A spectroscopy-based proof-of-concept (POC) for developing loading of pathogen analyzer (LOPA) for dairy products

Detection of bacterial contamination in dairy products of daily use is a challenge worldwide. We have utilized Methylene Blue Reduction Test (MBRT) for quantification of the microbial count in dairy products (milk) and developed a proof-of-concept (POC) based on this for in-filed applications. In this study, we have used pasteurized milk contaminated with model bacteria <i>Escherichia coli</i>, and <i>Staphylococcus aureus</i> for the calibration and validation of the developed POC. The conversion of MB to Leuco-MB i.e., the colorimetric change due to the reduction of MB to Leuco-MB in presence of microbes has been utilized as the tool to detect presence of microbes in milk. The absorbance peak for methylene blue (MB) at 664 nm decreases significantly in presence of microbes and the blue color becomes faded. In our study, we have employed methylene blue (MB) discolouration phenomenon to estimate the microbial count in milk samples using our developed spectroscopy based POC. The limit of detection (LOD) and the limit of quantitation (LOQ) of the POC were found to be 0.32 CFU/mL and 0.97 CFU/mL. The end users of the developed POC are primarily those involved in the production, processing, testing, regulation, and research of dairy products to ensure they meet safety standards and protect public health. These include retailers, dairy farmers, dairy processors, quality control laboratories, regulatory agencies and research institutions. In our experiment, we have observed a significant change in MB absorption in the milk contaminated with microbes. The indigenously developed sensor strips designed for the working of the POC turn to colorless Leuco-MB compared to milk without the microbes. The analysis of the strips has been measured in the developed device.