CN115024382B - 一株抗动物腹泻动物联合乳杆菌zjuids-r2及其应用 - Google Patents
一株抗动物腹泻动物联合乳杆菌zjuids-r2及其应用 Download PDFInfo
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Abstract
本发明提供一株抗动物腹泻动物联合乳杆菌ZJUIDS‑R2及其应用。所述动物联合乳杆菌ZJUIDS‑R2(Ligilactobacillus animalis)保藏号为:CGMCC NO.24560。本发明从健康宠物兔粪便中分离获得的益生菌中筛选出来具有预防腹泻功能的动物联合乳杆菌ZJUIDS‑R2。该菌株具有较强的胆盐水解酶活力,在耐酸耐胆盐方面比其他乳酸菌有明显的优势,适合肠胃环境并具有增殖能力。该菌株无抗生素耐性,具有抗菌活性,能抑制肠内有害的病原菌。所述动物联合乳杆菌ZJUIDS‑R2可在制备预防宠物腹泻产品中的应用。本发明中的动物联合乳杆菌可广泛用于开发预防腹泻相关益生功能产品。
Description
技术领域
本发明属于动物微生物技术领域,涉及具有抗腹泻功能的动物联合乳杆菌,尤其涉及一株抗动物腹泻动物联合乳杆菌ZJUIDS-R2及其应用。
背景技术
在家兔和一些动物饲养过程中,因其脆弱的消化系统常发生胃肠道疾病。腹泻的发生通常与肠道微生物的区系变化有关,约80%的患病兔会出现腹泻这一症状。出现该症状的原因可以分为环境因素、营养因素和病原因素,在宠物兔的饲养过程中,占比最大的原因是营养因素,例如饲喂高蛋白、高纤维或含高碳水化合物饲料,以及饲料发霉、饲料中随意添加抗生素等都会引起兔子发生腹泻。
目前,市面上用于预防家兔、猫狗等动物腹泻的疫苗较少,进行治疗时主要采用药物调控和生物调控。随着禁止使用抗生素的进程,腹泻等疾病防治开始寻找新的出路,益生菌、寡聚糖和酶制剂等具有巨大的应用前景,其中益生菌相关的研究最多。
2001年,FAO和WHO联合专家咨询会将益生菌定义为“活的微生物,当摄入足够的数量时,会给宿主带来未指明的健康益处”。机体摄入的外来有益细菌,通过与胃肠道免疫系统和微生物菌群相互作用,对宿主健康产生积极的影响。当受到外界刺激后,肠道内菌群失调,有害菌大量繁殖,有益菌难以对抗,自身稳态被打破,此时补充益生菌事半功倍,能在较短时间内发挥作用。益生菌在动物机体内通过免疫调节、增加肠道屏障功能、维持肠道菌群平衡、调节营养物质等作用机制影响宿主健康,能有效降低幼龄动物的发病率和死亡率、提高成年动物生产性能、改善肉质、提高饲料转化率等。益生菌作为一种天然高效环保的添加剂,是目前为止最有效的抗生素替代品。
发明内容
本发明的目的是提供一株抗动物腹泻动物联合乳杆菌ZJUIDS-R2,所述动物联合乳杆菌ZJUIDS-R2(Ligilactobacillus animalis)已于2022年3月21日保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏号为:CGMCC NO.24560。本发明所提供的动物联合乳杆菌ZJUIDS-R2(Ligilactobacillus animalis)的16s rDNA全序列如SEQ ID No.1所示。
本发明从健康宠物兔的粪便中筛选出一株动物联合乳杆菌ZJUIDS-R2(Ligilactobacillus animalis),通过细菌形态学、生理学、培养特性和16S rDNA测序等对该菌进行了鉴定。
本发明所提供动物联合乳杆菌ZJUIDS-R2(Ligilactobacillus animalis)的菌落形态学特征为:在MRS琼脂培养基上形成明显的菌落,直径1~2mm、圆形、凸起、边缘整齐、表面光滑、乳白色、不透明;革兰氏染色阳性,两端钝圆,短棒状,单个排列或短链状排列,无芽孢,无鞭毛。
本发明的另一个目的是提供所述动物联合乳杆菌ZJUIDS-R2在制备预防宠物腹泻产品中的应用。本发明涉及的宠物主要有:犬、猫、兔、豚鼠、龙猫、仓鼠等。本发明主要是针对动物的细菌性腹泻,且偏向多种致病菌混合感染导致的腹泻。导致动物腹泻的病原微生物主要又分为细菌、真菌、病毒、寄生虫等。引起动物腹泻的细菌常见于大肠杆菌、沙门氏菌、魏氏梭菌、巴氏杆菌、产气荚膜梭菌等,其中以大肠杆菌为病因的腹泻占首位,单一病原菌可以引起动物腹泻,也可以与其他病原菌混合感染发病,在生产和生活中混合感染引起的腹泻占比最大。
所述产品为具有抗腹泻功能的药品、保健品、宠物饲料和宠物零食等。
本发明所述的具有预防腹泻功能的药品包括具有抗腹泻功能的活菌制剂和菌液,该活菌制剂中动物联合乳杆菌ZJUIDS-R2的活菌数为1.0×1010~5.0×1010CFU/g。
所述具有抗腹泻功能的保健品是具有预防腹泻功能的宠物奶粉(ZJUIDS-R2有效菌保证值1×108CFU/g)和滴剂(ZJUIDS-R2有效菌保证值1×109CFU/ml)。
本发明所述的具有抗动物腹泻功能的饲料由动物联合乳杆菌ZJUIDS-R2单独或与其他菌株混合发酵制备。所述饲料优选为具有预防腹泻功能的草粉(例如为提摩西草草粉)混合物。
本发明所提供的动物联合乳杆菌ZJUIDS-R2(Ligilactobacillus animalis)在动物试验中腹泻预防率达到87.5%(n=8)。其具有以下能力:(1)具有较强的胆盐水解酶活力;(2)能耐受酸和胆盐,并且具有一定的增殖能力;对常见的抗生素敏感;具有抗菌活性;具有较高的凝集率和疏水性。即,能耐受胃肠道环境、无抗生素耐性、能抑制肠内有害的病原菌,与肠上皮细胞具有较高的亲和力,能阻碍致病菌在肠道的定植的和感染。
本发明从健康宠物兔粪便中分离获得的益生菌中筛选出来具有预防动物腹泻功能的动物联合乳杆菌。该菌株具有较强的胆盐水解酶活力,该菌株在耐酸耐胆盐方面比其他乳酸菌有明显的优势,适合肠胃环境并具有增殖能力。无抗生素耐性,具有抗菌活性。本发明中的动物联合乳杆菌可广泛用于开发预防动物腹泻相关益生功能产品。
附图说明
图1为动物联合乳杆菌ZJUIDS-R2的菌落形态图。
图2为动物联合乳杆菌ZJUIDS-R2革兰氏染色的菌体形态图(放大倍数100×40)。
图3为动物联合乳杆菌ZJUIDS-R2的16S rDNA的电泳鉴定图。其中1:ZJUIDS-R2;N:阴性对照;M:5000bp DNA Marker。
条带从上自下分别为100bp,250bp,500bp,750bp,1000bp,2000bp,3000bp,5000bp。
图4为动物联合乳杆菌ZJUIDS-R2抗菌活性的平板示例。其中A:ZJUIDS-R2对大肠杆菌的抗菌活性;B:ZJUIDS-R2对鼠伤寒沙门氏菌的抗菌活性;C:ZJUIDS-R2对金黄色葡萄球菌的抗菌活性;D:ZJUIDS-R2对单核细胞增生李斯特菌的抗菌活性。
图5为动物联合乳杆菌ZJUIDS-R2抗生素敏感性的平板示例。其中1:氨苄西林;2:青霉素;3头孢曲松;4:庆大霉素;5:四环素;6:红霉素;7:环丙沙星;8:林可霉素;9:氯霉素;10:复方新诺明。
图6为动物联合乳杆菌ZJUIDS-R2腹泻粪便形状图。
图7为腹泻兔组织剖检图。
图8为动物联合乳杆菌ZJUIDS-R2抗腹泻肠道切片图。
具体实施方式
下面结合附图和具体实施例对本发明进行进一步描述,但本发明的保护范围并不仅限于此。
实施例1、动物联合乳杆菌ZJUIDS-R2的筛选及鉴定:
1.动物联合乳杆菌ZJUIDS-R2的筛选
1.1样品来源
本发明所使用的菌株均从花鸟市场、健康宠物兔中采集到的各种兔子粪便样品中分离获得。粪便样品共采集53份,其中所有采样对象在被采样前的至少一个月时间内未出现腹泻等胃肠道疾病。
1.2菌株的分离纯化
每份样本取2颗新鲜粪便,用无菌EP管收集,立即送至实验室进行菌株分离。取1颗粪便放入1mL的无菌生理盐水中,涡旋混匀后静置10min,3000r离心5min;然后在超净台中吸取富集液100uL,用无菌生理盐水进行十倍梯度稀释,选取10-5、10-6、10-7、10-8四个稀释梯度,每个梯度取菌液100μL涂于MRS琼脂培养基上,于37℃培养24h。培养结束后,从琼脂培养基中选择生长有50~150个单菌落的平板,挑取典型菌落,在MRS琼脂平板上多次划线纯化,直至整个平板上的菌落形态一致,挑取单菌落到MRS液体培养基增菌培养。其中共得到97株乳酸菌,得到的菌株均在含40%甘油的MRS液体培养基中于-80℃冷冻保存。
将分离得到的97株乳酸菌进行抗菌活性鉴定,在5株抗菌活性结果较好的菌株中,结合耐酸、耐胆盐试验结果筛选出1株,命名为动物联合乳杆菌ZJUIDS-R2。
2.动物联合乳杆菌ZJUIDS-R2的鉴定
2.1菌落特征
动物联合乳杆菌ZJUIDS-R2在MRS琼脂培养基培养24h后,在MRS琼脂培养基上形成明显的菌落,直径1~2mm、圆形、凸起、边缘整齐、表面光滑、乳白色、不透明(见图1)。
2.2显微镜下形态:
动物联合乳杆菌ZJUIDS-R2菌落涂片:革兰氏染色呈阳性,两端钝圆,短棒状,单个排列或短链状排列(见图2)。
2.3 16S rDNA鉴定
使用细菌基因组DNA抽提试剂盒提取目标菌株基因组DNA,将提取的基因组DNA作为PCR扩增的模板,采用细菌通用引物27F和1492R进行16S rDNA的PCR实验,PCR反应扩增结束后,取PCR产物进行琼脂糖凝胶检测拍照,扩增片段长度为1.5kb左右(见图3)。
将PCR产物送至生工生物工程(上海)有限公司进行测序,结果如SEQ ID NO:1所示,在NCBI网站上进行BLAST序列比对,结果显示该序列与动物联合乳杆菌的16S rDNA序列同源性超过99%。
根据菌株ZJUIDS-R2的序列比对结果,确定筛选的乳酸菌ZJUIDS-R2为动物联合乳杆菌(Ligilactobacillus animalis)
实施例2动物联合乳杆菌ZJUIDS-R2抗腹泻能力的确认
1.菌株发酵液的制备
将甘油管中保存的菌株在MRS琼脂平板上划线活化2~3次,然后挑取单菌落在MRS液体培养基中扩大培养,37℃静置培养24h,菌液的浓度达到108CFU/mL左右,取出后4℃离心机8000rmp,离心15min,收集上清液。
2.体外抗菌能力的测定
2.1指示菌株培养
将四种指示菌株(大肠杆菌ATCC 25922、鼠伤寒沙门氏菌ATCC 13311、金黄色葡萄球菌CMCC 26003、单核细胞增生李斯特菌CMCC5400均购自中国工业微生物菌种保藏中心)从-80℃冰箱取出,放于室温溶解,分别挑取少量甘油管菌液于LB培养基上活化,24h后挑取LB培养基上的指示菌株单菌落再划线传代3次。将最后一次活化的单菌落接种于LB液体培养基中,37℃静置培养24小时后取出,4℃离心机8000rmp,离心15min,弃去上清液保存下层菌体沉淀并重悬,使活菌数达到108CFU/mL。
2.2琼脂扩散法
采用国际通用琼脂扩散法测量乳酸菌代谢产物的抑菌活性。
使用光口锥形瓶配置LB固体培养基,待培养基灭菌后置于55℃水浴保温,等温度降下来后放于超净台中,将指示菌菌液按照1%的量接入锥形瓶中,摇晃至混合菌液,将指示菌悬液与LB固体培养基混合好后倒入已经放置牛津杯的平皿中制成15mL/皿的LB平板。待LB平板凝固后,用无菌镊子轻轻拔掉牛津杯。每孔定量加入200μL,以恩诺沙星溶液为阳性对照,接入乳酸菌悬液和发酵上清液,将接好液体的平板放于37℃,培养24h。培养结束后,用游标卡尺测量抑菌圈直径并记录。
表1.ZJUIDS-R2对病原菌抑制能力的结果
表1的结果显示,ZJUIDS-R2对常见的致病菌都有较好的抗菌效果,其中对大肠杆菌和鼠伤寒沙门氏菌的抗菌效果最好。抑菌圈的结果也可见图4。(说明菌株来源)
实施例3动物联合乳杆菌ZJUIDS-R2的耐酸性、耐胆盐性和疏水性的确认
乳酸菌酸耐受性的测定
挑取长势最好的ZJUIDS-R2单菌落在MRS液体培养基中扩大培养24h,以10%(v/v)的量将活菌数为108CFU/mL的乳酸菌菌悬液接种于PH为1.5的MRS液体配培养基中(液体培养基PH使用1M的NaOH与HCl调节),分别在试验进行的0h、3h采样。为保证数值准确,使用倾注法进行活菌数的计数。取100μL的乳酸菌菌悬液注入15mL的55℃左右的MRS固体培养基内,冷却至平皿内培养基定型,37℃静置培养培养24h后计数,并基于所得数值分别计算乳酸菌3h后的酸耐受率,酸耐受计算公式如下:
酸耐受率(%)=A3/A0×100
A3=经酸处理3h的乳酸菌株的活菌数;A0=乳酸菌株初始溶液的活菌数
乳酸菌胆盐耐受性的测定
同样,挑取长势最好的ZJUIDS-R2单菌落在MRS液体培养基中扩大培养24h,以10%(v/v)的量将活菌数为108CFU/mL的乳酸菌菌悬液接种于0.3%牛胆盐(v/v)的MRS液体配培养基中,分别在试验进行的0h、3h采样。为保证数值准确,使用倾注法进行活菌数的计数。取100μL的乳酸菌菌悬液注入15mL的55℃左右的MRS固体培养基内,冷却至平皿内培养基定型,37℃静置培养培养24h后计数,并基于所得数值分别计算乳酸菌3h后的胆盐耐受率,胆盐耐受计算公式如下:
胆盐耐受率(%)=T3/T0×100
T3=胆盐处理3h的ZJUIDS-R2的活菌数;T0=ZJUIDS-R2菌株初始溶液的活菌数。
乳酸菌疏水性的测试
取2mLZJUIDS-R2培养菌液和2mL二甲苯彻底混合,37℃水浴充分震荡5min,并在2h后测量水相的吸光值,ZJUIDS-R2疏水性计算公式如下:
疏水作用力(%)=(A0-A2)/A0×100
A0=ZJUIDS-R2菌株0h的吸光值;A2=ZJUIDS-R2菌株处理后2h的吸光值。
表2.ZJUIDS-R2对酸、胆盐耐受性和疏水性结果
菌株 | 酸耐受率(%) | 胆盐耐受率(%) | 疏水性(%) |
ZJUIDS-R2 | 20.9 | 0.9 | 29.8 |
益生菌必须能够耐受胃肠道中胃酸和胆汁等一系列不良环境而活着才能发挥其益生作用。本发明提供的动物联合乳杆菌ZJUIDS-R2在pH 1.5的条件下能够生长增殖,其能顺利通过胃中的酸性环境到达小肠。同时,动物联合乳杆菌ZJUIDS-R2能耐受胆盐,在肠道内存活,进而能有效地预防腹泻的作用。动物联合乳杆菌ZJUIDS-R2疏水率为29.8%,属于中等疏水性菌株。
实施例4动物联合乳杆菌ZJUIDS-R2的抗生素感受性的确认
将培养24h的浓度约为1010CFU/mL动物联合乳杆菌ZJUIDS-R2菌悬液按1%的量加入冷却至55℃左右的灭过菌的MRS琼脂培养基中,充分混合,定量加入15mL/皿。待凝固后,用镊子取药敏纸片放于培养基上。将培养皿正面朝上放于37℃培养箱中培养24h。以无抗生素的纸片为空白对照。测量抑菌圈直径。每个重复三次。
动物联合乳杆菌ZJUIDS-R2对抗生素感受性抑菌圈直径如表3所示,图片的结果见图5。参考CLSI(2017)药敏试验标准可得,动物联合乳杆菌ZJUIDS-R2对庆大霉素、氨苄西林、红霉素、氯霉素等均表现出极高的敏感性,对青霉素、四环素和环丙沙星表现出敏感性。实验结果表明,动物联合乳杆菌ZJUIDS-R2对常见的抗生素敏感。
表3动物联合乳杆菌ZJUIDS-R2对抗生素敏感性结果
注:S,敏感;I,中介;R,耐药
随着抗生素在临床治疗的广泛应用,乳酸菌的耐药性也越来越严重,长期摄入耐药性的乳酸菌会给临床治疗带来很大的困难。本发明所提供的动物联合乳杆菌ZJUIDS-R2对常见的抗生素敏感,不会对人体健康造成危害。
实施例5.动物联合乳杆菌ZJUIDS-R2对宠物兔腹泻的预防作用
1、试验动物和试验设计
试验动物选取24只体重接近、健康程度一致的30日龄新西兰大白兔,随机分为3组,每组雌雄各半,分别记为N(空白对照)组、A(抗生素饲喂)组、T(动物联合乳杆菌ZJUIDS-R2饲喂)组。试验周期28天,前三周,N组、A组和T组分别饲喂无菌生理盐水、抗生素(恩诺沙星)、动物联合乳杆菌ZJUIDS-R2,最后一周饲喂常见肠道致病菌(大肠杆菌、鼠伤寒沙门氏菌、单核细胞增生李斯特菌和金黄色葡萄球菌),初步探索动物联合乳杆菌ZJUIDS-R2对细菌性腹泻的预防作用。
2、基础日粮与饲养管理
本实验断奶幼兔采用笼养方式,每天饲喂2次,根据幼兔日龄调整饲喂量,每晚将未吃完的饲料进行称量,自由饮水,每天打扫兔舍的卫生,保证幼兔良好的生活环境。其中A组为基础日粮+生理盐水;B组为基础日粮+拜有利(3mg/Kg/d);T组为基础日粮+动物联合乳杆菌(1mL/kg/d,1×108cfu/mL)。其他按照兔场的日常管理进行。
基础日粮的原料有:玉米、麸皮、豆粕、苜蓿草粉、碳酸钙、氯化钠、鱼粉、复合维生素、复合微量元素等。基础日粮营养成分见表4。
表4基础日粮营养成分表
注:表中营养成分均为实测值。
3、测定指标
在试验周期内按时记录兔的采食量、体重,试验结束时,每只断奶幼兔进行耳源静脉采血,取1mL放入肝素钠采血管,取1.5mL放于无菌空白EP管。随后,对断奶幼兔进行剖检取样,取内脏器官肝脏、脾脏和肾脏;每段肠管取样三份;取样前,快速测量兔蚓突长度。试验结束后从兔生长性能、血液生化、血液炎症、血液抗体浓度等指标分析饲喂动物联合乳杆菌ZJUIDS-R2对断奶幼兔的影响。
(1)腹泻发生率
试验后期,断奶幼兔饲喂肠道致病菌后,观察并记录断奶幼兔腹泻发生情况,拍照记录腹泻幼兔粪便形态,计算各组断奶幼兔腹泻发生率。腹泻的粪便形状见图6。图6显示腹泻初期可观察到兔肛门周围存在粪便内容物,粪便形状不规则,出现一头带尖或者两头都带尖,粪便普遍变小且带有胶冻样物质(见图6A)。严重者排出稀软粪便,粪便不成形,排出疑似脱落的肠黏膜(见图6B)。
由表5可以看出,N组的腹泻率在所有分组中最高,且明显高于A组、T组的腹泻率,而A组、T组腹泻率基本无差异。即给断奶幼兔饲喂抗生素和ZJUIDS-R1均能降低幼兔的腹泻率。
表5益生菌对断奶幼兔腹泻率的影响
腹泻发生率(100%) | |
N组 | 37.5 |
A组 | 12.5* |
T1组 | 12.5* |
*代表差异显著;
(2)生长性能
通过计算每组断奶幼兔的采食量、日增重和饲料转化率初步评估益生菌对断奶幼兔生长性能的影响。
从表6结果可以看出,N组日采食量与A组存在明显差异,N组日采食量最高,其次是T组、A组;在3个分组中,N组平均日增重最多,其次是A组和T组,但3组之前无显著性差异;计算饲料转化率后发现,A组饲料转化率最高,其次是N组、T组。即在试验期间,N组日采食量最高,同样日增重也最高,但A组料重比最低,益生菌对断奶幼兔生长性能无显著性影响。
表6益生菌对断奶幼兔生长性能的影响
测定指标 | 日采食量(g) | 日增重(g) | 饲料转化率 |
N组 | 139.0±10.6<sup>a</sup> | 36.1±3.3<sup>a</sup> | 3.9±0.26<sup>a</sup> |
A组 | 114.3±6.9<sup>b</sup> | 31.9±5.6<sup>a</sup> | 3.7±0.74<sup>a</sup> |
T组 | 127.0±8.3<sup>ab</sup> | 28.3±4.3<sup>a</sup> | 4.6±0.5<sup>b</sup> |
注:同一列未共用同一上表字母的均值间存在显著差异(P<0.05)。
(3)血液生化指标
试验结束时,每只断奶幼兔采集血液,使用爱德士全自动生化仪测定血浆葡萄糖(GLU)、血液肌酐(CREA)、血液尿素氮(BUN)、总蛋白(TP)、白蛋白(ALB)、球蛋白(GLOB)、白球比(ALB/GLOB)、丙氨酸氨基转移酶(ALT)、碱性磷酸酶(ALKP)等生化指标。
由表7可知,不同处理下N组、A组、T组这3组GLU、BUN、TP、ALB、GLOB、ALT、ALKP等生化指标均无显著性差异(P>0.05)。但与N组对比分析发现,N、A两组血液内的GLU、BUN、TP、ALB降低,CREA增高。
表7益生菌对断奶幼兔血液生化的影响的影响
检测项目 | 单位 | N组 | A组 | T组 |
葡萄糖GLU | mg/dL | 108.20±28.03 | 95.67±26.90 | 70.33±29.49 |
肌酐CREA | mg/dL | 0.94±0.09<sup>b</sup> | 1.23±0.29<sup>a</sup> | 1.05±0.24<sup>ab</sup> |
尿素氮BUN | mg/dL | 22.4±7.16 | 20.00±10.14 | 17.83±7.91 |
尿素氮/肌酐BUN/CREA | 17.80±4.44<sup>a</sup> | 10.35±4.89<sup>b</sup> | 12.83±3.37<sup>ab</sup> | |
总蛋白TP | g/dL | 34.94±37.51 | 26.28±30.39 | 26.88±29.93 |
白蛋白ALB | g/dL | 18.62±19.63 | 14.78±17.22 | 14.30±16.86 |
球蛋白GLOB | g/dL | 16.48±18.55 | 11.65±13.46 | 12.77±13.49 |
白球比ALB/GLOB | 1.28±0.22<sup>a</sup> | 1.25±0.08<sup>a</sup> | 0.97±0.24<sup>b</sup> | |
谷丙转氨酶ALT | U/L | 61.4±30.53 | 68.67±15.59 | 54.50±9.40 |
碱性磷酸酶ALKP | U/L | 119.80±45.32 | 119.17±29.53 | 94.17±26.70 |
注:同一列未共用同一上表字母的均值间存在显著差异(P<0.05)。
(4)血液炎症指标
试验结束时,每只断奶幼兔进行耳源静脉采血,取1.5mL放于无菌空白EP管内,空白管里的血液,3000r离心10min,分离出上层的血清,通过酶联免疫吸附实验(ELISA)检测血清里的C反应蛋白(CRP)指标。
由表8可知,T组CRP显著低于N、A两组(P<0.05),A组CRP相对N组出现下降,但两组差异不显著(P>0.05)。总体而言,A组、T组都呈下降趋势,即断奶幼兔饲喂抗生素与ZJUIDS-R1均能降低幼兔全身炎症指标,且ZJUIDS-R1降低全身炎症的效果明显好于抗生素。
表8益生菌对断奶幼兔血清C反应蛋白的影响
CRP(mg/L) | |
N组 | 8.51±0.73<sup>a</sup> |
A组 | 8.15±0.73<sup>a</sup> |
T组 | 6.61±0.78<sup>b</sup> |
注:同一列未共用同一上表字母的均值间存在显著差异(P<0.05)。
(5)血液抗体指标
使用上述分离得到的血清,通过酶联免疫吸附实验(ELISA)检测血清抗体浓度(IgG、IgM、IgA)。由表9可知,T组的三种抗体水平都显著高于N组和A组(P<0.05)。即断奶幼兔补充ZJUIDS-R2能显著提高血液内IgA、IgG和IgM抗体水平。
表9益生菌对断奶幼兔血清抗体浓度的影响
注:同一列未共用同一上表字母的均值间存在显著差异(P<0.05)。
(6)益生菌对断奶幼兔盲肠菌群的影响
由表10可知,各分组的coverage指数都等于0.99,表明各组之间样本中序列被测出的概率一样高,同样表明本次测序结果可以代表样本中微生物的真实情况。
通过对比不同分组间的四个指数发现,A组OTU、chaos、Shannon指数最高,T组Simpson指数最低。三组之间α多样性指数无显著性差异。
表10益生菌对断奶幼兔肠道菌群α多样性的影响
注:同一列未共用同一上表字母的均值间存在显著差异(P<0.05)。
断奶幼兔盲肠内菌群在门水平的变化如表11所示。在所有分组中厚壁菌门(Firmicutes)丰度最高,其次是拟杆菌门(Bacteroidetes)、放线菌门(Actinobacteria)和变形菌门(Proteobacteria)。饲喂益生菌后,T组中的Bacteroidetes、Bacteroidetes、Actinobacteria增加,与N组相比,疣微菌门(Verrucomicrobia)、单糖菌门(Saccharibacteria)、蓝藻菌门(Cyanobacteria)等数量增加,表明饲喂益生菌后肠道菌群有所改善。
表11断奶幼兔盲肠菌群在门水平的变化
N组 | A组 | T组 | |
Firmicutes | 40811 | 36755 | 40062 |
Bacteroidetes | 7003 | 7918 | 7761 |
Actinobacteria | 902 | 1767 | 1436 |
Proteobacteria | 472 | 1463 | 492 |
Verrucomicrobia | 252 | 409 | 319 |
Saccharibacteria | 55 | 140 | 149 |
Tenericutes | 125 | 45 | 111 |
Cyanobacteria | 38 | 89 | 145 |
(7)十二指肠组织切片
不同组的剖检和组织切片见图7和图8。图7可见腹泻兔肠道鼓气,肠道积液,肠壁变薄呈透明状,多段肠管空虚,偶见肠套叠现象(见图7A),结肠粪便变小且大小不均,粪便有少量黏液包裹,蚓突可见大量白色突起,触摸有明显颗粒感(见图7B)。图中可以看下益生菌组在组织切片的效果接近与抗生素组,显著优于空白组,表明益生菌处理可以缓解对组织的损伤。
绒毛高度(VH)越高,与肠道内容物的接触面积越大;隐窝深度(CD)越浅表明细胞的成熟度越好,分泌功能和吸收功能也越好;绒毛高度与隐窝深度比值(V/C值)与动物的生长速度呈正相关。由表12可以看出,与N组相比,益生菌组均显著提高了断奶幼兔十二指肠VH和V/C,降低了CD(P<0.05),其作用程度与抗生素组无显著性差异。上述结果表明益生菌对断奶幼兔十二指肠发育存在有益作用。
表12益生菌对断奶幼兔十二指肠的影响
益生菌对断奶幼兔盲肠内短链脂肪酸(SCFAs)的影响
从表13可以看出,在所有分组中,A组盲肠内SCFAs浓度最低,但与N组无显著性变化(P>0.05)。T组中乙酸、丙酸、丁酸浓度均显著高于A组(P<0.05);T组中丙酸浓度显著高于N组(P<0.05)。表明动物联合乳杆菌可以显著提高断奶幼兔盲肠内VFA浓度。进而改善肠道健康,达到抗腹泻的效果。
表13益生菌对断奶幼兔盲肠内SCFAs的影响
乙酸(mol/L) | 丙酸(mol/L) | 丁酸(mol/L) | |
N组 | 8.21±1.19<sup>ab</sup> | 0.67±0.11<sup>b</sup> | 1.72±0.76<sup>ab</sup> |
A组 | 7.21±2.01<sup>b</sup> | 0.59±0.08<sup>b</sup> | 1.16±0.53<sup>b</sup> |
T组 | 10.29±1.90<sup>a</sup> | 0.94±0.17<sup>a</sup> | 2.44±0.79<sup>a</sup> |
注:同一列未共用同一上表字母的均值间存在显著差异(P<0.05)。
实施例6利用动物联合乳杆菌ZJUIDS-R2制备抗腹泻菌粉
1.动物联合乳杆菌ZJUIDS-R2菌泥的制备
挑取动物联合乳杆菌ZJUIDS-R2单菌落接种于50mL MRS液体培养基中,放置37℃培养箱中培养24h。再次按5%的接种量于250mL MRS液体培养基中活化,放置37℃培养箱中培养24h。最后将活化好的动物联合乳杆菌ZJUIDS-R2以5%接种量于10L发酵罐中进行高密度厌氧培养,于37℃、pH 6.8的条件下培养24h。之后在8000r/min、4℃条件下离心15min,弃去上清液,收集菌体沉淀,用无菌磷酸盐缓冲液(pH 7.0)漂洗菌体2次。即可得到动物联合乳杆菌ZJUIDS-R2菌泥。
2.保护剂的制备
冻干保护剂含有15%的脱脂乳粉,5%的海藻糖,3%的谷氨酸钠,1%的甘油,0.5%的半胱氨酸盐酸盐。水作为溶剂。于110℃灭菌备用。
3.动物联合乳杆菌ZJUIDS-R2菌粉的制备
将上述制备的动物联合乳杆菌ZJUIDS-R2菌体沉淀按1:5的比例与保护剂溶液充分混匀。于-40℃条件下预冻5h,使其均匀冻结在容器内壁上,然后进行真空冷冻干燥,干燥18~20h后,即可得到动物联合乳杆菌ZJUIDS-R2菌粉。用生理盐水复水后,洗涤两次,测得动物联合乳杆菌ZJUIDS-R2菌粉中活菌数为1.0×1010~5×1010CFU/g。
制备的菌粉可直接用于猫、狗、兔及豚鼠等宠物,或和宠物的食物或水等混合后使用。
动物联合乳杆菌ZJUIDS-R2菌粉在宠物上的抗腹泻效果
将上述制备好的菌粉(0.1-1g/天,依据体重确定剂量)饲喂给腹泻的兔子、猫、狗和仓鼠,经过2天后其观察腹泻率下降和粪便形状改变,结果如下表所示。饲喂抗腹泻的动物联合乳杆菌ZJUIDS-R2菌粉后,兔子、猫、狗和仓鼠的腹泻率均有显著的下降,同时动物粪便的形状及软硬程度也有明显的改善。表明该菌株对这些动物的腹泻具有明显的改善效果。
表14饲喂菌粉后动物的腹泻率(%)
菌株 | 兔子 | 猫 | 狗 | 仓鼠 |
ZJUIDS-R2 | 26.7 | 33.3 | 31.3 | 41.7 |
实施例7利用动物联合乳杆菌ZJUIDS-R2制备预防兔腹泻草粉
动物联合乳杆菌ZJUIDS-R2预防兔腹泻提摩西草粉的步骤如下:
(1)原料准备:提摩西草收割后,保证清洁,无腐败变质,将其切断至1~2cm,再打磨成粉;
(2)将实施例6所制备的动物联合乳杆菌ZJUIDS-R2菌粉按0.02‰接种量接种到原料中,混合均匀;
(3)储藏条件:避光保存,温度为15~25℃,时间为2年。
实施例8利用动物联合乳杆菌ZJUIDS-R2制备预防兔腹泻滴剂
预防兔腹泻滴剂,由以下质量比的原料制备而成:低聚半乳糖1%、油酸1%、植物油10%、卵磷脂1%、维生素2%、ZJUIDS-R1菌粉、纯净水35%、丙三醇50%。
将实施例6所制备的动物联合乳杆菌ZJUIDS-R2菌粉按1%的比例添加进去,混合后活菌数达到1.0×108CFU/ml以上。产品密封、避光低温保存,保质期为1年。
实例例9利用动物联合乳杆菌ZJUIDS-R2制备抗腹泻宠物奶粉
宠物兔专用防腹泻奶粉,包括以下百分比的原料:羊奶粉65-70%、混合植物油25%,ZJUIDS-R2有效菌保证值108CFU/g,矿物质2%,预混剂(维生素、微量元素等)2%、葡萄糖和麦芽糊精3%。上述防腹泻奶粉可增强机体免疫力,从根源上解决断奶幼兔肠道功能不适、肠道致病菌感染等引起的腹泻问题。
实施例10利用动物联合乳杆菌ZJUIDS-R2制备犬猫胃肠道处方膏
主要包括以下百分比的原料:麦芽糖15%、茯苓10%、陈皮15%、山楂5%、白术(炒)5%、鸡内金5%、预混剂(维生素和微量元素等)5%、纯净水35%,ZJUIDS-R2有效菌保证值108CFU/mL,制备成牙膏状。本产品可以促进肠道免疫,增加机体抵抗力,适用于犬猫疫苗或驱虫不完善、消化不良、食欲不振等。使用方法:体重小于5kg:每天两次,每次3mL;体重5-15kg:每天两次,每次4mL;体重大于15kg:每天两次,每次5mL。本产品可直接饲喂,可拌在犬猫粮食内混合饲喂,也可涂抹在爱宠鼻子上让其舔食。
最后,还需要注意的是,以上列举的仅是本发明的若干个具体实施例。显然,本发明不限于以上实施例,还可以有许多变形。本领域的普通技术人员能从本发明公开的内容直接导出或联想到的所有变形,均应认为是本发明的保护范围。
序列表
<110> 浙江大学
<120> 一株抗动物腹泻动物联合乳杆菌ZJUIDS-R2及其应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1415
<212> DNA
<213> 动物联合乳杆菌(Ligilactobacillus animalis)
<400> 1
ctcaggatga acgctggcgg cgtgcctaat acatgcaagt cgaacgaaac ttctttatca 60
ccgagtgctt gcactcaccg ataaagagtt gagtggcgaa cgggtgagta acacgtgggc 120
aacctgccca aaagaggggg ataacacttg gaaacaggtg ctaataccgc ataaccatag 180
ttaccgcatg gtaactatgt aaaaggtggc tatgctaccg cttttggatg ggcccgcggc 240
gcattagcta gttggtgagg taaaggctta ccaaggcaat gatgcgtagc cgaactgaga 300
ggttgatcgg ccacattggg actgagacac ggcccaaact cctacgggag gcagcagtag 360
ggaatcttcc acaatgggcg aaagcctgat ggagcaacgc cgcgtgggtg aagaaggtct 420
tcggatcgta aaaccctgtt gttagagaag aaagtgcgtg agagtaactg ttcacgtttc 480
gacggtatct aaccagaaag ccacggctaa ctacgtgcca gcagccgcgg tcatacgtag 540
gtggcaagcg ttatccggat ttattgggcg taaagggaac gcaggcggtc ttttaagtct 600
gatgtgaaag ccttcggctt aaccggagta gtgcattgga aactgggaga cttgagtgca 660
gaagaggaga gtggaactcc atgtgtagcg gtgaaatgcg tagatatatg gaagaacacc 720
agtggcgaaa gcggctctct ggtctgtaac tgacgctgag gttcgaaagc gtgggtagca 780
aacaggatta gataccctgg tagtccacgc cgtaaacgat gaatgctaag tgttggaggg 840
tttccgccct tcagtgctgc agctaacgca ataagcattc cgcctgggga gtacgaccgc 900
aaggttgaaa ctcaaaggaa ttgacggggg cccgcacaag cggtggagca tgtggtttaa 960
ttcgaagcaa cgcgaagaac cttaccaggt cttgacatct tctgacaatc ctagagatag 1020
gactttccct tcggggacag aatgacaggt ggtgcatggt tgtcgtcagc tcgtgtcgtg 1080
agatgttggg ttaagtcccg caacgagcgc aacccttatt gttagttgcc agcattaagt 1140
tgggcactct agcaagactg ccggtgacaa accggaggaa ggtggggatg acgtcaaatc 1200
atcatgcccc ttatgacctg ggctacacac gtgctacaat ggacggtaca acgagtcgca 1260
agaccgcgag gtttagcaaa tctcttaaag ccgttctcag ttcggattgt aggctgcaac 1320
tcgcctacat gaagtcggaa tcgctagtaa tcgcggatca gcatgccgcg gtgaatacgt 1380
tcccgggcct tgtacacacc gcccgtcaca ccatg 1415
Claims (8)
1. 一株抗动物腹泻动物联合乳杆菌(Ligilactobacillus animalis)ZJUIDS-R2,其特征在于,所述动物联合乳杆菌ZJUIDS-R2的保藏号为:CGMCC NO. 24560。
2.权利要求1所述的动物联合乳杆菌ZJUIDS-R2在制备预防宠物腹泻产品中的应用。
3.根据权利要求2所述的应用,其特征在于,所述产品为具有抗腹泻功能的药品、保健品、宠物饲料或宠物零食。
4.根据权利要求3所述的应用,其特征在于,所述药品包括具有抗腹泻功能的活菌制剂和菌液,该活菌制剂中动物联合乳杆菌ZJUIDS-R2的活菌数为1.0×1010~5.0×1010CFU/g。
5.根据权利要求3所述的应用,其特征在于,所述保健品是具有预防腹泻功能的宠物奶粉和滴剂。
6.根据权利要求3所述的应用,其特征在于,所述的饲料由动物联合乳杆菌ZJUIDS-R2单独或与其他菌株混合发酵制备。
7.根据权利要求6所述的应用,其特征在于,所述的饲料为具有预防腹泻功能的草粉混合物。
8.根据权利要求2所述的应用,其特征在于,所述宠物为兔子、猫、狗、豚鼠和仓鼠。
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