三叶木通果皮果胶抑菌作用的研究

安徽农业科学,Journal of Anhui Agri. Sci. 2009,37(17) :7835-7836,7849　　　　　　　　　　　　　　　　　　责任编辑　罗芸　责任校对　卢瑶

三叶木通果皮果胶抑菌作用的研究

张孟琴, 田野, 李兴平, 田爱琴　(河南科技大学林业职业学院, 河南洛阳471002)

摘要　[目的]探讨三叶木通果皮果胶的抑菌和杀菌性能。[方法]从三叶木通果皮中提取出果胶, 采用圆纸片扩散法对提取的果胶进行抑菌和杀菌性能研究。[结果]三叶木通果皮果胶的红外光谱图显示1700cm -1处存在酯化的羰基, 且在1700cm -1处的吸收峰强度较强, 表明提取的三叶木通果皮果胶为高酯果胶。所得果胶的红外光谱图与G nanasambanadam 的果胶谱图主要特征峰基本相同。从三叶木通果皮中得到的果胶制品和标准桔皮果胶, 在大肠杆菌DH 5α和金黄色葡萄球菌的平板上均没有检测出抑菌效果, 或作用效果不明显, 难以在平板上检测出。[结论]三叶木通果皮果胶没有抑菌及杀菌的作用。关键词　三叶木通果皮; 果胶; 抑菌性能中图分类号　Q946. 887　　文献标识码　A 　　文章编号　0517-6611() 17--Study on B acteriostasis of P ectins from Akebia trifoliate koiaz P ZH ANG M eng 2qin et al 　(F orestry V ocational C enan , Lu oyang , H enan 471002) Abstract 　[Objective]T he aim was to ance of pectins from Akebia trifoliate peels. [Meth od]Pectins were extracted from A. perform ance of extracted pectins were studied by disk diffusion m eth od.

-1

[Result ]T he in frared peels sh owed that esterifiable carb onyl existed in 1700cm and intensity of abs orption peak in 1700cm -1was stronger , owed the extracted pectins from A. trifoliate peels was high m eth oxyl pectin. T he m ain characteristic peaks of in frared spectrum of obtained were basically sam e as that of G nanasambanadam pectin spectra. T he antibacterial effect was n ot detected in E s 2cherichia coli DH 5αand S taphylococcus aureus plate contained pectin products obtained from A. trifoliate peels and standard orange peel pectin , or the antibacterial effect was n ot obvious and it was hard to detect in plate. [C onclusion]T he pectins from A. trifoliate peels had n o antibacterial and bacterici 2dal effect. K ey w ords 　Akebia trifoliate peels ; Pectin ; Antibacterial perform ance

　　果胶多糖是一种相对分子量在50～300kD 的酸性多糖, 是细胞壁的一种组成成分, 广泛存在于植物的果实、根、茎、叶中。随着功能性多糖的开发研究, 果胶作为水溶性膳食纤维, 越来越受到研究和加工行业的重视[1]。另外, 一些研究表明果胶具有抗菌、止血、消肿、止痛、解毒、止泻、抗辐射等作用[1-2], 特别对一些革兰氏阴性细菌非常敏感, 接触2h 后杀菌效果非常显著, 因此在应用上, 果胶与多种抗菌药物配合, 达到抗菌增效作用[1,3]。

野生三叶木通(Akebia trifoliate koiaz ) 属于木通属落叶木

[4]质藤本植物。果实肉质浆果, 形似香焦, 故称“野香蕉”。

水分, 然后用去离子水洗涤4～5次, 用穆立虚反应检验至无糖分为止, 按固液比(三叶木通质量与水的质量比列) 1∶25用稀硫酸调溶液pH 为1. 2左右, 在95℃条件下恒温水解120

m in [5], 趁热过滤并洗涤滤渣3次, 合并滤液。滤液用活性碳

脱色(100m l 浸提液中加入0. 7～0. 9g 活性炭) 过滤后得澄清液, 置于旋转蒸发仪中浓缩至果胶含量约50mg/m l , 然后用

80%的酒精沉析约60m in , 沉析的pH 为4. 0, 沉淀物置于45

℃烘箱中干燥3h [6]。

1.2. 2　果胶制品的鉴别。参照有关果胶制品的鉴定方

法[7-8]:①称取1. 5g 果胶试样于烧杯中, 并加入去离子水60

m l , 不断搅拌使其溶解, 溶液呈粘稠状, 并有浅黄色絮状悬浮

笔者前期从三叶木通中提取出了高质量的果胶, 但由于不同植物中果胶的特性有所差异, 为了提高其应用价值进行了该研究, 旨在验证三叶木通果皮果胶的抑菌、杀菌性能。

1　材料与方法

1. 1　材料、试剂及仪器　

1. 1. 1　材料。三叶木通果皮采于河南淅川县境内。

物。②称取果胶试样0. 1g , 加水50m l , 再加乙醇20m l , 不断搅拌, 出现灰白色絮凝状半透明凝胶沉淀。③取1%的果胶水溶液5m l , 加入2m ol/L 的氢氧化钠1m l , 搅拌均匀, 室温下放置10m in , 出现灰白色絮状沉淀, 加热后呈黄色, 产生棉花状白色沉淀或凝胶。④把果胶研成粉末, 与K Br 混合压片制样, 采用傅立叶变换红外光谱仪对三叶木通果皮果胶样品进行红外分析, 制作果胶的红外光谱图。

1. 2. 3　LB 培养基配制。牛肉膏10g , 蛋白胨5g ,NaC l 10g , 琼

α1. 1. 2　试剂。硫酸G R 、2萘酚AR 、无水乙醇AR 、活性炭

AR 、大肠杆菌(E scherichia coli ) 、金黄色葡萄球菌等, 均由湖南

农业大学生物科学技术学院微生物实验室提供。

1. 1. 3　仪器。pHS 23C 型pH 计; 万分之一电子天平; 离心沉

脂粉15g , 蒸馏水500m l , 用1m ol/L NaOH 调pH 为7. 0, 高压灭菌, 冷却至50～60℃左右, 倒在直径为8cm 的无菌平板上, 厚度约为4～6mm ,4℃下保存备用。

1. 2. 4　菌悬液的制备。在超净工作台上, 用加样枪吸取200

淀机;ZK 282B 电热鼓风干燥箱;SHZ 2D 多功能循环水式真空泵;RE 2522旋转蒸发器; 傅立叶转换红外光谱仪; 高压灭菌锅

280B 型; 电热恒温培养箱HZ Q 2X 100; SW 2C J 21F 型超净工作

台等。

1. 2　试验方法

1. 2. 1　果胶的制备。三叶木通果皮置于气流烘干箱内80℃

μl 培养20h 左右的受试菌, 置于10m l 带无菌玻璃珠的无菌水中制成菌体浓度为107～108cfu/L 的均匀菌悬液。

1. 2. 5　抑菌活性试验[9-10]。用灭菌涂布环将上述菌液涂布

左右烘干过40目筛网, 用8～10倍的水浸泡1h 左右后, 去除

基金项目　河南省省属科研院所研究专项资金项目(0441100102) 。作者简介　张孟琴(1969-) , 女, 河南洛阳人, 硕士, 讲师, 从事植物化学研究。收稿日期　2009203209

于制作的LB 平板上, 在无菌工作台上自然干燥后, 待用。把

7mm 直径大小的无菌滤纸圆片经无菌水浸湿, 用无菌镊子

夹住粘些三叶木通果胶样品粉末, 把此滤纸片放在上述含菌平板上, 于37℃恒温箱中倒置培养16～20h , 测量抑菌圈直径, 同时以在无菌蒸馏水中浸泡过的晾干滤纸片为阴性对

7836　　　　　　　　　　　　　安徽农业科学　　　　　　　　　　　　　　　　　　　　　　　　2009年

照, 以在6μm ol/L 氨苄青霉素(Am p ) 浸泡过的晾干滤纸片为阳性对照, 测定抑菌圈直径的大小, 衡量果胶抑菌效果[9]。

2　结果与分析

2. 1　提取物检测　对从三叶木通果皮中提取的制品性质进

用的菌种质量没有问题。同无菌纸片效果相似, 从三叶木通果皮中得到的果胶制品和标准桔皮果胶, 在2种菌种的平板上均没有检测出抑菌效果, 或作用效果不明显, 难以在平板

上检测出。

行检测, 结果表明其符合果胶制品的性质, 证明提取的为果胶制品。

2. 2　三叶木通果皮果胶的红外光谱图　由图1可见,1700cm -1处存在酯化的羰基, 且在1700cm -1处的吸收峰强度较

强, 因此提取的三叶木通果皮果胶为高酯果胶。1400cm -1处的峰为C OO -的对称伸缩振动,3384cm -1处的峰为O -H 的伸缩振动。3000～2800cm -1处的峰为C -H 对称伸缩、不对称伸缩。所得果胶的红外光谱图与G nanasambanadam 胶谱图主要特征峰基本相同。

2. 3　果胶对大肠杆菌DH 5α果　由图2可见, 浸有DH 5α和金黄, 表明该试验所采

图1　三叶木通果皮果胶的红外光谱

Fig. 1　I nfrared spectroscopy o f Akebia trifoliate koiaz peels

pectin

注:①a. 沾有三叶木通果皮果胶的纸片;b. 对照, 浸过无菌水的纸片;c. 浸过6μm ol/L 氨苄青霉素的纸片;d. 沾有S igm a 公司生产的标准品桔皮果胶; ②图A 、C 平板上的菌种为大肠杆菌DH 5α; 图B 、D 平板上的菌种为金黄色葡萄球菌; ③图C 、D 上第1区为打孔法检验果胶的抑菌效果, 其余为圆片法。

N ote :①a ,Paper with Akebia trifoliate koiaz peel pectin ; b ,C ontrol paper with im m ersing with aseptic water ; c ,Paper s oaked with 6μm ol/L am picillin ; d ,Paper

) and that on B and D are with the standard orange peel pectin produced by S igm a com pany ; ②,T he strains on A and C plate are E scherichia coli (DH 5α

Staphylococcus aureus ; ③,T he first region in figure C and D is the antibacterial effect of test peel pectin by punch m eth od ; T hat in other regions are disc

m eth od.

图2　果胶对大肠杆菌DH 5α和金黄色葡萄球菌的抑菌效果

Fig. 2　The antib acterial effect o f peel pectin on Escherichia coli (DH 5α) and Staphylococcus aureus

3　结论与讨论大肠杆菌DH 5α(阴性) 和金黄色葡萄球菌(阳性) 为例, 对三叶木通果皮的果胶进行了抑菌试验, 结果表明三叶木通果皮

(下转第7849页)

一些研究者认为, 果胶具有抑菌作用, 为了验证笔者从三叶木通果皮中提取的果胶是否也具有抑菌或杀菌作用, 以

37卷17期　　　　　　　　　　　　　　　赵祥强　植物耐盐性分子机理研究进展P lant Physiol P lant M ol Biol ,2001,52:469-497. [68]W A NG B ,LUT TG E U ,R A T A JCZ AK R. E ffects o f salt treatm ent and osm otic

stress on V 2A T Pase and V 2PPase in leaves o f the halophy te Suaeda salsa [J].J E x p B ot ,2001,52(365) :2355-2365. [69]G AXI O LA R A ,LI J ,U N D URR AG A S ,et al. Drought and salt tolerant plants

result from overex pression o f the A VP 1H +2pum p [J].P N AS ,2001,98(20) :11444-11449. [70]G U O S ,Y I N H ,ZH A NG X,et al. M olecular cloning and characterization o f a

vacu olar H 2pyroph osphatase gene , SsVP ,from the halophyte Suaeda salsa and its overex pression increases salt and drought tolerance o f Arabidop sis [J].P lant M ol Biol ,2006,60(1) :41-50. [71]G A O F , G A O Q ,D U A N X,et al. C loning o f an H +2PPase gene from Thel 2

lungiella halophila and its heterolog ous ex pression to im prove tobacco salt toler 2ance[J].JE x p B ot ,2006,57(12) :3259-3270.

[72]BRI NI F ,H A NI N M ,MEZG H A NI I ,et al. Overex pression o f wheat Na +/H +

antiporter T aNH X 1and H +2pyroph osphatase T VP 1im prove salt and drought stress tolerance in Arabidop sis thaliana plants[J].JE x p B ot ,2007,58(2) :301-308. [73]MIT T LER R. Ox idative stress ,antiox idants and stress T S ci ,2002,7(9) :405-410.

[74]SHI H ,XI O NGL ,STE VE B ,et 4

m utants uncover a critical in in salt tolerance[J].P lant C ell ,2002,14(3) :575-588. [75]Y A M A M OT O A ,BH UIY A N M N ,W A DITEE R ,et al. Suppressed ex pression o f

the apoplastic ascorbate ox idase gene increases salt tolerance in tobacco and Arabidop sis plants[J].JE x p B ot ,2005,56(417) :1785-1796. [76]R O DRIG UES S M ,A N DR A DE M O ,G O MES A P ,et al. Arabidop sis and tobac 2

co plants ectopically ex pressing the s oybean antiquitin 2like ALDH 7gene display enhanced tolerance to drought ,salinity ,and ox idative stress [J].J E x p B ot , 2006,57(9) :1909-1918. [77]H U A NGM D ,W U WL. Overex pression o f T M AC 2,a n ovel negative regulator

o f abscisic acid and salinity responses ,has pleiotropic e ffects in Arabidop sis thaliana [J].P lant M ol Biol ,2007,63(4) :557-569. [78]H U H ,D AI M ,Y A O J ,et al. Overex pressing a N A M ,A T AF ,and C UC (N AC )

transcription factor enhances drought resistance and salt tolerance in rice[J].P N AS ,2006,103(35) :12987-12992. [79]Y A M AG UCHI 2SHI N OZ AK I K,SHI N OZ AK I K . A n ovel cis 2acting elem ent in an

Arabidop sis gene is inv olved in responsiveness to drought ,low 2tem perature ,or hight 2salt stress[J].P lant C ell ,1994,6(2) :251-264. [80]MI L LA M A ,T OW NSE N D J ,CH A NG I F ,et al. T he Arabidop sis AtDi 19gene

fam ily encodes a n ovel ty pe o f C ys2/H is2zinc 2finger protein im plicated in A BA independent dehydration ,high 2salinity stress and light signaling path ways [J].P lant M ol Biol ,2006,61(1-2) :13-30.

[81]ZH U J K . S alt and drought stress signal transduction in plants[J].Annu Rev

P lant Biol ,2002,53:247-273. [82]LI U J ,ISHIT A NI M ,H A LFTER U ,et al. T he Arabidop sis thaliana SOS 2gene

encodes a protein kinase that is required for salt tolerance[J].P N AS ,2000,97(7) :3730-3734. [83]ISHIT A NI M ,LI U J ,H A LFTER U ,et al. SOS 3function in plant salt tolerance

requires N 2m yristoy lation and calcium binding[J].P lant C ell ,2000,12(9) :1667-1678. [84]QI U Q S ,G U O Y,Q UI NTER O F J ,et al. Regulation o f vacu olar Na +/H +ex 2

7849

change in Arabidop sis thaliana by the salt 2overly 2sensitive (S OS ) path way[J].

J Biol Chem ,2004,279(1) :207-215. [85]G U O Y,QI U Q S ,Q UI NTER O FJ ,et al. T ransgenic evaluation o f activated m u 2

tant alleles o f S OS 2reveals a critical requirem ent for its kinase activ ity and C 2term inal regulatory d om ain for salt tolerance in Arabidop sis thaliana [J].P lant C ell ,2004,16(2) :435-449. [86]CHE NGN H ,PIT T M A NJ K,ZH UJ K,et al. T he protein kinase S OS 2activates

the Arabidop sis H +/C a 2+antiporter C AX 1to integrate calcium transport and salt tolerance[J].JBiol Chem ,2004,279(4) :2922-2926. [87]K A TIY AR 2AG ARW A L S , ZH U J , K I M K, et al. T he plasm a m em brane

Na +/H +antiporter S OS 1interacts w ith RC D1and functions in ox idative stress tolerance in Arabidop sis [J].P N AS ,2006,103(49) :18816-18821. [88]AH LF ORS R ,LA NG S ,O VER MY K,et al. Arabidop sis R ADIC AL 2INDUCE D

CE LL DE ATH 1belongs to protein 2protein interaction d om ain protein fam ily m lene ,and m ethy l jasm onate responses [JC ell NG ,LI A al. T ranscriptional pro filing im plicates n ovel

abiotic stress and h orm onal responses in Thellungiella ,a relative o f Arabidop sis [J].P lant Physiol ,2006,140(4) :1437-1450. [90]C A O W H ,LI U J ,HE XJ ,et al.M odulation o f ethy lene responses a ffects plant

salt 2stress responses[J].P lant Physiol ,2007,143(2) :707-719. [91]M ARTI NEZ 2A TIE NZ A J , J I A NG X,G ARCI A DE B LAS B ,et al. C onservation o f the salt overly sensitive path way in rice[J].P lant Physiol ,2007,143(2) :1001

-1012. [92]K A W AS AK I S ,BORCHERT C ,DEY H O LOS M ,et al. G ene ex pression pro files

during the initial phase o f salt stress in rice[J].P lant C ell ,2001,13(4) :889-905. [93]S U NK AR R ,ZH U J K . N ovel and stress 2regulated m icroR N As and other sm all

R N As from Arabidop sis [J].P lant C ell ,2004,16(8) :2001-2019. [94]BORS A NI O ,ZH U J ,VERS LUES P E ,et al. End ogen ous siR N As derived from

a pair o f natural cis 2antisense transcripts regulate salt tolerance in Arabidop sis [J].C ell ,2005,123(7) :1279-1291. [95]LISK A AJ ,SHE VCHE NK O A ,PICKU ,et al. Enhanced ph otosynthesis and re 2

d ox energy production contribute to salinity tolerance in Dunaliella as revealed by h om ology 2based proteom ics[J].P lant Physiol ,2004,136(1) :2806-2817. [96]K ORE 2E D A S ,C USH M N M A ,AK SE LR O D I ,et al. T ranscript pro filing o f

salinity stress responses by large 2scale ex pressed sequence tag analysis in Mesembryanthemum crystallinum [J].G ene ,2004,341:83-92. [97]ZH U J K . G enetic analysis o f plant salt tolerance using Arabidop sis [J].P lant

Physiol ,2000,124(3) :941-948. [98]ZH O U J ,W A NG X, J I A O Y,et al. G lobal gen om e ex pression analysis o f rice in

response to drought and high 2salinity stresses in sh oot ,flag lea f ,and panicle [J].P lant M ol Biol ,2007,63(5) :591-608. [99]A M T M A N N A ,BOH NERT H J ,BRESS A N R A. Abiotic stress and plant

gen om e ev olution. S earch for new m odels[J].P lant Physiol ,2005,138(1) :127-130. [100]BR OW N C E ,PEZESHK I S R. T hreshh old for recovery in the m arsh halophy te

Spartina alterniflora grown under the com bined e ffects o f salinity and s oil dry 2ing[J].JP lant Physiol ,2007,164(3) :274-282. [101]F LOWERS TJ. Im prov ing crop salt tolerance[J].JE x p B ot ,2004,55(396) :

307-319.

(上接第7836页)

果胶没有抑菌、杀菌的作用, 同时发现桔皮果胶同样也没有抑菌、杀菌的功能。已有研究报道, 从不同植物中提取的果胶化学组成与化学构造有一定差异[11], 因此推测, 可能是由于这些差异导致了从不同植物中提取的果胶的抑菌与杀菌效果不同, 这也可能是该试验结果与已往报道结果[3]不同的原因。参考文献

[1]魏亚冉. 果胶多糖的提取、分离与应用研究进展[J].中国食品添加剂,

2008(4) :82-86.

[2]田三德, 任红涛. 果胶生产技术工艺现状及发展前景[J].食品科技,

2003(1) :53-55.

[3]陈靖, 陈孔荣. 果胶———一种有开发前途的药物制剂基质[J].现代应

用药学,1997,14(2) :22.

[4]刘孟军. 中国野生果树[M].北京:中国农业出版社,1999.

[5]张孟琴, 田爱琴, 刘占朝, 等. 酸解法从三叶木通果皮中提取果胶的研

究[J].安徽农业科学,2007,35(16) :4757-4759.

[6]张孟琴, 孔繁伦, 田爱琴, 等. 三叶木通果皮果胶沉析条件的研究[J].

食品工业科技,2007,28(12) :79-82.

[7]凌关庭. 食品添加剂手册[M].北京:化学工业出版社,2000. [8]樊明涛. 食品分析与检验[M].北京:世界图书出版公司,1998.

[9]雷崎, 庄东红, 吴奕瑞. 番石榴叶中总黄酮最佳提取条件的探讨及其抑

菌作用的研究[J].天然产物研究与开发,2006,18(S 1) :107-111.

[10]朱丹, 牛广财, 孙希云, 等. 马齿苋黄酮类物质抑菌作用的研究[J].安

徽农业科学,2006(1) :15-16.

[11]李拖平, 王娜, 李苏红, 等. 山楂果胶的黏度特性及其与化学构造的关

联性[J].食品科学,2008,29(1) :64-68.