3. FOOD ChEMISTRy & bIOTEChNOLOGy 3.1. Lectures

Chem. Listy, 102, s265–s1311 (2008) Food Chemistry & Biotechnology
P70 MONITORING OF bARLEy STARCh
AMyLOLySIS by GRAVITATIONAL FIELD
FLOw FRACTIONATION AND MALDI-TOF/
TOF MS
KAREL MAZAnEC and JAnETTE BOBáľOVá
Institute of Analytical Chemistry of the ASCR, v. v. i.Veveří
97, 602 00 Brno, Czech Republic,
mazanec@iach.cz
Introduction
In barley grain, starch occurs in form of granules with
bimodal size distribution. The oval large starch granules (A)
have diameters in the range from 10 to 40 µm and they prevail
in weight. On the other hand, the spherical small starch
granules (B) have diameters from 1 to 10 µm and they prevail
in number 1 . Enzymatic starch granule hydrolysis is one of
the most important reactions occurring during malting and
mashing.
In this study, the capacity of gravitational field flow
fractionation (GFFF) to monitor the amylolysis of starch granules
was investigated. In previous works 2-6 GFFF was successfully
used for study of size distribution of starch granules
in different barley varieties. Degradation of starch particles
from wheat was monitored by sedimentation FFF 7 . Lower
saccharides released during amylolysis can be studied by
MALDI-MS.
Experimental
Kernels of barley variety Jersey were used for isolation
of starch granules. Kernels were graded and the fractions
over 2.5 mm were used. The isolation procedure is described
in detail elsewhere 8 . It combines classical approaches
(incl. crushing of barley kernels by a roll crusher, steeping
in 0.02M HCl, repeated rubbing and filtering through sieve
0.08 mm) and present knowledge (treatment with ß-glucanase
and cellulase).
Starch granules were hydrolyzed by 2 units of
α-amylase mg –1 starch (Sigma-Aldrich) added to 2 ml of 3%
(w/v) starch suspension at 35 °C. Aliquots were removed in
time interval during hydrolysis and centrifuged. Supernatant
was mixed with 2,5-dihydroxybenzoic acid (DHB) and measured
by 4700 Proteomics Analyzer (Applied Biosystems,
USA) MALDI-TOF/TOF mass spectrometer (equipped with
nd/YAG laser; 355 nm).
Starch granules were resuspended in 10 -3 % sodium
dodecyl sulphate (SDS) (Fluka, Germany), sonicated and
analyzed by GFFF. Apparatus for GFFF is described in 6 .
The channel dimensions were 360 × 20 × 0.150 mm. The highpressure
pump HPP 4001 (Laboratory Instruments, Prague,
Czech Republic) was used to introduce the carrier liquid
into the channel via an inlet capillary situated at the channel
head. UV/VIS Spectra 100 (Spectra Physics, San Jose, USA)
operated at 470 nm was used as a detector. The samples were
injected at the stopped flow by using a Hamilton microsyringe.
Just after injection, a loading flow rate of 0.2 ml min –1
s728
was applied for 10 s. Then a relaxation time (stopped-flow
period) was 1.5 min. After this time period, a linear flow rate
of 0.8 ml min –1 was applied.
Results
Experimental conditions of GFFF separation were chosen
in order to elute starch particles in focusing or lift-hyperlayer
elution mode 6 . In this elution mode, starch granules do
not interact with the channel bottom; therefore particle-wall
interactions are negligible.
Fig. 1. shows a representative GFFF elution fractogram
obtained for native starch of barley variety Jersey. Three
major peaks can be seen: the first corresponding to unretained
species (void volume), the second corresponding to the
large A starch granules and the third peak corresponding to
the small B starch granules.
Fig. 1. Representative fractogram of native starch from barley
variety jersey after GFFF elution
The α-amylase type VIII-A from barley (Sigma) was
chosen for starch granule attack. In the case of bimodal starch
population, the amylolysis mechanisms are more complex
than those for small monomodal starches. Amylases start
enzymatic digestion on specific points of the large A starch
granules. Degradation then continues towards the centre of
the granule by the formation of channels leading to particle
Fig. 2. GFFF monitoring of barley starch enzymatic hydrolysis.
Starch was isolated from barley variety jersey