Comparison of Standardized X-gal, ONPG and MUG Assay Methods with IS Methods by Analyzing Milk Samples

Abstract

Aims: Food Safety Standards Authority of India (FSSAI) in 2011 has adopted a conventional IS-5887 (Part-I) 1976 and IS-5401 Part-1 (2012) protocol for monitoring of E. coli and coliforms in dairy products respectively. These methods are time consuming and sometimes requires further isolation and confirmation to finalize the true contaminant. The current investigation was carried out to compare these methods with developed chromogenic and fluorogenic assay methods to access their suitability in actual analysis in terms of time saving, reliability and reproducibility. Place and Duration of Study: Department of Dairy Microbiology, SMC College of Dairy Science, Anand Agricultural University, Anand, June 2019 to June 2020. Methods: Ten samples of raw and pasteurized milks were inoculated with formulated selective broth. After incubation for 10 hrs crude enzymes were extracted to detect the presence of coliforms. Similarly 0.1 ml 4-Methylumbelliferyl-β-D-Glucuronide (MUG) solution was added in Formulated selective broth for the detection of E. coli. Enzyme extract procedure was not required after incubation of sample for detection of E. coli. Then after presence of coliforms were detection by the X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside) impregnated strips and quantification was done by the chart developed using optical density measurement in o-nitrophenyl-ß-d-galactopyranoside (ONGP) assay. For E. coli detection, blue fluorescence generated was measured under UV light at 350 nm and quantification was done by measuring relative light unit (RLU) generated by Fluorescence spectrophotometer. Same sample was also analyzed by standard IS 5401: part-1 (2012) and IS 5887: Part - I (1976; reaffirmed 2005) procedure and compared with standardized methods for enumeration of coliforms and E. coli respectively. Results: This study made clear that the results of analysis of raw and pasteurized milk samples by developed chromogenic and fluorogenic assay method were in accordance with the conventional IS methods. Conclusion: Developed chromogenic and fluorogenic assay method indicating their suitability, ease in operation, time saving and preciseness over the conventional methods and can be opt as a suitable alternative for monitoring presence of E. coli and coliforms in fluid milks.