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 À1 concentration in pH 5 acetate buffer for 6 h at 40°C. Flax stems treated with 0.5 or 0.7 ml l À1 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 À1 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 À1 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 À1 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
Fibres were placed in unsterile commercial planting soil known to contain active soft rot fungi and incubated at 20-22°C for one month. After incubation, the fibres were removed from the soil and washed before undertaking light microscopic observations. Degradation of kapok fibres in liquid culturesMicroscopic observations were also made of kapok fibres degraded under liquid culture conditions by the soft rot fungus Humicola alopallonella Meyers & Moore. The fungus was grown for 3 weeks at 25°C in 100 ml Erlenmeyer flasks containing 25 ml of the following basal salts media: NH 4 NO 3 , 1.0 g; KH 2 PO 4 , 1.0 g; MgSO 4 . 7H 2 O, 0.5 g; FeSO 4 . 7H 2 O, 0.1 g; ZnSO 4 . 7H 2 O, 0.01 g; plus yeast extract (0.01 g); glucose (2.5 g), and asparagine (1.0g). Small tussocks of kapok fibres were added prior to autoclaving and the fungus inoculated using 3 * 1 cm -2 agar pieces removed from the fungus actively growing on 2 % w/v malt agar plates. Partially decayed kapok fibres were removed for both light and electron microscopic observations. Light microscopyFibres were stained with either 1 % (w/v) aqueous safranin or with 0.02 % (w/v) aniline blue-lactic acid and observed using polarized light microscopy. With light microscopy, it was often difficult to determine the exact location of cavities in the cell walls, and careful and accurate focusing was therefore done on either the upper or the lower cell walls to avoid shadow effects. Measurements of the angle of soft rot cavities with respect to the longitudinal axis of the fibres were carried out on 30 fibres using polarized light microscopy in conjunction with image analysis. Digital images (512 * 512 pixels, dpi: 300) were acquired from a Leitz Orthoplan light microscope (mag. 40 *) using a CCD camera using the software program AnalySIS 2.0 from Soft-Image Software GmbH purchased from Philips, Holland. Transmission electron microscopy (TEM)Partially degraded and control kapok fibres were fixed for 3 hrs in a fixative solution containing 3 % (v/v) glutaraldehyde prepared in 0.1M sodium cacodylate buffer (pH 7.2) and 2 % (v/v) paraformaldehyde at room temperature. After rinsing in buffer, fibres were post fixed overnight at 4°C in 2 % (w/v) osmium tetroxide in the same buffer. Some control fibres were also fixed in the above fixative in the presence of 0.05 % (w/v) ruthenium red for visualization of pectin-rich regions. Following washing in 3
Extended abstract of a paper presented at Microscopy and Microanalysis 2004 in Savannah, Georgia, USA, August 1–5, 2004.
Overnight incubation of flax straw in a weakly acidic (pH 2) solution decreases the amount of enzyme needed for virtually complete retting by two to three orders of magnitude, as judged by a modified Fried test. This effect is probably based on removal of Ca2+ crosslinking pectin chains in the middle lamella. Washing the enzyme-retted fibers in ethanol and then in acetone or in 0.1 M KOH reduces their stickiness and matting, thus apparently eliminating dry-back. The methods show potential usefulness for the development of a commercial enzyme retting process.
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