A kind of dog and cat oral care spray containing lysozyme and preparation method thereof
A technology for oral care and egg white lysozyme, which is applied to skin care preparations, medical preparations containing active ingredients, oral care, etc., can solve the problems of less application of persimmon tannin, achieve non-toxic side effects, eliminate bad breath, The effect of improving oral inflammation
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Embodiment 1
[0021] Example 1 Efficient preparation of egg white lysozyme
[0022] (1) Wash the fresh eggs, control the water, break the small heads, collect the egg whites, stand at 4°C for more than 4 hours, and obtain the egg white lysozyme aqueous solution by the salting-out method; (2) Add Na to the egg white lysozyme aqueous solution 2 SO 3 Solution mixing reaction, egg white lysozyme aqueous solution and Na 2 SO 3 The reaction mass ratio of the solution is 20:1; (3) Put the mixed solution in step (2) into a water bath and stir it with a magnetic stirrer for 30min-100min to make it evenly mixed; (4) Stir the mixture in step (3) evenly The resulting mixture was placed in a dialysis bag and placed in a 4°C refrigerator for dialysis; (4) after dialysis, it was placed in a vacuum freeze dryer for freeze-drying, and the obtained lysozyme product was stored in a dryer.
[0023] The egg white lysozyme sequence extracted by the method of the present invention was searched through the NCBI...
Embodiment 2
[0024] Example 2 Antibacterial test (MIC) of egg white lysozyme spray
[0025] (1) Inoculate bacteria (Escherichia coli, bad breath-producing bacteria, tooth decay-producing bacteria, etc.) on the MH broth solid medium, and cultivate until a single colony grows; (2) Pick a single colony and inoculate it into 3 mL of MH broth medium (3) Transfer 30 μL of bacterial suspension to 3 mL of fresh MH broth medium, and culture with constant temperature shaking to OD 600 = about 0.5; (4) Transfer 10 μL of bacterial suspension to 10 mL of fresh MH broth medium, vortex and mix, and control the concentration at 1×10 5 —5×10 5 CFU / mL, used for the determination of MIC; (5) First add 90 μL of the prepared bacterial suspension to the sterile 96-well round bottom plate, and then add 10 μL of oral care solution or lysozyme in different proportions of corresponding concentrations one by one, and then different The solutions of the groups were serially diluted to a final concentration of 1280...
Embodiment 3
[0029] Example 3 Enzyme activity detection of egg white lysozyme
[0030](1) Dilute different lysozyme samples to 10-1~10-3. According to the operation method of egg white lysozyme test kit, test the enzyme activity of different test groups. The test is divided into three groups: blank wells (4 μL double-distillation water+40 μL buffer+40 μL matrix solution), standard well (4 μL phenol standard application solution+40 μL buffer+40 μL matrix solution), test well (4 μL test sample+40 μL buffer+40 μL matrix (2) Transfer the 96-well plate to a water bath at 37°C for 30 min. (3) Calculate the enzyme activity formula of the sample, lysozyme activity (U / L) = (measured OD value - blank OD value) / (standard OD value - blank OD value) × phenol standard concentration (0.1 mg / mL) ×100 mL × dilution factor of the sample before measurement × 7.14. The definition of lysozyme activity unit: 100 mL of serum or liquid reacted with the substrate for 30 minutes at 37 °C to produce 1 mg of phen...
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