CN108220212B - 一株广谱抑菌植物乳杆菌 - Google Patents
一株广谱抑菌植物乳杆菌 Download PDFInfo
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Abstract
一株广谱抑菌植物乳杆菌，该植物乳杆菌(Lactobacillus plantarum)MG‑1的保藏编号为CCTCC NO：M2018029。本发明菌株MG‑1及其发酵液对细菌和霉菌具有明显抑制作用，能有效抑制葡萄灰霉病菌、葡萄白腐病菌、白菜黑斑病菌、辣椒炭疽病菌、猕猴桃间座壳病菌、辣椒白娟病菌、马铃薯白娟病菌、大肠杆菌和金黄色葡萄球菌，具有广谱抑菌性。
Description
技术领域
本发明涉及一株广谱、抑菌活性较强的乳酸菌，具体涉及一株植物乳杆菌。
背景技术
乳酸菌作为益生菌，能产生一些可抑制其他微生物生长的代谢产物，如细菌素、多肽、酸性产物(如乙酸、乳酸及其他有机酸)等，该些产物对腐败微生物和致病微生物具有抑制作用。截至目前，已发现的乳酸菌细菌素有300多种，分别由乳杆菌、乳球菌、片球菌、明串球菌、肠球菌等属菌株产生。食品工业中广泛使用的化学防腐剂严重影响人体健康，而乳酸菌细菌素、多肽等代谢产物具有无毒、安全、稳定等特点，因此被用作天然食品防腐剂以控制某些腐败菌和病原菌的生长。
但生物防腐的相关研究主要是针对抗细菌和黄曲霉菌，而乳酸菌对果蔬采后其他真菌抑制作用的研究相对较少。目前研究发现只有少数乳酸菌能产生一系列抗真菌物质，而大部分乳酸菌没有此特性。Baek等研究发现Leuconostoc citreum和Weisella confuse可以有效抑制米糕中Cladosporium sp YS1，Neurospora sp YS3和Penicilliumcrustosum YS2的生长，经试验得出抗真菌物质为乳酸和乙酸；Cabo等学者对56株荷兰乳品研究所的乳酸菌及抑制各种青霉菌的效果进行了研究，证实大部分乳杆菌对变色青霉(Penicillium discolor)和杂色曲霉(A.versicolor)都表现出了不同程度的抑制效果，但对普通青霉(P.commune)和娄地青霉(P.roqueforti)的生长，抑制效果相对不明显，并且抑制作用只发生在pH 5.5以下。因此利用广谱乳酸菌和其代谢产物作为安全的生物防腐剂来代替化学防腐剂防止果蔬采后病菌，进行果蔬采后防腐保鲜很有必要。
发明内容
本发明的目的是，针对上述现有技术的不足，提供一种广谱、抑菌活性较强的植物乳杆菌(Lactobacillus plantarum MG-1)MG-1，对细菌和霉菌具有明显的抑制作用。
上述提及的植物乳杆菌(Lactobacillus plantarum MG-1)MG-1，于2018年1月12日保藏于中国典型培养物保藏中心，保藏编号为CCTCC NO：M2018029，保藏地址为中国武汉武汉大学。
本发明菌株MG-1是利用牛津杯双层平板法从发酵酸白菜中筛选分离得到。该菌株MG-1在MRS固体平板上培养24h呈乳白色、圆形、表面光滑、湿润、边缘整齐、隆起菌落。显微镜下观察，菌株菌体圆短直杆状、成对，参见图1。液体培养浑浊度大，革兰氏染色阳性，无芽孢，荚膜和鞭毛。
本发明菌株MG-1的常规生理生化实验鉴定结果如下表1所示(表中，+呈阳性，-呈阴性)。
表1本发明菌株MG-1常规生理生化实验鉴定结果
根据细菌基因组提取试剂盒的说明提取本菌株基因组DNA，以基因组DNA为模板，PCR扩增16S rDNA片段，PCR产物纯化后委托上海美吉生物医药科技有限公司测序。所得16SrDNA序列在Gene Bank数据库中进行Blast比对，本菌株MG-1与Lactobacillus plantarumMF966541.1有99％的同源性。结合形态学特征及生理生化实验结果，鉴定菌株MG-1为植物乳杆菌(Lactobacillus plantarum)，命名为植物乳杆菌(Lactobacillus plantarum)MG-1。
将本发明菌株MG-1接种1-3％至MRS液体培养基中，36-38℃静置培养16-20h，能制得该菌株的发酵液。经抑菌谱测定，本菌株MG-1及其发酵液对细菌和霉菌具有明显抑制作用，能有效抑制葡萄灰霉病菌、葡萄白腐病菌、白菜黑斑病菌、辣椒炭疽病菌、猕猴桃间座壳病菌、辣椒白娟病菌、马铃薯白娟病菌、大肠杆菌和金黄色葡萄球菌，具有广谱抑菌性。
附图说明
图1是本发明菌株MG-1菌体的显微(×100)形态图。
具体实施方式
下面实施例中提及的培养基如无特别说明皆为常规培养基。
实施例1本菌株MG-1的分离筛选
取长沙市自制的自然发酵白菜10g，用无菌生理盐水梯度稀释与含碳酸钙的50℃MRS琼脂培养基混合倒平板，38℃恒温培养，48h后挑选溶钙圈大单菌落反复纯化。
采用牛津杯双层平板法：1.5％琼脂10mL为培养基下层，上层含葡萄灰霉病菌孢子的半固体PDA培养基；上层均匀放置2个牛津杯(直径1cm)，晾干，取出牛津杯，每孔加入60μL无菌发酵上清液(制备同实施例2中所述，采用的菌株为前述纯化得到的菌株)，放置2h，37℃恒温培养16h，测量抑菌圈直径，取抑菌直径最大的乳酸菌菌株斜面保存。
实施例2抑菌谱测定
发酵上清液的制备：将保存的菌株活化后接种2％到MRS液体培养基，37℃静置培养18h，12000r/min离心20min，上清液经0.22μm滤膜过滤除菌，4℃保存。
方法：按照牛津杯双层平板方法，制作含不同指示菌的指示平板，上层半固体培养基中细菌终浓度为106CFU/mL，真菌、酵母菌细胞或孢子10 4个/mL，取菌株发酵上清液60μL加入孔内，于各指示菌最适生长温度条件下培养，以无菌水为阴性对照，以恶霉灵(使用时稀释1000倍作为原液)为阳性对照，待阴性对照长到孔径边缘时，测量抑菌圈直径，每组3次重复。
指示菌为：黄瓜霜霉病菌(Pseudoperonosporacubensis)、白菜黑斑病菌(Alternaria brassicae)、辣椒炭疽病菌[Colletotrichumcapsici(syd.)Butl]、辣椒白娟病菌(Sclerotium rolfsiiSacc)、辣椒疫病菌(Phytophthora capsici)、葡萄白腐病菌(Coniothyriumdiplodiella)、葡萄灰霉病菌(Botrytis cinerea)、猕猴桃座腔病菌(Actinidiachinensis)、番茄早疫病菌(Alternaria sonali)、猕猴桃间座壳病菌(Diaporthaceae)、柑橘青霉病菌(Penicillium Citrus)、马铃薯白娟病菌(SclerotiumrolfsiiSacc)、金黄色葡萄球菌(Staphylococcus aureus)、大肠杆菌(Escherichiacoli)、假丝酵母菌(Candida albicans)、酿酒酵母菌(Saccharomyces cerevisiae)，均由湖南农业大学实验室提供。
结果见下表2，可知本菌株MG-1的发酵上清液对葡萄灰霉病菌、葡萄白腐病菌、白菜黑斑病菌、辣椒炭疽病菌、猕猴桃间座壳病菌、辣椒白娟病菌、马铃薯白娟病菌、大肠杆菌、金黄色葡萄球菌和大部分霉菌有抑制作用，但对酵母无抑制效果，本菌株MG-1的发酵上清液具有良好的广谱抑菌效果。
表2本菌株MG-1发酵上清液抑菌谱
| 指示菌 | 抑菌效果 | 指示菌 | 抑菌效果 |
| 黄瓜霜霉病菌 | + | 葡萄灰霉病菌 | +++ |
| 白菜黑斑病菌 | +++ | 猕猴桃座腔病菌 | – |
| 辣椒炭疽病菌 | ++ | 猕猴桃间座壳病菌 | ++ |
| 辣椒白娟病菌 | ++ | 番茄早疫病菌 | + |
| 辣椒疫病菌 | + | 柑橘青霉病菌 | – |
| 葡萄白腐病菌 | +++ | 马铃薯白娟病菌 | ++ |
| 金黄色葡萄球菌 | ++ | 大肠杆菌 | ++ |
| 酿酒酵母菌 | – | 假丝酵母菌 | – |
注：“－”表示无抑菌活性；“+”表示抑菌圈直径0-5mm；“++”表示抑菌圈直径5-10mm；“+++”表示抑菌圈直径>10mm。
Claims (5)
1.植物乳杆菌(Lactobacillus plantarum)MG-1，保藏号为CCTCC NO：M2018029。
2.权利要求1所述的植物乳杆菌MG-1在抑制致病菌中的应用，其中，致病菌为葡萄灰霉病菌、葡萄白腐病菌、白菜黑斑病菌、辣椒炭疽病菌、猕猴桃间座壳病菌、辣椒白娟病菌和马铃薯白娟病菌。
3.权利要求1所述植物乳杆菌MG-1发酵制得的发酵液，其中，发酵条件为：接种量1-3％、MRS液体培养基、36-38℃静置培养16-20h。
4.如权利要求3所述的发酵液，其中，发酵条件为：接种量2％、MRS液体培养基、37℃静置培养18h。
5.权利要求3或4所述的发酵液在抑制致病菌中的应用，其中，致病菌为葡萄灰霉病菌、葡萄白腐病菌、白菜黑斑病菌、辣椒炭疽病菌、猕猴桃间座壳病菌、辣椒白娟病菌和马铃薯白娟病菌。
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| CN111887293B (zh) * | 2020-07-14 | 2022-10-28 | 江苏大学 | 一种酿酒酵母用于娃娃菜采后病害控制及贮藏保鲜的用途 |
| CN112358987B (zh) * | 2020-11-10 | 2022-09-23 | 广西壮族自治区农业科学院 | 植物乳杆菌菌株ldvs005及其应用 |
| CN112841225A (zh) * | 2021-02-20 | 2021-05-28 | 东北农业大学 | 一种防控窖藏马铃薯腐烂的微生物制剂及其应用 |
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| CN114794227B (zh) * | 2022-04-06 | 2024-01-09 | 湖南农业大学 | 植物乳杆菌o2在辣椒采后防腐保鲜中的应用及防腐保鲜方法 |
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