KR20130134914A - Complex strain for purifing water and improving barn environment and its making method - Google Patents

Abstract

The present invention relates to a complex strain for purifying water and improving a barn environment, and a method for manufacturing the same. The complex strain for purifying water and improving a barn environment according to the present invention comprises Bacillus subtilis CH, Pseudomonas chlororaphis, Enterobactor intermedius, Klebsiella oxytoca, Lactobacillus acidophilus and Saccharomyces cerevisiae.

Description

Complex strain for improving water quality and livestock environment and its manufacturing method {COMPLEX STRAIN FOR PURIFING WATER AND IMPROVING BARN ENVIRONMENT AND ITS MAKING METHOD}

The present invention relates to a complex strain and a method for producing the same, and more particularly, to a complex strain for improving water quality and livestock environment and a method for producing the same.

As the eco-friendly agriculture and livestock industry has been in the spotlight recently, the market for eco-friendly materials and biological environment improvement is gradually being activated. The global market for biological materials is on the rise, and more than 12 billion won in the trillion pesticide market is expected to be replaced by biological materials. In particular, it is essential to develop various eco-friendly materials in policy projects such as eco-friendly livestock product certification farm support project and eco-friendly livestock product certification system to prepare for the FTA and strengthen the competitiveness of livestock farmers.

Among the environmentally friendly materials, the most researched is the development of microbial preparations using biologically useful bacteria. In particular, the development and commercialization of various soil microorganisms that exist in the plant root zone and have many useful traits among the biologically useful bacteria are in progress. It inhibits the influx of strains or indirectly promotes water purification and the improvement of livestock environment. In recent years, studies on the environmental purification and the improvement of livestock quality of these useful microorganisms have been actively conducted, and eco-friendly materials of new materials have been commercialized accordingly.

However, although most of the bioavailable bacteria developed so far have an effective mechanism of action, there is a substantial reluctance in livestock farms, which means that the control of microbial products is lower than chemicals, while the cost is higher than chemicals. Because it is variable.

On the other hand, Patent Registration No. 10-403146 of the prior art discloses a complex microbial agent for removing the odor of livestock manure in the livestock environment, but the patent registration publication contains Monascus perpurius and Rhodobacter encapsulators As a complex microbial agent, only the odor removal effect of livestock manure is disclosed, and there is no mention of the effect of inhibiting the growth of pathogens harmful to the livestock house. In addition, the complex strain of the present application and its species are completely different.

In addition, Patent Registration No. 10-541844 of the prior art discloses a method for treating dyed wastewater by a complex process, but it is disclosed that a complex strain may be used in a complex process for treating dyed wastewater. The complex strain includes Bacillus cerius cabling UEL 1 and Bacillus cerius cabling UEL 2, which is completely different from the complex strain of the present application.

Therefore, it is possible to improve the environment of water purification and livestock raising in the environment of agriculture / livestock industry, to be industrially mass-produced, and to be able to cultivate strains by using inexpensive culture medium instead of expensive culture medium to enjoy economic effects. There is a need for microbial agents.

Accordingly, an object of the present invention is to provide a complex strain and a method for producing the same that can improve the water purification and the environment of the livestock house. The complex strain can suppress the growth of pathogens harmful to livestock which may be present in the barn, and also provides a complex strain and a method for producing the same having the effect of improving the odor of the barn.

The object is, according to the present invention, Bacillus subtilis CH, Pseudomonas chlororaphis, Enterobactor intermedius, Klebsiella oxytoca, Lactobacillus exix It is achieved by a water purification complex consisting of Lactobacillus acidophilus and Saccharomyces cerevisiae.

In addition, the above object, according to the present invention, Bacillus subtilis CH, Pseudomonas chlororaphis, Enterobactor intermedius, Klebsiella oxytoca, Lactobacillus It can be achieved by a complex strain for improving the barn environment composed of Lactobacillus acidophilus and Saccharomyces cerevisiae.

The complex strain can improve the odor environment by having a malodor improvement and growth inhibition effect of animal pathogens.

In addition, the above object, according to the present invention, in the production method of the complex strain, the first strain to isolate the strain for water purification and harmful gas generation through centrifugation after shaking culture in the nutrient medium of the strains isolated from the soil. Steps; It can be achieved by a method for producing a complex strain for water purification and barn environment improvement comprising the step of identifying and reconstituting the strain to remove the malodor gas by injecting malodor gas to the separated strains.

In addition, the above object, in the cultivation method of mass culture of the complex strain, the complex strain is cultured for 24 to 72 hours in a medium containing 1 to 3% molasses, 1 to 3% beer foil and 0.5 to 1% salt Including the steps, the complex, Bacillus subtilis (Bacillus subtilis) CH, Pseudomonas chlororaphis, Enterobactor intermedius, Enterobactor intermedius, Klebsiella oxytoca, Lactobacillus It can be achieved by a mass culture method of a complex strain consisting of Lactobacillus acidophilus and Saccharomyces cerevisiae.

As described above, according to the present invention, it is possible to improve the water purification and the livestock environment in the environment of agriculture / livestock industry, can be industrially mass-produced, strain culture using an inexpensive culture medium instead of expensive culture medium It is to provide a complex strain for improving the water quality and / or livestock environment with good economic effect and a manufacturing method thereof. Accordingly, the use of the complex strain according to the present invention can not only be economical, but also environmentally friendly to improve the water purification and livestock environment.

Figure 1 shows the effect on the growth curve of Salmonella enteritidis (Salmonella enteritidis) of the complex according to an embodiment of the present invention,
Figure 2 shows the effect on the growth curve of Bacillus cereus complex strain according to an embodiment of the present invention,
Figure 3 is a graph showing the odor gas removal effect of the complex strain according to an embodiment of the present invention.

Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings, which will be readily apparent to those skilled in the art to which the present invention pertains. The present invention may be embodied in many different forms and is not limited to the embodiments described herein.

Complex strain according to an embodiment of the present invention, Bacillus subtilis CH, Pseudomonas chlororaphis, Enterobactor intermedius, Enterobactor intermedius, Klebsiella oxytoca, Lactobacillus acidophilus and Saccharomyces cerevisiae. The complex strain has the effect of water purification and / or livestock environment improvement. The complex strain not only suppresses the growth of pathogens present in the barn, but also has the effect of improving the odor of the barn.

In addition, the manufacturing method of the complex strain according to an embodiment of the present invention, the first step of separating the strain for inhibiting water quality purification and harmful gas through centrifugation after shaking culture in the nutrient medium of the strains isolated from the soil and ; Injecting the malodorous gas into the separated strains may comprise the step of identifying and reconstructing the strain to remove the malodorous gas.

In addition, according to one embodiment of the present invention, the mass culture method of the complex strain, the complex strain in a medium containing 1 to 3% molasses, 1 to 3% beer foil and 0.5 to 1% saline 24 to 72 hours Culturing during. In particular, more preferably comprises culturing the complex strain for 24 to 72 hours in a medium containing 1% molasses, 3% beer foil, 1% salt.

Hereinafter, exemplary embodiments of the present invention will be described with reference to the accompanying drawings.

Example  One. Complex  detach

The following steps were carried out to isolate strains that exhibited excellent effects on water purification and odor improvement.

Step 1: Collect the soil and smear it on agar media

Soil was collected, suspended in sterile water and plated in LB (Luria Bertani) medium.

Step 2. Isolation of Single Colonies

Strains showing the respective characteristics on the LB agar medium were single colony isolation.

Step 3. Cultivation and Recovery

Each of the single colony isolated strains was shaken in an LB liquid medium at about 30 ° C. for about 24 hours at 200 revolutions per minute. Thereafter, centrifugation was performed to recover the bacteria and suspended in the same amount of sterile water.

Step 4. Strain Selection

Strains having an effect of removing the ammonia gas were selected by injecting ammonia gas affecting water purification and odor, and the strains were Bacillus subtilis CH, Pseudomonas chlororaphis, and enterobacter. Enterobactor intermedius, Klebsiella oxytoca, Lactobacillus acidophilus and Saccharomyces cerevisiae.

Example  2. Complex  Sympathy

2-1. Bacillus Subtilus ( Bacillus subtilis ) CH

By confirming the 16S rRNA base sequence of the strains isolated in Example 1, one of the strains was identified as Bacillus subtilis CH.

The isolated Bacillus subtilis CH strain was subjected to shaking culture at 200 rotations per minute for about 24 hours in an LB liquid medium at about 30 ° C., and then cells were recovered to separate genomic DNA. The 16S rRNA of the strain was cloned by Polymerase Chain Reaction using 5'-AGAGTTTGATCCTGGCTCAG-3 'as the forward primer and 5'-ACGGCTACCTTGTTACGACTT-3' as the reverse primer. The base sequence of the analyzed 16S rDNA is the same as SEQ ID NO: 1 in the Sequence Listing, the contents of which are as follows.

Bacillus subtilis strain S64 16S ribosomal RNA gene, partial sequence Length = 1454

Score = 2662 bits (1441), Expect = 0.0

Identities = 1443/1444 (99%), Gaps = 0/1444 (0%)

Strand = Plus / Plus

2-2. Pseudomonas Chloro Lapis Pseudomonas chlororaphis )

By confirming the 16S rRNA base sequence of the strains isolated in Example 1, one of the strains was identified as Pseudomonas chlororaphis O6.

The isolated Pseudomonas chloro rapi strain was incubated for about 24 hours in an LB liquid medium at about 30 ° C., and then cells were recovered to separate genomic DNA. 16S rRNA of the strain was cloned by Polymerase Chain Reaction using 5'-AGAGTTTGATCCTGGCTCAG-3 'as the forward primer and 5'-ACGGCTACCTTGTTACGACTT-3' as the reverse primer. The base sequence of the analyzed 16S rDNA is the same as SEQ ID NO: 2 in the Sequence Listing, the contents of which are as follows.

Pseudomonas sp. lip35 16S ribosomal RNA gene, partial sequence Length = 1530

Score = 2697 bits (1460), Expect = 0.0

Identities = 1467/1470 (99%), Gaps = 1/1470 (0%)

Strand = Plus / Plus

2-3. Enterobacter intermediaus ( Enterobactor intermedius )

By confirming the 16S rRNA base sequence of the strains isolated in Example 1, one of the strains was identified as Enterobactor intermedius B1.

The isolated Enterobacter intermediaus B1 strain was shaken for 24 hours in an LB liquid medium at 30 ° C., and then cells were recovered to separate genomic DNA. 16S rRNA of the strain was cloned by Polymerase Chain Reaction using 5'-AGAGTTTGATCCTGGCTCAG-3 'as the forward primer and 5'-ACGGCTACCTTGTTACGACTT-3' as the reverse primer. The base sequence of the analyzed 16S rDNA is the same as SEQ ID NO: 3 in Sequence Listing, and the contents are as follows.

Enterobacter intermedius 16S ribosomal RNA gene, partial sequence Length = 1453

Score = 2663 bits (1442), Expect = 0.0

Identities = 1444/1445 (99%), Gaps = 0/1445 (0%)

Strand = Plus / Plus

2-4. Krebscheiler  Oxytoca Klebsiella oxytoca )

By confirming the 16S rRNA base sequence of the strains isolated in Example 1, one of the strains was identified as Klebsiella oxytoca Cl036.

The isolated Bacillus Krebsiella oxytoca Cl036 strain was shaken for 24 hours in an LB liquid medium at 30 ° C., and then cells were recovered to separate genomic DNA. 16S rRNA of the strain was cloned by Polymerase Chain Reaction using 5'-AGAGTTTGATCCTGGCTCAG-3 'as the forward primer and 5'-ACGGCTACCTTGTTACGACTT-3' as the reverse primer. The base sequence of the analyzed 16S rDNA is the same as SEQ ID NO: 4 in the Sequence Listing, the contents of which are as follows.

Klebsiella oxytoca 16S ribosomal RNA gene, partial sequence Length = 1502

Score = 2641 bits (1430), Expect = 0.0

Identities = 1441/1446 (99%), Gaps = 1/1446 (0%)

Strand = Plus / Plus

2-5. Lactobacillus  Excidophilus ( Lactobacillus acidophilus )

By confirming the 16S rRNA base sequence of the strains isolated in Example 1, one of the strains was identified as Lactobacillus acidophilus AP.

The isolated Lactobacillus exidophilus AP strain was shaken for 24 hours in an LB liquid medium at 30 ° C., and then cells were recovered to separate genomic DNA. 16S rRNA of the strain was cloned by Polymerase Chain Reaction using 5'-AGAGTTTGATCCTGGCTCAG-3 'as the forward primer and 5'-ACGGCTACCTTGTTACGACTT-3' as the reverse primer. The base sequence of the analyzed 16S rDNA is the same as SEQ ID NO: 5 in the Sequence Listing, the contents of which are as follows.

Lactobacillus acidophilus 16S ribosomal RNA gene, partial sequence Length = 1449

Score = 2235 bits (1210), Expect = 0.0

Identities = 1212/1213 (99%), Gaps = 0/1213 (0%)

Strand = Plus / Plus

2-6. Sakaromayses  Cereje ( Saccharomyces cerevisiae )

By confirming the 18S rRNA base sequence of the strains isolated in Example 1, one of the strains was identified as Saccharomyces cerevisiae.

The isolated Saccharomyces cerevise strain was shaken for 24 hours in an LB liquid medium at 30 ° C., and then cells were recovered to separate genomic DNA. The 18S rRNA of the strain was cloned by Polymerase Chain Reaction using 5'-GTAACCCGTTGAACCCCATT-3 'as the forward primer and 5'-CCATCCAATCGGTAGTAGCG-3' as the reverse primer. The base sequence of the analyzed 18S rDNA is the same as SEQ ID NO: 6 in the Sequence Listing, the contents of which are as follows.

Saccharomyces cerevisiae gene for 18S rRNA, partial sequence Length = 1714

Score = 2130 bits (1153), Expect = 0.0

Identities = 1153/1153 (100%), Gaps = 0/1153 (0%)

Strand = Plus / Plus

Experimental Example  One. Complex  Complex culture

Each of the strains isolated through Example 1 and Example 2 were shaken for 24 hours in an LB liquid medium at about 30 ° C., followed by replacement culture to culture the strains that do not inhibit the growth of each other. The result of measuring the viable cell count of each microorganism by complex culture is shown in Table 1 below.

Strain name Viable cell count Used badge Pseudomonas chlororaphis 2.4 × 10 ⁸ cfu / g LB Bacillus subtilis 3.7 × 10 ⁸ cfu / g LB Enterobactor intermedius 1.4 × 10 ⁸ cfu / g LB Klebsiella oxytoca 4.8 × 10 ⁸ cfu / g LB Lactobacillus acidophilus 6.3 × 10 ⁸ cfu / g LB Saccharomyces cerevisiae 1.8 × 10 ⁷ cfu / g YPD

Experimental Example  2. Complex  Growth Inhibitory Effects on Animal Pathogenic Bacteria

As an animal pathogen, Salmonella enteritidis and Bacillus cereus, which cause food poisoning in animals, were used to test the growth inhibitory effect on the animal pathogenic bacteria of the complex strain according to the present invention.

For the growth of pathogens, shaking culture was performed for 24 hours at 30 ° C., which is a temperature at which various microorganisms grow well in LB (Luria Bertani) liquid medium.

In addition, after centrifuging the mixed strain cultured as in Experimental Example 1 in a 50ml conical tube, using the supernatant Salmonella enteritidis and Bacillus cereus (Salmonella enteritidis), which is an animal food poisoning strain cereus) were replaced in two cultures.

As a result of the replacement culture, the effect on the growth of Salmonella enteritidis and Bacillus cereus of the complex strain of Experimental Example 1 is as shown in Figs.

The control (control) is the growth curve of each pathogen alone, the growth curve of the pathogen when Saccharomyces cerevisiae is added as a comparative example, EM-Complex shows the growth curve of the pathogen when the complex strain of Example 1 is added have.

Figure 1 shows the effect on the growth curve of the pathogen Salmonella enteritidis of the complex strain of Experimental Example 1. 1, it was confirmed that the growth curve of Salmonella enteritidis (Salmonella enteritidis) is inhibited by more than 90% in 12 hours, which is the secondary metabolite of the complex strain according to the present invention of Salmonella enteritidis It can be confirmed that growth is suppressed.

Figure 2 shows the effect on the growth curve of the pathogen Bacillus cereus of the complex strain of Experimental Example 1. Referring to Figure 2, the growth curve of Bacillus cereus (Bacillus cereus) was confirmed that the inhibition of more than 80% in 24 hours, which is the growth of Bacillus cereus by the secondary metabolite of the complex according to the present invention It can be confirmed that it is suppressed.

Experimental Example  3. Complex  Hazardous Gas Suppression Effect

Odor gas removal effect of the composite microorganism fermentation broth prepared according to Experimental Example 1 was tested. As a test method for confirming the odor gas removal effect was used a modification method of KS M 0001 and KS M 0062 gas detection tube method, the test conditions were used in the test chamber 20ℓ Tedlar bag, the applied sample amount 4ml. % Removal was calculated by the following equation.

Removal rate

(%) = (Blank Test-Sample) / Blank Test × 100

Most of the components exhibiting odor in the barn were ammonia and trimethylamine, and thus the removal ability of the two gases was confirmed, and the results are shown in Table 2 below.

Test gas Test time (hr) Blank test (ppm) Sample (ppm) Removal rate (%) Ammonia 0 60 60 - One 60 4.5 92.5 3 60 2 96.6 5 60 N.D. 99.0 < Trimethylamine 0 60 60 - One 60 11 81.6 3 60 3.5 94.2 5 60 N.D. 99.0 <

As shown in Table 2, the ammonia gas showed a removal rate of 90% or more after 1 hour of application of the fermentation broth of the complex strain according to the present invention, it was confirmed that no ammonia gas was detected after 5 hours. In addition, trimethylamine showed a removal rate of 90% or more in 3 hours after application of the fermentation broth of the complex according to the present invention, and it was confirmed that trimethylamine was not detected in 5 hours.

Experimental Example  4. Complex  Hazardous Gas Inhibition Effect

The odor gas removal effect in the system of the mixed microbial fermentation broth prepared according to Experimental Example 1 was tested. Uric acid is a harmful toxin in the body and is generally caused by algae feces, which is more severe than barns or pig odors. Therefore, in order to confirm the harmful gas inhibitory effect of algae, the poultry was taken from the poultry farm and the odor removal effect in the poultry of the fermentation broth of the complex microorganism of the present invention was tested.

As a test method for confirming the odor gas removal effect in the system, 5 individuals of each treatment group were selected for the average range of the living body, and the excreted minute was collected for 1 day. After removing the feathers and foreign matter, and put in a glass container of 500ml, it was stored in a 30 ℃ incubator. Hazardous gas emissions were measured using H2S and NH3 gas detector tubes (Gastec, Kanagawa, Japan) at intervals of 12 hours after 36 hours.

The results of performing the experiment are as shown in FIG. 3. As a control of FIG. 3, no treatment was performed, and as a comparative example, the odor removal effect in the flour when Saccharomyces cerevisiae was treated, and EM-Complex in the fermentation solution of the fermentation broth of the complex of Experimental Example 1 Odor removal effect is shown.

Referring to FIG. 3, the ammonia level in the system powder began to show a difference of about 30% after 24 hours of application of the fermentation broth of the complex strain according to the present invention and showed a difference of 50% or more after 48 hours. Through this, it was possible to confirm the odor gas suppression effect in the system of the fermentation broth of the complex according to the present invention.

Experimental Example  5. Complex  Water Quality Improvement Effect

In order to confirm the water quality improvement effect of the composite microorganism prepared according to Experimental Example 1 was tested by the Institute of Agricultural Science and Technology. Sample 500 L of contaminated stagnant water in agricultural irrigation reservoir located in Changjeong-ri, Eokseong-gun, Gokseong-gun, Jeonnam, and administer the composite microorganisms cultured according to Experimental Example 4 at weekly intervals. , BOD (biological oxygen demand), COD (chemical oxygen demand), TN (total nitrogen), TP (total phosphorus)) were investigated and analyzed for the difference.

Suspended Solid (SS), Biochemical Oxygen Demand (BOD), Chemical Oxygen Demand (COD), TN (Total Ammonia), as described in the Water Pollution Process Test Method. Content) -continuous automatic measurement method, TP (total phosphorus content) -continuous automatic measurement method, and the results are shown in Table 3.

process SS BOD COD T-N T-P Before processing 53.4 34.5 14.5 13.21 0.32 After processing 39.1 26.8 9.3 9.64 0.16

"Before treatment" in Table 3 indicates the water quality test value measured before the complex strain of the present application, "after treatment" represents the water quality test value measured after the treatment of the complex strain of the present application. As shown in Table 3, SS, BOD, COD, TN showed a total reduction of about 30%, TP value of 50% showed a reduction rate of the composite strain of the present application was found to show an effect on water quality improvement.

Experimental Example  6. Complex Investigation

100 ml of LB medium (Bacto Tryptone 20g, NaCl 5g, Bacto Yeast Extract 15g, pH7.5) was prepared, and the mixed strain of Experimental Example 1 was incubated at about 30 ° C. for about 24 hours at a rotational speed of 200 rpm. The shaken, pig, and poultry farms were inoculated with the shake-cultured complex strain to determine whether the complex strain exhibited pathogenicity to the livestock. The bacteria cultured for the inoculation were recovered by centrifugation, diluted in sterile water to an appropriate inoculation concentration (1.6 × 10 9 CFU / mL), and then treated in a barn, pig house, and poultry farm for 2,000 ml each. Were examined for a week in a 25 ~ 30 ℃ incubator.

The results are shown in Table 4 below.

Barn Donsa poultry farm Weakness - - -

(-: Weak; +: weak)

As a result of the experiment, the complex strain according to the present invention was confirmed that it does not show any damage to the barn, pigs, poultry farm, and this can be confirmed that the complex strain of the present application can be safely used in the barn, pigs, poultry farm there was.

Experimental Example  7. Complex  For mass culture Culture medium  Furtherance

Experiments were carried out for the composition of the culture medium which can culture the complex strain of Experimental Example 1. To this end, a medium for mass cultivation using various industrial by-products was constructed. As the main components of the medium for mass culture, the molasses and fermented beer gourd produced during the production of sugar were used as the main raw material, and the composition ratio of the present invention showed the best culture effect at the fastest time.

Medium composition condition 24 hour culture 48 hours incubation 72 hours culture ① LB (control) 1.4 × 10 ⁷ 8.7 × 10 ⁷ 3.2 × 10 ⁸ ② molasses 1% + beer foil (dry powder) 1% 1.5 × 10 ⁸ 2.1 × 10 ⁸ 3.4 × 10 ⁸ ③ 3% molasses + 3% beer foil (dry powder) 2.3 × 10 ⁶ 1.9 × 10 ⁶ 1.4 × 10 ⁸ ④ molasses 1% + beer foil (dry powder) 1% + salt (NaCl) 1% 1.7 × 10 ⁸ 5.1 × 10 ⁸ 5.7 × 10 ⁸ ⑤ 3% molasses + 3% beer foil (dry powder) + 1% salt (NaCl) 1.2 × 10 ⁶ 1.2 × 10 ⁷ 3.7 × 10 ⁸ (6) Molasses 1% + Beer foil (hot water extraction) 1% 7.8 × 10 ⁶ 1.8 × 10 ⁷ 4.2 × 10 ⁷ ⑦ 3% molasses + 3% beer foil (hot water extraction) 7.4 × 10 ⁵ 1.6 × 10 ⁷ 2.8 × 10 ⁸ (8) Molasses 1% + Beer foil (hot water extraction) 1% + Salt (NaCl) 1% 2.4 × 10 ⁷ 1.1 × 10 ⁸ 3.9 × 10 ⁸ ⑨ 3% molasses + 3% beer foil (hot water extraction) + 1% salt (NaCl) 4.8 × 10 ⁶ 7.0 × 10 ⁶ 2.4 × 10 ⁸

(Unit: cfu / ml)

As shown in Table 5, the medium containing 1% molasses, 1% dried beet meal and 1% salt (NaCl) as a culture medium for mass culturing the complex strain of the present application was found to be the most efficient.

Although several embodiments of the present invention have been shown and described, those skilled in the art will appreciate that various modifications may be made without departing from the principles and spirit of the invention . The scope of the invention will be determined by the appended claims and their equivalents.

<110> ECOTECHSEED CO., LTD <120> COMPLEX STRAIN FOR PURIFING WATER AND IMPROVING BARN ENVIRONMENT          AND ITS MAKING METHOD <130> DPP-2012-5086-KR <160> 6 <170> Kopatentin 2.0 <210> 1 <211> 1444 <212> RNA <213> Bacillus subtilis <400> 1 ggtgctatac atgcagtcga gcggacagat gggagcttgc tccctgatgt tagcggcgga 60 cgggtgagta acacgtgggt aacctgcctg taagactggg ataactccgg gaaaccgggg 120 ctaataccgg atggttgttt gaaccgcatg gttcagacat aaaaggtggc ttcggctacc 180 acttacagat ggacccgcgg cgcattagct agttggtgag gtaacggctc accaaggcga 240 cgatgcgtag ccgacctgag agggtgatcg gccacactgg gactgagaca cggcccagac 300 tcctacggga ggcagcagta gggaatcttc cgcaatggac gaaagtctga cggagcaacg 360 ccgcgtgagt gatgaaggtt ttcggatcgt aaagctctgt tgttagggaa gaacaagtgc 420 cgttcaaata gggcggcacc ttgacggtac ctaaccagaa agccacggct aactacgtgc 480 cagcagccgc ggtaatacgt aggtggcaag cgttgtccgg aattattggg cgtaaagggc 540 tcgcaggcgg tttcttaagt ctgatgtgaa agcccccggc tcaaccgggg agggtcattg 600 gaaactgggg aacttgagtg cagaagagga gagtggaatt ccacgtgtag cggtgaaatg 660 cgtagagatg tggaggaaca ccagtggcga aggcgactct ctggtctgta actgacgctg 720 aggagcgaaa gcgtggggag cgaacaggat tagataccct ggtagtccac gccgtaaacg 780 atgagtgcta agtgttaggg ggtttccgcc ccttagtgct gcagctaacg cattaagcac 840 tccgcctggg gagtacggtc gcaagactga aactcaaagg aattgacggg ggcccgcaca 900 agcggtggag catgtggttt aattcgaagc aacgcgaaga accttaccag gtcttgacat 960 cctctgacaa tcctagagat aggacgtccc cttcgggggc agagtgacag ggggtgcatg 1020 gttgtcgtca gctcgtgtcg tgagatgttg ggttaagtcc cgcaacgagc gcaacccttg 1080 atcttagttg ccagcattca gttgggcact ctaaggtgac tgccggtgac aaaccggagg 1140 aaggtgggga tgacgtcaaa tcatcatgcc ccttatgacc tgggctacac acgtgctaca 1200 atggacagaa caaagggcag cgaaaccgcg aggttaagcc aatcccacaa atctgttctc 1260 agttcggatc gcagtctgca actcgactgc gtgaagctgg aatcgctagt aatcgcggat 1320 cagcatgccg cggtgaatac gttcccgggc cttgtacaca ccgcccgtca caccacgaga 1380 gtttgtaaca cccgaagtcg gtgaggtaac ctttaggagc cagccgccga aggtgacaga 1440 aagg 1444 <210> 2 <211> 1472 <212> RNA Pseudomonas sp. <400> 2 tcctgggtca attgaacgct ggcggcaggc ctaacacatg caagtcgagc ggtagagagg 60 tgcttgcacc tcttgagagc ggcggacggg tgagtaatgc ctaggaatct gcctggtagt 120 gggggataac gttcggaaac ggacgctaat accgcatacg tcctacggga gaaagcaggg 180 gaccttcggg ccttgcgcta tcagatgagc ctaggtcgga ttagctagtt ggtgaggtaa 240 tggctcacca aggcgacgat ccgtaactgg tctgagagga tgatcagtca cactggaact 300 gagacacggt ccagactcct acgggaggca gcagtgggga atattggaca atgggcgaaa 360 gcctgatcca gccatgccgc gtgtgtgaag aaggtcttcg gattgtaaag cactttaagt 420 tgggaggaag ggtacttacc taatacgtga gtattttgac gttaccgaca gaataagcac 480 cggctaactc tgtgccagca gccgcggtaa tacagagggt gcaagcgtta atcggaatta 540 ctgggcgtaa agcgcgcgta ggtggttcgt taagttgaat gtgaaatccc cgggctcaac 600 ctgggaactg catccaaaac tggcgagcta gagtatggta gagggtggtg gaatttcctg 660 tgtagcggtg aaatgcgtag atataggaag gaacaccagt ggcgaaggcg accacctgga 720 ctgatactga cactgaggtg cgaaagcgtg gggagcaaac aggattagat accctggtag 780 tccacgccgt aaacgatgtc aactagccgt tgggagcctt gagctcttag tggcgcagct 840 aacgcattaa gttgaccgcc tggggagtac ggccgcaagg ttaaaactca aatgaattga 900 cgggggcccg cacaagcggt ggagcatgtg gtttaattcg aagcaacgcg aagaacctta 960 ccaggccttg acatccaatg aactttccag agatggattg gtgccttcgg gaacattgag 1020 acaggtgctg catggctgtc gtcagctcgt gtcgtgagat gttgggttaa gtcccgtaac 1080 gagcgcaacc cttgtcctta gttaccagca cgtaatggtg ggcactctaa ggagactgcc 1140 ggtgacaaac cggaggaagg tggggatgac gtcaagtcat catggccctt acggcctggg 1200 ctacacacgt gctacaatgg tcggtacaga gggttgccaa gccgcgaggt ggagctaatc 1260 ccataaaacc gatcgtagtc cggatcgcag tctgcaactc gactgcgtga agtcggaatc 1320 gctagtaatc gcgaatcaga atgtcgcggt gaatacgttc ccgggccttg tacacaccgc 1380 ccgtcacacc atgggagtgg gttgcaccag aagaagctag tctaaccttc gggaggacgg 1440 ttaccacggt gtgattcatg actggggtgc ca 1472 <210> 3 <211> 1455 <212> RNA <213> Enterobacter intermedius <400> 3 aattacggac cggcaggcct aacacatgca agtcgaacgg tagcacagag agcttgctct 60 tgggtgacga gtggcggacg ggtgagtaat gtctgggaaa ctgcccgatg gagggggata 120 actactggaa acggtagcta ataccgcata acgtcgcaag accaaagtgg gggaccttcg 180 ggcctcacac catcggatgt gcccagatgg gattagctag taggtggggt aatggctcac 240 ctaggcgacg atccctagct ggtctgagag gatgaccagc cacactggaa ctgagacacg 300 gtccagactc ctacgggagg cagcagtggg gaatattgca caatgggcgc aagcctgatg 360 cagccatgcc gcgtgtatga agaaggcctt cgggttgtaa agtactttca gcgaggagga 420 aggcattgtg gttaataacc gcagtgattg acgttactcg cagaagaagc accggctaac 480 tccgtgccag cagccgcggt aatacggagg gtgcaagcgt taatcggaat tactgggcgt 540 aaagcgcacg caggcggtct gtcaagtcgg atgtgaaatc cccgggctca acctgggaac 600 tgcattcgaa actggcaggc tagagtcttg tagagggggg tagaattcca ggtgtagcgg 660 tgaaatgcgt agagatctgg aggaataccg gtggcgaagg cggccccctg gacaaagact 720 gacgctcagg tgcgaaagcg tggggagcaa acaggattag ataccctggt agtccacgcc 780 gtaaacgatg tcgacttgga ggttgtgccc ttgaggcgtg gcttccggag ctaacgcgtt 840 aagtcgaccg cctggggagt acggccgcaa ggttaaaact caaatgaatt gacgggggcc 900 cgcacaagcg gtggagcatg tggtttaatt cgatgcaccg cgaagaacct tacctactct 960 tgacatccag agaacttagc agagatgctt tggtgccttc gggaactctg agacaggtgc 1020 tgcatggctg tcgtcagctc gtgttgtgaa atgttgggtt aagtcccgca acgagcgcaa 1080 cccttatcct ttgttgccag cggttcggcc gggaactcaa aggagactgc cagtgataaa 1140 ctggaggaag gtggggatga cgtcaagtca tcatggccct tacgagtagg gctacacacg 1200 tgctacaatg gcatatacaa agagaagcga cctcgcgaga gcaagcggac ctcataaagt 1260 atgtcgtagt ccggatcgga gtctgcaact cgactccgtg aagtcggaat cgctagtaat 1320 cgtagatcag aatgctacgg tgaatacgtt cccgggcctt gtacacaccg cccgtcacac 1380 catgggagtg ggttgcaaaa gaagtaggta gcttaacctt cgggagggcg cttaccactt 1440 tgaaattcat gactg 1455 <210> 4 <211> 1452 <212> RNA <213> Klebsiella oxytoca <400> 4 ttagacgctg gcggcaggcc taacacatgc aagtcgaacg gtagcacaga gagcttgctc 60 tcgggtgacg agtggcggac gggtgagtaa tgtctgggaa actgcccgat ggagggggat 120 aactactgga aacggtagct aataccgcat aacgtcgcaa gaccaaagag ggggaccttc 180 gggcctcttg ccatcggatg tgcccagatg ggattagctt gtaggtgagg taacggctca 240 cctaggcgac gatccctagc tggtctgaga ggatgaccag ccacactgga actgagacac 300 ggtccagact cctacgggag gcagcagtgg ggaatattgc acaatgggcg caagcctgat 360 gcagccatgc cgcgtgtatg aagaaggcct tcgggttgta aagtactttc agcggggagg 420 aagggagtga ggttaatacc tcattcattg acgttacccg cagaagaagc accggctaac 480 tccgtgccag cagccgcggt aatacggagg gtgcaagcgt taatcggaat tactgggcgt 540 aaagcgcacg caggcggtct gtcaagtcgg atgtgaaatc cccgggctca acctgggaac 600 tgcattcgaa actggcaggc tggagtcttg tagagggggg tagaattcca ggtgtagcgg 660 tgaaatgcgt agagatctgg aggaataccg gtggcgaagg cggccccctg gacaaagact 720 gacgctcagg tgcgaaagcg tggggagcaa acaggattag ataccctggt agtccacgct 780 gtaaacgatg tcgacttgga ggttgttccc ttgaggagtg gcttccggag ctaacgcgtt 840 aagtcgaccg cctggggagt acggccgcaa ggttaaaact caaatgaatt gacgggggcc 900 cgcacaagcg gtggagcatg tggtttaatt cgatgcaacg cgaagaacct tacctactct 960 tgacatccac agaacttagc agagatgctt tggtgccttc gggaactgtg agacaggtgc 1020 tgcatggctg tcgtcagctc gtgttgtgaa atgttgggtt aagtcccgca acgagcgcaa 1080 cccttatcct ttgttgccag cgattaggtc gggaactcaa aggagactgc cagtgataaa 1140 ctggaggaag gtggggatga cgtcaagtca tcatggccct tacgagtagg gctacacacg 1200 tgctacaatg gcatatacaa agagaagcga cctcgcgaga gcaagcggac ctcataaagt 1260 atgtcgtagt ccggattgga gtctgcaact cgactccatg aagtcggaat cgctagtaat 1320 cgtggatcag aatgccacgg tgaatacgtt cccgggcctt gtacacaccg cccgtcacac 1380 catgggagtg ggttgcaaaa gaagtaggta gcttaacctt cgggagggcg cttaccactt 1440 tgtgattcag ta 1452 <210> 5 <211> 1213 <212> RNA <213> Lactobacillus acidophilus <400> 5 atcatgcaag tcgaacgaac tctggtattg attggtgctt gcatcatgat ttacatttga 60 gtgagtggcg aactggtgag taacacgtgg gaaacctgcc cagaagcggg ggataacacc 120 tggaaacaga tgctaatacc gcataacaac ttggaccgca tggtccgagt ttgaaagatg 180 gcttcggcta tcacttttgg atggtcccgc ggcgtattag ctagatggtg gggtaacggc 240 tcaccatggc aatgatacgt agccgacctg agagggtaat cggccacatt gggactgaga 300 cacggcccaa actcctacgg gaggcagcag tagggaatct tccacaatgg acgaaagtct 360 gatggagcaa cgccgcgtga gtgaagaagg gtttcggctc gtaaaactct gttgttaaag 420 aagaacatat ctgagagtaa ctgttcaggt attgacggta tttaaccaga aagccacggc 480 taactacgtg ccagcagccg cggtaatacg taggtggcaa gcgttgtccg gatttattgg 540 gcgtaaagcg agcgcaggcg gttttttaag tctgatgtga aagccttcgg ctcaaccgaa 600 gaagtgcatc ggaaactggg aaacttgagt gcagaagagg acagtggaac tccatgtgta 660 gcggtgaaat gcgtagatat atggaagaac accagtggcg aaggcggctg tctggtctgt 720 aactgacgct gaggctcgaa agtatgggta gcaaacagga ttagataccc tggtagtcca 780 taccgtaaac gatgaatgct aagtgttgga gggtttccgc ccttcagtgc tgcagctaac 840 gcattaagca ttccgcctgg ggagtacggc cgcaaggctg aaactcaaag gaattgacgg 900 gggcccgcac aagcggtgga gcatgtggtt taattcgaag ctacgcgaag aaccttacca 960 ggtcttgaca tactatgcaa atctaagaga ttagacgttc ccttcgggga catggataca 1020 ggtggtgcat ggttgtcgtc agctcgtgtc gtgagatgtt gggttaagtc ccgcaacgag 1080 cgcaaccctt attatcagtt gccagcatta agttgggcac tctggtgaga ctgccggtga 1140 caaaccggag gaaggtgggg atgacgtcaa atcatcatgc cgcttatgac ctgggctaca 1200 cacgtgctac aat 1213 <210> 6 <211> 1153 <212> RNA <213> Saccharomyces cerevisiae <400> 6 agctcgtagt tgaactttgg gcccggttgg ccggtccgat tttttcgtgt actggatttc 60 caacggggcc tttccttctg gctaaccttg agtccttgtg gctcttggcg aaccaggact 120 tttactttga aaaaattaga gtgttcaaag caggcgtatt gctcgaatat attagcatgg 180 aataatagaa taggacgttt ggttctattt tgttggtttc taggaccatc gtaatgatta 240 atagggacgg tcgggggcat cagtattcaa ttgtcagagg tgaaattctt ggatttattg 300 aagactaact actgcgaaag catttgccaa ggacgttttc attaatcaag aacgaaagtt 360 aggggatcga agatgatcag ataccgtcgt agtcttaacc ataaactatg ccgactaggg 420 atcgggtggt gtttttttaa tgacccactc ggcaccttac gagaaatcaa agtctttggg 480 ttctgggggg agtatggtcg caaggctgaa acttaaagga attgacggaa gggcaccacc 540 aggagtggag cctgcggctt aatttgactc aacacgggga aactcaccag gtccagacac 600 aataaggatt gacagattga gagctctttc ttgattttgt gggtggtggt gcatggccgt 660 tcttagttgg tggagtgatt tgtctgctta attgcgataa cgaacgagac cttaacctac 720 taaatagtgg tgctagcatt tgctggttat ccacttctta gagggactat cggtttcaag 780 ccgatggaag tttgaggcaa taacaggtct gtgatgccct tagacgttct gggccgcacg 840 cgcgctacac tgacggagcc agcgagtcta accttggccg agaggtcttg gtaatcttgt 900 gaaactccgt cgtgctgggg atagagcatt gtaattattg ctcttcaacg aggaattcct 960 agtaagcgca agtcatcagc ttgcgttgat tacgtccctg ccctttgtac acaccgcccg 1020 tcgctagtac cgattgaatg gcttagtgag gcctcaggat ctgcttagag aagggggcaa 1080 ctccatctca gagcggagaa tttggacaaa cttggtcatt tagaggaact aaaagtcgta 1140 acaaggtttc cgt 1153

Claims (6)

Bacillus subtilis CH, Pseudomonas chlororaphis, Enterobactor intermedius, Klebsiella oxytoca, Lactobacillus exocphilus and Lactobacillus acidophilus Water purification complex consisting of Saccharomyces cerevisiae. Bacillus subtilis CH, Pseudomonas chlororaphis, Enterobactor intermedius, Klebsiella oxytoca, Lactobacillus exocphilus and Lactobacillus acidophilus Complex strain for improving barn environment composed of Saccharomyces cerevisiae. 3. The method of claim 2,
The complex strain is a complex strain that can improve the barn environment by having the odor improvement and growth inhibition effect of animal pathogens. In the manufacturing method of the complex strain,
Firstly separating the strains for purifying water and generating noxious gases through centrifugation after culturing the strains separated from the soil in a nutrient medium;
Injecting the malodorous gas into the separated strains to identify and reconstruct the strains to remove the malodorous gas refining method of producing a complex strain for improving water quality and livestock environment. 5. The method of claim 4,
The complex strain,
Bacillus subtilis CH, Pseudomonas chlororaphis, Enterobactor intermedius, Klebsiella oxytoca, Lactobacillus exocphilus and Lactobacillus acidophilus Method for producing a complex strain consisting of Romeis serejese (Saccharomyces cerevisiae). In the culture method of mass culture of complex strains,
Culturing the complex strain for 24 to 72 hours in a medium containing 1 to 3% molasses, 1 to 3% beer foil, and 0.5 to 1% saline,
The complex strain, Bacillus subtilis CH, Pseudomonas chlororaphis, Enterobactor intermedius, Enterobactor intermedius, Klebsiella oxytoca, Lactobacillus exidophilus Lactobacillus acidophilus) and Saccharomyces cerevisiae is a mass culture method of a complex strain consisting of.

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