CN103228161A - 一种含抗菌肽的组合物作为食品防腐剂的应用 - Google Patents
一种含抗菌肽的组合物作为食品防腐剂的应用 Download PDFInfo
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Abstract
本发明提供了一种含抗菌多肽的组合物在防止或抑制微生物引起的食品腐败变质中的应用,所述抗菌多肽包含Blad或其活性变体。本发明还提供了一种防止或抑制微生物引起的食品腐败变质的方法,包括向有需要的食品食用有效量的含抗菌多肽的组合物,其实所述抗菌多肽包含Blad或其活性变体。
Description
技术领域
本发明涉及针对使食物腐败变质的微生物体的抗菌剂领域。
背景技术
食品保藏是预防或抑制由内源性化学降解/酶降解引起的和/或由微生物导致或微生物加速的食品腐败变质的食品处理方法。现有技术已有大量关于食品保藏技术,有些可以抑制内源过程(如抗氧化剂),有些是抑菌过程(如抗菌剂),还有一些同时抑制这两种过程(如冷冻)。用于抑制食品腐败变质的化合物通常被称为防腐剂,例如,可能是抗氧化剂或抗菌剂。
特殊的食品保藏技术包括干燥,加热,冷藏,冷冻,渗透抑制(如使用糖或盐),真空包装,罐头和瓶装,胶冻化,装罐,装壶,电离辐射,脉冲电场处理,高压食品保藏,以及超高水压食品保藏,使用抗氧防腐剂,和/或使用抗菌性防腐剂(如二氧化硫,二氧化碳,乙醇,醋酸,柠檬酸,乳酸,山梨酸,苯甲酸盐,硝酸盐和亚硝酸盐,亚硫酸盐,丙酸钙盐和甲基氯异塞唑啉酮)。
尽管现今使用了相对大量的食品保藏技术,但仍有需要开发一种新的抗菌防腐剂。这是因为许多现有技术中能有效针对微生物的防腐技术还很欠缺,还因为尤其是现有许多抗菌防腐剂的效力和/或安全的问题。
许多食品防腐技术试图建立一个不利于微生物生长的环境,这对极端环境可生存的微生物是无效的(如假单胞菌(Pseudomonas)能在低温下生长;凝结芽孢杆(Bacilluscoagulans)是耐高温且酸耐受的;许多曲霉(Aspergillus)表现贫营养状态;接合酵母(Zygosaccharomyces)具有高干燥耐受性)。
许多现有抗菌防腐剂具有适度活性,特别针对先天或后天获得性抗性的微生物,和/或具有窄谱的微生物。例如,接合酵母对乙醇,醋酸,山梨酸,苯甲酸和二氧化硫都具有高耐受性。此外,现有抗菌防腐剂都与各种副作用相关,如呼吸问题或衰老和残疾(ADD)。在特殊实例中,二氧化硫对气喘患者的支气管具刺激性,亚硝酸盐是潜在致癌的,苯甲酸盐与各种过敏、哮喘、皮疹以及脑损伤有关。
此外,抑制食品中微生物生长的有效的技术如低pH值或低水活性往往因为不被消费者接受(如有酸味)或对健康有负面影响(如高盐或高糖)。
本发明目的在于探求所有以上问题的解决方案,并尤其例如,提供一种针对微生物具有有效和广谱活性并同时具有低毒性的新的抗菌剂。
发明内容
发明人惊讶的发现来自羽扇豆属(Lupinus)的Blad多肽,针对能导致食品腐败变质的多种不同的细菌和真菌生物表现出有效的抗菌活性。本发明还发现Blad多肽是非毒性的,因此Blad是作为抗菌食品防腐剂的极佳的化合物。
因此,本发明提供了一种包括抗菌多肽的组合物在预防或抑制微生物引起的食品腐败变质中的应用,其中所述抗菌多肽包含Blad或其活性变体。优选的,所述微生物为细菌(优选的选自假单胞菌(Pseudomonas)或芽孢杆菌(Bacillus)的导致食品腐败的种属)或为真菌(优选的选自以下导致食品腐败的种属:链格孢菌(Alternaria),曲霉(Aspergillus),镰刀霉(Fusarium),葡萄孢菌(Botrytis),炭疽菌(Colletotrichum),酵母菌(Saccharomyces),克鲁维酵母(Kluyveromyces)和接合酵母(Zygosaccharomyces))。
在优选的具体实施例中,所述食品源自、或提供、或为,水果、坚果、蔬菜、种子、糖、奶制品、液体食品或糊状食品、肉、鱼或面包。
在优选的具体实施例中,所述食品为草莓,优选的,其中所述微生物为灰葡萄孢(Botrytis cinerea)或尖孢炭疽菌(Colletotrichum acutatum),优选的为灰葡萄孢(Botrytis cinerea)。
在优选的具体实施例中,所述组合物进一步包括螯合剂。
本发明还提供了一种预防或抑制微生物引起的食品腐败变质的方法,包括给有需要的食品施以有效剂量的包含抗菌多肽的组合物,其中所述抗菌多肽包含Blad或其活性变体的。
附图说明
本发明将参考附图进行说明,其中:
图1显示白花羽扇豆(Lupinus albus)β-球蛋白前体编码序列(SEQ ID NO:1);以及
图2显示对应于Blad的β-球蛋白前体的内部片段的编码序列(SEQ ID NO:3)。
具体实施方式
Blad
Blad(″banda de Lupinus albus doce″-band from sweet L.albus,名称源自白花羽扇豆)是以β-球蛋白的稳定的中间的分解产物命名的,是羽扇豆属(Lupinus)的种子中主要的储存蛋白。Blad为一个20kD的多肽,由173个氨基酸残基组成,并由羽扇豆(Lupinus)中的β-球蛋白前体的编码基因(1791个核苷酸,GenBank中储存的收录编号为AAS97865)的内部片段(519个核苷酸,GenBank中储存的收录编号为ABB13526)所编码。当编码Blad末端序列的引物用于扩增羽扇豆DNA基因组序列时,可以得到一个大约620bp的产物,表明在编码Blad的基因片段中有一个内含子存在。天然存在的Blad是210kD的低聚糖的主要成分,该低聚糖在开始发芽之后的4-12天,特异的聚集于羽扇豆的子叶中(随后β-羽扇豆球蛋白发生密集的限制性蛋白水解)。同时,所述低聚物被糖基化,天然存在的Blad是非糖基化的。含Blad的寡聚糖由多个多肽组成,其中主要的几个分子量分别为14、17、20、32、36、48和50kD。该20kD的多肽Blad,是至今为止低聚物中最丰富的多肽,并且可能是唯一一个具有凝集素活性的多肽。天然存在的Blad在8天龄植物子叶总蛋白中占大约80%。
图1显示所述白花羽扇豆(L.albus)β-球蛋白前体的编码序列(SEQ ID NO:1)。β-球蛋白母亚基编码序列位于70-1668位残基。编码的533氨基酸残基β-球蛋白母亚基(SEQ ID NO:2)为:
MGKMRVRFPTLVLVLGIVFLMAVSIGIAYGEKDVLKSHERPEEREQEEWQPRRQRPQSRREEREQEQEQGSPSYPRRQSGYERRQYHERSEQREEREQEQQQGSPSYSRRQRNPYHFSSQRFQTLYKNRNGKIRVLERFDQRTNRLENLQNYRIVEFQSKPNLILPKHSDADYVLVVLNGRATIIVNPDRRQAYNLEYGDALRIPAGSSYILNPDDNQKLRWKLAIPINNPGYFYDFYPSSKDQQSYFSGFSRNTLEAFNTRYEEIQRIILGNEDEQEYEEQRRGQEQSDQDEGVIVIVSKKQIQKLTKHAQSSSGKDKPSDSGPFNLRSNEPIYSNKYGNFYEIPDRNPQVQDLNISLTYIKINEGALLLPHYNSKAIYWWDEGEGNYELVGIRDQQRQQDEQEEKEEEVIRYSARLSEGDIFVIPAGYPISINASSNLRLLGFGINADENQRNFLAGSKDNVIRQLDRAVNELTFPGSAEDIERLIKNQQQSYFANGQPQQQQQQQSEKEGRRGRRGSSLPF
图2显示了对应Blad的β-球蛋白前体内部片段的编码序列(SEQ ID NO:3)。该Blad多肽(SEQ ID NO:4)为:
RRQRNPYHFSSQRFQTLYKNRNGKIRVLERFDQRTNRLENLQNYRIVEFQSKPNTLILPKHSDADYVLVVLNGRATIIVNPDRRQAYNLEYGDALRIPAGSSYILNPDDNQKLRWKLAIPINNPGYFYDFYPSSKDQQSYFSGFSRNTLEAFNTRYEEIQRIILGNED
本发明涉及一种包含含有Blad或其活性变体的抗菌多肽的组合物。因此,其涉及一种含抗菌多肽的组合物,该抗菌多肽含SEQ ID NO:4的多肽序列或其活性变体。在其中可选的具体实施例中,该组合物主要由含blad或其活性变体的抗菌多肽组成,和/或所述抗菌多肽主要由blad或其活性变体组成。在进一步的具体实施例中,所述含(或主要包含)blad或其活性变体的抗菌多肽可能以单独的方式使用。
Blad的活性变体是指保持了作为抗菌剂的能力(即具有抗菌活性-参照以下关于该活性水平和如何测量的说明)的Blad变体。“blad的活性变体”包括在其范围内的一个SEQ ID NO:4片段。在优选的具体实施例中,SEQ ID NO:4片段为选自为SEQ ID NO:4序列长度的至少10%,优选的为至少其长度的20%,优选的为至少其长度的30%,优选的为至少其长度的40%,优选的为至少其长度的50%,优选的为至少其长度的60%,优选的为至少其长度的70%,优选的为至少其长度的80%,优选的为至少其长度的90%,以及最优选的为至少SEQ ID NO:4序列长度的95%。Blad或其变体通常具有至少10个氨基酸残基的长度,如至少20个、25个、30个、40个、50个、60个、80个、100个、120个、140个、160个或173个氨基酸残基的长度。
“Blad的活性变体”还包括在其范围内的一个与SEQ ID NO:4同源的多肽序列,比如至少40%相同,优选的至少60%相同,优选的至少70%相同,优选的至少80%相同,优选的至少85%相同,优选的至少90%相同,优选的至少95%相同,优选的至少97%相同,以及最优选的至少99%相同,例如,超过全序列或超过至少20个连续氨基酸残基区域,优选的超过至少30个连续氨基酸残基区域,优选的超过至少40个连续氨基酸残基区域,优选的超过至少50个连续氨基酸残基区域,优选的超过至少60个连续氨基酸残基区域,优选的超过至少80个连续氨基酸残基区域,优选的超过至少100个连续氨基酸残基区域,优选的超过至少120个连续氨基酸残基区域,优选的超过至少140个连续氨基酸残基区域,以及最优选的超过至少160或更多的连续氨基酸残基的区域。测量蛋白同源性的方法是本领域公知常识,而且在本文内容中本领域技术人员可以理解同源性是基于相同氨基酸而计算的(有时也称为“硬同源”)。
具有同源活性的Blad变体典型的是通过取代,插入或缺失从而与SEQ ID NO:4多肽序列区别开来的,例如,通过1、2、3、4、5到8或更多位取代,缺失或插入的方式。该取代更多为“保守的”,也即,一个氨基酸可能被另一个相似氨基酸取代,相似氨基酸可以借由为以下组的其中一个:芳香残基(F/H/W/Y),非极性脂肪族残基(G/A/P/I/L/V),极性不带电脂肪族残基(C/S/T/M/N/Q)和极性带电脂肪族残基(D/E/K/R)。优选的亚族包括:G/A/P;I/L/V;C/S/T/M;N/Q;D/E;和K/R。
一种含Blad或其活性变体的抗菌多肽(如上所述)可能由在N末端和/或C末端添加了任意数量的氨基酸的Blad或其活性变体组成,并且该抗菌多肽保持了抗菌活性(再次参照以下关于所述活性水平和如何测量的说明)。优选的,不超过300个氨基酸残基添加至Blad或其活性变体的一个末端或者两个末端,更优选的,不超过200个氨基酸残基添加至Blad或其活性变体的一个末端或者两个末端,更优选的,不超过150个氨基酸残基添加至Blad或其活性变体的一个末端或者两个末端,更优选的,不超过100个氨基酸残基添加至Blad或其活性变体的一个末端或者两个末端,更优选的,不超过80、60、或40个氨基酸残基添加至Blad或其活性变体的一个末端或者两个末端,最优选的,不超过20氨基酸残基添加至Blad或其活性变体的一个末端或者两个末端。
一种包括(或主要包括)Blad或其活性变体的抗菌多肽(如上所述)可以以纯化的形式(例如,从植物、动物或微生物来源中提取)和/或重组蛋白形式。重组形式的产品使得能够产生Blad的活性变体。
本领域已经公开了纯化天然存在的Blad的方法(如,Ramos et al.(1997)Planta203(1):26-34和Monteiro et al.(2010)PLoS ONE5(1):e8542)。天然存在的Blad的一个合适的来源为一种羽扇豆属植物,如白色羽扇豆(Lupinus albus),优选的,所述为所述植物的子叶,优选的,取在发芽开始后大约4到14天收获,更优选的,取在发芽开始后大约6到12天收获(如,发芽开始后的8天收获)。本领域已经公开了获得含Blad的粗提的全蛋白提取物的方法,以及对上述提取物纯化从而获得部分纯化提取物的蛋白纯化方法,如包含Blad的低聚糖构成的Blad。
可以用聚丙烯酰胺凝胶电泳(SDS-PAGE)法和/或,优选的,采用C-18柱反相高效液相色谱法(RP-HPLC)分离Blad本身。
另一种获得含Blad的低聚糖的部分纯化提取物的方法是利用了Blad的甲壳素绑定活性。所述低聚糖与甲壳素柱以强有力的形式结合,作为甲壳素亲和色谱纯化的一部分,以0.05N HCl洗脱。所述纯化方法的实施例详细说明如下:
收获8天龄的羽扇豆植物的子叶并在含10mM CaCl2和10mM MgCl2的Milli-Q plus中加水匀浆(pH调至8.0)。匀浆通过粗棉布过滤并30,000转,4C离心1小时。随后取沉淀物并悬浮于pH7.5的100Mm三羟甲基氨基甲烷-盐酸(Tris-HCl)缓冲液中,该缓冲液含重量体积比为10%(w/v)的NaCl,10mM EDTA和10mM EGTA,4℃搅拌1小时,并在4℃,3000转离心1小时。上清中的总球蛋白片段用硫酸铵(561g/L)沉淀,低温搅拌1小时并在4℃,3000转离心30分钟。所得的颗粒沉淀物溶解在pH7.5,50mM的Tris-HCl缓冲液中,在用相同缓冲液平衡过的PD-10柱中脱盐并通过在相同缓冲液中预平衡了的甲壳素亲和层析柱。该柱用pH7.5,50mMTris-HCl缓冲液冲洗,并且将该结合蛋白用0.05NHCl洗脱。该洗脱物片段立即用2M Tris中和,并用SDS-PAGE收集其峰值区的洗脱物片段,冻干并分析。
甲壳素柱的制备,从Sigma公司购买粗甲壳素并做以下处理:甲壳素样品用Milli-Qplus加水充分洗涤,然后用0.05NHCl充分洗涤。然后用重量体积比为1%(w/v)的碳酸钠洗涤,之后用乙醇洗涤,直至洗到光吸收度小于0.05。将甲壳素装入移液管端部并用pH7.5,50mMTris-HCl平衡。
生产重组蛋白的方法为本领域公知常识。其在本发明中使用的方法涉及将编码含Blad或其活性变体的多肽的多核苷酸插入到合适的表达载体上,使得所述多核苷酸与一个或多个启动子(如诱导性启动子,如T71ac)以及其他目的多核苷酸或目的基因并置,将表达受体引入到合适的细胞或者组织中(比如,大肠杆菌E.Coli),在转化细胞或组织中表达该多肽,并将表达的重组多肽从细胞或组织中提取。表达载体可以包含多核苷酸额外编码的构建以辅助纯化,例如,一个末端标签可以辅助纯化:例如,用于亲和纯化的一个组氨酸残基标签。一旦该重组多肽被纯化,纯化标签可以从多肽中去除。比如,通过溶蛋白性裂解。
在一个包含含有(或主要含有)Blad或其活性变体的抗菌多肽的组合物中,所述多肽优选的为以部分纯化的形式,更优选的以纯化的形式。当在缺少一种或多种天然与之相连的多肽的环境中和/或为至少10%总蛋白的多肽代表时,所述多肽为部分纯化形式。当在一个缺少全部,或多数与之天然相连的其他多肽的环境中时,所述多肽为纯化的形式。例如,纯化的Blad是指组合物中Blad代表了至少50%的总蛋白,至少60%、至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少97%、至少98%、至少99%的总蛋白。
在一个包含含(或主要包括)Blad或其活性变体的抗菌多肽的组合物中,所述羽扇豆蛋白含量可能主要由含多肽的Blad-低聚糖组成,该多肽含(或主要包含)Blad或其变体。
一个含(或主要包含)Blad的抗菌多肽的组合物其也可以是包含由本领域技术人员添加了其他化合物的配方。
食品腐败变质和食品
通过微生物的食品腐败变质是指微生物对食品的任何改变,其结果是在以下方面改变,例如,味道,气味或外观(如,形状、颜色、质地、硬度),从而降低了其营养和/或商业价值。通过食品是指为营养或令人愉悦的目的被人类或动物消费的任何液体或固体物质。该食品可以是直接或间接消耗(如,烹饪或处理之后,如谷物精制)。
在适用的情况下,该食品优选的是一种从其天然环境中分离的形式,如收获植物食品(如水果、蔬菜、种子)和从动物中分离的产品(如肉类、鱼类、奶类)。
该食品可以源自,可以提供,或可以是,一种水果、坚果、蔬菜、种子、糖、奶制品、液体食品或糊状食品、肉、鱼或面包。源自水果的食品包括酒和果汁。提供种子的植物包括谷类(如玉米、小麦、大麦、高粱、小米、大米、燕麦和黑麦)以及豆类(如黄豆、豌豆和扁豆)。糖,优选蔗糖,可以源自甜菜或甘蔗。奶制品包括奶、奶油、奶酪和酸奶。液体或糊状食物包括汤、调味料、咸菜、蛋黄酱、色拉酱和其他色拉调味料、饯、糖浆和婴儿食品。所述肉类和/或鱼类食品估计可以是处理过或未处理过的,以及可以是烹饪过的或未烹饪过的。进一步优选的食品预计可以在下一部分涉及可能被微生物腐败变质的食品例子中找到,其可能是本发明的应用和方法的针对对象。
食品还包括预处理的复合食品,如三明治、派、乳蛋饼等,尤其是那些一般用来冷冻保藏的食品。
目标微生物
本发明提供了含抗菌多肽的组合物在预防或抑制微生物引起食品腐败变质中的应用,该抗菌多肽含Blad或其活性变体。可以导致食品腐败变质的微生物,即食品腐败微生物,包括,尤其是,细菌和真菌。在该应用中,该抗菌多肽可以视为抗菌食品防腐剂。
该抗菌多肽可以用来预防或抑制革兰氏阳性细菌或革兰氏阴性细菌引起的食品变质腐败。尤其优选的目标细菌(可能被腐败变质的食品的例子在括号中给出)包括:食品腐败假单胞菌(Pseudomonas)如,铜绿假单胞菌(Pseudomonas aeruginosa)(拟水芹和莴苣),丁香假单胞菌(Pseudomonas syringae)(各种取自植物的食品,如甜菜、小麦和大麦),托拉斯假单胞杆菌(Pseudomonas tolaasii)(蘑菇),蘑菇假单胞菌(Pseudomonas agarici)(蘑菇),莓实假单胞菌(Pseudomonas fragi)(奶制品)和隆德假单胞菌(Pseudomonas lundensis)(奶、奶酪、肉类和鱼类),最优选的铜绿假单胞菌;和食品腐败芽孢杆菌(Bacillus)种类,如枯草芽孢杆菌(Bacillus subtilis)(番茄、土豆、面包)和凝结芽孢杆菌(Bacillus coagulans)(奶类、番茄汁)。
所述抗菌多肽可用来防止或抑制单细胞(酵母)或多细胞(丝状霉菌)真菌引起的食品腐败变质。尤其优选的目标酵母(可能被腐败变质的食品的例子在括号中给出)包括:食品腐败酵母菌类(Saccharomyces)如,酿酒酵母(Saccharomyces cerevisiae)(糖、糖浆、酒、和软饮如果汁);食品腐败克鲁维酵母类(Kluyveromyces)如马克斯克鲁维酵母(Kluyveromyces marxianus)(奶酪);以及食品腐败接合酵母类(Zygosaccharomyces),如拜氏接合酵母(Zygosaccharomyces bailii)(酒、果汁、色拉调味料和番茄酱)和鲁氏接合酵母(Zygosaccharomyces rouxii)(糖浆、果汁、果酱和色拉调味料)。尤其优选的目的霉菌(可能被腐败变质的食品的例子在括号中给出)包括:食品腐败链格孢菌类(Alternaria)如互隔交链孢菌(Alternaria alternata)(马铃薯)、树状链格孢菌(Alternaria arborescens)(番茄)、果园链格孢菌(Alternariaarbusti)(亚洲梨)、芸苔链格孢菌(Alternaria brassicae)(蔬菜)、甘蓝链格孢菌(Alternaria brassicicola)(甘蓝类蔬菜)、Alternaria carotiincultae(胡萝卜)、刺蛾链格孢菌(Alternaria conjutncta)(欧洲萝卜)、胡萝卜链格孢菌(Alternaria dauci)(胡萝卜)、大戟生链格孢菌(Alternaria euphorbiicola)(甘蓝类蔬菜)、,梨黑斑链格孢菌(Alternariagaisen)(梨)、感染链格孢菌(Alternaria infectoria)(小麦)、日本链格孢菌(Alternaria japonica)(甘蓝类蔬菜)、洋芫荽链格孢菌(Alternaria petroselini)(欧芹)、Alternaria selini(欧芹)、索兰尼链格孢.(Alternaria solani)(马铃薯,番茄)、和Alternaria smyrnii(alexanders,欧芹);食品腐败曲霉类(Aspergillus)如烟曲霉(Aspergillus fumigatus)(坚果、马铃薯、大米和面包)、黑曲霉(Aspergillus niger)(水果和蔬菜如葡萄和洋葱)、黄曲霉(Aspergillus flavus)(谷物、花生);食品腐败镰刀霉类(Fusarium)如尖孢镰刀霉(Fusarium oxysporum)(水果)和禾谷镰刀霉(Fusarium graminearum)(大麦、小麦和玉米);食品腐败葡萄孢菌类(Bottytis)如灰葡萄孢菌(Bottytis cinerea)(草莓、葡萄和番茄);和食品腐败炭疽菌类(Colletotrichum)如尖孢炭疽菌(Colletotrichum actuatum)(草莓、芹菜、苹果、油梨、茄子、咖啡喝番石榴)、马铃薯炭疽菌(Colletotrichum coccodes)(番茄、马铃薯)、辣椒炭疽菌(Colletotrichum capsici)(紫苏、鹰嘴豆、胡椒)、壳皮炭疽菌(Colletotrichum crassipes)(百香果)、荔枝炭疽菌(Colletotrichum gloeosporioides)(蔬菜和水果如柑橘和苹果)、禾生炭疽菌(Colletotrichum graminicola)(谷类)、卡哈瓦刺盘孢菌(Colletotrichum kahawae)(咖啡)、菜豆炭疽菌(Colletotrichumlindemuthianum)(豆类)、香蕉炭疽菌(Colletotrichum musae)(香蕉)、黑刺盘孢炭疽菌(Colletotrichum nigrum)(番茄)、西瓜炭疽菌(Colletotrichum orbiculare)(甜瓜、黄瓜)、豌豆炭疽菌(Colletotrichum pisi)(豌豆)和亚线孢炭疽菌(Colletotrichumsublineolum)(大米)。
在优选具体实施例中,所述抗菌多肽用来防止或抑制微生物引起的水果腐败变质,优选的为草莓,以及优选的其中所述微生物为灰葡萄孢菌或炭疽菌,优选的为灰葡萄孢菌。
本领域技术人员能够通过常规方法,将含(或主要含)Blad(或其活性变体)的抗菌多肽用来在任何特定情况下预防或抑制腐败变质,确定一个合适的剂量浓度。优选的,例如,Blad的浓度至少为1μg/ml,浓度至少为5μg/ml,浓度至少为10μg/ml,浓度至少为50μg/ml,浓度至少为100μg/ml,浓度至少为150μg/ml,直到浓度为350μg/ml,直到浓度为500μg/ml,直到浓度为600μg/ml,直到浓度为1mg/ml,直到浓度为2.5mg/ml,直到浓度为5mg/ml,直到浓度为10mg/ml。优选的,使用Blad的浓度选自10μg/ml和5mg/ml之间,更优选的50μg/ml和2.5mg/ml之间,更优选的100μg/ml和1mg/ml之间,以及更优选的150μg/ml和600μg/ml之间(如大约250μg/ml)。本发明提供证据证明了Blad直到浓度至少为400μg/ml,对宿主无毒(参照例4和5)。
本发明惊讶的发现,将Blad与螯合剂(如EDTA)结合能产生协同抗菌作用。因此,优选的螯合剂用于改善所述含(或主要包含)Blad(或其活性变体)多肽的抗菌活性,以及该螯合剂的使用可能降低达到防止或抑制食品腐败变质的特定水平所需的抗菌多肽的浓度。螯合试剂(也称螯合剂,络合剂或多价螯合剂)可以是与金属离子结合的以形成非共价复合物以及减少离子活性的任意组合物。合适的螯合剂包括多胺羧酸盐,如EDTA(乙二胺四乙酸)和EGTA(乙二醇-双-(β-氨基乙醚)-N,N,N’,N’-四乙酸)。优选的,螯合剂为EDTA,优选的EDTA浓度至少为10μg/ml,至少为50μg/ml,至少为100μg/ml,至少为500μg/ml,直到浓度为1mg/ml,直到浓度为5mg/ml,直到浓度为10mg/ml,直到浓度为20mg/ml。优选的,EDTA的浓度在0.1mg/ml,和1mg/ml之间。
结果
该抗菌多肽可以用于抑制食品上的食品腐败微生物的生长(即其具有抑菌活性)和/或杀死所述微生物(即其具有杀菌活性),如此以致防止或抑制了被该微生物引起的所述食品的腐败变质。本领域技术人员能够确定一个合适的剂量和/或浓度从而实现生长抑制或杀死该微生物的特定的需要。
优选的,当作为抑菌剂使用时,与相同条件下但无抗菌多肽时相比所述抗菌多肽降低了10%的生长率,更优选的降低了50%的生长率,更优选的降低了75%的生长率,更优选的降低了90%的生长率,更优选的降低了95%的生长率,更优选的降低了98%的生长率,更优选的降低了99%的生长率,以及更优选的,降低了99.9%的生长率。最优选的,抗菌多肽能防止任何微生物的生长。
优选的,当用作杀菌剂时,与相同条件下无抗菌多肽时相比,该抗菌多肽能杀死微生物总数的10%,更优选的,能杀死微生物总数的50%,更优选的,能杀死微生物总数的75%,更优选的,能杀死微生物总数的90%,更优选的,能杀死微生物总数的95%,更优选的,能杀死微生物总数的98%,更优选的,能杀死微生物总数的99%,以及更优选的,能杀死微生物总数的99.9%,最优选的,抗菌多肽杀死所有的微生物。
当用于防止或抑制微生物引起的食品腐败变质时,所述抗菌多肽优选的使用有效剂量,也就是说,该剂量水平是指能够杀死和/或抑制微生物生长,能达到防止或抑制腐败的可检测水平(如降低腐败变质率)优选的,是同与相同条件的不加该抗菌多肽时对比。所述抗菌多肽的有效剂量对人或动物主体是无毒的。
应用和方法
本发明提供了一种含抗菌多肽的组合物的在防止或抑制微生物引起的食物腐败变质中的应用,所述抗菌多肽包含Blad或其活性变体。为此,本发明还提供了一种防止或抑制微生物引起的食品腐败变质的方法,包括给有需要的食品施用有效量的含抗菌多肽的组合物,其中所述抗菌多肽含Blad或其活性变体。优选的,所述抗菌多肽的有效量对人或动物是无毒的。所述防止或抑制腐败变质可以发生于食品的储存过程、运输过程、处理操作过程、加工过程或展示过程。
含抗菌多肽的组合物可以例如混合掺入食品中,或例如可以应用于食品表面(如作为液态膜或喷雾)。食品也可以浸入(并可选的保持于)所述组合物中。对于任意特殊的食品,所述组合物作为防腐剂的应用可以与任意其他公知的食品防腐技术、抗菌剂或相反的技术结合,包括干燥、加热,冷藏,冷冻,渗透抑制(如使用糖或盐),真空包装,罐头,瓶装,胶冻化,装罐,装壶,电离辐射,脉冲电场处理,高压食品保藏,以及超高水压食品保藏,使用抗氧防腐剂,和/或使用抗菌性防腐剂(如二氧化硫,二氧化碳,乙醇,醋酸,柠檬酸,乳酸,山梨酸,苯甲酸盐,硝酸盐和亚硝酸盐,亚硫酸盐,丙酸钙盐和甲基氯异塞唑啉酮)。
实施例,
在以下的例子中,BLAD是指天然形成的含Blad的低聚糖,其包含20kD的Blad多肽,按“per Ramos et al.(1997)Planta203(1):26-34”中的方法纯化,参照该文献中材料和方法部分的“植物材料和生长条件”以及“蛋白纯化”的内容。
定义:
MIC-最小抑制浓度:抑制微生物可见生长的抗菌剂的最低浓度
MFC/MBC-最低杀真菌浓度/最低杀细菌浓度(或最小致死浓度):在标准化条件下24小时后,杀死99.9%初始接种菌数所需的最低杀菌剂浓度。
实施例1Blad的杀菌活性
不同种类的细菌的BLAD的MIC和MBC(使用Mueller-Hinton培养基)
尤其的,铜绿假单胞菌和枯草芽孢杆菌可以导致食品腐败变质。对铜绿假单胞菌,BLAD在浓度为100μg/ml时具有抑菌作用,浓度为250μg/ml时具有杀菌作用。对铜绿假单胞菌,BLAD在浓度为50μg/ml时或EDTA浓度为1mg/ml抑制菌生长(即,两者都均有抑菌作用)但是两者结合具有杀菌作用。
实施例2-BLAD的杀真菌活性
各种丝状真菌BLAD的MIC和MFC(使用RPMI培养基)
各种丝状真菌BLAD的MIC(使用各种培养基)
BLAD对灰葡萄孢菌BM的抑菌圈(直径)数据,0.6%或0.9%(w/v)马铃薯葡萄糖琼脂(PDA)培养基(25℃培养3天):
在0.6%琼脂上,随BLAD的量从20μg到200μg的增加,灰葡萄孢菌的生长逐渐被抑制。在0.9%琼脂上,5mg/ml和10mg/ml的显示抑制作用不明显。
200μg/盘的BLAD对多种酵母菌的抑菌圈(直径)数据,0.9%(w/v)马铃薯葡萄糖琼脂(PDA)培养基(25℃培养3天到5天):
200μg/盘的BLAD对所有能导致食品腐败变质的酵母菌均表现明显的生长抑制。
实施例3-草莓防腐/去污染分析
BLAD处理后草莓对灰葡萄孢菌污染的敏感性研究。商业供应商提供的未处理的草莓中高达38%的草莓被污染(样品大小:十个500g箱),显示了草莓的污染问题。
分析操作-
1.准备同类匀质的代表性样品。
2.水洗并用70%v/v的乙醇去污染
3.用水冲洗两次
4.用不同浓度的BLAD处理(不加BLAD的作为对照):浸入处理溶液1分钟后,保持在培养皿中干燥(培养皿中含无菌水处理的湿海绵,过滤纸和塑料网丝)。
6.用10μl,1-5x106孢子/ml的灰葡萄孢菌孢子溶液培养每个草莓。
7.在以下的环境中培养5天:
温度:25℃(+/-3℃)
相对湿度:80-90%
光照:免受直射阳光
8.隔特定时间间隔,收集1g样品并用涡旋匀浆。
9.递减稀释处理
l0.分散于固体培养基并做菌落计数
所有操作技术在无菌条件下进行,采用高压灭菌材料。
以下数据显示与灰葡萄孢菌孢子溶液处理培养后,被灰葡萄孢菌污染的草莓的比例:
浓度150μg/ml到350μg/ml的BLAD显著地延迟了灰葡萄孢菌的污染的开始,并与对照组相比被污染的草莓的总比例数减少。
实施例4-BLAD在豚鼠中的皮肤毒性研究
里斯本理工大学兽医系高等农学院以Instituto Superior de Agronomia的名义(2006年6月18日-2006年8月1日)进行了保密研究,根据国际经济合作与发展组织(OECD)化学物质测试指南,No.402,急性皮肤毒性。该实验室按照实验室管理规范和动物福利进行实施的。
分别将单独剂量的BLAD给豚鼠给药之后,检测其急性皮肤毒性,豚鼠为皮肤毒性研究中广泛接受的合适的实验动物。将200μg/ml和400μg/ml的BLAD分别对两组实验动物中给药,每组10只。BLAD给药之后监测实验动物15天,并记录包括体重,病状和死亡数。
材料和方法:
1.材料
试验项目:5mg/ml的BLAD(淡黄色不透明液体,0-4℃)并在-80℃储存。
实验动物:白化豚鼠;品系:顿金哈德莱豚鼠(HsdPoc:DH)(来源:Harlan Iberica,巴塞罗那)。
使用动物数量:30;动物体重:400-449克;动物年龄:6周。
动物住所:所述动物单独的置于含灭菌木屑的聚乙烯箱(Lignocel)。
周围环境:
a)光照期:采用每12小时进行光照/黑暗周期循环。
b)受控的环境:平均温度19/22℃和平均湿度60%。
适应:实验开始前将所述动物在实验环境条件下保持7天。
食物:2014全球饮食,巴塞罗那Harlan Iberica提供的啮齿类动物常规饮食;水任意。
2方法
给药:试验前48小时将动物剃光,仅选取无皮肤损伤的动物用于试验。用1ml小份(浓度为200μg/ml或400μg/ml)施用于每只动物的剃光的皮肤上。
实验设计:试验的30只动物分成4组,其中两组每组10只动物,另外两组每组5只动物。其中一组10只动物的组暴露在200μg/ml的BLAD下(实验组1),另外一组10只动物的组暴露在400μg/ml的BLAD下(实验组2)。每组5只动物的两组作为对照:一组暴露在水中(1ml量)同时另一组不加任何处理但与其他组同时处理。
结果:在暴露之后,每天观察所有动物15天以记录病状或甚至死亡的任何表象。其中关于病状尤其关注暴露位点的皮肤损伤的可能症状,以及一般毒性的可能表象,如正常行为模式的变化。在暴露之前和测试期结束时分别测量动物的体重。
结果:
BLAD以以上任何浓度给药后,皮肤给药区域均没有任何物理变化、或饮水/进料、或一般行为也没有发生变化。BLAD给药后没有发生不良反应或死亡。所有组中体重增加相似(并且与该青年实验动物的正常生长的预期体重增加一致)。
结论:
BLAD在直到400μg/ml(并可能更高)的浓度时不显现皮肤毒性。
实施例5-白化大鼠中BLAD的口服实验
里斯本理工大学兽医系高等农学院以Instituto Superior de Agronomia的名义进行了保密研究,根据国际经济合作与发展组织(OECD)化学物质测试指南,No.40l,急性口服毒性。该实验室按照实验室管理规范和动物福利进行实施的。
分别将单独剂量的BLAD给大鼠施用之后,分别检测其急性皮肤毒性,大鼠为口服毒性研究中广泛接受的合适的实验动物。将200μg/ml和400μg/ml的BLAD分别采用填喂法给两组实验动物给药,每组10只。给药之后监测观察实验动物15天,并在此期间记录体重,病状和死亡数。观察期之后,将动物安乐处死并进行解剖。
材料和方法-
1材料
试验项目:5mg/ml的BLAD(浅黄色不透明液体,0-4℃)并在-80℃储藏。
动物:褐家鼠;品种:Wistar Hannover大鼠,(来源:里斯本理工大学兽医系饲养室从巴塞罗那Harlan Iberica获得)
使用动物数量:30;动物的体重:250-300g;动物的年龄:10周。
动物住所:所述动物单独的放置在含灭菌木屑的聚乙烯箱子中(Lignocel)。
周围环境:
a)光周期:采用每12小时进行光照/黑暗周期循环。
b)受控的环境:平均温度19/22℃和平均湿度60%。
适应:实验开始前将所述动物在实验环境条件下保持7天。
食物:(2014全球饮食,巴塞罗那Harlan Iberica提供的啮齿类动物常规饮食;水任意。
2方法
给药:将1ml(浓度为200μg/ml或者400μg/ml)的药物以插管口服(口-食管)的方式,也即填喂法给实验动物给药,采用适合该动物品种的金属探针进行给药实验。实验动物在给药之前需18小时禁食,给药之后3小时进料。
实验设计:试验的30只动物分成4组,其中两组每组10只动物,另外两组每组5只动物。其中一组10只动物的组暴露在200μg/ml的BLAD下(实验组1),另外一组10只动物的组暴露在400μg/ml的BLAD下(实验组2)。每组5只动物的两组作为对照:一组暴露在水中(1ml量)同时另一组不加任何处理但与其他组同时处理。
结果:在暴露之后,每天观察所有动物15天以记录病状或甚至死亡的任何表象。在暴露之前和测试期结束时分别测量动物的体重。观察期之后,将动物安乐处死(通过饱和二氧化碳环境窒息致死)随后进行解剖实验。
结果:
BLAD以以上任何浓度给药后,没有任何物理变化表征、或饮水/进料、或一般行为也均没有发生变化。BLAD给药后没有发生不良反应或死亡。所有组中体重增加相似(并且与该青年实验动物的正常生长的预期体重增加一致)。此外胸腔和腹腔通过解剖实验/肉眼观察组织均未发生变化。
结论:BLAD在直到400μg/ml(并可能更高)的浓度时不显现口服毒性。
Claims (9)
1.一种包含抗菌多肽的组合物在防止或抑制微生物引起的食品腐败变质中的应用,其中所述抗菌多肽含Blad或其活性变体。
2.根据权利要求1所述的应用,其中所述微生物位细菌或真菌。
3.根据权利要求2所述的应用,其中所述细菌是选自假单胞菌或芽孢杆菌的食品腐败种类细菌。
4.根据权利要求2所述的应用,其中所述真菌是选自以下种属的食品腐败种类真菌:链格孢菌、曲霉菌、镰刀霉、葡萄孢菌、炭疽菌、酵母、克鲁维酵母和接合酵母。
5.根据前述任意一项权利要求所述的应用,其中所述食品为源自、提供、或为,水果、坚果、蔬菜、种子、糖、奶制品、液态或糊状食品、肉类、鱼类或面包。
6.根据前述任意一项权利要求所述的应用,其中所述食品为草莓。
7.根据权利要求6所述的应用,其中所述微生物是灰葡萄孢菌或尖孢炭疽菌,优选的为灰葡萄孢菌。
8.根据前述任意一项权利要求所述的应用,其中所述组合物进一步包括螯合剂。
9.一种防止或抑制微生物引起的食品腐败变质的方法,包括给有需要的食品使用含有有效剂量抗菌多肽的组合物,所述抗菌多肽含Blad或其活性变体。
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PCT/EP2011/067821 WO2012049213A1 (en) | 2010-10-12 | 2011-10-12 | Use of a composition comprising an antimicrobial peptide as a food preservative |
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