A rumen methane inhibitor and its application

Abstract

This invention provides a rumen methane inhibitor and its application, belonging to the field of ruminant farming. The active ingredients of the rumen methane inhibitor of this invention include: *Lactobacillus reuteri* inoculum, *Lactobacillus gasseri* inoculum, *Lactobacillus casei* inoculum, amylase, phytase, xylanase, cellulase, and β-glucanase. The rumen methane inhibitor can effectively regulate the intestinal microbiota of dairy cows, promote the decomposition of fibrous material in the rumen, optimize the rumen fermentation environment, thereby controlling rumen function, reducing methane production, and improving the productivity of ruminants. The rumen methane inhibitor is safe and environmentally friendly, which is conducive to its application in ruminant farming, agriculture, and other fields, and has good practicality.

Description

Technical Field

[0001] This invention belongs to the field of ruminant animal breeding, specifically relating to a rumen methane inhibitor and its application. Background Technology

[0002] Against the backdrop of global climate change, the contribution of livestock farming, especially ruminant animal husbandry, to greenhouse gas emissions is receiving increasing attention. Studies show that global methane emissions from livestock farming are generally on the rise, with methane produced by fermentation in the gastrointestinal tracts of ruminants accounting for 97.5% of total methane emissions, approximately 20% of global methane emissions. Methane's greenhouse effect is dozens of times greater than that of carbon dioxide, and the environmental impact of greenhouse gases emitted by ruminants is drawing growing attention. Therefore, controlling methane emissions from ruminants through appropriate measures is of great significance.

[0003] Currently, it is generally believed that rumen methanogenesis in ruminants is the result of the combined action of various microorganisms within the rumen, with methanogenic archaea being the direct methane-producing microorganisms. Various rumen methane regulation measures have been developed to address the mechanisms of methane production. These measures primarily include adjusting feed composition and altering rumen fermentation patterns, such as increasing the proportion of concentrate; increasing other electron donors to competitively inhibit methane production, such as nitrates, malic acid, and fumaric acid; and using chemical inhibitors or plant secondary metabolite inhibitors to suppress the activity of microorganisms involved in the methane production pathway, such as tannins, lipids, halogenated compounds, and ion carriers. While these measures can reduce methane production to some extent, they suffer from high costs, poor sustained effectiveness, and potential side effects. In contrast, probiotic agents can effectively improve the structure of the rumen fermentation microbiota with fewer side effects, demonstrating significant potential in the field of methane control in ruminants.

[0004] Currently, existing technologies disclose the application of reducing acetic acid-producing bacteria, plant lactic acid bacteria, and Candida tropicalis in inhibiting or reducing methane production in ruminants. However, these methods only use a single probiotic or compound to inhibit methane production, and their effectiveness still has significant room for improvement. Therefore, there is an urgent need to develop a rumen methane inhibitor for dairy cattle that can effectively regulate rumen function, reduce methane production, improve ruminant productivity, and is safe and environmentally friendly. Summary of the Invention

[0005] To address some shortcomings in existing technologies, this invention provides a rumen methane inhibitor and its application. The active ingredients of the rumen methane inhibitor include: *Lactobacillus reuteri*, *Lactobacillus gasseri*, *Lactobacillus casei*, amylase, phytase, xylanase, cellulase, and β-glucanase. This rumen methane inhibitor effectively regulates the intestinal microbiota of dairy cows, promotes the decomposition of fibrous material in the rumen, optimizes the rumen fermentation environment, thereby controlling rumen function, reducing methane production, and improving the productivity of ruminants. The rumen methane inhibitor is safe and environmentally friendly, facilitating its application in ruminant farming and agriculture, and possesses excellent practicality.

[0006] To achieve the above-mentioned technical objectives, the present invention employs the following technical means.

[0007] The present invention provides a rumen methane inhibitor, wherein the active ingredients of the rumen methane inhibitor include probiotics and a complex enzyme that promotes the degradation of fibrous tissue.

[0008] Preferably, the probiotics include one or more of Lactobacillus reuteri, Lactobacillus gasseri, or Lactobacillus casei.

[0009] The complex enzyme that promotes fibrous degradation includes any two or more of amylase, phytase, xylanase, cellulase, or β-glucanase.

[0010] Preferably, the probiotic is a mixture of Lactobacillus reuteri, Lactobacillus gasseri, or Lactobacillus casei, and the complex enzyme that promotes cellulose degradation is a mixture of amylase, phytase, xylanase, cellulase, and β-glucanase.

[0011] Preferably, the amount of active ingredient in each 1g rumen methane inhibitor is: Lactobacillus reuteri 1×10 8 ~1×10 10 CFU, Lactobacillus gasseri 2×10 7 ~2×10 9 CFU, Lactobacillus casei 1×10 8 ~1×10 10 CFU, amylase 10-150U, phytase 200-2000U, xylanase 500-5000U, cellulase 10-100U, β-glucanase 100-1000U.

[0012] Preferably, the amount of active ingredient in each 1g rumen methane inhibitor is: Lactobacillus reuteri 2×10 9 CFU, Lactobacillus gasseri 1×10 9 CFU, Lactobacillus casei 2×10 9CFU, amylase 90U, phytase 1000U, xylanase 2000U, cellulase 50U, β-glucanase 500U.

[0013] Preferably, the preparation method of the rumen methane inhibitor includes: mixing the active ingredients one by one into a veterinary drug carrier.

[0014] Preferably, the veterinary drug carrier includes any one of glucose, inorganic salt, soluble starch, and dextrin; more preferably, it is corn starch.

[0015] The present invention also provides the application of the above-mentioned rumen methane inhibitor in reducing the methane content in the rumen of ruminants.

[0016] Preferably, the ruminant animals include animals with ruminant stomachs belonging to the Camelidae, Cervidae, and Bovidae families, and are preferably dairy cows.

[0017] Preferably, 0.2-2g of rumen methanogen inhibitor is added per kilogram of feed; more preferably, 0.5g.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] This invention uses corn starch as a carrier, to which *Lactobacillus reuteri*, *Lactobacillus gasseri*, *Lactobacillus casei*, amylase, phytase, xylanase, cellulase, and β-glucanase are added. Among these, *Lactobacillus reuteri*, *Lactobacillus gasseri*, and *Lactobacillus casei*, among other probiotics, synergistically regulate the balance of the rumen microbial community after entering the rumen, thereby maintaining the stability of rumen fluid pH, inhibiting the activity of methanogenic bacteria, and reducing methane production. Furthermore, the addition of probiotics can promote intestinal peristalsis in dairy cows, maintain intestinal homeostasis, inhibit harmful pathogens, maintain the ecological balance of the host's intestinal flora, and have a positive impact on dairy cow health, enhancing their immunity, improving feed digestibility and energy utilization efficiency, and contributing to improved overall farming efficiency.

[0020] The complex enzyme combination of amylase, phytase, xylanase, cellulase, and glucanase described in this invention not only promotes the breakdown of fiber in the rumen of dairy cows, helping animals improve the digestibility and utilization of their diet, but also optimizes the rumen fermentation environment, inhibits the activity of methanogenic bacteria, and reduces methane production. Among these enzymes, amylase has direct nutrient digestion function, promoting animal growth; xylanase, cellulase, and glucanase can reduce dietary stickiness, reducing the proliferation of harmful microorganisms; xylanase and cellulase control harmful microorganisms by producing functional oligosaccharides and oligopeptides; and phytase can improve the digestibility of proteins and amino acids, providing more usable nutrients. Simultaneously, the combination of the complex enzyme preparation and probiotics can effectively improve the survival rate of probiotic lactic acid bacteria in the rumen, promote probiotic growth, and inhibit methanogenic bacteria.

[0021] Therefore, the inhibitor of the present invention has the dual advantages of probiotics and complex enzymes, which can effectively regulate the intestinal microbial population of dairy cows and achieve the goal of reducing methane emissions.

[0022] This invention verifies its effectiveness through experiments. The described rumen methanogen inhibitor effectively reduces the number of methanogens, thereby decreasing methane emissions. It also effectively regulates rumen function, improves ruminant productivity, and is safe and environmentally friendly. Compared to the method without the rumen methanogen inhibitor, dairy cows showed an average reduction of 58.3% in methanogen count and 52.6% in methane emissions, while milk production increased by 3.3%. This rumen methanogen inhibitor fills a gap in the development of rumen methanogen inhibitor technology for ruminants and has significant practical applications. Detailed Implementation

[0023] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto. In the following embodiments, various processes and methods not described in detail are all conventional methods known in the art. The source, trade name, and components of the reagents used are indicated when they first appear, and subsequent use of the same reagents are not otherwise specified and are from the same source as initially indicated; the reagents and materials involved are all obtained for commercial use unless otherwise specified.

[0024] The rumen methanogen inhibitor of the present invention has the dual advantages of probiotics and complex enzymes; the rumen methanogen inhibitor mainly acts on ruminant animals, preferably dairy cows.

[0025] In the following examples, the *Lactobacillus reuteri* (BNCC192190), *Lactobacillus gasseri* (BNCC339385), and *Lactobacillus casei* (BNCC134415) strains used were purchased from Beina Biotechnology; amylase (SDG-2420) and phytase (

[0026] SDG-2436), xylanase (SDG-2412), cellulase (SDG-2423), and β-glucanase (SDG-2402) are feed enzyme preparations purchased from Xiasheng Biotechnology Development Co., Ltd., and can be used directly in the formulation of inhibitors.

[0027] The fermentation medium for Lactobacillus reuteri was: sucrose 90 g / L, yeast extract 60 g / L, ammonium citrate 3.5 g / L, sodium acetate 7.0 g / L, K2HPO4 4.0 g / L, MnSO4·H2O 0.4 g / L, MgSO4 0.6 g / L, and Tween 80 1.0 g / L.

[0028] The fermentation medium for Lactobacillus gasseri was: glucose 50 g / L, yeast extract 20 g / L, peptone 10 g / L, ammonium citrate 3.5 g / L, sodium acetate 7.0 g / L, K2HPO4 4.0 g / L, MnSO4·H2O 0.4 g / L, MgSO4 0.6 g / L, and Tween 80 1.0 g / L.

[0029] The fermentation medium for Lactobacillus casei is as follows: enzymatically defatted milk is used as the base carbon and nitrogen source, with the addition of 2% glucose, 2% tryptone, and 1% yeast extract.

[0030] Stabilizers: 10 wt% skim milk powder, 8 wt% glucose, 7 wt% maltose, and the remainder is water.

[0031] Example 1: Preparation of rumen methane inhibitors

[0032] (1) Preparation of probiotics:

[0033] Lactobacillus reuteri was anaerobically fermented in a 5L tank using Lactobacillus reuteri fermentation medium. The fermentation conditions were: 65% liquid volume, 2% inoculum, 100 rpm, 37℃, pH 6.5, with added ammonia, for 24 hours. The final fermentation broth yielded 5 × 10⁶ Lactobacillus reuteri bacteria. 10 CFU. Then, the fermentation broth was divided into 50mL centrifuge tubes, and each tube was centrifuged at 8000g for 10min. After centrifugation, the bacterial sludge was collected, and the sludge was mixed with the stabilizer at a mass ratio of 1:1. The mixture was then freeze-dried at -56℃ and a vacuum degree of less than 5pa for 24h to obtain Lactobacillus reuteri freeze-dried powder.

[0034] Lactobacillus gasseri was anaerobically fermented in a 5L tank using Lactobacillus gasseri fermentation medium. The fermentation conditions were: 65% liquid volume, 2% inoculum, 100 rpm, 37°C, pH 6.5, with added ammonia, for 24 hours. The final fermentation broth contained 1 × 10⁻⁶ Lactobacillus gasseri. 10 CFU. Then, the fermentation broth was divided into 50mL centrifuge tubes, each containing 30mL of the broth, and centrifuged at 8000g for 10min. After centrifugation, the bacterial sludge was collected, and the sludge was mixed with the stabilizer at a mass ratio of 1:1. The mixture was then freeze-dried at -56℃ and a vacuum degree below 5pa for 24h to obtain Lactobacillus gasseri freeze-dried powder.

[0035] Lactobacillus casei was anaerobically fermented in a 5L tank using Lactobacillus casei fermentation medium. The fermentation conditions were: 65% liquid volume, 2% inoculum, 100 rpm, 37°C, pH 6.5, with added ammonia, for 24 hours. The final fermentation broth contained 1 × 10⁻⁶ Lactobacillus casei. 10CFU. Then, the fermentation broth was divided into 50mL centrifuge tubes, and each tube was centrifuged at 8000g for 10min. After centrifugation, the bacterial sludge was collected, and the sludge was mixed with the stabilizer at a mass ratio of 1:1. The mixture was then freeze-dried at -56℃ and a vacuum degree of less than 5pa for 24h to obtain Lactobacillus casei freeze-dried powder.

[0036] (2) Preparation of rumen methane inhibitors:

[0037] (a) Formula:

[0038] Veterinary drug carrier: corn starch

[0039] Active ingredient: Lactobacillus reuteri 1×10 8 CFU, Lactobacillus gasseri 2×10 7 CFU, Lactobacillus casei 1×10 8 CFU, amylase 10, phytase 200U, xylanase 500U, cellulase 10U, β-glucanase 100U.

[0040] (b) Preparation steps:

[0041] The lyophilized powders of Lactobacillus reuteri, Lactobacillus gasseri, and Lactobacillus casei, along with amylase, phytase, xylanase, cellulase, and β-glucanase, were mixed one by one into the carrier corn starch and mixed evenly to obtain the rumen methane inhibitor.

[0042] Example 2: Preparation of rumen methane inhibitors

[0043] The preparation of Lactobacillus reuteri lyophilized powder, Lactobacillus gasseri lyophilized powder, and Lactobacillus casei lyophilized powder is the same as step (1) in Example 1.

[0044] (a) Formula:

[0045] Veterinary drug carrier: corn starch

[0046] Active ingredient: Lactobacillus estrus 2×10 9 CFU, Lactobacillus gasseri 1×10 9 CFU, Lactobacillus casei 2×10 9 CFU, amylase 90U, phytase 1000U, xylanase 2000U, cellulase 50U, β-glucanase 500U.

[0047] (b) Preparation steps:

[0048] The lyophilized powders of Lactobacillus reuteri, Lactobacillus gasseri, and Lactobacillus casei, along with amylase, phytase, xylanase, cellulase, and β-glucanase, were mixed one by one into the carrier corn starch and mixed evenly to obtain the rumen methane inhibitor.

[0049] Example 3: Preparation of rumen methane inhibitors

[0050] The preparation of Lactobacillus reuteri lyophilized powder, Lactobacillus gasseri lyophilized powder, and Lactobacillus casei lyophilized powder is the same as step (1) in Example 1.

[0051] (a) Formula:

[0052] Veterinary drug carrier: corn starch

[0053] Active ingredient: Lactobacillus reuteri 1×10 10 CFU, Lactobacillus gasseri 2×10 9 CFU, Lactobacillus casei 1×10 10 CFU, amylase 150U, phytase 2000U, xylanase 5000U, cellulase 100U, β-glucanase 1000U.

[0054] (b) Preparation steps:

[0055] The lyophilized powders of Lactobacillus reuteri, Lactobacillus gasseri, and Lactobacillus casei, along with amylase, phytase, xylanase, cellulase, and β-glucanase, were mixed one by one into the carrier corn starch and mixed evenly to obtain the rumen methane inhibitor.

[0056] Example 4: Application Investigation of Rumen Methanogens

[0057] This embodiment uses dairy cows as an example to investigate the effects of rumen methanogen inhibitors on the number of methanogens, methanogenesis, and milk production in ruminants, thereby reflecting the effectiveness of rumen methanogen inhibitors. The specific steps are as follows:

[0058] The rumen methanogen inhibitor prepared in Example 2 was added to the feed and mixed thoroughly to obtain feed with added rumen methanogen inhibitor. Eighteen Holstein dairy cows of similar age, weight (650±20 kg), lactation period (110±20 kg), and milk yield (24±1.5 kg) were selected, with nine cows in each of the experimental and control groups. The experimental group was fed the feed with added rumen methanogen inhibitor, while the control group was fed the feed without methanogen inhibitor. The experiment lasted for 60 days, and the changes in the number of methanogenic bacteria, methanogenesis, and milk yield of the dairy cows before and after the addition of the rumen methanogen inhibitor to the feed were measured.

[0059] Before the formal experiment began, all dairy cows underwent a 10-day pre-trial period to ensure their physiological state was largely similar. During the observation period, all cows were milked twice daily at 6:00 and 8:00 AM, and fed at 7:00 and 7:00 PM. Throughout the experiment, the cows had free access to feed and water. Intestinal methane emissions were measured using sulfur hexafluoride tracer gas technology. The daily intestinal methane emissions of the cows were calculated using the following formula: QCH4 = QSF6 × ([CH4]y - [CH4]b) / [SF6]. The methane emission rate (QCH4) could be calculated from the measured concentrations of [CH4]y and SF6, the background concentration of [CH4]b, and the known QSF6 release rate. On days 59 and 60 of the experiment, rumen digestive fluid was collected via rumen cannulation and mixed. The mixed rumen digestive fluid was then filtered through two layers of filters to obtain filtered rumen fluid samples for microbial analysis. Meanwhile, the daily milk production was recorded, and the results are shown in Table 1.

[0060] Table 1. Effects of rumen methanogen inhibitors on the number of methanogens, methanogenesis, and milk yield in dairy cows.

[0061] control group experimental group p-value Methanogen count in dairy cows <![CDATA[3.6×10 8 CFU / mL]]> <![CDATA[1.5×10 8 CFU / mL]]> P<0.01 Methane emissions from dairy cows 960±15L / d 455±10L / d P<0.01 Milk production of dairy cows 27.6±0.3L / d 28.5±0.3L / d P<0.01

[0062] As shown in Table 1, under the same feeding conditions, the experimental group of dairy cows had an average reduction of 58.3% in methanogen count and 52.6% in methane emissions compared to the control group, while milk production increased by 3.3%. This demonstrates that adding the rumen methanogen inhibitor described in this invention can effectively reduce the number of methanogens, thereby decreasing methane emissions, and effectively regulate rumen function, improving the productivity of ruminants, while also being safe and environmentally friendly.

[0063] In summary, the rumen methanogen inhibitor of this invention possesses the dual advantages of probiotics and complex enzymes, effectively regulating the intestinal microbiota of dairy cows to reduce methane emissions. Under the same feeding conditions, the experimental group of dairy cows showed a 58.3% reduction in the average number of methanogenic bacteria and a 52.6% reduction in methane emissions compared to the control group; milk production increased by 3.3%. This rumen methanogen inhibitor effectively regulates rumen function, reduces methane production, and improves the productivity of ruminants. Furthermore, it is safe and environmentally friendly, facilitating its application in ruminant farming and agriculture, and demonstrating significant practicality.

[0064] The embodiments described above are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments. Any obvious improvements, substitutions or modifications that can be made by those skilled in the art without departing from the essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. A rumen methane inhibitor, characterized in that, The active ingredients of the rumen methane inhibitor include probiotics and a complex enzyme that promotes the degradation of fibrous tissue. The probiotics are a mixture of Lactobacillus reuteri, Lactobacillus gasseri, and Lactobacillus casei, and the complex enzyme that promotes cellulose degradation is a mixture of amylase, phytase, xylanase, cellulase, and β-glucanase. The amount of active ingredient in each 1g rumen methane inhibitor is: Lactobacillus reuteri 1×10 8 ~1×10 10 CFU, Lactobacillus gasseri 2×10 7 ~2×10 9 CFU, Lactobacillus casei 1×10 8 ~1×10 10 CFU, amylase 10-150 U, phytase 200-2000 U, xylanase 500-5000 U, cellulase 10-100 U, β-glucanase 100-1000 U.

2. The rumen methane inhibitor according to claim 1, characterized in that, The dosage of the active ingredient in each 1g rumen methane inhibitor is: Lactobacillus reuteri 2×10 9 CFU, Lactobacillus gasseri 1×10 9 CFU, Lactobacillus casei 2×10 9 CFU, amylase 90U, phytase 1000U, xylanase 2000U, cellulase 50U, β-glucanase 500U.

3. The rumen methane inhibitor according to claim 1, characterized in that, The preparation method of the rumen methane inhibitor includes: mixing the active ingredients one by one into a veterinary drug carrier.

4. The rumen methane inhibitor according to claim 3, characterized in that, The veterinary drug carrier includes any one of glucose, inorganic salts, and soluble starch.

5. The use of the rumen methane inhibitor according to any one of claims 1-4 in reducing the methane content in the rumen of ruminants; wherein the ruminant is a dairy cow; and 0.2-2g of rumen methane inhibitor is added to each kilogram of feed.