# Improving Oxygen Barrier Performance of Polylactide Film by Using Bleached and Unbleached Nanocellulose From Grape Pomace

Jingwen Chen, Nadia O Okamoto-schalch, Maria Gabriela B Dalben, Sandra Domenek, Chloé Chevigny, Giana Almeida, Milena Martelli-tosi

► To cite this version:

Jingwen Chen, Nadia O Okamoto-schalch, Maria Gabriela B Dalben, Sandra Domenek, Chloé Chevigny, et al.. Improving Oxygen Barrier Performance of Polylactide Film by Using Bleached and Unbleached Nanocellulose From Grape Pomace. Journal of Applied Polymer Science, In press, pp.e70401. 10.1002/app.70401. hal-05467873

HAL Id: hal-05467873

https://hal.inrae.fr/hal-05467873v1

Submitted on 20 Jan 2026

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons CC BY 4.0 - Attribution - International License
<figure><img src="image_2.png" /></figure>

RESEARCH ARTICLE

OPEN ACCESS

# Improving Oxygen Barrier Performance of Polylactide Film by Using Bleached and Unbleached Nanocellulose From Grape Pomace

Jingwen Chen<sup>1,2</sup> | Nadia O. Okamoto-Schalch<sup>1</sup> | Maria Gabriela B. Dalben<sup>1</sup> | Sandra Domenek<sup>2</sup> | Chloé Chevigny<sup>2</sup>
Giana Almeida<sup>2</sup> | Milena Martelli-Tosi<sup>1,3</sup>

<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>

**Correspondence:** Milena Martelli-Tosi (mmartelli@usp.br)

Received: 29 August 2025 | Revised: 9 January 2026 | Accepted: 9 January 2026

**Keywords:** biodegradable | biopolymers and renewable polymers | cellulose and other wood products | fibers | packaging

## ABSTRACT

This study investigated two types of lignin-containing nanocelluloses derived from grape pomace and their applications as coatings on PLA. First, bleached and unbleached grape pomace-derived nanocelluloses (BGNC and UGNC) were extracted. Atomic force microscopy (AFM) and transmission electron microscopy (TEM) revealed a mix of nanofibrils and well-defined nanocrystals from BGNC and only nanofibrils from UGNC. BGNC exhibited a higher crystallinity (25.2%) compared to UGNC (20.2%), both with preserved native cellulose type I structure. Thermogravimetric analysis (TGA) showed that residual lignin was present in both samples although strongly reduced after bleaching. BGNC or UGNC coatings were applied via bar-coating at theoretical thicknesses of 90, 140, and 1540 nm. Due to its higher lignin content, the UGNC-coated surface was more hydrophobic, an interesting property for packaging, while the BGNC-coated film showed better oxygen barrier properties thanks to its higher crystallinity and more uniform coating. BGNC-coated film exhibited a good oxygen barrier with a permeability (OP) of $5.91 \times 10^{-19} \text{ m}^3 \text{ m}^2 \text{ s} \text{ Pa}$ at 90 nm thickness, while the UGNC-coated film only reached $9.40 \times 10^{-19} \text{ m}^3 \text{ m}^2 \text{ s} \text{ Pa}$. The oxygen barrier and hydrophobicity are both promising features, highlighting the potential application of by-product-derived nanocelluloses for bio-based, compostable food packaging and the interest of lower-impact chemical extractions to develop original properties.

## 1 | Introduction

The agriculture and food industry creates a high amount of by-products (peels, shells, leaves...) [1], which are rich in bioactive ingredients and lignocellulosic biomass and could be a sustainable source of active compounds and nanocelluloses [2]. Grape pomace is a high-quality residue of the wine industry, composed of pressed grape skins, seeds, and stems. According to the International Organization of Vine and Wine (OIV) data, grape production for winemaking reached 30.8 million tons in 2023, and the grape

pomace accounts for roughly 20% of this total [3]. The grape pomace is traditionally used to produce spirits or fertilizers. Some applications of grape pomace are animal feed, biogas production, and also the cosmetic and pharmaceutical industry [4]. The latter industries are interested in its bioactive components such as phenols, flavonoids, and anthocyanins that possess interesting biological properties including antioxidant, antimicrobial, and anti-inflammatory activities [5]. Moreover, its polyphenols compounds have also been recognized as valuable ingredients in the formulation of functional foods [6]. After the extraction of these

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

© 2026 The Author(s). *Journal of Applied Polymer Science* published by Wiley Periodicals LLC.

<footnote n="1"><sup>1</sup>Department of Food Engineering, Faculty of Animal Science and Food Engineering, University of São Paulo, Pirassununga, São Paulo, Brazil | <sup>2</sup>Université Paris-Saclay, INRAE, AgroParisTech, UMR SayFood, Palaiseau, France | <sup>3</sup>Department of Chemistry, Faculty of Philosophy, Sciences and Letters of Ribeirão Preto, Ribeirão Preto, São Paulo, Brazil</footnote>
compounds, a significant amount of the lignocellulosic biomass remains. This residual fraction (exhausted grape pomace) could be valorized into nanocellulose, offering a supplementary pathway for the production of higher value-added products.

Nanocellulose, extracted from cellulose by mechanical and chemical treatments, has low density, excellent mechanical properties, and outstanding oxygen barrier properties, making it a very promising compound for packaging materials [7, 8]. It can form a dense network due to strong hydrogen bonding, which is an efficient barrier reducing diffusion of gas molecules. However, the application of nanocellulose in food packaging remains challenging due to its inherent hydrophilicity and poor compatibility with hydrophobic materials [9]. Lignin-containing nanocellulose, obtained from unbleached biomass, has thus increasingly attracted interest, since recent studies have demonstrated its superior thermal stability, water resistance, UV-blocking properties, and antioxidant activity compared to bleached nanocellulose [10, 11]. Furthermore, the compatibility of nanocellulose with polymers, such as polylactide (PLA) or poly(vinyl alcohol) (PVA), improved in the presence of lignin, owing to lignin's amphiphilic structure [9, 12]. Conventional nanocellulose production process typically begins with pre-treatments (alkaline treatment and bleaching process) to remove hemicelluloses and lignin, followed by acid and mechanical treatments for cellulose hydrolysis. To obtain the lignin-containing nanocellulose, the pre-treatments are skipped, which is more environmentally friendly and economically viable [11]. Studies have also reported that unbleached nanocellulose provides good barrier properties while applied as food packaging materials. For example, Espinosa et al. [12] investigated the water vapor and oxygen transmission rate (OTR) of PVA-based nanocomposite films, which decreased from approximately $7 \times 10^{-7}$ to $1 \times 10^{-7}$ g s$^{-1}$ m$^{-1}$ Pa$^{-1}$ and from around 3.46 to 3.37 $cm^{-2}$ day$^{-1}$ at 50% relative humidity (RH) respectively, with just 1% addition of TEMPO-oxidized unbleached nanocellulose from wheat straw. Another study found that pure cellulose films fabricated from unbleached nanocellulose exhibited fourfold lower OTR values than their bleached equivalent [13].

Bio-based packaging materials are gaining significant interest as a sustainable alternative to petroleum-based plastics, aiming to solve plastic environmental pollution issues. Among these, PLA, a bio-based polyester, is one of the most promising materials already in commercial production. It is biocompatible, compostable, and has good mechanical and thermal properties. However, PLA oxygen barrier properties are medium, which prevents its application in high-value-added packaging, such as modified atmosphere packaging [14]. There are extensive research efforts to overcome this disadvantage, notably using natural or bio-based solutions. PLA/nanocelluloses composites have been widely studied, and shown significant, but not sufficient, reduction in oxygen permeation with less than 50% barrier improvement from pure PLA [15, 16]. For example, Fortunati et al. [16] demonstrated a maximum oxygen permeability (OP) reduction of 48% for the PLA biocomposite reinforced with 5 wt% surfactant-modified nanocrystals. Alternatively, the multilayers approach appears much more efficient with several systems reporting a barrier improvement of two orders of magnitude: Svagan et al. [17], using LbL-coating to deposit 200 nm of chitosan/montmorillonite bilayers onto PLA, and Guivier

et al. [18], employing the bar-coating method to deposit commercial nanofibers (CNF), oxidized nanofibers (CNF TEMPO), and nanocrystals (CNC) onto PLA surface with comparable thicknesses.

This study hypothesizes that the higher content of lignin in unbleached nanocellulose could improve various performances of multilayer PLA-based films, for example contact angle or thermal resistance [10, 11]. To validate this hypothesis, bleached and unbleached grape pomace-derived nanocelluloses (BGNC and UGNC) were extracted, characterized, and applied as coatings on PLA through the bar-coating method to develop fully bio-based packaging materials. The obtained BGNC and UGNC were analyzed in terms of morphology, electrostatic stability, chemical structure, crystallinity, as well as thermal stability. Finally, the impact of the two nanocellulose types (bleached and unbleached) on the properties of the resulting PLA bio-based multilayer films has been investigated.

## 2 | Material and Methods

### 2.1 | Materials

The by-product (skins and seeds) from the 2022 harvest of Bordeaux grapes (*Vitis labrusca*) was kindly donated by Adega Familia Ferragut, located in the city of Vinhedo-SP (Brazil). This material was collected after the pressing step for the production of red wine and dried in an air circulation oven (TE 394/2, Tecnal, Brazil) at 40°C for 48 h. The dried grape pomace has been ground in a vertical rotor mill (MA090, Marconi, Brazil), with stainless steel sieves with a mesh size of 10. It was stored in polyethylene bags at -18°C.

The dry grape pomace contained about 13.1% ± 0.1% cellulose, 28.8% ± 0.4% acid-soluble lignin, and 7.7% ± 0.3% hemicellulose. This analysis was conducted by the Animal Nutrition Laboratory at the School of Animal Science and Food Engineering, University of São Paulo (FZEA/USP) following the methodologies described by Silva and Queiroz [19] and Van Soest et al. [20].

Ethanol was purchased from Éxodo Científica (São Paulo, Brazil). Hydrogen peroxide, sodium hydroxide, magnesium sulfate heptahydrate, and sulfuric acid were purchased from Dinâmica Química Contemporânea LTDA (Indaiatuba, São Paulo, Brazil). Chitosan of medium molecular weight ((1.9–3.1)×$10^5$ Da) with a degree of deacetylation between 75% and 85%, and acetic acid (99%–100%) were purchased from Sigma-Aldrich (USA). Transparent PLA films (L-lactide, density: 1.25 $cm^{-3}$, thickness 50 µm) were bought from Goodfellow (UK).

### 2.2 | Production of Nanocelluloses From Exhausted Grape Pomace

The bioactive compounds from dried grape pomace grounds were extracted using 40% ethanol in water as solvent, with an ultrasound amplitude of 70% for 10 min (Branson 450, Switzerland). The suspension was then filtered and the residue in the filter was dried (50°C for 24 h in an air circulation oven, TE 394/2, Tecnal, Brazil) and then used to produce nanocelluloses.
BGNCs were produced according to Martelli-Tosi et al. [21]. The first step is a pre-treatment including alkaline and bleaching processes. Ten grams of lignocellulosic residue is suspended in 100 mL of 5% NaOH aqueous solution under magnetic stirring at room temperature. After 15 h of stirring, the sample is washed with water over a 38 µm-aperture sieve until the pH of wash water is neutral (measured by a pH paper indicator). The sample retained on the sieve is then added to a bleaching solution ($H_2O_2$ 4% (v/v) + NaOH 2% (w/v) + $MgSO_4·7H_2O$ 0.3% (w/v)) and heated at 90°C for 3 h. Then, the sample is washed with water over the same 38 µm-aperture sieve until a neutral pH is achieved in the rinse water. Finally, the last wash is done with ethanol. Only BGNC undergoes this pre-treatment.

The second step and third steps are common to both BGNC and UGNC preparation. For the second step, acid hydrolysis of grape pomace (either bleached grape pomace residue or exhausted grape pomace), 1 g of sample is mixed with 30 mL of $H_2SO_4$ 64% (g/100 mL of solution) at 70°C for 40 min under vigorous stirring. To stop the reaction, cold distilled water is added at a ratio of 1:10 and the suspension is then left to decant for 24 h. Finally, the suspension is placed inside a MEMBRA-CEL dialysis membrane with a molecular weight cut-off of 14 KDa (D9402-100FT, Sigma-Aldrich, USA) and left in a container with running water until pH is neutral, which usually takes 5–7 days. The third step, mechanical homogenization, is performed on the neutralized suspension at 14,000 rpm using an Ultra turrax disperser (T18 IKA, China) for 5 min, followed by 3 min of tip sonication at 50% amplitude (Branson 450, Switzerland). The final colloidal suspensions were eventually freeze-dried for transportation to UMR SayFood in France where the films preparation and characterization were done. Two batches were made using the same protocol. The nanocellulose extraction process is summarized in Figure 1. Five samples were studied: grape pomace, exhausted grape pomace, bleached grape pomace residue, BGNC and UGNC.

## 2.3 | Nanocelluloses Characterization

### 2.3.1 | Morphology and Zeta Potential

AFM measurements were performed using an NT-MDT Solver microscope (Solver Next Brand, Russia) in semi-contact mode with a 150 kHz resonance frequency probe and a scanning speed of 0.7 Hz. The nanocellulose suspensions were subjected to treatment in an ultrasonic bath for 2 min and subsequently diluted with Milli-Q water to a concentration appropriate for AFM analysis. The diluted suspensions were dripped onto freshly cleaved mica substrate and allowed to evaporate at room temperature for subsequent analysis. AFM images were processed using Image Analysis 3.2.5 software.

The morphology of the fibers was analyzed using TEM. Suspensions were subjected to treatment in an ultrasonic bath for 2 min, followed by 1:30 and 1:60 dilutions in milli-Q water. Diluted samples were dropped onto a carbon-coated grid and stained with 1.5% uranyl acetate (aqueous solution). After drying for 24 h at room temperature, they were analyzed with a JEM 100CXII microscope (Tokyo, Japan) with an accelerating voltage of 80 kV. Fibril diameters were determined

<figure><img src="image_5.png" /><figcaption>**FIGURE 1** | Production of bleached and unbleached grape pomace-derived nanocellulose (BGNC and UGNC, respectively). [Color figure can be viewed at wileyonlinelibrary.com]</figcaption></figure>

by analyzing 10 TEM images for each sample. From each image, individual nanofibers were randomly selected, and 15 diameter measurements were taken using ImageJ software (National Institutes of Health, USA). A total of 150 measurements per sample were used to calculate the average fibril diameter and standard deviation.

Zeta potentials of the nanocellulose suspensions (BGNC and UGNC) were measured twice: first right after the mechanical homogenization process and second after resuspension following freeze-drying (the resuspension procedure is described in Section 2.4.1). The first measurement was performed by electrophoretic light scattering (ELS) using a Zetasizer Advance Lab (Blue Label, Malvern Instruments Ltd., UK), with a detection angle of 173° and a wavelength of 633 nm. The second measurement was conducted at UMR SayFood after sample transportation using a Malvern Zetasizer Nano ZS under the same detection angle and wavelength. Each sample was measured at least three times.

### 2.3.2 | Attenuated Total Reflectance—Fourier Transform Infrared Spectroscopy (ATR-FTIR)

FTIR analyses were carried out using a Spectrum-One spectrophotometer (Perkin Elmer, Shelton, USA) with a universal
transmittance attenuation accessory (ATR). Sixteen scans were carried out between 550 and 4000 cm⁻¹, with a resolution of 2 cm⁻¹.

### 2.3.3 | X-Ray Diffraction (XRD)

The crystallinity of BGNC and UGNC was determined in a Miniflex 600 X-ray diffractometer (Rigaku, Japan) with Cu radiation, at 40 kV voltage, 15 mA and 600 W tube current. The measurements were carried out in the 2θ range from 5° to 40°, at a scan rate of 1° min⁻¹.

The crystallinity index (CrI, %) was calculated by doing the ratio of crystalline peaks area over the total diffracted area using the deconvolution method. Individual crystalline peaks were extracted by a curve-fitting process from the I=f(2θ) curves [22]. A peak fitting program (PeakFit, Systat) was used, assuming Gaussian functions for each peak, while a broad peak at around 21.5° was assigned to the amorphous contribution. The fit was deemed adequate when R² was > 0.99.

Crystallites sizes were determined using Scherrer's equation:

$$ \tau = \frac{K\lambda}{\beta \cos \theta} $$

With τ being the crystallite size, K is a constant equal to 0.9, λ the wavelength in nm, β the full width at half maximum of the XRD peak corresponding to the 2θ plane (around 22°), and θ the diffraction angle [23].

### 2.3.4 | Thermogravimetric Analysis (TGA)

The thermal properties of grape pomace, exhausted grape pomace, and bleached grape pomace residue were evaluated in a STA 449 F3 Jupiter simultaneous thermal analyzer (NETZSCH, German), using 10 mg of each sample sealed in platinum pans. The samples were heated from 20°C to 600°C at a heating rate of 10°C·min⁻¹ under a nitrogen flow of 60 mL·min⁻¹. The thermal stability of lyophilized BGNC and UGNC was evaluated at UMR SayFood using a TGA-Q500 thermogravimetric analyzer (TA Instruments, USA). The samples (5–10 mg) were heated from room temperature to 575°C under the same conditions using the High-Resolution-Dynamic mode (resolution number 4.00 and sensitivity value 1.00), and each sample was measured at least three times.

## 2.4 | Multilayer Films Production and Characterization

### 2.4.1 | Preparation of Chitosan Solution and Nanocellulose Resuspensions

Cationic chitosan coating solution (1.0 wt%) was prepared by dissolving chitosan in 1.0 wt% acetic acid under magnetic stirring (500 rpm) for 4 h at ambient temperature, followed by an ultrasonic treatment (50% amplitude, 5 min) using an ultrasonic processor (model: 75186, Bioblock Scientific, France). To obtain 0.25 wt% aqueous BGNC and UGNC resuspensions, freeze-dried nanocelluloses were redispersed in Milli-Q water using 20 min

of ultrasound treatment (50% amplitude), followed by 10 min mixing (12,000 rpm, Ultra Turrax), and finally 20 min ultrasound treatment (50% amplitude) again.

### 2.4.2 | Bio-Based Multilayer Films Preparation Using Bar Coating

The fabrication method of nanocellulose multilayer films was done following the method of Guivier et al. [18]. Chitosan and nanocellulose suspensions were coated using a bar-coater (Automatic Film Applicator, Industrial Physics, Netherlands). A 4 µm Mayer bar was selected to obtain nanometric layers. Coatings were done with a bar speed of 30 mm s⁻¹ and a bed temperature of 40°C. First, the PLA film (A5 size) was exposed to a Corona (Mini Corona, Boussey-Control, Belgium) treatment to enhance adhesion, at a frequency of 5 MHz, a voltage of 48 kV and using a 6.5 cm length electrode. Then the was deposited using a 4 µm Mayer bar. Finally, the nanocelluloses were deposited. Three films were prepared for each nanocellulose type, with theoretical thicknesses of 90 nm (5 layers with 4 µm bar), 140 nm (10 layers with 4 µm bar), and 1540 nm (6 layers with 100 µm bar). Between each layer deposit, samples were kept on the bar coater bed at least 15 min for drying. After the final coating, multilayer films were dried at 40°C for 48 h.

The theoretical thickness of the coated chitosan/nanocellulose (CS/NC) layer was calculated by following Equation (1):

$$ L_{th} (\text{CS/NC}) = C(\text{CS}) \times h_{\text{Mayer}} + C(\text{NC}) \times h_{\text{Mayer}} \times n \quad (1) $$

where Lth (CS/NC) is the theoretical thickness (nm), C(CS) is the concentration of chitosan and C(NC) is the concentration of the BGNC or UGNC resuspensions (wt%), hMayer is the Mayer bar height (4 or 100 µm), and n is the number of deposits. In a previous study, it had been investigated how well theoretical thickness related to actual, experimentally measured thickness (from both AFM scratching method and SEM cross-sections observations), and it was found to be fairly accurate [18].

Multilayer films with the three different theoretical thicknesses of coating layer are abbreviated as PLA/CS/(BGNC₄µm)₅ (90 nm), PLA/CS/(UGNC₄µm)₅ (90 nm), PLA/CS/(BGNC₄µm)₁₀ (140 nm), PLA/CS/(UGNC₄µm)₁₀ (140 nm), PLA/CS/(BGNC₁₀₀µm)₆ (1540 nm), and PLA/CS/(UGNC₁₀₀µm)₆ (1540 nm). After the oven drying, films were stored in a desiccator over SiO₂ gel until testing.

### 2.4.3 | Scanning Electron Microscopy (SEM)

The morphology of multilayer film surface was observed by an environmental SEM (ESEM Quanta-FEI-200, USA) operated at an accelerating voltage of 12.5 kV, a spot size of 3.5, a work distance of approximately 10 mm, and a chamber pressure of 0.75 Torr. The samples fixed on the double-sided carbon tape were coated with a carbon layer at a thickness of 10 nm before SEM observation.
### 2.4.4 | Contact Angle Measurements

The contact angle was measured by the sessile drop technique (KRUSS DSA 100 goniometer, France) under ambient conditions. The water drop volume of 0.8 µL was placed on the film sample surface from a micro-syringe. Contact angles were measured for 5 min each 0.5 s. The instrument software applies a numerical solution of the circle equation to calculate the contact angle. Contact angle values are determined randomly at three different places on each sample.

### 2.4.5 | Oxygen Permeability

The oxygen barrier properties of the prepared multilayer films were characterized by measuring OP according to ASTM D 3985 at 23°C and 0% RH [18], using a PSt6-oxygen sensor from PreSens Precision Sensing GmbH (Germany). Each sample was measured at least twice. The OTR and normalized OP were calculated using the following Equations (2) and (3):

$$OTR = \frac{dPO_2}{dt} \frac{V_{cell} \times T_{std}}{P_{std} \times A \times P_m \times T_m} \quad (2)$$

$$OP = OTR \times L \quad (3)$$

where $dPO_2/dt$ is the oxygen partial pressure change over time in the upper chamber (Pa·s⁻¹), $V_{cell}$ (1.1×10⁻⁴ m³) and A (6.81×10⁻³ m²) are the volume of the upper chamber and the permeation area in PreSens test cell; $P_{std}$ (1.013×10⁵ Pa) and $T_{std}$ (273 K) are standard pressure and temperature; $P_m$ and $T_m$ represent the average atmospheric pressure (Pa) and the average ambient temperature (K) during measurement, respectively. L represents the film thickness (m), measured with a Minigauge (ElektroPhysik, Germany).

## 2.5 | Statistical Analysis

The experimental results are presented as mean ± SD values. Data analysis was performed using SPSS 29.0 (IBM, USA). ANOVA (with Tukey's test, p < 0.05) was conducted to analyze the data.

# 3 | Results and Discussion

## 3.1 | Characterization of Grape Pomace-Derived Nanocelluloses

### 3.1.1 | Yield and Morphology

The dry matter content of the nanocellulose suspensions was considered as the fiber yield, relative to the initial amount of raw material (exhausted grape pomace residue). The BGNC suspensions yielded approximately 11.1 ± 2.5 g/100 g, while the UGNC suspensions had a higher yield of 24.1 ± 0.6 g/100 g. During the production of BGNC, it was shown previously that the alkaline treatment primarily decreased hemicellulose from exhausted grape pomace, and the bleaching process after alkaline delignification mostly reduced the remaining lignin

[21]. Without these chemical pre-treatments, UGNC thus contains more lignin, hemicellulose, and other impurities, resulting in a higher yield of nanocellulose from exhausted grape pomace.

AFM micrographs (Figure 2A,C) show that both nanocellulose extraction methods (with and without alkaline-bleaching pre-treatments) were effective in obtaining cellulose nanofibrils. It is evident that the fibrils from BGNC and UGNC exhibit microscale lengths. However, closer examination of TEM micrographs revealed the significant morphological differences between the two nanocelluloses, as depicted in Figure 2B,D. Except for the cellulose nanofibers, the individual cellulose nanocrystals, with nanometric diameter and length, are also observed in BGNC, whereas the UGNC sample exhibited a complex, entangled, and web-like morphology, with only longer cellulose nanofibers and thick bundles visible in Figure 2D. The nanofibers from both samples exhibited relatively similar diameters of 12 ± 5 nm for BGNC and 13 ± 6 nm for UGNC (p > 0.05), while the diameter of those bundles ranged from 31 to 116 nm in UGNC. These morphological differences indicate that alkaline and bleaching pre-treatments contributed to producing more homogeneous nanocelluloses and extracting cellulose nanocrystals from exhausted grape pomace. The lignocellulosic biomass structure consists of cellulose bundles containing amorphous and crystalline regions, which are surrounded and interconnected by hemicellulose and lignin [7]. Without these pre-treatments, acid molecules could not easily diffuse into cellulose amorphous regions due to the abundant hemicellulose and lignin. This hindered the removal of the amorphous regions and the fibrillation of cellulose bundles during sulfuric acid hydrolysis. Consequently, individual nanocrystals could not form, and entangled nanofibers and nanofiber bundles were obtained in the UGNC sample instead.

### 3.1.2 | Zeta Potential

Zeta potential is a good indicator to evaluate the electrostatic stability of dispersed systems. The initial (right after extraction) zeta potential values of BGNC and UGNC were −22.70 ± 5.15 mV and −19.17 ± 3.19 mV, respectively (Table 1). Sulfate groups (SO₄²⁻) were introduced on the nanocelluloses surface via sulfation of surface hydroxyl groups during acid hydrolysis, accounting for the zeta potential negative values [7]. However, the absolute values significantly increased after freeze-drying followed by redispersion in water, indicating the stability of the resuspended samples (Res-BGNC and Res-UGNC) had been enhanced. This enhancement can be explained by the ultrasonic and homogenization treatments applied during redispersion, which effectively broke fiber bundles, increased the exposed surface area of fibers, and thus increased the surface charge of samples.

### 3.1.3 | ATR-FTIR Study

The functional groups of grape pomace and its processed forms, including grape pomace after extraction, bleached grape pomace residue as well as derived nanocelluloses were analyzed by FTIR, as presented in Figure 3. The ethanolic
<figure><img src="image_6.png" /><figcaption>**FIGURE 2** | AFM and TEM micrographs of grape pomace-derived nanocelluloses: (A, B) bleached grape pomace-derived nanocellulose (BGNC) and (C, D) unbleached grape pomace-derived nanocellulose (UGNC). [Color figure can be viewed at wileyonlinelibrary.com]</figcaption></figure>

<table><caption>**TABLE 1** | Zeta potential of grape pomace-derived nanocelluloses (BGNC and UGNC), measured immediately after fabrication and after resuspension (Res-BGNC and Res-UGNC) prior to bar coating.</caption><thead><tr><td>Sample</td><td>Zeta potential (mV)</td></tr></thead><tbody><tr><td>BGNC</td><td>-22.70 ± 5.15<sup>b</sup></td></tr><tr><td>UGNC</td><td>-19.17 ± 3.19<sup>b</sup></td></tr><tr><td>Res-BGNC</td><td>-32.35 ± 4.97<sup>a</sup></td></tr><tr><td>Res-UGNC</td><td>-31.27 ± 5.23<sup>a</sup></td></tr></tbody></table>

Note: Values are presented as mean ± standard deviation. Different letters indicate significant differences among groups (Tukey, p < 0.05).

extraction process did not change the chemical structure of the grape pomace, since the spectral bands of the exhausted grape pomace remained similar to those of grape pomace before the extraction.

The band 1740 cm⁻¹ corresponds to the carbon-oxygen double bond (C=O), associated with ketone, carbonyl and aliphatic groups from hemicellulose and lignin [24–27]. This band was diminished in bleached grape pomace residue, UGNC and BGNC, indicating the reduction of hemicellulose and lignin during chemical treatments. The band at 1605 cm⁻¹, which corresponds to the carbonyl stretching (C=O) conjugated with the aromatic ring [24, 26], shifted to 1590 cm⁻¹ in bleached grape pomace residue, and a broad band appeared at 1621 cm⁻¹ for both BGNC and UGNC. Additionally, at 1515 cm⁻¹, the band

<figure><img src="image_7.png" /><figcaption>**FIGURE 3** | ATR-FTIR spectra of grape pomace, exhausted grape pomace, bleached grape pomace residue, UGNC and BGNC. [Color figure can be viewed at wileyonlinelibrary.com]</figcaption></figure>

corresponding to C=C stretching from the aromatic skeletal structure [26, 27] of phenolics and lignin was observed. This band shifted to 1525 cm⁻¹ for UGNC and disappeared for BGNC, suggesting that BGNC was probably fully delignified. These changes indicate that the delignification and phenolic hydrolysis occurred during alkaline and acid hydrolysis.

The spectral profile of FTIR fingerprint region (below 1500 cm⁻¹) differed between samples. The band around 1430 cm⁻¹ corresponding to the CH₂ scissoring motion of cellulose structure [26],
showed higher intensity in BGNC and UGNC compared to grape pomace and its other processed forms. Another notable observation was the intense bands at 1159 and 1101 cm⁻¹ in BGNC and UGNC, attributed to the C—O—C antisymmetric bridge stretching mode and C—O deformation in secondary alcohols and aliphatic esters [26]. The two bands are related to glycosidic ether vibrations from cellulose [28]. The increased intensities of these bands reflect the higher cellulose content in BGNC and UGNC after chemical extraction. The most intense band in the spectrum of BGNC was centered around 1030 cm⁻¹ and was ascribed to the contribution of several vibrations associated with the CH asymmetric vibration and with the CO stretching of the β-glycosidic linkages between β-D-glucopyranosyl units in cellulose [29].

### 3.1.4 | X-Ray Diffraction

The diffractograms of BGNC and UGNC are shown in Figure 4. They present typical diffraction patterns of semicrystalline cellulosic materials, with a broad amorphous band and more defined crystalline peaks. The main diffraction peaks at around 2θ = 16° (planes 1–10 and 110, overlapped) and 2θ = 22° (plane 200) are characteristic of cellulose type I [29, 30]. Besides, a smaller peak at 2θ = 34.6° (plane 040) was also detected in both samples, which is indicative of cellulose Iβ [30]. Cellulose type I (consisting of cellulose Iα and Iβ) is the crystalline form of native cellulose [30]. It indicates the chemical treatments, including alkaline and bleaching treatments and acid hydrolysis, did not change the cellulose polymorph. However, it should be noted that those diffractograms present relatively broad and small crystalline peaks. Indeed, the CrI values of BGNC and UGNC were 25.2% and 20.2%, respectively, and the crystallite size was found to be 3.7 and 3.2 nm, respectively. Those relatively small crystal sizes are easily explained by the large amount of amorphous fiber remaining.

These values are rather low, especially for the BGNC, although they are coherent with the general fibrous aspect of the obtained particles (see Figure 2) and the visibly mostly amorphous spectra. We used deconvolution through peak fitting as a method to calculate CrI, while the majority of literature still uses the semi-empirical Segal method (1959) [31] which has long been proven

<figure><img src="image_8.png" /><figcaption>**FIGURE 4** | X-ray diffractograms of BGNC and UGNC. [Color figure can be viewed at wileyonlinelibrary.com]</figcaption></figure>

to give overestimated and non-quantitative values [32, 33]. Using Segal's equation, CrI(Segal) is found to be 45.9% for BGNC and 25.5% for UGNC. While still low, these values are in line with published CrI(Segal) values for cellulose nanofibers [29]. Besides, we have used gentler extraction methods in this study: compared to previous extraction studies, our alkaline treatment is milder [21], and the purification steps are simplified. For example, the alkaline treatment was conducted at room temperature rather than high temperature (e.g., 90°C) in other reported studies [8, 34], and only single-pass alkaline and bleaching treatments were used to pre-treat exhausted grape pomace, while previous studies have used multiple pre-treatment steps to purify the nanocellulose, including acid pre-treatment, and repeated alkaline and bleaching processes [8]. And while grape pomace nanocelluloses extracted with the much longer and heavier process were found to have a CrI(Segal) of 62% [8], the difference with ours is then easily explained by the milder process resulting in the incomplete removal of amorphous regions from cellulose bundles, causing a lower crystallinity of BGNC and UGNC.

### 3.1.5 | Thermal Properties

The thermogravimetric (TG) and derivative thermogravimetric (DTG) curves of grape pomace, exhausted grape pomace, bleached grape pomace residue, BGNC and UGNC samples are shown in Figure 5. In general, nanocellulose degrades in three steps during the TGA test: first, elimination of low molecular weight compounds or loosely bound volatile matter such as water, then thermal degradation of hemicellulose, cellulose and lignin, and finally degradation of charred residue into gaseous products [35]. The initial weight loss, less than 10%, observed in all samples between 20°C and 150°C is mainly attributed to the evaporation of water and volatile compounds. After that, samples of grape pomace, exhausted grape pomace and UGNC exhibited a second weight loss ranging from 200°C to 300°C, with several shoulder peaks in their DTG curves. This loss is attributed to thermal degradation of hemicellulose [34, 35]. This phenomenon did not occur in the bleached grape pomace residue and BGNC samples, indicating the alkaline treatment they underwent effectively removed the hemicellulose. The main degradation event for bleached grape pomace residue, UGNC and BGNC samples occurred in the range of 300°C to 350°C, with corresponding mass losses of 33.4%, 38.0% and 67.8%, respectively, primarily attributed to cellulose decomposition. The higher mass loss observed in BGNC reflects its higher cellulose content, which is coherent with its most purified state. Finally, between 360°C and 500°C, a last weight loss is observed in all samples. The ones without alkaline/bleaching treatment, that is, grape pomace, exhausted grape pomace and UGNC, present the highest weight losses at 18.5%, 18.6% and 14.7%, respectively. This loss is mainly attributed to the thermal degradation of lignin. It is also observed in the bleached samples, that is, bleached grape pomace residue and BGNC, although at a lower scale, 13.7% and 8.9% respectively. This suggests that, unlike what was observed with FT-IR (see Section 3.1.3), lignin was actually not completely removed after the alkaline and bleaching processes, even if its proportions are significantly reduced. Besides hemicellulose and lignin, the alkaline treatment and
<figure><img src="image_9.png" /><figcaption>**FIGURE 5** | TGA (A, C) and DTG (B, D) thermograms of grape pomace, exhausted grape pomace, bleached grape pomace residue, BGNC, and UGNC. [Color figure can be viewed at wileyonlinelibrary.com]</figcaption></figure>

bleaching processes also removed other sources of ash (non-cellulosic materials) [36], leading to a reduced amount of residual mass (14.7%) at 590°C in BGNC group compared to UGNC group (29.6%).

## 3.2 | Characterization of Bio-Based Multilayer Films

### 3.2.1 | Observation of Nanocellulose-Coated Samples

Figure 6 shows the surface images of PLA film coated with BGNC and UGNC layers at different theoretical coating thicknesses (90, 140 and 1540 nm), obtained by SEM. Fiber agglomerates and particles were observed in all BGNC and UGNC-coated films. However, fewer and smaller aggregates were observed in BGNC-coated films (Figure 6A–C) compared to the UGNC-coated ones (Figure 6D–F), indicating the BGNC-coated films displayed more homogenous surfaces after the coating process. For UGNC-coated films, the thicker the deposited layer, the less homogenous the surface, with agglomerates forming in the size range of approximately 1 µm (Figure 6D, 90 nm layer) to hundreds of micrometers (Figure 6F, 1540 nm layer). According to the TEM images (Figure 2B,D), UGNC nanofibers were shown to be longer and more entangled, with more variability in size than BGNC, and also contained more lignin,

whose hydrophobicity could facilitate aggregation in aqueous suspension. This explains the difference in aggregation between BGNC and UGNC coatings. This trend of increasing surface heterogeneity with thickness, as observed in the SEM images, has been reported in similar systems like polyelectrolyte multilayers [37], and is commonly explained by a “multiplication” of surface defects with every new layer. It is observed in systems where deposited objects are larger than the size of the “holes and hills” on the surface. Here, with a suspension that is quick to aggregate, every new deposited layer contains larger objects. Comparatively, the surface of BGNC-coated films appeared, at the observed micro-scale, unaffected by the thickness, with the thickest sample (Figure 6C) appearing as smooth as the thinnest one at the observed scale. It is of course possible that smaller, nano-sized aggregates or defects exist in the BGNC-coated films. Some pieces of grape seeds are however visible in all samples, including BGNC-coated ones.

### 3.2.2 | Contact Angle Measurements of Multilayer Films

The water contact angles of PLA and coated samples are shown in Table 2. Contact angle provides a measure of surface hydrophobicity, with higher contact angles indicating a more hydrophobic surface. The pure PLA film exhibited a contact angle
<figure><img src="image_10.png" /><figcaption>**FIGURE 6** | SEM surface images of PLA/CS/(BGNC<sub>4µm</sub>)<sub>5</sub> (A), PLA/CS/(BGNC<sub>4µm</sub>)<sub>10</sub> (B), PLA/CS/(BGNC<sub>100µm</sub>)<sub>6</sub> (C), PLA/CS/(UGNC<sub>4µm</sub>)<sub>5</sub> (D), PLA/CS/(UGNC<sub>4µm</sub>)<sub>10</sub> (E), and PLA/CS/(UGNC<sub>100µm</sub>)<sub>6</sub> (F).</figcaption></figure>

of 80.1° ± 3.9°, indicating a rather hydrophobic surface. The contact angle of chitosan-coated PLA increased to 87.2° ± 3.7°. Studies show that the contact angle of chitosan films is affected by many factors including arrangement of structural units, molecular weight, degree of de-acetylation, and crystallinity degree of chitosan [38–40]. Films made from low molecular weight chitosan (60 kDa) are more hydrophilic than films made from high molecular weight (350 kDa) chitosan, with corresponding water contact angles of 68° and 89°, respectively [38]. Hamilton et al. [39] found that the water contact angle decreased as the degree of de-acetylation increased, due to the higher proportion of hydrophilic amine groups: the contact angle of chitosan films reduced from 89.5° to 62.1° as the degree of de-acetylation increased from 76.1% to 95.6%. Our result is comparable to these reported contact angles of chitosan films, as the degree of deacetylation and molecular weight of chitosan used in this study were confirmed by the literature: 76% and 361 ± 32 kDa [41] or 78% and 583 ± 87 kDa [42].

After coating with BGNC, the contact angle significantly decreased to 80.0° ± 2.7° (*p* > 0.05), compared to PLA/CS, although it was higher than that of pure CNCs reported in the literature (around 65°) [43]. This is mainly attributed to the presence of residual lignin in BGNC, as its nonpolar hydrocarbon and benzene groups contribute to the hydrophobicity of the BGNC layer surface [9]. As previously discussed, TGA results already revealed BGNC was not fully delignified. The UGNC-coated film exhibited a much higher contact angle (91.1° ± 5.4°, *p* < 0.05) than that of BGNC-coated samples, owing to its higher lignin content. Several studies have shown that the higher the lignin content, the more hydrophobic the film surface becomes [11, 44, 45]. The

surface inhomogeneity observed in SEM can also explain the hydrophobicity.

### 3.2.3 | Oxygen Barrier Properties of Multilayer Films

Extensive studies have shown that lignin-containing nanocelluloses are good oxygen barrier materials [12, 13, 46], making them highly promising for applications in food packaging. However, lignin-containing nanocelluloses are primarily studied either for direct fabrication of neat films or as a reinforcing agent in polymer-based composite films. For example, Dou et al. [46] produced lignin-containing cellulose nanofibrils from willow bark through hot water extraction and microfluidization, and the OP (3.47 × 10<sup>-20</sup> m<sup>3</sup> m/m<sup>2</sup> s Pa at 50% RH) of the resulting film is comparable to commercial synthetic polyvinylidene chloride (PVDC) films (0.46–5.90 × 10<sup>-20</sup> m<sup>3</sup> m/m<sup>2</sup> s Pa). In this article, we investigated multilayers, specifically the effects of the deposition of lignin-containing nanocellulose coatings on PLA on the oxygen barrier properties. The OTR of these films, as a function of their respective CS/NC layer theoretical thicknesses, and the normalized oxygen permeabilities of multilayer film samples are presented in Figure 7A,B, respectively.

CS/NC coatings (90 nm, 5 deposits) effectively improved oxygen barrier performance of PLA, reducing its permeability from 1.84 × 10<sup>-18</sup> to 5.91 × 10<sup>-19</sup> m<sup>3</sup> m/m<sup>2</sup> s Pa in BGNC-coated samples and to 9.40 × 10<sup>-19</sup> m<sup>3</sup> m/m<sup>2</sup> s Pa in UGNC-coated samples. This decrease in oxygen permeation is a good indirect indication that the deposited layers are homogeneous, because they are able to provide a gas barrier. BGNC layers are more homogeneous than
UGNC, which is in adequation with SEM surface observations and expected stronger aggregation in particles with higher lig-nin content. After this, the behavior was different for bleached and unbleached samples.

For bleached samples, as the number of deposition cycles increased to 10, corresponding to a theoretical coating thickness of 140nm, the OP increased to $1.59 \times 10^{-18} m^3/m^2s$ Pa in PLA/CS/(BGNC$_{4\mu m}$)$_{10}$ samples. This behavior can be mainly attributed to the inhomogeneity (see Figure 6B) induced by grape seed particles. Indeed, all nanocellulose suspensions still

contained some micrometric pieces of grape seeds, which are very stable in suspension and are thus very difficult to completely remove from the sample. As the number of NC deposition cycles increased, the accumulation of grape seed particles on the PLA surface could have created localized cracks on the already coated layer due to the repeated rolling of the 4µm Mayer bar, cracks which decreased the oxygen barrier. The permeability, however, then decreased again to $7.95 \times 10^{-19} m^3/m^2s$ Pa in PLA/CS/(BGNC$_{100\mu m}$)$_{6}$, when the 100µm Mayer bar was used to produce thicker coatings of 1540nm (theoretically). This improvement was probably due to the thicker layer deposited

<table><caption>TABLE 2 | Comparison of initial contact angle of grape pomace-derived nanocellulose coated PLA multilayer films under ambient conditions.</caption><thead><tr><th>Sample</th><th>Contact angle (°)</th><th>Image</th></tr></thead><tbody><tr><td>PLA</td><td>80.1 ± 3.9<sup>b</sup></td><td></td></tr><tr><td>PLA/CS</td><td>87.2 ± 3.7<sup>a</sup></td><td></td></tr><tr><td>PLA/CS/(BGNC<sub>4µm</sub>)<sub>5</sub></td><td>80.0 ± 2.7<sup>b</sup></td><td></td></tr><tr><td>PLA/CS/(UGNC<sub>4µm</sub>)<sub>5</sub></td><td>91.1 ± 5.4<sup>a</sup></td><td></td></tr></tbody></table>

Note: Values are presented as mean ± standard deviation. Different letters indicate significant differences among groups (Tukey, *p* < 0.05).

<figure><img src="image_11.png" /><figcaption>FIGURE 7 | Comparison of oxygen transmission rate (OTR) of grape pomace-derived nanocellulose coated PLA multilayer films as a function of their respective CS/NC layer theoretical thicknesses (in nm) (A) and oxygen permeability (OP) of grape pomace-derived nanocellulose coated PLA multilayer films at 23°C and 0% RH (B). *n* is the number of grape pomace-derived nanocellulose deposits. Different letters indicate significant differences among groups (Tukey, *p* < 0.05). [Color figure can be viewed at wileyonlinelibrary.com]</figcaption></figure>
during each coating cycle: 100 µm exceeds the characteristic size of the grape seed particles, which are then less likely to be dragged across the coating surface and thus induce fewer flaws in the coatings than a 4 µm Mayer bar. However, the longer drying times required for thicker deposits also induce more nanofibers aggregation, the two effects competing and resulting in similar oxygen barrier performance as 90 nm BGNC layer.

For unbleached samples, OP consistently increased with increasing deposited layer thickness, to the point that no significant decrease in OP was observed for PLA/CS/(UGNC₁₀₀µm)₆ films compared to unmodified PLA film (p < 0.05). The evident non-uniform surface features observed in SEM (Figure 6D–F), powered by increasing UGNC particles aggregation and grape seeds-induced defects in the coating, create more and more inhomogeneous layers as thickness increases, which damages the gas barrier.

These findings are not consistent with previous studies on a similar setup but using commercial nanocellulose-coated PLA films, where an enhancement in oxygen barrier properties was observed as the theoretical coating layer thickness increased [18]. The difference could be attributed to the presence of grape seed particles from grape pomace, as well as the aggregates of nanocelluloses, both of which disturbed the coating process as detailed above. These factors can hinder the formation of homogenous nanocellulose layers, thereby negatively influencing the oxygen barrier ability of the coatings. The difference in crystallinity is also worth mentioning: BGNC crystallinity index (25.2%) is higher than UGNC (20.7%), but much lower than commercial CNC (88%–94%) [47]. It is recognized that homogenous nanomaterials with high crystallinity and polarity are more effective in reducing gas permeability through their dense and ordered structure [48]. This would explain why the BGNC coatings with fewer deposits (n = 5 or 6) showed lower OP values than UGNC coatings but still showed far higher OP compared to commercial CNC coatings. The latter achieved a two-order-of-magnitude reduction in OP for PLA-based samples [18].

## 4 | Conclusions

The most studied nanocelluloses are lignin-free nanocelluloses derived from bleached wood pulps. The extraction of lignin-containing nanocelluloses (BGNC and UGNC) from exhausted grape pomace and their application as oxygen barrier enhancements on PLA films were successful. UGNC requires less processing and yields more product compared to BGNC, as it skips alkaline and bleaching treatments. Although the retained lignin brought higher hydrophobicity to the nanocelluloses, it also prevented isolating cellulose nanocrystals from exhausted grape pomace, producing only lower-crystallinity nanofibers containing micrometric grape seed particles. Consequently, the lower crystallinity, stronger aggregation during coating, and flaws induced by the seed particles collectively led to less homogeneous and dense deposited layers, finally resulting in a poorer oxygen barrier performance of UGNC coatings compared to BGNC coatings. The deposition of a 90 nm-thin layer of UGNC resulted in a 49% reduction of oxygen permeation, while a 90 nm BGNC layer exhibited a 68% OP reduction compared to pure PLA. Although

these performances were lower than what was obtained with commercial CNC, the change is still significant and promising for food packaging applications with a lower environmental impact. The other important result of this study is that, thanks to the higher content of lignin, films coated with UGNC were significantly more hydrophobic than those of BGNC. This could have a positive impact on water vapor barrier properties and on the durability of the film, both very relevant for food packaging applications. Future studies will focus on the water-related properties like water uptake and water transport, to better understand the influence of the lignin content in nanocellulose layers. In addition, more advanced structure studies such as cross-sectional images of multilayer films will be included to directly evaluate coating layer continuity and interfacial adhesion, which will provide more comprehensive mechanistic understanding of the oxygen barrier performance. Furthermore, the fabrication process of nanocelluloses can be applied to a variety of other agricultural by-products from the fruits and vegetables industry, which is a potential way to valorize them for the development of fully bio-based, high-performance food packaging materials through the fabrication of nanoscale coatings.

## Author Contributions

**Jingwen Chen:** conceptualization (supporting), formal analysis (lead), investigation (lead), validation (lead), writing – original draft (lead), writing – review and editing (equal). **Nadia O. Okamoto-Schalch:** conceptualization (supporting), formal analysis (equal), methodology (lead), validation (equal), writing – original draft (supporting), writing – review and editing (equal). **Maria Gabriela B. Dalben:** formal analysis (supporting), investigation (equal), methodology (supporting), validation (supporting), writing – original draft (supporting), writing – review and editing (supporting). **Sandra Domenek:** conceptualization (lead), data curation (lead), formal analysis (supporting), methodology (equal), project administration (supporting), supervision (lead), validation (lead), visualization (supporting), writing – original draft (equal), writing – review and editing (lead). **Chloé Chevigny:** conceptualization (equal), data curation (supporting), formal analysis (lead), investigation (lead), project administration (supporting), supervision (lead), writing – original draft (lead), writing – review and editing (lead). **Giana Almeida:** conceptualization (lead), data curation (supporting), funding acquisition (lead), investigation (supporting), project administration (lead), resources (lead), validation (supporting), visualization (supporting), writing – original draft (equal), writing – review and editing (lead). **Milena Martelli-Tosi:** conceptualization (lead), data curation (supporting), formal analysis (supporting), funding acquisition (lead), investigation (supporting), project administration (lead), resources (lead), supervision (lead), validation (lead), visualization (equal), writing – original draft (equal), writing – review and editing (lead).

## Acknowledgments

The authors acknowledge the French National Research Agency (ANR) and the Brazilian funding agency FAPESP (São Paulo, Brazil); this work was funded by ANR (ref. ANR-22-CE43-0016-01) and FAPESP (2022/03469-9, 2023/05286-1 and 2023/10790-0). The authors would like to thank Rodrigo Vinicius Lourenço, Séverine Keller, Sarrah Ghorbal and Gilles Gerlot for their valuable technical assistance.

## Funding

This work was funded by the Agence Nationale de la Recherche (ANR) (ref. ANR-22-CE43-0016-01) and Brazilian funding agency FAPESP (São Paulo, Brazil) (2022/03469-9, 2023/05286-1, 2023/10790-0).
Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

The data that support the findings of this study are available from the
corresponding author upon reasonable request.

References

1. F. Dilucia, V. Lacivita, A. Conte, and M. A. Del Nobile, "Sustainable Use of Fruit and Vegetable By-Products to Enhance Food Packaging Performance," *Food* 9, no. 7 (2020): 857, https://doi.org/10.3390/foods9070857.

2. R. Sirohi, A. Tarafdar, S. Singh, et al., "Green Processing and Biotechnological Potential of Grape Pomace: Current Trends and Opportunities for Sustainable Biorefinery," *Bioresource Technology* 314 (2020): 123771, https://doi.org/10.1016/j.biortech.2020.123771.

3. P. Ferreira-Santos, C. Nobre, R. M. Rodrigues, Z. Genisheva, C. Bo-
telho, and J. A. Teixeira, "Extraction of Phenolic Compounds From
Grape Pomace Using Ohmic Heating: Chemical Composition, Bioactiv-
ity and Bioaccessibility," *Food Chemistry* 436 (2024): 137780, https://doi.
org/10.1016/j.foodchem.2023.137780.

4. A. Ferrara, G. D'Auria, D. Barile, et al., "The Valorization of Grape Pomace From Montepulciano Winemaking: A New Source of Functional Ingredients for Sustainable Food Industry," *Foodservice Research International* 200 (2025): 115443, https://doi.org/10.1016/j.foodres.2024.115443.

5. A. Pop, C. Bogdan, I. Fizesan, et al., “In Vitro Evaluation of Biological Activities of Canes and Pomace Extracts From Several Varieties of *Vitis vinifera* L. for Inclusion in Freeze-Drying Mouthwashes,” *Antioxidants* 11, no. 2 (2022): 218, https://doi.org/10.3390/antiox11020218.

6. K. Sridhar and A. L. Charles, "Fortification Using Grape Extract Polyphenols – A Review on Functional Food Regulations," *International Journal of Food Science and Technology* 56, no. 8 (2021): 3742–3751, https://doi.org/10.1111/ijfs.15001.

7. N. Lin and A. Dufresne, "Nanocellulose in Biomedicine: Current Status and Future Prospect," *European Polymer Journal* 59 (2014): 302–325, https://doi.org/10.1016/j.eurpolymj.2014.07.025.

8. C. C. S. Coelho, M. Michelin, M. A. Cerqueira, et al., "Cellulose Nanocrystals From Grape Pomace: Production, Properties and Cytotoxicity Assessment," *Carbohydrate Polymers* 192 (2018): 327–336, https://doi.org/10.1016/j.carbpol.2018.03.023.

9. S. S. Nair, H. Chen, Y. Peng, Y. Huang, and N. Yan, "Polylactic Acid Biocomposites Reinforced With Nanocellulose Fibrils With High Lignin Content for Improved Mechanical, Thermal, and Barrier Properties," *ACS Sustainable Chemistry & Engineering* 6, no. 8 (2018): 10058–10068, https://doi.org/10.1021/acssuschemeng.8b01405.

10. S. Hong, Y. Song, Y. Yuan, H. Lian, and H. Liimatainen, "Produc-
tion and Characterization of Lignin Containing Nanocellulose From
Luffa Through an Acidic Deep Eutectic Solvent Treatment and System-
atic Fractionation," *Industrial Crops and Products* 143 (2020): 111913,
https://doi.org/10.1016/j.indcrop.2019.111913.

11. A. Kumar, A. Sood, P. Maiti, and S. S. Han, "Lignin-Containing Nanocelluloses (LNCs) as Renewable and Sustainable Alternatives: Prospects, and Challenges," *Current Opinion in Green and Sustainable Chemistry* 41 (2023): 100830, https://doi.org/10.1016/j.cogsc.2023.100830.

12. E. Espinosa, I. Bascón-Villegas, A. Rosal, F. Pérez-Rodríguez, G. Chinga-Carrasco, and A. Rodríguez, "PVA/(Ligno)Nanocellulose Bio-composite Films. Effect of Residual Lignin Content on Structural, Mechanical, Barrier and Antioxidant Properties," *International Journal of Biological Macromolecules* 141 (2019): 197–206, https://doi.org/10.1016/j.ijbiomac.2019.08.262.

13. R. O. Almeida, A. Ramos, E. Kimiaei, M. Österberg, T. C. Maloney, and J. A. F. Gamelas, "Improvement of the Properties of Nanocellulose Suspensions and Films by the Presence of Residual Lignin," *Cellulose* 31, no. 18 (2024): 10951–10967, https://doi.org/10.1007/s10570-024-06222-y.

14. S. Domenek, S. Fernandes-Nassar, and D. V. Rheology, “Mechanical Properties, and Barrier Properties of Poly(Lactic Acid),” in *Synthesis, Structure and Properties of Poly(Lactic Acid)*, vol. 279. Advances in Polymer Science., ed. M. L. Di Lorenzo and R. Androsch (Springer International Publishing, 2017), 303–341, https://doi.org/10.1007/12_2016_17.

15. H. Faraj, N. Follain, C. Sollogoub, et al., “Gas Barrier Properties of Polylactide/Cellulose Nanocrystals Nanocomposites,” *Polymer Testing* 113 (2022): 107683, https://doi.org/10.1016/j.polymertesting.2022.107683.

16. E. Fortunati, M. Peltzer, I. Armentano, L. Torre, A. Jiménez, and J. M. Kenny, "Effects of Modified Cellulose Nanocrystals on the Barrier and Migration Properties of PLA Nano-Biocomposites," *Carbohydrate Polymers* 90, no. 2 (2012): 948–956, https://doi.org/10.1016/j.carbpol.2012.06.025.

17. A. J. Svagan, A. Åkesson, M. Cárdenas, et al., “Transparent Films Based on PLA and Montmorillonite With Tunable Oxygen Barrier Properties,” *Biomacromolecules* 13, no. 2 (2012): 397–405, https://doi.org/10.1021/bm201438m.

18. M. Guivier, G. Almeida, S. Domenek, and C. Chevigny, “Resilient High Oxygen Barrier Multilayer Films of Nanocellulose and Polylactide,” *Carbohydrate Polymers* 312 (2023): 120761, https://doi.org/10.1016/j.carbpol.2023.120761.

19. D. J. Silva and A. C. Queiroz, *Análise de Alimentos: Métodos Químicos e Biológicos*, 3rd ed. (UFV, 2006).

20. P. J. Van Soest, J. B. Robertson, and B. A. Lewis, "Methods for Di-
etary Fiber, Neutral Detergent Fiber, and Nonstarch Polysaccharides in
Relation to Animal Nutrition," *Journal of Dairy Science* 74, no. 10 (1991):
3583-3597, https://doi.org/10.3168/jds.S0022-0302(91)78551-2.

21. M. Martelli-Tosi, O. B. G. Assis, N. C. Silva, B. S. Esposto, M. A. Martins, and D. R. Tapia-Blácido, "Chemical Treatment and Characterization of Soybean Straw and Soybean Protein Isolate/Straw Composite Films," *Carbohydrate Polymers* 157 (2017): 512–520, https://doi.org/10.1016/j.carbpol.2016.10.013.

22. S. Park, J. O. Baker, M. E. Himmel, P. A. Parilla, and D. K. Johnson, "Cellulose Crystallinity Index: Measurement Techniques and Their Impact on Interpreting Cellulase Performance," *Biotechnology for Biofuels* 3, no. 1 (2010): 10, https://doi.org/10.1186/1754-6834-3-10.

23. J. Gong, J. Li, J. Xu, Z. Xiang, and L. Mo, "Research on Cellulose Nanocrystals Produced From Cellulose Sources With Various Polymorphs," *RSC Advances* 7, no. 53 (2017): 33486–33493, https://doi.org/10.1039/C7RA06222B.

24. X. Zhao, H. Zhu, G. Zhang, and W. Tang, "Effect of Superfine Grinding on the Physicochemical Properties and Antioxidant Activity of Red Grape Pomace Powders," *Powder Technology* 286 (2015): 838–844, https://doi.org/10.1016/j.powtec.2015.09.025.

25. H. Shurvell, "Spectra—Structure Correlations in the Mid- and Far-IR Infrared," in *Handbook of Vibrational Spectroscopy* (John Wiley & Sons, Ltd, 2006), https://doi.org/10.1002/0470027320.s4101.

26. D. J. Krasznai, P. Champagne, and M. F. Cunningham, "Quantitative Characterization of Lignocellulosic Biomass Using Surrogate Mixtures and Multivariate Techniques," *Bioresource Technology* 110 (2012): 652–661, https://doi.org/10.1016/j.biortech.2012.01.089.

27. H. Chen, C. Ferrari, M. Angiuli, J. Yao, C. Raspi, and E. Bramanti, "Qualitative and Quantitative Analysis of Wood Samples by Fourier Transform Infrared Spectroscopy and Multivariate Analysis," *Carbohydrate Polymers* 82, no. 3 (2010): 772–778, https://doi.org/10.1016/j.carbpol.2010.05.052.
28. J. I. Morán, V. A. Alvarez, V. P. Cyras, and A. Vázquez, "Extraction of Cellulose and Preparation of Nanocellulose From Sisal Fibers," *Cellulose* 15, no. 1 (2008): 149–159, https://doi.org/10.1007/s10570-007-9145-9.

29. M. Martelli-Tosi, M. M. Masson, N. C. Silva, et al., "Soybean Straw Nanocellulose Produced by Enzymatic or Acid Treatment as a Reinforcing Filler in Soy Protein Isolate Films," *Carbohydrate Polymers* 198 (2018): 61–68, https://doi.org/10.1016/j.carbpol.2018.06.053.

30. A. Santmartí and K. Y. Lee, "Crystallinity and Thermal Stability of Nanocellulose," in *Nanocellulose and Sustainability* (CRC Press, 2018).

31. L. Segal, J. J. Creely, A. E. Martin, Jr., and C. M. Conrad, "An Empirical Method for Estimating the Degree of Crystallinity of Native Cellulose Using the X-Ray Diffractometer," *Textile Research Journal* 29, no. 10 (1959): 786–794, https://doi.org/10.1177/004051755902901003.

32. N. Terinte, R. Ibbett, and K. Schuster, "Overview on Native Cellulose and Microcrystalline Cellulose I Structure Studied by X-Ray Diffraction (WAXD): Comparison Between Measurement Techniques," *Lenzinger Berichte* 89 (2011): 118–131.

33. P. H. F. Pereira, H. L. Ornaghi Júnior, L. V. Coutinho, B. Duchemin, and M. O. H. Cioffi, "Obtaining Cellulose Nanocrystals From Pineapple Crown Fibers by Free-Chlorite Hydrolysis With Sulfuric Acid: Physical, Chemical and Structural Characterization," *Cellulose* 27, no. 10 (2020): 5745–5756, https://doi.org/10.1007/s10570-020-03179-6.

34. K. S. Prado and M. A. S. Spinacé, "Isolation and Characterization of Cellulose Nanocrystals From Pineapple Crown Waste and Their Potential Uses," *International Journal of Biological Macromolecules* 122 (2019): 410–416, https://doi.org/10.1016/j.ijbiomac.2018.10.187.

35. H. Yang, R. Yan, H. Chen, C. Zheng, D. H. Lee, and D. T. Liang, "In-Depth Investigation of Biomass Pyrolysis Based on Three Major Components: Hemicellulose, Cellulose and Lignin," *Energy & Fuels* 20, no. 1 (2006): 388–393, https://doi.org/10.1021/ef0580117.

36. X. Chen, J. Yu, Z. Zhang, and C. Lu, "Study on Structure and Thermal Stability Properties of Cellulose Fibers From Rice Straw," *Carbohydrate Polymers* 85, no. 1 (2011): 245–250, https://doi.org/10.1016/j.carbpol.2011.02.022.

37. M. Lösche, J. Schmitt, G. Decher, W. G. Bouwman, and K. Kjaer, "Detailed Structure of Molecularily Thin Polyelectrolyte Multilayer Films on Solid Substrates as Revealed by Neutron Reflectometry," *Macromolecules* 31, no. 25 (1998): 8893–8906, https://doi.org/10.1021/ma980910p.

38. T. S. Demina, M. G. Drozdova, M. Y. Yablokov, et al., "DC Discharge Plasma Modification of Chitosan Films: An Effect of Chitosan Chemical Structure," *Plasma Processes and Polymers* 12, no. 8 (2015): 710–718, https://doi.org/10.1002/ppap.201400138.

39. V. Hamilton, Y. Yuan, D. A. Rigney, et al., "Bone Cell Attachment and Growth on Well-Characterized Chitosan Films," *Polymer International* 56, no. 5 (2007): 641–647, https://doi.org/10.1002/pi.2181.

40. S. Jagodzinska, B. Palys, and D. Wawro, "Effect of Chitosan Film Surface Structure on the Contact Angle," *Progress in Chemistry and Application of Chitin and Its Derivatives* 26 (2021): 89–100, https://doi.org/10.15259/PCACD.26.008.

41. K. Ssekatawa, D. K. Byarugaba, E. M. Wampande, et al., "Isolation and Characterization of Chitosan From Ugandan Edible Mushrooms, Nile Perch Scales and Banana Weevils for Biomedical Applications," *Scientific Reports* 11, no. 1 (2021): 4116, https://doi.org/10.1038/s41598-021-81880-7.

42. N. L. Vanden Braber, L. I. Díaz Vergara, F. E. Morán Vieyra, et al., "Physicochemical Characterization of Water-Soluble Chitosan Derivatives With Singlet Oxygen Quenching and Antibacterial Capabilities," *International Journal of Biological Macromolecules* 102 (2017): 200–207, https://doi.org/10.1016/j.ijbiomac.2017.04.028.

43. E. Espino-Pérez, S. Domenek, N. Belgacem, C. Sillard, and J. Bras, "Green Process for Chemical Functionalization of Nanocellulose With

Carboxylic Acids," *Biomacromolecules* 15, no. 12 (2014): 4551–4560, https://doi.org/10.1021/bm5013458.

44. Q. Qin, X. Zhang, B. Gao, et al., "Insight Into the Effect of Different Nanocellulose Types on Protein-Based Bionanocomposite Film Properties," *International Journal of Biological Macromolecules* 257 (2024): 127944, https://doi.org/10.1016/j.ijbiomac.2023.127944.

45. S. He, F. Shu, X. Liu, et al., "Production of Lignin-Containing Nanocellulose by FeCl3-Catalyzed Ternary Deep Eutectic Solvent Pretreatment System: Structural Characteristics and Emulsification Capabilities," *Industrial Crops and Products* 203 (2023): 117200, https://doi.org/10.1016/j.indcrop.2023.117200.

46. J. Dou, T. Vuorinen, H. Koivula, N. Forsman, M. Sipponen, and S. Hietala, "Self-Standing Lignin-Containing Willow Bark Nanocellulose Films for Oxygen Blocking and UV Shielding," *ACS Applied Nano Materials* 4, no. 3 (2021): 2921–2929, https://doi.org/10.1021/acsanm.1c00071.

47. M. S. Reid, M. Villalobos, and E. D. Cranston, "Benchmarking Cellulose Nanocrystals: From the Laboratory to Industrial Production," *Langmuir* 33, no. 7 (2017): 1583–1598, https://doi.org/10.1021/acs.langmuir.6b03765.

48. A. S. Michaels and H. J. Bixler, "Solubility of Gases in Polyethylene," *Journal of Polymer Science* 50, no. 154 (1961): 393–412, https://doi.org/10.1002/pol.1961.1205015411.