Matrix Extension with Fitness for Purpose and Stability Assessment of DHA and Additional Fatty Acids in Individual Whole Chicken Eggs

The consumption of long chain polyunsaturated fatty acids (LC-PUFA) is associated with several human health benefits. Most notable of these LC-PUFA is docosahexaenoic acid (DHA C22:6) whose inclusion is considered essential for optimum human health. Biofortification of common foods such as eggs with DHA has emerged as a specific approach to increase the intake of DHA in human populations. This can be achieved by supplementing poultry rations with feeds like microalgae or fish oil that are rich in DHA, which results in an increased uptake in the egg. Gas chromatography with flame ionization detection (GC-FID) is the method of choice when analyzing food such as eggs for DHA and other fatty acids. For regulatory studies it is desirable to demonstrate that the method is specifically suitable for the analysis of DHA and fatty acids in eggs. The purpose of this paper is to further extend the scope of the AOAC 996.06 methodology examined in the paper by Dillon et al., and to demonstrate the fitness for purpose of the method by examining specific validation parameters. It is a further objective to investigate the stability of DHA and other fatty acids of short and long timepoints. A validation of the method for the determination of DHA and three other fatty acids in eggs is thus presented.

Analytical Method Assessment for the Determination of DHA and Fatty Acids Present in Unextracted <i>Aurantiochytrium limacinum</i>Biomass

Research into long-chain polyunsaturated fatty acids (LC-PUFA), such as docosahexaenoic acid (DHA C22:6 n-3), has shown that their inclusion in the human diet is linked with many health benefits. This has led to an increased interest in the enrichment of certain foodstuffs with DHA by supplementing animal fed with DHA-rich ingredients which can lead to an increased uptake in the meat, milk and eggs animal by-products. The microalgae Aurantiochytrium limacinum has been found to be especially useful in this pursuit. It is subsequently desirable to availably have a simple and robust method for the routine analysis of DHA and other fatty acids in the algal biomass. The AOAC method 996.06 is often followed for the analysis of fatty acids in foods and demonstrating that its fitness for purpose in the analysis of DHA and additional fatty acids in Aurantiochytrium limacinum is therefore the objective of this paper. A validation of the method for the determination of DHA and three other fatty acids in Aurantiochytrium limacinum is presented. The method was found to be linear over the following ranges for each fatty acid methyl ester (FAME) analyte;50 to 15,000 μg/ml (C14:0), 300 to 95,000 (C16:0), 25 to 15,000 (C18:0) and 300 to 59,375 (C22:6). The accuracy, precision and LOD and LOQ of the method were confirmed and its robustness tested. The application of the method to assess the stability of Aurantiochytrium limacinum containing two alternative antioxidants was further examined. The investigation showed that DHA was stable over six months with the inclusion of either Duralox? or ethoxyquin as an antioxidant and ethoxyquin could additionally stabilize DHA in Aurantiochytrium limacinum up to 24 months.

Identity and Safety of a Novel <i>Aurantiochytrium sp.</i>for Terrestrial Heterotrophic Docosahexaenoic Acid Production

The objective of the studies in this paper was to expand on the published toxicological assessment of Aurantiochytrium limacinum (AURA) with further strain characterization and to investigate the potential for the biomass or extracted oil to have antimicrobial properties or undesirable substances. AURA is being investigated as a novel source of the omega-3 long-chain polyunsaturated fatty acid docosahexaenoic acid (DHA) for enriching foods of animal origin by means of feed supplementation. In the first studies, we provided the 18S rRNA identification of the novel marine isolated thraustochytrid, established the nutritional composition of AURA biomass for application as a food or feed ingredient including proximate analysis and fatty acid profiling, and confirmed the DHA production potential of the strain. We determined through minimum inhibitory concentration (MIC) analysis that the unextracted AURA biomass was safe, showing no antimicrobial influence and no evidence of any deleterious effects of this product or its extracts at concentrations up to 1% w/w on the reference human intestinal bacteria tested. This would indicate that AURA should not stimulate selective pressure on the commensal microbiota and is therefore unlikely to aid development of antimicrobial resistance and the concomitant harm to humans and animals. Further analysis revealed that the AURA biomass produced through industrial heterotrophic fermentation was free from undesirables;toxic marine microalgal metabolites, heavy metals, pesticides, microbial contaminants, and mycotoxins. Including heterotrophically-grown AURA in food or feed, up to 1% w/w, is a safe and environmentally beneficial strategy for DHA supplementation.