A Novel Approach to Protect Brazil Nuts from Lipid Oxidation: Efficacy of Nanocellulose–Tocopherol Edible Coatings

Abstract

High levels of unsaturated fatty acids in Brazil nuts compromise their sensory quality through lipid oxidation. To mitigate this reaction, it is crucial to package nuts under a vacuum and in aluminate packaging. An alternative method is the application of an edible coating with antioxidant properties. This study aimed to develop an edible coating composed of carboxymethylcellulose and sorbitol, physically reinforced with nanocellulose, and chemically fortified with tocopherol. The edible coating was characterized based on its physical properties, mechanical strength, biodegradability, optical light transmission properties, color parameters, and water vapor permeability. Formulations CC5 (Carboxymethyl cellulose (CMC) + sorbitol + 5% nanocellulose) and CCT5 (CMC + sorbitol + tocopherol + soy lecithin + 5% nanocellulose) showed enhanced mechanical strength. The combination of nanocellulose with tocopherol in formulations CCT3 (CMC + sorbitol + tocopherol + soy lecithin + 3% nanocellulose) and CCT5 developed superior barriers to visible and ultraviolet light, a desired characteristic for coatings intended to increase the shelf life of Brazil nuts. The nuts coated with CC5 and CCT3 showed the lowest PV values at the end of the accelerated oxidation test conducted at 60 °C.

1. Introduction

Brazil nuts are oilseeds included in the daily diet to address selenium deficiency, as they have high concentrations ranging from 0.2 to 512 mg/kg [1]. Selenium is an essential element for the body’s metabolism, as it regulates thyroid hormones and assists in modulating the immune system [2,3].

Brazil nuts consist of 60% fatty acids, primarily unsaturated lipids, such as oleic acid and linoleic acid. Unsaturated fatty acids have a hydrogen atom adjacent to double bonds that requires minimal energy to be removed, allowing the formation of lipid radicals. In the presence of atmospheric oxygen, the reaction continues until aldehydes, ketones, alcohols, and hydrocarbons are produced, which alter the sensory quality of the nuts [4,5,6].

Sensorial deterioration caused by lipid oxidation in Brazil nuts results in a rancid taste and yellowish tones. This can be controlled using aluminized packaging and vacuum sealing. A recently developed alternative for oilseeds rich in unsaturated fatty acids is the application of edible coatings with antioxidant activity during the processing of Brazil nuts [7,8,9].

Edible coatings are a low-cost primary packaging produced with biopolymers and additives that improve the quality of the film formed on the food. These coatings may also have functionalities such as an antioxidant that captures free radicals and prevents the progress of lipid oxidation reactions to increase the shelf life of the food [10,11,12].

Carboxymethyl cellulose (CMC) is a widely used biopolymer due to its low cost, high production, biodegradability, and edibility [13]. However, this biopolymer has low mechanical resistance, necessitating the addition of plasticizers and other components, such as nanocellulose, to provide an edible coating with a physical barrier and greater mechanical resistance [14,15,16].

Fibrillated nanocellulose (CNF) contains free hydroxyl groups that form bonds with the polymer matrix, creating crosslinking agents in the polymer matrix. This cross-linking between the CNF molecules and the biopolymer produces a structure with mechanical resistance, higher tensile strength (TS), and reduced elongation at break (EB). In addition to these properties, CNF provides excellent barrier properties against water vapor permeability and ultraviolet light, which has garnered interest from the food industry due to its potential for incorporation into biopolymer matrices. This incorporation results in packaging with enhanced mechanical resistance and barrier properties, making it a promising alternative to petrochemical-based packaging [17,18,19,20,21].

Applying nanomaterials in edible packaging or coatings requires a thorough assessment of their safety for human health [22]. In a study conducted by Jung et al. [23], which analyzed the interaction between nanocellulose and human pancreatic cancer cells in vitro, it was found that coating solutions containing nanocellulose did not exhibit cytotoxicity, with cell viability remaining above 90%. Similarly, Sarwar et al. [24] evaluated the viability and proliferation of human HepG2 liver cells using the MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide). They reported no cytotoxicity in edible films containing nanocellulose, with cell viability exceeding 90%.

Tocopherols are antioxidants that capture free radicals. Incorporating this compound into a polymeric matrix allows for the development of an edible coating with a chemical barrier capable of slowing lipid oxidation reactions [25,26,27,28]. Tocopherol is a fat-soluble compound and, to be incorporated into the carboxymethylcellulose polymeric matrix, it is necessary to use a surfactant. This agent reduces the surface tension between two compounds with different molecular structures. Soy lecithin is a natural surfactant that has a hydrophilic end, allowing it to bind to carboxymethylcellulose, and a hydrophobic end, enabling it to bind to tocopherol [29].

Few studies have focused on edible coatings to extend the shelf life of Brazil nuts and reduce waste. Bonilla et al. [30] developed an edible coating using gelatin and boldo extract as antioxidant agents, applied it to Brazil nuts, and found that the edible coating reduced oxygen gas permeability, thereby increasing the conservation of Brazil nuts. Leme et al. [31] produced active packaging composed of thermoplastic starch/poly (butylene adipate–co–terephthalate) with the incorporation of pine nut aqueous extract and curcumin to enhance the conservation of Brazil nuts. The results showed that chestnuts stored in the thermoplastic starch/poly (butylene adipate–co–terephthalate) formulation with curcumin for 15 d exhibited greater oxidative stability.

This study aimed to develop an effective strategy to protect Brazil nuts against lipid oxidation using an innovative edible coating. The coating was composed of CMC and sorbitol, physically reinforced with nanocellulose, and chemically protected by tocopherol. The formulation was designed to explore different concentrations of nanocellulose to identify the concentration that provides the best physical barrier against oxidizing agents. In addition, a constant concentration of tocopherol was incorporated into each CNF formulation to evaluate its combined efficacy in forming physical and chemical barriers. The goal was to determine the most effective formulation that maximizes the protection of Brazil nuts and extends their quality and shelf life.

2. Materials and Methods

2.1. Materials

The raw materials used to make the film solutions were carboxymethyl cellulose (Adicel, Belo Horizonte, MG, Brazil), tocopherol (Inlab Confiança, Sao Paulo, SP, Brazil), soy lecithin (Grings, Brazil), and fibrillated nanocellulose (CNF) produced by Suzano (Aracruz, ES, Brazil). The Brazil nuts were bought from the Vale do Amanhecer Farmers’ Cooperative (COOPAVAM, Juruena, MT, Brazil). The study using B. excelsa has been registered in the National System for the Management of Genetic Heritage and Associated Traditional Knowledge (SisGen, code AF5E472).

2.2. Characterization of the CNF

2.2.1. Transmission Electron Microscopy

A JEOL JEM 1200EXII transmission electron microscope (TEM) (Peabody, MA, USA) (600,000×) was used at the Electron Microscopy Center of the Federal University of Paraná. The CNF suspension was diluted in deionized water and dripped onto the surface of a screen for observation under the microscope. The samples were left at room temperature to evaporate the solvent, dried, and analyzed. Twenty regions were measured per image using ImageJ software 1.53e version, yielding a total of 60 measurements.

2.2.2. X-ray Diffraction

The crystallinity indices of the samples were determined at the X-ray Optics and Instrumentation Laboratory of the Federal University of Paraná, Brazil, using a SHIMADZU XRD-7000 X-ray diffractometer (Shimadzu Corp., Kyoto, Japan). The configuration adopted for the analysis was a monochromator with slits (1, 1, 0.3), operated at 40 kV and a current of 20 mA. The scanning speed was 1°/min using Cu-Ka radiation with a wavelength of 0.15418 nm.

The crystallinity index was calculated using a previously described method by Creely et al. [32]. Data were processed with Origin Pro8.5 software using Fourier transform smoothing with a 20% cut-off to obtain the intensities of the crystalline and amorphous peaks.

2.3. Preparation of Filmogenic Solution for Edible Films

Filmogenic solutions for casting drying films and edible coatings were prepared as follows: CNF was added at a concentration of 1 g per 100 g of biopolymer in a beaker containing 70% of the total required water volume. The mixture was heated to 80 °C to facilitate solubilization. Once the CNF was fully dissolved by mechanical stirring, CMC (1 g) and sorbitol (30 g per 100 g of biopolymer) were added and stirred until completely dissolved.

Following this, tocopherol and soy lecithin were added at concentrations of 50 g and 75 g per 100 g of biopolymer, respectively, to the remaining 30% of the water. The mixture was homogenized using a high-shear disperser (Ika-Werke, Ultra-Turrax T25, Breisgau, BW, Germany). Soy lecithin, an amphiphilic substance, ensured a stable emulsion without phase separation, given the polar nature of CMC and the lipophilic properties of tocopherol.

The aqueous biopolymer phase containing CNF was then combined with the lipophilic phase containing tocopherol. This blend was homogenized using a heated magnetic stirrer (Solab, SL-91, Piracicaba, SP, Brazil) at 40 °C to achieve a uniform film solution.

Eight formulations were tested, primarily varying in nanocellulose concentration (1%, 3%, and 5% w/w of biopolymer) and the inclusion of antioxidants. The formulations were as follows: C01 (CMC + Sorbitol), CC1 (CMC + Sorbitol + 1% CNF), CC3 (CMC + Sorbitol + 3% CNF), CC5 (CMC + Sorbitol + 5% CNF), C02 (CMC + Sorbitol + Tocopherol + Soy Lecithin), CCT1 (CMC + Sorbitol + Tocopherol + Soy Lecithin + 1% CNF), CCT3 (CMC + Sorbitol + Tocopherol + Soy Lecithin + 3% CNF), and CCT5 (CMC + Sorbitol + Tocopherol + Soy Lecithin + 5% CNF.

After preparing these solutions, the samples from each treatment were analyzed for stability. The remaining solution was used to produce biodegradable films by the casting method. The film solutions were evenly poured into smooth containers, such as Petri dishes (0.31 g film solution per cm²) and subjected to a drying process in an oven with forced air circulation, following the protocol described in the literature Silva et al. [33].

After production, the films were stored alternately on sheets of non-stick paper in a desiccator maintained at 27 °C with a relative humidity of 75 ± 3%, regulated by a saturated solution of NaCl, as outlined in Ruggeri et al. [34].

2.4. Characterization of Film Solutions and Edible Films

2.4.1. Creaming Index

The creaming index (CI) was determined following the methodology proposed in the literature by Sepeidnameh et al. [35]. After preparing each treatment, 2 mL of each sample was added to a 4.5 mL test tube, sealed, and the lower height (H₀) was measured. Every 24 h, the samples were checked for phase separation, and if observed, the upper height (H) was measured to calculate the CI using Equation (1).

$$\mathrm{CI}=\mathrm{H}/{\mathrm{H}}_0.$$

(1)

2.4.2. Water Content

To determine the water content (WC) of the edible films, each sample was cut to 2 cm × 2 cm, the initial masses weighed, and placed in an oven with air circulation and renewal (Solab, Sl-102, Piracicaba, SP, Brazil) at 105 ± 2 °C for 24 h until a constant mass was reached. The WC was quantified using Equation (2) [36].

$$\mathrm{WC} (\%)=\left(\right({\mathrm{M}}_0-{\mathrm{M}}_{\mathrm{f}})/{\mathrm{M}}_0) \ast 100$$

(2)

2.4.3. Film Thickness

The thickness was measured using a digital micrometer (Mitutoyo, MDC-25PX, Takatsu-ku, KP, Japan) with a capacity of 0–25 mm and a resolution of 0.001 mm. Thickness measurements were taken at five different points: the center and four extreme points, and the average thickness of each edible film was obtained [37].

2.4.4. Color

CIELab color parameters were measured using a colorimeter (Linshang, LS-171, Shenzhen, China) to obtain luminosity (L) values ranging from 0 (dark) to 100 (white), and chromaticity coordinates a* (redness/greenness) and b* (yellowness/blueness). Measurements were taken at three different points on a single sample, and the average values for L, a*, b*, chroma (C*), and hue (h*) were recorded if it conveys the same meaning [38].

2.4.5. Optical Properties of Light Transmission

To determine the efficiency of the visible and ultraviolet light barriers of the edible films, light transmission was determined using a transmission meter (Linshang, LS162, Shenzhen, China). Measurements were taken at the side and center of the film to simultaneously measure the visible light transmittance (VLT) at 380–760 nm, the UV rejection (UVR) at a peak wavelength of 365 nm, and the infrared rejection rate (IRR) at a peak wavelength of 940 nm [39].

2.4.6. Water Vapor Permeability

The water vapor permeability (WVP) was quantified using cylindrical glass jars (2.4 cm diameter and 5.0 cm height), with a permeability area of 4.52 cm². Each sample contained 7 g of silica gel and was sealed to produce permeable cells. These cells were placed in a desiccator with relative humidity maintained at 100% and a temperature of 25 °C. Mass gain was measured every 30 min for 8 h, and subsequently every 24 h for three days. The WVP was calculated according to the ASTM-E96/E96M-10 [40] method, using Equation (3):

$$\mathrm{WVP} (\mathrm{g} \mathrm{mm}{\mathrm{h}}^{-1}{\mathrm{cm}}^{-2}{\mathrm{Pa}}^{-1})=(\mathrm{w}/\mathrm{t}) \ast (\mathrm{x}/\mathrm{A}.∆\mathrm{p}),$$

(3)

where w is the water loss from the permeation cup (g), t is the analysis time (h), x is the thickness (mm), A is the film area (m²), and ∆p is the difference in the water vapor pressure between the inside and outside of the cup (Pa).

2.4.7. Tensile Test

The mechanical properties were evaluated in terms of TS and EB, following the methodology described in the literature ASTM D88-12 [41]. Specimens were cut into rectangular shapes (12 cm × 1 cm) and placed between the grips of a texturometer (Stable Micro Systems, TA.XT plus, Godalming, UK) with pneumatic traction control and a load cell set to 500 N capacity. The films were subjected to a traction force with a load of 1 kN, a speed of 12 mm.min⁻¹, and a claw separation setting of 100 mm. The TS and EB values were determined using Equations (4) and (5).

$$\mathrm{TS} \left(\mathrm{MPa}\right)=\mathrm{F}/\mathrm{A},$$

(4)

$$\mathrm{EB} (\%)=(\mathrm{L}-{\mathrm{L}}_0/{\mathrm{L}}_0) \ast 100,$$

(5)

where F is the force (N), A is the cross-section area (mm²), L is the length of the film at break (mm), and L₀ is the original length of the film (mm).

2.4.8. Puncture Test

The perforation resistance of the films was measured ASTM F1342-05 [42] using a texturometer (Stable Micro Systems, TA. XT Plus, Godalming, UK) to quantify the perforation force and deformation, using Equation (6). The 8.0 mm diameter films were fixed in the film support rig (HDP/FSR) geometry and perforated by a spherical steel probe (P/5S, Ø = 5 mm), operating at a capacity of 50 N and a penetration speed of 0.2 mm s⁻¹. The deformation percentage (D) was calculated as follows:

$$\mathrm{D} (\%)=\sqrt{{\mathrm{d}}^2+{\mathrm{L}}_0^2}-{\mathrm{L}}_0/{\mathrm{L}}_0,$$

(6)

where d is the maximum distance penetrated by the probe (m) and L₀ is the radius of the film surface (m).

2.4.9. Biodegradability

To assess the biodegradability of the edible films, samples with a diameter of 6.5 cm were prepared. Fertile soil was collected in the municipality of Santo Antonio do Leverger, Mato Grosso, Brazil (15°51′09.6″ S 56°04′15.4″ W and 15°50′59.3″ S 56°04′16.2″ W) and prepared according to the method described in the literature ASTM G60 [43]. Each edible film was placed in a net-type polyethylene container. The films were buried 5 cm deep and placed at 25 ± 5 °C, 60 ± 5% RH. Degradation was assessed visually by taking samples every 24 h over a period of 7 d.

2.5. Evaluation of the Edible Coating Applied to Unshelled Brazil Nuts

To assess the efficiency of the edible coating, the film solutions were applied to unshelled Brazil nuts (ready-to-eat) and subjected to an accelerated oxidation test for 11 d, as described below.

2.5.1. Edible Coating Application on Brazil Nuts

The Brazil nuts were coated using the dipping method, following the protocol described in the literature [44]. Approximately 30 ± 5 g of nuts from each treatment batch were submerged in 300 mL of filmogenic solution for one minute to ensure an even coating. After immersion, the nuts were carefully removed and placed on steel sieves to drain the excess solution effectively. Subsequently, the coated nuts were transferred to a circulating air oven (Solab model SL-102, Brazil) set at 35 °C and dried for 30 min to fix the coating onto their surfaces. This coating procedure was repeated three times to ensure the formation of a flawless protective layer.

2.5.2. Accelerated Oxidation Test for Brazil Nuts Protected by Edible Coating

Accelerated oxidation tests were conducted on both coated and uncoated Brazil nuts to assess the protective efficacy of the edible coating. Approximately 30 ± 5 g of nuts were placed in 8 cm diameter Petri dishes and subjected to elevated temperature conditions. The samples were placed in a circulating air oven (Solab model SL-102, Brazil) set at 60 °C, maintained in the absence of light to simulate accelerated storage conditions, as referenced in the literature [45]. Sampling was performed at predefined intervals: initially (0 h), followed by 12 h, 24 h, 72 h, 144 h, 192 h, and finally at 264 h. At each time point, nuts were removed for oil extraction, following a modified protocol Folch et al. [46], where ethanol was replaced with methanol to enhance safety and environmental compatibility. The extracted oil samples were then transferred to amber glass bottles to minimize light-induced degradation and stored at a controlled temperature of −1 °C to preserve the integrity until subsequent analytical assessments were performed.

2.5.3. Acid Value (AV)

The acid index of the oil extracted from the coated nuts was determined according to the literature AOCS Cd 3a-63 [47].

2.5.4. Peroxide Value (PV)

The peroxide index values of the oil extracted from the uncoated and coated nuts were determined using the method described in the literature AOCS Cd 8-53 [48].

2.5.5. Conjugated Dienes (CD)

The amount of conjugated dienes in the oils of uncoated and coated nuts was determined using the method described in the literature AOCS Ti 1a-64 [49].

2.6. Statistical Analysis

The Levene homogeneity test (p > 0.05) and Shapiro-Wilk normality test (p > 0.05) were applied to the data obtained from the analyzed parameters. When a normal distribution was observed, the data were considered parametric and subjected to analysis of variance (ANOVA) followed by Tukey’s test (p < 0.05). For non-parametric data, they were subjected to Kruskal-Wallis analysis (p < 0.05). Correlation, principal component, and hierarchical cluster analyses were performed according to the literature Soka et al. [50]. Significant eigenvalues and each principal component (PC) were equal to or above 70%, following established guidelines Wairegi et al. [51]. All the data and graphs were analyzed using the statistical program R^® version 4.3.3 (R Development Core Team, 2023). The biodegradability parameters of the films were analyzed qualitatively based on their visual characteristics, and photographic records were obtained.

3. Results

3.1. Fribillated Nanocellulose Characterization

TEM and X-ray Diffraction

Figure 1 shows the micrographs obtained through TEM for nanocellulose obtained from the industrial processing of bleached Eucalyptus sp. Cellulose pulp. The mechanical process led to the defibrillation of the cell wall, producing CNFs. The micrographs revealed that the bleached CNF had an average diameter of 21.40 nm in image (a) and 18 nm in image (b). In image (a), microfibrils were also visible. Both images show fibrils several micrometers in length.

According to Zimmermann, Bourdeanu, and Strub [52] hydroxyl groups were distributed along the length of the nanofibrillated cellulose chains, facilitating various hydrogen bonds and physical interactions. This led to a nanonetwork structure composed of intertwined and disorganized nanofibrils, making the isolation of single nanocellulose challenging.

As to Pääkkö et al. [53] observed, the length of the CNF chains significantly expanded the network zones, enabling the formation of numerous hydrogen bonds with the nanofibers, thereby enhancing the reinforcement of composites [54].

Figure 2 demonstrates that bleached nanocellulose from Eucalyptus sp. is characteristic of type I cellulose, where the amorphous halo and the crystalline peak are located between the angles 18° ≤ 2θ ≤ 19° and 22° ≤2θ ≤ 23°. Analyzing the highest intensity peak and the amorphous halo in the XRD graph identified a Segal crystallinity index of 77.51%. Two peaks not characteristic of cellulose were identified at 20 and 28°, possibly due to residues from the disc used during fibrillation.

3.2. Characterization of the Filmogenic Solution and Edible Films

3.2.1. Creaming Index of Filmogenic Solution

The creaming index evaluates the stability of the coating solution under gravitational forces, indicating the separation of the constituent phases of the emulsion through sedimentation or flocculation of the components. Figure 3 and Figure 4 present the results obtained after five days of analysis.

At the end of the test, no phase separation was observed. This result indicates that all compounds were successfully incorporated into the film-forming solution.

3.2.2. Water Content and Thickness of Films

Figure 5a shows the water content results for edible films, ranging from 8.86 to 13.59%. The presence of tocopherol (0.5 g) in the formulation of films C02, CCT1, CCT3, and CCT5 did not significantly differ (p < 0.05) from the treatments without tocopherol (C01, CC1, CC3, and CC5).

To effectively protect unshelled Brazil nuts, the almonds should have a water content between 11 and 15%, reducing the chances of survival of toxin-producing fungi. All edible films met this requirement regardless of the formulation [55,56].

Figure 5b shows a significant difference (p < 0.05) in thickness between the treatments without tocopherol (C01, CC1, CC3, and CC5) and those with tocopherol (C02, CCT1, CCT3, and CCT5), ranging from 0.04 to 0.082 mm. The thicknesses of edible films and coatings are influenced by the density, viscosity, and surface tension of the film-forming solution [57]. Tocopherol formulations, consisting of a mixture of a polar phase (CMC + nanocellulose + sorbitol) and a non-polar phase (lecithin + tocopherol), exhibit greater density and viscosity due to hydrophobic interaction between the two phases, increasing the thickness of edible films and coatings [58].

3.2.3. Color Parameters

Table 1. Results of the analysis of color properties of edible films.

Samples	L	a*	b*	C*	°Hue
C01	89.7 ± 0.2 A	1.6 ± 0.2 A	−2.8 ± 0.1 D	3.2 ± 0.2 CD	300.0 ± 2.5 B
CC1	89.6 ± 0.2 A	1.5 ± 0.1 A	−2.6 ± 0.1 D	3.0 ± 0.1 D	301.2 ± 2.2 AB
CC3	89.4 ± 0.2 A	1.6 ± 0.1 A	−2.6 ± 0.1 D	3.1 ± 0.1 D	301.9 ± 1.8 AB
CC5	89.4 ± 0.1 A	1.5 ± 0.1 A	−2.2 ± 0.1 D	2.7 ± 0.1 E	304.7 ± 3.0 A
C02	87.5 ± 0.3 B	1.5 ± 0.2 A	3.6 ± 0.2 B	3.9 ± 0.3 B	67.1 ± 2.1 C
CCT1	87.3 ± 0.25 B	1.5 ± 0.1 A	3.0± 0.1 C	3.4 ± 0.1 C	62.0 ± 3.3 E
CCT3	87.4 ± 0.09 BC	1.6 ± 0.1 A	4.0 ±0.1 A	4.3 ± 0.3 A	67.3 ± 0.6 CD
CCT5	87.0 ± 0.13 C	1.7 ± 0.1 A	3.5 ± 0.3 B	3.9 ± 0.3 AB	63.6 ± 0.6 DE

C01 (CMC + sorbitol); CC1 (CMC + sorbitol + 1% CNF); CC3 (CMC + sorbitol + 3% CNF); CC5 (CMC + sorbitol + 5% CNF); C02 (CMC + sorbitol + tocopherol + soy lecithin); CCT1 (CMC + sorbitol + tocopherol + soy lecithin + 1% CNF); CCT3 (CMC + sorbitol + tocopherol + soy lecithin + 3% CNF); CCT5 (CMC + sorbitol + tocopherol + soy lecithin + 5% CNF). Different capital letters indicate a significant difference (p < 0.05) using the Kruskal–Wallis test.

presents the color parameters L* (brightness), a* (redness), b* (negative values for blueness and positive values for yellowness), chroma (saturation), and the °hue angle of the edible films.

Luminosity is related to the transparency of the edible film; higher L* values are desired for greater acceptability of food coated with the film-forming solution [59]. The L* values in Table 1 show that the edible films containing tocopherol in the formulation reduced the L* value, which was expected since tocopherol is not colorless. Figure 6 shows that tocopherol did not significantly influence the transparency of the film, making the background of the image difficult to observe. The addition of 3 and 5% nanocellulose, combined with tocopherol, reduced luminosity, indicating interaction between these additives and the CMC film.

No variation was observed in the a* parameter related to the red (+a) and green (−a) tones. The b* color parameter, related to blue (−b) or yellow (+b) degree [60]. The edible films with tocopherol showed a significant difference (p < 0.05) compared to other formulations. The yellow hue of tocopherol imparted this color to the films (C02, CCT1, CCT3, and CCT5), as shown in Figure 6 and the higher b* values in Table 1. The colors of films and edible coatings significantly impacts the sensory quality and consumer acceptance of foods [61].

The parameter c describes the color intensity of the edible films. Films with reinforced physical barriers and the addition of CCT3 tocopherol showed higher C* values. The °hue parameter varied most significantly for films without the addition of tocopherol (C01, CC1, CC3, and CC5), indicating an increased intensity in the purple-blue range [62].

3.2.4. Optical Light Transmission Properties

Visible light transmission value (VLT) measures the amount of visible light passing through the film. The average VLT values for different formulations are listed in

Table 2. Average values of VLT, UVR, and IRR parameters for edible films.

Samples	VLT (%)	UVR (%)	IRR (%)
C01	88.5 ± 1.8 a	15.5 ± 0.7 e	11.1 ± 0.4 e
CC1	86.0 ± 0.9 ab	20.1 ± 1.3 e	11.1 ± 0.4 e
CC3	79.6 ± 3.5 bc	29.1 ± 1.0 d	21.3 ± 2.4 cd
CC5	76.4 ± 3.5 c	36.4 ± 5.9 c	26.4 ± 0.8 c
C02	62.5 ± 2.8 d	58.7 ± 3.6 ab	42.7 ± 3.1 b
CCT1	62.8 ± 4.0 d	56.0 ± 3.1 b	42.7 ±4.1 b
CCT3	54.1 ± 6.0 e	63.1 ± 1.9 a	49.5 ± 6.3 a
CCT5	52.9 ± 4.0 e	65.3 ± 2.0 a	50.3 ± 2.6 a

C01 (CMC + sorbitol); CC1 (CMC + sorbitol + 1% CNF); CC3 (CMC + sorbitol + 3% CNF); CC5 (CMC + sorbitol + 5% CNF); C02 (CMC + sorbitol + tocopherol + soy lecithin); CCT1 (CMC + sorbitol + tocopherol + soy lecithin + 1% CNF); CCT3 (CMC + sorbitol + tocopherol + soy lecithin + 3% CNF); CCT5 (CMC + sorbitol + tocopherol + soy lecithin + 5% CNF). Different lowercase letters indicate significant differences (p < 0.05) by Tukey’s test.

. The addition of different concentrations of nanocellulose and the addition or absence of tocopherol significantly reduced VLT values (p < 0.05).

The increase in CNF concentration in the CC5 and CCT5 formulations provided a more significant barrier against visible light, with transmission rates of 76.4% and 52.9%, respectively. These results are attributed to the excellent interactions between the CNF and the polymer matrix [63,64].

For an edible coating intended to delay lipid oxidation in Brazil nuts, a formulation allowing less light penetration is desirable, as visible light initiates free radicals that propagate oxidation. Therefore, nuts coated with the CCT3 or CCT5 formulations, which had lower VLT values, were expected to provide a more significant barrier against visible light.

A significant difference (p < 0.05) in ultraviolet light rejection rate (UVR) was observed among the different formulations, with a more significant barrier against ultraviolet radiation as CNF concentration increased. When incorporated into a polymeric matrix, CNFs can disperse ultraviolet light, reducing its passage into food and creating a barrier [65].

Formulations CCT3 and CCT5 showed higher rejection values for ultraviolet radiation, indicating that the incorporation of tocopherol and CNF enhances the barrier against ultraviolet light. Tocopherol, an aromatic compound capable of absorbing ultraviolet light, prevents its passage into food, thus controlling one of the pro-oxidant effects and inhibiting lipid oxidation reactions initiated by lipid radicals [66].

The infrared light rejection rate (IRR) decreased with increasing nanocellulose content in the matrix. The CCT3 and CCT5 formulations showed a higher IRR. Infrared light is associated with heat, and a barrier against it can reduce molecular vibrations and prevent rapid heating effects caused by this type of electromagnetic radiation [67,68].

3.2.5. Water Vapor Permeability of Edible Films

Water vapor permeability (WVP) is a crucial characteristic of edible films and coatings that indicates their ability to control the transport of water vapor into the food [69]. Figure 7 shows the significant difference (p < 0.05) in WVP results between treatments, ranging from 0.46 ± 0.02 to 0.34 ± 0.03 (g h⁻¹ m⁻² kPa⁻¹).

Edible films and coatings produced only with CMC have high WVP values as biopolymers rich in hydroxyl groups that allow intermolecular interactions with water molecules [70]. This can be improved by adding edible chemical compounds with hydrophobic characteristics. In this study, tocopherol was added to provide antioxidant functionality to edible films and coatings, thereby reducing WVP [71,72,73].

Comparing treatments without tocopherol (C01, CC1, CC3, and CC5) to those with tocopherol (C02, CCT1, CCT3, and CCT5), a reduction in WVP was observed for formulations with tocopherol. Fabra et al. [74] created edible films with the addition of tocopherol and obtained a WVP equal to 0.50 (g h⁻¹ m⁻² kPa⁻¹), similar to the values obtained in this study.

The lowest WVP values obtained for films with tocopherol can be attributed to the effective incorporation of the lipid phase in the biopolymer. In hydrocolloidal edible films and coatings, smaller particle size, homogeneity, and distribution in the hydrophilic phase result in lower WVP values [75,76].

3.2.6. Tensile Test of Film

Table 3. Result of the analysis of mechanical tensile properties.

Samples	Tensile Strength (MPa)	Elongation at Break (%)
C01	20.57 ± 3.83 B	11.14 ± 7.44 BC
CC1	24.02 ± 4.79 B	16.33 ± 2.64 A
CC3	22.99 ± 6.70 B	15.92 ± 4.08 A
CC5	33.43 ± 9.07 A	17.20 ± 4.54 A
C02	6.58 ± 1.44 CD	9.87 ± 3.94 AB
CCT1	6.16 ± 2.00 C	9.44 ± 6.03 C
CCT3	7.47 ± 1.38 C	9.10 ± 5.56 C
CCT5	8.05 ± 1.93 C	13.36 ± 2.17 A

C01 (CMC + sorbitol); CC1 (CMC + sorbitol + 1% CNF); CC3 (CMC + sorbitol + 3% CNF); CC5 (CMC + sorbitol + 5% CNF); C02 (CMC + sorbitol + tocopherol + soy lecithin); CCT1 (CMC + sorbitol + tocopherol + soy lecithin + 1% CNF); CCT3 (CMC + sorbitol + tocopherol + soy lecithin + 3% CNF); CCT5 (CMC + sorbitol + tocopherol + soy lecithin + 5% CNF). Different capital letters indicate a significant difference (p < 0.05) using the Kruskal–Wallis test.

shows significant differences (p < 0.05) between treatments with and without tocopherol for tension strength and elongation analyses.

Increasing the concentration of CNF in the formulations increased their tensile strength and elongation. For the CC5 and CCT5 treatments, the values were 33.43 ± 9.07 and 8.05 ± 1.93 (MPa), 17.20 ± 4.54 and 13.36 ± 2.17 (%), respectively. This increase is due to the ability of nanocellulose to incorporate into the CMC biopolymeric matrix, intercalating or anchoring in its structure [77,78].

The decrease in tensile strength and elongation values with the addition of tocopherol can be explained by the difference in polarity between the biopolymer phase and the lipid phase rich in tocopherol, preventing the formation of strong bonds between the two phases. To reduce this, surfactants, such as soy lecithin, are used, and the lipid phase is mechanically processed to reduce particle size. Shen and Kamdem [79], and Song, Zuo and Chen [80] added essential oil to synthesized edible films to impart antioxidant and antimicrobial activities. They reported a decrease in the tensile strength and elongation after incorporating these additives. These results suggest that the reduction in mechanical traction properties may not influence the coated Brazil nuts since the antioxidant action of tocopherol is expected to remain effective.

3.2.7. Puncture Test of Film

Figure 8 presents the results of the puncture analysis in terms of force and deformation. Figure 8a shows a significant difference in puncture force (p < 0.05), with a reduction in resistance to the force applied by the spherical probe, with an increase in the concentration of CNF in the CC1, CC3, and CC5 formulations. According to Lago et al. [81], the direction in which a force is applied to edible films with the addition of nanocellulose can influence mechanical resistance. In addition to overcoming the friction between the nanocellulose and biopolymer interactions, the probe can break these bonds and reduce the resistance to the applied force.

For the puncture deformation analysis, a significant difference was observed between treatments (p < 0.05), Figure 8, varying between 44.63 ± 24.38 and 6.39 ± 0.45 (%), and a decrease in deformation as the concentration of CNF increased. The incorporation of CNF increased the rigidity of the formed film, reducing its resistance to deformation when punctured by the probe [82].

The films with tocopherol did not differ from each other in terms of puncture force and deformation (p < 0.05) and presented lower values of deformation caused by the probe. Tocopherols are lipophilic compounds that are found in plants. García-Betanzos et al. [83] reported that the addition of fat-soluble compounds to a hydrocolloid emulsion negatively affected the mechanical resistance of edible films to perforation. The polymeric matrix of carboxymethylcellulose is polar, whereas that of tocopherol is nonpolar, which can cause discontinuities in the biopolymer matrix and reduce its resistance to perforation [84,85,86].

3.2.8. Biodegradability

Biodegradability tests showed that all formulations of edible films and coatings were partially biodegradable at the end of the analysis (7 days), verifying only residues of the material used in the experiment, Figure 9. With this result, it is expected that after 30 days, edible films and coatings are fully degradable.

Carboxymethylcellulose is known as a biodegradable biopolymer; the incorporation of nanocellulose, tocopherol, sorbitol, and soy lectin did not interfere with this characteristic [87,88,89].

3.2.9. Principal Component Analysis and Hierarchical Clustering

Principal component analysis was conducted to verify the distribution of formulations according to the variables of mechanical properties, barriers to visible light, ultraviolet light, and water vapor, as shown in Figure 10. The main two components accounted for 90.1% (PC 1 = 77.3%, PC 2 = 12.8%) of the variability among formulations, considering the concentration gradient and addition of tocopherol.

Tensile and elongation strengths were positively correlated with the CC1 and CC5 formulations. The incorporation of CNF into the CMC biopolymer matrix acts as a cross-linking agent, producing a structure with excellent mechanical resistance and elongation through cross-links between the hydroxyls of CNF and carboxymethylcellulose.

The negative correlation between the CCT5 formulation and the WVP variable has practical implications. Tocopherol, a hydrophobic component, reduces the interactions between the hydroxyls of CMC and water vapor, thereby increasing the barrier and reducing water vapor transmission into food.

The combination of CNF and tocopherol in the CCT5 and C02 formulations led to a positive correlation between the UVR and IRR variables. This finding suggests that these formulations have the potential to delay lipid oxidation reactions initiated by ultraviolet light and reduce molecular agitation caused by infrared radiation, potentially extending the shelf life of food products.

One of the objectives of this study is to incorporate edible coatings with added CNF and tocopherol into the Brazil nut production chain. Although adding CNF shows promising potential, its application is contingent upon regulation and food safety evaluations, including toxicity and migration testing. Globally, there is no uniform specific regulation for using nanocellulose in contact with food packaging. In the European Union, the use of nanomaterials in food is regulated by Regulation (EC) in 1333/2008 on food additives and Regulation (EC) No 258/97 on novel foods. Additionally, the Novel Foods Regulation (EU) 2015/2283 defines the criteria for nanomaterials [90,91].

In the United States, the Food and Drug Administration (FDA) regulates food additives. Several companies involved in the production of nanocellulose, such as Fiberlean Technologies (USA) and Celluforce (Canada), have received FDA approval for its use in food packaging applications. This approval allows nanocellulose to be incorporated at levels of up to 5% in contact with paper and paperboard packaging, as well as in banana coatings, where its concentration must not exceed 0.3% by dry weight in the coating formulation (aqueous suspension) [90]. For CNC, Celluforce has received regulatory clearances in multiple jurisdictions, including Canada’s Domestic Substances List (DSL) and the Toxic Substances Control Act (TSCA) regulations in the United States [92].

Currently, there are no food packaging materials utilizing nanocellulose, whether nanocrystalline or nanofibrillated, in direct contact with food due to the need for standardized characterization norms in accordance with regulatory bodies. However, governmental agencies such as the U.S. Forest Service, normative organizations like the Technical Association of the Pulp and Paper Industry (TAPPI), and companies such as Vireo Advisors are collaborating with research committees and manufacturing companies to develop and standardize techniques for the characterization and regulation of nanocellulose in packaging and food applications.

3.3. Comparative Analysis of Lipid Oxidation in Coated and Uncoated Brazil Nuts

3.3.1. Acidity Index

Figure 11 shows the acidity index (AV) of the oils extracted from the coated and uncoated Brazil nuts subjected to accelerated oxidation. The formation of free fatty acids indicates hydrolytic rancidity; the formulations prepared in the present study after 264 h of the accelerated oxidation test did not differ from each other in reducing hydrolytic rancidity. However, coatings C01, CC1, CC3, CC5, CCT3, and CCT5 exhibit AV values lower than 4 mg KOH/g. Normative Instruction No. 87 of 15 March 2021, from ANVISA [93] establishes that the AV allowed for vegetable oils extracted from Brazil nuts cannot exceed 4 mg KOH/g, and that these coatings can be considered suitable for controlling the hydrolytic rancidity of Brazil nuts [94].

Researchers Kowalczyk et al. [95] developed an edible coating from the biopolymer carboxymethylcellulose with the antioxidants ascorbic acid and candelilla wax to increase the shelf life of Persian walnuts. To evaluate oxidative stability, Persian walnuts were stored under commercial conditions (23 ± 1 °C, 35 ± 5% RH) for 16 weeks, and it was possible to observe through acid index analysis that the edible coating with ascorbic acid and candelilla wax in the last period presented 0.39 mg KOH/g while the control sample presented 0.45 mg KOH/g.

Researchers Razavi et al. [96] have increased the shelf life of hazelnuts and produced an edible coating with carboxymethylcellulose and Thymus vulgaris L. extract by varying the concentration of the biopolymer to 0.5 and 1.5 m/v, and the extract 0.5 and 1.0 v/v. Hazelnuts were stored at room temperature (25–27 °C and 30–40% RH) for 147 days and were evaluated for their oxidative stability in terms of hydrolytic rancidity. The formulation with 1.5 m/v CMC and 1.0 v/v thyme extract showed better activity in reducing free fatty acids. According to the authors, the CMC coating with the antioxidant extract prevented the absorption of moisture and oxygen in the hazelnut tissue, preventing hydrolytic rancidity and the activity of the lipase enzyme, which would lead to the production of free fatty acids.

3.3.2. Peroxide Value (PV)

The peroxide value (PV) changes during the oxidation process accelerate at 60 °C of the oils extracted from coated and uncoated Brazil nuts are shown in Figure 12. Normative Instruction No. 87 of 15 March 2021 from ANVISA states that the peroxide value must not exceed 15 mEq/kg for oil samples extracted from Brazil nuts. The accelerated oxidation test at 60 °C is a mechanism to understand the effects of lipid oxidation reactions on coated samples. Above 60 °C, unsaturated fatty acids are in pro-oxidant conditions, producing more peroxides and hydroperoxides. Therefore, it is possible to record PV values higher than those established by regulations in this study [94,97,98,99].

Figure 12a shows the PV of the formulations with reinforced physical barriers C01, CC1, CC3, and CC5.

An effect observed in formulations with a CNF gradient was that, in CC3-coated nuts, after 24 h in the accelerated oxidation test, there was an increase in PV. This indicates excellent oxygen permeability for Brazil nuts. Increasing the concentration of nanocellulose can increase the rigidity of the edible coating matrix, particularly when the nanoparticles are not evenly distributed throughout the biopolymer matrix. The aggregation of nanoparticles can reduce barrier properties and increase the passage of oxygen molecules [100,101,102].

In their study, Fang et al. [103] incorporated nanocellulose into biodegradable polyurethanes, working with 1%, 5%, and 9% concentrations of nanocellulose. They aimed to determine the concentration that would reduce the permeability to oxygen gas. The authors observed that increasing the concentration of nanocellulose reduced its permeability to oxygen gas. However, the same effect was not observed for the formulation with 9%.

At 264 h, the formulation with the highest CC5 concentration had the lowest PV. This effect can be explained by the efficient interaction between CNF and CMC, which hinders the diffusion of oxygen molecules in the polymeric matrix [104,105]. Based on these results, the reinforced physical barrier proved effective in reducing Brazil nut degradation via lipid oxidation reactions.

Figure 12b shows the PV results for the oils extracted from nuts coated with a physical barrier reinforced by adding nanocellulose and chemistry with C02, CCT1, CCT3, and CCT5 in the formulation. After 264 h of the accelerated oxidation test, the CCT3 formulation presented a lower PV value. This indicates that the compounds incorporated into the CMC matrix created a reinforced physical and chemical barrier with the capacity to reduce the permeability to oxygen gas. Consequently, this reduces hydroperoxides and increases the conservation of Brazil nuts.

Carboxymethylcellulose is a polar compound and tocopherol is lipophilic. Soy lecithin is necessary for the incorporation of tocopherol into the carboxymethylcellulose matrix, and an amphiphilic compound is capable of binding to both compounds and producing a film-forming solution without phase separation, as determined by the creaming index analysis. The PV results obtained at 264 h for formulations CCT1 and CCT5 showed better oxygen permeability, which may have been influenced by microphase separation in the coating solution [106,107].

3.3.3. Conjugated Dienes

The results of the analysis of conjugated dienes (CDs) in the oils extracted from coated and uncoated Brazil nuts after the accelerated oxidation test are shown in

Table 4. Conjugated dienes (CDs) from oils extracted from coated Brazil nut subjected to accelerated oxidation.

Samples	Conjugated Dienes (g L−1)
	12 h	24 h	72 h	144 h	192 h	264 h
SR	0.34	NA	0.41	0.36	0.44	0.50
C01	0.34	0.40	0.25	0.48	0.40	0.34
CC1	0.45	0.40	0.30	0.34	0.40	0.40
CC3	0.15	0.15	0.10	0.14	0.05	0.12
CC5	NA	0.42	NA	0.44	0.28	0.45
C02	0.30	0.30	0.26	0.23	0.21	0.52
CCT1	0.39	0.39	0.20	0.29	0.42	0.56
CCT3	0.21	0.31	0.11	0.42	0.39	0.45
CCT5	0.36	0.47	0.30	0.36	0.49	0.48

SR (uncoated); C01 (CMC + sorbitol); CC1 (CMC + sorbitol + 1% CNF); CC3 (CMC + sorbitol + 3% CNF); CC5 (CMC + sorbitol + 5% CNF); C02 (CMC + sorbitol + tocopherol + soy lecithin); CCT1 (CMC + sorbitol + tocopherol + soy lecithin + 1% CNF); CCT3 (CMC + sorbitol + tocopherol + soy lecithin + 3% CNF); CCT5 (CMC + sorbitol + tocopherol + soy lecithin + 5% CNF). NA: not evaluated due to lack of samples for analysis.

. From the data obtained from CDs as in PV, the samples with tocopherol in the edible coating formulation, C02, CCT1, CCT3, and CCT5, showed higher CD values after 264 h at 60 °C, respectively, 0.52, 0.56, 0.45, and 0.48 gL⁻¹. The formulation with the lowest value was CC3 with 0.12 g L⁻¹.

The process of producing a lipid radical (R∙) that causes the movement of the double bond of polyunsaturated fatty acids, producing conjugated dienes, can be accelerated with the presence of high temperatures [108,109]. Based on the results obtained, the CC3 formulation contributed to the edible coating’s ability to delay the formation of conjugated dienes after 264 h of exposure at 60 °C.

The research was developed by Sartori et al. [110], who stored Brazil nuts in a vacuum and obtained conjugated dienes above three g L⁻¹. CNF is not an antioxidant agent capable of capturing lipid radicals or preventing the formation of conjugated dienes. However, the incorporation of 3% in the polymer matrix of the CC3 formulation produced a physical barrier that could delay the formation of lipid radicals that trigger lipid oxidation reactions.

4. Conclusions

Regarding the characterization of the edible films, the CCT3 and CCT5 formulations presented the greatest barriers to visible and ultraviolet light. Thus, these formulations, in addition to presenting a physical barrier through the incorporation of CNF and a chemical barrier through the incorporation of tocopherol, provide a barrier against visible and ultraviolet light. Furthermore, the CCT3 formulation was the best barrier against water vapor.

The CNF gradient in the CC1, CC3, and CC5 formulations played a crucial role in enhancing the tensile and elongation resistance of the edible films. However, the addition of the tocopherol mixture to the CCT1, CCT3, and CCT5 formulations significantly reduced mechanical resistance. Despite this, the study suggests that a reduction in the mechanical tensile properties may not significantly affect the results of coated Brazil nuts, considering that they are sold with packaging.

The Brazil nuts coated with the CC5 and CCT3 formulations showed the lowest PV values at the end of the test and accelerated oxidation at 60 °C, leading to the conclusion that edible coatings are agents capable of delaying lipid oxidation reactions in Brazil nuts.

The incorporation of CNFs enhanced protection against lipid oxidation in Brazil nuts. However, to ensure consumer safety and verify that CNFs do not pose potential health risks, future research should focus on conducting comprehensive cytotoxicity and genotoxicity assessments. These studies are crucial in validating edible coatings containing nanocellulose and tocopherol within the Brazil nut production chain.