Optimisation of medium composition for probiotic biomass production using response surface methodology Masumeh Anvari 1 *, Gholam Khayati 2 and Shora Rostami 3 1 Department of Microbiology, Faculty of Sciences, Islamic Azad University, Rasht Branch, P.O. Box 41335-3516, Rasht, Iran 2 Department of Chemical Engineering, Faculty of Eng., Guilan University, P. O. Box 3756-41635, Rasht, Iran 3 Department of Microbiology, Sciences and Research Branch, Islamic Azad University, Guilan, Iran Received 25 April 2013; accepted for publication 16 October 2013 This study was aimed to optimise lactose, inulin and yeast extract concentration and also culture pH for maximising the growth of a probiotic bacterium, Bifidobacterium animalis subsp. lactis in apple juice and to assess the effects of these factors by using response surface methodology. A second-order central composite design was applied to evaluate the effects of these independent variables on growth of the microorganism. A polynomial regression model with cubic and quadratic terms was used for analysis of the experimental data. It was found that the effects involving inulin, yeast extract and pH on growth of the bacterium were significant, and the strongest effect was given by the yeast extract concentration. Estimated optimum conditions of the factors on the bacterial growth are as follows: lactose concentration = 9·5 g/l; inulin concentration = 38·5 mg/l; yeast extract concen- tration = 9·6 g/l and initial pH = 6·2. Keywords: Bifidobacterium animalis subsp. Lactis, probiotic, response surface methodology, apple juice. Probiotics are defined as viable microorganisms which, upon ingestion in sufficient amounts, exert health benefits to the host beyond inherent basic nutrition (Guarner & Schaafsma, 1998). They have been used for the treatment of various types of diarrhoea (Szymanski et al. 2006), urogenital infections (Reid et al. 2003), and gastrointestinal diseases such as Crohn’s disease (Bousvaros et al. 2005) and pouchitis (Kuehbacher et al. 2006), although there is still no consensus about their effectiveness (Lin, 2003). Probiotic bacteria, often belonging to the Lactobacillus and Bifidobacterium genera (Weinbreck et al. 2010). Lactic acid bacteria are commercialised mainly as food supplements with dairy products being the most often used vehicle (Heller, 2001; Lourens-Hattingh & Viljoen, 2001). However, recent studies have suggested fruit juices as an alternative vehicle for the incorporation of probiotics (Mousavi et al. 2011; Pereira et al. 2011; Fonteles et al. 2012; Costa et al. 2013). Fruit juices are rich in nutrients and do not contain starter cultures that compete for nutrients with probiotics. Furthermore, fruit juices contain high amounts of sugars, which could encourage probiotic growth (Ding & Shah, 2008). The optimal growth of probiotic bacteria is affected by fermentation conditions such as pH, temperature, medium formulation and the others. Study of the individual and interactive effects of these factors will help in efforts to optimise biomass production of the probiotic microorganism (Du Toit et al. 2011). According to Oliveira & Damin (2003), to evaluate the growth of lactic acid bacteria, it is necessary to know the substrates applied for the microbial growth, as well as, the optimal temperature and pH values because these factors are the most important for the microbial development. Response surface methodology (RSM) is a useful model for studying the effect of several factors influencing the responses by varying them simultaneously and carrying out a limited number of experiments. In addition, response surface methodology is an efficient strategic experimental tool by which the optimal conditions of a multivariable system may be determined (Khayati & Kiyani, 2012; Khayati, 2013). Lactobacilli are also extensively used as probiotics, but no information is available on the growth of the species Bifidobacterium in apple juice. The objective of this study was to determine the suitability of apple juice as a raw material for production of probiotic by Bifidobacterium spp.. Thus, the use of apple juice as substrate to produce a pro- biotic was studied herein. So in this paper, the growth of probiotic Bifidobacterium animalis subsp. lactis in apple juice (as a basement medium) with a function of four affec- ting parameters including lactose (g/l), inulin (mg/l) and yeast *Corresponding author; e-mail: anvari@iaurasht.ac.ir Journal of Dairy Research (2014) 81 59–64. © Proprietors of Journal of Dairy Research 2013 doi:10.1017/S0022029913000733 59