CN115109730B - 一种枯草芽孢杆菌菌株、菌粉及其应用 - Google Patents
一种枯草芽孢杆菌菌株、菌粉及其应用 Download PDFInfo
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- CN115109730B CN115109730B CN202210872869.XA CN202210872869A CN115109730B CN 115109730 B CN115109730 B CN 115109730B CN 202210872869 A CN202210872869 A CN 202210872869A CN 115109730 B CN115109730 B CN 115109730B
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Abstract
本发明提供一株枯草芽孢杆菌菌株，为枯草芽孢杆菌(Bacillus subtilis)HY‑001，保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO.M 2022622，保藏日期为2022年05月13日。由广东省微生物菌种保藏中心采购编号为GDMCC1.372，经过紫外诱变、50℃高温诱导及高产纤维素酶、淀粉酶的筛选获得。该枯草芽孢杆菌HY‑001能耐受50℃高温，且对多种弧菌具有明显抑制效果，故在堆肥腐熟及水产防治弧菌具有重要意义。
Description
技术领域
本发明涉及一株能耐受高温的枯草芽孢杆菌菌株及其制备的菌粉和应用。
背景技术
枯草芽孢杆菌(Bacillus subtilis)，是一种好氧的革兰氏阳性芽孢杆菌，其菌体生长过程中产生的枯草菌素与多粘菌素等活性物质，能对致病菌或内源性感染的条件致病菌有明显的抑制作用。枯草芽孢杆菌代谢可合成α-淀粉酶、蛋白酶、纤维素酶等多种酶类，在消化道中提高消化能力，且能分解有机物及有毒有害物质。
从目前现有的研究报道中可见，枯草芽孢杆菌的生长温度一般在10-40℃，而耐高温的报道主要是其代谢酶类或其形成的芽孢状态，至于非芽孢状态的菌体能耐受高于50℃的报道基本没有。故筛选一种能在高温下能正常生长代谢的枯草芽孢杆菌，在堆肥腐熟的运用具有重大意义。
农业部于2015年10月起禁止使用蛭弧菌微生态制剂，故在水产弧菌病原菌的防治上急需安全可靠的替代品。而枯草芽孢杆菌在水产中的运用主要体现在水质调节方面，在抑菌水体病原菌的运用较少，故筛选一种能有效抑制水产病原菌的枯草芽孢杆菌具有重大意义。
发明内容
本发明为了弥补现有枯草芽孢杆菌存在的不足之处，从而提供了一种枯草芽孢杆菌HY-001，该菌株具有能耐受高温培养、抑制多株水产病原菌的能力，将在堆肥腐熟及水产养殖具有广泛的运用价值。
本发明提供的枯草芽孢杆菌(Bacillus subtilis)HY-001，为广东省微生物菌种保藏中心、采购编号为GDMCC1.372的枯草芽孢杆菌，经过紫外诱变、50℃高温诱导及高产纤维素酶、淀粉酶一系列筛选获得，保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO.M 2022622，保藏日期为2022年05月13日。
在对枯草芽孢杆菌HY-001的产酶能力对比试验中发现，其产纤维素酶、淀粉酶的酶活力高于枯草芽孢杆菌GDMCC1.372，且在温度耐受方面也明显优于枯草芽孢杆菌GDMCC1.372。
在对水产病原菌抑制试验检测中发现，本发明的枯草芽孢杆菌HY-001对副溶血弧菌、溶藻弧菌、铜绿假单胞菌、嗜水气单胞菌等水产病原菌上具有明显抑制效果，其对水产病原菌的抑制能力高于枯草芽孢杆菌GDMCC1.372。
本发明的一株枯草芽孢杆菌菌粉的制备方法，包括以下步骤：将枯草芽孢杆菌HY-001摇瓶活化后接入10L种子罐，装发酵液4L，发酵结束后按4％接入200L发酵罐，装发酵液100L。发酵温度40℃，发酵25h，搅拌150r/min，溶氧80％。发酵结束通过碟式离心机离心收集菌泥后通过离心式喷雾干燥机喷雾制备成菌粉，其中控制进风温度110℃，出风温度70℃。
进一步的，所述发酵液原料包括：葡萄糖25g/L，酵母浸膏5g/L，复合氨基酸粉2g/L，氯化铵2g/L，硫酸镁0.1g/L，硫酸锰0.05g/L，碳酸钙3g/L，pH自然。
本发明的枯草芽孢杆菌HY-001具有以下优点：
1.遗传性质稳定，能够在50-55℃环境中生存并进行正常生长代谢，有利于在高温环境状态下存活发挥其功能，从而扩展了枯草芽孢杆菌的运用范围。
2.能有效抑制多种水产病原菌，从而在功能上由普遍调水功能向防治水产动物疾病转变，从而部分替代蛭弧菌及抗生素产品。
3.能产生大量高效的纤维素酶、淀粉酶，在堆肥腐熟应用中效果显著。
附图说明
图1为枯草芽孢杆菌HY-001在不同温度下的生长曲线；
图2为枯草芽孢杆菌HY-001菌株耐高温测试试验涂布平板；
图3为枯草芽孢杆菌GDMCC1.372菌株耐高温测试试验涂布平板；
图4为枯草芽孢杆菌HY-001抑制大肠杆菌、副溶血弧菌抑菌圈照片；
图5为菌株在对虾养殖防治弧菌的运用中试验前所涂布的平板，稀释度为10-1；
图6为菌株在对虾养殖防治弧菌的运用中试验5天所涂布的平板，稀释度为10-1。
具体实施方式
为了对本发明的技术方案做进一步的描述，故做如下具体实施例，但本发明的内容不仅仅局限于实施例所述范围，凡是不背离本发明的构思及等同替代均在本发明的保护范围之内。
实施例1紫外诱变及高温诱导培养
取培养24h的枯草芽孢杆菌GDMCC1.372培养液，采用10倍稀释至10-3制成菌悬液。取直径9cm无菌平皿1套，加入上述10-3菌悬液2mL，用无菌涂布棒涂成薄薄一层。置于在距离为30cm，功率为15W的紫外线灯下照射3min，在照射前将紫外线灯开关打开预热约20min。用无菌涂布棒蘸取菌液快速涂布于牛肉膏蛋白胨琼脂培养基平板中，操作过程不开白炽灯，保持暗光线操作。将上述涂匀的平板，用黑布包好，置50℃培养48h。将50℃培养后在平板上长出来的枯草芽孢杆菌菌落，全部挑取至30mL营养肉汤培养基，100mL三角瓶装，50℃摇床培养24h备用。
本发明提供的枯草芽孢杆菌(Bacillus subtilis)HY-001，为广东省微生物菌种保藏中心、采购编号为GDMCC1.372的枯草芽孢杆菌，经过紫外诱变及50℃高温诱导及高产纤维素酶、淀粉酶一系列筛选获得，保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO.M 2022622，保藏日期为2022年05月13日。
实施例2产淀粉酶、纤维素酶筛选
1.产淀粉酶筛选
将50℃高温诱导培养后的菌液，进行10倍稀释，选取10-4、10-5、10-6各0.1mL涂布淀粉酶培养基，50℃培养48h，将碘液滴加平板上，观察测量透明圈大小。将产生较大透明圈的菌株进行纯化分离备用。其中淀粉酶筛选培养基：可溶性淀粉10g、牛肉膏3g、蛋白胨10g、氯化钠5g、琼脂20g。
2.产纤维素酶筛选
将在淀粉酶筛选试验中纯化后的菌株进行活化培养18h，进行10倍稀释，选取10-4、10-5、10-6各0.1mL涂布纤维素酶筛选培养基，50℃培养48h，观察测量透明圈大小。将产生较大透明圈的菌株进行纯化分离备用。其中纤维素酶筛选培养基：羧甲基纤维素钠10g、酵母膏1g、刚果红0.2g、硫酸铵4g、磷酸二氢钾2g、硫酸镁0.5g、硫酸锰0.5g、琼脂20g、pH 5.5。
实施例3筛选菌株复筛
将经过一系列的筛选后获得的菌株进行划线纯化，获得纯种枯草芽孢杆菌HY-001，将该菌株进行采用营养琼脂进行划线，于50℃、55℃进行培养24h，观察有无菌落长出。采用发酵培养基培养液体24h，以NY/T 2321-2013做纤维素酶活检测，以NY/T 1847-2010做蛋白酶活检测，并与枯草芽孢杆菌GDMCC1.372进行对比。其中发酵培养基为葡萄糖25g/L，酵母浸膏5g/L，复合氨基酸粉2g/L，氯化铵2g/L，硫酸镁0.1g/L，硫酸锰0.05g/L，碳酸钙3g/L，pH自然。
表1 2株枯草芽孢杆菌对比试验结果
由上表1可知，枯草芽孢杆菌HY-001能在50℃、55℃正常生长，而枯草芽孢杆菌GDMCC1.372则无法生长，枯草芽孢杆菌HY-001在耐温上就优于枯草芽孢杆菌GDMCC1.372。在产酶能力上也远比枯草芽孢杆菌GDMCC1.372好。
实施例4菌株生长温度检测
将筛选获得的枯草芽孢杆菌HY-001接种30mL发酵培养基，100mL三角瓶装，分别于25℃、30℃、35℃、40℃、45℃、50℃、55℃、60℃条件下培养30h，每隔6h取样检测OD600nm(吸光值OD600nm正比于溶液中的吸光物质的浓度)，并与枯草芽孢杆菌GDMCC1.372进行对比。其中发酵培养基为葡萄糖25g/L，酵母浸膏5g/L，复合氨基酸粉2g/L，氯化铵2g/L，硫酸镁0.1g/L，硫酸锰0.05g/L，碳酸钙3g/L，pH自然。
枯草芽孢杆菌HY-001其最适生长温度为40℃，且在50℃、55℃条件下还能缓慢正常生长，60℃则无法生长，呈凋亡状态。而枯草芽孢杆菌GDMCC1.372，其最适生长温度为35℃，但在高于50℃无法正常生长，具体其他温度菌株的活性见表2、表3和图1。故枯草芽孢杆菌HY-001在对高温环境的耐受上优于枯草芽孢杆菌GDMCC1.372。
表2枯草芽孢杆菌HY-001在不同温度下的生长情况
表3枯草芽孢杆菌GDMCC1.372在不同温度下的生长情况
实施例5遗传稳定性测试
以最初筛选获得的枯草芽孢杆菌HY-001作为起始第1代，每天进行划线营养琼脂平板传代培养测试，以第1天划线第2天长出后，重新划线接种下一个平板为1代算，连续以上班时间计算，测试半年，合计约150多次，以最后一次重新检测该菌株的能否耐受50℃培养生长，及纤维素酶、淀粉酶活力的能力是否减弱，检测方法同实施例3。
表4枯草芽孢杆菌HY-001遗传稳定性测试结果
| 温度耐受50℃ | 温度耐受55℃ | 纤维素酶U/mL | 淀粉酶U/mL |
| + | + | 164±3.65 | 858±1.64 |
由上表4可知，经过150多次的划线传代培养，经过复测，菌株依旧能在50℃、55℃正常生长，且纤维素酶活力、淀粉酶活力跟菌株复筛实施例3数据相差不大，说明经过150多次的划线传代培养未改变其菌株特性，该菌的遗传性较为稳定。
实施例6菌株耐高温测试
将菌株活化后的培养液放置60℃、80℃、100℃水浴保温2min、5min、10min、20min、30min、1h、2h后进行活菌检测，吸取0.1mL涂布营养琼脂平板，以60℃，保温30min后的活菌数为起始菌体浓度算，并与枯草芽孢杆菌GDMCC1.372进行对比。
由试验结果数据可知，枯草芽孢杆菌GDMCC1.372在经过80℃处理1h、100℃处理5min以上就全部失活，而枯草芽孢杆菌HY-001在该相同处理条件下任具有活性，试验结果具体详见表5以及图2和图3，表明枯草芽孢杆菌HY-001菌株具有良好的耐高温能力。
表5菌株耐高温测试结果对比
实施例7菌株抑制水产病原指示菌测试
取1mL水产病原指示菌悬液加入50℃100mL海水制备的营养琼脂培养基中摇匀(菌终浓为105CFU/mL)，倾注平板，待冷却凝固后将牛津杯置于平板中，往牛津杯中分别加入枯草芽孢杆菌HY-001、枯草芽孢杆菌GDMCC1.372发酵离心后的发酵上清液0.1mL，置于30℃下培养24h，并与枯草芽孢杆菌GDMCC1.372进行对比。观察有无抑菌圈产生，并用游标卡尺测量抑菌圈直径大小。
菌株发酵液在水产病原指示菌的抑制试验中，结果可知枯草芽孢杆菌HY-001的发酵液对试验的病原指示菌敏感性都达中敏感以上，而枯草芽孢杆菌GDMCC1.372敏感性大部分为低敏感，具体相关指示菌的抑菌结果如表6所示和图4，说明枯草芽孢杆菌HY-001发酵所产生的相关抑菌物质量高于枯草芽孢杆菌GDMCC1.372，其抑菌效果优于枯草芽孢杆菌GDMCC1.372。
表6菌株对水产病原指示菌抑制测试结果
注：抑菌圈直径(mm)：不敏感：0，低敏感：<10，中敏感：10-14，高敏感：15-20，极敏感：>20
实施例8菌株在防治弧菌的运用
在某对虾养殖场中，挑选6个养殖对虾水塘，其中水深、面积、放养量以及管理模式一致的池塘作为试验。分为3组，每组2个养殖塘。在2组试验组中分别使用枯草芽孢杆菌HY-001、枯草芽孢杆菌GDMCC1.372的菌粉，菌粉规格为200亿CFU/g，按使用量10g/亩，对照组则不做任何处理。试验时间为5天，在试验期间，6个池塘管理模式一致。5天后对这6个池塘水质中的弧菌总数指标进行检测分析，弧菌总数采用TCBS培养基平板稀释涂布检测。
在没运用枯草芽孢杆菌的对照组中，前后弧菌总数变化不大，使用枯草芽孢杆菌HY-001的弧菌总数平均下降98.8％，枯草芽孢杆菌GDMCC1.372的弧菌总数平均下降59.3％，具体试验结果数具详见表7以及图5和图6。说明枯草芽孢杆菌在防治弧菌上具有一定效果，且枯草芽孢杆菌HY-001的效果优于枯草芽孢杆菌GDMCC1.372。
表7弧菌活菌总数前后对比
实施例9菌株在鸡粪堆肥腐熟的运用
以鸡粪主料与辅料米糠按3:1比例混合，控制水分在50-60％，手抓物料成团无水滴的状态。按500g/吨料的使用比例分别混合枯草芽孢杆菌HY-001、枯草芽孢杆菌GDMCC1.372的菌粉，菌粉规格为200亿CFU/g。控制每堆堆料5吨，高度1米，当堆温升至60℃时开始每天一次翻倒一次，腐熟为期30天，每隔10天取样检测并记录相关试验数据，其中粪大肠菌群数检测依据GB/T19524.1，蛔虫卵死亡率检测依据GB/T19524.2，同时以不加枯草芽孢杆菌做为对照组。
试验结果可知，能耐受高温的枯草芽孢杆菌HY-001，能在4天时间使温度快速提升至50℃以上，且持续时间长达25天，通过持续的高温作用，使粪大肠菌群、蛔虫卵快速死亡，从而臭味得以消失，而对照组及枯草芽孢杆菌GDMCC1.372则无法使鸡粪腐熟的温度上升至50℃以上，其腐熟效果差或无法达到腐熟效果，具体相关试验项目数据如表8所示。试验结果表明枯草芽孢杆菌HY-001的腐熟效果远优于枯草芽孢杆菌GDMCC1.372。
表8菌株在鸡粪腐熟的试验结果数据
以上描述了本发明的部分实施方式，但本发明并不限于上述实施方式中的具体细节，在本发明的技术构思范围内，可以对本发明的技术方案进行多种简单变型，这些简单变型均属于本发明的保护范围。
Claims (8)
1.一株枯草芽孢杆菌菌株，其特征在于，所述枯草芽孢杆菌菌株为枯草芽孢杆菌(Bacillus subtilis)HY-001，保藏于中国典型培养物保藏中心（CCTCC），保藏编号为CCTCCNO.M 2022622，保藏日期为2022年05月13日。
2.根据权利要求1所述的一种枯草芽孢杆菌菌株，其特征在于，所述枯草芽孢杆菌HY-001，由广东省微生物菌种保藏中心采购编号为GDMCC1.372，经过紫外诱变、50℃高温诱导及高产纤维素酶、淀粉酶的筛选获得。
3.根据权利要求2所述的一种枯草芽孢杆菌菌株，其特征在于，紫外诱变：取培养24 h的枯草芽孢杆菌GDMCC1.372培养液，采用10倍稀释至10-3制成菌悬液；取直径9 cm无菌平皿1套，加入上述10-3菌悬液2 mL，用无菌涂布棒涂成薄薄一层；置于在距离为30 cm，功率为15W的紫外线灯下照射3 min，在照射前将紫外线灯开关打开预热约20 min；用无菌涂布棒蘸取菌液快速涂布于牛肉膏蛋白胨琼脂培养基平板中，操作过程不开白炽灯，保持暗光线操作。
4.根据权利要求2所述的一种枯草芽孢杆菌菌株，其特征在于，产淀粉酶筛选：将50℃高温诱导培养后的菌液，进行10倍稀释，选取10-4、10-5、10-6各0.1mL涂布淀粉酶培养基，50℃培养48h，将碘液滴加平板上，观察测量透明圈大小；将产生较大透明圈的菌株进行纯化分离备用；其中淀粉酶筛选培养基：可溶性淀粉10g、牛肉膏3g、蛋白胨10g、氯化钠5g、琼脂20g。
5.根据权利要求2所述的一种枯草芽孢杆菌菌株，其特征在于，产纤维素酶筛选：将在淀粉酶筛选试验中纯化后的菌株进行活化培养18h，进行10倍稀释，选取10-4、10-5、10-6各0.1mL涂布纤维素酶筛选培养基，50℃培养48h，观察测量透明圈大小；将产生较大透明圈的菌株进行纯化分离备用；其中纤维素酶筛选培养基：羧甲基纤维素钠10g、酵母膏1g、刚果红0.2g、硫酸铵4g、磷酸二氢钾2g、硫酸镁0.5g、硫酸锰0.5g、琼脂20g、pH 5.5。
6.一种枯草芽孢杆菌菌粉，其特征在于，将权利要求1所述的一种枯草芽孢杆菌HY-001摇瓶活化后接入10L种子罐，装发酵液4L，发酵结束后按4%接入200L发酵罐，装发酵液100L；发酵温度40℃，发酵25h，搅拌150r/min，溶氧80%；发酵结束通过碟式离心机离心收集菌泥后通过离心式喷雾干燥机喷雾制备成菌粉，其中控制进风温度110℃，出风温度70℃。
7.根据权利要求6所述的一种枯草芽孢杆菌菌粉，应用在抑制副溶血弧菌、溶藻弧菌、铜绿假单胞菌、嗜水气单胞菌。
8.根据权利要求1所述的一种枯草芽孢杆菌菌株，应用在抑制副溶血弧菌、溶藻弧菌、铜绿假单胞菌、嗜水气单胞菌。
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