栀子叶、茎挥发油成分及其抑制豆腐致腐细菌作用研究
2016-07-21卫强徐飞
卫强,徐飞
(安徽新华学院 药学院,安徽 合肥,230088)
栀子叶、茎挥发油成分及其抑制豆腐致腐细菌作用研究
卫强,徐飞
(安徽新华学院 药学院,安徽 合肥,230088)
摘要研究了栀子叶、茎中挥发油的化学成分及其对豆腐的抑菌作用。采用超临界二氧化碳和环己烷、乙醚分别萃取挥发油,以气相色谱-质谱联用(GC-MS)法鉴定其化学成分,共鉴定153个化合物,栀子叶中挥发油中主要有1,2,3,4,5,6,7,8α -八氢化-1,4-二甲基-7-(1-甲基乙烯基)薁、甲苯、[1S-(1α,4α,7α)]-1,2,3,4,5,6,7,8-八氢化-1,4-二甲基-7-(1-甲基乙烯基)薁、4,8,12,15,15-五甲基双环[9.3.1]十五烷-3,7-二烯-12-醇、1,1-二乙氧基乙烷、2-乙基-1,3-二氧戊烷;栀子茎中挥发油主要有4,8,12,15,15-五甲基双环[9.3.1]十五烷-3,7-二烯-12-醇、1,2,3,4,5,6,7,8α -八氢化-1,4-二甲基-7-(1-甲基乙烯基) 薁、龙脑、1,1-二乙氧基乙烷。栀子叶(乙醚)挥发油对豆腐中菌群有较强抑制力,可使豆腐保鲜期延长6天。
关键词栀子叶、茎;挥发油;化学成分;豆腐;抑菌活性
豆腐是我国传统的大豆食品,营养丰富,富含蛋白质、不饱和脂肪酸、卵磷脂、脑磷脂等营养成分。食用豆腐具有降低胆固醇、降血压、降血糖、减少动脉硬化、预防骨质疏松、保护心血管系统等保健功能[1-2]。但是,大豆制品营养成分极易引起微生物生长繁殖,引起腐败。豆腐腐败主要是由细菌引起的,在加工过程中易受到各种细菌的污染,如坚强芽孢杆菌、屎肠球菌[3]、枯草芽孢杆菌和凝结芽孢杆菌[4]、团肠杆菌、短小芽孢杆菌、巨大芽孢杆菌和溶酪葡萄球菌[2]等大量增殖,在腐败菌中占据主导地位[5]。
栀子(GardeniajasminoidesEllis)来源于茜草科(Rubiaceae)栀子属植物,分布于我国大部分地区,其果实始载于《神农本草经》,列为中品[6],具有泻火除烦,清热利湿,凉血解毒的功能,外用消肿止痛。现代研究表明,栀子具有保肝[7-8]、抗抑郁[9]、降血糖[10]、抗氧化[11]等多种生物活性。栀子叶在我国四川、广西等地民间广泛应用,有治疗跌打损伤、血淤、烫伤等症[12]。栀子叶、茎挥发油鲜有报道,本文对其挥发油成分及抑制豆腐致腐细菌进行研究,以期寻找天然防腐有效成分。
1材料与方法
1.1材料与试剂
栀子叶、茎于2014年5月采自安徽合肥大蜀山地区。市售普通新鲜南豆腐,大小齐整,每块约100 g。坚强芽孢杆菌(Bacillusfirmus)、屎肠球菌(Enterococcusfaecium)、凝结芽孢杆菌(Bacilluscoagulans)、成团肠杆菌(Enterococcusagglomerans)、短小芽孢杆菌(Bacilluspumilus)、巨大芽孢杆菌(Bacillusmegaterium)、溶酪葡萄球菌(Staphylococcaceaecaseolyticus)、枯草芽孢杆菌(Bacillussubtilis)购于中国菌种保藏中心。所用试剂为分析纯。
1.2仪器与设备
HA221-50-01型超临界萃取设备,江苏南通华安超临界有限公司;Agilent 6890-5973N气相色谱仪,美国安捷伦公司。
1.3方法
1.3.1挥发油的提取
精密称取干燥粉碎后的栀子叶、茎各220 g装入料筒,放入超临界二氧化碳萃取釜内,设置萃取压力40 MPa,萃取温度25 ℃,萃取流量20 L/h,萃取2 h后,打开分离釜排料阀,收集萃取物。萃取物经减压蒸馏后分别以环己烷、乙醚萃取,氮气吹去溶剂,得到挥发油。叶、茎环己烷层挥发油几近无色,叶、茎乙醚层挥发油呈淡黄色,3次萃取,以油重/样品重得栀子叶环己烷、乙醚萃取挥发油得率为0.79%、0.98%,茎环己烷、乙醚萃取挥发油得率为0.33%、0.51%。
1.3.2色谱条件
色谱柱:HP-5 MS石英毛细管柱(30 m×0.25 mm,0.25 μm);升温程序:程序升温,柱起始温度为40 ℃,保持1 min后以5 ℃/min升至150 ℃,保持2 min,再以 10 ℃/min升至280 ℃,保持7 min,再以5 ℃/min升至300 ℃,保持至完成分析;载气:高纯He;进样量1 μL;不分流。
1.3.3质谱条件
电子轰击离子源;电子轰击能量60 eV;扫描范围m/z25-550;离子源温度220 ℃;接口温度280 ℃;全离子扫描。通过面积归一化法测定鉴定的化合物的相对含量,检索为NIST2011数据库。
1.3.4抑菌实验
以苯甲酸钠为对照,以蒸馏水溶解。制备苯甲酸钠、栀子叶、茎挥发油肉膏平板培养基,质量浓度(ρ)依次为3.000、1.500、0.750、0.375、0.188、0.094、0.047 mg/mL。在以上培养基上分别接种坚强芽孢杆菌、屎肠球菌、凝结芽孢杆菌、成团肠杆菌、短小芽孢杆菌、巨大芽孢杆菌、溶酪葡萄球菌、枯草芽孢杆菌,移入培养箱中37 ℃培养48 h,根据苯甲酸钠、栀子叶、茎挥发油的最小抑菌浓度(minimum inhibitory concentration, MIC)选择最优抑菌挥发油。
将栀子叶挥发油(乙醚)以喷瓶喷入新鲜豆腐(约100 g),喷入量分别为豆腐质量分数(ω)的0.05%、0.1%、0.15%;以苯甲酸钠为阳性对照组喷入质量分数(ω)为0.1%。放入保鲜袋内,置于4 ℃冰箱中。采用平板菌落计数法(参照GB4789.2—2010《食品微生物学检验菌落总数测定》),分别按照一定的稀释梯度稀释,贮藏于冰箱冷藏一定时间(0,3,6,9,12,15 d),计算菌落总数:
2结果与分析
2.1栀子叶、茎挥发油化学成分
结果见图1、表1和表2所示。由表1,表2可知,从栀子叶、茎挥发油中共鉴定出153种化学成分,主要有烃类、酯类、苯类、醇类、醛类、酮类、酸类、醚类、杂环类等。其中从栀子叶环己烷萃取挥发油以烃类成分为主,栀子叶乙醚萃取挥发油和茎环己烷、乙醚萃取挥发油均以烃类和醇类成分为主。4,8,12,15,15-五甲基双环[9.3.1]十五烷-3,7-二烯-12-醇为栀子叶乙醚萃取挥发油和茎环己烷、乙醚萃取挥发油的主要成分。
图1 栀子叶(AB)、茎(CD)中挥发油总离子流Fig.1 Total ion chromatogram of volatile oil of leaf and stem from Gardenia jasminoides Ellis
2.2抑菌实验
由表3可知,4个萃取物中,栀子叶(乙醚)挥发油对豆腐中腐生菌坚强芽孢杆菌等8种菌有较强抑制力,其MIC值范围为0.047~0.188 mg/mL;栀子叶(环己烷)挥发油、栀子茎(环己烷)挥发油和茎(乙醚)挥发油抑制力较弱,其MIC值范围为0.094~3.000 mg/mL。由表4可知,与空白组对照,在4 ℃冰箱中放置3~15 d,随着栀子叶(乙醚)挥发油添加浓度的提高,对豆腐抑菌作用明显增强。根据GB2711—2003[13]要求,散装豆腐菌落总数(CFU/ mL)≤1×105,由表4可知,豆腐自然放置超过3 d,菌落数超标;而使用栀子叶(乙醚)挥发油进行抑菌处理后,可放置9 d,菌落数在正常范围内。
续表1
序号保留时间/min分子式化合物相对含量/%叶(环)叶(醚)茎(环)茎(醚)3525.770C15H241,2,3,4,5,6,7,8α-八氢化-1,4-二甲基-7-(1-甲基乙烯基)薁 1,2,3,5,6,7,8,8α-Octahydro-1,4-dimethyl-7-(1-methylethenyl)-azulene6.960.725.894.823625.905C15H24O白菖油萜环氧化物 Calareneepoxide1.890.780.900.773725.976C15H24Oα-环氧柏木烷 α-Cedreneepoxide0.423826.560C15H24cis-α-甜没药烯 cis-α-Bisabolene0.803926.690C15H24α-石竹烯 α-Caryophyllene4027.410C15H24O氧化双环外雪松烯 Diepicedrene-1-oxide0.540.330.280.444127.621C15H24O环氧异香树烯 Isoaromadendreneepoxide0.330.244227.751C15H24O喇叭烯氧化物-(II) Ledeneoxide-(II)0.480.410.264327.843C16H34十六烷 Hexadecane0.680.574429.689C17H362,6,10-三甲基十四烷 2,6,10-Trimethyl-tetradecane0.420.810.564532.465C21H442,6,10,15-四甲基十七烷 2,6,10,15-Tetramethyl-heptadecane0.664632.931C20H32西松烯 [S-(E,Z,E,E)]-3,7,11-Trimethyl-14-(1-methylethyl)-1,3,6,10-cy-clotetradecatetraene3.624733.883C26H543-乙基-5-(2-乙基丁基)-十八烷 3-Ethyl-5-(2-ethylbutyl)-octadecane0.324834.214C20H325α,8α,9α,10α,12α-Atis-16-ene0.184934.625C18H37Cl氯代十八烷 1-Chloro-octadecane1.715035.291C16H33NO十六碳酰胺 Hexadecanamide1.651.685135.458C26H543-乙基-5-(2-乙基丁基)-十八烷 3-Ethyl-5-(2-ethylbutyl)-octadecane0.720.725237.115C18H37NO十八酰胺 Octadecanamide0.330.625336.341C21H44二十一烷 Heneicosane1.650.971.655437.185C24H50二十四烷 Tetracosane2.010.365538.008C25H52二十五烷 Pentacosane1.935638.219C31H64三十一烷 Hentriacontane0.805738.825C28H58二十八烷 Octacosane1.983.360.422.375840.898C27H56二十七烷 Heptacosane0.661.040.190.665941.082C30H50反式角鲨烯 (all-E)-2,6,10,15,19,23-Hexamethyl-2,6,10,14,18,22-tetra-cosahexaene0.14酯类605.326C9H16O2乙酸(6-庚烯-1-基)酯 6-Heptenylacetate616.398C6H12O2醋酸正丁酯 Aceticacid,butylester0.19628.801C10H20O4二乙二醇丁醚醋酸酯 2-(2-Butoxyethoxy)-ethanol,acetate0.05639.218C7H14O22-甲基戊酸甲酯 2-Methyl-pentanoicacid,methylester0.986411.513C8H16O33-羟基-4-甲基-戊酸乙酯 3-Hydroxy-4-methyl-pentanoicacid,ethylester0.180.126512.087C12H20O21-甲基-4-(1-异丙烯基)环己醇乙酯 1-Methyl-4-(1-methylethenyl)-cyclohex-anolacetate0.126617.142C8H8O3水杨酸甲酯 Methylsalicylate0.540.856717.754C15H26O2异戊酸香叶酯 Geranylisovalerate0.056819.610C12H24O43-(1-乙氧乙氧基)-2-乙基-丁酸乙酯 3-(1-Ethoxy-ethoxy)-2-ethyl-butyricacid,ethylester0.236919.968C26H50O22-辛基-环己烷十四烷酸甲酯 2-Octyl-cyclopropanetetradecanoicacid,methylester0.247023.881C16H26O23,7,11-三甲基-1,6,10-十二烷-3-基甲酸酯 Formicacid,3,7,11-trimethyl-1,6,10-dodecatrien-3-ylester0.277124.038C17H28O2醋酸橙花椒酯 Nerolidylacetate7224.260C15H24O2新戊酸-6-烯酯 Limonen-6-ol,pivalate0.427327.058C17H32O27-甲基-Z-十四烯-1-醇乙酯 7-Methyl-Z-tetradecen-1-olacetate0.390.597429.873C16H28O3Z-(13,14-环氧)十四烷基-11-烯-1-醇乙酯 Z-(13,14-Epoxy)tetradec-11-en-1-olacetate0.297531.854C16H22O4邻苯二甲酸二异丁酯 1,2-Benzenedicarboxylicacid,bis(2-methylpropyl)es-ter1.057631.929C26H42O4丁基邻苯二甲酸十四酯 Phthalicacid,butyltetradecylester7731.935C22H34O4邻苯二甲酸丁酯8-甲基壬酯 1,2-Benzenedicarboxylicacid,butyl8-methyl-nonylester0.410.98
续表1
序号保留时间/min分子式化合物相对含量/%叶(环)叶(醚)茎(环)茎(醚)7832.260C19H32O32-羟基-9,12,15-十八碳三烯酸甲酯 Methyl2-hydroxy-octadeca-9,12,15-trienoate0.607932.687C17H34O2十六酸甲酯 Hexadecanoicacid,methylester0.630.478034.316C21H36O4亚麻酸甘油酯 (Z,Z,Z)-9,12,15-Octadecatrienoicacid,2,3-dihydroxypropylester0.871.168135.399C33H54O33,5-二氢-6-甲氧基-胆甾-22-烯-21-新戊酸酯 3,5-Dehydro-6-methoxy-cho-lest-22-ene-21-ol,pivalate1.088238.273C24H38O4邻苯二甲酸二异辛酯 1,2-Benzenedicarboxylicacid,diisooctylester1.650.920.40苯类835.537C7H8甲苯 Toluene6.721.930.23847.475C8H10乙苯 Ethylbenzene1.050.750.30857.708C8H10间二甲苯 1,3-Dimethyl-benzene867.946C8H10邻二甲苯 o-Xylene0.660.610.11878.341C8H10对二甲苯 p-Xylene0.198811.210C9H121,2,3-三甲苯 1,2,3-Trimethyl-benzene8911.215C9H121-乙基-3-甲基苯 1-Ethyl-3-methyl-benzene0.069012.433C9H102,3-二氢茚 Indane9121.548C10H12O2丁香酚 Eugenol2.550.360.360.189224.352C15H26O1,2,3,4,4α,7,8,8α-八氢-1,6-二甲基-4-(1-甲基乙基)-1-萘酚 1,2,3,4,4α,7,8,8α-Octahydro-1,6-dimethyl-4-(1-methylethyl)-1-naphthalenol0.29醇类934.163C6H14O21-丙氧基-2-丙醇 1-Propoxy-2-propanol0.08944.866C7H16O2,2-二甲基-1-戊醇 2,2-Dimethyl-1-pentanol0.29957.313C6H12O(Z)-3-己烯-1-醇 (Z)-3-Hexen-1-ol2.220.31967.681C6H14O正己醇 1-Hexanol0.340.93977.881C6H14O21-(1-甲基乙氧基)异丙醇 1-(1-Methylethoxy)-2-propanol0.089811.242C6H12O56-脱氧-D-半乳糖 6-Deoxy-D-galactose0.719911.854C6H14O23-甲基-2,4-戊二醇 3-Methyl-2,4-pentanediol0.4810012.260C8H16O4α-(1-乙氧乙氧基)环氧乙醇 α-(1-Ethoxyethoxy)-oxiraneethanol0.2910112.531C9H20O23-(1-甲基丁氧基)-2-丁醇 3-(1-Methylbutoxy)-2-butanol0.4710212.568C13H18O5甲基2-O-苄基-D-阿拉伯呋喃糖苷 Methyl2-O-benzyl-D-arabinofuranoside0.5010312.861C8H18O2,4-二甲基-3-己醇 2,4-Dimethyl-3-hexanol0.0510413.505C10H18O2α-甲基-α-[4-甲基-3-戊烯基]环氧基甲醇 α-Methyl-α-[4-methyl-3-pentenyl]oxiranemethanol0.070.0910513.862C10H18O2cis-α,α-5-三甲基-5-乙烯基四氢化呋喃-2-甲醇 cis-5-Ethenyltetrahydro-α,α,5-trimethyl-2-furanmethanol0.0910614.003C9H20O23-(2,2-二甲基-丙氧基)-2-丁醇 3-(2,2-Dimethylpropoxy)-2-butanol0.3910714.349C10H18O3,7-二甲基-1,6-辛二烯-3-醇 3,7-Dimethyl-1,6-octadien-3-ol2.430.740.4110814.669C8H10O苯乙醇 Phenylethylalcohol0.181.010.6010914.918C7H16O2,4-二甲基-3-戊醇 2,4-Dimethyl-3-pentanol0.330.1811015.702C8H15ClO1-氯-3,5-二甲基-1-己烯-4-醇 1-Chloro-3,5-dimethyl-1-hexen-4-ol0.3511117.256C10H18O松油醇 (S)-α,α,4-Trimethyl-3-cyclohexene-1-methanol0.870.270.3511218.733C10H18O香叶醇 (E)-3,7-Dimethyl-2,6-octadien-1-ol0.210.2411321.916C9H16O4-环辛烯-1-甲醇 4-Cyclooctene-1-methanol0.270.1511428.135C19H36O2-甲基-E,E-3,13-十八烷二烯-1-醇 2-Methyl-E,E-3,13-octadecadien-1-ol0.2011529.158C15H26O蓝桉醇 Globulol1.500.832.381.7311629.857C15H22O3,5,6,7,8,8α-六氢-4,8α-二甲基-6-(1-羟甲基乙烯基)-(1H)-萘-2-酮 3,5,6,7,8,8α-Hexahydro-4,8α-dimethyl-6-(1-methylethenyl)-2(1H)naphthalenone0.2811729.981C15H24O22-甲基-9-(丙-1-烯-3-醇-2-基)-双环[4.4.0]癸-2-烯-4-醇 2-Methyl-9-(prop-1-en-3-ol-2-yl)-bicyclo[4.4.0]dec-2-ene-4-ol0.4111831.399C20H40O2(Z)-2-(9-十八碳烯基氧基)乙醇 (Z)-2-(9-Octadecenyloxy)-ethanol11932.189C19H34O2E,E,Z-1,3,12-十九碳三烯-5,14-二醇 E,E,Z-1,3,12-Nonadecatriene-5,14-diol0.38
续表1
注:“环”为环己烷萃取,“醚”为乙醚萃取。
表2 栀子叶、茎挥发油化学成分结果比较
表3 栀子叶、茎挥发油抗菌实验的MIC值(n=3) 单位:mg/mL
表4 栀子叶(乙醚)挥发油处理前后对豆腐中微生物中菌落数的影响
3结论
从栀子叶、茎挥发油中共鉴定出153种化学成分,主要有烃类、酯类、苯类、醇类、醛类、酮类、酸类、醚类、杂环类等。叶环己烷萃取挥发油以烃类成分为主,达到36.78%,而叶乙醚萃取挥发油以烃类和醇类成分为主,分别达到34.22%和30.77%。茎环己烷、乙醚萃取的挥发油也以烃类和醇类成分为主,分别达到34.54%、39.56%和46.73%、29.94%。4,8,12,15,15-五甲基双环[9.3.1]十五烷-3,7-二烯-12-醇为栀子叶乙醚萃取挥发油和茎环己烷、乙醚萃取挥发油的主要成分。对以上挥发油进行抑制豆腐致腐细菌实验表明,栀子叶(乙醚萃取)挥发油可明显延长豆腐冷藏时间,使其保鲜时间延长6 d。由于气味芳香,不影响其自然风味。
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Chemical constituents of essential oil of the leaf and stem fromGardeniajasminoidesEllis and their antimicrobial activities in the bean curd
WEI Qiang, XU Fei
(Pharmacology College, Anhui Xinhua University, Hefei 230088, China)
ABSTRACTThe chemical constituents of essential oils of the leaf and stem from Gardenia jasminoides Ellis and their biological activities in the bean curd were studied herein. The volatile oils of leaf and stem were extracted by supercritical CO2, cyclohexane and ethyl ether extraction, then separated and identified by GC-MS. 153 components were identified from the volatile oil, wherein main constituents were 1,2,3,5,6,7,8,8α-octahydro-1,4- dimethyl-7-(1-methylethenyl)-azulene, toluene, [1S-(1α,4α,7α)]- 1,2,3,4,5,6,7,8-octahydro-1,4- dimethyl-7- (1- methylethenyl)- azulene, 4, 8, 12, 15, 15-pentamethyl-bicyclo[9.3.1] pentadeca-3,7-dien-12-ol, 1,1-diethoxy- ethane, 2-ethyl-1,3-dioxolane (5.46%) from the leaf, and 4, 8, 12, 15, 15- pentamethyl-bicyclo[9.3.1]pentadeca- 3,7- dien-12-ol, 1, 2, 3, 5, 6, 7, 8, 8α-octahydro-1,4-dimethyl-7-(1- methylethenyl)-azulene, borneol, 1,1-diethoxy- ethane from the stem. The volatile oils of leaf (aether) showed apparent antimicrobial activities in the bean curd, which could prolong the storage time.
Key wordsleaf and stem from Gardenia jasminoides Ellis; volatile oil; chemical components; bean curd; antimicrobial activities
DOI:10.13995/j.cnki.11-1802/ts.201606022
基金项目:安徽省质量工程项目(2014zy078)
收稿日期:2015-12-03,改回日期:2016-01-19
第一作者:硕士,副教授。