CN114908013A - 一株产ddp-iv抑制剂的厦门希瓦氏菌及其应用 - Google Patents
一株产ddp-iv抑制剂的厦门希瓦氏菌及其应用 Download PDFInfo
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Abstract
一株产DDP‑IV抑制剂的厦门希瓦氏菌及其应用,涉及一株菌株及其应用。本发明产DDP‑IV抑制剂的厦门希瓦氏菌,其为厦门希瓦氏菌(Shewanella piezotolerans)CX‑4,保藏在中国微生物菌种保藏管理委员会普通微生物中心,保藏号为CGMCC No.24198。本发明上述产DDP‑IV抑制剂的厦门希瓦氏菌在鱼类养殖中的用途。本发明产DDP‑IV抑制剂的厦门希瓦氏菌加入鱼饲料中用于鱼类养殖。厦门希瓦氏菌(Shewanella piezotolerans)CX‑4可以产DDP‑IV抑制剂,从而具有降血糖、提高饲料消化利用率、增强生长性能的作用。
Description
技术领域
本发明涉及一株菌株及其应用。
背景技术
人和动物血糖含量受多种激素如胰岛素、胰高血糖素、生长抑素等激素的调节。对哺乳动物来说,胰岛素可以促进组织、细胞对葡萄糖的摄取和利用,促进葡萄糖合成糖原,并贮存于肝和肌肉中,同时抑制糖异生,促进葡萄糖转变为脂肪酸,贮存于脂肪组织,从而降低血糖含量。胰高血糖素的作用与胰岛素相反。因此,胰岛素被认为是哺乳动物调节和维持血糖含量最为重要的一种激素,但在鱼类情况并非如此。一些研究指出,鱼类胰岛素含量低下,而且胰岛素受体数量少,亲和力弱,但是Thorpe和Ince观察到太平洋鳕鱼(Gadousmacrocephaius)和欧洲鳗鲡(Anguilla anguilla)血浆胰岛素含量均高于人类,随着饲料中糖类含量的增加肝糖原含量随之增加。这说明鱼类同哺乳动物一样可以通过肝糖原合成途径来降低血糖负荷,以保持血糖稳定。饲料中的糖类进入鱼体后先要经过消化和降解,鱼类尤其是肉食性鱼类对糖的利用能力低下,似乎先天性地具有糖尿病患者的某些生理特征。饲料中过高的糖水平会导致鱼类抗病力下降、生长速度减缓、死亡率增加等症状。总体表现为鱼类解除葡萄糖负荷的能力有限从而表现为对糖的利用能力低下。
发明内容
本发明提供了一株产DDP-IV抑制剂的厦门希瓦氏菌及其应用。
本发明产DDP-IV抑制剂的厦门希瓦氏菌,其为厦门希瓦氏菌(Shewanellapiezotolerans)CX-4,保藏在中国微生物菌种保藏管理委员会普通微生物中心,保藏号为CGMCC No.24198。
本发明上述产DDP-IV抑制剂的厦门希瓦氏菌在鱼类养殖中的用途。
本发明产DDP-IV抑制剂的厦门希瓦氏菌加入鱼饲料中用于鱼类养殖。
本发明厦门希瓦氏菌(Shewanella piezotolerans)CX-4菌落呈白色,圆形,不透明,表面光滑,有光泽,边缘整齐,经革兰氏染色为革兰氏阴性菌。
厦门希瓦氏菌(Shewanella piezotolerans)CX-4为能产生高铁鳌合能力的铁载体,37℃环境中产铁载体的Su值为68%。在缺铁条件下,厦门希瓦氏菌(Shewanellapiezotolerans)CX-4可通过产生更高亲和力的铁载体与致病菌竞争铁元素达到抑菌效果,提高鲤免疫防御能力,降低病原菌在肠道菌群中的比例。
在37℃培养环境中厦门希瓦氏菌(Shewanella piezotolerans)CX-4在SKM固体培养基上乳蛋白的水解圈直径D为23.72mm,菌落直径d为5.02mm,D/d为4.73;在37℃环境中DDP-IV抑制剂产率高达67.48%。厦门希瓦氏菌(Shewanella piezotolerans)CX-4可以产DDP-IV抑制剂,从而具有降血糖、提高饲料消化利用率、增强生长性能的作用。
本发明厦门希瓦氏菌(Shewanella piezotolerans)CX-4是同时兼具降血糖和产铁载体的菌株。将本发明厦门希瓦氏菌(Shewanella piezotolerans)CX-4加入鱼饲料用于鱼类养殖,可以提高饲料消化利用率,并产生抑菌效果。
在37℃条件下本发明厦门希瓦氏菌(Shewanella piezotolerans)CX-4在胆盐浓度为0.6%的环境中存活率仍保持为80%;且厦门希瓦氏菌(Shewanella piezotolerans)CX-4具有优异的耐酸性。因此,厦门希瓦氏菌(Shewanella piezotolerans)CX-4可以在鱼肠道内生存、发挥作用。且厦门希瓦氏菌(Shewanella piezotolerans)CX-4溶血实验结果为阴性,使用安全性高。
厦门希瓦氏菌(Shewanella piezotolerans)CX-4为厦门希瓦氏菌,属于希瓦氏菌属(Shewanella);保藏在中国微生物菌种保藏管理委员会普通微生物中心(CGMCC),保藏地址是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CGMCCNo.24198,保藏日期为2021年12月27日。
附图说明
图1是厦门希瓦氏菌(Shewanella piezotolerans)CX-4在SKM培养基上乳蛋白的水解试验结果图;
图2是厦门希瓦氏菌(Shewanella piezotolerans)CX-4在CAS固体检测培养基上的铁载体筛选试验结果图;
图3是由厦门希瓦氏菌(Shewanella piezotolerans)CX-4构建的系统发育树。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅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的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。
需要说明的是,在不冲突的情况下,本发明中的实施例及实施例中的特征可以相互组合。
具体实施方式一:本实施方式一株利于稻渔共生的菌株为厦门希瓦氏菌(Shewanella piezotolerans)CX-4,保藏在中国微生物菌种保藏管理委员会普通微生物中心,保藏号为CGMCC No.24198。
2021年9月,选取黑龙江省鸡西市密山市兴凯湖区养殖的鲤,将鲤肠道内含物置于含玻璃珠和50mL无菌水的锥形瓶中,以180r/min的转速室温振荡30min,再进行梯度稀释,将10-3、10-4、10-5梯度取100μL涂布于SKM培养基(2%脱脂乳培养基)平板上,每个梯度重复3次,置于28℃培养24~48h。采用乳蛋白水解圈的方法进行筛选,挑取产生透明圈的菌株进行纯化。然后,将纯化的菌株接种于96孔板,每孔准确滴加25μL、1.6mmol/L的甘氨酰-脯氨酰-对硝基苯胺和25μL的CFS或CFE;在37℃下反应15min,再加入50μL、0.01U/mL的DDP-IV(二肽基肽酶IV),在37℃继续反应1h,之后再加入100μL、1mol/L的醋酸钠缓冲溶液(pH=4.0)终止反应,使用酶标仪在405nm处检测反应溶液的吸光值,获取菌株DDP-IV抑制率。
其中,DDP-IV抑制率计算公式为:
A待测样品:25μL样品+25μLGly-pro-phy+50μLDDP-IV+100μL醋酸钠;
A样品空白:25μL样品+50μLTris-HCl+25μLGly-pro-phy+100μL醋酸钠;
A阴性对照:25μLTris-HCl+25μLGly-pro-phy+50μLDDP-IV+100μL醋酸钠;
A阴性空白:75μLTris-HCl+25μLGly-pro-phy+100μL醋酸钠。
挑选DDP-IV抑制率较高的菌株。其中,菌株CX-4在SKM固体培养基上乳蛋白的水解圈直径D为23.72mm,菌落直径d为5.02mm,D/d为4.73(如图1所示);菌株CX-4在37℃环境中DDP-IV抑制剂产率高达67.48%。
将具有较高DDP-IV抑制率的分离纯化菌株重新活化后接种到LB平板上培养24h,再用灭菌的牙签挑取单菌落接到CAS固体检测培养基,37℃倒置培养2~3d,观察菌落周围变色圈大小。其中,菌株CX-4在CAS培养基上培养一段时间后菌落周围形成明显的变色圈,说明菌株CX-4能产生高铁鳌合能力的铁载体(如图2所示)。
对菌株CX-4进行铁载体含量试验:
(1)将活化的菌苔接种于限铁SA液体培养基中,37℃摇床培养48h;
(2)将生长48h的菌株CX-4菌悬液转移到已灭菌的10mL离心管中,13000rpm离心15min;
(3)将上清液转移到经浓盐酸处理过的试管中,加入一定量现配的CAS检测液使上清液与检测液的体积比为1:1,充分混匀后室温静置1h;
(4)测定630nm波长处的吸光度值(A),取双蒸水作为对照调零,以同法测定的未接种的SA限铁培养基与检测液混合后630nm(Baakza et al.,2004)波长处的吸光度值作为参比值(Ar),并以下式表示铁载体活性单位:
Su≈(Ar-As)/Ar×100;
式中:Su为铁载体含量;Ar为所测未接种的SA限铁培养基与检测液上清液的OD值;As为所测培养基上清液的OD值。铁载体活性单位小于10时,一般认为是阴性的,并且铁载体与检测液的混合物无颜色变化。
经检测,菌株CX-4在37℃环境中产铁载体的Su值为68%,说明菌株CX-4具有较强的产铁载体能力。
菌株CX-4的16S rRNA鉴定:采用北京索莱宝生物科技公司的细菌基因组DNA提取试剂盒,提取分离纯化后的菌株DNA。采用细菌通用引物27F/1492R进行PCR扩增,PCR扩增体系为25μL体系:10×缓冲液2.5μL、Taq酶0.5μL、dNTPs 2μL、引物27F 0.5μL、引物1492R0.5μL、DNA模板1μL、ddH2O 18μL。PCR扩增反应程序设定为:95℃预变性5min;94℃变性50s、56℃退火30s、72℃延伸1.5min,循环次数30次;72℃再延伸10min,4℃保存。将PCR扩增产物送往RuiBiotech公司测序,菌株CX-4的16S rRNA与厦门希瓦氏菌(Shewanella piezotolerans)基因序列相似度为99%。通过NCBI数据库比对菌株16S rRNA的测序结果,并构建系统进化树(如图3所示)。由图3菌株CX-4的系统发育树可以看出菌株CX-4与厦门希瓦氏菌(Shewanella piezotolerans)(NR 076454.1)同处最小的一个分支,进化距离较近。
菌株CX-4的生理生化鉴定:将菌株CX-4在固体LB培养基平板上三区划线,分离出单菌落并对其形态进行描述,根据《常用细菌系统鉴定手册》对菌株进行革兰氏染色以及生理生化鉴定。菌株CX-4菌落呈白色,圆形,不透明,表面光滑,有光泽,边缘整齐,经革兰氏染色为革兰氏阴性菌。菌株CX-4的部分生理生化指标如表1所示。
表1
CX-4与厦门希瓦氏菌(Shewanella piezotolerans)模式种的生理生化具有相同特征,由各项生理生化以及16S rRNA鉴定结果,确定菌株CX-4为厦门希瓦氏菌(Shewanellapiezotolerans),将其命名为厦门希瓦氏菌(Shewanella piezotolerans)CX-4。
具体实施方式二:本实施方式配制厦门希瓦氏菌(Shewanella piezotolerans)CX-4菌液。
厦门希瓦氏菌(Shewanella piezotolerans)CX-4菌液培养基由6.34%的蔗糖、2.76%的蛋白胨、0.78%的碳酸钙和余量的蒸馏水组成。
以0.6%的接种量将厦门希瓦氏菌(Shewanella piezotolerans)CX-4种子液接种于厦门希瓦氏菌(Shewanella piezotolerans)CX-4菌液培养基,37℃、130r/min(装液量为100mL/500mL)、培养24h,厦门希瓦氏菌(Shewanella piezotolerans)CX-4菌液的活菌数达到3.29×109cfu/mL,(而CX-4种子液中活菌数为1.92×109cfu/mL,CX-4种子液由厦门希瓦氏菌(Shewanella piezotolerans)CX-4接种LB液体培养基制成)。
实施例1
厦门希瓦氏菌(Shewanella piezotolerans)CX-4耐酸实验:
配制LB液体培养基并用0.1mol/L盐酸调节液体培养基pH值分别为2.0、3.0、4.0,再以2%的接种量将厦门希瓦氏菌(Shewanella piezotolerans)CX-4菌液(菌液活菌数3.29×109cfu/mL)分别接种于上述液体培养基中,并设置不加盐酸的培养基作为对照。37℃培养3h,之后活菌计数。选择合适的稀释度,吸取100μL液体涂布平板,每个稀释度两个平板,在超净台内吹干。
存活率=酸调节培养液的活菌数/不加盐酸培养液的活菌数×100%。
厦门希瓦氏菌(Shewanella piezotolerans)CX-4耐酸实验结果如表2所示。说明厦门希瓦氏菌(Shewanella piezotolerans)CX-4具有优异的耐酸性。
表2
实施例2
厦门希瓦氏菌(Shewanella piezotolerans)CX-4耐胆盐实验:
将厦门希瓦氏菌(Shewanella piezotolerans)CX-4菌液(菌液活菌数3.29×109cfu/mL)以2%的接种量分别接种于不同胆盐浓度0.2%、0.4%和0.6%的液体培养基中,设置无胆盐培养基做对照,37℃培养4h,之后活菌计数。每个稀释度两个平板,在超净台内吹干。
存活率=胆盐培养液的活菌数/不加胆盐培养液的活菌数×100%。
厦门希瓦氏菌(Shewanella piezotolerans)CX-4耐胆盐实验结果如表3所示。说明厦门希瓦氏菌(Shewanella piezotolerans)CX-4具有优异的耐胆盐性能。
表3
实施例3
在每个新鲜的血平板上划线接种厦门希瓦氏菌(Shewanella piezotolerans)CX-4,30℃培养48h后观察。溶血分为α溶血(不完全性溶血,产生草绿色溶血环)、β溶血(完全性溶血,呈界限分明、无色透明的溶血环)以及不溶血。
厦门希瓦氏菌(Shewanella piezotolerans)CX-4溶血实验结果为阴性,可初步说明厦门希瓦氏菌(Shewanella piezotolerans)CX-4没有人畜致病的风险。
实施例4
用LB培养基培养活化厦门希瓦氏菌(Shewanella piezotolerans)CX-4,然后在37℃、180rpm条件下挑取厦门希瓦氏菌(Shewanella piezotolerans)CX-4单菌落于改良后的培养基(改良后的培养基由6.34%的蔗糖、2.76%的蛋白胨、0.78%的碳酸钙和余量的蒸馏水组成)中培养24h,制成种液;再按1%接种量将种液接种至改良后的培养基(6.34%的蔗糖、2.76%的蛋白胨、0.78%的碳酸钙和余量的蒸馏水)中进行发酵,37℃、180rpm条件下培养48h;之后再将厦门希瓦氏菌(Shewanella piezotolerans)CX-4发酵液加入无菌水进行稀释到菌数为1×108cfu/mL,按2%的比例拌入饲料,阴干后投喂。
在中国水产科学院黑龙江水产研究所实验室循环水池内进行本实施例。试验所用的鲤购于辽宁省丹东市景波养殖场。选取当年繁殖的初始体重为66g左右的鲤200尾,于4.3m×1.5m×1.2m的水泥池中暂养1周以适应试验环境,暂养期间投喂对照组饲料(通威181锦鲤鱼饲料)。暂养后选取体重相近、活力较好、体表无伤的鲤120尾,随机分为2组,每组3个平行,每个平行20尾置于6个1.3m×0.8m×0.7m的循环水饲养箱中,水体覆盖饲养箱80%以上,以曝气24h以上的自来水作为试验水体,试验期间采用气石24h充气,每日08:00和16:00投喂2次,试验组投喂混有厦门希瓦氏菌(Shewanella piezotolerans)CX-4菌液的对照组饲料(饲料:CX-4菌液比为1:0.2),对照组投喂对照组饲料;表观饱食,每日吸底1次、换水1次,换水量约为1/2,试验期间水温(23±1)℃,溶氧为(6±0.5)mg/L,pH为(7.1±0.5),养殖周期为6周。取样前,试验鱼停食24h,放入冰水中冷休克,称重测量体长、体重,剥离内脏和肠系膜脂肪并称重,计算其生长指标。
特定生长率(SGR,%/d)=100×(ln终末体质量-ln初始体质量)/42
增重率(WGR,%)=100×(平均终末体质量-平均初始体质量)/平均初始体质量饲料系数(FCR)=平均投饵量/(终末平均体质量-初始平均体质量)
肥满度(CF,g/cm3)=100×终末体质量/体长3
成活率(SR,%)=100×(终末个体数/初始个体数)
肠脂比(ISI,%)=100×肠脂质量/体质量
脏体比(VSI,%)=100×内脏团质量/体质量
试验组和鲤的成活率在各组间均无显著差异(P>0.05)。试验组饲料系数低于对照组,其余各项指标均有一定程度的升高(如表4所示),说明厦门希瓦氏菌(Shewanellapiezotolerans)CX-4可以显著提升饲料的利用率。
表4
肠促胰岛素具有促进胰岛素具有促进胰岛素分泌和抑制胰高血糖素的分泌的功能,从而可以达到降血糖的作用,但是肠促胰岛素易被DDP-IV降解失去活性而无法起效。DDP-IV抑制剂能够抑制二肽基肽酶,延长肠促胰岛素胰高血糖素样肽(GLP-1)和胰岛素释放肽(GIP)的半衰期。因此,分析认为厦门希瓦氏菌(Shewanella piezotolerans)CX-4间接达到降低血糖的目的。
我国的渔业资源非常丰富,养殖业尤为发达,但在饲养过程中不可避免的会因为饲料中含有的糖类物质导致鱼体中血糖含量上升。鱼类对糖类物质分解能力有限,从而导致饲料的浪费且不利于鱼体健康,本发明厦门希瓦氏菌(Shewanella piezotolerans)CX-4加入投喂饲料,抑制了鱼体中DDP-IV降解肠促胰岛素,提高了鱼类对糖类物质分解能力,达到了降糖作用,提高了饲料的利用率。
Claims (2)
1.一株产DDP-IV抑制剂的厦门希瓦氏菌,其为厦门希瓦氏菌(Shewanellapiezotolerans)CX-4,保藏在中国微生物菌种保藏管理委员会普通微生物中心,保藏号为CGMCC No.24198。
2.如权利要求1所述产DDP-IV抑制剂的厦门希瓦氏菌在鱼类养殖中的用途。
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