Oil-in-water Pickering emulsions stabilized by nanostructured cellulose: comparison of cellulose nanocrystals and nanofibrils

Oil-in-water Pickering emulsions stabilized by nanostructured cellulose: comparison of cellulose nanocrystals and nanofibrils | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Oil-in-water Pickering emulsions stabilized by nanostructured cellulose: comparison of cellulose nanocrystals and nanofibrils Annachiara Pirozzi, Paolo Bettotti, Giovanna Ferrari, Tiziano Facchinelli, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2530423/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Nanostructured celluloses, in the form of nanofibrils (CNFs) and nanocrystals (CNCs), obtained through TEMPO-mediated oxidation, by controlling the intensity of the process (changing catalyst concentration and processing time), were tested in the stabilization of Pickering emulsions, fabricated through high-pressure homogenization (HPH). Results showed that both CNFs and CNCs provided an efficient steric and electrostatic stabilization of the oil-in-water emulsions. Remarkably, the strong inter-droplet interactions, observed when CNFs were used as stabilizers, because of fibrils entanglement in the continuous phase, resulted in a 3D fibrous network emulsion, with higher viscosity than CNCs-stabilized emulsions, and higher tendency towards flocculation. However, the HPH treatment significantly affected the nanofibrils interfacial layer, promoting the emulsifying ability of CNFs, and increasing stability against coalescence. In contrast, CNCs-stabilized emulsions exhibited, along with lower viscosity, higher interfacial activity and emulsion stabilization capability, without any phase separation during 10 months of refrigerated storage. Remarkably, the HPH treatment did not significantly change the emulsifying ability of CNCs. Therefore, it can be concluded that nanocelluloses with tailored emulsifying properties can be easily obtained through the regulation of the process intensity of TEMPO-mediated oxidation of pulp cellulose, opening the way to the production of new ingredients for the food and cosmetic industries. Nanocellulose Emulsion stability Emulsifying property TEMPO oxidation High-pressure homogenization Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction Pickering emulsions, stabilized by particles at the oil-water interface are attracting increasing interest because they do not require the use of surfactants, which is matter of health and environmental concerns. Moreover, Pickering emulsions are highly stable, due the nearly irreversible absorption of the particles at the liquid interface, and their organization into a compact and dense layer, which reduce droplet coalescence because of efficient steric repulsion (Chevalier & Bolzinger, 2013 ). The unique features of colloidal particles as stabilizers and the applications of Pickering emulsions in different fields, in particular in pharmaceutical (as topical and oral drug delivery systems or as templates to prepare materials for biomedical purposes) and food (as vehicles of nutraceuticals and component of edible films) manufacturing, have been extensively reviewed (Bruno et al., 2020 ; H. Jiang et al., 2020 ). However, in spite of the general agreement on the potential perspectives of use of Pickering emulsions, much is still to be investigated to enable their practical implementation. Indeed, particles characteristics play a fundamental role on Pickering emulsion preparation and stabilization (Wu & Ma, 2016 ): their specific features affect their behavior at interfaces and self-assembling tendency, as well as thickness and cohesion of the interfacial particle layer. Besides the nature of the constitutive chemical components and the presence of surface functional groups, also physical parameters, such as size, shape and crystallinity are relevant for tuning the Pickering emulsions characteristics and performance as a function of the desired application. Several solid particle materials of inorganic or organic sources have been used to obtain stable Pickering emulsions (Y. Yang et al., 2017 ). However, in the last years, the interest has mainly converged toward nanoparticles obtained from natural sources, which combine the advantage of self-assembling, driven by nanoscale forces (Bishop et al., 2009 ) with the limited environmental impact and lack of toxicity concerns, differently from inorganic nanomaterials (Kipen & Laskin, 2005 ). Different organic particles of natural origin have been tested, made of biopolymers (polysaccharides, lignin, and proteins), microorganisms, or molecular assemblies. In general, only few of them were able to stabilize Pickering emulsions in pristine form, whereas in most of the cases a modification process was needed to provide the required techno-functional properties (Dupont et al., 2021 ). Cellulose-derived nanomaterials, usually indicated as nanocellulose (NC), are among the most promising polysaccharide stabilizers, because they are abundant, sustainable, environmentally-friendly (Xue et al., 2017 ) and non-toxic to human cells (Meschini et al., 2020). NC has been successfully used for the preparation of films, hydrogels (De France et al., 2017) and emulsions (Jiménez Saelices & Capron, 2018 ). Moreover, thanks to the reactive cellulose hydroxyls, NC can be easily chemically modified (Habibi, 2014 ), complexed through coordination with metal ions (Maestri et al., 2017 ) and hybridized with inorganic nanoparticles (Voisin et al., 2021 ). NC is highly dispersible in water, its colloidal suspensions being very stable, and it shows an amphiphilic structure since it exposes crystalline faces with largely different polarity, which is of interest in Pickering emulsion stabilization (F. Jiang & Hsieh, 2016; Johansson et al., 2011 ; Medronho & Lindman, 2014 ). Several methods have been developed to obtain NCs from different sources (Kargarzadeh et al., 2017 ) but the most versatile one is the TEMPO-mediated oxidation (Isogai et al., 2011 ), through which nanostructures with variable morphology can be obtained by small adjustment of the reaction conditions (Saito & Isogai, 2004 ). NCs include cellulose nanofibrils (CNFs) and nanocrystals (CNCs), with high and low aspect ratios, respectively. In particular, while the diameter of CNFs is in the nanoscale, i.e., less than 100 nm, their length is typically up to a few micrometers, and disordered regions are still present in the fibrils. Conversely, CNCs are rod-like crystalline structures, with diameters of 2–25 nm and lengths from 100 to 750 nm (Rajinipriya et al., 2018 ). The tuning of the structure of this nanomaterials, while preserving the similar chemical nature, offers an interesting opportunity to investigate the effect of the physical (i.e. morphological) parameters on the characteristics and stability of the Pickering emulsions. More specifically, the exploitation of different types of cellulose-based nanomaterials, such as CNFs and CNCs, represents a promising strategy to increase the technologic and profitable potential of NCs in the fabrication of food-grade Pickering emulsions, due to their unique properties and excellent sustainability, biocompatibility, and renewability. This study aimed to investigate the structure effect of different cellulose nanoparticles, such as CNFs and CNCs, obtained by TEMPO-mediated oxidation from cellulose pulp, on the production and stabilization of oil-in-water Pickering emulsions. In particular, the correlation between the physicochemical properties of different NCs with emulsion properties (mean diameter, ζ-potential, microstructure and rheological properties) are investigated to understand how to improve emulsions behavior and stability over time. 2. Material And Methods 2.1 Materials Northern bleached softwood kraft pulp Celeste 85 was a kind gift of SCA (Sundsvall, Sweden). This pulp was produced by a totally chlorine-free thermomechanical process and flush-dried. Peanut oil, purchased from a local market (Olio di Semi di Arachide Giglio Oro, Firenze, Italy), was used as the oil phase. Its composition (on weight basis, based on manufacture’s specification) comprises 18 wt% of saturated fatty acids, 46 wt% of monounsaturated fatty acids, and 28 wt% of polyunsaturated fatty acids. Sodium dodecyl sulfate (C 12 H 25 NaO 4 S, ACS GR, 99.0%, PanReac, Barcelona, Spain) was used as received without further purification. Milli-Q water (obtained with Barnstead™ Pacific TII Water, Thermo Scientific, Waltham, MA, USA) was used throughout this work. 2.2 Preparation of nanocellulose TEMPO-mediated oxidation of cellulose pulp was performed according to the method of Saito and Isogai (Saito & Isogai, 2004 ) slightly modified as herein reported. In order to obtain CNCs, 5 g of Celeste 85 pulp were swollen in 400 mL H 2 O. 83 mg of 2,2,6,6-Tetramethylpiperidine-1oxyl (TEMPO, C 9 H 19 N, ≥ 98.0%, Alfa Aesar Chemicals, Ward Hill MA, USA), the reaction catalyst, and 0.5 g of sodium bromide (NaBr, ≥ 99.99%, Carlo Erba, Milan, Italy), the catalyst re-cycling, were solubilized in 100 mL H 2 O and added to the pulp suspension. The reaction was started adding 18 mL of sodium hypochlorite containing 6–14% active chlorine (NaClO, Sigma Aldrich, Milan, Italy) which is the primary oxidant. The pH of the reaction mixture was kept in the interval 10.5–11 by adding NaOH until it remained constant as occurred after 4 h. Then, the suspension was rinsed several times with H 2 O and tip sonicated at 80% power delivered in the suspension aliquots of 35 mL volume by a Bandelin Sonuplus ultrasonicator system (Berlin, Germany). CNFs were obtained with the same procedure except that a lower amount of TEMPO (12.5 mg) was used and the reaction was stopped after 2 h. 2.3 Preparation of Pickering emulsions Coarse oil-in-water emulsions were prepared by mixing peanut oil (5 wt%) with the aqueous phase (95 wt%) containing 0.5 wt% of CNCs or CNFs in ultrapure H 2 O, in a high-shear mixer (HSM) (MIULab MT-30K Handheld Homogenizer, Hangzhou, China) at 35,000 rpm for 5 min in an ice bath. The obtained coarse emulsions were then treated through a high-pressure homogenization (HPH) process at 80 MPa for 15 passes, with the heat exchangers set at 5°C, in a lab set-up (orifice-type homogenizer) previously described in details by Pirozzi et al. ( 2021 ) (Pirozzi et al., 2021 ). 2.4 Nanocellulse characterization Atomic Force Microscopy (AFM) images were acquired using a NT-MDT P47H probe microscope (Apeldoorn, Netherlands) scanning operated in the semi contact mode (tip spring constant = 40 N m − 1 ). Detailed information of on the size and morphology of the CNCs and CNFs produced by the TEMPO-mediated oxidation procedure was obtained by analyzing these images with the Gwyddion 2.59 software. Infrared spectra of CNCs and CNFs were acquired by a Nicolet IN10 microFTIR instrument (Thermo Fisher scientifica Waltham MA, USA) operating in transmittance mode. To this purpose, a drop of CNCs’ and CNFs’ suspensions was deposited on a ZnSe optical window and left to dry in an oven. The carboxylation of CNCs and CNFs was determined by performing conductometric titration by a Delta Ohm 2256 instrument (Padova, Italy). The samples were brought to pH 2.8 by addition of HCl then the solution was titrated by 0.5 M NaOH. The degree of oxidation (OD), which is given by the carboxyl content per weight of CNCs and CNFs (µmol mg − 1 ), was calculated from Eq. 1 : \(OD=\frac{C\bullet V}{m}\) (Eq. 1) where C is the concentration of the NaOH solution, and m is the total mass of CNCs and CNFs measured after oven drying the suspensions. V is the volume of the NaOH solution added to reach complete ionization of COOH groups, which was obtained by fitting the experimental titration curve (Da et al., 2003 ). The interfacial tensions of NCs (0.5 wt%) were measured by using the pendant drop method using a contact-angle meter (KSV Instruments LTD CAM 200, Helsinki, Finland), equipped with an image analysis software. Briefly, a syringe equipped with a stainless-steel needle (0.71 mm in diameter) filled with the aqueous phase containing nanoparticles was submerged into the oil phase within a glass cuvette. The initial volume of the formed drop during the whole experiment was about 30 µL. The optical contact angle meter recorded the change of the oil/water interface and the Young-Laplace equation was used to calculate the interfacial tension. Distilled water was used as the control. The interfacial tension (γ) measurements were performed for 2,500 s, and the equilibrium interfacial tension values were estimated using the exponential decay model of Eq. 2 (Mauriello et al., 2021 ): \(\gamma ={\gamma }_{{\infty }}+\left({\gamma }_{0}-{\gamma }_{{\infty }}\right){e}^{-\frac{t}{{\tau }_{r}}}\) (Eq. 2) where γ ∞ is the asymptotic interfacial tension, γ 0 is the initial interfacial tension, τ r is the characteristic time for the arrangement of the molecules at the water-oil interface, and t is the time variable. The ζ-potential of the cellulose nanoparticles in water were measured by dynamic light scattering (DLS) and electrophoretic mobility using a Zetasizer Nano (ZEN3600 Malvern Instruments Ltd., Malvern, UK) at 25°C. 2.5 Pickering emulsions characterization The microscopic structure of Pickering emulsions prepared with CNCs and CNFs was observed using the optical inverted microscope Nikon Eclipse (TE 2000S, Nikon instruments Europe B.V., Amsterdam, The Netherlands). The emulsions size distributions were measured by laser diffraction using a Mastersizer 2000 instrument (Malvern instrument Ltd., Malvern, UK), using the Fraunhofer approximation, which does not require knowledge of the optical properties of the sample. The temperature of the cell was maintained at 25 ± 0.5°C. Characteristic diameters d(0.1), d(0.5), and d(0.9), corresponding to the 10th, 50th (median value), and 90th percentile of the cumulative size distribution of the suspensions, were evaluated. Additionally, the surface-weighted mean diameter D[3,2] and volume-weighted mean diameter D[4,3] were determined according to Eqs. 3 and 4 , respectively: \(D\left[\text{3,2}\right]=\frac{{\sum }_{i}{n}_{i}{d}_{i}^{3}}{{\sum }_{i}{n}_{i}{d}_{i}^{2}}\) (Eq. 3) \(D\left[\text{4,3}\right]=\frac{{\sum }_{i}{n}_{i}{d}_{i}^{4}}{{\sum }_{i}{n}_{i}{d}_{i}^{3}}\) (Eq. 4) where n i is the number of particles of diameter d i . The relative span factors were calculated according to Eq. 5 , to express the distribution width of the droplet size distribution: \(Span=\frac{d\left(0.9\right)-d\left(0.1\right)}{d\left(0.5\right)}\) (Eq. 5) The surface coverage (SC) of NCs of the internal phase of the Pickering emulsions was given by the theoretical maximum surface area by the particles (S P ) divided by the total surface displayed by the oil droplets (S d ) according to the following equation (He et al., 2020 ; Hu et al., 2015 ; Kalashnikova et al., 2011 ): \(SC=\frac{{S}_{P}}{{S}_{d}}=\frac{{m}_{P}\bullet D\left[\text{3,2}\right]}{6\bullet h\bullet {\rho }_{P}\bullet V}\) (Eq. 6) where m P is the mass of NC in the Pickering emulsion (g), D[3,2] is the surface-weighted mean diameter, h is the thickness of cellulose fibers (the average thickness of CNCs and CNFs fibers is 3.0 ± 0.5 nm and 10 nm, respectively), as estimated through AFM, ρ P is the cellulose density (1.6 g cm − 3 ) and V is the volume of oil used in the Pickering emulsion (mL). The rheological properties of Pickering emulsions were characterized by using a rotational rheometer (AR 2000 rheometer, TA instruments, Newcastle, DE, USA), equipped with a concentric cylinder (15 mm stator inner diameter, 28 mm rotor outer diameter, 42 mm cylinder immersed height, 2° cone angle). Viscosity curves were obtained by changing the shear rate from 0.1 s − 1 up to a rate of 200 s − 1 and the temperature was set at 20°C. The emulsion’s stability was determined according to the turbidimetric method (De Maria et al., 2016 ). The absorbance values of freshly prepared emulsions (t 0 ) and after 30 min (t 30 ) were read at 500 nm against a blank (dilution solution) following the introduction of 50 µL of emulsion into 5 mL of 0.1 wt% sodium dodecyl sulfate (SDS) solution. The emulsifying activity index (EAI) and the emulsion stability index (ESI) were calculated using Eqs. 7 and 8 , respectively. \(EAI \left(\frac{{m}^{2}}{g}\right)=2\bullet \frac{T}{\left(1-\vartheta \right)\bullet C\bullet 1000}\) (Eq. 7) \(ESI \left(-\right)=\frac{EA{I}_{{t}_{0}}}{EA{I}_{{t}_{0}}-EA{I}_{{t}_{30}}}\bullet 100\) (Eq. 8) where T is the turbidity, \(\vartheta\) is the volume fraction of oil used to form the emulsion, and C (g mL − 1 ) is the initial concentration of NCs. EAI t0 and EAI t30 are the emulsifying activity indexes calculated at 0 and 30 min, respectively. The turbidity was calculated by using Eq. 9 : \(T=2.303\bullet \frac{A\bullet DF}{OP}\) (Eq. 9) where A is the absorbance of the sample at t 0 and 500 nm, DF is the dilution factor, and OP is the optical path (1 cm, in the used equipment). 2.6 Statistical analysis Experiments were repeated in triplicates and the values were expressed as mean ± standard deviation. Significant differences at p < 0.05 were assessed with SPSS 20 (SPSS Inc., Chicago, IL, USA) statistical package through one-way analysis of variance (ANOVA) and Tukey's test. The data were normally distributed. 3. Results And Discussion 3.1 Morphology and interfacial properties of NCs Two types of nanostructured cellulose, CNCs and CNFs, obtained through different treatments of the same cellulose raw material, were used to stabilize Pickering emulsions. The morphological, topological, and size features of CNCs and CNFs were evaluated through AFM. Representative images of the CNCs and CNFs are reported in Fig. 1 , with CNCs characterized by a needle-like structure, and CNFs by a fibrous network. The statistical analysis of the CNCs’ size, performed on a data set of 900 CNCs individually resolved in the AFM images, indicated a length of 170 ± 90 nm and a width of 3.0 ± 0.5 nm. A rigorous statistical analysis of CNFs size was not possible, because of the entanglement propensity of the flexible and disordered regions still present in the fibers, which are clearly visible in the AFM images. However, an average fiber width of the order of 10 nm and a length of a few micrometers can be estimated. FT-IR spectroscopy was used to analyse the functional groups of the NC samples. The representative spectra of CNCs (black trace) and CNFs (red trace) reported in Fig. 2 show the typical features of cellulose nanostructures. In particular, the C-H rocking at 910 cm − 1 , the C-O bond vibrations in the range 1030–1100 cm − 1 , the antisymmetric stretching vibration of the C-O-C glycosidic bond at 1160 cm − 1 , the stretching vibration of the C-H bond at 2900 cm − 1 and a large band ascribable to the stretching vibrations of the O-H bonds of the primary and secondary hydroxyl groups in the range 3200–3500 cm − 1 can be observed (Foster et al., 2018 ). The strong peak at 1620 cm − 1 can be assigned to the carboxylates introduced by the TEMPO mediated oxidation. The different extent of oxidation of CNCs and CNFs appears evident when comparing the carboxylate peak intensities of the traces of Fig. 2 normalized to the peak at 2900 cm − 1 , which should be unaffected by the TEMPO-mediated reaction. The normalized spectra show a larger oxidation of the CNCs with respect to CNFs, according to the milder oxidation conditions used to obtain the larger size and preserve some amorphous regions of CNFs. The FT-IR data were confirmed by the conductometric titrations which provided OD values of 0.6 ± 0.2 µmol mg − 1 and 1.4 ± 0.1 µmol mg − 1 , of CNFs and CNCs, respectively. Figure 3 shows the dynamic adsorption behavior of CNCs and CNFs at the water-oil interface, investigated by monitoring the change of the interfacial tension (γ) with adsorption time (0–2,500 s) at 25°C. The addition of both CNCs and CNFs to deionized water caused a significant reduction in the interfacial tension of oil-water interface. More specifically, a higher reduction was observed for CNFs than for CNCs. The interfacial tension of water rapidly decreased during the initial 500 s, because of the adsorption of the more hydrophilic oil components, before gradually reaching equilibrium. The interfacial tension of CNCs and CNFs decreased over time, tending towards equilibrium values (γ ∞ ), which were estimated using an exponential decay model (Eq. 2) at 16.7 and 13.8 mN m − 1 , respectively. The γ ∞ value for CNCs’ was slightly lower than the one estimated for pure water (19.2 mN m − 1 ), indicating the reduced ability of the CNCs particles to act onto the oil-water interface. In contrast, CNFs particles effectively adsorbed at the O/W interface, reducing its free energy, suggesting that, according to interfacial tension measurements, CNFs particles have the potential to be more effective in stabilizing O/W Pickering emulsions. The ζ-potential of the CNCs and CNFs was also measured, to obtain indications about the colloidal stability of the different cellulose suspensions. Generally, a stable particle suspension should be characterized by ζ-potential values greater than 30 mV of absolute value, to promote sufficient interparticle electrostatic repulsion forces for their stabilization (Hunter, 2013 ). Both CNCs and CNFs suspensions, whose visual appearance is shown in Fig. 4 , are characterized by high ζ-potential values, of -37.0 ± 2.35 mV at a natural pH = 6.4 ± 0.2 and − 52.97 ± 4.55 mV at a natural pH = 6.8 ± 0.4 for CNCs and CNFs, respectively. These high ζ-potential values support the hypothesis that both suspensions might exhibit good colloidal stability. CNFs exhibited a higher absolute ζ-potential value than CNCs. It must be highlighted that, based on FT-IR results, which show a higher presence of carboxylate groups for CNCs particles, it would be expected an opposite effect on ζ-potential values of CNCs and CNFs, with higher absolute values for the formers. Further studies are needed to better elucidate the reasons that might the influence electro-kinetic phenomena of NCs, such as pKa of the carboxylate groups, counter ions effects, etc…. It can be concluded that the CNCs and CNFs are characterized by similar chemical composition, except for a different content of carboxylate groups. In contrast, the particles dimension and morphology are clearly different: CNCs are needle-shaped, while CNFs are elongated, and partly disordered fibers, with a nanosized, roughly spherical cross section. 3.2 Characterization and stability of Pickering emulsions The emulsifying capacity, size distribution, mean diameters, and optical microscopy of Pickering emulsions stabilized with CNCs and CNFs were evaluated. Figure 5 shows the microstructure of freshly prepared Pickering emulsions stabilized by using CNCs (Fig. 5a) and CNFs (Fig. 5b) at 5 wt% oil phase. The oil droplets of CNFs-stabilized emulsions showed a spherical structure with a uniform size of about 3 µm. When the stabilizer was CNCs, the emulsion was still uniform in size, but the particles, characterized by a greater rigidity, were not able to completely cover the oil droplets, remaining partly dispersed in the aqueous phase. This can be related to the difference in morphological properties (shape and size) of nanoparticles cellulose chains: flexible and defibrillated nanocellulose (CNFs) are likely to diffuse faster, and better cover smaller oil droplets. Another possible reason for this phenomenon was the aggregation of nanofibrillated cellulose and thickening effect of the aqueous phase due to the longer cellulose chains: therefore, entrapping of the oil droplets was more efficient, and a 3D emulsified structure was promoted. It can be speculated that the improved capacity of entanglement allows to bridge the neighbouring droplets to create an entangled droplet-CNFs fiber 3D network. In general, the micrographs of Fig. 5 agree with previous observations for the preparation of Pickering emulsions stabilized by cellulose nanofibrils (Pirozzi et al., 2021 ). Generally, the differences in interfacial tension (14 mN m − 1 for CNFs vs. 19 mN m − 1 for CNCs at 2,000 s) and ζ-potential (-53 mV for CNFs vs. -37 mV for CNCs) can be related to the observed difference in emulsifying performance, such as ability to act onto the oil-water interface, the promotion of droplet formation, and the increase of droplet stability induced by electrostatic repulsion (Dong et al., 2021 ). Remarkably, despite the reported differences in interfacial tension and ζ-potential, both CNCs and CNFs displayed outstanding stability against droplet coalescence, without any phase separation under refrigerated storage at 4°C, likely because of the efficient steric and electrostatic stabilization. In order to further understand the mechanisms of Pickering emulsion stabilization with NCs, the droplet size distribution (Fig. 6 ) and mean droplet diameters (Table 1 ) of the emulsions was measured for the coarse (after HSM) and the final emulsions (after HPH). It can be observed that the measured size distributions are coherent with the micrographs of Fig. 5 . Obviously, the process intensity has a significant effect on the size distribution. The droplet characteristic diameters and the distribution span decreased sharply after the application of HPH treatment. As results, the particle size distribution of emulsions treated by HSM was multimodal, with two peaks in the distribution curve within a range from 1 to 100 µm. In contrast, after HPH treatment, the emulsions stabilized by NCs presented a monomodal and narrower droplet size distribution (span 1.2 and 1.4 for CNCs and CNFs, respectively). The droplet size distribution of HPH treated samples are characterized by d(0.9) of 2.23 and 2.92 µm for CNCs and CNFs, respectively. Similarly, d(0.1) was reduced to sub-micrometric, and d(0.5) to the micrometric size range for both HPH emulsions. The volume mean diameters D[4,3] were in the range of 1.4–1.8 µm, the surface mean diameters D[3,2] in the range of 1.2–1.4 µm (Table 1 ). Low D[3,2] values are generally associated with higher kinetic stability because of the reduction of the effects of the gravitational phase separation, this, in turn, is due to the fact that the droplets separation velocity is proportional to the square radius (Martins et al., 2022 ). Table 1 . Particle size distribution (expressed as characteristic diameters and span) of Pickering emulsions homogenized by HSM or HPH treatment and stabilized by CNCs and CNFs. O/W Pickering emulsion stabilized by CNCs O/W Pickering emulsion stabilized by CNFs HSM HPH HSM HPH d(0.1) (µm) 2 1 3 1 d(0.5) (µm) 70 1 30 2 d(0.9) (µm) 180 2 79 3 D[4,3] (µm) 77 1 42 2 D[3,2] (µm) 6 1 8 1 Span (-) 3 1 3 1 In addition, to evaluate the influence of NCs particle types on the stabilization of emulsion, the surface coverage has been determined, by investigating the relationship between the size of the emulsions as a function of the volume fraction of dispersed phase (i.e. oil) and estimating the emulsion coverage as the maximum number of NCs entrapped at the droplet interface. The percentage coverage of the emulsions stabilized with different type of NCs was higher than 100% (377% and 131% for CNCs and CNFs, respectively). This could be ascribed to the content, physical size and morphology of NCs, whose fibrous particles linked or overlapped at the oil-water surface. Based on these results, it can be inferred that the NCs particles had a significant influence on the O/W interface, where they likely associate and form a three-dimensional network entrapping the emulsion droplets and inhibiting them from freely moving, hence contributing to improving emulsion stability. The strong interaction between the cellulosic fibers and the oil droplets, as suggested by the present data, can promote the physical stability of the Pickering emulsions, by (i) increasing the viscosity of the continuous phase, (ii) reducing the emulsion droplets size, and/or (iii) reducing the difference in density between the continuous phase and the disperse one (Dong et al., 2021 ; Kalashnikova et al., 2013 ; Tang, 2020 ; Varanasi et al., 2018 ; H. Yang et al., 2020 ). Therefore, the rheological properties of the emulsions have been evaluated for interpreting the effect of morphological properties of nanocellulose on the viscosity of the emulsions, in relation to their stability. The Pickering emulsions stabilized by CNCs and CNFs exhibited the typical shear-thinning behaviour, i.e. the apparent viscosity decreased at increasing shear rate, with a in the entire shear rate range of 0.1 to 100 s − 1 in the case of CNFs, while in the case of CNCs it reached a plateau at shear rates higher than 1 s − 1 . This behaviour is related to the structural deformation of emulsion, since the breakdown of entangled three-dimensional networks and the orientation along flow lines upon the shear force is easier for CNCs emulsions (Fig. 7 ) (Costa et al., 2022 ; Reid et al., 2016 ). Moreover, CNFs favored the increase of emulsion apparent viscosity values (Yuan et al., 2021 ) with respect to the same amount (mg mL − 1 ) of CNCs, due to their peculiar morphological properties. The long, flexible and defibrillated fibers interacted strongly in the O/W emulsion system and consequently formed internal entanglements in the continuous phase. These results suggest that CNFs particles are prone to strongly interact through Van der Waals forces and/or hydrogen bonds in the continuous phase, with a consequent increase in viscosity. The strong 3D emulsion network formed leads to restricted movement of the oil droplets and improved emulsion stability. The emulsifying activity and emulsion stability are recognized as the most important parameters to estimate the emulsifier ability to form a stable emulsion. EAI and ESI, evaluated through Equations 7 and 8 and reported in the Table 2 , are greatly dependent on the hydrophobicity and the ionic charge of CNCs and CNFs. A significantly ( p < 0.05 ) higher value of the index of the interfacial area stabilized per unit of weight (EAI = 186 ± 4 m 2 g − 1 ) was observed for CNCs. This phenomenon was attributed to the smaller molecules that could quickly diffuse and adsorb onto the oil-water interface (Bao et al., 2022 ; Y. Liu et al., 2022 ). Simultaneously, ESI was 0.14 ± 0.03 and 0.33 ± 0.09% for CNCs and CNFs-stabilized emulsions, respectively. ESI of CNFs emulsion was significantly ( p < 0.05 ) higher than for the CNCs-stabilized emulsion, likely because of the higher molecular flexibility of CNFs, an important factor for forming a compact interfacial layer and promoting emulsion stability. Remarkably, the results of Table 2 show that the effect of HPH was significantly higher for CNFs-stabilized emulsions than for CNCs-stabilized ones: EAI values increased ten folds for the former and only twice for the latter upon HPH processing, likely due to the previously reported defibrillation effect of the HPH treatment on CNFs, which increased their emulsification ability (Pirozzi et al. 2021 ). Therefore, thanks to HPH treatment, the performance of CNFs in terms of emulsification ability (EAI) becomes comparable to the performance of CNCs. The capability to stabilize emulsions (ESI) was higher in CNFs-stabilized emulsions mainly because of the higher viscosity of the continuous phase, which slowed down gravitational separation phenomena. Table 2 Emulsifying activity (EAI) and emulsion stability (ESI) parameters of Pickering emulsions homogenized by HSM or HPH treatment and stabilized by CNCs and CNFs. O/W Pickering emulsion stabilized by CNCs O/W Pickering emulsion stabilized by CNFs HSM HPH HSM HPH EAI (m 2 /g) 89.49 ± 6.60 b 186.03 ± 4.40 d 16.60 ± 1.54 a 150.02 ± 2.98 c ESI (%) 0.05 ± 0.01 a 0.14 ± 0.03 a 0.15 ± 0.03 a 0.33 ± 0.09 b Different letters denote significant differences ( p < 0.05 ) among the different samples within each row (n = 3). To examine the long-term stability of different types of nanocellulose on Pickering emulsions, the change in the microstructure and the mean droplet diameter upon aging for 10 months at 5°C was monitored. Figures 8 displays microscope images of the emulsions stabilized by CNCs and CNFs. Visual observations show that CNCs could efficiently stabilize the Pickering emulsions, without showing any sign of precipitation or flocculation during the entire storage period. The appearance of CNCs-stabilized emulsion suggested no occurrence of phase separation after 10 months. Emulsion droplets size distribution exhibited some changes over the storage period, with the appearance of a peak at smaller size and the reduction in volume of the initially observed peak. This phenomenon can be attributed to the onset of coalescence, which caused an initial increase in droplet size, associated with the reduction of the surface area to be covered by the CNC particles, with the suspension in water of CNC particles no more adsorbed at the O/W surface. At the same time, the reduction of the O/W specific surface is also reported to increase the surface coverage of droplets, whereby a network structure is formed that prevents oil droplets from further condensing together into larger droplets (Ojala et al., 2018 ). In contrast, flocculation of the emulsions prepared with CNFs can be clearly observed at the end of the storage period, through the formation of aggregates visibly by naked eye and through optical microscopy (Fig. 8). This instability phenomenon can be ascribed to bridging flocculation, in agreement with the observed increase in CNF emulsion viscosity because of fiber entanglements in the continuous phase. The formation of aggregates is clearly visible also from the particle size distribution data in Fig. 9 , which might be due to CNFs chain interactions which led to droplet flocculation through particles bridging mechanism at O/W interface. Moreover, also the emulsifying activity index (EAI) and emulsion stability index (ESI) of the emulsions were determined after 10 months of storage. Emulsions, after the storage period, exhibited lower EAI values than those freshly prepared (54.27 ± 4.02 m 2 g − 1 and 45.23 ± 4.16 m 2 g − 1 for CNCs and CNFs, respectively), but constant ESI values (017 ± 0.10% and 0.32 ± 0.02% for CNCs and CNFs, respectively). The droplet size distribution measurements confirmed the microscopy observations and showed the decrease of droplet volume diameter for CNCs and the increase for CNFs after 10 months of storage (Fig. 9 ). Remarkably, both CNCs and CNFs stabilized emulsions exhibited initially a monomodal distributions, characterized by relatively small span values (1.17 and 1.40 for CNCs and CNFs, respectively) and turned to bimodal during storage, with significantly larger span values (8.28 and 5.82 for CNCs and CNFs, respectively). 4. Conclusions This study focused on the application of TEMPO-mediated oxidation to softwood kraft pulp Celeste 85 to obtain CNCs or CNFs, which, because of their distinctive properties in terms of morphology, carboxylate group content, and wettability, are suitable for the stabilization of Pickering emulsions. The results show that the obtained CNCs, with a needle-like structure of ~ 3 nm in thickness and ~ 170 nm in length, and CNFs, characterized by a fibrous structure of ~ 10 nm in thickness and a few micrometers in length, were both able to stabilize 5% wt. sunflower oil-based Pickering emulsions at a dosage of 0.5% wt. Despite the significant morphological difference between CNCs and CNFs, both contribute to forming an inter-connected network structure of the emulsion droplets, promoting their efficient steric and electrostatic stabilization. Remarkably, when the emulsions are produced via high-pressure homogenization, the treatment had a more significant effect on emulsification ability and stability on CNFs, which are likely to be due to a defibrillation process, improving the CNF performance to the level of CNC’s one. The present results, hence, provide specific and relevant outcomes about how tailoring the properties of the nanostructured cellulose during fabrication, through TEMPO-oxidation and high-pressure homogenization, may represent a simple strategy to affect the behavior and stability of Pickering emulsions. More specifically, CNCs can be exploited in the stabilization of emulsions for low-viscosity applications, such as drinks and beverages, due to the finer attainable emulsion size and span values. In contrast, CNFs could be exploited in creams, sauces and dressings, due to the higher apparent viscosity and stronger 3D-network formed by entangled fibers. Declarations Foundings This work was supported by the Italian Ministry of University (MUR) call PRIN 2017 with the project 2017LEPH3M “PANACEA: A technology PlAtform for the sustainable recovery and ad-vanced use of NAnostructured CEllulose from Agro-food residues”. Authors and Affiliations Department of Industrial Engineering, University of Salerno, Via Giovanni Paolo II, 132, 84084 Fisciano, SA, Italy Annachiara Pirozzi, Giovanna Ferrari, & Francesco Donsì Department of Physics, University of Trento, Via Sommarive, 14, 38123 Trento, Italy Paolo Bettotti, Tiziano Facchinelli, Elvira D’Amato, & Marina Scarpa ProdAl scarl, University of Salerno, via Giovanni Paolo II, 132, 84084 Fisciano, SA, Italy Giovanna Ferrari Author Contributions Conceptualization: Francesco Donsì, Paolo Bettotti, Marina Scarpa, Annachiara Pirozzi; Methodology: Annachiara Pirozzi, Marina Scarpa; Formal analysis and investigation: Francesco Donsì, Paolo Bettotti, Marina Scarpa, Tiziano Facchinelli, Elvira D’Amato, Annachiara Pirozzi; Writing - original draft preparation: Annachiara Pirozzi; Writing - review and editing: Annachiara Pirozzi, Francesco Donsì, Paolo Bettotti, Marina Scarpa; Funding acquisition: Francesco Donsì, Marina Scarpa; Resources: Francesco Donsì, Marina Scarpa; Supervision: Francesco Donsì, Marina Scarpa Corresponding author Correspondence to Francesco Donsì. 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The spectra were normalized to the intensity of the peak at 2900 cm\u003csup\u003e-1\u003c/sup\u003e, taken as internal reference.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2530423/v1/5c1b1cc44368612473c21d88.jpg"},{"id":32390807,"identity":"67f9cad2-384d-43cd-a432-49b899004e4e","added_by":"auto","created_at":"2023-02-02 16:15:57","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":18551,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-2530423/v1/d35d6f931abf8a35a49a2370.png"},{"id":32391435,"identity":"d5c241e8-0594-4c8c-9d3f-f0698ab854a5","added_by":"auto","created_at":"2023-02-02 16:23:57","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":52193,"visible":true,"origin":"","legend":"\u003cp\u003eSuspensions of CNCs (on the left) and CNFs (on the right).\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2530423/v1/f8f13aa26567332b5107563c.jpg"},{"id":32391437,"identity":"d1b99eb7-c651-425f-b360-9df8eb6a13da","added_by":"auto","created_at":"2023-02-02 16:23:57","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":81788,"visible":true,"origin":"","legend":"\u003cp\u003eVisual observation and micrographs of Pickering emulsion stabilized with (a) CNCs and (b) CNFs at two different magnifications (10x on the lefthand side and 40x on the righthand side).\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2530423/v1/c9daeb3f755b9a1782b36b52.jpg"},{"id":32392195,"identity":"6d9380b2-2227-435c-a021-bae85bdcb6ba","added_by":"auto","created_at":"2023-02-02 16:31:57","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":18994,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-2530423/v1/7f7947184182d8964f37d95b.png"},{"id":32390810,"identity":"2ea08ce8-beb3-4bc4-806f-15456db3ee98","added_by":"auto","created_at":"2023-02-02 16:15:57","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":18597,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-2530423/v1/3ab184f825cbb639e43900e4.png"},{"id":32391440,"identity":"84c43226-aa57-4277-aa0c-d30162fa943e","added_by":"auto","created_at":"2023-02-02 16:23:57","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":53726,"visible":true,"origin":"","legend":"\u003cp\u003eVisual observation and micrographs of (a) freshly prepared Pickering emulsions and (b) emulsions after 10 months of storage at 5 °C (10x of magnification) stabilized by CNCs and CNFs at first and second row, respectively.\u003c/p\u003e","description":"","filename":"Figure8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2530423/v1/9c7f9e2f796a8ddf8a6bdde0.jpg"},{"id":32392196,"identity":"40ad8de9-7ba8-469b-84c8-e9765d9ba56d","added_by":"auto","created_at":"2023-02-02 16:31:57","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":258087,"visible":true,"origin":"","legend":"\u003cp\u003eDroplet size distributions of CNCs and CNFs stabilized emulsions on day 0 (··· dotted line) ) and 10 months (⸺ solid line).\u003c/p\u003e","description":"","filename":"Figure9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2530423/v1/0b8283e9e3d8265b3e2df12b.jpg"},{"id":33714461,"identity":"090c0995-e5ba-4dea-94f8-e1ad3bcdb577","added_by":"auto","created_at":"2023-03-02 23:44:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":863538,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2530423/v1/47af70b9-e8e2-43ae-bfa3-23740ff7cf10.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Oil-in-water Pickering emulsions stabilized by nanostructured cellulose: comparison of cellulose nanocrystals and nanofibrils","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003ePickering emulsions, stabilized by particles at the oil-water interface are attracting increasing interest because they do not require the use of surfactants, which is matter of health and environmental concerns. Moreover, Pickering emulsions are highly stable, due the nearly irreversible absorption of the particles at the liquid interface, and their organization into a compact and dense layer, which reduce droplet coalescence because of efficient steric repulsion (Chevalier \u0026amp; Bolzinger, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe unique features of colloidal particles as stabilizers and the applications of Pickering emulsions in different fields, in particular in pharmaceutical (as topical and oral drug delivery systems or as templates to prepare materials for biomedical purposes) and food (as vehicles of nutraceuticals and component of edible films) manufacturing, have been extensively reviewed (Bruno et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; H. Jiang et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). However, in spite of the general agreement on the potential perspectives of use of Pickering emulsions, much is still to be investigated to enable their practical implementation. Indeed, particles characteristics play a fundamental role on Pickering emulsion preparation and stabilization (Wu \u0026amp; Ma, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2016\u003c/span\u003e): their specific features affect their behavior at interfaces and self-assembling tendency, as well as thickness and cohesion of the interfacial particle layer. Besides the nature of the constitutive chemical components and the presence of surface functional groups, also physical parameters, such as size, shape and crystallinity are relevant for tuning the Pickering emulsions characteristics and performance as a function of the desired application.\u003c/p\u003e \u003cp\u003eSeveral solid particle materials of inorganic or organic sources have been used to obtain stable Pickering emulsions (Y. Yang et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). However, in the last years, the interest has mainly converged toward nanoparticles obtained from natural sources, which combine the advantage of self-assembling, driven by nanoscale forces (Bishop et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) with the limited environmental impact and lack of toxicity concerns, differently from inorganic nanomaterials (Kipen \u0026amp; Laskin, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Different organic particles of natural origin have been tested, made of biopolymers (polysaccharides, lignin, and proteins), microorganisms, or molecular assemblies. In general, only few of them were able to stabilize Pickering emulsions in pristine form, whereas in most of the cases a modification process was needed to provide the required techno-functional properties (Dupont et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCellulose-derived nanomaterials, usually indicated as nanocellulose (NC), are among the most promising polysaccharide stabilizers, because they are abundant, sustainable, environmentally-friendly (Xue et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) and non-toxic to human cells (Meschini et al., 2020). NC has been successfully used for the preparation of films, hydrogels (De France et al., 2017) and emulsions (Jim\u0026eacute;nez Saelices \u0026amp; Capron, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Moreover, thanks to the reactive cellulose hydroxyls, NC can be easily chemically modified (Habibi, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), complexed through coordination with metal ions (Maestri et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) and hybridized with inorganic nanoparticles (Voisin et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). NC is highly dispersible in water, its colloidal suspensions being very stable, and it shows an amphiphilic structure since it exposes crystalline faces with largely different polarity, which is of interest in Pickering emulsion stabilization (F. Jiang \u0026amp; Hsieh, 2016; Johansson et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Medronho \u0026amp; Lindman, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Several methods have been developed to obtain NCs from different sources (Kargarzadeh et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) but the most versatile one is the TEMPO-mediated oxidation (Isogai et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), through which nanostructures with variable morphology can be obtained by small adjustment of the reaction conditions (Saito \u0026amp; Isogai, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2004\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNCs include cellulose nanofibrils (CNFs) and nanocrystals (CNCs), with high and low aspect ratios, respectively. In particular, while the diameter of CNFs is in the nanoscale, i.e., less than 100 nm, their length is typically up to a few micrometers, and disordered regions are still present in the fibrils. Conversely, CNCs are rod-like crystalline structures, with diameters of 2\u0026ndash;25 nm and lengths from 100 to 750 nm (Rajinipriya et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The tuning of the structure of this nanomaterials, while preserving the similar chemical nature, offers an interesting opportunity to investigate the effect of the physical (i.e. morphological) parameters on the characteristics and stability of the Pickering emulsions. More specifically, the exploitation of different types of cellulose-based nanomaterials, such as CNFs and CNCs, represents a promising strategy to increase the technologic and profitable potential of NCs in the fabrication of food-grade Pickering emulsions, due to their unique properties and excellent sustainability, biocompatibility, and renewability.\u003c/p\u003e \u003cp\u003eThis study aimed to investigate the structure effect of different cellulose nanoparticles, such as CNFs and CNCs, obtained by TEMPO-mediated oxidation from cellulose pulp, on the production and stabilization of oil-in-water Pickering emulsions. In particular, the correlation between the physicochemical properties of different NCs with emulsion properties (mean diameter, ζ-potential, microstructure and rheological properties) are investigated to understand how to improve emulsions behavior and stability over time.\u003c/p\u003e"},{"header":"2. Material And Methods","content":"\u003cdiv class=\"Section2\" id=\"Sec3\"\u003e\n \u003ch2\u003e2.1 Materials\u003c/h2\u003e\n \u003cp\u003eNorthern bleached softwood kraft pulp Celeste 85 was a kind gift of SCA (Sundsvall, Sweden). This pulp was produced by a totally chlorine-free thermomechanical process and flush-dried.\u003c/p\u003e\n \u003cp\u003ePeanut oil, purchased from a local market (Olio di Semi di Arachide Giglio Oro, Firenze, Italy), was used as the oil phase. Its composition (on weight basis, based on manufacture\u0026rsquo;s specification) comprises 18 wt% of saturated fatty acids, 46 wt% of monounsaturated fatty acids, and 28 wt% of polyunsaturated fatty acids.\u003c/p\u003e\n \u003cp\u003eSodium dodecyl sulfate (C\u003csub\u003e12\u003c/sub\u003eH\u003csub\u003e25\u003c/sub\u003eNaO\u003csub\u003e4\u003c/sub\u003eS, ACS GR, 99.0%, PanReac, Barcelona, Spain) was used as received without further purification. Milli-Q water (obtained with Barnstead\u0026trade; Pacific TII Water, Thermo Scientific, Waltham, MA, USA) was used throughout this work.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec4\"\u003e\n \u003ch2\u003e2.2 Preparation of nanocellulose\u003c/h2\u003e\n \u003cp\u003eTEMPO-mediated oxidation of cellulose pulp was performed according to the method of Saito and Isogai (Saito \u0026amp; Isogai, \u003cspan class=\"CitationRef\"\u003e2004\u003c/span\u003e) slightly modified as herein reported. In order to obtain CNCs, 5 g of Celeste 85 pulp were swollen in 400 mL H\u003csub\u003e2\u003c/sub\u003eO. 83 mg of 2,2,6,6-Tetramethylpiperidine-1oxyl (TEMPO, C\u003csub\u003e9\u003c/sub\u003eH\u003csub\u003e19\u003c/sub\u003eN, \u0026ge; 98.0%, Alfa Aesar Chemicals, Ward Hill MA, USA), the reaction catalyst, and 0.5 g of sodium bromide (NaBr, \u0026ge; 99.99%, Carlo Erba, Milan, Italy), the catalyst re-cycling, were solubilized in 100 mL H\u003csub\u003e2\u003c/sub\u003eO and added to the pulp suspension. The reaction was started adding 18 mL of sodium hypochlorite containing 6\u0026ndash;14% active chlorine (NaClO, Sigma Aldrich, Milan, Italy) which is the primary oxidant. The pH of the reaction mixture was kept in the interval 10.5\u0026ndash;11 by adding NaOH until it remained constant as occurred after 4 h. Then, the suspension was rinsed several times with H\u003csub\u003e2\u003c/sub\u003eO and tip sonicated at 80% power delivered in the suspension aliquots of 35 mL volume by a Bandelin Sonuplus ultrasonicator system (Berlin, Germany). CNFs were obtained with the same procedure except that a lower amount of TEMPO (12.5 mg) was used and the reaction was stopped after 2 h.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec5\"\u003e\n \u003ch2\u003e2.3 Preparation of Pickering emulsions\u003c/h2\u003e\n \u003cp\u003eCoarse oil-in-water emulsions were prepared by mixing peanut oil (5 wt%) with the aqueous phase (95 wt%) containing 0.5 wt% of CNCs or CNFs in ultrapure H\u003csub\u003e2\u003c/sub\u003eO, in a high-shear mixer (HSM) (MIULab MT-30K Handheld Homogenizer, Hangzhou, China) at 35,000 rpm for 5 min in an ice bath. The obtained coarse emulsions were then treated through a high-pressure homogenization (HPH) process at 80 MPa for 15 passes, with the heat exchangers set at 5\u0026deg;C, in a lab set-up (orifice-type homogenizer) previously described in details by Pirozzi et al. (\u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e) (Pirozzi et al., \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec6\"\u003e\n \u003ch2\u003e2.4 Nanocellulse characterization\u003c/h2\u003e\n \u003cp\u003eAtomic Force Microscopy (AFM) images were acquired using a NT-MDT P47H probe microscope (Apeldoorn, Netherlands) scanning operated in the semi contact mode (tip spring constant\u0026thinsp;=\u0026thinsp;40 N m\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). Detailed information of on the size and morphology of the CNCs and CNFs produced by the TEMPO-mediated oxidation procedure was obtained by analyzing these images with the Gwyddion 2.59 software.\u003c/p\u003e\n \u003cp\u003eInfrared spectra of CNCs and CNFs were acquired by a Nicolet IN10 microFTIR instrument (Thermo Fisher scientifica Waltham MA, USA) operating in transmittance mode. To this purpose, a drop of CNCs\u0026rsquo; and CNFs\u0026rsquo; suspensions was deposited on a ZnSe optical window and left to dry in an oven.\u003c/p\u003e\n \u003cp\u003eThe carboxylation of CNCs and CNFs was determined by performing conductometric titration by a Delta Ohm 2256 instrument (Padova, Italy). The samples were brought to pH 2.8 by addition of HCl then the solution was titrated by 0.5 M NaOH. The degree of oxidation (OD), which is given by the carboxyl content per weight of CNCs and CNFs (\u0026micro;mol mg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), was calculated from\u0026nbsp;\u003cstrong\u003eEq.\u0026nbsp;1\u003c/strong\u003e:\u003cbr\u003e\u0026nbsp;\u003c/p\u003e\n \u003ctable border=\"1\" id=\"Taba\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(OD=\\frac{C\\bullet V}{m}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(Eq.\u0026nbsp;1)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003cp\u003ewhere \u003cem\u003eC\u003c/em\u003e is the concentration of the NaOH solution, and \u003cem\u003em\u003c/em\u003e is the total mass of CNCs and CNFs measured after oven drying the suspensions. \u003cem\u003eV\u003c/em\u003e is the volume of the NaOH solution added to reach complete ionization of COOH groups, which was obtained by fitting the experimental titration curve (Da et al., \u003cspan class=\"CitationRef\"\u003e2003\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eThe interfacial tensions of NCs (0.5 wt%) were measured by using the pendant drop method using a contact-angle meter (KSV Instruments LTD CAM 200, Helsinki, Finland), equipped with an image analysis software. Briefly, a syringe equipped with a stainless-steel needle (0.71 mm in diameter) filled with the aqueous phase containing nanoparticles was submerged into the oil phase within a glass cuvette. The initial volume of the formed drop during the whole experiment was about 30 \u0026micro;L. The optical contact angle meter recorded the change of the oil/water interface and the Young-Laplace equation was used to calculate the interfacial tension. Distilled water was used as the control. The interfacial tension (\u0026gamma;) measurements were performed for 2,500 s, and the equilibrium interfacial tension values were estimated using the exponential decay model of \u003cstrong\u003eEq.\u0026nbsp;2\u003c/strong\u003e (Mauriello et al., \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e):\u0026nbsp;\u003c/p\u003e\n \u003ctable border=\"1\" id=\"Tabb\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\gamma ={\\gamma }_{{\\infty }}+\\left({\\gamma }_{0}-{\\gamma }_{{\\infty }}\\right){e}^{-\\frac{t}{{\\tau }_{r}}}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(Eq.\u0026nbsp;2)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003cp\u003ewhere \u003cem\u003e\u0026gamma;\u003c/em\u003e\u003csub\u003e\u003cem\u003e\u0026infin;\u003c/em\u003e\u003c/sub\u003e is the asymptotic interfacial tension, \u003cem\u003e\u0026gamma;\u003c/em\u003e\u003csub\u003e\u003cem\u003e0\u003c/em\u003e\u003c/sub\u003e is the initial interfacial tension, \u003cem\u003e\u0026tau;\u003c/em\u003e\u003csub\u003e\u003cem\u003er\u003c/em\u003e\u003c/sub\u003e is the characteristic time for the arrangement of the molecules at the water-oil interface, and \u003cem\u003et\u003c/em\u003e is the time variable.\u003c/p\u003e\n \u003cp\u003eThe \u0026zeta;-potential of the cellulose nanoparticles in water were measured by dynamic light scattering (DLS) and electrophoretic mobility using a Zetasizer Nano (ZEN3600 Malvern Instruments Ltd., Malvern, UK) at 25\u0026deg;C.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec7\"\u003e\n \u003ch2\u003e2.5 Pickering emulsions characterization\u003c/h2\u003e\n \u003cp\u003eThe microscopic structure of Pickering emulsions prepared with CNCs and CNFs was observed using the optical inverted microscope Nikon Eclipse (TE 2000S, Nikon instruments Europe B.V., Amsterdam, The Netherlands).\u003c/p\u003e\n \u003cp\u003eThe emulsions size distributions were measured by laser diffraction using a Mastersizer 2000 instrument (Malvern instrument Ltd., Malvern, UK), using the Fraunhofer approximation, which does not require knowledge of the optical properties of the sample. The temperature of the cell was maintained at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u0026deg;C. Characteristic diameters d(0.1), d(0.5), and d(0.9), corresponding to the 10th, 50th (median value), and 90th percentile of the cumulative size distribution of the suspensions, were evaluated. Additionally, the surface-weighted mean diameter D[3,2] and volume-weighted mean diameter D[4,3] were determined according to \u003cstrong\u003eEqs.\u0026nbsp;3 and 4\u003c/strong\u003e, respectively:\u0026nbsp;\u003c/p\u003e\n \u003ctable border=\"1\" id=\"Tabc\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(D\\left[\\text{3,2}\\right]=\\frac{{\\sum }_{i}{n}_{i}{d}_{i}^{3}}{{\\sum }_{i}{n}_{i}{d}_{i}^{2}}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e(Eq.\u0026nbsp;3)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(D\\left[\\text{4,3}\\right]=\\frac{{\\sum }_{i}{n}_{i}{d}_{i}^{4}}{{\\sum }_{i}{n}_{i}{d}_{i}^{3}}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(Eq.\u0026nbsp;4)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003cp\u003ewhere \u003cem\u003en\u003c/em\u003e\u003csub\u003e\u003cem\u003ei\u003c/em\u003e\u003c/sub\u003e is the number of particles of diameter \u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003ei\u003c/em\u003e\u003c/sub\u003e.\u003c/p\u003e\n \u003cp\u003eThe relative span factors were calculated according to \u003cstrong\u003eEq.\u0026nbsp;5\u003c/strong\u003e, to express the distribution width of the droplet size distribution:\u0026nbsp;\u003c/p\u003e\n \u003ctable border=\"1\" id=\"Tabd\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(Span=\\frac{d\\left(0.9\\right)-d\\left(0.1\\right)}{d\\left(0.5\\right)}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(Eq.\u0026nbsp;5)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003cp\u003eThe surface coverage (SC) of NCs of the internal phase of the Pickering emulsions was given by the theoretical maximum surface area by the particles (S\u003csub\u003eP\u003c/sub\u003e) divided by the total surface displayed by the oil droplets (S\u003csub\u003ed\u003c/sub\u003e) according to the following equation (He et al., \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e; Hu et al., \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e; Kalashnikova et al., \u003cspan class=\"CitationRef\"\u003e2011\u003c/span\u003e):\u0026nbsp;\u003c/p\u003e\n \u003ctable border=\"1\" id=\"Tabe\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(SC=\\frac{{S}_{P}}{{S}_{d}}=\\frac{{m}_{P}\\bullet D\\left[\\text{3,2}\\right]}{6\\bullet h\\bullet {\\rho }_{P}\\bullet V}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\"\u003e\u003cp\u003e(Eq.\u0026nbsp;6)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003ewhere \u003cem\u003em\u003c/em\u003e\u003csub\u003e\u003cem\u003eP\u003c/em\u003e\u003c/sub\u003e is the mass of NC in the Pickering emulsion (g), \u003cem\u003eD[3,2]\u003c/em\u003e is the surface-weighted mean diameter, \u003cem\u003eh\u003c/em\u003e is the thickness of cellulose fibers (the average thickness of CNCs and CNFs fibers is 3.0 \u0026plusmn; 0.5 nm and 10 nm, respectively), as estimated through AFM, \u003cem\u003e\u0026rho;\u003c/em\u003e\u003csub\u003e\u003cem\u003eP\u003c/em\u003e\u003c/sub\u003e is the cellulose density (1.6 g cm\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e) and \u003cem\u003eV\u003c/em\u003e is the volume of oil used in the Pickering emulsion (mL).\u003c/p\u003e\u003cp\u003eThe rheological properties of Pickering emulsions were characterized by using a rotational rheometer (AR 2000 rheometer, TA instruments, Newcastle, DE, USA), equipped with a concentric cylinder (15 mm stator inner diameter, 28 mm rotor outer diameter, 42 mm cylinder immersed height, 2\u0026deg; cone angle). Viscosity curves were obtained by changing the shear rate from 0.1 s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e up to a rate of 200 s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and the temperature was set at 20\u0026deg;C.\u003c/p\u003e\u003cp\u003eThe emulsion\u0026rsquo;s stability was determined according to the turbidimetric method (De Maria et al., \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e). The absorbance values of freshly prepared emulsions (t\u003csub\u003e0\u003c/sub\u003e) and after 30 min (t\u003csub\u003e30\u003c/sub\u003e) were read at 500 nm against a blank (dilution solution) following the introduction of 50 \u0026micro;L of emulsion into 5 mL of 0.1 wt% sodium dodecyl sulfate (SDS) solution. The emulsifying activity index (EAI) and the emulsion stability index (ESI) were calculated using \u003cstrong\u003eEqs.\u0026nbsp;7 and 8\u003c/strong\u003e, respectively.\u0026nbsp;\u003c/p\u003e\u003ctable border=\"1\" id=\"Tabf\"\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\"\u003e\u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(EAI \\left(\\frac{{m}^{2}}{g}\\right)=2\\bullet \\frac{T}{\\left(1-\\vartheta \\right)\\bullet C\\bullet 1000}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e(Eq.\u0026nbsp;7)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(ESI \\left(-\\right)=\\frac{EA{I}_{{t}_{0}}}{EA{I}_{{t}_{0}}-EA{I}_{{t}_{30}}}\\bullet 100\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(Eq.\u0026nbsp;8)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003cp\u003ewhere \u003cem\u003eT\u003c/em\u003e is the turbidity, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\vartheta\\)\u003c/span\u003e\u003c/span\u003e is the volume fraction of oil used to form the emulsion, and \u003cem\u003eC\u003c/em\u003e (g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) is the initial concentration of NCs. EAI\u003csub\u003et0\u003c/sub\u003e and EAI\u003csub\u003et30\u003c/sub\u003e are the emulsifying activity indexes calculated at 0 and 30 min, respectively. The turbidity was calculated by using \u003cstrong\u003eEq.\u0026nbsp;9\u003c/strong\u003e:\u0026nbsp;\u003c/p\u003e\n \u003ctable border=\"1\" id=\"Tabg\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(T=2.303\\bullet \\frac{A\\bullet DF}{OP}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(Eq.\u0026nbsp;9)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003cp\u003ewhere A is the absorbance of the sample at t\u003csub\u003e0\u003c/sub\u003e and 500 nm, DF is the dilution factor, and OP is the optical path (1 cm, in the used equipment).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec8\"\u003e\n \u003ch2\u003e2.6 Statistical analysis\u003c/h2\u003e\n \u003cp\u003eExperiments were repeated in triplicates and the values were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Significant differences at \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e were assessed with SPSS 20 (SPSS Inc., Chicago, IL, USA) statistical package through one-way analysis of variance (ANOVA) and Tukey\u0026apos;s test. The data were normally distributed.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3. Results And Discussion","content":"\u003cdiv class=\"Section2\" id=\"Sec10\"\u003e\n \u003ch2\u003e3.1 Morphology and interfacial properties of NCs\u003c/h2\u003e\n \u003cp\u003eTwo types of nanostructured cellulose, CNCs and CNFs, obtained through different treatments of the same cellulose raw material, were used to stabilize Pickering emulsions.\u003c/p\u003e\n \u003cp\u003eThe morphological, topological, and size features of CNCs and CNFs were evaluated through AFM. Representative images of the CNCs and CNFs are reported in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, with CNCs characterized by a needle-like structure, and CNFs by a fibrous network. The statistical analysis of the CNCs\u0026rsquo; size, performed on a data set of 900 CNCs individually resolved in the AFM images, indicated a length of 170\u0026thinsp;\u0026plusmn;\u0026thinsp;90 nm and a width of 3.0 \u0026plusmn; 0.5 nm. A rigorous statistical analysis of CNFs size was not possible, because of the entanglement propensity of the flexible and disordered regions still present in the fibers, which are clearly visible in the AFM images. However, an average fiber width of the order of 10 nm and a length of a few micrometers can be estimated.\u003c/p\u003e\n \u003cp\u003eFT-IR spectroscopy was used to analyse the functional groups of the NC samples. The representative spectra of CNCs (black trace) and CNFs (red trace) reported in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e show the typical features of cellulose nanostructures. In particular, the C-H rocking at 910 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, the C-O bond vibrations in the range 1030\u0026ndash;1100 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, the antisymmetric stretching vibration of the C-O-C glycosidic bond at 1160 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, the stretching vibration of the C-H bond at 2900 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and a large band ascribable to the stretching vibrations of the O-H bonds of the primary and secondary hydroxyl groups in the range 3200\u0026ndash;3500 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e can be observed (Foster et al., \u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e). The strong peak at 1620 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e can be assigned to the carboxylates introduced by the TEMPO mediated oxidation. The different extent of oxidation of CNCs and CNFs appears evident when comparing the carboxylate peak intensities of the traces of Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e normalized to the peak at 2900 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, which should be unaffected by the TEMPO-mediated reaction. The normalized spectra show a larger oxidation of the CNCs with respect to CNFs, according to the milder oxidation conditions used to obtain the larger size and preserve some amorphous regions of CNFs.\u003c/p\u003e\n \u003cp\u003eThe FT-IR data were confirmed by the conductometric titrations which provided OD values of 0.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 \u0026micro;mol mg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 1.4 \u0026plusmn; 0.1 \u0026micro;mol mg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, of CNFs and CNCs, respectively.\u003c/p\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e shows the dynamic adsorption behavior of CNCs and CNFs at the water-oil interface, investigated by monitoring the change of the interfacial tension (\u0026gamma;) with adsorption time (0\u0026ndash;2,500 s) at 25\u0026deg;C. The addition of both CNCs and CNFs to deionized water caused a significant reduction in the interfacial tension of oil-water interface. More specifically, a higher reduction was observed for CNFs than for CNCs. The interfacial tension of water rapidly decreased during the initial 500 s, because of the adsorption of the more hydrophilic oil components, before gradually reaching equilibrium. The interfacial tension of CNCs and CNFs decreased over time, tending towards equilibrium values (\u0026gamma;\u003csub\u003e\u0026infin;\u003c/sub\u003e), which were estimated using an exponential decay model (Eq.\u0026nbsp;2) at 16.7 and 13.8 mN m\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively. The \u0026gamma;\u003csub\u003e\u0026infin;\u003c/sub\u003e value for CNCs\u0026rsquo; was slightly lower than the one estimated for pure water (19.2 mN m\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), indicating the reduced ability of the CNCs particles to act onto the oil-water interface. In contrast, CNFs particles effectively adsorbed at the O/W interface, reducing its free energy, suggesting that, according to interfacial tension measurements, CNFs particles have the potential to be more effective in stabilizing O/W Pickering emulsions.\u003c/p\u003e\n \u003cp\u003eThe \u0026zeta;-potential of the CNCs and CNFs was also measured, to obtain indications about the colloidal stability of the different cellulose suspensions. Generally, a stable particle suspension should be characterized by \u0026zeta;-potential values greater than 30 mV of absolute value, to promote sufficient interparticle electrostatic repulsion forces for their stabilization (Hunter, \u003cspan class=\"CitationRef\"\u003e2013\u003c/span\u003e). Both CNCs and CNFs suspensions, whose visual appearance is shown in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e, are characterized by high \u0026zeta;-potential values, of -37.0\u0026thinsp;\u0026plusmn;\u0026thinsp;2.35 mV at a natural pH\u0026thinsp;=\u0026thinsp;6.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 and \u0026minus;\u0026thinsp;52.97\u0026thinsp;\u0026plusmn;\u0026thinsp;4.55 mV at a natural pH\u0026thinsp;=\u0026thinsp;6.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4 for CNCs and CNFs, respectively. These high \u0026zeta;-potential values support the hypothesis that both suspensions might exhibit good colloidal stability. CNFs exhibited a higher absolute \u0026zeta;-potential value than CNCs. It must be highlighted that, based on FT-IR results, which show a higher presence of carboxylate groups for CNCs particles, it would be expected an opposite effect on \u0026zeta;-potential values of CNCs and CNFs, with higher absolute values for the formers. Further studies are needed to better elucidate the reasons that might the influence electro-kinetic phenomena of NCs, such as pKa of the carboxylate groups, counter ions effects, etc\u0026hellip;.\u003c/p\u003e\n \u003cp\u003eIt can be concluded that the CNCs and CNFs are characterized by similar chemical composition, except for a different content of carboxylate groups. In contrast, the particles dimension and morphology are clearly different: CNCs are needle-shaped, while CNFs are elongated, and partly disordered fibers, with a nanosized, roughly spherical cross section.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec11\"\u003e\n \u003ch2\u003e3.2 Characterization and stability of Pickering emulsions\u003c/h2\u003e\n \u003cp\u003eThe emulsifying capacity, size distribution, mean diameters, and optical microscopy of Pickering emulsions stabilized with CNCs and CNFs were evaluated.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eFigure 5\u003c/strong\u003e shows the microstructure of freshly prepared Pickering emulsions stabilized by using CNCs (Fig. 5a) and CNFs (Fig. 5b) at 5 wt% oil phase. The oil droplets of CNFs-stabilized emulsions showed a spherical structure with a uniform size of about 3 \u0026micro;m. When the stabilizer was CNCs, the emulsion was still uniform in size, but the particles, characterized by a greater rigidity, were not able to completely cover the oil droplets, remaining partly dispersed in the aqueous phase. This can be related to the difference in morphological properties (shape and size) of nanoparticles cellulose chains: flexible and defibrillated nanocellulose (CNFs) are likely to diffuse faster, and better cover smaller oil droplets. Another possible reason for this phenomenon was the aggregation of nanofibrillated cellulose and thickening effect of the aqueous phase due to the longer cellulose chains: therefore, entrapping of the oil droplets was more efficient, and a 3D emulsified structure was promoted. It can be speculated that the improved capacity of entanglement allows to bridge the neighbouring droplets to create an entangled droplet-CNFs fiber 3D network. In general, the micrographs of \u003cstrong\u003eFig.\u0026nbsp;5\u003c/strong\u003e agree with previous observations for the preparation of Pickering emulsions stabilized by cellulose nanofibrils (Pirozzi et al., \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eGenerally, the differences in interfacial tension (14 mN m\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for CNFs vs. 19 mN m\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for CNCs at 2,000 s) and \u0026zeta;-potential (-53 mV for CNFs vs. -37 mV for CNCs) can be related to the observed difference in emulsifying performance, such as ability to act onto the oil-water interface, the promotion of droplet formation, and the increase of droplet stability induced by electrostatic repulsion (Dong et al., \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eRemarkably, despite the reported differences in interfacial tension and \u0026zeta;-potential, both CNCs and CNFs displayed outstanding stability against droplet coalescence, without any phase separation under refrigerated storage at 4\u0026deg;C, likely because of the efficient steric and electrostatic stabilization.\u003c/p\u003e\n \u003cp\u003eIn order to further understand the mechanisms of Pickering emulsion stabilization with NCs, the droplet size distribution (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e) and mean droplet diameters (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e) of the emulsions was measured for the coarse (after HSM) and the final emulsions (after HPH). It can be observed that the measured size distributions are coherent with the micrographs of \u003cstrong\u003eFig.\u0026nbsp;5\u003c/strong\u003e. Obviously, the process intensity has a significant effect on the size distribution. The droplet characteristic diameters and the distribution span decreased sharply after the application of HPH treatment. As results, the particle size distribution of emulsions treated by HSM was multimodal, with two peaks in the distribution curve within a range from 1 to 100 \u0026micro;m. In contrast, after HPH treatment, the emulsions stabilized by NCs presented a monomodal and narrower droplet size distribution (span 1.2 and 1.4 for CNCs and CNFs, respectively). The droplet size distribution of HPH treated samples are characterized by d(0.9) of 2.23 and 2.92 \u0026micro;m for CNCs and CNFs, respectively. Similarly, d(0.1) was reduced to sub-micrometric, and d(0.5) to the micrometric size range for both HPH emulsions. The volume mean diameters D[4,3] were in the range of 1.4\u0026ndash;1.8 \u0026micro;m, the surface mean diameters D[3,2] in the range of 1.2\u0026ndash;1.4 \u0026micro;m (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Low D[3,2] values are generally associated with higher kinetic stability because of the reduction of the effects of the gravitational phase separation, this, in turn, is due to the fact that the droplets separation velocity is proportional to the square radius (Martins et al., \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e. Particle size distribution (expressed as characteristic diameters and span) of Pickering emulsions homogenized by HSM or HPH treatment and stabilized by CNCs and CNFs.\u003c/p\u003e\n \u003cdiv align=\"\"\u003e\n \u003ctable border=\"0\" cellpadding=\"0\" cellspacing=\"0\" width=\"484\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"21.90082644628099%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" width=\"39.049586776859506%\"\u003e\n \u003cp\u003e\u003cstrong\u003eO/W Pickering emulsion stabilized by CNCs\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" width=\"39.049586776859506%\"\u003e\n \u003cp\u003e\u003cstrong\u003eO/W Pickering emulsion stabilized by CNFs\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"21.946169772256727%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"19.461697722567287%\"\u003e\n \u003cp\u003e\u003cstrong\u003eHSM\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"19.66873706004141%\"\u003e\n \u003cp\u003e\u003cstrong\u003eHPH\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"19.461697722567287%\"\u003e\n \u003cp\u003e\u003cstrong\u003eHSM\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"19.461697722567287%\"\u003e\n \u003cp\u003e\u003cstrong\u003eHPH\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"21.946169772256727%\"\u003e\n \u003cp\u003ed(0.1) (\u0026micro;m)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"19.461697722567287%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"19.66873706004141%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"19.461697722567287%\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"19.461697722567287%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"21.946169772256727%\"\u003e\n \u003cp\u003ed(0.5) (\u0026micro;m)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"19.461697722567287%\"\u003e\n \u003cp\u003e70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"19.66873706004141%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"19.461697722567287%\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"19.461697722567287%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"21.946169772256727%\"\u003e\n \u003cp\u003ed(0.9) (\u0026micro;m)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"19.461697722567287%\"\u003e\n \u003cp\u003e180\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"19.66873706004141%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"19.461697722567287%\"\u003e\n \u003cp\u003e79\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"19.461697722567287%\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"21.946169772256727%\"\u003e\n \u003cp\u003eD[4,3] (\u0026micro;m)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"19.461697722567287%\"\u003e\n \u003cp\u003e77\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"19.66873706004141%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"19.461697722567287%\"\u003e\n \u003cp\u003e42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"19.461697722567287%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"21.946169772256727%\"\u003e\n \u003cp\u003eD[3,2] (\u0026micro;m)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"19.461697722567287%\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"19.66873706004141%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"19.461697722567287%\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"19.461697722567287%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"21.946169772256727%\"\u003e\n \u003cp\u003eSpan (-)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"19.461697722567287%\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"19.66873706004141%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"19.461697722567287%\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"19.461697722567287%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eIn addition, to evaluate the influence of NCs particle types on the stabilization of emulsion, the surface coverage has been determined, by investigating the relationship between the size of the emulsions as a function of the volume fraction of dispersed phase (i.e. oil) and estimating the emulsion coverage as the maximum number of NCs entrapped at the droplet interface. The percentage coverage of the emulsions stabilized with different type of NCs was higher than 100% (377% and 131% for CNCs and CNFs, respectively). This could be ascribed to the content, physical size and morphology of NCs, whose fibrous particles linked or overlapped at the oil-water surface. Based on these results, it can be inferred that the NCs particles had a significant influence on the O/W interface, where they likely associate and form a three-dimensional network entrapping the emulsion droplets and inhibiting them from freely moving, hence contributing to improving emulsion stability.\u003c/p\u003e\n \u003cp\u003eThe strong interaction between the cellulosic fibers and the oil droplets, as suggested by the present data, can promote the physical stability of the Pickering emulsions, by (i) increasing the viscosity of the continuous phase, (ii) reducing the emulsion droplets size, and/or (iii) reducing the difference in density between the continuous phase and the disperse one (Dong et al., \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e; Kalashnikova et al., \u003cspan class=\"CitationRef\"\u003e2013\u003c/span\u003e; Tang, \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e; Varanasi et al., \u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e; H. Yang et al., \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eTherefore, the rheological properties of the emulsions have been evaluated for interpreting the effect of morphological properties of nanocellulose on the viscosity of the emulsions, in relation to their stability. The Pickering emulsions stabilized by CNCs and CNFs exhibited the typical shear-thinning behaviour, i.e. the apparent viscosity decreased at increasing shear rate, with a in the entire shear rate range of 0.1 to 100 s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in the case of CNFs, while in the case of CNCs it reached a plateau at shear rates higher than 1 s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. This behaviour is related to the structural deformation of emulsion, since the breakdown of entangled three-dimensional networks and the orientation along flow lines upon the shear force is easier for CNCs emulsions (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e) (Costa et al., \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e; Reid et al., \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e). Moreover, CNFs favored the increase of emulsion apparent viscosity values (Yuan et al., \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e) with respect to the same amount (mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) of CNCs, due to their peculiar morphological properties. The long, flexible and defibrillated fibers interacted strongly in the O/W emulsion system and consequently formed internal entanglements in the continuous phase. These results suggest that CNFs particles are prone to strongly interact through Van der Waals forces and/or hydrogen bonds in the continuous phase, with a consequent increase in viscosity. The strong 3D emulsion network formed leads to restricted movement of the oil droplets and improved emulsion stability.\u003c/p\u003e\n \u003cp\u003eThe emulsifying activity and emulsion stability are recognized as the most important parameters to estimate the emulsifier ability to form a stable emulsion. EAI and ESI, evaluated through \u003cstrong\u003eEquations 7\u003c/strong\u003e and \u003cstrong\u003e8\u003c/strong\u003e and reported in the Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e, are greatly dependent on the hydrophobicity and the ionic charge of CNCs and CNFs. A significantly (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e) higher value of the index of the interfacial area stabilized per unit of weight (EAI\u0026thinsp;=\u0026thinsp;186\u0026thinsp;\u0026plusmn;\u0026thinsp;4 m\u003csup\u003e2\u003c/sup\u003e g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was observed for CNCs. This phenomenon was attributed to the smaller molecules that could quickly diffuse and adsorb onto the oil-water interface (Bao et al., \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e; Y. Liu et al., \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e). Simultaneously, ESI was 0.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 and 0.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09% for CNCs and CNFs-stabilized emulsions, respectively. ESI of CNFs emulsion was significantly (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e) higher than for the CNCs-stabilized emulsion, likely because of the higher molecular flexibility of CNFs, an important factor for forming a compact interfacial layer and promoting emulsion stability.\u003c/p\u003e\n \u003cp\u003eRemarkably, the results of Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e show that the effect of HPH was significantly higher for CNFs-stabilized emulsions than for CNCs-stabilized ones: EAI values increased ten folds for the former and only twice for the latter upon HPH processing, likely due to the previously reported defibrillation effect of the HPH treatment on CNFs, which increased their emulsification ability (Pirozzi et al. \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e). Therefore, thanks to HPH treatment, the performance of CNFs in terms of emulsification ability (EAI) becomes comparable to the performance of CNCs.\u003c/p\u003e\n \u003cp\u003eThe capability to stabilize emulsions (ESI) was higher in CNFs-stabilized emulsions mainly because of the higher viscosity of the continuous phase, which slowed down gravitational separation phenomena. \u0026nbsp;\u003c/p\u003e\n \u003ctable border=\"1\" id=\"Tab2\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eEmulsifying activity (EAI) and emulsion stability (ESI) parameters of Pickering emulsions homogenized by HSM or HPH treatment and stabilized by CNCs and CNFs.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eO/W Pickering emulsion stabilized by CNCs\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eO/W Pickering emulsion stabilized by CNFs\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eHSM\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eHPH\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eHSM\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eHPH\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEAI (m\u003csup\u003e2\u003c/sup\u003e/g)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e89.49\u0026thinsp;\u0026plusmn;\u0026thinsp;6.60\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e186.03\u0026thinsp;\u0026plusmn;\u0026thinsp;4.40\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16.60\u0026thinsp;\u0026plusmn;\u0026thinsp;1.54\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e150.02\u0026thinsp;\u0026plusmn;\u0026thinsp;2.98\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eESI (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003cp\u003eDifferent letters denote significant differences (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e) among the different samples within each row (n\u0026thinsp;=\u0026thinsp;3).\u003c/p\u003e\n \u003cp\u003eTo examine the long-term stability of different types of nanocellulose on Pickering emulsions, the change in the microstructure and the mean droplet diameter upon aging for 10 months at 5\u0026deg;C was monitored. Figures 8 displays microscope images of the emulsions stabilized by CNCs and CNFs. Visual observations show that CNCs could efficiently stabilize the Pickering emulsions, without showing any sign of precipitation or flocculation during the entire storage period. The appearance of CNCs-stabilized emulsion suggested no occurrence of phase separation after 10 months. Emulsion droplets size distribution exhibited some changes over the storage period, with the appearance of a peak at smaller size and the reduction in volume of the initially observed peak. This phenomenon can be attributed to the onset of coalescence, which caused an initial increase in droplet size, associated with the reduction of the surface area to be covered by the CNC particles, with the suspension in water of CNC particles no more adsorbed at the O/W surface. At the same time, the reduction of the O/W specific surface is also reported to increase the surface coverage of droplets, whereby a network structure is formed that prevents oil droplets from further condensing together into larger droplets (Ojala et al., \u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e). In contrast, flocculation of the emulsions prepared with CNFs can be clearly observed at the end of the storage period, through the formation of aggregates visibly by naked eye and through optical microscopy (Fig.\u0026nbsp;8). This instability phenomenon can be ascribed to bridging flocculation, in agreement with the observed increase in CNF emulsion viscosity because of fiber entanglements in the continuous phase.\u003c/p\u003e\n \u003cp\u003eThe formation of aggregates is clearly visible also from the particle size distribution data in Fig. \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e, which might be due to CNFs chain interactions which led to droplet flocculation through particles bridging mechanism at O/W interface. Moreover, also the emulsifying activity index (EAI) and emulsion stability index (ESI) of the emulsions were determined after 10 months of storage. Emulsions, after the storage period, exhibited lower EAI values than those freshly prepared (54.27\u0026thinsp;\u0026plusmn;\u0026thinsp;4.02 m\u003csup\u003e2\u003c/sup\u003e g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 45.23\u0026thinsp;\u0026plusmn;\u0026thinsp;4.16 m\u003csup\u003e2\u003c/sup\u003e g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for CNCs and CNFs, respectively), but constant ESI values (017\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10% and 0.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02% for CNCs and CNFs, respectively).\u003c/p\u003e\n \u003cp\u003eThe droplet size distribution measurements confirmed the microscopy observations and showed the decrease of droplet volume diameter for CNCs and the increase for CNFs after 10 months of storage (Fig. \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e). Remarkably, both CNCs and CNFs stabilized emulsions exhibited initially a monomodal distributions, characterized by relatively small span values (1.17 and 1.40 for CNCs and CNFs, respectively) and turned to bimodal during storage, with significantly larger span values (8.28 and 5.82 for CNCs and CNFs, respectively).\u003c/p\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4. Conclusions","content":"\u003cp\u003eThis study focused on the application of TEMPO-mediated oxidation to softwood kraft pulp Celeste 85 to obtain CNCs or CNFs, which, because of their distinctive properties in terms of morphology, carboxylate group content, and wettability, are suitable for the stabilization of Pickering emulsions.\u003c/p\u003e \u003cp\u003eThe results show that the obtained CNCs, with a needle-like structure of ~\u0026thinsp;3 nm in thickness and ~\u0026thinsp;170 nm in length, and CNFs, characterized by a fibrous structure of ~\u0026thinsp;10 nm in thickness and a few micrometers in length, were both able to stabilize 5% wt. sunflower oil-based Pickering emulsions at a dosage of 0.5% wt. Despite the significant morphological difference between CNCs and CNFs, both contribute to forming an inter-connected network structure of the emulsion droplets, promoting their efficient steric and electrostatic stabilization. Remarkably, when the emulsions are produced via high-pressure homogenization, the treatment had a more significant effect on emulsification ability and stability on CNFs, which are likely to be due to a defibrillation process, improving the CNF performance to the level of CNC\u0026rsquo;s one.\u003c/p\u003e \u003cp\u003eThe present results, hence, provide specific and relevant outcomes about how tailoring the properties of the nanostructured cellulose during fabrication, through TEMPO-oxidation and high-pressure homogenization, may represent a simple strategy to affect the behavior and stability of Pickering emulsions. More specifically, CNCs can be exploited in the stabilization of emulsions for low-viscosity applications, such as drinks and beverages, due to the finer attainable emulsion size and span values. In contrast, CNFs could be exploited in creams, sauces and dressings, due to the higher apparent viscosity and stronger 3D-network formed by entangled fibers.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFoundings\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Italian Ministry of University (MUR) call PRIN 2017 with the project 2017LEPH3M \u0026ldquo;PANACEA: A technology PlAtform for the sustainable recovery and ad-vanced use of NAnostructured CEllulose from Agro-food residues\u0026rdquo;.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors and Affiliations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDepartment of Industrial Engineering, University of Salerno, Via Giovanni Paolo II, 132, 84084 Fisciano, SA, Italy\u003c/p\u003e\n\u003cp\u003eAnnachiara Pirozzi, Giovanna Ferrari, \u0026amp; Francesco Dons\u0026igrave;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDepartment of Physics, University of Trento, Via Sommarive, 14, 38123 Trento, Italy\u003c/p\u003e\n\u003cp\u003ePaolo Bettotti, Tiziano Facchinelli, Elvira D\u0026rsquo;Amato, \u0026amp; Marina Scarpa\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eProdAl scarl, University of Salerno, via Giovanni Paolo II, 132, 84084 Fisciano, SA, Italy\u003c/p\u003e\n\u003cp\u003eGiovanna Ferrari\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: Francesco Dons\u0026igrave;, Paolo Bettotti, Marina Scarpa, Annachiara Pirozzi; Methodology: Annachiara Pirozzi, Marina Scarpa; Formal analysis and investigation: Francesco Dons\u0026igrave;, Paolo Bettotti, Marina Scarpa, Tiziano Facchinelli, Elvira D\u0026rsquo;Amato, Annachiara Pirozzi; Writing - original draft preparation: Annachiara Pirozzi; Writing - review and editing: Annachiara Pirozzi, Francesco Dons\u0026igrave;, Paolo Bettotti, Marina Scarpa; Funding acquisition: Francesco Dons\u0026igrave;, Marina Scarpa; Resources: Francesco Dons\u0026igrave;, Marina Scarpa; Supervision: Francesco Dons\u0026igrave;, Marina Scarpa\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding author\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence to Francesco Dons\u0026igrave;.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors declare no conflicts of interest.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors kindly thank Luigi Esposito for particle size measurements.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAw YZ, Lim HP, Low LE, Surjit Singh CK, Chan ES, Tey BT (2022). 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Carbohydrate Polymers 269:118339. https://doi.org/10.1016/j.carbpol.2021.118339\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Nanocellulose, Emulsion stability, Emulsifying property, TEMPO oxidation, High-pressure homogenization","lastPublishedDoi":"10.21203/rs.3.rs-2530423/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2530423/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eNanostructured celluloses, in the form of nanofibrils (CNFs) and nanocrystals (CNCs), obtained through TEMPO-mediated oxidation, by controlling the intensity of the process (changing catalyst concentration and processing time), were tested in the stabilization of Pickering emulsions, fabricated through high-pressure homogenization (HPH). Results showed that both CNFs and CNCs provided an efficient steric and electrostatic stabilization of the oil-in-water emulsions. Remarkably, the strong inter-droplet interactions, observed when CNFs were used as stabilizers, because of fibrils entanglement in the continuous phase, resulted in a 3D fibrous network emulsion, with higher viscosity than CNCs-stabilized emulsions, and higher tendency towards flocculation. However, the HPH treatment significantly affected the nanofibrils interfacial layer, promoting the emulsifying ability of CNFs, and increasing stability against coalescence. In contrast, CNCs-stabilized emulsions exhibited, along with lower viscosity, higher interfacial activity and emulsion stabilization capability, without any phase separation during 10 months of refrigerated storage. Remarkably, the HPH treatment did not significantly change the emulsifying ability of CNCs. Therefore, it can be concluded that nanocelluloses with tailored emulsifying properties can be easily obtained through the regulation of the process intensity of TEMPO-mediated oxidation of pulp cellulose, opening the way to the production of new ingredients for the food and cosmetic industries.\u003c/p\u003e","manuscriptTitle":"Oil-in-water Pickering emulsions stabilized by nanostructured cellulose: comparison of cellulose nanocrystals and nanofibrils","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-02-02 16:15:52","doi":"10.21203/rs.3.rs-2530423/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"121682c9-48e0-4823-b89c-aab5c8b25a2a","owner":[],"postedDate":"February 2nd, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-03-02T23:44:19+00:00","versionOfRecord":[],"versionCreatedAt":"2023-02-02 16:15:52","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2530423","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2530423","identity":"rs-2530423","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}