CN104207135A - 乳杆菌用于增加选自铁、锌、钙的金属及其离子的吸收的用途 - Google Patents
乳杆菌用于增加选自铁、锌、钙的金属及其离子的吸收的用途 Download PDFInfo
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- CN104207135A CN104207135A CN201410290210.9A CN201410290210A CN104207135A CN 104207135 A CN104207135 A CN 104207135A CN 201410290210 A CN201410290210 A CN 201410290210A CN 104207135 A CN104207135 A CN 104207135A
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Classifications
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- A—HUMAN NECESSITIES
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- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
- A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
- A23C9/1234—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt characterised by using a Lactobacillus sp. other than Lactobacillus Bulgaricus, including Bificlobacterium sp.
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- C—CHEMISTRY; METALLURGY
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Abstract
本发明涉及乳杆菌用于增加选自铁、锌、钙的金属及其离子的吸收的用途。特别是,本发明涉及至少一种选自下列的植物乳杆菌菌株及其部分在制备组合物中的用途,所述菌株选自植物乳杆菌299,DSM6595、植物乳杆菌299v,DSM9843、植物乳杆菌HEAL9,DSM15312、植物乳杆菌HEAL19,DSM15313、植物乳杆菌HEAL99,DSM15316,所述组合物用于在哺乳动物优选人中增加至少一种金属/金属离子的吸收。
Description
本申请是申请日为2006年7月4日,申请号为200680031271.7(国际申请号为PCT/SE2006/000846),发明名称为“乳杆菌用于增加选自铁、锌、钙的金属及其离子的吸收的用途”的发明专利申请的分案申请。
发明技术领域
本发明涉及至少一种植物乳杆菌特效菌株在增加哺乳动物的金属/金属离子吸收中的用途。本发明还涉及至少一种植物乳杆菌特效菌株在制备药用组合物中的用途以及包含至少一种植物乳杆菌特效菌株的组合物。
背景技术
在西方和发展中国家,婴儿、青少年和育龄女性都普遍存在铁缺乏症和铁储备低。铁缺乏症的原因之一是来自食物的铁的生物利用度低,部分归因于膳食中的抑制因素,如植酸和酚类化合物。其它因素增强铁吸收。这些因素包括肌肉组织、抗坏血酸和某些其它有机酸。
植酸主要位于谷类、蔬菜和水果的纤维部分。植酸的抑制效应是由于在肠内pH与铁形成了不溶性络合物。减少这些食物的植酸含量或者抑制与铁络合键合将解决从富含铁或者被视为健康和有营养的食物中铁吸收少的问题。植酸由某些植物、微生物和动物组织的植酸酶水解。大部分谷类植酸酶的最佳pH在5.0-5.6范围。通过降低食物的pH,可激活谷类和蔬菜的内源性植酸酶,从而减少植酸含量,如在酵母发酵时。
EP1 003 532描述乳杆菌在制备非发酵肠内组合物中的用途,所述组合物用于促进或增加膳食矿物质的吸收。其中支持所述权利要求的吸收的唯一实验是用Caco-2肠细胞系(致癌细胞系)进行的钙反应或钙转运体外模型。
根据本发明,已经在人体进行金属/金属离子吸收的体内研究。令人惊奇地发现并非所有乳杆菌都具有如EP1 003 532要求的所需吸收。还发现某些植物乳杆菌特效菌株在体内令人惊奇地实现了所述金属/金属离子的良好吸收。
发明概述
本发明一方面涉及至少一种植物乳杆菌菌株及其部分在制备组合物中的用途,所述植物乳杆菌选自植物乳杆菌299,DSM6595、植物乳杆菌299v,DSM9843、植物乳杆菌HEAL9,DSM15312、植物乳杆菌HEAL19,DSM15313、植物乳杆菌HEAL99,DSM15316,所述组合物用于增加至少一种金属/金属离子在哺乳动物、优选人中的吸收。
通过使用上述组合物,可通过增加所述金属/金属离子在体内的吸收,明显增强哺乳动物、优选人且特别优选女性和儿童的一般健康状况。因此,人体可利用消耗的食物中大部分金属/金属离子,获得更好的健康状况,个人感觉更好。本发明的又一个优点是为了获得所需吸收,个体不需要增加摄入各种金属/金属离子如Fe。不需要添加补充量的各种金属/金属离子即可发生这种情况。因此,可避免以前用于增加某些金属吸收时增加如Fe消耗的情况可能出现的不良反应。通过本发明可避免的此类不良反应的实例包括结肠癌,也就是说已经显示过高消耗Fe可导致结肠癌,参阅如″Iron intake and the risk ofcolorectalcancer″,Wurzelmann JI等,Cancer Epidemiol Biomarkers Prev.1996Jul;5(7):503-7。
本发明另一方面涉及通过给予包含至少一种植物乳杆菌菌株及其部分的组合物,增加至少一种金属/金属离子在哺乳动物优选人的吸收的方法,所述植物乳杆菌选自植物乳杆菌299,DSM6595、植物乳杆菌299v,DSM9843、植物乳杆菌HEAL9,DSM15312、植物乳杆菌HEAL19,DSM15313和植物乳杆菌HEAL99,DSM15316。
本发明又一方面涉及至少一种选自植物乳杆菌299,DSM6595、植物乳杆菌299v,DSM9843、植物乳杆菌HEAL9,DSM15312、植物乳杆菌HEAL19,DSM15313和植物乳杆菌HEAL99,DSM15316的植物乳杆菌菌株及其部分在制备药用组合物中的用途,所述药用组合物用于治疗贫血、骨质疏松症或任何其它其中存在至少一种选自Fe、Zn、Ca和Mg及其离子的金属/金属离子缺乏问题的疾病。
可用本发明的药用组合物治疗或预防其中缺乏一种上述金属/金属离子的任何紊乱或疾病。
本发明还有另一方面涉及一种组合物,所述组合物包含至少一种选自植物乳杆菌299,DSM6595、植物乳杆菌299v,DSM9843、植物乳杆菌HEAL9,DSM15312、植物乳杆菌HEAL19,DSM15313、植物乳杆菌HEAL99,DSM15316的植物乳杆菌菌株及其部分以及至少一种选自Fe、Zn、Ca、Mg及其离子的金属/金属离子。
发明详述
在本发明的一个实施方案中,所述至少一种金属/金属离子选自Fe、Zn、Ca和Mg及其离子。还可能所述至少一种金属/金属离子与另一种元素缔合或者与另一种元素络合键合。因此,即使任何上述金属/金属离子以另一种形式存在,也可增加金属/金属离子的吸收。
在本发明的另一个实施方案中,所述至少一种菌株可以是有活力的、灭活或抑制或者死亡的,只要获得金属/金属离子吸收的所需增加。而且,所述至少一种菌株可以是基因修饰的。
在本发明的另一个实施方案中,所述组合物包含载体材料。所述载体材料可选自麦片粥、乳酸发酵食物、抗性淀粉、菊粉、果聚糖和糖醇、膳食纤维、碳水化合物、蛋白质、糖基化蛋白和脂类。载体还可以是上述载体的组合,例如脂类、碳水化合物和蛋白质的混合物。可将所述载体材料与一种或多种选自植物乳杆菌299,DSM6595、植物乳杆菌299v,DSM9843、植物乳杆菌HEAL9,DSM15312、植物乳杆菌HEAL19,DSM15313和植物乳杆菌HEAL99,DSM15316的菌株发酵。
在本发明的又一个实施方案中,所述组合物选自食品、膳食补充剂、营养品、功能食品和医疗食品。所述食品可选自饮料、酸奶、果汁、冰淇淋、面包、饼干、谷类、健康棒和酱。上述组合物可以发酵或非发酵,优选发酵组合物。
在还有另一个实施方案中,所述组合物包含至少一种选自Fe、Zn、Ca和Mg及其离子的金属/金属离子。通过将金属/金属离子包含在待摄入的组合物内,可在增加的浓度下观察到甚至更高的吸收。
在另一个实施方案中,所述至少一种菌株在组合物中的量为约1×106-约1×1014CFU,优选1×108-1×1012,更优选1×109-1×1011。
在本发明的又一个实施方案中,所述药用组合物是液体制剂或固体制剂。固体制剂可选自片剂、含片(sucking tablet)、糖、咀嚼片、口香糖、胶囊剂、香囊剂、散剂、颗粒剂、包衣颗粒和包衣片、肠溶片和胶囊剂以及熔解条带(strips)和薄膜。所述液体制剂可选自口服溶液、混悬液、乳液和糖浆剂。
在另一个实施方案中,所述药用组合物包含至少一种选自Fe、Zn、Ca和Mg及其离子的金属/金属离子。只要获得所需吸收,还可使所述至少一种金属/金属离子与另一种元素缔合或者与另一种元素络合键合。
所述至少一种在药用组合物中的菌株可以是有活力的、灭活或抑制或者死亡的。所述至少一种菌株也可以是基因修饰的。使用的菌株也可以是上述的组合,也就是说例如一种菌株有活力而另一种菌株是灭活的。
在另一个实施方案中,所述至少一种菌株在药用组合物中的量为约1×106-约1×1014CFU,优选1×108-1×1012,更优选1×109-1×1011。
在还有另一个实施方案中,提供一种组合物,所述组合物包含至少一种选自植物乳杆菌299,DSM6595、植物乳杆菌299v,DSM9843、植物乳杆菌HEAL9,DSM15312、植物乳杆菌HEAL19,DSM15313、植物乳杆菌HEAL99,DSM15316的植物乳杆菌及其部分以及至少一种选自Fe、Zn、Ca、Mg及其离子的金属/金属离子,其中所述至少一种金属/金属离子与另一种元素缔合或者与另一种元素络合键合。所述至少一种菌株可以有活力、灭活或抑制或者死亡的。所述至少一种菌株也可以是基因修饰的。通过给予本发明组合物,可提高人的一般健康状况。通过使用本发明组合物,特别可提高经期女性或其它铁储备低的人的健康状况。虽然所述人不一定贫血,但可增强一般健康状况。
在又一个实施方案中,所述组合物包含载体材料,其中所述载体材料选自麦片粥、乳酸发酵食品、抗性淀粉、膳食纤维、菊粉、果聚糖和糖醇、碳水化合物、蛋白质、糖基化蛋白和脂类。还可将所述载体材料用一种或多种选自植物乳杆菌299,DSM6595、植物乳杆菌299v,DSM9843、植物乳杆菌HEAL9,DSM15312、植物乳杆菌HEAL19,DSM15313和植物乳杆菌HEAL99,DSM15316的菌株发酵。
在本发明的一个实施方案中,所述组合物选自食品、膳食补充剂、营养品、功能食品和医疗食品。所述食品可选自饮料、酸奶、果汁、冰淇淋、面包、饼干、谷类、健康棒和酱。所述至少一种菌株在组合物中的量可为约1×106-约1×1014CFU,优选1×108-1×1012,更优选1×109-1×1011。
在本说明书中菌株的“部分”指细菌细胞部分如DNA、细胞壁、细胞膜部分,或者具有增加上述金属/金属离子吸收需要的活性的细菌细胞的任何其它部分。在本文中菌株的部分也可以是全部或部分匀浆。
用于本文时,“金属/金属离子”分别指纯金属和金属离子,如Fe、Zn、Ca和Mg以及Fe2+、Fe3+、Zn2+、Ca2+和Mg2+。即使所述金属/金属离子处于另一种形式如与其它元素络合键合或缔合,本文描述的吸收也可自然发生。
“与元素络合键合”或“与元素缔合”在本文指金属/金属离子可存在并且将发生所需吸收的任何形式。
本发明实验实施方案的详细描述
实验1
在本研究中,用交叉设计研究植物乳杆菌299v及其发酵产物、乳酸和乙酸对从低铁生物利用度膳食中吸收非血红素铁的作用。用四种不同的麦片粥测试植物乳杆菌299v和有机酸的特殊作用:含活性植物乳杆菌299v的发酵麦片粥、含发酵制品但灭活细菌的巴氏发酵麦片粥、pH调节的非发酵麦片粥以及含添加有机酸的非发酵麦片粥。
受试者和方法
受试者
在研究前对70名女性志愿者筛选2-4周,选择24名女性进行研究,选择的基础是无贫血但铁储备相对低,即血清铁蛋白浓度≤40μg/L和血红蛋白浓度≥110g/L。24名志愿者是健康年轻女性,平均年龄(±SD)为25±4岁,平均体重为62±7kg,平均体重指数为21.3±1.9kg/m2。所有受试者都不吸烟,无妊娠或哺乳,或者在研究前≥2月或研究期间没有摄入任何维生素或矿物质补充剂。18名受试者使用口服避孕药,但受试者没有常规摄入任何其它药物。研究前≥2月或研究期间没有捐血。对每个参加者告知关于本研究的口头和书面信息,然后获得知情同意书。本研究得到Municipal Ethical Committee ofCopenhagen and Frederiksberg,Denmark(档案号KF01-219/03)和National Institute of Radiation Hygiene,Denmark的批准。
实验设计
本研究是完全随机化、双盲交叉试验,其中每个受试者进食4种试验餐:(A)发酵麦片粥,(B)巴氏消毒发酵麦片粥,(C)非发酵麦片粥(用乳酸调节pH),和(D)含添加有机酸(乳酸和乙酸)的非发酵麦片粥。
用双重标签外标记法测定4种试验餐的铁吸收。使用这种方法时,通过在两个周期内每个周期同时从两种试验餐吸收的铁测定来自4种试验餐的铁吸收。将每个周期内两种不同的试验餐分别用55Fe和59Fe外标记,连续4个早晨服用两次,使每天变化的潜在效应最小,例如以ABBA的顺序。总计使用12种服用顺序,随机分配给受试者,以便所有试验餐在本周期作为第一餐服用的次数相等。这对于能够确认在周期内含活定居菌的发酵麦片粥的可能后续效应很重要。
进食18天后测定血液样品内两种同位素的活性,然后用其余试验餐实施第二周期。用第二周期血液样品的同位素活性水平减去第一周期残留的同位素活性。
试验餐的构成和食用程序
用麦片和水混合制备麦片粥,然后用酶和巴氏消毒处理(通过Probi AB获得)。然后将麦片粥A用植物乳杆菌299v(DSM9843,活菌计数1.1×109cfu/g)发酵[20]。麦片粥B是巴氏消毒的麦片粥A(活菌计数<10cfu/g),麦片粥C是非发酵的碱性麦片粥,用L-乳酸酸化至与麦片粥A、B和D相等的pH,麦片粥D是非发酵的碱性麦片粥,加入预期在麦片粥A发酵时产生的等量有机酸DL-乳酸和乙酸。对于每份试验餐,100g麦片粥(A、B、C或D)与140g全麦小面包(60.0g小麦面粉,20.0g全麦面粉,2.0g盐,2.0g酵母,16.0g菜籽油,40.0g超纯水)和10g黄油以及一玻璃杯超纯水(200mL)一起进食。制备一批麦片粥,冷藏(4℃)待用。制备一批全麦小面包,冷冻储藏,食用前在200℃烤箱再加热10分钟。
在禁食12小时后于早晨食用试验餐。整夜允许摄入最多0.5L水。在摄入试验餐前12小时内不允许进行中度或强烈的体力活动或者摄入任何酒精或药物。食用试验餐后,受试者不允许进食或喝水2小时,禁止摄入酒精24小时。受试者填写与每份试验餐有关的调查表,确保她们坚持完成所有程序,她们按照指示交替进食和喝水,用水彻底冲洗装麦片粥的玻璃杯,确保摄入全部同位素剂量。工作人员保证她们要吃完所有东西。在实验期间,受试者填写关于其日常进食习惯的详细调查表。
同位素和标记程序
在同位素交换食用前18小时,将1mL同位素溶液[55FeCl3(NENLife Science Products,Inc.,Boston,MA)或59FeCl3(AmershamBiosciences Corp.,Piscataway,NJ)在0.1mol/L HCl中]直接加入麦片粥内,从外部标记所有试验餐。在第一周期每剂量包含37kBq55FeCl3或59FeCl3,第二周期包含74kBq55FeCl3或59FeCl3。
食谱分析
将4种麦片粥和面包冷冻干燥,匀浆处理,对总铁、钙、锌和植酸进行双重分析。用国家食物构成数据库(Danish Tables of FoodComposition,DANKOST2000,1.20版,Herlev,Denmark)计算能量含量。在含65%(w/v)超纯硝酸(Merck KgaA,Darmstadt,Germany)的MES1000Solvent Extraction System(CEM Corp.,Matthews,NC)中湿式灰化后通过原子吸收光谱法(Spectra-AA200,Varian,Mulgrave,Australia)测定总铁、钙和锌。用典型膳食标准参考物1548a(National Institute ofStandards and Technology,Gaithersburg,MD)作为铁(均数±SD:35.3±3.77μg/g)、钙(1.96±0.11mg/g)和锌(24.6±1.79μg/g)的参考,分析值分别是33.38μg/g、2.00mg/g和23.25μg/g。通过高效离子层析分析作为单独肌醇三-至六磷酸盐(IP3-6)1的植酸。通过毛细管气相层析测定麦片粥中的有机酸浓度。
铁状态的确定
在抽取血液样品之前的摄入和活动限制如对试验餐所述。让受试者仰卧位休息10分钟后从肘静脉抽取血液样品。用Sysmex KX-21自动血液分析仪(Sysmex America Inc.,Mundelein,IL)和适当的对照(Eight check-3WP,22490822,Sysmex America Inc.)对收集在装有溶解EDTA的管(Vacutainer system,Becton Dickinson,Franklin Lakes,NJ)内的静脉血(3.5mL)进行血红蛋白分析。测定内和测定间差异分别是0.5%(n=12)和0.6%(n=27)。对收集在光滑管(Vacutainer system,Becton Dickinson)内的静脉血(5.0mL)进行血清铁蛋白和α1-抗胰凝乳蛋白酶(ACT)分析。用Immulite1000分析仪(Diagnostic ProductsCorporation,Los Angeles,CA)化学发光免疫分析测定血清铁蛋白,也分析适当的参考血清(3rd铁蛋白国际标准(80/578),WHO,NIBSC,South Mimms,United Kingdom)。测定内和测定间差异分别是2.7%(n=15)和5.0%(n=15)。用免疫比浊技术在Cobas Mira analyzer(RocheDiagnostic Systems,F.Hoffman-La Roche Ltd.,Basel,Switzerland)上测定ACT,也分析适当的参考血清(欧洲委员会批准的参考物470,11924号,IRMM,Geel,Belgium)。测定内和测定间差异分别是1.4%(n=12)和3.2%(n=14)。
非血红素铁吸收的确定
测定收集在装有肝素作为抗凝剂的管(Vacutainer system,BectonDickinson)中的血液样品(60mL)的55Fe和59Fe的活性。同时通过干式灰化,接着再结晶和溶解,然后通过上文描述的方法用自动猝灭校正在Tricarb2100TR液体闪烁分析仪(Packard Instruments,Meriden,CT)中计数,测定55Fe和59Fe。
统计学分析
将非血红素铁吸收数据转化为对数,然后进行统计学分析,将结果再转化为反对数。用于统计分析的所有数据都呈正态分布,残差曲线检验呈方差齐性。使用线性混合模型比较不同餐的非血红素铁吸收,用log(非血红素铁吸收)作为因变量,餐、交替餐和铁蛋白作为固定的自变量,受试者和受试者×周期相互作用作为随机效应:
Log(非血红素铁吸收)=μ(餐i)+α(交替餐i)+b×铁蛋白i+A(受试者i)+B(受试者i×周期i)+εi
将数据用最小二乘平均估计值表示,平均估计值之间的差异用95%CI表示。用SAS统计软件包,8.2版(SAS Institute Inc.,Cary,NC)进行统计学分析,将P<0.05的值视为显著差异。
结果
试验餐的构成
试验餐的构成和麦片粥中有机酸的含量在以下表1列出。
表1包括含黄油的全麦小面包在内试验餐的构成,以及麦片粥中有机酸的pH和浓度
1代表单独肌醇四-至六磷酸盐
铁状态和非血红素铁吸收
受试者的血红蛋白浓度在111-137g/L范围,血清铁蛋白浓度在12-40μg/L范围。因为血清铁蛋白浓度对炎症敏感,所以将血清中测定的急性期蛋白ACT作为急性期反应的标志。浓度在0.20-0.37g/L范围内,提示无急性期反应(ACT<0.6g/L),因此有效测定受试者的铁状态。
通过混合线性模型分析计算的4种试验餐的非血红素铁吸收在以下表2显示。
表2来自含有4种不同麦片粥的试验餐的非血红素铁吸收
1混合线性模型分析最小二乘估计值的几何均数,括弧内为95%CI,n=24
2混合线性模型分析差异估计值的几何均数,括弧内为95%CI,n=24
*该数值与各列内所有其它数值显著差异(P<0.0001)
当比较非血红素铁吸收的绝对值和相对于pH调节的非发酵麦片粥餐的比值时,结果显示含发酵麦片粥试验餐具有非常显著的作用(P<0.0001),其中考虑了个体间差异。
因为植物乳杆菌299v可在人肠粘膜定居约2周和因为含发酵麦片粥的试验餐增加非血红素铁吸收,所以评估该试验餐对周期内接着摄入的试验餐的特殊后续效应。虽然没有观察到试验餐的广泛后续效应,但就含发酵麦片粥试验餐的特殊效应而言,该后续效应几乎达到统计学意义(P=0.06)。
不同试验餐的吸收率显示含乳酸发酵麦片粥的试验餐的非血红素铁吸收非常显著地增加,而作为不同对照食用的巴氏消毒发酵麦片粥餐和非发酵麦片粥餐则无效应。因为铁和植酸盐在4种试验餐中的含量是恒定不变的,所以这种显著效应可直接是植物乳杆菌299v发酵的作用。应该通过比较含发酵麦片粥餐的吸收率与含灭活植物乳杆菌299v(巴氏消毒发酵麦片粥)和添加有机酸的非发酵麦片粥餐的吸收率,确定是活性植物乳杆菌299v的作用或是发酵时产生的有机酸的作用,因为加入后者的是发酵处理时正常产生的浓度的乳酸和乙酸。有机酸的分析结果显示在进食3种麦片粥时不可能达到有机酸的相似水平(表2)。与发酵麦片粥的有机酸浓度差异最小的试验餐是巴氏消毒发酵麦片粥,其中乳酸和乙酸的浓度分别降低19%和8%。当比较这2种餐的铁吸收率时,巴氏消毒发酵麦片粥的吸收率减少至50%。因为添加有机酸的麦片粥中乳酸水平比巴氏消毒发酵麦片粥低52%而吸收率只减少9%,所以发酵麦片粥铁吸收的增加不可能主要归因于有机酸的作用。因此本研究的结果提示活性乳酸菌,植物乳杆菌299v(1.1×1011cfu)能够增加年轻女性从低铁生物利用度餐吸收非血红素铁。
通常认为在十二指肠和近端小肠发生铁吸收。食物中的小有机酸,如巴氏消毒发酵麦片粥和添加有机酸的麦片粥中的乳酸和乙酸,在胃肠道内非常迅速地吸收。对发酵麦片粥增强非血红素铁吸收的可能解释可以是植物乳杆菌299v定居在大部分近端小肠或者可能在结肠粘膜,其中活性菌局部产生的有机酸可能降低两者的局部pH,乳酸可能与铁形成可溶性络合物,如Derman等描述。含发酵麦片粥餐的后续效应几乎达到显著性(P=0.06),表明在细菌仍定居于肠道的接下来的几天内植物乳杆菌299v对从摄入的餐内吸收非血红素铁的作用,通过这一事实可强化这种假设。
当将含发酵麦片粥、巴氏消毒发酵麦片粥和添加有机酸麦片粥的试验餐(如上文描述)的吸收率相比,显然不可将含发酵麦片粥试验餐吸收率的增加指定为只是有机酸的作用,如上文假设,但清楚看到活性植物乳杆菌299v的特殊作用。
实验2
试剂。除非另外标明,否则所有试剂购自GTF(Sweden)。
Caco-2细胞培养物。第17代Caco-2细胞购自美国模式培养物保藏中心(Rockville,MD),在第20-35代用于实验。在37℃和95%空气-5%CO2的潮湿气氛下,将培养物原液保存在补充20%(v/v)胎牛血清(FSC)、100单位/L青霉素G和100mg/L链霉素的Dulbecco′s改良α必需培养液(DMEM)中。每2-3天更换生长培养液。用含0.5mmol/LEDTA的0.5g/L胰蛋白酶在Dulbecco′s磷酸盐缓冲液(PBS)中使~80%融合的细胞分离。在实验之前,将100000个细胞/0.5mL补充DMEM种在0.4μm微孔聚碳酸酯膜插入物(1cm2Tranwell inserts;Corning,Acton,MA)上。底外侧室装有1.5mL补充DMEM。每2-3天更换滤芯两侧的培养液。在接种后14-17天进行所有铁摄入和转运实验。
细菌培养物。将植物乳杆菌299v(DSM9843),(5)、植物乳杆菌299,(1)、植物乳杆菌Heal9(DSM15312),(2)、植物乳杆菌Heal19(DSM15313),(4)、植物乳杆菌299v突变体(AMJ1277),(3)和罗伊乳杆菌,(6)培养在旋转振荡器上的MRS肉汤(37℃,200rpm)中。在指数期收集细菌(OD600,max=1.3)。用预定标准曲线计算对应于一定细胞数的细胞培养液体积。使细胞以5000rpm旋转(Sorvall heraeus,multifuge)2分钟,然后再悬浮于HBSS(PAA)、HEPES2.5%(1M,PAA)和FeCl310μM转运溶液中。所有样品都以6.7×107个细胞/ml的细胞浓度进行试验,但罗伊乳杆菌的添加浓度为3.35×107个细胞/ml。将试验重复2次。
细胞55Fe摄入和跨单细胞层转运的测定。在摄入和转运测定前1天将新鲜补充的DMEM提供给细胞。为了研究Caco-2细胞的Fe(III)摄入和跨上皮转运,用55Fe(Perkin Elmer)追踪细菌悬液。将0.5mL体积的悬液置于Caco-2细胞顶侧,同时底外侧室包含1.5mlHBSS/HEPES。在37℃和95%空气-5%CO2的潮湿气氛下培养细胞。培养2小时后,将细胞用冰冷洗涤缓冲液(150mmol/L NaCl,10mmol/LHEPES,1mmol/L EDTA,pH7)洗涤4次,在0.5M NaOH中匀浆化。用液体闪烁计数测定转运至底外侧室或者与Caco-2细胞溶解液缔合的55Fe。在测定之前和之后通过测量TEER监测单细胞层的完整性。
加入不同菌株后在HBSS/HEPES中FeCl3(10μmol/l)的转运(参阅方法的描述)。
对照:同上但无细菌。
转运% | |
对照 | 0.06 |
1.植物乳杆菌299 | 0.58 |
2.植物乳杆菌Heal9 | 0.23 |
3.植物乳杆菌299v突变体 | 0.32 |
4.植物乳杆菌heal19 | 0.69 |
5.植物乳杆菌299v | 0.38 |
6.罗伊乳杆菌 | 0.11 |
结果:与净Fe溶液相比,菌株1-5影响Fe的转运。可见增加3-9倍。虽然在含罗伊乳杆菌的样品中显示转运增加,但与不同的植物乳杆菌菌株无法相比。
因此,在不同植物乳杆菌菌株观察到转运的增加,显示铁吸收的类似增加,如上述人体研究所示。
Claims (24)
1.至少一种选自下列的有活力的植物乳杆菌菌株在组合物中用于与不含所述有活力的植物乳杆菌菌株的组合物相比增加哺乳动物对Fe及其离子中至少一种的体内的吸收的用途,所述菌株选自植物乳杆菌299,DSM6595、植物乳杆菌299v,DSM9843、植物乳杆菌HEAL9,DSM15312、植物乳杆菌HEAL19,DSM15313、植物乳杆菌HEAL99,DSM15316。
2.至少一种选自下列的有活力的植物乳杆菌菌株在制备组合物中的用途,所述菌株选自植物乳杆菌299,DSM6595、植物乳杆菌299v,DSM9843、植物乳杆菌HEAL9,DSM15312、植物乳杆菌HEAL19,DSM15313、植物乳杆菌HEAL99,DSM15316,所述组合物用于在哺乳动物中增加至少一种选自Fe及其离子的金属/金属离子的吸收。
3.至少一种选自下列的有活力的植物乳杆菌菌株在组合物中用于增加哺乳动物对至少一种选自Fe及其离子的金属/金属离子的吸收的用途,所述菌株选自植物乳杆菌299,DSM6595、植物乳杆菌299v,DSM9843、植物乳杆菌HEAL9,DSM15312、植物乳杆菌HEAL19,DSM15313、植物乳杆菌HEAL99,DSM15316。
4.权利要求1的用途,其中所述Fe及其离子中至少一种与另一种元素缔合或者与另一种元素络合键合。
5.权利要求1-4中任一项的用途,其中所述组合物包含载体材料。
6.权利要求5的用途,其中所述载体材料选自麦片粥、乳酸发酵食物、抗性淀粉、膳食纤维、碳水化合物、蛋白质、糖基化蛋白和脂类。
7.权利要求5或6的用途,其中所述载体材料用一种或多种选自植物乳杆菌299,DSM6595、植物乳杆菌299v,DSM9843、植物乳杆菌HEAL9,DSM15312、植物乳杆菌HEAL19,DSM15313和植物乳杆菌HEAL99,DSM15316的菌株发酵。
8.权利要求1-7中任一项的用途,其中所述组合物选自食品、膳食补充剂、营养品、功能食品和医疗食品。
9.权利要求8的用途,其中所述食品选自饮料、酸奶、果汁、冰淇淋、面包、饼干、谷类、健康棒和酱。
10.权利要求1-9中任一项的用途,其中所述组合物包含至少一种选自Fe及其离子的金属/金属离子。
11.权利要求10的用途,其中所述至少一种金属/金属离子与另一种元素缔合或者与另一种元素络合键合。
12.权利要求1-11中任一项的用途,其中所述至少一种菌株在组合物中的量为约1×106-约1×1014CFU。
13.权利要求1的用途,其中所述哺乳动物是人。
14.权利要求12的用途,其中所述至少一种菌株在组合物中的量为约1×108-约1×1012CFU。
15.权利要求12的用途,其中所述至少一种菌株在组合物中的量为约1×108-约1×1012CFU。
16.至少一种选自下列的有活力的植物乳杆菌菌株在制备用于治疗贫血的药用组合物中的用途,所述菌株选自植物乳杆菌299,DSM6595、植物乳杆菌299v,DSM9843、植物乳杆菌HEAL9,DSM15312、植物乳杆菌HEAL19,DSM15313和植物乳杆菌HEAL99,DSM15316。
17.权利要求16的用途,其中所述药用组合物是液体制剂或固体制剂。
18.权利要求17的用途,其中所述固体制剂选自片剂、含片、糖、咀嚼片、口香糖、胶囊剂、香囊剂、散剂、颗粒剂、包衣颗粒和包衣片、肠溶片和胶囊剂以及熔解条带和薄膜。
19.权利要求17的用途,其中所述液体制剂选自口服溶液、混悬液、乳液和糖浆剂。
20.权利要求16-19中任一项的用途,其中所述组合物包含至少一种选自Fe及其离子的金属/金属离子。
21.权利要求20的用途,其中所述至少一种金属/金属离子与另一种元素缔合或者与另一种元素络合键合。
22.权利要求16-21中任一项的用途,其中所述至少一种菌株在药用组合物中的量为约1×106-约1×1014CFU。
23.权利要求22的用途,其中所述至少一种菌株在药用组合物中的量为优选约1×108-1×1012。
24.权利要求23的用途,其中所述至少一种菌株在药用组合物中的量为更优选约1×109-1×1011。
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BRPI0612590A2 (pt) | 2010-11-23 |
CN101252943A (zh) | 2008-08-27 |
WO2007004966A1 (en) | 2007-01-11 |
ZA200800108B (en) | 2009-08-26 |
US9687513B2 (en) | 2017-06-27 |
CA2614323A1 (en) | 2007-01-11 |
DK1904074T3 (da) | 2013-07-08 |
NO338795B1 (no) | 2016-10-17 |
SE0501556L (sv) | 2007-01-06 |
KR101467306B1 (ko) | 2014-12-01 |
BRPI0612590B1 (pt) | 2018-07-17 |
EP1904074B1 (en) | 2013-05-01 |
PL1904074T3 (pl) | 2013-12-31 |
SE529199C2 (sv) | 2007-05-29 |
PT1904074E (pt) | 2013-08-02 |
US10105403B2 (en) | 2018-10-23 |
KR20140131508A (ko) | 2014-11-13 |
CA2614323C (en) | 2011-11-22 |
KR101554960B1 (ko) | 2015-09-23 |
RU2402341C2 (ru) | 2010-10-27 |
KR20080039399A (ko) | 2008-05-07 |
ES2423497T3 (es) | 2013-09-20 |
AU2006266492B2 (en) | 2011-07-28 |
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JP2008544753A (ja) | 2008-12-11 |
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AU2006266492A1 (en) | 2007-01-11 |
US20090098190A1 (en) | 2009-04-16 |
RU2008104145A (ru) | 2009-08-10 |
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US20170360854A1 (en) | 2017-12-21 |
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