J. Dairying, Foods & H.S., 29 (1) : 8-14, 2010 AGRICULTURAL RESEARCH COMMUNICATION CENTRE www.arccjournals.com / indianjournals.com VALIDATION OF ULTRA-VIOLET AND VISIBLE SPECTROSCOPIC METHODS FOR DETECTION OF MILK FAT ADULTERATION Arun Kumar 1, Amit Kumar 2 Darshan Lal*, Raman Seth and Vivek Sharma Dairy Chemistry Division, National Dairy Research Institute, Karnal-132 001, India. ABSTRACT An investigation was carried out to explore the possibility of using ultra-violet and visible spectroscopy in the detection of adulteration of milk fat with cheaper oils and fats (vegetable oils and fats, and animal body fats). No characteristic differences were observed in the absorption behavior of different oils and fats including milk fat when scanned in the ultra violet (200-320 nm) and visible (400-800 nm) region and therefore ultraviolet and visible spectroscopy failed to give any conclusive evidence of detection of adulteration in ghee with vegetable oils, body fats and vanaspati. Key Words: Adulteration, UV and visible spectroscopy, Body fats, Vegetable oils Milk fat. INTRODUCTION Lipids form one of the most important constituents of milk and milk products. Major part of milk lipids is comprised of triglycerides (generally called fats). Minor components of milk lipids include partial glycerides (mono- and di- glycerides), phospholipids, fat soluble vitamins, cholesterol, squalene, waxes, etc. In India, milk fat is mostly consumed in the form of ghee (clarified butterfat). However, its supply falls short of demand particularly in the lean (summer) season. Further, due to its short supply and more demand, expensiveness (costing 3 to 4 times as much as edible vegetable oils) and variable chemical composition, ghee falls prey to adulteration with vegetable oils and fats, and animal body fats by the unscrupulous traders in the market. Extensive survey of the literature reveals that several methods have been developed in the past based on parameters like fatty acid composition and the physico-chemical constants to detect the adulteration in ghee. But very few attempts have been made to detect the adulteration on the basis of interaction of radiation. Therefore, techniques of Ultra-Violet and Visible spectroscopy have been explored in the present investigation. MATERIALS AND METHODS 1. Collection of milk and preparation of butter Milk used for the preparation of ghee samples was collected from the Institute s cattle yard. Cow milk was a mixture of the milk obtained from the herd of Karan Swiss, Karan Fries, Sahiwal and Tharparkar breeds. Buffalo milk used was also the herd milk from Murrah breed only. Cows and buffaloes were maintained under identical conditions of feeding and management. Soon after the collection of milk, it was warmed to 40 C and separated into cream using mechanical cream separator. The cream was pasteurized at 77 C for 5 minutes, cooled to room temperature and then kept in a refrigerator (5 to 10 C) for 3 to 5 hours for ageing. Butter was prepared under standard conditions (9 C in summer and 13 C in winter) by churning the cream using hand churn. 2. Collection of adulterants The body fats (pig and goat), vegetable oils (sunflower, soy bean and ground nut) and vanaspati (hydrogenated vegetable oils) were collected from the local market. *Corresponding Author: NDRI, Karnal- 132 001, India. 1 Department of Dairy Food Science & Technology, Maharana Pratap Univ. of Agriculture & Technology, University Campus, Udaipur-313001 (Rajasthan). 2 Dairy Chemistry Division, College of Dairy Science and Technology, GADVASU, Ludhiana -141004 (Punjab).
3. Preparation of adulterated ghee samples The adulterants described above were added individually to ghee (buffalo / cow) at the butter stage on the basis of its fat content at 5%, 10% and 15% level. The butter samples admixed with the adulterants were clarified on direct flame in a stainless steel vessel under continuous stirring at temperature of 120 C/flash and finally filtered through Whatman No.4 filter paper. Simultaneously, pure ghee sample (control) was also prepared under similar conditions from the same lot of butter without adding any of the adulterants. 4. Ultra-violet and visible spectroscopy Ultra-violet spectroscopy Ultraviolet (UV) spectroscopy measurements were made according to the method of Rego et al., (1964), using double beam spectrophotometer, Model Specord 200, equipped with UV and visible ranges of Analytikjena make, Germany. 0.1 per cent solution of fat sample was prepared in purified n-hexane and was scanned for absorption maxima over a UV range of 200 to 320 nm using a 1 cm quartz cell. Visible spectroscopy 0.1 per cent solution of fat sample was prepared in purified n-hexane and was also scanned for absorption maxima over a visible range of 400 to 800 nm using 1 cm cell under similar conditions as described above. RESULTS AND DISCUSSION Spectroscopic methods using ultra-violet (200 to 400 nm) and visible (400 to 800 nm) regions have been used for characterizing fats and oils. In the present study, samples of pure ghee (buffalo and cow), vegetable oils (soy bean, sunflower and groundnut oil), vanaspati, animal body fats (goat and pig), and ghee samples adulterated with vegetable oils and animal body fats were scanned in the Ultraviolet (200 to 320 nm) and Visible (400 to 800 nm) regions. The results are presented in Figures 1 to 8. Ultra-violet Spectroscopy Pure ghee (buffalo and cow), animal body fats (goat and pig), vegetable oils (soy bean, sunflower and groundnut oil) and vanaspati samples dissolved in n-hexane and scanned on double beam spectrophotometer, between 200 to 320 nm showed first absorption maxima between 220 and 230 nm Vol. 29, No. 1, 2010 (Figures 1 to 4). All these oils and fats showed second absorption maxima at around 270 to 280 nm, except vanaspati which showed no such second maxima (Figure 3B). Thus, it is apparent from the results that the ghee, animal body fats, vegetable oils and vanaspati behaved in almost similar fashion. Therefore, it can be inferred from the above findings that Ultra-violet (UV) spectroscopic examination of fats and oils did not give any indication for demonstrating the adulteration in ghee. Consequently, ghee samples adulterated with body fats or vegetable oils or vanaspati, individually or in combinations at 5, 10 and 15 percent levels also did not show any difference from pure ghee in terms of first or second absorption maxima. The results obtained in the present investigation are in full agreement with those of Singhal (1973) and Sharma (1989) who also opined that UV spectroscopy did not offer any help in detecting adulteration in milk fat. Visible Spectroscopy Pure ghee (buffalo and cow), body fats (goat and pig), vegetable oils (soybean, sunflower and groundnut oil) and vanaspati samples dissolved in n-hexane were also scanned for visible spectrum between 400 to 800 nm and showed no positive absorption maxima, rather showed somewhat increasing absorption on the negative side (Figures 5 to 8). However, buffalo and cow pure ghee samples showed some sign of positive absorption in the region of 430 to 440 nm and 400 to 500 nm, respectively. Thus, like UV spectroscopic scanning, visible spectroscopic examination could not be used in detecting adulteration of ghee as adulterated ghee samples also showed similar behaviour in visible (400 to 800 nm) region studied. Jha (1981) also used visible spectroscopy for the detection of Cheuri (Madhuca butyracia) fat in ghee, a common adulterant of ghee in Nepal, in a narrow visible region of 600 to 700 nm, and reported that pure ghee showed no absorption band whereas Cheuri fat showed an absorption band with maxima between 640 and 680 nm. Using this technique, as low as 5 percent Cheuri fat added to ghee was detectable. On the other hand, vegetable oils and fats used as an adulterant in the present study did not show the similar kind of absorption behaviour as has been reported for Cheuri fat. 9
10 J. DAIRYING, FOODS & H.S. A - Pure buffalo ghee B - Pure cow ghee Fig. 1: UV absorption spectra of pure buffalo ghee (A) and pure cow ghee (B). A - Sunflower oil B - Soy bean oil Fig. 2: UV absorption spectra of sunflower oil (A) and soy bean oil (B).
Vol. 29, No. 1, 2010 11 A - Groundnut oil B - Vanaspati Fig. 3: UV absorption spectra of groundnut oil (A) and vanaspati (B). A - Pig body fat B - Goat body fat Fig. 4: UV absorption spectra of pig body fat (A) and goat body fat (B).
12 J. DAIRYING, FOODS & H.S. A - Pure buffalo ghee B - Pure cow ghee Fig. 5: Visible spectra of pure buffalo ghee (A) and pure cow ghee (B). A - Sunflower oil B - Soy bean oil Fig. 6: Visible spectra of sunflower oil (A) and soy bean oil (B).
Vol. 29, No. 1, 2010 13 A - Groundnut oil B - Vanaspati Fig. 7: Visible spectra of groundnut oil (A) and vanaspati (B). A - Pig body fat B - Goat body fat Fig. 8: Visible spectra of pig body fat (A) and goat body fat (B).
14 J. DAIRYING, FOODS & H.S. CONCLUSION The study revealed that the UV and Visible spectroscopy did not offer any help in detecting the adulteration in ghee because no characteristic differences were observed in the absorption behaviour of different oils and fats including milk fat studied in the UV (200-400 nm) and visible (400-800 nm) region, indicating that there are common functional groups present in all these oils and fats. ACKNOWLEDGEMENT The first author thanks Indian Council of Agricultural Research and National Dairy Research Institute for the award of Senior Research Fellowship during the course of this study. REFERENCES Arun Kumar et al ( 2002). Indian J. Dairy Sci. 55(6):319-330. Jha,J.S.(1981). J. Amer. Oil Chem. Soc. 58: 843-845. Rego, M.D. et al (1964). Ann. Bromat., 16: 455 460. Sharma, S.K. (1989). Ph.D. Thesis, National Dairy Research Institute (Deemed University), Karnal, India. Singhal, O.P. (1973). Ph.D. Thesis, Punjab University, Chandigarh.