水溶性壳聚糖/没食子酸衍生物对鲜切果蔬活性氧自由基的清除及机理研究

The demand of fresh-cut fruits and vegetables is increasing as the improvement of people’s living standards. However, the mechanical injury of fresh-cut fruits and vegetables in the cutting process will cause accumulation of active oxygen radicals. It will accelerate the occurrence of browning and affect the shelf life and quality of product ultimately. Therefore, how to scavenge active oxygen radicals generated by the cutting of fresh-cut fruits and vegetables become to a key problem in the field of preservation of fresh-cut fruits and vegetables. At present, the synthetic antioxidants are used to scavenge active oxygen radicals of fresh-cut fruits and vegetables,but it would pose a threat to food safety of fruits and vegetables. A novel chitosan/gallic acid derivative is prepared by the simple conjugating reaction of chitosan and gallic acid with the catalyzing of enzyme. The study also demonstrates the active oxygen radicals scavenging capability and the effect on fruits and vegetables’ antioxidant defense system of the derivative.The products of chitosan/gallic acid derivative deoxidized active oxygen radicals are separated, purified and identified via TLC and HPLC-MS. On this basis, the structure-activity relationships of chitosan/gallic acid derivative for scavenging active oxygen radicals and its mechanism are researched. The study has magnificent meaning to improvement of new and natural preservative for fresh-cut fruits and vegetables.

随着人们生活水平的提高，鲜切果蔬产品的需求量日益增加。但是新鲜果蔬在切割过程中产生的机械伤会引起活性氧自由基的积累，加速褐变的发生，最终影响产品的货架期及食用品质。因此，如何清除鲜切果蔬切割后产生的活性氧自由基成为鲜切果蔬保鲜领域的一个关键问题。目前常用化学合成抗氧化剂来清除鲜切果蔬产生的活性氧自由基，但是会对果蔬的食用安全造成威胁。本项目以酶法催化壳聚糖与没食子酸的共价结合，通过调整酶催化反应条件，筛选出合成效率较高、水溶性较好的壳聚糖/没食子酸衍生物；并研究该衍生物对鲜切果蔬活性氧自由基的清除作用及对果蔬自身抗氧化防御系统的影响；在此基础上通过薄层层析、高效液相色谱/质谱联用等技术分离、纯化、鉴定衍生物还原活性氧自由基的产物，进一步对壳聚糖/没食子酸衍生物清除活性氧自由基的机理及构效关系进行了研究。本项目对于开发新型、天然的鲜切果蔬保鲜剂具有重要意义。

目的：合成新型壳聚糖/没食子酸衍生物（CTS-GA），对衍生物的结构进行研究，确定壳聚糖与没食子酸的结合方式和作用基团，探究其对体外自由基的清除作用，并研究衍生物对鲜切苹果的保鲜效果。方法：利用漆酶、辣根过氧化物酶在均相和非均相反应体系中催化壳聚糖与没食子酸反应，采用正交试验确定最佳反应条件；综合运用紫外光谱、红外光谱、X-射线衍射、核磁共振等技术对合成得到的衍生物结构进行鉴定；通过对鲜切苹果生理指标的测定评价其保鲜效果。结果：采用均相方法可以制备得到接枝率与水溶性较高的衍生物，最佳合成条件为：温度25℃，漆酶用量4U，时间5h，CTS:GA=1:3；没食子酸分子中-COOH与壳聚糖分子中-NH2通过酰胺键形式成功接枝；壳聚糖/没食子酸衍生物可明显降低EC50值，具备较强的抗氧化活性，抗氧化效果优于没食子酸。初步断定壳聚糖/没食子酸衍生物通过断裂分子主链，暴露更多活性基团来达到的抗氧化能力。同时细胞毒理学实验表明，采用没食子酸对壳聚糖改性，并没有增加其细胞毒性，在100μg/mL-1000μg/mL浓度范围内，CTS-GA细胞毒性为1级，属安全等级。将CTS-GA应用于鲜切苹果中，能维持较高的硬度及可溶性固形物含量，能有效抑制苹果中VC、酚类物质及还原型谷胱甘肽的氧化，亦能抑制多酚氧化酶活性，延缓褐变，同时能提高超氧化物歧化酶、过氧化氢酶等防御系统酶的活性，保持鲜切苹果品质，延长货架期。