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，微囊化酶可有效加速干酪成熟过程。然而，微囊化酶的释放难以精准调控，常导致干酪加工阶段即发生酶释放，进而引发干酪质地与风味缺陷。本研究旨在解决上述问题，开发负载氨肽酶（aminopeptidase）的pH响应型壳聚糖微球（A-CMs），以实现干酪成熟阶段的酶精准释放。本研究选取等电点为pH 5.4（与干酪成熟的pH环境相近）的氨肽酶，通过静电相互作用将其负载于壳聚糖微球之上。浊度滴定检测结果显示，pH 6.5为酶与微球结合的最优条件，此时负载效率可达58.16%的峰值。多种表征技术，包括扫描电子显微镜（scanning electron microscopy）、能量色散X射线能谱（energy dispersive spectroscopy）及傅里叶变换红外光谱（fourier-transform infrared spectroscopy），均证实氨肽酶分子已成功负载于壳聚糖微球表面。模拟干酪生产过程的体外释放实验表明，A-CMs负载的氨肽酶释放行为具有pH响应性：该微球可在干酪凝乳与切达工序（pH 5.5~6.5）阶段保留酶活性，仅在进入干酪成熟阶段（pH 5.0~5.5）后才释放酶类。通过将氨肽酶负载于壳聚糖微球，干酪乳清中的酶流失率降低了约79%。此外，与未添加氨肽酶的干酪及直接添加游离氨肽酶的干酪相比，采用A-CMs制备的干酪具有最高的蛋白水解水平，且在口感与风味感官评分上表现更优。A-CMs组干酪中的关键香气物质（如2/3-甲基丁醛与丁酸乙酯）含量较其他两组高出15倍以上。本研究为利用微囊化酶加速干酪成熟提供了可行方案。