CN104522058A - 一种含有藻胆蛋白的光敏杀菌剂及其制备方法及其杀菌的用途 - Google Patents
一种含有藻胆蛋白的光敏杀菌剂及其制备方法及其杀菌的用途 Download PDFInfo
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Abstract
本发明公开了一种含有藻胆蛋白的光敏杀菌剂及其制备方法及其杀菌的用途,本发明用蓝藻中提取的藻胆蛋白,利用其良好的光敏特性开发光敏杀虫剂,由0.025-0.4克藻胆蛋白和95-99mL的PBS组成。该消毒剂用量小,使用方便,生产工艺简单,不需要特殊设备,成本低廉,对环境安全,微生物不易产生抗性;而且本发明光敏杀菌剂见效快,为水华蓝藻的综合治理及资源利用开辟了新途径。
Description
技术领域
本发明属于“消毒剂”新技术的研究,具体涉及一种含有藻胆蛋白的光敏杀菌剂及其制备方法及其杀菌的用途。
背景技术
目前常用的消毒剂产品以成分分类主要有:含氯消毒剂、醛类消毒剂、醇类消毒剂、含碘消毒剂、酚类消毒剂、季铵盐类消毒剂,大部分属于化学类消毒剂,也称抗微生物剂,会让微生物把这些毒性化学物质从它们体内有效清除掉,这样就有可能会对一些抗生素产生耐药。
20世纪初始,人们发现光动力对细胞具有灭活作用。此后,便有很多学者致力于光动力对肿瘤细胞的损伤作用研究,目前光动力效应在医疗上已经有了广泛应用,称之为光动力学疗法( Photodynamictherapy,PDT) ,主要用于恶性肿瘤和一些良性疾病的治疗。
光动力灭菌技术( ant-imicrobial photodynamictechnology,APDT)是在光动力技术基础上发展起来的一种新技术,它是利用光敏剂对细菌的优先聚集特性,在适当波长光激发下,使光敏剂吸收能量后产生单线态氧、自由基等活性氧物质进而通过氧化作用杀伤微生物。
近年来,四吡咯化合物的光敏性质逐渐受到人们的关注,这类天然化合物具备光敏剂的优良特性:它们在紫外和可见光区的所有波长都有吸收;能够产生高量子产率的长寿命的三重激发态,因此光敏效率很高;在无光照的条件下没有光毒性,在光照条件下迅速降解且降解产物对生物体无毒,降低了环境持久污染的风险。
藻胆蛋白是藻类中捕光色素蛋白的总称,主要包括藻红蛋白、藻红蓝蛋白、藻蓝蛋白、别藻蓝蛋白4种,为红藻和蓝藻特有。藻胆蛋白主要依靠四吡咯结构的色基(藻胆素)而发出荧光,是一种荧光发射强烈的高效荧光物质。它还可作为食品或化妆品的色素、示踪剂、抗氧化剂、光合作用机理和进化研究、免疫增强剂等。尤其值得关注的是,藻胆蛋白还是一种有效的光敏剂,在受光照射后产生活性氧等自由基,每2-3个自由基可杀伤1个细胞。藻胆蛋白作为光敏杀菌剂尚未见报道,由于其安全性高,对环境无副作用,其作为“消毒剂”研发意义重大。
蓝藻(包括水华蓝藻)中藻胆蛋白含量可达细胞干重的10%~20%,收集蓝藻,将提取的藻胆蛋白用作消毒剂不仅能减少蓝藻生物量、降低内源水体中氮、磷营养水平并能加快水体生态恢复,而且能克服化学消毒剂带来的环境问题和害虫耐药性问题,可谓一举两得。
发明内容
为了缓解现有消毒剂的不足和缺陷,本发明提供一种低毒、环保、高效的含有藻胆蛋白的光敏杀菌剂制备方法及其杀菌用途,本发明用量小,使用方便,生产工艺简单,成本低廉,而且该产品对环境安全,对非靶标生物安全,微生物不易产生抗性。
为了实现上述目的本发明采用如下技术方案:
一种含有藻胆蛋白的光敏杀菌剂,其特征在于:其是由下述重量份的原料制得:藻胆蛋白0.075-0.3g和PBS 100mL。
所述的一种含有藻胆蛋白的光敏杀菌剂,其特征在于:PBS缓冲液的配方为:NaCl 137mmol/L,KCl 2.7mmol/L,Na2HPO4 10mmol/L, KH2PO4 2mmol/L;PBS缓冲液的pH7.2~7.4。
所述的含有藻胆蛋白的光敏杀菌剂的制备方法,其特征在于:室温下,将0.075-0.3g藻胆蛋白加入容器中,加PBS100mL混匀,避光保存,即得。
所述的含有藻胆蛋白的光敏杀菌剂的杀菌用途。
所述的一种含有藻胆蛋白的光敏杀菌剂的杀菌用途,其特征在于:其对金黄色葡萄球菌、枯草杆菌、黄曲霉、黑曲霉具有抑制作用。
所述的含有藻胆蛋白的光敏杀菌剂的制备方法包括以下步骤:
(1)藻胆蛋白的提取
藻胆蛋白生产沿用已经公开的实验室方法,即收集蓝藻晾干,通过采用PBS抽提、硫酸铵分级沉淀、冷冻干燥等提取纯化方法从蓝藻中提取藻胆蛋白,此处所用的蓝藻可以是水华蓝藻、螺旋藻中的任意一种。
(2)光敏杀菌剂的配制
室温下,将0.075-0.3g藻胆蛋白加入容器中,加PBS至100mL混匀,避光保存。
PBS缓冲液的配方:
PBS缓冲液(pH7.2~7.4):NaCl 137mmol/L,KCl 2.7mmol/L,Na2HPO4 10mmol/L, KH2PO4 2mmol/L
本发明的有益效果:
本发明所用的藻胆蛋白是从蓝藻中提取的藻胆蛋白,藻胆蛋白只能在生物体受光照的作用下才对生物体产生破坏作用,因此它的使用不会破坏环境,该消毒剂成分都是可溶性的,便于包装和运输,使用前溶于PBS即可形成真溶液,不沉淀;用量小,所用藻胆蛋白浓度为μg/ml单位量级的,使用方便,生产工艺简单,不需要特殊设备,一般农药企业均可加工,成本低廉;而且该产品对环境安全,对非靶标生物安全,微生物不易产生抗性。
对微生物的杀伤作用较强,光照下藻胆蛋白浓度在75 μg/mL以上时就能杀死80%以上的微生物。
本发明光敏杀菌剂可用于杀灭传播媒介上病原微生物,使其达到无害化要求,将病原微生物消灭于人体之外,切断传染病的传播途径,达到控制传染病的目的。具体实施方法为:将光敏杀菌剂采用喷雾法直接喷洒到待灭菌物体的表面,光照半小时即可达到消毒灭菌的目的。
附图说明:
图1藻胆蛋白光敏作用对金黄色葡萄球菌的抑制率
图2藻胆蛋白光敏作用下金黄色葡萄球菌生长延滞曲线
图3藻胆蛋白光敏作用对枯草杆菌的抑制率;
图4藻胆蛋白光敏作用下枯草杆菌生长延滞曲线。
图5藻胆蛋白光敏作用对黄曲霉的抑制率;
图6藻胆蛋白光敏作用下黄曲霉生长延滞曲线。
图7藻胆蛋白光敏作用对黑曲霉的抑制率;
图8藻胆蛋白光敏作用下下黑曲霉生长延滞曲线。
具体实施方式
实施例1: 7.5mg藻胆蛋白,余量为PBS,配成100ml溶液,搅拌均匀,避光保存。
实施例2:15mg藻胆蛋白,余量为PBS,配成100ml溶液,搅拌均匀,避光保存。
实施例3:30mg藻胆蛋白,余量为PBS,配成100ml溶液,搅拌均匀,避光保存。
对实施例1、2、3进行光敏杀菌剂的效果测试:
每组藻胆蛋白浓度为75、150、300μg/mL,将各处理的菌悬液放在灭菌的离心管中于37 ℃避光孵育30min,光照组移入灭菌具塞试管中在太阳光下(平均光强26klx)光照时间30 min。不同浓度的藻胆蛋白菌悬液孵育光照后取200 μL 分别接种到20mL新鲜的LB 液, 37 ℃温度下170 r/min振荡培养。稀释涂布平板法计数并绘制抑制率柱形图;同时每隔60min取样测650 nm 处的吸光值,绘制细菌生长延滞曲线。实验重复三次。
配制LB培养基:胰蛋白胨(Tryptone) 10g/L; 酵母提取物(Yeast extract) 5g/L;氯化钠(NaCl) 10g/L;10g/L琼脂粉(固体培养基)。调PH值至7.4。
配制PDA培养基:将马铃薯洗净去皮,再称取200g马铃薯切成小块,加水煮烂(煮沸20~30分钟,能被玻璃棒戳破即可),用八层纱布过滤,加热,固体培养基加1-10g琼脂,继续加热搅拌混匀,待琼脂溶解完后,加入葡萄糖,搅拌均匀,稍冷却后再补足水分至1000毫升,分装试管或者锥形瓶,加塞、包扎,(121℃)灭菌20分钟左右后取出试管摆斜面或者摇匀,冷却后贮存备用。
金黄色葡萄球菌杀灭效果实验
挑取菌种单菌落, 接种于LB液,扩大培养后离心(4000r/min,10 min),弃上清。悬浮菌体于以实施例1、实施例2、实施例3所制得的光敏杀菌剂为样品,使得菌夜浓度为107CFU/mL 左右,将菌悬液放在灭菌的EP管中于37 ℃避光孵育30min,移入灭菌具塞试管中在太阳光下(平均光强26klx),光照时间30 min。稀释涂布平板法计数并绘制抑制率柱形图(抑制率( %) =( N- n)/N ×100 式中: N 为对照组个数平均值; n 为实验组个数平均值)。不同浓度的藻胆蛋白菌悬液孵育光照后1:10接种于LB培养基, 37 ℃温度下170 r/min振荡培养。每隔60min取样测650 nm 处的吸光值。绘制细菌生长延滞曲线。对照组中不加藻胆蛋白,其余成分和实验组完全一致。实验重复3 次,取平均值。
其中图1显示了藻胆蛋白对金黄色葡萄球菌的抑制率;图2为藻胆蛋白作用下金黄色葡萄球菌生长延滞曲线。
检测枯草杆菌杀灭效果:
做法与金黄色葡萄球菌杀灭实验相同,将菌种替换为枯草杆菌。图3显示藻胆蛋白对枯草杆菌的抑制率;图4为藻胆蛋白作用下枯草杆菌生长延滞曲线。
检测黄曲霉杀灭效果:
挑取菌种单菌落, 接种于PDA液,扩大培养后离心(4000r/min,10 min),弃上清。悬浮菌体于以实施例2、实施例3、实施例4所制得的光敏杀菌剂为样品,使得菌夜浓度为107CFU/mL 左右,将菌悬液放在灭菌的EP管中于37 ℃避光孵育30min,移入灭菌具塞试管中在太阳光下(平均光强26klx),光照时间30 min。稀释涂布平板法计数并绘制抑制率柱形图(抑制率( %) =( N- n)/N ×100 式中: N 为对照组个数平均值; n 为实验组个数平均值)。不同浓度的藻胆蛋白菌悬液孵育光照后1:10接种于LB培养基, 37 ℃温度下170 r/min振荡培养。每隔60 min取样测650 nm 处的吸光值。绘制细菌生长延滞曲线。对照组中不加藻胆蛋白,其余成分和实验组完全一致。实验重复3 次,取平均值。图5显示藻胆蛋白对黄曲霉的抑制率;图6为藻胆蛋白作用下黄曲霉生长延滞曲线。
检测黑曲霉杀灭效果:
与黄曲霉杀灭效果实验相同,将菌种替换为黑曲霉。图7显示藻胆蛋白对黑曲霉的抑制率;图8为藻胆蛋白作用下下黑曲霉生长延滞曲线。
实验结果表明对微生物的杀伤作用较强,光照下藻胆蛋白浓度在75 μg/mL以上时就能杀死80%以上的微生物。
Claims (5)
1.一种含有藻胆蛋白的光敏杀菌剂,其特征在于:其是由下述重量份的原料制得:藻胆蛋白0.075-0.3g和PBS 100mL。
2.根据权利要求1所述的一种含有藻胆蛋白的光敏杀菌剂,其特征在于:PBS缓冲液的配方为:NaCl 137mmol/L,KCl 2.7mmol/L,Na2HPO4 10mmol/L, KH2PO4 2mmol/L;PBS缓冲液的pH7.2~7.4。
3.一种如权利要求1所述的含有藻胆蛋白的光敏杀菌剂的制备方法,其特征在于:室温下,将0.075-0.3g藻胆蛋白加入容器中,加PBS100mL混匀,避光保存,即得。
4.一种如权利要求1所述的含有藻胆蛋白的光敏杀菌剂的杀菌用途。
5.根据权利要求1所述的一种含有藻胆蛋白的光敏杀菌剂的杀菌用途,其特征在于:其对金黄色葡萄球菌、枯草杆菌、黄曲霉、黑曲霉具有抑制作用。
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