利用虾壳清洁化生产不同脱乙酰度壳寡糖及其抗TMV效果研究

Cleaner Production of Chitooligosaccharides with Different Degrees of Deacetylation from Shrimp Shells and Their Effects on TMV Resistance

中文关键词: 清洁化生产  壳寡糖  脱乙酰度  烟草花叶病毒

英文关键词: Cleaner production  Chitooligosaccharide  Deacetylation degree  Tobacco mosaic virus

中文摘要:

      壳寡糖的传统生产工艺是采用HCl和NaOH处理甲壳类制备壳聚糖，再酶解获得壳寡糖，生产过程中含高浓度Cl–和Na+的废水对环境造成严重污染。本研究采用H3PO4和KOH为反应溶液建立壳聚糖的绿色生产工艺，并制得不同脱乙酰度的壳寡糖，探究了不同脱乙酰度壳寡糖抗烟草花叶病毒(TMV)的效果。结果显示，通过该生产工艺制得壳寡糖的脱乙酰度分别为63.79%、72.12%、79.34%和88.15%，分子量均为1500 Da左右。脱乙酰度为79.34%和88.15%的壳寡糖诱导植株对TMV产生抗病性，表现出对TMV进行体外钝化、抑制TMV在寄主内的复制和提高植物体内过氧化氢酶、过氧化物酶和多酚氧化酶的活性。

英文摘要:

      Shrimp shells contain a number of components, including chitin, proteins, and minerals. Chitooligosaccharides, derivatives of chitin, are homo- or hetero-oligomers of N-acetyl-glucosamine and d-glucosamine that are linked by β-1,4-glycosidic bonds. The production of chitooligosaccharides from shrimp shells involves demineralization, deproteinization, deacetylation, and depolymerization. The traditional chemical processes involved in the extraction of chitosan from shrimp shells mostly use HCl for the demineralization and NaOH for the deproteinization and deacetylation processes; the chitosan is then oxidatively degraded to chitooligosaccharide. Although these methods are highly economic and effective, they present several disadvantages, such as energy wastage, long processing times, and environmental pollution. Chitooligosaccharides have been widely used in agriculture and it has been proven that their degree of deacetylation (DDA) affects the physiology of the plants. However, limited attention has been paid to understanding the relationship between the DDA of a chitooligosaccharide and its effects on plant resistance to tobacco mosaic virus (TMV). In the present study, an eco-friendly method was established for the production of chitooligosaccharides by using H3PO4 for the demineralization and KOH for the deproteinization and deacetylation processes. Additionally, we studied the effects of chitooligosaccharides with different DDAs on the resistance to TMV. The effects of chitooligosaccharides with different DDAs on inactivating TMV in vitro and inhibiting its multiplication were detected by the Evans blue method. Changes in the activities of defensive enzymes induced by chitooligosaccharides with different DDAs were tested with tobacco leaves. The degrees of demineralization and deproteinization were 94.04%±0.52% and 85.65%±1.06%, respectively. Chitosans and chitooligosaccharides with different DDAs were obtained by microwave-assisted KOH metathesis and oxidative degradation. The DDAs of the obtained chitooligosaccharides were 63.79%, 72.12%, 79.34%, and 88.15%, and their molecular weights were approximately 1500 Da. The chitooligosaccharides with 79.34% and 88.15% DDAs improved leaf resistance to TMV by inactivating the virus in vitro, inhibiting its multiplication, and increasing the activities of the CAT, POD, and PPO enzymes. The liquid waste generated during the demineralization, deproteinization, and deacetylation processes was found to be rich in phosphorus, potassium, and nitrogen, the main constituents of fertilizers. The chitooligosaccharides with 79.34% and 88.15% DDAs could thus be applied as resistance inducers. Therefore, the liquid waste and the chitooligosaccharides generated could be recovered and used in fertilizers, which would significantly decrease environmental pollution and improve the economic value of shrimp shells.