贺菊萍，赵 勇，孙晓红，吴启华，3，潘迎捷*

1上海海洋大学食品学院，上海 201306;2徐州工程学院食品工程学院，徐州 221008;3缅因大学食品科学与人类营养系，美国 缅因，04469-5735

Introduction

Burdock(Arctium Lappa L．)，which is a popular vegetable in China and Japan，has been extensively studied for its components of root，leaf and seed and biochemical activities［1］．Antimicrobial activity of its leaves extraction was studied and results showed the leaves extraction was a potential source of effective and safe antibacterial ingredients for food industry［2］．However，there is less information of antimicrobial activity about roots of burdock．Vibrio parahaemolyticus is a halophilic bacterium that is widely distributed in marine environments and frequently isolated from seafood［3］．In Shanghai，outbreaks of V．parahaemolyticus infection were reported as high as 60%of all food poisoning cases caused by bacteria［4-5］．The objectives of the present research were to study the antibacterial properties，including the bactericidal and suppressive effects，of burdock concentrated extract on V．parahaemolyticus in vitro．

Materials and Methods

Materials

The roots of two varieties of burdock named Liuchuan and Huangji were collected from Feng county，Jiangsu province．Three strains of V．parahaemolyticus(ATCC 17802，33846 and 33847)were used in the experiments．Shrimps was purchased from a local grocery store in Shanghai，China．Tryptic soy agar(TSA)，TryptIC Soya Broth(TSB)and thiosulphate citrate bile salts(TCBS)were obtained from Sangon Biotech(Shanghai)Co．，Ltd．．

Preparation of burdock extract

Peel and peeled root of burdock were dried at room temperature for 48 h and the dried materials were powdered．Peel and peeled root of Liuchuan and Huangji(50 g)were extracted with 1000 mL of 80% ethanol at 60℃for 4 h to obtain crude extracts and each extract was with the same volumes of 10 mL，which were classed as Liuchuan peel(LP)，Liuchuan root(LR)，Huangji peel(HP)，Huangji root(HR)respectively．The extracts were concentrated in a rotary evaporator．The yield concentrated extracts were used as the test substances．

Antibacterial test

10 mL of TSA were poured into sterile plate and allowing them to settle and approximately 106each bacterial cells were inoculated onto it．Ten microliters of plant extract was applied to each paper disc(6 mm，Xinhua)．The discs were air dried and placed on top of the agar layer．Three replicates of each extract were tested with a erythromycin disc(0．2 mg/mL)as a reference(four discs per plate)．The plates were then incubated for 10-18 h at37℃．Antibacterial activity was defined by measuring the diameter of the growth inhibition zone．

Antibacterial test of burdock concentrate

For each of the four bacteria used，100 μL of concentrate was two-fold serially diluted with 100μL sterile distilled water in a sterile 96-well micro-plate．One hundred microliters of each bacterial culture was added to each well．The plates were covered and incubated overnight at 37 ℃．To indicate bacterial growth，50 μL of0．2 mg/mL p-iodonitrotetrazolium violet(INT)was added to each well and the plates incubated at 37℃for 30 min．Bacterial growth in the wells was indicated by a red colour，whereas clear wells indicated inhibition by the tested substances．

Time-kill assay of burdock concentrate on V． parahaemolyticusin distilled water ( DW) and BHI

The method used to evaluate antibacterial effects of burdock concentrate on V．parahaemolyticus in distilled water(DW)and Brain Heart Infusion(BHI)broth referred to Vivian Chi-Hua Wu with slight modification［6］．Burdock concentrate was prepared at four concentration levels(250，500，750，and 1000 μL/mL)in 9 mL of sterilized DW to study its bactericidal effect with limited nutrients，and in BHI broth to gauge the suppressive effect．Both DW and BHI at each concentration level were inoculated with V．parahaemolyticus(ATCC17802，ATCC33846 and ATCC33847)suspension to achieve an initial inoculums level of approximately 4-5 log CFU/mL，which provides a wide range for observation of bacterial reduction．Each sample was mixed well by vortexing，then incubated at25 and 4 ℃for 0，1，3，5，7，12，and 24 h(DW samples)and 0，1，2，3，4，and 5 d(BHI samples)．V．parahaemolyticus inoculated in DW and BHI withoutburdock concentrate was analyzed as control(0μL/mL)using the same procedure．At each sampling time，the samples were serially diluted with 1 mg/mL sterile peptone water and the appropriate dilution was spiral plated onto the TCBS plates．After incubation at37 ℃ for 18 h，viable cells were determined．

Statistical analysis

All experiments were repeated three times．Analysis of variance(ANOVA)was performed on cell counts using SPSS software 18．0．The results were averaged and expressed as

Results

Antibacterial test of burdock concentrate

The results of antibacterial activity of burdock concentrate against V．parahaemolyticus by disk diffusion method were displayed in table 1．Erythromycin was employed as a positive control．LP and HP were found to have significant antibacterial activity against V．parahaemolyticus although their activities were less potent than those of erythromycin．The antibacterial activity of LP against V．parahaemolyticus was more effective than that of HP(P ＜0．05)．LR also had antibacterial activities against V．parahaemolyticus，but it was much weaker than LP and HP(P ＜0．05)．HR showed no antibacterial activity against V．parahaemolyticus in this study．

Determination of MIC of burdock concentrate againstV． parahaemolyticus

As shown in Table 2，LP inhibited the growth of V p17802 at6．250 μL/mL and inhibited the growth of Vp33846 and Vp33847 at3．125 μL/mL，LR inhibited the growth of all bacterial cells at 50 μL/mL，HP inhibited the growth of all bacterial cells at 6．250 μL/mL，while HR didn’t inhibit any bacterial cells at1000 μL/mL．

Table 2 Minimum inhibitory concentration(MIC)of burdock concentrate against V．parahaemolyticus(μL/mL)

Time-kill assay of burdock concentrate on V． parahaemolyticusin DW and BHI

The results above showed that LP exhibited the highest antibacterial activity．Thus，a time-kill experiment was carried out to further investigate the bactericidal activity of LP．The time-kill assay was carried out against V．parahaemolyticus to see the concentration and time-dependent killing effect at the initial inoculum of 105 CFU/mL．

Fig.1 Time-kill kinetics of LP against V．parahaemolyticus in DW．(a)4℃ (b)25 ℃

V．parahaemolyticus was very sensitive to the burdock concentrate．At 4 ℃ (Fig 1)，when compared to the control，the 500 μL/mL treatments resulted in 1．27 log CFU/mL reductions at 24 h．The 750 μL/mL treatments resulted in 2．97 log CFU/mL reductions at 24 h．The 1000 μL/mL treatments resulted in 2．33 log and 2．98 log CFU/mL reductions at5 and 7 h respectively and bacteria cells could not be detected after 12 h treatment．

At25 ℃ (Fig 1 b)，when compared to the control，the 500 μL/mL treatments resulted in 1．75 log and 2．40 log CFU/mL reductions at 12 and 24 h respectively．The 750 μL/mL treatments resulted in 1．29 log，2．39 log and 3．55 log CFU/mL reductions at 5，7 and 12 h respectively，and bacteria cells could not be detected after 24 h treatment．The 1000 μL/mL treatments resulted in 1．59 log and 3．31 log CFU/mL reductions at 3 and 5h respectively，when compared to the control and bacteria cells could not be detected after 7 h treatment．

V．parahaemolyticus were also sensitive to the burdock concentrate at both 4 ℃ and 25 ℃ in BHI．At0 d(Fig 2)，0．56 log and 0．51 log CFU/mL bacteria cells reduction was observed in the 500μL/mL burdock concentrate treatments at4℃ and 25℃ respectively，and no V．parahaemolyticus was detected in 750 and 1000 μL/mL treatments．After 1 d，no V．parahaemolyticus was detected in all treatments at both 4℃and 25℃．

Fig.2 Time-kill kinetics of LP against V．parahaemolyticus in BHI(a)4℃ (b)25℃

Discussion

Burdock plants are used as the well-known traditional Chinese medicine which has recognized healthy to humanbeings．Phenolic compounds，in particular phenolic acids(caffeic acid，chlorogenic acid and cynarin)，arctiin，luteolin and quercetin rhamnoside were strong radical scavengers［7］．Chlorogenic acid and caffeic acid existed mainly in the skin of burdock root，and the content of chlorogenic acid was much higher than that of caffeic acid．Peeling greatly decreased these two active components of burdock and its free radical scavenging activity，which resulted from elimination of most of these two components in the skin of the root［8］．Chlorogenic acid in bourdock leaf were confirmed with antibacterial activity against food-related bacteria［2，19］and pathogenic fungi［10］．Peel of Arctium Lappa showed greater antibacterial activity against Escherichia coil，Staphylococcus aureus，Bacillus subtili，Aspergillus niger and Saccharomyces cerevisiae than that of peeled root［11］．

In this study，LP and HP showed effectively antibacterial effects on V．parahaemolyticus with different virulence gene．LP and HP showed antibacterial effects on three culture of V．parahaemolyticus．LR also showed antibacterial effects，but it was much weaker than LP and HP．The results indicated that the components with antibacterial effects in burdock were mainly contained in burdock peel．

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