Investigating carotenoid loss after drying and storage of
Investigating carotenoid loss after drying and storage of
Investigating carotenoid loss after drying and storage of
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7. Involvement <strong>of</strong> enzymes<br />
7.3.3. Enhancement <strong>of</strong> β-carotene oxidation using a free radical generator<br />
Bleaching <strong>of</strong> β-carotene was tested in presence <strong>of</strong> horseradish peroxidase, hydrogen<br />
peroxide, linoleic acid <strong>and</strong> 2,4 Dichlorophenol (DCP) free radical generator (Table 7-4).<br />
Table 7-4: Instantaneous* effect <strong>of</strong> 2,4 Dichlorophenol addition on β-carotene<br />
absorbance value at 450nm at ambient temperature (about 22ºC)<br />
Absorbance at<br />
Sample DCP H2O2 LA POD<br />
450nm**<br />
1 - - - - 0.698 (0.021)<br />
2 x - - - 0.746 (0.039)<br />
3 x x - - 0.739 (0.033)<br />
4 x - x - 0.723 (0.028)<br />
5 x - x x 0.585 (0.018)<br />
6 x x - x 0.174 (0.014)<br />
*the reading was carried out as quickly as possible <strong>after</strong> the addition <strong>of</strong> reagents<br />
(maximum time <strong>of</strong> 5 minutes).<br />
**n=3 – average (st<strong>and</strong>ard deviation)<br />
x indicates addition <strong>of</strong> 5% substrate or enzyme. DCP: 2,4 Dichlorophenol 1.8mM in<br />
solution (4 ml); LA: Linoleic acid 0.4mM in solution; H2O2 1mM in solution. POD:<br />
Horseradish peroxidase; at pH= 7.4, POD activity = 0.746±0.046 (mmol ABTS.min -<br />
1.ml -1 ).<br />
Instantaneous bleaching <strong>of</strong> β-carotene occurred when DCP was added to peroxidase <strong>and</strong><br />
hydrogen peroxide (absorbance= 0.174). It can be noted that addition <strong>of</strong> DCP biased the<br />
reading <strong>of</strong> the blank; absorbance was increased with DCP (0.746; blank 0.698).<br />
Instantaneous autoxidation <strong>of</strong> β-carotene in presence <strong>of</strong> DCP <strong>and</strong> or H2O2 (0.739) or<br />
linoleic acid (0.723) did not occur. Bleaching efficiency was therefore attributed to the<br />
ability <strong>of</strong> horseradish peroxidase to use DCP to generate free radicals using H2O2 as a<br />
substrate. It was demonstrated that the oxidation <strong>of</strong> <strong>carotenoid</strong>s in the presence <strong>of</strong><br />
peroxidase was dependent on the presence <strong>of</strong> DCP (Matilde <strong>and</strong> Martinoia 1982) but it<br />
was not dependent on the addition <strong>of</strong> H2O2. Indeed DCP plays a crucial role in<br />
regenerating peroxidase back to its native state (Harvey P., Pers. Comm.). When linoleic<br />
acid replaced hydrogen peroxide as substrate the effect was slower (0.585). Bleaching in<br />
presence <strong>of</strong> linoleic acid might result from linoleic acid autoxidation that could then be<br />
used by the peroxidase as a substrate to generate free radicals with DCP. Oxidation <strong>of</strong> !-
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