Preparation of pH-responsive chitosan microspheres containing aminopeptidase and their application in accelerating cheese ripening

Microencapsulated enzymes have been found to effectively accelerate cheese ripening. However, microencapsulated enzyme release is difficult to control, often resulting in enzyme release during cheese processing, and causing texture and flavor defects. This study aims to address this issue by developing aminopeptidase-loaded pH-responsive chitosan microspheres (A-CMs) for precise enzyme release during cheese ripening. An aminopeptidase with an isoelectric point (pH 5.4) close to the pH value of cheese ripening was loaded on chitosan microspheres through electrostatic interaction. Turbidity titration measurements revealed that pH 6.5 was optimal for binding aminopeptidase and microspheres, affording the highest loading efficiency of 58.16%. Various characterization techniques, including scanning electron microscopy, energy dispersive spectroscopy, and fourier-transform infrared spectroscopy confirmed the successful loading of aminopeptidase molecules on the chitosan microspheres. In vitro release experiments conducted during simulated cheese production demonstrated that aminopeptidase release from A-CMs was pH-responsive. The microspheres retained the enzyme during the coagulation and cheddaring processes (pH 5.5–6.5), and only released it after entering the cheese ripening stage (pH 5.0–5.5). By loading aminopeptidase on chitosan microspheres, the loss rate of the enzyme in cheese whey was reduced by approximately 79%. Furthermore, compared with cheese without aminopeptidase and cheese with aminopeptidase added directly, the cheeses made with A-CMs exhibited the highest proteolysis level and received superior sensory ratings for taste and smell. The content of key aroma substances, such as 2/3-methylbutanal and ethyl butyrate in cheese with A-CMs was more than 15 times higher than the others. This study provides an approach for accelerating cheese ripening through the use of microencapsulated enzymes.