No abstract

An improved system for measuring antioxidant activity via thiobarbituric acid reactive substances and ferric thiocyanate assays is reported, on the basis of oxidation of a linoleic acid (LA) emulsion. Oxidation times were reduced from 20 h to 5 h by increasing the reaction temperature from 37 °C to 50 °C and with an acceptable precision of <10% coefficient of variation (CV). Antioxidants varying in polarity and chemical class—250 µM Trolox, quercetin, ascorbic acid and gallic acid—were used for method optimisation. Further reductions in reaction time were investigated through the addition of catalysts, oxygen initiators or increasing temperature to 60 °C; however, antioxidant activity varied from that established at 37 °C and 20 h reaction time—the method validation conditions. Further validation of the method was achieved with catechin, epicatechin, caffeic acid and α-tocopherol, with results at 50 °C and 5 h comparable to those at 37 °C and 20 h. The improved assay has the potential to rapidly screen antioxidants of various polarities, thus making it useful in studies where large numbers of plant extracts require testing. Furthermore, as this assay involves protection of a lipid, the assay is likely to provide complementary information to well-established tests, such as the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay.

The thiobarbituric acid reactive substances (TBARS) assay is widely used to measure antioxidant activity in the presence of lipid substrates such as linoleic acid (LA). In one version of this assay, LA is oxidized with and without antioxidant in a multi‐phase system in which LA lies over the surface of the aqueous layer of catalyst and antioxidant solution, with dissolved and atmospheric oxygen. Results from this assay showed low precision and therefore we conducted a systematic study to identify possible sources of variability including: order of addition of reactants; pre‐formed peroxides in LA (as precursors of TBARS); and the effect of different concentrations of added antioxidant, Trolox. These investigations showed that all contributed to variability of both formation of oxidation products and the antioxidant activity of Trolox. Furthermore, through the use of LA with pre‐formed peroxides removed, it was discovered that antioxidant activities reported in other studies using this system were most likely not due to the protective effect of antioxidant against formation of primary oxidation products. Rather, the antioxidant is likely to interfere with the radical scission of pre‐formed peroxides, resulting in lower TBARS. Practical applications: The results obtained from the TBARS assay have been used to interpret the potential antioxidant value of compounds added to lipid‐containing food products, such as in oil and meat. Alternatively, the TBARS assay has been used to assess multiple compounds or extracts to rank their effectiveness as antioxidants, thus selecting the most active ones for further study. In both cases, pre‐formed peroxides in the system may not be routinely measured. The results from this study suggest that pre‐formed peroxides may have a large impact on the results of antioxidant studies when TBARS are the measure of oxidation. Therefore, further research in other systems is necessary to ensure that robust antioxidant measurements are being performed. TBARS variability: absorbance values at 532 nm for different batches of linoleic acid. Absorbance values show TBARS variability in a multi‐phase oxidation system in measuring antioxidant activity of 250 and 1000 µM Trolox.

Solid state fermentation (SSF) utilizing filamentous fungus Amylomyces rouxii was investigated as a bio-processing strategy to enhance the bioactive properties of broken rice. Fermentation was carried out for 18 days and samples were withdrawn at 2-days interval. Established methods were deployed to assess the changes in bioactive properties and compounds content in fermented broken rice. The bioactive properties studied were total phenolic content (TPC), total flavonoid content (TFC), DPPH-radical scavenging activity and ferric-reducing antioxidant power (FRAP). Additionally, tyrosinase inhibition activity, which represents anti-pigmentation/browning property, was evaluated. Free phenolic acids and organic acids content were determined through high performance liquid chromatography (HPLC). The results showed that fermentation significantly increased the total phenolic content of broken rice from 0.03 mg GAE/g sample to 3.94 mg GAE/g sample and total flavonoid content from 0.04 to 1.71 mg QE/g sample. By the end of the fermentation, DPPH-radical scavenging of fermented broken rice was enhanced to 94.22%, compared to 9.03% in the unfermented sample. It was also observed that FRAP and tyrosinase inhibition activity of fermented broken rice were improved up to 39- fold and 50-fold, respectively. Kojic acid, a potent antioxidant and tyrosinase inhibitor, was detected in fermented broken rice, along with oxalic and ascorbic acid. Gallic, protocatechuic and 4-hydroxybenzoic acids were enhanced upon fermentation. This study manifested the positive effect of broken rice after fermentation with A. rouxii and thus revealed the potential of fermented broken rice as a promising natural bio-ingredients in food, cosmetics and medicinal products.