Detection method of the hottest lysozyme content

2022-08-18
  • Detail

Lysozyme is a small molecule viscous protein synthesized and secreted by phagocytes, which belongs to acetyl polysaccharide enzyme. It exists in saliva, milk, tears, nasal and tracheal secretions and can dissolve Gram-positive bacteria. Due to its high electric point (ph11.0), it can firmly bind with bacteria, hydrolyze bacterial cell wall peptidoglycans, and make bacteria cleave and die. This experiment was carried out on a plate. Salivary lysozyme is a small molecular viscous protein synthesized and secreted by phagocytes, which belongs to acetyl polysaccharide enzyme. It exists in saliva, milk, tears, nasal and tracheal secretions and can dissolve Gram-positive bacteria. Due to its high electric point (ph11.0), it can firmly bind with bacteria, hydrolyze bacterial cell wall peptidoglycans, and make bacteria cleave and die

in this experiment, salivary enzymes were measured on a plate. After lysozyme interacts with Bacillus subtilis, the bacteria can be dissolved due to the destruction of cell wall, resulting in a lysozyme ring around the sampling hole of agar plate. There is a linear relationship between the diameter of the lysozyme ring and the logarithm of the lysozyme content in the sample

[materials]

1 sterile PBS (ph6.4, 1/15mol/l), 5mol/lkoh, 3% PBS agar, lysozyme standard, saliva of the subject

2 12 hour culture of Bacillus subtilis

3 10100 test tubes, test tube racks, sterile plates, puncher, micro sampler

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[method]

1 Preparation of bacterial solution: add PBS to the inclined plane of Bacillus subtilis, and make bacterial suspension after leaving it at room temperature for minutes; At the wavelength of 640nm, measure the concentration of bacterial suspension, and adjust the concentration of bacterial suspension to the light transmittance%

2. Preparation of lysozyme standard solution: weigh the lysozyme standard, prepare it into 1000 m g/ml with PBS, freeze it in the refrigerator, and then dilute it into 100, 50, 25 and 10 m g/ml when using it temporarily

3. Collect saliva samples to improve the quality of the whole system: after the tester rinses with water, collect saliva in a sterile plate for use

4. Heat and melt 3% agar, cool it to 60 -70 ℃, and mix it with the preheated Bacillus subtilis suspension system with the actual maximum pressure of 28Mpa and the same volume to the plate. Use a punch to punch holes on the flat plate with a spacing of about 1.5cm. Each flat plate can be punched

5. Lysozyme standard and saliva sample were added into each well in turn, 20 ml per well

6. Measure the diameter of bacteriolytic ring after being placed at 24 -26 ℃ for hours

[result judgment]

record the diameter (mm) of the bacteriolytic ring in the following table, draw the standard curve, and find out the lysozyme content of the measured sample from the standard curve

[operation]

1 record the diameter of the lysozyme ring of the standard lysozyme sample and draw the standard curve

2 record and report the lysozyme content of saliva samples

figure lysozyme action in saliva samples and lysozyme ring produced by Bacillus subtilis. The upper row is the lysozyme ring produced by the lysozyme standard solution

, and the lower row is the lysozyme ring produced by the lysozyme in saliva samples

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