CN117327628A - 一株可发酵中草药抗HPV病毒的格氏乳杆菌Mia及其应用 - Google Patents
一株可发酵中草药抗HPV病毒的格氏乳杆菌Mia及其应用 Download PDFInfo
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- CN117327628A CN117327628A CN202311527288.3A CN202311527288A CN117327628A CN 117327628 A CN117327628 A CN 117327628A CN 202311527288 A CN202311527288 A CN 202311527288A CN 117327628 A CN117327628 A CN 117327628A
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Abstract
本发明公开了一株可发酵中草药抗HPV病毒的格氏乳杆菌Mia及其应用,所述格氏乳杆菌Mia(Lactobacillus gasseri Mia),保藏于中国典型培养物保藏中心,保藏日期为2022年12月23日,保藏编号为CCTCC NO:M20222048。本发明还公开了所述格氏乳杆菌及利用其发酵所得中药发酵物在制备抗HPV病毒产品中的应用。与现有技术相比,本发明提供了一株全新的格氏乳杆菌Mia,其自身以及利用其发酵所得中药发酵物,具有抑制脊髓灰质炎病毒或HPV病毒感染的作用,并且与菌株相比,利用其发酵所得中药发酵物MiaMed对于病毒的抑制作用显著更优。此外,该格氏乳杆菌Mia也能够抑制多种致病菌类,包括大肠杆菌、咽颊炎链球菌等,同样的,利用其发酵所得中药发酵物对于细菌的抑制作用显著更优。
Description
技术领域
本发明属于格氏乳杆菌技术领域,尤其涉及一株可发酵中草药抗HPV病毒的格氏乳杆菌Mia及其应用。
背景技术
人乳头瘤病毒(HPV病毒)是一种属于乳多空病毒科的乳头瘤空泡病毒A属,是球形DNA病毒,它能引起人体皮肤黏膜的鳞状上皮增殖。表现为寻常疣、生殖器疣(尖锐湿疣)等症状。随着性病中尖锐湿疣的发病率急速上升和宫颈癌、肛门癌等的增多,HPV病毒感染越来越引起人们的关注。目前还没有理想的方法能够在治疗手术上将HPV病毒彻底清除,因此目前抗HPV病毒主要有方法主要治疗加药物加自身免疫力抵抗。
格氏乳杆菌是一种常见的益生菌,属于革兰氏阳性杆菌的一种。它是乳酸菌属中的一员,广泛存在于自然界中,包括人体内的消化道、口腔、生殖道以及各种发酵食品中,如乳制品和发酵蔬菜。格氏乳杆菌对人体有益,它在肠道内起着重要的生态平衡和保护作用。它能够通过产生乳酸和其他有益物质,调节肠道的酸碱度,抑制有害菌的生长,维持肠道的正常菌群结构。格氏乳杆菌还有助于增强免疫系统功能,改善消化和吸收,促进营养物质的代谢,预防腹泻和便秘等肠道问题。由于格氏乳杆菌的益生作用,它常被加入到乳制品和益生菌补充剂中,作为一种益生菌成分。人们常常食用含有格氏乳杆菌的乳制品,如酸奶、发酵乳和乳饮料,以增加有益菌群在肠道中的数量,提供健康的益生菌供给。
目前已经有报道,格氏乳杆菌能够用于抑制HPV感染,但是往往也存在抑制效果不够理想的问题。
发明内容
发明目的:针对现有技术中存在的问题,本发明提供了一株可发酵中草药抗HPV病毒的格氏乳杆菌Mia及其应用。
技术方案:为实现上述发明目的,本发明采用以下技术方案:
第一方面,本发明提供了一株格氏乳杆菌,所述格氏乳杆菌为格氏乳杆菌Mia(Lactobacillus gasseri Mia),保藏于中国典型培养物保藏中心,保藏日期为2022年12月23日,保藏编号为CCTCC NO:M20222048。
第二方面,本发明提供了一种中草药发酵物,所述中草药发酵物主要是由包含中草药提取物的培养基中接种权利要求1所述的格氏乳杆菌进行发酵所得,所述中草药选自蒲公英、金银花、甘草和鱼腥草的一种或几种。
作为具体实施方案,所述中草药提取物的制备方法包括如下步骤:
取中草药,按干重加入3-15倍的水,80℃-95℃浸泡1-5h,所得浸出液即为所述中草药提取物。
作为优选方案,所述中草药包括如下重量份的原料:
1-5份蒲公英、1-5份金银花、1-5份甘草和1-5份鱼腥草。
作为具体实施方案,所述包含中草药提取物的培养基的组分包括:
大豆蛋白胨8-12g/L,酵母浸粉6-10g/L,水解乳蛋白4-8g/L,无水葡萄糖18-22g/L,磷酸氢二钾1-3g/L,柠檬酸氢二胺1-3g/L,乙酸钠4-6g/L,硫酸锰0.4-0.6g/L,L-半胱胺酸盐酸盐0.4-0.6g/L,吐温80 0.5-1.5ml/L,中草药提取物的添加量为培养基总量的1-10%。
作为具体实施方案,所述发酵的条件为:37±2℃发酵,时长为24-48h;所述发酵结束后,进行过滤浓缩,对浓缩液进行真空冷冻干燥,即得所述中草药发酵物。
第三方面,本发明提供了一种组合物,所述组合物包含所述的格氏乳杆菌或所述的中草药发酵物。
第四方面,本发明提供了所述的格氏乳杆菌、所述的中草药发酵物、所述的组合物在制备抗脊髓灰质炎病毒或HPV病毒产品中的应用。
第五方面,本发明提供了所述的格氏乳杆菌、所述的中草药发酵物、所述的组合物在制备抑菌剂中的应用。
进一步的,所述抑菌剂能够抑制大肠杆菌、金黄色葡萄球菌、白色念珠菌和/或咽颊炎链球菌。
本发明中所述产品包括食品、抑菌剂或者药物等。
有益效果:与现有技术相比,本发明提供了一株全新的格氏乳杆菌Mia,其自身以及利用其发酵所得中药发酵物,具有抑制脊髓灰质炎病毒或HPV病毒感染的作用,并且与菌株相比,利用其发酵所得中药发酵物对于病毒的抑制作用显著更优。此外,该格氏乳杆菌Mia也能够抑制多种菌类,包括大肠杆菌、咽颊炎链球菌等,同样的,利用其发酵所得中药发酵物对于细菌的抑制作用显著更优。
附图说明
图1为革兰氏染色结果。
图2为格氏乳杆菌Roea生物进化树。
具体实施方式
下面结合附图及实施例对本发明的技术方案做进一步详细说明。
实施例1本实施例对从健康女性生殖道分泌物样品的采集及保存
为获取健康女性生殖道中的有益菌种,采集健康女性生殖道分泌物样品36例,健康样本标准为:1月内无口服或阴道局部应用抗菌药物,无月经不调,避开月经期,无宫内置节育器,3天内存在性生活及阴道灌洗,阴道PH值≤4.5,清洁度Ⅰ-Ⅱ度。每例女性生殖道样品用无菌棉签采集三份样本,立即放入厌氧袋中,然后置入冰盒保存,及时返送实验室作后进行冻存和测序核算提取处理。其中对各例健康生殖道样品中选取1份,加入15-20%的无菌甘油,充分涡旋震荡后保存至冻存管,采用阶梯预冷法,将冻存管置于4℃预冷2小时,再置于-20℃预冻8小时,最后放置于-80℃或液氮进行保存,用于优势菌株的筛选。
实施例2耐中草药成分菌株的筛选
为发掘能够发酵中草药的菌种,将茵陈(Z1)、川黄连(Z2)、黄芪(Z3)、鱼腥草(Z4)、蒲公英(Z5)、金银花(Z6)、甘草(Z7)等中草药切碎成长度1cm以内的小段,各取2g分别将其分别浸入30ml水中,加热至80℃保持1h,然后冷却至室温备用。取100μL生殖道分泌物悬浮液以10倍梯度进行稀释,每个MRS-GMA-中草药提取物琼脂培养基涂布3-5个。MRS-GMA-中草药提取物培养基配置方法为MRS肉汤与改良GMA培养基按1:1使用,此外每200ml培养基中加入无菌0.1%维生素K1溶液1ml、无菌氯化血红素2mg和中草药提取物上清液2ml,MRS-GMA-中草药提取物培养固体培养基为钱数液体培养基中按每1000ml液体培养基添加15g琼脂比例进行配置。将涂布好的培养基装入厌氧培养罐,并用三元混合气(氮气80%、氢气10%和二氧化碳10%)对罐内气体进行置换,使培养罐内最终氧气浓度≤5%,将培养罐在37℃恒温箱中培养48h。挑选独立、色泽光亮、形态饱满的菌落,采用基质辅助激光解吸电离飞行时间质谱(Matrix-assisted laser desorption/ionization time-of-flightmassspectrometry,MALDI-TOF MS,简称飞行质谱)技术进行高通量菌种初步鉴定。
实施例3高活性高耐酸菌株筛选
对MALDI-TOF MS设备初步鉴定为非致病的单菌落挑至液体10ml MRS-GMA培养基中37℃培养24h。MRS-GMA培养基制备方法为MRS-GMA-中草药提取物培养基中不添加中草药提取物成分。通过测发酵液OD600和显微镜对比观察发酵液菌量,丢弃生长效果差或无明显菌株,将剩余18份发酵液各取2ml加2ml 40%的甘油溶液预冷后冻存至-80℃用于备选菌种保存,剩余部分作为种子液。将种子液分别接入15ml MRS-GMA液体培养基和MRS-GMA-中草药提取物液体培养基,接种量为3%-8%,置于37℃摇床发酵培养。
MRS-GMA培养基组和MRS-GMA-中草药提取物培养基组每隔6h测一次pH值和OD600,连续测10次。最终发酵液pH最低的可达3.2。选取能够在MRS-GMA-中草药提取物培养基中正常发酵、且对应MRS-GMA培养基发酵液pH值下降较快、OD600较高、最终发酵pH值在3.2-3.8的样品作为待选菌株,共3个菌株,根据MALDI-TOF MS初步鉴定结果分别是格氏乳杆菌、鼠李糖乳杆菌和格氏乳杆菌,依次编号为1号-3号。并留取1号-3号菌种对应MRS-GMA-中草药提取物培养上清液。
实施例4发酵中草药对病毒的杀灭试验
根据《消毒技术规范》中的病毒灭活试验方法,以脊髓灰质炎病毒1型(poliovirus-Ⅰ,PV-Ⅰ)疫苗株作为实验病毒株,VERO细胞系作为测试细胞。对各MRS-GMA-中草药提取物培养上清液进行病毒灭活实验,利用间接免疫荧光试验检测细胞培养物内的病毒含量,发酵液原液作为测试浓度,MRS-GMA-中草药提取物培养基作为对照,用Reed-Muench法计算各样本病毒的细胞半数感染量(TCID50)。杀灭病毒能力评价结果显示1号菌通过发酵可以提升原中草药提取物的杀毒能力,特别是对蒲公英、金银花、甘草和鱼腥草的杀毒能力提升明显。同时通过对中草药发酵产生的发酵液显示出优异的杀毒能力。
表1各中草药提取物培养上清液对脊髓灰质炎病毒的杀灭作用(处理时间5min)
注:“*”为未达到绝对100%杀灭率,按保留两位小数四舍五入计数法为100%
实施例5菌株的保存、鉴定和菌种的典藏
综合实施例1-4筛选菌株过程中的评价结果,既具备高活性、高产酸/耐酸,又能够发酵中草药且提升其杀毒能力的为1号菌株,作为目标菌株。
(1)菌株的保存
对筛选出的格氏乳杆菌在MRS-GMA琼脂培养基上划线培养48h后,挑选单菌落置于10ml液体培养基中。将该培养基置于37℃恒温培养12h-16h,测得培养液OD600值≥0.6时,培养液中按1:1体积加入甘油溶液,该甘油溶液浓度为30%-40%,用吸液器吹打混合均匀后分装至2ml无菌冻存管中,并置于4℃预冷2h,然后置于-20℃预冻4h,最后转至-80℃冰箱或液氮中保存。
(2)进一步形态鉴定
菌体在琼脂培养基上形态为棒菌落圆形,边缘整齐,乳白色,表面湿润光滑,中间凸起。取纯化菌液在显微镜下进行观察可知,革兰氏阳性,短棒状,单个或成链出现,无鞭毛,无芽孢。
(3)革兰氏染色
革兰氏染色结果为革兰氏阳性,如图1所示。
(4)16SrRNA分子生物学鉴定
利用已发表的16S通用引物(引物序列为:8F:5’-AGAFTTTGATCCTGGCTCA-3’;1510R:5’-GGTTACCTTGTTACGACTT-3’),对1号菌株的16S rDNA基因序列进行扩增和测序,PCR扩增产物送上海生工生物工程股份有限公司进行测序鉴定。1号菌株的16SrDNA的核苷酸序列为序列表中的序列1;经过16SrDNA基因比对,与Genebank中的Lactobacillusgasseri比对相似率达99%;结合微生物系统鉴定,1号菌株确定为一株格氏乳杆菌,命名为格氏乳杆菌Mia(Lactobacillus gasseri Mia),其16S rDNA如SEQ ID NO.1所示,生物进化树如图2所示。
对生物学鉴定的格氏乳杆菌Mia(Lactobacillus gasseri Mia)进行典藏,该菌株于2022年12月23日保藏于中国典型培养物保藏中心(简称CCTCC),保藏编号CCTCC M20222048,保藏地址为中华人民共和国湖北省武汉市武汉大学。
实施例6体外抑菌实验
格氏乳杆菌Mia(Lactobacillus gasseriMia)分别接种在50ml MRS-GMA液体培养基(同实施例7)和MRS-GMA-中草药提取物培养基中,其中中草药提取物选择鱼腥草、蒲公英、金银花和甘草(提取物制备方法如下:取中草药,按干重加入8倍量的水,85℃浸泡3h,所得浸出液即为所述中草药提取物),将其对应的培养基标注为MG-Z4、MG-Z5、MG-Z6、MG-Z7,37℃培养24h后,将发酵液10000r/min离心10min,分别收集上清液和菌体。菌体用0.9%无菌盐水洗涤两次,再用0.9%无菌盐水制备成OD600为0.5-0.8的菌悬液,4℃保存备用留存备用。
选取大肠杆菌8099、白色念珠菌ATCC10231、金黄色葡萄球菌ATCC 6538、咽峡炎链球菌ATCC 9895作为体外抑菌实验测试菌株,采用琼脂打孔法对各菌株的发酵上清液及菌悬液进行抑菌圈试验。用乳酸调节MRS培养液基使其与上清液PH值相同作为空白对照,浓度为0.05mg/mL的甲硝唑溶液为抑制大肠杆菌和金黄色葡萄球菌的阳性对照,浓度为0.05mg/mL的克霉唑溶液为抑制白色念珠菌的阳性对照。结果如表2所示。
格氏乳杆菌Mia(Lactobacillus gasseri Mia)的菌悬液和上清液对大肠杆菌8099、白色念珠菌ATCC10231、金黄色葡萄球菌ATCC 6538和咽峡炎链球菌ATCC 9895均有明显的抑制作用。格氏乳杆菌Mia与中草药提取进行发酵后的发酵液抑菌能力显著提升,特别是与蒲公英和金银花提取物的培养。
表2抑菌效果评价
实验组 | 大肠杆菌 | 金黄色葡萄球菌 | 白色念珠菌 | 咽峡炎链球菌 |
阳性对照(甲硝唑) | +++ | +++ | ++ | +++ |
阳性对照(克霉唑) | ++ | ++ | +++ | ++ |
空白对照(MRS-GMA) | - | - | - | - |
格氏乳杆菌Mia(菌悬液) | + | ++ | ++ | + |
MRS-GMA培养上清液 | ++ | ++ | ++ | ++ |
MG-Z4培养基 | + | + | - | + |
MG-Z4培养上清液 | ++ | ++ | ++ | ++ |
MG-Z5培养基 | + | + | + | + |
MG-Z5培养上清液 | +++ | +++ | ++ | ++ |
MG-Z6培养基 | - | ++ | ++ | ++ |
MG-Z6培养上清液 | ++ | +++ | +++ | ++ |
MG-Z7培养基 | - | + | + | + |
MG-Z7培养上清液 | ++ | ++ | ++ | ++ |
注:“-”为无明显抑菌圈出现,“+”为抑菌圈直径介于5mm-15mm,“++”为抑菌圈直径介于15mm-25mm,“+++”为抑菌圈直径>25mm。
实施例7格氏乳杆菌Mia的发酵冻干粉制备
(1)培养基配置。
提供工业化高密度发酵格氏乳杆菌Mia培养基配置方案为:大豆蛋白胨10g/L,酵母浸粉8g/L,水解乳蛋白6g/L,无水葡萄糖20g/L,磷酸氢二钾2g/L,柠檬酸氢二胺2g/L,乙酸钠5g/L,硫酸锰0.5g/L,L-半胱胺酸盐酸盐0.5g/L,吐温80 1ml/L进行配置,原料均采用食品级原料进行配置。
(2)液体培养
培养基配置后调节pH值为6.5,115℃灭菌30min,冷却至38℃-39℃时,按3%-10%接种量,本实施例中优选为8%进行发酵罐接种培养,培养温度为35℃-37℃,pH值为6.2-6.5,发酵过程中密封不通气体,用氮气保持发酵罐压力,慢速搅拌。连续培养12-20小时,在12h时测得发酵液活菌量为3.5×108CFU/mL,在20h时测得发酵液活菌量为1.2×109CFU/mL,发酵活菌量水平已经较高,达到了工业化高密度发酵生产需求。
(3)离心
为避免发酵时间过长导致菌种老化,影响冻干粉菌种活性,在培养12-20小时,活菌量达到108CFU/mL-109CFU/mL,根据OD600值测得生长曲线进入平台期后快速将发酵液降温至15℃以下,使用管式离心机5000rpm进行离心,收获菌泥。
(4)保护剂乳化及冻干
收获菌泥稀释为含水量60%-80%,等体积加入菌泥保护剂进行乳化;菌泥保护剂成分为:水70%,脱脂乳粉10%,蔗糖5%,甘露糖醇5%,吐温-80 1%,甜菜碱3%,谷氨酸钠0.5%,可溶性淀粉5%。物料干燥的特点为水分含量低于5%,水活度在0.08-0.20aw。
冻干后的菌粉活性可达1.5×1011CFU/g,可便于存放。也便于用于食品添加乳酸菌粉或用于制备微生态干预制剂。
实施例8混合中草药发酵产物MiaMed的制备
将鱼腥草(Z4)3份、蒲公英(Z5)3份、金银花(Z6)3份、甘草(Z7)3份,按干重加入8倍的水,85℃浸泡3h,将浸出液进行过滤加入实施例7的培养基中,添加比例为6%,然后接种格氏乳杆菌Mia进行发酵,发酵时长为36h。对发酵液用纳米陶瓷膜进行过滤浓缩,对浓缩液进行真空冷冻干燥,保持发酵产物的有效活性,即为MiaMed。
原料不同用量及不同工艺可以选择如下方法:
将鱼腥草(Z4)1份、蒲公英(Z5)5份、金银花(Z6)1份、甘草(Z7)5份,按干重加入3倍的水,95℃浸泡1h,将浸出液进行过滤加入实施例9的培养基中,添加比例为3%,然后接种格氏乳杆菌Mia进行发酵,发酵时长为24h。对发酵液用纳米陶瓷膜进行过滤浓缩,对浓缩液进行真空冷冻干燥,保持发酵产物的有效活性,
将鱼腥草(Z4)5份、蒲公英(Z5)1份、金银花(Z6)5份、甘草(Z7)1份,按干重加入10倍的水,80℃浸泡5h,将浸出液进行过滤加入实施例9的培养基中,添加比例为10%,然后接种格氏乳杆菌Mia进行发酵,发酵时长为48h。对发酵液用纳米陶瓷膜进行过滤浓缩,对浓缩液进行真空冷冻干燥,保持发酵产物的有效活性。
其他中草药发酵类似,产出的发酵代谢产物冻干粉可以用于制备粉剂、片剂、液体、凝胶等剂型的加工使用,不同形式的制剂可以用于抑菌、抗HPV的干预制剂。
实施例9生殖道黏膜细胞的刺激实验
依据原国家卫生部《消毒技术规范》中的阴道粘膜刺激实验标准,选用健康初成年并处在非动情期的雌性新西兰兔,试验前检查动物阴道口有无分泌物、充血、水肿和其他损伤情况。分别取MiaMed(实施例8所得)和格氏乳杆菌Mia冻干粉各1.2g(1.5×1011CFU/g)溶于100ml无菌生理盐水作为受试液,各随机选用3只雌性新西兰兔染毒,每次注入受试液2ml。对照组动物用生理盐水作同样处理。每次间隔24h,连续5天。末次染毒后24h,采用空气栓塞法处死动物,完整切下阴道后纵向切开,肉眼观察粘膜有无水肿、充血等表现,然后放入10%甲醛溶液中固定,进行组织病理学检查。
根据《消毒技术规范》中阴道粘膜侧记评分标准和刺激强度分级,格氏乳杆菌Mia冻干粉阴道粘膜刺激指数为0.89<1,混合中草药发酵液冻干粉MiaMed阴道黏膜刺激指数为0.85<1,判定为对阴道粘膜无刺激性。显示出格氏乳杆菌Mia冻干粉和混合中草药发酵液冻干粉MiaMed的安全性,可以作为直接用于阴道微生态干预的生物制剂。
实施例10HPV病毒抑制评价
同样按实施例4的试验方法,使用HPV假病毒(HPV-6)作为测试毒株对格氏乳杆菌Mia及其中草药发酵冻干粉对HPV病毒杀灭能力测试。测试组分为4组,分别为中草药提取物干预对照组HC1(参照实施例8中MiaMed的制备方法制备中药浸出液,浸出液直接浓缩冻干所制成的冻干粉,给药剂量为20g/L),格氏乳杆菌Mia发酵冻干粉组HC2(实施例7所得,1.5×1011CFU/g活性冻干粉,给药剂量为60g/L),中草药发酵液冻干粉MiaMed(实施例8所得)组HC3(给药剂量为20g/L)和格氏乳杆菌Mia接种中草药培养基后未发酵对照组HC4(参照实施例8中MiaMed的制备方法,接种格氏乳杆菌Mia后不进行发酵,直接冻干所得,给药剂量为200ml/L)。杀灭HPV病毒能力评价结果显示原中药提取物杀灭HPV病毒能力较弱,而格氏乳杆菌Mia发酵的中草药冻干粉MiaMed可以显著提高对HPV病毒的杀灭能力。
表3HPV病毒抑制评价
实施例11对HPV感染的治疗
参照《妇产科学》高危型HPV感染的诊断标准,筛选年龄20-60岁,月经周期规律和性生活史,对研究知情且能积极配合治疗随访,且符合高危型西医诊断标、中医辨证分型标准或HPV高危型持续感染1年以上条件之一者。按照随机对照试验(RCT)原则,选取经检测确诊高危型HPV感染患者45例,随机分为5组。干预对照组DZ1给予辛复宁(重组人干扰素α2b阴道泡腾胶囊;上海华新生物高技术有限公司)治疗,中草药提取物干预对照组DZ2(参照实施例8中MiaMed的制备方法制备中药浸出液,浸出液直接浓缩冻干所制成的冻干粉,每次给药0.1克)。实验组分别为格氏乳杆菌Mia冻干粉组GY1(实施例7所得,1.5×1011CFU/g活性冻干粉,每次给药0.05g),格氏乳杆菌Mia接种中草药培养基对照组GY2(参照实施例8中MiaMed的制备方法,接种格氏乳杆菌Mia后不进行发酵,直接冻干所得,每次给药0.1g)和中草药发酵液冻干粉MiaMed(实施例8所得)组GY3(每次给药0.1g)。治疗3个月后均门诊复查,对各组高危型HPV的感染分型进行统计,因为存在感染1种或1种以上HPV分型的情况,因此进行必要归类统计。取宫颈脱落细胞进行HC2检测,观察HPV转阴情况。HPV转阴率=HPV感染分型转阴数量/HPV感染分型的总频次×100%。
表3各组患者HPV转阴情况比较
注:对各组间HPV综合转阴率进行统计分析,a为与对照组DZ1间有显著性统计学差异,b为与对照组DZ2相比转阴率有显著性差异,c为与GY1组相比转阴率有显著性差异,d为与GY2组相比转阴率有显著差异。
根据对比实验结果,结合实施例4的筛选过程,MiaMed对高危型HPV感染均具有转阴效果,将HPV综合转阴率显著提升至72.22%,显著高于人干扰素α2b的治疗转阴率。因此格氏乳杆菌Mia和MiaMed可以作为高危型HPV感染的可靠治疗方案,且简单、安全,适合在临床的推广应用,对预防子宫颈癌也有很好的社会价值。
Claims (10)
1.一株格氏乳杆菌,其特征在于,所述格氏乳杆菌为格氏乳杆菌Mia(Lactobacillusgasseri Mia),保藏于中国典型培养物保藏中心,保藏日期为2022年12月23日,保藏编号为CCTCC NO:M20222048。
2.一种中草药发酵物,其特征在于,所述中草药发酵物主要是由包含中草药提取物的培养基中接种权利要求1所述的格氏乳杆菌进行发酵所得,所述中草药选自蒲公英、金银花、甘草和鱼腥草的一种或几种。
3.根据权利要求2所述的中草药发酵物,其特征在于,所述中草药提取物的制备方法包括如下步骤:
取中草药,按干重加入3-15倍的水,80℃-95℃浸泡1-5h,所得浸出液即为所述中草药提取物。
4.根据权利要求2所述的中草药发酵物,其特征在于,所述中草药包括如下重量份的原料:
1-5份蒲公英、1-5份金银花、1-5份甘草和1-5份鱼腥草。
5.根据权利要求2所述的中草药发酵物,其特征在于,所述包含中草药提取物的培养基的组分包括:
大豆蛋白胨8-12g/L,酵母浸粉6-10g/L,水解乳蛋白4-8g/L,无水葡萄糖18-22g/L,磷酸氢二钾1-3g/L,柠檬酸氢二胺1-3g/L,乙酸钠4-6g/L,硫酸锰0.4-0.6g/L,L-半胱胺酸盐酸盐0.4-0.6g/L,吐温80 0.5-1.5ml/L,中草药提取物的添加量为培养基总量的1-10%。
6.根据权利要求2所述的中草药发酵物,其特征在于,所述发酵的条件为:37±2℃发酵,时长为24-48h;所述发酵结束后,进行过滤浓缩,对浓缩液进行真空冷冻干燥,即得所述中草药发酵物。
7.一种组合物,其特征在于,所述组合物包含权利要求1所述的格氏乳杆菌或权利要求2-6任一项所述的中草药发酵物。
8.权利要求1所述的格氏乳杆菌、权利要求2-6任一项所述的中草药发酵物、权利要求7所述的组合物在制备抗脊髓灰质炎病毒或HPV病毒产品中的应用。
9.权利要求1所述的格氏乳杆菌、权利要求2-6任一项所述的中草药发酵物、权利要求7所述的组合物在制备抑菌剂中的应用。
10.根据权利要求9所述的应用,其特征在于,所述抑菌剂能够抑制大肠杆菌、金黄色葡萄球菌、白色念珠菌和/或咽颊炎链球菌。
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WO2002080946A1 (en) * | 2001-04-02 | 2002-10-17 | Wakamoto Pharmaceutical Co.,Ltd. | Compositions for preventing and/or treating oral diseases |
WO2016167489A1 (ko) * | 2015-04-16 | 2016-10-20 | 주식회사 고바이오랩 | 질 내 병원성 미생물에 대한 증식억제활성을 갖는 락토바실러스 속 균주 |
CN106957811A (zh) * | 2017-05-22 | 2017-07-18 | 泰安大凡神农制药有限公司 | 具有抑菌作用的格式乳杆菌与中药复方及其在治疗妇科炎症中的应用 |
CN108004187A (zh) * | 2018-01-11 | 2018-05-08 | 广东龙创基药业有限公司 | 一种格氏乳杆菌及其用于制备阴道抑菌药物的应用 |
WO2022100633A1 (zh) * | 2020-11-10 | 2022-05-19 | 深圳华大生命科学研究院 | 用于预防和/或治疗生殖道菌群紊乱相关疾病的格氏乳杆菌 |
CN113862188A (zh) * | 2021-10-15 | 2021-12-31 | 广东一元兰欣生物科技有限公司 | 一株格氏乳杆菌ls03及其应用 |
CN114129601A (zh) * | 2021-12-22 | 2022-03-04 | 南方医科大学第七附属医院(佛山市南海区第三人民医院) | 格氏乳杆菌lgv03在制备预防或治疗hpv感染药物中的应用 |
KR20230120267A (ko) * | 2022-02-09 | 2023-08-17 | (주)지에프씨생명과학 | 항염증 및 항균 효능을 갖는 신규의 락토바실러스 가세리 gfc_1220 균주 및 이를 이용하여 제조된 발효물 |
KR102554125B1 (ko) * | 2022-10-24 | 2023-07-13 | (주)헥토헬스케어 | 질염 예방 또는 개선 효과를 가지는 락토바실러스 가세리 belg08 및 이를 포함하는 조성물 |
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