CN115414389B - 一种枯草芽孢杆菌口服药物 - Google Patents
一种枯草芽孢杆菌口服药物 Download PDFInfo
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Abstract
本发明公开了一种枯草芽孢杆菌口服药物,通过以下方法制备:S1、诱导枯草芽孢杆菌产生生物被膜,并对其进行阳性聚合物修饰,使其表面电位为正;S2、水凝胶包被外膜修饰阳性聚合物的枯草芽孢杆菌,制备枯草芽孢杆菌耐酸水凝胶。本发明通过对枯草芽孢杆菌诱导,产生生物被膜,提高细菌在肠道中的黏附性;同时利用阳性聚合物对细胞进行修饰,结合水凝胶的包载,能够有效抵抗胃蛋白膜的攻击,提高枯草芽孢杆菌的存活率,且包载过程在中性条件下即可完成,进一步保留了枯草芽孢杆菌的活力。
Description
技术领域
本发明属于生物医药技术领域,具体涉及一种枯草芽孢杆菌口服药物,能有效提高益生菌口服递送过程中存活率和肠道黏附率。
背景技术
许多疑难疾病,如阿尔茨海默病、糖尿病和一些癌症,已被证明与肠道细菌的代谢有关。益生菌的补充具有抑制病原体定植和发挥有益作用的能力,是积极调节肠道菌群平衡的有效策略。虽然粪便菌群移植在预防和治疗方面取得了成功,但侵入性手术和不确定的成分在很大程度上限制了手术的实施,这不可避免地导致患者依从性低,以及胃肠道刺激和潜在的并发症。因此,一种非侵入性的方法,如口服将益生菌定植到肠道中是很有意义的,可以为益生菌治疗提供一种替代方法。但是益生菌口服过程中容易受到胃蛋白酶的分解,使得绝大部分失活或死亡,使得治疗结果下降。
枯草芽孢杆菌(BS)作为一种非常有用的益生菌,分泌的多种胞外酶已应用到许多不同领域中,其中脂肪酶和丝氨酸纤溶蛋白酶(即纳豆激酶)被广泛应用于医药行业。枯草芽孢杆菌脂肪酶具有多种催化能力,其在动物或人体的消化道中与原本存在的消化酶类共同发挥作用,使消化道处于健康的平衡状态。但是直接口服细菌,能够到达肠道的微乎其微,从而限制了枯草芽孢杆菌的功效。
发明内容
本发明的目的是为了提供一种在口服递送过程中存活率和黏附率较高的益生菌。
为了达到上述目的,本发明提供了一种枯草芽孢杆菌口服药物,通过以下方法制备:
S1、诱导枯草芽孢杆菌产生生物被膜,并对其进行阳性聚合物修饰,使其表面电位为正;
S2、水凝胶包被外膜修饰阳性聚合物的枯草芽孢杆菌,制备枯草芽孢杆菌耐酸水凝胶。
本发明通过对枯草芽孢杆菌诱导,产生生物被膜,提高细菌在肠道中的黏附性;同时利用阳性聚合物对细胞进行修饰,结合水凝胶的包载,能够有效抵抗胃蛋白膜的攻击,提高枯草芽孢杆菌的存活率,且包载过程在中性条件下即可完成,进一步保留了枯草芽孢杆菌的活力。
本发明枯草芽孢杆菌口服药物的具体制备方法如下:
S1、诱导生物被膜并进行表面修饰
在MSgg液体培养基中培养细菌,条件为37℃恒温振荡箱,转速220rpm,培养12h。然后取10ul涂板于MSgg培养平板上,37℃恒温培养箱中培养48h,得到具有生物外膜的细菌菌液。
配置25mg/mL的阳性聚合物溶液(EDAC水溶液,浓度为25mg/mL,并调节到pH7.4或PAH溶液(100mg溶于4ml 1M NaOH中),并向具有生物外膜的细菌菌液中加入阳性聚合物溶液,细菌菌液:阳性聚合物溶液为1mL:200μL。
其中阳性聚合物溶液优选PAH溶液,且阳性聚合物溶液的加入量均以过量为宜。
S2、耐酸水凝胶的制备
向阳性聚合物修饰后的细菌菌液中加入羧甲基壳聚糖溶液:配置2%的羧甲基壳聚糖水溶液5ml,将约5×104~1.0×106个外膜修饰有阳性聚合物的细菌(即步骤S1中制备获得的阳性聚合物修饰后细菌菌液约0.05ml~1ml)加入羧甲基壳聚糖溶液中搅拌均匀。
再向溶液中加钙离子络合,具体为:2%的羧甲基壳聚糖溶液与25℃下的饱和氯化钙溶液(约为10g氯化钙溶于10ml去离子水)的加入比例为10:1~5:1(V/V)。
搅拌,静置,形成包裹菌体的凝胶:2%的羧甲基壳聚糖水溶液与25℃下的饱和氯化钙溶液混合后磁力搅拌15min,搅拌条件:磁力搅拌器、温度为常温,转速为450rpm,搅拌15min;并在搅拌完成后,pH调节到7.0,将溶液在常温条件下静置,去除上清液,取絮状沉淀,得到凝胶包裹的细菌。
其中,细菌优选枯草芽孢杆菌枯草芽孢杆菌WB800N,作为一种常用的工程菌,在提高细菌存活率和黏附性的同时,还能够更好的应用于基因工程。
在部分实施例中,作为优选的,羧甲基壳聚糖溶液与外膜修饰阳性聚合物的枯草芽孢杆菌的加入比例为5ml:5×105个细菌。
本发明相比现有技术具有以下优点:
1、本发明对枯草芽孢杆菌诱导,产生生物被膜,口服至小鼠体内能够明显增加细菌的黏附数量。
2、本发明中的方法在中性pH下完成包载的枯草芽孢杆菌,包载后的枯草芽孢杆菌具有更强的活力;并通过对阳性聚合物的筛选,进一步提升枯草芽孢杆菌的包封率。
3、本发明中的方法完成羧甲基壳聚糖凝胶包载细菌修饰后,能够抵抗胃蛋白酶的攻击,提高细菌的存活率,更好的使益生菌在肠道发挥作用。
4、本发明中的枯草芽孢杆菌WB800N为一种常用的工程菌,在提高细菌的生存黏附后,使得该细菌能够更好的应用于基因工程。
5、本发明方法的制备工艺方便快捷、载菌量大、毒副作用低,不影响益生菌活性。
附图说明
图1为本发明枯草芽孢杆菌生物被膜诱导前后的原子力显微镜图;经过诱导后,可以看出表面厚度增加,说明本发明提供的方法能够成功诱导枯草芽孢杆菌能够产生生物被膜;
图2为本发明生物膜诱导、包覆凝胶过程中Zeta电势变化;其中,羧甲基壳聚糖包载产生生物被膜的枯草芽孢杆菌在pH值为7.2下进行;
图3为本发明生物膜诱导、包覆凝胶过程中Zeta电势变化;其中,羧甲基壳聚糖包载产生生物被膜的枯草芽孢杆菌在pH值为2下进行;
图4为本发明生物膜诱导、包覆凝胶过程中Zeta电势变化;其中,羧甲基壳聚糖包载修饰后的枯草芽孢杆菌于EDAC修饰到细菌表面后进行;
图5为本发明生物膜诱导、包覆凝胶过程中Zeta电势变化;其中,羧甲基壳聚糖包载修饰后的枯草芽孢杆菌于PAH修饰到细菌表面后进行;
图6为经不同处理后细菌的包封效果;
图7为不同处理后细菌的活力结果;
图8为不同量的细菌对羧甲基壳聚糖包载量的影响;
图9为羧甲基壳聚糖包载量对不同量的细菌进行包载效率变化;
图10为本发明生物膜诱导过程中及包覆凝胶后的SEM图;可以看出枯草芽孢杆菌产生生物被膜及凝胶包载后形态的变化;
图11为本发明生物膜诱导过程中及包覆凝胶后的TEM图;
图12为本发明凝胶在人工胃液(pH1.2)处理后的TEM图;水凝胶良好的包覆枯草芽孢杆菌;
图13为本发明水凝胶在不同pH条件下表面形态(SEM图);在pH 1.2时,水凝胶表面孔道关闭,为细菌避免胃酸侵蚀,起到保护细菌的作用;
图14为本发明凝胶包覆、不包覆的细菌在人工胃液中处理不同时间凝胶中细菌活死染色图;
图15为本发明凝胶包覆、不包覆的细菌在人工胃液中处理不同时间后,取出凝胶研磨涂板计数结果;
图16为本发明凝胶包覆、不包覆的细菌在人工胃液中处理不同时间后,取出凝胶研磨培养,细菌活力结果;凝胶包覆枯草芽孢杆菌后,能够提高细菌活力;
图17为本发明凝胶包覆细菌与未包覆细菌在小鼠口服24h后在胃肠道中的黏附结果;枯草芽孢杆菌诱导被膜、包覆水凝胶后,能够提高在肠道内的黏附。
具体实施方式
下面结合具体实施例和附图对本发明进行详细说明。
实施例1
制备水凝胶包覆的枯草芽孢杆菌,其经过以下步骤制得:
(1)诱导细菌产生生物被膜:在MSgg液体培养基中培养细菌(本实施例采用枯草芽孢杆菌WB800N),条件为37℃恒温振荡箱,转速220rpm,培养12h。然后取10ul涂板于MSgg培养平板上,37℃恒温培养箱中培养48h,得到具有生物外膜的细菌菌液。此时菌液浓度为1×106cfu/mL。
如图1所示,枯草芽孢杆菌经诱导后,其表面厚度增加,说明本发明提供的方法能够成功诱导枯草芽孢杆菌产生生物被膜。
(2)产生生物膜的细菌表面修饰:
配置2%的羧甲基壳聚糖溶液5ml,按以下不同方案分别进行细菌表面修饰。羧甲基壳聚糖溶液与步骤(1)获得的具有生物外膜细菌菌液的加入比例为:5ml羧甲基壳聚糖溶液中加入约0.5×106个细菌(即步骤(1)中制备获得的阳性聚合物修饰后细菌菌液约0.5ml)。
a)方案一:产生生物膜的细菌菌液,直接采用羧甲基壳聚糖-钙络合凝胶修饰。
b)方案二:将羧甲基壳聚糖溶液调节到pH2.0,使得其表面电位为正值,再修饰产生生物膜的细菌,最后加入氯化钙进行络合,标记为SCBS@CMC(pH2.0)。
c)方案三:配置EDAC溶液,pH7.4,并与步骤(1)中加入EDAC溶液,细菌:EDAC溶液为1mL:200μL;修饰后的水凝胶记为EDAC-SCBS@CMC。
d)方案四:配置PAH溶液,100mg溶于4ml 1MNaOH中,并在步骤(1)中加入PAH溶液,细菌:PAH溶液为1mL:200μL;修饰后的水凝胶记为PAH-SCBS@CMC。
按照以上方案分别将产生生物膜的细菌菌液加入羧甲基壳聚糖溶液中,搅拌均匀;再加入钙离子络合,具体为:2%的羧甲基壳聚糖溶液与25℃下的饱和氯化钙溶液(约为10g氯化钙溶于10ml去离子水)的加入量比例为:10:1~5:1(V/V)。
搅拌,静置,形成包裹菌体的凝胶:加入饱和氯化钙溶液混合后,磁力搅拌15min,搅拌条件:磁力搅拌器、温度为常温,转速为450rpm,搅拌15min;并在搅拌完成后,将溶液在常温条件下静置,pH调节到7.0,去除上清液,取絮状沉淀,得到凝胶包裹的细菌。
我们观察了以不同方案进行凝胶包覆细菌制备过程中Zeta电势变化结果(如图2~5所示),枯草芽孢杆菌产生生物膜自涂层后,Zeta电势降低。羧甲基壳聚糖的Zeta电势,在pH7.2时Zeta电位为负值。结合不同方案对细菌的包封结果,如图6,可以看出:直接采用羧甲基壳聚糖包载细菌【SCBS@CMC(pH7.2)】,只有少量的细菌被包载。
由于枯草芽孢杆菌产生生物膜自涂层后,其Zeta电位仍然为负值,而羧甲基壳聚糖当pH处于2~3之间时(如图3所示)Zeta电势呈正值,因此采用方案一直接进行羧甲基壳聚糖包载时需在pH 2~3的条件下进行,结合不同处理方案所获得的细菌包封效果和细菌活力对比可以看出(如图6、图7所示):羧甲基壳聚糖在pH 2条件下,能够提高细菌的包载量;但由于酸性包载条件的影响,细胞活力不高。
方案三中采用Zeta电势呈正值的EDAC修饰细菌(如图4所示),对细菌进行修饰后,细菌表面电位未能翻转为正值,细菌包封率(如图6所示)提高不显著。
方案四中使用阳性聚合物PAH修饰时,细菌表面Zeta电位转为正值,此时细菌包封效率提高(如图6所示),且不影响细菌活力,如图7所示。
实施例2
采用实施例1中方案四进行PAH-SCBS@CMC制备,其中羧甲基壳聚糖溶液与步骤(1)获得的具有生物外膜细菌菌液的加入比例为:5ml羧甲基壳聚糖溶液中加入约5.0×104~1.0×106个细菌(即步骤(1)中制备获得的阳性聚合物修饰后细菌菌液0.05~1ml),随着菌液加入量的增加,PAH-SCBS@CMC中枯草芽孢杆菌的包载量随之增加,当加入的细菌量超过0.5×106,包载量增加幅度很小,基本保持在3.0×105cfu/ml左右;当加入的细菌量达1.0×106时,包载量达到3.2×105cfu/ml左右,但包载效率大幅下降。如图8,9所示。
实施例2:
选择实施例1的方案四中PAH修饰产生被膜的枯草芽孢杆菌,再进行羧甲基壳聚糖与钙离子络合凝胶包载,构建PAH-SCBC@CMC,其抵抗胃酸攻击效果如下。
我们通过对上述凝胶在不同pH环境的SEM、TEM图像(如图10、11所示)分析可知:本发明条件下成功构建了PAH-SCBC@CMC。
在pH为7.2的环境中完成的凝胶,转入人工胃液(pH1.2)中,pH大幅降低后,凝胶体积缩小,凝胶中的孔隙缩小,质地变得更加密实。将在37℃人工胃液中处理2h的凝胶取出,更换至pH=7.2的环境中,凝胶又会逐渐变得疏松,恢复成絮状,(如图12、13所示)将细菌释放出来。
实施例3:
未进行凝胶包覆的细菌与凝胶包覆细菌在人工胃液中的对比
不包覆凝胶的对照组细菌Raw-BS与有凝胶包覆细菌PAH-SCBC@CMC(实施例1方案四制备所得)在人工胃液中处理不同时间活死染色图(如图14所示),不做任何处理,从0.5h荧光图像就可以看到细菌出现部分死亡。4h后,细菌基本都死亡。而有凝胶包覆的细菌,存活率得到大幅提高。在37℃人工胃液(pH 1.2)中处理不同时间后将凝胶取出研磨,涂板计数,结果如图15、16所示,可以看到随着时间的延长,PAH-SCBC@CMC细菌数量虽然会随着凝胶的损耗而减少一部分,但损耗不大,对细菌进行活力测试,发现细菌活力变化不大。未包覆凝胶的对照组细菌则直接置于37℃的胃液中,随着时间的延长,不管是细菌的数量还是活力,都在大幅的下降。
实施例4:
将细菌口服给小鼠
选择4-6周龄BALB/c小鼠,饲养2天后,取实施例1中制备的PAH-SCBC@CMC,通过灌胃的方法,口服递送到小鼠体内。24h后取不同肠段做切片,进行HE染色,结果如图17所示,与服用对照组细菌的小鼠相比,服用凝胶包覆细菌的小鼠在肠道中有更多的黏附。
虽然结合实施例对本发明的具体实施方式进行了详细地描述,但不应理解为对本专利的保护范围的限定。在权利要求书所描述的范围内,本领域技术人员不经创造性劳动即可作出的各种修改和变形仍属本专利的保护范围。
Claims (5)
1.一种枯草芽孢杆菌口服药物,其特征在于,所述枯草芽孢杆菌口服药物通过以下方法制备:
S1、诱导枯草芽孢杆菌产生生物被膜,并采用阳性聚合物PAH对其进行修饰,使其表面电位为正;
所述修饰采用如下方法:配置25mg/ml的阳性聚合物PAH溶液,向具有生物被膜的枯草芽孢杆菌菌液中加入所述阳性聚合物PAH溶液,所述阳性聚合物PAH溶液与枯草芽孢杆菌菌液体积比为200μl:1ml;
S2、水凝胶包被经阳性聚合物修饰的枯草芽孢杆菌,制备枯草芽孢杆菌耐酸水凝胶具体步骤包括:
配置2%的羧甲基壳聚糖水溶液5ml,向含有5×104~1.0×106个PAH修饰的细菌的菌液加入羧甲基壳聚糖溶液中搅拌均匀,再向溶液中加饱和氯化钙溶液进行络合,搅拌,静置,形成包裹菌体的凝胶,所述2%的羧甲基壳聚糖溶液与25℃下的饱和氯化钙溶液的加入比例为10:1~5:1(V/V)。
2.根据权利要求1所述的枯草芽孢杆菌口服药物,其特征在于,所述枯草芽孢杆菌耐酸水凝胶中,枯草芽孢杆菌的包载量为4.7×104~3.2×105cfu/ml。
3.根据权利要求1或2所述的枯草芽孢杆菌口服药物,其特征在于,所述步骤S1中诱导枯草芽孢杆菌产生生物被膜的具体步骤为:在MSgg液体培养基中培养细菌,条件为37℃恒温振荡箱,转速220rpm,培养12h;然后取10ul涂板于MSgg培养平板上,37℃恒温培养箱中培养48h,得到具有生物被膜的枯草芽孢杆菌。
4.根据权利要求1所述的枯草芽孢杆菌口服药物,其特征在于,所述步骤S2中饱和氯化钙溶液加入后,采用磁力搅拌,搅拌条件:磁力搅拌器、常温搅拌,转速为450rpm,搅拌时间15min。
5.根据权利要求1所述的枯草芽孢杆菌口服药物,其特征在于,所述枯草芽孢杆菌采用WB800N菌种。
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