Green methods for methyl esters determination in foodstuffs

Abstract

In this thesis development of novel, green methods for methyl esters in foodstuffs is described. Methods are based on enzymatic reactions and fluidics. Study focuses on two methyl esters: pectin methyl ester and aspartame, a methyl ester of aspartic acid/phenylalanine dipeptide. Pectin and aspartame are enzymatically hydrolysed by pectin methylesterase or _- chymotrypsin, respectively. Methanol is released and quantified. Several methanol determination methods have been tested, with the method with 4-AAP and phenol showing the best prospects for automation. Method was optimized and its robustness was investigated. Ascorbic acid interference removal with 4-hydroxy TEMPO was tested. Development of two automated methods for methyl esters determination is described: spectrophotometric pectin methyl esters determination and chemiluminescent aspartame determination.The method for pectin methyl esters is the first work on pectin analysis through flow injection. Detection limit down to 1.47 mM was achieved at the analysis rate of 7 samples h-1. The method provides identical results with manual off-line method. The development of the aspartame analyzer was preceded by the development of a spectrophotometric method, which showed good results in samples containing higher aspartame concentrations than expected in beverages. In order to improve method for possible application in beverages chemiluminescent detection was selected for the automated method. The chemistry from kinetic study was modified to accommodate luminol chemiluminescent detection and optimization of the system was performed. Several manifolds were constructed and the effect of following parameters was tested: flow rate, mixing coils length, location and number, preincubation time, alcohol oxidase concentration, use of separate solutions of AOX and HRP. 0.8 ml/min/line flow rate in combination with one 100 cm mixing coil, 60 s preincubation and use of separated solutions of AOX an HRP resulted in sufficient sensitivity that allowed for construction of a calibration curve within the range of aspartame concentration found in diet drinks. Additionally, following side projects related to the main topic of the study are described: development of PME activity assay and galacturonic acid determination.