膳食纤维虽然不能直接被人体消化和利用,但是可以调节肠道菌群及代谢物,潜在地影响宿主健康[1]。燕麦β-葡聚糖(oat β-glucan, OG)是存在于燕麦胚乳和糊粉层细胞壁的一种非淀粉多糖,具有混合的β-(1→3)和β-(1→4)糖苷键[2]。OG作为一种有益的膳食纤维,因在辅助治疗2型糖尿病[3-5]、肥胖[6-7]、结肠炎[8-9]以及生物节律紊乱引起的代谢综合征[10]等疾病中的潜力受到人们关注。

越来越多的研究表明,生物节律紊乱与肠道菌群的改变密切相关[11-12]。慢性睡眠剥夺会显著改变小鼠肠道微生物群的组成,导致乳杆菌科(Lactobacillaceae)、阿克曼氏菌属(Akkermansia)的丰度显著降低,这些菌在调节宿主免疫、增强肠上皮屏障和维持肠道稳态中起着至关重要的作用[11-12]。OG可能通过调节肠道菌群和菌群代谢物,从而缓解宿主生物节律的紊乱[8, 13]。研究表明,OG可以被肠道微生物消化,有助于小鼠肠道微生物群的重塑和对昼夜节律基因表达的调节,从而缓解高脂饮食诱导的小鼠胰岛素抵抗,消化产生的短链脂肪酸(short-chain fatty acids,SCFAs),还可以调节宿主的免疫反应[8, 13]。动物研究表明,OG可以逆转生物节律紊乱诱导的小鼠体重和血浆瘦素浓度的增加,减轻由光暗周期移位诱导的小鼠代谢综合征[10]。OG和菊粉联用还可以调节小鼠肝脏中节律基因和炎症因子(TNFα和TGFβ1)的表达,缓解昼夜节律紊乱引起的非酒精性脂肪性肝病[7]。

OG虽然已经被证明有调节肠道菌群或外周组织生物节律的潜力,可以缓解多种生物节律紊乱导致的代谢性疾病,但是关于肠道菌群在OG调节生物节律方面发挥的作用,目前只局限于门和属水平的分析,对菌群相关代谢物的研究也主要是针对SCFAs,缺乏对整体代谢轮廓转变的解析[7, 10, 13]。此外,目前尚未发现有研究揭示肠道菌群和代谢物转变是如何影响生物节律的,肠道菌群改变与节律基因表达变化之间是否存在因果关系目前也尚不清楚,因此有必要进一步探究OG调节生物节律的潜在机制。同时,还未见到有研究讨论这种调节作用对生物钟及核心节律基因表达的影响。因此,本研究拟通过体外粪菌发酵实验,探究OG对肠道菌群组成及菌群代谢物产生的影响,分析与生物节律相关的关键菌群和代谢物变化以及这些关键微生物-代谢物之间的关联。此外,本研究拟采用Caco-2细胞模型,在体外验证OG体外粪菌发酵产物对肠道细胞核心节律基因表达的影响,为OG预防宿主生物节律紊乱提供理论依据。

1 材料与方法

1.1 材料与试剂

OG(质量分数为85%),上海博飞美科化学科技有限公司;褪黑素(Melatonin),阿拉丁生化科技股份有限公司;mGAM培养基,青岛海博生物技术有限公司;人克隆结肠腺癌细胞(Caco-2细胞),上海中科院细胞库;DMEM细胞培养基、胎牛血清、胰酶,美国Thermo Fisher Scientific公司;细胞双抗(青霉素-链霉素),上海博光生物科技有限公司。

RNA提取试剂盒(RC112-01)、逆转录试剂盒(R433)、qPCR酶(Q711-02),南京诺唯赞生物科技股份有限公司;粪便DNA提取试剂盒,美国MP Biomedicals公司;DNA凝胶回收试剂盒,美国Biomiga公司。

1.2 仪器与设备

AW400SG型厌氧工作站,英国Electrotek公司;CFX384型荧光定量PCR系统,美国伯乐公司;二氧化碳培养箱、NanoDrop 2000型超微量光度计、Vanquish Flex型液相色谱仪,美国Thermo Fisher Scientific公司。

1.3 实验方法

1.3.1 体外粪便菌群发酵实验

共有6名健康的成年志愿者(3女3男,年龄为21～28岁,居住在中国无锡)为本研究提供新鲜粪便,所有志愿者在样本采集前至少6个月没有接受过抗生素、益生菌或益生元治疗。粪便收集过程符合人类福利要求和伦理规范,所有志愿者都已签署知情同意书。根据Pan等[14]的描述进行粪便处理,并对方法做了一些改进。将来自6名志愿者的新鲜粪便称重并等量混合,在含1 g/L半胱氨酸的生理盐水中均质化,充分混匀,其中粪便质量(g)与生理盐水体积(mL)的比例为1∶7,随后用4层无菌纱布过滤,以去除不溶性小颗粒和杂质。将过滤后的粪便匀浆立即放入厌氧工作站作为用于接种的细菌悬浮液,从粪便收集到接种的时间应控制在2 h内,所有操作尽可能在厌氧工作站中进行。

Rettedal等[15]曾报道mGAM培养基可以在体外有效地还原人肠道细菌群落,因此本研究选用mGAM培养基作为体外发酵的培养基,并对配方进行改进。mGAM培养基主要由15 g/L䏡胨,10 g/L胰酪蛋白胨,3 g/L大豆蛋白胨,13.5 g/L消化血清,5 g/L酵母浸膏,2 g/L牛肉浸膏,1.2 g/L肝脏浸膏,3 g/L葡萄糖,3 g/L氯化钠,0.3 g/L可溶性淀粉,0.3 g/L L-半胱氨酸,0.15 g/L硫代乙醇酸钠和 2.5 g/L 磷酸二氢钾组成,在115 ℃下,高温高压灭菌20 min。待培养基冷却至50 ℃左右时,将1 μg/mL 维生素K和5 μg/mL氯化血红素过膜除菌(0.22 μm)后加入。正式实验时将培养基中主要碳源(包括葡萄糖和可溶性淀粉,3.3 g/L)替换为等量的OG(OG发酵组)或无碳源发酵(CON组)。

将粪便悬浮液与浓缩mGAM培养基(浓缩1.67倍,目的是确保与粪便细菌悬浮液混合后的完全培养基各组分浓度正确)以2∶3的体积比混合,然后在37 ℃厌氧培养。分别在0 h和24 h收集样品,8 000 r/min离心15 min,收集上清液进行细胞实验和非靶向代谢组学分析,沉淀用于进行细胞实验和16S rDNA基因测序分析[16-17]。

1.3.2 OG发酵后粪便微生物群的16S rDNA基因扩增子测序

按照DNA提取试剂盒操作说明,提取OG(0 h)和OG(24 h)组粪便菌群的DNA。根据报道的引物(341F: 5′-CCTAYGGGRBGCASCAG-3′, 806R: 5′-GGACTACNNGGGTATCTAAT-3′)进行V3～V4区PCR扩增[16]。PCR条件包括:1)在95 ℃初始变性5 min; 2)在95 ℃变性30 s;3)在52 ℃退火30 s;4)在72 ℃延伸30 s;5)反应在72 ℃下维持10 min;6)在12 ℃下维持10 min。其中2)至6)步骤重复30个循环,同时设置一组以去离子无菌水为模板的阴性对照。通过凝胶电泳纯化DNA,用DNA凝胶回收试剂盒回收PCR产物(465 bp),所有纯化的DNA样本使用Illumina测序平台进行测序,最后用Quantitative Insights into Microbial Ecology 2(QIIME2)中的DADA2包进行下机数据处理和质量控制。Beta多样性使用未加权Unifrac距离(unweighted Unifrac distance)计算,并在联川云网站(https:∥www.omicstudio.cn/tool)上进行主坐标分析(principal coordinates analysis,PCoA)图的可视化。线性判别分析效应大小(linear discriminant analysis effect size,LEfSe)分析在Bioinfo Intelligent Cloud网站(https:∥www.bic.ac.cn/BIC/#/analysis)上进行,并基于线性判别分析(linear discriminant analysis,LDA)结果识别差异菌属或菌种。

1.3.3 OG体外粪菌发酵上清液的非靶向代谢组分析

将150 μL发酵上清液与600 μL 4 ℃预冷的乙腈-甲醇溶液(乙腈、甲醇与水三者体积比为2∶2∶1)混合,于冰水浴中超声提取10 min。于-20 ℃静置40 min沉淀蛋白,再以13 000 r/min的转速低温(4 ℃)离心15 min,最后取上清液用于LC-MS分析。色谱柱条件:Thermo超高效液相色谱和HILIC色谱柱(2.1 mm×100 mm,1.7 μm),流动相A和B分别为水相和乙腈。正离子模式的洗脱液组成:100% H2O、25 mmol/L CH3COONH4、25 mmol/L NH4OH(流动相A)。负离子模式的洗脱液组成:100% 乙腈(流动相B)。色谱条件:温度40 ℃,流速0.3 mL/min,进样体积为2 μL。原始数据的处理是通过Compound Discoverer 3.3(Thermo Fisher Scientific,USA)软件完成的,来自正离子模式和负离子模式的所有数据被合并成一个单一的特征表。偏最小二乘判别分析(partial least squares discriminant analysis,PLS-DA)、火山图、聚类图分析、KEGG通路富集分析都在分析网站(https:∥www.metaboanalyst.ca)上进行。差异代谢物的选择基于variable importance in projection(VIP)值(来自PLS-DA)、差异表达倍数(fold change,FC)值和t检验的显著性。最后基于Spearman相关性构建微生物-代谢物关联网络,用R语言Hmisc安装包进行分析,将Padj<0.05且r>0.8定义为统计学上显著,并用Gephi软件进行绘图。

1.3.4 Caco-2细胞-菌群/上清液共培养模型的建立

Caco-2细胞按照Zhai等[18]的方法进行培养。当细胞达到85%(面积占比)的融合率时,用胰酶在37 ℃下消化2～3 min并传代。将细胞以105个/孔的浓度接种在12孔板中,37 ℃孵育24 h。按照文献[19]和[20]报道的方法进行细胞同步化处理,先用磷酸盐缓冲液(pH值为7.0)洗涤2次,加入含体积分数为0.05%的胎牛血清的DMEM培养基,进行过夜的细胞饥饿处理;再用含体积分数为50%的胎牛血清的DMEM培养基进行2 h的血清休克处理[19-20]。用磷酸盐缓冲液洗涤2次后,再替换为无血清的1 mL DMEM培养基,并按照4%的体积比加入发酵上清液(过膜除菌)或加入107 CFU/mL的发酵菌体于各组细胞中[21]。空白对照组(CON组)不做任何处理,阳性对照组(Mel组)在培养基中加入2 mmol/L的褪黑素(过膜除菌),单糖对照组(OG组)在培养基中加入500 μg/mL的OG(过膜除菌),在37 ℃孵育12 h后收集细胞沉淀,并进行RNA提取实验[22-24]。

1.3.5 细胞节律基因mRNA 转录水平的测定

使用RNA提取试剂盒提取细胞总RNA,用超微量光度计进行浓度和RNA质量评估,用逆转录试剂盒将RNA逆转录成cDNA。再使用qPCR酶在BioRad-CFX 384仪器上进行实时荧光定量PCR,分析目标基因的mRNA转录水平。以GAPDH为内参基因,通过2-ΔΔCt计算目标基因的相对变化水平,ΔΔCt=(Ct目的基因-Ct内参基因)实验组-(Ct目的基因-Ct内参基因)对照组,目标基因引物序列见表1[19, 22, 25]。

1.4 数据处理

所有实验数据用SPSS software (version 19.0)和GraphPad Prism 7.0软件进行处理。对于两组间数据的比较,使用学生双尾非配对t检验。涉及两组以上的数据,使用单因素方差分析(one-way ANOVA),再用Tukey’s多重比较检验。

2 结果与分析

2.1 OG对粪便菌群的影响

OG具有调节肠道微生物群及其代谢物,缓解宿主生物节律紊乱的潜力,但是未见有研究对肠道关键微生物群转变进行深入的节律关联性分析,或是针对肠道微生物群种水平的分析。为了探究OG对肠道微生物群组成的影响,本研究通过体外粪便菌群发酵实验,对加入OG发酵后的粪便微生物群进行16S rDNA基因扩增子测序分析,结果如图1所示。由图1(a)可见,与Bai等[26]报道的研究结果一致,经过OG发酵后,粪便菌群的Shannon指数极显著上升(P<0.01),说明肠道微生物多样性增加。使用未加权Unifrac距离和PCoA对粪便菌群进行Beta多样性可视化分析,由图1(b)可知,与CON组相比,OG组菌群结构发生偏移,说明OG发酵前后菌群结构差异明显。

从门水平看,由图1(c)可知,OG发酵前后粪便微生物相对丰度发生显著变化。由图1(d)和图1(e)可知,与对照组相比,OG发酵后粪便菌群中厚壁菌门(Firmicutes)相对丰度极显著升高(P<0.01),变形菌门(Proteobacteria)相对丰度极显著降低(P<0.01)。Proteobacteria是一类可以产生内毒素的病原菌,与慢性炎症、节律位移有关[27-28]。F/B值为Firmicutes与拟杆菌门(Bacteroidetes)相对丰度的比值,是衡量肠道稳态的一个重要指标,较高的F/B值通常被认为跟肥胖有关。由图1(f)可知,在本研究中OG可以降低肠道菌群F/B值至接近CON组的正常水平(P<0.01),这说明OG对肠道菌群可以产生有益影响[29-30]。

此外,本研究还对OG发酵后的粪便菌群变化进行了属水平和种水平的分析,结果见图2。由图2(a)可知,OG可导致粪便菌群组成和丰度发生显著变化。使用LEfSe分析观察两组肠道微生物群的生物标志物,以P<0.05和LDA >3为筛选标尺列出了主要差异菌属。值得一提的是,一般研究通常是基于LDA >2进行差异菌筛选[31-32],但是本研究中差异菌数量较多,因此为了优先分析和讨论更显著的结果,本研究将筛选标准提高到LDA>3。由图2(b)可知,与CON组相比,OG组包含的差异菌属主要有双歧杆菌属(Bifidobacterium)、副拟杆菌属(Parabacteroides)、粪杆菌属(Faecalibacterium)和另枝杆菌属(Alistipes)等,而Dorea和Ruminococcus torques group等差异菌属在CON组占优势。对这些关键菌属的相对丰度变化分析,由图2(c)和图2(d)可以看出,潜在有益菌Bifidobacterium、Parabacteroides、Alistipes和Faecalibacterium的相对丰度在OG发酵后极显著增加(P<0.01),潜在有害菌Dorea和Ruminococcus torques group的相对丰度在OG发酵后极显著下调(P<0.01),这些结果表明,OG可以显著调节肠道微生物的结构和丰度。Dorea和Ruminococcus torques group通常在夜班人群中具有较高的丰度,Dorea丰度升高往往与肠道通透性增加有关,这预示着OG可能具有维持肠道屏障功能的作用[33-34]。肠道核心菌属Faecalibacterium是肠道丁酸盐的主要产生者之一,丁酸盐可以通过抑制细胞中的组蛋白脱乙酰酶(histone deacetylase,HDAC)来调控生物钟基因的表观遗传修饰[35-36]。此外,研究显示,Bifidobacterium和Parabacteroides也跟宿主昼夜节律紧密相关,在经历光照/黑暗周期颠倒后,Parabacteroides可以通过降低粪便次级胆汁酸的相对水平,促进宿主生理状态(核心体温、睡眠)昼夜节律的重新调整[37]。Bifidobacterium可以通过调节肠道中的异戊酸和γ-氨基丁酸以及血清中的嘌呤代谢物的生成,缓解睡眠剥夺诱导的昼夜节律失调[38]。

对两组粪便菌群进行种水平上的LEfSe分析,此处本研究选用的是大多数文献选择的差异菌筛选标尺:P<0.05和LDA>2[31-32]。由图2(e)可知,与CON组相比,检测到长双歧杆菌(Bifidobacterium longum)、短双歧杆菌(Bifidobacterium breve)和两歧双歧杆菌(Bifidobacterium bifidum)等菌种在OG组富集。Bifidobacterium breve可以调节肠道异戊酸和γ-氨基丁酸水平,这些代谢物具有调节褪黑激素分泌和生物钟基因表达的潜力[39]。由图2(f)的种水平相对丰度变化分析可知,与对照组相比,OG发酵后粪便菌群中的Bifidobacterium longum的相对丰度极显著升高(P<0.01),该菌种已被报道具有提升睡眠质量的作用[40]。OG对这些肠道关键菌的调节作用,可能是OG能够调节生物节律紊乱诱导的肠道菌群失衡的重要原因。

2.2 OG对肠道菌群代谢物的影响

为了探究OG对肠道菌群代谢物的调节作用,本研究对OG发酵后的上清液进行非靶向代谢组分析,结果见图3。在本研究中,使用LC-MS在正、负离子模式下测定发酵24 h的样品。图3(a)的主成分分析结果显示,CON和OG组分离明显,说明OG对粪便菌群代谢物的转变效果显著。再采用多因素和单因素分析来评估实验组之间的差异。通过PLS-DA分析得到VIP值,以VIP>1为筛选标准,图3(b)的筛选结果列出了VIP排名前50的代谢物。此外,还结合t检验(P<0.05)和Fold Change值(FC>2或FC<0.5)来筛选差异物质,并进行了火山图分析[图3(c)]和聚类热图分析[图3(d)],最后将筛选到的差异代谢物进行KEGG通路富集分析,图3(e)列出了显著性排名前15的代谢通路。与CON组相比,OG组5-羟色氨酸(5-hydroxytryptopha,5-HTP)、黄豆黄素(glycitein)、L-色氨酸(L-tryptophan)、D-(+)-色氨酸[D-(+)-tryptophan]的含量升高,而尿刊酸(urocanic acid)、硫胺素(thiamine)、3-乙酰氨基苯酚(3-acetamidophenol)的含量降低。glycitein可以通过SIRT5-NF-κB(sirtuin 5-nuclear factor-κB)轴影响类风湿性关节炎炎症节律,据报道,glycitein含量的提高很可能跟高丰度的Parabacteroides有关[39]。KEGG通路富集分析结果显示[图3(e)],色氨酸代谢通路在OG的调节通路中富集。色氨酸代谢已经被证明可以调节昼夜节律稳态,5-HTP作为色氨酸代谢的中间产物,可以由 L-tryptophan 转化而来,并最终被肠道微生物转化为血清素[41-42]。血清素是一种神经递质,血清素水平过低会影响大脑功能发育,并且与昼夜节律基因的延迟表达有关[43]。褪黑素也是生物节律的一个重要调节信号,虽然不能由肠道微生物直接生成,但是5-HTP是褪黑素合成的重要前体物质,因此OG可能通过提高肠道5-HTP水平,从而影响褪黑素分泌和调节生物节律[44-45]。

2.3 OG转变的关键微生物-代谢物的交互作用网络分析

基于肠道微生物组和代谢组的整体性,肠道微生物组的改变可能会导致代谢组的显著变化,因此本研究进行了OG转变的关键微生物-代谢物的交互作用网络分析,以整合和可视化肠道微生物组与代谢物之间的关联,结果见图4[46]。由图4(a)可见,在26个代谢物节点中,glycitein(25个连接)和L-tryptophan(63个连接)已经被报道跟生物节律相关[43, 47-48]。其中glycitein水平与巨单胞菌属(Megamonas)丰度呈负相关,如2.1中图2(a)和图2(b)的16S rDNA分析结果可知,Megamonas的丰度在OG发酵后下降,因此glycitein水平升高可能和OG调节的肠道Megamonas丰度降低有关[49]。

从差异菌属看,Fusicatenibacter的丰度,与8个代谢物存在相关性,其中与L-tryptophan水平呈正相关。在Bifidobacterium和Alistipes的连接中,与6个代谢物节点有关联,其中与D-(+)-tryptophan水平呈正相关。L-tryptophan和D-(+)-tryptophan都可参与色氨酸代谢,在维持和调节宿主生物节律中起着重要作用[43, 47]。从OTU水平上看,Bifidobacterium longum(OTU71)与7个代谢物相关,其中与油酸(oleic acid)水平呈正相关。Oleic acid是脂肪组织昼夜节律的调节剂,可用作预防肥胖及其并发症的饮食干预策略[50]。结合16S rDNA分析可知,上述差异菌群的丰度都在OG组升高,因此OG可能会诱导肠道微生物组成的转变,从而影响一些具有节律调节潜力的代谢物水平变化,特别是升高色氨酸代谢通路相关代谢物的水平。

基于文献调研结果,本研究总结了OG的分解代谢过程[图4(b)]。OG可能由来自肠道微生物的β-葡糖苷酶(GH1和GH3,EC 3.2.1.21)和β-葡聚糖酶(GH16,EC 3.2.1.73)分解为葡萄糖,后经过糖酵解途径生成丙酮酸,最终转化为SCFAs(包括乙酸、丙酸、丁酸)[51-53]。SCFAs水平的升高已经被证明可以缓解生物节律紊乱,因此转变SCFAs水平可能是OG调节生物节律的主要机制之一[54]。

2.4 OG体外粪菌发酵产物对Caco-2细胞节律基因表达的影响

为了验证OG发酵转变的差异菌群和代谢物是否是其调节宿主生物节律的关键因素,本研究探究了不同发酵产物对Caco-2细胞模型节律基因mRNA表达水平的影响(图5)。Caco-2细胞类似于未分化的小肠上皮细胞,将发酵产物与细胞接触可以在体外模拟肠道内的相互作用[55]。将OG及其体外粪便微生物发酵产物与Caco-2细胞共培养,以CON组为空白对照、Mel组作为阳性对照组,接触12 h后测定了7种核心节律基因表达量的变化,包括Bmal1、Clock、Per2、Per3、Cry1、Cry2、Rev-erbα[56]。

由图5(a)至图5(g)可知,对比CON组和OG组,发现单纯将OG加入细胞培养基中只能极显著改善细胞Rev-erbα基因的表达(P<0.01)。同样地,由图5(a)至图5(g)可知,与CON组相比,将粪便菌群直接与细胞接触也只对Rev-erbα基因有极显著调节效果(P<0.01),对其他基因的表达基本没有影响。但是加入OG发酵24 h后的粪便菌群可以显著促进大部分测定节律基因的表达,包括Bmal1(P<0.01)、Clock(P<0.01)、Per2(P<0.05)、Per3(P<0.01)、Cry2(P<0.05)和Rev-erbα(P<0.01)。此外,OG发酵后的菌群上清液也可以刺激细胞,显著影响大部分核心节律基因的表达,包括Bmal1(P<0.01)、Clock(P<0.05)、Per2(P<0.05)、Per3(P<0.05)、Cry2(P<0.05)和Rev-erbα(P<0.05),而不加碳源的发酵上清液不能得到预期结果,说明菌群上清液的生物节律调节作用可能归因于粪便菌群对OG的利用而产生的特异代谢物。

此外,研究表明,这些核心时钟基因还调控着肠道健康,Bmal1表达水平下降的小鼠对 DSS 诱导的结肠炎更敏感,Rev-erbα可以靶向调控肠道炎性细胞因子(IL-1β、IL-18和IL-6等)的表达[57-58]。因此,OG可能通过调节肠道菌群,从而调节肠道节律基因表达及肠道健康。

3 结 论

本研究发现,补充OG可以增加粪便菌群物种多样性,增加Firmicutes、Bifidobacterium、Parabacteroides、Faecalibacterium、Bifidobacterium longum等菌群的丰度,提高5-HTP、L-tryptophan和glycitein等代谢物的含量,转变后的菌群及代谢物可以在体外显著调节Caco-2细胞节律基因的表达。对菌群和代谢物进行网络关联分析发现,OG诱导的肠道菌群组成转变关联着L-tryptophan等具有生物节律调节潜力的代谢物水平变化,调节通路在色氨酸代谢通路显著富集。因此,推测OG可能通过调节肠道菌群及代谢物(特别是上调色氨酸通路相关代谢物),从而影响肠道细胞节律基因的表达,但该结论还需通过体内实验进行验证。本研究对OG调节肠道菌群和核心节律基因的作用进行了进一步探讨,希望能够为OG缓解宿主生物节律紊乱的机制研究提供理论依据。有关核心菌群与代谢物影响宿主节律基因表达的机制还有待进一步的研究和验证。

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Study on Effect of Oat β-Glucan on Host Circadian Rhythm by Altering Gut Microbiota

HUANG Lijuan1, ZHANG Yu1, GUO Min1, LU Wenwei1,2, XIAO Yue1,2, ZHAO Jianxin1,*

(1.School of Food Science and Technology, Jiangnan University, Wuxi 214122, China;2.National Engineering Research Center for Functional Food, Jiangnan University, Wuxi 214122, China)

Abstract： To examine the fermentation characteristics of oat β-glucan (OG) and its impact and mechanism on host circadian rhythms, an in vitro fecal microbial fermentation experiment was performed. 16S rDNA and non-targeted metabolomic analyses were conducted on the fecal flora and culture supernatant of OG fecal fermentation system, and a microbe-metabolite association network was constructed. The influence of altered gut microbiota and metabolites after OG treatment on the expression of rhythmic genes in Caco-2 cells were explored through in vitro cellular experiments. The results of the study demonstrated that OG induced notable alterations in the composition and structure of the gut microbiota. OG supplementation led to an increase in the abundance of Firmicutes, Faecalibacterium, Parabacteroides, and Bifidobacterium longum, along with other gut microbiota. The changed microbiota induced by OG was observed to produce more metabolites with rhythm-regulating potential, such as 5-hydroxytryptophan, L-tryptophan, and glycitein. The metabolic pathway based on differentially present metabolites after OG intervention was significantly enriched in the tryptophan metabolic pathway. The analysis of the microbe-metabolite interaction network revealed a positive correlation between the abundance of Fusicatenibacter and the level of L-tryptophan, while a negative correlation was observed between the abundance of Megamonas and the level of glycitein. Furthermore, the OG-treated fecal microbiota and associated culture supernatant were found to significantly regulate the expression of the majority of tested core clock genes in the in vitro Caco-2 cells. The findings indicated that OG might exert an indirect influence on the expression of rhythm genes in intestinal cells through the regulation of the composition of gut microbiota and associated metabolites, especially the metabolites related to the tryptophan metabolic pathway. The study would provide theoretical guidance for the development and future application of OG in alleviating circadian rhythm-related disorders.

Keywords： oat β-glucan; in vitro fecal fermentation; gut microbiota; microbiota-associated metabolites; circadian rhythm; intestinal cells

文章编号：2095-6002(2025)03-0034-12

doi：10.12301/spxb202400662

引用格式：黄丽娟,张宇,郭敏,等.燕麦β-葡聚糖调节肠道菌群影响宿主生物节律研究[J]. 食品科学技术学报,2025,43(3):34-45. HUANG Lijuan, ZHANG Yu, GUO Min, et al. Study on effect of oat β-glucan on host circadian rhythm by altering gut microbiota[J]. Journal of Food Science and Technology, 2025,43(3):34-45.

中图分类号： TS201.4; TS202.1

文献标志码： A

收稿日期： 2024-10-12

基金项目： 国家自然科学基金资助项目(32202059)。

Foundation: National Natural Science Foundation of China (32202059).

第一作者： 黄丽娟,女,硕士研究生,研究方向为益生菌、肠道菌群与健康。

*通信作者： 赵建新,男,教授,博士,主要从事益生菌资源的开发与利用、传统发酵食品工业化方面的研究。

(责任编辑：叶红波)