KR20180044051A - A feed composition of ruminant animals for reducing methane produced in rumen comprising lysozyme and a method for reducing methane produced in rumen by using the same - Google Patents

Abstract

The present invention relates to a ruminant feed composition for reducing methane gas production in the rumen of ruminant animals, and to a method for suppressing methane production in the rumen by using the same. The ruminant feed composition for reducing methane gas production in the rumen of ruminant animals, comprising lysozyme of the present invention as an effective component reduces methane gas production in the rumen; has few side effects on a human body and animals, and thus is safe; and fundamentally reduces the causes of methane formation in the rumen, thereby suppressing methane production. Accordingly, the feed composition comprising lysozyme according to the present invention, suppresses methane production in the rumen of the ruminant animals when fed to ruminant animals; and increases feed efficiency, and thus can create additional monetary profits.

Description

TECHNICAL FIELD The present invention relates to a ruminant feed composition having methane gas reducing ability in rumen containing lysozyme as an active ingredient and a method for reducing methane gas in rumen using the same. for reducing methane produced in rumen by using the same}

The present invention relates to a ruminant feed composition having methane gas-reducing ability in a rumen, and a method for inhibiting methane production in a rumen using the composition. More particularly, the present invention relates to a feed composition comprising lysozyme as an active ingredient, The present invention relates to a ruminant feed composition capable of improving rumen fermentation of an animal and lowering the production of methane gas, and a method of using the same to inhibit methane production in rumen.

Global interest in global warming caused by greenhouse gases has been increasing worldwide in recent years. In 2010, the Korean government enacted the Basic Law of Low Carbon Green Growth to promote various policies such as systematic adaptation of climate change prediction and response capacity and foster environment friendly industry as a new growth engine in order to reduce greenhouse gas emissions.

Causing global warming, a major greenhouse gas, six greenhouse gases carbon dioxide (CO _2), methane (NH _4), nitrous oxide (N ₂ O) is specified in the United Nations Framework Convention on Climate Change (UNFCC), perfluorocarbons (PFCS), Hydrogen fluoride (HFCS) and sulfur hexafluoride (SF ₆ ). As these greenhouse gases increase, the average global temperature and sea level rise, and desertification, drought, flood, and typhoon are changing. 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 rumen microbes 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 and ruminant microorganisms, which are important for livestock productivity. However, carbon dioxide and hydrogen are produced by methane-producing bacteria at the last stage of fermentation The production of succinic acid by fumaric acid-reducing microorganisms competitively occurs, and in particular, when methane is produced, it is not absorbed in the body of ruminants and is released into the atmosphere at about 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 ionophores or antibiotics, which are compounds that give toxicity to methanogenic microorganisms. When administered over a long period of time, methane-producing microorganisms adapt to decrease the effect of inhibiting methanogenesis. 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. feed additives and research and development are being made, but a majority of laboratory methods by in vitro, methane abatement methods actually looks a lasting effect on livestock to benefit from has not been developed.

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.

On the other hand, lysozyme is a common enzyme found in living organisms with various roles ranging from digestion to immune response. Lysozyme (for example, 1,4- [beta] -N-acetyl muramidase) has both enzymatic and bactericidal activity. They cleave glycosidic bonds in the peptidoglycan component of the Gram-positive bacterial cell wall, which ultimately leads to apoptosis (Ellison and Giehl, 1991). Interestingly, besides inducing autolysis of microorganisms, lysozyme is also known to degrade microbial cells from the outside (Ibrahim et al., 2001). Gram-positive bacteria are sensitive to the action of lysozyme, but most of the gram-negative bacteria are not due to their thick outer membrane on the peptidoglycan layer. In the field of animal production, antimicrobial agents such as antibiotics are used at low doses as growth promoters for their germicidal effect. However, the side effects of indiscriminate use of antibiotics in animals and environments (eg, antibiotic resistance) make the animal industry vulnerable. Therefore, livestock and animal science workers are researching and developing alternatives to antibiotics for sustainable livestock systems. The use of lysozyme as an antimicrobial agent is recommended to improve the growth ability of lysozyme due to its enzymatic and bacterial degradation properties (Nyachoti et al., 2012). It has been shown that lysozyme tends to reduce diarrhea and increase average daily gain (ADG) during calves' weaning period (Goncu et al., 72 2012). They were also found to have ADG-digestibility in pigs (May 73 et al., 2012).

However, there is no information available on the effects of lysozyme on rumen fermentation in vitro and the effects of methane release in ruminants.

Therefore, the present inventors have been studying to develop a method for inhibiting the production of methane, a kind of greenhouse gas, in the rumen rumen, while investigating the lysozyme which reduces the methane production in the in vitro fermentation parameters The present invention has been completed.

SUMMARY OF THE INVENTION Accordingly, it is an object of the present invention to provide a feed composition for ruminants that can improve rumen fermentation and reduce methane gas.

Another object of the present invention is to provide a method for inhibiting methane production in the rumen of a ruminant using the feed composition.

In order to solve the above technical problems, the present invention provides a ruminant feed composition for reducing methane gas in rumen of a ruminant containing lysozyme as an active ingredient.

According to another aspect of the present invention, there is provided a method for inhibiting methane production in a rumen of a ruminant by feeding the feed composition for a ruminant to a ruminant.

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 methane is the most important factor in global warming after carbon dioxide. When the ruminant feeds the carbohydrate-containing feed, it is produced in the rumen by the methanogenic microorganism, , It is important to inhibit methane production in rumen rumen.

Lysozyme, an active ingredient of the ruminant feed composition of the present invention, is an antimicrobial enzyme capable of decomposing bacterial cell walls by decomposing polysaccharide components (Salton, 1957).

In accordance with one specific embodiment of the present invention, in vitro experiments with ruminal components isolated from cows showed that the pH of the group treated with lysozyme was increased after 24 hours of incubation, (Owens et al., 1998), which may be helpful in the growth and proliferation of rumen animals.

According to one specific embodiment of the present invention, in vitro experiments with ruminal components isolated from cows resulted in significantly more acetic acid, propionic acid and total volatile fatty acids (VFA) after incubation in the lysozyme treated group for 24 hours And is supported by Giraldo et al. (2007) which observed increased total VFA using fiber-degrading enzymes (cellulase and xylanase) additions in these test tubes. This increased VFA may be due to the degradation of the feed material through hydrolysis and the resolution of these feed components is an important factor for methane production (Kim et al., 2013).

According to one specific embodiment of the invention, in which process the lysozyme in vitro experiments with the rumen component separated from the group of cows after incubation for 12 and 24 hours it was found a significant decrease in the CH ₄ concentration.

In accordance with one specific embodiment of the present invention, in vitro experiments with ruminal components isolated from cows showed no difference in the number of normal bacterial DNA copies in lysozyme treated groups.

According to one specific embodiment of the present invention, lysozyme, and results in a significant increase and CH ₄ concentration decrease in the in vitro total VFA, the increased total VFA and reduced CH ₄ concentration of dry matter (Dry material, DM ) it may be the lysozyme contained in the feed substrate improving ruminal fermentation in vitro and reduce the CH ₄ release.

According to a preferred embodiment of the present invention, the lysozyme as an active ingredient of the feed composition of the present invention has 4000 to 150,000 U (unit), and when the lysozyme is 150,000 U, % ≪ / RTI > by weight of the composition.

 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.

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.

In the present invention, when the animal feed is consumed, it is possible to prevent the global warming effect by inhibiting the formation of methane in the rumen of the ruminant animal, and the feed efficiency can be increased.

In the present invention, the ruminant refers to an animal having rumen and ruminating and digesting food. Examples of the animal include camel, camel, deer, deer, giraffe, cow, goat, ox, buffalo , Bison, deer, camel, sheep, and the like, and is preferably, but not limited to, cow.

In the present invention, the above-mentioned feeding refers to providing the ruminant animal with an animal feed containing the lysozyme, but the method is not limited thereto as long as it provides lysozyme to the ruminant animal. For example, the composition containing lysozyme may be liquidified with water or the like, and the liquid formulation may be administered into the rumen using a syringe or the like, or a feed containing lysozyme may be provided.

The livestock feed of the present invention is a diet fed to a ruminant to grow a ruminant containing the composition comprising the lysozyme, and its characteristics and content are as described above.

When the ruminant feed according to the present invention is fed to the ruminant animal, the ruminant can inhibit the formation of methane in the rumen of the ruminant animal to prevent global warming and increase the feed efficiency.

As described above, the feed composition containing lysozyme of the present invention reduces the methane gas in the rumen, reduces side effects to humans and animals, is safe, and substantially reduces the cause of methane production in the rumen, . Therefore, feeding a feed composition containing lysozyme according to the present invention to a ruminant can inhibit methane production in the rumen rumen of the ruminant animal and increase the efficiency of feed utilization, thereby providing additional economic benefits.

Figure 1 shows the methane concentration (mM / ml) from ruminal fermentation in vitro by addition of lysozyme.

Hereinafter, embodiments of the present invention will be described in detail to facilitate understanding of the present invention. However, the embodiments according to the present invention can be modified into various other forms, and the scope of the present invention should not be construed as being limited to the following embodiments. Embodiments of the invention are provided to more fully describe the present invention to those skilled in the art.

Materials and methods

An in vitro ruminal fermentation technique using commercially available pelletized concentrate (Nonghyup co.) And rice straw (8: 2) was adopted as a substrate. In the present experiment, the liquid lysozyme collected from Celltech co., Korea, containing 150,000 units / ml was used in the present invention. Treatments of 2000, 4000, and 8000 U lysozyme (hereinafter referred to as T1, T2 and T3, respectively) were applied in addition to the control (lysozyme was not added) and this was added to 1 g of dry matter (DM) substrate.

Analysis of in vitro fermentation parameters

Fermentation parameters were checked at the end of each incubation period. The pH was measured using a Pinnacle series M530p meter (Schott Instruments, Mainz, Germany). Before opening the serum bottles, TG production from each sera was measured using EA-6 (Sun Bee instrument, Inc. Korea) pressure sensor. Two 1 ml samples of fermentation from each of the sera bottles were immediately centrifuged at 13,000 rpm for 10 minutes at 4 占 폚 using Micro 17TR centrifuge (Hanil Science Industrial Co. Ltd., 120 Korea). Supernatant 2 1.5ml Eppendorf tube was transferred to (Eppendorf tube) and stored at that temperature to completely frozen at -80 ℃ until analysis of NH ₃ -N and VFA and also the rest of the pellets ratings DNA copy. NH ₃ -N and VFA, samples contained in eppendorf tubes were thawed at room temperature and used to determine NH ₃ -N, individual VFA, ie, acetic acid, propionic acid, butyric acid, and total VFA. NH ₃ -N was carried out using a Libra S22 127 spectrophotometer (Biochrom Ltd., CB40FJ, UK) at an optical density (OD) of 630 nm according to the method developed by Chaney and Marbach (1962) . VFA measurements were performed according to the methods described in Han et al. (2005) and Tabaru et al. (1988). The authors used high performance liquid chromatography (Agilent Technologies 1200 series) using METACARB87H (Varian, Germany) column using a UV detector set at 210 and 220 nm and a 0.0085 NH ₂ SO ₄ solvent as buffer at a flow rate of 0.6 ml / Were used. The concentration of each VFA in mM was calculated as part per million by molecular weight. The total VFA was calculated as the sum of the individual VFA values and the A / P value was calculated as acetic acid divided by propionic acid.

Statistical analysis

Data were analyzed for variance analysis (ANOVA) using a general linear model (GLM) for complete randomized design. All treatments were performed 3 times. Duncan's multiple range test and orthogonal polynomial contrast were used to identify differences between treated and control groups and among them. p <0.05 was considered to be statistically significant. All analyzes were performed using Statistical Analysis System (SAS, 2004) (Version 9.1; SAS Inst., Inc., Carrie, NC).

<Example 1> Measurement of in vitro fermentation effect on lysozyme rumen

The rumen components were rumen-cannulated Holstein cow (650 kg) with cannula at 48 month-old rumen fed twice a day with rice straw and commercially available pelleted concentrate (8: 2 ratio) &Lt; / RTI &gt; The fed rice straw contained 4.45% crude protein (CP) and 38.29% total digestible nutrient (TDN), while the concentrate contained 12% CP and 72% TDN. The pooled rumen components were squeezed out and the extracted fluid was filtered through a four-fold cheese cloth and collected in a glass bottle. The bottle was subsequently capped and immediately transferred to the laboratory while maintaining the temperature at 39 [deg.] C. In the laboratory, the bottle was placed in a water bath at 39 占 폚. The bottle was vigorously shaken by hand and then mixed with the buffer. Particle-free rumen fluid was transferred to buffer medium (pH 6.9) with 1: 3 rumen fluid: buffer ratio. Buffer medium was prepared according to the method described in the literature (Asanuma et al. (1999)). Under a constant flow of N ₂ gas, 100 ml of buffer rumen fluid was anaerobically transferred to 160 ml of 96 sera (1 mm particle size) containing 1 g substrate with 2000, 4000, and 8000 U concentrations of lysozyme. Serum bottles were subsequently sealed with a rubber septum stopper and an aluminum lid and placed in a shaking incubator at 39 占 폚 for 0, 6, 12, and 24 hours at 120 rpm in Hattori and Matsui (2008) And incubated as described. Three repeated experiments were performed during each incubation period.

pH, total gas (TG), methane (CH ₄ ), ammonia nitrogen (NH ₃ -N), acetic acid, propionic acid, butyric acid and total volatile fatty acids (VFA) were analyzed at each incubation time. Collecting a sample into a 15ml Falcon tube (falcon tube) from each serum sickness, and was kept at -80 ℃ until further analysis of VFA, NH ₃ -N, and molecular studies. The gas produced in the interior of the bottle were evaluated for the sample gas trapped in the TG and the vacuum tube and stored in a refrigerator until the measurement of CH _4. The in vitro dry matter (DM) and organic (OM) resolutions were evaluated after incubation for 0, 6, 12 and 24 hours.

The results of measurement of pH, total gas (TG) and ammonia nitrogen (NH ₃ -N) are shown in Table 1 below.

As shown in Table 1, there was no significant difference in pH after incubation for 6 hours and 12 hours. After 24 hours of incubation, the pH in the control group was lowest (p < 0.01) and the maximum pH was in the T3 treated group. In the case of TG, no significant difference was observed after 12 and 24 hours. However, a trend toward higher TG in the treatment group was observed after 12 and 24 hours compared to the control. There was no difference in NH ₃ -N after 12 and 24 hours of incubation. Nevertheless, in a 6 hour incubation, NH ₃ -N is the maximum in the phase were significantly (p = 0.05), T3 treatment group between treatment groups was observed.

The measurement results of acetic acid, propionic acid, butyric acid and total volatile fatty acid (VFA) are shown in Table 2 below.

As shown in Table 2 above, there was a significant difference in acetic acid over all incubation periods except after 6 hours of incubation. Maximum (p < 0.01) acetic acid was observed in T3 (41.61 mM) after 24 hours of incubation followed by the order of T2, T2 and control. Similarly, significant differences (p < 0.01) between 12.21 mM and 12.01 mM in propionic acid after 12 and 24 hours of incubation, respectively. Maximum and lowest propionic acid concentrations were found in T3 and control groups after 12 and 24 hours of incubation, respectively. In the case of butyric acid, there was no significant difference after 24 hours, but there was a tendency towards maximum T3 with butyric acid. Significant differences in total VFA (p <0.05) were found in all treatment groups except 6 hours of incubation. The maximum total VFA was observed at T3 after 12 and 24 hours of incubation. Although there was a tendency toward maximum A / P values in the control group after 12 and 24 hours of incubation, there was no significant difference in A / P values after 6, 12 or 24 hours of incubation.

<Example 2> Measurement of dry matter and organic matter decomposition ability in vitro from lysozyme rumen

At the onset of in vitro ruminal fermentation, the substrates DM and OM were measured by drying for 16 hours at 105 ° C and ashing for 12 hours at 550 ° C, respectively. The DM and the initial DM of the substrate using the OM percent (DM _i) and OM (OM _i) the content (g) obtained was calculated. After each specified incubation period, all treatments with three repeated fermentation samples from each sera were dried, pre-weighed and drained in a nylon bag knotted in nylon thread. Thereafter, they were washed in flowing water until the turbidity of the washing water disappeared. Final DM (DM _f) of the substrate and OM (OM _f) the DM _i And it was determined using the same conditions as applied when measuring the OM _i. DM and OM Resolution (%) of each was calculated by ([DM DM _{f i]} / DM _i) X 100.

In Table 3, there was no significant difference in DM or OM resolution, but there was a tendency toward higher DM resolution in T3 compared to the control group after 24 hours of incubation. Similarly, a trend toward maximum OM resolution after 24 hours of incubation was found at T3.

&Lt; Example 3 > Evaluation of methane in vitro from lysozyme rumen

The sample contained in the vacuum tube by using TCD detector and the car boksen (Carboxen) 150 1006PLOT capillary column 30 m X 0.53 mm gas chromatography (Agilent Technologies HP 5890) using the (Supelco) was analyzed for CH _4. The produced CH ₄ was evaluated using the equations described in the literature (ㅨ rskov and McDonald (1979)).

Figure 1 shows the methane concentration (mM / ml) from ruminal fermentation in vitro by addition of lysozyme. As can be seen, in the case of CH ₄ production, significant differences were observed at all incubation times, except after 6 hours of incubation. After 24 hours, the minimum concentration of methane followed by treatment group on the T3, T1 and T2 were observed at the maximum concentration of CH ₄ was detected in the control group. Although not significant, the typical bacterial DNA copy number tended to be lower in the treatment group (T1-T3) as compared to the control group.

Example 4 Measurement of Bacteria by Quantitative Real-Time PCR (qRT-PCR)

Common bacterial, methine, and protozoan DNA copies were evaluated using the protocol of Denman and McSweeney (2006). Was determined using a microplate spectrophotometer porch (Epoch Microplate Spectrophotometer) (BiotekR ^ㄾ 157) in the DNA concentration from a gDNA extracted from each of the pelletized samples. For quantitative PCR, the target-specific forward and reverse PCR primers shown in Table 4 below were used. Amplification was performed using Eco Real-Time PCR (Illumina, Inc.) with 10 μl of 2 × qPCRBIO SyGreen 160 mixture in sterile distilled water, 0.8 μl of each forward and reverse PCR primer and 50 μg / μl of 8.4 μl template DNA In a total reaction volume of 20 占 퐇 per reaction mixture containing the compound shown in Table 5 below.

Target microorganism  Primer sequence Annealing temperature
(° C) Product size  references Common bacteria
1114F: 5'-CGG CAA CGA GCG CAA CCC-3 ' 60
130
Denman and McSweeney, 2006 1275R: 3'-CCA TTG TAG CAC GTG TGT AGC C-5 ' Methane bacteria
qmcraA-F: 5'-GGA TTA GAT ACC CSG GTA GT-3 ' 60
140
Denman and McSweeney, 2006 qmcrA-R: 3'-GTT GAR TCC AAT TAA ACC GCA-5 ' Protist
F: 5'-GCT TTC GWT GGT AGT GTA TT-3 ' 60
223
Denman and McSweeney, 2006 R: 3'-CTT GCC CTC YAA TCG TWC T-5 '

As shown in Table 5, there was no significant difference in the number of methine bacterial DNA copies. The protoplast DNA copy number was also not significantly different, but the lowest DNA copy number was found in the T3 treatment group.

Claims (3)

A ruminant feed composition for mitigating methane gas in rumen of a ruminant comprising lysozyme as an active ingredient. The method according to claim 1,
Wherein the lysozyme is contained in an amount of 2.6 to 99% by weight based on the total weight of the composition when the lysozyme is 150,000 U (unit), and the rheumatic feed composition for reducing methane gas in rumen . A method for inhibiting methane production in the rumen of a ruminant by feeding the ruminant feed composition of claim 1 or 2 to the ruminant.

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