CN116515686A - 一株治疗女性生殖感染和调节经前心理症状的卷曲乳杆菌及其应用 - Google Patents
一株治疗女性生殖感染和调节经前心理症状的卷曲乳杆菌及其应用 Download PDFInfo
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- CN116515686A CN116515686A CN202310337582.1A CN202310337582A CN116515686A CN 116515686 A CN116515686 A CN 116515686A CN 202310337582 A CN202310337582 A CN 202310337582A CN 116515686 A CN116515686 A CN 116515686A
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Abstract
本发明公开了一株卷曲乳杆菌,所述卷曲乳杆菌为卷曲乳杆菌Roea(Lactobacillus crispatus Roea),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:M20222049。本发明还公开了所述卷曲乳杆菌在制备治疗女性生殖感染和调节经前心理症状产品中的应用。与现有技术相比,本发明提供了一株全新的卷曲乳杆菌Roea,其具有高产酸和耐酸特性,能够抑制多种菌类,包括化脓性链球菌、咽颊炎链球菌等;对***粘膜无刺激性,可以作为直接用于***微生态干预的生物制剂,可以有效治疗***炎等女性生殖感染;另外,该卷曲乳杆菌Roea能够用来改善女性经前心理症状,效果显著。
Description
技术领域
本发明属于卷曲乳杆菌技术领域,尤其涉及一株治疗女性生殖感染和调节经前心理症状的卷曲乳杆菌及其应用。
背景技术
人体女性***内环境是指女性***腔内有寄居着的微生物群所构成的微生态环境。正常情况下,女性***腔内寄居着大量的微生物,这些微生物在局部中发挥重要的作用,维持女性***健康状态。女性***微生物群以乳酸杆菌为主要优势菌,同时存在少量的其他厌氧菌,乳杆菌能通过产酸、过氧化氢(H2O2)维持***弱酸性环境从而达到抑制病原菌的生长,维持***正常内环境。当***内的乳杆菌丰度下降或者乳酸菌功能下降时,***中的其他厌氧菌过度增殖,使***微生态失衡,导致内环境紊乱,可促进病原体的感染从而导致局部炎症、病变;同时,***局部病原体感染可增加***、早产、流程等风险。如B族链球菌是造成孕妇产褥期脓毒血症和新生儿脑膜炎的一个重要原因,寄生在产妇生殖道内可致婴儿感染的发生,也可引起产后感染、菌血症、心内膜炎、皮肤和软组织感染及骨髓炎;化脓性链球菌感染女性生殖道会引起发热和红疹,且会引起脓肿病;咽颊炎链球菌感染女性生殖***可引起产后女性的身体免疫力低下,引起产褥感染,表现为宫腔感染、盆腔感染,引起腹痛、腰痛、白带增多、发热等不适症状。近年来,有研究通过给予病人使用乳杆菌,以其达到改善和重塑***内环境的目的,例如卷曲乳杆菌,现有的卷曲乳杆菌可以抑制多种菌,例如常见的大肠杆菌、白色念珠菌、金黄色葡萄球菌等,但是对B族链球菌、化脓性链球菌、咽颊炎链球菌能够抑制的菌类较少,限制了其应用。
发明内容
发明目的:针对现有技术中存在的问题,本发明提供了一株治疗女性生殖感染和调节经前心理症状的卷曲乳杆菌及其应用。
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,本发明提供了一株卷曲乳杆菌,所述卷曲乳杆菌为卷曲乳杆菌Roea(Lactobacillus crispatus Roea)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:M20222049。
第二方面,本发明提供了上述卷曲乳杆菌的应用,包括如下:
所述的卷曲乳杆菌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、白色念珠菌、金黄色葡萄球菌、B族链球菌、化脓性链球菌和/或咽颊炎链球菌。
或者,所述的卷曲乳杆菌在制备***微生态干预剂或治疗女性生殖感染产品中的应用;优选的,所述女性生殖感染为***炎;所述卷曲乳杆菌能有效治疗***炎,并且还能预防其复发。
或者,所述的卷曲乳杆菌在制备改善女性经前心理症状产品中的应用。进一步的,所述女性为20-35岁的健康女性。
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卷曲乳杆菌。
第四方面,本发明提供了所述的组合物的应用,包括如下:
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、白色念珠菌、金黄色葡萄球菌、B族链球菌、化脓性链球菌和/或咽颊炎链球菌。
或者,所述的组合物在制备***微生态干预剂或治疗女性生殖感染产品中的应用;优选的,所述女性生殖感染为***炎;所述卷曲乳杆菌能有效治疗***炎,并且还能预防其复发。
或者,所述的组合物在制备改善女性经前心理症状药物中的应用。进一步的,所述女性为20-35岁的健康女性。
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,本发明提供了一种全新的卷曲乳杆菌Roea,其具有高产酸和耐酸特性,能够抑制多种菌类,包括化脓性链球菌、咽颊炎链球菌等;对***粘膜无刺激性,可以作为直接用于***微生态干预的生物制剂,可以有效治疗***炎等女性生殖感染;另外,该卷曲乳杆菌Roea能够用来改善女性经前心理症状,效果显著。
附图说明
图1为革兰氏染色结果。
图2为卷曲乳杆菌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
本实施例对从健康人体中筛选的乳酸菌进行生殖道菌群调节生长的筛选,样品的采集及保存
为获取健康女性生殖道中的卷曲乳杆菌,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***局部应用抗菌药物,无***,避开月经期,无宫内置节育器,3天内存在性生活及***灌洗,***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高耐酸卷曲乳杆菌菌株的筛选
将实施例1中采集保存的样本速溶后,取100μL生殖道分泌物悬浮液以10倍梯度进行稀释,各涂布3-5个MRS琼脂培养基,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。
对MALDI-TOF MS设备初步鉴定为卷曲乳杆菌的单菌落挑至液体15ml MRS培养基中37℃培养8h后,获取发酵液。
通过测发酵OD600和显微镜对比观察发酵液菌量,丢弃液体发酵效果差或无明显菌株,将剩余45份发酵液各取0.5ml用于菌种测序鉴定备用,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,剩余发酵液继续置于37℃摇床发酵培养每隔6h测一次pH值,连续测4次。再次发酵24h后发酵液pH最低的可达3.0。选取发酵过程中发酵液pH值下降较快、OD600较高、最终发酵pH值在3.0-3.8的样品作为待选菌株,共5个菌株,编号为1号-5号。
实施例3体外抑菌实验
鼠李糖乳杆菌GR-1和罗伊氏乳杆菌RC-14在针对生殖道菌群干预上研究成果较多且效果较好,选择作为对照菌株,与5株筛选的备选菌株一起做抑菌能力评价,分别接种在50ml MRS液体培养基中,37℃培养16h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和金黄色葡萄球菌ATCC 6538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。测量抑菌圈大小,以待测样本产生的抑菌圈半径大于MRS对照液的为有抑菌活性。除去部分抑菌效果不明显菌株,具有显著抑菌效果的菌株评价结果抑菌结果如表1所示。
2号菌株的菌悬液和上清液对大肠杆菌8099、白色念珠菌ATCC10231和金黄色葡萄球菌ATCC 6538均有显著的抑制作用,且抑菌能力显著优于鼠李糖乳杆菌GR-1和罗伊氏乳杆菌RC-14。
表1抑菌效果评价
注:a与GR-1相比具有显著性,b与RC-14相比具有显著性
实施例4菌株的保存、鉴定和菌种的典藏
综合实施例1-3筛选菌株过程中的评价结果,既能够高产酸/耐酸,抑制引起妇科疾病致病菌,又具备黏膜高粘附能力的为2号菌株,作为目标菌株。
(1)菌株的保存
对筛选出的卷曲乳杆菌Roea在MRS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,该液体培养基为含有4.5%大豆卵磷脂和2%碳酸钙的MRS液体培养基。将该培养基置于37℃恒温培养12h-16h,测得培养液OD 600值≥1.2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
取纯化菌液在显微镜下进行观察可知,革兰氏阳性,细长杆,微弯成链出现,无鞭毛,无芽孢,菌落白色,边缘不整齐,片状,中央凸起。
(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5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。2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Lactobacilluscrispatus比对相似率达99%;结合微生物***鉴定,2号菌株确定为一株卷曲乳杆菌,命名为卷曲乳杆菌Roea,其16S rDNA如SEQ ID NO.1所示,生物进化树如图2所示。
对生物学鉴定的卷曲乳杆菌Roea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 NO:
M20222049,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5Roea对B族链球菌抑制
按照实施例3的方法制备Roea、GR-1和RC14的菌悬液及上清液,作为抑制B族链球菌实验成分。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。采用B族链球菌显色培养基对获取的4个B族链球菌阳性生殖样本进行涂布培养观察抑菌圈,统计结果显示Roea菌株的菌悬液及上清液对B族链球菌具有较强的抑制能力。
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6Roea对A族链球菌(化脓性链球菌)及咽颊炎链球菌抑制性
按照实施例3的方法制备Roea、GR-1和RC14的菌悬液及上清液,选取化脓性链球菌ATCC19615和咽峡炎链球菌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的青霉素溶液为抑制化脓性链球菌和咽峡炎链球菌的阳性对照。统计抑菌圈大小,结果显示Roea菌株的菌悬液及上清液对化脓性链球菌和咽峡炎链球菌具有较强的抑制能力。
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生殖道黏膜细胞的刺激实验
依据原国家***《消毒技术规范》中的***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***口有无分泌物、充血、水肿和其他损伤情况。将卷曲乳杆菌Roea冻干粉0.8g(2.7×1011CFU/g)溶于100ml无菌生理盐水作为受试液,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***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***粘膜侧记评分标准和刺激强度分级,卷曲乳杆菌Roea冻干粉***粘膜刺激指数为0.89<1,判定为对***粘膜无刺激性。可以作为直接用于***微生态干预的生物制剂。
实施例8对***炎的治疗
选取某医院收治的***炎患者98例,采用随机数字表法分为观察组和对照组,观察组47例,病程6天~60天,平均17.62±0.71天;复发31例,初发16例;***pH值5.42±1.52;其中细菌性***炎38例,滴虫性***炎9例。对照组51例,病程6天~60天,平均17.68±0.78天;复发34例,初发17例;***pH值5.41±1.56;其中细菌性***炎39例,滴虫性***炎12例。2组临床资料比较无显著差异(P>0.05)。
对照组中细菌性***炎患者采取甲硝唑***泡腾片每次1片***置入治疗,每晚1次,治疗7d;滴虫性***炎患者给予甲硝唑片400mg口服,每天2次,治疗7d。观察组患者在对照组治疗的基础上给予***用卷曲乳杆菌Roea活菌制剂(含卷曲乳杆菌Roea 5.8×109CFU/片),每次1粒***置入,每晚1次,治疗7天。
7天对照干预后,对照组显效15例、有效21例、无效14例,总有效率72%;观察组显效43例、有效7例、无效0例,总有效率100%。观察组临床疗效显著高于对照组。
***清洁级别比较。治疗前两组患者***清洁级别比较差异无统计学意义(P>0.05);治疗后,两组***清洁级别均降低,且观察组(清洁度为1.01±0.13)低于对照组(清洁度为2.3±0.47)(P<0.05)。
实施例9对***炎的干预及复发性预防
本实施例中对实施例8中对照组治疗后,经过下一次***后,再进行随访对照组,甲硝唑治疗的对照组仍有9例未获得临床治愈,而只有2例观察组患者仍未获得临床治愈。同时,有5例甲硝唑临床治愈的病例(7/50)在第二个随访周期复发,而卷曲乳杆菌Roea活菌制剂临床治愈的BV患者仅有1例出现复发现象(1/50)。结果显示Roea干预妇科炎症可有效提高治愈率及防止复发。
实施例10改善健康年轻女性的经前心理症状
肠脑轴通过双向交流在维持肠道稳态和大脑功能方面起着至关重要的作用。育龄妇女也可能在月经前几周感到生理和/或情绪不适,超过90%的年轻女性在某种程度上患有经前症状,症状因人而异。***依赖性生理或心理症状与大脑中生殖激素***(E2)和孕酮(P4)、垂体激素(***)、***素和神经递质水平的周期性变化有关。E2和P4也可以影响女性的情绪和睡眠模式。E2调节各种神经递质***,包括血清素和多巴胺,影响情绪。生殖激素已被证明可以调节女性在整个成年期的睡眠模式。这些激素还影响了啮齿动物模型中的焦虑样行为。本发明中观察到的卷曲乳杆菌可影响经前E2和P4水平的变化,进而缓解经前心里症状。
选择20-35岁的女性、未使用药物、***通常短于25天或长于38天、自我评估抑郁量表(SDS)上得分超过60的83名受试者,随机分配到对照组(38名)或观察组(45名)。观察组给予卷曲乳杆菌Roea制备的乳酸菌(2g/袋,含卷曲乳杆菌Roea 1.5×1010CFU/袋,辅料为麦芽糊精),对照组给予相同剂量的麦芽糊精(2g/袋,成分为麦芽糊精),每日早晚各食用1袋,同时要求参与者不食用发酵牛奶等含有活乳酸菌的食物或其他含有益生菌或益生元产品,干预周期约180天。在第1个月经期及前3-6天(黄体期)及月经后3-6天(***)的14时-16时之间采集唾液,第1个***结束后7天开始进行对照干预,采集从干预开始的第5次月经期前3-6天(黄体期)及月经后3-6天(***)唾液,唾液样品储存在-80℃直至检测分析。分别采用唾液孕酮ELISA试剂盒(Salimetrics)和唾液17β-***ELISA检测试剂盒(Salimetrics)对采集唾液样品中的***(E2)和孕酮(P4)进行检测。开始干预后,每次月经前根填写经前紧张综合征(PMTS)-视觉模拟量表(VAS)问卷,对每个***的经前症状评估。
*差异有统计学意义(p<0.05)
PMTS-VAS问卷列出的12种经前症状中,Roea观察组在“情绪低落”、“焦虑”、“对活动的兴趣下降”、“容易疲劳”和“睡眠更多”方面的得分明显低于安慰剂组。数据表明,每天摄入Roea可改善心理和睡眠相关的经前症状。
月经后的***唾液E2和P4水平在干预前和干预后受到Roea给药的影响不显著。与对照组相比,Roea组月经前的黄体期唾液P4水平显著增加,E2维持相对稳定。结合经前症状,卷曲乳杆菌Roea可影响黄体期的E2和P4分泌,从而改善经前症状。
实施例11卷曲乳杆菌Roea的工业发酵及冻干粉制备
(1)培养基配置。
提供工业化高密度发酵卷曲乳杆菌Roea培养基配置方案为:大豆蛋白胨10g/L,酵母浸粉8g/L,无水葡萄糖28g/L,低聚半乳糖3g/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1g/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,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8-16小时,在8h时测得发酵液活菌量为5.8×108CFU/mL,在16h时测得发酵液活菌量为3.1×109CFU/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8-12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%。搅拌均匀后装入真空冷冻干燥机进行冷冻真空干燥,冻干物料厚度≤1cm。冻干机温度曲线为:50℃保持4h,每隔2h温度提高5℃,到第24h时温度为0℃,之后每隔2h温度提高3℃,最终最高温度为30℃保持不变,直至物料干燥,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2.7×1011CFU/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。
Claims (10)
1.一株卷曲乳杆菌,其特征在于,所述卷曲乳杆菌为卷曲乳杆菌Roea(Lactobacilluscrispatus Roea),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:M20222049。
2.权利要求1所述的卷曲乳杆菌在制备抑菌剂中的应用。
3.根据权利要求2所述的应用,其特征在于,所述抑菌剂能够抑制大肠杆菌、白色念珠菌、金黄色葡萄球菌、B族链球菌、化脓性链球菌和/或咽颊炎链球菌。
4.权利要求1所述的卷曲乳杆菌在制备***微生态干预剂或治疗女性生殖感染产品中的应用;优选的,所述女性生殖感染为***炎。
5.权利要求1所述的卷曲乳杆菌在制备改善女性经前心理症状产品中的应用。
6.一种组合物,其特征在于,所述组合物包含权利要求1所述的卷曲乳杆菌。
7.权利要求6所述的组合物在制备抑菌剂中的应用。
8.根据权利要求7所述的应用,其特征在于,所述抑菌剂能够抑制大肠杆菌、白色念珠菌、金黄色葡萄球菌、B族链球菌、化脓性链球菌和/或咽颊炎链球菌。
9.权利要求6所述的组合物在制备***微生态干预剂或治疗女性生殖感染产品中的应用;优选的,所述女性生殖感染为***炎。
10.权利要求6所述的组合物在制备改善女性经前心理症状产品中的应用。
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