3. FOOD ChEMISTRy & bIOTEChNOLOGy 3.1. Lectures

Chem. Listy, 102, s265–s1311 (2008) Food Chemistry & Biotechnology
L05 PhySIOLOGICAL REGuLATION OF
bIOTEChNOLOGICAL PRODuCTION OF
CAROTENOID PIGMENTS
VLADIMíRA HAnUSOVá a , MARTInA ČARnECKá b ,
AnDREA HALIEnOVá b , MILAn ČERTíK a , EMíLIA
BREIEROVá c and IVAnA MáROVá b
a Department of Biochemical Technology, Faculty of Chemical
and Food Technology, Slovak University of Technology,
Radlinského 9, 812 37 Bratislava, Slovak Republic;
b Faculty of Chemistry, Brno University of Technology, Purkyňova
118, 612 00 Brno, Czech Republic;
c Institute of Chemistry, Slovak Academy of Sciences, Dúbravská
cesta 9, 845 38 Bratislava, Slovak Republic,
milan.certik@stuba.sk
Introduction
Carotenoids represent one of the broadest group of
natural antioxidants (over 600 characterized structurally)
with significant biological effects and numerous of industrial
applications. Because the application of synthetically prepared
carotenoids as food additives has been strictly regulated
in recent years, huge commercial demand for natural carotenoids
has focused attention on developing of suitable biotechnological
techniques for their production.
There are many microorganisms including bacteria,
algae, yeast and fungi, that are able to accumulate several
types of pigments; but only a few of them have been exploited
commercially 1 . From the view of yeasts, a range of species
such as Rhodotorula, Rhodosporidium, Sporidiobolus, Sporobolomyces,
Cystofilobasidium, Kockovaella and Phaffia
have been screened for carotenoids formation. Yeast strains
of Rhodotorula and Sporobolomyces formed β-carotene as
the main pigment together with torulene and torularhodine as
minor carotenoids. In contrast, Phaffia strains accumulated
astaxanthin as a principal carotenoid. Comparative success
in yeast pigment production has led to a flourishing interest
in the development of fermentation processes in commercial
production levels. However, in order to improve the yield of
carotenoid pigments and subsequently decrease the cost of
this biotechnological process, optimizing the culture conditions
including both nutritional and physical factors have
been performed. Factors such as carbon and nitrogen sources,
minerals, vitamins, pH, aeration, temperature, light and
stress showed a major influence on cell growth and yield of
carotenoids.
This paper summarizes our experience with physiological
regulation and scale-up of biotechnological production of
carotenoid pigments by yeasts.
Experimental
M i c r o o r g a n i s m s a n d C u l t i v a t i o n
C o n d i t i o n s
All strains investigated in this study (Sporobolomyces
roseus CCY 19-6-4, S. salmonicolor CCY 19-4-10, Rhodotorula
glutinis CCY 20-2-26, R. glutinis CCY 20-2-31, R. glu-
s547
tinis CCY 20-2-33, R. rubra CCY 20-7-28, R. aurantiaca
CCY 20-9-7 and Phaffia rhodozyma CCY 77-1-1) were
obtained from the Culture Collection of Yeasts (CCY; Institute
of Chemistry, Slovak Academy of Sciences, Bratislava)
and maintained on malt slant agar at 4 °C.
The basic cultivation medium for flasks experiments
for Rhodotorula and Sporobolomyces strains consisted of
(g dm –3 ): glucose – 20; yeast extract – 4.0; (nH 4 ) 2 SO 4 – 10;
KH 2 PO 4 – 1; K 2 HPO 4 . 3H2 O – 0.2; naCl – 0.1; CaCl 2 – 0.1;
MgSO 4 . 7H2 O – 0.5 and 1 ml solution of microelements
[(mg dm –3 ): H 3 BO 4 – 1.25; CuSO 4 . 5H2 O – 0.1; KI – 0.25;
MnSO 4 . 5H2 O – 1; FeCl 3 . 6H2 O – 0.5; (nH 4 ) 2 Mo 7 O 24 . 4H2 O
– 0.5 and ZnSO 4 . 7H2 O – 1]. The basic cultivation medium
for flasks experiments for Phaffia strain consisted of
(g dm –3 ): glucose – 20, yeast autolysate – 2.0, KH 2 PO 4 – 0.4,
(nH 4 ) 2 SO 4 – 2.0, MgSO 4 . 7H2 O – 0.5, CaCl 2 – 0.1, naCl
– 1.0. All strains grew under a non-lethal and maximally tolerated
concentration of ni 2+ , Zn 2+ , Cd 2+ and Se 2+ ions. Also,
stress conditions were induced by addition of various conventrations
of naCl and H 2 O 2 . The cultures were cultivated
in 500 ml flasks containing 250 ml cultivation medium on
a rotary shaker (150 rpm) at 28 °C to early stationary grow
phase. All cultivation experiments were carried out at triplicates
and analyzed individually.
Flasks results were verified in bioreactors and these
scale-up experiments were carried out in 2 L fermentor (B.
Braun Biotech), 20 L (SLF-20) and 100 L (Bio-la-fite) fermentors
with an agitation rate of 250–450 rpm and a temperature
of 20–22 °C. The pH was controlled at pH 5.0 by the
addition of nH 4 OH and the dissolved oxygen concentration
was maintained by supplying sterile air at a flow rate equivalent
to 0.3–0.7 vvm.
P i g m e n t I s o l a t i o n a n d A n a l y s i s
Pigments from homogenized bioproducts were isolated
by organic extraction and analyzed by high-performance
liquid chromatography (HPLC). Analysis was carried out
with an HP 1100 chromatograph (Agilent) equipped with a
UV-VIS detector. Pigments extracts (10 μl) were injected
onto LiChrospher ® 100 RP-18 (5 μm) column (Merck). The
solvent system (the flow rate was 1 ml min –1 ) consisted of
solvent A, acetonitrile/water/formic acid 86 : 10 : 4 (v/v/v),
and B, ethyl acetate/formic acid 96 : 4 (v/v), with a gradient
of 100 % A at 0 min, 100 % B at 20 min, and 100 % A at
30 min.
G e l E l e c t r o p h o p h o r e s i s
1D PAGE-SDS electrophoresis of proteins was carried
out by common procedure using 10% and 12.5% polyacrylamide
gels. Proteins were staining by Coomassie Blue and by
silver staining. For comparison, microfluidic technique using
1D Experion system (BioRad) and P260 chips was used for
yeast protein analysis too. 2D electrophoresis of proteins
was optimized in cooperation with Laboratory of Functional
Genomics and Proteomics, Faculty of Science, Masaryk
University of Brno. 2D gels were obtained from protein pre-