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adapted cells for dryness and elevated temperature for short
time were used for the industrial production process. The
purpose of this study was to optimise new cultivation strategy
for the production of cells in high cell density culture.
This step is considered as the first step for industrialization
process for this type of biotherapeutic yeast.
Materials and Methods
Microorganism
The yeast strain Saccharomyces boulardii ATCC-MYA-796
obtained from (American Type Culture Collection, Manassas,
VA, USA) was used in this study. This strain was
adapted to dryness by successive adaptation method. Cells
were grew on solid medium and allowed to dry on agar
plate. After complete drying of culture, broth medium was
added and the cells grew thereafter were isolated for further
growth on solid medium. This process was repeated
for more than 8 times.
Media
Growth medium
For cell propagation on solid medium, yeast-peptone-dextrose
(YPD) medium was used. This medium composed of
(g l -1 ): glucose, 10; yeast extract, 3; peptone, 3 and agar, 20.
The pH was adjusted to 4.5 before sterilization.
Production medium
Two types of broth media were used in this study. The first
medium used in this study was complex (YMG) medium.
This medium was composed of (g l -1 ): Malt extract, 10.0;
yeast extract, 4.0; glucose, 4.0. The pH was adjusted to 5.5
before sterilization. Unless otherwise mentioned, the yeast
production medium applied was complete synthetic medium
(CSM). This medium was composed of (g l -1 ): Glucose,
15.0; (NH 4 ) 2 SO 4 , 5.0; KH 2 PO 4 , 3.0.; MgSO 4 x7H 2 O,
1.0. The pH was adjusted to pH 5.5 before autoclaving.
Glucose was sterilized separately by autoclaving at 110 ºC
and added to the medium aseptically before inoculation.
Cultivation conditions
Shake flask cultivation and inoculum preparation
In case of shake flask culture, the submerged cultivations
were carried out in 250 ml Erlenmeyer flasks containing
50 ml of growth medium. After inoculation, the flasks were
incubated aerobically on a rotary shaker (Annova 4330,
New Brunswick Scientific Co., NJ, USA) at 200 rpm and
30 °C for 24 h. Cells were used thereafter to inoculate the
bioreactor with inoculum concentration of 5 % (v v -1 )
Bioreactor cultivation
For bioreactor experiments, cultivations were carried out
in 3.0 l stirred tank bioreactor, Bioflo III (New Brunswick
Scientific Co., New Brunswick, NJ, USA), with a work-
ing volume of 1.5 l. Agitation was performed using a three
4-bladed Rushton turbine impellers (d i(impeller diameter) = 65 mm;
d t(tank diameter) = 135 mm, d id t -1 = 0.48) at 300 rpm. Aeration
was performed by filtered sterile air [1 v v -1 min -1 ]. Dissolved
oxygen concentrations were analyzed by polarographic
electrode (Ingold, Switzerland). Foam was suppressed,
when necessary, by the addition of silicon antifoam reagent
(Fluka, Switzerland). In controlled pH batch cultures and in
fed-batch experiments, pH was controlled at 5.5 by addition
of 2.5 Mol l -1 NH 4OH.
Sample preparation and cell dry weight determination
In case of bioreactor cultivations, aliquots (in form of
20 ml) of the culture were taken from the bioreactor vessel
through a sampling system. Cell concentration was determined
immediately by spectrophotometer (Pharmacia Biotech,
Cambridge, England). The optical density of the culture
at 600 nm after diluting the samples into the optical density
range was of 0–0.5. The samples were then filtered using preweighed
filter paper and the filtered biomass was washed
twice by distilled water and subsequently dried in an oven at
110 °C for a constant weight. The relation between OD and
cell dry weight was then determined using standard curve between
the OD and cell dry weight. The filtrate was frozen at
–20 °C and used for glucose determination.
Analysis
Cell mass determination
Optical density of the cell cultures was determined by an
absorbance measurement at 600 nm with a spectrophotometer.
The OD of culture was converted to dry cell mass
through a linear correlation established for the instrument.
Biomass concentration is defined as dry cell mass per unit
volume of culture (g l -1 ).
Determination of glucose
The glucose concentration in the cultivation medium was
determined enzymatically using glucose determination kit
(Biocon GmbH, Germany). The intensity of developed
color was determined at 500 nm using spectrophotometer
(Pharmacia Biotech, Cambridge, England).
Results and Discussions
The production of S. boulardii cell mass was studied in different
cultivation media to improve the production process.
Therefore, three different media namely: complex media,
chemically defined media and semi-defined medium (defined
medium supplemented by yeast extract) were used for
yeast cultivations in batch mode in both shake flasks and
bioreactor. The data of these experiments were used as platform
to design fed-batch cultivation process with different
feeding strategies to cultivate the cells in high cell density
culture.
424 ı Originalarbeiten Deutsche Lebensmittel-Rundschau ı 104. Jahrgang, Heft 9, 2008