Technology Details:
The technology involves the application of specific enzymatic reaction in selective medium. The marker enzyme which participates in unique biochemical pathways of specific genera or strain hydrolyze chromogenic substrate complex and release free chromogen which can be detected visually by color change. Currently, commercially available media like citrate azide agar requires an incubation period of 72-96 h for detection of Enterococci in milk. The current investigation was carried out keeping in consideration that current techniques for enumeration of hygiene indicators are time-consuming and industry is looking forward to rapid assay. The developed technology is two-stage enzyme assay for detection of Enterococci within 18±1.0h of incubation at 37˚C in stage-1 employing EBSAM as a selective medium in lyophilized form and its confirmation within 3:30±0.30h in stage-2 using specific enzyme substrate mixture (Patent Reg.119/DEL/2012). The appearance of black color in stage-1 indicates the presumptive presence of Enterococci in milk. The appearance of yellow and orange color in T-1 & T-2 respectively in stage-2 confirms the presence of Enterococci in milk sample as depicted in the above fig. EBSAM medium is highly selective and specific for the growth of Enterococci and allows its detection in the single working day. Developed assay can detect 1.0 log Enterococci counts in milk within 18.0±1.0h of incubation at 37°C based on the appearance of black color. The technology has potential to replace the existing medium for Enterococci for being cost effective (Rs 98.3 per liter as against Citrate azide agar (CAA) available @ Rs 262.5 per litre, Bile Esculin azide agar available @ Rs 493.5per litre. The working performance of enzyme based bio-assay was validated in-house laboratory with IS: 5887 Part-2 (3-days protocol). The technology was validated at M/s SGS India Pvt. Ltd, Gurgaon, Certificate no. SGS GG12-009685.001 dated 09-11-2012. The wide scope of application to raw, pasteurized and dried milk for routine as well as for regulatory standard compliance.