Antifungal Activity Evaluation of Mexican Oregano (<I>Lippia

2192

Journal of Food Protection, Vol. 74, No. 12, 2011, Pages 2192–2198 doi:10.4315/0362-028X.JFP-11-308 Copyright G, International Association for Food Protection

Research Note Antifungal Activity Evaluation of Mexican Oregano (Lippia berlandieri Schauer) Essential Oil on the Growth of Aspergillus flavus by Gaseous Contact

AI´DA GO´ MEZ-SA´ NCHEZ, ENRIQUE PALOU, AND AURELIO LO´ PEZ-MALO* Downloaded from http://meridian.allenpress.com/jfp/article-pdf/74/12/2192/1684923/0362-028x_jfp-11-308.pdf by guest on 23 September 2021 Departamento de Ingenierı´a Quı´mica, Alimentos y Ambiental, Universidad de las Ame´ricas, Puebla, Cholula, Puebla 72810, Mexico

MS 11-308: Received 22 June 2011/Accepted 15 August 2011

ABSTRACT The antifungal activity of Mexican oregano (Lippia berlandieri Schauer) essential oil by gaseous contact on the growth of Aspergillus flavus at selected essential oil concentrations (14.7, 29.4, 58.8, or 117.6 ml of essential oil per liter of air) and temperatures (25, 30, or 35uC) was evaluated in potato dextrose agar formulated at water activity of 0.98 and pH 4.0. Mold growth curves were adequately fitted (0.984 , R2 , 0.999) by the modified Gompertz model. The effect of the independent variables (concentration of essential oil and temperature) on the estimated model parameters (reciprocal of growth rate [1/nm] and lag time [l]) were evaluated through polynomial equations. Both nm and l were significantly (P , 0.05) affected by the independent variables; nm decreased and l increased as essential oil concentration increased and temperature decreased, which suggests that Mexican oregano essential oil retards or inhibits mold germination stage. Further, minimum fungistatic and fungicide essential oil concentrations at 30 and 35uC were determined. Mexican oregano essential oil applied in gas phase exerts important antifungal activity on the growth of A. flavus, suggesting its potential to inhibit other food spoilage molds.

The use of antimicrobials in foods has been studied for components, the monoterpenes carvacrol and thymol, have many decades. Herbs, plants, and spices (and their important antibacterial and antifungal activities (9, 11, 43). components and essential oils) have bactericide and It has been suggested to use natural antimicrobials in fungicide properties, besides acting as traditional ingredients combination with other antimicrobials or environmental and flavorings in foods (6, 16, 21). Nowadays, consumers factors in order to improve their effectiveness while demand natural and healthy food products, so research diminishing their concentrations to be utilized in foods regarding natural antimicrobials is of increasing importance (20, 31–33). (37, 40, 47). The antimicrobial activity of oregano essential oils is Among the most well known spices that have high mainly attributed to the monoterpenes carvacrol, thymol, r- antimicrobial activity are clove, cinnamon, oregano, and cimene, and c-terpinene (9, 43). Paster et al. (39, 40) stated thyme (15). Many plants of several taxonomic families are that the essential oil of O. vulgare completely inhibited the known as ‘‘oregano,’’ but the ones of main economic mycelial growth of Aspergillus species A. niger, A. flavus, importance are Greek or Mediterranean oregano (Origanum and A. ochraceus. Portillo-Ruiz et al. (41) demonstrated that vulgare), Turkey oregano (Origanum onites), Spanish the essential oil of Mexican oregano (L. berlandieri oregano (Thymus capitatus), and Mexican oregano (Lippia Schauer) exerted a strong antifungal effect in the growth berlandieri Schauer or Lippia graveolens HBK). Mexican of Penicillium, Geotrichum, Aspergillus, and Bipolaris. oregano belongs to the Verbenaceae, and it is commonly A´ vila-Sosa et al. (4) observed that this essential oil had a found in Texas and North and Central America; research strong inhibitory effect on the lag phase and the growth of regarding Mexican oregano is still scarce in contrast to that A. niger and Penicillium spp. by incorporating this essential of Turkey and Mediterranean oregano (44). oil to edible films. Daferera et al. (14) reported that Oregano has been widely utilized; it is one of the most carvacrol and thymol have an additive effect in the important culinary spices in the world. It is utilized in inhibition of Penicillium digitatum. Further, they compared different types of foods, such as bakery products, meat and the antifungal activities of O. vulgare (71% carvacrol- cured meat products, processed vegetables, and snacks, thymol), Origanum dictamus (78% thymol), and Thymus among others. Likewise, it has been reported that the vulgaris (66% carvacrol-thymol) and observed that O. essential oils of several types of oregano and their volatile vulgare and T. vulgaris were more effective, due to the synergy of carvacrol and thymol. * Author for correspondence. Tel: z52 (222) 229-2409; Fax: z52 (222) Antimicrobial activity of the essential oils has been 229-2727; E-mail: [email protected]. evaluated and extensively proved in aqueous systems by J. Food Prot., Vol. 74, No. 12 EVALUATION OF ANTIFUNGAL ACTIVITY BY GASEOUS CONTACT 2193 direct contact. However, there is scarce research regarding solution to give a final spore concentration of 106 spores/ml and gaseous contact of essential oils (23–25). Inouye (23) was utilized the same day (30). reported that essential oils of cinnamon, thyme, and lavender inhibited molds more effectively by gaseous Experimental design. A complete factorial design was utilized to assess the effects of incubation temperature (25, 30, or contact than by direct contact in aqueous systems and 35uC) and essential oil concentration (0 [control], 14.7, 29.4, 58.8, explained that the diminished activity of the essential oil in or 117.6 ml of essential oil per liter of air) on mold radial growth aqueous media is due to the presence of detergents and rate and lag time. Thus, 15 systems were prepared and monitored lypophilic materials. Inouye (23) also pointed out that in triplicate. growth inhibition of Aspergillus by these essential oils occurred at the three stages of their life cycle: conidial Preparation of model systems. Potato dextrose agar systems germination, vegetative mycelium elongation, and sporula- were prepared, adding the necessary quantity of commercial tion of the reproductive mycelium. Arras and Usai (3) sucrose to reach a water activity (aw) of 0.98, sterilized for 15 min at 121uC, cooled, and acidified with (0.45-mm-pore-size) mem- observed alterations in the morphology of P. digitatum Downloaded from http://meridian.allenpress.com/jfp/article-pdf/74/12/2192/1684923/0362-028x_jfp-11-308.pdf by guest on 23 September 2021 caused by thyme essential oil applied by gaseous contact, brane-sterilized hydrochloric acid (0.1 N) to reach pH 4.0. Agar and Lo´pez et al. (28) established that oregano essential oil solutions were poured into sterile petri dishes (60 by 15 mm) (30). Triplicate petri dishes of each system were centrally inoculated by exerts antifungal activity by gaseous contact on A. flavus, pouring 2 ml of the spore suspension to form circular inocula. Three attributing this activity mainly to carvacrol (48). control plates of each system without inoculation were prepared for The objective of this study was to evaluate the measuring aw and pH at the beginning and at the end of the antifungal activity by gaseous contact of Mexican oregano experiment. The uncovered inoculated plates and controls were essential oil on the growth of A. flavus at selected placed on a perforated plastic sheet inside hermetically closed concentrations of essential oil and different incubation plastic chambers (1.7-liter capacity) with transparent lids, leaving temperatures in a model system. sufficient headspace. A glass plate containing the essential oil (0, 25, 50, 100, or 200 ml) was located under the perforated plastic MATERIALS AND METHODS sheet, and chambers were incubated at the experimental temperatures (25, 30, or 35uC). Plant material and essential oil. Dried Mexican oregano (L. berlandieri Schauer) leaves and flowers were acquired from a Determination of aw and pH. Water activity was determined commercial supplier at Puebla city in Mexico. The essential oil of by triplicate with a hygrometer (CX-1, Decagon, Pullman, WA), Mexican oregano was obtained by using a steam distillation calibrated and operated following the procedure described by apparatus (V2000, EO Lab, Puebla, Me´xico) for essential oils Lo´pez-Malo et al. (34). The pH was determined in triplicate with a during 3 h, yielding 3.0% (vol/wt). The essential oil was stored at pH meter (model 50, Beckman, Brea, CA). 4uC in an amber closed bottle until being utilized. Measurement of mold radial growth. The diameter of the Essential oil chemical analysis. Essential oil chemical growing mold colonies was daily measured in two directions at composition was determined by using a Network GC System right angles to each other through the transparent lid of the (6850, Agilent Technologies, Santa Clara, CA) gas chromatograph chamber in order to not disturb the internal system equilibrium. coupled to a mass selective detector (5975C VL, Agilent Observations were carried out daily or as necessary until a Technologies) with Triple-Axis, and a split-splitless injector maximum growth diameter of 48 mm (equivalent to the petri dish (1:10 split ratio). An HP-5MS (5% phenyl–95% polydimethylsi- diameter) was reached, or for a maximum of 3 months when no loxane) fused silica capillary column (30 m by 0.250 mm; film visible or too slow growth was observed (30). thickness, 0.25 mm) was utilized. The carrier gas was helium, at a flow rate of 1.1 ml/min. Samples were prepared by dilution of the Modeling growth kinetics. Growth data were modeled using essential oil at 5:100 (vol/vol) in ethanol, and the injection volume the modified Gompertz equation (12, 46) because nonlinear was 1 ml. The column oven temperature was programmed from behavior was observed for most of the tested cases, and growth 60uC (4 min) to 240uC (10 min) at 4uC/min. Injector and detector parameters were obtained by means of nonlinear regression using temperatures were set at 250 and 280uC, respectively. The the software Kaleidagraph V.3.51 (Synergy Software, Reading, identification of the essential oil components was based on the PA). The modified Gompertz equation is the following: identification of individual compounds on comparison of their hihi D v :e retention times with those of pure compounds, and also by ln t ~A exp { exp m ðÞl{t z1 ð1Þ comparing their mass spectra with the NIST08.L library spectra D0 A database, as well as literature data (2). Analysis was made in where Dt is the average colony diameter at time t, D0 is the average triplicate, and the average is reported. colony diameter at initial time, A is the maximum mold growth achieved during the stationary phase, nm is the mold growth rate, Microorganism and preparation of inocula. A. flavus and l is the lag time.

(ATCC 18672) was obtained from the Food Microbiology Second-order polynomial equations for 1/nm and l were Laboratory of the Universidad de las Ame´ricas Puebla and proposed as secondary models, as functions of essential oil cultivated on potato dextrose agar (Merck, Naucalpan de Jua´rez, concentration and temperature as follows: Mexico) slants at 25uC, with periodic subculturing to maintain Y~ b z b x z b x z b x2z b x2z b x x ð2Þ mold viability. Preparation of inocula was performed by incubation 0 1 i 2 j 3 i 4 j 5 i j of the microorganism on potato dextrose agar slants for 10 days at where Y is the response (1/nm or l); xi and xj are the independent 25uC, and the spores were harvested with 10 ml of 0.1% Tween 80 variables, essential oil concentration and temperature, respectively; (Merck) solution sterilized through membrane (0.45-mm pore size) b0 is a constant; and b1, b2, b3, b4, and b5 are regression filtration. The spore suspension was adjusted with the same coefficients. Analysis of variance was performed for the resulting 2194 GO´ MEZ-SA´ NCHEZ ET AL. J. Food Prot., Vol. 74, No. 12

TABLE 1. Main compounds in Mexican oregano (L. berlandieri Schauer) essential oil determined by gas chromatography–mass spectrometry analysis

Retention time % of total Compound (min) composition b-Myrcene 8.431 2.00 a-Terpinene 9.312 1.51 r-Cimene 9.781 26.34 Cineol 9.913 3.95 c-Terpinene 10.846 2.17 Terpinene-4-ol 15.143 2.41 Thymol 19.240 3.39

Carvacrol 19.921 41.53 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/74/12/2192/1684923/0362-028x_jfp-11-308.pdf by guest on 23 September 2021 Caryophyllene 23.417 5.29 4-tert-Butylchatecol 24.144 1.79 a-Humulene 24.470 3.45 3-tert-Butyl-4- hydroxyanisole 25.437 2.36 Caryophyllene oxide 28.372 3.82 equations in order to select the best equation by means of a backward selection procedure. Finally, the model was validated by means of its coefficient of determination, R2, and analysis of residuals with the software Design-Expert V.6.0.6 (Stat-Ease Inc., FIGURE 1. Effect of the concentration (N, control; %, 14.7 Minneapolis, MN). ml/liter; z, 29.4 ml/liter; ., 58.8 ml/liter; n, 117.6 ml/liter) of Mexican oregano (L. berlandieri Schauer) essential oil by gaseous Determination of inhibitory concentrations. In order to contact on radial growth of A. flavus at 25uC. evaluate the minimum fungistatic and fungicide concentrations of Mexican oregano essential oil, higher essential oil concentrations (294.1, 588.2, 882.4, or 1,470.6 ml of essential oil per liter of air) for Greek oregano there was also a chemotype in which were tested at 30 or 35 C, and the minimal concentrations for no u thymol was the most abundant compound. visible mold growth observed during 7 days were recorded. Then, regrowth of the mold in an essential oil–free atmosphere was Several studies (18, 43) exhibited that the composition observed during 42 days. of the essential oil of a particular plant is affected by several factors, among others, its geographic origin, harvest season, RESULTS AND DISCUSSION and the part of the plant. Silva and Dunford (44) reported that the percentage of thymol-carvacrol in Mexican oregano Chemical composition of the essential oil. Table 1 essential oil was significantly affected by plant maturation. exhibits the main compounds identified in Mexican oregano essential oil by gas chromatography–mass spectrometry A. flavus growth curves. The radial growth of A. analysis. Carvacrol was the most abundant compound, flavus as a function of time at studied oregano essential oil followed by r-cimene; thymol, cineol, and c-terpinene were concentrations applied by gaseous contact and at incubation also present. Several reports regarding the composition of tested temperatures is exhibited in Figure 1. Growth curves Mexican oregano also indicate that the main compounds displayed linear kinetics at 0-ml/liter concentration (control) found in its essential oil are carvacrol, thymol, r-cimene, and nonlinear behavior at the other studied concentrations. cineol, and c-terpinene (4, 44). Kokkini et al. (27) reported Lag time increased as essential oil concentrations increased. that the main components of Greek oregano essential oil are Similar behavior was observed at 30 and 35uC. At every carvacrol and thymol and their biologic precursors, the tested temperature, mold growth decreased as essential oil monoterpenes c-terpinene and r-cimene. concentration increased, exhibiting its inhibitory effect. In general, it has been reported that carvacrol is the Lo´pez et al. (28) obtained similar results regarding increased most abundant compound present in the Greek oregano effectiveness of oregano essential oil by gaseous contact on essential oil and represents about 50 to 60% of its total the growth of A. flavus while increasing its concentration composition (7). Oliveira et al. (38) identified carvacrol as from 17.5 to 175 ml/liter. Inouye et al. (24, 25) suggest that the main compound (38.6%) in the essential oil of Lippia essential oil vapors act by their accumulation at the mold origanoides. However, Morais et al. (36), Gallino (19), and mycelium due to its lipophilic nature and thus extend in a Vicun˜a et al. (49) found that thymol was its most abundant wide surface area, diminishing mold growth. compound (20.6 to 38.4%), whereas carvacrol was present only in trace amounts (0 to 0.4%). So, it can be concluded Modeling of A. flavus growth. Growth data were that there are different chemotypes for this type of oregano modeled using equation 1 (12), and growth parameters A, (17, 42, 45). Similarly, Daferera et al. (14) determined that nm, and l were obtained (Table 2). The maximum specific J. Food Prot., Vol. 74, No. 12 EVALUATION OF ANTIFUNGAL ACTIVITY BY GASEOUS CONTACT 2195

TABLE 2. Gompertz model estimated parameters for A. flavus growth at different incubation temperatures and concentrations of Mexican oregano (L. berlandieri Schauer) essential oil by gaseous contacta Concn of essential oil 21 2 Temp (uC) (ml/liter of air) A nm (h ) l (h) R

0 2.279 0.05387 23.13 0.999 25 14.7 2.654 0.00559 85.85 0.990 29.4 2.601 0.00574 228.65 0.998 58.8 3.501 0.00362 294.13 0.984 117.6 3.463 0.00208 563.91 0.986 30 0 3.307 0.03772 2.84 0.989 14.7 2.377 0.01279 21.90 0.999 29.4 2.052 0.01157 33.61 0.998

58.8 2.090 0.00716 46.06 0.996 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/74/12/2192/1684923/0362-028x_jfp-11-308.pdf by guest on 23 September 2021 117.6 1.831 0.00542 98.55 0.995 35 0 2.466 0.04806 2.85 0.995 14.7 2.182 0.02912 3.36 0.994 29.8 2.135 0.02108 4.61 0.990 58.8 1.981 0.02745 6.97 0.992 117.6 1.757 0.00497 15.70 0.992 a 2 A, maximum mold growth; nm, growth rate; l, germination time; R , adjusted determination coefficient.

growth parameter (A) depends on the maximum growth Secondary models for l and nm. Linear and quadratic diameter, which in our case is the petri dish diameter (12). secondary polynomial models for 1/nm and l, respectively, Mold growth was adequately adjusted by equation 1 were developed to evaluate the effects of the independent (R2 between 0.984 and 0.999). Figure 2 exhibits a Gompertz variables, temperature and essential oil concentration, on model fit to the experimental data of A. flavus growth at these A. flavus growth parameters; their estimated coeffi- every studied temperature. The inhibitory effect of an cients are exhibited in Table 3. Statistical analysis of the increasing concentration of essential oil vapors at each models demonstrated that the effects of the variables of temperature demonstrates a decrease in growth rates while temperature and essential oil concentration were significant increasing lag times. The inhibitory effect was best observed (P , 0.05) for the parameters 1/nm and l, while the at an incubation temperature of 25uC, with l values from interaction of temperature and concentration was significant 85.85 h at a concentration of 14.7 ml/liter to 563.91 h at a only for l (P , 0.05). Determination coefficients (R2) for concentration of 117.6 ml/liter; this difference represents an the 1/nm and l models were 0.824 and 0.948, respectively, increase of lag time of nearly 560%, whereas at 30 and 35uC indicating an adequate fit. these differences were close to 350 and 241%, respectively. The effect of essential oil concentration on lag time for Char et al. (12) utilized equation 1 for Eurotium chevalieri mold growth can be explained by the action of the essential growth data at different conditions of aw, pH, and potassium oil at the germination phase of the mold conidia. Burnett (8) sorbate concentrations at a temperature of 25uC and reported and Carlile and Watkinson (10) observed that for Aspergil- that the inhibitory effect of the antimicrobial increased as its lus fumigatus the germination phase consists of two stages: concentration did, causing the mold growth rate to decrease first, a spherical growth phase of the conidia, during which and the lag time to increase, thus suggesting that this model the membrane hydrates and causes the reconstruction of a is adequate to describe the mold growth behavior. Stu¨ckrath lipid bilayer and the synthesis of a new cell wall, resulting in and Petzold (46) also utilized this model for growth data of swelling of the conidia (26); finally, the emergent phase, at molds and yeasts in blueberries at different pHs and which the conidium polarizes due to the accumulation of potassium sorbate and glycerol concentrations at 25uC and vesicles inside the cell wall, from which then emerges the reported values of growth rate between 0.023 and 0.108 h21 germinative tube (5). Inouye (23) determined that the in their controls (at pH values of 2.48 and 4.3, respectively); essential oils of lavender (Lavandula officinalis) and tea tree 21 the value obtained for nm in our experiment (0.054 h ) for (Melaleuca alternifolia) applied by gaseous contact did not A. flavus at 25uC in the control falls into this interval. inhibit the spherical growth phase, although they did so at As the incubation temperature increased from 25 to the emergent phase of the germinative tube; likewise, 35uC, l decreased and growth rates increased. A. flavus Inouye (23) stated that inhibition by death of the conidia growth was favored at the high studied temperatures due to was realized at the emergent phase of the germinative tube. the proximity of these temperatures to its optimal growth Therefore, it is suggested that at the tested concentrations of temperature of 33uC (22). The effect of increasing the essential oil in our study, there could be an effect of delay of temperature in the control systems is reflected in the the spherical growth phase, but without inhibition of the reduction of l, and its effect on the nm is not very clear, germination emergent phase. since mold grew relatively quickly at the three evaluated The effect of temperature on A. flavus growth temperatures. parameters 1/nm and l agrees with the results obtained by 2196 GO´ MEZ-SA´ NCHEZ ET AL. J. Food Prot., Vol. 74, No. 12 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/74/12/2192/1684923/0362-028x_jfp-11-308.pdf by guest on 23 September 2021

FIGURE 2. Growth of A. flavus at 25uC (A), 30uC (B), or 35uC (C) exposed to selected concentrations (N, control; %, 14.7 ml/liter; z, 29.4 ml/liter; ., 58.8 ml/liter; n, 117.6 ml/liter) of Mexican oregano (L. berlandieri Schauer) essential oil by gaseous contact, adjusted (lines) with the modified Gompertz model.

Abellana et al. (1). In their case, the effect of the effects of individual factors aw, temperature, and mold, as independent variables of temperature and aw and their well as their interactions on the growth rates of Aspergillus interaction on the growth of Penicillium spp. were spp. and Penicillium spp., among other molds, were significant (P , 0.01). Also, Carrillo et al. (11) determined significant (P , 0.01). They also reported that growth rates that the individual factors of temperature, aw, and pH were greater as temperature increased. exerted a significant effect (P , 0.001) on the growth parameters (nm and l)ofRhizopus oryzae, finding that the Determination of minimum fungicide and fungi- growth of this mold was primarily affected by temperature, static concentrations. Table 4 exhibits the inhibitory then by aw, and to a lesser extent by pH. Similarly, Marin et effects of Mexican oregano essential oil by gaseous contact al. (35) and Lopez Dı´az et al. (29) determined that the at the concentrations at which there were observed J. Food Prot., Vol. 74, No. 12 EVALUATION OF ANTIFUNGAL ACTIVITY BY GASEOUS CONTACT 2197

TABLE 3. Polynomial equation coefficients for 1/nm and l TABLE 4. Inhibitory fungicide or fungistatic effect of Mexican secondary models as functions of temperature and concentration oregano (L. berlandieri Schauer) essential oil by gaseous contact of Mexican oregano (L. berlandieri Schauer) essential oil by on A. flavus growth at different incubation temperaturesa gaseous contact Temp (uC): Equation coefficientsb Concn of 30 35 a Factor 1/nm (h) l (h) essential oil (ml/liter of air) Growth Regrowth Growth Regrowth Constant 462.15312 3,042.39918 T 213.89370 2201.48558 294.1 2 z 2 z EO 1.82950 14.94762 588.2 2 z 2 z 2 z z T 3.293 882.4 2 2 T | EO 20.43850 1,470.6 222 2 a a T, temperature; EO, concentration of essential oil in microliters Growth experiments were conducted in an atmosphere with the Downloaded from http://meridian.allenpress.com/jfp/article-pdf/74/12/2192/1684923/0362-028x_jfp-11-308.pdf by guest on 23 September 2021 per liter of air. essential oil at gas phase for 7 days; regrowth experiments were b Significant coefficients (P , 0.05). conducted in an atmosphere without the essential oil for 42 days. z, growth; 2, no growth. fungistatic or fungicide effects at incubation temperatures of 30 and 35uC. It was determined at both studied temperatures 3. Arras, G., and M. Usai. 2001. Fungitoxic activity of 12 essential oils that a concentration of 1,470.6 ml of the essential oil per liter against four postharvest citrus pathogens: chemical analysis of exerted fungicide activity since there was no mold growth Thymus capitatus oil and its effect in sub-atmospheric pressure conditions. J. Food Prot. 64:1025–1029. during a period of 42 days. Besides, at concentrations 4. A´ vila-Sosa, R., E. Hernańdez-Zamoran, I. Lo´pez-Mendoza, E. Palou, greater than 294.1 ml/liter but less than 882.4 ml/liter, the M. Jimeńez, G. Neva´rez-Moorilloń, and A. Lo´pez-Malo. 2010. effect of the essential oil was fungistatic, since inhibition of Fungal inactivation by Mexican oregano (Lippia berlandieri Schauer) mold growth was observed during 7 days in the presence of essential oil added to amaranth, chitosan, or starch edible films. 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