3. FOOD ChEMISTRy & bIOTEChNOLOGy 3.1. Lectures

Chem. Listy, 102, s265–s1311 (2008) Food Chemistry & Biotechnology
L03 SOLID STATE FERMENTATION AS A TOOL
FOR PREPARATION OF bIOPRODuCTS
ENRIChED wITh POLyuNSATuRATED
FATTy ACIDS
MILAn ČERTíK, ZUZAnA ADAMECHOVá and LInDA
néMETH
Department of Biochemical Technology, Faculty of Chemical
and Food Technology, Slovak University of Technology, Radlinského
9, 812 37 Bratislava, Slovak Republic,
milan.certik@stuba.sk
Introduction
Increasing demand for high-value lipids has focused
commercial attention on the provision of suitable biosynthetic
framework for their production. One of the main target for
microbial oil transformation is construction of healthy and
dietary important polyunsaturated fatty acids, such as γ-linolenic
acid (18 : 3 ω-6; GLA), dihomo-γ-linolenic acid (20 : 3
ω-6; DGLA), arachidonic acid (20 : 4 ω-6; AA), eicosapentaenoic
acid (20 : 5 ω-3; EPA) and docosahexaenoic acid (22 : 6
ω-3; DHA). Their applications in biomedical, nutritional and
pharmaceutical fields coupled with their inadequacy from
conventional agricultural and animal sources has looked for
developing suitable biotechnologies to produce these compounds
1 .
Particularly active in the synthesis of PUFAs are species
of fungi belonging to Zygomycetes 2 . Oleaginous fungi
producing PUFA could be economically valuable because
the most of their PUFAs occur in the triacylglycerol fraction
of their lipids. Two basic processes have been developed for
microbial production of PUFAs: submerged and solid state
fermentations 3,4 . However, the principal difficulty that has
been experienced with submerged PUFA-riched oil production
has been in its marketing rather than in developing
the large-scale fermentation and oil extraction process. Therefore,
the association of oleaginous fungi with solid state
fermentations (SSF) has been developed in order to improve
commercial potential of microbial oils and thus to create new
perspectives for the economic competitiveness and market of
microbial polyunsaturated fatty acids (PUFAs). Solid state
fermentation is a process in which microorganisms grow
on a moist solid substrate in the absence of free water 5 . SSF
simulates fermentation reactions occurring in the nature and
allows microbial utilization of raw agro-materials or byproducts
of the agro-food industries. Because some oleaginous
fungi simultaneously decrease anti-nutrient compounds in
the substrates (e.g. phytic acid) and partially hydrolyze substrate
biopolymers, prefermented mass with a high content
of PUFAs may be used as inexpensive food and feed supplement
6 . Thus, SSF might provide the other opportunity to fill
marketing claims in food, feed, pharmaceutical, veterinary
and environmental fields.
This paper deals with effectivity of several lower filamentous
fungi to synthesize various PUFAs during their utilization
of cereals by solid state fermentations.
s544
Experimental
M i c r o o r g a n i s m s
Thamnidium elegans CCF 1456, Cunninghamella echinulata
CCF-103, Cunninghamella elegans CCF-1318, Mortierella
isabelina CCF-14, Mortierella isabelina CCF-1098,
Mortierella alpina CCF 185 were obtained from the Culture
Collection of Fungi (Charles University, Prague, Czech
Republic). The culture was maintained on modified Czapek-
Dox agar slants with yeast extract (2.5 g dm –3 ) at 4 °C.
S u b s t r a t e 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
Depending on the microorganism, various types of substrates
were employed during SSF experiments. Spent malt
grains (SMG) were added to some substrates. Autoclavable
microporous polypropylene bags (160 × 270 mm 2 ) were filled
with 10 g of dry substrate, moistened by the addition of 10 ml
distilled water, soaked for 2 h at laboratory temperature and
sterilized in autoclave (120 kPa, 120 °C, 20 min). In order
to increased yield of PUFAs, sunflower or linseed oils were
added to some substrates. In addition, various amounts of
10% acetone or ethanol solutions of selected plant extracts
were tested with the aim to activate enzymes involved into
PUFA biosynthesis. The substrates were inoculated and
mixed with 2 ml of spore suspension (1–2 × 10 6 spores per
ml). Then each bag was closed with sterile cotton plugs,
inoculated substrate was spread in the bags to obtain substrate
layer of about 1 cm and incubated statically at 25 °C
for 4–6 days (T. elegans, C. echinulata, C. elegans, M. isabellina)
and 10–14 days (M. alpina). Triplicate SSF experiments
for each substrate were prepared to assess reproducibility and
average results are presented.
L i p i d E x t r a c t i o n a n d F a t t y A c i d
A n a l y s i s
Prefermented cereal materials (bioproducts) were gently
dried at 65 °C for 10 h and weighed. Lipids from homogenized
bioproducts were isolated with chloroform/methanol
(2 : 1, v/v) and purified according to Čertík et al. 7 and total
lipids were determined gravimetrically. Fatty acids of total
lipids were analyzed as their methyl esters 8 by gas chromatography
according to Čertík et al 9 . Gas chromatograph (GC-
6890 n, Agilent Technologies) was equipped with a capillary
column DB-23 (60 m × 0.25 mm, film thickness 0.25 μm,
Agilent Technologies) and a FID detector (constant flow,
hydrogen 35 ml min –1 , air 350 ml min –1 , 250 °C). Analyses
were carried out under a temperature gradient (130 °C for
1 min; 130–170 °C at program rate 6.5 °C min –1 ; 170–215°C
at program rate 2.7°C min –1 ; 215 °C for 7 min; 220–240 °C at
program rate 2 °C min –1 ; 240 °C for 2 min) with hydrogen as
a carrier gas (flow 2.1 ml min –1 , velocity 49 cm s –1 , pressure
174 kPa) and a split ratio of 1/50 (inlets: heater 230 °C, total
hydrogen flow 114 ml min –1 , pressure 174 kPa). The fatty
acid methylester peaks were identified by authentic standards
of C 4 –C 24 fatty acid methylesters mixture (Supelco, USA)
and quantified by an internal standard of heptadecanoic acid