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0 引言
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霉菌病害是馆藏文物,如棉、麻、毛、丝、纸质、竹木、皮革等的主要微生物病害之一。霉菌以有机质文物为养分,发达的菌丝体覆盖文物,新陈代谢产物有机酸会腐蚀文物,还会代谢出色素污染文物,还能产生消化分解有机物质的纤维素酶,致使文物受到损坏。因此,防治文物霉菌病害,研发天然、安全、环境友好型的抗菌防霉剂成了国内外文物保护工作者的研究热点。
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植物精油又称为香精油、挥发油,是萃取植物特有的芳香味儿油状液体,取自于草本植物的花、叶、根、树皮、果实、种子、树脂等,以蒸馏、压榨和溶剂萃取等方式提炼出来[1]。植物精油具有多种生物学活性,如抗菌、抗氧化、杀虫等,在食品、制药、香料等行业具有广泛的应用。大量研究表明,植物精油具有广谱的抗微生物活性,能抑杀细菌、真菌和病毒[2]。植物精油及其活性成分对真菌的抑制作用表现在抑制真菌的生长和真菌毒素合成[3-5]。
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本工作研究了罗勒、香叶和山苍子三种精油对供试霉菌的抑菌活性,采用GC-MS分析了三种精油的主要化学成分,以期为植物精油及活性成分作为新型绿色防霉抑菌剂在博物馆馆藏文物的微生物病害防治工作中应用提供新的思路和参考依据。
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1 材料与方法
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1.1 材料与试剂
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罗勒精油、香叶精油、山苍子精油(上海维塔化学试剂有限公司)。纱纸(广西贡川),宣纸(安徽泾县),A4纸。
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霉菌液体培养基:北京索莱宝科技有限公司;葡萄糖琼脂(PDA)固体培养基:北京陆桥技术股份有限公司;蛋白胨:北京索莱宝科技有限公司。
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1.2 仪器与设备
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真菌提取试剂盒购自天根生化科技(北京)有限公司,引物由生工生物工程(上海)股份有限公司合成,DNA聚合酶购自宝日医生物技术(北京)有限公司。所用仪器名称和型号如表1。
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1.3 方法
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1.3.1 真菌分离
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霉菌的分离、纯化用沙氏液体培养基和葡萄糖琼脂(PDA)固体培养基。培养基分别灭菌后备用。
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霉菌的取样采用自然沉降法[6]。从展厅带回的平板置于28℃、70%RH的培养箱中培养5~6d。提取优势菌落进行多次划线,直至得到纯菌落。
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1.3.2 真菌DNA的提取
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将平板上纯化的单菌落接种于液体培养基中,置于28℃振荡器中180r·min-1培养。将过滤后的菌丝体烘干,使用液氮研磨后采用天根试剂盒提取霉菌DNA。将获得的DNA用1%的琼脂糖凝胶电泳检测。
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1.3.3 真菌ITS
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基因片段的PCR扩增及测序采用真菌核糖体rDNA转录间隔区(ITS)通用引物ITS1和ITS4,对提取的基因组DNA进行PCR扩增,真菌ITS基因片段扩增的引物序列[7-8]为ITS1,5’-TCC GTA GGT GAA CCT GCG G-3’和ITS4,5’-TCC TCC GCT TAT TGA TAT GC-3’。
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PCR循环参数:94℃预变性5min;PCR变性94℃ 30s,53℃退火30s,72℃延伸45s,35个循环;72℃延伸5min。PCR扩增体系如下:Premix PCR Taq酶12.5 μL,浓度为10 μmol/L的引物ITS1、ITS4各0.5 μL,模板DNA 2 μL,ddH2O 9.5 μL,共25 μL。PCR产物与载体连接克隆,挑取阳性克隆子测序,测序由生工生物工程(上海)股份有限公司完成。
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1.3.4 真菌18S rDNA序列分析
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测序结果经BLAST与NCBI数据库中已知序列进行同源性比较,根据比较结果,结合形态学特征,鉴定霉菌。
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1.3.5 抑菌活性试验
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孢子悬液、试验平板的制备和抑菌圈大小的测量见参考文献[9]。
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1.3.6 植物精油最低抑菌浓度(MIC)和最小杀菌浓度(MBC)的测定
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在5mL的霉菌液体培养基中加入100 μL孢子悬液。将无水乙醇稀释成10%的精油添加到试管中,使植物精油稀释成不同浓度,用无水乙醇做对照。试管置于180r/min 28℃的恒温振荡器中进行培养,120h后观察生长情况,不长菌的最小植物精油浓度定义为该植物精油的最低抑菌浓度(MIC)。MBC为在MIC基础上吸取100 μL培养液进行涂布,培养后完全无菌生长的最低精油浓度。实验处理重复3次。
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1.3.7 植物精油用于纸张防霉性评价
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将3 000 μL/L的待试精油分别均匀喷于纱纸、宣纸和A4纸的表面并密封,30min后取出,待精油晾干后将孢子悬液均匀喷涂待试纸张,然后将其置于温度28℃,95%RH的恒温恒湿箱内,培养28d,记录试样纸长霉情况。
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1.3.8 香叶精油的GC-MS分析
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色谱条件:HP-5MS 30m×0.25mm×0.25 μm弹性石英毛细管柱;载气为高纯氦气。
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程序升温:初始柱温50℃,保持2min,然后以5℃/min速率升至250℃(41min)。进样口温度230℃;进样量为1 μL;分流比为100∶1。
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质谱条件:EI离子源,电子能量70eV,离子源温度230℃,四极杆温度150℃,接口温度280℃;扫描范围为29~350u;溶剂延迟3min。
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数据库:采用NIST 08谱库检索。
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1.3.9 罗勒和山苍子精油的GC-MS分析
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色谱条件:毛细管色谱柱HP-35MS 30m×0.25mm,0.25 μm,载气为氦气。
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程序升温:初始柱温50℃,保持5min,然后以8℃/min升温速率升至290℃,保持5min,进样口温度280℃,载气为高纯氦气,流速1.0mL/min,进样量为1 μL,分流比50∶1。
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质谱条件:EI离子源,电子能量70eV。离子源温度300℃,MS四极杆温度,质谱接口温度为250℃,扫描范围为35~400u,溶剂延迟5min。
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数据库:采用标准谱库NIST2014检索。
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2 结果与讨论
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2.1 霉菌的分离纯化结果
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2.1.1 霉菌形态观察
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菌株AM01在PDA培养基上(图1A-1)28℃培养7d,直径50~60mm,菌落平坦或近于平坦,质地绒状;菌丝初为白色后逐渐变为暗绿色,分生孢子易脱落,分生孢子结构大量产生,分生孢子面深橄榄绿色,渗出液缺乏,反面近于无色,可溶性色素缺乏。
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显微镜下观察青霉菌AM01的分生孢子结构如图1A-2所示,分生孢子梗发生于基质,壁平滑,帚状枝通常双轮生,偶有三轮生或单轮生;梗基每轮2~4个,彼此通常较紧贴;瓶梗每轮5~8个或更多,幼龄时呈现瓶状或披针状,成熟时近圆柱状,梗颈通常明显;分生孢子见图1A-3,分生孢子典型的椭圆形,光滑;分生孢子集成链圆柱状。
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菌株AM02在PDA培养基(图1B-1)28℃培养7d,直径25~40mm,中部较厚而其他部分平坦,或有不规则的皱纹;质地绒状至絮状;菌丝体黄色;分生孢子结构较少或较多,分生孢子面绿色、灰绿色,近于灰橄榄绿色;渗出液缺乏;反面黄绿色或黄褐色;可溶性色素类似反面颜色。
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图1 博物馆空气分离获得的霉菌在PDA培养基上的菌落及形态特征
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Fig.1 Filamentous growth and morphological characteristics of airborne mould isolated from museum on PDA solid media
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显微镜下观察AM02的分生孢子结构如图1B-2所示。分生孢子梗发生于基质和气生菌丝,帚状枝典型双轮生,偶有单轮生或不规则者;梗基每轮5~8个或更多,彼此紧贴且长短基本一致;瓶梗每轮5~10个,瓶状,梗颈短;分生孢子见图1B-3,分生孢子通常呈现椭圆形,壁平滑或稍粗糙;分生孢子链疏松而不规则纠缠。
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菌株AM03在PDA培养基上(图1C-1)生长迅速,28℃培养3d菌落直径达6.0~8.5cm,菌落质地疏松平坦、呈丝绒状、无突起,菌落呈圆环状,气生菌丝较多,中心下凹、四周突起、边缘平坦、背面平坦呈放射状,生长初期呈白色,后期表面产生深褐色干粉,中心呈黑褐色至黑色、菌落质地紧密平坦、呈丝绒状、无突起,菌落中心呈黑色、四周黄至深褐色,菌落周围有一圈白色菌丝,背面乳白色,菌落表面有干粉,无渗出液。
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显微镜下观察AM03的分生孢子结构如图1C-2所示。AM03顶囊初生时为球形或辐射形,后呈球形或椭圆形;孢梗茎无色或淡褐色,表面平滑;分生孢子见图1C-3,分生孢子圆或椭圆形淡褐色,表面粗糙有刺突,串生于小梗顶端,小梗单层,全部表面可育。
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根据以上菌落培养形态和显微形态特征,参照《真菌鉴定手册》和《中国真菌志》[10-12]进行对比分析,菌株AM01和AM02符合青霉属的生长特性,可初步鉴定两株菌为青霉属(Penicillium)。菌株AM03符合曲霉属的生长特性,可初步鉴定该菌株为曲霉属(Aspergillus)。
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2.1.2 分子生物学鉴定结果
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对获得的3株真菌的ITS序列测序,得到大小合适的序列3条,提交至NCBI数据库,获得序列号MK811098、MK811099和MK811100。获得的ITS序列在GenBank中进行BLAST序列比对,所有序列对应Genbank中已知的参考序列的同源性均大于99%,确定了所得序列相似度最高的序列所属科属、物种等信息,具体比对结果见表2。因此,形态学特征分析和rDNA-ITS序列分析,确定分离的三株霉菌为草酸青霉菌(Penicillium oxalicum),梅花状青霉菌(Penicillium herquei),棘孢曲霉菌(Aspergillus aculeatus)。
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2.2 植物精油对霉菌的抑菌效果
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测得的抑菌圈大小结果如表3、图2所示。抑菌圈直径＞20mm为极敏感,10~20mm为中度敏感,5~10mm为低度敏感,无抑制作用者(≤5mm)为不敏感[13]。由表3可知,三种精油对供试霉菌的抑菌圈直径均大于20mm,表明供试霉菌对精油的抑菌敏感性为极度敏感,即罗勒精油、香叶精油和山苍子精油对三种霉菌的抗菌效果明显。图2可知,植物精油对霉菌的抑制作用除了抑制菌丝的生长还能抑菌霉菌的孢子生成。
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注:实验结果表示为平均值±标准差,孔径(5mm)包含在测量结果中,P＜0.01。“—”表示平皿不长菌或仅有平皿边缘有少量菌落。
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图2 抑菌圈照片
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Fig.2 Photos of inhibition zone test
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2.3 MIC和MBC的测定
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罗勒精油、香叶精油和山苍子精油对供试霉菌的MIC和MBC测定结果见表4。MIC和MBC值越低,表明该精油的抗菌效力越强。由表2可知,同一种霉菌对不同精油的抑菌敏感性不同,同一种精油对不同霉菌的抑菌活性也不同,综合抗菌效果最好的是香叶精油,MIC值均≤600 μL/L,其次是罗勒精油和山苍子精油。
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2.4 植物精油用于纸张的防霉效果
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精油处理过的待试纸张和对照组纸张在恒温恒湿箱培养箱中连续培养28d后,对纸张表面霉变情况进行观察并记录,结果见表5。
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由表5可知,对照组的宣纸、A4纸和纱纸均发生了明显霉变,其中霉变较严重的是A4纸,霉变面积达到20%~50%,宣纸和纱纸次之。A4纸处理组的霉变面积小于其对照组。宣纸对照组霉变面积为20%~30%,其试验组中山苍子精油组霉变情况较为严重,霉变面积约为10%,对照组霉变面积大于试验组。待试纸样中霉变较轻的是纱纸,霉变面积小于5%,低于对照组的10%~20%。所有处理组的纸张霉变程度均比对照组小,精油具有增强纸张防霉效果的作用。
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2.5 植物精油抗菌成分GC-MS分析
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按照前文条件,对三种精油分别检测后进行分析,以峰面积归一化法测定各成分的相对含量。香叶精油总离子流谱图共显示56个峰(图3),经质谱库检索与标准谱图共同对照分析,共鉴定出23种化合物,其含量占色谱总流出峰相对百分含量的98.39%,结果见表6。罗勒精油总离子流谱图见图4所示,共鉴定出12种化合物,其含量占色谱总流出峰相对百分含量的99.77%,结果见表7。山苍子精油总离子流谱图见图5,共鉴定出44种化合物,其含量占色谱总流出峰相对百分含量的98.07%,结果见表8。
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图3 香叶精油GC-MS分析总离子流谱图
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Fig.3 The TIC of GC-MS of essential oil from geranium
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图4 罗勒精油GC-MS分析总离子流图谱
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Fig.4 TIC of GC-MS of essential oil from basil
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(续表8)
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图5 山苍子精油GC-MS分析总离子流图谱
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Fig.5 TIC of GC-MS of essential oil from Litsea cubeba
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由表6可知,从香叶精油中分离出的挥发性化合物中,相对含量最高的是香叶醇(56.73%),其余相对含量较高的依次为橙花醇(38.31%),α-柠檬醛(0.71%),约占总化学成分的95.75%。由表7可知,罗勒精油中相对含量最高的是丁香酚(83.14%),其次是己二醇(16.15%),约占鉴定出化学成分的99.29%。由表8可知,山苍子精油中相对含量最高的是柠檬醛(36.01%),其余相对含量较高的依次为(Z)-3,7-二甲基-2,6-辛二烯醛(29.71%),柠檬烯(16.67%),桉树醇(2.10%),香茅醛(1.41%),石竹烯(1.33%),芳樟醇(1.21%),6-甲基-5-庚烯-2-酮(1.17%),约占鉴定出化学成分的89.61%。
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精油的成分受很多因素影响,不同产地来源[14-16]、不同提取方法[17],不同叶龄[18],不同季节[19],不同器官、不同品种、采集时间、加工方法、样品储藏方式、储藏时间和不同检测仪器设备[20]等因素都会影响精油的含油率和组分。由表6~8可见,经GC-MS分析鉴定,本研究中使用的香叶、罗勒和山苍子精油主要化学成分别为醇类、酚类和醛类物质。
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近年来,植物精油或其他植物源提取物受到了文物保护工作者的广泛关注。国内多家单位均进行了植物精油抑菌防霉、杀虫等的应用研究。唐欢[21]等利用5种植物源提取物,评价植物源提取物在不同使用剂量下对博物馆展厅空气来源细菌的抑制效果,实验结果表明,植物源提取物抑菌效果良好。中国罗勒油、香叶油、山苍子油等均被我国《食品安全国家标准食品添加剂使用标准》列入允许使用的食品天然香料名单[22],也就是说在正常使用的剂量范围内,三种精油不会对人体健康和生命安全造成威胁。但植物源提取物对文物本体的安全性是值得考虑的。王克华等[23]研究了柠檬醛、肉桂醛和香茅醛三种植物成分的防霉活性及其对纸张、颜料的影响,结果表明三种化合物对球毛壳、黑曲霉和橘青霉有很好的抑制作用,但对文物材质的影响主要表现在色差上,香茅醛不会造成纸张、颜料的色差改变,柠檬醛对纸张的的色差基本没有影响、对颜料则存在一定程度影响,而肉桂醛则会对纸张及颜料都存在一定程度的影响。由此,植物精油等植物源提取物对文物材质的强度和颜料的色差影响等还需进行相关研究。
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3 结论
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经形态学特征分析和分子生物学鉴定,从广西民族博物馆空气中分离的三株霉菌为草酸青霉菌(Penicillium oxalicum),梅花状青霉菌(Penicillium herquei),棘孢曲霉菌(Aspergillus aculeatus)。比较罗勒精油、香叶精油、山苍子精油对三种霉菌的抑制作用,发现三种精油的抑菌效果均较明显,抑菌效果大小依次为:香叶精油＞罗勒精油＞山苍子精油,综合抗菌效果最好的是香叶精油。精油用于纸张抗霉菌具有明显效果。经GC-MS分析,罗勒精油、香叶精油和山苍子精油的主要活性成分分别为丁香油酚、香叶醇和柠檬醛,精油对霉菌的抗菌活性与精油含有的主要化学成分有关,三种精油对霉菌的抑菌机理仍需进一步研究。植物精油对霉菌的抑菌研究结果表明,精油在博物馆馆藏文物的展存环境微生物污染霉菌的防治中具有良好的开发潜力。
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摘要
从广西民族博物馆易滋生霉菌的展柜里分离、纯化及鉴定了三株霉菌,并研究香叶精油、罗勒精油和山苍子精油对霉菌的抑菌效果,测试这些植物精油喷洒于纸张表面后纸样的防霉性及鉴定三种精油的主要抑菌活性成分。实验结果显示,三种供试植物精油对霉菌均具有显著的抑菌效果,在纸张上的防霉效果也很明显,其综合抑菌效果大小为:香叶精油＞罗勒精油＞山苍子精油。GC-MS分析表明,香叶精油的主要活性成分为:香叶醇(56.73%)、橙花醇(38.31%)。罗勒精油的主要活性成分为:丁香酚(83.14%),己二醇(16.15%)。山苍子精油的主要活性成分为:柠檬醛(36.01%),(Z)-3,7-二甲基-2,6-辛二烯醛(29.71%)和柠檬烯(16.67%)。植物精油作为博物馆展存环境的空气防霉剂具有良好的应用前景。
Abstract
Cultural relics are reminders of the precious cultural heritage of mankind. Microorganisms, such as fungi, can cause severe destruction to items in museums. The impact of fungal activities on the deterioration of cultural relics is a serious problem and thus the preservation of cultural relics is a challenging task. Physical treatments such as ultraviolet radiation and γ-radiation can induce deterioration of microorganisms, but they can also affect most heritage materials. Physical treatments have short-term effects, and the surfaces of cultural relics are readily recolonized. Furthmore, many chemical compounds (biocides) are not used for antifungal because of their possible harm to human body and their fungal resistance.In the light of these problems, there is a growing need to develop effective alternatives. The search for natural and environmentally-friendly alternatives, which show negligible toxicity to human, was a main goal for the present studies. As extracts of aromatic plants, essential oils (EOs) have many advantages: 1) having a wide range of antifungal efficacy; 2) being easy to use and extract from a wide variety of sources; 3) having strong volatility and being environmentally-friendly. Botanical fungicides based on essential oils are undoubtedly popular to prevent fungal growth and control fungal decay on culture relics.Three frequently-occurring airborne fungal species from exhibition halls in Anthropology Museum of Guangxi were isolated, purified and identified. The antifungal activities of basil, myrcia and Litsea cubeba essential oils were examined against the three fungal species and also compared to determine the potential uses of plant essential oils as novel antifungal agents. Isolation, morphological identification and molecular identification were carried out for highly frequent species. With a drilling method and according to minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC), the essential oils were evaluated for their efficacy in reducing the fungi isolated in museum air. The anti-fungal activities of these essential oils were also tested on paper. After uniform spraying of plant essential oils on the surface of paper, the paper was incubated in a constant temperature and humidity incubator for 28 days. Then, the mildewed area on the surface of paper samples in the treatment group and the control group were observed. In addition, the major components of these essential oils were analyzed and identified using gas chromatography-mass spectrometry (GC/MS).The three fungi species were identified as Penicillium oxalicum (the accession number MK811098), Penicillium herquei (the accession number MK811099) and Aspergillus aculeatus (the accession number MK811100). They were susceptible to the tested essential oils. According to antifungal activity, MIC and MBC of the tested essential oils with different concentrations, the most efficient one was myrcia oil (MBC ranging from 700~900 μL/L) followed by basil oil (MBC ranging from 700~1000 μL/L) and Litsea cubeba oil (MBC ranging from 700~800 μL/L). The mildewed areas on the paper of the treatment group were all smaller than those of the control group, which shows that all of tested essential oils had mildew-proof activities on paper. These results suggest that basil, myrcia and Litsea cubeba oils have potential as natural antifungal agents against fungi. The antifungal activity order of the three essential oils was: myrcia oil＞basil oil＞Litsea cubeba oil. Geraniol (57%) and 2,6-Octadien-1-ol, 3,7-dimethyl-, (Z)-(38.31%) were the major active components of myrcia oil, eugenol (83.14%) and hexylene glycol (16.15%) were the major active components of basil oil, and citral (36.01%), 2,6-Octadienal,3,7-dimethyl-,(Z)-(29.71%) and D-Limonene1(6.67%) were the major active components of Litsea cubeba.The inhibitory effects of plant essential oils on moulds were studied by laboratory methods, and also verified by comparing the antifungal effects on paper. The results show that plant essential oils had significant antifungal effects. This study has indicated that, as a kind of purified agent against airborne moulds, plant essential oils have good prospects as antifungal agents for the museum storage and exhibition environment.
Keywords
Museum ； Airborne moulds ； Plant essential oil ； Antifungal activity ； Component analysis
