-
Lebensm.-Wiss. u.-Technol., 255}260 (1999)
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Starch is the main component of bread and its gelatiniza-
protein interfaces and on the changes upon staling.
tion induces major structural changes during bakingof wheat
bread. The swollen granules and partiallysolubilized starch act as
essential structural elements ofbread (1). The other important
component of wheat isprotein, which is responsible for the
formation of theviscoelastic gluten in dough. On heating, the
glutentransforms from a gel to a coagel by polymerization,which
means that the gel looses its cohesiveness (2). Thus,the
transformation from dough to bread involveschanges both in the
starch and the protein fraction. Atthe macroscopic level, baking
induces the solidi"cationof dough and a change from a foam type
system with gascells to an open pore system, i.e. a sponge (2). On
coolingand ageing of bread, rearrangements in the starch frac-tion
lead to a series of changes including gelation andcrystallization.
This transformation is called starch retro-gradation and is thought
to be the major cause of breadcrumb "rming on ageing, commonly
referred to as breadstaling (3).Light microscopy presents a
valuable method for thestudy of the microstructural changes of
starch. Severalauthors described the microstructure of dough and
breadas shown by light microscopy (4}10). Nevertheless, there
A classical method to monitor starch gelatinization is theloss
of starch granule birefringence during heating. Na-tive starch
granules show strong birefringence in the formof Maltese crosses,
which disappear during heating dueto the loss of long range
molecular order (11). On bakingthe starch granules of bread crumb
also lose the typicalMaltese cross pattern of native starch but
still retainslight birefringence (4).Several authors (6, 8, 12, 13)
have pointed out the di$-culty involved in preparing dough and
bread samples formicroscopy. The disruption of the protein network
anda distorted image of the bread crumb due to hydrationduring
"xation (8) and staining (6) are common artefacts.The shrinkage of
starch and protein as consequence ofdehydration was also described
(12}14).The objective of this study were to characterize starchand
protein distribution in wheat dough and breadcrumb and to localize
the two starch polymers, amyloseand amylopectin in the bread crumb.
Finally, the micro-structural changes upon staling of bread were of
interestwith emphasis on the amylose fraction. The microstruc-ture
of dough and bread crumb was assessed by bright-"eld and
polarized-light microscopy. As few preparatorysteps as possible
were introduced in order to minimizethe risk of artefacts such as
structural distortion andChanges in Starch Micand Staling of
Susanna Hug-Iten, Stephan Handschin,
Institute of Food Science, Swiss Federal Institute o(Received
October 27, 1998;
The microstructure of starch in dough and in fresh and aged
bcryosectioned and stained with Light Green and iodine to
localizestarch from the protein phase is observed. On baking,
starch was gelstarch fraction itself was inhomogeneous and
consisted of swollen anand amylopectin, were found to phase
separate and amylose wamicroscopy of fresh bread crumb showed that
starch gelatinizationcrumb regained birefringence. The combination
of light microscopybirefringent structures. The most intense
birefringence was observeamylopectin rich zones showed slight
birefringence. It is concluded tamylopectin. It is hypothesized
that the reorganization of the intra-gbread staling.
( 1999 Academic Press
Keywords: microstructure; bread crumb; starch; light
microscop
Introduction*To whom correspondence should be addressed
0023-6438/99/050255#06 $30.00/0( 1999 Academic Press All ar
25rostructure on BakingWheat BreadeH atrice Conde-Petit* and
Felix Escher
Technology (ETH), CH-8092 Zurich (Switzerland)ccepted February
23, 1999)
ead crumb was studied by light microscopy. The samples
wereprotein and starch, respectively. In dough a partial
segregation oftinized and led to the formation of a continuous
starch network. Thed interconnected starch granules. The two starch
polymers, amyloses accumulated in the centre of starch granules.
Polarized-lightas accompanied by the loss of birefringence. On
ageing the bread
in the bright-xeld and polarized mode allowed identixcation of
thed in the amylose rich centre of starch granules, whereas the
outerat the ordered structures result from the reordering of
amylose andanular amylose fraction enhances the rigidity of starch
granules on
y
is still a lack of information on the localization ofamylose and
amylopectin in bread crumb, on the starchswelling of starch and
protein.
Article No. fstl.1999.0544ticles available online at
http://www.idealibrary.com on
5
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were examined in an Axioplan photomicroscope (ZeissLtd.,
D-Oberkochen)
Results and Discussion
The methods of sample preparation for microscopy weredeveloped
based on preliminary experiments (data notshown) and information
from the literature (16, 17). Thepreparation of specimens from
dough was similar toa method used by Cunin (18) for pasta and
consisted of
lwt/vol. 00 (1999) No. 5Experimental
Preparation of breadWhite pan bread was used for the
investigations. Breaddoughs were prepared from low-extraction wheat
#our(Coop MuK hle ZuK rich CMZ, CH-Zurich). According tothe
manufacturer, the #our contained 11.8 to 12.1 g/100g(dry base)
protein and 0.35 to 0.38 g/100 g (db) ash. Yeast(42 g) was
dispersed in a small amount of water andadded to the #our (1 kg).
The optimal amount of waterfor dough preparation was determined by
recordingfarinograms (15) and varied between 64 to 67 gwater/100 g
#our (wet base). The dough was kneaded ina mixer (Artofex AG,
CH-GraK nichen) for 3 min at lowspeed, followed by 4 min at high
speed. Salt (20 g) wasadded after 2 min mixing at low speed. Dough
pieces of600 g each were placed in greased (Boeson
Trennwax,Boehringer, D-Ingelheim) moulds. Proo"ng was ac-complished
in a ventilated oven (UWS 880, Wyrsch Ing.,CH-Meggen) during 90 min
at 25 3C, the moulds beingcovered with a wet cloth. The loaves were
baked at2203C for 35 min in a ventilated oven (Blodgett Co.
Inc.,USA-Burlington). The breads were cooled 4.5 h to
roomtemperature, sealed in plastic bags and stored for 0 to 7 dat
20 3C.
Light microscopySmall pieces of dough and crumb (approx. 7]7]7
mm)were cut from the centre of the dough and bread, respec-tively.
Dough samples were immediately frozen with car-bon dioxide. The
bread crumb samples were soaked for15 min under vacuum in
Tissue-Tek O.C.T. compound(Miles, USA-Kankakee) diluted 1 : 4 with
20% sucrosesolution, and then frozen with carbon dioxide.
Thesamples were cut in a Reichert-Jung cryostat (Leica,A-Vienna) at
!20 3C. Cryostat was displayed for sec-tions of 10 km thickness.
The samples were then mountedin the frozen state to microscope
slides, which had beencovered with a solution of glycerol/gelatine
to improvethe adhesiveness of the cryosections during staining.
Noglycerol/gelatine coating of the microscope slides wasused for
samples which were determined for polarizedmicroscopy. The thin
sections were either directly ob-served under polarized light to
assess the degree of starchbirefringence or "rst stained in order
to localize theprotein and the two starch fractions, amylose
andamylopectin, as well as to improve contrast. Protein wasstained
with aqueous Light Green solution (1 g/L, FlukaChemie AG, CH-Buchs)
for 30 min, starch with iodine ina diluted 1 : 10 (v/v) Lugols
solution (stock solutionI2"14 mM, KI"44 mM, Fluka Chemie AG,
CH-
Buchs) for 10 to 20 s. After each staining step the slideswere
rinsed in water. Before examining the samples underthe microscope
they were covered by a droplet ofglycerol/water solution (1 : 1
v/v) and a cover glass. Whenonly starch had to be stained the thin
sections wereexposed to iodine vapour (Lugols solution I
2"14 mM,
KI"44 mM, Fluka Chemie AG, CH-Buchs) for 1 to2 min. Then the
sections were covered with the glycer-ol/water solutions as
described above. All specimens25cFig. 1 Light micrograph of a
cryosection of proofed doughstained with Light Green and Lugols
solution (bar"25 km)Fig. 2 a, b Light micrographs of cryosections
of bread crumbat two di!erent magni"cations. Samples are stained
with LightGreen and Lugols solution (a) bar"50 km (b) bar"10 kmFig.
3a, b Polarized-light micrographs of cryosections of freshand aged
bread crumb. Samples are not stained (bar"50 km)(a) fresh, 0 d (b)
aged, 7 dFig. 4a, b Light micrographs of cryosections of bread
crumb.Samples are stained with iodine vapour (bar"10 km)
(a)bright-"eld mode (b) polarized-light mode
freezing the sample without prior "xation. Bread crumbwas soaked
in diluted O.C.T. compound for 15 min be-fore freezing. The aim was
to stabilize the porous struc-ture and to prevent a distortion of
the pores duringcryosectioning. A rather short soaking time was
selected,since the artefacts as described by Moss (8) are
mostprobably promoted by long hydration times (18 to 24 h).However,
in spite of the short hydration time, the swell-ing of bread crumb
could not be inhibited completely. Onthe other hand, shrinkage of
protein and starch due todehydration was avoided by using the
cryosectioningtechnique. Carbon dioxide was used as cryogen to
limitthe temperature gradient within the sample which pre-vented
the piece of bread from stress cracking. The risk ofice crystal
formation was considered to be negligiblesince O.C.T. acts as a
cryoprotectant. Iodine was selectedfor staining starch because it
allows di!erentiation be-tween amylose and amylopectin. However,
stainingintensity is known to vary between the starch
granules.Staining with aqueous solution presents a potentialsource
of artefacts. Samples which had been stained withaqueous solutions,
that is, Light Green and Lugols solu-tion, were compared to samples
which had only beenexposed to iodine vapour. The aqueous staining
solu-tions were found to slightly increase the swelling ofstarch.
Overall, the changes caused by hydration duringstabilization and
staining did not induce major changesin the starch fraction. The
starch granules maintainedtheir shape. Neither the localization of
starch in theprotein matrix nor the distribution of the two
starchpolymers were a!ected. It is therefore concluded that
thesample preparation techniques were suitable for the pur-pose of
the investigation.
Microstructure of doughFigure 1 presents a light micrograph of a
cryosection ofproofed wheat dough stained with iodine and
LightGreen. The native starch granules stain slightly violet6
-
lwt/vol. 00 (1999) No. 5
257
-
lwt/vol. 00 (1999) No. 5with iodine while the protein fraction
appears green ascoloured with Light Green. The starch granules show
thecharacteristic shape of native wheat starch granules. Thetypical
bimodal size distribution of wheat starch granulesis also
recognizable. Protein and starch are not evenlydistributed in the
dough and regions are found whereseveral starch granules are
accumulated.The literature data regarding the distribution of
starchand protein in bread dough is controversial. Based
onmicroscopy, several authors (6, 7) concluded thateach starch
granule is surrounded by protein. A morerecent structural model
described dough as a bicontinu-ous starch-protein system (2). The
starch granules havea surface coat of &free water and tend to
fuse into a con-tinuous phase. The gluten gel "lls the space
between thewater-fused starch granules. The results of the
presentinvestigation on the microstructure of dough are consis-tent
with the latter model of dough. However, it remainsto be
investigated how the processing steps (mixing,proo"ng and
reshaping) and the protein quality of wheatin#uence the
microstructure of dough. According toKie!er and Stein (19),
starch-protein segregation occursduring the reshaping of the dough
after a rest period.This is thought to be the reason for the large
increase ofresistance in uniaxial extension, called strain
hardening.Although, starch is by far the largest fraction
oflow-extraction wheat #our by weight, with a concentra-tion of
approx. 70 g/100 g, it only "lls about 60% of thedough volume due
to the high swelling capacity of gluten(20). The latter fraction
determines the rheological prop-erties of dough whereas starch is
considered to act asa "ller (20).
Microstructure of bread crumbFigures 2a and b show cross
sections of pore walls of freshbread crumb at two di!erent
magni"cations. Again,starch and protein are stained in order to
localize the twofractions. At low magni"cation (Fig. 2a) the starch
gran-ules appear swollen and elongated, but still retain
theirgranular identity. Most granules are aligned parallel tothe
pore surface. The protein phase is distinguishable asgreen areas
throughout the bread crumb. Details of thestarch fraction are
recognizable at higher magni"cation(Fig. 2b). Clearly, the starch
granules are oriented andpartly fused with neighbouring granules.
The iodinestaining reveals that amylose and amylopectin, whichstain
blue and brown/violet, respectively, are not homo-geneously
distributed in the granules. The blue, elon-gated areas in the
inner zone of the large starch granulescorrespond to accumulations
of amylose whereas thebrown/violet surrounding phase corresponds
toamylopectin rich structures. In contrast, the small gran-ule
fraction of starch does not show this phase separ-ation. Outside
the starch granules, amylose rich zonescan be observed as dark
lines along the starch proteininterface. In Fig. 2b, concentric
growth rings for oneparticular aspect of the internal structure of
starch gran-ules are visible for one highly swollen starch
granule.At the microscopic resolution level studied, the porewalls
of bread crumb may be described as a bicontinuous25structure, which
is built up by starch and protein, respec-tively. The starch
fraction itself is inhomogeneous andconsists of swollen &phase
separated granules andleached starch, mainly amylose. The
accumulation ofamylose in the starch granule centre is also
documentedfor rye bread, but not further commented by the
authors(9). Phase separation of starch is explained by the
ther-modynamic incompatibility of amylose and amylopectin(21) and
was also observed in wheat starch pastes (22).The elongated form of
starch granules and their orienta-tion in bread crumb was reported
in previous studies(4}6). This is thought to arise from the
extension of thelamellae due to the growing gas cells in the
initial stage ofbaking. The fact that growth rings of starch are
recogniz-able in bread crumb proves that in spite of the
mor-phological changes and the partial amylose-amylopectinphase
separation the native organization of starch gran-ules, which is
mainly determined by the packing densityand the radial orientation
of amylopectin (23), is largelypreserved. The transformation of
starch during bakingleads to a continuous starch network. It is
therefore notsurprising to "nd that the macroscopic properties
aredetermined by the gelatinized starch. As shown by Sand-stedt
(24), a typical bread crumb structure cannot beobtained when starch
is replaced by an inert "ller such asglass beads. This experiment
led to the conclusion thatstarch does not simply act as a "ller
that dilutes gluten toa suitable consistency. On the other hand, it
is possible togenerate starch systems with a sponge structure by
com-bining starch with other polymers (e.g. xanthan,pregelatinised
starch) which replace gluten (1, 25). Theresulting mechanical
properties are similar to breadcrumb and are determined by the
swelling state of starchgranules and by the distribution of the
thickness of thepore walls (1).The investigations with regular
bright-"eld microscopywere complemented by polarized-light
microscopy.Figures 3a and b show fresh and 7 d old bread crumb
atlow magni"cation. The samples were not stained. Asexpected, fresh
bread crumb exhibited almost no birefrin-gence, since starch
granule swelling upon gelatinization isaccompanied by a loss of
order of starch molecules (11).Interestingly, aged bread crumb
showed a marked in-crease of birefringence. At low magni"cation,
the biref-ringence is visible as bright irregular longish
structures,and no recurrence of the typical Maltese cross pattern
ofnative starch is observed. The circular spots should
bedisregarded as they may be the result of birefringent dustlaying
in planes out of focus. Figures 4a and b show thesame "eld of
stored bread crumb (7 d) at high magni"ca-tion in bright-"eld and
polarized-light mode, respective-ly. The protein staining was
omitted, and starch wasstained by exposing the cryosection to
iodine vapour.This technique was adopted to prevent the washing
outof amylose and swelling of the starch granules by aque-ous
staining solutions. The bright-"eld micrograph(Fig. 4a) clearly
reveals accumulations of amylose (bluecoloured) inside the large
starch granules and anamylopectin rich phase (violet/brown
coloured) at theouter zone of starch granules. On the
correspondingpolarized-light micrograph (Fig. 4b) the amylose
within8
-
lwt/vol. 00 (1999) No. 5the phase separated granules is
birefringent. A less in-tense birefringence is observed at the
outer amylopectinrich zones. This congruence of bright-"eld and
polarized-light microscopy allows the localization and
identi-"cation of birefringent structural elements. Itcon"rms that
reorganizations in the starch fractions, thatis, starch
retrogradation, is responsible for the
observedbirefringence.Birefringent zones as detected with polarized
lightmicroscopy correspond to anisotropic structures but
notnecessarily to crystallinity. The slight birefringence at
theouter zones of starch granules can be attributed to retro-graded
amylopectin which forms organized anisotropicregions. This
observation is consistent with the results ofJacobson et al. (26).
They describe that granule remnantsof low-concentrated starch
pastes regained slight biref-ringence upon storage near the outer
edges of intactswollen granules. The fact that the
intra-granularamylose becomes birefringent implies that amylose is
inan ordered state in aged bread crumb. From studies onpure amylose
systems and on starch gels with rheologicalmeasurements and X-ray
di!raction (27}29) it was con-cluded that the initial stages of
starch retrogradation aredominated by the gelation of the
solubilized amylose.The development of a three-dimensional network
resultsfrom a phase separation into polymer-rich and
polymer-de"cient phases (27). This is followed by a slow
crystalli-zation presumed to occur in the polymer-rich phase.Thus,
in connection with the observed birefringence ofstarch granules in
bread crumb it is conceivable thatthe amylose in the starch granule
centre rearranges onageing and eventually partly crystallizes by
lateralchain association. The possibility that the reorganizationof
amylose is induced by iodine complexation can beexcluded, since the
samples of Fig. 3 were not stained.However, the ordering of the
amylose fraction may havebeen promoted by complexation with
endogenous wheatstarch lipids. Finally, as the gelatinized granules
cannotbe simply considered as amylopectin rich structures,
thequestion of how the intra-granular amylose in#uencesthe
mechanical properties of starch granules arises. Stud-ies on pure
amylose solutions showed that their recrys-tallization did not
in#uence the complex modulus (27).Conversely, Conde-Petit (30)
concluded that therheological behaviour of wheat starch dispersions
overa period of 30 d is dominated by changes in the
amylosefraction, as determined by the loss of iodine
bindingcapacity. Although none of the model systems may befully
transferable to bread, it is hypothesized thatthe changes in the
intra-granular amylose fraction en-hance the rigidity of starch
granules on bread staling.
Acknowledgements
Thanks are due to Claudia Meyer (Department of Anat-omy, Prof.
M. MuK ntener, University of Zurich) for hertechnical assistance,
and to Chantal Bussmann (Instituteof Food Science, ETH Zurich) for
preparing micro-graphs.25References
1 KEETELS, C. J. A. M., VISSER, K. A., VAN VLIET, T.,JURGENS, A.
AND WALSTRA, P. Structure and mechanics ofstarch bread. Journal of
Cereal Science, 24, 15}26 (1996)
2 ELIASSON, A.-C. AND LARSSON, K. Cereals in breadmaking2 A
molecular colloidal approach. New York: MarcelDekker Inc.
(1993)
3 ZOBEL, H. F. AND KULP, K. The staling mechanism. In:HEBEDA, R.
E. AND ZOBEL, H. F. (Eds), Baked Goods Fresh-ness: echnology,
Evaluation and Inhibition of Staling. NewYork: Marcel Dekker, Inc.,
pp. 1}64 (1996)
4 VARRIANO-MARSTON, E., KE, V., HUANG, G. AND PONTE,J.
Comparison of methods to determine starch gelatiniz-ation in bakery
foods. Cereal Chemistry, 57, 242}248 (1980)
5 BURHANS, M. E. AND CLAPP, J. A microscopic study ofbread and
dough. Cereal Chemistry, 19, 196}216 (1942)
6 SANDSTEDT, R. M., SCHAUMBURG, L. AND FLEMING, J.
Themicroscopic structure of bread and dough. Cereal Chem-istry, 31,
43}49 (1954)
7 BECHTEL, D. B., POMERANZ, Y. AND DE FRANCISCO, A.Breadmaking
studied by light and transmission electronmicroscopy. Cereal
Chemistry, 55, 392}401 (1978)
8 MOSS, R. Bread microstructure as a!ected by cysteine,potassium
bromate and ascorbic acid. Cereal Foods =orld,20, 289}292
(1975)
9 AUTO, K., HAG RKOG NEN, H., PARKKONEN, T., FRIGARD,
T.,POUTANEN, K., SUKA-AHO, M. AND AMAN, P. E!ects ofpuri"ed
endo-beta-xylanase and endo-beta-glucanase onthe structural and
baking characteristic of rye doughs.ebensmittel-=issenschaft
und-echnologie, 29, 18}27 (1996)
10 POMERANZ, Y. AND MEYER, D. Light and scanning
electronmicroscopy of wheat- and rye-bread crumb. Interpretationof
specimens prepared by various methods. Food Micro-structure, 3,
159}164 (1984)
11 SEIDEMAN, J. StaK rke Atlas. Grudlagen der StaK
rkemikroskopieund Beschreibung der wichtigsten StaK rken. Berlin:
Paul Parey(1966)
12 CHABOT, J. F. Preparation of food science samples for
SEM.Scanning Electron Microscopy, 3, 279}286 (1979)
13 VERSCHAFFELT, E. AND VAN TEUTEM, F. E. Die AG nderungder
mikroskopischen Struktur des Brotes beim Altbacken-werden.
Hoppe-Seyler1s Zeitschrift fuK r PhysiologischeChemie, 95, 130}135
(1915)
14 LEE, R. M. K. W. A critical appraisal of the e!ects
of"xation, dehydration and embedding on cell volume.In: REVEL,
J.-P., BARNARD, T. AND HAGGIS, G. H. (Eds),Proceedings of the 2nd
Pfe+erkorn conference. Chicago:SEM Inc., pp. 61}70 (1983)
15 International Association for Cereal Science and Techno-logy.
ICC-Standards, No. 115/1, SchaK fer, Detmold (1992)
16 FLINT, O. Food microscopy: a manual of practical
methods,using optical microscopy. Oxford: BIOS Scienti"c
PublishersLtd, pp. 17 (1994)
17 BARTHEL, L. K. AND RAYMOND, P. A. Improved method
forobtaining 3-km cryosections for immunocytochemistry.heJournal of
Histochemistry and Cytochemistry, 38, 1383}1388(1990)
18 CUNIN, C., HANDSCHIN, S., WALTHER, P. AND ESCHER,
F.Structural changes of starch during cooking of durum wheatpasta.
ebensmittel-=issenschaft und-echnologie, 28,323}328 (1995)
19 KIEFFER, R. AND STEIN, N. Investigations on the phenom-enon
of dough hardening during the processing of wheatdoughs. In:
WINDHAB, E. J. AND WOLF, B. (Eds), Proceed-ings of the 1st
International Symposium on Food Rheologyand Structure. Hannover:
Vincentz Verlag, pp. 292}296(1997)
20 BLOKSMA, A. H. Dough structure, dough rheology, andbaking
quality. Cereal Foods =orld, 35, 237}244 (1990)
21 KALICHEVSKY, M. T. AND RING, S. G. Incompatibility ofamylose
and amylopectin in aqueous solution. Carbohy-drate Research, 162,
323}328 (1987)9
-
22 LANGTON, M. AND HERMANSSON, A.-M. Microstructuralchanges in
wheat starch dispersion during heating and cool-ing. Food
Microstructure, 8, 29}39 (1989)
23 GALLANT, D. J., BOUCHET, B. AND BALDWIN, P. M. Micros-copy of
starch: evidence of a new level of granule orgniz-ation.
Carbohydrate Polymers, 32, 177}191 (1997)
24 SANDSTEDT, R. M. The function of starch in the baking
ofbread. he Bakers Digest, 35, 36}44 (1961)
25 MORGAN, K. R., HUTT, L., GERRARD, J., EVERY, D., ROSS,M. AND
GILPIN, M. Staling in starch breads: the e!ect ofantistaling
alpha-amylase. Starch/StaK rke, 49, 54}59 (1997)
26 JACOBSON, M. R., OBANNI M. AND BEMILLER, J. N.
Retro-gradation of starches from di!erent botanical sources.Cereal
Chemistry, 74, 511}518 (1997)
27 MILES, M. J., MORRIS, V. J. AND RING, S. G. Gelation
ofamylose. Carbohydrate Research, 135, 257}269 (1985)
28 MILES, M, J., MORRIS, V. J., ORFORD, P. D. AND RING, S. G.The
roles of amylose and amylopectin in the gelation andretrogradation
of starch. Carbohydrate Research, 135,271}281 (1985)
29 CLARK, A. H., GIDLEY, M. J., RICHARDSON, R. K.
ANDROSS-MURPHY, S. B. Rheological studies of aqueousamylose gels:
the e!ect of chain length and concentration ongel modulus.
Macromolecules, 22, 346}351 (1989)
30 CONDE-PETIT, B. Interaktionen von StaK rke mit Emul-gatoren
in wasserhaltigen Lebensmittel-Modellen, Ph.D.Thesis No. 9785,
Swiss Federal Institue of TechnologyZurich (1992)
lwt/vol. 00 (1999) No. 5260
IntroductionExperimentalResults and DiscussionFigure 1Figure
2Figure 3Figure 4
AcknowledgementsReferences
