CN115353989A - 一种类布式乳杆菌及其用途 - Google Patents
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- CN115353989A CN115353989A CN202210715992.0A CN202210715992A CN115353989A CN 115353989 A CN115353989 A CN 115353989A CN 202210715992 A CN202210715992 A CN 202210715992A CN 115353989 A CN115353989 A CN 115353989A
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Abstract
本发明的类布氏乳杆菌（Lactobacillus parabuchneri）已于2022.5.20保藏于中国典型培养物保藏中心（CCTCC），保藏编号：CCTCC M 2022688。本发明的类布氏乳杆菌经过耐药基因检测、氨基酸脱羧酶活性及靛基质检测（有毒代谢产物检测）、致病性试验、溶血活性检测等安全性评价以及耐酸试验、耐胆盐试验、人工胃肠液耐受性试验、体外抑菌试验、表面性质测定、细胞粘附试验等相关试验后，结果均符合联合国粮农组织/世界卫生组织（FAO/WHO）工作组关于食品益生菌的要求。同时，在进行防治犊牛消化不良的试验中，类布氏乳杆菌MH3211与短乳杆菌MH3111配合使用，能够达到防治犊牛消化不良的效果。
Description
技术领域
本发明涉及犊牛养殖技术领域，尤其涉及一种类布式乳杆菌及其用途。
背景技术
犊牛消化不良是哺乳期犊牛胃肠消化机能障碍的总称，又称消化不良性腹泻，其主要临床特征是明显的消化机能障碍和不同程度的腹泻。根据临床表现和发病经过，可将犊牛消化不良分为单纯性消化不良和中毒性消化不良。单纯性消化不良多发生于15日龄内的新生犊牛，主要表现为消化和营养的急性障碍和轻微的全身症状。新生犊牛神经系统的调节机能较弱，消化系统发育尚不完善，在饲养管理不当、初乳或常乳饲喂不及时或过量、常乳或代乳品质量差、营养不良、畜舍卫生差、应激等因素的作用下，极易造成胃肠功能和肠道菌群紊乱，出现单纯性消化不良。如果单纯性消化不良得不到有效救治就会进一步发展为中毒性消化不良，呈现出严重的消化障碍、明显的自体中毒和重剧腹泻等全身症状。消化不良严重影响新生犊牛对营养物质的吸收，导致犊牛发育迟缓，抵抗力下降，易于继发大肠杆菌、轮状病毒、冠状病毒、隐孢子虫等病原的感染，发生感染性腹泻；消化不良的新生犊牛还易于继发瘤胃臌气、皱胃炎、皱胃阻塞和扭转、呼吸道感染等，严重的甚至发生死亡，每年给奶牛和肉牛养殖业带来严重的经济损失。
目前，规模化牧场主要通过加强犊牛新生期饲养管理、应用抗菌药物和中草药等来预防和治疗新生犊牛消化不良及腹泻。但预防性应用抗菌药物会带来潜在的抗生素滥用和细菌耐药性问题，此外，长期使用抗菌药物会扰乱新生犊牛肠道菌群和消化系统的发育，可能对其生长发育和生产性能的发挥产生长期的负面影响。近年研究表明，胃肠道菌群在人和动物胃肠道健康的维持、营养物质的消化和代谢、宿主免疫机能的调节等方面发挥着重要作用。基于肠道菌群开发的具有精准治疗或预防作用的新型微生物制剂已经成为人和动物疾病预防和治疗的新途径，具有广阔的市场前景。在犊牛出生后的早期阶段，肠道菌群能够促进消化系统和免疫系统的发育、成熟，并对成年后的健康及生产性能产生长远影响。另有研究表明，肠道菌群组成与犊牛的健康和日增重紧密相关，发生腹泻、肺炎或日增重低的犊牛，其肠道菌群多样性也较低，乳杆菌等具有益生作用的细菌显著减少。另据报道，消化不良犊牛肠道菌群中梭菌属、埃希氏菌属等有害菌的丰度显著增加，而乳杆菌属、黄杆菌属、颤螺菌属、 Desulfonauticus属等有益菌的丰度显著下降，表明肠道中上述细菌组成的变化与犊牛出血性腹泻相关。Jang JY等进一步分析了轮状病毒感染的腹泻犊牛肠道菌群的多样性指数和乳杆菌属、Subdoligranulum、Blautia、拟杆菌属等的丰度显著下降。上述研究均表明，乳杆菌等益生菌在犊牛肠道健康的维护中发挥着重要作用。近年来，不同来源和类型的益生菌被逐步应用到犊牛腹泻和消化不良的预防中，其中包括植物乳杆菌、干酪乳杆菌、屎肠球菌、乳酸片球菌、酿酒酵母、枯草芽孢杆菌等。临床应用研究表明，上述益生菌均能不同程度的降低犊牛腹泻发病率，改善腹泻症状。
犊牛消化不良的临床表现是食欲不振至厌食；粪便减少或增多以及粪便干燥、黏稠或腹泻。现阶段治疗犊牛消化不良的药物主要为西药和一部分中药，而长期使用西药会造成肝脏等脏器的损伤，虽然中药的使用副作用较小，但是中药的炮制以及制备过程复杂繁琐，且具有较好药物疗效的中药需要道地药材，费用昂贵。在早期饲料添加剂的生产中，存在着滥用抗生素与激素的问题。长期使用抗生素或磺胺类药物来防止畜禽的疾病或消化不良等消化系统疾病，会导致家畜肠道中病原菌产生耐药性，破坏机体胃肠道稳态。虽然犊牛的肠道中存在一定量的有益菌，但是不能直接将分离到的益生菌培育后作为犊牛的饲料添加剂。养殖用含可遗传耐药基因的益生菌大规模的使用将导致耐药基因向病原体转移，将造成患病动物对药物产生耐药性，降低患病动物的治愈率。
目前，我国农业部已规定了饲料添加剂中可添加的有益微生物菌种有34种，根据有益菌的不同生理生化特性，它们会以不同方式被应用于动物。近年来，随着微生物组学、培养组学技术的不断发展，越来越多的新的功能益生菌逐步被用于人类和动物疾病的预防和治疗中。根据联合国粮农组织/世界卫生组织 (FAO/WHO)关于食品益生菌评价指南的联合报告中指出，可用于商品化的益生菌菌株需要经过胃酸耐受试验、胆汁酸耐受试验、粘附试验或/和人上皮细胞粘附试验、对条件致病菌的拮抗活性试验、降低致病菌对消化道粘膜的粘附能力试验、胆汁盐水解酶活性试验和对杀精子剂的耐药性试验(适用于阴道用益生菌)等体外和在体试验来评估其益生特性。同时，还要对候选菌株进行溶血活性、有毒代谢产物、耐药基因及其药物敏感性及有无致病性、细胞和动物实验等来评价其安全性。
目前，用作防治犊牛消化不良的微生物制剂的益生菌还很有限，不能满足市场需求。
发明内容
有鉴于此，有必要提供一种符合食品益生菌要求的类布氏乳杆菌。
一种类布氏乳杆菌(Lactobacillus parabuchneri)已于2022.5.20保藏于中国典型培养物保藏中心(CCTCC)，保藏编号：CCTCC M 2022688。
上述保藏编号为CCTCC M 2022688的类布氏乳杆菌应用于犊牛的微生物制剂。
上述保藏编号为CCTCC M 2022688的类布氏乳杆菌应用于防治犊牛消化不良的微生物药物制剂。
上述保藏编号为CCTCC M 2022688的类布氏乳杆菌应用于犊牛的微生物饲料添加剂。
有益效果：本发明的类布氏乳杆菌经过耐药基因检测、氨基酸脱羧酶活性及靛基质检测(有毒代谢产物检测)、致病性试验、溶血活性检测等安全性评价以及耐酸试验、耐胆盐试验、人工胃肠液耐受性试验、体外抑菌试验、表面性质测定、细胞粘附试验等相关试验后，结果均符合微生物饲料添加剂的要求。同时，在进行防治犊牛消化不良的试验中，类布氏乳杆菌MH3211与短乳杆菌 MH3111配合使用，能够达到防治犊牛消化不良的效果。
附图说明
图1为本发明的基于本试验菌株MH321116S rRNA基因序列构建的系统发育进化树。
图2为本试验菌株急性口毒性脏器病理切片结果；其中；A、C、E、G分别为培养基空白对照组小鼠心肝脾肾脏的病理切片结果；B、D、F、H分别为本试验菌株五倍浓缩液灌腹小鼠心肝脾肾脏病理切片结果。
图3为本发明的MH3211溶血性试验对照图，其中A为肠球菌ATCC29212菌落周围出现了溶血环，发生溶血现象的阳性对照，B为本试验菌株菌落周围未出现溶血环，没有发生溶血现象。B为本试验菌株菌落周围未出现溶血环，没有发生溶血现象。
图4为MH3211黏附在人结直肠癌细胞(Caco-2)周围的图。
具体实施方式
以下将结合具体实施例对本发明的方案进行详细说明。实施例中所使用的试验方法如无特殊说明，均为常规方法。
本发明中出现的各英文缩写表示的中英文含义如表1所示，后续为了便于统计及叙述，试验中的有关词汇将通过英文缩写代替。
本发明的试验菌株涉及到的编号与菌株类型对应关系如下：
上述菌种都是从哺乳期荷斯坦健康犊牛新鲜粪便中分离出的，除了MH3211，其它菌种都作为对照菌，MH3211即为本发明要保护的菌种。MH3211已于 2022.5.20在中国典型培养物保藏中心(CCTCC)进行了保藏，保藏编号为CCTCC M 2022688。
除此之外，文中涉及到的菌种ATCC393是干酪乳杆菌购自广东省食品微生物安全工程技术研究开发中心。试验中所用到的埃希氏大肠杆菌ATCC8739 (Escherichia coli)、金黄色葡萄球菌ATCC33591(Staphylococcus aureus)、鼠伤沙门氏菌ATCC14028(Salmonella typhimurium)购自中国药品生物制品鉴定所。人结直肠腺癌细胞(Caco-2细胞)来源于宁夏大学生命科学学院。
试验中所需主要培养基见表2。
表2：培养基及其来源
| MRS肉汤 | 青岛高科园海博生物技术有限公司 |
| MRS肉汤琼脂 | 青岛高科园海博生物技术有限公司 |
| LB肉汤 | 青岛高科园海博生物技术有限公司 |
| LB肉汤琼脂 | 青岛高科园海博生物技术有限公司 |
| 心脑浸出液肉汤 | 北京陆桥技术股份有限公司 |
| 心脑浸液琼脂肉汤 | 北京陆桥技术股份有限公司 |
| MH肉汤 | 北京陆桥技术股份有限公司 |
| MH肉汤琼脂 | 北京陆桥技术股份有限公司 |
本发明试验中用到的类布氏乳杆菌通过以下方式获得。将筛选后的类布式乳杆菌接种于MRS琼脂平板中，并在37℃下，厌氧培养48h后挑取单菌落接种至新鲜无菌的MRS肉汤培养基中厌氧培养获得。
MRS肉汤培养基采用以下方式制备。蛋白胨10.0g、牛肉浸粉5.0g、酵母浸粉4.0g、葡萄糖20.0g、磷酸氢二钾2.0g、柠檬酸三铵2.0g、醋酸钠5.0g、硫酸镁0.2g、硫酸锰0.05g、琼脂15.0g、吐温801.0g，将上述成分加入蒸馏水中，定容至1000mL，调节pH为6.2±0.2，于121℃高压灭菌15～20min。
以下为类布氏乳杆菌MH3211的安全性检测的具体实施过程。
类布氏乳杆菌MH3211的可转移的耐药基因检测：
乳杆菌的耐药基因主要有四环素耐药基因tet(W)、tet(M)、tet(S)、tet(Q)、 tet(O)、tet(W)、tet(L)、tet(P)，环丙沙星耐药基因gyrA，古霉素耐药基因vanX，卡那霉素耐药基因aph(3“)-III.，甲氧苄啶耐药基因dfrD，利福平耐药基因 rpoB，克林霉素耐药基因lsa，氨基糖苷耐药基因aac(3)、aph(3)、aph(3′)-III，aadA，aadE、ant(6)、ant(9)，氯霉素耐药基因cat、cmlA和红霉素抗性基因 erm(B)、mef、mef(E)、mef(B)，β内酰胺类耐药基因blaCTX-M、blaSHV、blaTEM、 blaZ、blaOXA-1、blaOXA-48。
其中目前能确定可转移的耐药基因有tet(W)、tet(M)、aphA-1、aphA-2、aadA、addE、gyrA、aac6'-lb-cr、aac(6′)-aph(2″)、erm(B)、str(A)、str(B)、sul2、sul3、 mef(A)、blaIMP、和blaTEM耐药基因，对本发明的类布式乳杆菌进行检测后，未发现上述可转移的耐药基因。
同时，四环素耐药基因将通过性信息素介导的偶联系统进行广泛传播，本发明对类布氏乳杆菌MH3211的性信息素毒力基因cpd、cob、ccf进行检测，结果不含有性信息素毒力基因，即不存在四环素耐药基因的传播途径。
类布氏乳杆菌MH3211的氨基酸脱羧酶活性及靛基质检测(有毒代谢产物检测)：
(1)氨基酸脱羧酶活性检测
挑取MRS琼脂培养基培养48h的单个菌落分别接种于鸟氨酸、精氨酸、酪氨酸氨脱羧酶肉汤试验管和对照管中，并在培养基表面覆盖无菌液体石蜡，置 37℃厌氧培养18～24h。对照管为黄色，若试管为紫色，则为阳性，若试管为黄色，则为阴性。
挑取MRS琼脂培养基培养48h的单个菌落接种于苯丙氨酸安培瓶中，置 37℃厌氧培养18～24h，然后滴加质量分数为10％的FeCl3溶液4～5滴，2min 后判定结果，若显绿色为阳性，不变色为阴性。
(2)靛基质检测
将单菌落接种于蛋白胨水培养基中，置37℃培养24～48h，先在培养基中滴加适量二甲苯振荡混匀，萃取培养基中菌株生长代谢产生的吲哚，再滴加 Kovacs靛基质试剂2～3滴静置。若液体分层后，上层出现红色则为阳性，否则为阴性。
如表3所示，氨基酸脱羧酶活性检测和靛基质检测试验结果表明MH3211 不具有鸟氨酸和苯丙氨酸氨基脱羧酶活性，不会产生有毒物质吲哚。同时，也检测到MH3211具有精氨酸、色氨酸和赖氨酸氨基脱羧酶活性，它们产生的生物胺是否具有毒性要通过小鼠体内致病性试验进一步评价。
表3类布氏乳杆菌MH3211试验氨基酸脱羧酶活性检测和靛基质检测结果
致病性试验
购买SPF级ICR健康成年小鼠，雌雄各半，体重18.0g～22.0g分别通过腹腔注射和经口灌胃两种途径，给予小鼠类布氏乳杆菌MH3211培养液，以评价不同受试物暴露途径对动物的致病性。方法及计量均参考《保健食品原料用菌种安全性检验与评价技术指导原则(2020年版)》。
(1)乳杆菌MH3211培养物的活菌数计算
由表4可知，小鼠腹腔注射的MH3211菌株培养物中的菌的浓度为80.9± 1.31×107CFU/mL，因此给小鼠注射量为0.1mL时，菌株存活率可达到不少于 107CFU/mL。
表4腹腔注射菌量计算
注：1、2、3、X^±s分别表示第一个值、第二个值、第三个值和平均值加减标准差。
(2)腹腔注射试验
小鼠40只，雌雄各半，随机分成4组(每组10只)，分为211雄性小鼠无菌生理盐水对照组、211雄性小鼠菌悬液组、211雌性小鼠无菌生理盐水对照组、211雌性小鼠菌悬液组每只小鼠注射0.2mL，即受试物组每只小鼠注射菌量不少于1.0×107CFU。
(3)灌胃试验
小鼠80只，雌雄各半，分别随机分成8组(每组10只)，分别为MH3211 雄性小鼠培养基对照组、MH3211雄性小鼠紫外灭活菌悬液组、MH3211雄性小鼠 5倍浓缩培养基对照组、MH3211雄性小鼠5倍浓缩紫外灭活菌悬液组；MH3211 雌性小鼠培养基对照组、MH3211雌性小鼠紫外灭活菌悬液组、MH3211雌性小鼠 5倍浓缩培养基对照组、MH3211雌性小鼠5倍浓缩紫外灭活菌悬组各组均以 20mL/kg·BW的体积给小鼠灌胃，连续灌胃3d，首次灌胃前小鼠应禁食过夜 (16h)，灌胃后3h～4h喂食。
(4)动物腹腔注射和灌胃后每天观察1次，至少连续观察21d。观察并记录小鼠皮肤和毛、眼睛和粘膜、呼吸情况、肢体活动、行为方式等有无异常。特别注意观察是否出现振颤、抽搐、腹泻、嗜睡、流涎和昏迷等现象。试验前称量并记录所有小鼠的体重，试验结束后称量并记录所有存活小鼠的体重。对于试验期间死亡的小鼠，尽可能精确记录小鼠的死亡时间，同时称重并记录。试验结束后，尾静脉采集各组小鼠的血液，并用全自动血常规检测仪进行血常规检测。剖解灌腹了MH3211五倍浓缩菌悬液试验组及培养基对照组小鼠的心肝脾肺肾脏器进行观察。试验结果表明，腹腔注射乳杆菌MH3211培养物对小鼠体重的影响如表5和表6所示。试验期间，小鼠初始体重、受试7天后体重及终末体重在培养物试验组和生理盐水对照组组间比较均无显著差异(P＞0.05)，未观察到实验小鼠明显的活动或行为变化、疾病或死亡。灌腹乳杆菌MH3211培养物对小鼠体重影响见表7和表8。试验期间，雄性小鼠初始体重、终末体重在培养物试验组和培养基对照组组间比较均无显著差异(P＞0.05)，受试7d后 MH3211悬液试验组体重显著降低(P＜0.01)。雌性小鼠初始体重、7d后体重和终末体重在菌株培养物试验组和培养基对照组间比较均无显著差异(P＞0.05)，未观察到实验小鼠明显的活动或行为变化、疾病或死亡。急性口毒性小鼠脏器病理学检测结果如2所示，培养基对照组和浓缩菌悬液组小鼠心、肝、脾、肾均未出现坏死、纤维化、炎细胞浸润、正常结构缺失和萎缩等病变。腹腔注射小鼠一周后血常规检测结果如表10所示。类布氏乳杆菌MH3211菌株培养物小鼠腹腔注射试验组与生理盐水对照组的各项血常规组间差异不显著 (P>0.05)。MH3111和MH3211培养物灌服小鼠血常规检测结果见表10。类布氏乳杆菌乳杆菌MH3211培养物和浓缩培养物试验组与培养基对照组中小鼠的各项血常规组间差异不显著(P＞0.05)。
综上，类布式乳杆菌MH3211未发现有致病性。在试验过程中，小鼠也未表现出中毒症状，说明类布式乳杆菌MH3211的代谢产物无毒或毒性对小鼠未造成影响。
表5乳杆菌MH3211培养物对雄鼠体重的影响
表6乳杆菌MH3211培养物对雌鼠体重的影响
表7乳杆菌MH3211培养物对雄鼠体重的影响
表8乳杆菌MH3211培养物灌腹对雌鼠体重的影响
表9乳杆菌MH3211培养物腹腔注射雌性小鼠血常规
| 项目 | MH3211悬液 | 空白对照 | P值 |
| WBC(×109)/L | 4.55±2.758 | 6.45±0.354 | 0.759 |
| Lymph(×109)/L | 3.85±2.475 | 4.15±1.768 | 0.993 |
| Mon(×109)/L | 0.15±0.071 | 0.5±0.566 | 0.619 |
| Gran(×109)/L | 0.55±0.212 | 1.8±1.556 | 0.479 |
| Lymph％ | 84.05±3.041 | 64.6±30.830 | 0.617 |
| Mon％ | 2.45±0.071 | 8.05±8.556 | 0.588 |
| Gran％ | 13.5±3.111 | 27.35±22.274 | 0.63 |
| RBC×(×10<sup>12</sup>)/L | 9.475±4.236 | 11.175±0.841 | 0.806 |
| HGB(g/L) | 149.5±70.004 | 183±21.213 | 0.752 |
| HCT％ | 52.2±22.910 | 60.85±5.586 | 0.828 |
| MCV(fL) | 55.3±0.566 | 54.45±0.919 | 0.615 |
| MCH(pg) | 15.65±0.354 | 16.3±0.707 | 0.62 |
| MCHC(g/L) | 283.5±9.192 | 300±7.071 | 0.283 |
| RDW(％) | 15.55±0.354 | 13.95±2.192 | 0.551 |
| PLT(×109)/L | 948±353.553 | 672±120.208 | 0.777 |
| MPV(fL) | 5.9±0.566 | 6.05±0.212 | 0.915 |
| PDW | 17.1±0.141 | 17±0.566 | 0.965 |
| PCT(％) | 0.191±0.271 | 0.4075±0.087 | 0.687 |
表10乳杆菌MH3211培养物灌服雌性小鼠血常规
溶血活性检测
在无菌条件下，挑取活化后的乳杆菌菌悬液划线接种于哥伦比亚培养基，置37℃厌氧培养48h后观察平板中菌落周围是否产生α－溶血或β－溶血。用粪肠球菌ATCC29212作为阳性对照。试验结果如图3所示，表明MH3211不具有溶血活性。
进一步的，本发明还对类布氏乳杆菌MH3211进行耐受性试验。
试验一、耐酸性试验
以不加盐酸的MRS培养基作为对照，用1mol/L盐酸调节各培养管MRS液体培养基pH，使其pH分别为4.0、5.0、6.0，然后高压灭菌。将菌液调OD600 为0.85±0.05后按1％(V/V)分别接种于不同pH的MRS液体培养基中，37℃培养24h，分别测定OD600，每组设置3个平行重复。试验结果如表11所示，表明菌株在pH4时仍然具有生长活性，说明本发明的MH3211耐酸性良好。
表11：菌株MH3211耐酸试验结果
| 培养基空白组 | pH6 | pH5 | pH4 |
| 0.687 | 0.68 | 0.632 | 0.435 |
| 0.723 | 0.691 | 0.683 | 0.41 |
| 0.715 | 0.673 | 0.66 | 0.422 |
试验二、耐胆盐试验
在MRS液体培养基中分别加入0、0.1g/L、0.2g/L和0.3g/L牛胆盐，使其质量分数为0％、0.1％、0.2％和0.3％后高压灭菌。将菌液浓度调节至OD600 为1±0.02按1％(V/V)分别接种于0％、0.1％、0.2％和0.3％牛胆盐的MRS液体培养基中，分别于0h，4h将各管培养液取出并做10倍系列稀释，取适当稀释度涂布于MRS固体培养基培养48h，通过平板计数法计算活菌数和存活率(％)，每组同时设置3个平行重复。存活率＝N1/N0×100％。N1：胆盐处理4h后的活菌数；N0：0h的活菌数。菌株MH3211耐胆盐试验结果见表12，试验结果表明类布氏乳杆菌MH3211在胆盐浓度为0.1％的培养基中可以不断的生长繁殖，在 0.2％胆盐的培养基中存活率较低，在0.3％胆盐的培养基中不生长。
表12：菌株MH3211胆盐耐受性检测
试验三、人工胃肠液耐受性检测
人工胃液的制备：称取胃蛋白酶(1：10000)0.32g，NaCl 0.2g溶于100 mL去离子水中，HCl调pH至2.0，用0.22μm无菌滤膜过滤。
人工肠液的制备：称取胰蛋白酶1.0g，磷酸二氢钾0.68g溶于100mL去离子水中，NaOH调pH至7.5±0.1，用0.22μm无菌滤膜过滤。
人工胃液耐受性测定：挑取试验菌株单菌落接种于MRS液体培养基中培养至对数生长期，然后将菌液混匀，10倍系列稀释，取适当稀释度涂板计数，记录为N0，每组设置3个平行重复。再取1mL菌液5000r/min，4℃离心5min，弃去培养基；用无菌PBS重悬乳杆菌后5000r/min，4℃离心5min，弃去PBS 溶液，重复冲洗3次；弃去液体后，加入配制好的1mL人工胃液并将菌液混匀，置37℃厌氧培养4h后将菌液取出，10倍系列稀释，取适当稀释度涂板计数，每组设置3个平行重复。记录N1，计算存活率(％)。存活率＝N1/N0×100％。N1：胆盐处理4h后的活菌数；N0：0h的活菌数；
人工肠液耐受性测定：挑取试验菌株单菌落接种于MRS液体培养基中培养至对数生长期，然后将菌液混匀，梯度稀释后涂板计数，记录为N0，每组设置 3个平行重复。再取1mL菌液用离心机5000r/min，4℃离心5min，弃去培养基；用无菌PBS重悬乳杆菌后5000r/min，4℃离心5min，弃去PBS溶液，重复冲洗3次；弃去液体后，加入配制好的1mL人工肠液并将菌液混匀，置37℃厌氧培养17h后将菌液取出，10倍系列稀释，取适当稀释度涂板计数，每组设置3个平行重复。记录N1，计算存活率(％)。存活率＝N1/N0×100％。N1：胆盐处理17h后的活菌数；N0：0h的活菌数。
菌株MH3211胃肠道耐受性结果见表13，经过模拟胃液处理3h后菌株MH3211 存活率为0.23±0.03％，模拟肠液处理17h后，存活率为25.93±1.21％，可知该菌能很好地耐受胃肠道环境。
表13：菌株MH3211胃肠道耐受性检测
进一步的，本发明还进行了达到微生物饲料添加剂要求的其它项试验。
试验四、体外抑菌试验
发酵上清的制备：挑取单菌落接种于MRS液体培养基培养48h，菌液12000 r/min离心1min后取上清，用0.22μm无菌滤膜过滤后备用。
抑菌试验：直径10cm的平皿中倒入高压灭菌好的1.5％琼脂的LB、MHB 和BHI培养基20mL，将过夜培养的大肠杆菌ATCC8739、金黄色葡萄球菌 ATCC33591、鼠伤沙门氏菌ATCC14028稀释到106CFU/mL，用棉签菌液涂布于琼脂表面，待完全吸收后，每皿摆放3个牛津杯，牛津杯内加入200μL乳杆菌上清液，置4℃冰箱2h后，转入37℃温箱孵育24h后用游标卡尺对抑菌圈直径进行测量，结果如表14所示。体外抑菌试验结果表明MH3211对大肠埃希氏菌ATCC8739、金黄色葡萄球菌ATCC3359和鼠伤寒沙门氏菌 ATCC114028具有一定的抑制作用。
表:14菌株MH3211体外抑菌试验结果
| 大肠埃希氏菌ATCC8739 | 金黄色葡萄球菌ATCC3359 | 鼠伤寒沙门氏菌ATCC114028 | |
| 抑菌直径mm | 14.00±0.00 | 10.83±0.29 | 10.70±0.26 |
疏水性测定：将过夜培养的菌液离心，用PBS洗涤2次，调节OD600为 0.5±0.02，记录为A0；分别将2mL二甲苯或氯仿加入到同等体积的菌悬液中，涡旋混匀1min；37℃静置孵育2h；待两相分层后取水相，测定其600nm波长处的吸光值并记录为A2；进行三次重复试验，计算三次试验各菌株疏水率，最后计算三次试验疏水率的平均值±标准差。菌株疏水率(％)表示为：(A0-A2) /A0×100％，结果如表15所示。
自聚率测定：将过夜培养的菌液离心，用PBS洗涤两次，调节OD600为 0.5±0.02，记录为A0；取4mL菌悬液于37℃静置放置24h；取培养基上清液于600nm波长处测定吸光度，记录为A1；进行三次重复试验，计算各菌株自聚率，最后计算三次试验自聚率的平均值±标准差。菌株自聚率(％)表示为：(A0-A1)/A0×100％。实验结果表明类布氏乳杆菌MH3211的疏水性在氯仿中为60.28±0.95％，在二甲苯中的疏水性为43.58±11.47％，自聚率为45.91± 10.87。
表15：类布氏约氏乳杆菌MH3211表面性质测定结果
细胞粘附试验
细胞培养：Caco-2细胞复苏后，用10％胎牛血清培养基重悬细胞并接种于细胞培养瓶中，置于37℃，5％CO2细胞培养箱中，培养观察并拍照记录，第 3天换一次液，待细胞生长至铺满培养瓶底部，进行1比4传代，待细胞传代至 5代以上进行下列试验。
菌悬液的制备：待测乳杆菌活化后，取200μL接种于5mL装有4mL MRS 液体培养基的离心管中静置培养12h，4000r/min离心10min收集菌体，用PBS 洗涤3次。然后用不含双抗和血清的DMEM完全培养液重悬细菌，调节菌液浓度在1×109CFU/mL(OD600约为1.0)，进行黏附试验。
细胞黏附及观察试验：
把准备好的细菌缓缓加入准备好的细胞，将孔板置于37℃培养箱，静置培养2h后用吸管吸去菌液，用温育好PBS的轻轻洗三遍，洗去未粘附在细胞上的细菌。用细胞裂解液0.1％TritonX-100裂解细胞，并用1mL的PBS重悬裂解好的细胞，取摇匀的菌液稀释一定倍数，取稀释后的菌液用平板计算菌落形成单位，根据稀释程度计算菌液浓度。
粘附率表示为：(粘附在细胞上的细菌数目/加入的细菌总数目)×100％，结果如表16所示
细胞黏附观察：把准备好的菌悬液(1mL)缓缓加入培养好的细胞中，将12孔细胞板置于37℃培养箱，静置培养4h后用吸管吸去菌液，用温育好PBS 的轻轻洗三遍，洗去未粘附在细胞上的细菌，将细胞爬片取出，用PBS冲洗3 遍后用细胞固定液固定15min，再次用PBS冲洗2～3遍后用革兰氏染色试剂盒上的方法对细胞进行染色，用油镜观察并拍摄照片。如图4所示，试验结果表明乳杆菌MH3211可以黏附在Caco-2细胞的周围。
表:16类布氏乳杆菌MH3211的黏附率
| 菌株 | 黏附率(％) |
| 类布氏乳杆菌MH3211 | 5.57％ |
以上试验结果表明，本发明的类布氏乳杆菌MH3211可作为犊牛的微生物饲料添加剂。
为了验证类布氏乳杆菌MH3211在防治犊牛消化不良中的效果，本发明将类布氏乳杆菌MH3211与实验室分离的短乳杆菌MH3111的冻干粉按质量比1:1 的比例配伍成复合微生态制剂，应用于犊牛消化不良的防治。结果如表17所示，分别饲喂宁夏地区某规模化奶牛场中12～28日龄健康犊牛15头，消化不良犊牛 15头，连续饲喂7天，每头犊牛每餐掺喂1.75～2.25克复合微生态制剂，饲喂后连续观察21天。观察并记录犊牛皮肤和毛、眼睛和粘膜、呼吸情况、肢体活动、行为方式等有无异常。特别注意观察是否出现振颤、抽搐、腹泻、嗜睡、流涎和昏迷等现象。消化不良犊牛饲喂第二天，消化不良症状(厌食、粪便呈黏稠黄色或透明、腹泻)明显缓解，连续饲喂3-5天后，病畜基本痊愈，饲喂期间无动物死亡发生，食欲大增，粪便正常，无腹泻，且出现抢食现象，动物治愈率达99％，其中有一头犊牛因脱水严重而死亡。健康犊牛无消化不良现象发生，且犊牛皮肤和毛、眼睛和粘膜、呼吸情况、肢体活动、行为方式等无异常，没有出现振颤、抽搐、腹泻、嗜睡、流涎和昏迷等现象。上述饲喂试验中，益生菌制剂在对犊牛消化不良治愈中效果显著，且未出现康复动物病情复发等现象。此外，将上述复合微生态制剂初步应用于腹泻犊牛中，均有缓解并治愈腹泻的效果。因此，本发明类布氏乳杆菌MH3211可作为复合微生态制剂复配菌株应用于犊牛消化不良病的防治。
以上所揭露的仅为本发明较佳实施例而已，当然不能以此来限定本发明之权利范围，本领域普通技术人员可以理解实现上述实施例的全部或部分流程，并依本发明权利要求所作的等同变化，仍属于发明所涵盖的范围。
序列表
<110> 宁夏大学
<120> 一种类布式乳杆菌及其用途
<141> 2022-05-26
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1155
<212> DNA
<213> 类布氏乳杆菌（Lactobacillus parabuchneri ）
<400> 1
gcaacggcgg cggcaacagc aagcgaacgc gcggagagaa gggcgcaaac gaaacagaga 60
cgagggcgaa cgggagaaca cggggaaccg cccgaagagg ggaaacacgg aaacagggca 120
aaccgaaaca accaaaacca ccggggaaaa gaggccggca cacaggagga cccgcggcga 180
agcgggaagg aacggccacc aaggcaagaa cgagccgacc gagagggaac ggccacaggg 240
acgagacacg gcccaaaccc acgggaggca gcagagggaa cccacaagga cgaaagcgag 300
gagcaacgcc gcggaggaga agggcggccg aaaaccgggg agaagaacag gggagagaac 360
gcacacgacg gaccaaccag aaagccacgg caacacggcc agcagccgcg gaaacgaggg 420
gcaagcggcc ggaagggcga aagcgagcgc aggcggcagg cgaggaaagc ccggcaaccg 480
gagaaggcac ggaaaccagg agacgaggca gaagaggaca gggaacccag gagcgggaaa 540
gcgagaaagg aagaacacca gggcgaaggc ggcgcggcga acgacgcgag gccgaaagca 600
gggagcgaac aggaagaacc cggagccagc cgaaacgaga ggcaagggga gggccgcccc 660
aggcgcagca acgcaaagca cccgccgggg agacgaccgc aagggaaacc aaaggaagac 720
gggggcccgc acaagcgggg agcagggaac gagcacgcga agaaccacca ggcgacaccg 780
ccaaccaaga gaaggcgccc cggggacaga agacaggggg cagggcgcag ccggcggaga 840
ggggaagccc gcaacgagcg caacccagag gccagcacag gggcaccagc aagacgccgg 900
gacaaaccgg aggaaggggg gagacgcaaa cacagcccca gaccgggcac acacggcaca 960
aggacggaca acgagcgcga aaccgcgagg caagcaacca aagccgccag cggagaggcg 1020
caaccgccac agaagggaac gcagaacggg acagcagcca cgggaaacgc ccgggccgac 1080
acaccgcccg cacaccagag aggaacaccc aaagccggga ggaacccggg gaccagccgc 1140
aaggggacaa agggg 1155
Claims (4)
1.一种类布式乳杆菌，其特征在于：类布氏乳杆菌（Lactobacillus parabuchneri ），已于2022.5.20保藏于中国典型培养物保藏中心（CCTCC），保藏编号：CCTCC M 2022688。
2.如权利要求1所述的一种类布式乳杆菌，应用于犊牛的微生物制剂。
3.如权利要求1所述的一种类布式乳杆菌，应用于防治犊牛消化不良的微生物药物制剂。
4.如权利要求1所述的一种类布式乳杆菌，应用于犊牛的微生物饲料添加剂。
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120020943A1 (en) * | 2010-07-20 | 2012-01-26 | China Medical University | Lactobacillus Plantarum and Uses Thereof |
| CN103517991A (zh) * | 2010-10-27 | 2014-01-15 | 昆特拜克股份公司 | 通过包括嵌入物分子的探针捕获目标dna和rna |
| CN103734479A (zh) * | 2014-01-10 | 2014-04-23 | 孙震海 | 降低畜禽体内雌激素和胆固醇含量的饲料添加剂生产方法 |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120020943A1 (en) * | 2010-07-20 | 2012-01-26 | China Medical University | Lactobacillus Plantarum and Uses Thereof |
| CN103517991A (zh) * | 2010-10-27 | 2014-01-15 | 昆特拜克股份公司 | 通过包括嵌入物分子的探针捕获目标dna和rna |
| CN103734479A (zh) * | 2014-01-10 | 2014-04-23 | 孙震海 | 降低畜禽体内雌激素和胆固醇含量的饲料添加剂生产方法 |
Non-Patent Citations (1)
| Title |
|---|
| 戴青;赵述淼;谢树贵;梁运祥;: "一株凝结芽孢杆菌的分离筛选及生物学特性研究", 饲料工业, no. 12 * |
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