Application of allicin in promoting conversion of urea nitrogen into nitrogen element in nucleotide or amino acid

Abstract

The invention discloses an application of allicin in promoting urea nitrogen to be converted into a nitrogen element in nucleotide or amino acid. Experiments show that the plant source compound allicin has the effect of promoting rumen urea nitrogen to be converted into nitrogen in nucleotide, sugar nucleotide or amino acid; when the garlicin is directly fed to ruminants or added as a feed additive into basic ration of the ruminants to feed the ruminants, urea nitrogen in feed can be promoted to be converted into nitrogen elements in nucleotide, sugar nucleotide or amino acid; the invention further provides a non-therapeutic method for promoting the ruminant to convert urea nitrogen into nitrogen in nucleotides, sugar nucleotides or amino acids, comprising feeding the ruminant with allicin directly or as a feed additive. The method has application prospects in the aspects of providing high-quality protein sources for livestock and poultry, reducing feed cost, improving livestock and poultry production performance and the like.

Description

Technical Field

[0001] The invention relates to a new use of allicin in animal nutrition, in particular to the use of allicin in promoting the conversion of urea nitrogen into nitrogen elements in nucleotides, sugar nucleotides or amino acids, and belongs to the field of new uses of allicin in animal nutrition. Background Art

[0002] The inside of the rumen is a highly complex microbial ecosystem, inhabited by a variety of microorganisms including bacteria, fungi, protozoa and archaea. In the process of nitrogen metabolism in the rumen, microbial communities such as bacteria, protozoa and fungi play a synergistic role in promoting the biotransformation of nitrogen-containing substances and the synthesis of microbial proteins. Non-protein nitrogen compounds such as urea play a vital role in the growth of microorganisms. As an important nitrogen source, they can meet the needs of microorganisms to synthesize biomacromolecules such as proteins and nucleic acids and promote the reproduction of microorganisms. In animal husbandry, non-protein nitrogen compounds such as urea are used as feed additives and converted into microbial proteins through microbial fermentation, providing livestock and poultry with a high-quality protein source, reducing feed costs and improving production performance.

[0003] Nitrogen-containing compounds in microorganisms are mainly involved in key pathways such as energy production, amino acid synthesis, and nucleotide synthesis. They can use inorganic nitrogen compounds (such as ammonium salts, nitrates) or organic nitrogen compounds (such as proteins, polypeptides) as nitrogen sources to synthesize amino acids through specific metabolic pathways, and then synthesize proteins and other nitrogen-containing organic matter. In addition, nitrogen-containing compounds such as purine and pyrimidine are also the basic units that make up nucleotides. Microorganisms can use these compounds to synthesize nucleotides and then synthesize nucleic acids (DNA and RNA), which is essential for the growth, reproduction, and transmission of genetic information of microorganisms. At the same time, some inorganic nitrogen compounds can also be oxidized to release energy under the action of specific microorganisms (such as nitrifying bacteria), providing microorganisms with the energy source required for life activities.

[0004] ATP, ADP, NAD and NADP are the main energy suppliers for microbial energy metabolism, participate in enzyme-catalyzed reactions, and affect the distribution of material metabolic flows. They directly affect the secretion of small molecule metabolites, affect the growth of microbial cells and metabolic reaction rates, and have an important impact on the anabolism of proteins, lipids, nucleotides and amino acids. Amino acids play a key role in the growth of rumen microorganisms and are a key component for rumen microorganisms to synthesize proteins and maintain functions. Nucleotides are the basic units that make up DNA and RNA, and are essential for the storage and transmission of genetic information. They are not only the basic components of organisms, but also participate in growth, development, reproduction and heredity. Nucleotides are also components of important coenzymes and participate in the transfer of hydrogen atoms or electrons in biological oxidation systems. Nucleotides play many key roles in the growth and reproduction of microorganisms, and are indispensable from genetic information transmission to metabolism and immune regulation.

[0005] Allicin is a natural organic sulfide of garlic and an active ingredient in garlic essential oil. Adding allicin to feed can improve nutrient digestion, reduce methane production, and enhance animal immunity. However, there are no reports on whether allicin has a regulatory effect on the conversion of urea nitrogen to amino acids, sugar nucleotides, or nitrogen elements in nucleotides of rumen microorganisms. Summary of the invention

[0006] One of the main purposes of the present invention is to provide the use of allicin in promoting the conversion of urea nitrogen into nitrogen elements in nucleoside sugars, nucleotides or amino acids;

[0007] The second object of the present invention is a method for promoting ruminants to convert urea nitrogen into nitrogen elements in nucleotides, sugar nucleotides or amino acids for non-therapeutic purposes.

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

[0009] One aspect of the present invention is to provide the use of allicin in promoting the conversion of urea nitrogen in animal feed into nitrogen elements in nucleotides, sugar nucleotides or amino acids.

[0010] In a preferred embodiment of the present invention, the animal is preferably a ruminant, and further preferably, the ruminant includes cattle, sheep, alpacas or deer.

[0011] In a preferred embodiment of the present invention, the nucleotides include guanosine monophosphate, uridine monophosphate (UMP), adenosine monophosphate (AMP) or guanosine monophosphate (GMP); the sugar nucleotides are preferably uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc); and the amino acids include various nitrogen-containing amino acids, most preferably L-alanine.

[0012] Another aspect of the present invention is to provide a method for promoting ruminants to convert urea nitrogen into nitrogen elements in nucleotides, sugar nucleotides or amino acids for non-therapeutic purposes, comprising: directly feeding allicin to ruminants; or adding allicin as a feed additive to the basic diet of ruminants, and feeding the basic diet added with allicin to ruminants; or mixing allicin or its salt with trace elements and vitamins to prepare a premixed material and feeding it to ruminants; or mixing allicin or its salt with protein and energy feed to prepare a concentrated feed mixture and feeding it to ruminants.

[0013] In a preferred embodiment of the present invention, when allicin is directly fed to ruminants or fed to ruminants as a feed additive, the concentration of allicin in the rumen of the ruminants is preferably 1-4 mmol / L.

[0014] The structural formula of allicin described in the present invention is shown in Formula I:

[0015]

[0016] The present invention has found through experiments that the plant-derived compound allicin has the effect of promoting the conversion of rumen urea nitrogen into nitrogen elements in nucleotides, sugar nucleotides or amino acids. Therefore, directly feeding allicin to ruminants or adding it as a feed additive to the basic diet of ruminants and feeding them to ruminants can promote the conversion of urea nitrogen in feed into nitrogen elements in nucleotides, sugar nucleotides or amino acids. The present invention thus provides a method for promoting ruminants to convert urea nitrogen into nitrogen elements in nucleotides, sugar nucleotides or amino acids for non-therapeutic purposes. The present invention has application prospects in providing high-quality protein sources for livestock and poultry, reducing feed costs and improving livestock and poultry production performance.

[0017] Detailed description of the overall technical solution of the present invention

[0018] Allicin promotes the synthesis of urea nitrogen into nucleotide and amino acid nitrogen compounds by rumen microorganisms

[0019] The present invention collects rumen fluid and prepares anaerobic diluent for in vitro fermentation. 20 mL of filtered rumen fluid and 40 mL of anaerobic diluent are added into a 100 mL cell culture bottle by a syringe, CO 2 Deaeration and deoxygenation, fermentation for 24h. Each bottle contains exactly 1g of feed. The experiment was divided into 3 groups, each with 4 replicates, namely conventional urea group ( 14 N 14 The initial concentration of N-urea was 16.67mmol / L), control group ( 15 N 15 The initial concentration of N-urea was 16.67mmol / L. 15 N 15 N-urea abundance: 99.08atom%, Shanghai Stable Isotope Engineering Technology Research Center, China) and allicin group ( 15 N 15The initial concentration of N-urea was 16.67mmol / L, and the concentration of allicin was 2mmol / L), and the fermentation was carried out for 12h. At 12h, 10mL of fermentation broth was taken. Then, centrifuged at 12000×g for 10min, the supernatant was removed, and the precipitate was resuspended by adding 10mL PBS buffer. The precipitate was centrifuged again at 12000×g for 10min, and the supernatant was removed. The process was repeated 3 times, and the precipitate was retained, 0.6mL PBS was added, and the liquid nitrogen was repeatedly frozen and thawed 3 times. After thawing, grinding beads were added, and the mixture was ground on ice for 1min. After mixing, 0.3mL was taken, 1.2mL methanol was added, and the mixture was centrifuged at 12000×g for 10min. The supernatant was retained. Polar metabolites were extracted from 50μL of samples washed with sterile PBS and treated with methanol using 200μL of ice-cold methanol containing 8μg of phenylhydrazine. The sample was vortexed and stored at -20℃ for 1h for derivatization of α-keto acids. After derivatization, the samples were incubated at 1500×g for 30 min at 4°C. After incubation, the samples were centrifuged at 12 000×g for 10 min at 4°C. The supernatant was transferred and dried in a vacuum concentrator (SpeedVac, Thermo ScientificTM, China). Metabolites were analyzed by LC-MS / MS. The collision energy was set to (±) 35±10 eV. TF 1.7.1 software (AB Sciex, Concord, Canada) was used for data acquisition and processing. All detected ions were extracted into Excel in the format of a dimensional matrix using MarkerView 1.3 (AB Sciex, Concord, Canada), including mass-to-charge ratio (m / z), retention time, and peak area. PeakView 2.2 (AB Sciex, Canada) was used to extract LC-MS / MS data and compared with the Metabolite Database (AB Sciex, Canada), HMDB, and standard references to annotate ion properties. L-leucine-d10 was used as an internal standard to correct endogenous metabolites in the samples and normalized according to the protein content. The results showed that allicin promoted the urea-N flow to microbial nucleotide nitrogen-containing compounds (GMP, AMP, UMP, and guanylate) and amino acid nitrogen-containing compounds (L-alanine), but had no significant effect on the energy compounds ADP and NAD. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1Effect of allicin on urea nitrogen metabolites of rumen microorganisms; (a) relative content of GMP in the control group and the allicin treatment group; (b) relative content of UDP-GlcNAc in the control group and the allicin treatment group; (c) relative content of AMP in the control group and the allicin treatment group; (d) relative content of UMP in the control group and the allicin treatment group; (e) relative content of L-alanine in the control group and the allicin treatment group; (f) relative content of guanylate in the control group and the allicin treatment group; (g) relative content of ADP in the control group and the allicin treatment group; (h) relative content of NDP in the control group and the allicin treatment group. DETAILED DESCRIPTION

[0021] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0022] Example 1 Allicin regulates the synthesis of nitrogen-containing compounds in ruminant rumen urea nitrogen into nucleotides, sugar nucleotides or amino acids

[0023] 1. Test methods

[0024] Step 1: Collection of rumen fluid Three fistula Holstein cows were selected, 100 mL of rumen contents were collected, filtered through 4 layers of sterile gauze, the filtrate (rumen fluid) was dispensed into 50 mL sterile centrifuge tubes, centrifuged (300 g, 5 min, 4 °C), and the supernatant was collected into new centrifuge tubes.

[0025] Step 2: Prepare anaerobic dilution. Anaerobic medium composition (mg / L): CaCl 2 6.1, KH 2 PO 4 228.0, NaCl 45.6, MgSO 4 7H 2 O 9.5, NaHCO 3 500, L-cysteine ​​50, using CO 2 Deoxygenation.

[0026] Step 3: In vitro fermentation. The experiment was divided into 3 groups, each with 4 replicates, namely conventional urea group ( 14 N 14 The initial concentration of N-urea was 16.67mmol / L), control group ( 15 N 15The initial concentration of N-urea was 16.67mmol / L. 15 N 15 N-urea abundance: 99.08atom%, Shanghai Stable Isotope Engineering Technology Research Center, China) and allicin treatment group ( 15 N 15 The initial concentration of N-urea was 16.67 mmol / L, the concentration of allicin was 2 mmol / L), and the fermentation was carried out for 12 h.

[0027] Step 4: Sample collection and processing. At 12h, take 10mL of fermentation broth. Then, centrifuge at 12000×g for 10min, remove the supernatant, add 10mL PBS buffer to resuspend the precipitate, centrifuge again at 12000×g for 10min, remove the supernatant, repeat this process 3 times, retain the precipitate, add 0.6mL PBS, repeat freeze-thaw with liquid nitrogen 3 times, add grinding beads after thawing, grind on ice for 1min, mix well, take 0.3mL, add 1.2mL methanol, centrifuge at 12000×g for 10min, and retain the supernatant.

[0028] Step 5: Sample determination. Polar metabolites were extracted from 50 μL of sample washed with sterile PBS and treated with methanol using 200 μL of ice-cold methanol containing 8 μg of phenylhydrazine. The sample was vortexed and stored at -20°C for 1 h for derivatization of α-keto acids. After derivatization, the sample was incubated at 1500 × g for 30 min at 4°C. After the incubation, centrifuged at 12 000 × g for 10 min at 4°C. The supernatant was transferred and dried in a vacuum concentrator (SpeedVac, Thermo ScientificTM, China), and the metabolites were analyzed by LC-MS / MS with the collision energy set to (±) 35 ± 10 eV.

[0029] Step 6: Data and results analysis; use TF 1.7.1 software (AB Sciex, Concord, Canada) was used for data acquisition and processing. All detected ions were extracted into Excel in a dimensional matrix format using MarkerView 1.3 (AB Sciex, Concord, Canada), including mass-to-charge ratio (m / z), retention time, and peak area. PeakView 2.2 (AB Sciex, Canada) was used to extract LC-MS / MS data and compare with the Metabolite Database (AB Sciex, Canada), HMDB, and standard references to annotate ion characteristics. L-leucine-d10 was used as an internal standard to correct for endogenous metabolites in the samples and normalize them according to the protein content.

[0030] 2 Test results

[0031] The test results are shown in Figure 1 ; where a is guanylate monophosphate (GMP), b is uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc), c is adenylate monophosphate (AMP), d is L-alanine, e is uridine monophosphate (UMP), f is guanylate, g is adenosine diphosphate, and h is nicotinamide adenine dinucleotide (NAD); M+0 means no 15 N marks, M+1 means there is one 15 N mark, M+2 / 3 / ... and so on. 15 The N abundance was the background value, and the control group and allicin were analyzed after deducting the background value.

[0032] according to Figure 1 The experimental results show that allicin promotes the flow of urea-nitrogen to nitrogen elements in microbial nucleotides (GMP, AMP, UMP and guanosine), sugar nucleotides (UDP-GlcNAc) or amino acids (L-alanine), but has no significant effect on the energy compounds ADP and NAD.

Claims

1. The use of allicin in promoting the conversion of urea nitrogen in animal feed into nitrogen elements in nucleotides, sugar nucleotides or amino acids.

2. The use according to claim 1, characterized in that The animal is a ruminant.

3. The use according to claim 2, characterized in that: The ruminant includes cattle, sheep, alpacas or deer.

4. The use according to claim 1, characterized in that The nucleotides include guanylate, uridine monophosphate, adenosine monophosphate or guanosine monophosphate; the sugar nucleotide is uridine diphosphate-N-acetylglucosamine; and the amino acid is L-alanine.

5. The use according to claim 1, characterized in that: The structural formula of the allicin is shown in Formula I:

6. A method for promoting ruminants to convert urea nitrogen into nitrogen in nucleotides, sugar nucleotides or amino acids for non-therapeutic purposes, characterized in that: include: The allicin is fed directly to the ruminant or the allicin is fed to the ruminant as a feed additive.

7. The method according to claim 6, characterized in that The method of feeding allicin as a feed additive to ruminants comprises: adding allicin as a feed additive to the basic diet of the ruminants, and feeding the basic diet supplemented with allicin to the ruminants.

8. The method according to claim 6, characterized in that The method of using allicin as a feed additive to feed ruminants comprises: mixing allicin or its salt with trace elements and vitamins to prepare a premixed material for feeding the ruminants.

9. The method according to claim 6, characterized in that The method of using allicin as a feed additive to feed ruminants comprises: mixing allicin or its salt with protein and energy feed to prepare a concentrated feed mixture and feeding the ruminants.

10. The method according to any one of claims 6 to 9, characterized in that: When allicin is fed directly to ruminants or fed to ruminants as a feed additive, the concentration of allicin in the rumen of ruminants is 1 to 4 mmol / L.

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