Elsevier

Talanta

Volume 54, Issue 5, 21 June 2001, Pages 879-885
Talanta

A flow system with a conventional spectrophotometer for the chemiluminescent determination of lactic acid in yoghurt

https://doi.org/10.1016/S0039-9140(01)00360-5Get rights and content

Abstract

A flow-based analytical procedure for lactate determination in yoghurt by chemiluminescence using a conventional UV-Vis spectrophotometer as detector is described. The radiation source was switched off. The flow cell was machined in acrylic with a 1 mm path length and a 80 mm2 surface and was positioned 2 mm distal of the photodetector (100 mm2 sensible area) in order to improve detection. The flow network computer-controlled comprised a set of three-way solenoid valves assembled to implement the multicommutation approach. The chemiluminescence was obtained by using the reaction of luminol with hydrogen peroxide, catalyzed by hexacyanoferrate (III) after enzymatic reaction with lactate. The lactate oxidase enzyme was immobilized on porous silica beads (glass aminopropyl, SIGMA). The signal generated by the spectrophotometer reaction was read by the microcomputer and stored as a function of time for further treatment. Immobilization condition, enzyme concentration, temperature, pH, stability of the enzymatic reactor, and flow rates were investigated. The feasibility of the system was ascertained by analyzing a set of yoghurt samples. Results were in agreement with those obtained by a conventional method (Boehringer UV-Kit), and no significant difference at 95% confidence level was observed. A linear response within 10–125 mg l−1 l-lactate, a 1.9% standard deviation (n=10), and an analytical throughput of 55 determinations per hour were achieved.

Introduction

Lactic acid in foodstuffs is produced by bacterial fermentation and it is an essential component related to manufacturing of cheese, yoghurt, fermented meat products, butter milk, etc. Some food quality tests such as taste and flavor are nowadays carried out by skilled experts, nevertheless, other important parameters require chemical analyses. The quality control in the industrial manufacture of these products became mandatory mainly with the requirement established by the ISO-9000 norm [1]. Therefore, there is a high demand for lactate determination in products derived from milk and also in biologic fluids for clinical diagnostic [2]. Thus, to attain this requirement some analytical procedures have been proposed [2], [3].

Flow injection analysis has been employed to monitor fermentation processes presenting as advantages a simple hardware, a low reagent consumption and robustness [3], [4]. Several flow procedures for lactate determination in milk derivatives, blood, wine, etc., based on enzymatic reactions have been proposed employing as detection technique amperometry [5], potentiometry [6], spectrophotometry [7], chemiluminescence [1], [8], fluorimetry [9], etc.

Chemiluminescence (CL) presents as an advantage high sensitivity and the analytical procedure can be implemented with simple instrumentation. These features have been aroused the interest of the analytical chemists since 1970. In this context, several approaches have been proposed and among them worth be mentioned the determination of lactate in food industry [2] and in human blood [9]. Also, chemiluminescence procedures implemented with flow systems present, high analytical throughput and low reagent consumption. The basic set-up comprised the flow manifold and the light measurement device [10]. In order to improve detection, flow cells with different geometries have been used [10], [11].

In this paper, a multicommutated flow analysis procedure for l-lactate determination in yoghurt by chemiluminescence is described. The chemiluminescence radiation produced by the reaction of luminol with after enzymatic reaction, will be detected using conventional spectrophotometer.

Section snippets

Reagents and solutions

All solutions were prepared with analytical grade chemicals and freshly distilled/deionized water. As carrier stream (C), a buffer solution consisting of 0.1 mol l−1 tris(hydroxymethyl)-aminomethane (TRIS) adjusted to pH 7.0 with 0.1 mol l−1 HCl, was used. A 10 000 mg l−1 lactate stock solution was prepared by dissolving 1.077 g of solid lithium lactate (Sigma, ref. L-2250) in 100 ml of water. Reference solutions within the range 1.0–200 mg l−1 L(+) lactate were prepared in water by

Results and discussion

The luminol reaction occurred in an alkaline medium (pH 10.5), thus, its solution was prepared in this medium and a hexacyanoferrate (III) solution was used as catalyst and for co-oxidant of luminol [17]. To optimize concentrations of luminol and hexacyanoferrate( III) solutions, experiments were carried out by using a 5×10−4 mol l−1 H2O2 solution. For hexacyanoferrate (III) the concentration was varied from 0.01 to 0.30 mol l−1 and the maximum response was obtained within a narrow

Acknowledgements

Partial support from PRONEX, and grants CNPq and FAPESP. The authors express their gratitude to E.A.G. Zagatto for critical comments.

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