Investigating carotenoid loss after drying and storage of

7.2.2. pH measurement
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7. Involvement of enzymes
The pH value was measured on flour samples that were homogenised with 10ml
deionised water using ultra-turax for 5s and topped up to a total volume of 30ml with
deionised water. Determination was done in triplicate using Hanna Instruments pH210
Microprocessor pHmeter.
7.2.3. Enzyme assay on !-carotene
!-carotene enzymatic oxidation was evaluated spectrophotometrically following an
adaptation of the method of Fleischmann et al. (2003) and Baldermann et al. (2005).
!-carotene stock solution was prepared : 25mg of !-carotene (Type I approx. 95% UV
Sigma, UK) was dissolved in a small quantity of chloroform. Tween 20 (2.5ml) and
Chloroform (3.5ml) were slowly added to obtain an emulsion made up to a total volume
of 50ml with sodium phosphate buffer (pH 7.4). The solution was homogenised using an
ultra-turax for about 10s. For assays, the stock solution was diluted 1/40 in phosphate
buffer, which pH value was adjusted to 6.0 or 7.4 with HCl. A transparent solution,
which absorbance could be read by UV-visible spectrophotometer, was obtained..
For the assays, 0.2ml substrate (H2O2 (20mM), linoleic acid (4mM)), and/or enzyme
were added to the !-carotene solution (3ml) in a cuvette. The total volume was adjusted
with deionised water to be the same in each tube of the assay so each substrate or
enzyme represented 5% of total solution. The reaction mix was incubated at 40ºC and
reading of absorbance taken every 5 minutes at 450nm. Blank (!-carotene + deionised
water) and positive control (!-carotene + commercial soybean lipoxygenase + linoleic
acid) were prepared for each assay. All operations were performed under dim light.
7.2.4. Enzymatic extraction
Sweet potato extract for measurement of peroxidase and lipoxygenase activity was
prepared according to Isamah et al. (2004). Sweet potato flour (2g) or puree (10g) was
homogenised with 20ml of sodium phosphate buffer (pH 7.4) using an ultra-turax
homogeniser (IKA Janke and Kunkel Labortechnik) at 290 x g. for 1 minute and
immediately maintained on an ice bath. The homogenate was strained through one layer
of cheese cloth to remove solid particles. 10ml of buffer was added for rinsing. The