CN114703107A - 一株副干酪乳酪杆菌及其在预防婴幼儿链球菌感染中的应用 - Google Patents
一株副干酪乳酪杆菌及其在预防婴幼儿链球菌感染中的应用 Download PDFInfo
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- CN114703107A CN114703107A CN202210489912.4A CN202210489912A CN114703107A CN 114703107 A CN114703107 A CN 114703107A CN 202210489912 A CN202210489912 A CN 202210489912A CN 114703107 A CN114703107 A CN 114703107A
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明属于益生菌筛选与应用技术领域,具体涉及一株新的副干酪乳酪杆菌(Lacticaseibacillus paracasei)及其应用。所提供的副干酪乳酪杆菌分离自健康婴儿的新鲜粪便,已于2021年5月24日保藏于中国典型培养物保藏中心,其保藏号为CCTCC NO:M2021590。该菌株对β‑溶血性链球菌具有很强的抗菌效果,能够提高宿主免疫力,减轻炎症反应,有效预防和治疗链球菌引发的感染,尤其适用于婴幼儿链球菌感染的预防。
Description
技术领域
本发明属于益生菌筛选与应用技术领域,具体涉及一株副干酪乳酪杆菌及其在预防婴幼儿链球菌感染中的应用。
背景技术
B族链球菌(GBS)是一种β溶血性革兰染色阳性链球菌,其致病性多发生于免疫缺陷患者、老人、孕妇及新生儿中。据统计约20%的健康女性携带GBS,妊娠后可引发胎膜早破、晚期流产、早产、胎儿宫内发育不良等一系列不良妊娠结局,也可引起新生儿败血症、肺炎、脑膜炎,甚至死亡等不良预后,故 GBS 在围生期感染性疾病中占据重要地位。另外由于婴儿免疫功能低下,极易通过皮肤或黏膜感染环境中的链球菌,同样可导致婴儿败血症、脑膜炎等。因此,产前筛查 GBS 、良好的卫生习惯以及抗生素预防性使用能够有效降低 GBS引发的婴幼儿早期侵入性感染、院内感染或家庭接触感染等。但由于预防性抗生素的广泛应用,近年来全球 GBS 的耐药性逐年上升,且抗生素的使用增加了患儿坏死性小肠炎、肝肾损伤或者听力损伤等的发病率。
益生菌作为活微生物,如果摄入足够数量,将有益于宿主健康。益生菌可以在肠道内定植,和有害菌作斗争,维护肠道菌群平衡,可通过增强肠道黏膜屏障功能、抑制致病菌生长和黏附、调控免疫细胞活性及促进产生免疫因子等方式调节机体免疫功能。随着对益生菌功效的进一步研究,产生了多种用于治疗或预防的适应症,如炎性肠病、尿路感染、乳腺炎等,并且益生菌在儿科的应用越来越广泛,如可以改善新生儿喂养不耐受、预防过敏性疾病、小儿肠绞痛、早产儿坏死性小肠结肠炎等。
已有研究报道,链球菌感染如阴道感染GBS的妊娠晚期妇女肠道菌群发生紊乱,增加了其体内的炎症状态;败血症患者肠道菌群严重失调,使用一些特殊的益生菌可以调节肠道菌群,降低败血症的发病率。LisaHanson等针对B族链球菌定植阳性或者状态不明的产前女性使用益生菌干预,荟萃分析的结果显示,产前益生菌的使用能够使GBS阴性的概率增加79%。一项RTC研究显示,给予印度农村4556名母乳喂养的婴儿,植物乳杆菌PP11-217口服制剂和低聚果糖,可以预防新生儿败血症和死亡。尽管如此,Garland SM等研究发现益生菌对早产儿败血症的发生率或严重程度的影响是模棱两可的,在一项针对1310名早产儿的试验中,对婴儿配方奶粉中添加短双歧杆菌BBG-001,并没有显示对坏死性小肠结肠炎或败血症有预防作用。这说明益生菌能否发挥作用可能与益生菌菌株的选择、给药方法(剂量、频率、持续时间等)、患者(如与出生体重、体质)及研究样本量等有关。因此,筛选抑制链球菌效果突出的菌株,寻求最佳的给药方式仍是目前研究的热点。
发明内容
本发明的目的是提供一株新的副干酪乳酪杆菌(Lacticaseibacillus paracasei)及其应用;所提供的副干酪乳酪杆菌分离自健康婴儿的新鲜粪便,对β-溶血性链球菌具有很强的抗菌效果,能够提高宿主免疫力,减轻炎症反应,有效预防和治疗链球菌引发的感染,尤其适用于婴幼儿链球菌感染的预防。
本发明所提供的一株副干酪乳酪杆菌,为副干酪乳酪杆菌VHProbi F11(Lacticaseibacillus paracasei VHPribo F11),已于2021年5月24日保藏于中国典型培养物保藏中心,其保藏号为CCTCC NO:M2021590。
本发明所提供的副干酪乳酪杆菌VHPribo F11株,其16s rDNA序列如SEQ ID NO:1所示。
本发明所提供的副干酪乳酪杆菌VHPribo F11株,其Riboprinter 指纹图谱如图2所示;其RAPD指纹图谱如图3所示,rep-PCR指纹图谱如图4所示。
本发明所提供的副干酪乳酪杆菌VHPribo F11株在制备链球菌抗菌剂中的应用。
本发明所提供的副干酪乳酪杆菌VHPribo F11株在制备具有抗氧化功能的制品中的应用。
本发明所提供的副干酪乳酪杆菌VHPribo F11株在制备具有预防链球菌感染功能的制品中的应用。
所述的制品,为功能性食品、保健品、药品或护肤品。
本发明从健康婴儿粪便中筛选出的副干酪乳酪杆菌VHProbi F11对红霉素等常见的抗生素敏感,不产生溶血素,不能够溶解血细胞,具有良好的生物安全性;对胃液具有很强的耐受性。
所述副干酪乳酪杆菌VHProbi F11的抗氧化能力较强,菌体抗脂质过氧化抑制率菌体为23.99%、上清液为71.73%;DPPH清除率达到11.04%,HRS清除率达到43.59%。该菌株还能有效降解胆固醇,降解率达到23.65%;此外,该菌株对黄曲霉素B1吸附能力达到10.41%。
所述副干酪乳酪杆菌VHProbi F11对β-溶血性链球菌的拮抗作用明显,能有效抑制链球菌的生长和繁殖,因此可以广泛应用于链球菌感染的预防和治疗。副干酪乳酪杆菌VHProbi F11能显著降低链球菌感染大鼠炎症因子水平,减轻机体的炎症状态,保持大鼠肠系膜结构的完整性,维持正常的脾组织结构,减轻炎性细胞浸润,尤其是通过在体外环境中对链球菌进行拮抗处理,能显著降低链球菌的感染水平,与正常组差异极小,预防效果突出。
本发明提供的副干酪乳酪杆菌VHProbi F11可用于制备具有预防链球菌感染功能的食品、保健品、药品或护肤品,应用前景广阔。
附图说明
图1为F11菌株凝集现象图;
图2为F11菌株Riboprinter 指纹图谱;
图3为F11菌株的RAPD指纹图谱;
图4为F11菌株的rep-PCR指纹图谱;
图5为各组别大鼠炎症因子水平结果;
图6为各组别大鼠小肠HE染色结果;其中,A 为正常组,B 模型组,C 阳性组,D 益生菌预处理组,E 益生菌后处理组,F 益生菌体外拮抗组;
图7为各组别大鼠脾脏HE染色结果;其中,A 为正常组,B 模型组,C 阳性组,D 益生菌预处理组,E 益生菌后处理组,F 益生菌体外拮抗组。
具体实施方式
本发明提供的副干酪乳酪杆菌VHProbi F11符合法规要求,可以作为一种食品原料来源使用,长期服用不会有副作用及过量的风险。经多相分类学鉴定,副干酪乳酪杆菌VPHrobi F11为一株新发现的菌株。本发明提供的副干酪乳酪杆菌VHProbi F11具有预防链球菌感染的功效,单独使用该菌株且无需与益生元和/或其它益生菌复配即可对链球菌感染,尤其是婴幼儿链球菌感染起到预防功效,具有重要的应用价值。
申请人于2021年5月24日将所述副干酪乳酪杆菌VHProbi F11保藏于武汉大学的中国典型培养物保藏中心,其保藏号为CCTCC NO:M2021590。
本发明所述筛选方法并不局限于实施例所述,已知的能够达到筛选目的的方法均可以,实施例的筛选说明只是对本发明的说明,并不是对本发明保护范围的限制。在不背离本发明精神和实质的情况下,对本发明方法、步骤或条件所作的修改或替换,均属于本发明的范围。
下面结合具体实施例对本发明做详细的描述。
实施例1 副干酪乳酪杆菌VHProbi F11的筛选
1、菌株分离
配制MRS (Man Rogosa Sharpe) 肉汤:纯水1L,蛋白胨10g,牛肉浸取物10g,酵母提取物 5.0g, 乙酸钠5g,葡萄糖5g,磷酸二氢钾2g,吐温80 1.0mL,柠檬酸二胺 2.0g, 碳酸钙20g,七水硫酸镁0. 58g,七水硫酸锰0. 25g,调pH 6.2-6.5。
配置MRS琼脂培养基:1LMRS肉汤添加15g琼脂。
依据2019版《人类遗传资源库伦理规范》,与样本提供者签订项目承诺书和知情同意书后,按照生物样本库标准操作规范,取1g半年内未食用过益生菌制剂的健康婴儿的新鲜粪便样本,经无菌生理盐水稀释后放入无菌样品袋中,用匀浆仪拍打混匀;取100μL混匀液梯度稀释,涂布于MRS琼脂培养基后于37℃厌氧培养48h,待平板长出单菌落镜检。根据镜检结果,申请人共筛选出20株潜在乳酸杆菌,分别命名为F01、F02、……、F18、F19、F20。
2、抗β-溶血性链球菌乳酸菌筛选
乳酸菌菌液制备:将分离得到的20株潜在乳酸菌分别接种至MRS肉汤,37℃静置氧培养48h。
致病菌活化:β-溶血性链球菌(CMCC(B)32210)接种至BHI+5%牛血清培养基中,37℃培养48h。
铺下层培养基,营养琼脂灭菌后倒入平板中,铺满平板即可。待琼脂凝固后,均匀放置无菌牛津杯。铺上层培养基,将培养48h的β-溶血性链球菌取0.4%(v/v)加入BHI牛血清半固体培养基中。菌与培养基混匀后,取14mL倾注到下层培养基上。待凝固后取出牛津杯,并取混匀的100μL乳酸菌发酵液加入牛津杯孔中。在37℃下,培养24h后观察有无抑菌圈。
结果显示,本发明分离得到的20株乳酸菌中,抑菌圈直径超过15mm的有5株菌,分别为F03、F11、F17、F19、F20。
2、凝集吸附试验
进一步采用共孵育法对上述5株具有抑菌效果的乳酸菌进行凝集吸附实验。取活化好的乳酸菌菌悬液和β-溶血性链球菌新鲜菌液一起混合加入24孔板的孔中。将24孔板置于微孔板恒温振荡器,振荡孵育。振荡孵育至少24h,期间观察是否有凝集现象。
结果显示,F11菌株对β-溶血性链球菌的凝集吸附作用最强,抑菌圈和凝集现象如图1所示。
综上,本发明筛选出的F11菌株对β-溶血性链球菌的抗菌效果最佳。
实施例2 F11菌株鉴定
1、菌落形态鉴定
将F11菌株接种于MRS琼脂培养基上,37℃厌氧培养24h后,可见F11单菌落呈乳白色,菌落直径在1-2mm左右,表面光滑,边缘圆整、凸起,显微镜下菌株形态为杆菌。
2、生理生化特性鉴定
本实施例中接种液的准备如下:在无菌条件下,取适量新鲜F11菌液,5000rpm/min离心5 min,用PBS缓冲液洗2次,再用同体积PBS缓冲液重菌体后稀释50倍,作为接种液。
2.1、盐度耐受性试验
在无菌条件下,向96孔板中分别加入190μL盐浓度为1%、2%、3%、4% 、5%、6%、7%、8%的BSM液体培养基,每个盐浓度做3个平行,然后再加入10μL接种液,不接菌的孔作为对照。每孔加入50μL高压灭菌过的石蜡油以防止培养过程中水分蒸发。置于37℃恒温培养,观察培养基是否变浑浊。结果显示F11菌株最大耐受盐浓度为6%。
2.2、温度耐受性试验
取适量新鲜菌液(24h,37℃),5000rpm 离心5 min ,用PSP溶液洗一次,再用同体积PSP溶液重悬后稀释50倍,作为接种液。
将接种液按10% 的接种量接种到10mL MRS液体培养基中,不接菌的5mL MRS液体培养基作为对照,分别置于15℃恒温培养箱培养7天,45℃恒温培养箱培养2天,观察菌液是否变浑浊。
结果显示,F11 菌株在15℃生长良好,45℃有少量菌生长。
2.3、碳源代谢试验
2.3.1、本实施例中所用的基础培养基配方如下:
蛋白胨 1.5g;酵母提取物 0.6g;吐温 80 0.1g;盐溶液 0.5mL;酚红 18mg;蒸馏水 100mL;pH7.4±0.2。盐溶液成分:MgSO4·7H2O 11.5g,MnSO4·4H2O 2.8g,蒸馏水100mL。
配制 10g/100mL 的糖、醇和苷类碳水化合物溶液,并用 0.22μm 的无菌过滤器进行过滤。在无菌条件下,向 96 孔板中加入 20μL 除菌后的碳水化合物溶液,每种碳水化合物 4 个平行,然后加入 170μL 灭菌后含酚红的基础培养基,再加入 10μL 接种液,不接菌反应孔作为对照。每孔加入 50μL 液体石蜡以防止培养过程中水分蒸发。37℃厌氧培养,以酚红为指示剂,观察培养基颜色变化;具体结果见表 1。
表 1 F11菌株碳源代谢结果表
纤维二糖 | 蜜二糖 | 棉子糖 | 甘露醇 | 苦杏仁苷 |
+ | - | - | + | + |
半乳糖 | 乳糖 | 麦芽糖 | 甘露糖 | 水杨苷 |
+ | - | + | + | + |
阿拉伯糖 | 葡萄糖酸钠 | 松三糖 | 核糖 | 山梨醇 |
- | + | + | + | + |
蔗糖 | 海藻糖 | 木糖 | 鼠李糖 | / |
+ | + | - | - | / |
注:“+”阳性反应;“-”阴性反应。
2.4、葡萄糖产酸产气试验
本实施例中所用的培养基配方如下:
蛋白胨 0.5g;酵母提取物 0.3g;吐温 80 0.1mL;盐溶液 A 0.5ml;盐溶液 B0.5ml;乙酸钠 0.5g;葡萄糖 2.5g;2%溴甲酚绿(w/v) 0.05mL;蒸馏水 100ml;
pH6.8~7.0。
将配制好的培养基分装至含有倒置小试管的大试管中,3mL/管,121℃,高
压灭菌 15min。
盐溶液 A 成分:KH2PO4 10g、K2HPO4 1.0g,溶于蒸馏水,定容至 100mL。
盐溶液 B 成分:MgSO4·7H2O 11.5g、MnSO4·2H2O 2.4g、FeSO4·7H2O 0.68g,溶于蒸馏水,定容至 100mL。
在无菌条件下,将接种液按 10%的接种量接种培养基,不接菌的培养基作为对照,然后用 2mL 无菌液体石蜡封住顶部,置于 37℃培养24h,观察培养基颜色有无变化。
结果显示:37℃培养 24h后,培养基由绿色变为黄色,小倒管内无气体,说明 F11菌株发酵葡萄糖产酸,不产气。
2.5、精氨酸产氨试验
在PY基础培养液中加入配制好的精氨酸溶液。
精氨酸溶液的成分及制备如下:
精氨酸1.5g;半胱氨酸(1g/10ml H2O) 0.05ml;蒸馏水10ml;
调pH至7.0,灭菌后加3滴至3ml培养基中。
取适量新鲜菌液(24h,37℃),5000rpm 离心5 min ,用PSP溶液洗一次,再用同体积PSP溶液重悬后稀释50倍,作为接种液。
将接种液按10% 的接种量接种于10mL含精氨酸的培养基中,并同时接种不含精氨酸的培养基作为对照。置适温培养1~3d。
取已生长好的培养液1滴于洁净载玻片表面,加入1~2滴奈氏试剂,若黄色变为棕色,为阳性反应,产氨。
结果显示:奈氏试剂检测结果为阴性,说明F11菌株不产氨。
3、分子生物学鉴定
3.1 16s rDNA 基因序列分析
3.1.1、基因组DNA提取
参照天根细菌基因组DNA提取试剂盒(目录号:DP302)操作。
3.1.2、16s rDNA 基因扩增
引物序列:
27F:AGAGTTTGATCCTGGCTCA;
1492R:GGTTACCTTGTTACGACTT。
通过测序获得F11菌株的16s rDNA序列SEQ ID NO:1,并将该序列在NCBI 数据库中进行比对,初步确定F11菌株为副干酪乳酪杆菌。
3.2 Riboprinter指纹图谱
用一根取菌棒从琼脂培养基平板上沾取已纯化好的单菌落,将其放入有缓冲液的样品管中,用手持搅拌器搅拌使其在缓冲液中悬浮,然后将样品架放入加热器中灭活后放入Riboprinter 系统中,样品经过DNA制备、转膜、成像检测及数据处理后,得到细菌鉴定结果。鉴定结果显示,F11菌株为副干酪乳酪杆菌,其Riboprinter指纹图谱结果见图2。
3.3 RAPD和rep-PCR指纹图谱鉴定
3.3.1、RAPD指纹图谱鉴定
引物序列: GAGGGTGGCGGTTCT。
表2 RAPD 反应体系
反应成分 | 体积 |
TaqDNA 聚合酶(5U/μL) | 0.2 μl |
10×Buffer(含Mg2+) | 2 μl |
引物(10 uM) | 1 μl |
dNTPs(2.5 mM) | 0.8 μl |
DNA模板 | 2 μl |
无菌双蒸水 | 14 μl |
总体积 | 20 μl |
制备1.5%的琼脂糖凝胶板,DL2000DNA Marker 作为结果对照,稳压100V电80min,最后利用凝胶成像系统检测电泳图。F11菌株的RAPD指纹图谱如图3所示。
3.3.2、rep-PCR指纹图谱
引物序列:CTACGGCAAGGCGACGCTGACG。
表3 rep-PCR 的反应体系
反应成分 | 体积 |
r TaqDNA聚合酶 | 0.2 μl |
10×Ex Taq DNA Buffer(含Mg2+) | 2 μl |
引物(10 uM) | 1 μl |
dNTPs(2.5 mM) | 2 μl |
DNA模板 | 2 μl |
无菌双蒸水 | 12.8 μl |
DL2000 DNA Marker 作为结果对照。电压100 V,电泳时间80min 检测扩增结果。F11菌株的的rep-PCR指纹图谱如图4所示。
3.4全基因组测序
取新鲜F11菌液按照1%的体积比例接种到500 mL MRS肉汤培养基中,37 ℃培养20h,8000rpm离心10 min,收集菌体。菌体送到测序中心,得到该菌的全基因序列,基因序列上传至NCBI基因数据库,GenBank 登录号为CP094946-CP094949,包含1个染色体 3个质粒,GenBank登录号分别是CP094946、CP094947、CP094948、CP094949。
将F11菌株的菌落形态以及生理生化特性结果上传至网站http://www.tgw1916.net/bacteria_logare_desktop.htmL, 同时结合文献De Clerck E,et al.Systematic and applied microbiology, 2004, 27(1)50公布的结果,进行比对。综合分子生物学的鉴定结果,确定F11菌株为一株新的副干酪乳酪杆菌,将其命名为副干酪乳酪杆菌VHProbi F11(Lacticaseibacillus paracasei VHPribo F11)。
实施例3 副干酪乳酪杆菌VHProbi F11对人工胃液的耐受性试验
1、人工胃液的配制
分别称取蛋白胨 5g、酵母提取物 2.5g、葡萄糖1g和NaCl 2g,加入1000mL蒸馏水,用稀盐酸调pH3.0,然后115℃灭菌20min。然后使用前加入3.2g猪粘膜胃蛋白酶,摇匀溶解,置37℃水浴摇床中温水浴1h,以模拟人体温度。
2、试验方法
取2mL新鲜菌液,5000rpm/min 离心5min 收集菌体,菌体用生理盐水洗涤3次,再用2mL 生理盐水重悬,作为接种液。取1mL接种液,加入到24mL人工胃液中,置于37℃水浴摇床(200rpm/min)3h,取样1mL,检测活菌量。
活菌计数方法按照国标《GB4789.35-2016-食品微生物检验乳酸菌检验》测定菌量,该菌株经过人工胃液消化后的活菌量(Log CFU/mL)见表4。
表4人工胃肠液消化后的活菌量
消化前 | 人工胃液消化后 |
8.06±0.02 | 8.03±0.03 |
从表4可知,本发明提供的副干酪乳酪杆菌 VHProbi F11 对胃液有很强的耐受性。
实施例4 副干酪乳酪杆菌VHProbi F11的溶血性及抗生素耐受性试验
1、溶血性试验
称取TBS基础培养基的各种组分,溶解,121°C高压灭菌15 min,等培养基冷却到50℃的时候加入5%的无菌脱纤维绵羊血,混匀,倒平板。将测试菌株划线接种于准备好的血细胞平板,37℃培养箱培养,24 ~ 48h观察测试菌是否有溶血现象。
结果显示:副干酪乳酪杆菌 VHProbi F11不能生长,血细胞平板没有变化,说明副干酪乳酪杆菌 VHProbi F11不产生溶血素,不能够溶解血细胞。
2、抗生素耐受性试验
微量肉汤稀释法测定抗生素对副干酪乳酪杆菌 VHProbi F11的最小抑菌浓度MIC值具体结果见表5。
表5 副干酪乳酪杆菌 VHProbi F11的抗生素MIC值
MIC单位μg/mL。
从表5的结果可以看出,本发明提供的副干酪乳酪杆菌 VHProbi F11对红霉素、四环素等常见抗生素敏感,生物安全性良好。
实施例5 副干酪乳酪杆菌VHProbi F11抗氧化功能测定
1、菌株清除DPPH(1,1-二苯基-2-三硝基苯肼)能力测定
取1mL待测菌株的PBS菌悬液,加入1mL 0.4 mM的现配的DPPH自由基溶液,混合均匀后然后置于室温温度下遮光反应30 min,然后测定样品在波长 517nm处的吸光度A样本,测3次平行。对照组样品以等体积PBS溶液和DPPH·乙醇混合液,并以等体积PBS菌悬液和乙醇混合液空白调零。清除率按下列公式计算:清除率%=[1-(A样品-A空白)/A对照]×100%。结果见表6。
表6 DPPH自由基清除率表
菌株 | 清除率% | 标准差 |
副干酪乳酪杆菌 VHProbi F11 | 11.04% | 1.20% |
2、菌株清除HRS能力的测定
将100μL 5mM的水杨酸钠-乙醇溶液,100μL 5mM的硫酸亚铁,500μL去离子水和200μL乳酸菌PBS菌悬液混匀后加入100μL过氧化氢溶液(3mM),37℃水浴15min后在510nm波长处测量样品吸光度。羟自由基清除率按照下列公式进行计算。
清除率%=(A样品-A控制)/(A空白-A控制)×100%,其中A控制为去离子水替代样品,A空白为去离子水替代样品和H2O2,结果见表7。
表7 HRS自由基清除率表
菌株 | 清除率% | 标准差 |
副干酪乳酪杆菌 VHProbi F11 | 43.59% | 0.70% |
3、菌株抗脂质过氧化实验鉴定
亚油酸乳化液的制备:0.1mL亚油酸,0.2mL Tween 20,19.7mL去离子水。
0.5 mL的PBS溶液(pH 7.4)中加入1 mL亚油酸的乳化液, 1 mL FeSO4 (1%),再加入0.5 mL样品,37℃水浴1.5 h,混合液加入0.2 mL TCA(4%),2 mL TBA(0.8%),100 ℃水浴30 min,迅速冷却,4000 rpm/min离心15 min,收集上清液。在532 nm下测吸光度即为A;对照组以0.5 mL蒸馏水代替样品即为A0。抑制率/% =(A0-A)/ A0×100%。
注: A为样品组吸光度;A0为对照组吸光度,结果见表8。
表8副干酪乳酪杆菌VHProbi F11的抗脂质过氧化抑制率
抑制率 | 标准差 | |
菌体 | 23.99% | 0.25% |
发酵上清液 | 71.73% | 0.60% |
实施例6 副干酪乳酪杆菌VHProbi F11体外胆固醇降解试验
1、胆固醇胶束溶液的配制:
准确称取1g 胆固醇,溶于无水乙醇中,并定容至100 mL,在无菌条件下用 0.22 µm 微孔滤膜过滤除菌。
2、称取蛋白胨 10.0 g 牛肉膏 10.0 g 酵母膏 5.0 g 柠檬酸氢二铵2.0 g 葡萄糖20.0 g, 吐温80 1.0 mL,乙酸钠 5.0 硫酸镁 0.1 硫酸锰 0.05 ,磷酸氢二钾 2.0 g ,胆盐1g,蒸馏水1000mL调节pH值 7.3,115℃灭菌30min,然后加入胆固醇溶液使胆固醇终浓度为0.1%。
按照0.1%的接种量接种新鲜菌液,37℃静止培养48h,然后取0.2mL菌液,加入1.8mL无水乙醇,混匀,静止10分钟,3000转离心5分钟,取上清液用于测定胆固醇含量。胆固醇测定方法按照GB/T 5009.128-2003 <食品中胆固醇的测定>。
结果显示:本发明提供的副干酪乳酪杆菌 VHProbi F11对胆固醇的降解率达到23.65%(此为不含胆盐的数据)。
实施例7 副干酪乳酪杆菌VHProbi F11的黄曲霉毒素B1吸附能力
1、配制黄曲霉毒素B1浓度为1μg/mL的PBS溶液,简称AFB1-PBS溶液。
2、接种吸附:
取1mL副干酪乳酪杆菌 VHProbi F11新鲜菌液(24h,37℃),8000rpm离心5min,弃上清,用同体积PBS缓冲液清洗菌体2次,8000rpm离心5min,弃上清,然后将菌体重悬于1mL上述AFB1-PBS溶液,置于37℃恒温培养箱,1h后取出,8000rpm离心10min,取上清待测。
3、按照黄曲霉毒素B1检测试剂盒说明书测定上清液中黄曲霉毒素B1浓度。
测定前,将上清液用甲醇稀释100倍。
结果显示:本发明提供的副干酪乳酪杆菌 VHProbi F11 对黄曲霉毒素B1的吸附率为10.41%,标准差为0.07。
实施例9 副干酪乳酪杆菌VHProbi F11在预防乳鼠链球菌感染中的应用
1、实验动物
2周龄SPF级SD大鼠48只,雌雄各半。10只哺乳SD雌鼠,由青龙山动物繁殖中心提供,许可证号 SCXK(苏)2017-0001。大鼠专用颗粒饲料(江苏协同生物有限公司)饲喂,饲养于SPF级动物房。12h/12h光照/黑暗循环,自由摄食饮水,温度维持20-26℃,相对湿度40-70%。
2、试剂耗材
TNF-α ELISA kit:BioSource ,批号:MBS175904;
IL-6 ELISA kit :BioSource ,批号:MBS175908;
IFN-γ ELISA kit: BioSource ,批号:MBS176492;
HE 染色试剂盒:Promega。
3、实验方法
3.1、菌液制备
3.1.1 益生菌菌液制备
将副干酪乳酪杆菌 VHProbi F11划线 MRS平板上,37℃培养24~48h,挑取单菌落于MRS肉汤培养基扩大培养24h后,收集菌液。使用前计数调整其浓度为 1×109CFU/mL 。
3.1.2 链球菌菌液制备
将冻干的溶血性链球菌菌种于超净工作台内,用适量营养肉汤培养液反复吹打,使菌种融化分散。取少许菌种悬液,滴入含5mL营养肉汤培养基的试管中,37℃培养过夜。取第一代培养的菌悬液,划线接种于血琼脂培养基,37℃培养过夜。使用前挑取典型菌落无菌PBS稀释计数调整为备用浓度 1×106CFU/mL 。
3.1.3 体外拮抗菌液制备
配制模拟皮肤环境同时又能满足乳酸菌和链球菌生长需求的培养液,即每100ml培养液添加蛋白胨1g、酵母粉0.4g、牛肉粉0.8g、葡萄糖2g、K2HPO4 0.2g、角鲨烯20mg、辛酸/癸酸甘油三酯10mg 、丙氨酸1mg、尿素23mg、NaCl 100mg、MgSO4 20mg、MnSO4 4mg,调节pH5.7-6.0。
将副干酪乳酪杆菌 VHProbi F11菌液、溶血性链球菌菌液分别按1×107CFU/mL和1×105CFU/mL的比例接种于上述培养液中,体外共培养24h,收集菌液,备用。
3.2分组
大鼠适应性饲养7天后随机分为正常组(Normal)、阳性组(Positive)、模型组(Model)、益生菌预处理组(PreG)、益生菌后处理组(PosG)和益生菌体外拮抗组(ComG),每组8只。
3.3 造模及干预措施
链球菌造模:乙型溶血性链球菌菌液浓度 1×106CFU/ml,体积0.1ml,连续灌胃2天;
正常组:不做特殊处理;
模型组:1-7d适应性生长,8-17d常规饲养,18-19d链球菌造模,20-29d常规饲养,30d处理取材;
阳性组:1-7d适应性生长,8-17d常规饲养,18-19d链球菌造模,20-29d氨苄青霉素给药,浓度150mg/kg体重,体积0.1ml,30d处理取材;
益生菌预处理组:1-7d适应性生长,8-29d每天两次灌胃副干酪乳酪杆菌 VHProbiF11菌液,浓度为1×109 CFU/ml,体积0.1ml,18-19d链球菌造模,30d处理取材;
益生菌后处理组:1-7d适应性生长,8-17d常规饲养,18-19d链球菌造模,20-29d每天灌胃两次副干酪乳酪杆菌 VHProbi F11菌液,浓度为 1×109 CFU/ml,体积0.1ml,30d处理取材;
益生菌体外拮抗组:1-7d适应性生长,8-16d常规饲养,17-19d灌胃上述制备的体外拮抗菌液,体积0.1ml,20-29d常规饲养,30d处理取材。
4、检测指标
4.1一般观察
观察动物有无萎靡、躁动、腹泻,并观察其呼吸、饮食和活动情况。
4.2 Elisa检测大鼠血清细胞因子水平
取终末血清用于IL-6 、TNF-α及IFN-γ含量测定。
4.3组织病理学
大鼠处死后取小肠组织、脾脏组织多聚甲醛固定,脱水,石蜡包埋,切片,HE 染色观察组织病理变化。
5、数据统计处理方法
应用SPSS 22.0进行数据统计分析,所有实验数据以均数±标准差表示,两组间比较采用独立样本 t 检验,多组间差异比较采用单因素方差分析,以P<0.05判定为有显著性差异。
6、实验结果
6.1 一般观察
溶血性链球菌造模后大鼠逐渐出现行动迟缓、毛糙、摄水摄食减少、精神萎靡等症状。
6.2 IL-6 、TNF-α及IFN-γ含量测定结果
IL-6是一种功能广泛的多效性细胞因子,可调节多种细胞的生长与分化,具有调节免疫应答、急性期反应及造血功能,并在机体的抗感染免疫反应中起重要作用。TNF-α是一种涉及系统性炎症的细胞因子,同时也是属于引起急性反应的众多细胞因子中的一员。INF-γ是水溶性二聚体的细胞因子,是Ⅱ型干扰素的唯一成员,被称为巨噬细胞活化因子。实验结束后,血清中三种细胞因子水平的变化情况如图5所示。
从图5可知,链球菌造模后,大鼠炎性因子IL-6、TNF-α及IFN-γ含量比正常组分别提高了25.4%、26.8%和23.8%,差异显著(p<0.05)。治疗后,与模型组相比,阳性组和益生菌体外拮抗组各因子水平显著降低(p<0.05)且两组间差异不显著(p>0.05),其中益生菌体外拮抗组IL-6 、TNF-α及IFN-γ分别降低了18.0%、26.4%和23.9%;而益生菌预处理组TNF-α和IFN-γ也分别降低了19.9%和19.0%,益生菌后处理组TNF-α降低了19.5%。从而说明副干酪乳酪杆菌 VHProbi F11能有效缓解链球菌感染大鼠机体的炎症状态,且益生菌预处理组和体外拮抗组的效果优于后处理组。
6.3大体解剖观察
模型组大鼠解剖可见腹腔肠管肿胀,肠系膜有轻微出血,脾脏肿大,肾脏稍肿大。益生菌处理组未见肠管明显胀气,未见肠系膜出血,脾脏比正常对照组暗红,肿大相比模型组好转,肾脏无淤血点。益生菌体外拮抗组未发现肠管粘连及肠道坏死,未可见明显胀气发生,未发现肠系膜出血,脾脏可见肿大,肾脏形态圆润,说明副干酪乳酪杆菌 VHProbi F11改善了感染大鼠的器官损伤。
6.4 组织病理检测
6.4.1小肠HE 染色结果:
如图6所示,在光学显微镜下观察,正常组肠粘膜各层结构完整,肠绒毛排列整齐,肠粘膜微绒毛形态正常,排列整齐,肠上皮细胞结构完整;模型组黏膜层有大量炎性细胞浸润,肠绒层结构不完整,有严重黏膜上皮脱落;与模型组相比,各益生菌处理组黏膜层脱落程度明显减轻,且肠系膜结构完整,炎性细胞浸润减少;其中益生菌体外拮抗组的改善效果最明显。
6.4.2脾脏HE 染色结果:
如图7所示,在光学显微镜下观察,正常脾脏组织切片 HE 染色镜下见红髓白髓界限清晰,白髓淋巴细胞丰富,生发中心明显。模型组脾脏组织结构不清,白髓萎缩,脾小体结构不清。阳性组,红白髓界限清晰,淋巴细胞减少,白髓萎缩;益生菌处理组大鼠脾组织结构尚清,白髓萎缩,脾小体结构不清。益生菌体外拮抗组脾组织结构可见,红髓、白髓结构尚清,较正常组病理结构无差异。典型病理病变见图7。
上述动物实验结果表明,本发明提供的副干酪乳酪杆菌VHProbi F11能显著降低链球菌感染大鼠的炎症因子水平,对感染大鼠的小肠和脾脏等器官损伤有一定的改善作用,尤其是通过在体外环境中对链球菌进行拮抗处理,能显著降低链球菌的感染水平,与正常组差异极小,预防效果突出。
本发明所述副干酪乳酪杆菌VHProbi F11可广泛用于预防和治疗链球菌引发的感染,尤其适用于婴幼儿链球菌感染的预防。具体使用方式包括:(1)婴幼儿持续服用该益生菌预防链球菌感染;(2)当婴幼儿受到链球菌侵袭时,服用该益生菌控制感染的蔓延和加重;(3)围产期孕产妇通过在皮肤或乳房持续外用该益生菌,能在体表抑制链球菌的生长繁殖,最大程度避免或减轻婴幼儿通过吸吮等途径感染链球菌带来的损伤。
综上所述,本发明提供的副干酪乳酪杆菌VHProbi F11 对常见抗生素敏感,不产溶血素,生物安全性良好。经动物实验验证了副干酪乳酪杆菌VHProbi F11能有效缓解链球菌感染大鼠的炎症水平,减轻机体器官损伤,效果显著。所述副干酪乳酪杆菌VHProbi F11可广泛用于制备具有预防链球菌感染功能的食品、保健品、药品或护肤品。
序列表
<110> 山东百沃生物科技有限公司
<120> 一株副干酪乳酪杆菌及其在预防婴幼儿链球菌感染中的应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1433
<212> DNA
<213> 副干酪乳酪杆菌(Lacticaseibacillus paracasei)
<400> 1
ctcgctccct aaaagggtta cgccaccggc ttcgggtgtt acaaactctc atggtgtgac 60
gggcggtgtg tacaaggccc gggaacgtat tcaccgcggc gtgctgatcc gcgattacta 120
gcgattccga cttcgtgtag gcgagttgca gcctacagtc cgaactgaga atggctttaa 180
gagattagct tgacctcgcg gtctcgcaac tcgttgtacc atccattgta gcacgtgtgt 240
agcccaggtc ataaggggca tgatgatttg acgtcatccc caccttcctc cggtttgtca 300
ccggcagtct tactagagtg cccaactaaa tgctggcaac tagtcataag ggttgcgctc 360
gttgcgggac ttaacccaac atctcacgac acgagctgac gacaaccatg caccacctgt 420
cattttgccc ccgaagggga aacctgatct ctcaggtgat caaaagatgt caagacctgg 480
taaggttctt cgcgttgctt cgaattaaac cacatgctcc accgcttgtg cgggcccccg 540
tcaattcctt tgagtttcaa ccttgcggtc gtactcccca ggcggaatgc ttaatgcgtt 600
agctgcggca ctgaagggcg gaaaccctcc aacacctagc attcatcgtt tacggcatgg 660
actaccaggg tatctaatcc tgttcgctac ccatgctttc gagcctcagc gtcagttaca 720
gaccagacag ccgccttcgc cactggtgtt cttccatata tctacgcatt tcaccgctac 780
acatggagtt ccactgtcct cttctgcact caagtttccc agtttccgat gcgcttcctc 840
ggttaagccg agggctttca catcagactt aaaaaaccgc ctgcgctcgc tttacgccca 900
ataaatccgg ataacgcttg ccacctacgt attaccgcgg ctgctggcac gtagttagcc 960
gtggctttct ggttggatac cgtcacgccg acaacagtta ctctgccgac cattcttctc 1020
caacaacaga gttttacgac ccgaaagcct tcttcactca cgcggcgttg ctccatcaga 1080
cttgcgtcca ttgtggaaga ttccctactg ctgcctcccg taggagtttg ggccgtgtct 1140
cagtcccaat gtggccgatc aacctctcag ttcggctacg tatcatcgcc ttggtgagcc 1200
attacctcac caactagcta atacgccgcg ggtccatcca aaagcgatag cttacgccat 1260
ctttcagcca agaaccatgc ggttcttgga tctatgcggt attagcatct gtttccaaat 1320
gttatccccc acttaagggc aggttaccca cgtgttactc acccgtccgc cactcgttcc 1380
atgttgaatc tcggtgcaag caccgatcat caacgagaac tcgttcgact gca 1433
Claims (7)
1.一种副干酪乳酪杆菌,其特征在于,所述副干酪乳酪杆菌的保藏号为CCTCC NO:M2021590。
2.如权利要求1所述的副干酪乳酪杆菌,其特征在于,所述副干酪乳酪杆菌的16s rDNA序列如SEQ ID NO:1所示。
3.如权利要求1所述的副干酪乳酪杆菌,其特征在于,所述副干酪乳酪杆菌的Riboprinter 指纹图谱如图2所示;其RAPD指纹图谱如图3所示,rep-PCR指纹图谱如图4所示。
4.权利要求1所述的副干酪乳酪杆菌在制备链球菌抗菌剂中的应用。
5.权利要求1所述的副干酪乳酪杆菌在制备具有抗氧化功能的制品中的应用。
6.权利要求1所述副干酪乳酪杆菌在制备具有预防链球菌感染功能的制品中的应用。
7.如权利要求5或6所述的应用,其特征在于,所述的制品为功能性食品、保健品、药品或护肤品。
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