Surfactant-free emulsions stabilized by tempo-oxidized bacterial cellulose

Carbohydrate Polymers 151 (2016) 907–915

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Surfactant-free emulsions stabilized by tempo-oxidized bacterial cellulose Yuanyuan Jia a,b , Xiaoli Zhai b , Wei Fu c , Yang Liu b , Fei Li d , Cheng Zhong a,∗ a

Key Laboratory of Industrial Microbiology, Ministry of Education, Tianjin University of Science and Technology, TEDA, Tianjin 300457, PR China College of Chemical Engineering and Materials Science, Tianjin University of Science & Technology, TEDA, Tianjin 300457, PR China Cargill Food (Tianjin) Co. LTD., 29 Huashan Road, Hangu Modern Industrial Park, TEDA, Tianjin, PR China d College of Food Engineering and Biotechnology, Tianjin University of Science and Technology, TEDA, Tianjin 300457, PR China b c

a r t i c l e

i n f o

Article history: Received 1 May 2016 Received in revised form 24 May 2016 Accepted 27 May 2016 Available online 2 June 2016 Keywords: Pickering emulsion Bacterial cellulose TEMPO oxidation Particle size distribution Wettability Emulsion stability

a b s t r a c t In order to seek a safe, biodegradable, and sustainable solid stabilizer for food, topical and pharmaceutical emulsions, individualized cellulose nanofibers were prepared by oxidizing bacterial cellulose (BC) in a Tempo-mediated system; their ability to stabilize oil/water interface was investigated. Significant amounts of C6 carboxylate groups were selectively formed on each cellulose microfibril surface, so that the hydrophilicity was strengthened, leading to lower contact angles. Meanwhile, both the length and width of fibrils were decreased significantly, by partial cleavage of numerous numbers of inter- and intrafibrillar hydrogen bonds. Tempo-oxidized BC (TOBC) was more effective than BC in stabilizing oil-water interface, attributing to the much smaller size. Fibril dosage and oxidation degree exerted a great influence on the stability and particle size distribution of emulsion samples. When the fibril dosage was 0.7 wt.%, the sample was so stable that it did not experience creaming and coalescence over 8 months. The 2-TOBC coated droplets showed the greatest stability, although both the zeta potential and the electric repulsion were the largest for the 10-TOBC analogue, which was manipulated by the wettability of fibrils. In addition, the stability of samples was analyzed from the viewpoint of particle size distribution. Consequently, fibril size and wettability are two counterbalanced factors influencing the stability of TOBC-stabilized emulsions; a combination of suitable wettability and size imparts TOBC-stabilized emulsion high stability. As a kind of biomass-based particle stabilizer, TOBC showed great potential applications in food, topical and pharmaceutical formulations. © 2016 Elsevier Ltd. All rights reserved.

1. Introduction In the early 20th century, Ramsden (1903) in 1903 and Pickering (1907) in 1907 published that solid colloidal particles could stabilize the interface between two immiscible phases. Afterwards, systematic researches have been carried out by Pickering, thus this type of emulsions has been named as ‘Pickering Emulsions’. In the last twenty years, a concurrent increase in both theoretical and commercial interest has been found in the field of Pickering emulsions, arisen from the concern on health and safety, as well as the development of nanotechnology (Marku et al., 2014). Among the advantages provided by Pickering emulsions, reduction and removal of synthetic surfactants is the most attractive,

∗ Corresponding author. E-mail addresses: [email protected] (Y. Jia), [email protected] (X. Zhai), [email protected] (W. Fu), [email protected] (Y. Liu), [email protected] (F. Li), [email protected] (C. Zhong). http://dx.doi.org/10.1016/j.carbpol.2016.05.099 0144-8617/© 2016 Elsevier Ltd. All rights reserved.

since they are known to cause irritative, toxical and environmental issues (Mekhloufi, Bouyera, Rosilio, Grossiord, & Agnely, 2012). For instance, the increasing demand for clean label excipients calls for replacing synthetic surfactants with natural and degradable biopolymer specifically in pharmaceutical and topical products. Most surfactants elicit irritant reactions when applied to the skin, partially due to their relative ability to solubilize lipid membranes (Maibach & Effendy, 1995). Anionic surfactants cause misfunction possibly by binding to various bioactive components, such as proteins, peptides and DNA, or by inserting into various cell fragments (i.e. phospholipid membranes) (Forgács, Cserháti, & Oros, 2002). Generally, cationic surfactants are more toxic than anionic ones, since anionic surfactants generally exhibit higher IC50 (50% inhibition concentration) values than cationic ones; while the irritation potential of non-ionic surfactants is considered the lowest (Maibach & Effendy, 1995; Touraud, Vlachy, Heilmann, & Kunz, 2009).

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Biomass-based particles with partial dual wettability have been isolated to stabilize emulsions, such as proteins (soy protein (Tang & Liu, 2013), keratin protein (Nonomura & Hikima, 2011)) and polysaccharides (starch (Li, Li, Sun, & Yang, 2014a; Li et al., 2014c; Xu et al., 2014), cellulose (Bizot, Kalashnikova, Bertoncini, Cathala, & Capron, 2013; Bizot, Kalashnikova, Cathala, & Capron, 2011; Bizot, Kalashnikova, Cathala, & Capron, 2012; Cathala, Tasset, Bizot, & Capron, 2014; Cathala and Capron, 2013a), and chitin (Moschakis, Tzoumaki, Kiosseoglou, & Biliaderis, 2011)). Microcrystalline cellulose was used to stabilize O/W emulsions (Lee, Blaker, Li, Menner, & Bismarck, 2009; Stenius & Andresen, 2007), while hydrophobic modification to cellulose was adopted to stabilize W/O type emulsions (Kim, Wege, Paunov,; Zhong, & Velev, 2008Lee et al., 2009; Stenius & Andresen, 2007;). Cellulose whiskers are capable of forming ultra- stable emulsions (Bizot et al., 2013; Bizot et al., 2011; Cathala & Capron, 2013b). However, the manufacturing process of acid-hydrolyzed cellulose whiskers is relatively complex and time consuming.It also suffers from harsh condition and low yield. TEMPO (2, 2, 6, 6-tetramethylpiperidine-1-oxyl radical) and its analogues are stable nitroxyl radicals, being negative to the Ames test (Lee et al., 2009). TEMPO-mediated oxidation reactions are capable of conversing primary hydroxyls of celluose to carboxylate groups under mild conditions. It is a novel method to prepare individualized cellulose nanofibers. The nanofibrils within the fibres can be released since a part of hydrogen bonds holding the nanofibrils together are damaged (Durfresne et al., 2010). The high stability of Pickering-type emulsions arises from the irreversible adsorption of the particles at the oil–water interface, creating a physical barrier and preventing contact between droplets. Both particle size and wettability determine the stability of Pickering emulsions. The energy detaching a sphere from the interface is related to its radius and oil-water-particle contact angle:



2

G = R2 OW 1 − |cos OW |

(1)

where, G is the detach energy, J; R is the radius of spherical particles, m;  OW is the oil-water interface tension, N/m;  OW is the water-oil-particle contact angle ◦ . The oil-water-particle contact angle is calculated according to the following equation: cosOW =

␥ ␥W cos␪W − O cos␪O ␥OW ␥OW

(2)

where,  W and  O are surface tensions of water and oil, respectively, mN/m.  O is the contact angle of particle on the air/oil interface and  w is the contact angle of particle on the air/water interface. According to Eq. (1), for an oil-water interface intension  OW of 36 mN/m, if the particle radius R is 1000 nm with the contact angle 20◦ , the detach energy reaches as high as 105 kT; while for the same particle, when the detach energy reaches 103 kT, the contact angle is only 6◦ . At the same detach energy, for a 10 nm particle, its contact angle has to be 67◦ and for a 100 nm particle, the contact angle needs to be 20◦ . Therefore, small size or a three phase contact angle approaching 90◦ not always leads to strong stability. In comparison, the detachment energy of traditional surfactant is several kT only (Clint, Aveyard, & Horozov, 2003). So that, particles adsorbing irreversibly at fluid–fluid interface is the origin of high stability offered by Pickering emulsions. However, many biomass-based particles are not spherical, which makes the situation complex. For example, cellulose whiskers are nano-rods derived from cellulose with aspect ratio ranging from 13 to 160 (Bizot et al., 2013). Capillary interactions between anisotropic particles play an important role to stabilize interface (Lewandowski, Botto, Jr, & Stebe, 2012).

Bacterial cellulose is synthesized by some microorganisms, such as Glucose Acetobacter. It is manufactured into traditional food ‘nata de coco’ or dessert, consumed widely in Southeast Asia. It is an origin of dietary fiber, which is the seventh nutrient after proteins, lipids, hydrocarbons, vitamins, minerals, and water. In this paper, bacterial cellulose was modified by introducing carboxyl groups in a TEMPO/NaBr/NaClO mixed system. At the same time, the fibril size was reduced significantly. Although the hydrophilicity was strengthened, the moderately oxidized bacterial cellulose (TOBC) was capable of stabilizing oil-water interface, more efficiently than bacterial cellulose; size and wettability were balanced to get excellent effect. The interfacial stabilizing effect of microfibrils is also attributed to an interconnected network (Bizot et al., 2013); the viscoelastic characteristics of TOBC-stabilized emulsions have been investigated and will be published later. Prospect of using Tempooxidized cellulose to stabilize food, topical and pharmaceutical emulsions are forseeable, thanks to its safety, biodegradability, and renewability. 2. Experimental 2.1. Materials Gluconacetobacter xylinus (CGMCC NO.2955) used in this study, as described previously (Li et al., 2014b; Zhang et al., 2013), was screened from a traditional Chinese drink by our group and stored in China General Microbiological Culture Collection Center with the registered number, No. 2955. All other reagents were analytical grade. Deionized water was used for all tests. 2.2. Preparation and characterization of bacterial cellulose pellicles G. xylinus was activated in the medium at 30 ◦ C for 24 h; the medium contains: 25 g/l anhydrous dextrose, 10 g/l yeast powder, 7.5 g/l peptone and 10 g/l disodium hydrogen phosphate, and the pH was adjusted to 5.0 by acetic acid. Then G. xylinus suspension was inoculated at 5% (v/v) in the same medium. BC pellicles were formed after 5–8 days. The harvested gel-like membranes were purified by washing with deionized water, and then by boiling in 0.1 M NaOH to remove remaining medium and cells. Finally, they were washed until they were pH neutral. BC pellicles were dispersed preliminarily by a disintegrator (T-100, Adirondack Machine Corporation, America) and then homogenized using a high pressure homogenizer (NS1001 L Panda, GEA Process Engineering Inc.) for 10 times at 100 MPa to obtain a nano-scale dispersion 2.3. Preparation and characterization of TEMPO-oxidized bacterial cellulose(TOBC) BC was suspended in water (100 ml/g dry BC) containing TEMPO (0.1 mmol/ g dry BC) and sodium bromide (1 mmol/g dry BC). The oxidation reaction was started by adding an amount of 10% NaClO under continued stirring at ambient temperature. The pH was maintained at 10 ± 0.1 by adding 0.1 M NaOH. When the pH did not drop any more, 10 ml ethanol was added to quench the reaction. The reaction product was centrifuged at 10000 rpm (TGL–16 M, Hunan Instrument, China) for 10 min. The supernatant was removed, and the precipitate was cleaned repeatedly. The oxidized product TOBC was dialyzed against deionized water to remove ions. 1% AgNO3 was used to detect Cl− in the dialysate. The carboxyl content of TOBC samples was assayed by electric conductivity titration method similar to Arkai (Wada, Araki, & Kuga, 2001) with a slight modification as follows: 150 ml of TOBC (0.1 wt.%) or BC (0.1 wt.%) as control was mixed with 10 ml of 0.05 M NaCl. The pH was maintained at 3 by the addition of 0.1 M HCl.

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with an oil/water ratio of 1:1. Equal volumes of liquid paraffin and TOBC were sonicated with a high intensity ultrasonic vibracell processor (VCX150, Sonics & Materials Inc., America) at a power of 40 W for 5 min with 50% ultrasonic pulse. Emulsions were prepared under pH = 7. The emulsion stability was expressed by emulsion stability index (ESI), which was calculated as Eq. (4):





ESI (%) = HE /HT × 100

Fig. 1. Titration curve of 10-TOBC sample.

Sodium hydroxide with a concentration of 0.05 M was added by titration. The conductivity versus the volume of NaOH was plotted to give the titration curve. The carboxyl content was calculated according to Eq. (3):





Carboxylcontent mmol/gcellulose =

(V1 − V2 ) × c1 m×ω

(3)

where, V1 , volume consumption of NaOH in point B, ml; V2 , volume consumption of NaOH in point A, ml; c1 , the molar concentration of NaOH, mol/l; m, the weight of 150 ml TOBC (0.1 wt.%) or 150 ml BC (0.1 wt.%); ω, fibril dosage (0.1 wt.%). The oxidized products were named as 10-TOBC, 6-TOBC, and 2TOBC, corresponding to NaClO dosage 10 mmol/g, 6 mmol/g, and 2 mmol/g, respectively. Take the titration curve of 10-TOBC as example (Fig. 1); from the curve, V2 , corresponding to the amount of added HCl, and V1 corresponding to the total amount of HCl and carboxylic acid can be read. Therefore the carboxyl content of 10-TOBC was 1.15 mmol/g from Eq. (3). The TOBC suspension was adjusted to pH 3.0 with 0.5 M HCl and left dry at ambient temperature. Fourier transform Infrared (FTIR) spectrometer (Vector 22, Brooke, Germany) under transmission mode from 400 to 4000 cm−1 with a resolution of 4 cm−1and 16 scans was used to record these films by the KBr press. Contact angles of BC and TOBC samples were assayed by a dynamic contact angle measuring device (PGX 3.4a, Fibro System AB, Swiss) equipped with the PG software. Dried, flat and uniform BC and TOBC sheets were required and the volume of water droplet was 4 ␮l. The pictures were captured automatically; all the measurements were performed at least in duplicate. A scanning electron microscope (SU-1510, Hitachi, Japan) was used at a high voltage (20 kV) and a magnification of 10000 × to capture the size and morphology of freeze-dried BC and TOBC. The samples were coated with gold by the ion sputter instrument (SBC-12, KYKY, China). The images were processed by ImageJ 1.45 s software (National Institutes of Health Corporation, America). The morphology of air-dried BC and TOBC membranes was observed with an atomic force microscope (JSPM-5200, Japanese Electronics Corp.). Ambient atmosphere measurements were made in tapping mode. The BC and TOBC dispersions were diluted with water to 0.1 wt.% solid content, and the sizes of the dispersions were measured by a Laser-Doppler-Electrophoresis size analyzer (DelsaTM Nano C, Beckman Coulter, America). However, the size of BC exceeded the range of the instrument. 2.4. Preparation and characterization of pickering emulsions The Pickering emulsions were prepared using liquid paraffin and BC or TOBC dispersion at different fibril dosages (0.18–0.70 wt.%),

(4)

where, HE , the height of the emulsified layer; HT , the total height of the emulsion. Class Zetasizer Nano (90PLUS/BI, Brookhaven, America) was used to measure the Zeta potential of TOBC-stabilized emulsion samples. Immediately prior to measurements, the samples of 200 ␮l were diluted 20 times and dispersed evenly by an ultrasonic processor. The amount of samples tested was 1.5 ml. Each sample was performed in triplicate. The charge density was tested by a Particle charge detector (PCD-T3, BTG, United Kingdom). 200 ␮l emulsion was poured in a cylinder sample cup and dispersed by driving the piston for 3 min. The polydiallyl dimethyl ammonium chloride (PolyDADMAC) cation ion standard solution was dropped in the sample until the surface potential became zero, then the titration was automatically stopped. The consumption of Poly-DADMAC standard solution was recorded as V, which was calculated as following (5):





Chargedensity mmol/g =

V × c2 m1

(5)

Where, V, volume consumption of Poly-DADMAC cation standard solution, mlc2 , the molar concentration of Poly −DADMAC cation standard solution, mol/lm1 , the absolutely dry weight of the sample, g The particle size distribution (PSD) of the emulsions was measured by Laser diffraction particle size analyzer (LS 13–320, Beckman Coulter, America). A drop of the sample was diluted to 2 ml and dispersed evenly. A sample was added slowly until the sheltering coefficient reached 9–12%, then the testing was set out. Each sample was measured in triplicate. According to Mie theory, the particle size measurements were reported as areaweighted mean diameter D (3,2) and volume-weighted diameter D (4,3) , respectively, and calculated as follows (Ibarz, Augusto, & Cristianini, 2012): D(3,2) =

 n × d3  i i2

(6)

D(4,3) =

 n × d4  i i3

(7)

ni × di ni × di

where, ni is the number of the particles with the diameter di . 3. Results and discussion 3.1. Carboxylate content, wettability and fibril size of BC and TOBC samples TEMPO and its derivatives are water-soluble and stable compounds with nitroxyl radicals. Catalytic oxidation by TEMPO opens a new field that alcoholic hydroxyls are efficiently and selectively converted into aldehydes, ketones and carboxyls under a mild condition. C6 primary hydroxyl groups on the cellulose Dglucose units can be selectively oxidized into carboxylate groups by TEMPO/NaBr/NaClO mixed system. Slurries of regenerated, mercerized fibrils and the like were oxidized by TEMPO. Almost all of the C6 primary hydroxyl groups were oxidized to water-soluble carboxylate groups (Kato & Isogai, 1998). However, for native cellulose such as cotton linters, hardwood or softwood bleached Kraft

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Fig. 2. Relationship between the dosage of NaClO in the TEMPO/NaBr/NaClO oxidation system and the carboxylate contents of TOBC.

Table 1 Static contact angles and sizes of BC and TOBC samples. Samples

BC

2-TOBC

6-TOBC

10-TOBC

Contact angel(◦ ) Size(nm)

56.6 ± 2.75 >20,000a

42.7 ± 1.35 4580.4 ± 220

41.9 ± 2.05 3173.0 ± 150

<5 2993 ± 140

a BC length was beyond the detect limit of particle sizer. 20 ␮m were estimated from optical graph of BC (data not shown).

Fig. 3. SEM images of TEMPO-oxidized BC with different oxidation degrees: (a) 2TOBC, (b) 6-TOBC, (c) 10-TOBC, and (d) BC.

In a word, after oxidized reaction, the hydrophilicity of fibrils was strengthened and their size was cut down. 3.2. The morphologies of BC and TOBC nanostructure

pulps and bacterial cellulose, the oxidation process was slowed down because of their high degree of crystallinity. Thus, almost no or only a small amount of water-soluble products were obtained even under harsh oxidation conditions or with extended reaction times (Kato & Isogai, 1998; Yanagisawa, Isogai, & Saito, 2005). Trace carboxyl (about 0.10 mmol/g) was found in BC suspension, even after full dialyzation, since acetic acid was the by-product of biosynthesis. Fig. 2 exhibits the relationship between dosages of NaClO and carboxyl contents of TOBC samples. When the amount of NaClO varied from 2 to 10 mmol/g, carboxyl content of TOBC increased from 0.58 to 1.15 mmol/g. Under the same condition, hardwood bleached Kraft pulp oxidized by TEMPO had a higher carboxyl group content (1.50 mmol/g) (Satio, Kimura, & Nishiyama, 2007). Since BC has a relatively higher crystallinity (namely, less amorphous regions), in comparison with other native celluloses, it is difficult for oxidized TEMPO molecules or nitrosonium ions to penetrate into crystallized regions. Therefore, only the fibrils exposed on the surface can be oxidized. However the disordered amorphous region among the fibrils may be easily attacked by the oxidant (Saito, Isogai, & Fukuzumi, 2011). The wettability of BC and TOBC samples was evaluated by contact angles. As shown in Table 1, static contact angles of water droplet on the interface of air and TOBC sheets, represented by  W , were much smaller than that of BC sheets; furthermore, the contact angles were declined with increasing carboxylate contents of TOBC samples due to higher hydrophilicity, resulted from higher polarity of carboxylate groups in contrast with hydroxyl groups. Besides contact angles, Table 1 indicates that the size of TOBC was significantly cut down, compared with original BC, and TOBC size decreased according to carboxylate content increasing. Noticeably, it has to be pointed out that the model of particle size meter is based on ideally spherical particles. Therefore, the results are less accurate with high-aspect-ratio particles. It, however, is sufficient to distinguish significant dimension differences of BC and TOBCs. Fibril size reduction might be attributed to the cleavage of glycoside bonds by hydroxyl radicals formed in situ, so called ␤-elimination reaction (Hirota et al., 2009). At the same time, depolymerization occurred (data not shown) (Hirota et al., 2009).

Surface morphology of BC and TOBCs was characterized by SEM, shown in Fig. 3. It is seen from Fig. 3 that, BC and TOBC nanofibrils packed irregularly to form a dense porous network. The fibril size was gained from Fig. 3 by the image analysis software ImageJ 1.45s. Compared with BC, TOBC showed a much smaller size. Coarse fibril bundles were found everywhere in BC network, with a width around 100–250 nm. While for TOBC, individualized nanofibrils existed, with the width of 5–10 nm; the width of TOBC bundles was around 50 nm, which was consisted of nanofibrils. After oxidization, partial amorphous regions among the fibrils were dissolved, so that fibril bundles were separated into free nanofibrils (Saito et al., 2011). However, it was difficult to obtain reliable data about fibril length from the SEM images, since fibrils were entangled with each other. Either BC or TOBCs had a large aspect ratio and tended to bend. However, arisen from the chemical nature, the Tempooxidized hardwood fibril had a width of 3–4 nm and a length of a few microns, and a transparent and highly viscous dispersion was formed, probably due to the higher accessibility to oxidant molecules (Saito et al., 2011). From Fig. 3, the unique ultra-fine three dimensional network structure of bacterial cellulose was maintained even after TEMPO treatments, which was perfectly agreed with the work of Satio et al. (2007). Meanwhile, micro fibrils of TOBC in Fig. 4(a) and (b) were ‘cleaved’ after oxidative reaction, indicated by the arrows, which indicates that a part of inter-fibrillar hydrogen bonds were damaged, resulting in much smaller fibril and fibril bundle width [33]. 3.3. FT-IR spectra of the TOBC-COOH and BC Fig. 5 presents the FT-IR spectra of BC and TOBC. TOBC-COOH sample was obtained by adjusting the pH of cellulose-COONa sample to 3. The BC spectroscopy in Fig. 5 shows that the absorption band at about 1420 cm−1 is attributed to the symmetric bending of CH2 , which is in accordance with Barud et al. (2008). The band at 1060 cm−1 is due to C O symmetric stretching of primary alcohol; and, the band at 1163 cm−1 corresponds to C O C asymmetric

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Fig. 4. AFM graph of (a) 2-TOBC and (b) zoom-in of (a).

Fig. 6. ESIs of emulsions stabilized by BC fibrils with different oxidation degrees against storage time. The fibril dosage was 0.35 wt.%, and the ratio of oil and water was 1: 1 for all samples. Emulsion picture from left to right: BC, 2-TOBC, 6-TOBC, and 10-TOBC stabilized emulsions.

Fig. 5. FT-IR spectra of the TOBC-COOH and BC samples.

stretching. The peak at 1738 cm−1 is attributed to −COOH stretching vibration (Okita, Fujisawa, Fukuzumi, Saito, & Isogai, 2011). Small molecules and ions, including acetic acid were removed after sufficient dialysis; the band at 1720 cm−1 is subjected to C O stretching vibration of carboxyls due to the chemical shift, since free carboxyl groups without hydrogen bonds exhibit C O peak at 1740 cm−1 (Duevel & Corn, 1992; Nishiyama, Saito, Putaux, Vignon, & Isogai, 2006; Creager & Steiger, 1995). Thus intra-nanofibril or inter-nanofibril hydrogen bonds most probably exist among carboxyl groups or between carboxyl groups and hydroxyl groups. 3.4. Relationship between the emulsion ESIs and fibril oxidation degree, zeta potential and charge density TOBC oxidation degree directly affects the wettability and size of fibrils, thereby the stability of Pickering emulsions. The emulsion stability was expressed by emulsion stability index (ESI), which was defined by the fraction of emulsified volume to the total amount mixed initially. ESIs of emulsions stabilized by BC, 2-TOBC, 6-TOBC, and 10-TOBC during storage time are shown in Fig. 6. At the same fibril dosage (0.35 wt.%), the ESIs of emulsions were ranked as: 2-TOBC > 6-TOBC > BC > 10-TOBC on the 30th day. Additionally, 2-TOBC and 6-TOBC stabilized samples did not experience creaming after storing for 8 months at ambient temperature. From the photos of 30-day emulsions, the sample stabilized by BC showed grainy appearance and coagulation among droplets formed from eyes’ inspection, while the TOBC-stabilized sample exhibited uniform status with fine droplets. Fig. 7 shows the effects of the carboxylate content of TOBCs on Zeta potential and charge density of TOBC dispersions, in Fig. 7a, and Zeta potential, charge density, and ESI of emulsion samples, in Fig. 7b. Absolute values of the Zeta potential and charge density of samples were positively correlated. The absolute values of Zeta potential and charge density of TOBC dispersions increased

Fig. 7. Effects of carboxylate content of TOBCs on a, Zeta potential and charge density of TOBC dispersions, and b, Zeta potential, charge density and ESI of emulsions (The fibril dosage was 0.35 wt.% and ratio of water to oil was 1:1).

with increasing carboxyl contents. However, in Fig. 7b, when the carboxyl group content increased from 0.1 mmol/g (BC) to 0.58 mmol/g (2-TOBC), the absolute value of Zeta potential and charge density of the emulsion samples increased and achieved the maximum at 0.58 mmol/g (2-TOBC); then the absolute value of Zeta potential and charge density of the samples improved with rising carboxylate content. Since a majority of 2-TOBC nanofibrils in the emulsion sample anchored on the oil–water interface, some of the carboxyl and hydroxyl groups were hardly be exposed, leading to lower Zeta potential. When the carboxylate content increased from 0.58 mmol/g (2-TOBC) to 1.15 mmol/g (10-TOBC), the absolute Zeta potential and charge density got raised, but the emulsion stability got dropped. On the one hand, electrostatic repulsion

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Fig. 8. Relationships between ESI and fibril dosage: emulsifier BC and 2-TOBC in (a) and (b), respectively. Inset photos show the appearance of samples after 30- days-standing with different fibril dosage (from left to right), 0.18, 0.35, and 0.70 wt.%, respectively. The volume ratio of water phase and oil phase was 1:1. The fibril dosage was indicated by the weight percentage ratio of water phase.

and 6-TOBC were more effective emulsifiers than BC, while the more hydrophilic 10-TOBC was less effective. 3.5. Relationships between ESI of emulsions and fibril dosage

Fig. 9. Associations between droplet diameter (a) D(3,2) , (b) D(4,3) and carboxylate content. The dosage of TOBC was 0.35 wt.%, and the oil/water ratio was 1: 1.

among the emulsion droplets could be enhanced by Zeta potential; it was favorable for stabilizing emulsion to some extent. On the other hand, high hydrophilicity of the particles lowered the detach energy of TOBCs from the interface, resulting in unstable emulsions. For droplets coated by 10-TOBC fibrils, the negative impact raised from strong hydrophilicity dominated. In summary, 2-TOBC was the most effective particle stabilizer, verified by Fig. 7b. BC fibrils’ coating the interface of styrene and water was visualized by SEM in our previous study (Jia et al., 2013). The position of particles embedded on the interface depends on their wettability, often presented by a three-phase contact angle  ow . It is necessary to point out that  ow is different from the contact angle  w in Table 1. The relation is expressed by Eq. (2). For hydrophilic particles, e.g. starch,  ow is typically less than 90◦ and a large fraction of the particles resides in water phase rather than in oil phase. They can stabilize O/W emulsions. For hydrophobic particles, e.g. the hydrophobically modified silica,  ow is generally greater than 90◦ and the particles reside mostly in oil phase. BC is hydrophilic thanks to large amount of hydroxyl groups; and TOBC is more hydrophilic due to the introduction of carboxyl groups, which may have negative influence on the emulsion stability when the hydrophilicity is too high. Meanwhile, it was mentioned above that the size of TOBCs was reduced significantly during oxidation treatment (data shown in Table 1), which may have positive influence on the emulsion stability due to lower detachment energy. Fibril size and wettability are, therefore, two conditionally counterbalanced factors, which also well interprets the reason that 2-TOBC

Associations between ESI of emulsions and fibril dosage (0.18 wt.%, 0.35 wt.%, 0.70 wt.%) are shown in Fig. 8. For all samples, stratification occurred upon 5 min’s preparation; then they tended to be stable in 24 h. Watanabe, Ougiya, Matsumura, & Yoshinaga (1998) and Li, Li, Sun, & Yang (2013) also reported that stratification occurred for freshly prepared Pickering emulsions stabilized by BC and starch granules, which might be attributed to relatively large size droplets. Both Fig. 8 (a) and (b) present that stratification did not occur to the sample stabilized by 0.7 wt.% TOBC in 30 days, with 100% ESI, while the ESI of the sample stabilized by BC was 98.1% under the same conditions. Although the ESIs of two samples were close, their droplet diameters showed different ranges, which will be further discussed in 3.7. When the fibril dosage was 0.35 wt.%, ESI of the emulsion stabilized by TOBC were much higher than that of the BC-stabilized emulsion. When the fibril dosage was 0.18 wt.%, all of the samples became unstable after just 1 day. For both TOBC and BC stabilized emulsions, their stability was significantly enhanced with increasing fibril dosage, which can be interpreted as following two points: (1) monolayer or multilayer films constituted by fibrils adsorbed on the oil-water interface formed physical barriers; (2) a network structure was created by entangled fibrils. The droplets were embedded in its threedimensional array. Both physical barrier and 3D fibril network effectively and efficiently prevented the emulsion droplets from contacting each other, thereby achieved superior emulsion stability. The stability of emulsions stabilized by TOBC were higher than that of BC at both 0.35 and 0.7 wt.% fibril dosage, which likely depends on suitable size and wettability of solid particles. Binks (2002) have reported the relationship between the energy of detachment and the radius of particles with certain wettability and interfacial tension. Tiny particles, whose radius are close to the size of surfactant molecules (namely, less than 0.5 nm), are easily detached and may not act as stabilizers; similarly, particle size beyond some threshold is disadvantageous to stabilize Pickering emulsion. In our study, BC fibril length and width were cut down significantly by oxidative reaction, which resulted in more fibrils covering the surface of droplets to form rigid barrier. Furthermore, it has been visualized that fibrils bended and accommodated the

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curvature of the droplets in our previous study (Jia et al., 2013). The high water content, 99%, endowed bending stiffness to BC and TOBC fibrils. After oxidization, it became easier to bend and cover the droplets, since the width of fibrils was reduced noticeably (shown in Fig. 3 and Fig. 4). When the fibril dosage was low (0.18 wt.%), we assume that the small amount of fibril was not sufficient to cover the oil/water interface, so the samples stabilized by TOBC or BC exhibited unstable. 3.6. Associations between droplet diameter (droplet size distribution) and carboxylate content Phenomena like Ostwald ripening, creaming, aggregation, coalescence or partial coalescence might take place during the destabilization of the emulsions (Walstra, Fredrik, & Dewettinck, 2010). Apart from visualized phase separation, change in size distribution of emulsion droplets occurred also. Therefore, the evolution of particle size distribution is helpful to comprehend the instability mechanisms. By recording the droplet size distribution of samples stabilized by 2-TOBC, 6-TOBC, and 10-TOBC standing at 1, 7, and 30 days, the relevance between droplet diameter and the carboxylate content of fibrils was investigated. The droplet size of BC stabilized emulsion samples could not be measured by the sizer, since the sheltering coefficient was too small, probably due to the relatively large size of BC-coated droplets, or possible creaming during testing. Fig. 9 shows the relationship between droplet diameter and carboxylate content of BC. At the same fibril dosage, 0.35 wt.%, the droplet sizes D(3,2) were different; at each observation point, both the D(3,2) and D(4,3) of 2-TOBC coated droplets were the largest, followed by the 6-TOBC sample, and then the 10-TOBC sample, which can be explored from two facets. The fibril size decline occurred during the Tempo-mediated oxidation process, and it dropped progressively with oxidation degree, as shown in Table 1; so that a larger oil/water interface was covered by the fibrils and fibril flocs, resulting in smaller droplets. This might explain the sizes of the first day, when little creaming happened. Secondly, when the carboxyl content varied from 0.58 to 1.15 mmol/g, the stabilities of 2-, 6-, 10-TOBC samples went down successively (from Fig. 6). Serious creaming was observed in 10-TOBC sample after standing for 30 days; only small droplets were persisted. Carboxylation endows cellulose with more hydrophilic carboxyl groups, accompanied by distinct fibril size reduction, both fibril width and length. For 2-TOBC, the hydrophilicity changed little, but the fibril size dropped significantly (shown in Table 1) during the TEMPO oxidation process. However, when the carboxyl group content went higher, the dissociation occurred easily at the oilwater interface, due to the rigid hydrophilicity, resulting in droplet coagulation and creaming; at the same time, smaller droplets were preserved.After 30-day standing, stratification did not occur in 2TOBC emulsion sample, and the D(3,2) remained almost constant; however, the D(4,3) slightly increased from 7 to 30 days, which indicated that limited amount of large droplets formed in this period, resulted from slight coalescence, so we conclude that 2-TOBC fibrils can prevent droplets from coalescing. The D(3,2) of 6-TOBC sample increased progressively, and the D(4,3) increased rapidly within 30 days, which indicated that slow coagulation happened inside the 6-TOBC sample from the initial period, and the 6-TOBC fibrils only can prevent coalescence to a certain degree. The 10-TOBC fibrils were not capable of effectively preventing droplet coalescence, and then resulting in serious creaming. It can be verified from Fig. 9, both the D(3,2) and D(4,3) got rising from the 1st day to the 7th day, and then got dropping from the 7th day to 30th day; as mentioned earlier, the D(4,3) varied a much wider range. The distribution pattern is related closely to the emulsion stability. It is shown in Fig. 10 that the three samples showed unimodal

Fig. 10. Particle size distributions of emulsions stabilized by (a) 2-TOBC, (b) 6-TOBC, and (c) 10-TOBC respectively at different periods. The fibril dosage was 0.35 wt.% and the oil: water was 1:1.

distribution on the first day upon preparation. On the 7th day, 10TOBC sample experienced obvious layering, so a bimodal pattern appeared. While 2-TOBC and 6-TOBC emulsion maintained their original unimodal distribution, shown in Fig. 10a and b. On the 30th day, two peaks appeared on the distribution curve of 6-TOBC emulsion; the peak at 150 ␮m represented the coagulated droplets. However, for the 10-TOBC sample, serious layering happened; only

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Y. Jia et al. / Carbohydrate Polymers 151 (2016) 907–915 Table 2 Droplet diameter of emulsions stabilized by BC fibrils with different fibril dosage and storage time. Fibril dosage (wt.%)

0.18 0.35 0.70

Fig. 11. Droplet diameters of emulsions stabilized by 2-TOBC with different fibril dosages and storage time (The ratio of oil and water was 1:1).

small particles were remained, so that the distribution curve came back to a single peak. 3.7. Associations between droplet diameter and fibril dosage Relationship between droplet diameter D(3,2) and D(4,3) stabilized by 2-TOBC with fibril dosage of 0.18 wt.%, 0.35 wt.%, 0.7 wt.% is shown in Fig. 11.The D(3,2) s of the fresh emulsion samples were bigger than that of the samples stored for 1, 7, and 30 days, and from Fig. 11a, the D(3,2) diameters of the latter three (1, 7, and 30 d) were close to each other. The D(3,2) diameters of the emulsions varies from 2 to 8 ␮m, in terms of fibril dosage increase. Theoretically, increasing fibril dosage leads to smaller droplets diameter. However, the size of the fibril aggregates, formed by hydrogen bonds, was larger than that of the droplets, so that the tested droplet size was larger than theoretical values due to fibril aggregates resulted from hydrogen bonds. Balejko, Bortnowska, Tokarczyk, Romanowska-Osuch, &

Droplet diameter of emulsions,D(3,2) /D(4,3) (␮m) 0d

7d

30 d

60.75/119.20 57.36/111.80 70.13/106.20

24.93/140.70 22.08/131.70 32.32/201.70

/ / /

´ Krzeminska (2014), Kadkhodaee and Koocheki (2011), Zaritzky, Lorenzo, & Califano (2008), Xu, Liu, & Guo (2007) also reported highly similar results. With prolonged storage time, Pickering emulsions may experience destabilization, which is mainly caused by the droplet coalescence. Coalescence begins among the big droplets, which are not fully covered by nanofibrils, and smaller droplets will be saved. When coalescence reaches to a certain extent, there will be delamination of the oil and aqueous phase, until that the interface is sufficiently stabilized by the fibrils irreversibly adsorbed at the interface. So far, the instability trend is slowed down and the stability can be maintained for a long period. The D(3,2) is sensitive to small particles, while D(4,3) is susceptible to large particles. The variation of the D(4,3) is larger from Fig. 11b, compared with Fig. 11a, since the coalescence phenomenon occurred easily in large droplets. For the sample stabilized by 0.35 wt.% TOBC, after 30 days’ storage, the ESI was 95.5%, and the variation of D(4,3) was 6.1 ␮m, while the variation of D(3,2) was only 1.2 ␮m, compared to the size of the fresh sample. Therefore, the instability process can be further verified by the variation trend of D(4,3) . Table 2 shows D(3,2) and D(4,3) of BC emulsions with different fibril dosage at 0 and 7 days. Data at the 30th day was non-detectable, since the samples standing for 30 days experienced obvious cream-

Fig. 12. Particle size distribution of emulsions stabilized by 2-TOBC with different fibril dosage and storage time: (a) 0 day, (b) 1 day, (c) 7 days, (d) 30 days.

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ing. As the control, BC emulsion had rough appearance, and the particle size was significantly larger than that of 2-TOBC emulsion sample. Emulsification effect was, therefore, markedly improved by carboxylate modification to bacterial cellulose. When the amount of the fibril was increased, a dense cellulose film formed at the interface; the obtained compact fibrous network made the resistance of coalescence increase. It is shown in Fig. 12 that, for samples with dosage 0.35 wt.% and 0.7 wt.%, the particle size distribution hardly changed within 0–7 days upon preparation, which was the period that coalescence occurred most; and it changed little within 30 days, with the peak at 32 ␮m and 40 ␮m, respectively. However, the particle size distribution changed greatly at 0.18 wt.% fibril dosage during 30 days; the multimodal particle size distribution indicated the presence of droplet agglomeration in the system (Sobral, Palazolo, & Wagner, 2011). 4. Conclusions TOBC nanofibrils were obtained through a TEMPO/NaBr/NaClO system. The degree of oxidation was determined by electric conductivity titration method and increased with the dosage of NaClO. The carboxyl group content was in the range of 0.58–1.15 mmol/g at the dosage of the oxidizing agent, 2–10 mmol/g. Meanwhile, the absolute value of Zeta potential, charge density, and wettability of TOBC were reinforced by increase of carboxyl group contents. The carboxylation reaction on C6 site of cellulose during TEMPOoxidation was confirmed by FT-IR analysis. The width and length of TOBC fibrils were markedly cut down during TEMPO oxidation, which was observed by SEM and AFM. Oil/water type Pickering emulsions were prepared by using TOBCs as interfacial stabilizers. By static standing analysis, we can conclude that the stability of the emulsion was improved according to the increase of fibril dosage, but it decreased with the carboxyl content of TOBC increasing, since both fibril size and wettability played important roles in stabilizing emulsions. The particle size of the fibril-coated droplets increased with increasing fibril dosage. Eventually, 2-TOBC was considered as the most effective stabilizer for Pickering emulsions evaluated by particle size distribution. It has been stored at room temperature for 8 months with excellent stability. An interconnected network contributes well to the interfacial stability of Pickering emulsions. Rheological related investigation is carried out and will be reported elsewhere. Acknowledgements The authors are grateful for the financial support from the National Natural Science Foundation of China (project No. 31470610, and No. 21576212) as well as 2014 Chinese National Training Programs of Innovation and Entrepreneurship for Undergraduates under the grant 201410057007. References ´ Balejko, J., Bortnowska, G., Tokarczyk, G., Romanowska-Osuch, A., & Krzeminska, N. (2014). Food Hydrocolloids, 36, 229–237. Barud, H. S., Júnior, A. M. A., Santos, D. B., Rosana, M. N. A., Meireles, C. S., Cerqueira, D. A., et al. (2008). Thermochimica Acta, 471, 61–69. Binks, B. P. (2002). Current Opinion in Colloid and Interface Science, 7, 21–41.

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