CN101257910A - 胆固醇吸收抑制剂 - Google Patents
胆固醇吸收抑制剂 Download PDFInfo
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- CN101257910A CN101257910A CNA200680032921XA CN200680032921A CN101257910A CN 101257910 A CN101257910 A CN 101257910A CN A200680032921X A CNA200680032921X A CN A200680032921XA CN 200680032921 A CN200680032921 A CN 200680032921A CN 101257910 A CN101257910 A CN 101257910A
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- bifidobacterium
- yit
- acid
- cholesterol
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- A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
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Abstract
本发明提供在消化道内的生存性优异、在肠道内具有胆固醇吸收抑制作用、降低血中胆固醇等的脂质代谢改善作用优异、并且保存后的生存性高的双歧杆菌属细菌,以及使用该细菌的胆固醇吸收抑制剂。本发明的胆固醇吸收抑制剂以选自动物双歧杆菌动物亚种YIT 10394、动物双歧杆菌乳酸亚种JCM 1253、动物双歧杆菌乳酸亚种JCM 7117和假长双歧杆菌球形亚种中的1种以上的微生物作为有效成分。
Description
技术领域
本发明涉及胆固醇的吸收抑制作用优异的双歧杆菌属细菌和以这些双歧杆菌属细菌作为有效成分的胆固醇吸收抑制剂。
背景技术
胆固醇存在于许多细胞中,其主要的生理功能为,作为脂蛋白质和生物膜的构成成分而维持细胞的功能,并作为胆汁酸和各种激素的原材料等发挥作用。但是,已知过度摄取胆固醇含量多的食品是血清中胆固醇增加和动脉硬化的原因,在胆固醇摄取机会多的现代饮食生活中,需求抑制其在机体内的浓度。
有报告,双歧双歧杆菌、青春双歧杆菌、短双歧杆菌、婴儿双歧杆菌、长双歧杆菌的菌体有改善血中脂质的作用(专利文献1)。特别是关于长双歧杆菌进行了研究,有报告长双歧杆菌SBT 2933R(FERMP-8743)具有优异的效果(非专利文献1),并提出将该菌体或培养物作为胆固醇升高抑制剂而利用(专利文献2)。但是,一般而言,由于双歧杆菌属细菌不耐氧和体内的胃酸及胆汁酸,因此,当口服摄取该菌体和培养物时,存在有在消化道的生存性差、大多不能得到充分的效果的问题。因此,需求生存性优异的具有抑制血清胆固醇升高作用的双歧杆菌属细菌。
但是,关于这样的生存性优异的双歧杆菌属细菌,已知的是,长双歧杆菌SBT 10254(FERM P-14820)(专利文献3)和长双歧杆菌(FERM BP-7787)(专利文献4)的生存性优异,显示出抑制血清胆固醇升高的作用;而长双歧杆菌BB536、短双歧杆菌ATCC 15700、动物双歧杆菌ATCC 25527(非专利文献2)只不过显示胆固醇沉降作用,目前的现状是,这种生存性优异且具有胆固醇抑制作用的微生物的选择性小。
另外,使用这些微生物的药剂、食品,因为经常不得不长期保存,所以要求高的保存稳定性,但在现有已知的微生物中,多数在长期保存后生存性降低,特别是在非厌氧条件下保存后,生存性降低的居多。
专利文献1:日本特开昭61-271223号公报
专利文献2:日本特公平6-96537号公报
专利文献3:日本专利第3384907号
专利文献4:日本特开平2003-238423号公报
非专利文献1:第6回日本ビフイズス菌センタ一学衍集会予稿集,18页,1987
非专利文献2:Letters in Applied Microbiology,Vol.21,149-151,1995
发明内容
因此,本发明的目的在于提供在消化道内的生存性优异、在肠道内具有胆固醇吸收抑制作用、降低血中胆固醇等的脂质代谢改善作用优异、并且保存后的生存性高的双歧杆菌属细菌,以及使用该细菌的胆固醇吸收抑制剂。
本发明的发明人为了达到上述目的而进行了反复深入的研究,结果意外地发现,动物双歧杆菌动物亚种YIT 10394、动物双歧杆菌乳酸亚种JCM 1253、动物双歧杆菌乳酸亚种JCM 7117和假长双歧杆菌球形亚种的胆固醇清除作用优异,并且显示出强耐酸性和耐胆汁酸性,在肠道内也具有优异的胆固醇吸收抑制作用,另外,保存后的生存性高,特别是即使在非厌氧条件下保存后也显示出高生存性,从而完成了本发明。另外,在本发明使用的微生物中,动物双歧杆菌动物亚种YIT 10394、假长双歧杆菌球形亚种YIT 10392和假长双歧杆菌球形亚种YIT 10393是本发明的发明人新发现的微生物。
即,本发明提供一种以选自动物双歧杆菌动物亚种YIT 10394、动物双歧杆菌乳酸亚种JCM 1253、动物双歧杆菌乳酸亚种JCM 7117和假长双歧杆菌球形亚种中的1种以上的微生物作为有效成分的胆固醇吸收抑制剂。
另外,本发明提供一种以选自动物双歧杆菌乳酸亚种JCM 1253和动物双歧杆菌乳酸亚种JCM 7117中的1种以上的微生物作为有效成分的胆固醇吸收抑制剂。
另外,本发明提供一种以选自动物双歧杆菌动物亚种YIT 10394、假长双歧杆菌球形亚种YIT 10392和假长双歧杆菌球形亚种YIT 10393中的1种以上的微生物作为有效成分的胆固醇吸收抑制剂。
进而,本发明提供作为新微生物的动物双歧杆菌动物亚种YIT10394(FERM ABP-10662)、假长双歧杆菌球形亚种YIT 10392(FERMABP-10660)或假长双歧杆菌球形亚种YIT 10393(FERM ABP-10661)。
本发明的动物双歧杆菌动物亚利YIT 10394、动物双歧杆菌乳酸亚种JCM 1253、动物双歧杆菌乳酸亚种JCM 7117和假长双歧杆菌球形亚种,不仅具有优异的胆固醇消除作用,而且耐酸性和耐胆汁酸性优异,所以即使在肠道内也发挥优异的胆固醇吸收抑制作用,能够用于降低血中胆固醇等改善脂质代谢的目的。另外,这些微生物保存后的生存性高,特别是在非厌氧条件下保存后也显示出高的生存性,所以本发明的吸收抑制剂能够长期保存,而且也能够非厌氧保存。
具体实施方式
作为本发明中所使用的假长双歧杆菌球形亚种(Bifidobacteriumpseudolongum subsp.globosum),例如,可以列举假长双歧杆菌球形亚种YIT 10392、假长双歧杆菌球形亚种YIT 10393等。
这些假长双歧杆菌球形亚种YIT 10392、假长双歧杆菌球形亚种YIT 10393、和本发明中所使用的微生物之一的动物双歧杆菌动物亚种(Bifidobacterium animalis subsp.animalis)YIT 10394,是本发明的发明人分离得到的新菌株,其为具有如下性质的菌株:胆固醇消除活性为70%以上;在人工胃液中的生存率为20%以上;并且在含有胆汁酸的培养基中的增殖性以克莱特值(Klett value)(菌体浊度)计,为100以上。该菌株具有以下的细菌学性质。即,这些菌的细菌学性质均为革兰氏阳性多形性杆菌、不形成芽胞、非运动性、专性厌氧性、在37℃能够生长,除以上所示的胆固醇消除活性、在人工胃液中的生存率和在含有胆汁酸的培养基中的增殖性以外,分别依次显示出假长双歧杆菌球形亚种、假长双歧杆菌球形亚种、动物双歧杆菌动物亚种具有的性质。
关于该新菌株的种的鉴定,根据如下:在使用16S rDNA碱基序列的菌种鉴定中,YIT 10392的16S rDNA碱基序列与假长双歧杆菌球形亚种(accession No.D86194)显示出99.6%的同源性;YIT 10393的16SrDNA碱基序列与假长双歧杆菌球形亚种(accession No.D86194)显示出99.6%的同源性;YIT 10394的16S rDNA碱基序列与动物双歧杆菌动物亚种(accession No.D86185)显示出99.7%的同源性。这里,使用16S rDNA碱基序列的菌种鉴定通过下述方法进行:离心洗净使用添加有0.5%葡萄糖的GAM培养基、在37℃下厌氧(二氧化碳置换)培养24h的菌液,得到菌体,将从该菌体提取的DNA作为模板,通过PCR法扩增16S rDNA的全长序列,由终止物标记(Dye Terminator)法测定扩增产物的碱基序列,将得到的碱基序列在数据库中进行检索。另外,这些菌株为新菌株的判断根据为:16S rDNA碱基序列与标准株不同,由RAPD(Random Amplified Polymorphic DNA:随机扩增多态性DNA)法得到的染色体DNA多态性分析的结果不同。
动物双歧杆菌动物亚种YIT 10394以微生物保藏编号FERM ABP-10662、假长双歧杆菌球形亚种YIT 10392以微生物保藏编号FERMABP-10660、假长双歧杆菌球形亚种YIT 10393以微生物保藏编号FERM ABP-10661,在平成18年8月14日移管至独立行政法人产业技术综合研究所专利生物保藏中心。移管前的保藏者也是相同的,保藏日是平成17年8月18日。另外,该保藏中心的地址为:日本国茨城县津轻市东1丁目1番地中央第6,邮政编码305-8566。
另外,本发明中所使用的动物双歧杆菌乳酸亚种(Bifidobacteriumanimalis subsp.lactis)JCM 1253和动物双歧杆菌乳酸亚种JCM 7117均能够从JCM(Japan Collection of Microorganisms)和ATCC(AmericanType Culture Collection)得到,各自的ATCC编号是ATCC27536和ATCC27674。
其中,作为本发明中所使用的微生物,优选的是使用微生物制造发酵乳饮食品,在10℃、非厌氧条件(好氧条件)下保存21日后,该微生物的生存率为50%以上的微生物。
另外,作为本发明的微生物,优选胆固醇消除活性为70%以上的双歧杆菌属细菌,更优选胆固醇消除活性为70%以上、在人工胃液中的生存率为20%以上、并且在含有胆汁酸的培养基中的增殖性以克莱特值(菌体浊度)计,为100以上的双歧杆菌属细菌。一般的双歧杆菌属细菌在人工胃液中的生存率是0~几个%左右,在含有胆汁酸的培养基中的增殖性以克莱特值(菌体浊度)计,为0~几十左右。由上述值筛选出来的具有高耐酸性和耐胆汁酸性的细菌,多数菌能够以活菌状态到达肠道内。
这里,胆固醇消除活性的算出,例如,能够通过下述方法进行:将微生物菌体与人工脂质微团一起静置时,按照通常方法测定上清的胆固醇,与不含菌体的上清的胆固醇量进行比较,代入下式中。
胆固醇消除活性(%)=100-(含有菌体的上清的胆固醇量)/(不含菌体的上清液的胆固醇量)×100
另外,在人工胃液中的生存率,例如,能够定义为在pH3.0的人工胃液中,37℃保存1小时时的生存率。该值高表示在消化道内的生存率高。
另外,在含有胆汁酸的培养基中的增殖性,例如,能够定义为以克莱特值(Klett-Summerson比色计,No.66滤色镜)表示的在含有0.2%胆汁酸的培养基中,37℃培养24小时时的浊度。该值高表示在消化道内的生存率、增殖率高。
另外,微生物的属名、种名和株名,因命名者不同而多种多样,还因菌的再分类等,也是不确定的,虽然菌属名、菌种名、菌株名不同,但作为微生物在实质上相同的微生物,也包含在本发明的微生物中。即,例如,虽然动物双歧杆菌乳酸亚种也曾被作为其他种,但是Masco等报告,将它们归纳为动物双歧杆菌,作为其下位分类,分为动物双歧杆菌动物亚种和动物双歧杆菌乳酸亚种2个亚种(Int.j.Syst.Envol.Microbiol 54,1137-1143,2004),延用至今。因此,在本发明的动物双歧杆菌动物亚种或动物双歧杆菌乳酸亚种的范围中,也分别包含以前被称为动物双歧杆菌或乳酸双歧杆菌的菌。
另外,动物双歧杆菌乳酸亚种ATCC 27536与动物双歧杆菌乳酸亚种JCM 1253相同,动物双歧杆菌乳酸亚种ATCC 27674与动物双歧杆菌乳酸亚种JCM 7117相同,分别包含在本发明的微生物中。另外,在JCM和ATCC中,所有的菌株都被分类为动物双歧杆菌动物亚种,但由于这2种菌株的16S rDNA的碱基序列与动物双歧杆菌乳酸亚种(accession No.X89513)100%一致,所以在本发明中作为动物双歧杆菌乳酸亚种。
如后述的实施例所示的那样,本发明的微生物具有胆固醇消除活性,使胆固醇、特别使血中胆固醇浓度下降,所以,以有效量含有本发明微生物的物质作为胆固醇吸收抑制剂、特别是作为由肠道吸收胆固醇的抑制剂是有用的。本发明的胆固醇吸收抑制剂期待能够用于降低血中胆固醇、降低甘油三酯、降低VLDL和LDL胆固醇、降低动脉硬化指数、升高HDL胆固醇等脂质代谢的改善;因闭经后的女性激素欠缺而频发的高脂血症的改善;动脉硬化发病危险的降低。
另外,如后述的实施例所示的那样,本发明的微生物显示出耐酸性和耐胆汁酸性,所以,以有效量含有本发明微生物的物质能够作为口服用胆固醇吸收抑制剂使用。
如后述的实施例所示的那样,本发明的微生物在发酵乳饮食品中,即使在好氧条件下保存(非厌氧保存)时,也具有高生存率,因此,以有效量含有本发明微生物的物质能够作为好氧条件下保存(非厌氧保存)的胆固醇吸收抑制剂使用。保存温度例如可以为-80~10℃。
在本发明的胆固醇吸收抑制剂中,作为有效成分含有的微生物,为选自动物双歧杆菌动物亚种YIT 10394、动物双歧杆菌乳酸亚种JCM1253、动物双歧杆菌乳酸亚种JCM 7117和假长双歧杆菌球形亚种中的1种以上即可,这里,作为可以使用的微生物组合,例如,可以列举选自动物双歧杆菌乳酸亚种JCM 1253和动物双歧杆菌乳酸亚种JCM7117中的1种以上的组合;选自动物双歧杆菌动物亚种YIT 10394、假长双歧杆菌球形亚种YIT 10392和假长双歧杆菌球形亚种YIT 10393中的1种以上的组合。
另外,在本发明的胆固醇吸收抑制剂中,除了单独或组合使用动物双歧杆菌动物亚种YIT 10394、动物双歧杆菌乳酸亚种JCM 1253、动物双歧杆菌乳酸亚种JCM 7117和假长双歧杆菌球形亚种以外,也可以将它们与其它微生物并用。作为这样的微生物,例如,可以列举动物双歧杆菌动物亚种、动物双歧杆菌乳酸亚种、假长双歧杆菌球形亚种、青春双歧杆菌、角双歧杆菌、星状双歧杆菌、双歧双歧杆菌、短双歧杆菌、链状双歧杆菌、齿双歧杆菌、高卢双歧杆菌、婴儿双歧杆菌、长双歧杆菌、假小链双歧杆菌、假长双歧杆菌假长亚种、猪双歧杆菌、嗜热双歧杆菌等双歧杆菌属细菌;嗜酸乳杆菌、加氏乳杆菌、植物乳杆菌、干酪乳杆菌、约氏乳杆菌、卷曲乳杆菌、鼠李糖乳杆菌、高加索酸奶乳杆菌、德氏乳杆菌德氏亚种、德氏乳杆菌保加利亚亚种、瑞士乳杆菌、唾液乳杆菌、路氏乳杆菌、马里乳杆菌、食淀粉乳杆菌、消化乳杆菌、布氏乳杆菌、短乳杆菌、鸡乳杆菌、发酵乳杆菌、麦芽香乳杆菌、类干酪乳杆菌、戊糖乳杆菌等乳杆菌属细菌;嗜热链球菌等链球菌属细菌;乳酸乳球菌乳脂亚种、乳酸乳球菌乳亚种等乳球菌属细菌;肠膜明串珠菌乳脂亚种等明串珠菌属细菌;啤酒片球菌等片球菌属细菌;粪肠球菌等肠球菌属细菌;醋化醋杆菌等醋杆菌属细菌;氧化葡糖杆菌等葡糖杆菌属细菌;枯草芽孢杆菌等芽孢杆菌属细菌;啤酒糖酵母等糖酵母属酵母;戴尔凯氏有孢圆酵母等有孢圆酵母属酵母;乳酒假丝酵母等假丝酵母属酵母;马克斯克鲁维氏酵母等克鲁维氏酵母属酵母;汉逊氏德巴利氏酵母等德巴利氏酵母属酵母;异常毕赤氏酵母等毕赤氏酵母属酵母;鲁氏结合糖酵母等结合糖酵母属酵母;米曲霉、日本毛霉、紫色红曲霉、沙门氏柏干酪青霉、微小根毛霉、少根根霉等曲霉属、毛霉属、红曲霉属、青霉菌属、根毛霉属、根霉属的霉菌等。
另外,本发明的胆固醇吸收抑制剂中所含的微生物,也可以是经冷冻干燥的微生物,或者也能够作为含有这些微生物的培养物利用,但不论是何种方式,微生物都优选是活菌的状态。
本发明的胆固醇吸收抑制剂,能够将上述微生物与固体或液体的医药用无毒载体混合,以常用的医药品制剂的剂型给药。作为这样的制剂,例如,可以列举片剂、颗粒剂、散剂、胶囊剂等固体制剂;溶液剂、悬浮剂、乳剂等液剂;冻干制剂等。这些制剂能够通过制剂上的常用方法调制。作为医药用无毒载体,例如,可以列举葡萄糖、乳糖、蔗糖、淀粉、甘露醇、糊精、脂肪酸甘油酯、聚乙二醇、羟乙基淀粉、乙二醇、聚氧乙烯山梨糖醇酐脂肪酸酯、氨基酸、明胶、白蛋白、水、生理食盐水等。另外,根据需要,也能够适当添加稳定剂、湿润剂、乳化剂、粘合剂、等渗剂等常用的添加剂。
另外,本发明的胆固醇吸收抑制剂,除直接摄取上述微生物或通过直接添加在食品中而摄取之外,能够以饮食品的方式摄取。作为饮食品的优选例子,可以列举以活菌状态含有本发明微生物的发酵乳、发酵豆乳、发酵果汁、发酵植物液、发酵米汁、发酵麦汁和来自动植物的材料的发酵饮食品(咸菜、豆酱、酱油、发酵茶、发酵香肠、发酵蛋黄酱、乳酪、咸鱼肉等),这些饮食品能够通过通常方法制造。例如,在制造发酵乳时,首先,在已灭菌的乳培养基中单独或与其它微生物同时接种培养动物双歧杆菌动物亚种YIT 10394、动物双歧杆菌乳酸亚种JCM 1253、动物双歧杆菌乳酸亚种JCM 7117和假长双歧杆菌球形亚种中的至少1种以上,将其均质化处理,得到发酵乳基料。接着,添加混合另外调制的糖浆溶液,利用均质器等均质化,再添加香料,完成最终制品即可。这样操作所得到的本发明的发酵乳,也能够制造成为普通式、软式、水果香料式、固体状、液体状、冷冻状等任意形态的制品。另外,饮食品中也包括动物的饲料。发酵乳中的本发明微生物浓度,例如能够设为103~1013cfu/ml。
此时,在本发明的胆固醇吸收抑制剂中,能够配合在通常的饮食品中所使用的食品材料,例如,能够配合糖类、蛋白质、肽、脂质、维生素类、矿物质类、蔬菜、谷物、果实等植物成分,血液、乳、肝脏、骨、肌肉等动物成分,细菌、霉菌、酵母、真菌等微生物成分或培养物成分,胶凝剂、固化剂、增粘剂、香料、着色剂、双歧杆菌属细菌增殖促进剂、乳酸菌增殖促进剂等。具体而言,可以列举葡萄糖、蔗糖、果糖、麦芽糖、葡萄糖果糖液糖、木糖、帕拉金糖、蜂蜜、枫糖浆、甜米酒等各种甜味料;山梨糖醇、木糖醇、赤藓醇、乳糖醇、益寿糖、还原水饴、还原麦芽糖水饴等各种糖醇;三氯蔗糖、阿斯巴甜等各种高甜度的甜味料;甘草、甜菊、甘草酸糖苷等各种天然甜味料;蔗糖脂肪酸酯、甘油糖脂肪酸酯、卵磷脂等各种乳化剂;琼脂、角蛋白、角叉藻聚糖、瓜尔胶、阿拉伯胶、黄原胶、果胶、刺槐豆胶等各种增粘(稳定)剂。
此外,可以列举多含塔格糖、乳糖、海藻糖、海藻糖(trehallose)、琼脂寡糖、黑曲霉寡糖、半乳寡聚糖、寡聚果糖、寡聚木糖、棉子糖、水苏糖、乳果糖、麦芽三糖、异麦芽寡糖、环糊精、葡糖胺、N-乙酰葡糖胺等各种糖质;藻酸、藻酸钠、墨角藻聚糖、马尾藻多糖、furceran、海萝聚糖、紫菜聚糖、aminaran、芽霉菌糖、塔拉胶、魔芋甘露糖胶、菊糖、壳多糖、壳聚糖、聚右旋糖、透明质酸、硫酸软骨素、β-葡聚糖、甘露聚糖、半乳聚糖、果聚糖、木聚糖、阿拉伯聚糖、阿拉伯半乳聚糖、葡糖甘露聚糖、半乳甘露聚糖、甜菜纤维、燕麦纤维、小麦纤维、大豆纤维、米纤维、大麦纤维、黄原胶、玉米纤维、苹果纤维、柑桔纤维、欧车前纤维、菠萝纤维、洋李纤维、豌豆纤维、香蕉纤维、醋酸菌细菌纤维素、乳酸菌菌体细胞壁、双岐杆菌属细菌菌体细胞壁、酵母菌体细胞壁、纳豆果聚糖、胶原、纳豆聚谷氨酸等各种食物纤维或这些食物纤维的各种水解物;小麦麸、大麦麸、米麸、野燕麦麸、燕麦麸、黑麦麸、欧车前、米糠、糙米、菊苣、大豆豆腐渣、苹果浆、抗性淀粉、大麦麦芽、玉米种子外皮、乳酸菌菌体、双歧杆菌属细菌菌体、啤酒酵母菌体、葡萄酒酵母菌体、葡萄酒粕、酒粕、酱油粕、啤酒粕、米曲、麦曲、豆曲、红曲、黄曲、纳豆粘质物、葡萄种子提取物、蜂王浆、蜂巢蜡胶、小球藻、螺旋藻、裸藻、裙带菜、海带、马尾藻、黑海带、爱森藻、紫菜、肠浒苔、羊栖菜、石莼、海蕴等难消化性的膳食纤维的各种材料。
还可以列举钙、镁、锌、铁、锰、碘、硒、铜、钴、白云石等各种矿物质类或这些矿物质的各种盐类,柠檬酸、苹果酸、酒石酸、丙酮酸、葡糖酸、琥珀酸、富马酸、抗坏血酸、乳酸、醋酸、丙酸、丁酸、磷酸、肌酸、蛋氨酸、半胱氨酸、谷氨酸等氨基酸等各种酸类或这些酸类的各种盐类,谷胱甘肽、植酸钙镁、植酸、木质素、聚-γ-谷氨酸及其分解物、皂苷、阿魏酸、γ-氨基丁酸、γ-谷维素、查耳酮、二氢黄酮、黄酮、黄酮醇、异黄酮、花色素、儿茶素、原花色素、茶叶多酚、姜黄、辣椒素、芝麻素酚、芝麻木酚素、茶黄素、β二酮类、类胡萝卜素类、烯丙基硫化合物、异硫氰酸酯类、萜烯类、叶绿素类、神经鞘脂、神经节苷脂、n-3多不饱和脂肪酸类、n-6多不饱和脂肪酸类、共轭亚油酸类、磷脂质类、植物甾醇类等各种成分、大豆球蛋白、伴大豆球蛋白等大豆蛋白质、卵清蛋白、卵类粘蛋白等卵蛋白、酪蛋白、乳白蛋白、乳酰肝褐质等乳蛋白和乳清蛋白、酪蛋白、乳清蛋白、乳铁蛋白等乳蛋白和乳清蛋白、酪蛋白磷酸肽、米胶蛋白等米蛋白、麦谷蛋白、麦醇溶蛋白等小麦蛋白、鱼肉蛋白等各种蛋白类及其酶分解肽和酸分解肽类,维生素A、维生素B类、维生素C、维生素D类、维生素E、维生素K类、β胡萝卜素、视黄酸、叶酸等各种维生素类,黑升麻、西洋南瓜种子、石榴种子、西洋小连翘、西番莲、缬草、野葛根、迷迭香、薄荷、荷兰芹、金盏花、柠檬香油、艾蒿、红花、萝卜种子、咖啡树、五加、葫芦果实、柑橘科果皮、银杏叶、枣、枸杞、甘草、灵芝、人参、巴西可可等的各种提取物类,绿茶、红茶、乌龙茶、武靴叶茶、番石榴叶等各种植物提取物,胡椒、花椒、桂皮、沉金、鼠尾草、麝香草、罗勒、辣椒、肉豆蔻等各种香辣调味料。
另外,可以列举米、糙米、大麦、小麦、燕麦、黑麦、薏苡、笕、粟、稷、荞麦、高粱、玉米等各种谷物的成分或这些谷物种子的各种发芽物成分,小豆、白小豆、金时豆、菜豆、豌豆、紫花豆、鹰嘴豆、黑大豆、绿大豆、绿豆、蚕豆、大福豆、明日叶、羽衣甘蓝、郁金、马铃薯、甘薯、紫甘薯、山芋、南瓜、茄子、西红柿、苦瓜、柿子椒、芝麻、卷心菜、花茎甘蓝、菜花、莴苣、毛豆、姜、牛蒡、芹菜、萝卜、辣根、鳄梨、胡萝卜、菠菜、洋葱、蒜、百合、韭葱、紫苏、葱、韭菜、荷兰防风草、蕨菜、笋、香菇、蘑菇等各种蔬菜成分,柠檬、苹果、葡萄、草莓、橙、柿子、番石榴、香蕉、蓝莓、黑莓、红莓苔、木莓、越橘、杨梅、费约果、树番茄、黄褥花果、酸橙、扁平橘、甜瓜、桃、芒果、柚子、木瓜、波罗、梨、李子、葡萄柚、木梨、杏、橘子、石榴、西瓜、洋李、猕猴桃等各种果实成分,落花生、杏仁、椰子、腰果、澳洲坚果、可可、栗子、白果、核桃等各种坚果成分,牛奶、脱脂奶、乳清、奶油、发酵乳、酸奶、乳蛋白、酪蛋白、乳清蛋白等各种乳制品及它们成分,清酒、葡萄酒、绍兴酒、啤酒等酿造酒类,威士忌、白兰地、伏特加等蒸馏酒类等。
在利用双歧杆菌属细菌的发酵乳饮食品中,出于提高保存时的生存性的目的,主要使用以玻璃和铝涂布纸等不透氧性的包装剂构成的容器,但如在后述实施例中所示的那样,由于本发明的微生物具有高耐氧性,不需要严密的厌氧状态,所以,本发明的胆固醇吸收抑制剂的容器材料也能够使用透氧性高的树脂(聚苯乙烯、聚乙烯、聚对苯二甲酸乙二醇酯等)。相比于以不透氧性包装剂构成的容器,使用这些树脂的容器具有成本低、成型自由度高等优点。
作为本发明的胆固醇吸收抑制剂有效成分的微生物,如后述实施例中所显示的那样,显示出优异的耐酸性,所以,本发明的胆固醇吸收抑制剂能够制成酸性,例如,能够使其在25℃下的pH为2~7、特别是为3~6。
作为本发明的胆固醇吸收抑制剂有效成分的双歧杆菌属细菌,以往一直作为食品被利用,其安全性也是被确认的,所以当将其作为胆固醇吸收抑制剂使用时,给药量没有严格的限制,但适当的给药量是作为活菌数每日105cfu~1013cfu,特别优选108cfu~1012cfu。
实施例
以下,列举实施例和试验例对本发明进行更具体地说明,但本发明并不限于此。
〔试验例1〕
(1)胆固醇消除活性
以12000rpm、15分钟、4℃的条件离心在m-ILS培养基(Int.J.FoodMicrobiol.81.131-1362003)中37℃厌氧培养24小时的培养液,以pH5.5的150mM磷酸缓冲液洗净菌体。反复3次离心洗净后,悬浮在150mM的磷酸缓冲液中,使浊度(在660nm的吸光度)为3(菌数为108~109cfu/ml),调制菌体悬浮液。另外,将菌体悬浮液以121℃高压灭菌处理15分钟,调制死菌体悬浮液。
在150mM的磷酸缓冲液(pH7.0)中悬浮2g牛胆汁粉末、921mg胆固醇、135mg溶血卵磷脂、90.2mg单油酸、702.2mg油酸,进行12分钟超声波处理后,以100,000G在25℃超离心18小时,回收微团层,调制人工脂质微团。相对1ml菌体悬浮液(菌数为108~109cfu)加入150μl人工脂质微团,在37℃静置。18小时后,以Determiner TC555(Kyowa Medics生产)测定以12000rpm、4℃、离心15分钟的上清的胆固醇。作为对照,对不含菌体的磷酸缓冲液也进行同样的操作,由下式算出胆固醇消除活性。另外,对死菌体悬浮液也同样算出了胆固醇消除活性。
胆固醇消除活性(%)=100-(含有菌体的上清的胆固醇量)/(不含菌体的上清的胆固醇量)×100
(2)耐酸性
在以盐酸调制为pH3的50mM的Na2HPO4溶液10ml中加入0.1ml以GAM培养基(日水)培养到固定时间的菌液,在37℃下处理1小时,由下式算出生存率。
生存率(%)=(酸处理后的活菌数)/(酸处理前的活菌数)×100
(3)耐胆汁酸性
在添加有0.2%的胆汁酸的GAM培养基3ml中,接种30μl以GAM培养基培养到固定时间的菌液。24小时后,以Klett-Summerson比色计(No.66滤色镜)测定菌体浊度。
上述(1)~(3)的试验结果表示在表1中。
[表1]
各菌的胆固醇消除活性、耐酸性、耐胆汁酸性的测定结果
活菌体的胆固醇消除活性(%) | 加热死菌体的胆固醇消除活性(%) | 耐酸性(生存率%) | 耐胆汁酸性(克莱特值) | |
动物双歧杆菌动物亚种YIT 10394 | 85 | NT | 97 | 310 |
动物双歧杆菌乳酸亚种JCM 1253 | 86 | NT | 117 | 256 |
动物双歧杆菌乳酸亚种JCM 7117 | 79 | NT | 92 | 177 |
假长双歧杆菌球形亚种YIT 10392 | 77 | 2 | 100 | 153 |
假长双歧杆菌球形亚种YIT 10393 | 86 | 4 | 100 | 112 |
青春双歧杆菌ATCC 15703(标准株) | 20 | 6 | <1 | 198 |
动物双歧杆菌动物亚种JCM 1190(标准株) | 66 | 4 | 100 | 152 |
角双歧杆菌ATCC 27535(标准株) | 8 | NT | 12 | 108 |
婴儿双歧杆菌ATCC 15697(标准株) | 39 | NT | <1 | - |
链状双歧杆菌ATCC 27539(标准株) | 10 | 6 | <1 | 80 |
假小链双歧杆菌JCM 1200(标准株) | 19 | 11 | <1 | 126 |
双歧双歧杆菌IFO 14252(标准株) | 13 | NT | <1 | - |
短双歧杆菌ATCC 15700(标准株) | 27 | 9 | <1 | 116 |
长双歧杆菌ATCC 15707(标准株) | 34 | 12 | <1 | - |
NT表示未试验。
在耐酸性中,生存率为1%以下时,表示为<1。
在耐胆汁酸性中,在添加有胆汁酸的GAM培养基中几乎不产生时,以目测判断,用-表示。
如表1中所示的那样,本发明的动物双歧杆菌动物亚种、动物双歧杆菌乳酸亚种和假长双歧杆菌球形亚种显示出高的胆固醇消除活性,耐酸性、耐胆汁酸性优异。另外,由于死菌体不具有胆固醇消除活性,所以可知这些菌以活菌状态显示活性。
其次,为了了解胆固醇从上清消除是由沉淀引起的还是由转变和分解引起的,测定了沉淀部分的胆固醇,明确从上清消除的胆固醇没有被转变和分解,而是存在于沉淀物中。以上结果提示,上清中的胆固醇被菌体摄取而沉淀、或者通过菌体具有的胆汁酸解离作用等使脂质微团崩解、沉淀的可能性。
〔试验例2〕对动物血中脂质和肝脏脂质的影响
将含有0.03%酵母提取物的10%脱脂奶粉培养基以121℃灭菌15分钟,接种1%假长双歧杆菌球形亚种YIT 10393,厌氧培养24小时。在同样的培养基中接种2%该培养液,以37℃厌氧培养46~54小时。这样调制的发酵乳中所含的活菌数是3.7×108cfu/ml。将该发酵乳冷冻干燥,以表2所示的组成调制食饵。这里,食饵中的活菌数为是1.0×106cfu/ml。另一方面,作为对照,将含有0.03%酵母提取物的10%脱脂奶粉培养基以121℃灭菌15分钟后,将不接种菌而冷冻干燥的物质作为未发酵乳,以表2表示的组成调制食饵。
[表2]
给予动物的食饵的组成(%)
寡聚糖 | 无 | 有 |
酪蛋白 | 19.66 | 19.66 |
玉米油 | 5 | 5 |
维生素混合物(AIN-93G) | 1 | 1 |
盐类混合物(AIN-93G) | 3.5 | 3.5 |
酒石酸氢胆碱 | 0.2 | 0.2 |
蔗糖 | 14.34 | 11.84 |
α-玉米淀粉 | 50 | 50 |
纤维素 | 5 | 5 |
DL-蛋氨酸 | 0.3 | 0.3 |
发酵乳或未发酵乳 | 1 | 1 |
半乳寡聚糖 | 0 | 2.5 |
接着,将以8周龄购入的Wistar系的卵巢摘除大鼠(日本SLC)或假手术大鼠预饲养1周后,以AIN-93G精制饲料驯化1周,根据体重分为表3所示的实验组。
[表3]
实验组
动物 | 乳 | 半乳寡聚糖% | 只数 |
Sham | SM | 0 | 4 |
OVX | SM | 0 | 5 |
OVX | FSM | 0 | 5 |
OVX | SM | 2.5 | 5 |
OVX | FSM | 2.5 | 5 |
Sham:假手术,OVX:卵巢摘除,SM:未发酵乳,FSM:发酵乳
在室温24±1℃、湿度55±5℃、能够自由摄取各自的组对应的食饵和水的条件下,将这样分组的大鼠在各支架笼中用试验饲料饲养34日。每日的活菌数摄取量是约107cfu/只。
接着,取出投饵器,4小时后进行戊巴比妥麻醉,采取腹主动脉血,并冲洗后摘取肝脏。肝脏在分析前保存在-20℃,血液以3000rpm离心15分钟,分离血浆。肝脏在冷冻干燥后,采用Folch的方法(J.Biol.Chem.226,497-509,1957)提取。
分别使用Determiner TC555(Kyowa Medics)、Triglyceride E-testWako(和光纯药)求出在这样得到的血浆和肝脏提取物中所含有的总胆固醇、甘油三酯的量。另外,使用HDL-cholesterol E-test Wako(和光纯药)求出在血浆中所含的HDL胆固醇量。再通过从总胆固醇量减去HDL胆固醇量,求出血浆中所含的VLDL+LDL胆固醇量。
以得到的结果为基础,对卵巢摘除组进行双因素方差分析,求出p。另外,针对与假手术组给予同样食饵的卵巢摘除组进行t检验。得到的结果表示在表4~6中。值以平均值±SD表示。另外,在表4~6中,所谓交互作用,表示发酵乳和半乳寡聚糖之间的交互作用。
[表4]以假长双歧杆菌球形亚种YIT 10393调制的发酵乳对生长的影响
**、***:在假手术/未发酵乳/无寡聚糖组和卵巢摘除/未发酵乳/无寡聚糖组之间有0.01、0.001的有意义的差别。
NS:无有意义的差别
如表4中所示,卵巢摘除的大鼠和假手术组比较,如已报告(J.Comp.Physiol.Physicol 88:183-193,1975)的那样,体重和摄食量增大,但在卵巢摘除组中,不能确认寡聚糖的有无和给予发酵乳所带来的影响,成长良好。
[表5]以假长双歧杆菌球形亚种YIT 10393调制的发酵乳对血中脂质的影响
*:在假手术/未发酵乳/无寡聚糖组和卵巢摘除/未发酵乳/无寡聚糖组之间,有0.05的有意义的差别。
TC:总胆固醇(mg/dl),TG:甘油三酯(mg/dl),HDL-C:HDL胆固醇(mg/dl),VLDL+LDL-C:VLDL+LDL胆固醇(mg/dl),AI:动脉硬化指数(VLDL+LDL-C/HDL-C)
NS:无有意义的差别
另外,如在表5中所示,以假长双歧杆菌球形亚种YIT 10393调制的发酵乳的效果,在血中的胆固醇、甘油三酯、VLDL+LDL胆固醇、动脉硬化指数中均得到确认,所有项目均降低。特别是VLDL+LDL胆固醇的下降显著。另外,在统计学上没有观察到寡聚糖对这些项目的效果和发酵乳与寡聚糖的交互作用,但寡聚糖和发酵乳并用给药组的血中总胆固醇、甘油三酯、VLDL+LDL胆固醇、动脉硬化指数的值显示最低值。已知半乳寡聚糖被双歧杆菌属细菌特异地利用。因此,通过给予含有半乳寡聚糖的发酵乳,假长双歧杆菌球形亚种YIT 10393在肠道内增殖,表现出有效降低血中脂质的效果。另外,已知摘除卵巢使血中总胆固醇升高,作为闭经后高脂血症模型是有用的。根据卵巢摘除/未发酵乳/无寡聚糖组和假手术组的数据比较,显示本次试验中的卵巢摘除手术是成功的。
[表6]以假长双歧杆菌球形亚种YIT 10393调制的发酵乳对肝脏脂质的影响
*、**:在假手术/未发酵乳/无寡聚糖组和卵巢摘除/未发酵乳/无寡聚糖组之间,依次有0.05、0.01的有意义的差别。
NS:无有意义的差别
如在表6中所示,有无寡聚糖和是否给予发酵乳对肝脏胆固醇含量没有影响,脂质没有向肝脏积蓄。虽然给予发酵乳在肝脏中没有观察到甘油三酯在统计学上的有意义的差别,但每1g肝脏的甘油三酯含量下降。
如上所述,根据以假长双歧杆菌球形亚种YIT 10393调制的发酵乳使血中总胆固醇、甘油三酯、VLDL+LDL胆固醇、动脉硬化指数下降,可知其不仅对胆固醇代谢具有改善功能,而且对各种血中脂质的脂质代谢具有改善功能,并具有降低动脉硬化发病危险的效果。
〔试验例3〕在发酵乳中的生存率的研究
在10%脱脂乳中添加0.03%酵母提取物,灭菌,分别接种2%的本发明细菌(动物双歧杆菌动物亚种YIT 10394、动物双歧杆菌乳酸亚种JCM 1253、动物双歧杆菌乳酸亚种JCM 7117、假长双歧杆菌球形亚种YIT 10392、假长双歧杆菌球形亚种YIT 10393),在37℃下培养至pH5.1±1.0,制造5种发酵乳。另外,作为对照,使用长双歧杆菌ATCC15707,同样地制造发酵乳。
在厌氧保存时向玻璃试管中、在非厌氧保存(好氧保存)时向聚丙烯管中分别注入10ml调制的发酵乳,在10℃保存12周。在厌氧保存时,在N2气流下以丁基橡胶栓密封,在非厌氧保存时,松弛地盖上聚丙烯管的盖子。培养结束时的pH和活菌数表示在表7中,非厌氧保存下的菌数变化表示在表8中,厌氧保存下的菌数变化表示在表9中。
[表7]各菌株在培养结束时的pH和活菌数
pH | 活菌数(cfu/ml) | 培养时间(hr) | |
动物双歧杆菌动物亚种YIT 10394 | 5.06 | 9.1×108 | 27 |
动物双歧杆菌乳酸亚种JCM 1253 | 5.13 | 5.9×108 | 30 |
动物双歧杆菌乳酸亚种JCM 7117 | 5.19 | 1.4×109 | 30 |
假长双歧杆菌球形亚种YIT 10392 | 5.19 | 3.4×108 | 30 |
假长双歧杆菌球形亚种YIT 10393 | 5.14 | 6.2×108 | 30 |
长双歧杆菌ATCC 15707 | 5.01 | 3.3×108 | 27 |
[表8]10℃/非厌氧条件保存(好氧条件保存)下的发酵乳中各菌株的菌数变化
培养结束时 | 3周后 | 8周后 | 12周后 | |
动物双歧杆菌动物亚种YIT 10394 | 9.1×108 | 5.0×108 | 4.5×107 | 4.1×106 |
动物双歧杆菌乳酸亚种JCM 1253 | 5.9×108 | 4.2×108 | 3.9×108 | 3.7×108 |
动物双歧杆菌乳酸亚种JCM 7117 | 1.4×109 | 1.3×108 | 1.0×109 | 7.0×108 |
假长双歧杆菌球形亚种YIT 10392 | 3.4×108 | 2.6×108 | 7.6×107 | 1.7×107 |
假长双歧杆菌球形亚种YIT 10393 | 6.2×108 | 3.9×108 | 3.9×107 | 3.0×106 |
长双歧杆菌ATCC 15707 | 3.3×108 | 9.2×103 | - | - |
-:未被检出
[表9]10℃/厌氧条件保存下的发酵乳中各菌株的菌数变化
培养结束时 | 3周后 | 12周后 | |
动物双歧杆菌动物亚种YIT 10394 | 9.1×108 | 1.0×109 | 4.7×108 |
动物双歧杆菌乳酸亚种JCM 1253 | 5.9×108 | 5.9×108 | 1.0×109 |
动物双歧杆菌乳酸亚种JCM 7117 | 1.4×109 | 1.4×109 | 1.4×109 |
假长双歧杆菌球形亚种YIT 10392 | 3.4×108 | 3.3×108 | 1.8×108 |
假长双歧杆菌球形亚种YIT 10393 | 6.2×108 | 4.1×108 | 1.0×108 |
长双歧杆菌ATCC 15707 | 3.3×108 | 4.5×107 | 4.2×106 |
如在表8中所示的那样,在非厌氧条件(好氧条件)保存中,对照中使用的长双歧杆菌ATCC 15707在3周后减少至9.2×103cfu/ml,在8周后不能被检出。与此相对,本发明的双歧杆菌属细菌,对于任意的菌株,即使保存3周后也不低于1×108cfu/ml,与培养结束时比较的生存率为50%以上。另外,即使保存12周,动物双歧杆菌乳酸亚种JCM
1253、动物双歧杆菌乳酸亚种JCM 7117也不低于1×108cfu/ml,其余3种菌株(假长双歧杆菌球形亚种YIT 10392、假长双歧杆菌球形亚种YIT 10393、动物双歧杆菌动物亚种YIT 10394)也仅减少1~2个数量级左右。
如在表9中所示的那样,在厌氧条件保存下,保存3周后,长双歧杆菌ATCC 15707的菌数减少到1/10左右,但本发明的双歧杆菌属细菌几乎维持培养结束时的菌数。假长双歧杆菌球形亚种YIT 10392、假长双歧杆菌球形亚种YIT 10393、动物双歧杆菌动物亚种YIT 10394即使保存12周,菌数也仅减少1/2~1/6左右,动物双歧杆菌乳酸亚种JCM 1253、动物双歧杆菌乳酸亚种JCM7117即使保存12周后,菌数也几乎是相同。
〔处方例1〕片剂的制造
以下述处方混合各种成分,造粒、干燥、整粒后,压片制造片剂。
(处方) (mg)
本发明的细菌菌体的干燥物1) 20
微晶纤维素 100
乳糖 80
硬脂酸镁 0.5
甲基纤维素 12
1):冷冻干燥假长双歧杆菌球形亚种YIT 10392的活菌体而得到。
〔处方例2〕发酵乳饮料的制造
在15%的脱脂乳中添加3%的葡萄糖,杀菌,接种1%的假长双歧杆菌球形亚种YIT 10393,以35℃厌氧培养24小时,得到210g发酵乳基料。另一方面,在水中溶解97g砂糖、0.2g乳化铁,使总重量为790g,灭菌,得到糖浆。将如上述操作得到的发酵乳基料与糖浆混合,添加1g香料后,以15Mpa均质化,填充在容器中,得到发酵乳饮料。得到的发酵乳饮料的外观、风味都很良好,刚制造后的活菌数为2.5×108cfu/ml。另外,10℃保存21日后的活菌数为1.4×108cfu/ml,保存稳定性也很良好。
〔处方例3〕清凉饮料的制造
根据下述处方,按照通常方法混合各种成分,均质化而得到清凉饮料。将得到的清凉饮料充填在褐色瓶中后,以铝盖封口,实施加热处理。得到的清凉饮料的外观、风味都很良好,保存稳定性也很良好。
(处方) (g)
本发明的细菌菌体的干燥物1) 5
香料 0.8
水 100
还原淀粉糖化物 24
果糖 18
1):冷冻干燥动物双歧杆菌动物亚种YIT 10394的活菌体而得到。
Claims (4)
1.一种胆固醇吸收抑制剂,其特征在于:
以选自动物双歧杆菌动物亚种YIT 10394、动物双歧杆菌乳酸亚种JCM 1253、动物双歧杆菌乳酸亚种JCM 7117和假长双歧杆菌球形亚种中的1种以上的微生物为有效成分。
2.一种胆固醇吸收抑制剂,其特征在于:
以选自动物双歧杆菌乳酸亚种JCM 1253、动物双歧杆菌乳酸亚种JCM 7117中的1种以上的微生物为有效成分。
3.一种胆固醇吸收抑制剂,其特征在于:
以选自动物双歧杆菌动物亚种YIT 10394、假长双歧杆菌球形亚种YIT 10392和假长双歧杆菌球形亚种YIT 10393中的1种以上的微生物为有效成分。
4.动物双歧杆菌动物亚种YIT 10394(FERM ABP-10662)、假长双歧杆菌球形亚种YIT 10392(FERM ABP-10660)或假长双歧杆菌球形亚种YIT 10393(FERMABP-10661)。
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CN101503667B (zh) * | 2009-03-05 | 2010-12-01 | 浙江大学 | 一种耐氧性双歧杆菌 |
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BRPI0615785B8 (pt) | 2021-05-25 |
BRPI0615785B1 (pt) | 2019-07-02 |
WO2007029773A1 (ja) | 2007-03-15 |
US20090170185A1 (en) | 2009-07-02 |
BRPI0615785A2 (pt) | 2011-05-24 |
US7993903B2 (en) | 2011-08-09 |
JPWO2007029773A1 (ja) | 2009-03-19 |
ES2355064T3 (es) | 2011-03-22 |
JP5116473B2 (ja) | 2013-01-09 |
KR20080048468A (ko) | 2008-06-02 |
AU2006288252B2 (en) | 2012-01-12 |
EP1923065A1 (en) | 2008-05-21 |
AU2006288252A1 (en) | 2007-03-15 |
DE602006018627D1 (en) | 2011-01-13 |
EP1923065B1 (en) | 2010-12-01 |
CN101257910B (zh) | 2012-05-23 |
KR101270962B1 (ko) | 2013-06-11 |
EP1923065A4 (en) | 2009-08-26 |
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