Nitrocefin-Based β-Lactam Antibiotic Detection in Milk

The propagation medium was prepared by following the protocol in our recent work^{24 (link)}. The stock solution of the chromogenic substrate nitrocefin was prepared in 500 µL organic solvent with the addition of 5 mg nitrocefin (Sigma Aldrich, U.S.A; HiMedia, Mumbai, India). ß-lactam antibiotics (HiMedia) solution was prepared by diluting standard stock solution in milk and stored at − 20 °C. Potassium phosphate buffer (Hi-Media, India) (pH 6.8) was prepared by dissolving di-potassium hydrogen phosphate (K₂HPO₄) (0.174 g) and potassium di-hydrogen phosphate (KH₂PO₄) (0.136 g) in 100.0 mL of distilled water and stored at room temperature. Raw milk samples were collected from the dairy farms near by Karnal and pasteurized milk samples were collected from the local market of Karnal, stored under refrigerated conditions, without further treatment before test.