Myoglobin (Mb)-loaded poly(ε-caprolactone) (PCL) microparticles were prepared by multiple emulsion with solvent extraction/evaporation method under more or less deleterious operating conditions. The protein integrity was monitored using both UV/Vis absorbance ratio method at specific wavelengths and a conductometric bi-enzymatic biosensor based on proteinase K and pronase. Under standard operating conditions, Mb remained in native conformation, while different degrees of protein denaturation were observed by changing the encapsulation conditions. It was shown that solvent elimination under reduced pressure and in a lower extent addition of a higher molecular weight PCL led to protein alteration. In the first case, the loss of protein integrity can be attributed to residual solvent entrapped in particles whose solidification was accelerated. In the second case, denaturation may be explained by an increase in the protein exposure time at water/organic solvent interface due to an increase in organic phase viscosity.
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