Journal of the Science of Food and Agriculture J Sci Food Agric 82:685±696 (online: 2002)

DOI: 10.1002/jsfa.1090

The use of FT-IR microspectroscopic mapping

to study the effects of enzymatic retting of flax
(Linum usitatissimum L) stems†
David S Himmelsbach,1* Sadia Khalili2 and Danny E Akin1
1
United States Department of Agriculture, Agricultural Research Service, Richard B Russell Agricultural Research Center, PO Box 5677,
Athens, GA 30604-5677, USA
2
Department of Pulp and Paper Chemistry, Royal Institute of Technology, Box 10044, Stockholm, Sweden

Abstract: Fourier transform infrared (FT-IR) microspectroscopic mapping was investigated as a tool
to study the effects of enzymatic retting of ¯ax stems. The FT-IR technique permitted the elucidation
of the relative loss or changes in the distribution of key chemical components after treatment with
enzymes or enzyme/chelator mixtures in association with visible changes in structure. Cross-sections
of Ariane ¯ax stems were treated with SP 249 (a pectinase-rich enzyme mixture from Novo Nordisk) at
0.5, 0.7 or 1.0 ml l 1 concentration in pH 5 acetate buffer for 6 h at 40 °C. Flax stems treated with 0.5 or
0.7 ml l 1 SP 249 and 50 mM oxalic acid as a chelator were also investigated by the technique. The results
indicated that treatment with 0.5 ml l 1 SP 249 alone was ineffective in releasing the ®bre bundles from
the surrounding tissue, but the release was increased by the addition of 50 mM oxalic acid as a likely
chelator for the cations of pectate salts. However, the IR spectra of the bundles indicated that an
insoluble oxalate salt remained on the tissue after this treatment. Increasing the concentration of SP
249 to 0.7 ml l 1 plus 50 mM oxalic acid was effective in releasing the ®bre bundles and generating some
ultimate ®bres with no detectable oxalate expectate salt residues. Increasing the SP 249 concentration
to 1.0 ml l 1 without using oxalic acid was effective in separating the ®bre bundles into ultimate
(individual) ®bres, leaving no pectate salt residue and only a trace of pectic esters and/or acids. The use
of infrared mapping, or so-called chemical imaging, is shown to have advantages over visible imaging
alone in that it can detect and locate the chemical species present after each treatment in relation to the
anatomical features of the ¯ax stem. This analytical tool shows promise as a technique by which to
study the effects of enzymatic treatment of natural ®bre materials.
Published in 2002 for SCI by John Wiley & Sons, Ltd

Keywords: Fourier transform; infrared; microscopy; spectroscopy; ¯ax; retting; enzymes; chelator

INTRODUCTION reduced the amount of enzymes required from 2.5 to

Retting is a postharvest processing step which med-
iates the release of stem ®bres from bast plants such as
¯ax (Linum usitatissimum L). The economics and
uniformity of retting are critical to the overall viability
of an industry, and therefore improved retting would
make ¯ax a more economically competitive and
reliable source of textile ®bre or raw material for
composites. An enzymatic method could be a simple,
ef®cient, uniform and environmentally friendly solu-
tion to the problem. The method could also offer a
more consistent quality ®bre than dew-retting tech-
niques.1 One disadvantage of enzymatic methods is the
cost of enzymes. However, it has been shown that
combining chelating agents with enzymes can sub-
stantially reduce the amount of enzymes required to ret
¯ax.2 Combining enzymes with oxalic acid or EDTA
0.5 ml l 1 to achieve the same degree of retting of ¯ax
stems based on the Fried test.3 Pectin degradation has
been found to be essential to ¯ax retting.1 Evaluation
of ®bres resulting from different formulations (pecti-
nolytic enzymes with and without chelators) has
suggested that pectin degradation varies for different
regions of bast tissue.4 In ¯ax hypocotyls the amounts
of acidic polygalacturonans and calcium have been
reported to be greater in the epidermal than cortical
regions of the cell walls.5 Transmission infrared
(point) microspectroscopy has been employed to study
the removal of pectic polymers from the cell walls of
onion by cyclohexanediaminetetraacetic acid.6 The
changes in cell wall structure and composition of the
epidermal tissue of ¯ax hypocotyls during develop-
ment have been also been studied by this technique.7,8

* Correspondence to: David S Himmelsbach, USDA-ARS, RB Russell Agricultural Research Center, PO Box 5677, Athens, GA 30604-5677,
USA
E-mail: dshimmel@qaru.ars.usda.gov
†
Reference to company or trade names is for the purpose of description only and does not imply endorsement by the United States
Department of Agriculture. This article is a US Government work and is in the public domain in the USA
Contract/grant sponsor: Wenner–Gren Foundation of Sweden
(Received 12 November 2000; accepted 10 January 2002)

Published in 2002 for SCI by John Wiley & Sons, Ltd. J Sci Food Agric 0022±5142/2002/$30.00 685
DS Himmelsbach, S Khalili, DE Akin
In our previous report, using transmission FT-IR
microspectroscopic mapping, the relationship between
chemical composition and morphology of untreated
¯ax stems was studied.9 We were able to distinguish
the primary chemical components of stem tissues:
waxes in the cuticular and epidermal tissues, pectate
salts in the epidermal and parenchymal tissues,
cellulose in the core and ®bres, aromatics in the core,
and acetylated polysaccharides in the ®bres and core.
Now we report on the potential of the transmission
infrared technique to analyse semi-quantitatively the
process of enzymatic retting of ¯ax stems using a ¯ax-
retting enzyme mixture with or without oxalic acid as a
chelating agent. The results of Henriksson et al 2
indicated that the use of Flaxzyme, a liquid prepara-
tion containing cellulases, hemicellulases and pecti-
nases,1 with oxalic acid produced the greatest degree
of retting over other systems investigated. Using SP
249 (Novozymes, Franklinton, NC, USA), a currently
available enzyme mixture similar to Flaxzyme, we
extend the previous results of Henriksson et al 1 by
identifying and locating the chemical components
affected by the enzyme and/or chelator retting of ¯ax.
The results also demonstrate the utility of using
infrared mapping for this purpose.
EXPERIMENTAL
Reference samples
The wax reference sample was stearyl stearate,
obtained from NuCheck-Prep (Philadelphia, PA,
USA). The commercially available carbohydrate
reference samplesÐcellulose, as microcrystalline cel-
lulose PH-105, obtained from FMC (Princeton, NJ,
USA); acetylated glucomannan, from konjac, ob-
tained from Megazyme (Wicklow, Ireland); polyga-
lacturonic acid (890 g kg 1 purity), from orange peel;
and esteri®ed pectin (900 g kg 1 purity), from citrus
fruit, both from Sigma (St Louis, MO, USA)Ðwere
used without further puri®cation. Calcium pectate was
prepared by titrating polygalacturonic acid with
Ca(OH)2 as previously described.9 The aromatic
reference sample was lignin, obtained as ball-milled
enzyme-treated lignin from Eastern cottonwood
(Populus deltoides), supplied by the National Renew-
able Energy Laboratory (Golden, CO, USA). All
reference samples were powdered solids.
Transmission IR spectroscopy of reference samples
Transmission IR spectroscopy was performed on
reference samples using a Magna System 8502 FT-
IR bench (Nicolet Instruments Corporation, Madi-
son, WI, USA). The bench was con®gured with a
globar source, a KBr beamsplitter and a deuterated
triglycine sulphate (DTGS) detector. Spectra were
collected in the range of 4000±400 cm 1 at a resolution
of 8 cm 1 with 128 scans co-added, a mirror physical
velocity of 0.95 cm s 1, an aperture setting of 100
(10 mm) at the interferometer and a gain of 8. All
reference samples were dried under a 250 W Hot Spot
686
IR heat lamp (I2R, Cheltenham, PA, USA) for 5 min
and diluted to 5 mg g 1 with dry KBr (FT-IR grade,
Aldrich Chemical Co, Milwaukee, WI, USA). Diluted
samples and the KBr reference material were pressed
into pellets using a bolt and die device (McCarthy
Scienti®c Co, Fullerton, CA, USA) to produce clear
windows. Samples were then placed in the purged
sample compartment for 30 min prior to data collec-
tion. The spectral data were collected, apodised with a
Happ±Genzel function,10 ratioed against the back-
ground spectrum of KBr and displayed in the
absorbance mode. A linear baseline correction was
applied to generate the ®nal spectra.
Flax samples and treatments
Ariane ¯ax was grown from the end of April 1994 for
90 days under commercial conditions in The Nether-
lands and supplied by Van de Bilt zaden en vlas, bv
(Sluiskil, The Netherlands). For analysis, samples
were cut (7±8 cm in length) from the middle part of
two different unretted stems that were originally
25±30 cm in total length. These sections were then
hydrated by placing one end into a beaker of doubly
deionised water and drawing the water up the stem
under vacuum. After 1 h the stems were removed from
the beaker, wrapped in Para®lm1 `M' (American
National Can, Greenwich, CT, USA) and freehand
cut into 1 cm lengths. The 1 cm sections were then
frozen in doubly deionised water at 12 °C onto object
discs in a cryostatic microtome Minotome Plus,
Triangle Biomedical Sciences, Inc, Durham, NC,
USA and subsequently cut into thin cross-sections
(6±8 mm thick) using disposable razor blade micro-
tome knives. The cross-sections were transferred
directly from the microtome knives to individual
circular BaF2 discs (13 mm  2 mm). Seven sets, with
two repetitions each, of cross-sectioned samples were
then generated for evaluation as follows: (1) untreated;
(2) treated with 0.05 M acetate buffer (pH 5) alone; (3)
treated with 50 mM oxalic acid in acetate buffer; (4)
treated with 0.5 ml l 1 SP 249 in acetate buffer; (5)
treated with 0.5 ml l 1 SP 249 in acetate buffer and
50 mM oxalic acid; (6) treated with 0.7 ml l 1 SP 249 in
acetate buffer and 50 mM oxalic acid; and (7) treated
with 1.0 ml l 1 SP 249 in acetate buffer. The pH was
adjusted to 5.0 with 10 M sodium hydroxide when
oxalic acid was utilised with the acetate buffer. One
drop of the treatment solution was added to each
sample on the discs so as to completely cover the
section. The discs were placed on water-saturated
paper towels in covered Petri dishes. The dishes were
then placed in an incubator at 40 °C for 6 h. After
incubation, each sample was washed three times with a
drop of deionised water and dried. A crystal of dry KBr
was placed near the sample on each disc and a second
BaF2 disc was placed on top. The samples and the dry
KBr crystal were then ¯attened by compressing them
between the discs in a microcompression cell (Spectra-
Tech, Shelton, CT, USA) that ®t into the mounting
J Sci Food Agric 82:685±696 (online: 2002)

plate of the microscope stage. Infrared chemical maps
of each sample were collected as described below.
FT-IR mapping
FT-IR mapping was performed using a NIC-PLAN2
microscope (Nicolet Instruments Corporation, Madi-
son, WI, USA) attached to a Magna System 8502 FT-
IR bench (Nicolet Instruments Corporation). The
same bench con®guration was used for the macro-
sampling transmission spectroscopy, except that the
IR beam was directed through the infrared micro-
scope, which was equipped with its own liquid
nitrogen-cooled mercury cadmium telluride/narrow-
band (MCT/A) detector for signal collection. The
microscope was equipped with an automated
motorised stage that was operated by a microposition-
ing stage controller interfaced to a Pentium-based
microcomputer. Data collection, stage control and
image data processing were performed using OMNI-
Figure 1. KBr pellet transmission IR
spectra in absorbance mode (3010–
1170cm 1) of stearyl stearate (a),
polygalacturonic acid (b), esterified
pectin (c), calcium pectate (d),
cottonwood lignin (e), cellulose (f) and
acetylated glucomannan (g). Scale
from 2000 to 1200 cm 1 is expanded
two times.
J Sci Food Agric 82:685±696 (online: 2002)
Enzymatic retting of ¯ax stems
C1Atlms2 version 2.0 software (Spectra-Tech, Shel-
ton, CT, USA). Both visible and infrared images were
obtained in the transmission mode through a
32  Cassegrain objective and a 10  Cassegrain con-
denser. Visible images, obtained using a CCD camera
attached to the microcomputer by way of a video card/
frame grabber, were digitally linked to the infrared
images.
Each pixel was collected so as to contain an infrared
spectrum over the range of 3900±850 cm 1 at a
spectral resolution of 8 cm 1 with 256±384 scans, a
mirror physical velocity of 1.8988 cm s 1, a bench
aperture setting of 100 (10 mm) and an instrument
gain of 8. The IR radiation was redundantly apertured
at the microscope using 20 mm  20 mm upper and
25 mm  25 mm lower projection apertures. Collection
at each pixel was a result of stepping in 10 mm
increments (avoiding convolution of the image with
the microscope apertures) in both the X and Y
687
DS Himmelsbach, S Khalili, DE Akin
directions of the sample over areas of (110±180) mm to
(60±140) mm. The total data collection time varied
between 4 and 7 h, depending on the size of the area
mapped. The interferograms obtained were apodised
with a Happ±Genzel function10 prior to Fourier
transformation to the frequency domain spectrum.
Spectral data from the samples were ratioed against
the background spectrum of the KBr crystal. The
background was updated after every 10th pixel. The
resulting spectra were displayed in the absorbance
mode. After the mapping procedure was completed,
the spectra were truncated to the range of 3020±
1180 cm 1 in order to avoid regions of total absor-
bance (due to detector saturation by totally absorbing
bands). After an automatic baseline adjustment was
applied, all data sets were normalised by setting the
maximum response at 1335  2 cm 1 (that of cellu-
lose) to 0.40 absorbance units to provide the ®nal data
matrix.
Chemical pro®les were produced from the data
matrix by extracting and displaying speci®c absorp-
tions on the basis of their band intensities, versus local
baselines, over the entire mapped area. This procedure
produced chemically speci®c grey-scale 2D contour
maps of the selected components for band intensities
between 0.05 and 0.5 absorbance units.
RESULTS AND DISCUSSION
Fig 1 shows the KBr pellet transmission spectra of
seven `pure' reference materials used to represent the
major chemical components found in ¯ax stems. The
frequencies (in wavenumbers) that are diagnostic for
these types of components are marked on the spectra.
Only the CH stretch and the `®ngerprint' regions are
shown (3020±1180 cm 1), since bands outside this
region became totally absorbing and were not usable in
the microspectroscopic mapping. In addition, the so-
called `®ngerprint' region of the spectra (below
2000 cm 1) was expanded two times to better visualise
the bands in this region. The potentially diagnostically
important band maxima were 2850 and 1732 cm 1 for
waxes, 1745 cm 1 for pectic acid, 1748, 1445 and
1234 cm 1 for pectic ester, 1615 and 1425 cm 1 for
calcium pectate, 1505 cm 1 for aromatics, 1335 cm 1
for cellulose and 1732 and 1250 cm 1 for acetylated
polysaccharides.
The only bands for waxes that can be considered
diagnostic are the CH2 symmetrical stretch at
2850 cm 1 and the C=O band at 1732 cm 1. Of
these two, only the band at 2850 cm 1 is useful, owing
to the presence of C=O bands in many other of the
key components of ¯ax stems.
Since the removal of pectins is essential in ¯ax
retting,1 three types of pectin were considered here as
potentially important. Pectin is known to be a
heteropolysaccharide composed of a backbone of
1,4-linked a-D-galacturonic acid units with various
degrees of methyl esteri®cation and various attached
side chains.11,12 Both highly and less methylated
688
pectins are produced during ¯ax maturation.13 Also,
calcium and/or other cations can cross-link pectins
through non-methoxylated carboxyl groups of galac-
turonic acid and thereby stabilise pectin molecules.12
Inspection of the spectra for polygalacturonic acid
(Fig 1(b)) and its methyl ester (Fig 1(c)) revealed that
these two components could not be distinguished from
each other by their C=O bands, which occur at 1745
and 1748 cm 1 respectively. These bands for the acid
and ester have been reported to occur in D2O solution
at 1730 and 1740 cm 1 respectively.14 In mixtures of
these two forms of pectin, only by ensuring that all of
the free acid is converted to the anion can the band in
this region be used for the measurement of ester. The
potential does exist for the methyl ester to be
distinguished by its CH3 antisymmetric stretch at
1445 cm 1 plus its CÐO stretch at 1235 cm 1.15
Unfortunately, these bands are also not well separated
from other bands to make them very diagnostic and,
further, the maxima for the C=O bands are within
about 15 cm 1 of those for the waxes (Fig 1(a)), acetyl
groups in acetylated hemicelluloses (Fig 1(g)) and
acids and/or esters in lignin (Fig 1(e)). In a mixture,
none of these components can be distinguished using
only the 1750±1730 cm 1 spectral region. Only in
cases where the lack of interfering components can be
established by other information is this region diag-
nostically useful. For example, the existence of a band
maximum around 1750±1740 cm 1 and the lack of a
sharp band at 2850 cm 1, an aromatic ring stretch at
1505 cm 1 and a weak CÐO stretch at 1250 cm 1
would indicate that pectic acid is the likely compound
present. The additional lack of absorbance at
1445 cm 1 would support the argument that only the
pectic acid, not the ester, was present. Although these
situations would seem unlikely to exist, in fact they can
and do exist when speci®c compounds become
compartmentalised in anatomically distinct tissues.
Fig 1(d) shows the spectrum of calcium pectate.
Here the pectin is in the anionic form. Two broad
bands primarily characterise this spectrum. The
stronger one occurs with its maximum at 1615 cm 1
and is the asymmetric COO stretch vibration.15,16
This absorbance is observed at 1610 cm 1 when the
cation is sodium (not shown). The other band occurs
at 1425 cm 1 and is the symmetric COO stretch
vibration.15,16 However, the symmetric stretch band is
not useful as a diagnostic band owing to interferences
from other components. The asymmetric stretch is
also susceptible to interference from bound water at
1630 cm 1. This water band can be kept to a
minimum by drying the samples and retaining them
in a purged environment, thus permitting the use of
the 1615 cm 1 band as diagnostic for pectate salt.
Fig 1(e) reveals that a single band at 1505 cm 1
can selectively detect aromatics such as lignin without
interference from other components. Fig 1(f) shows
the spectrum for microcrystalline cellulose and the
location of the band that was used to detect cellulose at
1335 cm 1.9 Flax ®bres are known to contain appreci-
J Sci Food Agric 82:685±696 (online: 2002)

able amounts of O-acetylated glucomannans and
xylans as part of the hemicellulose component.17 Thus
the acetyl functional group provides a means of
detection of this component. Fig 1(g) shows the
spectrum of an acetylated glucomannan in which the
acetyl group is observed by the C=O band at
1732 cm 1, the CH3 symmetrical deformation at
1370 cm 1 and the asymmetric CÐCÐO stretch at
1250 cm 1.16 Since this C=O band occurs at nearly
the same location as other C=O bands and the CH3
overlaps many other bands, the CÐCÐO band is
more diagnostically useful for the detection of hemi-
cellulose. However, it is within 15 cm 1 of the
CH3ÐO stretch band observed for the pectic ester.
A band due to bound water is observed in all the
carbohydrate reference spectra between 1650 and
1630 cm 1.
In some cases, shifts were observed in the maxima of
infrared bands in the actual ¯ax stem tissue relative to
those of pure components. In order to identify such
band maxima shifts, spectra from single pixels in
selected tissues from the map of a cross-section of
untreated Ariane ¯ax were displayed (Fig 2). Fig 2(a)
indicates that there is no shift for the symmetrical CH2
stretch at 2850 cm 1 for waxes in epidermal tissue. Fig
2(b) shows that the carbonyl band for these waxes also
occurs at the same position as in the pure component
(1732 cm 1), as does the CÐCÐO stretching band
Figure 2. Transmission IR spectra in
absorbance mode of a cross-section of
untreated Ariane flax sandwiched
between two BaF2 crystals obtained
through an IR microscope equipped
with a 32 Cassegrain objective and a
10 Cassegrain condenser from
selected pixels (located at centre of
white cross-hairs in photomicrographs
on right) of map from 3220 to
2580cm 1 in epidermis (a) and from
1820 to 1180 cm 1 in epidermis (b),
fibre (c) and core (d) tissues.
J Sci Food Agric 82:685±696 (online: 2002)
Enzymatic retting of ¯ax stems
for acetyl groups (1250 cm 1). However, the strongest
band for the anion of carboxylic acids (antisymmetric
COO stretch) occurs at 1608 cm 1 in the ¯ax stem
matrix, while the weaker accompanying band (sym-
metric COO stretch) occurs at 1430 cm 1, versus
1615 and 1425 cm 1 respectively in the pure compo-
nent spectrum (Fig 1(d)). Fig 2(c) shows that there is
only a slight shift for the CÐO band for cellulose in the
®bres to 1336 cm 1, versus 1335 cm 1 in microcrystal-
line cellulose (Fig 1(f)). The C=O band observed at
this same spatial location displays a maximum at
1735 cm 1, which is at a frequency between those of
the C=O bands in acid and ester pectins (Figs 1(b)
and 1(c)) and the C=O band of the acetyl group (Fig
1(g)). In this case the C=O band may be due to
contributions from all three of these components. The
shoulder at 1645 cm 1 is most likely due to residual
bound water in the ®bres. This feature still interferes
somewhat with the band at 1608 cm 1 due to the
pectate anion. The spectrum shown in Fig 2(d),
obtained from the core tissue, is essentially that of
lignin or a closely related structure.18 There is a 3 cm 1
shift in the band maximum displayed here at
1508 cm 1 for the aromatic ring semicircle stretch15
relative to that same vibration in the cottonwood lignin
spectrum at 1505 cm 1 (Fig 1(e)). The exact position
of this band depends not only on the matrix that
surrounds the component but also on the exact nature
689
DS Himmelsbach, S Khalili, DE Akin
of substitution on the aromatic ring. Mono, ortho and
meta substitution gives rise to a band at 1500 cm 1,
whereas para substitution gives a band closer to
1510 cm 1.15 In the infrared spectrum of mixed
softwoods, this band appears at 1510 cm 1.9 The
band that appears at 1595 cm 1 is due to the quadrant
aromatic stretch. For mono- and disubstituted aro-
matic rings the quadrant stretch gives two bands at
1600 and 1580 cm 1 which can coalesce to form one
band at an intermediate position.15,16 It should be
noted that the strong set of bands observed at
1247 cm 1 in the spectrum shown in Fig 2(d) is
likely due to a combination of aromatic-OCH3
(1270 cm 1), acetate (1250 cm 1) and aromatic-OH
(1230 cm 1) CÐO stretches.16 This set of bands
overlaps the absorbance of the acetyl CÐCÐO stretch
in hemicelluloses and thus prevents the selective
detection of acetylated hemicelluloses in the core
tissue. The C=O band observed at 1740 cm 1 in Fig
2(d) appears, by its position, to be primarily indicative
of acids and/or esters with a shoulder at 1730 cm 1,
suggesting the presence of acetyl groups. It has been
stated that the observation of bands at 1730 and
900 cm 1 in isolated milled wood lignin preparations
should be considered as indicative of the presence of
associated hemicelluloses.19 Since the 900 cm 1 band
is not accessible in the instrumental set-up we have
employed, this situation cannot be veri®ed, but it is a
possibility. However, the main focus here is to
investigate the release of ®bres from bast tissue and
not the detailed nature of the core tissue of the ¯ax
stem.
Fig 3 shows the results of complete mapping of the
same untreated cross-section of Ariane ¯ax stem as
shown in Fig 2. The white box overlaid on the visible
image (Fig 3(a)) shows the location of the region
(120 mm  90 mm) that was selected for mapping the
tissues of interest. The letters and arrows on this ®gure
indicate the anatomical tissues that are contained
within the region (epidermis, cuticle, parenchyma,
®bre and core). Figs 3(b)±3(g) are grey-scale 2D
contour maps showing the intensity pro®les produced
from six of the diagnostic infrared bands. The bar in
the centre of the ®gure indicates the relationship of
band absorbances to grey-scale intensity over the
range from 0.050 to 0.500 absorbance units. The
intensity of the band at 2850 cm 1 was greatest in the
cuticular and epidermal tissues (Fig 3(b)). Thus the
location of waxes appears to be restricted to these
tissues. Fig 3(c) shows the result of mapping the
1740 cm 1 band. This band was most intense in the
core tissue, where it is most likely due to hemicellu-
loses associated with lignin. However, moderate
intensity is also shown for this band in the parench-
ymal tissue that surrounds the ®bres. In that tissue the
absorbance is more likely associated with pectic esters
and acids. However, a contribution from the acetyl
group carbonyl is also possible. Fig 3(d) shows the
map for the band with its maximum at 1608 cm 1. In
the absence of water the carboxylate anion is located
690
by this band. The greatest intensity is shown in the
epidermal tissue. Moderate absorbance is shown in the
parenchymal tissue, while the lowest absorbance is in
the region of the ®bres. Fig 3(e) shows the map of the
band with its maximum at 1508 cm 1, indicating the
location of the aromatic components. It is only intense
in the core tissue. The map of the band at 1336 cm 1
(Fig 3(f)) shows the greatest intensity in the ®bre
tissue, with lower intensity in the core tissue. This
®nding is consistent with the expected location of
cellulose in the bast tissues. Fig 3(g) shows the map of
the 1250 cm 1 band, which has it greatest intensity in
the core tissue, displays a moderate response in the
®bre region and has low intensity in the epidermal
tissue. In the core tissue this band is suggestive of
hemicelluloses and lignin (see Figs 1(e) and 2(d)). In
the other regions of the sample this band is more likely
a speci®c indication of the presence of acetylated
hemicelluloses. All the results shown here for the
untreated ¯ax (Fig 2) con®rm results previously
reported.9 The plots in Figs 3±7 have all been been
scaled to that of cellulose at 0.40 absorbance units.
This adaptation overcomes unavoidable differences in
sample thickness, which varied in this study from 6 to
8 mm, and permits the comparison of subsequent
treatments on an equivalent basis.
Treatment of cross-sections with the buffer solution
alone showed no change in the visible appearance
relative to the untreated sections that are shown in Fig
3. However, the infrared mapping indicated some loss
of pectate salts and acetylated materials (not shown).
The visible image of cross-sections treated with oxalic
acid alone indicated that the cuticular layer and
epidermal tissue were removed from the exterior of
the samples. Thus an infrared band for wax could no
longer be detected. However, bands for other compo-
nents still displayed appreciable absorbance.
Signi®cant changes in the visible images of cross-
sections did not occur until the SP 249 enzyme
mixture was applied to them. The visible image of the
resulting material (Fig 4(a)) shows that the ®bre
bundles have become separated from each other by the
action of the enzyme mixture at a concentration of
0.5 ml l 1 in acetate buffer at pH 5. The cuticular and
epidermal tissues have been removed from the
juncture of two ®bre bundles. However, they are still
clinging to the bundle shown, and bands indicative of
waxes can still be observed in Fig 4(a). Obviously, the
enzyme solution at this concentration was not effective
in removing the cuticular layer. Figs 4(b)±4(g) were
generated in the same manner as those shown in Fig 3.
Fig 4(b) shows that the cuticle and epidermal tissue
that remain attached to the ®bre contain no less wax
than the untreated tissue. However, most other maps
show decreases in intensity, except for Fig 4(f), which
indicates similar intensities for the band at 1336 cm 1.
This intensity indicates that the cellulose in the ®bre
tissue is retained while pectic acids, esters, salts and
acetylated hemicelluloses appear to be reduced by the
action of the enzyme preparation. Aromatic com-
J Sci Food Agric 82:685±696 (online: 2002)

Figure 3. Untreated Ariane flax cross-
section, showing photomicrograph
from a CCD camera obtained as in Fig
2(a), indicating epidermis (E) with
protective cuticle (white arrow),
parenchyma (black arrows), fibre (F)
and core (C) tissues, plus associated
2D infrared transmission contour
profiles at 2850 cm 1 (b), 1740cm 1
(c), 1608 cm 1 (d), 1508 cm 1 (e),
1336cm 1 (f) and 1250 cm 1 (g).
pounds also appear to be unaffected by the action of
SP 249 (Fig 4(e)).
Fig 5 shows the results after treatment of a cross-
section of ¯ax with 0.5 ml l 1 SP 249 and 50 mM oxalic
acid in buffer solution adjusted to pH 5. The visible
image (Fig 5(a)) clearly shows that the ®bre bundles
are now completely separated from the rest of the bast
and the core tissue. A few elementary ®bres are also
separated from the bundle (upper left corner of Fig
5(a)). The cuticle and epidermal and parenchymal
tissues are entirely removed, with no waxes or
aromatics being detected (above 0.05 absorbance
units) in the remaining ®bre bundles, and thus no
contours are shown for these components. Some
pectic acids and/or esters remain in the interior of
the selected ®bre bundle as indicated from Fig 5(b),
which shows the contour map of the band whose
J Sci Food Agric 82:685±696 (online: 2002)
Enzymatic retting of ¯ax stems
maximum occurs at 1736 cm 1. The location of the
maximum of this C=O band is some indication that
there are contributions from acid and/or ester pectins
and possibly from acetyl groups. The relative con-
tributions from these components are suggested by the
intensities displayed in Figs 5(b) and 5(e). A strong
band exists at 1615 cm 1 whose map is displayed in
Fig 5(c). The intensities displayed for this band appear
to be too great to arise just from residual pectate salts
and appear to be due to another source of COO
anion. The map of the absorbance at 1335 cm 1 (Fig
5(d)) displays the strongest intensity of any contour
map for this sample. This band again indicates that
cellulose is retained in the treatment. In order to try to
understand the nature of the strong absorbance at
1615 cm 1, a section (1825±1550 cm 1) of the single-
pixel spectrum taken at the point indicated by the
691
DS Himmelsbach, S Khalili, DE Akin

Figure 4. Ariane flax cross-section

treated with 0.5 ml l 1 SP 249 solution
in pH 5 buffer, showing
photomicrograph from a CCD camera
obtained as in Fig 2(a), indicating
tissues as in Fig 3 (except for cuticle),
plus associated 2D infrared
transmission profiles at 2851 cm 1 (b),
1739cm 1 (c), 1607cm 1 (d),
1508cm 1 (e), 1337 cm 1 (f) and
1250cm 1 (g).

cross-hairs in Fig 5(a) is shown in Fig 5(e). This
spectrum reveals three resolved bands above
1500 cm 1, at 1736, 1665 and 1615 cm 1. The ®rst
band appears to be primarily due to pectic acids/esters.
The band at 1665 cm 1 is probably due to bound
water, even though it is somewhat out of the range that
is normally observed for bound water (1650±
1630 cm 1). However, the band at 1615 cm 1, which
has its band maximum as that observed for the
antisymmetric COO stretch of the calcium pectate
salt (Fig 1(b)), is much narrower, suggesting a
different but similar source. One likely probability is
that the band is due to the calcium salt of oxalic acid.
This salt would be produced in the chelation of
calcium by the oxalic acid treatment. The calcium salt
is insoluble in water and thus would not have been
washed away by the water washes of the treated ¯ax
stem cross-section. In any event the product of the
treatment with 0.5 ml l 1 SP 249 and 50 mM oxalic
acid is visually but not chemically what is desired.
In order to improve the results, two strategies were
attempted. One approach was to increase the SP 249
concentration to 0.7 ml l 1 with 50 mM oxalic acid, and
the other was to not employ oxalic acid at all and to
increase the SP 249 concentration to 1.0 ml l 1.
Fig 6 shows the result of treating a ¯ax stem cross-
section with 0.7 ml l 1 SP 249 and 50 mM oxalic acid in
acetate buffer at pH 5. The visual image (Fig 6(a))
shows that only ®bre and core tissues remain and
many, but not all, ®bres are separated into individual
®bres. Part of the remaining ®bre bundle shown is
adjacent to the core tissue, but there is no remaining

692 J Sci Food Agric 82:685±696 (online: 2002)

 Enzymatic retting of ¯ax stems

Figure 5. Ariane flax cross-section

treated with 0.5 ml l 1 SP 249 and
50mM oxalic acid in buffer solution
adjusted to pH 5, showing
photomicrograph from a CCD camera
obtained as in Fig 2(a), indicating only
fibre tissue (F) of a fibre bundle, plus
associated 2D infrared profiles at
1736cm 1 (b), 1616 cm 1 (c),
1335cm 1 (d) and 1250 cm 1 (e)
together with a section of the IR
spectrum from 1820 to 1180 cm 1 (f)
from the point indicated by the white
cross-hairs in (a).

parenchymal connecting tissue. The 2D contour maps
displayed, analogous to those in Fig 5, show low
intensity at 1736 cm 1 (Fig 6(b)). The C=O band
that is observed in the spectrum taken in the region of
the ®bres (Fig 6(f)) is shifted to 1733 cm 1, which
makes it more closely aligned with that of acetyl
groups. With the map obtained at 1250 cm 1 (Fig
6(e)) still indicating intensity in this same sample
region, some acetylated hemicelluloses appear to
remain. No map could be produced that showed any
intensity >0.05 absorbance units above the local
baseline at 1615 cm 1, indicating that there was little
COO anion present (map not shown). Thus the
problem of chemical impurity experienced using only
0.5 ml l 1 SP 249 with oxalic acid was overcome by
slightly increasing the enzyme solution concentration
to 0.7 ml l 1 with oxalic acid. Possibly enzyme treat-
ment at the 0.5 ml l 1 level is only suf®cient enough to
open up the structure of the ¯ax stem to allow the
chelator to reach and attack the calci®ed pectin
between the ®bres, but not enough to proceed further
to break down the resulting matrix compounds.
Alternatively, all of the enzyme could be utilised in
releasing ®bre bundles from the bast tissue, leaving no

J Sci Food Agric 82:685±696 (online: 2002) 693

DS Himmelsbach, S Khalili, DE Akin

Figure 6. Ariane flax cross-section

treated with 0.7 ml l 1 SP 249 and
50mM oxalic acid in buffer solution
adjusted to pH 5, showing
photomicrograph from a CCD camera
obtained as in Fig 2(a), indicating only
core (C) and fibre (F) tissues, plus
associated 2D infrared transmission
profiles at 1739 cm 1 (b), 1508cm 1
(c), 1337 cm 1 (d) and 1250cm 1 (e)
together with a section of the IR
spectrum from 1820 to 1180 cm 1 (f)
from the point indicated by the white
cross-hairs in (a).

available enzyme to degrade the more entrenched
pectins. Whatever the explanation, only a very small
increase in enzyme concentration is needed to give an
acceptable result.
Treatment of ¯ax stem cross-sections with 1.0 ml l 1
SP 249 without chelator avoids the problem that
appears to be caused by the chelator remaining
attached to the remaining tissue. Fig 7 reveals that
such a treatment gives an even more satisfactory result
in terms of chemical purity of the residue tissue. Fig
7(a) shows that that this treatment breaks up the ®bre
bundles and produces ultimate (individual) ®bres with
no trace of cuticular, epidermal or parenchymal tissue.
All that remains visibly is mostly ultimate ®bres and
core tissue. Fig 7(b) shows a map produced from the
band at 1739 cm 1 with no C=O intensity above 0.05
absorbance units, except in the core tissue. Thus no
detectable pectic acids and/or esters are present in the
®bres. The only remaining C=O band is shifted to
1730 cm 1, indicative of some remaining acetylated
hemicelluloses. As with the 0.7 ml l 1 SP 249 plus
oxalic acid treatment, no absorbance at 1608 cm 1 was
detectable above the lower limit of absorbance,
indicating that no appreciable COO remained. Thus
this map also is not shown. Fig 7(c) indicates that the
only aromatic intensity occurs in the core tissue. The
remaining ®bre tissue gives very strong absorbences at
1335 cm 1 (Fig 7(d)), indicative of cellulose at the
location of the individual ®bres. Fig 7(e), the map
obtained at 1250 cm 1, shows only sporadic indica-

694 J Sci Food Agric 82:685±696 (online: 2002)

 Enzymatic retting of ¯ax stems

Figure 7. Ariane flax cross-section

treated with 1.0 ml l 1 SP 249 solution
in pH 5 buffer, showing
photomicrograph from a CCD camera
obtained as in Fig 2(a), indicating only
core (C) and fibre (F) tissues, plus
associated 2D infrared transmission
profiles at 1739 cm 1 (b), 1508cm 1
(c), 1337 cm 1 (d) and 1250cm 1 (e)
together with a section of the IR
spectrum from 1820 to 1180 cm 1 (f)
from the point indicated by the white
cross-hairs in (a).

tions of acetyl absorbance in the ®bres, indicative that
acetylated hemicelluloses have essentially been re-
moved. Most of the indications for hemicelluloses are
limited to the core tissue. The spectrum in Fig 7(f)
con®rms that cellulose, with small amounts of
acetylated hemicelluloses, characterises the ®bres that
remain after the treatment with 1.0 ml l 1 SP 249.
CONCLUSIONS
FT-IR microspectroscopic mapping has been shown
to be an extremely valuable tool for the analysis of the
effects of enzyme or enzyme/chelator retting of ¯ax
stems. The method relates the alterations in physical
structure to changes in speci®c chemical components,
which is a relationship dif®cult to obtain but important
for optimising enzymatic treatments in terms of ®bre
quality and economics. Speci®cally, the results ob-
tained here have con®rmed and extended our earlier
results that utilised more conventional methods.9
However, the technique is generally useful for in-
vestigations aimed at improving the enzymatic retting
of ¯ax, maximising its economic feasibility and
perhaps tailoring enzymes to obtain speci®c ®bre
properties. The results shown here also indicate that
FT-IR microspectroscopic mapping is a unique tech-
nique for evaluating the action of puri®ed enzymes and
new enzyme systems for the retting of ¯ax.
ACKNOWLEDGEMENTS
The authors wish to thank Dr Jeff Buhr for his
assistance with cryotoming, Luanne Rigsby for her
assistance with sample preparation and Farris Poole
for his technical assistance with FT-IR mapping. The
Wenner±Gren Foundation of Sweden is acknowl-
edged for the support of the work of Dr Sadia Khalili.
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