JP2016533755A5 - - Google Patents

Description

Microorganism capable of producing propionic acid and roughage composition containing the same

  The present invention relates to a microorganism capable of producing propionic acid and a roughage composition containing the microorganism.

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

  Patent Document 1 relates to a method for producing a fermented feed for livestock using a mushroom by-product as a main raw material. Fermentation is performed by adding lactic acid bacteria, Bacillus subtilis and yeast used as probiotics to a mushroom waste medium. It is a feature.

  Patent Document 2 relates to a method for producing pork feed using a mushroom waste medium, and is characterized by fermenting using a complex microorganism fermenter that does not contain microorganisms capable of producing propionic acid. To do.

  However, there is a need for an effective feed that can promote fattening without using side effects of antibiotics and hormones while utilizing abundant fiber.

Korean Patent No. 1144473 Korean Patent No. 1138934

  In addition, the present inventors conducted research on feed for promoting fattening of ruminants and fermented roughage in the rumen to increase the production of propionic acid, thereby increasing the ability to produce propionic acid that can promote fattening. The present invention was completed based on the microorganisms possessed.

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

  Another object of the present invention is to provide a roughage composition having an excellent fattening promotion effect.

  Another object of the present invention is to provide a method for fattening ruminants using roughage containing ruminant microorganisms.

  As an example of the present invention, a microorganism capable of producing propionic acid isolated from rumen is provided.

  As an example of the present invention, the microorganism is Lactobacillus mucosae KCCM11440P.

  Lactobacillus mucosae KCCM11440P has the base sequence of 16S rRNA of SEQ ID NO: 1, and was identified based on this sequence. FIG. 1 shows a system diagram prepared based on the base sequence of 16S rRNA.

  Propionic acid is the most important product of carbohydrate degradation in the ruminant and is converted to glucose in the ruminant liver, promoting ruminant fattening and contributing to improving meat quality.

  Lactobacillus mucosa KCCM11440P isolated based on the ability to produce propionic acid in the rumen grows in MRS medium. In particular, it was confirmed that the medium containing vitamin B12 and sodium lactate has high propion production ability.

  As another example of the present invention, a roughage composition comprising Lactobacillus mucosae KCCM11440P is provided.

  The term “rough feed composition” as used herein means a feed composition having a high fiber content and a low content of fat, protein, starch and the like, as in pasture, hay, silage and the like.

  The roughage composition according to an example of the present invention increases the production of propionic acid during fermentation of roughage in the rumen, including Lactobacillus mucosae KCCM11440P, which has the ability to produce propionic acid. This promotes fattening of ruminants and improves meat quality. Propionic acid is the only volatile fatty acid produced in the rumen, which is converted solely into glucose and involved in energy metabolism, etc., and the promotion of fattening of ruminants including cattle depends on the propionic acid produced in the rumen.

  As an example of the present invention, the forage composition includes a mushroom waste medium.

  The mushroom waste medium means a material obtained after culturing mushrooms, in which the main raw material is sawdust and the auxiliary raw material contains agricultural by-products. Mushroom waste medium sawdust is used as a carbon source for cellulolytic microorganisms in the rumen. Lactobacillus mucosae KCCM11440P is cultured in mushroom waste medium to increase the production of propionic acid. This can promote fattening of ruminants. In particular, the mushroom waste medium can be used as an excellent digestible roughage because it uses as a raw material the waste medium obtained by decomposition of mushrooms with cellulose-degrading bacteria.

  As an example of the present invention, the roughage composition includes at least one of vitamin B12 and lactate.

  When Lactobacillus mucosa KCCM11440P is supplemented with vitamin B12 or lactate such as sodium lactate in the medium, the amount of propionic acid produced is greatly increased. Therefore, when either the vitamin B12 or lactate is included in the roughage composition, the production of propioic acid is increased, thereby promoting fattening.

  As an example of the present invention, the roughage composition promotes fattening of ruminants. When Lactobacillus mucosae KCCM11440P, which has the ability to produce propionic acid, is included in the ruminant stomach, the production of propionic acid is increased in the rumen, thereby promoting the fattening of ruminants and improving the meat quality. Improve.

  As an example of the present invention, the roughage composition may further include probiotics.

  Antibiotics are unavoidable when raising livestock, but probiotics have been widely used since the problems caused by misuse have become serious. Probiotics feed beneficial microorganisms into the intestines of livestock, suppress the growth of pathogenic microorganisms, prevent disease outbreaks, and increase livestock productivity. Currently, microorganisms widely used are lactic acid bacteria, Bacillus subtilis, yeasts, and the like. Lactic acid bacteria produce organic acids to lower the pH, thereby suppressing harmful bacteria that are weak against acidity and promoting the activity of digestive enzymes. In particular, reduce the incidence of diarrhea. Bacillus subtilis produces enzymes that break down highly active carbohydrates, proteins, lipids, etc., thus promoting digestion and absorption of feed in the intestine and improving feed efficiency. It also reduces the stress associated with digestion and promotes livestock growth. It also has the effect of regulating the intestines and has a role to prevent diseases by energizing the intestines of livestock. Moreover, yeast exists in the form which can be easily digested in the digestive tract of livestock, produces natural flavor components, such as alcohol and glutamic acid, and increases the palatability of livestock feed.

  As an example of the present invention, the rough feed composition can be provided by mixing with general feed. For example, the roughage composition can be mixed with a commercial formula feed in a 1: 1 ratio and fed to ruminants twice a day. Appropriate mixing ratios, feeding amounts, and feeding frequencies can be easily determined by those skilled in the art in consideration of the age, weight, health status, etc. of the ruminant.

  As another example of the present invention, as a method for fattening ruminants, a method including a roughage feeding stage containing Lactobacillus mucosae KCCM11440P is provided.

  In one example of the present invention, the roughage may include a mushroom waste medium as a main component.

  As an example of the present invention, the roughage may further contain probiotics.

  In one example of the present invention, the roughage may include one or more of vitamin B12 and sodium lactate.

  In one example of the present invention, the roughage can be fed with a normal blended feed.

  In one example of the present invention, ruminants include but are not limited to cows, sheep, goats and deer.

  Hereinafter, the present invention will be described in more detail through examples. However, the following examples are illustrative of the present invention and should not be construed as limiting the present invention.

  Lactobacillus mucosae KCCM11440P according to a detailed example of the present invention and a roughage composition containing the same increase the production of propionic acid in the ruminant ruminant, thereby providing excellent fattening promotion and improving meat quality .

It is a systematic diagram created based on the 16S rRNA gene of Lactobacillus mucosae KCCM11440P of the present invention. It shows the amount of dry burnout of fermented feed, wheat waste, mushroom by-products and brewing by-products depending on the culture time.

1. Isolation and identification of propionic acid-producing bacteria 1-1. Isolation of strains To isolate a strain having the ability to produce propionic acid, rumen fluid was collected from three conventional black goats weighing 40 ± 5 kg. In order to maintain an anaerobic state, it performed in the anaerobic chamber and followed the method of Hattori and Matsui (Anarobe, Vol.14, pp.87-93, (2008)).

  Bacteria cultured from rumen gastric juice were diluted from 10-3 to 10-9 per ml of medium, then inoculated into Hungate Roll A tubes and cultured for 24 to 48 hours. A single colony was isolated from the culture, inoculated into liquid medium (MRS), and cultured at 120 rpm for 24 hours. All cultures were performed at 37 ° C. under anaerobic conditions (Lee, et al., Applied Microbiology and Biotechnology, Vol. 58, pp. 663-668, 2002), medium and buffer (Bryant and Burkey, Journal of Science, Journal of Science). Vol. 82, pp. 780-787, 1953) were used after sterilization at 121 ° C. for 15 minutes.

  A total of 51 strains were isolated, and the amount of volatile fatty acids produced was measured while culturing them in MRS medium. Among them, the strain showing the highest production amount of propionic acid was selected.

1-2. Identification of strains The strains isolated in 1-1 were cultured in MRS medium, and then chromosomal DNA was extracted using Wizard Genomic DNA Purification Kits (Promega, USA). In order to amplify a gene encoded by 16S rRNA using this as a template, PCR was performed using primers 27F (AGAGTTTGATCMTGGCTCAG) and 1492r (GGTTACCTTGTTACGACTT). The conditions for PCR were initial denaturation at 94 ° C for 5 minutes, denaturation at 94 ° C for 45 seconds, annealing at 65 ° C for 45 seconds, 32 cycles of 1 minute extension at 72 ° C and 10 minutes at 72 ° C. Is an extension of

  The amplified ribosomal DNA was analyzed for similarity by the method of ARDRA (Amplified Ribosomal DNA Restriction Analysis). The PCR product was treated with restriction enzymes (Takara, Japan) HaeIII and HhaI at 37 ° C. for 5 hours. DNA samples treated with restriction enzymes were separated by electrophoresis at 170 V for 80 minutes by MetaPhor (registered trademark) agarose (Takara, Japan). The DNA separated by electrophoresis was confirmed by Kodak Gel Logic 200 imaging system (Eastman Kodak Company, Rochester, NY, USA). Bands obtained from ARDRA were purified using QIA quick PCR Purification Kit.

  The purified 16S rDNA was confirmed to be SEQ ID NO: 1 by analysis of the base sequence of the PCR product (Macrogen, Korea). The analyzed base sequence information was combined using SeqMan program (DNA Star, Lasergene software, Madison, WI, USA), and the base sequence was NCBI (http://www.ncbi.nlm.nih.goV/BLAST ) And EzTaxon (http://147.47.212.35:8080/index.jsp) using the GeneBank BLAST program. The approximate phylogenetic classification was determined by comparing the closest species to the base sequence using CLUSTRAL W version 1.6. A system diagram was prepared according to the method of Kimura (1980) using the method of NJ (neighbor-joining) having the function of double-standard gap removal (pair-wise gap removal). Finally, in order to evaluate the stability of the system diagram using the two-parameter NJ method in the PHYLIP package, 1000 times of data was collected and the bootstrap analysis method was used. It was then chosen to exhibit a bootstrap value of 50% or higher. FIG. 1 shows the created system diagram.

  The isolated strain showed 96% similarity to Lactobacillus mucosae S32 (T). Based on this, the isolated strain was named Lactobacillus mucosae BR-PP, which was deposited on July 26, 2013 at the Korean Microbiology Conservation Center and received the deposit number KCCM11440P.

1-3. Selection of control strain
Selenomonas bobby known to have the ability to produce propionic acid in order to select a control group for comparing the ability to produce propionic acid of Lactobacillus mucosae KCCM11440P separated and identified in 1-1 and 1-2 Selenomonas bovis 15154 (SB), Veillonella parvula 5019 (VP), Propionibacterium acidipropionici 5020 (PAci) (PAcn) was used separately from KCTC and KACC, respectively.

  Each strain was measured for OD (Optical Density) value and pH while culturing at 37 ° C. for 48, 72, 96, 120 and 144 hours. Each experiment was performed in triplicate. In addition, a sample collected from the culture medium every hour was obtained by using a MetaCarb 87H (Varian, Germany) column with a high performance liquid chromatography (HPLC, Agilent Technologies 1200 series) with a UV detector, and a flow rate of 0.6 ml / min. And eluted with 0.0085 N · H 2 SO 4 buffer. Samples were detected at 210 nm and 220 nm, and the production of volatile fatty acids (VFA) was measured (Tabaru et al., Japan Journal of Veterinary Science Vol. 50, pp. 1124-1126 (1988) and Han et al. Process Biochemistry, Vol. 40, pp. 2897-2905 (2005)).

  Propionibacterium esidi propionis showed high production of propionic acid after 144 hours of culture. In addition, Propionibacterium esidi propionis showed facultative anaerobicity, unlike other propionic acid producing strains that were obligately anaerobic. A high production amount of propionic acid and facultative anaerobic propionibacterium ecidi propionis were used as a standard microorganism for propionic acid production and used as a control group.

Propionic acid production ability of Lactobacillus mucosae KCCM1140P The production ability of Lactobacillus mucosae propionate isolated and identified in Example 1 was evaluated. The cells were cultured for 48 hours in anaerobic condition with agarose containing MRS medium. The medium was placed in a Hungate tube, filled with pH 6.5, O2-free, 20% CO2, 80% N2 gas, and then autoclaved at 121 ° C for 15 minutes. In order to confirm the influence on the ability to produce propionic acid, biotin (0.5 mg / L), lactic acid (2%), vitamin B12 (50 μg / L) or glycerol (2%) were added to agarose containing MRS medium, respectively. Added. The control group for evaluating the ability to produce propionic acid was Propionibacterium ecidi propionis selected in Example 1.

  Samples cultured for 24 hours, 48 hours and 72 hours were taken to measure the production of volatile fatty acids including OD, pH and propionic acid.

  The pH was not measured in an incubator, but was measured with an M503P meter (wrks, Medifield, MA, USA) after bringing the medium to room temperature.

The amount of volatile fatty acid produced was analyzed by centrifuging the culture at 1000 × g and 4 ° C. for 10 minutes at each incubation time, collecting the supernatant, and purifying it with a 0.2 μm microfilter. Analysis was performed at 35 ° C. using a HPLC (Agilent technologies 1200 series) with a METACARB 87H (Varian, Germany) column. The UV wavelengths of the detector 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.
Tables 1 and 2 below show the results.

  As a result, it was confirmed that Lactobacillus mucosae KCCM11440P produces the largest amount of propionic acid when cultured in MRS medium containing vitamin B12 or sodium lactate.

Production of roughage Using ruminant fluid and buffer solution, the fermentation effect was evaluated in rumen in vitro.

Ruminal fluid collection and buffer production
In order to conduct an in vitro test, gastric juice was collected using a tube from a Holstein cow weighing about 600 ± 47 kg being bred on a farm attached to Suncheon University. Korean beef, the official test animal, is Italian ryegrass and concentrated feed (55% corn, 15% wheat, 8% defatted rice bran, 5% corn gluten feed, 10% soybean meal, 0.2% molasses on a weight basis) %, Calcium carbonate 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%} were mixed at a ratio of 2: 8, and about 2% of the body weight was fed twice a day. Then I was allowed to take water freely. Two hours after feeding the feed, gastric juice was collected and used using a cheese cloth. Buffers (Hino et al., 1992) were 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. 19 g / L, Tripticase 1.0 g / L, yeast extract 1.0 g / L, cysteine / HCl 0.6 g / L (pH 6.9) -containing basic medium (basal media).

  Ruminal fluid and buffer were mixed at a ratio of 1: 3 (ruminal fluid: buffer) and filled with nitrogen gas (N2 gas). Each 2% dry matter fermented feed was placed in a 160 ml serum bottle and 100 ml of buffer was added. While maintaining an anaerobic state with O 2 free-N 2, it was sealed with a rubber cap and an aluminum cap. The culture was carried out at 39 ° C. in an anaerobic state at 100 rpm while holding the horizontal (Hattori and Matsui, Anaerobe, Vol. 14, pp. 87-93, (2008)). In vitro cultures were tested in triplicate at 0, 12, 24 and 48 hours. For the characteristics of rumen fermentation, pH, total gas generation amount, methane, ammonia and VFA were measured.

Change in pH The pH was measured using an M503P meter (wrks, Medifield, MA, USA) after stabilization at room temperature, without measuring directly from the incubator.

Total gas generation amount The total gas generation amount was measured using an EA-6 (Inc, Sun Bee instrument) pressure sensor measuring instrument after the sample was stabilized. After measuring the generation amount of the total gas, in order to measure the generation amount of methane and carbon dioxide, the generated gas was collected using a vacuum tube. Based on the total gas generation amount obtained for each culture time, the gas generation amount was estimated by the formula of Qrskov and McDonald (1979).

Volatile fatty acid content The VFA measurement was carried out by centrifuging the culture at 1000 × g and 4 ° C. for 10 minutes at each incubation time, collecting the upper solution, and using a 0.2 μm microfilter. Purified and analyzed. Analysis was performed at 35 ° C. using HPLC (Agilent technologies 1200 series) with a METACARB 87H (Varian, Germany) column. The UV wavelengths of the detector were 210 nm and 220 nm. The mobile phase solvent was 0.0085N H2SO4 and the flow rate was 0.6 ml / min.

Ammonia Nitrogen (NH3-N) Concentration Ammonia concentration was measured by centrifuging the collected sample at 13000 rpm and then using the method of Chany and Marbach (Clinical Chemistry, Vol. 8, pp. 130-132, (1962)). It was. Ammonia in the sample was colored with a phenol solution, and the absorbance (Spectronics 21D) was measured at 630 nm.

3-1. By-product fermentation Most by-products are generally discarded after the main product is produced. Among these by-products, brewing by-products with high fiber content and mushroom by-products (mushroom waste medium) were collected and evaluated for use as ruminant feed. That is, using these as fermented feeds, as described above, fermentation was performed in vitro in a mixture of rumen fluid and buffer solution.

  FIG. 2 shows the amount of dry matter disappeared from the fermented feed according to the culture time. A combination of bran and brewing by-products or mushroom by-products, used as fermented feed. In the case of bran, the amount of disappearance was higher in the mushroom by-product than in the brewing by-product. Consequently, mushroom by-products are more digestible in the rumen than brewed by-products.

3-2. Fermentation of feed supplemented with microorganisms capable of producing propionic acid.
Silage provides feed quality and maximum dry matter. Also, the microorganisms in the feed increase the rate of fermentation and enhance the silage results. Therefore, microorganisms capable of producing propionic acid were added and the effect of fermentation was measured in vitro.

  After combining Lactobacillus mucosae KCCM11440P or Propionbacterium akland propion onishi in combination with bran and brewing by-products or mushroom by-products, dry matter disappearance, water content, pH, volatile fatty acid production The total gas generation amount and ammonia production amount were measured.

  Bran and brewing by-products or mushroom by-products (waste medium) are mixed at a ratio of 1: 1, 1% molasses is added, and then Lactobacillus mucosae or Propionibacterium ecidipropionis (positive control group) is cultured. 10% of the liquid was inoculated. All samples were incubated at 37 ° C. for 72 hours after vacuum packaging.

  After silage production, the brewing by-product had a higher moisture content than the mushroom by-product (P <0.05), but the pH value was lower (P <0.05). Table 3 shows the measured water content and pH.

  Table 4 below represents the disappearance of dry matter (dry matter) by fermentation time. The disappearance of mushroom by-product dry matter was lower than the brew by-product (P <0.05). As a result, it is shown that the increase in the disappearance of the dry matter of the silage brewing by-product is due to the addition of Lactobacillus mucosae.

  Table 5 represents the VFA production by fermentation. The disappearance of dry matter appeared to be lower for the mushroom by-products, but total VFA and propionic acid were higher for the brew by-products. In addition, the fermentation with Lactobacillus mucosae added to the by-product during the treatment showed that the production amount of propionic acid and the total volatile fatty acid (TVFA) were high. This indicates that the mushroom by-product acid change was higher nutritionally than the brew by-product. The large amount of acid that is produced when silage is made means a higher energy source for feed fed ruminants.

  Table 6 shows the total gas production, pH and ammoniacal nitrogen content from in vitro fermentation of brewing and mushroom by-products.

Statistical Methods All results obtained in this study were analyzed by analysis of variance (ANOVA) using a randomly designed general linear model (GLM). In all the treatments, the specificity of the treatment interval was confirmed by DMRT (Duncan's Multiple Range Test) method in 3 iterations. Statistical significance was expressed as P <0.05, and all analyzes were performed with SAS (Statistical Analysis Systems) version 9.1 (SAS, 2002).