CN115261426A - 一种具有抗氧化能力的两歧双歧杆菌e3胞外多糖 - Google Patents
一种具有抗氧化能力的两歧双歧杆菌e3胞外多糖 Download PDFInfo
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- CN115261426A CN115261426A CN202211169944.2A CN202211169944A CN115261426A CN 115261426 A CN115261426 A CN 115261426A CN 202211169944 A CN202211169944 A CN 202211169944A CN 115261426 A CN115261426 A CN 115261426A
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- bifidobacterium bifidum
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Abstract
本发明提供了一种具有抗氧化能力的两歧双歧杆菌E3胞外多糖。所述两歧双歧杆菌E3的完整菌体(IC)、无细胞上清液(CFS)和无细胞提取物(CFE)都具有较好的抗氧化性能，其中CFS的抗氧化活性最好。所有的EPSs都是HePSs（主要含有葡萄糖、甘露糖、鼠李糖等）。FT‑IR显示了EPS典型的特征吸收峰。源自两歧双歧杆菌E3的EPS是一种很有前景的天然抗氧化剂，可在功能食品中应用，为未来在抗氧化剂结合基因组学方法中研究EPS的结构和功能特征提供范例。
Description
技术领域
本发明属于微生物技术领域，具体涉及一种具有抗氧化能力的两歧双歧杆菌E3胞外多糖及其应用。
背景技术
双歧杆菌是革兰氏阳性菌株，是人类胃肠道(GIT)菌群的常见成员，也是母乳喂养婴儿主要肠道菌群的一部分。双歧杆菌对人体健康有益，它可以维持肠道生态系统，提高免疫力，预防腹泻，降低血液胆固醇等。一些双歧杆菌通常被认为是安全的，作为益生菌在制药和乳制品中使用。随着全基因组测序和强大的生物信息学工具的出现，系统发育进入了一个新时代。与此同时，益生菌基因组学旨在为益生菌的多样性和进化提供见解，同时也试图揭示它们促进健康的分子基础。双歧杆菌被认为是哺乳动物肠道的共生微生物，因此它们的基因组中含有大量被预测为编码碳水化合物代谢相关酶的基因。从基因组的角度来看，参与粗胞外多糖(cEPS)生物合成的基因通常聚集在双歧杆菌属成员内的特定遗传位点(通常称为EPS基因簇)。不同双歧杆菌物种的比较基因组分析显示，所有分析的类群都至少有一个EPS位点，除了一些两歧双歧杆菌菌株。
胞外多糖(EPS)是由微生物在生长代谢过程中分泌到细胞壁外的多糖。乳酸菌(LAB)产生的EPS已经引起了广泛的关注。EPS有两种类型，一种是由葡萄糖或半乳糖一类单糖组成的同型多糖(HoPS)，另一种是由几种不同类型的单糖组成的杂多糖(HePS)。一般情况下，同型多糖是由蔗糖大量生产，而杂多糖是由少量葡萄糖、半乳糖、果糖、鼠李糖、N -乙酰-D-氨基葡萄糖、半乳糖胺和多元醇合成。EPS作为益生菌的代谢物，不仅会影响发酵产品的风味和质地，还会影响益生菌的益生功能。据报道，实验室制备的EPS具有增强免疫、抗氧化、抗癌、抗炎、抗病毒和降低胆固醇的作用。事实上，多糖的功能性质(生物活性和物理化学性质)与其结构特征和复杂性密切相关。其结构主要集中在单糖组成、分子量(Mw)、糖苷键、官能团和取代基的研究。此外，利用扫描电镜(SEM)评价EPS的物理性质。到目前为止，对EPS结构与其功能性质之间的机理的认识还不清楚。因此，表征LAB-EPS的化学结构对探究其功能性质至关重要。
氧化态是指抗氧化剂和促氧化剂的含量。过度氧化引起的氧化应激会加速衰老、引发炎症、降低自身免疫能力、引起氧化损伤等。近年来，许多乳酸菌LAB菌株被发现具有抗氧化活性。而且，乳酸菌产生的胞外多糖也具有抗氧化特性。而寻找不影响人体健康的天然抗氧化剂具有重要的意义。
发明内容
本发明提供了一种生产具有抗氧化能力的两歧双歧杆菌E3胞外多糖的方法，其特征在于，发酵所述两歧双歧杆菌E3得到胞外多糖，所述两歧双歧杆菌E3为Bifidobacteriumbifidum E3，保藏于中国典型培养物保藏中心，武汉，保藏编号为CCTCC NO: M 2022384，保藏日期2022年4月6日。
具体的，将所述两歧双歧杆菌E3接种到含L-半胱氨酸盐酸盐的MRS肉汤培养基中孵育；优选的，是在37℃孵育24 h，进行2次传代后，离心(5000 × g)10 min；CFS作为实验上清液，采用冷乙醇沉淀法提取EPS。
所述胞外多糖是同型多糖或杂多糖；
所述胞外多糖含有甘露糖和葡萄糖，优选的，甘露糖:葡萄糖的摩尔比为1:5.04；
进一步还包含鼠李糖，优选的，鼠李糖:甘露糖:葡萄糖的摩尔比为0.32:1:1 .56；
进一步还包含半乳糖和葡萄糖醛酸，优选的，鼠李糖：甘露糖：葡萄糖胺：半乳糖：葡萄糖：葡萄糖醛酸的摩尔比为1.10:1:0.82:1.05:1.39:0.68。
所述胞外多糖组合物可以用来制备抗氧化药物。
本发明的有益效果：
本发明提供的两歧双歧杆菌E3的研究表明，完整菌体(IC)、无细胞上清液(CFS)和无细胞提取物(CFE)具有良好的抗氧化性能，其中CFS的抗氧化性能最好。同时对EPS进行提取纯化，所有的EPSs都是HePSs（主要含有葡萄糖、甘露糖、鼠李糖等）。FT-IR显示了EPS典型的特征吸收峰。因此，EPS是一种很有前景的天然抗氧化剂，可在功能食品中应用。此外，源自两歧双歧杆菌E3的EPS可为未来在抗氧化剂结合基因组学方法中研究EPS的结构和功能特征提供范例。
附图说明
图1两歧双歧杆菌E3基因组环状图谱(A)、两歧双歧杆菌E3基因组COG功能分类(B)、两歧双歧杆菌E3基因组KEGG通路分类(C)；
图2两歧双歧杆菌E3糖核苷酸(A)和基因簇(B)的合成；
图3 EPS-1、EPS-2、EPS-3的紫外光谱(A)和FT-IR光谱(B)；
图4 EPS-1、EPS-2、EPS-3在5000 ×、10000 ×放大后的场发射扫描电子显微镜；
图5 刚果红EPS-1、EPS-2、EPS-3的测定；
图6 EPS-1、EPS-2、EPS-3的热分析；
图7 IC、CFE、CFS抗氧化活性；
图8 cEPS的抗氧化活性；
图9 EPS-1、EPS-2和EPS-3对DPPH自由基(A)、羟基自由基(B)和超氧阴离子自由基(C)的清除能力。
具体实施方式
下面结合具体实施例和附图对本发明进行进一步的阐述。
本发明所述两歧双歧杆菌E3为Bifidobacterium bifidum E3，由东北农业大学储存提供，记载在CN202210668198.5中，保藏于中国典型培养物保藏中心，武汉，保藏编号为CCTCC NO: M 2022384，保藏日期2022年4月6日。
实施例1菌株培养
两歧双歧杆菌E3由东北农业大学储存提供。将两歧双歧杆菌E3接种到含L-半胱氨酸盐酸盐(2%v/v)的MRS肉汤培养基中，37℃孵育24 h，进行2次传代后开始实验，继代培养后离心(5000 × g) 10 min。CFS作为实验上清液，将离心后的沉淀用PBS缓冲液洗涤3次，计算后加入PBS缓冲液中重悬两歧双歧杆菌E3至浓度为109 CFU/mL。本实验通过采用IC超声破碎法提取CFE，超声条件为800 W，超声3 s，停止5 s，持续15 min。
实施例2 两歧双歧杆菌E3的全基因组测序、糖运输系统及基因簇
提取两歧双歧杆菌E3的DNA，制备DNA文库。该文库在Illumina和PacBio RS II平台进行测序。两歧双歧杆菌E3基因组无质粒，只有一条环状染色体(2,245,491 bp)，G+C含量为62.75%。该基因组共预测了1878个基因，总长度为1945738 bp，编码区总长度占全基因组的86.65%。其中编码基因的平均长度为1036 bp。此外，在基因组中预测了1818个CDSs。有完整的53个tRNAs，1个tmRNA， 6个rRNA (16s和23s)基因操纵子，没有sRNA(图1A)。
两歧双歧杆菌E3的糖转运系统分析如表1所示。在基因组中，E3_01768和E3_01767编码HPr和EI。两歧双歧杆菌E3有4个PTS系统相关EII。分别为运输糖EIIB(E3_01751)、葡萄糖EIIA(E3_00280)、N -乙酰氨基葡萄糖EIIABC(E3_00281)、果糖EIIABC(E3_01538)。两歧双歧杆菌E3中，1个基因(E3_01046)编码ABC型糖15转运系统的渗透酶，3个基因(E3_01045、E3_01047和E3_01393)编码多种糖转运系统的渗透酶蛋白。此外，E3_01493基因编码的半乳糖渗透酶可以转运乳糖，E3_01538基因编码的果糖渗透酶可以转运果糖，E3_01046基因编码的乳糖渗透酶可以转运乳糖。上述分析表明，两歧双歧杆菌E3具有转运乳糖、果糖、葡萄糖、N -乙酰氨基葡萄糖和半乳糖的能力，这些物质构成了合成糖核苷酸的底物，它激活了EPS合成的前体。
表1 两歧双歧杆菌E3的糖转运系统
EPS的合成过程分为两个阶段：前体物质糖核苷酸的合成和EPS基因簇合成EPS。如图2A所示，β-半乳糖苷酶(E3_00203)可以将乳糖水解为葡萄糖和半乳糖。半乳糖通过半乳糖激酶(E3_00446)和UDP-葡萄糖-己糖-1-磷酸尿苷基转移酶(E3_00445)转化为葡萄糖-1-磷酸。通过转运系统将葡萄糖转运到细胞内，葡萄糖激酶(E3_00603)形成葡萄糖-6-磷酸。部分葡萄糖-6-磷酸通过磷酸葡萄糖变位酶(01543)转化为葡萄糖-1-磷酸，通过UDP葡萄糖焦磷酸化酶(00967)形成UDP-葡萄糖。UDP-葡萄糖和UDP-半乳糖在UDP-半乳糖-4-差向异构酶的作用下可以相互转化(E3_0047)。dTDP-葡萄糖4, 6-脱水合酶(E3_00046)、dTDP-4-脱水合鼠李糖3,5-表二酶(E3_00071)、16 dTDP-4-脱水合鼠李糖还原酶(E3_00749)也可以催化葡萄糖-1-磷酸合成dTDP-l-鼠李糖。通过PTS系统将果糖转运到细胞质中，再通过谷氨酰胺-6-磷酸脱氨酶(E3_00604)将果糖-1-磷酸转化为果糖-6-磷酸。果糖-6-磷酸通过磷酸氨基葡萄糖突变酶(E3_00477)、氨基葡萄糖-1-磷酸n -乙酰转移酶(E3_01113)和氨基葡萄糖-乙酰氨基焦磷酸化酶(E3_01113)转化为UDP-N-乙酰-α-D -氨基葡萄糖。同时，在甘露糖-6-磷酸异构酶(E3_00393)、甘露糖-6-磷酸异构酶(E3_00477)和甘露糖-1-磷酸鸟苷转移酶(E3_00879)作用下，果糖-6-磷酸转化为GDP-甘露糖。这些核苷酸糖可以组装成EPS的重复单元。
编码EPS合成的基因大多聚集在EPS产生微生物的基因组或大质粒上。如图所示2B, 两歧双歧杆菌 E3有1个由20个基因组成的EPS合成基因簇，该EPS基因簇涉及转座酶(E3_00050、E3_00052、E3_00054-57)、鼠兔前体(E3_00046、E3_00071)、ABC转运酶(E3_00065、E3_00066)、酰基转移酶(E3_00060)、UDP-半乳糖突变酶(E3_00068)、糖基转移酶(E3_00049、E3_00061、E3_00067)的编码。糖基转移酶在测定EPS单糖组成中起着关键作用。转座酶促进胞外多糖基因簇的稳定，这表明两歧双歧杆菌E3与其他菌株之间存在水平基因转移的可能性。同时，我们也在这个基因簇中发现了5个假设的蛋白质。
实施例3 cEPS的分离纯化
cEPS的提取采用冷乙醇沉淀法。简单来说就是将两歧双歧杆菌E3的CFS在沸水中灭活10分钟，然后冷却到室温，加入80% (w/v)三氯乙酸至终浓度为4% (w/v)沉淀蛋白，在4℃下孵育过夜，然后离心（5000 × g）20 min，最后收集上清液。将上清液旋转蒸发，然后加入3体积的无水乙醇，得到沉淀。然后将沉淀溶于去离子水中，在4℃下透析(8-14 kDa) 48小时，然后进行冻干。经DEAE-Cellulose-52和Sephadex G-100柱纯化得到EPS-1、EPS-2和EPS-3。
采用高效液相色谱法研究了两歧双歧杆菌E3生产的EPS的单糖组成。ESP-1、ESP-2、ESP-3的单糖组成分析结果如表2所示。EPS-1主要由甘露糖和葡萄糖组成，其摩尔比约为1:5.04。EPS-2主要由鼠李糖、甘露糖和葡萄糖组成，其摩尔比约为0.32:1:1.56。EPS-3主要由鼠李糖、甘露糖、葡萄糖胺、半乳糖、葡萄糖、葡萄糖醛酸组成，其摩尔比约为1.10:1:0.82:1.05:1.39和0.68，表明它可能是一种酸性多糖。本实验中EPS-1、EPS-2和EPS-3的分子量分别为4.15 kDa、3.67 kDa和5.89 kDa。分子量是EPS的一个重要性质，可能会影响益生菌的性能。高分子量EPS可以改善发酵乳的质地，可能比低分子量EPS具有更强的抗肿瘤活性。此外，EPS的分子量值的差异可能与其特定功能有关。
表2单糖组成及分子分析
EPS-1、EPS-2、EPS-3的FT-IR光谱如图3B所示。EPS-1、EPS-2和EPS-3具有典型多糖的特征吸收峰3396.85 cm-1、3377.78 cm-1和3365.99 cm-1，与碳水化合物羟基有关。结果表明，EPS-1、EPS-2和EPS-3的FT-IR光谱具有多糖的主要特征。
扫描电镜结果如图4所示，10000倍放大的显微图像，可以看到EPS微观结构的额外细节，显示出粗糙和块状的表面。EPS-1由形状不规则的颗粒组成的致密多孔的海绵结构，使多糖具有良好的保水能力。EPS-2具有许多均匀的片状和棒状结构，结构致密，表面粗糙。EPS-3的微观形貌为块状结构，表面凹凸不平。
刚果红实验结果显示，EPS-1和EPS-2没有明显的变色位移，表明没有三螺旋排列（图5）。从肠膜明串珠菌DRP105中提取的多糖具有相同的构象。而EPS-3+刚果红与刚果红的最大吸收波长差为9 nm。表明EPS-3可能具有三螺旋结构。
EPS-1、EPS-2、EPS-3的TGA-DTG分析如图6所示。TGA曲线显示EPS的失重分两步进行。第一步在25-100℃时，EPS-1、EPS-2和EPS-3的失重率分别为8.59%、8.09%和8.89%，这可能与水分含量和挥发性物质有关，羧基可以与更多的水分子结合，EPS中的高水平羧基增加了第一步的降解。第二降解阶段为260℃-500℃，失重速率最大。EPS的降解主要是由于C-O键和C-C键的解离，随着温度的升高，形成了多核芳香族和石墨碳结构。最后，EPS-1、EPS-2、EPS-3的权重逐渐降低至不变(17.01%、14.97%、10.06%)。如图6B所示，EPS-1、EPS-2、EPS-3的降解温度（Td）分别为314.78℃、324.07℃、320.44℃。这些结果高于植物乳杆菌HM4741的最大EPS降解温度（273.6°C）。结果表明，EPS-1、EPS-2、EPS-3具有较高的热稳定性和物理性能，可应用于食品工业。
实施例4 IC、CFE和CFS的抗氧化活性
清除DPPH自由基的实验：将1 mL的样品(IC、CFE、CFS、cEPS、EPS-1、EPS-2和EPS-3)加入1 mL DPPH (0.2 mmol/L)中。在37℃避光条件下放置30 min。将上清液6000 r/min离心10 min，测定517 nm下的吸光度。清除计算公式如下：
DPPH自由基清除(%)=(1-
)×100
公式中，空白组中加入无水乙醇溶解的DPPH溶液，对照组用PBS来代替样品，其他反应条件不变。
清除羟基自由基实验：采用试剂盒(No. A018-1-1；NanjingJianchengBioengineering Institute, Nanjing, China)清除羟基自由基测定样品(IC,CFE, CFS, cEPS, EPS-1, EPS-2 and EPS-3)对羟基自由基清除能力。清除能力计算公式如下:
羟基自由基清除能力(%)=
×100
清除超氧阴离子自由基的实验：反应溶液里含2.8 mL Tris-HCl (0.05 mol/L，pH8.2)、0.1 mL邻苯三酚(0.05 mol/L)和0.1 mL (IC、CFE、CFS、cEPS、EPS-1、EPS-2和EPS-3)，在室温下避光反应4 min，然后用1 mL盐酸(8 mol/L)终止反应，测定最终溶液在320 nm处的吸光度。在所有抗氧化试验中，维生素C（Vc）与EPS平行作为阳性对照。清除能力的计算公式如下:
超氧阴离子清除能力(%) = (1- 
)×100
公式中，空白组为去离子水。
所有数据均以均数±标准差(n = 3)表示，并使用SPSS 18.0软件(SPSS Inc.,Chicago, IL, USA)进行单因素方差分析(ANOVA)。使用GraphPad Prism 8.0.2 (GraphPadSoftware, La Jolla, CA, USA)绘制图表。P < 0.05表示为差异显著，P > 0.05表示为差异不显著。
图7显示了IC、CFE和CFS的抗氧化活性。IC、CFE和CFS对DPPH自由基、羟基自由基和超氧阴离子自由基的清除能力为18.33%-98.07%。CFS对DPPH自由基、羟基自由基和超氧阴离子自由基的清除能力最强，分别为83.08% ± 2.02%、95.68% ± 1.60%和48.20% ±0.55%，显著高于IC和CFE(p < 0.05)。IC对DPPH自由基、羟基自由基和超氧阴离子自由基的清除活性分别为37.07% ± 0.78%、77.63% ± 0.74%和28.72% ± 1.11%。3种清除活性均显著高于CFE(p < 0.05)。
实施例5 cEPS的抗氧化能力
如图8所示，cEPS对DPPH自由基、羟基自由基和超氧阴离子的清除能力随cEPS浓度的增加而显著增加(p < 0.05)，呈剂量依赖性影响。当浓度为4 mg/mL时，cEPS对DPPH自由基的清除能力最强。Vc的抗氧化能力显著高于cEPS(p < 0.05)，但不随Vc浓度的增加而发生显著变化(p > 0.05)。cEPS具有较高的清除自由基能力可能是由于cEPS中存在其他抗氧化成分，如蛋白质和微量元素。结果表明，cEPS具有良好的抗氧化性能，这与我们的研究结果一致。因此，cEPS可能有助于减少自由基和活性氧等物质引起的氧化应激，并有潜力成为一种天然的抗氧化剂。
EPS-1、EPS-2、EPS-3的抗氧化能力：EPS-1、EPS-2、EPS-3抗氧化活性呈剂量依赖性增加(p < 0.05)，其中EPS-3清除DPPH自由基的能力最强（图9）。在0.5-4 mg/mL浓度范围内，EPS-2、EPS-3清除DPPH自由基的能力从2.8%显著提高到53.64%(p < 0.05)。而EPS-1在1-4 mg/mL浓度下无显著差异(p > 0.05)。DPPH是一种稳定的自由基，通过接受电子或氢基团来保持其稳定性，这意味着EPS-1、EPS-2和EPS-3可能是良好的电子或氢受体。
EPS-1、EPS-2和EPS-3对羟基自由基的清除活性从0.5 mg/mL提高到2 mg/mL (p <0.05)。在4.0 mg/mL浓度下，EPS-1、EPS-2和EPS-3对羟基自由基的清除能力分别为22.95± 0.26%、22.62±1.93%和31.15 ± 1.60%。由此可见，EPS-1、EPS-2和EPS-3可能有助于减轻羟基自由基引起的细胞氧化损伤，延缓人类慢性疾病的进展。
EPS-1、EPS-2和EPS-3在浓度为0.5 mg/mL和4 mg/mL时具有显著性差异，在浓度为4mg/mL时对超氧阴离子自由基的清除能力分别为14.50±1.24%、14.71±1.7%和14.89±0.88%。对超氧阴离子的清除能力与EPS浓度直接相关，在相同浓度下，三种组分对超氧阴离子的清除能力差异不大。EPS对超氧阴离子具有较强的清除能力，在减轻氧化应激和氧化损伤中发挥重要作用。
以上所述仅为本发明的实施例，并非因此限制本发明的专利范围，凡是利用本发明说明书内容所作的等同变换，或直接或间接运用在相关的技术领域，均同理包括在本发明的专利保护范围内。
Claims (10)
1.一种生产具有抗氧化能力的两歧双歧杆菌E3胞外多糖的方法，其特征在于，发酵两歧双歧杆菌E3得到胞外多糖，所述两歧双歧杆菌E3为Bifidobacterium bifidum E3，保藏于中国典型培养物保藏中心，武汉，保藏编号为CCTCC NO: M 2022384，保藏日期2022年4月6日。
2.权利要求1所述的生产具有抗氧化能力的两歧双歧杆菌E3胞外多糖的方法，其特征在于，将所述两歧双歧杆菌E3接种到含L-半胱氨酸盐酸盐的MRS肉汤培养基中孵育。
3.权利要求2所述的生产具有抗氧化能力的两歧双歧杆菌E3胞外多糖的方法，其特征在于，所述胞外多糖是杂多糖。
4.权利要求2所述的生产具有抗氧化能力的两歧双歧杆菌E3胞外多糖的方法，其特征在于，所述胞外多糖含有甘露糖和葡萄糖。
5.权利要求4所述的生产具有抗氧化能力的两歧双歧杆菌E3胞外多糖的方法，其特征在于，所述胞外多糖还含有鼠李糖。
6.权利要求5所述的生产具有抗氧化能力的两歧双歧杆菌E3胞外多糖的方法，其特征在于，所述胞外多糖还含有半乳糖和葡萄糖醛酸。
7.权利要求1所述方法获得的具有抗氧化能力的胞外多糖组合物，其特征在于，其通过发酵两歧双歧杆菌E3得到，包含甘露糖和葡萄糖，且甘露糖:葡萄糖的摩尔比为1:5.04。
8.权利要求1所述方法获得的具有抗氧化能力的胞外多糖组合物，其特征在于，其通过发酵两歧双歧杆菌E3得到，包含鼠李糖、甘露糖和葡萄糖，且鼠李糖:甘露糖:葡萄糖的摩尔比为0.32:1:1.56。
9.权利要求1所述方法获得的具有抗氧化能力的胞外多糖组合物，其特征在于，其通过发酵两歧双歧杆菌E3得到，包含鼠李糖、甘露糖、葡萄糖胺、半乳糖、葡萄糖、葡萄糖醛酸，且鼠李糖：甘露糖：葡萄糖胺：半乳糖：葡萄糖：葡萄糖醛酸的摩尔比为1.10:1:0.82:1.05:1.39:0.68。
10.权利要求7-9任一项所述胞外多糖组合物在制备抗氧化食品或抗氧化药物中的应用。
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