CN113604387A - 一株耐盐、耐高温罗伊氏乳杆菌及其在畜禽水产养殖中防治病原菌的应用 - Google Patents
一株耐盐、耐高温罗伊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
本发明公开了一株耐盐、耐高温罗伊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：罗伊氏乳杆菌(Lactobacillus reuteri)SB‑2，保藏号为GDMCC No.61275。该罗伊氏乳杆菌(Lactobacillus reuteri)SB‑2具有耐盐、耐酸、耐高温等优良特性，在盐浓度为4％的培养基中处理5h，该菌株的存活率仍有113％；在55℃下处理15min，存活率达137％。菌株SB‑2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，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，具体涉及一株耐盐罗伊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：
抗生素的过度使用使得人类健康遭受严重威胁，已引起全球的广泛关注与重视。寻找绿色健康、高效无毒的抗生素替代品具有重要意义。目前，益生菌应用于饲料行业中只有20多年历史，但在养殖和疾病治疗方面表现出巨大的潜力。罗伊氏乳杆菌(Lactobacillus johnsonii)是一种栖息于人和动物的肠道，具有肠道表皮粘附能力的专性异型乳酸发酵菌，也是乳酸菌中重要的种群之一。罗伊氏乳杆菌可抑制胃肠道病原菌繁殖，调节动物免疫机能，降低动物应激，减少动物发病率，调节肠道菌群，预防或减轻腹泻，同时还具有提高饲料利用效率，促进动物生长，降低料重比等重要作用。我国农业部也于2013年批准罗伊氏乳杆菌作为益生菌可用作饲料添加剂应用于动物养殖。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、耐盐、耐高温具有高效拮抗常见畜禽水产养殖病原菌能力的罗伊氏乳杆菌(Lactobacillus reuteri)SB-2，为畜禽水产养殖病原菌防治提供了一种良好的生物材料。
所述的罗伊氏乳杆菌(Lactobacillus reuteri)SB-2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，邮编：510070，保藏编号为GDMCC No：61275。
本发明的第二个目的是提供所述的罗伊氏乳杆菌SB-2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，其含有罗伊氏乳杆菌SB-2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罗伊氏乳杆菌SB-2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)本发明的罗伊氏乳杆菌SB-2有较强的耐酸、耐盐、耐高温能力。
(2)本发明的罗伊氏乳杆菌SB-2是个耐盐菌株，在盐浓度为4％的条件下处理5h存活率仍有113％以上。
(3)本发明的罗伊氏乳杆菌SB-2是个耐高温菌株，在温度为55℃的条件下处理15min存活率仍有137％以上。
(4)本发明的罗伊氏乳杆菌SB-2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，本发明的罗伊氏乳杆菌SB-2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。
Lactobacillus reuteri SB-2，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，邮编：510070，保藏编号为GDMCC No.61275。
附图说明
图1为罗伊氏乳杆菌SB-2形态的照片。
图2为罗伊氏乳杆菌SB-2对5种病原菌抑制效果的柱形示意图。
图3为罗伊氏乳杆菌SB-2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)GDMCC1.244抑菌圈照片，其中，a为菌株GDMCC 1.122，b为菌株GDMCC 1.244。
图4为罗伊氏乳杆菌SB-2在不同pH处理条件下的存活率。
图5为罗伊氏乳杆菌SB-2在不同盐浓度处理条件下的存活率。
图6为罗伊氏乳杆菌SB-2在不同牛胆盐浓度处理条件下的存活率。
图7为罗伊氏乳杆菌SB-2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罗伊氏乳杆菌(Lactobacillus reuteri)SB-2的富集培养与分离纯化及保存
(1)罗伊氏乳杆菌(Lactobacillus reuteri)SB-2的分离、筛选与保存
本发明的罗伊氏乳杆菌(Lactobacillus reuteri)SB-2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80 1g，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SB-2。肉眼观察菌落形态，菌落呈乳白色或灰白色、表面光滑、边缘整齐、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。结果见图1。
实施例2罗伊氏乳杆菌(Lactobacillus reuteri)SB-2的16S rDNA鉴定
提取菌株SB-2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SB-2是罗伊氏乳杆菌。将其命名为：罗伊氏乳杆菌(Lactobacillus reuteri)SB-2。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5楼，邮编：510070，保藏编号为GDMCC No：61275。
实施例3罗伊氏乳杆菌(Lactobacillus reuteri)SB-2抑菌效果测定
(1)供试菌上清液制备：将活化好的菌株罗伊氏乳杆菌SB-2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
吸取罗伊氏乳杆菌(Lactobacillus reuteri)SB-2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.罗伊氏乳杆菌(Lactobacillus reuteri)SB-2抑菌效果试验供试菌株
从图2和图3可以看出，罗伊氏乳杆菌(Lactobacillus reuteri)SB-2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罗伊氏乳杆菌(Lactobacillus reuteri)SB-2抗逆性评估
取培养24h的新鲜罗伊氏乳杆菌SB-2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的新鲜罗伊氏乳杆菌SB-2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pH 2.0、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 2、3、4条件下处理30min，存活率均在40％以上，在pH4条件下存活率最高，生长最旺盛，存活率达166％。
(2)不同的氯化钠浓度条件下的耐受性
取培养24h的新鲜罗伊氏乳杆菌SB-2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，罗伊氏乳杆菌SB-2是个耐盐菌株，在盐度为4％的条件下处理5h存活率仍有113％以上。
(3)牛胆盐的耐受性
取培养24h的新鲜罗伊氏乳杆菌SB-2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0.1％、0.2％、0.3％、0.4％、0.5％(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，存活率如图6所示。从图6可以看出，罗伊氏乳杆菌SB-2是个耐牛胆盐菌株，在牛胆盐度为0.1％、0.2％、0.3％的条件下处理2h，存活率均在55％以上。
(4)不同温度条件下的耐受性
取培养24h的新鲜罗伊氏乳杆菌SB-2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7所示。在50℃、55℃下处理15min，存活率仍在80％以上，在55℃条件下存活率最高，生长最旺盛，存活率达137％，是一株耐热的菌株。
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）
<120> 一株耐高温、耐盐罗伊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> 1438
<212> DNA
<213> 罗伊氏乳杆菌SB-2(Lactobacillus reuteri)
<400> 1
ctcctccata atggttaggc caccgacttt gggcgttaca aactcccatg gtgtgacggg 60
cggtgtgtac aaggcccggg aacgtattca ccgcggcatg ctgatccgcg attactagcg 120
attccgactt cgtgtaggcg agttgcagcc tacagtccga actgagaacg gctttaagag 180
attagcttac tctcgcgagc ttgcgactcg ttgtaccgtc cattgtagca cgtgtgtagc 240
ccaggtcata aggggcatga tgatctgacg tcgtccccac cttcctccgg tttgtcaccg 300
gcagtctcac tagagtgccc aacttaatgc tggcaactag taacaagggt tgcgctcgtt 360
gcgggactta acccaacatc tcacgacacg agctgacgac gaccatgcac cacctgtcat 420
tgcgtccccg aagggaacgc cttatctcta aggttagcgc aagatgtcaa gacctggtaa 480
ggttcttcgc gtagcttcga attaaaccac atgctccacc gcttgtgcgg gcccccgtca 540
attcctttga gtttcaacct tgcggtcgta ctccccaggc ggagtgctta atgcgttagc 600
tccggcactg aagggcggaa accctccaac acctagcact catcgtttac ggcatggact 660
accagggtat ctaatcctgt tcgctaccca tgctttcgag cctcagcgtc agttgcagac 720
cagacagccg ccttcgccac tggtgttctt ccatatatct acgcattcca ccgctacaca 780
tggagttcca ctgtcctctt ctgcactcaa gtcgcccggt ttccgatgca cttcttcggt 840
taagccgaag gctttcacat cagacctaag caaccgcctg cgctcgcttt acgcccaata 900
aatccggata acgcttgcca cctacgtatt accgcggctg ctggcacgta gttagccgtg 960
actttctggt tggataccgt cactgcgtga acagttactc tcacgcacgt tcttctccaa 1020
caacagagct ttacgagccg aaacccttct tcactcacgc ggtgttgctc catcaggctt 1080
gcgcccattg tggaagattc cctactgctg cctcccgtag gagtatggac cgtgtctcag 1140
ttccattgtg gccgatcagt ctctcaactc ggctatgcat catcgccttg gtaagccgtt 1200
accttaccaa ctagctaatg caccgcaggt ccatcccaga gtgatagcca aagccatctt 1260
tcaaacaaaa gccatgtggc ttttgttgtt atgcggtatt agcatctgtt tccaaatgtt 1320
atcccccgct ccggggcagg ttacctacgt gttactcacc cgtccgccac tcactggtga 1380
tccatcgtca atcaggtgca agcaccatca atcagttggg ccagtgcgta cgactgca 1438
Claims (5)
1.罗伊氏乳杆菌(Lactobacillus reuteri)SB-2，保藏编号为GDMCC No：61275。
2.权利要求1所述的罗伊氏乳杆菌SB-2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，所述的病原菌为金黄色葡萄球菌、大肠杆菌、猪霍乱沙门氏菌、化脓链球菌或痢疾志贺氏菌。
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所述的罗伊氏乳杆菌SB-2或其发酵液作为活性成分。
5.权利要求1所述的罗伊氏乳杆菌SB-2在制备食品添加剂或动物饲料添加剂中的应用。
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