3. FOOD ChEMISTRy & bIOTEChNOLOGy 3.1. Lectures

Chem. Listy, 102, s265–s1311 (2008) Food Chemistry & Biotechnology
P19 ChARACTERIZATION OF bETA-CAROTENE
ENRIChED bIOMASS PRODuCTION by RED
yEASTS
MARTInA ČARnECKá a , AnDREA HáROnIKOVá a ,
TEREZIE DVOřáKOVá a , AnDREA HALIEnOVá a ,
IVAnA MáROVá a and EMíLIA BREIEROVá b
a Brno University of Technology, Faculty of Chemistry,
Department of Food Chemistry and Biotechnology, Purkyňova
118, 612 00 Brno, Czech Republic,
b Institute of Chemistry, Slovak Academy of Sciences, Dúbravská
cesta 9, 845 38 Bratislava, Slovak Republic,
carnecka@fch.vutbr.cz
Introduction
During the last decades fast progress has been made
within the field of biochemistry of carotenoid biosynthesis
in bacteria, fungi, and plants. Although more than 600 different
carotenoids have been identified in nature, only a few
are used industrially. There are many yeast strains able to
produce different carotenoids. However, the heterobasidiomycetous
yeast Xanthophyllomyces dendrorhous, are the
only microbial systems with commercial potentials for the
production of astaxanthin. Several genes involved in the astaxanthin
biosynthetic pathway of X. dendrorhous have been
cloned and characterized recently. Analysis of DnA in other
red yeast is very complicated and only several genes were
described yet.
There are many yeast strains able to produce different
carotenoids, mainly as a part of stress response. In this work
three different red yeast strains (Sporobolomyces roseus,
Rhodotorula glutinis, Rhodotorula rubra) were enrolled into
a comparative study. To increase the yield of carotenoid pigments
at improved biomass production, several types of exogenous
as well as nutrition stress were tested. Each strain was
cultivated at optimal growth conditions and in medium with
modified carbon and nitrogen sources. Synthetic media with
addition of complex substrates (e.g. yeast extract) and vitamin
mixtures as well as some waste materials (whey, potato
extract) were used as nutrient source. Some types of exogenous
stress – peroxide, salt were applied too. The production
of carotene-enriched biomass was carried out in flasks as well
as in laboratory fermentor. Changes in yeast cells on metabolome
level were studied using LC/MS techniques to carotenoid
analysis.
Materials and Methods
S t r a i n s
In the study following red yeast strains were tested: Rhodotorula
glutinis CCY 20-2-26, Sporobolomyces roseus CCY
19-4-8; Rhodotorula rubra CCY 20-7-31; Phaffia rhodozyma
CCY 77-1-1, Sporobolomyces salmonicolor CCY 19-4-10.
C u l t i v a t i o n
Red yeasts were cultivated in a simple glucose medium
aerobically at 28 °C. Physilogical stress was induced by nutri-
s615
tion components (C and n source) and by addition of 5 mM
peroxide and 2% and/or 5 % naCl. Stress cultivations with S.
salmonicolor, R. glutinis and P. rhodozyma were realized in
flasks as well as in 2 L-laboratory fermentor (B.Braun Biotech).
Three series of cultivations were realized with each
strain. Two-step inoculation was done. All strains were firstly
inoculated into a medium containing yeast autolysate (7 g),
(nH 4 ) 2 SO 4 (5 g), glucose (40 g), KH 2 PO 4 (5 g), MgSO 4
(0.34 g) per liter (InO I) and cultivated at 28 °C for 24 hours
at permanent shaking and lighting. Second inoculum (InO II)
was prepared similarly, in 1 st series was used the same
medium as InO I, in 2 nd series lyophilized whey was added
(7 g dm –3 ) and in 3 rd series potato extract (7 g dm –3 ) was
added into InO II. Cultivation in InO II undergo at 28 °C
for 24 hours at permanent shaking and lighting. Production
media contained (nH 4 ) 2 SO 4 (5 g), glucose (40 g), KH 2 PO 4
(5 g), MgSO 4 (0.34 g) per liter. Several waste substrates were
added and cultivation was done for 80 hours at 28 °C under
permanent lighting and shaking. Production media were pre-
pared according to following scheme:
•
•
•
1 st series: InO I --- InO II --- production: 1 – control,
2 – 5 mM peroxide, 3 – 2% naCl, 4 – 5% naCl,
5 – lyophillized whey non-processed (7 g dm –3 ), 6
– lyophillized whey processed by deproteination agent
(7 g dm –3 ), 7 – liquid whey (250 ml dm –3 ), 8 – potato
extract (Hi Media; 7 g dm –3 )
2 nd series: InO I --- InO II (whey, 7 g dm –3 ) --- production:
1 – control, 2 – 5 mM peroxide, 3 – 2% naCl,
4 – 5% naCl, 5 – lyophillized whey non-processed
(7 g dm –3 ), 6 – lyophillized whey processed by deproteination
agent (7 g dm –3 ), 7 – liquid whey (250 ml dm –3 )
3 rd series: InO I --- InO II (potato extract 7 g dm –3 ) --
- production: 1 – control, 2 – 5 mM peroxide, 3 – 2%
naCl, 4 – 5% naCl, 5 – potato extract (7 g dm –3 ).
A n a l y z e d P a r a m e t e r s
In all samples biomass and carotenoid content were evaluated.
Biomass was determined gravimetrically. Levels of
carotenoids – lycopene and beta-carotene were analyzed using
HPLC/VIS (450 nm) and verified by HPLC/MS. Ergosterol
and phytoene were analyzed by RP-HPLC (280 nm).
Results
In this work the growth of some red yeasts on waste substrates
and subsequent effect of these substrates on beta-carotene
production was studied. It was observed that addition
of non-processed or deproteined whey or potato extract to
media can increase beta-carotene production.
In Rhodotorula glutinis addition of deproteined whey
into production medium led to 3.5 × increased production of
beta-carotene without changes in biomass. non-processed
whey or potato extract added to production media led to
about 3 × increase of beta-carotene production but it was
accompanied by lost in biomass. The highest yield was reached
after addition of lyophillized whey to InO II as well as