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Inhibition of growth and mycotoxin production of Aspergillus


International Journal of Food Microbiology 98 (2005) 271 – 279 www.elsevier.com/locate/ijfoodmicro

Inhibition of growth and mycotoxin production of Aspergillus flavus and Aspergill

us parasiticus by extracts of Agave species
Eduardo Sanchez, Norma Heredia, Santos Garc?a* ? ?
Facultad de Ciencias Biologicas, Universidad Autonoma de Nuevo Leon, Apdo. Postal 124-F, San Nicolas, N.L. 66451, Mexico ? ? ? ? Received 22 March 2004; received in revised form 9 July 2004; accepted 15 July 2004

Abstract In this work, the effect of ethanolic, methanolic and aqueous extracts of Agave asperrima and Agave striata on growth and production of aflatoxin (in A&M medium) and cyclopiazonic acid (CPA; in Czpaek-Dox medium) and on growth in corn under storage conditions was determined. Aspergillus strains were inoculated (106 conidia per ml of medium or per 6 g of corn), then plant extracts were added and incubated without shaking at 28 8C for 8 days (for aflatoxin-producing analysis) or for 12 days (for CPA-producing analysis). Aflatoxin was assayed by HPLC and cyclopiazonic acid by absorbance at 580 nm using the Erlich reagent. The extracts that most effectively inhibited growth were those from the flowers of both plants. These exhibited an MIC from 0.5 to 2 mg/ml in culture media. Extracts from scape showed an MIC from 15 to 30 mg/ml in culture media. The MIC of the flower extracts was higher (N30 mg/g) when examined in corn. However, concentrations lower than the MIC drastically inhibited production of aflatoxins in culture medium or in corn. Half of the MIC inhibited 99% of the production of aflatoxins and 85% of cyclopiazonic acid. D 2004 Elsevier B.V. All rights reserved.
Keywords: Aflatoxins; Aspergillus; Cyclopiazonic acid; Natural products

1. Introduction Mycotoxins are secondary metabolites produced by specific filamentous fungi that contaminate agricultural commodities. They are toxic to humans and animals, cause significant reductions in crop yield and cause economic losses (Gourama and Bullerman,

* Corresponding author. Tel./fax: +52 81 8376 3044. E-mail address: santos@microbiosymas.com (S. Garc?a). ? 0168-1605/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2004.07.009

1995; Gqaleni et al., 1996). Their occurrence in various countries has been well documented (Bathnagar and Garc?a, 2001). ? Aspergillus flavus and Aspergillus parasiticus are important contaminants of certain foods and animal feeds because of their ability to produce aflatoxins (Farr et al., 1989). When these fungi invade and grow in commodities such as peanuts, corn and cottonseed, the resulting contamination with aflatoxins often makes the commodities unfit for consumption (Vardon, 2003). Aflatoxins are considered the most carcinogenic,

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mutagenic and teratogenic substances found naturally in foods and feeds (Conner, 1993). These metabolites cause liver damage to humans and to most experimental animal species tested (Gradelet et al., 1997). Consumption of mycotoxin-contaminated foods has been associated with several cases of human poisoning, or mycotoxicosis, sometimes resulting in death (Bathnagar and Garc?a, 2001). ? Cyclopiazonic acid (CPA) is another toxic secondary metabolite produced by A. flavus (Gqaleni et al., 1996). It causes necrosis of liver or gastrointestinal tissue and necrotic changes in skeletal muscle and kidney (Dorner et al., 1984). However, there are suggestions that the teratogenic potential of CPA is low (Morrisey et al., 1984). Isolates of A. flavus have been reported to co-produce aflatoxins and CPA, as well as other toxins in different amounts (Martins and Martins, 1999). The co-production of these compounds in foods and feeds may result in additive or synergistic effects on consumers, thus increasing the toxigenic potential of A. flavus (Martins and Martins, 1999). Many consumers demand foods without preservatives and associate healthful and safe foods with fresh or minimally processed products (Malo et al., 1997). Due to the increasing public awareness of the pollutive, residual, carcinogenic and phytotoxic effects of many synthetic fungicides, the importance of alternative indigenous products to control phytopathogenic fungi is gaining popularity (Gould, 1996; Bankole, 1997). Laboratories throughout the world have found thousands of phytochemicals that have inhibitory effects on all types of microorganisms in vitro (Cowan, 1999). Much effort has been devoted to the search for new antifungal materials obtained from natural sources for use in food and grain, and many naturally occurring antimicrobials have been identified in plants (Beuchat and Golden, 1989). However, disappointingly few of them have been developed for use in foods (Shelef, 1984). Plants of the genera Agave, known in Mexico as bmagueyQ, are widely distributed, exhibit an exceptional adaptation to drought environments and provide useful products such as natural fibers, beverages and potted plants (Garc?a-Mendoza, 1995). Furthermore, a ? report from our laboratory has shown that the species Agave lecheguilla (lecheguilla) exhibits antifungal activity (Verastegui et al., 1996).

Maize is ranked second to wheat among the world cereal crops (Goodman, 1995) and is often invaded before harvest by A. flavus and A. parasiticus that produce mycotoxins (Ellis et al., 1991). The present study was undertaken to investigate the antifungal activity of Agave asperrima and Agave striata against A. flavus and A. parasiticus in vitro and in corn under storage conditions.

2. Materials and methods 2.1. Plant material and extract preparation Two wild agave species, A. asperrima Jacobi (maguey cenizo) and A. striata Gentry (espadin) were collected in the Huasteca and Fraile’s hills, Nuevo Leon, Mexico. The voucher specimens of the plant ? ? samples were deposited in the Herbarium of the Botanical Department, Facultad de Ciencias Biolog? icas, Universidad Autonoma de Nuevo Leon. A total ? ? of 20 g of freshly collected leaves, roots, flower and scape (a steam rising the root and bearing nothing but flowers) of A. asperrima and A. striata were separately ground in 100 ml of sterile distilled water (aqueous) ethanol or methanol. Aqueous extracts were soaked in water at 4 8C for 16 h and alcoholic extracts were soaked overnight. The suspensions were centrifuged at 3000 rpm for 20 min and filtered through Whatman No. 1 filter paper. The supernatant fluid was evaporated to dryness under reduced pressure on a rotary evaporator, resuspended in distilled water (4–5 ml) and sterilized by filtration using nitrocellulose membranes (0.45 Am Millipore). Samples were stored at ?20 8C until used. An aliquot was used to determine dry weight. 2.2. Organisms and inocula The fungal strains used in this study were A. flavus SRRC 1273 and 1299 and A. parasiticus 148 and Su1 (SRRC 143), obtained from Dr. Deepak Bhathnagar, USDA Southern Regional Research Center, New Orleans, USA. All cultures were maintained on potato dextrose agar (PDA, Difco) slants, as previously reported (Hitokoto et al., 1980). The strains were cultivated on PDA slants at 28 8C until they were sporulated, about 7 days. Spores were harvested by

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adding 10 ml of sterile distilled water containing 0.05% Tween 20 and scraping the surface of the culture to free the spores. The spore suspension was further adjusted with sterile 0.05% Tween to give a final concentration of 2.5?106 spores/ml. Spore concentration was determined with a hemacytometer. 2.3. Growth media The culture media used in this study were A&M broth (Adye and Mateles, 1964) to produce aflatoxin and Czapek-Dox (DIFCO) to produce CPA (Goto et al., 1996). The media were dispensed in 25-ml aliquots into 250-ml Erlenmeyer flasks and sterilized by autoclaving at 121 8C for 15 min. 2.4. Antifungal activity assay All extracts underwent preliminary testing for antifungal activity using the hole-plate diffusion method (Navarro et al., 1996). Petri dishes (150 mm) were filled with 25 ml of PDA. Aliquots of fungal strain (1?106 spores) were homogeneously inoculated onto the agar. Holes (6 mm diameter) were made in the seeded agar plate. Holes were then filled with 100 Al of each extract. The antifungal activity was evaluated by measuring the diameters of the clear zone around the hole. Extracts that exhibited the strongest inhibitory activity were tested using the macrobroth dilution method (Freiburghaus et al., 1996). For these experiments 250 Al of a suspension containing 1?106 spores/ml were added to 250-ml Erlenmeyer flasks containing 25 ml of broth with various concentrations of extracts. Cultures were incubated without shaking at 28 8C for 8 days (for aflatoxin-producing analysis) and for 12 days (for CPA-producing analysis). Growth of mold was observed visually throughout the incubation period. The minimal inhibitory concentration (MIC) was defined as the concentration of the plant extract that prevented growth in the media as determined visually. 2.5. Effect of extracts on toxin formation A total of 250 Al of a suspension containing 1?106 spores/ml were added to 250-ml Erlenmeyer flasks containing 25 ml of broth in the presence of plant

extracts at concentrations lower than the MIC (75%, 50% and 25% MIC). Dried weight of the mycelia mat was determined at the end of the incubation period (8 and 12 days for aflatoxin and CPA production, respectively) as follows: after the culture was filtered through filter paper (Whatman No. 1), the fungal mat was dried al 50 8C for 72 h, and weight of the dried fungal mat was determined. Supernatants were used for toxin determination. For aflatoxin measurement, the method described by Wheeler et al. (1989) was used. Fifty milliliters of acetone and 75 ml of dichloromethane were added to A&M cultures. Following the addition of each solvent, the cultures were shake-agitated at 300 rpm for 30 min. Mycelial pellets were removed from the mixture by filtration using Whatman No. 1 paper and then washed with 25 ml of additional dichloromethane. Pellets were dried overnight at 45 8C on filter paper and dry weight was recorded. The filtrate was partitioned in a preparatory funnel into an aqueous phase and a dichloromethane phase that contained most of the aflatoxin and aflatoxin precursors. The aqueous phase was partitioned again with 75 ml of fresh dichloromethane. The two dichloromethane fractions were combined and partitioned against 50 ml of H2O, saturated with NaCl. Residual H2O was removed from the final dichloromethane solution with 3 g Na2SO4 and the solution was evaporated at 40 8C to dryness. The residue was dissolved in 5 ml acetonitrile for HPLC analysis of aflatoxins. Samples were separated by a 15-cm LC 18 column (Supelco). The mobile phase flow was 1 ml/ min, and detection and quantitation were performed at 365 nm in a Shimadzu Liquid Chromatograph LC10vp with Fluorescence Detector RF-10Axl. Retention times of detected toxins were compared with those of standards (Sigma, St. Louis, MO). For CPA measurement at the end 12 days, the supernatant was extracted twice from the culture with 25 ml of methanol and filtered (Whatman No. 1). CPA was measured using the colorimetric method described by Rathinavelu et al. (1984). Aliquots (1 ml) of the extracted CPA were placed in separate test tubes containing 2.0-ml p-dimethylaminobenzaldehyde solution and mixed thoroughly. Then, 10 ml of 5 N HCl were added and the resultant color was allowed to develop for 2 min. Absorbance was measured at 560 nm with a spectrophotometer (Sequoia-Turner). The amount of CPA was determined from a standard curve

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generated from 1 ml aliquots of 5, 10, 20, 30, 40 and 50 Ag/ml of CPA (Sigma) in methanol. 2.6. Effect on corn under storage conditions

in Petri dishes ranged from 0.9 to 1.5 cm (flower extract) and from 2.5 to 3.1 cm (scape extract) (Table 1). 3.2. MIC

This assay was performed using maize kernels of the variety Cargell Roy Trevino that did not exhibit ? signs of mold contamination as observed in the stereoscope (10?). For each analysis, 25 kernels (6 g) were surface sterilized with 1% NaOCl solution. After the corn was washed twice with sterile distilled water, it was mixed by rotation with 100 Al of a suspension containing 1?106 spores/ml and different concentrations of extracts. Samples were placed in petri Y-dishes containing a piece of wet cotton and incubated for 14 days at 28 8C (Bankole, 1997). MIC in maize was defined as the concentration of the plant extract that inhibited mycelial development on the corn as determined by microscopic observation (10?). Extracts at concentrations lower than MIC (75%, 50% and 25% of MIC) were added to the kernels, followed by 100 Al of fungal spore suspension (1?106 spores/ml) as described above. Kernels without extracts were used as control. After incubation, the kernels were milled in the presence of chloroform, centrifuged at 3500 rpm for 10 min and filtered using Whatman No. 1. Supernatants were collected for toxin quantification as described above.

Since the methanolic extracts of flower and scape exhibited the stronger inhibitory activity, the MIC was determined only for these parts. The macrobroth dilution method in A&M broth medium and CzapekDox medium were used. The MIC values of the extracts were first determined in A&M medium. Flowers from A. asperrima exhibited the lowest MIC values with 0.5–1.0 mg/ml against strains of A. flavus and 1.0 mg/ ml against A. parasiticus strains (Table 2). Flower extracts from A. striata were less active ranging from 1.0 to 2.0 mg/ml for all fungal strains. Extracts from scape of both plants were the least effective, ranging from 19 to 30 mg/ml against strains of A. flavus and 22 to 25 mg/ml against strains of A. parasiticus. MIC values determined in Czapek-Dox showed no significant difference ( pz0.05) when compared to those obtained in A&M Broth (data not shown). 3.3. MIC in corn under storage conditions The MIC values of corn under storage conditions were higher than those of the culture media. The flower extract of A. asperrima exhibited the lowest MIC with 33–40 mg/ml against A. flavus strains and 35–42 mg/ml against A. parasiticus strains. Extracts of A. striata were less active, ranging from 40 to 45 mg/ml for all fungal strains. In terms of practical application, the MIC of methanolic extracts of scape were high (N60 mg/ml) (Table 3).
Table 1 Diameter of inhibition zones (cm) of growth of A. flavus and A. parasiticus by extracts from scape and flower of A. asperrima and A. striata Plant extract A. flavus 1273 1299 A. parasiticus 148 Su-I

3. Results 3.1. Inhibitory effect of Agave on Aspergillus A. asperrima and A. striata extracts had inhibitory effects on growth of A. flavus and A. parasiticus. Antifungal activity was confirmed for both plants, but not for all their parts. The most active parts were the flower and scape. The methanolic, ethanolic and aqueous extracts from roots and leaves did not exert any detectable inhibitory action on fungal strains. However, ethanolic and methanolic extracts from flower and scape did show antifungal activity against all fungal strains. The methanolic extract from flower exhibited the highest activity. Diameters of inhibition zones of growth of A. flavus and A. parasiticus

A. asperrima Flower 1.0F0.01* 0.9F0.1 1.2F0.2 1.2F0.1 Scape 3F0.8 2.9F0.2 3.1F0.5 3.0F0.7 A. striata Flower 1.1F0.1 1.4F0.4 1.5F0.2 1.4F0.2 Scape 2.7F0.5 2.5F0.8 2.8F0.6 2.9F0.4 A diffusion technique in solid media was used. * Standard deviation ( pz0.05).

E. Sanchez et al. / International Journal of Food Microbiology 98 (2005) 271–279 ? Table 2 Minimum inhibition concentration (mg/ml) of methanolic extracts from scape and flower of A. asperrima and A. sriata against growth of different stains of A. flavus and A. parasiticus in A&M broth Plant extract A. flavus 1273 A. asperrima A. striata Scape Flower Scape Flower 20F3* 0.5F0.1 20F2 1.5F0.5 1299 19F6 1.0F0.3 30F2 2.0F0.4 A. parasiticus 148 22F2 1.0F0.2 23F3 1.0F0.1 Su-I 23F3 1.0F0.1 25F2 1.0F0.2 A. asperrima A. flavus 1273

275

Table 4 Effect of A. asperrima and A. striata extracts on mycelial growth (mg/ml) of A. flavus and A. parasiticus strains in culture media Plant extract Fungal strain Plant extract mg/ml (% MIC)a Control (0) 0.125 (25) 0.25 (50) 0.325 (75) Control (0) 0.25 (25) 0.50 (50) 0.75 (75) Control (0) 0.25 (25) 0.5 (50) 0.75 (75) Control (0) 0.25 (25) 0.5 (50) 0.75 (75) Control (0) 0.375 (25) 0.75 (50) 1.125 (75) Control (0) 0.5 (25) 1.0 (50) 1.5 (75) Control (0) 0.25 (25) 0.50 (50) 0.75 (75) Control (0) 0.25 (25) 0.50 (50) 0.75 (75) Mycelial production mg/ml (% reduction) 304F5* 270F3 (11) 175F2 (42) 91F1 (70) 281F4 272F3 (3) 244F4 (13) 117F4 (58) 358F4 310F2 (11) 238F1 (34) 218F6 (40) 316F2 300F2 (5) 230F1 (27) 214F4 (32) 295F1 298F3 (0) 205F4 (30) 92F1 (69) 275F3 268F4 (3) 246F4 (11) 163F1 (41) 291F3 286F3 (2) 237F2 (19) 59F2 (80) 309F5 291F1 (6) 301F6 (3) 65F1 (79)

* Standard deviation ( pz0.05).

1299

3.4. Effect of different concentrations of extract on mycelial growth Extracts of A. asperrima and A. striata at 25% of the MIC in media (125–500 Ag/ml, respectively) exhibited a slight reduction (0–11%) in mycelial production of A. flavus and A. parasiticus. Fifty percent of the MIC (0.250–1.0 mg/ml) exhibited a reduction in mycelial production from 2.6% to 42%, and 75% of the MIC (0.325–1.5 mg/ml) exhibited a reduction of 32% to 79% (Table 4). 3.5. Effect of extracts on aflatoxin production When different concentrations of methanolic extracts of both plants were added to the cultures, a significant reduction in aflatoxin synthesis was observed. A. asperrima extract caused a significant ( pz0.05) toxin reduction (N97%) at 50% of the MIC in A. flavus 1273 and A. parasiticus 148, and greater than 65% reduction for the remaining strains. Extracts of A. striata also showed a similar pattern of inhibition of aflatoxin production with these strains (Table 5).

A. parasiticus

148

Su-I

A. striata

A. flavus

1273

1299

A. parasiticus

148

Su-I

Fraction (in %) of the minimal inhibitory concentration of extracts. * Standard deviation ( pz0.05).

a

Table 3 Minimum inhibitory concentrations (mg/ml) of methanolic extracts of scape and flower on growth of strains of A. flavus and A. parasiticus in storage simulated conditions Plant extract A. flavus 1273 A. asperrima A. striata Scape Flower Scape Flower N60 33F5* N60 40F6 1299 N60 40F6 N60 45F4 A. parasiticus 148 N60 42F7 N60 45F4 Su-I N60 35F5 N60 43F6

3.6. Effect of extract on aflatoxin production in storage conditions Production of aflatoxin in corn under storage conditions was very low compared to liquid media. Addition of extracts of A. asperrima and A. striata to corn at concentrations lower than the MIC greatly inhibited aflatoxin production of A. flavus and A. parasiticus. At 50% of the MIC value of both agaves, there was a N99% reduction in production of all types of aflatoxins in corn in all strains and 75% of MIC

* Standard deviation ( pz0.05).

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Table 5 Percentage of reduction of aflatoxin production of A. flavus and A. parasiticus by methanolic extracts of A. asperrima and A. striata in liquid media Plant extract A. asperrima Fungal strain A. flavus 1273 Plant extract added in mg/ml (% MIC)a Control (0) 0.125 (25) 0.25 (50) 0.325 (75) Control (0) 0.25 (25) 0.50 (50) 0.75 (75) Control (0) 0.25 (25) 0.5 (50) 0.75 (75) Control (0) 0.25 (25) 0.5 (50) 0.75 (75) Control (0) 0.375 (25) 0.75 (50) 1.125 (75) Control (0) 0.5 (25) 1.0 (50) 1.5 (75) Control (0) 0.25 (25) 0.50 (50) 0.75 (75) Control (0) 0.25 (25) 0.50 (50) 0.75 (75) Afl production in Ag/ml (% of reduction) Afl B1 1.17 (0) 0.75 (35) 0.2 (98) 0.009 (99) 0.72 (0) 0.608 (16) 0.249 (65) 0.07 (90) 0.87 (0) 0.04 (95) b0.00008 (N99) b0.00008 (N99) 1.02 (0) 0.58 (43) 0.22 (78) 0.043 (96) 1.31 (0) 0.53 (60) 0.013 (99) 0.011 (99) 0.682 (0) 0.387 (45) 0.14 (80) 0.006 (99) 1.81 (0) 0.88 (51) 0.31 (83) 0.074 (96) 0.69 (0) 0.21 (70) 0.071 (90) b0.00008 (N99) Afl B2 2.49 (0) 1.91 (23) 0.025 (99) b0.00008 (N99) 0.95 (0) 0.76 (20) 0.12 (87) 0.044 (95) 0.52 (0) 0.29 (44) 0.005 (99) b0.00008 (N99) 2.23 (0) 1.145 (37) 0.17 (93) b0.00008 (N99) 4.87 (0) 3.6 (26) 0.49 (90) 0.04 (99) 0.91 (0) 0.51 (44) 0.29 (68) 0.001 (99) 1.55 (0) 0.27 (83) 0.15 (90) 0.01 (N99) 1.21 (0) 0.85 (30) 0.1 (92) b0.00008 (N99) Afl G1 – – – – – – – – 1.9 (0) 0.3 (85) 0.06 (97) b0.00008 (N99) 0.61 (0) 0.23 (62) 0.027 (96) b0.00008 (N99) – – – – – – – – 0.04 (0) b0.00008 (N99) b0.00008 (N99) b0.00008 (N99) 0.94 (0) 0.38 (60) b0.00008 (N99) b0.00008 (N99) Afl G2 – – – – – – – – b0.00008 b0.00008 b0.00008 b0.00008 0.19 (0) 0.08 (42) b0.00008 b0.00008 – – – – – – – – 0.69 (0) 0.07 (90) b0.00008 b0.00008 0.008 (0) b0.00008 b0.00008 b0.00008

1299

A. parasiticus

148

(0) (NDb) (ND) (ND)

Su-I

(N99) (N99)

A. striata

A. flavus

1273

1299

A. parasiticus

148

(N99) (N99) (N99) (N99) (N99)

Su-I

a b

Fraction (in %) of the minimal inhibitory concentration of extracts. ND: not determined.

reduced the aflatoxin synthesis to undetectable levels (N80 pg, Table 6). 3.7. Effect of extracts on CPA production in liquid medium CPA was produced in detectable quantities only by A. flavus 1299 strain. In this case, the extract of A. asperrima more effectively inhibited CPA production since 0.5 mg/ml (50% MIC) produced a 98% reduction of CPA, while 1.5 mg/ml (75% MIC) of A. striata exhibited a similar reduction (Table 7).

4. Discussion Scientists from divergent fields are investigating plants for antimicrobial usefulness. To determine the antifungal effect of two Agave species, antimicrobial tests using diffusion agar techniques were employed with extracts made from distilled water, ethanol and methanol (Cowan, 1999). For both agaves methanolic extracts exhibited the best antifungal effect, in comparison with ethanolic and aqueous extracts. This parallels published reports that methanol can extract compounds with antimicro-

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Table 6 Percentage of reduction of aflatoxin production of A. flavus and A. parasiticus in corn in storage conditions by methanolic extracts of A. asperrima and A. striata Plant extract Fungal strain Plant extract added in mg/ml (% MIC)a 1273 Control (0) 8.25 (25) 16.5 (50) 24.75 (75) Control (0) 10 (25) 20 (50) 30 (75) Control (0) 10.5 (25) 21 (50) 31.5 (75) Control (0) 8.75 (25) 17.5 (50) 26.25 (75) Control (0) 10 (25) 20 (50) 30 (75) Control (0) 11.25 (25) 22.5 (50) 33.75 (75) Control (0) 11.25 (25) 22.5 (50) 33.75 (75) Control (0) 10.75 (25) 21.5 (50) Afl production in Ag/ml (% of reduction) Afl B1 0.042 (0) 0.03 (28) b0.00008 (N99) b0.00008 (N99) 0.09 (0) b0.00008 (N99) b0.00008 (N99) b0.00008 (N99) 0.003 (0) b0.00008 (N99) b0.00008 (N99) b0.00008 (N99) 0.01 (0) b0.00008 (N99) b0.00008 (N99) b0.00008 (N99) 0.016 (0) 0.003 (20) b0.00008 (N99) b0.00008 (N99) 0.005 (0) b0.00008 (N99) b0.00008 (N99) b0.00008 (N99) 0.071 (0) 0.007 (90) b0.00008 (N99) b0.00008 (N99) 0.039 (0) 0.00008 (N99) b0.00008 (N99) Afl B2 0.053 (0) 0.025 (47) b0.00008 (N99) b0.00008 (N99) 0.13 (0) 0.11 (84) b0.00008 (N99) b0.00008 (N99) 0.01 (0) 0.0054 (52) b0.00008 (N99) b0.00008 (N99) 0.09 (0) b0.00008 (N99) b0.00008 (N99) b0.00008 (N99) 0.049 (0) 0.005 (10) b0.00008 (N99) b0.00008 (N99) 0.01 (0) b0.00008 (N99) b0.00008 (N99) b0.00008 (N99) 0.045 (0) 0.01 (77) b0.00008 (N99) b0.00008 (N99) 0.11 (0) 0.00008 (N99) 0.00008 (N99) Afl G1 – – – – – – – – b0.00008 (0) b0.00008 (NDb) b0.00008 (ND) b0.00008 (ND) 0.014 (0) b0.00008 (N99) b0.00008 (N99) b0.00008 (N99) – – – – – – – – 0.0001 (0) 0.00008 (N99) b0.00008 (N99) b0.00008 (N99) 0.1 (0) 0.00008 (N99) b0.00008 (N99) Afl G2 – – – – – – – – b0.00008 (0) b0.00008 (ND) b0.00008 (ND) b0.00008 (ND) 0.12 (0) b0.00008 (N99) b0.00008 (N99) b0.00008 (N99) – – – – – – – – 0.003 (0) 0.00008 (N99) b0.00008 (N99) b0.00008 (N99) 0.001 (0) 0.00008 (N99) b0.00008 (N99)

A. asperrima

A. flavus

1299

A. parasiticus

148

Su-I

A. striata

A. flavus

1273

1299

A. parasiticus

148

Su-I

a b

Fraction (in %) of the minimal inhibitory concentration of extracts. ND: not determined.

Table 7 Reduction of CPA production of A. flavus 1299 by methanolic extracts of A. asperrima and A. striata Fungal strain A. flavus 1299 Plant extract Plant extract mg/ml (% MIC)a Control (0) 0.25 (25) 0.50 (50) 0.75 (75) Control (0) 0.5 (25) 1.0 (50) 1.5 (75) CPA production in mg/ml (% of reduction) 0.37 (0) 0.24 (35) 0.007 (98) 0.003 (99) 0.41 (0) 0.23 (45) 0.08 (80) 0.004 (99)

A. asperrima

A. striata

a Fraction (in %) of the minimal inhibitory concentration of extracts.

bial activities better than ethanol (Cowan, 1999) and that aqueous extractions usually show less antimicrobial activity (Zhang and Lewis, 1997). In 1998, Eloff (1998) examined the ability of a variety of solvents, such as methylene dichloride, methanol, ethanol and water, to solubilize natural antimicrobials. He found that most of the active compounds were more efficiently extracted with methanol and ethanol. The antimicrobial activity of plant extracts is sometimes found in specific parts, e.g., ginseng contains saponins and essential oils only in roots whereas Eucalyptus contains essential oils in leaves (Thomson, 1978). In the balsamic poplar, on the other hand, active components were found in leaves, stems and sprouts

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(Thomson, 1980). In the present study, the antimicrobial activity of the Agave species was found mainly in the flower extracts. The period of flowering has been shown to agree with an increase in the content of antimicrobial compounds in several plants (Thomson, 1978). Accordingly, methanolic extracts of flowers of A. asperrima and A. striata exhibited the lowest MIC (0.5–2.0 mg/ ml). Antimicrobial activity was also found in scape, but at lower levels, and no activity was detected in roots or leaves of the plants analyzed. The MIC values of the flower extracts obtained in corn under storage conditions were greater (32–45 mg/ml) than those observed in the liquid medium. Since the MIC of the methanolic extracts of the scapes of both Agave species was higher than 60 mg/ml in liquid media, no effort was taken to determine its value in corn under storage conditions. The levels of antimicrobials required to inhibit microorganisms in foods have sometimes been found to be much higher than those determined using laboratory cultures (Farbood et al., 1976). When different concentrations of extracts were added to the fungal cultures in liquid media and in corn, a remarkable reduction in aflatoxin synthesis was observed. The reduction in growth and toxin production was dependent on the concentration of extract. It has been reported that A. flavus can coproduce CPA with aflatoxins and that this commonly occurs in agricultural commodities (Urano et al., 1992). Reports have indicated a positive correlation between CPA and aflatoxin production, but considerable variation exists in the toxin synthesis between fungal strains (Horn et al., 1996). In this work, CPA was only detected in A. flavus 1299 and was coproduced with aflatoxins. The important reduction of mycotoxin production suggests that phytochemical compounds could be used alone or in conjunction with other substances or processes to control the presence of toxic metabolites in corn. These extracts must be subjected to further study to characterize the active compound, define toxicity and evaluate economic feasibility.

Primer Programa de Apoyo a la Investigacio n ? OMNILIFE 2000 bAlimentar con CienciaQ. We thank Dr. Deepak Bhathnagar (USDA Southern Regional Research Center, New Orleans, USA) for generously providing fungal strains. E. Sanchez was a CON? ACYT fellow.

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