Ultrasonic assisted water-in-oil emulsions encapsulating macro-molecular polysaccharide chitosan: Influence of molecular properties, emulsion viscosity and their stability

Journal Pre-proofs Ultrasonic assisted water-in-oil emulsions encapsulating macro-molecular polysaccharide chitosan: Influence of molecular properties, emulsion viscosity and their stability Kunming Zhang, Zhijuan Mao, Yongchun Huang, Yun Xu, Chengdu Huang, Yan Guo, Xian'e Ren, Chunyou Liu PII: DOI: Reference:

S1350-4177(19)31504-4 https://doi.org/10.1016/j.ultsonch.2020.105018 ULTSON 105018

To appear in:

Ultrasonics Sonochemistry

Received Date: Revised Date: Accepted Date:

5 October 2019 3 February 2020 8 February 2020

Please cite this article as: K. Zhang, Z. Mao, Y. Huang, Y. Xu, C. Huang, Y. Guo, X. Ren, C. Liu, Ultrasonic assisted water-in-oil emulsions encapsulating macro-molecular polysaccharide chitosan: Influence of molecular properties, emulsion viscosity and their stability, Ultrasonics Sonochemistry (2020), doi: https://doi.org/10.1016/j.ultsonch. 2020.105018

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Research article

Ultrasonic assisted water-in-oil emulsions encapsulating macromolecular polysaccharide chitosan: Influence of molecular properties, emulsion viscosity and their stability Kunming Zhanga,b,c,*, Zhijuan Mao a,b, Yongchun Huang a,b,c,d, Yun Xu a,b, Chengdu Huang a,b, Yan Guo a,b, Xian’e Ren a,b,c, Chunyou Liu a,b,c

aSchool

of Biological and Chemical Engineering, Guangxi University of Science and Technology,

Liuzhou 545006, China bGuangxi

Key Laboratory of Green Processing of Sugar Resources, Liuzhou 545006, China

cGuangxi

Liuzhou Luosifen Research Center of Engineering Technology, Liuzhou 545006, China

dProvince

and Ministry Co-sponsored Collaborative Innovation Center of Sugarcane and Sugar

Industry, Nanning 530004, China

*Corresponding author at: School of Biological and Chemical Engineering, Guangxi University of Science and Technology, Liuzhou 545006, China. Tel.: +86 772 2687033; Fax: +86 772 2687033. E-mail address: [email protected] (K. Zhang)

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Abstract An ultrasonic technique was applied to formulation of two-phase water-in-paraffin oil emulsions loading a high-molecular polysaccharide chitosan (CS) and stabilized by an oil-soluble surfactant (Span80) at different operational conditions. The influence of chitosan molecular properties, phase volume ratio (φw), Span80 volume fraction (φs) and ultrasonic processing parameters were systemically investigated on the basis of mean droplet diameter (MDD) and polydispersity index (PDI) of emulsions. It was observed that the molecular weight (Mw) of CS was an important influential factor to MDD due to the non-Newtonian properties of CS solution varying with Mw. The minimum MDD of 198.5 nm with PDI of 0.326 was obtained with ultrasonic amplitude of 32% for 15 min at an optimum φw of 35%, φs of 8%, probe position of 2.2 cm to the top of emulsion, while CS with Mw of 400 kDa and deacetylation degree of 84.6% was used. The rise of emulsion viscosity and the reduction of negative zeta potential at φw increasing from 5% to 35% were beneficial to obtain finer droplets and more uniform distribution of emulsions, and emulsion viscosity could be represented as a monotonically-decreasing power function of MDD at the same φw. FTIR analysis indicated that the molecular structure of paraffin oil was unaffected during ultrasonication. Moreover, the emulsions exhibited a good stability at 4 oC with a slight phase separation at 25 oC after 24 h of storage. By analyzing the evolution of MDD, PDI and sedimentation index (SI) with time, coalescence model showed better fitting results as comparison to Ostwald ripening model, which demonstrated that the coalescence or flocculation was the dominant destabilizing mechanism for such W/O 2/55

emulsions encapsulating CS. This study may provide a valuable contribution for the application of a non-Newtonian macromolecule solution as dispersed phase to generate nano-size W/O emulsions via ultrasound, and widen knowledge and interest of such emulsions in the functional biomaterial field. Keywords: Ultrasonication, W/O emulsion, Polysaccharide, Chitosan, Emulsification, Emulsion stability

1. Introduction Emulsions are typically defined as that the droplets of one liquid are dispersed into another immiscible liquid, with a mean droplet diameter (MDD) generally ranging from 0.1 μm to 100 μm [1, 2]. There often exists two forms of emulsion, i.e., oil-in-water (O/W) emulsion and water-in-oil (W/O) emulsion. For W/O emulsion, it can be used as a popular delivery system for active-substance encapsulation [3], antioxidant protection [4], drug-controlled release [5], flavor maintenance [6], micro-reactor [7], etc., which, thus, is widely applied in industry fields of foods, chemicals, medicine, cosmetics, polymerization [1-7]. Nevertheless, the performance of W/O emulsion is greatly dependent on the emulsifying parameter and the emulsion stability which is the key step for its industrial utilization [8, 9]. Therefore, the manufacturing of relatively stable W/O emulsion-based systems during storage, transportation and micro-reaction process has recently attracted much attention in public [10-13]. For instance, it is reported that the demineralized water was dispersed in pumpkin seed oil with polyglycerol polyricinoleate (PGPR) of 3 wt%, and the MDD was within the range of 400~850 nm by Nikolovski et al. [10], who found that the creaming index 3/55

of emulsion was 5.6% when it was stored at 25 oC for 15 days. Also, Bhatti et al. [11] reported a W/O emulsion encapsulating four types of amino acids (methionine, lysine, threonine and tryptophan) at a content of 1 wt% respectively, and found that such emulsions containing a surfactant of 5 wt% were stable at 4 oC after 30 days of storage with MDD ranging between 4~5 μm. Accordingly, Rabelo et al. [12] observed that W/O emulsions loading berry anthocyanins presented no phase separation at 4 oC after 30 days of storage, and the MDD varied between 131.5~814.8 nm with polydispersity index (PDI) ranging from 0.2 to 0.6. Raviadaran et al. [13] used an ultrasonic processor to assist in preparing water-in-palm oil emulsion at optimized condition, and found that such an emulsion showed no sedimentation when it was stored at 4 oC for 14 days with MDD increasing from 143.1 to 151.2 nm and with PDI increasing from 0.131 to 0.156. Nevertheless, to date, a lot of studies about W/O emulsions have taken liquid water or aqueous solution of small-molecule compounds as dispersed phase which essentially belongs to a Newtonian liquid [3, 4, 10-23], and very few work has been carried out on W/O emulsions with a non-Newtonian pseudoplastic liquid as dispersed phase, mostly like aqueous solution of high-molecular polymer (e.g., polysaccharide chitosan or soluble starch) [24, 25]. In the literature, Wardhono et al. [24] reported a water phase loading high-molecular carboxymethyl cellulose (CMC) at a content of 3.5 wt% which was dispersed in rapeseed oil methyl ester, and found that the rise of phase volume ratio led to a better stability and the MDD decreased from 18 μm to 12 μm when PGPR content was increased from 12 wt% to 16 wt%. Clausse et al. [25] reported W/O emulsions loading three types of polysaccharides, i.e., CMC, guar and xanthan, and 4/55

observed that such emulsions loading CMC exhibited a higher stability than those loading guar or xanthan, and the MDD of emulsions with PGPR used as surfactant was smaller than that of emulsions with lecithin used as surfactant. However, in their studies, the MDD of such emulsions prepared by using high pressure homogenizer is at micrometer level. To the best our knowledge, until now, there has been no published report on the formation of W/O emulsions encapsulating chitosan (CS), which is a linear aminopolysaccharide obtained by the deacetylation of naturally occurring polysaccharide chitin [26, 27]. By contrast with chitin, CS is readily soluble in aqueous acidic media below pH 6.0, which makes CS a water-soluble cationic polyelectrolyte [26]. In general, the aqueous solution of macro-molecular CS is a typical non-Newtonian pseudoplastic liquid, which possesses viscoelasticity and shear-thinning properties [28, 29]. Nonetheless, the non-Newtonian properties of macro-molecular CS solutions are significantly affected by chitosan molecular weight, degree of deacetylation, chitosan concentration, and additional salt content, etc. [28-30]. Especially at the same condition, for instance, the viscosity of CS solutions with a content of 1.0 wt% at 30 oC can be up to 192 mPa·s [31], which is far more than that of conventional Newtonian liquid (e.g., NaCl solution). Thus, it is not yet known the stability of such W/O emulsions encapsulating macro-molecular CS. On the other hand, chitosan as a basic skeleton has been explored as the forms of chitosan microcapsules, microspheres or nanoparticles for drug delivery systems [32], and the W/O emulsion-based cross-linking method has been widely applied to prepare 5/55

such microspheres or nanoparticles, during which process the reactive functional -NH2 groups of CS in dispersed water phase is used to cross-link with aldehyde groups of cross-linking agent (e.g., glutaraldehyde) [33]. Thus, the size of dispersed water phase in such W/O emulsions loading CS is a key factor to determine the particle size and size distribution of chitosan microspheres or nanoparticles, and simultaneously it is also a key step to obtain a stable W/O emulsion ahead of cross-linking reaction between CS and cross-linking agent [32, 33]. Hence, it would be of great significance to formulate ultra-fine (e.g., submicron or nano-scale) and stable W/O emulsions entrapping CS. As aforementioned, ultrasonic irradiation has been effectively applied to formulate nano-size W/O emulsions loading NaCl at a content of 0.5 M [13], thereby suggesting its potential to obtain an ultra-fine W/O emulsion loading macro-molecular CS. In this study, the water-in-paraffin oil emulsions entrapping macro-molecular CS were prepared using ultrasound and stabilized by an oil-soluble surfactant (Span80). The main objective of the present study was to evaluate the long-term stability of such emulsions prepared at the optimized operating condition, and to study the effect of various factors such as chitosan molecular properties, phase volume ratio (φw), Span80 volume fraction (φs) and ultrasonic parameters on emulsion properties (e.g., droplet diameter, size distribution or viscosity). The influence of ultrasound-induced chemical effects on the molecular structure of oil in prepared emulsions, and the mechanism of instability in water-paraffin oil-Span80 emulsion system loading CS were also studied.

2. Materials and methods 6/55

2.1. Chemical reagents Chitosan, molecular weight (Mw) 400~5000 kDa, degree of deacetylation (DD) 84.6% and 87.7%, was purchased from Zhongfayuan Biological Technology Co. Ltd. (Guangdong, China). Paraffin oil characterized by a low viscosity of 32 mPa·s and a density of ca. 0.85 g/cm3 at 20 oC was used as oil phase, which was purchased from Xilong Scientific Technology Co. Ltd. (Guangdong, China). The lipophilic surfactant sorbitan monooleate (Span80, HLB=4.3, C.P.) used in this study was purchased from Tianjin Damao Chemical Reagent Factory (Tianjin, China). All other chemicals including glacial acetic acid (CH3COOH) and sodium acetate (CH3COONa) were of analytical grade. De-ionized water prepared by a Millipore apparatus (Millipore, Billerica, USA) was employed during all the experimental runs. 2.2. Preparation of W/O emulsions entrapping macro-molecular CS For the preparation of W/O emulsions encapsulating high-molecular polysaccharide CS, both CS solution and paraffin oil were used as dispersed and continuous phases respectively, and the formulations were stabilized by surfactant (Span80). A total volume of mixed emulsion system was fixed at ca. 180 mL in the preparation of all the W/O formulations, and thus the liquid height in the vessel was about 6.6 cm. First, highmolecular CS was dissolved in sodium acetate-acetic acid buffer with a pH value of 4.0 to prepare non-Newtonian solutions with a high content of 10 g/L during all the experimental runs [28], and then Span80 was continuously added with stirring into the oil liquid at 30 oC for 5 minutes. Stirring was used to dissolve the Span80 and ensure homogeneity of the continuouse phase. Subsequently, CS solution was added dropwise 7/55

with stirring into the continuous oil phase by using a manual pipette at 30 oC. The stirring rate stated above was controlled at 500 rpm using a mechanical stirrer with a digital display (S312, Yuxiang Instrument Ltd., China). The MDD of ca. 1.53 μm and PDI of 1.0 were observed for the prepared coarse emulsions. Then, the prepared coarse emulsions were further subjected to ultrasonic irradiation to reduce the droplet size and PDI. For this reason, an ultrasonic processor (SCIENTZ950, SCIENTZ, China) with a probe diameter of 6 mm, a frequency of 25 kHz and a maximum power of 950 W was employed, and a cylindrical vessel with an inner diameter of ca. 6 cm was used. The experimental set-up used in this study was schematically depicted in Fig. 1. The ultrasonic probe was placed at a distance (h) to the top of the emulsions according to the experimental design. The ultrasonic process was stopped for 40 s after each 20 s run, during which a thermostat was applied to keep the temperature of ultrasonic emulsification at 30±5 oC. Finally, the generated W/O submicron or nano-size emulsions were characterized and the long-term stability of such emulsions entrapping CS could be systemically investigated. All the experiments were typically repeated three times to minimize the operating error. 2.3. Characterization of the W/O emulsions loading polysaccharide CS 2.3.1. Analysis of droplet size, polydispersity index (PDI), and zeta potential Droplet diameter, size distribution and zeta potential of freshly-prepared W/O emulsions loading high-molecular CS were measured by using dynamic light scattering (DLS) on a Zetasizer nano S90 (Malvern Instruments, UK). In brief, prior to conducting the analysis, samples were diluted to the ratio of 1:40 with paraffin oil to ensure free 8/55

Brownian motion of droplets, and each sample was equilibrated for 2 min, which then was analyzed at a scattering angle of 90o with a refractive index of 1.46 at 25 oC. The Z-average diameter represents the mean droplet diameter, and polydispersity index (PDI) represents the accumulated analysis of width measurements for droplet size distribution. The surface charge of emulsion droplets was measured from the electrophoretic mobility at 25 oC and values of zeta potential provided from the Zetasizer were expressed in mV. 2.3.2. Viscosity measurements The viscosity of freshly-prepared W/O emulsions loading CS was measured by using a digital viscometer (NDJ-5S, Brookfield Engineering Laboratories Inc., USA). The temperature of all samples during the test were kept at 30 oC, and the revolution rate was controlled at 60 rpm. The values of viscosity were recorded in mPa·s. 2.3.3. Sedimentation index (SI) measurements The instability of emulsions can be reflected from the phase separation of emulsions occurring with time. In this study, the ultrasonically-prepared W/O emulsions loading high-molecular CS was placed in the colorimetric cylinder with a plug, and stored at a certain condition. Then, the stability of the ultrasonically-prepared emulsions could be evaluated by using a sedimentation index (SI) as follows [13, 34]:

SI (%) 

H 100 Ho

(2)

where Ho represents the initial height of emulsion, and H represents the height of upper oil phase. From the eq. (1), it suggests that the value of SI is smaller, and the stability of such emulsions is better. 9/55

2.3.4. Physico-chemical characteristics The change in chemical bonds of compounds existed in paraffin oil was tested using fourier transform infrared spectroscopy (FTIR, Perkin Elmer, USA). Briefly, the FTIR spectrum of pure paraffin oil, coarse W/O emulsion and ultrasonically-prepared W/O emulsion at optimal conditions were studied respectively, and FTIR spectrophotometer was carried out with a scan range of 4000~600 cm-1. The aim of this test was to clarify whether the molecular structure of paraffin oil used as continuous phase was changed in presence of ultrasound-induced chemical effects during emulsification process. 2.4. Long-term stability of W/O emulsions The W/O emulsions encapsulating high-molecular polysaccharide CS prepared at the optimum operating conditions were stored at 4 oC and 25 oC, respectively. The stability of W/O emulsions loading CS was evaluated by measuring the sedimentation index, droplet size and polydispersity index for 30 days.

3. Result and discussion 3.1. Influence of chitosan molecular properties on W/O emulsions From the microcosmic point of view, both droplet size and polydispersity index (PDI) which represents the size distribution of dispersed droplets in the continuous phase are two key indicators for testing the stability of emulsion systems [6, 35]. Whilst, chitosan molecular properties refer to chitosan molecular weight (Mw) and degree of deacetylation (DD), which have an important influence on the properties of CS solution such as rheological behavior [29], solution viscosity [30] and surface tension [36]. Hence, such molecular properties probably have a close relationship with droplet size 10/55

and PDI of ultrasonically-prepared W/O emulsions loading high-molecular CS. Fig. 2a and b illustrates the observed variations of MDD and PDI of W/O emulsions encapsulating macro-molecular CS with Mw ranging from 400 kDa to 5000 kDa at DD of 84.6% and 87.7% respectively, while φw, φs and h were kept at 35%, 8% and 2.2 cm. As can be seen, the Mw of CS was an important influential factor to MDD, and the MDD of such emulsions increased with the increment of Mw of CS. This result can be attributed to the fact that when CS solution is at the same concentration, the increment of Mw results in the increase of viscosity in CS solution according to the Mark-Houwink equation given as follows [30, 37]:

  kM w a

(3)

where μ is the intrinsic viscosity of CS solution, Mw is the molecular weight, a and k are constants for given solute-solvent system and temperature. Subsequently, the nonNewtonian behavior of CS solution increases due to the increment of viscosity in CS solution [29, 30], which in turn leads to the greater ultrasonic-cavitation resistance in breaking up such a liquid into a fine emulsion droplet, and thus obtaining a lower droplet disruption rate. Therefore, the MDD increased with the rise of Mw. Moreover, even though the Mw of CS with DD of 84.6% was increased to 5000 kDa, the MDD obtained was still kept at 343.9 nm with PDI value of 0.435, which can be due to the fact that both the CS solution and the mixed water-paraffin oil-Span80 emulsion system loading CS are typically equipped with non-Newtonian pseudoplastic properties, respectively [28, 38], which are with the characteristics of viscoelastic shear-thinning property, i.e., the reduction of apparent viscosity triggered by the existed shear effect 11/55

generated from ultrasonic cavitation, and this property is beneficial to improve micromixing performance of ultrasonic-cavitation effect among CS solution, paraffin oil and Span80 [39], and thus the nano-sized emulsion droplets can be still obtained. Similarly, it can be observed that the PDI value of emulsions also increased with the increment of Mw of CS. It is possible that when Mw of CS increased, the increased droplet size in such emulsions stated above could lead to easier occurrence of collision among the dispersed water droplets due to the increase of gravity and the reduction of collision distance in the limited space of the vessel, which facilitated the coalescence or flocculation of the newly disrupted-droplets, and thus increasing the polydispersity of dispersed water droplets in the continuous oil phase. Nonetheless, the PDI of emulsions ranged between 0.326~0.435 even though the Mw of CS with DD of 84.6% was increased to 5000 kDa under the experiment condition, which is still less than 0.7, suggesting a narrow size distribution of the W/O emulsions loading macro-molecular CS. Herein, it should be mentioned that the PDI value is greater than 0.7, indicating a very broad droplet size distribution for the emulsion system [21, 40]. In addition, as shown in Fig. 2a and b, when the Mw of CS ranged from 400 kDa to 1000 kDa, the MDD value of emulsions ranged from 198.5 nm to 260.1 nm for DD at 84.6% and from 221.3 nm to 271.4 nm for DD at 87.7%, respectively. Correspondingly, the PDI value of emulsions ranged from 0.326 to 0.367 for DD at 84.6% and from 0.341 to 0.382 for DD at 87.7%, respectively. These results indicate that at the same Mw of CS, the increase of DD for CS can lead to the rise of MDD and PDI, and thus it is not conducive to obtain finer emulsion droplets and more narrow size distribution of such 12/55

W/O emulsions at a high DD of CS. This is mainly ascribed to the fact that both the viscosity and the non-Newtonian properties of CS solution increase with the rise of DD at the same Mw of CS reported by Wang et al. [29]. 3.2. Optimization of major emulsification process parameters 3.2.1. Effect of phase volume ratio (water to oil) Fig. 3a shows the variations of MDD and PDI of W/O emulsions entrapping highmolecular CS with φw ranging from 5% to 50%, while Mw, φs and h were kept at 400 kDa, 8% and 2.2 cm. As can be observed, there existed an optimum value of φw, i.e., φw=35%, for W/O emulsions entrapping polysaccharide CS to obtain a minimum value of MDD at 198.5 nm and PDI at 0.326 during ultrasonically-prepared process, which is beneficial to obtain a good stability performance for emulsions when both MDD and PDI of emulsions are at low values [7, 35]. More specifically, both MDD and PDI values of W/O emulsions loading CS presented a decreasing trend with the increment of φw varying from 5% to 35%, and whereafter as φw increased continually and greater than 35%, then significant increases in MDD and PDI could be found at the value of φw ranging from 35% to 50%, which is as shown in Fig. 3a and Table 1. Table 1 gives the effect of φw on the physico-chemical properties of such W/O emulsions loading macro-molecular CS formulated via ultrasound. It can be observed that as φw increased from 5% to 10%, the emulsion viscosity gradually increased from 71.8 mPa·s to 113.5 mPa·s, and subsequently the emulsion viscosity increased dramatically with the increment of φw, from which an important conclusion can be drawn that as phase volume ratio, i.e., φw, increases, the viscosity of W/O emulsions 13/55

loading macro-molecular CS actually increases, especially when the value of φw is greater than 10%, indicating that the viscosity of such W/O emulsions loading polysaccharide CS is significantly dependent on the value of φw. A similar trend was also found in such W/O emulsions where a small-molecular compound, i.e. water, was dispersed in the diesel fuel, which has been reported by Kojima et al. [34]. Thus, the decline of both MDD and PDI values at φw increasing from 5% to 35% can be understood by the fact that the rise of viscosity for such W/O emulsions loading CS with the increment of φw analyzed above led to a higher shear stress at the same emulsification strength during ultrasonic process [35, 41], which consequently became more favorable for water phase to disperse in the continuous paraffin oil phase. Furthermore, the rise of viscosity for such W/O emulsions could greatly increase the resistance to the dispersed water droplets approach each other [20, 42], and simultaneously, as φw increased from 5% to 35%, the zeta potential gradually dropped down from -35.7 mV to -38.4 mV (Table 1), and thus increasing the repulsive forces in the emulsion droplets, both of which were capable of preventing the occurrence of aggregation or coalescence among the particles. Therefore, a finer and a more uniform distribution of dispersed droplets could be formed in the W/O emulsions loading CS. Similar outcome has also been reported by Lin et al. [41], who found that the increase of φw was beneficial to the stability of water-in-oil petroleum ether emulsions due to the existence of smaller size emulsion droplets. Wardhono et al. [24] observed that a better stability could be obtained by increasing φw from 60% to 80% in W/O emulsions, in which the water phase loading polysaccharide CMC was dispersed in the rapeseed 14/55

oil methyl ester. The same trend was also given by Clausse et al. [25]. Nevertheless, the rapid increases in MDD and PDI values at φw increasing from 35% to 50% could be probably due to the fact that as φw increased continually to be at greater values, e.g., φw=40%, the distribution of dispersed water droplets in oil phase became more locally and more densely, which resulted in the water droplets getting close enough in the limited space of the vessel, and then the coalescence or aggregation among the particles could easily occur regardless of the increased emulsion viscosity and the reduced negative zeta potential (Table 1). Whereas, the reduction of negative zeta potential (Table 1) was a result of the enhanced adsorption of CH3COO- and OHions at the W/O interface owing to a thinning of nonionic surfactant (Span80) adsorption onto the interface during the increase of inner water content [43]. 3.2.2. Effect of Span80 volume fractions Fig. 3b presents the observed variations of MDD and PDI for W/O emulsions entrapping high-molecular CS with φs ranging from 1% to 50%, while Mw, φw and h were kept at 400 kDa, 35% and 2.2 cm. As can be seen, the value of φs has a significant influence on the values of MDD and PDI for W/O emulsions loading polysaccharide CS during ultrasonic process, and both MDD and PDI values of such emulsions significantly decreased with the increment of φs from 1% to 8%, especially when the Span80 was used at a low concentration, e.g., at 1%, the descending trend in MDD and PDI values became more significant. This result was expected. Nevertheless, when the amount of Span80 was added greater than 8%, both MDD and PDI values of such emulsions presented a gradually increasing trend with φs varying from 8% to 50%, 15/55

which indicates that it is not always beneficial to obtain finer droplets and more uniform distribution of W/O emulsions loading macro-molecular CS for a high-concentration Span80 used as surfactant during the water-paraffin oil emulsification process. The reduction of both MDD and PDI values at φs increasing from 1% to 8% can be due to the fact that when the Span80 used was at a low concentration such as at 1%, the quantity of Span80 was insufficient to form a thick surfactant film, and thus difficult to cover and stabilize the surface of newly-formed water droplets before their eventual coalescence [23, 24]. Nevertheless, as the amount of φs continued to increased, the interfacial area correspondingly increased and the interfacial tension between paraffin oil and CS solution decreased, and then a thicker and a higher strength surfactant film could be formed, which was beneficial to avoid the occurrence of coalescence between the newly-formed droplets [7, 25, 35]. Hence, both MDD and PDI values decreased while the value of φs increased from 1% to 8%. This is in agreement with the studies of Wardhono et al. [24], where they demonstrated that the increment of surfactant (PGPR) in W/O emulsions loading polysaccharide CMC from 12% to 18% could lead to the reduction in MDD from 18 μm to 12 μm. The increment of both MDD and PDI values at φs greater than 8% can be explained by the fact that the Span80 used reached its saturation adsorption plateau, and any further increase of the amount of Span80, i.e., φs, could lead to the rise of MDD owing to the reduction of the surface excess (T) [25, 35], which represents the number of moles of surfactant adsorbed per unit area of the interface. These results are in accordance with that reported by Alzorqi et al. [42], Chanana and Sheth [44], who observed that an 16/55

excess of surfactant could affect the diffusion rate of surfactant as well as the adsorption of surfactant-droplet effectively, and thus leading to the increase of coalescence between the newly-formed droplets [44, 45]. Moreover, Schmitt et al. [46] observed that a Span80-driven bush-like flocculated microstructure could be more fastly and easily produced by spontaneous emulsification (SE) at the water/paraffin oil interfaces when Span80 used was at a large concentration, during which process the water microdroplets/microfibers were covered with submicron droplets that were themselves covered with even smaller droplets, and therefore resulting in the increase in droplet size and non-uniform distribution of the W/O emulsions loading CS. As illustrated in Fig. 3b, the minimum value of MDD obtained was at 198.5 nm with PDI value of 0.326 after 15 min of sonication, indicating a narrow size distribution of such W/O emulsions at this experimental condition. 3.2.3. Effect of ultrasonic probe position (h) on emulsification Fig. 4a illustrates the variations of MDD and PDI for W/O emulsions loading macro-molecular CS with probe position (h) ranging from 0.5 cm to 5.5 cm, while Mw, φw and φs were kept at 400 kDa, 35% and 8%. As can be observed, there existed an optimum ultrasonic probe position to obtain a finer emulsion droplet and a more narrow size distribution of W/O emulsions entrapping CS during ultrasonic process. More specifically, as ultrasonic probe was placed at the height of h less than 2.2 cm, both MDD and PDI values of such emulsions were found to be reduced as h increased, and whereafter as ultrasonic probe was further continued to placed at greater depths of the W/O emulsions loading CS in the vessel, i.e., the heights of h was larger than 2.2 cm, 17/55

then, both MDD and PDI values of such emulsions presented an increasing trend obviously with the rise of h from 2.2 cm to 5.5 cm. The decline of both MDD and PDI values at h increasing from 0.5 cm to 2.2 cm in the emulsions is probably understood by the fact that as probe position, i.e., the heights of h increased from a shallow depth, e.g., at 0.5 cm, in the emulsions, a more homogeneous cavitation development could be formed in the directions from the probe center to the bottom of the vessel [35, 47], which thus could generate a more uniform shear-stress field in such emulsion systems, i.e., CS solution, paraffin oil and Span80, in the vessel. Furthermore, while the position of probe, i.e., the heights of h gradually increased from a shallow depth such as at 0.5 cm in the emulsions, the down-acoustic streaming generated from the ultrasonic probe was increasingly easy to make such emulsions form effective convection current in the limited region of vessel, which became more favorable to obtain a sufficient agitation for such emulsion systems in the vessel [34, 35]. Consequently, both MDD and PDI values decreased while the heights of h increased from 0.5 cm to 2.2 cm. Nevertheless, when the probe was placed too deep, e.g., at 5.5 cm, in the emulsions, the efficient-function region of down-acoustic streaming generated from the ultrasonic probe decreased significantly, which in turn hindered the emulsions to form a finer emulsion droplet and a more narrow size distribution to some extent [34, 35, 47]. 3.2.4. Effect of ultrasonic amplitude on emulsification Fig. 4b illustrates the variations of MDD and PDI of W/O emulsions encapsulating high-molecular CS with ultrasonic amplitude ranging from 6% to 64%, while Mw, φw, 18/55

φs and h were respectively kept at 400 kDa, 35%, 8% and 2.2 cm. As can be seen, both MDD and PDI values of such W/O emulsions loading CS progressively decreased with the increment of ultrasonic amplitude from 6% to 32%, and subsequently it can be observed that both MDD and PDI values of such W/O emulsions presented an increasing trend at the ultrasonic amplitude ranging from 32% to 64%. These results stated above were expected, and can be understood by the fact that as ultrasonic amplitude increased, the irradiation power would increase, and then more energy would be delivered into the emulsification systems, which could produce a higher cavitational collapse pressure at the end of asymmetric cavity collapse [13, 48]. Thus, the overall cavitational intensity increased, which resulted in primary largedispersed droplets breaking up into smaller droplets, and forming a more narrow size distribution of the emulsions. This result is in agreement with that obtained from other ultrasonic-assisted W/O emulsion process in which a small-molecule compound, e.g., NaCl solution or the liquid water, was used as dispersed phase [13, 49]. Consequently, both MDD and PDI values decreased with the rise of ultrasonic amplitude while it was less than 32%. However, when ultrasonic amplitude increased too high, the “overprocessing” effect would occur in such W/O emulsions loading CS [49], which was caused by an increase in emulsion droplet coalescence at the higher shear rates, and this trend agrees well with the findings reported by Kentish et al. [49] and Jafari et al. [50], in which the ultrasonic radiation forces (referred to as Bjerknes forces) increased with the rise of acoustic power, and then the second Bjerknes forces would drive emulsion droplets to the nodes and antinodes of the acoustic field, and finally such closer19/55

proximity droplets at these positions would lead to the increase of droplet coalescence and thus the observed “over-processing” effect [49, 50]. 3.2.5. Effect of ultrasonication time on MDD，PDI and viscosity Fig. 5 presents the variations of MDD and PDI for W/O emulsions entrapping highmolecular CS with ultrasonication time varying between 0~30 min, while Mw, φw , φs and h were respectively kept at 400 kDa, 35%, 8% and 2.2 cm. It can be observed that as ultrasonic processing time increased, both MDD and PDI values of such W/O emulsions loading CS presented a remarkable decreasing trend, especially when ultrasonic irradiation progressively increased to 10 min. Subsequently, a plateau in MDD and PDI values could be found beyond ultrasonication of 15 min. These results indicate that during earlier ultrasonication process, the primary large-dispersed water droplets can be dramatically broken up into smaller water droplets by cavitational effects that are generated from ultrasonic irradiation to the emulsions, and thus the size distribution of such W/O emulsions become more narrow. These results are well in agreement with the findings reported by Raviadaran et al. [13] and Kojima et al. [34]. The plateau in MDD and PDI values after ultrasonication exceeding 15 min can be explained by the fact that the droplet size of an emulsion was the result of an equilibrium of droplet break-up and its recoalescence [13, 35]. The high shear stresses generated from ultrasound could lead to large-dispersed droplets disruption, followed by the adsorption of Span80 onto the fresh interface to prevent the coalescence of newlyformed droplets [13, 47]. Hence, beyond the optimized irradiation time at the same Span80 concentration, the droplets break-up and recoalescence in emulsions would 20/55

reach their equilibrium, which thus could not lead to a further reduction in MDD and PDI values [13, 47]. As also seen from Fig. 5, while ultrasonic processing time was less than 15 min, the measured viscosity of such W/O emulsions loading macro-molecular CS at 30 oC increased significantly with ultrasonic processing time, and whereafter presented little change. This result could be due to the close non-linear relationship between droplet size and emulsion viscosity, which is as shown in Fig. 6. It can be observed that the emulsion viscosity could be represented as a monotonicallydecreasing power function of MDD, which can be given as follows:

y  axb

(4)

where a and b are constants, dependent on the type of macro-molecular polysaccharide. The values of a and b for macro-molecular polysaccharide CS are as follows: a=75323.5, b=-0.786; R2=0.99. This result can be mainly ascribed to the fact that the increased droplet size could lead to a decrease in the total surface area of such W/O emulsions loading macro-molecular CS, and thus obtaining a decreasing trend in viscosity [42, 51]. A similar result that the decreased droplet size could lead to an increase in emulsion viscosity has also been found in O/W emulsion loading β-D-glucan polysaccharides, which was reported by Alzorqi et al. [42]. Table 2 shows the optimized process parameters for the formulation of paraffin oilbased W/O emulsions loading CS with Mw of 400 kDa and DD of 84.6% and using the oil-soluble Span80 as surfactant. As can be seen, the smallest MDD of 198.5 nm with PDI of 0.326 were obtained using ultrasound at an ultrasonic amplitude of 32% for 15 min with an optimum φw of 35%, φs of 8%, h of 2.2 cm in the formation of such W/O 21/55

emulsions encapsulating macro-molecular polysaccharide CS. 3.3. Effect of ultrasonication on microstructure of oil As confirmed by many studies, ultrasonically-prepared submicron or nanoscale emulsions are produced by the physical effects generated from acoustic cavitation in liquids, e.g., acoustic streaming, high-speed microjets, microstreaming and high localized turbulence as a result of violent cavity collapse [35, 47]. Besides, the highly reactive free radicals (e.g., ·OH) can be effectively generated from the dissociation of water molecules due to the cavity collapse under the extreme conditions [35, 47], which can act on the hydrocarbon bonds in the molecules of compounds existed in the paraffin oil during ultrasonic process. Thus, to find out the potential influence of ultrasoundinduced chemical effects upon the molecular structure of paraffin oil during ultrasonic process, FTIR spectra of such W/O emulsions after ultrasonication at the optimized process condition (Table 2) using CS at Mw of 400 kDa and DD of 84.6% was compared with that of emulsions ahead of ultrasonication and pure paraffin oil, which is as shown in Fig. 7. As illustrated in Fig. 7b and c, it can be observed that all of the peaks of W/O emulsions ahead of ultrasonic process in FTIR spectra were the same as those obtained in pure paraffin oil except two peaks positioned at 3363.5 cm-1 and 1640.1 cm-1. Apparently, the peaks observed at 3363.5 cm-1 corresponding to H-OH stretch indicate the presence of water in emulsion, and the peaks observed at 1640.1 cm-1 corresponding to C=O stretch indicate the presence of carbonyl-containing compounds in emulsion, which is well in accordance with the fact that the CS solution used as dispersed phase 22/55

was prepared by using the sodium acetate-acetic acid buffer in this study. These results from FTIR analysis stated above indicate that the molecule structure of paraffin oil has no change in such W/O emulsions loading CS ahead of ultrasonic process, which agrees well with the fact that the use of single mechanical agitation to prepare coarse W/O emulsions can not lead to any changes in molecule structure of paraffin oil. Similary, as shown in Fig. 7a and b, FTIR spectra of W/O emulsions loading CS after ultrasonication at the optimized condition was the same as that observed in such W/O emulsions ahead of ultrasonication, suggesting that the chemical effects can not affect the molecular structure of paraffin oil which, even though, is used as continuous phase in the emulsions, and there are no secondary oxidation products produced during the whole ultrasonic emulsification process. The FTIR spectra of paraffin wax-in-water nanoemulsion was reported by Jadhav et al. [52], and they found that only one extra peak H-OH stretch in the FTIR spectra, indicating no chemical changes occurred in paraffin wax during ultrasonic process. Raviadaran et al. [13] has also provided similar results for the formation of water-inpalm oil nanoemulsion by ultrasound. The FTIR spectra remained unchanged between before and after ultrasonication and they made a conclusion that no oxidation products were formed due to processing under ultrasound. 3.4. Long-term stability of W/O emulsions encapsulating polysaccharide CS As is known, the performance of W/O emulsions is greatly dependent on the stability of emulsions, and it is necessary to evaluate their stability ahead of applications in the required fields [6, 9]. In present study, the macro-molecular CS with Mw and DD 23/55

of 400 kDa and 84.6% respectively was used for the preparation of such W/O emulsions via ultrasound at the optimized process condition (Table 2), and then the stability of such ultrasonically-prepared W/O emulsions entrapping CS was assessed by monitoring the changes in sedimentation index (SI), droplet size and size distribution with storage time under different storage conditions (4 oC, 25 oC). Fig. 8 represents the variations of such ultrasonically-prepared W/O emulsions in SI, MDD and PDI values with storage time at a temperature of 4 oC or 25 oC, respectively. As illustrated in Fig. 8a, the SI of emulsions stored for one day, i.e., 24 h, was 0.5% and 2.1% for the emulsions stored at 4 oC and 25 oC respectively, suggesting that such W/O emulsions loading CS exhibit a good physical stability at 4 oC with a slight phase separation at 25 oC after 24 h of storage. This could be confirmed from the changes of droplet size and PDI, which are as shown in Fig. 8b and c. The MDD in emulsions stored at 4 oC for 24 h increased from 198.5 nm to 204.6 nm with the rise of PDI from 0.326 to 0.331, while the MDD in emulsions stored at 25 oC for 24 h increased from 198.5 nm to 225.7 nm with the rise of PDI from 0.326 to 0.343. Subsequently, both droplet size and PDI of such W/O emulsions were kept repid growth during the long-term process lasting for ca. 10~12 days regardless of the storage temperature, and then the phase separation, i.e., SI value, of the emulsions stored at 4 oC and 25 oC unexpectedly became more obvious with storage time lasting for ca. 10~12 days, which is as illustrated in Fig. 9. This is probably attributed to the occurrence of flocculation or coalescence among the droplets triggered by Span80-driven SE process with time [46], and then resulting in a rapid increase in particle size, which, thus, accelerated the 24/55

rate of gravitational separation of such W/O emulsions. Whereafter, as the long-term test was continually performed for more than 10~12 days, the MDD, PDI and SI of such emulsions presented a slow-increase trend with storage time. These results indicate that droplet size and size distribution of emulsions can significantly affect the stability of W/O emulsions loading macro-molecular CS. Besides, as seen from Figs. 8 and 9, the SI of W/O emulsions encapsulating CS stored at 4 oC was lower than that of W/O emulsions stored at 25 oC during the whole storage days, suggesting that it is more suitable for the W/O emulsions entrapping CS to be stored at a low temperature. This can be understood by the fact that the Brownian motion of dispersed droplets increase with the rise of temperature and as a result obtaining a higher collision probability among the droplets, which, then, leads to the increase of droplet coalescence or aggregation in the emulsions, and thus accelerating the sedimentation of such W/O emulsions loading macro-molecular CS [7, 35]. Simultaneously, the ultrasonically-prepared W/O emulsions loading CS formed at the optimized process condition in this study presented an opaque gel-like liquid ( Fig. 9) with a high measured viscosity of ca. 1132.7 mPa·s, which was conducive to the stability of such emulsions owing to the increase of resistance to the dispersed-droplets approach each other and thus preventing the occurrence of coalescence [42, 51]. Consequently, It can be concluded that such W/O emulsions loading macro-molecular polysaccharide CS exhibit an acceptable kinetic stability for 24 h mainly due to their initial small droplet size, low PDI and high-viscosity properties. Thus, from the standpoint of practical uses, it would be better for the newly-prepared W/O emulsions 25/55

loading macro-molecular CS to be stored at 4 oC or 25 oC for less than 24 h while they are used for the preparation of chitosan microcapsules, microspheres or nanoparticles. As aforementioned, the phase separation of ultrasonically-prepared W/O emulsions entrapping CS occurred when they were stored for over 24 h even though the emulsions were stored at a low temperature of 4 oC. This may be ascribed to the fact that such W/O emulsions loading CS are non-equilibrium systems, and they have a tendency to reduce their interfacial area and free energy through several breakdown processes, e.g., coalescence and Ostwald ripening [46], both of which are the main instability mechanisms to the break of W/O emulsions [21, 53, 54]. For coalescence, the following equation holds true [55, 56]. 1 1 8  3 2 r ro 3t

(5)

where r is the mean droplet size of W/O emulsions at t, and ro is the initial droplet size of W/O emulsions at t=0, ω is the frequency of rupture per unit surface area of emulsion droplets. If the experimental values are represented by a linear plot of 1/r2 versus the reciprocal of storage time (t-1), the value of ω can be regarded as a constant with respect to change in droplet size and the coalescence rate can be determined. For Ostwald ripening, the Lifshitz-Slezov and Wagner (LSW) theory provides a linear relationship between r3 and t as follows [21, 53-56].

dr 3 8 C Vm D =  ( ) dt 9  RT

(6)

where ρ is the density of dispersed phase, R is the universal gas constant, T is the temperature of W/O emulsion system; C∞ is the bulk phase solubility of an infinitely 26/55

large droplet, γ is the interfacial tension, Vm is the molar volume of the dispersed phase and D is the diffusion coefficient of the dispersed phase in the continuous phase. If the experimental values fit a linear plot of r3 versus storage time (t) during the rupture process, the ripening rate can be determined. In order to ascertain the main destabilization mechanism to the W/O emulsions loading polysaccharide CS during the long-term storage process, both of the theories were used and the Fig. 10a and b was drawn. As shown in Fig. 10a and Table 3, the variations of 1/r2 as a function of the reciprocal of storage time (t-1) presented a good linear relationship (R2=0.963~0.975) according to the coalescence model as seen in eq. (4) regardless of the storage temperature at 4 oC or 25 oC during the long-term process. Similarly, as shown in Fig. 10b and Table 3, the variations of r3 as a function of storage time (t) also presented an acceptable linear relationship (R2=0.921~0.946) according to the LSW model as seen in eq. (5) regardless of the storage temperature at 4 oC or 25 oC during the 30-day storage process. Thus, the instability mechanism of such W/O emulsions should include the Ostwald ripening, which arises due to the solubility difference of dispersed droplets with different size [46], and in such a process the destabilization causes the larger droplets to grow at the expense of the smaller ones [2, 21]. However, the coalescence model showed better fitting results as comparison to the Ostwald ripening model (Table 3), which indicates that the droplet coalescence or flocculation is the dominant role to cause the increasing droplet radius and non-uniform distribution of such W/O emulsions loading CS. In addition, according to the slope of each straight line in Fig. 10, both of the 27/55

coalescence rate and the Ostwald ripening rate during the 30-day storage test at different storage temperature were calculated and listed in Table 3. As can be observed, both of the coalescence rate and the Ostwald ripening rate for such W/O emulsions stored at the temperature of 25 oC were greater than those stored at the temperature of 4 oC. This confirms the earlier result that such W/O emulsions stored at a low temperature (e.g., at 4 oC) are more stable and it is not conducive to obtain a high-stability performance for such W/O emulsions loading polysaccharide CS stored at a high temperature. To sum up, it could be surmised that both coalescence and Ostwald ripening might have occurred simultaneously, but coalescence was the dominant destabilization mechanism for such W/O emulsions encapsulating macro-molecular polysaccharide CS regardless of storage temperature during the long-term storage process.

4. Conclusions In this study, an ultrasonic technique was applied to preparation of water-in-paraffin oil emulsions and stabilized by an oil-soluble surfactant (Span80) at different operating conditions, during which process a non-Newtonian macro-molecular polysaccharide solution, i.e., chitosan (CS) solution, was used as a dispersed phase. The following conclusions could be drawn from this study based on data analysis: 1. The molecular weight (Mw) of CS was an important influential factor to mean droplet diameter (MDD) of such W/O emulsions due to the non-Newtonian properties of CS solution varying with Mw. Whilst, both MDD and polydispersity index (PDI) of the emulsions increased with an increase in Mw of CS from 400 kDa to 5000 kDa due to the increment of non-Newtonian behavior of CS solution. The rise of deacetylation 28/55

degree (DD) of CS from 84.6% to 87.7% also led to an increase in MDD and PDI of such emulsions. 2. The viscosity of W/O emulsions loading high-molecular CS was significantly dependent on the value of phase volume ratio (φw), and the rise of emulsion viscosity and the reduction of negative zeta potential at φw increasing from 5% to 35% were beneficial to obtain finer droplets and more uniform distribution of emulsions. As the Span80 volume fraction (φs) was varied in range of 1%-50%, the minimum value of MDD was found at φs of 8%, which was regarded as the optimum φs, and it was not always beneficial to obtain finer droplets and more uniform distribution of W/O emulsions loading CS for a high-concentration Span80 used as surfactant during the water-paraffin oil emulsification process. 3. There existed an optimum ultrasonic probe position to obtain a finer emulsion droplet and a more narrow size distribution of such W/O emulsions, and the droplet size was found to be the lowest when ultrasonic probe position was placed at the height of h equal to 2.2 cm. As ultrasonic amplitude was varied from 6% to 64%, an “overprocessing” effect on MDD and PDI could be found, and ultrasonic amplitude of 32% was the most suitable for preparing such W/O emulsions loading CS. 4. The droplet size and PDI decreased with the rise of ultrasonication time, and they could approach each limiting value while ultrasonic processing time were greater than 15 min. Whilst, the emulsion viscosity presented an increasing trend within 15 min of ultrasonication. It was observed that while the φw of emulsion was fixed, the emulsion viscosity could be represented as a monotonically-decreasing power function of MDD. 29/55

5. The minimum MDD of 198.5 nm with PDI of 0.326 was obtained with ultrasonic amplitude of 32% for 15 min at an optimum φw of 35%, φs of 8%, probe position of 2.2 cm to the top of emulsion, while CS with Mw of 400 kDa and deacetylation degree of 84.6% was used. FTIR analysis indicated that the molecular structure of paraffin oil was unaffected by ultrasonically-induced chemical effects during ultrasonication. 6. In the long-term stability test, the emulsions exhibited a good stability at 4 oC with a slight phase separation at 25 oC after 24 h of storage, and such an emulsion stored at 4 oC presented a higher stability in comparison with that stored at 25 oC during 30 days of storage. The variations in droplet size and sedimentation index (SI) with storage time demonstrated that droplets coalescence and Ostwald ripening might have occurred simultaneously, but coalescence was the dominant destabilizing mechanism for the W/O emulsions encapsulating macro-molecular polysaccharide chitosan.

Acknowledgements The authors gratefully acknowledge the financial support of the National Natural Science Foundation of China (No. 31701646), the Natural Science Foundation of Guangxi Province (No. 2017GXNSFBA198036), the Special Foundation of Scientific Base and Talent of Guangxi Province (No. GUIKEAD19110075), the High-LevelInnovation Team and Outstanding Scholar Project of Guangxi Higher Education Institutes (No. GUIJIAOREN[2014]7) and Research Program of Science at Universities of Guangxi Province (No. 2017KY0344). The authors are grateful to the helpful suggestions and assistance from Prof. Zhihua Qiao, Prof. Changcan Shi and Prof. Hui Su. 30/55

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Captions of Figures and Tables Figures Fig. 1. Schematic presentation of the experimental set-up. Fig. 2. Variations of MDD and PDI of W/O emulsions encapsulating macro-molecular CS with Mw of CS at DD of (a) 84.6% and (b) 87.7%, prepared at an amplitude of 32% and ultrasonication of 15 min (φw=35%, φs=8%, h=2.2 cm). Fig. 3. Variations of MDD and PDI of W/O emulsions entrapping high-molecular CS with (a) φw and (b) φs at CS with DD of 84.6%, prepared at an amplitude of 32% and ultrasonication of 15 min (Mw =400 kDa, h=2.2 cm). Fig. 4. Variations of MDD and PDI of W/O emulsions loading macro-molecular CS with (a) h and (b) ultrasonic amplitude at CS with DD of 84.6%, prepared at ultrasonication of 15 min (Mw =400 kDa, φw=35%, φs=8%). h represents the distance of ultrasonic probe to the top of emulsion. Fig. 5. Variations of MDD, PDI and viscosity of W/O emulsions entrapping highmolecular CS with ultrasonication time at CS with DD of 84.6% (Mw =400 kDa, φw=35%, φs=8%, h=2.2 cm). The emulsion viscosity was measured at 30 oC. Fig. 6. Relationship between emulsion viscosity and droplet size of ultrasonicallyprepared W/O emulsions loading macro-molecular polysaccharide CS. Fig. 7. Comparison of FTIR spectra in the formation of W/O emulsions loading macromolecular CS with Mw of 400 kDa and DD of 84.6% at different conditions: (a) after ultrasonication at optimized condition; (b) before ultrasonication; (c) pure paraffin oil. 39/55

Fig. 8. Dependent variables of ultrasonically-prepared W/O emulsions encapsulating macro-molecular CS with Mw of 400 kDa and DD of 84.6% prepared at optimized condition with storage time: (a) SI; (b) MDD; (c) PDI. The emulsions were stored at 4 oC

and 25 oC respectively.

Fig. 9. Typical visual observation of the stability of ultrasonically-prepared W/O emulsions entrapping polysaccharide CS with Mw of 400 kDa and DD of 84.6% stored at different temperature for 30 days: (a) 4 oC; (b) 25 oC. The emulsions were all prepared at the optimum process conditions. Fig. 10. Variations of (a) 1/r2 as a function of t-1 and (b) r3 as a function of t for ultrasonically-prepared W/O emulsions encapsulating macro-molecular polysaccharide CS with Mw of 400 kDa and DD of 84.6% produced at optimum process condition and stored at 4 oC and 25 oC respectively. Tables Table 1 Effect of phase volume ratio (φw) on the physico-chemical properties of W/O emulsions loading macro-molecular CS formulated via ultrasound. Table 2 Optimized process parameters for the formation of paraffin oil-based W/O emulsions encapsulating macro-molecular CS with Mw of 400 kDa and DD of 84.6% via ultrasound. Table 3 Coalescence and Ostwald ripening rate of dispersed water droplets loading macro-molecular polysaccharide CS in W/O emulsions at different storage temperature.

Conflict of Interest 40/55

The authors declare that they have no conflicts of interest.

Fig. 1. Schematic presentation of the experimental set-up.

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(a)

(b) 42/55

Fig. 2. Variations of MDD and PDI of W/O emulsions encapsulating macro-molecular CS with Mw of CS at DD of (a) 84.6% and (b) 87.7%, prepared at an amplitude of 32% and ultrasonication of 15 min (φw=35%, φs=8%, h=2.2 cm).

(a)

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(b) Fig. 3. Variations of MDD and PDI of W/O emulsions entrapping high-molecular CS with (a) φw and (b) φs at CS with DD of 84.6%, prepared at an amplitude of 32% and ultrasonication of 15 min (Mw =400 kDa, h=2.2 cm).

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(a)

(b) Fig. 4. Variations of MDD and PDI of W/O emulsions loading macro-molecular CS 45/55

with (a) h and (b) ultrasonic amplitude at CS with DD of 84.6%, prepared at ultrasonication of 15 min (Mw =400 kDa, φw=35%, φs=8%). h represents the distance of ultrasonic probe to the top of emulsion.

Fig. 5. Variations of MDD, PDI and viscosity of W/O emulsions entrapping highmolecular CS with ultrasonication time at CS with DD of 84.6% (Mw =400 kDa, φw=35%, φs=8%, h=2.2 cm). The emulsion viscosity was measured at 30 oC.

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Fig. 6. Relationship between emulsion viscosity and droplet size of ultrasonicallyprepared W/O emulsions loading macro-molecular polysaccharide CS.

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(a)

(b)

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(c) Fig. 7. Comparison of FTIR spectra in the formation of W/O emulsions loading macromolecular CS with Mw of 400 kDa and DD of 84.6% at different conditions: (a) after ultrasonication at optimized condition; (b) before ultrasonication; (c) pure paraffin oil.

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Fig. 8. Dependent variables of ultrasonically-prepared W/O emulsions encapsulating macro-molecular CS with Mw of 400 kDa and DD of 84.6% prepared at optimized condition with storage time: (a) SI; (b) MDD; (c) PDI. The emulsions were stored at 4 oC

and 25 oC respectively.

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(a)

(b) 51/55

Fig. 9. Typical visual observation of the stability of ultrasonically-prepared W/O emulsions entrapping polysaccharide CS with Mw of 400 kDa and DD of 84.6% stored at different temperature for 30 days: (a) 4 oC; (b) 25 oC. The emulsions were all prepared at the optimum process conditions.

(a)

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(b) Fig. 10. Variations of (a) 1/r2 as a function of t-1 and (b) r3 as a function of t for ultrasonically-prepared W/O emulsions encapsulating macro-molecular polysaccharide CS with Mw of 400 kDa and DD of 84.6% produced at optimum process condition and stored at 4 oC and 25 oC respectively.

Highlights  Cavitationally prepared a non-Newtonian macromolecule chitosan solution in oil.  Molecular weight of chitosan was an important influential factor to droplet size.  Emulsion viscosity represented as a decreasing power function of droplet size.  The minimum droplet size of emulsion was 198.5 nm with PDI of 0.326.  Such emulsions exhibited a good stability at 4 oC after 24 h of storage. 53/55

Table 1 Effect of phase volume ratio (φw) on the physico-chemical properties of W/O emulsions loading macro-molecular CS formulated via ultrasound. Fraction of phase Mean

droplet Polydispersity Zeta potential Viscosity*

volume ratio (%)

diameter (nm)

index

(mV)

(mPa·s)

0**

—

—

—

34.2±1.5

5

319.1±17.5

0.424±0.039

-35.7±4.28

71.8±4.3

10

293.9±14.1

0.377±0.027

-36.4±4.55

113.5±5.1

20

241.5±18.9

0.344±0.036

-37.2±5.26

365.9±4.8

35

198.5±13.2

0.326±0.021

-38.4±5.57

1132.7±5.6

40

239.6±17.9

0.346±0.043

-38.7±4.98

1201.2±6.4

45

326.7±21.3

0.391±0.047

-39.1±5.82

1283.5±5.2

50

418.4±19.4

0.435±0.035

-39.3±6.35

1329.1±6.7

* The viscosity of W/O emulsions loading chitosan was measured at 30 oC. ** The continuous oil phase contained 8% of surfactant Span80, but contained no dispersed water droplet.

Table 2 Optimized process parameters for the formation of paraffin oil-based W/O emulsions encapsulating macro-molecular CS with Mw of 400 kDa and DD of 84.6% via ultrasound. Parameters

Optimized values

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Phase volume refraction (%)

35

Surfactant volume fraction (%)

8

Ultrasonic Probe to the top of emulsion (cm)

2.2

Ultrasonic amplitude (%)

32

Ultrasonication time (min)

15

Mean droplet diameter (nm)

198.5±13.2

Polydispersity index

0.326±0.021

Table 3 Coalescence and Ostwald ripening rate of dispersed water droplets loading macro-molecular polysaccharide CS in W/O emulsions at different storage temperature. Storage

Initial

droplet

Rate of Coalescence

Rate of Ostwald

temperature (oC) radius, r (nm)

ripening ω(105nm2/d)

R2

ω(106nm3/d)

R2

4

198.5

4.18

0.963

2.71

0.946

25

198.5

5.23

0.975

3.58

0.921

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