ORIGINAL ARTICLE Enhancement of viability of a probiotic Lactobacillus strain for poultry during freeze-drying and storage using the response surface methodology Anahita KHORAMNIA, 1 Norhani ABDULLAH, 1,2 Siew Ling LIEW, 3 Chin Chin SIEO, 1,2 Kalavathy RAMASAMY 4 and Yin Wan HO 2 1 Department of Microbiology, Faculty of Biotechnology and Biomolecular Sciences, 2 Institute of Bioscience, Universiti Putra Malaysia, 3 Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 4 Faculty of Pharmacy, Universiti Teknologi MARA Malaysia, Selangor Darul Ehsan, Malaysia ABSTRACT A rotatable central composite design (CCD) was used to study the effect of cryoprotectants (skim milk, sucrose and lactose) on the survival rate of a probiotic Lactobacillus strain, L. reuteri C10, for poultry, during freeze-drying and storage. Using response surface methodology, a quadratic polynomial equation was obtained for response value by multiple regression analyses: Y = 8.59546 - 0.01038 X1 - 0.09382 X2 - 0.07771 X3 - 0.054861 X1 2 - 0.04603X3 2 - 0.10938 X1X2. Based on the model predicted, sucrose exerted the strongest effect on the survival rate. At various combinations of cryoprotectants, the viability loss of the cells after freeze-drying was reduced from 1.65 log colony forming units (CFU)/mL to 0.26–0.66 log CFU/mL. The estimated optimum combination for enhancing the survival rate of L. reuteri C10 was 19.5% skim milk, 1% sucrose and 9% lactose. Verification experiments confirmed the validity of the predicted model. The storage life of freeze-dried L. reuteri C10 was markedly improved when cryoprotectants were used. At optimum combination of the cryoprotectants, the survival rates of freeze-dried L. reuteri C10 stored at 4°C and 30°C for 6 months were 96.4% and 73.8%, respectively. Total viability loss of cells which were not protected by cryoprotectants occurred after 12 and 8 weeks of storage at 4°C and 30°C, respectively. Key words: cryoprotectants, freeze-drying, probiotic, response surface methodology, storage. INTRODUCTION During the past 50 years, antibiotics have been used in livestock production at therapeutic levels to treat bac- terial infections, as well as at sub-therapeutic levels to promote growth. Many of the antibiotics used as growth promoters in the livestock industry are also used in human medicine. Shortly after the initiation of widespread use of antibiotics in the livestock industry, particularly the poultry industry, the practice was placed under increased scrutiny because of concern over the development of bacterial resistance to the usual microbiocidal effects of the antibiotics. Since then, the indiscriminate use of antibiotics in poultry production has become a cause for concern (Edens 2003). An understanding of the importance of the intesti- nal microflora in the maintenance of health and the prevention of disease has led to the suggestion that probiotics could be used as an alternative to antibiotic growth promoters in livestock. Probiotics are defined as viable microorganisms that when administered in adequate amounts, promote or support a beneficial balance of the autochthonous microbial population of the gastrointestinal tract (Holzapfel & Schillinger 2001; Fung et al. 2008). As the viability of the probiotic microorganisms is essential to exert beneficial effects on the host, the viability and stability of the probiotic microorganisms during storage are critical factors to be considered by manufacturers and consumers. Drying methods are the most common techniques used for preservation of microorganisms (Carvalho et al. 2003) and preservation of large quantities of bac- teria by freeze-drying is frequently used. However, the Correspondence: Sieo Chin Chin, Department of Microbiol- ogy, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor Darul Ehsan, Malaysia. (Email: ccsieo@biotech.upm.edu.my) Received 12 July 2009; accepted for publication 14 February 2010. Animal Science Journal (2011) 82, 127–135 doi: 10.1111/j.1740-0929.2010.00804.x © 2010 The Authors Journal compilation © 2010 Japanese Society of Animal Science