CN113575612A - 细菌纤维素负载金纳米片的抗菌薄膜及其制备方法和应用 - Google Patents

细菌纤维素负载金纳米片的抗菌薄膜及其制备方法和应用 Download PDF

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CN113575612A
CN113575612A CN202110814479.2A CN202110814479A CN113575612A CN 113575612 A CN113575612 A CN 113575612A CN 202110814479 A CN202110814479 A CN 202110814479A CN 113575612 A CN113575612 A CN 113575612A
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bacterial cellulose
gold
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周满
孟珂卉
李忠玉
梁倩
徐松
梁玄
薛苏婷
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Changzhou University
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Abstract

本发明属于微生物技术以及无机材料领域，涉及一种细菌纤维素负载金纳米片的抗菌薄膜及其制备方法和应用，制备方法包括以下步骤：将细菌纤维素膜浸入0.1M 90℃～95℃NaOH溶液中，然后用蒸馏水洗至中性，真空干燥即可得到细菌纤维素纯样品；然后将获得的细菌纤维素纯样品浸入氯金酸溶液中并用紫外光照射，紫外光照后用去离子水水洗，再真空干燥即得所述细菌纤维素负载金纳米片的抗菌薄膜。本发明紫外光诱导细菌纤维素还原金纳米片的制备方法简单环保，可重复使用，且由于细菌纤维素具有良好的生物可降解性，因此细菌纤维素负载的金纳米片有着较好的抗菌能力和自降解能力。

Description

细菌纤维素负载金纳米片的抗菌薄膜及其制备方法和应用
技术领域
本发明涉及一种细菌纤维素负载金纳米片的抗菌薄膜及其制备方法和应用，属于微生物技术以及无机材料领域。
背景技术
抗菌材料对于解决当今传染病以及细菌危害人类生命健康具有重要意义，因此开发可重复使用且对环境友好的抗菌薄膜已成为生物化学和环境化学等许多其他相关领域的研究热点。细菌纤维素(BC)是一种自然界中储备丰富的天然纳米生物材料，具有可生物降解，生物相容性好。但是细菌纤维素它的缺点是本身不具有抗菌的能力，对于细菌来说细菌纤维素不能抑制或杀灭细菌，而且易被微生物腐蚀，难以应对被细菌感染所造成的伤口。因此，赋予细菌纤维素独特的抗菌性在生命科学中具有重要意义。目前，抗菌材料更多的是指通过添加一定的抗菌物质(抗菌剂)，从而使材料具有抑制或杀灭其表面细菌能力的一类新型功能材料，如抗菌塑料、抗菌纤维、抗菌陶瓷等。近年来，我们团队已经报道了利用细菌纤维素进行光催化剂等应用。然而，就我们所知，使用细菌纤维素进行抗菌防病的进展甚微。
金是一种过渡金属，被溶解后可以形成三价或者一价正离子(+1/+3/零价)。金对红外线的反射能力接近100％。部分金的盐类有防止发炎的性质，被用作治疗关节炎，研究表明，金纳米颗粒的抗菌谱广,抗菌机制多样,且生物相容性好。
金纳米颗粒(AuNPs)通常是通过还原金盐(例如四氯金酸(HAuCl4))或使用种子生长方法原位合成的，其中，颗粒逐步增大。根据其浓度和粒径以及周围的表面官能团(配体)，金纳米粒子可以是生物相容性的，并且是化学惰性的，可以用于医疗应用，例如细胞成像和光热疗法。
在过去的10年中，研究表明AuNPs在不与商业抗生素结合的情况下，也可以非常有效地对抗细菌，包括耐多药的细菌。总之，AuNPs的抗菌谱广,抗菌机制多样,且生物相容性好。我们利用细菌纤维素表面的羟基具有还原性，将氯金酸还原成形貌可控的金纳米片，内嵌于细菌纤维素的三维骨架中，将其应用于抑制常见的大肠杆菌(E.coli)和金黄色葡萄球菌(S.aureus)的生长。
发明内容
本发明的目的是提供一种细菌纤维素负载金纳米片的抗菌薄膜及其制备方法和应用。在紫外光照下，进行时间调控，诱导细菌纤维素能够自还原氯金酸，生成形貌可控的金纳米片，简单处理后能重复对大肠杆菌，金黄色葡萄球菌产生较好的抑制作用。
一种细菌纤维素负载金纳米片抗菌薄膜的制备方法，包括步骤如下：
a、细菌纤维素的纯化：将细菌纤维素膜浸入0.1M 90℃～95℃NaOH溶液中(浸泡时间优选2h)，用蒸馏水洗至中性，真空干燥即可得到细菌纤维素纯样品。
b、制备细菌纤维素金纳米片抗菌薄膜(简称AuNSs@BC)：将步骤a获得的细菌纤维素纯样品浸入氯金酸溶液中(作为优选，氯金酸溶液的浓度为30～100mM)并用紫外光照射，紫外光照后用去离子水水洗，再真空干燥即得所述AuNSs@BC。
作为优选，步骤b中每100ml的所述氯金酸溶液中浸入细菌纤维素的质量为6～8g。
作为优选，步骤b中紫外光波长为254nm～365nm紫外光照时间为12h。
一种细菌纤维素负载金纳米片抗菌薄膜，基于如上方法制得。
如上所述的细菌纤维素负载金纳米片抗菌薄膜的应用，用于抑制大肠杆菌和金黄色葡萄球菌。
本发明的有益效果是：细菌纤维素负载金纳米片抗菌薄膜的制备方法简单环保，可重复使用，且由于细菌纤维素具有良好的生物可降解性，因此细菌纤维素负载的金纳米片有着较好的抗菌能力和自降解能力。此外，AuNSs@BC相比于纯BC膜具有更好的抗拉强度和延伸率。本方法制得的金纳米片分散性高，无明显团聚现象，金纳米片的边长范围为4-12nm。
附图说明
图1为实施例一中紫外光诱导细菌纤维素还原金纳米片获得的AuNSs-S@BC的场发射扫描电镜图。
图2为实施例二中细菌纤维素浸渍30mM、60mM、100mM氯金酸溶液制得AuNPs@BC、AuNSs-S、AuNSs-L@BC的X射线衍射(XRD)谱图，(实施例2抑菌测试后的AuNSs@BC)。
图3为实施例二中紫外光诱导细菌纤维素还原金纳米片AuNSs-S@BC的透射电子显微镜图。
图4为实施例二中光学显微镜图像统计AuNSs边长尺寸分布柱状图。
图5为实施例二中AuNPs@BC、AuNSs-S@BC、AuNSs-L@BC对金黄色葡萄球菌、大肠杆菌的抑制带。
图6为实施例三中重复使用三次AuNSs-S@BC对金黄色葡萄球菌、大肠杆菌的抑制带。
图7为实施例二中AuNPs@BC、AuNSs-S@BC、AuNSs-L@BC对金黄色葡萄球菌、大肠杆菌的生长曲线图。具体实施方式
现在结合具体实施例对本发明做进一步说明，以下实施例旨在说明本发明而不是对本发明的进一步限定。
以下实施例中细菌纤维素为从桂林奇宏科技公司购买。
以下实施例制得的细菌纤维素负载金纳米片的抗菌薄膜的抗菌效果评价采用圆盘扩散法，其中固体培养基制备方法如下：
固体培养基制备：使用电子天平称取营养肉汤、琼脂粉溶于去离子水中，放入高压蒸汽灭菌锅中灭菌30min形成原始无菌培养基。在超净工作台上将温热的原始无菌培养基倒入经紫外灭菌30min的培养皿中，待其冷却凝固后形成固体培养基。其中，营养肉汤的质量为3.6g和琼脂粉的质量均为3.0g，去离子水的体积为200mL。
配制材料大肠杆菌和金黄色葡萄球菌：使用划线平板法进行培养，各取金黄色葡萄球菌、大肠杆菌一株分别置于营养纯肉汤中(无琼脂)，于37℃恒温细菌培养箱中振荡培养。其中营养肉汤的质量为9g，去离子水的体积为500mL，细菌孵育时间为12h。
配制材料对大肠杆菌和金黄色葡萄球菌的抗菌效果：将裁剪成圆片的AuNPs@BC、AuNSs-S@BC、AuNSs-L@BC分别加入金黄色葡萄球菌、大肠杆菌稀释液1mL，加入固体培养基中，于37℃恒温细菌培养箱中振荡培养。其中AuNPs@BC、AuNSs-S@BC、AuNSs-L@BC裁成尺寸为6mm的圆片，细菌孵育时间为24h。抑菌带宽度H＝(D－d)/2，式中，H—抑菌带宽度(mm)；D—抑菌带外径的平均值(mm)；d—试样直径(mm)；
表1抗菌效果评价
Figure BDA0003169460580000041
Figure BDA0003169460580000051
说明：细菌在培养皿中，能够迅速生长，但由于敷料样品具有抗菌能力，细菌在样品周围会生成一个明显的细菌阻滞圈，即抑菌圈。
实施例一：
细菌纤维素负载金纳米片的抗菌薄膜的制备包括以下几个步骤：
(1)称取细菌纤维素质量为0.6～0.8g的样品置于烧杯中，随即加入100mL0.1mol的NaoH溶液，95℃恒温煮沸2h，将其冷却至室温，透明溶液变成不透明的悬浮液，真空干燥24h即可得细菌纤维素(BC)纯样品。
(2)用移液枪移取质量含量为10％的氯金酸溶液1mL、2mL、3.4mL，分别转移至10mL容量瓶中，并配置30mM、60mM、100mM的氯金酸溶液，将步骤(1)中的细菌纤维素纯样品浸渍在30mM氯金酸溶液中紫外灯光照12h记为AuNPs@BC、60mM氯金酸溶液中紫外灯光照12h记为AuNSs-S@BC和100mM的氯金酸溶液中紫外灯光照12h记为AuNSs-L@BC，紫外灯波长为254nm，功率8W光照后取出用蒸馏水洗净，真空干燥即可得细菌纤维素负载金纳米片抗菌薄膜(AuNPs@BC、AuNSs-S@BC、AuNSs-L@BC)的样品。
附图1为细菌纤维素纯样品浸渍60mM氯金酸溶液中紫外灯光照12h获得的细菌纤维素负载金纳米片抗菌薄膜记为AuNSs-S@BC，AuNSs-S@BC的场发射扫描电镜图，从附图1看出紫外光诱导细菌纤维素还原的金纳米片的形状包括规则的多边形，四边形及三角形形貌，需要说明的是，紫外光照时间的改变，可以影响金纳米片的尺寸，金纳米片的形状依然包括规则的多边形、四边形及三角形形貌，且大量的BC纤维吸附在金纳米片的顶部和底部，可以阻止纳米片的聚集。
实施例二：
将实施例一制备得到的AuNPs@BC、AuNSs-S@BC、AuNSs-L@BC置于含有菌液的固体培养基中，在恒温培养箱中孵育24h，温度为37℃，培养箱震荡频率为78r/min，结果表明，所制得的AuNSs-S@BC具有明显的抑菌效果。见图5，为细菌纤维素纯样品浸渍60mM氯金酸溶液后紫外光照12h获得的细菌纤维素负载金纳米片抗菌薄膜(记为AuNSs-S@BC的抑菌效果)，从附图5图中可以看出，AuNSs-S@BC抑菌效果好，有明显的抑菌圈。
实施例三：
将实施例二置于含有菌液的固体培养基孵育24h后的AuNSs@BC取出，用去离子水和乙醇冲洗后，60℃烘干得到可重复使用的AuNSs@BC。在实施例二同等条件下进行重复实验，结果证明可重复使用的依然具有较好的抑菌效果，见图6，为清洗后可重复使用的AuNSs-S@BC的抑菌效果，从附图6图中可以看出，仍具有明显的抑菌效果。
实施例四：
将实施例一制备得到的AuNSs@BC以及BC分别置于含有菌液的固体培养基中在恒温培养箱中孵育24h得到抗菌效果，温度为37℃，培养箱震荡频率为78r/min，测试结果显示，AuNSs@BC相比于BC具有更加明显的抑菌圈，见图6具体给出了AuNSs-S@BC与BC的抑菌效果比对，从附图6图中可以看出，与BC相比，AuNSs-S@BC抑菌效果好，有明显的抑菌圈。
见图7为大肠杆菌和金黄色葡萄球菌的生长抑制带，图中AuNSs-S@BC为细菌纤维素纯样品浸渍60mM氯金酸溶液后紫外光照12h获得的AuNSs@BC。
此外，陈燕，陈仕艳等在《合成纤维》上发表的文章《细菌纤维素负载金纳米颗粒复合膜的制备及催化性能》，经验证其复合膜并不适用于本申请，其复合膜上超声下来的AuNPs结构基本呈球形，粒径均匀且形状较规则，具有较小的粒径以及较高的分散性，没有明显团聚现象出现。AuNPs的平均粒径大小为5.3nm，标准偏差为1.23nm，晶面间距为0.23nm；然而本申请紫外光照方法下的AuNSs结构基本呈片状，粒径分布较广且形状可控，AuNSs的平均粒径大小为7.4nm(见图7)，晶面间距为0.21nm。该文献方法的使用效果为在催化还原4-NP试验中表现出较高的催化效率；然而本申请方法的使用效果用于抗大肠杆菌和金黄色葡萄球菌。
以上所述仅是本发明的优选实施方式，应当指出：对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (7)

1.一种细菌纤维素负载金纳米片的抗菌薄膜的制备方法，其特征在于：包括如下步骤：
a、细菌纤维素的纯化：细菌纤维素的纯化，将细菌纤维素膜浸入0.1M 90℃～95℃NaOH溶液中，然后用蒸馏水洗至中性，真空干燥即可得到细菌纤维素纯样品；
b、制备细菌纤维素负载金纳米片的抗菌薄膜：将步骤a获得的细菌纤维素纯样品浸入氯金酸溶液中并用紫外光照射，紫外光照后用去离子水水洗，再真空干燥即得所述细菌纤维素负载金纳米片的抗菌薄膜。
2.根据权利要求1所述的细菌纤维素负载金纳米片的抗菌薄膜的制备方法，其特征在于：步骤a中在NaOH溶液中浸泡时间为2h。
3.根据权利要求1所述的细菌纤维素负载金纳米片的抗菌薄膜的制备方法，其特征在于：步骤b中氯金酸溶液的浓度为30～100mM。
4.根据权利要求1所述的细菌纤维素负载金纳米片的抗菌薄膜的制备方法，其特征在于：步骤b中紫外光波长为254nm～365nm，紫外光照时间为12h。
5.根据权利要求1所述的细菌纤维素负载金纳米片的抗菌薄膜的制备方法，其特征在于：步骤b中每100ml的所述氯金酸溶液中浸入细菌纤维素的质量为6～8g。
6.一种细菌纤维素负载金纳米片抗菌薄膜，其特征在于：由上述权利要求1至5中任一项所述的细菌纤维素负载金纳米片的抗菌薄膜的制备方法制得。
7.如权利要求6所述的细菌纤维素负载金纳米片抗菌薄膜的应用，其特征在于：用于抑制大肠杆菌和金黄色葡萄球菌。
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