WO2024106663A1 - Composition for reducing methane emission from ruminants containing thiamine triphosphate - Google Patents

Abstract

The present invention relates to a feed additive composition for reducing methane from ruminants and a method for reducing methane from ruminants, wherein by feeding the feed additive composition comprising thiamine triphosphate to the ruminants, it is possible to effectively reduce the amount of methane produced by the ruminants, and the feed additive composition does not affect the digestibility, energy efficiency, and rumen fermentation characteristics of the ruminants.

Description

Composition for reducing methane production in ruminants containing thiamine triphosphate

The present invention relates to a composition for reducing methane production, and more specifically, to a feed additive composition for reducing methane production in ruminant animals containing thiamine triphosphate and a method for suppressing methane production in ruminant animals using the same.

Due to the increase in the concentration of global warming gases, global warming is accelerating, resulting in abnormal weather conditions, crop production and food shortages. Global warming is a phenomenon in which the average temperature of the Earth's surface increases. It is known to be caused by various greenhouse gases entering the atmosphere and remaining there, absorbing or re-emitting infrared radiation. Currently, the Intergovernmental Panel on Climatic Change (IPCC) estimates that carbon dioxide (CO2) ), methane (CH4), nitrous oxide (N2O), hydrofluorocarbons (HFCs), perfluorocarbons (PFCs), and sulfur hexafluoride (SF6) were selected as the six major greenhouse gases. After industrialization, the Earth's temperature continues to rise due to an increase in greenhouse gas concentration due to excessive use of fossil fuels.

Of these, methane (CH4) gas is produced in a small amount compared to carbon dioxide, but its impact on global warming is 21 times higher, so it is known to be the second largest cause of global warming after carbon dioxide. This methane gas is produced by microbial fermentation such as intestinal fermentation and decomposition of manure in ponds, wetlands, rice fields, and ruminant livestock, or is also produced by natural and industrial activities such as coal, oil, natural gas, and exhaust gas. In particular, methane is generated during the normal digestion process (intestinal fermentation) in the stomach of ruminants, and methane gas emitted by livestock farming accounts for up to 115 Tg per year, accounting for 13-19% of the total methane production.

Ruminants are artiodactyls (artiodactyls) that digest plant-based foods by initially softening them in the animal's first stomach, known as the rumen, and then regurgitating the semi-digested mass, known as the cud, and chewing it again. It is a mammal of the order Artiodactyla. The process of chewing the cud again to further break up the plant material and stimulate digestion is referred to as "ruminating."

Ruminants, including cattle, buffalo, sheep, and goats, have a large potential for methanogenic fermentation to occur. The ruminal digestive tract consists of four gastric compartments, the rumen, the mesoplasty, the rumen stomach, and the mesoplasty. The largest and most important of these is the rumen. The rumen functions as a fermentation compartment. It contains a high community of microorganisms, including methanogenic archaea, which destroy plant material. The microorganisms mentioned are commonly referred to as methanogenic microorganisms. Archaeal communities use hydrogen and carbon dioxide, products of anaerobic microbial fermentation, to generate energy for growth, producing methane as the end product. Finally, methane is expelled from the rumen through belching.

Methanogenesis is the main route of hydrogen disposal during rumen fermentation (Beauchemin et al, 2008) and removal of H2 from the rumen milieu. While is essential for the efficient continuation of rumen fermentation, methane from methanogenic reactions is an important greenhouse gas contributing to global warming (Steinfeld et al, 2006) as well as dietary energy for animals. It is shown that this is the cause of loss (Johnson and Johnson, 1995). The above-mentioned subjects have led to a global search for feed additives to mitigate methane production from ruminants.

Prior art regarding compounds that reduce methane production in ruminant animals includes Korean Patent No. 10-1808957, “Feed additive composition for reducing methane production in ruminant animals containing cinigreen,” which describes methane in ruminant animals containing cinigreen. A feed additive composition for reducing methane production and a method for suppressing methane production in ruminants using the same are disclosed, and Korean Patent No. 10-1911465, “Nitrate coating composition for reducing methane gas in the rumen of ruminants and method for manufacturing the same,” discloses soybeans. It contains 100 parts by weight of protein powder, 300 to 600 parts by weight of nitrate, 200 to 550 parts by weight of oil, 80 to 200 parts by weight of stabilizer, and 400 to 600 parts by weight of distilled water. It suppresses the production of methane gas in the rumen of ruminants and uses pure nitrate. A nitrate coating composition that does not generate nitrite, a toxic substance generated by nitrite, is disclosed.

To reduce methane production, methods using chemicals such as halogen compounds, ionophore antibiotics, 2-bromoethane sulphonate, and lumazine were mainly used in the past. However, the methods proposed to date not only have a minimal effect on reducing methane gas generation, but also have a problem in that the methane suppression effect varies depending on the composition of the feed. In addition, when treated for a long time, there was a problem that the effect was not sustainable because the ruminant microorganisms adapted to the material or decomposed the material.

Therefore, there is an urgent need to develop substances effective in increasing the productivity of ruminants and at the same time reducing methane production in ruminants.

The present inventors have made extensive research efforts to develop a composition that suppresses fermentation gas production without affecting rumen nutrient digestibility and other rumen fermentation characteristics. As a result, when feeding a feed additive composition containing thiamine triphosphate, rumen digestibility decreases. The present invention was completed by confirming that it has the effect of reducing rumen fermentation gas production without affecting .

The purpose of the present invention is to provide a feed additive composition for reducing methane production in ruminant animals containing thiamine triphosphate and a method for suppressing methane production in ruminant animals using the same.

In order to achieve the above purpose,

The present invention provides a feed additive composition for reducing methane production in ruminants containing thiamine triphosphate.

In addition, the present invention provides a feed composition for reducing methane production in ruminants containing the feed additive composition containing thiamine triphosphate.

Additionally, the present invention provides a method for inhibiting methane production in ruminants, comprising feeding a feed additive composition containing thiamine triphosphate to the animal.

Feeding the feed additive composition according to the present invention to ruminants has the effect of reducing the generation of greenhouse gases that cause global warming by reducing methane generation due to fermentation in the rumen. Additionally, feeding the feed additive composition of the present invention can help productivity by reducing energy loss due to methane production in livestock and increasing practically usable energy.

Figure 1 is a diagram showing the pharmacophore of thiamine triphosphate of the present invention.

Figure 2 is a diagram showing the Fit score of thiamine triphosphate of the present invention.

Figure 3 is a diagram showing ΔG _bind of thiamine triphosphate of the present invention.

Hereinafter, the present invention will be described in detail.

The present invention provides a feed additive composition for reducing methane production in ruminants containing thiamine triphosphate.

Thiamine triphosphate of the present invention is a biomolecule found in most organisms, including bacteria, fungi, plants and animals, and chemically refers to a triphosphate derivative of the vitamin thiamine. Thiamine triphosphate plays a specific role in nerve excitability. It has been proposed but not yet confirmed, and is known to be involved in cellular energy metabolism. The relationship between the level of thiamine triphosphate and Leighs disease has been revealed, and in E. coli, thiamine triphosphate accumulates in the presence of glucose during amino acid starvation, but it is known that inhibition of the carbon source results in the accumulation of adenosine thiamine triphosphate (AThTP).

In the brain, thiamine triphosphate of the present invention is synthesized in mitochondria through a chemismotic mechanism similar to ATP synthase. It is hydrolyzed to ThDPl and can be converted to thiamine pyrophosphate (ThDP) by thiamine-diphosphate.

Thiamine triphosphate, a methane reduction compound of the present invention, can be defined by the following formula (1).

[Formula 1]

The molecular formula of thiamine triphosphate, a methane reduction compound of the present invention, is C ₁₂ H ₁₉ N ₄ O ₁₀ P ₃ .

The CAS number of the methane reduction compound thiamine triphosphate of the present invention is 3475-65-8.

The molecular weight of thiamine triphosphate, a methane reduction compound of the present invention, is 504.288.

XLop3 of thiamine triphosphate, a methane reducing compound of the present invention, is -2.6.

The hydrogen bond donor of the methane reduction compound thiamine triphosphate of the present invention is 1.

The hydrogen bond acceptor of thiamine triphosphate, a methane reduction compound of the present invention, is 14.

The number of rotational bonds of the methane reduction compound thiamine triphosphate of the present invention is 9.

The polar surface area of the molecule of thiamine triphosphate, a methane reduction compound of the present invention, is 255 Å ² .

The fit score of the methane reducing compound thiamine triphosphate of the present invention is 4.60.

The △G binding energy of thiamine triphosphate, a methane reduction compound of the present invention, is -132.39 kcal/mol.

The term "ruminant" in the present invention is a part of the vertebrate phylum Artiodactyla (herbivores with two hooves) and has ruminating characteristics, that is, a special digestive organ (stomach) capable of chewing the cud. That's why it was given the name. Ruminants are the most evolved animals among artiodactyls, the most numerous among ungulates, and the most widespread worldwide. Because rumination has a very unique digestive system, it is believed that it was able to survive even in poor vegetation conditions and thus thrive. All ruminants are divided into four chambers, including the hump stomach, honeycomb stomach, fold stomach, and fold stomach. Among these, the first and second stomachs are involved in rumination, so they are called the ruminant stomach. Spend more than 12 hours a day eating and ruminating

The stomach of a ruminant animal usually consists of four stomachs, and various types of microorganisms live within the rumen. These microorganisms effectively decompose, ferment, and digest high-fiber grass feed to supply necessary nutrients to the animal.

1st stomach (rumen): The largest of the four stomachs (150-200 liters in cows). All ingested feed is first collected here and sent directly to the 2nd to 4th chambers depending on roughness, size, specific gravity, and shape. The rough feed returned through the second stomach is lumped together and then expelled, chewed 40 to 60 times, and then swallowed again. The contents of the first stomach returned after chewing go directly to the second stomach. The cow repeats this action for 40 to 50 minutes and several times throughout the day until it is completely digested. First, there are many bacteria in the stomach that break down cellulose. Among fermentation products, carbon dioxide and methane gas are discharged out of the body through exhalation, and volatile fatty acids (VFA) and ammonia are absorbed into the blood vessels through papillae of the stomach wall. Here, the absorbed ammonia is converted into urea in the liver and then circulates and flows into the stomach through saliva and the gastric wall, where it is used to propagate microorganisms.

2nd stomach (reticulum): The inner wall is divided like a honeycomb, so it is also called a honeycomb stomach (see picture below). Among the feeds that come in from the first stomach, those of a certain size or larger are sent back to the first stomach through contraction and relaxation movements, and those of a certain size or less are continuously fermented and then sent to the third stomach.

3rd stomach - fold fold stomach (omasum, heavy stomach): The mucosal muscles are made up of numerous wrinkles, so they are named fold fold stomach and middle plate stomach. Before the digested matter from the second stomach enters the fourth stomach, the particle size of the digested matter is reduced and excess water is absorbed.

4th stomach - Abomasum: Some of the finely broken feed and microorganisms in the 3rd stomach move to the 4th stomach and are digested by gastric juice secreted from the stomach glands. The fourth stomach is also called the true stomach because it has the same function as the stomach of monogastric animals. It secretes digestive enzymes such as gastric acid, pepsin, and renin.

The ruminant animal of the present invention may be a cow, goat, bull, buffalo, bison, deer, moose, camel, or sheep, but is not limited thereto.

The ruminant animal of the present invention may be Korean beef.

The term "Korean beef" in the present invention refers to a native breed of cattle that has been traditionally raised on the Korean Peninsula for transportation or agricultural purposes.

The term "feed additive" of the present invention is used for various effects such as improving productivity or promoting growth, supplementing nutrients and preventing weight loss, improving digestibility of fiber in feed, improving oil quality, preventing reproductive disorders and improving conception rate, and preventing high temperature stress in the summer. It can be defined as a non-nutrient auxiliary material mixed in small amounts in feed. Amino acids, vitamins, antibiotics, probiotics, enzymes, organic acids, flavoring agents, sweeteners, antioxidants, various natural substances and functional substances can be classified as feed additives. You can.

The feed additive composition of the present invention may contain thiamine triphosphate at a concentration of 50 to 150 ppm, preferably 60 to 120 ppm.

The feed additive composition of the present invention includes sodium sulfite, methyl paraoxybenzoate, propyl paraoxybenzoate, propylene glycol, polyethylene glycol, ethanol, polysorbate, castor oil, benzyl alcohol, triethanolamine, glycerin, lactic acid, povidone, and dimethyl sulfoxide. , it may further include one or more excipients selected from the group consisting of N-methylpyrrolidone and sodium benzoate.

The feed additive composition of the present invention may further include purified water.

The feed additive composition of the present invention may be a formulation selected from the group consisting of liquid and tablets, but is not limited thereto.

The feed additive of the present invention can be manufactured by adding organic copper in an appropriate effective concentration range according to various feed manufacturing methods known in the art.

In embodiments of the invention, thiamine triphosphate inhibits methane gas production without affecting dry matter digestibility.

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

In the present invention, in measuring the amount of methane generated, methane (%) of the total gas amount represents the content of methane in the total amount of gas generated from Korean beef, and mL is the methane content converted to mL. In addition, mL/dry matter (g) is the conversion of methane (mL) generated from Korean beef into the amount generated per feed (dry matter basis), and mL/digested dry matter (g) is the methane (mL) generated from Korean beef converted to the amount generated per feed (dry matter basis). Standard) Converted to the amount generated per digested sugar.

In the present invention, dry matter digestibility (%) represents the digestibility of feed (dry matter basis) in the rumen of Korean cattle.

In addition, the present invention provides a feed composition for reducing methane production in ruminants containing the feed additive composition containing thiamine triphosphate.

The term "feed" in the present invention refers to something that is nutritious to livestock, animals, fish, etc. or necessary for health maintenance or growth, such as sweet feed, compound feed, supplementary feed, and concentrated feed.

The term “compounded feed” means a product in which single feed, supplementary feed, etc. are mixed or processed in an appropriate ratio.

The above “single feed” refers to a vegetable, animal or mineral substance that is used directly as feed or as a raw material for compound feed.

The above “supplementary feed” refers to what is added to feed to prevent deterioration of feed quality or increase the utility of feed.

The combined feed of the present invention may include, but is not limited to, corn, soybean hulls, wheat bran, corn gluten feed, soybean meal, lupine seeds, vitamins, salt, limestone, sodium bicarbonate, and rice straw silage.

Additionally, the present invention provides a method for inhibiting methane production in ruminants, comprising feeding a feed additive composition containing thiamine triphosphate to the animal.

The thiamine triphosphate may be fed at a concentration of 50 to 150 ppm, and preferably at a concentration of 60 to 120 ppm.

The cow may be Korean beef, but is not limited thereto.

In a specific example, the present inventors added a feed additive containing thiamine triphosphate at a concentration of 60 or 120 ppm, cultured the rumen fluid in an in vitro analysis for 24 hours, and measured the total gas in the rumen fluid culture and the amount of methane generated in the total amount of gas. and the effect on dry matter digestibility were measured, and as a result, it was confirmed that the amount of methane gas generated was reduced in the 60 or 120 ppm thiamine triphosphate treatment group without a decrease in dry matter digestibility compared to the control group, thereby completing the present invention.

Hereinafter, the present invention will be described in detail through examples.

However, the following examples illustrate the present invention, and the scope of the present invention is not limited thereto, and various modifications and improvements made by those skilled in the art using the basic concept of the present invention defined in the claims are also included in the scope of the present invention. belongs to

<Example 1>

By searching for compounds similar to 3-NOP (3-Nitrooxypropanol, C ₃ H ₇ NO ₄ ), which has previously been reported to have a methane reduction effect, we searched for compounds that interfere with the methane production process.

The molecular formula is C ₁₂ H ₁₉ N ₄ O ₁₀ P ₃ , the molecular weight is 504.288, the CAS number is 3475-65-8, XLop3 is -2.6, the hydrogen bond donor is 1, and the hydrogen bond acceptor is 14. Thiamine triphosphate was discovered, with a rotational bond number of 9 and a polar surface area of 255Å ² (Figure 1).

The fit score and ΔG binding energy of the methane reducing compound thiamine triphosphate of the present invention are shown in Figures 2 and 3.

<Experimental Example 1> Ruminal fluid collection and sample culture

Rumen fluid was collected from 3 Korean cattle (rice straw/concentrated feed ratio = 2:8, live weight 736 kg) equipped with a cannula after removing feed particles with 8 layers of gauze. The collected rumen fluid was mixed with sterilized McDougall buffer (Table 1) at a ratio of 1:4, stirred and maintained in an anaerobic state at 39°C, and 0.5 g of substrate (rice straw/concentrated feed) was added to a serum bottle for in vitro experiments. Ratio = 2:8) and a filter bag containing thiamine triphosphate (60 ppm or 120 ppm) of Example 1 was placed, 50 mL of rumen fluid (culture fluid) mixed with buffer was dispensed, and cultured for 24 hours.

composition Volume (g/L) NaHCO ₃ 9.80 NaHPO ₄ ·12H ₂ O 9.30 NaCl 0.47 KCl 0.57 CaCl ₂ anhyd. 0.04 MgCl ₂ anhyd. 0.06

McDougall buffer mixing ratio (McDougall, 1948) After the culture, the amount of gas generated was measured, the filter bag was taken out and used for digestion rate analysis, and the culture medium was used for fermentation characteristics analysis. The roughage used in the experiment was rice straw, and the concentrated feed was manufactured and used. The mixing ratio of the mixed feed is shown in Table 2 below, and the nutritional components of the concentrated feed and rice straw are shown in Table 3.

type %DM crushed corn 58.1 soybean husk 16.6 Corn Gluten Feed 11.7 wheat winter 5.8 soybean meal 3.5 lupine 2.3 limestone 0.8 sodium bicarbonate 0.6 salt 0.4 Vitamin & Mineral Mixture 0.2

Concentrated feed rice straw building, % 87.8 69.1 Crude protein, %DM 18.9 4.76 Crude fat, %DM 3.38 0.72 Non-fibrous carbohydrates, % DM 47.1 - Neutral fat-insoluble fiber, % DM 25.1 43.9 Acidic fat-insoluble fiber, % DM 10.8 29.1 Views, % DM 5.51 7.07

<Experimental Example 2> Analysis of rumen gas generation The amount of gas generated (mL) was measured using a 50 mL syringe (H13.SYS050, DH, South Korea) in a serum bottle where culture was completed. The gas collected in the syringe was transferred to a vacuum tube (BD 367953, 8.5 mL) and analyzed by GC (gas chromatography, NL/450 GC, Bruker, USA). 1.0 mL of gas was injected into the GC using a gas-tight syringe, and 22.4 μM, 63.5 μM, and 242.2 μM methane were used as standard gases.

The GC analysis conditions for methane production analysis were oven temperature at 90℃, injector temperature at 260℃, split ratio at 10, total flow at 55.8ml/min, column flow at 5ml/min, and helium was used as the mobile phase. , the column was analyzed using GS-GasPro 113-4332 (Agilent, 30 m×0.32 mm, -80~260°C).

As a result of the experiment, when thiamine triphosphate (60ppm and 120ppm) of Example 1 was cultured in vitro for 24 hours, the amount of methane generated (mL/digested dry matter) was significantly reduced (Table 4).

Through the above results, thiamine triphosphate could be determined as a candidate substance (medicinal crop) for reducing methane production.

<Experimental Example 3> Dry matter digestibility analysis

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 24 hours and then weighed. At this time, dry matter digestibility was calculated by comparing the existing sample weight and the dry weight after culture.

As a result of the experiment, there was no significant difference in dry matter digestibility when comparing the thiamine triphosphate (60ppm and 120ppm) treatment group of Example 1 and the control group.

The above results indicate that thiamine triphosphate has the effect of reducing methane production due to rumen fermentation without having a negative effect on dry matter digestibility.

division Addition level Methane production reduction rate ¹⁾
(Based on building fire extinguishment volume, %) Methane generation
Decrease rate significance Decrease in dry matter digestibility ²⁾ (%) Significance of reduced digestibility Positive control group (monensin) 5μM 28.4 ○ 3.1 × Positive control (3-NOP) 60ppm 0.7 × 1.2 × Thiamine triphosphate 28.5 ○ -0.2 × Positive control (3-NOP) 120 ppm 21.6 ○ -3.9 × Thiamine triphosphate 39.5 ○ 2.2 ×

Methane generation reduction rate = (control group - treatment group) / control group * 100, building digestibility reduction = (control group - treatment group) / control group * 100 (-): Methane generation amount / building digestion rate increase.

Claims (11)

A feed additive composition for reducing methane production in ruminants containing thiamine triphosphate. According to paragraph 1, The feed additive composition, wherein the thiamine triphosphate is defined by the following formula (1). [Formula 1]

According to paragraph 1, A feed additive composition, wherein the ruminant animal is a cow, goat, bull, water buffalo, bison, deer, moose, camel or sheep. According to paragraph 3, A feed additive composition, characterized in that the ruminant animal is Korean beef. According to paragraph 1, A feed additive composition, characterized in that the thiamine triphosphate is added at a concentration of 50 to 150 ppm. According to paragraph 1, A feed additive composition, characterized in that the composition does not affect the dry matter digestibility of ruminants. A feed composition for reducing methane production in ruminants containing the feed additive composition of claim 1. In clause 7, The feed composition further comprises dry matter, crude protein, crude fat, non-fiber carbohydrates, neutral fat-insoluble fiber, acidic fat-insoluble fiber, and ash. A method for inhibiting methane production in ruminants, comprising feeding the feed additive composition containing the thiamine triphosphate of claim 1 to the animal. According to clause 9, The method is characterized in that the thiamine triphosphate is fed at a concentration of 50 to 150 ppm. According to clause 9, A method, characterized in that the cow is Korean beef.

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