CN112813009B - 一种解淀粉芽孢杆菌及其应用 - Google Patents
一种解淀粉芽孢杆菌及其应用 Download PDFInfo
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Abstract
本发明涉及微生物技术领域，尤其涉及一种解淀粉芽孢杆菌及其应用。本发明所述解淀粉芽孢杆菌(Bacillus amyloliquefaciens)，将其命名为菌株HD‑1，从条纹斑竹鲨肠道内分离得到，其保藏编号为CGMCC No.18080。实验证明，该菌株能显著抑制溃疡性结肠炎(UC)小鼠的体重下降，减轻结肠的缩短程度，减轻UC小鼠结肠部位的炎症细胞浸润和粘膜破坏程度，对UC具有良好的治疗和预防作用。
Description
技术领域
本发明涉及微生物技术领域，尤其涉及一种解淀粉芽孢杆菌及其应用。
背景技术
溃疡性结肠炎(Ulcerative colitis，UC)是一种累及回肠、直肠和结肠粘膜的慢性非特异性肠道炎症性疾病，可发生于任何年龄，多见于10-40岁，临床表现包括腹泻(可伴粘液脓血)、腹痛等腹部症状以及贫血、发热、体重减轻等全身症状，严重影响患者生存质量。目前，针对UC的主要治疗目的是抑制粘膜炎症，常用药物包括氨基水杨酸、糖皮质激素、抗生素和一些免疫抑制剂。尽管这些治疗方法在很多患者中均取得较好的治疗效果，但上述药物都存在着不同的局限性和一定的毒副作用。
芽孢杆菌是兼性厌氧或好氧的产芽孢的革兰氏阳性杆菌，通常存在于土壤、水体、空气以及动物肠道中。芽孢杆菌抗逆性强，孢子细胞壁厚且含水量低，可有效抵抗干燥、酸碱、挤压、有毒化学物质等不良环境。目前缺少能耐受胃肠道的高酸、高胆盐等复杂的理化环境的菌株。
发明内容
有鉴于此，本发明提供了一种解淀粉芽孢杆菌及其应用。本发明提供的菌株能显著抑制溃疡性结肠炎(UC)小鼠的体重下降，减轻结肠的缩短程度，减轻UC小鼠结肠部位的炎症细胞浸润和粘膜破坏程度，对UC具有良好的治疗和预防作用。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了保藏编号为CGMCC No.18080的解淀粉芽孢杆菌(Bacillusamyloliquefaciens)。
本发明所述的解淀粉芽孢杆菌从浅海层的条纹斑竹鲨肠道内分离得到，将其命名为菌株HD-1。
本发明还提供了所述解淀粉芽孢杆菌在制备治疗、缓解或预防肠道疾病的药物中的应用。
本发明中，所述肠道疾病为结肠炎或肠道组织损伤，或由结肠炎引起的腹泻以及肠道组织损伤引起的腹泻。一些具体实施例中，所述肠道疾病为结肠炎，具体为溃疡性结肠炎。
本发明还提供了所述解淀粉芽孢杆菌在制备调节肠道菌群的保健食品中的应用。
本发明中，所述保健食品为饮品。
本发明还提供了所述的解淀粉芽孢杆菌在制备抑菌剂中的应用。
本发明中，所述抑菌为抑制革兰氏阳性菌或格兰仕阴性菌。
本发明还提供一种药物，包括解淀粉芽孢杆菌和药学上可接受的辅料。
本发明还提供一种调节肠道菌群的饮品，包括所述的解淀粉芽孢杆菌。
本发明提供一种抑菌剂，包括本发明所述的解淀粉芽孢杆菌的发酵液。
一些实施例中，芽孢杆菌HD-1发酵液制备方法包括：
将保存的菌种以1:100的比列接种于5ml LB培养基中，37℃220rpm培养10h，取1ml种子液接种于盛有100ml LB培养基的250ml锥形瓶中(接种量为1％)，培养条件同上。将摇瓶中的发酵种子液接种到200L发酵罐中(基础发酵培养基)，接种量为5％，温度为37℃，搅拌转速为150rpm，以1:2000(V/V装液量)的比例在培养基中加入消泡剂。在接种后8～10h，OD600达到1.8～2.0时可停止发酵，得到发酵液。
本发明提供的解淀粉芽孢杆菌HD-1能显著抑制溃疡性结肠炎(UC)小鼠的体重下降，减轻结肠的缩短程度，减轻UC小鼠结肠部位的炎症细胞浸润和粘膜破坏程度，对UC具有良好的治疗和预防作用。
生物保藏说明
生物材料HD-1，分类命名：解淀粉芽孢杆菌(Bacillus amyloliquefaciens)，于2019年07月08日保藏在中国微生物菌种保藏管理委员会普通微生物中心，地址为：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，保藏编号为CGMCC No.18080。
附图说明
图1示单菌落形态图；
图2示解淀粉芽孢杆菌HD-1的PCR鉴定电泳图；
图3示解淀粉芽孢杆菌HD-1处理G+枯草芽孢杆菌BS168后的抑菌效果图；
图4示解淀粉芽孢杆菌HD-1处理G-大肠杆菌TG1后的抑菌效果图；
图5示解淀粉芽孢杆菌HD-1处理实验动物后的动物体重变化图；
图6示解淀粉芽孢杆菌HD-1处理实验动物后的动物结直肠长度变化图；
图7示解淀粉芽孢杆菌HD-1处理实验动物后动物结直肠的HE染色图。
具体实施方式
本发明提供了一种解淀粉芽孢杆菌及其应用。本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
本发明采用的试材皆为普通市售品，皆可于市场购得。
下面结合实施例，进一步阐述本发明：
实施例1芽孢杆菌HD-1的分离、鉴定
购自广东海域的条纹斑竹鲨，购回后放于遮光水箱内暂养数日后，对其进行手术取其肠道，75％的酒精擦拭外表面后于紫外超净台内解剖其肠道，取出肠道内容物及粘膜液，使用生理盐水稀释，得到原菌液。以稀释涂布的方法涂布于海水LB固体培养基内，置于37℃培养箱培养，挑取单菌落，获得菌株HD-1，单菌落形态图见图1。
其中，海水LB固体培养基成分：酵母提取物5g，胰蛋白胨10g，氯化钠10g，琼脂13g，1000mL人工海水，摇动容器直至溶质溶解，在121℃下灭菌20min，每个玻璃平板内倒入约20mL培养基。人工海水为本实验室自配，由蒸馏水加适量海盐溶解的母液配制而成，pH约为7.0。
芽孢杆菌HD-1菌株的遗传学特征：
用细菌基因组试剂盒提取细菌基因DNA后，利用通用引物进行PCR扩增，扩增产物经琼脂糖凝胶回收纯化后，连接质粒载体，然后将连接产物转化到感受态细胞中，进行阳性克隆的筛选，培养后测序。
PCR扩增产物的电泳图见图2，该菌株的16S rDNA基因序列为：
GACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTTTGAACCGCATGGTTCAGACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCCGTTCAAATAGGGCGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTA。将该序列与GenBank中已登录的部分菌株的16S rDNA基因序列进行相似性比较，同源性达到99％，结合上述形态学鉴定结果，表明该菌株HD-1属于解淀粉芽孢杆菌(Bacillusamylotrophicus)。
实施例2芽孢杆菌HD-1发酵液的制备
将HD-1菌株以1:100的比列接种于5ml LB培养基中，37℃220rpm培奍10h，取1ml种子液接种于盛有100ml LB培养基的250ml锥形瓶中(接种量为1％)，培养条件同上。将摇瓶中的发酵种子液接种到200L发酵罐中(基础发酵培养基)，接种量为5％，温度为37℃，搅拌转速为150rpm，以1:2000(V/V装液量)的比例在培养基中加入消泡剂。在接种后8～10h，OD600达到1.8～2.0时可停止发酵，得到发酵液。
经检测，芽孢杆菌HD-1发酵液中含有多种抑菌成分及人体必氨需基酸组份，通过蛋白纯化、质谱分析以及氨基酸定量分析目前已知其中抑菌成分有β-1.3-1.4-葡聚糖甘酶，氨基酸组份及含量如表1所示。
表1
实施例3芽孢杆菌HD-1对UC小鼠的疗效实验
1.菌种：
芽孢杆菌HD-1菌株
2.实验动物：
C57BL6小鼠，雄性，7-8周龄，购买于浙江中医药大学
3.主要试剂：
葡聚糖硫酸钠(Dextran Sulfate Sodium Salt,DSS)购于美国MP公司、蛋白胨(Tryptone)、酵母提取物(Yeast Extract)购于英国Oxoid公司；其余试剂为国产分析纯。
种子培养基：5g酵母提取物，10g胰化蛋白胨，10g氯化钠，1L去离子水。
基础发酵培养基：8.5g磷酸氢二钠，3g磷酸二氢钾，1g氯化铵，0.5g氯化钠，50g蔗糖，0.492g七水合硫酸镁，1L去离子水。
PBS缓冲液：0.24g磷酸二氢钾，1.44g磷酸氢二钠，8g氯化钠，0.2氯化钾，1L去离子水
2.5％DSS溶液：25gDSS，1L去离子水
中性福尔马林固定液：100mL甲醛，加PBS定容至1L
4.样品的制备：
接种5mL芽孢杆菌HD-1菌液至500mL LB液体培养基中培养，得到HD-1种子液；再接种500mL种子液至加有10L LB液体培养基的发酵罐中扩大培养，得到HD-1菌制剂。
5.实验方法：C57BL/6小鼠以3％DSS浓度(w/v)的饮用水喂养七天，诱导UC动物模型，造模前三天开始灌胃HD-1菌直至实验结束，对照组灌胃生理盐水或发酵培养基(表2)。
表2实验动物分组及给药情况
| 实验分组 | N(只) | 培养基(mL/kg) | HD-1(mL/kg) | 给药途径 |
| Control | 9 | 10 | - | 灌胃，1次/日 |
| HD-1(10mL/kg) | 9 | - | 10 | 灌胃，1次/日 |
| DSS | 9 | 10 | - | 灌胃，1次/日 |
| DSS+HD-1(2.5mL/kg) | 9 | 7.5 | 2.5 | 灌胃，1次/日 |
| DSS+HD-1(10mL/kg) | 9 | - | 10 | 灌胃，1次/日 |
6.判定标准
各组动物体重变化，结直肠长度，HE染色观察结肠部位的炎症细胞浸润和粘膜破坏程度
7.实验结果
由图5可知，与正常组相比，模型组体重不断下降。给药组体重下降缓慢，与模型组相比存在显著性差异。
由图6结果可知，与正常组相比，模型组小鼠结肠明显缩短。两组给药组相比模型组均有明显好转。
由图7的HE染色结果可知，正常组小鼠结肠组织结构完整，杯状细胞完整，模型组小鼠组织粘膜受损，杯状细胞丢失，可见大量炎性细胞浸润，两组给药组均有明显好转。显示HD-1菌制剂具有一定的治疗作用。
以上结果显示，HD-1菌株能显著抑制溃疡性结肠炎(UC)小鼠的体重下降，减轻结肠的缩短程度，减轻UC小鼠结肠部位的炎症细胞浸润和粘膜破坏程度，对UC具有良好的治疗和预防作用。
以上仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
Claims (2)
1.解淀粉芽孢杆菌在制备治疗、缓解或预防肠道疾病的药物中的应用，所述解淀粉芽孢杆菌的保藏编号为CGMCC No. 18080；
所述肠道疾病为结肠炎及其引起的腹泻或肠道组织损伤及其引起的腹泻。
2.解淀粉芽孢杆菌在制备保健食品中的应用；所述解淀粉芽孢杆菌的保藏编号为CGMCC No. 18080；
所述保健食品为饮品。
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