CN105535030A - 鼠李糖乳杆菌和体重控制 - Google Patents
鼠李糖乳杆菌和体重控制 Download PDFInfo
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Abstract
本发明涉及肥胖症领域。具体而言,本发明涉及益生菌用以治疗肥胖症的用途。本发明的一个实施方案涉及鼠李糖乳杆菌CGMCC?1.3724和/或鼠李糖乳杆菌NCC?4007用于制备帮助体重控制、促进体重减轻和/或治疗肥胖症的组合物的用途。
Description
本申请是申请人于2008年7月24日提交的题为“鼠李糖乳杆菌和体重控制”的中国专利申请200880110900.4的分案申请。
本发明一般地涉及肥胖症领域。具体而言,本发明涉及使用益生菌来帮助体重控制、促进体重减轻和/或治疗肥胖症。
在近几十年来肥胖症的发病率在世界范围内已提高到流行病的程度。全世界约有十亿人超重或肥胖,这是提高死亡率、行动和经济费用的疾病。肥胖症在能量摄入高于能量消耗时发生,过剩的能量主要作为脂肪存储在脂肪组织中。体重减轻和防止体重增加可通过减少能量摄入或降低生物利用率、提高能量消耗和/或减少脂肪存储来实现。肥胖症对健康造成严重威胁,因为它与一系列慢性病相关,包括糖尿病、动脉粥样硬化、退行性障碍、呼吸道疾病和一些癌症。
最近已将肠道菌群的改变与肥胖症关联起来。这些改变在肥胖小鼠中已证实影响肠道小型生物群的代谢势(metabolicpotential),导致从膳食中获取能量的能力提高(TurnbaughPJ,LeyRE,MahowaldMA,MagriniV,MardisER,GordonJl.Anobesity-associatedgutmicrobiomewithincreasedcapacityforenergyharvest.Nature.2006;LeyRE,TurnbaughPJ,KleinS,GordonJl.Microbialecology:humangutmicrobesassociatedwithobesity.Nature.2006)。已经提出,这些肠道小型生物群的改变对肥胖症的病理生理学有所贡献。已知益生菌(存在于食品或食品补充剂中的有益细菌)可改变肠道小型生物群(FullerR&GibsonGR.Modificationoftheintestinalmicroflorausingprobioticsandprebiotics.ScandJ.Gastroenterol.1997)。
WO2006019222公开了具有减少机体脂肪之活性的鼠李糖乳杆菌(Lactobacillusrhamnosus)菌株PL60KCCM-10654P,其过度产生t10c12-十八碳二烯酸。
然而,对于对t10c12-十八碳二烯酸反应敏感的患者来说,t10c12-十八碳二烯酸的过度产生可能是有问题的。
US7001756和CN1670183提供了分离的微生物菌株——鼠李糖乳杆菌GM-020,发现其有效治疗肥胖症。
基于这些现有技术,本发明的目的是鉴定不依赖于过表达t10c12-十八碳二烯酸和/或赋予可用于治疗肥胖症的诱人效果的替代益生细菌,其克服了现有技术菌株的缺点。
该目的通过权利要求1的用途来实现。
本发明人(意外地)发现,鼠李糖乳杆菌CGMCC1.3724菌株或鼠李糖乳杆菌NCC4007菌株实现了这一目的。
因此,本发明的一个实施方案是鼠李糖乳杆菌CGMCC1.3724或鼠李糖乳杆菌NCC4007用于制备治疗动物肥胖症的组合物的用途。
本发明的另一实施方案是鼠李糖乳杆菌CGMCC1.3724或鼠李糖乳杆菌NCC4007用于制备促进体重减轻的组合物的用途。
本发明的又一实施方案是鼠李糖乳杆菌CGMCC1.3724或鼠李糖乳杆菌NCC4007用于制备支持体重控制的组合物的用途。
本发明框架中描述的组合物对于长期应用而言特别有益。例如,本发明人在动物模型中显示,用本发明所示组合物处理的小鼠的体重将显著轻于对照小鼠。这一效果在施用组合物的时间更长时甚至更为显著。该实验持续了约两个月,观察到的效果随时间的增加而增加。
因此,在本发明的一个优选实施方案中,所述组合物被施用至少2周、至少3周、至少4周、至少5周、至少6周、至少7周和/或至少8周。
在本说明书中,以下术语具有以下含义:
“动物”指包括人在内的动物。
术语“鼠李糖乳杆菌CGMCC1.3724”旨在包括该细菌、含有该细菌的细胞培养基或者培养有鼠李糖乳杆菌CGMCC1.3724的细胞生长培养基。
术语“鼠李糖乳杆菌NCC4007”旨在包括该细菌、含有该细菌的细胞培养基或者培养有鼠李糖乳杆菌NCC4007的细胞生长培养基。
“体重指数”或“BMI”是指体重(Kg)除以身高(米)的平方所得的比值。
“超重”定义为BMI为25至30的成人。
“肥胖症”这样的病症:动物(特别是人和其他哺乳动物)脂肪组织中的天然能量储备提高至与某些健康状况或死亡率提高相关的点。“肥胖”定义为BMI大于30的成人。
“益生菌”指对宿主的健康或康乐具有有益效果的微生物细胞制备物或微生物细胞组分。(SalminenS,OuwehandA.BennoY.等"Probiotics:howshouldtheybedefined"TrendsFoodSci.Technol.1999:10107-10)。
“益生素(prebiotic)”指旨在促进肠道中益生菌生长的食物。
“食品级细菌”指用于食品中并且一般认为在用于食品时是安全的细菌。
“体重减轻”在本发明上下文中指总体重的减轻。例如,体重减轻可以指为了增强体质、健康和/或外貌而减轻总体重。
“体重控制”或“体重维持”指保持总体重。例如,体重控制可涉及将BMI保持在被认为正常的18.5-25区间。
本发明的组合物还可含有保护性水状胶体(如胶质、蛋白质、改性淀粉)、粘合剂、成膜剂、包封剂/材料、壁/壳材料、基质化合物、包衣、乳化剂、表面活性剂、增溶剂(油、脂肪、蜡、卵磷脂等)、吸附剂、载体、填充剂、复合化合物(co-compound)、分散剂、湿润剂、操作助剂(溶剂)、流动剂、口味掩蔽剂、增重剂、成胶剂、凝胶形成剂、抗氧化剂和抗微生物剂。所述化合物还可含有常规药物添加剂和佐剂、赋形剂和稀释剂,包括但不仅限于水、任何来源的明胶、植物胶、木素磺酸盐、滑石、糖、淀粉、阿拉伯树胶、植物油、聚(亚烷基)二醇、调味剂、防腐剂、稳定剂、乳化剂、缓冲剂、润滑剂、着色剂、湿润剂、填充剂等。在所有情况下,这些其他组分都将根据其对于预期受者的适用性来进行选择。
所述组合物可以是全营养配方。
本发明的组合物可包含蛋白质来源。
任何合适的膳食蛋白均可使用,例如动物蛋白(如乳蛋白、肉蛋白和卵蛋白);植物蛋白(如大豆蛋白、小麦蛋白、稻蛋白和豌豆蛋白);游离氨基酸的混合物或其组合。特别优选的是乳蛋白(例如酪蛋白和乳清)以及大豆蛋白。
蛋白质可以是完整的或水解的,或者是完整蛋白与水解蛋白的混合物。可以期望提供部分水解的蛋白(水解程度为2至20%),例如用于认为有发生牛奶过敏之风险的动物。如果需要水解蛋白,则可根据需要和本领域所知进行水解过程。例如,可通过在一个或多个步骤中酶促水解乳清级分来制备乳清蛋白水解产物。发现如果用作起始材料的乳清级分基本不含乳糖,则蛋白质在水解过程中发生的酪氨酸阻断少得多。这使得酪氨酸阻断的程度可从总酪氨酸重量的约15%减少至酪氨酸重量的约10%,例如酪氨酸重量的约7%,这极大地提高了该蛋白质来源的营养品质。
所述组合物还可含有糖类来源和脂肪来源。
如果所述混合物包含脂肪来源,则该脂肪来源优选提供该组合物中5%至40%的能量,例如20%至30%的能量。合适的脂肪谱可使用芸苔油、玉米油和高油酸葵花籽油的掺合物(blend)来获得。
可向所述组合物中加入糖类来源。
糖类来源优选提供该组合物中40%至80%的能量。任何合适的糖类均可使用,例如蔗糖、乳糖、葡萄糖、果糖、玉米糖浆固体、麦芽糖糊精及其混合物。需要时还可加入可食纤维。可食纤维通过小肠而不被酶消化,发挥天然填充剂和轻泻药的功能。可食纤维可以是可溶的或不溶的,一般优选这两种类型的掺合物。合适的可食纤维来源包括大豆、豌豆、燕麦、果胶、瓜尔胶、阿拉伯树胶、果寡糖、半乳寡糖(galacto-oligosacchahdes)、唾液酸-乳糖以及来自动物乳的寡糖。优选的纤维掺合物是具有较短果糖寡糖链的菊粉混合物。优选地,如果存在纤维,则纤维含量为2至40g/l所摄入组合物,更优选4至10g/l所摄入组合物。
根据政府部门如USRDA的推荐,所述组合物还可含有矿物质和微量营养物,如微量元素和维生素。例如,所述组合物在日剂量中可含有以下范围内的以下微量营养物:300至500mg钙、50至100mg镁、150至250mg磷、5至20mg铁、1至7mg锌、0.1至0.3mg铜、50至200ug碘、5至15ug硒、1000至3000ugβ胡萝卜素、10至80mg维生素C、1至2mg维生素B1、0.5至1.5mg维生素B6、0.5至2mg维生素B2、5至18mg烟酸、0.5至2.0ug维生素B12、100至800ug叶酸、30至70ug生物素、1至5ug维生素D、3至10ug维生素E。
需要时,可向组合物中掺入一种或多种食品级的乳化剂,例如甘油单酯和甘油二酯的二乙酰基酒石酸酯、卵磷脂以及甘油单酯和甘油二酯。类似地,也可包含合适的盐和稳定剂。
所述组合物优选可经口或经肠施用,例如用奶或水重建的粉末形式。
优选地,所述组合物以粉末形式提供,例如可稳定储存的粉末。储存稳定性可通过如提供水活度小于0.2、例如0.19-0.05、优选小于0.15的组合物来提供。
水活度或aw是系统中水能量状态的度量。其定义为水蒸汽压除以同温度下纯水的蒸汽压,因此,纯蒸馏水的水活度恰好为1。
上述组合物可以任何合适的方式制备。例如,可通过将蛋白质、糖类来源和脂肪来源以适当的比例掺合在一起来制备。如果使用乳化剂,则可在此时加入。维生素和矿物质可在此时加入,但通常随后加入,以避免热降解。任何亲脂性维生素、乳化剂等均可在掺合之前溶于脂肪来源中。接着可以液体混合物的形式混入水,优选经过反渗透的水。水温通常为约50℃至约80℃,以帮助成分分散。可使用市售液化剂形成液体混合物。然后将液体混合物拌匀,例如在两个阶段中进行。
接着将所述液体混合物进行热处理,以减少细菌负荷(load),这通过例如将液体混合物迅速加热至约80℃至约150℃约5秒至约5分钟来实现。这可通过蒸汽注入、高压灭菌或热交换器(如平板热交换器)来实现。
接着,可将液体混合物冷却至约60℃至约85℃,例如通过瞬时冷却来进行。接着再将液体混合物拌匀,例如在两个阶段中进行,第一个阶段为约10MPa至约30MPa,第二个阶段为2MPa至约10MPa。接着可使拌匀后的混合物进一步冷却,以加入任何对热敏感的组分,如微生物和矿物质。通常在这时调节拌匀后混合物的pH和固体含量。
将拌匀后的混合物转移至合适的干燥设备(如喷雾干燥器或冷冻干燥器)中并转化成粉末。粉末应具有低于5wt%的含水量。
鼠李糖乳杆菌CGMCC1.3724或鼠李糖乳杆菌NCC4007可根据任何合适的方法培养,并通过如冷冻干燥或喷雾干燥制备,以添加到组合物中。用于鼠李糖乳杆菌CGMCC1.3724或鼠李糖乳杆菌NCC4007的合适的培养方法为本领域技术人员已知。或者,可从专门的供应商(ChristianHansen和Danisco)购买已制备成适于加入食品(如营养及婴儿配方)之形式的细菌制备物。益生菌可以合适的量加入配方中,优选102至1012cfu/g粉末,更优选107至1012cfu/g粉末。
在本发明的一个实施方案中,待使用根据本发明制备之组合物进行处理的动物为至少两岁。该年龄限制特别适用于人。如果待使用根据本发明制备之组合物进行处理的动物为狗或猫,则狗或猫应为至少4个月大。
在本发明的一个实施方案中,所述组合物为药物。作为药物,鼠李糖乳杆菌CGMCC1.3724和/或鼠李糖乳杆菌NCC4007的剂量可根据医生的推荐仔细进行调整。
根据本发明制备的组合物也可以是食物产品。作为食物产品,鼠李糖乳杆菌CGMCC1.3724或鼠李糖乳杆菌NCC4007的有益效果将可用于任何人。对肥胖症的治疗可以在早得多的阶段开始。此外,食物产品中的鼠李糖乳杆菌CGMCC1.3724或鼠李糖乳杆菌NCC4007将使人更喜欢进食。可适用于本发明的食物产品为用于人的酸乳、乳、调味乳、冰淇淋、即食点心、用例如乳或水重建的粉末、巧克力乳饮料、麦芽饮料、即食菜品、方便菜品或饮料,或者代表用于宠物或家畜的完全或部分膳食的食品组合物。
因此,在一个实施方案中,本发明的组合物是用于人、宠物或家畜的食物产品。特别地,所述组合物旨在用于选自狗、猫、猪、牛、马、山羊、绵羊、家禽或人的动物,在优选的实施方案中,该组合物是旨在用于成年物种(特别是成人)的食物产品。
本发明的组合物还可含有至少一种其他类型的其他食品级细菌或酵母。所述食品级细菌可以是益生菌,优选选自乳酸菌、双歧杆菌(bifidobacteria)、丙酸杆菌(propionibacteria)或其混合物。所述益生菌可以是具有确定的益生特征的任何乳酸菌或双歧杆菌。例如,它们可以还能够促进双歧肠道小型生物群的产生。合适的益生双歧杆菌菌株包括由丹麦ChristianHansen公司以商标Bb12销售的乳酸双歧杆菌(Bifidobacteriumlactis)CNCM1-3446,由日本MorinagaMilkIndustryCo.Ltd.以商标BB536销售的长双歧杆菌(Bifidobacteriumlongum)ATCCBAA-999,由Danisco以商标Bb-03销售的短双歧杆菌(Bifidobacteriumbreve)菌株,由Morinaga以商标M-16V销售的短双歧杆菌菌株以及由InstitutRosell(Lallemand)以商标R0070销售的短双歧杆菌菌株等。可以使用合适的益生乳酸菌与双歧杆菌的混合物。
可以使用以下作为食品级酵母,例如酿酒酵母(Saccharomycescerevisiae)和/或布拉氏酵母(Saccharomycesboulardii)。
在本发明的一个优选实施方案中,所述组合物还含有至少一种益生素。益生素能促进某些食品级细菌(特别是益生菌)在肠道中的生长,因而增强鼠李糖乳杆菌CGMCC1.3724和/或鼠李糖乳杆菌NCC4007的效果。此外,一些益生素对例如消化有积极影响。
优选地,所述益生素选自寡糖,并任选地含有果糖、半乳糖、甘露糖、大豆和/或菊粉、可食纤维或其混合物。
本发明的一个优点是鼠李糖乳杆菌CGMCC1.3724和/或鼠李糖乳杆菌NCC4007作为活细菌和失活细菌物种都是有效的。因此,即便是不允许活菌存在的条件也不会消除鼠李糖乳杆菌CGMCC1.3724或鼠李糖乳杆菌NCC4007的有效性。
然而,优选地,组合物中的至少一部分鼠李糖乳杆菌CGMCC1.3724和/或鼠李糖乳杆菌NCC4007是活的,并优选活着到达肠中。这样,它们可在肠中建群,并通过增殖来提高其效力。
然而,对于特定的无菌食品或药物来说,可能优选组合物中的鼠李糖乳杆菌CGMCC1.3724和/或鼠李糖乳杆菌NCC4007不是活的。因此,在本发明的一个实施方案中,组合物中的至少一部分鼠李糖乳杆菌CGMCC1.3724和/或鼠李糖乳杆菌NCC4007不是活的。
鼠李糖乳杆菌CGMCC1.3724或鼠李糖乳杆菌NCC4007在任何浓度下都将是有效的。如果鼠李糖乳杆菌CGMCC1.3724或鼠李糖乳杆菌NCC4007活着到达肠中,一个细菌就足以通过建群和增殖来获得强力的效果。
然而,对药物而言,一般优选日剂量的药物中含有102至1012cfu的鼠李糖乳杆菌CGMCC1.3724或鼠李糖乳杆菌NCC4007。特别合适的鼠李糖乳杆菌CGMCC1.3724或鼠李糖乳杆菌NCC4007日剂量为105至1011集落形成单位(cfu),更优选107至1010cfu。
对于失活鼠李糖乳杆菌CGMCC1.3724或鼠李糖乳杆菌NCC4007的请客,一般优选日剂量的药物中含有102至1012个鼠李糖乳杆菌CGMCC1.3724或鼠李糖乳杆菌NCC4007细胞。特别合适的鼠李糖乳杆菌CGMCC1.3724或鼠李糖乳杆菌NCC4007日剂量是105至1011个细胞,更优选107至1010个细胞。
对于食物组合物,一般优选每克食物组合物干重中含有103至1012cfu的鼠李糖乳杆菌CGMCC1.3724或鼠李糖乳杆菌NCC4007。特别合适的鼠李糖乳杆菌CGMCC1.3724或鼠李糖乳杆菌NCC4007的量是每克食物组合物干重105至1011cfu,更优选每克食物组合物干重107至1010cfu。
对于失活鼠李糖乳杆菌CGMCC1.3724或鼠李糖乳杆菌NCC4007的情况,一般优选每克食物组合物干重中含有103至1012个鼠李糖乳杆菌CGMCC1.3724或鼠李糖乳杆菌NCC4007细胞。特别合适的鼠李糖乳杆菌CGMCC1.3724或鼠李糖乳杆菌NCC4007的量是每克食物组合物干重105至1011个细胞,更优选每克食物组合物干重107至1010个细胞。
组合物中鼠李糖乳杆菌CGMCC1.3724或鼠李糖乳杆菌NCC4007的日剂量将取决于待处理的具体人或动物。要考虑的重要因素包括年龄、体重、性别和健康状况。
例如,组合物中鼠李糖乳杆菌CGMCC1.3724或鼠李糖乳杆菌NCC4007的典型日剂量将为每天104-1012cfu和/或个细胞,优选每天106-1010cfu和/或个细胞,优选每天107-109cfu和/或个细胞。
本发明包含鼠李糖乳杆菌CGMCC1.3724或鼠李糖乳杆菌NCC4007的组合物的另一用途是帮助减轻体重和/或体重维持。
由于建立和维持正常体重(特别是可接受的身体脂肪重量百分比)是治疗或预防代谢疾病的关键步骤,因此本发明包含鼠李糖乳杆菌CGMCC1.3724或鼠李糖乳杆菌NCC4007的组合物的另一用途是治疗或预防代谢疾病。
具体地,本发明包含鼠李糖乳杆菌CGMCC1.3724或鼠李糖乳杆菌NCC4007的组合物可用于治疗或预防肥胖症、高血压和/或心血管疾病,因此,可在许多国家(特别是发达国家)的现代人群的康乐作出显著贡献。
本领域技术人员很清楚,本说明书中描述的任何特征都可以自由组合,而不偏离本发明所公开的范围。
以下实施例和附图中显示本发明的其他特征和优点:
图1显示接受CGMCC1.3724菌株的大鼠与对照组相比体重增加显著减少(11%)。随时间测量体重的增加。
图2显示图1小鼠的脂肪量增加。
图3显示图1小鼠的葡萄糖耐量测试结果。
图4显示肥胖及瘦小鼠随时间的体重增加。
图5显示肥胖及瘦小鼠的脂肪量增加。
实施例1
将以饲料饲喂的6至8周龄雄性肥胖Zucker大鼠(n=12)每天用109-1010cfu的鼠李糖乳杆菌CGMCC1.3724进行处理,共60天。施用在含有NaCl9/1000的饮用溶液中的,以(deManRogosaSharpe)MRS培养基稀释的鼠李糖乳杆菌NCC-4007生物量,对照组接受盐溶液,其中含有益生菌制备物中相应量的MRS培养基。在研究过程中跟踪体重,并在处理前和处理结束时测量以NMR评估的体重组成。在干预结束时通过进行腹膜内葡萄糖耐量测试(IPGTT)来评估葡萄糖耐量,该测试由通过向禁食16小鼠的大鼠腹膜内注射20%D-葡萄糖溶液并在注射后0、15、30、60和120分钟从尾静脉中采集血样组成。接着测量不同时间点的葡萄糖。
图1显示接受CGMCC1.3724菌株的大鼠的体重增加与对照组相比显著减少(11%)。
对体重增加的影响与脂肪量增加较低有关(图2)。
CGMCC1.3724菌株的施用提高了这些肥胖大鼠的葡萄糖耐量,如更小的曲线下面积所示(图3)。
用鼠李糖乳杆菌NCC4007进行的相同实验获得了相似的结果。
实施例2
将7至8周龄的雄性肥胖ob/ob小鼠或其瘦杂合同窝ob/+小鼠用饲料喂饲,并每天用109-1010cfu的鼠李糖乳杆菌NCC-4007进行处理,共60天。施用在含有NaCl9/1000的饮用溶液中的,以MRS培养基稀释的鼠李糖乳杆菌1.3724生物量,对照组接受盐溶液,其中含有益生菌制备物中相应量的MRS培养基。在研究过程中跟踪体重,并在处理前和处理结束时测量以NMR评估的体重组成。
在饮用溶液中接受CGMCC1.3724的肥胖(ob/ob)及瘦(ob/+)小鼠的体重增加与其相应对照组相比分别减少37%和22%(图4)。
与对照动物相比,CGMCC1.3724菌株的施用诱导了肥胖小鼠中脂肪量增加的减少,并在瘦小鼠中取得了程度较低的相同效果(图5)。
用鼠李糖乳杆菌NCC4007进行的相同实验给出了相似的结果。
Claims (17)
1.鼠李糖乳杆菌CGMCC1.3724用于制备在人和/或动物中支持体重减轻和/或维持体重的组合物的用途。
2.权利要求1的用途,其特征在于所述人为至少2岁。
3.权利要求2的用途,其特征在于所述人为成人。
4.权利要求1的用途,其特征在于所述组合物为药物或食物产品。
5.权利要求2的用途,其特征在于所述组合物为药物或食物产品。
6.权利要求3的用途,其特征在于所述组合物为药物或食物产品。
7.权利要求1-6中任一项的用途,其特征在于所述组合物含有至少一种其他类型的其他食品级细菌和/或酵母。
8.权利要求7的用途,其特征在于所述食品级细菌选自乳酸菌、双歧杆菌、丙酸杆菌或其混合物。
9.权利要求1-6中任一项的用途,其特征在于所述组合物还含有至少一种益生素。
10.权利要求9的用途,其特征在于所述益生素选自寡糖,并任选的含有果糖、半乳糖、甘露糖、大豆和/或菊粉、可食纤维或其混合物。
11.权利要求1的用途,其特征在于该组合物中至少一部分的鼠李糖乳杆菌CGMCC1.3724是活的。
12.权利要求1的用途,其特征在于该组合物中至少一部分的鼠李糖乳杆菌CGMCC1.3724不是活的。
13.权利要求4-6中任一项的用途,其特征在于所述药物的每日剂量中含有102至1012cfu的鼠李糖乳杆菌CGMCC1.3724,或者所述药物的每日剂量中含有102至1012个鼠李糖乳杆菌CGMCC1.3724细胞。
14.权利要求4-6中任一项的用途,其特征在于所述食物产品的每克食物产品干重中含有103至1012cfu的鼠李糖乳杆菌CGMCC1.3724,或者所述食物产品的每克食物产品干重中含有103至1012个鼠李糖乳杆菌CGMCC1.3724细胞。
15.权利要求1的组合物在制备用于治疗肥胖症的药物中的用途。
16.权利要求1的组合物在制备用于治疗代谢障碍的药物中的用途。
17.权利要求16的用途,其特征在于所述代谢障碍选自糖尿病、高血压和心血管疾病。
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CN102215848A (zh) | 2011-10-12 |
BRPI0814934A2 (pt) | 2015-02-03 |
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EP2022502A1 (en) | 2009-02-11 |
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WO2009021824A1 (en) | 2009-02-19 |
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US9101651B2 (en) | 2015-08-11 |
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US20130224168A1 (en) | 2013-08-29 |
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AU2008288581B2 (en) | 2013-07-25 |
CL2008002350A1 (es) | 2009-09-25 |
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