The present application relates to a feed composition and a method for producing the feed additive containing the feed additive, said additive comprising a Bacillus subtilis and Bacillus piece nipo miss.
[2]
BACKGROUND
[3]
Ruminants, such as cattle, if about 20%, dairy Wu in the case of the amount of the consumed roughage fed cattle feed accounts for about 60%, fed roughage is decomposed by microorganisms in the first ranking of the rumen needed nutrients and energy It is absorbed. From a nutritional / physiological aspects, ruminants must consume a large portion of the protein, energy, fatty acids, such as minerals and vitamins needed by the organization of micro-organisms and animals that live in the rumen from roughage. Science may contain grazing, hay or silage or more 20 to 30% crude fiber, and benefit from the dry state to the animal in the form of a consists of a relatively hard to break down hemicellulose, lignin, cellulose. Increasing the digestibility of this Science jyeotneunde are many studies in the past made to enhance the nutritive value representatively method physical treatment (immersion, grinding, pressurized steam treatment, swelling, gamma irradiation, pelleting) and how the chemical treatment (NaOH, element , the ammonia, lime, calcium hydroxide, potassium hydroxide, sodium carbonate, chlorine, ozone, hydrogen peroxide, etc.), such as biological treatment (fermentation, enzymatic treatment, silage). However, one method of physical treatment, there is a problem costly processing, chemical methods have a problem that can give the subject the risk of increase in processing costs as well as handling contaminating soil or changes in normal physiology of animals a situation that is avoided. So take advantage of a lot of biological treatment methods for adding microorganisms that produce enzymes and many such enzymes to break down cellulose and has been made a lot of research, especially for increasing the degradation rate of cellulose.
[4]
Wow dairy cattle to meet the energy demand and lack of forage alone, and pay with the concentrate has high volumetric energy content is less well digested than roughage to improve productivity. However, if a lot of benefits to concentrate and increase the concentration of lactic acid in the course of the starch is decomposed, which by drastically reducing the pH rumen and adversely affect the growth of the rumen beneficial microorganisms, and acidosis extends all the way to the feed intake decrease, extinguishing thereby causing the disorder, severe dehydration and diarrhea.
[5]
Therefore, the development of a feed additive to increase the resolution, cellulose and lactic acid are required.
[6]
[Prior art document]
[7]
[Patent Document]
[8]
Republic of Korea Patent No. 10-1721900 No.
[9]
Detailed Description of the Invention
SUMMARY
[10]
One purpose of the present application will be helpful in improving milk fat containing the Bacillus subtilis and Bacillus piece nipo miss, to increase the flow rate to provide a valuable feed additives.
[11]
Another object of the present application is to provide a feed composition comprising a feed additive comprising a Bacillus subtilis and Bacillus piece nipo Miss described herein.
[12]
Problem solving means
[13]
It will now be described in more detail for the present application. Information that are not listed herein is sufficiently recognized and understood if one skilled in the art or similar field of the present application and the description thereof will be omitted.
[14]
[15]
One aspect of the present application, the present invention relates to Bacillus subtilis and Bacillus milk fat and increase the flow rate for the feed to be processed comprises a nipo miss additives.
[16]
Bacillus subtilis seuneun digestive enzymes, for example, to produce cellulase, and / or meet azepin long as it can decompose cellulose, etc. may be used herein. Bacillus subtilis seuneun mainly habitat in the soil, miso, or red pepper paste, and Gram-positive bacteria, and aerobic bacillus. Examples of Bacillus subtilis, Bacillus subtilis may be mentioned CJBS62 (Bacillus subtilis CJBS62), or Bacillus subtilis CJBS16. In particular, you can use the Bacillus subtilis CJBS62. Bacillus subtilis is CJBS62 KCCM: - 2017. on (Korea Culture Center of Microorganisms Republic of Korea Seoul Seodaemun Hongje My -2 road, Yoo - Lim Building 45) 22. 6. deposited as people: was deposited to KCCM12039P. Bacillus subtilis 16s ribosomal nucleotide sequence of DNA (5 '→ 3') of CJBS62 strain is shown in SEQ ID NO: 3.
[17]
Bacillus piece nipo misses as long as they can be synthesized by the acid decomposition of lactic acid can be used herein. In one embodiment, the Bacillus piece case where the nipo misses in lactate containing medium 9 hours to about 48 hours, the Bacillus piece against the initial lactic acid amount acetate conversion of nipo miss is 30% to 70%, specifically 50% to 65% of one may have, or the Bacillus piece acetate conversion rate compared to lactic acid consumption by the nipo misses may be at least 50%, at least 60%, at least 70%, or 80%. The initial amount of lactate compared to the conversion rate may refer to expression 1 below, acetate conversion rate compared to the lactate consumption may refer to expression 2 below:
[18]
[Equation 1]
[19]
Initial lactate compared acetate conversion rate = (amount of acetic acid / lactate initial amount) * 100
[20]
[Formula 2]
[21]
Lactate consumption compared to the conversion rate (%) = (production amount of acetic acid / lactic acid consumed) * 100
[22]
[23]
Bacillus piece Miss nipo can be added to, cellulases and / or meet strains that can produce Ajay.
[24]
And Bacillus piece nipo Miss Rich, mainly in soybean or pepper paste, and Gram-positive bacteria, and aerobic bacillus. Examples of Bacillus piece nipo miss, Bacillus piece nipo miss CJBL215 ( Bacillus licheniformis CJBL215) and Bacillus piece nipo miss CJBL219 ( Bacillus licheniformis can be given CJBL219). In particular, you can use a piece of Bacillus nipo Miss CJBL219. Bacillus piece nipo Miss CJBL215 and Bacillus piece nipo Miss CJBL219 each KCCM: - Accession 22/06/2017-letter (Korea Culture Center of Microorganisms Republic of Korea Seoul Seodaemun Hongje My -2 road, Yoo - Lim Building 45) people: a KCCM12040P and KCCM12041P It was deposited. Bacillus piece nipo miss CJBL215 and Bacillus piece nipo miss CJBL219 of 16s ribosomal DNA of the nucleotide sequence (5 '→ 3') are shown in each of SEQ ID NOS: 1 and 2.
[25]
Feed additives containing, Bacillus subtilis and Bacillus piece nipo misses according to the present application, it is possible to stabilize the pH of the rumen ruminants, ruminant can be increased digestibility of animal feed. Further, the feed additive is not only effective for the improvement of milk fat in milk produced chakyuwoo, it is possible to increase the flow rate to be produced efficiently.
[26]
Feed additive Bacillus subtilis Bacillus piece nipo misses are, each independently per 1g, 1 x 10 7 cfu or more, for example 1 x 10 8 or more cfu, more specifically 1 x 10 9 may be included in more than cfu. When the concentration, it is possible to decompose fibrin effectively and provide effective feed additives in the milk fat increased.
[27]
The amount of the feed additive is, 0.1g to about 1kg / tablets / day, in particular 5g to 900g / tablets / day, more specifically from 10 g to 800 g / grain / 1, per day.
[28]
Bacillus weight ratio subtilis Bacillus piece nipo miss from 1: 9 to 9: 1, in particular from 2: 8 to 8: 2, in particular from 3: 7 to 7: 3, and more particularly 1: 2 to 2 : The first days.
[29]
Feed additive may be in liquid or solid, when liquefied, Bacillus subtilis, Bacillus piece nipo Miss, can be present in the cells, the culture thereof, or a concentrated liquid form thereof in Bacillus subtilis cells, Bacillus piece nipo misses the bus. When the solid phase of Bacillus cells of subtilis, Bacillus cells of the piece nipo miss, the culture thereof, or can include its concentrate such as, for example, a powder, tablet, pellet the manufacture by thermal or freeze-dried and it may have the form of granules, the coating agent.
[30]
Another aspect of the present application, relates to a feed composition comprising a feed additive as described herein. Content of the feed composition of the feed additive is in the range of 0.1% to 50% by weight, based on the weight of the feed composition, and specifically from 0.1% to 40% by weight, more specifically, it may be included in the range of 1% by weight to 20% by weight have.
[31]
Feed composition may comprise additional optional components such as In addition to feed additives of the present application, the effective ingredient in the growth promotion of animals, nutrients, nutritional supplements ingredient component to increase the storage stability, coating agent component. Feed composition, on the other probiotics; It enzymes such as amylase, lipase; Vitamins, such as L- ascorbic acid, choline chloride, inositol; Potassium citrate, iron oxide, magnesium oxide, minerals such as phosphates and the like; Amino acids lysine, alanine, methionine and the like; Acid, an organic acid or a salt thereof such as butyric acid, lactic acid; Antifungal agents such as antioxidants, calcium propionate such as vitamin C, vitamin E; Emulsifiers such as lecithin, glycerol fatty acid esters; It may include a pigment or the like.
[32]
Herein may be a food animal feed, may be a ruminant feed Specifically, examples of the ruminant may be a cow, buffalo, goat, sheep, goats, deer, or, without being limited thereto. The amount of that type of animal feed intake, body weight, age, gender, health status, and can be adjusted appropriately depending on the composition of the feed.
[33]
[34]
Yet another aspect of the present application, relates to a method of producing a feed additive as described herein, wherein the manufacturing method is Bacillus subtilis Bacillus piece nipo cultured miss, and culture of the strain cells, the culture thereof, or a It may include drying the concentrate. The culture may comprise from 30 to 50 ℃ ℃ After specifically inoculated with the strain, for 2 hours to 60 hours, e.g., from 32 ℃ to 40 ℃, cultured for 5 hours to 50 hours. The incubation is more specifically, it was inoculated a strain during the 30 ℃ to 50 ℃, 2 hours to about 60 hours, for example, the first at 32 ℃ to 40 ℃ for 5 hours to 50 hours incubation with the strain culture 1g 1 x 10 per 7 in cfu, and so that they are at, since the culture of maize, wheat blood, soybean meal, 30 ℃ to 50 ℃ by the addition of one or more raw materials of molasses in, for 2 hours to 60 hours, e.g. , it can include secondary cultured at 32 ℃ to 40 ℃ 5 hours to about 50 hours. The drying is, for the cultured cells, the culture thereof, or a concentrated solution thereof in 40 ℃ to 70 ℃ for 5 hours to 120 hours, e.g., from 40 ℃ to 60 ℃, may be to dry for 5 hours to 60 hours . The manufacturing method may include a step of pulverizing further. In one embodiment, after the drying.
[35]
Yet another aspect of the present application, relates to a method for increasing the milk fat and the flow rate of an animal comprising administering a feed additive comprising a Bacillus subtilis and Bacillus nipo piece misses the animal. A detailed description of this embodiment are the same as described in the other embodiments described herein.
[36]
Yet another aspect of the present application, relates to a method of stabilizing the pH of the rumen, animals, especially ruminants, which comprises administering a feed additive comprising a Bacillus subtilis and Bacillus nipo piece misses the animal. A detailed description of this embodiment are the same as described in the other embodiments described herein.
[37]
Yet another aspect of this application, to a animal, in particular increase the digestibility of ruminant method comprising administering a feed additive comprising a Bacillus subtilis and Bacillus nipo piece misses the animal. A detailed description of this embodiment are the same as described in the other embodiments described herein.
[38]
Effects of the Invention
[39]
Feed additives according to the present application may improve fat reduction phenomenon of the summer heat stress, as well as to help the animal, especially milk fat, the improvement of ruminant animals. Moreover, it is effective in the rumen within the lactate decomposition is expected to be useful in ruminal pH decreased and this gosanjeung prevention caused due to lactic acid.
[40]
Brief Description of the Drawings
[41]
1 is an electron micrograph of a Bacillus subtilis strain CJBS62, Bacillus piece nipo miss CJBL215, CJBL219 strain in accordance with an embodiment of the present application.
[42]
Figure 2 shows a color change of the medium used for selection of the lactic acid consuming isolates In one embodiment of the present application. Here, the color of the medium turns purple (RHS) in the yellow (left side) was judged by the consumption of the medium the strain of lactic acid.
[43]
Figure 3 is a bar graph showing the conversion rates according to the embodiment of the present application, in each strain per acetate.
[44]
Figure 4 shows the conversion rate (right y axis) in the present In an embodiment of the application, 9h, according to the 48h incubation, Bacillus piece nipo miss CJBL215, lactic amount of CJBL219 strain acetate quantity (left y axis) and the acetate a graph.
[45]
5 is a photograph showing the results of hemolytic make Bacillus subtilis CJBS62 strain, Bacillus piece nipo miss CJBL215, CJBL219 strain in accordance with an embodiment of the present application.
[46]
6 is a graph showing the change in lactate concentration due to changes in the incubation time when added to a food enriched feed additive in accordance with one embodiment of the present application.
[47]
7 is a graph showing the change in acetate concentration in accordance with changes in the incubation time when added to a food enriched feed additive in accordance with one embodiment of the present application.
[48]
Figure 8 is a bar graph showing the addition of a pH stabilizing effect on the feed enriched feed additive in accordance with one embodiment of the present application compared to the control.
[49]
Figure 9 is a bar graph showing the degree of improvement in digestibility added to the feed enriched feed additive in accordance with one embodiment of the present application.
[50]
Figure 10 is a bar graph showing the improved digestibility when added to the feed additive to feed TMR degree in accordance with an embodiment of the present application.
[51]
Mode for the Invention
[52]
Hereinafter, one would like to be described in detail with reference to examples of the present application, this is not a may make the present application for a better understanding of the present application is not limited thereto.
[53]
[54]
EXAMPLES
[55]

[56]
(1) sample acquisition and isolates
[57]
To obtain a sample, such as the traditional soy sauce paste, hot pepper paste, by diluting the samples obtained in steps BHI (Brain Heart Infusion, Difco) at 37 ℃ then plated on solid medium and allowed to incubate for 24 hours. They were separated and the pure isolates from each sample in a way to move the culture in fresh medium, separately packed a pure culture in a 20 weight% glycerol, based on the weight of the total addition of the medium was collected and stored at below -70 ℃. The method of decomposing the cellulase activity and is excellent in strain, the lactic acid of the following to separate the superior strains ability to produce acetic acid.
[58]
[59]
(2) Morphological and biochemical characterization
[60]
For the identification of the separated strain it was primarily perform the morphological and biochemical research. Were positive for all of the separated Gram stain results in morphological characteristics Gram strain, it was confirmed that the electron microscope photography result, bacilli (Fig. 1).
[61]
In order to analyze the biochemical properties of the strain by using the API 50 CHB system (biomerieux Vitek, Inc, France), to analyze the sugar fermentation pattern of each strain it is shown in Table 1 below.
[62]
TABLE 1
Party result Party result
CJBL215 CJBL219 CJBS62 CJBL215 CJBL219 CJBS62
control - - - Esculine + + +
Glycerol + + + salicine + + -
Ertythritol - - - Cellobiose + + -
D-Arabinose - - - Maltose + + +
L-Arabinose + + + Lactose - + -
Ribose + + + Melibiose - - -
D-Xylose + - - Saccharose + + +
L-Xylose - - - Trehalose + + +
adonitol - - - Inuline - - +
β Methyl-xyloside - - - Melezitose - - -
Galactose - + - D-Raffinose + - +
D-Glucose + + + Starch + + +
D-Fructose + + + Glycogene + + -
D-Mannose + + - Xylitol - - -
L-sorbose - - - β Gentiobiose - - -
Rhamnose - + - D-Turanose + + -
Dulcitol - - - D-Lyxose - - -
Inositol + - + D-Tagatose + + -
Mannitol + + + D-Fucose - - -
Sorbitol - + + L-Fucose - - -
α Methyl-D-mannoside - - - D-Arabitol - - -
α Methyl-D-glucoside + + - L-Arabitol - - -
N Acetyl gllucosamine - - - Gluconate - - -
Amygdaline + + - 2 ceto-gluconate - - -
Arbutine + + - 5 class-gluconate - - -

[63]
+ Positive, - negative
[64]
[65]
Analysis of the fermentation pattern of each strain, CJBL215 was found to belong to the were found to belong to the Bacillus piece nipo miss (99.7% confidence), CJBL219 FIG Bacillus piece nipo miss (99.9% confidence). Also CJBS62 was found to belong to the Bacillus subtilis / amyl Lowry kwipen Precious (99.6% confidence).
[66]
[67]
(3) Strain Identification
[68]
For accurate identification than the strain it was subjected to molecular taxonomic system method according to the DNA base sequence. Sequencing using a PCR premix (Bioneer, Republic of Korea) and universal primers 27F (5 'AGAGTTTGATCMTGGCTCAG 3') and 1492R (5 'GGTTACCTTGTTACGACTT 3') was the gene amplification of the 16s rDNA. To the 72 ℃ 1 minutes bun 94 ℃, 1 minutes 56 ℃, according to the total reaction solution is 20 ㎕ during gene amplification repeat of 30 times, and analyzing the amplified DNA sequence. Nucleotide sequence of the 16s rDNA analysis of the isolated strains are shown in SEQ ID NO: 1-3. The analysis results, and CJBL215 CJBL219 is Bacillus piece nipo miss, CJBS62 was identified as a microorganism having a Bacillus subtilis 99% homology. In the above the strains were each designated as "Bacillus piece nipo CJBL215 miss", "Bacillus piece nipo CJBL219 miss", "Bacillus subtilis CJBS62 ' . The novel microorganism of the present invention as identified in the same manner as described above were deposited with the Bacillus piece nipo miss CJBL215, Bacillus piece nipo miss CJBL219, Bacillus subtilis CJBS62 respectively June 22, dated 2017 in Korea Culture Center of Microorganisms (KCCM) Name It was deposited by, KCCM12040P, KCCM12041P, KCCM12039P.
[69]
[70]

[71]
(1) digestive enzyme activity of the strains
[72]
To screen a strain with a compound digestive enzyme activity, to meet for the separated soy sauce-derived strain was subjected to activity evaluation of the kinase (mannase), cellulase in combination. Enzyme activity was evaluated measuring the activity capability in accordance with the clear zone (clear zone) formed approximately by using a medium containing the substrate for the respective enzyme.
[73]
Wherein a strain isolated BHI (Brain Heart Infusion, Difco) 24 hours after culture in liquid medium were collected and the resulting culture was used as a crude enzyme solution for enzyme activity analysis, using a medium containing the respective substrates for each enzyme as shown below It was evaluated by the degree of matrix degradation.
[74]
1) meet dehydratase activity measurement
[75]
1% met (logust bean gum, sigma, USA ) is added to a substrate medium (Yeast extract 3g / L, Peptone 5g / L, KH 2 PO 4 to 1g / L, Agar 20g / L , Ph 5) was prepared. After instillation of the crude enzyme solution to 1.5 by ㎕ substrate medium, after 15 ~ 18 hours at 37 ℃ according to the degree of clear zone formation was measured the ability of the enzyme activity, the results are shown in Table 2.
[76]
2) The cellulase activity was measured
[77]
1% CMC (carboxymethylcellulose) was prepared in the substrate is added to YM medium. By dropwise the crude enzyme solution to a substrate medium by ㎕ 1.5 was reacted for 15 to 18 hours at 37 ℃. For measuring the clear zone it was stained for 30 minutes with 0.2% Congo Red (Congo red) solution was decolorized with 1M NaCl solution. Depending on the transparent ring is formed around the substrate occurs around the strain decomposes We measured the ability of the enzyme activity, the results are shown in Table 2.
[78]
Be of the starter strains were selected on the basis of the evaluation of the cellulase activity the best Bacillus subtilis CJBS16, CJBS62 strain and excellent dehydratase activity.
[79]
Table 2 digestive enzyme activity of the selected strain (unit: mm)
　 CJBS16 CJBL215 CJBL219 CJBS62
Meet Ajay 5 0 2.5 3.5
Cellulase 2 3 2 3.5

[80]
(2) cellulose-degrading activity in the culture supernatant
[81]
The supernatant to determine the resolution of the cellulose in the culture supernatant of the selected strain, culturing the strain for 24 hours and separated for 5 minutes and centrifuged at 10,000rpm was filtered using a syringe filter 0.2㎛. The supernatant dropping a drop Cellulose is 20㎕ a containing medium (Carboxymethylcellulose sodium salt 10g / L, Bacto agar 15g / L) and stained after 1 days reaction at 37 ℃, pour 0.4% Congo red solution for 20 minutes. After staining with decolorized with 1M NaCl solution to measure the transparent ring size was confirmed cellulose resolution in the culture supernatant, the results are shown in Table 3. The starter CJBS62 Bacillus subtilis strain was confirmed in the culture supernatant of the most excellent evaluation.
[82]
Table 3 in a cellulose-decomposing activity of the culture supernatant starting strain (unit: mm)
　 CJBS16 CJBL215 CJBL219 CJBS62
Cellulose-decomposing activity 16.5 16.5 14 24

[83]

[84]
To screen a strain that consumes lactic acid, and then cultured using a medium which was jeotsanreul determine the presence or absence of lactic acid consumption by the BCP coloring method with a qualitative method. A medium (15mM lactate, Yeast extract 10g / L, Peptone 20g / L, NaCl 10g / L, Bromophenol blue (BCP) 0.0004g / L) containing Bromophenol blue (BCP) and 15mM lactate for color development was prepared. After dispensing the medium to 1.5ml by Microcentrifuge tube, were each inoculated 50㎕ the former strain cultured in BHI (Brain Heart Infusion, Difco) medium. At 37 ℃ of the strain in the tube and incubated 4-5 days value. It determines turns purple in the medium of the color yellow by observing the color of the medium that consume lactic acid in the medium was first screened lactic acid consuming bacteria away. After a culture of the strain isolated from a sample note 177, he was selected CJBS16, CJBL215, CJBL219, CJBS62 of four weeks (Fig. 2).
[85]
The starter was inoculated into the CJBS16, CJBL215, CJBL219 and CJBS62 in BHI (Brain Heart Infusion, Difco) medium and activated with the bacteria for 16 hours incubation at 37 ℃, 200rpm. To a medium containing 15mM of lactic acid (lactic acid 15mM, Yeast extract 10g / L, Peptone 20g / L, NaCl 10g / L) inoculated with the fungus 5%, 37 ℃, was 48 hours at 200rpm. Addition of 10% BCP in the culture liquid after completion of culture was confirmed by the presence of lactic acid consuming once again. It was confirmed that all the 4 strains of lactic acid consuming (culture media color change: yellow -> violet).
[86]
[87]

[88]
(1) Measurement acetate yield
[89]
The starter was inoculated into the CJBS16, CJBL215, CJBL219 and CJBS62 in BHI (Brain Heart Infusion, Difco) medium and activated with the bacteria for 16 hours incubation at 37 ℃, 200rpm. To a medium containing 15mM of lactic acid (lactic acid 15mM, Yeast extract 10g / L, Peptone 20g / L, NaCl 10g / L) inoculated with the fungus 5%, 37 ℃, was 48 hours at 200rpm.
[90]
Of 25% metaphosphoric acid 0.2ml to 1ml supernatant was recovered by separating the culture medium centrifuged is added. After filtering, the supernatant was recovered and separated for 5 minutes and centrifuged at 10,000rpm with 0.2um filter was analyzed acetate content using a GC (Agilent Technologies 7890A).
[91]
To consume lactic acid strains for the 4 species which consume lactic content was measured by generating an acetate, it was converted by the conversion to the formula (1). As a result, the conversion rate was of CJBL219 strains the highest conversion rate by 59.4%, the conversion of CJBL215 strain was 44.9% (Table 4, Fig. 3).
[92]
[Equation 1]
[93]
Initial lactate compared acetate conversion rate = (amount of acetic acid / lactate initial amount.) * 100
[94]
TABLE 4 medium acetate production and the conversion rate after in lactate consumption
Strain No. Acetate content (mM) Conversion by the formula (1) (%)
CJBL219 8.91 59.4
CJBL215 6.74 44.9
CJBS16 3.74 24.9
CJBS62 1.99 13.3

[95]
(2) Measurement of lactic acid consumption and production of acetate starter strain
[96]
It was to quantify the consumption of lactate and acetate production in culture medium of selected strains.
[97]
The starter was inoculated with CJBS16, CJBL215, CJBL219 and CJBS62 in BHI (Brain Heart Infusion, Difco) medium, was activated with the bacteria for 16 hours incubation at 37 ℃, 200rpm.
[98]
Was inoculated to the cultured bacteria containing 15mM lactate medium (15mM lactate, Yeast extract 10g / L, Peptone 20g / L, NaCl 10g / L) 5% by weight, while 48 hours at 37 ℃, 200rpm, 9 sigan , were each sampled at 48 hours.
[99]
1) Determination of Lactic Acid
[100]
Was prepared by filtering a supernatant was recovered by centrifuging the culture solution to 0.2um filter. Put the filtered culture supernatants to the microcentrifuge tube using a bio lactate (ROCHE) equipment of Bio Cedex (ROCHE) were measured lactic acid content, the results are shown in Table 5.
[101]
[102]
Determination of 2) acetate
[103]
Of 25% metaphosphoric acid 0.2ml to 1ml supernatant was recovered by separating the culture medium centrifuged is added. After filtering, the supernatant was recovered and separated for 5 minutes and centrifuged at 10,000rpm with 0.2um filter were analyzed acetate content using a GC (Agilent Technologies 7890A), the results are shown in Table 5, FIGS.
[104]
[Formula 2]
[105]
The lactate consumption compared acetate conversion rate = (amount of acetic acid / lactic acid consumption) x 100
[106]
Referring to Table 5, CJBL215 strain during 48 hours, was from the initial amount of lactic acid consuming lactic acid of 49.8%, was converted about 91.1% of the lactic acid consuming acetate. CJBL219 strain was consumed during 48 hours of incubation, 48.6% of lactic acid from the initial amount of lactic acid, it was converted to the acetate, both the consumption of lactic acid (Fig. 4).
[107]
Table 5 lactic acid content in the culture medium with the passage of time, acetate content and conversion rates
　 0h 9h 48h
CJBL215 Lactic acid amount (mM) 13.12 8.35 6.59
Acetate amount (mM) 0 2.50 5.94
Conversion by the formula (2) (%) 　 52.4 91.1
CJBL219 Lactic acid amount (mM) 13.12 8.52 6.74
Acetate amount (mM) 0 2.87 6.49
Conversion by the formula (2) (%) 　 62.4 101.8

[108]
[109]
< Example 5: Safety of the strain>
[110]
(1) Identification of hemolytic strains
[111]
β- hemolytic is the function of hemolysis of red blood cells by the hydrolysis of phospholipids which is supplied by the red blood cells to produce a phospholipid enzyme of harmful bacteria. Blood agar plate medium (sheep blood 5%, ㈜ one day co-Med, Republic of Korea) was used to examine the hemolytic isolates. After inoculation linear (streaking) on ​​the prepared blood agar plate medium, a result confirming whether hemolysis by 24 hours incubation at 37 ℃, it was confirmed that no exhibit hemolysis as shown in FIG.
[112]
[113]
(2) determine antibiotic sensitivity of strain
[114]
To determine the antibiotic susceptibility of the selected strains was the experiment. Strain starter was inoculated in BHI (Brain Heart Infusion, Difco) medium and cultured for 16 hours at 37 ℃, 200rpm. And smeared on a wet sterile the culture swab Mueller Hinton Ⅱ Agar plate (Difco). And incubated for 18 hours at 15 ~ 37 ℃ the bacteria are plated plate medium up an antibiotic disc. For testing antibiotics Prepare Ampicillin, Clindamycin, Gentamicin, Kanamycin, Tetracyclin, Vancomycin, Erythromycin, Ampcillin / Sulbactam, Chloramphenicol, Streptomycin disk (OXOID) experiment was performed. After incubation with the growth inhibition zone (clear zone) formed approximately around the antibiotic disks it was confirmed that antibiotic sensitivity. Result, the starter strain for antimicrobial susceptibility testing of selected strains were identified as not having resistance to the above antibiotics (Table 6).
[115]
Table 6 Growth inhibition of the strains according to antibiotics
Antibiotics (Antibiotics) A growth inhibition zone around the antibiotic radius (mm)
CJBL215 CJBL219 CJBS62
Amp10 (Ampicillin) 10 8 10
C30 (Clindamycin) 14 4 9
CN120 (Gentamicin) 18 12 12
K30 (Kanamycin) 13 9 10
TE30 (Tetracycline) 5 8 12
VA30 (Vancomycin) 9 6 7
E15 (Erythromycin) 13 13 11
SAM20 (Ampicillin/Sulbactam) 15 12 13
S10 (Chloramphenicol) 3 3 6
DA2 (Streptomycin) 6 7 9

[116]
[117]
< Example 6. Preparation of a feed additive containing the strain>
[118]
The Bacillus strain CJBL215, CJBL219 and CJBL62 ( Bacillus licheniformis 2 jong, Bacillus subtilis 1 species) each Tryptic Soy broth which was then incubated at 36 ℃ 36 sigan inoculated to 9L. Tryptic, 1 x 10 per 1g of the strain when the strain and plated to Soy agar medium measure the number of colonies 9 each strain was cultured until the above cfu. In addition to the solid-state fermentation ingredients are prepared the corn 20kg, wheat blood 30kg, 45kg soybean meal, molasses and 5kg mixed mixture. Making a total of 27L of culture liquid mixture was mixed by 9 L each of the culture media of the cultured strain Bacillus three kinds, was added to the solid phase fermentation ingredients 100kg. With stirring to allow the strain to be evenly fermented in the solid state fermentation ingredients it was fermented at 34 ℃ temperature for 48 hours. After fermentation is complete, after the rough drying process for 48 hours at 50 ℃ temperature pulverized to obtain a feed additive.
[119]
[120]
< Example 7. The feed substrate specific rumen effect measurements on the aqueous phase of the fermentation additives>
[121]
To, dry matter digestibility, and improve promotion ethyl effect The experiments were conducted using the static culture rumen model system as follows.
[122]
* Treatment group: was added to the substrate of the feed additive and 0.5g 50mg prepared in Example 6 in 200ml serum bottle and maintain an anaerobic condition in which the buffer and 37.5ml 12.5ml ruminant gastric juice reduced to carbon dioxide gas to carbon dioxide gas. Was filled with a carbon dioxide gas for about 30 seconds, the rubber stopper pieces serum blocking the bottle inlet and sealed with aluminum caps. This was incubated for 24 hours at 39 ℃ value incubator. At this time substrate was used as a concentrate or TMR (Total Mixed Ration, Total Mixed Ration), feed additive was added as a substrate ratio of 10 wt% per 0.5g. Buffer solution sodium hydrogen phosphate (Sodium Phosphate, monobasic, NaH 2 PO 4 -2H 2 O) 9.3g / L, sodium bicarbonate (Sodium bicarbonate, NaHCO 3 ) 9.8g / L, NaCl (Sodium chloride, NaCl) 0.47g / L, potassium (potassium chloride, KCl) 0.57g / L, magnesium chloride (magnesium chloride, MgCl 2 ) 0.256g / L, calcium chloride (calcium chloride, CaCl 2) A 0.106g / L, casein (Caseine, NZ-Amine) 2.5g / L, and Reza hungry (resazurine) solution 1.25ml / L was prepared by mixing.
[123]
* Control: the non-feed additive is added to the substrate was prepared, and was incubated under the same conditions as the treatment group.
[124]
After the treatment groups and the control were incubated at 39 ℃ incubator value, it was measured for each of the lactic acid content, acetate content, pH, and the dry matter digestibility in the following way:
[125]
Lactic acid content after filtering the supernatant was recovered by centrifuging the culture solution to 0.2um filter put in the microcentrifuge tube was measured using a lactate bio (ROCHE) equipment of Bio Cedex (ROCHE). Acetate content is then filtered and the supernatant was added to 25% metaphosphoric acid to a 0.2ml 1ml supernatant recovered by centrifuging the culture broth, and recovering the separated for 5 minutes and centrifuged at 10,000rpm with 0.2um filter using a GC (Agilent Technologies 7890A) It was measured. Dry matter digestibility was calculated by the following equation (3).
[126]
[Equation 3]
[127]
Dry matter digestibility (%) = (amount of culture before substrate - after the culture substrate volume) / I culture substrate amount * 100
[128]
The "culture substrate prior amount" or "amount after the culture substrate" is, after the culture broth after the culture or before the filter using a vacuum pump to Filter paper was measured by drying (60 ℃, overnight).
[129]
[130]
In the treatment group and control group, and showed a change in the concentration of lactate and acetate variation of the measurement depending on the incubation time in Fig. 6 and 7, respectively.
[131]
[132]
Referring to Figure 6, the minimum concentration of lactic acid in the treatment is about 10% lower than the minimum concentration of lactic acid in the control group. Further, referring to Figure 7, the higher the maximum concentration of acetate in the treatment is about 2.5 times the maximum concentration of acetate in the control group.
[133]
Each of the treatment groups and the control of pH and dry matter digestibility (%), Figure 8, are shown in FIGS.
[134]
Referring to FIG. 8, pH of 0.5, but the control was reduced compared to the initial, pH of the treatment groups could be a bar, feed additives according to the present application hayeotneun 0.42 decreased compared to the initial check indicates the effect on the rumen stability.
[135]
In the case 9, and referring to Figure 10, with the concentrate and TMR respectively on the substrate, it was determined to be dry matter digestibility of dry matter digestibility in the control treated group compared with the high 4% and 1.3%. That is, the feed additive according to the present application means that can have a positive effect on the bar, which increases milk fat which can not only to improve the digestibility in the rumen, to increase the amount of acetic acid synthesis.
[136]
[137]
< Example 8. The feed additive for promoting milk fat of chakyuwoo Effects on Performance>
[138]
To, the, feed additives according to the present application used for chakyuwoo feeding trial was produced by the method mentioned in Example 6. Via the well-known dairy feeding trial to evaluate the effect of the present application, feed additives on the milk productivity of chakyuwoo. Was placed a chakyuwoo 72 both to the control (36 both) and treatment (36 two), the control during the 4-week test period, by administering only conventional feed and treatment, the per capita 20g / day the feed additive according to the present application as a top dressing form It was fed conventional feed mix. Disclosed before by measuring the milk fat, milk protein and a flow rate after benefit results are shown in Table 7, Table 8, Table 9.
[139]
The feed additive was found to be a continuous increase of per capita 20g / day by the sustain discharge as shown in a benefit to the results, and Table 7 to 0.3% p chakyuwoo. Also as seen in Table 8. In the control group there were no changes in the milk proteins, it was possible to make a 0.1% p improvement in the treated group. In the control group in the measurement of flow rate, there was no change in the treated group increased 0.4kg (Table 9).
[140]
Through the above results, feed additives according to the present application was confirmed that the milk fat, milk protein and flow rate may both be improved positively, as a result, to increase the milk production and milk quality of chakyuwoo.
[141]
TABLE 7 Improvement of milk fat feed additive effect
　 Control Treatment
Before the start (3 Average) 4.7 % 4.7 %
After salary (average of 4 weeks) 4.7 % 5.0 %
Milk fat improving effect ( % p ) 0.0 0.3

[142]
[143]
[Table 8] milk protein improving effect of the feed additive
　 Control Treatment
Before the start (3 Average) 3.4 % 3.2 %
After salary (average of 4 weeks) 3.4 % 3.3 %
Milk protein improving effect ( % p ) 0.0 0.1

[144]
[145]
Table 9 Flow improvement in feed additives
　 Control Treatment
Before the start (3 Average) 27.9 kg 31.9 kg
After salary (average of 4 weeks) 28.0 kg 32.3 kg
Flow rate improving effect (kg) 0.1 0.4

[146]
[147]
< Example 9. The feed additive for promoting fat summer high temperature stress to chakyuwoo productivity Effect>
[148]
To, the, feed additives according to the present application used for chakyuwoo feeding trial was produced by the method mentioned in Example 6. Via the well-known dairy specification test, the feed additive according to the present application in a high temperature environment stress were evaluated for effect on the milk production of chakyuwoo. Typically the dairy reversed heat stress by selection of a period of exposure to the environment (May 26 to June 23, four weeks), feed according to the present application to chakyuwoo 150 both during the period additive is fed a feed containing 0.2wt% salaries were compared before / after.
[149]
For 4 weeks in the absence of heat stress environment, any feed additives also have been feed sets the salary chakyuwoo as controls are not included, for 4 weeks in the heat stress environment, the feed additive of the feed including 0.2wt% of Feeding chakyuwoo a processing port was set. The basal diet composition of the benefit to each treatment group and the control group was the same.
[150]
[Table 10] Effect of feed additives on the oil production in summer
butterfat flux
Control 3.7 % 36.2 kg
Treatment 3.9 % 36.7 kg
Improvements 0.2 %p 0.5 kg

[151]
Referring to Table 10, compared to the milk fat, and the flow rate measured in the control group, the milk fat and the flow rate measured in the treatment groups could be confirmed that each increase of 0.2% p and 0.5kg.
[152]
Typically when chakyuwoo is exposed to heat stress environment, it is common to reduce the flow rate and the milk fat is produced from chakyuwoo. However, in the chakyuwoo feed additive is provided in accordance with the present invention, even when exposed to a high temperature environment, stress does not appear and the flow rate of milk fat and reduced development, but rather there is a milk fat, and the flow rate can be improved.

WE Claims

[Claim 1]
Bacillus subtilis and milk fat and flow promoting feed additive comprising a Bacillus piece nipo miss.
[Claim 2]
The method of claim 1, wherein the Bacillus subtilis Bacillus subtilis seuneun CJBS62 ( Bacillus subtilis CJBS62 (KCCM12039P)), feed additive for milk fat and increase the flow rate comprising a.
[Claim 3]
The method of claim 1, wherein the Bacillus piece nipo Miss Bacillus piece nipo miss CJBL215 ( Bacillus licheniformis CJBL215 (KCCM12040P)) and Bacillus piece nipo miss CJBL219 ( Bacillus licheniformis , including at least one selected from the group consisting of CJBL219 (KCCM12041P)) feed additive for, milk fat and increase the flow rate.
[Claim 4]
2. The method of claim 1, wherein the feed additive Bacillus subtilis and Bacillus piece nipo miss each 1g per 1 x 10 7 cfu, feed additive for milk fat and increase the flow rate comprises at least.
[Claim 5]
The method of claim 1, wherein the Bacillus piece nipo miss, is to lactic acid is contained cultured in a medium 9 to 48 hours generate acetates, acetate conversion rate compared to the initial lactic acid rate of the Bacillus piece nipo miss is 30% to 70%, milk fat and increase the flow rate for the feed additive.
[Claim 6]
The method of claim 1, wherein the Bacillus subtilis and Bacillus said piece is blended with a weight ratio of nipo Miss 1: additive 1, feed for milk fat and increase flow rate: 9 to 9.
[Claim 7]
Wherein the first to fourth feed composition comprising a feed additive according to any one of items.