黑曲霉柠檬苦素脱苦酶组分解析、作用机理及其脱苦柚子果汁的动力学

Pummelo is an important citrus fruit of great economic value. Its juice tastes bitter, which is hard to remove, restraining the commercial practice of processing this fruit. It has been reported in previous studies that naringin and limonin are two major bitter compounds in pummelo juice, and the extracellular enzymes from Aspergillus niger can effectively debitter pummelo juice. Some researchers have reported that Aspergillus niger excretes naringinase which is responsible to debitter naringin, but the enzyme constituent and the mechanism for transforming limonin are not clarified yet. The proposal is planned to purify the key enzyme constituent for transforming limonin from the fermentation broth of Aspergillus niger, followed by further in-depth investigation of the chemical and enzymatic mechanism. The reaction products which are going to be purified by solvent extraction, chromatographic separation and recrystallization, will be structurally identified by HPLC-DAD-MS and NMR. Meanwhile,its bitter taste is going to be characterized according to the food chemical methods. This experiment helps to reveal the structural change in limonin caused by the enzyme reaction,and the debittering effect in relation to the limonin structure change, illustrate the active constituent of the limonin-debittering enzyme. Furthermore, its enzymatic properties will be analyzed, and the enzyme structure and the active site will be identified by HPLC-MS-MS, X-ray diffraction and bioinformatic methods. Moreover, the purified enzyme constituent will applied to transform the limonin in pummelo juice, which allows the construction of the debittering kinetics. Through this experiment, the active constituent of the limonin-debittering enzyme will be clarified, and the structure-function relationship of the enzyme will be charaterized. In addition, the debittering kinetics will be revealed. The research results are expected to facilitate the in-depth processing of pummelo fruit, which has important significance and application value.

柚子是具有重要经济价值的柑橘类水果，其果汁苦味难以去除是制约柚子深加工的关键技术难点。已有研究发现，柚子汁含有柚皮苷和柠檬苦素两种关键苦味成分，黑曲霉分泌的胞外酶可有效脱苦柚子汁；尽管相关研究已经明确了黑曲霉柚苷酶的组分构成及其对柚皮苷的脱苦作用机理，但其柠檬苦素脱苦酶的组分及作用机理尚不明确。本项目拟从黑曲霉发酵液中纯化对柠檬苦素具有转化作用的酶组分；制备酶组分的反应产物，鉴定其化学结构及苦味，分析酶反应引起的化学成分变化及由成分变化引起的脱苦作用，解析该柠檬苦素脱苦酶的组分构成及各组分的功能；测定酶组分的结构及酶学性质，鉴定活性中心，解析其作用机理；测定酶组分对柚子果汁中柠檬苦素的转化速度，解析其脱苦柚子果汁的动力学。通过本研究，将明确该柠檬苦素脱苦酶的组分构成，阐明其结构与功能性质的关系，并建立其脱苦柚子汁的动力学模型，为柚子深加工奠定理论基础，具有十分重要的科学意义。

针对前期发现黑曲霉发酵粗酶液具有去除蜜柚果汁苦味的活性，但相关机理尚不明确，本项目从柠檬苦素去除活性组分分离纯化、作用产物结构鉴定、关键组分结构特征分析及去除柠檬苦素机理等方面开展研究。实验结果显示，黑曲霉胞外酶液中的硫酸铵具有显著的柠檬苦素沉淀效果，而大分子物质却没有降低柠檬苦素的作用；沉淀的柠檬苦素与果汁中存在的柠檬苦素都是闭环柠檬苦素，它们的结构和苦味特征相同；用硫酸铵处理果汁后苦味程度降低是由于柠檬苦素沉淀析出，从而降低了果汁中柠檬苦素含量，但并没有改变柠檬苦素的化学结构和苦味特征；硫酸根对柠檬苦素的沉淀作用很强，而铵根、氢离子及其他离子对柠檬苦素的沉淀能力很弱，硫酸根是去除柠檬苦素活性组分的关键结构特征；硫酸根沉淀柠檬苦素的机理是破坏柠檬苦素与水、果胶的结合，从而使柠檬苦素沉淀析出。本研究首次发现了硫酸根具有很强的柠檬苦素沉淀能力，可以高效沉淀果汁及水提液中的柠檬苦素，不仅揭示了一种全新的柠檬苦素脱除理论，还为设计绿色柠檬苦素吸附材料，开发新型、绿色、安全方法，高效去除柑橘类果汁柠檬苦素及回收柑橘加工副产物中柠檬苦素提供了新途径，具有重要理论和应用价值。.项目开展国际合作交流2人次，国内合作交流4人次，省内合作交流3人次；培养研究生5名，发表SCI论文5篇（其中农业大类一区论文3篇），中文核心论文6篇，授权发明专利1项。部分成果已经实现产业化应用。.综上，项目完成了各项研究内容，并达到了“发表科研论文6-8篇，其中SCI论文4篇，申报国家发明专利1项，培养研究生3-5名”的预期成果指标。