CN114381400A - 一种大豆异黄酮微生物转化菌株及其应用 - Google Patents
一种大豆异黄酮微生物转化菌株及其应用 Download PDFInfo
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Abstract
本发明涉及一种大豆异黄酮微生物转化菌株及其应用,属于微生物研究技术领域。转化菌株为菌株DZU‑LM12,该菌株的保藏编号为CGMCC No.22779,保藏日期为2021年06月24日,保藏地址为北京市朝阳区北辰西路1号院3号,命名为DZU‑LM12;以及该菌株在转化大豆异黄酮、制备O‑Dma中的应用。本发明菌株属于斯奈克氏菌属(Slackia exigua),在厌氧条件下可将大豆苷元代谢为去氧甲基安哥拉紫檀素(O‑Dma),为大豆异黄酮的转化提供新的菌源。
Description
技术领域
本发明涉及一种大豆异黄酮微生物转化菌株及其应用,属于微生物研究技术领域。
背景技术
大豆异黄酮是豆类植物如大豆、葛根等产生的代谢产物,它不仅能够抑制癌症,还具有抗氧化、预防心律不齐、延缓衰老、治疗抑郁症、防辐射的作用。近二十年来,人们对于饮食中的植物激素越来越感兴趣,其中大豆异黄酮通过多种肠道微生物的氧化和还原代谢途径可以转化为多种具有生物活性的功能性衍生物。目前,人们无法从自然界中萃取得到各种异黄酮代谢产物,人工化学合成所需成本又极高。大豆异黄酮中主要的两种活性成分为大豆苷元(daidzein)和染料木黄酮(genistein)。
本发明从人类新鲜粪样中筛出对大豆异黄酮具有转化功能的细菌菌株,为异黄酮的转化过程提供新的肠道微生物来源,推动异黄酮转化菌株在益生菌剂方面的应用,旨在解决大豆异黄酮微生物转化产品资源匮乏的问题。
发明内容
本发明提供了一种大豆异黄酮微生物转化菌株及其应用,具体涉及菌株DZU-LM12,经检测本发明菌株Slackia exigua DZU-LM12能把底物大豆苷元开环转化成O-Dma。菌株DZU-LM12的保藏编号为CGMCC No.22779,保藏日期为2021年06月24日,保藏地址为北京市朝阳区北辰西路1号院3号,命名为DZU-LM12。
本发明从人新鲜粪样中分离得到一株可以将大豆苷元底物进行转化的专性厌氧斯奈克氏菌属菌株Slackia exigua DZU-LM12。并将该菌株的转化产物分离纯化后经过HPLC检测,与O-Dma标品对比初步推测该产物,以及利用ESI-MS准确鉴定代谢产物。最终,确定DZU-LM12在厌氧条件下能将底物大豆苷元代谢为O-Dma。
本发明与现有技术相比具有以下优点:
本发明菌株,在厌氧条件下可将大豆苷元代谢为去氧甲基安哥拉紫檀素(O-Dma),为异黄酮的转化提供新的菌源。自2000年以来,已从不同动物粪样菌群中分离筛选了大量对大豆异黄酮具有特定转化功能的细菌菌株,其中分离报道的O-Dma产生菌共涉及Clostridium、Eubacterium、Enterococcus三个属,本发明菌株DZU-LM12为属于Slackia属,为大豆异黄酮的转化过程提供新的肠道微生物来源。
附图说明
图1为专性厌氧菌株DZU-LM12代谢大豆苷元的高效液相色谱图;
图2为厌氧菌DZU-LM12的16S rDNA基因序列绘制的的系统进化树;
图3为DZU-LM12对大豆苷元的转化产物质谱图。
图4为O-Dma的化学结构。
具体实施方式
下面结合具体实例来进一步描述本发明,本发明的优点和特点将会随着描述而更为清楚。但实施例仅是范例性的,并不对本发明的范围构成任何限制。本领域技术人员应该理解的是,在不偏离本发明的精神和范围下可以对本发明技术方案的细节和形式进行修改或替换,但这些修改和替换均落入本发明的保护范围内。
1.菌株来源
取自德州学院大学生新鲜粪样。
2.试验方法
2.1菌株的分离
用提前灭过菌的若干棉棒采集少量的人新鲜粪便,并将样品放到已灭过菌的试管中,该试管中装有预先配好的2mL BHI液体培养基,并由2mL无菌石蜡油覆盖。将粪样用无菌BHI培养基从10-3-10-5进行梯度稀释。预备一些提前灭菌的BHI固体琼脂板,再依次用100μL移液枪取50μL稀释液在固体板上进行均匀涂布。将涂好的固体琼脂板倒放于厌氧工作室中(10%H2、10%CO2和80%N2),在37℃下培养2d。
等到固体琼脂板长满菌后,分别挑取形态不同的菌落,以每组十个菌随机组合进行分组标号,依次将它们重新接种到内含0.5mmol L-1大豆苷元的全新BHI液体培养基中,厌氧条件下培养3d。待菌株完全长出后,用3mL乙酸乙酯提取细菌培养物1mL,每种提取液取2mL,干燥后溶解在甲醇中,样品通过0.45μm微孔膜过滤,每个样品取20μL经HPLC检验。筛选出具有转化活性的菌落组合再一次重新在固体板上进行涂布、分离。经过筛选,最终得到了一株具有异黄酮转化活性的菌株DZU-LM12。菌株DZU-LM12的保藏编号为CGMCC No.22779,保藏日期为2021年06月24日,保藏地址为北京市朝阳区北辰西路1号院3号,命名为DZU-LM12。
2.2高效液相色谱检测
使用Kromasil C18分析柱(250×4.6mm,5μm)分离20μL样品。流动相为水-乙腈(1‰冰乙酸),并将温度设为30℃,流速保持在每分钟0.6mL,于波长270nm处检测。
2.3转化菌株的菌种鉴定
参照试剂盒的步骤,提取专性厌氧菌株DZU-LM12的基因组DNA。提取完成后,进行PCR扩增,并把纯化的PCR产物送往青岛睿博兴科生物科技有限公司测序。通过BLAST程序比对测序结果,并使用MEGA7.0软件绘制DZU-LM12的亲缘关系图来确定其菌属。
2.4代谢产物的分离纯化与鉴定
2.4.1代谢产物初步鉴定
首先,把菌株DZU-LM12接种在2mL BHI液体培养基(内含0.5mmol L-1大豆苷元)中在厌氧室内静置培养3天。用移液枪吸取发酵液100μL,将其置于干净无污染的EP管内,再迅速往里面添加入0.5mL乙酸乙酯进行萃取,并把离心转速设成每分钟8000r,时间为10min,结束后自EP管中取上清液0.5mL,再用旋转蒸发仪将剩余乙酸乙酯蒸发至干,收集到的残留物溶解于90μL甲醇中,透过0.45μm的有机滤膜过滤。取20μL样品经HPLC检测。2.4.2代谢产物的制备与分离纯化
为了获得大量的代谢物,把提前培养好的DZU-LM12的种子液取20mL接种在内含180mL BHI无菌培养基的三角瓶中,并用移液枪向瓶中加入45mmol L-1底物大豆苷元500μL,采用铝箔纸封住瓶口,在厌氧条件下培养3天。将发酵液用200mL的乙酸乙酯萃取3次,静置2h后进行分液,并用滤纸过滤乙酸乙酯,在60℃的真空下将萃取液进行浓缩蒸干。最后,把干燥后的代谢物溶解在100%的甲醇中,通过0.45μm微孔滤膜,采用HPLC色谱柱对代谢产物进行分离制备,并进行收集代谢产物峰。
先在60℃下用旋转真空蒸发仪将乙腈蒸干,再用180mL乙酸乙酯进行萃取,离心后取EP管上清液,最后将剩余的萃取剂乙酸乙酯蒸干,并溶解在1mL甲醇中,用高效液相分析柱检测所收集产物的纯度,并将该代谢产物储存于4℃冰箱用于后续分析。
2.4.3代谢产物的结构鉴定
本试验拟采用电喷雾电离质谱(ESI-MS)结合HPLC保留时间、紫外吸收光谱等进行代谢产物的鉴定。
3试验结果与分析
3.1大豆苷元转化菌株的分离与鉴定
经高效液相色谱(HPLC)检测,发现除了添加的大豆苷元底物在保留时间8.5min出现峰值外,在17.5min内又出现了一个新的物质峰,如图1所示。新物质峰是在大豆苷元的峰后出现,峰面积随着底物的消耗而增大,表明此峰为菌株DZU-LM12对大豆苷元的代谢产物。并通过对比发现此物质与本实验室保存的O-Dma标品的HPLC保留时间基本相吻合,如图1所示。对代谢物质的紫外吸收图谱进行分析,发现大豆苷元代谢产物在276nm、315nm处分别出现最大紫外吸收,这与本实验室保存的O-Dma标品紫外图谱中的最大紫外吸收相符合,如图1所示。因此,可以推测DZU-LM12能把底物大豆苷元开环转化成O-Dma。
对测序结果的序列进行编辑,并利用NCBI数据库进行BLAST比对。DZU-LM12的16SrDNA序列长度为1417bp,使用MAGA软件构建菌株系统进化树,如图2所示,通过亲缘关系图能够观察到DZU-LM12与所选菌株亲缘关系的远近,并发现DZU-LM12与Slackia exiguaATCC 700122(NR024952)相似度最高,相似性高达99.50%,结果可信度为100,可将其归为Slackia属的一个新物种,并将其命名为Slackia exigua DZU-LM12。
4.2大豆苷元转化产物的结构鉴定
本试验采用的离子源是ESI,源内裂解电压为150V,并将源温度设定在120℃,选择正离子模式下分析,其他条件也依次选用该产物测定的最优设定,并使用m/z单位进行质量选择。选取保留时间为2.788-3.386min的MS的解析结果如图3所示,标准大豆苷元的相对分子质量为254,质谱图上最大的峰的是目标分子的分子量,由ESI-MS对该峰的分析结果得知转化物质的分子量应当是258,显然与O-Dma的分子量相吻合,m/z为259.0868(图3)。与本实验室储存的O-Dma进行紫外图谱和HPLC保留时间的比对,并结合代谢产物质谱图分析能够确认代谢产物的分子量,故可以正确判定菌株DZU-LM12把底物大豆苷元转化成的代谢物质为O-Dma,其结构如图4所示。
Claims (5)
1.一种大豆异黄酮微生物转化菌株,其特征在于,所述转化菌株为菌株DZU-LM12,该菌株的保藏编号为CGMCC No.22779,保藏日期为2021年06月24日,保藏地址为北京市朝阳区北辰西路1号院3号,命名为Slackia exigua。
2.如权利要求1所述的大豆异黄酮微生物转化菌株在转化大豆异黄酮中的应用。
3.如权利要求1所述的大豆异黄酮微生物转化菌株在制备O-Dma中的应用。
4.如权利要求1所述的大豆异黄酮微生物转化菌株在制备用于转化大豆异黄酮制剂中的应用。
5.含有如权利要求1所述的大豆异黄酮微生物转化菌株的制剂在O-Dma中的应用。
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