CN115011527A - 一种基于解淀粉芽孢杆菌的复合菌制剂及其制备方法 - Google Patents
一种基于解淀粉芽孢杆菌的复合菌制剂及其制备方法 Download PDFInfo
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Abstract
本发明涉及一种微生物菌剂技术领域,尤其涉及一种基于解淀粉芽孢杆菌的复合菌制剂及其制备方法。本发明要解决的技术问题是提供一种能够有效抑制弧菌和蓝藻,提高水产免疫力的一种基于解淀粉芽孢杆菌的复合菌制剂及其制备方法。本发明所述的复合菌剂,通过组合配比发酵,能够有效抑制弧菌、蓝藻的繁殖;同时可以调节藻相和菌相以及降低氨氮、亚硝酸盐水质指标,各菌株之间相互影响,还能更充分的利用培养基中的各种营养成分,添加到水产饲料中后,可以调节水产肠道的菌群平衡,提高水产免疫力。
Description
技术领域
本发明涉及一种微生物菌剂技术领域,尤其涉及一种基于解淀粉芽孢杆菌的复合菌制剂及其制备方法。
背景技术
每到夏天,是虾塘蓝藻爆发的时候,近几年来,对虾早期死亡综合症,中后期白便、慢料、偷死、甚至暴死等问题频发,也一直反复困扰着养殖户。国内外专家经过长期研究普遍认为,新兴的对虾早期死亡综合症的爆发与弧菌有关,弧菌超标往往是对虾偷死病的前兆,也是其他水产养殖的病害之一。弧菌是普遍存在于水体、虾体内的一种正常菌群之一,也是一种条件性的致病菌。在我们的养虾过程当中,如果一些养殖条件变差,比如:水体环境恶化、对虾体质变差等情况发生,那么水体当中、虾体内的弧菌就会迅速繁殖,进而诱发各种对虾疾病。
很多虾农都试过以菌抑菌的防治方法,以优势菌群抵制弧菌的生存,但是市面上很多液态的菌种都携带有糖的成份,而糖能促进弧菌的繁殖,而且没有提高水产免疫力的功能,鉴于此,亟需研发一种能够有效抑制弧菌和蓝藻繁殖,提高水产免疫力,益于水产生长的复合菌制剂。
发明内容
本发明为了克服弧菌和蓝藻爆发,危害水产健康,市面上抑菌制剂药效不佳的缺点,本发明要解决的技术问题是提供一种能够有效抑制弧菌和蓝藻,提高水产免疫力,益于水产生长的基于解淀粉芽孢杆菌的复合菌制剂及其制备方法。
为了解决上述技术问题,本发明提供了这样一种基于解淀粉芽孢杆菌的复合菌制剂及其制备方法,能够有效抑制弧菌、蓝藻的繁殖;同时可以调节藻相和菌相和降低氨氮、亚硝酸盐水质指标,提高水产免疫力和生长能力。
本发明通过以下技术方案实现:
一种复合菌剂,以细胞个数记,包括以下组分:解淀粉芽孢杆菌1-10份,地衣芽孢杆菌1-10份、酵母菌1-15份,丁酸梭菌1-15份、嗜酸乳杆菌1-15份。
一种基于解淀粉芽孢杆菌的复合菌制剂及其制备方法,包括以下步骤:
(1)配制各菌株发酵所需的培养基,分别活化制得菌株发酵液;
(2)将上述菌株分别活化制得菌株发酵液,取制得的菌株发酵液,按配方量混合并与载体混合均匀、干燥后即得混合菌剂发酵粉。
进一步的,所述的步骤(1)中,分别以如下培养条件为最佳:
所述解淀粉芽孢杆菌发酵菌液的制备条件如下:
培养基为复合氨基酸营养液1%、蛋白胨1.5%、糖蜜5%、可溶性淀粉2.5%,设置pH为7.0,温度37℃、摇床转速200r/min、发酵时间36h。
所述地衣芽孢杆菌发酵菌液的制备条件如下:
培养基为玉米粉0.5%,蛋白胨1.0%,豆饼粉0.3%,酵母膏0.5%,硫酸锰0.001%,磷酸二氢钾0.1%,硫酸镁0.02%,起始pH值为7.5,温度为35℃,摇床转速180r/min、发酵时间40h。
所述酵母菌发酵菌液的制备条件如下:
培养基为蛋白胨1.0%、葡萄糖0.2%、酵母提取物0.5%、氯化钠1.0%,水100ml,起始pH值为4.7,温度为30℃,摇床转速210r/min,发酵时间24h。
所述丁酸梭菌发酵菌液的制备条件如下:
培养基为葡萄糖1.0%,豆粕1.0%,玉米粉0.5%,硫酸镁0.02%,硫酸铵0.02%,磷酸氢二钾0.05%,磷酸二氢钾0.05%,碳酸钙0.03%,蒸馏水1000mL,pH值调为5.0,温度为37℃,摇床转速160r/min,发酵时间24h。
所述嗜酸乳杆菌发酵菌液的制备条件如下:
培养基为大豆蛋白胨0.6%,胰蛋白胨0.6%,蛋白胨0.6%,牛肉浸膏1.2%,葡萄糖1%,低聚糖0.5%,乳糖0.5%,pH值为5.5,温度为37℃,静置培养,发酵时间24h。
进一步的,所述发酵液中,按质量份数记,解淀粉芽孢杆菌发酵菌液10-30份、地衣芽孢杆菌发酵菌液10-40份、酵母菌发酵菌液10-40份、丁酸梭菌发酵菌液10-40份、嗜酸乳杆菌发酵菌液10-40份、载体30-60份。
进一步的,所述步骤(2)中,复合菌剂载体为小麦粉、花生壳粉、玉米酒糟、脱脂糠粉、花生秧粉、玉米蛋白粉、豆秸粉、玉米粉、草粉、树叶粉、淀粉渣、玉米胚芽粕粉、稻壳粉、大豆粉、玉米芯粉中至少一种,上述载体均为有机载体,可用于与水产饲料混合。
进一步的,所述步骤(2)中,复合菌剂的干燥方式为真空冷冻干燥或喷雾干燥。
进一步的,可以根据需要将复合菌制剂调配成各种颜色。
与现有技术相比,本发明有以下优点:
1、本发明所述的复合菌剂,通过组合配比发酵,能够有效抑制弧菌、蓝藻的繁殖;同时可以调节藻相和菌相以及降低氨氮、亚硝酸盐水质指标,各菌株之间相互影响,还能更充分的利用培养基中的各种营养成分,与有机载体混合添加到水产饲料中后,可以调节水产肠道的菌群平衡,提高水产免疫力。
2、解淀粉芽孢杆菌,在生长过程中可以产生一系列能够抑制真菌和细菌活性的代谢物,产生拮抗作用,营养与空间的竞争,诱导寄主产生抗性。
3、地衣芽孢杆菌,可调整菌群失调达到治疗目的,可促使机体产生抗菌活性物质、杀灭致病菌。同时能够调节肠道内微生物之间、微生物与宿主之间处于微生态平衡,参与肠道内物质代谢,增强免疫力和抗应激能力。
4、丁酸梭菌能够在底部厌氧繁殖,抑制细菌和寄生虫繁殖,分解池塘淤泥、残饵、粪便和动植物尸体等有机物,消除恶臭,使养殖池塘COD的含量降低30-50%,平衡养殖水体生态,改善各项水质指标,同时能够调节肠道微生态平衡,修复肠道粘膜提高水生动物的免疫能力,提高饲料利用率。
5、酵母菌能够为水产提供营养,促进营养物质的吸收,抑制病原菌,调节肠道微生态平衡、增强免疫力,嗜酸乳杆菌能分泌乳酸,并产生蛋白酶、淀粉酶、脂肪酶等消化酶,分解水中糖类,抑制弧菌生长繁殖,还能降低养殖水体氨氮等有害物质的含量,分解鱼类残饵、粪便及水中有机质,促进鱼虾类健康生长。
附图说明
图1为复合菌剂制备简易流程示意图。
具体实施方式
下面结合附图和实施例对本发明作进一步的说明。
实施例1
一种复合菌剂,以细胞个数记,包括以下组分解淀粉芽孢杆菌5份,地衣芽孢杆菌5份、酵母菌2份,丁酸梭菌2份、嗜酸乳杆菌2份。
一种基于解淀粉芽孢杆菌的复合菌制剂及其制备方法,包括以下步骤:
(1)配制各菌株发酵所需的培养基,分别制取发酵菌液:
取5份解淀粉芽孢杆菌放入含1%复合氨基酸营养液、1.5%蛋白胨、5%糖蜜、2.5%可溶性淀粉的培养基中,设置pH为7.0,温度调为37℃、摇床转速200r/min、发酵36h,制得解淀粉芽孢杆菌发酵菌液;
取5份地衣芽孢杆菌菌株放入含0.5%玉米粉,1.0%蛋白胨,0.3%豆饼粉,0.5%酵母膏,0.001%硫酸锰,0.1%磷酸二氢钾,0.02%硫酸镁的培养基中,起始pH值调为7.5,温度设为35℃,摇床转速调为180r/min、发酵40h,制得地衣芽孢杆菌发酵菌液;
取2份酵母菌菌株放入含1.0%蛋白胨、0.2%葡萄糖、0.5%酵母提取物、1.0%氯化钠和100ml水的培养基中,起始pH值为4.7,温度为30℃,摇床转速210r/min,发酵24h,制得酵母菌发酵菌液;
取2份丁酸梭菌菌株放入含1.0%葡萄糖,1.0%豆粕,0.5%玉米粉,0.02%硫酸镁,0.02%硫酸铵,0.05%磷酸氢二钾,0.05%磷酸二氢钾,0.03%碳酸钙,1000mL蒸馏水的培养基中,pH值调为5.0,温度调为37℃,摇床转速设置为160r/min,发酵24h,制得丁酸梭菌发酵菌液;
取2份嗜酸乳杆菌菌株放入含0.6%大豆蛋白胨,0.6%胰蛋白胨,0.6%蛋白胨,1.2%牛肉浸膏,1%葡萄糖,0.5%低聚糖,0.5%乳糖的培养基中,pH值调为5.5,温度调为37℃,静置培养,发酵24h,制得嗜酸乳杆菌发酵菌液。
(2)取制得的各菌株发酵液,按质量份数记,取30份解淀粉芽孢杆菌发酵菌液、30份地衣芽孢杆菌发酵菌液、10份酵母菌发酵菌液、10份丁酸梭菌发酵菌液、10份嗜酸乳杆菌发酵菌液和50份淀粉渣混合均匀,真空冷冻干燥后即得混合菌剂发酵粉。
上述制得的复合菌剂可投放到鱼塘中用于抑制弧菌和蓝藻的繁殖。下面结合实验数据对实施例进行说明。
实施例1采用对照实验对鱼塘弧菌蓝藻的抑制情况和水产生长情况进行分析,具体设置有实验组和对照组一、二、三、四、五和六,所述七组的培养基条件均保持一致,各组按质量份数记,各组分构成进行差异化设置,参数见下
表1(其中各菌液构成份数不局限于表格中的配比,仅取部分做对比参考用):
表1
首先分别取鱼塘受弧菌蓝藻的污染水样,平均分装在七个实验鱼缸中(鱼缸大小适应实验需要,七组鱼缸容量和污染水量均一致),鱼缸中均添加等量小龙虾,对各鱼缸中的弧菌和蓝藻数量级进行测定,保持弧菌蓝藻,数量级一致,分别投放各组的复合菌剂,同等气候环境和饲料量下养殖6个月,实验结果如下表2:
表2
通过数据可以看出,投放复合菌剂的实验组的鱼缸对弧菌蓝藻的生长有明显的抑制作用。
实施例2
一种复合菌剂,以细胞个数记,包括以下组分解淀粉芽孢杆菌1份,地衣芽孢杆菌1份、酵母菌3份,丁酸梭菌3份、嗜酸乳杆菌3份。
一种基于解淀粉芽孢杆菌的复合菌制剂及其制备方法,包括以下步骤:
(1)配制各菌株发酵所需的培养基,分别制取发酵菌液:
取1份解淀粉芽孢杆菌放入含1%复合氨基酸营养液、1.5%蛋白胨、5%糖蜜、2.5%可溶性淀粉的培养基中,设置pH为7.0,温度调为37℃、摇床转速200r/min、发酵36h,制得解淀粉芽孢杆菌发酵菌液;
取1份地衣芽孢杆菌菌株放入含0.5%玉米粉,1.0%蛋白胨,0.3%豆饼粉,0.5%酵母膏,0.001%硫酸锰,0.1%磷酸二氢钾,0.02%硫酸镁的培养基中,起始pH值调为7.5,温度设为35℃,摇床转速调为180r/min、发酵40h,制得地衣芽孢杆菌发酵菌液;
取3份酵母菌菌株放入含1.0%蛋白胨、0.2%葡萄糖、0.5%酵母提取物、1.0%氯化钠和100ml水的培养基中,起始pH值为4.7,温度为30℃,摇床转速210r/min,发酵24h,制得酵母菌发酵菌液;
取3份丁酸梭菌菌株放入含1.0%葡萄糖,1.0%豆粕,0.5%玉米粉,0.02%硫酸镁,0.02%硫酸铵,0.05%磷酸氢二钾,0.05%磷酸二氢钾,0.03%碳酸钙,1000mL蒸馏水的培养基中,pH值调为5.0,温度调为37℃,摇床转速设置为160r/min,发酵24h,制得丁酸梭菌发酵菌液;
取3份嗜酸乳杆菌菌株放入含0.6%大豆蛋白胨,0.6%胰蛋白胨,0.6%蛋白胨,1.2%牛肉浸膏,1%葡萄糖,0.5%低聚糖,0.5%乳糖的培养基中,pH值调为5.5,温度调为37℃,静置培养,发酵24h,制得嗜酸乳杆菌发酵菌液。
(2)取制得的各菌株发酵液,按质量份数记,取10份解淀粉芽孢杆菌发酵菌液、10份地衣芽孢杆菌发酵菌液、20份酵母菌发酵菌液、20份丁酸梭菌发酵菌液、20份嗜酸乳杆菌发酵菌液和60份玉米蛋白粉混合均匀,喷雾干燥后即得混合菌剂发酵粉。
上述制得的复合菌剂可与鱼虾饲料混合,投放给鱼虾,用于调整鱼虾的肠道微生物平衡,增强鱼虾的免疫力。
实施例2同样采用对照实验对鱼塘弧菌蓝藻的抑制情况和水产生长情况进行分析,具体设置有实验组和对照组一、二、三、四、五和六,所述七组的培养基条件均保持一致,各组按质量份数记,各组分构成进行差异化设置,参数见下表3(其中各菌液构成份数不局限于表格中的配比,仅取部分做对比参考用):
表3
首先分别取鱼塘受弧菌蓝藻的污染水样,平均分装在七个实验鱼缸中(鱼缸大小适应实验需要,七组鱼缸容量和污染水量均一致),鱼缸中均添加等量小龙虾,对各鱼缸中的弧菌和蓝藻数量级进行测定,保持弧菌蓝藻,数量级一致,分别投放各组的复合菌剂,同等气候环境和饲料量下养殖6个月,实验结果如下表4:
表4
通过数据可以看出,投放复合菌剂与饲料混合的实验组的鱼缸对提高小龙虾免疫力有明显的促进作用。
以上所述实施例仅表达了本发明的优选实施方式,其描述较为具体和详细,但并不能因此而理解为对本发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形、改进及替代,这些都属于本发明的保护范围。因此,本发明专利的保护范围应以所附权利要求为准。
Claims (7)
1.一种基于解淀粉芽孢杆菌的复合菌制剂,其特征在于,所述复合菌制剂中,以细胞个数记,包括以下组分解淀粉芽孢杆菌1-10份,地衣芽孢杆菌1-10份、酵母菌1-15份,丁酸梭菌1-15份、嗜酸乳杆菌1-15份。
2.根据权利要求1所述的一种基于解淀粉芽孢杆菌的复合菌制剂,其特征在于,所述复合菌制剂中的菌株取自中国农业微生物菌种保藏管理中心的解淀粉芽孢杆菌(编号为ACCC60382)和地衣芽孢杆菌(编号为ACCC19747),以及取自北京中科质检生物技术有限公司的酵母菌(编号为ZW5031)、丁酸梭菌(编号为ZKCC-676)和嗜酸乳杆菌(编号为CICC-6074)。
3.一种基于解淀粉芽孢杆菌的复合菌制剂的制备方法,其特征在于,包括以下步骤:
步骤1,分别制备解淀粉芽孢杆菌发酵菌液、地衣芽孢杆菌发酵菌液、酵母菌发酵菌液、丁酸梭菌发酵菌液和嗜酸乳杆菌发酵菌液;
步骤2,以质量份数称取各组分,将发酵菌液混合并与载体混合均匀、干燥,得到所述微生物复合菌剂。
4.根据权利要求3所述的一种基于解淀粉芽孢杆菌的复合菌制剂的制备方法,其特征在于:
所述步骤1中解淀粉芽孢杆菌发酵菌液的制备条件如下:
培养基为复合氨基酸营养液1%、蛋白胨1.5%、糖蜜5%、可溶性淀粉2.5%,设置pH为7.0,温度37℃、摇床转速200r/min、发酵时间36h。
所述步骤1中地衣芽孢杆菌发酵菌液的制备条件如下:
培养基为玉米粉0.5%,蛋白胨1.0%,豆饼粉0.3%,酵母膏0.5%,硫酸锰0.001%,磷酸二氢钾0.1%,硫酸镁0.02%,起始pH值为7.5,温度为35℃,摇床转速180r/min、发酵时间40h。
所述步骤1中酵母菌发酵菌液的制备条件如下:
培养基为蛋白胨1.0%、葡萄糖0.2%、酵母提取物0.5%、氯化钠1.0%,水100ml,起始pH值为4.7,温度为30℃,摇床转速210r/min,发酵时间24h。
所述步骤1中丁酸梭菌发酵菌液的制备条件如下:
培养基为葡萄糖1.0%,豆粕1.0%,玉米粉0.5%,硫酸镁0.02%,硫酸铵0.02%,磷酸氢二钾0.05%,磷酸二氢钾0.05%,碳酸钙0.03%,蒸馏水1000mL,pH值调为5.0,温度为37℃,摇床转速160r/min,发酵时间24h。
所述步骤1中嗜酸乳杆菌发酵菌液的制备条件如下:
培养基为大豆蛋白胨0.6%,胰蛋白胨0.6%,蛋白胨0.6%,牛肉浸膏1.2%,葡萄糖1%,低聚糖0.5%,乳糖0.5%,pH值为5.5,温度为37℃,静置培养,发酵时间24h。
5.根据权利要求4所述的一种基于解淀粉芽孢杆菌的复合菌制剂的制备方法,其特征在于,按以下重量份数称取各组分:解淀粉芽孢杆菌发酵菌液10-40份、地衣芽孢杆菌发酵菌液10-40份、酵母菌发酵菌液10-40份、丁酸梭菌发酵菌液10-40份、嗜酸乳杆菌发酵菌液10-40份、载体30-60份。
6.根据权利要求5所述的一种基于解淀粉芽孢杆菌的复合菌制剂的制备方法,其特征在于,所述载体为以下有机载体:小麦粉、花生壳粉、玉米酒糟、脱脂糠粉、花生秧粉、玉米蛋白粉、豆秸粉、玉米粉、草粉、树叶粉、淀粉渣、玉米胚芽粕粉、稻壳粉、大豆粉、玉米芯粉中至少一种。
7.根据权利要求6所述的一种基于解淀粉芽孢杆菌的复合菌制剂的制备方法,其特征在于,复合菌剂的干燥方式为真空冷冻干燥或喷雾干燥,且复合菌制剂可根据需要调配成各种颜色。
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