5:00 PM - 5:15 PM
[2Dp-09] Development of Novel Colorimetric Assay Kits for Sucrose and Lactose Using Enzymatic Method
Keywords:sucrose, lactose, enzymatic method, colorimetric determination, assay kit
[Purpose] Sucrose or lactose is commonly measured by HPLC, but the HPLC method requires complicated pretreatment, and is not suitable for quantifying one type of free sugar in many samples. In this study, we aimed to develop a simple colorimetric measurement that can directly quantify sucrose or lactose at room temperature.
[Method] Sucrose or lactose kit which is composed of two types of solution, A and B, is designed to measure the end point of the color reaction. At first, the sample was reacted at room temperature with Solution A for 10 minutes to remove the coexisted glucose and ascorbic acid. By adding Solution B subsequently, sucrose is hydrolyzed by invertase, and lactose is hydrolyzed by β-galactosidase respectively producing glucose. The produced glucose is rapidly oxidized to gluconic acid and hydrogen peroxide by glucose oxidase in the presence of mutarotase. As the result of the peroxidase-mediated coupling reaction of the coupler compound and Trinder's reagent, the equimolar hydrogen peroxide produced from sucrose or lactose can be measured colorimetrically. We also measured sucrose or lactose in foods and drinks using the new kits and conventional kits to compare the results.
[Results] Both of the kit for sucrose and lactose can determine their target compound in the range of 10 to 600 mg/L, and the color reactions completed within 10 minutes after the addition of Solution B at room temperature. The kits showed high substrate specificity and are stable for 8 weeks at 28°C. The comparison results of new kits and conventional kits showed high correlate coefficients. This indicates that our new kits are reliable.
[Method] Sucrose or lactose kit which is composed of two types of solution, A and B, is designed to measure the end point of the color reaction. At first, the sample was reacted at room temperature with Solution A for 10 minutes to remove the coexisted glucose and ascorbic acid. By adding Solution B subsequently, sucrose is hydrolyzed by invertase, and lactose is hydrolyzed by β-galactosidase respectively producing glucose. The produced glucose is rapidly oxidized to gluconic acid and hydrogen peroxide by glucose oxidase in the presence of mutarotase. As the result of the peroxidase-mediated coupling reaction of the coupler compound and Trinder's reagent, the equimolar hydrogen peroxide produced from sucrose or lactose can be measured colorimetrically. We also measured sucrose or lactose in foods and drinks using the new kits and conventional kits to compare the results.
[Results] Both of the kit for sucrose and lactose can determine their target compound in the range of 10 to 600 mg/L, and the color reactions completed within 10 minutes after the addition of Solution B at room temperature. The kits showed high substrate specificity and are stable for 8 weeks at 28°C. The comparison results of new kits and conventional kits showed high correlate coefficients. This indicates that our new kits are reliable.