CN100455203C - 包含至少两种能定居在胃肠道中以及具有肠存活特性,肠结合特性,感染保护特性和纤维发酵特性的乳酸菌株的益生菌组合物 - Google Patents
包含至少两种能定居在胃肠道中以及具有肠存活特性,肠结合特性,感染保护特性和纤维发酵特性的乳酸菌株的益生菌组合物 Download PDFInfo
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- CN100455203C CN100455203C CNB2004800168064A CN200480016806A CN100455203C CN 100455203 C CN100455203 C CN 100455203C CN B2004800168064 A CNB2004800168064 A CN B2004800168064A CN 200480016806 A CN200480016806 A CN 200480016806A CN 100455203 C CN100455203 C CN 100455203C
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Abstract
在一种包括至少两种特异性乳酸菌株的益生菌组合物中，该菌株能定居在人和动物的胃肠道中，而且同时具有至少两种有益特性。该有益特性包括肠存活特性，肠结合特性，感染保护特性和纤维发酵特性。
Description
技术领域
本发明涉及一种新的益生菌组合物。更具体地说，本发明涉及一种益生菌组合物，其包含至少两种具有保持肠微生物生态系统，预防和治疗胃肠紊乱和能在胃肠道中定居中的至少两个显著特性的乳酸菌株。
背景技术
肠道菌群包括人体中细胞总数的大约95％。肠微生物区系，更具体地说它的组成，在成人胃肠道的生理学以及病理生理学过程中的重要性已经变得越来越明显。
与肠微生物区系改变相关的健康影响归因于对宿主的健康具有有益影响的活微生物(细菌或酵母)。业已发现乳酸菌的存在对保持肠微生物生态系统是重要的。这些微生物，由于长时间称为益生菌，通常定义为当它们被摄取时，对宿主的健康显示有益影响的活微生物。因此，益生菌可以定义为活的微生物单一培养物或混合培养物，其通过改善胃肠道中固有微生物区系的特性来影响宿主。目前，许多商品可有效地预防和治疗多种胃肠紊乱。
在EP 1 020 123 A1中，描述了与无牛奶基质中的冻干活乳酸菌混合物结合的饮料。冻干活乳酸菌混合物包括Brevibacterium breve，B.infants，B.longum，B.bifidum，嗜酸乳杆菌(lactobicillusacidophilus)，保加利亚乳杆菌(L.bulgaricus)，干酪乳杆菌(L.casei)，植物乳杆菌(L.plantarum)，嗜热链球菌(Streptococcusthermophilus)和粪链球菌(Streptococcus faecium)中的至少三种。这些饮料的目的是为消费者补充和平衡肠内菌群，以及为消费者提供其它的有益补充物，如维生素和抗氧化剂。
然而，这些文献在它们提出的益处中含有许多相矛盾的观察结果，而且相应的作用机理常常是不确定的。
已经将可能成功的益生菌菌株传统地掺入到发酵的奶制品中。在新微生物和改良的生物体的情况下，必须评估它们的安全问题与摄取风险之间的受益比率。食品中的乳酸菌具有悠久的安全使用历史。
在最近几年间，这些生物体已经包含于功能性食品和保健相关产品中。随着对益生菌影响人体健康的机制的理解增加，益生菌的定义已经逐渐地改变。虽然科学家和消费者普遍接受健康主张，但是许多声称的益生菌特性的基础分子机制仍然是有争论的。
已经成功地分离和鉴定了显示有益的益生菌性状的乳酸菌。这些特性包括证明的胆汁耐受性，耐酸性，附着到宿主上皮组织，和体外对可能致病的微生物或那些与促进炎症相关的微生物的拮抗作用。
市场上，存在几种包含乳酸菌的基于牛奶和水果的产品。常常较少描述其与胃肠道之间的特异性相互作用，而且很少限定其用量。
益生菌微生物必须能在工业条件下制备。而且，它们在贮存期间以及在它们将被掺入的食品中必须存活并保持它们的功能，而不产生消极作用。很多研究已经显示了销售的制品中益生菌的存活力较低。
对于在人或动物中施用乳酸菌作为益生菌的情况，它们应该适应成人胃肠道的特定条件，成人的胃肠道条件显著不同于新生儿的胃肠道条件。然而，迄今为止，存在很少成功移植给定菌株到健康成人个体的优势菌群中的证据。仅仅在出生时，或当例如在延长的抗生素治疗之后，施用益生菌生物体给患有极端失衡的肠微生物区系时，这种移植可能取得成功。
各种益生菌的不同生产者确实常常声称提供不同的益生菌能获得健康的益处。在大多数情况下，这是未经证实的，而且是不正确的。事实是只有很少数的乳酸菌具有所需的健康潜能。市场上出售的大多数益生菌在胃酸或小肠的胆汁酸内含物中不能存活，而且它们也不附着于结肠粘膜上，甚至也不能暂时定居在胃中。
发明内容
本发明的目的是，根据本领域的状况，通过提供益生菌组合物来避免上面所述的缺点，其中该益生菌组合物具有不仅在人，而且在那些具有相似的消化道并因此具有相应的肠微生物区系的动物的胃肠道中存活并定居的最佳能力。
为了达到该目的，根据本发明的方法已经获得了一种包括至少两种乳酸菌株的益生菌组合物，其特征在于所述的至少两种乳酸菌株能定居在人和动物的胃肠道中，而且共同具有至少两种有益的特性，其中的有益特性为肠存活特性，肠结合特性，感染保护特性和纤维发酵特性，所述的至少两种乳酸菌株选自植物乳杆菌F5，其BCCM保藏号为LMG P-20604，植物乳杆菌F26，其BCCM保藏号为LMG P-20605，植物乳杆菌2592，其BCCM保藏号为LMG P-20606，戊糖片球菌16∶1，其BCCM保藏号为LMG P-20608，和肠膜明串珠菌77∶1，其BCCM保藏号为LMGP-20607，植物乳杆菌50∶1，其BCCM保藏号为P-20609，和类干酪乳杆菌类干酪亚种F19，其BCCM保藏号为LMG P-17806。
本发明的组合物是基于两种或多种不同种的乳酸菌(LAB)菌株一起应具有利于人体摄入，同时模拟移植菌群的作用的特性。由于同时具有至少两种特性，这些特性限定了在胃肠中存活和/或定居的大家公认的标准，从而增强了可能的健康益处和对消化道菌群的影响。根据本发明，提供了新的活的有益生物体的益生菌LAB菌株，其具有特异性的有利功能特征。
用于本发明组合物的不同种的LAB菌株分离自死者的人大肠粘膜，该死者死于除胃肠道疾病以外的原因，而且在死亡之前的至少两个月期间没有通过抗生素进行治疗(F-菌株和2362)。其它的菌株分离自发酵的黑麦。
不同种的LAB菌株，根据API 50CH以及核糖分型(瑞典食品和生物技术研究所)分类为各种种类级别。
用于本发明益生菌组合物的特异性菌株是植物乳杆菌(Lactobacillus plantarum)F5，植物乳杆菌F26，植物乳杆菌2592，类干酪乳杆菌类干酪亚种(Lactobacillus paracasei subsp.paracasei)F19，戊糖片球菌(Pediococcus penosaceus)16∶1，植物乳杆菌50∶1和肠膜明串珠菌(Leuconostoc mesenteroides)77∶1。
按照，而且为满足，用于专利程序目的的国际公认微生物保藏布达佩斯条约的要求下列菌株于2001年6月19日保藏于比利时Gent的比利时微生物协调保藏中心(BCCM)：植物乳杆菌F5，保藏号为LMGP-20604；植物乳杆菌F26，保藏号为LMG P-20605；植物乳杆菌2592，保藏号为LMG P-20606；肠膜明串珠菌77∶1，保藏号为LMG P-20607；戊糖片球菌16∶1，保藏号为LMG P-20608。
植物乳杆菌50∶1于2001年6月21日保藏于比利时微生物协调保藏中心，保藏号为P-20609。
类干酪乳杆菌类干酪亚种F19更早保藏于比利时微生物协调保藏中心，保藏号为LMG P-17806。
具体实施方式
不同种的所有LAB-菌株于37℃在MRS琼脂上有氧生长24小时。在更进一步的实验中，确定了在不同温度的生长。所有菌株都可以在+4℃到40℃或45℃的温度繁殖。如通过脱脂乳琼脂测定法(24小时，有氧，37℃)所测定的，所有菌株都产生蛋白酶。没有菌株产生亚硝酸盐或硝酸盐。
如通过利用益生素这样的纤维作为唯一碳源，在YNB培养基上生长所测定的，不同种的所有LAB-菌株都可以利用益生素，如菊粉和支链淀粉，但不能利用β-葡聚糖。由于菌株能发酵这些纤维，它们将在结肠菌群中发挥有益的作用。增强的纤维降解也增加了粗纤维的消化率。
在本发明的组合物中，球菌和杆菌的混合物是最佳的，因为球菌和杆菌具有不同的世代时间，杆菌增殖比球菌快得多。优选通过组合物中包括至少一个乳酸杆菌菌株和至少一个乳酸球菌菌株来实现最佳生长。
为了促进乳酸菌株的生长，并使它在宿主的胃肠道上皮细胞中定居，以及发挥对传染病的抗性，本发明的组合物应具有肠存活特性，肠结合特性，感染保护特性和纤维发酵特性。
一种显著的肠存活特性是能够在胆汁存在时生长。所有上述的LAB-菌株都能在20％胆汁(人和猪)存在时生长，然后在选择压力已经除去和再应用之后，能保持它们的胆汁耐受性。
不同种的所有LAB菌株，当它们遭受pH 2.0-3.0的酸性时，也确实能存活。而且，随着添加0.3％的胃蛋白酶(37℃，24小时)，它们中的3个(植物乳杆菌F5，植物乳杆菌F26，和植物乳杆菌50∶1)仍然保持这种耐酸性，如下面的表1所述。
表1
菌株 pH 胃蛋白酶
植物乳杆菌F5 2.0 +
类干酪乳杆菌类干酪亚种F19 2.5
植物乳杆菌F26 2.0 +
植物乳杆菌2592 2.5
戊糖片球菌16∶1 2.5
植物乳杆菌50∶1 2.0 +
肠膜明串珠菌77∶1 3.0
因此，酸和胆汁耐受性菌株具有在胁迫条件下生长优于亲本菌株的优点。
Ba1bC小鼠经胃内喂饲本发明的乳酸菌。使用的LAB菌株为类干酪乳杆菌类干酪亚种F19，植物乳杆菌2592，戊糖片球菌16∶1和肠膜明串珠菌77∶1。通过培养获得给定的4种菌株的分泌物。可以在施用后6周期间检测到这些菌株的分泌物，这表明这些菌株在宿主的胃肠道定居。
当暴露于亚致死适应性酸条件(pH 5.0，60分钟)时，发现这些LAB菌株的酸应答能赋予显著水平的对随后的暴露于致死pH(pH 3.0)以及不同的环境胁迫(氧化胁迫，乙醇暴露和冷冻)的保护。
在适应pH 5.0期间产生的耐酸性应答影响了对抗环境胁迫的细胞存活。适应性培养产生了对乙醇(20％)，冷冻(-20℃)和氧化激发(10mM H2O2)的耐受性，但是没有产生对加热(60℃)和渗透压休克(3 M NaCl)的耐受性。在适应性阶段期间，氯霉素的存在部分地抑制了细胞抗性。发现这种适应性依赖于重新的蛋白质合成。
暴露于pH 5.0的酸胁迫1小时，如通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳所测定的，导致诱导了分子量(MW)从10.1到68.1kDa的9种新蛋白质，通过该方法也可确定这些蛋白质的过表达。所有其它的蛋白质被抑制，该蛋白质显示碱性或中性特性。
植物乳杆菌2592产生大量的分子量为19kDa的特征性蛋白质。
在耐酸性应答期间诱导的蛋白质毫无疑问是活性的，因为氨基酸类似物的掺入抑制了该应答。
也发现几种诱导的和过表达的蛋白质与前面描述的热激蛋白交叉反应。分子量为10，24和43kDa的蛋白质分别与cochaperons GroES，GrpE和DnaJ交叉反应。分子量为70和55kDa的较少过表达的蛋白质分别与GrpE和GroES交叉反应。
因此，发现在酸胁迫条件下的存活与适应性胁迫应答的表达相关。这种应答，其特征在于特异性蛋白质和生理学变化的瞬时诱导，增强了不同种的LAB菌株抵抗严酷环境条件的能力。上述的LAB菌株在酸性环境，如在胃中，诱导的连续的特异性蛋白质的蛋白质合成，然后将增强已存在蛋白质的稳定性。
不同种的LAB菌株更进一步的感染保护特性是它们的抗氧化特性，通过该特性，自由基的作用，其为炎症的产物，被抵消。
如通过分光光度测定法所测定的，在乳酸菌株的溶菌产物中，LAB菌株产生了抗氧化剂(总的抗氧化剂试剂盒，产品号NX 2332，购自Randox，San Diego，CA，USA.)。可以表明产生了抗氧化剂，其能有效对抗自由基并终止氧化链反应。所有的LAB菌株都产生了抗氧化剂，其总量为每升2.7到8.9毫克蛋白质。戊糖片球菌16∶1和植物乳杆菌F26菌株产生了最大量的抗氧化剂，紧接着是类干酪乳杆菌(类干酪)F19。
患糖尿病的小鼠每天以相同剂量给予这些LAB菌株，并进行12天。小鼠经胃内喂饲这些菌株中4个菌株(类干酪乳杆菌类干酪亚种F19，植物乳杆菌2592，戊糖片球菌16∶1，和肠膜明串珠菌77∶1)的1010个细胞每天两次，进行12天。小鼠的胆固醇水平没有降低。通过在研究结束时，取小鼠的血液培养物，证实了菌株的安全，其显示不生长。
同样，52个健康的人(年龄为14-87岁)每天消费不同种的4种LAB-菌株的1010个细菌，并进行3个月，没有产生副作用。
优选地，本发明组合物的感染保护特性为免疫增强作用，通过该作用获得阳性免疫应答。如通过斑点印迹(Wilson L，et al.，Gastroenterol 1999；117：106-114；and Splecker M，et al.，JImmunol 2000；15(March)：3316-22)所测定的，发现菌株具有把NF-κB转录到细胞核的不同能力。NF-κB的诱导导致细胞因子应答，其可以是促炎的或者抗炎的。
乳杆菌菌株，尤其是类干酪乳杆菌类干酪亚种F19，而不是球菌，在巨噬细胞细胞系U973中转录NF-κB，其导致白细胞介素IL-1β和IL-8的合成。诱导的细胞因子是促炎类型的。
本发明组合物的另一重要的方面是，本发明的益生菌LAB菌株应具有肠结合特性以便能发挥它们的功能特性。一种这种结合特性是它们显示高附着于肠道上。为了充当益生菌，乳酸菌株必须能在肠粘液层定居。
上述的菌株也可通过在ELISA方法(Tuomola EM，et al.，FEMSImmunology and Med Microbiol 26(1999)137-142)中筛选其对猪粘蛋白(II型，Sigma Chem Co，St Louis，CO，USA)的结合来获得。结果显示于下面的表2中。把该结果与商业菌株，鼠李糖乳杆菌(Lactobacillus rhamnosus)GG(Valio)的结果进行比较。
表2
菌株 粘蛋白结合 SAT
植物乳杆菌F5 0.557 1M
类干酪乳杆菌类干酪亚种F19 0.515 0.1M
植物乳杆菌F26 0.582 1M
植物乳杆菌2592 0.707 1M
戊糖片球菌16∶1 1.052 ＜0.1M
植物乳杆菌50∶1 0.883 2M
肠膜明串珠菌77∶1 1.265 ＜0.1M
鼠李糖乳杆菌GG 0.2 4M
除了显示粘蛋白结合，本发明组合物中的益生菌LAB菌株应能在人胃肠道中表现细胞表面疏水性。细胞表面疏水性是通过盐聚集测试(SAT)(Rozgoynyi F，et al.，FEMS Microbiol Lett 20(1985)131-138)测定的。在下面的表3中，显示了对玻连蛋白(Vn)，pH 3.5的玻连蛋白，胎球蛋白(Ft)，脱唾液酸胎球蛋白和pH 3.5的脱唾液酸胎球蛋白的附着的比较。
表3
菌株 Vn Vn pH 3.5 Ft* 脱唾液酸 脱唾液酸
-Ft -Ft pH 3.5
植物乳杆菌F5 ++ +++ - + +++
类干酪乳杆菌F19 +++ +++ +++ +++ +++
植物乳杆菌F26 + +++ + +++ +
植物乳杆菌2592 - + + +++ -
戊糖片球菌16∶1 +++ + - + -
植物乳杆菌50∶1 +++ + - ++ +
肠膜明串珠菌77∶1- ++ - + +
*表示pH7和pH 3.5时结果相同
发现粘附的微生物紧紧地与固定的粘液结合。5个菌株表现显著的细胞表面疏水性，另外3个菌株表现中等的细胞表面疏水性。没有观察到细胞表面疏水性与LAB菌株的附着能力之间的相关。
还在颗粒凝集测定法(PAA)(Paulsson M，et al.，J Clin Microbiol30(1992)2006-20012)中，测试了不同种的LAB菌株对牛颌下腺和猪II型粘蛋白(Sigma)的结合。通常，发现与牛粘液结合的表现更强烈。
由于多细胞生物体组织中许多细胞包埋在由分泌的蛋白质和多糖组成的胞外基质中，检测了本发明的不同种的LAB菌株与胃肠胞外基质蛋白之间的相互作用。以上述的固定形式(PAA测定法)研究了胞外基质蛋白和葡糖胺聚糖的结合。所有的LAB菌株都表现了能与I和III型胶原蛋白，粘连蛋白，纤维蛋白原和肝素，以及pH 3.5的I和III型胶原蛋白，粘连蛋白，纤维蛋白原和肝素结合。菌株类干酪乳杆菌(类干酪)F19，植物乳杆菌F26，和植物乳杆菌2592表现能与胎球蛋白，还有pH 3.5的胎球蛋白结合。
而且，除植物乳杆菌2592和肠膜明串珠菌77∶1之外，不同种的所有LAB菌株都表现能与玻连蛋白结合。不过，pH 3.5时，这些菌株表现了弱的结合。
本发明益生菌组合物中LAB菌株的另一个显著的肠存活特性是具有感染保护特性。
幽门螺杆菌(Heliobacter pylori)是急性和慢性胃炎的病原体，是全世界最流行的感染之一，可转为萎缩性胃炎，腺癌和MALT淋巴瘤。
根据乳酸的浓度和pH，本发明的乳酸杆菌体外确实抑制10种幽门螺杆菌菌株的生长。当pH调整到6.0时，抑制作用消失。更进一步地分析表明，L-乳酸，而不是D-乳酸或乙酸，在浓度为60到100mM时抑制生长。没有观察到幽门螺杆菌的CagA表型与对乳酸的耐受性之间的相关。发现抑制是菌株特异性的。
通过细菌素介导的相互作用，表明了进一步的感染保护特性。除了植物乳杆菌F5外，不同种的所有LAB菌株都分泌到无细胞的上清液中，其是一种具有抗微生物活性的产物。产生的该产物是热稳定的抗微生物化合物，其显示本质上是蛋白质性的，因此称为细菌素。该细菌素显示抗革兰氏阳性生物体的活性。一些具有抗革兰氏阴性生物体的活性(幽门螺杆菌菌株)，而一些具有抗酵母的活性(念珠菌属菌株)。该抗微生物作用不与产酸直接相关。
而且，幽门螺杆菌的形状是杆菌状的而不是球菌状的，这表明观察到的抑制与杀菌作用相关，而不与活的但是不可培养的球菌状的形式的诱导相关。发现杀菌作用是由于一种细胞内28kDa蛋白质，其在溶解后释放。这种细胞内蛋白质的蛋白水解处理导致抗菌活性的丧失。可利用十二烷基硫酸钠聚丙烯酰胺凝胶电泳，通过复性来消除这种丧失。
在一个建立的小鼠幽门螺杆菌胃炎模型中，施用本发明的不同种的LAB菌株，导致抑制的感染，同时伴随降低的炎症。这种抑制作用好象是菌株特异性的，而不是种特异性的。LAB菌株的体外活性与抗幽门螺杆菌活性非常相关。
本发明的益生菌组合物制品包括至少1010CFU/g的冷却，冷冻，或冻干的活细菌，作为食品或饲料中的益生菌添加剂。
Claims (11)
1.一种包括至少两种乳酸菌株的益生菌组合物，其特征在于所述的至少两种乳酸菌株能定居在人和动物的胃肠道中，而且共同具有至少两种有益的特性，其中的有益特性为肠存活特性，肠结合特性，感染保护特性和纤维发酵特性，所述的至少两种乳酸菌株选自植物乳杆菌F5，其BCCM保藏号为LMG P-20604，植物乳杆菌F26，其BCCM保藏号为LMG P-20605，植物乳杆菌2592，其BCCM保藏号为LMG P-20606，戊糖片球菌16∶1，其BCCM保藏号为LMG P-20608，和肠膜明串珠菌77∶1，其BCCM保藏号为LMG P-20607，植物乳杆菌50∶1，其BCCM保藏号为P-20609，和类干酪乳杆菌类干酪亚种F19，其BCCM保藏号为LMG P-17806。
2.权利要求1的益生菌组合物，其特征在于所述的乳酸菌株是至少1010CFU/g的活细菌。
3.权利要求1的益生菌组合物，其特征在于所述的肠存活特性是在胃蛋白酶存在时在低pH下存活的能力。
4.权利要求1的益生菌组合物，其特征在于所述的肠存活特性是产生应激蛋白的能力。
5.权利要求4的益生菌组合物，其特征在于所述的应激蛋白与热激蛋白交叉反应。
6.权利要求1的益生菌组合物，其特征在于所述的感染保护特性是产生细菌素的能力。
7.权利要求6的益生菌组合物，其特征在于所述的细菌素具有抗革兰氏阳性细菌的活性。
8.权利要求6的益生菌组合物，其特征在于所述的细菌素具有抗革兰氏阴性细菌的活性。
9.权利要求6的益生菌组合物，其特征在于所述的细菌素具有抗酵母的活性。
10.权利要求1的益生菌组合物，其特征在于所述的感染保护特性是诱导促炎细胞因子应答的能力。
11.乳酸菌株作为食品或饲料中的益生菌添加剂的用途，所述的乳酸菌株选自植物乳杆菌F5，其BCCM保藏号为LMG P-20604，植物乳杆菌F26，其BCCM保藏号为LMG P-20605，植物乳杆菌2592，其BCCM保藏号为LMG P-20606，戊糖片球菌16∶1，其BCCM保藏号为LMG P-20608，和肠膜明串珠菌77∶1，其BCCM保藏号为LMG P-20607，和植物乳杆菌50∶1，其BCCM保藏号为P-20609，单独或其组合。
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| SE529185C2 (sv) * | 2005-10-07 | 2007-05-22 | Arla Foods Amba | Användning av probiotiska bakterier för tillverkning av livsmedel eller läkemedel för förhindrande av övervikt |
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| DK1624762T3 (da) | 2007-06-04 |
| CA2526158A1 (en) | 2004-12-02 |
| DE602004005029D1 (de) | 2007-04-12 |
| EP1624762A1 (en) | 2006-02-15 |
| JP2007502858A (ja) | 2007-02-15 |
| AU2004241895A1 (en) | 2004-12-02 |
| NZ544116A (en) | 2008-03-28 |
| AU2004241895B2 (en) | 2008-05-01 |
| WO2004103083A1 (en) | 2004-12-02 |
| DE602004005029T2 (de) | 2007-11-08 |
| EP1624762B1 (en) | 2007-02-28 |
| ATE354964T1 (de) | 2006-03-15 |
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