CN116268419A - 一种益生菌纳米囊泡及其制备方法和在包埋生物活性物质中的应用 - Google Patents
一种益生菌纳米囊泡及其制备方法和在包埋生物活性物质中的应用 Download PDFInfo
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Abstract
一种益生菌纳米囊泡及其制备方法和在包埋生物活性物质中的应用。本发明属于食品科学领域。本发明提供一种益生菌纳米囊泡及其制备方法和在包埋生物活性物质中的应用。通过超速离心联合超声提供一种基于益生菌类囊泡的纳米载运体系的制备方法及其在生物活性物质的包埋中的应用。本发明制备的囊泡‑活性物质载运体系可提高生物活性物质的稳定性,减少胃液,肠液,热,紫外等因素造成生物活性物质结构的破坏,并且具有多种功能保健作用,如对于细胞的氧化应激反应具有保护作用和对脂多糖诱导的鼠源巨嗜细胞(RAW264.7)炎症具有的抑制作用。
Description
技术领域
本发明属于食品科学领域,具体涉及到一种益生菌纳米囊泡及其制备方法和在包埋生物活性物质中的应用。
背景技术
胞外囊泡是从动物身体组织和体液中自然释放的纳米级膜囊泡。它是生物活性化合物纳米递送系统的理想载体,因为它们具有良好的免疫学和药代动力学特性以及穿透生理屏障的能力。目前,胞外囊泡的分离主要是通过超速离心,然而,复杂的分离步骤和低的分离效率是其大规模应用的主要限制,这对其用于食品活性因子的封装是巨大的挑战。
为了突破胞外囊泡生产率低的限制,人们广泛研究了各种刺激产生或人工合成胞外囊泡,并将其应用于大规模胞外囊泡的生产。目前的研究提出了挤压或超声处理,以扩大细胞或细菌合成囊泡的生成,用于封装食品活性化合物。然而,不同来源的细胞外囊泡具有不同的功能,这些囊泡通常用于注射,不适合口服的食品活性化合物的封装。
益生菌已被证明对人类是安全的,并且它们还被证明通过与不同细胞类型相互作用来改善生物失调并调节宿主的免疫反应。此外,益生菌衍生胞外囊泡已被证明可以作为一种新的抗炎佐剂配方来增强抗炎效果,因为其脂质、蛋白质、核苷酸和代谢物可以刺激人体的免疫系统并调节巨噬细胞的极化。目前,传统方法主要采用培养后高速离心制备益生菌囊泡,然而该方法分离步骤复杂,生产效率低。
发明内容
为解决上述技术问题,本发明提供了如下技术方案:一种益生菌纳米囊泡及其制备方法和在包埋生物活性物质中的应用。通过超速离心联合超声提供一种基于益生菌类囊泡的纳米载运体系的制备方法及其在生物活性物质的包埋中的应用。本发明制备的囊泡-活性物质载运体系可提高生物活性物质的稳定性,减少胃液,肠液,热,紫外等因素造成生物活性物质结构的破坏,并且具有多种功能保健作用,如对于细胞的氧化应激反应具有保护作用和对脂多糖诱导的鼠源巨嗜细胞(RAW264.7)炎症具有的抑制作用。
本发明的目的通过如下技术方案实现:
本发明的目的之一在于是提供一种益生菌纳米囊泡的制备方法,包括,
S1:将益生菌培养后离心,收集益生菌细胞重悬于磷酸盐缓冲液,加入溶菌酶处理,然后离心,收集原生质体沉淀;
S2:将原生质体沉淀重悬于磷酸盐缓冲液中,在低温条件下,依次进行超声破碎和逐步离心,获得菌膜碎片沉淀;
S3:将菌膜碎片沉淀重悬于无菌磷酸盐缓冲液中,得到菌膜碎片溶液,低温超声,得到益生菌纳米囊泡,超低温贮存。
作为本发明纳米囊泡制备方法的一种优选方案,其中:S1中益生菌包括但不限于植物乳杆菌,干酪乳杆菌,副干酪乳杆菌,鼠李糖乳杆菌,乳双歧杆菌。
作为本发明纳米囊泡制备方法的一种优选方案,其中:S1中溶菌酶为蛋清溶菌酶。
作为本发明纳米囊泡制备方法的一种优选方案,其中:S1中溶菌酶的添加量为益生菌细胞重悬液的1-10mg/mL。
作为本发明纳米囊泡制备方法的一种优选方案,其中:S1中加入溶菌酶,在35-40℃
处理12-48h。
作为本发明纳米囊泡制备方法的一种优选方案,其中:S1中溶菌酶处理后离心的参数为:3000-10000×g下离心5-20min。
作为本发明纳米囊泡制备方法的一种优选方案,其中:S2中低温是指0-4℃。
作为本发明纳米囊泡制备方法的一种优选方案,其中:S2中超声破碎5-30min。
作为本发明纳米囊泡制备方法的一种优选方案,其中:S2中逐步离心具体过程:先于3000-10000×g下离心5-20min,然后于15000-25000×g下离心30-60min,最后在100000-200000×g下,离心30-120min。
作为本发明纳米囊泡制备方法的一种优选方案,其中:S3中低温超声为0-4℃下超声15-60min。
本发明的目的之二在于是提供一种按上述方法制得的益生菌纳米囊泡,所述益生菌纳米囊泡为球形纳米颗粒,直径为100-130nm。
本发明的目的之三在于是提供一种按上述方法制得的益生菌纳米囊泡在包埋生物活性物质中的应用。
作为本发明应用的一种优选方案,其中:生物活性物质为食品活性化合物或药物。
本发明的目的之四在于是提供一种囊泡-生物活性物质载运体系的制备方法,包括,
S1:将生物活性物质溶解于溶剂中,然后与上述方法得到的菌膜碎片溶液混合,低温超声,得到混合液;
S2:将混合液离心去除溶剂,收集沉淀并重悬于无菌磷酸盐缓冲液中,得到囊泡-生物活性物质载运体系,于超低温贮存。
作为本发明载运体系制备方法的一种优选方案,其中:S1中溶剂为极性溶剂或非极性溶剂中的一种。
作为本发明载运体系制备方法的一种优选方案,其中:S1中溶剂中生物活性物质的浓度为1-20mg/mL。
作为本发明载运体系制备方法的一种优选方案,其中:S1中菌膜碎片溶液的浓度为1-10mg/mL。
作为本发明载运体系制备方法的一种优选方案,其中:S1中生物活性物质与菌膜碎片的质量比为1:(1-10)。
作为本发明载运体系制备方法的一种优选方案,其中:S2中离心为在100000-200000×g下离心30-120min。
本发明的目的之五在于是提供一种按上述方法制得的囊泡-生物活性物质载运体系。
与现有技术相比,本发明具有如下有益效果:
(1)本发明以益生菌作为原料,使用溶菌酶处理以去除细胞壁,高效获得原生质体,利用超声与超速离心方法联用,获得质膜碎片,再通过超声自组装的原理,形成囊泡,这种制备方式极大的提高了益生菌衍生胞外囊泡的产量。
(2)本发明制备的囊泡进一步应用于生物活性化合物的包载,制备的囊泡-活性物质载运体系可提高生物活性物质的稳定性,减少胃液,肠液,热,紫外等因素造成生物活性物质结构的破坏。
(3)本发明制备的囊泡-活性物质载运体系具有诸多功能保健作用:对于细胞的氧化应激反应具有保护作用;对脂多糖诱导的鼠源巨嗜细胞(RAW264.7)炎症具有的抑制作用。
附图说明
图1为本发明对比例1提取的天然纳米囊泡的扫描电镜图;
图2为本发明对比例1提取的天然纳米囊泡的粒径分布图;
图3为本发明实施例1制备的纳米囊泡的扫描电镜图;
图4为本发明实施例1制备的纳米囊泡的粒径分布图;
图5为本发明实施例1与对比例1制备的纳米囊泡的产量对比图;
图6为本发明实施例1制备的纳米囊泡的细胞毒性测定。
图7为本发明应用例1制备的囊泡-岩藻黄质载运体系的扫描电镜图;
图8为本发明应用例1制备的囊泡-岩藻黄质载运体系的粒径分布图;
图9为本发明应用例1制备的囊泡-岩藻黄质载运体系与岩藻黄质在水溶液中的溶解性对比图;
图10为本发明应用例1制备的囊泡-岩藻黄质载运体系在胃液,小肠液,加热和紫外照射下的稳定性;
图11为本发明应用例1制备的囊泡-岩藻黄质载运体系在小肠液中的稳定性;
图12为本发明应用例1制备的囊泡-岩藻黄质载运体系在加热条件下的稳定性;
图13为本发明应用例1制备的囊泡-岩藻黄质载运体系在紫外照射下的稳定性;
图14为本发明应用例1制备的囊泡-岩藻黄质载运体系的细胞毒性测定。
具体实施方式
为使本发明的上述目的、特征和优点能够更加明显易懂,下面结合说明书实施例对本发明的具体实施方式做详细的说明。
在下面的描述中阐述了很多具体细节以便于充分理解本发明,但是本发明还可以采用其他不同于在此描述的其它方式来实施,本领域技术人员可以在不违背本发明内涵的情况下做类似推广,因此本发明不受下面公开的具体实施例的限制。
其次,此处所称的“一个实施例”或“实施例”是指可包含于本发明至少一个实现方式中的特定特征、结构或特性。在本说明书中不同地方出现的“在一个实施例中”并非均指同一个实施例,也不是单独的或选择性的与其他实施例互相排斥的实施例。
实施例1本实施例的益生菌纳米囊泡的制备方法按以下步骤进行:
S1:
植物乳酸菌的培养:配制MRS液体培养基,灭菌后按1%接入植物乳酸菌,37℃培养24h;
制备益生菌原生质体:植物乳杆菌悬浮液以8000×g离心10分钟,收集益生菌细胞重悬于10mL磷酸盐缓冲液溶液中,加入40mg的蛋清溶菌酶,35℃处理24小时,除去细胞壁,然后将混合液以80000×g离心10分钟,原生质体沉淀。
S2:
制备菌膜碎片沉淀:将200mgS1得到的原生质体沉淀重悬于40mL磷酸盐缓冲液中,在0℃下,先超声破碎30分钟,然后进行逐步离心,获得菌膜碎片沉淀;
所述逐步离心具体是:先在3500×g下离心10分钟,收集上清液A。将上清液A在20000×g条件下离心30分钟,收集上清液B。最后将上清液B在100000×g条件下,离心60分钟。
S3:
制备囊泡:将40mgS2得到的菌膜碎片沉淀重悬于5mL无菌磷酸盐缓冲液中,得到菌膜碎片溶液,在0℃条件下超声30分钟,得到益生菌纳米囊泡,置于-80℃条件下贮存。
对比例1益生菌衍生天然囊泡的提取分离
利用传统的超速离心方法,从植物乳杆菌培养液中分离益生菌衍生的胞外囊泡,形成囊泡,包括步骤:
S1、植物乳酸菌的培养:配制MRS液体培养基,灭菌后按1%接入植物乳酸菌,37℃培养24h。
S2、益生菌衍生的胞外囊泡的分离:植物乳杆菌悬浮液8000×g离心15min,并收集上清液。随后,将收集的上清液过0.22μm滤膜后,以150000×g离心180min,收集沉淀物并重新悬浮在无菌PBS中,将收集的样品通过0.22μm过滤器以获得纯化的益生菌衍生天然囊泡,并在-80℃下储存。
检测试验(一):
使用扫描电子显微镜观察本发明对比例1得到的益生菌衍生天然囊泡,结果如图1所示,是球形纳米颗粒,直径超过100nm。使用激光粒度仪测定本发明对比例1分离的天然益生菌囊泡的粒径分布,结果如图2所示,囊泡的平均粒径为142nm,与扫描电镜观察结果相对应。
使用扫描电子显微镜观察本发明对实施例1制备的益生菌纳米囊泡,结果如图3所示,是球形纳米颗粒,直径在100nm左右,与对比例1中分离天然益生菌衍生囊泡的结构大小类似。使用激光粒度仪测定本发明实施例1制备的益生菌纳米囊泡的粒径分布,结果如图4所示,囊泡的平均粒径为124nm,略小于对比例1分离天然益生菌衍生囊泡。
检测试验(二):
比较了实施例1与对比例1制备囊泡的产量,结果如图5所示,实施例1制备的益生菌类囊泡产量是对比例1分离天然益生菌衍生囊泡产量的150倍。
检测试验(三):
测定实施例1制备的益生菌纳米囊泡的生物安全性;选用RAW264.7细胞和含有10%(V胎牛血清/VDMEM培养基=1/9)的胎牛血清的高糖DMEM培养基。将细胞以1×104个细胞/孔的密度接种于96孔板,在体积分数为5%的CO2培养箱中孵育12h。将制备的类囊泡添加于培养基中,使得类囊泡在培养基中的终浓度为0、10、25、50、100、150、200μg/mL。将含有囊泡的培养基加入细胞中,培养12h。之后每孔加入MTT(0.5mg/mL)溶液20μL,继续培养4h。之后去除培养基,在每孔加入150μL二甲基亚砜,并在摇床上低速振荡10min,使结晶物充分溶解,在酶联免疫检测仪OD490nm处测量各孔的吸光值。同时设置调零孔(培养基、MTT、二甲基亚砜),对照孔(细胞、相同浓度的药物溶解介质、培养液、MTT、二甲基亚砜)。通过以下公式计算细胞活力。
细胞活力=[(对照-空白)-(给药-空白)]/(对照-空白)×100%
图6为不同浓度类对RAW264.7细胞的毒性作用,结果表明,在浓度为100μg/mL的类囊泡的作用下,RAW264.7细胞的活力与对照组并无显著性差异,证明本发明所制备的益生菌类囊泡在低浓度下不存在细胞毒性,具有良好的生物相容性,这是保证其应用于食品和药品领域应用的必要条件。
应用例1:囊泡-岩藻黄质载运体系的制备方法按以下步骤进行:
S1:将5mg岩藻黄质溶解于0.5mL乙醇中,然后与5mL实施例1S3得到的菌膜碎片溶液混合,在0℃条件下超声30分钟,得到混合液;
S2:将混合液在100000×g条件下,离心60分钟,收集沉淀,重悬于无菌磷酸盐缓冲液中,得到囊泡-岩藻黄质载运体系,于-80℃条件下贮存。
检测试验(四):
使用扫描电子显微镜观察本发明对应用例1制备的囊泡-岩藻黄质载运体系,结果如图7所示,是表面光滑的球形纳米颗粒。使用激光粒度仪测定本发明应用例1制备的囊泡-岩藻黄质载运体系中的囊泡的粒径分布,结果如图8所示,囊泡的平均粒径为422nm,与单独的类囊泡相比,粒径增大了3倍以上。
检测试验(五):
图9是应用例1制备的囊泡-岩藻黄质载运体系在不同消化液中的溶解性,结果可见,游离的岩藻黄质在水中呈聚集沉淀的状态,而囊泡-岩藻黄质载运体系在水中溶解,这是因为在结合过程中,岩藻黄质嵌入到囊泡磷脂分子层的疏水内部,并且外部磷脂的亲水头可以分散在水中。这种组合可以提高岩藻黄质在水中的溶解度,有利于其在食品基质中的应用,提高其生物利用度。
检测试验(六):
岩藻黄质由于其独特的化学结构,稳定性较差。因此,测定了游离岩藻黄质和囊泡-岩藻黄质载运体系在胃液和肠液中的保留量(图10和图11)。游离岩藻黄质在胃液中的相对含量在30分钟内迅速降至23%,2小时后降至20%以下。口服时,游离岩藻黄质易被消化液中的pH值和酶降解。当暴露于消化液中,应用例1制备的囊泡-岩藻黄质载运体系可保持较高的岩藻黄质含量,在胃液中达到58.09%,在肠液中达到86.45%。这主要是由于囊泡的脂质双层,保护FX免受消化液的低pH和人类消化过程中蛋白酶的酶降解。
检测试验(七):
进一步评估了应用例1制备的囊泡-岩藻黄质载运体系在加热和紫外照射下对岩藻黄质稳定性的增强作用。如图12所示,在80℃的加热条件下,盐藻黄质的相对含量在60分钟时急剧下降至41.43%。这表明,在加热条件下,囊泡可有效防止岩藻黄质的降解。
检测试验(八):
岩藻黄质在紫外照射下的稳定性测试表明,在紫外照射150分钟后,应用例1制备的囊泡-岩藻黄质载运体系中岩藻黄质的相对含量与游离岩藻黄质相比增加了23%(图13)。这些结果表明,囊泡可以有效地防止岩藻黄质在加热和紫外线照射下的降解。
检测试验(九):
测定应用例1制备的囊泡-岩藻黄质载运体系对细胞的毒性作用。图14为不同浓度囊泡-岩藻黄质载运体系处理细胞24小时后的细胞活力。结果显示,当浓度低于100μg/mL时,细胞活力与对照组无显著性差异,这表明在此浓度范围内,囊泡-岩藻黄质载运体系对细胞并未表现出明显的细胞毒性。而当囊泡-岩藻黄质载运体系浓度达到150μg/mL和200μg/mL时,细胞活力分别为92.81%和91.14%。综合以上结果,囊泡-岩藻黄质载运体系对RAW264.7细胞均具有良好的生物相容性且无毒副作用。
应说明的是,以上实施例仅用以说明本发明的技术方案而非限制,尽管参照较佳实施例对本发明进行了详细说明,本领域的普通技术人员应当理解,可以对本发明的技术方案进行修改或者等同替换,而不脱离本发明技术方案的精神和范围,其均应涵盖在本发明的权利要求范围当中。
Claims (10)
1.一种益生菌纳米囊泡的制备方法,其特征在于,包括,
S1:将益生菌培养后离心,收集益生菌细胞重悬于磷酸盐缓冲液,加入溶菌酶处理,然后离心,收集原生质体沉淀;
S2:将原生质体沉淀重悬于磷酸盐缓冲液中,在低温条件下,依次进行超声破碎和逐步离心,获得菌膜碎片沉淀;
S3:将菌膜碎片沉淀重悬于无菌磷酸盐缓冲液中,得到菌膜碎片溶液,低温超声,得到益生菌纳米囊泡,超低温贮存。
2.根据权利要求1所述的方法,其特征在于,S1中益生菌包括但不限于植物乳杆菌,干酪乳杆菌,副干酪乳杆菌,鼠李糖乳杆菌,乳双歧杆菌,溶菌酶为蛋清溶菌酶,溶菌酶的添加量为益生菌细胞重悬液的1-10mg/mL,加入溶菌酶,在35-40℃处理12-48h,溶菌酶处理后离心的参数为:3000-10000×g下离心5-20min。
3.根据权利要求1所述的方法,其特征在于,S2中低温是指0-4℃,超声破碎5-30min,逐步离心具体过程:先于3000-10000×g下离心5-20min,然后于15000-25000×g下离心30-60min,最后在100000-200000×g下,离心30-120min。
4.根据权利要求1所述的方法,其特征在于,S3中低温超声为0-4℃下超声15-60min。
5.权利要求1-4任一项所述的方法制得的益生菌纳米囊泡,其特征在于,它为球形纳米颗粒,直径为100-130nm。
6.权利要求1-4任一项所述的方法制得的益生菌纳米囊泡在包埋生物活性物质中的应用。
7.根据权利要求6所述的应用,其特征在于,生物活性物质为食品活性化合物或药物。
8.一种囊泡-生物活性物质载运体系的制备方法,其特征在于,包括,
S1:将生物活性物质溶解于溶剂中,然后与权利要求1-4任一项所述的方法得到的菌膜碎片溶液混合,低温超声,得到混合液;
S2:将混合液离心去除溶剂,收集沉淀并重悬于无菌磷酸盐缓冲液中,得到囊泡-生物活性物质载运体系,于超低温贮存。
9.根据权利要求8所述的方法,其特征在于,S1中溶剂为极性溶剂或非极性溶剂中的一种,溶剂中生物活性物质的浓度为1-20mg/mL,菌膜碎片溶液的浓度为1-10mg/mL,生物活性物质与菌膜碎片的质量比为为1:(1-10),S2中离心为在100000-200000×g下离心30-120min。
10.权利要求8或9所述的方法制得的囊泡-生物活性物质载运体系。
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