CN111690560B - 一种屎肠球菌及其在水产养殖中的应用 - Google Patents
一种屎肠球菌及其在水产养殖中的应用 Download PDFInfo
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- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/46—Streptococcus ; Enterococcus; Lactococcus
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- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K61/00—Culture of aquatic animals
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Abstract
本发明涉及功能微生物筛选与应用技术领域,具体提供了一株新型屎肠球菌(Enterococcus faecium),并提供了其在水产养殖中的应用。所述屎肠球菌筛选自健康鲫鱼的肠道,保藏编号为CCTCC NO:M2020088,能够显著抑制病原菌,提高水产动物的免疫力、抗应激能力、促进水产动物生长,提高水产动物繁殖性能,并能调节水质,对水产动物益生作用显著。
Description
技术领域
本发明涉及功能微生物筛选技术领域,具体涉及一株屎肠球菌及其在水产养殖中的应用。
背景技术
1974年Parker首创益生菌一词,最早提出益生菌有利于肠道微生态平衡。目前普遍认为,益生菌的作用主要是改善动物新陈代谢、提高营养物质吸收利用率和免疫力、减少环境污染等。益生菌因具有无毒、无残留、无抗药性、无污染等特点,逐渐被广泛应用。
随着水产养殖业的发展和养殖强度的增加,水产动物病害爆发频繁。传统的抗生素药物虽然对治疗疾病有积极效用,但长期使用会产生巨大的负面效应:病原菌产生耐药性,效力下降;药物残留干扰水产动物肠道内有益微生物菌群的生长和繁殖,影响水产动物的健康;残留在水体中的抗生素破坏养殖生态环境,水产品质量下降。
自从Kozasa发现分离自土壤的芽抱杆菌可以提高养殖鳗鲡抵御爱德华氏弧菌(Edward siellatarda)的能力,有益微生物在水产养殖中的应用研究得到了快速发展。水产益生菌作用的发挥主要是通过调节肠道微生物群落结构,增强养殖生物对营养的吸收利用,进而促进其生长;或通过增强养殖生物非特异性免疫功能,从而提高其健康水平。目前应用于水产养殖上的益生菌种类很多,其中以芽孢杆菌属和酵母菌属的应用最为普遍。例如,2013年Mirbakhsh等在虾苗培养中使用枯草芽孢杆菌SI02和花域枯草芽孢杆菌IS03(B.vallismortis IS03),提高了幼苗的生长率及成活率。2014年王芸等发现了一种分离自凡纳滨对虾的肠道的解淀粉芽孢杆菌SIP0902,该菌株对包括副溶血弧菌、鳗弧菌、溶藻弧菌等7种水产动物致病弧菌均具有较强的拮抗效果。2017年,荣克明等发现了一株酿酒酵母F2,该菌株能够降解氨氮,可在养殖水体中快速转化氨氮成蛋白质,同时产生大量菌体为水生动物提供饵料,维持水产动物肠道微生态平衡,提高水产动物存活率和产量。2019年薛明洋等发现了一种含有甲基营养型芽孢杆菌(Bacillus methylotrophicus)对维氏气单胞菌、嗜水气单胞菌、温和气单胞菌、爱德华氏菌、霉实假单胞菌、无乳链球菌均有抑菌效果,可应用于水产动物疾病的治疗。
水产用益生菌除了考虑益生菌在肠道内定殖以及定殖的时间外,还应注意其耐酸、耐盐、耐热等特性,这些因素均会影响益生菌的活性及作用效果。所以,筛选的益生菌应具有如下特点:(1)在低pH值和胆汁中可以存活,可在肠内定殖,竞争优势强;(2)易于获得,在体内易繁殖,体外易培养,不与病原菌杂交;(3)产生的抑菌物质对机体本身没有危害;(4)能够提高机体免疫力;(5)经高热加工后,益生菌活性好,稳定性高;(6)不带耐药因子或所含耐药因子少。
随着人们对水产品安全和品质的要求不断提高,以及水产养殖疾病问题日益突出,益生菌成了可以替代抗生素及化学药品,防止水产动物病害的最佳选择,所以筛选出适用于水产养殖的优质益生菌是当前急需解决的问题。
发明内容
本发明的目的是提供一株新型屎肠球菌(Enterococcus faecium),并提供了其在水产养殖中的应用。所述屎肠球菌筛选自健康鲫鱼的肠道,能够显著抑制病原菌,提高水产动物的免疫力、抗应激能力、促进水产动物生长,提高水产动物繁殖性能,并能调节水质,对水产动物益生作用显著。
本发明一方面提供了一种屎肠球菌CD01(Enterococcus faecium CD01),于2020年4月29日保藏于中国武汉武汉大学的中国典型培养物保藏中心,其保藏编号为CCTCC NO:M 2020088。
本发明一方面提供了所述屎肠球菌在水产养殖中的应用。
本发明一方面提供了所述屎肠球菌在水产发酵饲料中的应用。
本发明还提供了一种微生物制剂,包括上述屎肠球菌。
所述微生物制剂进一步包含枯草芽孢杆菌、地衣芽孢杆菌、嗜酸乳杆菌、粪肠球菌、乳酸片球菌、酿酒酵母、植物乳杆菌、双歧杆菌中的任意一种或两种或多种的组合。
所述微生物制剂中屎肠球菌的活菌量不低于108U/ml。
本发明还提供了所述微生物制剂在水产养殖中的应用。
有益效果
本发明筛选到的屎肠球菌CD01对灿烂弧菌和副溶血弧菌两种致病菌具有很强的抑制效果;对青霉素、四环素、氯霉素、诺氟沙星敏感,而对庆大霉素表现耐药,对环丙沙星表现中等耐药;对酸碱的耐受性强。
所述屎肠球菌CD01可作为饲料添加剂,显著促进养殖动物生长,提高免疫力。与对照组相比,添加了屎肠球菌CD01的益生菌组中鲤鱼苗的增重率、SOD活力、LZM活力、POD活力、AKP活力、分别提高了61.9%、42.0%、64.3%、14.3%,同时应对副溶血弧菌攻毒试验的成活率提高了26.2%。
所述屎肠球菌CD01还可以应用于发酵饲料的生产。与对照组相比,饲喂屎肠球菌CD01发酵饲料的实验组对虾增重率和特定生长率分别提高了16.0%、24.2%,饵料系数降低了14.0%;而实验组对虾血液中超氧化物歧化酶、溶菌酶、过氧化物酶活力分别提高了31.9%、279.8%、21.7%,总抗氧化力提高了1.47倍。效果非常显著。
本发明筛选到的屎肠球菌CD01生长性能优秀,抗逆性强,能有效抑制病原菌,促进养殖动物生长,并且能够提高水产动物免疫力,提高全周期存活率,有助于提高养殖户的经济效益,在水产养殖中应用前景广阔。
具体实施方式
本发明实施例中选用的设备和试剂可以选自市售任意一种。对于实施例中所用到的具体方法或材料,本领域技术人员可以在本发明技术思路的基础上,根据已有的技术进行常规的替换选择,而不仅限于本发明实施例的具体记载。
其中实施例中选用的培养基,具体配方如下:
MRS培养基:蛋白胨10g,牛肉膏10g,葡萄糖20g,酵母粉5g,吐温-80lml,磷酸二氢钾2g,柠檬酸二铵2g,乙酸钠5g,硫酸镁0.58g,硫酸锰0.15g,蒸馏水1000ml,pH6.2-6.4,加入15-20g的琼脂以配制固体培养基,115℃灭菌20min。
营养肉汤培养基(1L):蛋白胨10g,牛肉膏3g,氯化钠5g。将以上成分加入蒸馏水中,定容到1000mL,加热溶解,调节pH为7.3±0.2;分装后121℃高压灭菌20min。(注:固体培养基在此基础上加1.5%琼脂粉)。
2216E海水培养基:蛋白陈5g,酵母浸出物1g,磷酸铁0.01g,海水1000ml,pH7.6-7.8,配制固体培养基加入15-20g的琼脂,121℃灭菌20min。
TSB培养基:胰蛋白胨大豆肉汤30g,氯化钠15g,蒸馏水1000ml,pH7.4-7.6,配制固体培养基加入15-20g的琼脂,121℃灭菌20min。
下面结合具体实施例进一步阐述本发明。
实施例1菌株的分离、筛选与鉴定
1、样品
采集于青岛市胶州市九龙镇中河道的鲫鱼。
2、筛选方法
将鲫鱼体表用无菌水冲洗后进行解剖,取出肠道,去除肠系膜等附着物,剪取有肠道内容物的肠道,然后加入2mL预冷的无菌生理盐水,充分匀浆,离心,取上清液10倍系列稀释。取原液、10-1、10-2三个稀释度的样品各100uL,进行MRS平板涂布,每个稀释梯度设置3个平行,35℃恒温培养48h。挑取生长良好的单菌落,并通过多次划线纯化,编号CD01、CD02、CD03…、CD18,纯化后的菌株用40%甘油和MRS液体培养基1:1制成菌悬液,-80℃保藏备用。
将活化好的菌株菌液稀释后在MRS平板上涂布,置于10℃、55℃培养观察生长情况;并在调节pH到pH4.0、pH9.6在MRS平板上涂布,置于35℃培养观察生长情况;同样在加入6.5%NaCl的MRS平板上涂布,置于35℃培养观察生长情况。通过生长观察比较试验,筛选出了CD01、CD04、CD06、CD07、CD11、CD14、CD15、CD17、CD18共9株较优秀的菌株。
以致病菌——灿烂弧菌(Vibrio splendidus)和副溶血弧菌(VibrioParahemolyticus)为指示菌,采用平板拮抗法筛选益生菌。在涂有指示菌的平板上点种待筛选的细菌菌液,28℃恒温培养,48h内观察点种区周围是否出现抑菌透明区或覆盖区,分别测量抑菌圈直径和菌落直径,评价各菌株的抑菌作用效果,具体结果见表1。
表1待筛选菌株的抑菌情况
从表1的数据可以看出,本发明筛选到的9株菌对于灿烂弧菌和副溶血弧菌均有不同程度的抑制作用,其中CD01菌株对这两种致病菌的抑制效果最强。
3、菌株鉴定
(1)菌落生物学特性:
CD01菌株在MRS琼脂平板上生长性能良好,30h即可形成0.5-1mm大小乳白色圆形菌落;菌落圆整、光滑、***;生长增殖能力强,可兼性厌氧生长,在10℃-55℃范围内均能生长,最适生长温度28℃-40℃;生长酸碱度范围pH4-10,最适pH值是5.3-6.8。
CD01菌株部分生化特性如表2所示:
表2CD01菌株的部分生化特性
注:“+”表示阳性反应,“-”表示阴性反应
(2)分子生物学鉴定
提取上述CD01菌株的基因组DNA,利用PCR技术扩增16SrRNA序列,将测序结果进行NCBI BLAST比对分析。结果显示,CD01菌株与已公布的多株屎肠球菌的16S rRNA序列相似度高达98.8%,鉴定证实为屎肠球菌(Enterococcus faecium),与生化鉴定结果一致。
申请人将上述CD01菌株命名为屎肠球菌CD01(Enterococcus faecium CD01),已于2020年4月29日保藏于中国武汉武汉大学的中国典型培养物保藏中心,其保藏编号为CCTCC NO:M 2020088。
实施例2屎肠球菌CD01耐酸碱性实验
将活化的屎肠球菌CD01以2%的接种量分别接种于pH2.5的盐酸溶液、pH10.0的氢氧化钠溶液及pH6.8的蒸馏水37℃静置培养取接0h,1h,2h,4h的培养液,分别作连续10倍稀释,从每一稀释度取0.1mL涂于MRS平板,观察生长情况并进行菌落计数,计算活菌保留率,具体结果见表3。
活菌保留率(%)=(某h时的活菌量-0h时的活菌量)/0h时的活菌量×100%。
表3屎肠球菌株CD01对不同pH环境的耐受性
从表3的结果可以看出,本发明提供的屎肠球菌CD01在pH2.5条件下孵育2h、4h后,活菌数仅有少量减少,活菌保留率超过95%;在pH10.0条件下孵育2h、4h后,活菌数维持在3.78×107CFU/mL,活菌保留率超过97%;而在pH6.8条件下孵育,屎肠球菌CD01的菌量增加了39.8%和103.1%。从而说明,本发明提供的屎肠球菌CD01对酸碱性条件具有很强的耐受性,为其广泛应用打下基础。
实施例3屎肠球菌CD01对不同抗生素敏感性试验
将MRS固体培养基融化后冷却至50℃,每30mL加入活化好的屎肠球菌CD01菌液0.5mL(约108CFU/mL)倾倒平板,待平板表面略干后,放入常见抗生素(青霉素、庆大霉素、四环素、氯霉素、诺氟沙星、环丙沙星)药敏纸片35℃培养,观察生长情况,测量抑菌圈直径。
表4屎肠球菌株CD01对不同抗生素的敏感性试验结果
从表4的结果可以看出,本发明提供的屎肠球菌CD01对青霉素、四环素、氯霉素、诺氟沙星敏感,而对庆大霉素表现耐药,对环丙沙星表现中等耐药。
实施例4屎肠球菌CD01对鲤鱼生长、免疫及抗病力的影响
1、菌粉的制备
将屎肠球菌CD01进行活化,扩大培养,经液态发酵、离心、浓缩、冻干,制成活菌含量约为1010CFU/g的菌粉。
2、实验设计
实验设对照组和益生菌组,每组分别设置三个平行。每个平行鲫鱼苗30尾(初始体重6.98±0.14g)。其中,对照组以市售饲料为基础饲料,益生菌组是在基础饲料中按3.5‰添加上述屎肠球菌CD01菌粉,混合均匀投喂鲤鱼苗。养殖实验在室内循环水养殖***开展,共进行30天,每天投饵两次,pH 7.6±0.4,水温20-22℃,溶氧充足。
养殖实验结束后,对鲤鱼进行计数并称重,计算其特定生长率。每组随机抽取3尾鱼。用100mL/L的丁香油麻醉,采用尾静脉采血法抽取鲤鱼血液,37℃静置1h,5000r/min离心10min,收集血清于-80℃保存;检测血清中的超氧化物歧化酶(SOD)、溶菌酶(LZM)、过氧化氢酶(POD)、碱性磷酸酶(AKP)活力。以副溶血弧菌为病原菌,对鲤鱼进行腹腔注射,进行2周的观察,及时捞出死亡的鲤鱼苗,计算存活率。具体结果见表5。
表5屎肠球菌CD01对鲤鱼生长、免疫及抗病力的影响
对照组 | 益生菌添加组 | |
初始体重g | 6.99±0.14 | 6.97±0.13 |
终末体重g | 13.25±0.41 | 14.37±0.47 |
增重率% | 89.56±1.79<sup>a</sup> | 106.17±2.07<sup>b</sup> |
SOD活力mU/L | 0.42±0.03<sup>a</sup> | 0.68±0.04<sup>b</sup> |
LZM活力mU/L | 0.50±0.03<sup>a</sup> | 0.71±0.05<sup>b</sup> |
POD活力mU/L | 0.28±0.02<sup>a</sup> | 0.46±0.04<sup>b</sup> |
AKP活力mU/L | 0.49±0.05 | 0.56±0.04 |
存活率% | 67.8±5.1<sup>a</sup> | 85.6±1.9<sup>b</sup> |
注:不同的字母表示差异显著(P<0.05)。
从表5的结果可以看出,与对照组相比,添加了屎肠球菌CD01的益生菌组中鲤鱼苗的增重率、SOD活力、LZM活力、POD活力、AKP活力、分别提高了61.9%、42.0%、64.3%、14.3%,同时应对副溶血弧菌攻毒试验的成活率提高了26.2%。从而说明本发明所述屎肠球菌CD01对鲤鱼苗的生长有显著的促进作用,并能有效提高鲤鱼苗的免疫力及抵抗副溶血弧菌的能力,因此可以作为益生菌广泛应用于鱼类的养殖生产过程中。
实施例5采用屎肠球菌CD01发酵的饲料对南美白对虾生长的影响
本实验设对照组和发酵饲料组,以市售的对虾商业配合饲料为基础饲料。其中,对照组饲喂基础饲料,发酵饲料处理如下:将屎肠球菌CD01以1%的接种量接种在营养肉汤培养基中,在35℃条件下静置培养20h,培养后确认菌的浓度达到108CFU/ml以上,然后将菌液以2%的接种量接种在基础饲料中,加无菌水调整至最终水分29-30%。将混合物装入呼吸发酵袋封口,置于35℃环境下恒温发酵96小时。发酵完成后具有特殊的发酵风味,冻存于-20℃冰箱。
每组分别设置三个平行,每个平行50尾凡纳滨对虾。养殖实验持续8周,每日按虾体重的3-4%投喂,根据摄食量情况随时对投饵量进行调整,对照组和发酵饲料组投喂饲料的干物质重保持一致;每天早晚各投喂一次,吸底排污一次。实验期间溶氧≥7mg/l,温度28±2℃,盐度20-22‰,pH 8.0±0.3。养殖实验结束后,对凡纳滨对虾进行计数并称重,计算其存活率、特定生长率、增重率等。每平行取15—20尾虾!用无菌注射器***心脏取血,离心后移出血清分装保存备用;采用试剂盒测定血清超氧化物歧化酶、溶菌酶、过氧化物酶活力及总抗氧化力等。具体结果见表6和表7。
表6屎肠球菌CD01发酵的饲料对南美白对虾生长和存活的影响
对照组 | 发酵饲料组 | |
初始体重g | 1.42±0.05 | 1.41±0.05 |
终末体重g | 5.95±0.30 | 6.49±0.24 |
存活率% | 86.67±4.16<sup>a</sup> | 96.67±2.31<sup>b</sup> |
增重率% | 319.05±21.79<sup>a</sup> | 370.09±15.36<sup>b</sup> |
特定生长率% | 2.36±0.08<sup>a</sup> | 2.93±0.06<sup>b</sup> |
饵料系数 | 1.57±0.10<sup>a</sup> | 1.35±0.07<sup>b</sup> |
注:不同的字母表示差异显著(P<0.05)。
表7屎肠球菌CD01发酵的饲料对南美白对虾免疫力的影响
对照组 | 发酵饲料组 | |
超氧化物歧化酶活力 | 204.42±17.03<sup>a</sup> | 269.68±23.04<sup>b</sup> |
溶菌酶活力 | 1.24±0.93<sup>a</sup> | 4.71±2.05<sup>b</sup> |
过氧化物酶活力 | 57.57±2.32 | 70.08±1.03 |
总抗氧化力 | 3.46±0.65<sup>a</sup> | 8.56±0.74<sup>b</sup> |
注:不同的字母表示差异显著(P<0.05)。
从表6和表7的结果可以看出,与对照组相比,饲喂屎肠球菌CD01发酵饲料的实验组对虾增重率和特定生长率分别提高了16.0%、24.2%,饵料系数降低了14.0%;而实验组对虾血液中超氧化物歧化酶、溶菌酶、过氧化物酶活力分别提高了31.9%、279.8%、21.7%,总抗氧化力提高了1.47倍。从而说明,本发明提供的屎肠球菌CD01能显著促进养殖动物的存活生长,提高了养殖动物的免疫力和抗病能力,取得了意料不到的技术效果。
综上,本发明筛选到的屎肠球菌CD01,生长性能优秀,抗逆性强,能有效抑制病原菌,能作为饲料添加剂促进养殖动物生长,并且能够提高水产动物免疫力,提高全周期存活率,有助于提高养殖户的经济效益,在水产养殖中应用前景广阔。
Claims (5)
1.一种屎肠球菌(Enterococcus faecium),其特征在于,所述的屎肠球菌的保藏编号为CCTCC NO: M 2020088。
2.权利要求1所述的屎肠球菌在水产发酵饲料中的应用。
3.一种微生物制剂,其特征在于,所述的微生物制剂包含权利要求1所述的屎肠球菌。
4.如权利要求3所述的微生物制剂,其特征在于,所述的微生物制剂还包含枯草芽孢杆菌、地衣芽孢杆菌、嗜酸乳杆菌、粪肠球菌、乳酸片球菌、酿酒酵母、植物乳杆菌、双歧杆菌中的任意一种或两种或多种的组合。
5.如权利要求3或4所述的微生物制剂,其特征在于,所述的微生物制剂中屎肠球菌的活菌量不低于108 U/ml。
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