CN114438138A - 用生孢梭菌制备的代谢组合物及其生产方法、检测方法和应用 - Google Patents

用生孢梭菌制备的代谢组合物及其生产方法、检测方法和应用 Download PDF

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CN114438138A
CN114438138A CN202210173539.1A CN202210173539A CN114438138A CN 114438138 A CN114438138 A CN 114438138A CN 202210173539 A CN202210173539 A CN 202210173539A CN 114438138 A CN114438138 A CN 114438138A
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clostridium
metabolic composition
indole
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齐仁立
杜蕾
邱小宇
王敬
王琪
刘作华
杨飞云
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Chongqing Academy of Animal Sciences
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Abstract

本发明属于微生物领域，具体涉及用生孢梭菌制备的代谢组合物及其生产方法、检测方法和应用。本发明公开了一种用Clostridium sporogenes制备的代谢组合物，所述代谢组合物由吲哚丙酸、氢化肉桂酸、琥珀酸、吲哚丙烯酸、色胺、色氨酸、N‑乙酰苯丙氨酸、脱氢莽草酸、吲哚乙酰胺和吲哚乙酸按13‑16：2‑6：3‑6：1‑2：0.5‑1.5：0.2‑1：0.1‑0.4：0.1‑0.3：0.1‑0.3：0.1‑0.3的平均峰面积比组成。本发明还公开了上述代谢组合物的生产以及检测方法。所述生产方法为用Clostridium sporogenes发酵芳香族氨基酸得到代谢组合物。所述检测方法采用液相色谱串联质谱法。本发明还通过细菌培养、体外发酵、小鼠体内和肌管细胞体外试验，结合代谢组和转录组等多组学技术揭示Clostridium sporogenes及其代谢产物对动物肌肉发育的促进作用和炎症发生的缓解作用。

Description

用生孢梭菌制备的代谢组合物及其生产方法、检测方法和 应用
技术领域
本发明属于微生物领域，具体涉及用生孢梭菌制备的代谢组合物及其生产方法、检测方法和应用。
背景技术
肠道微生物产生大量的代谢产物，通过“微生物-肠-肌肉轴”交互途径影响宿主的肌肉组织发育和健康。炎症性肠病(inflammatory bowel disease，IBD)是人和动物常见的一种慢性胃肠疾病，这种慢性炎症状态通常会引起患者肌肉炎症损伤和肌肉萎缩等病理症状。据报道，42％的IBD患者会出现肌肉减少和炎症等症状。这种炎症状态通常是由宿主微生物紊乱引起的肠道屏障损伤，循环脂多糖(lipopolysaccharide，LPS)增加以及蛋白质和抗氧化营养物质摄入不足导致。
Clostridium sporogenes是一种肠道共生菌，它可以特异性分解芳香族氨基酸产生大量抗炎性吲哚类物质。吲哚丙酸(indole popionic acid，IPA)是其中含量最丰富的物质，大量报道显示IPA在肠道、肝脏和肺组织中均具有较好的抗炎作用。
发明内容
本发明拟通过细菌培养、体外发酵、小鼠体内和肌管细胞体外试验，结合代谢组和转录组等多组学技术揭示Clostridium sporogenes及其功能性代谢产物对动物肌肉发育的促进作用和炎症发生的缓解作用，并建立优化一种通过微生物代谢来减轻肌肉炎症的方法，为保障人和动物健康生长和改善畜禽肉品质提供帮助。
本发明的目的之一，在于提供一种用Clostridium sporogenes制备的代谢组合物，该代谢组合物能有效缓解动物机体炎症的发生以及促进动物体肌肉的发育。
为实现上述目的，本发明采取以下技术方案：
所述代谢组合物由吲哚丙酸、氢化肉桂酸、琥珀酸、吲哚丙烯酸、色胺、色氨酸、N-乙酰苯丙氨酸、脱氢莽草酸、吲哚乙酰胺和吲哚乙酸组成。
进一步，所述代谢组合物由吲哚丙酸、氢化肉桂酸、琥珀酸、吲哚丙烯酸、色胺、色氨酸、N-乙酰苯丙氨酸、脱氢莽草酸、吲哚乙酰胺和吲哚乙酸按照13-16：2-6：3-6：1-2：0.5-1.5：0.2-1：0.1-0.4：0.1-0.3：0.1-0.3：0.1-0.3的平均峰面积比组成。
本发明的目的之二，在于提供一种上述代谢组合物的生产方法，该方法为代谢组合物的生产提供了新思路。
为实现上述目的，本发明采取以下技术方案：
所述代谢组合物的生产方法为用Clostridium sporogenes发酵芳香族氨基酸得到代谢组合物。
进一步，所述Clostridium sporogenes复苏所用培养基为添加了芳香族氨基酸的液体硫乙醇酸盐培养基。
进一步，所述Clostridium sporogenes活化所用培养基为强化梭菌固体培养基。
进一步，所述Clostridium sporogenes的菌株号为
Figure BDA0003519531770000021
15579TM。
本发明的目的之三，在于提供一种上述代谢组合物的检测方法，该方法为代谢组合物的检测提供了新思路。
为实现上述目的，本发明采取以下技术方案：
所述检测方法采用液相色谱串联质谱法，其中液相色谱条件：用含体积分数为0.1％甲酸的水溶液作为流动相A，用含体积分数为0.1％的甲酸的乙腈溶液作为流动相B，按下表进行梯度洗脱，流动相流速为0.4ml/min，色谱柱柱温为35℃,色谱柱为WatersACQUITY UPLC HSS T3 C18柱1.8m×2.1mm×100mm。
Figure BDA0003519531770000031
进一步，所述检测方法的质谱条件为：电喷雾离子源(electrosprayionization，ESI)温度500℃，质谱电压5500V(阳离子)，-4500V(阴离子)，离子源气体Ⅰ(GSⅠ)55psi，气体II(GS II)60psi，气帘气(curtain,CUR)25psi，碰撞诱导电离(collision-activateddissociation，CAD)，参数设置为高。在三重四极杆(Qtrap)中，每个离子对是根据优化的去簇电压(declustering potential，DP)和碰撞能(collision energy，CE)进行扫描检测。
进一步，所述检测方法的检测对象为Clostridium sporogenes厌氧培养12-24h的发酵液。
进一步，所述发酵液的pH值为6.0±0.5。
进一步，所述发酵液的OD600值为2.0±0.5。
上述代谢组合物在制备肌细胞增殖剂中的应用。
本发明的有益效果在于：
1)提供了一种用Clostridium sporogenes制备的代谢组合物。
2)提供了一种生产上述代谢组合物的方法，此方法创新性应用Clostridiumsporogenes发酵芳香族氨基酸产生有益代谢物质。
3)提供了一种检测上述代谢组合物的方法，此方法可以同时对多达十种物质进行定性以及定量检测。
4)本发明还通过细菌培养、体外发酵、小鼠体内和肌管细胞体外试验并结合代谢组和转录组等多组学技术，揭示Clostridium sporogenes及其代谢产物对动物体肌肉发育的促进作用和炎症发生的缓解作用。
附图说明
图1为Clostridium sporogenes的生长和产酸曲线；
图2为Clostridium sporogenes发酵液内的芳香族氨基酸代谢物组成；
图3为Clostridium sporogenes处理对小鼠体重的影响；
图4为Clostridium sporogenes处理对小鼠骨骼肌组织重量的影响；
图5为Clostridium sporogenes处理对小鼠骨骼肌肌纤维发育的影响；
图6为Clostridium sporogenes处理对小鼠血液代谢途径的影响；
图7为Clostridium sporogenes处理对小鼠血液芳香族氨基酸代谢物的影响；
图8为Clostridium sporogenes处理对小鼠肌肉组织代谢的影响；
图9为Clostridium sporogenes处理对小鼠体内促炎因子表达的影响；
图10为肌肉内促炎因子表达与色氨酸代谢物的相关性；
图11为Clostridium sporogenes和IPA减轻LPS诱导的小鼠体重下降(试验第8天小鼠进行LPS腹腔注射，#代表与LPS组比较)；
图12为Clostridium sporogenes和IPA减轻LPS诱导的肌肉组织重量下降(*代表与对照组比较，#代表与LPS组比较，下同)；
图13为Clostridium sporogenes和IPA减轻LPS对小鼠肌肉力量的影响；
图14为Clostridium sporogenes和IPA降低小鼠体内促炎因子水平；
图15为IPA增加小鼠肌肉前体细胞C2C12得增殖活力；
图16为吲哚丙酸抑制脂多糖(LPS)诱导的小鼠肌管细胞炎症；
图17为吲哚丙酸通过miR-26a-2-3p(简称miR-26A)缓解小鼠肌细胞炎症；
图18为吲哚丙酸通过靶向miR-26a-2-3p抑制IL-1β的表达。
具体实施方式
下面将结合具体的实施例对本发明的技术方案进行更进一步地清楚、完整地描述。显然，所描述的实施例仅仅是本发明的一部分实施例，而不是全部实施例。因此，基于本发明中的实施例，本领域技术人员在没有付出创造性劳动前提下所获得的其他所有实施例都属于本发明的保护范围。
实施例1.Clostridium sporogenes的培养与代谢芳香族氨基酸能力的检测
Clostridium sporogenes的培养：本试验所用Clostridium sporogenes菌株是购买于ATCC官网的标准菌株(菌株号：
Figure BDA0003519531770000051
15579TM)，采用比浊法测定Clostridiumsporogenes的生长曲线。活化后的菌液，按1％的接种量分别接种于3个含有30mL厌氧微生物培养基FT的三角锥形瓶内，37℃条件下进行厌氧培养。间隔3h取出3mL培养液进行菌液OD值测定，结果显示15-24h为Clostridium sporogenes生长的平台期，如图1所示。
Clostridium sporogenes代谢芳香族氨基酸能力的检测：取Clostridiumsporogenes对数生长期末期(厌氧培养12h后)的发酵液，3000rpm离心10分钟得到菌液上清。接着通过液相色谱串联质谱(LC-MS/MS)准确定性和定量Clostridium sporogenes发酵产生的芳香族氨基酸代谢物，其中包含吲哚丙酸、氢化肉桂酸、琥珀酸、吲哚丙烯酸、色胺、色氨酸、N-乙酰苯丙氨酸、脱氢莽草酸、吲哚乙酰胺和吲哚乙酸等10种物质，它们的含量分别为：2.72×108±7.55×106、6.53×107±2.08×106、7.73×107±2.65×106、3.14×107±5.15×105、1.67×107±8.33×105、1.11×107±5.06×105、4.13×106±1.47×105、1.65×106±4.76×104、1.63×106±2.96×104、1.45×106±3.25×104平均峰面积。取300μL的甲醇溶液加入50μL的上清液样品进行萃取后，再用LC-ESI-MS/MS系统分析提取的150μL上清液。测定结果显示，Clostridium sporogenes发酵液内含有大量的芳香族氨基酸代谢物，尤其是色氨酸代谢物吲哚丙酸(IPA)，含量高达2×108平均峰面积，如图2所示。
实施例2.Clostridium sporogenes处理对小鼠体重和肌肉发育的影响
小鼠灌喂试验选取20只6周龄雄性C57BL/6小鼠，分为对照组和Clostridiumsporogenes处理组。Clostridium sporogenes厌氧培养12h后，用于处理试验组小鼠，每只小鼠每次灌喂200μL菌液，活菌数为1×108CFU/mL，间隔灌喂，每周2次，试验持续42天结束。对照组小鼠使用生理盐水进行相同处理。最终，试验结果显示，Clostridium sporogenes处理小鼠35天后，小鼠体重开始显著增加，如图3所示。
试验结束时，CO2麻醉后处死小鼠。分离小鼠的股四头肌、腓肠肌和胫骨前肌等骨骼肌组织，称量组织重量后分别放入中性甲醛固定液内固定和液氮内进行保存，称重结果表明Clostridium sporogenes处理显著增加股四头肌重量，如图4所示。对甲醛固定的股四头肌进行切片和HE染色，结果显示Clostridium sporogenes处理显著增加了骨骼肌纤维直径和纤维面积，如图5所示。
实施例3.Clostridium sporogenes处理对小鼠代谢和炎性水平的影响
Clostridium sporogenes处理小鼠42天后，麻醉小鼠后，通过眼眶静脉采集小鼠血液。血液在室温静置2小时后，3000rpm，4℃离心15分钟收集血清。血清经甲醇萃取后用实施例1所述体系进行代谢物检测。差异代谢物的KEGG分析结果显示，Clostridiumsporogenes处理后显著改变了小鼠体内苯丙氨酸、酪氨酸和色氨酸等芳香族氨基酸的合成代谢，如图6所示。与对照组相比，试验组小鼠血液内莽草酸(吲哚丙酸合成前体)、吲哚乙酸和琥珀酸的含量显著增加，如图7所示。小鼠肌肉内的色氨酸代谢物检测结果也显示，Clostridium sporogenes处理显著增加了小鼠肌肉内吲哚丙酸和吲哚乙酸的含量，如图8所示。
同时，本发明通过实时荧光定量PCR对小鼠肌肉组织内的促炎因子标志物CCL2、CCL5、IL-1β、TNFα和NLRP3的mRNA表达水平进行检测。结果显示，与对照组相比，Clostridium sporogenes处理组小鼠肌肉内促炎因子表达水平显著下调，如图9所示。将小鼠肌肉内色氨酸代谢物和促炎因子mRNA表达量进行相关性分析，结果表明IPA与促炎因子表达显著负相关，如图10所示。
实施例4.Clostridium sporogenes及吲哚丙酸对脂多糖诱导的肌肉炎症的缓解作用
试验选取40只C57小鼠，预饲1周后，随机分为4组，每组10只，试验为期11天。
1)生理盐水对照组：小鼠每天灌喂生理盐水200ul，连续7天，最后一次灌喂后1h再次腹腔注射200ul生理盐水；
2)生理盐水+LPS组：小鼠每天灌喂生理盐水200ul，连续7天，最后一次灌喂后1h进行腹腔注射1mg/kg.BW LPS；
3)IPA+LPS组：小鼠每天灌喂30mg/kg.BW IPA 200uL，连续7天，最后一次灌喂后1h进行腹腔注射1mg/kg.BW LPS；
4)CS+LPS组：小鼠每天灌喂1×108CFUs/mL Clostridium sporogenes发酵液，连续处理7天，最后一次灌喂后1h进行腹腔注射1mg/kg.BW LPS。
所有小鼠在最后一次处理后4h取尾静脉血检测炎性因子TNFα和IL1β的表达。24h后称量小鼠体重，观察小鼠腹泻状况。注射前和注射后48h测量小鼠前肢握力，每天称量小鼠体重。于第11天麻醉处死小鼠，采集小鼠股四头肌、胫骨前肌和腓肠肌，称重，中性甲醛固定，冻存-80℃待测。
Clostridium sporogenes和IPA减轻脂多糖对小鼠骨骼肌组织发育的影响：小鼠体重结果表明LPS处理显著降低小鼠体重，而IPA和Clostridium sporogenes可以不同程度缓解这种影响，如图11所示。小鼠骨骼肌组织称重结果显示，LPS显著降低小鼠股四头肌重量，并且Clostridium sporogenes处理可以减轻LPS造成的这种影响，如图12所示。小鼠抓力测定结果显示，LPS处理显著降低了小鼠四肢力量，并且IPA+LPS处理组小鼠在2天后肌肉力量显著大于LPS处理组，如图13所示。上述结果表明IPA在肌肉修复过程中扮演着重要角色。
Clostridium sporogenes和IPA减轻脂多糖对小鼠体内炎症水平的影响：小鼠血清中促炎因子TNFα的检测结果表明，IPA预处理小鼠可以有效预防LPS引起的促炎因子水平的升高，但Clostridium sporogenes处理对机体内炎症水平的缓解效果不显著，如图14所示。
实施例5.IPA缓解脂多糖诱导的肌细胞炎症的作用机制
IPA增加小鼠肌肉前体细胞C2C12增殖活力：将C2C12细胞按10000/孔的接种量接种于96孔板，使细胞粘附，然后用不同浓度的IPA(0.1、0.25和0.5mM)孵育24小时。最后，使用cell Counting Kit-8(碧云天)，参照试剂盒说明书检测细胞活力。检测结果显示低浓度IPA(0.1mM)显著增加了肌细胞增殖活力，如图15所示。
IPA抑制TLR4/MyD88/NF-κB炎症信号通路减轻肌管细胞炎症：C2C12前体细胞购自American Type Culture Collection(ATCC官网)，并用加入10％热灭活胎牛血清、青霉素(100U/mL)和链霉素(100μg/mL)的DMEM培养基进行培养。细胞在37℃,CO2浓度为5％的环境中生长，培养基每24h更换一次。C2C12细胞培养至约80％的融合状态后，将培养基更换为加入2％(v/v)马血清的DMEM分化培养基，开始向肌管细胞分化。C2C12细胞在6孔板上用分化培养基培养3天后，然后用不同浓度的IPA(0.1、0.25、0.5mM)进行预处理48小时。最后，再用1000ng/mL LPS处理12小时后，收集细胞进行westernblot检测。检测结果表明1000ng/mL的LPS可有效诱导肌管细胞的炎症，并且0.1mM IPA可以缓解LPS引起的TLR4、NF-κB、IL-1β和NLRP3等炎症蛋白的上调，如图16所示。
吲哚丙酸通过调控miR-26a-2-3p(简称miR-26A)直接靶向促炎因子IL-1β：C2C12细胞接种于6孔细胞培养板上，培养至约80％的融合度后，将培养基改为含有2％(v/v)马血清的分化培养基进行分化。细胞在分化培养基的上培养4天后，转染miR-26A模拟物和突变体过夜，miR-26A模拟物和突变体的工作浓度分别为20nM。最后，用1000ng/mL的LPS处理细胞12小时，然后收集细胞进一步分析。细胞蛋白westernblot结果显示，miR-26A有效降低LPS引起的炎症通路蛋白TLR4、NF-κB、IL-1β和TNFα水平的上调，如图17所示。
吲哚丙酸通过调控miR-26A直接靶向促炎因子IL-1β：miR-26A的靶标主要通过Targetscan进行预测，再通过双荧光素酶试验进行验证。将人肾上皮细胞293T细胞接种于6孔板连续培养12h，融合度大约80％时用miR-26A模拟物或突变体进行转染24小时。表达质粒的工作浓度为0.4μg/mL，miR-26A模拟物和突变体的浓度分别为20nM。最后，收集小鼠细胞进行裂解和检测，具体步骤参照双荧光素酶检测试剂盒(Promega)。检测结果显示，miR-26A可直接靶向促炎因子IL-1β得3’UTR，如图18所示。

Claims (10)

1.用Clostridium sporogenes制备的代谢组合物，其特征在于，所述代谢组合物由吲哚丙酸、氢化肉桂酸、琥珀酸、吲哚丙烯酸、色胺、色氨酸、N-乙酰苯丙氨酸、脱氢莽草酸、吲哚乙酰胺和吲哚乙酸组成。
2.根据权利要求1所述的代谢组合物，其特征在于，所述代谢组合物由吲哚丙酸、氢化肉桂酸、琥珀酸、吲哚丙烯酸、色胺、色氨酸、N-乙酰苯丙氨酸、脱氢莽草酸、吲哚乙酰胺和吲哚乙酸按照13-16：12-15：3-6：1-2：0.5-1.5：0.2-1：0.1-0.4：0.1-0.3：0.1-0.3：0.1-0.3的平均峰面积比组成。
3.权利要求1所述代谢组合物的生产方法，其特征在于，所述生产方法为用Clostridium sporogenes发酵芳香族氨基酸得到代谢组合物。
4.根据权利要求3所述的生产方法，其特征在于，所述Clostridium sporogenes复苏所用培养基为添加了芳香族氨基酸的液体硫乙醇酸盐培养基。
5.根据权利要求3所述的生产方法，其特征在于，所述Clostridium sporogenes活化所用培养基为强化梭菌固体培养基。
6.权利要求1所述代谢组合物的检测方法，其特征在于，所述检测方法采用液相色谱串联质谱法，其中液相色谱条件：用含体积分数为0.1％甲酸的水溶液作为流动相A，用含体积分数为0.1％的甲酸的乙腈溶液作为流动相B，按下表进行梯度洗脱，流动相流速为0.4ml/min，色谱柱柱温为35℃,色谱柱为Waters ACQUITY UPLC HSS T3 C18柱1.8m×2.1mm×100mm。
Figure FDA0003519531760000011
7.根据权利要求6所述的检测方法，其特征在于，所述检测方法的检测对象为Clostridium sporogenes厌氧培养12-24h的发酵液。
8.根据权利要求7所述的检测方法，其特征在于，所述发酵液的pH值为6.0±0.5。
9.根据权利要求7所述的检测方法，其特征在于，所述发酵液的OD600值为2.0±0.5。
10.权利要求1所述代谢组合物在制备肌细胞增殖剂中的应用。
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