Trehalose Extraction and Quantification

A medium containing 0.5 g of casamino acid hydrolysate, 0.5 g of yeast extract, and 0.5 g of peptone per liter was developed to produce trehalose (Difco™ R2A agar, Fisher scientific). The carbon source to produce trehalose in this medium was added as 2% glucose. The culture cell pellets were boiled at 95°C for 20 min to remove the trehalose from the pellets. After centrifuging the resulting mixes, the supernatant was collected. By continuing to boil the supernatant at 95°C until it lost half of its volume, the supernatant containing the extracted trehalose was concentrated. Furthermore, the trehalose extract sample was mixed with 495 mL of 50 mM sodium citrate buffer (pH 6.0) to create a total volume of 1,000 mL for the trehalose assay. Purified trehalase (Megazyme), with an enzyme concentration of 0.0009 UmL⁻¹, was added in a volume of 5 μL to start the reaction. The 3,5-dinitro salicylic acid colorimetric method (DNS method) (Miller et al., 1960 (link)) was used to evaluate the degradation of trehalose to glucose molecules. In this procedure, 1,000 μL of the reacted sample and 300 μL of the DNS solution were mixed and boiled for 5 min. The mixture was allowed to cool at room temperature, and the absorbance was measured at 540 nm using a spectrophotometer. Subsequently, by measuring the absorbance of a standard solution that contained 20 nmol of glucose instead of trehalose, glucose production was ascertained. A control sample devoid of trehalose was used in the measurement to gauge the level of pre-existing glucose in the tested sample. Additionally, a blank sample without trehalose extract was included as a reference.