KR102608235B1 - Probiotic composition for improving environment of livestock barn - Google Patents

Abstract

One embodiment of the present invention relates to a probiotic composition for improving the livestock environment containing a culture medium of the strain KBNP-BS3 (Bacillus Subtilis KBNP-BS3; KCTC 18446P), which has a deodorizing effect on livestock odor-causing substances and an antibacterial activity against pathogenic microorganisms. .
Through this, the present invention has a high deodorizing ability against livestock odor-causing substances, a wide antibacterial spectrum against harmful bacteria, and excellent antibacterial activity, so that it can efficiently improve the livestock environment, strengthen the immunity of industrial animals, and improve productivity. A probiotic composition for environmental improvement is provided. Furthermore, the probiotic composition for improving the livestock environment of the present invention can reduce the mortality rate and disease incidence of livestock, increase the efficiency of improving livestock productivity, and increase the efficiency of improving livestock productivity through the effect of removing substances that cause livestock odor and the antibacterial effect on harmful bacteria. It helps reduce civil complaints by reducing the level of pollution.

Description

Probiotic composition for improving livestock environment {PROBIOTIC COMPOSITION FOR IMPROVING ENVIRONMENT OF LIVESTOCK BARN}

The present invention relates to a probiotic composition for improving the livestock environment, which has a deodorizing effect against substances causing livestock odor and an antibacterial effect against pathogenic microorganisms.

The Ministry of Food, Agriculture, Forestry and Fisheries has been pursuing a policy to reduce antibiotics used in compound feed manufacturing since 2005. Accordingly, the use of antibiotics in compounded feeds has been completely banned from July 2011 in accordance with the scope and standards of hazardous feeds (Ministry of Agriculture, Food and Rural Affairs Notification No. 2010-142, December 27, 2010) as a revised notice related to the Feed Management Act.

In order to respond to this policy, a plan is being used to introduce alternative raw materials that can replace antibiotics in feed, but these alternative raw materials have the disadvantage of being very dependent on imports and having high costs, making it difficult to generate profits. These economic difficulties are leading to a worsening of the management balance of livestock farms.

In addition, the odor inside the livestock shed generated during the mass and dense breeding process of livestock is another factor that increases the difficulty of livestock farm management. The causes of bad odors in livestock houses are mainly harmful gases and odor-causing substances produced by microorganisms in manure.

Examples of odor-causing substances in livestock houses include ammonia, trimethylamine, and hydrogen sulfide. These odor-causing substances pollute the soil and air, causing physical and mental damage to people living in the area around the livestock farm, and are a factor in raising complaints about the operation of the livestock farm. In addition, odor-causing substances also cause diseases such as bloodshot eyes, respiratory irritation, pneumonia, loss of appetite, and dermatitis in livestock, resulting in a decrease in productivity in the livestock industry as a whole. In particular, the ammonia concentration in the livestock shed shows a correlation with the mortality rate and respiratory disease incidence of livestock, and it is thought that reducing it will have the effect of lowering the livestock mortality rate and respiratory disease prevention rate and increasing productivity.

Regarding odor-causing substances in livestock farms, the Ministry of Environment is continuously strengthening livestock manure and odor discharge standards for livestock farms through the odor prevention law and recommendations for designation standards for livestock breeding restricted areas. In order to respond to these policies, breeding technologies that utilize livestock manure as resources and animal welfare are being proposed in the livestock industry, but applying them to actual sites requires very high investment costs, making it difficult for them to be widely popularized.

Therefore, it is possible to reduce the cost and cost of popularization to a realistic level, enhance the immunity of livestock, prevent contamination of the area around livestock farms, and at the same time effectively reduce the mortality and disease incidence rates of livestock, thereby contributing to the creation of profits for livestock farms. The demand for existing technologies is increasing.

One object of the present invention is a probiotic for improving the livestock environment, which has a high deodorizing ability against livestock odor-causing substances, has a wide antibacterial spectrum against harmful bacteria, has excellent antibacterial activity, and is effective in enhancing the immunity of industrial animals and improving livestock productivity. A composition is provided.

One embodiment of the present invention is a probiotic for improving the livestock environment containing a culture medium of the Bacillus Subtilis KBNP-BS3 (KCTC 18446P) strain, which has a deodorizing ability against livestock odor-causing substances and an antibacterial activity against pathogenic microorganisms. It relates to composition.

The culture medium is one or more species selected from the group consisting of Bacillus Licheniformis KBNP-DJ1 (KCTC 18447P) and Pichia Kudriavzevii KBNP-BF5 (KCTC 18445P) It may further include culture medium of the strain.

The probiotic composition for improving the livestock environment may further include one or more natural products selected from the group consisting of green tea leaves, mugwort, and turmeric.

The substance causing the livestock odor may be one or more of ammonia, trimethylamine, hydrogen sulfide, and methyl mercaptan.

The pathogenic microorganism may be one or more of Bacillus cereus, Pseudomonas aeruginosa, and Salmonella enteritidis.

The present invention has a high deodorizing ability against substances causing livestock odor, has a wide antibacterial spectrum against harmful bacteria, and has excellent antibacterial activity, so it can efficiently improve the livestock environment, strengthen the immunity of industrial animals, and improve livestock productivity. A probiotic composition for improvement is provided.

Figure 1 shows an experiment evaluating livestock environment improvement in Examples and Comparative Examples of the present invention.
Figure 2 shows the results of PCR confirmation prepared in Preparation Example 1 of the present invention.
Figure 3 shows the results of phylogenetic tree analysis of the strain isolated in Preparation Example 1 of the present invention.
Figure 4 shows the results of Gram staining of the strain isolated in Preparation Example 1 of the present invention.
Figure 5 shows the apo staining results of the strain isolated in Preparation Example 1 of the present invention.
Figure 6 is an SEM image of the strain isolated in Preparation Example 1 of the present invention.
Figure 7 shows the results of API 50 CH strip analysis of the strain isolated in Preparation Example 1 of the present invention.
Figure 8 shows the results of enzyme activity analysis of the strain isolated in Preparation Example 1 of the present invention.

One embodiment of the present invention is a probiotic for improving the livestock environment containing a culture medium of the strain Bacillus Subtilis KBNP-BS3 (KCTC 18446P), which has a deodorizing effect against livestock odor-causing substances and an antibacterial activity against pathogenic microorganisms. It relates to composition.

Through this, the present invention provides a probiotic composition for improving the livestock environment that can enhance the immunity and productivity of livestock, has a high deodorizing ability against livestock odor-causing substances, has a wide antibacterial spectrum against harmful bacteria, and has excellent antibacterial activity. . Furthermore, the probiotic composition for improving the livestock environment of the present invention not only has the effect of removing substances causing livestock odor and has an antibacterial effect against harmful bacteria as a probiotic, but can also be applied as a livestock environment improvement agent at the same time. Through this, it is possible to lower the mortality and disease incidence rates of livestock, increase the efficiency of improving livestock productivity, and reduce the level of soil and air pollution, helping to reduce civil complaints.

The probiotic composition containing the culture medium of the Bacillus subtilus KBNP-BS3 strain of the present invention is fed as a feed additive or supplementary feed to livestock, thereby enhancing the digestive power and immunity of livestock and improving resistance to harmful microorganisms to increase livestock productivity. You can. In addition, the probiotic composition of the present invention is characterized in that it can be used as a probiotic composition for improving the livestock environment by acting on livestock manure through its deodorizing ability against substances causing livestock odor and antibacterial activity against pathogenic microorganisms.

In the present invention, in order to identify microorganisms that can be used as a livestock environment improvement agent, Cheonggukjang is suspended in sterilized saline solution, diluted, smeared on NA agar medium, and the colonies formed after culturing at 30°C for 24 hours are subcultured to the livestock farm. A strain with excellent deodorizing ability against odor-causing substances and antibacterial activity against pathogenic microorganisms was purified. The strain isolated as described above was identified as a new strain belonging to Bacillus subtilus, and the present inventors named the selected strain as Bacillus subtilus KBNP-BS3 and sent it to the Microbial Resource Center of the Korea Research Institute of Bioscience and Biotechnology in 2016. It was deposited on the 17th of March and was given the accession number KCTC18446P.

The Bacillus subtilus KBNP-BS3 strain is a strain isolated from Cheonggukjang, and was selected through a non-pathogenicity screening test using blood agar and a livestock probiotic activity screening test (enzyme activity for amylase, protease, cellulase, etc.). It has been done.

This Bacillus subtilis KBNP-BS3 strain was confirmed to be a congeneric strain with more than 99% homology to Bacillus Subtilis through phylogenetic, morphological, and physiological characterization results.

For the phylogenetic characteristics analysis, 16S rDNA sequence analysis and phylogenetic tree analysis using PCR were performed. For the morphological characteristics analysis, Gram staining, endospore staining, and scanning electron microscopy (SEM) imaging methods were performed. For the physiological characteristics analysis, API® 50 CH strip kit analysis method, antibiotic susceptibility test method, enzyme activity analysis method, and fatty acid analysis method for strain identification were performed. As such, analysis of the Bacillus subtilus KBNP-BS3 strain was performed under the same conditions as the method described in Preparation Example 1.

This Bacillus subtilus KBNP-BS3 strain, identified by the above-described analysis methods, has excellent antibacterial activity against pathogenic microorganisms (harmful bacteria) and a deodorization rate against substances that cause livestock odor. By using this Bacillus subtilus KBNP-BS3 strain as a probiotic composition for improving the livestock environment, the present invention can reduce the distribution cost and manufacturing cost, and take advantage of both a probiotic and a livestock environment improvement agent.

The livestock odor-causing substance may be a compound generally known as a livestock odor-causing substance, and its type is not limited. Specifically, the substance causing the livestock odor may be one or more of ammonia, trimethylamine, hydrogen sulfide, and methyl mercaptan.

In one embodiment, the Bacillus subtilus KBNP-BS3 strain can achieve a deodorization rate of 40% or more for all ammonia, trimethylamine, hydrogen sulfide, and methyl mercaptan. In another embodiment, the Bacillus subtilus KBNP-BS3 strain can achieve a deodorization rate of more than 96% for all ammonia, trimethylamine, and hydrogen sulfide, and at the same time, a deodorization rate for methyl mercaptan of more than 40%. In another embodiment, the Bacillus subtilus KBNP-BS3 strain achieves a deodorization rate of more than 99% for both ammonia and hydrogen sulfide, and at the same time has a deodorization rate of more than 96% for trimethylamine and a deodorization rate for methyl mercaptan of 40%. The above can be implemented. By including the culture medium of the Bacillus subtilus KBNP-BS3 strain, the probiotic composition for improving the livestock environment is excellent in deodorizing various types of livestock odor-causing substances and is more effective in preventing soil and air pollution around the livestock barn. Sheep productivity can be increased by lowering the mortality and disease incidence rates caused by irritants such as ammonia.

In addition, the Bacillus subtilus KBNP-BS3 strain contains ester hydrolase C4 (esterase), ester-lipid hydrolase C8 (esterase-lipase), acid phosphatase, naphthol-AS-Bi- It may have activity against one or more of Naphthol-AS-BI-phosphohydrolase and alpha-glucosidase. By including the culture medium of this Bacillus subtilus KBNP-BS3 strain, the probiotic composition for improving the livestock environment can improve the intestinal flora, improve the digestive power of livestock, and enhance immunity. In addition, at the same time, the enzyme activity can be used to additionally decompose volatile ester compounds, volatile fatty acids, and acidic phosphoric acids generated in livestock manure, thereby reducing a wider variety of livestock odors and disease-causing irritants with high efficiency. You can.

In one embodiment, the Bacillus subtilus KBNP-BS3 strain contains esterase C4 (esterase), esterase-lipase C8, acid phosphatase, naphthol-AS- The active effect on both Naphthol-AS-BI-phosphohydrolase and α-glucosidase can be achieved simultaneously. When containing the culture medium of this Bacillus subtilus KBNP-BS3 strain, the probiotic composition for improving the livestock environment has the effect of deodorizing a wider variety of livestock odor-causing substances, has a greater effect on preventing soil and air pollution around the livestock barn, and ammonia. It can achieve the effect of increasing livestock productivity by lowering the mortality and disease incidence rates caused by irritants such as irritants.

The pathogenic microorganism may be one or more of Bacillus cereus, Pseudomonas aeruginosa, and Salmonella enteritidis. By including the culture medium of the Bacillus subtilus KBNP-BS3 strain, the probiotic composition for improving the livestock environment can act antagonistically on pathogenic microorganisms (harmful bacteria) to improve intestinal flora and exert an antibacterial effect. Through this, the probiotic composition for improving the livestock environment can prevent food poisoning, diarrhea, vomiting, Pseudomonas aeruginosa infection, acute enteritis, etc., which infect livestock with weakened immunity, and enhance the immunity of livestock.

The pathogenic microorganisms (harmful bacteria) may be, for example, Bacillus cereus KCTC1012, Pseudomonas aeruginosa KCTC1750, and Salmonella enteritidis KCCM12400.

In one embodiment, the probiotic composition for improving the livestock environment containing the culture medium of the Bacillus subtilus KBNP-BS3 strain is effective against all of Bacillus cereus, Pseudomonas aeruginosa, and Salmonella enteritidis. The activation effect can be implemented simultaneously. In this case, the probiotic composition for improving the livestock environment has a wide antibacterial spectrum, so it can achieve a better effect of improving the livestock environment and preventing livestock diseases.

The culture medium of the Bacillus Subtilis KBNP-BS3 (KCTC 18446P) strain of the present invention is obtained by inoculating the above-described Bacillus Subtilis KBNP-BS3 (Bacillus Subtilis KBNP-BS3; KCTC 18446P) strain into the medium. This can be obtained by subculturing.

The medium may be, for example, one or more of TSB (Tryptic Soy broth), BHI (Brain Heart Infusion), LB (Luria Bertani), MRS medium, and NB (Nutrient broth). In this case, the growth efficiency of the Bacillus subtilus KBNP-BS3 strain can be increased.

The medium may be cultured, for example, at pH 5 to pH 9 and 30°C to 42°C for 24 to 72 hours. These culture conditions are very effective in improving strain yield. Through this, the productivity and cost-effectiveness of the probiotic composition for improving livestock environment can be further improved.

In one embodiment, the culture medium may be obtained by culturing in TSB (Tryptic Soy broth) medium at pH 7 and 37°C for 48 hours. Within the above range of culture conditions, strain yield and viable cell count can be increased to a very excellent level.

The culture medium is one or more species selected from the group consisting of Bacillus Licheniformis KBNP-DJ1 (KCTC 18447P) and Pichia Kudriavzevii KBNP-BF5 (KCTC 18445P) It may further include culture medium of the strain.

In the present invention, in order to identify microorganisms that can be used as a livestock environment improvement agent and do not have the effect of inhibiting the activity of Bacillus subtilus KBNP-BS3, Cheonggukjang and Nuruk were each suspended in sterile saline solution and diluted, then placed on NA agar medium. and subcultured the colonies formed after culturing for 24 hours at 30°C. It has excellent deodorizing and antibacterial activity against pathogenic microorganisms against livestock odor-causing substances, and has no antagonistic inhibitory effect with Bacillus subtilus KBNP-BS3. The strain was pure isolated.

Among the strains isolated above, the strain isolated from Cheonggukjang was identified as a new strain belonging to Bacillus licheniformis, and the present inventors named the selected strain as Bacillus licheniformis KBNP-DJ1, and Korea Biotechnology It was deposited at the Research Institute's Microbial Resource Center on February 17, 2016, and was given accession number KCTC18447P.

In addition, among the strains isolated as above, the strain isolated from yeast was identified as a new strain belonging to Pichia kudriabsvi, and the present inventors named the strain selected as Pichia kudriapsvi KBNP-BF5. , was deposited at the Microbial Resource Center of the Korea Research Institute of Bioscience and Biotechnology on February 17, 2016, and was given the accession number KCTC18445P.

The Bacillus Licheniformis KBNP-DJ1 (KCTC 18447P) strain is isolated from Cheonggukjang and was selected through a protease screening experiment using skim milk agar medium. This Bacillus subtilus KBNP-BF5 strain is an isogenic strain with more than 99% homology to Bacillus Licheniformis KBNP-DJ1 (Bacillus Licheniformis) through phylogenetic, morphological, and physiological characterization results. It has been confirmed that

The Bacillus Licheniformis KBNP-DJ1 (KCTC 18447P) strain has excellent protease activity and deodorizing effects on substances that cause livestock odor. By using this Bacillus Licheniformis KBNP-DJ1 (KCTC 18447P) strain as a probiotic composition for improving livestock environment, the present invention can achieve better probiotic effect and improved livestock environment.

The Pichia Kudriavzevii KBNP-BF5 (KCTC 18445P) strain is a strain isolated from yeast, and through phylogenetic and physiological characterization results, Pichia Kudriavzevii (Pichia Kudriavzevii) ) was confirmed to be a congeneric strain with more than 99% homology.

The Pichia Kudriavzevii KBNP-BF5 (KCTC 18445P) strain has excellent antibacterial activity against harmful bacteria and a deodorizing effect against substances that cause livestock odor. By using this Pichia Kudriavzevii KBNP-BF5 (KCTC 18445P) strain as a probiotic composition for improving the livestock environment, the present invention can achieve a more excellent probiotic effect and improved livestock environment.

In one embodiment, the probiotic composition for improving livestock environment of the present invention is Bacillus Subtilis KBNP-BS3 (KCTC 18446P) strain, Bacillus Licheniformis KBNP-DJ1 (Bacillus Licheniformis KBNP-DJ1; KCTC) 18447P) strain and Pichia Kudriavzevii KBNP-BF5 (KCTC 18445P) strain. In this case, not only is the antibacterial effect and protease activity effect against harmful bacteria more excellent, but it can also deodorize a wider variety of livestock odor-causing substances, and is more effective in preventing soil and air pollution around the livestock barn, and is more effective in preventing irritants such as ammonia. Sheep productivity can be increased by lowering the mortality and disease incidence rates caused by substances.

The probiotic composition for improving the livestock environment may further include one or more natural products selected from the group consisting of green tea leaves, mugwort, and turmeric. In this case, by using natural products as a probiotic composition for improving the livestock environment, high-quality, eco-friendly livestock products can be produced. Through this, it is possible to satisfy consumers' desire to consume safe agricultural products, while at the same time realizing the effects of improving the livestock environment, strengthening the immunity of industrial animals, and improving productivity as described above, contributing to increasing the income of livestock farmers.

In one embodiment, when the probiotic composition for improving the livestock environment includes Bacillus subtilus KBNP-BS3 strain and turmeric, the deodorizing ability for ammonia and triethylamine can be achieved at an excellent level at the same time. In another embodiment, when the probiotic composition for improving the livestock environment includes Bacillus subtilus KBNP-BS3 strain and green tea leaves, it can simultaneously achieve an excellent level of deodorizing ability against ammonia, triethylamine, and methyl mercaptan. In another embodiment, when the probiotic composition for improving the livestock environment includes Bacillus subtilus KBNP-BS3 strain and Artemisia mugwort, deodorization ability against ammonia, triethylamine, hydrogen sulfide, and methyl mercaptan can be achieved at an excellent level at the same time. there is.

Example

Hereinafter, the configuration and operation of the present invention will be described in more detail through preferred embodiments of the present invention. However, this is presented as a part of the example of the present invention and should not be construed as limiting the present invention in any way.

Any information not described here can be technically inferred by anyone skilled in the art, so description thereof will be omitted.

Manufacturing example 1. Preparation and identification of strains

<Preparation of strains>

3 g of Cheonggukjang was suspended and diluted in sterile saline solution, spread on NA agar medium, and cultured at 30°C for 24 hours. The colonies formed after culturing were subcultured to pure isolate Bacillus subtilus KBNP-BS3.

<Identification of strains>

1) Phylogenetic characterization

After extracting DNA for 16S rDNA sequence analysis from the pure isolated strain above, PCR was performed using 27F and 1492R primers. The PCR was subjected to electrophoresis to confirm that a PCR product of approximately 1.5 kb was obtained. The results of this PCR product confirmation are shown in Figure 2.

The PCR product identified above was purified and analyzed by a DNA analysis agency (Solgent Co., Ltd.), and it was confirmed that it had 99% homology with Bacillus subtilis. Based on these results, 16S rDNA sequence-based phylogenetic tree analysis was performed, and as a result, it was confirmed that the pure isolated strain (Bacillus subtilus KBNP-BS3) belongs to B. subtilis (subsp. subtilis). did. The results of this phylogenetic tree analysis (neighbor-joining tree) are shown in Figure 3.

2) Morphological characteristic analysis

As a result of performing Gram staining on the pure isolated strain above, the strain (B. subtilis KBNP-BS3) was confirmed to be a Gram positive strain. These Gram staining results are shown in Figure 4.

Endospore staining was performed on the pure isolated strain above, and the strain (B. subtilis KBNP-BS3) was confirmed to be a strain that forms endospores. These Gram staining results are shown in Figure 5. In Figure 5, endospores are stained and appear green (arrow).

As a result of observing the shape of the pure isolated strain above by taking a scanning electron microscope (SEM) image, it was confirmed that it was a typical Bacillus strain in the form of a bacillus of about 3 to 4 ㎛. These scanning electron microscopy images are shown in Figure 5.

3) Analysis of physiological characteristics

The pure isolated strain was cultured to obtain a single colony, and then suspended in API 50 CHB medium to adjust the suspension to 2 McFarland to prepare a strain suspension. The strain suspension was added to the API 50 CH strip kit, and the activity status was confirmed after 24 hours. Confirmation of the active state was observed through the color change of the phenol red indicator, which was confirmed to change to yellow. The results of changes in the kit were written on a record sheet and entered into the website (http://apiweb.biomerieux.com) to analyze the physiological characteristics of the pure isolated strain (B. subtilis KBNP-BS3) and identify the strain. As a result of this identification, it was confirmed that it had 99.9% homology with Bacillus subtilis, and the results are shown in Table 1 and Figure 6.

sample Identification result %ID T Index Test against Manufacturing Example 1 Baculus subtilis/
amyloliquefaciens 99.9 0.72 RIB 91% AMD 78% GLYG 79%

4) Antibiotic susceptibility profile

The strain isolated above was cultured by spreading inoculation on NA medium. A paper disk containing a total of 17 antibiotics was placed on the culture medium and further cultured at 37°C for 24 hours. Susceptibility to antibiotics was determined based on the size of the transparent ring generated from the paper disk of each antibiotic, and the results are shown in Table 2 below.

division Antibiotic Disk Potency (㎍) sensibility One Amoxicillin 30 S 2 Ampicillin 10 S 3 Apramicin 15 I 4 Cefotamime 30 I 5 Chloramphenicol 30 S 6 Ciprofloxacin 5 S 7 Colistin 10 R 8 Doxycycline 30 S 9 Enrofloxacin 5 S 10 Erythromycin 15 S 11 Gentamicin 10 R 12 Neomycin 30 R 13 Norfloxacin 10 S 14 Oxytetracycline 30 S 15 Streptomycin 10 S 16 Tetracycline 30 S 17 Trimethoprim/Sulfamethoxazole 1.25/23.7 S R: resistant
I: intermediate
S: susceptible

Manufacturing example 2. Preparation of strains (2)

<Preparation of strains>

3 g of Cheonggukjang was suspended and diluted in sterile saline solution, spread on NA agar medium, and cultured at 30°C for 24 hours. The colonies formed after culturing were subcultured to pure isolate Bacillus Licheniformis KBNP-DJ1 (KCTC 18447P).

Manufacturing example 3. Preparation of strains (3)

<Preparation of strains>

3 g of yeast was suspended and diluted in sterile saline solution, spread on NA agar medium, and cultured at 30°C for 24 hours. The colonies formed after culturing were subcultured to pure isolate Pichia Kudriavzevii KBNP-BF5 (KCTC 18445P).

Example One.

The Bacillus subtilus KBNP-BS3 strain isolated in Preparation Example 1 was inoculated into TSB medium and cultured to prepare a culture medium (200 ml). As a result of measuring the number of bacteria in the culture medium, it was confirmed that it was 7.5 × 10 ⁷ CFU/ml.

Example 2 to 7

The Bacillus subtilus KBNP-BS3 strain isolated in Preparation Example 1 was inoculated into TSB medium and cultured for 48 hours at pH 7 and 35°C to prepare a culture medium (200 ml). As a result of measuring the number of bacteria in the culture medium, it was confirmed that it was 1.0 × 10 ⁸ CFU/ml. 10% of the culture medium, green tea leaf powder, mugwort powder, and turmeric powder were mixed and stirred according to the mixing ratio in Table 3 below to prepare a livestock environment improvement agent.

Comparative example 1 (distilled water)

Instead of inoculating the strain in TSB medium, an equal amount of distilled water was added and cultured for 48 hours at pH 7 and 35°C to prepare a culture medium.

inoculation strain culture medium Distilled water green tea leaves Mugwort Turmeric Example 1 Manufacturing Example 1 10 90 0 0 0 Example 2 Manufacturing Example 1 10 85 5 0 0 Example 3 Manufacturing Example 1 10 80 10 0 0 Example 4 Manufacturing Example 1 10 85 0 5 0 Example 5 Manufacturing Example 1 10 80 0 10 0 Example 6 Manufacturing Example 1 10 85 0 0 5 Example 7 Manufacturing Example 1 10 80 0 0 10 Comparative Example 1 doesn't exist 0 100 0 0 0

Example 8 to 10

The Bacillus subtilus KBNP-BS3 strain purely isolated in Preparation Example 1 was cultured under the same conditions as Example 1 in the range of 30°C to 42°C (30°C, 37°C, 42°C), and the OD according to time at each temperature ₆₀₀ and viable cell count were investigated to confirm the optimal growth temperature. The OD ₆₀₀ and viable cell count results of Examples 8 and 10 are shown in Table 4 below. Through the results in Table 4, it was confirmed that the optimal growth temperature was 37°C. (Viable bacterial count unit: (Log CFU/ml))

Example 8 (30°C) Example 9 (37°C) Example 10 (42°C) Elapsed time OD ₆₀₀ Probiotic count OD ₆₀₀ Probiotic count OD ₆₀₀ Probiotic count 0 0.017 0.000 0.007 0.000 0.010 0.000 3 0.027 4.146 0.027 4.146 0.013 3.114 6 0.040 4.230 0.033 4.415 0.013 4.000 9 0.043 4.255 0.043 4.663 0.017 4.114 12 0.047 5.146 0.047 5.892 0.027 5.362 15 0.047 7.204 0.130 7.079 0.053 7.146 18 0.057 7.204 0.220 7.176 0.113 7.204 24 0.170 7.279 0.513 7.580 0.297 7.230 27 0.223 7.255 0.543 7.633 0.290 7.146 30 0.280 7.176 0.530 7.556 0.263 6.863 33 0.290 6.982 0.517 7.556 0.263 6.863 36 0.283 7.204 0.503 7.556 0.263 6.863

Example 11 to 13

The Bacillus subtilus KBNP-BS3 strain purely isolated in Preparation Example 1 was cultured under the same conditions as Example 1 in the range of pH 5 to pH 9 (pH5, pH7, pH9), and the OD ₆₀₀ and number of viable cells over time for each pH were measured. The optimal growth pH was confirmed by investigation. The OD ₆₀₀ and viable cell count results of Examples 11 and 13 are shown in Table 5 below. Through the results in Table 5, it was confirmed that the optimal growth pH was pH 7.

Example 11 (pH5) Example 12 (pH7) Example 13 (pH9) Elapsed time OD ₆₀₀ Probiotic count OD ₆₀₀ Probiotic count OD ₆₀₀ Probiotic count 0 0.010 0.000 0.010 0.000 0.010 0.000 3 0.015 4.146 0.035 4.146 0.010 3.000 6 0.055 6.000 0.130 7.591 0.020 6.301 9 0.185 7.000 0.255 8.041 0.020 6.505 15 0.285 7.146 0.500 7.594 0.160 6.845 18 0.450 7.079 0.535 7.778 0.260 7.000 24 0.525 7.000 0.505 7.699 0.375 7.699 27 0.500 7.114 0.485 7.602 0.385 7.699 30 0.480 7.204 0.455 7.663 0.360 7.863

Example 14 to 18

The Bacillus subtilus KBNP-BS3 strain isolated in Preparation Example 1 was grown in five different media (Brain Heart Infusion (BHI), Luria Bertani (LB), MRS medium, Nutrient broth (NB), and Tryptic Soy Broth (TSB). ) was inoculated and cultured under the same conditions as in Example 1, and the optimal growth medium was confirmed by examining OD ₆₀₀ and viable cell count over time. The OD ₆₀₀ and viable cell count results of Examples 14 and 18 are shown in Tables 6 and 7 below. Through the results in Tables 6 and 7, the optimal growth medium was confirmed to be TSB medium.

OD ₆₀₀ Example 14 Example 15 Example 16 Example 17 Example 18 BHI M.R.S. LB TSB N.B. 0 hours 1.74 0.04 0.88 1.80 0.18 24 hours 1.32 1.74 0.93 0.86 0.32 48 hours 0.94 1.93 0.82 0.62 0.16 72 hours 0.55 1.53 0.32 0.37 0.14

Probiotic count Example 14 Example 15 Example 16 Example 17 Example 18 (Log CFU/ml) BHI M.R.S. LB TSB N.B. 0 hours 8.85 6.78 7.90 8.26 7.90 24 hours 8.48 8.70 8.30 9.60 7.48 48 hours 7.60 7.00 8.48 9.86 7.30 72 hours 6.60 5.60 7.30 7.00 6.00

Example 19 to 20

It was performed in the same manner as Example 1, except that the pure isolated strain in Preparation Example 2 was used (Example 19) and the purely isolated strain in Preparation Example 3 was used (Example 20).

<Evaluation>

1) Confirmation of non-pathogenicity

The Bacillus subtilus KBNP-BS3 strain culture medium of Example 1 was streaked on blood agar, cultured at 37°C for 48 hours, and then β-hemolysis, which is the decomposition of red blood cells around the bacterial cells, was checked. As a result, the non-pathogenicity of the Bacillus subtilus KBNP-BS3 strain was confirmed.

2) Confirmation of enzyme activity

The culture medium of the Bacillus subtilus KBNP-BS3 strain of Example 1 was analyzed for enzyme activity using the API ZYM kit. As a result, it was confirmed that the Bacillus subtilus KBNP-BS3 strain had activities against esterase (C4), esterase-lipase (C8), acid phosphatase, Naphthol-AS-BI -phosphohydrolase, and α-glucosidase. The results of this enzyme activity analysis are shown in Figure 8.

3) Confirmation of the effect of improving the livestock environment and reducing odor

In order to prove the objectivity of the odor removal ability test, the Korea Institute of Construction and Living Environment, a testing institution, was requested to evaluate the deodorization effect using the culture solution prepared in Example 1.

(Test method EL608-2006/2/2012/36)

20 ml of liquid sample containing the culture medium of Example 1 was placed into four 10-liter reactors each and sealed. The initial concentration of the test gas was injected as ammonia 100 μmol/mol (ppm), trimethylamine 30 μmol/mol (ppm), hydrogen sulfide 50 μmol/mol (ppm), and methyl mercaptan 4 μmol/mol (ppm). The concentration of the test gas was measured at 120 minutes, and this was set as the concentration of the sample. The concentration of the test gas was measured using the gas detection tube method (KS I 2218), and the temperature was maintained at 25.6℃ ± 0.6℃ and humidity at 41.7% ± 0.7% during the test. Separately, the same test as above was performed in the absence of a sample and this was evaluated as blank concentration. The test gas concentration measured above was calculated using Equation 1 below to calculate the removal rate (deodorization rate) of the test gas. The results are shown in Table 8 below.

[Equation 1]

Removal rate of test gas (%) = [{(blank concentration)-(sample concentration)/(blank concentration)}

The deodorization rate for livestock odor-causing substances was 99.6% for ammonia, 96.0% for trimethylamine, 99.3% for hydrogen sulfide, and 40.0% for methyl mercaptan, which is judged to be at a level that can be used as a livestock odor improver.

ammonia trimethylamine hydrogen sulfide Methyl mercaptan Example 1 99.6% 96.0% 99.3% 40.0%

5) Evaluation of antibacterial activity

Six types of harmful bacteria (B. cereus KCTC1012, E. coli KCTC1467, E. faecalis KCTC2011, L. monocytogenes KCTC3567, P. aeruginosa KCTC1750, S. enteritidis KCCM12400) were each cultured in NA medium. A paper disk soaked with the culture solution of Example 1 was placed on each medium, and further cultured at 37°C for 24 hours. Antibacterial activity was determined based on the size of the transparent ring generated from the paper disk of each medium, and the results are shown in Table 9 below. As a result of the experiment, it was confirmed that Example 1 had antibacterial activity against B. cereus KCTC1012, P. aeruginosa KCTC1750, and S. enteritidis KCCM12400 strains.

Harmful bacteria Antibacterial activity (with +, without -) B. cereus KCTC1012 + E. coli KCTC1467 - E. faecalis KCTC2011 - L. monocytogenes KCTC3567 - P. aeruginosa KCTC1750 + S. enteritidis KCCM12400) +

6) Evaluation of the effect of improving the livestock environment

The livestock environment improvement agents prepared in Examples 1 to 7 and Comparative Examples 1 to 2 were prepared. After purchasing SPF chickens, they were raised in an animal shelter, and 500 g of chicken feces was placed in a 2 L sealed rotary bottle. After the livestock environment improvement agent was sprayed and treated on chicken feces, the final odor reduction effect was tested by comparing the initial odor concentration and the 24-hour concentration. The results are shown in Table 10 below.

unit(%) ammonia Triethylamine hydrogen sulfide Methyl mercaptan Example 1 99.6 96 99.3 40 Example 2 60 90 -400 -13 Example 3 71 95 -846 47 Example 4 80 90 10 41 Example 5 90 97 88 100 Example 6 0 98 -691 -163 Example 7 88 92 -1366 -3 Example 19 99.6 96 80 30 Example 20 99.6 96 26.7 16.7 Comparative Example 1 -23 -200 -471 0

Korea Research Institute of Bioscience and Biotechnology Microbial Resource Center KCTC18446P 20160217 Korea Research Institute of Bioscience and Biotechnology Microbial Resource Center KCTC18447P 20160217 Korea Research Institute of Bioscience and Biotechnology Microbial Resource Center KCTC18445P 20160217

Claims (5)

A probiotic composition for improving the livestock environment, which has a deodorizing effect against methyl mercaptan, a substance causing livestock odor, and an antibacterial effect against Bacillus cereus, Pseudomonas aeruginosa , and Salmonella enteritidis , A probiotic composition for improving the livestock environment containing Bacillus subtilis KBNP-BS3 ( Bacillus Subtilis KBNP-BS3; KCTC 18446P) strain culture medium and mugwort. According to paragraph 1,
The culture medium is one or more species selected from the group consisting of Bacillus Licheniformis KBNP-DJ1 (KCTC 18447P) and Pichia Kudriavzevii KBNP-BF5 (KCTC 18445P) A probiotic composition for improving the livestock environment further comprising a culture medium of the strain.
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