KR102598346B1 - Feed additive for reducing methane gas in rumen, and feed composition for ruminant containing same - Google Patents

Abstract

The present invention relates to a feed additive for reducing methane gas in the rumen and a feed composition for ruminants containing the same. More specifically, a feed additive for reducing methane gas in the rumen, comprising garlic extract and attapulgite, and It relates to a feed composition for ruminants containing a feed additive for reducing methane gas in the rumen.
The feed additive for reducing methane gas in the rumen of the present invention and the feed composition for ruminants containing the same have the effect of suppressing the production of methane gas in the rumen and solving atmospheric environmental problems by reducing greenhouse gas effects . In addition, the feed additive of the invention and the feed composition for ruminants containing the same reduce methane gas in the rumen, thereby increasing feed efficiency of feed energy, so that most of the feed energy can be used for the growth of ruminants.

Description

Natural extract feed additive for reducing methane gas emitted by ruminants and feed composition containing the same {FEED ADDITIVE FOR REDUCING METHANE GAS IN RUMEN, AND FEED COMPOSITION FOR RUMINANT CONTAINING SAME}

The present invention relates to a feed additive for ruminants and a feed composition containing the same. Specifically, it relates to a feed additive for reducing methane gas in the rumen and a feed composition for ruminants containing the same.

Due to the influence of international industrialization, greenhouse gas emissions are continuously increasing, causing serious global warming. The world's temperature has risen by about 1 degree over the past 100 years, and the rate of increase is expected to accelerate by 1.5 degrees before 2040 if the current trend continues. In particular, methane is known to produce a greenhouse effect 25 to 30 times greater than that of carbon dioxide [IPCC, 2001].

Accordingly, in 1992, an international consultative body was formed mainly in developed countries to respond to global warming, and specific action plans and efforts are continuing to reduce greenhouse gas emissions. 121 countries joined the Alliance to Raise Climate Goals, and at the Climate Summit held in April 2021, the US, EU, UK, Japan, etc. announced raised national greenhouse gas reduction goals in line with the 2050 carbon neutrality goal.

Korea has also established a 2050 carbon neutral scenario and aims to reduce net emissions to zero by matching carbon emissions and carbon absorption. To this end, we aim to achieve the assigned goals in each industry sector, and specifically, we aim to account for 37.7% of the carbon emissions in the agricultural, livestock and fisheries sector from 24.7 million tons to 15.4 million tons in 2018, of which the amount is due to enteric fermentation of livestock. The methane produced will increase from 4.5 million tons in 2018 to 2.8 million tons in 2050, with the goal of reducing about 1.7 million tons of methane in the rumen.

Methanogenic bacteria, which produce methane in the rumen, are taxonomically microorganisms belonging to archaea, which are fundamentally different from general bacteria (b acteria). Bacteria have a peptidoglycan layer, and archaea have a pseudopeptidoglycan layer, and the enzyme systems involved in metabolism and the components of the cell wall are very different from ordinary bacteria. According to previous studies, methanogenic archaea are composed of three taxa, and in the case of dairy cattle, they are classified into the genus Methanobrevibacter , Methanomesilicoccus, and Methanospiera (Korea Institute of Engineering and Life Science et al. ., 2017)

In this way, methane generated from livestock not only affects global warming, but the production and emission of methane gas through intestinal fermentation is a loss of about 2 to 12% of the total energy that ruminants obtain from feed, so the production of methane gas causes This may result in reduced feed use efficiency and reduced productivity (Johnson et al., 1991; Ha et al., 2005). In other words, reducing methane emissions from ruminants can kill two birds with one stone by improving the environmental aspect of slowing global warming and the nutritional aspect of increasing feed efficiency of feed energy (Lee and Ha, 2009).

The development of technology to reduce methane production through enteric fermentation is focused around the world on two directions: “additive development” and “feed development,” but continuous field application technology has not yet been developed. It can be broadly divided into the four methods below. It can be summarized.

① Method of directly suppressing methane-producing microorganisms that cause methane gas production

② A method of indirectly suppressing methane-producing bacteria by preventing the generation of hydrogen, which causes methane gas.

③ A method of indirectly suppressing methane-producing bacteria by promoting other methods of consuming hydrogen, such as propionate fermentation, in addition to methane fermentation.

④ There is a way to reduce methane bacteria activity by changing the community (diversity) of rumen microorganisms.

Methane-reducing feed additives developed to date are based on chemical products and single substances such as seaweed, nitrate, 3-nop, and antibiotics, and suppress methane generation through simple feeding. However, direct feeding of chemical products or antibiotics has the problem of causing toxicity to ruminant animals, remaining in secondary production materials or causing resistance problems, thereby interfering with basic physiology. Feeding a single substance may be effective in reducing methane, but it is believed that it causes problems by changing the nature of fermentation in the ruminant stomach and disrupting the physiological balance of cattle, so a complex feed additive that can offset the palatability problem and physiological problems of ruminants is recommended. We wanted to develop it, and it is expected that the development technology will help improve palatability and maintain the balance of fermentation characteristics. Therefore, there is a need for a material that suppresses the production of methane, a type of greenhouse gas, in the rumen of ruminants and does not harm humans or animals.

Prior art literature

patent literature

(Patent Document 001) Republic of Korea Patent Application No. 2021-0189023

(Patent Document 002) Republic of Korea Patent Application No. 2019-0177357

(Patent Document 003) Republic of Korea Patent Application No. 2018-0061694

(Patent Document 004) Republic of Korea Patent Application No. 2017-0101990

The purpose of the present invention is to provide a feed additive for reducing methane gas in the rumen of ruminants containing natural substances.

In addition, another object of the present invention is to provide a feed composition for ruminants containing the feed additive for reducing methane gas in the rumen of ruminants.

In order to achieve the above object, the present invention provides a feed additive for reducing methane gas in the rumen, which contains garlic extract and attapulgite as natural substances.

Preferably, the feed additive for reducing methane gas in the rumen may further include one or more of seaweed meal, green tea meal, linseed, and persimmon peel.

Preferably, the weight ratio of the garlic extract and attapulgite in the feed additive may be 0.5 to 5.5:0.5 to 5.5.

Preferably, in the feed additive, the weight ratio of the garlic extract, attapulgite, and one or more of seaweed meal, green tea meal, linseed, and persimmon may be 0.5 to 5.5:0.5 to 5.5:0.5 to 5.5.

In addition, in order to achieve the above-described other objects, the present invention provides a feed composition for ruminants, including a feed additive for reducing methane gas in the rumen, including garlic extract and attapulgite.

Preferably, the feed additive for reducing methane gas in the rumen may further include one or more of seaweed meal, green tea meal, linseed, and persimmon peel.

Preferably, based on 100% by weight of the feed composition, the amounts of the garlic extract and attapulgite may each be 0.5 to 5.5% by weight.

Preferably, based on 100% by weight of the feed composition, the amount of the garlic extract may be 0.5 to 2.5% by weight, and the amount of attapulgite may be 2.5 to 5.5% by weight.

Preferably, based on 100% by weight of the feed composition, the amount of the garlic extract may be 2.5% by weight, and the amount of the attapulgite may be 5.5% by weight.

Preferably, based on 100% by weight of the feed composition, the amount of one or more of the seaweed meal, green tea meal, linseed, and persimmon may be 0.5 to 5.5% by weight.

The feed additive for reducing methane gas in the rumen of the present invention and the feed composition for ruminants containing the same have the effect of slowing global warming by suppressing the production of methane gas in the rumen.

In particular, the large amount of methane gas generated in the rumen means that part of the energy of the fed feed is released into the atmosphere as methane gas, so if the amount of methane gas generated is large, the productivity of ruminant animals also decreases. However, the feed additive of the present invention and the feed composition for ruminants containing the same reduce methane gas in the rumen, thereby increasing feed efficiency of feed energy, so that most of the feed energy can be used for the growth of ruminants.

Figure 1 is a graph showing the efficacy of allicin on methane production (mL/g DM).
Figure 2 is a graph showing the efficacy of attapulgite on methane production (mL/g DM).
Figure 3a is a schematic diagram of a laser methane detector (kyewon kang et al. 2020), a methane measurement device used in the present invention.
Figure 3b is a graph showing the results of measurements for 6 minutes at 0.5 second intervals using a methane measuring device.
Figure 4 is a graph showing the amount of methane emissions from animals in the control and experimental groups over 4 weeks.

In one aspect, the present invention provides a feed additive for reducing methane gas in the rumen, including the natural substances allicin and attapulgite.

Typically, ruminants are animals belonging to the order Artiodactyla/Arminozoa, have a rumen, and have the characteristic of regurgitating and chewing food once swallowed. Also called ruminating animals, they include animals of the ruminant and camelid orders and may be selected from the group consisting of cattle, sheep, goats, buffalo, deer, camelids, giraffids, antelopes, bison and yaks. You can.

Allicin, a natural component of the feed additive of the present invention, is a component contained in garlic or onions and is a type of aryl sulfide, a sulfur compound with a strong odor. Generally, foods containing allicin include garlic, onions, green onions, and chives. Allicin inhibits coenzyme activity or has antimicrobial effects. There are two mechanisms for antibacterial activity: Garlic produces active oxygen, which causes cells to die from oxidative stress, or it reacts with intracellular -SH substances that protect cells through redox buffering, destroying the cell membrane. It can damage the structure and induce apoptosis. It quickly penetrates the cell membrane and binds to the thiol protein of microorganisms to inhibit metabolism, and the S-allyl part (S-CH 2 -CHCH 2) of allicin _reacts with _the -SH group of the enzyme protein essential for metabolism and becomes inactive. . Except for certain bacteria, antibacterial activity cannot be expected, and methane-producing gram-positive bacteria (Methanobacterium) exhibit antibacterial activity through a more direct effect than gram-negative bacteria [Ziletao Tao., 2020; Ziletao Tao.,2020]. In this way, the antimicrobial action of the complex feed additive can directly control methanogenic bacteria that produce methane.

In addition, attapulgite, another natural component of the feed additive of the present invention, is a mineral with the highest porosity among minerals on Earth and has an adsorption capacity, and is manufactured by fractionating sedimentary loess from the lower reaches of rivers. A clay mineral used as a supplement in animal feed. It has a unique structure compared to other clays and is composed of 2:1 ribbons of phyllosilicate, each ribbon being an inversion of a SiO ₄ tetrahedron along a set of Si-O-Si bonds. connected to the other by This structure results in a fibrous form and has high viscosity and adsorption capacity to bind toxins. Common effects include: It has natural diarrhea-preventing properties in both humans and animals, reduces inflammation, and improves antioxidant capacity. Attapulgite directly adsorbs and reduces methane gas and simultaneously releases Ca ²⁺ , Mg ²⁺ , K ⁺ , and Fe ³⁺ metal ions, which can reduce enzyme activity in the hydrolysis and methane production stages.

The weight ratio of allicin and attapulgite in the feed additive of the present invention may be 0.5 to 5.5:0.5 to 5.5, preferably 0.5 to 2.5:2.5 to 5.5, and more preferably 2.5:5.5. there is. If it is outside the above range, the effect of reducing methane gas in the rumen is reduced, and the best effect of the present invention cannot be obtained.

In addition to allicin and attapulgite, the feed additive of the present invention may further include one or more of seaweed meal, green tea meal, linseed, and gampi.

Seaweed meal can be obtained by washing general seaweed raw materials to remove contaminants and impurities, removing salt and moisture through soaking and pressing, securing inedible parts through cutting and sorting, and artificially drying and crushing them. Contains halogenated compounds such as bromoform.

In addition, green tea peel, linseed, and persimmon are commonly used and can be obtained by appropriately processing and washing raw materials obtained from nature, or they can be obtained commercially.

When containing such additional ingredients, the weight ratio of allicin, attapulgite, and one or more of seaweed meal, green tea meal, linseed, and persimmon is preferably 0.5 to 5.5:0.5 to 5.5:0.5 to 5.5, and the weight ratio outside the above range is preferably In this case, the effect of reducing methane gas in the rumen is low, and the best effect of the present invention cannot be obtained.

As can be seen through the examples and experimental examples below, the feed additive for reducing methane gas in the rumen complements the problems of single substances by combining natural substances in optimal ratios and exhibits an excellent methane reduction effect in the rumen. You can.

In addition, in another aspect, the present invention provides a feed composition for ruminants, including a feed additive for reducing methane gas in the rumen, including allicin and attapulgite.

In the present invention, the feed composition for ruminants can be any commonly used feed composition for ruminants. For example, it may be a feed composition obtained by mixing roughage, concentrated feed, supplementary feed, compound feed, etc. in an appropriate ratio, The present invention is not limited to its specific components and ratios.

The amounts of allicin and attapulgite, which are components of the feed composition for ruminants of the present invention, may each be 0.5 to 5.5% by weight based on 100% by weight of the feed composition of the present invention, preferably, based on 100% by weight of the feed composition. The amount of allicin may be 0.5 to 2.5% by weight, the amount of attapulgite may be 2.5 to 5.5% by weight, and more preferably, the amount of allicin may be 2.5% by weight based on 100% by weight of the feed composition. The amount of attapulgite may be 5.5% by weight.

When allicin and attapulgite are included in the above range, it can exhibit the best effect of reducing methane gas in the rumen, and when it is outside the above range, this effect is significantly reduced.

In addition, the feed additive for reducing methane gas in the rumen may further include one or more of seaweed meal, green tea meal, linseed, and persimmon peel in addition to allicin and attapulgite, and preferably, 100% by weight of the feed composition. It may additionally include one or more of seaweed meal, green tea meal, linseed, and gampi in an amount of 0.5 to 5.5% by weight.

If it is within the above range, it can exhibit a synergistic effect to show the best methane gas reduction effect in the rumen and can have an excellent effect in terms of nutrition for ruminants, but if it is outside the above range, this effect is greatly reduced. represents.

As described in Experimental Example 4 below, the feed composition for ruminants of the present invention showed that the difference between the additive treatment group and the control group was statistically significant when respiration and belching were calculated separately among the amount of methane generated in the rumen (P <0.05) This can be interpreted that the complex additive of attapulgite and allicin inhibits anti-methanogen and methanogenic enzymes. More specifically, as shown in Figure 4 and Table 19, the methane production amount from cow's burp (ppm/m) is 40.79% in the first week > 32.80% in the second week > 45.83% in the third week, and the methane production amount in respiration is > 13.82% in the first week. It was verified that it decreased from 39.62% in the 2nd week to 57.72% in the 3rd week.

Depending on the amount of methane production over time, respiration tended to decrease in methane in the first week and significantly decreased from the second week (p<0.05), and burping decreased from the first week, increasing the feeding period. It was confirmed that the methane reduction effect was greater (p<0.001). In other words, compared to the control group, all treatment groups showed excellent results in reducing methane production.

Hereinafter, preferred embodiments are presented to aid understanding of the present invention. However, the following examples are merely illustrative of the present invention, and it is clear to those skilled in the art that various changes and modifications are possible within the scope and spirit of the present invention. It is natural that such variations and modifications fall within the scope of the attached patent claims.

<Example> Preparation of feed composition for reducing methane gas

item Ingredients (based on 100% by weight of feed composition for methane gas reduction) Example 1 Allicin (5%) Example 2 Attapulgite (5%) Example 3 Seaweed meal (5%) Example 4 Allicin (2.5%) + Attapulgite (2.5%) Example 5 Allicin (2.5%) + seaweed meal (2.5%) Example 6 Attapulgite (2.5%) + seaweed meal (2.5%) Example 7 Allicin + Attapulgite + Seaweed Meal Comparative example No added ingredients

The feed composition of the present invention was prepared by adding and mixing the ingredients listed in Table 1 to the feed composition at a total of 5% DM (Isaac A. Aboagye, 2019).

As a comparative example, a feed composition without the above ingredients was used.

<Experimental Example 1> Verification of methane reduction effect of ingredients of the present invention

1. Sample preparation

go. order of experiment:

1) Natural materials were ground to the same particle size (1 mm or less).

2) For DM measurement, it was dried in a drying oven.

3) Using 'Closed batch culture [McBee, 1953]', the natural candidate material was divided into 0.3 g in a nylon bag.

me. experiment material

- Nylon bag

- 300 ml bottle

all. laboratory equipment

- electronic scale

- Drying oven

2. Shaking incubation

go. order of experiment:

1) Preparation of rumen fluid

1-1) Before sampling, the materials (nylon filter, pipette 50 ml) were set at 39°C.

1-2) Cannula The rumen of two cows was sampled from three areas: rumen direction, ventral direction (ventral side), and dorsal direction (caudal side).

1-3) Rumen fluid was filtered using a 250 ㎛ nylon filter.

2) Manufacture of Menke's buffer [Menke and Steingass, 1988]

Total fluid (L) 1.2 RF 0.6 badge 0.6 menker buffer Added amount (ml) macro solution 426.6 micro solution 0.072 buffer 142.2 Resazurin 0.732 reducing agent 30

3) Rumen fluid and Mencon buffer were mixed at the specified ratio (1:1-4).

4) CO ₂ was injected and manufactured in an anaerobic state.

5) After putting 50 ml of rumination solution in each bottle, Ar gas was injected into each bottle.

6) After being sealed with a crimper, it was fermented in a shaking incubator (39°C, 120 rpm) for 24 hours.

me. experiment material

- Ruminal fluid of the tested animal

- McDougall's buffer

- Flushing gas (Ar gas, CO ₂ )

- Thermos bottle

- 20 mn rubber stopper

- Aluminum seal

- Crimper/Decapper

all. laboratory equipment

- Shaking incubator

- Magnetic stirrer

3. TGP measurement

go. order of experiment:

1) The syringe needle was connected to the pressure transducer.

2) After setting the atmospheric pressure zero point, a needle was inserted into the culture bottle and the pressure was recorded.

3) Gas was injected into a vacuum vessel to measure methane.

me. experiment material

- 24g syringe

all. laboratory equipment

- Gas converter

4. pH measurement

go. order of experiment:

1) Transferred to a 50 ml tube.

2) pH was measured and recorded.

3) Centrifugation (500 × g for 5 minutes at 4°C) was performed to measure VFA.

me. experiment material

- 50 ml tube

all. laboratory equipment

- pH meter

5. Digestibility measurement

go. order of experiment:

1) After washing the filter bag, moisture was removed with a paper towel and the weight was measured.

me. experiment material

- Nylon bag for culture bottle

all. laboratory equipment

- electronic scale

6.CH ₄ for gas sampling

go. order of experiment:

1) 1 ml of methane gas contained in a vacuum container was put into a syringe and the sample was injected into the chromatography inlet.

2) After 4 to 5 minutes, the amount of methane, carbon dioxide, and other gases were measured and recorded (peak order ETC → CH4 → CO2).

me. experiment material

- Confidential syringe

- Vacuum container

- Standard gases: H2 1.0%, CH4 10.1%, CO2 20.1% and N2 19.9% in He.

all. laboratory equipment

- Gas chromatography

▶ Experiment results

This Experimental Example 1 was conducted to determine the effect on ruminal fermentation characteristics and carbon dioxide and methane emissions by combining preparations of natural substances (allicin, attapulgite, and seaweed meal). Allicin (A) did not affect the digestibility and pH of the rumen, and it was confirmed that it reduced methane gas along with carbon dioxide emissions by about 46% (P<0.05). In addition, AB (allicin + attapulgite) showed no statistically significant change, but was confirmed to have the highest methane reduction rate (23%) after allicin.

Therefore, the amount of methane generated is allicin (4.8 ml) < allicin + attapulgite (6.85 ml) < allicin + attapulgite + seaweed meal (7.93 ml) < attapulgite (8.55 ml) < attapulgite + seaweed meal (9.02 ml) Methane emissions were confirmed in the following order: ml) < allicin + seaweed meal (10 ml) < seaweed meal (10.96 ml). The methane amount was reduced in the combinations of allicin (A), allicin + attapulgite (A + B), allicin + attapulgite + seaweed meal (A + B + C), and attapulgite (B) compared to the control group, while in other groups No significant difference was confirmed. In the case of combinations or substances outside the group, it is believed that methane emissions increased due to enhanced fermentation in the rumen due to excessive nutrients rather than a methane reduction mechanism.

<Experimental Example 2> Efficacy test of natural methane reduction additive

item Added amount B C 2.5% 0.0075g allicin
(A) attapulgite
(B) seaweed gourd
(C) green tea powder
(D) flaxseed
(E) 5% 0.015g (A) (B) (C) (D) (E) 7.5% 0.0225g (A) (B) (C) (D) (E)

The ingredients listed in Table 4 were tested for methane reduction efficacy using the method and machine of Experimental Example 1.

▶ Experiment results

1. Allicin

The results of changes in pH according to in vitro rumen fermentation characteristics are shown in Table 5 above. The appropriate pH range for digestion of rumen feed is 6.0 to 6.8 (McCullough et al., 1968). The pH range of all additives after 12 hours of fermentation was 6.55 to 6.86, slightly increased at the 7.5% addition amount, but mostly within the appropriate pH range in the rumen. Results belonging to were found.

Through later statistical analysis, When allicin was added at a 2.5% addition amount, it was confirmed that methane emissions were significantly (P<0.05) reduced, and when calculated with the TGP ratio, only methane emissions from the total gas generation decreased by 21% at a 2.5% addition amount. You can check that.

As a result of comparing the CH4 ratio (%) of the control group, the garlic extract showed a significant (p < 0.05) linear decrease as the amount added increased.

Methane emissions decreased by 21% at 2.5% addition amount and by 34% at 7.5% addition amount.

Allicin, one of the components of garlic extract, inhibits the activity of enzymes required for the methanogenesis stage, and the disulfide bond, S-S (disulfide group), binds to proteins in the outer cytoplasmic membrane, thereby preventing lumen methanogene genes without affecting lumen bacteria. It has the potential to specifically inhibit archaea.

2. Attapulgite

(pH) The results of changes in pH according to in vitro rumen fermentation characteristics are shown in Table 6 above. The appropriate pH range for digestion of rumen feed is 6.0 to 6.8 (McCullough et al., 1968). After 12 hours of fermentation, the pH range of all added groups slightly increased to 6.83, but most results were within the appropriate pH range in the rumen. .

Later, a statistical analysis was conducted as the decrease in methane emissions was large, and it was confirmed that methane emissions decreased by 3.3% when the amount was added at 2.5% and significantly (P<0.05) by 8.3% when the amount was added at 7.5%.

The statistical linearity of attapulgite is a significant result (P<0.05), and it can be predicted that the methane reduction effect increases as the amount added increases.

The results of total gas, carbon dioxide, and methane production according to the in vitro rumen fermentation characteristics of the four natural extracts are shown in Tables 7 to 10 above.

(Seaweed meal) Seaweed meal is made from seaweed by-products after extracting edible seasoning and alginic acid from seaweed, drying them, and making additives using discarded agricultural products.

Methane emissions (ml/g DM) in the seaweed meal experimental group were confirmed to have significantly decreased linear statistics, so a synergistic effect can be expected with higher addition amounts or with other methane reduction agents. In addition, considering that IVDMD digestibility has increased, it can be expected that it is also used as TMR basic energy feed (H.G.LEE et al., 2005)

(Green tea foil) First, green tea foil contains 791 mg/kg of condensed tannin, one of the ingredients that reduces methane. It is very useful as it is made from green tea by-products left after brewing, like green tea bags.

It can be seen that the methane emissions of the green tea cake experimental group were reduced by 4.7% at the 7.5% addition amount. Since the total gas generation (TGP) did not decrease, it was verified that methane generation was reduced without affecting the total gas generation as well as microorganisms in the rumen.

(Linseed) Linseed is made by grinding the original seed, so the amount of methane produced is slightly reduced. The possibility of re-experimenting with linseed as a processed and manufactured material was confirmed in the future.

(Persimmon peel) In the persimmon experimental group, IVDMD digestibility increased and TVFA (Total volatile fatty acid), which is used as energy, both increased slightly, so it can be predicted that productivity improved because persimmon was used as energy. There was a slight difference in methane emissions from the control group, but significance was not recognized.

<Experimental Example 3> Verification of natural methane reduction additive concentration (added amount)

The ingredients listed in Table 11 were tested for methane reduction efficacy using methods 1 to 5 of Experimental Example 1 and a machine.

In addition, the following experiment was additionally performed.

6. Ammonia nitrogen (NH ₃ -N)

go. order of experiment:

1) Sample preparation

① The sample prepared in the previous chapter was centrifuged at 20,000 g at 4°C for 20 minutes.

② 0.5 mL of supernatant was transported into a 1.5 mL tube.

2) Standard formulation

① Prepare 1 mL of NH ₃ -N standard solution using NH ₄ Cl.

② 0.2 mL of HgCl ₂ (wt/vol) solution and 25% meta-phosphoric acid (wt/vol) solution were added.

3) NH ₃ -N analysis

① The plate layout was designed (the layout changes to the number of samples, replications, and standards).

② After transporting 2 ㎕ of sample and standard, the analysis was repeated with the number of replicates.

③ 125 ㎕ of alkali-hypochlorite reagent and 147 ㎕ of phenol colorant were transported.

⑤ The plate was gently shaken and heated at 50 to 57°C for 3 to 7 minutes.

⑥ Turn on the spectrophotometer and software and set the wavelength to 630 nm.

⑦ The accuracy of measurement was improved by analyzing the plate more than three times.

4) Calculation

① The average OD (nm) between analyzes was calculated.

② A linear regression equation was applied based on the results of the standard solution.

③ The concentration of NH ₃ -N was calculated from the standard curve, and the average NH ₃ -N between analysis replicates was calculated.

me. experiment material

- HgCl2 1% (wt/vol) and 25% meta-phosphoric acid (wt/vol) solution

-NH4Cl

-NaOH

-NaOCl

-Pheno

- Sodium - Nitrophericinide

- Alkali-hypochlorite reagent

- Phenol Color - Reagent

all. laboratory equipment

- Gas chromatography

- Spectroscopy

- 96-well plate

7. Volatile fatty acids (VFA)

go. order of experiment:

1) Sample preparation

① The sample was centrifuged at 20,000g for 20 minutes at 4°C.

② Transfer 0.5 mL of supernatant to a 1.5 mL tube.

2) Standard preparation

① 1 mL of NH ₃ -N standard solution was prepared using NH ₄ Cl.

② 0.2 mL HgCl ₂ (wt/vol) and 25% meta-phosphoric acid (wt/vol) were added to the standard solution.

me. experiment material

-Orange wide opening screw top (write-on spot)

- Blue cap, PRFE/Red silicone rubber septum

- GC standard syringe

- Ultra-inert liner

- Green Septum

- 2% pivalic acid

- Standard VFA solution (wt/vol)

all. laboratory equipment

- Centrifuge

- Gas chromatography

(pH) The results of changes in pH according to in vitro rumen fermentation characteristics are shown in Table 12. The appropriate pH range for digestion of feed with high ruminal fiber content is 6.0 to 6.8 (McCullough et al., 1968). After 12 hours of fermentation, the pH range for all groups was 6.55 to 6.73, slightly increasing at the 2.5% addition amount, but most The results were found to be within the appropriate pH range in the rumen.

It was confirmed that methane emissions (mL/g DM) decreased by 9.2% at the addition amount of 2.5% allicin. It was verified that methane begins to decrease significantly (P<0.05) from 2.5%, and given the statistically significant linear value (p=0.01), it is predicted that methane emissions will decrease as the amount added increases.

It was confirmed that there was no change in total gas production (TGP) and no particular change in TVFA (mM) productivity. This means that it specifically targets only methanogenic microorganisms to reduce methane or inhibits only the enzymes involved in methane production, which is an encouraging result as it only reduces methane without affecting the fermentation characteristics in the rumen.

(IVDMD) As an indicator of dry matter digestibility, IVDMD was significantly higher in all experimental groups compared to the control group (P<0.05)

(CH4) Compared to total gas generation (TGP), methane emissions specifically decreased significantly (P<0.05) from 2.5%, and as the amount of attapulgite added increased, methane emissions significantly decreased linearly. (P<0.05).

(TVFA) The TVFA results according to the in vitro rumen fermentation time of attapulgite are shown in Table 13. It can be seen that attapulgite increased significantly (P<0.05) in all experimental groups compared to the control group at 12 hours of fermentation. As the acetate concentration decreases and the propionate concentration increases, it can be assumed that hydrogen is used to produce propionate, and the hydrogen required for methane synthesis decreases, resulting in a decrease in methane production.

<Experimental Example 4> Verification of changes in methane production (in vivo) in ruminants fed with the methane reduction additive of the present invention

1. Experimental design (simultaneous axis and specification management)

The research procedure and feeding management of the test animals were conducted on 10 Holstein (380±20 kg) 15-16 month old cows, and the test animals were given 6 kg of concentrated feed and 5 kg of roughage twice a day at 10:00h and 17:00h. Water was allowed to be consumed freely. The experiment period was a total of 5 weeks from October to November 2022, with 1 week of adaptation period and 4 weeks of experiment period where complex feed additives (garlic extract, attapulgite) were fed. As for the feed additive, based on previous experiments, 0.48 g of a combination of 2.5% garlic extract and 5.5% attapulgite was given based on the concentrated feed, and the concentrated feed was top-dressed and fed at 10:00 h. Body weight and methane production (week 0) were measured and grouped by individual.

2. Analysis of feed ingredients and NRC requirements

Garlic extract (allicin) and attapulgite used in the experiment were manufactured by Gaya Bio Co., Ltd. (Green up).

In the current experiment, as a result of analyzing the composition of roughage and concentrated feed, the nutrient requirements were compared based on the NRC with DMI 9.7, TDN 65.08%, and CP 9.64%. The lowest body weight at the start of the experiment was 344 kg and the highest was 420 kg, so when comparing 0.5 to 1.1 kg/day of ADG based on the NRC's daily nutrient requirement BW of 400 kg, the total daily intake of TDN was not insufficient even at 1.1 kg of ADG. Confirmed. Crude protein showed a slightly lower CP content based on ADG of 0.8 kg/day, but it can be considered that the requirement was almost met, falling short by -0.05 kg.

3. Methane measuring instrument

The methane measuring device is the LMD method using a laser methane detector (LMD, mini-G (50A), Tokyo Gas Engineering Co., Japan) (Figure 3a), which detects methane in the air by near-infrared absorption spectroscopy using a semiconductor laser. It was utilized. After fixing the blank axes to the starchion, methane emission concentration was measured using LMD. The laser emitted from the LMD at a distance of 1 m from the test animal was positioned below the animal's nose and reflected back.

Methane emission concentration was measured 4 times a day for 6 minutes at intervals of 0.5 seconds, based on morning feed feeding (±1, 2 h) for a total of 2 days, 1 week before the start of the experiment and 1, 2, and 4 weeks after the start of the experiment. The reason for measuring for 6 minutes is that burps are the main route that emits methane gas and occur irregularly every 1 to 3 minutes, so by measuring for 6 minutes, measurements can be made including at least one burp peak (Figure 3b). If the measured data was 0 or exceeded 1,000 ppm-m, it was excluded from the data, and based on this concentration, daily respiration was calculated using the AMPD (automatic multi-scale peak detection) algorithm package (R software version 4.0.2). It was calculated by calculating the average methane emissions between and. The calculated methane production amount was expressed as methane production amount according to DMI and methane production amount according to ADG.

4. Test items and statistical analysis

(Dry matter intake and body weight measurement) Dry matter intake was measured during the experiment period, and the remaining amount of the mixed feed was measured 30 minutes after feeding. Body weight was measured before and 4 weeks after the start of the experiment.

Test items include body weight, daily intake, daily gain, blood CBC, and MPT. CBC provides information on anemia, infection, blood coagulation ability, and immune system response ability, and MPT is a metabolic judgment index that measures the concentration of metabolites in the blood to determine whether metabolic activity is abnormal. Garlic extract is known to contain a large amount of sulfur-containing amino acids, which are essential for the production of glutathione synthesized in the liver, and is very good for antioxidant effects.

This study was processed according to PROC MIXED and T-TEST of the Statistical Analysis System (version 9.4). Statistical significance was evaluated at a 5% significance level, and a tendency was evaluated when it was between 5% and 10%.

5.MPT

(GOT, GPT) concentration is a type of enzyme used in liver function tests. When liver cells are destroyed or damaged, GGT and AST are released. These enzymes increase under the influence of energy intake, stress, and disease, but there is no significant difference. Since there was no difference, it is judged that there was no abnormality in liver function.

A decrease in (BUN) levels can be seen as a lack of protein intake or lack of rumen decomposed protein (reduced rumen microbial growth, decreased hormone production, etc.), but total protein increased, and there was no statistically significant difference, so it is judged that there is no problem.

(TCHO) is mainly absorbed along with other substances in the ileum. It plays a role in performing metabolic functions as a quasi-sphere of major steroid hormones, a precursor of bile acids, and an essential component of cell membranes. It indicates the overall nutritional status and liver function. It decreases when nutrient intake is insufficient or increases when fat intake is excessive, but there was no significant difference, indicating that there was no abnormality in liver function.

(Glucose) A hypoglycemic effect can be seen due to an increase in insulin levels, but there is no difference between the control group and the added group.

As a result, there was no difference in metabolic abnormality indicators between the control and treatment groups, so it can be determined that there was no problem with metabolism.

6. Methane concentration analysis

The concentration of methane emitted by the animal was expressed by correcting the amount of methane produced due to the difference in intake and daily weight gain. The methane concentration (ppm-m/kg DMI) of each animal refers to the methane concentration per dry matter intake divided by the value measured by LMD (ppm-m) by the average dry matter intake. Additionally, the animal's methane concentration (ppm-m/kg ADG) refers to the methane concentration per daily body weight divided by the value measured by LMD (ppm-m) by the average daily weight gain.

When the amount of methane produced in the rumen was calculated separately from respiration and belching, the difference between the additive treatment group and the control group was statistically significant (P<0.05). This means that the combined additive of attapulgite and allicin inhibits anti-methanogen and methanogenic enzymes. It can be interpreted as doing.

It was verified that the amount of methane produced by burping (ppm/m) decreased from 40.79% in the 1st week > 32.80% in the 2nd week > 45.83% in the 3rd week, and the amount of methane produced in respiration decreased from 13.82% in the 1st week > 39.62% in the 2nd week > 57.72% in the 3rd week ( (see Figure 4 and Table 19).

Depending on the amount of methane production over time, respiration tended to decrease in methane in the first week and significantly decreased from the second week (p<0.05), and burping decreased from the first week, increasing the feeding period. It was confirmed that the methane reduction effect was greater (p<0.001). In other words, compared to the control group, all treatment groups showed excellent results in reducing methane production.

In the present invention, combinations and addition amounts of various natural substances were systematically tested to develop a feed additive for reducing rumen methane derived from natural extracts. As a result, the composite feed additive of garlic extract and attapulgite showed positive results in reducing methane production, so the addition of 2.5% of allicin and 5.5% of attapulgite showed the potential as a feed additive that can reduce methane production. presented.

Therefore, in the present invention, the effect of reducing methane production was verified through the selection of complex natural extracts and appropriate dosage ratio without negative effects on rumen microbial fermentation, feed use efficiency, and metabolism.

Claims (10)

Contains allicin, attapulgite and seaweed meal and linseed,
The weight ratio of allicin: attapulgite: seaweed meal and linseed is 0.5 to 2.5:2.5 to 5.5:0.5 to 5.5, a feed additive for reducing methane gas in the rumen. delete delete delete Contains allicin, attapulgite and seaweed meal and linseed,
Based on 100% by weight of feed composition
The amount of allicin is 0.5 to 2.5% by weight,
The amount of attapulgite is 2.5 to 5.5% by weight,
The combined amount of the seaweed meal and linseed contains a feed additive for reducing methane gas in the rumen of 0.5 to 5.5% by weight,
Feed composition for ruminants. delete delete delete delete delete