# Improved Food-Processing Techniques to Reduce Isoflavones in Soy-Based Foodstuffs
## 4 Discussion
### Comparison of ELISA and Folin-Ciocalteu Methods  
As already mentioned, comparing the ELISA and the Folin-Ciocalteu assay makes sense given the time involved, including the extraction steps, in each case. Thus, after sample collection, the latter technique gives a result in a few hours, while the ELISA requires at least three whole days and one night.  
The results of the correlation study showed that the Folin-Ciocalteu method was not able to assess the reduction of IFs in soy matter. The reason is undoubtedly linked to the presence of many insoluble polyphenols in soy products, and to the fact that the amount of water fixed on the material changes with the particle size (more water fixed on the smaller particles). The main discrepancy observed between the two methods is based on differences in their specificity. Indeed, the ELISA used in this study targets specifically GEN or DAI [38; 39] and the extraction process triggers the water-soluble fraction, whereas the Folin-Ciocalteu method recognizes all polyphenols based on the chemical reaction of phenol rings with the assay reagents. The extraction performed prior to the analysis also includes non-water-soluble compounds. However, soybeans contain many phenolic compounds, some of which are soluble in water, some of which are not. Indeed, insoluble tannins, saponins, and also proteins, amino-acids, sugars and vitamins, among other food components, were reported to be detected with a Folin-Ciocalteu assay [45]. Thus, it was not really surprising to observe strong discrepancies between the two assay techniques.  
On the contrary, the very good correlation between the ELISA and Folin-Ciocalteu results on water analysis was crucial, as it confirmed that the Folin-Ciocalteu method, by measuring water-soluble phenolic compounds, allowed an indirect evaluation of the total IF content in the treatment water samples. As the IF content in these waters is correlated with that in the corresponding treated soybeans, the Folin-Ciocalteu method seems adequate to simply and quickly evaluate the effectiveness of the IF reduction process.  
Interestingly, and confirming this result, the correlation between the reduced concentration of IFs in dehulled beans found by ELISA and the amounts of polyphenols in treatment waters determined by the Folin-Ciocalteu assays was significant. A plausible explanation could be that after centrifugation, the water only contains soluble polyphenols, and so potential interference is reduced. Consequently, the Folin-Ciocalteu method could be advantageously used to quickly compare the efficiency of water treatments of different processes designed for IF removal.  
It is important to acknowledge here that there is also a sensitivity difference between the two methods. The ELISA's sensitivity is 1 ng per well, and the detection limit is 0.48 ng per well. The 50% competition points vary from 8 to 50 ng/mL, depending on the assays. The Folin-Ciocalteu standard curve linearity lies between 35 and 250 ug/mL of gallic acid. The detection limit is 0.863 ug/mL, and the sensitivity is 0.0073 OD/ug/mL. These data are given in equivalent gallic acid.