JP7531195B2 - Methane production suppression composition for ruminants - Google Patents

Description

Application of Article 30, Paragraph 2 of the Patent Act March 28, 2018 Disclosure in "Abstracts of Lectures at the 124th Annual Meeting of the Japanese Society of Animal Science" [Publications, etc.] March 29, 2018 Disclosure due to presentation at the "124th Annual Meeting of the Japanese Society of Animal Science"

The present invention relates to a methane production suppression composition for ruminants, which is used to suppress methane production from ruminants.

A large number of microorganisms live in the rumen of ruminant animals such as cows. These microorganisms break down the feed they ingest and use it as an energy source, while they expel carbon dioxide and methane (gases) from their bodies, mainly through exhalation.
Carbon dioxide and methane gas generated from exhaled breath are said to have a large impact on global warming, and in particular, reducing the emission of methane gas, which has a greenhouse effect 25 times greater than that of carbon dioxide, is a major environmental issue. It has been reported that the amount of methane gas emitted from ruminants on earth accounts for 15-20% or approximately 40% of the total methane gas emissions, and therefore reducing the emission of methane gas from ruminants is an important issue from the perspective of environmental protection.
Conventionally, methods reported for suppressing methane gas emissions from ruminants include the use of antibiotics and specific compounds as feed additives (Patent Document 1), the addition of cyclodextrin inclusion compounds of components such as eucalyptus oil to feed (Patent Document 2), and the addition of flavanone glycosides extracted from citrus fruits, such as neohesperidin, to feed (Patent Document 3).

WO2012/160191 JP 2002-281912 A WO2013/156574

An object of the present invention is to provide a composition for addition to ruminant feed that can inhibit methane gas production in ruminants, and a method for producing the same.
A further object of the present invention is to provide a composition for adding to ruminant feed, which can suppress methane gas production without suppressing the digestibility of the feed or the amount of fatty acids due to feed decomposition, and a method for producing the same.

The present inventors discovered that methane production in ruminants can be suppressed by using a composition containing a saponified oil or fat, with fatty acid calcium as the main component, and silica gel as an excipient, and thus completed the present invention.
That is, the present invention provides the following.
<1> A methane production inhibitor composition for ruminants, comprising a saponified oil or fat containing fatty acid calcium as a main component, and silica gel.
<2> The methane production inhibitor composition for ruminants according to <1>, wherein the saponification product of fats and oils containing fatty acid calcium as a main component is a saponification product of a mixture of (a) fats and oils, (b) calcium hydroxide, (c) water, and (d) lipase.
<3> The methane production inhibitor composition for ruminants according to <2>, wherein in the mixture, a ratio of the number of moles of (a) total fatty acids derived from oils and fats to the number of moles of (b) calcium hydroxide is 1 or more.
<4> The methane production inhibitor composition for ruminants according to <2> or <3>, wherein the mixture further contains (e) caramel having antioxidant properties.
<5> The methane production inhibiting composition for ruminants according to any one of <2> to <4>, wherein (a) triglyceride in the oil or fat contains unsaturated fatty acids as main constituent fatty acids.
<6> A feed composition for ruminants, comprising the methane production inhibiting composition for ruminants according to any one of <1> to <5>.
<7> (a) fats and oils,
(b) calcium hydroxide;
(c) water, and (d) a lipase;
a step of reacting a mixture containing the above-mentioned components to produce a saponified oil and fat containing fatty acid calcium as a main component, and a step of adding silica gel to the saponified oil and fat containing fatty acid calcium as a main component;
A method for producing a methane production inhibiting composition for ruminants, comprising:
<8> The method according to <7>, wherein the ratio of the number of moles of the total fatty acids derived from (a) oils and fats to the number of moles of the calcium hydroxide (b) in the mixture is 1 or more.
<9> The method according to <7> or <8>, wherein the mixture further contains (e) caramel having antioxidant properties.
<10> The method according to any one of <7> to <9>, wherein (a) the triglyceride in the oil or fat contains unsaturated fatty acids as major constituent fatty acids.

By using the methane production inhibiting composition for ruminants of the present invention, it is possible to inhibit methane production that accompanies the digestive activity of ruminants. Furthermore, since the methane production inhibiting composition for ruminants of the present invention is in granular form, it can be easily added to ruminant feed as an energy source.

1 shows the amount of methane produced when each sample was added in Example 1. 1 shows the amount of carbon dioxide produced when each sample was added in Example 1. 1 shows the change over time in the amount of methane produced when each sample was added in Example 2. 1 shows the change over time in the amount of carbon dioxide produced when each sample was added in Example 2. 1 shows the amount of methane produced when each sample was added in Example 3. 1 shows the amount of carbon dioxide produced when each sample was added in Example 3. 4 shows the amount of methane produced when each sample was added in Examples 4 and 5. The amount of carbon dioxide produced when each sample was added in Examples 4 and 5 is shown.

The methane production inhibitor composition for ruminants according to the first aspect of the present invention contains a saponified oil and fat containing fatty acid calcium as a main component, and silica gel as an excipient.

In this specification, the term "methane production-inhibiting composition for ruminants" refers to a composition for inhibiting methane production during the digestion process of feed for ruminants by indirectly administering the composition to ruminants by mixing the composition with feed for ruminants or by directly administering the composition to ruminants without mixing the composition with feed. Ruminants include, but are not limited to, ruminants such as cows, goats, and sheep.
There are no particular limitations on the raw materials and other additives of the ruminant feed to which the "methane production inhibiting composition for ruminants" of the present invention is added, so long as they are commonly used.

In this specification, "saponified oils and fats containing fatty acid calcium as a main component" refers to a saponified oil and fat containing fatty acid calcium as a main component, obtained by a saponification reaction of an oil and fat containing triglyceride as a main component with an alkaline solution containing at least calcium such as calcium hydroxide. The saponified oil and fat containing fatty acid calcium as a main component may contain residues derived from the raw oil and fat, such as triglyceride, mono- or diglyceride, fatty acid, and glycerin.
"Containing fatty acid calcium as a main component" means that fatty acid calcium is contained in the largest amount (as the main component) among all the components in the saponified product.
In addition, in this specification, "oils and fats" refers to those containing triglycerides as a main component. "Containing triglycerides as a main component" means that among the components in oils and fats, triglycerides of fatty acids are contained in the largest amount (as the main component).
The saponification product of fats and oils containing fatty acid calcium as a main component may further contain lipase for promoting the saponification reaction, caramel having antioxidant properties, and the like, as described below.

In this specification, the term "main component" in expressions such as "mainly the component", "contained as the main component", and "contained as the main constituent fatty acid" means that the component contained in the largest amount among all the constituent components. Furthermore, it is preferable that the amount is 50% by mass or more among all the constituent components.

In the present invention, the saponification product of fats and oils containing fatty acid calcium as a main component is preferably a saponification product of a mixture of (a) fats and oils, (b) calcium hydroxide, (c) water, and (d) lipase.
The mixture for obtaining the saponified product containing fatty acid calcium as a main component may further contain (e) caramel having antioxidant properties.

In this specification, the (a) oil and fat is not particularly limited and various types can be used. For example, the raw oil and fat may be oil and fat of animal, vegetable, or other origin. Examples include vegetable oils such as lard, tallow, linseed oil, canola oil, sunflower oil, safflower oil, cottonseed oil, canola oil, olive oil, and corn oil.

In order to further reduce methane production, in the present invention, the triglyceride in the (a) oil or fat is preferably a "triglyceride containing unsaturated fatty acids as the main constituent fatty acids." A triglyceride containing unsaturated fatty acids as the main constituent fatty acids means a triglyceride containing at least one unsaturated fatty acid as the main constituent fatty acid.
The amount of unsaturated fatty acids relative to the mass of the triglyceride containing unsaturated fatty acids is preferably 50% by mass or more, more preferably 70% by mass or more, even more preferably 75% by mass or more, and even more preferably 80% by mass or more.

At least one type of unsaturated fatty acid is preferably a long-chain unsaturated fatty acid having 16 or more carbon atoms, more preferably 18 or more carbon atoms. The number of unsaturated bonds is preferably 2 or more. More preferably, the number of unsaturated bonds is 3 or more. Most preferably, it is an n-3 fatty acid. Specific examples of n-3 fatty acids include α-linolenic acid, eicosapentaenoic acid, and docosahexaenoic acid. Examples of fats and oils containing the above-mentioned unsaturated fatty acids as constituent fatty acids include linseed oil, perilla oil, rapeseed oil, soybean oil, fish oil, chia seed oil, green nut oil, and the like, or mixtures thereof, specifically, for example, a mixture of rapeseed oil and linseed oil, hard palm oil, and a mixture of rapeseed oil and soybean oil.
Furthermore, the n-6/n-3 mass ratio in the fatty acid composition of the raw material oil or fat is 30 or less, preferably 10 or less, more preferably 5 or less, and most preferably 1 or less.

For example, the raw material oil and fat may contain 20 to 95% by mass, preferably 50 to 95% by mass, of unsaturated fatty acids.Preferred examples include those containing preferably 1 to 70% by mass, more preferably 5 to 70% by mass, even more preferably 10 to 70% by mass, and most preferably 50 to 70% by mass of n-3 fatty acids.
The raw material oil or fat may further contain saturated fatty acids. The amount of saturated fatty acids is not particularly limited, but is preferably in the range of about 5 to 80% by mass, and more preferably about 5 to 50% by mass.

(a) The fat or oil is preferably 65 to 85% by mass, and more preferably 70 to 80% by mass, of the total mass of the raw material mixture for producing the saponified fat or oil, containing fatty acid calcium as the main component.

(b) Any calcium hydroxide may be used. In addition, since calcium oxide becomes calcium hydroxide in the presence of water, in the present invention, "calcium oxide" (quicklime) may be used instead of "calcium hydroxide" (Ca(OH) ₂ ) (slaked lime).
In the production of a saponified oil containing fatty acid calcium as a main component, the ratio of the total fatty acid moles derived from the triglyceride to the calcium hydroxide moles is preferably 0.8 or more. More preferably, it is 1.8 or more, even more preferably 2.5 or more, and even more preferably 2.7 or more. There is no particular upper limit, but from the viewpoint of not lowering the activity of ruminal microorganisms due to unsaponified oils and fats, it is preferably 4 or less.

The molecular weight of fats and oils used in calculating the molar ratio is calculated by multiplying the molecular weight of each fatty acid by its mass percentage based on the analysis value of the fatty acid composition in the fat and oil, and calculating the average fatty acid molecular weight, assuming that the fatty acid with the average fatty acid molecular weight is in the form of a triglyceride. For example, the average fatty acid molecular weight of fats and oils containing 50% by mass each of palmitic acid (C16:0) and stearic acid (C18:0) is 270 (256 x 0.5 + 284 x 0.5), so the molecular weight calculated as a triglyceride is 848 (270 x 3 - 92 - (18 x 3)).

The amount of calcium hydroxide (b) added is preferably 3 to 20 mass % based on the total mass of components (a) to (c) or the total mass of components (a) to (c) and (e), more preferably 4 to 15 mass %, and even more preferably 5 to 10 mass %.
When "calcium oxide" is added in place of "calcium hydroxide", "calcium oxide" (molecular weight 56) is added so that the amount becomes the above amount when converted from the molecular weight to "calcium hydroxide" (molecular weight 74).

In the present invention, when producing the "saponified oil or fat containing fatty acid calcium as a main component", (c) water may be added. (c) Water (added water) also plays a role as a reaction solvent. When calcium oxide is used, it reacts with calcium oxide to provide calcium hydroxide. The "water" may be distilled water, deionized water, tap water, or the like, as appropriate.
The amount of water added is preferably 15% by mass or less, more preferably 12% by mass or less, and even more preferably 11% by mass or less, based on the total mass of components (a) to (c) or the total mass of components (a) to (c) and (e). The lower limit of the amount of water is preferably 1% by mass or more, more preferably 3% by mass or more, even more preferably 5% by mass or more, and even more preferably 6% by mass or more.

In the present invention, the mass ratio of (c) water to (a) fats and oils may affect the reaction efficiency of the fatty acid to the divalent metal salt, the moldability of the obtained composition, and other properties. In particular, since the aqueous phase containing the oil and caramel is likely to separate when placed in a mold, the mass ratio of (c) "water" to (a) fats and oils is preferably 0.05 or more, more preferably about 0.05 to 0.50, even more preferably 0.07 to 0.30, even more preferably 0.08 to 0.20, and even more preferably 0.10 to 0.18.

In the present invention, when producing a "saponified oil or fat containing fatty acid calcium as a main component," (d) lipase may be added. In this specification, the term "lipase" refers to any lipase of animal, vegetable, or microbial origin, and is not limited thereto, but it is preferable that the lipase has a strong ability to decompose oil or fat in an alkaline condition and is highly heat-resistant. Examples of lipases of microbial origin include Amano Enzyme Lipase AY "Amano" 30SD, Lipase R "Amano", Lipase A "Amano" 6, Lipase MER "Amano", Lipase DF "Amano" 15, Lipase G "Amano" 50, Meito Sangyo Lipase MY, Lipase OF, Lipase PL, Lipase PLC, Lipase QLM, Lipase QLC, Novozyme Lipozyme TL 100L, Lipozyme CALB L, Palatase 2000L, etc. The amount of lipase added can be adjusted depending on the fat-decomposing power.

In the present invention, when producing a "saponified oil or fat containing fatty acid calcium as a main component", (e) caramel having antioxidant properties may be added. "Caramel having antioxidant properties" refers to caramel having antioxidant properties obtained by heat-treating sugars, and a representative example is the caramel described in JP-A-8-312224.
(e) When caramel having antioxidant properties is added, its content is preferably 1 to 10 mass %, more preferably 2 to 7 mass %, based on the total mass of the above-mentioned components (a) to (c) and (e).

In this specification, the antioxidant properties of caramel are evaluated based on the reactivity of caramel with 1,1-diphenyl-2-picrylhydrazyl (DPPH). That is, an appropriate amount of caramel was dissolved in 4 ml of ethanol, to which 1 ml of 0.5 mM DPPH ethanol solution was added, and the mixture was kept at 37°C for 30 minutes, after which the absorbance at 517 nm was measured to determine the amount of absorbance reduction per mg of caramel. Similarly, the DPPH antioxidant properties of a solution of Trorox were measured to determine the Trorox equivalent per mg of caramel. From the viewpoint of the stability against oxidation of unsaturated fatty acids in a composition containing fatty acid calcium, it is preferable that the Trorox equivalent is 3000 nmol Trorox equivalent/ml or more, more preferably 4000 nmol Trorox equivalent/ml or more, and even more preferably 5000 nmol Trorox equivalent/ml or more. This is because caramel with low antioxidant properties (less than 3000 nmol Torox equivalent) needs to be added in large amounts to a composition containing fatty acid calcium, making it difficult to produce fatty acid calcium that can be molded into powder form.

"Caramel having antioxidant properties" can be obtained by heating a high-concentration aqueous solution of glucose in the presence of a basic compound, preferably at 100° C. or higher, more preferably at 120 to 150° C. The heating time is preferably about 1 to 10 hours, more preferably about 2 to 8 hours.
The pH of the resulting caramel is preferably 5 or less.

Examples of basic compounds include alkali metal hydroxides such as sodium hydroxide and potassium hydroxide, alkali metal carbonates such as sodium carbonate and potassium carbonate, alkali metal bicarbonates such as sodium bicarbonate and potassium bicarbonate, alkali metal salts (e.g., sodium salts and potassium salts) or ammonium salts of organic acids such as acetic acid and citric acid, and ammonium hydroxide.

The pH of the high concentration aqueous glucose solution before heat treatment is preferably 7 or more, more preferably 8 or more, and most preferably 9 or more. If the reaction temperature (heat treatment temperature) is lower than 120°C, the reaction does not proceed sufficiently. If it is higher than 150°C, not only is there a high risk of the reaction system solidifying and carbonizing, but the antioxidant properties of the resulting caramel are also reduced, which is not preferred. The preferred reaction temperature is 125 to 135°C, and the most preferred reaction temperature is around 130°C. If the amount of basic compound is less than 1 part by mass, the reaction does not proceed sufficiently. In other words, caramel with antioxidant properties cannot be obtained. If the amount is more than 10 parts by mass, the reaction proceeds vigorously, a large amount of bubbles are generated in a short time, and there is a risk of the reaction vessel boiling over, creating a risk of danger. If the reaction time is less than 1 hour, the reaction may not proceed sufficiently. As the reaction time increases, the reaction proceeds and the viscosity tends to gradually increase.

In the present specification, the term "excipient" refers to an additive that is added to granulate the saponified oil and fat containing the fatty acid calcium as a main component. The present invention includes silica gel as an excipient. Other excipients, such as diatomaceous earth, vermiculite, bentonite, zeolite, talc, kaolin, silicic acid, calcium silicate, magnesium silicate, silicon anhydride, and natural aluminum silicate, may be added to the silica gel to such an extent that the effect of the silica gel is not impaired.
In a first embodiment of the present invention, the excipient is silica gel. The silica gel may have an average particle size, preferably between 0.1 and 200 μm, more preferably between 1 and 25 μm. In this specification, "average particle size" means the particle size measured by laser diffraction (according to conditions ISO 13320).
The average particle size of the silica gel is more preferably 1 to 20 μm, even more preferably 0.5 to 10 μm, and even more preferably 1 to 10 μm or 10 to 18 μm.

The silica gel contained as an excipient in the present invention can be a natural product, a synthetic product, or crystalline or amorphous silica, and synthetic amorphous silica sold under the trade names Carplex (Evonik) and Sipernat (Evonik) can be used.

In the composition of the first aspect of the present invention, the amount of silica gel used is preferably about 5 to 100 parts by mass, and more preferably about 5 to 50 parts by mass, relative to the amount of saponified substance (the total mass of components (a) to (c) or the total mass of components (a) to (c) and (e)) (100 parts by mass).
In the present invention, when the composition according to the first aspect of the present invention is used by mixing it with a feed composition, an appropriate amount may be added. For example, the amount of fat or oil in the saponified fat or oil product may be preferably 1 to 10% by mass, more preferably 1 to 5% by mass, based on the mass of the substrate (e.g., barley) in the feed composition.

A second aspect of the present invention is a feed composition for ruminants, which comprises the above-mentioned methane production inhibiting composition for ruminants.
The composition of the feed is not particularly limited, but includes, for example, roughage such as hay or rice straw made by drying the stems and leaves of plants, and silage that has been fermented with lactic acid to enhance storage properties, as well as concentrated feed that is a feed with increased energy and protein content by blending grains such as barley, corn, and koryan, oil cake from the residue of pressed soybeans, bran, rice bran, etc. Roughage and concentrated feed may be appropriately mixed.

A third aspect of the present invention is
(a) fats and oils,
(b) calcium hydroxide;
(c) water, and (d) a lipase;
a step of reacting a mixture containing the above-mentioned components to produce a saponified oil and fat containing fatty acid calcium as a main component, and a step of adding silica gel to the saponified oil and fat containing fatty acid calcium as a main component.
A method for producing a methane production inhibiting composition for ruminants, comprising:
It is.

In the above method, the definitions and preferred embodiments of (a) fats and oils, (b) calcium hydroxide, (c) water, (d) lipase, "excipient,""saponified fats and oils containing fatty acid calcium as a main component," and "methane production inhibitor composition for ruminants" are as described above.
The mixture containing (a) to (d) may further contain (e) caramel having antioxidant properties, and the definition and preferred embodiments of (e) caramel having antioxidant properties are as described above.

The above method includes a step of reacting a mixture containing (a) fats and oils, (b) calcium hydroxide, (c) water, and (d) lipase, and preferably further containing (e) caramel having antioxidant properties, at an appropriate temperature, preferably a temperature of 60°C or lower, to produce a saponified product of fats and oils containing fatty acid calcium as a main component.
The methane production inhibiting composition for ruminants of the present invention can be produced by adding silica gel to the saponified oil or fat.
Although not limited to the following, in more detail, the method of the present invention includes, for example, mixing (a) fats and oils, and (b) calcium hydroxide, adding (c) water, and (d) lipase to the mixture, and optionally adding (e) caramel having antioxidant properties, and carrying out a reaction at an appropriate temperature, and then adding silica gel.

[Production Example 1]
25 parts by mass of water, 80 parts by mass of a sugar (glucose), and 5 parts by mass of a basic compound (sodium citrate) were mixed and reacted at 130°C for 3 hours, and finally 40 parts by mass of water was added to obtain 120 parts by mass of Caramel 1.

[Example 1]
To 78 parts by mass of linseed oil, which was obtained by deacidifying, decolorizing and refining oil extracted from linseed seeds, 7 parts by mass of slaked lime (calcium hydroxide) were added, and the mixture was mixed and stirred in a mixing tank.
To the mixture, 5 parts by mass of caramel having antioxidant properties (Caramel 1 produced in Production Example 1), 10 parts by mass of water, and 0.015 parts by mass of lipase (Amano AK) were mixed, the liquid temperature was raised to 40° C., and the mixture was stirred for a further 30 minutes. Further, 27.3 parts by mass of silica gel was added and mixed uniformly to produce Sample 1.

[Production Examples 1 to 3 and Comparative Example 1 of Saponified Oils and Fats Containing Fatty Acid Calcium as the Main Component]
The saponified oils and fats of Production Reference Examples 1 to 3 and Comparative Example 1 were produced in the same manner as in Example 1, except that the amounts of each component were changed to those shown in Table 1 and no silica gel was added (see Table 1). The particle size of the rolled barley powder in Comparative Example 1 was too large to be measured by the laser method, and was approximately 1 mm or more. The amount of rolled barley powder in Table 1 (75 parts by mass) is the minimum amount required to make the composition into a solid form (powder). The control was a feed containing only rolled barley and no saponified oils and fats.

Table 1

The digestibility, pH and nitrogen content of each saponified product (Table 2), as well as the total VFA (volatile fatty acid) concentration (Table 3) and the amount of each gas produced (Table 4) were measured. The measurement methods are described below. In Table 2, "Amount added to substrate (%)" refers to the amount of fat (oil) equivalent in the added saponified oil product relative to the mass of rolled barley in the feed.

Table 2

Table 3

Table 4

<Adjustment of culture medium>
Rumen fluid (120 ml each) was collected from four sheep fitted with a ruminal cannula and fed a diet of concentrated feed and ryegrass straw in a 6:4 ratio, filtered through quadruple gauze, centrifuged at 500 x g, and the supernatants were mixed in equal amounts and diluted five-fold with artificial saliva to prepare a culture medium.
Each preparation was added to 40 ml of culture solution using 0.5 g (dry weight) of rolled barley as a substrate, and cultured in a 50 mL centrifuge tube at 39°C for 48 hours (three replicates per treatment).

Measurement item 1) Amount of gas produced The amount of gas produced from 0 to 48 hours after cultivation was recorded using the following method. The atmosphere inside the culture centrifuge tube was replaced with 99.9% _CO2 gas to create anaerobic conditions, and the tube was closed with a silicone stopper with a 50 mL syringe. The amount of gas produced at 3, 6, 9, 12, 18, 24, 30, 36, 42, and 48 hours after cultivation was read using the scale on the syringe. The syringe was replaced 18 hours after cultivation when it was full of gas.

2) Gas Composition The concentrations of _CO2 and _CH4 in the gases generated during 18 hours of culture were measured by gas chromatography (conditions as follows).
Equipment: Inolga Portable TCD gas chromatograph manufactured by GLC Sciences Column: 1/16 x 1.0 mm φ x 3 m
Stainless steel packed column (packing material Porapak Q 80/100mesh)
Carrier gas: He 4 mL/min Detector: TCD

3) Digestibility The digestibility of dry matter (DM) and crude protein was measured by the following method.
The disappearance of dry matter and crude protein in the culture medium after 48 hours of culture under anaerobic conditions was measured by the following method.
The dry matter intake was determined by measuring the weight of the residue after drying at 105° C. for 3 hours before and after the culture, and the change from the weight before the culture was calculated as the amount lost.
The amount of crude protein was determined by the Kjeldahl method before and after the culture, and the change after the culture from the amount of protein before the culture was calculated as the amount of loss.

4) Fermentation products The pH, ammonia nitrogen and VFA concentrations of the supernatant after cultivation were measured using the following apparatus and method.
pH: Horiba glass electrode pH meter Ammonia nitrogen: Conway unit microdiffusion method (Conway 1962)
VFA concentration: The VFA concentration was analyzed by gas chromatography according to the "Manual for Clinical Biochemical Tests for Biochemical Disease Identification in Livestock" by the National Agriculture and Food Research Organization.
http://www.naro.affrc.go.jp/laboratory/niah/disease/biochem/gc_vfa/index.html
The samples were treated and prepared for injection into the gas chromatograph according to the procedure described above.

Gas chromatograph conditions and equipment: Shimadzu GC-2014 FID gas chromatograph column, packed glass column (Thermon 3000-2% Shimalite TPA60/80 3.2 mmφ×2.1 m; Shimadzu)
Column temperature: 120°C
Injector and detector temperature: 200° C.
Carrier gas: _N2
Combustion gas; _H2
Detector: FID

Statistical processing The measurement data were analyzed using the GLM procedure of SAS (2008).
Yijk=μ+Ti+Lj+(TL)ij+eijk
Yijk: variable, μ: total mean, Ti: effect of fatty acid, Lj: effect of dosage, (TL)ij+: effect of interaction between fatty acid and dosage, eijk: error.
Test for differences between least squares means: Tukey and Kramer's multiple comparison procedure (Kramer, 1956).
In addition, a separate multiple test was performed with the addition of a control group.

[Example 2 and Comparative Examples 2 to 7]
Granules were prepared in the same manner as in Example 1, except that the amount of fats and oils, and the type and amount of excipients were changed as shown in Table 5 (see Table 5). Comparative Example 7 is a case where only silica gel was added in an amount equivalent to the amount contained in the formulation. In Table 5, the amounts of silica gel, diatomaceous earth, vermiculite, bentonite, and zeolite added are the minimum amounts required to solidify (particulate or powder) the saponified material.

Table 5

The digestibility, pH and nitrogen content (Table 6) of each saponified product of Example 2 and Comparative Examples 2 to 7, as well as the total VFA (volatile fatty acid) concentration (Table 7) and each gas production amount (Tables 8 to 9) were measured. The measurement methods were as described above. In Tables 6 to 9, the control was the feed containing rolled barley alone with no saponified oils or fats added.

Table 6

Table 7

Table 8

Table 9

The changes over time in the total gas production, methane production, and carbon dioxide production are shown in graphs in Figures 3 and 4. As is clear from Figures 3 and 4, methane production inhibition was observed only when the silica gel of Example 2 was used, and no inhibition of methane production was observed when other excipients were used.
The methane production inhibiting composition for ruminants of the present invention can provide a feed for ruminants that is excellent in oxidation stability, odor, and palatability in addition to inhibiting methane production.

[Example 3]
Granules were prepared in the same manner as in Example 1, except that the amount of linseed oil and the type and amount of silica gel in Manufacturing Reference Example 2 were changed as shown in Table 10 (Example 3). In Tables 11 and 12, the control is a case in which rolled barley was used as feed only and no saponified oil or fat was added. The methane production and carbon dioxide production were measured in the same manner as in Example 2 (Tables 11 and 12).

Table 10

Table 11

Table 12

The amounts of methane and carbon dioxide produced after 48 hours in Example 3 were graphed (FIGS. 5 and 6).
When silica gel with a particle size of 8 μm was used at 10% (Example 3), the effect was slightly lower, but methane production was suppressed. From this result, it is presumed that the degree of methane production suppression varies depending on the amount of silica gel added to the saponified oils and fats (Example 3).
The methane production inhibiting composition for ruminants of the present invention in Example 3 was able to provide a feed for ruminants that was excellent in oxidation stability, odor, and palatability in addition to inhibiting methane production.

[Examples 4 and 5]
Granules were prepared in the same manner as in Example 1, except that the amount of linseed oil and the type and amount of silica gel in Manufacturing Reference Example 3 were changed as shown in Table 13 (Examples 4 and 5). Granules were also prepared in the same manner as in Example 4, except that lipase was not added (Comparative Example 8). In Tables 14-15, the control is a case in which rolled barley was used as feed only and no saponified oil or fat was added. Comparative Examples 2-3 were used in which no excipient was used. Methane production and carbon dioxide production were measured in the same manner as in Example 2 (Tables 14 and 15).

Table 13

Table 14

Table 15

The amounts of methane and carbon dioxide produced after 48 hours in Examples 4 and 5 and the Comparative Example were graphed (FIGS. 5 and 6).
When silica gel with a particle size of 8 μm was used (Example 4), the effect was less than that of 125 μm (Example 5), but both suppressed methane production.
The methane production inhibiting compositions for ruminants of the present invention in Examples 4 and 5 were able to provide feed for ruminants that was excellent in oxidation stability, odor, and palatability in addition to inhibiting methane production.

[Example 6]
Four steers in the late fattening stage were used in the test. Two steers were divided into two groups, one fed the formulation prepared in Example 4 and the other not fed the formulation, and the animals were allowed to acclimate for 12 days, after which each animal's breath was measured for three days. This test was repeated twice in a crossover test.
The fatty acid calcium preparation of the present invention (saponified oils and fats containing fatty acid calcium as the main component) was top-dressed on normal feed in an amount of 2% by mass per dry matter intake. Water and minerals were freely available.

According to the body weight of each individual, the following concentrated feed was fed at 80% by mass and roughage at 20% by mass.

Feed was given in a fixed amount, weighed, and the amount of remaining feed was measured periodically. The TDN ingested was calculated from the amount of feed ingested.
Body weight was measured on the first and last days of the test period. For the first test (first half), the body weight on the first day was used, and for the second test (second half), the body weight on the last day was used.
Gas measurements were made using a measurement system consisting of an in-line filter, a suction pump, a flow meter, a suction bottle, a laser-type portable gas analyzer, and a PC. A transparent acrylic panel (50 cm long x 80 cm wide (feeding tank width) x 50 cm deep) was placed on the feeding tank to cover three sides (left, right, and front) and the top, and exhaled gases were collected from two points, the top and bottom of the feeding tank.

The measurement items are as follows.
1. Feed intake (DMI kg/day)
2. Methane/carbon dioxide concentration ratio ( _CH4 / _CO2 )
3. Body weight (first and last day of the test period) (BW(kg))

<Estimation of methane production>
The methane/carbon dioxide concentration ratio (CH ₄ /CO ₂ ) was substituted into the following estimation formula to calculate the "methane production amount."
Methane production (L/day) = (heat production/4.9) _{x CH4} / _CO2 /100 x 1000
In the above formula, the daily heat production (Mcal) of a dairy cow / daily carbon dioxide amount (L/day) was calculated as an average value of 4.9 based on a measurement example of the amount of carbon dioxide production and heat production when dry dairy cows were fed hay (https://ocw.kyoto-u.ac.jp/ja/faculty-of-agriculture-jp/5240000/lecturenote).

The "heat production" (HP) (Mcal) was calculated based on the following concept.
Heat production (HP) = Energy used for growth + Maintenance energy = (Energy intake - Maintenance energy) x (1 - 0.52) + Maintenance energy = Energy intake x 0.48 + Maintenance energy x 0.52

In the above formula, the efficiency of energy intake to be used as maintenance energy was set at 0.52.
Energy used for growth: HPgrowth
Energy intake: ME intake (Mcal) = TDN (total digestible nutrients) intake x 3.61
Maintenance energy: ME maintenance (Mcal) = AE x BW ^0.75
BW (kg): cow's weight AE (Mcal): amount of active energy per beef cow's weight BW ^0.75 (kg) (= 0.1124) (From the Japan Feeding Standard "Beef Cattle" (2008 edition) Central Livestock Association)

Table 16

^*1乾物摂取量当りのメタン産生量 ^*2 体重当りのメタン産生量 Table 17

^*1 Methane production per unit of dry matter intake ^*2 Methane production per unit of body weight

When actually fed to cattle, the composition made from the saponified oils and fats of the present invention was able to suppress methane production in the breath.

Claims (10)

The present invention includes a saponification product of fats and oils containing fatty acid calcium as a main component, and silica gel,
A methane production inhibitor composition for ruminants, wherein the saponification product of fats and oils containing the fatty acid calcium as a main component is a saponification product of a mixture containing at least (a) fats and oils and (b) calcium hydroxide, and the ratio of the number of moles of total fatty acids derived from (a) fats and oils to the number of moles of (b) calcium hydroxide in the mixture is 2.5 or more. The methane production inhibiting composition for ruminants according to claim 1, wherein the mixture containing at least (a) a fat or oil and (b) calcium hydroxide further contains (c) water, and (d) a lipase. 3. The methane production inhibitor composition for ruminants according to claim 1 or 2 , wherein the ratio of the number of moles of (a) total fatty acids derived from oils and fats to the number of moles of (b) calcium hydroxide in the mixture is 2.7 to 4 . The methane production inhibiting composition for ruminants according to any one of claims 1 to 3 , wherein the mixture further comprises (e) caramel having antioxidant properties. The methane production inhibiting composition for ruminants according to any one of claims 1 to 4, wherein (a) triglyceride in the oil or fat contains unsaturated fatty acids as main constituent fatty acids. A feed composition for ruminants comprising the methane production inhibitor composition for ruminants according to any one of claims 1 to 5. (a) fats and oils,
(b) calcium hydroxide;
(c) water, and (d) a lipase;
a step of reacting a mixture containing the above-mentioned components to produce a saponified oil and fat containing fatty acid calcium as a main component, and a step of adding silica gel to the saponified oil and fat containing fatty acid calcium as a main component;
Including ,
In the mixture, the ratio of the number of moles of (a) total fatty acids derived from fats and oils to the number of moles of (b) calcium hydroxide is 2.5 or more.
A method for producing a methane production inhibitor composition for ruminants. The method according to claim 7, wherein the ratio of the number of moles of (a) total fatty acids derived from fats and oils to the number of moles of (b) calcium hydroxide in the mixture is 2.7 to 4 . The method according to claim 7 or 8, wherein the mixture further contains (e) caramel having antioxidant properties. The method according to any one of claims 7 to 9, wherein (a) the triglyceride in the oil or fat contains unsaturated fatty acids as the main constituent fatty acids.

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