WO2015030423A1 - Microorganism having propionic acid production capability and feed composition comprising same - Google Patents

Microorganism having propionic acid production capability and feed composition comprising same Download PDF

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WO2015030423A1
WO2015030423A1 PCT/KR2014/007770 KR2014007770W WO2015030423A1 WO 2015030423 A1 WO2015030423 A1 WO 2015030423A1 KR 2014007770 W KR2014007770 W KR 2014007770W WO 2015030423 A1 WO2015030423 A1 WO 2015030423A1
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propionic acid
forage
acid production
feed
products
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PCT/KR2014/007770
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French (fr)
Korean (ko)
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정승기
조경현
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주식회사 바이오리쏘스
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Publication of WO2015030423A1 publication Critical patent/WO2015030423A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/12—Animal feeding-stuffs obtained by microbiological or biochemical processes by fermentation of natural products, e.g. of vegetable material, animal waste material or biomass
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
    • A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
    • A23K10/37—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms from waste material
    • A23K10/38—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms from waste material from distillers' or brewers' waste
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/105—Aliphatic or alicyclic compounds
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/10—Feeding-stuffs specially adapted for particular animals for ruminants
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
    • Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
    • Y02P60/87—Re-use of by-products of food processing for fodder production

Definitions

  • the present invention relates to a microorganism having a proonic acid producing ability and a forage composition comprising the same.
  • Volatile fatty acids are the most important end products of carbohydrate breakdown in the rumen. Volatile fatty acids are an important source of energy (70%) for ruminants and also affect the protein and fat content of milk.
  • the main volatile fatty acids produced by the metabolism in the rumen are three types of acetic acid, propionic acid and butyric acid, depending on the type of feed fed and the degree of digestion. Only propionic acid among volatile fatty acids in ruminants contributes to glucose synthesis, and quantitatively glucose is a very important single precursor. Propionic acid makes up 18-20% of the total volatile fatty acids and is converted to blood sugar in the liver to provide energy and used for lactose synthesis. Increasing propionic acid increases blood flow in ruminant epithelial cells, stimulates angiogenesis and increases epithelial cells, promotes the growth of ruminants, including cattle, and improves meat quality.
  • Fiber is the most abundant energy source on the planet. However, at present, a large part of the survey fee usage in Korea depends on imports. The high dependence on imports of forages is also a task that must be resolved in order to secure the competitiveness of domestic livestock farmers. After mushroom production, various attempts have been made to utilize resources such as fiber-rich waste media as byproducts.
  • Korean Patent No. 1144473 relates to a method for preparing fermentation fertilizer for livestock using mushroom by-products as a main raw material, and is characterized by fermentation by adding lactic acid bacteria, Bacillus subtilis and yeast bacteria used as probiotics to mushroom waste medium.
  • Korean Patent No. 1138934 relates to a method for producing a pig feed using a waste mushroom medium, characterized in that the fermentation using a complex microbial fermentation agent that does not contain a microorganism having a propioic acid producing ability.
  • the present inventors conducted a study on the feed for promoting the ruminant feed, the fermentation of the fertilizer in the rumen to increase the production of propioic acid and thereby the present invention based on a microorganism having a propionic acid production capacity that can promote the fattening Completed.
  • An object of the present invention is to provide a ruminant microorganism having a propionic acid production capacity.
  • the present invention also aims to provide a forage composition having an excellent fattening promoting effect.
  • One aspect of the invention provides a microorganism having a propionic acid production ability isolated from the rumen.
  • the microorganism is Lactobacillus mucosae KCCM11440P.
  • KCCM11440P in Lactobacillus mucosa has the 16S rRNA nucleotide sequence of SEQ ID NO: 1 and was identified based on this sequence. 1 shows a phylogenetic tree based on 16S rRNA sequences.
  • Propionic acid is the most important product of carbohydrate breakdown in the rumen, which is converted from ruminant liver to glucose to promote ruminant fattening and contribute to meat improvement.
  • KCCM11440P Lactobacillus mucosa isolated from ruminant on the basis of propionic acid production capacity, KCCM11440P was grown in MRS medium, especially in medium added with vitamin B 12 or sodium lactate was confirmed to have high propion production capacity.
  • Another aspect of the invention provides a forage composition comprising Lactobacillus mucosa KCCM11440P.
  • the term "irradiant composition” refers to a feed composition having a high fiber content, low fat, protein, starch and the like, such as grasses, hay, silage, and the like.
  • the fertilizer composition according to one aspect of the present invention includes KCCM11440P in Lactobacillus mucosa having propionic acid producing ability, thereby increasing the production of propionic acid during fermentation of the fertilizer in the rumen, thereby promoting the fattening of ruminants and improving meat quality.
  • propionic acid is the only volatile fatty acid produced in the rumen, it is converted into glucose and contributes to energy metabolism. Thus, promotion of the fat of ruminants including cattle is determined by propionic acid produced in the rumen.
  • the forage composition comprises mushroom waste medium.
  • Mushroom waste medium refers to a medium obtained by using a sawdust, the main raw material is a sawdust, a secondary material is obtained by using a culture medium for the culture of mushrooms.
  • Sawdust in mushroom waste media is used as a carbon source by fibrinolytic microorganisms in the rumen.
  • KCCM11440P in Lactobacillus mucosa can be cultured in mushroom lung medium to increase propionic acid production, thereby promoting the rearing of ruminants.
  • the mushroom waste medium can be used as a feedstock having excellent digestibility since the waste medium obtained after decomposition by the fibrinolytic bacteria of mushrooms is used as a raw material.
  • the forage composition comprises at least one of vitamin B 12 and lactate.
  • Lactobacillus mucosa KCCM11440P significantly increases propionic acid production when supplemented with vitamin B 12 or lactate, such as sodium lactate, in the medium. Therefore, the inclusion of at least one of vitamin B 12 and lactate in the forage composition increases propioic acid production, thereby promoting fattening.
  • the forage composition promotes the fattening of ruminants.
  • Lactobacillus mucosa which contains propioic acid producing ability, contains KCCM11440P, thereby increasing propioic acid production in the rumen, thereby promoting the rearing of ruminants and contributing to improved meat quality.
  • the forage composition may further comprise a probiotic.
  • Antibiotics are inevitable when raising livestock, but probiotics are widely used due to serious problems caused by misuse. Probiotics contribute to the uptake of beneficial microorganisms in the intestines of animals, thereby inhibiting the growth of pathogenic microorganisms, preventing the occurrence of diseases, and increasing the productivity of livestock.
  • beneficial microorganisms are lactic acid bacteria, subtilis bacteria, yeasts and the like. Lactic acid bacteria produce organic acids, lower the pH to inhibit harmful bacteria that are weak to acid, enhance the activity of digestive enzymes, and in particular, reduce the frequency of diarrhea.
  • Bacillus subtilis produces enzymes that break down high-active carbohydrates, proteins, and lipids, thereby enhancing feed digestion and absorption in the intestine, improving feed efficiency, and reducing stress from digestion, making livestock growth easier. In addition, it has a formal effect, strengthening the intestines of livestock and preventing disease.
  • yeast is present in a form that can be easily digested in the digestive tract of the livestock, by producing a natural flavor component such as alcohol, glutamic acid to enhance the palatability of the feed of the livestock.
  • the forage composition may be provided mixed with a general feed.
  • the forage composition may be mixed with a commercially formulated feed in a ratio of 1: 1 and fed to ruminants twice a day.
  • Appropriate mixing ratios, feeding amount and feeding frequency can be easily determined by those skilled in the art in consideration of the age, weight, health status, etc. of the subject ruminant.
  • Another aspect of the present invention provides a method of raising a ruminant, the method comprising feeding a forage comprising KCCM11440P to Lactobacillus mucosa.
  • the forage may include mushroom waste medium as a main component.
  • the forage may further comprise a probiotic.
  • the forage may further comprise one or more of vitamin B 12 and sodium lactate.
  • the feed may be fed by mixing with a conventional blended feed.
  • ruminants may be, but are not limited to, cattle, sheep, goats, and deer.
  • Lactobacillus mucosa KCCM11440P and a forage composition comprising the same according to one embodiment of the present invention increases the propioic acid production in the rumen of ruminants thereby resulting in excellent fattening and meat improvement.
  • 1 is a phylogenetic tree based on Lactobacillus mucosa of the present invention based on 16S rRNA gene of KCCM11440P.
  • Figure 2 shows the dry matter loss of fermented feed, bran, mushroom by-products, and brewing by-products with time of incubation.
  • Bacteria cultured from rumen juice were diluted to 10 ⁇ 3 to 10 ⁇ 9 per ml of medium and inoculated in Hungate roll tubes, followed by incubation for 24 to 48 hours. Cultured single colonies were isolated and incubated at 120 rpm for 24 hours after inoculation in liquid medium (MRS). All cultures were performed anaerobic at 37 ° C. (Lee, et al., Applied Microbiology and Biotechnology, Vol. 58, pp. 663-668, 2002), and all media and buffers used (Bryant and Burkey, Journal of Dairy). Science, Vol. 82, pp. 780-787, 1953) were sterilized at 121 ° C. for 15 minutes and O 2 and N 2 gases were used.
  • PCR reaction was performed using 27F primer (AGAGTTTGATCMTGGCTCAG) and 1492R primer (GGTTACCTTGTTACGACTT) for amplification of the gene encoding 16S rRNA.
  • PCR conditions consisted of 32 cycles of a cycle consisting of an initial denaturation step of 5 min at 94 ° C., 45 sec denaturation at 94 ° C., 45 sec annealing at 65 ° C., and 1 min elongation at 72 ° C., and 10 min at 72 ° C. It was kidney.
  • the amplified ribosomal DNA was analyzed for similarity by ARDRA (Amplified Ribosomal DNA Restriction Analysis) method.
  • ARDRA Analog Ribosomal DNA Restriction Analysis
  • the PCR products and the HaeIII HhaI restriction enzymes (Takara, Japan) at 37 °C for 5 hours, and were separated for 80 minutes the obtained DNA sample as a metaphor 170v through the electrophoresis using a gel with agar. Thereafter, 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 a QIA quick PCR Purification Kit.
  • the purified 16S rDNA PCR product was identified by SEQ ID NO: 1 by analyzing the nucleotide sequence in Macrogen (Korea).
  • the analyzed sequencing information was combined using SeqMan program (DNA Star, Lasergene software, Madison, WI, USA), and the base sequences were NCBI ( http://www.ncbi.nlm.nih.gov/BLAST ) and EzTaxon Comparison was made using the BLAST program at GeneBank ( http://147.47.212.35:8080/index.jsp ).
  • Approximate phylogenetic classification was determined using CLUSTRAL W version 1.6 to compare sequences with the nearest species.
  • the phylogenetic tree was prepared as disclosed in Kimura (1980) using a neighbor-joining (NJ) method with pair-wise gap removal. Finally, the two-parameter NJ method in the PHYLIP package was used, and the bootstrap analysis method was used to collect 1000 times more data to evaluate the stability of the tree. Only bootstrap values of 50% or more are shown. 1 shows the phylogenetic tree created.
  • the isolated strain showed 96% similarity to S32 (T) in Lactobacillus mucosa. Based on this, the isolated strain was named BR-PP to Lactobacillus mucosa, and it was deposited with the Korea Microorganism Conservation Center on July 26, 2013 and received an accession number of KCCM11440P.
  • Propionibacterium axidipropionis showed high propionic acid production after 144 hours. In addition, Propionibacterium axidipropionis showed conditional anaerobic unlike other propionic acid producing strains that are either complete or essential anaerobic. High propionic acid production and conditional anaerobic propionibacterium axidipropionis were selected as the propionic acid producing standard microorganisms and used as controls.
  • the propionic acid production capacity of Lactobacillus mutosa isolated and identified in Example 1 was evaluated.
  • the agar was cultured in anaerobic state for 48 hours in MRS medium.
  • the medium was placed in a Hungate tube and autoclaved at 121 ° C. for 15 minutes after filling with pH 6.5, O 2 -free 20% CO 2 -80% N 2 gas.
  • Biotin 0.5 mg / L
  • vitamin B 12 50 ⁇ g / L
  • glycerol (2%) was added to agar of MRS medium to confirm the effect on propionic acid production capacity, respectively.
  • the control for the evaluation of propionic acid production capacity was propionibacterium acidipropioni selected in Example 1.
  • Samples were taken at 24, 48 and 72 hours of culture to determine the production of volatile fatty acids, including OD, pH and propionic acid.
  • the pH was not measured directly in the incubator but stabilized at the same temperature as room temperature, and then measured using an M503P meter (wrks, Medififield, MA, USA).
  • the amount of volatile fatty acid produced was analyzed after centrifugation of the culture for each time period at 1000 ⁇ g 4 ° C. for 10 minutes, and then the supernatant was collected and purified with a 0.2 ⁇ m micro filter.
  • HPLC analysis was carried out 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 0.0085 NH 2 SO 4 and the flow rate was 0.6 ml / min.
  • Lactobacillus mucosa KCCM11440P produces the highest amount of propionic acid when cultured in MRS medium supplemented with vitamin B 12 or sodium lactate.
  • Rumen and buffer were used to evaluate the fermentation effect in the rumen in In Vitro .
  • Gastric fluid was collected for in vitro testing using 600 ⁇ 47 kg Holstein cows equipped with rumen fistulas from Sunchon National University farm.
  • Hanwoo a domestic animal, is Italian ly grass and rich feed (55% corn, 15% wheat, 8% skim rice, 5% corn gluten feed, 10% soybean meal, 0.2% molasses, calcium carbonate (limestone) ) 2.0%, salt 0.5%, calcium phosphate 1.3%, vitamin-mineral mixture (vitamin A 3000 IU, vitamin D 6000 IU, vitamin E 30 IU, Cu 25 mg, Fe 150 mg, Zn 200 mg, Mn 100 mg, Co 0.5 mg, and I 1.5 mg) 1.0%) was fed twice daily to 2% of body weight in a ratio of 2: 8 and free water was taken.
  • Buffer (Hino et al., 1992) is K 2 HPO 4 0.45 g / L , KH 2 PO 4 0.45 g / L, (NH 4) 2 SO 4 0.9 g / L, CaCl 2 ⁇ 2H 2 O 0.12 g / L , Basal media comprising MgSO 4 7H 2 O 0.19 g / L, Trypticase 1.0 g / L, yeast extract 1.0 g / L, cysteine HCl 0.6 g / L (pH 6.9) was prepared.
  • Rumen and buffer were mixed at a ratio of 1: 3 (rumen: buffer) and filled with nitrogen gas (N 2 gas).
  • N 2 gas nitrogen gas
  • Each fermented feed was placed in a 160 ml serum bottle with 2% dry matter, 100 ml of the prepared buffer was kept anaerobic with O 2 free-N 2 , sealed with a rubber stopper and an aluminum cap, and The mixture was incubated at 100 rpm in an anaerobic state (Hattori and Matsui, Anaerobe, Vol. 14, pp. 87-93, (2008)).
  • In vitro culture was performed in three replicate experiments for 0, 12, 24, and 48 hours, and pH, total gas evolution, methane, ammonia, and VFA were measured as characteristics of the rumen fermentation.
  • the pH was not measured directly in the incubator but stabilized at the same temperature as room temperature, and then measured using an M503P meter (wrks, Medififield, MA, USA).
  • the total gas generation amount was stabilized and used EA-6 (Inc, Sun Bee instrument) pressure sensor measuring instrument. After the total gas generation was measured, the generated gas was collected using a vacuum tube to measure the amount of methane and carbon dioxide generated. On the basis of the total gas generation amount obtained for each incubation time, the gas generation amount was estimated by the formula of Qrskov and McDonald (1979).
  • the VFA measurement was analyzed after centrifugation of the culture by time incubation at 1000 ⁇ g 4 °C for 10 minutes to extract the supernatant and purified with a 0.2 ⁇ m micro filter.
  • HPLC analysis was carried out 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 0.0085 NH 2 SO 4 and the flow rate was 0.6 ml / min.
  • Ammonia concentration was measured by centrifuging the sample at 13000rpm and spectroscopy was carried out by developing ammonia in the sample with phenol solution according to the method of Chany and Marbach (Clinical Chemistry, Vol. 8, pp. 130-132, (1962)). Absorbance was measured and measured at 630 nm using a photometer (Spectronics 21D).
  • Figure 2 shows the dry matter loss of the fermented feed according to the incubation time.
  • a combination of bran and brew by-products or mushroom by-products was used as fermented feed.
  • the amount of loss in mushroom by-products was higher than for brewing by-products.
  • mushroom byproducts are easier to digest in the rumen than brewing byproducts.
  • Silage provides for feed quality and maximum building. Microorganisms in the feed also speed up the fermentation and enhance the results of silage. Therefore, the fermentation effect was measured in vitro by adding microorganisms having propionic acid production capacity.
  • Table 4 shows the dry matter (buildings) loss with fermentation incubation time.
  • the loss of building by mushroom by-products was lower than by brewing by-products (P ⁇ 0.05).
  • the increase in dry matter loss of brewing by-products of silage is indicated by the addition to Lactobacillus mucosa.
  • Table 5 shows the VFA production by fermentation. Dry matter loss was observed to be low in mushroom byproducts, while total VFA and propionic acid were high in brewing byproducts. In addition, fermentation by adding Lactobacillus mucosa to the by-product during the treatment showed high propionic acid production and total volatile fatty acids (TVFA). This shows that the acid change of mushroom by-products occurs higher than brewing by-products due to nutritional composition. The large amount of acid produced while silage is produced means that feed is a higher energy source for ruminants.
  • Table 6 shows the total gas production, pH, and ammonia nitrogen production by in vitro fermentation of brewing by-products and mushroom by-products.

Abstract

The present invention relates to a microorganism having a propionic acid production capability and a feed composition comprising the same.

Description

프로피온산 생산능을 갖는 미생물 및 그를 포함하는 조사료 조성물Microorganisms having a propionic acid producing ability and a forage composition comprising the same
본 발명은 프로온산 생산능을 갖는 미생물 및 그를 포함하는 조사료 조성물에 관한 것이다. The present invention relates to a microorganism having a proonic acid producing ability and a forage composition comprising the same.
휘발성 지방산(VFA)은 반추위 내에서 탄수화물 분해의 가장 중요한 최종산물이다. 휘발성 지방산은 반추동물을 위한 에너지의 중요한 자원(70%)이고, 우유 중 단백질과 지방의 함량에도 영향을 미친다. 반추위 내의 대사에서 생성되는 주요한 휘발성 지방산은 아세트산, 프로피온산 및 부티르산의 3종으로, 급이된 사료의 종류 및 소화 정도에 따라 결정된다. 반추동물에서 휘발성 지방산 중 프로피온산만이 포도당 합성에 기여하며, 양적으로 포도당은 매우 중요한 단독의 전구체이다. 프로피온산은 총 휘발성 지방산의 18 내지 20%를 구성하며, 간에서 혈당으로 전환되어 에너지를 제공하고, 유당 합성에 사용된다. 프로피온산의 증가는 반추 상피세포에서 혈류를 증가시켜 혈관 생성을 자극하고 상피세포를 증가시키며, 소를 포함한 반추동물의 비육을 촉진하고 육질 개선에 기여한다.Volatile fatty acids (VFA) are the most important end products of carbohydrate breakdown in the rumen. Volatile fatty acids are an important source of energy (70%) for ruminants and also affect the protein and fat content of milk. The main volatile fatty acids produced by the metabolism in the rumen are three types of acetic acid, propionic acid and butyric acid, depending on the type of feed fed and the degree of digestion. Only propionic acid among volatile fatty acids in ruminants contributes to glucose synthesis, and quantitatively glucose is a very important single precursor. Propionic acid makes up 18-20% of the total volatile fatty acids and is converted to blood sugar in the liver to provide energy and used for lactose synthesis. Increasing propionic acid increases blood flow in ruminant epithelial cells, stimulates angiogenesis and increases epithelial cells, promotes the growth of ruminants, including cattle, and improves meat quality.
섬유소는 지구상에서 가장 풍부한 에너지원이다. 그러나, 현재 우리나라에서 조사료 사용량 중 상당 부분이 수입에 의존하고 있다. 조사료의 높은 수입 의존도는 국내 축산농가의 경쟁력 확보를 위해서도 해결되어야 하는 과제이다. 버섯 생산 후 부산물인 섬유소가 풍부한 폐배지 등의 자원을 사료로 활용하기 위한 다양한 시도가 이루어졌다. Fiber is the most abundant energy source on the planet. However, at present, a large part of the survey fee usage in Korea depends on imports. The high dependence on imports of forages is also a task that must be resolved in order to secure the competitiveness of domestic livestock farmers. After mushroom production, various attempts have been made to utilize resources such as fiber-rich waste media as byproducts.
한국 특허 제1144473호는 버섯 부산물을 주원료로 이용한 가축용 발효 조사료 제조방법에 관한 것으로, 버섯 폐배지에 생균제로 사용되고 있는 유산균, 고초균 및 효모균을 첨가하여 발효시키는 것을 특징으로 한다. Korean Patent No. 1144473 relates to a method for preparing fermentation fertilizer for livestock using mushroom by-products as a main raw material, and is characterized by fermentation by adding lactic acid bacteria, Bacillus subtilis and yeast bacteria used as probiotics to mushroom waste medium.
한국 특허 제1138934호는 폐 버섯 배지를 사용하여 돼지 사료를 제조하는 방법에 관한 것으로, 프로피오산 생산능을 갖는 미생물을 포함하지 않는 복합 미생물 발효제를 이용하여 발효시키는 것을 특징으로 한다. Korean Patent No. 1138934 relates to a method for producing a pig feed using a waste mushroom medium, characterized in that the fermentation using a complex microbial fermentation agent that does not contain a microorganism having a propioic acid producing ability.
그러나, 풍부한 섬유소를 활용하면서, 항생제나 호르몬 등의 사용에 따른 부작용 없이 비육 촉진에 기여할 수 있는 효과적인 사료에 대한 요구가 여전히 존재한다. However, there is still a need for an effective feed that can contribute to the promotion of fat without taking the side effects of using antibiotics or hormones while utilizing abundant fiber.
이에, 본 발명자들은 반추동물의 비육 촉진용 사료에 대한 연구를 수행하여, 반추위에서 조사료를 발효시켜 프로피오산 생성을 증가시키고 이에 의해 비육을 촉진할 수 있는 프로피온산 생산능을 갖는 미생물에 기반한 본 발명을 완성하였다. Thus, the present inventors conducted a study on the feed for promoting the ruminant feed, the fermentation of the fertilizer in the rumen to increase the production of propioic acid and thereby the present invention based on a microorganism having a propionic acid production capacity that can promote the fattening Completed.
본 발명은 프로피온산 생산능을 갖는 반추 미생물을 제공하는 것을 목적으로 한다. An object of the present invention is to provide a ruminant microorganism having a propionic acid production capacity.
본 발명은 또한 탁월한 비육 촉진 효과를 갖는 조사료 조성물을 제공하는 것을 목적으로 한다. The present invention also aims to provide a forage composition having an excellent fattening promoting effect.
본 발명은 또한 반추 미생물을 포함하는 조사료를 이용하여 반추동물을 비육하는 방법을 제공하는 것을 목적으로 한다.It is another object of the present invention to provide a method of raising ruminants using a forage comprising ruminant microorganisms.
본 발명의 일 양태는 반추위로부터 분리된 프로피온산 생산능을 갖는 미생물을 제공한다. One aspect of the invention provides a microorganism having a propionic acid production ability isolated from the rumen.
본 발명의 일 구체예에서, 상기 미생물은 락토바실러스 뮤코사에(Lactobacillus mucosae) KCCM11440P이다. In one embodiment, the microorganism is Lactobacillus mucosae KCCM11440P.
락토바실러스 뮤코사에 KCCM11440P는 서열번호 1의 16S rRNA 염기서열을 가지며, 이 서열에 근거하여 동정되었다. 도 1은 16S rRNA 염기서열에 근거하여 작성된 계통수를 보여준다. KCCM11440P in Lactobacillus mucosa has the 16S rRNA nucleotide sequence of SEQ ID NO: 1 and was identified based on this sequence. 1 shows a phylogenetic tree based on 16S rRNA sequences.
프로피온산은 반추위 내에서 탄수화물 분해의 가장 중요한 산물로서, 반추동물의 간에서 포도당으로 전환되어 반추동물의 비육을 촉진하며, 육질 개선에 기여한다. Propionic acid is the most important product of carbohydrate breakdown in the rumen, which is converted from ruminant liver to glucose to promote ruminant fattening and contribute to meat improvement.
반추위로부터 프로피온산 생산능에 근거하여 분리된 락토바실러스 뮤코사에 KCCM11440P는 MRS 배지에서 성장하며, 특히, 비타민 B12나 젖산 나트륨이 첨가된 배지에서 높은 프로피온 생산능을 갖는 것으로 확인되었다. Lactobacillus mucosa isolated from ruminant on the basis of propionic acid production capacity, KCCM11440P was grown in MRS medium, especially in medium added with vitamin B 12 or sodium lactate was confirmed to have high propion production capacity.
본 발명의 또 다른 양태는 락토바실러스 뮤코사에 KCCM11440P를 포함하는, 조사료 조성물을 제공한다. Another aspect of the invention provides a forage composition comprising Lactobacillus mucosa KCCM11440P.
본 명세서에서 사용된 용어 "조사료 조성물"은 목초, 건초, 사일리지, 등과 같이, 섬유질 함량이 높고, 지방, 단백질, 전분 등의 함량이 낮은 사료 조성물을 의미한다. As used herein, the term "irradiant composition" refers to a feed composition having a high fiber content, low fat, protein, starch and the like, such as grasses, hay, silage, and the like.
본 발명의 일 양태에 따른 조사료 조성물은 프로피온산 생산능을 갖는 락토바실러스 뮤코사에 KCCM11440P를 포함하여, 반추위에서 조사료의 발효시 프로피온산의 생산을 증가시키고, 이에 의해 반추동물의 비육을 촉진하며 육질을 개선시킬 수 있다. 프로피온산은 반추위에서 생성되는 휘발성 지방산 중 유일하게 포도당으로 전환되어 에너지 대사 등에 기여하므로, 소를 포함한 반추동물의 비육 촉진은 반추위에서 생성되는 프로피온산에 의해 결정된다.The fertilizer composition according to one aspect of the present invention includes KCCM11440P in Lactobacillus mucosa having propionic acid producing ability, thereby increasing the production of propionic acid during fermentation of the fertilizer in the rumen, thereby promoting the fattening of ruminants and improving meat quality. You can. Since propionic acid is the only volatile fatty acid produced in the rumen, it is converted into glucose and contributes to energy metabolism. Thus, promotion of the fat of ruminants including cattle is determined by propionic acid produced in the rumen.
본 발명의 일 구체예에서, 상기 조사료 조성물은 버섯 폐배지를 포함한다.In one embodiment of the invention, the forage composition comprises mushroom waste medium.
버섯 폐배지는 주원료가 톱밥이며, 부원료가 농산부산물로 이루어진 배지를 버섯 배양에 사용한 후 수득하는 배지를 의미한다. 버섯 폐배지 중 톱밥은 반추위에서 섬유소 분해 미생물에 의해 탄소 공급원으로 이용된다. 락토바실러스 뮤코사에 KCCM11440P는 버섯 폐배지에서 배양되어 프로피온산 생산을 증가시키고, 이에 의해 반추동물의 비육을 촉진시킬 수 있다. 특히, 버섯 폐배지는 버섯류의 섬유소 분해균에 의해 분해된 후 수득된 폐배지를 원료로 하므로 소화성이 우수한 조사료로 이용될 수 있다. Mushroom waste medium refers to a medium obtained by using a sawdust, the main raw material is a sawdust, a secondary material is obtained by using a culture medium for the culture of mushrooms. Sawdust in mushroom waste media is used as a carbon source by fibrinolytic microorganisms in the rumen. KCCM11440P in Lactobacillus mucosa can be cultured in mushroom lung medium to increase propionic acid production, thereby promoting the rearing of ruminants. In particular, the mushroom waste medium can be used as a feedstock having excellent digestibility since the waste medium obtained after decomposition by the fibrinolytic bacteria of mushrooms is used as a raw material.
본 발명의 일 구체예에서, 상기 조사료 조성물은 비타민 B12 및 젖산염 중 하나 이상을 포함한다. In one embodiment of the present invention, the forage composition comprises at least one of vitamin B 12 and lactate.
락토바실러스 뮤코사에 KCCM11440P는 배지 중에 비타민 B12 또는 젖산염, 예를 들면, 젖산 나트륨이 보충된 경우 프로피온산 생산량을 크게 증가시킨다. 따라서, 조사료 조성물 중에 비타민 B12 및 젖산염 중 하나 이상을 포함시키면 프로피오산 생산이 증가되고, 이에 의해 비육이 촉진된다. Lactobacillus mucosa KCCM11440P significantly increases propionic acid production when supplemented with vitamin B 12 or lactate, such as sodium lactate, in the medium. Therefore, the inclusion of at least one of vitamin B 12 and lactate in the forage composition increases propioic acid production, thereby promoting fattening.
본 발명의 일 구체예에서, 상기 조사료 조성물은 반추동물의 비육을 촉진시킨다. 통상적인 조사료 조성물에 비해, 프로피오산 생산능을 갖는 락토바실러스 뮤코사에 KCCM11440P를 포함하므로, 반추위에서 프로피오산 생산을 증가시키고, 이에 의해 반추동물의 비육을 촉진하며, 육질 개선에 기여한다. In one embodiment of the invention, the forage composition promotes the fattening of ruminants. Compared to conventional forage compositions, Lactobacillus mucosa, which contains propioic acid producing ability, contains KCCM11440P, thereby increasing propioic acid production in the rumen, thereby promoting the rearing of ruminants and contributing to improved meat quality.
본 발명의 일 구체예에서, 상기 조사료 조성물은 생균제를 더 포함할 수 있다.In one embodiment of the present invention, the forage composition may further comprise a probiotic.
가축 사육시에는 항생제의 사용이 불가피하나, 이의 오남용으로 인한 문제가 심각해지면서 생균제가 널리 이용되고 있다. 생균제는 가축의 장내에 유익한 미생물을 섭취시켜 병원성 미생물의 증식을 억제하고, 질병의 발생을 예방하며, 가축의 생산성을 높이는 데 기여한다. 현재 널리 사용되는 미생물은 유산균, 고초균, 효모균 등이다. 유산균은 유기산을 생성하여 pH를 저하시켜 산성에 약한 유해세균을 억제하며, 소화효소의 활동을 증진시키며, 특히, 설사발생빈도도 감소시킨다. 고초균은 고활성의 탄수화물, 단백질, 지질 등을 분해하는 분해효소를 생성하므로, 장내에서 사료의 소화, 흡수를 증진시켜 사료 효율을 향상시키며, 소화에 따른 스트레스를 감소시켜 가축의 생육을 원활하게 하며, 정장 효과가 있어 가축의 장을 튼튼하게 하여 질병을 예방하는 역할을 한다. 또한, 효모균은 가축의 소화관에서 용이하게 소화될 수 있는 형태로 존재하며, 알코올, 글루타민산 등 천연 향미성분을 생산하여 가축의 사료에 대한 기호성을 증진시킨다. Antibiotics are inevitable when raising livestock, but probiotics are widely used due to serious problems caused by misuse. Probiotics contribute to the uptake of beneficial microorganisms in the intestines of animals, thereby inhibiting the growth of pathogenic microorganisms, preventing the occurrence of diseases, and increasing the productivity of livestock. Currently widely used microorganisms are lactic acid bacteria, subtilis bacteria, yeasts and the like. Lactic acid bacteria produce organic acids, lower the pH to inhibit harmful bacteria that are weak to acid, enhance the activity of digestive enzymes, and in particular, reduce the frequency of diarrhea. Bacillus subtilis produces enzymes that break down high-active carbohydrates, proteins, and lipids, thereby enhancing feed digestion and absorption in the intestine, improving feed efficiency, and reducing stress from digestion, making livestock growth easier. In addition, it has a formal effect, strengthening the intestines of livestock and preventing disease. In addition, yeast is present in a form that can be easily digested in the digestive tract of the livestock, by producing a natural flavor component such as alcohol, glutamic acid to enhance the palatability of the feed of the livestock.
본 발명의 일 구체예에서, 상기 조사료 조성물은 일반 사료와 혼합되어 제공될 수 있다. 예를 들면, 조사료 조성물은 시판 배합사료와 1:1의 비율로 혼합되어 1일 2회 반추동물에게 급이될 수 있다. 적절한 혼합 비율이나 급이량 및 급이 빈도는 당업자가 대상 반추동물의 연령, 체중, 건강 상태 등을 고려하여 용이하게 결정할 수 있다. In one embodiment of the present invention, the forage composition may be provided mixed with a general feed. For example, the forage composition may be mixed with a commercially formulated feed in a ratio of 1: 1 and fed to ruminants twice a day. Appropriate mixing ratios, feeding amount and feeding frequency can be easily determined by those skilled in the art in consideration of the age, weight, health status, etc. of the subject ruminant.
본 발명의 또 다른 양태는 반추동물을 비육하는 방법으로서, 락토바실러스 뮤코사에 KCCM11440P를 포함하는 조사료를 급이하는 단계를 포함하는 것인 방법을 제공한다. Another aspect of the present invention provides a method of raising a ruminant, the method comprising feeding a forage comprising KCCM11440P to Lactobacillus mucosa.
본 발명의 일 구체예에서, 상기 조사료는 버섯 폐배지를 주성분으로 포함할 수 있다. In one embodiment of the present invention, the forage may include mushroom waste medium as a main component.
본 발명의 일 구체예에서, 상기 조사료는 생균제를 더 포함할 수 있다. In one embodiment of the present invention, the forage may further comprise a probiotic.
본 발명의 일 구체예에서, 상기 조사료는 비타민 B12 및 젖산 나트륨 중 하나 이상을 더 포함할 수 있다. In one embodiment of the invention, the forage may further comprise one or more of vitamin B 12 and sodium lactate.
본 발명의 일 구체예에서, 조사료는 통상적인 배합사료와 혼합하여 급이될 수 있다. In one embodiment of the invention, the feed may be fed by mixing with a conventional blended feed.
본 발명의 일 구체예에서, 반추동물은 소, 양, 염소, 및 사슴일 수 있으나, 이에 한정되지 않는다.In one embodiment of the invention, ruminants may be, but are not limited to, cattle, sheep, goats, and deer.
이하, 실시예를 통해 본 발명을 보다 상세하게 설명하고자 한다. 그러나, 하기 실시예는 본 발명을 예시하기 위한 것이며, 본 발명을 한정하는 것으로 해석되어서는 안 된다. 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.
본 발명의 일 구체예에 따른 락토바실러스 뮤코사에 KCCM11440P 및 이를 포함하는 조사료 조성물은 반추동물의 반추위에서 프로피오산 생산을 증가시키고 이에 의해 우수한 비육 촉진 및 육질 개선 효과를 가져온다. Lactobacillus mucosa KCCM11440P and a forage composition comprising the same according to one embodiment of the present invention increases the propioic acid production in the rumen of ruminants thereby resulting in excellent fattening and meat improvement.
도 1은 본 발명의 락토바실러스 뮤코사에 KCCM11440P의 16S rRNA 유전자를 근간으로 작성한 계통수이다.1 is a phylogenetic tree based on Lactobacillus mucosa of the present invention based on 16S rRNA gene of KCCM11440P.
도 2는 배양 시간에 따른 발효 사료, 밀기울, 버섯 부산물, 및 양조 부산물의 건물 소실량을 보여준다. Figure 2 shows the dry matter loss of fermented feed, bran, mushroom by-products, and brewing by-products with time of incubation.
실시예 1. 프로피온산 생성균의 분리 및 동정Example 1 Isolation and Identification of Propionic Acid Producing Bacteria
1-1. 균주의 분리1-1. Isolation of Strains
프로피오산 생산능을 갖는 균주를 분리하기 위해, 40±5kg의 재래 흑염소 3두의 반추위액을 채취하였다. 혐기 상태를 유지하기 위해 혐기 챔버 안에서 수행하였으며, Hattori 와 Matsui(Anaerobe, Vol. 14, pp. 87-93, (2008)) 방법을 이용하였다. To isolate strains with propioic acid production, three rumens of 40 ± 5 kg of conventional black goat 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)) were used.
반추위액으로부터 배양된 균을 배지 ml 당 10-3 내지 10-9로 희석한 후 Hungate 롤 튜브에 접종한 후 24 내지 48시간 동안 배양하였다. 배양된 단일 콜로니를 분리하여 액상 배지(MRS)에 접종 후 24시간 동안 120 rpm에서 배양하였다. 모든 배양은 37℃에서 혐기상태에서 수행하였으며(Lee, et al., Applied Microbiology and Biotechnology, Vol. 58, pp. 663-668, 2002), 사용된 모든 배지와 버퍼(Bryant and Burkey, Journal of Dairy Science, Vol. 82, pp. 780-787, 1953)는 121℃에서 15분 동안 멸균시켰고 O₂와 N₂가스를 사용하였다.Bacteria cultured from rumen juice were diluted to 10 −3 to 10 −9 per ml of medium and inoculated in Hungate roll tubes, followed by incubation for 24 to 48 hours. Cultured single colonies were isolated and incubated at 120 rpm for 24 hours after inoculation in liquid medium (MRS). All cultures were performed anaerobic at 37 ° C. (Lee, et al., Applied Microbiology and Biotechnology, Vol. 58, pp. 663-668, 2002), and all media and buffers used (Bryant and Burkey, Journal of Dairy). Science, Vol. 82, pp. 780-787, 1953) were sterilized at 121 ° C. for 15 minutes and O 2 and N 2 gases were used.
총 51개의 균주를 분리하고, 이들을 MRS 배지에서 배양하면서 휘발성 지방산의 생산량을 측정하였다. 가장 높은 프로피온산 생산량을 보이는 균주를 선택하였다. A total of 51 strains were isolated and these were cultured in MRS medium to measure the production of volatile fatty acids. The strain showing the highest propionic acid yield was selected.
1-2. 균주의 동정1-2. Identification of Strains
1-1에서 분리된 균주를 MRS 배지에서 배양한 후 Wizard Genomic DNA Purification Kits (Promega, USA)를 사용하여 염색체 DNA를 추출하였다. 이를 주형으로 이용하여 16S rRNA를 코딩하는 유전자의 증폭을 위해 27F 프라이머(AGAGTTTGATCMTGGCTCAG)와 1492R 프라이머(GGTTACCTTGTTACGACTT)를 사용하여 PCR 반응을 수행하였다. PCR 조건은 94℃에서 5분의 초기 변성 단계, 94℃에서 45초의 변성, 65℃에서 45초의 어닐링, 및 72℃에서 1분의 신장으로 이루어진 사이클의 32회 반복, 및 72℃에서 10분의 신장이었다.Strains isolated in 1-1 were cultured in MRS medium and chromosomal DNA was extracted using Wizard Genomic DNA Purification Kits (Promega, USA). Using this as a template, PCR reaction was performed using 27F primer (AGAGTTTGATCMTGGCTCAG) and 1492R primer (GGTTACCTTGTTACGACTT) for amplification of the gene encoding 16S rRNA. PCR conditions consisted of 32 cycles of a cycle consisting of an initial denaturation step of 5 min at 94 ° C., 45 sec denaturation at 94 ° C., 45 sec annealing at 65 ° C., and 1 min elongation at 72 ° C., and 10 min at 72 ° C. It was kidney.
증폭된 리보솜 DNA을 ARDRA(Amplified Ribosomal DNA Restriction Analysis) 방법을 통하여 유사성을 분석하였다. PCR 산물을 HaeⅢ 및 HhaⅠ 제한효소(Takara, Japan)를 37℃에서 5시간 동안 처리하고, 수득된 DNA 시료를 메타포 아가로스겔을 이용한 전기영동을 통해 170v로 80분 동안 분리하였다. 그 후, Kodak Gel Logic 200 화상 시스템(Eastman Kodak Company, Rochester, NY, USA)을 이용하여 시각화하고, ARDRA로부터 얻어진 밴드들은 QIA quick PCR Purification Kit를 사용하여 정제하였다.The amplified ribosomal DNA was analyzed for similarity by ARDRA (Amplified Ribosomal DNA Restriction Analysis) method. The PCR products and the HaeⅢ HhaⅠ restriction enzymes (Takara, Japan) at 37 ℃ for 5 hours, and were separated for 80 minutes the obtained DNA sample as a metaphor 170v through the electrophoresis using a gel with agar. Thereafter, 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 a QIA quick PCR Purification Kit.
정제된 16S rDNA PCR 산물은 Macrogen(한국)에서 염기서열을 분석하여 서열번호 1로 확인하였다. 분석된 염기서열 정보는 SeqMan program(DNA Star, Lasergene software, Madison, WI, USA)을 이용하여 조합하였으며, 염기서열은 NCBI(http://www.ncbi.nlm.nih.gov/BLAST)와 EzTaxon(http://147.47.212.35:8080/index.jsp)의 GeneBank에 있는 BLAST 프로그램을 이용하여 비교하였다. 대략적인 계통학적 분류는 CLUSTRAL W version 1.6을 이용하여 가장 가까운 종과 염기서열을 비교함으로써 결정하였다. 쌍대-갭 제거(pair-wise gap removal) 기능을 갖는 NJ(neighbor-joining) 방법을 이용하여 Kimura(1980)에 개시된 바에 따라 계통수를 작성하였다. 최종적으로, PHYLIP 패키지에 있는 two-parameter NJ 방법을 이용하고 계통수의 안정성을 평가하기 위하여 1000배에 이르는 자료를 더 수집하여 부트스트랩(bootstrap) 분석방법을 이용하였다. 50% 이상의 부트스트랩 값만 나타내었다. 도 1은 작성된 계통수를 보여준다. The purified 16S rDNA PCR product was identified by SEQ ID NO: 1 by analyzing the nucleotide sequence in Macrogen (Korea). The analyzed sequencing information was combined using SeqMan program (DNA Star, Lasergene software, Madison, WI, USA), and the base sequences were NCBI ( http://www.ncbi.nlm.nih.gov/BLAST ) and EzTaxon Comparison was made using the BLAST program at GeneBank ( http://147.47.212.35:8080/index.jsp ). Approximate phylogenetic classification was determined using CLUSTRAL W version 1.6 to compare sequences with the nearest species. The phylogenetic tree was prepared as disclosed in Kimura (1980) using a neighbor-joining (NJ) method with pair-wise gap removal. Finally, the two-parameter NJ method in the PHYLIP package was used, and the bootstrap analysis method was used to collect 1000 times more data to evaluate the stability of the tree. Only bootstrap values of 50% or more are shown. 1 shows the phylogenetic tree created.
분리된 균주는 락토바실러스 뮤코사에 S32(T)와 96%의 유사도를 보였다. 이에 근거하여, 분리된 균주를 락토바실러스 뮤코사에 BR-PP로 명명하고, 이를 2013년 7월 26일자로 한국미생물보존센터에 기탁하여 KCCM11440P의 수탁번호를 부여받았다. The isolated strain showed 96% similarity to S32 (T) in Lactobacillus mucosa. Based on this, the isolated strain was named BR-PP to Lactobacillus mucosa, and it was deposited with the Korea Microorganism Conservation Center on July 26, 2013 and received an accession number of KCCM11440P.
1-3. 대조군 균주의 선택1-3. Selection of Control Strains
1-1 및 1-2에서 분리되고 동정된 락토바실러스 뮤코사에 KCCM11440P의 프로피온산 생산능을 비교하기 위한 대조군을 선택하기 위해, 프로피온산 생산능을 갖는 것으로 알려진 셀레노모나스 보비스(Selenomonas bovis) 15154(SB), 베일리오넬라 파르불라(Veillonella parvula) 5019(VP), 프로피오니박테리움 액시디프로피오니시(Propionobacterium acidipropionici) 5020(PAci) 과 프로피오니박테리움 아크네스(Propionibacterium acnes) 11946 (PAcn)를 각각 KCTC 및 KACC에서 분양받아 이용하였다. To select a control for comparing propionic acid production capacity of KCCM11440P to Lactobacillus mucosa isolated and identified in 1-1 and 1-2, Selenomonas bovis 15154 (SB known to have propionic acid production capacity) ) , Veillonella parvula 5019 (VP) , Propionobacterium acidipropionici 5020 (PAci) and Propionibacterium acnes 11946 (PAcn) was used by KCTC and KACC, respectively.
각 균주를 37℃에서 48, 72, 96, 120 및 144시간 동안 배양하면서 OD(Optical density) 값과 pH를 측정하였다. 각 실험은 3회 반복실시하였다. 또한, 채취된 각 배양 시간의 시료를 UV 검출기가 장착된 고성능 액체 크로마토 그래피(HPLC) (Agilent Technologies 1200 series)에서 MetaCarb 87H (Varian, Germany) 컬럼 상에서 유동 속도 0.6ml/min로 0.0085N·H2SO4 완충액으로 용리시켜 분리하고 210nm 및 220nm에서 검출하여 휘발성 지방산(VFA)의 생산량을 측정하였다(Tabaru et al., Japanese Journal of Veterinary Science Vol.50, pp. 1124-1126 (1988) 및 와 Han et al., Process Biochemistry, Vol. 40, pp.2897-2905 (2005)). Each strain was incubated at 37 ° C. for 48, 72, 96, 120 and 144 hours, and the optical density (OD) value and pH were measured. Each experiment was repeated three times. In addition, samples of each incubation time taken were 0.0085 N · H 2 at a flow rate of 0.6 ml / min on a MetaCarb 87H (Varian, Germany) column in high performance liquid chromatography (HPLC) (Agilent Technologies 1200 series) equipped with a UV detector. Elution was performed by elution with SO 4 buffer and detection at 210 nm and 220 nm to determine the production of volatile fatty acids (VFA) (Tabaru et al . , Japanese Journal of Veterinary Science Vol. 50, pp. 1124-1126 (1988) and and Han et al., Process Biochemistry, Vol. 40, pp. 2897-2905 (2005)).
프로피오니박테리움 액시디프로피오니시는 144시간 후에 높은 프로피온산 생산량을 보였다. 또한, 프로피오니박테리움 액시디프로피오니시는 완전하거나 필수적 혐기성인 다른 프로피온산 생성 균주와 달리 조건적 혐기성을 보였다. 높은 프로피온산 생산량과 조건적 혐기성인 프로피오니박테리움 액시디프로피오니시를 프로피온산생성 표준 미생물로 선택하여 대조군으로 이용하였다.Propionibacterium axidipropionis showed high propionic acid production after 144 hours. In addition, Propionibacterium axidipropionis showed conditional anaerobic unlike other propionic acid producing strains that are either complete or essential anaerobic. High propionic acid production and conditional anaerobic propionibacterium axidipropionis were selected as the propionic acid producing standard microorganisms and used as controls.
실시예 2. 락토바실러스 뮤코사에 KCCM1140P의 프로피온산 생산능Example 2 Propionate Production Capacity of KCCM1140P in Lactobacillus Mucosa
실시예 1에서 분리하고 동정한 락토바실러스 뮤토사에의 프로피온산 생산능을 평가하였다. MRS 배지에 기반한 아가에서 48시간 동안 혐기 상태로 배양하였다. 배지를 Hungate 튜브를 넣고, pH 6.5, O2-free 20% CO2-80% N2 가스 충진 후 121℃에서 15분 동안 고압 멸균하였다. 프로피온산 생산능에 미치는 효과를 확인하기 위해 MRS 배지의 아가에 각각 비오틴(0.5mg/L), 소디움 락테이트 (2%), 비타민 B12 (50 ㎍/L) 또는 글리세롤 (2%)을 첨가하였다. 프로피온산 생산능의 평가를 위한 대조군은 실시예 1에서 선택된 프로피오니박테리움 액시디프로피오니시였다. The propionic acid production capacity of Lactobacillus mutosa isolated and identified in Example 1 was evaluated. The agar was cultured in anaerobic state for 48 hours in MRS medium. The medium was placed in a Hungate tube and autoclaved at 121 ° C. for 15 minutes after filling with pH 6.5, O 2 -free 20% CO 2 -80% N 2 gas. Biotin (0.5 mg / L), sodium lactate (2%), vitamin B 12 (50 μg / L) or glycerol (2%) was added to agar of MRS medium to confirm the effect on propionic acid production capacity, respectively. The control for the evaluation of propionic acid production capacity was propionibacterium acidipropioni selected in Example 1.
배양 24시간, 48시간 및 72시간에 시료를 채취하여 OD, pH 및 프로피온산을 포함한 휘발성 지방산의 생성량을 측정하였다. Samples were taken at 24, 48 and 72 hours of culture to determine the production of volatile fatty acids, including OD, pH and propionic acid.
pH는 인큐베이터에서 바로 측정하지 않고 상온과 동일한 온도로 안정화시킨 후 M503P 미터(wrks, Medififield, MA, USA)를 이용하여 측정하였다.The pH was not measured directly in the incubator but stabilized at the same temperature as room temperature, and then measured using an M503P meter (wrks, Medififield, MA, USA).
휘발성 지방산의 생성량은 배양시간대별 배양물을 1000×g 4℃에서 10분간 원심분리 후 상등액을 채취하여 0.2㎛ 마이크로 필터로 정제한 후 분석하였다. METACARB87H(Varian, Germany) 컬럼을 장착한 HPLC(Agilent technolgies 1200 series)를 이용하여 35℃에서 분석했고, 검출기의 UV의 파장은 210nm와 220nm였다. 이동상 용매는 0.0085N H2SO4이고 유속은 0.6ml/min이었다. The amount of volatile fatty acid produced was analyzed after centrifugation of the culture for each time period at 1000 × g 4 ° C. for 10 minutes, and then the supernatant was collected and purified with a 0.2 μm micro filter. HPLC analysis was carried out 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 0.0085 NH 2 SO 4 and the flow rate was 0.6 ml / min.
하기 표 1 및 2는 그 결과를 요약한다. Tables 1 and 2 below summarize the results.
표 1
Figure PCTKR2014007770-appb-T000001
 Table 1
Figure PCTKR2014007770-appb-T000001
표 2
Figure PCTKR2014007770-appb-T000002
 TABLE 2
Figure PCTKR2014007770-appb-T000002
수득된 결과는 락토바실러스 뮤코사에 KCCM11440P는 비타민 B12 또는 젖산 나트륨이 보충된 MRS 배지에서 배양 시 가장 많은 양의 프로피온산을 생산한다는 것을 보여준다. The results obtained show that Lactobacillus mucosa KCCM11440P produces the highest amount of propionic acid when cultured in MRS medium supplemented with vitamin B 12 or sodium lactate.
실시예 3. 조사료의 제조Example 3. Preparation of Forage
반추위액 및 완충액을 이용하여 인 비트로(In Vitro)에서 반추위에서의 발효 효과를 평가하였다. Rumen and buffer were used to evaluate the fermentation effect in the rumen in In Vitro .
반추위액 채취 및 완충액 제조Ruminant Sampling and Buffer Preparation
순천대학교 부설농장에서 사육중인 반추위 누관이 장착된 600 ± 47kg 홀스타인 젖소를 이용하여 in vitro 시험을 위한 위액을 채취하였다. 공시동물인 한우는 이탈리안 라이 그라스(rye grass)와 농후사료(중량 기준으로 옥수수 55%, 밀 15%, 탈지 미강 8%, 옥수수 글루텐 사료 5%, 대두박 10%, 당밀 0.2%, 탄산칼슘(limestone) 2.0%, 염 0.5%, 인산칼슘 1.3%, 비타민-미네랄 혼합물(비타민 A 3000 IU, 비타민 D 6000 IU, 비타민 E 30 IU, Cu 25 mg, Fe 150 mg, Zn 200 mg, Mn 100 mg, Co 0.5 mg, 및 I 1.5 mg) 1.0%)를 2:8의 비율로 체중의 2% 수준으로 1일 2회 분할 급여하였고 물은 자유 섭취하도록 하였다. 사료 급여 2시간 후 치즈 직포(cheese cloth)를 이용하여 위액을 채취하여 사용하였다. 완충액(Hino et al., 1992)은 K2HPO4 0.45 g/L, KH2PO4 0.45 g/L, (NH4)2SO4 0.9 g/L, CaCl2·2H2O 0.12 g/L, MgSO4·7H2O 0.19g/L, 트립티카제(Trypticase) 1.0 g/L, 효모 추출물 1.0 g/L, 시스테인·HCl 0.6 g/L (pH 6.9)을 포함하는 기본 배지(basal media)로 제조하였다. Gastric fluid was collected for in vitro testing using 600 ± 47 kg Holstein cows equipped with rumen fistulas from Sunchon National University farm. Hanwoo, a domestic animal, is Italian ly grass and rich feed (55% corn, 15% wheat, 8% skim rice, 5% corn gluten feed, 10% soybean meal, 0.2% molasses, calcium carbonate (limestone) ) 2.0%, salt 0.5%, calcium phosphate 1.3%, vitamin-mineral mixture (vitamin A 3000 IU, vitamin D 6000 IU, vitamin E 30 IU, Cu 25 mg, Fe 150 mg, Zn 200 mg, Mn 100 mg, Co 0.5 mg, and I 1.5 mg) 1.0%) was fed twice daily to 2% of body weight in a ratio of 2: 8 and free water was taken. After 2 hours of feeding, gastric juice was collected using a cheese cloth and used. Buffer (Hino et al., 1992) is K 2 HPO 4 0.45 g / L , KH 2 PO 4 0.45 g / L, (NH 4) 2 SO 4 0.9 g / L, CaCl 2 · 2H 2 O 0.12 g / L , Basal media comprising MgSO 4 7H 2 O 0.19 g / L, Trypticase 1.0 g / L, yeast extract 1.0 g / L, cysteine HCl 0.6 g / L (pH 6.9) Was prepared.
반추위액과 완충액을 1:3(반추위액: 완충액)의 비율로 혼합한 후 질소 가스(N2 gas)로 충진시켰다. 각각의 발효 사료를 건물 2%로 160 ml 배양 병(serum bottle)에 넣은 후 준비된 완충액 100 ml를 넣고 O2 free-N2로 혐기 상태를 유지하며 고무 마개와 알루미늄 캡으로 밀봉하고, 39℃의 혐기성 상태에서 100 rpm으로 수평이 되도록 섞어주면서 배양하였다(Hattori and Matsui, Anaerobe, Vol.14, pp.87-93, (2008)). 인 비트로 배양은 0, 12, 24, 및 48시간을 3개의 반복 실험으로 수행하고, 반추위 발효의 특성으로 pH, 총 가스발생량, 메탄, 암모니아, 및 VFA를 측정하였다. Rumen and buffer were mixed at a ratio of 1: 3 (rumen: buffer) and filled with nitrogen gas (N 2 gas). Each fermented feed was placed in a 160 ml serum bottle with 2% dry matter, 100 ml of the prepared buffer was kept anaerobic with O 2 free-N 2 , sealed with a rubber stopper and an aluminum cap, and The mixture was incubated at 100 rpm in an anaerobic state (Hattori and Matsui, Anaerobe, Vol. 14, pp. 87-93, (2008)). In vitro culture was performed in three replicate experiments for 0, 12, 24, and 48 hours, and pH, total gas evolution, methane, ammonia, and VFA were measured as characteristics of the rumen fermentation.
pH의 변화pH change
pH는 인큐베이터에서 바로 측정하지 않고 상온과 동일한 온도로 안정화시킨 후 M503P 미터(wrks, Medififield, MA, USA)를 이용하여 측정하였다.The pH was not measured directly in the incubator but stabilized at the same temperature as room temperature, and then measured using an M503P meter (wrks, Medififield, MA, USA).
총 가스발생량Total gas generation
총 가스발생량은 시료를 안정화시킨 후 EA-6(Inc, Sun Bee instrument) 압력센서 측정기를 사용하였다. 총 가스발생량 측정 후 메탄과 이산화탄소 발생량을 측정하기 위해 진공관을 이용하여 발생된 가스를 포집하였다. 각 배양시간대별로 얻어진 총 가스발생량을 기초로 하여 Qrskov와 McDonald(1979)의 공식에 의해 가스발생량을 추정하였다.The total gas generation amount was stabilized and used EA-6 (Inc, Sun Bee instrument) pressure sensor measuring instrument. After the total gas generation was measured, the generated gas was collected using a vacuum tube to measure the amount of methane and carbon dioxide generated. On the basis of the total gas generation amount obtained for each incubation time, the gas generation amount was estimated by the formula of Qrskov and McDonald (1979).
휘발성 지방산(volatile fatty acids) 함량Volatile fatty acids content
VFA 측정은 배양시간대별 배양물을 1000×g 4℃에서 10분간 원심분리 후 상등액을 채취하여 0.2㎛ 마이크로 필터로 정제한 후 분석하였다. METACARB87H(Varian, Germany) 컬럼을 장착한 HPLC(Agilent technolgies 1200 series)를 이용하여 35℃에서 분석했고, 검출기의 UV의 파장은 210nm와 220nm였다. 이동상 용매는 0.0085N H2SO4이고 유속은 0.6ml/min이었다. The VFA measurement was analyzed after centrifugation of the culture by time incubation at 1000 × g 4 ℃ for 10 minutes to extract the supernatant and purified with a 0.2 ㎛ micro filter. HPLC analysis was carried out 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 0.0085 NH 2 SO 4 and the flow rate was 0.6 ml / min.
암모니아태 질소(NH3-N) 농도Ammonia nitrogen (NH 3 -N) concentration
암모니아 농도측정은 채취한 시료를 13000rpm으로 원심분리시킨 후 Chany와 Marbach(Clinical Chemistry, Vol. 8, pp. 130-132, (1962))의 방법에 따라 페놀 용액으로 시료 중의 암모니아를 발색시킨 후 분광광도계(Spectronics 21D)를 이용하여 630nm에서 흡광도를 측정하여 계산하였다.Ammonia concentration was measured by centrifuging the sample at 13000rpm and spectroscopy was carried out by developing ammonia in the sample with phenol solution according to the method of Chany and Marbach (Clinical Chemistry, Vol. 8, pp. 130-132, (1962)). Absorbance was measured and measured at 630 nm using a photometer (Spectronics 21D).
3-1. 부산물의 발효3-1. Fermentation of By-Products
대부분의 부산물은 주요 생산물의 생산 후 일반적으로 폐기된다. 이러한 부산물 중 섬유소 성분이 여전히 높은 양조 부산물과 버섯 부산물(버섯 폐배지)을 수집하여 반추동물의 사료로 사용될 수 있는지 여부를 평가하였다. 즉, 이들을 발효 사료로 이용하여 전술된 바와 같이, 반추위액과 완충액의 혼합물 중 인 비트로 발효를 수행했다. Most by-products are usually discarded after the production of the main product. Among these by-products, fiber nutrients were still collected and the mushroom by-products (mushroom waste medium) were collected to evaluate whether they could be used as feed for ruminants. That is, using these as a fermentation feed, fermentation was performed in vitro in a mixture of rumen and buffer as described above.
도 2는 배양 시간에 따른 발효 사료의 건물 소실량을 보여준다. 밀기울과 양조 부산물 또는 버섯 부산물의 조합을 발효 사료로 이용했다. 밀기울의 경우 양조 부산물보다 버섯 부산물에서 소실량이 더 높았다. 결과적으로 버섯부산물은 양조 부산물보다 반추위 내에서 소화가 더 쉽다는 것을 나타낸다.Figure 2 shows the dry matter loss of the fermented feed according to the incubation time. A combination of bran and brew by-products or mushroom by-products was used as fermented feed. For bran, the amount of loss in mushroom by-products was higher than for brewing by-products. As a result, mushroom byproducts are easier to digest in the rumen than brewing byproducts.
3-2. 프로피온산 생산능을 갖는 미생물을 첨가한 사료의 발효3-2. Fermentation of Feed with Microorganisms Producing Propionic Acid
사일리지는 사료의 질과 최대의 건물을 제공한다. 또한 사료 중 미생물은 발효의 속도를 높이고 사일리지의 결과를 증진시킨다. 그러므로 프로피온산 생산능을 갖는 미생물을 첨가하여 인 비트로 발효 효과를 측정하였다. Silage provides for feed quality and maximum building. Microorganisms in the feed also speed up the fermentation and enhance the results of silage. Therefore, the fermentation effect was measured in vitro by adding microorganisms having propionic acid production capacity.
밀기울과 양조 부산물 또는 버섯 부산물의 조합에 락토바실러스 뮤코사에 KCCM11440P 또는 프로피오니박테리움 액시디오프로피오니시를 첨가한 후, 인 비트로 배양하면서 건물 소실량, 수분 함량, pH, 휘발성 지방산의 생산량, 총 가스발생량 및 암모니아 생산량을 측정하였다. After adding KCCM11440P or Propionibactium acidiotropioni to Lactobacillus mucosa to a combination of bran and brewing by-products or mushroom by-products, cultivated in vitro, dry matter loss, moisture content, pH, production of volatile fatty acids, total gas generation And ammonia production was measured.
밀기울과 양조 부산물 또는 버섯 부산물(폐배지)을 각각 1:1의 비율로 섞고 1%의 당밀을 첨가한 후 10%의 배양된 미생물, 락토바실러스 뮤코사에 또는 프로피오니박테리움 액시도프로피오니시(양성 대조군)를 접종하였다. 모든 시료는 진공포장한 후 37℃에서 72시간 배양하였다. Mix bran and brew by-products or mushroom by-products (waste medium) in a ratio of 1: 1 and 1% molasses, followed by 10% cultured microorganisms, Lactobacillus mucosae or propionibacterium apsidopropionis (Positive control) was inoculated. All samples were incubated for 72 hours at 37 ℃ after vacuum packaging.
사일리지 제조 후에 양조 부산물은 버섯 부산물보다 수분함량이 높았으나(P<0.05) pH 수치는 낮았다(P<0.05). 하기 표 3은 측정된 수분 함량 및 pH를 보여준다. After silage production, brewing by-products had higher moisture content than mushroom by-products (P <0.05) but lower pH values (P <0.05). Table 3 below shows the measured moisture content and pH.
표 3
Figure PCTKR2014007770-appb-T000003
 TABLE 3
Figure PCTKR2014007770-appb-T000003
하기 표 4는 발효 배양 시간에 따른 건조 물질(건물) 소실을 보여준다. 버섯 부산물의 건물의 소실은 양조 부산물보다 낮았다(P<0.05). 결과적으로 사일리지의 양조 부산물의 건물 소실의 증가는 락토바실러스 뮤코사에의 첨가에서 기인된다는 것을 나타낸다.Table 4 below shows the dry matter (buildings) loss with fermentation incubation time. The loss of building by mushroom by-products was lower than by brewing by-products (P <0.05). As a result, the increase in dry matter loss of brewing by-products of silage is indicated by the addition to Lactobacillus mucosa.
표 4
Figure PCTKR2014007770-appb-T000004
 Table 4
Figure PCTKR2014007770-appb-T000004
표 5는 발효에 의한 VFA 생산량을 보여준다. 건물 소실은 버섯 부산물에서 낮은 것으로 관찰되었으나, 총 VFA와 프로피온산은 양조 부산물에서 높았다. 또한, 처리 중에 부산물에 락토바실러스 뮤코사에를 첨가한 발효는 프로피온산 생산량과 총 휘발성 지방산(TVFA)이 높게 나타났다. 이는 영양학적 구성상 양조 부산물보다 버섯 부산물의 산의 변화가 더 높게 일어난다는 것을 보여준다. 사일리지가 제조될 동안 생산된 다량의 산은 사료 급여시 반추동물에게 더 높은 에너지원이 된다는 것을 의미한다.Table 5 shows the VFA production by fermentation. Dry matter loss was observed to be low in mushroom byproducts, while total VFA and propionic acid were high in brewing byproducts. In addition, fermentation by adding Lactobacillus mucosa to the by-product during the treatment showed high propionic acid production and total volatile fatty acids (TVFA). This shows that the acid change of mushroom by-products occurs higher than brewing by-products due to nutritional composition. The large amount of acid produced while silage is produced means that feed is a higher energy source for ruminants.
표 5
Figure PCTKR2014007770-appb-T000005
 Table 5
Figure PCTKR2014007770-appb-T000005
표 6은 양조 부산물과 버섯 부산물의 인 비트로 발효에 의한 총 가스생산량, pH, 및 암모니아 질소 생산량을 보여준다.Table 6 shows the total gas production, pH, and ammonia nitrogen production by in vitro fermentation of brewing by-products and mushroom by-products.
표 6
Figure PCTKR2014007770-appb-T000006
 Table 6
Figure PCTKR2014007770-appb-T000006
통계적 방법 Statistical method
본 연구에서 얻어진 모든 결과들의 무작위 디자인 일반적인 선형 모델(GLM)을 사용한 분산분석(ANOVA)에 의해 분석되었다. 모든 처리는 DMRT(Duncan's Multiple Range Test) 방법을 이용하여 처리구간의 특이성을 확인하였고, 3회 반복 실시하였다. 통계적 유의성은 P<0.05로 나타냈으며, 모든 분석은 SAS(Statistical Analysis Systems) 버전 9.1 (SAS, 2002)을 사용하여 수행했다.Random design of all results obtained in this study was analyzed by analysis of variance (ANOVA) using a general linear model (GLM). All treatments were confirmed for specificity of treatment using DMRT (Duncan's Multiple Range Test) method and repeated three times. Statistical significance was indicated as P <0.05, and all analyzes were performed using Statistical Analysis Systems (SAS) version 9.1 (SAS, 2002).
Figure PCTKR2014007770-appb-I000001
Figure PCTKR2014007770-appb-I000001

Claims (7)

  1. 프로피온산 생산능을 갖는 락토바실러스 뮤코사에(Lactobacillus mucosae) KCCM11440P. Lactobacillus mucosae KCCM11440P with propionic acid production capacity.
  2. 락토바실러스 뮤코사에 KCCM11440P를 포함하는, 조사료 조성물.  A forage composition comprising Lactobacillus mucosa KCCM11440P.
  3. 청구항 2에 있어서, 상기 조사료 조성물은 버섯 폐배지를 포함하는 것인 조사료 조성물.The forage composition of claim 2, wherein the forage composition comprises mushroom waste medium.
  4. 청구항 2에 있어서, 상기 조사료 조성물은 비타민 B12 및 젖산염 중 하나 이상을 포함하는 것인 조사료 조성물.The forage composition of claim 2, wherein the forage composition comprises at least one of vitamin B 12 and lactate.
  5. 청구항 2에 있어서, 상기 조사료 조성물은 반추동물의 비육촉진용인 것인 조사료 조성물.The forage composition of claim 2, wherein the forage composition is for promoting the ruminant's meat.
  6. 반추동물을 비육하는 방법으로서, 락토바실러스 뮤코사에 KCCM11440P를 포함하는 조사료를 급이하는 단계를 포함하는 것인 방법. A method of raising a ruminant, the method comprising feeding a forage comprising KCCM11440P to Lactobacillus mucosa.
  7. 청구항 6에 있어서, 상기 조사료는 버섯 폐배지를 포함하는 것인 방법.The method of claim 6, wherein the forage comprises mushroom waste media.
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