Name of the invention: Bacillus subtilis CJBS303 and composition comprising the same
Technical field
[One]
The present application relates to Bacillus subtilis CJBS303 having an effect of reducing odor derived from livestock manure and a composition comprising the same.
Background
[2]
Recently, odors occurring in livestock farms are a cause of environmental pollution, as well as an increase in civil complaints in neighboring villages, and diseases, and have become a major social problem. The main cause of odor in livestock farms is gas generated by anaerobic fermentation during the production of livestock manure and storage and treatment. Accordingly, the Ministry of Environment and Korea Handon Association designated 12 odor substances, including ammonia, methyl mercaptan, and hydrogen sulfide, and in 2010, the final 22 designated odor substances and emission standards were established. In addition, based on the results of analyzing 133 farms for two years from 2015 to 2016, the Korea Handy Pig Association has suggested the standard for odor management of livestock farms, and as one of these, the dilution factor of the complex odor inside the farm is 1000 times. It is demanding the following management. Here, the term “complex odor” refers to a odor that causes discomfort and disgust by stimulating a person's sense of smell while two or more odor substances are present in combination.
[3]
Ammonia (NH 3 ) , which is one of the typical harmful gas and odorous substances in pig houses , is an irritating, colorless, fluid gas that can be detected even at a relatively low concentration. Ammonia is known to be produced in the process of amino acid deamination, and other sources of ammonia are urea and nitric acid. Ammonia gas irritates the moist body tissues, sore eyes of workers and congestion of pigs' eyes. According to the study data on the effect of pig productivity according to the concentration of ammonia in the pig house, when ammonia is 50 ppm or more, about 12% of the piglets are delayed in growth, and at 100-150 ppm, 30% of pigs inhibit growth and show symptoms of respiratory disease. .
[4]
Another major odor gas is volatile sulfur compounds, such as hydrogen sulfide (H 2 S) and methyl mercaptan (CH 3 SH). These volatile sulfur compounds are produced in the process of reducing sulfates and metabolizing amino acids containing sulfur. Hydrogen sulfide is a toxic gas, and is heavier than air and is known to be laid on the surface of pig slurries.It causes headaches, dizziness, and nausea of ​​workers, and in pigs, it causes loss of appetite and respiratory diseases. In addition, odor gases from livestock farms include indole and phenols such as indole, scatol, and p-cresol, and volatile fatty acids (VOCs) such as propionic acid, butyric acid, and acetic acid. It is a necessary situation.
[5]
Under this background, the present inventors have isolated and identified a strain of Bacillus genus that can significantly reduce odor gases such as ammonia and hydrogen sulfide derived from livestock manure as a result of diligent efforts to reduce odors occurring in livestock farms. On the basis of this, the present application was completed.
[6]
[Prior technical literature]
[7]
[Patent Literature]
[8]
(Patent Document 1) Korean Patent Registration No. 10-1229865
Detailed description of the invention
Technical challenge
[9]
An object of the present application is to provide a Bacillus subtilis CJBS303 strain deposited under accession number KCCM12435P .
[10]
Another object of the present application is to provide a composition comprising the Bacillus subtilis CJBS303 strain, a culture of the strain, a concentrate of the culture, or a dried product of the concentrate.
[11]
Another object of the present application is to provide a feed composition comprising the composition.
[12]
Another object of the present application is to provide a microbial preparation for removing odor from livestock containing the Bacillus subtilis CJBS303 strain.
[13]
Another object of the present application is to provide a method for reducing odor of individual manure, comprising administering the Bacillus subtilis CJBS303 strain, the composition, or the microbial agent to the individual.
[14]
Another object of the present application is the step of culturing the Bacillus subtilis CJBS303 ( Bacillus subtilis CJBS303) strain deposited as KCCM12435P; And mixing the recovered strain with an additive.
[15]
Other objects and advantages of the present application will become more apparent by the following detailed description in conjunction with the appended claims and drawings. Contents not described in the present specification will be omitted because they can be sufficiently recognized and inferred by those skilled in the technical field or similar technical field of the present application.
Means of solving the task
[16]
Each description and embodiment disclosed in the application may be applied to each other description and embodiment. That is, all combinations of various elements disclosed in this application belong to the scope of the present application. In addition, it cannot be seen that the scope of the present application is limited by the specific description described below.
[17]
In one aspect, the present application provides a Bacillus subtilis CJBS303 strain deposited under accession number KCCM12435P .
[18]
The strain may be one having an effect of reducing odor derived from livestock manure. For example, the strain may reduce the level of at least one malodorous gas selected from the group consisting of ammonia, hydrogen sulfide, methylmercaptan, indole, phenols, and volatile fatty acids (VOCs).
[19]
As used herein, the term "malodor derived from livestock manure" may be used interchangeably with "malodor derived from livestock manure" or "malodor derived from livestock manure". The odor may be generated from malodorous gas derived from livestock manure, for example, may be generated from malodorous gas caused by anaerobic fermentation during the processing of livestock manure.
[20]
According to an embodiment, the CJBS303 strain having the highest ammonia consumption rate was selected from a total of 456 strains isolated from samples such as doenjang, red pepper paste, and meju, which are Korean traditional pastes. 16s rDNA sequencing was performed on the selected strain to confirm the nucleotide sequence of SEQ ID NO: 1, which showed 99% homology with Bacillus subtilis. Therefore, the strain was named Bacillus subtilis CJBS303, and the Bacillus subtilis CJBS303 strain was deposited with the Korea Microbial Conservation Center on February 13, 2019, and was given accession number KCCM12435P.
[21]
In one embodiment, the Bacillus subtilis CJBS303 strain may reduce the level of odor gas derived from livestock manure. The odor gas is ammonia; Hydrogen sulfide; Methyl mercaptan; Indole and phenols such as indole, scatol, and p-cresol; And volatile fatty acids (VOCs) such as propionic acid, butyric acid, and acetic acid. For example, it may be at least one odor gas selected from the group consisting of ammonia, hydrogen sulfide, methyl mercaptan, indole, phenols, and volatile fatty acids. .
[22]
According to an embodiment, it was confirmed that the addition of the Bacillus subtilis CJBS303 strain reduced the level of ammonia derived from pig fecal slurry, and the feeding of the strain also reduced the level of ammonia and hydrogen sulfide derived from manure. Accordingly, the present application can provide a new strain having an effect of reducing odor derived from livestock manure.
[23]
[24]
In another aspect, the present application provides a composition comprising the Bacillus subtilis CJBS303 strain, a culture of the strain, a concentrate of the culture, or a dried product of the concentrate.
[25]
The Bacillus subtilis CJBS303 strain is as described above.
[26]
In one embodiment, the culture of the Bacillus subtilis CJBS303 strain is inoculated with the strain in a microorganism culture medium, and prepared according to a microorganism culture method known in the art (eg, stationary culture, stirring culture). I can. In addition, concentrates of the strain culture, and dried products thereof may also be easily prepared according to a method of treating, concentrating or drying microorganisms or cultures known in the art.
[27]
According to an embodiment, feeding of the Bacillus subtilis CJBS303 strain reduces the levels of ammonia and hydrogen sulfide derived from manure, thereby contributing to the improvement of the livestock environment and the productivity of livestock. Therefore, the Bacillus subtilis CJBS303 strain, the culture of the strain, the concentrate of the culture, or the dried product of the concentrate may be included in the composition and used as a feed additive.
[28]
The composition of the present application may be prepared in powder or granular form, and if necessary, organic acids such as citric acid, humic acid, adipic acid, lactic acid, malic acid, or phosphates such as sodium phosphate, potassium phosphate, acid pyrophosphate, polyphosphate, etc. , Polyphenols, catechins, alpha-tocopherol, rosemary extract, vitamin C, green tea extract, licorice extract, chitosan, tannic acid, phytic acid, and other natural antioxidants.
[29]
The composition of the present application may include grains such as crushed or crushed wheat, oats, barley, corn and rice; Vegetable protein feeds such as feeds based on rape, soybeans, and sunflowers; Animal protein feeds such as blood meal, meat meal, bone meal and fish meal; It may further include a dry ingredient composed of sugar and dairy products, such as various milk powders and whey powder, and may further include nutritional supplements, digestion and absorption enhancers, growth promoters, and the like.
[30]
The composition of the present application may be administered to an animal alone or may be administered in combination with other feed additives in an edible carrier. In addition, the composition may be easily administered to an animal as a top dressing or directly mixed with feed or in an oral formulation separate from feed. When the composition is administered separately from feed, it can be prepared in an immediate release or sustained release formulation by combining it with a pharmaceutically acceptable edible carrier as well known in the art. Such edible carriers can be solid or liquid, for example corn starch, lactose, sucrose, soy flakes, peanut oil, olive oil, sesame oil and propylene glycol. When a solid carrier is used, the composition may be a tablet, a capsule, a powder, a troche or a sugar-containing tablet, or a top dressing in a microdispersible form. When a liquid carrier is used, the composition may be a gelatin soft capsule, or a formulation of a syrup, suspension, emulsion, or solution.
[31]
The composition of the present application may contain, for example, a preservative, a stabilizer, a wetting or emulsifying agent, a solution accelerator, a cryoprotectant, or an excipient. The cryoprotectant may be one or more selected from the group consisting of glycerol, trehalose, maltodextrin, skim milk powder and starch.
[32]
The preservative, stabilizer, or excipient may be included in the composition in an effective amount sufficient to reduce deterioration of the Bacillus subtilis CJBS303 strain contained in the composition. In addition, the cryoprotectant may be included in the composition in an effective amount sufficient to reduce the degradation of the Bacillus subtilis CJBS303 strain contained in the composition when the composition is in a dried state.
[33]
The composition may be used by salivating, spraying or mixing and adding to animal feed.
[34]
The composition of the present application can be applied to a plurality of animal diets including mammals and poultry. As the mammal, it can be used for pigs, cows, sheep, goats, laboratory animals, or pets (eg, dogs, cats), and can also be used for chickens, turkeys, ducks, geese, pheasants, and quails as poultry have.
[35]
[36]
The present application provides a feed composition comprising the composition in another aspect.
[37]
The feed composition of the present application may be formulated in the form of a conventional feed, and may include a common feed ingredient.
[38]
As used herein, the term "feed composition" refers to food fed to animals. The feed composition refers to a material that supplies organic or inorganic nutrients necessary for maintaining animal life or producing meat, milk, and the like. The feed composition may additionally contain nutrients necessary for maintaining animal life or producing meat, milk, and the like.
[39]
The content of the composition including the Bacillus subtilis CJBS303 strain, the culture of the strain, the concentrate of the culture, or a dried product of the concentrate contained in the feed composition of the present application is the type and age of the applied livestock, the application type, It can be appropriately adjusted according to the desired effect, for example, 0.01 to 1% (w/w), 0.01 to 0.5% (w/w), or 0.15 to 0.5% (w/w).
[40]
In one embodiment, the feed composition of the present application may include the Bacillus subtilis CJBS303 strain in an amount of 1.0×10 7 to 1.0×10 11 cfu/kg based on the total weight of the composition . The content of the strain is 1.0×10 7 to 5.0×10 10 , 1.0×10 7 to 1.0×10 10 , 1.0×10 7 to 5.0×10 9 , 1.0×10 7 to 1.0×10 9 , 1.0×10 7 to 5.0×10 8 , 1.0×10 7 to 1.0×10 8 , 1.0×10 7 to 5.0×10 7 , 1.0×10 8To 5.0×10 10 , 1.0×10 8 to 1.0×10 10 , 1.0×10 8 to 5.0×10 9 , 1.0×10 8 to 1.0×10 9 , 1.0×10 8 to 5.0×10 8 , 1.0×10 9 To 5.0×10 10 , 1.0×10 9 to 1.0×10 10 , or 1.0×10 9 to 5.0×10 9 cfu/kg, but appropriately adjusted according to the type and age of livestock, application form, and desired effect It is possible.
[41]
The feed composition of the present application further comprises organic acids such as citric acid, fumaric acid, adipic acid, and lactic acid for administration; Phosphates such as potassium phosphate, sodium phosphate, and polymerized phosphate; One or more of natural antioxidants such as polyphenol, catechin, tocopherol, vitamin C, green tea extract, chitosan, and tannic acid can be mixed and used, and other conventional additives such as anti-influenza agents, buffers, and bacteriostatic agents are added as needed. can do. In addition, the feed composition of the present application may be formulated into a dosage form for injection such as an aqueous solution, suspension, emulsion, capsule, granule or tablet by additionally adding a diluent, a dispersant, a surfactant, a binder or a lubricant. In addition, the feed composition of the present application includes various auxiliary agents such as amino acids, inorganic salts, vitamins, antioxidants, antifungal agents, antibacterial agents, etc. as auxiliary ingredients, and vegetable protein feed such as crushed or crushed wheat, barley, corn, blood meal, meat meal, fish meal. In addition to the main ingredients such as animal protein feed, animal fat and vegetable fat, it can be used together with nutritional supplements, growth promoters, digestion and absorption promoters, and disease prevention agents.
[42]
The feed composition of the present application may be mixed with livestock feed in an amount of about 10 to 500 g per 1 kg, for example, 10 to 100 g, based on dry weight, and supplied as a mash after thoroughly mixing, or an additional processing process Through the palletization, expansion or extrusion process can be carried out.
[43]
[44]
In another aspect, the present application provides a microbial preparation for removing odor from livestock houses including the Bacillus subtilis CJBS303 strain.
[45]
According to an embodiment, feeding of the Bacillus subtilis CJBS303 strain was able to reduce the levels of ammonia and hydrogen sulfide derived from manure, and significantly reduced ammonia, hydrogen sulfide, methylmercaptan, volatile organic compounds, and complex odors in the shed. Could be reduced. Therefore, the strain of the present application can be utilized as a microbial preparation for removing odor in livestock houses.
[46]
In one embodiment, the microbial preparation of the present application may be to reduce the level of at least one malodorous gas selected from the group consisting of ammonia, hydrogen sulfide, methyl mercaptan, volatile fatty acids, and combinations thereof.
[47]
In one embodiment, the microbial preparation of the present application may be in the form of a solid or liquid prepared according to a method known in the art. When the microbial preparation of the present application is, for example, in a solid form, after attaching the strain to a carrier, drying so that the moisture content is 0.1 to 10% by weight based on the total weight of the microbial preparation, and in the form of beads It can be commercialized or pulverized to be commercialized in powder form. As the carrier, at least one selected from the group consisting of powdery clay, activated carbon, coke, volcanic ash and combustion ash may be used, and as the clay, zeolite, vermiculite, diatomaceous earth, kaolin, pottery earth, feldspar, and clay And one or more selected from the group consisting of talc may be used, but the present invention is not limited thereto. In addition, an excipient may be added to the microbial preparation in the solid form. In the case of the excipient, amino acids, vitamin C, vitamin E, chitosan, and glucose may be used alone or in combination of two or more, but is not limited thereto.
[48]
When the microbial preparation of the present application is, for example, in a liquid form, it may be prepared by mixing the culture of the strain and mixing glucose or glycerin to stabilize the microorganism. Based on the total weight of the microbial preparation, it may be prepared so that the final concentration of the culture is 5 to 40% by weight, for example, it may be prepared so that the final concentration is 10 to 20% by weight, but is not limited thereto.
[49]
[50]
In another aspect, the present application provides a method for reducing odor of individual manure, comprising administering the Bacillus subtilis CJBS303 strain, the composition, or a microbial agent to the individual.
[51]
As used herein, the term "individual" refers to an object to reduce the odor of manure, and more specifically, may be a non-human animal. For example, the non-human animal may include mammals, and poultry, and the mammal may include pigs, cows, sheep, goats, dogs, cats, etc., and may include laboratory rodents or pets. The poultry may include, but is not limited to, chicken, turkey, duck, goose, pheasant, and quail.
[52]
[53]
In another aspect, the present application includes culturing the Bacillus subtilis CJBS303 strain deposited as KCCM12435P ; And it provides a method for producing a microbial preparation comprising; and mixing the recovered strain with an additive.
[54]
The cultivation of the strain may be performed according to a method known in the art, and the cultivation method may include at least one cultivation selected from the group consisting of batch, continuous, and fed-batch cultivation.
[55]
The medium used for the culture may be a medium capable of satisfying the requirements of a specific microorganism. The medium may be a medium selected from the group consisting of a carbon source, a nitrogen source, a trace element component, and a combination thereof.
[56]
The carbon source may be a carbon source selected from the group consisting of carbohydrates, fats, fatty acids, alcohols, organic acids, and combinations thereof. The carbohydrate may be glucose, sucrose, lactose, fructose, maltose, starch, cellulose, and combinations thereof. The fat may be soybean oil, sunflower oil, pajama oil, coconut oil, and combinations thereof. The fatty acid may be palmitic acid, stearic acid, linoleic acid, or a combination thereof. The alcohol may be glycerol or ethanol. The organic acid may include acetic acid.
[57]
The nitrogen source may include an organic nitrogen source, an inorganic nitrogen source, or a combination thereof. The organic nitrogen source may be selected from the group consisting of peptone, yeast extract, meat juice, malt extract, corn steep liquor (CSL), soybean meal, and combinations thereof. The inorganic nitrogen source may be selected from the group consisting of urea, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, ammonium nitrate, and combinations thereof.
[58]
The medium may include one selected from the group consisting of phosphorus, metal salts, amino acids, vitamins, precursors, and combinations thereof. The source of phosphorus may include potassium dihydrogen phosphate, dipotassium hydrogen phosphate, or a sodium-containing salt corresponding thereto. The metal salt may be magnesium sulfate or iron sulfate.
[59]
The medium or individual components constituting it may be added in batch, continuous or fed-batch culture.
[60]
The method of preparing the microbial preparation may further include recovering the strain from the culture obtained by culturing the strain after the step of culturing the strain. Recovery of the strain may be performed by a method known in the art, for example, may be performed by a method of recovering the precipitate generated after centrifugation of the culture.
[61]
In the method of manufacturing the microbial preparation, the additive may be a cryoprotectant, and after the step of mixing the recovered strain with the additive, it may further include a step of freeze-drying the obtained mixture. The freeze drying may be performed by a method known in the art.
[62]
The strain in the freeze-dried microbial preparation obtained in the freeze-drying step may be in a viable state. In addition, the freeze-dried microbial preparation may include a cryoprotectant in an effective amount sufficient to reduce the degradation of the strain contained in the formulation, thereby reducing the degradation of the viable strain.
[63]
In the method of manufacturing the microbial preparation, the microbial preparation may include a Bacillus subtilis CJBS303 strain of 1.0×10 7 to 1.0×10 11 cfu/kg based on the total weight of the microbial preparation , eg For example, 1.0×10 7 to 5.0×10 10 , 1.0×10 7 to 1.0×10 10 , 1.0×10 7 to 5.0×10 9 , 1.0×10 7 to 1.0×10 9 , 1.0×10 7 to 5.0× 10 8 , 1.0×10 7 to 1.0×10 8 , 1.0×10 7 to 5.0×10 7 , 1.0×10 8To 5.0×10 10 , 1.0×10 8 to 1.0×10 10 , 1.0×10 8 to 5.0×10 9 , 1.0×10 8 to 1.0×10 9 , 1.0×10 8 to 5.0×10 8 , 1.0×10 9 To 5.0×10 10 , 1.0×10 9 to 1.0×10 10 , or 1.0×10 9 to 5.0×10 9 cfu/kg of Bacillus subtilis CJBS303 strain, but the type of livestock to be applied and It can be appropriately adjusted according to age, application form, and desired effect.
Effects of the Invention
[64]
Bacillus subtilis CJBS303 strain according to the present application can effectively reduce the generation of odor gases derived from livestock manure or livestock shed.
[65]
Therefore, the composition and the microbial preparation containing the strain can not only contribute to the improvement of the livestock environment through reduction of odor, but also improve the production of livestock.
Brief description of the drawing
[66]
1 shows a result of evaluation of ammonia consumption ability according to an embodiment, and is a result of visually confirming the color development reaction of a culture medium according to whether or not ammonia is consumed.
[67]
2 is a result of quantitatively comparing the ammonia consumption rate of the CJ109, CJ251, CJ268, and CJBS303 strains cultured in ammonia-containing medium.
[68]
3 is a result of confirming the morphological characteristics of the CJBS303 strain through an electron microscope.
[69]
4 is a result of visually checking whether the CJBS303 strain is hemolytic.
[70]
5 is a result of confirming the change in the amount of ammonia generated from pig fecal slurry according to the addition of the CJBS303 strain.
[71]
6 is a result of confirming the change in the amount of ammonia and hydrogen sulfide generated from manure according to the feeding of the CJBS303 strain.
[72]
7a to 7e show the effect of reducing odor gas in the pig house according to the feeding of the CJBS303 strain, and FIG. 7a shows the level of complex odor in the pig house, FIG. 7D is a result of confirming the level of volatile fatty acids (VOCs) in the pig house, and FIG. 7E is a result of confirming the level of methyl mercaptan in the pig house over time.
[73]
8A to 8D are to verify the effect of reducing odor gas in the pig house according to the feeding of the CJBS303 strain, and FIG. 8A shows the level of ammonia in the pig house, FIG. , Figure 8d is a result of comparing the level of complex odor in the pig house.
Mode for carrying out the invention
[74]
Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.
[75]
Example 1. Isolation and identification of strains having the effect of reducing odor derived from livestock manure
[76]
(1) Securing samples and separation of strains
[77]
After securing samples of Korean traditional soybean paste, red pepper paste, and meju, dilute the sample step by step, spread it on BHI (Brain Heart Infusion, Difco) solid medium added with 3% sodium chloride, and then 37°C Incubated for 24 hours. Strains isolated from each sample were grouped and separated according to colonies. The selected colonies were purely separated by transferring them to a new medium for 3 times and culturing, and a total of 465 strains purified and cultured were stored in a medium to which 20% glycerol was added and stored at -70°C or below.
[78]
(2) Selection of strains according to evaluation of ammonia consumption ability
[79]
In order to select strains that consume ammonia, which is the main cause of odor, the ammonia consumption ability of the strains was evaluated. Ammonia consumption was confirmed by a qualitative method using a reagent (sulfanilic acid, N, N-Dimethyl-1-naphthylamine) that reacts with nitrous acid, an oxide of ammonia. Specifically, ammonia-added ion medium (4.95 g/L of (NH 4 ) 2 SO 4 , 8.82 g/L of K 2 HPO 4 , 1.1 ml/L of 1M MgSO 4 solution, 0.3 ml/L of 1M CaCl 2 solution, 0.5 ml/L of 30 mM FeSO 4 solution, 0.04 ml/L of 50 mM CuSO 4 solution, 0.7 g/L of NaH 2 PO 4 , and 12 ml/L of 5% (W/V) anhydrous Na 2 CO 3Including) was prepared, the strains were inoculated at a concentration of 0.01%, respectively, in the prepared 10 ml of the medium, and cultured at 30° C. for 14 days. Thereafter, the culture solution was centrifuged to collect 1 ml of the supernatant, respectively, and then 100 μl of the indicator for color development with nitrous acid was added thereto. The culture supernatant to which the indicator was added was reacted at 25° C. for 10 minutes to induce a color reaction, and then the color development of the culture solution was observed. Meanwhile, Bacillus subtilis KCCM11143P known in Korean Patent Application Publication No. 10-2012-0088436 was used as the standard strain in the following examples.
[80]
As shown in FIG. 1, when nitrous acid was produced due to consumption or oxidation of ammonia, the culture medium was colored dark purple, and based on this color reaction, strains having ammonia consumption ability were primarily selected. As a result, four strains having significant ammonia consumption ability, specifically CJ109, CJ251, CJ268, and CJBS303, were selected out of a total of 456 strains isolated from the sample.
[81]
In addition, for the four strains primarily selected, their consumption of ammonia was quantitatively compared. Specifically, after 0.1% of the strain was inoculated into the BHI liquid medium, it was cultured at 37° C. at 200 rpm for 15 hours to activate the strain. Ammonia-containing medium (0.5g/L (NH 4 ) 2 SO 4 , 13.5 g/L NaH 2 PO 4 , 0.7 g/L K 2 HPO 4 , 0.1 g/L MgSO 4 ㆍ7H 2 O, 0.18 g/L of CaCl 2 ㆍ2H 2 O, 0.5 g/L of NaHCO 3 , 0.014 g/L of FeCl 3 ㆍ6H 2O, including 0.5 g/L glucose) was inoculated with 1% of the activated strain, and then cultured at 37° C. at 200 rpm for 6 hours. After the culture was completed, the culture solution was centrifuged, and only the culture supernatant was collected therefrom. The ammonia content in the initial medium and the ammonia residual amount in the recovered culture supernatant were quantified, and the ammonia consumption rate was calculated according to Equation 1.
[82]
[Equation 1]
[83]
Ammonia consumption rate (%) = (AB / A) Х 100
[84]
(A: initial ammonia content, B: ammonia content after completion of culture)
[85]
As a result, as shown in FIG. 2, the ammonia consumption rate of the CJBS303 strain was observed to be the highest at 74.3% among the four strains that were primarily selected. Therefore, CJBS303, which has the highest ammonia consumption rate, was finally selected as a strain having the effect of reducing odor derived from livestock manure.
[86]
(3) Morphological and biochemical characteristics analysis
[87]
Morphological and biochemical characteristics were analyzed for the CJBS303 strain. Specifically, the morphological characteristics of the strain were analyzed through Gram staining and electron microscopic observation, and the biochemical characteristics of the strain were determined by confirming the sugar fermentation pattern of the strain through the API 50 CHB system (biomerieux Vitek, Inc, France). Confirmed.
[88]
As a result, it was confirmed that the CJBS303 strain was a Gram-positive strain, and as shown in FIG. 3, it was a bacillus. In addition, as shown in Table 1, it was confirmed that the CJBS303 strain exhibited a sugar fermentation pattern similar to Bacillus subtilis.
[89]
[Table 1]
sample Fermentation sample Fermentation
Control - Esculine +
Glycerol + Salicine +
Erythritol - Cellobiose +
D-Arabinose - Maltose +
L-Arabinose - Lactose +
Ribose + Melibiose -
D-Xylose + Saccharose +
L-Xylose - Trehalose +
Adonitol - Inuline -
ß Methyl-xyloside - Melezitose -
Galactose - D-Raffinose +
D-Glucose + Amidon -
D-Fructose + Glycogene -
D-Mannose + Xylitol -
L-Sorbose - ß Gentiobiose -
Rhamnose - D-Turanose -
Dulcitol - D-Lyxose -
Inositol + D-Tagatose -
Mannitol + D-Fucose -
Sorbitol + L-Fucose -
α Methyl-D-mannoside - D-Arabitol -
α Methyl-glucoside + L-Arabitol -
N Acetyl glucosamine - Gluconate -
Amygdaline + 2 keto-gluconate -
Arbutine + 5 keto-gluconate -
[90]
(4) Identification of strain
[91]
Molecular taxonomic analysis by DNA sequence was performed to identify the strain. For this, PCR premix (Bionia, Korea), universal primer 27F (5' AGAGTTTGATCMTGGCTCAG 3'), and 1492R (5' GGTTACCTTGTTACGACTT 3') were used to amplify the gene of 16s rDNA. During the gene amplification, the total reaction solution was set to 20 µl, and a total of 30 times was repeated under conditions of 94°C for 1 minute, 56°C for 1 minute, and 72°C for 1 minute. The 16s rDNA nucleotide sequence of the CJBS303 strain was the same as SEQ ID NO: 1, which showed 99% homology with Bacillus subtilis. In the following examples, the isolated strain was named'Bacillus subtilis CJBS303' or'CJBS303 strain' (accession number KCCM12435P).
[92]
Example 2. Safety evaluation of CJBS303 strain
[93]
In order to confirm the safety of the CJBS303 strain, the hemolytic properties of the strain were evaluated. Specifically, after linear inoculation (streaking) the CJBS303 strain on a blood agar plate medium (sheep blood 5%, Hanil Comed, Korea), it was cultured at 37°C for 24 hours. Then, whether or not hemolysis in the cultured medium was observed with the naked eye.
[94]
As a result, as shown in FIG. 4, the CJBS303 strain did not show hemolytic properties, and through this, the safety of the Bacillus subtilis CJBS303 strain was confirmed.
[95]
Example 3. Evaluation of odor reduction effect of livestock manure of CJBS303 strain
[96]
(1) Effect of reducing odor gas in fecal slurry
[97]
To compare the effect of reducing the odor gas in the pig fecal slurry of CJBS303 strain with the standard strain. Specifically, after culturing the CJBS303 strain in TSB (trypticase soy broth) medium, 4 ml of a solution containing 1 Х10 8 cfu/kg of CJBS303 strain was added to 400 ml of pig manure slurry, and this was performed at 120 rpm and 39° C. Shake culture was carried out for a total of 48 hours under conditions. At 24 hours and 48 hours from the time of incubation, the amount of ammonia gas was measured using a portable odor gas measuring device (MultiRAE) over a total of two times. The total amount of ammonia was compared by summing the gas amount measured twice for each treatment.
[98]
As a result, as shown in FIG. 5, the CJBS303 strain reduced the total amount of ammonia gas generated by 41% compared to the control without addition, which had a reduction effect of more than 4 times compared to the standard strain.
[99]
(2) Reduction of odor gas from manure
[100]
When pigs were fed with a probiotic containing the CJBS303 strain, the purpose of this study was to determine the effect on the amount of odor gas derived from manure. A total of 32 finishing pigs were classified into two groups and a feeding test was conducted. The group fed the additive-free feed was set as the control, and the control feed was fed to 4 pens of fattening pigs at 4 heads per pen for 5 weeks. On the other hand, the group fed with the feed containing the CJBS303 strain of 1x10 9 cfu/kg in the feed was set as a treatment group, and the feed was fed to 4 heads per pen for a total of 4 pens for 5 weeks. From the 2nd week of feeding, manure from the control and treatment groups was sampled weekly, and odor gases generated therefrom were measured. After mixing the manure sampled from each of the control and treatment groups at a certain ratio (600g feces + 1.2L diluted urine 12.5%), it was subdivided into 300ml bottles and cultured for a total of 42 hours at 39°C under aerobic conditions. At 12 hours, 18 hours, 36 hours, and 42 hours from the time of incubation, the amount of gas generated over a total of 4 times, that is, hydrogen sulfide and ammonia gas, was measured using a portable odor gas measuring device (MultiRAE). The manure cultivation experiment was carried out each time the manure was sampled every week, and data up to the 5th week were averaged to calculate the reduction rate compared to the control.
[101]
As a result, as shown in Table 2 and Figure 6, the treatment group was observed to have a lower concentration of ammonia and hydrogen sulfide gas compared to the control group from the 2nd week of feeding, and when the concentration of the gas generated up to the 5th week was averaged and compared, the CJBS303 strain It was confirmed that the reduction rate of odor gas according to the salary was 38.3% for ammonia and 88.9% for hydrogen sulfide.
[102]
[Table 2]
　 Ammonia (ppm) Hydrogen sulfide (ppm)
　 Control CJBS303 0.1% Control CJBS303 0.1%
Week 2 207 140 286 14
Week 3 278 152 166 22
Week 4 212 135 52 20
Week 5 180 113 91 10
Average 219 135 149 16
Reduction rate compared to control 　 38.30% 　 88.90%
[103]
(3) Effect of reducing odor gas in pig house
[104]
When pigs were fed with a probiotic containing the CJBS303 strain, it was attempted to determine the effect on the amount of odor gas generated in the pig house. A total of 96 heads of 74-day-old finishing pigs were classified into two groups and a feeding test was conducted. The group fed the additive-free feed was set as the control, and the control feed was fed to 4 heads per pen, 12 pens in total, for 4 weeks. On the other hand, the group fed with the feed containing the CJBS303 strain of 1 × 10 9 cfu/kg in the feed was set as a treatment group, and the feed was fed to a total of 12 pen breeding pigs at 4 heads per pen for 4 weeks. The control and treatment were raised in separate pig houses, and in order to exclude the effect of the existing slurry in the pig house, the slurry of the control and treatment pigs was simultaneously emptied before the start of the test and this test was conducted. From 4 days before the start of the test, gas in the pig house was measured at 9 am and 4 pm every day for control and treatment pigs. Ammonia, hydrogen sulfide, complex odor, and volatile fatty acids (VOCs) were measured using a precision odor measuring instrument (Odor Catch, SLC-OP-1350, Science and Technology Analysis Center), and methylmercaptan was measured using a portable odor gas measuring device (MultiRAE). ) Was used, and the data measured every day were calculated for each week.
[105]
As a result, as shown in Table 3 and FIGS. 7a to 7e, in all measurement items, the effect of reducing odor gas according to the feeding of the CJBS303 strain was observed, and this effect was more pronounced at 4 weeks after feeding each feed. . In particular, hydrogen sulfide and methyl mercaptan, which are sulfur-based odor gases, were reduced by more than 50% (Duncan, p>0.01), and complex odors decreased by more than 20% (Duncan, p>0.05), and ammonia and volatile fatty acids were also reduced. It showed a reduction rate of 17% and 20%, respectively.
[106]
[Table 3]
　 Complex odor (OU) Ammonia (ppm) Hydrogen sulfide (ppm) VOCs (ppm) Methylmercaptan (ppm)
Control 591 a 7.7 0.88 a 2.13 1.6 a
CJBS303 456 b 6.4 0.41 b 1.7 0.6 b
Reduction rate (%) 22.80% 17.40% 53.70% 20.10% 60.30%
[107]
Example 4. Evaluation of pig farming productivity according to feeding of CJBS303 strain
[108]
When pigs were fed with a probiotic containing the CJBS303 strain, it was attempted to determine the effect on pig pig productivity. Under the same conditions as in Example 3 (3), a total of 96 74-day-old finishing pigs were classified into two groups to conduct a feeding test. Before the start of the feeding test and after the end of the test at the 4th week, the average daily gain (ADG) was calculated by measuring the individual's body weight, and the average daily feed intake (ADFI) and feed were measured by measuring the daily feed feed amount and the remaining amount. The efficiency (feed conversion ratio, FCR) was calculated.
[109]
As a result, as shown in Table 4, the treatment group fed with the CJBS303 strain improved the daily feed intake by 7.3% (Duncan, p<0.1) and the daily weight gain by 9.0% (Duncan, p<0.03) compared to the control group. FCR also improved by 0.03. These experimental results indicate that feeding of the CJBS303 strain not only reduces the amount of odor gas, but also shows a positive effect on pig farming productivity.
[110]
[Table 4]
　　 Control CJBS303
BW(kg) Initiate 38.69 38.66
Week 4 63.77 66.01
ADG(kg) 0.90 b 0.98 a
ADFI(kg) 2.35 b 2.52 a
FCR 2.62 2.59
[111]
Example 5. Verification of the effect of reducing odor gas in pig farms according to the feeding of CJBS303 strain
[112]
In order to confirm the reproducibility of the effect of reducing odor gas in the pig house according to the feeding of CJBS303 strain, a feeding test was conducted on two pig farms. The first farmhouse (A) was selected as a growing pig house (A-1) and a finishing pig house (A-2), and the second farmhouse (B) was selected as a finishing pig house, and the number of test subjects and individuals for each farm is shown in Table 5.
[113]
[Table 5]
　 A-1 A-2 B
Test subject Upbringing money Fattening money Fattening money
Population 650 heads 800 heads 944 heads
[114]
Both farms were fed a test feed containing 2x10 9 cfu/kg of CJBS303 strain in feed to growing or finishing pigs for 3 weeks. Ammonia, hydrogen sulfide, and methyl mercaptan were measured using a portable odor measuring device (MultiRAE), and the complex odor was measured at 9 am using a precision odor measuring instrument (Odor Catch, SLC-OP-1350, Science and Technology Analysis Center). It was measured at.
[115]
As a result, as shown in Figures 8a to 8d, in the treatment group fed with the CJBS303 strain, ammonia was 39% or more, hydrogen sulfide was 26% or more, methylmercaptan was 24% or more, and complex odor was reduced by 21% or more. In addition, this odor gas reduction effect could be confirmed from the third week of salary regardless of the growing stage, which is the growth stage of pig farming, or the fattening period.
[116]
The above description of the present invention is for illustrative purposes only, and those of ordinary skill in the art to which the present invention pertains will be able to understand that other specific forms can be easily modified without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative and non-limiting in all respects.
[117]
[118]
Name of deposit institution: Korea Microorganism Conservation Center (overseas)
[119]
Accession number: KCCM12435P
[120]
Consignment Date: 20190213
[121]

[122]
[123]
[124]
Claims
[Claim 1]
Bacillus subtilis CJBS303 ( Bacillus subtilis CJBS303) strain deposited with accession number KCCM12435P
[Claim 2]
The method according to claim 1, wherein the strain has an effect of reducing odor derived from livestock manure, Bacillus subtilis CJBS303 strain.
[Claim 3]
The method according to claim 1, wherein the strain is to reduce the level of at least one malodorous gas selected from the group consisting of ammonia, hydrogen sulfide, methyl mercaptan, indole, phenols, and volatile fatty acids (VOCs), Bacillus subtilis CJBS303 strain .
[Claim 4]
A composition comprising the Bacillus subtilis CJBS303 strain of claim 1, a culture of the strain, a concentrate of the culture, or a dried product of the concentrate.
[Claim 5]
The composition of claim 4, wherein the composition further comprises a cryoprotectant or excipient.
[Claim 6]
The composition of claim 5, wherein the cryoprotectant is at least one selected from the group consisting of glycerol, trehalose, maltodextrin, skim milk powder and starch.
[Claim 7]
A feed composition comprising the composition of claim 4.
[Claim 8]
The feed composition according to claim 7, comprising a Bacillus subtilis CJBS303 strain of 1.0×10 7 to 1.0×10 11 cfu/kg based on the total weight of the composition.
[Claim 9]
Bacillus subtilis CJBS303 strain of claim 1, comprising a microbial preparation for removal of odor in cattle.
[Claim 10]
The method of claim 6, wherein the microbial preparation is to reduce the level of at least one odor gas selected from the group consisting of ammonia, hydrogen sulfide, methyl mercaptan, indole, phenols, and volatile fatty acids (VOCs).
[Claim 11]
A method for reducing odor of individual manure, comprising administering to the individual the strain, composition, or microbial preparation of any one of claims 1 to 10.
[Claim 12]
Culturing the Bacillus subtilis CJBS303 strain deposited as KCCM12435P; And mixing the recovered strain with an additive.
[Claim 13]
The method of claim 12, wherein the additive is a cryoprotectant, and further comprising freeze drying the mixture obtained after the mixing step.
[Claim 14]
The method of claim 13, wherein the strain in the freeze-dried microbial preparation obtained in the freeze-drying step is in a viable state.
[Claim 15]
The method of claim 12, wherein the microbial preparation comprises a Bacillus subtilis CJBS303 strain of 1.0×10 7 to 1.0×10 11 cfu/kg based on the total weight of the microbial preparation.