CN116584533A - 一种双靶点抑菌光敏化合物的制备方法及其应用 - Google Patents
一种双靶点抑菌光敏化合物的制备方法及其应用 Download PDFInfo
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Abstract
本发明公开了一种双靶点抑菌光敏化合物的制备方法及其应用,其是采用食品级的姜黄素和食品级的橙皮苷复配作为光敏剂复合物,联合ε‑聚赖氨酸作为载体,经真空冷冻干燥法制成双靶点光敏化合物粉末。由该粉末介导的光动力技术抑菌效果好,灭菌率高达99.9%;由其介导的光动力技术处理新鲜黄色金针菇后,能高效降低金针菇菌落总数,抑菌效果显著,且一定程度上可减缓黄色金针菇的水分、色差、可溶性固形物、可溶性蛋白等品质劣变,延长新鲜黄色金针菇的贮藏期,保障其食品安全问题,极大地提高经济效益。
Description
技术领域
本发明属于非热力减菌领域,具体涉及一种具有双靶向作用的姜黄素光敏化合物粉末。
背景技术
光动力杀菌技术是一种极具潜力的新型非热杀菌技术,光动力反应是光敏剂在特定波长光源激发下,产生活性氧引起生物分子氧化和细胞损伤的过程,达到杀灭微生物(包括细菌、真菌、病毒)的目的,同时还能最大限度地减少食品中各种营养成分的损失,保持食品的原有风味,延长食品的货架期,以满足广大消费者日益增长的物质生活的需要。在光动力杀菌过程中光源和光敏剂起关键性作用,理想的光敏剂应该具备安全可靠、成本低并且能够被适当波长的光激活的特点。目前,以姜黄素为代表的天然光敏物质及其衍生物是食品抑菌领域最具潜力的光敏剂。
姜黄素(Curcumin,Cur)是从姜科植物姜黄根茎中提取的小分子质量多酚类化合物,是一种安全性高、来源广、无污染的天然光敏剂具有抗菌、抗肿瘤等功能。姜黄素已被批准作为安全的着色剂及添加剂应用于食品。作为食品着色剂,姜黄素已被广泛应用于肠类制品、罐头、酱卤制品、面条、饮料、糖果等;作为食品调味剂,姜黄素在咖喱粉等调味料中发挥着举足轻重的作用。此外,姜黄素具有良好的光敏活性,可被400~480 nm蓝光高效激发产生大量活性氧,对单增李斯特菌、金黄色葡萄球菌、大肠杆菌、沙门氏菌等有强烈的杀灭效果。由姜黄素介导的光动力技术,具有高效杀菌、原料环保、能耗低、成本低等优点,已成为最炙手可热的杀菌技术之一,已被应用于哈密瓜、马铃薯等果蔬;鲍鱼、海蜇等水产品;银耳等食用菌的保鲜中,起到维持食品品质、延长货架期的作用。姜黄素具有的水溶性差、生物利用率低等缺陷限制了其在食品抑菌保鲜等领域的发展,故需将其与水溶性强的分子结合,以提高溶解性。
研究表明,大肠杆菌等食源性致病菌中存在一种姜黄素还原酶(CurA 蛋白),当细菌受到姜黄素光动力技术所造成的生存胁迫时,会通过上调CurA基因,产生大量姜黄素还原酶(CurA 蛋白)促进姜黄素还原代谢成光敏活性极弱的二氢姜黄素 (DHC)、四氢姜黄素(THC)等还原产物代谢物,并将其排出体外,降低姜黄素光动力杀菌效率,严重限制了光动力技术在杀灭食源性致病菌中的应用。
基于此,本发明引入了靶向抑制姜黄素还原酶(CurA 蛋白)活力的食品添加剂——橙皮苷,以提高杀菌效果。橙皮苷是陈皮黄酮类的主要活性成分之一,主要存在于芸香科植物橘及其栽培变种的成熟果皮中,具有极高的安全性和抗炎、抗氧化、抗辐射、改善血糖等生物活性,对金黄色葡萄球菌、大肠杆菌、铜绿假单胞菌和枯草芽孢杆菌的生长有抑制作用并被广泛应用于医药、食品等科学领域。通过分子对接技术探究橙皮苷和姜黄素还原酶(CurA 蛋白)的结合程度,结果(如图1)显示:2D 图中外侧为姜黄素还原酶(CurA 蛋白)分子,内测为橙皮苷分子。从图1可知,橙皮苷与姜黄素还原酶(CurA 蛋白)形成4个氢键作用、1个π-π作用和疏水作用;羰基作为氢键受体与 ILE284 形成1个氢键作用,距离为2.3Å;糖环上的羟基可以与SER65、TYR99、ASP112 形成 3 个氢键作用,距离分别为 2.7 Å、1.9Å、1.9 Å;此外,橙皮苷与VAL250、TYR253、VAL162、PRO160等氨基酸残基形成疏水作用。即橙皮苷与姜黄素还原酶(CurA 蛋白)结合紧密,可高效靶向抑制其活性。
由于细菌的等电点pI=2~5,低于生理条件pH值,所以细菌表面常带有负电荷。若将阳离子引入姜黄素-橙皮苷光敏复合物中,能提高对细菌的靶向吸附效率,进而增强杀菌效果。ε-聚赖氨酸是一种生物聚合物,主要由25−35个L-赖氨酸通过α-羧基和ε-氨基形成的酰胺键连接而成,其水溶液带正电荷。我国在2014年将ε-聚赖氨酸列为食品添加剂新品种。目前ε-聚赖氨酸盐酸盐应用于水果、蔬菜、肉制品、大米、杂粮、饮料等一系列食品的保鲜,具有极高的安全性。研究表明,带正电荷的ε-聚赖氨酸易与带负电荷的微生物细胞膜发生结合吸附,进而破坏膜结构的完整性,最终导致细胞对底物失去选择性而自溶死亡。所以,本发明引入ε-聚赖氨酸,以高效靶向带负电荷的细菌,从而促进姜黄素光敏剂复合物与细菌的结合,提高姜黄素光动力杀菌效果。
发明内容
本发明的目的是提供一种含姜黄素的双靶点光敏化合物粉末,提高光动力杀菌效果,并将其应用于金针菇保鲜中,维持金针菇品质,延长货架期。
为实现上述目的,本发明采用如下技术方案:
一种含姜黄素的双靶点抑菌光敏化合物粉末,所述双靶点抑菌光敏化合物能通过正负电荷靶向吸附细菌、特异性结合并抑制姜黄素还原酶(CurA 蛋白)表达以避免光敏剂降解这两个途径协同增强抑菌效果,其制备方法包括以下步骤:
(1)将橙皮苷溶解于DMSO中,姜黄素粉末溶解于丙酮中,并在连续搅拌下将姜黄素溶液加入橙皮苷溶液中,制成姜黄素-橙皮苷光敏复合物溶液;
(2)将ε-聚赖氨酸溶解于无菌PBS缓冲液中,并利用去离子水将其稀释至100 ug/mL,在连续搅拌下加入步骤(1)中的姜黄素-橙皮苷光敏复合物溶液,室温下,混合液于避光条件下搅拌24 h,期间不用加盖子配合30min氮吹以彻底去除丙酮和DMSO;
(3)将步骤(2)得到的溶液离心(1000 rpm,5 min),通过真空冷冻干燥得到具有高度水溶性的姜黄素-橙皮苷-ε-聚赖氨酸双靶点抑菌光敏化合物,于4 ℃冷藏备用。
步骤(1)中姜黄素-橙皮苷光敏复合物溶液中姜黄素的含量为100 μmol/L,橙皮苷的含量为0.02 wt%。
步骤(2)中ε-聚赖氨酸的pH值为7.0~7.4。
步骤(3)中离心转速为1000 rpm,时间5 min。冷冻干燥温度为-50 ℃,真空度0.1kPa。
所述的双靶点抑菌光敏化合物用于食品抑菌的方法:新鲜黄色金针菇经双靶点抑菌光敏化合物处理后,采用LED可见光进行照射,以实现抗菌效果。
采用LED可见光进行照射的时间为30 min。
所述LED可见光来源于波长为425 nm的LED蓝色光源。
本发明具有如下优点:
(1)本发明制备的姜黄素光敏化合物具有双靶向特性。采用所述双靶向是指一方面橙皮苷高效靶向抑制姜黄素还原酶的活力,降低其对姜黄素的代谢;另一方面ε-聚赖氨酸水溶液带正电荷,可高效靶向带负电荷的细菌,使姜黄素易与细菌结合紧密,从而大幅度提高杀菌效率。
(2)本发明将食品级的姜黄素和食品级的橙皮苷复配成光敏剂复合物,联合ε-聚赖氨酸,制成具有双靶向作用的姜黄素光敏化合物粉末,最后与LED蓝光配合使用,可最大程度地杀灭微生物,经过试验证明,灭菌率高达99.9%,抑菌效果好。
(3)由本发明制备的姜黄素光敏化合物粉末介导的光动力技术,可以有效杀灭黄色金针菇的微生物,并维持水分、色差、可溶性固形物、可溶性蛋白等品质,延长黄色金针菇的货架期。
(4)姜黄素、橙皮苷、ε-聚赖氨酸,被广泛用作食品添加剂,来源安全、可靠;三者结合制备的化合物介导的光敏化非热力抑菌技术相对于传统的灭菌方法更加安全。
(5)采用价格低廉且普通LED光源即可激发姜黄素-橙皮苷-ε-聚赖氨酸光敏化合物,很大程度上降低了灭菌成本。
附图说明
图1是分子对接技术探究橙皮苷和姜黄素还原酶(CurA 蛋白)的结合程度。
图2是本发明实施例1双靶点光敏化合物对大肠杆菌抑菌效果的影响。
图3是本发明实施例3双靶点光敏化合物光动力处理对黄色金针菇的外观颜色的影响。
图4是本发明实施例2双靶点光敏化合物光动力处理对黄色金针菇的菌落总数的影响。
图5是双靶点光敏化合物光动力处理的黄色金针菇贮藏0~7天外观实物图。
具体实施方式
为了使本发明所述的内容更加便于理解,下面结合具体实施方式对本发明所述的技术方案做进一步的说明,但是本发明不仅限于此。
实施例1:
在28 ℃条件下,将0.2 mg橙皮苷溶解于DMSO(5 mL),3.68mg姜黄素粉末溶解于丙酮(95 mL)中,并在连续搅拌下将姜黄素溶液加入橙皮苷溶液中,制成姜黄素-橙皮苷光敏复合物溶液(姜黄素终浓度为100 μmol/L,橙皮苷的终含量为0.02 wt%)。
将8 mg ε-聚赖氨酸溶解于80 mL无菌PBS缓冲液中,并利用去离子水将其稀释至100 ug/mL。在连续搅拌下将步骤(1)中的姜黄素-橙皮苷复合物溶液加入100ug/mL ε-聚赖氨酸水溶液中。室温下,混合液于避光条件下搅拌24 h,期间不用加盖子以蒸发丙酮和DMSO。
将去除丙酮和DMSO的溶液离心(1000 rpm,5 min),通过真空冷冻干燥得到具有高度水溶性的姜黄素-橙皮苷-ε-聚赖氨酸双靶点光敏化合物,于4℃冷藏备用。
活化大肠杆菌,并将其培养至对数生长期备用。制备浓度均为100 μg/mL的姜黄素双靶点光敏化合物溶液,同时制备单纯姜黄素溶液、单纯橙皮苷溶液以及单纯ε-聚赖氨酸溶液。设置10个组别:空白对照组(不做任何处理)、单纯橙皮苷组(单纯橙皮苷处理)、单纯ε-聚赖氨酸组(单纯ε-聚赖氨酸处理)、单纯姜黄素组(单纯姜黄素处理)、双靶点光敏化合物(单纯双靶点光敏复合物处理)、单纯光照组(只进行光照处理)、ε-聚赖氨酸+光照组(进行ε-聚赖氨酸和光照处理)、橙皮苷+光照组(进行橙皮苷和光照处理)、姜黄素+光照组(进行姜黄素和光照处理)和双靶点光敏化合物+光照组(进行双靶点光敏化合物和光照处理)。将大肠杆菌菌悬液分别与各溶液1:1混合,避光孵育30 min,然后置于距离波长为425 nm的LED蓝色光源20 cm处光照30min。每组设置三个平行。平板计数法对上述实验组和空白对照组的大肠杆菌菌落总数的计算(灭菌效果评价),结果见图2。
由图2可知,由空白对照组可得原始菌落总数为9.27 Log(CFU/mL)。无光照时,相较于其他组,单纯双靶点光敏化合物能显著降低大肠杆菌菌落总数(灭菌率为96.4%)。引入光照因素后,姜黄素光照组和双靶点光敏化合物光照组的菌落总数均明显下降,且双靶点光敏化合物光照组能达到99.9%的灭菌效果(菌落总数下降3.4 Log(CFU/mL))显著优于姜黄素光照组(菌落总数下降2.9 Log(CFU/mL)),说明双靶点光敏化合物能够有效提高姜黄素光动力抑菌作用。
实施例2:
姜黄素双靶点光敏化合物制备参照上述实施例1。
称取适量黄色金针菇,实验组采用制备双靶点光敏化合物制成浓度约为0.1 mg/mL的水溶液对新鲜金针菇进行喷洒处理,对照组不采用任何处理(菌落数约为6.57 Log(CFU/mL)。将实验组和对照组样品避光孵育30min,然后置于距离波长为425 nm的LED蓝色光源20 cm处光照30 min。每组设置三个平行。采用平板计数法对上述实验组和对照组的黄色金针菇对第0~7天进行菌落总数的计算(灭菌效果评价),结果见表1与图4。
表1双靶点光敏化合物介导的光动力技术对黄色金针菇菌落总数的影响
由表1与图4可知,与对照组比较,利用双靶点光敏化合物介导的光动力技术处理黄色金针菇后,菌落总数显著降低。贮藏7天后,实验组的菌落总数仍低于第3天对照组的菌落总数,说明经双靶点光敏化合物光动力处理,黄色金针菇的贮藏期可以从3天延长至7天。
实施例3:
双靶点光敏化合物制备参照上述实施例1。
称取适量黄色金针菇,实验组采用双靶点光敏化合物介导的光动力技术对黄色金针菇进行处理,并设置对照组。将实验组和对照组样品避光孵育30 min,置于距离波长为425 nm的LED蓝色光源20 cm处光照30min。每组设置三个平行。比较双靶点光敏化合物介导的光动力技术对新鲜黄色金针菇的失重率值、可溶性固形物含量值、可溶性蛋白质含量值和色差值,分析结果见表2以及实物图见图3。
表2双靶点光敏化合物介导的光动力技术对黄色金针菇理化特性的影响
由表2可知:双靶点光敏化合物介导的光动力处理前后,对照组和实验组的水分含量、色差、可溶性固形物含量以及可溶性蛋白含量基本持平。说明双靶点光敏化合物光动力处理基本上不会对黄色金针菇的理化品质造成影响。
实施例4:
双靶点光敏化合物的制备参照上述实施例1。
称取适量黄色金针菇,实验组采用双靶点光敏化合物对新鲜金针菇进行处理,并设置对照组。将实验组和对照组样品避光孵育30 min,置于距离波长为425 nm的LED蓝色光源20 cm处光照30min。每组设置三个平行。对第0天到第7天黄色金针菇的水分、色差、可溶性固形物含量和可溶性蛋白质含量进行评估,结果见表3,实物图见图5。
表3双靶点光敏化合物介导的光动力技术对贮藏0~7天黄色金针菇的理化特性的影响
由表3和图5可知,贮藏第3天时,对照组在已出现轻微失水和褐变,可溶性固形物和可溶性蛋白含量已明显降低,而实验组几乎没有劣变;贮藏第7天时,对照组已出现明显的失水和褐变,溶性固形物和可溶性蛋白进一步损失;而实验组仅出现轻微劣变,其水分、色差、可溶性固形物含量和可溶性蛋白含量均维持在较优良水平,说明在7天内,双靶点光敏化合物光动力处理可以维持黄色金针菇的理化品质。
以上所述仅为本发明的较佳实施例,凡依本发明申请专利范围所做的均等变化与修饰,皆应属本发明的涵盖范围。
Claims (7)
1. 一种双靶点抑菌光敏化合物的制备方法,其特征在于,所述双靶点抑菌光敏化合物能通过正负电荷靶向吸附细菌、特异性结合并抑制姜黄素还原酶CurA 蛋白表达以避免光敏剂降解这两个途径协同增强抑菌效果,其制备方法包括以下步骤:
(1)将橙皮苷溶解于DMSO中,姜黄素粉末溶解于丙酮中,并在连续搅拌下将姜黄素溶液加入橙皮苷溶液中,制成姜黄素-橙皮苷光敏复合物溶液;
(2)将ε-聚赖氨酸溶解于无菌PBS缓冲液中,并利用去离子水将其稀释至100 ug/mL,在连续搅拌下加入步骤(1)中的姜黄素-橙皮苷光敏复合物溶液,室温下,混合液于避光条件下搅拌24 h,期间不用加盖子配合30 min氮吹以彻底去除丙酮和DMSO;
(3)将步骤(2)得到的溶液离心,通过真空冷冻干燥得到具有高度水溶性的姜黄素-橙皮苷-ε-聚赖氨酸双靶点抑菌光敏化合物,于4 ℃冷藏备用。
2. 根据权利要求1所述的制备方法,其特征在于,姜黄素-橙皮苷光敏复合物溶液中姜黄素的含量为100 μmol/L,橙皮苷的含量为0.02 wt%。
3. 根据权利要求1所述的制备方法,其特征在于,步骤(3)离心转速为1000 rpm,时间5min。
4. 根据权利要求1所述的制备方法,其特征在于,步骤(3)冷冻干燥温度为-50 ℃,真空度0.1 kPa。
5.根据权利要求1所述的制备方法制得的双靶点抑菌光敏化合物用于新鲜黄色金针菇的抑菌保鲜,其特征在于,新鲜黄色金针菇经双靶点抑菌光敏化合物处理后,采用LED可见光进行照射,以实现抗菌效果。
6. 根据权利要求5所述的方法,其特征在于,采用LED可见光进行照射的时间为30min。
7. 根据权利要求5所述的方法,其特征在于,所述LED可见光来源于波长为425 nm的LED蓝色光源。
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CN117379574A (zh) * | 2023-10-13 | 2024-01-12 | 中国热带农业科学院农产品加工研究所 | 一种ε-聚赖氨酸联合姜黄素为介导的光动力灭菌方法及应用 |
CN117379574B (zh) * | 2023-10-13 | 2024-05-31 | 中国热带农业科学院农产品加工研究所 | 一种ε-聚赖氨酸联合姜黄素为介导的光动力灭菌方法 |
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