Joint Effects of Granule Size and Degree of Substitution on

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Joint effects of granule size and degree of substitution on octenylsuccinated sweet potato starch granules as Pickering emulsion stabilizers Li Jinfeng, Fayin Ye, Lin Lei, Yun Zhou, and Guohua Zhao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05507 • Publication Date (Web): 17 Apr 2018 Downloaded from http://pubs.acs.org on April 17, 2018

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Journal of Agricultural and Food Chemistry

Graphic abstract 366x219mm (300 x 300 DPI)

ACS Paragon Plus Environment


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Joint effects of granule size and degree of substitution on

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octenylsuccinated sweet potato starch granules as Pickering emulsion stabilizers

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Jinfeng Li†, Fayin Ye†, Lin Lei†, Yun Zhou†, Guohua Zhao*†,‡

4 5 6

†

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Republic of China

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‡

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China

College of Food Science, Southwest University, Chongqing 400715, People’s

Chongqing Sweet Potato Research Centre, Chongqing 400715, People’s Republic of

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*Corresponding author

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College of Food Science

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Southwest University

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Tiansheng Road 2, Chongqing, 400715

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PR China

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Tel.: +86 23 68 25 19 02

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Fax: +86 23 68 25 19 47

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E-mail address: [email protected] (G. Zhao)

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ABSTRACT

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The granules of sweet potato starch were size-fractionated into three portions with

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significantly different median diameters (D50) of 6.67 (small-sized), 11.54

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(medium-sized) and 16.96 µm (large-sized), respectively. Each portion was

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hydrophobized at the mass-based degrees of substitution (DSm) of approximately

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0.0095 (low), 0.0160 (medium) and 0.0230 (high). The Pickering emulsion stabilizing

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capacities of modified granules were tested, and the resultant emulsions were

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characterized. The joint effects of granule size and DSm on emulsifying capacity (EC)

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were investigated by response surface methodology. For small-, medium- and

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large-sized fractions, their highest emulsifying capacities are comparable but

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respectively encountered at high (0.0225), medium (0.0158) and low (0.0095) DSm

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levels. The emulsion droplet size increased with granule size, and the number of

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freely scattered granules in emulsions decreased with DSm. In addition, the term of

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surface density of octenyl succinic group (SD-OSG) was firstly proposed for modified

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starch granules and it was proved better than DSm in interpreting the emulsifying

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capacities of starch granules with varying sizes. The present results implied that, as

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the particulate stabilizers, the optimal DSm of modified starch granules is size specific.

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KEYWORDS: sweet potato starch, particle size, degree of substitution, Pickering

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emulsion, emulsifying capacity, joint effect

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■ INTRODUCTION

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In contrast to the ordinary emulsion stabilized by molecular emulsifiers,

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the Pickering emulsion is stabilized instead by solid particles, which can adsorb onto

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the interface between the two immiscible phases.1 Previous research indicated that

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Pickering emulsions are often more stable against droplet coalescence and Ostwald

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ripening than emulsions stabilized by molecular surfactants.2,3 Apparently, particles

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suitable for Pickering stabilization of emulsions should be simultaneously wetted by

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the two immiscible phases, usually oil as droplets dispersed in water. In this regard,

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these particles must be amphipathic like molecular surfactants to allow their

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adsorption onto the interface. This, in turn, results in a particle layer on the surface of

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the oil droplets. The resultant particle layer prevents the contact between adjacent

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oil-droplets via steric hindrance and maintains their suspension in water.4 To date,

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various particles have been verified with Pickering stabilizing capacity. According to

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chemical attributes, they can be classified into two categories: inorganic and organic

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particles. The inorganic particles are often represented by silicon dioxide, calcium

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carbonate and titanium dioxide,5,6 while the organic particles can be further grouped

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into carbohydrate-,7-9 protein-10-12 and lipid-based particles13 such as microcrystalline

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cellulose, lactoferrin and glycerol monostearate.

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To formulate carbohydrate-based particles, starch is an excellent candidate

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substrate due to its high abundance, low price and easy availability. Recent studies

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have demonstrated the feasibility of preparing starch-based particles with Pickering

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stabilizing capacity.14-17 In these cases, two strategies were adopted based on the 3


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stabilization energy (∆E)-determining equation of ∆E=πr2γow(1-|cosθow|)2, where r is

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the particle radius, γow is the interfacial tension, and θow is the three-phase contact

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angle. On the one hand, nanoscale starch particles were prepared to reduce ∆E by

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decreasing r. On the other hand, naturally occurring micron-scale particles were

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hydrophobicized to a certain extent by chemical modification to cause θow to approach

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90o because intact micron-scale starch particles are not wettable by the oil phase

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because of their poor hydrophobicity.18 In this context, any chemical modification

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with the ability to increase the hydrophobicity of intact starch particles could favor

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their Pickering stabilizing capacities such as the esterification by octenyl succinic

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anhydride (OSA), acetic anhydride or phthalic anhydride.19,20 In this way,

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hydrophobic groups are attached mainly on the surface of the starch particles,

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conferring a wettability by the oil phase.21 Among these anhydrides, OSA was most

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widely used due to its negligible impact on the crystalline pattern of the particles, high

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efficiency in changing hydrophobicity and the reliable safety of modified

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products.22-24

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Although nano-scale starch particles were observed super capacities in

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stabilizing Pickering emulsion, the naturally occurring starch granules were also

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investigated as the Pickering stabilizers25,26. This is due to the fact that these granules

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are easy available and the large particles in Pickering emulsion stabilization certainly

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resulted in an insurmountable energy barrier to droplet shrinkage beyond a tightly

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packed monolayer of small particles27. Regarding the Pickering stabilization of

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octenylsuccinated starch granules, extensive studies have focused on the effects of 4



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emulsion environmental parameters, the plant origin of starch granules and the degree

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of substitution (DS)28-30. In reviewing these references, the following problems persist:

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1) the heterogeneity in granule size of natural starches was not fully considered in

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these investigations, although recently Saar et al.31 reported the difference between

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small and large octenylsuccinated waxy barley starch granules in stabilizing a

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Pickering emulsion; 2) the emulsion stabilization capacity of the modified granules

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was jointly controlled by the particle size and DS, but their joint effects are seldom

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determined; 3) the rationality in applying traditional mass-based DS (DSm) to weight

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the Pickering stabilization capacity should be seriously reconsidered. In principle, the

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Pickering stabilization capacity of an octenylsuccinated granule is highly dependent

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on its surface hydrophobicity or the surface density of attached hydrophobic groups

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other than simply DSm. In detail, given a specific DSm, large particles certainly present

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a higher surface hydrophobicity than their small counterparts. In this regard, we

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hypothesized that the surface density of octenyl succinic groups (SD-OSG) would be

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better in this case. However, information on these problems, and notably the

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rationality of DSm or SD-OSG is scarce to nonexistent. In this study, the effects of

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granule size, DS (octenylsuccinated) and homogenization time on the emulsifying

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capacity (EC), emulsifying stability (ES) and median droplet size (D50) of O/W

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Pickering emulsion were investigated. The sweet potato starch was selected as the

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staring material in view of its wide size distribution of granules,32,33 which is easy to

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be fractioned into three parts with significantly different particle sizes.

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■ MATERIALS AND METHODS 5


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Materials. The sweet potato starch used for this study was provided by Yangtian

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Food Co. Ltd. (Sichuan, China) with a proximate composition of amylose (25.37

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g/100 g), protein (0.27 g/100 g), lipid (0.78 g/100 g) and ash (0.25 g/100 g). Canola

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oil was obtained from Yihai Kerry Food Co. Ltd. (Chongqing, China). OSA was

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purchased from Sigma-Aldrich Chemical Co. (St. Louis. MO, USA). The other

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chemicals were all analytical grade and used as received.

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Size-fractionation of Sweet Potato Starch. According to the Stokes’ law, the

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sweet potato starch granules were fractionated into large-, medium- and small-sized

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parts by a repeated sedimentation process as described by Takeda et al.34 Starch (40 g)

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was placed in a cylinder (2.0 L, 9.0 cm inside diameter), and water was added to

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achieve a final volume of 2.0 L. By manual stirring, the starch granules were totally

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suspended, and the resultant suspension was left undisturbed at room temperature for

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8 h to allow the intended sedimentation. Then, the turbid layer above the

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sedimentation was sucked out through a pipette connected to a peristaltic pump. Water

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with an equal volume of sucked turbid layer was supplemented in the cylinder. The

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above-mentioned suspension-sedimentation-suck was repeated six times. All the

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sucked layers were combined and subjected to centrifugation at 3000 g for 10 min.

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The obtained pellet was washed in triplicate with 200 mL absolute ethanol per

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washing and then air-dried at room temperature for 48 h to obtain a small-sized

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fraction (1.7 g). The remaining sediments in the cylinder were treated in the same way

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as done for the separation of the small-sized fraction instead of a sedimentation time

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of 3 h. The medium-sized fraction (11.7 g) was obtained from the turbid layers, while 6



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the final sediment was collected as a large-sized fraction (26.6 g). The size

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distribution patterns of the starch and its fractions were recorded using a Mastersizer

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2000 (Malvern Instruments Ltd., UK) with ultrapure water as the disperse medium

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and a circulation pump operating at 3000 g .28 The results were expressed as D10, D50,

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D90 and Span.

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Octenylsuccination of Sweet Potato Starch. At present, the starch samples are

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octenylsuccinated according to the procedure described by Song et al.35 The dried

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starch sample (30 g, db) was introduced into a conical flask (150 mL) containing 70

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mL distilled water. The flask equipped with a magnetic stirrer was settled in a 35 oC

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water bath. When the starch mixture was temperature-equilibrated, a pre-settled

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amount of 25% (w/w) alcoholic OSA solution was added dropwise over 2 h.

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Subsequently, the resultant slurry was further incubated for 5 h to complete the

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intended hydrophobic modification. During OSA addition and extended incubation,

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the pH of the starch slurry was maintained at 8.4 by adding 3% (w/v) NaOH solution.

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When the modification was completed, the pH of the slurry was adjusted to 7.0 using

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3% (v/v) HCl solution. Then, the starch slurry was centrifuged at 3000 g for 10 min.

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The pellet that was obtained was washed in triplicate with distilled water. After further

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triplicate washing with absolute alcohol, the pellet was dried at room temperature for

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48 h to obtain octenylsuccinated starch. A series of octenylsuccinated products with

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varying DSm for each starch sample was obtained by applying different amounts of

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OSA (3.0%, 6.0%, 9.0%, w/w, on dry basis of starch) in the modification. The size

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distribution patterns of the modified starches were determined as were did for native 7


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starches.

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Characterization of Octenylsuccinated Starch. The DSm of the modified starch

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was determined using a titration method.22 The modified starch sample (1.0 g, db) was

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weighed accurately and dispersed in 5 mL of 2.5 M HCl-isopropyl alcohol solution by

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magnetic stirring for 30 min. Then, 20 mL 90% (v/v) aqueous isopropyl alcohol was

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added to the slurry and stirred for 10 min. Then, the slurry was filtered through a G3

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sintered funnel, and the residue was washed with 90% (v/v) aqueous isopropyl

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alcohol until Cl- was absent from the filtrate (detected by 0.1 M AgNO3 solution). The

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residue was dispersed in 60 mL distilled water and cooked in a boiling water bath for

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20 min. After cooling to ambient temperature, the starch slurry solution was titrated

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with 0.1 M standard NaOH solution with phenolphthalein as the indicator. Native

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starch was used as the blank sample. DSm was calculated using the following

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equation:

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DS m =

0.162 ×（A1 − A0）× M / W 1 − [0.210 × ( A1 − A0 ) × M / W ]

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where A1 and A0 refer to the standard NaOH solution consumed by the sample and the

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blank, respectively (mL); M is the molarity of the standard NaOH solution (mol/L);

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W is the sample weight applied (g); 0.162 refers to the molar mass of anhydroglucose

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unit (kg/mol); and 0.210 is the net increase in the mass of starch with one mole of

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octenyl succinic group substituted (kg/mol).

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The SD-OSG, defined as the molar number of octenyl succinic groups attached per

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square meter of granule surface (mol/m2), was calculated on the basis of mean

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diameter (R, m), DSm and density of starch (ρ). In order to simplify the calculation of 8



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SD-OSG, the following assumptions were introduced: 1) after the size-fraction, the

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granules in each portion are supposed with the same size; 2) the octenyl succinic

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groups were predominately and evenly decorated on the surface of the modified starch

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granules, which had been evidenced by the previous studies36-38; 3) the densities of

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various starch fractions were unified as 1.5 g/cm3 due to the insignificant difference

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among them, namely, 1.48 g/cm3, 1.50 g/cm3 and 1.44 g/cm3 for small-, medium- and

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large-sized fractions, respectively. For a specific octenylsuccinated starch with a

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quality of m (g), the number of granules contained could be calculated as

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m/[4/3π(R/2)3×ρ] = 6m/πR3ρ. Then, the total surface area of these granules was

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6m/πR3ρ×4π(R/2)2 = 6m/Rρ. Thus, the molar number of octenyl succinic groups on

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these granules could be calculated as (m/162×DSm), where 162 refers to the molar

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mass

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(m/162×DSm)/(6m/Rρ) = 1.54×103×DSm×R (mol/m2).

per

glucosyl

unit.

As

a

result,

SD-OSG could

be

expressed

as

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The three-phase contact angle (θow) of the starch sample with or without

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octenylsuccination was measured by a contact angle-measuring device (JC20000D,

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Zhongchen, China), as described previously.39 The starch sample (0.2 g) was

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compressed into a disc (10 mm diameter, thickness 1 mm) by using a tablet machine.

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Then, the resultant disc was completely dipped into a canola oil for 10 s. Then, the

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disc was picked out, and the excessive oil on its surface was removed by using a piece

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of filter paper. Subsequently, the disc was transferred onto the object platform of the

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contact angle-measuring device. A drop of MilliQ water (2 µL) was deposited on the

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surface of the starch disc via the high-precision injector mounted on the device. After 9


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a short equilibration (3 s), the drop image was recorded, and the profile of the droplet

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was numerically solved and fitted to the Laplace-Young equation. The contact angle

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was measured with three discs per sample and five measurements were performed for

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each disc.

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Octenylsuccinated size-fractioned granules were totally dispersed in water at a

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concentration of 1.0 mg/mL. The zeta potential was measured using a zetasizer

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instrument (Malvern Nano-ZS90, England), equipped with a HeNe laser beam at a

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wavelength of 637 nm and a dynamic light scattering of 90̊. Approximately 0.8 mL

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sample was added to the zeta potential cell and measured after 60 s equilibration at 25

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ºC.

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Determination of Emulsifying Capacity and Stability. To prepare O/W

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Pickering emulsion, octenylsuccinated starch was first suspended in distilled water at

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a concentration of 3% (w/v). Then, the resultant starch suspension was mixed with the

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same volume of canola oil, and the mixture obtained was homogenized at 11000 r/min

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for a preset time. The starch concentration and the volume ratio of water and oil

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phases were optimized in preliminary experiments according to their effects on the

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emulsifying capacity (EC) and particle size stability in D50 of the emulsions during

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storage (See the Supporting Information 1). When the preset time had elapsed, the

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resultant emulsion (V0, 10 mL) was immediately transferred into a 15-mL graduated

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glass cylinder (i.d. 1.5 cm). The cylinder was left undisturbed at room temperature,

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and the volume of the emulsion layer in milliliters was recorded at 24 h (V1d) and 30

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days (V30d). At the same time, the size distribution of the emulsion at 24 h was 10



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recorded as was done for the starch samples. The emulsifying capacity (EC) was

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calculated by the equation of EC=V1d/V0, while the emulsifying stability (ES) was

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defined as the volume percentage of the emulsion remaining upon 30 days of storage,

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which was calculated via the equation of ES (%)=V30d/V1d×100%.

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Recording of the Bulk Appearance and Microstructure of Emulsions. The

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bulk appearance of the resultant Pickering emulsions prepared under varying

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conditions was recorded with a camera (Sony a7, Japan). The microstructure of the

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Pickering emulsions was captured by confocal laser scanning microscopy (ZEISS

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LSM 800, Germany), which was operated in fluorescence mode. The sample

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subjected to confocal laser scanning microscopy was stained in advance according to

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the method described by Yusoff et al. with a slightly modification.40 A mixed

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fluorescent solution was applied containing Nile blue A and Nile red. They were able

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to stain starch granules and the oil phase, respectively. Nile blue A and Nile red were

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dissolved in MilliQ water in advance, and 1,2-propanedio at the same concentration of

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0.01 mg/mL and the resultant solutions were stored in a dark place. Immediately prior

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to use, the Nile blue A and Nile red solutions were mixed in a volume ratio of 1:2

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obtaining the mixed fluorescent solution. An aliquot of the mixed fluorescent solution

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(20 µL) was spiked into 1 mL of Pickering emulsion, and they were thoroughly mixed.

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Then, the stained sample (2 µL) was immediately placed into a concave slide, and the

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coverslip was carefully placed ensuring no air gap entrapped between the sample and

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coverslip. The fluorescent dyes were excited by an argon laser at 488 nm and a He-Ne

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laser at 633 nm. Images were recorded at a resolution of 1024 × 1024 pixels at 20 × 11


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magnification.

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The microstructure of the emulsion droplets was further analyzed by a Keyence

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VHX-600 super depth 3D digital microscope fitted with VH-Z20R or VH-Z100R lens

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(Keyence ,Inc., Charlotte, NC,USA). The emulsions were stained by iodine solution,

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which prepared by dissolving 0.2 g iodine and 2.0 g potassium iodide in 100 mL

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distilled water.29 A drop of the stained emulsion was carefully transferred into the

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cavity of a concave slide. Micro-images of the emulsions and single droplet were

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recorded at 150× (VH-Z20R ) and 500 × (VH-Z100R) magnifications, respectively.

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Statistical Analysis. All the measurements were conducted in triplicate unless

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specified. The results were expressed as means with standard deviation. The statistical

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significance of the difference between groups was tested by one-way analysis of

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variance (ANOVA) using SPSS19.0（SPSS Inc., Chicago, IL, USA). In all statistical

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analyses, p

Joint Effects of Granule Size and Degree of Substitution on

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