柠檬香蜂草精油的气相色谱-质谱联用分析及抑菌活性研究

郭峰,董明辉,高梦园,舒方,孙冬冬,汪维云*

(安徽农业大学 生命科学学院,安徽 合肥,230036)

摘 要 该文采用水蒸气蒸馏萃取法(steam distillation, SD)对柠檬香蜂草提取精油,气相色谱-质谱联用(gas chromatograph-mass spectrometry, GC-MS)分析精油成分,制备成不同体积分数稀释液,通过纸片扩散法(kirby-bauer, K-B)测定柠檬香蜂草精油对3种食源性致病菌(金黄色葡萄球菌、大肠杆菌、四联球菌)的药物敏感性,筛选敏感菌株,最后稀释涂布平板法测定半数抑制浓度(50% inhibitory concentration, IC50)。测得柠檬香蜂草精油中脂肪族占25.91%,芳香族占20.16%,萜类占53.93%。且其对金黄色葡萄球菌抑制作用最弱,对四联球菌抑制作用最强,在实验浓度范围内,抑制强度随精油浓度升高而升高,经计算,柠檬香蜂草精油对四联球菌的IC50为457 μg/mL。最终判断柠檬香蜂草精油对四联球菌有较好抑制作用,且起抑菌作用的物质为β-石竹烯、香茅醇和柠檬醛。

关键词 柠檬香蜂草精油;四联球菌;大肠杆菌;金黄色葡萄球菌;气相色谱-质谱联用(gas chromatography-mass spectrometry,GC-MS)

作为现代食品工业的灵魂,食品添加剂对于食物在改善口感、提升质量、延长保质期等方面是必不可少的,化学食品添加剂常被广泛使用于食品生产,但这种食品添加剂的安全隐患问题一直存在,因而寻找天然食品添加剂代替化学食品添加剂已成为食品安全关注的焦点[1-2]。植物精油作为一类具有广谱抗细菌、抗真菌以及抗氧化等作用且安全、低毒、高效的植物源食品保鲜剂,逐渐开始进入大众的视野[3-5]

柠檬香蜂草(Melissa officinalis L.)又称蜜蜂花、蜂香脂、皱叶薄荷,原产于南欧和地中海地区,唇形科多年草本植物,因其具有清新的柠檬香气和清爽香甜的口感,常被作为调味料用在食品及饮料中[6-7]。此外,作为一种享有声誉的药食两用植物,柠檬香蜂草曾有被制成汤剂用于治疗银屑病的记载[8],以及将柠檬香蜂草与其他药草混合食用用于预防痴呆、延缓记忆障碍的记载[9]。现有大量研究证明柠檬香蜂草精油还可作为抗菌剂、抗氧化剂、抗炎剂、镇静剂等用于治疗各种疾病,且不同的柠檬香蜂草精油表现出对包含芽孢杆菌、金黄色葡萄球菌、大肠杆菌、铜绿假单胞菌在内的多种微生物的抑菌效果[6]。清新的味道、良好的功效、低毒性和易于种植的特点使得柠檬香蜂草精油具有成为理想的天然食品添加剂的潜质[10-11]

基于不同的柠檬香蜂草精油成分受产地与溶剂的影响[12-13],本研究采用水蒸气蒸馏萃取法(steam distillation,SD)提取国产柠檬香蜂草的精油,纸片扩散法(kirby-bauer,K-B)初步筛选敏感菌株,稀释涂布平板法测定半数抑制浓度(50% inhibitory concentration,IC50),初步判断出该精油的抑菌能力,气相色谱-质谱联用(gas chromatography-mass spectrometry,GC-MS)分析该精油组成成分,结合目前研究结果分析该精油中的抑菌成分。旨在为柠檬香蜂草精油在食品加工领域的开发应用提供新的思路。

1 材料与方法

1.1 试验材料

1.1.1 材料与试剂

新鲜柠檬香蜂草(市售),经鉴定为唇形科薄荷属柠檬香蜂草(Melissa officinalis L.)全草。除去枝干,将叶片经粉碎机粉碎后备用。

NaCl、无水硫酸钠、无水乙醇、乙酸乙酯(均为分析纯),天津国药集团。

1.1.2 供试菌株

大肠杆菌(Escherichia coli)、金黄色葡萄球菌(Staphylococcus aureus)、四联球菌(Micrococcus tetragenus),由安徽农业大学生命科学学院微生物实验室提供。

1.1.3 培养基的制备

蒸馏水1 000 mL;蛋白胨10 g;琼脂15~25 g;牛肉膏3 g;NaCl 5 g;调节pH至7.2~7.5,放入高压蒸汽灭菌锅内120 ℃灭菌2 h后,将培养基倒入无菌空白培养皿,待培养基冷却凝固备用。

1.2 试验方法

1.2.1 SD法提取精油

将粉碎后的柠檬香蜂草装入圆底烧瓶中,水蒸气蒸馏2 h以上得到水油混合物,用乙酸乙酯溶解后,加入无水硫酸钠静置12 h,抽滤,放入旋转蒸发仪蒸发,得到密度为0.71 g/mL的柠檬香蜂草挥发油,即柠檬香蜂草精油。

1.2.2 菌悬液的制备

蒸馏水1 000 mL;蛋白胨10 g;牛肉膏3 g;NaCl 5 g;调节pH至7.2~7.5,高压蒸汽灭菌锅120 ℃灭菌2 h。将活化后的细菌接种于该培养液中,摇床37 ℃恒温培养24 h,即得菌悬液。

1.2.3 柠檬香蜂草精油稀释液的制备

柠檬香蜂草精油以无水乙醇为溶剂,设置从10%~100%共10个浓度梯度的精油稀释液,并设置溶剂空白对照试验[13]

1.2.4 抑菌活性测定

移液枪分别吸取1 mL 3种菌悬液于固体培养基中,将菌悬液均匀涂布于培养基表面,夹取柠檬香蜂草精油稀释液滤纸片置于培养基上,设置3组平行实验,37 ℃恒温培养24 h。同时设置溶剂空白对照,通过测定抑菌圈直径大小以评估抑菌活性。

1.2.5 柠檬香蜂草精油对四联球菌IC50测定

取3 mL四联球菌菌悬液,用无菌水将其稀释,浓度梯度为10-1、10-2、10-4、10-5、10-6 CFU/mL,分别将菌液、无水乙醇、纯水按照表1加入到6根试管中作为对照组。

表1 稀释涂布对照组

Table 1 Control group of spread plate method

编号菌液/(CFU·mL-1)无水乙醇/μL纯水/μL110-4020210-5020310-6020410-4200510-5200610-6200

将菌液与柠檬香蜂草稀释液按照表2加入到20根试管中作为实验组。

将这26根试管放入摇床37 ℃恒温培养48 h,稀释涂布后,37 ℃恒温培养48 h。用平板菌落计数法计算菌落数,以抑菌率为纵坐标,柠檬香蜂草精油体积分数为横坐标绘制标准曲线,计算IC50值。

表2 稀释涂布实验组

Table 2 Experimental group of spread plate method

编号菌液/(CFU·mL-1)柠檬香蜂草精油体积分数/%710-410810-510910-4201010-5201110-4301210-5301310-1401410-2401510-1501610-2501710-1601810-260191702010-170211802210-180231902410-1902511002610-1100

2 结果与分析

2.1 柠檬香蜂草精油的GC-MS结果分析

采用GC-MS技术对柠檬香蜂草精油挥发性成分进行分析,得到柠檬香蜂草精油的总离子流图如图1所示。

图1 柠檬香蜂草挥发性成分总离子流图

Fig.1 Total ion GC-MS of the volatile compounds in leaves of Melissa officinalis L.

由图1可知,用SD法提取出的柠檬香蜂草精油含有众多不同类型的挥发物,成分复杂,根据每个峰在质谱库中进行检索,与标准谱库(NIST11)比对,设定相似度阈值(>85%)进行化合物甄别,最终共得到30种挥发性成分,解析结果如表3所示[14-15]

表3 柠檬香蜂草精油挥发性成分

Table 3 Volatile composents of essential oil from Melissa officinalis L.

名称化学式分数保留时间/minCAS相对含量/%1,6-octadien-3-ol,3,7-dimethylC11H18O287.2218.515115-99-12.86citronellalC10H18O92.7619.867106-23-02.42bicyclo[3.1.1]hept-3-en-2-ol,4,6,6-trimethyl-,[1S-(1α,2β,5α)]C10H16O86.5720.5918881-04-42.396-octen-1-ol,3,7-dimethyl-,(R)C11H20O291.0321.723105-85-12.892,6-octadienal,3,7-dimethyl-,(Z)C10H16O89.0922.1065392-40-52.392,6-octadien-1-ol,3,7-dimethyl-,(Z)C10H18O83.322.326106-25-22.42(S)-(-)-citronellic acid,methyl esterC11H20O291.4122.4261000333-55-12.892,6-octadienal,3,7-dimethyl-,(E)C10H16O86.1622.85392-40-52.392,6-octadienoic acid,3,7-dimethyl-,methyl ester,(E)C11H18O287.8124.011189-09-92.864-hexen-1-ol,5-methyl-2-(1-methylethenyl)-,acetateC12H20O289.525.24425905-14-03.08α-cubebeneC15H2490.8825.59317699-14-83.20bicyclo[5.3.0]decane,2-methylene-5-(1-methylvinyl)-8-methylC15H2491.7825.8181000159-39-33.20β-ylangeneC15H2489.126.58413744-15-53.20caryophylleneC15H2494.7726.667242794-76-93.20(+)-epi-bicyclosesquiphellandreneC15H2485.2626.8454274-73-63.20cis-β-farneseneC15H2487.1426.96428973-97-93.201,4,7,-cycloundecatriene,1,5,9,9-tetramethyl-,Z,Z,ZC15H2493.0427.441000062-61-93.201,3,6,10-dodecatetraene,3,7,11-trimethyl-,(Z,E)C15H2491.1927.73917699-05-73.20(+)-epi-bicyclosesquiphellandreneC15H2489.7227.95554274-73-63.20α-farneseneC15H2488.128.059502-61-43.20α-muuroleneC15H2491.9228.20331983-22-93.20naphthalene,1,2,4a,5,8,8a-hexahydro-4,7-dimethyl-1-(1-methylethyl)-,[1S-(1α,4aβ,8aα)]C15H2489.6628.593483-76-13.20γ-muuroleneC15H2491.7329.91330021-74-03.20caryophyllene oxideC15H24O92.5230.1231139-30-63.46tau-cadinolC15H26O90.2331.1855937-11-13.49α-cadinolC15H26O90.6631.41819912-62-03.49phthalic acid,isobutyl 4-octyl esterC20H30O489.4334.8461000314-84-75.24dibutyl phthalateC19H28O484.6136.4561000356-78-45.02pentacosaneC21H4481.2844.601629-94-74.65squaleneC30H5082.3851.438111-02-46.44

这30个挥发性成分中脂肪族化合物有8个占比为25.91%,芳香族化合物有5个占比为20.16%,萜类化合物有17个占比为53.93%。

根据表3可知,角鲨烯(squalene)的相对含量最高,可达6.44%。角鲨烯是一种天然的脂溶性三萜化合物,具有很强的携氧能力,还具有抗氧化、抗辐射、解毒、调节胆固醇代谢等生物活性,所以被广泛运用在化妆品和医疗保健方面[16-17]。其他具有抗菌、抗氧化等功效的挥发性物质分别为β-石竹烯(caryophyllene oxide)3.46%、香茅醇(citronellal)2.42%、柠檬醛(2,6-octadienal, 3,7-dimethyl)2.39%。

2.2 柠檬香蜂草精油抑菌实验结果分析

按照1.2.5中抑菌性测定方法,以无水乙醇溶剂空白试验为参照,采用K-B法进行抑菌活性测定,以抑菌圈直径大小评估抑菌活性。抑菌圈直径>20 mm 时为极敏;16~20 mm为高敏;11~15 mm为中敏;8~10 mm为低敏;<8 mm为不敏感。以无水乙醇为溶剂的柠檬香蜂草精油对金黄色葡萄球菌、大肠杆菌、四联球菌的抑菌效果如表4所示[14]

表4 柠檬香蜂草精油对3种待试菌的抑菌活性

Table 4 Antimicrobial activities of essential oil from Melissa officinalis L.against 3 tested microbes

注:试验用滤纸片直径为6 mm,抑菌圈直径以滤纸片中心为原点计算

编号金黄色葡萄球菌/mm大肠杆菌/mm四联球菌/mm162326231419

由表4可知,柠檬香蜂草精油对这3种细菌的抑制效果由强到弱为四联球菌>大肠杆菌>金黄色葡萄球菌。抑菌圈直径最大达26 mm,且在实验浓度范围内随着精油稀释液浓度的升高,抑菌圈直径增大,抑菌能力增强。

2.3 柠檬香蜂草精油对四联球菌IC50值测定

本研究采用平板菌落计数法,检测了10个不同浓度的柠檬香蜂草精油对四联球菌的抑菌活性,并以概率单位为纵坐标,样品不同稀释液体积分数对数为横坐标,绘制曲线图,计算IC50值为457 μg/mL。半数抑菌浓度越小,样品的抑菌活性越高,从该结果可知,柠檬香蜂草精油对四联球菌有较好的抑制效果。

3 讨论

从柠檬香蜂草精油GC-MS分析结果可以看出,柠檬香蜂草精油中萜类化合物占比较大,可达50%以上,其中角鲨烯的相对含量在所有化合物中最高;从柠檬香蜂草精油的抑菌实验结果可以看出,该精油对于四联球菌有着较好的抑制作用,且通过该作用能进一步延缓食物腐败。结合王静等[18-19]、刘晓生等[20]对柠檬香蜂草精油和精气的挥发性成分分析结果,可初步判断在该柠檬香蜂草精油中,对四联球菌的生长繁殖起抑制作用的挥发性物质是β-石竹烯、香茅醇和柠檬醛。

4 结论

本研究通过SD法制备柠檬香蜂草精油,以无水乙醇为溶剂配制不同浓度的柠檬香蜂草稀释液。通过K-B法测定不同体积分数的稀释液对于金黄色葡萄球菌、大肠杆菌和四联球菌抑菌效果,筛选敏感菌株。该实验结果显示,柠檬香蜂草精油对于四联球菌的抑制作用最好,且在实验浓度范围内,随稀释液体积分数增加,抑制作用也随之增强,而对于金黄色葡萄球菌则几乎没有抑制作用。通过稀释涂布平板法测得柠檬香蜂草精油对四联球菌的IC50为457 μg/mL。通过GC-MS对柠檬香蜂草精油中挥发性成分进行分析,可确定精油中各类化合物占比,初步筛选出柠檬香蜂草精油中对四联球菌具有抑制作用的化合物。本研究结果为研发绿色健康的食物添加剂提供了更多理论基础,拓宽了研究思路。

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GC-MS analysis of lemon balm essential oil and its anti-pathogen activity

GUO Feng,DONG Minghui,GAO Mengyuan,SHU Fang,SUN Dongdong,WANG Weiyun*

(College of Life Sciences, Anhui Agricultural University, Hefei 230036, China)

ABSTRACT To expand natural food additives in replacement of chemicals, steam distillation was applied to extract essential oil of lemon balm, followed by gas chromatography-mass spectrometry(GC-MS) to clarify components of the essential oil. After dilution, an antimicrobial screening was performed with three pathogenic bacteria (Staphylococcus aureus, Escherichia coli, Micrococcus tetragenus) using kirby-bauer method. IC50 was determined by dilution plating procedure subsequently. The results showed that the 25.91% of lemon balm essential oil were aliphatic compounds, 20.16% were aromatic compounds and 53.93% were terpenoids. The lemon balm essential oil showed the weakest inhibition on S. aureus and the strongest inhibition on M. tetragenus. The inhibition intensity increased with the concentration of essential oil. The IC50 of lemon balm essential oil to M. tetragenus was 457 μg/mL. The essential oil of lemon balm had strong inhibitory effect on M. tetragenus, and the antimicrobial components were caryophyllene oxide, citronellal and 2,6-octadienal, 3,7-dimethyl.

Key words lemon balm essential oil; Staphylococcus aureus; Escherichia coli; Micrococcus tetragenus; gas chromatography-mass spectrometry(GC-MS)

DOI:10.13995/j.cnki.11-1802/ts.024679

引用格式:郭峰,董明辉,高梦园,等.柠檬香蜂草精油的气相色谱-质谱联用分析及抑菌活性研究[J].食品与发酵工业,2021,47(2):109-113.GUO Feng,DONG Minghui,GAO Mengyuan, et al.GC-MS analysis of lemon balm essential oil and its anti-pathogen activity[J].Food and Fermentation Industries,2021,47(2):109-113.

第一作者:博士研究生,讲师(汪维云教授为通讯作者,E-mail:weiyunw@ahau.edu.cn)

收稿日期:2020-06-08,改回日期:2020-09-01