CN111896655A - 一种用于分析与发掘消化道益生菌产生的功能性代谢物的方法 - Google Patents
一种用于分析与发掘消化道益生菌产生的功能性代谢物的方法 Download PDFInfo
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Abstract
本发明公开一种用于分析与发掘消化道益生菌产生的功能代谢物的方法,所述方法联合运用完全合成培养基的体外培养方法与比较代谢组学方法进行研究与分析,所述方法包括如下步骤:(1)复苏益生菌,富集培养益生菌,并传代培养于完全合成培养基;(2)取益生菌菌液,将菌液重悬于完全合成培养基,作为接种物备用;(3)将步骤(2)接种物接种于添加胆盐溶液的完全合成培养基,培养,采集样品;(4)提取益生菌发酵液中的代谢物;(5)利用比较代谢组学方法分析步骤(4)的代谢物。本发明的方法适用于人、畜禽以及伴侣动物消化道益生菌的功能比较研究。可为深入研究消化道益生菌的功能、发掘新型益生化合物、开发新型日粮功能性添加物、调节肠道功能与健康提供基础。
Description
技术领域
本发明涉及营养学消化道微生物代谢物评价技术领域,尤其涉及一种用于分析与发掘消化道益生菌产生的功能代谢物的方法。
背景技术
近年来,代谢组学在营养研究领域已有诸多应用。He Q等通过检测肥胖猪和瘦肉型猪血清中代谢物的差异表明,肥胖猪血清中高密度脂蛋白、极低密度脂蛋白、饱和脂类、不饱和脂类、糖蛋白、肌醇、丙酮酸、苏氨酸、酪氨酸和肌酸相对丰度均高于瘦肉型猪;但血糖、尿素水平较低,并且肥胖猪血清中的肠道微生物群相关代谢物浓度(三甲胺-N-氧化物和胆碱)也发生变化。肖英平等通过代谢组学分析日粮中补充谷氨酰胺对断奶仔猪血浆代谢物谱的影响发现,谷氨酰胺可引起与精氨酸和脯氨酸代谢、碳水化合物代谢和脂肪酸代谢通路相关物质的改变。何庆华等运用代谢组学技术研究精氨酸对育肥猪的影响,发现血清中脂质、柠檬酸、胆碱等物质水平降低;而甜菜碱、甲胺、丙酮酸、琥珀酸和酮体的相对丰度增加,这表明精氨酸参与调节育肥猪体内能量代谢过程。贺邵君等通过气相色谱-质谱联用技术研究急性热应激状态下肉鸡血清物质的代谢变化表明,应激状态下机体内三羧酸循环和脂肪酸生物合成等代谢通路受到显著影响。程英显通过代谢组学技术研究日粮中添加解淀粉芽孢杆菌可引起肉鸡血清中50余种代谢物的变化,并影响机体蛋白质和氨基酸代谢过程。
益生菌在人和动物生产中使用多年,但是作用效果不一致,其中的重要原因之一就是对特定益生菌的作用方式与机制的研究不彻底,导致实际使用中的方式和阶段不合理,从而作用效果不明显。以上问题对于生产中使用肠道来源的益生菌尤其突出。一方面,源自肠道的益生菌(如,乳酸杆菌、双歧杆菌)由于不耐加工和日粮配制过程中的高温高湿以及有氧环境,导致到达肠道特定部位的有效活菌数达不到预期的数量;或加工过程中导致益生菌生理的改变,不能在肠道中产生有益代谢物,起到调节肠道内环境以及维持肠道发育和正常功能的作用。
此外,已有研究显示,微生物可通过参与肠道中营养物质的代谢,并通过产生有益的代谢产物发挥益生作用。如,乳酸菌可产生细菌素抑制肠道致病菌的生长,埃氏巨球菌可利用乳酸菌产生的乳酸生成丁酸调控肠细胞的生长。然而,目前有关乳酸杆菌和埃氏巨球菌对营养物质的代谢以及产生的代谢物的差异鲜有报道。
因此本研究通过优化体外培养方法并使用复合胆盐处理为研究策略,用于培养消化道来源的益生菌,并结合比较代谢组学技术和方法,比较了受胆盐调节的消化道优势乳酸杆菌和埃氏巨球菌产生的代谢物的异同,初步探明所研究益生菌的代谢物和代谢通路的异同,为生产中开发新型功能性日粮添加物提供基础。
发明内容
本发明公开一种用于分析与发掘消化道益生菌产生的功能代谢物的方法,所述方法联合运用完全合成培养基的体外培养方法与比较代谢组学方法进行研究和分析,具体地,所述方法包括如下步骤:
(1)复苏益生菌,富集培养益生菌,并传代培养于完全合成培养基;
(2)取益生菌菌液,将菌液重悬于完全合成培养基,作为接种物备用;
(3)将步骤(2)接种物接种于添加胆盐溶液的完全合成培养基,培养,采集样品;
(4)提取益生菌发酵液中的代谢物;
(5)利用比较代谢组学方法分析步骤(4)的代谢物。
优选地,所述步骤(3)中培养时间为3-12小时,优选为6小时。
所述步骤(3)的完全合成培养中胆盐溶液包含甘胆酸钠、甘氨鹅脱氧胆酸钠、牛胆酸钠、牛磺鹅去氧胆酸钠。
所述胆盐溶液的配制方法为:称取甘氨酸钠0.1-0.4g、甘氨鹅脱氧胆酸钠0.1-0.4g、牛胆酸钠0.02-0.06g、牛磺鹅去氧胆酸钠0.02-0.06g,使用磷酸缓冲液溶解并准确定容至10mL,过滤灭菌;优选地,称取甘胆酸钠0.22g、甘氨鹅脱氧胆酸钠0.20g、牛胆酸钠0.04g、牛磺鹅去氧胆酸钠0.04g。
所述胆盐溶液在完全合成培养基中添加量为0.2-0.8g/L,优选为0.5g/L。
所述完全合成培养基中包含氨基酸组分,氨基酸组分包含:天冬氨酸、谷氨酸、天冬酰胺、丝氨酸、谷氨酰胺、组氨酸、甘氨酸、苏氨酸、瓜氨酸、精氨酸、牛磺酸、丙氨酸、酪氨酸、色氨酸、蛋氨酸、缬氨酸、苯丙氨酸、异亮氨酸、亮氨酸、鸟氨酸盐酸盐、赖氨酸、脯氨酸、半胱氨酸。
所述完全合成培养基中氨基酸各组分含量为:
优选地,所述完全合成培养基中氨基酸各组分含量为:
所述完全合成培养基中每升培养基还包含:葡萄糖5-20g、乳酸钠2.0-3.0g、氯化钾0.5-0.7g、氯化钠0.5-0.7g、磷酸氢二钾0.9-1.1g、磷酸二氢钾4.5-5.5g、氯化钙0.1-0.2g、七水合硫酸镁0.45-0.55g、乙酸钠0.9-1.1g、柠檬酸铵0.5-0.7g、抗坏血酸0.4-0.6g、腺嘌呤0.01-0.02g、鸟嘌呤0.01-0.02g、肌苷0.005-0.01g、乳清酸0.005-0.01g、胸苷0.005-0.01g、尿嘧啶0.005-0.015g、黄嘌呤0.005-0.15g、9-11mL血红素溶液(0.01%,w/v)、9-11mL脂肪酸溶液、9-11mL还原剂溶液、9-11mL微量元素溶液、9-11mL维生素母液、45-55mL碳酸氢钠溶液及0.9-1.1mL刃天青溶液(1%,w/v);
优选地,所述完全合成培养基中每升培养基还包含:葡萄糖10g、乳酸钠2.7g、氯化钾0.6g、氯化钠0.6g、磷酸氢二钾1g、磷酸二氢钾5g、氯化钙0.15g、七水合硫酸镁0.5g、乙酸钠1g、柠檬酸铵0.6g、抗坏血酸0.5g、腺嘌呤0.01g、鸟嘌呤0.01g、肌苷0.005g、乳清酸0.005g、胸苷0.005g、尿嘧啶0.01g、黄嘌呤0.01g、10mL血红素溶液(0.01%,w/v)、10mL脂肪酸溶液、10mL还原剂溶液、10mL微量元素溶液、10mL维生素母液、50mL碳酸氢钠溶液及1mL刃天青溶液(1%,w/v)。
所述脂肪酸溶液配制方法为:准确量取6.85mL乙酸、3.00mL丙酸、1.84mL丁酸及0.55mL戊酸溶解于0.2M NaOH溶液中,并定容至1L;
所述还原剂配制方法为:称取20.5g Na2S·9H2O溶于1L去离子水中,并持续通入N2;
所述微量元素溶液配制方法为:准确称量25mg MnCl2·4H2O、25mg ZnCl2、20mgFeSO4·7H2O、25mg CuCl2·2H2O、50mg SeO2、50mg CoCl2·6H2O、250mg NiCl2·6H2O、250mgNa2MoO4·2H2O、31.4mg NaVO3和250mg H3BO3,溶解于20mL 0.02M盐酸溶液中,使用去离子水定容至1L;
所述维生素母液制备方法为:准确称取泛酸钙1.6mg、生物素2.5mg、烟酸1.6mg、对氨基苯甲酸0.2mg、盐酸吡哆胺5mg、盐酸吡哆醇2mg、核黄素1.6mg及盐酸硫胺素1.6mg,定容于10mL去离子水中,过滤灭菌。
所述步骤(5)具体为:代谢样品经液质联用仪鉴定,对差异代谢物筛选后进行比较代谢组学方法进行;优选,所述比较代谢组方法包含但不限于以下任意一种或多种分析方法:火山图分析、偏最小乘法判别分析、变量投影重要性分析、热图分析和代谢通路拓扑分析。
所述益生菌为乳酸杆菌和/或埃氏巨球菌;优选地,所述乳酸杆菌为粘膜乳酸杆菌(Lactobacillus mucosae)、罗伊氏乳酸杆菌(L.reuteri)、嗜淀粉乳酸杆菌(L.amylovorus)。
本申请方法优化了完全合成培养基,添加的氨基酸及维生素组成成分全面、配比合理,且添加了已知成分的胆盐处理消化道益生菌,从而能够研究益生菌产生的胞外代谢物,为代谢组学研究益生菌分泌的代谢物提供可能,为发掘新型功能性代谢物提供策略。
本研究通过体外培养联合比较代谢组学方法分析了胆盐对消化道优势乳酸杆菌和乳酸利用菌代谢的影响,由于分离自不同人和动物种属的益生菌具有相似地生长和代谢特性,如乳酸杆菌都产生乳酸,所有的埃氏巨球菌都可以利用乳酸产生丁酸,因此本申请方法适用于可以产生胆盐的所有动物消化道益生菌代谢的研究,包括但不限于人和畜禽以及伴侣动物,可为深入研究消化道益生菌的功能、发掘新型益生化合物、开发新型日粮功能性添加物、调节肠道功能与健康提供基础。
本申请添加完全合成培养基组与对照组相比:
1、添加胆盐显著增加了L.mucosae、L.reuteri和L.amylovorus三株乳酸杆菌培养液中2-正戊基呋喃(2-pentylfuran)等化合物的相对丰度(P<0.05);增加了L.reuteri、L.amylovorus和M.elsdenii三株细菌培养液中1-(甲硫基)乙基2-丙烯基二硫化物[1-(methylthio)ethyl 2-propenyl disulfide]等化合物的相对丰度(P<0.05);并增加了M.elsdenii培养液中十一碳二酸(undecanedioic acid)、二氢-5-戊基-2(3氢)-呋喃酮[dihydro-5-pentyl-2(3H)-furanone]和7-羟基去氢表雄酮(7a-hydroxydehydroepiandrosterone)的相对丰度(P<0.05)。
2、添加胆盐显著降低了L.mucosae、L.reuteri和L.amylovorus三株乳酸杆菌培养液中丙氨酰精氨酸(alanyl-arginine)的相对丰度(P<0.05);降低了L.mucosae和L.reuteri培养液中2-羟基-3-甲基戊酸(2-hydroxy-3-methylpentanoic acid)和L-γ-谷氨酰-L-异亮氨酸(L-gamma-glutamyl-L-isoleucine)等化合物的相对丰度(P<0.05);并降低了M.elsdenii培养液中丙氨酰精氨酸(alanyl-arginine)和甲基吡嗪(methylpyrazine)等化合物的相对丰度(P<0.05)。
3、添加胆盐后,L.mucosae和L.reuteri受影响的代谢通路主要有:精氨酸生物合成及精氨酸和脯氨酸代谢;L.amylovorus和M.elsdenii受影响的代谢通路主要有:丙氨酸、天冬氨酸和谷氨酸代谢。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1.L.mucosae的差异代谢物峰强度归一化处理
图2.L.reuteri的差异代谢物峰强度归一化处理
图3.L.amylovorus的差异代谢物峰强度归一化处理
图4.M.elsdenii的差异代谢物峰强度归一化处理
图5.乳酸杆菌与埃氏巨球菌的差异代谢物偏最小二乘法判别分析;A为L.mucosae,B为L.reuteri,C为L.amylovorus,D为M.elsdenii;+代表对照组,Δ代表添加胆盐组,每个点代表一个样本,n=6。
图6.乳酸杆菌与埃氏巨球菌差异代谢物变量投影重要性分析;A中L.M为L.mucosae,B中L.R为L.reuteri,C中L.A为L.amylovorus,D中M.E为M.elsdenii;每个点代表一种代谢物,图右侧为该代谢物在各组中浓度含量对比,n=6。
图7.L.mucosae的差异代谢物火山图分析;图中每一个点代表一种化合物,灰色大圆点代表添加胆盐组与对照组相比化合物丰度变化两倍以上(fold change>2)且P<0.05,图中横坐标大于0代表倍数增加,横坐标小于0代表倍数下降,图中对于重点化合物名称已做出标注。
图8.L.reuteri的差异代谢物火山图分析;图中每一个点代表一种化合物,灰色大圆点代表添加胆盐组与对照组相比化合物丰度变化两倍以上(fold change>2)且P<0.05,图中横坐标大于0代表倍数增加,横坐标小于0代表倍数下降,图中对于重点化合物名称已做出标注。
图9.L.amylovorus的差异代谢物火山图分析;图中每一个点代表一种化合物,灰色大圆点代表添加胆盐组与对照组相比化合物丰度变化两倍以上(fold change>2)且P<0.05,图中横坐标大于0代表倍数增加,横坐标小于0代表倍数下降,图中对于重点化合物名称已做出标注。
图10.M.elsdenii的差异代谢物火山图分析;图中每一个点代表一种化合物,灰色大圆点代表添加胆盐组与对照组相比化合物丰度变化两倍以上(fold change>2)且P<0.05,图中横坐标大于0代表倍数增加,横坐标小于0代表倍数下降,图中对于重点化合物名称已做出标注。
图11.添加胆盐后L.reuteri的差异代谢物热图分析;图中每列代表一组的平均值,每行代表一种代谢物,图中颜色的深浅代表代谢物的相对丰度,丰度的变化趋势见图中右上方颜色条旁的数字标注。图右侧为代谢物名,图左侧为代谢物聚类树,两种代谢物分支越近,说明这两种化合物的相对丰度越接近,n=6。
图12.添加胆盐后L.mucosae的差异代谢物热图分析;图中每列代表一组的平均值,每行代表一种代谢物,图中颜色的深浅代表代谢物的相对丰度,丰度的变化趋势见图中右上方颜色条旁的数字标注。图右侧为代谢物名,图左侧为代谢物聚类树,两种代谢物分支越近,说明这两种化合物的相对丰度越接近,n=6。
图13.添加胆盐后L.amylovorus的差异代谢物热图分析;图中每列代表一组的平均值,每行代表一种代谢物,图中颜色的深浅代表代谢物的相对丰度,丰度的变化趋势见图中右上方颜色条旁的数字标注。图右侧为代谢物名,图左侧为代谢物聚类树,两种代谢物分支越近,说明这两种化合物的相对丰度越接近,n=6。
图14.添加胆盐后M.elsdenii的差异代谢物热图分析;图中每列代表一组的平均值,每行代表一种代谢物,图中颜色的深浅代表代谢物的相对丰度,丰度的变化趋势见图中右上方颜色条旁的数字标注。图右侧为代谢物名,图左侧为代谢物聚类树,两种代谢物分支越近,说明这两种化合物的相对丰度越接近,n=6。
图15.L.mucosae受胆盐影响的差异代谢通路分析;图中每个点代表一种代谢通路,X轴代表代谢通路的影响值,Y轴代表富集分析P值的负对数,图中点的形状越大颜色越深,分别代表代谢通路的影响值越大以及富集程度越强。
图16.L.reuteri受胆盐影响的差异代谢通路分析;图中每个点代表一种代谢通路,X轴代表代谢通路的影响值,Y轴代表富集分析P值的负对数,图中点的形状越大颜色越深,分别代表代谢通路的影响值越大以及富集程度越强。
图17.L.amylovorus受胆盐影响的差异代谢通路分析;图中每个点代表一种代谢通路,X轴代表代谢通路的影响值,Y轴代表富集分析P值的负对数,图中点的形状越大颜色越深,分别代表代谢通路的影响值越大以及富集程度越强。
图18.M.elsdenii受胆盐影响的差异代谢通路分析;图中每个点代表一种代谢通路,X轴代表代谢通路的影响值,Y轴代表富集分析P值的负对数,图中点的形状越大颜色越深,分别代表代谢通路的影响值越大以及富集程度越强。
具体实施方式
以下结合具体实施例,对本发明作进一步说明。应理解,以下实施例仅用于说明本发明而非用于限定本发明的范围。所述方法如无特别说明均为常规方法,所述原料如无特别说明均能从公开商业途径获得。
主要仪器与设备
全自动高压蒸汽灭菌器(MLS-3750):Sanyo,日本;离心机(5810R):Eppendorf,德国;隔水式电热恒温培养箱(PYX-DHS-500BS);超低温冰箱(MLT):Thermo Scientific,美国;多功能酶标仪(SpectraMax M5):Molecular Devices,美国;流式细胞仪(Cytoflex):BECKMEN公司,美国;超净工作台(S SW-CJ-2F);超高效液相色谱:ACQUITY UPLC I-Class,Waters,美国;高分辨质谱:Q-Exactive,Thermo Scientific,美国。
实施例1、培养基的制备
1、培养基母液的制备
微量元素溶液:准确称量25mg MnCl2·4H2O、25mg ZnCl2、20mg FeSO4·7H2O、25mgCuCl2·2H2O、50mg SeO2、50mg CoCl2·6H2O、250mg NiCl2·6H2O、250mg Na2MoO4·2H2O、31.4mg NaVO3和250mg H3BO3,溶解于20mL 0.02M盐酸溶液中,使用去离子水定容至1L。
碳酸氢钠溶液(现配现用):称取4.1g Na2CO3于50mL沸水并持续通入CO2约20分钟。
还原剂溶液:称取20.5g Na2S·9H2O溶于1L去离子水中,并持续通入N2。
脂肪酸溶液:准确量取6.85mL乙酸、3.00mL丙酸、1.84mL丁酸及0.55mL戊酸溶解于0.2M NaOH溶液中,并定容至1L。
维生素母液(100×)制备的方法,准确称取:泛酸钙1.6mg、生物素2.5mg、烟酸1.6mg、对氨基苯甲酸0.2mg、盐酸吡哆胺5mg、盐酸吡哆醇2mg、核黄素1.6mg及盐酸硫胺素1.6mg,定容于10mL去离子水中,过滤灭菌备用。
上述溶液均置于4℃冰箱保存。
2、胆盐溶液的制备
磷酸缓冲液配制方法:称取磷酸二氢钠2.76g及二水合磷酸氢二钠3.56g,使用去离子水溶解并准确定容至100mL,调节pH值为7.4,过滤灭菌备用,储存于4℃冰箱。
配制胆盐溶液(100×):称取甘胆酸钠0.22g(G7132,Sigma)、甘氨鹅脱氧胆酸钠0.2g(G0759,Sigma)、牛胆酸钠0.04g(86339,Sigma)、牛磺鹅去氧胆酸钠0.04g(T6260,Sigma),使用磷酸缓冲液溶解并准确定容至10mL(胆盐组培养基中结合型胆盐的终浓度为0.05%,即0.5g/L),过滤灭菌备用,储存于4℃冰箱。
3、完全合成培养基的制备
配制完全合成培养基(即化学成分确定的培养基),每升培养基包含:葡萄糖10g、乳酸钠2.7g、氯化钾0.6g、氯化钠0.6g、磷酸氢二钾1g、磷酸二氢钾5g、氯化钙0.15g、七水合硫酸镁0.5g、乙酸钠1g、柠檬酸铵0.6g、抗坏血酸0.5g、腺嘌呤0.01g、鸟嘌呤0.01g、肌苷0.005g、乳清酸0.005g、胸苷0.005g、尿嘧啶0.01g、黄嘌呤0.01g、10mL血红素溶液(0.01%,w/v)、10mL脂肪酸溶液、10mL还原剂溶液、10mL微量元素溶液、10mL维生素母液、50mL碳酸氢钠溶液及1mL刃天青溶液(1%,w/v);氨基酸组分详见表1,其中天冬酰胺和谷氨酰胺过滤灭菌后加入经高压灭菌的培养基中;维生素母液为培养基灭菌后加入。
培养基配制过程中,持续向装有培养基的容器中通入CO2,直至培养基呈淡粉色至淡黄色。之后将完全合成培养基分装于通入CO2的Hungate厌氧管内,每管分装9mL,加塞旋紧螺口盖后115℃高压灭菌15min,灭菌冷却后的培养基均呈淡黄色表明厌氧管中环境处于厌氧状态。
表1完全合成培养基中氨基酸组分含量
4、乳酸杆菌培养基(MRS)的制备
MRS液体培养基方法配制,培养基组成如下:蛋白胨10g、葡萄糖20g、牛肉膏10g、酵母提取物5g、磷酸氢二钾2g、柠檬酸铵2g、乙酸钠5g、硫酸镁0.1g、硫酸锰0.05g、吐温80 1g,使用去离子水溶解,并定容至1L。为便于后续试验操作,将MRS液体培养基分装于Hungate厌氧管内,每管9mL,该过程应向培养基所在容器与待分装厌氧管持续通入CO2,115℃,高压灭菌15min。
MRS固体培养基的配置:在上述配方基础上,加入琼脂20g/L,配制时应使用沸水完全溶解琼脂,115℃,高压灭菌15min,置于超净工作台,冷却至60℃左右,倒入每个无菌培养皿中约20mL,凝固后倒置存放。
实施例2、体外培养联合比较代谢组学方法分析
1、肠道来源乳酸杆菌的复苏与计数
将实验室保存的三株分离自仔猪肠道优势乳酸杆菌:Lactobacillus mucosae、L.reuteri、L.amylovorus复苏,接种于MRS液体培养基,37℃恒温培养24h,传代培养一次。为统一后续培养试验各菌株的接种菌体量,应提前对各菌株在完全合成培养基中的生长情况进行计数及统计。按10%接种量传代于完全合成培养基,37℃恒温培养24h,取1mL菌液2000×g离心2min,去除上清液,将菌体重悬于1mL完全合成培养基(避免原MRS培养基中的营养成分对后续试验中氨基酸及代谢物测定造成干扰),对重悬菌液进行梯度稀释,选取10-6、10-7、10-8三个梯度进行涂板计数,每个梯度设置3次重复。三株乳酸杆菌的计数均按照上述步骤进行。
2、肠道来源的埃氏巨型球菌的复苏与计数
将实验室保存的猪肠道来源的埃氏巨型球菌(Megasphaera elsdenii)复苏,接种于YCFA液体培养基,37℃恒温培养24h,传代培养一次。为统一后续培养试验各菌株的接种菌体量(CFU),应提前对埃氏巨型球菌在完全合成培养基中的生长情况进行计数及统计。按10%接种量传代于完全合成培养基,37℃恒温培养24h。试验结合使用Hungate滚管计数法和流式细胞仪计数法,对培养液中的埃氏巨球菌进行计数。
(1)Hungate滚管计数:取1mL菌液2000×g离心2min,去除上清液,将菌体重悬于1mL完全合成培养基(避免原YCFA培养基中的营养成分对后续试验中氨基酸及代谢物测定造成干扰),对重悬菌液进行梯度稀释,选取10-6、10-7、10-8三个梯度进行Hungate滚管计数,每个梯度设置3次重复。
(2)流式细胞仪计数细菌:按照上述方法使用PBS缓冲液制备重悬菌液2mL,取1mL重悬菌液,用1mL 75%乙醇处理30min作为阴性对照,取重悬菌液与阴性对照菌液各800μL,加入200μL PI染料(碘化丙啶),PI终浓度为10μg/mL,混合均匀后置于4℃冰箱染色10min,上机测定前再次混匀。检测前使用蒸馏水执行机器清洗程序,并校正流速为10μL/min,样品的收集时间为5min,取进样稳定后单位时间内通过的细菌数量进行计算。
3、乳酸杆菌与埃氏巨球菌的培养
(1)分别在MRS和YCFA液体培养基中富集培养三株乳酸杆菌及埃氏巨球菌,并传代于完全合成培养基。
(2)根据前期的计数结果,各菌株取适量菌液,2000×g离心2min,去除上清液,将菌体重悬于完全合成培养基,作为接种物备用。
(3)本试验设置对照组和添加0.5g/L胆盐组,将上述接种物接种于完全合成培养基,使发酵初始细菌浓度约为1.0×107CFU/mL,静置于37℃恒温培养。于0h、3h、6h、9h、12h每组分别取出6管用于样品采集,采集的培养液用于测定OD600值、活菌数量、乳酸含量、游离氨基酸含量。
4、细菌发酵液代谢物提取
将在-80℃预冷的HPLC级甲醇与培养液上清1:1(v/v)混合,涡旋1min,混合均匀后置于-20℃冰箱中静置30min,取出后21,000×g,-20℃,离心10min,取100μL上清液过滤后保存于-80℃,待测。
5、比较代谢组学方法分析
代谢物分析的仪器方法
细菌发酵液代谢物样品经ACQUITY UPLC I-Class超高效液相色谱分离后,通过电喷雾离子源(HESI),加热离子化后进入Q-Exactive高分辨质谱收集数据。每进样10个细菌发酵液代谢物样品,使用1个所有样品的混合样品作为质控进样(QC)。
5.1代谢组学数据处理
使用Progenesis QI软件(Waters,美国)对液质联用仪收集到的原始数据进行分析处理,将图谱保留时间对齐、剔除背景、提取特征质谱峰,并以所有化合物的峰强度为基础对原始峰强度进行归一化处理、进行显著性分析(ANOVA),得到一个包含化合物样本名称、保留时间、质荷比及峰面积等信息的矩阵。使用Simca 14.1(Umetrics公司,瑞典)对化合物矩阵进行统计分析,选取变量投影重要性(variable importance in projection,VIP)大于1且P<0.05的化合物为培养基添加0.05%胆盐对试验菌株代谢影响显著的差异化合物,并对其进行二级质谱鉴定确定化学式和结构式。根据化合物的质核比(m/z)和保留时间(retention time,RT)可确定其化学式,使用Progenesis QI软件将化合物的精准分子量与实验室内部数据库进行比对,检索获取相关候选化合物列表。根据化合物的碎片信息对候选化合物进行图谱预测,将实际图谱与预测图谱比对得到候选化合物的准确率,并使用Xcalibur 2.0软件(Thermo Scientific公司,美国)查看化合物的二级图谱,将其与人类代谢组数据库(HMDB)、Metlin、京都基因和基因组百科全书(KEGG)公共数据库中标准品二级图谱比对,确定差异化合物的结构式。
5.2数据统计与分析
通过MetaboAnalyst网站(https://www.metaboanalyst.ca)对确定结构式的化合物进行数据归一化处理(normalization)、基于多维模型的偏最小二乘法判别分析(partial least squares discriminant analysis,PLS-DA)、变量投影重要性分析(variable importance in projection,VIP)、基于一元统计分析检验P值和变化倍数(fold change)的火山图分析(volcano plot)、热图分析(heatmap)和代谢通路拓扑分析(pathway analysis)。
数据归一化处理:将数据限定在一定范围内便于后续分析,每组6个生物学重复,对化合物的峰强度进行log转换及帕累托缩放(paretoscaling)。
偏最小二乘法判别分析:PLS-DA是一种有监督的学习方法,可最大化数据的组间差异,具有较好的分离效果,本试验展示了每组95%的置信区间。
变量投影重要性分析:VIP值是PLS-DA模型的变量权重值,可用于衡量各代谢物积累差异对各组样本分类判别的影响强度和解释能力,通常选用VIP≥1为差异代谢物筛选标准,本试验中变量投影重要性分析用于展示PLS-DA分析中主成分1的VIP值排名前25的化合物及每组化合物的相对丰度。
火山图分析:火山图常用于展示组间差异显著的代谢物(P<0.05),差异表达一般按照变化倍数(fold change)≥2.0为标准,对倍数进行log2的转化,对P值进行-log10的转化。
热图分析:使用Euclidean算法对归一化后的数据进行距离计算,并使用Ward聚类算法对化合物进行层次聚类,展示每组的平均值,通过颜色变化反映不同处理之间的化合物丰度差异。
代谢通路拓扑分析:对化合物数据进行归一化处理后,使用超几何检验算法(hypergeometric test)进行过度表达分析(over representation analysis),以及用relative-betweeness centrality算法进行通路拓扑分析(pathway topologyanalysis),P值由通路富集分析(enrichment analysis)得到,覆盖率的P应小于0.05;通路影响值(pathway impact)由通路拓扑分析得到,一般认为通路影响值大于0.1代表其中的差异代谢物在通路内具有重要调控意义。
5.3结果
5.3.1数据归一化处理
对数据分别进行归一化,使之更趋近于正态分布,便于后续分析,处理后结果如图1、图2、图3、图4所示。
5.3.2偏最小二乘法判别分析
为进一步分析添加胆盐对组间代谢物差异的影响,对归一化后的数据进行了偏最小二乘法判别分析,该方法可最大程度地忽略组内差异,并突出反映处理组之间的差异,结果如图5所示。由图可知,四种试验菌株培养基添加胆盐后产生的代谢物与对照组相比具有显著差异(P<0.05)。
5.3.3变量投影重要性分析
为进一步研究添加胆盐处理后不同代谢物对组间差异的影响作用,对各组的VIP值进行分析。VIP值是偏最小二乘法判别分析的重要度量,该参数可反映代谢物对处理组之间差异的贡献,VIP值越大表示该化合物在处理组之间的差异越大,本研究展示每组中VIP值位列前25(VIP>1)的代谢物,结果如图6所示。
根据变量投影重要性分析可知,与对照组相比,L.mucosae、L.reuteri和L.amylovorus三株细菌添加胆盐的培养液中2-正戊基呋喃(2-pentylfuran)等化合物的相对丰度显著上升(P<0.05)。L.reuteri、L.amylovorus和M.elsdenii三株细菌添加胆盐的培养液中1-(甲硫基)乙基2-丙烯基二硫化物[1-(methylthio)ethyl 2-propenyldisulfide]等化合物的相对丰度显著上升(P<0.05),丙氨酰精氨酸(alanyl-arginine)等化合物相对丰度显著下降(P<0.05)。L.mucosae和L.reuteri两株细菌培养基添加胆盐的培养液中2-羟基-3-甲基戊酸(2-hydroxy-3-methylpentanoic acid)和L-γ-谷氨酰-L-异亮氨酸(L-gamma-glutamyl-L-isoleucine)等化合物相对丰度显著下降(P<0.05)。L.reuteri和L.amylovorus两株细菌添加胆盐的培养液中2,6-二叔丁基-4-甲基苯酚(2,6-Di-tert-butyl-4-methylphenol)、3-叔丁基-4-羟基茴香醚(3-tert-butyl-4-hydroxyanisole)、藏花醛(safranal)、反式-4,5-环氧-2(E)-癸烯[trans-4,5-epoxy-2(E)-decenal]和紫苏酸(perillic acid)等化合物的相对丰度显著上升(P<0.05),4-O-α-D-吡喃半乳糖苷-D-半乳糖醛酸(4-O-alpha-D-Galactopyranuronosyl-D-galacturonic acid)等化合物相对丰度显著下降(P<0.05)。M.elsdenii添加胆盐的培养液中十一碳二酸(undecanedioic acid)、7-羟基去氢表雄酮(7a-hydroxydehydroepiandrosterone)和二氢-5-戊基-2(3氢)-呋喃酮[dihydro-5-pentyl-2(3H)-furanone]等化合物的相对丰度显著升高(P<0.05),而丙氨酰精氨酸(alanyl-arginine)和甲基吡嗪(methylpyrazine)等化合物的相对丰度显著下降(P<0.05)。
5.3.4火山图分析
对归一化后化合物数据分别进行火山图分析,结果如图7、图8、图9、图10所示。
5.3.5热图分析
热图可直观地显示各组代谢物的差异程度及整体变化,图中颜色深浅代表化合物在该组中的相对丰度,结果如图11、图12、图13和图14所示。与对照组相比,L.reuteri添加胆盐的培养液中,共有17种化合物(VIP>1)相对丰度显著上升(P<0.05),包括2,5-呋喃二甲酸(2,5-furandicarboxylic acid)等,而1,4,6-三羟基-5-甲氧基-7-异戊烯酮(1,4,6-trihydroxy-5-methoxy-7-prenylxanthone)、戊二酰甘氨酸(glutarylglycine)和丙氨酰精氨酸(alanyl-arginine)等在内的8种化合物(VIP>1)相对丰度显著下降(P<0.05)。
L.mucosae添加胆盐的培养液中,2-正戊基呋喃(2-pentylfuran)、pipercitine和胸苷(thymidine)等8种化合物(VIP>1)相对丰度显著上升(P<0.05),拉米夫定亚砜(lamivudine sulfoxide)、甲羟戊酸-5磷酸(mevalonic acid-5P)和酪蛋白(taxiphyllin)等30种化合物相对丰度显著下降(P<0.05)。
L.amylovorus添加胆盐的培养液中,N-(1-脱氧-1-果糖基)丝氨酸[N-(1-deoxy-1-fructosyl)serine]、D-七庚糖7-磷酸(D-sedoheptulose 7-phosphate)和腺苷(adenosine)等相对丰度显著上升(P<0.05)的化合物(VIP>1)有15种,氟苯甲酸酯(felbamate)和半胱氨酰谷氨酰胺(cysteinyl-glutamine)等相对丰度显著下降(P<0.05)的化合物(VIP>1)有32种。
M.elsdenii添加胆盐的培养液中,十一碳二酸(undecanedioic acid)、阿替洛尔(atenolol)、7a-羟基去氢表雄酮(7a-hydroxydehydroepiandrosterone)和N-甲酰基-L-谷氨酸(N-formyl-L-glutamic acid)等35种化合物(VIP>1)相对丰度显著上升(P<0.05),丙氨酰精氨酸(alanyl-arginine)、甲基吡嗪(methylpyrazine)和2-羟基腺嘌呤(2-hydroxyadenine)等15种化合物(VIP>1)相对丰度显著下降(P<0.05)。
5.3.6代谢通路预测分析
分析代谢物所参与的代谢通路有助于进一步研究胆盐对乳酸杆菌和乳酸利用菌代谢的影响。根据鉴定出的差异代谢物与KEGG数据库比对,综合考虑覆盖率的P应小于0.05和通路影响值(pathway impact)大于0.1两个参数,确定各菌株有价值的代谢通路并标注于对应的代谢通路点,结果如图15、图16、图17、图18所示。培养基添加胆盐后,预测L.mucosae受到影响的代谢通路包括:精氨酸生物合成(Arginine biosynthesis)、精氨酸和脯氨酸代谢(Arginine and proline metabolism)、戊糖和葡萄糖醛酸酯的相互转化(Pentose and glucuronate interconversions)、牛磺酸和牛磺酸代谢(Taurine andhypotaurine metabolism)、抗坏血酸和藻酸盐代谢(Ascorbate and aldaratemetabolism)和萜类骨架的生物合成(Terpenoid backbone biosynthesis)等6条代谢通路;预测L.reuteri受到影响的代谢通路包括:精氨酸和脯氨酸代谢(Arginine andproline metabolism)、精氨酸生物合成(Arginine biosynthesis)和三羧酸循环(Citratecycle)等3条代谢通路;预测L.amylovorus受到影响的代谢通路包括:丙氨酸,天冬氨酸和谷氨酸代谢(Alanine,aspartate and glutamate metabolism)、戊糖和葡萄糖醛酸酯的相互转化(Pentose and glucuronate interconversions)、萜类骨架的生物合成(Terpenoidbackbone biosynthesis)和抗坏血酸和藻酸盐代谢(Ascorbate and aldaratemetabolism)等4条代谢通路;预测M.elsdenii受到影响的代谢通路包括:丙氨酸,天冬氨酸和谷氨酸代谢(Alanine,aspartate and glutamate metabolism)、α-亚麻酸代谢(alpha-Linolenic acid metabolism)、戊糖和葡萄糖醛酸酯的相互转化(Pentose andglucuronate interconversions)、抗坏血酸和藻酸盐代谢(Ascorbate and aldaratemetabolism)、萜类骨架的生物合成(Terpenoid backbone biosynthesis)等5条代谢通路。
通过比较分析,乳酸杆菌L.mucosae和L.reuteri的精氨酸生物合成、精氨酸和脯氨酸代谢通路均受到添加胆盐影响。L.mucosae和L.amylovorus的戊糖和葡萄糖醛酸酯的转化、抗坏血酸和藻酸盐代谢和萜类骨架的生物合成等3条通路均受到添加胆盐影响。L.amylovorus和M.elsdenii的丙氨酸,天冬氨酸和谷氨酸代谢通路也同时受到胆盐的影响作用。
本试验结果表明胆盐可以影响乳酸杆菌内精氨酸和脯氨酸代谢途径,其原因可能是胆盐对精氨酸琥珀酸转移酶途径中相关酶的活性具有抑制作用。添加胆盐后,L.reuteri培养过程中与三羧酸循环相关的代谢通路可能受到影响。
Claims (10)
1.一种用于分析与发掘消化道益生菌产生的功能代谢物的方法,其特征在于,所述方法联合运用完全合成培养基的体外培养方法与比较代谢组学方法进行研究与分析,具体地,所述方法包括如下步骤:
(1)复苏益生菌,富集培养益生菌,并传代培养于完全合成培养基;
(2)取益生菌菌液,将菌液重悬于完全合成培养基,作为接种物备用;
(3)将步骤(2)接种物接种于添加胆盐溶液的完全合成培养基,培养,采集样品;
(4)提取益生菌发酵液中的代谢物;
(5)利用比较代谢组学方法分析步骤(4)的代谢物。
2.根据权利要求1所述的方法,其特征在于,所述步骤(3)的完全合成培养基中的胆盐溶液的组分包含甘胆酸钠、甘氨鹅脱氧胆酸钠、牛胆酸钠、牛磺鹅去氧胆酸钠。
3.根据权利要求2所述的方法,其特征在于,所述胆盐溶液的配制方法为:称取甘氨酸钠0.1-0.4g、甘氨鹅脱氧胆酸钠0.1-0.4g、牛胆酸钠0.02-0.06g、牛磺鹅去氧胆酸钠0.02-0.06g,使用磷酸缓冲液溶解并准确定容至10mL,过滤灭菌;
优选地,称取甘胆酸钠0.22g、甘氨鹅脱氧胆酸钠0.20g、牛胆酸钠0.04g、牛磺鹅去氧胆酸钠0.04g。
4.根据权利要求2或3所述的方法,其特征在于,所述胆盐溶液在完全合成培养基中添加量为0.2-0.8g/L,优选为0.5g/L。
5.根据权利要求1-4任一项所述的方法,其特征在于,所述完全合成培养基中包含氨基酸组分,氨基酸组分包含:天冬氨酸、谷氨酸、天冬酰胺、丝氨酸、谷氨酰胺、组氨酸、甘氨酸、苏氨酸、瓜氨酸、精氨酸、牛磺酸、丙氨酸、酪氨酸、色氨酸、蛋氨酸、缬氨酸、苯丙氨酸、异亮氨酸、亮氨酸、鸟氨酸盐酸盐、赖氨酸、脯氨酸、半胱氨酸。
7.根据权利要求1-6任一项所述的方法,其特征在于,所述完全合成培养基中每升培养基还包含:葡萄糖5-20g、乳酸钠2.0-3.0g、氯化钾0.5-0.7g、氯化钠0.5-0.7g、磷酸氢二钾0.9-1.1g、磷酸二氢钾4.5-5.5g、氯化钙0.1-0.2g、七水合硫酸镁0.45-0.55g、乙酸钠0.9-1.1g、柠檬酸铵0.5-0.7g、抗坏血酸0.4-0.6g、腺嘌呤0.01-0.02g、鸟嘌呤0.01-0.02g、肌苷0.005-0.01g、乳清酸0.005-0.01g、胸苷0.005-0.01g、尿嘧啶0.005-0.015g、黄嘌呤0.005-0.15g、9-11mL血红素溶液(0.01%,w/v)、9-11mL脂肪酸溶液、9-11mL还原剂溶液、9-11mL微量元素溶液、9-11mL维生素母液、45-55mL碳酸氢钠溶液及0.9-1.1mL刃天青溶液(1%,w/v);
优选地,所述完全合成培养基中每升培养基还包含:葡萄糖10g、乳酸钠2.7g、氯化钾0.6g、氯化钠0.6g、磷酸氢二钾1g、磷酸二氢钾5g、氯化钙0.15g、七水合硫酸镁0.5g、乙酸钠1g、柠檬酸铵0.6g、抗坏血酸0.5g、腺嘌呤0.01g、鸟嘌呤0.01g、肌苷0.005g、乳清酸0.005g、胸苷0.005g、尿嘧啶0.01g、黄嘌呤0.01g、10mL血红素溶液(0.01%,w/v)、10mL脂肪酸溶液、10mL还原剂溶液、10mL微量元素溶液、10mL维生素母液、50mL碳酸氢钠溶液及1mL刃天青溶液(1%,w/v)。
8.根据权利要求7所述的方法,其特征在于,所述脂肪酸溶液配制方法为:准确量取6.85mL乙酸、3.00mL丙酸、1.84mL丁酸及0.55mL戊酸溶解于0.2M NaOH溶液中,并定容至1L;
所述还原剂配制方法为:称取20.5g Na2S·9H2O溶于1L去离子水中,并持续通入N2;
所述微量元素溶液配制方法为:准确称量25mg MnCl2·4H2O、25mg ZnCl2、20mg FeSO4·7H2O、25mg CuCl2·2H2O、50mg SeO2、50mg CoCl2·6H2O、250mg NiCl2·6H2O、250mgNa2MoO4·2H2O、31.4mg NaVO3和250mg H3BO3,溶解于20mL 0.02M盐酸溶液中,使用去离子水定容至1L;
所述维生素母液配制方法为:准确称取泛酸钙1.6mg、生物素2.5mg、烟酸1.6mg、对氨基苯甲酸0.2mg、盐酸吡哆胺5mg、盐酸吡哆醇2mg、核黄素1.6mg及盐酸硫胺素1.6mg,定容于10mL去离子水中,过滤灭菌。
9.根据权利要求1-8任一项所述的方法,其特征在于,所述步骤(5)具体为:代谢样品经液质联用仪鉴定,对差异代谢物筛选后进行比较代谢组学方法进行;优选,所述比较代谢组方法包含但不限于以下任意一种或多种分析方法:火山图分析、偏最小乘法判别分析、变量投影重要性分析、热图分析和代谢通路拓扑分析。
10.根据权利要求1-9任一项所述的方法,其特征在于,所述益生菌为乳酸杆菌和/或埃氏巨球菌;优选地,所述乳酸杆菌为粘膜乳酸杆菌(Lactobacillus mucosae)、罗伊氏乳酸杆菌(L.reuteri)、嗜淀粉乳酸杆菌(L.amylovorus)。
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