Antimicrobial Activity of Plant Extracts Against Salmonella Typhimurium, Escherichia coli O157:H7, and Listeria monocytogenes on Fresh Lettuce

Sung-Youn Kim, Dong-Hyun Kang, Jin-Ki Kim, Yong-Geun Ha, Ju Young Hwang, Taewan Kim, and Seon-Ho Lee
Abstract: Plant extracts have been found to be effective in reducing microorganisms. This study evaluated antimicrobial

activity of 12 plant extracts against Salmonella Typhimurium, Escherichia coli O157:H7, and Listeria monocytogenes by using a disk diffusion assay, and Syzygium aromaticum (clove) showed the highest inhibitory effect. To investigate the efficacy of clove extract that inactivates pathogens on lettuce, inoculated lettuce with S. Typhimurium, E. coli O157:H7, and L. monocytogenes was treated with diluted clove extracts or distilled water for 0, 1, 3, 5, and 10 min. Clove extract treatment significantly reduced populations of the 3 tested pathogens from the surface of lettuce.
Keywords: antimicrobial agent, disinfectant, foodborne pathogen, fresh produce, plant extract

of foodborne pathogens on fresh lettuce. It also might be a natural antimicrobial for reducing or replacing chemical sanitizers in food preservation.

Introduction

Fresh fruits and vegetables are an essential part of the diet of people around the world (Beuchat 1996). For the last 3 decades, a number of outbreaks caused by foodborne pathogens associated with fresh produce consumption have been reported to the Centers for Disease Control and Prevention (Sivapalasingam and others 2004). Salmonella Typhimurium, Escherichia coli O157:H7, and Listeria monocytogenes have all been implicated in minimally processed fruits and vegetables outbreaks of foodborne illness (Beuchat 1996; Jablasone and others 2005). Growing in fields or orchards, fresh produce can be readily contaminated with pathogenic microorganisms. And contamination with pathogens may also occur during harvesting, postharvest handling, processing, and distributing (Beuchat 1996; Takeuchi and others 2000). Traditionally, aqueous chlorine at concentrations between 50 and 200 ppm is used to wash fruits and vegetables, which result in a microbial reduction of less than 2 log on fresh produce (Beuchat 1992; Brackett 1992; Lee and others 2006). Furthermore, harmful by-products, such as chloramines and trihalomethanes, may be produced after treatment with strong solutions of chlorine (Lee and

others 2004). Organic acids, chlorine dioxide, and trisodium phosphate are generally recognized as safe (GRAS) sanitizers (Cutter 2000). Although these sanitizers have been found to be effective, researchers are continually investigating the use of other antimicrobial agents to reduce foodborne pathogens or improve quality in fresh produce (Cutter 2000). A variety of natural plant antimicrobials have potential to control foodborne pathogens. Extracts from many types of plants have been used as flavoring and seasoning agents in foods and beverages and also as folk medicines and food preservatives, since ancient time (Elgayyar and others 2001; Shan and others 2007). It adds characteristic flavor and prolongs the shelf life of foods by their antioxidant, bacteriostatic, and bactericidal activity (Shan and others 2007). Numerous researches have been assessed and conducted on their antimicrobial properties. Plant extracts, including their essential oils and essences, have been recognized that they possess antimicrobial properties against bacteria, moulds, and yeast (Smith-Palmer and others 1998). This study examined the antimicrobial properties of 12 plant ethanolic extracts against foodborne pathogens, S. Typhimurium, E. coli O157:H7, and L. monocytogenes. The plant extract that has the strongest bacteriostatic and bactericidal properties among 12 was further researched for treatment in the inoculated lettuce MS 20100557 Submitted 5/21/2010, Accepted 9/30/2010. Authors Kim and surface to decrease foodborne pathogens.
Kang are with Dept. of Food and Animal Biotechnology, Seoul National Univ., San 56-1, Sillim-dong, Gwanak-gu, Seoul 151-742, Korea. Author Kang is with Dept. of Food Science and Human Nutrition, Washington State Univ., Pullman, WA Materials and Methods 99164-6376, U.S.A. Authors Kim, Ha, Hwang, and Kim are with Dept. of Food Science and Biotechnology, Andong National Univ., 388 Songchon-dong, Andong Bacterial strains 760-749, Korea. Author Lee is with Inst. of Marine Biotechnology, Andong National Three strains each of S. Typhimurium (ATCC 19586, ATCC Univ., 388 Songchon-dong, Andong 760-749, Korea. Direct inquiries to author Lee 43174, DT 104), E. coli O157:H7 (ATCC 35150, ATCC 43889, (E-mail: lotte@daum.net).

ATCC 43890), and L. monocytogenes (ATCC 15313, ATCC
C 2010 Institute of Food Technologists R doi: 10.1111/j.1750-3841.2010.01926.x

Vol. 76, Nr. 1, 2011 r Journal of Food Science M41

Further reproduction without permission is prohibited

M: Food Microbiology & Safety

Practical Application: This result indicated that clove extract is a useful antimicrobial agent to reduce the microbial level

Canada) containing 225 mL of BPW and homogenized for 2 min with a stomacher (EASY MIX. Mulberry.01 2. Each strain of S. For inoculation. Table 1–Description of plant extracts. Statistical analysis and washed 3 times with buffered peptone water (BPW. All cultures were maintained on tryptic soy agar (Difco. After that. Thuja orientalis L.5 McFarland barium sulfate standards.Antimicrobial activity of plant extract . The pH values of each extract have been recorded by a Mettler Toledo 7 Multi pH-meter at room temperature. France). clove extracts were diluted with sterile distilled water (DW). The adjusted suspensions were swabbed onto the entire surface of Mueller Hinton agar (Difco). Agrimony.84 6.75 ± 0. A 24 h culture of the each strain was diluted with sterile saline buffer until it achieves the turbidity of the 0. Three replicates were done for each for data analysis. Pinus densiflora. Black myrobalan.05 2.04 2. ATCC 19115) were obtained from the bacterial culture collection of Seoul Natl.20% and pH ranged from 4.A. Korea). Common name Black myrobalan Lightyellow Sophora Chaulmoogra Rosemary Hemp Myrrh Mulberry Agrimony Korean asarum Red pine Clove Franco Source Fruit Root Seed Leaf and branch Seed Stem Root bark Leaf and branch Root Pine needle Bud Leaf and branch Soluble solid content (%) 7.27 ± 0. Xylose Lysine Desoxycholate agar (Difco). Rennes. E.88 5. Syzygium aromaticum. Hydnocarpus anthelmintica Pierre.. Treatments of lettuce with clove extracts For treatment of lettuce. The plates were incubated at 37 ˚C for 24 to 48 h.66 ± 0. Franco) used in this study were purchased from the Omniherb (Taegu.. Samples (100 g) of edible parts (Table 1) were homogenized and mixed with 70% ethanol (1000 mL). Separated lettuce leaves were placed on aluminum foil in 0.80 4.. monocytogenes. Triplicate data were analyzed by analysis of variance using StatisThe final pellets were resuspended in BPW. and Oxford agar base with “BactoTM Oxford antimicrobic supplement” (Difco) were used as selective media for the enumeration of S. coli O157:H7.22 5. and L. and 10% clove extracts for 0.41 2.45 M42 Journal of Food Science r Vol.91 4. Botanical name Terminalia chebula Retz. 19114.56 ± 0.16 ± 0. Cary.01 2. The homogenates were continuously stirred for 12 h at room temperature and filtered over Whatman nr 1 filter paper.22. M: Food Microbiology & Safety Determination of Hunter’s color values In order to identify the changes in lettuce color during storage following treatments. 3. respectively.S. coli O157:H7. Red pine. Typhimurium. Preparation of plant ethanolic extracts Dried plants (Terminalia chebula Retz.. Agrimonia pilosa Ledeb. Osaka. Becton Dickinson. and L. Bacterial enumeration The treated lettuce leaves were transferred into sterile stomacher bags (Labplas Inc. Assay for antimicrobial activity To measure antimicrobial activity of each plant extract. Asarum sieboldii. 1.S. Asarum sieboldii Pinus densiflora Syzygium aromaticum Thuja orientalis L. (Seoul. Rosmarinus officinalis.20 ± 0. Hemp. Morus alba L. Fifty microliter of each plant extracts added to the paper disk (Advantec. 2011 . Commiphora molmol.64 to 6. Cannabis sativa.) slants at 4 ◦ C and subcultured monthly.92 5. Sophora flavescens Ait. After incubation at 37 ◦ C for 24 h. Sparks. 5%. AES Chemunex. P < 0.. a. which supplies medicinal herbs.87 4.64 5. South extracts. a. DW was also included as a control treatment. 76. corresponding to ap. homogenate was 10-fold serially diluted in BPW.redness. Cultures and cell suspension and b values. Twenty-five gram of lettuce samples inoculated with culture cocktails were immersed in 1 L of 1%. 5. . Color change of lettuce leaves was measured with a Minolta colorimeter (model CR300. Korean asarum. N. all treated samples were rinsed with DW and stored at 4 ˚C for 7 d. 8 mm. Minolta Co. and 0. Myrrh. lettuce leaves were cut into square pieces approximately 5 by 5 cm..63 ± 0. each species of the of means by Duncan’s multiple range test at a probability level of 3 foodborne pathogens were mixed to produce culture cocktails.1 mL of sample or dilution was spread-plated onto selective medium. and 10 min at room temperature.61 5. Sorbitol MacConkey agar (Difco). U.98 ± 0. Results and Discussion Sample preparation and inoculation Table 1 showed the soluble solid content and pH of 12 plant Lettuce was purchased from a local grocery store (Seoul. the disk diffusion assay was carried out following the instructions of the National Committee for Clinical Laboratory Standards (NCCLS 2000). . Colonies were counted and calculated as log CFU/g in lettuce samples. Hydnocarpus anthelmintica Pierre Rosmarinus officinalis Cannabis sativa Commiphora molmol Morus alba L. Agrimonia pilosa Ledeb. a biosafety hood.08 1..03 2.05.70% to 7.02 0. Md. Lightyellow Sophora.tical Analysis System (SAS Inst. The samples after inoculation were dried for 2 h and used in clove extracts treatment.05 1.) and separation proximately 106 to 107 CFU/mL. thick) placed on the inoculated plate.62 ± 0. cytogenes was cultured in tryptic soy broth (Difco) at 37 ◦ C for 24 h. respectively.08 5. Chaulmoogra. U. Hunter L. Typhimurium.00 1. and b values indicate color lightness. Univ. Japan) at 3 locations on each leaf and expressed as Hunter L. The filtrates were used in this study. Sainte-Julie. harvested by centrifugation at 4000 × g for 20 min at 4 ◦ C. and yellowness of the sample.79 ± 0. Nr. and the inoculated plate was dried for 5 to 10 min.A.70 ± 0. and 0. mono. Sophora flavescens Ait.. South Korea).1 mL of culture cocktail was applied to leaf surface by depositing droplets at 15 to 20 locations with a micropipettor. The soluble solid content of plant extracts ranged from Korea). After homogenization.. E. 1. the inhibition zones were measured and the assay was carried out 3 times for each extract.. These culture cocktails were used in subsequent experiments.08 1. Rosemary.75 6.58 ± 0.C. Clove. Quebec.02 pH 4. Difco).

8 ± 1. Extracts of S.0 ± 0.3 ± 0. Lettuce leaves were inoculated with 6.0∗ 9.6 10.0 9. Clove extract has shown inhibiting activity against Mean 10.6 14.3 ± 0.1 to 15.0∗ 9.8 ± 0.0 8.0 ± 1.3 8.5 8.3 ± 0.2 ± 2.5 ± 2.8 20.0∗ 8. 76.6 8.3 8. all treated samples had no significant effect on taste and flavor (data not shown).0∗ 9.5 9.2 8.7 ± 1.0∗ 8.8 8.2 ± 0. C.3 ± 1.8 ± 0. and 5. S.3 ± 0. Typhimurium and E.4 9.8 ± 1.05) between DW and 1% clove extract treatment. Nr.7 ± 2.1 10. Botanical name 10. Syzgium aromaticum (Clove) was the most effective in microbial reduction for 5 min treatment (data not shown). Agrimonia pilosa Ledeb.0∗ 8.7 15.2 ± 1. the other 5 extracts had no inhibitory activity against 3 strains of E. Typhimurium. Plant-derived antimicrobial compounds have been recognized as a means of inhibiting undesirable bacteria and numerous research articles have described the antimicrobial properties of plant extracts.3 10.0∗ 8. monocytogenes.8 ± 1.6 8. aromaticum (9.6 ATCC 19115 Table 2–Zone of inhibition showing antimicrobial activity against foodborne pathogens.5 11.0∗ 16.0 9.0 12. In case of 10% extract.0. For L.7 ATCC 35150 ∗ Vol.2 15.0 10.0 9.6 10.0 mm.6 to 10.0 8.9 ATCC 43889 ∗ .0 ATCC 19114 8.0 ± 2.6 8. and more than 3 log reductions were observed after 3 min treatment. For E.8) except H.0∗ 8.0∗ 8.3 ± 1.9 8. and b values between DW treated and clove extracts treated lettuce.3 ± 0. 1. Mean S.3 8.80. monocytogenes using the disk diffusion assay (Table 2).2 ± 1.3 ± 1.1 8.1 8.2 10. E.8 ± 0.5 ± 2. Table 3 shows the Hunter’s color values of lettuce stored at 4 ◦ C for 7 d following treatment with DW (control) and 1%. coli O157:H7. S. significant reduction occurred in just 1 min. officinalis (9.3 8. After 5 min treatment.0 ± 3.0∗ 8.5 9.3 ± 1.4 8.4). monocytogenes Diameter of inhibition zone (DIZ. A culture cocktail (0.2 8.6 14.0∗ 8. less than 1 log reduction occurred in all 3 pathogens when inoculated lettuce leaves were treated with the DW.6 14.2 8.3 ATCC 43174 8.2 9.1).4 12. Sophora flavescens Ait.8. coli O157:H7. 5%. and L.9 11.0∗ 8.0∗ 8.0 ± 1.4 9. aromaticum exhibited the strongest inhibitory activity (mean diameter of inhibition zone [DIZ] = 10.8 10.7 12. Typhimurium and 3.1 10.3 8.6 13.4 10. Typhimurium and E.6 8. For S.1 8. followed by R.0∗ 8. coli O157:H7 Mean ATCC 43890 8. officinalis.2 8.3 ± 1.7 ± 1. the reduction levels of 5% and 10% clove extract were similar.68 log by E. Commiphora molmol showed the strongest antimicrobial activity (10.0∗ 8.5 8.3 ± 0.7 ± 1.6 8.2 ± 0. flavescens (9.5 9.3 ± 3. Asarum sieboldii Pinus densiflora Syzygium aromaticum Thuja orientalis L.0∗ 8. In L.3 ± 0. Both treatments showed significant difference in microbial levels compared with initial cell count after 1 min treatment. monocytogenes.0 9. Typhimurium. C.0).2 8. Typhimurium Terminalia chebula Retz. 10-fold serial dilution were prepared in BPW.1 9.9 15.2 ± 3.0∗ 9.3 8.9 10.0 ± 1.0 13.Antimicrobial activity of plant extract .0∗ 10.3 ± 2.8 9.6 8.2 ± 0.0 9.0 8. .0 mm (∗ ).3 9. which means the extract has no inhibitory activity against this bacterium.0 ± 0.5 11.0 DT104 9. anthelmintica (8.3 ± 3.3 ± 0. sieboldii (9.0∗ 9.0 8. respectively. H.5 8.7 ± 2.6 ∗ 11.0∗ 9.2 8. flavescens.3 ± 0.3 ± 2.9 10.8 9.5 ± 0.5 ± 0.3 ± 2.7 ± 1.6).2 9. Because of physical removal effect.2 ± 1. There were no significant differences (P < 0.7 ± 1.0 mm and the diameter of inhibition zone (DIZ) of negative control for each bacterium is also 8.4 10.1 9.5 12.3 ± 1. molmol.7 8.5 8.0 ± 1. sativa. and S. anthelmintica and A. Hydnocarpus anthelmintica Pierre Rosmarinus officinalis Cannabis sativa Commiphora molmol Morus alba L.0 ± 2. And significant further reduction was not observed as the treatment time was increasing.3 ± 0.5 ± 0.70.5 ± 0. These 5 plant extracts were tested for its efficacy of inactivating pathogens.5 ± 0.3 8. a.5 8.3 ∗ The diameter of paper disk is 8. 6. sativa (10. Typhimurium.8 ATCC 19586 9. The antimicrobial activities of 12 plant extracts were evaluated against foodborne pathogens that were S. pilosa showed no inhibition against 3 strains of S.7 ± 0. coli O157:H7.1 12.7 ± 0.7 ± 2. Even 10% of clove extract did not significantly change lettuce color during 7 d storage compared with DW control treatment. However.5 ± 0.3 ± 2.0 ± 2.5 8.0 9.0 ± 2.3 ± 1. there were no significant differences on L. And also.2 ± 0.0∗ 8.0 8.0∗ 10.1 11.4 10. coli O157:H7.5 ± 0.0 ± 0.1 mL) was added to 10 mL of diluted extracts for 5 min at room temperature.0 8.8 ± 1. If the DIZ value is 8.31 log CFU/g of S.5 8.6 11.5 ± 0. molmol (10.9 8. E. Treatment with 5% clove extract significantly reduced microbial level of S.6 9. 2011 r Journal of Food Science M43 a M: Food Microbiology & Safety 8.0 8.9 10.04 log by S. and 10% clove extracts for 10 min (Table 3).7 ± 0.2 10. 10 among 12 extracts produce relatively high levels of inhibition zone (10.4) and C. Although the color degradation was observed in all treated samples after 7 d.8 ± 1.3 ± 1.3 8. monocytogenes.6 14.6 8.8 10. L.7) and A. coli O157:H7.5 ± 1.1).5 11.3 ± 1.6 ATCC 15313 10.2 9.9 10. mm) (Mean ± SD)a E.7 8.1).5 ± 0. The remaining plant extracts produced inhibition zones between 8.3 ± 0. coli O157:H7 after 3 min and more than 2 log reductions were observed for 10 min treatment.3 ± 2.5 mm).0∗ 11.4 and 9.7 11. aromaticum effectively inhibited growth of the all 3 pathogens tested. The efficacy of clove extract for inactivating foodborne pathogens on fresh lettuce was evaluated (Figure 1 to 3). R. The maximum reduction achieved was 4. .4 8.0∗ 8.3 11.0∗ 8. and S.0 ± 1. followed by C. and L. coli O157:H7. Typhimurium.6 11.3 ± 2. the mean DIZ values of 7 plant extracts ranged from 8.1 12.7 ± 2. The effect of clove extract treatments shows similarity in S. The result showed the wide variation in the antimicrobial activities of plant extracts.8 10.3 ± 2.5 ± 2. and 1 mL of appropriate dilutions were spread onto Petrifilm aerobic count plates (3 M) for enumeration of survived cell populations.7 ± 1.

monocytogenes. This was consistent with the previous studies on other plantderived compounds. bacteria (Lis-Balchin and Deans 1997. Wan and others (1998) applied basil essential oils to wash fresh lettuce against natural microbial flora and the effectiveness was comparable with the treatment of 125 ppm chlorine. ◦ = 1% clove extract. But most of study is limited to performing on laboratory media. did not completely follow the trend described above. 8 6 Log10 CFU/g Log10 CFU/g M: Food Microbiology & Safety 4 2 0 2 4 6 8 10 12 Treatment time (min) Figure 2–Survival curves for Escherichia coli O157:H7 on lettuce leaves treated with distilled water or diluted clove extracts (1%. which showed the strongest antibacterial property applied in fresh lettuce. then verified in fresh produce by microbial reductions. cereus and L. Dorman and Deans Figure 1– Survival curves for Salmonella Typhimurium on lettuce leaves treated with distilled water or diluted clove extracts (1%. = 5% clove extract. Smith-Palmer and others 1998.Antimicrobial activity of plant extract . The error bars indicate 95% confidence intervals ( r = distilled water. 8 6 4 2 0 2 4 6 8 10 12 Treatment time (min) M44 Journal of Food Science r Vol. = 10% clove extract). although the effect on food samples may be different (Burt 2004). This result showed that the antimicrobial effect of clove extracts was immediate (within 10 min) and effective. gram-negative bacteria are more resistant to plant oil because it has hydrophilic cell wall. Shan and others 2007). . Mytle and others 2006. anatum were greater than the effects on B. . 76. 5%. Burt and Reinders 2003. and 10%) at room temperature (22 ± 2 ◦ C). and 10%) at room temperature (22 ± 2 ◦ C). = 5% clove extract. and the clove extract was selected among 12. 1. Nr. Our results suggest that gram-positive bacteria are generally more sensitive to the plant extracts than gram-negative bacteria. = 10% clove extract). clove extract. 5%. Shan and others (2007) also found that the inhibitory effect of clove extracts against S. 12 plant extracts that had antimicrobial activity were first screened by a disk diffusion assay. However. 2011 . Menon and Grag 2001. Hammer and others 1999. And the efficacy of clove extract for inactivating foodborne pathogens on fresh produce has not been investigated prior to present study. The error bars indicate 95% confidence intervals ( r = distilled water. In the present study. ◦ = 1% clove extract. which showed the strongest activity. Commonly.

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