CN114836338B - 一株鼠李糖乳杆菌a5及其应用 - Google Patents
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Abstract
本发明公开了一株鼠李糖乳杆菌A5及其应用，该鼠李糖乳杆菌(Lactobacillus rhamnosus)A5已保藏于中国典型微生物保藏中心，保藏编号为CCTCC NO：M2018335，保藏时间为2018年6月1日。本发明提供的鼠李糖乳杆菌A5有以下几点功效：1)对细菌性病原菌具有较好抑制作用；2)具有防治鱼类诺卡氏菌病，增强鱼类免疫力，降低死亡率的作用；3)在优化的工业培养基方案使其能以较低成本制成益生制剂，本发明鼠李糖乳杆菌A5可以防治加州鲈鱼病害，在制备加州鲈鱼饲料应用方面具有广阔的前景。
Description
技术领域
本发明属于生物技术应用领域，具体涉及一株鼠李糖乳杆菌A5及其应用。
背景技术
益生菌的概念首次于1986年被Kozasa提出。目前，中国使用的益生菌主要属于五类：放线菌(actinobacteria)、拟杆菌类(bacteroidetes)、厚壁菌门(firmicutes)、变形菌门(Proteobacteria)和酵母菌(yeast)。其中乳酸菌，如鼠李糖乳杆菌，属于厚壁菌门，已被广泛用于水产养殖业益生菌。鼠李糖乳杆菌LGG(Lactobacillus rhamnosus GG)，是20世纪80年代，由Gorbach和Goldin从健康人的肠道中分离而得，并命名为鼠李糖乳杆菌LGG(Lactobacillus rhamnosus GG)，是目前最受全球关注的益生菌之一。大量研究显示，鼠李糖乳杆菌肠道黏着率高，定植能力强，并具有高效的抑菌活性，提高机体免疫力，无毒副作用的特点。然而到目前为止，尚未见将鼠李糖乳杆菌应用于加州鲈鱼诺卡氏病菌治理或添加于饲料中防治的相关报道。
诺卡氏菌病是一种死亡率很高的慢性疾病，其特征是形成肉芽肿、皮肤溃疡，以及在各种器官中形成大量的白色结节结构。目前，诺卡氏菌病正在成为海洋和淡水养殖业中最具系统性和破坏性的疾病，其发病率也在逐年上升。诺卡氏菌属的成员是革兰氏阳性、需氧、无运动的放线菌，到目前为止，该属的110多个物种已被鉴定和报告。诺卡氏菌病已成为包括加州鲈鱼在内的水产养殖中最严重的经济威胁之一。因此，努力研究开发有效的手段来控制这种疾病是非常重要的。
传统的药物治疗鱼类诺卡氏菌病的方法包括使用多种敏感抗生素，这可能导致严重的问题，如：病原菌的抗药性、药物在鱼体中的残留、环境污染等。与抗生素相比，疫苗接种具有安全性、环境友好性、长期疗效保护性的优势，被认为是另一种有效的诺卡菌病管理策略。尽管在开发针对诺卡氏菌的有效疫苗方面已经尝试了几种抗原，但最终的治疗效果仍然有限。近年来，直接口服抗生素替代溶液——免疫添加剂，或将其添加到饲料中成为一种可行的方法。抗菌肽、草药提取物、益生菌和益生元等均已作为免疫添加剂广泛用于加州鲈鱼以及其他水产养殖动物的水产饲料中。它们可以改善水生动物的免疫状态和健康状况，从而进一步保护养殖水生动物免受病原体的影响。特别是水生益生菌被认为是一种广泛应用于农业和水产养殖的环保型、绿色、可持续发展的模式。
发明内容
发明目的：针对现有技术存在的问题，本发明提供一株鼠李糖乳杆菌A5，该菌株对细菌性病原菌具有较好抑制作用，如放线菌诺卡氏菌，可以具有防治鱼类诺卡氏菌病，增强鱼类免疫力，降低死亡率的作用，可以制成益生制剂或者饲料。
本发明还提供所述一株鼠李糖乳杆菌A5的筛选方法和应用。
技术方案：为了实现上述目的，本发明所述一株鼠李糖乳杆菌(Lactobacillusrhamnosus)A5，鼠李糖乳杆菌A5已保藏于中国典型微生物保藏中心，保藏编号为CCTCC NO：M2018335，保藏时间为2018年6月1日，保藏地址：中国武汉，武汉大学。
本发明所述的鼠李糖乳杆菌A5的筛选方法，包括如下步骤：
取泡菜水用无生理盐水稀释，取稀释后的试液滴加在MRS固体培养基上，涂抹均匀，倒置过夜培养得到初筛菌株，以分离筛选得到的初筛菌株为供试菌，以诺卡氏菌为指示菌，采用牛津杯法筛选出具有诺卡氏菌拮抗效果最好的鼠李糖乳杆菌A5。
本发明所述的鼠李糖乳杆菌A5或者其在发酵产物在制备益生制剂或饲料中的应用。
其中，所述鼠李糖乳杆菌A5接种于培养基中进行发酵，获得发酵产物。
作为优选，所述培养基为：豆粕18-20g/L、玉米汁8-12g/L、尿素0.6-0.8g/L、乙酸钠3.0g/L、磷酸氢二钾0.5-0.8g/L、吐温80 1.0g/L、VB1、VB3、VB5、VB12、VH各1-3mg/L。
作为优选，所述发酵条件为通气比5.8-6.2vvm，转速130-160r/min，接种量1.8-2.0％，温度28-40℃，培养时间12-20h。
本发明所述的鼠李糖乳杆菌A5或者其在发酵产物在制备益生制剂或饲料中的应用。
其中，所述鼠李糖乳杆菌A5在制备抑制细菌性病原菌的益生制剂或饲料中的应用。
作为优选，所述鼠李糖乳杆菌A5在制备防治加州鲈鱼病害的益生制剂或饲料中的应用。
进一步地，所述鼠李糖乳杆菌A5的发酵液添加基础饲料中制备防治加州鲈鱼诺卡氏菌病害的益生制剂或饲料。
本发明提供了所述的鼠李糖乳杆菌A5在加州鲈鱼饲料中的应用。饲喂含有鼠李糖乳杆菌A5的饲料，感染诺卡氏菌的加州鲈鱼的存活率可达70％。
有益效果：与现有技术相比，本发明具有如下优点：
本发明提供一种全新的鼠李糖乳杆菌A5，该菌株在优化的培养基条件下使其能以较低成本制成益生制剂，更为重要的是本发明对鰤鱼诺卡氏菌的有明显的抑制效果。
本发明鼠李糖乳杆菌，首次报道可以治疗加州鲈鱼诺卡氏病。本发明将注射鲈鱼诺卡氏菌的加州鲈鱼分别喂食基础饲料和添加了含鼠李糖乳杆菌A5的基础饲料，结果显示喂食饲料中添加鼠李糖乳杆菌A5发酵液，感染鰤鱼诺卡氏菌病的加州鲈鱼存活率远远高于喂食普通饲料，证明鼠李糖乳杆菌A5对加州鲈鱼诺卡式菌病有很好的防治作用，可以有效应用在制备防治加州鲈鱼病害的益生制剂或饲料中。本发明鼠李糖乳杆菌A5对加州鲈鱼的相关疾病具有良好的防治效果，可用于制成相关的益生制剂和饲料。
附图说明
图1为鼠李糖乳杆菌A5形态图；
图2为用牛津杯法测定鼠李糖乳杆菌A5对鰤鱼诺卡氏菌、金黄色葡萄球菌、大肠杆菌的抑菌活性(从左到右)；
图3为基于鼠李糖乳杆菌A5 16sDNA片段基因序列构建进化树；
图4为鼠李糖乳杆菌A5抑菌活性测定；
图5为在MRS培养基及优化培养基中鼠李糖乳杆菌A5的活菌数；
图6为含鼠李糖乳杆菌A5的菌悬液的混合饲料及基础饲料；
图7为加州鲈鱼诺卡氏菌挑战实验死亡率测定。
具体实施方式
以下结合附图和实施例对本发明作进一步说明。
本发明实施例中所使用的材料、试剂等，如无特殊说明，均可从商业途径得到。实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂家建议的条件。其中使用的鰤鱼诺卡氏菌由淮阴工学院生物质转化过程与集成实验室提供，也可以使用其他野生型的鰤鱼诺卡氏菌。
实施例1
株鼠李糖乳杆菌A5的分离筛选和抑菌活性的测定
从江苏淮安的市售泡菜汁中分离获得，分离过程如下：用移液枪吸取泡菜汁1mL，加入装有9mL生理盐水的试管中，振摇均匀，完成10倍稀释度稀释，依照上述操作将样品做梯度稀释并标记好。选择适宜的稀释浓度梯度(10-4，10-5，10-6)用涂布棒涂布在MRS平板上，在37℃培养温度下培养24h。通过无菌操作观察培养基上单个菌落的形态，包括菌落的颜色、大小、菌落边缘以及表面粗糙情况，获得286株初筛菌株。将培养的初筛菌株为供试菌，以鰤鱼诺卡氏菌为指示菌，采用牛津杯法筛选出对鰤鱼诺卡氏菌具有良好拮抗效果的菌株，将该菌株命名为A5。无菌操作观察培养基上单个菌落的形态，菌株A5在MRS培养基上为圆形，乳白色，中心突起，边缘整齐，表面光滑，如图1所示。
采用牛津杯法进行抑菌活性的测定，具体步骤包括：取菌株单菌落于MRS液体培养基中在37℃静止培养48h后得到菌液，将以对数期的金黄色葡萄球菌、大肠杆菌和鰤鱼诺卡氏菌分别为指示菌平板三等分，无菌操作夹取3个牛津杯垂直放置在平板上，分别取0.2mL的菌液打入到牛津杯中，进行实验对比，37℃条件下静置培养24h，观察牛津杯周围抑菌圈，抑菌圈直径/mm＝总直径/mm-牛津杯直径/mm，结果如图2所示，菌株A5对革兰氏阳性菌、革兰氏阴性菌和鰤鱼诺卡氏菌均具有良好的抑制效果。综上，菌株鼠李糖乳杆菌A5对细菌性病原菌具有较好的抑制作用。
实施例2
菌株鼠李糖乳杆菌A5的种属鉴定
将筛选出的A5培养至对数生长期，提取其基因组DNA。利用细菌的通用引物扩增出它的16srDNA片段，进行测序，通过序列比对，采用BLAST分析法，对该基因的16srDNA片段进行比对，序列全长为1620bp，见序列表SEQ ID NO.1。将A5的序列与NCBI注册的鼠李糖乳杆菌等序列比较，同源性为99％，基因构建进化树如图3所示，结合菌株A5生理生化实验结果，鉴定为鼠李糖乳杆菌(Lactobacillus rhamnosus)，命名为鼠李糖乳杆菌A5(Lactobacillus rhamnosus A5)，该菌株已于2018年6月1日保藏于中国典型培养物保藏中心(CCTCC)，保藏地址：中国武汉，武汉大学，保藏编号为CCTCC NO：M2018335。
实施例3
对鼠李糖乳杆菌A5发酵物对鰤鱼诺卡氏菌的抑制效果鉴定
鼠李糖乳杆菌A5的基础培养基为MRS培养基，包括：牛肉膏10g，酵母膏5g，蛋白胨10g，柠檬酸氢二铵2g，乙酸钠5g，葡萄糖20g，吐温80 1mL，硫酸镁0.58g，磷酸氢二钾2g，硫酸锰0.25g，蒸馏水1000mL，121℃灭菌15min。
以上述培养基及培养条件，培养鼠李糖乳杆菌A5，并将获得的发酵液于4℃，8000rpm离心10min，取上清液；按体积比2％的量将对数期的鰤鱼诺卡氏菌菌液接种至100mL冷却至46℃的LB固体培养基中，混匀后倒入无菌平板中，无菌操作下夹取牛津杯垂直放置在无菌平板中，取200uL上清液加入到牛津杯中，进行实验对比，静置平放于37℃培养箱中培养24h，观察牛津杯周围抑菌圈，抑菌圈直径(mm)＝测量总直径(mm)-牛津杯直径(mm)。
如图4所示，菌株鼠李糖乳杆菌A5对鰤鱼诺卡氏菌有良好的抑制效果，抑菌圈直径为14.8±0.3mm。
实施例4
菌株鼠李糖乳杆菌A5在液态MRS培养基及优化工业培养基中生长曲线的测定
取鼠李糖乳杆菌A5划线培养于MRS平板培养基上。在37℃下培养24h后取单菌落分别接种于5mL MRS基础培养液(牛肉膏10g，酵母膏5g，蛋白胨10g，柠檬酸氢二铵2g，乙酸钠5g，葡萄糖20g，吐温80 1mL，硫酸镁0.58g，磷酸氢二钾2g，硫酸锰0.25g，蒸馏水1000mL，121℃灭菌15min)和优化的工业培养基(豆粕20g/L、玉米汁12g/L、尿素0.6g/L、乙酸钠3.0g/L、磷酸氢二钾0.5g/L、吐温80 1.0g/L、VB1、VB3、VB5、VB12、VH各2mg/L)，通气比6.0vvm，转速150r/min，接种量1.8％，37℃静置培养20h，制备种子液，按照体积比2％接种量将种子液接入含有50ml MRS培养液的250mL锥形瓶中，37℃静置培养48h，每两小时取出一瓶测定菌悬液的活菌数，绘制生长曲线。如图5所示，0～2h，鼠李糖乳杆菌细胞的分裂繁殖及生长缓慢，处于延滞期；2～16h菌体生长速率最快，代时最短，进入生长对数期；16～24h，随着营养物质的消耗，乳酸含量升高，环境pH值下降，活菌数上升趋势逐渐平缓，说明此时为菌体生长的稳定期。如图5所示，优化工业培养基中鼠李糖乳杆菌A5的活菌数为7.5×109cfu/ml，MRS基础培养基的活菌数为5×109cfu/ml，优化工业培养基中鼠李糖乳杆菌A5的活菌数与MRS基础培养基的活菌数接近。本发明提出的优化的工业培养基可以较低成本制成产物。
实施例5
取上述于优化培养基中，培养时间为16h的鼠李糖乳杆菌A5的发酵液(7.5×109cfu/ml)，喷洒入基础饲料(膨化饲料，粗蛋白质42.2％，粗脂肪11.9％，粒径为2.5mm)，使鼠李糖乳杆菌A5的菌悬液和基础饲料混合均匀，每1kg基础饲料添加180g鼠李糖乳杆菌A5发酵液，制成7组混合饲料(如图6)，28℃静置培养7天，每天同一时间取一组混合饲料，准确称取待测样品10g，放入装有90mL无菌水并放有小玻璃珠的250ml三角瓶中，置摇床上振荡20min，使微生物细胞分散，静置20-30s，即成10-1稀释液；再用1ml无菌吸管，吸取10-1稀释液lmL，移入装有9mL无菌水的试管中，吹吸3次，让菌液混合均匀，即成10-2稀释液；以此类推，连续稀释，制成10-3、10-4、10-5、10-6、10-7、10-8、10-9等一系列稀释菌液。取0.1mL菌液对号接种在不同稀释度编号的MRS基础培养基上，37℃静置培养，观察平板上的菌落数，每个平板上的菌落数在30-300个，可以用于防治加州鲈鱼病害。
实施例6
首先随机取三条加州鲈鱼，无菌操作下解剖，取鳃部分涂板确定无细菌感染。选取体重为125±10g，大小一致，健康的加州鲈鱼共30条，实验鱼分三组，每组十条：一个感染组，一个实验组，一个空白对照组。感染组与实验组中使用的诺卡氏菌接种在BHI培养基中，后在BHI肉汤中培养，在30℃培养6h后，4000rpm离心10分钟获得的细菌，将其稀释为2.5×107cfu/mL菌悬液，采取腹腔注射，感染组与实验组的每条加州鲈鱼注射0.2mL。空白对照组对腹腔注射相同量的无菌水。感染组喂食基础饲料(膨化饲料，粗蛋白质42.2％，粗脂肪11.9％，粒径为2.5mm，饲喂量为体重的2％)，实验组在基础饲料中添加实施例5制备的鼠李糖乳杆菌A5发酵液(每1kg基础饲料添加180g鼠李糖乳杆菌A5发酵液)，饲喂量同感染组。实验共饲养21d，水温28℃，记录加州鲈鱼的存活率。存活率计算如下：存活率(％)＝(存活的鱼数/初始鱼数)×100。
通过活体防治实验证明鼠李糖乳杆菌A5对于诺卡氏菌导致的加州鲈鱼病害有良好的防治效果，如图7所示，实验组的发病速度远慢于感染组，感染组加州鲈鱼全部死亡时，添加了鼠李糖乳杆菌A5的实验组存活率为70％，鼠李糖乳杆菌A5作为加州鲈鱼益生菌具有良好前景，可以有效应用于制备防治加州鲈鱼病害的益生制剂或饲料中。
序列表
<110> 淮阴工学院
<120> 一株鼠李糖乳杆菌A5及其应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1620
<212> DNA
<213> 鼠李糖乳杆菌A5(Lactobacillus rhamnosus A5)
<400> 1
tttccttaga aaggaggtga tccagccgca ggttctccta cggctacctt gttacgactt 60
caccctaatc atttgtccca ccttagacgg ctcgctccct aaaagggtta cgccaccggc 120
ttcgggtgtt acaaactctc atggtgtgac gggcggtgtg tacaaggccc gggaacgtat 180
tcaccgcggc gtgctgatcc gcgattacta gcgattccga cttcgtgtag gcgagttgca 240
gcctacagtc cgaactgaga atggctttaa gagattagct tgacctcgcg gtctcgcaac 300
tcgttgtacc atccattgta gcacgtgtgt agcccaggtc ataaggcatg catgatgatt 360
tgacgtcatc cccaccttcc tccggtttgt caccggcagt cttactagag tgcccaacta 420
aatgctggca actagtcata agggttgcgc tcgttgcggg acttaaccca acatctcacg 480
acacgagctg acgacaacca tgcaccacct gtcattttgc ccccgaaggg gaaacctgat 540
ctctcaggtg atcaaaagat gtcaagacct ggtaaggttc ttcgcgttgc ttcgaattaa 600
accacatgct ccaccgcttg tgcgggcccc cgtcaattcc tttgagtttc aaccttgcgg 660
tcgtactccc caggcggaat gcttaatgcg ttagctgcgg cactgaaggg cggaaaccct 720
ccaacaccta gcattcatcg tttacggcat ggactaccag ggtatctaat cctgttcgct 780
acccatgctt tcgagcctca gcgtcagtta cagaccagac agccgccttc gccactggtg 840
ttctccatat atctacgcat ttcaccgcta cacatggagt tccactgtcc tcttctgcac 900
tcaagtttcc cagtttccga tgcacttcct cggttaagcc gagggctttc acatcagact 960
taaaaaaccg cctgcgctcg ctttacgccc aataaatccg gataacgctt gccacctacg 1020
tattaccgcg gctgctggca cgtagttagc cgtggctttc tggttggata ccgtcacgcc 1080
gacaacagtt actctgccga ccattcttct ccaacaacag agttttacga cccgaaagcc 1140
ttcttcactc acgcggcgtt gctccatcag acttgcgtcc attgtggaag attccctact 1200
gctgcctccc gtaggagttt gggccgtgtc tcagtcccaa tgtggccgat caacctctca 1260
gttcggctac gtatcattgc cttggtgagc cgttacctca ccaactagct aatacgccgc 1320
gggtccatcc aaaagcgata gcttacgcca tctttcagcc aagaaccatg cggttcttgg 1380
atttatgcgg tattagcatc tgtttccaaa tgttatcccc cacttaaggg caggttaccc 1440
acgtgttact cacccgtccg ccactcgttc aaaattaaat caagatgcaa gcacctttca 1500
ataatcagaa ctcgttcgac ttgcatgtat taggcacgcc gccagcgttc atcctgagcc 1560
aggatcaaac tctcatataa atatgagctg ttcgaatagc tcgatttgtt gttctagcga 1620
Claims (8)
1.一株鼠李糖乳杆菌（Lactobacillus rhamnosus）A5，其特征在于，鼠李糖乳杆菌A5已保藏于中国典型微生物保藏中心，保藏编号为CCTCC NO：M2018335，保藏时间为2018年6月1日。
2.一种权利要求1所述的鼠李糖乳杆菌A5或者其发酵产物在制备益生制剂或饲料中的应用。
3.根据权利要求2所述的应用，其特征在于，所述鼠李糖乳杆菌A5接种于培养基中进行发酵，获得发酵产物。
4.根据权利要求3所述的应用，其特征在于，所述培养基为：豆粕18-20g/L、玉米汁8-12g/L、尿素0.6-0.8g/L、乙酸钠3.0 g/L、磷酸氢二钾0.5-0.8g/L、吐温80 1.0g/L、VB1、VB3、VB5、VB12、VH各1-3mg/L。
5.根据权利要求3所述的应用，其特征在于，所述发酵条件为通气比5.8-6.2vvm，转速130-160r/min，接种量1.8-2.0%，温度28-40℃，培养时间12-20h。
6.根据权利要求2所述的应用，其特征在于，所述鼠李糖乳杆菌A5在制备抑制病原菌的益生制剂或饲料中的应用，所述病原菌包括金黄色葡萄球菌、大肠杆菌和鰤鱼诺卡氏菌中的一种或者多种。
7.根据权利要求2所述的应用，其特征在于，所述鼠李糖乳杆菌A5在制备防治加州鲈鱼诺卡氏菌病害的益生制剂或饲料中的应用。
8.根据权利要求2所述的应用，其特征在于，所述鼠李糖乳杆菌A5的发酵液添加基础饲料中制备防治加州鲈鱼诺卡氏菌病害的益生制剂或饲料。
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