1. Introduction
Fungal diseases that affect agricultural crops require special attention, not only for damage to health and the environment, but also because they affect the economy, particularly in developing countries. In 2020, Brazil exported approximately 8 tons of Brazil nuts (about US
$20 million) [
1]. However, Brazil’s exportation of this product is decreasing due to some factors, such as reduced production (by replacement of native Brazil nuts areas for other planting crops), commercialization of Brazil nuts by other countries, and delay in the improvement of extraction, storage, and export techniques regarding aflatoxin contamination [
2,
3,
4]. In addition, Brazil has a large area under a tropical climate that favors contamination by deteriorating and toxigenic fungi, which makes it difficult to maintain grains and oilseeds under suitable conditions [
5,
6].
Aspergillus nomius is a fungus producer of aflatoxins type B and G, which is capable of contaminating food at harvest, storage, and after processing [
7,
8]. These aflatoxins are extremely toxic and harmful to health when chronically ingested, being classified as Group 1 (carcinogenic to humans) by IARC (International Agency for Research on Cancer) [
9]. To prevent and control the attack of these fungi, the agricultural sector is encouraged to make use of synthetic fungicides. Nevertheless, the exacerbated use of these compounds represents a risk for non-target organisms and for the environment, in addition to causing fungi resistance [
10,
11]. In order to reduce the negative impacts on the environment and human and animal health, some natural and biodegradable alternatives have been studied to replace synthetic fungicides, for example, the use of essential oils [
12,
13].
Palmarosa (
Cymbopogon martinii) is a perennial and aromatic plant, whose leaves and flowers produce an essential oil (EO) considered safe by the Food and Drug Administration (FDA) [
14,
15]. Palmarosa essential oil (PEO) is recognized for its antifungal, antibacterial, and antioxidant properties [
16,
17,
18]. Although very versatile, EOs contain labile and volatile compounds that evaporate or decompose easily under high temperatures, low pressure, and the presence of air and light, which shortens its useful life [
19]. In this context, one of the possibilities to improve the effectiveness of EOs is the use of encapsulation techniques, such as lipid nanoparticles, which can protect these compounds from degradation and loss of biological activity [
20].
Among lipid systems, there are two main types, solid lipid nanoparticles (SLNs) and nanostructured lipid carriers (NLCs) [
21]. The use of NLCs has significantly increased due to their ability to encapsulate hydrophobic molecules with high efficiency, increase drug stability/availability during storage, and overcome some limitations of SLNs (e.g., comparatively lower loading capacity and core material (encapsulate) loss during storage) [
22,
23,
24]. Considering the food industry, lipid nanoparticles have some advantages over other systems, such as biocompatibility, low toxicity, ease for large-scale production, and no need to use organic solvents to prepare the formulation [
22,
25]. Previous studies reported the successful loading of EOs into NLCs and showed they were efficient for protection, production of fortified food products, and as a potential natural food preservative [
21,
22,
24,
25,
26,
27,
28]. Furthermore, since these nanoparticles are composed of lipids, they have the ability to interact with several bacterial and fungal cell types and can also facilitate the antimicrobial activity of EOs by improving the diffusion properties through biological membranes due to the nanosized and lipophilic nature [
28].
Unlike SLNs, the NLC matrix is formed by a blend of solid and liquid lipids; the presence of liquid oil creates larger empty spaces through the solid lipid, which can provide higher solubility and entrap bioactive compounds [
22,
25]. In this work, cocoa butter and sesame oil were used as lipid materials. Considering the wide acceptance of natural products as food preservatives, such as EO, combined with the necessity of increasing their stability, the aim of this work was to develop an NLC containing PEO as a formulation to avoid the contamination of nuts by
A. nomius. To the best of our knowledge, there is no other study involving encapsulation of PEO into NLCs with this purpose.
3. Materials and Methods
3.1. Materials
The PEO (Cymbopogon martinii) was purchased from the company BySamia® (Perdizes, SP, Brazil). Geraniol (98% of purity) was obtained from Sigma-Aldrich® (CAS: 106-24-1, Saint Louis, MO, USA). N-hexane (HPLC grade) was purchased from EM SCIENCE® (CAS: 110-54-3, Gibbstown, NJ, USA) and n-alkanes were obtained from Sigma-Aldrich® (C8-C20, Saint Louis, MO, USA). Sesame oil (Sesamum indicum, CAS: 8008-74-0) and Phospholipon® 80H (P80H; hydrogenated soy lecithin; CAS 92128-87-5) were kindly donated by the companies Veris Brasil (Vinhedo, SP, Brazil) and LIPID Ingredients & Technologies (Ribeirão Preto, SP, Brazil), respectively. Cocoa butter was purchased from Mapric® (Theobroma cacao, CAS: 8002-31-1, Ipiranga, Brazil) and Tween 80 from Synth® (polyoxyethylene sorbitan monooleate, CAS: 9005-65-6, Diadema, SP, Brazil).
3.2. Chemical Analysis of Palmarosa Essential Oil
The PEO was analyzed by a gas chromatography/mass spectrometry (GC-MS) system with a quadrupole detector (Thermo Electron Corporation DSQ II, TLC, Thermo Fisher, Waltham, MA, USA) equipped with a column HP-5 (30 m × 0.25 mm), in full scan mode, for 76 min. A solution of PEO in n-hexane (1746 µg mL−1) was prepared and then diluted in n-hexane (5:1000) for injection (1µL) in the equipment with a split ratio of 1:10. The chromatographic conditions were as follows: the oven temperature increased from an initial temperature of 50 °C min−1 to 230 °C (3 °C min−1); the injector and detector temperatures were 250 °C and 270 °C, respectively; the carrier gas (helium) flow rate was 1 mL min−1; and the temperature of the ionization source was 270 °C.
The characterization was based on the retention time, which was compared with the main compounds using the Kovats retention index [
62]. The PEO components were identified by comparison of the mass spectra with those of the NIST version 2.0 device library, literature, and Kovats retention index obtained from a mixture of
n-alkanes (C
8–C
24). The chemical compounds were confirmed according to Adams (2007) and presented in a relative area (%) [
63].
3.3. Development of a GC-MS Method for Quantification of Geraniol in the Nanostructured Lipid Carriers Containing Palmarosa Essential Oil
The analyses were performed using the same GC-MS system and the same conditions described before, except the temperature, which increased from 80 to 120 °C min−1 (3 °C min−1), followed by other heating from 50 °C min−1 to 280 °C (2 min). After lyophilization, the samples were diluted in n-hexane (1 mL) and 1 µL was injected in the equipment (split ratio of 1:10). The selection of ions present in the PEO and in standard geraniol was performed in TIC (total ion chromatogram) mode after 1:100 dilution in n-hexane from the initial concentration of 1746 and 1758 µg mL−1, respectively. In this new chromatographic condition, the running time was reduced to 20 min. From the TIC mode and using GC–MS-SIM (selected ion monitoring), the following selected ions were monitored for a more sensitive and selective quantification of geraniol in the NLC containing PEO: 41, 44, and 69 (11.50 and 14.00 min).
3.4. Antifungal Activity
3.4.1. Determination of the Minimum Inhibitory Concentration (MIC) and Minimum Fungicidal Concentration (MFC) of Palmarosa Essential Oil
The A. nomius was obtained from the Collection of Reference Fungi on Health Surveillance (Fiocruz/CFRVS/) (Access number: 40010, Lot: 068740010). The fungus was cultivated in a tube containing Potato Dextrose Agar (PDA) (Neogen® Co., Lansing, MI, USA) and maintained for 10 days, at 25 °C, in a BOD incubator (Ethik®, 411 FDP, Vargem Grande Paulista, SP, Brazil). The broth microdilution assay using a serial dilution was used to determine the MIC of PEO (M38-A, Clinical and Laboratory Standards Institute [CLSI], 2008). The wells of a 96-well microplate were filled with 5 µL of a suspension of A. nomius (105 conidia mL−1), previously standardized with 0.9% saline solution in the Neubauer chamber, and 100 µL of RPMI 1640 medium (Sigma-Aldrich, Saint Louis, MO, USA) containing L-glutamine without bicarbonate and buffered with a 0.165 mol L−1 MOPS solution (Sigma-Aldrich, Saint Louis, MO, USA). The PEO was diluted in a sterile Tween 80 (1%, v/v) aqueous solution and concentrations from 3.91 to 4000 μg mL−1 were tested. The microplates were incubated in a BOD incubator, at 25 °C, for 72 h. The well with the least concentration was determined as the MIC value. The MFC was determined after the MIC test. For this, aliquots of 5.0 µL were taken from the wells corresponding to the concentration immediately above the MIC, the MIC, as well as three concentrations lower than the MIC, and added to sterile Petri dishes containing PDA medium. The plates were incubated in a BOD incubator for 24 h, at 25 °C, and the MFC was determined as the lowest concentration without visual fungal growth. All assays were carried out in triplicate.
3.4.2. Inhibition of Mycelial Growth (IMG)
About 5.0 μL of the suspension containing
A. nomius (10
5 conidia mL
−1) were inoculated in the center of a Petri dish containing 20 mL of PDA medium and one of the following samples: (1) 250 µg mL
−1 (MIC previously determined) of Carbendazim (positive control, PC); (2) non-encapsulated PEO at a concentration of 250 µg mL
−1 (MIC value); and (3) the NLC formulations (F1 to F13 and optimized formulations OF1, OF4, and OF12). The concentration of PEO-loaded NLC used in the test was higher than the PEO MIC [
64], i.e., 3.0 mL of the formulations were added to their respective plate containing 17 mL of PDA medium. A Petri dish containing the medium and the microorganism without any treatment was used as the fungal control (FC). In addition, blank NLCs (negative control, NC) were also tested to check whether the excipients used in the preparation of the NLCs would not interfere in the results. All tests were performed in triplicate and incubated in a BOD incubator, at 25 °C, for 10 days. The diameter of the fungus colony was measured in two directions, at right angles to each other. The percentage of IMG was calculated using Equation (2), where
C2 is the mean diameter of growth in the FC plate and
C1 is the mean diameter of fungal growth in the treatment plates (PC, pure PEO, and PEO-loaded NLCs) [
65]:
3.5. NLC Development Based on the Design of Experiments (DoE)
The nanocarriers were prepared with food-grade solid and liquid lipids (cocoa butter and sesame oil (SO), respectively) by a melt-emulsification method [
25,
66]. Firstly, the aqueous phase was prepared by dispersing the aqueous surfactant Tween 80 (2.5%,
w/
w) in water and heating to a temperature of 75 °C. The oil phase, comprising the cocoa butter, SO, PEO, and the lipophilic surfactant P80H, was also heated to the same temperature. Then, the aqueous phase was added over the oil phase under constant agitation, at 5400 rpm, for 2 min, using an Ultra-Turrax T25 homogenizer (IKA
® Works, Wilmington, NC, USA). The temperature was kept at 75 °C during the process. Afterwards, the formulations were sonicated using an ultrasonic tip (model CV334 (500 W), Cole Parmer
®, Vernon Hills, Illinois, IL, USA), set at 50% of the amplitude, for 10 min, with cycles of 59/30 s, at room temperature. Subsequently, the formulations were cooled down in the fridge for 30 min for solidification of the particles. Blank formulations (without PEO) were prepared using the same method.
After some preliminary tests, a ternary mixture design was used to evaluate the effect of some components of the formulation on the dependent variable (antifungal activity–IMG), where X
1 is the concentration of cocoa butter, X
2 is the concentration of PEO:SO (in a proportion of 1:3), and X
3 is the concentration of P80H. The total amount of these components was 5.75 g for each 100 g of the formulations. The values for each component in amount (%,
w/
w), proportion, and pseudocomponents are shown in
Table 6.
From the results obtained in the IMG test, the Scheffe model was tested in the following order: linear, quadratic, full cubic, special cubic, and special quartic order. The value of the adj R2 and the ANOVA results were used for validation/acceptance of the mathematical model. With a 95% confidence level (α = 0.05), the statistical significance of each term of the model was analyzed by the probability value (p-value) and F-value. Parameters with p-values less than 0.05 were considered statistically significant. In addition, the “Adeq Precision” parameter was used to assess the signal-to-noise ratio, where a value greater than 4 is desirable, indicating that the model is adequate and can be used within the planning limits. The Design-Expert software version 11 (Stat-Ease, Inc., Minneapolis, MN, USA) was used for generation and statistical analysis of the mixture design.
3.6. Size, Polydispersity Index, and Zeta Potential
The nanocarriers were characterized regarding the particle diameter and PDI by using the dynamic light scattering (DLS) method. The zeta potential (ζ-potential) was measured by means of electrophoretic mobility. These analyses were done in the NanoPlus/Zeta Particle Analyzer equipment (Micrometirics Instrument Corporation, Georgia, GA, USA). Before the measurements, the NLC formulations (F1 to F13 and the optimized formulations) were diluted with ultra-purified water in the proportion 1:100 [
67], and it was performed at a temperature of 25 ± 1 °C and a scattering angle of 90˚. The analyses were carried out at least in triplicate.
3.7. Entrapment Efficiency (EE) and PEO Content (PEOC)
The method of ultrafiltration was used for determination of the EE [
68]. An aliquot of 500 µL of the PEO-loaded NLC dispersions was added to Amicon ultra-filtration devices (Microcon MWCO 10kDa, Millipore Co., Billerica, MA, USA) and centrifuged at 7000 rpm for 30 min (Hettich
®–164 Universal 320R, Tuttingen, BW, Germany). From the filtrate, 30 µL were lyophilized (Martin Christ
®-Alpha 1-4 LD, Osterode am Harz, Germany), diluted in 1 mL of n-hexane, and then the geraniol quantification was determined by the GC-MS-SIM method (see
Section 3.3). Non-filtered NLC dispersions (20 µL) were also lyophilized and diluted for total geraniol quantification. The EE was calculated by using Equation (3):
where
Ctotal is the total geraniol concentration in the NLCs containing PEO (encapsulated and non-encapsulated PEO),
Cfiltrate is the geraniol concentration found in the filtrate after ultrafiltration (non-encapsulated PEO), and
Ctheoretical is the theoretical concentration of geraniol according to the amount of PEO present in each formulation. The PEOC was expressed as mass of PEO (in mg) per mass of formulation (in g). The analyses were carried out in triplicate.
3.8. In Situ Test with Brazil Nuts
The antifungal activity of PEO-loaded NLCs was also tested in Brazil nuts after inoculation with
A. nomius. Briefly, Brazil nuts were washed with distilled water to remove the excess organic matter and sterilized in an autoclave, at 121 °C, for 20 min [
69]. Fungus inoculation was performed according to Ribeiro et al. (2020), with some modifications. Each nut was inoculated in five different points with 5 μL of the suspension containing
A. nomius (10
5 conidia mL
−1) and kept under a laminar flow hood for 1 h to fix the fungi [
70]. After this, the contaminated nuts were treated with one of the following treatments (1.0 mL): (1) optimized NLC formulations containing PEO, (2) optimized blank NLC formulations (negative control, NC), (3) aqueous solution of Tween 80 (1%,
w/
w) containing 250 µg mL
−1 of PEO, (4) aqueous solution of Tween 80 (1%,
w/
w) containing Carbendazim at a concentration of 250 µg mL
−1 (positive control, PC), or 5) a 0.9% saline solution (fungal control, FC). After 2 h under the laminar flow hood for drying, the nuts were placed in sterilized tubes and incubated in a BOD incubator, at 25 °C, for 10 days. The test was performed in triplicate, where each tube contained one (treatments with non-encapsulated PEO, PC, and FC) or two (blank and PEO-loaded NLC formulations) Brazil nuts. The nuts were visually checked for deterioration.
3.9. Stability Study
The optimized formulations were added into glass tubes with screw caps and stored at room temperature, in the absence of light, for 90 days. Analyses of the size, PDI, ζ-potential, as well as pH, EE, IMG, and in situ antifungal test with Brazil nuts were conducted at 30-day intervals. The pH value of the NLC dispersions was determined at 25 ± 1 °C using a pH meter (Digimed Digital Model DM-22) previously calibrated with pH 4.0 and pH 7.0 standard buffers. The data were analyzed by one-way ANOVA and post-hoc Tukey’s test using the Statistica 10 (StatSoft Inc., Tulsa, OK, USA) software. The results were considered statistically different for p-value ≤ 0.05.
3.10. Fourier Transform Raman Spectroscopy (FT-Raman)
The FT-Raman analyses of optimized formulations (lyophilized PEO-loaded NLC and blank NLC), physical mixture, PEO, and raw materials (P80H, Tween 80, sesame oil, and cocoa butter) were performed using a FT-Raman spectrometer (model RAM II, Bruker, Vertex 70v, Billerica, MA, USA) at a wavelength of 1064 nm from a Nd:YAG laser, at 80 mW of power, in the wavelength range of 600–3200 cm−1 and resolution of 4 cm−1.
3.11. Transmission Electron Microscopy (TEM)
The morphology of the optimized formulation OF12 was observed by using a transmission electron microscope (JEM-1400, JEOL, Peabody, MA, USA). The NLC dispersion was placed onto a 400-mesh copper grid coated with carbon film (Electron Microscopy Sciences, Hatfield, PA, USA) and stained with a 5% phosphotungstic acid solution. After drying at 25 °C, for 24 h, the analysis was performed at an accelerating voltage of 80 kV.
4. Conclusions
The fusion-emulsification technique proved to be effective in the development of NLCs containing PEO, resulting in particles with a homogeneous size distribution and high entrapment efficiency values. The three optimized formulations proposed by the special quartic order (Scheffe model) presented small particles with a uniform size distribution, and a zeta potential suggestive of a physically stable system. Moreover, the antifungal activity test indicated that NLCs allowed the release of incorporated PEO, which could inhibit the growth of the mycotoxigenic fungus A. nomius. The in situ test using pre-contaminated Brazil nuts showed the potential of PEO-loaded NLC to control the deterioration caused by A. nomius, which was not observed for blank NLCs. In addition, even after 90 days of storage at room temperature, the NLC containing PEO maintained the physicochemical characteristics and antifungal activity. Thus, the results showed the efficiency of NLCs containing the Palmarosa essential oil, suggesting the possibility of using these systems as a biofungicide in the food industry, or even in the pharmaceutical and cosmetic industries.