CN116064295A - 一株具有抑制流感嗜血杆菌功能的副干酪乳酪杆菌及其应用 - Google Patents
一株具有抑制流感嗜血杆菌功能的副干酪乳酪杆菌及其应用 Download PDFInfo
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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。所述副干酪乳酪杆菌的保藏号为CCTCC NO:M2022362,能显著抑制鼻窦炎致病微生物的生长,杀灭鼻窦炎致病微生物,对于防治鼻窦炎等呼吸道疾病具有重要的应用价值。
Description
技术领域
本发明属于功能益生菌筛选与应用技术领域,具体涉及一株能够抵抗流感嗜血杆菌引发的呼吸道感染的副干酪乳酪杆菌及其应用。
背景技术
鼻子是人体呼吸的门户,承担呼吸和免疫防御等关键性生理功能。遗传变异、环境致病因素、清除功能或免疫防御功能异常等均可引发各种鼻腔炎症。
鼻窦炎是鼻腔黏膜的炎症性疾病,多与鼻炎同时存在,所以也称为鼻-鼻窦炎。鼻窦炎分为急性鼻窦炎(ARS)和慢性鼻窦炎(CRS),感染是鼻窦炎发病的重要因素,流感嗜血杆菌是急性鼻窦炎主要病原菌之一,约占感染的35%,另外,化脓性球菌、大肠埃希菌、厌氧菌等也可能会引起急慢性鼻窦炎。我国传统中医学将鼻窦炎列为耳鼻喉科“三炎一聋”之一,这足以说明该病对人们日常生活影响之严重。
临床上抗生素的广泛应用,细菌的耐药性已成为鼻窦炎治疗中的难题。近年来,耐甲氧西林金黄色葡萄球菌(MRSA)的出现逐渐引起了耳鼻咽喉科医师的注意。有研究发现,CRS的MRSA感染率9.22%,他们认为抗生素滥用可能在MRSA在CRS中出现起了重要作用。另有资料表明,由于大量抗生素的使用,使得耐药菌株如抗青霉素菌株等的产生,使青霉素或头抱类常用抗生素变得无效,此外某些菌株能产生β-内酰胺酶如流感嗜血杆菌、卡他莫拉氏菌等,已使CRS的治疗变得更加困难。因此,寻找天然的、安全的新型抗菌剂具有重要意义。
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。该菌株能显著抑制鼻窦炎致病微生物的生长,杀灭鼻窦炎致病微生物,对于防治鼻窦炎等呼吸道疾病具有重要的应用价值。
本发明所提供的副干酪乳酪杆菌,命名为副干酪乳酪杆菌VHProbi M56(
Lacticaseibacillus paracasei VHProbi M56),已于2022年4月1日保藏于中国武汉大学的中国典型培养物保藏中心,其保藏号为CCTCC NO:M2022362。
本发明所提供的副干酪乳酪杆菌VHProbi M56株,其Riboprinter指纹图谱如图3所示;其MALDI-TOF核糖体蛋白分子量图谱如图4所示;其RAPD指纹图谱如图5所示;rep-PCR指纹图谱如图6所示。
本发明所提供的副干酪乳酪杆菌VHProbi M56株的16s rDNA序列为SEQ ID NO:1。
本发明所提供的副干酪乳酪杆菌可用于制备益生菌制剂,该制剂包括如上所述的副干酪乳酪杆菌活菌菌体、副干酪乳酪杆菌死菌菌体、副干酪乳酪杆菌的代谢产物及胞内提取物中的至少一种。
本发明提供的副干酪乳酪杆菌可用于制备具有预防或治疗上呼吸道疾病功能的制品。
所述的制品为功能性食品、保健品或药品。
所述的上呼吸道疾病为由细菌导致的鼻窦炎。
所述的细菌为流感嗜血杆菌、β溶血性链球菌、化脓性链球菌、大肠埃希氏菌及金黄色葡萄球菌。
本发明所提供的副干酪乳酪杆菌VHProbi M56,其活菌和发酵上清液都能够显著抑制鼻窦炎主要致病微生物流感嗜血杆菌的生长,含活菌的发酵液抑菌圈直径达到20.45±0.12mm;添加副干酪乳酪杆菌 VHProbi M56无细胞发酵上清液能显著抑制致病菌流感嗜血杆菌的生长,且抑制效果随添加量的增大而增强;当发酵上清液添加量达到15%时,对流感嗜血杆菌生长的抑制率高达39.59%。
所述副干酪乳酪杆菌VHProbi M56,对鼻腔上皮细胞有较强的粘附作用,为其在鼻腔中粘附和定植提供了有利条件;并且能够显著抑制鼻窦炎主要致病微生物流感嗜血杆菌对鼻腔上皮细胞的粘附,粘附抑制率达到74.25%。
所述副干酪乳酪杆菌VHProbi M56,其菌体具有较强的凝集能力,5h时自聚合率达到20%,与流感嗜血杆菌的共聚合率达到25.5%,为其在鼻腔环境中与其他微生物,尤其是致病微生物共凝集提供了必要的条件。
所述副干酪乳酪杆菌VHProbi M56,对其他大多数鼻窦炎致病微生物的生长也具有抑制作用,其中对β溶血性链球菌、化脓性链球菌、大肠埃希氏菌、金黄色葡萄球菌的抑菌圈直径最大达到19.52±0.23mm。
本发明提供的副干酪乳酪杆菌VHProbi M56,对机体无毒害作用,安全性好,可以添加在功能性食品、保健品或药品中,用于预防呼吸道疾病或改善细菌性鼻窦炎症状,具有广阔的应用前景。
附图说明
图1为菌株 M56的菌落图及革兰氏染色图;其中A为菌落图,B为革兰氏染色图;
图2为菌株 M56的API试验结果图;
图3为菌株 M56的Riboprinter指纹图谱;
图4为菌株 M56的MALDI-TOF核糖体蛋白指纹图谱;
图5为菌株 M56的RAPD指纹图谱;
图6为菌株 M56的rep-PCR指纹图谱;
图7为菌株 M56对流感嗜血杆菌的抑菌圈照片图;
图8为菌株 M56发酵上清液抑制流感嗜血杆菌的生长图;
图9为菌株 M56的自凝聚率和与流感嗜血杆菌的交互凝聚率图;
图10为菌株 M56与流感嗜血杆菌共凝集的宏观图片;
图11为菌株 M56与流感嗜血杆菌共凝集的显微图片(油镜,100×放大);
图12为菌株 M56对鼻咽癌细胞5-8F的粘附图;
图13为菌株 M56对流感嗜血杆菌对鼻咽癌细胞5-8F的粘附抑制图;
图14为菌株 M56对其他鼻窦炎致病菌的抑制作用图。
具体实施方式
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 M56的分离筛选
1.1初筛
从收集的3份传统发酵乳制品样品中各取1g,分别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,待平板长出单菌落进行镜检。
镜检结果显示,从发酵乳制品样品中,申请人共筛选出68株潜在乳酸杆菌,分别命名为M01、M02、……、M67、M68。
2.2复筛
将1L MRS液体培养基灭菌、冷却后,加入3.2g猪粘膜胃蛋白酶,摇匀溶解,置37℃水浴摇床中水浴1h制成耐酸性培养基。将初筛得到的68株乳酸杆菌按6%接种量分别接种于上述耐酸性培养基中,37℃条件下厌氧静置培养48h,取发酵液进行菌量计数。
结果显示,在所述68株乳酸杆菌发酵液活菌量的对数值中,M56菌株经耐酸性培养基复筛后活菌量最多,对数值高达9.72 Log CFU/mL,从而说明M56菌株耐酸能力最高。
实施例2 菌株鉴定
2.1 菌落和菌体形态鉴定
将M56菌株接种于MRS琼脂培养基上,37℃厌氧培养24h后,可见M56单菌落呈乳白色,表面光滑隆起,不透明,圆形或类圆形,菌落直径在0.5~1mm左右;菌体在显微镜下为短杆状,革兰氏染色阳性,两端钝圆,呈短链状排列,无芽孢。M56单菌落及光学显微镜下照片见图1。
2.2 碳源代谢试验
利用API 50CHL试剂验证菌株 M56的碳源代谢性能。API 50CHL试剂可用来鉴定菌株在属或种水平上的差异。实验方法及结果分析具体参见API 50CHL试剂盒说明书。分析结果显示菌株 M56与副干酪乳酪杆菌的ID值为94.9%,碳水化合物代谢活性基本相同,API试验结果见图2。
2.3分子生物学鉴定
2.3.1 16s rDNA基因序列分析
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。
2)16s rDNA基因扩增
引物序列:
27F:AGAGTTTGATCCTGGCTCA;
1492R:GGTTACCTTGTTACGACTT。
通过测序获得 M56菌株的16s rDNA序列,为SEQ ID NO:1。并将该序列在 NCBI 数据库中进行比对,初步确定 M56菌株为副干酪乳酪杆菌。
2.3.2 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,得到菌株M56的Riboprinter指纹图谱(图3)。
2.3.3MALDI-TOF-MS检测菌株核糖体蛋白表达
按照0.1%的接种量在MRS液体培养基中接种新鲜菌液,37℃,150rpm培养48h后,收集菌体,无菌水洗涤4次,晾干表面水分。然后取少量新鲜菌体以薄膜的形式均匀涂布于靶板上,加1μL裂解液覆盖样品,晾干后,再加1μL基质溶液覆盖样品,晾干后,将样品靶放入质谱仪进行鉴定。用激光照射样品与基质形成的共结晶薄膜,使样品中蛋白质电离,离子在10~20KV电场作用下加速飞过飞行管道,根据到达检测器的飞行时间不同检测蛋白质的分子量。利用Autofms 1000分析软件Autof Analyzer v1.0获取蛋白指纹图谱,菌株 M56主要的离子峰为:
m/z 3485.616、3619.586、4703.116、7240.508、9405.100等,鉴定结果如图4所示。
2.3.4 RAPD和rep-PCR指纹图谱鉴定
2.3.4.1 RAPD指纹图谱鉴定
1)引物序列:
5’- GAGGGTGGCGGTTCT-3’;
2)RAPD反应体系
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。
3)电泳
制备1.5 %的琼脂糖凝胶板,DL2000 DNA Marker作为结果对照,稳压100V电泳80min,最后利用凝胶成像系统检测电泳图。菌株 M56的RAPD指纹图谱如图5所示。
2.3.4.2 rep-PCR指纹图谱
1)引物序列:
CTACGGCAAGGCGACGCTGACG。
2)rep-PCR的反应体系
r TaqDNA聚合酶 0.2μL;10×Ex Taq DNA Buffer 2μL;引物(10 uM)1 μL;dNTPs(2.5 mM) 2 μL;DNA模板 2μL;无菌双蒸水12.8 μL。
3)电泳
DL2000 DNA Marker作为结果对照。电压100 V,电泳时间80min检测扩增结果。M56菌株的的rep-PCR指纹图谱如图6所示。
2.3.5全基因组测序
取新鲜 M56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CP094959-CP094960。
综上,结合菌株的菌落形态、碳源代谢和分子生物学的鉴定结果,可以得出结论,M56菌株为一株新发现的副干酪乳酪杆菌菌株,将其命名为副干酪乳酪杆菌VHProbi M56(
Lacticaseibacillus paracasei VHProbi M56)。
申请人于2022年4月1日将所述副干酪乳酪杆菌VHProbi M56保藏于中国武汉 武汉大学的中国典型培养物保藏中心,其保藏号为CCTCCNO: M2022362。
实施例3副干酪乳酪杆菌VHProbi M56对流感嗜血杆菌的抑菌试验
3.1培养基配制
MRS培养基和MRS肉汤:青岛海博生物。
脑心浸液巧克力液体培养基:脑心浸液肉汤,121℃高压灭菌15分钟,冷至 50℃左右时,加入7%无菌脱纤维羊血混匀,在80℃水浴中加热10min左右,并不停摇动,直至培养基颜色变成棕色(巧克力色),冷却至室温后4℃保存备用。
脑心浸液巧克力琼脂培养基:脑心浸液琼脂高压灭菌后,加入7%无菌脱纤维羊血,80℃水浴中加热10min直至培养基颜色变成棕色,冷至45~50℃时倾入无菌平皿,备用。
3.2菌株活化
副干酪乳酪杆菌 VHProbi M56:取冷冻保藏的甘油管,划线至MRS平板,37℃培养24~48h;待平板长出单菌落后,无菌条件下挑至MRS肉汤,37℃静置培养24h。
流感嗜血杆菌ATCC49766:取冷冻保藏的甘油管,划线至脑心浸液巧克力琼脂培养基,置于37℃、5%CO2条件下,培养24h;待平板长出单菌落后,无菌条件下挑至脑心浸液巧克力液体培养基中,置于37℃、5%CO2条件下,培养18~24h。
3.3抑菌试验
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,铺上层培养基,将脑心浸液巧克力琼脂培养基均匀铺在上层。待上层培养基凝固后,将培养好的流感嗜血杆菌菌液稀释后,取适宜稀释度涂布于培养基表面。打孔,并取混匀的100ul乳酸菌发酵液加入孔中。在37℃、5%CO2条件下培养24h后,测量抑菌圈大小。
经测量,副干酪乳酪杆菌 VHProbi M56对流感嗜血杆菌的抑菌圈直径达到20.45±0.12mm,说明副干酪乳酪杆菌 VHProbi M56对流感嗜血杆菌具有显著的抑制作用,结果如图7所示。
实施例4副干酪乳酪杆菌VHProbi M56对流感嗜血杆菌的生长抑制作用
1)流感嗜血杆菌ATCC49766:接种至脑心浸液巧克力液体培养基中,37℃、5% CO2条件下,培养16~24h。
将副干酪乳酪杆菌 VHProbi M56按1%接种量接于MRS培养基中,37℃静置培养16~24h,发酵液经4℃,10000 r/min离心30 min收集上清液,并用0.22μm微孔滤膜过滤得到副干酪乳酪杆菌 VHProbi M56无细胞发酵上清液。
2)将流感嗜血杆菌ATCC49766新鲜菌液按1%接种量分别接种至添加5%、10%、15%(v/v)副干酪乳酪杆菌 VHProbi M56无细胞发酵上清液的脑心浸液巧克力液体培养基中,不添加副干酪乳酪杆菌 VHProbi M56无细胞发酵上清液的脑心浸液巧克力液体培养基作为对照,37℃、5% CO2条件下,培养24h。在600 nm波长下测定流感嗜血杆菌生长情况。
结果如图8所示,与对照组相比,添加副干酪乳酪杆菌 VHProbi M56无细胞发酵上清液能显著抑制致病菌流感嗜血杆菌的生长,且抑制效果随添加量的增大而增强;当发酵上清液添加量达到15%时,对流感嗜血杆菌生长的抑制率高达39.59%,取得了意料不到的技术效果。
实施例5副干酪乳酪杆菌VHProbi M56的聚集性分析
5.1菌悬液的制备
将活化好的副干酪乳酪杆菌 VHProbi M56在MRS肉汤中培养18h,流感嗜血杆菌用脑心浸液巧克力液体培养基,在37℃、5%CO2条件下培养18h,培养完毕后用磷酸盐缓冲溶液中(PBS,pH7.4)洗涤3次,调节菌的浓度,使菌数达到107~108 CFU/mL,备用。
5.2 聚集性分析
分别将20mL副干酪乳酪杆菌 VHProbi M56菌悬液、流感嗜血杆菌菌悬液以及两者的菌悬液等体积混合后,充分混匀,置于37℃恒温箱,分别取0h、2h、4h、5h时的菌悬液测其600nm处的吸光值,计算自聚合率和共聚合率。
自聚合率(%)=[1-AL/A0]×100%。
共聚合率(%)=[(AL+AH)/2-Amix]/[( AL+ AP)/2]×100%。
A0:M56菌株0h的吸光度值;
AL:M56菌株单独静置2、4、5h后的吸光度值;
AH:流感嗜血杆菌单独静置2、4、5h后的吸光度值;
Amix:M56菌株和流感嗜血杆菌混合静置2、4、5h后的吸光度值。
结果如图9所示,副干酪乳酪杆菌 VHProbi M56的聚集效果随着时间逐渐增强,5h时自聚合率达到20%,与流感嗜血杆菌的共聚合率达到25.5%,效果显著。
实施例6副干酪乳酪杆菌VHProbi M56对流感嗜血杆菌的凝集吸附试验
取适量活化好的副干酪乳酪杆菌 VHProbi M56和流感嗜血杆菌新鲜菌液,8000rpm离心4min,用磷酸盐缓冲溶液(PBS,pH7.4)洗2次并且调整至OD600=4。取副干酪乳酪杆菌 VHProbi M56菌悬液300μL加入24孔板中,再加入300μL流感嗜血杆菌菌悬液作为实验组;另取等量的副干酪乳酪杆菌 VHProbi M56菌悬液和缓冲液混合作为对照组,每个对照及实验组设2个平行。将24孔板置于微孔板恒温振荡器,400rpm,室温,振荡孵育。显微地观察并拍照记录初始孔板状态及不同时间的孔板状态,观察是否有凝集现象出现。
宏观观察结果如图10所示,实验组中副干酪乳酪杆菌 VHProbi M56和流感嗜血杆菌结合出现明显的凝集物,而对照组中无明显凝集现象;显微镜下能观察到副干酪乳酪杆菌 VHProbi M56和流感嗜血杆菌的凝集结块,如图11所示。
实施例7副干酪乳酪杆菌VHProbi M56对鼻咽癌细胞的细胞毒性测试
6.1 菌悬液的制备
将副干酪乳酪杆菌 VHProbi M56用MRS液体培养基培养至稳定期,用磷酸盐缓冲溶液(PBS,pH7.4)洗涤3次,调节菌的浓度,使菌数达到5×107 CFU/mL(OD600吸光度值约为0.4),于70℃水浴中20min灭活备用。
6.2 细胞毒性试验
复苏鼻咽癌细胞5-8F,接种至含10%小牛血清细胞培养液的24孔培养板中,接种密度为2×105 cells/孔,细胞培养24h。将灭活后副干酪乳酪杆菌 VHProbi M56按MOI(Multiplicity of Infection,感染复数)值为10的比例加入细胞中,并设置不加菌的空白对照组,培养箱中继续培养24h。在待检测的每个细胞培养孔中加入MTT溶液,终浓度为0.3mg/ml,二氧化碳培养箱中孵育3h。小心弃掉上清,在每个24孔板细胞培养孔中加入500ul的DMSO,37℃下孵育30min,使紫色结晶充分溶解。检测490nm下的吸光度值。
由检测结果可知,与对照组相比,副干酪乳酪杆菌 VHProbi M56对细胞的增殖活性无显著影响,无细胞毒性,安全性良好。
实施例8 副干酪乳酪杆菌VHProbi M56对鼻咽癌细胞的粘附试验
7.1 菌悬液的制备
将副干酪乳酪杆菌 VHProbi M56用MRS液体培养基培养至稳定期,用磷酸盐缓冲溶液(PBS,pH7.4)洗涤3次,用细胞培养液重悬,并调节菌的浓度,使菌数达到1×108 CFU/mL,备用。
7.2 5细胞培养
从液氮罐中取出鼻咽癌细胞5-8F,进行复苏、传代培养,培养细胞数量扩增至所需用量。将5-8F细胞接种于内置细胞爬片的含10%小牛血清细胞培养液的六孔培养板中,每个孔的细胞铺板个数约为2×106 cells,将六孔板置于二氧化碳培养箱中24h。
7.3 粘附试验
6孔板中已贴壁的5-8F单细胞层,使用PBS缓冲液洗涤3次,加入上述制备好的副干酪乳酪杆菌 VHProbi M56菌悬液,放入二氧化碳培养箱中培养1h。将细胞爬片用PBS缓冲液反复洗涤3次,去除未粘附的细菌。用无水甲醇固定20分钟,取出细胞爬片晾干,进行革兰氏染色,于100倍油镜下镜检观察20个随机视野,共100个细胞上粘附的乳酸菌,计算平均每个细胞上粘附的乳酸菌的个数。
经统计分析可知,副干酪乳酪杆菌 VHProbi M56对鼻咽癌细胞的粘附量为7.87±0.55 CFU/cell,表明该菌株能较强地粘附在鼻咽癌细胞表面,效果显著,结果见图12。
实施例9 副干酪乳酪杆菌VHProbi M56对流感嗜血杆菌的粘附抑制实验
8.1 菌悬液的制备
将副干酪乳酪杆菌 VHProbi M56和流感嗜血杆菌的新鲜菌液,用磷酸盐缓冲溶液(PBS,pH7.4)洗涤3次,用细胞培养液重悬,并调节菌的浓度,使菌数达到1×108 CFU/mL,备用。
8.2细胞培养
操作同7.2。
8.3 粘附抑制试验
6孔板中已贴壁的5-8F单细胞层,使用PBS缓冲液洗涤3次,分别加入1mL上述副干酪乳酪杆菌 VHProbi M56和流感嗜血杆菌菌悬液,以不加副干酪乳酪杆菌 VHProbi M56菌悬液的细胞作为空白对照,放入二氧化碳培养箱中培养2h。将细胞爬片用PBS缓冲液反复洗涤3次,去除未粘附的细菌。用无水甲醇固定20min,取出细胞爬片晾干,进行革兰氏染色,于100倍油镜下镜检观察20个随机视野,共100个细胞上粘附的流感嗜血杆菌,计算平均每个细胞上粘附的流感嗜血杆菌的个数。比较副干酪乳酪杆菌 VHProbi M56存在和不存在的条件下流感嗜血杆菌粘附数的变化,以对照组流感嗜血杆菌的粘附率计100%,计算副干酪乳酪杆菌 VHProbi M56对实验组流感嗜血杆菌的粘附抑制率。
粘附抑制率(%)=(对照组流感嗜血杆菌粘附量-实验组流感嗜血杆菌粘附量)/对照组流感嗜血杆菌粘附量×100%。
结果显示,副干酪乳酪杆菌 VHProbi M56对实验组流感嗜血杆菌的粘附抑制率高达74.25%,从而说明副干酪乳酪杆菌 VHProbi M56能显著降低流感嗜血杆菌对细胞的粘附效果,结果见图13。
实施例10副干酪乳酪杆菌VHProbi M56对其他鼻窦炎致病菌的抑制作用
本实例中所用的鼻窦炎致病菌包括:
β溶血性链球菌:CMCC(B)32210,使用脑心浸液肉汤培养基添加5%(v/v)胎牛血清,37℃培养16~24h;
化脓性链球菌:BNCC337110、BNCC185918,使用哥伦比亚肉汤培养基添加5%(v/v)胎牛血清,37℃培养16~24h;
大肠埃希氏菌:BNCC337304、BNCC133264、BNCC269342,使用营养肉汤培养基,37℃培养16~24h;
金黄色葡萄球菌:ATCC29213、ATCC25923、ATCC6538,使用营养肉汤培养基,37℃培养16~24h。
将副干酪乳酪杆菌 VHProbi M56按1%接种量接于MRS培养基中,37℃静置培养24h。
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,每个平板均匀放置3个无菌牛津杯。铺上层培养基,将培养的β溶血性链球菌、化脓性链球菌(2株菌等体积混合)、大肠埃希氏菌(3株菌等体积混合)、金黄色葡萄球菌(3株菌等体积混合)充分混匀后,分别取0.2%(v/v)加入相应的半固体培养基中,取适量均匀铺到下层培养基上。待上层培养基凝固后,取出牛津杯,取混匀的100ul副干酪乳酪杆菌 VHProbi M56菌液加入牛津杯孔中,37℃静置培养24h,观察并测量抑菌圈大小。抑菌圈图片见图14,抑菌圈大小如表1所示:
表1副干酪乳酪杆菌 VHProbi M56对4种鼻窦炎致病菌的抑菌能力
致病菌 | 抑菌圈直径/mm |
β溶血性链球菌 | 14.05±0.11 |
化脓性链球菌 | 19.52±0.23 |
大肠埃希氏菌 | 13.83±0.08 |
金黄色葡萄球菌 | 12.55±0.15 |
从图14和表1的结果可知,副干酪乳酪杆菌 VHProbi M56对β溶血性链球菌、化脓性链球菌、大肠埃希氏菌及金黄色葡萄球菌均具有较强的抑制作用。
综上所述,本发明的副干酪乳酪杆菌VHProbi M56能有效抑制鼻窦炎致病微生物的生长、杀灭鼻窦炎致病微生物;且具有较强的凝集能力,为其在鼻腔环境中与其他微生物,尤其致病微生物共凝集提供了必要的条件;对鼻腔上皮细胞有较强的粘附作用,为其在鼻腔中粘附和定植提供了有利条件,并且能够显著抑制鼻窦炎主要致病微生物流感嗜血杆菌的粘附;对其他大多数鼻窦炎致病微生物也具有显著抑制作用。
副干酪乳酪杆菌VHProbi M56无细胞毒性,安全性好,可广泛用作液态食品、固态食品、胶状食品、鼻腔清洁用品和鼻腔治疗药品等的添加剂,能有效预防呼吸道疾病及改善细菌性鼻窦炎症状,应用前景广阔。
Claims (7)
1.一种副干酪乳酪杆菌,其特征在于,所述的副干酪乳酪杆菌的保藏号为CCTCC NO:M2022362。
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Riboprinter指纹图谱如图3所示;MALDI-TOF核糖体蛋白分子量图谱如图4所示;RAPD指纹图谱如图5所示;rep-PCR指纹图谱如图6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16s rDNA序列SEQ ID NO:1。
4.一种益生菌制剂,其特征在于,所述益生菌制剂中包含权利要求1所述副干酪乳酪杆菌的活菌菌体、死菌菌体、代谢产物或胞内提取物中的至少一种。
5.权利要求1所述的副干酪乳酪杆菌在制备具有预防或治疗呼吸道疾病功能的制品中的应用。
6.如权利要求6所述的应用,其特征在于,所述的制品为功能性食品、保健品或药品。
7.如权利要求5或6所述的应用,其特征在于,所述的呼吸道疾病为鼻窦炎。
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