JP6614673B2 - Compost using Biobalance (registered trademark) Y and manufacturing method thereof - Google Patents

Description

Cross reference

  This application claims the priority based on Japanese Patent Application No. 2014-190904 for which it applied in Japan on September 19, 2014, and all the content described in the said application is referred as it is by this specification. Incorporated. In addition, all the contents described in all patents, patent applications, and documents cited in the present application are incorporated herein by reference in their entirety.

  The present invention relates to the field of compost manufactured using animal (especially livestock) manure as raw material. The present invention also relates to the fields of return compost, fertilizer for growing plants (especially edible plants such as vegetables and rice), culture soil, or soil conditioner containing the compost.

  Manufacture of compost using manure of traditional animals (especially livestock animals) has been composted over a long period of time by mixing manure and rice straw. However, this method has a problem that problems such as bad odor occur. In addition, in the process of composting, fermentation by aerobic microorganisms such as Bacillus subtilis is used. Conventionally, by performing repeated stacking or stirring, the bad odor is removed and the contact surface with air and It was necessary to increase the contact time (Patent Documents 1 to 3).

  As another means for suppressing the generation of malodor and composting in a short period of time, a composting method in which microorganisms exhibiting the decomposition action of organic substances are added has been reported (Patent Documents 4 to 8). In addition, as another means for suppressing malodor in the composting process, it has been reported that compost is produced from livestock manure fed with feed containing microorganisms (Patent Documents 9 to 13). In most cases, composting of the obtained excreta requires a plurality of times of conventional stacking and stirring (Patent Documents 9 to 12). However, Patent Document 13 refers to microorganisms belonging to the genus Bacillus and genus Clostridium. It has been disclosed that composting can be performed with few bad odors even by single turnover by combining a mixed microorganism containing microorganisms belonging to it and a porous carrier and adding them to the feed.

  On the other hand, the present inventor has reported that malodours are suppressed in livestock manure ingested feed supplemented with lactic acid bacteria belonging to Lactobacillus delbricki species (Patent Document 14).

JP-A-2005-320181 JP 2012-246177 A JP2013-234097A JP 2008-127225 A JP2011-65 JP 2011-223883 A JP 2012-23994 JP2013-60378A JP 2004-91225 A JP2007-14241 JP2007-14243 JP 2007-15881 A JP 2008-253226 A JP-A-2005-245299

  In the method for producing compost that has already been reported, in particular, in the method for producing compost described in JP-A-2008-253226 (Patent Document 13), a porous carrier that is not originally edible is used in addition to useful bacteria. It was necessary to ingest the animal, and there were thought to be problems in maintaining the quality and health management of livestock. In addition, the water content of the compost (organic fertilizer) obtained in this document was as low as 44.2%, and the number of useful bacteria in the compost requiring water was thought to be reduced.

  As a result of intensive studies on a method for producing high-quality compost that is safer and easier for livestock, the present inventor uses manure derived from an animal ingesting a lactic acid bacterium belonging to the Lactobacillus delbrikkii species as a raw material. Thus, it was found that composting can proceed in a short period of time without causing malodor even when the number of turnovers is about once, and compost having abundant water content in the compost can be produced. In addition, the present inventor has conducted intensive studies to identify factors in compost that give such characteristics of compost. As a result, it was found that the number of actinomycetes exhibiting a high proliferation activity in a low temperature region to a medium temperature region (20 to 40 ° C.) is markedly large in such compost under aerobic conditions. Furthermore, the present inventors have succeeded in isolating a strain that seems to be characteristic among such actinomycetes, and completed the present invention.

That is, the present invention is derived from an animal that ingests a compost characterized by a markedly large number of actinomycetes exhibiting high growth activity in the middle temperature range under aerobic conditions, in particular, a lactic acid bacterium belonging to the Lactobacillus delbricki species Specifically, the present invention relates to the following invention.
(1) Compost produced from feces of livestock animals, and the number of actinomycetes growing under aerobic conditions in the middle temperature range is the number of actinomycetes growing under low temperature or high temperature aerobic conditions Compost characterized by more than.
(2) Compost manufactured from feces of livestock animals, characterized in that the number of actinomycetes growing under aerobic conditions in the middle temperature range is greater than the number of actinomycetes growing under the same conditions in aerobic composts Compost.
(3) The livestock animal is a livestock animal that has ingested a feed containing Lactobacillus delbrueckii ANTI MUFFA (FERM BP-10663), any of (1) to (2) The compost according to item 1.
(4) The compost according to (3), wherein the feed containing Lactobacillus delbrueckii (ANTI MUFFA (FERM BP-10663)) is Biobalance (registered trademark) Y.
(5) The compost according to any one of (1) to (4), which is negative for E. coli.
(6) The compost according to any one of (1) to (5), wherein the pH is 6.0 to 9.0.
(7) The compost according to any one of (1) to (6), wherein the water content is 45% or more.
(8) Compost according to any one of (1) to (7), wherein actinomycetes or materials containing actinomycetes are not added.
(9) A method for producing compost, comprising depositing feces obtained from a livestock animal ingesting a feed containing Lactobacillus delbrueckii (ANTI MUFFA (FERM BP-10663)).
(10) a) preparing stool moisture obtained from a livestock animal fed a feed containing Lactobacillus delbrueckii ANTI MUFFA (FERM BP-10663),
b) depositing the stool and standing still;
c) reversing the compost obtained by the process, and
d) settling the compost;
(9) The manufacturing method as described in (9).
(11) The method according to (10), wherein the number of actinomycetes is small during the period of step a).
(12) The manufacturing method according to (10) or (11), wherein an internal temperature of the stool or the compost deposit during the period of step b) and step d) is 30 to 60 ° C. .
(13) The manufacturing method according to any one of (10) to (12), wherein the water preparation in step a) is performed by adding a dry material.
(14) feed containing Lactobacillus delbrueckii (Lactobacillus delbrueckii) ANTI MUFFA (FERM BP-10663) , characterized in that a bio-balance (R) Y, either (9) - (13) 2. The production method according to item 1.
(15) The production method according to any one of (9) to (14), wherein no actinomycetes or materials containing actinomycetes are added.
(16) comprises depositing a stool obtained from Lactobacillus delbrueckii (Lactobacillus delbrueckii) ANTI MUFFA (FERM BP-10663) ingested livestock animals feed containing, under aerobic conditions intermediate temperature in compost A method of increasing the number of actinomycetes growing.
(17) The method according to (16), wherein the feed containing Lactobacillus delbrueckii (ANTI MUFFA (FERM BP-10663)) is Biobalance (registered trademark).
(18) The method according to (16) or (17), wherein no actinomycetes or a material containing actinomycetes is added.
(19) Actinomycetes according to any one of i) to ii) below: i) Streptomyces sp. A44 stock
ii) Streptomyces sp. A51 strain | stump | stock (20) The actinomycetes which grow under the aerobic condition of the said mesophilic range contain at least 1 of the actinomycetes of Claim 19, Comprising: Any one of (1)-(8) compost.
(21) Back compost containing the compost according to any one of (1) to (8) and (20) and a dry material.
(22) The production method according to any one of (9) to (14), comprising adding at least one actinomycete according to (19).
(23) A method for producing back compost, comprising a step of mixing the compost obtained by the production method according to any one of (9) to (14) and (20) and a dry material.
(24) The method according to any one of (16) to (17), comprising adding at least one actinomycete according to claim 19.
(25) Culture soil, fertilizer, or soil conditioner characterized by containing the compost according to any one of (1) to (8) and (20).
(26) A composition for eliminating harmful bacteria containing actinomycetes according to (19).
(27) A method for eliminating harmful bacteria in soil, compost or manure, comprising adding the actinomycetes according to (19).
(28) The composition according to (26) or the method according to (27), wherein the harmful bacteria are Escherichia coli and / or Streptococcus bacteria.
(29) The composition according to (26) or the method according to (27), wherein the harmful bacteria is one or more bacteria selected from Escherichia coli, Streptococcus dysgalactiae , Streptococcus uberis , and Streptococcus agalactiae .

  Therefore, in one aspect, the present invention relates to a compost that is produced from feces of livestock animals and has a large number of actinomycetes that grow under aerobic conditions in a medium temperature range. In this specification, livestock animals mean animals that are raised mainly for the purpose of using their products (milk, meat, eggs, hair, skin, fur, labor force, etc.). Including, buffalo, deer, sheep, goat, alpaca, pig, eagle, rabbit, chicken, moth, ostrich, turkey, goose, duck, pheasant, guinea fowl.

Preferably, the livestock animal is a livestock animal ingesting a feed containing Lactobacillus delbrueckii ANTI MUFFA (FERM BP-10663) (hereinafter referred to as “main feed” in this paragraph). Lactobacillus delbrueckii (Lactobacillus delbrueckii) ANTI MUFFA (FERM BP-10663) , at February 27, 2004, National Institute of Advanced Industrial Science and Technology, Tsukuba Center Patent Organism Depositary Center (Higashi, Tsukuba-shi, Ibaraki-ken, Japan 1-1-1 Center No. 6) is deposited under the deposit number FERM BP-10663 at the current patent administration depository center for product evaluation technology (2-5-8, Kazusa Kamashi, Kisarazu City, Chiba Prefecture). About this feed, its manufacturing method, and ingestion method, it describes in Unexamined-Japanese-Patent No. 2005-245299. The feed is preferably Biobalance (registered trademark) Y (Biobalance Inc., Nagoya, Aichi, Japan). In this specification, Biobalance (registered trademark) Y contains Lactobacillus delbruecki (ANTI MUFFA (FERM BP-10663)) sold by Biobalance Co., Ltd. (Nagoya, Aichi, Japan). Livestock feed.

  In the present specification, “actinomycetes that grow under aerobic conditions in the middle temperature range” means proliferation when cultured for a certain period (for example, one week to one month) in the temperature range belonging to the middle temperature range under aerobic conditions. It means actinomycetes with a high rate. By utilizing the composting method of the present invention, such actinomycetes can be sufficiently grown because stirring, forced aeration, and high temperature conditions are not used.

  In the present specification, the “medium temperature range” means, for example, a temperature range of 15 ° C. to 45 ° C., preferably 20 ° C. to 40 ° C., and most preferably 37 ° C. . In the present specification, the “low temperature range” means a temperature lower than the lower limit value of the intermediate temperature range, and may mean, for example, less than 15 ° C., less than 20 ° C., or less than 37 ° C. In the present specification, the “high temperature range” means a temperature higher than the upper limit value of the middle temperature range, for example, a temperature higher than 45 ° C., a temperature higher than 40 ° C., or a temperature higher than 37 ° C. May mean.

In this specification, the number of actinomycetes that grow under aerobic conditions in the middle temperature range is, for example, that the actinomycetes grow in other temperature ranges (low temperature range or high temperature range) included in the same compost. This means that the number of actinomycetes growing in the middle temperature range is large. Alternatively, “the number of actinomycetes” is “large” may mean that the number of actinomycetes growing in the middle temperature range contained per compost mass (eg, 1 g) is larger than that of aerobic compost. . Here, “aerobic compost” means compost cultivated by artificially increasing the contact surface and contact time with air as described above. The increase in contact surface and contact time with air may be performed mechanically or may be performed manually. In the compost production process, when the contact surface with air and the contact time are increased by human power, it is usually done by reversing compost (also called turning back). Means compost produced by being performed at least twice. Such aerobic compost may preferably be brewed under high temperature conditions (for example, 70 ° C. or higher). In the present specification, unless otherwise specifically understood, the “number” of actinomycetes means the unit volume (for example, 1 cm ³ ) or the unit weight (for example, 1 g). Means number.

In the present specification, the actinomycete is not particularly limited as long as it is a fungus classified into actinomycetes, but may be a fungus classified into the genus Streptomyces, for example. Preferably, the actinomycetes that grow under aerobic conditions in the middle temperature range in the present invention are Streptomyces sp. A44 strain, Streptomyces sp. A51 strain. These actinomycetes have been isolated from stationary fermentation type compost in Obihiro City, Hokkaido, Japan. As of September 9, 2014, these microorganisms were registered with the Patent Microorganism Depositary Center for Product Evaluation Technology (2-5-8 Kazusa-Kamashita, Kisarazu City, Chiba Prefecture) with the receipt number NITE BP-01934 ( Streptomycins sp. A44), and NITE BP-01935 ( Streptomyces sp. A51 strain).

  In particular, the compost of the present invention may have a feature that is negative for Escherichia coli, which can be harmful microorganisms for crops or those that eat them. Here, “negative for E. coli” does not mean that E. coli does not exist at all, but means that the number of E. coli per 1 g of compost is 100 or less. The number of E. coli per gram of compost can be determined by, for example, suspending 1 gram of compost in sterilized physiological saline, serially diluting it, seeding it on an agar medium, culturing at 37 ° C. for 24 hours, counting the number of colonies, And can be calculated by multiplying the number of colonies.

  Further, the compost of the present invention may be characterized by showing weak acidity to weak alkalinity, preferably pH 6.0 to 9.0, and more preferably pH 6.5 to 8.0. .

  Further, the compost of the present invention may be characterized by a high water content, for example, 45% or more, 50% or more, 55% or more, 60% or more. In particular, if the water content is 55% or more, it is preferable because the survival and growth of useful bacteria are not hindered. Therefore, the compost of the present invention may have a moisture content of 45 to 90%, 55 to 90%, 50 to 85%, 55 to 85%, 55 to 80%, and the like.

As will be described later, according to the present invention, the quality as described above is high even under relatively anaerobic conditions (switching once) without adding actinomycetes or Bacillus microorganisms in the composting process. Compost can be obtained. That is, the compost of the present invention may be characterized in that no useful microorganisms (including actinomycetes and Bacillus microorganisms) or materials containing useful microorganisms are added in the production process. Since there are a large number of microorganisms in nature, the term “not added” means that it is not added at least as a fungal culture (including a culture solution and a culture medium). as CFU / g-DM (dry weight) value for a mixture of manure and Inuizai, 10 ³ or more, 10 ⁴ or more, 10 ⁵ or more, 10 ⁶ or more, ¹⁰⁷ or more, ¹⁰⁸ or more, ¹⁰⁹ or more, ^{10 10} It may mean that the above or 10 ¹¹ or more useful microorganisms are not added.

  In another aspect, the present invention relates to back compost containing the compost of the present invention. The return compost of the present invention may be a mixture of the compost of the present invention and a dry material, or may contain the mixture as a main component. For example, the mixing ratio of the compost and dry material of the present invention can be compost: dry material = 7: 3 to 8: 2.

  In still another aspect, the present invention relates to a culture soil, a fertilizer, or a soil conditioner containing the compost of the present invention. The content ratio of the compost of the present invention in the culture soil, fertilizer, or soil conditioner can be any amount depending on the purpose, but for example, 100%, 10% to 90%, 20 to 80%, 30 % To 70%. In particular, the compost of the present invention is excellent in that it can be used as it is (100%) as culture soil.

In another aspect, the present invention relates to a composition for eliminating harmful bacteria containing the actinomycetes of the present invention. The composition of the present invention may be, for example, an actinomycete culture or a processed product thereof, and the processed product includes an actinomycete culture after being concentrated or dried. In the present specification, harmful bacteria are not particularly limited as long as they are harmful to human bodies, livestock, agricultural products, etc., but are preferably E. coli or Streptococcus spp. The bacteria Streptococcus, Streptococcus pyogenes, Streptococcus agalactiae, pyogenic group such as Streptococcus dysgalactiae; Streptococcus anginosus, Streptococcus constellatus , anginosus group such as Streptococcus intermedius; Streptococcus pneumoniae, Streptococcus mitis , mitis group such as Streptococcus sanguinis; Streptococcus uberis, Streptococcus spp. , Streptococcus cristatus, Streptococcus gordonii, Streptococcus oralis, Streptococcus parasanguinis, Streptococcus salibarius, Streptococcus bovis, mention may be made of Streptococcus mutans. As the harmful bacteria, E. coli , S. et al . dysgalactiae , S. et al . uberis , and S. agalactiae . For example, as the harmful bacteria, E. coli IID562, S. coli . dysgalactiae NRIC 1986, S. dysgalactiae . uberis NRIC 1153, and S. cerevisiae . agalactiae NRIC1137 may be used.

  INDUSTRIAL APPLICABILITY The present invention can provide compost of good quality that is safer for livestock and simple. Moreover, since the compost of this invention can be manufactured by a very simple method, the cost, time, etc. which are required for composting can be saved. In particular, the compost of the present invention has abundant moisture content in the compost, has a very small number of E. coli, and is very useful as a fertilizer for back compost and plant cultivation. The actinomycetes of the present invention are very useful in maintaining such excellent compost properties.

It is the graph which compared the number of actinomycetes in BB compost and general aerobic compost. The horizontal axis shows the culture temperature of actinomycetes, and the vertical axis shows the number of actinomycetes (log CFU / g compost). It is the photograph and table | surface which show the result of having measured the antibacterial activity with respect to Escherichia coli IID562 strain | stump | stock of two isolated actinomycetes strains ( Streptomyces sp. A44 strain, Streptomyces sp. A51 strain). It is a molecular phylogenetic analysis figure of A44 stock. It is a molecular phylogenetic analysis figure of A51 stock. Streptomyces sp. Strain A44 and Streptomyces sp. It is a graph showing the result of the colon_bacillus | E._coli exclusion test in a compost by A51 stock | strain. The vertical axis represents the number of E. coli (CFU / g), and the horizontal axis represents the number of days since E. coli inoculation. In the graph, diamonds are the control group, and white circles are the Streptomyces sp. Strain A44 and Streptomyces sp. A group to which 0.2% of the culture of the A51 strain was added, and black circles represent Streptomyces sp. Strain A44 and Streptomyces sp. The group to which 1% of the culture of the A51 strain was added is shown.

1. The present invention In the method aspect of the compost production method of composting comprising depositing a stool obtained from Lactobacillus delbrueckii (Lactobacillus delbrueckii) ANTI MUFFA (FERM BP-10663) to food containing the livestock animals About. As mentioned above, Lactobacillus delbrueckii ANTI MUFFA (FERM BP-10663) has been deposited under the accession number FERM BP-10663. A feed containing Lactobacillus delbrueckii ANTI MUFFA (FERM BP-10663) is not limited in form and composition as long as the feed contains the lactic acid bacteria. , Including feeds that can be taken orally by animals in all forms, such as powder, liquid, gel and gummi. Moreover, it does not ask | require whether the feed said here contains the component required for growth of a domestic animal. Therefore, for example, even if it is non-nutrition, if a domestic animal can be ingested orally, it will be included in the feed. Such feed is preferably Biobalance (registered trademark) Y (Biobalance Co., Ltd., Nagoya, Aichi, Japan).

More specifically, the method for producing compost of the present invention may comprise the following steps:
a) preparing stool water from a livestock animal fed a feed containing Lactobacillus delbrueckii ANTI MUFFA (FERM BP-10663);
b) depositing the stool and standing still;
c) reversing the compost obtained by the process, and
d) settling the compost;
A method for producing compost, comprising:

  The water preparation in the step a) can be performed by adding a dry material, for example. Examples of such a dry material include sawdust, cannabis, or chaff. The amount of water to be contained is the amount of water that allows the compost to mature, and may be, for example, 45% or more, 50% or more, 55% or more, or 60% or more. In particular, if the water content is 55% or more, it is preferable because the survival and growth of useful bacteria are not hindered. Therefore, the amount of water contained in the method of the present invention may be 45 to 90%, 55 to 90%, 50 to 85%, 55 to 85%, 55 to 80%, or the like. In the method of the present invention, the number of actinomycetes is small in the initial process of composting (sedimented feces), but the compost finally produced (without adding actinomycetes from the outside). Is rich in actinomycetes. Therefore, the number of actinomycetes in stool in step a) may be small. Here, the small number of actinomycetes preferably means that the number of actinomycetes per gram of feces or compost is less than 6.0 log CFU or 2.0 to 5.0 log CFU. good.

  The standing (deposition) of step b) and step d) can be performed at any place, and may be on artificial ground such as concrete or natural ground such as soil. Preferably, these steps are performed in a place not exposed to wind and rain, for example, under a rain guard such as a roof, inside a hut or the like, and the surface is covered with a water-impermeable sheet (eg, a vinyl sheet). Done. In addition, the internal temperature of the stool or compost deposit during these steps may be, for example, 30 to 60 ° C. or 30 to 55 ° C., and preferably the temperature is high in order not to kill useful bacteria. A temperature that is not too low (for example, 55 ° C. or lower) is selected. The standing period can be 1 to 3 months, preferably 1 to 2 months for step b). Since the aging has already progressed during the standing period of step d), it can be composted in a shorter time, for example, 2 weeks to 1 month. The height to be deposited can be any height, but can be 1 to 5 m as the height at which deposition and turning can be easily performed, or the height that provides an appropriate internal temperature. 3m.

  The inversion of the compost in step c) is a method called turning back in this technical field. By changing the inside and outside of the compost or mixing the inside and outside compost, the maturity of the whole compost is made uniform. To be done. The compost manufacturing method of the present invention can manufacture high-quality compost despite the small number of compost reversal steps (step c)). In the method for producing compost of the present invention, inversion of compost is usually required only once.

  Preferably, the method for producing compost of the present invention is compost manufactured from the feces of the livestock animals of the present invention, and has a large number of actinomycetes growing under aerobic conditions in a medium temperature range, or a low temperature range or a high temperature range. It is a manufacturing method of compost characterized by being more than the number of actinomycetes growing under aerobic conditions.

  Moreover, the method for producing compost of the present invention has a small number of actinomycetes at the start of composting. Nevertheless, even if no actinomycetes are added from the outside, a very large number of actinomycetes can be contained when composting is completed. That is, conventionally, in order to increase the number of actinomycetes in compost, actinomycetes or materials containing actinomycetes were artificially added during the production of compost, but in the method of the present invention, such addition is not It is unnecessary. Therefore, in one aspect, the production method of the present invention may be characterized in that, in the production process, no actinomycetes or materials containing actinomycetes are artificially added. Here, the material containing actinomycetes means a plant, plant processed product (fermented product, etc.), soil, culture medium or the like on which actinomycetes are grown or adhered.

Alternatively, in the present invention, Streptomyces sp., Which is useful for improving the properties of compost, is used. A44 strain, and Streptomyces sp. Since the A51 strain was found, in another embodiment, the method of the present invention may comprise adding at least one of these actinomycetes in the composting step.

2. Methods for Increasing Actinomycetes In another aspect, the present invention comprises depositing stool obtained from a livestock animal fed a diet containing Lactobacillus delbrueckii ANTI MUFFA (FERM BP-10663). The present invention relates to a method for increasing the number of actinomycetes that grow under aerobic conditions in the middle temperature range of compost. The feed containing Lactobacillus delbrueckii ANTI MUFFA (FERM BP-10663) is the same as the production method described above.

  In particular, the method of the present invention contains a large amount of actinomycetes at the time composting is completed without adding actinomycetes from the outside even though the number of actinomycetes is small at the start of composting. Can do. Therefore, in one aspect, the method of the present invention may be characterized in that actinomycetes or materials containing actinomycetes are not added. Here, the material containing actinomycetes means plants, plant products (fermented products, etc.), soil, or culture medium on which actinomycets are grown or adhered.

Alternatively, the methods of the present invention can be applied to the actinomycetes Streptomyces sp. That has been found to be particularly useful in the present invention. A44 strain, and Streptomyces sp. You may provide adding at least 1 strain of A51 stock | strain in a composting process.

3. Method for Producing Back Compost The compost of the present invention can be made back compost. Therefore, in one aspect, the present invention relates to a method for producing back compost, comprising the step of mixing the compost obtained by the method for producing compost and a dry material. Here, examples of the dry material include sawdust, cannabis, and rice crackers.

4). Method for Producing Various Products Containing Compost of the Present Invention The culture soil, fertilizer, or soil conditioner containing the compost of the present invention is appropriately utilized by methods well known to those skilled in the art for producing these products from compost. Can be manufactured.

5. Method for using various products containing the compost of the present invention, compost of the present invention, compost of the present invention Compost of the present invention, compost of the back, various products containing the compost of the present invention are usually used by those skilled in the art according to their respective applications. It can be used according to the method used.

6). Production of composition for eliminating harmful bacteria containing actinomycetes of the present invention A composition for eliminating harmful bacteria containing actinomycetes of the present invention is produced, for example, as an actinomycete culture or a processed product thereof. can do. Cultivation of actinomycetes can be performed in a medium suitable for actinomycetes. For example, a medium obtained by mixing soil (for example, vermiculite) in a liquid medium in which glucose and yeast extract are dissolved in water can be used. Streptomyces sp. A44 strain and / or Streptomyces sp. An actinomycete culture can be obtained by suspending the inoculum of the A51 strain in physiological saline and inoculating the actinomycetes medium and culturing for 10 days to 2 months. If necessary, the actinomycete culture may be concentrated or dried to obtain a processed product of the actinomycete culture.

7). Method for eliminating harmful bacteria in soil, compost or manure, including adding actinomycetes of the present invention Since actinomycetes of the present invention can eliminate harmful bacteria in soil, compost or manure, It can be used in a method for eliminating harmful harmful bacteria. In the method for eliminating harmful bacteria according to the present invention, the actinomycete composition can be directly added to the target soil, compost or manure, and may be mixed after the addition, if necessary. Alternatively, the actinomycetes composition may be sprayed or mixed in advance on a place where manure in a livestock housing facility such as a cowshed, a piggery, or a poultry house comes into contact or in return compost.

  Hereinafter, examples will be shown to describe the present invention in more detail, but the present invention is not limited to these examples. It should be noted that all documents cited throughout this application are incorporated herein by reference in their entirety.

(Example 1) BB compost production method Biobalance (registered trademark) Y (Biobalance Co., Ltd., Nagoya City, Aichi, Japan) fed cow dung, added sawdust and adjusted to 70% moisture, It was deposited at a height of 2 to 3 m under rain protection and left to stand for 1 month. When the center temperature of this period was measured, it was 40-55 degreeC. After that, the mixture was inverted and allowed to stand still for another month to complete composting. (Hereafter, the obtained compost is called “BB compost”)

(Example 2) Difference in the number of actinomycetes between aerobic compost and Biobalance (registered trademark) Y The BB compost obtained in Example 1 was dried at 105 ° C for 6 hours and 1 g each of aerobic compost was SDS-yeast. It was dispersed in 9 g of a pretreatment liquid containing an extract (Yeast extract) and pretreated at 40 ° C. for 20 minutes. Thereafter, serially diluted products up to 10000 times with sterile physiological saline were smeared on 50 μl HV plate (Humic acid-vitamin agar, HV agar). The smeared plate was cultured for 2 weeks in an incubator set at 55 ° C., 37 ° C., and 25 ° C. under aerobic conditions. Appearing colonies were counted as actinomycetes.

A pretreatment solution containing SDS-yeast extract (Yeast extract) was prepared by dissolving 0.5 g of sodium dodecyl sulfate (Kanto Chemical) and 60 g of yeast extract (OXOID) in 50 mM Phosphate buffer (pH 7.0). And then autoclaved for 15 minutes.
Moreover, the HV flat plate (Humic acid-vitamin agar, HV agar) which is an actinomycete isolation medium was autoclaved at 121 ° C. for 15 minutes after adjusting the following composition to pH 7.2, and a flat plate was prepared according to a conventional method.

Humic acid ^{* 1} (Wako Pure Chemical Industries) 1.0g
Na ₂ HPO ₄ (Kanto Chemical) 0.5g
KCl (Kanto Chemical) 1.7g
MgSO ₄ · 7H ₂ O (Kanto Chemical) 0.05g
FeSO ₄ · _7H 2 O (Kanto Chemical Co., Inc.) 0.01g
CaCO ₃ (Kanto Chemical) 0.02g
B-vitamins ^{* 2} 5mL
Cycloheximide ^{* 3} (Wako Pure Chemical Industries) 50mg
Nalidixic acid ^{* 4} (Wako Pure Chemical Industries) 20mg
Agar (Far East Chemical) 20g
Distributed water 1L

* 1 Humic acid solution 5 g / 100 ml 20 N of 0.2 N NaOH solution dissolved at 110 ° C. for 10 minutes was added to 1 L of medium.
* 2 B-Vitamins
The following reagents (all Wako Pure Chemical Industries) were dissolved and prepared, and then sterilized by filtration with a filter.
Thiamine-HCl 100mg
Rivoflavin 100mg
Niacin 100mg
Pyridoxin-HCl 100mg
Inositol 100mg
Ca-pantothenate 100mg
p-aminobenzoic acid 100mg
biotin 50mg
* 3 Cycloheximide solution (final concentration 50ppm)
It was prepared as a 1000 mg / 100 ml EtOH solution, and 5 ml was added to 1 L of the medium.
* 4 Nalidixic acid solution (final concentration 500 ppm)
It was prepared as a 2000 mg / 100 ml, 0.2N NaOH solution, and 1 ml was added to 1 L of the medium.

  The results are shown in FIG. The number of actinomycete colonies that emerged by culturing at 25-37 ° C. was 100 times greater in BB compost than in aerobic compost. Moreover, also in the comparison in the same BB compost, the number of colonies of actinomycetes that appeared by culturing at 25 to 37 ° C. was larger than the number of colonies of actinomycetes that appeared by culturing at other temperatures. Therefore, it was shown that BB compost is easier to utilize organic matter decomposition by actinomycetes than aerobic compost.

(Example 3) Isolation of actinomycetes having antibacterial activity Actinomycetes were caught from the HV medium cultured in Example 2, and streak-isolated with YM medium. Two isolated actinomycetes strains ( Streptomyces sp. A44 strain, Streptomyces sp. A51 strain) were cultured again in YM medium and HV medium, and the growth conditions were examined. A piece of YM medium on which actinomycetes were grown was placed on an NB medium smeared with Escherichia coli IID562 in advance, and formation of inhibition circles was confirmed after 24 hours.

The results are shown in Table 1 and FIG. The isolated actinomycetes grew at 25 ° C. and 37 ° C., but did not grow at 55 ° C. The isolated actinomycetes showed antibacterial activity against Escherichia coli IID562 strain. Thus, isolated actinomycetes are thought to suppress the growth of pathogenic bacteria during the deposition period.

(Example 4) Identification of actinomycete The obtained actinomycete was obtained from ISP medium No. 2 (ISP2) aerobic culture at 30 ° C. for 1 week on an agar medium (“Digo” (Nippon Pharmaceutical Co., Ltd.)) and simplified form using an optical microscope (BX50F4 (Olympus)) and a stereomicroscope (SZH10 (Olympus)) Observations were made. The 16S rDNA nucleotide sequence was analyzed based on BLAST homology search and molecular phylogenetic analysis based on Apollon DB-BA10.0 (Techno Suruga Lab) and international nucleotide sequence database (GenBank / DDBJ / EMBL). . The base sequences obtained from the A44 strain and the A51 strain are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. These sequences were compared with the homology rates of the top 30 database strains. The software used was Apollon 3.0 (Techno Suruga Lab). Molecular phylogenetic analysis was performed based on the comparison results, and the molecular phylogenetic positions of the A44 strain and A51 strain were confirmed.

The results of specifying the obtained actinomycetes are shown in Table 2 ( Streptomyces sp. A44 strain) and Table 3 ( Streptomyces sp. A51 strain). As a result, in comparison of homology rates, the A44 strain and A51 strain do not show a homology rate that completely matches the known species in the database, and the molecular phylogenetic position is different from any known species in the molecular phylogenetic position. showed that. The A44 strain and A51 strain molecular phylogeny analysis diagrams are shown in FIGS. 3 and 4, respectively. From these analysis results, the A44 strain and the A51 strain were identified as Streptomyces sp. Was identified. From these results, it was confirmed that both were new strains of Streptomyces.

(Example 5) Microflora of compost Bio-balance (registered trademark) Y (Bio-balance Co., Ltd., Nagoya City, Aichi Prefecture, Japan) did. After performing static deposition at 50 to 60 ° C. for one month, it was inverted and further static deposition was performed for one month to complete the fertilization. The completed compost was examined using the XMG medium for coliform bacteria and the HA medium for actinomycetes.

As a result, the coliform group was negative. Actinomycetes became 2.0 × 10 ⁵ CFU / g. This indicated that the compost was hygienic and free of coliforms that caused mastitis and bacterial diarrhea.

(Example 6) Exclusion of harmful bacteria by actinomycete culture (1) Preparation of actinomycete culture 4 g of glucose and 4 g of yeast extract were dissolved in 1 L of water to prepare a liquid medium. To 1 kg of vermiculite, 10 ml of water was added to obtain a solid medium. The liquid medium and the solid medium were each autoclaved at 121 ° C. for 15 minutes and then mixed 1: 1 to obtain an actinomycete medium. Streptomycins sp., Which has been smeared on Trypticase Soy Agar and fully grown. A44 strain and Streptomyces sp. What collected the colony of A51 stock | strain was suspended in the physiological saline, and was used as an inoculum. The inoculum was inoculated into the actinomycete medium, cultured for 28 days, and the number of actinomycetes contained in the cultured actinomycete culture was measured. The number of actinomycetes contained in the actinomycete culture obtained by culturing for 28 days was 8.9 × 10 ⁶ CFU / g.

(2) Test for elimination of harmful fungi by actinomycete cultures 0.2% or 1.0% of the actinomycete culture obtained in (1) above was mixed with Escherichia coli as harmful bacteria. The IID562 strain was inoculated at 10 ⁶ to 10 ⁷ CFU per gram. As a control group, compost not containing actinomycetes was similarly inoculated with Escherichia coli IID562 strain at 10 ⁶ to 10 ⁷ CFU per gram. Thereafter, all the compost was allowed to stand at 50 to 60 ° C. for 28 days, and Escherichia coli IID562 strain was examined using XMG medium.

The results are shown in FIG. The compost added with actinomycete cultures rapidly decreased the number of E. coli compared to the control group. Therefore, Streptomyces sp. Strain A44 and Streptomyces sp. The A51 strain was shown to have an effect of quickly eliminating E. coli contained in compost.

(Example 7) Examination of various properties of actinomycetes Based on the contents described in “Classification and identification of actinomycetes” (edited by the Japanese Society for Actinomycetes, Japan Society for Business Administration), Streptomyces sp. A44 strain and Streptomyces sp. The morphological properties, culture properties, and physiological properties of the A51 strain were examined. Streptomyces sp. Strain A44 and Streptomyces sp. The results of the A51 strain are shown in Table 4 and Table 5 below, respectively.

(Example 8) Examination of antibacterial activity of actinomycetes
Streptomyces sp. A44 strain or Streptomyces sp. The A51 strain was grown at 25 ° C. or 37 ° C. on TS agar medium or YM agar medium. Each growth medium was punched with a 5 mm straw, and agar pieces were collected. The bacteria of the subject for measuring the antibacterial activity (test bacteria), E. coli IID562, P. aeruginosa NRIC0201, S. aeruginosa . dysgalactiae NRIC 1986, S. dysgalactiae . uberis NRIC 1153, and S. cerevisiae . agalactiae NRIC1137 was used. The agar pieces were placed on the LB medium smeared with the test bacteria in advance, and the length of the growth inhibition portion was measured after 36 to 48 hours of culture.

Streptomyces sp. Strain A44 and Streptomyces sp. The results of measuring the inhibition circle for the A51 strain are shown in Table 6 and Table 7, respectively. From this result, Streptomyces sp. Strain A44 and Streptomyces sp. All of the A51 strains were shown to exhibit stronger antibacterial activity when cultured for 3 to 7 days in the middle temperature range (25 ° C.). Moreover, since the antibacterial spectrum and the strength of the antibacterial activity differed depending on the culture temperature and the culture medium, it was speculated that two or more types of antibacterial active substances were produced. Streptomyces sp. A44 strain, E. coli IID562, S. coli . dysgalactiae NRIC 1986, S. dysgalactiae . uberis NRIC 1153, and S. cerevisiae . Antibacterial activity against agalactiae NRIC1137, Streptomycins sp. A51 strain, S. uberis NRIC 1153, and S. cerevisiae . It was revealed that antibacterial activity was exhibited against agalactiae NRIC1137.

Claims (19)

Compost produced from feces of livestock animals fed a feed containing Lactobacillus delbrueckii ANTI MUFFA (FERM BP-10663) , which is an actinomycete that grows under aerobic conditions at 20-40 ° C A compost characterized in that the number is greater than the number of actinomycetes grown under aerobic conditions at a temperature lower than 20 ° C or higher than 40 ° C. The compost according to claim 1 , wherein the feed containing Lactobacillus delbrueckii ANTI MUFFA (FERM BP-10663) is Biobalance (registered trademark) Y. The compost according to claim 1 or 2 , which is negative for coliform group. The compost according to any one of claims 1 to 3 , wherein the pH is 6.0 to 9.0. The compost according to any one of claims 1 to 4 , wherein the water content is 45% or more. Compost according to any one of claims 1 to 5 , wherein no actinomycetes or materials containing actinomycetes are added. The manufacturing method of the compost of any one of Claims 1-6 . a) preparing stool water obtained from a livestock animal fed a feed containing Lactobacillus delbrueckii ANTI MUFFA (FERM BP-10663);
b) depositing the stool and standing still;
c) reversing the compost obtained by the process, and
d) settling the compost;
The manufacturing method of Claim 7 provided with these. The manufacturing method according to claim 8, wherein the inversion is performed once. The feed containing Lactobacillus delbrueckii ANTI MUFFA (FERM BP-10663) is Biobalance (registered trademark) Y, according to any one of claims 7-9. The manufacturing method as described. The production method according to any one of claims 7 to 10, wherein an actinomycete or a material containing actinomycetes is not added. a) preparing stool water obtained from a livestock animal fed a feed containing Lactobacillus delbrueckii ANTI MUFFA (FERM BP-10663);
b) depositing the stool and standing still;
c) reversing the compost obtained by the process once, and
d) settling the compost;
A method for increasing the number of actinomycetes grown under aerobic conditions in compost at 20 ° C. to 40 ° C., wherein no actinomycetes or materials containing actinomycetes are added . 13. The method according to claim 12 , characterized in that the feed containing Lactobacillus delbrueckii ANTI MUFFA (FERM BP-10663) is Biobalance (R) Y. Actinomycetes that grow under aerobic conditions at 20 ° C. to 40 ° C. are produced by Streptomyces sp. A44 strain and / or Streptomyces sp. The compost according to any one of claims 1 to 6 , comprising A51 strain . Back compost containing the compost according to any one of claims 1 to 6 and claim 14 , and a dry material. Streptomyces sp. A44 strain and / or Streptomyces sp. Comprising adding A51 strain, The method according to any one of claims 7 to claim 10. Claims 7 to claim 11, and comprising the step of mixing the resulting compost and dry material by the method according to any one of claims 16, back method for producing compost. Streptomyces sp. A44 strain and / or Streptomyces sp. The method of Claim 12 or Claim 13 including adding A51 stock | strain . Culture soil, a fertilizer, or a soil conditioner containing the compost of any one of Claims 1-6 and Claim 14 .