Discussion of Results:
1. Humidity Levels
As suspected, it was determined that the samples exposed to more humid
conditions had higher rates of enzyme activity. It was also determined that time
affected the levels of enzyme activity, as the amount of protease in the seven
day samples was significantly higher than that of the one day samples.
2. Inhibitors
The citric acid worked surprisingly well. It inhibited protease activity
significantly in almost every different rice sample. The known protease
inhibitors worked fairly well, although pepstatin and iodoacetamide were much
less effective in the inhibition of the enzymes in comparison to the others.
3. Protein Determination
It was possible to calculate the amount of sample to use in each well for the zymography gels. By comparing the level of protein in each sample to an established standard curve, the volume of each sample need to obtain 4.4 μg of protein in each well of the gel was found.
4. Zymography
It was possible to see some significant areas of protease in each of the gels. However, merit of performing this aspect of the project was found more in the understanding of its purpose in the study of protease qualities.
The applications of our discoveries are extensive. The greatest area of possibility lies in the industrial use of citric acid as an inhibitor of rice spoilage. The quality of citric acid that makes it superior for industrial use is the fact that it is food-grade or edible. Thus, it would be theoretically possible to develop some form of concentrated citric acid source which could be used in rice storage areas to prevent significant spoilage. Citric acid is also readily available and relatively inexpensive in comparison to the other known inhibitors, and would therefore be more practical for use in developing countries.
In taking this project further, many things could be redone or extended further. Although the inhibitor assay data was clear, several more trials with varying concentrations of citric acid could be conducted in an attempt to determine the most effective form. Further research could also be done surrounding the isolation and classification of the specific enzyme responsible for the degradation of rice. As a lack of sufficient available technology prevented us from observing the genetic properties of the enzyme, it was difficult to determine whether or not it had been previously isolated or studied.