壳聚糖酶解产物抑制真菌活性研究

为探究并提高壳聚糖抑制真菌的活性,本研究利用蜡状芽孢杆菌ncps116发酵所制备的壳聚糖酶来酶解壳聚糖,测定不同酶解时间壳聚糖产物的抑菌活性,并监测酶解产物中还原糖的含量;之后选取抑菌活性提高最显著的壳聚糖酶解产物为研究对象,测定最低抑菌活性和最适pH,并使用电子显微镜观察酶解产物对病原真菌菌丝和孢子萌发的抑制作用。结果表明,壳聚糖经0.5~12 h酶解能够显著提高市售壳聚糖的抑菌活性,在酶解24 h内抑菌活性呈先升高后降低的趋势,而且酶解产物的抑菌活性与还原糖浓度相关,还原糖浓度过低(≤44.24 μg·mL^-1),酶解不充分,抑菌圈直径13 mm;而还原糖浓度过高(≥1 900 μg·mL^-1),酶解完全,抑菌圈直径10 mm,抑菌活性均较差。其中,酶解6 h的壳聚糖产物抑菌活性提高最显著,对3种病原真菌(棉花黄萎病菌、苹果轮纹病菌、西瓜专化型尖孢镰刀菌)的最低抑菌活性提高4~8倍,还原糖浓度为383.34 μg·mL^-1,在pH值4.0~4.7时抑菌活性最高;此外,酶解产物能导致真菌菌丝断裂、褶皱、菌丝末端囊泡等畸形生长,并长期抑制孢子生长和菌丝伸长。综上所述,酶解是提高壳聚糖抗菌活性的有效手段,这为推进亮聚糖在防腐保鲜方面的应用奠定了基础。

Antifungal Activities of Chitosan Hydrolysates

In previous studies, the inhibiting activities of the commercially available chitosan on the fungus was significantly improved after hydrolysis. For the development of the fungicidal activities, chitosan hydrolysates was prepared using chitosanase from fermentation of Bacillus cereus ncps116 in this study. The fungicidal activities and reducing sugar content were analyzed in the process of enzymatic hydrolysis with time going. The most active hydrolysate was chosen to be tested the minimum antibacterial concentration and the optimum pH. Inhibition to the hyphae and spore was investigated under electron microscopy. The results showed that the activity was increased by the hydrolysis. It increased firstly and then decreased with time going within 24 h, which was related to sugar content. The hydrolysates with too low (≤44.24 μg·mL^-1) or too high (≥1 900 μg·mL^-1) reducing sugar content, which mean that hydrolysis was insufficient or complete, showed bad activities with he diameters of the inhibtion zone around 13 and 9 mm, respectively. The hydrolysate at 6 h showed the most significant fungicidal activities, which affected the growth of the tested pathogenic fungi, including Verticillium dahliae, Physalospora piricola, Fusarium oxysporum f.sp. niveum, with a 4 to 8-fold improvement at minimum fungicidal concentration around 0.125%~0.250%. The concentration of reducing sugar was tested to be 383.34 μg·mL^-1. Besides, the hydrolysates exhibited the most high antifungal bioactivity at pH 4.0~4.7. Furthermore, the hydrolysates could cause disrupt and abnormal growth of fungal hyphae, such as folds and vesicles. And the inhibition to the spore and mycelial growth was irreversible for a long time. In a word, the hydrolysis could significantly improve the fungicidal activities of chitosan, which provided a research basis for the further application on chitosan in agricuture.