CN109069521A - 粪杆菌属细菌增殖剂 - Google Patents
粪杆菌属细菌增殖剂 Download PDFInfo
- Publication number
- CN109069521A CN109069521A CN201780017016.5A CN201780017016A CN109069521A CN 109069521 A CN109069521 A CN 109069521A CN 201780017016 A CN201780017016 A CN 201780017016A CN 109069521 A CN109069521 A CN 109069521A
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- China
- Prior art keywords
- bacillus faecalis
- kine
- trisaccharide
- bacterium
- faecalibacterium
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
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Classifications
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Abstract
本发明的课题在于提供可以使粪杆菌属细菌有效地增殖,由此,可以有助于肠内炎症等疾病的预防、治疗的物质和方法。作为解决本发明的课题的手段为将1‑蔗果三糖作为有效成分的粪杆菌属细菌增殖剂。根据本发明,在生物体外和生物体内的任一者中,都可以使粪杆菌属细菌有效地增殖。特别是可以几乎不用担心副作用、安全性,而简便并且有效地增加人、动物的体内的粪杆菌属细菌。此外,通过增加人、动物的体内的粪杆菌属细菌,从而可以进行肠内炎症的预防或者治疗。
Description
技术领域
本发明涉及将1-蔗果三糖作为有效成分的粪杆菌(Faecalibacterium)属细菌增殖剂、粪杆菌属细菌增殖用食品组合物、粪杆菌属细菌的增殖方法、肠内炎症的治疗或预防方法、以及用于制造肠内炎症的治疗或预防用药品的1-蔗果三糖的应用。
背景技术
粪杆菌属细菌是常驻在肠内的粘液层的专性厌氧性杆菌,人中的保有率和保有数都高,是肠内细菌群的主要的构成菌种。在近年来的研究中,显示出粪杆菌属细菌具有向肠内的细胞供给能量的作用、良好地保持肠内环境的作用、强的抗炎症作用等优选的生理功能(非专利文献1)。进一步,关于抗炎症作用,报告了通过对肠炎模型动物、培养细胞施用粪杆菌属细菌、其培养上清液,从而可以预防或改善炎症状态(非专利文献2、3)。
另一方面,以疾病的预防、治疗或者增进健康作为目的,在非专利文献1和2所记载的那样的将粪杆菌属细菌其本身施用于生物体的方法中,具有下述(i)~(iv)那样的问题。(i)经口施用时受到胃酸、胆汁、消化液的作用,大多数的菌在到达肠内之前会死亡。(ii)一般而言,外源菌难以固定于肠内,大多数会穿过消化道,因此认为效果是暂时性的。(iii)粪杆菌属细菌没有食用经验的历史,因此在安全性方面存在担忧,(iv)粪杆菌属细菌为专性厌氧性,因此为了以活菌的状态配合于药品、食品中,需要特殊的加工、保存条件。
因此,为了解决上述(i)~(iv)的问题,尝试了增加生物体内在的粪杆菌属细菌,例如,专利文献1中公开了,通过摄取包含聚右旋糖或水溶性玉米纤维的组合物,从而增加粪杆菌属细菌的方法。
现有技术文献
专利文献
专利文献1:日本特开2014-532710号公报
非专利文献
非专利文献1:Yuan Gao等,Gastroenterology Reseach and Practice,Vol.2014,Art.ID 872725,2014年3月27日
非专利文献2:Miquel S.等,Current Opinion in Microbiology,Vol.16(3),第二55~261页,2013年6月
非专利文献3:Xinyun Qiu等,Journal of Crohn’s and Colitis,Vol.7,Issuel1,e558~e568,2013年12月
发明内容
发明所要解决的课题
然而,尚未充分地提供能够充分地增加粪杆菌属细菌的数目的物质或者方法,而要求开发这样的物质或者方法。本发明是为了解决这样的课题而提出的,其目的在于提供可以有效地增殖粪杆菌属细菌,由此,可以有助于肠内炎症等疾病的预防、治疗的物质和方法。
用于解决课题的方法
本发明人等深入研究的结果发现,1-蔗果三糖在生物体外和生物体内的任一者中,都显著地增加粪杆菌属细菌的数目。因此,基于该认识,完成了下述各发明。
(1)本发明涉及的粪杆菌属细菌增殖剂将1-蔗果三糖作为有效成分。
(2)本发明涉及的粪杆菌属细菌增殖剂可以用于肠内炎症的预防或治疗。
(3)本发明涉及的粪杆菌属细菌增殖剂可以适合用于肠内炎症中,特别是炎症性肠疾病的预防或治疗。
(4)本发明涉及的粪杆菌属细菌增殖剂可以适合用于炎症性肠疾病中,特别是克罗恩病或溃疡性大肠炎的预防或治疗。
(5)本发明涉及的粪杆菌属细菌增殖用食品组合物将1-蔗果三糖作为有效成分。
(6)本发明涉及的粪杆菌属细菌的增殖方法的第一方式具有下述工序:通过使人或动物摄取1-蔗果三糖,从而增加上述人或动物的体内的粪杆菌属细菌的数目的工序。
(7)本发明涉及的粪杆菌属细菌的增殖方法的第二方式具有下述工序:在包含粪杆菌属细菌的培养基中添加1-蔗果三糖来进行培养的工序。
(8)本发明涉及的肠内炎症的治疗或预防方法具有下述工序:通过使罹患有肠内炎症、或者有罹患的可能性的人或动物摄取1-蔗果三糖,从而增加上述人或动物的体内的粪杆菌属细菌的数目的工序。
(9)本发明涉及的1-蔗果三糖的应用为用于制造肠内炎症的治疗或预防用药品的1-蔗果三糖的应用。
发明的效果
根据本发明,在生物体外和生物体内的任一者中,都可以有效地增殖粪杆菌属细菌。
此外,1-蔗果三糖是也包含于洋葱、大蒜、大麦、黑麦等蔬菜、谷物中的寡糖的一种,是自古以来具有食用经验的物质;在致突变性试验、急性毒性试验、亚慢性毒性试验和慢性毒性试验的任一者中都没有观察到毒性,因此可以说安全性极其高(食品和开发,Vol.49,No.12,第9页,2014年)。此外,1-蔗果三糖的水溶性高,具有类似于砂糖的良好的甜味质,因此可以直接,或者作为甜味剂等调味料,日常地简便地摄取,此外也可以容易地配合至各种食品、药品等中。
因此,根据本发明,可以几乎不用担心副作用、安全性,简便并且有效地增加人、动物的体内的粪杆菌属细菌。此外,根据本发明,可以通过增加人、动物的体内的粪杆菌属细菌,从而进行肠内炎症的预防或者治疗。
附图说明
图1为表示使用各种寡糖来培养的普氏粪杆菌(Faecalibacterium prausnitzii)ATCC27768株的培养基的浊度(OD值)与培养时间的关系的折线图。
图2为表示使用各种寡糖,培养了72小时的普氏粪杆菌ATCC27768株的培养基的浊度(OD值)的条形图。
具体实施方式
以下,对于本发明涉及的粪杆菌属细菌增殖剂、粪杆菌属细菌增殖用食品组合物、粪杆菌属细菌的增殖方法、肠内炎症的治疗或预防方法、以及用于制造肠内炎症的治疗或预防用药品的1-蔗果三糖的应用进行详细地说明。
在本发明中,“粪杆菌属细菌”是指属于粪杆菌(Faecalibacterium)属的微生物。作为属于粪杆菌属的微生物,可举出普氏粪杆菌(Faecalibacterium prausnitzii)。
“1-蔗果三糖”为由1分子的葡萄糖和2分子的果糖形成的三糖类的寡糖。1-蔗果三糖可以通过将蔗糖作为基质,利用日本特开昭58-201980号公报所公开的那样的酶进行的酶反应来制作。具体而言,首先,将β-呋喃果糖苷酶添加至蔗糖溶液中,在37℃~50℃静置20小时左右,从而进行酶反应,获得含有1-蔗果三糖的反应液。将该含有1-蔗果三糖的反应液供于日本特开2000-232878号公报所公开的那样的色谱分离法,从而将1-蔗果三糖与其它糖(葡萄糖、果糖、蔗糖、4糖以上的寡糖)进行分离而精制,获得高纯度1-蔗果三糖溶液。接着,将该高纯度1-蔗果三糖溶液进行浓缩之后,利用日本特公平6-70075号公报所公开的那样的结晶化法进行结晶化,从而可以以结晶的形式获得1-蔗果三糖。
此外,1-蔗果三糖包含于市售的低聚果糖中,因此可以将它们直接使用,或者由低聚果糖,利用上述方法将1-蔗果三糖进行分离精制来使用。即,作为本发明的1-蔗果三糖,可以使用含有1-蔗果三糖的寡糖等含有1-蔗果三糖的组合物。在使用含有1-蔗果三糖的组合物的情况下,1-蔗果三糖的纯度优选为80质量%以上,更优选为85质量%以上,进一步优选为90质量%以上。另外,在本发明中,所谓特定的寡糖的“纯度”,是指将糖的总量设为100%的情况下的该寡糖的质量%。
1-蔗果三糖通过使人、动物摄取来使用。作为1-蔗果三糖的摄取量(施用量),可举出例如,0.01~0.34g/kg体重,优选为0.01~0.30g/kg体重,更优选为0.01~0.24g/kg体重等。这样的摄取量不限于1天1次,可以分成多次来摄取。
1-蔗果三糖如后述的实施例2所示那样,具有在生物体内(in vivo)增加粪杆菌属细菌的数目这样的功能。因此,作为使人、动物摄取的方法,只要是到达作为粪杆菌属细菌的栖息场所的肠内的方法即可。作为这样的方法,具体而言,可举出将1-蔗果三糖直接,或者以饮食品、药品的形态使人或动物经口摄取的方法。此外,可举出将1-蔗果三糖从***直接施用或经由***的管来施用的方法、将1-蔗果三糖添加至经肠营养剂,将它们经由***至胃、小肠等消化道的管,通过经肠营养法来施用的方法等。
另一方面,1-蔗果三糖如后述的实施例1所示那样,还可以用于增加生物体外(invitro)的粪杆菌属细菌的数目。在该情况下,例如,可举出在包含粪杆菌属细菌的培养基中添加1-蔗果三糖,在厌氧环境下,在37℃静置培养规定的时间的方法。
如上述那样,显示出粪杆菌属细菌通过菌体、其培养上清液的施用,从而可以预防或改善炎症状态。即,非专利文献2中记载了,通过在被2,4,6-三硝基苯磺酸(TNBS)诱导的肠炎模型小鼠中施用普氏粪杆菌A2-165株、其培养上清液,从而可以抑制肠炎。此外,非专利文献3中显示出,通过在人外周血单核细胞的培养基中添加普氏粪杆菌(ATCC27766)、其培养上清液,从而促进IL-10、TGF-β1和IL-12p70这样的抗炎症性细胞因子的产生,可以促进控制性T细胞的分化。进一步,非专利文献3中也显示出,通过在被TNBS诱导的肠炎模型大鼠中经口施用普氏粪杆菌(ATCC27766)、其培养上清液,从而除了抗炎症性细胞因子的产生促进和控制性T细胞的分化促进以外,可以促进肠粘膜上所观察到的炎症组织的治愈。
因此,通过使人、动物摄取1-蔗果三糖来增加其体内或者肠内的粪杆菌属细菌的数目,从而可以预防或治疗肠内炎症。此外,1-蔗果三糖可以用于制造肠内炎症的治疗或预防用药品。
这里,在本发明中,所谓“肠内炎症”,是指肠道产生了炎症的状态,还包含达到疾病的状态。作为由肠内炎症达到的疾病,可举出炎症性肠疾病。炎症性肠疾病具有以溃疡性大肠炎和克罗恩病作为指定,狭义地解释的立场,以及以肠道的所有炎症性疾病作为指定,广义地解释的立场(最新内科学大全<progress 8>,消化器官疾病,pp320,1997年,中山书店)。在本发明中炎症性肠疾病这样的情况下,是指后者的肠道中的所有炎症性疾病。本发明可以适合用于肠内炎症中,特别是,除了***反应以外的疾病的预防或治疗。
作为本发明的粪杆菌属细菌增殖剂的具体的方式,可举出例如,药品、准药品、食品添加物、补充剂等健康食品等。
含有1-蔗果三糖的药品、准药品、补充剂的剂型不受特别限定,可以适当选择适于施用方法的剂型。例如,在经口施用的情况下,可以制成散剂、片剂、糖衣剂、胶囊剂、颗粒剂、干糖浆剂、液剂、糖浆剂、滴剂、饮料剂等固体或液状的剂型。
上述各剂型的药品、准药品、补充剂可以是本领域技术人员利用公知的方法进行制造的。例如,如果是散剂,则可以将1-蔗果三糖800g和乳糖200g良好地混合之后,添加90%乙醇300mL使其湿润。接着,将湿润粉末进行造粒之后,在60℃通风干燥16小时,然后,进行整粒,获得适当的细度的散剂1000g(1-蔗果三糖含量800mg/1g)。此外,如果是片剂,则可以将1-蔗果三糖300g、粉末还原糖稀380g、米淀粉180g和糊精100g良好地混合之后,添加90%乙醇300mL使其湿润。接着,将湿润粉末挤出,进行造粒之后,在60℃通风干燥16小时,获得颗粒。然后,将该颗粒用850μm的筛进行整粒,接着在颗粒470g中添加蔗糖脂肪酸酯50g进行混合之后,使用旋转压片机(6B-2,菊水制作所制)进行压片,获得直径8mm、重量200mg的片剂5000片(1-蔗果三糖含量60mg/1片)。
作为本发明的粪杆菌属细菌增殖用食品组合物的具体的方式,可举出例如,供于饮料、乳制品、食用的颗粒;糊料、调味料、蒸煮袋食品、婴幼儿食品、发酵食品、罐头食品、水产加工品、肉制加工品、谷物加工品等加工食品、食品添加物、健康食品、动物饲料等。
1-蔗果三糖可以在各种饮食品、食品添加物、动物饲料的通常的制造过程中,添加来使用。1-蔗果三糖的甜味度为30,且其味质、物性、加工性与蔗糖接近,因此可以在各种饮食品的制造过程中,将砂糖的一部分或全部置换为1-蔗果三糖等,与砂糖同样地处理来制造各种饮食品、食品添加物、动物饲料。
以下,对于本发明,基于各实施例进行说明。另外,本发明的技术的范围不限定于通过这些实施例来表示的特征。此外,本实施例中,将以规定的纯度含有特定的寡糖的组合物以该寡糖的名称进行记载。
实施例
<实施例1>生物体外的粪杆菌属细菌增殖作用的研究
无菌地调制下述组成的液体培养基。作为各种寡糖,使用了1-蔗果三糖(纯度99质量%;物产食品科技股份有限公司)、蔗果四糖(纯度98质量%;和光纯药公司)、低聚木糖(纯度98质量%;物产食品科技股份有限公司)、棉子糖(纯度98质量%;明治food materia公司)或乳果糖(纯度89质量%;盐水港精糖公司)。此外,作为对照,同样地调制出下述组成中不含寡糖的液体培养基。
《液体培养基的组成/每1L》(最终pH:pH7.0)
酪蛋白分解物15.0g、酵母提取物5.0g、L-胱氨酸0.5g、氯化钠2.5g、巯基乙酸0.5g、色氨酸25mg、氯化血红素(Hemin)适量、维生素K1适量、各种寡糖5.0g。
以使预培养的普氏粪杆菌ATCC27768株在660nm的浊度成为0.05~0.06的OD(Optical Density)值的方式接种于调制的液体培养基直至铺满。将其在厌氧环境下,在37℃静置培养,在24小时后、48小时后和72小时后,使用分光光度计,测定在660nm的浊度(OD值)。将其结果示于表1中。此外,将OD值与培养时间的关系示于图1的折线图中。此外,将培养时间72小时的OD值示于图2的条形图中。
[表1]
1-蔗果三糖组
麦芽糖组
如表1、图1和图2所示那样,OD值在对照的液体培养基中,0小时为0.062,24小时为0.173,48小时为0.113,72小时为0.166。与此相对,添加有1-蔗果三糖的液体培养基中,0小时为0.063,24小时为0.345,48小时为0.885,72小时为1.803。另一方面,添加有蔗果四糖的液体培养基中,0小时为0.063,24小时为0.290,48小时为0.443,72小时为0.305。此外,添加有低聚木糖的液体培养基中,0小时为0.069,24小时为0.271,48小时为0.234,72小时为0.294。此外,添加有棉子糖的液体培养基中,0小时为0.052,24小时为0.251,48小时为0.191,72小时为0.246。此外,添加有乳果糖的液体培养基中,0小时为0.055,24小时为0.298,48小时为0.448,72小时为0.295。
即,添加有1-蔗果三糖的液体培养基中,与未添加寡糖的液体培养基(对照的液体培养基)以及添加有其它寡糖(蔗果四糖、低聚木糖、棉子糖和乳果糖)的液体培养基相比,在24小时、48小时和72小时的任一培养时间,OD值都显著地大。
培养基的浊度(OD值)反映微生物数的大小。因此,由上述结果明确了,在24小时、48小时和72小时的任一培养时间,添加有1-蔗果三糖的液体培养基中的普氏粪杆菌ATCC27768株的菌数与未添加寡糖的液体培养基以及添加有其它寡糖的液体培养基中的菌数相比,显著地大。由该结果明确了,1-蔗果三糖在生物体外,增殖粪杆菌属细菌的作用显著地大。
<实施例2>生物体内的粪杆菌属细菌增殖作用的研究
将6个月~6岁的乳幼儿58名分成2个组,每组29名,设为1-蔗果三糖组和麦芽糖组。试验期间中,1-蔗果三糖组每日经口摄取1-蔗果三糖(纯度98质量%以上;物产食品科技股份有限公司),麦芽糖组每日经口摄取麦芽糖(纯度92质量%以上;“sunmalt-S”粉末品;三和淀粉工业公司)。试验期间设为12周。1-蔗果三糖或麦芽糖的1天的摄取量在小于1岁时为1g(标准摄取量;0.121g/kg体重/天),1~3岁时为2g(标准摄取量;0.169g/kg体重/天),4~6岁时为3g(标准摄取量;0.170g/kg体重/天)。在试验期间的开始时(0周)、6周经过时(6周)和12周经过时(12周)回收粪便。
另外,上述标准摄取量是基于“厚生劳动省平成22年乳幼儿身体发育调查”所刊登的分年龄体重数据而算出的。即,由相当于本实施例2的试验参加者的年龄(月龄)的体重数据的最小值和最大值,分男女求出中心值,算出中心值的平均值。接着,对于中心值的平均值,算出男女的平均值,将它们设为标准体重。标准体重是小于1岁为8.26kg,1~3岁为11.85kg,4~6岁为17.62kg。将1-蔗果三糖或麦芽糖的1天的摄取量除以该标准体重而得的值设为标准摄取量。
通过实时PCR法来定量回收的粪便中的粪杆菌属细菌的菌数。具体而言,首先,使用基因组提取用试剂“Ultra Clean Soil DNA Isolation Kit”(Mo Bio Laboratories公司)和“High Pure PCR Template Preparation Kit”(Roche公司),从粪便中提取基因组DNA。将该基因组DNA作为模板,使用对于粪杆菌属细菌特异的引物(以下示出序列)、实时PCR试剂“SYBR GREEN Master Mix”(Applied Biosystems公司)和实时PCR装置“ABIPRISM7700 sequence detection system(Applied Biosystems公司)进行实时PCR,对于粪便每1g的粪杆菌属细菌的菌数(个)进行定量。检测范围为105个/g以上,相对于检测范围以下的检体,代入102.5个/g的数值。标准曲线使用将从纯粹培养而在显微镜下计数了的普氏粪杆菌ATCC27768株利用同样的方法提取得到的基因组DNA作为模板,基于在相同条件下进行实时PCR得到的结果而制作得到的标准曲线。
《对于粪杆菌属细菌特异的引物的序列》
正向引物;CCATGAATTGCCTTCAAAACTGTT(序列号1)
反向引物;GAGCCTCAGCGTCAGTTGGT(序列号2)
求得的菌数对于各组分别算出平均值。此外,对于6周和12周的菌数的平均值,相对于0周的菌数的平均值,通过威尔科克森符号秩检验来进行显著性差异检验,求出p值。将其结果示于表2中。
[表2]
如表2所示那样,1-蔗果三糖组的菌数在0周为3.7×106个/粪便1g,与此相对,6周为1.4×107个/粪便1g,12周为3.5×107个/粪便1g。即,与0周比较的菌数在6周时,虽然没有显著性差异,但增加至3.5倍以上,在12周时,显著地增加至9倍以上。
另一方面,麦芽糖组的菌数在0周为7.6×106个/粪便1g,与此相对,6周为5.8×106个/粪便1g,12周为1.3×107个/粪便1g。即,与0周比较的菌数在6周时略微减少,12周时稍微增加了,但没有显著性差异。
如以上那样,显示出粪杆菌属细菌的菌数在麦芽糖组中没有增加,与此相对,在1-蔗果三糖组中显著地增加了。由该结果明确了,1-蔗果三糖在生物体内,增殖粪杆菌属细菌的作用显著地大。
Claims (9)
1.一种粪杆菌(Faecalibacterium)属细菌增殖剂,其将1-蔗果三糖作为有效成分。
2.根据权利要求1所述的粪杆菌(Faecalibacterium)属细菌增殖剂,其特征在于,用于肠内炎症的预防或治疗。
3.根据权利要求2所述的粪杆菌(Faecalibacterium)属细菌增殖剂,肠内炎症为炎症性肠疾病。
4.根据权利要求3所述的粪杆菌(Faecalibacterium)属细菌增殖剂,炎症性肠疾病为克罗恩病或溃疡性大肠炎。
5.一种粪杆菌(Faecalibacterium)属细菌增殖用食品组合物,其将1-蔗果三糖作为有效成分。
6.一种粪杆菌(Faecalibacterium)属细菌的增殖方法,其具有下述工序:通过使人或动物摄取1-蔗果三糖,从而增加所述人或动物的肠内的粪杆菌(Faecalibacterium)属细菌的数目的工序。
7.一种粪杆菌(Faecalibacterium)属细菌的增殖方法,其具有下述工序:在包含粪杆菌(Faecalibacterium)属细菌的培养基中添加1-蔗果三糖来进行培养的工序。
8.一种肠内炎症的治疗或预防方法,其具有下述工序:通过使罹患有肠内炎症、或者有罹患的可能性的人或动物摄取1-蔗果三糖,从而增加所述人或动物的肠内的粪杆菌(Faecalibacterium)属细菌的数目的工序。
9.用于制造肠内炎症的治疗或预防用药品的1-蔗果三糖的应用。
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