CN115461442A - 抑制il-17的产生的双歧杆菌 - Google Patents
抑制il-17的产生的双歧杆菌 Download PDFInfo
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Abstract
本发明提供一种来源于婴儿肠道的双歧杆菌，其抑制IL‑17的产生并可适用于食品和饮料，以及含有该双歧杆菌的食品和饮料。提供了抑制IL‑17产生的长双歧杆菌物种。该双歧杆菌是长双歧杆菌菌株N714(NITE BP‑03004)。这可能会减少由增强的IL‑17引起的慢性炎症性疾病。
Description
技术领域
本发明涉及抑制IL-17的产生的源于婴儿肠道的双歧杆菌菌株，以及含有该菌株的食品和饮料产品。特别地，本发明涉及长双歧杆菌(Bifidobacterium longum)。
背景技术
已知的参与免疫的细胞包括参与细胞免疫的1型辅助T(Th1)细胞和参与体液免疫的Th2细胞。除了Th1和Th2细胞外，最近的研究还发现了一种新的T细胞亚群，称为Th17细胞。已发现这些Th17细胞在过敏反应、自身免疫和一些感染防御中发挥核心作用。Th17细胞因其产生白介素17(IL-17)的特征而得名。
白介素(IL)是免疫细胞如淋巴细胞、单核细胞和巨噬细胞产生的生物活性物质的总称，是一种介导与免疫反应相关的细胞间相互作用的蛋白质物质。其中，IL-17主要由Th17细胞产生，已知通过作用于成纤维细胞、上皮细胞、血管内皮细胞、巨噬细胞等细胞，诱导炎性细胞因子、趋化因子、细胞粘附因子等各种因子，引起炎症(参见NPL 1和2)。
IL-17包含几个亚群，Th17细胞产生IL-17A和IL-17F。IL-17A和IL-17F在氨基酸水平上具有50％的同源性，并且相应的受体相同，表明许多功能重叠。还已知与IL-17A相比人Th17细胞产生更多的IL-17F。
还发现IL-17的增强与几种慢性炎症性疾病有关，包括类风湿性关节炎、银屑病和多发性硬化症(参见NPL 3和4)。因此，正在探索靶向IL-17的疗法来治疗慢性炎症性疾病。
引用列表
非专利文献
[NPL 1]Aggarwal S,Gurney AL.IL-17:prototype member of an emergingcytokine family.J Leukoc Biol 2002；71:1-8.
[NPL 2]Kolls JK,Linden A.Interleukin-17family members andinflammation.Immunity 2004；21:467-76.
[NPL 3]Nakae S,Nambu A,Sudo K,et al.Suppression of immune inductionof collagen-induced arthritis in IL-17-deficient mice.J Immunol2003；171:6173-7.
[NPL 4]Komiyama Y,Nakae S,Matsuki T,et al.IL-17plays an importantrole in the development of experimental autoimmune encephalomyelitis.JImmunol 2006；177:566-73.
发明内容
技术问题
在该研究中，发明人研究了IL-17的产生是否可以通过饮食来抑制，并发现从婴儿粪便中分离的双歧杆菌含有抑制IL-17产生的双歧杆菌，从而完成了本发明。
问题的解决方案
为了解决上述问题，本发明的特征在于属于双歧杆菌属的双歧杆菌，其抑制IL-17的产生。
为了解决上述问题，本发明的特征还在于所述双歧杆菌为长双歧杆菌菌株N714(NITE BP-03004)。
为了解决上述问题，本发明的特征还在于含有双歧杆菌的食品和饮料。
发明的有益效果
本发明的双歧杆菌抑制IL-17的产生。这可以因此减少由增加的IL-17引起的慢性炎症性疾病。
附图说明
图1是比较本发明菌株和比较菌株对IL-17F产生的抑制作用的图。
图2是比较本发明菌株和模式菌株对IL-17F产生的抑制作用的图。
具体实施方式
现在将详细描述本发明。
1.长双歧杆菌菌株N714(NITE BP-03004)
本发明中的双歧杆菌为长双歧杆菌。本发明中的符号N714是日清食品控股株式会社(Nissin Foods Holdings Co.Ltd.)独立分配给菌株的编号。该菌株由发明人首次从婴儿粪便中分离。
长双歧杆菌菌株N714在以下条件下保藏：
(1)保藏机构名称：国家技术评估学会，专利微生物保藏中心(NITE)
(2)联系方式：日本千叶县木更津市上总镰足2-5-8号122室，邮编292-0818
(3)受托人编号：NITE BP-03004
(4)识别标识：N714
(5)保藏日期：2019年7月10日
长双歧杆菌菌株N714的细菌学特征如下表1和表2所示。细菌学特征是根据伯杰氏系统细菌学手册第2卷(1986年)中描述的方法确定的。表1显示了与该菌株有关的形状和其他特征，表2显示了使用Api 50CH和Api CHL (Biomérieux)的生理生化表征结果。在表1和表2中，符号“+”表示阳性的，符号“-”表示阴性的。
[表1]
+：阳性的，-：阴性的
[表2]
*发酵性能测试
+：阳性的-：阴性的
2.IL-17产生的抑制试验
本发明的长双歧杆菌菌株N714具有高抑制IL-17产生的能力，如下所述的实验实施例所示。IL-17产生的抑制通过以下方法确认。
<Il-17产生抑制试验中使用的样品的制备>
IL-17产生抑制试验中使用的样品是通过在表3所示的GAM培养基(Nippon SuisanKaisha，Ltd.)中在37℃培养双歧杆菌24小时制备的。然后通过离心收集增殖的细菌。收集的细菌用无菌水洗涤并用离心机收集。洗涤和收集重复3次。然后进行热灭菌，将热灭菌的样品用冷冻干燥机冷冻干燥，得到干燥的细菌粉末。
[表3]
<IL-17产生的抑制试验>
人外周血单核细胞(hPBMC，CTL)用于IL-17产生的抑制试验。使用补充有10％胎牛血清和4mM GlutaMAX(Gibco)的IMDM培养基(Sigma)，将细胞接种至1.0×106个细胞/孔中，并将上述制备的样品接种到96孔板上的10ng/孔中。作为Th17细胞分化刺激剂，加入25ng/mL rhIL-6(BioLegend)、12.5ng/mL rhIL-23(BioLegend)、25ng/mL rhIL-21(BioLegend)、2ng/mL rhTGF-β1(BioLegend)和25ng/mL rhIL-1β(BioLegend)，在37℃、5％CO2中孵育96小时。培养后，收集hPBMC的培养物上清液，用市售的免疫测定试剂盒(R&D)测定上清液中的IL-17F。
3.食品和饮料产品
本发明的双歧杆菌可以通过复合在食品和饮料产品中来使用。本发明的双歧杆菌特别适用于饮料，例如发酵乳和乳酸菌饮料。根据现行的《牛奶和乳制品成分标准部令》(Ministerial Ordinance on Standard of Ingredients for Milk and DairyProducts)，成分标准要求发酵乳(脱脂乳的固体含量为8.0％或更高)和奶制乳酸菌饮料(脱脂牛奶的固体含量为3.0％或更高)的最低含量为1.0×107cfu/mL，乳酸菌饮料(脱脂牛奶的固体含量小于3.0％)最低为1.0×106cfu/mL，但上述细菌计数可以通过在发酵溶液(例如牛奶)中生长或以最终产品的形式来实现。除了含有双歧杆菌的发酵乳和乳酸菌饮料之外，双歧杆菌还可以用于黄油等乳制品、蛋黄酱等加工蛋制品和奶油蛋糕等糕点。双歧杆菌也可以适当地用于加工食品，例如方便面和饼干。除了这些应用之外，本发明的食品产品可以与适当的载体和必要的添加剂一起制成双歧杆菌的制剂形式(例如，粉末、颗粒、胶囊和片剂)。
除了一般的饮料和食品以外，本发明的双歧杆菌还可以复合到特定保健用食品和营养补充剂中。除了食品之外，本发明的双歧杆菌还可应用于化妆品领域，例如乳液，医药领域，例如肠道调节剂，日用品领域，例如牙膏，以及动物饲料和植物肥料领域，例如青贮饲料、动物饲料，以及植物液体肥料。
工业实用性
双歧杆菌(长双歧杆菌菌株N714)在抑制L-17的产生方面具有高度活性。
实施例
现在将描述本发明的实施例，但本发明不限于以下实施例。
<实施例1>IL-17产生的抑制试验
对本发明的长双歧杆菌菌株N714和公司拥有的长双歧杆菌对照菌株进行IL-17产生抑制试验。
将本发明的菌株、下述的模式菌株和比较菌株各自在表3所示的GAM培养基(Nippon Suisan Kaisha,Ltd.)中于37℃培养24小时。AnaeroPac(Mitsubishi GasChemical Company,Inc.)用于在厌氧条件下培养细菌。然后通过离心收集细菌。收集的细菌用无菌水洗涤3次，加热灭菌，冷冻。然后用冷冻干燥机将细菌冷冻干燥以产生干燥的细菌粉末。将得到的干燥细菌粉末悬浮在PBS(-)中，作为双歧杆菌悬浮液。
将hPBMC以1.0×106个细胞/孔接种在96孔板中。然后将每个样品组添加至最终浓度为10ng/mL。加入25ng/mL rhIL-6(BioLegend)、12.5ng/mL rhIL-23(BioLegend)、25ng/mL rhIL-21(BioLegend)、2ng/mL rhTGF-β1(BioLegend)和25ng/mL rhIL-1β(BioLegend)的Th17细胞分化刺激剂，以在37℃和5％CO2中孵育96小时。孵育后，收集hPBMC的培养物上清液，用市售的免疫测定试剂盒(R&D)测定上清液中的IL-17。不含双歧杆菌悬浮液和Th17细胞分化刺激剂的样品被定义为“未经刺激的”，而仅含有Th17细胞分化刺激剂的样品被定义为“经刺激的”。
图1显示了在添加每个样品组的情况下培养物上清液中IL-17F的浓度(pg/mL)。
图1表明一些双歧杆菌抑制IL-17F的产生，而另一些则不。与其他双歧杆菌相比，菌株N714抑制IL-17F产生的活性最高，尽管IL-17F的产生高于未经刺激的样品。未经刺激的IL-17F浓度低可能是由于未能分化成Th17细胞。尽管我们不确定N714菌株中IL-17F产生的抑制的原因，但我们现阶段推测如下：一种可能性是菌株N714的添加抑制了Th17细胞分化本身，从而抑制了IL-17F的产生。另一种可能性是尽管Th17细胞是分化的，但由于某种原因抑制了IL-17的产生。这些原因中的一个或两个，或一些其他原因，可能是抑制IL-17产生的原因。
本研究以IL-17F作为评价IL-17的指标，因为IL-17F在IL-17亚群中产量最高且易于检测。然而，IL-17F以外的亚群的产生量非常低且难以检测。因此，IL-17F被用作本研究的评价指标。基于上述推论，假定IL-17F以外的亚群的产生也被长双歧杆菌N714抑制。
<实施例2>长双歧杆菌菌株N714的优越性评价
然后将长双歧杆菌菌株N714与模式菌株长双歧杆菌JCM1217进行比较，以确认长双歧杆菌菌株N714的优越性。试验与实施例1一样进行3次，取平均值与经刺激组进行比较。结果如图2所示。
图2明显地表明，与经刺激组相比，含有长双歧杆菌菌株N714的组中IL-17的产生被显著抑制。
如上所述，长双歧杆菌菌株N714具有有效抑制IL-17产生的功能。
Claims (3)
1.属于长双歧杆菌种的双歧杆菌，其抑制IL-17的产生。
2.根据权利要求1所述的双歧杆菌，其中，所述双歧杆菌为长双歧杆菌菌株N714(NITEBP-03004)。
3.含有权利要求1或2所述的双歧杆菌的食品或饮品。
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| JP2020-147208 | 2020-09-02 | ||
| PCT/JP2021/032090 WO2022050302A1 (ja) | 2020-09-02 | 2021-09-01 | Ｉｌ－１７の産生を抑制するビフィズス菌 |
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| CA3178100A1 (en) * | 2017-01-31 | 2018-08-09 | University-Industry Cooperation Group Of Kyung Hee University | Novel lactic acid bacteria and use thereof |
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