CN115125162A - 一种复合生物杀虫剂及其制备方法 - Google Patents
一种复合生物杀虫剂及其制备方法 Download PDFInfo
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- CN115125162A CN115125162A CN202210488391.0A CN202210488391A CN115125162A CN 115125162 A CN115125162 A CN 115125162A CN 202210488391 A CN202210488391 A CN 202210488391A CN 115125162 A CN115125162 A CN 115125162A
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Classifications
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- A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
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- C—CHEMISTRY; METALLURGY
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- C12N11/089—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
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Abstract
本发明提供一种复合生物杀虫剂及其制备方法,属于生物杀虫剂领域,具体是先将乳酸菌、苏力菌、白僵菌和绿僵菌分别进行发酵培养,发酵完成后每种菌株的发酵液中有效活菌数均应大于2.0×109cfu/mL,再按比混合并进行复合培养,最后加入负载载体进行菌体负载后冻干,制得所述复合生物杀虫剂;本发明利用微生物不仅可以改良土壤还可以提高蔬菜的品质,增产增收,同时又具有良好的保存稳定性,是有机蔬菜种植过程中的必备产品。
Description
技术领域
本发明涉及生物杀虫剂领域,具体涉及一种复合生物杀虫剂及其制备方法。
背景技术
生物杀虫剂主要分为苏云金杆菌、昆虫病毒和植物浸提液3大类,具有取材方便,成本低廉、控制期长,高效、经济、安全、无污染,与环境高度相容等特点,是当前生产无公害绿色蔬菜生产的最佳农药选择。随着时间的推移,已开发出由生物菌组合的多种生物杀虫剂组合以消灭害虫并减轻其造成的损坏。这些生物杀虫剂以活的真菌或细菌作为昆虫病原菌杀死昆虫,但杀虫菌剂在保存过程中易受环境条件影响其菌体活性,其稳定性不高,难以长期有效保存,甚至发生灭活失效。
发明内容
针对上述问题,本发明提供一种既能消灭地下害虫、又能对蔬菜的根系起到保护作用以 保证蔬菜的顺利生长,又不会对使用者或蔬菜食用者的身体造成毒害,同时具有高稳定性的 生物杀虫-生根双重保护剂及其制备方法。
本发明的目的采用以下技术方案来实现:
一种复合生物杀虫剂的制备方法,包括以下步骤:
(1)单菌种培养
将乳酸菌、苏力菌、白僵菌和绿僵菌分别进行发酵培养,发酵完成后每种菌株的发酵液 中有效活菌数均应大于2.0×109cfu/mL;
(2)复合培养
将分别发酵培养得到的乳酸菌、苏力菌、白僵菌和绿僵菌按比混合并进行复合培养;
(3)负载
加入负载载体进行菌体负载后冻干,制得所述复合生物杀虫剂。
优选的,所述乳酸菌、苏力菌、白僵菌和绿僵菌的质量比例为(2-6):(0.5-3.5):(1-5): (0.5-3.5),更优选为4:2:2:2。
优选的,所述负载载体的制备方法包括以下步骤:
S1、模板前处理
称取粒径小于150μm的氧化镁并干燥至恒重,将所述氧化镁分散在去离子水中,在60-80℃下恒温搅拌2-4h,分离沉淀后真空干燥制得模板材料;
S2、前体制备
称取三聚氰胺并溶解在去离子水中,加入植酸溶液,在常温下搅拌反应4-8h,分离沉淀 后干燥,得到产物A,分别称取芘、所述产物A、所述模板材料和氢氧化钾,混合后研磨至 均,将研磨混合物升温至160-180℃并保温0.5-1h,再在惰性气氛下加热至800-900℃并保温 1h,产物冷却后依次以稀的硫酸溶液和去离子水洗涤,干燥后制得载体前体;
其中,所述三聚氰胺与所述植酸溶液的质量比例为1:(2.8-3.2);所述芘与所述产物A、 所述模板材料和所述氢氧化钾的混合质量比例为(5-6):1:10:(14-16);
S3、外层制备
将所述载体前体和PEO-PPO-PEO三嵌段共聚物溶解在醇溶剂中,加入正硅酸乙酯和盐 酸溶液,在30-35℃下保温搅拌反应1-2h,分离沉淀并干燥,得到硅化产物,将所述硅化产 物在保护气氛下以5-10℃/min的速率升温至800-900℃,保温热处理2-3h,制得所述负载载 体;
其中,所述载体前体和PEO-PPO-PEO三嵌段共聚物、所述正硅酸乙酯的质量比例为(12-15):1:(1.8-2)。
优选的,所述复合培养的培养温度在22-28℃,培养时间6-10d。
优选的,按重量份数计,所述乳酸菌的培养基组成为:葡萄糖25-50份、酵母浸粉10-45 份、碳酸钙3-10份、可溶性淀粉10-50份、硫酸镁0.1-0.5份、醋酸钠0.1-0.3份、柠檬酸铵 0.1-0.3份、磷酸氢二钾0.2-0.8份、硫酸锰0.1-0.5份、吐温1-2份,去离子水溶解稀释。
优选的,按重量份数计,所述苏云金杆菌的培养基组成为:葡萄糖4-8份、酵母粉2-4 份、可溶性淀粉2-6份、蛋白胨1-4份、硫酸镁0.005-0.02份、硫酸锰0.005-0.01份、磷酸二氢钾0.1-0.3份、氯化钠0.3-0.8份,去离子水溶解稀释。
优选的,按重量份数计,所述白僵菌的培养基组成为:葡萄糖5-15份、酵母粉2-8份、 马铃薯汁3-10份、蛋白胨5-20份、蚕粉5-10份、硫酸二氢钾1-5份、头孢霉素1-5份,去离子水溶解稀释。
优选的,按重量份数计,所述绿僵菌的培养基组成为:葡萄糖5-15份、酵母粉4-8份, D-山梨醇2-6份,氯化钡0.01-0.1份,维生素B 0.01-0.02份,三醋酸甘油酯3-7份,去离子 水溶解稀释。
本发明的另一目的在于提供一种复合生物杀虫剂,所述复合生物杀虫剂由前述制备方法 制备得到。
优选的,所述复合生物杀虫剂按1-2公斤/亩并加水稀释至200-400倍重量使用。
本发明的有益效果为:
(1)本生物杀虫生根双重保护剂原理为利用白僵菌分解产生的抗生素杀死幼虫的卵体, 乳酸酸性很强而地下害虫是靠身体的气门进行呼吸,当接触乳酸菌后会使虫体溶化腐败到其 死亡;乳酸菌分解色氨酸形成吲哚,吲哚是天然的生根素能使作物迅速生根植株强壮,白僵 好氧能促进厌氧菌的生长,产生有机酸,提高群体免疫力,可与多种菌种混配,在土壤中需 求大量的氧气,促进了乳酸的生长环境,苏力菌为昆虫病原细菌,在营养缺乏或环境不良的 时候会进入不分裂的半静止期形成孢子和杀虫结晶蛋白,对一些害虫虫卵有特殊的毒效,进 入害虫体内迫使细胞破孔,肠道崩解迅速死亡,苏力菌对脊椎动物无毒害,是环境保护要求 的理想杀虫剂;绿僵菌是一种重要的植物根际促生细菌能够利用根系分泌物中大部分营养迅 速在植物根围定值,促进植物生长和防治病虫害的作用,也对菊酯农药有很好的降解功效。 利用这几种微生物的功效和菊酯农药共生培养,经过实验试用,证实了它对地下害虫的特效 作用和无毒无残留的机理,是理想的生物制剂。本生物杀虫生根双重保护剂不仅可以迅速(第 二天)起到杀灭消灭地下害虫如韭蛆的作用,还可以在一年内抑制地下害虫如韭蛆再次复发, 同时还是很好的生根护根产品,利用微生物不仅可以改良土壤还可以提高蔬菜的品质,增产 增收,既能消灭地下害虫如韭蛆、又能对蔬菜的根系起到保护作用以保证蔬菜的顺利生长, 同时又不会对使用者或蔬菜食用者的身体造成毒害,是有机蔬菜种植过程中的必备产品。
(2)针对杀虫菌剂的保存稳定性问题,本发明通过载体负载,对杀虫菌剂起到良好的稳 定作用,针对现有技术中的多孔载体与杀虫菌剂的亲和性和负载量不高的问题,本发明以氧 化镁为原料,通过水化法制备为氢氧化镁模板,以芘分子为碳源,以三聚氰胺-植酸超分子聚 合物为掺杂剂,以氢氧化钾为活化剂,得到掺杂剂相互连接的氮磷共掺杂多孔碳材料,经热 处理后,再以PEO-PPO-PEO三嵌段共聚物为致孔剂和模板,在所述载体前体上生成多孔硅 化层,经过二次热处理去除模板后制得氮磷共掺杂多孔碳材料和二氧化硅组成的多壳层负载 载体,一方面,相比常规多孔载体,本发明所述负载载体具有更高的生物亲和性,多壳层载 体结构为菌种担载提供了更多的负载点,提高了载体的单位负载量,同时也为所述杀虫菌剂 提供隔离缓冲作用,不仅提高其环境稳定性,同时为杀虫菌种的起始增殖提供良好的增殖环 境,促进生长繁殖并提高杀虫效果。
具体实施方式
结合以下实施例对本发明作进一步描述。
实施例1
一种复合生物杀虫剂的制备方法,包括以下步骤:
(1)单菌种培养
将乳酸菌、苏力菌、白僵菌和绿僵菌分别进行发酵培养,发酵完成后每种菌株的发酵液 中有效活菌数均应大于2.0×109cfu/mL;
按重量份数计,所述乳酸菌的培养基组成为:葡萄糖25-50份、酵母浸粉10-45份、碳 酸钙3-10份、可溶性淀粉10-50份、硫酸镁0.1-0.5份、醋酸钠0.1-0.3份、柠檬酸铵0.1-0.3 份、磷酸氢二钾0.2-0.8份、硫酸锰0.1-0.5份、吐温1-2份,去离子水溶解稀释;
按重量份数计,所述苏云金杆菌的培养基组成为:葡萄糖4-8份、酵母粉2-4份、可溶 性淀粉2-6份、蛋白胨1-4份、硫酸镁0.005-0.02份、硫酸锰0.005-0.01份、磷酸二氢钾0.1-0.3 份、氯化钠0.3-0.8份,去离子水溶解稀释;
按重量份数计,所述白僵菌的培养基组成为:葡萄糖5-15份、酵母粉2-8份、马铃薯汁 3-10份、蛋白胨5-20份、蚕粉5-10份、硫酸二氢钾1-5份、头孢霉素1-5份,去离子水溶 解稀释;
按重量份数计,所述绿僵菌的培养基组成为:葡萄糖5-15份、酵母粉4-8份,D-山梨醇 2-6份,氯化钡0.01-0.1份,维生素B 0.01-0.02份,三醋酸甘油酯3-7份,去离子水溶解稀释;
(2)复合培养
将分别发酵培养得到的乳酸菌、苏力菌、白僵菌和绿僵菌按比混合并进行复合培养;所 述乳酸菌、苏力菌、白僵菌和绿僵菌的质量比例为4:2:2:2,所述复合培养的培养温度在 22-28℃,培养时间7d。
(3)负载
加入负载载体进行菌体负载后冻干,制得所述复合生物杀虫剂;
所述负载载体的制备方法包括以下步骤:
S1、模板前处理
称取粒径小于150μm的氧化镁并干燥至恒重,将所述氧化镁分散在去离子水中,在60-80℃下恒温搅拌2-4h,分离沉淀后真空干燥制得模板材料;
S2、前体制备
称取三聚氰胺并溶解在去离子水中,加入植酸溶液,在常温下搅拌反应4-8h,分离沉淀 后干燥,得到产物A,分别称取芘、所述产物A、所述模板材料和氢氧化钾,混合后研磨至 均,将研磨混合物升温至160-180℃并保温0.5-1h,再在惰性气氛下加热至800-900℃并保温 1h,产物冷却后依次以稀的硫酸溶液和去离子水洗涤,干燥后制得载体前体;
其中,所述三聚氰胺与所述植酸溶液的质量比例为1:2.9;所述芘与所述产物A、所述 模板材料和所述氢氧化钾的混合质量比例为5:1:10:15;
S3、外层制备
将所述载体前体和PEO-PPO-PEO三嵌段共聚物溶解在醇溶剂中,加入正硅酸乙酯和盐 酸溶液,在30-35℃下保温搅拌反应1-2h,分离沉淀并干燥,得到硅化产物,将所述硅化产 物在保护气氛下以5-10℃/min的速率升温至800-900℃,保温热处理2-3h,制得所述负载载 体;
其中,所述载体前体和PEO-PPO-PEO三嵌段共聚物、所述正硅酸乙酯的质量比例为13: 1:1.8。
实施例2
一种复合生物杀虫剂的制备方法,同实施例1,区别在于,所述负载载体为轻质碳酸钙。
实施例3
一种复合生物杀虫剂的制备方法,同实施例1,区别在于,所述负载载体聚氨酯海绵。
实施例4
一种复合生物杀虫剂的制备方法,同实施例1,区别在于,不含所述负载载体。
实验例
1、对实施例1-3所述复合发酵菌剂的最大负载量和不同放置时间的活菌负载数进行测定, 测定结果如下:
将实施例1-4制备的生物杀虫剂进行杀虫效果检测,具体检测方法如下:将1300只草地 贪夜蛾3龄幼虫平均分成4组,并分别置于直径为10cm的圆形玻璃器皿中,器皿上方设置 透气纱网,以避免草地贪夜蛾逃逸,分别喷施实施例1-4的生物杀虫剂,喷施量为10mL/组。 另外一组喷施清水作为空白对照组,记录草地贪夜蛾幼虫存活数量,结果如下:
72h/只 | 96h/只 | 120h/只 | 144h/只 | |
实施例1 | 40 | 12 | 1 | 0 |
实施例2 | 68 | 52 | 28 | 19 |
实施例3 | 59 | 27 | 12 | 1 |
实施例4 | 79 | 61 | 44 | 19 |
空白 | 100 | 99 | 98 | 96 |
最后应当说明的是,以上实施例仅用以说明本发明的技术方案,而非对本发明保护范围 的限制,尽管参照较佳实施例对本发明作了详细地说明,本领域的普通技术人员应当理解, 可以对本发明的技术方案进行修改或者等同替换,而不脱离本发明技术方案的实质和范围。
Claims (10)
1.一种复合生物杀虫剂的制备方法,其特征在于,包括以下步骤:
(1)单菌种培养
将乳酸菌、苏力菌、白僵菌和绿僵菌分别进行发酵培养,发酵完成后每种菌株的发酵液中有效活菌数均应大于2.0×109cfu/mL;
(2)复合培养
将分别发酵培养得到的乳酸菌、苏力菌、白僵菌和绿僵菌按比混合并进行复合培养;
(3)负载
加入负载载体进行菌体负载后冻干,制得所述复合生物杀虫剂。
2.根据权利要求1所述的一种复合生物杀虫剂的制备方法,其特征在于,所述乳酸菌、苏力菌、白僵菌和绿僵菌的质量比例为(2-6):(0.5-3.5):(1-5):(0.5-3.5)。
3.根据权利要求1所述的一种复合生物杀虫剂的制备方法,其特征在于,所述负载载体的制备方法包括以下步骤:
S1、模板前处理
称取粒径小于150μm的氧化镁并干燥至恒重,将所述氧化镁分散在去离子水中,在60-80℃下恒温搅拌2-4h,分离沉淀后真空干燥制得模板材料;
S2、前体制备
称取三聚氰胺并溶解在去离子水中,加入植酸溶液,在常温下搅拌反应4-8h,分离沉淀后干燥,得到产物A,分别称取芘、所述产物A、所述模板材料和氢氧化钾,混合后研磨至均,将研磨混合物升温至160-180℃并保温0.5-1h,再在惰性气氛下加热至800-900℃并保温1h,产物冷却后依次以稀的硫酸溶液和去离子水洗涤,干燥后制得载体前体;
其中,所述三聚氰胺与所述植酸溶液的质量比例为1:(2.8-3.2);所述芘与所述产物A、所述模板材料和所述氢氧化钾的混合质量比例为(5-6):1:10:(14-16);
S3、外层制备
将所述载体前体和PEO-PPO-PEO三嵌段共聚物溶解在醇溶剂中,加入正硅酸乙酯和盐酸溶液,在30-35℃下保温搅拌反应1-2h,分离沉淀并干燥,得到硅化产物,将所述硅化产物在保护气氛下以5-10℃/min的速率升温至800-900℃,保温热处理2-3h,制得所述负载载体;
其中,所述载体前体和PEO-PPO-PEO三嵌段共聚物、所述正硅酸乙酯的质量比例为(12-15):1:(1.8-2)。
4.根据权利要求1所述的一种复合生物杀虫剂的制备方法,其特征在于,所述复合培养的培养温度在22-28℃,培养时间6-10d。
5.根据权利要求1所述的一种复合生物杀虫剂的制备方法,其特征在于,按重量份数计,所述乳酸菌的培养基组成为:葡萄糖25-50份、酵母浸粉10-45份、碳酸钙3-10份、可溶性淀粉10-50份、硫酸镁0.1-0.5份、醋酸钠0.1-0.3份、柠檬酸铵0.1-0.3份、磷酸氢二钾0.2-0.8份、硫酸锰0.1-0.5份、吐温1-2份,去离子水溶解稀释。
6.根据权利要求1所述的一种复合生物杀虫剂的制备方法,其特征在于,按重量份数计,所述苏云金杆菌的培养基组成为:葡萄糖4-8份、酵母粉2-4份、可溶性淀粉2-6份、蛋白胨1-4份、硫酸镁0.005-0.02份、硫酸锰0.005-0.01份、磷酸二氢钾0.1-0.3份、氯化钠0.3-0.8份,去离子水溶解稀释。
7.根据权利要求1所述的一种复合生物杀虫剂的制备方法,其特征在于,按重量份数计,所述白僵菌的培养基组成为:葡萄糖5-15份、酵母粉2-8份、马铃薯汁3-10份、蛋白胨5-20份、蚕粉5-10份、硫酸二氢钾1-5份、头孢霉素1-5份,去离子水溶解稀释。
8.根据权利要求1所述的一种复合生物杀虫剂的制备方法,其特征在于,按重量份数计,所述绿僵菌的培养基组成为:葡萄糖5-15份、酵母粉4-8份,D-山梨醇2-6份,氯化钡0.01-0.1份,维生素B 0.01-0.02份,三醋酸甘油酯3-7份,去离子水溶解稀释。
9.根据权利要求1-8之一所述的制备方法制得的复合生物杀虫剂。
10.根据权利要求9所述的复合生物杀虫剂,其特征在于,按1-2公斤/亩并加水稀释至200-400倍重量使用。
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