CN107151634B - 面包乳杆菌、抗菌肽及其应用 - Google Patents
面包乳杆菌、抗菌肽及其应用 Download PDFInfo
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- CN107151634B CN107151634B CN201710157633.7A CN201710157633A CN107151634B CN 107151634 B CN107151634 B CN 107151634B CN 201710157633 A CN201710157633 A CN 201710157633A CN 107151634 B CN107151634 B CN 107151634B
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- lactobacillus
- bacteria
- antibacterial
- antibacterial peptide
- bakeri
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Abstract
本发明涉及面包乳杆菌、抗菌肽及其应用，所公开的面包乳杆菌，Lactobacillus crustorum，在中国微生物菌种保藏管理委员会普通保藏中心的保藏号为CGMCC No.13613。本发明的面包乳杆菌能产多种抗菌肽，所产抗菌肽具有广谱抗菌活性且对多重耐药菌显示出同等的杀菌活性。同时，该益生菌菌株具有极强的环境压力耐受性，能耐受酸、胆盐、碱、热、冷、紫外辐射等。
Description
技术领域
本发明属于微生物技术领域，具体涉及一株产多种抗菌肽的面包乳杆菌益生菌及其应用。
背景技术
“益生菌”这一概念最早源自于希腊语“for life”，即对生命有益。2001年世界粮农组织(FAO)和世界卫生组织(WHO)对益生菌做出了如下定义：通过摄取适当的量、对食用者的身体健康能发挥有效作用的活菌。
目前，益生菌大致可分为如下四类：⑴乳酸菌，包括乳杆菌、双歧杆菌、链球菌；⑵不产乳酸型细菌，包括芽孢杆菌、丙酸菌；⑶非病原性酵母，包括酵母菌；⑷无芽孢和无鞭毛的一类杆菌或球杆菌(陈忠秀，2016)。乳杆菌是一类革兰氏阳性、兼性厌氧或者微好氧、杆状、不形成芽孢、不运动的乳酸菌。它能产生有利于食物保藏的代谢物，如CO2、双乙酰、过氧化氢、脂肪酸、乳酸、苯甲酸、细菌素、环二肽等(李婷等，2013)。乳酸菌通常被认为是食品级的微生物(Ross et al.2002)，并且获得了FDA“一般认为安全(GRAS)”等级认定。
近年来，随着抗生素在医药、兽药、农业种植、水产养殖等领域广泛而大量的使用，大量耐药菌不断涌现，且耐药谱逐渐扩大。当多耐药菌以食物为传播介质时，直接导致耐药菌感染人体，对人类健康是巨大威胁。我国目前在食品中也发现了大量耐药菌的存在，如：奶粉相关婴儿食品中的多重耐药沙门氏菌(Yang et al.2014)、原料奶和冰激凌中的多重耐药金黄色葡萄球菌(Gun et al.2006)等。因此急需一种新的、高效的、安全的抗菌物质来控制这些耐药菌。乳酸菌能够产生一类具有抗菌活性的多肽物质，即抗菌肽(antimicrobial peptide)，包括核糖体编码多肽和非核糖体合成多肽。前者称为细菌素，被广泛用作生物防腐剂研究。抗菌肽具有广谱抗菌特点，且具有高效、无毒、耐高温、无残留等优点。随着抗生素的大量使用，抗生素抗性逐步扩散和进化，目前我国已发现多种多重耐药菌，包括直接食用食物中的多耐药致病菌。因此，如何战胜耐药菌，维护人体健康越来越受关注。
发明内容
针对现有技术的缺陷或不足，本发明目的在于，提供一株产广谱抗菌肽的微生物菌株，该菌株能产生多种抗菌肽，尤其的优点是能杀死多重耐药致病菌。所提供菌株耐酸耐胆盐，具有多种有益人体健康的特性，是一株具有极大利用价值的益生菌。
本发明的菌株是从新疆牧民家庭传统发酵的酸马奶中分离取得。本菌株是一天然菌株，鉴定为面包乳杆菌，Lactobacillus crustorum，其保藏于中国微生物菌种保藏管理委员会普通保藏中心，保藏编号为CGMCC No.13613，保藏地址：北京市朝阳区北辰西路1号院3号中国科学院微生物研究所，保藏时间：2017年1月13日。
本发明首次公开了益生菌面包乳杆菌的全基因组序列，从遗传水平揭露其益生特性。通过联合第三代Pacbio RS II和第二代Illumina Hiseq 4000测序技术，测得本发明菌株的全基因组序列(0gaps)，其含有1条染色体(2,261,471bp)和2条质粒(46,788bp和8,220bp)。全基因组序列提交至NCBI的GenBank数据库，染色体和质粒的accession number分别是CP017996、CP017997和CP017998。
经申请人的研究表明，本益生菌能产生至少3种抗菌肽，且所产抗菌肽具有广谱抗菌活性，能同时杀死革兰氏阳性菌和革兰氏阴性菌，包括多重耐药致病菌。其中3种抗菌肽的氨基酸序列为：
MN047B:EGPVGLADPDGPASAPLGAP；
MN047C:GPVGLLSSPGSLPPVGGAP；
MN047D:KSIEEAMNKLLKMSA。
经申请人的研究表明，抗菌肽通过引起致病菌细胞壁/细胞膜破损、形成孔洞、细胞质物质流失导致致病菌死亡。
本发明的一个目的是提供面包乳杆菌的一种新的制药用途，本发明益生菌所产抗菌肽可以以活性菌体或多肽提取物形式用于治疗细菌性感染。活性菌体选用可食载体，可以是一种药物学合适的载体，如胶囊、片剂或粉末。优选可摄食载体是一种食品，如酸奶、酸乳酪、浓缩奶、饮料等。多肽提取物亦可以以液体制剂、冻干粉、片剂、胶囊等形式，也可以掺入到软膏、洗液、霜膏制剂中作为抗微生物剂。
本发明的益生菌所产抗菌肽可以用于治疗动物或者人类的病原菌感染，感染可以是由下面的任何一种或多种细菌引起：金黄色葡萄球菌(Staphylococcus aureus)、粪肠球菌(Enterococcus faecalis)、单核增生李斯特菌(Listeria monocytogenes)、大肠埃希氏菌(Escherichia coli)、沙门氏菌(Salmonella)、阪崎肠杆菌(Enterobacter sakazakii)、铜绿假单胞菌(Pseudomonas aeruginosa)。同时本发明益生菌所产抗菌肽其广谱抗菌活性不局限于以上病原菌种属。
本发明的益生菌所产抗菌肽用于治疗动物或者人类的病原菌感染时，当这些病原菌对抗生素具有抗性时，该抗菌肽依然有效，抗生素抗性包括下面一种或者多种：氨苄青霉素、阿莫西林、阿米卡星、氯霉素、环丙沙星、先锋霉素、头孢哌酮钠、克拉霉素、头孢曲松、头孢呋辛、头孢西丁、加替沙星、庆大霉素、卡那霉素、左氧氟沙星、二甲胺四环素、萘啶酸、妥布霉素、氧氟沙星、苯甲异噁唑青霉素、盘尼西林、哌拉西林、利福平、链霉素、复方新诺明、四环素、万古霉素。同时本发明益生菌所产抗菌肽作用的抗生素抗性谱不局限于以上抗生素种类。
本发明的另外一个目的在于益生菌在食品生产加工过程中的应用。本发明的益生菌所产抗菌肽可以用作生物防腐剂，防止食源性腐败菌导致的食物腐败，包括由芽孢杆菌类引起的食物腐败变质。本发明益生菌对外界环境压力具有极强的耐受性，除了酸和胆盐，还能耐碱、盐、高温、低温、高压、紫外辐射等，因此在生产加工过程中，能保持一定的活菌数。
本发明得到的微生物菌株，是一株新颖的益生菌菌株，它能产生至少3种广谱抗菌肽，抗菌肽能同时杀死革兰氏阳性菌和革兰氏阴性菌，包括多耐药菌。此外，该菌株具有良好的环境耐受性。因此，本发明面包乳杆菌MN047益生菌菌株具有极大的应用价值。
附图说明
为了更好地理解本发明及显示本发明是如何实施的，现将参考附图作为示例，其中：
图1面包乳杆菌MN047全基因组；a为染色体的圈图；b为质粒1的圈图；c为质粒2的圈图。
图2抗菌肽的分离纯化及抑菌。a1为HiPrep Q XL 16/10阴离子交换柱洗脱曲线及Q1抑菌圈，其中Q1、Q2、Q3、Q4和Q5分别为280nm下的5个吸收峰；a2为RESOURCE RPC反相柱洗脱曲线，其中R1、R2、R3和R4分别为280nm下的4个吸收峰；a3为SP Sepharose Fast Flow阳离子交换柱洗脱曲线，其中S1和S2分别为280nm下的2个吸收峰；a4为再一次RESOURCE RPC反相柱洗脱曲线及其抑菌圈,其中P为280nm下的吸收峰；b1为a2获得4个峰的抑菌圈；b2为a3获得的2个峰的抑菌圈及缓冲液的抑菌圈。
图3抗菌肽MN047B LC-MS/MS质谱序列。
图4抗菌肽MN047C LC-MS/MS质谱序列。
图5抗菌肽MN047D LC-MS/MS质谱序列。
图6金黄色葡萄球菌的扫描电镜和透射电镜图；A1和A2为未经抗菌肽处理；B1和B2为经过抗菌肽处理。
图7大肠杆菌的扫描电镜和透射电镜图；A1和A2为未经抗菌肽处理；B1和B2为经过抗菌肽处理。
具体实施方式
以下是结合具体实施例来对本发明进行详细说明，本发明的优点和特点将会随着描述而更为清楚。
实施例1：本发明微生物菌株MN047的筛选鉴定
酸马奶提供了丰富的乳酸菌资源，它是一种传统发酵的马奶饮料，流行于中国(内蒙古、新疆、青海)、蒙古、哈萨克斯坦、吉尔吉斯斯坦、俄罗斯等游牧地区(Danova etal.2005)。酸马奶以新鲜马奶为原料，在固有的天然微生物(乳酸菌和酵母菌为主)菌群的作用下自然发酵而成(Montanari et al.1996)。数百年以来，酸马奶以促进健康的饮料闻名，它拥有丰富的营养物并具有一定的医疗保健作用，属于功能性食品。酸马奶在我国饮用历史悠久，据记载两千多年前人们已经开始饮用酸马奶。大量研究表明酸马奶能够提高消化道系统、内分泌系统、肾功能、神经系统、免疫系统等，同时能够辅助治疗肝炎、慢性溃疡、肺结核等(Wang et al.2008,Mu et al.2012)。一般认为，酸马奶的医疗保健功能与它本身含有的乳酸菌所分泌的抗菌类物质有着直接的关系(Xie et al.2011)。
发明人从新疆传统发酵的酸马奶中，经分离、筛选，获得了一株微生物菌株，该菌株命名为面包乳杆菌MN047(Lactobacillus crustorum MN047)。
(1)微生物菌株的分离与筛选：
用灭菌生理盐水以10倍的稀释梯度对酸马奶样品进行稀释。分别取100μL 10-4、10-5、10-6稀释度稀释液涂布到MRS固体培养基上，37℃恒温培养48h。挑取乳酸菌单菌落并进一步采用划线分离法对特征菌落进行反复地分离纯化，记录各菌落的形态特征并采用革兰氏染色镜检，观察。
所述的MRS固体培养基主要成分组成：葡萄糖2％、蛋白胨1％、牛肉膏1％、酵母膏0.5％、乙酸钠0.5％、吐温80 0.1％、柠檬酸氢二胺0.1％、硫酸镁0.02％、硫酸锰0.005％、琼脂粉1.5％、蒸馏水。
菌落形态特征：在MRS琼脂平板上菌落形状为圆形、表明光滑且稍微凸起，菌落颜色为浅乳白色。
个体形态特征：革兰氏阳性(G+)，短杆状，单个或成对，无芽孢，无鞭毛。
(2)微生物菌株的鉴定：
将菌株MN047接种于MRS液体培养基中，37℃培养12h。按OMEGA的Bacterial DNAKit试剂盒所示方法提取细菌基因组DNA。以乳酸菌基因组DNA为模板、27F(5’-AGA GTT TGATCC TGG CTC AG-3’)和1495R(5’-CTA CGG CTA CCT TGT TAC GA-3’)为引物进行16s rDNA扩增，并对PCR扩增产物进行测序分析。
结果：共测得1479bp核苷酸，并将该16s rDNA序列提交至NCBI的GenBank(GenBankAccession No.KJ152778.1)，它与NCBI数据库中的面包乳杆菌属(Lactobacilluscrustorum)相似度为99％，因此本发明菌株鉴定为面包乳杆菌。
实施例2：本发明面包乳杆菌MN047的全基因组序列测定
使用第三代Pacbio RS II测序技术，构建20-Kb单分子实时(SMRT)文库。得到的原始数据使用SMRT Analysis v2.3.0软件的RS_HGAP Assembly 3(Pacific Biosciences,USA)进行从头组装。产生了3个contig。然后通过Illumina Hiseq 4000获得的数据使用soapSNP和soapIndel软件对contig进行校正。最后得到了一个环状染色体和2个环状质粒(0gaps)。使用Glimmer 3.02软件进行基因预测。
结果：面包乳杆菌MN047全基因组组成：染色体大小为2,261,471bp，平均GC含量为35.02％；质粒1大小为46,788bp，平均GC含量为37.91％；质粒2大小为8,220bp，平均GC含量为36.19％。全基因组共含有2218个蛋白编码基因、55个tRNA基因、12个rRNA基因和3个sRNA基因，具体统计如表1。
表1面包乳杆菌MN047基因组特性
实施例3：本发明面包乳杆菌MN047的基因功能注释
对获得的面包乳杆菌MN047全基因组DNA序列，使用NR(Non-Redundant ProteinDatabase),Swiss-Prot,TrEMBL,COG(the Clusters of Orthologous Groups ofproteins),KEGG(the Kyoto Encyclopedia of Genes and Genomes)和GO(GeneOntology)6个数据库进行BLAST比对，从而对相应基因进行功能注释。
结果：总共2176个基因进行了功能注释，分为988个COG分类、975个KEGG分类和294个GO分类(表4，图1)。其中包括14个耐酸和耐胆盐功能基因(表2)，28个碱、盐、高温、低温、高压、紫外辐射等外界环境压力耐受功能基因(表3)。
表2面包乳杆菌MN047耐酸和耐胆盐功能基因及相应基因产物。
表3面包乳杆菌MN047耐碱、盐、热、冷、高压和耐紫外辐射功能基因及相应基因产物。
表4面包乳杆菌MN047蛋白编码基因功能预测的COG分类
实施例4：本发明微生物菌株MN047产抗菌肽粗样的制备及分离纯化
(1)抗菌肽粗样的制备
配制6L的MRS液体培养基，按1％接种量接入MN047菌株，30℃静置培养72h，离心除去菌体(6000rpm,15min,4℃)，用l mol/L NaOH将上清液pH值调节到7.0，以中和有机酸的干扰。加入硫酸铵至饱和度为60％，4℃过夜沉淀，离心除沉淀(10000rpm,15min,4℃)。上清液继续加入硫酸铵至饱和度为80％，4℃过夜沉淀，离心(10000rpm,15min,4℃)获得沉淀，即抗菌肽粗样。
(2)抗菌肽的分离纯化
使用AKTA纯化系统(AKTA Purifier 100,GE,Sweden)对抗菌肽进行纯化。硫酸铵沉淀获得的粗抗菌肽首先使用Hiprep 26/10脱盐柱脱盐。脱盐后的样品使用HiPrep Q XL16/10阴离子交换柱分离，在含有1M NaCl的pH 6.0柠檬酸-磷酸盐缓冲液中洗脱。然后使用RESOURCE RPC反相柱分离，洗脱过程为100％buffer A(95％water,5％acetonitrile,0.1％TFA)到100％buffer B(100％acetonitrile)的线性洗脱。接着使用SP SepharoseFast Flow阳离子交换柱进行分离，缓冲液为pH 3.0柠檬酸-柠檬酸钠缓冲液。最后，再一次使用RESOURCE RPC反相柱进行分离。以上过程中，每步分离得到的抑菌活性最好的峰进行下一步分离，活性检测方法为牛津杯法抑菌实验(实施例6)，指示菌为金黄色葡萄球菌ATCC29213。
结果：如图2所示，在整个纯化过程中，在通过阴离子交换柱后，只有峰Q1具有抑菌活性；将Q1峰通过RPC反相柱时，峰R1和R2同时具有抑菌活性；将R2峰通过阳离子交换柱后，峰S1和S2同时具有抑菌活性。由此可见，峰R1、S1和S2三个峰分别含有某一种或多种抗菌肽，即本发明益生菌菌株能产生至少3种抗菌肽。
实施例5：抗菌肽的鉴定
将实施例3中制备的抗菌肽粗样使用截留分子量3500的透析袋，在4℃层析柜中透析，收集透过液，真空冷冻干燥浓缩。浓缩后的样品采用C18固相萃取柱进行纯化(活化、平衡、上样、洗涤、洗脱)，洗脱液冷冻抽干，进行LC-MS/MS串联质谱分析。同时，将实施例3中最后一步纯化得到的P峰进行LC-MS/MS串联质谱分析。
LC-MS/MS通过岛津公司LC-20AD型号的纳升液相色谱仪进行分离，偶联Q-Exactive(Thermo Fisher Scientific)质谱，离子源为纳喷离子源。样品上样量为30μL，所用的柱子包括Trap柱和分析柱两部分。分离程序如下：先以8μL/min的流速在4min内将样品loading到Trap柱上，紧接着一个总流速为300nL/min的分析梯度将样品带入分析柱，分离并传输至质谱系统。HPLC洗脱程序为：5％buffer B(95％CAN，0.1％FA)，5min；5％bufferB-35％buffer B，35min；35％buffer B-60％buffer B，5min；60％buffer B-80％bufferB，2min；80％buffer B，2min。质谱数据采集的主要参数设置如下：DDA(data dependentacquisition)模式进行一级质谱(MS)和二级质谱(MS/MS)之间自动切换扫描和数据采集；一级质谱扫描质荷比(m/z)范围为350-1800，采用分辨率为70000；二级质谱碎裂模式为HCD(high-energy collisional dissociation)，选择二级碎裂的电荷设置为+2价态以上，碎片离子扫描检测分辨率为17500；母离子动态排除设置为：在一半的出峰时间内(约8s)，相同母离子的碎裂不超过2次。
获得的原始质谱数据使用Maxquant和Mascot软件进行搜索鉴定，数据库使用MN047全基因组序列、UniProt antimicrobial peptide和UniProt bacteriocin。
结果：对MN047全基因组序列鉴定到的可疑抗菌肽序列进行化学合成(上海波泰生物科技有限公司)，并进行抗菌活性验证。最终确定以下3条抗菌肽：
MN047B:EGPVGLADPDGPASAPLGAP(图3)；
MN047C:GPVGLLSSPGSLPPVGGAP(图4)；
MN047D:KSIEEAMNKLLKMSA(图5)。
实施例6：抗微生物活性的测定
(1)抗微生物谱
食源性腐败菌和食源性致病菌标准菌株及其耐药菌经活化后接种于LB液体培养基中，37℃摇瓶培至对数期。使用牛津杯法测定抗菌肽对指示菌的抑菌活性。
牛津杯法步骤如下：
将5mL 2％无菌琼脂倒入无菌平皿中使其将平皿底部覆盖，待凝固后，将4个灭菌后的牛津杯用镊子等距离放到琼脂平板上。培养至对数期的指示菌，使用新鲜的LB液体培养基调节菌体浓度至108CFU/mL左右，取1mL指示菌稀释液加入到冷却至45℃左右的100mL含有0.75％琼脂的LB培养基中。混合均匀后，倒入20mL到平板中(不要倒入到牛津杯的孔里)，待完全凝固后，小心地用无菌镊子夹出牛津杯。用移液枪吸取样品100μL，分别加入到周围的3个小孔中，另一孔加入100μL pH 7.0的柠檬酸磷酸盐缓冲液作为对照。平板放到4℃下2h，使样品充分扩散后，转移到37℃下培养12h，观察抑菌现象，并测量抑菌圈直径。
上述LB液体培养基主要成分为：胰蛋白胨1％，酵母提取物0.5％，NaCl 1％，蒸馏水。
结果：如表5所示，面包乳杆菌MN047所产抗菌肽具有广谱抑菌活性。能同时杀死革兰氏阳性菌和革兰氏阴性菌，并且能杀死多重耐药菌。其对多耐药菌的杀死能力同标准菌相当，故这些耐药菌对面包乳杆菌MN047所产抗菌肽不具有耐药性。由此可见，本发明益生菌MN047所产抗菌肽具有极大的应用价值。
表5抗菌肽的抗微生物谱
AM氨苄青霉素、AMX阿莫西林、AN阿米卡星、C氯霉素、CIP环丙沙星、CF先锋霉素、CFP头孢哌酮钠、CLR克拉霉素、CRO头孢曲松、CXM头孢呋辛、FOX头孢西丁、GAT加替沙星、GM庆大霉素、K卡那霉素、LVX左氧氟沙星、MI二甲胺四环素、NA萘啶酸、NN妥布霉素、OFX氧氟沙星、OX苯甲异噁唑青霉素、P盘尼西林、PIP哌拉西林、RA利福平、S链霉素、SXT复方新诺明、TC四环素、VA万古霉素。
(2)电子显微镜观察
以标准金黄色葡萄球菌ATCC29213和标准大肠杆菌ATCC25922分别作为革兰氏阳性菌和革兰氏阴性菌的代表，利用扫描电镜来观察经抗菌肽处理后指示菌的形态变化。指示菌在LB液体培养基中培养至对数期后离心获得菌体，并重新悬浮到新鲜的LB液体培养基中，菌体密度约为109CFU/mL。加入抗菌肽至终浓度为10mg/mL后在37℃下培养2h。未加入抗菌肽的指示菌经相同处理作为空白对照。
金黄色葡萄球菌结果：如图6所示，未经处理的金黄色葡萄球菌具有正常的外部结构，细胞壁完整，细胞周围没有任何碎片，细胞壁外周轮廓完整光滑无毛刺，细胞膜紧密地附着在细胞壁内侧，细胞质物质的密度均匀。经抗菌肽处理2h后的金黄色葡萄球菌细胞形态发生明显的转变，细胞表面变得不规则、凹凸不平。一方面，细胞质物质围绕着细胞膜凝聚或细胞质物质浓缩，导致细胞内出现空白体积，细胞质物质不再均匀分布；另一方面，细胞内物质大量渗漏，甚至观察到有的细胞表面出现大的空洞。
大肠杆菌结果：如图7所示，未经处理的大肠杆菌具有完整饱满的外部结构，细胞壁厚度均匀，完整光滑，无褶皱变形，细胞膜紧密地附着在细胞壁内侧，细胞质物质的密度均匀。经抗菌肽处理2h后的大肠杆菌细胞壁出现大的凹洞，周围有小碎片，细胞壁和细胞膜严重分离，细胞质物质浓缩聚集，细胞内物质渗漏，甚至变空。
由以上之实施例可知，本发明所提供的益生菌面包乳杆菌MN047所产抗菌肽具有广谱杀菌活性(包括多重耐药菌)，具有产业上的利用价值。
核苷酸序列表电子文件
<110>西北农林科技大学
<120>面包乳杆菌、抗菌肽及其应用
<141>
<160>
<210>1
<211>20
<212>多肽
<213>MN047B
<220>
<223>
<400>1
EGPVGLADPDGPASAPLGAP
<210>2
<211>19
<212>多肽
<213>MN047C
<220>
<223>
<400>2
GPVGLLSSPGSLPPVGGAP
<210>3
<211>15
<212>多肽
<213>MN047D
<220>
<223>
<400>3
KSIEEAMNKLLKMSA
Claims (3)
1.一种抗菌肽，该抗菌肽的序列为：KSIEEAMNKLLKMSA。
2.权利要求1述的抗菌肽用于制备抗菌药物的应用。
3.权利要求1所述抗菌肽用于制备食品防腐剂的应用。
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| Purification and characterization of a novel bacteriocin produced by Lactobacillus crustorum MN047 isolated from koumiss from Xinjiang, China;Lanhua Yi等;《J. Dairy Sci.》;20161231;第99卷(第9期);摘要，7002页右栏最后1段至7003页左栏，表1，7014页右栏第2段，7003页"Purification of Bacteriocin"节 * |
| Two novel antimicrobial peptides purified from the symbiotic bacteria Xenorhabdus budapestensis NMC-10;Yao Xiao等;《Peptides》;20120403;第35卷;256页，第3.3节 * |
| 面包乳杆菌（Lactobacillus panis）C-M2 细菌素的分离纯化及特性研究;胡彦新等;《食品科学》;20170303;1-12 * |
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