CN116731899A - 一株警犬源唾液乳杆菌ca03及应用 - Google Patents
一株警犬源唾液乳杆菌ca03及应用 Download PDFInfo
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Abstract
本发明公开了一株耐酸、耐胆盐、抑菌效果好的唾液乳杆菌及其应用,并验证了其对动物生长、免疫和肠炎的影响。该菌株通过分离、功能筛选和鉴定而获得,该菌可作为犬食品添加剂使用,具有促进生长,抑制病原菌,提高免疫力和改善肠炎的潜力。为维护犬肠道健康提供新思路,也为犬源微生态制剂提供可靠的备用菌种。本发明适用于饲料厂和规模化养犬场。
Description
技术领域
本发明涉及一株经分离而获得的警犬源耐酸、耐胆盐、抑菌效果好且具有改善肠道炎症和增加机体免疫功能作用的唾液乳杆菌,属于微生物技术领域。
背景技术
近年来,随着人们经济水平的提高和饲养宠物意愿的增加,宠物行业迎来高速发展期。犬作为人类饲养数量最多的宠物,因其喜爱舔舐,吞咽异物的生活习性,常受到胃肠道疾病的侵扰。益生菌对动物胃肠道健康具有优秀的维护和改善作用,被逐渐应用于宠物犬的饲养中。但是部分市售犬用微生态在质量上存在一定问题,而且多数犬用微生态产品使用的核心菌株仍是人用益生菌常见菌株,如动物双歧杆菌BB12、鼠李糖乳杆菌LGG等,并非犬源肠道益生菌,其犬肠道定植能力存疑。而分离自犬肠道组织或粪便中的犬源益生菌,因其来源于犬肠道微生物组,一般具有较好的犬肠道定植能力,因此,使用从犬肠道中分离筛选性能优良的犬源益生菌制备微生态制剂或可进一步提高犬用微生态制剂中益生菌的定植能力,增强其应用效果。警犬是具有特种工作能力的工作犬。其通过严格的品种选育,且经过长期严格规律的管理和高强度的系统训练,相较于普通宠物犬,其具有更加的优秀运动性能。已有研究表明,人类专业运动员肠道菌群多样性和分度比普通人更高,且优秀水平运动员的肠道菌群多样性也显著高于普通水平运动员。经过长期训练,具有高运动能力的警犬是性能十分优秀的犬只,相比与普通宠物犬,在肠道菌群组成上很可能存在一定差异。从品种优良,训练得当的,身体条件优异的优秀警犬的肠道组织或粪便中分离筛选得到性能优良犬源益生菌的概率也更高。迄今为止,尚未见到有关警犬源唾液乳杆菌筛选及其在医生作用方面的报道。
发明内容
发明目的:鉴于背景所述,同时针对目前致病菌耐药性增强及抗生素禁用导致疾病很难治愈的情况,本发明的目的是提供一株具能够适应犬胃肠环境,在抑菌、抗炎维持肠道健康方面效果好的警犬源唾液乳杆菌及其应用。
技术方案:本发明解决其技术问题所采用的技术方案如下:本发明提供了一株警犬源唾液乳杆菌(Lactobacillus salivarius)CA03,所述警犬源唾液乳杆菌(Lactobacillus salivarius)CA03于2022年12月26日保藏于中国普通微生物菌种保藏管理中心,保藏编号为:CGMCC No.26329,保藏地址为:中国北京。
本发明内容还包括一种益生菌制剂,所述益生菌制剂包括所述的警犬源唾液乳杆菌(Lactobacillus salivarius)CA03。
本发明内容还包括一种动物食品添加剂,所述动物食品添加剂包括所述的警犬源唾液乳杆菌(Lactobacillus salivarius)CA03。
本发明内容还包括一种动物饲料,所述动物饲料包括所述的警犬源唾液乳杆菌(Lactobacillus salivarius)CA03。
本发明内容还包括所述的警犬源唾液乳杆菌(Lactobacillus salivarius)CA03或所述的益生菌制剂在制备治疗肠炎药物方面的应用。
其中,所述肠炎为动物肠炎。
本发明内容还包括所述的警犬源唾液乳杆菌(Lactobacillus salivarius)CA03或所述的益生菌制剂在制备降低鼠伤寒沙门氏菌含量的药物方面的应用。
本发明内容还包括所述的警犬源唾液乳杆菌(Lactobacillus salivarius)CA03或所述的益生菌制剂在制备增强机体免疫力或提高血清IgG水平药物方面的应用。
本发明内容还包括所述的警犬源唾液乳杆菌(Lactobacillus salivarius)CA03或所述的益生菌制剂在制备增加结肠隐窝深度和绒毛长度的药物中的应用。
本发明采集健康警犬的粪便,灭菌生理盐水冲悬后,利用MRS培养基进行分离培养和鉴定,得到一株对沙门氏菌有显著抑制作用的的警犬源唾液乳杆菌(Lactobacillussalivarius)CA03,然后进行耐酸、耐胆盐试验。最后利用该菌剂灌胃小鼠,检测益生菌处理后小鼠的生长性能、肠道炎症、肝脏和粪便载菌量以及血清免疫学指标的变化。
有益效果:与现有技术相比,本发明具备以下优点:本发明的警犬源唾液乳杆菌(Lactobacillus salivarius)CA03具备耐酸、耐胆盐的性能,而且能够有效的治疗动物的肠道炎症,提高血清免疫学的相关的指标,该菌可作为犬食品添加剂使用,具有促进生长,抑制病原菌,提高免疫力和改善肠炎的潜力。为维护犬肠道健康提供新思路,也为犬源微生态制剂提供可靠的备用菌种。本发明适用于饲料厂和规模化养犬场。
附图说明
图1为本发明的技术流程。
图2为本发明“唾液乳杆菌CA03”的耐酸、耐胆盐实验结果。
图3为本发明“唾液乳杆菌CA03”的抑菌实验结果。
图4为本发明“唾液乳杆菌CA03”饲喂后,小鼠的体重变化结果。
图5为本发明“唾液乳杆菌CA03”饲喂后,小鼠的结肠长度变化结果。
图6为本发明“唾液乳杆菌CA03”饲喂后,小鼠的空肠和结肠的病理组织学变化结果。
图7为本发明“唾液乳杆菌CA03”饲喂后,小鼠粪便中沙门氏菌载量的变化结果。
图8为本发明“唾液乳杆菌CA03”饲喂后,小鼠的血清的免疫学指标变化结果。
具体实施方式
实施例1本发明“唾液乳杆菌CA03”的获取与鉴定
1、本发明“唾液乳杆菌CA03”的获取与鉴定
(1)菌株分离
采集自于南京警犬新鲜粪便,立即送实验室检测。每份粪便样品取0.5g,使用5mL灭菌0.9%生理盐水冲悬,涡旋混匀仪混匀成悬浊液后使用灭菌生理盐水进行倍比稀释,稀释10-6,10-7,10-8梯度,各吸取100μL,在MRS固体平板上进行平板涂布,温箱37℃培育24h。使用灭菌移液枪枪头挑取形态大小不一致的单菌落,接种于10mL MRS液体培养基中,37℃培育12h。再用接种针在MRS固体平板上划线,37℃培育24h。挑取单株菌落,接种于10mL MRS液体培养基中,37℃培育24h,获得单株乳酸菌的纯培养液。
(2)菌株分子生物学鉴定
解冻-80℃中保存菌种,用接种针平板划线于MRS固体平板上,温箱37℃培育12h。用移液器枪头沾取平板上单菌落。以纯化后的细菌单菌落直接作为DNA模板,扩增该菌株16SrRNA基因。引物:27F:5′-AGAGTT TGATCCTGG CTCAG-3′和1492R:5′-TAC CTT GTT ACGACT T-3′。由上海生工生物有限公司合成。PCR扩增体系为50μL体系,其中包括25μL 2×TaqPCRMasterMix;19μL ddH2O;2μL正向引物27F(25μ mol/L)、2.0μL反向引物1492R(25μ mol/L)和2μL DNA模板。扩增条件为95℃5min;94℃30s,55℃30s,72℃2min,该部分30个循环;72℃7min,4℃终止反应。PCR产物全序列由上海生工生物有限公司测定。
(3)鉴定结果
测得16S rRNA基因后,在NCBI网站上进行BLAST序列比对,16S rRNA基因序列在NCBI进行BLAST序列比对后,确定所分离的菌为唾液乳杆菌,即为警犬源唾液乳杆菌(Lactobacillus salivarius)CA03,该菌株在2022年12月26日保藏于中国普通微生物菌种保藏管理中心,保藏编号为:CGMCC No.26329。
(4)唾液乳杆菌CA03株耐酸耐胆盐检测
为了检测从警犬粪便中分离得到的唾液乳杆菌CA03在低PH和高胆盐浓度下的生长状态,直接选用pH=2的生理盐水和0.3%胆盐浓度生理盐水作为耐酸耐胆盐检测条件。益生菌耐酸试验:解冻-80℃中保存菌种,吸取100μL菌液接种于30mlMRS液体培养基中,温箱37℃培育12h,活化菌株。吸取活化后菌液20mL并分为两份,每份各10mL,3000rpm 12min离心弃去上清液获得细菌沉淀。每份加入10mL生理盐水冲悬混匀,洗去MRS培养基残留。然后3000rpm 12min离心弃去上清液获得细菌沉淀。将其中一份细菌沉淀加入10mL生理盐水冲悬混匀,另一份加入10mL pH=2的生理盐水冲悬混匀,两份菌液在温箱37℃培育2h后,3000rpm 12min离心,弃去上清液,再各用10mL生理盐水冲悬洗,去除处理液残留。两管菌液进行稀释涂布,稀释梯度为106,107,108。温箱37℃培育12h后,进行菌落计数,计算菌液细菌浓度。再计算pH=2生理盐水处理的细菌存活率,计算公式为:酸性条件下细菌存活率=(pH=2生理盐水处理后的菌液浓度/普通生理盐水处理后的菌液浓度)%。益生菌耐胆盐试验:试验步骤与益生菌耐酸试验的试验步骤基本一致,需将pH=2的生理盐水换成0.3%猪胆盐浓度的生理盐水。0.3%猪胆盐生理盐水处理的细菌存活率计算公式为:胆盐条件下细菌存活率=(0.3%猪胆盐生理盐水处理后的菌液浓度/普通生理盐水处理后的菌液浓度)%。益生菌耐酸耐胆盐试验均完成后,计算益生菌耐酸耐胆盐性能指数=(胆盐条件下细菌存活率×酸性条件下细菌存活率)。
(5)耐酸耐胆盐筛选结果
通过耐酸耐胆盐性能检验,如图2所示,唾液乳杆菌CA03在低PH和高浓度胆盐中的存活率均高于30%,具有较强的耐酸耐胆盐性能。
(6)菌株抑制病原菌效果试验
解冻-80℃中保存的志贺杆菌、鼠伤寒沙门氏菌、小肠结肠炎耶尔森菌和金黄色葡萄球菌接种在LB培养基中,摇床37℃培育12h进行细菌活化,再将首次活化后的菌液接种到LB培养基,摇床37℃培育12h再次活化。使用稀释涂布平板法计算菌液中细菌浓度,稀释并配制成106CFU/mL菌液。采用牛津杯打孔方法,向含有50ml LB琼脂的离心管中缓缓倒入5ml106CFU/mL的病原菌,微微摇晃使其均匀分布。在培养基没有完全凝固时每个平板放入2个大小相同的牛津杯,等LB平板完全凝固后,其中一孔中加入100μL MRS液体培养基作为阴性对照,最后一孔倒入100μL 108CFU/mL待测乳酸菌菌液。各倒三个平板作为技术重复,温箱37℃培育8h后,平板出现抑菌圈,使用游标卡尺测量圈直径。
(7)菌株抑制病原菌效果试验结果
如图3所示,唾液乳杆菌CA03对鼠伤寒沙门氏菌和金黄色葡萄球菌都能形成明显抑菌圈,且抑菌圈直径均显著(P<0.05)高于阴性对照组。试验结果表明唾液乳杆菌CA03对鼠伤寒沙门氏菌和金黄色葡萄球菌的繁殖均有一定抑制作用,具备优秀犬源益生菌的能力。
(8)菌株超氧化物歧化酶活性检测
按照超氧化物歧化酶(Superoxide Dismutase,SOD)活性检测试剂盒(微量法)说明书检测SOD活性。
(8)菌株SOD活性检测结果
SOD广泛存在于动物、植物、微生物和培养细胞中,催化超氧化物阴离子发生岐化作用,生成H2O2和O2。SOD不仅是超氧化物阴离子清除酶,也是H2O2主要生成酶,在生物抗氧化系统中具有重要作用。通过黄嘌呤及黄嘌呤氧化酶反应系统产生超氧阴离子(O2 -),O2 -可还原氮蓝四唑生成蓝色甲臌,后者在560nm处有吸收;SOD可清除O2 -,从而抑制了甲臌的形成;反应液蓝色越深,说明SOD活性愈低,反之活性越高。如表1所示,唾液乳杆菌CA03组活性氧清除率为4.525%,表明该菌具有一定SOD活性。
表1唾液乳杆菌CA03 SOD活性检测
实施例2动物实验
(1)实验试剂准备
五周龄雄性C57BL/6小鼠购自扬州大学比较医学中心共64只。小鼠在SPF环境下饲养一周后进行试验。所用菌株唾液乳杆菌CA03由实施例1中筛选所得。鼠伤寒沙门氏菌SL1344由本实验室保存。
(2)实验动物分组
选择五周龄雄性C57BL/6小鼠共32只,将小鼠平均分为4组,每组8只,分别为:阴性对照组、沙门氏菌组、唾液乳杆菌组、唾液乳杆菌保护组。各组于每日14:00灌胃各组灌胃液,其中阴性对照组和沙门氏菌组每只小鼠每日灌胃200μL无菌PBS缓冲液;唾液乳杆菌组和唾液乳杆菌保护组每只小鼠每日灌胃200μL 5×108CFU/mL唾液乳杆菌灌胃液。试验第21d时,阴性对照组、唾液乳杆菌组改为灌胃200μL无菌PBS缓冲液;沙门氏菌组、唾液乳杆菌保护组每只小鼠改为灌胃200μL 5×109CFU/mL沙门氏菌攻毒液。第27d小鼠处死并采样。
(3)生长体重检测
每日灌胃之前对各小鼠进行体重测量与记录
(4)生长体重检测
如图4所示,试验第23d之前,4组小鼠的体重基本保持一致,无显著差异(P>0.05)。试验第25d,沙门氏菌组小鼠体重显著下降,而阴性对照组和唾液乳杆菌组的小鼠体重显著高于沙门氏菌组(P<0.05),唾液乳杆菌保护组和沙门氏菌组的小鼠体重无显著差异(P>0.05)。试验第27d,阴性对照组和唾液乳杆菌组小鼠体重显著高于沙门氏菌组(P<0.05);唾液乳杆菌保护组体重显著高于沙门氏菌组(P<0.05)。
(5)病理状态观察
21d攻毒后,随时监控小鼠日常精神体态,直至27d小鼠处死解剖采样,对其表观病理变化进行记录。27d采取小鼠组织样本,制作石蜡切片。对石蜡切片进行HE染色使用光学显微镜观察空肠绒毛和隐窝形态变化时,按照盲法对各组的肠道进行病理打分。病理打分共检测三个相关的指标,分别是炎性因子浸润水平(0-4:无,较轻,中度,严重,全部),隐窝损伤(0-4:无,1/3基底处受损,2/3基底处受损,全部隐窝受损,隐窝和上皮完全丢失),黏膜损伤(0-4:无,黏膜,黏膜下层,黏膜透壁,全部)。统计方法为各参数的评分乘以反应组织损伤程度的百分比(×1,0-25%;×2,26-50%;×3,51-75%;×4,76-100%),将所有分数相加取平均值,最高分数为4分,每组保证有30个视野进行形态打分。
(6)病理状态观察结果
如图5所示被益生菌处理过的小鼠结肠长度显著高于沙门氏菌组(P<0.05)。
如图6所示,沙门氏菌组空肠绒毛长度最低,显著低于益生菌组和益生菌保护组(P<0.05)。沙门氏菌组隐窝深度最高,唾液乳杆菌保护组显著低于沙门氏菌组(P<0.05)唾液乳杆菌组显著低于唾液乳杆菌保护组(P<0.05)。沙门氏菌组结肠病理得分最高,显著高于其他组(P<0.05),损伤最严重。
(7)粪便病原菌载菌量检测
27d采样之前,采集所有小鼠的新鲜粪便约0.1g,加入1mL无菌PBS缓冲液中混匀,使用稀释涂布平板法将粪便悬浊液稀释104、105、106,再涂布于链霉素LB培养基,24小时后计数菌落并统计。
(8)粪便病原菌载菌量检测结果
如图7所示,沙门氏菌组粪便中沙门氏菌载菌量最高。对照组和唾液乳杆菌组载菌量较低。
(9)血清学免疫指标检测
使用眼球采血采集每只小鼠血液,静置12h后,离心2000rpm 10min,得到小鼠血清样。使用双抗体夹心法ELISA检测小鼠血清中IgG含量。试验过程遵照ELISA试剂盒说明书操作指南进行。
(10)血清学免疫指标检测结果
如图8所示,沙门氏菌组血清IgG含量最低,显著低于除阳性对照组外的其余组,阴性对照组显著高于阳性保护组(P<0.05)。
Claims (9)
1.一株警犬源唾液乳杆菌(Lactobacillus salivarius)CA03,其特征在于,所述警犬源唾液乳杆菌(Lactobacillus salivarius)CA03于2022年12月26日保藏于中国普通微生物菌种保藏管理中心,保藏编号为:CGMCC No.26329。
2.一种益生菌制剂,其特征在于,所述益生菌制剂包括权利要求1所述的警犬源唾液乳杆菌(Lactobacillus salivarius)CA03。
3.一种动物食品添加剂,其特征在于,所述动物食品添加剂包括权利要求1所述的警犬源唾液乳杆菌(Lactobacillus salivarius)CA03。
4.一种动物饲料,其特征在于,所述动物饲料包括权利要求1所述的警犬源唾液乳杆菌(Lactobacillus salivarius)CA03。
5.权利要求1所述的警犬源唾液乳杆菌(Lactobacillus salivarius)CA03或权利要求2所述的益生菌制剂在制备治疗肠炎药物方面的应用。
6.根据权利要求5所述的应用,其特征在于,所述肠炎为动物肠炎。
7.权利要求1所述的警犬源唾液乳杆菌(Lactobacillus salivarius)CA03或权利要求2所述的益生菌制剂在制备降低鼠伤寒沙门氏菌含量的药物方面的应用。
8.权利要求1所述的警犬源唾液乳杆菌(Lactobacillus salivarius)CA03或权利要求2所述的益生菌制剂在制备增强机体免疫力或提高血清IgG水平药物方面的应用。
9.权利要求1所述的警犬源唾液乳杆菌(Lactobacillus salivarius)CA03或权利要求2所述的益生菌制剂在制备增加结肠隐窝深度和绒毛长度的药物中的应用。
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