On-the-spot trace lactose test for flavored and unflavored lactose-free dairy beverages

We previously reported a novel Bioluminescence Resonance Energy Transfer (BRET)-based biosensing construct able to estimate trace levels of lactose (>0.01% (w/v)), directly in lactose-modified white milk, without the need for sample pre-treatment. In that study, the method was performed by highly trained researchers using a BRETcapable microplate reader in a research laboratory. It is desirable for the method to be adapted for on-the-spot use by production staff in a very different setting, the quality control lab of a milk processing factory. In such a setting, there is a growing requirement to produce and quality-control lactose-modified milks that are colored and flavored. Measurement of trace lactose in these matrices presents additional challenges over white milk because colors, flavors and added sugars potentially interfere with existing electrochemical or colorimetric biosensors. In the current work we therefore describe the development and validation of a method that uses the aforementioned BRET-based lactose biosensing construct to measure trace lactose in white, colored and flavored milks using a dedicated user-friendly device. Milk specific calibrations allowed the estimation of lactose at concentrations ranging from approximate to 7% (w/v) "full-lactose" to 0.01% (w/v), which is the most stringent quantified "lactose-free" level currently mandated globally. Limits of detection were 0.004% (w/v) for white milk and 0.003% (w/v) for both strawberry- and coffee-flavored milks. The assay could be performed in less than 5 min. We demonstrated the use of this assay to follow, effectively in real-time, simulated commercial lactase hydrolyses in white, strawberry- and coffee-flavored milks where the milk was aseptically incubated with food-grade lactase enzyme at 4 degrees C. These experiments highlighted the potential value of such a point-of-need lactose test for monitoring production, expediting release of finished product and accelerating new product development of a range of lactose-modified beverages.