A rumen methane inhibiting composition comprising asparagus cochinchinensis and pterostilbene

Abstract

This invention relates to the field of agricultural biotechnology, specifically to a rumen methane inhibitory composition for ruminants containing *Asparagus yew* and *Pterostilbene*. The active components of the composition include *Asparagus yew* and its preparations, and *Pterostilbene*, which are used in a predetermined ratio to reduce rumen methane production and alleviate hydrogen accumulation in ruminants.

Description

Technical Field

[0001] This invention relates to the field of agricultural biotechnology, and more specifically to a rumen methane inhibitory composition for ruminants containing yew-like asparagus and pterostilbene. Background Technology

[0002] Rumen fermentation in ruminants such as cattle and sheep is the primary source of biogenic methane in agriculture. The rumen of ruminants harbors a large and interacting community of microorganisms whose metabolic activities and related enzymatic reactions significantly influence carbohydrate fermentation and methane production. During rumen fermentation, microorganisms break down substrates such as carbohydrates in feed, producing reducing equivalents such as hydrogen (H2). Methanogens utilize hydrogen to reduce substrates such as carbon dioxide (CO2) to methane (CH4), thereby maintaining a low hydrogen partial pressure in the rumen and contributing to the stability of electron flow and fermentation metabolism. When methanogenesis is inhibited through exogenous measures, the main pathway for hydrogen may be blocked, easily leading to hydrogen accumulation in the rumen. This, in turn, affects fermentation processes such as fiber degradation and volatile fatty acid (VFA) production, impacting rumen metabolic homeostasis and productivity. Therefore, there is an urgent need to develop a rumen regulation technology that can reduce methane production and alleviate hydrogen accumulation.

[0003] Existing technologies for regulating rumen methane production in ruminants mainly include seaweed additives, plant extract additives, and electron acceptor additives. Among these, the red algae *Asparagus purpureus*, rich in brominated compounds (such as bromoform), has been used to reduce rumen methane emissions. While this approach can reduce methane production, its application is limited. First, some methods require high dosages, affecting palatability. Second, while inhibiting methane production, the limited hydrogen utilization pathway may lead to increased hydrogen concentration in the rumen, thus affecting the VFA ratio and microbial stability. Third, the availability and large-scale supply of seaweed are limited, and the high cost of artificial cultivation and harvesting hinders sustainable application.

[0004] Plant extracts such as tannins, saponins, essential oils, flavonoids, and polyphenols have also been used in rumen methane emission reduction research. However, these additives usually have limited efficacy and relatively unclear regulatory mechanisms under low-dose conditions, making it difficult to achieve the emission reduction levels of seaweed inhibitors.

[0005] Electron acceptor additives such as malic acid, nitrates, and fumaric acid typically reduce the supply of substrates for methane formation by competing for hydrogen utilization pathways and diverting hydrogen flow to alternative metabolic pathways such as propionic acid. However, this approach has limited inhibitory efficacy at conventional doses; some inorganic electron acceptors may introduce intermediate products such as nitrites, and they generally lack direct inhibitory effects on the methanogenesis process.

[0006] In summary, existing technologies still suffer from drawbacks such as high addition amounts, significant risk of hydrogen accumulation, and the inability of a single mechanism to simultaneously address methane inhibition and hydrogen metabolism balance. Therefore, it remains necessary to provide a combined technical solution that can reduce methane formation and mitigate hydrogen accumulation under lower addition amounts. Summary of the Invention

[0007] This invention solves the problems of high dosage, hydrogen accumulation, and limited application of existing rumen methane inhibitors.

[0008] The purpose of this invention is to provide a rumen methane inhibitory composition for ruminants containing yew-like asparagus and pterostilbene.

[0009] This invention provides a rumen methane-inhibiting composition for ruminants, wherein the active component of the composition includes yew asparagus (… Asparagopsis taxiformis (and / or its preparations); pterostilbene and / or its feedable salts and feedable derivatives. The mass ratio of the yew-like asparagus to pterostilbene is 10:1 to 200:1, preferably 10:1 to 100:1, and more preferably 20:1.

[0010] In some embodiments, the yew-like asparagus can inhibit methanogenesis through its brominated compounds; the pterostilbene can regulate key enzymes and pathways related to methanogenesis, as well as hydrogen metabolism-related functions. When used in combination, the two can reduce methane production while decreasing high-dose dependence on yew-like asparagus, and to some extent alleviate hydrogen accumulation that occurs when yew-like asparagus is used alone.

[0011] In a further embodiment, the composition may further comprise an electron acceptor component to further direct hydrogen flow to a non-methanogenic pathway (e.g., propionic acid generation pathway), wherein the electron acceptor component is preferably malic acid and / or its feedable salt.

[0012] The composition provided by this invention can be used to reduce rumen methane production in ruminants and alleviate hydrogen accumulation to some extent, thus having application value as a feed additive for ruminants.

[0013] This invention achieves combined regulation of rumen methane generation and hydrogen metabolism by using *Asparagus cochinchinensis* in combination with *Pterocarya stenoptera*, and further adding malic acid in some embodiments. Compared with the prior art, this invention exhibits the following technical effects: (1) Two-way synergistic regulation: Simultaneously inhibiting methane and hydrogen, yew asparagus has a significant methane inhibition ability, but hydrogen accumulates in the rumen. After adding pterostilbene in this invention, the hydrogen concentration is significantly reduced to close to the control group level, reducing side effects from the source; without sacrificing the methane emission reduction efficiency, the regulation of hydrogen metabolism homeostasis is achieved, effectively ensuring the stable operation of the rumen fermentation system.

[0014] (2) The dosage is significantly reduced, and the palatability and safety of feed are better. The introduction of Pterostilbene enhances the synergistic effect of the methane inhibition system, so that the dosage of Taxus chinensis can reduce methane by 60% with only about 0.2% dosage. While reducing the dosage, the emission reduction effect is maintained, which not only reduces the cost of use, but also alleviates the resource dependence problem of Taxus chinensis and enhances the industrialization of the combined product.

[0015] (3) It is applicable to a variety of ruminants and has a wide range of practical prospects. The dosage can be adjusted according to the different animal diet structure to achieve a feeding promotion model that is compatible with individualization and standardization. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 A schematic diagram illustrating the effects of different treatment combinations on rumen microbial composition; Figure 2 A schematic diagram illustrating the effects of different treatment combinations on rumen volatile acid production and methanogenesis pathways; Figure 3 This is a schematic diagram illustrating the effect of different treatment combinations on rumen hydrogenase abundance. Detailed Implementation

[0018] The present invention will be further described below with reference to embodiments. Unless otherwise stated, all percentages (%) mentioned in this specification refer to the mass percentage based on dietary dry matter (DM).

[0019] The composition provided by this invention includes: (1) Purple-leaved asparagus ( Asparagopsis taxiformis (and / or its powder, dried form, extract or preparation. The raw material of *Asparagus purpurea* can be collected from the Wanshan Islands of Zhuhai City and identified as *Asparagus purpurea*.)

[0020] (2) Pterostilbene and / or its feedable salts and feedable derivatives; And optionally include: (3) Electron acceptor / organic acid component: malic acid and / or its feedable salts.

[0021] The above components, when combined, can form a combined system that addresses both methane suppression and hydrogen accumulation mitigation.

[0022] In some preferred embodiments, the purity of pterostilbene is ≥98%, the raw material of yew-like asparagus is freeze-dried, pulverized and mixed, and the purity of malic acid is ≥98%.

[0023] In some preferred embodiments, the composition can be prepared as a premix, concentrate, concentrate supplement, or total mixed ration (TMR) additive; it can also be further mixed with a carrier / excipient to form granules, tablets, or licks to improve mixing uniformity and ease of use. The carrier / excipient can be selected from starch, wheat bran, microcrystalline cellulose, zeolite powder, montmorillonite, calcium carbonate, or combinations thereof.

[0024] One preparation method includes: freeze-drying and pulverizing *Asparagus officinalis* raw material, adding it to a carrier along with *Pterostilbene strychnifolia* and optionally malic acid in a predetermined ratio, and mixing them using a stepwise premixing method to obtain the composition. For active components sensitive to oxygen or heat, stabilization treatment can be performed using coating or microencapsulation.

[0025] The key improvements of this invention include: In some implementations, the combined use of pterostilbene and yew asparagus can reduce CH4 generation while decreasing dependence on higher amounts of yew asparagus.

[0026] By combining *Asparagus purpurea* and *Pterocarya stenoptera* in a certain proportion, it is possible to simultaneously affect the methanogenesis and hydrogen metabolism processes in some embodiments.

[0027] This invention also provides a method of applying the composition in ruminant feeding, namely, adding the composition to ruminant diets, TMR, premixes, concentrate supplements, or licks for feeding, to reduce rumen methane production and alleviate hydrogen accumulation. The ruminants include dairy cows, beef cattle, sheep, goats, and buffalo.

[0028] In a preferred embodiment, the amount of red algae added, based on the dry matter of the diet, is 0.05% to 0.50%, more preferably about 0.20%; the amount of pterostilbene added is 0.001% to 0.02%, more preferably about 0.01%. The amount of malic acid added is 5%. Example 1: Combination of Pterocarya stenoptera and Asparagus officinalis

[0029] The purity of pterostilbene used was ≥98%, and the yew-like asparagus was freeze-dried and uniformly pulverized. The purity of malic acid was ≥98%, and the rumen fluid was derived from lactating dairy cows. Fermentation substrate: basal TMR diet. In vitro anaerobic culture system: ANKOM.

[0030] Experimental groups (6 replicates per group): CON: control group (no additive); AT: red algae group (0.20% Taxodium spp.); AP: red algae and malic acid combination group (0.20% Taxodium spp. + 5% malic acid); AZ: red algae and Pterostilbene combination group (0.20% Taxodium spp. + 0.01% Pterostilbene).

[0031] Prepare the fermentation system according to the experimental group (liquid rumen fluid and buffer solution are mixed, and substrate and additives are added). Under anaerobic conditions, incubate in a constant temperature incubator at 39℃ for 24h. The total gas production (TGP) is measured at the endpoint. Gas samples are taken to measure methane (CH4) and hydrogen (H2). Fermentation broth is taken to measure rumen fermentation parameters.

[0032] The results showed that, compared with the CON group, the AT group significantly reduced TGP and CH4 production and significantly increased H2 production; the AP group significantly reduced CH4 production and increased TGP, H2 and CO2 production; compared with the AT group, the AP group significantly increased TGP and significantly reduced H2 production; the CH4 and H2 production of the AZ group were significantly lower than those of the AT group, while the CO2 production was higher than that of the AT group. This indicates that under the conditions of this embodiment, the combined use of Pterostilbene spp. and Asparagus officinalis can reduce CH4 production and alleviate H2 accumulation.

[0033] As shown in Table 1, a portion of the data is as follows: The TGP (mL / g) values ​​in the CON, AT, AP, and AZ groups were 125.27, 116.55, 130.77, and 116.13, respectively. The concentrations of H2 (mL / g) were 0.76, 2.57, 2.10, and 1.41, respectively; the concentrations of CH4 (mL / g) were 7.87, 3.88, 5.01, and 1.79, respectively. The H2 / TGP (%) ratios were 0.61, 2.21, 1.61, and 1.21, respectively. The CH4 / TGP (%) ratios were 6.28, 3.34, 3.87, and 1.53, respectively.

[0034] The above results indicate that the AZ group significantly reduced H2 levels while simultaneously reducing CH4 levels compared to the AT group, demonstrating a synergistic regulatory advantage.

[0035] Table 1. Effects of different combinations on gas yield and composition. .

[0036] As shown in Table 2, compared with the CON group, the AT and AP groups significantly reduced the acetic acid ratio and increased the propionic acid ratio, and the ethyl / propionic acid ratio was significantly reduced; the AZ group significantly reduced the valeric acid and butyric acid ratios; compared with the AT group, the AZ group significantly increased the acetic acid ratio and the ethyl / propionic acid ratio, and significantly reduced the butyric acid and valeric acid ratios, suggesting that this combination has an improving effect on the fermentation mode under the background of methane inhibition.

[0037] Table 2 Effects of different combinations on fermentation parameters .

[0038] Fermentation broth from each group in Example 1, collected after 24 hours of fermentation, was immediately stored at -80°C for DNA extraction and metagenomic sequencing. Total DNA was extracted, libraries were constructed, and sequenced using Illumina NovaSeq PE150. After quality control and host removal of the raw sequencing data, MEGAHIT was used for assembly, Prodigal was used to predict genes, and KEGG was used to annotate functional pathways. The analysis focused on rumen microbial species variation and methanogenic pathways (such as methyl-CoM reduction, H2 metabolism, and the F420 pathway).

[0039] Metagenomic analysis showed that, compared with the control group, both the single-use (AT) and combined (AZ) groups of red algae significantly reduced methanogenic archaea. Methanobrevibacter Relative abundance ( Figure 1 At the functional gene level, the abundance of some functional genes related to hydrogen-trophic methanogenesis was reduced in the AZ group, including key enzyme genes (such as mcrA, mtr, etc.) from CO2 reduction and formyl transfer to terminal methyl coenzyme M reduction, which were significantly downregulated. Figure 2 This result is consistent with the decrease in methane production observed in Example 1.

[0040] The results of hydrogenase-related analysis showed that the abundance of some hydrogenase types changed in the AZ group, suggesting that the introduction of pterostilbene may be related to the relief of H2 accumulation. This is the core mechanism for alleviating hydrogen accumulation: (1) Inhibition of "retention-type" hydrogenases: The abundance of [NiFe]_4h and [NiFe]_1a type hydrogenases related to the high hydrogen partial pressure environment was significantly reduced in the AZ group. P <0.01); (2) Enrichment of "uptake-type" hydrogenases: The AZ group was significantly enriched with [NiFe]_4a and [FeFe]_A2 type hydrogenases ( P <0.01)( Figure 3 Following the blockade of the methanogenesis pathway, changes in community composition and hydrogenase abundance suggest that the introduction of pterostilbene may promote the transfer of H2 to other utilization pathways. Example 2: The effect of the ratio of red algae components to pterostilbene on the technical effect

[0041] To determine the optimal mass ratio range of red algae components and pterostilbene in this invention, and to screen for a preferred range that effectively inhibits methane and alleviates hydrogen accumulation, gradient experiments were conducted using an in vitro anaerobic culture system.

[0042] 1. Experimental design: The experimental materials, rumen fluid source, in vitro anaerobic culture system (ANKOM), culture conditions (39℃, 24 h), and gas collection and measurement methods were all the same as in Example 1.

[0043] With the red algae component added at a fixed amount of 0.20% (DM), a gradient dosage of pterostilbene was set up to form different mass ratios (red algae component: pterostilbene), with 6 replicates in each group, as follows: CON group: Control group (without the addition of red algae components and pterostilbene); AT group: Red algae group (0.20%); AZ-200:1 group: 0.20% red algae component + 0.001% pterostilbene (mass ratio 200:1); AZ-100:1 group: 0.20% red algae component + 0.002% pterostilbene (mass ratio 100:1); AZ-40:1 group: 0.20% red algae component + 0.005% pterostilbene (mass ratio 40:1); AZ-20:1 group: 0.20% red algae component + 0.01% pterostilbene (mass ratio 20:1); AZ-10:1 group: 0.20% red algae component + 0.02% pterostilbene (mass ratio 10:1).

[0044] 2. Experimental Results: The results are shown in Table 3. With the increase of the pterostilbene addition ratio (from 200:1 to 10:1), the CH4 production in the system showed a further decreasing trend. More importantly, the accumulation of H2 decreased significantly. Although the AT group significantly reduced CH4 (from 8.43 to 4.01 mL / g), it led to a surge in H2 (from 0.95 mL / g to 3.15 mL / g). When the ratio reached 20:1 (AZ-20:1 group), the H2 production dropped back to 1.67 mL / g, significantly lower than that of the AT group (…). P <0.05), and the CH4 yield was further reduced to 1.79 mL / g. Continuing to increase the ratio of pterostilbene to 10:1, although H2 levels decreased further, the marginal effect was diminishing compared to the 20:1 group. Considering both the CH4 reduction effect and the mitigation of H2 accumulation, the optimal mass ratio of *Asparagus officinalis* to pterostilbene under the conditions of this embodiment is 20:1.

[0045] Table 3. Effects of different red algae components and the ratio of pterostilbene to gas yield and composition. . Example 3: Verification of the synergistic effect of red algae components combined with pterostilbene

[0046] To verify whether the combination of red algae components and pterostilbene produces a synergistic effect, a two-way ANOVA design was used to compare the differences between single addition and combined addition.

[0047] 1. Experimental design: The source of rumen fluid, the in vitro anaerobic culture system, the culture conditions (39℃, 24 h), and the gas collection and measurement methods (H2, CH4, CO2) were all the same as in Example 1.

[0048] Each group has 6 repetitions, and the groups are as follows: CON group: No red algae components or pterostilbene added; AT group: only 0.20% DM of red algae component added; PT group: Only 0.01% DM of Pterostilbene was added; Group AZ (20:1): Red algae component 0.20% DM + Pterostilbene 0.01% DM (mass ratio 20:1).

[0049] 2. Experimental Results: The results are shown in Table 4. Both the AT and PT groups reduced methane levels, but the CH4 production in the AZ group (1.55 mL / g) was significantly lower than that in the AT group (4.28 mL / g) and the PT group (6.79 mL / g), and also lower than the theoretical sum of the effects of the two individual components. The AT group resulted in a significant increase in H2 (3.20 mL / g), exhibiting the typical "hydrogen blockage" side effect of red algae inhibitors; while the AZ group, by introducing pterostilbene, controlled the H2 level at 1.53 mL / g, significantly lower than that in the AT group (…). P <0.05. Two-way ANOVA showed that there was a significant interaction between red algae components and pterostilbene in terms of CH4 inhibition and H2 regulation ( P <0.05). The above results indicate that, under the conditions of this embodiment, the CH4 inhibition effect produced by the combination of the two is higher than the theoretical independent additive effect when used alone, and is accompanied by a decrease in H2 level, indicating that there is a synergistic effect between the two.

[0050] Meanwhile, according to the Bliss independent model, the measured CH4 inhibition effect Eobs (0.82) of the AZ group was higher than the theoretical independent additive effect EBliss (0.61), further indicating that the combination of the two has a synergistic effect under the conditions of this embodiment.

[0051] Table 4. Effects of different red algal components on gas yield and composition when combined with pterostilbene. . Example 4: Composition to reduce red algae dosage

[0052] This embodiment is used to investigate the effect of the composition of the present invention on methane inhibition when the amount of yew asparagus added is reduced.

[0053] 1. Experimental design: The source of rumen fluid, the in vitro anaerobic culture system, the culture conditions (39℃, 24 h), and the gas collection and measurement methods (H2, CH4, CO2) were all the same as in Example 1.

[0054] Each group has 6 repetitions, and the groups are as follows: CON group: No red algae components or pterostilbene added; AT-0.2 group: only 0.20% DM of red algae component added; AT-0.1 group: only 0.10% DM of red algae component added; PT group: Only 0.01% DM of Pterostilbene was added; AZ-low group: red algae component 0.10% DM + pterostilbene 0.01% DM.

[0055] Table 5. Effects of different doses of red algae on gas yield and composition when combined with pterostilbene. .

[0056] The results are shown in Table 5. Compared with the high-dose red algae group (AT-0.2), although the amount of red algae used in the AZ-low group was reduced by 50%, its CH4 production (4.37 mL / g) was not significantly different from that of the AT-0.2 group (4.36 mL / g). P >0.05), indicating that under the conditions of this embodiment, the addition of pterostilbene can maintain a low CH4 yield while reducing the amount of yew-like asparagus added. The bromoform content of the AZ-low group was 13.92 mg / g, which was lower than the 27.13 mg / g of the AT-0.2 group.

[0057] It should be understood that the present invention is not limited to the above embodiments. Equivalent substitutions or modifications made by those skilled in the art without departing from the spirit of the present invention should fall within the protection scope of the present invention.

Claims

1. A rumen methane-inhibiting composition for ruminants, characterized in that, The active components of the composition include red algae and pterostilbene.

2. The rumen methane inhibitor for ruminants according to claim 1, characterized in that, The red algae is *Asparagus purpurea* or its dried form, powder, extract, or preparation.

3. The composition according to claim 1, characterized in that, The mass ratio of the yew-like asparagus to the pterostilbene is 10:1 to 200:

1.

4. The composition according to claim 3, characterized in that, The preferred mass ratio of the yew-like asparagus to the pterostilbene is 10:1 to 100:

1.

5. The composition according to claim 4, characterized in that, The preferred mass ratio of the yew-like asparagus to the pterostilbene is 20:

1.

6. The composition according to claim 1, characterized in that, The composition also includes an electron acceptor component.

7. The composition according to claim 6, characterized in that, The electron acceptor component is malic acid and / or its feedable salt.

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