CN114874952A - 干酪乳杆菌hjd及其应用 - Google Patents
干酪乳杆菌hjd及其应用 Download PDFInfo
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- CN114874952A CN114874952A CN202210674625.0A CN202210674625A CN114874952A CN 114874952 A CN114874952 A CN 114874952A CN 202210674625 A CN202210674625 A CN 202210674625A CN 114874952 A CN114874952 A CN 114874952A
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- hjd
- lactobacillus casei
- escherichia coli
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- mouse
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Abstract
本发明涉及微生物技术领域,特别涉及干酪乳杆菌HJD及其应用,本发明筛选出的菌株干酪乳杆菌HJD对猪霍乱沙门氏菌和大肠埃希菌等肠道指示菌均表现出广谱和较高的抗菌活性。对低pH和0.30%的胆盐均有较好的耐受性,对Caco‑2细胞的粘附性较强,且不携带质粒编码的可转移的抗生素耐药基因。安全性试验显示,HJD株在血平板上不产生α或β溶血;小鼠经口毒性试验中,灌胃后小鼠未出现任何不良反应,同时,灌胃HJD株的小鼠与对照组相比,能显著增加小鼠的增重,由此可见,干酪乳杆菌HJD是潜在的益生菌候选菌株,具有能提高动物生长性能和免疫力的功效。
Description
【技术领域】
本发明涉及微生物技术领域,特别涉及干酪乳杆菌HJD及其应用。
【背景技术】
在养猪业中,抗生素能有效提高饲料转化率,降低临床病死率,但在养殖业中过度和不加选择地使用抗生素作为生长促进剂,增加了人类和动物病原体的抗生素耐药菌株的数量。为此,欧盟和中国分别从2006年1月和2020年1月开始禁止在动物饲料中使用抗生素生长促进剂。因此,迫切需要开发用于动物生产的抗生素替代品。到目前为止,一些潜在的抗生素替代品,如益生菌、有机酸、植物提取物和抗菌肽,已作为饲料添加剂在畜禽生产中被证明有利于动物健康。在这些添加剂中,益生菌被认为是较好的抗生素生长促进剂替代品,因为它们是安全的,可以通过调节宿主肠道微生物的平衡,保持宿主健康。益生菌被定义为活的微生物,当给予足够的剂量时,给宿主带来健康益处。近年来研究表明,益生菌具有抑制病原微生物生长、提高饲料转化率,改善肉品质、增强免疫应答等有益作用。益生菌的作用机制有多种,如产生有机酸、释放抗菌物质、竞争性地排除致病菌、产生消化酶、营养物质和生长因子以及刺激免疫应答等。
乳酸菌(Lactic acid bacteria,LAB)主要通过发酵碳水化合物产生乳酸,包括乳酸杆菌属、肠球菌属、芽孢杆菌属、链球菌属和其他一些微生物。乳酸菌作为益生菌在猪生产中得到了广泛的应用。
虽然对益生菌的研究较多,但目前在猪生产中应用的益生菌种类仍较少,有待开发出更多的益生菌株。此外,益生菌是菌株特异性的,不同菌株具有不同的有益特性和作用。因此,本研究旨在从猪粪便中分离乳酸菌,并对这些菌株进行体外评价,以期在猪生产中作为抗生素替代品。对具有抗菌活性的菌株,进一步评估它们的低酸和胆盐耐受性、Caco-2细胞粘附性、抗生素敏感性和安全性等益生菌特性。
【发明内容】
鉴于上述内容,有必要提供一种具有耐酸、耐胆盐、抑菌性强、抗生素耐药性强的益生菌,拥有这些特性的益生菌具有能很好的在动物体内定植,形成优势菌群,从而改善动物肠道环境、提高动物免疫力的功效。
为达到上述目的,本发明所采用的技术方案是:
一株干酪乳杆菌(Lactobacillus casei)HJD,该菌株保藏于中国微生物菌种保藏管理委员会普通微生物中心,地址:北京市朝阳区北辰西路1号院3号中国科学院微生物研究所,保藏编号为CGMCC NO:21367,保藏日期为2020年12月14日。
本发明还包括所述干酪乳杆菌(Lactobacillus casei)HJD在制备抑菌、耐胆盐、耐酸和/或提高动物生长性能相关制剂上的应用。
进一步的,所述抑菌抑制的细菌为:猪霍乱沙门氏菌(SalmonellaCholeraesuis)、金黄色葡萄球菌(Staphylococcus aureus)和/或大肠埃希菌(Escherichia coli)。
进一步的,所述猪霍乱沙门氏菌(Salmonella Choleraesuis)为:猪霍乱沙门氏菌CVCC 2139。
进一步的,所述大肠埃希菌(Escherichia coli)为:大肠埃希菌(O147:K89)CVCC199、大肠埃希菌(O141:K99)CVCC 223、大肠埃希菌(O139)CVCC 1496和/或大肠埃希菌(O157:H7) CICC 21530。
本发明具有如下有益效果:
1、本发明旨在筛选具有良好肠道致病菌抑制能力并有望在生猪生产中替代抗生素的潜在益生菌。从健康猪中分离到2株干酪乳杆菌(HJD和TH2)、1株鼠李糖乳杆菌(MRS1)和1 株屎肠球菌(S-3),并进行16S rDNA测序。对所有分离株进行抑菌活性、耐酸和胆盐耐受性、 Caco-2细胞粘附性、抗生素敏感性和小鼠经口毒性试验。结果表明,干酪乳杆菌HJD对猪霍乱沙门氏菌CVCC 2139、大肠埃希菌(O147:K89)CVCC 199、大肠埃希菌(O141:K99)CVCC 223和大肠埃希菌(O139)CVCC 1496等肠道指示菌均表现出广谱和较高的抗菌活性。对低pH (pH 2.5和pH 3.5)和0.30%的胆盐均有较好的耐受性,对Caco-2细胞的粘附性较强,且不携带质粒编码的可转移的抗生素耐药基因。安全性试验显示,HJD株在血平板上不产生α或β溶血;小鼠经口毒性试验中,灌胃后小鼠未出现任何不良反应,同时,灌胃HJD株的小鼠与对照组相比,能显著增加小鼠的增重,证明该菌株是安全的,HJD株还具有增加动物日增重的潜在特性。由此可见,干酪乳杆菌HJD是潜在的益生菌候选菌株,具有能提高动物生长性能和免疫力的功效。
【附图说明】
图1为本申请实施例的HJD菌株在平皿上的形态图。
【具体实施方式】
本说明书中公开的所有特征,或公开的所有方法或过程中的步骤,除了互相排斥的特征和/或步骤以外,均可以以任何方式组合。
本说明书(包括任何附加权利要求、摘要)中公开的任一特征,除非特别叙述,每个特征只是一系列等效或类似特征中的一个例子而已。
本实施例实验的过程使用的材料和方法如下:
细菌菌株和Caco-2细胞
猪霍乱沙门氏菌CVCC 2139、金黄色葡萄球菌CVCC 546、大肠埃希菌(O147:K89)CVCC 199、大肠埃希菌(O141:K99)CVCC 223和大肠埃希菌(O139)CVCC 1496均购于中国兽医微生物菌种保藏中心(China Veterinary Culture Collection Center,CVCC)。大肠埃希菌(O157:H7) CICC 21530购于中国工业微生物菌种保藏管理中心(China Center ofIndustrial Culture Collection,CICC),大肠埃希菌ATCC 25922购于中国典型培养物保藏中心(China Center for Type Culture Collection,CCTCC)。这些菌株在Luria-Bertani(LB)培养液中保存,37℃有氧培养24h。
Caco-2细胞(人结肠上皮癌细胞系)购自上海博谷生物技术有限公司。细胞在添加10%(v/v) 胎牛血清、青霉素G(100U/mL)和链霉素(100U/mL)的DMEM培养基中保存,在37℃,5%CO2 条件下培养。
实施例1:
本实施例为干酪乳杆菌(Lactobacillus casei)HJD的筛选方法:
本实施例的菌株从猪粪便中分离而得,具体方法如下:
采集外观健康猪的粪便样品,每份样品称取5g,与45mL无菌0.9%(w/v)NaCl混合,150rpm振荡30min,混合液用棉纱过滤,滤液10倍连续稀释至10-6,分别将100μL 10-4、10-5和10-6稀释液涂布在MRS琼脂平板上,37℃厌氧或有氧培养48h。
菌株的鉴定:
采用革兰氏染色和过氧化氢酶试验对分离菌进行初步鉴定。将初步鉴定为乳酸菌的菌株再进行16S rDNA鉴定,操作如下:采用细菌基因组DNA试剂盒从分离菌中提取基因组DNA,用通用引物16sRp1(5'-AGAGTTTGATCATGGCTCAG-3')和16sRp2(5'-GTGTGACGGGCGGTGTGTAC-3')从提取物中扩增16S rDNA序列。PCR反应总体积为25μL,其中PCR Master Mix(2×)12.5μL,上下游引物(10μM)各0.5μL,模板DNA 2μL,ddH2O 9.5μL。扩增程序为:94℃5min;94℃30s,57℃30s,72℃2min,共35个循环;72℃延伸10min。PCR 产物(~1380bp)纯化后送生工生物技术有限公司测序,序列提交NCBI GenBank。
其结果显示从MRS琼脂平板上分离得到4个菌株(HJD、TH2、MRS1、S-3)。所有分离菌均为革兰氏阳性、杆状或球菌状、过氧化氢酶阴性菌。16S rDNA扩增测序结果显示,HJD 和TH2为干酪乳杆菌,MRS1为鼠李糖乳杆菌,S-3为屎肠球菌。4个分离株16S rDNA序列已提交GenBank,登录号为MZ558169-MZ558174。
本申请要求保护的干酪乳杆菌HJD保藏于中国微生物菌种保藏管理委员会普通微生物中心,地址:北京市朝阳区北辰西路1号院3号中国科学院微生物研究所,保藏编号为CGMCC NO:21367,保藏日期为2020年12月14日。
本发明所述干酪乳杆菌(Lactobacillus casei)HJD在平皿上的形态特征如图1所示,该菌株菌落呈白色、边缘光滑、中间凸起呈半球状。
实施例2:
菌株抗菌活性的研究:
采用打孔琼脂扩散法测定分离株对猪霍乱沙门氏菌CVCC 2139、金黄色葡萄球菌CVCC 546、大肠埃希菌(O147:K89)CVCC 199、大肠埃希菌(O141:K99)CVCC 223、大肠埃希菌(O139) CVCC 1496和大肠埃希菌(O157:H7)CICC 21530的抗菌活性。操作如下:将营养琼脂融化,与过夜培养的指示菌混合,倒入90mm的培养皿中,待琼脂凝固后,在琼脂平板上打4个直径6mm孔。将过夜培养的分离菌在7000rpm离心10min,然后用孔径为0.22μm滤器过滤上清,得到无细胞上清(cell free supernatant,CFS)。将200μL的分离菌CFS加入孔中,培养板在37℃孵育24h。以孔周围的透明圈直径判定抗菌活性。每个样本进行3次重复。得到的结果参见表1:
表1不同分离菌株对肠道指示病原菌的抗菌活性 单位:mm
注:表中“—”表示无抑制作用,a,b,c,d:表示同一列数据差异显著(p<0.05)。
由表1可知,大肠杆菌(O141:K99)CVCC 223和大肠杆菌(O157:H7)CICC 21530的生长被所有分离的菌株抑制,而金黄色葡萄球菌CVCC 546的生长仅受到干酪乳杆菌TH2和屎肠杆菌S-3的抑制;在4株候选菌株中,干酪乳杆菌HJD对猪霍乱沙门氏菌CVCC 2139(17.83mm)和大肠杆菌(O147:K89)CVCC 199(15.65mm)的抗菌活性显著高于其它试验菌株。说明,干酪乳杆菌HJD对常见的猪肠道致病菌具有良好的抗菌特性,具有预防和治疗细菌感染的潜力。
实施例3:
pH值耐受性实验:
离心洗涤过夜培养的分离菌,沉淀分别在pH为2.5、3.5和4.5的0.9%NaCl中重悬90 min。取100μL悬液加入10mL MRS肉汤,37℃培养16h。将未加入分离菌的MRS肉汤(pH 6.2±0.2)作为空白对照,通过测量600nm处的光密度(OD)值评估菌株的pH耐受性。具体参见表2:
表2不同分离菌株的pH值耐受性
注:表中a,b,c:表示同一列数据差异显著(p<0.05)。
由表2可知,干酪乳杆菌HJD和鼠李糖乳杆菌MRS1的低酸(pH2.5和pH3.5)耐受力显著强于其它分离菌,而屎肠球菌S-3对酸敏感。即屎肠球菌S-3不耐酸。
实施例4:
胆盐耐受性实验
将100μL过夜培养的分离菌接种于含有不同浓度(0.03、0.3、0.5%)猪胆盐的MRS肉汤中,37℃培养16h。以未添加胆盐的MRS肉汤作为空白对照。通过测量600nm处的光密度(OD)值评估菌株的胆盐耐受性。结果见表3:
表3不同分离菌株的胆盐耐受性
注:a,b,c:表示同一列数据差异显著(p<0.05)。
由表3可知,干酪乳杆菌HJD、干酪乳杆菌TH2和鼠李糖乳杆菌MRS1对0.30%的胆盐耐受良好。与此相反,屎肠杆菌S-3对胆盐的耐受性较低。
实施例5:
Caco-2细胞粘附性实验
离心收集过夜培养的分离菌,用无菌PBS洗涤三次,并在DMEM中重悬,平板计数法测定初始细菌数。将500μL细菌悬液添加到长满Caco-2细胞单层的24孔细胞培养板。37℃培养90min后,移除细菌悬液,并用无菌PBS洗涤Caco-2细胞三次。每孔加入500μL 0.1% (v/v)Triton X-100洗脱附着在Caco-2细胞表面的细菌,并用平板计数法进行细菌计数。具体见表4:
表4不同分离菌株对Caco-2细胞的粘附率
注:a,b,c,d:表示同一列数据差异显著(p<0.05)。
由表4可见,试验菌株的粘附能力各不相同。干酪乳杆菌HJD的粘附率显著高于其它试验菌株,为36.93%。
实施例6:
抗生素敏感性测定
采用Kirby-Bauer法测定分离菌对抗生素的敏感性。质控菌株为大肠埃希菌ATCC25922。选用的抗生素为β内酰胺类(氨苄西林,10μg/片;头孢曲松,30μg/片)、大环内酯类(阿奇霉素,15μg/片)、林可酰胺(克林霉素,2μg/片)、氨基糖苷类(链霉素,10μg/片;庆大霉素,10 μg/片)、四环素类(四环素,30μg/片)、糖肽类(万古霉素,30μg/片)、喹诺酮类(环丙沙星,5μg/ 片;萘啶酸,5μg/片)。将过夜培养的分离菌和质控菌分别涂布于MRS琼脂平板和Mueller-Hinton琼脂平板上,37℃厌氧或好氧培养24h,测量抑菌圈直径。根据临床与实验室标准协会(CLSI)的指导方针,将试验菌株按药敏程度分为敏感(S)、中度敏感(I)和耐药(R)。具体见表5:
表5不同分离菌的抗生素敏感性检测
表中,各符号代表为:AMP氨苄西林,CEF头孢曲松,AZI阿奇霉素,CLI克林霉素,STR链霉素,GEN 庆大霉素,TET四环素,VAN万古霉素,CIP环丙沙星,NAL萘啶酸;
R耐药,I中毒敏感,S敏感
由表5可见,酪乳杆菌HJD对氨苄西林、链霉素、庆大霉素、万古霉素、环丙沙星、萘啶酸耐药,对头孢曲松和阿奇霉素中度敏感,对克林霉素和四环素敏感。
实施例7:
①质粒DNA提取
使用TIANprep Mini Plamid试剂盒从分离菌中提取质粒DNA,纯化的DNA产物进行1%琼脂糖电泳分析。电泳分析结果显示,所有分离株均未检测到质粒。
②溶血测定
将过夜培养的分离菌接种到含5%(w/v)羊血的MRS琼脂板上。37℃培养24h后,观察菌落周围的溶血情况。以金黄色葡萄球菌CVCC 546作为阳性对照。溶血分为三种类型:β-溶血(透明带)、α-溶血(绿色带)、γ-溶血(无溶血)。结果表明,试验菌株在羊血琼脂平板上生长时均不表现出α溶血和β溶血活性。
③小鼠体内安全性评价
将小鼠随机分为7组,包括6个实验组和1个阴性对照组(每组6只,雌雄各半)。将过夜培养的分离菌离心洗涤三次,并在无菌PBS中重悬至终浓度1×109CFU/mL。每只小鼠以10mL/kg的剂量灌胃分离菌,阴性对照组灌胃PBS。试验期14d,分别于第0天和第14天记录各组小鼠的体重,每天记录并分析每只小鼠的食欲、外表、精神状态、行为以及死亡率。具体见表6:
表6不同分离菌灌胃小鼠的安全性测定
注:*P<0.05:与PBS对照组相比,差异显著。
从表6可见,与PBS对照组相比,试验组小鼠的健康状况良好,未出现任何不良反应。如表6所示,灌胃干酪乳杆菌HJD组小鼠在第14天的体重增加显著高于对照组(P<0.05)。
上述实施例2-7采用的是SPSS生物统计软件(release 16.0standard edition;SPAA,Inc., Chicago)进行数据分析,数值以平均值±标准差(SD)表示,通过单向方差分析(one-way ANOVA) 进行数据比较。P<0.05表示显著性差异。
上述实验结果表明,集约化养猪增加了肠道疾病的风险,细菌性感染是仔猪腹泻的主要诱因。新生仔猪和断奶仔猪易受潜在致病菌(如大肠杆菌和沙门氏菌)的感染。乳酸菌在代谢过程中能释放乳酸、过氧化氢、细菌素等广谱抗菌物质,对致病菌有明显的抑制作用。本研究旨在从健康猪的粪便中筛选和鉴定对常见肠道致病菌具有抗菌活性的益生菌LAB菌株。筛选出的LAB菌株对不同指示肠道致病菌具有不同的抗菌活性,各菌株均对其中5株指示菌株具有抑菌活性,抑菌谱较宽。干酪乳杆菌HJD对猪霍乱沙门氏菌CVCC 2139(17.83mm) 和大肠杆菌(O147:K89)CVCC 199(15.65mm)的生长抑制作用较强,结果表明干酪乳杆菌HJD 对常见肠道致病菌具有良好的抗菌性能,具有预防和治疗细菌感染的潜力。
酸和胆汁的耐受性是选择潜在益生菌菌株的首要标准之一。体外酸和胆汁耐受性试验的结果往往能预测菌株在宿主体内环境中生存的能力。本研究中,干酪乳杆菌HJD和鼠李糖乳杆菌MRS1对低pH(pH 2.5和pH 3.5)和0.30%的胆盐耐受良好,表明它们有可能通过胃进入肠道。
对肠道上皮细胞的粘附性是益生菌筛选的重要指标。许多动物和人类细胞系可作为肠道模型,如Caco-2、T84、HT-29、IEC-6和IEC-18。Caco-2细胞系已被广泛用于评价菌株对肠道的粘附能力。在本研究中,干酪乳杆菌HJD的粘附率为36.93%。
抗生素敏感性是挑选益生菌的重要标准,抗生素耐药性是国内外关注的热点问题。乳酸菌对抗生素的耐药性分为固有耐药和获得性耐药,固有耐药的耐药基因存在于基因组中,不具有传播性;获得性耐药通常由细菌突变引起,或可能携带可移动的遗传元件(质粒和转座子),并可能传播给其他细菌。因此,确定分离乳酸菌的抗生素耐药基因是存在于基因组还是游离质粒是关键。
潜在的益生菌必须通过动物口服毒性试验进行安全性评估。本研究中,对所有挑选菌株均进行了小鼠安全性试验。结果表明,所有小鼠均保持良好的身体状况,无不良反应。此外,与对照组相比,灌胃干酪乳杆菌HJD显著增加了小鼠的体重。上述结果表明,所有试验菌株都具有潜在的食用安全性,干酪乳杆菌HJD具有提高动物采食量、转化率和增重等生产性能的潜力。
实施例8:
从实施例1-7的研究可知,本发明的菌株HJD有良好的耐胆盐、耐酸、抑菌、粘附性高的特性。
为此,可以选用合适的包埋剂用于加工上述菌株的冻干粉,该冻干粉应用到动物体内,由于内含菌株具有耐胆盐、耐酸、抑菌、粘附性高的特性,使得该冻干粉剂中的微生物在动物体内有良好的定植、生长能力,进而起到调节肠道,提高动物生长性能的效果。
综上,干酪乳杆菌HJD的体内外益生菌特性和安全性研究表明,该菌株是较好的益生菌候选菌株,具有广谱和较高的抗菌活性,并且在动物体内有良好的定植、生长能力,对常用抗生素有耐药性,在今后的动物体中更容易形成菌群优势调节动物肠道,能进一步加强动物的抗感染、生长性能、免疫等方面的有益作用。
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对本发明范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。因此,本发明的保护范围应以所附权利要求为准。
Claims (5)
1.一株干酪乳杆菌(Lactobacillus casei)HJD,其保藏编号为CGMCC NO:21367。
2.如权利要求1所述干酪乳杆菌(Lactobacillus casei)HJD在制备抑菌、耐胆盐、耐酸和/或提高动物生长性能相关制剂上的应用。
3.根据权利要求2所述的应用,其特征在于,所述抑菌抑制的细菌为:猪霍乱沙门氏菌(Salmonella Choleraesuis)和/或大肠埃希菌(Escherichia coli)。
4.根据权利要求2所述的应用,其特征在于,所述猪霍乱沙门氏菌(SalmonellaCholeraesuis)为:猪霍乱沙门氏菌CVCC 2139。
5.根据权利要求2所述的应用,其特征在于,所述大肠埃希菌(Escherichia coli)为:大肠埃希菌(O147:K89)CVCC 199、大肠埃希菌(O141:K99)CVCC 223、大肠埃希菌(O139)CVCC 1496和/或大肠埃希菌(O157:H7)CICC 21530。
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