CN116286447A - 一株枯草芽孢杆菌及其应用 - Google Patents
一株枯草芽孢杆菌及其应用 Download PDFInfo
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- CN116286447A CN116286447A CN202211227679.9A CN202211227679A CN116286447A CN 116286447 A CN116286447 A CN 116286447A CN 202211227679 A CN202211227679 A CN 202211227679A CN 116286447 A CN116286447 A CN 116286447A
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- bacillus subtilis
- vibrio harveyi
- spotted maigre
- culture
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Abstract
本发明公开了一株枯草芽孢杆菌及其应用。本发明所提供的枯草芽孢杆菌具体为枯草芽孢杆菌(Bacillussubtilis)B0E9,它在中国微生物菌种保藏管理委员会普通微生物中心的保藏编号为CGMCCNo.24950。本发明的枯草芽孢杆菌(Bacillussubtilis)B0E9CGMCCNo.24950能够有效提高黄姑鱼对红头病病原——哈维氏弧菌B0003的抗病力,增强感染哈维氏弧菌B0003后黄姑鱼的免疫功能,减少炎性因子产生,有效改善哈维氏弧菌B0003攻毒后黄姑鱼肝脏损伤,降低哈维氏弧菌B0003攻毒后的发病率和死亡率。同时,本发明的枯草芽孢杆菌可制成菌剂用于预防或治疗黄姑鱼的红头病,是一种绿色、安全的水产饲料添加剂。
Description
技术领域
本发明属于生物技术领域,尤其涉及一株枯草芽孢杆菌及其应用。
背景技术
黄姑鱼(Nibea albiflora)俗称黄姑子、黄婆鸡,在福建沿海地区俗称春子鱼,隶属于鲈形目(Perciformes),鲈亚目(Percoidei)、石首鱼科(Sciaenidae)、黄姑鱼属(Nibea),为近海中下层经济鱼类,主要分布于中国东海、日本南海,是我国主要的养殖经济鱼类之一。黄姑鱼年产量达6.7万吨以上,年养殖产量超过3万吨,具有较高的经济和养殖价值。近年来,随着东海渔业资源的枯竭,人工养殖黄姑鱼的规模不断扩大。与此同时,高密度的养殖环境加大了黄姑鱼疾病的风险。在黄姑鱼养殖中,红头病是黄姑鱼主要病害之一,患病鱼活性较弱、离群、游动缓慢,头部有明显红肿,感染后5-6天,死亡率可达50%以上,给养殖户造成巨大经济损失。实验室前期从患红头病的黄姑鱼病灶中分离出了哈维氏弧菌(Vibrio harveyi)B0003(GenBank登录号:ON076875),经回归感染试验验证,哈维氏弧菌B0003是黄姑鱼红头病的主要病原菌。
目前生产中,抗生素和化学药物仍然是防治黄姑鱼红头病的重要手段,但是,抗生素和化学药物在水产养殖中的大量应用存在抗药性问题、药物残留问题、破坏微生态环境和抑制水产动物免疫系统等副作用。因此,寻求无残留无污染的抗生素及化学药物的有效替代品具有重要意义。
发明内容
本发明的目的是提供一株枯草芽孢杆菌及其培养物、及其在制备菌剂或饲料添加剂中的应用。
为了实现本发明的上述目的,采用了以下技术方案:
一株枯草芽孢杆菌,该枯草芽孢杆菌保藏于中国普通微生物菌种保藏管理中心(简称:CGMCC,北京市朝阳区北辰西路1号院3号),其保藏编号为CGMCC No.24950。
上述的枯草芽孢杆菌的培养物。
上述的枯草芽孢杆菌或枯草芽孢杆菌的培养物在制备菌剂或饲料添加剂中的应用。
一种菌剂,其包括上述的枯草芽孢杆菌或其培养物。
一种饲料添加剂,其包括上述的枯草芽孢杆菌或其培养物。
术语“培养物”是指经人工接种和培养后,长有微生物群体的液体或固体产物(培养容器内的所有物质,发酵产物)的统称。即通过将微生物进行生长和/或扩增而获得的产物,其可以是微生物的生物学纯培养物,也可以含有一定量的培养基、代谢物或培养过程中产生的其他成分。
与现有技术相比,本发明的有益效果为:通过比较易感和耐受哈维氏弧菌B0003黄姑鱼的肠道微生物,在耐受哈维氏弧菌的黄姑鱼肠道中筛选出了一株对哈维氏弧菌B0003具有较强拮抗作用的枯草芽孢杆菌(Bacillus subtilis)B0E9 CGMCC No.24950,能够有效提高黄姑鱼对红头病病原——哈维氏弧菌B0003的抗病力,增强感染哈维氏弧菌B0003后黄姑鱼的免疫功能,减少炎性因子产生,有效改善哈维氏弧菌B0003攻毒后黄姑鱼肝脏损伤,降低哈维氏弧菌B0003攻毒后的发病率和死亡率。本发明将枯草芽孢杆菌干燥得到的枯草芽孢杆菌菌剂,可以作为添加剂用于制备黄姑鱼饲料。
保藏说明
保藏地址: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
保藏日期:2022年05月23日
菌种名称:枯草芽孢杆菌
拉丁名:Bacillus subtilis
菌株编号:B0E9
保藏机构:中国微生物菌种保藏管理委员会普通微生物中心
保藏机构简称:CGMCC
保藏中心登记入册编号:CGMCC No.24950
附图说明
下面参照附图结合实施例对本发明作进一步的说明。
图1为黄姑鱼红头病症状图。
图2为哈维氏弧菌B0003的电镜图。
图3为枯草芽孢杆菌B0E9对哈维氏弧菌B0003抑菌效果图。
图4哈维氏弧菌B0003攻毒后黄姑鱼肝脏组织形态(HE染色)
具体实施方式
为了更好地理解本发明,下面结合实施例和附图对本发明做进一步的详细说明,但本领域技术人员了解,下述实施例不是对本发明保护范围的限制,任何在本发明基础上做出的改变和变化,都在本发明的保护范围之内。
下述实施例中所使用的实验方法如无特殊说明,均为常规方法。
下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
实施例1
本实施例提供枯草芽孢杆菌B0E9的分离与鉴定
一、枯草芽孢杆菌B0E9菌株来源
通过易感和耐受哈维氏弧菌B0003(黄姑鱼红头病症状如图1所示,哈维氏弧菌B0003的电镜图如图2所示)的黄姑鱼肠道差异微生物,发现枯草芽孢杆菌B0E9(菌落形态:白色,菌落不透明,形状不规则,菌落较大且表面不光滑)仅存在于耐受哈维氏弧菌B0003的黄姑鱼肠道,提示其可能有利于提高黄姑鱼对哈维氏弧菌B0003的抗病力。
二、枯草芽孢杆菌B0E9的鉴定
S1.形态学鉴定
一方面,将处于对数生长期,且菌落大小稳定,上述步骤一分离并筛选得到的菌株B0E9进行单菌落状态描述,主要包括菌落的大小、颜色、透明度、湿润度、菌落表面状态、菌落边缘状态。另一方面,对处于对数生长期的所述菌株B0E9,经涂片染色后采用光学显微镜观察菌体的形态。
分离并筛选得到的单菌落为:白色,菌落不透明,形状不规则,菌落较大且表面不光滑。
分离并筛选得到的菌株B0E9的培养基为2216E琼脂培养基。
S2.16S rDNA序列同源性分析
挑取平板上的单菌落进行16S rDNA测序鉴定,所得序列与GenBank数据库中已知序列进行比对,发现其16S序列与数据库中已知枯草芽孢杆菌序列相识度达99%(如SEQ IDNO:1所示):
枯草芽孢杆菌B0E9的16S rDNA序列:
GCAGTCGAGCGGACAGATGGGAGCTTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTTTGAACCGCATGGTTCAAACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTACCGTTCGAATAGGGCGGTACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGcCGCGGTAATACGTAGGTGGCAAGCGtTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCT
CTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTAGGAGCCAGCCGCCG
上述形态特征和16srDNA序列同源性分析结果表明该菌为枯草芽孢杆菌(Bacillus subtilis)。该菌株已于2022年5月23日保藏于中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC,地址: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,邮编100101),保藏号为CGMCC No.24950,其分类命名为枯草芽孢杆菌(Bacillussubtilis)B0E9。
实施例2
本实施例提供枯草芽孢杆菌B0E9的体外抑菌实验
将指示菌哈维氏弧菌B0003和枯草芽孢杆菌B0E9分别在营养肉汤培养基中33℃、180r/min条件下培养24h。将培养好的B0003用生理盐水稀释1000倍,取100uL涂布于营养琼脂平板上,用内径为5mm的琼脂打孔器在营养琼脂平板上均匀打3个孔,每个孔加入B0E9菌液150uL。将平板置于33℃恒温培养箱中正置培养24h,用游标卡尺测量抑菌圈的直径,判断试验菌对指示菌的抑制效果。
结果如图3所示,打孔周围出现明显抑菌圈,抑菌圈直径为13.13±0.89mm。表明枯草芽孢杆菌B0E9对哈维氏弧菌B0003具有良好的抑菌效果。
实施例3
本实施例提供枯草芽孢杆菌B0E9的药物敏感性
采用纸片琼脂扩散法(K-B法)进行药物敏感性检测。选择常见的30种药敏纸片(杭州微生物试剂有限公司):米诺环素、新霉素、氧氟沙星、链霉素、卡那霉素、诺氟沙星、四环素、阿奇霉素、氟苯尼考、头孢他啶、氨苄西林、苯唑西林、头孢唑林、头孢氨苄、克拉霉素、头孢拉定、奈替沙星、头孢哌酮、庆大霉素、头孢曲松、利福平、复方新诺明、氟罗沙星、氯霉素、多粘菌素B、环丙沙星、洛美沙星、新生霉素、恩诺沙星、呋喃唑酮。
将枯草芽孢杆菌B0E9在33℃、180r/min条件下活化培养至浓度为1.5×108CFU/mL,分别取不同试验菌菌液100uL涂布于对应营养琼脂培养基中。使用无菌镊子将药敏纸片贴在培养基的表面,每张纸片的距离大于24mm,纸片的中心距离要距离平板边缘15mm以上,每个培养基平板放4个药敏纸片。于33℃恒温培养箱中培养24h后,使用游标卡尺测量其抑菌圈的大小,统计其药物敏感性。
枯草芽孢杆菌B0E9对所检测抗菌药物均具有敏感性,其中对利福平、多粘菌素B、呋喃唑酮具有中等敏感。
表1枯草芽孢杆菌B0E9的药物敏感性
名称 | B0E9 | 名称 | B0E9 | 名称 | B0E9 |
米诺环素 | S | 氨苄西林 | S | 利福平 | I |
新霉素 | S | 苯唑西林 | S | 复方新诺明 | S |
氧氟沙星 | S | 头孢唑林 | S | 氟罗沙星 | S |
链霉素 | S | 头孢氨苄 | S | 氯霉素 | S |
卡那霉素 | S | 克拉霉素 | S | 多粘菌素B | I |
诺氟沙星 | S | 头孢拉定 | S | 环丙沙星 | S |
四环素 | S | 奈替沙星 | S | 洛美沙星 | S |
阿奇霉素 | S | 头孢哌酮 | S | 新生霉素 | S |
氟苯尼考 | S | 庆大霉素 | S | 恩诺沙星 | S |
头孢他啶 | S | 头孢曲松 | S | 呋喃唑酮 | I |
注:抑菌圈直径d>15mm,为敏感(S);10mm<抑菌圈直径d<15mm,为中度敏感(I);抑菌圈直径d<10mm,为不敏感(R)。
实施例4
本实施例提供枯草芽孢杆菌B0E9的应用
菌株培养和制剂的制备:
S1.用接种环挑取适量斜面保存的枯草芽孢杆菌B0E9菌体接种到6-10个装有50mL培养基1的250mL三角瓶中进行培养。其中,培养基1的组成为:蛋白胨8-12g,牛肉膏4-6g,葡萄糖16-25g,氯化钠4-6g,蒸馏水1000mL;培养条件为:培养温度28-30℃;初始pH值6.9-7.2;摇床转速200-260rpm;培养时间为20-24h;得到300-500mL的一级种子培养液。
S2.按照2-4%的比例将一级种子培养液接种到20-30个装有400mL培养基2的2000mL三角瓶中。其中,培养基2的组成为:蛋白胨8-12g,牛肉膏4-6g,葡萄糖16-25g,氯化钠4-6g,蒸馏水1000mL;培养条件为:培养温度28-30℃;初始pH值6.9-7.2;摇床转速200-260rpm;培养时间为18-24h;得到8-12L的二级种子培养液。
S3.液体发酵具体为:按照3%-5%的比例将二级种子培养液接种到培养基3中,其中培养基3的组分为玉米粉10-16g,豆粕粉8-12g,玉米浆4-8g,硫酸铵5-8g,七水硫酸镁0.8-1.2g,三水合磷酸氢二钾2-4g,硫酸锰0.04-0.06g,自来水1000mL;在500L发酵罐进行液体发酵,发酵条件为:发酵温度28-30℃;初始pH值6.9-7.2;搅拌转速200-260rpm;装液系数0.5;发酵时间为20-28h。
S4.制备益生菌粉。按照S3.发酵后,离心收集菌体(转速为4000-5000rpm;离心时间为10min-20min),在菌体中加入玉米淀粉作为载体(质量比为1:2-1:4),经振动流化床干燥(干燥条件为:进风温度60℃-100℃,出风温度40℃-60℃)、粉碎、过80目筛、真空包装,获得可常温保存的益生菌粉,每克益生菌粉中枯草芽孢杆菌B0E9活菌含量1.0×1010CFU;
使用方法:
将枯草芽孢杆菌B0E9制备的益生菌粉按照0.1%-1%的比例添加到黄姑鱼基础饲料(表2)中,枯草芽孢杆菌B0E9在每克基础饲料中含量达到1.0×107-1.0×108CFU。优选每克基础饲料中的B0E9含量达到1.0×108CFU,可降低哈维氏弧菌B0003攻毒后黄姑鱼的累积死亡率(表3),且能显著提高黄姑鱼血清溶菌酶活性(表4)和显著上调皮肤相关免疫基因的表达(表5)。
表2基础饲料组成
原料 | 含量 |
鱼粉Fish meal | 45.00 |
豆粕soybean meal | 19.40 |
玉米蛋白粉Corn gluten | 8.00 |
鸡肉粉Chicken powder | 8.00 |
α-淀粉α-starch | 9.00 |
鱼油Fish oil | 3.00 |
豆油Soybean oil | 3.00 |
大豆卵磷脂Soybean lecithin | 1.50 |
磷酸二氢钙Ca(H2PO4) | 1.50 |
维生素预混料Vitamin premix | 0.60 |
矿物质预混料Mineral premix | 0.40 |
氯化胆碱Choline chloride | 0.50 |
维生素C VitaminC | 0.10 |
总计total | 100.00 |
营养水平Nutrient level | |
水分Moisture | 6.32 |
粗蛋白Crude protein | 50.85 |
粗脂肪Crude lipid | 8.43 |
粗灰分Crude ash | 10.90 |
实验结果(应用效果):
(1)枯草芽孢杆菌B0E9提高黄姑鱼免疫功能
以蒸汽鱼粉和豆粕为主要蛋白源,以鱼油、豆油、大豆卵磷脂为主要脂肪源配制基础饲料,试验组在基础饲料的基础上添加1%益生菌粉(每克饲料中的枯草芽孢杆菌B0E9含量为1.0×108CFU),进行为期42d的养殖试验,研究枯草芽孢杆菌B0E9对黄姑鱼免疫功能的影响。结果表明,试验组血清补体C3含量和溶菌酶活性显著高于对照组(P<0.05),试验组IgM含量与对照组无显著差异。提示枯草芽孢杆菌B0E9能显著提高黄姑鱼非特异性免疫功能。
表3枯草芽孢杆菌B0E9对黄姑鱼免疫功能的影响
溶菌酶(U/mL) | 补体C3(ug/mL) | IgM(ug/mL) | |
对照组 | 327.83±20.07b | 43.45±1.71b | 10.90±0.99 |
枯草芽孢杆菌B0E9组 | 400.95±3.84a | 50.66±3.40a | 10.75±0.58 |
(2)枯草芽孢杆菌B0E9提高黄姑鱼对哈维氏弧菌B0003攻毒抵抗力
42d的养殖试验结束后,利用黄姑鱼红头病病原——哈维氏弧菌B0003进行攻毒实验,记录攻毒后5天的红头症状发生和死亡情况。从表4中可以看出,饲料中添加1%益生菌粉黄姑鱼的红头症状发生率降低了43.74%,累积死亡率降低了33.34%。提示枯草芽孢杆菌B0E9能有效提高黄姑鱼抗红头病能力。
表4哈维氏弧菌B0003攻毒后黄姑鱼累积死亡率
红头症状发生率(%) | 累积死亡率(%) | |
对照组 | 72.63±12.56 | 53.57±15.15 |
枯草芽孢杆菌B0E9组 | 40.86±15.24 | 35.71±18.90 |
(3)枯草芽孢杆菌提高哈维氏弧菌B0003攻毒后黄姑鱼的免疫功能
从表5中可以看出,与对照组相比,饲料中添加0.1%益生菌粉组黄姑鱼血清中溶菌酶活性显著上升(P<0.05),补体C3和IgM含量略有上升,IL-1β含量明显下降,提示枯草芽孢杆菌B0E9可显著提高哈维氏弧菌B0003攻毒后黄姑鱼的血清免疫功能,减少炎性因子产生。
表5哈维氏弧菌B0003攻毒后黄姑鱼血清免疫指标
溶菌酶(U/mL) | 补体C3(ug/mL) | IgM(ug/mL) | IL-1β(pg/mL) | |
对照组 | 259.26±37.04b | 419.23±15.82 | 80.32±7.14 | 32.18±7.11 |
枯草芽孢杆菌B0E9组 | 493.83±42.77a | 428.87±29.81 | 86.07±4.40 | 25.12±2.15 |
由表6可知,与对照组相比,饲料中添加1%益生菌粉组黄姑鱼头部皮肤抗菌肽基因(NK-lysin)、溶菌酶基因(Lysozyme)和白细胞介素10(IL-10)表达量均显著提高(P<0.05),提示饲料中添加枯草芽孢杆菌B0E9可显著提高哈维氏弧菌B0003攻毒后黄姑鱼皮肤免疫性能。
表6哈维氏弧菌B0003攻毒后黄姑鱼皮肤免疫基因相对表达量
NK-lysin | Lysozyme | IL-10 | |
对照组 | 1.00±0.16b | 1.00±0.24b | 1.00±0.18b |
枯草芽孢杆菌B0E9组 | 4.67±1.29a | 6.58±0.71a | 5.14±2.44a |
哈维氏弧菌B0003攻毒后,对黄姑鱼肝脏组织HE染色后,观察肝脏组织形态,由图4可知,与对照组相比,枯草芽孢杆菌B0E9组肝细胞排列更为紧密,且细胞空泡化减少,提示枯草芽孢杆菌B0E9能有效改善哈维氏弧菌B0003攻毒后黄姑鱼肝脏损伤。
综上,本发明从黄姑鱼肠道分离筛选到对黄姑鱼红头病病原——哈维氏弧菌B0003具有较强拮抗作用的功能微生物——枯草芽孢杆菌B0E9。动物实验证实,饲料中添加枯草芽孢杆菌B0E9能显著提高黄姑鱼免疫功能,能有效改善哈维氏弧菌B0003攻毒后黄姑鱼肝脏损伤,降低哈维氏弧菌B0003攻毒后的发病率和死亡率。因此,枯草芽孢杆菌B0E9能有效增强黄姑鱼抗红头病能力,为黄姑鱼红头病的防治提供一种新的解决方案。
虽然以上描述了本发明的具体实施方式,但是熟悉本技术领域的技术人员应当理解,我们所描述的具体的实施例只是说明性的,而不是用于对本发明的范围的限定,熟悉本领域的技术人员在依照本发明的精神所作的等效的修饰以及变化,都应当涵盖在本发明的权利要求所保护的范围内。
Claims (5)
1.一株枯草芽孢杆菌,其特征在于,所述枯草芽孢杆菌为枯草芽孢杆菌B0E9(Bacillussubtilis B0E9),保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为CGMCC No.24950。
2.如权利要求1所述的枯草芽孢杆菌的培养物。
3.如权利要求1所述的枯草芽孢杆菌或权利要求2所述的枯草芽孢杆菌的培养物在制备菌剂或饲料添加剂中的应用。
4.一种菌剂,其特征在于,所述菌剂包括权利要求1所述的枯草芽孢杆菌或权利要求2所述的枯草芽孢杆菌的培养物。
5.一种饲料添加剂,其特征在于,所述饲料添加剂包括权利要求1所述的枯草芽孢杆菌或权利要求2所述的枯草芽孢杆菌的培养物。
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