CN117051080B - 一种微生态活菌制品丁酸代谢通路激活剂的筛选方法和应用 - Google Patents
一种微生态活菌制品丁酸代谢通路激活剂的筛选方法和应用 Download PDFInfo
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Abstract
本发明公开了一种微生态活菌制品丁酸代谢通路激活剂的筛选方法和应用，属于微生态活菌制品领域。提取完整的微生态活菌制品基因组，并对基因序列拼接及序列校正，获得环状基因组，进行基因注释和酶功能注释，经过基因注释的基因组和酶功能注释与代谢通路数据库比对，对丁酸代谢通路进行分析，获得完整的丁酸代谢通路，确认微生态活菌制品的代谢通路覆盖度，确认相关基因和化合物，即为微生态活菌制品丁酸代谢通路激活剂。本发明通过生物信息学对一种生物标志物代谢通路进行代谢通路的完整性分析，确定关键标志物和基因，采用补加方式补全完善通路，使现有微生态活菌产品发挥更多的益生作用，起到更好的临床治疗效果。
Description
技术领域
本发明属于微生态活菌制品技术领域，具体涉及一种微生态活菌制品丁酸代谢通路激活剂的筛选方法和应用。
背景技术
微生态活菌制品系由人体内正常菌群成员或具有促进正常菌群生长和活性作用的无害外籍细菌经培养，收集菌体、干燥成菌粉后，加入适宜辅料混合制成。微生态活菌制品纳入药品管理，我国最早的微生态活菌制品研发于上世纪50年代，目前中国药典收载了15种可用于生产微生态活菌制品的菌株。微生态活菌制品安全性好，疗效确切，在临床上广泛使用。微生态活菌的作用机理复杂，其中，有益的代谢产物发挥了重要作用，具有改善肠道环境、抑制有害菌、促进有益菌生长等作用。微生态活菌菌株有多重代谢通路可以产生有益代谢产物，但是，由于筛选方式方法的局限性，现有用于商业生产的菌株大多基于最优代谢法则原则，有些通路的部分代谢所需化合物缺失，代谢通路未完全畅通。因此，部分限制了菌株益生作用的发挥。
丁酸又名酪酸，是一种短链脂肪酸，它是人肠道上皮细胞的主要营养物质，在小肠中，丁酸是结肠细胞正常生长发育的重要物质，是快速分化细胞的能量源。丁酸用于补充能量可被直接吸收，不需借助胆汁盐的作用。在体内外的研究中已经表明，丁酸对结肠上皮细胞的生长发育具有最显著的作用。研究表明，丁酸可促使小肠吸收面积最大化。此外，体外培养人的大肠黏膜细胞，在没有循环神经因素作用下丁酸钠可直接刺激上皮细胞增殖。另外，以丁酸为代表的短链脂肪酸可刺激结肠吸收钠，从而满足机体贮存水分的需要，维持机体稳态。丁酸还能通过影响肠道pH值进而影响消化酶的活性。丁酸可调节胃肠道酸度、肠道菌群平衡，刺激结肠有益微生物的生长，抑制不耐酸的弯曲杆菌、沙门氏菌、大肠杆菌等有害菌，肠道菌群平衡得到改善。人体内超过95%的丁酸在结肠内产生和吸收，一定水平的丁酸能够使结肠细胞保持稳定，从而可防止或抑制癌变，调节肠道菌群失调和治疗肠易激综合征、抗生素相关性肠炎、急慢性腹泻等疾病。丁酸属于脂肪酸，当通过外界食物摄取时，在其到达大肠发挥作用之前就基本被吸收，因此无法在下消化道中发挥益生作用；而丁酸也是大肠中产丁酸细菌的主要代谢物，因此，大肠中产丁酸微生物对机体健康意义重大。
目前尚未有针对为生态活菌制品中的菌株代谢特点，加入代谢通路激活剂，打通特定代谢通路，产生更多益生代谢产物，增强微生态活菌制品益生功能的产品。代谢通路调节剂填补了市场空白，会对微生态活菌制品市场的发展产生积极的推动作用。
发明内容
针对现有技术中微生态活菌制品代谢通路未完全打开、限制了菌株益生作用的发挥的问题，本发明提供了一种微生态活菌制品丁酸代谢通路激活剂的筛选方法和应用，通过生物信息学对一种生物标志物代谢通路进行代谢通路的完整性分析，确定关键标志物和基因，采用补加方式补全完善通路，使现有微生态活菌产品发挥更多的益生效果，起到更好的临床作用效果。
本发明通过以下技术方案实现：
一种微生态活菌制品丁酸代谢通路激活剂的筛选方法，包括以下步骤：
（1）基因组提取：对微生态活菌制品的菌株进行培养、纯化，提取完整基因组；
（2）基因序列拼接及序列校正：将基因组测序得到的碱基序列通过无参序列拼接，组成完整的基因组，然后采用序列矫正对测序产生的低质量环化共有序列进行纠错，使所得序列能够完整正确反映目标菌株的实际遗传序列信息，拼接结果为环状基因组，无碎片；
（3）基因注释和酶功能注释；
（4）代谢通路分析：经过基因注释的基因组和酶功能注释与代谢通路数据库比对，对丁酸代谢通路进行分析，获得完整的丁酸代谢通路；丁酸代谢通路的相关基因为丙酮酸合成酶基因（EC 1.2.7.1）、乙酰辅酶A乙酰转移酶基因（EC 2.3.1.9）、羟丁酸辅酶A脱氢酶基因（EC 1.1.1.35）、3-羟基丁基-CoA脱水酶基因（EC 4.2.1.150）、丁酰基辅酶A脱氢酶基因（EC 1.3.8.1）、磷酸转丁酸酶基因（EC 2.3.1.19）以及丁酸激酶基因（EC 2.7.2.7）；
（5）根据步骤（4）的比对结果确认微生态活菌制品的代谢通路覆盖度，确认相关基因和化合物，即为微生态活菌制品丁酸代谢通路激活剂。
进一步地，步骤（1）采用磁珠法提取完整基因组。
进一步地，步骤（2）中以二代及三代测序结果互为参考，采用从头测序方法，原始数据通过FastQC进行质量评估，通过Trimmomatic对测序数据进行质量剪切，使用SPAdes拼接二代测序数据，采用GapFiller对拼接得到的contig补GAP，利用PrInSeS-G修正拼接过程中的剪辑错误及小片段的插入缺失。
进一步地，步骤（3）中采用Uniref作为基因注释及酶功能注释的数据库。
进一步地，步骤（4）中的代谢通路数据库为京都基因和基因组百科全书和MetaCyc。
进一步地，步骤（4）中获得的丁酸代谢通路的相关化合物包括丙酮酸盐、氧化铁氧还蛋白[铁硫]簇、CoA、乙酰CoA、还原铁氧还蛋白[铁硫]、乙酰乙酰CoA、3-羟基丁酰基-CoA、3-羟基丁酰基-CoA、巴豆酰CoA、丁酰基CoA、氧化的电子转移黄素蛋白+H+、还原的电子转移黄素蛋白、丁酰基磷酸。
本发明中，所述的微生态活菌制品丁酸代谢通路激活剂的筛选方法在筛选微生态活菌制品丁酸代谢通路激活剂中的应用。
进一步地，所述的微生态活菌制品蜡样芽孢杆菌活菌胶囊、蜡样芽孢杆菌活菌片、双歧杆菌四联活菌制剂，或其他含有蜡样芽孢杆菌的微生态活菌制品。
进一步地，蜡样芽孢杆菌活菌胶囊、蜡样芽孢杆菌活菌片、双歧杆菌四联活菌制剂，或其他含有蜡样芽孢杆菌的微生态活菌制品的丁酸代谢通路激活剂为3-羟基丁酰辅酶A脱氢酶和/或其产物巴豆酰辅酶A。
本发明取得的有益效果为：
本发明通过生物信息学对一种生物标志物代谢通路进行代谢通路的完整性分析，确定关键标志物和基因，采用补加方式补全完善通路，使产品功效得到提升，针对其中一种未打通的有益代谢通路，通过增加调节剂，使通路打开，从而强化了其益生效果。
附图说明
图1为丁酸代谢全局图及3-羟基丁酰辅酶A脱氢酶在其中的位置；
图2为双歧杆菌四联活菌中不同菌株的丁酸途径基因丰度图；
图3为丁酸对照品溶液色谱图；
图4为巴豆酰辅酶A对照品溶液色谱图；
图5为空白培养基对照色谱图；
图6为粪肠球菌未添加激活剂培养物色谱图；
图7为嗜酸乳杆菌未添加激活剂培养物色谱图；
图8为婴儿双歧杆菌未添加激活剂培养物色谱图；
图9为蜡样芽胞杆菌未添加激活剂培养物色谱图；
图10为添加巴豆酰辅酶A的空白培养基对照色谱图；
图11为粪肠球菌添加巴豆酰辅酶A培养物色谱图；
图12为嗜酸乳杆菌添加巴豆酰辅酶A培养物色谱图；
图13为双歧杆菌添加巴豆酰辅酶A培养物色谱图；
图14为蜡样芽胞杆菌添加巴豆酰辅酶A培养物色谱图。
具体实施方式
为了对本发明进行更进一步的详细描述，给出以下具体实施范例，但仅用于阐明本发明，使步骤更加清晰，而不是为了限制本发明的应用范围。
实施例1 一种微生态活菌制品丁酸代谢通路激活剂的筛选方法，具体为：
（1）基因组提取：对微生态活菌制品的菌株进行培养、纯化，采用磁珠法提取完整基因组；
（2）基因序列拼接及序列校正：将基因组测序得到的碱基序列通过无参序列拼接，组成完整的基因组，然后采用序列矫正对测序产生的低质量环化共有序列进行纠错，使所得序列能够完整正确反映目标菌株的实际遗传序列信息，拼接结果为环状基因组，无碎片；
测序的原始数据通过FastQC进行质量评估，通过Trimmomatic对测序数据进行质量剪切，得到相对准确的有效数据，使用SPAdes拼接二代测序数据，采用GapFiller对拼接得到的contig补GAP，利用PrInSeS-G修正拼接过程中的剪辑错误及小片段的插入缺失。基因组测序得到散在的reads通过无参序列拼接，即从头测序（Denovo Genome Sequencing，Denovo），组装成完整的基因组，三代及二代测序结果互为参考可有效避免“一致性序列”不足，之后采用序列矫正（Polish）对测序产生的低质量环化共有序列（Circular ConsensusSequencing，CCS）等进行纠错，所得序列能够完整正确反映目标菌株的实际遗传序列信息，拼接结果为环状基因组，无碎片。基于参考序列拼接方法获得的序列称为“一致性序列”，顾名思义，就是与参考序列保持一致。但这种方法得到的序列并不是真实存在的基因组序列，主要有以下原因：1）已发表出来的参考序列并不是100%准确的，如果参考序列中累积的错误，使用该方法，错误会被逐渐累积；2）该方法受参考序列影响很大，选择不同的参考序列会得到不同的一致性序列；3）如果参考序列与测序物种之间存在较大的结构变异，则无法突出这些结构变异。因此，本专利未使用参考基因组测序拼接，而使用从头测序（denovo）。
（3）基因注释和酶功能注释；基因注释及酶功能注释采用Uniref（https://ftp.uniprot.org/pub/databases/uniprot/uniref/，2023-05-03）作为数据库，本专利采用Uniref而未采用GO（Gene Ontology）、NR（NCBI non-redundant protein sequences）以及COG（Clusters of Orthologous Groups of proteins）等注释数据库的原因是UniRef是目前最全面的非冗余蛋白质序列数据库。
（4）代谢通路分析：经过基因注释的基因组和酶功能注释与代谢通路数据库比对，对丁酸代谢通路进行分析，获得完整的丁酸代谢通路；代谢通路数据库为京都基因和基因组百科全书（KEGG）和MetaCyc，使用的数据库版本为MetaCyc Pathway Database（2022年05月）。在通路的完整度上，KEGG更加注重将所有物种的通路汇总到一张图上，而MetaCyc更加注重不同物种通路的差异化。相较于MetaCyc，KEGG通路覆盖更全面，而MetaCyc相对会更特异，两者MetaCyc可以互为补充。
通过代谢通路分析，丁酸代谢全局图如图1所示：
通过对丁酸代谢通路进行分析，丁酸代谢通路涉及的相关基因为由丙酮酸合成酶（pfoA）基因（EC 1.2.7.1，KEGG：R01196）、乙酰辅酶A乙酰转移酶（thl）基因（EC 2.3.1.9，KEGG：R00238）、羟丁酸辅酶A脱氢酶（hbd）基因（EC 1.1.1.35，KEGG：R01975）、3-羟基丁基-CoA脱氢酶（crt）基因（EC 4.2.1.150，KEGG：R03026）、丁酰基辅酶A脱氢酶（NAD+，铁氧还蛋白）（etfA）基因（EC 1.3.8.1，KEGG：R01178）、磷酸转丁酸酶（ptb）基因（EC 2.3.1.19，KEGG：R01174）以及丁酸激酶（buk1）基因（EC 2.7.2.7，KEGG：R01688）7种核心基因用以实现微生物体内合成丁酸；
丁酸代谢通路涉及的化合物包括丙酮酸盐、氧化铁氧还蛋白[铁硫]簇、CoA、乙酰CoA、还原铁氧还蛋白[铁硫]、乙酰乙酰CoA、3-羟基丁酰基-CoA、3-羟基丁酰基-CoA、巴豆酰CoA、丁酰基CoA、氧化的电子转移黄素蛋白+H+、还原的电子转移黄素蛋白、丁酰基磷酸等。
丁酸代谢通路涉及涉及的酶促化学反应包括：1）丙酮酸盐+2氧化铁氧还蛋白[铁硫]簇+辅酶A↔乙酰CoA+CO2+2还原铁氧还蛋白[铁硫]簇+H+；2）乙酰CoA↔乙酰乙酰CoA+CoA；3）3-羟基丁酰基-CoA+NAD+↔乙酰乙酰CoA+NADH+H+；4）3-羟基丁酰基-CoA↔巴豆酰基CoA+H2O；5）丁酰基CoA+氧化的电子转移黄素蛋白+H+↔巴豆酰CoA+还原的电子转移黄素蛋白；6）丁酰基CoA+磷酸盐↔丁酰基磷酸+CoA；7）丁酸盐+ATP↔丁酰基磷酸+ADP。
丁酸代谢通路始于乙酰辅酶A C-乙酰转移酶，它催化两个乙酰辅酶A分子缩合形成乙酰乙酰辅酶A；乙酰基辅酶A通过NAD依赖性的（S）-3-羟基丁基辅酶A脱氢酶转化为（S）-3-羟基丁酰基辅酶A；3-羟基丁基-CoA脱水酶和丁酰基-CoA脱氢酶（NAD+，铁氧还蛋白）继续形成巴豆酰基CoA和丁酰基CoA；丁酰基辅酶A向丁酸的最后两步转化是细胞产生ATP的重要能量来源。这种两步转化是由磷酸反式丁酸酶和丁酸激酶催化的。在正常发酵条件下，反应仅在丁酸形成的方向上进行。
（5）根据步骤（4）的比对结果确认微生态活菌制品的代谢通路覆盖度，确认相关基因和化合物，即为微生态活菌制品丁酸代谢通路激活剂。
实施例2 双歧杆菌四联活菌制剂丁酸代谢通路激活剂的筛选：
（1）无菌称取3.0g成品（双歧杆菌四联活菌片，思连康），加入27ml 0.9%无菌氯化钠溶液或其他适宜稀释液中，振荡混匀，必要时可以加入无菌玻璃珠。充分混匀后即为10-1稀释液，取10-1稀释液1ml至9ml 0.9%无菌氯化钠溶液或其他适宜稀释液中，即为10-2稀释液，用相同方法10倍系列稀释至10-3和10-4稀释液，取10-3稀释液100μl，滴入BA琼脂平皿上，平行制备3份；取10-4稀释液100μl，分别滴入改良TPY琼脂培养基、改良MRS琼脂培养基、EC琼脂培养基平皿上，每种培养基平行制备3份，以无菌涂布棒涂匀。观察分析。进行多次菌株纯化。采用磁珠法提取完整基因组；
（2）根据实施例1步骤（4）丁酸代谢通路分析结果，对双歧杆菌四联活菌制剂中的四个菌株进行所述代谢通路分析，双歧杆菌四联活菌制剂四种菌株丁酸代谢通路线管基因丰度图如图2所示（白色基因位置表示基因缺失，灰度越深，基因丰度越高），结果显示各菌株代谢通路均存在差异，双歧杆菌四联活菌制剂四种菌株丁酸代谢通路覆盖率如下表1所示：
表1
通过对四个菌株的分析可知，蜡样芽孢杆菌的通路相关基因覆盖度达0.857，仅缺失编码EC 4.2.1.150的基因，该基因为3-羟基丁酰辅酶A脱氢酶（3-hydroxybutyryl-CoAdehydratase），其底物为3-羟异丁酰辅酶A（(S)-3-hydroxybutanoyl-CoA），产物为巴豆酰辅酶A（crotonyl-CoA）基因，3-羟基丁酰辅酶A在丁酸代谢全局图中的位置如图1圆形标出的位置所示。可通过添加3-羟基丁酰辅酶A脱氢酶和/或其产物巴豆酰辅酶A（crotonyl-CoA）促进丁酸合成。
实施例3 丁酸代谢通路激活剂3-羟基丁酰辅酶A脱氢酶和其产物巴豆酰辅酶A（crotonyl-CoA）促进丁酸合成的效果验证：
（1）HPLC法测定丁酸的方法：色谱条件：十八烷基硅胶键合相（镶嵌极性基团）为填充剂（色谱柱为Waters® XBridge Shield RP18（5 μm，4.6 mm×250 mm）），流动相为乙腈加入0.15%的磷酸溶液，流动相中乙腈与磷酸溶液的体积比为25:75，所述乙腈为色谱纯，流速为1.0ml/min，检测器为紫外检测器，波长为206nm，柱温为30℃，进样量为10 μL，所有进样溶液均摇匀后过0 .45μm微孔滤膜处理后进样。
（2）标准对照品溶液的制备步骤为：精密称定丁酸钠对照品约10mg，置于10ml容量瓶中，用0.1%的磷酸溶液定容，摇匀后过0.45μm微孔滤膜，制成浓度为1 mg/ml的溶液；精密称定巴豆酰辅酶A对照品约100mg，置于10ml容量瓶中，用0.1%的磷酸溶液定容，摇匀后过0.45μm微孔滤膜，制成浓度为10 mg/ml的溶液；上述两种溶液作为对照品溶液，将两种对照品溶液分别注入高效液相色谱仪进行检测，其中丁酸对照品溶液的色谱图如图3所示，巴豆酰辅酶A对照品溶液的色谱图如图4所示。
（3）对照培养物溶液的制备：选取胰酪大豆胨液体培养基等适宜的培养基，分装成100ml每瓶湿热灭菌，配置完成后，在4瓶培养基中分别接入4株不同菌株（蜡样芽胞杆菌、粪肠球菌、嗜酸乳杆菌和婴儿双歧杆菌），36℃，180rpm培养18小时，其中婴儿双歧杆菌厌氧培养，培养结束后8000rpm离心10分钟，取上清作为对照培养物溶液，另外取未加入微生物的空白培养基作为培养基对照。
将对照培养物溶液和空白培养基分别注入高效液相色谱仪进行检测，其中空白培养基的色谱图如图5所示，粪肠球菌、嗜酸乳杆菌、婴儿双歧杆菌和蜡样芽胞杆菌未添加激活剂（巴豆酰辅酶A）的对照培养物溶液的色谱图分别如图6、图7、图8和图9所示。
（4）供试品溶液的制备：选取胰酪大豆胨液体培养基等适宜的培养基，分装成100ml每瓶湿热灭菌，灭菌后加入0.45μm微孔滤膜处理的上述巴豆酰辅酶A对照品溶液2ml，使培养基中巴豆酰辅酶A终浓度约为200μg/ml，配置完成后，在4瓶培养基中分别接入4株不同菌株（蜡样芽胞杆菌、粪肠球菌、嗜酸乳杆菌和婴儿双歧杆菌），36℃，180rpm培养18小时，其中婴儿双歧杆菌厌氧培养，培养结束后8000rpm离心10分钟，取上清作为供试品溶液，另外取未加入微生物的含巴豆酰辅酶A的空白改良培养基作为培养物对照溶液。以上所有溶液均摇匀后过0 .45μm微孔滤膜处理后进样。
将供试品溶液和培养物对照溶液分别注入高效液相色谱仪进行检测，其中培养物对照溶液的色谱图如图10所示，粪肠球菌、嗜酸乳杆菌、婴儿双歧杆菌和蜡样芽胞杆菌添加激活剂（巴豆酰辅酶A）的供试品溶液的色谱图分别如图11、图12、图13和图14所示。
经过实验验证（图3-14），体外培养蜡样芽孢杆菌至指数期过程中补加3-羟基丁酰辅酶A脱氢酶和巴豆酰辅酶A，可以检测到丁酸分泌水平的提高，表明羟基丁酰辅酶A脱氢酶和巴豆酰辅酶A可作为蜡样芽孢杆菌菌剂或双歧杆菌四联活菌制剂丁酸通路激活剂。
Claims (1)
1.巴豆酰辅酶A在激活微生态活菌的丁酸代谢通路中的应用，其特征在于，所述的微生态活菌为思连康双歧杆菌四联活菌片中的蜡样芽孢杆菌，通过在培养基中添加巴豆酰辅酶A激活蜡样芽孢杆菌的代谢通路。
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