Transforming the Chemical Functionality of Nanocellulose for Applications in Food Pickering Emulsions: A Critical Review

ABSTRACT Pickering emulsions (PEs) have garnered wide attention due to their high physical stability as bioactive carrier agents in the food industry. PEs are highly stable against coalescence due to the irreversible adsorption of particles forming a physical barrier at the oil-water interface between the droplets. As a sustainable alternative to conventional emulsifiers, there is an increasing interest in developing food-grade colloidal particles by forming PEs. Cellulose-based nanoparticles have proven to be efficient materials due to their renewable, biodegradable, non-toxic and controllable surface-active characteristics. Nanocellulose can be modified by various topochemical functionalization to modulate wettability and stimuli-responsive behaviour, endowing its vast potential as Pickering stabilisers for food applications. This review highlights the recent studies of protein functionalized nanocellulose for stabilising food PEs. We hope this review will assist in improving the performance and development of nanocellulose in PEs, which is vital for developing new functional foods and delivery systems. GRAPHICAL ABSTRACT


Introduction
Many natural and processed foods are emulsions or have been in an emulsified state during their preparation process.Emulsification involves mixing two immiscible liquids, such as oil and water, into a stable colloid of improved stability against creaming or aggregation.Emulsion systems with good physical and chemical stabilities have been widely used in the food and nutraceutical industries to create functional foods by encapsulating biomolecules with nutritional and pharmaceutical properties (e.g., vitamins, flavours, essential oils and spices). [1,2]Food products as emulsions can be classified into two types: oil-in-water (O/W) emulsions, such as milk, salad dressing or mayonnaise, where oil droplets are dispersed in an aqueous solution (continuous phase); and water-in-oil (W/O) emulsion, such as butter, with water droplets present as the dispersed phase in the oil medium.Generally, the volume fraction of both phases (water and oil) and the emulsifiers are employed to determine the resultant type of emulsion. [3]In a Pickering emulsion (PE), solid (nano)particles replace the conventional small-molecule surfactants to stabilise the inclusion phase.[8] In addition, consumers' awareness of the environmental and health impact in recent years also demands more sustainable, non-toxic, food-grade alternatives to chemically synthesised emulsifiers. [9]mong the food-grade natural biopolymers such as polysaccharides and proteins, cellulose-based particles are of particular interest due to their inexhaustible availability, biodegradability, and biocompatibility with many fascinating structures. [10,11][14] However, the surfaces of CNC and CNF possess abundant hydroxyl groups, making them inherently hydrophilic, along with the negatively charged sulfate half-ester groups from sulfuric acid hydrolysis, unavoidably retarding their emulsifying properties. [15,16]There is a need to modulate the nanocellulose surface composition to optimise food emulsions for these reasons.This review focuses on the basic stabilisation and destabilisation mechanisms of PEs and how the properties of cellulose-based nanoparticles can be modulated with a series of strategic functionalization approaches of nanocellulose for specific food applications.Further, the discussion is extended to the applications and performance of functionalized-nanocellulose as food-grade stabilisers.Lastly, a perspective devoting the current challenges and outlook of PEs as functional foods is presented.

Stabilisation and destabilisation mechanisms
Common emulsifiers used in the food industry, such as surfactants, are amphiphilic.They can interact with water and oil due to the dual presence of hydrophilic and hydrophobic moieties.These emulsifiers are driven toward the oil-water interface. [17,18]Their partial wettability is the key determinant that controls their adsorption morphology at the interlayer between two separate phases in a liquid-liquid system.In comparison, solid particle emulsifiers are adsorbed at the oil-water interface, which then forms a dense layer of remarkable strength. [19]Solid particles with partial wettability can stabilise both O/W and W/O emulsions; their suitability can be determined by measuring the contact angle between oil-water interfaces.Particles with a contact angle lesser than 90° typically form O/W emulsion, while W/O emulsion is usually created by particles with a contact angle higher than 90°, [20] as depicted in Figure 1.The degrees of hydrophilicity and hydrophobicity of the solid particles, together with the volume ratio of oil/water ultimately determine the type of emulsion formed.
An emulsion is a thermodynamically unstable system because the oil and water phases tend to separate and form immiscible liquid layers over time.The separation state is the most stable configuration, possessing the minimum free energy for immiscible phases. [17]Several mechanisms contribute to the breakdown of emulsions, including coalescence, flocculation, creaming, sedimentation, and Ostwald ripening (Figure 2).Coalescence occurs when smaller droplets merge to reduce the contact area between the two phases. [21]This is very similar to Ostwald ripening, where the steady growth of larger droplets from the smaller ones leads to the eventual disappearance of the tiny droplets over time. [22]In addition, droplets tend to aggregate into a floc when the attractive forces overcome the repulsive forces between droplets, leading to flocculation. [23]When coalescence and flocculation occur, the droplets may either sink to the bottom (sedimentation) or float to the surface (creaming) depending on their densities, in which their continuation in destabilisation eventually results in phase separation. [3]The mechanism for Pickering stabilisation differs from that of conventional surfactants. [18,24]This is primarily due to the different adhesion energy profiles exerted by the solid particles, often of size within the nanoscale, as stabilisers.For instance, the adsorption of the particle at the interfaces is typically irreversible, owing to the high energy required to remove them from the interfaces.The desorption energy (i.e., the energy needed to remove a particle from the oil-water interface) of a spherical particle is denoted in Equation 1: Where r is the radius of the particle (m), γ is the oil-water interfacial tension (Nm −1 ) and θ is the threephase contact angle.This value is often in the order of 10 2 to 10 3 ~kB T (the thermal energy, where k B is the Boltzmann constant and T is the temperature in K). [25,26] For rod-like particles such as nanocellulose, this equation can be modified to include a term for the surface area of the particle. [27]herefore, the energy well is often much deeper due to the stretching and swelling of soft particles at the interface.This deformation effect can also be harnessed to introduce stimuli-responsiveness of PEs stabilised by soft particles.In contrast, surfactant molecules usually have desorption energies in the order of ~kB T; they are in dynamic equilibrium with the interface and the continuous phase.Therefore, droplet coalescence and Ostwald ripening are unavoidable in surfactants-stabilised emulsions but practically cease to exist in PEs. [28]n addition, solid particles stabilised emulsions lead to an effective physical barrier of mono-or multilayer of solid particles surrounding individual emulsion droplets, providing superior repulsion forces between droplets. [29]The repulsion forces are further governed by steric hindrance and/or electrostatic repulsion between the droplets stabilised by charged solid particles. [28]This combination of forces reduces the coalescence rate and collision of droplets, thus forming stable PEs.Capillary pressure resulting from the interactions between two immiscible fluids, and their surrounding solids also contribute to the increased stabilisation.For instance, the accumulation of particles can occur from the capillary forces between particles at the interface, increasing the build-up of mechanical barrier and thus improving emulsion stability. [30]Moreover, the emergence of inter-particle bridges between droplets strengthens the physical segregation of droplets, which resist coalescence and flocculation by steric repulsion and thickening of the continuous phase. [29]

Cellulose and nanocellulose
Cellulose is the most plentiful renewable natural polymer on Earth.It is widely available in plant cell walls.Cellulose is hierarchically organised into macro-, micro-, and nano fibrillated structures, all held together by well-defined networks of intra-and intermolecular hydrogen bonds. [31,32]Although plants are the primary source of cellulose, algae, bacteria, and sea animals like tunicates are also made up of cellulose. [33]Cellulose consists of glucopyranose units linked by β-(1→4)-glycosidic bonds into a high molecular weight linear homopolymer, naturally forming strong fibre [34] (Figure 3).Generally, the source of cellulose determines the degree of polymerisation, thus defining its molecular weight.For instance, wood-derived cellulose comprises 10,000 glucose units, while cotton-derived cellulose contains even more glucose units (15,000/chain). [35]Every glucopyranose unit bears three highly reactive hydroxyl groups on its surface, positioned at the 2nd, 3rd and 6th glycosidic carbon (C2, C3, C6), providing cellulose with some unique characteristics: high hydrophilicity, chirality, and biodegradability.The hydroxyl groups are responsible for the intense and 3-dimensional inter-and intramolecular hydrogen bonding network between cellulose chains to form micro-fibrillated structures, crystalline and amorphous fractions, contributing to its cohesive nature. [36][39] These cellulose fibres can be subjected to mechanical, chemical, or biological processing to yield micro and nano-scaled cellulose fibrils with unique surface, optical and mechanical properties.The formation of cellulose micro-and nano-fibrils can be achieved by mechanical treatments such as homogeniser, microfluidizer, and grinder. [40]The resulting fibres are commonly known as cellulose microfibril (CMF) or cellulose nanofibril (CNF), depending on the extent of fibrillation.These micro-/nanofibrils can assemble into an orderly packing, resulting in the crystalline structure, while some less-ordered sections form the amorphous regions.The packing of the micro-/nanofibrils and the degree of the hierarchical organisation, including the crystallinity (crystalline or amorphous) give rise to different polymorphs or allomorphs of cellulose, each having different properties. [41]Cellulose I is the structure of native cellulose used for micro and nanofibers. [42]The specific configuration of the cellulose I structure holding the micro-/nanofibrils is mainly attributed to inter-and intramolecular hydrogen bonds with some contribution of van der Waals attraction forces. [43]To achieve a higher level of crystallinity, the amorphous region can be eliminated by sulfuric acid hydrolysis to produce microcrystalline cellulose (MCC) which can then form cellulose nanocrystals (CNC) after prolonged hydrolysis. [38]Besides plant-derived cellulose, bacterial nanocellulose (BNC), composed of cellulose nanofibers secreted extracellularly by certain bacteria has also been investigated.This unique nanofibrillar structure confers excellent physical and mechanical characteristics such as high crystallinity and high elastic modulus. [44]These unique properties make nanocellulose one of the most promising food additives, especially when surfactants need to be avoided, typically for safety reasons.Overall, nanocellulose can be classified into three categories: CNF, CNC, and BNC, prepared via mechanical, biological, and chemical routes, respectively, as shown in Figure 4.

Cellulose nanofibril (CNF)
CNF are long and flexible fibrils of large aspect ratio consisting of bundles of stretched cellulose chains; they have unique properties such as high strength and stiffness, and a large surface area. [45,46]CNF can be synthesised from raw cellulose through mechanical de-structuring processes. [46]Wood-based CNF has dimensions in the range of <20 nm in diameter and<2 µm in length.The crystalline and amorphous regions of CNF remain unchanged since it is produced via mechanical shearing force without any chemical treatment.Hence, different mechanical treatment methods can generate different ratios of crystallinity to amorphous structure organisation for specific applications. [47]Due to the high hydrophilicity of cellulose, CNF can form a gel when sheared in water, often leading to poor dispersion in a non-polar polymer matrix, emulsions, or organic solvent suspensions, especially at low concentrations due to its tendency to undergo aggregation.This incompatibility with hydrophobic polymers causes significant challenges when developing composite matrices. [48]However, it can be overcome with the addition of surfactants, by surface modification of CNF, or by solvent exchange, leading to the uniform dispersibility of CNF in the polymeric matrix. [49]These CNF properties mentioned have been explored for applications in the food industry, including as food additives, packaging and films. [50]

Cellulose nanocrystals (CNC)
Unlike CNF, cellulose nanocrystals (CNC) are rigid, crystalline rod-shaped nanoparticles achieved by acid hydrolysis of cellulose or lignocellulosic fibres.Strong acids such as sulfuric acid and hydrochloric acid are generally used for the acid hydrolysis process. [2]CNC's typical dimension ranges from 5 to 30 nm in width and 100 to 500 nm in length, depending on the fibre's origin. [51]For instance, plantderived CNC are generally shorter than non-plant derived CNC, such as from algae or tunicates. [52]hese nanocrystals possess high strength, large surface area and exceptional liquid crystalline properties in suspension, which are beneficial for emulsion formation, food packaging and ultrathin film coating materials. [33]n most cases, CNC produced by sulfuric acid contain half-ester sulfate groups (OSO 3 − ) as surface functionalities, contributing to a high negative charge on the glucopyranose ring. [53]Although the density of CNC particles at the interface would be lower, sulfuric acid-hydrolysed CNC could form very stable emulsions due to the strong electrostatic repulsive charges.The high surface charge can also be screened by adding salt to favour stable PEs. [54]Additionally, the negatively charged surface groups of nanocellulose can be tuned with the addition of a predetermined amount of cationic molecules through electrostatic forces or chemical conjugation to further stabilise PEs. [55,56]However, a similar amount of oppositely charged materials could also lead to complex agglomeration, resulting in a broken emulsion.

Bacterial nanocellulose (BNC)
Besides plants, cellulose can also be obtained from the secretion of many bacteria such as Acetobacter, Rhizobium, Agrobacterium, Aerobacter, Gluconacetobacter and Sarcina. [57]The most common BNC producer is the gram-negative bacteria, Gluconobacter xylinus, commonly known as Acetobacter xylinum. [58]BNC excreted by G. xylinus has the identical chemical structure to plant cellulose with the molecular formula (C 6 H 10 O 5 ) n with repeating subunits of (1,4)-β-D-glucose [59] and the absence of hemicellulose, lignin, and pectin that typically cover cellulose.BNC forms flat and ribbon-like microfibrils of large aspect ratio, which are weakly charged as only a part of their surface is exposed.Additional desulfation or modification is not required for BNC to stabilise emulsions [60,61] BNCs have already been demonstrated in the food industry as raw materials for manufacturing creams, edible films, texture modifiers and Pickering stabilisers. [62]However, due to the expensive carbon sources required for large-scale production, [10] the development and potential applications of BNC are much less established compared to plant-based cellulose.

Biosafety of nanocellulose
Cellulose is regarded as a safe ingredient for food products by regulatory bodies such as the Food and Drug Administration (FDA).Other than as fillers and thickeners, cellulose and its derivative are widely used as chemical penetration enhancers, which are drug additives to improve bioavailability, in several FDA-approved medications. [63]Although promising for use in ingested products, nanocellulose is not yet classified as a safe material due to concerns of nanoscale materials possessing altered biological properties compared to the micron-sized counterparts. [64]In fact, the regulation for the use of nanoscale materials in food and drug formulations, including nanocellulose, is more complicated due to the lack of reliable testing methods. [65]As a result, nanomaterials are often reviewed by regulatory bodies on a case-by-case basis. [66]Nevertheless, countless studies have been done to evaluate the cytotoxicity of nanocellulose in vitro and in vivo and have been reviewed elsewhere. [67,68]Toxicological studies of nanocellulose in vitro using human intestinal cell lines and subsequent in vivo rat models reveal little to no cytotoxicity when nanocellulose is digested. [69]Few studies, however, noted some potential safety concerns due to the risk of self-aggregation of nanocellulose. [70,71]Therefore, the toxicity of specific nanocellulose based food and drug formulations need to be thoroughly assessed before they can be safely used in mass manufacturing and processing.

Functionalisation of nanocellulose for Pickering emulsification
Solid particles of edible natural substances are often used as stabilisers, creating Pickering emulsions equipped with good biocompatibility and functional properties for food. [19]The superior stability of Pickering emulsions presented potential opportunities for the delivery of bioactive ingredients.For example, successful encapsulation and delivery of a lipophilic compound via Pickering emulsions revealed excellent stability against oxidant, reducing agent, UV radiation and ionic strength up to 500 mM. [72]The controlled release and encapsulation efficiency is tuneable by modifying the solid particle properties and the oil-water ratio.Emulsions affect the physical structure and consumer requirements, such as the texture and shelf-life of food products.The critical factors for a successful food-based Pickering emulsion rely on the properties of the stabilisers employed.Current trends have focused on the functionalisation of nanocellulose as emulsifying agents with enhanced stability to improve nutrient bioavailability during food digestion.
To date, nanocellulose of numerous sources and types has been used to form PEs. For example, Xie et al. created beeswax-in-water PEs stabilised by CNF/carboxymethyl chitosan with uniform particlesized emulsion droplets of approximately 10 µm.They improved the creaming stability of PEs by increasing the content of CNF. [73]Paximada et al. reported that using BNC as a stabiliser can increase emulsion stability better than other types of cellulose derivatives such as carboxymethyl cellulose (CMC) and hydroxypropyl methylcellulose (HPMC). [74]The stability and microstructure of PEs by CNC can be modulated by adjusting the CNC concentration and oil loading. [75]Furthermore, delayed lipolysis is often expected when oil is trapped within a highly stable PE.This could be exploited to control the release rate of the encapsulated bioactives. [76]espite the various successful studies revealing the Pickering emulsification performance of nanocellulose, bare nanocellulose without any functionalisation or additives might be insufficient to prepare stable PE due to its poor dispersibility in non-polar organic solvent and polymer matrix. [77]NC derived from acid hydrolysis possesses an abundance of highly negative OH surface groups.This induces a strong image charge effect due to the repulsive forces between the solvent and particle. [78]odifying the hydrophilic nanocellulose surface is necessary to modulate the stability of nanocellulose-stabilised PE. [79] Alternatively, salt is needed as an additive to stabilise emulsions by dampening the large negative charge of CNC. [80]The addition of salt simultaneously neutralises the image charge effect at the oil-water interface, facilitating the adsorption of particles at the interface. [81]In turn, a low concentration of salt (≤100 mM) is able to produce smaller emulsion droplets, thus improving the emulsion stability without the risk of particle aggregation. [13,82,83]Li et al. observed a significant drop in zeta potential of CNF-stabilised PEs from −45.4 mV to −9.7 mV when the salt concentration is increased from 100 mM to 1000 mM. [84]The authors proposed that this reduction may be due to conversion of emulsions into gels at a salt concentration of 50 mM and above.In addition, although CNF promotes the formation of stable PE in their raw form up to several months against coalescence, they are still prone to flocculation and creaming after long-term storage. [85]86 This restricts their potential as smart emulsion carriers with controlled stabilisation for bioactive delivery.Fortunately, the surface of nanocellulose is rich in reactive hydroxyl groups which can be used for surface functionalisation or promoting the anchoring of ligands with different chemical functionality to yield nanocellulose of desired physicochemical properties. [72]Table 1 presents the methods of chemical modifications to introduce functionality into the surface of nanocellulose chains.

Physical modification
Surface modification of nanocellulose can be accomplished by both non-covalent (physical) and covalent surface functionalisation.For instance, non-covalent modification involves the physical adsorption of molecules or polymers onto nanocellulose. [96]The molecules interact by inter-or intramolecular forces of attraction, electrostatic interaction, and hydrogen bonding. [97]For example, Lv et al. developed a food-grade PE stabilised by blending the anionic CNF with the cationic chitin nanofibrils.They postulated that the CNF and chitin nanofibers adsorbed at the oil droplet surfaces, giving rise to highly stable PEs with a thick particulate layer protecting against coalescence.The stable PEs improved their creaming stability, where the nanofibers formed a network in the aqueous solution that prevented the oil droplets from moving. [1]The addition of water-soluble polymers such as hydroxyethyl cellulose (HEC) or methylcellulose (MC) with CNC produced smaller and more stable PE droplets that showed no significant creaming or phase separation for over several months.The results showed HEC and MC to be surface-active and adsorb onto CNC, covering 75% of the oil droplets interface and sterically stabilising the remaining by unbound HEC and MC.The PEs stabilised by CNC with HEC/MC displayed reversible thermogelation, where emulsion coalescence did not occur even after multiple cycles of heating/cooling. [98]The addition of cationic alkylammonium surfactants, such as didecyldimethylammonium bromide (DMAB) and cetyltrimethylammonium bromide (CTAB), changed the wettability of CNC, which is directly linked to the increase in emulsion stability and smaller droplet size. [99]Generally, non-covalent interactions are weaker than ↑ Surface positive charge [95]  covalent interactions, which can be reverted easily without much energy input, leading to good reversibility of the obtained materials. [97]The formation of nanocellulose composites via noncovalent approaches is essential in modulating the functionalised nanocellulose properties to cater to various applications.

Covalent modification
Covalent surface modification of nanocellulose includes conjugation with small molecules or polymers. [100]The advantage of chemical modification is that electrostatic charges can be introduced to nanocellulose, improving dispersion in many solutions.This modification further alters the surface features to enhance compatibility, particularly in tuning the nanocellulose interactions with water and non-polar solution as well as with different monomers and polymers. [100,101]Common covalent modification methods for nanocellulose include oxidation, esterification, amidation, etherification and nucleophilic substitution. [102]Click chemistry has been utilised to graft and modulate the orientation of bulky molecules onto CNC. [103]Although different modification methods are feasible to functionalise nanocellulose, the real challenge remains in preserving the original morphology, unique properties, and integrity of CNC.Several functionalisation routes of nanocellulose have been depicted in Figure 5.

Oxidation
In recent years, oxidation using 2,2,6,6-Tetramethylpiperidine-1-oxyl (TEMPO) as the oxidising agent to convert the hydroxyl groups on the nanocellulose surface into the carboxylic form has attracted much attention. [104]It was first introduced by De Nooy et al., where oxidation was performed on polysaccharides with TEMPO and hypobromite as the regenerating oxidant. [105]Araki et al. conducted TEMPO-mediated oxidation of CNC obtained from hydrolysing cellulose fibres using HCl. [106]This modification is essential as an intermediate stage to facilitate the grafting of another polymer.They have shown that the initial morphological integrity of CNC was maintained, and the well-dispersed oxidised CNC in water could form homogenous suspensions. [105,106]Besides, Saito et al. produced highly crystalline and individualised CNF with a dimension of 3-4 nm in width and a few micrometres in length dispersion via the TEMPO-mediated oxidation process. [107]Recently, Mendoza et al. increased and tuned the carboxylic concentration by combining TEMPO with periodate. [108]Furthermore, Li et al. fabricated TEMPO-oxidized BNC reinforced with polylactic acid (PLA) nanocomposite via the PE approach.The addition of BNC increased the crystallinity of PLA nanocomposites, where the BNC/PLA nanocomposites displayed good mechanical properties, which can be tuned accordingly. [109]terification Esterification involves converting hydroxyl groups into ester groups via sulfation, phosphorylation, and acetylation. [33,110102]Esterification is among cellulose's most versatile chemical transformations, providing complete synthetic access to form functional nanocellulose.Esterification is an acylation reaction involving the carboxylic acid as an acylating agent under strong acid catalysis or using activated derivatives such as acid anhydride or acid chloride .Braun et al. claimed that acetylated and butylated CNC were produced via Fisher esterification with acetic acid or butyric acid, a reaction catalysed by hydrochloric acid.The conversion of the hydroxyl into an ester group was almost complete via this route as confirmed quantitatively through FTIR.However, the authors demonstrated that a high degree of substitution on CNC surfaces (up to DS surf = 1.5) could damage the crystallinity, thus changing the physical and mechanical properties.This could be due to the loss of inter-chain hydrogen bonding networks arising from hydroxyl groups on the surface of CNC after substitution, resulting in decreased crystallinity. [111]In addition, Tang et al. successfully prepared hydrophobically modified CNC/CNF using cinnamoyl chloride (CC) or butyryl chloride (BC) as the esterification agents.The hydroxyl groups of CNC/CNF were grafted with CC/BC, forming ester bonds, which were subsequently employed to link the nanocellulose with hydrophobic alkyl and benzyl groups.The resulting modified nanocellulose exhibit larger contact angle than pristine nanocellulose, thus stabilising O/W emulsions. [80]herification Etherification of CNC is another essential reaction which involves the dehydration of an alcohol functionality (−OH) to form an ether (R-O-R').The most commonly used chemical agent for CNC etherification is glycidyltrimethylammonium chloride (GTMAC) or its derivatives to catonise the nanocellulose surface groups. [33,102]Zaman et al. reported the surface cationisation of CNC with GTMAC, leading to an increase in the surface charge density compared to unmodified CNC.This cationic modification of CNC involves the nucleophilic reaction between the alkali-activated hydroxyl groups of CNC and the epoxy groups of GTMAC.Due to the high tendency of undesirable hydrolysis of GTMAS, the dry environment is critical for the reaction.The modified CNC showed excellent dispersity, stability, and delayed gelation in an aqueous system due to enhanced cationic surface charge density (+63 ± 1.65 mV). [112]Han et al. demonstrated the preparation of cationic nanocellulose following different approaches, namely via acid hydrolysis, high-pressure homogenisation, and highintensity ultrasonication using (3-chloro-2-hydroxypropyl)trimethylammonium chloride (CHPTAC) modified-MCC.Cationic nanocellulose was obtained in high yields and formed stable suspensions thanks to their higher zeta potential compared to untreated MCC. [113]Hasani and co-workers combined the cationisation agent, epoxypropyltrimethylammonium chloride (EPTMAC), with a CNC suspension, which yielded CNC with cationic hydroxypropyltrimethylammonium chloride substituents on the surface (HPTMAC-CNC).This modification preserved the original CNC's crystalline morphology, leading to extensive hydrolyses of the anionic surface sulfate ester groups.The cationisation lowered the total cationic surface charge density, leading to a slightly lower electrostatic repulsion that contributed to consequent gelation. [114]lymer/Molecules grafting by amidation Amidation is performed on nanocellulose with carboxylic groups produced from oxidation and esterification reactions.It typically involves the activation of the carboxyl groups by selected coupling reagents to form the N-hydroxysuccinimide (NHS) ester, which is highly reactive against primary amine groups.The introduction of NHS ester on the CNC surface facilitates direct conjugation with amine groups of polymers/proteins to form amide products. [102,115]Araki et al. employed this technique using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and NHS as the coupling reagents to allow for the conjugation of oxidised cellulose with amine-terminated poly(ethylene glycol) (PEG-NH 2 ).The chemical binding of PEG to the cellulose was confirmed by a 20-30% decrease in the existing carboxyl groups. [106]Ruiz-Palomero et al. also functionalised TEMPOoxidised nanocellulose with amine groups using the combination of NHS and EDC before covalently coupling it to β-cyclodextrin.The modified nanocellulose exhibited excellent extraction capacity and reusability, hence demonstrating its usefulness as a sorbent material. [116]Calderón-Vergara et al. performed conjugation of octadecylamine (ODA) with TEMPO-oxidized CNF through the formation of amide bonds using O-(1 H-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU) as the coupling reagent.The coupling of ODA on CNF led to high contact angle (125º) and increased thermal decomposition temperature, indicating the effect of hydrophobic properties and the shielding effect of ODA alkyl chains on the surface of CNF. [117]o-inspired modification Inspired by how mussels attach themselves to hard surfaces such as rocks and boats, scientists discovered that this phenomenon is due to the liquid proteins secreted by the mussels.These proteins exhibit universal adhesive properties that attach to almost any organic and inorganic substrates. [118]his biomimetic surface modification is useful as an intermediate linker for further functionalisation procedures. [119]The powerful wet adhesive force is due to the proteins known as the Mytilus food proteins-3 and −5 (Mfp-3 and −5), secreted by the byssus of mussels which engages on the substrate surface. [120]These food proteins possess two key features (high catechol content and presence of lysine/ histidine residues) that inspired the possible practicality of dopamine as functional ligands for chemical cross-linking.Waite et al. speculated on the combination and intimate association of catechol and amine groups.They could be related to interfacial adhesion, as observed in mussels foot proteins. [121,122]opamine or its polymeric form, polydopamine (PDA), aroused great interest in surface functionalisation and was first reported in 2007. [123]PDA's emergence as one of the most powerful tools to modify surfaces is due to its simple preparation method, material independence in deposition, and unique adaptability, hence pragmatic for industries like biomedical, energy, consumers. [124]The widespread adsorption of PDA as a coating material can be attributed to the oxidative polymerisation of dopamine.The initial driving force for dopamine oxidation is the dissolved oxygen in an alkaline solution.The oxidation products, dopamine quinone, and 5,6-dihydroxyindole formed by the nucleophilic intramolecular cyclisation of the formal derivative are the critical building blocks for PDA responsible for its coating properties. [124]Nonetheless, its detailed formation mechanism remains an active area of exploration.Shang et al. demonstrated the grafting of castor oil onto CNC with the assistance of dopamine.The hydrophobicity of modified CNC was improved (contact angle of 95.6º), whereas the hydrophilic surface hydroxyl groups of CNC were grafted with PDA without significantly affecting the morphology and crystallinity. [125]Recently, Wong et al. fabricated hydrophobic CNC grafted with soy protein isolate (SPI) using L-dopa, the dopamine precursor, as the linker to allow direct amidation.The hydroxyl groups of CNC were functionalised with the hydroxyl groups via the self-polymerisation of L-dopa on the surface of CNC.The resulting PEs stabilised by the bio-inspired synthesised CNC-SPI nanoconjugate displayed excellent stability than the unmodified CNC and SPI. [92]he surface functionalisation of nanocellulose can be performed using various coupling reactions (covalent and non-covalent) to impart and tune the properties of nanocellulose.The desired characteristics of nanocellulose, such as their wettability, dispersibility, thermal stability and structural integrity, are important when choosing the most appropriate reaction route of functionalisation.The novel insight on the usage of bio-inspired linkers such as dopamine in the conjugation of nanocellulose with other polymers or molecules broadly opens the opportunities for future surface engineering of nanocellulose to achieve various applications in the nutraceutical and pharmaceutical fields.Following the introduction of diverse functionalisation of nanocellulose, the following section discusses the stabilisation of food-grade PEs using protein-modified nanocellulose.

Protein functionalised nanocellulose as Pickering stabilisers
Polysaccharides such as nanocellulose, i.e., CNC, CNF, and BNC, are biodegradable and sustainable biopolymers proven to be effective Pickering stabilisers.These polysaccharides form a physical barrier by both electrostatic and steric repulsion at the liquid-liquid interface.Their thickening and formation of a 3-dimensional network retards droplets coalescence and phase separation. [126]Proteins are another class of natural polyelectrolytes that possess inherent flexibility, allowing them to selfadsorb at the oil-water interface, forming a rigid protective layer of high viscoelasticity. [127]The stability of protein-based emulsions is directly related to the protein's ability to lower the interfacial tension, facilitating droplets breakage during homogenisation, hence imparting stability to the emulsion droplets. [128]Protein-stabilised emulsions are often susceptible to changes in pH, especially during gastrointestinal digestion. [129]This is due to the pH involved in the digestive track going below and above the protein isoelectric point, therefore reversing the charges.Hence, a combination of nanocellulose and protein is attractive to engineer PEs in food products.

Functionalised nanocellulose with plant-based protein
Soy protein isolate (SPI) is a highly refined and purified form of protein extracted from soybeans.It is extensively used in the food industry because of its functional properties, low cost, and high nutritional value. [141]Several studies have investigated PEs using SPI-functionalised nanocellulose and its influence on the stability and properties of the emulsions. [94,130,131]Wong et al. modified CNC with various concentrations of SPI via a bio-inspired approach and identified the optimum ratio of CNC to SPI to form stable PEs.They found that a ratio of 1:1 for CNC:SPI produces PEs the smallest droplet size with the highest emulsifying activity, in addition to providing the best stability with negligible changes in droplet size after 30 days of storage. [131]The hydrophobic bioactive compound, thymoquinone (TQ), was encapsulated via CNC/SPI stabilised PEs in alginate-chitosan hydrogel beads for controlled delivery.The TQ containing hydrogel demonstrated good stability during gastric digestion, with 83% TQ released after 2 h of gastric digestion and 4 h of intestinal digestion. [129]  [132]

CNC Electrostatic attraction
Outer: 25-100 Inner: 2-15 -A one-step emulsification produced stable water-in-oil-water emulsions co-stabilised by both CNC and ZCP.The contact angle of the binary mixture of ZCP/CNC was found to be 89.4 ± 2.9º [133]   /PPM CNC Electrostatic attraction 17.6 ± 4.9 −34 ± 3 CNC/PPM formed a combined layer at the oil-water interface.Increasing the CNC concentration caused the gelation of PE, delaying their gastric breakdown.Physical interaction ~300-2000 ~+25 -+32 CNF increased the stability of the HFBII-coated PE.The optimal formulation for sustained release contained 0.1% HFB, 0.15% oxidised CNF and 10% oil phase. [139] Zhang et al. adsorbed SPI onto TEMPO-oxidised BNC at pH 3 through electrostatic attraction.SPI modification increased the surface hydrophobicity of BNC, and the PEs stabilised by SPI/BNC complex exhibited smaller droplet size, higher viscosity, and better stability.Along with the increasing concentration of BNC/SPI as Pickering stabilisers, the PEs showed good anti-oxidative stability.The primary (peroxide values) and secondary (thiobarbituric acid reactive substances) products of lipid oxidation were significantly lower than PEs stabilised with lower BNC/SPI concentration. [94]Liu and co-workers stabilised high internal phase emulsions (HIPEs) comprising of 75% oil phase with different BNC/SPI ratios (1:25, 3:25, 5:25, 7:25 and 9:25 w/w).They found the stability of HIPE with a BNC/SPI ratio of 7:25 was the highest after a storage period of two months.This is due to the formation of a gel-like network at the oil-water interface by the colloidal particles.These stable foodgrade particles-stabilised PEs showed promising application in the food industry. [130]ein colloidal particle (ZCP) is a food-grade processed protein extracted from corn.It has been reported to act as efficient Pickering stabilisers. [25]Li and co-workers reported that ZCP and CNC together were able to successfully stabilise double emulsions. [133]The water-in-oil-in-water emulsion formed via a one-step emulsification process, where both emulsifiers were added to the formulation during homogenisation, was found to be more stable than emulsions with either ZCP or CNC alone, which rapidly flocculates and splits within 24 h.Interestingly, without ZCP, CNC-stabilised emulsions resulted in a temporary double emulsion, before transitioning into a single emulsion within 4 h.Wei et al. employed the same stabilisers through a different formulation process where each emulsification was first done with a single stabiliser followed by homogenisation with the second co-stabiliser.The resulting single oil-in-water emulsion delayed the lipolysis of β-carotene-loaded PEs during digestion in vitro.At a ZCP/CNC mass ratio of 1:4, PEs exhibited the best physical and photothermal stabilities.The retention rate of β-carotene in the PEs co-stabilised by ZCP/CNC increased from 9.29% to 60.23% compared to PEs stabilised only with ZCP after 28 days of storage at 55°C.The reduction in lipolysis was demonstrated by the free fatty acid release rate decreasing from 19.5% to 8.7%.In comparison, the bioavailability of β-carotene in PEs ranged from 9.1% to 27.3%, achieved by adjusting the mass ratio and addition sequence of particles during emulsification. [132]hang et al. showed the synergistic interactions of pea protein microgel (PPM) and CNC at the oilwater interface by delaying the gastric digestion of PEs.This suggests that PPM/CNC form a combined layer at the oil-water interface through the electrostatic attraction between the biopolymers, thereby creating a barrier against interfacial pepsinolysis.Increasing the concentration of CNC also was found to increase the viscosity of PPM/CNC stabilised emulsions, jellifying the emulsions and effectively delaying their enzymatic breakdown. [134]

Functionalised nanocellulose with animal-based protein
Besides plant-based proteins, sodium caseinate (NaCas) obtained from the acidification of casein and subsequent neutralisation by sodium hydroxide has been widely used as an emulsifier to stabilise emulsions.The presence of α s1 -and β-casein in NaCas with high surface activity, enable their rapid adsorption at the liquid-liquid interface.However, emulsions stabilised solely by NaCas are unstable under certain conditions, for example, when pH is close to the isoelectric point of NaCas (pH 4.6) and during high processing temperature. [142,143]A combination of nanocellulose with NaCas can improve the stability of caseinate emulsions against environmental factors.
The interaction between CNC and NaCas in stabilised O/W emulsions was reported by Pind'áková. [137]This study demonstrated the aggregation of CNC and NaCas at pH 3, forming emulsions with a droplet size of 8.3 µm, slightly smaller than that for emulsions stabilised by both CNC and NaCas alone.This indicates better emulsifying performance for the CNC/NaCas complex.The surface activity of NaCas was balanced by the irreversible adsorption of CNC in the final emulsions at pH 7.They concluded that the emulsion properties could be controlled with the interactions between stabilisers at different pH and the addition of multiple stabilisers during the emulsification process.Urbánková et al. described a similar finding where O/W emulsions of droplet sizes between 2.4-35.8µm were formulated with 0.3 wt% CNC and NaCas.The properties of PEs were modulated by the nature of the oil phase, together with the addition of stabilisers, where the amount and type of stabilisers are pivotal.In addition, the formation of liquid oil-based gels (oleogels) by CNC/NaCas-stabilised PEs as a template may be used as carriers for bioactive lipophilic substances. [138]nother milk protein, whey protein isolate (WPI), was used as O/W emulsifiers due to its amphiphilic properties. [144]Sarkar et al. presented a hybrid protein particle system consisting of WPI (1 wt%) and CNC (3 wt%) to stabilise O/W PEs.The emulsions stabilised by combined WPI/ CNC systems significantly reduced the rate and degree of lipolysis by providing a steric and electrostatic barrier to the interfacial displacement by bile salts.They also reported that CNC is firmly bound to the protein-coated droplets by hydrogen bonding.The combined WPI/CNC system can bridge several protein-coated droplets in the aqueous phase to reduce the overall surface area available for lipolysis. [136]Besides, Paximada et al. demonstrated that stable PEs containing extra virgin olive oil could be formed using BNC and WPI.BNC acts as a thickening agent through the formation of a fibril network, thus able to sterically stabilise emulsions.BNC was compared to locust bean gum (LGB) and xanthan gum (XG), both of which are commonly used thickeners in the food industry.The authors observed similar shear thinning behaviour of BNC as XG.Still, smaller amounts of BNG were required to achieve the same low shear viscosity, demonstrating BNC as a good thickener for food applications. [135]Liu et al. obtained BSA/CNC complexes by mixing at pH 3 which were then used to stabilise HIPEs with 80% oil loading.Stable HIPEs were formed by adding CNC at a concentration of 0.5% (w/v) with a low amount of BSA (0.01-0.1%, w/v) used as a stabiliser.The resulting HIPEs exhibited high stability against creaming after 30 days of storage.They also reported that the stiffness and microstructure of the HIPEs can be modulated for their food application with by altering the concentrations of BSA and CNC particles.The increased stiffness was attributed mainly to the enhanced droplet packing and interfacial bridging from the coverage of BSA and CNC. [15]

Functionalised nanocellulose with fungal protein
In addition to plant and animal-based proteins, hydrophobin protein (HFB) represents a group of relatively small amphiphilic fungal proteins (10 kDa) that can lower the interfacial tension driven by self-assembly at liquid-liquid interface, forming a monolayer. [139,145]HBF exhibits high surface activity for adhesion and surface modification or as a coating and protective agent. [145]Varjonen et al. combined the bi-functional protein HFB and CNF to form an O/W emulsion stable for several months.The addition of HFB encourages the self-assembly of CNF into tightly packed thin films at the oil-water interface resulting in synergistic improvements in the formation and stability of emulsions.The protein bi-functionality can bind hydrophobic substances to CNF, extending their stability under physiological conditions as a delivery carrier. [140]Paukkonen et al. developed hydrophobin class II (HFBII)/CNF stabilised O/W PEs to encapsulate and release poorly water-soluble model compounds.They found that the addition of CNF as a viscosity modifier further enhances the stability of the emulsion by entangling the oil droplets within the CNF fibre network.The stabilisation of the emulsions can be achieved at low concentrations of HFBII (0.1%) and CNF (0.15%), lower than with conventional surfactants.Furthermore, oxidised CNF with HFBII demonstrates a more controlled release profile, advocating the use of native and oxidised CNF as delivery vectors. [139]any studies revealed the long-term stability of the PEs is highly dependent on the properties of the particulate stabiliser, for instance, particle size, particle concentration, surface charges, wettability, and flexibility.Nanocellulose functionalised either by physical interaction or covalent modification helps define the relative contribution of the particles adsorbed at the oil-water interface and the networkinduced stabilisation of the emulsion.Modified nanocellulose are better stabilisers than pristine nanocellulose as their surface properties can be tailored for higher coverage, protecting droplets against coalescence and better texture with good rheological properties.The proper combination of functionalised nanocellulose with proteins from different sources can be synergistic in forming PEs with enhanced stability and properties, which is critical in designing functional foods for consumer choice and acceptability.

Conclusion
Functionalised nanocellulose is efficient at forming stable Pickering emulsions (PE)s, contrary to pristine nanocellulose.Properties such as excellent biocompatibility, biodegradability, and renewability of nanocelluloses make them attractive stabilisers for developing food-grade PEs.Different forms of nanocellulose, including CNC, CNF, and BNC, have been commonly used as PE stabilisers.Regardless of its structure, nanocellulose obtained from various sources can be successfully applied in constructing food-grade PEs with desired functions.The primary mechanism for Pickering stabilisation is the irreversible adsorption of particles at the liquid-liquid interface, leading to steric hindrance between emulsion droplets and the formation of a three-dimensional network that reduces droplet coalescence.The surface functionalisation of nanocellulose utilising physical interaction and covalent modification enhances the dispersibility of nanocellulose in non-polar solvents, offering better wettability with optimal surface charges to form O/W PEs.A combination of strategic surface modifications, including oxidation, esterification, etherification, bio-inspired conjugation, and polymer/molecules grafting, can modulate the nanocellulose adsorption and stabilisation at the liquid-liquid interface, ideal for various food applications.
Conventional surfactants, which pose health and environmental concerns, can be replaced by green macromolecules.Nanocellulose and protein are natural biopolymers with highly tuneable surface chemical properties can be employed as food-grade PE stabilisers for bioactive delivery applications.In pH-controlled systems, pH-responsive carriers offer excellent potential in oral delivery systems where good stability in the stomach and controlled release in the intestine are achieved.The abundance of hydroxyl groups allows for robust modifications of surface properties of nanocellulose-stabilised PEs, which can be used to promote stimuli-responsiveness.In addition, protein-functionalised cellulose can also be capitalised to introduce targeting abilities for site-specific drug delivery.This is key to expanding the functionalisation of nanocellulose in the preparation of functional foods and targeted delivery systems.Moreover, the droplet interfaces stabilised by layers of protein and nanocellulose particles provide strong steric hindrance to restrict the contact of lipase with the emulsified lipid droplets, thereby protecting the encapsulated bioactive agents against lipolysis.The slower lipid digestion offers a superior-sustained release profile as controlled delivery systems.
Despite many indications of protein-functionalised nanocellulose as effective Pickering stabilisers with enhanced physical properties, further research is required to establish the critical differences between protein functionalised nanocellulose by physical interaction and covalent modification as PE stabiliser.For instance, the stimuli-response behaviour and stability of PEs can vary significantly based on the bonding mechanisms, the types of biopolymer used, the solution composition, and the environmental conditions. [146]Thus, strong experimental models involving both modification methods (physical interaction and covalent conjugation) are required to examine the properties of the resultant PEs.This can be directed by a theoretical model involving Derjaguin−Landau−Verwey −Overbeek (DLVO) interactions such as Van der Waals and electrostatic interactions as well as other forces like hydrogen bonding and hydrophobic interaction, which can result in significant deviations in colloidal stability.These steps are necessary to establish the full potential of the different functionalisation mechanisms of nanocellulose in food and delivery applications.
Another feature worth exploring is the water-in-water (W/W) PEs comprising two immiscible aqueous solutions.Since CNC can stabilise W/W PEs, [147,148] further venturing into protein-stabilised CNC for W/W PE can promote the generation of fat-free food products with sustained release behaviour.Finally, despite the employment of food-grade particles in the preparation of PEs, the actual toxicity and gastrointestinal fate of the nanocellulose stabilised PEs remain unclear.This needs to be validated using in vivo models to unveil the safety and efficacy of the food-grade PEs for the delivery of bioactive compounds market-ready.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Nomenclature γ ow
Oil

Figure 1 .
Figure 1.Schematic diagram exhibiting the particles at the oil-water interface determined by the contact angle, θ.Hydrophilic particles (θ < 90°) and hydrophobic particles (θ > 90°) most likely form O/W and W/O emulsions, respectively.

Figure 3 .
Figure 3.The chemical structure of cellulose is linked by β-1,4 glycosidic bond and the schematic of crystalline and amorphous regions in a typical cellulose fiber.

Figure 5 .
Figure 5. Different functionalisation routes of nanocellulose via physical and covalent surface modification techniques.
of complexes had no layering phenomenon.Droplet size was hardly changed after with ratios of 3:25, 5:25 and 7:25 showed no significant difference in droplet size after 60 days.
with a ratio of 1:4 showed the best stability.They could inhibit lipolysis during gastrointestinal digestion.
with a ratio of 1:1 had a smaller droplet size than emulsions stabilised by CNC and NaCas alone.
the subsequent addition of NaCas, retained a more elastic character.Delayed lipids digestion was observed when 3% CNC was added with WPI-based PE, forming an electrostatic barrier covering the emulsion droplets.
second layer coating on WPI-stabilised PE, giving steric stabilisation to the emulsions.enhanced the fabrication of HIPEs with an oil loading of 80%.The viscoelastic properties of HIPEs were dependent on CNC to BSA ratio and particle concentration.
resulted in synergistic improvement in forming highly stable O/W PE for several months.

Table 1 .
Various functionalisation approaches of nanocellulose with reported particle size and zeta potential.

Table 2 .
Pes stabilised by nanocellulose-based conjugates with different proteins.High stability throughout 30 days storage with no creaming.CNC/SPI with a ratio of 1:1 displayed the smallest droplet size with enhanced emulsifying activity.