Determinación de aminas biógenas por métodos espectroscópicos

Abstract

ABSTRACT: The biogenic amines (BA) are organic bases of low molecular weight biologically active. Foods with favorable conditions for the growth of microorganisms with amino acid decarboxylase may present the formation of BA. Excessive oral intake of BA, may induce adverse reactions, such as toxicological effects and changes in blood pressure. BA analysis is important not only because of toxicity, but also to its use as a food quality indicators. Analytical methods commonly used for the determination of BA are the chromatographic, however, it is complex, expensive and it is time consuming during the process for this reason the aim of this work is to develop the construction of a biosensor coupled to an optical transducer (FT-IR) that allows the identification and quantification of BA in fish sample (tilapia). The immobilization of (DAO) diamine oxidase of pig kidney > 0.05 U / mg solid, for the construction of the biosensor, on the supports quartz silanized with 3-aminopropyltrimethoxysilane, was carried out using three different methods: by a first link amide EDC-NHS activation of the carboxyl group of DAO; the second by an electrostatic bond functionalization where 3′,4′-Dihydro-spiro[cyclohexane-1,1′(2′H)-pyrido[3,4-b]indole ]-3′-carboxylic acid implemented; and third means implemented by a covalent bond glutaraldehyde. The characterization of biosensors was carried out by analysis AFM and FT-IR. The amounts of immobilized DAO and its activity were also investigated. The methodology of DAO by activation assembly EDC / NHS carboxyl group of the DAO enzyme, was the only construction process biosensor demonstrating the immobilization of DAO on the surface of quartz silanized (2.07 mg protein). The decreased activity of the immobilized DAO is considered to be due to the reduced accessibility of BA molecules to the active sites of the enzyme and the conformational change of DAO molecules as a result of covalent immobilization. The biosensor implementation coupled to chemometric analysis (PLS and PCA) has proved a simple, useful and powerful technique to determine the concentration of AB (putrescine, cadaverine, histamine, spermine and spermidine) in tilapia samples, acid extracted trichloroacetic and methanol.