2.2. Methods
2.2.1. Preparation of lactose–zinc complex
The basic steps followed for the preparation of lactose–zinc complex were as described by Kearsley and Birch (1977) . 0.5 mol/L lactose solution (17.11 g lactose in 100 mL of distilled water) was prepared and to this solution zinc salt (zinc sulphate heptahydrate) was added slowly from the stock solution to obtain a final zinc concentration ranging from 1 to 10 mg/g of lactose (viz. 2.61–26.16 mmol/L) with constant stirring using magnetic stirrer (SPINOT MC 02, Tarsons Products Pvt. Ltd., Kolkata, India). The pH of the solution was adjusted to 7.0 by 2 mol/L NaOH and was left at room temperature (30 °C) for 2 h (the solution was constantly monitored for changes in pH and maintained at pH 7 with constant stirring). The solution was then centrifuged at 2000× g for 5 min to remove insoluble zinc and lactose as precipitate. The supernatant which contained soluble lactose and zinc was further concentrated 5 times (i.e. 20 mL) by a rotary evaporator (RV 10 DS 96, IKA, Wilmington, NC, USA) at 60 °C/20 min under 600 mm Hg vacuum. Absolute alcohol (lactose: alcohol at 1:10 ratio) was added immediately and the mixture was kept at freezing temperature (−18 to −20 °C) for 24 h to separate lactose crystals. Subsequently, the complex was washed (to remove unbound free zinc) by dissolving the crystals in water (100 mL) with constant stirring for another 1 h, concentrated and crystallized as mentioned above. Finally, the crystallized complex was dried at 50 °C/20 min in a hot air oven.