ilk β-lactamase detection method

No anti-milk, by definition, that antibiotic-free milk. As early as 2002, the concept has been proposed, without antibiotics words also boarded the dairy packaging. Why milk containing antibiotics

"No anti-milk", by definition, that antibiotic-free milk. As early as 2002, the concept has been proposed, "no antibiotics" message also boarded the dairy packaging.
Why milk containing antibiotics?
Antibiotics in milk from cows, dairy cows are injected antibiotics, primarily for the treatment of breast inflammation. Cow mastitis is common, the use of antibiotics is the industry's "open secret." Antibiotics in milk drinkers may cause allergies, or because of prolonged exposure to antibiotics and resistance.
How to achieve "no anti"?
Drug reasonable intervals after milking, milk can be produced without resistance, you can verify by examining the way antibiotics.
An indirect method
Method: the use of β-lactamase hydrolysis of penicillin to the principle of adding a certain amount of milk to penicillin, if present in a concentration of milk β-lactamase, then penicillin by β-lactamase enzyme concentration after will be reduced, in order to determine whether the milk β-lactamase.
Experimental Procedure: Weigh 20 g sample at 4 ℃, 16000rpm was centrifuged 10 min. Take the lower serum 10 g in 50 mL plastic centrifuge tube and placed in plastic centrifuge tubes were incubated at 37 ℃ pot oscillation 30 min. After the incubation tube were added ethanol 15 mL. After 30 min shaking extraction centrifugation, the supernatant was collected on filter paper, pear shaped flask. Concentrated under reduced pressure to evaporate ethanol. After rotary evaporation to Add 10 mL pear-shaped flask of phosphate buffer solution (pH = 8.5), vortex for 1 min pH was adjusted to 8.5. To 1 mL / min rate of the extract through a pretreated Oasis HLB solid phase extraction column with 2 mL of phosphate buffer (pH = 8.5) leaching extraction column, and then 2 mL water rinse. Finally with 3 mL of acetonitrile. The eluate was evaporated to dryness at 40 ℃ with 0.025 M phosphate buffer (pH = 7.0) the residue to constant volume 1 mL, pending the determination on the machine.
Chromatographic conditions: LabAlliance PC-2000 high-performance liquid chromatography with UV detector 500;
          lanbo Column: C18 (4.6mm × 150mm × 5μm);
          Mobile phase: methanol / 0.004M potassium dihydrogen phosphate (pH = 4.5) = 40/60;
          Detection wavelength: 268 nm
2, the direct method
Method: potassium penicillin in milk β-lactamase enzyme, the main generate penicilloic potassium, and generates a small amount of accompanying penicillamine, Penicillium aldehydes and other substances. After going fat, protein and other pre-treatment, it can be penicilloic potassium extracted by HPLC testing to confirm its existence, thereby determining whether the milk man "no anti-milk."
Experimental Procedure: Weigh 20 g sample at 4 ℃, 16000 rpm by centrifugation 10 min. Take the lower supernatant was 10 g in 50 mL plastic centrifuge tube, adding ethanol 15 mL, shaking extraction for 30 min centrifugation, the supernatant was filtered through 0.45 μm membrane filter, detected by HPLC.
Preparation of Stainless steel slewing bearing  Weigh 0.01 g (accurate to 0.001 g) in 100 ml of potassium penicillin flask, add 1ml commercially available antibiotics decomposing agent at room temperature for 2h after the complete hydrolysis of penicillin potassium to volume with deionized water to obtain a green thiazole mold potassium standard solution, the concentration of 100 μg / mL. Diluted with the actual situation.
Chromatographic conditions: LabAlliance PC-2000 HPLC equipped with 2000 UV detector;
 lanbo Column: C18 (4.6mm × 150mm × 5μm);
          Mobile phase: methanol / 0.004 M potassium dihydrogen phosphate (pH = 4.5) = 40/60;
          Detection wavelength: 230 nm