Supplementary material

Microencapsulated enzymes have been found to ef- fectively accelerate cheese ripening. However, microen- capsulated 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 chito- san microspheres through electrostatic interaction. Tur- bidity 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 demon- strated 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.<br>

研究表明，微囊化酶（microencapsulated enzymes）可有效加速奶酪成熟（cheese ripening）。然而，微囊化酶的释放难以精准调控，常于奶酪加工过程中提前释放酶，进而引发产品质地与风味缺陷。本研究旨在解决这一问题，开发负载氨肽酶（aminopeptidase）的pH响应型壳聚糖微球（pH-responsive chitosan microspheres, A-CMs），以实现奶酪成熟阶段的酶精准释放。选取等电点（isoelectric point）为pH 5.4、与奶酪成熟体系pH值相近的氨肽酶，通过静电相互作用（electrostatic interaction）将其负载至壳聚糖微球表面。浊度滴定测定（turbidity titration measurements）结果表明，pH 6.5为酶与微球结合的最优条件，此时可获得最高58.16%的负载率（loading efficiency）。采用扫描电子显微镜（scanning electron microscopy）、能量色散光谱（energy dispersive spectroscopy）与傅里叶变换红外光谱（fourier-transform infrared spectroscopy）等多种表征技术，证实氨肽酶已成功负载于壳聚糖微球之上。模拟奶酪生产流程开展的体外释放实验（in vitro release experiments）显示，A-CMs所载氨肽酶的释放具有pH响应特性：在凝乳与切达干酪加工阶段（pH 5.5~6.5），微球可留存酶活性，仅当进入奶酪成熟阶段（pH 5.0~5.5）后才释放酶。通过将氨肽酶负载于壳聚糖微球，奶酪乳清（cheese whey）中的酶损失率降低了约79%。此外，与未添加氨肽酶的奶酪及直接添加游离氨肽酶的奶酪相比，采用A-CMs制备的奶酪展现出最高的蛋白水解（proteolysis）水平，且在味觉与嗅觉维度获得了更优异的感官评分（sensory ratings）。其关键香气物质如2/3-甲基丁醛（2/3-methylbutanal）与丁酸乙酯（ethyl butyrate）的含量较其余两组高出15倍以上。本研究为利用微囊化酶加速奶酪成熟提供了可行策略。