Starch granule oxidation evaluation using laser confocal microscope-Raman spectroscopy: A novel analytical method

The total degree of oxidation (TDO) of oxidized starch for food applications is typically evaluated by determining the sum of the carboxyl and carbonyl contents using titrimetric methods. However, these methods are often time-consuming, complex and do not differentiate between intra- and inter-granule oxidation, limiting in-depth studies on oxidized starch. Herein, by extracting intensity values at 478 and 1401 cm⁻¹ in the starch Raman spectra, we reported a method based on laser confocal microscope-Raman spectroscopy (LCM-Raman) to rapidly and non-destructively analyze the oxidation level of starch. Using R_1401/478 as a color mapping factor, the oxidation level inside and outside the starch granules was visualized by the map-scanning of LCM-Raman, and a highly significant correlation between the average R_1401/478 and the TDO determined by titration was observed (R² = 0.99327, p < 0.0001). The error of this method for detecting the TDOs of commercial oxidized starches was within 3 % when compared to the titration method, indicating a good accuracy in determining the starch oxidation level. In addition, the oxidation homogeneity between starch granules of different sizes was assessed using the line-scanning of LCM-Raman. In summary, these findings provided a practical tool to study structural changes in starch (related to CO bond) at the granule scale.