柑橘果胶低聚糖抗真菌活性及其构效关系研究

Owing to their physiological activities including bifidus factor, anti-tumor and antioxidant, pectic oligosaccharides (POS) have been demonstrated as ideal functional compounds which are of high research value and application potential. Our preliminary study found the pectic oligosaccharides, enzymolyzed by pectinases, have obvious inhibitory effects on the growth of pathogenic fungi leading to rot of the stored citrus fruits. Moreover, our results suggested that the inhibitory effects of pectic oligosaccharides have much to do with their special structural characteristics. Thus, the illumination of structure-effect relationship could be helpful to revelation of the mechanism of pectic oligosaccharides’ antifungal activity. In order to learn the antifungal mechanism of obtained pectic oligosaccharides, this project aims at the in-depth study on the molecular structure of pectic oligosaccharides including molecular weight, degree of polymerization, charge characteristics and methylation characteristics, and demonstration of the morphological, physiological and biochemical effects on the fungal growth aroused from the inhibition function. By this, we would like to make clear of the structure-effect relationship of the obtained pectic oligosaccharides’ antifungal activity. We hope this study could provide theoretical basis of the application of pectin oligosaccharides in food storage.

果胶低聚糖被证实具有较好的双歧因子特性以及抗氧化、抗肿瘤等生理活性，是一种理想的功能性成分，具有较高的研究价值和应用潜力。我们前期研究发现果胶酶控制性酶解柑橘果胶产生的低聚糖对柑橘贮藏过程中腐烂致病真菌的生长具有明显的抑制作用，而且果胶低聚糖的聚合度太高或者太低都会影响其对病原菌的抑制效果。我们推断果胶低聚糖特殊的结构特征决定了其对一些食品腐败真菌的生长具有抑制效果，这种构效关系的研究有助于阐明果胶低聚糖抗真菌活性的作用机制。因此，本项目拟在前期研究基础上进一步研究通过果胶酶酶解柑橘果胶所得的抗真菌低聚糖的分子结构包括分子量、聚合度、电荷特性以及甲基化等特性，考察其对真菌生长过程中的形态学、生理生化及分子生物学等方面的影响，比较分析抗真菌活性的构效关系。项目研究目的在于阐释果胶低聚糖抗真菌活性的作用机制，为果胶低聚糖应用于食品贮藏保鲜提供理论依据。

果胶低聚糖具有较好的双歧因子特性以及抗氧化、抗肿瘤等生理活性，是一种理想的功能性成分。本项目从柑橘果园土样中筛选产果胶酶优良菌株。通过筛选获得一株高产耐酸性耐高温的青霉菌，基因序列测定和形态鉴定为皮壳青霉Penicillium crustosum，所产酶经验证能分解果胶得到果胶低聚糖。该菌株不仅产酶活高且具有较高的内切酶活性，而且有别于目前商业果胶酶主要生产菌株-黑曲霉，具有一定的菌种新颖性。通过单因素试验，确定了最优发酵条件。该酶纯化后测定其最适温度为45 ℃、最适pH为5.0。经过比较，该酶比商用果胶在水解制备果胶低聚糖具有明显优势，经过响应面优化水解条件，该酶在水解30 min左右能获得最大量的果胶低聚糖。经过初步分离纯化，组分5的果胶低聚糖具有最佳抗真菌活性，并在柑桔体内具有明显的真菌增值抑制效果。进一步从组分5中分离纯化得到一系列果胶低聚糖，通过分析鉴定了结构特征，比较分析了不同结构果胶低聚糖的抗真菌活性。