CN116640687B - 罗姆布茨菌my01及其应用 - Google Patents
罗姆布茨菌my01及其应用 Download PDFInfo
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- CN116640687B CN116640687B CN202310535844.5A CN202310535844A CN116640687B CN 116640687 B CN116640687 B CN 116640687B CN 202310535844 A CN202310535844 A CN 202310535844A CN 116640687 B CN116640687 B CN 116640687B
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Abstract
本发明公开了一种罗姆布茨菌MY01及其应用。罗姆布茨菌MY01于2021年4月13日保存于广东省微生物菌株保藏中心,保藏编号:GDMCC No:61558。本发明的罗姆布茨菌在开发产氨基酸类产品或促进鱼类,特别是罗非鱼的生长与健康的产品中具有重要的应用价值。
Description
技术领域
本发明属于微生物技术领域,特别涉及一种提高罗非鱼生长性能的罗姆布茨菌MY01及其应用。
背景技术
水产益生菌是活菌和灭活菌或菌细胞某些组分,通过饵料投喂或投入到养殖水体中,提高水产动物疾病抵抗力,改善健康状况,促进生长性能和饲料利用率,提高动物应激抵抗力和总体活力。益生菌以其环境友好、安全有效等优点在水产养殖行业中引起广泛关注。目前,鱼类养殖中常用益生菌有芽孢杆菌属(Bacillus)、乳酸杆菌属(Lactobacillus)、乳球菌属(Lactococcus)和酵母菌属(Saccharomyces)细菌等。但水产益生菌的应用还存在一定的盲目性。很多水产益生菌产品的使用效果并不稳定,其中一个很重要的原因可能是益生菌的选择和使用不当。许多益生菌株并非分离自鱼类消化道,而是来自于环境或恒温动物。且同一种益生菌在不同的鱼种上也有可能表现出不同功效。由于水产养殖业的益生菌制剂定植肠道的条件不同,在使用时应该具有针对性,分离筛选针对性强的益生菌。
鱼类肠道内定植着大量微生物,肠道微生物与宿主之间存在一种营养共生关系。肠道微生物能降解食糜,利用肠道营养物质进行生长繁殖,同时细菌在代谢过程中分泌生理活性物质,如氨基酸、维生素、脂肪酸、消化酶等,有利于鱼类的生长和健康。鱼类肠道有益微生物的筛选的研究,将为水产行业提供更高效的益生菌产品。
发明内容
本发明提供一种提高罗非鱼生长性能的罗姆布茨菌及其应用。该菌株于2021年4月13日保存于广东省微生物菌株保藏中心,命名为罗姆布茨菌(Romboutsialituseburensis)MY01,保藏地址:广州市先烈中路100号大院59号楼5楼广东省微生物研究所,保藏编号:GDMCC No:61558。
采用苛养厌氧培养基从健康罗非鱼肠道中分离获得一株罗姆布茨菌MY01,37℃培养48h,菌落白色光滑半透明、圆形,直径0.5-1.0mm,为厌氧革兰氏阳性菌。使用通用引物27F/1492R PCR扩增16S rRNA基因,测序获得目的序列进行NCBI BLAST比对分析,获得的菌株与已公布的Romboutsia lituseburensis具有最高的同源性。全基因组测序及比对结果表明MY01为罗姆布茨菌属的一个新种。该菌安全性能良好。
罗姆布茨菌MY01能促进罗非鱼的生长,显著提高罗非鱼的体重,提高罗非鱼血清赖氨酸、蛋氨酸、色氨酸和酪氨酸等的含量;且可以产生短链脂肪酸,降低空腹血糖,提高餐后胰岛素水平。投喂罗姆布茨菌MY01发酵饲料,可以提高肠道菌群在蛋氨酸、色氨酸、酪氨酸、苯丙氨酸、胱氨酸、丙氨酸、缬氨酸、亮氨酸和异亮氨酸等的合成代谢等功能。全基因组测序结果表明,该菌中12个基因参与赖氨酸合成代谢通路,9个基因参与苯丙氨酸、酪氨酸和色氨酸合成代谢通路,对应的表达产物分别为赖氨酸、苯丙氨酸和酪氨酸。本发明的罗姆布茨菌在开发产氨基酸类产品或促进鱼类,特别是罗非鱼的生长与健康的产品中具有重要的应用价值。
附图说明
图1为Romboutsia MY01在苛性厌氧培养基培养的菌落形态;
图2为Romboutsia MY01的透射电镜观察图;
图3为Romboutsia MY01基于16S rRNA基因序列的NJ系统进化树;
图4为Romboutsia MY01赖氨酸合成代谢通路;
图5为Romboutsia MY01苯丙氨酸、酪氨酸和色氨酸合成代谢通路;
图6为Romboutsia MY01对罗非鱼生长的影响图;
图7为不同组罗非鱼血清中氨基酸成分及含量图;
图8为Romboutsia MY01发酵饲料对罗非鱼血糖和胰岛素的影响;
图9为不同组罗非鱼肠道菌群Tax4Fun基因功能预测图。
具体实施方式
为让本领域的技术人员更加清晰直观的了解本发明,下面将结合附图,对本发明作进一步的说明。
一材料与方法
1.菌种的分离与纯化
将尼罗罗非鱼肠道内容物至于苛养厌氧液体培养基,37℃,厌氧培养48h。获得的培养液进行梯度稀释将其稀释至10-3、10-4、10-5,取稀释液各150μl,涂布至苛养厌氧固体培养基,置于37℃恒温培养箱中培养48h。选取菌落形态为单个、不规则的菌落进行反复纯化培养,直至获得纯种菌株。选择获得纯种菌株的单菌落进行16S rRNA基因序列分析、形态学及革兰氏染色观察。
苛养厌氧液体培养基配方:混合蛋白胨23.0g,氯化钠5.0g,可溶性淀粉1.0g,碳酸氢钠0.4g,葡萄糖1.0g,丙酮酸钠1.0g,半胱氨酸盐酸盐0.5g,氯化血红素0.01g,维生素K0.001g,L-精氨酸1.0g,可溶性焦磷酸0.25g,琥珀酸钠0.5g,pH7.2±0.2,双蒸水1000mL,混合均匀后,灭菌备用。
2.菌种鉴定
2.1形态学观察及染色
将筛选出的菌株划线接种于苛养厌氧固体培养基上,置于37℃恒温培养箱中厌氧培养24h,观察其菌落形态。挑选单菌落置于滴有生理盐水的载玻片上,涂布均匀后通过革兰氏染色法在光学显微镜下观察菌体形态。投射电镜观察菌体形态。
2.2生理生化鉴定
将纯化培养的待测菌株用梅里埃厌氧菌鉴定试剂盒进行生理生化指标检测,具体操作见试剂盒说明书。
2.3 16S rRNA基因序列分析
用北京天根生物细菌基因组DNA提取试剂盒提取待测菌株的细菌基因组DNA,具体操作步骤参照说明书。将提取的细菌基因组DNA作为模板,用通过引物27F:5′–GTTTGATCCTGGCTCAG–3′和1492R:5′–TACGGCTACCTTGTTACGACTT–3′来扩增细菌的16S rRNA基因。反应体系(25μL)为:模板DNA1μL,正反向引物各0.5μL,Mix 12.5μL,灭菌蒸馏水10.5μL,PCR扩增的反应条件:94℃3min;94℃30s,56℃30s,72℃90s,30个循环;72℃10min。PCR产物送至生工生物工程(上海)股份有限公司进行测序。获得目的系列之后,登录NCBI网站,通过GenBank中的BLAST程序进行同源性分析,同时下载相关细菌的16S rRNA基因序列,再通过BioEdit软件进行多重比对,用MEGA7.0软件中邻接法(neighbor joining,NJ)构建分子系统进化树并通过自建分析(boostrap)进行置信度检测,自检数据集为1000次。
2.4安全性实验
实验设置待测菌株注射组和阴性对照组,每组3个平行,以尼罗罗非鱼为实验用鱼(均重22.5±1.3g),每个平行随机选取罗非鱼15尾。将待测菌株设置三个浓度,分别为1.5×107CFU/ml、1.5×108CFU/ml和1.5×109CFU/ml,采取腹腔注射的方式对实验组和对照组中每尾鱼进行注射,实验组每尾鱼注射100μL,对照组注射等体积的0.85%生理盐水,实验观察周期为7d,期间按常规方法喂养,每天观察记录鱼的死亡数量。
2.5全基因组测序分析
将Romboutsia MY01单菌落接种至苛养厌氧液体培养基,37℃,厌氧培养24h,4℃,6000r/min离心5min收集菌体。用基因组提取试剂盒提取高质量的DNA并构建DNA文库,利用基于第三代测序技术Oxford Nanopore Technology的测序仪PromethION对DNA进行单分子测序,同时使用MGISEQ-2000系统在PE150读长模式下对菌株的全基因组进行双末端测序。
2.6Romboutsia MY01发酵饲料投喂对罗非鱼生长及生理的影响
将Romboutsia MY01单菌落接种至苛养厌氧液体培养基,37℃,厌氧培养24h,4℃,6000r/min离心5min收集菌体。随后将MY01接入到饲料中,含量为1.0×107cfu/g,控制含水量为45%,将饲料放入厌氧箱中,37℃培养48h。GC-MS分析饲料中短链脂肪酸含量。
实验在中国水产科学研究院珠江水产研究所池塘进行。实验选取质量约50g的罗非鱼鱼苗150尾,随机分配到6个方形网箱中,25尾/网箱,每个网箱实际水体积为3m3(L×W×H=1m×2m×1.5m)。设对照组(CK)1个,MY01发酵饲料组(MY),每组设3个重复。每天两次投饵,日投饵量为体质量的4%。实验时间为4周。
表1饲料配方
将罗非鱼冰浴麻醉后,用无菌生理盐水冲洗罗非鱼体表两遍,除去表面污物。解剖罗非鱼称重后抽血,收集血清,采集肠道内容物样品,液氮速冻后于-80度冰箱保存。基于液相色谱串联质谱法(LC-MS/MS)对血清进行氨基酸含量检测,对肠道内容物进行菌群的16SrRNA测序分析。血糖测定采用血糖检测试剂盒,胰岛素测定采用罗非鱼INS-ELISA试剂盒。
二结果与分析
1.菌株的分类与鉴定
1.1菌株
Romboutsia MY01在苛养厌氧固体培养基上的菌落形态为菌落白色光滑半透明、圆形,直径0.5-1.0mm,为厌氧革兰氏阳性菌。透射电镜下观察,菌体为杆状(图1;图2)。革兰氏染色结果表明,该菌为革兰氏阳性菌。
1.2生理生化鉴定
Romboutsia MY01的生理生化鉴定结果表明(表2),该菌的明胶酶、葡萄糖苷酶反应为阳性,尿素酶反应为阳性,产吲哚试验阴性,能利用葡萄糖、蔗糖、麦芽糖、海藻糖,不能利用甘露醇、乳糖、木糖、阿拉伯糖、丙三醇、纤维二糖、松三糖、棉子糖、山梨醇、鼠李糖、水杨苷、甘露醇,过氧化氢酶反应为阴性。
表2Romboutsia MY01的生理生化特征
+:阳性;-:阴性。
1.3 16S rRNA基因序列分析
Romboutsia MY01经PCR扩增,16S rRNA测序后获得目的序列,经BLAST与数据库中的基因序列进行比较(图3),结合菌株的生理生化特性,可以基本确定MY01为Romboutsia属细菌。
2.安全性试验
通过腹腔注射法来检测Romboutsia MY01对罗非鱼的安全性。注射实验结果表明,在给体重为(22.5±1.3)g罗非鱼注射100μL浓度分别为1.5×107CFU/ml、1.5×108CFU/ml和1.5×109CFU/mL的菌液的条件下,并没有出现死亡及其他异常情况。以上实验结果表明,Romboutsia MY01是一株安全性能好的菌株。
3.全基因组测序
通过比对KEGG库,Romboutsia MY01中该菌中12个基因(粗线框表示)参与赖氨酸合成代谢通路(图4),9个基因(粗线框表示)参与苯丙氨酸、酪氨酸和色氨酸合成代谢通路(图5),对应的表达产物分别为赖氨酸、苯丙氨酸和酪氨酸。
全基因组测序结果显示Romboutsia MY01基因组大小为3925608bp。将MY01和NCBI上最大相似度的Romboutsia lituseburensis参考基因组进行比较,分离菌与参考菌株的ANI为89.0-93.4%,低于物种划分的阈值(95-96%),且DDH为36.7-53.2%,低于物种划分的阈值70%,因为一般同物种间的ANI值在95%以上,DDH值在70%以上,因此MY01为Romboutsia属的一个新物种。
表3Romboutsia MY01与NCBI上的Romboutsia lituseburensis基因组平均核苷酸相似度和DNA-DNA杂交值比较
4.投喂Romboutsia MY01发酵饲料对罗非鱼生长和生理的影响
Romboutsia MY01发酵产生短链脂肪酸乙酸、异丁酸和异戊酸(表4),能促进罗非鱼的生长(图6),益生菌喂养4周后,显著提高罗非鱼体重和去内脏后体重,显著提高罗非鱼血清中赖氨酸、蛋氨酸、色氨酸和酪氨酸的含量(图7;表5),提高罗非鱼餐后血清胰岛素水平,降低空腹血糖水平(图8)。
表4发酵饲料中短链脂肪酸的含量
注:上标字母表示组间有显著性差异(P<0.05)
表5MY01对罗非鱼血清氨基酸成分的影响
通过在饲料中添加Romboutsia MY01,可以提高肠道菌群在蛋氨酸、色氨酸、酪氨酸、苯丙氨酸、胱氨酸、丙氨酸、缬氨酸、亮氨酸和异亮氨酸等的合成代谢等功能(图9)。
本发明中,全基因组测序结果显示Romboutsia MY01基因组大小为3925608bp,这里只列出Romboutsia MY01的16srRNA序列(1349bp):
CTTCTTCGGAAGAGAGCGGCGGACGGGTGAGTAACGCGTGGGTAACCTGCCCTGTACACACGGATAACATACCGAAAGGTATGCTAATACGGGATAACATACTTTTATCGCATGGTAGAAGTATCAAAGCTCCGGCGGTACAGGATGGACCCGCGTCTGATTAGCTAGTTGGTAAGGTAACGGCTTACCAAGGCGACGATCAGTAGCCGACCTGAGAGGGTGATCGGCCACATTGGAACTGAGACACGGTCCAAACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGAAAGCCTGATGCAGCAACGCCGCGTGAGCGATGAAGGCCTTCGGGTCGTAAAGCTCTGTCCTCAAGGAAGATAATGACGGTACTTGAGGAGGAAGCCCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGGGCTAGCGTTATCCGGAATTACTGGGCGTAAAGGGTGCGTAGGTGGTTTCTTAAGTCAGAAGTGAAAGGCTACGGCTCAACCGTAGTAAGCTTTTGAAACTAAGAGACTTGAGTGCAGGAGAGGAGAGTAGAATTCCTAGTGTAGCGGTGAAATGCGTAGATATTAGGAGGAATACCAGTTGCGAAGGCGGCTCTCTGGACTGTAACTGACACTGAGGCACGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTACTAGCTGTCGGGGGTTACCCCCCTCGGTGGCGCAGCTAACGCATTAAGTACTCCGCCTGGGAAGTACGCTCGCAAGAGTGAAACTCAAAGGAATTGACGGGGACCCGCACAAGTAGCGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCTAAGCTTGACATCCTTTTGACCTCTCCCTAATCGGAGATTTCCCTTCGGGGACAGAAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGCCTTTAGTTGCCAGCATTAAGTTGGGCACTCTAGAGGGACTGCCAGGGATAACCTGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGCTTAGGGCTACACACGTGCTACAATGGGTGGTACAGAGGGCGGCCAAGTCGTGAGGCGGAGCTAATCCCTTAAAGCCATTCTCAGTTCGGATTGTAGGCTGAAACTCGCCTACATGAAGCTGGAGTTACTAGTAATCGCAGATCAGAATGCTGCGGTGAATGCGTTCCCGGGTCTTGTACACACCGCCCGTCACACCACGGGAGTTGGAGGCGCCCGAAGCCGGATAGCTAACCTTTGGAAGC。
Claims (10)
1.罗姆布茨菌MY01,该菌株于2021年4月13日保存于广东省微生物菌株保藏中心,保藏编号:GDMCC No:61558。
2.罗姆布茨菌MY01在生产赖氨酸和/或苯丙氨酸和/或酪氨酸中作为产氨基酸菌的应用。
3.罗姆布茨菌MY01在制备促进鱼类生长的产品中的应用。
4.根据权利要求3所述的应用,其特征在于,所述鱼类为罗非鱼。
5.根据权利要求3所述的应用,其特征在于,促进鱼类生长表现为提高鱼类体重。
6.根据权利要求3所述的应用,其特征在于,所述产品为微生物制剂或饲料添加剂。
7.根据权利要求3所述的应用,其特征在于,产品中罗姆布茨菌MY01的含量为1.0×107cfu/g。
8.一种含有罗姆布茨菌MY01的产品。
9.根据权利要求8所述的产品,其特征在于,产品为微生物制剂或饲料添加剂或饲料。
10.罗姆布茨菌MY01的培养方法,其特征在于,先将其接种至苛养厌氧固体培养基上,然后置于37℃恒温培养箱中培养。
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