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

Abstract

The application discloses use of allicin in promoting urea nitrogen conversion into nitrogen element in nucleotide or amino acid. The application finds through experiments that plant source compound allicin has the effect of promoting rumen urea nitrogen conversion into nitrogen element in nucleotide, sugar nucleotide or amino acid, thus, feeding ruminants with allicin directly or adding allicin into the basic daily ration of ruminants as a feed additive can promote urea nitrogen conversion into nitrogen element in nucleotide, sugar nucleotide or amino acid in the feed; the application further provides a method for promoting ruminants to convert urea nitrogen into nitrogen element in nucleotide, sugar nucleotide or amino acid for non-treatment purposes, which comprises: feeding ruminants with allicin directly or as a feed additive. The application has application prospects in providing high-quality protein sources for livestock and poultry, reducing feed costs and improving production performance of livestock and poultry and the like.

Description

Technical Field

[0001] This invention relates to novel uses of allicin in animal nutrition, particularly the use of allicin in promoting the conversion of urea nitrogen into nitrogen in nucleotides, glyconucleotides, or amino acids, which falls within the field of novel uses of allicin in animal nutrition. Background Technology

[0002] The rumen is a highly complex microbial ecosystem, home to a diverse range of microorganisms, including bacteria, fungi, protozoa, and archaea. During rumen nitrogen metabolism, these microbial communities work synergistically to promote the biotransformation of nitrogenous substances and the synthesis of microbial proteins. Non-protein nitrogen compounds such as urea play a crucial role in microbial growth. As important nitrogen sources, they meet the needs of microorganisms in synthesizing proteins, nucleic acids, and other biomolecules, promoting microbial reproduction. In animal husbandry, non-protein nitrogen compounds like urea are used as feed additives, where they are fermented into microbial proteins, providing livestock with a high-quality protein source, reducing feed costs, and improving production performance.

[0003] Nitrogenous compounds in microorganisms are primarily involved in key pathways such as energy production, amino acid synthesis, and nucleotide synthesis. They can utilize inorganic nitrogen compounds (such as ammonium salts and nitrates) or organic nitrogen compounds (such as proteins and polypeptides) as nitrogen sources to synthesize amino acids through specific metabolic pathways, thereby synthesizing proteins and other nitrogen-containing organic substances. Furthermore, nitrogenous compounds such as purines and pyrimidines are also basic building blocks of nucleotides. Microorganisms can utilize these compounds to synthesize nucleotides, which in turn synthesize nucleic acids (DNA and RNA), crucial for microbial growth, reproduction, and the transmission of genetic information. Simultaneously, certain inorganic nitrogen compounds can be oxidized to release energy under the action of specific microorganisms (such as nitrifying bacteria), providing the energy source required for microbial life activities.

[0004] ATP, ADP, NAD, and NADP are the main energy-supplying substances in microbial energy metabolism, participating in enzyme-catalyzed reactions and influencing the distribution of metabolic flux. They directly affect the secretion of small molecule metabolites, influencing microbial cell growth and metabolic reaction rates, and have a significant impact on the synthesis and metabolism of proteins, lipids, nucleotides, and amino acids. Amino acids play a crucial role in the growth of rumen microorganisms, being key components for protein synthesis and function maintenance. Nucleotides are the basic building blocks of DNA and RNA, essential for the storage and transmission of genetic information. They are not only fundamental components of organisms but also participate in growth, development, reproduction, and heredity. Nucleotides are also components of important coenzymes, participating in the transfer of hydrogen atoms or electrons in biological oxidation systems. Nucleotides play multiple crucial roles in microbial growth and reproduction, indispensable from genetic information transmission to metabolic and immune regulation.

[0005] Allicin is a natural organosulfur compound found in garlic and is also an active ingredient in garlic oil. Adding allicin to animal feed can improve nutrient digestion, reduce methane production, and enhance animal immunity. However, there are no reports on whether allicin regulates the conversion of urea nitrogen into nitrogen in rumen microorganisms' amino acids, glyconucleotides, or nucleotides. Summary of the Invention

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

[0007] The second objective of this invention is to provide a non-therapeutic method for promoting the conversion of urea nitrogen into nitrogen in nucleotides, glyconucleotides, or amino acids in ruminants.

[0008] The above-mentioned objective of the present invention is achieved through the following technical solution:

[0009] One aspect of the present invention provides the use of allicin in promoting the conversion of urea nitrogen in animal feed into nitrogen in nucleotides, glyconucleotides or amino acids.

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

[0011] In a preferred embodiment of the present invention, the nucleotides include guanylic acid, uridine monophosphate (UMP), adenosine monophosphate (AMP), or guanosine monophosphate (GMP); the glyconucleotides 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 the conversion of urea nitrogen into nitrogen in nucleotides, glyconucleotides, or amino acids in ruminants for non-therapeutic purposes, comprising: directly feeding allicin to ruminants; or adding allicin as a feed additive to the basal diet of ruminants and feeding the basal diet containing allicin to the ruminants; or preparing a premixed feed by mixing allicin or its salt with trace elements and vitamins and feeding it to ruminants; or preparing a concentrate mixture by mixing allicin or its salt with protein and energy feed and feeding it to ruminants.

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

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

[0015]

[0016] This invention, through experiments, has discovered that the plant-derived compound allicin promotes the conversion of rumen urea nitrogen into nitrogen in nucleotides, glyconucleotides, or amino acids. Therefore, feeding allicin directly to ruminants or adding it as a feed additive to their basal diet can promote the conversion of urea nitrogen in the feed into nitrogen in nucleotides, glyconucleotides, or amino acids. This invention thus provides a method for promoting the conversion of urea nitrogen into nitrogen in nucleotides, glyconucleotides, or amino acids in ruminants for non-therapeutic purposes. This invention has promising applications 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 nitrogenous compounds, including nucleotides and amino acids, from urea nitrogen in rumen microorganisms.

[0019] This invention involves collecting rumen fluid and preparing an anaerobic diluent for in vitro fermentation. 20 mL of filtered rumen fluid and 40 mL of the anaerobic diluent are added to a 100 mL cell culture flask using a syringe, followed by CO2 degassing and fermentation for 24 hours. Each flask contains exactly 1 g of feed. The experiment is divided into three groups, with four replicates per group: a conventional urea group (…). 14 N 14 The initial N-urea concentration was 16.67 mmol / L, and the control group ( 15 N 15 The initial N-urea concentration was 16.67 mmol / L. 15 N 15 N-urea abundance: 99.08 atom%, Shanghai Stable Isotope Engineering Technology Research Center, China) and allicin group ( 15 N 15The initial concentration of N-urea was 16.67 mmol / L, and the concentration of allicin was 2 mmol / L. Fermentation was carried out for 12 h. At 12 h, 10 mL of fermentation broth was collected. Then, it was centrifuged at 12000×g for 10 min, the supernatant was removed, and the precipitate was resuspended in 10 mL of PBS buffer. The mixture was centrifuged again at 12000×g for 10 min, the supernatant was removed, and this process was repeated 3 times. The precipitate was retained, 0.6 mL of PBS was added, and the mixture was subjected to three freeze-thaw cycles with liquid nitrogen. After thawing, grinding beads were added, and the mixture was ground on ice for 1 min to mix thoroughly. 0.3 mL of the mixture was collected, 1.2 mL of methanol was added, and the mixture was centrifuged at 12000×g for 10 min. The supernatant was retained. Polar metabolites were extracted from 50 μL of the sample, which had been 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 the derivatization of α-keto acids. After derivatization, the sample was incubated at 1500×g at 4°C for 30 min. After incubation, it was centrifuged at 12,000×g for 10 min at 4°C. The supernatant was transferred and dried in a vacuum concentrator (SpeedVac, Thermo Scientific™, China). Metabolites were analyzed by LC-MS / MS. The collision energy was set to (±) 35±10 eV. Data acquisition and processing were performed using TF 1.7.1 software (AB Sciex, Concord, Canada). All detected ions were extracted into Excel in 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, which were compared with metabolite databases (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 was normalized according to protein content. The results showed that allicin promoted urea-N flow to microbial nucleotide nitrogenous compounds (GMP, AMP, UMP, and guanylic acid) and amino acid nitrogenous compounds (L-alanine), but had no significant effect on energy compounds ADP and NAD. Attached Figure Description

[0020] Figure 1Effects of allicin on rumen microbial urea nitrogen metabolites; (a) relative GMP content in the control group and allicin-treated group; (b) relative UDP-GlcNAc content in the control group and allicin-treated group; (c) relative AMP content in the control group and allicin-treated group; (d) relative UMP content in the control group and allicin-treated group; (e) relative L-alanine content in the control group and allicin-treated group; (f) relative guanylic acid content in the control group and allicin-treated group; (g) relative ADP content in the control group and allicin-treated group; (h) relative NDP content in the control group and allicin-treated group. Detailed Implementation

[0021] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as a result. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions to the details and form of the present invention can be made without departing from the spirit and scope of the invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0022] Example 1: Allicin regulates the synthesis of nitrogen-containing compounds in rumen urea nitrogen into nucleotides, glyconucleotides, or amino acids in rumen animals.

[0023] 1. Test Methods

[0024] Step 1: Rumen fluid collection. Select 3 Holstein cows with fistulas and collect 100mL of rumen contents. Filter the contents through 4 layers of sterile gauze and aliquot the filtrate (rumen fluid) into 50mL sterile centrifuge tubes. Centrifuge (300g, 5min, 4℃) and collect the supernatant into new centrifuge tubes.

[0025] Step 2: Prepare the anaerobic dilution solution. The composition of the anaerobic culture medium (mg / L): CaCl2 6.1, KH2PO4 228.0, NaCl 45.6, MgSO4·7H2O 9.5, NaHCO3 500, L-cysteine ​​50, with CO2 used for deoxygenation.

[0026] Step 3: In vitro fermentation. The experiment was divided into 3 groups, with 4 replicates in each group, namely the conventional urea group (… 14 N 14 The initial N-urea concentration was 16.67 mmol / L, and the control group ( 15 N 15 The initial N-urea concentration was 16.67 mmol / L. 15 N 15 N-urea abundance: 99.08 atom%, 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, and the concentration of allicin was 2 mmol / L. Fermentation lasted for 12 hours.

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

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

[0029] Step Six: Data and Results Analysis; Using Data acquisition and processing were performed using TF 1.7.1 software (AB Sciex, Concord, Canada). All detected ions were extracted into an Excel spreadsheet in 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, which were compared with metabolite databases (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 normalized according to protein content.

[0030] 2. Experimental Results

[0031] The test results are shown in Figure 1Where a is guanylate monophosphate (GMP), b is uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc), c is adenosine 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 indicates no 15 N is a marker, and M+1 indicates that there is one. 15 N-labeled, M+2 / 3 / ... and so on. (Using the conventional urea group) 15 N abundance was used as the background value, and the control group and allicin were analyzed after subtracting this 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), glyconucleotides (UDP-GlcNAc) or amino acids (L-alanine), but has no significant effect on energy compounds ADP and NAD.

Claims

1. The use of allicin in promoting the conversion of urea nitrogen in rumen feed of ruminants into nitrogen in nucleotides, glyconucleotides or amino acids; wherein the ruminants include cattle, sheep or deer; The nucleotide is guanylic acid, uridine monophosphate, or adenosine monophosphate; the glyconucleotide is uridine diphosphate-N-acetylglucosamine; the amino acid is L-alanine; when allicin is fed directly to ruminants or as a feed additive to ruminants, the concentration of allicin in the rumen of ruminants is 1-4 mmol / L.

2. The use according to claim 1, characterized in that, The guanylic acid mentioned is guanosine monophosphate.

3. A method for promoting the conversion of urea nitrogen in the rumen of ruminants into nitrogen in nucleotides, glyconucleotides, or amino acids for non-therapeutic purposes, characterized in that, include: Allicin is fed directly to ruminants or as a feed additive to ruminants; the ruminants include cattle, sheep or deer; The nucleotides are guanylic acid, uridine monophosphate, and adenosine monophosphate; the glyconucleotides are uridine diphosphate-N-acetylglucosamine; the amino acid is L-alanine; and the concentration of allicin in the rumen of ruminants is 1-4 mmol / L.

4. The method according to claim 3, characterized in that, The guanylic acid mentioned is guanosine monophosphate.

5. The method according to claim 3, characterized in that, Methods of feeding ruminants with allicin as a feed additive include: adding allicin as a feed additive to the basal diet of ruminants, and feeding ruminants with a basal diet containing allicin.

6. The method according to claim 3, characterized in that, Methods for feeding allicin as a feed additive to ruminants include: mixing allicin with trace elements and vitamins to prepare a premixed feed for feeding ruminants.

7. The method according to claim 3, characterized in that, Methods of feeding allicin as a feed additive to ruminants include: mixing allicin with protein and energy feed to prepare a concentrate mixture for feeding ruminants.

Images