CN116287163A - 一种评估肠道菌群对菊粉的动态响应方法 - Google Patents
一种评估肠道菌群对菊粉的动态响应方法 Download PDFInfo
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- CN116287163A CN116287163A CN202310129959.4A CN202310129959A CN116287163A CN 116287163 A CN116287163 A CN 116287163A CN 202310129959 A CN202310129959 A CN 202310129959A CN 116287163 A CN116287163 A CN 116287163A
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Abstract
本发明公开了一种评估肠道菌群对菊粉的动态响应方法,步骤如下:1)采集猪新鲜粪便;2)体外模拟菊粉进行胃‑小肠消化,获取未消化残渣作为发酵底物;3)37℃进行体外发酵;4)测定发酵过程中的发酵液中pH、乳酸含量、短链脂肪酸含量、β‑呋喃果糖苷酶活性;同时对猪粪便发酵后发酵液中微生物进行16s高通量测序;5)进行统计和生物信息分析,确定发酵底物的动态降解特性,优势菌群相对丰度的演替变化。本发明优化了体外发酵培养基参数,首次阐明了菊粉在发酵过程中的动态代谢情况和微生物的演替规律,同时证实了个体对相同纤维的响应差异,有助于更好地理解膳食纤维发酵的动力学过程,同时为靶向筛选纤维降解菌提供了事实基础。
Description
技术领域
本发明属于生物技术和畜牧领域,具体涉及利用体外发酵和微生物测序评估一种纤维原料动态发酵特性的方法。
背景技术
膳食纤维是一种自然来源的功能性物质,由肠道微生物选择性代谢,并在微生物-微生物和微生物-宿主相互作用中发挥主导作用。膳食纤维的营养价值与宿主健康之间的关联已得到广泛认可。了解膳食纤维如何调节肠道微生物,为改善宿主健康状况提供新方法。
菊粉作为一种不易消化的碳水化合物,选择性地刺激目标肠道微生物群的增殖。体内和体外研究表明,补充菊粉或菊粉型果聚糖可诱导肠道微生物组成的特异性改变,例如促进结肠中双歧杆菌和产丁酸盐的细菌增殖,并生成小分子活性产物。因此,菊粉作为“益生元”可以通过重塑肠道菌群和提高短链脂肪酸(SCFA)水平来改善代谢综合症并保护肠道屏障功能。但是,目前动物体内实验的方法对评估肠道微生物代谢菊粉的特性以及其在塑造微生物组成的确切作用的理解仍然有限。
纤维对受试者宿主代谢的这些异质性影响与其自身肠道微生物的多样性有关,肠道微生物组的组成决定了它是否可以获得膳食纤维。纤维干预前微生物组的特征可能有助于通过预测应答者和无应答者从而提高干预的功效。
膳食纤维在肠道中的发酵是一个短期过程,其中底物与微生物定植,底物降解以及细菌之间的交叉喂养导致生态位演替。目前关于膳食纤维对肠道微生物组和宿主健康影响的评价大多是基于长时期、几个时间点的,对纤维驱动肠道菌群重塑的短暂的动态变化过程的评估方式和分析方法仍不清晰。
CN114304653A提供了一种包含菊粉在内的具有调节肠道微生态的组合物,然而却并未评估该混合物被肠道菌群所利用的代谢特征,CN114657230A虽然利用仿生消化技术优化了体外发酵方法的高精准度和重复性,并对纤维原料的发酵特性进行了评估,但是却忽视了纤维原料在体内的动态降解变化,同时未考虑到个体对相同纤维的响应差异。
发明内容
为了克服现有技术的不足,本发明设计提供一种评估肠道菌群对菊粉的动态响应方法,评估猪肠道微生物代谢纤维能力,为日粮纤维的高效利用提供方法和指导,有助于靶向筛选关键菌的种类。
一种评估肠道菌群对菊粉的动态响应方法,步骤如下:
1)采集猪新鲜粪便;
2)体外模拟菊粉进行胃-小肠消化,获取未消化残渣作为发酵底物;
3)在厌氧培养箱中,将粪便接种液和发酵培养基加入装有发酵底物的发酵瓶中作为试验组,空白对照组不加发酵底物,将试验组和对照组于37℃进行体外发酵;
4)测定发酵过程中的发酵液中pH、乳酸含量、短链脂肪酸(SCFA)含量、β-呋喃果糖苷酶活性,其中pH值的降低,乳酸和短链脂肪酸含量的变化表示发酵底物是否被微生物所利用;同时对猪粪便发酵后发酵液中微生物进行16s高通量测序,测序所得结果显示发酵过程中微生物丰度变化;
5) 采用SPSS软件,GraphPad Prism 、STAMP软件和R进行统计和生物信息分析,确定发酵底物的动态降解特性,优势菌群相对丰度的演替变化。
所述的方法,步骤1)中粪便的采集为选择断奶后2个月健康的杜长大商品猪(DLY)和金华猪(JH)各6头,所有猪饲养管理一致,在采食后同一时间段内从猪直肠中收集粪便样本,并将其转移到无菌冻存管中,所有操作均在厌氧条件下进行,-80℃冷冻储存。
所述的方法,步骤2)所述的发酵底物制备方法为:所述的菊粉由β-(2→1)连接的D-果糖组成,聚合度大于23; 将25g菊粉溶于300 mL的PBS磷酸缓冲盐溶液(pH 7.2~7.4,2mM KH2PO4,8mM Na2HPO4,136mM NaCl,2.6mM KCl)中,随后加入2.25 ml α-淀粉酶,37℃,150 rpm,反应15 min;用1M HCl调整pH至2.5±0.1,加入10 ml 质量百分比10%的胃蛋白酶,37℃,150 rpm,反应30 min;随后加入50 mL 0.1M马来酸钠缓冲液,加入1 M NaHCO3调整pH至6.9±0.1;再加入50 mL 质量百分比12.5% 胰蛋白酶,最后加入2 mL淀粉葡萄糖苷酶,37℃,150 rpm,反应2 h;95%乙醇沉淀处理,透析24 h后冷冻干燥,最终得到消化后的菊粉用于体外厌氧发酵。
所述的方法,步骤3)中,
a)发酵培养基配方为:0.16 g/L 蛋白胨,0.1 g/L 酵母浸出物,0.16 g/L 吐温80,0.16 g/L NaHCO3,3.6 g/L NaCl,1.6 g/L K2HPO4,0.32 g/L L-半胱氨酸盐酸盐,0.36g/L CaCl2·6H2O,0.5 g/L MgSO4·7H2O,0.01 g/L 血红素;
b)体外厌氧发酵:将1 g发酵底物溶于25 mL无菌发酵培养基中得到待发酵菊粉溶液,100℃煮沸10 min;随后放入厌氧室中,冷却到室温,放置2 h;0.1 g粪便样本添加到5mL的无菌培养基中得到粪菌悬液,振荡涡旋混匀至均质状态;将2.5 mL粪菌液与2.5 mL待发酵菊粉溶液混合,最终发酵体系为5 mL,粪便浓度:1%,待发酵菊粉浓度:2%;37°C厌氧条件下以130 rpm振荡培养。
所述的方法,步骤4)中,在发酵的第1、3、6、9、12、15、18、21、24、48和72小时进行采样;采取冰浴终止发酵,将发酵液离心,上清液和沉淀物-80℃冻存。
所述的方法,测量各时间点pH值、气相色谱法定量分析短链脂肪酸含量、ELISA法测定β-呋喃果糖苷酶活性和乳酸含量;提取各时间点发酵液离心后沉淀物中基因组DNA,使用NanoDrop 2000测定提取DNA的浓度和纯度,所有样品均使用适用于细菌16S rDNA 的V3-V4区域进行PCR扩增,利用Illumina MiSeq平台测序。得到的以上数据,作为评估菊粉动态发酵性能的指标。
所述的方法,用于挖掘决定日粮纤维代谢响应能力差异的肠道微生态。
所述的方法,用于评估肠道微生物碳水化合物代谢与肠道健康关系。
与现有技术相比,本发明具有如下优点:
厌氧发酵培养基优化:与传统体外发酵培养基比较分析,测定体外发酵后的pH值、大肠杆菌数量以及产生的短链脂肪酸含量。阐明与传统体外发酵培养基相比,改良培养基粪菌厌氧培养后大肠杆菌数量更低,提高发酵结果的准确性。
本发明的有益效果在于:
本发明提供一种体外消化联合体外发酵技术评价菊粉动态发酵特性的方法,利用16s高通量测序分析不同品种肠道微生物发酵菊粉过程中菌群的动态变化,通过生信技术整合代谢结果挖掘决定日粮纤维代谢响应能力差异的肠道微生态特征,为肠道微生物碳水化合物代谢与肠道健康关系提供评估方法。
附图说明
图1是厌氧培养基优化前后大肠杆菌分布图。
图2是体外消化步骤示意图。
图3是体外发酵步骤示意图。
图4是体外消化前后菊粉对比图。
图5是pH、SCFA、乳酸和酶活性变化图。
图6是微生物多样性变化图。
图7是菊粉发酵阶段图。
图8是微生物在门和科水平的动态变化图。
图9是微生物共丰度响应群网络关系图。
具体实施方式
以下结合附图和实施例对本发明做进一步的阐述。
实施例1培养基的优化
表1. 培养基1号和培养基2号的配制参数
表1公开了培养基1号和培养基2号的配制参数。参见图1,与传统体外发酵培养基比较分析,测定体外发酵后的pH值、大肠杆菌数量以及产生的短链脂肪酸含量。结果验证了在传统体系发酵的过程中,大肠杆菌过度增殖进而影响后续试验结果准确性的问题;同时阐明与传统体外发酵培养基相比,改良培养基粪菌厌氧培养后大肠杆菌数量更低。优化后的培养基配方为:0.16 g/L 蛋白胨,0.1 g/L 酵母浸出物,0.16 g/L 吐温80,0.16 g/LNaHCO3,3.6 g/L NaCl,1.6 g/L K2HPO4,0.32 g/L L-半胱氨酸盐酸盐,0.36 g/L CaCl2·6H2O,0.5 g/L MgSO4·7H2O,0.01 g/L 血红素。
实施例2体外模拟消化菊粉
操作如图2所示,25g菊粉(比利时BENEO公司)溶于300 mL的PBS磷酸缓冲液中,随后加入2.25 ml α-淀粉酶,37℃,150 rpm,反应15 min。用1M HCl调整pH至2.5±0.1,加入10 ml 质量百分比10%的胃蛋白酶,37℃,150 rpm,反应30 min。随后加入50 ml 0.1M马来酸钠缓冲液,加入1 M NaHCO3调整pH至6.9±0.1。再加入50 ml 质量百分比12.5% 胰蛋白酶,最后加入2 ml淀粉葡萄糖苷酶,37℃,150 rpm,反应2 h。之后用95%乙醇沉淀处理,透析24 h后进行冷冻干燥。最终获得用于体外发酵的底物。图4扫描电子显微镜显示了体外消化前后菊粉的形态特征。
实施例3肠道菌群对菊粉的动态响应
1、粪便接种发酵
培养基配制按实施例1中的方法进行,选择断奶后2个月健康的杜长大商品猪(DLY)和金华猪(JH)各6头。采集其粪菌并进行发酵底物(预消化处理菊粉)体外肠道微生物发酵。具体操作如图3所示,将1 g已进行体外消化的菊粉置于25 mL无菌发酵培养基中,煮沸10 min,煮沸后的纤维溶液转移至厌氧室,冷却至室温。将0.1 g粪便样本加入5 mL无菌发酵培养基中,均质。取2.5 mL均质粪菌悬液与2.5 mL纤维溶液混合(最终粪菌浓度1%,纤维浓度2%),在厌氧条件下,37°C, 130 rpm振荡培养。所有发酵步骤都在厌氧室中进行。在体外发酵的第1、3、6、9、12、15、18、21、24、48和72h进行采样。
2、各时间点pH、乳酸、短链脂肪酸(SCFA)和β-呋喃果糖苷酶活性的测量
使用标准pH计测量每个采样时间点的pH值。取1 ml发酵液,20, 000× g离心15min,取上清液800 μL,与200 μL的25% (w/v)磷酸混合,随后取200 μL进行气相色谱上机,检测SCFA含量。样品8, 000 × g离心20 min后,取上清液,采用ELISA法测定β-果糖呋喃苷酶活性和乳酸浓度。
3、微生物测序
采用E.Z.N.A.®粪便DNA试剂盒提取微生物基因组DNA。提取的DNA浓度和纯度用NanoDrop 2000 (Thermo Scientific, Wilmington, USA)测定,用1%琼脂糖凝胶检测。所有样本均采用V3-V4区进行PCR扩增,同一样品PCR产物混合后用2%琼脂糖凝胶电泳检测扩增效果,目的条带用DNA切胶回收试剂盒进行回收。将已构建好的PCR产物文库参照电泳初步定量结果,采用AxyPrep DNA凝胶提取试剂盒和Quantus™ 荧光计进行DNA荧光定量检测,样品等量混合,构建基因组测序上机文库,通过 Illumina MiSeq 测序平台进行双末端测序。
4、生物信息分析
使用Illumina-utils 拼接质量控制后的序列,使用UCHIME移除 PCR 扩增过程中的嵌合体;使用UPARSE 聚类操作分类单元(OTU)。细菌从门到属的分类由Greengenes数据库进行对比注释,通过NCBI平台进一步确定感兴趣的序列,通过BLAST算法优化,筛选出高度相似序列的物种。采用QIIME1和QIIME2进行多样性分析。此外,利用PICRUSt2进行KEGG代谢功能注释。1)发酵阶段的分析:采用R语言中的vegdist包计算各个时间点的微生物群落相似性(Bray-Curtis距离),eclust进行层次聚类分析,根据K-means得到最佳分类簇。2)利用随机森林得到随时间响应的微生物,计算随发酵阶段变化的微生物丰度值。进行Spearman相关性分析,构建相关矩阵和层次聚类,采用Cytoscape (v3.8.2)构建共线性网络,最终得到影响菊粉降解的关键物种。
5、结果与分析
不同时间点表观指标变化
如图5所示,金华猪和杜长大猪体外发酵菊粉产生的SCFA水平有差异,与DLY相比,JH在12、24、48和72 h的乙酸含量增加幅度较大(P<0.05),9 h和15h时JH丙酸产量较高(P<0.05),JH中丁酸浓度在9 h时急剧升高(P<0.05)。总SCFA浓度JH在72 h显著高于DLY(P<0.05)。与DLY相比,JH乳酸含量在15 h迅速增加(P<0.05)。JH在3 h和15 h时的pH值显著低于DLY (P<0.05)。发酵过程中两个猪种酶活性的变化相似,JH在21 h时的β-呋喃果糖苷酶活性高于DLY(P<0.05)。可以说明本发明通过体外粪便发酵实验揭示了不同肠道微生物组成利用菊粉的代谢差异。
微生物多样性的动态变化
如图6所示,JH和DLY的微生物丰富度(OTUs数量)和多样性(Shannon指数)均呈下降趋势。除12、24和72 h外,JH微生物丰富度均高于DLY(P<0.05),Shannon指数仅在1 h和15h时大于DLY(P<0.05)。两个品种的β-多样性有显著差异,不同时间点微生物结构呈现出随时间分布的特征。JH的微生物群落相似性与DLY具有相似的变化趋势,表明两个猪种的肠道微生物对相同的纤维干预反应相似,进一步说明本发明通过体外发酵实验揭示了纤维的动态降解过程。
菊粉的发酵过程呈现阶段性变化
如图7所示,根据Bray-Curtis的群落结构相似性,JH可分为1 h、3-12 h、15-24 h和48-72 h 4个阶段,DLY分为1-6 h、9-15 h、18-24 h和48-72 h 4个阶段。为了进一步验证纵向划分的合理性,根据OTU和KEGG水平进行PCA分析,结果显示两猪种微生物群落根据上述时间点划分聚类。这些结果表明,菊粉的动态发酵过程可以分为4个阶段,分别代表细菌对纤维利用的初始、早期、中期和后期阶段。
微生物在门和科水平的动态变化
如图8所示,两个猪种体外发酵菊粉其微生物在门和科水平上随时间而明显变化,主要表现为早期以克里斯藤森菌、毛螺菌、肠球菌和肠杆菌占主导,后期以乳酸杆菌、双歧杆菌和巨球型菌为优势菌株。再次说明了肠道微生物利用菊粉过程中微生态群落的动态演替。
共丰度响应群(CARGs)的网络关系
如图9所示,共现网络分析用于识别相互关联的响应群,这些响应群可作为菊粉降解过程中发挥作用的主要微生物群落。总共得到五个不同的CARGs。CARG5与CARG1和CARG2的细菌类群呈负相关关系。CARG2中有6个属于Enterococcus属的OTUs,其中Enterococcuscecorum(OTU649)与Ligilactobacillusagilis(OTU656)呈正相关关系。Streptococcus gallolyticus(OTU273)与CARG1中的5个OTU呈强负相关关系。此外,CARG3中的Limosilactobacillusreuteri(OTU277)与OTU273呈正相关,与Lactobacillus delbrueckii(OTU282)和OTU656呈负相关。该结果为精准筛选出纤维靶向促进生长的益生菌种类提供了一定的研究基础。
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对发明范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。因此,本发明的保护范围应以所附权利要求为准。
Claims (8)
1.一种评估肠道菌群对菊粉的动态响应方法,其特征在于,步骤如下:
1)采集猪新鲜粪便;
2)体外模拟菊粉进行胃-小肠消化,获取未消化残渣作为发酵底物;
3)在厌氧培养箱中,将粪便接种液和发酵培养基加入装有发酵底物的发酵瓶中作为试验组,空白对照组不加发酵底物,将试验组和对照组于37℃进行体外发酵;
4)测定发酵过程中的发酵液中pH、乳酸含量、短链脂肪酸(SCFA)含量、β-呋喃果糖苷酶活性,其中pH值的降低,乳酸和短链脂肪酸含量的变化表示发酵底物是否被微生物所利用;同时对猪粪便发酵后发酵液中微生物进行16s高通量测序,测序所得结果显示发酵过程中微生物丰度变化;
5)采用SPSS软件,GraphPad Prism、STAMP软件和R进行统计和生物信息分析,确定发酵底物的动态降解特性,优势菌群相对丰度的演替变化。
2.根据权利要求1所述的方法,其特征在于,步骤1)中粪便的采集为选择断奶后2个月健康的杜长大商品猪和金华猪各6头,所有猪饲养管理一致,在采食后同一时间段内从猪直肠中收集粪便样本,并将其转移到无菌冻存管中,所有操作均在厌氧条件下进行,-80℃冷冻储存。
3.根据权利要求1所述的方法,其特征在于,步骤2)所述的发酵底物制备方法为:所述的菊粉由β-(2→1)连接的D-果糖组成,聚合度大于23; 将25g菊粉溶于300 mL的PBS磷酸缓冲盐溶液(pH 7.2~7.4,2mM KH2PO4,8mM Na2HPO4,136mM NaCl,2.6mM KCl)中,随后加入2.25 ml α-淀粉酶,37℃,150 rpm,反应15 min;用1M HCl调整pH至2.5±0.1,加入10 ml质量百分比10%的胃蛋白酶,37℃,150 rpm,反应30 min;随后加入50 mL 0.1M马来酸钠缓冲液,加入1 M NaHCO3调整pH至6.9±0.1;再加入50 mL 质量百分比12.5% 胰蛋白酶,最后加入2 mL淀粉葡萄糖苷酶,37℃,150 rpm,反应2 h;95%乙醇沉淀处理,透析24 h后冷冻干燥,最终得到消化后的菊粉用于体外厌氧发酵。
4.根据权利要求1所述的方法,其特征在于,步骤3)中,
a)发酵培养基配方为:0.16 g/L 蛋白胨,0.1 g/L 酵母浸出物,0.16 g/L 吐温80,0.16 g/L NaHCO3,3.6 g/L NaCl,1.6 g/L K2HPO4,0.32 g/L L-半胱氨酸盐酸盐,0.36 g/LCaCl2·6H2O,0.5 g/L MgSO4·7H2O,0.01 g/L 血红素;
b)体外厌氧发酵:将1 g发酵底物溶于25 mL无菌发酵培养基中得到待发酵菊粉溶液,100℃煮沸10 min;随后放入厌氧室中,冷却到室温,放置2 h;0.1 g粪便样本添加到5 mL的无菌培养基中得到粪菌悬液,振荡涡旋混匀至均质状态;将2.5 mL粪菌液与2.5 mL待发酵菊粉溶液混合,最终发酵体系为5 mL,粪便浓度:1%,待发酵菊粉浓度:2%;37°C厌氧条件下以130 rpm振荡培养。
5.根据权利要求1所述的方法,其特征在于,步骤4)中,在发酵的第1、3、6、9、12、15、18、21、24、48和72小时进行采样;采取冰浴终止发酵,将发酵液离心,上清液和沉淀物-80℃冻存。
6.根据权利要求5所述的方法,其特征在于,测量各时间点pH值、气相色谱法定量分析短链脂肪酸含量、ELISA法测定β-呋喃果糖苷酶活性和乳酸含量;提取各时间点发酵液离心后沉淀物中基因组DNA,使用NanoDrop 2000测定提取DNA的浓度和纯度,所有样品均使用适用于细菌16S rDNA 的V3-V4区域进行PCR扩增,利用Illumina MiSeq平台测序;得到的以上数据,作为评估菊粉动态发酵性能的指标。
7.根据权利要求1所述的方法,其特征在于,用于挖掘决定日粮纤维代谢响应能力差异的肠道微生态。
8.根据权利要求1所述的方法,其特征在于,用于评估肠道微生物碳水化合物代谢与肠道健康关系。
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