CN115181738B - 用于嗜油菌株包埋的新型有机胶及其制备方法和应用 - Google Patents
用于嗜油菌株包埋的新型有机胶及其制备方法和应用 Download PDFInfo
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Abstract
本发明属于酵母包埋领域，具体涉及一种用于嗜油菌株包埋的新型有机胶及其制备方法和应用。用于嗜油菌株包埋的新型有机胶包括有机胶以及包埋的酵母细胞；所述的有机胶包括甲基丙烯酸烷基酯单体、交联剂以及引发剂；所述的甲基丙烯酸烷基酯单体为C12‑C18烷基甲基丙烯酸甲酯。本发明所述的有机胶具有优良的油吸附能力；能够确保嗜油微生物存在于水油混合中的油层，能够持续降解石油。本发明可以长期保存酵母，并且二次加密携带DNA信息的菌株，实现信息的精准传递。
Description
技术领域
本发明属于酵母包埋领域，具体涉及一种用于嗜油菌株包埋的新型有机胶 及其制备方法和应用。
背景技术
固定化细胞技术是利用物理或化学手段将游离状态的细胞固定或定 位在限定的空间区域，并使其保持固有的催化活性。固定化生物细胞能 持续增殖、代谢和衰亡，其活性保持稳定。固定化生物细胞能持续增殖、 休眠和衰亡，其活性始终保持稳定。固定化的生物细胞除保持其原有的 识别、结合、催化活性，支撑保证细胞/微生物的生存，保护其不受恶劣 环境的影响外，还具有细胞密度高、反应速率快、耐毒性能力强、稳定 性高、产物分离容易、能实现连续操作等特点，可大大提高生产能力。细胞固定化技术经常用于各种生物医学和工业应用，包括益生菌递送， 生物催化剂，功能活性材料等。
目前，细胞固定化技术在人们的生活中发挥着越来越重要的作用。 比如在城市污水净化方面，目前城市污水的成分愈加复杂，对处理技术 提出更高的要求。城市污水的浊度较高，容易影响固定细菌的传质性， 且城市污水化学成分复杂，对固定载体稳定性破坏性很强。曾宇等分别 采用采用琼脂、海藻酸钠、海藻酸钠加活性炭粉、聚乙烯醇、聚乙烯醇加活性炭粉为载体，包埋固定光合细菌处理城市污水，静态试验结果表 明使用海藻酸钙包埋的菌体亦表现良好活性，对污水处理96h后，COD 去除率达90％以上。
除此之外，细胞固定化技术在食品生产工业上也有非常重要的意义。 采用菌体热固法制成的固定化细胞，实现了果葡糖浆生产的连续化。 Chihata等人用固定化聚丙烯酰胺水凝胶固定能够生产天冬氨酸酶的大 肠杆菌，生产L-天冬氨酸，使生产成本降低40％。日本田边制药厂用K- 角叉菜糖凝胶包埋菌体，并用戊二醛、已二胺进行硬化处理，由此制得 的固定化菌体半衰期达630d，其生产能力比聚丙烯酰胺凝胶包埋的菌体 提高14倍。生物微胶囊是一种新兴的细胞固定化技术。进入21世纪， 微胶囊技术在食品工业上的应用发展得很快，微胶囊技术应用于食品工 业上，极大地推动了食品工业由农产品初加工向高级产品的转化，微胶囊技术与超微粉碎技术、生物技术、膜技术和热压反应技术等相结合， 为食品工业开发应用高新技术展现了美好的前景。细胞固定化技术经常 用于各种生物医学和工业应用，包括益生菌递送，生物催化剂，人工器 官，和功能活性材料等。这种策略可以作为支撑保证细胞/微生物的生存， 保护其不受恶劣环境的影响，提高其稳定性和可重复使用性。使其易于操作，增加其在生物反应器中的密度，从而显著提高其功能。
迄今为止，科学家探索了多种有机和无机封装材料，包括水凝胶， 聚巴胺，聚电解质，聚电解质/氧化碳纳米管杂化，二氧化硅，和磷酸钙 /碳酸盐。它们都是亲水的，有几个原因。首先，成熟的技术使得生物相 容性和无毒亲水材料的制备变得容易，这可以保证被封装的细胞/微生物 的生存。其次，所有细胞和大多数微生物都生活在水环境中，亲水材料可以保证水溶性分子的有效传质。第三，生物相容性疏水封装材料的设 计仍然是一个挑战因此，亲水性材料，特别是水凝胶，似乎是目前封装 细胞/微生物的“金标准”。
然而，嗜油微生物对于工业应用是必不可少的。微藻、细菌、真菌 和酵母等产油微生物可以根据细胞内的细胞干重合成超过20％的脂质， 甚至可以在细胞干重基础上合成高达70％的脂质。
首先，它们可以降解油腻的食物垃圾和石油。嗜油微生物可直接利 用秸秆类农林废弃物、农副产品加工废弃物、工业废物及废水等廉价再 生资源经发酵转化为微生物柴油。例如，耶氏解脂酵母是一种嗜油酵母， 可以吸收各种油性物质，包括油脂废物、动物脂肪废物和烷烃；从石油 中分离出来的假单胞菌属可以将石油烃类降解成简单无毒的化合物；海洋产油微生物的酰基载体蛋白作为一类非常保守的转运蛋白,在脂肪酸 代谢中可作为酰基供体，是不可或缺的辅因子，研究酰基载体蛋白的结 构和功能,为指导脂肪酸合成途径的应用和改造提供依据(海洋产油微生物筛选及酰基载体蛋白基因克隆)；此外研究发现金属离子通过影响产油 微生物形态和生长，调节产油微生物关键酶活力，金属离子渗透压影响 产油微生物油脂积累而影响其产量。
第二，产油微生物可用于发酵和生物制造。例如，李建政等人以废 糖蜜为原料，利用粘红酵母作为出发菌株发酵产油；Xu等人报道，经过 工程改造的雅罗氏脂溶菌可以作为酵母生物炼制平台来生产类燃料分子 和油性化学品；Zhou等人发现Candidatusmethanoliparum可以将长链 烷烃转化为甲烷。因此，开发疏水性、亲脂性和生物相容性的包埋材料 是迫切需要的。
第三，产油微生物在合成多种胞内碳化合物方面具有巨大潜力。它 们可合成包括可转化为生物柴油和喷气燃料的甘油三酯(TAG)；也可合成 药物如类固醇激素和二十烷类；以及生产各种工业产品，如聚合物、蜡 酯和羟基脂肪酸。此外，产油微生物的脂肪酸组成、三酰基甘油酯类型 和次生代谢产物与植物中的同类具有相同的特征。据报道，产油微生物 合成的细胞内脂质不仅作为能量储存材料，而且在细胞的生物发生和细 胞膜的完整性方面起着重要作用。
因此，开发具有疏水性、亲脂性和生物相容性的包埋材料是迫切需 要的，以促进这些含油微生物的应用。并且，微生物作为生物样本，其 长期保存具有重大意义，而冻存是目前被公认的最有效的菌种长期保藏 技术之一。因此，能够长期冻存嗜油菌株的包埋材料具有重要应用前景。 有机凝胶是一种憎水材料，由三维聚合物网络渗透油或非极性有机溶剂。它们有很多有前途的性质，包括亲脂性和设计性，显示出作为含油微生 物封装的潜在候选人。然而，由于其制备通常需要使用有毒的有机溶剂、 非生物相容性的有机凝胶剂和苛刻的制备条件，其生物相容性不足。因 此，开发生物相容的有机凝胶需要避开这些有害因素。
发明内容
本发明的目的在于克服现有技术中的缺点，提供一种用于嗜油菌株包埋的 新型有机胶及其制备方法和应用。
为实现上述目的，本发明采用的技术方案为：
一种用于嗜油菌株包埋的新型有机胶，包括有机胶以及包埋的酵母细胞； 所述的有机胶包括甲基丙烯酸烷基酯单体、交联剂以及引发剂；其中甲基丙烯 酸烷基酯单体为含有长碳侧链的烷基甲基丙烯酸甲酯；所述酵母细胞为嗜油性 菌株。
所述的单体为C12-C18烷基甲基丙烯酸甲酯。
所述的有机胶的交联度为0.5-5％。
优选的，所述的有机胶的交联度为1-2％。
所述的交联剂为EDGMA或者TEGDMA。
所述的引发剂为IRGACURE 819或者IRGACURE TPO。优选的，所述的引发剂 与单体的摩尔配比为0.2：100。
本发明还包括一种所述的用于嗜油菌株包埋的新型有机胶的制备方法，包 括下述步骤：
S1：将引发剂溶解在甲基丙烯酸烷基酯单体溶液中，超声混合均匀；只有 向混合溶液中加入交联剂，通过摇匀使交联剂分散得到凝胶前体混合物；
S2：在凝胶前体混合物溶液中酵母细胞，将其混匀；
S3：将步骤S2得到的混合物转移到由聚四氟乙烯隔开的两个玻璃片之间； 将其放置在氙灯下照射，前体混合物通过光聚合形成包埋有酵母细胞的有机胶。
本发明还包括一种所述的新型有机胶的应用，用于嗜油菌株的包埋、保存 以及含有DNA信息的酵母的储存运输。
所述的嗜油菌株为解脂耶氏酵母。
本发明还包括一种所述的用于嗜油菌株包埋的新型有机胶的制备方法，包 括下述步骤：
S1：将引发剂溶解在甲基丙烯酸烷基酯单体溶液中，超声混合均匀；只有 向混合溶液中加入交联剂，通过摇匀使交联剂溶解得到凝胶前体混合物；
S2：在凝胶前体混合物溶液中酵母细胞，将其混匀；优选的，600μL凝胶 前体溶液中加入约1×108个酵母细胞；
S3：将步骤S2得到的混匀后的混合物转移到由聚四氟乙烯隔开的两个玻璃 片之间；将其放置在氙灯下照射，前体混合物通过光聚合形成包埋有酵母细胞 的有机胶。
本发明还包括一种所述的新型有机胶的应用用于嗜油菌株的包埋以及含有 DNA信息的酵母的储存运输。所述的嗜油菌株包括但不限于解脂耶氏酵母。
与现有技术相比，本发明的有益效果是：
本发明用于嗜油菌株包埋的新型有机胶克服了现有包埋材料全部为亲水性 材料的不足，具有生物相容性，疏水性以及吸收有机溶剂的能力。
其采用简单温和的制备方法、对酵母无毒的甲基丙烯酸类分子作为单体制 备得到的有机胶，使用最优化的配比，制备过程几乎不造成包埋的生物的明显 死亡。
本发明所述的有机胶具有优良的油吸附能力，能够保证包埋的嗜油酵母获 取足够的碳源以支撑生长。能够确保嗜油微生物存在于水油混合中的油层，能 够持续降解石油。
本发明利用合成生物学、生物材料学等多学科交叉技术，提供了一些高生 物相容性的有机胶作为嗜油菌株的载体，同时可以二次加密携带DNA信息的菌 株，实现信息的精准传递。
附图说明
图1：有机胶制备过程图；
图2：有机胶疏水性的示意图；
图3：有机胶对于有机分子的吸收性能图；
图4：有机胶的水接触角和油接触角示意图；
图5：酵母在不同培养基中生长48小时后的形态图；
图6：有机胶包埋酵母的成活率图；
图7：有机胶冷冻保存成活率图；
图8：有机胶信息存储示意图。
具体实施方式
为了使本技术领域的技术人员更好地理解本发明的技术方案，下面结合附 图和最佳实施例对本发明作进一步的详细说明。
实施例1-15：有机胶的设计与制备：如图1所示以五种不同的甲基丙烯酸 烷基酯(十二烷基/十三烷基/十四烷基/十六烷基/十八烷基甲基丙烯酸甲酯)、 EDGMA和IRGACURE819分别作为单体、交联剂和光引发剂；将0.2wt％的引发 剂(IRGACURE 819)溶解在600mL单体溶液中，30℃超声处理10min。超声之 后，向溶液中加入不同剂量的交联剂，通过摇匀使交联剂溶解于上述溶液中，交联密度(即交联剂与单体的摩尔比)分别为1％、1.5％和2％。将混匀后的凝胶前 体溶液转移到由聚四氟乙烯(PTFE)隔开的两个玻璃片(厚度为1.5mm)之间。将其 放置在氙灯(LAB-SQLAR500,2kW/m2)照射下，前体溶液通过光聚合形成有机胶。 调控不同的光照时间为10min，对应表1中。计算不同交联度、不同反应时间的单体反应率。
表1
将不同光照时间下的有机胶，切割成直径为8mm，厚度为1.5mm的圆盘形 状，测定完全干燥的样品的干重。随后，将干燥的样品在三氯甲烷中浸泡48小 时以达到吸收平衡。将样品取出，轻轻放置在称量纸上，然后置于通风橱中， 放置24小时。使有机胶中的三氯甲烷挥发完全。
称取最终干燥的有机胶的质量，计算单体反应率。
单体反应率(％)＝1-(Wa-Wb)/Wa×100％；其中，Wa—初始干凝胶的重量；Wa —最终干凝胶的重量。
测试：
1、有机胶疏水性能、吸收性能的表征
将有机凝胶(对应表1中配方1)置于油相中，水凝胶置于水相中，然后将 有机凝胶压入或拉入水相中，并记录有机凝胶的路径轨迹(图2)。
采用称重法测定有机凝胶的吸油性能。本实验所采用的有机胶对应表1中 不同的配方。将有机凝胶打孔成直径为8mm，厚度为1.5mm的圆盘，测定完全 干燥的样品的干重。随后，将干燥的样品在大豆油中浸泡72小时以达到吸收平 衡。最后，将样品取出，轻轻去除多余的油，在室温下测量湿重。吸油率计算 如下:
吸油率(％)＝(Wa-Wb)/Wb×100％
其中，Wb—干凝胶的重量；Wa—湿凝胶的重量。
采用同一方法测定有机凝胶(对应表1中1、4、7、10、13)对不同有机 溶剂的吸收能力，包括甲醇、ACN、乙醇、DMF、四氢呋喃、DMSO、DCM、正己烷、 甲苯和四氢呋喃(图3)。从图3中可以看出对于极性可与水互溶的分子，有机胶的吸收性能很低。对于不溶于水的非极性有机溶剂，有机胶具有优异的吸收 能力。
2、有机胶接触角测试。我们测试了制备得到的有机胶的水接触角和油接触 角。使用的有机胶对应表1中的不同实施例，我们使用水和大豆油进行接触角 的测试。当水滴或油滴接触到有机胶表面的一瞬间进行照片捕捉，然后进行角度测量(图4)。由图4看出，有接触角均小于45°，水接触角均大于106°， 表明有机胶的疏水亲油的性能。
3、单体毒性测试。将5种不同的单体，分别添加到YPO培养基(3.7％v/v) 中制成复合培养基。在YPO培养基中培养解酯耶氏酵母种子液，以终浓度 OD600＝0.2接种到5种不同的复合培养基和YPO培养基中。菌液在30℃，200rpm 的条件下培养。使用显微镜对于培养48小时后的酵母形态进行观察。(图5)， 从图5中可以看出5种单体对于酵母的生长没有明显影响，不会影响到酵母的形态。
4、有机胶包埋解酯耶氏酵母。在600μL不同实施例的凝胶前体溶液中加 入约1×108个酵母细胞，将其混匀。将含有酵母细胞的前体溶液转移到玻璃 片模具中，然后在氙灯(LAB-SQLAR500,2kW/m2)下照射，得到含有酵母细胞的有 机胶。
使用荧光酵母(基因组用红色荧光蛋白基因修饰)制备荧光样品用于显微镜 观察。其他鉴定实验中涉及的生物体均为脂解耶氏酵母(ATCC 201249)，没有进 一步的基因修饰。在干净的研钵中，将制备得到的含有酵母细胞的有机胶切碎， 使用1M1 PBS溶液反复冲洗，提取出有机胶中的酵母。对得到的含有酵母的PBS 溶液进行染色，使用的是7012活死染色试剂盒。染色30分钟后，在荧光显微 镜下观察酵母细胞的成活状态。如图6所示，交联剂的用量在1.5-2之间的时 候，有机胶具有较高的酵母存活率(表2示出具体的存活率数据)，其中由实施 例2制备得到的有机胶具有最高的酵母存活率，经过制胶过程后，酵母的成活率可以达到96％。
表2
5、有机胶用于酵母的冷冻保存。根据实施例方法制备得到含有酵母细胞的 有机胶。由五种不同的单体得到5种有机胶。由于细胞代谢速率随温度的降低 而降低，可达到代谢停滞状态。因此，冷冻保存是目前被公认的最有效的菌种长期保藏技术之一。
将5种含有酵母的有机胶直接放入-80℃或-196℃体系中。使用YPO培养 基作为从对照组用来冻存酵母。长时间冻存之后，将有机胶和对照组取出，有 机胶被切碎，用PBS溶液提取出冻融之后的酵母。从有机胶与对照组提取出来的酵母通过活死染色进行存活率的测试。结果如图7所示。
6、有机胶用于含有DNA信息的酵母的储存和运输。根据实施例的方法制备 得到含有酵母细胞的有机胶。由五种不同的单体得到5种有机胶。由于单体含 有不同长度的侧链，有机胶具有不同的相相变性质。C12-C14制备得到的有机胶 不随温度改变发生相变，C16和C18制备得到的有机胶随温度降低透明度降低，发生相变。由图8可以看出，在DNA用作信息存储的过程中，DNA信息的保存是 一个重要的环节。试验表明本申请的有机胶可用于含有DNA信息的酵母的储存 和运输。
以实施例8、以及实施例11的有机胶进行示例性说明；选择具有相变性质 的Gel16(实施例11)作为含有DNA信息的酵母细胞的载体，不具有相变性质的 Gel14(实施例8)用来存储其他不含DNA信息的酵母细胞。在运输的过程中，两 种胶不存在区别，可以很好的保护信息。当需要提取信息时，将有机胶在4℃冰 箱中放置约2分钟，可以轻松选择出含有目的信息的有机胶，进而提取有效DNA 信息，同时实验表明，经过4天的储存，从有机胶中提取得到的菌株为解酯耶 氏酵母(图8c)，表明有机胶存储之后，菌株不会被污染，保障了存储的安全性。
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 (3)
1.一种用于嗜油菌株包埋的新型有机胶，其特征在于，包括有机胶以及包埋的酵母细胞；
所述的有机胶包括甲基丙烯酸烷基酯单体、交联剂以及引发剂；其中甲基丙烯酸烷基酯单体为含有长碳侧链的烷基甲基丙烯酸甲酯；
所述酵母细胞为嗜油性菌株；所述的嗜油菌株为解脂耶氏酵母；
所述的甲基丙烯酸烷基酯单体为C12烷基甲基丙烯酸甲酯，交联度为1%、1.5%或者2%；
或者所述的甲基丙烯酸烷基酯单体为C13烷基甲基丙烯酸甲酯，交联度为1%、1.5%或者2%；
或者所述的甲基丙烯酸烷基酯单体为C14烷基甲基丙烯酸甲酯，交联度为1%或者1.5%；
或者所述的甲基丙烯酸烷基酯单体为C16烷基甲基丙烯酸甲酯，交联度为1%、1.5%或者2%；
或者所述的甲基丙烯酸烷基酯单体为C18烷基甲基丙烯酸甲酯，交联度为1%、或者1.5%；
所述的交联剂为EDGMA；
所述的引发剂为IRGACURE 819。
2.一种权利要求1所述的用于嗜油菌株包埋的新型有机胶的制备方法，其特征在于，包括下述步骤：
S1：将引发剂溶解在甲基丙烯酸烷基酯单体溶液中，超声混合均匀；只有向混合溶液中加入交联剂，通过摇匀使交联剂分散得到凝胶前体混合物；
S2：在凝胶前体混合物溶液中酵母细胞，将其混匀；
S3：将步骤S2得到的混合物转移到由聚四氟乙烯隔开的两个玻璃片之间；将其放置在氙灯下照射，前体混合物通过光聚合形成包埋有酵母细胞的有机胶。
3.一种权利要求1所述的新型有机胶的应用，其特征在于，用于嗜油菌株的包埋、保存以及含有DNA信息的酵母的储存运输。
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