CN116496945A - 一种烟草谷氨酸杆菌n1及其应用 - Google Patents
一种烟草谷氨酸杆菌n1及其应用 Download PDFInfo
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Abstract
本发明涉及一种烟草谷氨酸杆菌(Glutamicibacter nicotianae)N1，其保藏编号为CGMCC No.26330，其可分泌α‑葡萄糖苷酶、α‑半乳糖苷酶、脲酶、胃蛋白酶、胰蛋白酶和糜蛋白酶等，应用于海水养殖生产活动中，可有效解决对虾等水产养殖水体中有害无机氮污染等问题，保障养殖动物的健康生长，是一株良好的潜在水产用益生菌。
Description
技术领域
本发明涉及微生物领域，具体涉及一种烟草谷氨酸杆菌N1及其应用。
背景技术
烟草谷氨酸杆菌(Glutamicibacter nicotianae)属于放线菌门微生物，GenBank:BJNE01000038.1，生物学分类：细菌、放线菌门、放线菌纲、微球菌目、微球菌科、谷氨酸杆菌属。
放线菌是一类革兰氏阳性、高(G+C)含量(>55％)的细菌，因菌落呈放线状而得名。它是一个原核生物类群，在自然界中分布很广，主要以孢子繁殖，其次是断裂生殖。近年来在无脊椎动物，甚至脊椎动物消化道中发现了多种能够产生生物活性物质的放线菌，对宿主的消化吸收、免疫和疾病预防等方面有明显的促进作用。例如，白蚁的共生放线菌能够消化纤维素类物质，广泛存在于白蚁肠道和蚁巢。切叶蚁共生链霉菌可产生杀假丝菌素(Candicidin)抑制寄生真菌Escovopsis的生长，但对共生真菌的生长影响不大。欧洲土蜂共生放线菌Streptomyces spp.可产生9种抗生素保护幼虫免受病原真菌与细菌的感染。牛瘤胃中分离到的厌氧放线菌Denitrobacterium detoxificans可降解饲料中的3-硝基-1-丙醇及3-硝基-1-丙酸毒素，推测在瘤胃中具有解毒作用。
目前，国内关于烟草谷氨酸杆菌的研究相对空缺，国外也仅有少量研究。Ashtaad等(2020)从土壤中分离3株AHL降解放线菌，其中烟草谷氨酸杆菌AI5a对群体感应植物病原Pcc BR1的毒力抑制效果最好。Maulina等(2022)从禾本科植物根系中分离多株根际细菌，其中烟草谷氨酸杆菌Rg1显著(p<0.05)提高了玉米的养分吸收率、叶片叶绿素含量、净同化率和作物生长速率，对玉米生长有明显的促进作用。Haque等(2022)从65株耐盐根际细菌中筛选出21株能够形成生物膜的菌株，其中烟草谷氨酸杆菌ESK19、ESM8和ESM16等根际细菌能够促进植物生长(PGP)活性，包括产生IAA、ACC脱氨酶和铁载体，以及溶解磷酸盐。Purushothaman(2020)和Xuejun(2021、2022)报道了烟草谷氨酸杆菌在有毒重金属存在下具有较高的苯酚降解效率，可用于苯酚及其衍生物污染环境的生物修复。
以上研究表明烟草谷氨酸杆菌在防治病害、促进生长和分解有害物质等方面具有良好效果。然而，其在水产养殖中的功能应用尚未有报道。
发明内容
本发明的目的是提供一种可分泌α-葡萄糖苷酶、α-半乳糖苷酶、脲酶、胃蛋白酶、胰蛋白酶和糜蛋白酶且可有效去除海水养殖水体中氨态氮和亚硝态氮等有害无机氮化合物的烟草谷氨酸杆菌N1及其应用。
本发明采用如下技术方案：
一种烟草谷氨酸杆菌(Glutamicibacter nicotianae)N1，其保藏编号为CGMCCNo.26330，保藏日期为2022年12月26日，保藏单位为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。
进一步的，其可分泌α-葡萄糖苷酶、α-半乳糖苷酶、脲酶、胃蛋白酶、胰蛋白酶和糜蛋白酶。
进一步的，其可降低养殖水体中氨态氮的浓度。
进一步的，其可降低养殖水体中亚硝态氮的浓度。
一种上述烟草谷氨酸杆菌N1在海水养殖中的应用。
一种上述烟草谷氨酸杆菌N1在海水对虾养殖中的应用。
一种上述烟草谷氨酸杆菌N1在制备海水益生菌剂中的应用。
本发明的有益效果在于：本发明分离纯化得到的烟草谷氨酸杆菌N1具有高效去除海水养殖水体中氨态氮和亚硝态氮的特点，该菌兼具分泌α-葡萄糖苷酶、α-半乳糖苷酶、脲酶、胃蛋白酶、胰蛋白酶和糜蛋白酶等多种特性，可应用于海水对虾养殖中，有效解决水产养殖中氮污染等问题，保障养殖生物的健康生长，是一株良好的潜在水产用益生菌，为完善我国水产益生菌资源库和促进水产养殖可持续发展提供理论依据和技术保障。
附图说明
图1为烟草谷氨酸杆菌N1的系统发育树。
图2为对照组和N1组养殖水体中氨态氮浓度的变化图。
图3为对照组和N1组养殖水体中亚硝态氮浓度的变化图。
具体实施方式
下面通过附图和实施例对本发明进行详细的描述。
实施例1烟草谷氨酸杆菌N1的分离鉴定
步骤一：菌株的富集、分离纯化
(1)对冻存的生态池塘沉积物样品应用富集驯化培养基进行富集处理，相同条件下重复操作多次，得到富集菌液。
富集驯化培养基：C6H12O6·H2O 5g，NH4Cl 0.5g，NaNO2 1.0g，CaCO3 1.0g，NaH2PO4·2H2O 0.26g，FeSO4·7H2O 0.183g，K2HPO7·3H2O 0.655g，MgSO4 0.1g，酵母粉0.03g，陈海水1000mL。
(2)取100uL最后一次富集液稀释涂布于2216E琼脂培养基上，30℃培养2～3d，将平板上不同形态的单菌落反复划线纯化3次，直至平板上所有菌株的形态大小近乎一致。
(3)在菌种管中加入0.5mL的30％甘油，加入1mL的细菌培养液，保存于-80℃冰箱。
步骤二：菌株的复筛以检测菌株对氨态氮、亚硝态氮和硝态氮的综合去除效果作为复筛指标：实验前利用2216E液体培养基对实验菌株进行活化培养，取对数期菌液5000r/min，离心10min，用0.90％无菌生理盐水洗涤沉淀并重悬，接种浓度为1010CFU·mL-1。
将活化的初筛菌株培养到24h后，取5mL活化液转接含有氨态氮、亚硝态氮和硝态氮各14mg·L-1的海水降解培养基中，使降解液中细菌浓度控制在(1～2)×108CFU·mL-1，28℃、160r/min恒温震荡培养3d后，测定测试液中氨态氮、亚硝态氮和硝态氮的值，比较各菌株综合去除三氮能力，筛选具有较高去除率的菌株N1进行研究。
海水降解培养基的组成：C6H12O6·H2O 2.0808g，NH4Cl 0.0535g，NaNO2 0.069g，NaNO3 0.085g，陈海水1000mL。
步骤三：菌株的鉴定
(1)将菌株N1接种至2216E琼脂培养基，28℃恒温培养3d，待长出单个菌落后，进行革兰氏染色和形态学观察，结果见表1。
表1菌株N1在2216E琼脂培养基上的形态学特征
(2)由中国海洋微生物菌种保藏管理中心(Marine Culture Collection ofChina，CMCC)进行生理生化特征实验，具体参照常见细菌系统鉴定手册，结果见表2。
表2菌株N1的生理生化鉴定结果
备注：“+”：阳性反应；“-”：阴性反应；“W”：较弱反应
(3)16Sr DNA基因扩增：以细菌通用引物扩增16S rDNA基因，正向引物27F：5`-AGAGTTTGATCCTGGTCAG-3`，反向引物1492R：5`-GGTTACCTTGTTACGACTT-3`。
(4)PCR扩增体系(25μL)：SanTaq Plus PCR mix 12.5μL；细菌DNA模板2μL；正向引物27F 1μL；反向引物1492R 1μL；ddH2O 8.5μL。PCR扩增程序：95℃预电泳5min，95℃30s，50℃30s，72℃1min30s，35个循环，72℃延伸10min。
(5)系统发育分析：菌株N1的PCR扩增产物送交生工生物技术(上海)有限公司进行测序，获得16S rDNA基因序列，16S rDNA序列如SEQ ID No.1所示。将序列上传至EzBioCloud细菌基因序列数据库，选择相似度前10的菌株序列应用MEGA 11构建系统进化树，分别采用Neighbor-Joining算法和1000次Bootstrap统计检验进行构建，以确定菌株N1的分类地位，菌株N1系统发育树见图1。
经鉴定，菌株N1为烟草谷氨酸杆菌(Glutamicibacter nicotianae)，该菌株于2022年12月26日在中国微生物菌种保藏管理委员会普通微生物中心进行了保藏，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.26330。
实施例2酶活的测定
使用苏州科铭生物技术公司的α-半乳糖酶(α-Galactosidase,α-GAL)试剂盒、α-葡萄糖苷酶(α-Glucosidase，α-GC)试剂盒、脲酶(Urease，UE)测定试剂盒、胃蛋白酶(Pepsin)试剂盒、胰蛋白酶(Trypsin)试剂盒和糜蛋白酶(Chymotrypsin)试剂盒分别测定烟草谷氨酸杆菌N1在12h、16h和20h的α-葡萄糖苷酶、α-半乳糖苷酶、脲酶、胃蛋白酶、胰蛋白酶和糜蛋白酶的活性，烟草谷氨酸杆菌N1的各项酶活测定结果见表3。
表3酶活测定结果
注：数据以平均值±标准差(Mean±SD)表示。
实施例3烟草谷氨酸杆菌N1对水产养殖中的应用
一、实验材料
(1)凡纳滨对虾幼虾初始平均体重0.16±0.01g，健康无病害，暂养3～5天后开始试验，以适应新环境。
(2)2216E液体培养基：蛋白胨5.0g，酵母浸粉1.0g，FeC6H5O7 0.1g，NaCl19.45g，MgCl2 5.98g，Na2SO4 3.24g，CaCl2 1.8g，KCl 0.55g，Na2CO3 0.16g，KBr 0.08g，SrCl20.034g，H3BO3 0.022，Na2O·nSiO2 0.004g，NaF 0.0024g，NH4NO3 0.0016g，Na2HCO3 0.008g，纯水1000mL。
二、实验操作
在400L的养殖圆桶内(实际养殖水体280L)进行凡纳滨对虾养殖实验，养殖密度设置为500尾/m3。养殖试验为期28天。实验共设置2个处理组，分别为对照组和N1组，每个处理组设置3个重复。对照组在试验期间内正常喂养，N1组在养殖第14天开始向养殖水体间歇泼洒(每三天停止泼洒一天)烟草谷氨酸杆菌N1(2216E液体培养基活化，养殖水体中菌株N1浓度为2×108CFU·L-1)。
实验结束时，测定养殖对虾存活率(Survival rate，SR)、特定生长率(Specificgrowth rate，SGR)和饲料系数(Feed conversion ratio，FCR)进行表征，另外最终对虾的收获量(Final shrimp harves，FSH)也是衡量菌株养殖效果的重要指标。
实验开始后，每3天采集一次养殖水体，保存于-20℃冷库中以便于氨态氮和亚硝态氮含量的测定。每7天测定养殖水体的温度、溶解氧、盐度和pH。
三、实验结果
(1)对虾生长状况结果
对照组与N1组养殖对虾的生长参数如表4所示。在养殖结束时，对照组的对虾末体重显著高于N1组(p＜0.05)，但存活率又显著低于N1组(p＜0.05)，而两组之间的对虾最终收获量并无显著差异(p＞0.05)，说明在凡纳滨对虾养殖过程中，泼洒烟草谷氨酸杆菌N1能够提高养殖对虾的存活率，且对养殖对虾并无危害。
表4凡纳滨对虾生长状况
注：数据以平均值±标准误表示(n＝3)。表中不同字母间表示差异显著(p<0.05)。
(2)常见水体环境指标
养殖水环境的温度、溶解氧、盐度和pH在养殖过程中保持稳定，且对照组与N1组无显著性差异(p＞0.05)，如表5所示。
表5养殖水体环境状况
注：数据以平均值±标准误表示(n＝5)。
(3)养殖水体中氨态氮含量的变化
对照组和N1组在28天内养殖水体的氨态氮浓度变化如图2所示，不同时间各组间氨态氮浓度经方差齐性检验均有齐性(p＞0.05)，经ANOVA分析在18天和27天时有显著差异(p＜0.05)，N1组在第18天和27天时氨态氮浓度显著低于对照组。研究证明，在凡纳滨对虾养殖过程中，泼洒烟草谷氨酸杆菌N1能够显著降低养殖水体中氨态氮的浓度。
(4)养殖水体中亚硝态氮含量的变化
对照组和N1组在28天内养殖水体的亚硝态氮浓度变化如图3所示，不同时间各组间亚硝态氮浓度经方差齐性检验均有齐性(p＞0.05)，经ANOVA分析在27天时有显著差异(p＜0.05)，N1组在第27天时亚硝态氮浓度显著低于对照组。研究证明，在凡纳滨对虾养殖过程中，泼洒烟草谷氨酸杆菌N1能够显著降低养殖水体中亚硝态氮的浓度。
从上述实验结果可知，在凡纳滨对虾养殖过程，本发明提供的烟草谷氨酸杆菌N1对水产养殖中危害较大的氨态氮与亚硝态氮有明显的去除效果，能够保障养殖生物的健康生长。
本发明提供的烟草谷氨酸杆菌N1在海水氮降解培养基中表现出优秀的氮去除能力，拥有良好除氮效果，且同时能够分泌α-葡萄糖苷酶、α-半乳糖苷酶、脲酶、胃蛋白酶、胰蛋白酶和糜蛋白酶等多种酶，对养殖动物无不良影响。因此本发明提供的烟草谷氨酸杆菌N1在水产养殖中具有良好的应用前景。
Claims (7)
1.一种烟草谷氨酸杆菌(Glutamicibacter nicotianae)N1，其特征在于，其保藏编号为CGMCC No.26330。
2.根据权利要求1所述的烟草谷氨酸杆菌N1，其特征在于，其可分泌α-葡萄糖苷酶、α-半乳糖苷酶、脲酶、胃蛋白酶、胰蛋白酶和糜蛋白酶。
3.根据权利要求1所述的烟草谷氨酸杆菌N1，其特征在于，其可降低养殖水体中氨态氮的浓度。
4.根据权利要求1所述的烟草谷氨酸杆菌N1，其特征在于，其可降低养殖水体中亚硝态氮的浓度。
5.一种如权利要求1所述的烟草谷氨酸杆菌N1在海水养殖中的应用。
6.一种如权利要求1所述的烟草谷氨酸杆菌N1在海水对虾养殖中的应用。
7.一种如权利要求1所述的烟草谷氨酸杆菌N1在制备海水益生菌剂中的应用。
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