Bromelain composition for treating cow mastitis and application of bromelain composition

Abstract

The invention discloses a bromelain composition for treating cow mastitis and application of the bromelain composition, and belongs to the technical field of biological medicine. The composition is prepared by compounding the following components in parts by weight: 1-20 parts of bromelain, 1-20 parts of cystine and 1-20 parts of sodium gluconate. 4 to 40 parts of cystine; and 0.2 to 2 parts of sodium gluconate. The bromelain composition has the effects of diminishing swelling, dispersing blood stasis, clearing heat, detoxifying, diminishing inflammation and the like, and is small in toxic and side effects, not easy to generate drug resistance, high in bioavailability, high in curative effect on cow mastitis caused by pathogenic bacteria infection, free of residues, free of drug resistance, green and safe. The bromelain composition for treating cow mastitis can reduce the number of somatic cells in cow's milk with mastitis and the content of ALP, NAG and LDH in cow's milk and cow's serum in a short time, and has a good treatment effect on cow mastitis.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a bromelain composition for treating mastitis in dairy cows and its application. Background Art

[0002] Mastitis in dairy cows, also known as bovine mastitis, is one of the most common and frequently-occurring mammary gland diseases in dairy cows. Due to its high incidence rate and complex and diverse causes, it seriously hinders the development of the dairy cow breeding industry and restricts the health of the livestock and dairy industries. This disease is caused by the stimulation of the mammary gland tissue of dairy cows by various comprehensive factors such as physical, chemical, and microbial factors, resulting in inflammation of the udders of dairy cows. The main symptoms are pathological damage to the mammary gland tissue, an increase in the number of somatic cells in the milk, and abnormal changes in the milk composition. Invasion by pathogenic microorganisms, poor sanitary conditions, improper feeding management, and the factors of the dairy cows themselves are the main factors leading to mastitis.

[0003] Mastitis in dairy cows is mainly caused by the invasion and proliferation of pathogenic microorganisms (such as Staphylococcus aureus, Escherichia coli, Streptococcus dysgalactiae, Streptococcus agalactiae, Klebsiella pneumoniae, etc.) in the mammary gland tissue, resulting in a large amount of pathogenic microorganisms and toxins of certain pathogenic bacteria in the milk. If the milk is not processed thoroughly, it will cause serious harm to human health. At present, the treatment plan for mastitis in dairy cows mainly focuses on antibiotics. However, antibiotic treatment is prone to cause bacterial drug resistance. When large doses of antibiotics such as penicillin and streptomycin are applied, drug residues will appear in the milk. These residual antibiotics may cause allergic reactions in the body, and in severe cases, anaphylactic shock may occur, endangering human health and life. Moreover, there are a wide variety of pathogenic microorganisms causing mastitis, and the diagnosis of mastitis is difficult, resulting in the blind and abusive use of antibiotics in pastures, exacerbating the side effects brought by antibiotics. Therefore, how to reduce the use of antibiotics while ensuring the treatment effect of mastitis or find green and safe antibiotic alternative drugs is a hot issue that needs to be solved urgently at present. Summary of the Invention

[0004] The purpose of the present invention is to provide a bromelain composition for treating mastitis in dairy cows, which is green, safe, and has good curative effects, and its application. This composition has the effects of detumescence, stasis dissipation, heat clearing, detoxification, and anti-inflammation, with small toxic and side effects, not easily generating drug resistance, high bioavailability, high curative effect, no residue, no drug resistance, green, and safe for mastitis in dairy cows caused by pathogenic bacteria infection.

[0005] The technical solution adopted by the present invention is as follows: A bromelain composition for treating mastitis in dairy cows, which is composed of a compound of bromelain, cystine, and sodium gluconate. The weight part ratio of each component is: 1 - 20 parts of bromelain; 4 - 40 parts of cystine; 0.2 - 2 parts of sodium gluconate.

[0006] Further, the bromelain is preferably bromelain extracted from the stems of pineapple fruits over 2 years old.

[0007] The method of using the bromelain composition for treating mastitis in dairy cows of the present invention is as follows: when preventing and treating mastitis in dairy cows, for the powder, 0.2 g per kg of body weight; for the granule, 0.1 g per kg of body weight; for the oral liquid, 0.1 ml per kg of body weight; for the injection, 0.05 ml per kg of body weight, by intramuscular injection, once a day; all are used continuously for 3 - 5 days.

[0008] For the bromelain composition for treating mastitis in dairy cows, the preparation method of the solid composition is: mixing solid bromelain, cystine, and sodium gluconate in a weight ratio of: 1 - 20 parts of bromelain; 4 - 40 parts of cystine; and 0.2 - 2 parts of sodium gluconate.

[0009] For the bromelain composition for treating mastitis in dairy cows, the preparation method of the liquid composition is: dissolving the solid composition in a phosphate buffer solution with a concentration of 10 mM and a pH of 7.4, so that the concentrations (mass - volume) of bromelain, cystine, and sodium gluconate reach 1 - 20 mg / mL, 4 - 40 mg / mL, and 0.2 - 2 mg / mL respectively, or preparing a 4 - fold concentrated solution for use.

[0010] The present invention also discloses the application of the bromelain composition in the preparation of drugs for treating mastitis in dairy cows. The bromelain composition can be made into various pharmaceutically acceptable dosage forms for treating mastitis in dairy cows according to market demands, production conditions, and actual needs according to conventional processes. The dosage forms include suitable dosage forms such as powders, granules, oral liquids, and injections.

[0011] In the present invention, the bromelain used is the general name of a class of proteolytic enzymes present in the stems, fruits, and peels of pineapple fruits. Among them, the protease content in the pineapple stems is the highest, and the bromelain is obtained by pressing, refining, purifying, concentrating, enzyme immobilization, and freeze - drying of pineapple stems. At present, it is widely used in human clinical medicine and belongs to other antipyretic, analgesic, and anti - inflammatory drugs. It is mainly used for the adjuvant treatment of various inflammations, such as osteoarthritis, enteritis, mastitis, metritis, pancreatitis, etc. Clinically, it can be used as an anti - edema and anti - inflammatory drug. Bromelain can inhibit the aggregation of inflammatory factors and reduce vascular permeability, thereby reducing the inflammatory response. After taking bromelain, a part is absorbed in the form of active polypeptides, and these polypeptides can enhance the hydrolysis of fibrin and casein, remove fibrin or coagulation caused by inflammation or trauma, etc., improve local circulation, reduce vascular permeability, and have a good effect of detumescence and anti - inflammation.

[0012] The present invention mainly uses bromelain, and combines cystine, sodium gluconate with bromelain to prepare a composition for treating mastitis in dairy cows. After the combined composition is ingested into the human body with food, its main component bromelain, as a protein, can be hydrolyzed into small peptides and amino acids by proteases in the digestive tract, and then absorbed and utilized; while cystine itself is a nutrient component that can be absorbed and transformed by the human body, and can enhance the redox ability of body cells, promote the proliferation of white blood cells, and play a role in preventing the development of pathogenic bacteria in medicine. As a feed nutritional fortifier, cystine can promote the growth of the body, is beneficial to the development of animals, and enhances the liver and kidney functions. At the same time, cystine can also reduce the absorption ability of copper, prevent copper poisoning, and neutralize toxins. Sodium gluconate, as a new type of feed additive, because of its stable properties, inability to be absorbed by the intestine, safety and reliability, can be excreted from the body through the digestive tract, and has good chelating properties. Sodium gluconate is a chelating agent for divalent cations Mg 2+ and Ca 2+ in the bacterial cell surface. Mg 2+ and Ca 2+ can stabilize the negative charges carried by peptidoglycan, teichoic acid and lipopolysaccharide in bacterial cells, enhance the cross-linking and folding of these molecules, and improve the compactness of the bacterial surface. After Mg 2+ and Ca 2+ are chelated, not only the cross-linking and folding of the bacterial cell wall are weakened, but also a part of the lipopolysaccharide fragments on the cell surface are dissolved out to form pores. The present invention combines and uses bromelain, cystine and sodium gluconate, which greatly improves the effect of treating mastitis in dairy cows. The bromelain, cystine and sodium gluconate used in the present invention can all be obtained commercially.

[0013] The beneficial effects of the present invention are as follows: The antibacterial test of the present invention shows that the bromelain composition for treating mastitis in dairy cows of the present invention has strong antibacterial and bactericidal effects on Escherichia coli and Staphylococcus aureus, the main pathogenic bacteria of mastitis in dairy cows. Moreover, compared with the antibacterial effect of bromelain alone or the antibacterial effect of the composition prepared by combining bromelain with cystine or sodium gluconate, the composition prepared by combining bromelain, cystine and sodium gluconate in the present invention has the best antibacterial effect on Escherichia coli and Staphylococcus aureus, the bactericidal and antibacterial rate is greatly improved, the antibacterial effect is greatly enhanced, and it can more effectively kill various pathogenic bacteria in the mammary glands of dairy cows, and is more conducive to the treatment of mastitis in dairy cows.

[0014] Clinical trials of the present invention have shown that, compared with the effect of treating mastitis in dairy cows with bromelain alone, the bromelain composition of the present invention has a better therapeutic effect on mastitis in dairy cows. The composition for treating mastitis in dairy cows of the present invention can reduce the somatic cell count in the milk of dairy cows suffering from mastitis and the contents of ALP, NAG, and LDH in the milk and serum of dairy cows within a shorter period of time, bringing them back to the normal range values, and has a very good therapeutic effect on mastitis in dairy cows, with high efficacy and quick results. Moreover, the bromelain composition provided by the present invention is green, safe, and residue-free, and is suitable for clinical promotion to replace antibiotics in the treatment of mastitis in dairy cows.

[0015] A bromelain composition for treating mastitis in dairy cows provided by the present invention is composed of a compound of bromelain, cystine, and sodium gluconate. Compared with antibiotics, it has the characteristics of being green, residue-free, and safe. This composition has the effects of detumescence, stasis dissipation, heat clearing, detoxification, and anti-inflammation, with small toxic and side effects, is not prone to drug resistance, has high bioavailability, and has high efficacy, no residue, no drug resistance, is green, and is safe for the mastitis in dairy cows caused by pathogen infection. Brief Description of the Drawings

[0016] Figure 1 The bacteriostatic rate of the bromelain composition of Formulation 1 of the present invention and each comparative experiment against Escherichia coli ATCC25922; Figure 2 The bacteriostatic rate of the bromelain composition of Formulation 1 of the present invention and each comparative experiment against Staphylococcus aureus ATCC29213; Figure 3 The bacteriostatic rate of the bromelain composition of Formulation 2 of the present invention and each comparative experiment against Escherichia coli ATCC25922; Figure 4 The bacteriostatic rate of the bromelain composition of Formulation 2 of the present invention and each comparative experiment against Staphylococcus aureus ATCC29213. Detailed Description of the Invention Example 1

[0017] A bromelain composition for treating mastitis in dairy cows, which is prepared by compounding and mixing bromelain, cystine, and sodium gluconate. The weight ratio of each component is: 4 parts of bromelain; 16 parts of cystine and 0.8 parts of sodium gluconate. The bromelain is extracted from the stem of pineapple fruits over 2 years old. This composition is made into a powder or granule acceptable for veterinary clinical use for treating mastitis in dairy cows according to actual needs by conventional processes. Example 2

[0018] A bromelain composition for treating mastitis in dairy cows, which is composed of bromelain, cystine and sodium gluconate through compounding. The weight ratio of each component is: 15 parts of bromelain; 35 parts of cystine; 1.6 parts of sodium gluconate. This composition is made into an oral liquid or injection acceptable for veterinary clinical use for treating mastitis in dairy cows according to actual needs by conventional processes. Example 3

[0019] A bromelain composition for treating mastitis in dairy cows, which is composed of bromelain, cystine and sodium gluconate through compounding. The weight ratio of each component is: 10 parts of bromelain; 20 parts of cystine; 1 part of sodium gluconate. The bromelain is extracted from the stems of pineapples over 2 years old. This composition is made into a pharmaceutical dosage form acceptable for veterinary clinical use for treating mastitis in dairy cows according to market demand, production conditions and actual needs by conventional processes. Example 4

[0020] A bromelain composition for treating mastitis in dairy cows. The preparation method of the solid composition is: mixing solid bromelain, cystine and sodium gluconate in a weight ratio of: 18 parts of bromelain; 37 parts of cystine and 1.9 parts of sodium gluconate. Example 5

[0021] A bromelain composition for treating mastitis in dairy cows, which is compounded and mixed by bromelain, cystine and sodium gluconate. The weight ratio of each component is: 12 parts of bromelain; 25 parts of cystine; 1.2 parts of sodium gluconate. The bromelain is extracted from the stems of pineapples over 2 years old. Example 6

[0022] A bromelain composition for treating mastitis in dairy cows, which is compounded by bromelain, cystine and sodium gluconate. The bromelain is extracted from the stems of pineapples over 2 years old. The preparation method of the liquid composition is: dissolving the solid composition in a phosphate buffer solution with a concentration of 10 mM and a pH of 7.4, so that the concentrations of bromelain, cystine and sodium gluconate reach 1 mg / mL, 4 mg / mL and 0.2 mg / mL respectively, or preparing a 4-fold concentrated solution for use. Example 7

[0023] A bromelain composition for treating mastitis in dairy cows, which is prepared by compounding bromelain, cystine and sodium gluconate. The preparation method of the liquid composition is as follows: Dissolve the solid composition in a phosphate buffer solution with a concentration of 10 mM and a pH of 7.4, so that the concentrations of bromelain, cystine and sodium gluconate reach 10 mg / mL, 20 mg / mL and 1 mg / mL respectively, or prepare a concentrated solution with a 4-fold concentration for use. Example 8

[0024] A bromelain composition for treating mastitis in dairy cows, which is prepared by compounding bromelain, cystine and sodium gluconate. The bromelain is extracted from the stems of pineapples over 2 years old. The preparation method of the liquid composition is as follows: Dissolve the solid composition in a phosphate buffer solution with a concentration of 10 mM and a pH of 7.4, so that the concentrations of bromelain, cystine and sodium gluconate reach 18 mg / mL, 40 mg / mL and 1.8 mg / mL respectively.

[0025] Example 9 Pharmacodynamic Test The bromelain is a product of Nanning Pangbo Bioengineering Co., Ltd., with an enzyme activity of 20,000 u / g. Its production process is as follows: pineapple stem → pressing → plate and frame separation → centrifugate → microfiltration → ultrafiltration → concentrated solution → freeze-drying → pulverization → mixing → packaging → finished product. Cystine and sodium gluconate are domestic analytical pure reagents. Escherichia coli ATCC25922 and Staphylococcus aureus ATCC29213 are respectively purchased from the National Institute for the Control of Pharmaceutical and Biological Products.

[0026] I. In vitro antibacterial test of the bromelain composition for treating mastitis in dairy cows of the present invention (I) Antibacterial effect of the bromelain composition of Formula 1 of the present invention on Escherichia coli ATCC25922 Weigh 4 mg of bromelain, 16 mg of cystine and 0.8 mg of sodium gluconate respectively, mix them evenly, dissolve them in 4 ml of a phosphate buffer solution with a concentration of 10 mM and a pH of 7.4 to prepare a bromelain composition solution. The concentrations of bromelain, glycine and sodium gluconate in the solution are 1 mg / ml, 4 mg / ml and 0.2 mg / ml respectively. Add 200 μl of the prepared bromelain composition solution to 200 μl of Escherichia coli ATCC25922 bacterial solution with a bacterial concentration of 10 6 CFU / ml and 400 μl of LB medium and act for two hours. The antibacterial rate against Escherichia coli ATCC25922 is 10 2.78 .

[0027] The calculation method of the bacteriostatic rate is as follows: the bacteriostatic rate is expressed as N / Y, where N represents the number of colonies formed by the sample without adding the composition solution of the present invention, and Y represents the number of colonies formed by the sample adding the composition solution of the present invention. The same applies hereinafter.

[0028] Comparative experiment: ① Dissolve 4 mg of bromelain alone in 4 mL of phosphate buffer with a concentration of 10 mM and pH 7.4. The concentration of bromelain is 1 mg / ml. Take 200 μL of it and add it to 200 μL of Escherichia coli ATCC25922 bacterial solution with a bacterial concentration of 10 6 CFU / mL and 400 μL of LB medium and act for two hours. The bacteriostatic rate against Escherichia coli ATCC25922 is 10 0.94 ; ② Dissolve 4 mg of bromelain and 16 mg of cystine in 4 mL of phosphate buffer with a concentration of 10 mM and pH 7.4. The concentrations of bromelain and cystine are 1 mg / mL and 4 mg / mL respectively. Take 200 μL of it and add it to 200 μL of Escherichia coli ATCC25922 bacterial solution with a bacterial concentration of 10 6 CFU / mL and 400 μL of LB medium and act for two hours. The bacteriostatic rate against Escherichia coli ATCC25922 is 10 1.37 ; ③ Dissolve 4 mg of bromelain and 0.8 mg of sodium gluconate in 4 mL of phosphate buffer with a concentration of 10 mM and pH 7.4. The concentrations of bromelain and sodium gluconate are 1 mg / mL and 0.2 mg / mL respectively. Take 200 μL of it and add it to 200 μL of Escherichia coli ATCC25922 bacterial solution with a bacterial concentration of 10 6 CFU / mL and 400 μL of LB medium and act for two hours. The bacteriostatic rate against Escherichia coli ATCC25922 is 10 1.19 .

[0029] In this bacteriostatic experiment, the bacteriostatic rates of the bromelain composition of the present invention's formula 1 and each comparative experiment against Escherichia coli ATCC25922 are shown in the appendix Figure 1 . Compared with the bromelain in comparative experiment ①, the bacteriostatic rate of the bromelain composition of the present invention's formula 1 is increased by 69.18 times; compared with the composition in comparative experiment ②, the bacteriostatic rate is increased by 25.71 times; compared with the composition in comparative experiment ③, the bacteriostatic rate is increased by 38.90 times.

[0030] (2) Bacteriostatic effect of the bromelain composition of the present invention's formula 1 against Staphylococcus aureus ATCC29213 Weigh 4 mg of bromelain, 16 mg of cystine, and 0.8 mg of sodium gluconate separately, mix them evenly, dissolve them in 4 ml of 10 mM, pH 7.4 phosphate buffer to prepare a bromelain composition solution. The concentrations of bromelain, glycine, and sodium gluconate in the solution are 1 mg / ml, 4 mg / ml, and 0.2 mg / ml respectively. Add 200 μl of the prepared bromelain composition solution to 200 μl of Staphylococcus aureus ATCC 29213 bacterial solution with a bacterial concentration of 10 6 CFU / ml and 400 μl of LB medium and act for two hours. The antibacterial rate against Staphylococcus aureus ATCC 29213 is 10 2.62 .

[0031] Control experiment: ① Dissolve 4 mg of bromelain alone in 4 mL of 10 mM, pH 7.4 phosphate buffer. The concentration of bromelain is 1 mg / ml. Take 200 μL of it and add it to 200 μL of Staphylococcus aureus ATCC 29213 bacterial solution with a bacterial concentration of 10 6 CFU / mL and 400 μL of LB medium and act for two hours. The antibacterial rate against Staphylococcus aureus ATCC 29213 is 10 0.81 ; ② Dissolve 4 mg of bromelain and 16 mg of cystine in 4 mL of 10 mM, pH 7.4 phosphate buffer. The concentrations of bromelain and cystine are 1 mg / mL and 4 mg / mL respectively. Take 200 μL of it and add it to 200 μL of Staphylococcus aureus ATCC 29213 bacterial solution with a bacterial concentration of 10 6 CFU / mL and 400 μL of LB medium and act for two hours. The antibacterial rate against Staphylococcus aureus ATCC 29213 is 10 1.36 ; ③ Dissolve 4 mg of bromelain and 0.8 mg of sodium gluconate in 4 mL of 10 mM, pH 7.4 phosphate buffer. The concentrations of bromelain and sodium gluconate are 1 mg / mL and 0.2 mg / mL respectively. Take 200 μL of it and add it to 200 μL of Staphylococcus aureus ATCC 29213 bacterial solution with a bacterial concentration of 10 6 CFU / mL and 400 μL of LB medium and act for two hours. The antibacterial rate against Staphylococcus aureus ATCC 29213 is 10 1.24 .

[0032] In this antibacterial experiment, the antibacterial rates of the bromelain composition of Formula 1 of the present invention and each control experiment against Staphylococcus aureus ATCC 29213 are shown in the appendix Figure 2, when comparing the bromelain composition of Formula 1 of the present invention with the bromelain in Comparative Experiment ①, the bacteriostatic rate increased by 64.57 times; when compared with the composition in Comparative Experiment ②, the bacteriostatic rate increased by 18.20 times; when compared with the composition in Comparative Experiment ③, the bacteriostatic rate increased by 23.99 times.

[0033] (III) Bacteriostatic effect of the bromelain composition of Formula 2 of the present invention on Escherichia coli ATCC25922 Weigh 40 mg of bromelain, 80 mg of cystine, and 4 mg of sodium gluconate respectively, mix them evenly, dissolve them in 4 ml of 10 mM, pH 7.4 phosphate buffer to prepare a bromelain composition solution. The concentrations of bromelain, glycine, and sodium gluconate in the solution are 10 mg / ml, 20 mg / ml, and 1 mg / ml respectively. Add 200 μl of the prepared bromelain composition solution to 200 μl of Escherichia coli ATCC25922 bacterial solution with a bacterial concentration of 10 6 CFU / ml and 400 μl of LB medium and act for two hours. The bacteriostatic rate against Escherichia coli ATCC25922 is 10 3.26 .

[0034] Comparative experiment: ① Dissolve 40 mg of bromelain alone in 4 mL of 10 mM, pH 7.4 phosphate buffer. The concentration of bromelain is 10 mg / ml. Take 200 μL of it and add it to 200 μL of Escherichia coli ATCC25922 bacterial solution with a bacterial concentration of 10 6 CFU / mL and 400 μL of LB medium and act for two hours. The bacteriostatic rate against Escherichia coli ATCC25922 is 10 0.89 ; ② Dissolve 40 mg of bromelain and 80 mg of cystine in 4 mL of 10 mM, pH 7.4 phosphate buffer. The concentrations of bromelain and cystine are 10 mg / mL and 20 mg / mL respectively. Take 200 μL of it and add it to 200 μL of Escherichia coli ATCC25922 bacterial solution with a bacterial concentration of 10 6 CFU / mL and 400 μL of LB medium and act for two hours. The bacteriostatic rate against Escherichia coli ATCC25922 is 10 1.41 ; ③ Dissolve 40 mg of bromelain and 4 mg of sodium gluconate in 4 mL of 10 mM, pH 7.4 phosphate buffer. The concentrations of bromelain and sodium gluconate are 10 mg / mL and 1 mg / mL respectively. Take 200 μL of it and add it to 200 μL of Escherichia coli ATCC25922 bacterial solution with a bacterial concentration of 10 6The Escherichia coli ATCC25922 bacterial solution at a concentration of CFU / mL was incubated with 400 μL of LB medium for two hours, and the antibacterial rate against Escherichia coli ATCC25922 was 10 1.21 .

[0035] In this antibacterial experiment, the antibacterial rates of the bromelain composition of Formula 2 of the present invention and each comparative experiment against Escherichia coli ATCC25922 are shown in the appendix Figure 3 . Compared with bromelain in Comparative Experiment ①, the antibacterial rate of the bromelain composition of Formula 2 of the present invention increased by 234.5 times; compared with the composition in Comparative Experiment ②, the antibacterial rate increased by 70.81 times; compared with the composition in Comparative Experiment ③, the antibacterial rate increased by 112.19 times.

[0036] (4) Antibacterial effect of the bromelain composition of Formula 2 of the present invention against Staphylococcus aureus ATCC29213 Weigh 40 mg of bromelain, 80 mg of cystine, and 4 mg of sodium gluconate respectively, mix them evenly, dissolve them in 4 ml of 10 mM, pH 7.4 phosphate buffer to prepare a bromelain composition solution. The concentrations of bromelain, cystine, and sodium gluconate in the solution are 10 mg / ml, 20 mg / ml, and 1 mg / ml respectively. Add 200 μl of the prepared bromelain composition solution to 200 μl of the Staphylococcus aureus ATCC29213 bacterial solution with a concentration of 10 6 CFU / ml and 400 μl of LB medium and incubate for two hours. The antibacterial rate against Staphylococcus aureus ATCC29213 is 10 3.17 .

[0037] Comparative experiment: ① Dissolve 40 mg of bromelain alone in 4 mL of 10 mM, pH 7.4 phosphate buffer. The concentration of bromelain is 10 mg / ml. Take 200 μL of it and add it to 200 μL of the Staphylococcus aureus ATCC29213 bacterial solution with a concentration of 10 6 CFU / mL and 400 μL of LB medium and incubate for two hours. The antibacterial rate against Staphylococcus aureus ATCC29213 is 10 0.83 ; ② Dissolve 40 mg of bromelain and 80 mg of cystine in 4 mL of 10 mM, pH 7.4 phosphate buffer. The concentrations of bromelain and cystine are 10 mg / mL and 20 mg / mL respectively. Take 200 μL of it and add it to 200 μL of the Staphylococcus aureus ATCC29213 bacterial solution with a concentration of 10 6 CFU / mL and 400 μL of LB medium and incubate for two hours. The antibacterial rate against Staphylococcus aureus ATCC29213 is 101.32 ; ③ Dissolve 40 mg of bromelain and 4 mg of sodium gluconate in 4 mL of phosphate buffer with a concentration of 10 mM and pH 7.4. The concentrations of bromelain and sodium gluconate are 10 mg / mL and 1 mg / mL respectively. Take 200 μL of it and add it to 200 μL of Staphylococcus aureus ATCC29213 bacterial solution with a bacterial concentration of 10 6 CFU / mL and 400 μL of LB medium and act for two hours. The antibacterial rate against Staphylococcus aureus ATCC29213 is 10 1.22 .

[0038] In this antibacterial experiment, the antibacterial rates of the bromelain composition of Formula 2 of the present invention and each comparative experiment against Staphylococcus aureus ATCC29213 are shown in the appendix Figure 4 . Compared with bromelain in Comparative Experiment ①, the antibacterial rate of the bromelain composition of Formula 2 of the present invention is increased by 218.8 times; compared with the composition in Comparative Experiment ②, the antibacterial rate is increased by 70.8 times; compared with the composition in Comparative Experiment ③, the antibacterial rate is increased by 89.13 times.

[0039] From the above 4 antibacterial experiments, it can be seen that the bromelain compositions of Formula 1 and Formula 2 of the present invention for treating cow mastitis have strong antibacterial and bactericidal effects on the main pathogenic bacteria of cow mastitis: Escherichia coli ATCC25922 and Staphylococcus aureus ATCC29213. Moreover, compared with the antibacterial effect of bromelain alone or the antibacterial effect of the composition prepared by compounding bromelain with cystine or sodium gluconate, the antibacterial rates of the bromelain composition prepared by compounding bromelain, cystine and sodium gluconate of the present invention against Escherichia coli and Staphylococcus aureus are greatly improved, the antibacterial effect is greatly enhanced, the antibacterial effect is the best, and it can more effectively kill the pathogenic bacteria in the cow mammary gland, which is more beneficial to the treatment of cow mastitis.

[0040] II. Application effect In order to verify the treatment effect of the bromelain composition of the present invention for treating cow mastitis on cow mastitis, we conducted a curative effect test of the composition of the present invention on cow mastitis in a farm in Nanning where cow mastitis occurred.

[0041] The cows with 2-4 births, similar body weight, lactation period and milk production, and the same feeding environment and management level were selected. First, the somatic cell count of milk samples of all lactating cows was detected by somatic cell electronic counting method, and 20 healthy cows with somatic cell count <500,000 / ml were randomly selected as the control group (the control group was not treated in any way), and 40 cows with somatic cell count >1 million / ml and no other clinical diseases except clinical mastitis were randomly selected as the clinical mastitis group: 20 cows in the test group I were intramuscularly injected with the bromelain composition of the present invention (0.05 ml per kg body weight, intramuscular injection, once a day at 9:00), and the test group II was intramuscularly injected with bromelain alone ((0.05 ml per kg body weight, intramuscular injection, once a day at 9:00)). The data of the number of somatic cells in the milk of each group of cows, the content of ALP, NAG, LDH in the milk of each group of cows, and the content of ALP, NAG, LDH in the serum of each group of cows were detected and recorded on the 0th day, the 3rd day and the 5th day, so as to evaluate the therapeutic effect of the composition of the present invention.

[0042] (I) Test Group I 10 parts of bromelain, 20 parts of cystine and 1 part of sodium gluconate were mixed in a weight ratio to prepare the bromelain composition of the present invention. 5 parts of the bromelain composition were added with 10 parts of 0.9% physiological saline, stirred evenly, filtered through a microporous filter membrane (pore size 0.22 μm-0.24 μm), sterilized, and filled in a glass ampoule to prepare an injection.

[0043] (II) In Experiment II, 10 portions of bromelain were taken separately, and 10 portions of 0.9% normal saline were added, stirred evenly, filtered through a microporous filter membrane (pore size 0.22μm-0.24μm), sterilized, and filled into glass ampoules to prepare injections.

[0044] The number of somatic cells in the milk of cows in each group is shown in Table 1 below.

[0045]

[0046] Test results: The changes in the number of somatic cells in the milk of each group of dairy cows, the changes in the ALP, NAG, and LDH contents in the milk of each group of dairy cows, and the changes in the ALP, NAG, and LDH contents in the serum of each group of dairy cows are shown in Tables 2, 3, and 4, respectively.

[0047]

[0048]

[0049]

[0050] As can be seen from the above clinical trial results, for dairy cows suffering from clinical mastitis, after injection with the composition of the present invention, within 5 days, the somatic cell count in milk decreased, and the contents of ALP, NAG, and LDH in milk and dairy cow serum also decreased, and all the values decreased to the normal values of healthy cows.

[0051] For dairy cows suffering from clinical mastitis, when bromelain was injected intramuscularly alone, within 5 days, the somatic cell count in milk decreased, and the contents of ALP, NAG, and LDH in milk and dairy cow serum decreased numerically, but did not decrease to the normal values of healthy cows.

[0052] From the results, the effect of the injection of the bromelain composition at an equal dose is significantly better than that of using bromelain injection alone. The bromelain composition obtained by compounding in the present invention has a better therapeutic effect on treating mastitis in dairy cows. The bromelain composition injection of the present invention has basically healed after 5 days of intramuscular injection in mastitis dairy cows, replacing antibiotics. The somatic cell count in milk has decreased, and the contents of ALP, NAG, and LDH in milk and dairy cow serum have reached the levels of healthy dairy cows. It can be seen that the bromelain composition provided by the present invention can effectively treat mastitis in dairy cows, quickly eliminate symptoms, enable dairy cows to recover health, increase the yield and quality of dairy products, have stable quality, and is green, safe and residue-free, and can be used for clinical promotion to replace antibiotic treatment.

Claims

1. A bromelain composition for treating bovine mastitis, characterized in that: The composition is prepared by compounding bromelain, cystine and sodium gluconate, and the weight proportion of each component is: 1-20 parts of bromelain; 4-40 parts of cystine; and 0.2-2 parts of sodium gluconate.

2. The bromelain composition for treating bovine mastitis according to claim 1, characterized in that: The bromelain is extracted from the stems of fruit pineapples over 2 years old.

3. The bromelain composition for treating bovine mastitis according to claim 1 or 2, characterized in that: The preparation method of the solid composition is as follows: solid bromelain, cystine and sodium gluconate are mixed in a weight ratio of 1-20 parts of bromelain, 4-40 parts of cystine and 0.2-2 parts of sodium gluconate.

4. The bromelain composition for treating bovine mastitis according to claim 1 or 2, characterized in that: The preparation method of the liquid composition is as follows: dissolving the solid composition in a phosphate buffer with a concentration of 10 mM and a pH of 7.4, so that the concentrations of bromelain, cystine, and sodium gluconate reach 1-20 mg / mL, 4-40 mg / mL, and 0.2-2 mg / mL, respectively, or preparing a concentrated solution with a concentration of 4 times the concentration for use.

5. Use of the bromelain composition as claimed in claim 1 or 2 in the preparation of a medicament for treating bovine mastitis.

6. The use according to claim 5, characterized in that The bromelain composition is prepared into a pharmaceutical dosage form for treating bovine mastitis that is clinically acceptable to veterinarians according to actual needs and conventional processes. The pharmaceutical dosage form includes powder, granules, oral solution and injection.

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