In ternationa l Scholars Journa ls African Journal of Food Science Research ISSN 2375-0723 Vol. 3 (10), pp. 222-225, October, 2015. Available online at www.internationalscholarsjournals.org © International Scholars Journals Author(s) retain the copyright of this article. Full Length Research Paper Detection of dilution of milk with the help of glass transition temperature by differential scanning calorimetry (DSC) T. K. Goswami* and S. K. Gupta Agricultural and Food Engineering Department, Indian Institute of Technology, Kharagpur-721302, West Bengal, India. Accepted 23 April, 2015 Glass transition temperature of pure milk, milk diluted with water, and pure milk in which sugars like glucose, fructose and sucrose were investigated by differential scanning calorimetry (DSC). The glass transition temperature Tg of pure milk (containing 89.2 kg water and 10.8 kg total solids per 100 kg milk) varied between - 114 to -118 o C. Effect of change in composition of the milk varying the amounts of water and total solids content (77.47, 81.56, 83.81, 84.31, 85.76, 86.69, 89.43, 92.15, 93.16, 95.71 and 96.38 kg water to make each 100 kg milk sample) on the glass transition temperature of milk showed a linear relationship between glass transition temperature and composition of the milk expressed in %moisture content (w.b.). The addition of sugars (glucose, froctose and sucrose) also showed a significant change in glass transition temperature. Under applied conditions, adulterations in milk by dilution with water only and water and sugars were detectable even at 0.5 and 5 % respectively. Key words: Milk, glass transition temperature, differential scanning calorimetry, adulteration. INTRODUCTION The term quality, as applied to milk and products made out of milk, embraces a variety of features. These include such diverse properties as absence of dirt, antibiotics, off-flavours, pathogenic organisms and abnormal numbers of body cells; evidence of cleanliness and care in production and handling as indicated by microbiological analysis; chemical analysis to check for dilution with water, remo-val of fat, and any added adulterants; possession of desi-rable aroma and flavor; and adequate amounts of those constituents which are of nutritional importance. The forms of adulteration like addition of water, skimming or removal of fat and addition of fluid skim milk can be detected from specific gravity and fat content (Singhal et al., 1997). Glucose, cane sugar, urea, ammonium sulphate and other substances have been encountered as additives for the purpose of masking the effects of dilution of water. Even a sensitive test like freezing point of milk fails to *Corresponding author. E-mail: tkg@agfe.iitkgp.ernet.in. Fax: - +91-3222-255303. Tel: +91-3222-283122. unmask this adulteration (Dharmarajan et al., 1953). Den- sity differences have been noted on addition of glucose, urea and ammonium sulphate, but again the increase in density is not exactly proportional to the molar concen- tration of the added solute. Also, rise in milk density due to glucose is greater than that due to urea and lower than that due to ammonium sulphate, and is not in molar proportions. So detection of such types of adulteration are generally based on composition and structure (mole-cular mobility) or physical properties measured by molecular refraction, differential thermal analysis, differ-rential scanning calorimetry (DSC) or IR spectroscopy (Singhal et al., 1997; Kantor et al., 1999). Most of the physical or chemical transformations in-volve heat exchanges. Therefore, DSC analysis has a very broad field of application. Even when no heat ex-change occurs, as observed in glass transition, the phe-nomenon gives rise to a baseline deviation of the thermoanalytical curve, related to a variation of heat capacity. Glass transition and heat capacity measure-ments provide detailed information about both the physical and calorimetric properties of a substances Goswami and Gupta 223 Figure 1. DSC thermograph for 87.5% moisture content (w.b.) sample. (Bhandari and Howes, 2002; Laaksonen et al., 2001). The textural change of food materials is also measured with the help of glass transition temperature using DSC (Ross et al., 2002). The state diagram of date flesh was developed by measuring its freezing points, glass transition temperatures, maximal-freeze-concentration condition, and solute melting points by DSC (Rahman, 2004). The glass transition of a polymer is described as a transition from a flexible rubber to a hard brittle solid (Shen and Eisenberg, 1966). For a low molecular weight material the glass transition is marked by an increase in viscosity of several orders of magnitude as the material is cooled from a super cooled liquid to a glass (Ollett and Parker, 1990; Truong et al., 2004). Glass transition temperature (Tg) is a terminology to characterize the transition from a liquid to a glassy state (or vice versa) of a substance. The onset or midpoint values of the tran- sition in the thermographs are normally used for this terminology (Hawalkar, 1990). Glass transitions of food material have gained in importance in recent years, particularly in the case of dried and frozen products. Cor- della et al. (2002) reported that Glass transition tempe- rature variation is a powerful technique for characterizing the thermal behavior of honey and for detecting the effect of adulteration on physicochemical and structural proper- ties of samples. Considering that the detection of adulteration would require knowledge of the food products’ physical and chemical properties, the approach of this present study followed two steps, firstly DSC was used to determine the thermal behavior of pure and diluted milk, and, secondly, DSC was used to analyze change in glass transition tem- perature due to addition of sugars like glucose, fructose and sucrose. MATERIALS AND METHODS Sample preparation Milk of a commercial brand (Thacker Dairy, Midnapur an ISO 9001:2000 certified company) was purchased from a local market. According to the producer’s specifications, the composition of milk was (for 100 ml.): 3.3 g milk fat, 3.0 g milk protein, 4.1 g lactose, 117 mg calcium, 88 mg phosphorus, 157 mg vitamin. So by calcu- lation it was found that total milk solid was 10.8 / 100kg milk and water was 89.2 / 100 kg milk. Moisture content of the milk was measured by using Mettler moisture analyzer (Mettler LJ16, 0.01). The sample was kept on the sample pan of the instrument and evaporation of moisture was carried out setting the temperature of the infrared-drying chamber of the instrument at 120 o C. The moisture content was obtained as percentage moisture in wet basis. Moisture content of milk at various levels was determined by vacuum oven (Annapurna Chemical Industries, Kolkata, ± 0.1 o C and ± 1 mmHg) technique. The sample was kept on a Petri dish and evaporation of moisture was carried out overnight by setting the temperature at 65 o C and vacuum level at 650 mm of Hg (AOAC. Official Methods of Analysis, 1997). Milk solutions with different soluble sugars like glucose, fructose and sucrose were made by dissolving these solutes in the pro- portion of 5, 10, 15, 20, and 25% on the basis of milk weight. These solutions were maintained at the constant water content by adding the calculated amount of water externally. Differential scanning calorimetry A differential scanning calorimeter (DSC 204Phoenix Netzsch – Germany) was used for the calorimetric analysis of glass transition temperature. The purge gas used was dry nitrogen gas at a rate of 10 mlmin -1 . The protective gas for balancing the chamber used was also nitrogen gas at a flow rate of 20 mlmin -1 . Indium and Zinc (Netzsch standards) were used for temperature and flow calibration. Before measuring the Tg, the instrument was calibrated with two empty, closed Aluminium crucibles (Roos, 1995). One of the cru- cibles was used as the reference and the other was used for keeping the sample. About 20 - 25 mg of conditioned sample was taken in the sample crucible for the measurement of Tg. The sam- ple was cooled to about -150 o C and then heated to 40 o C at 10 o C min -1 heating rate (Jaya, 2002). The glass transition temperature was determined from the endothermic DSC thermograph as shown in Figure 1. RESULTS AND DISCUSSION Thermal behavior of milk with water DSC thermographs were used to follow the effect of dilu- tion of milk with water on the thermal behavior of milk. Lactose and water were the main constituents (>93%) of milk. Fat, protein, salt and minerals were minor consti- tuents of milk. In the DSC heating curve as shown in Figure 2. it is found that the glass transition temperature of milk de- creases continuously with increase in water content. This gives a clear indication that this thermophysical property of the milk can be used as an indicator of adulteration of milk diluted with water. The glass transition temperture of pure milk varied from -114 and -118 o C. Figure 2 indicates linear relationship between 224 Afr. J. Food Sci. Res. -130 C -120 o te m pe ra tu re , -110 tr an si ti o n -100 -90 G la ss -80 -70 -60 75 80 85 90 95 100 Moisture content, % (w.b.) Figure 2. Glass transition Vs moisture content. 120 (% ) 110 100 ri se 90 T e m p e r at u re 80 70 Tr an si tio n 60 50 40 G la ss 30 20 10 0 0 5 10 15 20 25 30 35 40 45 Water added (%) Figure 3. Rise in glass transition temperature by adding water. Glass transition temperature and moisture content which is expressed as: Tg = - 3.3262 M + 194.28 R 2 = 0.9977 (1) where M is the moisture content on wet basis of milk and R 2 is the coefficient of determination of the tendency curve. It is clear from the eq.(1) that the addition of water was detected by statistically significant temperature gap. From curve fitting a direct relationship for calculatiing the added water percentage was found out by knowing the rise in the glass transition temperature. The equation is: Tg  2.332 A  2.1429 R 2 = 0.9978 (2) where A is the percentage of water added and Tg is the rise in glass transition tamprature in percentage. The adulteration of milk diluted with water was detectable at a dilution level of even 0.5% (The minimum difference in change of water content between two successive levels 0 5 10 15 20 25 30 -116 -118 (o C ) -120 Fructose te m pe ra tu re -122 T ra n s it io n -124 -126 Sucrose G la ss -128 Glucose -130 -132 Weight fraction (%) Figure 4. Tg vs. Weight fraction of sugar added in milk. of 84.81 and 85.31%). Table 1 illustrates the glass tran- sition temperature at different composition of milk with respect to moisture content and total solids. Thermal behavior of milk with dissolved sugars When the DSC thermographs of milk were analyzed at different weight fraction of fructose having T g = 4.8 o C, maintaining the constant weight fraction of water, its glass transition temperatures were found to vary between - 118.02 and -123.02 o C. It is clear that the change in Tg with weight fraction of fructose is very small as shown in Figure 4. Almost similar type of phenomenon was depicted by glucose and sucrose. In case of glucose having Tg = 31.5 o C, and sucrose having Tg = 58.5 o C, glass transition temperature varied from -127.97 to -131.31 o C and - 123.61 to - 129.2 o C respectively. But with glucose, it was found that the values of glass transition temperature attained almost a constant value with increased amount of weight fraction. At the same time in case of fructose and sucrose Tg decreased continuously. Since the differ- rence between the additions of two successive sugars to the milk samples was 5%, the adulteration in milk by sugars could be measured at a minimum impurity level of 5%. Similar results were obtained by researchers in case of honey (Cordella et al., 2002; Kantor et al., 1999). Conclusion Glass transition temperature is one of the most potentially useful parameter powerful for characterizing the thermal behavior of milk and for detecting the effect of adultera- tion on physicochemical and structural properties of milk. In the DSC heating curve it was found that the glass transition temperature of milk rose continuously with de- Goswami and Gupta 225 Table 1. Glass transition temperatures of milk with various composition. Sl. No. Water content (kg/100 kg milk) Total solids (kg/100 kg milk) Glass transitiontemperature, Tg ( o C) 1. 77.47 22.53 -64.1 2. 81.56 18.44 -77.1 3. 83.81 16.19 -84.2 4. 84.31 15.69 -87.4 5. 85.76 14.24 -90.6 6. 86.69 13.31 -92.1 7. 89.43 10.57 -102.1 8. 92.15 7.85 -113.5 9. 93.16 6.84 -115.3 10. 95.71 4.29 -124.2 11. 96.38 3.62 -126.6 crease in water content, which gave a clear indication that this thermophysical property of the milk could be used as an indicator of adulteration of milk by the dilution of water. So the addition of water could be detected by statistically significant temperature gap, which was 4.4779 o C for 1% added water. When soluble sugars like glucose, fructose and sucrose were added, it caused in- evitable changes in Tg due to implicit structural modifi- cation and change in composition. So these sugars can not be used for hiding the milk adulteration by dilution of milk with water. The observed effect could be used for developing a new technique for detection of dilution of milk. REFERENCES Singhal RS, Kulkarni PR, Rege DV (1997). Milk and milk products. In Handbook of Indices of Food Quality and Authenticity, Wood Head Publishing Limited, Cambridge England. pp. 130-176. Dharmarajan CS, Venkateswara RR, Dastur NN (1953). Milk adulteration by adding different chemical substances for hiding the freezing point depression. Indian J. Vet. Sci. 23: 249-267. Kantor Z, Pitsi G, Thoen J (1999). Glass transition temperature of honey as a function of water content as determined by Differential Scanning Calorimetry. J. Agri. food Chem. 47: 2317-2330. Bhandari BR, Howes T (2002). Glass transition in processing and stability of food. Food Australia. 26:1-15. Laaksonen TJ, Roos YH, Labuza TP (2001). Comparisons of the use of desiccators with or without vacuum for water sorption and glass transition studies. Inter. J. Food Properties. 4 (3): 545- 563. Ross KA, Campanella OH, Okos MR (2002). The effect of porosity on glass transition measurement. Inter. J. Food Properties. 5(3): 611- 628. Rahman MS (2004). State Diagram of Date Flesh Using Differential Scanning Calorimetry (DSC). Inter. J. Food Properties .7(3):407- 428. Shen MC, Eisenberg A (1966). Glass transition in polymers. Progress in solid state chemistry. 3: 407-481. Ollett AL, Parker R(1990). The viscosity of supercooled fructose and its glass transition temperature. J. Texture studied. 21: 355-362. Truong V, Bhandari BR, Howes T, Adhikari B (2004). Glass transition behavior of fructose. Inter. J. Food sci. Technol. 39: 569-578. Hawalkar VR, Ma C (1990). In Thermal Analysis of foods; Elsevier Applied science. London, U.K. pp. 92-98. Cordella C, Antinelli JF, Aurieres C (2002). Use of Differential Scanning Calorimetry (DSC) as a New Technique for adulteration in honeys. 1. Study of Adulteration on honey Thermal Behavior. J. Agric. Food Chem. 50: 203-208. AOAC (1997). Official Methods of Analysis (16 th Ed.) Association of official analytical chemists, Washington DC. Roos YH (1995). Glass transition-related physio-chemical changes in foods. Food Technol. 49: 97-102. Jaya S(2002). Vacuum drying of mango pulp. Unpublished Doctoral Thesis, Indian Institute of Technology Kharagpur, India.