Urease inhibitor for preventing / treating ammonia poisoning of ruminants

Abstract

The invention relates to application of trimethylglycine and salts thereof as urease inhibitors in preventing / treating ammonia poisoning of ruminants. Trimethylglycine and salts thereof have an efficient inhibition effect on ruminant rumen urease, can be used as a novel urease inhibitor to improve the safety of urea as a non-protein nitrogen feed, and effectively prevent / treat ammonia poisoning caused by too fast urea decomposition and accumulation of ammonia in blood.

Description

Technical Field

[0001] The present invention relates to trimethylglycine and its salts as novel rumen urease inhibitors for ruminants, and is used for preventing / treating ammonia poisoning in ruminants. Background Art

[0002] Rumen microorganisms of ruminants (such as cattle and sheep) can use non-protein nitrogen (NPN) as a nitrogen source to synthesize bacterial protein and be used by animals. The most commonly used NPN in ruminant feeding is urea. Urea has a high nitrogen content of about 46%. The nitrogen content of 1 kg of urea is approximately equivalent to the nitrogen content of 2.8 kg of crude protein, or the amount of crude protein contained in 6.5 kg of soybean meal. Therefore, compared with protein feeds such as soybeans and soybean meal that are more expensive, urea is a cheap nitrogen source feed resource for ruminants (Sun Meijie. Effects of different urea addition levels on rumen fermentation and microbial flora structure of fattening Hu sheep. Journal of Nanjing Agricultural University, 2022, 45 (2): 323-332).

[0003] Ruminants can use urea because the bacteria in the rumen of ruminants can produce urease (also known as urease). Urease is a hydrolase that catalyzes the decomposition of urea into ammonia and carbon dioxide. Rumen microorganisms use the ammonia released by the decomposition of urea to combine with the decomposition products of carbohydrates in the feed to form amino acids, and then synthesize microbial proteins. These bacterial proteins enter the true stomach and small intestine and are digested and absorbed to synthesize proteins in the animal body and its products.

[0004] Urease produced by rumen microorganisms can catalyze the rapid hydrolysis of urea into ammonia. Studies have shown that 1g of urea can be decomposed into ammonia within 1 hour for every 1kg of rumen contents, that is, the rate at which urea decomposes to produce ammonia is 4 times the rate at which rumen microorganisms utilize ammonia. If urea decomposes too quickly, a large amount of ammonia will be produced that cannot be utilized by rumen microorganisms, resulting in reduced urea utilization efficiency. In addition, excessive ammonia is absorbed into the blood by the rumen wall and the posterior gastrointestinal tract and cannot be converted by the liver in time, which can easily cause animal ammonia poisoning, resulting in symptoms such as forced spasms, dyspnea, and foaming at the mouth. In severe cases, it can lead to animal death (Niu Ying. New Explorations to Improve the Utilization of Urea by Cattle and Sheep. Modern Agriculture, 2020, 7: 62). It can be seen that the main limiting factor for ruminants to utilize urea is the rapid degradation of urea into ammonia by urease in the rumen, resulting in ammonia poisoning. Therefore, the prevention / treatment of ammonia poisoning in ruminants is an important topic in current ruminant feeding research, among which urease inhibitors are a technology with good application prospects.

[0005] Currently, there are more than 100 urease inhibitors that have been patented, which are mainly divided into three categories: metal salts, organic small molecules and plant extracts. At present, only a few urease inhibitors such as acetohydroxamic acid, n-butylthiophosphoric acid triamide (NBPT), n-propylthiophosphoric acid triamide (NPPT) and hydroquinone (HQ) have been put into practical use. However, the existing urease inhibitors still have many problems in use, such as low biological activity, low cost performance and biosafety, and have not been widely used in the prevention / treatment of ammonia poisoning in ruminants.

[0006] Trimethylglycine, also known as betaine and glycine betaine, is a quaternary ammonium alkaloid. Its pure form is prism-shaped or leaf-shaped white crystals, soluble in water, methanol and ethanol. As a natural plant ingredient, trimethylglycine has the advantages of being safe, green, non-toxic and pollution-free. It has been synthesized in large quantities and is inexpensive.

[0007] This study discovered for the first time that trimethylglycine and its salts have significant rumen urease inhibitory activity, and have the advantages of high cost-effectiveness and good biosafety. They can be used as new rumen urease inhibitors for ruminants and applied in the prevention / treatment of ammonia poisoning in ruminants, thereby improving the safety of urea as a non-protein nitrogen feed. Summary of the invention

[0008] The purpose of the present invention is to provide a novel rumen urease inhibitor trimethylglycine and its salts for use in the prevention / treatment of ammonia poisoning in ruminants. The structural formula of trimethylglycine is: .

[0009] The urease inhibitor also includes acceptable salts, including hydrochloride, citrate, malate, phosphate, maleate, fumarate, sulfonate or methanesulfonate.

[0010] A urease inhibitor for preventing / treating ammonia poisoning in ruminants, wherein the ruminants include various types of ruminants such as cattle, sheep, deer, and camels, and the ammonia poisoning in ruminants refers to the poisoning symptoms caused by excessive ammonia concentration in the blood due to improper consumption of urea by the ruminants. The urease inhibitor comprises trimethylglycine or an acceptable salt thereof, and the usage ratio of the urease inhibitor is 0.1 wt % to 30 wt % of the quality of the urea fed; more preferably 1 wt % to 10 wt %.

[0011] The present invention comprises the use of the urease inhibitor in preparing various types of products for preventing / treating ammonia poisoning in ruminants.

[0012] 1. Preparation of rumen urease solution The rumen contents of Yiling cattle were collected through rumen fistula, mixed evenly, and centrifuged at 4°C, 26,000 g for 15 min. The rumen bacterial pellet was collected and stored in a -80°C refrigerator. Before the experiment, 20 g of rumen bacterial pellet was resuspended in HEPES buffer (pH 7.5, 50 mmol / L) and then crushed with a high-pressure cell disruptor at low temperature and high pressure (22,000 psi (cell pressure)). After centrifugation at 4°C, 12,000 g for 10 min, the supernatant was the rumen urease solution.

[0013] 2. Determination of rumen urease inhibition activity The basic principle of urease inhibitor activity test is to determine the activity of urease inhibitor by detecting the change in the ability of urease to catalyze the decomposition of urea and release ammonia after the urease inhibitor and urease interact. The most commonly used test method is the indophenol method. First, the inhibitor is allowed to react with urease for a period of time, and then it is allowed to catalyze the decomposition of urea. The ammonia produced is dissolved in the buffer solution and treated with indophenol for color development. The OD value is then tested with a spectrophotometer. Finally, the inhibition rate of the inhibitor on urease is calculated. The calculation formula is as follows: ; Blank: No inhibitor and urea are added to the above test solutions; Control: No inhibitor was added to the above test solutions.

[0014] The activity test of rumen urease was carried out according to the method reported by Weatherburn. The specific steps are as follows: add 25 μL of urease solution and 25 μL of test compound (the concentration is set according to the experiment and prepared with DMSO-phosphate buffer solution) to a 96-well plate, co-culture at 37°C for 30 minutes, add 50 μL of phosphate buffer solution containing 25 mmol of urea, co-culture at 37°C for 30 minutes, then add 50 μL of reagent A (127 mM phenol and 0.168 mM sodium nitroprusside) and 50 μL of reagent B (125 mM NaOH and 11.3 mM NaOCl), co-culture at 37°C for 30 minutes, and measure the absorbance value at 620 nm. A blank group and a normal control group were set up separately, and 3 replicates were set up for each group, from which the inhibition rate and half inhibition concentration IC of the inhibitor were calculated. 50 .

[0015] 3. Ruminant feeding experiment A number of ruminants with similar body weights were selected and three treatment groups were set up in the experiment: (1) normal diet; (2) diet + urea (added at 1% to 3% of the diet); (3) diet + urea (added at 1% to 3% of the diet) + rumen urease inhibitor (trimethylglycine or its salts). Urease inhibitors can be used alone or mixed with urea in feeding, and the usage ratio is 1% to 20% of the quality of urea used. The experimental period is 4 weeks. The animals were observed daily to check whether they had symptoms of ammonia poisoning, and their weight was weighed to calculate the average daily weight gain.

[0016] This study found that trimethylglycine and its salts have significant rumen urease inhibitory activity, with a half inhibitory concentration IC 50 The concentration of 28.5 µM to 36.3 µM can be used as a new type of ruminant urease inhibitor, applied to the prevention / treatment of ammonia poisoning in ruminants, and improve the safety and economic benefits of ruminant farming.

[0017] The symptoms of ammonia poisoning described in the present invention (i.e., when the peripheral blood exceeds about 1 mg ammonia / 100 ml blood) are accompanied by symptoms including muscle contraction, ataxia, excessive salivation, swelling, respiratory failure, etc. DETAILED DESCRIPTION

[0018] Example 1 Add 25 μL of rumen urease solution and 25 μL of trimethylglycine solution (1 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 80 μM, 100 μM, prepared with DMSO-phosphate buffer solution) to a 96-well plate, co-culture at 37°C for 30 min, add 50 μL of phosphate buffer solution containing 25 mmol of urea, co-culture at 37°C for 30 min, then add 50 μL of reagent A (127 mM phenol and 0.168 mM sodium nitroprusside) and 50 μL of reagent B (125 mM NaOH and 11.3 mM NaOCl), co-culture at 37°C for 30 min, and measure the absorbance at 620 nm. Set up a blank group and a normal control group, and calculate the half-maximum inhibitory concentration IC of trimethylglycine on rumen urease 50 is 31.7 µM.

[0019] Example 2 Add 25 μL of rumen urease solution and 25 μL of trimethylglycine hydrochloride solution (1 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 80 μM, 100 μM, prepared with DMSO-phosphate buffer solution) to a 96-well plate, co-culture at 37°C for 30 min, add 50 μL of phosphate buffer solution containing 25 mmol of urea, co-culture at 37°C for 30 min, then add 50 μL of reagent A (127 mM phenol and 0.168 mM sodium nitroprusside) and 50 μL of reagent B (125 mM NaOH and 11.3 mM NaOCl), co-culture at 37°C for 30 min, and measure the absorbance at 620 nm. Set up a blank group and a normal control group, and calculate the half-maximal inhibitory concentration IC of trimethylglycine hydrochloride on rumen urease 50 is 28.5 µM.

[0020] Example 3 Add 25 μL of rumen urease solution and 25 μL of trimethylglycine citrate solution (1 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 80 μM, 100 μM, prepared with DMSO-phosphate buffer solution) to a 96-well plate, co-culture at 37°C for 30 min, add 50 μL of phosphate buffer solution containing 25 mmol of urea, co-culture at 37°C for 30 min, then add 50 μL of reagent A (127 mM phenol and 0.168 mM sodium nitroprusside) and 50 μL of reagent B (125 mM NaOH and 11.3 mM NaOCl), co-culture at 37°C for 30 min, and measure the absorbance at 620 nm. Set up a blank group and a normal control group, and calculate the half-maximum inhibitory concentration IC of trimethylglycine citrate on rumen urease 50 is 33.8 µM.

[0021] Example 4 Add 25 μL of rumen urease solution and 25 μL of trimethylglycine malate solution (1 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 80 μM, 100 μM, prepared with DMSO-phosphate buffer solution) to a 96-well plate, co-culture at 37°C for 30 min, add 50 μL of phosphate buffer solution containing 25 mmol of urea, co-culture at 37°C for 30 min, then add 50 μL of reagent A (127 mM phenol and 0.168 mM sodium nitroprusside) and 50 μL of reagent B (125 mM NaOH and 11.3 mM NaOCl), co-culture at 37°C for 30 min, and measure the absorbance at 620 nm. Set up a blank group and a normal control group, and calculate the half-maximum inhibitory concentration IC of trimethylglycine malate on rumen urease 50 is 36.3 µM.

[0022] Example 5 Yiling cattle of similar weight were selected and three treatment groups (7 in each group) were set up in the experiment: (1) normal diet (corn starch, the same below); (2) diet + urea (added at 1% of the diet); (3) diet + urea (added at 1% of the diet) + trimethylglycine, with the proportion of trimethylglycine being 1% of the urea quality. The experimental period was 4 weeks, and the animals were observed daily for symptoms of ammonia poisoning and weighed. No ammonia poisoning symptoms appeared in any group during the entire experimental period. The average daily weight gain of group 1 was 820±73g, the average daily weight gain of group 2 was 1015±106g, and the average daily weight gain of group 3 was 1272±68g.

[0023] Example 6 Yiling yellow cattle with similar weight were selected, and three treatment groups (7 heads in each group) were set up in the experiment: (1) normal feeding diet (the same group as Group 1 in Example 5); (2) feeding diet + urea (added at 1.5% of the diet); (3) feeding diet + urea (added at 1.5% of the diet) + trimethylglycine hydrochloride, and the use ratio of trimethylglycine hydrochloride was 2% of the urea quality. The experimental period was 4 weeks, and the animals were observed daily for symptoms of ammonia poisoning and weighed. During the entire experimental period, one cow in Group 2 showed symptoms of ammonia poisoning in the third week. After timely feeding of trimethylglycine hydrochloride, the symptoms were relieved and the experiment was removed. No other groups showed symptoms of ammonia poisoning. The average daily weight gain of Group 1 was 820±73g, the average daily weight gain of Group 2 was 1076±96g, and the average daily weight gain of Group 3 was 1429±72g.

[0024] Example 7 Yiling yellow cattle with similar body weight were selected, and three treatment groups (7 in each group) were set up in the experiment: (1) normal feeding diet (the same group as Group 1 in Example 5); (2) feeding diet + urea (added amount was 2% of the diet); (3) feeding diet + urea (added amount was 2% of the diet) + trimethylglycine, and the addition ratio of trimethylglycine was 3% of the urea quality. The experimental period was 4 weeks, and the animals were observed daily for symptoms of ammonia poisoning and weighed. During the entire experimental period, three cattle in Group 2 showed symptoms of ammonia poisoning in the second week. After timely feeding of trimethylglycine, the symptoms were relieved and the experiment of Group 2 was stopped. No symptoms of ammonia poisoning appeared in the other groups. The average daily weight gain of Group 1 was 820±73g, and the average daily weight gain of Group 3 was 1482±95g.

[0025] Example 8 Yiling yellow cattle with similar body weight were selected, and three treatment groups (7 in each group) were set up in the experiment: (1) normal feeding diet (the same group as Group 1 in Example 5); (2) feeding diet + urea (added at 2.5% of the diet); (3) feeding diet + urea (added at 2.5% of the diet) + trimethylglycine citrate, and the addition ratio of trimethylglycine citrate was 5% of the urea quality. The experimental period was 4 weeks, and the animals were observed daily for symptoms of ammonia poisoning and weighed. During the entire experimental period, 5 cattle in Group 2 showed symptoms of ammonia poisoning in the first week. After timely feeding of trimethylglycine citrate, the symptoms were relieved and the experiment of Group 2 was stopped. No symptoms of ammonia poisoning appeared in the other groups. The average daily weight gain of Group 1 was 820±73g, and the average daily weight gain of Group 3 was 1506±83g.

[0026] Example 9 White goats of similar weight were selected and three treatment groups (9 in each group) were set up in the experiment: (1) normal diet; (2) diet + urea (added at 1.5% of the diet); (3) diet + urea (added at 1.5% of the diet) + trimethylglycine, with trimethylglycine used at a ratio of 2% of the urea quality. The experimental period was 4 weeks, and the animals were observed daily for symptoms of ammonia poisoning and their body weights were weighed. During the entire experimental period, one goat in Group 2 showed symptoms of ammonia poisoning in the fourth week. After timely feeding of trimethylglycine, the symptoms were relieved and the goat was removed from the experiment. No other groups showed symptoms of ammonia poisoning. The average daily weight gain of Group 1 was 197±45g, the average daily weight gain of Group 2 was 245±67g, and the average daily weight gain of Group 3 was 288±59g.

[0027] Example 10 White goats with similar body weight were selected, and three treatment groups (9 in each group) were set up in the experiment: (1) normal diet (the same group as Group 1 in Example 9); (2) diet + urea (added at 2% of the diet); (3) diet + urea (added at 2% of the diet) + trimethylglycine hydrochloride, with the addition ratio of trimethylglycine hydrochloride being 3% of the urea quality. During the 4-week experiment, the animals were observed daily for symptoms of ammonia poisoning and their body weights were weighed. During the entire experimental period, three goats in Group 2 showed symptoms of ammonia poisoning in the third week. After timely feeding of trimethylglycine hydrochloride, the symptoms were relieved and the experiment was removed. No other groups showed symptoms of ammonia poisoning. The average daily weight gain of Group 1 was 197±45g, the average daily weight gain of Group 2 was 268±53g, and the average daily weight gain of Group 3 was 321±46g.

Claims

1. A ruminant rumen urease inhibitor, characterized in that: The urease inhibitor is trimethylglycine, and the structural formula of trimethylglycine is: 。 2. A urease inhibitor for preventing / treating ammonia poisoning in ruminants, characterized in that: The urease inhibitor is trimethylglycine, and the structural formula of trimethylglycine is: 。 3. The urease inhibitor according to any one of claims 1 to 2, characterized in that The inhibitor includes acceptable salts, including any of hydrochloride, citrate, malate, phosphate, maleate, fumarate, sulfonate or methanesulfonate.

4. The urease inhibitor according to claim 3, characterized in that The inhibitor includes acceptable hydrates.

5. The urease inhibitor for preventing / treating ammonia poisoning in ruminants according to claim 2, characterized in that: The ruminants include ruminants such as cattle, sheep, deer, and camels.

6. The urease inhibitor for preventing / treating ammonia poisoning in ruminants according to claim 2, characterized in that: The ruminant ammonia poisoning refers to the symptoms of ammonia poisoning caused by excessive ammonia concentration in the blood of ruminants due to excessive or accumulated urea.

7. A urease inhibitor composition for preventing / treating ammonia poisoning in ruminants, characterized in that: The invention comprises the urease inhibitor according to any one of claims 1 to 6, urea and corn starch.

8. The urease inhibitor composition for preventing / treating ammonia poisoning in ruminants according to claim 7, characterized in that: The urease inhibitor is used in an amount of 0.1 wt % to 30 wt % of the quality of the urea fed.

9. The urease inhibitor composition for preventing / treating ammonia poisoning in ruminants according to claim 7, characterized in that: The urea usage ratio is 0.1wt% to 5wt% of the mass of the fed diet.

10. Use of the urease inhibitor according to any one of claims 1 to 6 in the preparation of a product for preventing / treating ammonia poisoning in ruminants, or use of the composition according to any one of claims 7 to 9 in the preparation of a product for preventing / treating ammonia poisoning in ruminants.

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