KR20120126917A - Bacillus subtilis IN-55, Lactobacillus plantarum BT-77 and Method for Manufacturing Silage Using the Same - Google Patents

Abstract

PURPOSE: A novel Bacillus subtilis IN-55, Lactobacillus plantarum BT-77, and a method for preparing plant silage using the same are provided to be used as an additive for silage fermentation and to improve livestock product quality. CONSTITUTION: A microorganism additive for fermenting plant silage contains Bacillus subtilis IN-55 (KCTC 11914BP) or Lactobacillus plantarum BT-77 (KCTC 11915BP). The plant is rice straw, ryegrass, whole barley, whole rice, wheat, or corn. A method for producing the plant silage comprises a step of adding Bacillus subtilis IN-55 (KCTC 11914BP) or Lactobacillus plantarum BT-77 (KCTC 11915BP) to a plant. [Reference numerals] (AA) Rice straw; (BB) Rice straw + TGY media; (CC) Rice straw + TGY media + IN-55; (DD) Rice straw + TGY media + BT-77; (EE) Rice straw + TGY media + IN-55 + BT-77

Description

Bacillus subtilis IN-55 and Lactobacillus plantarum BT-77 and Method for Manufacturing Silage Using the Same

The present invention relates to a novel Bacillus subtilis IN-55, Lactobacillus plantarum BT-77 and a method for producing plant silage using the same.

Forages are produced in large quantities at one time, so they need to be stored for stable feeding throughout the year. The dehydrated storage is hay, while the silage is silage.

Silage sharply lowers the acidity by producing normal lactic acid fermentation in the anaerobic state of lactic acid bacteria in plants or soil, thereby inhibiting the reproduction of other aerobic bacteria in the early stages of fermentation and continuing anaerobic initiation. By inhibiting fermentation of butyric acid, the shelf life, nutrition and palatability of the feed are enhanced. However, it is difficult to obtain a silage of uniform quality depending on the growth state of the crop during the silage production, weather conditions, and the like. Therefore, in order to solve this drawback is used inocurant (additives for silage), by adding the inoculant can improve the quality of the silage and improve the productivity of the livestock (milk and meat productivity).

Innocurant, an additive for silage, has been mostly used in foreign countries, depending on imports.In recent years, as the preparation and use of silage has increased, awareness of the need for additives to improve silage quality has spread. Research is underway to develop additives. In particular, it is necessary to develop additives that effectively lower the initial pH during silage fermentation to promote normal lactic acid fermentation, thereby improving fermentation efficiency and feed storage, as well as improving nutrition, palatability and digestibility of feed.

The present invention provides a novel silage fermentation microorganism and a plant silage using the same for producing excellent feed having a favorable palatability improvement effect, an antimicrobial effect on pathogenic bacteria and an improvement in digestibility due to enzyme products at low cost in manufacturing plant silage. To provide a manufacturing method.

In order to solve the above problems, the present invention Bacillus subtilis ( Bacillus subtilis) IN-55 (KCTC 11914BP ) and Lactobacillus strains Planta Room (Lactobacillus plantarum ) provides the BT-77 (KCTC 11915BP) strain.

The present invention also provides a microbial additive for plant silage fermentation comprising at least one of Bacillus subtilis IN-55 (KCTC 11914BP) and Lactobacillus plantarum BT-77 (KCTC 11915BP).

In another aspect, the present invention provides a method for producing plant silage, characterized by adding at least one of Bacillus subtilis IN-55 (KCTC 11914BP) and Lactobacillus plantarum BT-77 (KCTC 11915BP) to the plant.

Bacillus subtilis IN-55 according to the present invention And by using Lactobacillus plantarum BT-77 as an additive for plant silage fermentation, it is possible to improve the quality of livestock by improving the digestibility of plant silage, antibacterial effect and palatability, and also to prevent the environmental pollution that occurs during livestock raising. It is accompanied by such effects.

1 is a diagram showing 2192 bp of 16S rRNA sequences analyzed for identification of Bacillus subtilis IN-55 strain.
Figure 2 shows the 601 bp of 16S rRNA sequences analyzed for the identification of Lactobacillus plantarum BT-77 strain.
3 is a diagram showing the protease, cellulase and amylase enzyme activity of Bacillus subtilis IN-55.
Figure 4 shows the cellulase enzyme activity of Lactobacillus plantarum BT-77.
Figure 5 Listeria of Bacillus subtilis IN-55 culture supernatant This figure shows the antibacterial activity against monocytogenes .
Figure 6 shows the antimicrobial activity of various pathogenic bacteria of the Lactobacillus plantarum BT-77 culture supernatant.
7 is an electron micrograph showing the surface of rice straw after silage preparation using Bacillus subtilis IN-55 and Lactobacillus plantarum BT-77.
Figure 8 is a photograph of observation of the decomposition of rice straw by the strain after the production of silage using Bacillus subtilis IN-55 and Lactobacillus plantarum BT-77 by electron microscope.

The present invention Bacillus subtilis ( Bacillus) is a novel microorganism suitable for plant silage fermentation subtilis ) IN-55 (KCTC 11914BP) and Lactobacillus plantarum BT-77 (KCTC 11915BP).

The inventors have isolated the novel Bacillus subtilis and Lactobacillus plantarum strains showing strong cellulase enzyme activity from food and soil; Assaying the enzyme activity of the isolated Bacillus subtilis and Lactobacillus plantarum strains; Identifying the isolated new microorganisms Bacillus subtilis and Lactobacillus plantarum strains; Assaying plant silage fermentation using the isolated Bacillus subtilis and Lactobacillus plantarum strains; Pot experiment using the separated silage fermentation Bacillus subtilis and Lactobacillus plantarum and assay step through electron micrograph; And through the mass culturing of the isolated Bacillus subtilis and Lactobacillus plantarum strains, a new microorganism suitable for plant silage fermentation, Lactobacillus plantarum BT-77 (Accession No. KCTC 11915BP) and Bacillus subtilis IN -55 (Accession No. KCTC 11914BP) was obtained.

As can be seen through the following examples, Bacillus subtilis IN-55 (KCTC 11914BP) and Lactobacillus plantarum BT-77 (KCTC 11915BP) according to the present invention has an excellent pH lowering effect when used as an additive for plant silage fermentation In addition, due to the increase in fermentation efficiency, it is possible to effectively suppress the propagation of other pathogenic bacteria, thereby improving feed storage, and inhibiting the growth of pathogenic bacteria to reduce proteolysis, thereby inhibiting the production of ammonia nitrogen and the like. Enhances palatability, and excellent enzyme activity has the effect of effectively improving the digestibility of livestock. And it was confirmed that this effect is particularly increased when using the Bacillus subtilis IN-55 (KCTC 11914BP) and Lactobacillus plantarum BT-77 (KCTC 11915BP) together.

Accordingly, the present invention provides a microbial additive for plant silage fermentation comprising at least one of Bacillus subtilis IN-55 (KCTC 11914BP) and Lactobacillus plantarum BT-77 (KCTC 11915BP).

In one embodiment of the present invention, the plant to which the microbial additive for plant silage fermentation is added may be, for example, rice straw, rygrass, whole barley, whole rice, rye or corn, but is not limited thereto. It is not intended to be appropriately selected by those skilled in the art as necessary.

The microbial additive for plant silage fermentation is Bacillus subtilis IN-55 (KCTC 11914BP) or Lactobacillus plantarum BT-77 (KCTC 11915BP) strain itself, the culture stock solution containing the strain, or dilution of the culture stock solution It can be added in the form.

In another aspect, the present invention provides a method for producing plant silage, characterized by adding at least one of Bacillus subtilis IN-55 (KCTC 11914BP) and Lactobacillus plantarum BT-77 (KCTC 11915BP) to the plant.

In the present invention, the method for producing plant silage is a general method used in the art, for example, characterized in that it comprises a plurality of fermentation steps for fermentation of plants. The plurality of fermentation steps may include, for example, fermentation of acetic acid bacteria which are anaerobic bacteria and subsequent fermentation of lactic acid bacteria, and may be performed by those skilled in the art under appropriate conditions.

Therefore, according to one embodiment of the present invention, the present invention includes a plurality of fermentation steps for fermentation of a plant, the plant Bacillus subtilis IN-55 (KCTC 11914BP) to the plant in at least one fermentation step of the plurality of fermentation steps ) And Lactobacillus plantarum BT-77 (KCTC 11915BP) is added to provide a method for producing plant silage.

In this case, the plant used for silage production may be, for example, rice straw, rygrass, ryegrass, whole barley, whole barley, rye or corn, but is not limited thereto. Can be selected.

Hereinafter, the configuration of the present invention will be described in more detail by the following examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited to the description of these examples.

Example  1: Isolation and Identification of Strains

1-1. Isolation of strain

In this example, the cellulase was used as a test strain, and samples were collected from traditional fermented foods and soils from all over the country in order to isolate microorganisms having enzymatic activity thereto. 1 g of the sample was mixed with 10 ml of sterile water, shaken at 30 ° C. for 30 minutes, diluted by 10 ⁴ times by dilution agar plate method, and 0.14 ml of the diluted solution was dispensed on the LB medium (composition in Table 1) and evenly spread on the entire surface of the medium. After incubation for 3 days at 30 ℃ incubator, a single colony of fungi (single colony) was purely separated and transferred to a slope medium and stored at 4 ℃.

Badge Ingredient Concentration (g / L) Yeast extract 5 Tryptone 10 Sodium chloride 10

In addition, 10 g of sterile water was mixed with 1 g of the sample collected for screening of Lactobacillus plantarum strain, shaken at 30 ° C. for 30 minutes, and diluted to 10 ⁴ times by diluting agar plate method to dilute 0.14 ml of MRS medium ( Difco Co.) was evenly spread over the medium, and then cultured in a 36 ° C. incubator for 2 days, and then single colonies of bacteria were purely separated, transferred to a slope medium, and stored at 4 ° C.

Each selected strain was selected one of the best Bacillus subtilis and Lactobacillus plantarum strains showing strong antimicrobial activity using cellulase. Identification of the selected strains was carried out as follows.

1-2: Morphological and Physiological Properties

Selected strains Bacillus subtilis IN-55 and Lactobacillus plantarum BT-77 were inoculated in nutrient medium and incubated at 30 ° C. for 3 days, and then morphological and physiological characteristics were observed. As a result, the selected strains were produced as a single colony, and the surface of the colonies was smooth. When observed under a microscope, they were shaped as bacilli. In addition, the physiological characteristics of Bacillus subtilis IN-55 and Lactobacillus plantarum BT-77 were examined. Detailed characteristics of Bacillus subtilis IN-55 and Lactobacillus plantarum BT-77 are shown in Tables 2 and 3 below.

Bacillus subtilis
IN-55 Bacillus subtilis
Standard Gram / Shape + / R + / R Spore + + Motility + D Growth in aerobic conditions
(Growth in air) + + Growth in Anaerobic Conditions
(Growth anaerobically) + D Catalase Activity + + Oxidase Activity + d Glucose (acid) + D O / F /- -/- O / F /- Spore shape X X Spore position U U Growth at 45 ° C + + Growth at 65 ° C - - growth at pH 5.7 + + Growth at 7% NaCl + + Citric acid use
(Utilization of citrate) + + Anaerobic Growth in Glucose Broth
Anaerobic growth in glucose broth - - carbohydrate
(Carbohydrates, acid from :)
Glucose

+

+ Arabinose - + Mannitol + + Xylose w + VP test + + Starch hydrolysis
(Starch hydrolysis) + + Nitrate reduction - + Indole - - Gelatin hydrolysis
(Gelatin hydrolysis) + + Casein hydrolysis
(Casein hydrolysis) + + Urease - d LV d - Lysozyme sensitivity
Lysozyme sensitivity r d

Different reactions for different D-homologous species, different reactions for different d-strains

O-Oxidation, F-Fermentation

T-terminal spore, X-spore oval

V-subterminal spore, U-central spore

r-resistant, s-sensitive

R-rod-shaped ( Bacillus )

w-weak reaction

Lactobacillus plantarum
(BT-77) Lactobacillus plantarum
Standard Gram / Shape + / R + / R Spore - - Motility - - Growth in aerobic conditions
(Growth in air) + + Growth in Anaerobic Conditions
(Growth anaerobically) + + Catalase Activity - - Oxidase Activity - - Glucose (acid) + + O / F /- F F Spore shape - - Gas from glucose - - Growth at 15 + + Growth at 45 - - VP test - - Nitrate reduction - - Gelatin hydrolysis
(Gelatin hydrolysis) - - Esculin hydrolysis
(Aesculin hydrolysis) + + Arginine Hydrolysis
Arginine hydrolysis - - carbohydrate
(Carbohydrates, acid from :)
Amygdalin

+

+ Arabinose + d Cellobiose + + Esculin + + Fructose + + Galactose + + Glucose + + Gluconate - + Lactose + + Maltose + + Mannitol + + Mannoose + + Melezitose + d Melibiose + + Raffinose + + Ramnose - - Ribose + + Salincin + + Sorbitol + + Sucrose + + Trehalose + + Xylose - d

Different reactions per d-strain

O-Oxidation, F-Fermentation

1-3: 16S rRNA  Sequencing

The rRNA sequences were analyzed to identify the phylogenetic location of the selected strains. In the case of Bacillus subtilis IN-55, the nucleotide sequence of 2192 bp was analyzed (FIG. 1), and in the case of Lactobacillus plantarum BT-77, 601 bp corresponding to 30% or more of the total 16S rRNA sequences Was analyzed (FIG. 2). 16S rRNA fragments from the selected strains were amplified by PCR with a primer set, and then the amplification products were purified and inserted into the TOPO TA Cloning kit (Invitrogen), which was transformed into E. coli. After extracting the plasmid from the transformed E. coli, the nucleotide sequence was analyzed using an Applied Biosystems model 373A automatic sequencer and a kit (BigDye Terminator Cycle Sequencing kit, Perkin-Elmer Applied Biosystems). 16S rDNA of the selected strains were analyzed for homology using the gene bank blast program.

As a result, the selected strains showed 99% homology with Bacillus subtilis and Lactobacillus plantarum, respectively, and the strains of the present invention were finally identified as Bacillus subtilis and Lactobacillus plantarum, respectively. Therefore, as of April 8, 2011, it was deposited as a patent strain at the Genetic Resource Center in the Korea Research Institute of Bioscience and Biotechnology and deposited with the accession numbers KCTC 11914BP (Bacillus subtilis IN-55) and KCTC 11915BP (Lactobacillus plantarum BT-77). Was granted.

Example  2: enzyme Bow performance  And active range tests

Proteases, cellulases and amylases were assayed for assaying the resolution of various organic substances. After sterilization of each skim milk, carboxymethyl cellulose (CMC) and starch medium, the medium was completely cured, and then the culture medium of Bacillus subtilis IN-55 and Lactobacillus plantarum BT-77 was incubated with paper disc ( Buried in 8 mm diameter) and placed on a petri dish to confirm enzyme activity. After activating for 12 hours at 25 ℃ to verify the activity of various enzymes, the resolution of the skim milk, CMC, starch was investigated by the diameter of the clear zone (Table 4).

As can be seen in Table 4, the Bacillus subtilis IN-55 strain of the present invention was excellent in all protease, cellulase, amylase enzyme activity, Lactobacillus plantarum BT-77 strain only cellulase enzyme activity Appeared strongly. In particular, both strains were confirmed to have excellent cellulase enzyme activity, which has a large effect on silage production.

Assay enzyme activity Activity level
(Bacillus subtilis
IN-55) Activity level
(Lactobacillus plantarum BT-77) Protease ++++ - Cellulase +++ +++ Amylase +++ -

++++: size of clear zone 15-20 mm, +++: 10-15 mm

Figure 3 shows the protease and cellulase enzyme activity of Bacillus subtilis IN-55, it can be seen that it shows a very good enzyme activity.

Figure 4 shows the cellulase enzyme activity of Lactobacillus plantarum BT-77, it can be seen that it shows a very good enzyme activity.

Example  3: antibacterial Bow performance  And active range tests

Syracuse Hancock Staphylococcus aureus (Staphylococcus aureus , BRC Biomaterials Center, KCTC 1928), Bacillus cereus ( Bacillus cereus , KCTC 3624), and the like on the paper disk of the prepared solid medium inoculated with the pathogenic bacteria described in Table 5. Incubate the culture supernatants of Bacillus subtilis IN-55 and Lactobacillus plantarum BT-77 at 100 μm and incubate at the optimal culture temperature of Bacillus cereus to observe whether the inhibitory rings were formed on the NB plate. The activity was confirmed.

Indicator strain Bacillus subtilis IN-55
(Inhibition ring diameter, mm) Lactobacillus plantarum BT-77
(Inhibition ring diameter, mm) Eshericlria coli
ATCC 700928 - 18 Salmonella typhimurium
KCTC 2057 - 18 Salmonella choleraesuis
KCTC 2929 - 18 Streptococcus suis
KCTC 3557 - 18 Bacillus cereus
KCTC 3624 - 20 Listeria monocytogenes
KCTC 3710 14 -

As a result, as shown in Table 5, for Bacillus subtilis IN-55 It was confirmed that only Listeria monocytogenes showed antimicrobial activity (FIG. 5), and in case of Lactobacillus plantarum BT-77, Listeria All of the other pathogenic bacteria except monocytogenes showed strong antimicrobial activity (FIG. 6), and when the two strains were used together, it was confirmed that the antimicrobial activity was highly complementary.

Example  4: New strain  Microbial assay for the production of used silage

To test silage production using Bacillus subtilis IN-55 and Lactobacillus plantarum BT-77, rice straw was cut into 2.5 cm and placed into a 500 ml flask by 20 g each, followed by Bacillus subtilis and Lactobacillus plantarum. TGY medium useful in the growth of both strains (Table 6 composition) was added and sterilized.

Badge Ingredient Concentration (g / L) Yeast extract 3 Tryptone 5 Glucose 15 Calcium carbonate 2 pH 5.5

Then, silage was prepared using a control group, an experimental group to which only Bacillus subtilis IN-55 was added, an experimental group to which only Lactobacillus plantarum BT-77 was added, and an experimental group to which both strains were added. Each sample was obtained by incubating at 30 ° C. for 36 hours to obtain each sample, and 10 g of the sample was taken in 100 ml of distilled water, and stored at 4 ° C. for 24 hours. The results are shown in Table 7.

In addition, each sample was diluted 10-fold to confirm the number of bacteria of the Lactobacillus plantarum and Bacillus subtilis strains according to the present invention in each sample, and the fungi and the total number of bacteria in the samples were measured. As shown in 7.

pH Total bacteria IN-55 BT-77 mold straw - 1.75 × 10 ⁷ - - 4.2 × 10 ⁶ Straw + TGY Badge 6.56 5.0 × 10 ⁸ - - - Straw + TGY Badge + IN-55 6.53 9.8 × 10 ⁸ 9.0 × 10 ⁸ - - Straw + TGY Badge + BT-77 4.12 2.65 × 10 ⁷ - 2.65 × 10 ⁷ - Straw + TGY Badge
+ IN-55 + BT-77 4.06 2.04 × 10 ⁷ 1.0 × 10 ⁵ 2.04 × 10 ⁷ -

As can be seen from Table 7, in the experimental group to which the Lactobacillus plantarum BT-77 according to the present invention was added, the pH showed weak acidity. In the total number of bacteria, the existing silage manufacturing group using only straw showed high mold count and low total number of bacteria, and the experimental group treated with straw and TGY medium showed higher levels of bacteria than the straw-treated group. The growth of microorganisms in the basic rice straw was active due to the addition of, showing a higher total bacterial count than the experimental group treated with straw only. In the experimental group to which Bacillus subtilis IN-55 or Lactobacillus plantarum BT-77 according to the present invention was added, no bacteria other than the treated strains were identified, and only strains of the present invention, which were microorganisms useful for silage production, were identified. . Through this, it was confirmed that treatment of Bacillus subtilis IN-55 or Lactobacillus plantarum BT-77 was significantly significant in promoting feed storage by effectively inhibiting the growth of other pathogenic bacteria during silage preparation.

Example  5: Silage via electron micrograph Manufacturing ability  black

Electron micrographs were taken using the experimental groups of Example 4 to assay silage production using Bacillus subtilis IN-55 or Lactobacillus plantarum BT-77. After pretreatment, electron micrographs were taken through the immobilization process for each experimental group.

Figure 7 is a photograph of the decomposition degree of rice straw surface for each experimental group of Example 4 by the electron microscope, Figure 8 is a photograph of the rice straw decomposed by the treated strain for each experimental group of Example 4 by an electron microscope. When the strains of the present invention were treated through this, the degree of degradation of rice straw was superior to the control group, and the resolution was more excellent when the Bacillus subtilis IN-55 and Lactobacillus plantarum BT-77 strains of the present invention were treated together. It can be seen.

Example  6: New strain  Culture condition

6-1: Bacillus Subtilis IN Culture condition of -55

Bacillus subtilis IN-55 strain, a new strain for producing silage, was cultured using a 2.5 L fermenter, and the medium used was prepared by using a conventional liquid medium having a pH of 7.0 adjusted to the composition shown in Table 1. Species were cultured using a 100 ml liquid medium by adjusting the pH to 7.0 in a composition as shown in Table 1 in a 500 ml Erlenmeyer flask. The culture conditions were shaken at 30 ° C. and 200 rpm for 24 hours, followed by inoculation of 5% (v / v) seed culture solution containing Bacillus subtilis to a 2.5 L fermenter containing 1.5 L production medium. Incubated for 72 hours under conditions.

6-2: Lactobacillus Flora Room IT Culture conditions of -77

Lactobacillus plantarum BT-77, a new strain for silage production, was cultured using a 5 L Erlenmeyer flask, and the medium used was purchased by MRS media (Difco Co.), and the cultivation was carried out in a 500 mL Erlenmeyer flask. The pH was adjusted to 6.5 with the same composition and cultured using 100 ml of liquid medium. Culture conditions were incubated for 24 hours at 36 ℃, 5% (v / v) of the culture medium containing Lactobacillus plantarum was inoculated into a 5 liter Erlenmeyer flask containing 1.5 liter production medium and incubated for 48 hours at 36 ℃. .

Korea Biotechnology Research Institute KCTC11914BP 20110408 Korea Biotechnology Research Institute KCTC11915BP 20110408

Claims (6)

Bacillus subtilis ) IN-55 (KCTC 11914BP). Lactobacillus ( Lactobacillus) plantarum ) BT-77 (KCTC 11915BP). A microbial additive for plant silage fermentation comprising at least one of Bacillus subtilis IN-55 (KCTC 11914BP) and Lactobacillus plantarum BT-77 (KCTC 11915BP). The method of claim 3,
The plant is rice straw, rygrass (ryegrass), total barley, total rice, rye or corn microbial additive for plant silage fermentation. A method for producing plant silage, comprising adding at least one of Bacillus subtilis IN-55 (KCTC 11914BP) and Lactobacillus plantarum BT-77 (KCTC 11915BP) to the plant. The method of claim 5,
The plant is rice straw, rygrass (ryegrass), total barley, total rice, rye or corn plant silage manufacturing method.

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