Studies on the Lipophilisation of 3,4-Dihydroxyphenylacetic Acid and its Application in Enhancing the Oxidative Stability of Fish Oil

Abstract

Antioxidants are important food additives that can enhance the storage stability by significantly delaying oxidation. Synthetic antioxidants can be potentially toxic and carcinogenic, which has led to the exploration of various natural antioxidants as alternatives. Phenolic acids are hydrophilic in nature and have excellent antioxidative properties in addition to other beneficial effects, including anti-microbial, anti-cancer, and anti-inflammatory properties. 3,4-dihydroxyphenylacetic acid, is one such rarely studied phenolic acid which has not been utilized for retarding oxidation in lipids. The study aims to lipophilise this hydrophilic antioxidant to obtain an ester with improved hydrophobicity for application in fish oils. Lipases from Candida antarctica and Thermomyces lanuginosus was used in immobilized form under different reaction conditions for esterification. The enzyme was only efficient in synthesizing methyl ester, while the increase in length of the acyl chain significantly reduced the esterification efficiency. Homogeneous catalyst p-toluenesulfonic acid gave higher yields of the ester but the purity of the ester could not be improved despite our best efforts. The methyl, butyl, and hexyl esters of 3,4-DHPA were synthesized in 81.1%, 69.3%, and 78.% conversion using 15% (w/w) Amberlyst-15 as the catalyst in a binary solvent system comprising n-hexane:2-butanone in 75:25 (v/v) ratio. The methyl ester was obtained through direct esterification in 1:60 molar ratio of phenolic acid: alcohol while the butyl and hexyl esters were obtained through trans-esterification reaction in 1:1 molar ratio. The obtained esters were studied in refined bulk fish oil for a period of 30 days at 35°C in contact with atmospheric air. TOTOX values of fish oil samples indicated that the antioxidative potential of 3,4-DHPA was on par with the synthetic antioxidant BHT. Among the esters, the methyl ester showed a similar efficacy to that of 3,4-DHPA. The phenolic acid and its esters were also evaluated separately and in various combinations in bulk fish oil containing trace amounts of water. A significant synergistic effect was observed between 3,4-DHPA and its hexyl ester in 2:1 molar ratio in this study. This indicates the role of reverse micelles formed in the presence of trace water in controlling lipid oxidation in a bulk oil system. The relative partitioning of the hydrophilic and hydrophobic antioxidants in a combination among the oxidation sites at the water-oil interfaces significantly impacts their overall efficacy in retarding lipid oxidation.