Aspergillus flavus growth in the presence of chemical preservatives and naturally occurring antimicrobial compounds

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1 International Journal of Food Microbiology 99 (2005) Aspergillus flavus growth in the presence of chemical preservatives and naturally occurring antimicrobial compounds Aurelio López-Malo a, *, Stella Maris Alzamora b, Enrique Palou a a Departamento de Ingeniería Química y Alimentos, Universidad de las Américas, Puebla, Cholula, Puebla, 72820, Mexico b Departamento de Industrias, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria 1428, Buenos Aires, Argentina Received 26 April 2004; received in revised form 6 August 2004; accepted 11 August 2004 Abstract The combined effects of water activity ([a w ] 0.99 or 0.95), ph (4.5 or 3.5) and antimicrobial agent (potassium sorbate, sodium benzoate, sodium bisulfite, carvacrol, citral, eugenol, thymol, or vanillin) concentration (0, 100, 200 up to 1800 ppm) on the growth of Aspergillus flavus were evaluated in potato dextrose agar (PDA). Mold spore germination time and radial growth rates (RGR) were significantly ( pb0.05) affected by the variables. For equal antimicrobial concentration, reduction in ph or a w had important effects, lowering RGR and delaying germination time. Depending on a w and ph, increase in antimicrobial concentration slightly reduced RGR until a critical concentration where RGR was drastically reduced or mold growth was inhibited. Germination time increased as antimicrobial agent concentration increased and when a w and ph decreased. Important antimicrobial differences were observed, being, in general, the natural antimicrobials less ph-dependent than chemical preservatives. A. flavus exhibited higher sensitivity to thymol, eugenol, carvacrol, potassium sorbate, sodium bisulfite, and sodium benzoate (at ph 3.5) than to vanillin or citral. D 2004 Elsevier B.V. All rights reserved. Keywords: Aspergillus flavus; Preservatives; Natural antimicrobials 1. Introduction Fungal growth is influenced by a variety of complex interactions between intrinsic and extrinsic * Corresponding author. Tel.: ; fax: address: amalo@mail.udlap.mx, aurelio.lopezm@udlap.mx (A. López-Malo). factors. Temperature, time, moisture, gaseous composition, and antimicrobials agents are some factors that are being evaluated in combination as fungistatic or fungicidal systems. Mold growth is commonly controlled using synthetic antimicrobials; however, natural antimicrobials have also demonstrated important antifungal properties (López-Malo et al., 2000). Naturally occurring antimicrobials are perceived as raising fewer concerns among consumers and regu /$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi: /j.ijfoodmicro

2 120 A. López-Malo et al. / International Journal of Food Microbiology 99 (2005) latory agencies or within the food industry (Nychas, 1995; López-Malo et al., 1997). Interest in the use of natural antimicrobials is growing, especially for herbs, plants, and spices (or their components), which are traditional ingredients and flavor enhancers. Conner and Beuchat (1984a, b) suggested that the antimicrobial activity of essential oils of herbs and spices or their constituents such as thymol, carvacrol, vanillin, etc. could be the result of damage to enzymatic cell systems, including those associated with energy production and synthesis of structural compounds. Nychas (1995) indicated that phenolics could denature the enzymes responsible for spore germination or interfere with amino acids involved in germination. Several studies have attempted to determine the efficacy of extracts from selected plants as antimicrobial and antifungal agents (López-Malo et al., 2000, in press). Some studies have shown that specific essential oils and phenolic compounds can control the growth rate and spore germination time of spoilage fungi (Hope et al., 2003). Paster et al. (1994) found that oregano and thyme essential oils inhibited the growth of Aspergillus niger, Aspergillus ochraceus, and Aspergillus flavus. Growth was fully inhibited with 400 ppm of oregano and 600 ppm of thyme, these oil concentrations also prevented spore germination. However, few studies have taken into account the effect that different environmental factors such as water activity and ph may have on the effectiveness of phenolic compounds and synthetic antimicrobials in relation to fungal growth. The objective of this study was to determine the effect of several antimicrobial agents (chemical preservatives and naturally antimicrobial compounds) on the growth of A. flavus in media formulated with selected ph and a w values. 2. Materials and methods 2.1. Microorganism and spore suspension preparation A. flavus ATCC strain was cultivated on potato dextrose agar (PDA) slants (Merck, Merck- Mexico) for 10 days at 25 8C, and the spores were harvested with 10 ml of 0.1% Tween 80 (Merck, Merck-Mexico) solution sterilized through membrane (0.45 Am) filtration. Spore suspension was adjusted with the same solution to give a final spore concentration of 10 6 spore/ml and was utilized the same day Experimental design Factorial designs (Montgomery, 1984) were employed to assess the effects of water activity (0.99 or 0.95), ph (4.5 or 3.5), and antimicrobial agents (potassium sorbate, sodium benzoate, sodium bisulfite, carvacrol, citral, eugenol, thymol, or vanillin) and concentrations (0, 100, 200 up to 1800 ppm) on mold radial growth rates (RGR) and germination time. Systems prepared with the resulting variable combinations were replicated three times Preparation of the systems Following experimental designs, PDA systems were prepared with the necessary quantity of commercial sucrose to reach a w 0.99 or 0.95, sterilized for 15 min at 121 8C, cooled, and acidified with hydrochloric acid 0.1 N to the desired ph (amounts of sucrose and HCl needed in each case were previously determined). Sterilized and acidified agar solutions were aseptically divided, and then the necessary amount of every antimicrobial agent (Sigma-Aldrich Química, Toluca, México) was added and mechanically incorporated and dissolved under sterile conditions. Agar solutions were poured into sterile Petri dishes Inoculation and incubation Triplicate Petri dishes of every system were centrally inoculated by pouring 2 Al of the spore suspension (approximately spores/ml) to give circular inocula of 2 mm in diameter. For a w and ph measurements, three plates of each system were maintained without inoculation. Growth controls without antimicrobials were prepared and inoculated as above. The inoculated plates and the controls were stored in hermetically closed plastic containers (over sodium chloride solutions of the same a w ), to avoid dehydration, for 2 months at 25F0.5 8C in incubation chambers. Sufficient headspace was left in the containers to avoid anoxic conditions. Periodically, inoculated plates were removed briefly to be observed and to measure

3 A. López-Malo et al. / International Journal of Food Microbiology 99 (2005) Table 1 Water activity (a w ), ph, radial growth rate (RGR), and germination time of Aspergillus flavus inoculated in potato dextrose agar and incubated at 25 8C a w ph RGR (mm/h) Germination time (h) F F F F F F F F3.1 colony diameter (in two directions at right angles to each other) and immediately reincubated Measurements of water activity and ph Water activity, a w, was measured with a Decagon CX-1 (Decagon Devices, Pullman, WA), calibrated, and operated following the procedure described by López-Malo et al. (1993). The ph was determined with a Beckman ph meter model 50 (Beckman Instruments, Fullerton, CA). Every measurement was made by triplicate, and the average value was reported Colony radial growth The inoculated systems were daily examined using a stereoscopic microscope (American Optical, model Forty). After the colonies were confluent, their increase in size was followed by measuring the colony diameter. Increase in diameter of each plate was plotted as function of incubation time, and radial growth rate was obtained from the slope by linear regression of the linear phase of growth (Horner and Anagnostopoulos, 1973, López-Malo et al., 1995). Mean radial growth rate for every experiment was calculated and reported. To calculate germination time, the linear equation was extrapolated to a zero increase in diameter (2 mm diameter), and the intercept on the time axis was defined as germination time (López-Malo et al., 1995) Statistical analysis Analysis of variance (ANOVA) of the effect of the independent variables and their interactions on radial growth rates and germination times in the different systems was performed (Gacula and Singh, 1984). Statisticak software (Statsoftk, Tulsa, OK) was used to analyze experimental results. 3. Results and discussion The ph and a w of the PDA systems without inoculation determined at the beginning and at the end of incubation (60 days) demonstrated that the desired values remained constant under the storage conditions. The results of the control systems (without antimicrobials) demonstrated that A. flavus grew in every a w ph studied combination (Table 1). Fig. 1 presents the increase in colony diameter during the incubation of A. flavus inoculated in PDA with a w 0.99 and 800 ppm vanillin at ph 4.5 and 3.5. In the conditions where the mold grew, after a lag period that varied with the conditions tested, the colony diameter exhibited a constant increase with time (zero-order kinetics). Similar results (not shown) were obtained for every other evaluated condition. Regression coefficients greater than 0.98 were obtained for the linear growth phase, with variability coefficients (standard deviation/mean value) lower than 10%. Horner and Anagnostopoulos (1973), González et al. (1987), andlópez-malo et al. (1995, 1997, 1998) also reported a linear colony diameter increase as function of incubation time for molds in solid systems and found significant differences among radial growth when evaluating different conditions. Several environmental factors determine and affect mold growth, germination time, and RGR in solid media. Brancato and Golding (1953) demonstrated that mold colony diameter could be used as a growth response to evaluate growth rate and compare the Colony diameter (mm) ph Time (h) ph 3.5 Fig. 1. Aspergillus flavus colony diameter in potato dextrose agar formulated with a w 0.99, 800 ppm of vanillin, and selected ph values (5 n, replicate 1;. o, replicate 2; 4 E, replicate 3).

4 122 Table 2 Mean radial growth rate (mm/h) of Aspergillus flavus in potato dextrose agar formulated with selected a w and ph values and different concentrations (ppm) of natural and synthetic antimicrobials ppm a w ph a w ph a w ph a w ph a w ph a w ph a w ph a w ph a w ph a w ph a w ph a w ph a w ph a w ph a w ph a w ph Vanillin Sodium benzoate Sodium bisulfite Potassium sorbate Eugenol Carvacrol Thymol Citral A. López-Malo et al. / International Journal of Food Microbiology 99 (2005)

5 A. López-Malo et al. / International Journal of Food Microbiology 99 (2005) influence of several factors (additives, environmental conditions) on mold response. These authors reported colony diameter as a satisfactory measurement of growth inasmuch as growth rate is constant over time, and after germination, many fungal species reach a constant radial growth rate. RGR may be considered as the result of several rates related with mold physiology and morphology, apical cell division, branch formation, and cell enlargement (Brancato and Golding, 1953). Table 1 presents RGR and germination times of A. flavus inoculated in PDA without antimicrobials (control systems) formulated with selected a w and ph values. A. flavus grew in the four a w - and phtested combinations; this mold was resistant to the combined effects of a w 0.95 and ph 3.5. Important effects on RGR were observed when decreasing a w and ph, while slight effect was observed on germination time. At a w 0.99 and ph 4.5, A. flavus RGR was significantly ( pb0.05) higher than for other combinations. It is known that many fungal species are capable of growth in a wide ph range but also that this parameter affect growth rates and other extrinsic and intrinsic growth limits (Holmquist et al., 1983). Brancato and Golding (1953) reported 5.5 as optimum ph at 25 8C for six mold species, including A. flavus. Holmquist et al. (1983) observed that ph of media had an important effect on Aspergillus growth and reported differences at ph between 4.0 and 7.0 and significant ( pb0.05) growth reduction at ph 3.0. Thompson (1990) suggested that ph might affect bin vitroq growth of Aspergillus strains; for six different strains of A. flavus, ph reduction from 6.0 to 4.0 reduced 13% mycelial production, while for two strains of Aspergillus parasiticus, the reduction was 9%. Arroyo et al. (2003) reported that A. ochraceus growth was faster as a w increased while ph (4.5, 6.0 or 7.5) did not affect mold growth in the same extent, its effect was more noticeable at higher a w levels. Similarly, López-Malo et al. (1998) demonstrated faster growth rates of A. flavus at a w 0.98 and ph 5.5 than at ph 3.0. Table 2 presents the effect of the studied variables on the mean RGR of A. flavus. For the conditions where no growth was observed after 60 days, RGR was reported as zero. In systems formulated with antimicrobial agents that allowed mold growth, RGR varied from (a w 0.95; ph 3.5; 500 ppm eugenol) to mm/h (a w 0.99; ph 4.5; 100 ppm sodium benzoate) with standard deviation variability from to mm/h. For every antimicrobial tested, an analysis of variance demonstrated that antimicrobial concentration (below the minimal inhibitory concentration), a w, and ph significantly ( pb0.05) affected A. flavus RGR. As examples, Table 3 presents the ANOVA results when vanillin and sodium benzoate were tested, similar results were obtained for the other antimicrobials (data not shown). Depending on a w and ph, the increase in antimicrobial concentration slightly reduced (or did not affect) RGR until a critical concentration, where RGR was drastically reduced or mold growth was stopped (Table 2). Juven et al. (1994) reported that increasing thyme essential oil, thymol, or carvacrol concentration was not always directly related with a greater antimicrobial effect. However, concentrations higher than a critical one caused a fast and considerable reduction in survivals. Juven et al. (1994) attributed this behavior to the fact that phenolic compounds hit specific sites on cell membrane, and only when these sites are saturated, a severe damage can be observed Table 3 Analysis of variance of Aspergillus flavus radial growth rate (mm/h) inoculated in potato dextrose agar formulated with selected concentrations of vanillin or sodium benzoate (A), a w (B), and ph (C) Source of variation Degrees of freedom Quadratic mean Vanillin A B C A B A C B C A B C Error Sodium benzoate A B C , A B A C B C A B C Error F p

6 124 Table 4 Mean germination time (h) of Aspergillus flavus in potato dextrose agar formulated with selected a w and ph values and different concentrations (ppm) of natural and synthetic antimicrobials ppm a w ph a w ph a w ph a w ph a w ph a w ph a w ph a w ph a w ph a w ph a w ph a w ph a w ph a w ph a w ph a w ph Vanillin Sodium benzoate Sodium bisulfite Potassium sorbate N N N N N1440 N N1440 N N N N1440 N N1440 N N1440 N1440 N N1440 N N1440 N N1440 N1440 N N1440 N N1440 N N1440 N1440 N N1440 N N1440 N1440 N N1440 N1440 N N1440 N N1440 N1440 N1440 N1440 N1440 N1440 N N1440 N1440 N1440 N1440 N1440 N1440 N1440 N1440 N1440 N N1440 N1440 N1440 N1440 N1440 N1440 N1440 N1440 N1440 N N N1440 N1440 N1440 N1440 N1440 N1440 N1440 N1440 N1440 N1440 N N N1440 N1440 N1440 N1440 N1440 N1440 N1440 N1440 N1440 N1440 N N1440 N1440 N1440 N N1440 N1440 N1440 N1440 N1440 N1440 N1440 N1440 N1440 N1440 N N1440 N1440 N1440 N N1440 N1440 N1440 N1440 N1440 N1440 N1440 N1440 N1440 N1440 N N1440 N1440 N1440 N1440 N1440 N1440 N1440 N1440 N1440 N1440 N1440 N1440 N1440 N1440 N1440 N1440 Eugenol Carvacrol Thymol Citral N N1440 N1440 N N N N1440 N1440 N1440 N1440 N1440 N1440 N1440 N N1440 N N1440 N1440 N1440 N1440 N1440 N1440 N1440 N N1440 N1440 N1440 N1440 N1440 N1440 N1440 N1440 N1440 N1440 N1440 N N1440 N1440 N1440 N1440 N1440 N1440 N1440 N1440 N1440 N1440 N1440 N N1440 N1440 N1440 N1440 N1440 N1440 N1440 N1440 N1440 N1440 N1440 N N1440 N1440 N1440 N1440 N1440 N1440 N1440 N1440 N1440 N1440 N1440 N N1440 N1440 N1440 N1440 N1440 N1440 N1440 N1440 N1440 N1440 N1440 N N1440 N1440 N1440 N1440 N1440 N1440 N1440 N1440 N1440 N1440 N1440 N N1440 N1440 N1440 N1440 N1440 N1440 N1440 N1440 N1440 N1440 N1440 N N1440 N1440 N1440 N1440 N1440 N1440 N1440 N1440 N1440 N1440 N1440 N N1440 N1440 N1440 N1440 N1440 N1440 N1440 N1440 N1440 N1440 N1440 N N N1440 N1440 N1440 N1440 N1440 N1440 N1440 N1440 N1440 N1440 N1440 N N1440 A. López-Malo et al. / International Journal of Food Microbiology 99 (2005)

7 A. López-Malo et al. / International Journal of Food Microbiology 99 (2005) as the result of cytoplasmatic membrane collapse and lose of cell constituents. Table 4 presents the effects of antimicrobial agent concentration on A. flavus germination time at different a w and ph values. Germination time increased as concentration increased. In those conditions where no growth was observed after 60 days at 25 8C, germination time was reported as higher than 1440 h. An important effect of antimicrobial concentration on germination time can be observed; as an example, growth was delayed for 1400 h (55 days) at a w 0.99 and ph 3.5 when PDA was formulated with 300 ppm of thymol in comparison with 328 h (14 days) when 200 ppm of thymol was added. For every antimicrobial, analysis of variance results demonstrated the significant effect ( pb0.05) of the studied variables (a w, ph, antimicrobial agent concentration) and their interactions on A. flavus germination time. As an example, Table 5 presents ANOVA results when vanillin and sodium benzoate were tested; similar results were obtained for the other studied antimicrobials (data not shown). Table 5 Analysis of variance of Aspergillus flavus germination time (h) inoculated in potato dextrose agar formulated with selected concentrations of vanillin or sodium benzoate (A), a w (B), and ph (C) Source of variation Degrees of freedom Quadratic mean Vanillin A 12 31, B 1 79, , C 1 34, , A B 12 22, A C 12 13, B C 1 45, , A B C 12 14, Error Sodium benzoate A , B , C ,315, A B , A C , B C , A B C Error F p In general, those conditions that delayed germination time resulted also in a reduction of RGR. Delayed mold spore germination time by the effect of subinhibitory antimicrobial concentrations has been reported previously by Buchanan and Shepherd (1981), Karapinar (1985), Thompson (1986), and López-Malo et al. (1995, 1997, 1998, 2002). López- Malo et al. (1995) observed an important effect of vanillin concentration on mold spore germination time in PDA at ph 3.5 and 25 8C and reported that the germination time was extended to around 250 h for A. flavus and A. parasiticus when 1000 ppm of vanillin was present. Delay of the onset of mold growth by the addition of natural antimicrobials has been also reported for A. parasiticus with 100 ppm of thymol in laboratory media (ph 4.5 and incubation temperature 28 8C) by Buchanan and Shepherd (1981) and with 2% of thyme (laboratory media, ph , incubation temperature 25 8C) by Karapinar (1985). Mahmoud (1994) reported that thymol and cinnamic aldehyde concentrations lower than the inhibitory ones delayed for 8 days at 28 8C A. flavus germination time in nutritive broth at ph 5.5. Rusul and Marth (1987) also observed that aflatoxigenic molds growing in liquid media with subinhibitory antimicrobial (potassium sorbate, sodium benzoate) concentrations delayed the onset of growth. A. flavus grew in a larger number of combinations (66 of 122) when PDA was formulated with a w 0.99 and ph 4.5, followed by inoculation in media with a w 0.95 ph 4.5 (51 combinations), a w 0.99 ph 3.5 (41 combinations), and a w 0.95 ph 3.5 where only 38 combinations allowed mold growth. Analyzing growth results by antimicrobial agent, carvacrol only permitted mold growth in 8 combinations of 60 evaluated, followed by thymol (10 combinations), potassium sorbate (13 combinations), sodium bisulfite (16 combinations), eugenol (18 combinations), sodium benzoate (27 combinations), vanillin (46 combinations), and citral in which growth was observed in 58 combinations of a total of 68 evaluated for this antimicrobial. For the media formulated with thymol at both evaluated ph levels, increase in antimicrobial concentration caused an important increase in germination time; for potassium sorbate and sodium benzoate, the concentration needed to obtain the same effect on mold spore germination time was higher at ph 4.5 than the value required at ph

8 126 A. López-Malo et al. / International Journal of Food Microbiology 99 (2005) , demonstrating the antimicrobial action of undissociated organic acids (Davidson, 2001). Table 4 presents mold spore germination time to selected natural antimicrobials and chemical preservatives; the patterns observed differed depending on the antimicrobial, citral and vanillin being less effective to inhibit mold growth inasmuch as higher concentrations were needed. However, a relatively high vanillin or citral concentration caused an important delay in mold growth. As antimicrobial concentration increased, longer germination times were obtained. Important differences were observed among antimicrobials, with the natural antimicrobials being, in general, less ph-dependent than chemical preservatives. A. flavus presented higher sensitivity to thymol, eugenol, carvacrol, potassium sorbate (at ph 3.5), sodium bisulfite (ph 3.5), and sodium benzoate (ph 3.5) than to vanillin or citral. It is also noticed that increasing antimicrobial concentration in the case of thymol, carvacrol, eugenol, potassium sorbate, and sodium benzoate had a dramatic effect on A. flavus germination time. Minimal inhibitory concentrations (Table 6) for A. flavus growth were in the range of ppm depending on the antimicrobial agent; some of them are higher than those reported for common organic acids traditionally used as antimicrobials but could be sensory compatible and acceptable with some fruit and vegetable products (Cerrutti and Alzamora, 1996; Cerrutti et al., 1997; López-Malo et al., 2000; Alzamora et al., 2003). The obtained results suggest that compounds from natural origin Table 6 Antimicrobial minimal inhibitory concentrations a (ppm) for Aspergillus flavus in potato dextrose agar formulated with selected a w and ph values Antimicrobial a w 0.99 a w 0.95 ph 3.5 ph 4.5 ph 3.5 ph 4.5 Vanillin Benzoate Bisulfite Sorbate Eugenol Carvacrol Thymol Citral a Defined as those where mold growth was not observed after 60 days of incubation at 25 8C. (vanillin, eugenol, carvacrol, thymol, and citral) can be used as antimicrobial agents to avoid fungal growth. Natural antimicrobial agent addition in combination with a w and ph reduction may result in an interesting and promising food preservation approach. Minimal inhibitory concentrations of sodium benzoate, potassium sorbate, and sodium bisulfite (Table 6) depended on ph value, being more noticeable at high a w (0.99). At a w 0.95, an interaction between 300 ppm potassium sorbate and ph 4.5 can be observed. Potassium sorbate, carvacrol, and thymol were the most effective antimicrobials at a w 0.99 ph 4.5. Minimal inhibitory concentrations for common antimicrobials (Table 6) were similar to those reported by other authors (Parra et al., 1992, 1993). Kabara and Eklund (1991) reported that weak lipophilic acids, such as sorbic, benzoic, and propionic, were effective as antimicrobials due to their solubility in the nondissociated form inside the cell membrane, relating this with their ph dependence on activity. Our results demonstrate an interaction between the evaluated variables inasmuch as a w and ph reduction or their combinations were not enough to inhibit mold growth (Table 1). Water activity (a w ) ph and antimicrobial agents combinations inhibited A. flavus. The concentration needed for a particular case depends on the evaluated antimicrobial. Thompson (1990) reported for eight toxigenic strains of A. flavus and A. parasiticus that the addition of 1.0 M of carvacrol in liquid media inhibited (at 27 8C) mold growth at least for 7 days when ph was adjusted to 4.0 or 8.0; at ph 6.0, only reductions (around 50 80%) of mycelial production were observed. This author reported also that thymol (1.0 M) inhibited mold growth at the evaluated ph levels (4.0, 6.0 and 8.0). Juven et al. (1994) found an increased antimicrobial activity of essential oil of thyme and thymol at ph 5.5 in comparison with ph 6.5 and attributed these effects to changes in polar group s distribution of phenolic constituent of oils between the cytoplasm membrane and the external medium. At low ph, thymol molecule is largely undissociated and therefore more hydrophobic and can be joined better to hydrophobic regions of membrane proteins and also dissolved more easily in the lipidic phase. Kabara (1991) mentioned that undissociated phenolic groups were more active as antimicrobials than dissociated

9 A. López-Malo et al. / International Journal of Food Microbiology 99 (2005) forms, suggesting that phenols can act on a wide ph range ( ). Possible modes of action of phenolic (and terpens) compounds have been reported in different reviews (Wilkins and Board, 1989; Beuchat, 1994; Nychas, 1995; Sofos et al., 1998; Davidson and Naidu, 2000; Davidson, 2001; López-Malo et al., 2000, in press). However, the mechanisms have not been completely elucidated. Prindle and Wright (1977) mentioned that the effect of phenolic compounds is concentrationdependent. At low concentration, phenols affected enzyme activity, especially of those enzymes associated with energy production, while at greater concentrations, caused protein denaturation. The effect of phenolic antioxidants on microbial growth and toxin production could be the result of the ability of phenolic compounds of altering microbial cell permeability, permitting the loss of macromolecules from the interior. They could also interact with membrane proteins, causing a deformation in its structure and functionality (Fung et al., 1977). Lis-Balchin and Deans (1997) reported that strong antimicrobial activity could be correlated with essential oils containing high percentage of monoterpenes, eugenol, cinnamic aldehyde, and thymol. Conner and Beuchat (1984a, b) suggested that antimicrobial activity of essential oils on yeasts could be the result of disturbance in several enzymatic systems involved in energy production and structural components synthesis. Once the phenolic compound crossed the cellular membrane, interactions with membrane enzymes and proteins would cause an opposite flow of protons, affecting cellular activity. Davidson (2001) reported that the exact cause effect relation for the mode of action of phenolic compounds, such as thymol, eugenol, carvacrol, and vanillin, has not been determined, but they may inactivate essential enzymes, react with the cell membrane, or disturb genetic material functionality. A. flavus exhibited higher sensitivity to thymol, eugenol, carvacrol, potassium sorbate, sodium bisulfite, and sodium benzoate (at ph 3.5) than to vanillin or citral. Plant-derived antimicrobials are not yet fully exploited. The use of spices, herbs, plants, essential oils, and related phenolic compounds as antimicrobials is limited due to the high MICs required in foods with high protein and/or fat contents (López-Malo et al., 1995, 2000, in press; Castañón et al., 1999), which also may impart objectionable flavors and/or aromas. These undesirable effects can be minimized if the natural compound is used in combination with other environmental stress factors, as in our case, reduced a w and ph. Acknowledgements Authors López-Malo and Palou acknowledge the financial support from CONACyT of Mexico (Project No B), CYTED Program (Project XI.15), and Universidad de las Américas, Puebla (Mexico). References Alzamora, S.M., López-Malo, A., Guerrero, S., Palou, E., Plant antimicrobials combined with conventional preservatives for fruit products. In: Roller, S. (Ed.), Natural Antimicrobials for the Minimal Processing of Foods. Woodhead Publishing, UK, pp Arroyo, M., Aldred, D., Magan, N., Impact of environmental factors and preservatives on growth and ochratoxin A production by Aspergillus ochraceus in wheat-based media. Aspects of Applied Biology 68, Beuchat, L.R., Antimicrobial properties of spices and their essential oils. In: Dillon, V.M., Board, R.G. (Eds.), Natural Antimicrobial Systems and Food Preservation. CAB Intl., Wallingford, England, pp Brancato, F.P., Golding, N.S., The diameter of the mold colony as a reliable measure of growth. Mycologia 45, Buchanan, R.L., Shepherd, A.J., Inhibition of Aspergillus parasiticus by thymol. Journal of Food Science 46, Castañón, X., Argaiz, A., López-Malo, A., Effect of storage temperature on the microbial and color stability of banana purées prepared with the addition of vanillin or potassium sorbate. Food Science and Technology International 5, Cerrutti, P., Alzamora, S.M., Inhibitory effects of vanillin on some food spoilage yeasts in laboratory media and fruit purées. International Journal of Food Microbiology 29, Cerrutti, P., Alzamora, S.M., Vidales, S.L., Vanillin as an antimicrobial for producing shelf-stable strawberry purée. Journal of Food Science 62, Conner, D.E., Beuchat, L.R., 1984a. Effects of essential oils from plants on growth of food spoilage yeasts. Journal of Food Science 49, Conner, D.E., Beuchat, L.R., 1984b. Sensitivity of heat-stressed yeasts to essential oils of plants. Applied and Environmental Microbiology 47, Davidson, P.M., Chemical preservatives and naturally antimicrobial compounds. In: Doyle, M.P., Beuchat, L.R., Montville, T.J. (Eds.), Food Microbiology. Fundamentals and Frontiers, 2nd ed. ASM Press, Washington, DC, pp

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