A composite methane inhibitor, a sustained-release methane inhibitor micro-particle, and a preparation method and application thereof

Abstract

This invention relates to a composite methane inhibitor, sustained-release methane-inhibiting microparticles, their preparation method, and applications. The active ingredient of the composite methane inhibitor includes lauric acid monoglyceride and a composite additive, wherein the composite additive includes at least two of malic acid, fumaric acid, and tea saponin. This invention is the first to combine lauric acid monoglyceride, malic acid, fumaric acid, and tea saponin to prepare a composite methane inhibitor. It exhibits excellent methane emission inhibition by utilizing the synergistic effect between the components, and effectively avoids the adaptive evolution of rumen microorganisms to a single inhibitor through multi-target regulation. Furthermore, it uses safe and conventional raw materials, has low production costs, and possesses broad prospects for industrial application and research significance.

Description

Technical Field

[0001] This invention relates to the field of feed additive technology, specifically to a compound methane inhibitor, a sustained-release methane inhibitor microparticle, its preparation method, and its application. Background Technology

[0002] Against the backdrop of global climate change, methane (CH4), as the second largest anthropogenic greenhouse gas, has a warming potential 28 times greater than carbon dioxide (CO2), becoming a significant bottleneck restricting the sustainable development of animal husbandry. In dairy farming, CH4 mainly originates from rumen microbial fermentation. Rumen microorganisms convert carbohydrates and cellulose into digestible substances, including short-chain fatty acids such as acetic acid, propionic acid, and butyric acid, while simultaneously producing large amounts of CO2 and hydrogen. Methanogenic bacteria in the rumen use CO2 and hydrogen as substrates to produce methane through a reduction reaction. CH4 production not only causes a 2%–12% loss of feed energy but also significantly increases the industry's carbon footprint, reflecting the urgent need for optimization and upgrading of current farming technologies.

[0003] Although various feed additives with the potential to inhibit methane emissions in ruminants have been identified, such as algae (seaweed, brown algae, etc.), plant extracts (flavonoids, tannins, cinnamaldehyde, allicin, etc.), vegetable oils (lauric acid, coconut oil, etc.), and organic compounds (malic acid, fumaric acid, sulfates, nitrates, etc.), the addition of single components still has significant drawbacks. These additives either suffer from poor palatability, interference with normal rumen fermentation, limited and unstable inhibitory effects, or pose toxicity risks, are subject to regulatory restrictions, and are costly, making it difficult to meet the needs of industrial application. For example, 3-nitrooxypropanol, monensin, and nitrates, while effective, are constrained by regulations and safety concerns; lauric acid and its monoglycerides affect feed intake and fiber degradation; organic acids (such as fumaric acid and malic acid) and Schizochytrium powder are costly and have inconsistent and unstable effects in dairy cows; tea saponins, essential oils, and tannins have variable and unsustainable effects. These limitations severely restrict their industrial application, necessitating the development of novel compound inhibitors to overcome these technological bottlenecks.

[0004] Synergistic regulation of rumen fermentation through the combination of additives with complementary mechanisms has become an important research direction for overcoming existing technological bottlenecks. Theoretically, medium-chain fatty acids (such as lauric acid or its monoglycerides), organic acids, and tea saponins can respectively produce synergistic emission reduction effects by inhibiting specific microorganisms, promoting propionic acid production (competitively consuming hydrogen), and disrupting the interaction between methanogens and protozoa. While existing additive technologies (such as Schizochytrium powder using encapsulation processes) provide a framework, their targets are relatively singular, and the long-term stability and economic viability of their application remain to be verified.

[0005] To address the technical challenges of methane inhibitors, such as high cost, poor palatability affecting feed intake, and unstable and unsustainable inhibitory effects, there is an urgent technical need to develop a novel composite additive composed of safe and natural ingredients that can achieve stable and efficient emission reduction through synergistic effects of multiple mechanisms without affecting or even improving animal production performance. Summary of the Invention

[0006] The purpose of this invention is to address at least one deficiency in the prior art by providing a sustained-release methane inhibitor, microparticles, its preparation method, and its applications. This aims to solve problems in related technologies such as high cost, poor palatability affecting feed intake, and unstable and unsustainable inhibitory effects.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first aspect of the present invention is to provide a composite methane inhibitor, wherein the active ingredient of the composite methane inhibitor includes monoglyceride laurate and a composite additive, wherein the composite additive includes at least two of malic acid, fumaric acid and tea saponin; preferably, the composite additive includes three of malic acid, fumaric acid and tea saponin.

[0008] Furthermore, the mass ratio of monoglyceride laurate to the composite additive is (2~4):(2~7.5), preferably (2~4):(2.5~7).

[0009] Furthermore, in the composite additive, the mass ratio of malic acid, fumaric acid and tea saponin is (1~3):(1~3):(0.5~1.5).

[0010] Furthermore, the mass ratio of lauric acid monoglyceride, malic acid, fumaric acid, and tea saponin is 4:3:3:1.

[0011] A second aspect of the present invention provides a sustained-release methane inhibitor microparticle, the sustained-release methane inhibitor microparticle comprising: The core comprises a compound additive and excipients, wherein the compound additive comprises at least two of malic acid, fumaric acid, and tea saponin; An inner coating, the inner coating covering the surface of the core, wherein the inner coating comprises monoglyceride lauryl ester and a first flow aid; An outer coating, which covers the surface of the inner coating, wherein the outer coating comprises hydrogenated palm oil, a palatability enhancer, and a second flow aid.

[0012] Furthermore, in the composite additive, the mass ratio of malic acid, fumaric acid and tea saponin is (1~3):(1~3):(0.5~1.5).

[0013] Furthermore, the excipients include pregelatinized starch, microcrystalline cellulose, and binders.

[0014] Furthermore, the adhesive comprises sodium carboxymethyl cellulose.

[0015] Furthermore, the first flow aid comprises nano-silica.

[0016] Furthermore, the second flow aid comprises nano-silica.

[0017] Furthermore, the palatability enhancer includes γ-nonanolide, ethyl vanillin, and ethyl lactate, wherein the mass ratio of γ-nonanolide: ethyl vanillin: ethyl lactate is (2~4.5): (1~2.5): (0.1~1).

[0018] Furthermore, the mass ratio of monoglyceride laurate to the compound additive is (2~4):(2~7.5).

[0019] Furthermore, the raw materials for the sustained-release methane inhibitor microparticles, by mass percentage, include: 10%~30% laurate monoglyceride, 21%~50% compound additives, 0.1%~0.5% binder, 5%~15% pregelatinized starch, 5%~15% microcrystalline cellulose, 10%~20% hydrogenated palm oil, 0.1%~0.5% nano silica, and 0.1%~0.5% palatability enhancer.

[0020] Furthermore, in the composite additive, the mass ratio of malic acid: fumaric acid: tea saponin is (1~3): (1~3): (0.5~1.5).

[0021] Furthermore, the adhesive includes sodium carboxymethyl cellulose.

[0022] Furthermore, the palatability enhancer includes 20%~45% γ-nonanolactone, 10%~35% ethyl vanillin, and 15%~30% lactic acid.

[0023] In one specific embodiment, each kilogram of the sustained-release methane inhibitor microparticles comprises the following components by weight: 240 g laurate monoglyceride, 180 g malic acid, 180 g fumaric acid, 60 g tea saponin, 90 g pregelatinized starch, 90 g microcrystalline cellulose, 150 g hydrogenated palm oil, 2 g sodium carboxymethyl cellulose, 5 g nano silica, and 3 g palatability enhancer (1.5 g γ-nonalactone, 1 g ethyl vanillin, and 0.5 g ethyl lactate).

[0024] A third aspect of the present invention is to provide a method for preparing sustained-release methane inhibitor microparticles as described in the second aspect, comprising the steps of: S1. The composite additives and excipients are mixed, granulated, and dried to obtain core particles; S2. Melt laurate monoglyceride by heating, add the first gliding agent, stir evenly to obtain the first sustained-release coating solution, and keep it warm for later use; S3. Heat and melt hydrogenated palm oil, add palatability enhancer and second flow aid, disperse ultrasonically to obtain second sustained-release coating solution, and keep warm for later use; S4. Place the core particles prepared in step S1 into a fluidized bed coating machine, and spray the first slow-release coating liquid obtained in step S2 at an air inlet temperature of 25~30℃. After curing, inner coated microparticles are obtained. S5. Adjust the air inlet temperature to 35~40℃, spray the inner coating microparticles obtained in step S4 with the second sustained-release coating liquid prepared in step S3, cool and sieve to obtain sustained-release composite methane inhibitor microparticles.

[0025] Furthermore, in step S1, the particle size of the core particles ranges from 0.5 to 1.0 mm.

[0026] Furthermore, in step S1, the water content of the core particles is ≤5%.

[0027] Further, in step S2, the stirring parameters are: stirring at 100-300 rpm for 1-3 minutes; preferably, stirring at 200 rpm for 2 minutes.

[0028] Further, in step S3, the ultrasonic dispersion processing parameters are 20~25 kHz for 1~3 min; preferably, 20 kHz for 2 min.

[0029] Furthermore, in step S4, the inner coating increases in weight by 15% to 30% based on wet weight.

[0030] Furthermore, in step S4, the outer coating increases in weight by 10% to 15% based on wet weight.

[0031] In one specific embodiment, the preparation method includes the following steps: dissolving 2 kg of sodium carboxymethyl cellulose in deionized water to prepare an aqueous solution with a mass concentration of 0.25%; adding 180 kg of malic acid, 180 kg of fumaric acid, and 60 kg of tea saponin into a high-speed mixer and starting the stirring device to premix for 10 min; sequentially adding 90 kg of pregelatinized starch and 90 kg of microcrystalline cellulose into the high-speed mixer, starting the stirring device, and simultaneously spraying the sodium carboxymethyl cellulose aqueous solution at a uniform speed through an atomizing nozzle to obtain a mixed soft material; transferring the mixed soft material to a gyratory granulator, extruding and granulating it through a screen to obtain mixed particles with a particle size of 0.5~1.0 mm; placing the mixed particles in a spherical shot blasting machine for rounding treatment for 5~20 minutes, and then transferring them to a fluidized bed dryer to dry at 70°C until the moisture content is ≤5% to obtain core particles; 240 kg of glyceryl monolaurate was melted into a liquid state at a constant temperature of 90°C, and 3 kg of nano-silica was added. The mixture was stirred continuously at 200 rpm for 2 min to obtain the first sustained-release packaging material. 150 kg of hydrogenated palm oil was heated to 90°C to undergo phase change melting to form a vegetable oil-based liquid carrier. 2 kg of nano-silica and 3 kg of palatability enhancer were added to the liquid carrier, and the mixture was ultrasonically treated at 20 kHz for 2 min to obtain the second sustained-release packaging material. The first and second sustained-release packaging materials were loaded into the reaction chamber of the fluidized bed coating machine, respectively. The inlet air temperature was set to 25°C. The first sustained-release packaging material was sprayed onto the core particles and allowed to stand and cure for 25 minutes. Then the inlet air temperature was adjusted to 39°C and the second sustained-release packaging material was sprayed. After cooling to 25°C, the particles were passed through a 30-mesh vibrating sieve to obtain sustained-release methane inhibitor microparticles.

[0032] A fourth aspect of the present invention is to provide a feed additive comprising a compound methane inhibitor as described in the first aspect, or a slow-release methane inhibitor microparticle as described in the second aspect, or a slow-release methane inhibitor microparticle prepared by the preparation method described in the third aspect.

[0033] The fifth aspect of the present invention is to provide an application of the compound methane inhibitor as described in the first aspect, or the sustained-release methane inhibitor microparticles as described in the second aspect, or the preparation method as described in the third aspect, or the feed additive as described in the fourth aspect, the application including at least one of the following applications: application in the preparation of a formulation to inhibit methane emissions from ruminants, application in the preparation of ruminant feed, and application in the preparation of a formulation to increase milk yield in dairy cows.

[0034] Furthermore, the ruminants include dairy cows.

[0035] In the preparation of ruminant feed, 10-20g of the slow-release compound methane inhibitor microparticles are added per kilogram of total mixed ration (TMR, dry matter basis).

[0036] The present invention adopts the above technical solution and has the following technical effects compared with the prior art: (1) Synergistic effect of multiple pathways, significantly improving methane inhibition rate: This invention is the first to combine lauric acid monoglyceride (destroys methanogenic bacterial membrane / inhibits protozoa), malic acid and fumaric acid (competitive hydrogen consumption), and tea saponin (lyses protozoa and cuts off symbiosis), and utilizes the synergistic effect between the components to show excellent methane emission inhibition effect.

[0037] (2) Antimicrobial adaptation and stable emission reduction effect: Through multi-target regulation, the adaptive evolution of rumen microorganisms to a single inhibitor is effectively avoided.

[0038] (3) Precise delivery and taste masking to improve palatability and rumen environment: A unique double-layer encapsulation process is used to achieve "oral taste masking and rumen slow release". The outer layer of hydrogenated palm oil and palatability enhancer effectively masks the sour taste of organic acids and the irritating odor of tea saponins, significantly improving the palatability of the additive and preventing a decrease in feed intake. At the same time, the inner layer of lauric acid monoglyceride coating enables the gradient release of active ingredients in the rumen (2h release rate <50%), avoiding drastic fluctuations in rumen pH caused by rapid release of organic acids and maintaining the stability of rumen fermentation function.

[0039] (4) Optimization of rumen environment and immune regulation: The functional components of the compound sustained-release inhibitor microparticles are slowly released in the rumen, avoiding a sharp drop in rumen pH due to rapid absorption of organic acids, and maintaining the stability of rumen fermentation function.

[0040] (5) Raw materials are readily available and costs are controllable: The raw materials used in this invention are all conventional and safe food or feed grade raw materials. Compared with special raw materials such as Schizochytrium powder, the production cost is greatly reduced and it has broad prospects for industrial application. Attached Figure Description

[0041] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this invention, illustrate exemplary embodiments of the invention and are for illustrative purposes only, and do not constitute an undue limitation of the invention. In the drawings: Figure 1 This is a graph showing the effect of methane inhibitors on milk yield in lactating cows in one embodiment of the present invention; Figure 2 This is a graph showing the effect of methane inhibitors on methane emissions from lactating dairy cows in one embodiment of the present invention; Figure 3 This is a graph showing the effect of an unencapsulated sustained-release methane inhibitor on fresh milk in one embodiment of the present invention; Figure 4 This is a graph showing the effect of methane inhibitor particles on methane emissions from lactating dairy cows in one embodiment of the present invention. Detailed Implementation

[0042] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. Experimental materials in the following embodiments that do not specify their source are all commercially available raw materials. The equipment used in each step of the following embodiments is conventional equipment. If there is no corresponding national standard, it is carried out according to general international standards, conventional conditions, or conditions recommended by the manufacturer. Unless otherwise stated, all parts are parts by weight. Unless otherwise defined or stated, all professional and scientific terms used in the present invention have the same meaning as those skilled in the art. In addition, any methods and materials similar or equivalent to those described can be applied to the methods of the present invention.

[0043] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other. The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but this is not intended to limit the scope of the invention.

[0044] Unless otherwise specified, all raw materials and reagents used in the examples can be obtained through commercial purchases.

[0045] The present invention will be described below by way of example.

[0046] Example 1 - Composite methane inhibitor and sustained-release methane inhibitor microparticles This embodiment relates to a specific formulation of a compound methane inhibitor.

[0047] This embodiment provides a composite methane inhibitor, the active ingredients of which include monoglyceride laurate and a composite additive, wherein the composite additive includes at least two of malic acid, fumaric acid and tea saponin.

[0048] Furthermore, the compound additives include malic acid, fumaric acid, and tea saponin.

[0049] Furthermore, the mass ratio of lauric acid monoglyceride, malic acid, fumaric acid and tea saponin is (2~4):(1~3):(1~3):(0.5~1.5).

[0050] Furthermore, the mass ratio of lauric acid monoglyceride, malic acid, fumaric acid, and tea saponin is 4:3:3:1.

[0051] Example 2 - Sustained-release methane inhibitor microparticles This embodiment relates to a specific formulation of a compound methane inhibitor.

[0052] This embodiment provides a sustained-release methane inhibitor microparticle, which includes a core, an inner coating, and an outer coating. The core includes a composite additive and excipients, wherein the composite additive includes at least two of malic acid, fumaric acid, and tea saponin. The inner coating coats the surface of the core and includes monoglyceride laurate and a first flow aid. The outer coating coats the surface of the inner coating and includes hydrogenated palm oil, a palatability enhancer, and a second flow aid.

[0053] In this embodiment: In the compound additive, the mass ratio of malic acid, fumaric acid, and tea saponin is (1~3):(1~3):(0.5~1.5). The excipients include pregelatinized starch, microcrystalline cellulose, and binder, wherein the binder is sodium carboxymethyl cellulose; The first flow aid includes nano-silica; The second flow aid includes nano-silica; The palatability enhancers include γ-nonanolide, ethyl vanillin, and ethyl lactate, wherein the mass ratio of γ-nonanolide: ethyl vanillin: ethyl lactate is (2~4.5): (1~2.5): (0.1~1).

[0054] Specifically, the raw materials of the sustained-release methane inhibitor microparticles include, by mass percentage: 10%~30% lauric acid monoglyceride, 10%~20% malic acid, 10%~20% fumaric acid, 1%~10% tea saponin, 0.1%~0.5% sodium carboxymethyl cellulose, 5%~15% pregelatinized starch, 5%~15% microcrystalline cellulose, 10%~20% hydrogenated palm oil, 0.2%~0.4% primary gliding agent, 0.1%~0.3% secondary gliding agent, and 0.1%~0.5% palatability enhancer.

[0055] Example 3 - Preparation method of sustained-release methane inhibitor microparticles This embodiment relates to a specific method for preparing sustained-release methane inhibitor microparticles. Using the formulation of Example 1, sustained-release methane inhibitor microparticles are prepared, including the following steps: (1) Dissolve sodium carboxymethyl cellulose in deionized water to prepare an aqueous solution with a mass concentration of 0.25%.

[0056] (2) According to the formula, malic acid, fumaric acid and tea saponin are added to the high-speed mixer and the stirring device is started to premix for 10 min; pregelatinized starch and microcrystalline cellulose are added to the high-speed mixer in sequence, the stirring device is started, and the sodium carboxymethyl cellulose aqueous solution prepared in step (1) is sprayed into the mixture at a uniform speed through the atomizing nozzle to obtain the mixed soft material.

[0057] (3) Transfer the mixed soft material to a swing granulator and granulate it by screen extrusion to obtain mixed particles with a particle size of 0.5~1.0 mm; place the mixed particles in a spherical shot blasting machine for 5~20 minutes to round them, and then transfer them to a fluidized bed dryer to dry them at 70℃ until the moisture content is ≤5% to obtain core particles.

[0058] (4) Melt monoglyceride of laurate into a liquid state at a constant temperature of 90°C, add nano-silica, and stir continuously at a speed of 100~300 rpm for 1~3 min to obtain the first sustained-release packaging material.

[0059] (5) Heat hydrogenated palm oil to 90°C to undergo phase change melting to form a vegetable oil-based liquid carrier. Add nano-silica and palatability enhancer to the liquid carrier and sonicate for 1-3 min to obtain a second sustained-release packaging material. Keep it warm for later use.

[0060] (6) Load the first slow-release packaging material and the second slow-release packaging material into the reaction chamber of the fluidized bed coating machine respectively, set the air inlet temperature to 25~30℃, spray the first slow-release packaging material onto the core particles, and according to the wet weight, the coating weight gain is 15%~30%, and then let it stand at 25~30℃ for 25 minutes to form the inner coating (the melting point of lauric acid monoglyceride is 45~55℃, and the air inlet temperature is controlled at 15~20℃ below the melting point to ensure that the coating layer solidifies quickly to form a complete coating layer).

[0061] (7) Adjust the air inlet temperature to 35~40℃ and continue to spray the second slow-release coating material. According to the wet weight, the coating weight gain is 10%~15%. After forming the outer coating layer, cool it to 25℃ and pass it through a 30-mesh vibrating sieve device to obtain slow-release methane inhibitor particles (the melting point of hydrogenated palm oil is 55~65℃, and the air inlet temperature is controlled at 15~25℃ below the melting point to maintain appropriate fluidity during the coating process while avoiding excessive penetration).

[0062] Example 4 This embodiment is based on the formulation of the sustained-release methane inhibitor microparticles in Example 1, and uses the preparation method of Example 3 to prepare a specific sustained-release methane inhibitor microparticle, including the following steps: 1. Formula: Each kilogram contains the following components by weight: 240 g laurate monoglyceride, 180 g malic acid, 180 g fumaric acid, 60 g tea saponin, 90 g pregelatinized starch, 90 g microcrystalline cellulose, 150 g hydrogenated palm oil, 2 g sodium carboxymethyl cellulose, 5 g nano silica, and 3 g palatability enhancers (1.5 g γ-nonalactone, 1 g ethyl vanillin, and 0.5 g ethyl lactate).

[0063] 2. Preparation steps: (1) Dissolve 2 kg of sodium carboxymethyl cellulose in deionized water to prepare an aqueous solution with a mass concentration of 0.25%; (2) Add 180 kg of malic acid, 180 kg of fumaric acid and 60 kg of tea saponin into a high-speed mixer and start the stirring device to premix for 10 min; (3) 90 kg of pregelatinized starch and 90 kg of microcrystalline cellulose are put into a high-speed mixer in sequence, the stirring device is started, and the sodium carboxymethyl cellulose aqueous solution obtained in step S1 is sprayed into the mixture at a constant speed through the atomizing nozzle to obtain a mixed soft material. (4) The mixed soft material is transferred to a gyratory granulator and granulated by extrusion through a screen to obtain mixed granules with a particle size of 0.5~1.0 mm; (5) Place the mixed particles in a spherical shot blasting machine and roll them into rounds for 5-20 minutes, then transfer them to a fluidized bed dryer and dry them at 70°C until the moisture content is ≤5%; (6) Melt 240 kg of monoglyceride of laurate into a liquid state at a constant temperature of 90°C, add 3 kg of nano-silica, and stir continuously at 200 rpm for 2 min; (7) Heat 150 kg of hydrogenated palm oil to 90°C to undergo phase change melting to form a vegetable oil-based liquid carrier. Add 2 kg of nano-silica and 3 kg of palatability enhancer to the liquid carrier and treat it with ultrasound at 20 kHz for 2 min. (8) Load the composite additive mixture into the reaction chamber of the fluidized bed coating machine, set the air inlet temperature to 25°C, spray the first slow-release coating material to form the inner coating; after standing and curing for 25 min, adjust the air inlet temperature to 39°C, and continue to spray the second slow-release coating material to form the outer coating. (9) After cooling to 25°C, the slow-release methane inhibitor microparticles are obtained by passing them through a 30-mesh vibrating sieve device.

[0064] Example 5 - Comparison of methane emission reduction effects between single and combined methane inhibitors based on in vitro simulated fermentation method This embodiment uses the formulation of the compound methane inhibitor provided in Example 1 to conduct experiments to verify the methane emission reduction effect of the compound methane inhibitor when applied to dairy cows.

[0065] 1. Experimental Materials and Design 1) Experimental animals: 3 healthy dairy cows weighing approximately 690 kg each, with permanent rumen fistulas.

[0066] 2) Fermentation substrate: Based on the standard TMR diet for high-yielding dairy cows, control groups (basal diet), single additive groups (experimental groups 1-6), compound inhibitor groups (compound groups 1-3), and control groups (control groups 1-3) were set up. The specific additives and dosages for each group are shown in the table below. Among them, the following methane inhibitors were prepared according to the weight percentage of the diet: Compound methane inhibitor A: contains four ingredients: lauric acid monoglyceride, fumaric acid, malic acid, and tea saponin, and the mass ratio of lauric acid monoglyceride: fumaric acid: malic acid: tea saponin is 4:3:3:1.

[0067] Compound methane inhibitor B: without fumaric acid, containing monoglyceride laurate, malic acid, and tea saponin, with a mass ratio of monoglyceride laurate: malic acid: tea saponin = 4:3:1; Compound methane inhibitor C: malic acid-free, containing lauric acid monoglyceride, fumaric acid, and tea saponin, with a mass ratio of lauric acid monoglyceride: fumaric acid: tea saponin = 4:3:1; Compound methane inhibitor D: Free of tea saponins, containing monoglyceride laurate, fumaric acid, and malic acid, with a mass ratio of monoglyceride laurate: fumaric acid: malic acid = 4:3:3; Compound methane inhibitor E: without laurate monoglyceride, containing fumaric acid, malic acid, and tea saponin, with a mass ratio of fumaric acid: malic acid: tea saponin = 3:3:1.

[0068] Note: a. The mass percentage before the additive is based on the dry matter content (DM) of the diet.

[0069] b. The addition amounts of control groups 1-3 were set to be consistent with the addition amounts of other components in combination group 2, except for the missing component, so as to compare the effects of the missing component on the basis of the same dosage.

[0070] 3) Microbial culture medium: Dissolve 8.75 g NaHCO3, 1.00 g NH4HCO3, 1.43 g Na2HPO4, 1.55 g KH2PO4, 0.15 g MgSO4·7H2O, 0.52 g Na2S, 0.015 g MnCl2·4H2O, 0.002 g CoCl2·6H2O, 0.012 g FeCl3·6H2O, 0.017 g CaCl2·2H2O, and 1.25 mg resazurin in 1 L of distilled water. Slowly pass high-purity CO2 into the bottom of the solution until the pH reaches 6.8.

[0071] 2. Test Methods In vitro rumen fermentation was used, and gas production and methane production were measured after 24 h of culture. Rumen fluid from three cattle was used and then mixed. Each treatment group had four replicates. The entire experiment was performed in triplicate.

[0072] 1) On the day of the experiment, rumen fluid was collected from 3 healthy Holstein dairy cows with rumen fistulas 1 hour before morning feeding. The fluid was filtered through 4 layers of gauze into a thermos (the thermos was preheated with 39°C hot water beforehand), and quickly brought back to the laboratory. The thermos was then placed in a 39°C constant temperature water bath, and CO2 was continuously introduced above the liquid surface until the inoculation was completed.

[0073] 2) Accurately weigh 0.5 g of each group of substrate samples and place them in the corresponding culture flask (100 mL), with 4 replicates for each group.

[0074] 3) Add 50 mL of microbial culture medium and 25 mL of rumen fluid to each culture flask. Introduce CO2 into the flask, and after about 5 seconds (to create an anaerobic environment), immediately put on the stopper. Attach a plastic three-way valve to the stopper opening, connect a gas collection bag, check for airtightness, and then incubate at 39°C with shaking in a water bath.

[0075] 4) After 24 hours, remove the corresponding culture flasks and quickly place them in an ice-water bath to terminate fermentation. Measure the gas pressure inside the culture flasks using a digital pressure sensor (DPG 1000B15PSIG-5, Cecomp Electronics, Libertyville, USA) and convert it to gas production. Calculate the gas production using the method of OSMOND et al.: Gas production = Volume of the top space of the gas-producing flask × Measured gas pressure × 0.068.

[0076] 5) Collect 10 mL of gas from the fermentation flask and store it in a gas collection bag. Detect the CH4 content in the gas using a gas chromatograph (chromatographic reference conditions: TCD detector, TDX-01 packed column, 1 mm × 3 mm × 2 mm, injection port temperature 120℃, detector temperature 150℃, carrier gas helium, flow rate 50 mL / min, injection volume 0.1 mL). Calculate the CH4 yield based on the gas production and CH4 content.

[0077] 6) The experimental data were statistically analyzed using a one-way ANOVA with a general linear model (GLM) in SPSS 16.0, with a significance level set at P < 0.05. The experimental data are expressed as mean and standard error (SEM). The criteria for judging differences were: P < 0.05 indicates a significant difference, P < 0.01 indicates a highly significant difference, and P < 0.1 indicates a trend of difference.

[0078] 3. The test results are shown in Table 1.

[0079] Table 1. In vitro methane emission reduction effects of single and combined methane inhibitors Note: 1. Different lowercase letters after the data in the same column indicate significant differences (P<0.05).

[0080] 2. The P-value between experimental group 2 (fumaric acid) and the control group was 0.062, showing a decreasing trend.

[0081] As can be seen from Table 1, (1) The methane inhibition rate of compound methane inhibitor A (compound group 2) reached 18.9% at the addition of 1.1% DM, which was significantly higher than that of any single component (the highest inhibition rate of experimental groups 1-6 was 12.3%). It is worth noting that, under the premise that the addition of other components is basically the same as that of compound methane inhibitor A, the compound groups that only lacked fumaric acid (comparison group 1), malic acid (comparison group 2), or tea saponin (comparison group 3) had an inhibition rate of 13.2%~14.7%, which was significantly lower than that of compound group 2 (18.9%), compound group 3 (16.7%), and compound group 4 (16.3%) which contained four components. This proves that fumaric acid, malic acid, and tea saponin have a synergistic effect, and changing any component will affect the methane inhibition rate.

[0082] (2) The control group 4, which lacked only monoglyceride laurate, had an inhibition rate of only 11.5%. Compared with control groups 1-3, the inhibition rate of methane was significantly reduced, indicating that monoglyceride laurate plays an important role in inhibiting methane emissions and is an essential component.

[0083] (3) From the results of compound group 1 (inhibition rate of 14.3%), compound group 2 (inhibition rate of 18.9%), compound group 3 (inhibition rate of 16.7%), and compound group 4 (inhibition rate of 16.3%), it can be seen that compound group 2 has the most significant inhibitory effect on methane when only the addition ratio is different. This indicates that the addition ratio of compound group 2 at 1.1% is the better ratio.

[0084] (4) The above results strongly demonstrate that there is a significant synergistic effect among lauric acid monoglyceride, malic acid, fumaric acid and tea saponin, and its effect is far superior to any combination of the three.

[0085] (5) In addition, the addition of allicin failed to significantly reduce methane production. Although the addition of Schizochytrium powder could reduce methane by 9.5%, it was expensive and not cost-effective.

[0086] Example 6 - In vivo verification of the methane emission reduction effect of methane inhibitors In this embodiment, a compound methane inhibitor A was used, and an in vitro experiment was conducted at the optimal addition amount of the compound methane inhibitor 1.1% DM to verify the methane emission reduction effect.

[0087] 1. Experimental Design and Feeding Management Thirty healthy mid-lactation Holstein dairy cows with similar lactation days (180.6 ± 10.1 days) and milk yield were selected and randomly divided into two groups of 15 cows each, according to a randomized block design. The control group was fed a pasture basal diet, while the experimental group was fed a diet supplemented with 1.1% DM compound methane inhibitor A. The experiment lasted for 70 days. The cows were tethered and fed a TMR diet three times a day, with free access to feed and water.

[0088] 2. Measurement Indicators and Methods 1) Feed intake: Dry matter intake was measured every two weeks starting from the first day of the trial period.

[0089] 2) Milk yield and milk composition: The milk yield of each cow was measured in the morning, noon and evening for two consecutive days each week using a split meter, and milk samples were collected according to a 4:3:3 ratio. The milk composition was measured using a milk composition analyzer.

[0090] 3) Methane Measurement: Methane emissions from dairy cows were measured using an AHC (automated head-chamber) monitoring device starting at weeks 4 and 9 of the trial period. A 3-day acclimatization period was implemented before data collection, during which cows were induced to enter the head chamber of the device using a small amount of alfalfa pellets (less than 5% of their daily dry matter intake). Measurements were taken at 8 time points, each at least 6 hours apart, with each monitoring session lasting at least 5 minutes.

[0091] 4) Rumen fluid collection and VFA composition determination: Rumen fluid was collected orally at weeks 5 and 9, filtered through four layers of gauze, and stored at -80°C. Quantitative analysis of VFA was performed using gas chromatography (GC-2010, Shimadzu Corporation, Japan).

[0092] 5) Microbial analysis: metagenomic sequencing was used to analyze the rumen microbiota structure.

[0093] 3. Statistical Analysis The analysis was performed using a repeated-measures mixed model (PROC MIXED procedure) in SAS 9.4 software. Treatment, week, and their interaction were set as fixed effects, while blocks were set as random effects. Except for rumen fermentation parameters, bacterial genus abundance, and methane emission indicators, the initial sampled values ​​of all other indicators were included as covariates in the model for correction. The optimal fit structure was determined based on the Schwarz Bayesian Information Criterion (BIC).

[0094] 4. Analysis of experimental results, as shown in Table 2. Figure 1 , Figure 2 As shown.

[0095] Table 2. Effects of compound methane inhibitors on lactating cow productivity and methane emissions. From Table 2, Figure 1 , Figure 2 It can be seen that, (1) The compound methane inhibitor A used in this embodiment has virtually no effect on the dry matter intake of dairy cows.

[0096] (2) In the early stage of the experiment (first 4 weeks), the milk production of the experimental group was not significantly different from that of the control group; from the 5th week onwards, the milk production showed a significant increasing trend compared with the control group, and the average milk production increased by 1 kg / d throughout the experiment.

[0097] (3) Compared with the control group, the experimental group reduced the methane production of dairy cows by 20.0% in the fourth week and by 17.2% in the ninth week. The methane emission reduction rate decreased by ≤3%, indicating that the compound methane inhibitor A can effectively delay the adaptive evolution of rumen microorganisms.

[0098] (4) Methane emissions decreased by 18.6% throughout the entire experimental period, methane emissions per unit of milk production decreased by 20.3%, and methane emissions per unit of dry matter intake decreased by 19.6%.

[0099] Table 3. Effects of compound methane inhibitors on rumen fermentation parameters in lactating dairy cows Table 4. Effects of compound methane inhibitors on the rumen microbiota structure of lactating dairy cows The effects of the compound methane inhibitor on rumen fermentation parameters and microbial structure in lactating dairy cows are shown in Tables 3 and 4. The results indicate that compound methane inhibitor A significantly shifted rumen fermentation towards propionic acid-based fermentation, manifested as an increase in propionic acid proportion and a decrease in the acetic acid / propionic acid ratio, while the total volatile fatty acid concentration, pH, and ammonia nitrogen concentration remained stable. Microbial analysis (as shown in Table 4) revealed that the compound methane inhibitor specifically increased the abundance of propionic acid-producing bacteria (such as *Succinosporium* and *Macrococcus*) and competitive hydrogen-consuming bacteria (*Desulfovibrio*), while decreasing the overall abundance of archaea. The community structure of core fiber-degrading bacteria (such as *Ruminococcus* and *Fibrobacterium*) remained stable. This change suggests that the compound methane inhibitor works by "inhibiting hydrogen production." By strengthening the microbial regulation mechanism that "enhances hydrogen consumption", methane emission reduction was achieved without affecting the basic fermentation function.

[0100] Example 7 - Rumen Sustained-Release Effect Test This embodiment uses the sustained-release methane inhibitor microparticles prepared in Example 4 as experimental material to explore their sustained-release effect in the rumen.

[0101] 1. Experimental group design: The experimental group used the sustained-release methane inhibitor microparticles prepared in Example 4; the control group used a composite methane inhibitor (containing monoglyceride laurate, fumaric acid, malic acid, and tea saponin in a weight ratio of 4:3:3:1, without coating or granulation).

[0102] 2. Experiment: Three Holstein dairy cows (690±25kg) with rumen fistulas were used as experimental animals. The rumen degradation rate of the finished product was determined at 1, 2, 4, and 8 hours after sample administration using the nylon bag method. The experimental results are shown in Table 1. Table 5. Release rate (%) of sustained-release compound methane inhibitor microparticles in the rumen of dairy cows. The results showed that the sustained-release methane inhibitor microparticles prepared in this invention had a release rate of 49.73% in the rumen at 2 h and 88.35% at 8 h, while the control group (uncoated) had a release rate of 82.54% at 1 h, proving that the encapsulated sustained-release methane inhibitor microparticles have a good sustained-release effect.

[0103] Example 8 - Palatability Assessment of Sustained-Release Methane Inhibitors This embodiment uses the sustained-release methane inhibitor microparticles prepared in Example 4 as the experimental subject to evaluate their palatability.

[0104] (1) Experimental design: Fifteen calves aged 7-8 days were randomly divided into three groups of five each. The control group was fed regular fresh milk (3 kg each time), the compound group was fed fresh milk with 15 g / d of compound additive (the same formula as in Example 4, but without coating or granulation), and the slow-release compound group was fed an equal amount (15 g / d) of the slow-release methane inhibitor microparticles prepared in Example 4. The experiment lasted for 3 days, with feeding three times a day.

[0105] (2) Monitor and record the feed intake of each group of calves. The results are shown in Table 6.

[0106] Table 6. Effects of sustained-release compound methane inhibitor microparticles on milk intake in lactating calves Note: Different lowercase letters after the data in the same column indicate significant differences (P<0.05).

[0107] Results analysis: The organic acids in the composite group (unencapsulated) caused a decrease in the pH of the fresh milk, leading to denaturation and coagulation of milk proteins and the formation of flocculent precipitates (such as...). Figure 3 As shown in the figure, it significantly reduced the calves' willingness to drink milk, and their feed intake was significantly lower than that of the control group.

[0108] In contrast, the feed intake of the slow-release compound group (encapsulated treatment) was 8.81 kg, which was 25.5% higher than that of the compound group and basically the same as that of the control group (8.85 kg). The results show that the slow-release encapsulation process can effectively mask the unpleasant odor of organic acids and tea saponins in the compound additives, avoid their adverse effects on milk properties, and thus ensure normal feed intake for calves.

[0109] Example 9 - Animal Feeding Validation Trial of Sustained-Release Methane Inhibitor Microparticles This embodiment involves a specific verification experiment to verify the methane emission reduction effect of the sustained-release methane inhibitor particles prepared in Example 4.

[0110] 1. Experimental Design: Fifteen healthy Holstein cows in late lactation with similar lactation days (average 280 days) and milk yield were selected and randomly divided into three groups of five cows each. The control group received a basal diet supplemented with 300 g / d of a compound methane inhibitor (containing lauric acid monoglyceride, fumaric acid, malic acid, and tea saponin in a mass ratio of 4:3:3:1, without coating or granulation). The slow-release group 1 received a basal diet supplemented with 200 g / d of the slow-release methane inhibitor microparticles prepared in Example 4. The slow-release group 2 received a basal diet supplemented with 300 g / d of the slow-release methane inhibitor microparticles prepared in Example 4.

[0111] 2. Experiment: The experiment lasted 14 days. During the experiment, dairy cows were tethered and fed a TMR diet three times a day, with free access to feed and water. Methane emissions from the cows were measured using an AHC (automated head-chamber) monitoring device two days before and two days after the start of the experiment. The results are as follows: Figure 4 As shown.

[0112] 3. Results Analysis: From Figure 4 It can be seen that the methane emission reduction effect achieved by adding 200 g of sustained-release methane inhibitor microparticles daily in the control group is comparable to that of sustained-release group 1 (adding 300 g of compound methane inhibitor daily); while the methane emission reduction effect of sustained-release group 2 (adding 300 g of sustained-release methane inhibitor microparticles daily) is significantly better than that of the control group and sustained-release group 1, and the methane production of sustained-release group 2 is comparable to that of the control group.

[0113] In summary, this invention innovatively proposes a composite methane inhibitor composed of safe and conventional raw materials with synergistic effects. In vitro fermentation experiments demonstrate that the methane inhibition rate of the four-component combination (18.9%) is significantly higher than that of any single component or the control group lacking any key component (13.2%~14.7%), exhibiting excellent synergistic effects among the components. In vivo trials in dairy cows lasting up to 10 weeks show that the methane emission reduction rate of this composite methane inhibitor at the end of the trial (week 9) (17.2%) is ≤3% lower than that at the beginning of the trial (week 4, 20.0%), addressing the pain point of the difficulty in maintaining the effect in existing technologies.

[0114] This invention also innovatively prepares sustained-release methane inhibitor microparticles. A unique double-layer encapsulation process for the composite methane inhibitor achieves "oral masking and rumen sustained release." The outer layer of hydrogenated palm oil and palatability enhancers effectively masks the acidic taste of organic acids and the pungent odor of tea saponins, significantly improving palatability and solving the problem of poor palatability and reduced animal feed intake caused by acidic and bitter components in existing methane inhibitors. The inner layer of lauric acid monoglyceride coating enables gradient release of the active ingredient in the rumen, avoiding drastic pH fluctuations caused by rapid release of organic acids and maintaining the stability of rumen fermentation function. This not only achieves gradient release of the active ingredient in the rumen, prolonging the duration of action, delaying microbial adaptation, and ensuring a continuous and stable methane emission reduction effect, but also ensures readily available raw materials, controllable costs, and broad prospects for industrial application.

[0115] The above description is merely a preferred embodiment of the present invention and does not limit the implementation and protection scope of the present invention. Those skilled in the art should realize that any equivalent substitutions and obvious changes made based on the description and illustrations of the present invention should be included within the protection scope of the present invention.

Claims

1. A composite methane inhibitor, characterized by, The active ingredient of the compound methane inhibitor includes monoglyceride of laurate and a compound additive, wherein the compound additive includes at least two of malic acid, fumaric acid, and tea saponin.

2. The composite methane inhibitor of claim 1, wherein, The mass ratio of monoglyceride laurate to the compound additive is (2~4):(2~7.5).

3. The composite methane inhibitor according to claim 1 or 2, characterized in that, In the composite additive, the mass ratio of malic acid, fumaric acid and tea saponin is (1~3):(1~3):(0.5~1.5).

4. A sustained-release methane inhibitor microparticle, characterized in that, The sustained-release methane inhibitor particles include: The core comprises a compound additive and excipients, wherein the compound additive comprises at least two of malic acid, fumaric acid, and tea saponin; An inner coating, the inner coating covering the surface of the core, wherein the inner coating comprises monoglyceride lauryl ester and a first flow aid; An outer coating, which covers the surface of the inner coating, wherein the outer coating comprises hydrogenated palm oil, a palatability enhancer, and a second flow aid.

5. The sustained-release methane inhibitor microparticles according to claim 4, characterized in that, In the composite additive, the mass ratio of malic acid, fumaric acid, and tea saponin is (1~3):(1~3):(0.5~1.5); and / or The excipients include pregelatinized starch, microcrystalline cellulose, and binders; and / or The first flow aid comprises nano-silica; and / or The second flow aid comprises nano-silica; and / or The palatability enhancer includes γ-nonanolide, ethyl vanillin, and ethyl lactate, wherein, by mass ratio, γ-nonanolide:ethyl vanillin:ethyl lactate = (2~4.5):(1~2.5):(0.1~1); and / or The mass ratio of monoglyceride laurate to the compound additive is (2~4):(2~7.5).

6. The sustained-release methane inhibitor microparticles according to claim 4 or 5, characterized in that, The raw materials for the sustained-release methane inhibitor microparticles, by mass percentage, include: 10%~30% laurate monoglyceride, 21%~50% compound additives, 0.1%~0.5% binder, 5%~15% pregelatinized starch, 5%~15% microcrystalline cellulose, 10%~20% hydrogenated palm oil, 0.2%~0.4% first flow aid, 0.1%~0.3% second flow aid, and 0.1%~0.5% palatability enhancer.

7. A method for preparing sustained-release methane inhibitor microparticles as described in any one of claims 4 to 6, characterized in that, Including the following steps: S1. The composite additives and excipients are mixed, granulated, and dried to obtain core particles; S2. Melt laurate monoglyceride by heating, add the first gliding agent, stir evenly to obtain the first sustained-release coating solution, and keep it warm for later use; S3. Heat and melt hydrogenated palm oil, add palatability enhancer and second flow aid, disperse ultrasonically to obtain second sustained-release coating solution, and keep warm for later use; S4. Place the core particles prepared in step S1 into a fluidized bed coating machine, and spray the first slow-release coating liquid obtained in step S2 at an air inlet temperature of 25~30℃ to form an inner coating. After curing, inner coated microparticles are obtained. S5. Adjust the air inlet temperature to 35~40℃, spray the inner coating microparticles obtained in step S4 with the second sustained-release coating liquid prepared in step S3 to form an outer coating, cool and sieve to obtain sustained-release composite methane inhibitor microparticles.

8. The method according to claim 7, characterized in that, In step S1, the particle size of the core particles ranges from 0.5 to 1.0 mm; and / or In step S1, the water content of the core particles is ≤5%; and / or In step S2, the stirring parameters are: stirring at 100-300 rpm for 1-3 minutes; and / or In step S3, the ultrasonic dispersion processing parameters are 20~25 kHz for 1~3 min; and / or In step S4, the inner coating, based on wet weight, increases in weight by 15% to 30%; and / or In step S4, the outer coating increases in weight by 10% to 15% based on wet weight.

9. A feed additive, characterized in that, The feed additive comprises: The composite methane inhibitor as described in any one of claims 1 to 4; or The sustained-release methane inhibitor microparticles as described in any one of claims 5-6; or The sustained-release methane inhibitor microparticles prepared by the preparation method according to any one of claims 7 to 8.

10. The application of a composite methane inhibitor as described in any one of claims 1-4, or a sustained-release methane inhibitor microparticle as described in any one of claims 5-6, or a preparation method as described in any one of claims 7-8, or a feed additive as described in claim 9, characterized in that, The application includes at least one of the following: application in the preparation of formulations that inhibit methane emissions from dairy cows, and application in the preparation of formulations that increase milk yield from dairy cows.

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