CN117678597A - 一种生防微生物种衣剂及其制备方法与应用 - Google Patents
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Abstract
本发明公开了一种生防微生物种衣剂及其制备方法与应用。本发明提供的生防微生物种衣剂,包括如下组成:生防微生物及其载体;所述载体为花粉微凝胶。本发明利用花粉微凝胶作为生防微生物种衣剂中的微生物载体及成膜剂,更有利于提高生防微生物的贮存稳定性,且所用原料来源广泛,绿色环保,制备工艺简单,在微生物种衣剂的开发领域具有较大的应用前景。
Description
技术领域
本发明属于播种技术及微生物制剂技术领域,主要涉及一种生防微生物种衣剂及其制备方法与应用。
背景技术
随着现代农业的发展,利用生防微生物进行包衣已逐渐成为国内外重要的病害防治策略。与化学防治相比,该技术具有绿色环保,高效价廉的优势,其关键在于生防微生物的开发与应用。虽然越来越多的功能微生物被发掘出来,但真正能形成商业化产品的菌株却很少,其中一个主要原因就是生防菌种衣剂稳定性差,主要表现为货架期短、加工过程容易造成菌体死亡或者活性降低。
将生防微生物进行包埋再进行包衣处理是目前提高微生物包衣性能的有效策略,即利用物理或者化学方法将微生物细胞包埋在有机高分子聚合物的凝胶或微囊中,从而降低环境因素对微生物活性造成的影响,可极大提高功能微生物在包衣过程和贮存过程的存活率,延长货架期,并可通过调整壁材的性能达到控制释放的效果。
目前应用较多的包埋材料主要包括合成高分子材料以及天然高分子材料。其中化学合成的高分子材料性能可控,能较大程度的满足生防菌对载体功能需求,但普遍存在制备工艺严格,成本较高的问题。与合成材料相比,大多数天然来源的高分子材料具有生态友好,可生物降解等特点,如木质素类,淀粉,海藻酸钠,壳聚糖及其衍生材料等都受到了广泛的关注,并且发展了对应的药物装载方法,包括复凝聚法,喷雾干燥法,溶剂蒸发法等,然而大多数工艺较为复杂或存在适用范围有限等问题。因此,发展新颖的良好的生物相容性的包埋材料以及简洁的制备工艺成为了该领域的研究热点。
近年来,植物花粉微凝胶作为一种新型的生物质材料,以其独特的形貌结构和理化特性受到了越来越多的关注。花粉微凝胶是以植物的花粉为原料,经过脱脂、去除胞浆质和碱煮等处理后形成的具有花粉外壳的微凝胶材料。目前,花粉微凝胶主要报道应用于3D打印、印刷材料等领域,但在生物防治领域尚未有相关研究。
发明内容
本发明的目的是提供一种生防微生物种衣剂,其利用花粉微凝胶作为微生物载体及成膜剂,所得种衣剂有利于提高生防微生物的贮存稳定性,且所用原料来源广泛,绿色环保,制备工艺简单,在微生物种衣剂开发领域具有较大的应用前景。
为实现上述目的,本发明提供如下技术方案:
第一方面,本发明提供一种生防微生物种衣剂,包括如下组成:生防微生物及其载体;所述载体为花粉微凝胶。
所述花粉微凝胶是由天然花粉制得。所述天然花粉为油菜花粉、向日葵花粉、蒲公英花粉、茶花花粉和松花粉中的一种或多种,优选为茶花花粉。
所述生防微生物为枯草芽胞杆菌、多粘类芽孢杆菌、解淀粉芽孢杆菌或蜡质芽孢杆菌中的一种或多种。示例性地,所述生防微生物为枯草芽胞杆菌ZF71(Bacillus subtilis ZF71)。
所述生防微生物种衣剂中,所述生防微生物的含量为107-1011CFU/g。
第二方面,本发明进一步提供所述生防微生物种衣剂的制备方法,包括如下步骤:
S1、将天然花粉脱脂,碱处理,得到花粉微凝胶;
S2、将所述花粉微凝胶加入含有生防微生物的菌悬液中分散,涡旋处理,孵育,得到负载生防微生物的种衣剂。
步骤S1中,所述脱脂使用的有机溶剂为丙酮、乙酸乙酯、乙醇和甲醇中的一种或多种,优选为丙酮。
所述有机溶剂的体积浓度范围为80-100%,优选为100%。
所述天然花粉与所述有机溶剂的比例为:1 g:8-50 mL;优选为1 g:10 mL。
所述脱脂的条件为:30-80 ℃下搅拌1-3h,优选为50℃下搅拌1h,搅拌速度为100-1000转/分钟,优选为800转/分钟。
所述碱为氢氧化钠、氢氧化钾和氢氧化钙中的一种或多种,优选为氢氧化钠。
所述碱处理使用的碱液的浓度为1(w/v)%-15(w/v)%,优选为10(w/v)%。
所述脱脂花粉与所述碱液的比例为:1g:5-20 mL。
所述碱处理的条件为:60-90 ℃下搅拌3-5h,优选为80℃下搅拌3h。
所述花粉微凝胶的pH为7-7.5。
步骤S2中,所述菌悬液是由生防微生物加入液体培养基中培养形成的;其中:
所述液体培养基由如下重量百分比组分组成:酵母提取物0.1-0.8%,胰蛋白胨0.5-2%,葡萄糖0.2-0.5%,氯化钠0.5 -1.5%,pH 7.2-7.5,余量为水。
所述培养的时间为10-36h,优选为20h。
所述菌悬液的浓度范围为1亿-1000亿CFU/g,优选为1000亿CFU/g。
所述花粉微凝胶与所述菌悬液的质量比为1 g:10-50 g,优选为1 g:40 g。
所述涡旋处理的条件为:速度为1000-3000转/min,优选为2000转/min,时间为10-30min,优选为10min。
所述孵育的条件为:温度为20-30℃,时间为5-48h,优选为12-24h。
步骤S2中,将完成孵育的菌悬液过滤、洗涤,加入助剂,得到生防微生物种衣剂。
第三方面,本发明进一步提供一种包衣种子,包括如下组成:种子和所述生防微生物种衣剂。
所述生防微生物种衣剂和所述种子的质量比为1:5-1:200,优选为1:5。
所述包衣种子的制备方法,包括如下步骤:将所述生防微生物种衣剂加入水中,得到分散液;将种子浸入所述分散液中,晾干,得到所述包衣种子。
本发明取得的有益效果如下:
1、本发明首次将植物花粉微凝胶作为微生物种衣剂的载体及成膜剂,既可以促进生防菌在微凝胶表面形成生物膜,增强生防菌抗逆能力,提高种衣剂贮存稳定性,又可以利用微凝胶的成膜特性将装载微生物的花粉微凝胶包裹于种子表面,无需使用合成的高分子成膜剂,所用原料来源广泛且绿色环保,是一种绿色环保的微生物种子包衣技术。
2、本发明巧妙的利用了芽孢杆菌趋向于在花粉微凝胶表面形成生物膜的特点,通过简单的孵育就可以有效的对生防菌进行装载,与喷雾干燥,复凝聚法等常见的生防菌包埋技术相比,工艺简单,制备条件温和。
附图说明
图1为实施例1所提供的茶花粉微凝胶的外观及其微观形貌图;图中,A代表茶花粉微凝胶的外观,B、C代表茶花粉微凝胶的扫描电子显微镜照片,200倍和2000倍放大照片。
图2为天然茶花粉的微观形貌图。
图3为实施例2所提供的微生物种衣剂的外观及其微观形貌图;图中,A代表微生物种衣剂的外观,B、C代表微生物种衣剂的扫描电子显微镜照片,红色箭头指示的圆柱体为枯草芽孢杆菌ZF71。
图4为实施例2所提供的微生物种衣剂在不同温度下的贮存稳定性对比图。
图5为实施例3中未包衣的豇豆种子和包衣种子的电子显微镜照片。
具体实施方式
下面结合具体实施例对本发明作进一步阐述,但本发明并不限于以下实施例。
下述实施例中所使用的实验方法如无特殊说明,均为常规方法。
下述实施例中所用的试剂、材料、仪器等,如无特殊说明,均可从商业途径得到。
实施例1、茶花粉微凝胶的制备
将天然茶花花粉(10g)置于水(200mL)中,用玻璃棒搅拌至完全溶于水,抽滤除水后加入热丙酮(80 mL,50℃)搅拌(800转/分)1h,抽滤除去丙酮,加入10(w/v)%氢氧化钠溶液(110 mL)搅拌(80℃)3h以除去花粉内容物,同时形成茶花粉微凝胶,抽滤除去氢氧化钠溶液,并加入水(150 mL)涡旋分钟后抽滤,如此反复清洗3-5遍,直至茶花粉微凝胶pH7-7.5。
实施例2、生防菌(多粘类芽孢杆菌ZF129)种衣剂的制备
将活化后的枯草芽胞杆菌ZF71(Bacillus subtilis ZF71)在LB液体培养基中摇培20小时,所得菌悬液的浓度为1000亿CFU/g;之后加入实施例1所得的茶花粉微凝胶(10g),涡旋混匀(2000转/分,10分钟),之后置于培养箱(25℃)培养12小时,对所得样品溶液进行离心(3000转/分,6分钟),倒掉上清液,加入水(100 mL)离心(3000转/分,6分钟)清洗两次,最后倒掉上清液,得到凝胶状的微生物种衣剂。
实施例3、包衣种子的制备
将10g豇豆种子用10%的次氯酸钠溶液浸泡3分钟后晾干;称取10g实施例2所得的微生物种衣剂,加入50g蒸馏水,轻微搅拌混匀,得到微生物种衣剂分散液;再将晾干的豇豆种子浸入到微生物种衣剂分散液中,取出,自然晾干,得到包衣种子。
效果验证:
1、茶花粉微凝胶的形貌特征
如图1A所示,所制备的花粉微凝胶经过离心后团聚成凝胶状,外观呈淡黄色,具有一定的粘附性,放在倒立的玻璃样品瓶中不会滑落下来。
如图1B、图1C所示,通过扫描电子显微镜观察,可以看出花粉微凝胶颗粒已经呈现出片状结构,三个花粉管萌发孔的三个裂缝将整个片状结构分成三瓣。这种变化是因为花粉壳在碱溶液处理过程中逐渐变软形成微凝胶形态,吸附了大量水分,而在电镜观察之前需要将样品先干燥处理,样品在常温干燥过程中逐渐失水,导致花粉颗粒结构塌陷形成片状。这也从一方面证明了花粉微凝胶干燥后的成膜特性。
2、天然茶花粉的微观形貌
如图2所示,天然茶花粉呈圆球形,多数颗粒粒径在30-40微米之间,某些花粉已经裂开,可以看出是花粉管萌发孔的位置。颗粒表面可以看出花粉鞘的结构,该结构主要由脂质、类脂质和少量蛋白等成分组成。从放大的图片来看,颗粒表面分布了很多孔隙,孔径多在1μm以下。
3、微生物种衣剂的形貌特征
如图3所示,实施例2中制备的微生物种衣剂外观呈黄褐色,从扫描电镜图像来看,花粉微凝胶的颗粒表面布满了大量的生防菌(箭头所指),由此可知,生防菌可以很好的吸附于花粉微凝胶上。
4、微生物种衣剂的贮存稳定性测定
将50g实施例2所得的微生物种衣剂置于血清瓶中,于25℃条件进行贮存,每隔一定时间测定其中的活菌数。将生防菌发酵液作为对照。
结果如图4所示,花粉微凝胶种衣剂的活菌数在贮存42天后,由7.2logCFU/g减少到6.7logCFU/g,而ZF71发酵液的活菌数载贮存42天后,7.2logCFU/g减少到0.2logCFU/g。由此说明,花粉微凝胶明显增强了生防菌的贮存稳定性。
5、包衣豇豆种子的结构特征
如图5所示,豇豆裸种表面比较平滑,经过微生物种衣剂包衣后,可以看到种子表面形成了一层均匀的薄膜,放大倍数的照片可以看出该薄膜是花粉微凝胶干燥成片状后堆叠而成。由此可知,该种衣剂具有良好的包衣成膜性。
虽然,上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述,但在本发明基础上,可以对之作一些修改或改进,这对本领域技术人员而言是显而易见的。因此,在不偏离本发明精神的基础上所做的这些修改或改进,均属于本发明要求保护的范围。
Claims (10)
1.一种生防微生物种衣剂,包括如下组成:生防微生物及其载体;所述载体为花粉微凝胶。
2.根据权利要求1所述的生防微生物种衣剂,其特征在于:所述花粉微凝胶由天然花粉制得。
3.根据权利要求2所述的生防微生物种衣剂,其特征在于:所述天然花粉为油菜花粉、向日葵花粉、蒲公英花粉、茶花花粉和松花粉中的一种或多种。
4.根据权利要求1所述的生防微生物种衣剂,其特征在于:所述生防微生物为枯草芽胞杆菌、多粘类芽孢杆菌、解淀粉芽孢杆菌或蜡质芽孢杆菌中的一种或多种。
5.根据权利要求1所述的生防微生物种衣剂,其特征在于:所述生防微生物种衣剂中,所述生防微生物的含量为107-1011CFU/g。
6.权利要求1-5任一项所述的生防微生物种衣剂的制备方法,包括如下步骤:
S1、将天然花粉脱脂,碱处理,得到花粉微凝胶;
S2、将所述花粉微凝胶加入含有生防微生物的菌悬液中分散,涡旋处理,孵育,得到负载生防微生物的种衣剂。
7.根据权利要求6所述的制备方法,其特征在于:步骤S1中,所述脱脂使用的有机溶剂为丙酮、乙酸乙酯、乙醇和甲醇中的一种或多种;
所述天然花粉与所述有机溶剂的比例为:1 g:8-50 mL;
所述脱脂的条件为:30-80 ℃下搅拌1-3h,搅拌速度为100-500转/秒。
8.根据权利要求6所述的制备方法,其特征在于:步骤S1中,所述碱为氢氧化钠、氢氧化钾和氢氧化钙中的一种或多种;
所述碱处理使用的碱液的浓度为1(w/v)%-15(w/v)%;
所述脱脂花粉与所述碱液的比例为:1g:5-20 mL;
所述碱处理的条件为:60-90 ℃下搅拌3-5h。
9.根据权利要求6所述的制备方法,其特征在于:步骤S2中,所述菌悬液是由生防微生物加入液体培养基中培养形成的;
所述液体培养基由如下重量百分比组分组成:酵母提取物0.1-0.8%,胰蛋白胨0.5-2%,葡萄糖0.2-0.5%,氯化钠0.5-1.5%,pH 7.2-7.5,余量为水;
所述培养的时间为12-36h;
所述菌悬液的浓度范围为1亿-1000亿CFU/g;
所述花粉微凝胶与所述菌悬液的质量比为1 g:10-50 g;
所述涡旋处理的条件为:速度为1000-3000转/min,时间为10-30min;
所述孵育的条件为:温度为20-30℃,时间为5-48h。
10.一种包衣种子,包括如下组成:种子和权利要求1-5任一项所述生防微生物种衣剂。
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