KR20200071317A - Feed additive composition for reducing methane emission from ruminants comprising Allium fistulosum and tannic acid - Google Patents

Abstract

The present invention relates to a feed additive composition for reducing ruminant methane, which includes green onion and tannic acid as active ingredients. In the present invention, green onion and tannic acid reduce total gas generation, methane generation and carbon dioxide generation, and reduce methane production in rumen. It has been confirmed that it increases propionic acid and reduces methane-producing bacteria and fibrinolytic bacteria. Green onion and tannic acid having the above-mentioned effects can promote the growth of livestock and increase feed efficiency, and are safe synthetic additives for long-term use. Alternatively, it may be used as an antibiotic substitute, and may be used as a feed additive as a natural methane reducing agent that reduces methane gas production.

Description

Feed additive composition for reducing methane emission from ruminants comprising Allium fistulosum and tannic acid

The present invention relates to a feed additive composition for reducing methane in ruminants containing green onion and tannic acid as an active ingredient, a synthetic additive used to reduce methane gas produced in ruminants of ruminants, for reducing methane to replace antibiotics It relates to a feed additive composition and its use.

Natural substances, anti-synthetic substances, or synthetic substances having antimicrobial action are collectively called antibiotics, and the antibiotics are administered orally or by injection or used topically to treat or prevent diseases caused by microorganisms. These antibiotics have been used to treat patients with infectious diseases for the past 70 years, and are currently used for the purpose of treating or preventing human diseases as well as animal diseases.

On the other hand, antibiotics are also used as growth promoters to promote animal growth in addition to the purpose of treating or preventing animal diseases. In the domestic livestock industry, ionophore-based antibiotics such as monensin, chloroform and halogen compounds are used as typical antibiotics. When an antibiotic is added to the feed as a growth accelerator and administered to animals, it promotes the growth of animals, improves feed utilization, and lowers the prevalence of infectious diseases caused by bacteria in animals kept in groups. Can be. In other words, antibiotics prevent infectious diseases in animals, reduce the prevalence and mortality rate for infectious diseases, and increase the growth rate, thereby lowering the price of meat, eggs, milk, etc. It also has a positive effect on the pharmaceutical industry. In addition, antibiotics as growth promoters inhibit the production of undesirable fermentation products by intestinal bacteria, lactic acid, volatile fatty acids, ammonia, amines, methane, etc., by stimulating beneficial microorganisms and inhibiting harmful microorganisms in the gastrointestinal tract of animals. In particular, methane is the main cause of global warming, and the amount of methane gas emitted by animals per year is 80 to 11.5 million tons, accounting for 15 to 20% of global methane emissions. It has been reported that the animal antibiotic and growth promoter monensin reduces the production of methane, a major culprit of global warming, by increasing the fermentation of propionate in the stomach of rumen animals.

As described above, in the livestock industry, antibiotics have greatly contributed to improving the productivity of livestock, but the use of such antibiotics has recently been restricted due to problems such as residual antibiotics and emergence of multi-drug resistant super bacteria in livestock products. In particular, the inclusion of antibiotics in the blended feed for the purpose of improving livestock productivity, not for the purpose of treatment, is entirely prohibited.

Therefore, for the production of eco-friendly livestock and organic livestock products, the development of substances that can replace the existing antibiotics and the development of related technologies are recognized as very important tasks. Among the substances that are most likely to replace antibiotics are natural extracts derived from plants. Plants produce secondary metabolites to protect themselves from the environment, and since these secondary metabolites exhibit various physiologically active effects, they can be used as medicines to prevent or treat diseases of humans or animals. The antimicrobial effect on pathogenic microorganisms is also one of the physiological activities that can be exhibited by secondary metabolites of plants. In addition, studies have been reported that secondary metabolites of plants increase fermentation in rumen and increase the efficiency of feed energy while reducing methane gas generation in rumen animals, and among the secondary metabolites produced by plants, It has been demonstrated that some plant extracts containing phenolic substances, essential oils, flavonoids, and sarsaponin, which are representative components of action, reduce methane production.

There are two main mechanisms of action for the reduction of rumen methane gas. The first is a hydrogen substitution method that allows the generated hydrogen to be metabolized in a direction other than methane gas production. The second method is to reduce methane gas production through direct antibacterial action against methane gas-producing bacteria. It cannot be concluded that any of these two methods is efficient, but it is more important to find the effect of ultimately reducing methane gas.

Accordingly, the present technology aims to search for plants or plant extracts capable of reducing methane gas in the rumen and to use them as feed additives.

Republic of Korea Patent Publication No. 10-2015-0004836 (2015. 01. 13 published)

An object of the present invention is to provide a composition for adding feed for reducing ruminant methane.

Another object of the present invention is to provide a method for inhibiting methane gas production in the rumen.

In order to achieve the above object, the present invention provides a feed additive composition for reducing methane in ruminants, including leek (Allium fistulosum) and tannic acid (tannic acid) as an active ingredient.

In addition, the present invention provides a feed composition comprising a feed additive composition for reducing ruminant methane.

In addition, the present invention provides a method for inhibiting methane gas production in a rumen comprising the step of administering a feed composition to a ruminant.

In the present invention, it was confirmed that par and tannic acid reduce total gas generation, methane generation, and carbon dioxide generation, increase propionic acid to reduce methane production in the rumen, and reduce methane-producing bacteria and fibrinolytic bacteria. Eggplant green onion and tannic acid can promote the growth of livestock and increase feed efficiency, can be used as a safe synthetic additive or antibiotic substitute even after long-term use, and can be used as a feed additive as a natural methane reducing agent to reduce methane gas production. have.

1 is a result confirming the change in the rumen microbial community according to the methane reduction additive and fermentation time (a, b, c, etc. = P <0.05).

The term "ruminant" as used in the present invention is a receding animal belonging to mammalian cattle, including cows, goats, bulls, buffaloes, bison, deer, camels, sheep, etc., having one of four compartments, one of which It is rumen. Ruminants In the digestive system called rumen, cellulose is converted to volatile fatty acids through the fermentation of microorganisms and used as nutrients. That is, volatile fatty acids, hydrogen, carbon dioxide, etc. are generated through the decomposition of carbohydrates in the rumen, and the hydrogen and carbon dioxide cause rumen acidity to fall. The hydrogen and carbon dioxide are converted to methane and lost.

The term "methane" used in the present invention is a factor that has the greatest influence on global warming after carbon dioxide, and is produced in the rumen by methane gas-producing microorganisms when ruminants ingest feed containing carbohydrates. As one of the reasons for deteriorating utilization efficiency, it is important to suppress methane gas production in the rumen of ruminants.

The term "active ingredient" used in the present invention is a substance or group of substances (such as a crude drug whose pharmacologically active ingredient is not known) that is expected to directly or indirectly express the efficacy and/or the effect of the drug by the inherent pharmacological action. It includes) as a main component.

The present invention provides a feed additive composition for reducing ruminant methane, which includes leek (Allium fistulosum) and tannic acid as active ingredients.

The composition may reduce total gas generation, methane generation and carbon dioxide generation, and may reduce methane-producing bacteria and fibrinolytic bacteria.

The ruminants include cattle, goats, sheep, giraffes, American bison, European bison, yak, buffalo, deer, camel, alpaca, llama, wildebeest, antelope, pronghorn and It may be selected from the group consisting of nilgai nutrition, but is not limited thereto.

In addition, the present invention provides a feed composition comprising a feed additive composition for reducing ruminant methane.

The feed composition may include a feed additive composition for reducing methane and other ingredients added to a known feed composition, and the feed composition is prepared by a known method, and detailed description thereof will be omitted. do. In addition, the feed includes general rice straw, weeds, grasses, ansiridge, hay, and wild grass, but is not limited thereto, and may be any feed used for breeding livestock.

The composition for adding feed for reducing methane may be added in an amount of 0.1 to 15 parts by weight based on 100 parts by weight of the dry feed, but is not limited thereto.

In addition, the present invention provides a method for inhibiting methane gas production in the rumen comprising the step of administering the feed composition to ruminants.

Hereinafter, the present invention will be described in more detail through examples. These examples are only intended to illustrate the present invention more specifically, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example 1: Disclosed animals and rumen fluid

The study procedure and specification management of the public axis were performed in accordance with the National Institute of Economics and Animal Welfare and Ethics, and the ruminant fluid was a cow with a cannula mounted on the rumen that is bred at the National Institute of Animal Wildlife Protection (450 kg±30 weight). kg).

For specification management of public animals, thick feed (No. BBVMRO 158) and timothy are mixed at a weight ratio of 6:4, and 2% of body weight is divided twice a day (09:00 and 17:00) Water and mineral blocks were freely consumed.

The rumen gastric juice was collected through a rumen cannula just before the morning feeding, filtered using 4 layers of cheese cloth, and then quickly transferred to a laboratory in an anaerobic 2 L glass bottle. The rumen solution was left in an 39°C water bath, Lab Companion (BS-21) for 1 hour, and then used for in vitro testing after removing the feed particles with a vacuum pump.

Example 2: Disclosed materials and in vitro exam

Timothy crushed to 2 mm was dried in a dry oven at 65° C. for 24 hours, and used as a test material. 0.95 g of test material in a nylon bag (Ankom Forage bag: R1020) manufactured by itself with 3 cm×5.5 cm. Was added and used in batches for all treatment groups, and 0.05 g was used as a raw material for methane reduction for each treatment group.

The control group was wheat bran (wheat barn), treatment group 1 (MRA-1) was leek ( Allium fistulosum L., 0.05 g), treatment group 2 (MRA-2) was sodium lauryl sulfate (SLS, 0.025 g)+ Wheat bran (0.025 g), treatment group 3 (MRA-3) is sodium dodecyl sulfate (SDS, 0.025 g) + wheat bran (0.025 g), treatment group 4 (MRA-4) is green onion (0.02 g) + Tannic acid (0.02 g) + bran (0.01 g) was used as a raw material.

The raw material for each treatment group, timothy, was placed in a nylon bag, heat sealed, and then administered to a 125 mL serum bottle. Subsequently, 60 mL of the culture solution in which the rumen fluid and McDougall buffer (Mcdougall, 1948) were mixed at a ratio of 1:2 was dispensed into an anaerobic state (O ₂ -free N ₂ ), followed by butyl rubber and an aluminum seal. ) Was used to seal the serum bottle. Incubation for each treatment group was performed in vitro by repeating 3 times in a time zone (3, 6, 9, 12, 24 and 48 hours) in a shaking incubator at 39°C (shaking incubator, Jeio Tech; SI-900R, 120 rpm). Did. Chemical composition of the test material, control and treatment are shown in Table 1 below.

* NDF: Neutral detergent fiber

ADF: Acid detergent fiber

Example 3: Analysis items and methods

3-1. pH analysis

For pH analysis, the butyl rubber and the aluminum seal of the serum bottle were removed using Weaton decapper (Weaton Co., USA), and then the pH of the culture was measured using a pH meter (Mettler Toledo, MP230).

3-2. Building digestibility analysis

For analysis of building digestibility, a nylon bag in a serum bottle was collected and placed in a water-filled water tank, and washed three times at 100 rpm for 20 minutes using Heidolphs Rotamax 120 (Heidolph Instrument, Germany), followed by a dryer at 65°C (Jeio tech , Korea) for about 24 hours to measure the remaining amount of the building. Subsequently, after obtaining the difference from the air mass before fermentation, the difference was converted into a percentage of the air mass before fermentation to calculate the building digestibility.

3-3. Microbial growth analysis

For the analysis of microbial growth, 1 mL of culture was taken, and then the feed particles were removed by centrifugation at 3,000 rpm for 3 minutes. Subsequently, the supernatant was re-centrifuged at 14,000 rpm for 3 minutes to precipitate microbial pellets, and after removal of the supernatant, 1 mL of sodium phosphate buffer (pH 6.5) was added to the pellets and stirred with vortex. The washing process was repeated 3 times. Thereafter, a microscopic growth amount was calculated by measuring an optical density (O.D) value at 550 nm with a spectrophotometer (BIO-RAD Model 680).

3-4. Total gas generation analysis

The total gas generation was performed with reference to the method of Theodorou et al. (1994). The gas in the upper space of the serum bottle was measured using a detachable pressure transducer and a digital readout voltmeter (Laurel Electronics, Inc., CA, USA), after which gas was collected in a 9 mL vacuum tube to perform gas chromatography (gas Chromatography; GC, HP 5890 Gas Chromatography, USA) was used to measure the amount of methane and carbon dioxide generated.

3-5. Volatile fatty acid analysis

For the analysis of volatile fatty acids, 1 mL of culture was taken, and then the feed particles were removed by centrifugation at 12,000 rpm for 3 minutes. Subsequently, the supernatant was filtered using a 0.20 μM syringe filter and then measured using high performance liquid chromatography (HPLC, Agilent-1200, Germany). The injection amount of the sample was 20 μL, and the mobile phase solution used 0.0085 NH ₂ SO ₄ and the flow rate was 0.6 mL/min. As a column, 300 mm×7.8 mm Id MetaCarb 87H (Varian, USA) was used at 35°C.

3-6. Microbial community change analysis

To analyze the changes in the microbial community, real-time PCR was performed. After taking 1 mL of the culture solution, the feed particles are removed by centrifugation at 3,000 rpm for 3 minutes, and then DNA is extracted using NucleoSpin Soil (Macherey-Nagel, Duren, Germany), and Nano-drop (Thermo Scientific, Wilmington, Delaware) USA) was used to measure the concentration of DNA and correct it to 10 mg/μL to use in the experiment.

Primers used in the real-time polymerase chain reaction are total bacteria (Total bacteria, Denman and McSweeney, 2006), methane producing bacteria (Ciliate-associated methanogens, Skillman et al., 2006), and methane bacteria (Methanogenic archaea, Denman) et al., 2007), Fibrobacter succinogenes and Ruminococcus flavefaciens , Denman and McSweeney, 2006), and Ruminococcus albus , Koike and Kobayashi, 2001 ).

Before use in the experiment, 10 μL of forward primer (FW) and 10 μL of reverse primer (RW) were mixed with 180 μL of distilled water and diluted to 10 pmol. 0.2 μl PCR tube (PCR-0208-FCP-C, Corning Axygen, New York, USA) was mixed with 10 mg/μL of DNA and distilled water and dispensed 9 μL, diluted 1 μL of primer and SYBR Green (Code: QPK-201, Toyobo Co., LTD., Japan) 11 μL of pre-mixture mixed with 10 μL was dispensed so that the total volume was 20 μL.

Since then, Real-Time PCR (CFX96TM Real-Time system, BIO RAD) was performed with reference to the methods of Denman and McSweency (2006) and Denman et al. (2007). ( Ruminococcus albus , Ruminocuccus flavefaciens , Fibrobacter succinogenes ), the expression rate of methane bacteria and methane producing bacteria was measured, and the control group was regarded as 1 to analyze the change of the microbial community by relative quantification of other treatment groups.

Example 4: Statistical processing

Statistical processing was processed according to the General Linear Model (GLM) procedure of SAS package program (2002), and after analyzing the variance to verify the significance of each processing section, Duncan's multiple range test (Duncan, 1955) at 5% level. Significance was tested ( P <0.05).

Test Example: Analysis of the effects of green onion and tannic acid on ruminant methane reduction

The effects of green onion and tannic acid on pH, building digestibility and microbial growth during fermentation in vitro rumen are shown in Table 2 below.

The pH was maintained at an appropriate level in both the control and the addition, and tended to decrease as the fermentation time passed. In general, the pH in the rumen is consistent with the results of previous studies (Ha et al., 2013), which maintains a state of 5.0 to 7.8.

In the case of building digestibility, there was no significant difference ( P >0.05) when comparing the control and treatment groups.

In the case of microbial growth, the microbial growth was significantly ( P <0.05) higher in the 4 treatment groups (MRA-4) in the fermentation 6 and 9 hours compared to the control, and at the maximum in the 12 hours and 48 hours in all treatment groups. It was confirmed that it reached. These results indicate that green onion and tannic acid do not show the growth delay of microorganisms.

That is, the green onion and tannic acid used in the present invention maintain a pH range in an appropriate range, and confirm that they do not negatively affect the building digestibility and microbial growth, and are suitable for the conditions of natural additives capable of reducing total gas and methane. It was judged.

* a, b, c etc = P <0.05

The effects of green onion and tannic acid on total gas generation in rumen in vitro are shown in Table 3 below.

Total gas generation increased in all treatment groups over the course of the fermentation time, and decreased significantly ( P <0.05) in the treatment group 4 (MRA-4) in the 3, 9, 24, and 48-hour periods compared to the control. Confirmed.

The amount of methane generated increased in all treatment groups as the fermentation time elapsed, and compared to the control, treatment 2 (MRA-2), treatment 3 (MRA-3) and treatment 4 (MRA-4) in the 24-hour fermentation period. It was confirmed to decrease significantly ( P <0.05).

The amount of carbon dioxide generated also increased in all treatment groups over the course of the fermentation time, and compared to the control, treatment 2 treatment groups (MRA-2), treatment treatment 3 treatment groups (MRA-3), and treatment treatment treatment 4 (MRA-) It was found to decrease significantly ( P <0.05) at 4).

That is, the green onion and tannic acid used in the present invention showed a methane reduction effect similar to that of the synthetic additive, and in particular, it was confirmed that the carbon dioxide reduction effect was superior to the synthetic additive in terms of sustainability.

The effects of green onion and tannic acid on volatile fatty acids in rumen in vitro are shown in Table 4 below.

In the case of total volatile fatty acids, fermentation was significantly higher in 4 treatment groups (MRA-4) in the 24-hour fermentation period ( P <0.05) compared to the control, and in the case of acetic acid content, fermentation when compared to the control 6 , It was significantly lower in treatment group 4 (MRA-4) at 9 hours ( P <0.05) and significantly lower in treatment group 1 (MRA-1) at 48 hours than fermentation ( P <0.05). In the case of propionic acid, compared to the control, it was confirmed that the fermentation was significantly higher ( P <0.05) in 4 treatment groups (MRA-4) at 12 and 24 hours of fermentation. Propionic acid in the rumen is known to be produced using hydrogen, a precursor of methane (Grobner et al., 1982), and it is thought that tannic acid reduces methane production by using hydrogen in the rumen.

The effect of green onion and tannic acid on the microbial community changes in the rumen in vitro is shown in FIG. 1. Compared to the control, fibrinolytic bacteria ( Ruminococcus albus, Fibrobacter succinogenes ) The fermentation was significantly lower ( P <0.05) in all treatments at 12 hours, and Ruminococcus albus was The fermentation 24-hour treatment group (MRA-1), treatment group 2 (MRA-2), and treatment group 3 (MRA-3) were significantly higher ( P <0.05), and fibrobacter succinogenes ( Fibrobacter succinogenes ) was significantly higher ( P <0.05) in treatment 2 (MRA-2), treatment 3 (MRA-3) and treatment 4 (MRA-4).

The above results show that the anionic surfactant SLS reduces the activity of carboxy-methyl cellulase enzyme in the rumen and the number of ciliate protozoa, thereby reducing the decomposition rate of organic matter and fibrin (Santra Et al., 2007). However, it was different from the results of previous studies (McAllister et al., 2000) that increasing the concentration of surfactants prevents the adhesion of enzymes to feed.

In addition, compared to the control, methane-producing bacteria (ciliate-associated methanogens) were significant in treatment group 1 (MRA-1), treatment group 2 (MRA-2), and treatment group 4 (MRA-4) in the 24-hour fermentation period ( It was confirmed that P <0.05) was low, and it was confirmed that green onion and tannic acid can be used as a substitute for a synthetic additive showing a methane reducing effect.

The specific parts of the present invention have been described in detail above, and it is obvious that for those skilled in the art, these specific techniques are only preferred embodiments, and the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

The scope of the present invention is indicated by the following claims, and all modifications or variations derived from the meaning and scope of the claims and their equivalent concepts should be interpreted to be included in the scope of the present invention.

Claims (7)

A feed additive composition for reducing ruminant methane, which includes leek (Allium fistulosum) and tannic acid as active ingredients. According to claim 1, The composition is a feed additive composition for reducing ruminant methane, characterized in that for reducing the total gas generation, methane generation and carbon dioxide generation. According to claim 1, wherein the composition is a feed additive composition for reducing ruminant methane, characterized in that to reduce methane-producing bacteria and fibrinolytic bacteria. The method of claim 1, wherein the ruminant is a cow, goat, sheep, giraffe, American bison, European bison, yak, buffalo, deer, camel, alpaca, llama, wildebeest, antelope, eggplant Feed additive composition for reducing ruminant methane, characterized in that selected from the group consisting of horn antelope (pronghorn) and nilgai. A feed composition comprising a feed additive composition for reducing ruminant methane according to any one of claims 1 to 4. According to claim 5, The feed composition for reducing methane, the feed composition, characterized in that added to 0.1 to 15 parts by weight based on 100 parts by weight of the dry feed. Method of inhibiting methane gas production in the rumen comprising the step of administering to the ruminant the feed composition according to claim 5.

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