CN115505546A - 一株抑制嗜水气单胞菌的耐盐芽孢杆菌及其应用 - Google Patents
一株抑制嗜水气单胞菌的耐盐芽孢杆菌及其应用 Download PDFInfo
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Abstract
本发明涉及微生物技术领域,尤其涉及一株抑制嗜水气单胞菌的耐盐芽孢杆菌及其应用。本发明提供的耐盐芽孢杆菌(Bacillus halotolerans)LM32,其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.25631。本发明的耐盐芽孢杆菌能够产蛋白酶、淀粉酶、脂肪酶,能够提高锦鲤的抗氧化能力,调节锦鲤免疫反应,对嗜水气单胞菌具有拮抗作用,是一种潜在益生菌。
Description
技术领域
本发明涉及微生物技术领域,尤其涉及一株抑制嗜水气单胞菌的耐盐芽孢杆菌及其应用。
背景技术
嗜水气单胞菌(Aeromonas hydrophila)属类弧菌科,革兰染色阴性,单鞭毛而有动力,无荚膜,不形成芽胞。嗜水气单胞菌普遍存在于淡水、污水、淤泥和土壤中,是一种典型的***共患病病原,对鱼、蛙等冷血动物和小鼠、豚鼠、家兔等温血动物均有致病性,可以引起人类腹泻和多种水产动物的败血症。
锦鲤也称绯锦鲤的原始品种,为红色鲤鱼(Cyprinus carpio),是欣赏价值非常高的观赏鱼类。锦鲤感染嗜水气单胞菌会出现典型出血性败血症症状。
拮抗作用是细菌或其他微生物相互作用的方式之一,是指一种微生物在生长过程中通过某种代谢产物或改变生活环境来抑制其他微生物的生长发育,甚至杀死他们的现象。与药物控制相比,益生菌具有无毒高效的优点。因此,利用拮抗性能强的菌株抑制病原菌的生长不仅能预防或治疗疾病,还会减少对抗生素、消毒剂等的使用和依赖,开发能够抑制病原菌生长的菌株具有非常重要的意义。但是,现有技术中缺少一种能够抑制嗜水气单胞菌的菌剂。
发明内容
本发明提供一株抑制嗜水气单胞菌的耐盐芽孢杆菌及其应用,该菌株可以提高锦鲤的抗氧化能力,还能调节免疫反应,对嗜水气单胞菌具有拮抗作用。
本发明提供耐盐芽孢杆菌(Bacillus halotolerans)LM32,其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.25631。保藏日期:2022年8月31日;保藏单位地址: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,邮政编码:100101。
本发明还提供一种菌剂,其含有所述的耐盐芽孢杆菌(Bacillus halotolerans)LM32。
根据所述的菌剂,所述菌剂中耐盐芽孢杆菌(Bacillus halotolerans)LM32的有效活菌数≥108CFU/g。
在一些实施例中,所述菌剂中耐盐芽孢杆菌(Bacillus halotolerans)LM32的有效活菌数108-109CFU/g。
在一些实施例中,所述菌剂的剂型包括:固体制剂、液体制剂;所述固体制剂包括颗粒剂、粉末剂(优选冻干粉);所述液体制剂包括注射剂、口服剂。
本发明还提供一种动物的饲喂方法,利用上述的耐盐芽孢杆菌(Bacillushalotolerans)LM32或上述的菌剂进行饲喂。
在一些实施例中,耐盐芽孢杆菌(Bacillus halotolerans)LM32与基础日粮的比例为0.6-6×105CFU/g。
在一些实施例中,将菌剂制备成冻干粉末,利用上述菌剂进行饲喂,所述菌剂的添加量为基础日粮的0.06-0.6wt%。
饲料添加剂、动物饲料或药物,包括上述的耐盐芽孢杆菌(Bacillushalotolerans)LM32或上述的菌剂或其发酵物或发酵提取物。
上述耐盐芽孢杆菌(Bacillus halotolerans)LM32或上述的菌剂或其发酵物或发酵提取物在制备饲料添加剂、动物饲料或药物中的应用,所述饲料添加剂、动物饲料或药物被用于以下至少一方面:
(1)抑制病原菌;
(2)提高动物免疫能力;
(3)增加动物重量;
(4)提高动饲料转化率;
(5)提高动物对营养物质的利用能力;
(6)促进或抑制动物生长性能;
(7)促进肠道发育或维持肠道微生物的稳态。
根据上述的应用,所述病原菌包括嗜水气单胞菌(Aeromonas hydrophila)。
根据上述的应用,所述动物为水生动物。
根据上述的应用,所述动物为鱼类或蛙类。
在一些实施例中,所述动物为鲤科鱼类。
在一些实施例中,所述动物包括鲤鱼、鲫鱼、鲢鱼、团头鲂、鲢鳙鱼、草鱼、鲫鱼、麦穗鱼、鳊鱼。
根据上述的应用,所述增加动物重量具体通过提高动物对营养物质的利用能力来实现。
和/或,所述提高动饲料转化率具体通过提高动物对营养物质的利用能力来实现。
和/或,所述提高动物免疫能力具体通过调节TP、SOD、LZM、ALP、ACP中的一种或几种实现。
在一些实施例中,所述饲料添加剂、动物饲料或药物被用于提高TP、SOD、LZM或降低ALP、ACP。
本发明筛选出的耐盐芽孢杆菌LM32能产多种酶、对常见条件性致病菌具有拮抗作用、耐热性较好。
本发明所述的耐盐芽孢杆菌LM32可以提高锦鲤的抗氧化能力,调节锦鲤免疫反应,对嗜水气单胞菌具有拮抗作用,是一种潜在益生菌。
附图说明
为了更清楚地说明本发明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一简单地介绍,显而易见地,下面描述中的附图是本发明的一些实施例,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,还可以根据这些附图获得其他的附图。
图1是本发明耐盐芽孢杆菌LM32对嗜水气单胞菌拮抗能力试验结果;
图2是本发明耐盐芽孢杆菌LM32的血平板划线;
图3是本发明耐盐芽孢杆菌LM32的菌落形态图;
图4是本发明饲喂不同浓度耐盐芽孢杆菌LM32对锦鲤生长指标的影响图;
图5是本发明饲喂不同浓度耐盐芽孢杆菌LM32对锦鲤血清免疫指标的影响图。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚,下面将结合本发明中的附图,对本发明中的技术方案进行清楚、完整地描述,显然,所描述的实施例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作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。
1菌株的分离、纯化
解剖锦鲤肠道,将其肠道内容物及黏膜溶于1ml pbs溶液中充分混匀,作为原液,将一部分原液进行梯度稀释,直至10-6。分别取100ul10-1、10-2、10-3、10-4、10-5、10-6稀释样品涂布于MRS和LB固体培养基上,每个梯度三个平行,于37℃恒温培养1-3d。选择菌落清楚、疏密适当且菌落数在30~300之间的平板,随机挑取形态大小不同的菌落进行分离,通过多次划线对菌株进行纯化,直至获得形态相同的单一菌落。
1.1产酶能力测定
用无菌枪头挑取纯化后的菌株,接种于1ml LB和MRS液体培养基中,37℃、180rpm/min培养24h,用滤纸片法分别接种在产蛋白酶、产淀粉酶、产脂肪酶培养基上,28℃分别培养2d后观察,若菌落周围出现透明环,表示该细菌能产淀粉酶和脂肪酶,为阳性,反之则为阴性。对于产淀粉酶的检测,28℃培养2d后加入卢氏碘液,若菌落周围出现透明的圈则表示淀粉被分解,为阳性,反之则为阴性。以水解圈直径(Dh)与菌落直径(Dc)之比(Dh/Dc),判断该菌株产酶能力。
以产消化酶为筛选指标,筛选出产淀粉酶、脂肪酶和蛋白酶的菌株,产酶结果如表1-1
表1-1产酶能力结果
注:“+”表示有产酶效果;“-”表示无产酶效果
注:Dh/Dc表示水解圈直径与菌落直径之比
1.2致病菌拮抗能力测定
以嗜水气单胞菌为指示菌,应用滤纸片法筛选具有抑菌活性的菌株。将指示菌经生理盐水稀释,调节细菌浓度为106CFU/mL,取100ul指示菌,用灭菌棉签均匀涂布于LB固体培养基上,待平板稍干后,用灭菌镊子夹取沾有菌液的滤纸片,放入培养基中,37℃培养1-3d,观察滤纸片周围是否出现抑菌圈。
通过拮抗试验筛选出抑制嗜水气单胞菌的菌株LM32,嗜水气单胞菌拮抗能力试验结果如图1所示。
1.3溶血性试验
将该菌种进行血平板划线,37℃培养,观察其是否产生透明圈。产生透明圈的为严重溶血,没有溶血环产生的为不溶血。结果表明LM32周围没有产生溶血环,证明LM32不溶血。菌株LM32血平板划线图如图2所示。
1.4安全性实验
将25尾健康的锦鲤于水族箱暂养,期间饲喂基础饲料,待锦鲤适应养殖环境后,开始试验。将LM32号菌株浓度调为109CFU/mL,离心弃上清,溶于等量生理盐水中,每日上午9:00灌服500ul活菌悬液(108CFU/mL),连续灌喂7d,每天观察死亡情况,灌喂后在相同养殖条件下继续饲养21d,每天记录死亡情况。结果表明灌喂LM32菌悬液7d后成活率100%,继续养殖21d后成活率100%,证明LM32菌株具有安全性。
2细菌鉴定
2.1形态观察
LB培养基,37℃培养24h后,LM32单菌落呈淡黄色,圆形,不透明菌落,边缘不整齐,表面粗糙,有***。本发明LM32的菌落形态图如图3所示。
2.2生理生化检测
利用法国BioMerieux公司生产的API50CHB细菌鉴定盒对拟选菌株进行生理生化指标鉴定。
结果表明LM32可利用丙三醇、山梨醇、肌醇、苦杏仁苷、七叶灵柠檬酸铁、D-土伦糖、D-甘露醇、D-蔗糖、D-密二糖、D-麦芽糖、D-纤维二糖、D-果糖、D-葡萄糖、D-木糖、D-核糖、L-***糖、甲基-α-D-吡喃葡萄糖苷、D-蔗糖、D-海藻糖、菊粉、淀粉。具体结果见下表。
表1-2LM32菌株的的API 50CHB生化试验结果
注:+阳性;-阴性
2.3 16S rDNA序列分析
进行16S rDNA基因测序后,测序结果经blast比对,判定该菌为耐盐芽孢杆菌(Bacillus halotolerans)。
3耐盐芽孢杆菌LM32对锦鲤生长、非特异性免疫及免疫相关基因表达量的影响
3.1实验设计:
将耐盐芽孢杆菌LM32制成冻干粉,添加至饲料中进行锦鲤的养殖实验。将108尾锦鲤分成四组,每组3个重复,每个重复9尾锦鲤。对照组(CT)为基础日粮,不添加耐盐芽孢杆菌;低浓度组(0.1%),每100g基础日粮添加0.1g耐盐芽孢杆菌LM32冻干粉末;中浓度组(0.3%),每100g基础日粮添加0.3g耐盐芽孢杆菌LM32冻干粉末;高浓度组(0.5%),每100g基础日粮添加0.5g耐盐芽孢杆菌LM32冻干粉末。耐盐芽孢杆菌LM32冻干粉末浓度为108CFU/mL。
3.2饲养管理
试验开始前对锦鲤进行称重、记录。试验期间每天9:00和18:00定时投喂,投喂量为前30d按鱼体重1%饲喂,后30d按鱼体重2%饲喂,连续投喂60d。试验期间每天换水,溶氧8-8.5mg/L,水温20-22℃,pH 6.9±0.2。
3.3生长指标测定
对锦鲤的初末体重进行称重并记录,计算生长指标。
增重(WG,g)=Wt-W0
增重率(WGR,%)=100×(Wt-W0)/W0
特定生长率(SGR,%/d)=100×[Ln(Wt)-Ln(W0)]/t
饲料转化率(FCR,%)=F/(Wt-W0)
注:W0为锦鲤初始体重(g);Wt为锦鲤终末体重(g);t为试验饲喂天数(d);F为饲喂量(g)。
结果表明饲喂含有LM32益生菌饲料对锦鲤的生长性能无明显影响。0.5%组增重高于对照组与0.1%和0.3%组,但差异不显著,对于增重率而言,饲喂浓度为0.3%和0.5%LM32益生菌和对照组之间差异不显著,但饲喂0.1%LM32益生菌显著降低锦鲤的增重率。0.1%组显著降低了锦鲤的特定增长率,其他三组之间差异不显著。饲喂不同浓度LM32对锦鲤生长指标的影响如图4所示。
3.4血清非特异性免疫指标测定
饲养60d后,采样前空食24h,将每条锦鲤称重并做记录,置于MSS中麻醉,使用无菌注射器,在鱼臀鳍位置的侧线偏下位置进行静脉采血,采血完成后,4℃,12000rpm/min离心10min,收集血清,测定血清总蛋白、碱性磷酸酶、酸性磷酸酶、超氧化物歧化酶和溶菌酶活性。
结果表明饲养60d后,血清中各组之间TP含量相近,差异不显著。0.1%、0.3%、0.5%组血清中SOD含量与对照组相比得到了极显著提高,但0.5%组SOD含量显著高于0.1%组。对于LZM活性来说,饲喂不同浓度的LM32益生菌均可极显著提高LZM的含量,与0.1%和0.3%组相比,0.5%组显著提高了LZM的活性。由ALP图可知,对照组与0.5%组差异显著,0.5%组显著降低了锦鲤ALP的水平,对照组与0.1%和0.3%组之间无显著差异。对于ACP含量而言,饲喂了益生菌组极显著降低了ACP含量,三个处理组之间差异不显著。饲喂不同浓度LM32对锦鲤血清免疫指标的影响如图5所示。
4结论
本试验从健康锦鲤肠道内容物及其黏膜中分离筛选出能产多种酶、对常见条件性致病菌具有拮抗作用以及耐热性较好的潜在益生菌耐盐芽孢杆菌LM32,进而评估投喂LM32菌株对锦鲤生长性能和非特异性免疫功能的影响,结果表明饲喂浓度为0.1%、0.3%和0.5%的耐盐芽孢杆菌LM32可以提高锦鲤的抗氧化能力,调节锦鲤免疫反应,对其生长性能无明显影响。
最后应说明的是:以上实施例仅用以说明本发明的技术方案,而非对其限制;尽管参照前述实施例对本发明进行了详细的说明,本领域的普通技术人员应当理解:其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换;而这些修改或者替换,并不使相应技术方案的本质脱离本发明各实施例技术方案的精神和范围。
Claims (10)
1.耐盐芽孢杆菌(Bacillus halotolerans)LM32,其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.25631。
2.一种菌剂,其特征在于,其含有权利要求1所述的耐盐芽孢杆菌(Bacillushalotolerans)LM32。
3.根据权利要求2所述的菌剂,其特征在于,所述菌剂中耐盐芽孢杆菌(Bacillushalotolerans)LM32的有效活菌数≥108CFU/g。
4.一种动物的饲喂方法,其特征在于,利用权利要求1所述的耐盐芽孢杆菌(Bacillushalotolerans)LM32或权利要求2-3任一项所述的菌剂进行饲喂。
5.根据权利要求4所述的动物的饲喂方法,其特征在于,耐盐芽孢杆菌(Bacillushalotolerans)LM32与基础日粮的比例为0.6-6×105CFU/g。
6.饲料添加剂、动物饲料或药物,其特征在于,包括权利要求1所述的耐盐芽孢杆菌(Bacillus halotolerans)LM32或权利要求2-3任一项所述的菌剂或其发酵物或发酵提取物。
7.权利要求1所述的耐盐芽孢杆菌(Bacillus halotolerans)LM32、权利要求2-3任一项所述的菌剂或其发酵物或发酵提取物在制备饲料添加剂、动物饲料或药物中的应用,其特征在于,所述饲料添加剂、动物饲料或药物被用于以下至少一方面:
(1)抑制病原菌;
(2)提高动物免疫能力;
(3)增加动物重量;
(4)提高动饲料转化率;
(5)提高动物对营养物质的利用能力;
(6)促进或抑制动物生长性能;
(7)促进肠道发育或维持肠道微生物的稳态。
8.根据权利要求7所述的应用,其特征在于,所述病原菌包括嗜水气单胞菌(Aeromonashydrophila)。
9.根据权利要求7或8所述的应用,其特征在于,所述动物为水生动物。
10.根据权利要求7-9任一项所述的应用,其特征在于,所述增加动物重量具体通过提高动物对营养物质的利用能力来实现;
和/或,所述提高动饲料转化率具体通过提高动物对营养物质的利用能力来实现;
和/或,所述提高动物免疫能力具体通过调节TP、SOD、LZM、ALP、ACP中的一种或几种实现。
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