Profiling Hydrolysis Rates of αS1‐Casein to αS1‐I‐Casein During Cheese Ripening by Intact‐Protein Liquid Chromatography–High Resolution Mass Spectrometry
Sebastiaan Dolman, Marjolein van der Eijk, Loes Bevers, Kees Muijlwijk, Mee Loke Lim, Margriet Hendriks, Nicolas Abello, Rob van der HoevenABSTRACT
Rationale
The proteolytic behavior of chymosins during cheese ripening impacts cheese texture, flavor, and quality. Especially, the specific hydrolysis of αS1‐casein to αS1‐I‐casein is relevant for early cheese texture development, and this activity varies between chymosin variants. Accurate profiling of αS1‐casein and αS1‐I‐casein during cheese ripening is crucial for understanding these differences between chymosins and the impact on cheese quality.
Methods
Intact‐protein liquid chromatography–high‐resolution mass spectrometry (LC‐HRMS) was employed to monitor the hydrolysis of αS1‐casein to αS1‐I‐casein during mozzarella cheese ripening. Cheese samples, produced with both bovine and camel chymosin variants, were collected at multiple ripening stages. Protein extraction, separation, and quantification were performed using optimized LC‐HRMS protocols to assess hydrolysis kinetics and phosphorylation variants.
Results
LC‐HRMS analysis revealed that the tested bovine chymosins displayed significantly higher rates of αS1‐casein hydrolysis to αS1‐I‐casein compared to camel chymosins throughout mozzarella cheese ripening. Distinct phosphorylation variants were confidently detected and quantified. Kinetic profiles demonstrated consistent proteolytic activity differences between coagulants.
Conclusions
LC‐HRMS allows tracking of αS1‐casein hydrolysis kinetics throughout mozzarella cheese ripening. This approach not only highlights the pronounced and faster proteolytic activity of bovine chymosins compared to camel chymosins, resulting in more rapid αS1‐I‐casein formation, but also facilitates the monitoring of phosphorylation variants present in the cheese. These findings emphasize the enzyme‐specific effects on cheese texture and quality, demonstrating the importance of advanced protein analysis for comprehensively understanding cheese production and maturation processes.