Redalyc.Effect of Chili (Capsicum Annuum L.) Extracts and Derived Compounds on Growth of Erwinia Carotovora Subsp. Carotovora (J

Revista Mexicana de Fitopatología ISSN: 0185-3309 [email protected] Sociedad Mexicana de Fitopatología, A.C. México

Acero Ortega, Claudia; Dorantes Alvarez, Lidia; Jaramillo Flores, María Eugenia; Hernández Sánchez, Humberto; López Malo, Aurelio Effect of Chili (Capsicum annuum L.) Extracts and Derived Compounds on Growth of Erwinia carotovora subsp. carotovora (Jones) Bergey, Harrison, Breed, Hammer and Huntoon Revista Mexicana de Fitopatología, vol. 21, núm. 2, julio-diciembre, 2003, pp. 233-237 Sociedad Mexicana de Fitopatología, A.C. Texcoco, México

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Effect of Chili (Capsicum annuum L.) Extracts and Derived Compounds on Growth of Erwinia carotovora subsp. carotovora (Jones) Bergey, Harrison, Breed, Hammer and Huntoon

Claudia Acero-Ortega, Lidia Dorantes-Alvarez, María Eugenia Jaramillo-Flores, Humberto Hernández-Sánchez, Depto. de Graduados e Investigación en Alimentos, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Carpio y Plan de Ayala, México, D.F., México CP 11340; and Aurelio López-Malo, Depto. de Ingeniería Química, Alimentos y Ambiental, Universidad de las Américas, Sta. Catarina Mártir, Cholula, Puebla, México CP 72820. Correspondencia: [email protected]

(Received: December 19, 2002 Accepted: January 29, 2003)

Acero-Ortega, C., Dorantes-Alvarez, L., Jaramillo-Flores, Orto-cumárico, ácido Meta-cumárico, ácido Trans-cinámico, M.E., Hernández-Sánchez, H., and López-Malo, A. 2003. Capsaicina, Dihidrocapsaicina. Effect of chili (Capsicum annuum L.) extracts and derived compounds on growth of Erwinia carotovora subsp. Erwinia carotovora is a soil bacterium that affects vegetables, carotovora (Jones) Bergey, Harrison, Breed, Hammer and such as carrots [Daucus carota subsp. sativus (Hoffm.) Huntoon. Revista Mexicana de Fitopatología 21:233-237. Arcang.], potatoes (Solanum tuberosum L.), cucumbers Abstract. Extracts from Habanero, Serrano and bell pepper (Cucumis sativus L.), onions (Allium cepa L.), tomatoes (Capsicum annuum) varieties were evaluated for its natural (Lycopersicon esculentum Mill.), lettuce (Lactuca sativa L.), antimicrobial compounds on growth of the phytopathogenic and green peppers (Capsicum annuum L.). Crops can be bacterium Erwinia carotovora subsp. carotovora. Bacterial infected in the field as well as during storage. Affected tissues suspensions were prepared and inoculated into culture media become soft and watery, turning slimy and foul-smelling. This on which sterile filter paper discs saturated with the extract disease known as bacterial soft rot is caused by E. c. subsp. were placed. Growth of the microorganism was inhibited by atroseptica (van Hall) Dye, and E. carotovora subsp. extracts from the three varieties and by some of the carotovora Jones (Bergey et al., 1923). However, the latter compounds found in the extracts, like meta-coumaric and is more common and has a wide host range. Although Erwinia trans-cinnamic acids. Capsaicin and dihydrocapsaicin did not carotovora is usually present on the surface of fruits or tubers, affect the growth of the bacterium. they require a wound to invade the interior (Kaláb, 2002). Germination of contaminated seeds is reduced and seedlings Additional keywords. Antimicrobial effect, Ortho-coumaric can easily be infected (Tegene and Korobko, 1985). It is acid, Meta-coumaric acid, Trans-cinnamic acid, Capsaicin, important to develop disease management strategies for soft Dihydrocapsaicin. rot. Currently, it is recommended: a) the use of high quality, pathogen-free seeds or tubers (for potatoes), b) to avoid Resumen. Se evaluaron extractos de los chiles (Capsicum mechanical injury, c) to avoid over-irrigation, and d) the use annuum) habanero, serrano y pimiento por el efecto de sus of proper postharvest storage conditions. Genetic engineering compuestos antimicrobianos naturales en el crecimiento de has been tested in potatoes for control of soft rot by attaching la bacteria fitopatógena Erwinia carotovora subsp. the promoter portion of the ubiquitin7 gene to an anti-rot carotovora. Se prepararon e inocularon las suspensiones gene effective, when tubers are damaged; this method has bacterianas en medios de cultivo en los que se colocaron been 85 to 96% effective (Wood, 1998). Although chemical discos de papel filtro estériles saturados con los extractos. control is not usually recommended, the post-plant application Los extractos de las tres variedades de chile y algunos de sus of some copper-based compounds was effective in reducing constituyentes como los ácidos meta-cumárico y trans- soft rot; however, these compounds also reduce the overall cinámico inhibieron el crecimiento del microorganismo. La plant growth (Gracia-Garza et al., 2002). Over the years, capsaicina y la dihidrocapsaicina no afectaron el crecimiento antimicrobial properties of substances of natural origin have de la bacteria. been recognized, including those derived from animals, plants, and microorganisms. It is known that spices and Palabras clave adicionales. Efecto antimicrobiano, ácido aromatic herbs have different properties, in addition to those 234 / Volumen 21, Número 2, 2003 for which they are grown. Furthermore, due to their in Trypticase Soy Broth (Dibico, Mexico, DF) and grown to bacteriostatic, bactericidal, and fungicidal properties, certain a concentration of 6 x 108 bacteria/ml at 28ºC (around 24 h) species increase the useful life of foods, and some even to be used as inoculum in the inhibition tests. prevent food spoilage by their antioxidant activity (Shelef et Inhibition tests with the extracts. Chili extracts were al., 1980). Antimicrobial components in plants are frequently evaluated in their ability to inhibit growth of E. c. subsp. found in essential oil fractions (Deans and Ritchie, 1987; Jay carotovora. The procedure was carried out using the standard and Rivers, 1984; Morris et al., 1979). Various plant zone inhibition test on Trypticase Soy Agar (Dibico, México, compounds have been found to exert a large spectrum of DF) as described by Conner and Beuchat (1984). Petri dishes antimicrobial effects, including thymol from thyme and were inoculated with 0.1 ml of inoculum prepared as already oregano, and cinnamic aldehyde, and eugenol from cinnamon described. The inoculum was spread uniformly over the and clove, respectively (Beuchat and Golden, 1989). Vanillin, culture medium using a sterile cell spreader. Next, 0.6 cm the main component of vanilla, is structurally similar to sterile filter paper discs were soaked individually with 20 ml eugenol and has shown to inhibit the growth of molds and of each of the three chili extracts (Morris et al., 1979). Extracts yeasts (López-Malo et al., 1995, 1997). Many of the were allowed to absorb completely before placing the discs compounds mentioned are related to capsaicin, the flavor and in the center of each inoculated Petri dish (one disc per plate). pungent principle in hot peppers, which are present in different All tests were repeated in triplicates. A control was included concentrations. They are also called capsaicinoids and are in which a filter paper disc soaked with 20 ml of isopropanol synthesized by the phenylpropanoid pathway (Dorantes et was used to see whether or not the alcohol in the extract al., 2000). Also, Kim and Ryeom (1979) reported antibacterial contributed to the inhibitory effect. Plates were incubated at effects of capsaicin from Korean hot pepper. The majority of 28ºC for 24 h, and the inhibition zone was measured with a studies on the antimicrobial activity of spices and their vernier (Morris et al., 1979). essential oils have been carried out with food-borne Inhibition tests using individual components of extracts. microorganisms. Few reports have been published on After testing the effect of each extract on the microorganism, antimicrobial compounds against Erwinia. Thyme essential another test was performed using the individual compounds oil has been reported to be an excellent antimicrobial agent previously identified by Dorantes et al. (2000) by HPLC in against E. carotovora and E. amylovora (Horváth et al., each extract (Table 1). Tests were carried out as described 2002); the essential oil of Rosa damascene has an effective before, but the filter paper discs were impregnated with 20 antibacterial activity against E. amylovora (Basim and Basim, ml of each individual compound (purchased from Sigma 2000); and the aqueous extracts of onion, garlic, and leek Chemical Company, St. Louis, USA) at a concentration equal were good antibacterials against E. carotovora subsp. to the one present in each extract. carotovora (Lirio et al., 1998). The objective of this study Inhibition tests using extract combinations. Two or more was to evaluate the effect of extracts from three varieties of of the identified compounds were combined to carry out chili (Capsicum sp.), their derived compounds, and the inhibition tests, to be able to establish if any of the combinations of some of these compounds on Erwinia combinations showed synergistic, antagonist, or additive carotovora subsp. carotovora. effects. Combinations included only compounds that Samples and extraction protocol. Chilies with different individually were proved to be inhibitory (Table 2). degrees of pungency, high, medium, and low (habanero, Statistic analysis. The means and the standard deviations of serrano, and bell pepper, respectively) were bought in a local inhibition halos were calculated, and a one-way analysis of market and ground in a food processor with isopropyl alcohol in a 1:1 (weight:volume) ratio. The mixtures underwent a Table 1. Compounds identified in the extracts from the three Soxhlet type of extraction for approximately 15 min, after varieties of chili studied. reaching the boiling point. They were filtered through a large Chili Compound Concentration pore filter paper and the procedure was repeated twice in (mg/ml) order to maximize the extraction. Active charcoal was added to the filtrate in a proportion of 15%, based on the weight of Habanero Ortho-coumaric acid 0.09 chili initially used. The mixture was shaken for 5 min and Capsaicin 6.00 filtered through Whatman filter paper No. 1. The cake was Dihydrocapsaicin 0.80 discarded and the alcohol was removed from the clear filtrate Serrano Meta-coumaric acid 0.30 by rotavaporation at a controlled temperature and pressure. Ortho-coumaric acid 0.90 Finally, the extract was frozen until further use (Dorantes et Trans-cinnamic acid 0.50 al., 2000). Capsaicin 0.08 Strain. Erwinia carotovora subsp. carotovora B-136 in Dihydrocapsaicin 0.05 freeze-dried form was obtained from the Microbial Properties Bell pepper Research of the National Center for Agricultural Research Meta-coumaric acid 0.20 (USDA, Peoria, Ill, USA). The microorganism was cultured Trans-cinnamic acid 0.20 Revista Mexicana de FITOPATOLOGIA/ 235

Table 2. Combinations of different compounds tested in the three Capsicum sp. extracts. Extract Compouns identified with Combination tested inhibitory effect Habanero Ortho-coumaric acid (0.09 mg/ml) Ortho-coumaric acid + meta-coumaric acid (Serrano) Ortho-coumaric + meta-coumaric acid (bell pepper) Ortho-coumaric acid + trans-cinnamic acid (Serrano) Ortho-coumaric acid + trans-cinnamic acid (bell pepper) Serrano Meta-coumaric acid (0.3 mg/ml) Meta-coumaric acid + trans-cinnamic acid Trans-cinnamic acid (0.5 mg/ml) Bell pepper Meta-coumaric acid (0.2 mg/ml) Meta-coumaric acid + trans-cinnamic acid Trans-cinnamic acid (0.2 mg/ml)

variance was performed for these results. In order to establish phytopathogen was inhibited by several compounds present the effect of the different derived compounds in the in the extracts of the chilies used in this study (Table 3). In concentrations found in each extract on the inhibition halo, a the case of bell pepper and serrano chili, meta-coumaric acid multiple regression analysis was performed. showed a good individual inhibitory effect (4 mm inhibition E. carotovora subsp. carotovora growth was highly inhibited halos). To determine the effect of different compounds, when by the three chili varieties tested, showing in all cases, a large combined in the proportions found in each extract, on the inhibition halo (habanero 10 ± 1 mm, serrano 10 ± 0 mm, diameter of the inhibition halo, a multiple regression analysis and bell pepper 10 ± 2 mm) on plates. No significant was applied, obtaining the following equation: IH = 16.68 difference (p < 0.05) was detected when one way ANOVA OCA + 15.39 MCA + 5.86 TCA + 21.33 OCA*MCA - 51.75 was used for the statistical analysis. Deans and Ritchie (1987) OCA*TCA + 16.36 MCA*TCA, with r2 = 0.956, where IH conducted a similar study with undiluted bell pepper essential is inhibition the halo in mm, OCA = ortho-coumaric acid oil, and could not find inhibitory effect in the case of E. concentration in mg/ml, MCA = meta-coumaric acid carotovora (no inhibition halo). These results confirm the concentration in mg/ml, and TCA = trans-cinnamic acid in finding of Dorantes et al. (2000) that chili extracts are mg/ml. The equation shows that all the components used inhibitory to some microorganisms. Also, growth of this separately inhibit E. carotovora. The interaction ortho- coumaric and meta-coumaric acids, and the combination of meta-coumaric and trans-cinnamic acids synergically favor Table 3. Inhibitory effect of compounds identified in the inhibition. On the other hand, combination of ortho-coumaric extracts of three varieties of chili (Capsicum spp.) against and trans-cinnamic acids has a synergistic effect on growth Erwinia carotovora subsp. carotovora. of the microorganism; this combination, then, must be Extract Compound identified Inhibition halo avoided. Table 4 shows results of the effects of compound (mm) combinations at different concentrations detected in the (mean ± sd)z extracts, on growth of E. carotovora. As predicted by the Habanero Ortho-coumaric acid 1 ± 1 a regression equation, the inhibition effect was low when ortho- Capsaicin 0 ± 0 coumaric and trans-cinnamic acids were used. In contrast, Dihydrocapsaicin 0 ± 0 meta-coumaric and trans-cinnamic acids gave results Serrano Meta-coumaric acid 4 ± 1 b comparable with any of the chili extracts, that is, a 10 mm Ortho-coumaric acid 0 ± 0 inhibition halo. This is also the exact value predicted by the Trans-cinnamic acid 3 ± 1 ab regression equation for those concentrations of the two Capsaicin 0 ± 0 compounds. It has been reported that cinnamic acid inhibits Dihydrocapsaicin 0 ± 0 growth of the food-borne pathogen Listeria monocytogenes, Bell pepper Meta-coumaric acid 4 ± 1 b possibly by interaction of the acid with cysteinyl residues Trans-cinnamic acid 1 ± 1 a (Kouassi and Shelef, 1998). Capsaicin, the major pungent Control (discs soaked with isopropanol) 0 ± 0 terpenoid compound in serrano and habanero chilies, has zNon-zero inhibition halos were analyzed by one way ANOVA been reported to be an antimicrobial compound which disrupts with Tukey test. Different letter indicate significant statistical the membrane of some microorganisms (Cowan, 1999), but differences (p < 0.05). it did not have any inhibitory effect on E. c. subsp. carotovora 236 / Volumen 21, Número 2, 2003

Table 4. Inhibition halos produced by the different combinations of the compounds present in the extracts of the three varieties of chili against Erwinia carotovora subsp. carotovora. Compound combination Inhibition halo ( mm) (mean ± sd)z Meta-coumaric acid (0.3 mg/ml) + trans-cinnamic acid (0.5 mg/ml) 10 ± 1 a Ortho-coumaric acid (0.09 mg/ml) + meta-coumaric acid (0.3 mg/ml) 6 ± 1 ab Ortho-coumaric acid (0.09 mg/ml) + meta-coumaric acid (0.2 mg/ml) 6 ± 2 ab Ortho-coumaric acid (0.09 mg/ml) + trans-cinnamic acid (0.5 mg/ml) 2 ± 1.732 b Ortho-coumaric acid (0.09 mg/ml) + trans-cinnamic acid (0.2 mg/ml) 2 ± 2 b Control (isopropanol-soaked discs) 0 ± 0 zNon-zero inhibition halos were analyzed by one way ANOVA with Tukey test. Different letter indicate significant statistical differences (p < 0.05).

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