Indian J Dairy Sci 69(4), 2016 433 RESEARCH ARTICLE Standardization of enzymatic diagnostic kit based method for cholesterol estimation in butter, kalakand, khoa and paneer Yogesh Parmar, Vivek Sharma, Sumit Arora, Anupama Rani and Tanmay Hazra Received : 09 July 2015 / Accepted: 12 January 2016 Vivek Sharma Dairy Chemistry Division ICAR - National Dairy Research Institute, Karnal -132001, (Haryana), INDIA Email: vishk12000@yahoo.com Mobile: +91 9416651314 Yogesh Parmar, Vivek Sharma, Sumit Arora, Anupama Rani and Tanmay Hazra Dairy Chemistry Division, National Dairy Research Institute, Karnal (Haryana) – India Abstract: The objective of this study was to standardize the protocol for cholesterol quantification in dairy based products (butter, kalakand, khoa and paneer) using enzymatic diagnostic kit (EDK) assay. Practically about 0.15-0.2 g product was saponified using different methanolic KOH concentrations (2.5, 5 and 7.5%) as well as at different saponification temperatures (80 0 , 85 0 and 90 0 C) for 20 min. followed by extraction of unsaponifiable matter with hexane. An aliquot of unsaponifiable fraction was evaporated at 50 0 -65 0 C and cholesterol estimation was done using EDK. For standardization of protocol, recovery trials were also conducted in dairy based products at different methanolic KOH concentratisons as well as at different saponification temperatures. The optimized conditions for rapid saponification of dairy products were found to be the use of 5% methanolic KOH concentration and heating at 90 0 C for 20 min., wherein the per cent recovery in case of butter, kalakand, khoa and paneer were to the tune of 100.13±0.70; 100.25±0.58; 100.54±0.51; 97.99±0.28, respectively. Recovery studies clearly indicated that the developed EDK based method is a choice for the rapid determination of cholesterol content in selected indigenous dairy products. On using the above said optimized saponification conditions, the average cholesterol content estimated using EDK method in butter, kalakand, khoa and paneer was 277.72, 76.34, 95.22 and 79.57 mg/100 g product, respectively. Developed EDK method can be used as a routine method to test the cholesterol content in indigenous dairy products to measure the batch to batch variation. Key words: Cholesterol, enzymatic diagnostic kit, dairy products, saponification Introduction Cholesterol, a lipid component, chemically a steroid alcohol, having chemical formula of C 27 H 45 OH. It has several functions like precursor of bile acids, steroid hormones, and pro-vitamin D 3 . Cholesterol is mainly found in different foods such as eggs, meat, fish and dairy products. Now, the quantitative determination of cholesterol content in foods as well as in dairy products has become a necessity mainly for two reasons. The first is its health concern about the role of excess plasma cholesterol on heart and heart related disease like Cardio vascular Disease (CVD). The second one deals with the nutritional labeling of foods and dairy products which has become mandatory due to legal regulations provided by many countries. Recently, interest has been increased in direct saponification methods for determination of cholesterol. This method eliminates the fat extraction step required in gas liquid chromatography (GLC) as well as in high performance liquid chromatography (Richardson et al., 1994). This direct saponification method has been applied to many foods such as frozen foods (Al Hasani et al., 1990); milk (Tsui, 1989); fish based products (Kovacs et al., 1979); egg yolk (Pasin et al., 1998) and pasta products (Kovacs, 1990). Many analytical approaches have been mentioned in the literature like gravimetric, colorimetric and gas chromatographic methods for cholesterol estimation in dairy products. Chromatographic techniques involve the use of gas-liquid chromatography (Fletouris et al., 1998), High performance liquid chromatography (Oh et al., 2001) and capillary super critical fluid chromatography (Novak et al., 1991). However, GLC based method was most accurate for cholesterol estimation but has some disadvantages like tediousness and labour intensiveness, and requires highly skilled person and costly instruments. Similarly Fourier transformed infrared spectroscopy and high performance liquid chromatography also requires expensive instruments and analytical skills. Therefore, in the present study, an enzymatic diagnostic kit, which is commonly used for cholesterol estimation in blood, has been evaluated for cholesterol estimation in the above mentioned dairy products to find an alternative and cost effective method at manufacturing level.