3. FOOD ChEMISTRy & bIOTEChNOLOGy 3.1. Lectures

Chem. Listy, 102, s265–s1311 (2008) Food Chemistry & Biotechnology
P78 INFLuENCE OF MODIFIED bIOCOMPOSITES
ON PRODuCTION OF ExTRACELLuLAR
POLySACChARIDES by IMMObILIZED
AureObAsiDiuM PullulANs
VLADIMíR OnDRUŠKA, IVAnA MáROVá, JAn
DAVID AnD LUCY VOJTOVá
Faculty of Chemistry, Brno University of Technology,
Purkyňova 118, 612 00 Brno, Czech Republic,
xcondruska@fch.vutbr.cz
Introduction
Pullulan is an extracellular water-soluble polysacharid
produced by yeast-like strain Aureobasidium pullulans. It is
a linear homopolysaccharide usually described as an a-(1–6)
lincagen polymer, consisting mainly of maltotriose units 1 .
The regular alternation of alpha-1,4 and alpha-1,6 bonds
results in two distinctive properties, such as structure flexibility,
enhanced water-solubility and excellent film- and fiber
forming properties 2 . Thanks these characteristic pullulan
can be used in low-calorie food aidtives, cosmetic emulsions,
oxygen-impermeable film for packaging, adhesives, and
thickening and extending agents. Recently, pullulan has been
getting renewed attention as an excellent material for pharmaceutical
and biomedical application 3 .
Fermentation can be affected by different types of carbon
and nitrogen source resulted in varying pullulan yields
during the culture growth process, since the philamentous
forms or chlamydospores are less productive than the yeast
or pigment-free blastospores 4 .
Fig. 1. Chemical structure of polysaccharide pullulan
In this work, several media with different type of carbon
and/or nitrogen source were used for A. pullulans cultivation.
PUR foams modified by 10 % of carboxymethyl cellulose,
2-hydroxyethyl cellulose, acetylated starch and acetyl
celulose, respectively were used as immobilization agents.
A. pullulans cells were cultivated in Erlenmeyer flasks at
28° C for 120 to 480 hours. First, all materials were tested
as potential carbon/nitrogen source. Further, microorganisms
s747
were cultivated for 4–6 weeks in presence of PUR under permanent
shaking. Samples were taken in 24-hour intervals,
quantitative changes of biomass and pullulan content were
determined gravimetrically and/or spectrophotometrically.
Additionally, surface microscopy of all degraded polyurethanes
was tested.
Material and methods
M i c r o o r g a n i s m a n d C u l t u r e
C o n d i t i o n s
Strain of A. pullulans, CCM F-148 was purchased from
Czech Collection of Microorganisms. The culture was stored
on medium with malt extract and yeast malt agar, respectively,
at 4° C. For biodegradation experiments 100 ml of
inoculum was prepared in 500 ml Erlenmayer flask. Cultivation
was performed at 28 °C and 150 rpm for 24 hours.
Inoculum as well as production medium contained (g dm –3 ):
yeast extract 7, potassium phosphate 5, ammonium sulphate
5, magnesium sulphate 0.34, glucose 40 was used as carbon
source, distilled water. Initial pH was 6.5 before autoclaving.
All flasks were incubated in an incubator shaker operating at
28–30 °C and 150 rpm for 120 hours.
M a t e r i a l s
General chemicals: polyether polyol, tolylene diisocyanate
80/20 (TDI), tin and amine based catalysts, surfactant
and water. Biodegradable fillers: acetylated starch (AS),
acetylcellulose (AC) Mn = 30,000 Da and 2-hydroxyethylcellulose
(HEC) Mn = 90,000 Da. The foams were prepared
by a three-step reaction process. The chemical composition
of the pulverized BIO-PU foams was proved by an infrared
spectroscopy on the nicolet Impact 400D Fourier Transform
InfraRed (FTIR) spectrometer using the KBr technique.
M e a s u r e m e n d o f D r y C e l l W e i g h t
a n d P u l l u l a n P r o d u c t i o n
Samples for analyses (10 ml) were taken from each flask
in regular 24-hour intervals. Total biomass (summ of mycelial
and yeast cells) was determined after centrifugation of the
culture sample at 12,000 × g for 20 min and washing the sediment
with distilted water gravimetrically (drying at 105 °C
for 2 hours).
To analysis of pullulan production, supernatant was
mixed with 2 volumes of absolute ethanol for 20 min. Precipited
polysaccharide was separated by centrifugation or
filtration and dried at 80 °C. Pullulan precipitate was purified
twice by hot water and by ethanol. Crude pullulan yield was
measured gravimetrically.
Pure polysaccharide content was determined by analysis
of products of its enzymatic hydrolysis (effect of pullulanase
resulted in maltotriose). The content of maltotriose was determined
by Somogyi-nelson method 5 , calibration with maltotriose
was performed.