CN111534452B - 具有广谱抑制多重耐药性食源性致病菌的乳酸菌菌株及其应用 - Google Patents
具有广谱抑制多重耐药性食源性致病菌的乳酸菌菌株及其应用 Download PDFInfo
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Abstract
本发明公开了具有广谱抑制多重耐药性食源性致病菌的乳酸菌菌株及其应用；所述菌株具有广谱抑制多重耐药菌和食源性致病菌活性的功能，命名为植物乳杆菌GS083、植物乳杆菌GS086、植物乳杆菌GS090和发酵乳杆菌GF110。本发明提供的乳酸菌菌株对多重耐药菌和食源性致病菌具有很好的抑菌作用，既能抑制革兰氏阴性菌，还可以抑制革兰氏阳性菌；所述菌株还具有胆盐水解酶活性、耐酸耐胆盐、低耐药性、不溶血、不存在毒力基因等特性，丰富了乳酸菌菌种资源库的同时，在食品、化妆品、药品等行业具有广阔的应用前景。
Description
技术领域
本发明属于微生物技术领域，涉及乳酸菌，具体涉及具有广谱抑制多重耐药性食源性致病菌的乳酸菌菌株及其应用。
背景技术
食品安全问题频繁爆发，其主要原因就是致病菌的生长繁殖。食源性致病菌可引起人类疾病，WHO估测全世界每年有成千上万例食源性疾病，危害人体健康。虽然，包括抗生素在内的抗菌剂拯救了无数人的生命，并且帮助人们提高了生活品质。但是，不幸的是，过度使用和误用抗菌剂导致了抗性微生物的出现和大量传播，抗菌剂抗性已经成为人类健康的重大威胁之一。因此，对于抗生素替代品和控制抗生素抗性传播的研究显得尤为迫切。
以往的研究表明乳酸菌可以作为生物防腐剂代替抗生素应用在食品、日化等行业中。例如，青贮饲料中添加植物乳杆菌(CGMCCNo.12849)既抑制了大肠杆菌等致病菌的生长，延长了饲料的贮藏期，又显著提高了其发酵品质；护肤产品中添加可抑制痤疮丙酸杆菌生长的植物乳杆菌(CGMCC No.8072)，避免了由于痤疮丙酸杆菌引起的皮肤问题。但目前所报道的乳酸菌大多是对某一种或某一个属的致病菌生长有抑制作用，同时对多种属(特别是多重耐药菌株，且包括革兰氏阴性菌和革兰氏阳性菌)有抑制效果的乳酸菌研究较少。乳酸菌作为抗生素的替代品不光是抑制了多重耐药菌的生长，还可以从一定程度上缓解抗性微生物的传播。
乳酸菌是一类厌氧或兼性厌氧的革兰氏阳性球菌或杆菌、不形成芽胞、无运动性、无细胞色素和过氧化氢酶活性，可从发酵食品、人体肠道、鸡肠道粘膜等样本中分离得到。长期的研究结果表明，大部分乳酸菌是人体必不可少的且具有重要生理功能的有益菌，其主要生理功能有：调节肠道菌群平衡、抗肿瘤、预防癌症和防止乳糖不耐症等功能。因此，探索具有抑制多重耐药菌和食源性致病菌活性的乳酸菌不但可以减少抗生素的使用和缓解抗性微生物的传播，还可以丰富乳酸菌菌种资源库，进一步的挖掘益生菌的功能，为防治多重耐药性和食源性致病菌开辟出新的途径和解决方案
发明内容
针对上述问题，本发明的目的是提供具有广谱抑制多重耐药菌和食源性致病菌活性的乳酸菌菌株及其应用。
为实现上述目的，本发明采取的技术方案为：乳酸菌菌株，具有广谱抑制多重耐药菌和食源性致病菌活性的功能，命名为植物乳杆菌(Lactobacillusplantarum)GS083、植物乳杆菌(Lactobacillus plantarum)GS086、植物乳杆菌(Lactobacillus plantarum)GS090和发酵乳杆菌(Lactobacillus fermentum)GF110，于2020年3月9日保藏于广东省微生物菌种保藏中心，地址：中国广州市先烈中路100号省微生物所，保藏编号分别为GDMCC 60972、GDMCC 60973、GDMCC 60974、GDMCC 60975。
本发明经过筛选得到四种对多种多重耐药菌和食源性致病菌(包括革兰氏阴性菌和革兰氏阳性菌)的生长均表现出强抑制作用的乳酸菌菌株，命名为植物乳杆菌(Lactobacillusplantarum)GS083、植物乳杆菌(Lactobacillus plantarum)GS086、植物乳杆菌(Lactobacillus plantarum)GS090和发酵乳杆菌(Lactobacillus fermentum)GF110。经试验证明，四株乳酸菌菌株均具有较强抑制多重耐药菌和食源性致病菌生长的活性、胆盐水解酶活性和耐酸耐胆盐能力，同时可在模拟胃肠道环境中可存活，并且耐药性较低，无溶血性，不含有毒力基因；通过生理生化、API试剂条和16S rDNA序列比对分析，将菌株分别鉴定为植物乳杆菌和发酵乳杆菌。
本发明还提供了所述乳酸菌的培养方法，包括以下步骤：将适量的所述乳酸菌接种于培养基中，37℃培养。
作为本发明的优选实施方式，所述培养基为MRS培养基。
本发明还要求保护所述的乳酸菌在制备微生物制剂中的应用。
作为本发明的优选实施方式，所述微生物制剂具有抑菌作用。
本发明还要求保护一种微生物制剂，所述微生物菌剂包括所述的乳酸菌中的至少一种。
作为本发明的优选实施方式，所述微生物制剂还包括辅料和/或载体。
本发明还要求保护所述微生物制剂在食品、药物或化妆品中的应用。
作为本发明的优选实施方式，所述微生物制剂作为抑菌剂在食品、药物或化妆品领域中应用。
作为本发明的优选实施方式，所述微生物制剂还可以为食品发酵剂。
本发明相对于现有技术，具有以下的优点及效果：
本发明提供的乳酸菌菌株对多重耐药菌和食源性致病菌具有很好的抑菌作用，既能抑制革兰氏阴性菌，还可以抑制革兰氏阳性菌；所述菌株还具有胆盐水解酶活性、耐酸耐胆盐、低耐药性、不溶血、不存在毒力基因等特性，丰富了乳酸菌菌种资源库的同时，在食品、化妆品、药品等领域具有广阔的应用前景。
附图说明
图1为本发明各乳酸菌的16SrDNA序列扩增片段电泳结果。
图2为本发明各乳酸菌毒力基因检测电泳结果。
具体实施方式
为更好的说明本发明的目的、技术方案和优点，下面将结合附图和具体实施例对本发明作进一步说明。
实施例1乳酸菌的分离、筛选
一、乳酸菌初步筛选
采集农户家自制泡菜作为样品，将各样品分别浸没于100mL MRS肉汤培养基中，37℃富集培养24h，10倍梯度稀释后，分别接种于MRS固体培养基中，37℃厌氧培养48h，挑取平板上的典型菌落，划线分离得到纯菌落，并进行镜检、革兰氏染色和过氧化氢酶活性试验。纯化后的菌种置于-80℃冰箱保存。
共筛选得到63株疑似菌株，其中革兰氏染色结果为阳性，过氧化氢酶活性试验结果为阴性。结果如表1所示(“+”为阳性，“-”为阴性)。
表1乳酸菌的筛选结果
二、耐酸耐胆盐乳酸菌筛选
①耐酸测试：将活化好的乳酸菌制成菌悬液，取1mL菌悬液加入9mLpH＝2.0的MRS液体培养基中，37℃孵育3h。通过测定600nm处的吸光度来监测细菌的生长情况。
②耐胆盐测试：将活化好的乳酸菌菌悬液按3％的接种量接种至含0.3％胆盐的MRS液体培养基中，37℃培养3h后，通过记录600nm处的吸光度来观察细菌的生长。
测试结果如表2(“+”为耐受，“-”为不耐受)。
表2乳酸菌的耐酸耐胆盐能力
由表2可知，63株分离株中有20株菌株在pH＝2.0和胆盐浓度为0.3％的条件下均可以存活，具有耐酸耐胆盐能力。
三、抑菌乳酸菌筛选
(1)指示菌的制备
6株多重耐药菌和8株食源性致病菌(如表3)的单菌落分别接种于营养肉汤液体培养基中，37℃，200r/min培养8h，调节浓度至1×106CFU/mL，4℃保存，备用。
表3多重耐药菌和食源性致病菌指示菌株
注：G+：革兰氏阳性菌；G-：革兰氏阴性菌；抗生素：氨苄青霉素(AMP)；阿莫西林(AMC)；头孢他啶(CAZ)；头孢噻肟(CTX)；头孢菌素(KF)；头孢西丁(FOX)；头孢替坦(CTT)；环丙沙星(CIP)；萘啶酸(NA)；诺氟沙星(NOR)；阿米卡星(AK)；庆大霉素(CN)；氯霉素(C)；复方新诺明(SXT)；链霉素(S)；卡那霉素(K)；四环素(TE)；红霉素(E)；克林霉素(DA)；利福平(RD)；强力霉素(DO)；替考拉宁(TEC)；青霉素(P)；奎奴普丁(QD)；呋喃妥因(FD)；恩诺沙星(ENR)。
(2)乳酸菌(上清液)的制备
将筛选出的20株耐酸耐胆盐乳酸菌菌株的单菌落接种于50mLMRS液体培养基中，37℃，静置培养24h，10000rpm，4℃，10min离心乳酸菌培养液，取上清通过0.22μm无菌滤膜，获得的滤液为抑菌实验备用，4℃保存。
(3)(上清液)抑菌活性筛选
取步骤(1)制备获得的指示菌菌液200μL均匀涂布于营养琼脂体培养基上，用镊子将无菌牛津杯放置于平板上，每只牛津杯加入200μL步骤(2)制备的上清液。将平皿置于4℃冰箱扩散4h，然后于37℃培养24h后，测量抑菌圈直径，结果见表4、5(“+”6-10mm，“++”10-14mm，“+++”14-18mm，“++++”>18mm。)。
表4乳酸菌发酵上清液对多重耐药菌指示菌的体外抑菌试验
表5乳酸菌发酵上清液对食源性致病菌指示菌的体外抑菌试验
| 菌株 | 大肠 | 沙门 | 阪崎 | 铜绿 | 单增 | 蜡样 | 枯草 | 金葡 |
| HZ009 | - | + | - | + | + | + | + | - |
| HB051 | - | - | + | + | +++ | + | + | - |
| HN057 | - | + | - | - | - | - | + | - |
| HN058 | + | - | + | + | +++ | ++ | + | - |
| HN069 | + | + | ++ | + | - | - | ++ | + |
| HN070 | - | - | - | - | + | - | - | - |
| HN071 | - | + | + | + | ++ | + | ++ | - |
| HN072 | + | ++ | ++ | + | +++ | ++ | +++ | + |
| GS083 | + | +++ | ++ | ++ | ++++ | ++ | +++ | + |
| GS086 | ++ | +++ | +++ | ++ | ++++ | +++ | +++ | + |
| GS089 | + | +++ | +++ | +++ | ++++ | +++ | +++ | + |
| GS090 | + | +++ | +++ | +++ | ++++ | +++ | +++ | + |
| XP097 | ++ | ++ | +++ | + | ++++ | ++ | ++ | + |
| GF106 | - | + | + | + | + | + | + | - |
| GF107 | - | - | + | + | ++ | + | + | - |
| GF108 | + | + | + | + | ++ | + | ++ | - |
| GF109 | + | - | ++ | + | ++ | ++ | - | + |
| GF110 | + | ++ | +++ | ++ | ++++ | ++ | +++ | + |
| GF111 | - | - | + | - | + | + | + | - |
| XZ123 | + | ++ | ++ | ++ | +++ | ++ | +++ | + |
由表4、5可知，菌株GS083、GS086、GS090和GF110对6株多重耐药菌和8株食源性致病菌均有一定的抑制能力；其中，对单增李斯特菌抑制效果最明显，抑菌圈直径均超过18mm。
将筛选得到的乳酸菌分别为于2020年3月9日保藏于广东省微生物菌种保藏中心，地址：中国广州市先烈中路100号省微生物所；
植物乳杆菌(Lactobacillus plantarum)GS083，保藏编号为GDMCC 60972；
植物乳杆菌(Lactobacillus plantarum)GS086，保藏编号为GDMCC 60973；
植物乳杆菌(Lactobacillus plantarum)GS090，保藏编号为GDMCC 60974；
发酵乳杆菌(Lactobacillus fermentum)GF110，保藏编号为GDMCC 60975。
实施例2乳酸菌的鉴定
(1)API鉴定
利用API50CHL系列鉴定试剂条对菌株GS083、GS086、GS090和GF110进行鉴定，鉴定结果见表6(“+”表示阳性，“-”表示阴性，“？”表示可疑)。
表6菌株的API50CHL系列鉴定结果
由表6及可知，本发明菌株GS083、GS086、GS090均为植物乳杆菌，鉴定率为99.9％；菌株GF110为发酵乳杆菌，鉴定率为99.9％
(2)菌株16S rDNA测序鉴定
使用基因组DNA提取试剂盒获得菌株GS083、GS086、GS090和GS090的DNA作为PCR扩增的模板，采用通用引物(正向引物为5’-agagtttgatcctggctcag-3’，反向引物为5’-ctacggctaccttgttacga-3’)PCR扩增各菌的16SrDNA序列。电泳检测扩增产物后(结果见图1)，送至深圳华大基因科技有限公司进行测序，测序结果如SEQ ID NO.1～4所示。将测序得到的序列与GenBank数据库中的序列进行同源性比对分析，结果见表7。
表7 16S rDNA比对结果
| 菌株 | 亲缘关系最近的菌株 | 同源性 |
| GS083 | 植物乳杆菌(Lactobacillus plantarum JCM 1149) | 99.93％ |
| GS086 | 植物乳杆菌(Lactobacillus plantarum CIP 103151) | 99.93％ |
| GS090 | 植物乳杆菌(Lactobacillus plantarum JCM 1149) | 99.93％ |
| GF110 | 发酵乳杆菌(Lactobacillus fermentum NBRC 15885) | 99.93％ |
实施例3乳酸菌的益生特性
(1)模拟胃液和胰液存活能力测试
模拟胃液：将0.35％(w/v)胃蛋白酶溶于磷酸缓冲液(PBS)中，用1M HCl酸化至pH＝2.0制得；模拟胰液：含有1.1％(w/v)NaHCO3、0.1％(w/v)胰蛋白酶的PBS溶液，用1M NaOH将其pH值调整为8.0制得。
过夜培养的菌液经离心后，收集菌体，用PBS洗涤2次后重悬于等量的人工胃液中，37℃培养，分别在0h和3h用平板计数法测定活菌数，计算其存活率(％)。收集的经人工胃液处理后的菌体悬浮于相同体积的模拟胰液中，37℃孵育24h，分别在0h和24h用平板计数法测定活菌数，计算其存活率(％)，计算方法如下，结果如表8。
表8乳酸菌在模拟人工胃/胰液中存活情况(Log10 CFU/mL)
| 菌株 | 0h人工胃液 | 3h人工胃液 | 24h人工胰液 | 存活率％ |
| GS083 | 9.9 | 7.5 | 6.8 | 68.7 |
| GS086 | 10.1 | 8.5 | 6.9 | 68.3 |
| GS090 | 9.9 | 8.2 | 7.3 | 73.7 |
| GF110 | 9.8 | 7.4 | 6.0 | 61.2 |
由表8可知，植物乳杆菌GS083、植物乳杆菌GS086、植物乳杆菌GS090和发酵乳杆菌GF110经过人工胃液、人工胰液处理后存活率分别为68.7％、68.3％、73.7％和61.2％，并且活菌数均超过106CFU/mL。
(2)胆盐水解酶活性测定
将植物乳杆菌GS083、植物乳杆菌GS086、植物乳杆菌GS090和发酵乳杆菌GF110的菌悬液点种在含0.5％牛去氧胆酸的MRS琼脂上37℃培养48h，检测菌株的胆盐水解酶活性。菌落周围出现白色沉淀物被标记为胆汁盐水解酶阳性，反之，为阴性。结果表明，菌落周围出现白色沉淀物。所以，植物乳杆菌GS083、植物乳杆菌GS086、植物乳杆菌GS090和发酵乳杆菌GF110具有胆盐水解酶活性。
实施例4乳酸菌的安全特性
(1)溶血分析分别取5μL植物乳杆菌GS083、植物乳杆菌GS086、植物乳杆菌GS090和发酵乳杆菌GF110的菌悬液点种于血平板上，37℃培养。48h后，观察菌落周围如出现透明环状标志为β-溶血，棕色或绿色为α-溶血，无现象为γ-溶血。
结果表明，各菌落周围无现象，即为γ-溶血。所以四株乳酸菌(植物乳杆菌GS083、植物乳杆菌GS086、植物乳杆菌GS090和发酵乳杆菌GF110)均不具有溶血活性。
(2)抗生素敏感性试验
使用微量肉汤稀释法检测四株乳酸菌对抗生素的敏感性。九种抗生素(氯霉素、红霉素、利福平、四环素、庆大霉素、克林霉素、亚胺培南、氨苄西林和万古霉素)溶解在相应的稀释剂中(不同浓度：0.5-1024μg/mL)。所有含抗生素的菌悬液(McFarland标准0.5)在37℃下培养48h。最小抑菌浓度(MIC)确定为抗生素完全抑制细菌生长的最低浓度。根据EFSA报告的断点来定义敏感株和耐药株，结果见表9(注：R为耐药，S为敏感)。
表9乳酸菌抗生素敏感性
| 编号 | 抗生素 | GS083 | GS086 | GS090 | GF110 |
| 1 | 氯霉素Chloramphenicol | S | S | S | S |
| 2 | 红霉素Erythromycin | S | S | S | S |
| 3 | 利福平Rifampicin | S | S | S | S |
| 4 | 四环素Tetracycline | S | S | S | S |
| 5 | 庆大霉素Gentamycin | S | S | S | S |
| 6 | 克林霉素Clindamycin | S | S | S | S |
| 7 | 亚胺培南Imipenem | S | S | S | S |
| 8 | 氨苄青霉素Ampicillin | S | S | S | S |
| 9 | 万古霉素Vancomycin | R | R | R | R |
由9可知，植物乳杆菌GS083、植物乳杆菌GS086、植物乳杆菌GS090和发酵乳杆菌GF110对氯霉素、红霉素、利福平、四环素、庆大霉素、克林霉素、亚胺培南、氨苄西林具有敏感性，仅对万古霉素表现出耐受。
(3)毒力因子检测
毒力基因主要包括asa1、cylLS、gelE、efaAfs和ace。提取四株菌株的基因组DNA，对上述五种常见的乳酸菌相关毒力基因进行PCR扩增(引物如表10)，所得产品用1％琼脂糖凝胶电泳检测。阳性对照：鼠乳杆菌(Lactobacillus murinus)GGF1107-4、粪肠球菌(Enterococcus faecalis)1004-9。
表10毒力因子基因PCR扩增引物
如图2所示(注：泳道1:阳性对照样品；泳道2:菌株GS083；泳道3：菌株GS086；泳道4：菌株GS090；泳道5:菌株GF110；泳道M：DNA marker。)，结果显示并未出现和预期片段大小相同的条带，即植物乳杆菌GS083、植物乳杆菌GS086、植物乳杆菌GS090和发酵乳杆菌GF110中均不含有上述五种毒力基因。
上述特性试验结果表明四株乳酸菌(植物乳杆菌GS083、植物乳杆菌GS086、植物乳杆菌GS090和发酵乳杆菌GF110)是可耐受胃肠道环境且安全的。
最后应当说明的是，以上实施例仅用以说明本发明的技术方案，而非对本发明保护范围的限制，尽管参照较佳实施例对本发明作了详细地说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的实质和范围。
SEQUENCE LISTING
<110> 广东省微生物研究所(广东省微生物分析检测中心)、广东环凯生物科技有限公司
<120> 具有广谱抑制多重耐药性食源性致病菌的乳酸菌菌株及其应用
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 1419
<212> DNA
<213> Lactobacillus plantarum
<400> 1
aaggttaccc caccgacttt gggtgttaca aactctcatg gcgtgacggg cggtgtgtac 60
aaggcccggg aacgtattca ccgcggcatg ctgatccgcg attactagcg attccgactt 120
catgtaggcg agttgcagcc tacaatccga actgagaatg gctttaagag attagcttac 180
tctcgcgagt tcgcaactcg ttgtaccatc cattgtagca cgtgtgtagc ccaggtcata 240
aggggcatga tgatttgacg tcatccccac cttcctccgg tttgtcaccg gcagtctcac 300
cagagtgccc aacttaatgc tggcaactga taataagggt tgcgctcgtt gcgggactta 360
acccaacatc tcacgacacg agctgacgac aaccatgcac cacctgtatc catgtccccg 420
aagggaacgt ctaatctctt agatttgcat agtatgtcaa gacctggtaa ggttcttcgc 480
gtagcttcga attaaaccac atgctccacc gcttgtgcgg gcccccgtca attcctttga 540
gtttcagcct tgcggccgta ctccccaggc ggaatgctta atgcgttagc tgcagcactg 600
aagggcggaa accctccaac acttagcatt catcgtttac ggtatggact accagggtat 660
ctaatcctgt ttgctaccca tactttcgag cctcagcgtc agttacagac cagacagccg 720
ccttcgccac tggtgttctt ccatatatct acgcatttca ccgctacaca tggagttcca 780
ctgtcctctt ctgcactcaa gtttcccagt ttccgatgca cttcttcggt tgagccgaag 840
gctttcacat cagacttaaa aaaccgcctg cgctcgcttt acgcccaata aatccggaca 900
acgcttgcca cctacgtatt accgcggctg ctggcacgta gttagccgtg gctttctggt 960
taaataccgt caatacctga acagttactc tcagatatgt tcttctttaa caacagagtt 1020
ttacgagccg aaacccttct tcactcacgc ggcgttgctc catcagactt tcgtccattg 1080
tggaagattc cctactgctg cctcccgtag gagtttgggc cgtgtctcag tcccaatgtg 1140
gccgattacc ctctcaggtc ggctacgtat cattgccatg gtgagccgtt accccaccat 1200
ctagctaata cgccgcggga ccatccaaaa gtgatagccg aagccatctt tcaagctcgg 1260
accatgcggt ccaagttgtt atgcggtatt agcatctgtt tccaggtgtt atcccccgct 1320
tctgggcagg tttcccacgt gttactcacc agttcgccac tcactcaaat gtaaatcatg 1380
atgcaagcac caatcaatac cagagttcgt tcgacttgc 1419
<210> 2
<211> 1438
<212> DNA
<213> Lactobacillus plantarum
<400> 2
ttaggcggct ggttcctaaa aggttacccc accgactttg ggtgttacaa actctcatgg 60
tgtgacgggc ggtgtgtaca aggcccggga acgtattcac cgcggcatgc tgatccgcga 120
ttactagcga ttccgacttc atgtaggcga gttgcagcct acaatccgaa ctgagaatgg 180
ctttaagaga ttagcttact ctcgcgagtt cgcaactcgt tgtaccatcc attgtagcac 240
gtgtgtagcc caggtcataa ggggcatgat gatttgacgt catccccacc ttcctccggt 300
ttgtcaccgg cagtctcacc agagtgccca acttaatgct ggcaactgat aataagggtt 360
gcgctcgttg cgggacttaa cccaacatct cacgacacga gctgacgaca accatgcacc 420
acctgtatcc atgtccccga agggaacgtc taatctctta gatttgcata gtatgtcaag 480
acctggtaag gttcttcgcg tagcttcgaa ttaaaccaca tgctccaccg cttgtgcggg 540
cccccgtcaa ttcctttgag tttcagcctt gcggccgtac tccccaggcg gaatgcttaa 600
tgcgttagct gcagcactga agggcggaaa ccctccaaca cttagcattc atcgtttacg 660
gtatggacta ccagggtatc taatcctgtt tgctacccat actttcgagc ctcagcgtca 720
gttacagacc agacagccgc cttcgccact ggtgttcttc catatatcta cgcatttcac 780
cgctacacat ggagttccac tgtcctcttc tgcactcaag tttcccagtt tccgatgcac 840
ttcttcggtt gagccgaagg ctttcacatc agacttaaaa aaccgcctgc gctcgcttta 900
cgcccaataa atccggacaa cgcttgccac ctacgtatta ccgcggctgc tggcacgtag 960
ttagccgtgg ctttctggtt aaataccgtc aatacctgaa cagttactct cagatatgtt 1020
cttctttaac aacagagttt tacgagccga aacccttctt cactcacgcg gcgttgctcc 1080
atcagacttt cgtccattgt ggaagattcc ctactgctgc ctcccgtagg agtttgggcc 1140
gtgtctcagt cccaatgtgg ccgattaccc tctcaggtcg gctacgtatc attgccatgg 1200
tgagccgtta cctcaccatc tagctaatac gccgcgggac catccaaaag tgatagccga 1260
agccatcttt caaactcgga ccatgcggtc caagttgtta tgcggtatta gcatctgttt 1320
ccaggtgtta tcccccgctt ctgggcaggt ttcccacgtg ttactcacca gttcgccact 1380
cactcaaatg taaatcatga tgcaagcacc aatcaatacc agagttcgtt cgacttgc 1438
<210> 3
<211> 1431
<212> DNA
<213> Lactobacillus plantarum
<400> 3
gctggttcct aaaaggttac cccaccgact ttgggtgtta caaactctca tggtgtgacg 60
ggcggtgtgt acaaggcccg ggaacgtatt caccgcggca tgctgatccg cgattactag 120
cgattccgac ttcatgtagg cgagttgcag cctacaatcc gaactgagaa tggctttaag 180
agattagctt actctcgcga gttcgcaact cgttgtacca tccattgtag cacgtgtgta 240
gcccaggtca taaggggcat gatgatttga cgtcatcccc accttcctcc ggtttgtcac 300
cggcagtctc accagagtgc ccaacttaat gctggcaact gataataagg gttgcgctcg 360
ttgcgggact taacccaaca tctcacgaca cgagctgacg acaaccatgc accacctgta 420
tccatgtccc cgaagggaac gtctaatctc ttagatttgc atagtatgtc aagacctggt 480
aaggttcttc gcgtagcttc gaattaaacc acatgctcca ccgcttgtgc gggcccccgt 540
caattccttt gagtttcagc cttgcggccg tactccccag gcggaatgct taatgcgtta 600
gctgcagcac tgaagggcgg aaaccctcca acacttagca ttcatcgttt acggtatgga 660
ctaccagggt atctaatcct gtttgctacc catactttcg agcctcagcg tcagttacag 720
accagacagc cgccttcgcc actggtgttc ttccatatat ctacgcattt caccgctaca 780
catggagttc cactgtcctc ttctgcactc aagtttccca gtttccgatg cacttcttcg 840
gttgagccga aggctttcac atcagactta aaaaaccgcc tgcgctcgct ttacgcccaa 900
taaatccgga caacgcttgc cacctacgta ttaccgcggc tgctggcacg tagttagccg 960
tggctttctg gttaaatacc gtcaatacct gaacagttac tctcagatat gttcttcttt 1020
aacaacagag ttttacgagc cgaaaccctt cttcactcac gcggcgttgc tccatcagac 1080
tttcgtccat tgtggaagat tccctactgc tgcctcccgt aggagtttgg gccgtgtctc 1140
agtcccaatg tggccgatta ccctctcagg tcggctacgt atcattgcca tggtgagccg 1200
ttacctcacc atctagctaa tacgccgcgg gaccatccaa aagtgatagc cgaagccatc 1260
tttcaaactc ggaccatgcg gtccaagttg ttatgcggta ttagcatctg tttccaggtg 1320
ttatcccccg cttctgggca ggtttcccac gtgttactca ccagttcgcc actcactcaa 1380
atgtaaatca tgatgcaagc accaatcaat accagagttc gttcgacttg c 1431
<210> 4
<211> 1440
<212> DNA
<213> Lactobacillus fermentum
<400> 4
cggctggctc ctaaaaggtt accccaccga ctttgggtgt tacaaactct catggtgtga 60
cgggcggtgt gtacaaggcc cgggaacgta ttcaccgcgg catgctgatc cgcgattact 120
agcgattccg acttcgtgca ggcgagttgc agcctgcagt ccgaactgag aacggtttta 180
agagatttgc ttgccctcgc gagttcgcga ctcgttgtac cgtccattgt agcacgtgtg 240
tagcccaggt cataaggggc atgatgatct gacgtcgtcc ccaccttcct ccggtttgtc 300
accggcagtc tcactagagt gcccaactta atgctggcaa ctagtaacaa gggttgcgct 360
cgttgcggga cttaacccaa catctcacga cacgagctga cgacgaccat gcaccacctg 420
tcattgcgtt cccgaaggaa acgccctatc tctagggttg gcgcaagatg tcaagacctg 480
gtaaggttct tcgcgtagct tcgaattaaa ccacatgctc caccgcttgt gcgggccccc 540
gtcaattcct ttgagtttca accttgcggt cgtactcccc aggcggagtg cttaatgcgt 600
tagctccggc actgaagggc ggaaaccctc caacacctag cactcatcgt ttacggcatg 660
gactaccagg gtatctaatc ctgttcgcta cccatgcttt cgagtctcag cgtcagttgc 720
agaccaggta gccgccttcg ccactggtgt tcttccatat atctacgcat tccaccgcta 780
cacatggagt tccactaccc tcttctgcac tcaagttatc cagtttccga tgcacttctc 840
cggttaagcc gaaggctttc acatcagact tagaaaaccg cctgcactct ctttacgccc 900
aataaatccg gataacgctt gccacctacg tattaccgcg gctgctggca cgtagttagc 960
cgtgactttc tggttaaata ccgtcaacgt atgaacagtt actctcatac gtgttcttct 1020
ttaacaacag agctttacga gccgaaaccc ttcttcactc acgcggtgtt gctccatcag 1080
gcttgcgccc attgtggaag attccctact gctgcctccc gtaggagtat gggccgtgtc 1140
tcagtcccat tgtggccgat cagtctctca actcggctat gcatcatcgc cttggtaggc 1200
cattacccca ccaacaagct aatgcaccgc aggtccatcc agaagtgata gcgagaagcc 1260
atcttttaag cgttgttcat gcgaacaacg ttgttatgcg gtattagcat ctgtttccaa 1320
atgttgtccc ccgcttctgg gcaggttacc tacgtgttac tcacccgtcc gccactcgtt 1380
ggcgaccaaa atcaatcagg tgcaagcacc atcaatcaat tgggccaacg cgttcgactt 1440
Claims (10)
1.乳酸菌菌株，其特征在于，所述菌株具有广谱抑制多重耐药菌和食源性致病菌活性的功能，所述菌株为植物乳杆菌GS083、GS086或GS090，或发酵乳杆菌GF110；分别为于2020年3月9日保藏于广东省微生物菌种保藏中心，地址：中国广州市先烈中路100号省微生物所，保藏编号分别为GDMCC 60972、GDMCC 60973、GDMCC 60974、GDMCC 60975。
2.如权利要求1所述乳酸菌的培养方法，其特征在于，将适量的所述乳酸菌接种于培养基中，37℃培养。
3.如权利要求2所述的培养方法，其特征在于，所述培养基为MRS培养基。
4.如权利要求1所述的乳酸菌在制备微生物制剂中的应用。
5.如权利要求4所述的应用，其特征在于，所述微生物制剂具有抑菌作用。
6.一种微生物制剂，其特征在于，包括如权利要求1所述乳酸菌中的至少一种。
7.如权利要求6所述的微生物制剂，其特征在于，还包括辅料和/或载体。
8.如权利要求6或7所述微生物制剂在食品、药物或化妆品领域中的应用。
9.如权利要求8所述的应用，其特征在于，所述微生物制剂作为抑菌剂在食品、药物或化妆品领域中应用。
10.如权利要求8所述的应用，其特征在于，所述微生物制剂为食品发酵剂。
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| CN114231462B (zh) * | 2021-12-29 | 2023-07-18 | 广东省科学院微生物研究所(广东省微生物分析检测中心) | 一种抑制耐药型食源性致病菌的活性菌株及应用 |
| CN115093990B (zh) * | 2022-06-01 | 2023-08-25 | 江南大学 | 一株可广谱抑制食品腐败菌的乳酸菌及其应用 |
| CN115851491A (zh) * | 2022-08-31 | 2023-03-28 | 西北农林科技大学 | 植物乳植杆菌及其应用 |
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