The first step in K-YBGL procedure is solubilisation/pre-hydrolysis in concentrated sulphuric acid (12M), therefore a sample should be in a powder form. We recommend freeze-drying of liquid samples prior to the actual analysis.
However if the sample is completely dissolved (clear solution) it can be analysed as is. First the sample is heated in a boiling water bath for 5 min to ensure complete dissolution of carbohydrates. The total carbohydrate content is determined, e.g. by using phenol-sulphuric method. Equivalent of ~ 100 mg of carbohydrate is mixed with 12 M sulphuric acid and distilled water to a final concentration of 2M sulphuric acid (e.g. 2 mL sample at approximately 46 mg of carbohydrates/mL+ 2 mL of 12 M sulphuric acid + 8 mL of water) and incubated in a boiling water bath for 2 hours. After the incubation the K-YBGL procedure is followed (KOH addition, volume adjustment, incubation with exo-1,3-β-glucanase/β-glucosidase and glucose determination with GOPOD Reagent).
The α-glucan (starch and glycogen) in such samples could be completely hydrolysed with just AMG/invertase incubation. Please ensure the pH of sample is approximately 4.5-5 before addition of enzyme mixture. The same volume should be used as in total glucan determination and the volume after the enzyme hydrolysis should be adjusted to 100 mL before glucose determination with GOPOD Reagent.
MegaCalc can be used to simplify the calculations; sample weight should be taken from carbohydrates concentration (mg/mL) and volume of sample used for the assay (e.g. 2 mL at 46 mg/mL- sample weight – 96 mg). Please note that total glucan, α-glucan and β-glucan in liquid samples will be expressed as g/100g of carbohydrate present in the sample. This can be converted to mg/mL since the volume used in the assay is known.