A composite methane inhibitor and its preparation method

Abstract

The present invention belongs to the technical field of feed additives, and specifically discloses a composite methane inhibitor and a preparation method thereof. The composite methane inhibitor comprises the following raw materials by weight: 16-21 parts of oligomeric schizophyllan, 6-10 parts of atractylodes lactone IV, 3-5 parts of loganin, 1-5 parts of mannose erythritol lipids, and 4-8 parts of modified montmorillonite. Experimental results show that the components of the composite methane inhibitor cooperate with each other to achieve the effect of regulating intestinal flora and reducing methane content. Specifically, atractylodes lactone IV and loganin synergistically inhibit the activity of methanogens, and oligomeric schizophyllan can inhibit the oxidation of unsaturated fatty acids due to its antioxidant activity, reducing side reactions that may promote methane production; and modified montmorillonite has a larger interlayer spacing and specific surface area, and has excellent adsorption, which is conducive to maintaining the activity of components such as oligomeric schizophyllan, thereby further enhancing the inhibitory effect.

Description

Technical Field

[0001] The invention belongs to the technical field of feed additives, and particularly relates to a composite methane inhibitor and a preparation method thereof. Background Art

[0002] During the digestion process, ruminants such as cattle and sheep produce large amounts of methane through fermentation by rumen microorganisms. Rumen methanogens primarily use the metabolic hydrogen produced by rumen fermentation to reduce carbon dioxide or formate to produce methane. This not only results in a feed energy loss of approximately 2%-15% of total energy intake, but also exacerbates the greenhouse effect. Therefore, the development of efficient and safe methane inhibitors is crucial for reducing carbon emissions from the livestock industry and improving feed utilization.

[0003] Currently, strategies for methane suppression primarily include adjusting feed formulations (such as adding oils and nitrates), using antibiotics (such as monensin), and using natural plant extracts. However, chemically synthesized inhibitors may remain in animal products, impacting food safety. Long-term use of antibiotics can easily lead to microbial resistance. Single plant-based active ingredients often have limited effectiveness, making it difficult to achieve both methane suppression and rumen microbial balance. Therefore, the development of a comprehensive, green, and safe methane inhibitor is urgently needed.

[0004] Among natural active ingredients, polysaccharides have attracted much attention due to their good biocompatibility and antioxidant properties. For example, patent CN110771742B discloses the use of Artemisia sphaerocarpa polysaccharides, active dry yeast, Artemisia annua extract, etc. as rumen fermentation regulators. In addition, Atractylodes macrocephala lactone compounds and iridoid ether glycosides have been shown to regulate rat intestinal flora. However, the effect of a single active ingredient in the complex rumen environment is often limited, and its stability, bioavailability and synergistic effect with other components still need to be further optimized. Montmorillonite, as a natural layered silicate material, has excellent adsorption properties and is mostly used for adsorbing heavy metals or as a drug carrier. There is little research on its application in the field of methane inhibitors.

[0005] Based on this, the present invention proposes a novel composite methane inhibitor and a preparation method thereof to solve the above problems. Summary of the Invention

[0006] In order to overcome the deficiencies of the prior art, the primary purpose of the present invention is to provide a composite methane inhibitor, in which the components cooperate with each other to achieve the effect of regulating intestinal flora and reducing methane content.

[0007] Another object of the present invention is to provide a method for preparing the above-mentioned composite methane inhibitor, which has a wide range of raw materials, a scientific and reasonable ratio, a simple preparation process, and is convenient for industrial production and practical application promotion.

[0008] The purpose of the present invention is achieved through the following technical solutions:

[0009] A composite methane inhibitor comprises the following raw materials, calculated by weight: 16-21 parts of oligomeric schizophyllan polysaccharide, 6-10 parts of atractylodes lactone IV, 3-5 parts of loganin, 1-5 parts of mannose erythritol ester, and 4-8 parts of modified montmorillonite.

[0010] According to the above composite methane inhibitor, further, 18 parts of oligomeric schizophyllan polysaccharide, 8 parts of atractylodes lactone IV, 4 parts of loganin, 3 parts of mannose erythritol ester, and 6 parts of modified montmorillonite.

[0011] According to the above composite methane inhibitor, the preparation process of the modified montmorillonite is as follows: montmorillonite is dispersed in water, a surfactant and calcium chloride are added, heated and stirred for reaction, and the modified montmorillonite is obtained after filtering, washing, drying and calcining.

[0012] According to the above composite methane inhibitor, further, the surfactant is octadecyltrimethylammonium chloride.

[0013] According to the above composite methane inhibitor, further, the mass ratio of the montmorillonite, surfactant and calcium chloride is 1: (0.02-0.06): (0.08-0.1).

[0014] According to the above composite methane inhibitor, further, the heating and stirring temperature is 80-90° C., and the time is 2-4 h.

[0015] According to the above composite methane inhibitor, further, the calcination temperature is 600-700° C. and the calcination time is 3-5 hours.

[0016] According to the above composite methane inhibitor, further, the molecular weight of the oligomeric Schizophyllan polysaccharide is 2-5 kDa.

[0017] The preparation method of the composite methane inhibitor is as follows: oligomeric schizophyllan polysaccharide, atractylodes lactone IV, loganin, mannose erythritol ester and modified montmorillonite are uniformly mixed according to the aforementioned parts by weight.

[0018] The present invention has the following effects compared to the prior art:

[0019] 1. The present invention provides a composite methane inhibitor composed of oligomeric schizophyllan, atractylodes lactone IV, loganin, mannoerythritol esters, and modified montmorillonite. Experimental results demonstrate that the components work together to regulate intestinal flora and reduce methane levels. Specifically, atractylodes lactone IV and loganin synergistically inhibit the activity of methanogens. Oligomeric schizophyllan, due to its antioxidant activity, can inhibit the oxidation of unsaturated fatty acids, reducing side reactions that may promote methane production. Mannoerythritol esters may play a supporting role by regulating microbial metabolism. Modified montmorillonite, with its large interlayer spacing and specific surface area, exhibits excellent adsorption, which helps maintain the activity of components such as oligomeric schizophyllan, thereby further enhancing the inhibitory effect.

[0020] 2. The composite methane inhibitor provided by the present invention has a wide range of raw materials, a scientific and reasonable ratio, and a simple preparation process, which is convenient for industrial production and practical application promotion. DETAILED DESCRIPTION

[0021] The technical solution of the present invention will be further described below in conjunction with specific embodiments. However, it should be understood by those skilled in the art that the following examples are only intended to illustrate the present invention and should not be construed as limiting the present invention. Specific conditions not specified in the examples are to be followed according to conventional conditions or the conditions recommended by the manufacturer. All reagents or instruments used, unless otherwise specified, are conventional products obtained from commercial channels.

[0022] Example 1

[0023] A composite methane inhibitor is composed of the following raw materials in parts by weight: 18 parts of oligomeric schizophyllan polysaccharide with an average molecular weight of 5 kDa, 8 parts of atractylodes lactone IV, 4 parts of loganin, 3 parts of mannose erythritol ester, and 6 parts of modified montmorillonite.

[0024] The preparation process of the modified montmorillonite is as follows:

[0025] Montmorillonite was dispersed in water, and then surfactant and calcium chloride were added, wherein the mass ratio of montmorillonite, surfactant and calcium chloride was 1:0.04:0.09, and the mixture was heated and stirred at 85°C for 3 h, filtered, washed, dried and calcined at 650°C for 4 h to obtain the product.

[0026] This embodiment also provides a method for preparing the composite methane inhibitor, which specifically comprises the following steps: uniformly mixing oligomeric schizophyllan polysaccharide, atractylodes lactone IV, loganin, mannose erythritol ester, and modified montmorillonite according to the aforementioned parts by weight.

[0027] Example 2

[0028] A composite methane inhibitor is composed of the following raw materials in parts by weight: 16 parts of oligomeric schizophyllan polysaccharide with an average molecular weight of 2 kDa, 6 parts of atractylodes lactone IV, 5 parts of loganin, 1 part of mannose erythritol ester, and 4 parts of modified montmorillonite.

[0029] The preparation process of the modified montmorillonite is as follows:

[0030] Montmorillonite is dispersed in water, and then a surfactant and calcium chloride are added, wherein the mass ratio of montmorillonite, surfactant and calcium chloride is 1:0.02:0.08. The mixture is heated and stirred at 80°C for 4 h, filtered, washed, dried and calcined at 600°C for 5 h to obtain the product.

[0031] The preparation method of the composite methane inhibitor of this embodiment is the same as that of Example 1.

[0032] Example 3

[0033] A composite methane inhibitor is composed of the following raw materials in parts by weight: 21 parts of oligomeric schizophyllan with an average molecular weight of 5 kDa, 10 parts of atractylodes lactone IV, 3 parts of loganin, 5 parts of mannose erythritol ester, and 8 parts of modified montmorillonite.

[0034] The preparation process of the modified montmorillonite is as follows:

[0035] Montmorillonite is dispersed in water, and then a surfactant and calcium chloride are added, wherein the mass ratio of montmorillonite, surfactant and calcium chloride is 1:0.06:0.1. The mixture is heated and stirred at 90°C for 2 h, filtered, washed, dried, and calcined at 700°C for 3 h to obtain the product.

[0036] The preparation method of the composite methane inhibitor of this embodiment is the same as that of Example 1.

[0037] Comparative Example 1

[0038] A methane inhibitor is composed of the following raw materials in parts by weight: 18 parts of oligomeric schizophyllan with an average molecular weight of 5 kDa, 4 parts of loganin, 3 parts of mannose erythritol ester, and 6 parts of modified montmorillonite; the rest of the ingredients are the same as those in Example 1.

[0039] Comparative Example 2

[0040] A methane inhibitor is composed of the following raw materials in parts by weight: 18 parts of oligomeric schizophyllan with an average molecular weight of 5 kDa, 8 parts of atractylodes lactone I, 4 parts of loganin, 3 parts of mannose erythritol ester, and 6 parts of modified montmorillonite; the remaining ingredients are the same as those in Example 1.

[0041] Comparative Example 3

[0042] A methane inhibitor is composed of the following raw materials in parts by weight: 18 parts of oligomeric schizophyllan with an average molecular weight of 5 kDa, 8 parts of atractylodes lactone IV, 3 parts of mannose erythritol ester, and 6 parts of modified montmorillonite; the remaining ingredients are the same as those in Example 1.

[0043] Comparative Example 4

[0044] A methane inhibitor is composed of the following raw materials in parts by weight: 18 parts of oligomeric schizophyllan with an average molecular weight of 5 kDa, 12 parts of atractylodes lactone IV, 3 parts of mannose erythritol ester, and 6 parts of modified montmorillonite; the remaining ingredients are the same as those in Example 1.

[0045] Comparative Example 5

[0046] A methane inhibitor is composed of the following raw materials in parts by weight: 12 parts of Atractylodes lactone IV, 3 parts of mannose erythritol ester, and 6 parts of modified montmorillonite; the remaining ingredients are the same as those in Example 1.

[0047] Comparative Example 6

[0048] A methane inhibitor is composed of the following raw materials in parts by weight: 18 parts of small molecule schizophyllan with an average molecular weight of 20 kDa, 12 parts of atractylodes lactone IV, 3 parts of mannose erythritol ester, and 6 parts of modified montmorillonite; the rest of the ingredients are the same as those in Example 1.

[0049] In vitro rumen fermentation experiment

[0050] 1.1 Donor animal management and rumen fluid collection

[0051] Three healthy adult goats with permanent rumen cannulas were used as donor animals. They were fed a mixed diet of 50% concentrate (70% corn meal + 25% soybean meal + 5% alfalfa) and 50% whole corn silage (DM 920 g / kg, CP 80.6 g / kgDM) at 08:00 and 20:00 daily, with a total feed intake of 2.2 kg DM.

[0052] The rumen contents of three rumen fistula goats were collected before morning feeding, and the rumen fluid was obtained by filtering with four layers of gauze. The collected rumen fluid was evenly mixed and placed in a thermos cup preheated at 39°C and filled with carbon dioxide, and used as rumen fluid for subsequent fermentation.

[0053] 1.2 Preparation of in vitro fermentation system

[0054] The culture substrate was consistent with the donor animal diet (50% concentrate + 50% silage) and was crushed and passed through a 2 mm sieve. 10 g of the composite inhibitor of Examples 1-3 or Comparative Examples 1-6 was added to each 1000 g culture substrate, mixed thoroughly, and 0.5 g was accurately weighed based on DM as a fermentation substrate for later use.

[0055] The experiment included 10 treatment groups, including Examples 1-3, Comparative Examples 1-6, and a control group, with six replicates per group. Each 125 mL fermentation flask contained 0.5 g of fermentation substrate, 45 mL of preheated rumen buffer (prepared by the Goering & Van Soest method), and 15 mL of rumen fluid. In the control group, no inhibitors were added to the fermentation substrate. The flasks were then filled with carbon dioxide for 30 seconds, sealed with a butyl rubber stopper, and fermented in a 39°C rotary incubator (125 rpm) for 24 hours.

[0056] 1.3 Sample collection and analysis

[0057] (1) After the fermentation is completed, cool the fermentation bottle with ice. When the gas temperature is close to the room temperature, measure the total gas production by connecting a barometer through a glass needle. Use gas chromatography (TDX-01 column, argon carrier) to analyze the hydrogen and methane contents. Take the fermentation liquid and use a pH meter to directly measure the acidity and alkalinity.

[0058] (2) Using the Qiagen Bacterial Genomic DNA Kit, total bacterial genomic DNA was extracted from the fermentation residues. Methanogens (mcrA gene) and cellulolytic bacteria (such as Ruminococcus flavefaciens and Fibrobacter succinogenes, based on 16S rDNA) were quantified by qPCR. The primer sequences for the target bacteria, Methanobrevibacter ruminantium (ATCC 35063), Ruminococcus flavefaciens (ATCC 19208), and Fibrobacter succinogenes (ATCC 51216), are shown in Table 1. The conventional PCR reaction system settings are shown in Tables 2 and 3. Based on the SYBR Green I fluorescent dye method, a standard curve was constructed on the LightCycler® 96 Real-Time PCR System (Roche Diagnostics) to achieve absolute quantitative detection of the 16S rDNA of the target bacteria.

[0059] Table 1 Sequence list of target bacterial species

[0060]

[0061] Table 2 Fluorescence quantitative PCR system

[0062]

[0063] Table 3 Fluorescence quantitative PCR reaction procedure

[0064]

[0065] 1.4 Results and Analysis

[0066] The effects of each group of inhibitors on the total gas production, methane and pH of rumen in vitro fermentation are shown in Table 4, and the effects of each group of inhibitors on the rumen microbial flora are shown in Table 5.

[0067] Table 4 Effects of inhibitors on total gas production, methane and pH of rumen fermentation in vitro

[0068]

[0069] As can be seen from Table 4, compared with the control group, the inhibitors prepared in Examples 1-3 can significantly reduce the total gas production and methane content. Compared with Example 1, the methane production of Comparative Examples 1-6 is higher, which shows that by compounding oligomeric schizophyllan polysaccharides, atractylodes lactone IV, loganin, mannose erythritol lipids, and modified montmorillonite, the components cooperate with each other to play a synergistic role and more effectively reduce the methane content. Specifically, atractylodes lactone IV and loganin may synergistically inhibit the activity of methanogens; oligomeric schizophyllan polysaccharides can inhibit the oxidation of unsaturated fatty acids due to their antioxidant activity, reducing side reactions that may promote methane production; mannose erythritol lipids may play an auxiliary role by regulating microbial metabolism; and modified montmorillonite has a larger interlayer spacing and specific surface area, and has excellent adsorption, which is conducive to maintaining the activity of components such as oligomeric schizophyllan polysaccharides, thereby further enhancing the inhibitory effect.

[0070] Table 5 Effects of each group of inhibitors on rumen microbial flora

[0071]

[0072] As can be seen from Table 5, the number of methanogens in the control group was higher than that in Examples 1-3, while the number of Ruminococcus xanthus and Succinicobacterium succiniciproducens did not vary much between the groups, indicating that the addition of the inhibitor prepared in Examples 1-3 of the present invention can specifically inhibit the activity of methanogens, adjust the structure of the rumen microbial community, and thus significantly reduce the methane emissions of ruminants.

[0073] Therefore, the present invention confirmed through in vitro rumen fermentation experiments that the addition of the composite methane inhibitor prepared in Examples 1-3 of the present invention can effectively inhibit the activity of methanogens and adjust the structure of rumen microbial communities, thereby significantly reducing methane emissions from ruminants, which is of great significance for reducing greenhouse gas emissions in animal husbandry.

[0074] Finally, it should be noted that the above embodiments are intended only to illustrate the technical solutions of the present invention and are not intended to limit the same. The basic principles and main features of the present invention have been described above using specific implementation schemes. Modifications or substitutions may be made based on the present invention, but such modifications or substitutions do not detract from the essence of the corresponding technical solutions from the scope of protection claimed by the present invention.

Claims

1. A composite methane inhibitor, characterized in that: The raw materials are as follows: 16-21 parts of oligomeric schizophyllan polysaccharide, 6-10 parts of atractylodes lactone IV, 3-5 parts of loganin, 1-5 parts of mannose erythritol ester and 4-8 parts of modified montmorillonite.

2. The composite methane inhibitor according to claim 1, characterized in that: 18 parts of oligomeric schizophyllan polysaccharide, 8 parts of atractylodes lactone IV, 4 parts of loganin, 3 parts of mannose erythritol lipids, and 6 parts of modified montmorillonite.

3. The composite methane inhibitor according to claim 1, characterized in that: The preparation process of the modified montmorillonite is as follows: montmorillonite is dispersed in water, a surfactant and calcium chloride are added, heated and stirred for reaction, and the modified montmorillonite is obtained after filtering, washing, drying and calcining.

4. The composite methane inhibitor according to claim 3, characterized in that: The surfactant is octadecyltrimethylammonium chloride.

5. The composite methane inhibitor according to claim 3, characterized in that: The mass ratio of the montmorillonite, the surfactant and the calcium chloride is 1: (0.02-0.06): (0.08-0.1).

6. The composite methane inhibitor according to claim 3, characterized in that: The heating and stirring is performed at a temperature of 80-90° C. for 2-4 h.

7. The composite methane inhibitor according to claim 3, characterized in that: The calcination temperature is 600-700° C. and the calcination time is 3-5 h.

8. The composite methane inhibitor according to claim 1, characterized in that: The molecular weight of the oligomeric Schizophyllan polysaccharide is 2-5 kDa.

9. The method for preparing the composite methane inhibitor according to any one of claims 1 to 8, characterized in that: According to the aforementioned parts by weight, the oligomeric schizophyllan polysaccharide, atractylodes lactone IV, loganin, mannose erythritol ester and modified montmorillonite are mixed evenly.

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