ANTIBACTERIAL ACTIVITY OF THAI HERB AND SPICE EXTRACTS AGAINST THE POTENT FOODBORNE BACTERIA

Seven herb and spice extracts of Allium cepa (onion), Allium sativum (garlic), Areca catechu (betel nut), Capsicum frutescens (chili), Psidium guajava (guava), Mentha cordifolia (mint) and Alpinia galanga (galangal) were chosen to screen for antibacterial activity against eight strains of Gram-positive and Gram-negative bacteria using a disc-diffusion method. Garlic extract exhibited an inhibition effect against five strains whereas guava and betel nut extracts showed antibacterial activity against four strains. Further studies showed that the minimum inhibitory concentration (MIC) of garlic, guava and betel nut extract against bacteria was ranging from 62.5-500 mg/ml. These results showed that herb and spice extracts may prove useful in inhibiting bacteria. INTRODUCTION Microbial activity is one of the primary modes of deterioration of many foods and is often responsible for food quality reduction, spoilage, and economic loss. The approaches to regulating microbial deterioration of foods are the direct addition of antimicrobials and the use of antimicrobials in food contact surfaces such as packaging materials. Despite the widespread use and effectiveness of chemicals interventions, researchers are continually investigating the use of other novel antimicrobial agents such as natural plants. Herbs and spices are known for antimicrobial properties (Pandit and Shelef, 1994; Cutter, 2000). In Thailand, there are hundreds of herbs and spices and our preliminary experiments showed that aqueous extract of Allium cepa (onion), Allium sativum (garlic), Areca catechu (betel nut) and Capsicum frutescens (chili) had inhibitory efficiency against two common foodborne bacteria: Escherichia coli and Staphylococcus aureus (Panutat and Vatanyoopaisarn, 2002). Thus, the following study was conducted to determine antibacterial activity of those herbs to more bacterial strains. In addition, three more variety of the popular herbs: Psidium guajava (guava), Mentha cordifolia (mint), Alpinia galanga (galangal) were also tested. MATERIALS AND METHODS Bacterial Strains The following bacterial cultures were obtained from the culture collection of Thailand Institute of Scientific and Technological Research, Bangkok, Thailand (MIRCEN): Bacillus cereus TISTR 037, Escherichia coli TISTR 073, Proteus mirabilis TISTR 100, Pseudomonas aeruginosa TISTR 357, Pseudomonas fluorescens TISTR 358 and Staphylococcus aureus TISTR 029. Additional cultures, Salmonella sp. was isolated from retail market chicken meat, whereas Ps. aeruginosa (Pk) was isolated from a dairy farm (Saraburee, Thailand). All cultures were maintained on nutrient agar (NA) slant at 4°C and were grown overnight on NA slant for 3 consecutive times at 37°C prior to the biocide testing. Proc. WOCMAP III. Vol. 4: Targeted Screening of MAPs, Economics & Law Eds. C. Franz, Á. Máthé, L.E. Craker and Z.E. Gardner Acta Hort. 678, ISHS 2005 192 Preparation of Herb and Spice Extracts Fresh herbs and spices were purchased from a local market. An aqueous crude extract was prepared as follows: 100 g of each spices and herbs were chopped and soaked in 200 ml distilled water for 1 h at 37°C with shaking (200 rpm). After grinding with the homogenizer, the homogenate was passed through the filter papers and the filtrate was then centrifuged at 5,000 g for 10 min. The supernatant was collected and sterilized by membrane filtration (0.45 μm in diameter). The extracts were kept at 4°C in refrigerator for inhibition assay. Determination of MIC by Paper Disc Diffusion Method The overnight grown cultures were suspended in normal saline solution to an OD600 of 0.5. The cell suspension was then diluted 1:100 to obtain the initial cell number of 10-10 CFU/ml. An aliquot (0.1 ml) of the diluted cultures were inoculated on plate count agar (PCA) using spread plate technique (Collins et al., 1995). All the extracts were tested at a concentration of 500 mg/ml. Twofold serial dilution of the extracts was prepared in distilled water down to 62.5 mg/ml. Paper discs (6 mm in diameter) were moistened with 20 μl of each diluted extract and placed on the surface of the assay plates, which were incubated for 24 h at 37°C. The diameter (mm) of the clear inhibition zone was measured and the MIC was assessed as the lowest concentration of the extract that prevented growth of the test bacteria (inhibition zone >10 mm) was recorded. Three replications were performed. RESULTS AND DISCUSSION Many methodologies have been employed for determination of antibacterial activity of herb and spice extracts. The widespread used methods included paper disc agar diffusion, agar well diffusion and addition of the extracts in agar media. The antibacterial activity of the extracts varied somewhat depending on the testing system (Wan et al., 1998). In order to determine the MIC that would be applied for edible films or solid food, herb and spice extracts were determined of antibacterial activity using a disc diffusion method. Fig. 1, 2 and 3 show the effect of herb and spice extracts on the antibacterial activity. With mint extract, no inhibitions could be observed in any strains. However, all other extracts showed various effects on following different strains of bacteria. Ps. fluorescens was inhibited by the guava leaves and betel nut extracts (Fig. 1a). The culture collection strain of Ps. aeruginosa (TISTR 357) was suppressed by garlic extract whereas the same species of the farm isolate (Pk) did not produce any detectable zone of growth inhibition (Fig. 1b). The garlic extract also showed an inhibitory effect to E. coli as well as effective against P. mirabilis and Salmonella sp. (Fig. 2). Furthermore, P. mirabilis was also sensitive to betel nut and guava leaves extracts. The Gram-positive bacterium B. cereus was sensitive to betel nut and garlic extracts, while S. aureus was only sensitive to betel nut extract. Onion, chili and galangal extracts showed slightly inhibition effects to only S. aureus. The MIC of each strain was then summarized in Table 1. It was apparent that the extracts of betel nut and guava leaves showed strong biocide activity against the similar strain of bacteria, whereas the extract of garlic exhibited an inhibition to the different strains. The antibacterial activity of garlic extract obtained in this study is similar to that reported by Kumar and Berwal (1998), showing the use of 1-10% fresh garlic in suppressing the growth of E. coli, Salmonella typhi, Listeria monocytogenes and S. aureus. Although our study has agreed upon the effect of garlic against E. coli and Salmonella sp. , the garlic extract, however, did not show the inhibition to S. aureus. This could be due to the highest concentration of the extract (500 mg/ml) used was not sufficient to suppress the growth of S. aureus. Commonly known, garlic produces Alliin and Allicins, which are board-spectrum antimicrobial agents, and seems to inhibit sulfhydryl enzyme in many species of bacteria (Council for Agricultural Science and Technology, 1998). The chemical compositions of betel nut and guava extract remained