【作者】 刘振华；

【导师】 李元广；

【作者基本信息】 华东理工大学 ， 生物化工， 2011， 博士

【摘要】 可湿性粉剂是我国微生物农药剂型的主要种类,但有关微生物农药可湿性粉剂配方及其加工工艺优化与放大方面的研究鲜有报道。具有我国自主知识产权的可以防治多种植物土传病害的多粘类芽孢杆菌细粒剂,是国内外唯一以类芽孢杆菌属菌株为生防菌的微生物农药,但由于使用不方便而难以大面积推广；此外,海洋微生物农药的研制尚未见报道,而以海洋芽孢杆菌为有效成分的微生物农药却具有广阔的应用前景,因此,针对这两种微生物农药,亟待开展可湿性粉剂研制与产业化方面的研究。本文以多粘类芽孢杆菌HY96-2和海洋芽孢杆菌B-9987为研究对象,在可湿性粉剂配方、粉碎、喷雾干燥以及多粘类芽孢杆菌抗菌物质的分离纯化等方面开展了较为系统的研究,建立了多粘类芽孢杆菌可湿性粉剂工业化生产工艺和海洋芽孢杆菌可湿性粉剂中试生产工艺。研究结果不仅为国内外首创的多粘类芽孢杆菌可湿性粉剂及海洋芽孢杆菌可湿性粉剂的产业化提供了技术依托,同时也可为其它微生物农药可湿性粉剂的创制与产业化提供参考。悬浮率是可湿性粉剂最重要的性质之一,但悬浮率较低曾是多粘类芽孢杆菌可湿性粉剂产业化的瓶颈。研究结果表明,气流粉碎和添加表面活性A可以协同提高多粘类芽孢杆菌可湿性粉剂M-1000-P1(载体M-1000和工厂多粘类芽孢杆菌HY96-2发酵液P1混合干燥后所得制剂)的悬浮率。另一方面,气流粉碎过程中M-1000-P1活菌含量没有受到明显影响,而表面活性剂A与多粘类芽孢杆菌HY96-2之间的生物相容性明显好于表面活性剂EFW(?)、D425(?)、十二烷基苯磺酸钠和茶皂素。以5%的比例添加表面活性剂A可使M-1000-P1气流粉碎制剂的悬浮率从58.3%提高到81.1%。在进风口温度为210-230℃、出风口温度为80～90℃的范围内,多粘类芽孢杆菌可湿性粉剂在小型干燥塔(最大水蒸发量5kg/h)、中型干燥塔(最大水蒸发量50kg/h)和大型干燥塔(最大水蒸发量800kg/h)上具有相同的干燥规律：制剂活菌存活率均大于75%,含水量均小于5%,表明小型或中型喷雾干燥塔中多粘类芽孢杆菌可湿性粉剂干燥工艺成功放大到工业生产规模。此外,在相同的粉碎条件(气流压力0.8MPa,粉碎腔温度为常温)下,多粘类芽孢杆菌可湿性粉剂的气流粉碎过程可成功地从小试规模(5kg/h)放大到工业规模(50kg/h)。在上述干燥和粉碎研究结果基础上,建立了多粘类芽孢杆菌可湿性粉剂工业化生产工艺,产品的活菌含量(2.1×109CFU/g)、水含量(3%)、润湿时间(95秒)、悬浮率(76.1%)和细度(100%通过325目筛)等指标均达到农药可湿性粉剂的质量要求,并且该产品对番茄青枯病、西瓜枯萎病、黄瓜细菌性角斑病和西瓜炭疽病的田间防效分别为91.66%、83.75%、81.14%和77.01%,表明它是一种广谱性植病生防微生物农药。进一步研究结果表明,多粘类芽孢杆菌HY96-2多糖粗提物可以明显提高高岭土、M和N这3种载体的悬浮率,且对多粘类芽孢杆菌芽孢具有热保护和紫外保护功能。据此,本文提出利用多粘类芽孢杆菌多糖提高其可湿性粉剂悬浮率,并将多糖作为制剂热保护和紫外保护助剂的策略。利用多糖助悬浮作用和新发现的助剂S改善制剂润湿性的作用,对多粘类芽孢杆菌可湿性粉剂配方重新进行了筛选,并首次将混料实验设计运用于微生物农药可湿性粉剂的配方优化,进而建立了多粘类芽孢杆菌可湿性粉剂新的配方和加工工艺,所加工出的可湿性粉剂的润湿时间(99秒)和悬浮率(71.7%)均达到农药可湿性粉剂要求。新工艺省去粉碎操作单元,可大幅降低生产成本。杀镰孢菌素(fusaricidin)是多粘类芽孢杆菌菌株产生的一种抗真菌脂肽类物质。本文首次从商业化菌株——多粘类芽孢杆菌HY96-2代谢产物中分离得到杀镰孢菌素A(fusaricidin A),并发现其对盆栽中的黄瓜灰霉病具有较好防治效果：浓度为250μg/ml和62.5μg/ml的杀镰孢菌素A对黄瓜灰霉病的防治效果分别为95.0%和63.3%,明显好于对照药剂嘧霉胺。上述研究结果为将多粘类芽孢杆菌制剂中代谢产物抑菌活性或抗生素含量作为一项新的质量控制指标奠定了基础。海洋芽孢杆菌可湿性粉剂配方研究结果表明,载体N有利于该制剂悬浮率的提高,而高岭土则会延长其润湿时间；有机硅与海洋芽孢杆菌B-9987之间的生物相容性较好并可以提高海洋芽孢杆菌可湿性粉剂(以N为载体)的悬浮率。干燥研究结果表明,小型喷雾干燥塔生产工艺可成功地放大至中型喷雾干燥塔；当中型干燥塔进风口温度为230℃,出风口温度在80～90℃时,海洋芽孢杆菌可湿性粉剂的活菌存活率高于70%,含水量低于5%。在上述研究结果基础上,建立了海洋芽孢杆菌B-9987可湿性粉剂中试生产工艺,产品的活菌含量(1.5×1010CFU/g)、水含量(3.5%)、润湿时间(56秒)、悬浮率(84.4%)和细度(100%通过325目筛)等指标均达到农药可湿性粉剂质量要求,并且该产品对番茄青枯病和甜瓜炭疽病的防效分别为74.42%和63.31%,表明其在植物病害防治方面具有广阔的应用前景。更多还原

【Abstract】 Wettable powder is the main type of microbial pesticide formulations in China. However, fewer studies were reported about the formulation, process optimization and scale up in microbial pesticide. Paenibacillus polymyxa fine granular (PPFG) formulation, with independent intellectual property rights in China, is the first microbial pesticide developed by Paenibacillus strain. PPFG can control many soilborne diseases; however, the large-scale application of PPFG is limited because of its inconveniences. In addition, although there is little study about microbial pesticide with marine microorganisms as biocontrol agents, Bacillus marinus B-9987 has broad application prospects as microbial pesticide. So studies about the preparation and industrial production of P. polymyxa and B. marinus microbial pesticide should be carried out urgently.With P. polymyxa and B. marinus as study objectives, formulation, milling, spray-drying of microbial pesticide wettable powder were investigated systematically in this paper, and antifungal substance of P. polymyxa was also investigated. Industrial production processes of P. polymyxa wettable powder and pilot production processes of B. marinus wettable powder were established together. The results in the paper not only provided technical support for the industrialization of P. polymyxa and B. marinus wettable powder, but also provided reference for other microbial pesticide.Suspensibility is an important property of wettable powder and poor suspensibility of P. polymyxa wettable powder had hindered its industrial production in larger scale. It was showed that jet milling and surfactant A could synergistically improve the suspensibility of P. polymyxa wettable powder M-1000-P1 (prepared by spray-drying the mixture of carrier M-1000 and P. polymyxa HY96-2 broth from factory). On the other hand, no obvious adverse effect of jet milling on P. polymyxa population in powder was observed. Besides, surfactant A has better biocompatibility than those of EFW(?), D-425(?), sodium dodecyl benzene sulfonate or tea saponin. When jet milling powder was added with surfactant A at 5%(w:w), its suspensibility was improved from 58.3% to 81.1%.When P. polymyxa wettable powder was spray-dried at small (maximum water evaporation of 5kg/h), medium (maximum water evaporation of 50kg/h) or large dryer (maximum water evaporation of 800kg/h) with inlet temperature 210～230℃and outlet temperature 80～90℃, bacteria survival rate above 75% and moisture content below 5% were obtained. It implies that the spray-drying process of P. polymyxa at small and medium dryer can be scaled up to production scale. On the other hand, jet milling process at laboratory scale (5kg/h) was successfully scaled up to production scale (50kg/h) under the same condition (air pressure was 0.8Mpa and grinding chamber was at room temperature). Thus, the preparation process of P. polymyxa wettable powder in production scale was established. The product has good quality properties with bacteria population of 2.1×109CFU/g, moisture content of 3%, wetting time of 95 seconds, suspensibility of 76.1% and fineness that all particles could pass through a 325-mush sieve. All the properties meet the requirements of pesticide wettable powder in China. P. polymyxa wettable powder had good control efficacies on tomato bacteria wilt (91.66%), watermelon fusarium wilt (83.75%), angular leaf spot of cucumber (81.14%) and watermelon anthracnose (77.01%), respectively, which indicates that P. polymyxa wettable powder is a microbial pesticide with broad control spectrum to plant diseases.Furthermore, P. polymyxa polysaccharide extract (PPPE) has obvious suspensibility improving effect on carrier kaolin clay, M-1000 and N-1000. In addition, PPPE has protective effect for P. polymyxa spores from heat and ultraviolet (UV). Based on the above results, a strategy, improving the suspensibility of P. polymyxa wettable powder and heat or ultraviolet stability of P. polymyxa spore by PPPE, was developed. Using the suspensibility improving effect of PPPE and wettability improving effect of adjuvant S, the components of P. polymyxa wettable powder were rescreened and further optimized with mixed experiment design. Then, a new formulation and preparation process were developed which had low producing cost because milling was no longer needed. The new product has eligible wettability (99 seconds of wetting time) and suspensibility (71.7%).Fusaricidin is a kind of lipopeptide produced by many P. polymyxa strains. Fusaricidin A was firstly obtained from a commercial strain—P. polymyxa HY96-2. Fusaricidin A has good control effect on gray mold of cucumber:250μg/ml and 62.5μg/ml of fusaricidin A had control effect of 95.0% and 63.3%, which were better than those of pyrimethanil (one chemical pesticide as control). The results of fusaricidin A indicate that the inhibition activity or antibiotic content of P. polymyxa formulation could be considered as a new product quality control indicator.B. marinus B-9987 formulation with carrier of N-1000 had better suspensibility and B-9987 formulation with carrier of kaolin clay had worse wettability. Organosilicon had better biocompatibility with B. marinus B-9987 and could improve the suspensibility of B. marinus B-9987 powder formulation (N as carrier). The spray-drying process at laboratory scale could be scaled up to pilot scale. When B. marinus wettable powder was spray-dried at medium dryer with inlet temperature at 230℃and outlet temperature 80～90℃, bacteria survival rate above 70% and moisture content below 5% were obtained. Then, pilot production process of B-9987 wettable powder was established and the product had a eligible quality performance with bacteria population of 1.5×1010CFU/g, moisture content of 3.5%, wetting time of 56 seconds, suspensibility of 84.4% and fineness that all particles could pass through a 325-mush sieve. The field incidence control efficacies of B. marinus powder on tomato bacterial wilt and melon anthracnose were 74.42% and 63.31%, respectively, which suggests that B. marinus wettable powder has broad application prospect in plant disease control.更多还原