“Discrimination of Gram-Positive and Gram-Negative Bacteria in Milk by Means of Voltammetry Analysis”

ASTRID HILDING-OHLSSON; NATALIA J. SACCO; M. CELINA BONETTO; JONATHAN A. FAUERBACH; EDUARDO CORTÓN

Tucuman

Congreso; VII congreso Argentino de Microbilogía General – SAMIGE; 2011

Milk represents the main dairy product, and it is consumed all around the world, playing a fundamental roll in international commerce; this gives milk a great economic importance. Electrochemical techniques have various applications and have been widely used in food analysis as wine, coffee, fruit juices or milk examination. Voltammetry is an electrochemical technique that seems to have several advantages such as high sensitive, versatility, simplicity and robustness. The current registered as a result of the potential applied depends on the composition of the sample and the presence of electro-active molecules. Cyclic voltammetry is an effective and versatile electro analytical technique available for samples studying that is being increasingly used. Moreover, this electrochemical method has been used for classification of Gram-negative and Gram-positive intestinal bacteria and in complex media like milk for monitoring milk freshness. Different electrochemical reactions could occur depending on the material of the working electrode (WE). In this work, two planar WE were done and tested. The metal wires exposed as WEs had a diameter of 2 mm (Au) and 0.7 mm (Pt). The primary objective of our investigation is to develop a method to detect udder colonizing bacteria and contribute to veterinary diagnosis. Milk samples were made with rehydrated powdered skimmed milk (La Serenisima™, Buenos Aires, Arg.) with reverse osmosis distilled water, as indicated by manufacturer's instructions (100gL-1). The microbial cells used, in order to conduct preliminary experiments for electrochemical determination of bacteria in order to find a practical method to be used at dairy farms were rapid identification could be an important advantage over traditional (culture) microbiological methods. As a probe concept, two bacteria were used: Escherichia coli K12 (Gram-negative) and Bacillus cereus (Gram-positive). Microorganisms were growth in LB medium at 37 ºC. After centrifugation, the pellet was resuspended in rehydrated skimmed milk in order to simulate contaminated milk samples with an OD of 4.5 (± 0.1). Electrochemical experiments were performed with a standard three electrode systems using Au and a Pt working electrode. A saturated Ag/AgCl was used as reference electrode, and a stainless steel helicoidally electrode (CrNi; DIN: 1.4310; resistivity 0.43 mΩ cm-1, 1 mm diameter) was used as counter electrode (CE). Cyclic voltammetries were performed with a potentiostat (Gamry 300) under control of its own software. Measuring conditions were a scan rate of 10 mV/seg and potential window from -0.5 to 1 V. Direct observation of cyclic voltammetries shows mayor complexity (number of peaks) when Au WE was used. The anodic peak at ca. 680 mV is more important at Gram-positive bacteria. Catodic peaks at ca. 0 mV shows more complexity is Gram-positive voltammogram. The anodic peak at ca 300 mV looks similar at both bacteria. When the data was analyzed by principal component both bacteria and milk control samples were separated in different groups. These preliminary results demonstrate that practical use of this method could be possible. More experiments with increasing number of Gram-positive and -negative strains will be accomplished to demonstrate and validate the suggested method.