Proceedings of the 27th West Indies Agricultural Economics Conference
In collaboration with
The Ministry of Agriculture and Fisheries, Belize,
Caribbean Agricultural Research and Development Institute (CARDI),
The University of the West Indies (UWI)
and
la Asociación de Latinoamérica y del Caribe de Economía Agrícola (ALACEA) Improving Marketing and Sustaining Natural Resource Systems in Latin America and the Caribbean
Belize City, Belize
23rd - 27th July, 2007
Sharon D. Hutchinson Editor
Copyright @ June 2009 by the Department of Agricultural Economics and Extension. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical, photocopy, recording or otherwise, without the prior consent of the publisher.
Antibacterial Efficacy of Eryngium foetidum (Culantro)- Peer Reviewed 179
Antibacterial Efficacy of Eryngium foetidum (Culantro) against Select Food-borne Pathogens
Sharon Homer, *Gail Baccus-Taylor & John Akingbala
Food Science and Technology Unit, Department of Chemical Engineering, Faculty of Engineering, University of the West Indies, St. Augustine, Trinidad and Tobago *Corresponding Author: 1-868-662-2002 Ext. 3408 [email protected] or [email protected]
Abstract
There is a growing trend of consumer preference for the use of natural food preservatives either to prevent the growth of food-borne pathogens, or to delay the onset of food spoilage. In this investigation, an in-vitro screening method was used to determine the antibacterial efficacy of a 10% w/v suspension of the natural leaves of the herb culantro (Eryngium foetidum), against Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Salmonella Typhimurium. The apparent sensitivity of the gram-positive bacteria and resistance of the gram-negative bacteria were distinct. Significant antibacterial activity was evident against S. aureus and B. subtilis, but no apparent antibacterial activity was evident against E. coli and S. Typhimurium. There was approximately 100% kill for both S. aureus and B. subtilis. The results obtained from this investigation suggest that culantro leaves can potentially be used as a food preservative, by increasing the safety and extending the shelf life of food products.
Keywords: natural food preservatives; food-borne pathogens; antibacterial efficacy; culantro leaves; in-vitro screening; % kill.
INTRODUCTION antimicrobials. Among the compounds having a wide spectrum of Internationally, approximately 1400 antimicrobial effectiveness are the herbs, spices and plants, have been essential oil thymol, from thyme and reported to be potential sources of oregano; eugenol from cloves;
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Antibacterial Efficacy of Eryngium foetidum (Culantro)- Peer Reviewed 180 cinnamic aldehyde from cinnamon attributes or residual toxicity, and allicin from garlic (Nychas 1996). substantiates the use of natural Although substantial literature exist preservatives like culantro, as a with respect to the antimicrobial desirable alternative in food properties of its relative coriander, applications (Nychas and Tassou there is very little information on the 1999). There is considerable interest antimicrobial properties of the herb in the possible use of such natural culantro, Eryngium foetidum, Family: alternatives as food additives, either Umbelliferae (or Apiaceae- parsley to prevent the growth of food-borne family); Common Names: long pathogens, or to delay the onset of coriander leaf, thorny coriander, food spoilage (Nychas 1995). culantro, recao, saw leaf, chandon There is need for more information beni [French], shado beni [English- on the antimicrobial efficacy of the speaking Caribbean] and bhandhania leaves of herbs. It has been [Trinidad & Tobago] (Ramcharan documented that Gram-positive 1999). bacteria such as Staphylococcus In the Caribbean Region, the aureus and Bacillus subtilis, are more harvested natural leaves of culantro sensitive to spices and herbs than are widely used as a food flavoring Gram-negative bacteria such as and seasoning for vegetable and meat Escherichia coli and Salmonella dishes, chutneys, preserves, sauces Typhimurium (Hirasa and Takemasa and snacks. Its medicinal value 1998). Therefore, the objective of this includes its use as a tea for flu, research was to determine the diabetes, constipation and fevers antibacterial efficacy of the freshly (Ramcharan 1999). Culantro is harvested leaves of the indigenous strongly flavored and pungent, and it herb, Eryngium foetidum, against the has been established by some common food-borne pathogens Gram researchers that the more aromatic positive Staphylococcus aureus and herbs exhibit greater antimicrobial Bacillus subtilis and Gram negative properties (Hirasa and Takemasa Escherichia coli and Salmonella 1998). Typhimurium.
Objectives of study MATERIALS AND METHODS
Increased consumer preference is Acquisition & storage of culantro occurring for minimally processed and leaves natural food products that are shelf- stable and safe for human Whole leaves of the culantro plant consumption. The excessive use of were obtained from a herbal garden chemical preservatives, many of located in the Sangre Grande region which are suspect because of their (Trinidad). The leaves were kept potential carcinogenic and teratogenic refrigerated (2 – 8ºC) in snap-sealed,
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Antibacterial Efficacy of Eryngium foetidum (Culantro)- Peer Reviewed 181 transparent, low-density polyethylene Bacillus subtilis, ATCC 6633 bags and used within 72h after (derived), Oxoid Quanti-Cult Plus harvesting. brand; (3) Escherichia coli, ATCC 35218 (derived), Oxoid Culti-Loop Preparation of culantro leaves brand and (4) Salmonella Typhimurium, ATCC 14028, Oxoid As shown if Figure 1, the culantro Culti-Loop brand. leaves were washed under cold, running tap water to remove any Preparation of bacterial cultures extraneous matter from the surface of the leaves. The leaves were then The bacterial cultures were immersed in a beaker of boiling water rehydrated according to the (100 + 1ºC) and blanched for 2 manufacturer’s instructions (Bridson minutes to reduce the microbial load. 1998). Duplicate tryptone soya broth After blanching, the leaves were tubes (10ml), [TSB - Oxoid, England] removed with sterile forceps, and were inoculated with 0.1 ml of each placed in a single layer on a sterile culture suspension, to provide the tray in a laminar flow cabinet to air working broth cultures to be used in dry, thereby maintaining sterile the test method. Duplicate tryptone conditions. soya agar plates [TSA - Oxoid, The leaves were visually examined England] were streaked with a loopful for the absence of moisture. Using of each culture suspension, to verify aseptic techniques, and within the purity of the test micro organisms by confines of the laminar flow cabinet, visual examination of colonies for the leaves were firstly cut into bits with uniformity in morphology. All tubes sterile, stainless steel scissors, to and plates were incubated at 35ºC for allow ease of grinding. A steam 24h and 48h, respectively. sterilized mortar and pestle were used Subcultures were prepared by to manually grind the bits of leaves, to streaking a loopful of broth culture release their antimicrobial active onto TSA test tube slants and components. incubating at 35ºC for 48h. Both subcultures and working cultures were refrigerated at 2-8ºC. Test micro organisms Quantitative suspension test The four (4) commercially obtained (Oxoid, Hampshire, England) food- Immediately after grinding, a 10% borne pathogenic bacteria used in the w/v suspension was prepared study were: (1) Staphylococcus according to Bagamboula et al. aureus, ATCC (American Type (2003). Aliquots (1g) of ground leaves Culture Collection) 6538 (derived), were aseptically weighed into sterile Oxoid Quanti-Cult Plus brand; (2) TSB (9 ml) [sterilized by autoclaving
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Antibacterial Efficacy of Eryngium foetidum (Culantro)- Peer Reviewed 182 at 121ºC for 15 minutes], contained in of the relevant dilutions were spread- wide-mouth universal bottles. plated onto pre-poured, solidified, Duplicate bottles were prepared for sterile TSA (autoclaved at 121ºC for each of the 4 test micro organisms 15 minutes) plates [disposable petri under investigation, the negative dishes, 90 x 15 mm]. Surface plating control and the unblanched leaves. All technique was used since the colonial bottles were vigorously hand-shaken morphology of surface colonies is to obtain an even suspension of herb. easily observed to distinguish any In a biological safety cabinet, each contaminating micro organisms from bottle was inoculated with the relevant typical colonies (Downes and Ito culture suspension (0.1 ml). No 2001). Sterility control plates of TSB, inoculum was added to the TSB BPW and TSA were included. The bottles containing the unblanched plates were inverted and incubated at minced leaves, and the negative 35 + 1ºC for 48 + 2 h. control, with the latter constituting only At the end of the incubation period, blanched ground leaves. Positive viable CFU/ ml were enumerated. The control bottles for comparative entire procedure, except for the analysis to evaluate efficacy of the unblanched leaves, was replicated 3 test samples, were also prepared by times for all 4 test bacteria, with all inoculating TSB (10 ml), with the analyses conducted in duplicate, in culture suspension (0.1 ml), for each each replication. test bacterium. A sterility control consisting of TSB only, to verify that Confirmatory tests the medium was free from contamination, was included. All Gram staining was performed on bottles were mixed and incubated at isolated colonies from each cultured 24 + 1ºC for 18-24 h, which was plate of the 4 test micro organisms. considered adequate conditions for The procedure was used to the experiential growth of these differentiate intact, morphologically bacterial broth cultures. similar bacteria based on cell colour, At the end of the contact time, the cell form, size and structural details bottles were removed from the (Becton, Dickinson and Company, incubator and the contents were 2001). The slide preparations were thoroughly hand-mixed. Using an examined microscopically under an oil Eppendorf pipetter and sterile tips, immersion lens, and compared with serial dilutions were prepared, with 1 colonies from the positive control ml of the TSB reaction mixture from plates. each bottle into 9 ml-sterile Buffered Peptone Water (autoclaved at 121ºC Staphylococcus aureus. for 15 minutes) diluent tubes [BPW – The coagulase test was conducted Oxoid, England]. The contents of the on the presumptive S. aureus colonies tubes were mixed and 0.1 ml volumes from both the test plates and the
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Antibacterial Efficacy of Eryngium foetidum (Culantro)- Peer Reviewed 183 corresponding +ve control plates. stabbing and streaking LIA [Lysine Colonies were picked from the TSA Iron Agar – Oxoid, England] slants. plate and inoculated into 0.5ml Slants were incubated at 35 + 1ºC for coagulase plasma with EDTA [BD 24 + 2 h. The agar slants were BBL, Maryland, U.S.A.] tubes, examined for changes in colour, incubated at 35 + 1°C, and observed indicative of acid/ alkaline production, over a six (6) hour period for and also for hydrogen sulphide coagulation. A positive control using production. A positive control using the reference control S. aureus, a the reference control S. Typhimurium, negative control using the reference a negative control using the reference control B. subtilis and a sterility control control E. coli and a sterility control without culture to verify that the were included (USFDA 1998). TSI medium was free from contamination, cultured slants were sent to a were included (Downes and Ito 2001). reference laboratory (Trinidad Public Health Laboratory) for serological Escherichia coli. confirmation of S. Typhimurium. Colonies were picked from the TSA plate and inoculated into EC broth IMViC tests (10ml), [E. coli Broth - Difco, Further biochemical tests were Maryland, U.S.A.] tubes containing an simultaneously conducted on inverted Durham vial. Tubes were presumptive E. coli and S. incubated in a circulating water bath Typhimurium colonies. Colonies were at 45 + 0.5°C for 48 + 2 h and picked from the respective TSA plates observed for gas production. A and IMViC (Indole, Methyl red, Voges- loopful of culture suspension was Proskauer and Citrate) biochemical aseptically taken from each gassing tests were conducted. Each bacterium tube and streaked unto Levine Eosin functioned as the negative control for Methylene Blue Agar [L-EMB, Oxoid, the other. An un-inoculated medium England] plates. Plates were control was used for each test to incubated at 35 + 1°C for 18 to 24 h eliminate any false positives. and then examined for typical For the Indole test, tubes of colonies. A positive control using the tryptone water (5 ml) [Oxoid, England] reference control E. coli, a negative were inoculated with isolated colonies control using the reference control S. and incubated 24 ± 2 hr at 35 ± 1.0ºC. typhimurium and a sterility control A volume of 0.2 – 0.3 ml Kovac’s were included (USFDA 1998). reagent (USFDA – BAM, 1998) was added to each tube and observed for Salmonella Typhimurium indole production, evident by the Colonies were picked from the TSA appearance of a distinct red color on plate and inoculated by streaking and the surface of the medium (USFDA stabbing TSI [Triple Sugar Iron – 1998). Oxoid, England] agar slants, and
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Antibacterial Efficacy of Eryngium foetidum (Culantro)- Peer Reviewed 184 For Voges-Proskauer-reactive replicates, according to the procedure compounds, tubes of MR-VP medium for computing and reporting by (6 ml) [Oxoid, England] were Downes and Ito (2001). inoculated with isolated colonies and incubated for 48 ± 2 hr at 35 ± 1.0ºC. After incubation, 0.6 ml of 5% alpha- RESULTS AND DISCUSSION naphthol solution [BDH, England] and 0.2 ml 40% KOH solution (potassium The mean number of CFU/ g in the hydroxide – commercially available) blanched (100 + 1ºC for 2 minutes) were added to a 1-ml culture leaves, the negative control, was suspension and vigorously hand- below the detection limit (<10 shaken. Few crystals of creatine CFU/ml), indicating that the blanching [Sigma, USA] were added, contents process effectively reduced were again shaken, and the tubes contaminating micro organisms on the kept at ambient temperature in BSC leaves. The absence of any viable for 2 hr. The contents were observed CFU from both the sterility controls for the development of an eosin pink and the negative control also colour throughout the medium indicated that aseptic techniques (USFDA 1998). employed throughout the procedure For Methyl Red-reactive were effective. compounds, the remaining contents of The mean population count for the the MR-VP tube were re-incubated for S. aureus positive control was an additional 48 ± 2 hr at 35 ± 1.0ºC. approximately 109 CFU/ ml. The mean After incubation, 5 drops of methyl red population count for the B. subtilis solution (methyl red in ethanol positive control was approximately 107 solution) were added to each tube and CFU/ ml. The mean population counts observed for a diffused red colour in for both the E. coli and S. the medium (USFDA 1998). Typhimurium positive controls were For utilization of Citrate, Simmon’s approximately 109 CFU/ ml. Citrate Agar [Oxoid, England], slants were inoculated by streaking and Antibacterial activity of 10 % (w/v) stabbing with an inoculating needle. culantro (Eryngium foetidum) The slants were incubated for 48 - 96 leaves on S. aureus and B. subtilis hr at 35 ± 1.0ºC and then observed for a colour change from green to blue As shown in Table 1, there was a (USFDA 1998). 99.99% kill of S aureus in the test sample, when compared with the number of viable organisms in the Statistics control. The reduction is similar to the findings of Nychas (1995), in which The mean CFU/ml was calculated almost all essential oils from herbs using data from all three (3)
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Antibacterial Efficacy of Eryngium foetidum (Culantro)- Peer Reviewed 185 tested against S. aureus, inhibited its As seen in Table 2, in contrast to growth. the significant antibacterial efficacy Reduction in the number of viable obtained against S. aureus and B. organisms was even greater for B. subtilis, there was no reduction in the subtilis, with approximately 100% kill counts of E. coli and S. Typhimurium. in the test sample, which was less Similarly, it was reported that E. coli than 1.0 * 102 CFU/ ml for data was relatively resistant to the volatile analysis. Counts of B subtilis oils of dill weed and thyme (Hirasa obtained for the positive control were and Takemasa 1998). According to lower than the counts obtained for the Nevas et al. (2004), S. Typhimurium other 3 test organisms. This could be and E. coli were among the gram- due to the optimal growth negative bacteria that were most requirements of B. subtilis, which resistant to the 13 essential oils grows better under agitation, and in a studied. growth medium that is richer than The CFU/ ml obtained was slightly TSB. TSB provides only the minimal greater for both E. coli and S. nutrients required for growth (Dahl Typhimurium test samples (3.7 x 109 2000). Additionally, B. subtilis is a and 3.6 x 109 CFU/ ml respectively), sporing bacterium and according to compared to the controls (1.3 x 109 Dahl (2000), the sporulation process and 1.6 x 109 CFU/ ml respectively). is initiated at the end of the Since the blanching process (negative exponential growth phase. External control) and the sterility (media) (and presumably also internal) controls showed no viable signals, force the cell to respond by contaminating micro organisms, the inhibiting cell division and initiating the increase in number of viable microbes sporulation process (Dahl 2000). could have been due to a possible In a study comparing the growth-promoting factor present in the susceptibility of microorganisms to the culantro leaves. volatile oils of herbs, B. subtilis was the most susceptible to the volatile Confirmation of purity of bacteria oils of dill weed and thyme. Hexane extracts of rosemary and sage were All colonies visually appeared to be found to possess especially strong of the same colony type on TSA inhibitory activity against S. aureus plates, for each individual bacterium. and B. cereus (Hirasa and Takemasa Microscopic examination of the Gram- 1998). stained colonies obtained from both the test plates, as well as from the Antibacterial activity of 10 % (w/v) corresponding positive control plates, culantro (Eryngium foetidum) revealed similar characteristics. leaves on E. coli and S. Typhimurium Morphological and biochemical characteristics of the bacterial
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Antibacterial Efficacy of Eryngium foetidum (Culantro)- Peer Reviewed 186 colonies from the test and control 2001). The negative control (E. coli) TSA plates produced an acid slant and butt (yellow throughout) and no H2S. The morphological characteristics There was no change in appearance observed for the 4 bacteria are of the sterility control (un-inoculated outlined in Table 3. For the coagulase medium). The reactions observed on test, coagulation was evident within 6 the LIA were also typical of hours, which is a reaction typical of Salmonella spp. that is, an alkaline coagulase-positive S. aureus. No (purple) butt from the decarboxylation coagulation was evident in the of lysine and production of H2S negative control (B. subtilis) and the (Downes and Ito 2001). The negative sterility control tubes. control produced an acid (yellow) butt The production of gas in EC and no H2S. There was no change in medium, when incubated at 45.5°C appearance of the sterility control (un- for 48h, is considered to be a positive inoculated medium). reaction for the presence of faecal coliforms, as well as presumptive E. IMViC reactions for E. coli and S. coli (Downes and Ito 2001). Both the Typhimurium test sample and the positive control gave positive reactions. No gas Table 4 reveals that the colonies production was evident in the negative on the TSA test plate were confirmed control (S. Typhimurium) and the as E.coli by the IMViC tests. IMViC sterility control tubes. tests are used to elicit specific Colonies on L-EMB selective agar biochemical reactions between certain from the test sample were typical of bacteria and the reagent(s) used. The the characteristic nucleated, dark- resulting interactions are considered centered colonies with green sheen, to be either positive (+) or negative (-), as that of the E. coli colonies and the combination of results is used observed on the positive control in identifying the particular bacterium. plates (Downes and Ito 2001). There The biochemical reactions obtained was no growth on the negative control were identical to that obtained from and sterility control plates. the colonies on the positive control The reactions from the TSA plates. All presumptive cultures presumptive S. Typhimurium colonies, that give IMViC patterns of ++-- when inoculated on the TSI and LIA (biotype 1) or -+-- (biotype 2) are slants, were identical for both the test considered to be E. coli (USFDA and the positive control. The reactions 1998). No visible reaction occurred in observed were typical of Salmonella the tests using the un-inoculated spp. on TSI agar, which are an medium, indicating that there were no alkaline (red) slant and acid (yellow) false positives. The reactions obtained butt with the production of H2S from the IMViC tests are represented [blackening of agar] (Downes and Ito in Table 4.
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Antibacterial Efficacy of Eryngium foetidum (Culantro)- Peer Reviewed 187 IMViC tests conducted on colonies components of their essential oils from the test plate and the +ve control (Hirasa and Takemasa 1998). plate produced identical reactions Herbs, especially their essential typical for Salmonella spp. that is -+-+ oils, are more active against gram (USFDA 1998). No visible reaction positive than gram negative bacteria occurred in the tests using the un- (Davidson and Zivanovic 2003). The inoculated medium, indicating that activity of essential oils of herbs there were no false positives. The against gram positive bacteria is reactions obtained from the IMViC bacteriocidal, while it is bacteriostatic tests are represented in Table 4. against gram negative bacteria Further biochemical and serological (Nychas and Tassou 2000). Rota et tests conducted by the reference al. (2004) reported that the MIC of the laboratory confirmed that the culture essential oils from aromatic plants on the TSI slant was Salmonella was lower for gram-positive S. aureus, Typhimurium. than for gram-negative bacteria, including S. Typhimurium and E. coli Comparison with referenced O157:H7. Culture medium, literature temperature and inoculum size also influence antimicrobial activity of In all three (3) replications, essential oils (Nychas and Tassou significant antibacterial activity was 2000). evident against the gram-positive S. aureus and B. subtilis. No CONCLUSION antibacterial activity was apparent against the gram-negative E. coli and The blanching process of 100 + 1ºC S. Typhimurium. These findings are for 2 minutes was effective in reducing similar with those of Nevas et al. the normal micro flora on the culantro (2004), in which the gram-positive leaves, to below the detection limit. bacteria appeared to be more The concentration of the leaves sensitive to the essential oils than the suspension, 10% w/v, and the mincing gram-negative bacteria. These process, were effective for researchers found that S. antimicrobial activity. The contact Typhimurium and E. coli were among period and time used, 18-24h at 24 + the gram-negative bacteria that were 1ºC respectively, provided adequate most resistant to the essential oils conditions to produce antimicrobial studied. Most pungent herbs like efficacy. culantro, have relatively strong The test bacteria and the method antibacterial and/or antifungal selected for this study proved to have properties. The majority of been very applicable since the antimicrobial components of these difference in viability, and therefore pungent herbs are found in volatile efficacy, was distinctly evident between the gram-positive and the
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Antibacterial Efficacy of Eryngium foetidum (Culantro)- Peer Reviewed 188 gram-negative bacteria. The v To determine the MIC of the sensitivity of the gram-positive essential oil on these test micro bacteria and the resistance of the organisms. gram-negative bacteria to the culantro v To determine the MIC of dried leaves were clearly apparent. culantro leaves on these test The antibacterial efficacy of the micro organisms. leaves of the indigenous herb, v To determine the effect on efficacy Eryngium foetidum, against these by varying parameters such as select common food borne pathogens contact time and temperature. Staphylococcus aureus, Bacillus v To determine the antimicrobial subtilis, Escherichia coli and efficacy on other food-borne Salmonella Typhimurium, was pathogenic and spoilage bacteria, determined. At 10% w/v, the culantro as well as fungi. leaves exhibited significant v To determine the antioxidant antibacterial efficacy against gram- properties of the active positive Staphylococcus aureus and components in the culantro plant. Bacillus subtilis, but not against gram- v To determine the antimicrobial negative Escherichia coli and efficacy in real food products, Salmonella Typhimurium. conduct sensory evaluations and Implications perform comparative analysis on impact on shelf life. The results obtained from this v To use other test methods for investigation indicated that culantro comparative analysis of efficacy. leaves do possess antimicrobial v To test and compare efficacy with components, with significant other locally grown herbs. antimicrobial activity. Therefore, in theory, culantro leaves can be used REFERENCES as a natural additive in the preservation of food, by increasing the Bagamboula, C.F., M. Uyttendaele, safety and extending the shelf life of and J. Debevere. 2003. food products. Culantro’s Antimicrobial effect of spices and characteristic intense flavour may also herbs on Shigella sonnei and enhance the organoleptic properties in Shigella flexneri. Journal of Food food products such as meat, sauces, Protection 66 (4): 668-673. dressings and condiments. Becton, Dickinson and Company, 2001. Leaflet. Gram Stain Kits Recommendations for future and Reagents. Maryland: Becton, research Dickinson and Company. v To determine the MIC of the Block, Seymour S. 2001. natural culantro leaves on these Disinfection,Sterilization, and test micro organisms. Preservation. 5th ed. Philadelphia:
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Antibacterial Efficacy of Eryngium foetidum (Culantro)- Peer Reviewed 189 Lippincott Williams & Wilkins. Nychas, G.J.E. 1995. Natural 1321. antimicrobials from plants. New Bridson, E. Y., comp. 1998. The Methods of Food Preservation. Oxoid Manual. 8th ed. Hampshire: Edited by G.W. Gould. London: Oxoid Limited. 207-209, 229-231. Blackie Academic & Professional. Dahl, Michael K. 2000. Bacillus 59-69. subtilis. In Encyclopedia of Food Nychas, G.J. E., and C.C. Tassou. Microbiology. Vol.1. Edited by 2000. Traditional preservatives – Richard K. Robinson. London: oils and spices. In Encyclopedia Academic Press. 135-141. of Food Microbiology. Vol.3. Davidson, P. M. 2000. Antimicrobial Edited by Richard K. Robinson. compounds. In Encyclopedia of London : Academic Press. 1717- Food Science and Technology. 1722. 2nd. Ed. Vol. 1. Edited by Ramcharan, C. 1999. Culantro: A Frederick J. Francis. New York: much utilized, little understood John Wiley & Sons, Inc. 63-75. herb. In Perspectives On New Davidson, P.M., and S. Zivanovic. Crops And New Uses. [online]. 2003. The use of natural Last updated 05 Nov. 2001 [cited antimicrobials. Food Preservation 28 February 2006]. Edited by J. Techniques. Edited by Peter Janick, 506–509. Virginia : ASHS Zeuthen and Leif Bogh-Sorensen. Press. Available from World Wide Cambridge: Woodhead Web: Publishing Limited and CRC
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Antibacterial Efficacy of Eryngium foetidum (Culantro) 190 Wash & blanch leaves (Blanch at 100 ºC/2 minutes)
Aseptically grind leaves with sterilized mortar and pestle
Weigh 1 g aliquots of ground leaves into TSB bottles (duplicates)
Vigourously hand-mix
Inoculate samples & positive controls with suspension of test bacterium
Incubate @ 24 ºC/24 hours
Serially dilute in BPW tubes prepared from samples & controls
Spread plate dilutions onto TSA plates
Incubate plates at 35ºC/48 hours
Count colonies (CFU/ ml)
Conduct confirmatory tests
Compare data analysis between samples and controls
Figure1. Flow Chart for Determining Antibacterial Efficacy of Eryngium foetidum (Culantro)
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Antibacterial Efficacy of Eryngium foetidum (Culantro) 191
Table 1: Antibacterial Activity of 10 % (w/v) Culantro (Eryngium foetidum) Leaves on S. aureus and B. subtilis.
S. aureus B. SUBTILIS Gram Positive Test Bacteria Test Control Test Control Mean CFU/ ml 1.2 x 105 2.0 x 109 1.0 x 102 3.9 x 107
% Kill 99.99 100
Source: Compiled by author
Table 2: Antibacterial Activity of 10 % (w/v) Culantro (Eryngium foetidum) Leaves on E. coli and S. Typhimurium.
E. coli S.TYPHIMURIUM Gram Negative Test Bacteria Test Control Test Control
Mean 3.7 x 109 1.3 x 109 3.6 x 109 1.6 x 109 CFU/ ml
% Kill 0 0
Source: Compiled by author
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Antibacterial Efficacy of Eryngium foetidum (Culantro) 192
Table 3: Morphological Characteristics of the Bacteria Colonies from both the Test and Control TSA Plates
Test and Positive Control TSA Gram Stain
Staphylococcus aureus Yellow colonies, Gram positive, large smooth margin cocci in clusters
Bacillus subtilis White colonies, Gram positive, irregular margin, dry, sporing rods, central flat spores
Escherichia coli Cream colonies, Gram negative, rods smooth margin
Salmonella Typhimurium Cream colonies, Gram negative, rods smooth margin Source: Compiled by author
Table 4: IMViC reactions for E. coli and S. Typhimurium
Test and Control I M V C
Test + + - - E. coli
Control + + - - E. coli
Test - + - + S. Typhimurium
Control - + - + S. Typhimurium
Sterility Control No No No No Un-inoculated change change change change Source: Compiled by author
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Antibacterial Efficacy of Eryngium foetidum (Culantro) 148
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