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(Above) Figure 2. Proposed cell wall architecture of canola revealed using stepwise degradation by enzymes. The canola cell wall consists of an outer layer of xyloglucan and an inner pectin layer surrounding the protein. Removal of the outer xyloglucan layer by a glucanase containg product (XG’ase) or a pure glucanase revealed the pectin layer. Removal of the pectin layer by a pectinase containing product resulted in an enhanced visualization and utilization of protein present in canola in vitro.


(Left) Figure 3. Visualization of canola cell walls by fluorescence microscopy. Canola cell walls consists of an outer layer of xyloglucan and an inner pectin layer surrounding the protein. Removal of the outer xyloglucan layer by a glucanase containing product revealed the pectin layer. Removal of the pectin layer by a pectinase containing product resulted in an enhanced visualization of protein present in canola.


3. Visualizing pectin degradation in SBM in vitro and in vivo Ruthenium red (Rr) is a dye commonly used in microscopy to stain pectin. Petri plates containing agarose with SBM appear pink when stained with Rr, this is due to a colour reaction between pectin in the SBM and the dye. The effect of a pectinase containing enzyme product on SBM was studied by punching holes in the agarose and adding either buffer or enzyme to these holes before leaving the plates to incubate 4 hours at 400


C. After incubation the pink colour around


the enzyme treated wells disappeared indicating that pectin had been degraded into smaller oligomers which can no longer bind to the dye to give the pink colouration. Using the same dye and binding principal,


(Right) Figure 4. (A) Agarose containing SBM and stained with Ruthedium red which stains pectin pink. Enzyme and buffer (control) was added to holes made in the agarose and plates were incubated 4 hours at 400


C and then washed to remove excess dye. Clearing zones (black dashed circles) were seen where the enzyme degraded the SBM pectin. (B and C) Ileum digesta samples from broilers fed a corn-SBM diet without (negative control) or with supplementation of a pectinase containing enzyme product. Samples were visualized by light microscopy after staining with Ruthenium red and it is evident that much less pink colour is present in the enzyme treated sample (C) compared to the negative control (B).


FEED COMPOUNDER MAY/JUNE 2018 PAGE 37


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