CN108815151A - Purposes of the lauroyl arginine ethyl ester as feed addictive - Google Patents

Abstract

The present invention relates to a novel feed additive, which is an antibacterial agent containing a condensate derived from fatty acid and esterified dibasic amino acid, capable of preventing and treating Riemerella anatipestifer in ducklings caused by Riemerella anatipestifer It can treat peritonitis caused by Escherichia coli and Staphylococcus aureus, and prevent fish sepsis caused by Aeromonas temperatus. The feed additive can effectively inhibit or kill bacteria, can greatly reduce the risk of poultry, livestock and aquatic animals infected with bacteria, and increase the survival rate of animals infected with pathogenic bacteria. Ethyl lauroyl arginate can be safely degraded in the human body and animals, with little toxic and side effects, avoiding the emergence of environmental drug-resistant bacteria caused by the discharge of residual antibacterial agents into the environment, and achieving effective antibacterial effects while harming the environment The negative impact is small.

Description

Use of ethyl lauroyl arginate as a feed additive

This application requires Chinese invention patent application 201711071008.7, the invention name is "Ethyl Lauroyl Arginate and its derivatives as feed additives” priority application.

technical field

The invention relates to the application of the compound lauroyl arginine ethyl ester in the preparation of veterinary antibacterial agents, feed additives or feed nutrition energy substances.

Background technique

Riemerella anatipestifer is a contagious disease caused by Riemerella anatipestifer (RA), also known as duck infectious serositis, duck septicemia, duck plague syndrome, and duck plague. bacillosis etc. It is more common in ducklings aged 1-8 weeks, and ducklings aged 2-4 weeks are most susceptible. It is acute or chronic sepsis, clinically manifested as increased eye and nasal secretions, panting, coughing, diarrhea, ataxia, and head and neck tremors, and a small number of chronic cases have symptoms such as head and neck skewing. The lesion is characterized by fibrinous pericarditis, perihepatitis, air sac inflammation, meningitis and arthritis in some cases, which often cause a large number of morbidity and death of ducklings. The incidence rate of the disease can reach more than 90%, and the mortality rate is related to the age of the diseased duck, the virulence of the strain, and adverse stress factors, up to 75%. In addition to causing the death of ducks aged 1 to 8 weeks, it can also cause salpingitis, resulting in a decline in egg production rate and growth retardation in adult ducks, causing great economic losses to farmers. Since it was first reported in 1982, the disease has become one of the most common bacterial diseases affecting the duck farming industry. Under natural conditions, the disease occurs throughout the year. The disease is mainly caused by entering poultry through contaminated feed, drinking water, dust, droplets, etc. through the respiratory tract, digestive tract or skin wounds (especially the skin of the webs). Different breeds of ducks such as Peking duck, Cherry Valley duck, Di Gao duck, teal duck, Muscovy duck, Muscovy duck, and Shelduck duck can be infected. From July to August 2012, random checks were conducted on 48 duck farms in Jiangsu Province. Among them, 45 duck farms had suspected cases of Riemerella anatipestifer, and 12 duck farms even went sour during the spot check. There were two outbreaks of the disease. In addition, if the disease occurs in a certain duck farm, the disease will also break out in the surrounding duck farms, which shows the seriousness of the damage caused by the disease to the duck industry.

Staphylococcus aureus disease is a general term for different diseases or disease types caused by Staphylococcus aureus. Pathogenic Staphylococcus aureus can cause diseases in various poultry and livestock. Young livestock and poultry are most susceptible to this disease, and they are mostly infected through the digestive tract. Chickens can also be infected through the respiratory tract. Common symptoms include diarrhea, enteritis, and liver necrosis. It often causes two types of diseases, one is suppurative disease, which mainly causes mastitis, arthritis, trauma infection and sepsis in animals, etc.; the other is toxic disease, and feed contaminated by pathogenic bacteria can cause poisoning in animals. Enteritis and human toxic shock syndrome. The pathogenicity of Staphylococcus aureus mainly depends on the virulence pathogenic factors it produces, mainly including plasma coagulase, enterotoxin, thermostable nuclease, hemolytic toxin and leukocidin.

Colibacillosis is a general term for different diseases or disease types of various animals caused by pathogenic Escherichia coli. There is no difference between pathogenic Escherichia coli and non-pathogenic Escherichia coli normally inhabiting human and animal intestines in terms of morphology, staining reaction, culture characteristics and biochemical reactions, but the antigenic structure is different. Pathogenic Escherichia coli can cause various poultry and livestock diseases, such as pigs, cattle, sheep, horses, chickens, rabbits, etc. Young livestock and poultry are most susceptible to this disease, and they are mostly infected through the digestive tract, and chickens can also be infected through the respiratory tract. Among them, porcine colibacillosis, depending on the growth period of piglets and the serotype of the pathogen, has different clinical manifestations in piglets, and can be divided into yellow dysentery, pullorum and edema. When yellow scour occurs in piglets, it often affects more than 90% of a litter of piglets, and the mortality rate is high, and some can reach 100%; the incidence of pullorum is 30% to 80%; the incidence of edema is 10% to 35%. Colibacillosis in chicks usually manifests as acute septicemia, yolk peritonitis, ophthalmia, air sacculitis and swollen head syndrome, etc. The morbidity rate can reach 30%～60%, and the case fatality rate can reach 100%.

Aeromonas sobria is a Gram-negative, facultative anaerobic bacterium that often exists in various water environments and soil environments, and is a zoonotic pathogen. Aeromonas sobria can produce a variety of pathogenic factors such as lysosin and extracellular enzymes, which can cause sepsis in aquatic animals, thereby causing the death of aquatic animals and seriously affecting the economic basis of aquaculture. In addition, pathogenic bacteria may also infect people through aquatic products, and patients will develop symptoms such as diarrhea and even develop food poisoning or sepsis.

In addition to improving feeding conditions, applying antibiotics is the main measure to prevent and treat aquatic bacterial diseases in livestock and poultry. However, in recent years, commonly used antibiotics such as aureomycin, oxytetracycline, tetracycline, and chloramphenicol have been widely used in livestock and poultry farming because of their disease resistance and growth-promoting effects. According to statistics, my country's annual production of antibiotic raw materials is about 210,000 tons, of which 97,000 tons of antibiotics are used in livestock and poultry breeding, accounting for 46.1% of the total production. Inappropriate use of antibiotics, insecure drug quality, incomplete supervision, and unscrupulous medication regulations have led to increased abuse of antibiotics, resulting in many serious problems such as the emergence of bacterial resistance, decreased immune function of animals, Drug residues in meat products, etc., directly endanger human health. According to a study, about 75% of antibiotics cannot be absorbed and metabolized by humans or animals, and some of them will remain in the body. 20%-50% of live or frozen chicken tissues can detect antibiotic residues; residual antibiotics can also be As excrement enters the environment, some will directly enter the river and affect the drinking water safety of downstream residents. Oxytetracycline, tetracycline, doxycycline, amoxicillin, and gold have been detected in the tap water of residents in many riverside cities in my country. Residues of antibiotics in the livestock and poultry industry such as toxin. In recent years, a study conducted antibiotic testing on the urine of 1,000 children in Shanghai, and 58% of the urine samples detected a variety of veterinary antibiotics (such as tylosin, chlortetracycline, and enrofloxacin, etc.) that are only used in the breeding industry. ). More importantly, these residual antibiotics will naturally select or induce genetic mutations in microorganisms in the environment, and the resistant bacteria will survive and continue to reproduce more resistant bacteria. In addition, bacteria can also transfer their drug resistance genes to other microorganisms of different species and genera through conjugation, transformation, transduction, transposition, etc. to acquire drug resistance. This leads to the enrichment of drug-resistant bacteria in the environment. Once the contaminated soil and water are contacted by people or livestock, it is very easy to cause diseases and accelerate the spread of drug-resistant bacteria. In addition to illegal addition of antibiotics or antibacterial agents, veterinary drugs and feed additives mainly include preservatives, anti-dust agents, antioxidants, anti-protozoal drugs, etc. These substances will cause harm to livestock and poultry and human health due to long-term use or improper use.

Therefore, in the breeding of aquatic products, animal husbandry, and poultry, especially in the breeding of poultry (such as chickens and ducks), there is an urgent need for a kind of antibacterial agent that can quickly sterilize, and is easily degradable in the body and has no residue. It can prevent the occurrence and spread of diseases in China, and can also avoid the impact of similar antibiotic residues on the human body and the environment.

Ethyl lauroyl arginate (LAE) is an organic compound formed by the condensation of fatty acids and dibasic amino acids. It is a white hygroscopic solid with stable chemical properties in the range of pH 3-7 and a melting point of 50-58°C. At this temperature, 247g of LAE can be dispersed in 1kg of water, and its distribution coefficient in water and oil is greater than 10, that is, it mainly exists in the water phase. Studies have found that lauroyl arginine ethyl ester LAE has the characteristics of strong antibacterial ability, low biological toxicity, good metabolism in vivo, and high compatibility with the environment. The most representative feature is that there is no residue in the metabolism of ethyl lauroyl arginine. Relevant studies have shown that ethyl lauroyl arginine can quickly undergo natural metabolism in humans and animals to generate lauric acid and arginine. Metabolized to ornithine, urea, carbon dioxide and water. All the primary metabolites and final products produced during the metabolism of ethyl lauroyl arginate are non-toxic and harmless, and are the same as the metabolites in the body of the daily intake of humans and animals.

For example, Chinese patent application CN201710056593, titled "A Fruit and Vegetable Antiseptic Preservative and Its Preparation Method and Application", discloses a combination of ethyl lauroyl arginate hydrochloride and sodium methylparaben as the main active ingredients. As a preservative for fruits and vegetables, it can effectively inhibit the growth of bacteria that cause fruits and vegetables to rot. However, the independent bacteriostatic effect of high-concentration sodium methylparaben (2000 μg/ml) is stronger than low-concentration LAE (1000 μg/ml) in this invention, and this is because it has a phenolic hydroxyl structure, and its antibacterial performance is far stronger than Benzoic acid, sorbic acid, so under the premise of ensuring antiseptic performance, this method clearly points out that using sodium methylparaben instead of LAE helps to reduce the dosage cost of preservatives.

Chinese patent application CN201510748675, titled "Method for Inhibiting Alcohol Fermentation-Contaminating Microorganisms Using Lauroyl Arginine Ethyl Ester", discloses a method for inhibiting alcohol fermentation-contaminating microorganisms using lauroyl arginine ethyl ester. The method includes LAE and its The salt compound, added to the fermentation broth of Saccharomyces cerevisiae at a concentration lower than 50 μg/ml, can effectively inhibit the growth of lactic acid bacteria and control the growth of other polluting microorganisms. However, the bacteriostatic agent slightly affected yeast growth to some extent and resulted in a 0.6% reduction in alcohol production.

Chinese patent application CN201610466729, the title of the invention is "a mild baby shampoo and bath bubble", which discloses a mild baby shampoo and bath bubble, which uses cocoyl glutamic acid two Sodium, cocamidopropyl betaine and sodium hydroxypropyl sulfonate lauryl glucoside cross-linked polymer compound are used as the surfactant system, and camellia seed oil, α-glucan oligosaccharide/inulin compound is selected As a conditioning component, wild chrysanthemum extract and ethyl lauroyl arginate HCl are compounded as a preservative system. In this invention, each raw material cooperates with each other, and the cleaning effect is good, mild and non-irritating.

Chinese patent application CN201280073013, titled "Synergistic Antimicrobial Agent", discloses synergistic antimicrobial agents by combining an effective amount of N-α-long-chain alkanoyl dibasic amino acid alkyl ester salt with glycerol monofatty acid ester Compositions resulting in more effective antimicrobials and food preservatives. At the same time, Chinese patent application CN200810131638, titled "microbicide composition", discloses the use of a composition of methylisothiazolinone and LAE for the preparation of antimicrobial agents and food preservatives. However, this method involves multiple antibacterial components including LAE, and the antibacterial effect of LAE alone was not studied. At the same time, the invention only teaches that the composition is used for daily products, cleaning agents, wound care compositions, various foods, various medical cleaning products, etc., and does not teach how to use a single LAE component for poultry Use of antibacterial feed for aquatic products.

Chinese patent application CN201280027864, titled "Cosmetic sunscreen preparation or dermatology sunscreen preparation with improved water resistance" discloses the use of LAE for the preparation of cosmetic sunscreen preparations or dermatology sunscreen preparations. Also includes the emulsifier polyglyceryl‐10 stearate.

As the closest prior art, the Chinese patent application CN200580051259, titled "Anti-corrosion System Including Cationic Surfactant", discloses for the first time the use of LAE and its hydrochloride in an anti-corrosion system, adding 0.2 This system of g/kg LAE thus acts as a preservative. This invention studies the antibacterial mechanism of LAE, and proposes how to use LAE for antiseptic applications such as food and cosmetics. Therefore, the U.S. Food Safety Administration approved ethyl lauroyl arginate for food preservatives in 2005; in 2012 The EU Food Safety Authority, Australia and New Zealand have also approved ethyl lauroyl arginate as a food preservative. At the same time, in view of the fact that the invention first proposed the application in cosmetics, it was found in follow-up studies that ethyl lauroyl arginate can be used in oral care products (such as US20100330136A1, EP2361606A2, EP231603A2), such as mouthwash, toothpaste, etc., which can effectively inhibit The formation of dental plaque in the oral cavity, it is compatible with other chemical ingredients in mouthwash and is chemically stable; ethyl lauroyl arginate can be used in cosmetics with topical therapeutic effects, these cosmetics have the following properties: antibacterial effect, low Toxic, no sensitization, no irritation to the skin. Currently, researchers are developing cleansing hand sanitizers and antibacterial agents for use on skin surfaces.

On the other hand, studies have found that LAE is rapidly degraded in vivo, the laurylamide bond or ester bond in LAE is broken, and the lauric acid part or ethanol part is removed to form arginine ethanol ester or laurylamide arginine, which are then removed respectively. Remove the ethanol moiety or the lauric acid moiety, forming the same intermediate L‐arginine. The lauric acid produced by this metabolic process is also the main fatty acid component of edible oils such as palm oil, mountain pepper oil, and coconut oil. Arginine is one of the 20 kinds of amino acids that make up protein. It exists in foods such as nuts, cheese and fish. According to the Ministry of Agriculture of the People's Republic of China Announcement No. 2045 - Feed Additives Category Catalog (2013), L-arginine It belongs to the category of amino acids, amino acid salts and their analogues, and is a feed additive that provides energy. In addition, L-arginine can be further hydrolyzed into ornithine and urea, wherein ornithine can synthesize organic matter through the urea cycle and citric acid (trihydroxy acid) cycle, and finally decompose into urea and carbon dioxide to be excreted. That is, the metabolic intermediates or final products produced during the metabolic process of LAE in animals are not only safe and non-toxic, but also produce a large amount of energy substances. Therefore, the application of LAE in feed supplementation can exert its antibacterial and metabolic energy functions at the same time, which is beneficial to the health and nutritional growth of animals.

Although LAE and its conventional derivatives have broad application prospects, their core patents are held in developed countries such as Europe and the United States. According to the needs of biomedicine and its related applications in the "Development Plan", there is an urgent need to study new uses of LAE, new derivatives and applications, so as to realize the overtaking of my country's biotechnology on European and American countries, so as to serve the great goal of a technological power.

Contents of the invention

In summary, the existing invention does not teach how to use a single lauroyl arginine ethyl ester (LAE) and its derivatives or its hydrate components as antibacterial drugs or feed additives for poultry and aquatic products. The appropriate concentration of ethyl lauroyl arginate and its derivatives or hydrates as antibacterial drugs or feed additives is not disclosed. Therefore, the present invention proposes for the first time that ethyl lauroyl arginate and derivatives thereof or hydrates thereof (preferably LAE) are used in the research of antibacterial drugs for poultry, livestock and aquatic products, and determine its suitable concentration for use by experiments to While achieving effective antibacterial and disease prevention effects, it has little negative impact on the environment, low toxic and side effects, and alleviates the consequences of severe bacterial resistance caused by the abuse of antibiotics in the aquaculture industry today. On the other hand, on the basis of the above research, the LAE component is used as a feed additive for the production of livestock and aquatic products. Finally, in the case of ensuring the bacteriostatic effect of LAE, the influence of LAE as an energy substance in different bacteriostatic feeds on the vegetative growth of poultry and aquatic animals was analyzed respectively, so as to determine the appropriate combination of antibacterial and disease prevention and energy nutrition. Matching.

The first object of the present invention is to provide the application of ethyl lauroyl arginate or its hydrate or pharmaceutically acceptable salt as shown in formula (I) in the preparation of feed additives for poultry, livestock and aquatic animals, wherein the The compound represented by the formula (I) is 0.01% to 1.0% according to the total weight of the feed,

Wherein, X is halogen or HSO ₄ ;

R ₁ is a straight-chain saturated fatty acid group containing 8-14 carbon atoms, or a straight-chain oxyacid group containing 8-14 carbon atoms.

R ₂ is a straight-chain fatty acid group containing 1-18 carbon atoms, or a branched-chain fatty acid group containing 1-18 carbon atoms, or an aromatic group containing 1-18 carbon atoms, or containing 1-4 A straight-chain alkyl group of carbon atoms.

R ₃ is one of the following structures:

The range of n is 0‐4.

Wherein, the compound (lauroyl arginine ethyl ester and derivatives thereof) represented by the formula (I) is an antibacterial agent comprising a condensate derived from a fatty acid and an esterified dibasic amino acid, and can prevent and treat diseases caused by duck plague. Riemerella anatipes in ducklings caused by Riemerella, peritonitis caused by Escherichia coli and Staphylococcus aureus, and fish sepsis caused by Aeromonas sobria; preferably used for the prevention and treatment of duck plague Riemerella anatipes in ducklings caused by Riemerella is used for the treatment of peritonitis caused by Escherichia coli and Staphylococcus aureus, and for the prevention and treatment of fish sepsis caused by Aeromonas temperatus. The feed additive can effectively inhibit or kill bacteria, can greatly reduce the risk of poultry, livestock and aquatic animals infected with bacteria, and increase the survival rate of animals infected with pathogenic bacteria.

In one embodiment, the X is Cl, and the compound shown in the formula (I) is lauroyl arginine ethyl ester hydrochloride, and its structural formula is shown in the following formula (II):

In one embodiment, the poultry and aquatic animals include, but are not limited to: chickens, ducks, geese, turkeys, quails, pigeons, pigs, cattle, sheep, horses, camels, cats, dogs, and fish, shrimps and crabs. In a specific embodiment, the poultry and aquatic animals are selected from ducks, and the pathogenic microorganisms are selected from pathogenic Riemerella anatipestifer, pathogenic Escherichia coli, and pathogenic Staphylococcus aureus. In another specific embodiment, the poultry and aquatic animals are selected from tilapia, and the pathogenic microorganisms are selected from pathogenic Aeromonas temperatus.

In any of the above embodiments, the active ingredient of the compound medicine represented by the formula (I) or (II) is 0.1-1.0%, preferably 1.0%, based on the total weight of the feed, and the poultry and aquatic animals are selected from Duck, the pathogenic microorganism is selected from pathogenic Riemerella anatipestifer.

In any of the above embodiments, the active ingredient of the compound medicine represented by the formula (I) or (II) is 0.1-1.0%, preferably 1.0%, based on the total weight of the feed, and the poultry and aquatic animals are selected from For tilapia, the pathogenic microorganisms are selected from pathogenic Aeromonas temperatus.

In any of the above embodiments, the use is to directly add this product to livestock and poultry aquatic feed; or mix this product with a carrier to make a premix; or mix it with other feed additives or feed ingredients to make a premix Feeding livestock, poultry and aquatic animals in the form of feed and concentrated feed.

In a preferred embodiment, the compound represented by the formula (I) or (II) can kill pathogenic microorganisms within 30 minutes without causing the emergence of drug-resistant bacteria. In another preferred embodiment, the compound represented by the formula (I) or (II) can stimulate pathogenic microorganisms for 30 consecutive days without triggering the emergence of drug-resistant bacteria. In a more preferred embodiment, the compound represented by the formula (I) or (II) has low toxicity to normal mammalian cells and does not cause red blood cell hemolysis at the minimum bactericidal concentration.

In another specific embodiment, the compound represented by the formula (I) or (II) has no effect on the survival rate of healthy ducklings, has no effect on the weight gain of healthy ducklings, has no toxicity to the organs of healthy ducklings, and It can reduce the level of inflammatory factor IL-1β and/or IL-1β protein in animals that rises due to bacterial infection.

The present invention also provides the application of the compound represented by the formula (I) or (II) in the preparation of a drug for reducing the level of inflammatory factor IL-1β and/or IL-1β protein in an animal body increased due to bacterial infection.

The beneficial effects of the present invention are:

1. The compound represented by formula (I) or (II) of the present invention can completely eliminate Escherichia coli, Staphylococcus aureus and Riemerella anatipestifer within 30 minutes.

2. Continuous administration of the compound represented by formula (I) or (II) for 30 days does not induce drug-resistant mutations of pathogenic microorganisms.

3. The compound represented by formula (I) or (II) of the present invention does not cause red blood cell hemolysis at the minimum bactericidal concentration.

4. The compound represented by formula (I) or (II) of the present invention can improve the survival rate of ducklings infected with Riemerella anatipestifer, reduce the risk of duckling infection with Riemerella anatipestifer, and have a positive effect on healthy ducklings. Duck survival was unaffected.

5. The compound represented by the formula (I) or (II) of the present invention not only does not affect the weight gain of ducklings, but can be used as an energy substance to promote the growth of ducklings.

6. The compound represented by the formula (I) or (II) of the present invention can restore the increase of protein levels of inflammatory factors IL-1β and TNF-α caused by bacterial infection in animals.

7. Experiments have shown that the compound shown in formula (I) or (II) of the present invention is administered orally to animals continuously for 3 months, and the body weight gain of the administration group has a significant increase relative to the control group, and the administration group is relatively There was no significant toxicity in important organs in the control group.

8. Experiments show that drug residues in hearts, livers, spleens, lungs, kidneys, small intestines, stomachs, and muscle tissues of animals in the drug-administered group meet EU requirements for unregulated veterinary drug residues after 3 months of drug withdrawal.

9. Experiments have proved that the compound represented by the formula (I) or (II) can promote the growth of poultry, especially the growth of duck, and the daily weight gain can be increased by 11.5%.

10. Experiments have proved that the compound represented by the formula (I) or (II) can not only prevent and treat bacterial diseases of aquatic animals, but also promote the vegetative growth of poultry aquatic animals, wherein: the results of anti-infection experiments show that edible The test fish of the compound feed shown in formula (I) or (II) all died, and the tilapia survival rate that edible contains the compound shown in formula (I) can reach 88.33%; For tilapia, can improve relative weight gain The feed rate was 3.03%, 6.23%, 3.88%, and the specific growth rate was 12.32%, and the feed coefficient was reduced by 3.15%.

11. Ethyl lauroyl arginate can be safely degraded in the human body and animals, with little toxic and side effects, and avoids the generation of environmental drug-resistant bacteria caused by the discharge of residual antibacterial agents into the environment, while achieving effective antibacterial effects Little negative impact on the environment.

Description of drawings

Formula (I) lauroyl arginine ethyl ester compound shown in Fig. 1 (A) is to the time bactericidal curve of Riemerella anatipestifer clinical isolation pathogenic strain RA‐11; Formula (I) lauroyl shown in (B) Arginine ethyl ester compound is to the time sterilization curve of Staphylococcus aureus ATCC29213; Formula (I) shown in (C) lauroyl arginine ethyl ester compound is to the time sterilization curve of Escherichia coli ATCC 25922.

Formula (I) lauroyl arginine ethyl ester compound shown in Fig. 2 (A) and chloramphenicol induce the result of Riemerella anatipestifer drug resistance; Formula (I) lauroyl arginine ethyl ester compound shown in (B) Ester compound and chloramphenicol induce the result of Staphylococcus aureus drug resistance; Formula (I) shown in (C) lauroyl arginine ethyl ester compound and chloramphenicol induce the result of Escherichia coli drug resistance.

The ethyl lauroyl arginate compound of formula (I) shown in Figure 3 is toxic to mammalian cells. Wherein, the cytotoxicity of the lauroyl arginine ethyl ester compound shown in (A) to mouse fibroblasts, mouse myofibroblasts, human fibroblasts; (B) the lauroyl arginine ethyl ester compound Hemolysis experiments on mammalian erythrocytes.

Figure 4 shows the toxicity results of the compound of ethyl lauroyl arginate of formula (I) in animals. Wherein, (A) is the survival status of ducklings after oral administration of ethyl lauroyl arginate compound for 5 days; (B) is the effect of ethyl lauroyl arginine compound on the weight gain rate of ducklings; (C) is the Oral lauroyl arginine ethyl ester compound three months drug residues in the body; (D) is the change of mice oral lauroyl arginine ethyl ester compound three months body weight; (E) is oral lauroyl arginine compound for mice Main organ index of arginine ethyl ester compound for three months; (F) H&E staining results of the organs on the 5th day of oral administration of lauroyl arginine ethyl ester compound to ducklings; (G) oral administration of lauroyl arginine ethyl ester compound H&E staining results of three-month organ of ester compound.

The effect of formula (I) ethyl lauroyl arginate compound shown in Fig. 5 on bacterial cell membrane. Wherein, (A) is the scanning electron micrograph of normal Escherichia coli; (B) is the scanning electron micrograph of Escherichia coli after lauroyl arginine ethyl ester compound is processed 15min; (C) is the effect of lauroyl arginine ethyl ester compound on duck Fluorescence spectrophotometer result of the influence of Lymerella epiphysis cell membrane polarity; (D) is the fluorescence spectrophotometer result of the influence of lauroyl arginine ethyl ester compound on Staphylococcus aureus cell membrane polarity; (E) is Fluorescence spectrophotometer results of the effect of ethyl lauroyl arginine compound on the polarity of Escherichia coli cell membrane; (F) is the effect of ethyl lauroyl arginine compound on the polarity of mammalian erythrocyte membrane; (G) is the effect of lauryl arginine Flow cytometric results of the effect of ethyl arginate compound on the polarity of the cell membrane of L. anatipestifer, (J) is its statistical graph; (H) is the effect of ethyl arginate compound on the membrane polarity of Staphylococcus aureus The flow cytometric result of the impact of polarity, (K) is its statistical diagram; (I) is the flow cytometric result of the influence of lauroyl arginine ethyl ester compound on the polarity of Escherichia coli cell membrane; (L) is its statistical diagram.

The lauroyl arginine ethyl ester compound of formula (I) shown in Figure 6 exerts a bactericidal effect by binding to phosphatidylserine. Wherein, (A) is the titration ITC result of lauroyl arginine ethyl ester compound and aqueous solution; (B) is the titration ITC result of lauroyl arginine ethyl ester compound and phosphatidylserine aqueous solution; (C) is lauroyl arginine The titration ITC result of ethyl arginate compound and phosphatidylcholine aqueous solution; (D) is the titration ITC result of ethyl arginine lauroyl ester compound and phosphatidylethanolamine aqueous solution; (E) is the protection of large intestine by different concentrations of phosphatidylserine The result of bacilli resisting lauroyl arginine ethyl ester compound, (G) is its statistical graph; (F) is the result of different concentrations of phosphatidylserine protecting Staphylococcus aureus against lauroyl arginine ethyl ester compound, (H ) is its statistical diagram; (I) is the result that phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine respectively protect Escherichia coli of the same concentration against lauroyl arginine ethyl ester compound, and (K) is its statistical diagram; ( J) is the result of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine respectively protecting Escherichia coli against lauroyl arginine ethyl ester compound at the same concentration, and (L) is its statistical graph.

Detailed ways

The present invention will be described in further detail in conjunction with the following specific examples and accompanying drawings, and the protection content of the present invention is not limited to the following examples. Without departing from the spirit and scope of the inventive concept, changes and advantages conceivable by those skilled in the art are all included in the present invention, and the appended claims are the protection scope. The process, conditions, reagents, experimental methods, etc. for implementing the present invention are general knowledge and common knowledge in the art except for the content specifically mentioned below, and the present invention has no special limitation content.

Embodiment one: micro-broth dilution method measures LAE to bacterium minimum inhibitory concentration (MIC)

Principle and purpose: According to the micro-broth dilution method stipulated by CLSI, after co-incubating the drug and bacteria in a 96-well plate for 24 hours, the minimum drug concentration at which bacterial growth is inhibited is the minimum inhibitory concentration of the drug.

Methods: LAE and bacteria (the clinical isolate of Riemerella anatipestifer RA‐11, Staphylococcus aureus ATCC 29213, and Escherichia coli ATCC 25922) were diluted with tryptone soy broth (TSB) and cultured. Added to the 96-well plate, and set up blank control medium CK1 without bacteria, medium CK2 added with LAE (1000 μg/ml) and control medium CK3 without drug LAE but containing normal growth of bacteria. The 96-well plate was incubated in a 37 °C incubator for 24 h, and then the absorbance value at 625 nm of each well was measured. Wells with the same OD ₆₂₅ value of the blank control medium CK1 were considered as having no obvious growth of bacteria. The lowest concentration of the drug without obvious growth of bacteria is the minimum inhibitory concentration MIC (Minimal Inhibitory Concentration) of LAE on bacteria. The liquid in each well was added to tryptone soybean agar medium (TSA) by the serial dilution method, and after the liquid was evaporated to dryness, it was placed upside down and placed in a 37°C incubator for 18-24h to observe the colony formation. The lowest concentration of drug without colony formation is the minimum bactericidal concentration MBC (Minimal Bactericidal Concentration) of LAE on bacteria.

The results are shown in Table 1 below. The minimum bactericidal concentration and minimum inhibitory concentration of LAE to RA‐11 are both ≥16 μg/ml, the minimum bactericidal concentration and minimum inhibitory concentration to ATCC25922 are both ≥16 μg/ml, and the minimum bactericidal concentration to ATCC 29213 Both the minimum bactericidal concentration and the minimum inhibitory concentration are ≥16 μg/ml.

The in vitro antibacterial effect of table 1LAE on three kinds of bacteria

Note: ++ means that the bacterial liquid is turbid with suspended matter; + means that the bacterial liquid is relatively turbid; +‐ means that the bacterial liquid is relatively clear; - means that the bacterial liquid is clear

Embodiment two: LAE is to the mensuration of effective bactericidal time of bacteria

Principle and purpose: Drugs and bacteria are co-incubated, and a part of the samples are taken out at regular intervals for bacterial plate counting, so as to obtain the growth curve of the bacteria under the action of the drug, which is the time-killing curve of the drug.

Method: The bacteria in the logarithmic phase were mixed with different concentrations of LAE solutions, samples were taken at 0, 0.5, 1, 1.5, and 2 hours after the addition of the drug, and the 10-fold serial dilution points were placed on the TSA plate, and cultured at 37°C for 18‐ 24h. The bacterial concentration of each dosing group at each time point was calculated by the number of colonies, and the bacterial survival curve shown in Figure 1 was obtained.

Result analysis: at a concentration of 16 μg/ml, LAE can completely kill Riemerella anatipestifer and Escherichia coli within 30 minutes, and reduce the number of Staphylococcus aureus by 6 orders of magnitude; within 30 minutes, at a concentration of 32 μg/ml , can completely kill Riemerella anatipestifer, Staphylococcus aureus and Escherichia coli. Therefore, LAE is an antibacterial compound that can quickly kill bacteria, and the advantage of its short action time is that it can eliminate bacteria before drug-resistant mutations occur, reducing the risk of bacteria being induced to develop drug resistance.

Example 3: LAE-induced bacterial drug resistance experiment

Principle and purpose: Bacteria can be induced to produce drug-resistant bacteria at sub-inhibitory concentrations, and the drug-induced bacteria can be detected by long-term transfer of bacteria cultured with sub-inhibitory concentrations of drugs to fresh drug-containing media Ability to generate resistance mutations.

Method: The bacteria in the logarithmic phase were mixed with the LAE solution in a glass test tube, sealed with a cotton plug, and placed in a constant temperature shaker at 37°C and 220 rpm for 24 hours. Afterwards, check the MIC of the drug every 24 hours, and add the bacterial solution with the highest drug concentration (that is, the highest drug concentration lower than the MIC) that can cultivate a turbid bacterial solution to fresh cultures containing different drug concentrations at a ratio of 1:100. In Kiri, culture at 37°C, 220rpm for 24h and last for 30 days. Chloramphenicol was used as the positive control group, and the operation was the same as that of the LAE group. The statistical chart shown in Figure 2 is obtained.

Result analysis:

As shown in Figure 2(A), for Riemerella anatipestifer, after 30 days of continuous administration stimulation, the MIC of chloramphenicol first showed a continuous upward trend, up to 6 times the initial MIC, and then fluctuated , but basically higher than the initial MIC. The MIC of LAE is basically stable, only one day appeared 1.5 times the initial MIC, and the rest of the time was lower than or equal to the initial MIC;

As shown in Figure 2(B), for Staphylococcus aureus, after 30 days of continuous administration stimulation, the MIC of chloramphenicol began to rise rapidly from the 18th day, up to 10 times the initial MIC, and finally stabilized 8 times the initial MIC. The MIC of LAE is only slightly improved, and finally reaches 2 times of the initial MIC;

As shown in Figure 2(C), for Escherichia coli, after 30 days of continuous administration stimulation, the MIC of chloramphenicol began to rise rapidly from the 16th day, and finally reached 12 times the initial MIC. The MIC of LAE is only slightly improved, and finally reaches 2 times of the initial MIC. The results show that chloramphenicol has a high ability to induce drug resistance of Riemerella anatipestifer, E. Bacteria have a slight effect of inducing drug resistance. This property of LAE suggests that it can be an antimicrobial agent superior to existing antibiotics.

Example 4: Study of LAE Toxicity to Mammalian Cells in Vitro

1.LAE cytotoxicity test on mammals:

Principle and purpose: MTS is a tetrazolium blue salt compound, which can be reduced to colored formazan products by dehydrogenase in living cells. The absorbance value of the sample is measured by a microplate reader and converted into the survival rate of cells in the sample.

Method: Cytotoxicity experiment: Cells in the logarithmic growth phase were inoculated in a 96-well plate at an amount of 5000/well. After the cells adhered to the wall, the medium was replaced with fresh medium containing different concentrations of LAE, and placed in Treat the cells at 37°C for 72 hours, add 20 μl MTS to each well, incubate at 37°C for 1 hour, measure the absorbance value at 490nm of each well with a microplate reader, when the OD ₄₉₀ of the control group without administration is between 0.8-1, the data is reliable Sex is the highest. The cell viability of each well was calculated and graphed as shown in Fig. 3(A). 2.LAE hemolytic test on rabbit erythrocytes:

Principle and purpose: Red blood cells will release content after hemolysis, including colored substances such as hemoglobin. After centrifugation, cell debris will settle, and the colored substances will remain in the supernatant. The absorbance value of the supernatant can be converted into the degree of cell hemolysis.

Methods: fresh rabbit whole blood was collected by ear artery blood collection, anticoagulated with heparin, centrifuged and washed, the accumulated red blood cells were taken to prepare a 2% red blood cell suspension, and 100 μl per well was added to a 96-well plate. An equal volume of LAE solutions of different concentrations was added to the experimental group, an equal volume of PBS was added to the blank control group, and an equal volume of 1% Triton X-100 was added to the positive control group, and placed in a 37°C incubator for 1 hour. Take the supernatant by centrifugation, measure the absorbance at 450nm with a microplate reader, and calculate the hemolysis rate according to the following formula:

A statistical graph as shown in Fig. 3(B) is obtained.

Result analysis:

As shown in Fig. 3(A), all the tested cells had a high survival rate after 72 hours of drug treatment, even under the condition that the administration concentration was much higher than the MIC, the survival rate of the cells was still more than 80%. According to the Pharmacopoeia, a sample with a hemolysis rate greater than 5% is hemolytic.

As shown in Figure 3(B), the half-haemolytic concentration of LAE was twice the bacterial MIC. This experiment shows that under in vitro conditions, LAE has very low toxicity to mammalian cells and has good selectivity to bacteria.

Embodiment five: LAE prevents and treats the bacterial disease effect of poultry and livestock

1. Oral therapeutic effect of LAE on ducklings infected with Riemerella anatipestifer

Principle and purpose: After the ducklings were infected with bacteria, LAE was administered orally to observe the survival of the animals, and to study the therapeutic effect of LAE on poultry with bacterial diseases.

Method: 25 ducklings were randomly divided into 5 groups, 5 in each group. Riemerella anatipestifer was inoculated subcutaneously in the legs of four groups of ducklings with 4×10 ⁶ CFU as the infection group, and the other group was not treated as the blank group. After 12 hours, the four infection groups were given different doses of LAE aqueous solution or blank aqueous solution control group in the form of intragastric administration, and observed and recorded the death situation at 1h, 7h, 12h, 24h, 48h, 72h, and 96h after administration, and recorded the death situation at 96h. The survival status of the animals is shown in Table 2.

Table 2: The therapeutic effect of oral administration of LAE on ducklings infected with L. anatipestifer

Note: The test group is animals infected with pathogenic bacteria and given different concentrations of LAE;

The blank group is animals that have not been exposed to pathogenic bacteria;

The control group was animals infected with pathogenic bacteria and only given blank aqueous solution but not LAE aqueous solution.

Result analysis: After the ducklings were infected with Riemerella anatipestifer, if no drug treatment was given, all the ducklings would die within 48 hours; if the LAE aqueous solution was given intragastrically at 12 hours after infection, the survival rate at 96 hours after infection would reach 60%. Considering the 4-8-day treatment cycle and the cost of LAE treatment solvent, LAE at an oral dose of at least 8-16 mg/kg bw can effectively reduce the incidence of ducklings infected with L. anatipestifer, which meets the needs of production. Therefore, LAE greatly improved the survival rate of infected ducklings. 2. The control effect of LAE on ducklings infected with Riemerella anatipestifer

Principle and purpose: Before infection with bacteria, ducklings were orally administered LAE, and after infection, LAE was continued to be administered daily to observe the survival of animals, and to study the effect of LAE as an antibacterial agent to protect poultry from bacterial infection.

Method: 50 ducklings were randomly divided into 5 groups, 10 in each group. Among them, 2 groups were given blank aqueous solution as the non-administration group in the form of gavage, and the other 3 groups were given LAE water solution as the test group in the form of gavage. 8 hours after the first administration, Riemerella anatipestifer was subcutaneously inoculated with 4×10 ⁶ CFU in the legs of ducklings in the three experimental groups and a control group without administration as the infection group, and the other group The non-administration group was treated as the non-infected group without any treatment (that is, no infection with bacteria and no administration as blank). At 12h, 24h, and 48h after infection, the blank aqueous solution was given to the uninfected group and the infected non-administered group in the form of gastric gavage, and LAE aqueous solutions of different concentrations were given to experimental groups 1, 2, and 3, respectively. 1h, 7h, 12h, 24h, 48h, 72h, and 96h after administration were observed and the death situation was recorded respectively. Table 3 shows the results of survival at 96h.

Table 3: The control effect of oral administration of LAE on ducklings against L. anatipestifer infection.

Note: The test group is the animals infected with pathogenic bacteria after being given different concentrations of LAE;

The blank group is the animals that have not been exposed to pathogenic bacteria and have not been given LAE;

The control group was animals infected with pathogenic bacteria but not given LAE.

Analysis of the results: Compared with administration of LAE after infection of ducklings before infection with bacteria, this way of intake can improve the survival rate of ducklings again. LAE at an oral dose of 8‐40 mg/kg bw has been effective in preventing ducklings from being infected with L. anatipestifer. Continuous drug administration can exert the effect of preventing and treating diseases to a greater extent. The 10 ducklings in the solvent control group all died within 96 hours, and 9 ducklings in the treatment group still survived at the end of the experiment, and the survival rate reached 90%. Considering the 4-8 day treatment cycle and the cost of the LAE treatment solvent, the dose range of 40 mg/kg bw is already the upper limit of production needs.

3. Study on the therapeutic effect of LAE intraperitoneal administration on mice infected with Escherichia coli

Principle and purpose: Escherichia coli was injected intraperitoneally into mice to simulate the infection of Gram-negative bacteria in livestock, and the survival rate of mice was observed by intraperitoneal administration after infection, the clearance rate of pathogenic bacteria in mice was detected by LAE, and the effect of drugs on infection was detected. Effects on the levels of inflammatory factors in mice. This experiment can detect the therapeutic effect of LAE on livestock infected with Gram-negative bacterial diseases.

Methods: 50 Balb/c mice were randomly divided into 5 groups, 10 in each group. The logarithmic phase of Escherichia coli was injected into the peritoneal cavity of 4 groups of mice in the amount of 10 ⁸ CFU, and the other group was set as the uninfected group without any treatment. One hour later, one infection group was injected with 0.5ml of normal saline intraperitoneally, and the other three infection groups were injected with different concentrations of LAE solution. The survival of the mice after 24 hours was counted, and the results in Table 4 were obtained.

Another 60 Balb/c mice were randomly divided into 6 groups, 10 in each group. The logarithmic phase of Escherichia coli was injected into the peritoneal cavity of 5 groups of mice in the amount of 10 ⁸ CFU, and the other group was set as the uninfected group without any treatment. One hour later, 0.5ml normal saline was injected intraperitoneally into one infection group, and different concentrations of LAE solutions were injected into the other four infection groups. The mice were sacrificed by cervical dislocation 24 hours after administration, the peritoneal cavity of the mice was washed with sterile PBS, and the peritoneal fluid was collected. The peritoneal fluid was spotted on the TSA plate by the serial dilution method, and incubated upside down at 37°C for 18‐24 hours, and the concentration of pathogenic bacteria in the peritoneal fluid of mice in each group was counted and calculated. The protein levels of IL-1β and TNF-α in peritoneal fluid were detected by ELISA kits for IL-1β and TNF-α, respectively. The results are shown in Table 5.

Table 4: The therapeutic effect of intraperitoneal administration of LAE on mammals infected with Escherichia coli

Note:

The test group was animals infected with pathogenic bacteria and administered orally with different concentrations of LAE;

The blank group is animals that have not been exposed to pathogenic bacteria;

The control group was infected with pathogenic bacteria but not given LAE.

Table 5: Effect of intraperitoneal administration of LAE on pathogenic bacteria in mammals infected with Escherichia coli

Note:

The test group was animals infected with pathogenic bacteria and given different concentrations of LAE;

The blank group is animals that have not been exposed to pathogenic bacteria;

The control group was animals infected with pathogenic bacteria but not given LAE.

Result analysis: After the mice were infected with Escherichia coli, all 10 animals in the saline injection group died within 11 hours, and the survival rate of the mice injected with LAE solution was 70% after 24 hours, indicating that LAE had a therapeutic effect on the animals and eliminated Pathogenic E. coli in mice reduced levels of inflammatory factors that were elevated due to bacterial infection.

Intraperitoneal injection of 2.5-25 mg/kg body weight of LAE can improve the survival rate of mice infected with Escherichia coli, and intraperitoneal injection of 1-25 mg/kg body weight of LAE can eliminate pathogenic bacteria in infected mice.

4. The therapeutic effect of oral administration of LAE on mice infected with Gram-negative bacteria and positive bacteria.

Principle and purpose: It is a high requirement for antibacterial agents to be able to take orally and exert curative effects. Therefore, we use mice to represent livestock, and infect small animals with Gram-negative bacteria Escherichia coli and Gram-positive bacteria Staphylococcus aureus. Rats were then administered orally with LAE to evaluate the therapeutic effect of the drug on the animals.

Methods: 60 Balb/c mice were randomly divided into 6 groups, 10 in each group. Staphylococcus aureus in the logarithmic phase was injected into the peritoneal cavity of the mice in an amount of 10 ⁸ CFU. After 1 hour, 0.5 ml of normal saline, LAE aqueous solution of different drug concentrations or cefazolin aqueous solution were administered to the stomach respectively. 24 hours after the administration, the mice were killed by cervical dislocation, and the peritoneal cavity of the mice was washed with sterile PBS, and the peritoneal fluid was collected. The peritoneal fluid was spotted on the TSA plate by the serial dilution method, and incubated upside down at 37°C for 18‐24h. The concentration of pathogenic bacteria in the peritoneal fluid of mice in each group was counted and calculated, and the results are shown in Table 6.

Table 6: The effect of oral administration of LAE on the elimination of Gram-positive pathogenic bacteria in mammals

Note:

The test group was animals infected with pathogenic bacteria and administered orally with different concentrations of LAE;

The cefazolin group is animals infected with pathogenic bacteria and orally administered cefazolin aqueous solution;

The blank group is animals that have not been exposed to pathogenic bacteria;

The control group was animals infected with pathogenic bacteria but not administered.

Thirty Balb/c mice were randomly divided into 6 groups with 5 mice in each group. Escherichia coli in the logarithmic phase was injected into the peritoneal cavity of the mice in an amount of 10 ⁸ CFU. One hour later, the five groups were orally administered 0.5ml of normal saline, different concentrations of LAE aqueous solution and ampicillin aqueous solution, and the mice were killed by cervical dislocation 24 hours after the administration. The peritoneal cavity of the mice was washed with sterile PBS, and the peritoneal fluid was collected. The peritoneal fluid was spotted on the TSA plate by the serial dilution method, and incubated upside down at 37°C for 18‐24h. The concentration of pathogenic bacteria in the peritoneal fluid of mice in each group was counted and calculated, and the results are shown in Table 7.

Table 7: The effect of oral administration of LAE on the elimination of Gram-negative pathogenic bacteria in mammals

Note:

The test group was animals infected with pathogenic bacteria and administered orally with different concentrations of LAE;

The ampicillin group is the animals infected with pathogenic bacteria and oral ampicillin aqueous solution;

The blank group is animals that have not been exposed to pathogenic bacteria;

The control group was animals infected with pathogenic bacteria but not administered.

Analysis of the results: After the mice were infected with bacteria orally, LAE could reduce the amount of Escherichia coli and Staphylococcus aureus in the infected mice, and oral administration of 2.5-25 mg/kg body weight of LAE could remove the pathogenic genes in the sick mice. The gram-positive bacterium Staphylococcus aureus; oral administration of 0.625‐10 mg/kg body weight of LAE cleared the pathogenic gram-negative bacterium Escherichia coli in diseased mice. The clearing effect is equivalent to the curative effect of antibiotics, indicating that LAE can treat Gram-negative bacteria and Gram-positive bacteria diseases in mammals in the form of oral administration.

In summary, LAE can be used orally to treat and prevent bacterial diseases in poultry; LAE can be used orally to treat livestock infected with Gram-negative bacteria and Gram-positive bacteria; LAE can be administered intraperitoneally to treat livestock infected with bacterial diseases.

Embodiment 6: LAE toxicity research on poultry

1. Toxicity study of oral administration of LAE on cherry duck ducklings

Principle and purpose: Orally administer LAE four times the effective therapeutic concentration once a day for one week, observe the animal's mental state, survival rate and body weight growth, and detect organ toxicity.

Method: 20 ducklings were randomly divided into 2 groups, 10 in each group. Among them, group A was given a blank aqueous solution in the form of gavage, and group B was given an aqueous solution of LAE four times the effective therapeutic concentration in the form of gavage, and the drug was given once every 24 hours for five consecutive days, and the observation was continued for 7 days and recorded. The clinical manifestations of the experimental animals were shown in Figure 4 (A), and the heart, liver, spleen, and kidney tissues of the experimental animals in the blank group and the highest drug dose group were taken for pathological analysis at the end of the experiment. The organ toxicity results shown in Figure 4(F) were obtained. And count the average daily weight gain of experimental animals in each group within 7 days as shown in Figure 4 (B).

2. Oral administration of LAE on acute and subacute toxicity and accumulation in animals

Principle and purpose: To study the long-term toxicity of LAE orally administered to animals and the accumulation of the drug in the body, so as to evaluate the safety of LAE. The study on the accumulation of drugs in animals analyzed whether LAE can be used as a residue-free additive.

Method: Acute toxicity test of ducklings: 20 ducklings were randomly divided into two groups, which were given intragastric administration every day, the experimental group was given LAE aqueous solution, and the blank control group was given the same volume of water. Oral administration for 7 days, body weight measurement, H&E staining of heart, liver, spleen, lung and kidney; mouse subacute toxicity test: 20 mice were randomly divided into two groups, 10 in each group. Mice were administered intragastrically every day, the experimental group received LAE aqueous solution, and the blank control group received the same volume of water. The mice were weighed every week, and the mice were killed 24 hours after the last administration after 90 days. The hearts, livers, spleens, lungs, and kidneys were weighed, H&E stained, and the residual LAE in various tissues and organs were detected by HPLC. quantity.

Result analysis:

(1) Acute toxicity test on ducklings: no experimental animals died, and all drug-administered groups showed no abnormalities with the control group in terms of morphology, mental state, coat color, etc.

(2) There was no abnormality in the appearance of the organs, and the H&E staining of the tissue sections of the heart, liver, spleen and kidney did not show any abnormality compared with the control group. The average daily gain of the blank control group was 21.38g, and the average daily gain of the highest drug dose group was 22.57g. The drug did not have a negative impact on the duck's weight gain. LAE has no oral toxicity to cherry duck ducklings at four times the dose of the drug effective in preventing and controlling Riemerella anatipestifer;

(3) Subacute toxicity test in mice: As shown in Figure 4(D), after 90 days of oral feeding, the weight gain of the mice in the experimental group was significantly higher than that in the blank group in the first week and the 4th to 10th week promote. Figure E shows that LAE has no significant effect on the organ coefficient, and the H&E staining results of various organs in Figure G show that LAE has no obvious toxicity to the organs of experimental animals. The residual amount of LAE in various tissues and organs (Figure C) is also lower than 10 μg/kg (the European Union's detection amount requirements for unregulated veterinary drugs).

The above results indicate that oral administration of LAE to mammals for 3 months has extremely low toxicity to animals, can promote animal weight gain to a certain extent, and has extremely low drug residues. The above results show that the acute and subacute toxicity of LAE to animals is extremely low, and the accumulation in the body is extremely low after long-term administration, which meets the safety and environmental protection requirements of antibacterial agents.

Embodiment 7: LAE compounds are used as feed additives for the prevention of diseases in mammals

The active ingredient of the LAE compound is directly added to the mouse feed at 0.01% to 1.0% based on the total weight of the feed; or the product is mixed with a carrier to make a premix; or mixed with other feed additives or Feed materials are mixed to make premix and concentrated feed to feed mice.

Fifty 6-week-old mice were selected and randomly divided into 5 treatment groups according to the principle of similar body weight. The control group was fed with corn-wheat-soybean meal-based diet, and the experimental group was fed with corn-wheat-soybean meal-based diet and added 0.01%, 0.1%, and 1% of the total weight of the feed. After feeding for 7 days, the disease resistance of the mice to Escherichia coli was determined. . The results are shown in Table 8.

Table 8: Disease prevention and control of mammals with feed supplemented with LAE

Note:

The test group was the feed that was exposed to pathogenic bacteria and added different doses of LAE compounds;

The blank group was not exposed to pathogenic bacteria;

The control group was the feed exposed to the pathogenic bacteria but without the addition of LAE compounds

As can be seen from the above table, the survival rate of mice in all test groups was significantly increased compared with the control group, and the best group was the test group 3, with a survival rate of 80%. The experimental animals in the control group without adding LAE all died. As a feed additive, it can prevent and treat mammalian diseases. Considering the treatment cycle and the cost of LAE therapeutic agents, the effective concentration of LAE as a drug or feed additive is 0.01-1%, which can effectively prevent and treat the disease and meet the needs of production.

Embodiment 8, LAE compounds are used as nutritional energy additives for the vegetative growth of mammals

According to the scope of application of the concentration of the LAE compound determined in Example 7, on the basis of the same ration formula in each group, the LAE compound was added to the full price in a mass ratio of 0.3%, 0.6%, and 0.9% in test 1, 2, and 3, respectively. In the compound feed, the control group did not add any medicine. Forty 6-week-old Balb/c mice were randomly divided into 4 groups. The body weight of each group was weighed on the first day at the beginning of the formal experiment, and the body weight of the mice was weighed again 15 days later. The results are shown in Table 9:

Table 9: Effects of feed supplementation with LAE on growth of mammals

Note:

The test group is the feed added with different doses of LAE compounds;

The control group was the feed without adding LAE compound

Result analysis: It can be seen from the above table that there is no significant difference in the initial weight of the test among the groups. Judging from the 15-day average individual net weight gain, Group 2 was significantly higher than Group 1 and Group 3.

This shows that during the test period, compared with the control group, adding 0.6% LAE compound to the mouse feed significantly increased the average weight gain of the mice by 9.64%. Therefore, feed supplementation with LAE compounds can promote the growth of mammals.

Embodiment 9: LAE compounds are used as feed additives for disease prevention and treatment of ducklings

The active ingredient of the LAE compound is 0.01% to 1.0% based on the total weight of the feed, and this product is directly added to duckling feed; or this product is mixed with a carrier to make a premix; or mixed with other feed additives Or feed raw materials are mixed to make premix and concentrate to feed ducklings.

Fifty 14-day-old cherry ducklings were selected and randomly divided into 5 treatment groups according to the principle of similar body weight. One repetition (column) was set up for each treatment, with 10 rats in each column (half male and half female). The control group was fed with corn-soybean meal-based diets, and the test group was fed with corn-soybean meal-based diets and added 0.01%, 0.1%, and 1% of the total weight of LAE compounds. After feeding for 7 days, the ducklings were tested for their resistance to Riemerella anatipestifer. disease ability. The results are shown in Table 10.

Table 10: The disease control effect of adding LAE to ducklings in feed:

Note:

The test group is the feed added with different doses of LAE compounds;

The blank group was not exposed to pathogenic bacteria;

The control group was the feed without adding LAE compound

Result analysis: It can be seen from the above table that the survival rate of ducklings in all test groups has increased significantly compared with the control group, and the best group is the test group 3, with a survival rate of 90%. The experimental animals in the control group without adding LAE all died. Explain that LAE compounds have preventive effect on duckling diseases as feed additives. Considering the treatment cycle and the cost of LAE therapeutic agents, LAE can effectively prevent and treat diseases when the effective concentration of LAE as a drug or feed additive is 0.01-1%, preferably 0.1-1%. Disease, in line with the needs of production.

Embodiment ten, LAE compound is used for disease prevention and vegetative growth of duckling as nutritional energy supplement

According to the scope of application of the concentration of the LAE compound determined in Example 9, on the basis of the same dietary formula of each group, the LAE compound was added to the full price of the compound in the test 1, 2, and 3 groups with a mass ratio of 0.3%, 0.6%, and 0.9% respectively. In the compound feed, the control group did not add any medicine. At the beginning of the experiment, the body weight of the animals in each group was weighed on the first day, and the body weight was weighed again 15 days later to measure the disease resistance of each group to Riemerella anatipestifer. The results are shown in Table 11:

Table 11: The effect of adding LAE on the disease control and nutritional growth of ducklings

Note:

The test group is the feed added with different amounts of LAE compounds;

The control group was the feed without adding LAE compound

Result analysis: This shows that during the test period, compared with the control group, adding 0.3%-0.9% LAE to the duckling feed can increase the average weight gain of ducklings, and the weight gain of 0.6% LAE compound is the most obvious. Compared with the control group, the average weight gain of ducklings was significantly increased by 11.5%. Therefore, the LAE compound can not only reduce the death of ducklings caused by Riemerella anatipestifer, but also improve the growth performance of ducklings and promote their vegetative growth.

Embodiment 11: LAE compounds are used as nutritional energy additives for disease prevention and vegetative growth of aquatic animals

240 tilapia with a body weight of 42.1±0.24g were selected and divided into 4 treatment groups, and different gradient levels of LAE compounds were added respectively, with 3 repetitions in each group and 20 fish in each repetition. The effects of LAE compounds on the growth performance of tilapia were evaluated by 8-week growth test. At the same time of feeding, in order to explore the ability of LAE to resist bacterial diseases in aquatic animals, tilapia fed with LAE feed for 8 weeks were infected with Aeromonas sobria, and the survival rate of tilapia after infection was used to reflect its disease resistance . The results are shown in Table 12.

Table 12 The effect of feed adding LAE on the vegetative growth of tilapia

Note:

The test group is the feed added with different doses of LAE;

The control group was the feed without adding LAE compound

Table 12 data shows that adding LAE compounds in the feed of tilapia can improve relative weight gain rate 3.03%, 6.23%, 3.88% and specific growth rate 5.69%, 12.32%, 7.1% (P＜0.05), reduce feed coefficient 2.36%, 3.15%, 1.57% (P<0.05), promote the growth of tilapia.

At the same time, the results of the anti-infection experiment showed that all the test fish that ate the diet without LAE died, and the survival rates of the tilapia that ate the feed containing LAE were 61.67%, 78.33%, and 88.33%, respectively. It shows that adding 0.01%‐1% LAE can indeed improve the anti-infection ability of fish.

Example 12: LAE triggers bacterial death by depolarizing bacterial cell membranes

1. SEM to observe the effect of LAE on bacterial morphology

Principle and purpose: The surface morphology of samples after fixation, dehydration, and gold spraying can be observed under the scanning electron microscope. This technique is often used to analyze the microscopic surface morphology of chemical materials and biological materials.

Method: Escherichia coli in logarithmic phase was treated with 1×MIC LAE for 15 minutes. Centrifuge at 8000rpm for 5min, discard the supernatant, add 2.5% glutaraldehyde for fixation for 2h, wash with PBS three times for 10min each, fix with 1% osmic acid for 1h, wash with PBS for three times for 10min each. Dehydrate with 50%, 70%, and 90% ethanol for 10 minutes each, and dehydrate with absolute ethanol for 5 minutes. Dry by critical point drying method for 2h. Stick the dried powder on the stage and spray gold for 40min. Observe and take pictures with a scanning electron microscope.

2. Fluorescent dye staining to study the effect of LAE on the polarity of prokaryotic and eukaryotic cell membranes.

Principle and purpose: DiSC ₃ (5) and DiSC ₂ (3) fluorescent dyes are polar dyes of cell membranes. Because of the potential difference between the inside and outside of the cell membrane, the cell membrane emits red fluorescence after staining with DiSC ₂ (3). Once the potential difference disappears, a depolarization reaction occurs , DiSC ₂ (3) will emit green fluorescence. By calculating the ratio of red and green fluorescence values, the intensity of cell depolarization can be obtained.

Method: RA‐11 in the logarithmic phase was diluted with PBS to OD ₆₀₀ =0.05, added DiSC ₃ (5) fluorescent dye, kept in the dark at 37°C for 1 hour, and then added KCl solution. Different concentrations of LAE solutions were added to make the final concentrations 0.5×MIC and MIC respectively, and polymyxin B, vancomycin and PBS were used as positive control, negative control and blank control respectively. Using light with a wavelength of 622nm as the excitation light, measure the fluorescence value at 670nm at 0, 2, 10, 20, 30, 40, 50, and 60 minutes after adding the drug, and make a statistical chart as shown in Figure 5(C).

Staphylococcus aureus in logarithmic phase was diluted with PBS to OD ₆₀₀ =0.05, DiSC3(5) fluorescent dye was added, and KCl solution was added after standing at 37° C. in the dark for 1 hour. Different concentrations of LAE solutions were added to make the final concentrations 0.5×MIC and MIC respectively, and polymyxin B, vancomycin and PBS were used as positive control, negative control and blank control respectively. Using light with a wavelength of 622nm as the excitation light, measure the fluorescence value at 670nm at 0, 2, 10, 20, 30, 40, 50, and 60 minutes after adding the drug, and make a statistical chart as shown in Figure 5(D).

Escherichia coli in logarithmic phase was diluted with PBS to OD ₆₀₀ =0.05, DiSC3(5) fluorescent dye was added, and KCl solution was added after standing at 37° C. in the dark for 1 hour. Different concentrations of LAE solutions were added to make the final concentrations 0.5×MIC and MIC respectively, and polymyxin B, vancomycin and PBS were used as positive control, negative control and blank control respectively. Using light with a wavelength of 622nm as the excitation light, measure the fluorescence value at 670nm at 0, 2, 10, 20, 30, 40, 50, and 60 minutes after adding the drug, and make a statistical chart as shown in Figure 5(E).

Fresh rabbit erythrocytes were counted and diluted to 5×10 ⁷ cells/ml, added with DiSC3(5) fluorescent dye, kept in the dark at 37°C for 1 hour, and then added with KCl solution. Different concentrations of LAE solutions were added to make the final concentrations 0.5×MIC and MIC of Escherichia coli, respectively, and the light with a wavelength of 622nm was used as the excitation light. The fluorescence value at 670nm was measured for 60 minutes, and a statistical diagram as shown in FIG. 5(F) was made.

The L. anatipestifer in the logarithmic phase was diluted to 5×10 ⁷ CFU/ml, and LAE solutions of different concentrations were added to make the final concentrations 0.5×MIC, MIC, and 2×MIC, respectively. In addition, the CCCP group was taken as the positive control, and the PBS group was taken as the negative control. DiOC ₂ (3) was added for 1 hour, and the red and green fluorescence values of each sample were detected by flow cytometry, as shown in Figure 5(G), and the degree of depolarization of the bacterial cell membrane was analyzed by calculating the ratio, as shown in Statistical graph of Figure 5(J).

Staphylococcus aureus in the logarithmic phase was diluted to 5×10 ⁷ CFU/ml, and LAE solutions of different concentrations were added to make the final concentrations 0.5×MIC, MIC, and 2×MIC, respectively. In addition, the CCCP group was taken as the positive control, and the PBS group was taken as the negative control. Add DiOC ₂ (3) to treat for 1 hour, flow cytometer detects the red and green fluorescence values of each sample, as shown in Figure 5(H), and analyzes the degree of depolarization of the bacterial cell membrane by calculating the ratio, as follows Statistical graph of Figure 5(K).

Escherichia coli in the logarithmic phase was diluted to 5×10 ⁷ CFU/ml, and LAE solutions of different concentrations were added to make the final concentrations 0.5×MIC, MIC, and 2×MIC, respectively. In addition, the CCCP group was taken as the positive control, and the PBS group was taken as the negative control. Add DiOC ₂ (3) to process for 1 hour, flow cytometry detects the red and green fluorescence values of each sample, and obtains as shown in Figure 5 (I), analyzes the degree of depolarization of the bacterial cell membrane by calculating the ratio, and makes as follows Statistical graph of Fig. 5(L). Result analysis: Scanning electron microscope results showed that after treating E. coli with 1×MIC LAE for 15 minutes, depressions and even cavities appeared on the surface of the bacteria, indicating that LAE can destroy the integrity of bacteria and cause bacterial death. For Riemerella anatipestifer and Escherichia coli, all LAE application groups and the positive control polymyxin B group can greatly increase the fluorescence value in a short time, indicating that LAE can depolarize the cell membrane, and the negative control The fluorescence values of the vancomycin group and the blank control group did not change significantly; for Staphylococcus aureus, all LAE application groups and the positive control polymyxin B group could greatly increase the fluorescence values in a short period of time, indicating that LAE can make Cell membrane depolarization occurred, and the fluorescence value of the negative control vancomycin group and the blank control group did not change significantly; for mammalian erythrocytes, slight depolarization occurred only when the concentration of LAE reached twice the MIC of Escherichia coli. The experimental results show that LAE may have a bactericidal effect by depolarizing the bacterial cell membrane to rupture the membrane, and the effect on prokaryotic cells is stronger than that of eukaryotic cells, which is related to its strong bactericidal effect and low toxicity of eukaryotic cells phenomenon is consistent. The results of flow cytometry were consistent with those detected by spectrophotometer.

Example 13: LAE triggers bacterial death by binding to phosphatidylserine on the bacterial cell membrane

1. Isothermal titration calorimetry to detect the binding effect of LAE and phospholipids

Principle and purpose: When two molecules are combined, heat will be absorbed or released. By detecting the heat change when two molecules are titrated with each other, it can be determined whether the two molecules have a binding reaction.

Method: Titrate phosphatidylcholine (PC) aqueous solution, phosphatidylethanolamine (PE) aqueous solution, and phosphatidylserine (PS) aqueous solution on the ITC200 instrument with LAE aqueous solution, record their respective heat change curves and calculate the ratio of LAE and each phospholipid molecule the binding constant. The curves shown in Figure 6A/B/C/D are obtained.

Results: Only the heat change diagrams of phosphatidylserine and LAE in the titration process conformed to the heat change of the combination of the two molecules, and the binding constant of the two molecules was about 2.95E5±2.48E5 M ^‐1 . However, no binding reaction was detected for phosphatidylcholine and phosphatidylethanolamine. It shows that LAE can combine with more acidic phospholipids on the surface of bacterial membranes, such as phosphatidylserine, but not with neutral phospholipids. 2. Phospholipid protection test

Principle and purpose: According to the above experiments, we can preliminarily judge that there is a binding reaction between LAE and phosphatidylserine molecules. If this combination is a necessary factor for LAE to exert its bactericidal function, then when we add a certain amount of LAE to the co-incubation system of bacteria Adding more PS will reduce the bactericidal effect of LAE, and adding PE or PC will not have the same effect.

Methods: + We added low or high concentrations of phosphatidylserine PS to Gram-negative bacteria Escherichia coli and Gram-positive bacteria Staphylococcus aureus respectively. Afterwards, LAE aqueous solution of the same concentration was added and cultured at 37° C. for 24 h. Count the viable bacteria on the plate for each sample, and incubate it upside down in a 37°C incubator for 18-20 hours, and then take pictures to obtain the situation of Escherichia coli in Figure 6E and Staphylococcus aureus in Figure 6F under the protection of PS, respectively, against LAE, Figure 6G/H for their respective statistics. In addition, we repeated the above experiments with the same concentrations of phosphatidylcholine PC, phosphatidylethanolamine PE, and phosphatidylserine PS, and obtained Escherichia coli in Figure 6I and Staphylococcus aureus in Figure 6J respectively resisting LAE under the protection of phospholipids. In the case of , Figure 6K/L is its respective statistical chart.

RESULTS: As expected, only phosphatidylserine PS was able to protect bacteria from the killing effect of LAE. It can be concluded that LAE plays a bactericidal effect through combining with PS.

Embodiment 14, LAE and the comparison of the therapeutic effect of antibiotic to diarrhea piglet

The applicant commissioned a large pig farm in Henan Province to conduct an experiment of administering SY (ie LAE of the present invention) to 118 piglets with diarrhea in the farm.

Another gentamicin administration group was set up as a parallel control.

The sick pigs were irrigated three times a day at morning (7:00), middle (12:00) and evening (18:00).

Evaluation criteria: Animals that do not need medication after the diarrhea is cured are counted as the remaining surviving animals, that is, the test is stopped;

Animals that died after the piglets failed to use the drug were counted as dead animals, that is, the test was stopped.

The results are shown in Table 13.

Table 13

As shown in the table above, the average cure rate of piglets treated with gentamicin was 77.5%. However, this dosage has far exceeded the dosage of conventional antibiotics, indicating that in order to obtain this cure rate, the antibiotic residues in piglets will significantly exceed the standard.

Using LAE to treat piglets, the cure rate of the minimum dose of 10mg/kg was 72.2%, and the cure rate of the maximum dose of 50mg/kg was 78.57%. The cure rate is not shown), which shows that LAE has a higher cure rate than gentamicin at the same dosage in the treatment of piglet diarrhea, and because of its good biological metabolism characteristics, it will indicate that there is no Residues of any harmful drugs provide a good direction for alternatives to veterinary antibiotics.

Claims (11)

1. if formula (I) compound represented or its hydrate or pharmaceutically acceptable salt are in the feeding for preparing poultry, aquatic livestock Purposes in feed additives, which is characterized in that formula (I) structure is as follows：

Wherein, X is halogen or HSO₄；

R₁It is the linear saturated fatty acids group containing 8-14 carbon atom or the oxygen-containing acidic group of straight chain containing 8-14 carbon atom Group；

R₂Be the straight chain fatty acid groups containing 1-18 carbon atom or the Branched fatty acid groups containing 1-18 carbon atom or Aromatic group containing 1-18 carbon atom or the straight chained alkyl containing 1-4 carbon atom；

R₃It is one kind of having structure：

The range of n is 0-4.

2. purposes as described in claim 1, which is characterized in that the X is Cl, and formula (I) compound represented is laurel Acyl arginine ethyl ester hydrochloride, shown in structural formula such as following formula (II)：

3. purposes as claimed in claim 1 or 2, which is characterized in that the effective component of formula (I) compound represented according to Feed total weight is 0.01%~1.0%.

4. purposes as claimed in claim 1 or 2, which is characterized in that the poultry, aquatic livestock include：Chicken, duck, goose, fire Chicken, quail, pigeon, pig, ox, sheep, horse, camel, cat, dog and fish shrimp crab.

5. purposes as claimed in claim 1 or 2, which is characterized in that the feed includes such as formula (I) compound represented, energy Poultry, the disease of aquatic livestock caused by being enough in treatment or preventing and treating because of pathogenic microorganisms.

6. purposes as claimed in claim 5, which is characterized in that the poultry, aquatic livestock are selected from duck, the pathogenic microorganisms Selected from pathogenic riemerella anatipestifer, enteropathogenic E. Coli, pathogenic staphylococcus.

7. purposes as claimed in claim 5, which is characterized in that the poultry, aquatic livestock be selected from Tilapia mossambica, it is described cause a disease it is micro- Biology is selected from pathogenic Aeromonas sobria.

8. purposes as claimed in claim 5, which is characterized in that the effective component of formula (I) compound represented is according to feeding Material total weight is 0.1-1.0%, and the poultry, aquatic livestock are selected from duck, and the pathogenic microorganisms is selected from pathogenic pest of duck Mo Shi bacillus.

9. purposes as claimed in claim 5, which is characterized in that the effective component of formula (I) compound represented is according to feeding Material total weight is 0.1-1.0%, and the poultry, aquatic livestock are selected from Tilapia mossambica, and the pathogenic microorganisms is warm-natured selected from causing a disease And Aeromonas.

10. purposes as claimed in claim 1 or 2, which is characterized in that the purposes is will directly to change shown in the formula (I) It closes object and is added to livestock and poultry, in aquatic feeds；Or the formula (I) compound represented and carrier are mixed and made into pre-mixing agent；Or it will Formula (I) compound represented and other feed addictives or feedstuff are mixed and made into premix, concentrate feed form is fed Livestock and poultry, aquatic livestock.

11. purposes as claimed in claim 1 or 2, which is characterized in that formula (I) compound represented can be reduced due to thin Bacterium infection and rise animal body in inflammatory factor IL-1 β and/or IL-1 β protein level.