Use of lachnospiraceae bacteria in the preparation of microbial preparations for reducing methane emissions from ruminants

Abstract

The present invention discloses the use of bacteria of the Lachnospiraceae family in the preparation of a microbial preparation for reducing methane emissions in ruminants.

Description

Technical Field

[0001] This invention belongs to the fields of microbiology, animal nutrition and greenhouse gas emission reduction, and specifically relates to the use of Trichophyceae bacteria in the preparation of microbial preparations for reducing methane emissions from ruminants. Background Technology

[0002] Methane produced by rumen fermentation in ruminants is a significant source of greenhouse gases in agriculture. Current methane emission reduction strategies mainly focus on chemical additives that inhibit methanogens and altering dietary composition. However, chemical additives pose safety risks, easily lead to drug resistance, and affect production performance; while most dietary regulation methods are inconsistent in their effects, their underlying mechanisms are unclear, and precise application is difficult. In recent years, achieving functional emission reduction through rumen microbiota regulation has become a research hotspot, but existing technologies mostly focus on describing the overall microbial community structure, lacking the discovery, verification, and utilization of single key bacterial species or communities with clear emission reduction functions and their precise mechanisms of action. How to discover, enrich, and utilize functional bacteria capable of actively regulating hydrogen metabolism flow, and establish a microbially driven intrinsic emission reduction mechanism, is a pressing challenge in the current technological field. Summary of the Invention

[0003] The present invention aims to overcome the shortcomings of the prior art and provide the use of Trichophyceae bacteria in the preparation of microbial preparations for reducing methane emissions from ruminants.

[0004] To achieve the above objectives, the technical solution provided by this invention is as follows: First, this invention provides the use of Lachnospiraceae bacterium in the preparation of microbial preparations for reducing methane emissions from ruminants.

[0005] The present invention also provides the use of Trichophyceae bacteria in the preparation of feed or feed additives for reducing methane emissions from ruminants.

[0006] The present invention also provides the use of mannan oligosaccharides (MOS) in the preparation of formulations for promoting the growth of Trichophyceae bacteria in the rumen of ruminants and / or activating their methane emission reduction metabolic activity.

[0007] The present invention also provides a methane emission reduction feed additive for ruminants, wherein the methane emission reduction feed additive for ruminants contains live bacteria of the Trichophyceae family and / or its culture.

[0008] Preferably, the methane emission reduction feed additive for ruminants also contains mannan oligosaccharides.

[0009] The present invention also provides a method for reducing rumen methane emissions in ruminants, the method being to enrich and / or activate Trichophycete bacteria in the rumen of ruminants.

[0010] Preferably, the enrichment and / or activation of the Trichophyceae bacteria is achieved by directly administering a live Trichophyceae bacterial preparation to the ruminants; or by feeding the ruminants a diet containing mannan oligosaccharides or their precursors.

[0011] Preferably, the precursor is β-mannan, which can be degraded in the rumen environment to generate mannooligosaccharides.

[0012] More preferably, the dosage of the live bacterial preparation of the Trichophytonceae family is ≥10 mg / day per animal. 8 CFU; the feeding dosage of the mannan oligosaccharide is 10-50 grams per animal per day.

[0013] The present invention also provides a culture medium for isolating, culturing or proliferating bacteria of the Trichophyceae family, wherein the culture medium uses mannooligosaccharides as the main carbon and energy source.

[0014] The present invention will be further described below:

[0015] This invention discovers the core role of Trichophytoncidae bacteria in regulating rumen hydrogen metabolism and provides a technical solution to reduce methane emissions by enriching and enhancing the metabolic activity of these bacteria, thereby overcoming the shortcomings of existing technologies such as unclear targets and unstable effects.

[0016] This invention reveals, isolates, and verifies for the first time the core functional role of Trichophyceae bacteria in mediating rumen methane emission reduction in ruminants, and elucidates their unique mechanism of action.

[0017] This invention designed and conducted the following key in vitro validation experiments: Experimental Design: An in vitro rumen fermentation system was used. Two core treatments were set up: (A) Starch group: starch was used as the sole or main carbon source; (B) MOS group: mannan oligosaccharide (MOS) was used as the sole or main carbon source. Both groups were inoculated with the same background rumen microbial broth. Introduction of Trichophyceae bacteria: Before fermentation, equal amounts of pure cultures or high-purity enrichments of Trichophyceae bacteria isolated and purified from the rumen of high-producing animals were simultaneously inoculated into both groups A and B. Results: Methane production was measured after fermentation. The results showed that the MOS group (group B) inoculated with Trichophyceae bacteria had a significantly lower methane production than the starch group (group A) inoculated with the same bacteria. This result conclusively proves that Trichophyceae bacteria themselves are a necessary biological factor for producing emission reduction effects. The emission reduction efficiency of this bacterium is highly dependent on its fermentation substrate, and MOS is its "preferred functional substrate" for exerting the strongest emission reduction function. When this bacterium uses MOS as "fuel", it can most effectively drive rumen hydrogen flow to the non-methane pathway.

[0018] Specifically, the technical solution of the present invention is based on the following key findings and mechanisms that have been experimentally verified: 1. Identification and Functional Localization of Functional Bacteria: Through comparative experiments, this invention identifies bacteria from the Trichophyceae family as key methane-reducing bacteria in the complex rumen microbiota. These bacteria do not exert their effects by directly inhibiting methanogenic archaea, but rather indirectly influence methane production by altering the hydrogen production generated during their own fermentation metabolism.

[0019] 2. Substrate Specificity and Function Activation: This invention further reveals that the emission reduction function of the *Trichophyceae* bacteria exhibits significant substrate dependence. When the bacteria use mannooligosaccharides (MOS) as the primary fermentation substrate, their ability to reduce methane production is maximized. MOS, as a preferred functional substrate for these bacteria, guides their metabolic flux into a fermentation pathway with relatively lower hydrogen production (particularly butyrate fermentation). In contrast, when common carbohydrates such as starch are used as substrates, the emission reduction effect of these bacteria is significantly weakened. This indicates that *Trichophyceae* bacteria are the "executive unit" of the emission reduction function, and MOS is the "specific activation key" for initiating and optimizing the emission reduction performance of this unit.

[0020] 3. Construction of a Complete Mechanism of Action: This invention constructs a clear emission reduction mechanism, namely, in the rumen environment, by providing or creating conditions favorable to the growth of Trichophyceae bacteria and the presence of their preferred substrate MOS, selective enrichment and metabolic activation of this functional bacterium can be achieved. The enriched and activated Trichophyceae bacteria utilize MOS extensively for metabolism. Due to the low net hydrogen production of their dominant fermentation pathways (such as the butyrate pathway), the total amount of molecular hydrogen (H2) available for methanogenesis in the entire rumen fermentation ecosystem is directly reduced. The reduction in hydrogen supply leads to a substrate shortage for methanogenic archaea, ultimately resulting in a decrease in methane production.

[0021] In this invention, bacteria of the Trichophyceae family or their cultures can be directly used as microbial additives, introduced into the rumen of ruminants via oral administration, coating, or other methods to increase the abundance of these functional bacteria. Alternatively, a dietary nutrition strategy can be employed to provide specific substrates (such as MOS or its precursors) that can be efficiently utilized by Trichophyceae bacteria and stimulate their emission reduction function, thereby promoting their proliferation and functional expression in situ within the rumen. These precursors include, but are not limited to, β-mannan, which can be degraded in the rumen to generate MOS.

[0022] In summary, this invention discovers and utilizes the ability of Trichophyceae bacteria to perform a low-hydrogen-producing fermentation metabolism when utilizing their preferred substrate, mannooligosaccharide (MOS), thereby effectively reducing the supply of hydrogen, a precursor to methane synthesis, in the rumen. This invention provides two pathways to achieve this emission reduction mechanism: one is the direct application of Trichophyceae bacteria as a probiotic preparation; the other is the in-situ enrichment and activation of indigenous Trichophyceae bacteria in the rumen by feeding them MOS or its precursors. This invention clarifies the synergistic emission reduction effect produced by the combination of specific functional bacteria and specific substrates, providing core bacterial resources, key active substrates, and a clear mechanism of action for developing next-generation, targeted, and stable microbial emission reduction products. Attached Figure Description

[0023] Figure 1 The morphology of *Trichophyton spp.* under a microscope; Figure 2 Methane production after long-term feeding of preferred substrates; Figure 3 Methane and hydrogen production during in vitro fermentation after co-addition of preferred substrate and *Helicobacter pylori*; Figure 4 The methane and hydrogen production of pure bacteria was inhibited after co-addition of the preferred substrate and *Helicobacter pylori*. Detailed Implementation

[0024] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0025] To obtain, preserve, and cultivate on a large scale the core functional bacteria of this invention—Trichophyceae bacteria—it is necessary to provide suitable culture conditions that maximize their emission reduction activity. The culture medium formulations shown in Table 1 use MOS as the key selective carbon source.

[0026] Table 1. Special culture substrates for the isolation, culture, and proliferation of bacteria from the Trichophyceae family.

[0027] Example 1: Application of Trichophyceae bacteria as direct feed microbial additives Bacteria of the family Trichophyceae, isolated, identified, and cultured from rumen contents, were collected by centrifugation. The morphology of the bacterial cells was observed under a microscope. Figure 1 After being mixed with a preservative (such as skim milk, glycerin, etc.), a live bacterial preparation was prepared by freeze-drying. This preparation was administered at a dosage of ≥10 mg per animal per day. 8CFU is administered to ruminants (such as sheep (goats, sheep, dairy goats, dairy cows, beef cattle) via oral gavage or by mixing with concentrate for a certain period (e.g., 2-4 weeks) to colonize the rumen or increase its abundance, thereby achieving methane emission reduction. Figure 2 ).

[0028] Example 2: Application of in-situ enrichment of functional bacteria by adding specific substrates to diets Add a certain amount of mannan oligosaccharide (MOS) or its precursor (such as feed ingredients rich in β-mannan) to the basal diet of ruminants daily. The amount of MOS added can be 10-50 grams per animal per day. By continuously feeding (e.g., for more than 4 weeks), the preferred substrate of this functional bacteria is continuously provided in the rumen, thereby creating selective pressure in the rumen microecology, promoting the natural enrichment and functional activation of indigenous Trichophyceae bacteria, and establishing a stable low-methane fermentation pattern. Figure 3 ).

[0029] Example 3: Verification by pure culture of methanogens and spirochetes.

[0030] Pure cultures of *Bacillus methanogenus* and *Helicobacter spirochetes* were cultured separately in their respective anaerobic flasks. Based on the ratio of *Bacillus methanogenus* to *Helicobacter spirochetes* in rumen fluid from animal experiments, the bacterial concentration was calculated using OD values. The bacteria were then added at three different ratios: 1:1, 1:3, and 1:9. Methane and hydrogen concentrations were measured to determine the differences in methanogenesis between the two bacteria in MOS and starch. Figure 4 The results showed that 1x and 3x *Trichoderma* reduced methane emissions by 40-55%, while adding 9x *Trichoderma*, simulating the addition ratio of MOS in the animal rumen, reduced methane emissions by 85-90%. Furthermore, *Trichoderma* showed better performance and stronger decomposition capabilities in MOS substrates.

Claims

1. Use of Trichophyceae bacteria in the preparation of microbial preparations for reducing methane emissions from ruminants.

2. Use of Trichophyceae bacteria in the preparation of feed or feed additives for reducing methane emissions from ruminants.

3. Use of mannan oligosaccharides in the preparation of formulations for promoting the growth of Trichophyceae bacteria in the rumen of ruminants and / or activating their methane-reducing metabolic activity.

4. A methane emission reduction feed additive for ruminants, characterized in that, The methane emission reduction feed additive for ruminants contains live bacteria of the Trichophyceae family and / or its cultures.

5. The methane emission reduction feed additive for ruminants as described in claim 4, characterized in that, The methane emission reduction feed additive for ruminants also contains mannan oligosaccharides.

6. A method for reducing rumen methane emissions in ruminants, characterized in that, The method involves enriching and / or activating Trichophycete bacteria in the rumen of ruminants.

7. The method for reducing rumen methane emissions in ruminants as described in claim 6, characterized in that, The enrichment and / or activation of the Trichophyceae bacteria is achieved by directly administering a live Trichophyceae bacterial preparation to the ruminants; or by feeding the ruminants a diet containing mannan oligosaccharides or their precursors.

8. The method for reducing rumen methane emissions in ruminants as described in claim 7, characterized in that, The precursor substance is β-mannan, which can be degraded in the rumen environment to produce mannan oligosaccharides.

9. The method as described in claim 7 or 8, characterized in that, The dosage of the live bacterial preparation from the Trichophytonceae family is ≥10 mg / day per animal. 8 CFU; the feeding dosage of the mannan oligosaccharide is 10-50 grams per animal per day.

10. A culture medium for isolating, culturing, or proliferating bacteria of the Trichophyceae family, characterized in that, The culture medium uses mannooligosaccharides as the main carbon and energy source.

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