Adaptation of Lactobacillus rhamnosus riboflavin assay to microtiter plates

Riboflavin (vitamin B2) is essential to humans and must be obtained through the diet. It plays a significant role in the metabolism of carbohydrates, fatty acids and amino acids. The test microorganism, most commonly used to quantify riboflavin is Lactobacillus rhamnosus ATCC 7469 since this bacterium requires external B2 for growth. The objective of the current study was to reduce the time of the assay and volumes of assay media by adaptation to microtiter plates while still maintaining the repeatability of the original tube assay. A previously developed riboflavin tube assay was used as a guideline for adapting the method to a microtiter plate assay. The standard growth curve for the riboflavin assay was linear from 0 to 20 ng/mL (R2=0.99) and from 0 to 10 ng/mL (R2=0.97) when conducted in microtiter plates and tubes, respectively. The data showed no significant difference between the tube assay and microtiter plate assay (P>0.05) for the commercial maize sample. Commercial cereal and grain samples were analyzed to confirm repeatability among multiple independent trials performed with the microtiter plates. The microtiter assay reduced the amount of time required for sufficient bacterial growth response to generate linear standard curves from 16.5 to 10 h.