CN117297099A - 基于海藻酸钠和金属多酚网络的益生菌涂层制备方法 - Google Patents
基于海藻酸钠和金属多酚网络的益生菌涂层制备方法 Download PDFInfo
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- A23L29/30—Foods or foodstuffs containing additives; Preparation or treatment thereof containing carbohydrate syrups; containing sugars; containing sugar alcohols, e.g. xylitol; containing starch hydrolysates, e.g. dextrin
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Abstract
本发明公开了基于海藻酸钠和金属多酚网络的益生菌涂层制备方法,属于新型材料应用领域,将益生菌重悬在PBS缓冲液中,然后向其中加入多酚溶液,涡旋15s分散均匀后,向其加入金属离子溶液,涡旋15s,后加入PBS缓冲液来稳定体系pH,最后通过钙离子诱导海藻酸钠涂层在益生菌表面。所述金属多酚网络是通过多酚与金属离子络合配位形成的团聚体包覆在益生菌表面。本发明的金属多酚网络涂层能够自组装快速形成,绿色环保,方便快捷,对益生菌的正常生长没有明显影响。通过透射电镜和共聚焦激光显微镜观察证实了益生菌表面的金属多酚网络涂层成功,实现了益生菌的快速涂层,保护其免受恶劣环境的影响。
Description
技术领域
本发明涉及新型材料应用技术领域,具体为基于海藻酸钠和金属多酚网络的益生菌涂层制备方法。
背景技术
随着健康饮食理念的深入人心,益生菌食品受到了广泛关注。研究表明,益生菌能够发挥益生作用的最低浓度为106CFU/g,然而,益生菌通过口腔摄入后,经食管、胃,最终到达肠道,要经历胃酸、胆汁、消化酶等重重考验。胃液的强酸性环境能够杀死大多数益生菌,而肠道里的胆盐也可通过溶解脂质对细菌结构造成破坏,这大大限制了益生菌在体内的疗效,因此,采用适当的方法对益生菌进行保护至关重要。
金属多酚网络是基于多酚分子的邻苯二酚基团与金属离子配位络合形成的自组装超分子非晶态材料,通过双组分在均相溶剂中配位实现材料相分离而获取得到的,该自组装过程是一种反应动力学迅速、反应条件温和的组装制备过程。
益生菌经金属多酚网络涂层后,肠道粘附能力和体外胃肠道耐受能力都得到了提高,大大增强了益生菌的生理活性,从而更好的发挥体内益生活性,改善人体健康。
所以我们提出了基于海藻酸钠和金属多酚网络的益生菌涂层制备方法,以便于解决上述中提出的问题。
发明内容
本发明的目的在于提供基于海藻酸钠和金属多酚网络的益生菌涂层制备方法,以解决上述背景技术提出的目前市场上现有的制备方法在制备的过程中整体的工艺较为复杂,不便于有效的保证益生菌活性的问题。
为实现上述目的,本发明提供如下技术方案:基于海藻酸钠和金属多酚网络的益生菌涂层制备方法,包括以下步骤:
第一步、将培养好的益生菌用PBS缓冲液清洗三次,取适量108CFU/mL的益生菌重悬在4.75-9.5mLPBS缓冲液中,缓冲液的pH值为7.2,涡旋15s以使菌体分散均匀;
第二步、向其中加入50-200μL多酚溶液,涡旋15s以使多酚分散均匀;
第三步、然后向其中加入10-50μL金属离子溶液,迅速涡旋15s后,再向其中加入5-10mLPBS缓冲液,以稳定体系的pH;
第四步、将得到的菌悬液离心5000-12000rpm/min,5-10min后,得到益生菌和表面涂层;
第五步、用PBS缓冲液重悬两次并离心5000-12000rpm/min,5-10min后,以去除未结合的多酚;
第六步、然后向其中加入100-1000μLCaCl2溶液,CaCl2溶液为10-100mM,涡旋30s,接着加入100-1000μL海藻酸钠溶液,海藻酸钠溶液为1mg/mL,快速涡旋1min后,将菌悬液离心5000-12000rpm/min,5-10min后,弃上清液得到益生菌及涂层。
优选的,所述益生菌为植物乳杆菌、鼠李糖乳杆菌、双歧杆菌和嗜热链球菌。
优选的,所述多酚为单宁酸、没食子酸、原儿茶酸和EGCG,且多酚溶液为10-40mg/mL。
优选的,所述金属离子为铁离子、铜离子、锌离子、镁离子、铝离子、锰离子和钛离子,且金属离子溶液为10-40mg/mL。
与现有技术相比,本发明的有益效果是:该基于海藻酸钠和金属多酚网络的益生菌涂层制备方法,制备条件温和,工艺简单易行,保证了益生菌的活性。所采用的多酚和金属离子廉价易得,且金属和多酚配位形成的网络结构可以增强益生菌对外界环境的抗性,透射电镜和共聚焦显微镜表明涂层成功。
附图说明
图1为本发明经涂层后的益生菌尺寸变化示意图;
图2为本发明不同涂层包埋的益生菌透射电镜图示意图;
图3为本发明不同涂层包埋的益生菌生理活性示意图;
图4为本发明胃酸耐受实验示意图;
图5为本发明胆盐耐受实验示意图。
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。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
请参阅图1-图5,本发明提供如下三种实施例:
实施例1:本实施例中多酚采用原儿茶酸溶液,同时金属离子采用铁离子,且益生菌采用植物乳杆菌作为原料进行制备加工,具体的公开内容如下:
基于海藻酸钠和金属多酚网络的益生菌涂层制备方法,包括如下步骤:
第一步、将培养好的益生菌用PBS缓冲液清洗三次,取适量108CFU/mL的植物乳杆菌重悬在4.75-9.5mLPBS(pH:7.2)缓冲液中,涡旋15s以使菌体分散均匀;
第二步、向其中加入50-200μL原儿茶酸(10-40mg/mL)溶液,涡旋15s以使多酚分散均匀;
第三步、然后向其中加入10-50μL铁离子(10-40mg/mL)溶液,迅速涡旋15s后,再向其中加入5-10mLPBS缓冲液,以稳定体系的pH;
第四步、将得到的菌悬液离心5000-12000rpm/min,5-10min后,得到益生菌和表面涂层。
第五步、用PBS缓冲液重悬两次并离心5000-12000rpm/min,5-10min后,以去除未结合的多酚。
第六步、然后向其中加入100-1000μLCaCl2溶液(10-100mM),涡旋30s,接着加入100-1000μL海藻酸钠溶液(1mg/mL),快速涡旋1min后,将菌悬液离心5000-12000rpm/min,5-10min后,弃上清液得到益生菌及涂层。
实施例2:本实施例中技术方案与上述实施例一的区别是:在本实施例中益生菌采用鼠李糖乳杆菌,且多酚采用EGCG,同时金属离子采用铜离子作为原料进行制备加工,具体的公开内容如下:
基于海藻酸钠和金属多酚网络的益生菌涂层制备方法,包括如下步骤:
第一步、将培养好的益生菌用PBS缓冲液清洗三次,取适量108CFU/mL的鼠李糖乳杆菌重悬在4.75-9.5mLPBS(pH:7.2)缓冲液中,涡旋15s以使菌体分散均匀;
第二步、向其中加入50-200μLEGCG(10-40mg/mL)溶液,涡旋15s以使多酚分散均匀;
第三步、然后向其中加入10-50μL铜离子(10-40mg/mL)溶液,迅速涡旋15s后,再向其中加入5-10mLPBS缓冲液,以稳定体系的pH;
第四步、将得到的菌悬液离心5000-12000rpm/min,5-10min后,得到益生菌和表面涂层。
第五步、用PBS缓冲液重悬两次并离心5000-12000rpm/min,5-10min后,以去除未结合的多酚。
第六步、然后向其中加入100-1000μLCaCl2溶液(10-100mM),涡旋30s,接着加入100-1000μL海藻酸钠溶液(1mg/mL),快速涡旋1min后,将菌悬液离心5000-12000rpm/min,5-10min后,弃上清液得到益生菌及涂层。
实施例3:本实施例中技术方案与上述实施二的区别是:在本实施例中益生菌采用植物乳杆菌,且多酚采用单宁酸,同时金属离子采用铁离子作为原料进行制备加工,具体的公开内容如下:
基于海藻酸钠和金属多酚网络的益生菌涂层制备方法,包括如下步骤:
第一步、将培养好的益生菌用PBS缓冲液清洗三次,取适量108CFU/mL的植物乳杆菌重悬在4.75-9.5mLPBS(pH:7.2)缓冲液中,涡旋15s以使菌体分散均匀;
第二步、向其中加入50-200μL单宁酸(10-40mg/mL)溶液,涡旋15s以使多酚分散均匀;
第三步、然后向其中加入10-50μL铁离子(10-40mg/mL)溶液,迅速涡旋15s后,再向其中加入5-10mLPBS缓冲液,以稳定体系的pH;
第四步、将得到的菌悬液离心5000-12000rpm/min,5-10min后,得到益生菌和表面涂层。
第五步、用PBS缓冲液重悬两次并离心5000-12000rpm/min,5-10min后,以去除未结合的多酚。
第六步、然后向其中加入100-1000μLCaCl2溶液(10-100mM),涡旋30s,接着加入100-1000μL海藻酸钠溶液(1mg/mL),快速涡旋1min后,将菌悬液离心5000-12000rpm/min,5-10min后,弃上清液得到益生菌及涂层。
实验结果部分:LP为益生菌单体,PCA-LP为经金属多酚网络涂层包埋的益生菌,SA-PCA-LP为经海藻酸钠和金属多酚网络复合涂层的益生菌。
结果表明,海藻酸钠和金属多酚网络复合涂层包埋的益生菌成功构建,其生理活性不受影响,且相较于单独的益生菌,在胃肠道环境中表现出更强的耐性。
本说明书中未作详细描述的内容属于本领域专业技术人员公知的现有技术。
尽管参照前述实施例对本发明进行了详细的说明,对于本领域的技术人员来说,其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (4)
1.基于海藻酸钠和金属多酚网络的益生菌涂层制备方法,其特征在于,包括以下步骤:
(1)、将培养好的益生菌用PBS缓冲液清洗三次,取适量108CFU/mL的益生菌重悬在4.75-9.5mLPBS缓冲液中,缓冲液的pH值为7.2,涡旋15s以使菌体分散均匀;
(2)、向其中加入50-200μL多酚溶液,涡旋15s以使多酚分散均匀;
(3)、然后向其中加入10-50μL金属离子溶液,迅速涡旋15s后,再向其中加入5-10mLPBS缓冲液,以稳定体系的pH;
(4)、将得到的菌悬液离心5000-12000rpm/min,5-10min后,得到益生菌和表面涂层;
(5)、用PBS缓冲液重悬两次并离心5000-12000rpm/min,5-10min后,以去除未结合的多酚;
(6)然后向其中加入100-1000μLCaCl2溶液,CaCl2溶液为10-100mM,涡旋30s,接着加入100-1000μL海藻酸钠溶液,海藻酸钠溶液为1mg/mL,快速涡旋1min后,将菌悬液离心5000-12000rpm/min,5-10min后,弃上清液得到益生菌及涂层。
2.根据权利要求1所述的基于海藻酸钠和金属多酚网络的益生菌涂层制备方法,其特征在于:所述益生菌为植物乳杆菌、鼠李糖乳杆菌、双歧杆菌和嗜热链球菌。
3.根据权利要求1所述的基于海藻酸钠和金属多酚网络的益生菌涂层制备方法,其特征在于:所述多酚为单宁酸、没食子酸、原儿茶酸和EGCG,且多酚溶液为10-40mg/mL。
4.根据权利要求1所述的基于海藻酸钠和金属多酚网络的益生菌涂层制备方法,其特征在于:所述金属离子为铁离子、铜离子、锌离子、镁离子、铝离子、锰离子和钛离子,且金属离子溶液为10-40mg/mL。
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