Influence of fermentation time, cryoprotectant and neutralization of cell concentrate on freeze-drying survival, storage stability, and acid and bile exposure of Bifidobacterium animalis ssp. lactis cells produced without milk-based ingredients M. Saarela 1 , I. Virkaja ¨rvi 1 , H.-L. Alakomi 1 , T. Mattila-Sandholm 1 , A. Vaari 1 , T. Suomalainen 2 and J. Ma ¨tto ¨ 1 1 VTT Biotechnology, Espoo, Finland, and 2 Valio Ltd R&D, Helsinki, Finland 2004/0762: received 2 July 2004, revised 7 February 2005 and accepted 25 June 2005 ABSTRACT M. SAARELA, I. VIRKAJA ¨ RVI, H.-L. ALAKOMI, T. MATTILA-SANDHOLM, A. VAARI, T. SUOMALAINEN AND J. MA ¨ TTO ¨ . 2005. Aims: To investigate the stability of Bifidobacterium animalis ssp. lactis VTT E-012010 (¼Bb-12) during freeze- drying, storage and acid and bile exposure. The effect of harvesting time and composition and pH of the cryoprotectant on the survival was evaluated. The procedure was performed by using a milk-free culture medium and cryoprotectants to produce cells for nonmilk-based applications. Methods and Results: Bifidobacterial cells were grown in fermenters in general edible medium for 15 or 22 h. The cell mass was freeze-dried either as non-neutralized or neutralized using sucrose, betaine or reconstituted skim milk (control) as cryoprotectants. For stability studies freeze-dried powders were stored at 37, 5 and )20°C for 2– 6 months. In addition, acid and bile tolerance of the powders was tested. Sucrose-formulated B. animalis ssp. lactis preparations had an excellent stability during storage at refrigerated and frozen temperatures for 5–6 months. They also had a good survival during storage at 37°C for 2 months as well as during exposure to pH 3 and 1% bile acids. No difference was observed between 15 and 22 h grown cells or between non-neutralized and neutralized cells. Betaine proved to be a poor cryoprotectant compared with sucrose. Conclusions: Fermentation time and neutralization of cell concentrate before freeze-drying had no impact on the storage stability and bile and acid tolerance of freeze-dried bifidobacterial cells. The nonmilk-based production protocol using sucrose as a cryoprotectant yielded powdery preparations with excellent stability in adverse conditions (storage at elevated temperatures and during acid and bile exposure). Significance and Impact of the Study: The results indicate that it is feasible to develop nonmilk-based production technologies for probiotic cultures. This provides new possibilities for the development of nondairy- based probiotic products. Keywords: acid tolerance, Bb-12, Bifidobacterium, fermentation, freeze-drying. Correspondence to: Maria Saarela, VTT Biotechnology, PO Box 1500, FIN-02044 VTT, Espoo, Finland (e-mail: maria.saarela@vtt.fi). ª 2005 The Society for Applied Microbiology Journal of Applied Microbiology 2005, 99, 1330–1339 doi:10.1111/j.1365-2672.2005.02742.x