CN1209972C - Sterilization formulation and wet tissue, preparation method and use - Google Patents

Abstract

The present invention discloses a sterilization preparation which comprises 0.0001 to 0.1 wt% of lysostaphin, 10 to 50 wt% lysozyme, 1 to 15 wt% of stabilizing agent, 0.1 to 0.5 wt% of surface active agent, and water as the rest. The present invention also discloses a sterilization wet tissue, a preparation method for the sterilization wet tissue, and an application of the sterilization wet tissue to treating and preventing mammary gland infection of a milk cow. A sterilization wet tissue is obtained in a mode that the sterilization preparation is loaded on a carrier. The sterilization wet tissue provided by the present invention is used for sterilizing various bacteria causing mammary gland infection of a milk cow, and can obtain a very obvious effect. Particularly, the sterilization rate of the sterilization wet tissue on sterilizing escherichia coli, staphylococcus aureus, streptococcus agalactiae, streptococcus dysgalactiae, streptococcus uberis and corynebacterium pyogenes is higher than 99%.

Description

A kind of bactericidal preparation and bactericidal wet tissue and its preparation method and application

technical field

The invention relates to a bactericidal preparation and a bactericidal material, in particular to a bactericidal preparation and a bactericidal wet tissue for killing bacteria that cause milk cow mammary gland infection.

Background technique

Dairy cow mammary gland infection is mainly caused by Staphylococcus aureus, Escherichia coli, Streptococcus, Pseudomonas aeruginosa, etc. Herd surveys show that 30% to 70% of cows in the United States have at least one infected udder area. Mastitis caused by mammary gland infection in dairy cows causes a loss of 0.5 kg of milk production per day, and the reduced milk production is estimated to be 69%-80% of the total loss due to mastitis. The U.S. dairy industry loses more than $2 billion annually in direct losses from mastitis. It can be seen that the economic impact of mastitis on the dairy industry is huge.

At home and abroad, large doses of antibiotics (such as penicillin, streptomycin, gentamicin, chloramphenicol, neomycin, etc.) are often used to treat dairy cow mammary gland infection. Although antibiotics have certain curative effects, most of the above-mentioned pathogenic bacteria produce Drug-resistant strains have been eliminated, so the efficacy is not obvious. On the contrary, it causes antibiotic residues in the milk, and the consequences of mixing the milk produced by these cows into the edible milk are serious. Both the Food and Agriculture Organization of the United Nations (FAO) and the World Health Organization (WHO) recommend that the milk expressed within at least 3 days after the withdrawal of antibiotic treatment by cows should not be directly used as raw milk for consumption (it is best to stop the drug for 5-7 days) ). At present, there is no other medicine to deal with mastitis in the world except the use of antibiotics.

U.S. Patent No. 5,762,948 discloses a wet wipe for the prevention and treatment of cow mammary gland infection. Its composition is mainly based on an antibacterial peptide nisin, supplemented by other antibacterial ingredients such as alcohol. The wet tissue can remove various pathogenic bacteria that cause mammary gland infection, but the effect on drug-resistant Staphylococcus aureus is not obvious. At the same time, due to the complicated synthesis process and high production cost of antimicrobial peptides, it is difficult to popularize and apply.

Contents of the invention

One of the technical problems to be solved by the present invention is to provide a preparation for killing bacteria that cause mammary gland infection of dairy cows, so as to overcome the above-mentioned defects in the prior art;

The second technical problem to be solved by the present invention is to provide a bactericidal wet wipe that kills bacteria that cause milk cow mammary gland infection.

The third technical problem to be solved by the present invention is to provide a preparation method of sterilizing wet wipes.

Another technical problem to be solved by the present invention is to disclose the application of the bactericidal wet wipe.

The inventive concept of the present invention is such:

Lysostaphin is a zinc-containing metalloprotease that specifically degrades the glycine peptide bond of the cell wall, and can crack the cell wall of Gram-positive bacteria such as Staphylococcus aureus, thereby completely killing the bacteria. It is not easy to produce drug resistance, especially for pathogenic bacteria that are difficult to eradicate such as drug-resistant Staphylococcus aureus. But lysostaphin is only effective to Gram-positive bacteria, and the bacterium that causes milk cow mammary gland infection is except Gram-positive bacteria, and Gram-negative bacteria also account for a certain proportion, such as escherichia coli etc., so inventor will Lysozyme and lysostaphin are compounded, lysozyme can cleave the β-1, 4 glycosidic bond of the bacterial cell wall, and has a killing effect on both Gram-positive bacteria and Gram-negative bacteria. In addition to the bactericidal spectrum, it can also strengthen the bactericidal effect of lysostaphin. Both enzymes belong to proteins, without stimulation and without any toxic side effects, and at the same time, due to low production costs, the price is not expensive.

The more effective way to eliminate and control mammary gland infection in dairy cows is to kill all kinds of bacteria that cause infection, especially various pathogenic bacteria in the milk area, which not only cause mammary gland infection, but also infect other dairy cows. After many experiments, the inventor compounded lysostaphin and lysozyme in a certain proportion, and made sterilizing wet wipes from this material, which can easily and effectively kill various pathogenic bacteria in the milk area of cows, eliminate Infection, but also play a role in preventing recurrence and prevention.

Technical scheme of the present invention is such:

A bactericidal preparation comprises 0.0001-0.1wt% of lysostaphin, 10-50wt% of lysozyme, 1-15wt% of stabilizer, 0.1-0.5wt% of surface active agent and the balance of water.

The mentioned stabilizer includes one of polyethylene glycol or chitosan, and its main function is to maintain the stability of the enzyme in the aqueous solution state, that is, to maintain the activity of the enzyme.

The surfactants mentioned include nonionic surfactants or zwitterionic surfactants, preferably 20≥HLB≥6, such as polysorbate series, including polysorbate 20, polysorbate 40, polysorbate Sorbitan 60 or polysorbate 80, etc. and betaine, etc., are mainly used to remove the dirt in the milk area of cows, so that the active ingredients in the preparation can fully act on the skin surface, which is conducive to absorption.

The above-mentioned bactericidal preparation can be loaded on a carrier to make a bactericidal wet tissue, and the carrier includes non-woven material or cotton gauze.

The preparation method of described bactericidal wet tissue comprises the steps:

1. Weigh lysostaphin, lysozyme, stabilizer and surfactant and mix with water according to the above ratio;

2. Spray the prepared solution on the carrier of the required specification or soak the carrier in the solution, take it out, and obtain the bactericidal wet wipe of the present invention.

The formulation and antiseptic wipes of the present invention are used to kill various bacteria, including Gram-negative bacteria and Gram-positive bacteria, that cause mammary gland infection in dairy cows. In particular Escherichia coli, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis and Corynebacterium pyogenes. Various types of mastitis caused by these bacteria can be treated, and the occurrence of mastitis can also be prevented.

The preparation and wet wipes of the present invention can be smeared or wiped on the milk area of cows before and after milk production to carry out disinfection, so as to ensure the cleanliness of the milk area and reduce the possibility of infection.

The preparation or sterilizing wet tissue of the present invention can be applied or covered on the infected udder area of the cow, and the dose can be determined according to the age and condition of the cow, generally twice a day.

Bactericidal wet wipes have passed the in vitro bactericidal test, which proves that it has an ideal bactericidal effect on the main pathogenic bacteria that cause dairy cow mammary gland infection: Escherichia coli, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Corynebacterium pyogenes, The killing rate is more than 99%.

The results of the acute toxicity test of bactericidal wet wipes on Kunming mice showed that after 7 days of intramuscular injection, the animals had no abnormal reaction and their activities were normal. After 7 days of observation, all 60 mice survived. Proved to be non-toxic.

Sterilizing wipes were used to test the vaginal mucosa irritation of SD rats. The intensity of vaginal mucosa irritation was evaluated by referring to the scoring criteria for the intensity of skin irritation reactions and the classification of skin irritation intensity in the Disinfection Technical Specifications (Third Edition) 1999.11. The results showed that the sterilizing wipes had no irritation to the vaginal mucosa of rats, with the highest total integral mean value of 0.

The treatment test of bactericidal wet wipes on acute infection in the breast area, and using penicillin G potassium and streptomycin as controls, after 7 days of administration, the results show that bactericidal wet wipes can effectively kill various pathogenic bacteria that cause breast infection, especially for golden yellow Staphylococci were particularly effective, while the two antibiotics were not effective in removing pathogens from the dairy area.

The test of sterilizing wet wipes to prevent mammary gland infection shows that sterilizing wet wipes can effectively prevent bacterial infection in the udder area of cows and keep the udder area clean.

Detailed ways

Example 1

Preparation of antiseptic wipes

1. prepare 100g for the solution of bactericidal wet tissue, wherein lysostaphin content 0.001g (0.001wt%), lysozyme content 20g (20wt%), polyethylene glycol content 3g (3%), polysorbate Ester 20 content 0.2g (0.2wt%).

2. Disinfect tabletops, instruments, utensils, and ingredient barrels.

3. Accurately weigh lysostaphin, lysozyme, polyethylene glycol and polysorbate 20 on an electronic balance, place them in the batching bucket, add 76.80g of water in proportion, stir evenly, and heat to 60°C to dissolve, That is the desired solution.

4. Cut the non-woven fabric into 8cm×8cm, sterilize it with ultraviolet light for 20 minutes, immerse it in the above solution, remove it after soaking, drip off the excess solution, and then use it.

Example 2

In Vitro Bactericidal Test of Bactericidal Wet Wipes

1. Preparation of test samples:

(1) Sterilizing wet tissue sample: prepared according to the method of Example 1, wherein the content of lysostaphin is 0.001wt%, the content of lysozyme is 20wt%, the content of polyethylene glycol is 3%, and the content of polysorbate 20 is 0.2wt%. .

(2) Contrast wet tissue: wherein the content of polyethylene glycol is 3%, and the content of polysorbate 20 is 0.2 wt%.

2. Test strains: Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Escherichia coli and Corynebacterium pyogenes (the above strains are from the Military Horse Control Institute of Shenyang Military Region).

3. Test steps:

(1) Wash the fresh slant of the cultivated test strain with 1% peptone PBS, and dilute to an appropriate concentration, 10 ⁷ -10 ⁸ cfu/ml.

(2) The sample wet tissue and the control wet tissue were made into a size of 1×3 cm, and placed in a sterile plate.

(3) Take 20ul of the bacterial solution and add it dropwise to the wet tissue, spread evenly and start timing at the same time.

(4) Put the wet wipe into a test tube containing 5ml of neutralizing agent with sterile tweezers after the predetermined time, shake and mix well.

(5) Put the control wet wipe into the test tube of neutralizer, shake it fully and make appropriate dilution.

(6) Take 0.5 ml of the final solution and pour it into a plate, and incubate it upside down at a constant temperature of 37 for 48 hours.

(7) Calculate the amount of recovered bacteria in the control and the recovered bacteria in the sample to obtain the killing rate.

4. Test results

           Table 1: The results of the killing rate of germicidal wet wipes in vitro sterilization test

Test strains Control wipes

Escherichia coli 99.92% 0

Staphylococcus aureus 99.98% 0

Streptococcus agalactiae 99.92% 0

Streptococcus dysgalactiae 99.97% 0

Streptococcus uberis 99.56% 0

Corynebacterium pyogenes 99.46% 0

Example 3

In Vitro Bactericidal Test of Bactericidal Wet Wipes

1. Preparation of test samples:

(1) Sterilizing wet tissue sample: prepared according to the method of Example 1, wherein the content of lysostaphin was 0.001wt%, the content of lysozyme was 20wt%, the content of polyethylene glycol was 3%, and the content of betaine was 0.2wt%.

(2) Contrast wet tissue: wherein the content of polyethylene glycol is 3%, and the content of betaine is 0.2 wt%.

2. Test strains: Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Escherichia coli and Corynebacterium pyogenes (the above strains are from the Military Horse Control Institute of Shenyang Military Region).

3. Test procedure: Carry out according to embodiment 2.

4. Test results

           Table 2: The results of the killing rate of germicidal wet wipes in vitro

Test strains Control wipes

Escherichia coli 99.95% 0

Staphylococcus aureus 99.97% 0

Streptococcus agalactiae 99.95% 0

Streptococcus dysgalactiae 99.98% 0

Streptococcus uberis 99.62% 0

Corynebacterium pyogenes 99.67% 0

Example 4

Acute Toxicity Test in Animals

1. Preparation of samples: prepare the required solution for sterilizing wet wipes according to the method in Example 1, wherein: wherein the content of lysostaphin is 0.01wt%, the content of lysozyme is 30wt%, the content of polyethylene glycol is 5%, poly pear The content of behenyl ester 20 is 0.3wt%.

2. Experimental animals: 60 Kunming mice, half male and half male, weighing 33.5±0.25g, were provided by the Experimental Animal Department of Fudan University.

3. Test method: intramuscularly inject mice once a day for 7 consecutive days, and observe the toxic reaction of the animals at the maximum dose. The experimental results showed that after 7 days of intramuscular injection, the animals had no abnormal reaction and their activities were normal. After 7 days of observation, all 60 mice survived. Proved to be non-toxic.

Example 5

Irritation test of germicidal wipes on vaginal mucous membrane of experimental animals

1. Sample preparation: The preparation method of sterilizing wet wipes refers to Example 1, wherein: the content of lysostaphin is 0.001wt%, the content of lysozyme is 20wt%, the content of polyethylene glycol is 3%, and the content of polysorbate 20 is 0.2% wt%.

2. Experimental animals: SD rats, female, normal grade, 18 rats, weighing 230g-250g, were randomly divided into three groups, sterilizing wet tissue group, excipient group and blank control group, 6 rats in each group, provided by Fudan University Provided by the Laboratory Animal Department of the University.

3. Test method: Refer to Article 3.8 of Disinfection Technical Specifications (Third Edition) 1999.11. Put the sterilizing wipes into the animal's vagina and contact with the vaginal mucosa for 4 hours. The vehicle group used 0.01 g of tampon soaked in sterilized physiological saline for the same test. The blank control group received no treatment. The animals were sacrificed at 24 hours, and local vaginal tissues were taken out to observe whether there was congestion and edema. The vaginal mucosal irritation intensity was evaluated with reference to the scoring standard scoring and skin irritation intensity classification in the disinfection technical specification (third edition) 1999.11.

4. Test results:

        Table 3: Irritation test of bactericidal wet wipes on the vaginal mucosa of test animals

Stimulus Response Score

Group Number of animals (only)                         

                                            

Antiseptic Wipes Set 6 0 0 0 0

Excipient group 6 0 0 0 0

Blank control group 6 0 0 0

The sterilizing wet wipes had the highest total score mean value of 0 for the irritation reaction of the vaginal mucosa of rats, which was non-irritating.

Example 6

Therapeutic Experiment of Bactericidal Wet Wipes on Acute Infection in Udder Area

1. Sample preparation

5. Preparation of bactericidal wet wipes: the preparation method is according to Example 1, wherein the content of lysostaphin is 0.008wt%, the content of lysozyme is 20wt%, the content of polyethylene glycol is 2%, and the content of polysorbate 20 is 0.3wt%. .

Penicillin G Potassium: 800,000 units, North China Pharmaceutical Factory

Streptomycin sulfate: 1 million units, North China Pharmaceutical Factory

2. Preparation of experimental animals

A total of 20 young, high-yielding, black-and-white dairy cows aged 4-8 in the lactation period were selected. Acute mammary gland infection caused inflammation. The symptoms were breast swelling and milk abnormalities: clots, flakes or later flakes. But no pain, no other symptoms. They were randomly divided into 4 groups, group 1 was treated with antiseptic wet wipes, group 2 was treated with penicillin G potassium, group 3 was treated with streptomycin, and group 4 was blank control group.

3. Test plan:

Group 1: Every morning and evening, after squeezing the milk, wipe it repeatedly with a sterilizing wet tissue and cover the infected breast area until the skin absorbs it. Groups 2 and 3: Once a day in the morning and evening, after squeezing the milk dry, inject 100,000 units of penicillin and 100,000 units of streptomycin into the breast. Group 4: the control group, without any treatment, administered for 7 days.

4. Observation indicators:

(1) Recovery: All the symptoms of clinical mastitis disappeared, the breast tissue was soft, there was no clot in the milk, and the color of the milk was as white as usual.

Effective: the symptoms of clinical mastitis are relieved, or no longer worsen, the milk is normal, but the breast swelling has not completely disappeared.

(2) Bacteriological detection: isolate, culture, and identify pathogenic bacteria that infect the milk area before and after treatment, among which Escherichia coli, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, and Udder chain Coccus and Corynebacterium pyogenes are mainly six kinds of bacteria, so this detection test is mainly based on the above six kinds of bacteria. The specific operation steps are as follows:

A. Sampling: Same as conventional aseptic sampling, use 10ml test tubes, collect 5-7ml fresh milk from each dairy area of each cow, pay attention to discard the first 2-3 milks, mark them well, and collect them as soon as possible Store in a 4°C refrigerator.

B. Cultivation and separation: Cultivation is carried out as usual, the temperature is 37°C, and the relative temperature is above 50%. The medium used mainly includes: 7% sheep blood agar; sheep broth agar with 1% aescin added; Chapelman's agar; sodium azide crystal violet blood agar; meat flow medium, etc. (The above medium comes from the Military Horse Prevention and Control Research Institute of Shenyang Military Region)

C. Identification

Identification of Staphylococcus aureus: using test tube method and slide method, using citrate rabbit plasma coagulation test for determination.

Identification of Streptococcus: Use the Camp test. If the Camp test is positive but aescin is negative, it is Streptococcus agalactiae. If the Camp test is positive but aescin is positive, it is Streptococcus uberis. If the Camp test is negative, aescin is also Those who are negative are Streptococcus dysgalactiae.

Identification of Escherichia coli: Escherichia coli was isolated and cultured with eosin-methylene blue medium, and V, P test and M, R test were carried out. If the V, P test was negative, and the M, R test was positive, it was E. coli.

Identification of Corynebacterium pyogenes: cultured on blood agar medium, after 36 hours, if there are small colonies with a diameter of about 1-2cm, graphics, protrusions, milky white, smooth and shiny surface, and β-agar blood circles, that is Corynebacterium pyogenes.

5. Test results

            Table 4: The results of the treatment trial of antiseptic wet wipes on acute infection in the udder area

                                                                                                                                                                                                                          Period period period period over time

Clinically cured (number of heads) 4 2 1 0

Performance Effective (Number of Heads) 1 2 2 2 0

Escherichia coli 99.7 97.2 92.3 0

Pathogen Staphylococcus aureus 99.8 65.3 41.2 0

Bactericidal Streptococcus agalactiae 99.5 86.2 85.3 0

Kill rate Streptococcus dysgalactiae 99.3 78.2 86.4 0

(%) Streptococcus uberis 99.1 89.6 91.3 0

  Corynebacterium pyogenes 99.4 78.5 87.9 0

It can be seen from the above table that the sterilizing wipes can effectively kill various pathogenic bacteria that cause breast infection, especially for Staphylococcus aureus, while the two antibiotics cannot effectively remove the pathogenic bacteria in the breast area.

Example 7

Experiment of Preventing Breast Infection with Bactericidal Wet Wipes

1. Sample preparation

Preparation of bactericidal wet wipes: The preparation method was as in Example 1, wherein the content of lysostaphin was 0.001wt%, the content of lysozyme was 20wt%, the content of polyethylene glycol was 3%, and the content of polysorbate 20 was 0.2wt%.

Iododipine solution: 100ml, Zhejiang Agricultural University.

Sampling solution: 1% peptone, 0.5% sodium thiosulfate, 2% Tween-80, 0.5% lecithin in PBS, 10ml per tube.

2. Preparation of experimental animals

Select 4-8-year-old young, middle-aged high-yielding, black-and-white dairy cows in the lactation period, with no symptoms of infection, a total of 3 cows, 1 group is the antiseptic wet wipes prevention group, 2 groups are the iodide group, and 3 groups are the blank control group .

3. Test method:

Wet a sterile cotton swab with the sampling liquid in the milk collection area, and detect the natural colony count of each sampling sample in the milk area. After the first group wiped the adjacent parts of the original sampling with a sterilizing wet towel, they took samples for 1 minute, 30 minutes, and 2 hours each; the method of the second group was the same as that of the first group, detecting the number of natural bacteria remaining after the action, and calculating the natural bacteria before and after the action kill rate.

4. Test results

                                                                          

Killing rate (%)   Sterilizing wipes group   Iodide group   Blank control group

After 1 minute 99.9 94.2 13.5

After 30 minutes 99.2 92.1 15.2

After 2 hours 98.4 62.3 14.6

It can be seen from the above tests that the sterilizing wipes can effectively prevent the milk area of dairy cows from being infected by bacteria, and can keep the milk area clean.

Claims (3)

1. The application of a bactericidal wet wipe in killing bacteria that cause mammary gland infection in dairy cows, the bactericidal wet wipe is composed of a carrier and a bactericide loaded thereon, and the bactericide includes 0.0001 to 0.1wt of lysostaphin %, lysozyme 10-50wt%, stabilizer 1-15wt%, surfactant 0.1-0.5wt%, water balance; The stabilizer is polyethylene glycol, and the surfactant is polysorbate series or betaine. 2. The application of the bactericidal wet tissue according to claim 1, characterized in that said bacteria are Gram-negative bacteria and Gram-positive bacteria. 3. The application of the bactericidal wet wipe according to claim 2, characterized in that said bacteria include Escherichia coli, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis and Corynebacterium pyogenes.