CN110079469B - 一株植物乳杆菌及应用 - Google Patents
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Abstract
一种植物乳杆菌及应用，植物乳杆菌(Lactobacillus plantarum)STM1‑3，于2018年10月29日保藏于广东省微生物菌种保藏中心，保藏编号为GDMCC NO.60464。植物乳杆菌STM1‑3以菌液的形式加入到酸菜半成品中，制剂中活菌数为1.0×106CFU/mL，在酸菜中添加量是每100g酸菜加入所述菌液2mL。优点是：植物乳杆菌STM1‑3在袋装东北酸菜产品中对枯草芽孢杆菌的MIC为105CFU/mL，抑制率达到100.0％，能够有效抑制传统酸菜袋装产品中枯草芽孢杆菌的生长，达到了防腐保鲜的效果，有利于传统东北酸菜袋装产品在加工贮藏和运输销售等环节的保藏。
Description
技术领域
本发明属于微生物领域，特别涉及一种植物乳杆菌及应用。
背景技术
传统东北酸菜一般以秋季大白菜为主要原料，利用天然乳酸菌在5℃至15℃厌氧条件下经过35天-40天发酵而成，产品具有色泽金黄、质地鲜脆、气味酸香的特点。传统东北酸菜最大限度地保留了原有蔬菜的营养成份，富含各种维生素、氨基酸、膳食纤维等。另外，酸菜在发酵过程中产生的乳酸菌及其在代谢过程中产生的有机酸等可保持胃肠道正常生理功能，能够帮助消化、预防便秘等，而且酸菜具有酸香等独特风味物质，深受人们的喜爱。
传统东北酸菜在发酵过程中初始阶段在原料表面均附有大量的各类微生物，随着厌氧条件的逐步形成，乳酸菌在中后期占据主导地位成为优势菌群，通过形成酸性环境和拮抗性代谢产物使发酵原料中的它种微生物生长受到抑制，在最终酸菜成品中乳酸菌也占有统治地位。传统东北酸菜袋装产品加工过程中，经过清洗、切丝、包装等环节，来自生产环境、加工人员和器具等诸方面的细菌污染，致使东北酸菜袋装产品在保质期内发生涨袋腐败现象，严重地影响了东北酸菜产品在贮藏、运输、销售等环节的安全性和稳定性。
目前，解决东北酸菜产品中微生物污染腐败的主要方法是添加食品防腐剂。但是，国家标准《GB 2714-2015食品安全国家标准酱腌菜》中将东北酸菜划分为发酵蔬菜，《GB2760-2014食品安全国家标准食品添加剂使用标准》中发酵蔬菜不允许使用食品防腐剂。因此，由于酸菜产品受到国家标准的限制，在保质期(一般为10个月)内很难控制细菌性污染，产品货架期内“涨袋”是困扰酸菜产品生产企业发展的关键问题。解决发酵酸菜产品腐败变质的问题只能依赖生物防腐技术。
发明内容
本发明要解决的技术问题是提供一种植物乳杆菌制剂，对传统东北酸菜包装产品中酸菜中芽孢杆菌具有抑菌效果，避免袋装酸菜存放过程中的涨袋，可应用于传统东北酸菜包装产品的防腐保鲜。
发明的技术解决方案：
一株植物乳杆菌(Lactobacillus plantarum)STM1-3，于2018年10月29日保藏于广东省微生物菌种保藏中心，保藏编号为GDMCC NO.60464。
一株植物乳杆菌STM1-3在传统东北酸菜袋装产品中的应用。
所述的植物乳杆菌STM1-3在传统东北酸菜袋装产品中的应用，其特征在于：以菌液的形式加入到酸菜半成品中，菌液的活菌数为1.0×106CFU/mL，添加量为2.0mL/100g。
本发明通过平板初筛、牛津杯打孔法复筛，最终获得一株能够有效抑制传统发酵酸菜袋装产品中枯草芽孢杆菌生长的植物乳杆菌STM1-3，其达到效果是：
植物乳杆菌STM1-3在袋装酸菜产品中对芽孢杆菌的MIC为1.0×105CFU/mL，抑制率达到100.0％，能够有效抑制传统酸菜袋装产品中芽孢杆菌的生长，达到了防腐保鲜的效果，有利于传统酸菜袋装产品在加工和运输过程中的保藏。
植物乳杆菌(Lactobacillus plantarum)STM1-3，于2018年10月29日保藏于广东省微生物菌种保藏中心，保藏编号为GDMCC NO.60464。
附图说明
图1是本发明的乳酸菌对芽孢杆菌抑制作用结果；
图2是本发明的乳酸菌菌株STM1-3在光学显微镜下观察结果；
图3是本发明的乳酸菌菌株STM1-3的16S rDNA基因扩增电泳结果；
图4是本发明的乳酸菌菌株STM1-3的16S rDNA基因系统发育树分析结果。
具体实施方式
一、植物乳杆菌(Lactobacillus plantarum)STM1-3的获得
1、培养基
MRS固体培养基：蛋白胨10.0g，牛肉膏10.0g，酵母浸粉5.0g，琼脂15.0g，CaCO310.0g，葡萄糖20.0g，无水乙酸钠5.0g，柠檬酸二铵2g，吐温-80 1.0mL，MgSO4·7H2O 0.58g，MnSO4·4H2O 0.25g,磷酸氢二钾2.0g，蒸馏水1000mL，调节pH 6.4，121℃，灭菌20min。
MRS液体培养基：蛋白胨10.0g，牛肉膏10.0g，酵母浸粉5.0g，葡萄糖20.0g，无水乙酸钠5.0g，柠檬酸二铵2.0g，吐温-80 1.0mL，MgSO4·7H2O 0.58g，MnSO4·4H2O 0.25g，磷酸氢二钾2g，蒸馏水1000mL，调节pH 6.4，121℃，灭菌20min。
复筛培养基：(下层)素琼脂1.5％，(上层)蛋白胨10.0g，酵母提取物5.0g，氯化钠10.0g， 20.0g琼脂蒸馏水1000ml，121℃，灭菌20min。
2、乳酸菌菌株的分离
在无菌条件下，取辽宁沈阳酸汤子原料水面10.0g置于90.0mL无菌生理盐水中，充分振荡5min，用接种环挑取一环在1.0％CaCO3的MRS固体培养基上划线接种，37℃培养48h。挑取有溶钙圈的白色菌落进一步分离纯化，经革兰氏染色和过氧化氢酶试验，选择革兰氏阳性和过氧化氢酶阴性的菌落进行复筛，分离获得植物乳杆菌。
3.抑制传统东北酸菜中腐败菌拮抗性乳酸菌的筛选
将步骤2分离获得的乳酸菌对传统东北酸菜中芽孢杆菌进行抑菌活性测定。首先将分离的乳酸菌菌株在MRS液体培养基中活化3代得到菌悬液。分离自传统东北酸菜袋装腐败产品中的腐败菌包括枯草芽孢杆菌(Bacillus subtilis)和弯曲芽孢杆菌(Bacillusflexus)，在30℃恒温静置培养24h，活化至3代。无菌条件下取1.0mL菌液于9.0mL 0.85％NaCl中获得10 倍稀释液并进行10倍系列梯度稀释，取10-6、10-7稀释液各1.0mL置于平板中(每稀释度3 个平行)，倒入20～25mL 45±1℃的无菌LB琼脂培养基，摇匀后在30℃培养48h进行菌落计数，得到107CFU/mL菌液，4℃条件下保存。稀释到106CFU/mL枯草芽孢杆菌(Bacillus subtilis)和弯曲芽孢杆菌(Bacillus flexus)菌悬液为指示菌。利用牛津杯打孔法进行乳酸菌拮抗芽孢杆菌试验：将10.0mL琼脂倒入无菌平板中，将牛津杯放在凝固后的琼脂表面。吸取125μL 106CFU/mL芽孢杆菌菌悬液置于25mL 45±1℃LB琼脂培养基，摇匀，倒入置有牛津杯的平板中(不要倒进牛津杯的孔里)。凝固后取出牛津杯，在牛津杯孔中加入180μL 的乳酸菌菌悬液，37℃培养24h后记录抑菌圈直径。选取抑菌圈直径最大的菌株STM1-3。
采用上述菌株的分离筛选方法，从辽宁阜新酸黄瓜分离出一株植物乳杆菌命名为ZHG2-1，辽宁锦州大菱鲆分离出一株植物乳杆菌命名为LP1-3，从辽宁大连碱蓬草分离出一株植物乳杆菌命名为DLH2-2，从辽宁锦州发酵小麦面团分离出一株植物乳杆菌命名为LJ2-2；图1a和1b分别是上述植物乳杆菌和本发明植物乳杆菌STM1-3对枯草芽孢杆菌和弯曲芽孢杆菌的抑菌作用结果。
由图1可以看出，图1a中STM1-3、ZHG2 -1和LP1 -3对枯草芽孢杆菌的抑菌直径分别为15.88mm、13.79mm和14.02mm，STM1-3对枯草芽孢杆菌抑菌效果最好；图1b中STM1-3、DLH2-2和LJ2-2对枯草芽孢杆菌的抑菌直径分别为16.65mm、12.95mm和14.12mm，STM1-3 对枯草芽孢杆菌抑菌效果最好。
二、菌株STM1-3的鉴定
1.生理生化鉴定
参照《乳酸细菌分类鉴定及实验方法》，将筛选出的菌株STM1-3在MRS液体培养基中 37℃培养24h，用接菌环接入相应的细菌微量反应管中，37℃培养24h，记录反应管颜色变化，并参照《常见细菌系统鉴定手册》进行鉴定。图2a和2b为筛选菌株STM1-3的菌落形态及革兰氏染色后的光学显微镜观察结果，表1为菌株STM1-3的生理生化反应结果。
表1菌株STM1-3的生理生化反应结果
菌株STM1-3为革兰氏阳性杆菌，经生理生化反应结果初步鉴定为植物乳杆菌。
2.16S rDNA鉴定
用DNA快速抽提试剂盒提取植物乳杆菌STM1-3对数期培养物的菌株DNA。用正向引物为27f(5′-AGAGTTTGATCCTGGCTCAG-3′)及反向引物1492r (5′-TACGGYTACCTTTGTTACGACTT-3′)进行PCR扩增。PCR扩增反应体系为：上游引物、下游引物各加入1.0μL，DNA模板1.0μL，Taq PCR Master mix12.5μL，超纯水9.5μL。PCR 扩增反应条件为：94℃预变性2min；94℃变性1min，60℃退火1min，72℃延伸90s，循环30次；72℃延伸8min，4℃保温。经1.0％琼脂糖凝胶电泳检测验证后的PCR产物送生工生物工程(上海)股份有限公司测序。登陆GenBank并对测得序列进行BLAST同源性比较，应用MEGA7.0构建系统发育进化树。图3、图4分别为菌株STM1-3的16S rDNA基因扩增电泳结果以及16S rDNA基因系统发育树结果。
结果表明，16S rDNA基因电泳图中可看出1500bp左右出现特异性亮带，说明目标片段扩增成功。测序结果在GenBank数据库中进行BLAST比对表明，菌株STM1-3与植物乳杆菌MH779888.1在同一个分支上，置信度为98％。因此，鉴定菌株STM1-3为植物乳杆菌。此鉴定结果与生理生化鉴定结果相一致。命名为植物乳杆菌STM1-3，于2018年10月29日保藏于广东省微生物菌种保藏中心(简称GDMCC，地址为：广州市先烈中路100号大院59 号楼5楼，广东省微生物研究所，邮政编码：510075)，保藏编号为GDMCC NO.60464。三、植物乳杆菌STM1-3(GDMCC NO.60464)对传统东北酸菜袋装产品中芽孢杆菌抑菌活性的应用试验
为确定植物乳杆菌STM1-3在传统东北酸菜袋装产品中对枯草芽孢杆菌K、弯曲芽孢杆菌W的抑菌效果，将植物乳杆菌STM1-3添加到传统东北酸菜产品中进行应用性试验，并测定其最小抑制菌浓度(MIC)以及对枯草芽孢杆菌K、弯曲芽孢杆菌W的抑菌率。
1.最小抑菌浓度(MIC)测定
对实例1筛选出的植物乳杆菌STM1-3进行最小抑制浓度(MIC)测定，结果如表2 所示。将植物乳杆菌STM1-3菌悬液稀释至107、106、105、104CFU/mL和103CFU/mL，各取2mL接入100g传统东北酸菜袋装产品中，37℃培养24h后对酸菜产品中的芽孢杆菌菌落总数进行检测，选择能够抑制芽孢杆菌生长的乳酸菌MIC。
表2乳酸菌MIC结果
实施的结果从表中可以看出当乳酸菌1.0×105CFU/mL时，芽孢杆菌的生长活性受到抑制，此时即为植物乳杆菌STM1-3的MIC。
2.抑菌率测定
应用上述步骤1所得到的MIC的植物乳杆菌STM1-3加入到酸菜产品中，未加乳酸菌的酸菜产品作为对照，测定其中芽孢杆菌菌落数，并计算乳酸菌的抑菌率。计算公式如下：
X——抑菌率，％
A——对照平均菌落数
B——样品平均菌落数
表3乳酸菌抑菌率测定结果
结果分析
根据以上述结果进行综合评定，从辽宁沈阳酸汤子中筛选得到植物乳杆菌STM1-3。 STM1-3对芽孢杆菌具有良好的抑制活性。
植物乳杆菌STM1-3在传统东北酸菜袋装产品中，以菌液的形式加入到酸菜中，菌液的活菌数为1.0×106cfu/mL，添加量为2.0mL/100g。抑制率达到100.0％，能够有效抑制传统酸菜袋装产品中芽孢杆菌的生长，达到了防腐保鲜的效果。
以上仅为本发明的具体实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
SEQUENCE LISTING
<110> 渤海大学
<120> 一株植物乳杆菌及应用
<130> 2018
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1098
<212> RNA
<213> 植物乳杆菌（Lactobacillus plantarum）
<400> 1
ggaggtcccg tcatgctaat acatgcagtc gaacgaactc tggtattgat tggtgcttgc 60
atcatgattt acatttgagt gagtggcgaa ctggtgagta acacgtggga aacctgccca 120
gaagcggggg ataacacctg gaaacagatg ctaataccgc ataacaactt ggaccgcatg 180
gtccgagctt gaaagatggc ttcggctatc acttttggat ggtcccgcgg cgtattagct 240
agatggtggg gtaacggctc accatggcaa tgatacgtag ccgacctgag agggtaatcg 300
gccacattgg gactgagaca cggcccaaac tcctacggga ggcagcagta gggaatcttc 360
cacaatggac gaaagtctga tggagcaacg ccgcgtgagt gaagaagggt ttcggctcgt 420
aaaactctgt tgttaaagaa gaacatatct gagagtaact gttcaggtat tgacggtatt 480
taaccagaaa gccacggcta actacgtgcc agcagccgcg gtaatacgta ggtggcaagc 540
gttgtccgga tttattgggc gtaaagcgag cgcaggcggt tttttaagtc tgatgtgaaa 600
gccttcggct caaccgaaga agtgcatcgg aaactgggaa acttgagtgc agaagaggac 660
agtggaactc catgtgtagc ggtgaaatgc gtagatatat ggaagaacac cagtggcgaa 720
ggcggctgtc tggtctgtaa ctgacgctga agctcgaaag tatgggtagc aacaggattt 780
agataccctg gtagtccata ccgtaacgat gaatgctaag tgttggaggg tttccgccct 840
tcagtgctgc agctaaacgc attaagcatt tccgcctggg ggagtactgg ccgcaaagct 900
gaaacctcaa agggaattga ctgtgaggcc ccgccacaag ccggtggagc atgtgggtat 960
cattttcgaa gctacgtcga aagaaccctt accgagtctt tgacttatct cagctaaatc 1020
ctaacaagat taaaacgttc cccttcaggt gcacatcgat atatcaggtt ggatcgcatg 1080
gactgatagc tcatatcc 1098
Claims (3)
1.一株植物乳杆菌(Lactobacillus plantarum)STM1-3，于2018年10月29日保藏于广东省微生物菌种保藏中心，保藏编号为GDMCC NO.60464。
2.根据权利要求1所述的植物乳杆菌STM1-3在传统东北酸菜袋装产品中的应用。
3.根据权利要求2所述的植物乳杆菌STM1-3在传统东北酸菜袋装产品中的应用，其特征在于：以菌液的形式加入到传统东北酸菜半成品中，菌液的活菌数为1.0×106CFU/mL，添加量为2.0mL/100g酸菜。
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