CN106893687A - 生产抗菌、抗真菌及抗病毒剂的乳酸杆菌ir wee‑01 - Google Patents

生产抗菌、抗真菌及抗病毒剂的乳酸杆菌ir wee‑01 Download PDF

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CN106893687A
CN106893687A CN201611167764.5A CN201611167764A CN106893687A CN 106893687 A CN106893687 A CN 106893687A CN 201611167764 A CN201611167764 A CN 201611167764A CN 106893687 A CN106893687 A CN 106893687A
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李贞和
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Abstract

本发明提供一种具有抗菌活性的抗菌用乳酸菌株，植物乳杆菌IR WEE‑01以高水平生产包含从培养上清液中分离的环二肽及DL‑3‑苯基乳酸的多种抗菌、抗真菌及抗病毒物质。因此，利用本发明的菌株的情况下，能够以低廉的费用制备出高收率的抗菌、抗真菌及抗病毒物质。

Description

生产抗菌、抗真菌及抗病毒剂的乳酸杆菌IR WEE-01
技术领域
本发明涉及一种生产抗菌、抗真菌及抗病毒剂的乳酸杆菌IR WEE-01。
背景技术
贯穿本说明书的整体，参照了多个论文及专利文献，并表示出对其的引用。被引用的论文及专利文献的公开内容作为整体在本说明书中作为参照内容被引入，从而对本发明所属的技术领域的水平及本发明的内容更加明确地进行说明。
条件性(facultative anaerobic)厌氧性及非孢子形成(non-spore-forming)革兰氏阳性乳酸菌(lactic acid bacteria；LAB)对于微生物和病毒在体内(in vivo)和体外(in vitro)被用作生理活性物质(pro-bioactive substances)(Naidu et al.,1999)。因乳酸菌(LAB)分泌拮抗剂化合物(antagonistic materials)并通过代谢副产物来改变细胞外环境，从而一直被作为对于环境微生物组代谢竞争者(competitor)使用(Naidu et al.,1999；Rouse and van Sinderen，2008)。从动物和植物等的原料中自然起源的多个发酵食品和培养过滤液中共同发现了LAB的接抗性性质(Lindgren and Do brogosz，1990)。由LAB生产的具有生活性的二次代谢产物因包含有有机酸(organic acids)、过氧化氢(hydrogenperoxide)、二氧化碳(carbon dioxide)、二乙酰基(diacetyl)和乙醛(acetaldehyde)，从而在研究显示出抗细菌活性的小的化合物(small compound)方面具有重要性(Naidu etal.,1999；Rouse and van Sinderen，2008)。多个种类的LAB中，已知乳杆菌(Lactobacillus spp.)在代谢过程中生产分子量小的抗生物质(Messens and De，2002)。Kwak等研究过从乳酸杆菌LBP-10K培养过滤液中纯化得到的CDPs的抗病毒性及抗真菌性活性(Kwak et al.,2013；Kwak et al.,2014)。在现有的研究中，顺式-环(L-亮氨酸-脯氨酸)和顺式-环(L-苯基丙氨酸-L-脯氨酸)显示出了对抗流行性感冒A病毒(influenza Avirus)的生活性(Kwak et al.,2013)，证明了顺式-环(L-缬氨酸-L-脯氨酸)和顺式-环(L-苯基丙氨酸-L-脯氨酸)分别抑制作为植物和人的病原有机体的岛灵芝(Ganodermaboninense)和白色念珠菌(Candida albicans)(Kwat et al.,2014)。乳酸菌和其培养上清液是抑制细菌和真菌的有用的工具(Rouse and van Sinderen，2008)。报道有乳酸菌在啮齿类的腹部抑制酵母的生长，并在鼠的消化道(digestive tract)中对瑞斯特杆菌属(Ristella)具有拮抗效果(Ducluzeau et al.,1971)。包含嗜酸乳杆菌(Lactobacillusacidophilus)的发酵混合物抑制了痢疾杆菌(Shigella dysenteriae)、鼠伤寒沙门氏菌(Salmonella typhimurium)及大肠杆菌(Escherichia coli)的生长(Rani andKhetarpaul，1998)。C57BJ/6雌性小鼠中，罗伊氏乳杆菌(Lactobacillus reuteri)抑制了隐孢子虫(Cryptosporidium parvum)的感染(Alak et al.,1997)。
通过测定是否能够保护无胸腺(athymic)鼠和正常胸腺(euthymic)鼠免受全身性念珠菌病(systemic candidiasis)的侵害来调查嗜酸乳杆菌(Lactobacillus acidophilus)、罗伊氏乳杆菌(Lactobacillus reuteri)、干酪乳杆菌(Lactobacillus casei)GG及动物双歧杆菌(Bifidobacterium animalis)的抗真菌活性(Wagner et al.,1997)。棒状乳杆菌(Lactobacillus coryniformis)、植物乳杆菌(Lactobacillus plantarum)及戊糖片球菌(Pediococcus pentosaceus)对抗真菌而具有活性(Magnusson et al.,2003)。乳酸乳球菌(Lactococcus lactis)、乳脂链球菌(Streptococcus cremoris)R3、双醋酸乳球菌(Lactococcus diacetylactis)V1和嗜热链球菌(Streptococcus thermophiles)T2菌株相对于革兰氏阳性菌显示出了宽的抑制范围(Mezaini et al.,2009)。据报道，分离自黑麦米糊(rye sourdoughs)的乳酸菌生产对抗细菌和真菌的类细菌素(bacteriocin-like)抑制物质(Digaitiene et al.,2012)。
本发明人发现了乳酸杆菌IR WEE-01菌株与现有的已发现的乳酸菌相比，具有高的抗菌、抗真菌及抗病毒活性，从而完成了本发明。
(现有技术文献)
(专利文献)
作为相关的现有技术，韩国授权专利第10-2006-0080130号(利用分离自泡菜的抗真菌活性乳酸杆菌AF1和上述菌的培养液的产品)中公开了一种分离自泡菜的乳酸杆菌AF1菌株。通过将该菌株添加到食品、饲料及化妆品等中，能够实现提高储存性的用途，除此之外，能够用作抗菌剂。
韩国授权专利第10-2013-0002870(顺式-环(L-苯基丙氨酸-L-脯氨酸)的新用途)为本发明人的在先申请，记载了一种包含对担子菌类具有特异性抗真菌活性的顺式-环(L-苯基丙氨酸-L-脯氨酸)作为有效成分的组合物和其生产方法。
(非专利文献)
Scopel M等记载了作为青霉菌属生产的二肽的顺式-环(亮氨酰-酪氨酰)抑制表皮葡萄球菌(Staphylococcus epidermidis)的生物膜形成(Scopel et al.,2013)。
Strom K等报道了植物乳杆菌MiLAB 393菌株生产作为抗真菌的环二肽的环(L-苯基丙氨酸-L-脯氨酸)、环(L-苯基丙氨酸-反式-4-OH-脯氨酸)及3-苯基乳酸(Strom etal.,2013)。
发明内容
要解决的技术问题
本发明的目的在于，提供一种具有高的抗菌、抗真菌及抗病毒活性的乳酸菌株植物乳杆菌IR WEE-01。
技术方案
为了实现上述目的，本发明的一个实施方式的特征为，提供一种具有抗菌、抗真菌及抗病毒活性的乳酸菌株植物乳杆菌IR WEE-01。
发明的效果
根据本发明的植物乳杆菌IR WEE-01以高水平生产多种抗菌、抗真菌及抗病毒物质。因此，在利用本发明的菌株的情况下，能够以低廉的费用来制备高收率的抗菌、抗真菌及抗病毒物质。
具体实施方式
下面，通过实施例对本发明进行更详细的说明。这些实施例仅是为了更加具体地说明本发明，本领域技术人员应当知晓根据本发明的主旨，本发明的范围并不受限于这些实施例。
实施例1由植物物质分离及鉴定植物乳杆菌IR WEE-01菌株。
*菌株的分离及鉴定
从韩国传统的发酵泡菜中分离并培养植物乳杆菌IR WEE-01菌株，对分离的乳酸菌细胞进行PCR扩增，以及采用16s rDNA碱基序列法进行鉴定。将连续稀释的细菌细胞在mMRS培养基中培养，从而计算集落形成单位(CFU，colony forming unit)。
为了进行抗菌活性测定，准备菌株指标(bacterial indicators)、多药耐性(multidrug-resistant)革兰氏阳性及革兰氏阴性细菌菌株。在此使用的多药耐性菌株如下。对包含甲氧西林(methicillin)、双氯西林(dicloxacillin)、萘夫西林(nafcillin)及新青二(oxacillin)的青霉素(penicillins)的β-内酰胺抗生素(beta-lactamantibiotics)显示出抵抗性的新青二-抵抗性金黄色酿脓葡萄球菌(oxacillin-resistantStaphylococcus aureus；ORSA)11471，在青霉素、红霉素(erythromycin)、四环素(tetracycline)及克林霉素(clindamycin)中显示出抵抗性的肺炎链球菌(Streptococcuspneumonia)14596及对ACSSuT(氨苄青霉素(ampicillin)、氯霉素(chloramphenicol)、链霉素(streptomycin)、磺胺类(sulphonamides)和四环素(tetracycline))显示出抵抗性的鼠伤寒沙门氏菌(Salmonella typhimurium)12219。以上所有的细菌性病原菌均由韩国国立保健院(Korea National lnstiute of Health)提供。
抗菌活性测定(Antimicrobial assays)
所有的实验均是利用相当于没有有机酸(organic acids)或糖(sugars)或者没有有机酸及糖的培养液的植物乳杆菌IR WEE-01-I、II及III或单一的化合物测定的。
通过以下方式测定抗菌活性。以最小抑菌浓度(minimum inhibitoryconcentration；MIC)接种种菌(seed inoculation)后，每隔24小时进行测定(Huys etal.,2002；Paulo et al.,2010)。
统计分析(Statistical analysis)
结果以平均±标准偏差表示(表3)。各个差异的统计性显著度利用了微软办公电子表格处理软件(Microsoft Office Excel，2013)程序的学生t检验(Student’s-test)。在所有的比较中，视为p<0.05(*)值在统计性方面有效。
强有力的抗菌性菌株的特性调查
从韩国传统的三种泡菜中分离出400个左右的菌株，对其中的200个乳酸菌株进行鉴定(表1)。将具有强有力的抗菌活性的这些菌株接种到mMRS培养基中培养72小时(28℃)，通过16S rDNA定序方法来进行鉴定。从30个菌株中获得的培养过滤液对革兰氏阳性指标细菌显示出了显著性的抗菌活性(表2)。其中，植物乳杆菌IR WEE-01的培养过滤液被测定为具有最优异的抗菌活性的过滤液(表2)。将上述植物乳杆菌IR WEE-01于2015年10月26日保藏在韩国种菌中心，保藏编号为KCCM11779P。
为了使在植物乳杆菌IR WEE-01的过滤液中分离抗菌物质的实验条件最优化，测定细胞生长、菌落数、pH及抗菌活性。即使在吸光度增加的情况下，在32小时后CFU数也具有显著性而减小，pH为4.0左右。稳定期之后pH维持在3.7-3.8左右。因此，可以认为是因代谢产物的影响而使得CFU和pH减小，从而抗菌活性得到增加。如果在细胞生长期间CFU增加pH减小，则可以认为抗菌物质显著性地过多生产。基于这种结果，显示出富含CDP(CDP-rich)植物乳杆菌IR WEE-01培养过滤液显示出了最强的抗菌性，因此，在该研究中利用该菌株来纯化分离了单一的CDP和植物乳杆菌IR WEE-01菌株。
表1
表2植物供应院分离的LAB的培养上清液的抗菌活性比较
a标记：+，<15mm；++，<22mm；+++，>22mm(指示株系：枯草芽孢杆菌)
bMIC：最小抑菌浓度
c标记：+，1-倍；++，0.5-倍；+++，小于0.25-倍(指示株系：枯草芽孢杆菌)
*所有的试验代表了三个独立试验的平均值。
实施例2对于革兰氏阳性和革兰氏阴性细菌菌株的植物乳杆菌IR WEE-01的活性(bioactivity)
在该研究中，分别使用了枯草芽孢杆菌(Bacillus subilis)、金黄色酿脓葡萄球菌(Staphylococcus aureus)、李斯特菌(Listeria monocytogens)及肺炎链球菌(Streptococcus pneumonia)等的革兰氏阳性细菌。此外，分别使用了沙门氏菌(Salmonella typhimuruium)、大肠杆菌(Escherichia coli)及宋内志贺菌(Shigellasonnei)等的革兰氏阴性细菌。此外，使用了包括金黄色酿脓葡萄球菌11471，肺炎链球菌14596菌株的革兰氏阳性细菌和包括鼠伤寒沙门氏菌12219菌株的革兰氏阴性细菌作为多药耐性菌株。
显然地，通过MIC试验证明了植物乳杆菌IR WEE-01菌株对革兰氏阳性细菌及革兰氏阴性细菌和多药耐性细菌具有显著地抗菌活性(表3)。对于多药耐性细菌的活性所需的植物乳杆菌IR WEE-01菌株的浓度示于表3中。关于植物乳杆菌IR WEE-01菌株对于金黄色酿脓葡萄球菌11471、肺炎链球菌14596及鼠伤寒沙门氏菌12219的抗菌活性的情况，分别在表3中示出了它们的浓度和活性。
由于抗菌活性与其它菌株相比优异，因此认为植物乳杆菌IR WEE-01菌株为LAB培养液的最强的抗菌混合物。其结果，显示出植物乳杆菌IR WEE-01菌株的抗菌物质是通过该菌株自然发生而生产出的，可以与其它抗菌剂或LAB匹敌。
表3
植物乳杆菌IR WEE-01菌株中生产的顺式-环(L-Leu-L-Pro)的抗菌活性
aMIC:最小抑菌浓度.
b标记:+，1-倍；++，0.5-倍；+++，小于0.25-倍.
c多耐药性革兰氏阳性菌.
d多耐药性革兰氏阴性菌.
*这些值是3个独立试验的平均值.
参考文献
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