CN111876456A - 一种黑水虻抗菌肽的提取方法 - Google Patents
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Abstract
本发明涉及一种黑水虻抗菌肽的提取方法。包括：用米曲霉种子液与乳酸杆菌制备发酵液；用有机废弃物制备黑水虻养殖料；制备成发酵养殖料；黑水虻幼虫在发酵养殖料中养殖到5龄幼虫；黑水虻5龄幼虫与发酵养殖料分离；幼虫及发酵液一起粉碎均质得到降解液；离心去除非蛋白杂质得到降解物的上清液；上清液以三氯乙酸沉淀大分子蛋白，再次离心得去大分子上清液；用盐酸溶液洗脱吸附在海藻酸上的抗菌肽；用氯化钠沉析，透析，脱盐干燥得抗菌肽粗品；用超滤膜过滤得超滤液，冰冻干燥得目标抗菌肽。本发明应用低成本黑水虻制备广谱抗菌肽，成本低，适于大规模生产；抗菌肽为粗品，可以作为抗菌肽原料药；利用垃圾养殖黑水虻，使垃圾变废为宝。
Description
技术领域
本发明涉及一种黑水虻抗菌肽的提取方法，属于医药生物技术领域。
背景技术
黑水虻处理畜禽粪便、餐饮垃圾、蔬菜水果垃圾、作物秸秆等有机废弃物技术已得到广泛利用，而处理有机废弃物过程会生产大量黑水虻幼虫。
黑水虻不具备高等动物那样的免疫体系，以产生抗菌肽的方式构建免疫系统，抑制和杀灭入侵的微生物。
抗菌肽通常是由多个氨基酸残基组成的小分子多肽，具有较强的热稳定性、水溶性以及广谱高效的抗菌活性。抗菌肽的抗菌机理与抗生素不同，不易产生耐药性菌株。抗菌肽不仅对革兰阳性菌和革兰阴性菌有较强的抑菌作用，对某些真菌、病毒、癌细胞也具有杀灭作用。
利用黑水虻为原料提取抗菌肽是一条经济便捷的途径。
CN202010094406.6公开了一种黑水虻抗菌肽的提取方法及其在制备抗烟草病害的肥料中的应用，其利用蘸有烟草青枯病菌、烟草野火病菌和烟草角斑病菌诱导菌液的针刺破幼虫腹部后将其放入诱导菌液中浸泡产生抗菌肽。黑水虻虫体较小，针刺虫体在大规模的生产中难以实现。另外，诱导菌烟草青枯病菌、烟草野火病菌和烟草角斑病菌都是烟草有害菌，生产中很难防止有害菌的扩散，且防止措施成本相对较高。该申请方案的目的是生产防抗烟草青枯病菌、烟草野火病菌和烟草角斑病菌的肥料，应用领域较窄，限制了黑水虻抗菌肽的应用领域。
发明内容
本发明要解决的技术问题是提供一种黑水虻抗菌肽的提取方法，该方法以处理有机废弃物过程中生产的大量黑水虻幼虫为原料，经济，便捷。
本发明解决其技术问题采用的技术方案是：
一种黑水虻抗菌肽的提取方法，按下述步骤进行：
（1）按米曲霉制种方法制备一定量的米曲霉种子液，米曲霉种子液加入乳酸杆菌扩大培养24小时成为发酵液，发酵液中乳酸杆菌菌体密度2-4亿个/升；
（2）用有机废弃物制备黑水虻养殖料，有机废弃物包括畜禽粪便、餐饮垃圾、蔬菜水果垃圾、作物秸秆；
（3）接种米曲霉种子液于黑水虻养殖料中，米曲霉种子液与黑水虻养殖料的质量比为1：10-20，制备成发酵养殖料；
（4）将黑水虻幼虫投入步骤（3）制备的发酵养殖料中，控制养殖环境温度25-32℃，湿度65-75%，养殖幼虫达到5龄幼虫；
（5）将步骤（4）养殖的黑水虻5龄幼虫与发酵养殖料分离，将5龄幼虫置于步骤（1）制备的发酵液中再养殖24小时，米曲霉和乳酸杆菌诱导黑水虻产生更多抗菌肽；
（6）将步骤（5）养殖24小时的幼虫及发酵液一起置于胶体磨，粉碎均质10-20分钟后置于恒温反应器中，30℃下反应12-36小时得到降解液，米曲霉蛋白酶会将黑水虻部分大分子蛋白降解为小分子的类抗菌肽；
（7）将步骤（6）得到的降解液用10000转/分，离心去除非蛋白杂质，得到降解物的上清液；
（8）降解物上清液以0.5mol/L三氯乙酸沉淀大分子蛋白，静置30分钟后10000转/分再次离心，得到去大分子上清液；
（9）去大分子上清液用海藻酸过滤吸附，再用0.2mol/L盐酸溶液洗脱吸附在海藻酸上的抗菌肽；
（10）将洗脱液用氢氧化钠调解PH值为4.5后，用氯化钠沉析，透析，脱盐干燥得抗菌肽粗品；
（11）将抗菌肽粗品用超纯水溶解，经过0.22μm-0.45μm超滤膜过滤，得到超滤液，冰冻干燥得目标抗菌肽。
本发明中，刺激诱导黑水虻产生抗菌肽的菌体包括但不限于米曲霉、乳酸杆菌等非致病菌；刺激诱导方式包括但不限于菌剂饲喂、菌悬液浸泡。
采用上述技术方案的本发明，与现有技术相比，有益效果是：
本发明应用低成本黑水虻制备广谱抗菌肽，生产成本低廉，适用于大规模生产；本发明生产的抗菌肽为粗品，可以作为抗菌肽原料药。本发明利用垃圾养殖黑水虻，使垃圾变废为宝，产品价值得到提高。
具体实施方式
以下结合实施例详述本发明。
一种黑水虻抗菌肽的提取方法，按以下步骤进行：
（1）用畜禽粪便、餐饮垃圾、蔬菜水果垃圾、作物秸秆等有机废弃物制备黑水虻养殖料900公斤；
（2）制备米曲霉种子液100公斤，制备米曲霉乳酸杆菌发酵液200公斤；
（3）将米曲霉种子液100公斤加入到900公斤养殖料中制成发酵养殖料1000公斤；
（4）在1000公斤发酵养殖料中投放黑水虻虫卵100克，幼虫达到5龄期，分离虫体和虫砂，得到虫体180公斤；
（5）将180公斤幼虫虫体置于200公斤发酵液中再养殖24小时；
（6）将步骤（5）的180公斤幼虫虫体和200公斤发酵液置于胶体磨，粉碎均质10分钟，置于恒温反应器中，30℃下反应15小时得到降解液；
（7）10000转/分，离心步骤（6）的降解液得降解物的上清液180升；
（8）降解物上清液加入0.5mol/L三氯乙酸，静置30分钟后10000转/分再次离心沉淀大分子蛋白，得去大分子上清液170升；
（9）上清液用海藻酸吸附，过滤，再用0.2mol/L盐酸溶液洗脱吸附在海藻酸上的抗菌肽，得洗脱液40升；
（10）将洗脱液用氢氧化钠调解PH值为4.5后，用氯化钠沉析，透析，脱盐干燥得抗菌肽粗品90克；
（11）将抗菌肽粗品用超纯水溶解，经过0.22μm-0.45μm超滤膜过滤，得到超滤液，冰冻干燥得目标抗菌肽60克。
以上对本发明做了示例性的描述，应该说明的是，在不脱离本发明的核心的情况下，任何简单的变形、修改或者其他本领域技术人员能够不花费创造性劳动的等同替换均落入本发明的保护范围。
Claims (1)
1.一种黑水虻抗菌肽的提取方法，其特征在于，按下述步骤进行：
（1）按米曲霉制种方法制备一定量的米曲霉种子液，米曲霉种子液加入乳酸杆菌扩大培养24小时成为发酵液，发酵液中乳酸杆菌菌体密度2-4亿个/升；
（2）用有机废弃物制备黑水虻养殖料，有机废弃物包括畜禽粪便、餐饮垃圾、蔬菜水果垃圾、作物秸秆；
（3）接种米曲霉种子液于黑水虻养殖料中，米曲霉种子液与黑水虻养殖料的质量比为1：10-20，制备成发酵养殖料；
（4）将黑水虻幼虫投入步骤（3）制备的发酵养殖料中，控制养殖环境温度25-32℃，湿度65-75%，养殖幼虫达到5龄幼虫；
（5）将步骤（4）养殖的黑水虻5龄幼虫与发酵养殖料分离，将5龄幼虫置于步骤（1）制备的发酵液中再养殖24小时，米曲霉和乳酸杆菌诱导黑水虻产生更多抗菌肽；
（6）将步骤（5）养殖24小时的幼虫及发酵液一起置于胶体磨，粉碎均质10-20分钟后置于恒温反应器中，30℃下反应12-36小时得到降解液，米曲霉蛋白酶会将黑水虻部分大分子蛋白降解为小分子的类抗菌肽；
（7）将步骤（6）得到的降解液用10000转/分，离心去除非蛋白杂质，得到降解物的上清液；
（8）降解物上清液以0.5mol/L三氯乙酸沉淀大分子蛋白，静置30分钟后10000转/分再次离心，得到去大分子上清液；
（9）去大分子上清液用海藻酸过滤吸附，再用0.2mol/L盐酸溶液洗脱吸附在海藻酸上的抗菌肽；
（10）将洗脱液用氢氧化钠调解PH值为4.5后，用氯化钠沉析，透析，脱盐干燥得抗菌肽粗品；
（11）将抗菌肽粗品用超纯水溶解，经过0.22μm-0.45μm超滤膜过滤，得到超滤液，冰冻干燥得目标抗菌肽。
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| CN117887592A (zh) * | 2024-03-04 | 2024-04-16 | 江苏省农业科学院 | 米曲霉jaas-32及其在制备黑水虻虫浆蛋白肽中的应用 |
| CN117887592B (zh) * | 2024-03-04 | 2024-05-17 | 江苏省农业科学院 | 米曲霉jaas-32及其在制备黑水虻虫浆蛋白肽中的应用 |
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