Application of pinostilbene in inhibition of methane production in ruminant rumen

Abstract

The invention provides application of pine stilbene to inhibition of ruminant rumen methane production and ruminant feed containing pine stilbene. It is found for the first time that pinostilbene can be combined with the active site of methyl coenzyme M reductase, and on the premise that the total gas production rate and rumen fermentation parameters are not affected, rumen methane brevibacterium and key enzymes in a methanogenesis pathway are specifically inhibited, so that generation of rumen methane is inhibited, and the rumen hydrogen concentration cannot be increased.

Description

Technical Field

[0001] The present application belongs to the field of feed technology, and in particular, relates to the use of stilbene in inhibiting rumen methane production in ruminants, and ruminant feed comprising stilbene. Background Art

[0002] The production of rumen methane in ruminants not only causes the loss and waste of about 2%-12% of feed energy, but is also an important source of greenhouse gas emissions in my country's agricultural activities. Developing inhibitors to reduce rumen methane production can not only improve the energy utilization efficiency of animals and save feeding costs, but also alleviate the environmental problem of global warming caused by greenhouse gas emissions, achieving a win-win situation in economic and ecological benefits.

[0003] At present, it has been found that some feed additives, such as plant extracts, electron acceptors (nitrates and sulfates, etc.), plant essential oils, and large seaweeds can reduce rumen methane emissions from ruminants. Although these feed additives have a certain inhibitory effect on methane production and methane emission intensity, they also have some negative effects. For example, when adding electron acceptors such as nitrates, if they are added in high doses or for a long time, they may bring risks such as nitrite poisoning to dairy cows, and potential concerns about animal health hinder their application in animal production. Adding plant essential oils will have some negative effects on the digestibility of nutrients in the diet. Although some large seaweeds are one of the hot spots in current research, there are still controversies in terms of safety and long-term effects, which require further research. Plant extracts have the advantages of natural health and high safety, but the effect of plant extracts on reducing rumen methane production is limited, and most of the emission reduction effects are between 5% and 10%. China is the world's largest producer of plant extracts, with abundant plant resources and cost advantages. However, the plant extraction industry still has a lot of room in the field of green and low-carbon breeding, especially in the development and application of rumen methane inhibitors for ruminants. Summary of the invention

[0004] In view of the problems existing in the prior art, the present application provides the use of pine stilbene in inhibiting rumen methane production in ruminants, and ruminant feed comprising pine stilbene.

[0005] Specifically, this application involves the following aspects:

[0006] In one aspect, the present application provides use of matsostigmine in inhibiting rumen methane production in ruminants.

[0007] Further, matsostigmine reduces the relative abundance of Methanobrevibacterium and / or increases the relative abundance of Methanococcus in the rumen.

[0008] Furthermore, matsutane inhibits formylmethanofuran dehydrogenase (EC 1.2.7.12), formylmethanofuran-tetrahydromethanopterin N-formyltransferase (EC 2.3.1.101), methylenetetrahydromethanopterin cyclohydrolase (EC3.5.4.27), methylenetetrahydromethanopterin dehydrogenase (EC 1.5.98.1), Coenzyme F420 hydrogenase (EC 1.12.98.1), 5,10-methylenetetrahydromethanopterin hydrogenase (EC 1.12.98.2), 5,10-methylenetetrahydromethanopterin reductase (EC1.5.98.2), tetrahydromethanopterin S-methyltransferase (EC 2.1.1.86), methyl Coenzyme M reductase (EC 2.8.4.1), ferredoxin:CoB-CoM heterodisulfide reductase (EC 1.8.7.3), dihydromethanophenazine:CoB-CoM heterodisulfide reductase (EC 1.8.98.1), and formate:CoB-CoM heterodisulfide, ferredoxin reductase (EC 1.8.98.6).

[0009] On the other hand, the present application provides the use of matsostigmine in preparing a rumen methane inhibitor for ruminants.

[0010] Further, matsostigmine reduces the relative abundance of Methanobrevibacterium and / or increases the relative abundance of Methanococcus in the rumen.

[0011] Furthermore, matsutane inhibits formylmethanofuran dehydrogenase (EC 1.2.7.12), formylmethanofuran-tetrahydromethanopterin N-formyltransferase (EC 2.3.1.101), methylenetetrahydromethanopterin cyclohydrolase (EC3.5.4.27), methylenetetrahydromethanopterin dehydrogenase (EC 1.5.98.1), Coenzyme F420 hydrogenase (EC 1.12.98.1), 5,10-methylenetetrahydromethanopterin hydrogenase (EC 1.12.98.2), 5,10-methylenetetrahydromethanopterin reductase (EC1.5.98.2), tetrahydromethanopterin S-methyltransferase (EC 2.1.1.86), methyl Coenzyme M reductase (EC 2.8.4.1), ferredoxin:CoB-CoM heterodisulfide reductase (EC 1.8.7.3), dihydromethanophenazine:CoB-CoM heterodisulfide reductase (EC 1.8.98.1), and formate:CoB-CoM heterodisulfide, ferredoxin reductase (EC 1.8.98.6).

[0012] On the other hand, the present application provides a ruminant feed comprising stilbene as an additive.

[0013] Furthermore, the content of stilbene in the feed is 5 mg / kg-500 mg / kg.

[0014] Furthermore, the content of pine stilbene in the feed is 50mg / kg-300mg / kg.

[0015] Furthermore, the feed also includes one or more of the following ingredients: corn, wheat bran, soybean meal, rapeseed meal, cottonseed meal, whole corn silage, alfalfa, oat hay, and premix.

[0016] Compared with the prior art, this application has the following beneficial effects:

[0017] This application has discovered for the first time that pine stilbene can bind to the active site of methyl coenzyme M reductase, and specifically inhibits the key enzymes in the rumen Methanobacterium genus and the methanogenic pathway without affecting the total gas production and rumen fermentation parameters, thereby inhibiting the production of rumen methane, and will not cause the rumen hydrogen concentration to increase. In addition, the recommended dosage of pine stilbene is extremely low, which can reduce the cost of adding in animal production. Pine stilbene is non-toxic and harmless, has anti-inflammatory, anti-oxidative stress and other functions, and is of great significance to improving animal health and production performance. In addition, pine stilbene can be synthesized artificially, and production costs can be compressed by large-scale production to meet the carbon emission reduction needs of my country's animal husbandry. Experiments have shown that pine stilbene can effectively inhibit rumen methane production and will not affect rumen fermentation. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It was shown that matsostigmine binds to the active site of methyl-CoA reductase;

[0019] Figure 2 To show the effect of different supplementation amounts of matsutane on rumen methane production;

[0020] Figure 3 To show the effects of different supplementation amounts of matsutane on rumen methanogens;

[0021] Figure 4 Showing the effects of different supplemental amounts of spartosil on key enzymes in the rumen methanogenesis pathway. DETAILED DESCRIPTION

[0022] The present application is further described below in conjunction with examples. It should be understood that the examples are only used to further describe and illustrate the present application and are not used to limit the present application.

[0023] Unless otherwise defined, the technical and scientific terms in this specification have the same meaning as those generally understood by those skilled in the art. Although similar or identical methods and materials as described herein may be used in experiments or practical applications, materials and methods are described herein below. In the event of a conflict, the present specification including the definitions therein shall prevail. In addition, materials, methods and examples are provided for illustration only and are not restrictive. The present application is further described below in conjunction with specific embodiments, but is not intended to limit the scope of the present application.

[0024] In view of the problems existing in the prior art, the present application provides the use of pine stilbene in inhibiting the production of methane in the rumen of ruminants.

[0025] The present application also provides the use of stilbene in preparing a rumen methane inhibitor for ruminants.

[0026] The chemical structure of matsostigmine is shown below:

[0027]

[0028] Songqi is a natural active substance derived from the bark of pine trees. A large number of studies in the past have shown that Songqi has the effects of anti-oxidative stress, anti-inflammatory, antibacterial, etc., and is often developed as a health product, showing the characteristics of being non-toxic and harmless, having no side effects, and feeding animals without health risks. Songqi can be directly purchased commercially, or it can be extracted from plants by methods known in the art or prepared by chemical synthesis.

[0029] In this application, rumination refers to returning semi-digested food in the stomach to the mouth for re-chewing after a period of time. Ruminants are animals that have this digestion method of rumination. Exemplary ruminants include camels, giraffes, sika deer, buffaloes, cattle, cows, sheep, goats, antelopes, alpacas, roe deer, etc. In a specific embodiment, the ruminant is a cow.

[0030] The present application found that pine stilbene enables specific inhibition of rumen methane production without increasing the concentration of hydrogen in the rumen, because excessive rumen hydrogen will inhibit normal rumen fermentation function. From the perspective of rumen fermentation parameters, the addition of pine stilbene did not significantly affect the pH value of the rumen fermentation liquid and the concentration and ratio of short-chain volatile fatty acids. From the perspective of changes in the flora structure of rumen methanogens, the addition of pine stilbene can significantly reduce the relative abundance of Methanobacterium in rumen fermentation liquid, significantly increase the relative abundance of Methanococcus, and significantly reduce the gene expression of some key enzymes in the carbon dioxide reduction pathway (an important pathway for rumen methanogenesis). These key enzymes can be, for example, formylmethanofuran dehydrogenase (EC 1.2.7.12), formylmethanofuran---tetrahydromethanopterin N-formyltransferase (EC 2.3.1.101), methylenetetrahydromethanopterin cyclohydrolase (EC 3.5.4.27), methylenetetrahydromethanopterin dehydrogenase (EC 1.5.98.1), coenzyme F420 hydrogenase (EC1.12.98.1), 5,10-methylenetetrahydromethanopterin hydrogenase (EC 1.12.98.2), 5,10-methylenetetrahydromethanopterin reductase (EC 1.5.98.2), tetrahydromethanopterin S-methyltransferase (EC 2.1.1.86), methyl coenzyme M reductase (EC 2.8.4.1), ferredoxin: CoB-CoM heterodisulfide reductase (EC 1.8.7.3), dihydromethanophenazine:CoB-CoM heterodisulfide reductase (EC 1.8.98.1), and formate:CoB-CoM heterodisulfide, ferredoxin reductase (EC 1.8.98.6).

[0031] The amount of methane can be detected by methods known in the art, for example, by gas chromatography. Specifically, the chromatographic conditions can be: FID detector, carrier gas is nitrogen, flow rate is 45 mL / min, Aglient porapak Q G3591-80135:1 filling column, detector temperature is 300°C, column temperature is 80°C, injection volume is 1 mL.

[0032] The present application also provides a ruminant feed, comprising pine stilbene as an additive.

[0033] In a specific embodiment, the content of pine stilbene in the feed is 5mg / kg-500mg / kg, for example, it can be 5mg / kg, 10mg / kg, 20mg / kg, 30mg / kg, 40mg / kg, 50mg / kg, 60mg / kg, 70mg / kg, 80mg / kg, 90mg / kg, 100mg / kg, 110mg / kg, 120mg / kg, 130mg / kg, 140mg / kg, 150mg / kg, 160mg / kg, 170mg / kg, 180mg / kg, 190mg / kg, 200mg / kg, 210mg / kg, 220mg / kg, 230mg / kg, 240 470mg / kg, 480mg / kg, 490mg / kg, 500mg / kg, and any number between these values.

[0034] In a specific embodiment, the content of stilbene in the feed is 50 mg / kg-300 mg / kg.

[0035] Within the above content range, compared with feed without inhibitors, the addition of pine stilbene can inhibit methane production by up to 20%. Other existing methane inhibitors, such as rosmarinic acid, need to be added at 10g / kg to achieve an inhibition rate of about 15% when evaluated under the same conditions as in this example. Other studies have shown that the maximum inhibition effect of methane inhibitors such as tea saponin and allicin is usually less than 10%.

[0036] Those skilled in the art will appreciate that the feed may also include basic ingredients of feed known in the art, such as corn, wheat bran, soybean meal, rapeseed meal, cottonseed meal, whole corn silage, alfalfa, oat hay, premixes, and the like.

[0037] Example

[0038] Example 1

[0039] Test method:

[0040] Using the methyl-CoA reductase (MCR) of methanogens as the target protein, The Protein Preparation Wizard module in the software performs bond optimization, hydrogenation, disulfide bond distribution on the target protein, and protonation at pH 7.0 using the PROPKA method; the OPLS4 force field is used for restricted energy optimization to eliminate interatomic conflicts in the structure and make the RMSD of heavy atoms converge to The side chain position was optimized to obtain a good side chain structure. The optimized target protein was molecularly docked with the receptor-pine stilbene compound to evaluate whether the pine stilbene could bind to the methyl coenzyme M reductase (MCR) in methanogens.

[0041] Test results

[0042] The results showed that pine stilbene can pass through the narrow and closed channel inside methyl coenzyme M reductase (MCR) to reach the active site, and then form a hydrogen bond with the adjacent amino acid residue Tyr367, thereby forming a space occupancy and affecting the activity of methyl coenzyme M reductase (MCR). Figure 1 ).

[0043] Example 2

[0044] Test materials

[0045] The pinostilbene used in the experiment is a commercially available product with a purity of >98%, CAS: 42438-89-1, and its English name is Pinostilbene.

[0046] Experimental animals and rumen fluid collection

[0047] The experimental animals were three cows with permanent rumen fistulas, fed a basal diet containing concentrate, whole corn silage and alfalfa (Table 1). On the day of the experiment, fresh rumen fluid was obtained from the three rumen fistula cows before morning feeding and placed in a preheated thermos and brought back to the laboratory.

[0048] The collected rumen fluid was filtered through 4 layers of gauze and quickly poured into the artificial buffer prepared in advance. The buffer was configured according to the Menke method. The mixing ratio of rumen fluid and buffer was 1:2 (rumen fluid: buffer, v:v), and it was fully mixed by a magnetic stirrer. 150mL of the mixed fermentation liquid was divided into fermentation bottles. 2 grams of basal diet (organic basis) and different doses of pine stilbene were pre-added to the fermentation bottles. Six replicates were set for each treatment group. The fermentation bottles were placed in a constant temperature shaker at 39°C and cultured at 80rpm for 24 hours.

[0049] The specific composition of the basal diet is shown in Table 1.

[0050] Table 1 Basic diet composition and nutritional level (dry matter basis)

[0051]

[0052]

[0053] The experiment set up the following 8 treatment groups:

[0054] (1) Control group: Basic diet group

[0055] (2) Treatment group: 5 mg / kg of matsutane was added

[0056] (3) Treatment group: 25 mg / kg of matsutane was added

[0057] (4) Treatment group: 50 mg / kg of matsutane was added

[0058] (5) Treatment group: 75 mg / kg of pine

[0059] (6) Treatment group: 100 mg / kg of matsutane was added

[0060] (7) Treatment group: 300 mg / kg of pine

[0061] (8) Treatment group: 500 mg / kg of matsutane was added

[0062] Chemical analysis

[0063] The gas production was recorded in real time using the Ankom RF fermentation system. After the fermentation was completed, the gas was collected for gas composition analysis. The gas composition was determined using gas chromatography under the following chromatographic conditions: FID detector, nitrogen carrier gas, flow rate 45 mL / min, Aglient porapak Q G3591-80135:1 packed column, detector temperature 300 °C, column temperature 80 °C, injection volume 1 mL.

[0064] Data Statistical Analysis

[0065] The data were analyzed by one-way ANOVA using SAS 9.4 software, and Duncan's post hoc test was used. P < 0.05 indicated a significant difference.

[0066] result

[0067] like Figure 2 As shown, the addition of pine stilbene can significantly reduce the production of rumen methane. When the pine stilbene addition dose is 100 mg / kg, the inhibitory ability on rumen methane reaches the best, and as the dose increases to 300 mg / kg and 500 mg / kg, there is no further reduction in rumen methane production.

[0068] Other existing methane inhibitors, such as rosmarinic acid, need to be added at 10 g / kg to achieve an inhibition rate of about 15% when evaluated under the same conditions as in this example. In other studies, methane inhibitors such as tea saponin and allicin have been shown to have a maximum inhibition effect of less than 10%.

[0069] Example 3 Experiment on regulating rumen fermentation parameters and rumen microbial flora of ruminants by using pine needles

[0070] The test materials are the same as those in Example 1.

[0071] The experiment set up the following two treatment groups:

[0072] (1) Control group: Basic diet group

[0073] (2) Treatment group: 200 mg / kg of matsutane was added

[0074] Chemical analysis

[0075] The gas composition analysis was the same as in Example 1, and the results are shown in Table 2.

[0076] Table 2 Effects of pine needles on rumen gas production and gas composition

[0077]

[0078] At the end of the experiment, the fermentation broth was collected and stored at -20°C for the determination of ammonia nitrogen, microbial protein, and volatile fatty acid indicators. The results are shown in Table 3. In addition, the fermentation broth was collected and stored at -80°C for the determination of microbial flora. The results are shown in Table 3. Figure 3 and Figure 4 As shown in the figure, CON represents the control group and PIN represents the treatment group.

[0079] Ammonia nitrogen (NH3-N) was determined according to the method of Broderick and Kang (1980). Microbial protein (MCP) was determined according to the method of Makkar et al. (1982). Short-chain volatile fatty acids (VFAs) were determined according to the method of Zhang Junyu et al. (2017). The microbial flora analysis of rumen fermentation broth was sent to Shanghai Meiji Biotechnology Co., Ltd. for metagenomic sequencing, and the original sequences were quality controlled, host-free, spliced ​​and assembled, and species and functional annotations were performed to further analyze the changes in the abundance of key enzymes in the rumen microbial flora and rumen methanogen pathways.

[0080] Table 3 Effects of pine nuts on rumen fermentation parameters

[0081]

[0082]

[0083] result

[0084] The addition of matsutrazine significantly reduced the production of rumen methane and hydrogen, the total gas production tended to increase, and the proportion of rumen methane in the total gas was significantly reduced. From the perspective of rumen fermentation parameters, the addition of matsutrazine did not significantly affect the pH value of rumen fermentation liquid, the concentration of short-chain volatile fatty acids, and the proportion of each component. From the perspective of the changes in the flora structure of rumen methanogens, the addition of pine stilbene can significantly reduce the relative abundance of Methanobacterium in rumen fermentation broth, significantly increase the relative abundance of Methanococcus, and significantly reduce some key enzymes in the carbon dioxide reduction pathway (an important pathway for rumen methanogenesis), including: formylmethanofuran dehydrogenase (EC 1.2.7.12), formylmethanofuran-tetrahydromethanopterin N-formyltransferase (EC 2.3.1.101), methylenetetrahydromethanopterin cyclohydrolase (EC 3.5.4.27), methylenetetrahydromethanopterin dehydrogenase (EC 1.5.98.1), coenzyme F420 hydrogenase (EC 1.12.98.1), 5,10-methylenetetrahydromethanopterin hydrogenase (EC 1.12.98.2), 5,10-methylenetetrahydromethanopterin reductase (EC 1.5.98.2), tetrahydromethanophenazone S-methyltransferase (EC 2.1.1.86), methyl-CoA reductase (EC 2.8.4.1), ferredoxin:CoB-CoM heterodisulfide reductase (EC 1.8.7.3), dihydromethanophenazine:CoB-CoM heterodisulfide reductase (EC 1.8.98.1), and formate:CoB-CoM heterodisulfide, ferredoxin reductase (EC 1.8.98.6).

[0085] in conclusion

[0086] Songqi has a good rumen methane inhibition effect. It can inhibit the production of rumen methane by inhibiting the relative abundance of Methanobacterium in rumen fluid and the gene expression of key enzymes in the methanogenic pathway. While inhibiting rumen methane, Songqi does not affect rumen fermentation parameters. As a safe and healthy feed additive with strong methane inhibition specificity, it has good application value.

Claims

1. The use of pine stilbene in inhibiting the production of methane in the rumen of ruminants.

2. The use according to claim 1, wherein matsostigmine reduces the relative abundance of Methanobrevibacterium spp. in the rumen and / or increases the relative abundance of Methanococcus spp.

3. The method according to claim 1 or 2, wherein the styrylamine inhibits formylmethanofuran dehydrogenase (EC 1.2.7.12), formylmethanofuran-tetrahydromethanopterin N-formyltransferase (EC 2.3.1.101), methylenetetrahydromethanopterin cyclohydrolase (EC 3.5.4.27), methylenetetrahydromethanopterin dehydrogenase (EC 1.5.98.1), coenzyme F420 hydrogenase (EC 1.12.98.1), 5,10-methylenetetrahydromethanopterin hydrogenase (EC 1.12.98.2), 5,10-methylenetetrahydromethanopterin reductase (EC 1.5.98.2), tetrahydromethanopterin S-methyltransferase (EC 2.1.1.86), methyl coenzyme M reductase (EC 2.8.4.1), ferredoxin:CoB-CoM heterodisulfide reductase (EC 1.8.7.3), dihydromethanophenazine:CoB-CoM heterodisulfide reductase (EC 1.8.98.1), and formate:CoB-CoM heterodisulfide, ferredoxin reductase (EC 1.8.98.6).

4. Use of pine stilbene in the preparation of rumen methane inhibitors for ruminants.

5. The use according to claim 4, wherein matsostigmine reduces the relative abundance of Methanobrevibacterium spp. and / or increases the relative abundance of Methanococcus spp. in the rumen.

6. The method according to claim 4 or 5, wherein the styrylamine inhibits formylmethanofuran dehydrogenase (EC 1.2.7.12), formylmethanofuran-tetrahydromethanopterin N-formyltransferase (EC 2.3.1.101), methylenetetrahydromethanopterin cyclohydrolase (EC 3.5.4.27), methylenetetrahydromethanopterin dehydrogenase (EC 1.5.98.1), coenzyme F420 hydrogenase (EC 1.12.98.1), 5,10-methylenetetrahydromethanopterin hydrogenase (EC 1.12.98.2), 5,10-methylenetetrahydromethanopterin reductase (EC 1.5.98.2), tetrahydromethanopterin S-methyltransferase (EC 2.1.1.86), methyl coenzyme M reductase (EC 2.8.4.1), ferredoxin:CoB-CoM heterodisulfide reductase (EC 1.8.7.3), dihydromethanophenazine:CoB-CoM heterodisulfide reductase (EC 1.8.98.1), and formate:CoB-CoM heterodisulfide, ferredoxin reductase (EC 1.8.98.6).

7. A ruminant feed comprising pine stilbene as an additive.

8. The feed according to claim 7, wherein the content of stilbene in the feed is 5 mg / kg-500 mg / kg.

9. The feed according to claim 8, wherein the content of stilbene in the feed is 50 mg / kg-300 mg / kg.

10. The feed according to any one of claims 7 to 9, wherein the feed further comprises one or more of the following ingredients: corn, wheat bran, soybean meal, rapeseed meal, cottonseed meal, whole corn silage, alfalfa, oat hay, and premix.

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