KR101641755B1 - Microorganism for ruminal methane mitigation and ruminal enhancing feed efficiency of ruminant - Google Patents

Abstract

The present invention relates to a microbial agent for inhibiting methane production and improving fermentation of a ruminant comprising a yeast and a reductive acetic acid-producing bacterium, a feed for livestock containing the microbial agent, and a method for feeding the microorganism preparation or feed to a ruminant, To inhibit methane production and to improve fermentation. According to the present invention, the methane production by the methane-producing bacteria in the rumen rumen is inhibited and the production of the volatile fatty acids and the like by the reducing acetic acid-producing bacteria is increased and the feed efficiency of the ruminant is increased.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a microorganism for ruminal methane reduction and fermentation,

The present invention relates to a microorganism for improving fermentation in a rumen of a ruminant while reducing the occurrence of methane in rumen, and more particularly to a microorganism preparation for improving digestion efficiency and improving methane production by improving fermentation in rumen, And a method for improving fermentation in rumen while reducing methane by using the microorganism preparation and / or an animal husbandry feed.

The major greenhouse gases that cause global warming are the six greenhouse gases designated by the United Nations Framework Convention on Climate Change (UNFCCC), carbon dioxide (CO ₂ ), methane gas (NH ₄ ), nitrous oxide (N ₂ O), perfluorocarbon (PFCS) Hydrogen fluoride carbon (HFCS), and sulfur fluoride (SF ₆ ). Of these, methane gas is the biggest contributor to global warming after carbon dioxide, with more than 500 million tons annually emitted to the atmosphere worldwide, contributing 15 to 17% to global warming. In addition, methane gas has 21 times higher ability to absorb infrared rays than carbon dioxide, so it has been reported that the effect on methane production of digestion by intestinal fermentation of ruminant is 15% (Crutzen, 1995). In the case of domestic animals, the ratio of the total methane emissions produced by animals is close to 75% of ruminant animals, and the number of livestock breeding is increasing every year according to the increase of meat consumption, The increase in the amount of gas generation is also inevitable (Korea Energy Economics Institute, 2007).

On the other hand, feeds consumed by ruminants are decomposed by microorganisms in the rumen, resulting in the production of carbon dioxide, ammonia, hydrogen, and volatile fatty acids as final degradation products. Among these fermentation products, volatile fatty acids and ammonia are used by ruminant livestock or ruminal microorganisms, which ultimately play an important role in the productivity of livestock. However, carbon dioxide and hydrogen are produced from methane by the methanogenic bacteria at the last stage of fermentation Acetic acid is produced competitively by reductive acetic acid-producing bacteria. In particular, when methane is produced, it is not absorbed in the body of ruminants and is released into the atmosphere in 15 to 20 times per hour.

In this way, when methane is produced in rumen rumen, various problems are being researched to suppress the production of methane due to environmental problems caused by global warming and decrease of feed efficiency (Ha et al., 2009 (Kim et al., 2011), and the results of this study are summarized as follows.

Methods for inhibiting methane production in rumen rumen can be roughly divided into two categories. The first method is to use a compound such as nitrate or nitrite, which can use hydrogen to control the amount of hydrogen that is a precursor of methane generation. In this case, there is a possibility that the supply of oxygen necessary for the cell may become insufficient. The second method uses antibiotics, which are compounds that give toxicity to methanogens. When administered over a long period of time, methane-producing bacteria are adapted and the methane production inhibiting effect is reduced. In addition, since the above methods use a compound, if such a substance is left in the livestock product, the safety may be a problem, and since the growth of useful microorganisms in the rumen is also lowered, the feed utilization efficiency is lowered and the productivity is lowered. Have been studied for the development of additives and feeds.

With regard to the method for inhibiting ruminal methane production in ruminants, Korean Patent Laid-Open Publication No. 2006-0019062 selectively reduces the protozoa in rumen rumen, including one or more of ginger, leek extract and complex linoleic acid Korean Patent Laid-Open Publication No. 2011-0119370 suppresses the production of greenhouse gases in ruminants, which includes 35 to 45% by weight of concentrated feed, 35 to 45% by weight of timothy and 10 to 30% by weight of rice straw. And a method of raising a ruminant. Korean Patent Laid-Open Publication No. 2011-0036470 discloses a feed for reducing methane gas production of a ruminant including garlic, and Korean Patent Publication No. 2011-0039540 discloses a method for eliminating all the fat originating from animal origin, A method for limiting the amount of methane produced by a useful ruminant using a method of limiting the extrinsic intake of the vegetable oil contained therein, and the like. Japanese Patent No. 5192108 discloses a composition for inhibiting methane production for a ruminant, which comprises at least one member selected from the group consisting of lactic acid bacteria, yeast and oligosaccharides. In Chinese Patent Publication No. 102106462, And a saccharide that secretes germs, and a composition for inhibiting methane production for a ruminant.

Accordingly, the present inventors have made various efforts to prevent global warming by inhibiting the generation of methane, a kind of greenhouse gas, in rumen of ruminants, and to increase the efficiency of feed use of ruminants, and as a result, the present invention has been completed .

It is an object of the present invention to provide a microbial agent for inhibiting methane production and improving fermentation in rumen comprising yeast bacteria and reductive acetic acid-producing bacteria.

Another object of the present invention is to provide an animal feed comprising the microorganism or microbial agent.

It is another object of the present invention to provide a method for improving rumen inhibition of methane production and fermentation of a ruminant by feeding yeast and reductive acetic acid-producing bacteria to an animal.

In one aspect, the present invention relates to a microbial agent for inhibiting methane production and improving fermentation of rumen in a rumen containing yeast and a reductive acetic acid-producing bacterium.

In the present invention, the ruminant animals belong to the mammalian vine, and they are also called as a rearing animal as an animal having a chewing characteristic by rewinding the food once swallowed by the digestive form. The ruminant is not limited to an animal having a rumen such as a camel, a deer, a deer, a giraffe, a cow, a goat, a bull, a buffalo, a buffalo, a deer, a camel, a sheep, .

In the present invention, the rumen is the first stomach of the ruminant. There are various microorganisms in the rumen. These microorganisms in the rumen produce carbon dioxide, ammonia, hydrogen, volatile fatty acids, and so on, which cause fiber digestion to digest many fibrous materials that are not suitable for non-ruminants. Double volatile fatty acids are absorbed in the intestines of ruminants and used as energy sources. However, methanogens such as Metanobrevibacter smithii produce methane, which is one of the major causes of greenhouse gases as a result of fermentation activity in rumen. Therefore, it is important to increase the energy absorption of ruminants by increasing the fermentation efficiency by the microorganisms in the rumen while reducing the generation of methane, a fermentation product by microorganisms in the rumen.

In the present invention, the yeast is a yeast belonging to the genus Saccharomyces such as Saccharomyces cerevisiae , Saccaromyces carlsbergensis , Saccharomyces cerevisiae , And Saccharomyces cerevisiae . The yeast does not grow in the anaerobic rumen but is present in a metabolic active state. As a result, the product produced by the yeast is actively used as a source of reducing acetic acid-producing bacteria that compete with the methane-producing bacteria for the substrate, resulting in suppression of methane production by the methane-producing bacteria in the rumen (Examples 2-4 Reference).

In the present invention, the reductive acetic acid-producing bacterium is a bacterium that produces acetic acid using hydrogen or carbon dioxide among final decomposition products after fermentation and decomposition by microorganisms in rumen when ruminant feeds a feed containing carbohydrate . The reducing acetic acid-producing bacteria effectively compete with the methane-producing bacteria to produce acetic acid. Since the growth rate is faster than that of methane-producing bacteria, the hydrogen provided by the yeast is used from the early stage of growth, do. As a result, it results in suppression of methane production in rumen rumen by methanogens. In addition, since the volatile fatty acid including acetic acid produced by the reducing acetic acid-producing bacteria is used as an energy source of ruminants, fermentation efficiency of rumen and efficiency of ruminant feed utilization are increased. Examples of the reductive acid producing bacteria according to the present invention, Proteinase am peel William acetoxy Tatiana to Ness (Proteiniphilium acetatigenes) etc., and preferably a proteinase am peel William acetoxy Tatiana to Ness (Proteiniphilium acetatigenes) found by the present inventors SROD1 (Accession No. KCCM11219P) can be used.

In one specific embodiment, when the yeast according to the present invention and the reducing acetic acid-producing bacteria were co-cultured with methanogenic bacteria, respectively, the amount of methane produced was lowest and the production of volatile fatty acids including acetic acid was relatively excellent (See Example 2). In addition, when the expression of the methanogenic gene was confirmed, the expression amount of the methanogenic gene was relatively low when co-cultured (see Example 2).

From these results, it is possible to reduce the amount of methane produced by ruminants and improve the fermentation efficiency in rumen when yeast and reducing acetic acid-producing bacteria are provided to ruminants together according to the present invention.

Herein, the amount of methane generated by the ruminant is reduced by at least 1%, preferably at least 3%, more preferably at least 5%, even more preferably at least 10% .

In addition, the improvement in the fermentation efficiency in the rumen has the same meaning as the improvement in the feed efficiency of the ruminant. The efficiency of fermentation by the microorganism in the rumen according to the present invention is 1% or more, preferably 3% More preferably 5% or more, and even more preferably 10% or more.

In the present invention, the microorganism preparation refers to a preparation comprising yeast and a reducing acetic acid-producing bacterium. Such a microorganism preparation may be prepared as a dry powder form by mixing the yeast with a reducing acetic acid-producing bacterium, culturing it or cultivating it in a liquid form, or lyophilizing, hot air drying or vacuum drying. have.

When the formulation of the microorganism preparation is in the form of a liquid, the final concentration of the yeast and the reducing acetic acid-producing bacteria is 5 to 20% by weight after mixing glucose or glycerin to stabilize the mixture or dilute and stabilize the culture of the yeast and the reducing acetic acid- By weight, preferably 10 to 15% by weight. When the concentration of the yeast and the reducing acetic acid-producing bacteria is less than 5% by weight, the methanogenesis-inhibiting effect and the fermentation efficiency in the rumen may be insignificant. When the content of yeast and reducing acetic acid-producing bacteria exceeds 20% The inhibitory effect on the methanogenesis and the increase in the fermentation efficiency in the rumen are insignificant compared with the microbial agents containing the lower contents. When a microbial agent is used in the form of a liquid, it is more active and easier to handle than a dry powder form.

When the microorganism is in the form of a dry powder, the yeast and a reducing acetic acid-producing bacterium are adhered to a carrier and dried and pulverized to a moisture content of 0.1 to 10% by weight. When the moisture content is more than 10% by weight, the reactivation efficiency of the yeast and the reducing acetic acid-producing microorganism is lowered or the efficiency of inhibiting methanogenesis is lower than that of the microbial agent having a water content of less than that. The use of a microbial formulation in the form of a dry powder is advantageous in that the manufacturing process is simple, the production cost is low, and the preservability is good.

The carrier may be a clay, an activated carbon, a coke, a volcanic ash and a soft material. The clay may be zeolite, vermiculite, diatomaceous earth, kaolin, onionite, feldspar, It is not.

In addition, microbial preparations in the form of a dry powder may further contain excipients. The excipient may be an amino acid, vitamin C, vitamin E, chitosan, glucose, and the like, but is not limited thereto.

In another aspect, the present invention relates to a feed for animal husbandry comprising a microbial agent for inhibiting methane production and improving fermentation of rumen in rumen comprising yeast and a reductive acetic acid-producing bacterium.

In the present invention, the feed is not necessarily limited to any one, but may be any one or a mixture of two or more selected from the group consisting of corn, wheat, wheat, soybean meal, soybean meal, oats, Processed by-products such as rice bran, wheat bran, and rice bran obtained in the process of processing water and these can be used.

The livestock feed according to the present invention is characterized in that the microbial preparation contains 0.1 to 0.5% by weight, preferably 0.3% by weight based on the total weight of the feed. When the amount of the microorganism preparation is less than 0.1% by weight, the effect of inhibiting methane production of the animal feed may be insufficient. When the amount of the microorganism preparation is more than 0.5% by weight, There is an uneconomical problem because the increase in fermentation efficiency in the rumen is insignificant.

In the present invention, the animal feed includes, but is not limited to, ruminants such as camels, deer, deer, giraffe, and cattle belonging to cattle, goats, ox, buffalo, bison, deer, To an animal, preferably to a cow.

The microorganisms in animal feed according to the present invention are non-toxic and have a strong tolerance to heat, bile, gastric juice, and antibiotics, and thus survive the intestines. In addition, it can produce physiologically active substances even in an anaerobic environment, and can be adsorbed on intestinal epithelial cells or mucous membranes.

In the present invention, when the animal feed is consumed, the effect of global warming can be prevented by suppressing methane production in the rumen of the ruminant, and the fermentation efficiency of the reduced acetic acid-producing bacteria in the rumen is increased, Can be increased.

In another aspect, the present invention relates to a method for inhibiting methane production in rumen and improving fermentation by feeding yeast and reductive acetic acid-producing bacteria to a ruminant.

In the present invention, the feeding refers to providing a ruminant animal feed containing a microorganism preparation for inhibiting methane production and fermentation of rumen in a ruminant containing yeast and reductive acetic acid-producing bacteria or the microorganism preparation, But are not limited to, methods for providing yeast and reducing acetic acid-producing bacteria to ruminants. For example, a microorganism preparation containing yeast and reductive acetic acid-producing bacteria may be liquidified with water or the like, and the liquid microorganism preparation may be administered into a rumen using a syringe or the like or a feed containing a microorganism preparation may be provided.

When the ruminant feed according to the present invention is fed to a ruminant animal, the ruminant can inhibit the formation of methane in the rumen of the ruminant animal, thereby preventing global warming and increasing the fermentation efficiency of the reductive acetic acid-producing bacteria in the rumen It is possible to increase the feed efficiency of ruminants.

The yeast and reductive acetic acid-producing bacteria of the present invention compete with methanogens in the rumen rumen to inhibit methane production and increase the production of volatile fatty acids, thereby reducing the amount of methane released from the ruminant animal and increasing the feed efficiency Effect.

Figure 1 is a graphical representation of the expression levels of Saccharomyces cerevisiae (SC), Proteiniphilum acetatigenes (G3), and Metanobrevibacter smithii (MS) And the amount of methane produced in the case of cultivation was compared and observed.
Fig. 2 is a graph showing the results of the measurement of the activity of Saccharomyces cerevisiae (SC), Proteiniphilum acetatigenes (G3), and Metanobrevibacter smithii (MS) And the amount of methanogenic gene expressed when cultured.

The present invention will be explained in more detail through the following examples. It is to be understood that these embodiments are provided by way of illustration only, and are not intended to limit the scope of the invention.

Example 1: Microbial culture

Proteiniphilum acetatigenes , a reductive acetic acid-producing strain , and Metanobrevibacter smithii , a methanogenic strain , were purchased from the Korean Resource Center for Biological Resources. Yeast strain Saccharomyces cerevisiae was isolated from malt-yeast extract agar medium.

More specifically, the Proteinase am pilrum acetoxy Tatiana jeneseu (Proteiniphilum acetatigenes, G3) is _{80% H 2/20% CO} 2 gas (1 atm), with a temperature of 121 ℃ sterilized for 15 min AC-B1 media (LeVen et al., 1998) and incubated at 100 rpm for 24 hours in an incubator maintained at a temperature of 39 ° C.

The Metanobrevibacter smithii MS was inoculated into Balch's methanogen medium (1 g / L yeast extract and 2 g / L trypcase) mixed with 80% H ₂ /20% CO ₂ gas at 200 kPa, And incubated for 24 hours at 100 rpm in an incubator maintained at a temperature.

Saccharomyces cerevisiae (SC) was isolated from yeast, cultured at a temperature of 30 ° C, and then cultured to a concentration of 1 × 10 ⁸ cfu / ml.

Example 2: in vitro  Perform experiments on fermentation systems

2-1. Strain preparation

(G3 + SC, G3 + MS, MS) mixed with two different bacteria among the above-mentioned fungi (G3 + SC, G3 + MS, 5% of G3 + MS + SC mixed with all three strains were inoculated into 10 ml of Balch's methanogen medium (1 g / L yeast extract and 2 g / L trypcase) and then inoculated with 0.1% glucose Lt; / RTI > The gas in the medium was then exchanged with hydrogen / carbon dioxide (80% / 20%, vol / vol) using a gas vacuum pump and an anaerobic gassing system. Followed by agitation at 0, 24 and 48 hours at a temperature of 39 DEG C at a rate of 100 rpm. As a control, Balch's methanogen medium not inoculated with acetic acid-producing bacteria (G3), methanogenic bacteria (MS) or yeast (SC) was used.

2-2. Measurement of microbial absorbance (OD value)

(G3 + MS, G3MS), methanogens (MS), yeast (SC), acetic acid-producing bacteria + yeast bacteria (G3 + SC, G3SC) (Biochrom Libra S22, Biochrom Ltd, Cambrodege CB4 0FJ England) was used as the culture medium for the production of methanogens and yeasts (MS + SC, MSSC) and acetic acid bacteria / methanogenic bacteria + yeast bacteria (G3 + SC + MS, G3SCMS) The OD value was measured according to the incubation time. The Duncan 's multiple range test (DMRT) was used for the post - test. The results are shown in Table 1.

Strain Culture time (hr) 0 24 48 hours Control group 0.013 ^d 0.017 ^e 0.018 ^g G3 0.032 ^cd 0.495 ^d 0.278 ^e SC 0.668 ^a 0.563 ^c 0.546 ^c MS 0.027 ^cd 0.039 ^a 0.067 ^f G3SC 0.669 ^a 0.914 ^a 0.728 ^a MSG3 0.047 ^c 0.551 ^c 0.399 ^d MSSC 0.645 ^b 0.586 ^c 0.598 ^b G3SCMS 0.666 ^ab 0.843 ^b 0.748 ^a SEM 0.005 0.011 0.011 SEM = standard deviation, ^{a, b, c, d, e, f, g The} values with different superscripts indicate that there is a significant difference at p <0.05 level using Duncan's multiple test.

As a result, the viable cell counts of MS and MSSC increased with increasing incubation time. In case of G3 or G3 mixed culture, the number of viable cells increased up to 24 hours but decreased at 48 hours.

2-3. Measurement of pH

(G3 + MS, G3MS), methanogens (MS), yeast (SC), acetic acid-producing bacteria + yeast bacteria (G3 + SC, G3SC) ), Methane-producing bacteria (MS + SC, MSSC) and acetic acid bacteria + methanogenic bacteria + yeast bacteria (G3 + SC + MS, G3SCMS) were placed on ice kept at a temperature of 4 ° C to stop the fermentation, Respectively. The Duncan 's multiple range test (DMRT) was used for the post - test. The results are shown in Table 2.

Strain Culture time (hr) 0 24 48 Control group 6.80 ^ab 6.66 ^a 6.59 ^c G3 6.76 ^bc 6.38 ^c 6.38 ^d SC 6.68 ^c 6.50 ^b 6.49 ^cd MS 6.81 ^ab 6.71 ^a 6.76 ^b G3SC 6.71 ^c 6.50 ^b 6.45 ^d MSG3 6.87 ^a 6.52 ^b 6.86 ^ab MSSC 6.83 ^ab 6.72 ^a 6.89 ^a G3SCMS 6.75 ^bc 6.63a 6.90 ^a SEM 0.020 0.024 0.028 SEM = standard deviation, ^{a, b, c, and d.} Values with different superscripts indicate a significant difference at p <0.05 using Duncan's multiple test.

As a result, pH value of G3, SC and G3SC decreased significantly with increasing incubation time, and MS or MS - mixed bacteria showed higher pH value than other microorganisms.

2-4. Measurement of methane, hydrogen and carbon dioxide emissions

(G3 + MS, G3MS), methanogens (MS), yeast (SC), acetic acid-producing bacteria + yeast bacteria (G3 + SC, G3SC) ), Methane, hydrogen and carbon dioxide emissions of methanogens + yeast (MS + SC, MSSC) and acetic acid bacteria + methanogen + yeast (G3 + SC + MS, G3SCMS) were measured by gas chromatography (Agilent Technologies HP 5890) Respectively. The detection conditions were oven temperature 35 ° C, injection temperature 200 ° C, partial detector temperature 200 ° C at the outlet, and 3 ml of mobile phase gas N ₂ (TCD, column Carboxen 1006PLOT capillary column 30m x 0.53mm / min, and the amount of the cells was determined after 24 hours of incubation. Based on the amount of methane, hydrogen and carbon dioxide generated in total gas production, the amount of methane, hydrogen and carbon dioxide production was estimated by the formula of Qrskov and McDonald (1979). The Duncan 's multiple range test (DMRT) was used for the post - test. The results are shown in FIG. 1 and Table 3.

Strain Culture time (hr) 0 24 48 Hydrogen (mM / ml) Control group 661.03 511.43 ^d 595.40 ^ab G3 642.05 519.16 ^dc 379.83 ^bcd SC 741.37 617.61 ^bcd 655.25 ^a MS 744.39 624.18 ^abc 335.38 ^cd G3SC 635.68 582.17 ^bcd 574.44 ^ab MSG3 790.50 689.76 ^ab 262.18 ^d MSSC 640.99 670.58 ^abc 491.54 ^ab G3SCMS 872.27a 769.68 ^a 205.63 ^d SEM 97.18 34.46 59.64 Carbon dioxide (mM / ml) Control group 5.09 3.55 ^c 4.68 ^ab G3 5.24 4.49 ^abc 3.76 ^ab SC 5.88 4.91 ^bc 5.80 ^a MS 5.51 4.09 ^bc 3.58 ^ab G3SC 4.58 5.28 ^abc 6.19 ^a MSG3 6.25 6.41 ^a 2.71 ^b MSSC 6.27 5.10 ^abc 5.47 ^ab G3SCMS 6.80a 6.01 ^ab 4.44 ^ab SEM 0.73 0.46 0.78 SEM = standard deviation, ^{a, b, c, and d.} Values with different superscripts indicate a significant difference at p <0.05 using Duncan's multiple test.

As a result, the amount of hydrogen production tended to decrease with increasing incubation time. In particular, hydrogen production of G3SCMS decreased to 205.63mM / ml after 48 hours of culture, followed by MSG3, MS and G3 It was confirmed that the amount of hydrogen produced was 262.18 mM / ml, 335.38 mM / ml and 379.83 mM / ml, respectively. On the other hand, the hydrogen consumption of MS was higher than that of SC, but there was no significant difference (see Table 3).

As a result of measuring the amount of carbon dioxide generated, it was confirmed that the amount of carbon dioxide generated in MSG3 after 48 hours of culture was 2.71mM / ml, which is the greatest decrease (see Table 3).

MS, MSG3, MSSC and G3SCMS showed no significant difference in the methane production of MS, MSSC and G3SCMS, but MSG3 was the highest methane production after 24 hours and 48 hours of culture. In particular, it was confirmed that the amount of methane generated by G3SCMS after 48 hours of culture was 3.06mM / ml, generating the smallest amount of methane (see FIG. 1).

2-5. Real-time quantitative PCR (qRT-PCR) for methanogenic gene expression confirmation

(MS), methionine-producing bacteria (G3 + MS, G3MS), methanogens + yeast (MS + SC, MSSC) and acetic acid bacteria + methanogenic bacteria + yeast bacteria G3 + SC + MS, G3SCMS). Then, the extracted RNA was subjected to a reverse transcription reaction with a template to synthesize cDNA. Then, the above cDNA was used as a template, Met630F (5'-GGA TTA GAT ACC CSG GTA GT-3 ') primer and Met803R (5'-GTT GAR TCC AAT TAA ACC GCA-3') primer, 2x fluorescent material Sensimix plus SYBR & Fluorescein, Quantance), denatured at 94 ° C for 15 minutes using a PCR premix, amplified for 30 seconds at a temperature of 94 ° C, for 30 seconds at a temperature of 60 ° C and for 1 minute at a temperature of 72 ° C, Quantitative PCR was performed. Methane-producing mRNA expression analysis was performed by measuring the intensity of fluorescence every time the PCR sample was raised by 0.5 ° C between 60 ° C and 95 ° C. The results are shown in Fig.

The number of methanogenic genes in MS, MSSC, and G3SCMS was not significantly different, but MSG3 showed the highest number of methanogenic genes at 24 hours and 48 hours after culture.

2-6. Measurement of volatile fatty acid (VFA) content

(G3 + MS, G3MS), methanogens (MS), yeast (SC), acetic acid-producing bacteria + yeast bacteria (G3 + SC, G3SC) , Lactate, formate and acetate by methanogens + yeast (MS + SC, MSSC) and peroxidic acid + methanogen + yeast (G3 + SC + MS, G3SCMS) Fatty acid yields were determined using high performance liquid chromatography (HPLC, Agilent Technologies 1200 series). As the solvent, 0.0085 normal sulfuric acid was flowed at a rate of 0.6 l / min, and the temperature of the column (METACARB87H, Varian, Germany) was maintained at 35 ° C. The samples were centrifuged for 10 minutes at a temperature of 4 ° C at a rate of 13,000 rpm before use. The supernatant was filtered with a 0.2 μm filter (Milipore), and then 20 μl each was injected. The Duncan 's multiple range test (DMRT) was used for the post - test. The results are shown in Table 4.

Strain Culture time (hr) 0 24 48 Lactate Control group ND ND ND G3 ND 10.61 ^b 10.09 ^a SC ND ND ND MS ND ND 2.65 ^c G3SC ND 3.39 ^c 3.36 ^bc MSG3 ND 37.70 ^a 11.06 ^a MSSC ND ND 3.03 ^bc G3SCMS ND 3.94 ^c 4.02 ^b SEM - 1.23 0.30 Formate Control group 35.57 ^a 35.79 ^a 37.43 ^a G3 35.28 ^a 34.61 ^a 34.41 ^ab SC 33.26 ^b 35.34 ^a 34.23 ^ab MS 31.26 ^c 29.30 ^c 28.25 ^c G3SC 33.15 ^b 32.50 ^b 31.14 ^bc MSG3 31.45 ^c 30.41 ^c ND MSSC 29.37 ^d 27.30 ^d 21.81 ^d G3SCMS 29.33 ^d 25.43 ^e ND SEM 0.10 0.40. 0.94 Acetate (acetate) Control group 23.55 ^ab 22.95 ^d 24.03 ^b G3 23.78 ^a 32.69 ^b 35.65 ^a SC 21.99 ^d 22.90 ^d 22.22 ^bc MS 23.02 ^bc 22.66 ^d 22.36 ^bc G3SC 22.51 ^dc 31.53 ^e 34.80 ^a MSG3 23.42 ^ab 33.96 ^a 35.41 ^a MSSC 22.04 ^d 21.41 ^e 20.82 ^c G3SCMS 22.38 ^dc 31.15 ^c 34.16 ^a SEM 0.14 0.32 0.51 SEM = standard deviation, ^{a, b, c, d, and e The} values with different superscripts indicate that there is a significant difference at p <0.05 using Duncan's multiple test.

The amount of acetic acid produced was found to be acetic acid - producing G3, and the amount of acetic acid produced by mixing G3 alone or G3 was increased with increasing incubation time. MS and SC did not or did not produce acetic acid. However, G3SCMS was found to produce a large amount of acetic acid with increasing incubation time.

It was confirmed that lactic acid was produced only by G3 and MS alone or by culturing them with a mixture of these microorganisms.

Formic acid was found to decrease formate formation in microorganisms cultured by MS alone or MS.

Claims (7)

A microorganism preparation comprising Saccharomyces cerevisiae and Proteiniphilium acetatigenes , which inhibits methane production by methane-producing bacteria in the rumen rumen and improves fermentation A microbial agent.
delete delete A feed for animal husbandry comprising the microbial agent of claim 1.
The method of claim 4, wherein the animal feed is fed to a ruminant to inhibit methane production by the methane-producing bacteria in the rumen and improve the fermentation.
delete delete

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