高热致米谷蛋白聚集的分子机制及曲霉联合解聚研究

Heat treatment is often applied in food processing,but rice protein treated by heat is easy to highly aggregate,and futher to decrease its function, quality and digestibility, so aggregation is the bottleneck problem affecting research and application on it.Formation mechanism of rice protein aggregation has not been reported. In the project, heat aggregate of rice gluten is designed as the core, the gluten is purified from rice by its dissolution properties, and then its sturcture information such as subunits, structure, surface charge, hydrophobicity and disulfide bond is analysized systematically, and futher its subunits is also purified and stucture of the subunits are analysed.A single subunit system and different subunit combination are treated under the condition of high temperature with high humidity, and then molecular mechanism of rice gluten aggregate by high heat is clarified through dynamic analysis of its structure change in the process of treatment.Finally, with rice gluten aggregate as the only source of protein and aspergillus species in food security level, such as aspergillus oryzae, aspergillus niger, monascus and so on, degradation characteristics of gluten aggregate are analyzed in the conditions of single strain cultivation and multi-strain cultivation, which aims at exploring the law of the protein aggregate degradation and screens out aspergillus combination system and function conditions for aggregate degradation efficiently, and can provide a theoretical reference for basic and applied basic research of rice protein aggregation.

食品加工中常需热处理，大米蛋白热处理易形成高度聚集体，使蛋白质功能、品质及可消化性明显降低，因此聚集是影响大米蛋白研究和应用的关键问题。大米蛋白聚集体形成机理尚未见报道。本项目以大米谷蛋白热聚集为核心问题，首先主要利用溶解特性纯化获得谷蛋白，并对其亚基组成、空间结构、表面电荷、疏水性、二硫键等结构信息进行系统分析，然后对各亚基进行纯化和结构分析；利用高温湿热分别处理单一亚基体系和不同亚基组合体系，通过对处理过程结构变化的动态分析，阐明米谷蛋白分子高热聚集的分子机制；最后以大米蛋白热变性高度聚集体为唯一蛋白质来源，以米曲霉、黑曲霉、红曲霉等食品安全级曲霉为菌种，分析各单一菌种培养和多菌种耦合培养条件下蛋白聚集体的降解特性，探索聚集体降解规律，筛选出高效降解聚集体的曲霉组合体系和作用条件，为大米蛋白聚集体的基础和应用基础研究提供理论参考。

大米谷蛋白（RG）受热易聚集而更加难溶，限制了其应用和研究。本项目以RG热致聚集机理作为关键问题，并以大米谷蛋白聚集体（RGA）为主要氮源，以RGA酱油作为蛋白降解的载体，探讨几种曲霉及鲁氏酵母、植物乳杆菌等对RGA的作用规律，对阐明RG聚集机理、拓宽其应用范围、提高RGA的生物利用率和功能具有重要意义。主要研究内容、结果和关键数据如下：.（1）利用碱溶酸沉法得到纯度约95%的RG，亚基分析表明其含三个亚基，分子量分别约为16、25、38kDa。RG碱溶液亦属于不稳定体系（pH11.0），Zeta电位为-14～-16mV，溶解度14.2%，表面疏水性指数为330，游离巯基含量约为28.0 μmol/g，并且盐离子、温度、加热时间等均与疏水性有明显正相关。.（2）RG热致聚集机理。热处理导致RG可解离亚基明显减少、游离巯基数量减少，高热处理游离巯基降低更为明显；受热后，α-helix明显减少，β-sheet明显增加，高热会加剧这种变化。碱法脱酰胺的RG在中性条件下有理想的水溶性，脱酰胺度可达60%以上，溶解度达到80%以上。综合多种研究方法和结果表明，RG的热致聚集为无定形聚集，其分子机制为RG分子之间二硫键增加、疏水作用力增强和静电斥力减小。ThT荧光法和TEM法分析表明RG在酸性条件下可发生纤维化自组装聚集。.（3）RGA浓缩产品—米渣的微生物降解。三种红曲霉中M. anka产蛋白酶能力最强，得到电泳纯的M. anka酸性蛋白酶。米曲霉对RGA的降解能力较强，总氮转化率约为63～65%。黑曲霉作用30d后总氮转化率为42.7%。米曲霉耦合其它菌种发酵有利于提高RGA降解效率，其中耦合植物乳杆菌降解效率最高，游离氨基酸总量达5600 mg/100 mL。.（4）RGA降解产物的抗氧化活性和风味。在一定温度范围内，高温有利于抗氧化活性成分的积累，米曲霉、黑曲霉、鲁氏酵母耦合发酵降解产物具有很强的抗氧化活性，清除羟自由基（•OH）的能力达到等质量浓度Vc的175倍，对DPPH自由基清除能力达到Vc 的2倍以上，米曲霉对RGA的降解具有重要作用，而鲁氏酵母对抗氧化活性的形成有明显作用，鲁氏酵母和植物乳杆菌对降解产物的风味作用明显。