Detection of ethanol released from aged radish (Raphanus sativus L.) seeds using resazurin

Abstract

The release of volatiles from aged radish (Raphanus sativus L., var. Yechanmoo) seeds and from ethanol was evaluated using the resazurin reagent (RR, solution of resazurin and yeast mixture). The primary volatile released from the aged radish seeds was identified as ethanol, but no volatiles from non-aged seeds were detected by solid phase microextraction (SPME) followed by GC-MS analysis and ethanol standard confirmation. The volatiles from aged radish seeds and from ethanol changed the color of the RR from blue to pink after 8–10 hours of incubation at 35°C, and the absorbance of the RR was read at 570 nm. When ethanol was diluted with the RR at concentrations ranging from 0 to 10,000 ppm, the absorbance and ethanol concentrations were found to be negatively correlated in the linear regression analysis with r² = 0.91. For the seed test, the aged and non-aged radish seeds were soaked individually in the RR using a 96-well plate and incubated at 35°C for 4 hours. The absorbance of the RR soaked non-aged seeds did not change; however, the absorbance of the aged seeds rapidly decreased with incubation time. Also, the visual color of the RR soaked aged seeds visibly changed from blue to pink after two hour of incubation and was colorless after four hours of incubation. The color changes of the RR could be attributed to the fact that ethanol accelerated respiration of the yeast and resorufin was produced through the reduction of resazurin. In conclusion, RR was shown to be a highly sensitive reagent for the detection of volatiles (mainly ethanol) released from aged radish seeds and could be used to simply, rapidly and nondestructively classify the viable and non-viable seeds.