Growth kinetics of mycotoxigenic Aspergillus species in response to essential oil constituents

Abstract

Background

Mycotoxigenic Aspergillus species are primary contaminants in food and feed. Growing health concerns regarding synthetic chemical preservatives have driven interest in essential oil (EO) constituents as natural alternatives. However, the specific impact of these compounds on fungal growth dynamics remains insufficiently understood.

Aims

This study quantitatively evaluated the growth-kinetic responses of four major mycotoxigenic species, namely Aspergillus flavus, Aspergillus parasiticus, Aspergillus niger, and Aspergillus carbonarius, when exposed to selected essential oil constituents.

Methods

Fungal growth was monitored in vitro under treatment with D-limonene, thymol, 1,8-cineol, cuminaldehyde, and linalool (250–1000 mg/kg). Growth kinetics were analyzed using a two-phase linear model to estimate lag phase duration (λ) and specific growth rate (μ). Additionally, mycelial growth inhibition and conidial germination assays were conducted to validate antifungal efficacy.

Results

All tested constituents induced dose-dependent alterations in growth kinetics. Thymol and cuminaldehyde demonstrated the most potent inhibitory effects, significantly extending the lag phase and reducing growth rates across all species. Notably, thymol completely suppressed A. parasiticus growth at 750 mg/kg and extended the A. flavus lag phase to 6.01 days. Cuminaldehyde reduced the A. carbonarius growth rate to 6.83 mm/day. While D-limonene showed moderate inhibition, 1,8-cineol and linalool were the least effective. At 1000 mg/kg, both thymol and cuminaldehyde achieved 100% inhibition of conidial germination in all species.

Conclusions

Growth-kinetic modeling is a highly sensitive tool for differentiating the efficacy of natural antifungal agents. The significant disruption of growth patterns by phenolic (thymol) and aldehydic (cuminaldehyde) compounds underscores their potential for the targeted, predictive control of Aspergillus in food and feed systems.

Resumen

Antecedentes

Las especies de Aspergillus micotoxigénicas son uno de los principales contaminantes en alimentos y piensos. La creciente preocupación por los conservantes químicos sintéticos ha despertado el interés en los constituyentes de aceites esenciales como agentes antifúngicos naturales. Sin embargo, sus efectos sobre la dinámica del crecimiento fúngico aún no se han caracterizado suficientemente.

Objetivos

Este estudio tuvo como objetivo evaluar cuantitativamente las respuestas cinéticas de crecimiento de cuatro especies principales de Aspergillus (Asperillus flavus, Aspergillus parasiticus, Aspergillus niger y Aspergillus carbonarius) al ser expuestas a componentes seleccionados de aceites esenciales.

Métodos

Se monitorizó el crecimiento fúngico in vitro en presencia de D-limoneno, timol, 1,8-cineol, cuminaldehído y linalool en concentraciones de 250 a 1.000 mg/kg. La cinética de crecimiento se analizó mediante un modelo lineal de dos fases para estimar la duración de la fase de latencia (λ) y la tasa específica de crecimiento (μ). También se realizaron ensayos antifúngicos convencionales, incluyendo pruebas de inhibición del crecimiento micelial y de germinación conidial.

Resultados

Todos los constituyentes de los aceites esenciales indujeron alteraciones dependientes de la dosis en la cinética de crecimiento. El timol y el cuminaldehído ejercieron los efectos inhibitorios más potentes, prolongando marcadamente la fase de latencia y reduciendo significativamente las tasas de crecimiento en todas las especies. El timol suprimió completamente el crecimiento de A. parasiticus a una concentración de 750 mg/kg, y extendió la fase de latencia de A. flavus a 6,01 días, mientras que el cuminaldehído redujo la tasa de crecimiento de A. carbonarius a 6,83 mm/día. El D-limoneno mostró una inhibición moderada, mientras que el 1,8-cineol y el linalool presentaron los efectos más débiles. Se observó una inhibición completa de la germinación conidial con timol y cuminaldehído a 1.000 mg/kg en todas las especies evaluadas.

Conclusiones

El modelado de la cinética de crecimiento demostró ser una herramienta sensible para diferenciar la eficacia antifúngica de los constituyentes de aceites esenciales. La pronunciada alteración cinética inducida por los compuestos fenólicos y aldehídicos resalta su potencial para el control predictivo y dirigido de especies de Aspergillus micotoxigénicas en sistemas de alimentos y piensos.

Mycotoxigenic fungi, particularly members of the Aspergillus genus such as Aspergillus flavus, Aspergillus parasiticus, Aspergillus niger, and Aspergillus carbonarius, represent a significant threat to global food safety and public health. This is due to their ability to synthesize highly toxic secondary metabolites, most notably aflatoxins and ochratoxins. These fungi frequently contaminate a wide variety of agricultural commodities, including cereals, nuts, oilseeds, dried fruits, and spices. The risk of contamination is heightened by warm and humid conditions, which are becoming more common because of climate change and inadequate storage infrastructure in many regions.^12,28

Among these strains, A. flavus and A .parasiticus are the primary producers of aflatoxins, especially aflatoxin B1 (AFB1), which is the most potent naturally occurring hepatocarcinogen. The International Agency for Research on Cancer (IARC) has classified AFB1 as a Group 1 carcinogen, confirming its established role in human liver cancer.¹⁵ Chronic exposure to aflatoxins is linked not only to hepatocellular carcinoma but also to immunosuppression, stunted growth in children, and greater susceptibility to infectious diseases.^4,22 In contrast, A. niger and A. carbonarius are well-known for producing ochratoxin A (OTA). OTA is a nephrotoxic, immunotoxic, and possibly carcinogenic compound (Group 2B) that frequently contaminates dried fruits, cereal grains, coffee, cocoa, and wine.^8,9

Despite the various post-harvest practices and regulatory limits put in place, managing mycotoxin contamination remains a persistent challenge. Traditional chemical fungicides and preservatives, while effective, face increasing criticism due to their environmental persistence, potential health risks, and contribution to the emergence of resistant fungal strains.^17,24 Consequently, there is an urgent demand for novel, eco-friendly antifungal strategies that are both effective and acceptable to consumers.³²

In this context, essential oils (EOs) and their bioactive constituents have emerged as promising natural antifungal agents. These complex mixtures of volatile terpenes and phenolic compounds, derived from aromatic and medicinal plants, are widely recognized for their broad-spectrum antimicrobial properties.^29,31 Notably, many EO components are categorized as ‘Generally Recognized as Safe’ (GRAS) by the U.S. Food and Drug Administration, a regulatory status that significantly enhances their viability for application in food systems.^11,14,25

EOs components are particularly promising because of their multifaceted mechanisms of antifungal action. These include disrupting cell membrane integrity and permeability, inhibiting ergosterol biosynthesis, generating reactive oxygen species (ROS), interfering with mitochondrial function, and inhibiting spore germination and mycelial growth.^1,9,35,38,39 For instance, thymol and carvacrol, phenolic monoterpenes found in thyme and oregano oils, respectively, have shown potent antifungal activity by destabilizing the membrane and inducing oxidative damage.^18,23 Similarly, D-limonene, a major component of citrus peel oil, exerts antifungal effects by altering membrane permeability and disrupting fungal metabolism.¹⁰ Linalool, 1,8-cineol (eucalyptol), and cuminaldehyde are also widely reported to have strong inhibitory effects on various filamentous fungi, including A. flavus, by targeting multiple cellular sites and interfering with energy metabolism.^16,38

Importantly, EO compounds are increasingly being integrated into food-grade delivery systems, such as edible coatings, biodegradable films, and nanoemulsions. This is done to enhance their stability and controlled release while minimizing sensory alterations.^7,34 These applications not only improve the safety and shelf-life of food products but also align with the growing demand for sustainable and natural food preservatives.

Although the antifungal activity of EOs and their individual constituents against Aspergillus species has been extensively reported, most studies rely primarily on endpoint-based measurements such as inhibition percentages, MIC values, or qualitative growth suppression. While informative, these approaches provide limited insight into the temporal dynamics of fungal development and do not distinguish between delayed growth onset, reduced growth rates, or complete growth arrest. Quantitative growth-kinetic modeling offers a more informative approach by capturing key parameters such as lag phase duration and specific growth rate, which are directly relevant for predicting fungal behavior under food storage and processing conditions. However, the application of such kinetic approaches to evaluate EO constituents against mycotoxigenic Aspergillus species remains comparatively limited. Accordingly, the present study aimed to quantitatively compare the growth-kinetic responses of four mycotoxigenic Aspergillus species exposed to selected monoterpenes and aromatic aldehydes, using mathematical growth modeling, with conventional antifungal assays employed as complementary endpoints.

Materials and methods

Materials

D-limonene (99% purity), thymol (98% purity), 1,8-cineol (≥98% purity), cuminaldehyde (≥98% purity) and linalool (97% purity) were purchased from Sigma Aldrich Chimie (St. Quentin Fallavier, France) and stored in the dark at 4 °C until use. Tween 80 and dimethyl sulfoxide (DMSO) were also obtained from Sigma-Aldrich France. Potato dextrose agar (PDA) was supplied by Merck KGaA (Darmstadt, Germany).

Fungal strains

A. flavus NRRL 3251, A. parasiticus CBS100926, A. niger NRRL567 and A. carbonarius NRRL369 strains were kindly supplied by the Laboratory of Microbial Systems Biology (LBSM), École Normale Supérieure of Kouba (Algiers, Algeria). For inoculum preparation, spores were harvested from 7-day-old PDA cultures incubated at 25 °C. The spores were collected by rinsing each plate with 10 mL of sterile distilled water containing 0.1% Tween-80 (v/v), followed by gentle scraping with a sterile glass rod. The spore concentration was standardized to 10⁵ spores/mL using a hemocytometer (0.2 mm depth, 1/400 mm²) and examined under a light microscope (Motic BA210, China).

In vitro antifungal activity on mycelial growth

The inhibitory effects of D-limonene, thymol, 1,8-cineol, cuminaldehyde, and linalool on the mycelial growth of the tested fungal strains were assessed through direct contact assays on PDA plates. Stock solutions were prepared in 1% DMSO and incorporated into autoclaved PDA cooled to 55 °C, yielding final concentrations ranging from 250 to 1000 mg/kg. The concentration range of 250–1000 mg/kg was selected based on established literature for essential oil constituents against mycotoxigenic Aspergillus species, preliminary trials that identified this range as the biologically informative window for estimating kinetic parameters, and the need to maintain concentrations within limits that preserve the organoleptic quality of food products. After medium solidification, each plate was inoculated at the center with 10 μL of the conidial suspension. Plates containing PDA with 1% DMSO were inoculated as growth controls. Colony diameters were measured along two perpendicular axes at multiple time points during incubation (1, 2, 3, 5, 6 and 7 days) to capture the complete growth dynamics of each strain. The percentage of inhibition (I%) was calculated according to the following formula⁶:

D_Control: diameter of the control growth zone in mm; D_Test: diameter of the test growth zone in mm. All treatments and controls were performed in triplicate using independent plates.

Spore germination inhibition assay

To assess the effect on spore germination, essential oil components (D-limonene, thymol, 1,8-cineol, cuminaldehyde, and linalool) were first dissolved in DMSO as a solvent to obtain stock solutions. Then, 100 μL of each DMSO-dissolved compound solution (250–1000 mg/kg) were mixed with 100 μL of a conidial suspension (1 × 10⁵ spores/mL). The mixtures were placed on sterile glass depression slides and incubated at 25 °C for 24 h in sealed moist chambers lined with water-saturated absorbent paper.²⁷ This setup reliably maintains a near-saturated environment of approximately 95–100% relative humidity.

Control samples consisted of conidial suspension mixed with DMSO alone at the same final proportion used in the treatments, and no significant reduction in spore germination was observed in these controls. After incubation, spore germination was evaluated microscopically by counting 100 spores per treatment.⁶

G_Control and G_Test represent the number of germinated spores in control and treated slides, respectively. All treatments and controls were performed in six replicates.

Growth assessment and kinetic modelling

The diameter of the developed mycelia was measured along two orthogonal axes. Mean diameters at any time t were noted d^(t) and subsequently analyzed using a two-phase linear model proposed by Gougouli and Koutsoumanis¹³:

where μ is the growth rate (mm/day), λ the lag time (days) and d⁰ is the diameter of the inoculated spore suspension (d⁰ = 4–5 mm).

Parameter estimation and statistical methods

The parameters of the two-phase linear model were estimated using nonlinear regression with a curve fitting toolbox (MATLAB 6.5, The Math-Works Inc., Natick, MA, USA). The root mean squared error (RMSE) between all experimental and predicted data, adjusted coefficients of determination (Radjusted2) and confidence intervals (calculated with 95% of probability) were used as goodness-of-fit indicators for the estimated parameters.

A covariance analysis test (ANCOVA) was used to examine the significant effect of the type of antifungal substance on the percentage of inhibition and growth kinetic parameters while controlling for the effect of their concentrations. This was followed by a Bonferroni post-hoc test for multiple comparisons. A p-value <0.05 was considered statistically significant. This analysis was performed using SPSS Statistics software version 23 (IBM, New York, USA).

Results

Effect of essential oil compounds on the growth kinetics of Aspergillus species

Colony diameter data for all Aspergillus species, recorded at multiple incubation time points up to 7 days, were accurately described by the two-phase linear model, yielding high adjusted R² values (>0.97) and low RMSE values (mean 1.28 mm), indicating excellent model performance (Table 1 and Fig. 1). The repeated measurements allowed reliable estimation of lag phase duration (λ) and specific growth rate (μ) for all treatments.

Table 1.

Estimated lag time (λ in days) and growth rates (μ in mm/day) of Aspergillus flavus, Aspergillus parasiticus, Aspergillus niger and Aspergillus carbonarius at different concentration of D-limonene, thymol, 1,8-cineol, cuminaldehyde and linalool. Mean values ±95% confidence interval.

Compounds	Concentration	A. flavus				A. parasiticus				A. niger				A. carbonarius
	mg/kg	λ	μ	RMSE	R^2	λ	μ	RMSE	R^2	λ	μ	RMSE	R^2	λ	μ	RMSE	R^2
Control	0	0.34 ± 0.12	13.31 ± 0.19	1.13	0.996	0.08 ± 0.06	12.01 ± 0.30	1.35	0.993	0.12 ± 0.07	13.26 ± 0.19	1.15	0.995	0.033 ± 0.002	11.76 ± 0.58	2.533	0.991
D-limonene	250	1.95 ± 0.30	9.05 ± 0.57	1.98	0.988	2.59 ± 0.24	10.23 ± 0.71	1.86	0.989	0.53 ± 0.29	8.49 ± 0.59	2.57	0.982	3.36 ± 0.19	13.10 ± 0.39	1.666	0.992
	500	3.47 ± 0.22	8.23 ± 0.70	1.29	0.988	2.51 ± 0.18	8.17 ± 0.50	1.31	0.992	2.64 ± 0.24	8.70 ± 0.61	1.61	0.989	3.72 ± 0.16	9.00 ± 0.29	1.277	0.993
	750	5.26 ± 0.25	6.33 ± 1.15	0.68	0.974	4.94 ± 0.20	7.83 ± 1.07	1.25	0.961	5.46 ± 0.17	7.33 ± 1.01	0.59	0.981	5.33 ± 0.12	4.00 ± 0.19	0.530	0.997
	1000	NG	NG	–	–	NG	NG	–	–	NG	NG	–	–	NG	NG	–	–
Thymol	250	2.83 ± 0.20	8.40 ± 0.51	1.03	0.991	2.81 ± 0.22	6.83 ± 0.50	1.30	0.987	0.20 ± 0.13	8.16 ± 0.45	1.97	0.989	4.47 ± 0.20	10.17 ± 0.67	1.779	0.989
	500	4.62 ± 0.21	8.67 ± 0.73	1.20	0.988	5.42 ± 0.28	4.00 ± 0.81	0.53	0.972	3.49 ± 0.26	9.60 ± 0.94	1.74	0.983	5.25 ± 0.15	6.67 ± 0.51	1.043	0.987
	750	6.01 ± 0.31	3.69 ± 0.14	0.37	0.929	>7	ND	–	–	5.85 ± 0.23	6.67 ± 0.83	1.04	0.978	>7	ND	–	–
	1000	NG	NG	–	–	NG	NG	–	–	NG	NG	–	–	NG	NG	–	–
1,8-Cineol	250	0.23 ± 0.13	10.62 ± 0.69	2.03	0.991	0.27 ± 0.24	7.35 ± 0.39	1.70	0.990	1.45 ± 0.25	9.54 ± 0.48	2.37	0.986	0.15 ± 0.07	8.86 ± 0.52	1.669	0.993
	500	0.59 ± 0.17	7.33 ± 0.53	2.01	0.990	2.58 ± 0.23	8.00 ± 0.54	1.40	0.990	1.51 ± 0.14	7.92 ± 0.21	1.77	0.989	0.39 ± 0.11	7.75 ± 0.35	1.551	0.992
	750	4.57 ± 0.25	9.33 ± 1.02	1.55	0.970	3.74 ± 0.19	7.23 ± 0.57	1.06	0.987	4.61 ± 0.16	9.50 ± 0.38	1.03	0.987	4.22 ± 0.11	8.50 ± 0.27	1.486	0.995
	1000	NG	NG	–	–	NG	NG	–	–	NG	NG	–	–	NG	NG	–	–
Cuminaldehyde	250	2.79 ± 0.37	10.80 ± 0.71	2.16	0.970	0.19 ± 0.18	9.56 ± 0.37	1.63	0.994	0.12 ± 0.12	8.70 ± 0.45	1.96	0.990	0.18 ± 0.14	9.91 ± 0.55	2.426	0.989
	500	3.55 ± 0.33	8.88 ± 0.65	2.12	0.971	1.81 ± 0.20	8.19 ± 0.61	2.13	0.983	2.14 ± 0.28	9.10 ± 0.67	1.76	0.990	2.42 ± 0.13	8.03 ± 0.74	1.932	0.983
	750	4.55 ± 0.18	7.33 ± 0.51	0.84	0.986	3.96 ± 0.13	10.20	1.01	0.992	3.35 ± 0.20	7.20 ± 0.52	0.96	0.992	4.26 ± 0.22	6.83 ± 0.72	0.880	0.986
	1000	NG	NG	–	–	NG	NG	–	–	NG	NG	–	–	NG	NG	–	–
Linalool	250	1.39 ± 0.29	11.45 ± 0.82	2.17	0.986	0.19 ± 0.9	9.56 ± 0.37	1.63	0.996	0.41 ± 0.20	10.46 ± 0.48	2.86	0.988	0.28 ± 0.07	9.85 ± 0.36	1.567	0.995
	500	2.34 ± 0.25	8.57 ± 0.58	1.51	0.991	2.22 ± 0.15	8.73 ± 0.81	2.11	0.994	0.85 ± 0.21	9.36 ± 0.47	2.52	0.987	1.29 ± 0.23	9.29 ± 0.42	1.467	0.995
	750	3.46 ± 0.17	8.70 ± 0.54	1.00	0.993	3.96 ± 0.11	10.20 ± 0.58	1.08	0.992	3.62 ± 0.21	9.13 ± 0.35	1.38	0.989	3.41 ± 0.17	9.33 ± 0.59	1.084	0.993
	1000	NG	NG	–	–	NG	NG	–	–	NG	NG	–	–	NG	NG	–	–

RMSE: root mean squared error (in mm) between experimental and predicted colony diameters; NG: no growth; ND: not determined.

Fig. 1.

Effect of different concentrations (● 0, ■ 250, ▾ 500, ▴ 750 and ★ 1000 mg/kg) of D-limonene, thymol 1,8-cineol, cuminaldehyde and linalool on the growth diameter d kinetic of Aspergillus flavus, Aspergillus parasiticus, Aspergillus niger and Aspergillus carbonarius. Solid lines represent the fit of the two-phase linear model to the growth diameter data, represented by symbols.

All essential oil (EO) compounds induced dose-dependent alterations in fungal growth kinetics across the four Aspergillus species. Control cultures exhibited negligible lag phases (λ < 0.35 days) and high growth rates (μ > 11 mm/day). In contrast, EO-treated cultures showed significant extensions of the lag phase and reductions in growth rate.

Thymol and cuminaldehyde produced the strongest inhibitory effects. At 750 mg/kg, thymol extended the lag phase of A. flavus to 6.01 days and completely inhibited the growth of A. parasiticus. Cuminaldehyde reduced the growth rate of A. carbonarius to 6.83 mm/day at the same concentration. D-limonene showed moderate inhibitory effects, characterized by prolonged lag phases (>5 days in A. flavus and A. niger at 750 mg/kg) while maintaining growth rates above 6 mm/day. In contrast, 1,8-cineol and linalool exhibited weaker inhibition, with μ values generally exceeding 8.5 mm/day even at higher concentrations.

Species-dependent responses were observed. A. parasiticus showed the highest sensitivity to thymol and cuminaldehyde, whereas A. carbonarius displayed greater tolerance, particularly toward D-limonene and linalool.

ANCOVA analysis confirmed that EO compound type significantly influenced antifungal efficacy independently of concentration (p < 0.05). As shown in Fig. 2, thymol treatments formed statistically distinct groups across inhibition percentage, μ, and λ values, especially at 750 and 1000 mg/kg, for all species. Cuminaldehyde also showed significant separation from D-limonene and 1,8-cineol at concentrations ≥500 mg/kg. Linalool frequently shared statistical groupings with 1,8-cineol and D-limonene, indicating no significant differences among these compounds across several concentrations.

Fig. 2.

Effect of different concentrations of D-limonene, thymol 1,8-cineol, cuminaldehyde and linalool on percentage growth inhibition, growth rate μ and lag time λ of Aspergillus flavus, Aspergillus parasiticus, Aspergillus niger and Aspergillus carbonarius. Error bars represent standard deviation (of three independent experiments), and identical letters denote means that are not statistically different.

The heatmap (Fig. 3) clearly illustrates these dose-dependent inhibitory patterns across all tested compounds and species. Thymol and cuminaldehyde showed the strongest antifungal effects, whereas 1,8-cineol and linalool were consistently less effective. Differences in species sensitivity were also visually apparent, with A. parasiticus and A. flavus appearing more responsive to the treatments than A. niger and A. carbonarius, further supporting the trends obtained from growth kinetics analysis.

Fig. 3.

Heatmap representation of the in vitro antifungal activity of D-limonene, thymol, 1,8-cineol, cuminaldehyde and linalool against Aspergillus flavus, Aspergillus parasiticus, Aspergillus niger and Aspergillus carbonarius, based on percentage growth inhibition at 250, 500 and 750 mg/kg.

Inhibitory effects of essential oil compounds on spore germination

All tested EO compounds caused a clear, concentration-dependent inhibition of spore germination in the four Aspergillus species (Table 2). In control treatments, germination reached 100% for all species, confirming normal spore viability in the absence of EO exposure. Increasing concentrations of EO constituents progressively reduced germination percentages across all strains.

Table 2.

Effect of different concentrations of D-limonene, thymol 1,8-cineol, cuminaldehyde and linalool on the spore germination (%) of Aspergillus flavus, Aspergillus parasiticus, Aspergillus niger and Aspergillus carbonarius.

Concentration (mg/kg)	D-limonene	Thymol	1,8-Cineol	Cuminaldehyde	Linalool
A. flavus
0	100	100	100	100	100
250	53.7 ± 3.2^a	32.2 ± 3.1^b	67.3 ± 2.8^c	38.7 ± 2.2^d	44.8 ± 3.5^e
500	30.6 ± 4.2^a	17.7 ± 3.6^b	45.0 ± 2.6^c	24.0 ± 2.6^d	32.0 ± 2.1^a
750	13.0 ± 2.6^a	11.3 ± 2.8^a	23.7 ± 2.9^b	12.8 ± 1.5^a	23.3 ± 2.2^b
1000	0	0	0	0	0
A. parasiticus
0	100	100	100	100	100
250	48.8 ± 4.4^ab	57.7 ± 3.3^c	52.8 ± 2.3^ac	45.7 ± 3.7^b	57.7 ± 3.3^c
500	31.7 ± 3.8^a	35.8 ± 2.6^a	34.5 ± 3.1^a	35.3 ± 2.6^a	35.8 ± 2.6^a
750	20.5 ± 3.3^a	25.0 ± 2.4^bc	20.0 ± 2.4^a	23.0 ± 2.4^ac	25.0 ± 2.4^b
1000	0	0	0	0	0
A. niger
0	100	100	100	100	100
250	40.2 ± 2.6^a	55.0 ± 3.7^b	54.2 ± 2.9^b	54.8 ± 2.0^b	65.5 ± 2.4^c
500	30.0 ± 4.2^a	27.7 ± 2.8^a	39.8 ± 2.3^b	36.8 ± 2.9^b	55.5 ± 2.4^c
750	9.5 ± 2.7^a	17.0 ± 2.2^b	29.7 ± 3.3^c	20.7 ± 2.2^b	28.5 ± 3.0^c
1000	0	0	0	0	0
A. carbonarius
0	100	100	100	100	100
250	45.3 ± 4.3^a	26.8 ± 2.6^b	55.3 ± 3.8^c	63.5 ± 2.4^d	63.7 ± 2.7^d
500	25.0 ± 3.2^a	18.5 ± 2.4^b	40.0 ± 2.6^c	32.5 ± 1.9^d	51.7 ± 2.4^e
750	10.5 ± 2.7^a	0	20.2 ± 2.1^bc	18.7 ± 3.0^b	24.7 ± 2.9^c
1000	0	0	0	0	0

Values are the means of six determinations ± standard deviation.

Means followed by different letters in the same row are significantly different (p < 0.05).

At 250 mg/kg, germination was already markedly reduced, with thymol and cuminaldehyde showing stronger inhibitory effects than the other compounds. In A. flavus, thymol reduced germination to 32.2% and cuminaldehyde to 38.7%, compared with 53.7% for D-limonene and 67.3% for 1,8-cineol. Further increases in concentration (500 and 750 mg/kg) intensified germination inhibition. Complete suppression of germination was observed for all EO compounds at 1000 mg/kg in all tested species.

Across all strains and concentrations, thymol consistently exhibited the highest inhibitory activity against spore germination, followed by cuminaldehyde. D-limonene showed intermediate efficacy, whereas 1,8-cineol and linalool were the least effective. Notably, thymol completely inhibited germination of A. carbonarius at 750 mg/kg.

ANCOVA analysis did not indicate a significant overall strain effect on germination inhibition (p > 0.05). However, specific interspecies differences were detected, with A. flavus and A. niger responding significantly differently to thymol and linalool treatments (p = 0.023).

Discussion

The strong fit of the two-phase linear model to the colony diameter data, as shown by high adjusted R² values and low RMSE values, validates its appropriateness for describing Aspergillus growth dynamics under EO constituents treatment. The model reliably represented the two-phase character of fungal growth responses across all four species and all treatment conditions, allowing precise and reproducible estimation of both lag phase duration (λ) and specific growth rate (μ). The capacity to separately quantify these two kinetic parameters demonstrates the added value of growth kinetic modeling compared with conventional endpoint-based antifungal assessments, which usually report a single inhibition percentage without accounting for the temporal dynamics of fungal development. This dual-parameter characterization is important for food safety applications, where both the timing of fungal growth initiation and the rate of subsequent colonization are critical determinants of mycotoxin accumulation risk throughout storage.

The pronounced growth kinetic effects of thymol and cuminaldehyde establish these phenolic and aldehydic compounds as the strongest inhibitory constituents of those evaluated. Thymol led to the largest extensions of lag phase across all species. Cuminaldehyde demonstrated particularly strong inhibition of growth rate. These kinetic results align with previous reports describing strong inhibitory activity of thymol and aromatic aldehydes against Aspergillus spp.,^1,22,37 and extend those findings through providing quantitative kinetic parameters that more precisely define the nature, extent, and temporal profile of growth disruption. In terms of food preservation, the significant lag phase extensions induced by these compounds are of particular practical relevance, as delayed growth initiation directly reduces the risk of fungal colonization and mycotoxin accumulation during the critical early stages of food storage.

The moderate inhibition noted for D-limonene presents a kinetically distinct profile from that of thymol and cuminaldehyde. D-limonene caused extended lag phases with high growth rates, showing that this compound primarily disrupts early fungal adaptation and growth initiation instead of continuing inhibition throughout the exponential growth phase. This kinetic differentiation between D-limonene and the phenolic or aldehydic compounds would not have been detectable using conventional endpoint inhibition measurements, demonstrating better distinguishing ability of the kinetic modeling approach used in this study. These observations agree with previously reported antifungal properties of monoterpene hydrocarbons,^10,37 and the kinetic data presented here provide a more precise quantitative basis for defining the differential inhibitory profiles of chemically different EO constituents.

On the other hand, the lesser kinetic effects of 1,8-cineol and linalool, and comparatively modest lag phase extensions even at higher concentrations, indicate minor impact with fungal growth dynamics in these experiments. These compounds produced the smallest kinetic perturbations of the five EO constituents tested, clearly distinguishing them from thymol and cuminaldehyde with regard to antifungal potency according to growth kinetic parameters. The observed difference among species variability in kinetic responses, with A. parasiticus consistently displaying the highest susceptibility and A. carbonarius displaying higher resistance particularly toward D-limonene and linalool, further highlights species-specific differences in susceptibility that could indicate differences in stress-response capacity among Aspergillus species.^21,33 These differential responses emphasize the importance of evaluating antifungal compounds against multiple target species, given the frequent co-occurrence of several Aspergillus species in contaminated food and feed commodities.

The strong and consistent kinetic disruption caused by thymol in all four Aspergillus species is in alignment with its reported antifungal potency reported in the literature.^1,40 While the precise molecular basis of these kinetic effects was not directly explored in the present study, the quantitative extensions of lag phase and reductions in growth rate represent evidence of significant interference with fungal developmental processes, providing a measurable kinetic correlate of antifungal activity that complements previously reported findings on thymol efficacy. The concentration-dependent inhibition of spore germination observed across all species further demonstrates the broad antifungal capacity of the tested EO constituents. Since conidial germination represents the fundamental phase in fungal colonization of food substrates, inhibition at this developmental stage is of direct relevance for preventing fungal establishment and the associated risk of mycotoxin production in food and feed systems.^5,30

The remarked performance of thymol and cuminaldehyde against spore germination is consistent with and reinforces their strong effects on mycelial growth kinetics, indicating that compounds capable of disrupting early developmental processes also exert antifungal pressure during later growth stages. This parallel inhibitory ranking across both assays with thymol most effective, followed by cuminaldehyde, D-limonene, and then 1,8-cineol and linalool reinforces the relevance of kinetic parameters as dependable indicators of overall antifungal performance, and justifies the complementary use of growth kinetic modeling and germination inhibition assays as mutually consistent experimental approaches. The strong germination inhibition caused by thymol and D-limonene is consistent with previously reported antifungal effects of these compounds on conidial viability,^19,20,36 while the concentration-dependent responses confirm the dose-dependent nature of EO antifungal activity across developmental stages.

Although 1,8-cineol and linalool showed modest inhibitory activity when applied individually, as consistently reflected in both kinetic and germination assays, their comparatively lower potency as individual compounds may be complemented in practical applications through synergistic interactions with more potent EO constituents such as thymol or cuminaldehyde, a strategy that has been shown to enhance overall antifungal activity while potentially reducing the concentration of each individual component required for effective control.^2,3,26

Conclusion

This study demonstrates the strong, dose-dependent antifungal activity of thymol and cuminaldehyde against all tested Aspergillus species, reflected in delayed mycelial growth and inhibited spore germination. Although complete inhibition required relatively high in vitro concentrations, these results identify thymol and cuminaldehyde as promising candidates for antifungal applications. The main limitation of this study is its in vitro design, which may not fully represent the behavior of these compounds in real food systems. Future work should focus on validating the antifungal efficacy of these compounds in food matrices, evaluating their impact on mycotoxin production, and optimizing formulations to achieve effective fungal suppression at practical concentrations while maintaining sensory quality and safety.

Authors’ contributions

Conceptualization, Y.B.-M.; methodology, Y.B.-M.; investigation Y.B.-M., data curation, A.B., formal analysis, A.B.; validation, A.B. and Y.B.-M.; writing – original draft preparation, Y.B.-M. and B.A.; writing – review and editing, Y.B.-M., B.A., D.D., M.H. and A.A.; visualization, Y.B.-M., B.A., D.D., M.H. and A.A.; supervision, Y.B.-N., B.A. and D.D.; project administration, Y.B.-M., B.A. and M.H.; funding acquisition, Y.B.-M. and M.H. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Ministry of Higher Education and Scientific Research of Algeria and the contract OTRI 2022/0057 of the University of Zaragoza (Spain).

Conflicts of interest

The authors declare no conflicts of interest.

Acknowledgment

The authors wish to thank the support provided by the Gobierno de Aragón (grant Grupo AESA).