Enzyme-based online monitoring and measurement of antioxidant activity using an optical oxygen sensor coupled to an HPLC system.

It is estimated that up to 50% of the adult population take antioxidant products on a daily basis to promote their health status. Strangely, despite the well-recognized importance of antioxidants, currently there is no international standard index for labeling owing to the lack of standardized methods for antioxidant measurement in complex products. Here, an online high-performance liquid chromatography (HPLC)-based method to detect and measure the total antioxidant capacity of antioxidant samples is presented. In this approach, complex samples containing antioxidants are separated by the HPLC system, which is further coupled to an antioxidant measuring system consisting of an optical oxygen sensor, laccase, and tetramethoxy azobismethylene quinone (TMAMQ). The antioxidants, separated via HPLC, reduce TMAMQ to syringaldazine, which is then reoxidized by laccase while simultaneously consuming O(2). The amount of consumed oxygen is directly proportional to the concentration of antioxidants and is measured by the optical oxygen sensor. The sensor is fabricated by coating a glass capillary with an oxygen-sensitive thin layer made of platinum(II) meso-tetra(4-fluorophenyl)tetrabenzoporphyrin and polystyrene, which makes real-time analysis possible (t(90) = 1.1 s in solution). Four selected antioxidants (3 mM), namely, catechin, ferulic acid, naringenin (used as a control), and Trolox, representing flavonol, hydrocinnamic acid, flavanone, and vitamin E, respectively, were injected into the online antioxidant monitoring system, separated, and then mixed with the TMAMQ/laccase solution, which resulted in oxygen consumption. This study shows that, with the use of such a system, the antioxidant activity of individual antioxidant molecules in a sample and their contribution to the total antioxidant activity of the sample can be correctly assigned.

Antioxidant activity assay based on laccase-generated radicals.

A novel antioxidant activity assay was developed using laccase-oxidized phenolics. In a three-step approach, phenolic compounds were first oxidized by laccase. Laccase was then inhibited using 80% (v/v) methanol which also stabilized the oxidized phenolics which were then used to measure antioxidant activities of ascorbic acid and Trolox. From a number of laccase-oxidized phenolics screened for potential use in the measurement of antioxidant activities, syringaldazine emerged the best, giving results comparable to the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical, which is currently used in conventional methods. Like DPPH radicals, two moles of stoichiometric oxidized syringaldazine were reduced by one mole of either ascorbic acid or Trolox. For the first time we show that antioxidant activity can be correlated to oxygen consumption by laccase. Reduction of one molecule of oxygen corresponded to oxidation of four molecules of syringaldazine which in turn is reduced by two molecules of Trolox or ascorbic acid. This study therefore demonstrates the great potential of using laccase-oxidized syringaldazine for the measurement of antioxidant activity.

Enzymatic polymer functionalisation: advances in laccase and peroxidase derived lignocellulose functional polymers.

Enzymatic polymer functionalisation has entered its most fascinating period with development in this field largely at the basic research level and pilot scale applications. Development of enzymatic processes for the development of lignocellulose-based functional polymers has not been spared, ranging from textile fibres with novel properties (antimicrobials properties, hydrophobic properties, attractive shed colours, etc.) to fibreboards. Enzymatic processes are also being actively pursued aimed at developing functional polymers from lignin (a major by product of the pulp and process).

Polymerization of lignosulfonates by the laccase-HBT (1-hydroxybenzotriazole) system improves dispersibility.

The ability of laccases from Trametes villosa (TvL), Myceliophthora thermophila (MtL), Trametes hirsuta (ThL) and Bacillus subtilis (BsL) to improve the dispersion properties of calcium lignosulfonates 398 in the presence of HBT as a mediator was investigated. Size exclusion chromatography showed an extensive increase in molecular weight of the samples incubated with TvL and ThL by 107% and 572% from 28400 Da after 17h of incubation, respectively. Interestingly, FTIR spectroscopy, (13)C NMR and Py-GC/MS analysis of the treated samples suggested no substantial changes in the aromatic signal of the lignosulfonates, a good indication of the ability of TvL/ThL-HBT systems to limit their effect on functional groups without degrading the lignin backbone. Further, the enzymatic treatments led to a general increase in the dispersion properties, indeed a welcome development for its application in polymer blends.