CN113040390A - 一株益生、耐盐约氏乳杆菌及其在畜禽水产养殖中防治病原菌的应用 - Google Patents
一株益生、耐盐约氏乳杆菌及其在畜禽水产养殖中防治病原菌的应用 Download PDFInfo
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Abstract
本发明公开了一株益生、耐盐约氏乳杆菌及其在畜禽水产养殖防治病原菌中的应用。该乳酸菌命名为：约氏乳杆菌(Lactobacillusjohnsonii)BM‑1，保藏号为GDMCC No.61276。该约氏乳杆菌(Lactobacillusjohnsonii)BM‑1具有耐盐、耐酸、耐高温等优良特性，在盐浓度为8％的培养基中处理5h，该菌株的存活率仍有52％。菌株BM‑1对常见畜禽水产病原菌具有高效抑制能力，用于防治病原菌不会产生耐药性、无二次污染，且具有明显的益生作用，在畜禽水产养殖病害防治中有较大的应用潜力。
Description
技术领域：
本发明属于微生物技术领域，具体涉及一株益生、耐盐约氏乳杆菌及其在畜禽水产养殖中防治病原菌的应用。
背景技术：
约氏乳杆菌(Lactobacillusjohnsonii)分类于乳杆菌属(Lactobacillus)，是普遍存在于人和动物肠道中的有益乳酸菌，可承受胃酸和胆汁的作用而到达小肠的上部并粘附于小肠壁，因而能够有效地抑制肠道病原微生物的生长，从而用于预防腹泻，防治畜禽坏死性肠炎，调整肠道菌群平衡和调节机体免疫反应等，对人和动物无害，具有良好的市场前景。我国卫生部也于2003年批准约氏乳杆菌作为保健品益生菌种。此外，金黄色葡萄球菌(Staphylococcus aureus)、伤寒沙门氏菌(Salmonella typhimurium)、大肠杆菌(Escherichia coli)等是肠道中或饲料附带的常见病原菌，易被忽视但对养殖动物的健康影响很大。益生菌，因其安全无毒、无污染等优点，逐渐受到市场青睐。利用微生物间的拮抗作用开展生物防治，已成为防控的重要途径。
发明内容：
本发明的第一个目的提供一株耐酸、耐胆盐、耐高温具有高效拮抗常见畜禽水产养殖病原菌能力的约氏乳杆菌(Lactobacillusjohnsonii)BM-1，为畜禽水产养殖病原菌防治提供了一种良好的生物材料。
所述的约氏乳杆菌(Lactobacillusjohnsonii)BM-1于2020年11月6日保藏于广东省微生物菌种保藏中心(GDMCC)，地址为广东省广州市越秀区先烈中路100号大院59号楼，保藏编号为GDMCC No：61276。
本发明的第二个目的是提供所述的约氏乳杆菌BM-1在畜禽水产养殖中防治病原菌中的应用。
优选，是在制备畜禽水产养殖中防治金黄色葡萄球菌、大肠杆菌、猪霍乱沙门氏菌、化脓链球菌或痢疾志贺氏菌等制剂中的应用。
本发明的第三个目的是提供一种防治金黄色葡萄球菌、大肠杆菌、猪霍乱沙门氏菌、化脓链球菌或痢疾志贺氏菌的制剂，其含有约氏乳杆菌BM-1或其发酵液作为活性成分。
本发明的第四个目的是提供上述约氏乳杆菌BM-1在制备食品添加剂或动物饲料添加剂中的应用。
与现有技术相比，本发明具有以下有益效果：
(1)本发明的约氏乳杆菌BM-1有较强的耐高温、耐酸、耐盐能力。
(2)本发明的约氏乳杆菌BM-1是个耐盐菌株，在盐度为8％的条件下处理5h存活率仍有52％以上。
(3)本发明的约氏乳杆菌BM-1能产生抗菌类抑菌物质，对常见畜禽水产养殖病原菌有很好的抑菌效果。
综上所述，本发明的约氏乳杆菌BM-1在海水或咸水中生长适应性强，对常见畜禽水产养殖病原菌抑菌效果好，在畜禽水产养殖中有较大的应用潜力。
本发明的Lactobacillusjohnsonii BM-1，于2020年11月6日保藏于广东省微生物菌种保藏中心(GDMCC)，地址为广东省广州市越秀区先烈中路100号大院59号楼5楼，保藏编号为GDMCC No.61276。
附图说明
图1为约氏乳杆菌BM-1形态的照片。
图2为约氏乳杆菌BM-1对5种病原菌抑制效果的柱形示意图。
图3为约氏乳杆菌BM-1发酵液离心后的上清液对病原菌株金黄色葡萄球菌(Staphylococcus aureus)GDMCC 1.122和猪霍乱沙门氏菌(Salmonella cholemesuis)GDMCC 1.244抑菌圈照片，其中，a为菌株GDMCC 1.122，b为菌株GDMCC 1.244。
图4为约氏乳杆菌BM-1在不同pH处理条件下的存活率。
图5为约氏乳杆菌BM-1在不同盐浓度处理条件下的存活率。
图6为约氏乳杆菌BM-1在不同温度处理条件下的存活率。
具体实施方式
下面结合附图和实施例，对本发明的具体实施方式作进一步详细描述，以下实施例用于说明本发明，但不用来限制本发明的范围，实施例的参数、比例等可因地制宜做出选择而对结果并无实质性影响。若无特别说明，实施例中所述方法均为常规方法，所用试剂均为常规试剂或按常规方式配制的试剂。
实施例1约氏乳杆菌(Lactobacillusjohnsonii)BM-1的富集培养与分离纯化及保存
(1)约氏乳杆菌(Lactobacillusjohnsonii)BM-1的分离、筛选与保存
本发明的约氏乳杆菌(Lactobacillusjohnsonii)BM-1是由发明人于从猪粪中分离得到。
分离培养基(MRS肉汤培养基)：蛋白胨10g、牛肉粉8g、酵母粉4g，葡萄糖20g，磷酸氢二钾2g，柠檬酸氢二铵2g，乙酸钠5g，硫酸镁0.2g，硫酸锰0.04g，吐温801g，琼脂15g，加水定容至1000mL，调pH至5.7±0.2，分装至250mL三角瓶中，每瓶100mL；于121℃高温高压灭菌20min，倒平板。
将猪粪采用稀释涂布法涂布到含分离培养基的平板上，经纯化获得菌株BM-1。肉眼观察菌落形态，菌落呈乳白色、表面光滑、液滴状、边缘不规则、不透明；革兰氏染色呈紫色，为革兰氏阳性菌，在显微镜下观察菌体形状为杆状，单个、成对或成链状排列。结果见图1。
实施例2约氏乳杆菌(Lactobacillusjohnsonii)BM-1的16S rDNA鉴定
提取菌株BM-1的基因组DNA，用细菌16S rDNA基因扩增通用引物27F/1492R(5’-AGAGTTTGATCCTGGCTCAG-3’和5’-TACGACTTAACCCCAATCGC-3’)扩增得到PCR产物，并送至上海美吉生物医药科技有限公司(广州分公司)进行序列测序，测序后得到的16S rRNA序列长度为1500bp，序列见SEQ ID NO.1。将测序结果与NCBI及EzBioCloud数据库中的16S rDNA序列进行同源性比对分析，结合形态学观察，初步鉴定本发明实施例1分离得到的菌株BM-1是约氏乳杆菌。将其命名为：约氏乳杆菌(Lactobacillusjohnsonii)BM-1。该菌株于2020年11月6日保藏于广东省微生物菌种保藏中心(GDMCC)，地址：广东省广州市先烈中路100号大院59号楼，保藏编号为GDMCC No：61276。
实施例3约氏乳杆菌(Lactobacillusjohnsonii)BM-1抑菌效果测定
(1)供试菌上清液制备：将活化好的菌株约氏乳杆菌BM-1接到MRS肉汤培养基中，置于37℃三气培养箱中培养24h；培养液离心后取上清液，置于4℃中备用。
(2)含菌平板制作：将培养好的供试菌株(如下表1)，用紫外分光光度计调节OD600值到0.1，然后分别吸取适量菌液加入到45℃左右熔融的相应培养基中，充分摇匀后倒平板，待其自然凝固后，用打孔器分别在同一平皿上均匀打孔(孔径9mm)。
(3)抑菌试验
吸取约氏乳杆菌(Lactobacillusjohnsonii)BM-1离心后的上清菌液100μL分别加入到各个含供试病原菌平板的孔中，置于37℃培养箱中培养约24h后，观察抑菌效果(结果见图3)，并测量抑菌圈直径(结果见图2)。
表1.约氏乳杆菌(Lactobacillusjohnsonii)BM-1抑菌效果试验供试菌株
从图2和图3可以看出，约氏乳杆菌(Lactobacillusjohnsonii)BM-1的上清菌液对金黄色葡萄球菌、大肠杆菌、猪霍乱沙门氏菌、化脓链球菌、痢疾志贺氏菌都有较好的抑制作用。
实施例4约氏乳杆菌(Lactobacillusjohnsonii)BM-1抗逆性评估
取培养24h的新鲜约氏乳杆菌BM-1菌液，用生理盐水调节菌液活菌数至107-108cfu/mL，将菌液分别接种于pH、氯化钠、牛胆盐、温度等压力条件下，37℃三气培养箱恒温培养一定时间。平板菌落计数法计算存活率。
(1)不同pH条件下的耐受性
取培养24h的新鲜约氏乳杆菌BM-1菌液，用生理盐水调节活菌数至107-108cfu/mL，取1mL菌液用无菌生理盐水梯度稀释涂MRS培养基平板计活菌数。分别取1mL菌液调节到pH2.0、3.0、4.0、5.0、6.0，37℃三气培养箱恒温培养30min，用平板计数法计算存活率，存活率如图4所示。从图4可以看出，在pH 3、4、5条件下处理30min，存活率均在80％以上，在pH 3条件下存活率最高，生长最旺盛。
(2)不同的氯化钠浓度条件下的耐受性
取培养24h的新鲜约氏乳杆菌BM-1菌液，用生理盐水调节活菌数至107-108cfu/mL，取1mL菌液用无菌生理盐水梯度稀释涂MRS培养基平板计活菌数。分别取1mL菌液加入氯化钠2、4、6、8％(质量分数)，37℃三气培养箱恒温培养5h，用平板计数法计算存活率，存活率如图5所示。从图5可以看出，约氏乳杆菌BM-1是个耐盐菌株，在盐度为8％的条件下处理5h存活率仍有52％以上。
(3)牛胆盐的耐受性
取培养24h的新鲜约氏乳杆菌BM-1菌液，用生理盐水调节活菌数至107-108cfu/mL，取1mL菌液用无菌生理盐水梯度稀释涂MRS培养基平板计活菌数。取1mL菌液加入牛胆盐0.3％(质量分数)，37℃三气培养箱恒温培养2h，用平板计数法计算存活率，存活率为60.16％。
(4)不同温度条件下的耐受性
取培养24h的新鲜约氏乳杆菌BM-1菌液，用生理盐水调节活菌数至107-108cfu/mL，取1mL菌液用无菌生理盐水梯度稀释涂MRS培养基平板计活菌数。分别取1mL菌液分别置于50、55、60℃水浴处理培养15min。用平板计数法计算存活率，存活率如图6所示。在50℃、55℃下处理15min，存活率仍在80％以上，是一株耐热的菌株。
实施例5约氏乳杆菌(Lactobacillusjohnsonii)BM-1对水产动物生长性能的影响
实验动物：选用加州鲈鱼苗(体长5cm左右)，随机分为两个处理，每个处理2个重复(缸)，每缸35条加州鲈鱼苗。
实验日粮：对照组饲料由市场上销售的普通基础加州鲈鱼饲料提供，不添加其他任何菌剂。实验组采用益生菌(约氏乳杆菌BM-1)添加饲料组，添加推荐剂量益生菌(1×106CFU/g～1×108CFU/g饲料)，加入1％脱脂奶粉作为保护剂(对照组也添加)，均匀喷洒到饲料表面后自然晾干，4℃冰箱保存。
检测指标：平均日增重、死亡率统计。
最终试验数据如下：对照组平均日增重0.0873g，试验组平均日增重0.099g，实验组较对照组提高鲈鱼平均日增重13.4％。
对照组鱼苗死亡率为21.62％，实验组鱼苗死亡率为14.09％，实验组较对照组降低鲈鱼苗死亡率34.8％。
序列表
<110> 广东博沃特生物科技有限公司
广东省科学院微生物研究所（广东省微生物分析检测中心）
广州王道生物科技有限公司
<120> 一株益生、耐盐约氏乳杆菌及其在畜禽水产养殖中防治病原菌的应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1419
<212> DNA
<213> 约氏乳杆菌BM-1(Lactobacillus johnsonii)
<400> 1
acggctgact cctataaagg ttatcccacc ggctttgggt gttacagact ctcatggtgt 60
gacgggcggt gtgtacaagg cccgggaacg tattcaccgc ggcgtgctga tccgcgatta 120
ctagcgattc cagcttcgtg taggcgagtt gcagcctaca gtccgaactg agaacggctt 180
taagagatcc gcttgccttc gcaggttcgc ttctcgttgt accgtccatt gtagcacgtg 240
tgtagcccag gtcataaggg gcatgatgac ttgacgtcat ccccaccttc ctccggtttg 300
tcaccggcag tctcattaga gtgcccaact taatgatggc aactaatgac aagggttgcg 360
ctcgttgcgg gacttaaccc aacatctcac gacacgagct gacgacagcc atgcaccacc 420
tgtctcagcg tccccgaagg gaacacctaa tctcttaggt ttgcactgga tgtcaagacc 480
tggtaaggtt cttcgcgttg cttcgaatta aaccacatgc tccaccgctt gtgcgggccc 540
ccgtcaattc ctttgagttt caaccttgcg gtcgtactcc ccaggcggag tgcttaatgc 600
gttagctgca gcactgagag gcggaaacct cccaacactt agcactcatc gtttacggca 660
tggactacca gggtatctaa tcctgttcgc tacccatgct ttcgagcctc agcgtcagtt 720
gcagaccaga gagccgcctt cgccactggt gttcttccat atatctacgc attccaccgc 780
tacacatgga gttccactct cctcttctgc actcaagttc aacagtttct gatgcaattc 840
tccggttgag ccgaaggctt tcacatcaga cttattgaac cgcctgcact cgctttacgc 900
ccaataaatc cggacaacgc ttgccaccta cgtattaccg cggctgctgg cacgtagtta 960
gccgtgactt tctaagtaat taccgtcaaa taaaggccag ttactacctc tatctttctt 1020
cactaccaac agagctttac gagccgaaac ccttcttcac tcacgcggcg ttgctccatc 1080
agactttcgt ccattgtgga agattcccta ctgctgcctc ccgtaggagt ttgggccgtg 1140
tctcagtccc aatgtggccg atcagtctct caactcggct atgcatcatt gccttggtaa 1200
gccgttacct taccaactag ctaatgcacc gcaggtccat ccaagagtga tagcagaacc 1260
atctttcaaa ctctagacat gcgtctagtg ttgttatccg gtattagcat ctgtttccag 1320
gtgttatccc agtctcttgg gcaggttacc cacgtgttac tcacccgtcc gccgctcgct 1380
tgtatctagt ttcatttagt gcaagcacta aaatacatc 1419
Claims (5)
1.约氏乳杆菌(Lactobacillusjohnsonii)BM-1，保藏编号为GDMCC No：61276。
2.权利要求1所述的约氏乳杆菌BM-1在畜禽水产养殖中防治病原菌中的应用。
3.根据权利要求2所述的应用，其特征在于，是在制备畜禽水产养殖中防治金黄色葡萄球菌、大肠杆菌、猪霍乱沙门氏菌、化脓链球菌或痢疾志贺氏菌制剂中的应用。
4.一种防治金黄色葡萄球菌、大肠杆菌、猪霍乱沙门氏菌、化脓链球菌或痢疾志贺氏菌的制剂，其特征在于，含有权利要求1所述的约氏乳杆菌BM-1或其发酵液作为活性成分。
5.权利要求1所述的约氏乳杆菌BM-1在制备食品添加剂或动物饲料添加剂中的应用。
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