CN116059223B - 酪醇-β-半乳糖苷在调整肠道微生物中的应用 - Google Patents
酪醇-β-半乳糖苷在调整肠道微生物中的应用 Download PDFInfo
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Abstract
本发明涉及酪醇‑β‑半乳糖苷在调整肠道微生物中的应用。酪醇‑β‑半乳糖苷的分子式为C14H20O7，通过酶法以半乳糖和酪醇制备得到的酪醇‑β‑半乳糖苷可以显著抑制疾病相关菌瘤胃球菌和布劳特氏菌的生长，促进有益菌乳酸杆菌、双歧杆菌和艾克曼氏菌的增殖。并且酪醇‑β‑半乳糖苷还能使得小鼠的异黄酮类合成代谢通路明显增强，使得小鼠代谢物中存在大量具有优异的生物活性即抗癌、抗炎、抗氧化活性的异黄酮类化合物，促进肠道健康。
Description
技术领域
本发明涉及酪醇-β-半乳糖苷在调整肠道微生物中的应用，属于生物医药技术领域。
背景技术
肠道微生物与人类正常的生理活动和疾病的发生发展密切相关。肠道菌群在健康胃肠道中的功能包括抵抗潜在致病微生物的定植和增殖、调节肠道内分泌的功能、影响神经信号的传导、分解食物为宿主提供营养和能量、合成代谢物、对特定药物反应或修饰等作用。另一方面，肠道菌群也与多种慢性疾病的发生发展有关，包括癌症、炎症、代谢、心血管、免疫及神经类相关疾病等。因此，研发具有调节肠道菌群的药物或保健品对于维持人体肠道健康十分重要。
药用植物红景天在亚洲和欧洲国家通常被用作医疗保健的“食品补充剂”，以防止或缓解疲劳和虚弱等。目前在欧洲食品安全局的第13条健康声明综合清单中，红景天的功能声明被表述为“有助于优化精神和认知活动”。红景天在我国也具有悠久的使用历史，已经被广泛应用于保健品及护肤品中，应用潜力巨大。红景天苷(即酪醇-β-葡萄糖苷)是红景天的主要活性组分，具有重要的药理学功能，如神经保护、心血管保护、延缓衰老、抗疲劳、抗氧化等。人工合成的酪醇-β-半乳糖苷是红景天苷类似物，在神经保护和抗氧化方面更优于红景天苷，但是目前尚未发现其调整肠道菌群的功能。
发明内容
针对现有技术的不足，本发明提供酪醇-β-半乳糖苷在调整肠道微生物中的应用。
本发明的技术方案如下：
酪醇-β-半乳糖苷在制备调整肠道微生物的药物或保健品中的应用。
根据本发明优选的，所述酪醇-β-半乳糖苷的分子式为C14H20O7，结构式为：
根据本发明优选的，所述酪醇-β-半乳糖苷的使用剂量为40～60mg/kg/day。
根据本发明优选的，所述调整肠道微生物具体为：抑制瘤胃球菌(Ruminococcus)和布劳特氏菌(Blautia)的生长，促进乳酸杆菌(Lactobacillus)、双歧杆菌(Bifidobacterium)和艾克曼氏菌(Akkermansia)的生长，增强异黄酮类合成代谢通路。
根据本发明优选的，所述酪醇-β-半乳糖苷的制备方法，包括以下步骤：
构建半乳糖浓度为0.25～2M、酪醇浓度为50～400mM、β-半乳糖苷酶浓度为5～50U/mL的反应体系，然后在40～75℃条件下反应6～24h，升温至90～100℃终止反应，离心，将上清液经分离和干燥后，制得酪醇-β-半乳糖苷；
所述β-半乳糖苷酶的氨基酸序列如SEQ ID No.2所示，核苷酸序列如SEQ ID No.1所示。
根据本发明优选的，所述反应体系中半乳糖浓度为1M，所述反应体系中酪醇浓度为100mM，所述反应体系中β-半乳糖苷酶浓度为40U/mL。
根据本发明优选的，所述分离具体过程为：上清液经过真空冷冻干燥后，用硅胶柱进行色谱分离，以甲醇和乙酸乙酯为流动相，体积比例为1:6，样品上样量为5g冻干样品，洗脱流速为4～6mL/min，收集80min后的洗脱样品，再通过薄层层析检测合并产物，得到酪醇-β-半乳糖苷。
根据本发明优选的，所述β-半乳糖苷酶的制备方法如下：
(1)将如SEQ ID No.1所示的β-半乳糖苷酶基因序列克隆到大肠杆菌表达载体pET-22b中，构建重组表达载体pET-22-bga，然后将表达载体转化到大肠杆菌中，用氨苄青霉素抗性筛选出含β-半乳糖苷酶的重组菌株；
(2)将步骤(1)筛选出的重组菌株接种于LB液体培养基中，在37℃、200rpm下培养过夜，获得种子液；将种子液按照体积比0.5～1.5％接种于LB液体培养基中，待生长到OD600为0.6时，加入终浓度为1mM的IPTG进行诱导，诱导温度为16℃，诱导时间为12h，得到诱导后的菌细胞；
(3)收集步骤(2)诱导后获得的菌细胞，超声破碎后，离心去除沉淀，获得粗酶液；粗酶液通过镍亲和层析柱进行纯化，获得β-半乳糖苷酶。
有益效果
1、本发明证实了酪醇-β-半乳糖苷可以显著抑制疾病相关菌瘤胃球菌(Ruminococcus)和布劳特氏菌(Blautia)的生长，促进有益菌乳酸杆菌(Lactobacillus)、双歧杆菌(Bifidobacterium)和艾克曼氏菌(Akkermansia)的增殖。并且酪醇-β-半乳糖苷还能使得小鼠的异黄酮类合成代谢通路明显增强，使得小鼠代谢物中存在大量具有优异的生物活性即抗癌、抗炎、抗氧化活性的异黄酮类化合物，促进肠道健康。
2、本发明的酪醇-β-半乳糖苷是采用特定的β-半乳糖苷酶，以半乳糖单糖为糖基供体合成获得，相比于现有以乳糖为糖基供体的制备酪醇-β-半乳糖苷的方法，本发明的整个制备过程中不存在水解副反应，反应产物容易纯化，可通过廉价的硅胶柱层析进行分离，有效地降低了成本。
附图说明
图1为酪醇-β-半乳糖苷的氢谱图。
图2为酪醇-β-半乳糖苷的碳谱图。
图3为实施例2所述空白组与实验组小鼠的肠道微生物群落结构的PCoA分析图。
图4为实施例2所述空白组与实验组小鼠的肠道微生物在群落物种组成上的变化。
其中，图中所示具体为肠道微生物中检测到的属水平上的物种及其所占比例。
图5为实施例2所述空白组与实验组小鼠的肠道微生物群落中有益菌群占比情况。
其中，图中所示具体为经典的益生菌Lactobacillus、Bifidobacterium和新发现的有益菌Akkermansia在两组间的占比情况。
图6为实施例2所述空白组与实验组小鼠的肠道微生物的LDA Effect Size分析图。
图7为实施例3所述空白组与实验组小鼠肠道代谢物的PCA分析图。
图8为实施例3所述空白组与实验组小鼠233种差异代谢物的热图。
其中，深色区域表示代谢物在该组含量较高，浅色区域表示代谢物在该组含量较低。
图9为实施例3所述空白组与实验组小鼠差异代谢物KEGG通路富集拓扑学分析。
具体实施方式
下面结合实施例对本发明做进一步说明，但本发明所保护范围不限于此。
本发明通过16S rRNA Illumina测序技术测定粪便微生物多样性，采用LC-MS非靶向测定代谢物，具体操作均由上海美吉生物科技有限公司完成。
实施例1:酪醇-β-半乳糖苷的制备
1、人工合成如SEQ ID No.1所示的β-半乳糖苷酶核苷酸序列，然后克隆到大肠杆菌表达载体pET-22b中，构建重组表达载体pET-22-bga；将表达载体转化到大肠杆菌BL21(DE3)中，用氨苄青霉素抗性筛选出含β-半乳糖苷酶的重组菌株。再将重组菌株接种于LB液体培养基中，在37℃、200rpm下培养过夜，获得种子液；将种子液按照体积比1％接种于LB液体培养基中，待生长到OD600为0.6时，加入终浓度1mM的IPTG诱导，诱导温度为16℃，诱导时间为12小时，12000rpm离心5min，弃去上清，菌细胞用pH 7.0、50mM磷酸钾缓冲液洗涤2次，重悬于pH 7.0、50mM的磷酸钾缓冲液，采用超声波破碎仪进行细胞破壁，所得悬浮液于11000rpm离心30min，所得上清经过镍柱亲和层析纯化即获得β-半乳糖苷酶纯化酶。
2、采用浓度50mM、pH 6.5的磷酸缓冲液配制半乳糖浓度为1M、酪醇浓度为100mM、β-半乳糖苷酶浓度为40U/mL的反应体系，然后在70℃条件下反应15h，100℃煮沸5min终止反应，13000rpm离心10min，上清液进行真空冷冻干燥后，用规格5cm×50cm的硅胶柱进行色谱分离，以甲醇和乙酸乙酯为流动相，比例为1:6；样品上样量为5g冻干样品，洗脱流速为5mL/min，收集80min后的洗脱样品，薄层层析检测，合并迁移距离相同的糖苷产物，然后更换溶剂为水后冷冻干燥，制得酪醇-β-半乳糖苷。
将5mg上述制得的酪醇-β-半乳糖苷溶于氘代水中，进行核磁解析，氢谱图和碳谱图分别如图1和图2所示。核磁分析所用仪器为Bruker Daltonics 400MHz核磁共振波谱仪(美国)。
由图1和图2可知，本实施例成功合成了酪醇-β-半乳糖苷。
实施例2：酪醇-β-半乳糖苷对肠道微生物的调整作用
将7周龄C57BL/6J小鼠12只(重量约为21±2g)随机分为两组：空白组和实验组，每组6只，适应性喂养一周后开始正式实验。实验期间小鼠自由饮食、饮水，每周更换2-3次垫料。小鼠喂养环境的明暗交替周期为12h，室温24±2℃，湿度55％±5％。使用无菌灌胃针和一次性无菌注射器对各组小鼠灌胃，空白组和实验组分别灌胃生理盐水和50mg/kg/day酪醇-β-半乳糖苷。实验期间每日观察小鼠体重变化，精神行为及外形变化。实验共进行7天。7天后，收集小鼠新鲜粪便，立即存放于液氮中，测定肠道微生物变化及代谢物差异。
实验过程中，小鼠体重均逐步增高，精神状态正常，证明该剂量药物对小鼠无毒性影响。取小鼠粪便进行菌群分析。空白组与实验组小鼠的肠道微生物群落结构的PCoA分析图，如图3所示。空白组与实验组小鼠的肠道微生物在群落物种组成上的变化图，如图4所示。空白组与实验组小鼠的肠道微生物群落中有益菌群占比情况，如图5所示。空白组与实验组小鼠的肠道微生物的LDA Effect Size分析图，如图6所示。
由图3可知，空白组与实验组样本物种组成在OTU水平上不同，具体反映在图中的两样本数据距离较远，无交集，群落结构存在显著差异。
由图4和图5可知，相比于空白组，酪醇-β-半乳糖苷可以显著促进有益菌群增殖，抑制瘤胃球菌(Ruminococcus)、布劳特氏菌(Blautia)等与疾病相关的属的丰度。对人们熟知的乳酸杆菌(Lactobacillus)、双歧杆菌(Bifidobacterium)以及新发现的艾克曼氏菌(Akkermansia)，酪醇-β-半乳糖苷表现出显著的促进作用。
由图6可知，通过LDA Effect Size分析可以确定实验组和空白组间的显著差异物种及生物标志物。即在阈值设置为4的条件下，乳酸杆菌(Lactobacillus)在目、科、属水平上均被识别为酪醇-β-半乳糖苷作用后的生物标志物。
实施例3：酪醇-β-半乳糖苷增强小鼠的异黄酮类合成代谢通路
对实施例2所述两组小鼠的肠道内容物分别进行非靶向(LC-MS)代谢组学分析，空白组与实验组小鼠肠道代谢物的PCA分析图，如图7所示。空白组与实验组小鼠233种差异代谢物的热图，如图8所示。空白组与实验组小鼠差异代谢物KEGG通路富集拓扑学分析，如图9所示。
由图7可知，空白组与实验组的代谢物组成模式存在差异，具体表现在两组的代谢物在图上距离较远。
由图8和图9可知，两组间共存在233个差异代谢物。将两组的差异代谢物富集后进行KEGG通路富集分析发现，酪醇-β-半乳糖苷组的异黄酮类合成代谢通路明显增加，小鼠代谢物中存在大量异黄酮类物质，异黄酮类物质具有优异的生物活性，包括抗癌、抗炎、以及抗氧化活性等，能够促进肠道健康。
综上所述，酪醇-β-半乳糖苷可以通过增加益生菌以及有益菌群的丰度、降低疾病相关菌群丰度，进而调节肠道菌群，改善肠道功能；同时显著促进异黄酮类化合物的合成路径，增加具有抗癌、抗炎症活性的异黄酮类化合物的产生，促进肠道健康。
Claims (6)
1.酪醇-β-半乳糖苷在制备调整肠道微生物的药物中的应用，其特征在于，所述酪醇-β-半乳糖苷的结构式为：
；
所述调整肠道微生物具体为：抑制瘤胃球菌（Ruminococcus）和布劳特氏菌（Blautia）的生长，并促进乳酸杆菌（Lactobacillus）、双歧杆菌（Bifidobacterium）和艾克曼氏菌（Akkermansia）的生长。
2.如权利要求1所述的应用，其特征在于，所述酪醇-β-半乳糖苷的使用剂量为40~60mg/kg/day。
3.如权利要求1所述的应用，其特征在于，所述酪醇-β-半乳糖苷的制备方法，包括以下步骤：
构建半乳糖浓度为0.25~2M、酪醇浓度为50~400 mM、β-半乳糖苷酶浓度为5~50U/mL的反应体系，然后在40~75℃条件下反应6~24h，升温至90~100℃终止反应，离心，将上清液经分离和干燥后，制得酪醇-β-半乳糖苷；
所述β-半乳糖苷酶的氨基酸序列如SEQ ID No.2所示，核苷酸序列如SEQ ID No.1所示。
4.如权利要求3所述的应用，其特征在于，所述反应体系中半乳糖浓度为1M，所述反应体系中酪醇浓度为100mM，所述反应体系中β-半乳糖苷酶浓度为40U/mL。
5.如权利要求3所述的应用，其特征在于，所述分离具体过程为：上清液经过真空冷冻干燥后，用硅胶柱进行色谱分离，以甲醇和乙酸乙酯为流动相，体积比例为1:6，样品上样量为5g冻干样品，洗脱流速为4~6 mL/min，收集80min后的洗脱样品，再通过薄层层析，得到酪醇-β-半乳糖苷。
6.如权利要求3所述的应用，其特征在于，所述β-半乳糖苷酶的制备方法如下：
（1）将如SEQ ID No.1所示的β-半乳糖苷酶基因序列克隆到大肠杆菌表达载体pET-22b中，构建重组表达载体pET-22-bga，然后将表达载体转化到大肠杆菌中，用氨苄青霉素抗性筛选出含β-半乳糖苷酶的重组菌株；
（2）将步骤（1）筛选出的重组菌株接种于LB液体培养基中，在37℃、200rpm下培养过夜，获得种子液；将种子液按照体积比0.5~1.5%接种于LB液体培养基中，待生长到OD600为0.6时，加入终浓度为1 mM的IPTG进行诱导，诱导温度为16℃，诱导时间为12h，得到诱导后的菌细胞；
（3）收集步骤（2）诱导后获得的菌细胞，超声破碎后，离心去除沉淀，获得粗酶液；粗酶液通过镍亲和层析柱进行纯化，获得β-半乳糖苷酶。
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