Composition for reducing methane production and improving nitrogen utilization efficiency of ruminant animals comprising haloacetamide as active ingredient

Abstract

The present invention relates to a composition for reducing methane production and improving nitrogen utilization efficiency of ruminant animals, comprising haloacetamide as an active ingredient. The composition for reducing methane production and improving nitrogen utilization efficiency of ruminant animals according to the present invention not only has excellent effects of reducing methane production and improving nitrogen utilization efficiency, but is also characterized by preventing a decrease in feed digestibility in a methane reduction environment. The composition for reducing methane production and improving nitrogen utilization efficiency of ruminant animals according to the present invention is expected to contribute to the commercialization of feed additives capable of significantly alleviating environmental pollution.

Description

Composition for reducing methane production and improving nitrogen utilization efficiency in ruminants, containing haloacetamide as an active ingredient

[0001] The present invention relates to a composition for reducing methane production and improving nitrogen utilization efficiency in ruminants, which comprises haloacetamide as an active ingredient.

[0002] Methane (CH4) is the second most abundant greenhouse gas, accounting for 16% of total greenhouse gas emissions, after carbon dioxide (CO2). The global warming potential of methane is 28 times greater than that of carbon dioxide. Furthermore, methane emissions from ruminants account for 13% to 19% of global methane emissions. Therefore, ruminant feeding is a major contributor to global warming, and reducing ruminant methane emissions can reduce the rate of global warming, which is crucial for reducing global greenhouse gas emissions. Furthermore, methane emissions contribute to energy loss during ruminant rearing. On average, approximately 8-12% of the energy digested in feed is wasted as methane emissions.

[0003] Methane is produced in the rumen of ruminants by methanogens, a subgroup of the Archaea. During normal rumen function, plant material is broken down and fermented by fiber-degrading microorganisms, producing primarily volatile fatty acids, ammonia, hydrogen, and carbon dioxide. Rumen methanogens primarily use hydrogen to reduce carbon dioxide to methane in a series of reactions coupled with ATP synthesis.

[0004] Various attempts to inhibit the activity of methanogens in the rumen have failed or achieved limited success due to low efficiency, poor selectivity, toxicity of compounds to the host, or increased resistance to anti-methanogen compounds. Therefore, there is a pressing need for methods and effective, selective, and safe compositions that inhibit methanogens in ruminants.

[0005] Conventional representative methane reduction agents include 3-nitrooxypropanol (3-NOP) disclosed in European Patent No. 2,654,455 (Patent Holder: DSM) and PCT International Publication No. 2020 / 212,348 (Applicant: DSM); freeze-dried Asparagopsis feed, nitrates, essential oil mixtures, saponins, and tannins disclosed in U.S. Patent Nos. 9,980,995, 1,088,1697, and 1,188,3449. 3-NOP has a methane reduction rate of approximately 20-40%, but has safety issues and persistence in animals. Freeze-dried Asparagopsis (especially Asparagopsis taxiformis) feeds are not only unpalatable to ruminants but also have toxicity issues (e.g., bromoform contained in Asparagopsis taxiformis is a carcinogen). Nitrates are toxic due to the nitrite produced during the reduction process. Essential oil mixtures have low methane reduction efficiency, and saponins are not only toxic to ruminants but also have low methane reduction efficiency. Tannins, when fed at high concentrations, not only reduce intake and dry matter digestibility, but also have low methane reduction efficiency.

[0006] Meanwhile, ruminants have a lower nitrogen utilization efficiency (approximately 15-40%) than other domestic animals. This is because a significant portion of the crude protein supplied as feed is excreted in manure, which can have a negative impact on the environment. There is a need to develop methane-reducing agents that can be practically applied to ruminants and improve their nitrogen utilization efficiency. Protease inhibitors have been reported to significantly affect in vitro rumen fermentation and to be effective in suppressing rumen microbial communities, particularly rumen protozoa.

[0007] After extensive research, the present inventors have completed an invention regarding a composition for reducing methane production and improving nitrogen utilization efficiency in ruminants, and expect that this will contribute to the commercialization of feed additives that can drastically alleviate environmental pollution.

[0008] The purpose of the present invention is to provide a composition for reducing methane production and improving nitrogen utilization efficiency in ruminants, which contains haloacetamide as an active ingredient.

[0009] The present invention provides a composition for reducing methane production and improving nitrogen utilization efficiency in ruminants, which contains haloacetamide as an active ingredient.

[0010] In the composition for reducing methane production and improving nitrogen utilization efficiency in ruminants according to the present invention, it is preferable that the haloacetamide is chloroacetamide, iodoacetamide, or a combination of chloroacetamide and iodoamide.

[0011] In the composition for reducing methane production and improving nitrogen utilization efficiency in ruminants according to the present invention, haloacetamide is characterized in that it significantly reduces the copy number of 16S rRNA of methanogens, thereby reducing methane production in ruminants.

[0012] The mechanism of action for reducing methane production is 1) nickel (Ni)-containing coenzyme F as a prosthetic group. 430 1) electrophilic attack of halogen compounds on (tetrapyrrole) (see Fig. 8); 2) inhibition of transfer of methyl groups to coenzyme M (see Fig. 9); and 3) inhibition of heterodisulfide bond formation between methyl-coenzyme M and coenzyme B (see Fig. 10).

[0013] In the composition for reducing methane production and improving nitrogen utilization efficiency in ruminants according to the present invention, the haloacetamide is characterized in that it improves nitrogen utilization efficiency and prevents a decrease in feed digestibility in a methane reduction environment by increasing Fibrobacter succinogenes, Ruminobacter amylophilus, and Succinivibrio dextrinosolvens. Fibrobacter succinogenes, Ruminobacter amylophilus, and Succinivibrio dextrinosolvens are the main taxonomic drivers of haloacetamide-related functions related to various metabolic activities and microbial protein synthesis of rumen microbiota. This can explain the increased nitrogen utilization efficiency of the haloacetamide-treated ruminal microbiome and the feed digestibility similar to that of the control group (the group without haloacetamide addition).

[0014] In addition, in the composition for reducing methane production and improving nitrogen utilization efficiency in ruminants according to the present invention, chloroacetamide is characterized by increasing bacteria of the genus Fibrobacter, the genus Anaeroplasma, the genus Treponema, and the genus Succinivibrio, as well as the bacterium Ruminococcus albus.

[0015] In addition, in the composition for reducing methane production and improving nitrogen utilization efficiency in ruminants according to the present invention, iodoacetamide is characterized by increasing bacteria of the genus Fibrobacter, the genus Pseudobutyrivibrio, and the genus Treponema, and the bacterium Ruminococcus flavefaciens.

[0016] In addition, in the composition for reducing methane production and improving nitrogen utilization efficiency in ruminants according to the present invention, the combination of chloroacetamide and iodoacetamide is characterized in that it reduces bacteria of the genus Saccharofermentans and the genus Limimorpha, and increases Fibrobacter succinogenes, Prevotella rumincola, and Anaerovibrio lipolyticus.

[0017] The present invention provides a feed additive comprising a composition for reducing methane production and improving nitrogen utilization efficiency in ruminants.

[0018] The composition for reducing methane production and improving nitrogen utilization efficiency in ruminants according to the present invention is characterized by not only excellent methane production reduction and nitrogen utilization efficiency improvement effects, but also preventing a decrease in feed digestibility in a methane reduction environment.

[0019] It is expected that the composition for reducing methane production and improving nitrogen utilization efficiency in ruminants according to the present invention will contribute to the commercialization of feed additives that can drastically alleviate environmental pollution.

[0020] Figure 1 shows a non-metric multi-dimensional scaling (NMDS) plot based on the Bray-Curtis distance matrix and pairwise Q values ​​based on the Bonferroni correction for statistical significance between groups.

[0021] Figure 2 characterizes taxonomic drivers of the enriched MetaCyc pathway in the haloacetamide-associated rumen microbiome using Fish Taco.

[0022] Figure 3 shows the genus and species of differentially abundant microorganisms by haloacetamide treatment.

[0023] Figure 4 is about the mechanism of action of reducing methane production, nickel (Ni)-containing coenzyme F as a prosthetic group. 430 This shows the electrophilic attack of halogen compounds on (tetrapyrrole).

[0024] Figure 5 shows the mechanism of action for reducing methane production, which involves inhibition of the transfer of methyl groups to coenzyme M.

[0025] Figure 6 shows the mechanism of action for reducing methane production, which is inhibition of heterodisulfide bond formation between methyl-coenzyme M and coenzyme B.

[0026] The present invention is described in detail and illustratively through the following examples. However, the scope of the present invention should not be construed as being limited by the following examples.

[0027] Experimental method

[0028] Experimental design and in vitro rumen fluid incubation

[0029] Rumen fluid was sampled from Hanwoo cows at the Nonghyup Experimental Ranch in Anseong-si, Gyeonggi-do, and mixed with buffer at a ratio of 1:2 within 1 hour to prepare a buffer. Oat hay was used as forage, and pelleted feed for fattening cattle was used as a concentrate. After grinding, the feed passed through a 1 mm screen and mixed at a ratio of 1:1 were used. 50 ml of culture solution was dispensed onto 0.5 g of feed dry matter, and the control group and five experimental groups (acrylamide, chloroacetamide, glycolamide, iodoamide, and propionamide) were cultured for 24 hours, repeating five times each. The control group received 50 ml of culture solution, and the experimental groups received 49.9 ml of culture solution + 0.1 ml of deionized water solution. For the five experimental groups, the addition levels of amides were based on 0.2 mmol / L (final concentration), and acrylamide was 1.4216 mg / g, 2-chloroacetamide was 1.8702 mg / g, propionamide was 1.4618 mg / g, glycolamide was 1.5014 mg / g, and iodoacetamide was 3.6992 mg / g.

[0030] Sample collection and analysis

[0031] Gas pressure was measured at 0, 3, 6, 12, and 24 h (gas collection intervals), and the gas emitted during the measurement was captured to determine the total gas collected and the amount of methane produced up to the final 24 h. After 24 h of incubation, pH, ammonia nitrogen (NH3-N), dry matter digestibility, neutral detergent fiber digestibility, volatile fatty acids (VFA), and microbiome analyses were performed.

[0032] DNA extraction, qPCR, and metataxonomic analysis

[0033] After extracting metagenomic DNA using the RBB+C method, qPCR analysis and microbiome analysis were performed. Microbial functions were predicted using PICRUSt2. Taxonomic drivers of differentially abundant MetaCyc pathways were identified using FishTaco (Functional Shifts' Taxonomic Contributor).

[0034] Statistical analysis

[0035] Data that followed normal distribution and homogeneity of variance were analyzed using the Glimmix method in SAS and the Turkey's HSD test. Data that did not show normality or homogeneity of variance were analyzed using the nonparametric Kruskal-Wallis H test followed by Dunn's Post-hoc in R (a post-hoc analysis: to determine which group differences exist after confirming a significant difference between groups through ANOVA). Differences in the overall bacteriota and their functional profiles were analyzed using PERMANOVA. Differentially abundant microbial biomarkers were identified using multivariable association using the MaAsLin2 R package from the relatively abundant bacterial taxa and predicted microbial functions.

[0036] Experimental results

[0037] Ruminant fermentation characteristics

[0038] In vitro digestibility and rumen fermentation characteristics are presented in Table 1 below.

[0039]

[0040] In the above table, the terms are defined as follows:

[0041] CON: control

[0042] AA: Acrylamide

[0043] CLA: chloroacetamide

[0044] GA: glycolamide

[0045] IA: Iodoacetamide

[0046] PA: propionamide

[0047] DMD: dry matter digestibility

[0048] NDFD: Neutral Detergent Fiber Digestibility

[0049] NH3-N: Ammonia nitrogen

[0050] TGP: total gas production

[0051] CH4(mL / g dDM): methane production (mL) per gram (g) of degraded dry matter

[0052] Total VFA: Total volatile fatty acid

[0053] A:P ratio: acetate:propionate ratio

[0054] SEM: standard error of the mean

[0055] As shown in Table 1 above, it was confirmed that haloacetamides such as chloroacetamide and iodoacetamide have an excellent effect in reducing methane production.

[0056] Volatile fatty acid (VFA) profile

[0057] The volatile fatty acid (VFA) profiles for the control group and five experimental groups (acrylamide, chloroacetamide, glycolamide, iodoamide, and propionamide) are presented in Table 2 below.

[0058]

[0059] Quantitative real-time PCR data

[0060] For the control and five experimental groups (acrylamide, chloroacetamide, glycolamide, iodoamide and propionamide), the effect of amide compounds on the absolute quantification of total bacteria, methanogens, protozoa and specific cellulolytic bacterial species (Fibrobacter succinogenes, Ruminococcus flavefaciens, Ruminococcus albus and Prevotella ruminicola) is presented in Table 3 as quantitative real-time PCR data.

[0061]

[0062] Measuring alpha diversity

[0063] The alpha diversity measurements for the control group and five experimental groups (acrylamide, chloroacetamide, glycolamide, iodoamide, and propionamide) are presented in Table 4 below.

[0064]

[0065] Measuring beta diversity

[0066] The results of beta diversity measurements for the control group and five experimental groups (acrylamide, chloroacetamide, glycolamide, iodoamide, and propionamide) are shown in Figure 1.

[0067] Differentially abundant genera and species by haloacetamide treatment

[0068] Differentially abundant genera and species by haloacetamide treatment are shown in Fig. 3.

Claims

1. A composition for reducing methane production and improving nitrogen utilization efficiency in ruminants, comprising haloacetamide as an active ingredient.

2. In paragraph 1, A composition for reducing methane production and improving nitrogen utilization efficiency in ruminants, characterized in that the haloacetamide is chloroacetamide, iodoacetamide, or a combination of chloroacetamide and iodoamide.

3. In paragraph 1, A composition for reducing methane production and improving nitrogen utilization efficiency in ruminants, characterized in that the above haloacetamide significantly reduces the copy number of 16S rRNA of methanogens, thereby reducing methane production in ruminants.

4. In paragraph 1, A composition for reducing methane production and improving nitrogen utilization efficiency in ruminants, characterized in that the haloacetamide increases Fibrobacter succinogenes, Ruminobacter amylophilus and Succinivibrio dextrinosolvens, thereby preventing a decrease in nitrogen utilization efficiency and feed digestibility in a methane reduction environment.

5. In paragraph 2, The above chloroacetamide is a composition for reducing methane production and improving nitrogen utilization efficiency in ruminants, characterized in that it increases bacteria of the genus Fibrobacter, the genus Anaeroplasma, the genus Treponema, and the genus Succinivibrio, and the bacterium Ruminococcus albus.

6. In paragraph 2, The above iodoacetamide is a composition for reducing methane production and improving nitrogen utilization efficiency in ruminants, characterized in that it increases bacteria of the genus Fibrobacter, the genus Pseudobutyrivibrio, and the genus Treponema, and the bacterium Ruminococcus flavefaciens.

7. In paragraph 2, A composition for reducing methane production and improving nitrogen utilization efficiency in ruminants, characterized in that the combination of chloroacetamide and iodoacetamide increases bacteria of the genus Saccharofermentans and the genus Limimorpha, as well as Fibrobacter succinogenes, Provetella rumincola, and Anaerovibrio lipolyticus.

8. A feed additive comprising a composition for reducing methane production and improving nitrogen utilization efficiency in ruminants according to any one of claims 1 to 7.

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