍだけでも有効に効果を奏する場合もあり、特に、Ｔｒｉｃｈｏｄｅｒｍａ ｖｉｒｉｄｅに対しては、濃度の濃淡に関わり無く、非常に有効に作用することが分かった。

As can be seen from this result, regardless of the type of mold, when the iturin concentration is 138 ppm, it is effective for all molds. In addition, depending on the type of mold, even 9 ppm alone may have an effective effect. In particular, it has been found that Trichoderma via acts very effectively regardless of the density of the concentration.

In addition, this result is an effect in a crude fraction containing iturin and surfactin, and further purified iturin may have an effective effect at a concentration lower than the concentration performed in this example. Be expected.
(Example 2)
FIG. 7 shows the results of high-performance liquid chromatography (HPLC) analysis comparing the results obtained as an example of the extraction and purification method for producing a crude fraction of iturin with sigma Iturin A.

  In Example 2, the Bacillus subtilis DB9011 strain produced by the method described in Example 1 was cultured in a solid medium, extracted and purified, and a crude iturin fraction was obtained. As a specific method, first, the solid culture was autoclaved at 121 ° C. for 15 minutes, 10 times the amount of water was added thereto, and the pH was adjusted to 9.0 with NaOH. Subsequently, the extraction process was performed at 60-80 degreeC for 1 hour. At this stage, diatomaceous earth was added as a filter aid, filtration was performed using a cloth or the like, and the obtained filtrate was adjusted to pH 3 to 4 with HCl. Subsequently, this liquid was centrifuged and a precipitate was obtained as an iturin crude fraction.

  From this result, it can be seen that the obtained crude fraction is very similar to Sigma's Iturin A, and the content of Iturin is high.

  Therefore, as is clear from the above examples, a crude fraction having a high content of iturin, which is a specific antifungal substance, from the Bacillus subtilis DB9011 strain can be efficiently extracted and purified. The crude fraction can be used in place of chemical fungicides and fungicides.

  In addition, iturin alone has sufficient antifungal and antibacterial activity, but natural iturin has a reduced effect in 4-5 days, especially when paraoxybenzoate (paraoxybenzoate (paraben)) is added. As an antifungal agent, fungicide, etc., it is possible to exert further excellent effects.

  Furthermore, it can be provided as an antifungal agent that is safe to eat due to the properties of iturin.

  The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to such specific embodiments, and various modifications and changes can be made within the scope of the gist of the present invention described in the claims. It can be changed.

It is a composition figure of the culture medium which manufactures Bacillus subtilis DB9011 strain. It is a flowchart which manufactures Bacillus subtilis DB9011 strain. It is a flowchart which shows the process of manufacturing an iturin crude fraction from a solid culture medium. It is a flowchart which shows the process of manufacturing an iturin crude fraction from a liquid culture medium. It is a flowchart which shows the manufacturing process (3) of the iturin rough fraction following FIG. 3 or FIG. It is the graph which showed the anti-fungal effect by the area ratio with respect to control at the time of applying the crude fraction of iturin and surfactin of various iturin concentration with respect to 8 types of mold. It is a figure which shows the HPLC analysis result of the iturin crude fraction performed by the method of Example 2.

Claims (7)

Bacillus subtilis DB9011 strain is solid or liquid cultured,
A method for producing a crude fraction containing iturin, comprising extracting and purifying a crude fraction having a high iturin content from the culture solution of the Bacillus subtilis DB9011 strain.   The method according to claim 1, wherein a solid culture of the Bacillus subtilis DB9011 strain is extracted with water.   The method according to claim 1, wherein the crude fraction further contains surfactin.   The crude fraction containing the Bacillus subtilis DB9011 strain production Iturin manufactured by the method in any one of Claims 1-3.   The crude fraction according to claim 4, wherein paraoxybenzoic acid ester is added.   The fungal growth inhibitor which uses the antibacterial activity function of the rough fraction of Claim 4 or 5.   The fungal growth inhibitor according to claim 6, wherein the fungal growth inhibitor is at least one of a fungicide, a bactericide, a fungicide, or an edible detergent.

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