3. FOOD ChEMISTRy & bIOTEChNOLOGy 3.1. Lectures
3. FOOD ChEMISTRy & bIOTEChNOLOGy 3.1. Lectures
3. FOOD ChEMISTRy & bIOTEChNOLOGy 3.1. Lectures
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Chem. Listy, 102, s265–s1311 (2008) Food Chemistry & Biotechnology<br />
L05 PhySIOLOGICAL REGuLATION OF<br />
bIOTEChNOLOGICAL PRODuCTION OF<br />
CAROTENOID PIGMENTS<br />
VLADIMíRA HAnUSOVá a , MARTInA ČARnECKá b ,<br />
AnDREA HALIEnOVá b , MILAn ČERTíK a , EMíLIA<br />
BREIEROVá c and IVAnA MáROVá b<br />
a Department of Biochemical Technology, Faculty of Chemical<br />
and Food Technology, Slovak University of Technology,<br />
Radlinského 9, 812 37 Bratislava, Slovak Republic;<br />
b Faculty of Chemistry, Brno University of Technology, Purkyňova<br />
118, 612 00 Brno, Czech Republic;<br />
c Institute of Chemistry, Slovak Academy of Sciences, Dúbravská<br />
cesta 9, 845 38 Bratislava, Slovak Republic,<br />
milan.certik@stuba.sk<br />
Introduction<br />
Carotenoids represent one of the broadest group of<br />
natural antioxidants (over 600 characterized structurally)<br />
with significant biological effects and numerous of industrial<br />
applications. Because the application of synthetically prepared<br />
carotenoids as food additives has been strictly regulated<br />
in recent years, huge commercial demand for natural carotenoids<br />
has focused attention on developing of suitable biotechnological<br />
techniques for their production.<br />
There are many microorganisms including bacteria,<br />
algae, yeast and fungi, that are able to accumulate several<br />
types of pigments; but only a few of them have been exploited<br />
commercially 1 . From the view of yeasts, a range of species<br />
such as Rhodotorula, Rhodosporidium, Sporidiobolus, Sporobolomyces,<br />
Cystofilobasidium, Kockovaella and Phaffia<br />
have been screened for carotenoids formation. Yeast strains<br />
of Rhodotorula and Sporobolomyces formed β-carotene as<br />
the main pigment together with torulene and torularhodine as<br />
minor carotenoids. In contrast, Phaffia strains accumulated<br />
astaxanthin as a principal carotenoid. Comparative success<br />
in yeast pigment production has led to a flourishing interest<br />
in the development of fermentation processes in commercial<br />
production levels. However, in order to improve the yield of<br />
carotenoid pigments and subsequently decrease the cost of<br />
this biotechnological process, optimizing the culture conditions<br />
including both nutritional and physical factors have<br />
been performed. Factors such as carbon and nitrogen sources,<br />
minerals, vitamins, pH, aeration, temperature, light and<br />
stress showed a major influence on cell growth and yield of<br />
carotenoids.<br />
This paper summarizes our experience with physiological<br />
regulation and scale-up of biotechnological production of<br />
carotenoid pigments by yeasts.<br />
Experimental<br />
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<br />
C o n d i t i o n s<br />
All strains investigated in this study (Sporobolomyces<br />
roseus CCY 19-6-4, S. salmonicolor CCY 19-4-10, Rhodotorula<br />
glutinis CCY 20-2-26, R. glutinis CCY 20-2-31, R. glu-<br />
s547<br />
tinis CCY 20-2-33, R. rubra CCY 20-7-28, R. aurantiaca<br />
CCY 20-9-7 and Phaffia rhodozyma CCY 77-1-1) were<br />
obtained from the Culture Collection of Yeasts (CCY; Institute<br />
of Chemistry, Slovak Academy of Sciences, Bratislava)<br />
and maintained on malt slant agar at 4 °C.<br />
The basic cultivation medium for flasks experiments<br />
for Rhodotorula and Sporobolomyces strains consisted of<br />
(g dm –3 ): glucose – 20; yeast extract – 4.0; (nH 4 ) 2 SO 4 – 10;<br />
KH 2 PO 4 – 1; K 2 HPO 4 . 3H2 O – 0.2; naCl – 0.1; CaCl 2 – 0.1;<br />
MgSO 4 . 7H2 O – 0.5 and 1 ml solution of microelements<br />
[(mg dm –3 ): H 3 BO 4 – 1.25; CuSO 4 . 5H2 O – 0.1; KI – 0.25;<br />
MnSO 4 . 5H2 O – 1; FeCl 3 . 6H2 O – 0.5; (nH 4 ) 2 Mo 7 O 24 . 4H2 O<br />
– 0.5 and ZnSO 4 . 7H2 O – 1]. The basic cultivation medium<br />
for flasks experiments for Phaffia strain consisted of<br />
(g dm –3 ): glucose – 20, yeast autolysate – 2.0, KH 2 PO 4 – 0.4,<br />
(nH 4 ) 2 SO 4 – 2.0, MgSO 4 . 7H2 O – 0.5, CaCl 2 – 0.1, naCl<br />
– 1.0. All strains grew under a non-lethal and maximally tolerated<br />
concentration of ni 2+ , Zn 2+ , Cd 2+ and Se 2+ ions. Also,<br />
stress conditions were induced by addition of various conventrations<br />
of naCl and H 2 O 2 . The cultures were cultivated<br />
in 500 ml flasks containing 250 ml cultivation medium on<br />
a rotary shaker (150 rpm) at 28 °C to early stationary grow<br />
phase. All cultivation experiments were carried out at triplicates<br />
and analyzed individually.<br />
Flasks results were verified in bioreactors and these<br />
scale-up experiments were carried out in 2 L fermentor (B.<br />
Braun Biotech), 20 L (SLF-20) and 100 L (Bio-la-fite) fermentors<br />
with an agitation rate of 250–450 rpm and a temperature<br />
of 20–22 °C. The pH was controlled at pH 5.0 by the<br />
addition of nH 4 OH and the dissolved oxygen concentration<br />
was maintained by supplying sterile air at a flow rate equivalent<br />
to 0.3–0.7 vvm.<br />
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<br />
Pigments from homogenized bioproducts were isolated<br />
by organic extraction and analyzed by high-performance<br />
liquid chromatography (HPLC). Analysis was carried out<br />
with an HP 1100 chromatograph (Agilent) equipped with a<br />
UV-VIS detector. Pigments extracts (10 μl) were injected<br />
onto LiChrospher ® 100 RP-18 (5 μm) column (Merck). The<br />
solvent system (the flow rate was 1 ml min –1 ) consisted of<br />
solvent A, acetonitrile/water/formic acid 86 : 10 : 4 (v/v/v),<br />
and B, ethyl acetate/formic acid 96 : 4 (v/v), with a gradient<br />
of 100 % A at 0 min, 100 % B at 20 min, and 100 % A at<br />
30 min.<br />
G e l E l e c t r o p h o p h o r e s i s<br />
1D PAGE-SDS electrophoresis of proteins was carried<br />
out by common procedure using 10% and 12.5% polyacrylamide<br />
gels. Proteins were staining by Coomassie Blue and by<br />
silver staining. For comparison, microfluidic technique using<br />
1D Experion system (BioRad) and P260 chips was used for<br />
yeast protein analysis too. 2D electrophoresis of proteins<br />
was optimized in cooperation with Laboratory of Functional<br />
Genomics and Proteomics, Faculty of Science, Masaryk<br />
University of Brno. 2D gels were obtained from protein pre-