13 research outputs found
Rapid measurement of phytosterols in fortified food using gas chromatography with flame ionization detection
Rapid measurement of phytosterols in fortified food using gas chromatography with flame ionization detection
Antioxidants in food systems: influencing factors
For well over 20 years the Oxygen Radical Absorbance Capacity (ORAC) assay has been an important research tool in identifying antioxidant candidates in food and serum samples. The ORAC value is derived from a series of fluorescence intensity measurements taken over a period of time, usually a few hours. The kinetics of this reaction is controlled by the sustained release of peroxyl radicals at 37°C by an azo compound (AAPH). The peroxyl radicals attack the fluorescent probe causing a gradual decrease in fluorescence intensity. Ultimately, the test yields fluorescence decay curves over time, with the presence of any antioxidants in a sample having a protective effect, delaying probe decay and resulting in a greater area under the decay curve. This forms the basis of the ORAC measurement.
Concerns have been raised about the applicability of this assay, chemical interferences influencing the data, and the use of ORAC values to attribute health benefits of foods. In an effort to curb misrepresentation of health benefits from ORAC data, the USDA retracted its ORAC database in 2012 and published a statement by Dr Ronald Prior, a founding researcher for the ORAC assay. The explanatory statement by Dr Prior identified the misapplication of ORAC values and misleading perceived health benefits as major reasons for withdrawal of the database.
Despite this, ORAC remains a popular assay, and the method is widely used as a product development and marketing tool. Some researchers have reported that ORAC values can be influenced by factors such as choice of solvent, chemical constituents in matrices, and pH conditions. However, little has been published on antioxidant synergies in food systems and their contribution to the ORAC value.
Given the predisposition of the assay to interferences, a better understanding of antioxidant synergies is necessary to understand the contributing factors to measurement in real foods, and also to determine how these values may be manipulated. With the USDA caution in mind, this research was carried out to systematically investigate the factors influencing the ORAC measurement and its interpretation.
Firstly, an alternative mode of reporting antioxidant activity to consumers on product labeling was proposed. Calculations needed to convert ORAC units from μM Trolox equivalents (μM T.E/kg or μM T.E/L) to mass units (g T.E/kg or g T.E/L) of Trolox equivalents per kg or per L of foodstuff are provided. We propose that mass units are less misleading to consumers, by not reporting very large and impressive-looking values when a simple conversion reveals most of them to be much more modest when viewed as a mass of vitamin E equivalent. For example, the antioxidant activity of blueberries when measured by the ORAC assay was equivalent to over 71,000 μM of Trolox equivalents. When converted, the blueberries can be said to have the same antioxidant activity as 17.9g of vitamin E per kg of fruit (17.9g T.E/kg). This new mode of reporting was successfully applied to a range of commodities including fruit, confection and beverages.
Influencing factors, including environmental conditions, role of additives and nanoparticles and interactions between classes of chemical constituents were all investigated. Environmental conditions, specifically rainfall, were found to influence the levels of antioxidant compounds/bioactives in Australian wines. Six chemical constituents were identified as warranting further investigation; namely 6-methylcoumarin, protocatechuic acid, vanillic acid, p-coumaric acid, rutin and chlorogenic acid. Significant differences were also observed between the antioxidant capacity of wines by in vitro ORAC and ex vivo CAA-RBC assays, where wines with similar ORAC values had vastly different bioavailability and activity in the cellular system.
Amino acids and CuNPs additives were found to greatly influence the antioxidant measurements of “superfoods”. Results indicated strong enhancements and synergies related to the properties of the amino acids and complexes formed with Cu(I) and essentially matrix independent.. The order of antioxidant enhancement in bilberry, coffee berry, and apple concentrates was Tryptophan > Tyrosine > Methionine ≥ Histidine ≥ 4-Hydroxyproline. This order was also consistent with the order of calculated bond dissociation energies (BDEs), reflecting the inherent antioxidant potentials of the amino acids studied. Density Functional Theory (DFT) was used to support a proposed “substrate zone” and “antioxidant zone” postulate for amino acids and related additives and this concept assists in demonstrating potential mechanisms involved in achieving such extraordinary enhancements and synergies. Histidine was used as a model system for DFT calculations, and allowable species had homolytic BDEs ranging from high (deactivated) to very low (activated), in the case of species (b) the BDE was at a level well below that of vitamin E, making it an excellent and potentially potent antioxidant. DFT calculations revealed that the histidine-Cu(I) complex had a comparable BDE to that of Trolox, again demonstrating how interactions between chemical constituents can influence, and in this case enhance antioxidant activity measurements.
Synergies and antagonisms were also reported for eight classes of chemical constituents typically found in navel oranges. These mixtures were prepared based on the levels reported in nutritional data tables, and analyzed by ORAC and CAA-RBC assays. A correlation analysis revealed that the ORAC and CAA-RBC data did not correlate overall, however distinct clustering and several interesting outliers were noted. Cluster (a) had low ORAC and low CAA-RBC values, involving combinations of preservatives, sugars and CuNPs. Cluster (b) had low to moderate responses in both assays, and was made up primarily of vitamins in combination with CuNPs, preservatives, sugars and flavonoids. Cluster (c) was dominated by phenolics and their interactions with a number of groups, which gave high antioxidant activity in both ORAC and CAA-RBC assays, and amino acids are the main contributors in cluster (d). Organic acids featured in both outliers, firstly with a high antioxidant activity in both assays when combined with polyphenolics, and secondly as having an auto-oxidation effect in the CAA-RBC assay but a high ORAC value when analysed individually. Antioxidant activities of individual mixtures and combinations of classes of compounds showed antagonism/suppression of antioxidant activity between sugars and vitamins, and between polyphenolics and flavonoids in the ORAC assay. However, these same solutions resulted in antioxidant synergy in the CAA-RBC assay. In fact, the auto-oxidation effect of organic acids was reversed and synergies were noted in interaction with polyphenolics. A number of synergisms ex vivo involved polyphenolics in combination with other constituents such as vitamins, the amino acid Tryptophan, preservatives and CuNPs. These findings support the postulate that interactions at the “substrate zone” are influencing factors of antioxidant capacity at the molecular level. Computational chemistry was used to postulate mechanisms for antioxidant synergy, activation and deactivation of phenolic O-H groups, using quercetin and (-)-epicatechin-3-gallate as examples.
It was concluded that factors including rainfall, amino acid and CuNPs addition, and interactions between common classes of food constituents influenced antioxidant activity in food systems. Computational chemical calculations were used to postulate mechanisms for antioxidant enhancement and synergy, a major influencing factor in antioxidant measurements. This research describes the potential for unlocking new and powerful antioxidant synergies in food systems, nutrition and health and the medical/pharmaceutical fields
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Modes of handling Oxygen Radical Absorbance Capacity (ORAC) data and reporting values in product labelling
Antioxidant measurement assays are widely used and should be chosen based on their being fit for purpose. Likewise, the mode of reporting antioxidant measurements should also be fit for purpose. The Oxygen Radical Absorbance Capacity (ORAC) assay is widely used internationally for measuring the antioxidant capacity of commodities using the peroxyl radical. However, the current mode of reporting of the ORAC values is not obvious, especially for the consumer groups. In this mode, reporting of the ORAC values is the unit of micromoles of vitamin E analogue (VEA), known commercially as Trolox Equivalents per kilogram or per litre (μM T.E./kg or L). Unlike mass units, molar units are not widely used in nutrition information panels (NIP). This paper presents a simple mathematical model for conversion of ORAC values to mass units to facilitate better understanding of the antioxidant capacity quoted. Additionally, mass values are in keeping with current labelling practice in Australia. Unless legislation is passed for the regulation of ORAC data use in labelling and product marketing, mass units should be considered as a mode of reporting, limiting sensationalism of antioxidant capacity and keeping with current labelling practice