30% glycerol by zein weight BSA/Zein filmdissolution in 95% ethanol and turgidity in water40% glycerol by zein weight BSA/Zein film
There was negligible difference between the "40%" and "30%" glycerol films in terms of their strength. The decision was then made to try to incorporate some thickening food additives to make the films a bit more robust. That procedure appears below.
Another brief test made was to determine the effect of placing the film in deionized water. Scrapings from the film were placed in small flasks with deionized water, water of pH 9 and water of pH 5. The pH seemed to have no significant effect, but the water did not break down the film but rather distended it, causing it to become pale and thicker. This was to be suspected because the zein protein is precipitated in water. Placing the films in ethanol, the solvent into which the zein was dissolved, caused the films to very quickly break down into solution. The non-BSA film was not altogether different physically, though it was a bit thinner because it had less volume due to the absence of 500 μL of water with BSA. The role of BSA on its own is still questionable without the gold nanoparticles proposed in the previously referenced paper by Bakshi et al.
Method: Incorporating Food Additives
The additives considered in this procedure were chosen based on availability in the lab. They include xanthan gum, guar gum, and agar. The amount of additive to use was decided upon rather arbitrarily and turned our to be a bit too high. The zein solutions for three separate samples were: 0.60g zein, 5.40mL 95% ethanol, 240μL glycerol and 0.06g of x, where x = xanthan gum, guar gum or agar. The solutions with just ethanol and zein were agitated for 30 minutes with a stir bar, then the additives (as powder) were added to their respective solutions. They were stirred vigorously and then glycerol was added. Finally, the BSA in water solution was added (it had not been heated for long, but only warmed to 60°C in a few minutes). The samples had to be mixed extensively because the water made globs of zein precipitate out. When the solutions, now really suspensions, had been mixed as well as possible, they were poured into plates that were left uncovered in the lab to dry overnight at room temperature (between 25 and 27°C here). The soundness of the design that includes leaving the plates at room temperature and having the additives in suspension is questionable, but the results will be interesting. In the future, we would like to add less of the additives so that the solution is homogenous. As well, we will see what room temperature incubation does for the films outside the hood.
Protein Biofilms Exploration
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