In this research, whey protein/pullulan (WP/pullulan) microcapsules were developed in order to assess its protective effect on the viability of Lactobacillus acidophilus NRRL-B 4495 under in vitro gastrointestinal conditions. Results demonstrated that WP/pullulan microencapsulated cells exhibited significantly (p £ 0.05) higher resistance to simulated gastric acid and bile salt. Pullulan incorporation into protein wall matrix resulted in improved survival as compared to free cells after 3 h incubation in simulated gastric solution. Moreover WP/pullulan microcapsules were found to release over 70% of encapsulated L. acidophilus NRRL-B 4495 cells within 1 h. The effect of encapsulation during refrigerated storage was also studied. Free bacteria exhibited 3.96 log reduction while, WP/pullulan encapsulated bacteria showed 1.64 log reduction after 4 weeks of storage.Ó 2015, The Society for Biotechnology, Japan. All rights reserved.[Key words: Encapsulation; Pullulan; Whey protein; Lactobacillus acidophilus; Probiotic]Viability losses during storage and gastrointestinal transit due to detrimental conditions such as harsh acidic environment, oxygen stress and enzymatic reactions reduce the functionality of probiotics to exert health benefits (1,2). Thus, microencapsulation of probiotics is considered as an effective approach for their efficient survival under gastrointestinal conditions and to improve the viability during shelf life to maintain their health promoting effects.Additionally, mechanical protection of probiotic cells during incorporation process into food product is another advantage of microencapsulation (3,4). Despite wide applications of several microencapsulation materials for probiotics, it is a challenge to produce microcapsules for preservation of desired bacterial culture with high viability which does not cause non desirable texture in the final product. Proteins and polysaccharides are widely used materials for the microencapsulation of bioactive ingredients (5e8). Whey protein is one of the most widely used polymer for microencapsulation processes. It is proved to be an effective encapsulating agent in microencapsulation of bioactive ingredients because of their ability to form microcapsules easily under mild conditions using different techniques (9,10).Pullulan is an extracellular polysaccharide produced by Aureobasidium pullulans. It is especially used as a coating material in the food industry, paper industry and pharmaceutical area because of its non-toxic, water-soluble, colorless, tasteless, odorless and heat stable characteristics. Many different industrial wastes such as potato peel, grape skin and olive oil wastes found to be used as a carbon source for the synthesis of pullulan (11,12). More recently, combination of proteins, especially whey proteins, with polysaccharides has been studied for microencapsulation of bioactive substances. Whey protein and pullulan mixtures have been studied to form edible films to reduce moisture loss and increase the shelf life in food applications (13,14...