Application of the Spectroelectrochemistry of the Resazurin/Resorufin System to the Determination of Milk Quality

In the dairy industry, the resazurin (RZ) test is used as a measure of microbial reduction capability, and thus to determine the quality and shelf life of milk. Milk is mixed with RZ solution and the fluorescence of the resulting resorufin (RS), or colour change from blue (RZ) to magenta (RS), is measured. The more bacteria present in the milk, the more the conversion of RZ to RS. The RZ test is therefore used to check the hygiene of milk production and the freshness and quality of the milk. The following groups of bacteria are primarily responsible for the reduction of resazurin in milk: 1. Lactic acid bacteria (LAB): Lactococcus, Lactobacillus and Streptococcus (e.g. S. thermophilus). These bacteria are often found in raw milk and starter cultures. They are facultative anaerobes with an active redox system (e.g. NADH/H⁺), which can transfer electrons to resazurin. 2. Enterobacteria Genera: Escherichia, Enterobacter and Klebsiella. These bacteria occur in poor hygiene conditions or in the presence of faecal contamination. They have high reduction activity and can decolorise resazurin very quickly. 3. Pseudomonads genus: Pseudomonas (e.g. P. fluorescens): common psychrotrophs in refrigerated milk. They exhibit high activity in redox metabolism and can rapidly reduce resazurin, particularly during prolonged storage. 4. Clostridia: Strictly anaerobic spore formers (e.g. Clostridium butyricum). They can reduce resazurin under anaerobic conditions, particularly in silage-contaminated milk. 5. Bacillus species: Aerobic spore formers that can also contribute to reduction through their metabolic activity. In the present work, the RZ/RS system has been studied by fluorescence and electrogenerated surface-enhanced Raman-Spectroelectrochemistry (EC-SERS) to determine the quality of milk. In addition to the analytical background of the RZ/RS system, fluorovoltammograms demonstrate how fluorescence intensity changes during electrochemical transitions between RZ and RS, providing an indication of which bacteria can reduce RZ. The electrochemistry and fluorescence of RZ and RS are influenced by physically dissolved O2 in water which occurs at a similar potential as the reduction of RZ to RS. In addition, oxygen partially quenches the fluorescence. When using the RZ method, the oxygen content of the milk must first be measured. Correction factors are used to calculate the quenching effect. In the EC-SERS Raman voltammograms, the Raman intensities change during the electrochemical formation of the nanostructured Ag electrode surface. During the corresponding chemical reactions between RZ and RS, the Raman transitions shift, which has been explained in detail for the Raman transitions between 500 and 600 cm-1. We show that the change in SERS Raman spectrum from RZ to RS can also be used to investigate the increasing bacterial contamination of milk. As we cannot determine the quantitative bacterial contamination, we are not able to compare the sensitivity of the two methods: Fluorescence and SERS Raman.