KR20160047787A - Feed Ingredients Against Methane Gas Produced by the Rumen of Ruminant Animals - Google Patents

Abstract

The present invention relates to a livestock feed composition for reducing methane gas and, more specifically, to a feed composition controlling the generation of methane gas without influencing microorganism fermentation in the rumen. The composition comprises a garlic extract, nitrate, and fumarate.

Description

[0001] The present invention relates to a feed composition for inhibiting the production of methane gas in rumen rumen,

The present invention relates to a livestock feed composition for methane gas abatement, and more particularly, to a feed composition capable of inhibiting methane production without affecting microbial fermentation in rumen.

To cope with climate change globally, it concluded a climate agreement and obliges each country to reduce greenhouse gas emissions. Methane (CH ₄ ) is one of the strongest greenhouse gases emitted directly to the atmosphere in the livestock industry and various agricultural sectors. Considering the properties of methane that absorbs infrared light and the length of time it stays in the atmosphere, the greenhouse effect of methane is about 72 times that of carbon dioxide in 20 years and about 25 times in 100 years.

Methane gas is a natural by-product of the digestion of ruminants, with 75% of the ruminants in ruminants and the rest from water buffalo, sheep and goats (Crutzen et al., 1986). In the case of cattle, on average, it produces more than 50 L / hour of gas, 25-30% of which is methane.

It is also related to feed efficiency, as it results in the loss of 3 to 12% of the total energy consumed by the ruminant from the feed, due to methane production. As such, when methane is generated in rumen rumen, environmental problems due to global warming occur and the efficiency of feed utilization is decreased, and various methods for suppressing the generation of methane are being studied.

With respect to a method for inhibiting methane production in a rumen rumen, Korean Patent Laid-Open No. 10-2014-0022572 discloses a feed additive for ruminant methane reduction using an oat extract, and Korean Patent Laid- 2014-0055882 is effective for releasing the leaves and stems of plants and plants with low methane emission, medicinal herb medicine, weed seeds, and far-infrared radiation, and by recombining the livestock feeds for activating useful microorganisms, Although a livestock feed has been disclosed, its technical structure is different from that of the present invention.

SUMMARY OF THE INVENTION Accordingly, the present invention has been made keeping in mind the above problems occurring in the prior art, and an object of the present invention is to provide a livestock feed composition for reducing methane gas containing garlic extract, nitrate and fumarate.

Another object of the present invention is to provide a method for suppressing the production of methane gas in rumen by administering a livestock feed composition for methane gas abatement to a ruminant.

To achieve these and other advantages and in accordance with the purpose of the present invention, as embodied and broadly described herein, the present invention provides a livestock feed composition for reducing methane gas comprising garlic extract, nitrate salt and fumaric acid salt.

According to one embodiment of the present invention, the garlic extract, nitrate salt and fumaric acid salt are mixed at a ratio of 0.01 to 0.1: 0.5 to 2.0: 0.5 to 2.0 (g / L).

According to another embodiment of the present invention, the garlic extract may use garlic oil.

According to another embodiment of the present invention, the livestock feed composition for methane gas abatement is added at a weight ratio of 0.1 to 0.5% based on the feed weight.

The present invention also provides a method for inhibiting the production of methane gas in rumen by administering a livestock animal feed composition for methane gas abatement.

The methane gas reduction feed composition of the present invention can selectively inhibit methane production in ruminants without affecting microbial fermentation in the rumen.

, CON), 마늘유 50 mg/L (

, GO), 푸마르산염 15 mM (

, FM), 질산염 20 mM (

, NT) 및 마늘유 50 mg/L , 푸마르산염 15 mM, 질산염 20 mM의 혼합물 (

, GONF))
도 2 A 내지 D는 본 발명의 실시예에 따른 가축사료 조성물의 발효 시간에 따른 초산(acetate, A), 프로피온산(propionate, B), 뷰틸산(butyrate, C) 및 발레릭산(valerate, D) 생성량을 나타낸 그래프이다. (대조구 (

, CON), 마늘유 50 mg/L (

, GO), 푸마르산염 15 mM (

, FM), 질산염 20 mM (

, NT) 및 마늘유 50 mg/L , 푸마르산염 15 mM, 질산염 20 mM의 혼합물 (

, GONF))FIGS. 1A to 1D are graphs showing total biogas (A), methane (B), hydrogen (C), and ammonia nitrogen (D) production amounts according to fermentation time of a livestock feed composition according to an embodiment of the present invention. (Control

, CON), garlic oil 50 mg / L (

, GO), fumarate 15 m M (

, FM), 20 m M nitrate (

, NT) and a mixture of 50 mg / L of garlic oil, 15 m M of fumarate and 20 m M of nitrate (

, GONF))
2 A to D are graphs showing the effect of the fermentation time of acetate, A, propionate, B, butyrate, C and valerate (D) on the livestock feed composition according to the embodiment of the present invention, Fig. (Control

, CON), garlic oil 50 mg / L (

, GO), fumarate 15 m M (

, FM), 20 m M nitrate (

, NT) and a mixture of 50 mg / L of garlic oil, 15 m M of fumarate and 20 m M of nitrate (

, GONF))

In the present invention, a ruminant refers to an animal that induces it to its nutrients by converting cellulose into a volatile fatty acid through fermentation of the microorganism in a digestive system called rumen, These include bulls, buffalo, buffalo, deer, elk, deer, camel, sheep and more.

When the ruminant feeds on a feed containing carbohydrates, the carbohydrate breaks down and fermentation takes place in the rumen, resulting in the production of hydrogen gas and carbon dioxide. The hydrogen gas and carbon dioxide produced in this way produce methane as a methane-producing organism or as a precursor for producing acetic acid-producing acetic acid competitively.

The methanogen bacteria means a bacterium capable of generating methane. Methanococcus, Methanobacterium, Methanosarcina, Methanobacter, The present invention relates to a method for the treatment and / or prophylaxis of a disease or condition selected from the group consisting of Methanobrevibacter, Methanothermus, Methanothrix, Methanospirillum, Methanomicrobium, Methanococcoides, But are not limited to, Methanogenium, Methanoplanus, and the like.

The acetic acid-producing bacterium means a bacterium having an ability to produce acetic acid, and includes bacteria including Acetobacter, Agrobacterium, Rhizobium, Pseudomonas, and the like. It is not.

Methane gas is produced by methane-producing bacteria using hydrogen and carbon dioxide as precursors in the rumen. Therefore, in order to reduce the methane gas production amount, there is a method of controlling the generation amount of hydrogen which is a precursor of methane production in the rumen, , A method for controlling methanogens that directly generate methane gas using precursors, and a control method for converting metabolic pathways by dominating acetic acid-producing bacteria that compete with methanogenic bacteria using the same precursors to produce acetic acid And so on.

For example, antibiotics and halogen compounds such as monensin have been studied in order to reduce methane gas generation in cattle rumen, but there is a problem that these substances may remain in livestock products. Methane-producing microorganisms adapt to the effects temporarily and reduce the growth of useful microorganisms in the rumen together, thereby lowering feed efficiency and weakening productivity.

Accordingly, the present inventors have conducted studies on a livestock feed for mitigating methane gas which is eco-friendly but does not affect the digestion of ruminants in consideration of the above points. In the optimum mixed composition of garlic extract, nitrate and fumarate, It is possible to selectively inhibit the methane gas without affecting the fermentation of the microorganism. Thus, the present invention has been accomplished.

One aspect of the invention relates to a livestock feed composition for methane gas abatement comprising garlic extract, nitrate and fumarate salt.

Hereinafter, each configuration of the present invention will be described in more detail.

Garlic extract

The garlic according to the present invention is a perennial herb which is a perennial herbaceous plant with a monocotyledonous plant lily lily and contains 60% of water and 3% of protein and contains minerals such as K, Ca, Mg and P and vitamins B1, B2 and C have.

Garlic is known to have anticancer effects by inhibiting the development of bacteria by its excellent antibacterial action, and it also inhibits arteriosclerosis by promoting secretion of gastric juice, promoting blood circulation and lowering cholesterol in blood.

In particular, allicin, a sulfur organic compound in garlic, is effective in suppressing methane - producing bacteria in rumen. Therefore, when garlic is administered to ruminants, it is possible to reduce methane gas production by inhibiting the above-described antimicrobial activity, anticancer activity and cholesterol inhibition effect as well as methane-producing bacteria in the rumen.

In the present invention, the garlic extract may preferably be garlic oil obtained by compression or steam distillation of garlic, but is not limited thereto.

nitrate

Two mechanisms by which nitrate inhibits methane production in the rumen are either to take advantage of nitrate toxicity or deplete available precursors of methanogens (Allison et al., 1981).

Methane is very sensitive to nitrate and 3 mM nitrate inhibits methanogenesis (Iwamoto et al., 2002). Nitrate toxicity has been shown to inhibit the number of methanogens in most studies.

Another potential mechanism is that nitrates efficiently control the generation of hydrogen, a precursor of methane production.

On the other hand, nitrite formed as an intermediate due to the reduction of nitrate to ammonia in the rumen is known to reduce microbial activity, among which animal feed intake is reduced have.

Also, high doses of nitrate in ruminants have been reported to cause oxygen deficiency in animal tissues and cause methemoglobinemia.

Therefore, it is necessary to develop a substance capable of mitigating the toxicity of nitrate or an alternative substance.

Fumarate

Fumarate theoretically reduces the production of methane. In other words, fumarate is an inorganic compound that reduces intestinal methane production through redirecting H2 metabolism toward reduction of succinate and propionate in methane production.

However, according to the previous batch culture, the efficacy of fumarate to reduce methane is relatively small. In other words, according to existing studies, methane is reduced by 2.3% on average and the maximum is 18% (Ungerfeld et al., 2007).

The fumarate salt can enhance the feed efficiency by enhancing the nutrient digestibility and feed intake rate when added to the livestock feed in in vitro and in vivo tests, and is therefore preferable as the livestock feed composition according to the present invention.

In addition, it is possible to provide a safe methane production reducing agent by reducing the use amount of the nitrate, and the garlic extract and the nitrate reduce the growth of useful microorganisms in the rumen in inhibiting methane production, thereby lowering the feed efficiency and weakening the productivity Can be solved.

The livestock feed composition for methane gas reduction according to the present invention preferably contains garlic extract, nitrate and fumaric acid in a ratio of 0.01 to 0.1: 0.5 to 2.0: 0.5 to 2.0 (g / L).

The mixing ratio can bring about methane gas inhibiting effect without adversely affecting the rumen metabolic process.

Another aspect of the present invention relates to a livestock feed composition characterized in that the livestock feed composition is added in a weight ratio of 0.1 to 0.5% based on the feed weight.

If the amount of the animal feed composition is less than 0.1% by weight, the effect of inhibiting the methane production of the animal feed may be insignificant. If the animal feed composition exceeds 0.5% by weight, adverse effects such as building intake, nutrient digestibility, Can be expressed.

The livestock feed according to the present invention may be a feed used for breeding livestock, including, but not limited to, general rice straw, wild grass, grasses, anthiria, hay, and hay fever.

Still another aspect of the present invention relates to a method of inhibiting the production of methane gas in rumen by administering the animal feed composition to a ruminant.

Hereinafter, the present invention will be described in detail with reference to Examples. However, the following examples are for illustrative purpose only and are not intended to limit the scope of the present invention.

1. Materials and Methods

1) In vitro rumen fermentation test

One kilogram of Hanwoo steer (weighing 350 kg ± 50 kg) equipped with a rumen cannula was presented at the Livestock Hygiene Research Institute in Kimje, Jeollabuk-do. The public axes were fed twice a day (09:00) and afternoon (17:00) And 4 kg of fat-fed diets were fed. Mineral blocks and water were allowed to eat freely.

The ruminal fluid was collected by a cannula attached to the rumen 30 minutes before the feeding of the diary at the morning of the day, filtered with 4-ply cheese cloth, blocked with oxygen in the 2L flask filled with O ₂ -free CO ₂ , Respectively. To remove residual feed particles, ruminal inoculum was transferred to the laboratory and re-filtered with 8-ply cheese cloth. McDougall's buffer (Mcdougall, 1948) and rumen juice were mixed 4: 1 as rumen inoculum. The orchardgrass used as the substrate of the experiment was milled using a laboratory mill equipped with a 1 mm sieve (Cutter mill, IKA MF10.1, Staufen, Germany).

In Example 1, 0.5 g orchard grass was used, and in Example 2 and Example 3, 0.1 g orchard grass and 0.4 g poultry feed were mixed. Serum bottles were incubated for 24 hours in Example 1 and Example 2, and 48 hours in Example 3 in a 39 ° C incubator. All experiments were performed in triplicate according to the method of Tilley and Terry (1963).

2) Analysis items and analysis methods

Total gas production by culture time was measured by using a glass syringe for experimental purposes. The measured gas was collected in an aluminum pack equipped with a rubber stopper to measure hydrogen and methane emissions. Hydrogen and methane production were analyzed by gas chromatograph (HP7890, Agilant, CA, USA) equipped with Carboxen ^™ , fusedsilicacapillarycolumn (0.53 mmi.d. × 30 m length, SUPELCO, USA) 150 deg. C and 150 deg. After the fermentation was completed, the pH of the rumen juice was measured using a pH meter (S20 Seven Easy ^TM , Mettler-Toledo). Amniotic nitrogen content of rumen juice was determined according to the method of Chaney and Marbach (1962), and centrifuged at 4,000 rpm for 15 minutes to obtain 1 ml of phenol color reagent and alkali-hypochlorite 1 ml of the reagent was thoroughly mixed and reacted at 37 ° C for 15 minutes. Absorbance was measured at 630 nm using a spectrophotometer (Optizen UV2120, Mecasis, Korea). Volatile fatty acids were prepared according to the method of Erwin et al. (1961). The feed particles to a ruminant by adding metaphosphoric acid 200㎕ 1㎖ the supernatant of gastric juice to settle for 30 minutes to remove coarse pre-treatment to which the sample was centrifuged at 13,000rpm ^{Nukol TM, fusedsilicacapillarycolumn (0.25mmi.d. ×} 0.25㎛ film (HP7890, Agilant, CA, USA) equipped with oven, injector and detector at 180 ° C, 220 ° C and 200 ° C, respectively.

3) Measurement of microbial distribution change

For in vitro fermentation of rumen mixed microbes, pellets were used for centrifugation at 3000 × g for 15 minutes. In the analysis, QIAamp  DNAstoolminikit (50, QIAGEN) was used (Patra, 2011). DNA extraction was performed with GEN5 version 10.8 and Epoch / Biotech spectrophotometer.

Accurate methanogen analysis and total microbial counts of PCR (qPCR) were measured using a CFX96 Real-Time System (BIORAD). SBYR-based primers were MB1174F, 5'-GAGGAAGGAGTGGACGACGGTA-3 '; Arch 1406-1389R, 5'-ACGGGCGGTGTGTGCAAG-3 '(Methanogens) (Ohene-Adjei et al., 2008), 341f-GC, 5'-CCTACGGGAGGCAGCAG-3'; 534r-GC, 5'-ATTACCGCGGCTGCTGG-3 '(total bacteria) (Dar et al., 2005) were used. The cycling condition for methanogen extraction was denaturation at 95 ° C for 2 minutes, followed by 40 cycles at 95 ° C for 15 seconds and 60 ° C for 60 seconds. In total bacterial population analysis, denaturation was performed at 95 ° C for 3 minutes, followed by 41 cycles of 95 ° C for 10 seconds, 60 ° C for 20 seconds, and final extension at 72 ° C for 1 second.

2. Experimental design

< Example  1>: Garlic extract, nitrate and Fumarate  Effect Black

In vitro fermentation tests were carried out for 24 hours in 3 replicates per 6 treatments (Table 1).

Six treatments were garlic oil 300 mg / L (GO); Fumarate 21 mM (FM); Nitrate 21 mM (NT); A mixture of 300 mg / L of garlic oil and 21 mM of fumarate (GOF); (GON) mixture of 300 mg / L of garlic oil and 21 mM of nitrate.

Garlic oil blend, 98% (Sigma Aldrich, USA), fumaric acid (Sigma Aldrich, USA) and sodium nitrate (Daejung chemicals and metals Co. Ltd, South Korea) were used for the experiment. Respectively.

All results were analyzed by randomized one-way ANOVA using the GLM procedure of SAS 9.2 (SAS Inst., Inc., Cary, NC). The mean was calculated using Duncan's multiple range test (p <0.05).

As shown in Table 1 below, treatment of garlic oil, fumaric acid salt, and nitrate affected ruminal fermentation parameters. Methane production was decreased in garlic oil (GO), nitrate (NT), mixture of garlic oil and fumaric acid salt (GOF), and mixture of garlic oil and nitrate (GON), but addition of fumarate (FM) . Except for the mixture of garlic oil and nitrate (GON), garlic oil had higher hydrogen content than other treatments, and the treatments affected the respective VFA trend and total VFA. The mixture of nitrate (NT), garlic oil (GO) and garlic oil and nitrate (GON) decreased the total VFA but the mixture of fumarate (FM) and garlic oil and fumarate (GOF) . Nitrate (NT), garlic oil (GO), mixture of garlic oil and nitrate (GON) and mixture of garlic oil and fumarate (GOF) decreased acetate production. Fumarate (FM) increased propionate concentration but no difference in garlic oil (GO). A mixture of nitrate (NT) and garlic oil and nitrate (GON) had a negative effect on propionate production. Nitrate (NT), fumarate (FM) and a mixture of garlic oil and nitrate (GON) reduced the amount of butyrate.

Major VFA in the rumen were affected by various treatments. Acetate was consistently affected (p = 0.051) and significance was observed in propionate (p = 0.006) and butyrate (p = 0.001). Secondary effects were seen in propionate (p = 0.002) and butyrate (p = 0.001) but not in acetate (p = 0.266). The effect of the interaction between garlic oil and nitrate had a negative effect on propionate concentration (p = 0.008).

parameter Treatment SEM
P-value
Control GO FM NT GOF GON Biogas (ml) 76.00 ^b 45.67 ^d 89.33 ^a 44.67 ^d 70.67 ^c 36.33 ^e 4.69 <0.001 CH ₄ (ml) 6.35 ^a 0.40 ^c 4.36 ^b 1.07 ^c 0.28 ^c 0.26 ^c 0.59 <0.001 H ₂ (ml) 0.06 ^c 1.46 ^a 0.01 ^c 0.08 ^c 1.23 ^b 0.09 ^c 0.15 <0.001 pH 6.62 ^c 6.66 ^b 6.56 ^d 6.72 ^a 6.59 ^cd 6.74 ^a 0.02 <0.001 NH ₃ -N
(Mg / 100 ml) 2.89 ^b 1.04 ^bc 2.24 ^bc 9.13 ^a 0.48 ^c 9.77 ^a 0.84 <0.001 Acetate (mM) 38.46 ^a 26.84 ^c 38.11 ^a 32.67 ^b 26.79 ^c 27.64 ^c 1.30 <0.001 Propionate (mM) 12.64 ^b 11.86 ^b 17.76 ^a 8.72 ^c 17.05 ^a 7.64 ^c 0.93 <0.001 Butyrate (mM) 4.99 ^a 4.85 ^ab 4.38 ^b 1.38 ^c 4.42 ^b 1.41 ^c 0.38 <0.001 Valerate (mM) 1.37 ^a 1.04 ^b 1.27 ^a 0.88 ^c 1.03 ^b 0.87 ^c 0.05 <0.001 Total VFA (mM) 57.46 ^a 44.60 ^b 61.52 ^a 43.65 ^b 49.29 ^b 37.56 ^c 2.09 <0.001 C2: C3 3.04 ^c 2.26 ^d 2.14 ^e 3.75 ^a 1.57 ^f 3.62 ^b 0.19 <0.001

▷ Parameters: NH3-N, ammonia-N; TVFA, totalvolatilefattyacid; C2: C3, acetate: propionate.

▷ Treatment: GO = garlic oil at 300 mg / L; FM = fumarate at 21 mM; NT = nitrate at 21 mM; GON = garlic oil at 300 mg / L + Nitrate at 21 mM; GOF = garlic oil at 300 mg / L + fumarate at 21 mM.

▷ af within a row, means without a common superscript differ (p <0.05).

< Example  2>: rumen fermentation gas without affecting microbial fermentation Abatement Garlic extract, nitrate and Fumarate  Derive optimal ratio of mixture

In vitro fermentation tests were carried out for 24 h with 3 replicates per treatment (Table 2).

Low, medium and high concentrations of garlic oil (GO), nitrate (NT) and fumarate (FM) were applied in the experiment. Specifically, garlic oil (GO) 30, 165 and 300 mg / L; Fumarate (FM) 7,14 and 21 mM; And nitrate (NT) 7, 14 and 21 mM were applied. The experimental design was Box Behnken design (Box & Behken, 1960).

Experimental design to derive the optimum compounding ratio was performed using the Box Behnken design method, one of the fraction factorial design methods, and the optimal mixing ratio for each reaction surface model was calculated using Minitab v.16 (Ryan, 2012) .

The experimental design for measuring the total VFA (volatile fatty acids), total gas and methane production in the four control and 15 test groups used in the experiment is shown in Table 2 below. The ANOVA output values in the 15 runs showed an effect on biogas (p = 0.0004), methane (p = 0.053) and no effect on total VFA (p = 0.483). Secondary effects are observed only in biogas generation (p = 0.001). There was no correlation between treatments in biogas and methane, but total VFA showed interactions between treatments.

The following equations show models with regression coefficients.

▷ Y _biogas (㎖), Y _methane (㎖) and Y _totalVFA (mM) react in various ways.

X ₁ , X ₂ and X ₃ represent garlic oil, fumarate and nitrate.

▷ * Coefficient is significantly different (p <0.05)

Treatment
variable result GO FM NT Total VFA
(mM) Total gas
(Ml) CH ₄
(Ml) One 30 7 14 66.39 75.00 1.34 x 10 ^-1 2 300 7 14 57.73 68.67 1.65 x 10 ^-2 3 30 21 14 67.63 86.67 1.19 x 10 ^-1 4 300 21 14 70.20 78.33 4.04 x 10 ^-2 5 30 14 7 63.90 79.00 3.31 x 10 ^-1 6 300 14 7 69.29 73.33 6.74 x 10 ^-2 7 30 14 21 71.17 71.33 7.00 x 10 ^-4 8 300 14 21 64.44 68.67 7.00 x 10 ^-4 9 165 7 7 59.47 63.33 6.00 x 10 ^-4 10 165 21 7 69.13 80.33 3.86 × 10 ^-2 11 165 7 21 64.26 55.67 6.00 x 10 ^-4 12 165 21 21 72.02 70.33 7.00 x 10 ^-4 13 165 14 14 67.21 59.67 6.00 x 10 ^-4 14 165 14 14 65.44 61.00 6.00 x 10 ^-4 15 165 14 14 71.47 60.33 6.00 x 10 ^-4 Control 1 0 0 0 74.56 100.67 1.31 × 10 ¹ Control 2 30 0 0 70.50 98.67 1.34 × 10 ¹ Control 3 165 0 0 64.30 87.00 2.23 Control 4 300 0 0 59.55 79.67 6.95 x 10 ^-1

In the present invention, the reaction values (total volatile fatty acid, methane gas) obtained in Table 2 were approximated to the reaction surface model, and then the conditions for maximizing total volatile fatty acid production and simultaneously minimizing methane production were calculated. The results are shown in Table 3.

ingredient content GO 50 mg / L NT 20 mM FM 15 mM

< Example  3>: garlic extract, nitrate and Fumarate  Effect of mixture on rumen fermentation gas

This example was carried out for the purpose of verifying the optimum ratio of the garlic oil, nitrate salt and fumaric acid salt obtained in Example 1. [ In vitro fermentation tests were carried out using one control and four test strips (GO 50 ㎎ / L; GONF (GO 50 ㎎ / L + NT 20 mM + FM 15 mM); NT 20 mM; FM 15 mM. The time was measured at 0, 3, 6, 9, 12, 24, 48 hours (Figs. 1 to 8).

All results were analyzed by two-way ANOVA using the GLM procedure of SAS 9.2 (SAS Inst., Inc., Cary, NC).

Experimental results showed that the interaction between fermentation time and treatments was observed in Biogas production (p <0.0001), and the interaction can be seen in Fig. While fumarate tended to increase (p = 0.1259), garlic oil tended to decrease biogas production when compared to the control (p = 0.0992). The mixed treatment of nitrate, garlic oil, nitrate, and fumarate suppressed biogas (p = 0.0001) compared to the control (p <0.0001). Garlic oil and fumaric acid decreased methane production to 32% and 8%, respectively. Particularly in FIG. 1B, methane reduction effects of fumarate salt started to be seen after 24 hours of culture, and garlic oil (P <0.0001) and nitrate (P = 0.0308) accumulated a large amount of hydrogen. Garlic oil showed a unique pattern of evolution in hydrogen evolution (Fig. 1C). After 24 hours, the amount of hydrogen production in the mixed treatment and nitrate was increased similarly. The ammonia nitrogen concentration of the culture was increased in nitrate (p <0.0001) and mixed treatment (p <0.0001) (Fig. 1D). Fumarate decreased ammonium nitrogen concentration and garlic oil treatment decreased to the same trend (p = 0.1806).

Fumarate increased total VFA concentration, but nitrate and garlic oil decreased total VFA concentration. There was no difference in total VFA between control and control (Table 4). Figures 2A-D show the effects of treatments on each VFA. In fumarate, nitrate and mixed treatments, the proportion of acetate was increased (p <0.0001), whereas the concentration of acetate in garlic oil was decreased (p <0.0001). The concentration of propionate was increased to 13% and 11% in fumarate and mixed treatment (p <0.0001). On the other hand, nitrate (p = 0.3518) and garlic oil (p = 0.9987) showed no effect on propionate. Butyrate and valerate were inhibited in nitrate and mixed treatment (p <0.0001). The other treatments were not different from the control (p> 0.05). A / P ratio was affected by treatments; Fumarate <garlic oil <control <mixed treatment <nitrate (Table 4).

Table 4 also shows the effects of treatments on IVDMD and pH. Mixed treatments and nitrate showed lower digestibility of dry matter. Garlic oil and nitrate showed higher pH than control, while fumarate and mixed treatment showed lower pH than control.

variable
Treatment SEM
valence Control GO FM NT GONF Tr Tm I pH 6.69 ^c 6.70 ^b 6.57 ^e 6.71 ^a 6.60 ^d 0.01 *** *** *** IVDMD (%) 51.64 ^a 51.01 ^a 50.99 ^a 46.57 ^b 47.79 ^b 1.29 *** *** *** Total VFA (mM) 44.66 ^b 41.85 ^d 49.80 ^a 43.78 ^c 45.27 ^b 1.68 *** *** *** C2: C3 2.61 ^c 2.41 ^d 2.33 ^e 2.79 ^a 2.67 ^b 0.04 *** *** ***

▷ Variable: IVDMD, in vitro dry matter disappearance; VFA, volatile fatty acids; C2: C3, acetate: propionate.

▷ Treatment: Control = no additive, GO = garlic oil at 50 mg / L; FM = fumarate at 15 mM; NT = nitrate at 20 mM; GONF = a blend of garlic oil at 50 mg / L + fumarate at 15 mM + nitrate at 20 mM.

▷ significance: probability for Tr = treatment; Tm = incubation time; I = interaction between treatment and incubation time.

*** mean P <0.001.

▷ ae Means within a row with different superscripts differ (p <0.05).

Biogas, methane and total VFA production in the mixed treatment (GONF; mixture of garlic oil at 50 mg / L + fumarate at 15 mM + nitrate at 20 mM) are shown in Table 5 below.

As shown in Table 5, mixed treatments inhibited the methane production without adversely affecting the total VFA concentration, although there was a discrepancy with the predicted values from the response surface model in 24-hour in vitro fermentation.

Fermentation parameters Predicted value Observation value Total biogas (ml) 70.00 55.00 Methane (ml) 0.03 0.20 Total VFA (mM) 72.00 66.11

▷ Fermentation parameters: VFA, volatile fatty acids.

The effects of treatments on microbial communities are shown in Table 6.

As shown in Table 6, Methanogens was not significant in the 0 hour value of the treatment, and methanogens number was decreased 48 hours after the nitrate and mixed treatment. Total bacterial counts were not affected by treatments. In conclusion, nitrate and mixed treatments show significantly lower methanogens than other treatments.

microbe
Treatment SEM
valence Control GO FM NT GONF Tr Tm I Methanogens 2.61 ^a 2.49 ^a 2.48 ^a 1.91 ^b 1.65 ^b 0.06 *** NS *** Total bacteria 10.87 11.02 10.92 10.86 10.38 0.16 NS NS NS Met: Total bact 0.24 ^a 0.23 ^a 0.23 ^a 0.18 ^b 0.16 ^b 0.01 *** NS ***

Unit: mean copy number / 1g of pellet, log10 at 0h and after 48h in vitro batch fermentation.

▷ Microorganism: Met: Toatal bact, Methanogen: Total bacteria.

▷ Treatment: Control = No additive, GO = garlic oil at 50 mg / L; FM = fumarate at 15 mM; NT = nitrate at 20 mM; GONF = a blend of garlic oil at 50 mg / L + fumarate at 15 mM + nitrate at 20 mM.

▷ significance: probability of Trt = treatment; Time = incubation time; Trt × Time = interaction between treatment and incubation time.

▷ ae Means within a row with different superscripts differ (p <0.05).

3. Review

1) Methane and hydrogen production

The mixed treatment of the present invention reduced methane without affecting the overall VFA concentration. Mixed treatments and nitrate showed similar trends in biogas, methane and hydrogen production, and nitrate was suppressed to a higher level than other treatments. Mixed treatments and nitrates have a negative impact on the number of methanogen populations. Nitrate has been reported to be effective in methane reduction in vitro (Sar et al., 2005a; Zhou et al., 2012) and in vivo (Sar et al., 2004; Van Zijderveld et al., 2010) Nitrate treatments inhibited methane production in all experiments.

Two mechanisms by which nitrate inhibits rumen methane production are the use of nitrate toxicity or the depletion of methanogen precursors (Allison et al., 1981). Ruminal methane production is very sensitive to nitrate and about 3 mM of nitrite has been reported to inhibit methane production (Iwamoto et al., 2002).

Garlic oil selectively inhibits arthropods including methanogens at a sufficient concentration (Busquet et al., 2005a, b; Chaves et al., 2008). In the experiment with 15 mg / L, garlic oil decreased methane production by more than 30% without affecting the number of methanogenic bacteria. As the incubation time increased, the apparently contradictory results of garlic oil could be explained by the mechanism of the rumen microorganisms. The hydrogen concentration reached a maximum at 12 hours and was significantly higher at the previous time. By the time it became similar to the control, the concentration of hydrogen decreased. Garlic oil inhibited methanogens when cultured for a short time, but the hydrogen began to deplete as the microbial community adapted. Some of the garlic oil mixed treatments, however, did not show similar trends to hydrogen because they contained H2 sink, nitrate and fumarate.

Fumarate theoretically reduces the production of methane. However, studies of previous batch cultures have shown that the effect of fumarate on reducing methane is relatively small. Methane is reduced by an average of 2.3% and the maximum is 18% (Ungerfeld et al., 2007). The reduction of methane to less than 10% in fumarate is consistent with the results of this study. Fumarate increased the propionate fraction and maintained total VFA concentration.

2) Volatile fatty acids

The results of this study showed that VFA and butyrate were reduced in vitro, including nitrate, as in previous studies (Sar et al., 2005a, b; Takahashi, 1989). On the other hand, nitrate-containing propionate did not affect concentrations in vitro (Sar et al., 2005a, b) and in vivo (Allison & Reddy, 1984; Farra & Satter, 1971) This can be explained as a partial nitrate masking effect, using a rich fermented feed to produce high levels of propionate. Zhou et al. (2012) and Alaboudi and Jones (1985), the increase in the ratio of acetate to propionate through the addition of nitrate was consistent with the results of this study. Other effects of acetates and propionates resulted in an increase in C2: C3. Changes in these ratios were reported by Takahashi (1989). Previous studies have reported that the addition of garlic oil inhibits VFA production and has a high VFA concentration (Busquet et al., 2005b; Cardozo et al., 2005). Chaves et al. (2008) and Yang et al. (2007) did not show a negative effect on the total VFA of garlic in vivo results, which is in contrast to previous studies. The positive effects of fumarate counteract the negative effects of total VFA in nitrate, garlic oil and mixed treatment. Fumarate has been reported to increase propionate, molar proportion, total VFA and acetate in previous studies (Garcia-Martinez et al., 2005; Isobe & Shibata, 1993; Lopez et al., 1999). Ungerfeld et al. (2007) reported that fumarate can thermodynamically convert to acetate.

4. Conclusion

The mixture of the present invention in which garlic oil, nitrate and fumaric acid salt are mixed at a ratio of 0.01 to 0.1: 0.5 to 2.0: 0.5 to 2.0 (g / L) is used in a state in which the amount of methane produced in the rumen is not negatively affected Respectively. The mixed treatment inhibited the methanoginic archaea, but kept the total number of bacteria constant. Acetate and propionate ratios were significantly increased with addition of mixed treatment. Nitrate showed an equivalent level of its effect on reducing methane production and methanogens as a mixed treatment. However, the total VFA concentration was lower than that of the mixed treatment. 50 mg / L of garlic oil decreased methane production. However, microbial populations were not affected. Garlic oil also inhibited total VFA production. The fumarate salt inhibited a small amount of methane compared with the case of adding the fumarate salt alone. The fumarate salt also increased total VFA production and had no effect on viable cell count.

The mixed feed composition of garlic oil, nitrate, and fumarate of the present invention improved the methane suppressing effect negatively without affecting the VFA concentration.

The methane gas reduction feed composition containing garlic extract, nitrate salt and fumarate salt of the present invention has an effect of suppressing global warming by reducing methane production of ruminants.

In addition, the livestock feed composition of the present invention does not affect the microbial fermentation in the rumen, so that the efficiency of feed utilization is improved, and it can contribute to the development of the livestock-related industry, which is industrially applicable.

Claims (5)

Garlic extract, nitrate and fumaric acid salt. The composition of claim 1, wherein the garlic extract, nitrate, and fumarate are mixed at a ratio of 0.01 to 0.1: 0.5 to 2.0: 0.5 to 2.0 (g / L). The composition of claim 1, wherein the garlic extract is a garlic oil. 4. The livestock feed according to any one of claims 1 to 3, wherein the composition is added in a weight ratio of 0.1 to 0.5% based on the feed weight. A method for inhibiting the production of methane gas in rumen by administering a livestock feed composition according to any one of claims 1 to 3 to a ruminant.

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