Our proof of concept experiment consisted of experimentally determining extent of phytate breakdown in dough prepared using a millets, which have a high phytate content.
We used the method described by Heinonen and Lahti (Anal Biochem 1981 May 15;113(2):313-7. doi: 10.1016/0003-2697(81)90082-8) to determine phytate breakdown. This method is used to estimate free inorganic phosphate, which is released when phytate is broken down. Unlike the well-known Fiske-Subbarow method of inorganic phosphate determination, phytate does not cause interference in the method of Heinonen and Lahti.
We constructed a standard curve using KH2PO4 to establish the relationship between Pi and absorbance at 390 nm.
Using the equation determined from the above experiment, we determined the free Pi content of sorghum (locally known as jowar, a staple diet in rural areas of Maharashtra and Telangana states) and refined wheat flour.
We then proceeded to ferment dough using our constructed yeast strains to determine the extent of Pi liberated.
Dough was prepared using one part each of sorghum and refined wheat flour. The yeast strains were used to ferment the flour for four hours. Samples of the rising dough were taken and concentration of free Pi in the dough was determined.
As can be seen from the above table, dough fermented by strains expressing Aj and Ec phytases had 10 and 23 percent more Pi as compared to dough fermented by the control strain (P=0.005). This demonstrates that phytase breakdown was significantly higher when phytase expressing strains are used to ferment the dough.