ORIGINAL RESEARCH Lactic acid-mediated isolation of alpha-, beta- and kappa-casein fractions by isoelectric precipitation coupled with cold extraction from defatted cow milk SARANYA THEKKILAVEEDU, VENKATESHWARAN KRISHNASWAMI, DHILIN PATHAYAPPURAKKAL MOHANAN, SHANMUGARATHINAM ALAGARSAMY, SUBRAMANIAN NATESAN * and RUCKMANI KANDASAMY * Department of Pharmaceutical Technology, University College of Engineering, National Facility for Bioactive Peptides from Milk (NFBP), Centre for Excellence in Nanobio Translational Research (CENTRE), Anna University, BIT Campus, Tiruchirappalli, Tamil Nadu, India The present study focused on the isolation of individual caseins (a, b and j) from defatted cow’s milk using lactic acid-mediated isoelectric precipitation coupled with cold extraction technique, fol- lowed by purification using sucrose-based density gradient centrifugation method. In vitro charac- terisation of the isolated casein fractions confirmed preservation of micellar morphology throughout the experimental conditions with anionic surface charge and wider particle size range. The RP- HPLC analysis showed a peak at retention time of 25.13 min and 27.84 min for a-casein and b-ca- sein, respectively. Results confirmed isolation of pure casein fractions from cow’s milk which could serve as a rich source of bioactive peptides for nutraceutical/pharmaceutical applications. Keywords Alpha-casein, Beta-casein, Density gradient centrifugation, Kappa-casein, Milk protein etc. INTRODUCTION Milk is considered as a complete or ultimate food for mammals, as it contains rich proteins (20% casein and 80% whey protein), fat, miner- als and vitamins. It also serves as a potential source of bioactive peptides (Korhonen and Pih- lanto 2007; Nielsen et al. 2017; Zhang et al. 2017). Casein is a hydrophobic, thermostable (62–71 °C) and serine-rich phosphoprotein with limited alpha-helix and beta-sheet structure. Casein exists in milk as micelles (50 to 600 nm), composed of three different individual proteins viz. a, b and j caseins, vary from each other with amino acid sequences, molecular weight, phosphoserine units, and calcium ion sensitivity in the order of a > b > j and hydrophobicity in the order b > a > j (Hernan- dez and Harte 2009; Wang et al. 2013; Yen et al. 2015; Caira et al. 2016; Zhang et al. 2016). But the hydrophobic strength of b-casein weakens at a low temperature of 2 °C. j-casein also differs from other types of caseins due to its disulphide-linked polymers and carbohydrate side chains which surrounds the casein micelle that protects from interparticle aggregation (Phadungath 2005). Whole casein has been reported to be sepa- rated from whey protein by various techniques such as isoelectric precipitation (pH 4.6) using different acids (hydrochloric acid, acetic acid and citric acid etc.), coacervation by anionic polysaccharides (pectin, chitosan, guar gum etc.), enzymatic coagulation, salting out, high- resolution centrifugation and gel-filtration. At pH 4.6, the calcium phosphate gets dissolved, causing disruption of micellar arrangement and precipitation of casein occurs (Lachkar et al. 2008; Horne 2014). Apart from the well-established techno-func- tional applications of casein in textile, paper and plastic industries (Guo and Wang 2016), recently its bio functional applications are greatly diversified into molecular chaperone *Author for correspondence. E-mails: hodpharma.aut@ gmail.com; natesansubbu@gmail.com © 2019 Society of Dairy Technology Vol 70 International Journal of Dairy Technology 1 doi: 10.1111/1471-0307.12646