CN104039950A - 能够代谢草酸盐的细菌菌株 - Google Patents

能够代谢草酸盐的细菌菌株 Download PDF

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CN104039950A
CN104039950A CN201280034197.XA CN201280034197A CN104039950A CN 104039950 A CN104039950 A CN 104039950A CN 201280034197 A CN201280034197 A CN 201280034197A CN 104039950 A CN104039950 A CN 104039950A
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G·莫格纳
G·P·斯特罗兹
卢卡·蒙加
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Abstract

本发明涉及筛选能够代谢草酸的人肠道源乳酸菌和双歧杆菌的菌株。此外，本发明涉及包含所述细菌菌株的食品组合物或增补剂产品或药物组合物。

Description

能够代谢草酸盐的细菌菌株
技术领域
本发明涉及能够代谢草酸和/或其盐(草酸盐)的人肠道源乳酸菌菌株和双歧杆菌的筛选。此外，本发明涉及包含所述细菌菌株的食品组合物或增补剂产品或医疗设备或药物组合物。
背景技术
草酸盐(草酸的盐)为植物界中的遍在化合物，广泛存在于所有人类饮食中。日常摄取在70至920mg(平均495mg～5.6mM)范围内，但在素食主义者的饮食中这些值容易过量。
草酸(二羧酸)是最高度氧化的有机化合物之一，因此充当阳离子、特别是离子Ca2+的强有力的螯合剂。由于该性质，草酸的盐(草酸盐)在分解代谢过程和能量产生中几乎没有用处。此外，草酸对大多数生物体，特别是对哺乳动物是有毒的。
由于该原因，草酸和草酸盐在人体的积累可引发和恶化一系列病理状态，其中我们应提及高草酸盐尿、尿石病、肾衰竭、心肌病及其它心脏病。特别地，草酸与钙结合形成相应的草酸钙，草酸钙是一种造成超过70%的确诊的肾结石的不溶性盐。此外，草酸是影响肠粘膜的强有力的致炎因子。因此，这种酸在内腔中的过量存在可通过改变上皮组织的渗透性，因而刺激草酸盐的吸收增加，来危害上皮组织的天然屏障功能。特别地，结肠是草酸盐吸收的主要部位，正常病理状态下摄取3-5%。减少肠腔中的草酸盐可因而有助于减少吸收。然后这将导致草酸盐在血浆和尿液中的浓度下降，因而减少其危害。
此外，血液中高水平的草酸盐可导致憩室病或憩室炎。憩室病，也称为"憩室性疾病(diverticular disease)"，为以结肠中的憩室为特征的医学状况；结肠的粘膜和粘膜下层的外翻贯穿结肠壁中肌层的相对弱的区域。憩室毫无疑 问地在乙状结肠中更为常见，乙状结肠是肠的一部分，特征在于较大的压力，其为促进憩室形成因素。憩室炎是消化道的病理，其特征在于一种以上的憩室的炎症。大多数情况的憩室炎位于结肠(特别是降结肠和乙状结肠)。
因此，重要的是能够以避免与高草酸盐值相关的并发症，诸如高草酸盐尿、尿石病、肾衰竭、心肌病和其它心脏病、肾结石、憩室病和憩室炎等的方式减少肠腔、血浆和尿液中草酸盐的量。
特别地，期望能够减少两类受试者的尿液中草酸盐的水平：
·不倾向于高草酸盐含量的饮食的高草酸盐尿受试者。
·倾向于高草酸盐含量的饮食的正常草酸盐尿受试者。
发明内容
申请人已在繁复的研究活动后提供了上述所需的答案，最终从高度广泛的菌株组中鉴定出筛选的属于乳酸杆菌(Lactobacillus)属和双歧杆菌(Bifidobacterium)属的细菌菌株；这些菌株显示显著的定量降解草酸盐的能力。所选菌株示出使用草酸盐作为能量来源，从所述草酸盐初始发现的环境中将其除去的能力。因此，所选菌株能够降解草酸盐。
如所附独立权利要求所公开的，本发明的主题涉及属于乳酸杆菌属和双歧杆菌属并具有降解草酸盐的能力的细菌菌株。
如所附独立权利要求所公开的，本发明的主题还涉及包含所述细菌菌株的食品组合物或增补剂产品或医疗设备或药物组合物。
本发明的优选实施方案将示于以下详细描述中。
附图说明
图1示出在SPE纯化之前(A)和之后(B)包含5mM草酸盐的培养基的色谱图之间的比较。
图2示出包含5mM草酸盐的培养基的色谱图(阳性对照)。
图3示出细菌菌株短双歧杆菌(B.breve)BR03DSM16604的色谱图。
图4示出细菌菌株副干酪乳杆菌副干酪物种(L.paracasei spp.paracasei)LPC09 DSM 24243的色谱图。
图5示出当使菌株副干酪乳杆菌副干酪物种LPC 09 DSM 24243生长在无糖MRS培养基(碳源)，分别向其中添加其它碳源(纤维)时以时间(T=0、3、6、8和10小时)为函数获得的酸化曲线(pH值)。
具体实施方式
申请人已研发了能够鉴定和定量属于乳酸杆菌属和双歧杆菌属的菌株培养物的草酸盐降解能力的方法。
申请人已发现以下细菌菌株具有证实的使用草酸盐作为能量来源的能力：
(i)副干酪乳杆菌副干酪物种LPC 09，由诺瓦腊(意大利)的益生菌股份公司于2010年11月23日保藏并具有保藏号DSM 24243。
(ii)格氏乳杆菌(L.gasseri)LGS 01，由诺瓦腊(意大利)的益生菌股份公司于2006年05月24日保藏并具有保藏号DSM 18299。
(iii)格氏乳杆菌LGS 02，由诺瓦腊(意大利)的益生菌股份公司于2006年05月24日保藏并具有保藏号DSM 18300。
(iv)嗜酸乳杆菌(L.acidophilus)LA 07，由诺瓦腊(意大利)的益生菌股份公司于2010年11月23日保藏并具有保藏号DSM 24303。
(v)嗜酸乳杆菌LA 02由诺瓦腊(意大利)的益生菌股份公司于2008年08月06日保藏并具有保藏号DSM 21717。
(vi)植物乳杆菌(L.plantarum)LP 01，由诺瓦腊(意大利)的莫菲因有限公司(Mofin Srl)通过保藏机构BCCM-LMG(比利时)于2001年10月16日保藏并具有保藏号LMG-P-21021。
(vii)罗伊氏乳杆菌(L.reuteri)LRE 03，由诺瓦腊(意大利)的益生菌股份公司于2010年08月05日保藏并具有保藏号DSM 23879。
(viii)罗伊氏乳杆菌LRE 02，由诺瓦腊(意大利)的益生菌股份公司于 2010年08月05日保藏并具有保藏号DSM 23878。
(ix)短双歧杆菌BR 03，由诺瓦腊(意大利)的益生菌股份公司于2004年07月20日保藏并具有保藏号DSM 16604。
(x)长双歧杆菌(B.longum)BL 03，由诺瓦腊(意大利)的益生菌股份公司于2004年07月20日保藏并具有保藏号DSM 16603。
(xi)鼠李糖乳杆菌(L.rhamnosus)GG，ATCC 53103，于ATCC公共保藏中心获得。
(xii)罗伊氏乳杆菌LRE 04，由诺瓦腊(意大利)的益生菌股份公司于2010年08月05日保藏并具有保藏号DSM 23880。
(xiii)鼠李糖乳杆菌LR 06，由诺瓦腊(意大利)的益生菌股份公司于2008年11月14日保藏并具有保藏号DSM 21981。
(xiv)乳双歧杆菌(B.lactis)BA 05，由诺瓦腊(意大利)的益生菌股份公司于2006年06月15日保藏并具有保藏号DSM 18352。
(xv)干酪乳杆菌鼠李糖物种(L.casei spp.rhamnosus)LR 04，由诺瓦腊(意大利)的益生菌股份公司于2004年07月20日保藏并具有保藏号DSM16605。
在优选的实施方案中，组合物包括选自上述(i)至(xv)表示的那些中的至少一种菌株，或任选地由选自上述(i)至(xv)表示的那些中的至少一种菌株组成；优选菌株选自上述(i)至(viii)表示的那些。
在本发明的背景下，“细菌菌株”是指活的和/或死亡的细胞和/或其部分、成分/衍生物和/或酶。
所选细菌菌株属于乳酸杆菌属并具有以超过50%的量降解和使用草酸盐作为能量来源的能力。有利地，所述能力超过60%。有利地，所述能力超过70%。
所选细菌菌株属于副干酪乳杆菌(Lactobacillus paracasei)物种。优选的菌株为副干酪乳杆菌副干酪物种LPC 09 DSM 24243。
所选细菌菌株属于格氏乳杆菌(Lactobacillus gasseri)物种。几种优选的菌 株选自包括格氏乳杆菌LGS 01 DSM 18299和格氏乳杆菌LGS 02 DSM18300的组或任选地由格氏乳杆菌LGS 01 DSM 18299和格氏乳杆菌LGS02 DSM 18300组成的组。
所选细菌菌株属于嗜酸乳杆菌(Lactobacillus acidophilus)物种。几种优选的菌株选自包括嗜酸乳杆菌LA02 DSM 21717和嗜酸乳杆菌LA 07 DSM24303的组或任选地由嗜酸乳杆菌LA02 DSM 21717和嗜酸乳杆菌LA 07DSM 24303组成的组。
根据本发明的组合物包括至少一种细菌菌株，其用于治疗高草酸盐尿、尿石病、肾衰竭、心脏病、肾结石、憩室病和憩室炎。
组合物可为食品组合物或增补剂产品或医疗设备或药物组合物。
用于治疗高草酸盐尿、尿石病、肾衰竭、心脏病、肾结石、憩室病和憩室炎的组合物包括选自上述(i)至(xv)表示的那些中的至少两种菌株或任选地由选自上述(i)至(xv)表示的那些中的至少一种菌株组成，优选菌株选自上述(i)至(viii)表示的那些。
用于治疗高草酸盐尿、尿石病、肾衰竭、心脏病、肾结石、憩室病和憩室炎的组合物包括选自上述(i)至(v)表示的那些中的至少两种菌株或任选地由选自上述(i)至(v)表示的那些中的至少两种菌株组成。
用于治疗高草酸盐尿、尿石病、肾衰竭、心脏病、肾结石、憩室病和憩室炎的组合物包括下述菌株或任选地由下述菌株组成：
(a)副干酪乳杆菌副干酪物种LPC 09-DSM 24243；或
(b)副干酪乳杆菌副干酪物种LPC 09-DSM 24243和格氏乳杆菌LGS01-DSM 18299；或
(c)副干酪乳杆菌副干酪物种LPC 09-DSM 24243和格氏乳杆菌LGS02-DSM 18300；或
(d)副干酪乳杆菌副干酪物种LPC 09-DSM 24243、格氏乳杆菌LGS01-DSM 18299和格氏乳杆菌LGS 02-DSM 18300；或
(e)副干酪乳杆菌副干酪物种LPC 09-DSM 24243、格氏乳杆菌LGS 01-DSM 18299、格氏乳杆菌LGS 02-DSM 18300和嗜酸乳杆菌LA 07-DSM 24303；或
(f)副干酪乳杆菌副干酪物种LPC 09-DSM 24243、格氏乳杆菌LGS 01-DSM 18299、格氏乳杆菌LGS 02-DSM 18300和嗜酸乳杆菌LA 02-DSM21717；或
(g)副干酪乳杆菌副干酪物种LPC 09-DSM 24243、格氏乳杆菌LGS01-DSM 18299、格氏乳杆菌LGS 02-DSM 18300、嗜酸乳杆菌LA 07-DSM24303和嗜酸乳杆菌LA 02-DSM 21717。
所有上述组合物，特别是上述组合物(a)至(g)，可进一步包括低聚果糖(FOS)和/或菊粉。相对于组合物的重量低聚果糖(FOS)和/或菊粉的含量在1至30重量%范围内，优选3至15重量%，甚至更优选5至10重量%。
本发明的主题涉及属于副干酪乳杆菌或格氏乳杆菌物种并能够降解超过60%的量的草酸和/或其盐的细菌菌株。所述菌株能够降解超过70%的量的草酸和/或其盐。所述属于副干酪乳杆菌物种的菌株为副干酪乳杆菌副干酪物种LPC 09 DSM 24243。所述属于格氏乳杆菌物种的菌株选自包括菌株格氏乳杆菌LGS 01 DSM 18299和菌株格氏乳杆菌LGS 02 DSM 18300的组。所述属于格氏乳杆菌物种的菌株选自由菌株格氏乳杆菌LGS 01 DSM 18299和菌株格氏乳杆菌LGS 02 DSM 18300组成的组。
本发明的主题涉及包含含有如上所述的至少一种细菌菌株的细菌组合物的食品组合物或增补剂产品或医疗设备或药物组合物，用于预防性或治愈性治疗高草酸盐尿、尿石病、肾衰竭、心脏病、肾结石、憩室病和憩室炎。所述细菌组合物包括菌株副干酪乳杆菌副干酪物种LPC 09 DSM 24243。所述细菌组合物包括菌株格氏乳杆菌LGS 01 DSM 18299和菌株格氏乳杆菌LGS02 DSM 18300。所述细菌组合物进一步包括菌株嗜酸乳杆菌LA02 DSM21717和菌株嗜酸乳杆菌LA 07 DSM 24303。所述细菌组合物由副干酪乳杆菌副干酪物种LPC 09 DSM 24243、嗜酸乳杆菌LA02 DSM 21717和/或嗜酸乳杆菌LA 07 DSM 24303组成。所述组合物进一步包含低聚果糖和/或菊粉。
实施方案部分
1.待分析的细菌菌株
研究约70株属于双歧杆菌属和乳酸杆菌属的菌株；它们来自诺瓦腊的益生菌股份公司的内部菌株保藏中心(strain collection)和诸如DSMZ-德国等国际保藏中心，或者见于文献中。所选菌株示于表1，其示出被试验细菌菌株降低的草酸盐的百分比。使用包含5mM草酸铵的培养基进行实验。
表1
列于表1的细菌菌株(i)至(v)、(vii)至(x)和(xii)至(xv)由诺瓦腊(意大利)的益生菌股份公司保藏。菌株(vi)由诺瓦腊(意大利)的莫菲因有限公司保藏。菌株(xi)可由ATCC保藏中心获得。所有菌株可根据由布达佩斯条约确立的条件下供公众使用和获得。
2.所采用的培养条件
要提供分析的菌株的制备包括在37℃下温育的MRS肉汤(+1%cys-HCl，厌氧生活，针对双岐(bifid))中的一系列三次连续的继代培养(subculture)，直至实现足够的生长。该培养策略能够完全活化菌株。随后将菌株以相同的接种物百分比(2%)接种在补充有5mM草酸铵(相当于草酸的平均日摄取的量) 的实验培养基中，该培养基专门设计以确保乳酸杆菌和双歧杆菌的最大生长。由此制备的培养物在37℃下温育24小时。
3.样品的SPE(固相提取)纯化
在温育期结束时，离心肉汤培养物并通过0.22μm滤器过滤上清液。样品的HPLC注射出现不清楚的色谱图。特别地，出现的草酸的色谱峰与存在于样品中的葡萄糖峰重叠。为了纠正前述问题，使用有机酸特异性SPE(固相提取)柱纯化样品。
通过SPE柱纯化的操作手册需要优化步骤以便获得最佳的终产量。特别地，关于柱调节步骤和分析物的最终洗脱，使用不同的试剂。如图1A-B中可以看出，该固相纯化使得可获得草酸的明显更干净的色谱峰。所用操作手册如下：
SPE柱的类型：Phenomenex Strata-XA
活化：1ml甲醇
调节：2ml20mM甲酸钠
上样：1ml样品
杂质洗涤：1ml25mM醋酸铵+1ml甲醇
洗脱：2×500μl1M HCl+2×500μl3M HCl
4.HPLC分析
通过HPLC分析由各单独菌株降解的草酸盐的量，计算T0(发酵前)时存在于培养基(5mM)中的草酸盐浓度与微生物生长后的残余浓度之差。单独菌株的结果表示成百分比，将T0时草酸盐的浓度假定为100。例如，菌株副干酪乳杆菌副干酪物种LPC09DSM24243的结果，等于约70%，表明后者能够使用等于约3.5mM草酸盐(70%5mM)的草酸盐量。所用HPLC操作手册如下：
柱的类型：Phenomenex Hydro-RP250×4.6mm
检测器的类型：UV-Vis，220nm下读取
洗脱流速：0.7ml/min
注入量：20μl
柱温：30℃
洗脱类型：等度(isocratic)
流动相：20mM磷酸钾pH2.0
显示对草酸的高降解活性的属于乳酸杆菌属的细菌菌株为上述(i)至(v)表示的细菌菌株。
A.菌株副干酪乳杆菌副干酪物种LPC09DSM24243的酸化曲线的确定。
菌株LPC09在实验前通过在MRS中继代培养并在37℃好氧生活下温育而再活化。在实验前重复三次再活化步骤，并过夜温育。在第三次再活化步骤结束时，使细胞成球，用无菌水洗涤，并在接种至补充有纤维的培养基内前重悬。所用培养基基于无糖MRS(碳源)，分别补充有：
-葡萄糖(Glu)(通过加热处理灭菌的溶液，121℃15’)，对照培养基。
-低聚果糖-FOS(通过过滤灭菌的溶液，0.20μl滤器)。
-GOS-Gl-含葡萄糖残基的低聚乳糖(通过过滤灭菌的溶液，0.20μl滤器)。
-GOS-Gal,-含半乳糖残基的低聚乳糖(通过过滤灭菌的溶液，0.20μl滤器)。
-XOS-低聚木糖(通过过滤灭菌的溶液，0.20μl滤器)。
-Larex(Lar)-落叶松纤维(larch fibre)(通过加热处理灭菌的溶液，121℃15’)。
-菊粉(Inu)(通过加热处理灭菌的溶液，121℃15’)。
所有培养基的碳源的终浓度为20g/l。
然后由此获得的培养基以4%的百分比接种菌株LPC09，并在37℃好氧生活下温育。
在时间0和3、6、8和10小时时，进行pH测量，以便构建图5的图中所示的酸化曲线。
表2示出当使菌株副干酪乳杆菌副干酪物种LPC09DSM24243生长在 如上所述的培养基中时以时间(T=0、3、6、8和10小时)为函数获得的酸化曲线(pH值)。
表2

Claims (11)

1.一种细菌菌株，其属于副干酪乳杆菌或格氏乳杆菌物种并能够降解超过60%的量的草酸和/或其盐。
2.根据权利要求1所述的菌株，其中所述菌株能够降解超过70%的量的草酸和/或其盐。
3.根据权利要求1或2所述的菌株，其中所述属于副干酪乳杆菌物种的菌株为副干酪乳杆菌副干酪物种LPC 09 DSM 24243。
4.根据权利要求1或2所述的菌株，其中所述属于格氏乳杆菌物种的菌株选自包括菌株格氏乳杆菌LGS 01 DSM 18299和菌株格氏乳杆菌LGS 02DSM 18300的组。
5.根据权利要求1或2所述的菌株，其中所述属于格氏乳杆菌物种的菌株选自由菌株格氏乳杆菌LGS 01 DSM 18299和菌株格氏乳杆菌LGS 02 DSM18300组成的组。
6.一种食品组合物或增补剂产品或医疗设备或药物组合物，其包括细菌组合物，用于预防性和/或治愈性治疗高草酸盐尿、尿石病、肾衰竭、心脏病、肾结石、憩室病和憩室炎，所述细菌组合物包含至少一种根据权利要求1-5任一项所述的细菌菌株。
7.根据权利要求6所述的组合物，其中所述细菌组合物包含菌株副干酪乳杆菌副干酪物种LPC 09 DSM 24243。
8.根据权利要求6或7所述的组合物，其中所述细菌组合物包含菌株格氏乳杆菌LGS 01 DSM 18299和菌株格氏乳杆菌LGS 02 DSM 18300。
9.根据权利要求6-8任一项所述的组合物，其中所述细菌组合物进一步包含菌株嗜酸乳杆菌LA02 DSM 21717和菌株嗜酸乳杆菌LA 07 DSM 24303。
10.根据权利要求6-9任一项所述的组合物，其中所述细菌组合物由副干酪乳杆菌副干酪物种LPC 09 DSM 24243、嗜酸乳杆菌LA02 DSM 21717和嗜酸乳杆菌LA 07 DSM 24303组成。
11.根据权利要求6-10任一项所述的组合物，其中所述组合物进一步包含低聚果糖和菊粉。
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