CN116035123A

CN116035123A - Application of silybin in preparing feed additive for regulating and controlling rumen methane emission of ruminant

Info

Publication number: CN116035123A
Application number: CN202211707734.4A
Authority: CN
Inventors: 李洋; 刘蕊; 孙雨坤; 张广宁; 李白; 徐柏衡; 马浩凯; 张永根
Original assignee: Northeast Agricultural University
Current assignee: Northeast Agricultural University
Priority date: 2022-12-29
Filing date: 2022-12-29
Publication date: 2023-05-02

Abstract

The invention discloses an application of silybin in preparing a feed additive for regulating and controlling rumen methane emission of ruminants. The artificial simulation rumen in-vitro fermentation test is carried out by using silybin as a feed additive, and the species composition and community structure of rumen are changed by inhibiting prevolella (Prevotella), isotricha (Isodon), ophryosporex (Tocophyta), unclassified_Rotifera (unclassified rotifer), methanospira (Methanospira), orpinomyces (anaerobic fungus) and Neocallimastix (anaerobic fungus) in rumen, so that the metabolic pathway of rumen is changed, the metabolic products are changed, the rumen fermentation parameters are regulated, and the yield and total gas yield of hydrogen, carbon dioxide and methane are regulated; the silybin addition can reduce the generation of methane in the rumen of ruminants, and has good effect of reducing the emission of methane.

Description

Application of silybin in preparing feed additive for regulating and controlling rumen methane emission of ruminant

Technical Field

The invention relates to the technical field of feed additives, in particular to application of silybin in preparing a feed additive for regulating and controlling rumen methane emission of ruminants.

Background

Climate change is a global problem faced by human beings, and carbon dioxide, methane and other room gases in various countries are increased, and extreme climate disasters occur in time, so that the global life system is threatened. CH (CH) ₄ Is a major contributor to the total global greenhouse gas emissions, and has a warming potential 28 times that of carbon dioxide. Animal husbandry methane emissions account for about 33% of human activities.

Wherein, the gastrointestinal tract emission from livestock accounts for more than 90% of the total methane emission of livestock, and is the largest source of methane emission in human agriculture. In addition, CH produced by cows ₄ Also represents an energy loss, and the reduction of methane production by ruminant rumen is of great ecological and nutritional importance.

In previous studies, the feed additive milk ionophore (polyether ionophores such as monensin and salinomycin) can reduce CH ₄ The emissions are effective, but the European Union bans the use of these additives. Nitrite generated by nitrate in the metabolic process has toxicity, and excessive addition and improper use can damage the health of organisms, so that the wide application of the nitrite in production practice is limited. The alleviating effect of other feed additives, such as 3-nitro ester-1-propanol (3-NOP), shows that it is possible to reduce CH of cows ₄ Emissions, however, have been less studied for 3-NOP safety evaluation.

The plant extracts have no toxic or side effects, the most common natural plant functional components are phenolic compounds, the largest of which are flavonoids, the species of which exceeds 8000 species, and which are widely distributed in various plants.

The chemical nature of silybin (shown as formula (1)) is a flavonoid lignan compound.

The silymarin is prepared from silybum marianum seeds of the Compositae through the steps of squeezing, acetone extraction, concentration, degreasing, residue removal, drying and crushing, and the silymarin is purified in a series, and the product with the content of more than 97% of silybum marianum A, B related substances controlled within 2.5% is separated independently. Research on livestock and poultry has proved that flavonoid has biological activities such as antioxidation, antibiosis, anti-inflammatory and the like.

Disclosure of Invention

The invention discovers the new application of the silybin in preparing the feed additive for regulating and controlling the rumen methane emission of ruminants for the first time, and provides a new direction for solving the problem of rumen methane emission of ruminants.

The specific technical scheme is as follows:

the invention provides an application of silybin in preparing a feed additive for regulating and controlling rumen methane emission of ruminants.

The silybin is in powder form, can be a commercially available product, and can be obtained by purifying silymarin, and the purity is more than 97%.

Further, the regulation and control modes are as follows: reducing the generation amount of methane and reducing the emission amount of methane.

Further, the silybin reduces the generation amount of methane by inhibiting the variety of microorganisms in rumen, thereby reducing the emission amount of methane; the microorganisms include Prevolella (Prevoltella), homoptera (Isotricha), torulopsis (Ophryosocolox), methanosphaera (Methanosphaera), unclassified rotifers (unclassified_Rotifera), anaerobic fungi (Orpinomyces) and anaerobic fungi (Neocallimastix).

Furthermore, the silybin reduces the production amount of methane by changing the community composition of the archaea, bacteria, protozoa and fungi in the rumen, so that the emission amount of methane is reduced.

Further, the silybin regulates and controls the methane production by changing the metabolic pathway in the rumen; the metabolic pathway includes: phenylalanine metabolic pathway, flavonoid biosynthetic pathway, and folic acid biosynthetic pathway.

Further, the ruminant is a dairy cow.

The invention also provides a feed additive which comprises silybin.

The invention also provides a feed, which comprises a feed additive and basic ration, wherein the feed additive comprises silybin; the addition amount of the silybin is 0.075g/L to 0.6g/L based on basic ration.

The invention also provides a method for improving rumen methane emission of ruminants, which comprises the following steps: adding silybin as a feed additive into basic ration, and feeding ruminant;

the addition amount of the silybin is 0.075g/L to 0.6g/L based on basic ration.

Further, the ruminant is a dairy cow.

Compared with the prior art, the invention has the following beneficial effects:

the invention discovers the new application of silybin in preparing the feed additive for regulating and controlling rumen methane emission of ruminants for the first time, and uses silybin as the feed additive to carry out artificial simulation rumen in vitro fermentation test, changes the species composition and community structure of rumen, changes metabolic pathway of rumen, changes metabolite, regulates rumen fermentation parameters, regulates the output and total gas production of hydrogen, carbon dioxide and methane by inhibiting Prevotella (prasuvorexa), isotricha (all-caterpillar), ophryostex (Tocopsis), unclassified_rotifera (unclassis), methanosphaera (methane globus), orpinomyces (anaerobic fungus) and Neocilimastix (anaerobic fungus) in rumen; the silybin addition can reduce the generation of methane in the rumen of ruminants, and has good effect of reducing the emission of methane.

Drawings

FIG. 1 shows bacterial, protozoal, methanogen and fungal beta diversity in rumen microbiota without or with silybin addition;

wherein A is the beta diversity of methanogens of a control group and a silybin group added with 0.60 g/L; b is the bacterial beta diversity of the control group and the silybin group added with 0.60 g/L; c is the diversity of control group and added 0.60g/L protozoan beta; d is the fungus beta diversity of the control group and the silybin group added with 0.60 g/L;

note that: control means adding 0g/L silybin; silibinin indicates the addition of 0.60g/L silybin, each point in the figure representing one sample; the oval represents a 95% confidence oval (i.e., the sample set would have 95 if there were 100 samples in it). The abscissa represents the first principal component, and the percentage represents the contribution value of the first principal component to the sample difference; the ordinate represents the second principal component, and the percentage represents the contribution value of the second principal component to the sample variance.

Fig. 2 is a graph of analysis of the differential metabolite major components of the control group and the silybin-added group. Wherein the group of degrees of sample aggregation within the group expresses the reproducibility of the samples within the group. The more significant the inter-group separation, the more significant the variability of the inter-group samples.

Note that: CON means adding 0g/L silybin; SLB means adding 0.60g/L of silibinin, wherein the X-axis represents the first principal component, the Y-axis represents the second principal component, and the percentage of the coordinate axis represents the contribution of the principal component to the sample difference. Each point in the graph represents a sample, and a repetition of more than 3 within the group will reveal the 95% elliptical confidence interval of the group.

Detailed Description

The invention will be further described with reference to the following examples, which are given by way of illustration only, but the scope of the invention is not limited thereto.

Example 1

1. Test materials

The basal diet used in this experiment was from ruminant nutrition laboratory at northeast agricultural university and included alfalfa, silage, soybean meal, corn flakes, dried corn husks, DDGS, beet particles, premix, and the ingredients are shown in table 1.

Table 1 basic ration composition and nutrient level (dry matter basis)

1CP = crude protein; 2NEL = net energy of lactation; 3RDP = rumen degradable protein; 4ADF = acid wash fiber; 5NDF = neutral wash fiber; 6peNDF = physically effective neutral detergent fiber.

The silybin added in the test is prepared by squeezing silymarin, extracting with ethyl acetate, concentrating, degreasing, removing residue, using 80-85% ethanol as solvent, dissolving for two times in three steps, degreasing, standing, press filtering, and vacuum drying to obtain the silybin with content higher than 97%, and controlling the content of related substances within 2.5%. The silymarin is prepared from milk thistle seeds of Compositae by squeezing, extracting with acetone, concentrating, degreasing, removing residue, drying, and pulverizing to 30% by mass.

2. Test method

(1) Control group: only basic ration is placed in the culture flask, and the composition of the basic ration is shown in Table 1.

(2) Test groups 1 to 4: 0.075g/L, 0.15g/L, 0.3g/L, and 0.6g/L of silibinin, respectively, as feed additives, were added to a base ration (the base ration is TMR (total mixed ration) prepared using the materials of Table 1, and obtained by drying and grinding through a 0.45 mm sieve).

TABLE 2 daily ration and Silybin dosage

Rumen fluid was collected from chinese holstein cows with three rumen fistulas prior to morning feeding and placed in a vacuum flask flushed with anaerobic carbon dioxide. Three Holstein cows (days of lactation=120±11, milk yield=27.6±3.3 kg) were kept in separate tethered pens in the cowshed for free drinking. All cows were fed the same mixed diet, fed and milked 2 times per day at 6 o 'clock 30 minutes and 18 o' clock 30 minutes.

The rumen fluid is filtered through four layers of dry gauze and mixed with a pre-heated (39 ℃) buffer (buffer: rumen fluid, v: v) in a 1:2 ratio with continuous flow of carbon dioxide to ensure an anaerobic environment. After mixing, 150ml of the buffer gastric juice was transferred to a 200ml glass bottle containing 2 g of basal ration and different doses of silibinin (table 2). The bottles (8 replicates per treatment) were sealed with a silicone stopper, with an airtight collection bag, and incubated in triplicate in a shaking water bath at 39℃for 24 hours with 40-50 movements per minute.

The implementation and experimental procedure of anaerobic culture techniques are the same as described in literature (Xin, h.s., khan, n.a., liu, x., jiang, x., sun, f., zhang, s.z., sun, y.k., zhang, y.g., li, x.,2021.Profiles of odd-and welded-chain fatty acids and their correlations with rumen fermentation parameters, microbial protein synthesis, and bacterial populations based on pure carbohydrate incubation in vitro.

3. Test results

3.1 influence of Silybin on rumen fermentation parameters and digestibility of dry matter

0.5 ml of each treated gas subsamples were analyzed by gas chromatography (GC-8A;Shimadzu Co.Ltd, tokyo, japan) using specific methods such as literature (Kim, w.y., hanigan, m.d., lee, s.j., lee, s.m., kim, d.h., hyun, j.h., yeo, j.m., lee, S.S.,2014.Effects of Cordyceps militaris on the growth of rumen microorganisms and in vitro rumen fermentation with respect to methane emissions.J.Dairy Sci, 7065-7075). After the gas production was determined, a pH meter (Sartorius basic pH meter,

germany) to determine the pH of the culture broth.

To analyze NH ₃ -N andvolatile Fatty Acids (VFAs) 1 ml 25% metaphosphoric acid was added to 5 ml broth and stored at-20℃until analysis. The VFA concentration was determined by gas chromatography (GC-8A; shimadzu corporation, kyoto, japan) as described in the literature (Hu, W.L., liu, J.X., ye, J.A., wu, Y.M., guo, Y.Q.,2005.Effect of tea saponin on rumen fermentation in vitro.Anim.Feed Sci.Technol.120,333-339). NH (NH) ₃ N is determined according to the phenol/hypochlorous acid method, reference Broderick, G.A., kang, J.H.,1980.Automated simultaneous determination of ammonia and total amino acids in ruminal fluid and in vitro media.J.Dairy Sci.63,64-75. All assays were performed in triplicate. Rumen microbiology diversity and metabolite detection were performed by the hundred michael biotechnology company.

TABLE 2 influence of different doses of silibinin on rumen fermentation parameters and on dry matter digestibility after 24 hours of in vitro fermentation

¹ SEM＝Standarder error of mean； ² TVFA＝Total volatile fatty acids； ³ DMD＝Dry matter digestibility；*In the same row,values with different small letter superscripts mean significant difference(P<0.05),while with the same or no letter superscripts mean no significant difference(P>0.05).The same as below.

* The same row of data shoulders indicates that the difference is significant (P < 0.05), and the same or no letters indicate that the difference is not significant (P > 0.05).

From Table 2, it can be seen that the pH value increases linearly with the increase of the addition level of silybin (P<0.05 The pH of the control group was significantly lower than the other groups. The ammonia nitrogen concentration does not change with the addition level of the silybin. The TVFA concentration decreased linearly with increasing silybin addition level (P<0.05 A) is provided; acetic acid concentrationThe degree decreases in quadratic terms with increasing levels of silybin addition (P<0.05 A) is provided; propionic acid concentration increased linearly with increasing silybin addition level (P<0.05 A) is provided; the concentration of the ethylene-propylene ratio decreases linearly with the increase of the addition level of the silybin (P<0.05 A) is provided; DMD% decreases linearly with increasing levels of silybin addition (P<0.05). The TVFA concentration of the silybin added with 0.30g/L and 0.60g/L is obviously lower than that of the control group and other groups (P)<0.05 The concentration of the silybin group acrylic acid of 0.60g/L is obviously higher than that of the control group and other groups (P)<0.05). The ethylene-propylene ratio of the 0.60g/L silybin group is significantly lower than that of the control group and other groups (P<0.05). Indicating that the addition of silybin causes the decrease of TVFA concentration in the rumen and the increase of pH value of the rumen; adding silybin to reduce the concentration of acetic acid in rumen, and increasing the concentration of propionic acid to reduce the ratio of ethyl to propyl; adding silybin to rumen NH ₃ -N concentration has no effect; the addition of silybin reduces the digestibility of dry matter in the rumen. The decrease in methane yield was more pronounced with the addition of 0.30g/L and 0.60g/L of silybin groups.

3.2 total gas yield, methane yield, carbon dioxide yield, hydrogen yield and percentage thereof of silybin in vitro fermentation

At the end of the incubation time, the gas was transferred to an air collection bag and the volume of gas was measured by syringe. A0.5 ml gas sub-sample was analyzed by gas chromatography (GC-8A;Shimadzu Co.Ltd, tokyo, japan) to determine the hydrogen, carbon dioxide and methane content as previously described (Kim et al, 2014).

Kim,W.Y.,Hanigan,M.D.,Lee,S.J.,Lee,S.M.,Kim,D.H.,Hyun,J.H.,Yeo,J.M.,Lee,S.S.,2014.Effects of Cordyceps militaris on the growth of rumen microorganisms and in vitro rumen fermentation with respect to methane emissions.J.Dairy Sci.,7065-7075.

TABLE 3 total gas yield, methane yield, carbon dioxide yield, hydrogen yield and percentage thereof for in vitro fermentation of different doses of silybin

¹ The amount of silymarin added was 0.00g/L,0.075g/L,0.15g/L,0.30g/L，0.60g/L respectively； ² TG＝Total gas production； ³ CH ₄ (％)＝CH ₄ /Total gas production； ⁴ CO ₂ (％)＝CO ₂ /Total gas production； ⁵ H ₂ (％)＝H ₂ /Total gas production。

From table 3, it can be seen that the total gas yield, methane yield, carbon dioxide yield, hydrogen yield decreased linearly with increasing silybin addition level (P < 0.05). The methane yield percentage, the carbon dioxide yield percentage, and the hydrogen yield percentage decrease linearly with increasing silybin addition level (P < 0.05). The total gas yield, methane yield, carbon dioxide yield, hydrogen yield, methane yield percentage, carbon dioxide yield percentage and hydrogen yield percentage of the silybin-added group are all reduced compared with the control group, which shows that the total gas yield, methane yield, carbon dioxide yield and hydrogen yield in rumen can be reduced by adding the silybin. Methane production requires hydrogen and carbon dioxide as substrates, so a decrease in the content of hydrogen and carbon dioxide results in a decrease in methane production. As can be seen from the table, the total gas yield, methane yield, carbon dioxide yield, hydrogen yield, methane yield percentage, carbon dioxide yield percentage of the silybin group added with 0.60g/L are lower than those of the control group and other groups, and the maximum reduction degree of the methane yield of the silybin group added with 0.60g/L is shown.

3.3 rumen microbial diversity

The measurement method is as follows:

(1) Extraction of rumen microorganism DNA: the extraction of nucleic acid was accomplished using TGuide S96 magnetic bead method fecal genomic DNA extraction kit (remark: manufacturer: tiangen Biochemical technology (Beijing) Co., ltd., model: DP812 1.).

(2) Rumen microorganism diversity detection

Rumen DNA samples were sequenced for 16s, v4 rRNA gene, and bacterial diversity was analyzed;

the primer is as follows: 515F:5'-GTGYCAGCMGCCGCGGTAA-3';

806R2：5'-GGACTACNVGGGTWTCTAAT-3'。

carrying out 18s and v4 rRNA gene sequencing on the sample, and analyzing protozoan diversity;

the primer is as follows: TAReuk454FWD1:5'-CCAGCA (G/C) C (C/T) GCGGTAATTCC-3',

TAReukREV3：5'-ACTTTCGTTCTTGAT(C/T)(A/G)A-3'。

performing its1 rRNA gene sequencing on the sample, and analyzing fungus diversity;

the primer is ITS1F:5'-CTTGGTCATTTAGAGGAAGTAA-3' the number of the individual pieces of the plastic,

ITS2：5'-GCTGCGTTCTTCATCGATGC-3'。

carrying out 16s, v3+v4 rRNA gene sequencing on the sample, and analyzing the archaea diversity;

the primer is Arch349F:5'-GYGCASCAGKCGMGAAW-3',

Arch806R：5'-GGACTACVSGGGTA。

adding a sequencing joint at the tail end of the primer, and carrying out PCR amplification, wherein the PCR reaction program is that the primer is pre-denatured for 5min at 95 ℃; denaturation at 95 ℃,30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 40s, 25 cycles; extending at 72 ℃ for 7min, and preserving heat at 4 ℃. And (3) identifying the PCR products by adopting 1.8% agarose gel electrophoresis, purifying and recovering the amplified products, and carrying out fluorescent quantitative analysis on the PCR amplified recovered products.

According to the result, mixing according to the sequencing amount requirement of each sample and the corresponding proportion, and analyzing by using an Illumina Novaseq high-throughput sequencing platform. And after the sequencing is finished, performing quality primary screening on the original off-machine data of the high-throughput sequencing, and re-testing or complement testing the problem sample. The obtained original image data file is converted into an original sequencing sequence (Sequenced Reads) through Base recognition (Base sequencing) analysis, and the result is stored in a FASTQ (fq) file format, wherein the FASTQ file format comprises sequence information of the sequencing sequence (Reads) and corresponding sequencing quality information. Samples were sequenced to obtain 799,967 pairs of Reads, and the quality of the double-ended Reads was controlled to produce 797,868 clear Reads after splicing, each sample produced at least 79,313 clear Reads, and an average of 79,787 clear Reads was produced.

OTU clustering sequences were clustered at the level of similarity 97% (default) using USEARCH (Edgar Robert c.uparse: highly accurate OTU sequences from microbial amplicon reads. [ J ]. Nat. Methods,2013,10 (10): 996-8.) (version 10.0), defaults to sequence 0.005% of all sequences as threshold filter OTUs (Bokulich NA, sub-amannian S, faith JJ, gelers D, gordon JIs, knight R, mills DA, corporation JG: quality-filtering vastly improves diversity estimates from Illumina amplicon sequencing. Nature methods 2013,10 (1): 57-59.). And carrying out taxonomic annotation on the feature sequences by using a naive Bayesian classifier by taking SILVA as a reference database, so as to obtain species classification information corresponding to each feature, further counting the composition of each sample community at each level (phylum, class, family, genus), and generating species abundance tables at different classification levels by using QIIME software.

Alpha index analysis was performed using QIIME2 (Deng et al 2012) Molecular ecological network analyses BMC Bioinformatics 13:113. Beta diversity analysis was based on a variety of algorithms of binary jaccard, binary curtis, weighted unifera. Beta diversity analysis presents a species diversity matrix based on a variety of algorithms of binary jaccard, binary curtis, (un) weighted unifera. And drawing a sample Principal Component Analysis (PCA) and a principal coordinate analysis (PCoA) based on the R language platform.

TABLE 4 alpha-diversity index of archaea, bacteria, protozoa and fungi after 24 hours of in vitro fermentation without Silybum Marianum and with Silybin addition

¹ Con＝0g/L； ² SLB＝0.60g/L。

From Table 4, it can be seen that the control group (0 g/L) and the 0.60g/L group differ significantly in Simpson index and Shannon index of the rumen fungi (P < 0.05), indicating that the diversity of the colonies of fungi in the rumen of the group to which 0.60g/L silybin was added is higher than that of the control group. It was shown that a decrease in methane production in the rumen is associated with a diversity of fungal communities in the rumen.

TABLE 5 relative abundance of the most predominant bacteria, protozoa, methanogens and fungi in rumen microbiota without or with silybin added (%)

/>

¹ Con＝0g/L； ² SLB＝0.60g/L

**:P<0.01,*:P<0.05,NS:notsignificant.

As can be seen from Table 5, 0.60g/L of the silybin group and the control group were added to have significant differences in bacterial abundance (P < 0.05) among Prevotela (Prevotella), succiclysis (Succiniaceae), NK4A214_ group, candidatus _ Saccharimonas, unclassified _Lachnospirace (unclassiaceae), others, and the abundance of 0.60g/L of the silybin group Prevotela (Prevotela) was lower than that of the control group, whereas Succiclysis (Succinum), NK4A214_ group, candidatus _Saccharimonas (Candida), unclassified_Lachnospirace (unclassiaceae) were higher than that of the control group; it is shown that the addition of 0.60g/L of silybin group can inhibit the production of Prevolella (Prevotella). Prevolella (Prevotella) can degrade starch, indicating that the addition of 0.60g/L silybin group can reduce the degradation rate of starch. Succidioides (succinic acid bacteria) and NK4A214_group can produce degraded cellulose, which shows that the addition of 0.60g/L silybin group can reduce the degradation of starch and improve the degradation of cellulose.

The addition of 0.60g/L silybin group and control group showed significant differences in protozoan abundance (P < 0.05) among Isotricha (all Caterpillar), ophryosocolox, unclassified_Rotifera (unclassified rotifer). The abundance of the added 0.60g/L silybin group Isotricha (Homophila), ophryosocolox (Torula), unclassified_Rotifera (unclassified rotifer) was lower than that of the control group. Isotricha (genus Leptospira), ophryosocolox (genus Torulopsis) is a fermentation process that produces acetic acid and hydrogen from starch. It was shown that 0.60g/L silybin was added to inhibit Isotricha (Homoparatus), ophryosocolox (Tocopa), unclassified_Rotifera (unclassified rotifer) in the rumen, thereby reducing acetic acid and hydrogen production.

The addition of 0.60g/L silybin group and control group showed significant differences in Methanosphaera (Methanosphaera) abundance (P < 0.05). It was shown that 0.60g/L of silibinin inhibits Methanosphaera (Methanosphaera) in the rumen and reduces methane production.

The added 0.60g/L silybin group and the control group have significant differences (P < 0.05) in fungus abundance of Orpinomyces (anaerobic fungus) and Neocallimastix (anaerobic fungus). The addition of 0.60g/L of silibinin group fungi was lower in the genus level than the control group. Orpinomyces, neocallimastix all degrade cellulose to acetic acid. The addition of 0.60g/L of silibinin group was shown to inhibit the decrease of acetic acid production by rumen orinomycetes, neocalimastix.

The addition of 0.60g/L silybin group was shown to inhibit in-rumen Prevotella (Prevotella), isotricha (Isodon), ophryostex (Torula), unclassified_Rotifera (unclassified rotifer), methanosphaera (Methanofaciens), orpinomyces (anaerobic fungus), neocallimastix (anaerobic fungus), and reduce the production of acetic acid and methanogenic substrate hydrogen.

As can be seen from table 5, the composition of the communities of the rumen archaea, bacteria, protozoa and fungi is different (P < 0.05) when 0.60g/L of silybin group is added, which indicates that silybin regulates rumen fermentation by changing the composition of the rumen archaea, bacteria, protozoa and fungi, thereby regulating methane generation.

3.4 determination of rumen metabolite

The method for extracting the sample comprises the following steps: (1) Weighing 100 mu L of sample, adding 500 mu L of extracting solution containing internal standard (methanol acetonitrile volume ratio=1:1, internal standard concentration 20 mg/L), and vortex mixing for 30 seconds; (2) ultrasonic treatment for 10min (ice water bath);

the metabolite extraction method comprises the following steps:

the LC/MS system for metabonomics analysis consisted of a Waters acquisition I-Class PLUS ultra-efficient liquid tandem Waters Xevo G2-XS QTof high resolution mass spectrometer. The chromatographic column used was purchased from a Waters Acquity UPLC HSS T3 column (1.8 um 2.1 x 100 mm).

Positive ion mode: mobile phase a:0.1% formic acid in water; mobile phase B:0.1% acetonitrile formate; negative ion mode: mobile phase a:0.1% formic acid in water; mobile phase B:0.1% acetonitrile formate; the sample injection amount is 1 mu L;

LC-MS/MS analysis: the Waters Xevo G2-XS QTOF high resolution mass spectrometer can acquire primary and secondary mass spectrometry data in MSe mode under the control of acquisition software (MassLynx V4.2, waters). In each data acquisition cycle, two-channel data acquisition can be performed for both low and high collision energies. The low collision energy is 2V, the high collision energy is 10-40V, and the scanning frequency is 0.2 second mass spectrum. The parameters of the ESI ion source are as follows. Capillary voltage: 2000V (positive ion mode) or-1500V (negative ion mode); cone voltage: 30V; ion source temperature. 150 ℃; the desolvation gas temperature is 500 ℃; recoil gas flow. 50L/h; desolventizing gas flow rate. 800L/h.

Data preprocessing and annotation: the original data acquired by using MassLynx V4.2 are subjected to data processing operations such as peak extraction, peak alignment and the like by Progenesis QI software, and are identified based on an online METIN database of the Progenesis QI software and a self-built database of a Biomark, and simultaneously, theoretical fragment identification is carried out, and the quality deviation is within 100 ppm.

Data analysis: and carrying out subsequent analysis after carrying out normalization treatment on the original peak area information and the total peak area. Principal component analysis and Spearman correlation analysis were used to determine the reproducibility of samples within the group and to quantitatively control the samples. The identified compounds were classified and path information retrieved in KEGG, HMDB and lipidmap databases. Based on the grouping information, fold differences were calculated and compared, and the T-test was used to calculate the P-value of the difference significance for each compound. OPLS-DA modeling was performed using R language packages ropls and 200 permutation tests were performed to verify the reliability of the model. The VIP values of the model were calculated using multiple cross-validation. Differential metabolites were screened using a combination of fold difference, P-value and VIP-value for OPLS-DA model. The screening criteria were FC > P <0.05, VIP >1. And calculating the differential metabolites of the KEGG pathway enrichment significance by using a super-geometric distribution test method.

From FIG. 2, it can be seen that the added 0.60g/L silybin group has a difference in metabolite type and level (P < 0.05) from the control group. The result shows that the silybin is added to influence the composition of metabolites in rumen so as to regulate and control the generation of methane.

TABLE 6 significant kegg pathway for differences between groups with and without added Silybin in vitro fermentation

TABLE 7 metabolites with significant differences in kegg pathways between groups with and without added silybin by in vitro fermentation

As can be seen from tables 6 and 7, dihydrobioterin (Dihydrobiopterin), 2-amino-6- (hydroymethyl) -7, 8-dihydropteridin-4-ol (2-amino-6- (hydroxymethyl) -7, 8-dihydropteridin-4-ol), 3- (2-Hydroxyphenyl) propionicacid (3- (2-Hydroxyphenyl) propionic acid), 2-phenylglycide (2-Phenylacetamide), P-Coumaroyl quinic acid (P-coumaroyl quinic acid), N-phenylglycine (N-Phenylacetylglycine), N-Acetyl-D-phenylalanine), phozin (chlorohydroxypyridine), 5,6,7, 8-tetrahydrominochroman (5, 6,7, 8-tetrahydromethylpterin) metabolites have a significant difference in phenylalanine metabolism, flavonoid biosynthesis and the pathway of 3.05. The added 0.60g/L silybin group had a significant difference in phenylalanine metabolic pathway compared to the control group (P < 0.05). There is a differential trend in 2 metabolic pathways of flavonoid biosynthesis and folic acid biosynthesis (0.05 < P < 0.1). The addition of silybin affects the rumen metabolic pathway.

Flavonoids are the largest class of polyphenols, and the chemical components of silybin are also flavonoids, and ruminal microorganisms can cleave the glycoside components of polymeric flavonoids, making them available for intestinal absorption. Flavonoid metabolism in the animal digestive tract may involve O-and C-deglycosylation, demethylation, dehydroxylation, ester cleavage, reduction of carbon-carbon double bonds, isomerization, ring fission, fatty carbon chain extension and truncation, decarboxylation, and the like. These reactions convert flavonoids to simple phenolic acids, thereby enhancing the functional properties of flavonoids while improving their bioavailability in the gastrointestinal tract. The rumen microorganism of ruminant can synthesize folic acid, and the synthesis of folic acid is promoted after silybin is added.

Claims

1. The application of silybin in preparing feed additive for regulating and controlling rumen methane emission of ruminant is provided.

2. The use according to claim 1, wherein the regulation is by: reducing the generation amount of methane and reducing the emission amount of methane.

3. The use according to claim 1, wherein the silibinin reduces the amount of methane produced by inhibiting the species of microorganisms in the rumen, thereby reducing the amount of methane emitted; the microorganisms include Prevolella (Prevoltella), homoptera (Isotricha), torulopsis (Ophryosocolox), methanosphaera (Methanosphaera), unclassified rotifers (unclassified_Rotifera), anaerobic fungi (Orpinomyces) and anaerobic fungi (Neocallimastix).

4. The use according to claim 1, wherein the silibinin reduces the production of methane by changing the composition of the community of archaea, bacteria, protozoa, fungi in the rumen, and thus the emission of methane.

5. The use according to claim 1, wherein the silybin regulates methane production by altering the in-rumen metabolic pathway; the metabolic pathway includes: phenylalanine metabolic pathway, flavonoid biosynthetic pathway, and folic acid biosynthetic pathway.

6. The use according to claim 1, wherein the ruminant is a dairy cow.

7. A feed additive, characterized by comprising silybin.

8. A feed, comprising a feed additive and a basic ration, wherein the feed additive comprises silybin; the addition amount of the silybin is 0.075g/L to 0.6g/L based on basic ration.

9. A method of increasing rumen methane emissions from a ruminant animal, comprising: adding silybin as a feed additive into basic ration, and feeding ruminant;

the addition amount of the silybin is 0.075g/L to 0.6g/L based on basic ration.

10. The method of claim 9, wherein the ruminant is a dairy cow.