CN111358813B - 一种用于保护呼吸道的盐雾混合物 - Google Patents
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Abstract
本发明公开了一种用于保护呼吸道的盐雾混合物,其特征在于:按照质量百分比,其包含10‑20%的盐溶液,30‑35%的益生菌发酵上清液;所述益生菌为鼠李糖乳杆菌(Lactobacillus rhamnosus LC‑STH‑13)CGMCC NO.3994。盐雾本身就具有保护呼吸道,杀菌消毒的效果。本申请人研发团队发现,该鼠李糖乳杆菌(Lactobacillus rhamnosus LC‑STH‑13)CGMCC NO.3994的发酵上清液具有一定的保护呼吸道,抗病毒效果。盐雾和上述益生菌发酵上清液在保护呼吸道上具有一定的协同作用。
Description
技术领域
本发明涉及一种用于保护呼吸道的盐雾混合物,本发明属于生物技术领域,尤其是涉及一种用于保护呼吸道的盐雾与益生菌发酵上清液的混合物,其具有抗病毒保护呼吸道的效果。
背景技术
人体各部位微生物群的组成基于环境因素、先天免疫反应和遗传因素决定。肠道微生物群由100万亿个微生物组成,由1000多个不同的细菌组成,产生的细菌代谢产物通过血液进入肺部来调节肺部免疫。肠道微生物群可能通过这些循环产物,直接地诱导肺部的免疫反应,并且还能影响肺部微生物的组成成分。肺部微生物群在维持健康的免疫反应方面也有重要的作用。肺中的微生物群落的形成由上呼吸道吸入,或肠道微生物群影响。形成肺部的先天和获得的免疫反应。
益生菌是一类活体微生物。当适当的摄入时,可影响宿主机体的内部环境,最终会对宿主机体带来益处的微生物。近年来,益生菌的临床研究已成为很多学者关注的热点,相关研究发现在消化道疾病中应用益生菌制剂可明显改善疾病的进程,通过平衡消化道菌群改善内环境从而达到辅助治疗的目的。同时在一些感染疾病中也得到证实,适当摄入益生菌可降低宿主体内炎症介质,协同抗菌作用最终提高了机体免疫力。在呼吸系统疾病的治疗方面的研究也取得了较大的进展,在临床上给呼吸系统感染的患者摄入适量益生菌,可以达到辅助抗炎,改善预后的作用。
盐雾理疗是将盐卤水通过特殊设备雾化以后,供理疗者吸入以获得治疗效果的技术。盐雾理疗的机理是病人在盐雾条件下,自然吸入低浓度的盐雾气体。该盐雾气体不仅可影响支气管的分泌液体,还可减少管内凝聚分泌物形成,调节肺泡表面活性物质,改善肺泡表面张力,改善通气功能。盐微粒可使细菌蛋白质结构脱水,具有杀菌抗炎,溶解痰液,改善气管通气状况的功能等。
本发明旨在提供一种用于保护呼吸道的盐雾与益生菌发酵上清液的混合物,其具有抗病毒保护呼吸道的效果。
发明内容
本发明旨在提供一种用于保护呼吸道的盐雾与益生菌发酵上清液的混合物,其具有抗病毒保护呼吸道的效果。
为了实现上述目的,本发明采用了如下技术方案:
一种用于保护呼吸道的盐雾混合物,其特征在于:
按照质量百分比,其包含10-20%的盐溶液,30-35%的益生菌发酵上清液。
本发明所述的盐可以是纯氯化钠,也可以是含有其他矿物质的湖盐。
中国专利2012105787207公开了一株鼠李糖乳杆菌(Lactobacillus rhamnosusLC-STH-13)CGMCC NO.3994;该菌株的已知功能是产乳酸浓度高,能在肠道中定植和存活,增强胃肠道粘膜屏障的功能,具有改善胃肠道的功能,能抑制有害菌的定植,还可在低酸环境存活,忍受肠道环境(如胃酸、胆汁等)的能力强并可定殖于人体肠道。
本申请人研发团队发现,该鼠李糖乳杆菌(Lactobacillus rhamnosus LC-STH-13)CGMCC NO.3994的发酵上清液具有一定的保护呼吸道,抗病毒效果。本发明人进一步研究发现,在该菌株的发酵特定阶段加入少量蛇床子素,具有预料不到的提高其抗病毒效果的作用。经过对比试验,该功能并非蛇床子素本身带来了,推测是蛇床子素直接参与影响了菌株的生理功能,产生较多,较高生理活性的胞外多糖等物质,从而达到该效果。
鼠李糖乳杆菌是国际公认的安全益生菌,蛇床子也是传统的抗菌治疗湿疹的药材,总体而言本发明的方案十分安全。
本发明团队还发现,盐雾和上述益生菌发酵上清液在保护呼吸道上具有一定的协同作用。
将本发明制得的盐雾混合物可以通过专用的雾化设备雾化后,供人们理疗用,也可以浓缩浸泡多孔介质如海绵制成芯片置于口罩内理疗。
本发明益生菌发酵液的制备方法为:
(1)活化菌种;
(2)配置培养基:
配置含有蛇床子素的培养基;
(3)厌氧发酵后离心取上清液,灭菌。
作为优选,上述益生菌发酵液的制备方法为:
(1)活化菌种:
取一环保藏好的鼠李糖乳杆菌(Lactobacillus rhamnosus LC-STH-13)CGMCCNO.3994,接种到MRS固体培养基中划线,37℃厌氧培养24h,挑取长势好的单菌落接种至MRS液体培养基中活化,厌氧培养成1*108cfu/ml鼠李糖乳杆菌(Lactobacillus rhamnosusLC-STH-13)CGMCC NO.3994种子液,使用乳酸调节pH值6.0;
(2)配置培养基:
蛇床子素溶液配制:取蛇床子素,溶于适量二甲基亚砜,再溶于完全培养基,配制成10mmol/L的母液;
用MRS培养基配置,加入蛇床子素母液和海藻糖,配置成含有0.1mol/L的蛇床子素,3g/L海藻糖的MRS培养基。
(3)厌氧发酵:
接种5%质量分数的种子液,厌氧培养30h,100目过滤后,用1000r/min离心10min,取上清液进行紫外灭菌。
H3N2型甲型流感病毒株,由浙江省疾病预防控制中心保存并提供。病毒经小鼠增强毒力后,于鸡胚尿囊腔常规传代培养2次,分装,-70℃保存备用。
与现有技术相比,本发明的有益效果是:
(1)盐雾本身就具有保护呼吸道,杀菌消毒的效果。
(2)本申请人研发团队发现,该鼠李糖乳杆菌(Lactobacillus rhamnosus LC-STH-13)CGMCC NO.3994的发酵上清液具有一定的保护呼吸道,抗病毒效果。本发明人进一步研究发现,在该菌株的发酵特定阶段加入少量蛇床子素,具有预料不到的提高其抗病毒效果的作用。经过对比试验,该功能并非蛇床子素本身带来了,推测是蛇床子素直接参与影响了菌株的生理功能,产生较多,较高生理活性的胞外多糖等物质,从而达到该效果。鼠李糖乳杆菌是国际公认的安全益生菌,蛇床子也是传统的抗菌治疗湿疹的药材,总体而言本发明的方案十分安全。
(3)本发明团队还发现,盐雾和上述益生菌发酵上清液在保护呼吸道上具有一定的协同作用。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。
实施例1:
一种用于保护呼吸道的盐雾混合物,其特征在于:
按照质量百分比,其包含10%的湖盐,35%的益生菌发酵上清液,其余为水。
上述益生菌发酵液的制备方法为:
(1)活化菌种:
取一环保藏好的鼠李糖乳杆菌(Lactobacillus rhamnosus LC-STH-13)CGMCCNO.3994,接种到MRS固体培养基中划线,37℃厌氧培养24h,挑取长势好的单菌落接种至MRS液体培养基中活化,厌氧培养成1*108cfu/ml鼠李糖乳杆菌(Lactobacillus rhamnosusLC-STH-13)CGMCC NO.3994种子液,使用乳酸调节pH值6.0;
(2)配置培养基:
蛇床子素溶液配制:取蛇床子素,溶于适量二甲基亚砜,再溶于完全培养基,配制成10mmol/L的母液;
用MRS培养基配置,加入蛇床子素母液和海藻糖,配置成含有0.1mol/L的蛇床子素,3g/L海藻糖的MRS培养基。
(3)厌氧发酵:
接种5%质量分数的种子液,厌氧培养30h,100目过滤后,用1000r/min离心10min,取上清液进行紫外灭菌。
实施例2
对比例1,只含有湖盐:
一种用于保护呼吸道的盐雾混合物,按照质量百分比,其包含10%的湖盐,其余为水。
对比例2,只含有益生菌发酵上清液:
一种用于保护呼吸道的盐雾混合物,其特征在于:
按照质量百分比,35%的益生菌发酵上清液,其余为水,益生菌发酵上清液制备方法同实施例1。
对比例3,培养基不含蛇床子素:
其他同实施例1,益生菌发酵液的制备方法为:
(1)活化菌种:
取一环保藏好的鼠李糖乳杆菌(Lactobacillus rhamnosus LC-STH-13)CGMCCNO.3994,接种到MRS固体培养基中划线,37℃厌氧培养24h,挑取长势好的单菌落接种至MRS液体培养基中活化,厌氧培养成1*108cfu/ml鼠李糖乳杆菌(Lactobacillus rhamnosusLC-STH-13)CGMCC NO.3994种子液,使用乳酸调节pH值6.0;
(2)配置培养基:
用MRS培养基配置,加入海藻糖,配置成含有3g/L海藻糖的MRS培养基。
(3)厌氧发酵:
接种5%质量分数的种子液,厌氧培养30h,100目过滤后,用1000r/min离心10min,取上清液进行紫外灭菌。
对比例4,培养基不含蛇床子素,最后在混合液中加入蛇床子素:
益生菌发酵液的制备同实施例3不含蛇床子素,一种用于保护呼吸道的盐雾混合物,其特征在于:按照质量百分比,35%的益生菌发酵上清液,10%的湖盐,0.05%蛇床子素,其余为水。
实施例3效果测评
H3N2型甲型流感病毒株,由浙江省疾病预防控制中心保存并提供。病毒经小鼠增强毒力后,于鸡胚尿囊腔常规传代培养2次,分装,-70℃保存备用。
测定SPF级雌性BALB/C小鼠测定的流感病毒半数致死剂量(mouse influenzavirus median lethal dose,MLD50)为10-5/100ul。
流感病毒试验步骤
(1)小鼠80只,6-8周龄,体质量18~20g,分为8组,每组10只,各组之间无统计学差异。
(2)正常对照组首日给药0.9%生理盐水100ul,分2次给与小鼠滴鼻;除开正常对照组外,小鼠为首日给药H3N2型甲型流感病毒株浓度为0.1MLD50 100ul,分2次给与小鼠滴鼻。
(3)将小鼠分为8组,各组的给药分别如下所示,小鼠雾化使用的机器为小鼠雾化仪:
对比例1组:H3N2滴鼻(首日)+对比例1成分1.50g/(kg·d)雾化*D2-8天;
对比例2组:H3N2滴鼻(首日)+对比例2成分1.50g/(kg·d)雾化*D2-8天;
对比例3组:H3N2滴鼻(首日)+对比例3成分1.50g/(kg·d)雾化*D2-8天;
对比例4组:H3N2滴鼻(首日)+对比例4成分1.50g/(kg·d)雾化*D2-8天;
实施例1组:H3N2滴鼻(首日)+实施例1成分1.50g/(kg·d)雾化*D2-8天;
正常对照组:0.9%生理盐水滴鼻+生理盐水1.50g/(kg·d)雾化*D2-8天;
达菲组:H3N2滴鼻(首日)+达菲0.03g/kg·d灌胃*D2-8天;
模型组:H3N2滴鼻(首日)+生理盐水1.50g/(kg·d)雾化*D2-8天;
给药剂量和方法如上说示。各组小鼠从第2日连续给药7日。每日喂养全价饲料,自由饮水。
(4)第8天各组摘取眼球取血后处死。血液标本室温静置1h,3000r/min离心10min分离血清,按ELISA试剂盒说明书分别检测IL-2、IFN-α的含量,结果如表1所示。
表1不同组别小鼠血中IL-2、IFN-α、IFN-β的含量
IL-2由T细胞产生,以自分泌和旁分泌方式发挥效应,INF-α、IFN-β是免疫细胞抗病毒免疫应答时产生的细胞因子,通过干扰病毒基因转录或病毒蛋白组分的翻译,拮抗流感病毒。
结果显示,在小鼠血清中,实施例1组和达菲组小鼠血清中IL-2、INF-α、IFN-β含量升高最多,而正常组和模型组升高不多,说明并没有有效刺激免疫系统发挥作用。经过对比试验,发现对比例1和对比例2都有轻微的刺激作用,单独加入蛇床子也并没有更多的提高免疫因子的含量(对比例3、4)。说明该功能并非蛇床子素本身带来的,可能是蛇床子素直接参与影响了菌株的生理功能,产生较多,较高生理活性的胞外多糖等物质,从而达到该效果。
(5)D8将各组小鼠取血处死后,开胸分离左肺,加入磷酸盐缓冲液,研磨,离心取其上清液,以Qiagen试剂盒提取肺组织中的流感病毒RNA。采用Prime ScriptTM One StepRT-PCR试剂盒及甲型流感病毒通用引物、探针来检测病毒RNA合成量。甲型流感病毒通用引物和探针由浙江省疾病预防控制中心提供,引物序列见表2。采用Prime ScriptTM OneStep RT-PCR试剂盒及甲型流感病毒通用引物、探针来检测病毒RNA合成量。
反应体系共25μL,2×One Step RT-PCR BufferⅢ12.5μL,TaKaRa Ex TaqTM HS(5U·μL-1)0.5μL,Prime ScriptTM RT Enzyme MixⅡ0.5μL,20μmol·L-1的PCR上下游引物各0.6μL,20μmol·L-1的探针0.3μL,以RNase Free dH2O补足至25μL。反应条件为:42℃逆转录30min,95℃预变性2min,95℃变性5s,55℃退火35s,共40个循环。反应完成后,分析得出各个样品的Ct值。
Ct值来表示病毒载量,Ct值越小,则病毒载量越大,小鼠流感病毒感染程度越严重,结果如表3所示。
表2引物序列
序列(5’-3’) | |
上游引物 | GACCRATCCTGTCACCTCTGAC |
下游引物 | AGGGCATTYTGGACAAAKCGTCTA |
探针 | FAM-TGCAGTCCTCGCTCACTGGGCACG-BHQ |
表3不同组别小鼠RT-PCR第8天检测的CT值
CT值 | |
对比例1组 | 14.3±1.2 |
对比例2组 | 16.7±1.8 |
对比例3组 | 16.4±1.6 |
对比例4组 | 17.1±1.6 |
实施例1 | 23.3±1.2 |
达菲组 | 22.5±1.2 |
模型组 | 13.4±1.5 |
结果显示达菲组和实施例1的病毒载量最少,而模型组的病毒载量最高。对比例1和对比例2都有轻微的抗病毒作用,单独加入蛇床子也并没有更多的抗病毒作用(对比例3、4)。说明在该菌株的发酵特定阶段加入少量蛇床子素,具有预料不到的提高其抗病毒效果的作用。
综上所述:在小鼠血清中,实施例1组和达菲组小鼠血清中IL-2、INF-α、IFN-β含量升高最多,抗病毒效果最好。而正常组和模型组升高不多,抗病毒效果差。对比例组含有盐雾组别效果也较未含有盐雾的组别效果好,说明盐雾本身就具有保护呼吸道,杀菌消毒的效果。
当加入鼠李糖乳杆菌(Lactobacillus rhamnosus LC-STH-13)CGMCC NO.3994的发酵上清液时候,和盐雾一起在保护呼吸道上具有协同作用的抗病毒效果。而对比例和实施例的对比结果显示在该菌株的发酵特定阶段加入少量蛇床子素,具有预料不到的提高其抗病毒效果的作用。并且该功能并非蛇床子素本身带来了,推测是蛇床子素直接参与影响了菌株的生理功能,产生较多,较高生理活性的胞外多糖等物质,从而达到该效果。
将本发明制得的盐雾混合物可以通过专用的雾化设备雾化后,供人们理疗用,也可以浓缩浸泡多孔介质如海绵制成芯片置于口罩内理疗。
以上所述,仅为本发明较佳的具体实施方式,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明揭露的技术范围内,根据本发明的技术方案及其发明构思加以等同替换或改变,都应涵盖在本发明的保护范围之内。
Claims (3)
1.一种用于保护呼吸道的盐雾混合物,按照质量百分比,其包含10-20%的盐溶液,30-35%的益生菌发酵上清液; 所述益生菌为鼠李糖乳杆菌(Lactobacillus rhamnosus LC-STH-13)CGMCC NO .3994;
所述益生菌发酵上清液的制备方法为:
(1)活化菌种:
取一环保藏好的鼠李糖乳杆菌CGMCC NO.3994,接种到 MRS 固体培养基中划线,37℃厌氧培养24h,挑取长势好的单菌落接种至 MRS 液体培养基中活化,厌氧培养成1*108cfu/ml鼠李糖乳杆菌CGMCC NO.3994种子液,使用乳酸调节pH值6.0;
(2)配置培养基:
蛇床子素溶液配制:取蛇床子素,溶于适量二甲基亚砜,再溶于完全培养基,配制成10mmol/L的母液;
用MRS培养基配置,加入蛇床子素母液和海藻糖,配置成含有0.1mol/L的蛇床子素,3g/L海藻糖的MRS培养基;
(3)厌氧发酵:
接种5%质量分数的种子液,厌氧培养30h,100目过滤后,用1000r/min离心10min,取上清液进行紫外灭菌;
所述盐雾混合物具有抗甲型流感病毒的作用。
2.权利要求1所述的盐雾混合物,其特征在于:
所述的盐是纯氯化钠或者含有其他矿物质的湖盐。
3.权利要求1或2所述的盐雾混合物用于制备口罩芯片的应用,所述芯片的制作方法为:将权利要求1或2所述的盐雾混合物浓缩浸泡多孔介制成芯片置于口罩内理疗。
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