CN117568221A - 一株高产脂肪酶的枯草芽孢杆菌及其应用 - Google Patents
一株高产脂肪酶的枯草芽孢杆菌及其应用 Download PDFInfo
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- CN117568221A CN117568221A CN202311555505.XA CN202311555505A CN117568221A CN 117568221 A CN117568221 A CN 117568221A CN 202311555505 A CN202311555505 A CN 202311555505A CN 117568221 A CN117568221 A CN 117568221A
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Abstract
本发明公开了一株高产脂肪酶的枯草芽孢杆菌及其应用,涉及生物医药技术领域。一株高产脂肪酶的枯草芽孢杆菌,为枯草芽孢杆菌C1,枯草芽孢杆菌C1保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏号CGMCC NO.28693,保藏时间2023年10月20日,保藏地址北京市朝阳区北辰西路1号院3号,分类命名枯草芽孢杆菌Bacillus subtilis,其16S rRNA序列如SEQ ID NO.1所示。本发明采用上述的一株高产脂肪酶的枯草芽孢杆菌及其应用,产脂肪酶酶活力较高,同时可以分解橄榄油产生高不饱和脂肪酸(HUFA),通过添加一定浓度的枯草芽孢杆菌C1,可以缓解高脂质饲料导致的鱼体脂肪沉积、脂质代谢紊乱等负面影响。
Description
枯草芽孢杆菌C1保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏号CGMCC NO.28693,保藏时间2023年10月20日,保藏地址北京市朝阳区北辰西路1号院3号,分类命名枯草芽孢杆菌Bacillus subtilis。
技术领域
本发明涉及生物医药技术领域,尤其是涉及一株高产脂肪酶的枯草芽孢杆菌及其应用。
背景技术
由于优质蛋白质来源短缺,水产养殖生产中常通过提高饲料脂肪比例达到节约蛋白质并促进养殖对象生长的效果。然而,饲料中脂质含量过高可能导致鱼体肝脏中脂质沉积过多并伴随肝损伤、脂质代谢紊乱,导致养殖动物生长减缓,进而对水产养殖业以及市场消费者造成较大的负面影响。
通过营养手段缓解鱼类脂肪肝是目前养殖生产中的主要应用手段。许多研究发现,在饲料中添加肉碱、胆碱、中草药提取物等能够缓解鱼类肝脏中脂肪沉积的现象。然而,鉴于对这些抗脂肪肝因子的分子机制研究尚不充分,且受物种差异的影响,导致不同鱼种的剂量和结果不同。
已有研究表明,肉碱可通过生物合成方法可从赖氨酸及蛋氨酸两种氨基酸合成产生,并与脂肪代谢与能量合成有关。在大部分抗脂肪肝因子中肉碱的研究较为深入,且肉碱在鱼类饲料中的添加剂量以及相关功能性研究也较多,主要机制是能够携带长链脂肪酸进入线粒体内进行β氧化,加速脂类的消耗。但是肉碱对于鱼类脂肪沉积现象的调节作用可能会受到物种因素的影响。
槲皮素和白藜芦醇。槲皮素是植物界分布广泛,具有多种生物活性的黄酮醇类化合物。但是槲皮素在植物中含量较低,且提取分离较难、成本高。白藜芦醇是一种非黄酮类多酚有机化合物,是许多植物受到刺激时产生的一种抗毒素。天然白藜芦醇主要以虎杖苷的形式存在于虎杖植物中,该植物主要集中在湖南和四川,年开采量已经达到饱和,虎杖的人工栽培研究虽然已经起步,但由于技术及野生资源供应量、栽培种植的成本等方面的原因尚未进行大面积种植。现阶段,白藜芦醇的天然野生资源匮乏,这与市场的需求形成了极大的差距。因此,有必要开发更易于实际应用的调控鱼体脂质沉积的新策略和新途径。
益生菌作为饲料添加剂已广泛应用于畜牧业和水产养殖中,具有重要的研究价值和广阔的应用前景。因此,本发明从鱼体肠道中筛选了一株高产脂肪酶的枯草芽孢杆菌,用于防治高脂饲料引起的鱼类肝脏脂质沉积。
斑马鱼(Danio rerio)因为其繁殖快、生长周期短,已经成为最受重视的模式动物之一。同时也被广泛用于鱼类营养代谢以及肠道菌群与宿主相互作用的研究,是评价益生菌应用效果的一个较好动物模型。
发明内容
本发明的目的是提供一株高产脂肪酶的枯草芽孢杆菌及其应用,可以改善高脂饮食导致的鱼类脂质沉积,调节脂代谢,缓解鱼类脂肪肝发生的现象,从而弥补现有技术的不足。
为实现上述目的,本发明提供了一株高产脂肪酶的枯草芽孢杆菌,为枯草芽孢杆菌C1,枯草芽孢杆菌C1保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏号CGMCCNO.28693,保藏时间2023年10月20日,保藏地址北京市朝阳区北辰西路1号院3号,分类命名枯草芽孢杆菌Bacillus subtilis。
进一步地,枯草芽孢杆菌C1是从鱼类肠道中分离得到的,枯草芽孢杆菌C1的16SrRNA序列如SEQ ID NO.1所示。
本发明还提供了上述的枯草芽孢杆菌在制备改善高脂饮食导致的鱼类脂质沉积产品中的应用。
进一步地,应用方式包括水体添加和饲料添加。
进一步地,水体添加枯草芽孢杆菌C1的含量为105、106、107CFU/mL,饲料添加枯草芽孢杆菌C1的含量为105、106、107、108CFU/g。
更进一步地,水体添加枯草芽孢杆菌C1的含量为107CFU/mL,饲料添加枯草芽孢杆菌C1的含量为106CFU/g。
本发明还提供了上述的枯草芽孢杆菌在制备改善高脂饮食导致的肠道炎症产品中的应用。
本发明所述的一株高产脂肪酶的枯草芽孢杆菌及其应用的优点和积极效果是:
1、本发明中的枯草芽孢杆菌C1,不溶于血对宿主无害,对绝大部分抗生素敏感。
2、本发明中的枯草芽孢杆菌C1产脂肪酶酶活力较高,同时可以分解橄榄油产生高不饱和脂肪酸(HUFA)。
3、通过添加一定浓度的枯草芽孢杆菌C1,可以缓解高脂质水平的饲料导致鱼体脂肪沉积、脂质代谢紊乱等负面影响,该菌株可以作为水产益生菌。
下面通过附图和实施例,对本发明的技术方案做进一步的详细描述。
附图说明
图1为本发明中枯草芽孢杆菌C1的分离鉴定结果,其中A为显微镜在100X放大倍数下枯草芽孢杆菌C1的形态图,B为枯草芽孢杆菌C1的革兰氏染色图,C为枯草芽孢杆菌C1的系统发育树,D为枯草芽孢杆菌C1的生长曲线;
图2为本发明中枯草芽孢杆菌C1的安全性检测,其中A为溶血性实验结果,B为使用不同浓度(105、106、107CFU/mL)的枯草芽孢杆菌C1水浴斑马鱼仔鱼(3dpf)3天,统计存活率(n=3次重复/组,20尾仔仔鱼/重复)结果,C为通过纸片扩散法药敏试验检测枯草芽孢杆菌C1对12种抗生素的敏感性,并测量抑菌圈的直径,S表示该菌对抗生素敏感,I表示中等敏感;
图3为本发明中枯草芽孢杆菌C1对高脂饲料喂养的斑马鱼仔鱼脂质含量的影响,其中A为尼罗红染色,B为油红O(ORO)染色,C为尼罗红染色荧光强度image J统计(n=9),D为甘油三酯含量(n≥4次重复/组,5尾仔仔鱼/重复),#p<0.05,##p<0.01,Ctrl与HFD比较;*p<0.05,**p<0.01,***p<0.001,****p<0.0001,HFD与HFB比较;
图4为本发明中枯草芽孢杆菌C1对高脂饲料喂养的斑马鱼仔鱼脂质代谢相关基因的影响,其中A为pparα,B为pgc-1α,C为pparg,D为fas,##p<0.01,Ctrl与HFD比较;*p<0.05,**p<0.01,HFD与HFB比较;
图5为本发明中枯草芽孢杆菌C1减少高脂饲料喂养的斑马鱼脂质沉积,其中A为血清甘油三酯含量,B为血清总胆固醇含量,C为肝脏甘油三酯含量,D为肝脏总胆固醇含量,数据以平均值±标准差表示(n=6-7),#p<0.05,##p<0.01,Ctrl与HFD比较;*p<0.05,**p<0.01,***p<0.001,****p<0.0001,HFD与HFB比较;
图6为本发明中斑马鱼成鱼肝脏切片油红O染色结果,其中A为油红O染色,B为脂滴面积统计结果,数据以平均值±标准差表示(n=4),#p<0.05,Ctrl与HFD比较;*p<0.05,HFD与HFB比较;
图7为本发明中枯草芽孢杆菌C1调节高脂饲料喂养的斑马鱼脂质代谢相关信号通路,其中A为pparα,B为pgc-1α,C为hsl,D为atgl,E为pparg,F为acc-a,G为fas,H为dgat2,数据以平均值±标准差表示(n=6-7),#p<0.05,###p<0.001,Ctrl与HFD比较;*p<0.05,**p<0.01,***p<0.001,****p<0.0001,HFD与HFB比较;
图8为本发明中枯草芽孢杆菌C1改善高脂饲料喂养的斑马鱼肠道健康,其中A为il-1β,B为il-8,C为il-6,D为tnf-α,数据以平均值±标准差表示(n=6-8),#p<0.05,##p<0.01,Ctrl与HFD比较;*p<0.05,**p<0.01,***p<0.001,****p<0.0001,HFD与HFB比较。
具体实施方式
以下通过附图和实施例对本发明的技术方案作进一步说明。
除非另外定义,本发明使用的技术术语或者科学术语应当为本发明所属领域内具有一般技能的人士所理解的通常意义。
以下实施例中所使用的实验方法如无特殊说明,均为常规方法。下述实施例中所使用的材料、试剂等,如无特殊说明,均可从商业途径得到。
枯草芽孢杆菌C1,枯草芽孢杆菌C1保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏号CGMCC NO.28693,保藏时间2023年10月20日,保藏地址北京市朝阳区北辰西路1号院3号,分类命名枯草芽孢杆菌Bacillus subtilis。
高脂饲料是在正常饲料基础上添加豆油、猪油制成的。正常饲料和高脂饲料的成分、营养配比见表1。
表1
注:a维生素预混料由下列成分组成(mg/g预混料):硫胺素盐酸盐,5;核黄素,10;泛酸钙,10;D-生物素,0.6;盐酸吡哆醇,4;叶酸,1.5;肌醇,200;L-维生素C-2-磷酸镁,60;烟酸,6.05;α-维生素E醋酸酯,50;维生素K,4;视黄醇醋酸酯,2000IU和维生素D3,400IU,再用微晶纤维素添加至1g。
b矿物盐预混料由下列成分组成(mg/g预混料):磷酸二氢钙,135.8;乳酸钙,327;硫酸亚铁,2.125;硫酸镁,137;磷酸二氢钠,87.2;氯化钠,43.5;氯化铝,0.15;碘酸钾,0.125;氯化钾,75;氯化铜,0.1;硫酸锰,0.80;氯化钴,1和硫酸锌3,再用微晶纤维素添加至1g。
实施例1枯草芽孢杆菌C1的分离鉴定及生物学分析
1、枯草芽孢杆菌C1的分离鉴定
(1)枯草芽孢杆菌C1的分离
产脂肪酶菌株——枯草芽孢杆菌C1从鱼类肠道分离,在无菌条件下肠道匀浆后将其涂布在含有吐温80的LB琼脂平板(海博HB0129,中国青岛)和LB琼脂平板(海博HB0132,中国青岛),在28.5℃培养24h随机挑取单菌落,2次划线纯化培养。纯化后的菌株进行16SrRNA测序鉴定,并最终以终浓度为25%甘油保藏于-80℃。
(2)枯草芽孢杆菌C1的16S rRNA基因序列分析
采用16S rRNA序列的常用引物27F(SEQ ID NO.2):5'-AGAGTTTGATCCTGGCTCAG-3',1492R(SEQ ID NO.3):5'-CTACGGCTACCTTGTTACGA-3',对分离菌株PCR扩增(Bio-Rad,美国)。PCR反应体系:2×Taq12.5μL,27F和1492R各1μL,模板1μL,DEPC水补充至25μL。反应程序为:94℃预变性5min,94℃变性30s,51℃复性30s,72℃延伸1.5min,35个循环,72℃延伸5min。PCR扩增产物经过琼脂凝胶电泳检测合格后大约在1500bp,将其送至第三方公司进行测序。测序结果序列在NCBI-Blast上进行比对,从GenBank中下载相似性最高近缘物种的模式菌株序列,通过MEGA7.0.14软件构件系统发育树。
结果如图1所示,枯草芽孢杆菌C1形态呈现长杆状(图1中A)。通过革兰氏染色结果确定为该菌株为革兰氏阳性菌(图1中B)。在NCBI上进行BLAST序列比对,结果显示该菌株与枯草芽孢杆菌序列相似性达到99%,通过Mega构建系统发育树(图1中C),表明该菌株与枯草芽孢杆菌(Bacillus subtilis)的亲缘关系最近,命名为枯草芽孢杆菌C1(Bacillussubtilis C1)。此外,将该菌株在28.5℃培养6-10h为指数生长期,12h后趋于稳定(图1中D)。
(3)生理生化鉴定
使用细菌生理生化鉴定试剂盒(滨和,中国杭州)对枯草芽孢杆菌C1进行鉴定,测定20种生理生化指标(甘露糖、半乳糖、葡萄糖、果糖、蔗糖、麦芽糖、海藻糖、糊精、甘露醇、淀粉、阿拉伯糖、苦杏仁苷、棉子糖、鼠李糖、乳糖、纤维二糖、木糖、水杨苷、山梨醇、葡萄糖酸盐),每种设定3个平行,培养基作为空白对照,在28.5℃恒温培养箱中过夜培养后观察结果并记录。
结果如表2所示,生理生化实验结果表明,枯草芽孢杆菌C1能利用甘露糖、阿拉伯糖、葡萄糖、果糖、蔗糖、麦芽糖、纤维二糖、海藻糖、木糖、水杨苷、甘露醇、淀粉;对半乳糖、苦杏仁苷、棉子糖、鼠李糖、乳糖、糊精、葡萄糖酸盐不能利用。
表2
注:“+”代表阳性,“-”代表阴性。
2、枯草芽孢杆菌C1安全性检测
(1)溶血性
在血平皿平板(海博3400071,中国青岛)上对枯草芽孢杆菌C1进行平板划线,在28.5℃恒温培养箱中过夜培养后观察菌落周围是否有溶血环。
(2)枯草芽孢杆菌C1对斑马鱼存活率的影响
斑马鱼受精三天(3dpf)后,将仔鱼均匀放入含有8mL胚胎培养液的六孔板中,加入终浓度为105、106、107CFU/mL的枯草芽孢杆菌C1水浴3天。同时观察并统计死亡率,每组3个平行。
(3)药物敏感性
采用纸片扩散法得到枯草芽孢杆菌C1对抗生素敏感性的定性估计。将菌液涂布于LB琼脂平板上,过夜培养后,观察有无抑菌圈,并记录其直径。药敏纸片(滨和,中国杭州)包含卡那霉素、链霉素、氨苄西林、多粘菌素B、氯霉素、庆大霉素、四环素、青霉素G、头孢噻肟、克拉霉素、左氧氟沙星、万古霉素。
结果如图2所示,枯草芽孢杆菌C1不溶于血(图2中A),当使用不同浓度的枯草芽孢杆菌C1水浴斑马鱼仔鱼3天后,对斑马鱼仔鱼存活率无显著影响(图2中B)。枯草芽孢杆菌C1对卡那霉素、链霉素、氨苄西林、多粘菌素B、氯霉素、庆大霉素、四环素、头孢噻肟、克拉霉素、左氧氟沙星、万古霉素敏感,对青霉素G中度敏感(图2中C)。
实施例2枯草芽孢杆菌C1油脂分解能力
1、产脂肪酶能力的检测
(1)枯草芽孢杆菌C1在28.5℃过夜培养后,分别取100μL菌液和上清液置于含有吐温80/鱼油直径为7mm琼脂平板上的孔中,培养3天,测量沉淀圈和水解圈的直径,并计算沉淀圈/水解圈与孔径直径的比值。
(2)脂肪酶活力的测定
通过脂肪酶将底物pNPP降解为黄色的pNP原理,采用分光光度法检测发酵上清脂肪酶活。将分离菌株按1%接种量接种在LB肉汤培养基中,在28.5℃、180r/min培养48h,将菌体培养液按照12000r/min,离心2min,取上清即为粗酶液。配制6mmol/L对硝基苯酚(pNP),使用50mmol/L Tris-HCL(pH8.0)缓冲液稀释至不同浓度后加入10%三氯乙酸,410nm测定光吸收值,绘制对硝基苯酚标准曲线。
称取90mg棕榈酸对硝基苯酯(pNPP)溶于30mL异丙醇为底物A溶液,反应缓冲液为50mmol/L Tris-HCL(pH8.0),将试管中按顺序加入溶液B l.8mL及0.1mL底物溶液A,在37℃水浴保温5min后,加入待检测的粗酶液,水浴中准确反应10min,加入10%三氯乙酸,混匀,分光光度计下测定酶催化产生的对硝基苯酚在410nm的吸收值,计算其脂肪酶活力。脂肪酶活力单位(U)定义为:在以上条件下,每分钟释放1μmol对硝基苯酚(pNP)所需要的酶量为1U。
酶活计算公式:A=([At-A0]×K+C0)×V1/(V2×t)。
式中A:样品酶活(U/mL);At:反应后样品酶液的吸光度OD值;A0:对应酶液的空白吸光度OD值;K:对硝基苯酚标准曲线的斜率;C:对硝基苯酚标准曲线的截距;V1:反应液的体积/mL;V2:粗酶液的体积/mL;t:反应时间/min。
(3)枯草芽孢杆菌C1对橄榄油的降解率
分别配制不同浓度的橄榄油溶液(0、0.2、0.4、0.6、0.8、1.0、1.2、1.4和1.6mg/mL),以石油醚溶液作为空白组,在OD300分别测量以上不同浓度的橄榄油溶液的吸光度,并绘制橄榄油标准曲线。按照1%接种量将菌株接种在含有橄榄油的肉汤培养基中,培养48h后,8000rpm离心10min,取上清液于分液漏斗中,弃去沉淀。以未接种菌株,其余条件完全一致的培养基作为空白组,经过离心、萃取、测吸光值计算剩余油脂的浓度,计算得到油脂降解率。由此可得到油脂降解率的公式为:P=(C-Ctest)/C×100%,式中P为油脂降解率,%;C为初始油脂浓度,mg/mL;Ctest为剩余油脂浓度,mg/mL。
结果如表3所示,将枯草芽孢杆菌C1接种于含有吐温80的琼脂平板上,培养后可观察到沉淀圈,由此我们推断改菌株可以产生脂肪酶并具有分解吐温80的能力,测量并计算沉淀圈直径和孔径直径比为4.10±0.36cm。同样地,在含有鱼油的琼脂平板上,也可以观察到水解圈,且直径比为2.10±0.17cm,说明该菌株可以很好的分解油源。根据对硝基苯酚法测定粗酶液脂肪酶活力,通过绘制对硝基苯酚标准曲线得到标准方程为:y=112.09x-6.0467,通过分光光度计测定酶催化产生的对硝基苯酚在410nm的吸收值,计算其脂肪酶活力。结果显示,枯草芽孢杆菌C1脂肪酶活力为20.47±2.70U/mL。此外,根据油脂含量测定方法,绘制橄榄油标准曲线得到标准方程:y=11.111x-30.586。在含橄榄油的肉汤培养基中培养枯草芽孢杆菌C1,通过离心、萃取、计算剩余橄榄油浓度,进而计算菌株对橄榄油的降解率。结果表明,枯草芽孢杆菌C1具有降解橄榄油能力,降解率为41.98%。综上所述,结果均表明枯草芽孢杆菌C1具有较强的产脂肪酶能力。
表3
2、枯草芽孢杆菌C1产脂肪酸种类
按照1%接种量将枯草芽孢杆菌C1接种在含有橄榄油的LB肉汤培养基中,培养48h后,3000rpm离心15min,分别收集菌体沉淀和上清液,根据前期方法进行样品处理。即吸取1mL样品,加入KOH-甲醇3mL,于75-80℃水浴中加热20min,放置冷却。加HCL-甲醇溶液3mL,于75-80℃水浴中加热20min,冷却。加正己烷1mL,振荡萃取静置分层。吸取适量上层液,5000rpm离心5min,将样品保存于-20℃。使用GC-MS(Shimadzu,QP2010,日本)检测产脂肪酸种类。
结果如表4所示,使用GC-MS检测菌体和上清液脂肪酸种类。在不含橄榄油的肉汤培养基中培养菌株48h后,结果显示空白对照(LB肉汤培养基)脂肪酸全部为C4:0,为饱和脂肪酸(SFA);枯草芽孢杆菌C1脂肪酸种类主要为C16:1、C16:0、C15:1,少数为C14:0、C14:1,转化成更多的不饱和脂肪酸(MUFA)。
在含有橄榄油的培养基中培养菌株48h后,分别检测橄榄油、培养基、菌体和上清液脂肪酸种类,结果显示,橄榄油大约含有75%的油酸(C18:1CIs)、11%的棕榈酸(C16:0)、5%亚油酸(C18:2n-6CIs),加入菌株培养后,枯草芽孢杆菌C1菌体中减少了不饱和脂肪酸并转化成更多的多不饱和脂肪酸(PUFA),且含有少量的高不饱和脂肪酸(HUFA)——EPA,但是在枯草芽孢杆菌C1上清液中产生更多的饱和脂肪酸。
表4
实施例3枯草芽孢杆菌C1在高脂饮食下对斑马鱼脂代谢的影响
1、枯草芽孢杆菌C1在高脂饮食下对斑马鱼仔鱼脂代谢的影响
(1)实验设计
斑马鱼受精5天后,将斑马鱼仔鱼均匀放入含有8mL胚胎培养液的六孔板中,分别投喂正常饲料(Ctrl)和高脂饲料(HFD)3d,每天投喂2次,诱导斑马鱼仔鱼高脂模型后,加入终浓度为105(HFB-105)、106(HFB-106)、107(HFB-107)CFU/mL的枯草芽孢杆菌C1水浴24h。每5尾鱼一个样,全鱼匀浆,用于检测甘油三酯(TG)含量;每3尾鱼一个样,全鱼匀浆,用于检测基因相对表达量。
(2)尼罗红染色和荧光强度统计
尼罗红先溶解于丙酮中配成浓度为1mg/mL的工作母液,将仔鱼置于在以胚胎培养液稀释后浓度为0.1μg/mL尼罗红溶液中,保持黑暗及水温28.5℃,水浴1h后先用胚胎培养液润洗一遍,采用荧光显微镜观察(NIKON,SMZ25,日本),使用imageJ统计荧光强度。
(3)油红O(ORO)染色和甘油三酯含量测定
将仔鱼收集到1.5mL离心管中,4℃放置30min后去除水分,PBS清洗3遍,使用4%多聚甲醛固定4h,PBS清洗3遍去除多聚甲醛。60%异丙醇摇晃30min后去除异丙醇,使用ORO染色3h后去除ORO,60%异丙醇清洗3遍,PBS清洗3遍,显微镜下观察(OLYMPUS,BX43,日本)。依据制造商的说明书(南京建成生物工程研究所,南京,中国),检测斑马鱼仔鱼的甘油三酯(TG,A110-1-1)含量。
(4)基因水平检测
收集样品提取RNA,反转录得到cDNA,以cDNA为模板,使用荧光定量PCR(Bio-rad,美国)分析斑马鱼cDNA中脂代谢相关基因的表达量。其中pparα、pgc1α均为脂肪酸β氧化相关基因;pparg、fas均为脂合成相关基因。pparα、pgc1α、pparg、fas的上下游引物如表5所示。
表5
(5)数据分析
采用GraphPad Prism8.0(GraphPad,美国)对实验数据进行统计分析,使用单因素方差分析(one-way ANOVA)处理数据,采用Dunnett's检验进行多重比较,若p<0.05表示处理组间存在差异显著,实验数据均用平均值±标准误(Mean±SEM)表示。
结果如图3所示,尼罗红染色结果与对照组(Ctrl)相比,高脂饮食组(HFD)可以观察到更强的荧光,然而,当使用不同浓度的枯草芽孢杆菌C1(HFB)处理后降低了斑马鱼仔鱼的脂质含量(图3中A,C);此外,油红O染色显示,喂食高脂饲料的仔鱼有更多脂质积累,添加枯草芽孢杆菌C1后减少了脂质沉积(图3中B),进一步分析表明,枯草芽孢杆菌C1组(HFB)的斑马鱼仔鱼甘油三酯(TG)含量与高脂饮食组相比显著降低(图3中D)。
如图4所示,为研究枯草芽孢杆菌C1减少脂质积累的机制,检测脂质代谢相关基因的相对表达。与对照组相比,高脂饮食组降低了脂肪酸β氧化基因pparα、pgc-1α(图4中A、B)的表达,添加106、107CFU/mL的枯草芽孢杆菌C1(HFB-106、HFB-107)能够显著上调pgc-1α的表达,从而促进脂肪酸的β氧化。添加105CFU/mL的枯草芽孢杆菌C1(HFB-105)的脂合成基因pparg的表达水平相对于高脂饮食组显著降低,抑制了脂肪的合成;fas没有显著变化(图4中C、D)。以上结果表明枯草芽孢杆菌C1可以减少高脂饮食诱导的斑马鱼仔鱼脂质沉积。
2、枯草芽孢杆菌C1对斑马鱼成鱼脂代谢的调节
(1)实验设计与样品采集
三个月的野生型斑马鱼为实验对象,随机放入6个缸中,每种饲料处理组分别设置3个平行,每缸10尾鱼。所有实验用鱼均为雄鱼,喂食正常饲料1周。在高脂饲料添加不同含量的枯草芽孢杆菌C1,使其终浓度为105(HFB-105)、106(HFB-106)、107(HFB-107)、108(HFB-108)CFU/g。分别投喂正常饲料(Ctrl组)、高脂饲料(HFD组)以及补充菌株饲料(HFB组),每天9:00和18:00饱食投喂2次并换水,投喂6周。
投喂实验结束后,将各组实验用鱼禁食24h,即可取样,将所有鱼放入0.1%三卡因(MS-222)中进行麻醉。血清:通过断尾收集斑马鱼血液,然后以1467×g离心10min,获得上清液,即血清。解剖取肠道、肝脏等组织,分别置于离心管中,液氮冷冻,再放入-80℃冷冻保存,用于后续检测分析。
(3)甘油三酯和总胆固醇含量的测定
依据制造商的说明书(南京建成生物工程研究所,南京,中国),检测斑马鱼血清、肝脏的甘油三酯(TG,A110-1-1)、总胆固醇(TC,A111-1-1)含量。
结果如图5所示,与正常饲料(Ctrl组)相比,高脂饲料(HFD组)甘油三酯含量在斑马鱼肝脏中没有显著变化,在血清中显著上调;添加106、107、108CFU/g枯草芽孢杆菌C1后显著降低斑马鱼血清中甘油三酯的含量(图5中A)。对照组相比,HFD组的血清和肝脏总胆固醇显著增加,添加枯草芽孢杆菌(HFB组)后显著降低了其含量(图5中B、D),说明枯草芽孢杆菌具有降脂能力。
(4)油红O染色结果
斑马鱼肝脏样品从-80℃冰箱取出转入-20℃冰箱缓冲30min,使用OTC包埋剂包埋冷冻的肝脏组织块,迅速放到液氮中冷冻,在切片前保持在液氮中。用预冷的冷冻切片机将组织块切成7mm的切片,将样本切片转移至载玻片上用甲醇固定10s,-80℃冷冻过夜。油红O(ORO)染色15min,蒸馏水中流水冲洗20min,立刻用甘油明胶封片,显微镜下镜检。使用image J统计脂滴含量。结果如图6所示,HFD组肝脏脂滴较大且数量多,显著高于Ctrl组,添加枯草芽孢杆菌C1后,脂滴含量显著减少。统计后结果显示HFD组发生更多脂质积累,添加枯草芽孢杆菌C1缓解了这种现象。
(5)基因水平检测
收集斑马鱼肝脏组织样品提取RNA,反转录得到cDNA,以cDNA为模板,使用荧光定量PCR(Bio-rad,美国)分析斑马鱼cDNA中脂代谢相关基因的表达量。其中pparα、pgc1α、hsl、atgl均为脂分解相关基因;pparg、fas、acc-a、dgat2均为脂合成相关基因。pparα、pgc1α、pparg、fas的上下游引物如表4所示,hsl、atgl、acc-a、dgat2的上下游引物如表6所示。
表6
结果如图7所示,与Ctrl组相比,HFD组脂分解基因pparα、hsl、atgl有下降趋势,脂肪分解能力下降。与HFD组相比,添加106CFU/g枯草芽孢杆菌pgc-1a、hsl、atgl显著上调,可以促进的脂肪的分解。同样地,与Ctrl组相比,HFD组脂合成基因pparg、acc-a显著上调促进了脂肪生成。与HFD组相比,添加枯草芽孢杆菌抑制了脂肪的合成,从而缓解高脂饮食导致的脂肪沉积。
3、枯草芽孢杆菌C1对斑马鱼成鱼肠道健康的影响
(1)基因水平检测
收集斑马鱼肠道组织样品提取RNA,反转录得到cDNA,以cDNA为模板,使用荧光定量PCR(Bio-rad,美国)分析斑马鱼cDNA中脂代谢相关基因的表达量。i1-1β、il-8、il-6、tnf-α基因的上下游引物如表7所示。
表7
结果如图8所示,与Ctrl组相比,高脂饮食导致了斑马鱼肠道炎症,炎症基因显著上调。与HFD组相比,添加枯草芽孢杆菌C1后il-6、il-1β、il-8有不同程度的下调,缓解了炎症。
因此,本发明采用上述的一株高产脂肪酶的枯草芽孢杆菌及其应用,产脂肪酶酶活力较高,同时可以分解橄榄油产生高不饱和脂肪酸(HUFA),通过添加一定浓度的枯草芽孢杆菌C1,可以缓解高脂质水平的饲料导致鱼体脂肪沉积、脂质代谢紊乱等负面影响,还可以作为水产益生菌使用。
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进行限制,尽管参照较佳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:其依然可以对本发明的技术方案进行修改或者等同替换,而这些修改或者等同替换亦不能使修改后的技术方案脱离本发明技术方案的精神和范围。
Claims (7)
1.一株高产脂肪酶的枯草芽孢杆菌,其特征在于:为枯草芽孢杆菌C1,枯草芽孢杆菌C1保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏号CGMCC NO.28693,保藏时间2023年10月20日,保藏地址北京市朝阳区北辰西路1号院3号,分类命名枯草芽孢杆菌Bacillus subtilis。
2.根据权利要求1所述的一株高产脂肪酶的枯草芽孢杆菌,其特征在于:枯草芽孢杆菌C1是从鱼类肠道中分离得到,枯草芽孢杆菌C1的16S rRNA序列如SEQ ID NO.1所示。
3.如权利要求1所述的枯草芽孢杆菌在制备改善高脂饮食导致的鱼类脂质沉积产品中的应用。
4.根据权利要求3所述的应用,其特征在于:应用方式包括水体添加和饲料添加。
5.根据权利要求4所述的应用,其特征在于:水体添加枯草芽孢杆菌C1的含量为105、106、107CFU/mL,饲料添加枯草芽孢杆菌C1的含量为105、106、107、108CFU/g。
6.根据权利要求5所述的应用,其特征在于:水体添加枯草芽孢杆菌C1的含量为107CFU/mL,饲料添加枯草芽孢杆菌C1的含量为106CFU/g。
7.如权利要求1所述的枯草芽孢杆菌在制备改善高脂饮食导致的肠道炎症产品中的应用。
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