球孢白僵菌孢子粉生产、制剂和贮存技术的改进及其淀粉酶特性测定
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摘要
球孢白僵菌(Beauveria bassiana(Balsamo)Vuillemin)是一种经典的丝孢类昆虫病原真菌，在农林害虫的微生物防治中广受注目，国外已有多种商品制剂注册生产。然而，球孢白僵菌的孢子粉生产、制剂及保存方面的技术问题从未获得圆满解决，成为影响其深度开发和应用的基本因素。在本实验室对一杀蚜球孢白僵菌株(SG8702)的前期工作基础之上，本研究探索了改进该菌高纯度孢子粉生产、孢子乳悬剂配方及常温贮存的技术途径，并首次测定了其产淀粉酶的特性，以期有助于推动我国真菌杀虫剂技术的不断进步。
固体培养与产孢条件优化 以熟化度适中的大米作为固相产孢基质，研究了米料厚度、氮源营养添加剂及其浓度对球孢白僵菌气生分生孢子的产量、含水量及活孢率的影响。结果表明，在直径15cm的培养皿中用50g大米生产的孢子粉含水量最低。在大米中添加蛋白胨、硝酸钾及硝酸铵，3种氮源都能提高孢子粉产量及活孢率，但相互间差异不显著。浓度试验表明，在大米中添加0.1％的KNO_3最有利于产孢。大米连续二次发酵利用，产粉量达到57.3～67.9 mg／g，可大幅提高大米的利用率。在扩大培养中，大米最大产粉量达55.5mg／g，产孢数为5.83×10~9个孢子／g；所获孢子粉的含孢量达1.48;×10~(11)个孢子／g，含水量仅5.25％，活孢率达98.72％。这些指标说明，用此法生产球孢白僵菌分生孢子粉，具有产量高、粉质优、活力强、含水量低及经济实用等优点，为合理利用库存低值陈米生产杀虫真菌高纯度孢子粉开辟了新的途径。
胞外淀粉酶的产生及其特性 为探索该菌产淀粉酶的特性及条件，从二次旋转组合设计的13种培养液中，筛选出适合该菌产淀粉酶的培养液配方，其主要成分为可溶性淀粉0.3％，葡萄糖、蛋白胨和酵母粉各0.5％。将含10~6个分生孢子／mL的该培养液恒温(25±1℃)振荡(120r／min)培养3.5d，菌丝生物量为16.7mg／mL，胞外淀粉酶产量达527.1U／mg菌丝。培养液初始pH 4～6最有利于淀粉酶的产生，产酶量为1315.8～1439.2U／mg菌丝。在40℃、pH 4.0条件下最强，在pH 3～8范围内20℃下处理20min或在pH 4～6范围内37℃下处理1 h，淀粉酶的活性较为稳定；40℃下处理20min酶活保持在90％以上，50℃和60℃下处理相同时间则酶活分别丧失52％和91％。一定浓度的Ca~(2+)有利于酶活提高，但Cu~(2+)、Mn~(2+)、Na~+、Hg~(2+)、Fe~(2+)和Mg~(2+)等常见金属离子则不同程度地抑制酶活。
袍子乳悬剂助剂优化对本实验室研制的SG8702抱子乳悬剂的主要助剂(乳化剂
和增效剂)进行了改进和优化。轻质液体石蜡作为制剂载体，其本身对抱子存活的影响
很小，不含乳化剂的两个对照常温贮存10个月时活抱率仍达78.57%和81 .78%。筛选
出了乳化性能优异的乳化剂A，在2%的含量下能使抱子乳悬剂常温有效保存3个月
左右，活抱率仅略有下降(由98%下降至83.6%)，但含量越高有效贮存期越短。毗虫
琳10%可湿性粉剂与球抱白僵菌分生抱子的生物学相容性最好，按1一5%的比例(w/v)
加入抱子乳悬剂中在常温贮存下对抱子活力不产生明显影响。
袍子粉贮存生理将不同含水量(l .12%、4.73%、7.23%、9.84%及14.11%)的
球抱白僵菌抱子粉分别贮存于4℃和25℃下，每隔15d定期检测活抱率、内贮总糖和
蛋白含量，持续进行180d。结果表明，各处理内源总糖含量在头3个月贮存期间缓
慢下降，然后表现起伏较大;内贮总蛋白含量变化虽略有反复，但总体呈明显下降趋
势。在25℃下贮存的最高含水量抱子粉，在第30d时全部失活，其余含水量的饱子
粉在贮存的头90d活抱率仅轻微下降，而后下降幅度较大，且随含水量增高而下降加
剧。在4℃下，不同含水量抱子粉的活抱率始终比在25℃下降低缓慢。同一温度下活
抱率的下降随抱子粉含水量增大而加剧。贮存中抱子粉的活抱率与内源蛋白代谢的相
关性不达显著水平，而与内源总糖代谢呈显著负相关。根据对数残留定律y=a’e一bt的
拟合估计，贮存中抱子粉的活抱率切随贮存时间(t)和抱子粉含水量而变化，如在4℃
下贮存含水量1 .12%和14.n%的抱子粉，抱子失活50%所需的时间分别约需1734d
和388d，而在25℃下贮存上述含水量的抱子粉，抱子失活50%所需的时间分别约230
d和17d。
综合以上结果，本研究初步建立了利用库存低值陈米生产球抱白僵菌高纯度抱子
粉的固相产抱新工艺;加入适当助剂和降低抱子粉含水量，能有效提高抱子乳悬剂的
商品货架期。由于学习时间的限制，本研究中的遗憾和不足之处还有待研究完善。
The entomopathogenic fungus, Beauveria bassiana (Balsamo) Vuillemin, is a classic fungal agent highly potential for use in microbial control of insect pests and has been developed into a variety of commercial formulations called mycoinsecticides in the world. However, technical problems involved in mass production, formulation, and normal storage of B. bassiana conidia remain obstacles to intensive development and practical use of B. bassiana-based mycoinsecticides for pest control. Based on previous work on an aphid-derived isolate (SG8702) of B. bassiana in our laboratory, the present study was attempted to search for technical approaches to improving mass production of aerial conidia, optimizing components of emulsifiable formulation, and prolonging shelf life under normal storage conditions. Aspects on production of extracellular amylases by the fungal isolate were first assayed as well.
A modified technology for production of high-quality conidia powder on low-quality rice substrate. Low-quality rice used as solid substrate was tested to produce aerial conidia of B. bassiana SG8702 in 15-cm dishes or large trays. The thickness of rice layer piled in dishes, and the source and concentration of supplemented nitrogen were found affecting conidial yield, viability, and water content. The thinner the rice layer the lower the water content in the conidia powder produced. Adding a very small amount of peptone, KNO3 or NH4NO3 to the rice substrate significantly increased conidial yield and viability, but there was little difference among the nitrogen sources tested. A supplement of 0.1% KNO3 (w/w) was found most favorable to conidial production, resulting in a conidial yield of 57.3-67.9 mg per gram of rice in repeated solid fermentation. In an expanded experiment including the use of 6.5 kg rice, each gram of rice yielded a maximal conidial powder of 55.5 mg, equivalent to 5.83×109 conidia. The powdery product was in high quality with pureness of 1.48 ×1011 conidia/g, viability of 98.72%, and water content of 5.25%. The results indicate that the low-quality rice can be utilized for efficient production of high-quality B. bassiana conidia for microbial control of insect pests.
Production and activity of extracellular amylases in liquid culture. Cuticle-degrading enzymes of B. bassiana such as proteases and chitinases have been well investigated but little attention has been paid to production of its amylases. A liquid medium including 0.3% soluble starch, 0.5% glucose, 0.5% peptone, and 0.5% yeast extract (w/v) was optimal for production of an amylase in liquid culture of B. bassiana SG8702 among 13 media generated from central composite design. Shaking the liquid culture containing 106 conidia/mL for 3.5 d at the regime of 25 ?1@ and 120 r/min resulted in mycelial biomass of 16.7 mg/mL and amylase yield of 527.1 units/mg. An initial range of pH 4.0-6.0 in the liquid culture was found being most favorable to amylase production with the yield of 1315.8-1439.2 units/mg. The activity of B. bassiana amylase was highest at 40@ and pH 4.0. Its stability to pH apparently varied with temperature and time length for reaction. The amylase was considerably stable at pH 3.0-8.0 for 20 min at 20@ or at pH 4.0-6.0 for 1 h at 37@. The activity of the amylase remained >90% after 20-min maintenance at 40@, but lost 52% and 91% after the same period of maintenance at 50@ and 60@, respectively. Moreover, the presence of Ca2+ had little effect on the activity of the amylase whereas other cations including Cu2+, Mn2+, Na+, Hg2+, Fe2+ and Mg2+ could be significantly inhibitory to its activity at different levels. This is the first report on the production and features of amylase produced by B. bassiana.
Optimization of components for emulsifiable formulatons. Primary components of an emulsifiable formulation of B. bassiana SG8702 conidia developed previously were further optimized. As a formulation carrier, light liquid paraffin was found affecting little the viability of suspended conidia during long-term storage at ambient te
固体培养与产孢条件优化 以熟化度适中的大米作为固相产孢基质，研究了米料厚度、氮源营养添加剂及其浓度对球孢白僵菌气生分生孢子的产量、含水量及活孢率的影响。结果表明，在直径15cm的培养皿中用50g大米生产的孢子粉含水量最低。在大米中添加蛋白胨、硝酸钾及硝酸铵，3种氮源都能提高孢子粉产量及活孢率，但相互间差异不显著。浓度试验表明，在大米中添加0.1％的KNO_3最有利于产孢。大米连续二次发酵利用，产粉量达到57.3～67.9 mg／g，可大幅提高大米的利用率。在扩大培养中，大米最大产粉量达55.5mg／g，产孢数为5.83×10~9个孢子／g；所获孢子粉的含孢量达1.48;×10~(11)个孢子／g，含水量仅5.25％，活孢率达98.72％。这些指标说明，用此法生产球孢白僵菌分生孢子粉，具有产量高、粉质优、活力强、含水量低及经济实用等优点，为合理利用库存低值陈米生产杀虫真菌高纯度孢子粉开辟了新的途径。
胞外淀粉酶的产生及其特性 为探索该菌产淀粉酶的特性及条件，从二次旋转组合设计的13种培养液中，筛选出适合该菌产淀粉酶的培养液配方，其主要成分为可溶性淀粉0.3％，葡萄糖、蛋白胨和酵母粉各0.5％。将含10~6个分生孢子／mL的该培养液恒温(25±1℃)振荡(120r／min)培养3.5d，菌丝生物量为16.7mg／mL，胞外淀粉酶产量达527.1U／mg菌丝。培养液初始pH 4～6最有利于淀粉酶的产生，产酶量为1315.8～1439.2U／mg菌丝。在40℃、pH 4.0条件下最强，在pH 3～8范围内20℃下处理20min或在pH 4～6范围内37℃下处理1 h，淀粉酶的活性较为稳定；40℃下处理20min酶活保持在90％以上，50℃和60℃下处理相同时间则酶活分别丧失52％和91％。一定浓度的Ca~(2+)有利于酶活提高，但Cu~(2+)、Mn~(2+)、Na~+、Hg~(2+)、Fe~(2+)和Mg~(2+)等常见金属离子则不同程度地抑制酶活。
袍子乳悬剂助剂优化对本实验室研制的SG8702抱子乳悬剂的主要助剂(乳化剂
和增效剂)进行了改进和优化。轻质液体石蜡作为制剂载体，其本身对抱子存活的影响
很小，不含乳化剂的两个对照常温贮存10个月时活抱率仍达78.57%和81 .78%。筛选
出了乳化性能优异的乳化剂A，在2%的含量下能使抱子乳悬剂常温有效保存3个月
左右，活抱率仅略有下降(由98%下降至83.6%)，但含量越高有效贮存期越短。毗虫
琳10%可湿性粉剂与球抱白僵菌分生抱子的生物学相容性最好，按1一5%的比例(w/v)
加入抱子乳悬剂中在常温贮存下对抱子活力不产生明显影响。
袍子粉贮存生理将不同含水量(l .12%、4.73%、7.23%、9.84%及14.11%)的
球抱白僵菌抱子粉分别贮存于4℃和25℃下，每隔15d定期检测活抱率、内贮总糖和
蛋白含量，持续进行180d。结果表明，各处理内源总糖含量在头3个月贮存期间缓
慢下降，然后表现起伏较大;内贮总蛋白含量变化虽略有反复，但总体呈明显下降趋
势。在25℃下贮存的最高含水量抱子粉，在第30d时全部失活，其余含水量的饱子
粉在贮存的头90d活抱率仅轻微下降，而后下降幅度较大，且随含水量增高而下降加
剧。在4℃下，不同含水量抱子粉的活抱率始终比在25℃下降低缓慢。同一温度下活
抱率的下降随抱子粉含水量增大而加剧。贮存中抱子粉的活抱率与内源蛋白代谢的相
关性不达显著水平，而与内源总糖代谢呈显著负相关。根据对数残留定律y=a’e一bt的
拟合估计，贮存中抱子粉的活抱率切随贮存时间(t)和抱子粉含水量而变化，如在4℃
下贮存含水量1 .12%和14.n%的抱子粉，抱子失活50%所需的时间分别约需1734d
和388d，而在25℃下贮存上述含水量的抱子粉，抱子失活50%所需的时间分别约230
d和17d。
综合以上结果，本研究初步建立了利用库存低值陈米生产球抱白僵菌高纯度抱子
粉的固相产抱新工艺;加入适当助剂和降低抱子粉含水量，能有效提高抱子乳悬剂的
商品货架期。由于学习时间的限制，本研究中的遗憾和不足之处还有待研究完善。
The entomopathogenic fungus, Beauveria bassiana (Balsamo) Vuillemin, is a classic fungal agent highly potential for use in microbial control of insect pests and has been developed into a variety of commercial formulations called mycoinsecticides in the world. However, technical problems involved in mass production, formulation, and normal storage of B. bassiana conidia remain obstacles to intensive development and practical use of B. bassiana-based mycoinsecticides for pest control. Based on previous work on an aphid-derived isolate (SG8702) of B. bassiana in our laboratory, the present study was attempted to search for technical approaches to improving mass production of aerial conidia, optimizing components of emulsifiable formulation, and prolonging shelf life under normal storage conditions. Aspects on production of extracellular amylases by the fungal isolate were first assayed as well.
A modified technology for production of high-quality conidia powder on low-quality rice substrate. Low-quality rice used as solid substrate was tested to produce aerial conidia of B. bassiana SG8702 in 15-cm dishes or large trays. The thickness of rice layer piled in dishes, and the source and concentration of supplemented nitrogen were found affecting conidial yield, viability, and water content. The thinner the rice layer the lower the water content in the conidia powder produced. Adding a very small amount of peptone, KNO3 or NH4NO3 to the rice substrate significantly increased conidial yield and viability, but there was little difference among the nitrogen sources tested. A supplement of 0.1% KNO3 (w/w) was found most favorable to conidial production, resulting in a conidial yield of 57.3-67.9 mg per gram of rice in repeated solid fermentation. In an expanded experiment including the use of 6.5 kg rice, each gram of rice yielded a maximal conidial powder of 55.5 mg, equivalent to 5.83×109 conidia. The powdery product was in high quality with pureness of 1.48 ×1011 conidia/g, viability of 98.72%, and water content of 5.25%. The results indicate that the low-quality rice can be utilized for efficient production of high-quality B. bassiana conidia for microbial control of insect pests.
Production and activity of extracellular amylases in liquid culture. Cuticle-degrading enzymes of B. bassiana such as proteases and chitinases have been well investigated but little attention has been paid to production of its amylases. A liquid medium including 0.3% soluble starch, 0.5% glucose, 0.5% peptone, and 0.5% yeast extract (w/v) was optimal for production of an amylase in liquid culture of B. bassiana SG8702 among 13 media generated from central composite design. Shaking the liquid culture containing 106 conidia/mL for 3.5 d at the regime of 25 ?1@ and 120 r/min resulted in mycelial biomass of 16.7 mg/mL and amylase yield of 527.1 units/mg. An initial range of pH 4.0-6.0 in the liquid culture was found being most favorable to amylase production with the yield of 1315.8-1439.2 units/mg. The activity of B. bassiana amylase was highest at 40@ and pH 4.0. Its stability to pH apparently varied with temperature and time length for reaction. The amylase was considerably stable at pH 3.0-8.0 for 20 min at 20@ or at pH 4.0-6.0 for 1 h at 37@. The activity of the amylase remained >90% after 20-min maintenance at 40@, but lost 52% and 91% after the same period of maintenance at 50@ and 60@, respectively. Moreover, the presence of Ca2+ had little effect on the activity of the amylase whereas other cations including Cu2+, Mn2+, Na+, Hg2+, Fe2+ and Mg2+ could be significantly inhibitory to its activity at different levels. This is the first report on the production and features of amylase produced by B. bassiana.
Optimization of components for emulsifiable formulatons. Primary components of an emulsifiable formulation of B. bassiana SG8702 conidia developed previously were further optimized. As a formulation carrier, light liquid paraffin was found affecting little the viability of suspended conidia during long-term storage at ambient te
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