Determination of l-Lactic Acid Content in Foods by Enzyme- Based Amperometric Bioreactor Zsuzsanna Bori, a Gµbor Csiffµry ,* b Diµna Virµg, a Marianna Tóth-Markus , a Attila Kiss , b Nóra Adµnyi a a Central Food Research Institute, 1022 Budapest Herman Ottó fflt 15., Hungary b Eszterhµzy Kµroly College, EGERFOOD Regional Knowledge Center, 3300 Eger Leµnyka u. 6., Hungary *e-mail: csiffary.gabor@ektf.hu Received: July 29, 2011; & Accepted: November 2, 2011 Abstract A thermostable lactate oxidase (from Aerococcus viridans) based bioreactor was elaborated for l-lactate determina- tion in dairy products. The enzyme was immobilised in a thin-layer enzyme cell connected into a flow injection system with an amperometric detector. The linear measuring range was 0.1–1 mmol/L l-lactate (R 2 = 0.99; linear re- gression equation: y = 81.2x + 3.3). The detection limit was 0.05 mmol/L. 40 Samples were analyzed per hour. Con- sidering reproducibility: the signals measured were very stable for over 800 injections, after this period the activity of the cell decreased slowly. The method was used for the control of dairy products and for monitoring milk fresh- ness. Keywords: Enzymes, Biosensors, Thin-layer enzyme cell, Lactate oxidase, Aerococcus viridans DOI: 10.1002/elan.201100409 1 Introduction l-Lactate is produced from glucose in anaerobic metabo- lism mainly by Lactobacillus species and Lactococcus spe- cies . Its determination is relevant in clinical analysis, sport medicine and food analysis [1]. The level of l-lactate in blood is an important parameter. It can indicate different pathological states, such as cardiac dysfunction etc. [2], and also correlates to the status of anaerobic metabolism during muscle work [3]. In foodstuffs, l-lactate monitor- ing is important for the control of the fermentation of cheese, yoghurt, butter, pickles, sauerkraut, wine, cider and other products. Therefore the reliable and rapid mea- surement of lactic acid is substantial in food industry [4]. Many different analytical methods are now available for the determination of l-lactate with chromatographic, spectrophotometric, amperometric or by different enzy- matic methods [1], but they are time- and labour-inten- sive. In the last few years a lot of research has gone into es- tablishing rapid, routine methods for fast determination of l-lactate. The measurement of l-lactate is usually per- formed by employing immobilized enzymes, such as lac- tate dehydrogenase (LDH) and lactate oxidase (LOD) or several microorganisms in different types of bioreactors [5]. l-Lactic acid was determined during the production of mozzarella cheese by an electrochemical wall-jet cell bioreactor where the platinum (Pt) electrode covered with LOD enzyme [6]. Palmisano [7] investigated a dis- posable bioreactor, where the biosensing layer of LOD was cast on an underlying electropolymerized layer of overoxidized polypyrrole. The introduction of a micro- dialysis membrane-based sampler increased the sensitivity from 50 mmol/L to 7.9 Æ 0.2 mmol/L. Gomes et al. [8] de- scribed the construction and evaluation of lactate sol–gel bioreactors to accomplish the determination of lactate in pharmaceutical products. LOD was incorporated in a porous sol-gel film placed onto a Pt-based electrode. Huang et al. [9] prepared a highly sensitive amperometric l-lactate bioreactor based on LOD immobilized in sol-gel matrix and combined with multiwalled carbon nanotubes modified electrode with the linear measuring range of 0.2 mmol/L to 2 mmol/L l-lactate. In other constructions, bienzymatic multilayer system was used, where the inner layer of horseradish peroxidase (HRP) was crosslinked with poly(allylamine) containing an osmium complex, and the outer layer contained the LOD immobilized in polymeric matrixes [10, 11]. Suman et al. [12] developed a bioreactor where the LOD from Pediococcus species was immobilized by glutaraldehyde (GA) onto poly(aniline-co-fluoroaniline) (poly(An-co- FAn), which is a conducting copolymer film coating an indium tin oxide (ITO) plate. This poly(An-co-FAn) film with immobilized LOD on the ITO plate acted as work- ing electrode and Pt electrode as counter electrode joined through an electrometer. Carbon paste electrode was modified with bakers yeast Saccharomyces cerevisiae and was investigated as amperometric bioreactor for lactic acid. The yeast cells remained viable at least for 1 month. The electrode was suitable for the determination of lactic acid in the concen- tration range of 0.03 to 1 mmol/L [13]. Luong et al. [14] SPECIAL ISSUE 158  2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Electroanalysis 2012, 24, No. 1, 158 – 164 Full Paper