The Application of Cellulose Nanocrystals in Pickering Emulsion as the Particle Stabilizer

N.A.Michael Eskin, NI Yang, DUAN Hui, FAN Liu-ping

(1.Department of Food and Nutritional Sciences, University of Manitoba,Winnipeg, Manitoba R3T 2N, Canada; 2.School of Food Science and Technology,Jiangnan University, Wuxi, Jiangsu 214122, China)

Abstract: Pickering emulsions stabilized by solid particles have attracted considerable interest by the food,pharmaceutical, and cosmetic industries.Cellulose nanocrystals (CNCs), with their high aspect ratio,renewability, degradability and biocompatibility, proved to be excellent Pickering stabilizers.This paper will initially review the purification and extraction methods used for targeted cellulose nanocrystals.This will be followed by a discussion of factors influencing the stability of Pickering emulsions containing cellulose nanocrystals.The applications of Pickering emulsions stabilized with cellulose naocrystals will be briefly discussed as well as research ideas for their future uses.

Key words: cellulose nanocrystals; Pickering emulsions; Morphology; surface charge; hydrophobicity

1.INTRODUCTION

Compared to conventional emulsions stabilized by surfactants, Pickering emulsions stabilized by solid particles have attracted considerable interest in the food, pharmaceutical and cosmetic industries due to their outstanding stabilization against coalescence and creaming.This excellent stabilization mechanism is attributed to the accumulation of particles at the oil-water interface to form a densely packed layer.The general principles for solid particles to function as Pickering emulsion stabilizers can be summarized as[1]: i) particles should be partially wetted by both continuous and dispersed phase, yet should not be soluble in either phase; ii) particles should preserve proper partial wettability to gain sufficient interface absorption efficiency; iii) particle size should be substantially smaller than the targeted emulsion droplet size (at least by one order of magnitude).The solid particle stabilizers could be synthetic nano/micro-particles, or bio-based particles from natural resources (such as protein, cellulose, starch,and chitin).However, the inorganic particles such as Sio2and Tio2might be potentially harmful to health,which limited the applications of the resultant Pickering emulsions in the food field[2,3].Therefore,it is of significance to develop food-grade Pickering emulsion stabilizers from natural resources.

Cellulose, the most widely distributed and abundant biopolymer in nature, has captured more research attention in a variety of fields due to its low cost,renewability, sustainability, and biodegradability[4].Cellulose nanocrystals (CNCs), also named as nanocrystalline cellulose or cellulose nanowhiskers,are broadly rod- or needle-shaped particles exhibiting a high crystallinity (CrI) and a nanometer size with high aspect ratio[4,5].CNCs have been widely used in many fields, including biomedicine, nanocomposite materials, and food.Many studies have shown that CNCs are excellent candidates for a food-grade Pickering emulsion stabilizers.In this review,extraction methods for CNCs and factors affecting their emulsification performance and application in food-related research are presented.

2.EXTRACTION OF CNCs

Cellulosic materials are widely found in nature.These plant-derived fiber sources can be divided into six basic types[6], including bast fibers (e.g.jute, flax, and hemp), leaf fibers (e.g.abaca, sisal,and pineapple), seed fibers (e.g.coir, cotton, and ginkgo seed), core fibers (e.g.kenaf, hemp, and jute),grass and reed fibers (e.g.wheat, corn and rice) and all other types (wood and roots).Additionally,bacteria[7](Acetobacterxylinum) and sea animals[8](tunicates) can also be used as sources of cellulosic materials.In fact, the most easily obtained cellulosic biomass sources are mainly agricultural and forestry residues.These cellulosic residues should be considered an optimal sustainable and renewable source of cellulose.

Commonly, cellulosic sources mainly consist of cellulose, lignin, hemicellulose, and other components(e.g.pectins, proteins, and waxes).The presences of non-cellulosic components can reduce the CrI and influence mechanical and emulsifying properties of CNCs[5,9].Therefore, during the production of CNCs,purification of cellulose is a necessary step.Currently,the widely used purification method contains three steps[10,11]: first, the removal of dust and soluble impurities in raw materials using washing, grinding,and/or cooking; second, purifications of cellulose by the removals of hemicellulose and lignin with alkali treatments; third, the bleaching treatments using bleaching agents further removes the residual lignin.In the alkali treatments step, KOH and NaOH are mostly used.Sodium chlorite (NaClO2), sodium hypochlorite (NaClO), and hydrogen peroxide (H2O2)are all commonly bleaching agents which can be used during bleaching treatments.Ni[12]used 4% NaOH and 1.7 % NaClO to purify cellulose from ginkgo seed shells.Winuprasith[13]applied 25 % NaOH and 30 % H2O2to treat fibers from mangsteen.Pelissari[14]used 5 % KOH and 1 % NaClO2to purify cellulose from banana peels.Additionally, more pretreatment steps have also been reported as shown in Table 1.

Table 1 CNCs were obtained using different methods

The purified cellulose usually forms slender cellulose fibrils, alternately composed of crystalline regions and non-crystalline regions[4].These cellulose particles have a large number of hydroxyl groups,showing strong hydrophilicity and poor wettability,incapable of stabilizing Pickering emulsions.Therefore,it is necessary to further fragment the cellulose fibrils into CNCs.Among the different methods for preparing CNCs (Table 1), the acid hydrolysis method is the most well-known and widely used based on the fact that disordered or amorphous regions of cellulose are preferentially hydrolyzed,while crystalline regions are better able to resist acid attack[18,23,24].Sulfuric acid, hydrochloric acid,phosphoric acid and phosphotungstic acid are commonly used for acid hydrolysis treatments[10].However, it is worth noting that, during the acid hydrolysis process, the acid type, concentration,hydrolysis temperature, hydrolysis time, and the source of cellulose all affect the physical and chemical properties of CNCs[4,12,25].Sulfuric acid is most commonly used for preparing CNCs.The surface hydroxyl groups of CNCs react with sulfuric acid yielding negatively charged sulphate ester groups[4].These negatively charged groups provide a certain amount of electrostatic repulsion, which is beneficial for obtaining a stable CNCs suspension.However, too much negative charge can also affect the emulsification performance of CNCs as Pickering stabilizers[26].Since the CNCs obtained by sulfuric acid hydrolysis exhibit low yield, crystallinity, and poor thermal stability[27], hydrochloric acid or phosphoric acid are used as substitutes to prepare CNCs with better thermal stability, higher yield, and crystallinity.Kasiri et al[11]used 3 M hydrochloric acid to produce CNCs from pistachio nut shells.The resulting CNCs were spherical nanoparticles with a diameter of 68.8 ± 20.7 nm and high crystallinity(79.4%).Moreover, the yield of CNCs was as high as 77.1%.When hydrochloric acid steam hydrolysis was used to prepare CNCs[28], the yield was as high as 97.4% with CNCs 100～300 nm in length and 7～8 nm in diameter.Camarero et al.[29]compared the effects of sulfuric acid hydrolysis, hydrochloric acid hydrolysis and phosphoric acid hydrolysis on the properties of cotton CNCs and found that CNCs obtained from phosphoric acid hydrolysis exhibited better thermal stability.

Due to the harsh equipment corrosion and environmental pollution of strong acid hydrolysis,developing eco-friendly process deserves more attention such as the use of recyclable chemicals,including organic acid[30](e.g.oxalic acid, maleic acid, and formic acid), ionic liquid[22], solid acid[31].Besides, enzymatic hydrolysis[21,32], mechanical methods[33-35]can be also used to prepare CNCs.However, it is worth noting that the morphology and properties of CNCs depend on the preparation method and the source of cellulose.For example, in Fig.1 the same wood pulp was treated to prepare nanocellulose using enzymatic hydrolysis, mechanical destruction, and sulfuric acid hydrolysis, respectively.Atomic force microscopy (AFM) was used for morphological characterization.It is clear that the resulting nanocellulose from the different methods showed huge differences in morphology.In addition,the influence of cellulosic sources on the CNCs morphology was shown in Fig.2.Although all CNCs were obtained from sulfuric acid hydrolysis,the obvious variation in morphology of CNCs could be seen among different cellulose sources.

Fig.1 Nanocellulose from wood pulp were obtained from different methods

Fig.2 AFM images of CNCs obtained from various lignocellulosic materials.Asparagus[15]; Ginkgo seed shells[37];Softwood[36]; Garlic straw residues[38]; Pineapple leaf[23]; Bacterial cellulose[36].

3.FACTORS AFFECTING EMULSIFICATION PERFORMANCE OF CNCs

3.1 The influence of CNCs properties

3.1.1 The effect of morphology

CNCs differ from the spherical particles observed for starches and proteins, by being short,rod-shaped or needle-shaped particles with a high aspect ratio.The rod-liked CNCs can connect together and form bridge structures at the interface[39], thus showing a higher stability.Although the CNCs particles show a similar rod-like shape, there are certain differences in the particle length and aspect ratio depending on different sources and different preparation methods (as shown in Fig.1 and Fig.2).Moreover, this difference in morphology could affect the emulsification performance of CNCs.Kalashnikova et al.[40]prepared cellulosic nanorods of various aspect ratios based on three different biological sources, including cotton (CCN, aspect ratio 13),bacterial cellulose (BCN, aspect ratio 47), andCladophora(CLACN, aspect ratio 160).The authors prepared Pickering emulsions using CNCs with different aspect ratios, and found that the droplet sizes of the emulsions were similar, but the aspect ratios directly affected the surface coverage of CNCs on the surface of droplets.As shown in Fig.3A, the short CNC showed a dense coverage ratio(＞80%), while longer CLACN formed emulsions with a low coverage ratio (40%).This difference could be attributed to the fact that CNCs with low aspect ratio can be closely adsorbed at the oil-water interface, while adsorbed long CNCs at the interface caused steric hindrance due to the adjacent absorbed droplets.On the other hand, SEM (Fig.3A) clearly showed entanglement among the adsorbed long CNCs, which led to the formation of the entangled network structure.This network structure is good for enhancing the viscosity and improving the stability of emulsions.Moreover, Ni et al.[12]applied the high-pressure homogenization treatment as a post-treatment after acid hydrolysis to alter morphology and physical properties of CNCs.Their results showed that the short CNCs obtained from 70 MPa had the better emulsifying property.Similar results were reported with asparagus CNCs[15].

3.1.2 The effect of surface charge

The preparation of CNCs using acid hydrolysis,especially sulfuric acid and phosphoric acid, can lead to the introduction of negatively charged groups on the surface of CNCs.Although the negative charge provided electrostatic repulsion to hinder the aggregation of CNCs, the strong electrostatic repulsion also caused the poor performance in stabilizing Pickering emulsions[41].Kalashnikova et al.[42]modulated the surface charge density of CNCs ranging from 0.123～0.019 e/nm2by mild acidic treatment (1～10 h).The results (Fig.3B) showed that the surface charge density of CNCs influences the emulsifying efficiency during the formation of oil/water Pickering emulsions,as cellulose nanocrystals with a surface charge density above 0.03 e/nm2were unable to efficiently stabilize at the oil/water interface.Thus, in many studies on the application of CNCs in preparing Pickering emulsions, the interface adsorption behavior of CNCs is examined, with researchers adding a certain concentration of sodium chloride solution to reduce electrostatic repulsion[41-43].

3.1.3 The effect of CNCs wettability

As the Pickering stabilizer, solid particles could be partially wetted by both continuous and dispersed phase.This property is good for the adsorption of CNCs at the O/W interface.The wettability of particles is generally represented by a three-phase contact angle (θ) located at the oil-particle-water interface[10].The higher contact angle value indicated that the particles have higher hydrophobicity.The amphiphilic character of CNCs is closely related to the hydrophobic (200)β crystalline edge.As shown in Fig.3C, the (200)β crystalline edge directly interacts with the O/W interface,resulting in CNCs adsorption at the interface.However, compared to protein particles, unmodified CNCs showed the relatively low hydrophobicity.Therefore, the improvement of CNCs hydrophobicity has been attracting attention.Recently, some hydrophobic modification strategies of CNCs have been investigated for Pickering emulsions application such as oxidation, esterification and graft polymerization.Chen et al.[45]applied octenyl succinic anhydride(OSA) to modify CNCs, which resulted in an increase of the contact angle from 51.7°(unmodified CNCs) to 85.0° (OSA-modified CNCs).The resulting CNCs successfully prepared the stable O/W Pickering high internal phase emulsion.The same modification method was also conducted by Du Le et al.[46].The modified CNCs had a significant increase in contact angle from 56°to 80°.Additionally, physical methods could be also used to increase CNCs hydrophobicity.For example,Ni et al.[12,44]applied high pressure homogenization and ultrasonic treatment to treat cellulose particles,respectively.The static water contact angles were used to evaluate the particle wettability.Their results showed that high pressure homogenization and ultrasonic treatment both increased CNCs hydrophobicity.This may be attributed to collapse of cellulose structure induced by high-energy mechanical treatments.High-energy mechanical treatments disintegrated long cellulose nanoparticles into short rod-like nanoparticles while destroying partial crystal structure.This process may expose more (200)β crystalline planes, causing higher hydrophobicity of cellulose nanoparticles.Similar results were also reported by Costa et al.[21].

Fig.3 A represents the effect of CNCs aspect ratios on the surface coverage ratio of droplets[40]; B represents the effect of CNCs surface charged density on the stability of emulsions[42]; C represents Schematic representation of cotton CNCs stabilization at the oil/water interface, exposing the hydrophobic edge (200) to the oil phase[42].

3.2 The influence of environmental factors

Due to the charged characteristics of CNCs,change of ionic strength and pH in the system could affect the charge density and interaction of CNCs[47].Bertsch et al.[48]studied the effects of monovalent(Na+) and divalent (Ca2+) cations on CNCs suspensions.The results showed that the addition of cations could change the zeta potential and rheological properties of CNCs suspension.When the ionic strength reached a certain range, CNCs particles aggregated and entangled together and eventually formed the hydrogel.Compared to monovalent cations, divalent cations have a greater influence,and fewer divalent cations can induce the formation of hydrogel in CNCS system.This phenomenon is because cations can cause the electrostatic shielding effect of CNCs, which reduces the degree of electrostatic repulsion between initially charged CNCs particles, allowing more CNCs particles to aggregate, and eventually forming a hydrogel system.This indicates that in the process of emulsion preparation, a high ionic strength in the system is not conducive to the stabilization of CNCs emulsion.Wen et al[49]stabilized D-limonene Pickering emulsion using CNCs to study the effect of ionic strength on the stability of the emulsion.When the salt concentration in the emulsion system increased from 0 to 100 mmol/L NaCl, the absolute potential of the emulsion system decreased from 46.3 mV to 16.7mV, which decreased the stability of the emulsion.On the other hand, many studies have also found that a certain concentration of low ionic strength is conducive to the adsorption of CNCs at the oil-water interface.For example, Bertsch et al.[41,50]studied the adsorption behavior of unmodified CNCs at the oil-water/gas-water interface.They found that the addition of a certain concentration of NaCl solution into the CNCs system caused an electrostatic shielding effect, which is good for the easier adsorption of CNCs at the oil/water interface.

Similarly, the change of pH of the emulsion system can also affect the stability of CNCS emulsion.Mikulcova et al.[51]studied the influence of pH on the stability of Pickering emulsion stabilized by carboxylated CNCs particles.The results showed that the emulsion prepared at pH 4 and 7 had good stability without significant creaming, while the emulsion prepared at pH 2 had complete stratification in both aqueous and emulsion phases.Wen et al.[49]prepared a CNCs-stabilized emulsion.They found that when the pH of the system increased from 4.2 to 7.8, the absolute value of the emulsion potential increased which also improved the stability of the emulsion.In short, low pH or high salt concentration environments are not conducive to stabilizing CNCs Pickering emulsions.

4.FOOD-RELATED RESEARCH OF CNCs STABILIZED PICKERING EMULSIONS

4.1 Delivering bioactive compounds

The bioaccessibility and bioavailability of many fat-soluble active ingredients are commonly reduced due to their poor water solubility, incomplete release from food substrates, and degradation during digestion.A CNCs-stabilized Pickering emulsion can be used as an active substance delivery system to protect and improve the bioaccessibility of the active substance.Asabuwa et al.[52]prepared Pickering emulsion using aminated CNCS particles and successfully encapsulated coumarin and curcumin in the emulsion.The encapsulation efficiency was over than 90%.These emulsions exhibited excellentinvitrocytotoxicity for anticancer and antimicrobial effects with sustained release.Winuprasith et al.[53]encapsulated vitamin D3in Pickering emulsion stabilized by mangosteen shell nanocellulose, and studied its effects on fat digestibility and bioavailability of vitamin D3using a GIT model.The results showed that increased concentration of nanocellulose reduced the digestibility of oil and alleviate the degradation of vitamin D3.Le et al.[54]used OSA modified CNCs as emulsion stabilizer to deliver short-chain fatty acids.They hoped that the CNCS-stabilized emulsions would protect the short-chain fatty acids during intestinal digestion,thus allowing the majority of the short-chain fatty acids to be targeted for release in the colon.Invitrogastrointestinal lipid digestibility and fatty acid release rates of the emulsion were measured.It was found that approximately 65% of short-chain fatty acids were retained afterinvitrointestinal digestion,suggesting that CNCS-stabilized Pickering emulsion had great potential for the colon-targeted delivery of short-chain fatty acids.In contrast to proteins and starches, that can be digested within gastrointestinal conditions, celluloses are resistant to hydrolysis by digestive enzymes within the GIT[55].This feature can help the active substance embedded in the emulsion to be less affected by the stomach environment.In addition, nanocellulose obtained from acid hydrolysis is sensitive to changes in ionic strength and pH[54,56].Based on these properties,CNCs-stabilized emulsions undergo the formation of a gel structure when mixed with the gastric juice in the stomach.The gel structure formed could effectively reduce the degradation of active substances due to many entrapped droplets that were inaccessible to the lipase and GIT environment.

4.2 Improving the oxidation stability

A CNCS-stabilized Pickering emulsion can also be used to improve lipid oxidation stability.Foods with a high lipid content, especially in polyunsaturated fatty acids, is prone to lipid oxidation during storage, resulting in the loss of nutrients and development of off-odors, thus affecting shelf stability and the sensory characteristics of products[10].Wang et al.[57]prepared CNCs and tannic acid composite particles by a simple shear emulsification method,and successfully prepared an avocado oil high internal phase emulsion using composite particles.After conducting accelerated storage experiments,the emulsion showed good oil stability.It means that the composite particles effectively prevented oil oxidation in the emulsion.Angkuratipakorn et al.[58]prepared CNCs and arginine laurate composite particles through electrostatic interaction for the production of a Pickering emulsion.The results show that the composite particles formed by 0.2% CNCs and 0.1% laurate ester c effectively delayed oil oxidation in the emulsion.

5.SUMMARY AND FUTURE PERSPECTIVES

CNCs are widely available and can be used as an ideal Pickering emulsion stabilizer due to its excellent properties, including low cost, amphiphilic nature, renewable, biodegradable, high aspect ratio,and non-toxic.The preparation process of CNCs consists of several key steps, including raw material pretreatment, removal of lignin and hemicellulose,and separation of CNCs by chemical treatment or mechanical destruction.However, it is important to note that both the source of cellulose and the preparation method of CNCs affect the properties of CNCs.The preparation method and conditions should be chosen depending on the ultimate purpose.At present, the traditional preparation methods of CNCs are mainly concentrated on strong acid hydrolysis, high energy machinery and enzymatic methods.These methods have obvious disadvantages,such as the corrosion by strong acids and the resulting waste water, the high energy consumed by mechanical methods, and the long time period associated with enzymatic methods.Therefore, it is extremely important to develop simple and efficient green technology suitable for large-scale production of CNCs.

Many studies have proved that CNCs can be irreversibly adsorbed at the oil-water interface to form the interfacial film.This film could prevent emulsion droplets from aggregation and coalescence.However, the ability of CNCs to stabilize emulsions is affected by their own properties such as morphology,surface charge and hydrophobicity, as well as by pH and ionic strength of the environment.Compared with protein particles, there are few relevant studies on the adsorption behavior of CNCs at the oil-water interface.Moreover, the dynamic adsorption process and interaction between CNCs and other substances(such as proteins, polyphenols, surfactants, inorganic particles, etc.) at the oil-water interface still needs further clarification.CNCs a dietary fiber and its ability to form a stable Pickering emulsion has great potential for applications in food.At present, the application of CNCs emulsion is mainly focused on the delivery system of active substances and improving their stability.However, there are relatively few studies on the application of CNCs emulsions in actual food systems, such as cream, salad dressing,mayonnaise, ice cream.On the other hand, there are few studies on the preparation of responsive Pickering emulsions based on CNCs materials,particularly their application in food systems.Consequently there is an urgent need to explore the potential of CNCs based Pickering emulsions in food technology.

REFERENCE

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