CN109452284A - 一种氨基寡糖素和芽孢杆菌复配制剂的制备工艺 - Google Patents
一种氨基寡糖素和芽孢杆菌复配制剂的制备工艺 Download PDFInfo
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- A01N43/16—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom six-membered rings with oxygen as the ring hetero atom
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01N37/44—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a nitrogen atom attached to the same carbon skeleton by a single or double bond, this nitrogen atom not being a member of a derivative or of a thio analogue of a carboxylic group, e.g. amino-carboxylic acids
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Abstract
本发明公开了一种氨基寡糖素和芽孢杆菌复配制剂的制备工艺,采用蜡状芽孢杆菌和地衣芽孢杆菌混合发酵的方法得到固体发酵产物,将该固体发酵产物与植物生长调节剂氨基寡糖素、腐植酸、聚谷氨酸以一定的比例混合均匀,然后通过离心喷雾干燥得到芽孢杆菌与氨基寡糖素复配制剂。该菌剂施用于作物后,不仅可以起到防病、治病的作用,提高作物的抗逆性,还可以对植物的生长产生调节作用,大幅度提高农作物的产量。本生产工艺简单、设备投资少、无残留物污染,具有较高的应用价值。
Description
技术领域
本发明涉及了一种芽孢杆菌和氨基寡糖素复配制剂的制备工艺,属于生物技术领域。
技术背景
我国是农药生产量和使用量最大的国家,近年来,我国化学农药残留导致的食品安全问题非常突出。长期依赖和大量使用化学农药带来的环境污染和食品安全等重大问题,给农业可持续发展带来诸多不利的影响,成为社会经济发展中亟待解决的最大问题。芽孢杆菌是我国农业部正式批准生产生物制品的菌种,该菌株具有防病和促进植物生长的作用,在国内外已经获得大面积推广应用,广泛应用于饲料、农业、医药保健和食品等各领域。
蜡状芽孢杆菌是芽孢杆菌属的一种,是比较理想的微生态制剂菌种,由于其在自然界广泛分布,蜡状芽孢杆菌产生孢子时能分泌许多种酶类物质和生长因子,稳定性极好,具有耐氧化、抗挤压、耐高温、耐酸等特征此外,蜡状芽孢杆菌抗逆性很强,能够储藏的时间较长。此外,蜡状芽孢杆菌不污染环境,不破坏生态。地衣芽孢杆菌同蜡状芽孢杆菌一样可以产生多种活性物质,地衣芽胞杆菌可产生多种抑菌物质,主要为多肽类、抗菌蛋白以及几丁质酶等蛋白类抗菌物质,可以有效抑制一些植物病原菌。
但是尽管芽胞杆菌以内生孢子的形式存在,且对加工、运输过程中的干燥、高温、高压、氧化等不良环境因素的抵抗能力强、稳定性高,但目前芽孢杆菌制剂仍普遍存在在加工和运输过程中以及有效期内活菌含量降低、货架期短等问题。
氨基寡糖素誉为植物疫苗,是继人体疫苗、动物疫苗之后的第三类疫苗。寡糖素不但可以提高植物的抗逆性(包括抗病性、抗寒和抗旱性等),同时还可以促进作物的生长,提高作物的品质。同时,还具有调节植物生长的作用,在作物增产和提高品质方面也有很大的潜力。其具有环境相容性好、超高效、安全性好、避免抗药性、作用广泛等显著优势,在小麦、玉米等粮食作物及蔬菜瓜果上都具有广阔的应用前景。
发明内容
本发明的目的是提供一种氨基寡糖素与芽孢杆菌复配制剂的制备工艺,该复配制剂施用于作物后,不仅可以起到防病、治病的作用,提高作物的抗逆性,还可以对植物的生长产生调节作用,大幅度提高农作物的产量。
本发明的技术方案为:一种氨基寡糖素和芽孢杆菌复配制剂的制备工艺,其具体步骤为:
(1)将蜡状芽孢杆菌(Bacillus cereus CGMCC No.4348)和地衣芽孢杆菌(Bacillus licheniformis CGMCC No.6155)分别种接于各自的斜面培养基中培养20~24h,培养温度均为30~37℃,然后用灭菌生理盐水冲洗各自的斜面,分别制成浓度为2-4×106cfu/mL的菌悬液;
(2)将步骤(1)中制备的两种菌的菌悬液分别接种于各自的液体培养基中进行培养,菌悬液体积和液体培养基体积的比例为8~13%,培养时间为20~24h,培养条件均为30~37℃,转速为120~180rpm;
(3)取步骤(2)中培养好的蜡状芽孢杆菌和地衣芽孢杆菌接种于灭菌后的固体培养基中,蜡状芽孢杆菌和地衣芽孢杆菌的体积比为3~4:5,两种芽孢杆菌的总体积和固体培养基体积的比例35~40%,保持湿度60~70%,温度34~37℃,培养30~36h的到固体发酵产物;
(4)发酵结束,将固体发酵产物与氨基寡糖素、腐植酸、聚谷氨酸按比例混合,搅拌均匀,进行离心喷雾干燥,制得氨基寡糖素和芽孢杆菌复配制剂。
优选步骤(1)中的蜡状芽孢杆菌斜面培养基配方为:酵母膏3~5g/L,大豆蛋白胨7~10g/L,玉米淀粉12~15g/L,NaCl 7~10g/L,pH 7.0~7.5;地衣芽孢杆菌斜面培养基配方为:酵母膏3~5g/L,胰蛋白胨8~10g/L,琼脂粉15~20g/L,NaCl 3~5g/L,pH 7.0~7.5。
优选步骤(1)中灭菌生理盐水的质量浓度为10~20%。
优选步骤(2)中的蜡状芽孢杆菌的液体培养基配方为:蔗糖7.5~10g/L、蛋白胨10~15g/L、磷酸氢二钾1~2g/L、硫酸铵1~2g/L、维生素VH 1~2g/L,pH 7.0~7.5;地衣芽孢杆菌的液体培养基配方为:糖蜜15~20g/L、蛋白胨10~15g/L、酵母膏3~6g/L、磷酸二氢钾1~2g/L、硫酸镁1~2g/L、维生素VH 1~2g/L。
优选步骤(3)固体培养基成分及重量百分比分别为:米糠20~25%,菜粕20~25%,磷酸一铵1~2%,玉米皮渣5~10%,水41~51%。
优选步骤(4)中固体发酵产物、氨基寡糖素、腐植酸和聚谷氨酸的质量百分比分别为:固体发酵产物75~85%,氨基寡糖素10~15%,腐植酸2.5~5%,聚谷氨酸2.5~5%。
优选步骤(4)中的干燥条件为:进风温度210~230℃,出口温度60~80℃,离心喷雾转速为8000~12000转/分。
本发明所述的蜡状芽孢杆菌,其分类命名为蜡状芽孢杆菌,菌种的拉丁学名是Bacillus scereus,参据的微生物:NJYH63305,保藏日期是2010年11月15日,保藏中心登记入册编号是CGMCC No.4348。
所述的地衣芽孢杆菌,其分类命名为地衣芽孢杆菌,菌种的拉丁学名是Bacilluslicheniformis,参据的微生物是NJWGYH833051,保藏日期是2012年05月25日,保藏中心登记入册编号是CGMCC No.6155。以此菌种作为生产菌株。
CGMCC No.6155菌株具有下述性质:
1、培养特征:
该菌落在营养琼脂上37℃培养24h,形成圆形凸起菌落,菌落边缘整齐,有光泽,呈乳白色、微透明,菌落表面偏光滑。
2、菌体及芽孢的形态特征:
在营养琼脂培养基平板上37℃培养,16-25h形成脱落的芽孢,到25-35h芽孢大部分脱落。菌体呈链状杆菌,菌体大小约为0.8um×2.0um,椭圆形或柱状芽孢,芽孢位于杆菌中心或偏中生。
3、生理生化性质:
表1 CGMCC No.6155菌株的生理生化性质
+:为可利用;-:为不可利用
有益效果:
本发明通过对蜡状芽孢杆菌和地衣芽孢杆菌分别活化培养,种子培养,然后在液体发酵罐中进行混合发酵,提高两种菌的活菌数,再将混合发酵液接种到特定的固体培养基中进行混合菌种的固体扩大培养,可以有效提高菌体存活率,固体发酵产物与氨基寡糖素、腐植酸、聚谷氨酸按一定的比例复配,对农作物的抑菌效率比单纯使用蜡状芽孢杆菌或地衣芽孢杆菌芽孢显著提高;还可以对植物的生长产生调节作用,大幅度提高农作物的产量。本生产工艺简单、设备投资少、无残留物污染,更加适用于工业化生产的需求。
保藏信息
上述蜡样芽孢杆菌Bacillus cereus CGMCC No.4348是由本实验室选育并保藏于中国微生物菌种保藏管理委员会普通微生物中心(北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所),其简称为CGMCC,登记入册的编号是CGMCC No.4348,保藏日期是:2010年11月15日(见本发明人申请的专利,申请号为201010579133.0)。
上述地衣芽孢杆菌Bacillus licheniformis CGMCC No.6155是由本实验室选育并保藏于中国微生物菌种保藏管理委员会普通微生物中心(北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所),其简称为CGMCC,登记入册的编号是CGMCC No.6155,保藏日期是:2012年5月25日。
具体实施方式
下面结合实施例对本发明作进一步详尽的说明,但实施案例并不对本发明做任何形式的限定。
实施例1:
(1)芽孢杆菌的活化
将蜡状芽孢杆菌、地衣芽孢杆菌分别种接种于各自的斜面培养基中,在32℃下培养24h,然后用质量浓度为15%的灭菌生理盐水冲洗各自的斜面,分别制成浓度为2×106cfu/mL的菌悬液。蜡状芽孢杆菌的斜面培养基配方为:酵母膏3.5g/L,大豆蛋白胨8.5g/L,玉米淀粉12g/L,NaCl 8g/L,pH值为7.0;地衣芽孢杆菌的斜面培养基配方为:酵母膏3.5g/L,胰蛋白胨8g/L,琼脂粉15g/L,NaCl 3.5g/L,pH值为7.0。
(2)芽孢杆菌的摇瓶培养:
将步骤(1)中制备的两种菌的菌悬液分别接种于各自的液体培养基中进行培养,菌悬液体积和液体培养基体积的比例为10%,32℃下在摇瓶中培养24h,摇床转速为150rpm。所述蜡状芽孢杆菌摇瓶中液体培养基配方为:蔗糖7.5g/L、鱼蛋白胨10g/L、磷酸氢二钾1.5g/L、硫酸铵1.5g/L、维生素VH 1.5g/L,pH值为7.0。地衣芽孢杆菌摇瓶中液体培养基配方为:糖蜜15g/L、鱼蛋白胨10g/L、酵母膏3.5g/L、磷酸二氢钾1.5g/L、硫酸镁1.5g/L、维生素VH 1.5g/L。
(3)菌种混合发酵
将步骤(2)中培养好的蜡状芽孢杆菌和地衣芽孢杆菌接种于灭菌后的固体培养基中,蜡状芽孢杆菌和地衣芽孢杆菌的体积比为4:5,两种芽孢杆菌的总体积和固体培养基体积的比例35%,保持湿度60%,温度34℃,培养36h得到固体发酵产物。所述固体培养基成分及质量百分比为:米糠23%,菜粕21%,磷酸一铵1%,玉米皮渣5%,水51%。
(4)芽孢杆菌与氨基寡糖素的复配
发酵结束,将固体发酵产物与氨基寡糖素、腐植酸、聚谷氨酸以如下重量百分比混合:固体发酵产物85%,氨基寡糖素10%,腐植酸2.5%,聚谷氨酸2.5%。混合后搅拌均匀,进行离心喷雾干燥,干燥条件为:进风温度220℃,出口温度70℃,离心喷雾转速为10000转/分。
(5)效果:
抗病效果:施用该菌剂于农作物之后,对棉花枯黄萎病的抑制率可达到67.9%;对番茄晚疫病、番茄灰霉病的抑制率分别可达73.5%,78.2%;对小麦白粉病的抑制率可达65.7%。
促生效果:经拌种处理后的番茄苗的苗成活率、平均株高和株重都显著高于清水处理的番茄苗,与经清水处理的番茄苗相比其株干重增加了30.2%,根重增加了9.8%。
实施例2:
(1)芽孢杆菌的活化
将蜡状芽孢杆菌、地衣芽孢杆菌分别种接种于各自的斜面培养基中,在37℃下培养20h,然后用质量浓度为13%的灭菌生理盐水冲洗各自的斜面,分别制成浓度为4×106cfu/mL的菌悬液。所述蜡状芽孢杆菌的斜面培养基配方为:酵母膏5g/L,大豆蛋白胨10g/L,玉米淀粉15g/L,NaCl 10g/L,pH值为7.2;地衣芽孢杆菌的斜面培养基配方为:酵母膏5g/L,胰蛋白胨10g/L,琼脂粉20g/L,NaCl 5g/L,pH值为7.2。
(2)芽孢杆菌的摇瓶培养:
将步骤(1)中制备的两种菌的菌悬液接种于各自的液体培养基中进行培养,菌悬液体积和液体培养基体积的比例为8%,37℃下在摇瓶中培养20h,摇床转速为180rpm。所述蜡状芽孢杆菌摇瓶中液体培养基配方为:蔗糖10g/L、鱼蛋白胨15g/L、磷酸氢二钾2g/L、硫酸铵2g/L、维生素VH 2g/L,pH值为7.2。地衣芽孢杆菌摇瓶中液体培养基配方为:糖蜜18g/L、鱼蛋白胨12g/L、酵母膏5g/L、磷酸二氢钾1.8g/L、硫酸镁1.8g/L、维生素VH 1.8g/L。
(3)菌种的混合发酵
将步骤(2)中培养好的蜡状芽孢杆菌和地衣芽孢杆菌接种于灭菌后的固体培养基中,蜡状芽孢杆菌和地衣芽孢杆菌的体积比为3:5,两种芽孢杆菌的总体积和固体培养基体积的比例40%,保持湿度65%,温度37℃,培养30h得到固体发酵产物。所述固体培养基成分及质量百分比为:米糠20%,豆粕24%,磷酸一铵2%,玉米皮渣8%,水46%。
(4)芽孢杆菌与氨基寡糖素的复配
发酵结束,将固体发酵产物与氨基寡糖素、腐植酸、聚谷氨酸以如下重量百分比混合:固体发酵产物82%,氨基寡糖素12%,腐植酸3%,聚谷氨酸3%。混合后搅拌均匀,进行离心喷雾干燥,干燥条件为:进风温度210℃,出口温度60℃。离心喷雾转速为12000转/分。
(5)效果:
抗病效果:施用该菌剂于农作物之后,对棉花枯黄萎病的抑制率可达到69.3%;对番茄晚疫病、番茄灰霉病的抑制率分别可达76.2%,80.7%;对小麦白粉病的抑制率可达68.4%。
促生效果:经拌种处理后的辣椒苗的苗成活率、平均株高和株重都显著高于清水处理的番茄苗,与经清水处理的番茄苗相比其株干重增加了27.2%,根重增加了10.7%。
实施例3:
(1)芽孢杆菌的活化
将蜡状芽孢杆菌、地衣芽孢杆菌分别种接种于各自的斜面培养基中,在35℃下培养22h,然后用质量浓度为18%的灭菌生理盐水冲洗各自的斜面,分别制成浓度为3×106cfu/mL的菌悬液。蜡状芽孢杆菌的斜面培养基配方为:酵母膏3g/L,大豆蛋白胨7g/L,玉米淀粉12g/L,NaCl 7g/L,pH值为7.5;地衣芽孢杆菌的斜面培养基配方为:酵母膏3g/L,胰蛋白胨9g/L,琼脂粉18g/L,NaCl 3g/L,pH值为7.5。
(2)芽孢杆菌的摇瓶培养:
将步骤(1)中制备的两种菌的菌悬液接种于各自的液体培养基中进行培养,菌悬液体积和液体培养基体积的比例为13%,35℃下在摇瓶中培养22h,摇床转速为130rpm。所述蜡状芽孢杆菌摇瓶中液体培养基配方为:蔗糖8g/L、鱼蛋白胨12g/L、磷酸氢二钾1.2g/L、硫酸铵1.2g/L、维生素VH 1.2g/L,pH值为7.5。地衣芽孢杆菌摇瓶中液体培养基配方为:糖蜜20g/L、鱼蛋白胨15g/L、酵母膏6g/L、磷酸二氢钾2g/L、硫酸镁2g/L、维生素VH 2g/L。
(3)菌种的混合发酵
将步骤(2)中培养好的蜡状芽孢杆菌和地衣芽孢杆菌接种于灭菌后的固体培养基中,蜡状芽孢杆菌和地衣芽孢杆菌的体积比为7:10,两种芽孢杆菌的总体积和固体培养基体积的比例37%,保持湿度70%,温度35℃,培养32h得到固体发酵产物。所述固体培养基成分及质量百分比为:米糠25%,豆粕22.5%,磷酸一铵1.5%,玉米皮渣10%,水41%。
(4)芽孢杆菌与氨基寡糖素的复配
发酵结束,将固体发酵产物与氨基寡糖素、腐植酸、聚谷氨酸以如下重量百分比混合:固体发酵产物75%,氨基寡糖素15%,腐植酸5%,聚谷氨酸5%。混合后搅拌均匀,进行离心喷雾干燥,干燥条件为:进风温度230℃,出口温度80℃离心喷雾转速为8000转/分。
(5)效果:
抗病效果:施用该菌剂于农作物之后,对棉花枯黄萎病的抑制率可达到68.3%;对番茄晚疫病、番茄灰霉病的抑制率分别可达75.1%,79.4%;对小麦白粉病的抑制率可达66.8%。
促生效果:经拌种处理后的番茄苗的苗成活率、平均株高和株重都显著高于清水处理的番茄苗,与经清水处理的番茄苗相比其株干重增加了35.6%,根重增加了13.1%。
Claims (7)
1.一种氨基寡糖素和芽孢杆菌复配制剂的制备工艺,其具体步骤为:
(1)将蜡状芽孢杆菌和地衣芽孢杆菌分别种接于各自的斜面培养基中培养20~24h,培养温度均为30~37℃,然后用灭菌生理盐水冲洗各自的斜面,分别制成浓度为2-4×106cfu/mL的菌悬液;
(2)将步骤(1)中制备的两种菌的菌悬液分别接种于各自的液体培养基中进行培养,菌悬液体积和液体培养基体积的比例为8~13%,培养时间为20~24h,培养条件均为30~37℃,转速为120~180rpm;
(3)取步骤(2)中培养好的蜡状芽孢杆菌和地衣芽孢杆菌接种于灭菌后的固体培养基中,蜡状芽孢杆菌和地衣芽孢杆菌的体积比为3~4:5,两种芽孢杆菌的总体积和固体培养基体积的比例35~40%,保持湿度60~70%,温度34~37℃,培养30~36h的到固体发酵产物;
(4)发酵结束,将固体发酵产物与氨基寡糖素、腐植酸、聚谷氨酸按比例混合,搅拌均匀,进行离心喷雾干燥,制得氨基寡糖素和芽孢杆菌复配制剂。
2.如权利要求1所述的制备工艺,其特征在于,步骤(1)中的蜡状芽孢杆菌斜面培养基配方为:酵母膏3~5g/L,大豆蛋白胨7~10g/L,玉米淀粉12~15g/L,NaCl 7~10g/L,pH7.0~7.5;地衣芽孢杆菌斜面培养基配方为:酵母膏3~5g/L,胰蛋白胨8~10g/L,琼脂粉15~20g/L,NaCl 3~5g/L,pH 7.0~7.5。
3.如权利要求1所述的制备工艺,其特征在于,步骤(1)中灭菌生理盐水的质量浓度为10~20%。
4.如权利要求1所述的制备工艺,其特征在于,步骤(2)中的蜡状芽孢杆菌的液体培养基配方为:蔗糖7.5~10g/L、蛋白胨10~15g/L、磷酸氢二钾1~2g/L、硫酸铵1~2g/L、维生素VH 1~2g/L,pH 7.0~7.5;地衣芽孢杆菌的液体培养基配方为:糖蜜15~20g/L、蛋白胨10~15g/L、酵母膏3~6g/L、磷酸二氢钾1~2g/L、硫酸镁1~2g/L、维生素VH 1~2g/L。
5.如权利要求1所述的制备工艺,其特征在于,步骤(3)固体培养基成分及重量百分比分别为:米糠20~25%,菜粕20~25%,磷酸一铵1~2%,玉米皮渣5~10%,水41~51%。
6.如权利要求1所述的制备工艺,其特征在于,步骤(4)中固体发酵产物、氨基寡糖素、腐植酸和聚谷氨酸的质量百分比分别为:固体发酵产物75~85%,氨基寡糖素10~15%,腐植酸2.5~5%,聚谷氨酸2.5~5%。
7.如权利要求1所述的制备工艺,其特征在于,步骤(4)中的干燥条件为:进风温度210~230℃,出口温度60~80℃,离心喷雾转速为8000~12000转/分。
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