CN1065568C - 嗜热链球菌细菌素 - Google Patents
嗜热链球菌细菌素 Download PDFInfo
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- CN1065568C CN1065568C CN94190654A CN94190654A CN1065568C CN 1065568 C CN1065568 C CN 1065568C CN 94190654 A CN94190654 A CN 94190654A CN 94190654 A CN94190654 A CN 94190654A CN 1065568 C CN1065568 C CN 1065568C
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- bacteriocin
- nucleotide
- strains
- seq
- bacteriocins
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Abstract
本发明提供了两种具有SEQ ID NO∶1或SEQ ID NO∶2氨基酸顺序的新的嗜热链球菌细菌素、这两种细菌素的信号肽、编码这些细菌素的核苷酸序列,特别是编码具有SEQ ID NO∶3顺序细菌素的操纵子、产生至少一种该细菌素的菌株,特别是CNCMⅠ-1351菌株、含有至少一种该细菌素上清提取液的生产方法,该细菌素在食品,特别是乳酪和酸奶生产中以及在化妆器生产中用作抗病原体的活性剂。
Description
本发明涉及两种嗜热链球菌(Streptococcus(S.)thermophilus)细菌素、能产生这些细菌素的嗜热链球菌菌株、该菌株产生这些细菌素的方法以及这些细菌素和/或该菌株在食品或化妆品生产中的应用。
技术背景
细菌素是一种具有抗菌活性的抗细菌物质或药剂,它含有与抗菌作用或抗生作用有关的蛋白部分。一种细菌素的抗菌谱或抑菌谱一般很窄,通常局限于与产生该细菌素的细菌很近的种属。
目前已知有四种嗜热链球菌细菌素。
尤其是第一种,其分子量为10至20KD,90℃时对热不稳定,对胃蛋白酶敏感(Smaczny et al.,Deutsche Molkerei-Zeitung,105:15,460-464 1984)。
在EP 443543中主要介绍了第二种的抑菌谱,它的特点是能够抑制葡萄球菌属和假单胞菌属细菌的生长.但不能抑制乳酸球菌和肠球菌属以及仙人山杆菌属细菌的生长。
Pulusani等人(J.of Food Science,44:2,575-578,1979)介绍了第三种,它能强烈抑制假单胞菌,对胃蛋白酶不敏感,并含有糖残基。
最后,Gilano等人(Microbiologie-Aliment-Nutrition8,21-30,1990)介绍的第四对胃蛋白酶不敏感,含有糖残基,不能通过有100KD孔的膜。
目前嗜热链球菌在食品方面十分重要,特别与乳产品生产有关,例如酸乳酪和某些乳酪。此外,同时具有抗杆菌属、梭状芽孢杆菌属和李斯特杆菌属活性的细菌素几乎没有。因此,该细菌素可能更为有用,尤其是在这类产品的生产中,换句话说,即需要由这一种属的代表株所产生的多种细菌素,以便具有更广的抗菌谱。
本发明的目的就是要满足这种需求。
发明概述
本发明的主要内容之一就是对两种新的嗜热链球菌细菌素的氨基酸顺序和促使细菌素分泌的信号肽进行特性分析。
编码这两种细菌素的核苷酸序列也是本发明的另一个主要内容。
能产生至少一种本发明所述细菌素的嗜热链球菌菌株也是本发明的另一个主要内容,特别是下述的嗜热链球菌CNCM I-1351菌株,它能产生本发明所述的两种细菌素。
含有至少一种本发明所述的细菌素提取液的生产方法也是本发明的另一个主要内容。
本发明的最后一个主要内容是本发明所述细菌素的应用及其核酸序列和信号序列的应用。
发明详细描述
从捷克斯洛伐克发酵乳产品中分离出嗜热链球菌CNCM I-1351菌株,并且出乎意料地观察到该菌株具有明显抑制多种细菌生长的特性。根据布达佩斯条约,该菌株于1993年8月5日保存在CollectionNationale de Cultures de Microorganismes,PASTEURINSTITUTE,25,Rue du Docteur Roux,F-75724 PARISCEDEX 15,France,编号为No.I-1351。
下面列出了该菌株的详细情况,特别是有关形态学、糖发酵及类似特性:
形态学:
-无鞭毛链状排列球菌,无芽孢形成。
-革兰氏阳性菌,过氧化氢酶阴性,兼性厌氧菌。
糖发酵:
可分解D-葡萄糖、乳糖、蔗糖、棉子糖产生乳酸。不能分解甘露糖、果糖、半乳糖而产生乳酸。
其它:
该菌株可产生至少两种细菌素、一种对细菌素免疫有关的蛋白和具有结构特性的表面多糖。
CNCM I-1351菌株培养物上清液因而具有较宽的抗菌谱。在对该上清液敏感的细菌中,可以包括下列细菌:例如嗜热链球菌、乳酸乳球菌、乳酸乳球菌二乙酰乳酸变种、乳酪乳酸球菌、粪肠球菌、屎肠球菌、发酵乳杆菌、瑞士乳杆菌、保加利亚乳杆菌、嗜酸乳杆菌、短乳杆菌、乳酪明串球菌、肠膜明串球菌、短双歧杆菌、长双歧杆菌、两歧双歧杆菌、婴儿双歧杆菌、丙酸杆菌属、无毒李斯特杆菌、单核细胞增多性李斯特杆菌、易变微球菌和肉毒梭状芽孢杆菌、干酪酪酸梭状芽孢杆菌、双酶梭状芽孢杆菌、产芽孢梭状芽孢杆菌、枯草杆菌、短小杆菌及蜡样杆菌的芽孢和营养细胞(Bactéries Lactiques,Vol1,1994,Lorica edition)。
然后可以从CNCM I-1351菌株中分离出两种称为细菌素的与抗菌活性有关的蛋白因子。
因此,本发明所述的第一种细菌素在本发明中命名为“嗜热素1(thermophilin 1)”它具有下述顺序清单中介绍的SEQ ID NO:1顺序。
此外,当嗜热素1的顺序与SEQ ID NO:1顺序不同时,例如至少一个氨基酸发生替换、缺失和/或***,可以设想这种细菌素对于细菌的一个属或细菌的一个种的抗菌谱可能要比嗜热素1更宽或更特异。EP 521240的确已经证实乳链球菌素Z具有比乳链球菌素A更为实用的抗菌谱,而乳链球菌素Z与乳链球菌素A的差别只不过是一个氨基酸发生了替换。
在其原顺序SEQ ID NO:1中至少一个氨基酸发生替换、缺失和/或***的所有细菌素均可以看作是本发明的细菌素,上述内容便是其原因所在。
本发明的第二种细菌素在本发明中命名为“嗜热素2(thermophilin 2)”,它具有下述顺序清单中介绍的SEQ ID NO:2顺序。也可以设想当这种细菌素的顺序与SEQ ID NO:2顺序不同时,例如至少一个氨基酸发生替换、缺失和/或***,它可能会具有抗菌活性。因此,在其原顺序SEQ ID NO:2中具有至少一种上述改变的所有细菌素均可以看作是本发明的细菌素。
此外,编码嗜热素1和嗜热素2的核苷酸序列也是本发明的另一个主要内容,因为通过转化作用,这些核苷酸序列均可使例如细菌、酵母或植物具有抑制某些细菌的能力。由于遗传密码的简并,因而这些核苷酸序列可以发生相对的变异,特别是可以包含在CNCM I-1351菌株的操纵子中,该菌株具有下述顺序清单中介绍的SEQ ID NO:3核酸序列。
特别是可以采用含有SEQ ID NO:3序列中第221至475核苷酸的核酸序列,用其信号肽它可编码嗜热素1。然而,只用SEQ IDNO:3序列中从第221至288核苷酸这一编码信号肽的序列与所需基团相连,这样更为有利,可以促使嗜热链球菌菌株分泌由该所需基团编码的蛋白质。同样,只采用SEQ ID NO:3序列中从第289至475核苷酸这一编码分泌型嗜热素1的序列,这样更为有利,以便在表达质粒中能够将这一序列与信号序列相连,例如,可在嗜热链球菌以外的其它微生物中表达嗜热素1。
同样,可以采用含有SEQ ID NO:3序列中第495至686核苷酸的核酸序列,用其信号肽它可编码嗜热素2。然而,只用SEQ IDNO:3序列中从第495至557核苷酸这一编码信号肽的序列则更为有利,这样可以促使嗜热链球菌菌株分泌与该信号肽相连的任何蛋白质。同样,只采用SEQ ID NO:3序列中从第558至686核苷酸这一编码分泌型嗜热素2的序列,这样更为有利,以便在表达质粒中能够将这一序列与信号序列相连,例如,可在嗜热链球菌以外的其它微生物中表达嗜热素2。
最后,因为已经观察到CNCM I-1351菌株以外的一些嗜热链球菌菌株具有与CNCM I-1351菌株相似的抑菌谱,而且对后者具有抗性(尤其是下述的Sfi 12和25菌株),十分可能的是这些菌株能够产生至少一种本发明的细菌素,同时作为免疫蛋白使其产生抗性。因此,所有能够产生至少一种上述的细菌素的菌株均包括在本发明中。
在含有至少一种本发明所述的细菌素提取液的生产方法中,能产生至少一种所述细菌素的嗜热链球菌菌株在有利于嗜热链球菌生长的条件下用培养基培养,直到培养基中每毫升含有107-109个该菌株细菌,将所得培养物离心,这时便制得了含有至少一种所述细菌素的上清液的提取液。
为生产这种提取液,能产生至少一种本发明所述细菌素的嗜热链球菌菌株可以在有利于嗜热链球菌生长的条件下用培养基培养,尤其是可以用例如MSK培养基(脱脂牛奶中补充酵母提取物)或HJ培养基(牛奶超滤浸出液中补充酵母提取物和soytone)来培养。最好采用链球菌选择性培养基来培养,例如,P.E.Terzaghi et al.,J.Appl.Microbiol.,29,807-813(1975)介绍的M17培养基,补充0.5-2%可被嗜热链球菌发酵的糖,尤其是蔗糖、乳糖或葡萄糖等。
这种培养基的制备方法如下:将95ml基本培养基与每100ml水中含有10g可酵解糖的溶液5ml混合,可酵解糖的溶液和基本培养基都已经分别在121℃灭菌15分钟,基本培养基是将下列成分溶解于950ml开水中制成的:
胰蛋白酶酪蛋白水解物 2.5g
胃蛋白酶肉水解物 2.5g
番木瓜蛋白酶大豆水解物 5.0g
酵母提取物 2.5g
肉提取物 5.0g
β-甘油磷酸盐 19g
硫酸镁 0.25g
抗坏血酸 O.5g
所述的菌株可在有利于嗜热链球菌生长的上述培养基中于37-48℃培养,例如2-8小时,直到培养基中每毫升含有大约107-109个该菌株细菌,每毫升中约108个细菌这一数值一方面相当于在600nm测定时培养基的光密度(OD600)约为3.6,另一方面在培养菌株产生的酸化作用下相当于在牛奶中细菌浓度达到了使牛奶凝固的程度。
要制备上述上清液的粗制提取液,可以使用任何一种适宜的沉淀方法,例如用三氯醋酸沉淀法、盐析法或溶剂沉淀法等。为制备这种粗制提取液,最好用H3PO4将上清液的pH调整至1.0-2.0,除去沉淀物,用三氯醋酸进行一次或多次连续沉淀,每次沉淀后都在具有三氟醋酸的水悬浮液中再悬浮。
细菌素和/或能产生本发明的这些细菌素的嗜热链球菌菌株的用途在于食品或化妆品生产方面。
所述嗜热链球菌菌株的培养物尤其在乳酪制备中可用作促酵物,特别是干酪型乳酪(避免由多粘杆菌产生的空洞,其芽孢发酵后仍存活)、硬干酪型乳酪(例如,Gruyére或Emmental,以防止干酪酪酸梭状芽孢杆菌的污染)、vacherin型乳酪(以防止单核细胞增多性李斯特杆菌的污染)和“séré”型乳酪(法语名称,表示软乳酪或者奶油乳酪);或者该培养物可用于酸奶的制备,特别是例如婴儿配方的酸乳酪或奶粉的制备中。
所述嗜热链球菌菌株尤其可以在牛奶中与保加利亚乳杆菌菌株联合培养,后者对嗜热素轻度敏感(例如下述的YL5菌株),以避免由保加利亚乳杆菌引起的酸乳酪后酸化作用。
所述的细菌素,尤其是以粗提取物或纯化提取物形式的细菌素,或者所述的菌株均可以用作抗病原菌的添加剂或活性剂,尤其是在诸如奶油冻这样的肉产品的制备中作为抑制梭状芽孢杆菌特别是肉毒梭状芽孢杆菌芽孢生长的活性剂,或者在乳剂或洗剂的制备中用作抗皮肤病原菌的活性剂,或者在口腔保健品制备中可选作抗口颊病原菌,尤其是例如抗sobrinus链球菌的活性剂。
下面更为详细地描述本发明的细菌素的特性,采用各种微生物学、生物化学和遗传学数据说明其性能。百分比均按重量计。抗菌单位--“琼脂孔试验”
在本发明的研究方案中是以人为确定单位来定义抗菌活性的。
一个人为确定单位(au)定义为在称为“琼脂孔试验”条件下,在专业人员已知的试验中,样品仍能表现出抗菌活性的最高稀释度的倒数。“琼脂孔试验”英文表达的字面含义是在琼脂上打孔进行试验。
在实施例1中列举的标准条件下制备的本发明嗜热链球菌培养物上清液的标准样品,其体积为70μl,通常表现出32au的活性。因此这一结果说明其活性为460au/ml。
在实施例1中列举的培养物上清液经澄清,再用三氯醋酸连续进行两次沉淀,每次沉淀后都在具有三氟醋酸的水悬浮液中再悬浮,这样制得的细菌素标准粗制提取液的活性通常约为1.4×105au/ml。
正是借助了所述的琼脂孔试验才确定了样品在给定的稀释度是否仍具有抗菌活性。
为进行这一试验,将35ml M17培养基倾入培替氏培养皿(Petri dish),向其中加入1 %蔗糖和1.5%琼脂。
5ml M17培养基中加入1%蔗糖和0.75%琼脂,在其中接种5μl前夜制备好的嗜热链球菌菌株培养物,它是典型的对本发明细菌素敏感的菌株(代表性指示菌),在这种情况下例如Sfi3菌株。
将这5ml倾入上述35ml上面,使其在层流状态下干燥15分钟,在培养基上打出直径为5mm的孔。
将试验样品注入孔内,每孔加样量为70μl。厌氧条件下于42℃培养6小时。在培养过程中,代表性指示菌株已生长,可见抑菌环。样品不再表现出抗菌活性的稀释度就是抑菌环不再能辨认的那个稀释度。用酶灭活
借助所述的琼脂孔试验,在接种了上述代表性指示菌株的琼脂上来确定本发明细菌素是否可被各种酶灭活。
除脂酶之外,对于所有采用的酶均取1μg/ml至10mg/ml加入所述的标准粗制提取液中,该提取液已用酶提供者推荐的缓冲液稀释33倍,以便获得300au 70μl的样品,然后使酶在提供者推荐的温度下作用30分钟,然后再将全部样品注入琼脂孔试验的孔内。
另一方面,对于商品化脂酶,先取1μl抑制剂混合液(1.25MEDTA;0.25%胃酶抑素A(p4265 Sigma);0.25%E-64(E3132 Sigma);0.25%抑肽酶(A1153 Sigma))加入100μl含有200μg/ml脂酶的溶液中,使抑制剂室温下作用45分钟,然后加入5μl(450au)稀释的标准粗制提取液,使其37℃反应3D分钟,然后将70μl混合液置琼脂孔试验的孔内。用于稀释的缓冲液如下:
-pH2.0:100mM马来酸,用NaOH调整
-pH7.0:100mM磷酸盐缓冲液(K2 HPO4/KH2 PO4)
-pH7.5:100mM磷酸盐缓冲液(K2 HPO4/KH2 PO4)
-pH7.75:100mM Tris-Cl
将抑菌环直径与不加酶而得到的对照环直径相比较,对于每一种缓冲液在每一个培养温度下对照环直径约为14mm。
下面的表Ⅰ列出了用试验酶得出的结果。在该表中酶标出了其类型、提供者名称和提供者条款号。细菌素的灭活表示为加入的酶浓度的作用。数字0表示不再出现抑菌环,换句话说,就是本发明细菌素的抗菌活性经与酶一起培养之后被破坏了。数字14表示仍然存在14mm的抑菌环,这相当于本发明细菌素完全的抗菌活性。
表Ⅰ
酶 | 浓度(μg/ml) | 缓冲液的pH | 孵育温度(℃) | 灭活(mm) |
胃蛋白酶(SIGMA P-700) | 10 | 2.0 | 37 | 0 |
蛋白酶K(MRRCK 1000 144) | 4 | 7.0 | 37 | 0 |
无花果蛋白酶(SIGMA P-3266) | 10 | 7.0 | 37 | 0 |
链霉蛋白酶K(SIGMA P-8038) | 10 | 7.5 | 37 | 0 |
枯草菌蛋白酶(SIGMA P-4789) | 10 | 7.5 | 37 | 0 |
胰蛋白酶(SIGMA T-8128) | 10 | 7.5 | 25 | 0 |
α-胰凝乳蛋白酶(SIGMA C-7762) | 1 | 7.75 | 25 | 14 |
过氧化氢酶(SIGMA C-10) | 10000 | 7.75 | 25 | 14 |
α-淀粉酶(SIGMA A-0521) | 1 | 7.75 | 25 | 14 |
脂酶(SIGMA L-0382)+蛋白酶抑制剂 | 200 | 7.75 | 37 | 14 |
所有蛋白酶均抑制上清液的抗菌活性,这说明某蛋白部分与这种活性有关。
没有观察到过氧化氢酶对细菌素抗菌活性的影响,这种现象也说明代表性指示菌株的生长抑制作用并不是H2O2的抗菌活性所致,虽然目前已知H2O2与细菌素具有相似的抗菌活性,但H2O2应该是被过氧化氢酶分解的。
同样,也没有观察到α-淀粉酶的抗菌活性灭活作用,这种现象说明这种抗菌活性与α-淀粉酶可水解的糖类无关。抑菌谱
借助琼脂孔试验,在接种了各种芽孢或细菌菌株的琼脂上来测定产生本发明的两种细菌素的CNCM Ⅰ-1351菌株的培养物上清液是否具有抑制各种细菌生长的活性,换句话说,即测定该上清液的抑菌谱。
进行这一试验时取抑菌活性在pH7.0时为300au的上清液样品,观察该样品对试验菌株生长的抑制作用,并将其与事先灭活的同一样品的抑制作用相联系,灭活方法是在5μg/ml蛋白酶K存在时37℃孵育30分钟,灭活样品的抑菌作用正常情况下为零。
要进行这些试验,采用FALCON 3046 Multiwell组织培养皿。用700μl额外含有1%乳糖和0.6%琼脂的M17培养基复盖6ml额外含有1%乳糖和1.5%琼脂的M17培养基(M17L培养基),接种1%的试验菌株培养物,培养物在前夜制备并稀释至OD600值为0.1。
当需要由芽孢生长成试验菌株时,用每毫升上层培养基中含105-106个芽孢进行接种。
当试验菌株不是乳球菌属、链球菌属或肠球菌属时,用有利于试验细菌生长的标准培养基来替换M17L培养基。特别是额外含有2%葡萄糖的MRS培养基适用于乳杆菌属、小球菌属、明串球菌属和双歧杆菌属(Sanofi Diagnostics Pasteur,France);RCM培养基适用于梭状芽孢杆菌的芽孢和营养细胞(Oxoid,England);BHI培养基(Difco,USA)适用于其它试验细菌。
每个平皿上打出2个直径5mm深5mm的孔,在一孔内加入70μl含300au的本发明细菌素样品,在另一孔内加入事先灭活的相同样品。在有利于试验菌株生长的温度下进行培养,直至可见的菌苔复盖平皿。
以可观察到的抑菌环直径来判定抑菌作用或程度。如果抑菌环直径为16-18mm,则认为抑菌作用很强(++++),直径为11.5-15.5mm为强(++++),直径为7.5-11mm为一般(++),直径为5-、7.5mm时为弱(+),如果没有观察到抑菌环则认为抑菌作用为零。
至少74株乳酸菌的各个种和亚种进行了这样的试验,并观察到只有约7%的菌株对该上清液具有抗性。下面的表Ⅱ列出了这些试验的详细结果。像下面所有表格一样,在表Ⅱ中标出的菌株名称或编号是Nestlé菌库(地址:NESTEC S.A.,Research Center,Vers-chez-les-Blanc,CH-1000 Lausanne 26,Switzerland)中的编号。标出的温度是试验中的培养温度。
表Ⅱ
种 | No. | T(℃) | 抑菌作用 |
嗜热链球菌(Sfi12和25菌株表现出对CNCM I-1351菌株的抗性,而且其抗菌谱也与该菌株相似)(STII菌株仅对CNCM I-1351菌株表现出抗性;但是能表达这种抗性的链球菌似乎不超过5%) | YS3YS4YS11YS7YS8YS20Sfi3Sfi18Sfi19Sfi20Sfi16ST11Sfi12Sfi25 | 4242424242424242424242424242 | +++++++++++++++++++++++++++++++++--- |
乳酸乳球菌(乳酸链球菌素发酵菌) | SL2SL13SL16SL25SL31SL63 | 303030303030 | ++++++++++++ |
乳酸乳球菌 | SLP26SLP29SLP24SL64SL58SL40 | 303030303030 | ++++++++++++ |
乳酸乳球菌二乙酰乳酸变种 | SD39SD80SD57SD11SD113 | 3030303030 | ++++++++++ |
种 | No. | T(℃) | 抑菌作用 |
乳酪乳球菌 | SC20SC15SC11SC145SC63SC28 | 303030303030 | ++++++++++++ |
粪肠球菌 | SFS1SFS2SFS10 | 303030 | +++ |
屎肠球菌 | SFM1SFM3SFM6SFM10SFM14SFM9 | 303030303030 | ++++++++++ |
发酵乳杆菌 | 126150L28LF16LF15 | 3030303030 | ++++++++++ |
瑞士乳杆菌 | LH91LH2LH3LH1 | 40404040 | +++++++++++++ |
嗜酸乳杆菌 | L01LQ3LQ10LQ21LQ23LQ26 | 404040404040 | ++++++++ |
短乳杆菌 | LB2LB10LB13 | 303030 | ++++++ |
种 | No. | T(℃) | 抑菌作用 |
保加利亚乳杆菌 | YL12YL2YL5LB32 | 40404040 | ++++++++++ |
乳酪明串球菌 | LCC1LCC7LCC2 | 303030 | ++++++ |
肠膜明串球菌 | LCM9LCM10LCM18 | 303030 | ++++++ |
对于乳杆菌属的某些种,例如,嗜酸乳杆菌、短乳杆菌和保加利亚乳杆菌,其抑菌程度不一致,从这种意义上来讲在表Ⅱ中所观察到的上清液的抑菌谱是窄的。但是,对于其它菌种,例如发酵乳杆菌、瑞士乳杆菌和乳球菌属,抑菌程度是一致的。
同种菌株很难相互区别,从这一点上来看上述现象是有利的。因此可以设想上清液或纯化细菌素有利于区别工业用菌株。
也可以设想用能产生至少一种本发明所述细菌素的菌株与另一乳酸菌株一起培养,后者对培养基中产生的一种或两种细菌素具有天然抗性或轻度敏感。这样可以生产酸乳酪,特别是例如后酸化作用小的酸乳酪。
也观察到上清液能抑制乳酸乳球菌的六个乳酸链球菌素发酵菌株的生长。这说明本发明的细菌素并不是乳酸链球菌素。本发明细菌素可被10μg/ml的胰蛋白酶灭活,这一性质与乳酸链球菌素不同,由此可证实上述观点。
然而,产生本发明两种细菌素的培养物上清液的抑菌谱也很宽,因为它并不局限于乳酸菌的菌种,而且也扩展到革兰氏阳性菌的其它菌种,尤其是双歧杆菌这样的食品细菌,也扩展到丙酸杆菌属、无毒李斯德杆菌、单核细胞增多性李斯德杆菌和易变微球菌等有害菌或致病菌,还扩展到例如梭状芽孢杆菌属和芽孢杆菌属中许多致病菌的芽孢和营养细胞,如下面的表Ⅲ列出的结果所示。
表Ⅲ
种 | No. | T(℃) | 抑菌作用 |
短双歧杆菌 | BBR27BBR4BBR39 | 373737 | ++++++ |
长双歧杆菌 | BL20BL18BL22 | 373737 | +++++++++ |
两歧双歧杆菌 | BB7BB9BB12 | 373737 | ++++++++++ |
婴儿双歧杆菌 | B16B11 | 3737 | ++++++ |
丙酸丁菌属 | PP1 | 30 | +++ |
肉毒梭状芽孢杆菌(芽孢和营养细胞) | CB1CB2 | 3030 | ++++ |
干酪酪酸梭状芽孢杆菌(芽孢和营养细胞) | 107001107002 | 3030 | +++ |
下列细菌芽孢混合物产芽孢梭状芽孢杆菌发酵梭状芽孢杆菌梭状芽孢杆菌(6株) | 100021100022A-69;B-213;BKA40;B-73-211;A-80-124c1ovis;B-1-NCA | 30 | +++ |
种 | N0. | T(℃) | 抑菌作用 |
无毒李斯德杆菌 | 242527394041 | 303030303030 | ++++++ |
单核细胞增多性李斯德杆菌 | 575859606162 | 303030303030 | ++++++++++++ |
枯草杆菌(芽孢和营养细胞) | A2A3A13A14A15 | 3030303030 | ++++++++++ |
短小杆菌(芽孢和营养细胞) | B2 | 30 | ++ |
蜡样杆菌(芽孢和营养细胞) | C14 | 30 | ++ |
易变微球菌 | MCVl | 30 | ++ |
藤黄微球菌(乳酸链球菌素指示菌) | MCL1 | 30 | - |
除此之外,由表Ⅲ所列结果可以设想这种上清液或细菌素适合于用作食品生产中的添加剂作为抗病原菌的活性剂,特别是例如用于在肉制品中抗梭状芽孢杆菌属,在乳酪中抗单核细胞增多性李斯德杆菌和干酪酪酸梭状芽孢杆菌,或在鲜酱或鲜酱汁中抗芽孢杆菌属,上述菌株就是起源于芽孢杆菌属。
最后,根据下面表Ⅳ所列结果可以看出,本发明细菌素对革兰氏阴性菌不产生抑制作用。表Ⅳ
热抗性和热稳定性
种 | No. | T(℃) | 抑菌作用 |
大肠杆菌 | BZ234 | 37 | - |
鼠伤寒沙门氏杆菌 | 274273 | 3737 | -- |
绿脓杆菌 | 513 | 3737 | -- |
荧光假单孢杆菌 | 1112 | 3737 | -- |
在实施例1中所示的条件下获得的提取液如果事先不经加热,其中的细菌素在4℃保存时稳定性低。但是如果提取液急剧加热至少15分钟,例如90-121℃,再进行保存,则其稳定性高。
尤其是证实了如果该上清液事先加热20分钟,例如94℃水浴,4℃保存5个月后这种提取液的活性可保持50%以上。也证实了该提取液100℃加热60分钟后,其活性可保持100%(试验是在恒温油浴中进行的,用1ml本发明方法的嗜热链球菌菌株培养物的浓缩的上清液或其它方法制备的上清液)。
另一方面,本发明细菌素经121℃灭菌处理30分钟后仅保留三分之一(用本发明方法的嗜热链球菌菌株培养物的非浓缩上清液40ml进行试验)。
最后,用Amicon过滤器进行超滤试验,再进行凝胶电泳(SDS-PAGE),由此观察到嗜热链球菌培养物的上清液中,特别是实施例1中获得的标准培养物上清液中的本发明细菌素是以分子量(MW)大于10KDa的聚集物形式存在的,其中67%分子量在100KD以下,33%分子量在100KD以上。细菌素的纯化
在下面的介绍中三氟醋酸和乙腈的比例都是按体积计。
用补充1%蔗糖的M17培养基在厌氧条件下于42℃培养CNCMⅠ-1351菌株6小时,制成1升培养物。
将20g XAD-7树脂(Sigma)直接加入培养物,在4℃下轻轻搅拌1小时。然后用604号Schleicher&Schvell滤纸(德国)将混合物过滤,截留在滤纸上的树脂再用1升pH5.2的50mM醋酸溶液洗涤,目的是除去细菌。然后将树脂装入层析柱,用45ml含有70%乙腈和1%三氟醋酸(TFA)的溶液洗脱细菌素。这时便获得含有两种细菌素的洗脱液。
然后用下列方法分离这两种洗脱的细菌素。
先用离心/冻干(Speedvac,Savant设备)法将洗脱液体积减小到24ml,再将该体积调整至2M NaCl和250mM Tris.Cl的浓度,pH8,体积为50ml,然后将这一溶液体积注射入预先用含50mMTris.Cl,pH8和2M NaCl的缓冲液平衡过的具有疏水相互作用的苯基Superose HR 16/10层析柱(Pharmacia)中,然后用200ml上述缓冲液,100ml以上述缓冲液开始以50mM Tris.Cl溶液pH8结束的线性梯度液,100ml上述溶液、6Dml纯水、60ml50mM Tris.Cl溶液pH8、最后用60ml纯水顺序洗脱,速度为4ml/分钟。
从层析柱出口收集的各个组分均取50μl,再用50μl0.1%TFA将其稀释,然后用上述的琼脂孔试验测定各个混合液的抗菌活性。
由此观察到第470至第490ml的组分表现出抗菌活性。再将这些组分混合,将该混合液的体积用离心/冻干法减小至1ml,然后将这一减小的体积注射入用0.1%TFA溶液预平衡的Pep RPC 5/5层析柱(Parmacia)中,0.1%TFA溶液在本发明中称为“溶液A”。还制备了含有70%乙腈和0.097%TFA的洗脱液“B”。然后用1ml溶液A、9ml以溶液A开始以50/50溶液A和B的混合液结束的线性梯度液、2ml后者混合液、7ml以后者混合液开始以第二种20/80溶液A和B混合液结束的线性梯度液、2ml以第二种混合液开始以溶液B结束的线性梯度液,然后用2ml后者溶液顺序洗脱层析柱,速度为1ml/分钟。
然后采用上述的琼脂孔试验测定层析柱出口的组分的抗菌活性。从第14至第22ml的所有组分表现出抗菌活性。另一方面,在215nm的光密度观察到的两个蛋白质主峰可在组分15和21(按毫升计)中区分。细菌素的顺序分析
在组分15、18、20、21和22中所含蛋白质的N-末端部分采用Applied Biosystems 4774自动顺序分析仪进行顺序测定。
测定结果发现在组分15中存在具有48个氨基酸顺序的肽,就N-末端部分而言,它与顺序SEQ ID NO:1的氨基酸顺序相同。主要存在于组分21中的另一种肽也具有23个氨基酸顺序,就N-末端部分而言,它与顺序SEQ ID NO:2的氨基酸顺序相同。
因此,这些结果说明CNCM I-1351菌株产生两种具有抗菌活性的肽。然而,组分15和21之间出现不同的抑菌环可能会令人怀疑本发明的嗜热素1和嗜热素2之间抗菌活性不同。
另一方面,用6N HCl 100℃水解24小时后的组分15和21的氨基酸组成采用熟知的“氯化物衍生化作用”来分析。结果表明每一组分呈现与其各自肽顺序一致的氨基酸组成。
最后,对组分15和21也进行了质谱测定,发现嗜热素1的分子量为5800D左右,嗜热素2的分子量为3900D左右。细菌素基因的序列分析
用常规方法生产下面顺序清单中介绍的简并核酸序列SEQ ID NO:6和SEQ ID NO:7,它们与前面进行顺序分析的嗜热素1肽的N-末端和C-末端部分分别对应。
取一份SEQ ID NO:6序列混合物,再经实验室手册“分子克隆,实验室手册”(second edition,Sambrook et al.,ColdSpring Harbour,Laboratory Press,1989)中介绍的T4多核苷酸激酶作用使其具有放射活性,该手册在本发明中称为“Maniatis”。
用两种无放射活性的简并序列SEQ ID NO:6和SEQ ID NO:7混合物在由CNCM Ⅰ-1351菌株中制备的常染色体DNA上按照“PCR技术”手册(H.A.Erdlich editor,M Stockton Press,London)介绍的方法进行PCR(多聚酶链反应)。
这时在电泳凝胶上出现一条128个碱基对(bp)的带,然后按照Maniatis的方法将其洗脱。按照提供者推荐的方法将一份直接克隆到pGEM-T质粒(Promega)中,然后按照Maniatis的方法采用通用pUC19探针用“二脱氧核苷酸”法进行序列分析。由此可获得具有下面顺序清单中介绍的SEQ ID NO:8序列的,与编码嗜热素1第9至47位氨基酸的序列相对应的探针。最后,采用Maniatis所述的称为“随机引物”的方法使128bp洗脱带的其它部分具有放射活性。
另一方面,由CNCM Ⅰ-1351菌株制备的常染色体DNA用EcoRⅠ和HindⅢ按照酶提供者推荐的方法进行消化,然后取10μl消化产物在分析电泳凝胶上进行电泳,用碱性介质将DNA从凝胶转移到“Zeta探针”膜(Biorad)上,该膜是用含有6X SSC,1%SDS和1%脱脂牛奶54℃预平衡过夜,然后将该膜在预杂交介质中与具有放射活性的变性探针SEQ ID NO:6杂交,开始54℃18小时,每3小时温度降低2℃,然后42℃ 24小时。然后将该膜在室温下用6X SSC洗涤2分钟,连续3次,再用6X SSC 47℃洗涤1分钟。最后将膜曝光于放射自显影胶片。所有这些步骤都是按照Naniatis手册进行。
另外还发现一条3.6Kb的带,在与上述相同的条件下进行的CNCNⅠ-1351菌株常染色体DNA(300μg)的制备电泳凝胶中使我们有可能将含有所需DNA片断的凝胶部分定位。然后用常规方法将该凝胶部分切下并洗脱,洗脱的DNA与预先用EcoRⅠ和HindⅢ水解的载体pUC19(Nessing et al.,Methods Enzymol.,101:20,1983)相连接。这些步骤均按照Maniatis手册方法进行。
将事先激活的大肠埃希氏杆菌属BZ234菌株(Biocentrecollection University of B_le Switzerland)用连接介质以常规方法转化。然后用α-互补作用选择转化细胞。然后根据Maniatis按照称为“挑取菌落”的方法将300个转化的菌落转移到滤纸上,菌落溶解,将其与放射性序列SEQ ID NO:8杂交,然后将滤纸曝光于放射自显影胶片。
然后在胶片上观察含有能与序列SEQ ID NO:8杂交的质粒的13个菌落,再选择这些菌落中的两个,用常规方法从中提取质粒DNA,克隆到两种选择出的pUC19质粒中的DNA片断用“二脱氧核苷酸”法借助通用pUC19探针进行序列分析,由此便获得以这些序列为基础的探针。
由此可获得下面顺序清单中介绍的核酸序列SEQ ID NO:3,它与选择出的两种质粒相同。因此,该序列含有编码两种蛋白质的操纵子,这两种蛋白质具有与完成加工之前的嗜热素1相一致的氨基酸顺序SEQ ID NO:4和与完成加工之前的嗜热素2相一致的氨基酸顺序SEQ ID NO:5(见下面的顺序清单)。第三部分是开放解读框架,也是从这一序列的679位核苷酸开始的,它肯定是对应于免疫基因。
将纯化细菌素N-末端肽顺序与由操纵子SEQ ID NO:3编码框架编码的蛋白质的氨基酸顺序进行比较,可以确定氨基酸顺序SEQ IDNO:4(嗜热素1)的蛋白质有一个23个氨基酸的领头肽,它有一个甘氨酸-甘氨酸单位,这是乳酸菌这类细菌素的特征。最后,嗜热素1的分子量按照其核酸序列计算,根据光谱测定结果,为5800道尔顿(D)左右。
同样,氨基酸顺序SEQ ID NO:5(嗜热素2)的蛋白质有一个21个氨基酸的领头肽链,它有一个甘氨酸-甘氨酸单位,这是乳酸菌这类细菌素的特征。最后,嗜热素2的分子量按照其核酸序列计算,根据光谱测定结果,为3900D左右。不同细菌素的作用
在嗜热素1的顺序上发现了“LactococeinM”操纵子的第一个肽的同源序列(Klaenhammer et al.,FEMS Micro.Rew.,12,39-86,1993)。该同源序列与GA单位的重复有关。同样,采用GCG的TFASTA程序在GenEMBL数据库中在嗜热素2上发现了“LactacinF”操纵子基因的同源序列(Klaenhammer et al.,出处同上)。
LactococeinM和LactacinF这两个操纵子实际上是编码包含数种肽的poration复合物。因此,有可能上述的操纵子可以编码在poration复合物中联合作用的肽。
然而,并不排除两种细菌素独立作用,因为在前述的琼脂孔试验中观察到两种嗜热素的抑菌环稍有不同。
下面介绍实施例来说明本发明所述的细菌素的生产方法和应用。除另作说明外,这里所用的百分比按重量计。
实施例1
已加入1%蔗糖的M17培养基用1%(v/v)每毫升含有108个嗜热链球菌CNCM Ⅰ-1351菌株细菌的培养物来接种。在厌氧条件下42℃进行孵育6小时,培养之后培养基中每毫升含约108个该菌株细菌,OD600为3.6。
将由此得到的标准培养物进行离心,收集其上清液(标准)。用H3PO4将其酸化至pH1.5,用离心法除去产生的沉淀物并收集酸性沉淀上清液。
存在于后者中的细菌素用10%三氯醋酸来沉淀,收集沉淀的细菌素,然后用0.2%三氟醋酸(v/v)在水悬浮液中将其再悬浮。
用10%三氯醋酸将细菌素再沉淀,收集沉淀的细菌素,用100%丙酮洗涤,并于含有0.2%三氟醋酸(v/v)的水悬浮液中再悬浮。
制得的本发明细菌素的标准粗制提取液其活性为1.4105au/ml。
表Ⅳ列出了1升标准上清液和18ml标准粗制提取液特性的一些详细数据,换句话说,后者也就是从前者中制得的浓缩液,尤其是在蛋白质含量和抗菌活性方面。
表Ⅵ
体积(ml) | 总蛋白(PIRRCR试剂盒)(mg) | au/ml | au/mg蛋白质 | au/mg干重 | 总活性(au) | |
上清液 | 1000 | 6700 | 1.6×102 | 68 | 4.6×103 | |
粗制提取液 | 18 | 83 | 1.4×105 | 2.5×105 | 1.4×105 | 2.1×106 |
实施例2
制备的凝固型酸乳酪含有本发明菌株,即嗜热链球菌CNCM Ⅰ-1351、嗜热链球菌STⅡ菌株(该菌株对本发明所述细菌素具有抗性,但没有抗菌活性)和上述的保加利亚乳杆菌YL5菌株。
为此制备了以含有3.7%脂肪和2.5%脱脂奶粉的全乳为基础的牛奶。该牛奶40m192℃经巴氏灭菌6分钟,然后75℃150bar使其均化(两阶段),最后冷却至42℃左右。
嗜热链球菌CNCM Ⅰ-1351、嗜热链球菌STⅡ和保加利亚乳杆菌菌株然后在灭菌MSK培养基(10%重配成的粉末状脱脂牛奶,含有1%的商品酵母提取物)中用连续数次预培养再激活。
在培养基凝固阶段取出嗜热链球菌菌株,然后用一定量的1%(v/v)的各菌株第三次预培养物来接种灭菌牛奶;在培养基凝固阶段取出保加利亚乳杆菌菌株,用一定量的2%(v/v)的该菌株第三次预培养物来接种灭菌牛奶。然后将牛奶在42℃培养至pH约为4.65,再冷却至4℃。
为进行比较,按上述相同的方法,用前述的嗜热链球菌YS8和SFi3菌株和保加利亚乳杆菌YL18菌株制备了传统的凝固型酸乳酪,这些菌株都是在酸乳酪生产中长期使用的。
下表中介绍了制得产品的特性,特别是产品在4℃保存期间的pH。
实施例 | 酸化至pH4.65所需时间 | 1天后产品的pH(4℃) | 24天后产品的pH(4℃) |
实施例2 | 8h30 | 4.6 | 4.6 |
对照实施例 | 6h | 4.34 | 4.3 |
实施例3
用嗜热链球菌CNCM Ⅰ-1351菌株培养物按传统方法制备了干乳酪。
实施例4
在厌氧条件下42℃用补充了1%蔗糖的M17培养基培养6小时生产10升嗜热链球菌CNCM Ⅰ-1351菌株培养物。然后将200gXAD-7树脂(Sigma)直接加入培养物中,将其在4℃轻轻搅拌1小时。然后用604号Schleicher&Schvell滤纸(德国)将混合液过滤,再用10升50mM pH5.2的醋酸洗涤截留在滤纸上的树脂,目的是除去细菌。再向树脂中加入450ml含有100%乙醇和20mM醋酸铵的溶液,将含树脂的溶液过滤,目的是除去树脂,将滤液冻干,直至获得含有本发明所述细菌素的粉末,该粉末可用于食品工业。
该粉末先用水稀释,然后用上述的琼脂孔试验测定其抗菌活性。这种粉末的抗菌活性为107au/每克粉末。
最后,在用传统方法制备肉奶油冻过程中向其中加入0.5g/kg的上述粉末。由此制得的肉奶油冻含有5.103au/g的细菌素,能够完全抑制致病菌的生长,特别是梭状芽孢杆菌属。
实施例5
本实施例涉及到保持皮肤湿润的乳剂的制备,其中含有0.05g/kg的实施例4中所述的粉末,因此也就是说含有5102au/g的细菌素,能够抑制皮肤表面有害菌的生长。
要生产这种乳化液,将脂相A的成份混合,并加热至75℃。制备水相B,也将其加热至75℃,然后将其加入脂相A中,同时缓慢混合,再将混合液缓慢混合使其冷却至室温,即25℃左右。在室温下按配方顺序将添加剂C缓慢加入。
脂相A%
peg-6-硬脂酸酯,甘油酸酯和peg-20-+六烷基***(peg:聚乙二醇) | 15 |
凡士林油 | 5 |
用1%苯茚满(抗氧化剂)稳定的麦胚油和1%大豆磷脂(见EP94109355.1) | 3 |
甜杏仁油 | 2 |
十六烷醇 | 1 |
异硬脂酸异硬脂基酯 | 2 |
肉豆蔻酸2-辛基-十二烷基酯 | 1 |
羊毛脂 | 1 |
水相B%
添加剂C %
甲基异噻唑啉 | 0.1 |
去离子水 | 59.6 |
人胎盘蛋白 | 2 |
丙二醇和金盏花提取物 | 2 |
50%可溶性胶原去离子水溶液 | 5.8 |
香料 | 0.3 |
2.5%实施例4所述细菌素粉末的去离子水溶液 | 0.2 |
实施例6
将0.5g/kg的实施例4中所述的细菌素粉末加入液体洗牙水中。因此这种洗牙水能够抑制口颊部致病菌的生长,特别是sobrinus链球菌。
实施例7
含有实施例4的细菌素粉末的溶液用水稀释成总量为1%,将其喷到食品表面使其灭菌,目的是防止包装过程中的后污染。
顺序清单(1)一般情况
(ⅰ)申请人:
(A)名称:SOCIETE DES PRODUITS NESTLE S.A.
(B)街道:Case postale 353
(C)城市:Vevey
(E)国家:Suisse
(F)邮政编码(区号):1800
(G)电话:(021)924 47 60
(H)传真:(021)924 28 80
(ⅱ)发明题目:嗜热链球菌细菌素
(ⅲ)顺序数量:8
(ⅳ)计算机可读形式:
(A)介质类型:软盘
(B)计算机:IBM PC兼容机
(C)操作***:PC-DOS/MS-DOS
(D)软件:PatentIn Release #1.0,版本#1.25(EPO)
(ⅵ)优先申请资料:
(A)申请号:CH 2628/93-7
(B)存档日期:03-SEP-1993(2)SEQ ID NO:1的情况:(ⅰ)顺序特征:
(A)长度:62个氨基酸
(B)类型:氨基酸
(D)结构:线性(ⅱ)分子类型:蛋白质(ⅵ)来源:
(A)生物体:嗜热链球菌
(B)菌株:CNCM Ⅰ-1351(ⅹⅰ)顺序描述:SEQ ID NO:1:Tyr Ser Gly Lys Asp Cys Leu Lys Asp Met Gly Gly Tyr Ala Leu Ala1 5 10 15Gly Ala Gly Ser Gly Ala Leu Trp Gly Ala Pro Ala Gly Gly Val Gly
20 25 30Ala Leu Pro Gly Ala Phe Val Gly Ala His Val Gly Ala Ile Ala Gly
35 40 45Gly Phe Ala Cys Met Gly Gly Met Ile Gly Asn Lys Phe Asn
50 55 60(2)SEQ ID NO:2的情况:(ⅰ)顺序特征:
(A)长度:43个氨基酸
(B)类型:氨基酸
(D)结构:线性(ⅱ)分子类型:蛋白质(ⅳ)来源:
(A)生物体:嗜热链球菌
(B)菌株:CNCM Ⅰ-1351
(ⅹⅰ)顺序描述:SEQ ID NO:2Gln Ile Asn Trp Gly Ser Val Val Gly His Cys Ile Gly Gly Ala Ile1 5 10 15Ile Gly Gly Ala Phe Ser Gly Gly Ala Ala Ala Gly Val Gly Cys Leu
20 25 30Val Gly Ser Gly Lys Ala Ile Ile Asn Gly Leu
35 40(2)SEQ ID NO:3的情况:
(ⅰ)序列特征:
(A)长度:770个碱基对
(B)类型:核酸
(C)链:单链
(D)结构:线性
(ⅱ)分子类型:DNA(基因组)
(ⅵ)来源:
(A)生物体:嗜热链球菌
(B)菌株:CNCM Ⅰ-1351
(ⅸ)特性:
(A)名称/关键词:CDS
(B)位置:221..475
(ⅸ)特性:
(A)名称/关键词:sig_肽
(B)位置:221..289
(ⅸ)特性:
(A)名称/关键词:mat_肽
(B)位置:290..475
(D)其它情况:/功能=“编码嗜热素1”(ⅸ)特性:
(A)名称/关键词:CDS
(B)位置:495..686(ⅸ)特性:
(A)名称/关键词:sig_肽
(B)位置:495..557(ⅸ)特性:
(A)名称/关键词:mat_肽
(B)位置:558..686
(D)其它情况:/功能=“编码嗜热素2”(ⅹⅰ序列描述:SEQ ID NO:3AATGGCACGA ACGTCCTGAA TGGTTAAAAG ATATTTCGGA TCTTCCTAAA AAATACATAC 60TGAACGGTCG CTTTCCCTTC TTGAATGGTA AAATTTTCCC ATTAGGAAAG TTAAATGACT 120GTTCAAGAAA TGGGGAAATT ATTTTTTGAA GTAGTGCTAT ACTAGACTTG TCAAGGTTGC 180AACCCGACAA AATAAAAATA TTAGGTAGGA GATATTTACA ATG AAT ACA ATA ACT 235
Met Asn Thr Ile Thr
-23 -20ATT TGT AAA TTT GAT GTT TTA GAT GCT GAA CTT CTT TCG ACA GTT GAG 283Ile Cys Lys Phe Asp Val Leu Asp Ala Glu Leu Leu Ser Thr Val Glu
-15 -l0 -5GGT GGA TAC TCT GGT AAG GAT TGT TTA AAA GAC ATG GGA GGA TAT GCA 331Gly Gly Tyr Ser Gly Lys Asp Cys Leu Lys Asp Met Gly Gly Tyr Ala
1 5 10TTG GCA GGA GCT GGA AGT GGA GCT CTG TGG GGA GCT CCA GCA GGA GGT 379Leu Ala Gly Ala Gly Ser Gly Ala Leu Trp Gly Ala Pro Ala Gly Gly15 20 25 30GTT GGA GCA CTT CCA GGT GCA TTT GTC GGA GCT CAT GTT GGG GCA ATT 427Val Gly Ala Leu Pro Gly Ala Phe Val Gly Ala His Val Gly Ala Ile
35 40 45GCA GGA GGC TTT GCA TGT ATG GGT GGA ATG ATT GGT AAT AAG TTT AAC 475Ala Gly Gly Phe Ala Cys Met Gly Gly Met Ile Gly Asn Lys Phe Asn
50 55 60TAAGGAAGGA GTTTATATC ATG AAG CAG TAT AAT GGT TTT GAG GTT CTA CAT 527
Met Lys Gln Tyr Asn Gly Phe Glu Val Leu His
-21 -20 -15GAA CTT GAC TTA GCA AAT GTA ACT GGC GGT CAA ATT AAT TGG GGA TCA 575Glu Leu Asp Leu Ala Asn Val Thr Gly Gly Gln Ile Asn Trp Gly Ser-10 -5 1 5GTT GTA GGA CAC TGT ATA GGT GGA GCT ATT ATC GGA GGT GCA TTT TCA 623Val Val Gly His Cys Ile Gly Gly Ala Ile Ile Gly Gly Ala Phe Ser
10 15 20GGA GGT GCA GCG GCT GGA GTA GGA TGC CTT GTT GGG AGC GGA AAG GCA 671Gly Gly Ala Aia Ala Gly Val Gly Cys Leu Val Gly Ser Gly Lys Ala
25 30 35ATC ATA AAT GGA TTA TAAAAGTCTT TTATCGCTTT TATTATTCAT AATTCCCCSTT 726Ile Ile Asn Gly Leu
40GTAGTTATAC TAATCGTTCT TCGAAAGAAT AATCAGAAAC TAAT 770(2)SEQ ID NO:4的情况:
(ⅰ)顺序特征:
(A)长度:85个氨基酸
(B)类型:氨基酸
(D)结构:线性
(ⅱ)分子类型:蛋白质
(ⅹⅰ)顺序描述:SEQ ID NO:4Met Asn Thr Ile Thr Ile Cys Lys Phe Asp Val Leu Asp Ala Glu Leu-23 -20 -15 -10Leu Ser Thr Val Glu Gly Gly Tyr Ser Gly Lys Asp Cys Leu Lys Asp
-5 1 5Met Gly Gly Tyr Ala Leu Ala Gly Ala Gly Ser Gly Ala Leu Trp Gly10 15 20 25Ala Pro Ala Gly Gly Val Gly Ala Leu Pro Gly Ala Phe Val Gly Ala
30 35 40His Val Gly Ala Ile Ala Gly Gly Phe Ala Cys Met Gly Gly Met Ile
45 50 55Gly Asn Lys Phe Asn
60(2)SEQ ID NO:5的情况:
(ⅰ)顺序特征:
(A)长度:64个氨基酸
(B)类型:氨基酸
(D)结构:线性
(ⅱ)分子类型:蛋白质
(ⅹⅰ)顺序描述:SEQ ID NO:5Met Lys Gln Tyr Asn Gly Phe Glu Val Leu His Glu Leu Asp Leu Ala-21 -20 -15 -10Asn Val Thr Gly Gly Gln Ile Asn Trp Gly Ser Val Val Gly His Cys-5 l 5 10Ile Gly Gly Ala Ile Ile Gly Gly Ala Phe Ser Gly Gly Ala Ala Ala
15 20 25Gly Val Gly Cys Leu Val Gly Ser Gly Lys Ala Ile Ile Asn Gly Leu
30 35 40(2)SEQ ID NO:6的情况:
(ⅰ)序列特征:
(A)长度:17个碱基对
(B)类型:核酸
(C)链:单链
(D)结构:线性
(ⅱ)分子类型:DNA(基因组)
(ⅲ)假设序列:是
(ⅹⅰ)序列描述:SEQ ID NO:6GAYATGGGNG GNTAYGC(2)SEQ ID NO:7的情况:
(ⅰ)序列特征:
(A)长度:17个碱基对
(B)类型:核酸
(C)链:单链
(D)结构:线性
(ⅱ)分子类型:DNA(基因组)
(ⅲ)假设序列:是
(ⅹⅰ)序列描述:SEQ ID NO:7GCTATNGCNC CNACGTG(2)SEQ ID NO:8的情况:
(ⅰ)序列特征:
(A)长度:128个碱基对
(B)类型:核酸
(C)链:单链
(D)结构:线性
(ⅱ)分子类型:DNA(基因组)
(ⅵ)来源:
(A)生物体:嗜热链球菌
(B)菌株:CNCM Ⅰ-1351(ⅹⅰ)序列描述:SEQ ID NO:8GATTGTTTAA AAGACATGGG AGGATATGCA TTGGCAGGAG CTGGAAGTGG AGCTCTGTGG 60GGAGCTCCAG CAGGAGGTGT TGGAGCACTT CCAGGTGCAT TTGTCGGAGC TCATGTTGGG 120GCAATTGC 128
Claims (20)
1.具有氨基酸顺序SEQ ID NO:1或SEQ ID NO:2的嗜热链球菌细菌素。
2.编码权利要求1所述的嗜热链球菌细菌素之一的核苷酸序列。
3.根据权利要求2所述的序列,它具有编码权利要求1或2所述细菌素的核苷酸序列SEQ ID NO:3。
4.根据权利要求3所述的核苷酸序列,其特征在于它包括序列SEQ ID NO:3的第221至475位的核苷酸。
5.根据权利要求4所述的核苷酸序列,其特征在于它包括序列SEQ ID NO:3的第289至475位的核苷酸。
6.根据权利要求3所述的核苷酸序列,其特征在于它包括序列SEQ ID NO:3的第495至686位的核苷酸。
7.根据权利要求6所述的核苷酸序列,其特征在于它包括序列SEQ ID NO:3的第558至686位的核苷酸。
8.根据权利要求4所述的核苷酸序列,其特征在于它包括序列SEQ ID NO:3的第221至288位的核苷酸。
9.根据权利要求6所述的核苷酸序列,其特征在于它包括序列SEQ ID NO:3的第495至557位的核苷酸。
10.由权利要求8或9所述的核苷酸序列编码的嗜热链球菌信号肽。
11.能够产生至少一种权利要求1所述的细菌素的嗜热链球菌菌株。
12.按照权利要求11的嗜热链球菌菌株,所述菌株是能产生权利要求1所述的细菌素的嗜热链球菌CNCMI-1351菌株。
13.生产至少一种权利要求1所述的细菌素的方法,在该方法中,在有利于嗜热链球菌生长的条件下在培养基中培养能产生至少一种所述细菌素的嗜热链球菌菌株,直至培养基中含有每毫升107-109个该菌株细菌,将所得培养物离心,然后制备含有至少一种所述细菌素的上清液的提取液。
14.根据权利要求13所述的方法,其中,为了制备存在于所述上清液中的至少一种所述细菌素的提取液,用H3PO4将该上清液的pH调整至1.0-2.0,除去沉淀物,用三氯醋酸进行一次或多次相继的沉淀,每次沉淀后均于含有三氟醋酸的水悬浮液中再悬浮。
15.根据权利要求13所述的方法,在该方法中所述的嗜热链球菌菌株是能够产生所述细菌素的CNCMI-1351菌株。
16.至少一种权利要求1所述的细菌素在食品或化妆品生产方面的应用,所述的细菌素特别是以按权利要求13方法制得的提取液形式和/或能产生至少一种所述细菌素的嗜热链球菌菌株的形式应用。
17.根据权利要求16所述的应用,其中所述嗜热链球菌菌株培养物作为促酵物在乳酪生产和酸奶生产中的应用。
18.根据权利要求16所述的应用,其中至少一种所述的细菌素或至少一种所述的菌株作为添加剂或抗致病菌活性剂,特别是在例如奶油冻这样的肉制品生产中用作抗肉素梭状芽孢杆菌的活性剂,或在乳剂或洗剂生产中用作抗皮肤致病菌的活性剂,或在口腔保健品生产中用作抗口颊部致病菌的活性剂,尤其是抗sobrinus链球菌。
19.权利要求2至7中所述的至少一种核酸序列的用途,通过转化作用将抑制某些细菌的能力授与细菌、酵母菌或植物。
20.权利要求8或9所述的核酸序列之一的用途,即作为与所需基因相连的信号序列,通过转化作用将分泌该所需基因编码的蛋白质的能力授与嗜热链球菌菌株。
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CH2628/93-7 | 1993-09-03 | ||
CH2628/19937 | 1993-09-03 | ||
CH262893 | 1993-09-03 |
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PT759469E (pt) * | 1995-08-07 | 2003-08-29 | Nestle Sa | Bacteriocina |
WO1997023619A1 (en) * | 1995-12-22 | 1997-07-03 | Innogenetics N.V. | Sequences coding for new bacteriocins |
ATE473237T1 (de) * | 2001-11-29 | 2010-07-15 | Univ Bruxelles | Lantibiotikum von streptococcus macedonicus mit nahrungsmittelqualität und verwendungen |
US7556833B2 (en) * | 2003-11-26 | 2009-07-07 | Kraft Foods Global Brands Llc | Cheese flavoring systems prepared with bacteriocins |
US7988958B2 (en) * | 2005-04-05 | 2011-08-02 | The United States Of America As Represented By The Secretary Of Agriculture | Enterococcus and Streptococcus strains and bacteriocins |
US20100034924A1 (en) * | 2006-06-16 | 2010-02-11 | Christophe Fremaux | Bacterium |
EP2099818A2 (en) * | 2006-11-29 | 2009-09-16 | Novozymes Inc. | Bacillus licheniformis chromosome |
EP1972207B1 (en) * | 2007-03-21 | 2018-02-28 | Nestec S.A. | Safety System For Powdered Nutritional Compositions |
FR2916759B1 (fr) * | 2007-05-29 | 2009-07-10 | Adisseo France Sas Soc Par Act | Peptide rumc presentant une activite antimicrobienne |
CN102076220B (zh) * | 2008-06-30 | 2014-05-28 | 株式会社明治 | 发酵乳的制作方法以及发酵乳 |
DE202009011379U1 (de) * | 2009-08-24 | 2010-12-30 | Khalifa, Samir | Orale Präparate zur Mund- und Zahnpflege und Bekämpfung von Mundgeruch |
WO2012146953A1 (en) * | 2011-04-29 | 2012-11-01 | Compagnie Gervais Danone | Use of nisin resistant mutant strains of lactobacilli for reducing the post acidification in food products |
RU2492231C2 (ru) * | 2011-07-28 | 2013-09-10 | Федеральное бюджетное учреждение науки Государственный научный центр прикладной микробиологии и биотехнологии (ФБУН ГНЦ ПМБ) | Способ выделения бактериоцинов |
LT6142B (lt) | 2013-05-15 | 2015-04-27 | Uab "Biocentras" | Sėklinių grūdų ir sėklų apdorojimo būdas |
CN106010996B (zh) * | 2016-04-29 | 2020-04-24 | 周礼红 | 一种醋酸杆菌及其培养分离方法、筛选方法和应用 |
CN111248277A (zh) * | 2018-11-30 | 2020-06-09 | 内蒙古伊利实业集团股份有限公司 | 一种高蛋白低脂肪酸奶及其制备方法 |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
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EP0443543A2 (en) * | 1990-02-23 | 1991-08-28 | Snow Brand Milk Products Co., Ltd. | Novel lactic acid bacteria, antibacterial substance produced by the bacteria, fermented milk starter containing the bacteria, and process for producing fermented milk by using the starter |
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1994
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Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
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EP0443543A2 (en) * | 1990-02-23 | 1991-08-28 | Snow Brand Milk Products Co., Ltd. | Novel lactic acid bacteria, antibacterial substance produced by the bacteria, fermented milk starter containing the bacteria, and process for producing fermented milk by using the starter |
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