KR101470995B1 - Propionic acid producing microorganism and roughage comprising the same - Google Patents

Abstract

The present invention relates to a microorganism having an ability to produce propionic acid and a bulky feed composition comprising the same. The purpose of the present invention is to provide a bulky feed composition having excellent fatting promoting effects and a method for fatting a ruminant by using the bulky feed composition including rumen microbes. To this end, provided is a bulky feed composition including a microorganism having an ability to produce propionic acid separated from the ruminant stomach and Lactobacillus mucosae KCCM11440P.

Description

[0001] The present invention relates to a microorganism having propionic acid producing ability and a roughage composition comprising the microorganism,

The present invention is derived from research conducted by the Small and Medium Business Administration as part of the SME technology development project.

[Assignment number: S2063309, Project title: Development of waste microbial enriched mushroom waste medium with finishing promotion effect]

The present invention relates to a microorganism having proton acid producing ability and a calcination composition containing the same.

Volatile fatty acids (VFA) are the most important end product of carbohydrate degradation in the rumen. Volatile fatty acids are an important source of energy for ruminants (70%) and also affect protein and fat content in milk. The main volatile fatty acids produced in the metabolism in the rumen are acetic acid, propionic acid, and butyric acid, depending on the type and degree of digestion of feed. In ruminants, only propionic acid among volatile fatty acids contributes to glucose synthesis, and quantitatively glucose is a very important precursor. Propionic acid constitutes 18 to 20% of total volatile fatty acids and is converted from liver to blood sugar to provide energy and is used in the synthesis of lactose. Increase of propionic acid stimulates angiogenesis, increases epithelial cell by increasing blood flow in ruminant epithelial cells, promotes fattening of ruminants including bovine and contributes to improvement of meat quality.

Fibers are the most abundant energy source on the planet. However, at present, a large part of the use of the forage is dependent on imports in Korea. The dependence of forage on high imports is a problem that needs to be solved also to secure competitiveness of domestic farmers. Various efforts have been made to utilize resources such as waste paper, which is a byproduct of mushroom production and fiber-rich, as feed.

Korean Patent No. 1144473 relates to a method for producing a fermentation forage for livestock using mushroom by-products as a main raw material and is characterized in that lactic acid bacteria, Bacillus subtilis, and yeast bacteria used as a probiotic agent are added to the mushroom waste medium and fermented.

Korean Patent No. 1138934 relates to a method for producing a pig feed using a waste mushroom culture medium and is characterized in that fermentation is carried out using a complex microbial fermentation agent containing no microorganism capable of producing propionic acid.

However, there is still a need for an effective feed that can contribute to the promotion of fodder without the side effects associated with the use of antibiotics and hormones, while utilizing abundant fiber.

Accordingly, the present inventors conducted a study on ruminants for promoting finishing of ruminants, and found that the present invention based on a microorganism having a propionic acid-producing ability capable of promoting fattening by fermenting the forage in the rumen to increase propionic acid production Completed.

It is an object of the present invention to provide a ruminant microorganism having the ability to produce propionic acid.

It is also an object of the present invention to provide a forage composition having an excellent fattening effect.

The present invention also aims at providing a method for raising a ruminant using a foraging material containing a ruminant microorganism.

One aspect of the present invention provides a microorganism having the ability to produce propionic acid separated from the rumen.

In one embodiment of the invention, the microorganism is selected from the group consisting of Lactobacillus mucosae ) KCCM11440P.

KCCM11440P in Lactobacillus mucosa has the 16S rRNA nucleotide sequence of SEQ ID NO: 1 and is identified based on this sequence. Figure 1 shows the phylogenetic tree created based on the 16S rRNA nucleotide sequence.

Propionate is the most important product of carbohydrate degradation in the rumen, which converts rumen liver to glucose, promotes the flesh of ruminants, and contributes to meat quality improvement.

KCCM11440P in Lactobacillus mucosa isolated from rumen on the basis of propionic acid production ability was found to grow in MRS medium, especially in the medium supplemented with vitamin B ₁₂ or sodium lactate.

Another aspect of the present invention provides a trial composition comprising KCCM 11440P in Lactobacillus mucosa.

The term " roughage composition "as used herein means a feed composition having a high fiber content and a low content of fat, protein, starch, etc., such as grasses, hay, silage and the like.

The forage composition according to an embodiment of the present invention includes KCCM11440P in Lactobacillus mucosa having propionic acid producing ability to increase the production of propionic acid upon fermentation of the forage in the rumen, thereby promoting the fattening of ruminants and improving meat quality . Since propionic acid is the only volatile fatty acid produced in the rumen, it is converted to glucose and contributes to energy metabolism and so, the promotion of the fattening of the ruminants including the bovine is determined by the propionic acid produced in the rumen.

In one embodiment of the invention, the forage composition comprises mushroom waste media.

The mushroom waste medium means a medium in which the main raw material is sawdust and the supplementary raw material is a medium containing agricultural by-products, which is then used for mushroom cultivation. Sawdust in mushroom waste media is used as a carbon source by fibrinolytic microorganisms in the rumen. KCCM11440P in Lactobacillus mucosa may be cultivated in mushroom waste medium to increase production of propionic acid, thereby promoting the flesh of ruminants. In particular, the mushroom waste medium is decomposed by fibrinolytic bacteria of mushroom, and the obtained waste medium is used as a raw material.

In one embodiment of the invention, the forage composition comprises at least one of vitamin B ₁₂ and a lactate.

KCCM11440P at Lactobacillus mucosa significantly increases production of propionic acid when supplemented with vitamin B ₁₂ or lactate, for example, sodium lactate, in the medium. Thus, inclusion of at least one of vitamin B ₁₂ and lactate in the forage composition increases propionic acid production, thereby promoting fattening.

In one embodiment of the present invention, the forage composition promotes the fattening of ruminants. Compared to a conventional forage composition, it contains KCCM11440P in lactobacillus mucosa having propionic acid producing ability, thereby increasing the production of propionic acid in the rumen, thereby promoting the fattening of ruminants and contributing to the improvement of meat quality.

In one embodiment of the present invention, the forage composition may further comprise a probiotic agent.

The use of antibiotics is inevitable at the time of livestock breeding, but probiotics are widely used as the problems caused by abuse of them are serious. Probiotics contribute beneficial microbes to the intestines of livestock to inhibit the growth of pathogenic microorganisms, prevent disease outbreaks, and increase livestock productivity. Currently widely used microorganisms are lactic acid bacteria, Bacillus subtilis, and yeast bacteria. Lactic acid bacteria produce organic acids to lower the pH to inhibit harmful bacteria that are weak in acidity, promote the activity of digestive enzymes, and especially reduce the incidence of diarrhea. Bacillus subtilis produces decomposition enzymes that degrade highly active carbohydrates, proteins, and lipids. It enhances digestion and absorption of feed in the intestines, improves feed efficiency, reduces stress caused by digestion, facilitates the growth of livestock , And the effect of suits to strengthen the field of livestock to prevent disease. In addition, yeast exists in a form that can be easily digested in the digestive tract of livestock, and produces natural flavor components such as alcohol and glutamic acid, thereby improving the palatability of the livestock feed.

In one embodiment of the present invention, the forage composition may be provided in admixture with a common feed. For example, the forage composition may be mixed with commercially available feed at a ratio of 1: 1 to feed the ruminant twice a day. Appropriate mixing ratios, feed rates and feeding frequency can be easily determined by those skilled in the art taking into account the age, weight, and health status of the subject ruminants.

Yet another aspect of the present invention provides a method of raising a ruminant, comprising feeding raffia containing KCCM 11440P to Lactobacillus mucosa.

In one embodiment of the present invention, the forage may contain mushroom waste medium as a main component.

In one embodiment of the present invention, the forage can further comprise a probiotic agent.

In one embodiment of the present invention, the forage may further comprise at least one of vitamin B ₁₂ and sodium lactate.

In one embodiment of the invention, the forage can be fed by mixing with conventional compound feed.

In one embodiment of the invention, the ruminant can be, but is not limited to, cattle, sheep, goats, and deer.

Hereinafter, the present invention will be described in more detail with reference to examples. However, the following examples are intended to illustrate the invention and should not be construed as limiting the invention.

In accordance with one embodiment of the present invention, KCCM 11440P and a calcination composition containing the same in Lactobacillus mucosa increase the production of propionic acid in the rumen rumen and thereby provide excellent finishing and meat quality improving effects.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a phylogenetic tree based on the 16S rRNA gene of KCCM11440P in Lactobacillus mucosa of the present invention.
Figure 2 shows the loss of building fermented feed, wheat bran, mushroom by-products, and brewing by-products over time

Example  1. Propionic acid Bacillus  Isolation and identification

1-1. Isolation of strain

In order to isolate strains having propionic acid producing ability, 3 rats of 40 ± 5 kg of conventional black goats were collected. In order to maintain the anaerobic condition, it was performed in the anaerobic chamber and Hattori and Matsui (Anaerobe, Vol. 14, pp. 87-93, (2008)) method was used.

Bacteria cultured from rumen juice were diluted to 10 < ^-3 > to 10 < ^-9 > / ml of medium, inoculated into a Hungate roll tube and cultured for 24 to 48 hours. The cultured single colonies were separated and cultured at 120 rpm for 24 hours after inoculation into liquid medium (MRS). All cultures were performed in anaerobic conditions at 37 ° C. (Lee et al., Applied Microbiology and Biotechnology, Vol. 58, pp. 663-668, 2002) Science, Vol 82, pp. 780-787, 1953) was sterilized at 121 ° C for 15 minutes and O 2 and N 2 gas were used.

A total of 51 strains were isolated and the amount of volatile fatty acid production was measured by culturing them in MRS medium. The strains showing the highest production of propionic acid were selected.

1-2. Identification of the strain

The strain isolated from 1-1 was cultured on MRS medium and then the chromosomal DNA was extracted using Wizard Genomic DNA Purification Kits (Promega, USA). PCR was performed using 27F primer (AGAGTTTGATCMTGGCTCAG) and 1492R primer (GGTTACCTTGTTACGACTT) for amplification of the gene encoding 16S rRNA using this as a template. The PCR conditions consisted of 32 cycles of a cycle consisting of an initial denaturation step at 94 DEG C for 5 minutes, denaturation at 94 DEG C for 45 seconds, annealing at 65 DEG C for 45 seconds, extension at 72 DEG C for 1 minute, and 72 DEG C for 10 minutes It was kidney.

The similarity of amplified ribosomal DNA was analyzed by ARDRA (Amplified Ribosomal DNA Restriction Analysis) method. The PCR products were treated with Hae Ⅲ and Hha Ⅰ restriction enzymes (Takara, Japan) at 37 캜 for 5 hours, and the obtained DNA samples were separated by electrophoresis using a meta-agarose gel for 70 minutes at 170 v. Then, visualization was performed using a Kodak Gel Logic 200 imaging system (Eastman Kodak Company, Rochester, NY, USA), and bands obtained from ARDRA were purified using QIA quick PCR Purification Kit.

The purified 16S rDNA PCR product was identified as SEQ ID NO: 1 by analyzing the nucleotide sequence in Macrogen (Korea). The nucleotide sequence was analyzed using NCBI ( http://www.ncbi.nlm.nih.gov/BLAST ) and EzTaxon (Nasdaq, Nasdaq, Japan) using the SeqMan program (DNA Star, Lasergene software, Madison, WI, USA) ( http://147.47.212.35:8080/index.jsp ) using the BLAST program in GeneBank. The approximate phylogenetic classification was determined by comparing the closest species to the nucleotide sequence using CLUSTRAL W version 1.6. We constructed the phylogenetic tree as described in Kimura (1980) using a neighbor-joining method with pair-wise gap removal. Finally, we used a two-parameter NJ method in the PHYLIP package and used a bootstrap analysis method to collect 1000 times more data to evaluate the stability of the phylogenetic tree. Only 50% or more bootstrap values were shown. Figure 1 shows the tree number created.

The isolated strains showed 96% similarity to S32 (T) in Lactobacillus mucosa. Based on this, the isolated strain was named BR-PP in Lactobacillus mucosa, deposited with the Korean Society for Microbiological Research on July 26, 2013, and granted the deposit number of KCCM 11440P.

1-3. Selection of control strains

In order to select a control group for comparing the production ability of KCCM11440P with propionic acid to Lactobacillus mucosa isolated and identified in 1-1 and 1-2, Selenomonas bovis, which is known to have the ability to produce propionic acid bovis ) 15154 (SB) , Veilonella parvula ) 5019 (VP) , Propionibacterium acipidipropionis ( Propionobacterium acidipropionici ) 5020 (PAci) and Propionibacterium < RTI ID = 0.0 > acnes ) 11946 (PAcn) were purchased from KCTC and KACC, respectively.

Optical density (OD) and pH were measured while each strain was incubated at 37 ° C for 48, 72, 96, 120 and 144 hours. Each experiment was repeated three times. In addition, each incubation time for the sample by high performance liquid chromatography equipped with a UV detector (HPLC) the collected (Agilent Technologies 1200 series) in MetaCarb 87H (Varian, Germany) Flow rate 0.6ml / min with 0.0085N · H ₂ on a column SO ₄ (VFA) production was measured at 210 nm and 220 nm (Tabaru et al . , Japanese Journal of Veterinary Science Vol. 50, pp. 1124-1126 (1988) and Han et al., Process Biochemistry, Vol. 40, pp. 2897-2905 (2005)).

Propionibacterium acidic acid propionic acid showed high propionic acid production after 144 hours. In addition, Propionibacterium acidic propionice showed conditional anaerobic differently from other propionic acid producing strains which were complete or essential anaerobic. Propionibacterium acidic propionic acid, which is high in propionic acid production and conditional anaerobic, was selected as a standard microorganism producing propionic acid and used as a control.

Example  2. Lactobacillus Mucosa KCCM1140P Of propionic acid Production capacity

The production ability of propionic acid in Lactobacillus mythusa isolated and identified in Example 1 was evaluated. And cultured in agar based on MRS medium for 48 hours. The medium was placed in a Hungate tube and autoclaved at 121 ° C for 15 min at pH 6.5, O ₂ -free 20% CO ₂ -80% N ₂ gas filling. Biotin (0.5 mg / L), sodium lactate (2%), vitamin B ₁₂ (50 μg / L) or glycerol (2%) were added to the agar of the MRS medium to confirm the effect on the production of propionic acid. The control group for evaluation of the production ability of propionic acid was the propionibacterium acidic proteinide selected in Example 1.

The samples were collected at 24 hours, 48 hours and 72 hours after culturing and the amounts of volatile fatty acids including OD, pH and propionic acid were measured.

The pH was stabilized at the same temperature as the room temperature without measuring directly in the incubator and then measured using M503P meter (wrks, Medififield, MA, USA).

The amount of volatile fatty acid was determined by centrifugation at 1000 x g for 10 min at 4 ° C, and the supernatant was collected and analyzed by 0.2 μm microfilter. Was analyzed at 35 ° C using HPLC (Agilent technolgies 1200 series) equipped with a METACARB87H (Varian, Germany) column and the UV wavelengths of the detectors were 210 nm and 220 nm. The mobile phase solvent was 0.0085NH ₂ SO ₄ and the flow rate was 0.6 ml / min.

Tables 1 and 2 below summarize the results.

parameter Incubation time C
(MRS) B
(MRS + biotin) B _12
( MRS + vitamin B ₁₂ ) S
(MRS +
Sodium lactate) G
(MRS + glycerol) SEM P value OD 24 2.580 2.569 2.560 2.429 2.332 0.031 0.0053 48 2.655 2.659 2.603 2.424 2.293 0.004 <.0001 72 2.647 2.637 2.619 2.407 2.327 0.008 <.0001 pH 24 4.24 4.24 4.25 4.57 4.25 0.00 <.0001 48 4.21 4.22 4.22 4.53 4.23 0.01 <.0001 72 4.22 4.23 4.23 4.55 4.23 0.01 <.0001
Lactate Formate acetate Propionate Butyrate Total VFA MRS 13146.5 -550.8 -1190.1 678.2 0 -511.9 MRS + Biotin 12782.8 -502.1 -554.0 1012.2 0 458.2 MRS + sodium lactate 13993.8 0 3115.0 1810.6 0 4925.6 MRS + Vitamin B ₁₂ 15224.6 0 6002.7 1797.6 0 7800.3

delete

The results obtained show that KCCM11440P in Lactobacillus mucosa produces the highest amount of propionic acid when cultured in MRS medium supplemented with vitamin B ₁₂ or sodium lactate.

Example  3. Manufacture of forage

In vitro (In Vitro) using a ruminant stomach juices and the buffer solution was evaluated in the effect of the fermentation in the rumen.

Ruminal fluid collection and buffer preparation

Use the Sunchon National University is equipped with a 600 ± 47kg Holstein cow rumen fistula being bred in R & D in farm vitro The gastric juice for the test was taken. Hanwoo, a public animal, was fed with a mixture of Italian rye grass and concentrated feed (55% corn, 15% wheat, 8% defatted rice, 5% corn gluten feed, 10% soybean meal, 0.2% molasses, (Vitamin A 3000 IU, vitamin D 6000 IU, vitamin E 30 IU, Cu 25 mg, Fe 150 mg, Zn 200 mg, Mn 100 mg, and Co 0.5 mg, and I 1.5 mg) 1.0%) at a ratio of 2: 8 to 2% of body weight twice a day, and free access to water. Gastric juice was collected by using cheese cloth 2 hours after feeding. The buffer solution (Hino et al., 1992) contained 0.45 g / L K ₂ HPO _4, 0.45 g / L KH ₂ PO ₄ , 0.9 g / L (NH ₄ ) ₂ SO ₄ , 0.12 g / L CaCl ₂ .2H ₂ O , Basal medium containing 0.19 g / L of MgSO ₄ .7H ₂ O, 1.0 g / L of Trypticase, 1.0 g / L of yeast extract and 0.6 g / L of cysteine HCl (pH 6.9) .

Ruminal fluid and buffer were mixed at a ratio of 1: 3 (ruminal fluid: buffer) and filled with nitrogen gas (N ₂ gas). Each fermented feed was placed in a 160 ml serum bottle at 2% of the building, and 100 ml of the prepared buffer was added. The mixture was anaerobically maintained with O ₂ free-N ₂ , sealed with a rubber stopper and an aluminum cap, (Hattori and Matsui, Anaerobe, Vol. 14, pp. 87-93, (2008)). In vitro culturing was performed in three replicate experiments at 0, 12, 24, and 48 hours, and the pH, total gas production, methane, ammonia, and VFA were measured by the characteristics of rumen fermentation.

Change in pH

The pH was stabilized at the same temperature as the room temperature without measuring directly in the incubator and then measured using M503P meter (wrks, Medififield, MA, USA).

Total gas production

The total gas emission was measured by EA-6 (Inc, Sun Bee instrument) pressure sensor after the sample was stabilized. After measuring the total gas emission, the gas generated by the vacuum tube was collected to measure methane and carbon dioxide emissions. Gas production was estimated by the formula of Qrskov and McDonald (1979) based on the total gas production amount obtained for each culture time.

Volatile fatty acids content

The VFA measurement was performed by centrifuging the culture for 10 minutes at 1000 x g at 4 DEG C after culturing the culture supernatant. The supernatant was collected and purified with a 0.2 mu m microfilter and analyzed. Was analyzed at 35 ° C using HPLC (Agilent technolgies 1200 series) equipped with a METACARB87H (Varian, Germany) column and the UV wavelengths of the detectors were 210 nm and 220 nm. The mobile phase solvent was 0.0085NH ₂ SO ₄ and the flow rate was 0.6 ml / min.

Ammonia nitrogen (NH ₃ -N) concentration

Ammonia concentration was determined by centrifuging the collected samples at 13000 rpm and then developing the ammonia in the sample with a phenol solution according to the method of Chany and Marbach (Clinical Chemistry, Vol.8, pp. 130-132, (1962) Absorbance was measured at 630 nm using a spectrophotometer (Spectronics 21D).

3-1. Fermentation of by-products

Most by-products are generally discarded after production of the main product. Among these by-products, the fibrin component still collected high brewing by-products and mushroom by-products (mushroom waste media) and evaluated whether they could be used as feed for ruminants. In other words, in vitro fermentation was carried out in a mixture of ruminal juice and buffer, as described above, using them as a fermented feed.

Fig. 2 shows the amount of building loss in the fermented feed according to the incubation time. Bran and a combination of brewed by-products or mushroom by-products were used as fermented feed. The loss of bran in mushroom by - products was higher than that of brewed by - products. As a result, mushroom by-products indicate that digestion is easier within the rumen than by-product by-products.

3-2. Fermentation of feeds containing microorganisms with the ability to produce propionic acid

Silage provides the quality of feed and maximum building. In addition, microorganisms in the feed increase the rate of fermentation and improve the results of silage. Therefore, the fermentation efficiency of the in vitro was measured by adding a microorganism having the ability to produce propionic acid.

The addition of KCCM11440P or Propionibacterium acipiodopropionis to Lactobacillus mucosa in combination with wheat bran, by-products or by-products of mushroom byproducts was followed by incubation with in-vitro to determine the amount of building loss, moisture content, pH, volatile fatty acid production, And ammonia production were measured.

The bran and the brewed by-product or the mushroom by-product (waste medium) were mixed at a ratio of 1: 1, 1% molasses was added, and 10% of the cultured microorganism, Lactobacillus mucosa or Propionibacterium liquorpropionice (Positive control group). All samples were vacuum packed and incubated at 37 ° C for 72 hours.

The yield of byproducts after silage production was higher than that of by - products of mushrooms (P <0.05) but the pH value was lower (P <0.05). Table 3 below shows the measured moisture content and pH.

code process Water content (%) pH Con 1 Brewing byproduct + bran + distilled water 48.7869 3.84 T1 Brewing Byproduct + Bran + Lactobacillus mucosae KCCM11440P 47.9173 3.85 T2 Brewing by-product + Bran + Propionibacterium acuminata Propionii 47.6139 3.83 Con 2 Mushroom by-product + bran + distilled water 35.3198 4.86 T3 Mushroom by product + Bran + Lactobacillus mucosae KCCM11440P 36.8942 4.90 T4 Mushroom by-product + Bran + Propionibacterium acuminata Propionice 35.6735 4.85

Table 4 below shows the disappearance of dry matter (building) over fermentation incubation time. The disappearance of mushroom by - products was lower than that of brewed by - products (P <0.05). As a result, the increase in the disappearance of the brewing by-products of silage is attributed to the addition to Lactobacillus mucosa.

Table 5 shows VFA production by fermentation. The loss of building was observed to be low in mushroom byproducts, but total VFA and propionic acid were higher in brewing byproducts. In addition, fermentation with Lactobacillus mucosae as a byproduct during the treatment showed high production of propionic acid and total volatile fatty acid (TVFA). This shows that the change in acidity of mushroom byproducts is higher than that of brewed by-products due to nutritional composition. The large amount of acid produced during the manufacture of the silage means that it becomes a higher energy source for ruminants when fed.

Table 6 shows the total gas production, pH, and ammonia nitrogen production by in vitro fermentation of brewed by-products and mushroom by-products.

Time (h) Brewing byproduct Mushroom by-products Con1 T1 T2 Con2 T3 T4 SEM Total gas production (ml) 12 58.00 54.00b 59.00 65.00 65.33 66.67 1.926 24 76.33 84.33 88.00 107.50 110.00 112.50 1.461 48 116.00 116.50 105.50 112.50 120.67 109.33 1.542 pH 0 5.34 5.38 5.38 5.68 5.68 5.69 0.006 12 5.08 5.07 5.03 5.06 5.15 5.17 0.014 24 5.00 5.07 4.94 5.00 5.27 5.26 0.017 48 5.01 5.05 4.99 5.24 5.27 5.26 0.019 Ammonia-nitrogen (ppm) 0 253.90 260.54 252.95 247.95 255.69 250.41 5.548 12 319.11 361.12 271.52 363.12 360.80 386.27 8.841 24 408.71 479.95 418.95 505.03 486.07 514.53 15.904 48 450.72 511.19 378.21 491.59 510.13 509.81 15.576

Mushroom byproduct + distilled water (Con1), brewing byproduct + L. mucosae (T1), brewing byproduct + P. acidipropionici (T2, positive control), mushroom byproduct + distilled water (Con2) By-product + P. acidipropionici (T4, positive control)

Statistical method

All of the results obtained in this study were analyzed by ANOVA using the random design general linear model (GLM). All treatments were confirmed by Duncan's Multiple Range Test (DMRT) method. Statistical significance was P <0.05 and all analyzes were performed using SAS (Statistical Analysis Systems) version 9.1 (SAS, 2002).

Korea Microorganism Conservation Center (overseas) KCCM11440P 20130726

<110> BIORESOURCE INC. <120> Propionic acid-producing microorganism and roughage comprising          the same <130> PN102527 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 1446 <212> DNA <213> Lactobacillus mucosae <220> <221> gene &Lt; 222 > (1) .. (1446) <223> 16S rRNA <400> 1 ccgactttgg gtgttgcaaa cttttcatgg tgtgacgggc ggtgtgtaca aggcccggga 60 acgtattcac cgcggcatgc tgatccgcga ttactagcga ttccgacttc gtgcaggcga 120 gttgcagcct gcagtccgaa ctgagaacgg ttttaagaga ttagcttgcc ctcgcgagtt 180 cgcgactcgt tgtaccgtcc attgtagcac gtgtgtagcc caggtcataa ggggcatgat 240 gatctgacgt cgtccccacc ttcctccggt ttgtcaccgg cagtctcact agagtgccca 300 acttaatgct ggcaactagt aacaagggtt gcgctcgttg cgggacttaa cccaacatct 360 cacgacacga gctgacgacg accatgcacc acctgtcatt gcgttcccga aggaaacgcc 420 ctatctctag ggttggcgca agatgtcaag acctggtaag gttcttcgcg tagcttcgaa 480 ttaaaccaca tgctccaccg cttgtgcggg cccccgtcaa ttcctttgag tttcaacctt 540 gcggtcgtac tccccaggcg gagtgcttaa tgcgttagct gcggcactga agggcggaaa 600 ccctccaaca cctagcactc atcgtttacg gcatggacta ccagggtatc taatcctgtt 660 cgctacccat gctttcgagc ctcagcgtca gttgcagacc aggcagccgc cttcgccact 720 ggtgttcttc catatatcta cgcattccac cgctacacat ggagttccac tgtcctcttc 780 tgcactcaag tttgacagtt tccgatgcac ttcttcggtt aagccaaagg ctttcacatc 840 agacttagca aaccgcctgc gctctcttta cgcccaataa atccggataa cgcttgccac 900 ctacgtatta ccgcggctgc tggcacgtag ttagccgtga ctttctggtt aaataccgtc 960 actgcatgaa cagttactct catacacgtt cttctctaac aacagagctt tacgagccga 1020 aacccttctt cactcacgcg gtgttgctcc atcaggcttg cgcccattgt ggaagattcc 1080 ctactgctgc ctcccgtagg agtatggacc gtgtctcagt tccattgtgg ccgatcagtc 1140 tctcaactcg gctatgcatc atcgccttgg taggccgtta ccctaccaac aagctaatgc 1200 accgcaggtc catcccaaag tgatagccaa aaccatcctt taaatttgag tcatggcaat 1260 caaagtggta aggcggaata acatccggtt ccaaatggta gcccccggtt tgtggcaagg 1320 taactaagtg gtaattaacc cgccggccaa tcggtgggag accaacgtca agtccgtgca 1380 agcaccgttc aatcaattgg gccaacgcgt tcgactttgc atgtattaag gcacaccggc 1440 cggcgt 1446

Claims (7)

Lactobacillus mucosa ( Lactobacillus) having the ability to produce propionic acid mucosae ) KCCM11440P. Gt; KCCM11440P &lt; / RTI &gt; in Lactobacillus mucosa. 3. The calcite composition of claim 2, wherein the calcareous composition comprises mushroom waste medium. 3. The calcite composition of claim 2, wherein the forage composition comprises at least one of vitamin B ₁₂ and a lactate. [Claim 3] The roughage composition according to claim 2, wherein the roughage composition is for promoting the raising of the ruminant. A method of raising a ruminant, comprising the step of raising a forage containing KCCM 11440P to Lactobacillus mucosa. 7. The method of claim 6, wherein the forage comprises mushroom waste media.

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