Indian J Dairy Sci 73(6): 505-516 505 RESEARCH ARTICLE Application of physico-chemical and chromatographic techniques for detection of adulteration in ghee (Milk fat) Dnyaneshwar Shinde, Hriday Darji, Rajiv Chawla, Badal Patel, Chitrangana Joshi, Hima Thakkar, Sushil Gawande, Swati Patil and Rajesh R Nair CALF, National Dairy Development Board, Anand-388 001, Gujarat, India Dnyaneshwar Shinde() CALF, National Dairy Development Board Anand-388 001, Gujarat, India E-mail: drshinde@nddb.coop; Phone: +918758555183 Received: 06 July 2020 / Accepted: 29 August 2020 / Published online: 27 December 2020 Indian Dairy Association (India) 2020 © Abstract: Ghee (milk fat) due to its high price and demand is highly susceptible to adulteration for economic gains. Further, due to advanced practices of adulteration, its detection is becoming difficult by any single method. In the present study, methods based upon physico-chemical (BR reading and RM value) and chromatography (fatty acid, triglycerides, and plant sterols profiling) were evaluated to detect adulteration. Pure ghee was adulterated @ 1%, 2.5%, 5%, and 10% with coconut, soya bean, groundnut, and sunflower oil. Results of physico-chemical methods indicated that BR reading crossed regulatory limit only @ 10% of sunflower and soya bean oil adulteration while for the other two oils it was within the acceptable range (FSSAI standards for Gujarat i.e. 40 to 43.5). In the case of RM value, adulteration was not detected with any oil up to 10% level of adulteration with respect to FSSAI regulation (i.e. minimum 24) for Gujarat. Among chromatographic methods, fatty acids marker molecules like lauric for coconut and linoleic for soya bean, groundnut, and sunflower oil showed an increase upon adulteration even @ 1%. Marker fatty acids % values were falling outside the range for lauric (i.e.2.3 to 3.2%) and linoleic (i.e.1 to 2%) as specified by FSSAI in the manual of analysis for milk and milk products 2016 however, natural variations might affect the results. Triglyceride profiling was found to be capable of detecting adulteration @ 5% for all the oils except groundnut which was detected @ 10%. Plant sterols (β-sitosterol and stigmasterol) based method was found suitable to detect adulteration @ 1% with sunflower and soya bean oil, @ 2.5% with groundnut oil, and @ 5% with coconut oil. All the methods appear to have their strengths and limitations; hence laboratories may apply a combination of techniques to detect advanced adulteration. Keywords: Adulteration, BR reading, Ghee, GC-FID, HPLC, Milk fat, RM value, Sterols, Triglycerides Introduction Ghee (milk fat) is one of the most valuable and premium dairy product prepared by clarification of cream or butter at a temperature of 110 º C to 120 º C and is widely consumed in the Indian subcontinent. The production of ghee and butter in India is projected to rise from 5.8 MMT in 2019 to 6.1 MMT in 2020 (GAIN. 2020). Due to the high cost and demand for ghee, it is highly susceptible to economically motivated adulteration (Rani et al. 2016). Unscrupulous traders adulterate milk fat with low priced foreign fats for the maximization of profits. Adulteration of vegetable oil or animal body fat to milk and dairy products is old but illegal practice (Gutierrez et al. 2009; Molkentin et al. 2007). Major adulterants found in ghee are hydrogenated fats, vegetable oils, and animal body fats (Aparnathi et al. 2019). Conventional physico-chemical techniques like Butyro Refractometer reading at 40°C (BR reading) and Reichert Meissl (RM) value have their limitations in detecting adulteration at lower levels and are prone to overlap the values of natural milk fat (Jirankalgikar et al. 2014). RM modifiers/adjusters/RM cutting oil/adjuster oils are being used in the market to adjust the RM value of ghee adulterated with foreign fats. The RM adjusters probably contain acetic acid esters of short or medium-chain fatty acids with RM of 150 to 165 (Pathania et al. 2020). An attempt has been made to identify such modifiers at 0.5% level using ISO 17678:2010 (Pathania et al. 2020). Because of advancements in adulteration practices and low sensitivity of physico-chemical techniques, these tests fail to detect adulteration of milk fat with required sensitivity and specificity. Enormous efforts have been made in the last few decades to address milk fat adulteration by undertaking advancements in physico-chemical methods and the development of https://doi.org/10.33785/IJDS.2020.v73i06.001