Application of N-benzyloxycarbonyl-leucine in reducing rumen methane emission in ruminants

Abstract

The invention discloses an application of N-benzyloxycarbonyl-leucine in reducing rumen methane emission of ruminants. As a natural plant extract, N-benzyloxycarbonyl-leucine has a completely different structure type from existing inhibitors, is safe and non-toxic, and can be used as a feed additive.

Description

Technical Field

[0001] The invention belongs to the field of feed additives, and in particular relates to the application of N-benzyloxycarbonyl-leucine in reducing rumen methane emission of ruminants. Background Art

[0002] Rumen methane production in ruminants not only causes 2-12% of ruminant feed energy loss, but is also an important source of greenhouse gases. The research and application of ruminant methane inhibitors can not only improve the energy utilization efficiency and production performance of animals, but also have good ecological benefits. Although various feed additives (such as natural plant extracts, ion carriers, polyhalogen compounds, electron acceptors, oils and nitro compounds, etc.) have been studied and developed, they all have different problems, such as long-term effectiveness, safety and microbial tolerance. These defects limit the widespread application of existing additives, and it is urgent to develop new ruminant rumen methane reduction additives. The natural plant active substance N-benzyloxycarbonyl-leucine is present in plants such as silkworm feces, mung beans, humulus, and milk thistle. However, there has been no report on reducing rumen methane emissions in ruminants. Summary of the invention

[0003] Purpose of the invention: The purpose of the present invention is to provide an application of N-benzyloxycarbonyl-leucine in reducing rumen methane emission in ruminants.

[0004] Technical solution: The application of N-benzyloxycarbonyl-leucine of the present invention in reducing rumen methane emission of ruminants.

[0005] Wherein, the N-benzyloxycarbonyl-leucine is N-benzyloxycarbonyl-L-leucine.

[0006] The feed additive for reducing rumen methane emission of ruminants of the present invention comprises N-benzyloxycarbonyl-leucine as an active ingredient of the additive.

[0007] Wherein, the concentration of the N-benzyloxycarbonyl-leucine is 0.2-0.8 mg / mL.

[0008] Wherein, the concentration of the N-benzyloxycarbonyl-leucine is 0.2-0.4 mg / mL.

[0009] The invention discloses an application of N-benzyloxycarbonyl-leucine in the preparation of a methanogen inhibitor.

[0010] Wherein, the concentration of N-benzyloxycarbonyl-leucine in the inhibitor is 0.2-0.8 mg / mL.

[0011] Wherein, the concentration of N-benzyloxycarbonyl-leucine in the inhibitor is 0.2-0.4 mg / mL.

[0012] Beneficial effects: Compared with the prior art, the present invention has the following outstanding and significant advantages: The present invention discloses for the first time the application of N-benzyloxycarbonyl-leucine in reducing rumen methane emissions in ruminants. As a natural plant extract, N-benzyloxycarbonyl-leucine has a completely different structural type from the existing inhibitors, is safe and non-toxic, and can be used as a feed additive. DETAILED DESCRIPTION

[0013] The technical solution of the present invention is further described below.

[0014] Example 1: N-Benzyloxycarbonyl-leucine inhibits methane production in rumen fermentation in vitro

[0015] 1. Materials and Methods

[0016] 1.1 Experimental animals and rumen fluid collection

[0017] The experimental animals were raised at the Dairy Cow Experimental Base of the College of Animal Science and Technology of Nanjing Agricultural University (Nanjing, Jiangsu Province). Three multiparous Holstein cows were equipped with permanent rumen fistulas, with an average weight of 613±35kg. On the day of the experiment, rumen fluid was collected half an hour before morning feeding, quickly put into a thermos bottle pre-filled with carbon dioxide and brought back to the laboratory.

[0018] 1.2 Preparation of artificial rumen fermentation broth

[0019] The preparation of fermentation liquid was based on the method of Menke and Steingass (1988). The collected fresh rumen liquid was filtered through 4 layers of gauze, and 100 ml of rumen liquid was quickly poured into the prepared 900 ml artificial buffer (Table 1). The rumen liquid and buffer were mixed at a ratio of 1:9 (v / v) to prepare a mixed artificial rumen culture liquid. The mixed fermentation liquid was divided into 100 ml into 180 ml fermentation bottles. The temperature of the whole process was kept at 39 ° C, and carbon dioxide was continuously introduced to maintain an anaerobic environment. 1.0 g of substrate (24.4% corn, 6.2% DDGS, 17.2% soybean meal, 25.4% alfalfa, 15.6% straw, 11.3% corn silage) and inhibitor (N-benzyloxycarbonyl-L-leucine or N-benzyloxycarbonyl-L-valine) were added to the fermentation bottle in advance. All fermentation bottles were fermented at 39 ° C and 80 r / min for 24 hours.

[0020] Table 1 Buffer composition (1L)

[0021]

[0022] Preparation of specific solution:

[0023] (1) Trace element solution (Solution A):

[0024]

[0025]

[0026] Add deionized water to 100 mL.

[0027] (2) Buffer solution (Solution B)

[0028] NH4HCO3 4.0g

[0029] NaHCO3 35.0g

[0030] Add deionized water to 1000 mL.

[0031] (3) Constant element solution (Solution C)

[0032] Na2HPO4·12H2O 9.45g

[0033] KH2PO4 6.2g

[0034] MgSO4·7H2O 0.6g

[0035] Add deionized water to 1000 mL.

[0036] (4) 0.1% Resazurin solution (Solution D)

[0037] 100 mg of resazurin was added into 100 mL of deionized water.

[0038] (5) Reducing agent solution (liquid E)

[0039]

[0040] 1.3 Test methods

[0041] The experiment set up 4 groups

[0042] 1) Control group: no addition

[0043] 2) Treatment group: N-benzyloxycarbonyl-L-leucine 0.2 mg / mL (final concentration);

[0044] 3) Treatment group: N-benzyloxycarbonyl-L-leucine 0.4 mg / mL (final concentration);

[0045] 4) Treatment group: N-benzyloxycarbonyl-L-leucine 0.8 mg / mL (final concentration);

[0046] 5) Treatment group: N-benzyloxycarbonyl-L-valine 0.2 mg / mL (final concentration);

[0047] 6) Treatment group: N-benzyloxycarbonyl-L-valine 0.4 mg / mL (final concentration);

[0048] 7) Treatment group: N-benzyloxycarbonyl-L-valine 0.8 mg / mL (final concentration).

[0049] 1.4 Chemical analysis

[0050] Methane and hydrogen determination. The gas production in the fermentation bottle was determined using a pressure converter according to the method of Theodorou et al. (1994). The methane and hydrogen concentrations were determined using an Agilent gas chromatograph 8860. The chromatographic column (J&W HP-PLOT Molesieve, Agilent) was used at a column temperature of 80°C; the TCD detector temperature was 200°C; and the carrier gas was nitrogen.

[0051] 1.5 Data Statistical Processing

[0052] The data were analyzed using One-way ANOVA (Tukey) in SPSS20.0 software, and P<0.05 indicated a significant difference.

[0053] 2. Results

[0054] Table 2 Effects of N-benzyloxycarbonylleucine on total gas production, hydrogen, methane and pH of rumen in vitro fermentation

[0055]

[0056] a,b,c Groups marked with different letters are significantly different.

[0057] Table 3 Effects of N-benzyloxycarbonyl-L-valine on total gas production, hydrogen, methane and pH of rumen fermentation in vitro

[0058]

[0059] a,b Groups marked with different letters are significantly different.

[0060] As shown in Tables 2 and 3, N-benzyloxycarbonyl-L-leucine at a concentration of 0.2-0.8 mg / mL can significantly inhibit methane production during rumen in vitro fermentation (P<0.05), while reducing hydrogen accumulation (P<0.05), and the total gas production is also inhibited to a certain extent (P<0.05). However, N-benzyloxycarbonyl-L-valine with a similar structure has no effect on rumen methane production at the same concentration (P>0.05).

Claims

1. Application of N-benzyloxycarbonyl-leucine in reducing methane emission in artificial rumen fermentation liquid of ruminants, characterized in that: The N-benzyloxycarbonyl-leucine is N-benzyloxycarbonyl-L-leucine; the concentration of the N-benzyloxycarbonyl-L-leucine is 0.2-0.8 mg / mL.

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