Experimentally determining the relative impact of various significant bioprocess parameters on the daily kefir grain increase mass, during 24 h incubation in cow’s milk, based on Taguchi’s fractional factorial design approach, requires the performance of a series experiments. It was established (Harta et al., 2004; Schoevers and Britt, 2003) that culture medium temperature, 3, glucose mass concentration, yG, baker’s yeast mass concentration, y, and the rotational frequency of the stirrer, fm are the main influences bioprocess parameters. The bioprocess parameter in our case is a factor affecting daily kefir grain increase mass and its value is called the ‘level’. We examined the relative impact of the selected bioprocess parameters at four different levels, as shown in Table 1.

During the first stage of the experimental work, it is necessary to prepare the design of experiments. The DoE envisages determining the number of experiments, their performance conditions, and their sequence. Based on the assumption that the daily kefir grain increase mass would be affected by four bioprocess parameters being considered at four levels, we chose the L16 array as the most adequate OA requiring the performance of 16 experiments (Ranjit, 1990). The OA L16 is usually intended for the investigation of five bioprocess parameters at four levels; however, it may also be used in our case (four parameters at four levels) by ignoring the bioprocess parameter E. The DoE is presented in Table 2. The first column presents the experimental serial number. Each experiment was defined by the bioprocess parameters (A, B, C, D and E) marked at specific levels by numbers from 1 to 4. During the second stage of the experimental work, we implemented the proposed DoE by performing the 24 h kefir grain biomass incubations in the RC1 system. The incubation procedure was the same for all experiments. Individual experiments were implemented by means of first charging the reactor by 1 L of fresh HTP whole fat cow’s milk and adding the mass of glucose previously defined by the DoE. This fermentation medium was heated up to working temperature under the defined rotational frequency of the stirrer. After establishing the temperature steady state and dissolved glucose, we inoculated the fermentation medium with the mass of the baker’s yeast also defined by DoE and with 40 g of active kefir grains, which corresponds to initial kefir grain mass concentration, yKG = 40 g/L. After the 24 h incubation was completed, the kefir grain increase mass was determined using the gravimetric method.