Feed additives for reducing an enteric methane production from rumen fermentation of ruminants using deodeok

Abstract

The present invention relates to a feed additive composition for reducing methane in ruminants and a method for reducing methane in ruminants, which can effectively reduce methane production in ruminants by feeding the ruminants a feed additive composition containing Codonopsis lanceolata, without affecting the digestibility, energy efficiency, and rumen fermentation characteristics of the ruminants.

Description

Technology Field

[0001] The present invention relates to deodeok ( Codonopsis lanceolata The present invention relates to a feed additive composition for reducing methane derived from intestinal fermentation in ruminants using ), and provides a methane-reducing feed additive composition to replace synthetic additives currently used to reduce methane generated in the rumen of ruminants. Background Technology

[0003] Recently, climate change has rapidly emerged as a top global agenda, and pressure is mounting in Korea to participate in mandatory reductions for greenhouse gas emissions, including methane. The major greenhouse gases causing global warming are the six major gases designated by the Intergovernmental Panel on Climate Change (IPCC): carbon dioxide (CO2), methane (CH4), nitrous oxide (N2O), perfluorocarbons (PFCs), hydrofluorocarbons (HFCs), and sulfur hexafluoride (SF6). As these greenhouse gases increase, the Earth's average temperature and sea levels have risen, and extreme weather events such as desertification, droughts, floods, and typhoons are occurring. Among these, methane is the factor with the second-largest impact on global warming after carbon dioxide; over 8.3 billion tons are released into the atmosphere worldwide (international methane emissions), accounting for approximately 30% of total global warming (IPCC Sixth Assessment Report Working Group 1, 2021).

[0004] Once emitted, methane remains in the atmosphere for approximately 8.4 years before being removed. As a greenhouse gas with a greenhouse effect more than 20 times greater than carbon dioxide, it accounts for 16% of global greenhouse gas emissions and is a representative substance that destroys the ozone layer. According to national methane emission data (as of 2018), greenhouse gases emitted from the livestock sector amounted to 5.9 million tons CO2eq., accounting for approximately 21% of total national emissions, with 70–80% of this generated by ruminant livestock. Domestically, approximately 28 million tons CO2eq. of methane are emitted annually, with about 16% of this originating from the digestive systems of ruminants such as cattle, sheep, and goats. Therefore, in relation to the issue of global warming, reducing methane emissions to increase feed utilization efficiency for ruminants is a critical task. The government has indicated the need to develop technologies to reduce methane emissions from intestinal fermentation and has been pursuing the development of technologies to suppress methane generation by isolating and culturing methane-producing bacteria within the rumen.

[0005] Methane is a natural byproduct generated during the process of ruminants digesting feed. Ruminants, such as cows, do not digest feed using digestive enzymes in gastric juice; instead, they utilize digestive products produced by microorganisms within the stomach breaking down the feed. Cows repeatedly ruminate throughout the day, regurgitating feed stored in their stomachs, chewing it about 40 to 60 times, and then swallowing it again. The methane generated during this process is expelled through belching.

[0006] Meanwhile, feed consumed by ruminants is broken down by rumen microorganisms in the rumen, producing carbon dioxide, ammonia, hydrogen, and volatile fatty acids as final decomposition products. Among these fermentation products, volatile fatty acids and ammonia are utilized by ruminant animals and microorganisms, ultimately playing a crucial role in livestock productivity. However, carbon dioxide and hydrogen are converted into methane by methane-producing bacteria or competitively produced as succinic acid by fumarate-reducing microorganisms during the final stage of fermentation. In particular, the production of methane poses a problem because it is not absorbed within the ruminant's body and is released into the atmosphere approximately 15 to 20 times per hour. Cattle, on average, produce over 50 L / hour of gas, of which 25–30% is methane. Consequently, major countries, including Korea, are conducting research to reduce methane produced by microorganisms.

[0007] However, since the gas produced in the rumen is generated during the decomposition of ingested feed by rumen microorganisms, reducing fermentation gas is equivalent to lowering the digestibility of nutrients. Therefore, it is necessary to suppress only fermentation gas without affecting the digestibility of nutrients or other rumen fermentation characteristics.

[0009] Meanwhile, Korean Patent No. 10-1989130 (hereinafter referred to as the prior art) relates to a feed composition for cattle for increasing omega-3 content and discloses a feed composition for ruminants (cattle, Hanwoo) containing Codonopsis pilosula. However, it differs from the present invention in that it ferments and ages a mixture of Codonopsis pilosula, uses Codonopsis pilosula as a probiotic, and does not relate to the methane reduction effect caused by rumen fermentation.

[0010] Korean Patent No. 10-1834227 discloses a livestock feed composition for methane reduction comprising garlic extract, nitrate, and fumarate, and Korean Published Patent No. 10-2018-0044051 discloses a feed composition for ruminants for reducing methane in the rumen of ruminants comprising lysozyme as an active ingredient, but the technical configurations are different from the present invention.

[0011] Korean Patent No. 10-0679153 discloses a feed composition containing an additive that selectively reduces protozoa in the rumen of ruminants. Specifically, the prior art discloses that feeding a ruminant a feed composition containing at least one of ginger, chive extract, and complex linoleic acid can reduce methane production in the ruminant by inhibiting the growth of methane-producing bacteria, which in turn inhibits the growth of protozoa in the rumen. However, there is a problem in that removing protozoa to reduce methane production in ruminants, as in the prior art, slows down the degradation rate of feed proteins. Furthermore, the feed composition containing ginger, chive extract, and complex linoleic acid disclosed in the prior art is not readily available in the surrounding area, and there is a problem in that the manufacturing cost to produce said feed composition is high, and consequently, the purchase cost to buy said feed composition is also high.

[0013] Accordingly, the inventors of the present invention have completed the present invention by diligently researching to develop a composition that suppresses the generation of fermentation gas without affecting the nutrient digestibility and other rumen fermentation characteristics, and by confirming that when a feed additive composition containing Codonopsis pilosula is administered, it reduces the production of rumen fermentation gas without significantly inhibiting the rumen digestibility. The problem to be solved

[0015] The objective of the present invention is to provide a feed additive composition for reducing methane derived from intestinal fermentation in ruminants.

[0016] Another objective of the present invention is to provide the above feed additive composition to effectively reduce methane without affecting other metabolic processes, such as a decrease in feed digestibility. means of solving the problem

[0018] To solve the above problem, the present invention provides a feed additive composition comprising Codonopsis pilosula.

[0019] In addition, the present invention provides a method for reducing methane in ruminants, characterized by reducing the amount of methane produced by ruminants through the digestion of a feed additive composition for methane reduction containing Codonopsis pilosula in rumen fluid. Effects of the invention

[0021] When the feed additive composition according to the present invention is fed to ruminants, it has the effect of reducing the generation of greenhouse gases that cause global warming by reducing methane production caused by rumen intestinal fermentation. Furthermore, a high amount of methane production within the rumen implies that a portion of the energy from the fed feed is released into the atmosphere as methane; therefore, high methane production leads to a decrease in the productivity of ruminants. Accordingly, feeding the feed additive composition of the present invention allows most of the feed energy to be utilized for the growth of ruminants, thereby reducing energy loss due to methane production in livestock and enabling the efficient use of that energy for growth, which also has the effect of increasing energy efficiency. Brief explanation of the drawing

[0023] Figure 1 shows deodeok ( Codonopsis lanceolata Displays a photo of ). Figure 2 shows a graph of methane production during 48 hours of in vitro culture. Figure 3 shows a graph of dry matter digestibility of 48-hour in vitro culture. Specific details for implementing the invention

[0024] The present invention provides a feed additive composition for reducing methane gas generated by rumen fermentation during the digestion of feed in ruminants.

[0026] In the present invention, the rumen is a special digestive organ found in some mammals of the order Artiodactyla, also known as the rumen or ruminating stomach. Rumination refers to the process of regurgitating food that has been swallowed back into the mouth to chew it thoroughly before swallowing it again, and the stomach that enables this rumination is called the rumen. Meanwhile, microorganisms coexist in the rumen, giving it the ability to break down plant cellulose, which ordinary animals cannot digest, and convert it into energy.

[0027] In the present invention, ruminants refer to animals that convert cellulose into volatile fatty acids through microbial fermentation in the rumen digestive system, thereby inducing this into their nutrients, and include cattle, goats, bulls, water buffalo, bison, deer, moose, camels, sheep, etc. In one embodiment of the present invention, Hanwoo cattle were used, but the invention is not limited thereto.

[0028] Since these ruminants produce methane and are a major cause of global warming, it is necessary to reduce the amount of methane gas produced by ruminants.

[0029] Furthermore, in addition to the environmental aspect of mitigating global warming, there is also the aspect of increasing feed efficiency by reducing the feed energy released as methane.

[0031] In this regard, the present invention ( Codonopsis lanceolata This relates to a feed additive composition for reducing methane in ruminants, comprising ) and it was confirmed that adding the feed additive composition containing Codonopsis pilosula of the present invention to ruminant feed and feeding it has the effect of reducing the generation of methane derived from rumen fermentation.

[0033] Deodeok ( Codonopsis lanceolata Codonopsis pilosula is a perennial vine belonging to the Campahutaceae family that grows in forests throughout Korea. It thrives in environments with limited sunlight, abundant humus, and high ambient humidity. The plant reaches a height of 2 to 5 meters. Four leaves appear clustered closely together at the tips of short branches; they are elongated oval-shaped, measuring 3 to 10 cm in length and 1.5 to 4 cm in width. The margins of the leaves are smooth, and while the upper surface is green, the underside is powdery white. The flowers are light green on the outside with purplish-brown spots on the inside and bloom facing downwards. The fruit ripens around October to November, and the seeds are tiny. The roots are thick, similar to those of the balloon flower (Platycodon grandiflorus); cutting the vine releases a white sap, and the roots are used for both culinary and medicinal purposes.

[0034] Deodeok contains a large amount of saponins, which are beneficial for preventing vascular diseases and cancer. Additionally, it is known to be effective for coughs accompanied by chest pain or phlegm, asthma, high blood pressure, cholesterol removal, inflammation treatment, skin detoxification, and as a tonic; thus, it has been called Sasam since ancient times and has been widely used as a substitute for ginseng.

[0035] The history of Deodeok cultivation is not long, and while it was originally harvested from wild plants for medicinal and edible use, it has since been domesticated and cultivated. Types of Deodeok include wild Deodeok (mountain Deodeok) and artificially cultivated Deodeok (field Deodeok), but the types of Deodeok that can be used in this invention are not limited.

[0036] In the present invention, the Codonopsis pilosula may be any part, such as the above-ground part, the underground part, or the whole plant. In one embodiment of the present invention, the underground part is preferably used, but is not limited thereto.

[0037] The nutritional components of deodeok include abundant minerals such as potassium, iron, calcium, and phosphorus, and it is known to contain a large amount of saponin, and the leaves of deodeok contain antioxidant components such as phenols and flavonoids.

[0039] In the present invention, 1 to 10% can be added relative to the dry substrate of the deodeok, preferably 3 to 7%, most preferably 5%, but is not limited thereto.

[0040] For the substrate, 0.1g of rice straw and 0.4g of compound feed (Table 2) were used as roughage, and 25mg (5%) of Codonopsis pilosula was added to it. In the present invention, all types of basic feed can be used.

[0041] In the present invention, the total gas volume (mL) represents the total amount of gas generated during feed digestion.

[0042] In the measurement of methane production in this invention, methane (%) of total gas volume represents the methane content in the total gas volume generated by Hanwoo, and mL represents the methane content converted into mL units. Additionally, mL / dry matter (g) represents the methane (mL) generated by Hanwoo converted into the amount produced per feed (dry matter basis), and mL / digested dry matter (g) represents the methane (mL) generated by Hanwoo converted into the amount produced per digested feed (dry matter basis).

[0043] In this invention, pH refers to the pH in the rumen fluid and exhibits a positive correlation with the total volatile fatty acid production.

[0044] In this invention, total volatile fatty acids (mM) are utilized as a major metabolic energy source for Hanwoo cattle, where acetic acid (% of molar) is utilized as a substrate for methane production, exhibiting a positive correlation with methane output, and is used as a source for fat synthesis in cattle.

[0045] In addition, propionic acid (% of molar) shows a negative correlation with methane production by utilizing hydrogen in the production process and is resynthesized into glucose in the liver of cattle and used as a major energy source.

[0046] In addition, butyric acid and valeric acid (% of molar) are utilized as energy sources for the growth of rumen epithelial cells, and isobutyric acid and isovaleric acid (% of molar) are known to influence rumen fermentation, cellulose-degrading microorganisms, rumen enzyme activity, nutrient digestion, and bovine productivity; furthermore, the acetic acid:propionic acid ratio serves as a measure of feed energy utilization efficiency.

[0047] In the present invention, ammoniacal nitrogen (mg / dL) is used as a protein synthesis source for ruminants (cattle).

[0048] In the present invention, the dry matter digestibility (%) represents the digestibility of feed (based on dry matter) in the rumen of Hanwoo cattle.

[0050] In a specific embodiment of the present invention, a feed additive containing Codonopsis pilosula was added and rumen fluid was cultured for 48 hours in an in vitro analysis, and the effects of total gas and methane production among the total gas volume, fermentation characteristics (pH, total volatile fatty acids, acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, and acetic acid:propionic acid ratio) and dry matter digestibility were measured.

[0051] According to the results of in vitro culture of rumen fluid for 48 hours with 5% of Codonopsis pilosula added relative to the substrate, there were no significant differences in dry matter digestibility, pH, total volatile fatty acids, and acetic acid:propionic acid ratio between the Codonopsis pilosula treatment group and the control group (P>0.05).

[0052] Therefore, it was confirmed that when a feed additive composition containing Codonopsis pilosula was fed to ruminants, there was no negative effect on the digestibility, energy efficiency, and rumen fermentation characteristics of the feed (see Tables 7, 8, and 10).

[0053] Meanwhile, according to the results of in vitro culture of rumen fluid for 48 hours with 5% of Codonopsis pilosula added relative to the substrate, there was no significant difference in the ammonia nitrogen content, which is used as a protein synthesis source for ruminants, between the Codonopsis pilosula treatment group and the control group (P>0.05). Therefore, it was confirmed that feeding a feed additive composition containing Codonopsis pilosula to ruminants did not have a negative effect on protein synthesis (see Table 9).

[0054] In addition, according to the results of in vitro culture of rumen fluid for 48 hours with 5% of Codonopsis pilosula added relative to the substrate, the total gas volume (mL) decreased in the Codonopsis pilosula treatment group compared to the control group, and the amount of methane produced per feed (dry matter) digested was significantly reduced in the Codonopsis pilosula treatment group compared to the untreated group (control group) (P<0.05). Therefore, it was confirmed that feeding a feed additive composition containing Codonopsis pilosula to ruminants can significantly reduce the amount of methane produced by rumen fermentation (see Table 6).

[0056] The present invention is described in detail below by the following examples.

[0057] However, the following examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following examples.

[0059] Example 1. Rumen fluid collection and sample culture

[0060] Rumen fluid was collected from three Hanwoo cattle equipped with cannulas (rice straw to concentrate feed ratio = 2:8, body weight 736 kg) after removing feed particles using eight layers of gauze. The collected rumen fluid was mixed with sterilized McDougall buffer (Table 1) at a ratio of 1:4, stirred at 39°C, and maintained under anaerobic conditions. A filter bag containing 0.5 g of substrate (rice straw to concentrate feed ratio = 2:8) and 5% additive (Codonopsis pilosula 25 mg) was placed in an in vitro serum bottle, and 50 mL of the buffer-mixed rumen fluid (culture medium) was dispensed and incubated for 24 and 48 hours. After incubation, gas production was measured, the filter bag was removed for digestibility analysis, and the culture medium was used for fermentation characteristics analysis. The roughage used in the experiment was rice straw, and the concentrate feed was prepared for use. Tables 2 and 3 below show the composition of the feed used in the experiment.

[0062] Table 1 shows the mixing ratio of the above McDougall buffer.

[0063] McDougall buffer mixing ratio (McDougall, 1948) composition Amount (g / L) NaHCO3 9.80 NaHPO4·12H2O 9.30 NaCl 0.47 KCl 0.57 CaCl2anhyd. 0.04 MgCl2anhyd. 0.06

[0064] Compound feed formulation ratio type % DM Ground corn 58.1 Soybean scalp 16.6 Corn gluten feed 11.7 Wheat winter 5.8 Soybean meal 3.5 Lupin 2.3 limestone 0.8 sodium bicarbonate 0.6 salt 0.4 Vitamin & Mineral Mixture 0.2

[0065] Concentrated feed, rice straw nutritional components Concentrated feed rice straw building, % 87.8 69.1 Crude protein, %DM 18.9 6.89 Crude fat, %DM 3.38 1.04 Non-fiber carbohydrates, % DM 47.1 - Neutral detergent insoluble fiber, % DM 25.1 63.5 Acid detergent insoluble fiber, % DM 10.8 42.0 Views, % DM 5.51 10.2

[0067] Example 2. Analysis of rumen fermentation characteristics and digestibility after 48 hours of Codonopsis pilosula culture

[0068] deodeok After in vitro culture using [the method], the rumen fermentation characteristics and digestibility were measured. Rumen fluid collection and sample culture were performed in the same manner as in Example 1 above. Figure 1 shows a photograph of the Codonopsis pilosula used in the experiment.

[0069] Table 4 shows the results of the analysis of the nutritional components of the deodeok used in the experiment.

[0070] Nutritional components Deodeok building, % 89.69 Crude protein, % DM 7.77 Crude fat, % DM 2.30 Views, % DM 4.24 Neutral detergent insoluble cellulose, % DM 18.64 Acid detergent insoluble cellulose, % DM 15.82 Non-fiber carbohydrates, % DM 67.05

[0072] 2-1. Experimental Method

[0073] ① Analysis of rumen gas production

[0074] The amount of gas produced (mL) was measured in the serum bottle after culture was completed using a 50 mL syringe (H13.SYS050,DH, South Korea). The gas collected in the syringe was transferred to a vacuum tube (BD 367953, 8.5 mL) and analyzed using GC (gas chromatography, NL / 450 GC, Bruker, USA). Gas was injected into the GC at 1.0 mL a time using a gas-tight syringe, and 22.4 μM, 63.5 μM, and 242.2 μM methane were used as standard gases.

[0075] For the analysis of methane production, the GC analysis conditions were set with an oven temperature of 90℃, an injector temperature of 260℃, a split ratio of 10, a total flow of 55.8 mL / min, and a column flow of 5 mL / min, using helium as the mobile phase, and the analysis was performed using a GS-GasPro 113-4332 column (Agilent, 30 m × 0.32 mm, -80~260℃).

[0076] ② Culture solution pH analysis

[0077] After the culture was finished, the culture medium was transferred from the serum bottle to a conical tube for storage, and the pH of the culture medium was measured using a pH meter (pinnacle pH meter M540, Corning, NY, USA).

[0078] ③ Analysis of Volatile Fatty Acids in Culture Solution

[0079] The culture medium for volatile fatty acid analysis was frozen after treatment with 25% metaphosphoric acid. For analysis, the frozen sample was thawed and centrifuged at 12,000 rpm for 20 minutes using a microcentrifuge (Cyro1730MR, Labogene, Korea). The supernatant was collected in an analysis vessel and analyzed using GC (gas chromatography, 6890N, Agilent Technologies, USA). The standard solution used for analysis was a volatile fatty acid standard solution (Sigma-Aldrich, 46975-U, USA, acetate: 10 mM, propionate: 10 mM, isobutyrate: 10 mM, butyrate: 10 mM, isovarate: 10.1 mM, varate: 10.2 mM), and a fused silica capillary column (Supelco, Nukol™, 15 m × 0.53 mm × 0.5 µm) was used.

[0080] The GC analysis conditions were set with an oven temperature of 110℃, an injector temperature of 250℃, a split ratio of 20:11, a split flow of 62 mL / min, and a column flow of 11 mL / min, and nitrogen was used as the mobile phase.

[0081] ④ Analysis of ammoniacal nitrogen in the culture medium

[0082] The culture medium for ammonia nitrogen analysis was frozen after treatment with 25% metaphosphoric acid. After sufficiently thawing the samples at room temperature, the supernatant obtained by centrifuging at 12,000 rpm for 5 minutes was placed in 10 µl aliquots of ammonia standard solutions (50, 100, 200, and 400 ppm) and distilled water, respectively, into three 1.5 mL microtubes (60015, SPL, South Korea). To each test tube, 500 µl of phenol color reagent (Phenol (C6H5OH)) and 500 µl of alkali-hypochlorite reagent (sodium hydroxide (NaOH)) were added, followed by stirring. Subsequently, the microtubes were capped and incubated in a 50°C water bath for 7 minutes until color development was complete. Afterward, 200 µl of the sample was dispensed onto plates, and analyzed using a UV spectrophotometer (BIO-RAD 10582, Nebchmark Plus, Japan). The optical density (OD) was measured at 630 nm. At this time, the content of ammoniacal nitrogen in the sample was calculated by comparing it with the calibration curve of an ammonia standard solution.

[0083] ⑤ Building Fire Extinguishing Rate Analysis

[0084] The filter bag removed from the serum bottle was washed until impurities were removed, and the washed filter bag was dried in a drying oven at 60°C for 48 hours and then weighed. At this time, the dry matter digestibility was calculated by comparing the original sample weight with the dry weight after culture.

[0086] 2-2. Analysis Results

[0087] Table 5 shows the results of analyzing gas production, methane production, rumen fermentation characteristics, and dry matter digestibility after 48 hours of in vitro culture of Codonopsis pilosula.

[0088] Control group Deodeok SEM P-value Total gas generation Total gas production, mL 84.6 a 61.8 b 5.672 0.001 methane production % of methane in total gas volume 8.37 8.58 0.121 0.997 methane mL 7.08 a 5.42 b 0.418 0.033 Methane mL / dry weight (g) 14.1 a 10.3 b 0.957 < 0.001 methane mL / digested dry weight (g) 20.9 a 15.1 b 0.425 < 0.001 Rumen fermentation characteristics and dry matter digestibility pH 6.49 6.38 0.028 0.076 Total volatile fatty acids, mM 89.2 72.1 4.119 0.163 Acetic acid, % of molar 55.9 57.5 0.472 0.778 Propionic acid, % of molar 22.7 21.4 0.415 0.924 Butyric acid, % of molar 13.0 12.7 0.166 1.000 Isobutyric acid, % of molar 1.64 1.56 0.027 0.763 Valeric acid, % of molar 5.10 5.10 0.252 1.000 Isovaleric acid, % of molar 1.74 1.72 0.041 1.000 Acetic acid:propionic acid ratio 2.47 2.68 0.063 0.920 Ammonia nitrogen, mg / dL 28.8 26.9 1.228 4.857 Building fire extinguishing rate, % 67.6 68.2 2.150 1.000

[0089] The treatment group had 5% of the substrate (based on building weight) added, and a, b Superscripts indicate a significant difference according to the Tukey test (P<0.05).

[0090] As a result of the experiment, methane production (mL / digested dry weight) was significantly reduced in 48 hours of in vitro culture of Codonopsis pilosula (Fig. 2). Based on this, Codonopsis pilosula can be identified as a candidate substance (medicinal crop) for reducing methane production.

[0091] In addition, there was no significant difference in dry matter digestibility between the deodeok treatment group and the control group (P>0.05) (Fig. 3).

[0092] These results indicate that Codonopsis pilosula has the effect of reducing methane production resulting from rumen fermentation without having a negative impact on dry matter digestibility.

[0094] In the following examples, rumen fermentation characteristics and digestibility were analyzed by measuring rumen gas production, pH, volatile fatty acids, ammonia nitrogen, and dry matter digestibility using Codonopsis pilosula.

[0096] Example 3. Analysis of Rumen Gas Production

[0097] The amount of gas produced (mL) in the serum bottle after culture was completed was measured using a 50 mL syringe (H13.SYS050, DH, South Korea). The gas collected in the syringe was transferred to a vacuum tube (BD 367953, 8.5 mL) and analyzed using GC (gas chromatography, NL / 450 GC, Bruker, USA). Gas was injected into the GC at 1.0 mL a time using a gas-tight syringe, and 22.4 μM, 63.5 μM, and 242.2 μM methane were used as standard gases.

[0098] For the analysis of methane generation, the GC analysis conditions were set with an oven temperature of 90℃, an injector temperature of 260℃, a split ratio of 10, a total flow of 55.8 mL / min, and a column flow of 5 mL / min, using helium as the mobile phase, and the analysis was performed using a GS-GasPro 113-4332 column (Agilent, 30 m × 0.32 mm, -80~260℃).

[0099] Table 6 shows the results of measuring total gas and methane production in 48-hour in vitro rumen cultures with the addition of Codonopsis pilosula.

[0100] Control group Deodeok SEM P-value Total gas generation Total gas production, mL 84.6 a 61.8 b 5.672 0.001 methane production % of methane in total gas volume 8.37 8.58 0.121 0.997 methane mL 7.08 a 5.42 b 0.418 0.033 Methane mL / dry weight (g) 14.1 a 10.3 b 0.957 < 0.001 methane mL / digested dry weight (g) 20.9 a 15.1 b 0.425 < 0.001

[0101] The treatment group had 5% of the substrate (based on building weight) added, and a, b Superscripts indicate a significant difference according to the Tukey test (P<0.05).

[0102] In Table 6 above, the total gas volume (mL) represents the total amount of gas generated during respiration or belching of Hanwoo cattle. The total gas volume decreased in the deodeok treatment group compared to the control group, and the amount of methane produced per feed (dry matter) digested was significantly reduced in the deodeok treatment group compared to the untreated group (control group) (P<0.05). This indicates that adding deodeok to the feed can significantly reduce the amount of methane produced by rumen fermentation.

[0104] Example 4. Culture solution pH analysis

[0105] After the culture was finished, the culture medium was transferred from the serum bottle to a conical tube for storage, and the pH of the culture medium was measured using a pH meter (pinnacle pH meter M540, Corning, NY, USA).

[0106] Table 7 shows the results of measuring the pH in rumen fluid culture for 48 hours in vitro with the addition of Codonopsis pilosula.

[0107] Control group Deodeok SEM P-value pH 6.49 6.38 0.028 0.076

[0108] The treatment group had 5% of the substrate (based on building weight) added, and a, b Superscripts indicate a significant difference according to the Tukey test (P<0.05).

[0109] In Table 7 above, pH represents the pH of the rumen fluid and shows a positive correlation with the total volatile fatty acid production. When Codonopsis pilosula was added to the feed, there was no significant difference in rumen pH compared to the control group (P>0.05), so it can be concluded that there was no negative effect.

[0111] Example 5. Analysis of Volatile Fatty Acids in Culture Medium

[0112] The culture medium for volatile fatty acid analysis was frozen after treatment with 25% metaphosphoric acid. For analysis, the frozen sample was thawed and centrifuged at 12,000 rpm for 20 minutes using a microcentrifuge (Cyro1730MR, Labogene, Korea). The supernatant was collected in an analysis vessel and analyzed using GC (gas chromatography, 6890N, Agilent Technologies, USA). The standard solution used for analysis was a volatile fatty acid standard solution (Sigma-Aldrich, 46975-U, USA, acetate: 10 mM, propionate: 10 mM, isobutyrate: 10 mM, butyrate: 10 mM, isovarate: 10.1 mM, varate: 10.2 mM), and a fused silica capillary column (Supelco, Nukol™, 15 m × 0.53 mm × 0.5 µm) was used.

[0113] The GC analysis conditions were set with an oven temperature of 110℃, an injector temperature of 250℃, a split ratio of 20:11, a split flow of 62 mL / min, and a column flow of 11 mL / min, and nitrogen was used as the mobile phase.

[0114] Table 8 shows the results of measuring total volatile fatty acids, acetic acid, propionic acid, butyric acid, valeric acid, isovaleric acid, and the acetic acid:propionic acid ratio as fermentation characteristics in 48-hour in vitro rumen culture with the addition of Codonopsis pilosula.

[0115] Control group Deodeok SEM P-value Total volatile fatty acids, mM 89.2 72.1 4.119 0.163 Acetic acid, % of molar 55.9 57.5 0.472 0.778 Propionic acid, % of molar 22.7 21.4 0.415 0.924 Butyric acid, % of molar 13.0 12.7 0.166 1.000 Isobutyric acid, % of molar 1.64 1.56 0.027 0.763 Valeric acid, % of molar 5.10 5.10 0.252 1.000 Isovaleric acid, % of molar 1.74 1.72 0.041 1.000 Acetic acid:propionic acid ratio 2.47 2.68 0.063 0.920

[0116] The treatment group had 5% of the substrate (based on building weight) added, and a, b Superscripts indicate a significant difference according to the Tukey test (P<0.05).

[0117] In Table 8 above, total volatile fatty acids (mM) are utilized as a major metabolic energy source for Hanwoo cattle, and the acetic acid:propionic acid ratio serves as a measure of feed energy utilization efficiency. Since there were no significant differences (P>0.05) in each item when Codonopsis pilosula was added to the feed compared to the control group, it can be concluded that there were no negative effects on metabolic energy and energy utilization efficiency.

[0119] Example 6. Analysis of ammoniacal nitrogen in culture medium

[0120] The culture medium for ammonia nitrogen analysis was frozen after treatment with 25% metaphosphoric acid. After sufficiently thawing the samples at room temperature, the supernatant obtained by centrifuging at 12,000 rpm for 5 minutes was placed in 10 µl aliquots of ammonia standard solutions (50, 100, 200, and 400 ppm) and distilled water, respectively, into three 1.5 mL microtubes (60015, SPL, South Korea). To each test tube, 500 µl of phenol color reagent (Phenol (C6H5OH)) and 500 µl of alkali-hypochlorite reagent (sodium hydroxide (NaOH)) were added, followed by stirring. Subsequently, the microtubes were capped and incubated in a 50°C water bath for 7 minutes until color development was complete. Afterward, 200 µl of the sample was dispensed onto plates, and analyzed using a UV spectrophotometer (BIO-RAD 10582, Nebchmark Plus, Japan). The absorbance (OD, optical density) was measured at 630 nm. At this time, the content of ammoniacal nitrogen in the sample was calculated by comparing it with the calibration curve of an ammonia standard solution.

[0121] Table 9 shows the results of measuring ammonia nitrogen in rumen fluid cultures for 48 hours in vitro with the addition of Codonopsis pilosula.

[0122] Control group Deodeok SEM P-value Ammonia nitrogen, mg / dL 28.8 26.9 1.228 4.857

[0123] The treatment group had 5% of the substrate (based on building weight) added, and a, b Superscripts indicate a significant difference according to the Tukey test (P<0.05).

[0124] The ammonia nitrogen content, which is used as a protein synthesis source for ruminants, showed no significant difference in the Codonopsis pilosula treatment group compared to the control group (P>0.05), so it can be concluded that there is no negative effect on protein synthesis in cattle.

[0126] Example 7. Building Fire Extinguishing Rate Analysis

[0127] The filter bag removed from the serum bottle was washed until impurities were removed, and the washed filter bag was dried in a drying oven at 60°C for 48 hours and then weighed. At this time, the dry matter digestibility was calculated by comparing the original sample weight with the dry weight after culture.

[0128] Table 10 shows the results of measuring the dry matter digestibility in 48-hour in vitro rumen culture with the addition of Codonopsis pilosula.

[0129] Control group Deodeok SEM P-value Building fire extinguishing rate, % 67.6 68.2 2.150 1.000

[0130] The treatment group had 5% of the substrate (based on building weight) added, and a, b Superscripts indicate a significant difference according to the Tukey test (P<0.05).

[0131] The dry matter digestibility (%), which represents the digestibility of feed (on a dry matter basis) in the rumen of Hanwoo cattle, showed no significant difference in the Codonopsis pilosula treatment group compared to the control group (P>0.05), so it can be concluded that there is no negative effect on dry matter digestibility.

[0133] The above results indicate that when a feed additive composition containing Codonopsis pilosula is fed to Hanwoo cattle, it has the effect of reducing methane production caused by rumen fermentation without having a negative impact on digestibility and rumen fermentation characteristics.

Claims

Claim 1 Deodeok ( Codonopsis lanceolata A feed additive composition for methane reduction in ruminants comprising ). Claim 2 In Article 1, the above deodeok ( Codonopsis lanceolata A feed additive composition for methane reduction in ruminants characterized by using one or more parts selected from the group consisting of the underground part, the above-ground part, and the whole plant of Codonopsis pilosula. Claim 3 In Clause 2, the above deodeok ( Codonopsis lanceolata A feed additive composition for reducing methane in ruminants characterized by using the underground part of Codonopsis pilosula. Claim 4 A feed additive composition for methane reduction in ruminants, characterized in that, in claim 1, the ruminant is a cow, goat, bull, water buffalo, bison, deer, moose, camel, or sheep. Claim 5 A feed additive composition for methane reduction in ruminants, characterized in that, in claim 4, the ruminant is a cow. Claim 6 A feed additive composition for methane reduction in ruminants, characterized in that, in claim 1, the above-mentioned Codonopsis pilosula is added in an amount of 1 to 10% based on the total weight of the feed composition including roughage and concentrate feed. Claim 7 A feed additive composition for methane reduction in ruminants, characterized in that, in claim 6, the amount of added Codonopsis pilosula is based on dry matter. Claim 8 A feed additive composition for methane reduction according to claim 1, characterized in that the feed additive composition has no effect on the dry matter digestibility of ruminants. Claim 9 A feed additive composition for methane reduction according to claim 1, characterized in that the feed additive composition does not affect ruminal fermentation characteristics selected from the group consisting of ruminal pH, total volatile fatty acids, acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, and the ratio of acetic acid to propionic acid of ruminants. Claim 10 A feed additive composition for methane reduction according to claim 1, characterized in that the feed additive composition does not affect the ammoniacal nitrogen content in the rumen of ruminants. Claim 11 Deodeok pursuant to Article 1 ( Codonopsis lanceolata A method for reducing methane in ruminants, characterized by reducing the amount of methane produced by ruminants by feeding them a feed additive composition for methane reduction that includes ).

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