CN116536195A - 克劳氏盐碱芽孢杆菌及其在制备有机微生物菌肥中的应用 - Google Patents
克劳氏盐碱芽孢杆菌及其在制备有机微生物菌肥中的应用 Download PDFInfo
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- CN116536195A CN116536195A CN202310460572.7A CN202310460572A CN116536195A CN 116536195 A CN116536195 A CN 116536195A CN 202310460572 A CN202310460572 A CN 202310460572A CN 116536195 A CN116536195 A CN 116536195A
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- bacillus clausii
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明公开了克劳氏盐碱芽孢杆菌及其在制备有机微生物菌肥中的应用，属于微生物技术领域。本发明公开的克劳氏盐碱芽孢杆菌FJAT‑53071，其保藏编号为GDMCCNo：63291。克劳氏盐碱芽孢杆菌FJAT‑53071菌株具有安全性，该菌制备的水溶性有机微生物菌肥有机质和氨基酸含量高，能明显促进作物生长，提高产量。
Description
技术领域
本发明涉及微生物技术领域，更具体的说是涉及克劳氏盐碱芽孢杆菌及其在制备有机微生物菌肥中的应用。
背景技术
克劳氏盐碱芽孢杆菌具有多种功能，产生碱性蛋白酶、生产抗生素、降解抗生素头孢呋辛、头孢噻肟和头孢吡吡肟。咖啡渣是咖啡饮料制作过程中产生的废弃物。用克劳氏盐碱芽孢杆菌对废咖啡渣进行固态发酵后，生物活性酚类化合物、抗氧化和抗菌活性都有所提高。克劳氏盐碱芽孢杆菌和解淀粉芽孢杆菌HM618共培养可产生表面素，有效降解抗生素。
益生菌被定义为活的微生物，当施用适量时，对宿主的健康有益。克劳氏盐碱芽胞杆菌作为一种益生菌，已经在国外上市55年。克劳氏盐碱芽孢杆菌在有氧和无氧环境中快速生长是独特的。克劳氏盐碱芽孢杆菌作为益生菌补充剂越来越多地用于成人和儿科患者群体。已往研究表明，克劳氏盐碱芽孢杆菌可有效治疗腹泻、反复呼吸道感染和急性胃肠炎。
尽管克劳氏盐碱芽孢杆菌是重要的益生菌，被广泛用于商业生产，但到目前为止，国内关于克劳氏盐碱芽孢杆菌的研究较少，尤其在农业上的应用甚少报道。
因此，提供克劳氏盐碱芽孢杆菌及其在制备有机微生物菌肥中的应用是本领域技术人员亟需解决的问题。
发明内容
有鉴于此，本发明提供了克劳氏盐碱芽孢杆菌及其在制备有机微生物菌肥中的应用。
基因组学系统分类，Patel and Gupta(2019)对克劳氏芽胞杆菌(Bacillusclausii)进行重分类，将其从芽孢杆菌属中划分出来，建立新的属水平分类单元Alkalihalobacillus，克劳氏芽孢杆菌重分类为克劳氏盐碱芽孢杆菌(Alkalihalobacillus clausii)。
为了实现上述目的，本发明采用如下技术方案：
一株克劳氏盐碱芽孢杆菌(Alkalihalobacillus clausii)FJAT-53071，其保藏编号为GDMCC No：63291，已保藏于广东省微生物菌种保藏中心，简称GDMCC，地址广州市先烈中路100号大院59号楼5楼广东省科学院微生物研究所，保藏日期为2022年03月24日，分类命名为克劳氏盐碱芽孢杆菌Alkalihalobacillus clausii。
进一步，所述的克劳氏盐碱芽孢杆菌FJAT-53071在制备有机微生物菌肥中的应用。
进一步，一种有机微生物菌肥，含有所述的克劳氏盐碱芽孢杆菌FJAT-53071。
经由上述的技术方案可知，与现有技术相比，本发明公开提供了克劳氏盐碱芽孢杆菌及其在制备有机微生物菌肥中的应用，克劳氏盐碱芽孢杆菌FJAT-53071菌株具有蛋白酶活性，不具备淀粉和纤维素降解能力，且具有安全性；菌株FJAT-53071对头孢唑林、氨苄西林、氯霉素、丁胺卡那、卡那霉素、新霉素、庆大霉素、利福平、多西环素、四环素、万古霉素敏感，而对青霉素、克林霉素、苯唑西林、多粘菌素B、链霉素、阿奇霉素、红霉素不敏感；菌株FJAT-53071制备的水溶性有机微生物菌肥有机质和氨基酸含量高，能明显促进作物生长，提高产量。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据提供的附图获得其他的附图。
图1附图为本发明菌株FJAT-53071的菌落形态；
图2附图为本发明菌株FJAT-53071的16S rRNA基因系统发育树；
图3附图为本发明菌株FJAT-53071培养7天的溶血性检测正面图；
图4附图为本发明菌株FJAT-53071的抗生素抗药性检测；
其中，1：头孢唑啉(30μg/片)；2：青霉素(10μg/片)；3：克林霉素(2μg/片)；4:氨苄西林(10μg/片)；5：苯唑西林(1μg/片)；6：氯霉素(30μg/片)；7：丁胺卡那(30μg/片)；8：卡那霉素(30μg/片)；9：新霉素(30μg/片)；10：庆大霉素(10μg/片)；11：利福平(5μg/片)；12：多粘菌素B(300Iu/片)；13：多西环素(30μg/片)；14：链霉素(10μg/片)；15：四环素(30μg/片)；16：万古霉素(30μg/片)；17：阿奇霉素(15μg/片)；18：红霉素(15μg/片)。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
本发明基于基因组和实验研究，鉴定了1株克劳氏盐碱芽孢杆菌，并对其进行了适应性分析和安全性评价。生物有机肥指特定功能微生物与主要以动植物残体(如畜禽粪便、农作物秸秆等)为来源并经无害化处理、腐熟的有机物料复合而成的一类兼具微生物肥料和有机肥效应的肥料。本发明还检测了发酵的菌肥的营养成分。
实施例1克劳氏盐碱芽孢杆菌菌株的分离
(1)土壤样本采集：采样福建红树林生态系统沉积物，放置无菌采样袋中。
(2)土样稀释：称取沉积物样本10g，放置90ml无菌水震荡溶解，120rpm摇床震荡30min混匀，然后取1ml转移至9ml无菌水，依次稀释4次，得到土壤悬浮液。
(3)涂布分离：吸取200μL土壤悬浮液，涂布于2216E平板。
(4)培养：将涂布好的平板放置30℃恒温培养箱培养2d。
(5)纯化：挑取平板上培养的不同颜色的菌落，采用连续划线法进行纯化，直至获得单菌落FJAT-53071。菌落形态：乳白色，扁平，圆形，结果见图1。
实施例2克劳氏盐碱芽孢杆菌菌株的鉴定
1)DNA提取方法
(1)1.5mL EP管中加入400μL STE，用刮菌环从平板中刮取适量已纯化好的菌落，混匀，获得细胞悬液。
(2)1mL的细胞悬液在8000g下离心2min，弃去上清后用400μL STE Buffer冲洗细胞两次，8000g离心2min，弃去上清。(若为平板，则400μL STE悬浮，离心，弃上清)。
(3)用200μL TE Buffer悬浮细胞，然后用100μL的Tris-saturatedphenol(取下层)加到离心管中，涡旋混合60s(振荡用浮板)。
(4)在4℃下13000g离心5min以从有机相中分离出水相，取160μL上清液转移到干净的1.5mL EP管中。
(5)加40μL TE Buffer到EP管中，用100μL氯仿混合，4℃13000g离心5min，用氯仿提取的方法纯化裂解，直到没有白色的界面出现，此过程重复2～3遍，每次直接往里加100μL氯仿，离心。
(6)取160μL上清液到干净的1.5mL EP管中，再加40μL TE在37℃下放置10min，以分解RNA。
(7)将100μL氯仿加到离心管中混匀后，在4℃下13000g离心5min。
(8)将150μL上清液转移到干净的1.5mL EP管中，此时的上清液包含有纯化的DNA，且可以直接用于序列实验，并可在-20℃保存。
2)PCR扩增、测序和鉴定
利用细菌16S rRNA基因通用引物27F和1492R进行PCR扩增。
27F和1492R的引物序列如下：
27F：5’-AGAGTTTGATCCTGGCTCAG-3’；SEQ ID NO.1；
1492R：5’-CTACGGCTACCTTGTTACGA-3’；SEQ ID NO.2。
PCR扩增程序：94℃预变性5min；94℃变性30s，55℃退火60s，72℃延伸90s，共30个循环；最后72℃延伸10min。取5μL PCR产物，点样于1％的琼脂糖凝胶中，以100bp Marker作为标准分子量，110V电压，电泳30min，用凝胶成像系统观察结果。检测出有条带的菌株PCR产物送至铂尚生物技术有限公司进行测序。测序序列上传至EzBiocloud进行比对，获得菌株分类地位，结果见图2。
经比对，FJAT-53071鉴定为克劳氏盐碱芽孢杆菌(Alkalihalobacillusclausii)，16S rRNA相似性为99.7％。16S rRNA基因序列长度为1377bp，如SEQ ID NO.3所示。
>Alkalihalobacillus clausii FJAT-53071
GCTTGCTCCCGGACGTTAGCGGCGGACGGGTGAGTAACACGTGGGCAACCTGCCCCTTAGACTGGGATAACTCCGGGAAACCGGAGCTAATACCGGATAATCCCTTTCTCCACCTGGAGAGAGGGTGAAAGATGGCTTCGGCTATCACTAAGGGATGGGCCCGCGGCGCATTAGCTAGTTGGTAAGGTAACGGCTTACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGAGGAAGGCCTTCGGGTCGTAAAGCTCTGTTGTGAGGGAAGAAGCGGTACCGTTCGAATAGGGCGGTACCTTGACGGTACCTCACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGCTTCTTAAGTCTGATGTGAAATCTCGGGGCTCAACCCCGAGCGGCCATTGGAAACTGGGGAGCTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAGGTGTTAGGGGTTTCGATGCCCGTAGTGCCGAAGTTAACACATTAAGCACTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGACCCGCACAAGCAGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTTTGACCACCCAAGAGATTGGGCTTCCCCTTCGGGGGCAAAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTGAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAAAGGGCAGCGAAGCCGCGAGGTGAAGCCAATCCCATAAAGCCATTCTCAGTTCGGATTGCAGGCTGCAACTCGCCTGCATGAAGCCGGAATTGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGCAACC；SEQ ID NO.3。
3)基因组测序与鉴定
新鲜菌体采用细菌DNA提取试剂盒(Shanghai Generay Biotech Co.,Ltd)，委托北京诺禾致源有限公司进行基因组测序，获得菌株FJAT-53071的基因组序列。
经基因组比较分析，基因组大小为4,263,539bp，DNA G+C含量为44.62％。菌株FJAT-53071与最近模式菌株Alkalihalobacillus clausii DSM 8716T的平均核苷酸一致性(ANI)为97.7％，远高于细菌种的界定阈值(96％)。因此，确定该菌株为克劳氏盐碱芽孢杆菌(Alkalihalobacillus clausii)。
4)pH、温度和盐适应性
FJAT-53071的纯菌落无菌条件下，点接于LB平板上。温度适应性条件范围10,15,20,25,30,35,40,45,50,55,60和65℃，培养2天。pH适应性pH6-13(间隔1个单位)，45℃培养2天。盐耐受性，0％,1％,2％,3％,4％,5％,6％,7％,10％,15％和20％(w/v)NaCl梯度，pH9、45℃培养2天。观察并测量菌落直径大小。
结果表明，生长温度范围20-55℃、pH7-13、耐盐性0-15％NaCl。
5)淀粉酶、蛋白酶和纤维素酶水解能力
采用点接法检测FJAT-53071菌株产碱性淀粉酶、蛋白酶和纤维素酶的能力，将新鲜菌液分别接种于含1％淀粉、1％干酪素、1％纤维素羧甲基钠的LB培养基平板上，培养基的pH调整为9.0。培养2天后，观察菌落周围水解圈的直径大小。
结果表明，菌株FJAT-53071具有蛋白酶活性，不具备淀粉和纤维素降解能力。
6)溶血性检测
新鲜菌体接种至含5％羊血LB平板上，30℃恒温培养箱培养观察。
溶血性检测发现，培养3天和7天后，菌株FJAT-53071的菌落周围皆未产生透明圈，为γ型溶血菌株(图3)。表明该菌株为生物安全型菌株，可用于产品研发。
7)菌株FJAT-53071的抗生素抗药性检测
菌株FJAT-53071在LB液体培养基180rpm振荡培养2天后，取0.1mL菌液均匀涂布于LB固体平板上，待菌液完全被LB培养基吸收后，将商业购买的抗生素纸片(杭州微生物试剂有限公司)贴于上述平板上。倒置培养2天后观察菌株FJAT-53071的抗生素抗性。敏感：产生透明圈；不敏感：未产生透明圈。
图4结果显示，菌株FJAT-53071对头孢唑林、氨苄西林、氯霉素、丁胺卡那、卡那霉素、新霉素、庆大霉素、利福平、多西环素、四环素、万古霉素敏感，而对青霉素、克林霉素、苯唑西林、多粘菌素B、链霉素、阿奇霉素、红霉素不敏感。
实施例3水溶性有机微生物菌肥的制备和评价
1)水溶性有机微生物菌肥的制备
(1)菌种繁殖：将纯化好的菌株FJAT-53071接种于30mL LB液体培养基放置30℃摇床180rpm转速震荡培养2d，获得菌液，菌落数为108CFU/mL。
(2)有机微生物菌肥制备：将柑橘废果(柑橘废果指不能食用的，或者风味很差的柑橘)、红糖、水按质量3:1:10比例混合均匀，添加1.0％步骤(1)制备的菌液后，混匀发酵3个月，获得有机微生物菌肥。
(3)采用灌根法将有机微生物菌肥稀释300倍后施于芦柑果园，果实迅速膨大期至果实转色成熟期，每隔20天浇1次，共浇3次。每棵树每次浇灌3-5kg。
采集柑橘熟果，测定果实的糖酸比、重量、大小，评价FJAT-53071发酵的有机微生物菌肥的效力，结果见表1。
表1芦柑果指标
由表1可知，施加FJAT-53071发酵菌肥后，柑橘的糖酸比、重量和果实横茎之比明显高于对照未施菌肥(未浇灌菌株FJAT-53071制备的有机微生物菌肥，只浇水)的，提高了柑橘产量，改善了风味。
2)有机微生物菌肥的营养成分测定
采用国家标准NY/T 1121.6-2006测定发酵的液体有机微生物菌肥中的有机质含量，氨基酸的含量检测参考国标GB 5009.124-2016，蛋白质的检测参考GB 5009.5-2016。
结果表明，有机微生物菌肥中的有机质含量为16.5g/kg，氨基酸含量为1g/kg，蛋白质含量为2.1g/kg。
对所公开的实施例的上述说明，使本领域专业技术人员能够实现或使用本发明。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见的，本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下，在其它实施例中实现。因此，本发明将不会被限制于本文所示的这些实施例，而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。
Claims (3)
1.一株克劳氏盐碱芽孢杆菌(Alkalihalobacillusclausii)FJAT-53071，其特征在于，其保藏编号为GDMCCNo：63291。
2.权利要求1所述的克劳氏盐碱芽孢杆菌FJAT-53071在制备有机微生物菌肥中的应用。
3.一种有机微生物菌肥，其特征在于，含有权利要求1所述的克劳氏盐碱芽孢杆菌FJAT-53071。
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| CN117070394A (zh) * | 2023-04-20 | 2023-11-17 | 广东药科大学 | 产碱性蛋白酶的嗜碱性菌株、碱性蛋白酶及其应用 |
| CN117070394B (zh) * | 2023-04-20 | 2024-03-29 | 广东药科大学 | 产碱性蛋白酶的嗜碱性菌株、碱性蛋白酶及其应用 |
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