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Interface Components: Nanoparticles, Colloids, Emulsions, Surfactants, Proteins, Polymers

Properties of Ternary Biopolymer Nanocomplexes of Zein, Sodium Caseinate, and Propylene Glycol Alginate and Their Function of Stabilizing High Internal Phase Pickering Emulsions Cuixia Sun, Yanxiang Gao, and Qixin Zhong Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b01887 • Publication Date (Web): 06 Jul 2018 Downloaded from http://pubs.acs.org on July 6, 2018

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Properties of Ternary Biopolymer Nanocomplexes of Zein, Sodium Caseinate, and Propylene Glycol Alginate and Their Function of Stabilizing High Internal Phase Pickering Emulsions Cuixia Sun1,2, Yanxiang Gao*1, Qixin Zhong*2 1

Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing Laboratory

for Food Quality and Safety, Beijing Key Laboratory of Functional Food from Plant Resources, College of Food Science & Nutritional Engineering, China Agricultural University, Beijing 100083, China. 2

Department of Food Science, University of Tennessee, Knoxville, TN, 37996, USA.

*1Corresponding author. Tel.: + 86-10-62737034; Fax: + 86-10-62737986 E-mail: [email protected] *2Corresponding author. Tel.: +1 8659746196; Fax: +18659747332 E-mail: [email protected]

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ABSTRACT: A pH-cycle method based on preparing an alkaline solution of zein followed by neutralization with acid can be used to prepare zein nanoparticles. In the present work, partial alkaline hydrolysis of propylene glycol alginate (PGA) to lower pH was studied to prepare binary zein-PGA nanocomplexes and ternary complexes with additional sodium caseinate (NaCas). 0.5% or more PGA was sufficient to reduce pH to 7.5 or lower, eliminating the need of titration, and resulted in simultaneous nanocomplex formation. Addition of NaCas in alkaline zein-PGA solution resulted in smaller complexes with all biopolymers, whereas adsorption on binary zein-PGA complexes was observed when NaCas was added in the neutral zein-PGA dispersions. Formation of nanocomplexes involved with hydrophobic and electrostatic attractions and hydrogen bonds and was further affected by the amount of NaCas. The ternary nanocomplexes with equal masses of zein and NaCas had excellent capacity to prepare gel-like Pickering emulsions with as much as 80% v/v oil with characteristics suitable for texture modification and delivery systems of bioactive compounds in food and consumer products. Therefore, PGA can be used to possibly scale-up the pH-cycle method to produce zein-based nanoparticles with unique functional properties. KEYWORDS: Zein; propylene glycol alginate; sodium caseinate; nanocomplex; pH cycle; Pickering emulsions


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INTRODUCTION Zein, the major seed storage protein of maize, is a generally recognized as safe (GRAS) food ingredient with many advantages such as environmental friendliness, biodegradability, nontoxicity, edibility, and versatility in applications.1 Zein contains a large proportion (more than 50%) of non-polar amino acid residues2 and is thus water-insoluble. Zein is readily soluble in aqueous ethanol solutions,3 and therefore, anti-solvent precipitation (ASP) is commonly applied to prepare zein nanoparticles,4 which has long been investigated to be a vehicle for many bioactive compounds such as quercetagetin,5 curcumin,6 procyanidins,7 resveratrol,8 and vitamin D3.9 However, about 55%-95% (v/v) ethanol is used to dissolve zein and in turn it has to be removed after ASP, which may cause flammability concerns and increase the production cost. On the basis that zein is soluble in an alkaline aqueous solution at pH 11.3-12.7,10 a pH-cycle method was created in our previous study to encapsulate hydrophobic curcumin in sodium caseinate (NaCas)11 and to prepare zein-NaCas nanocomplexes,12 based on first increasing the mixture pH to dissolve zein and curcumin/NaCas followed by lowering acidity to neutral pH to precipitate zein as self-assembled nanoparticles. There are still drawbacks to be improved to scale up the pH-cycle method. Acidification of alkaline protein solutions by dropwise addition of an acid causes initial inhomogeneity in proton distribution and therefore possible structural heterogeneity of precipitated protein nanoparticles. In addition, the process of titrating the sample can be time-consuming and tedious. D-glucono-δ-lactone (GDL) is gradually hydrolyzed in water, and the produced gluconic acid lowers solution pH.13,14 Our recent study shows that hydrolysis of GDL results in smaller and more uniform zein nanoparticles and therefore better storage stability than those acidified with HCl.15 However, GDL acidification only lowers the pH


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of aqueous alkaline zein solution to form zein nanoparticles, and no modification of particle properties was found. Propylene glycol alginate (PGA) is a high molecular weight linear polysaccharide prepared from alginic acid by treatment under acidic conditions with propylene oxide.16 PGA shows a unique surface activity with the combination of emulsifying and thickening properties, is recognized as a harmless food additive, and is commonly applied in a variety of food manufacturing processes to produce the creamier foam of beer, stabilize emulsions, and suspend particles in liquids such as fruit pulp drinks.17 Martinez et al. 18 reported that the incorporation of PGA (0.5 wt%) strongly increased the foam stability of casein glycomacropeptide (4.0 wt%) against drainage and collapse. Besides its functional properties, interactions between PGA and proteins have been studied under different pH conditions. Dickinson & Euston19 reported experimental evidences of non-covalent interfacial complexation between PGA and milk proteins at pH 7.0. Weak electrostatic complexes between PGA and food proteins (β-casein, βlactoglobulin, gelatin or soy protein) were also reported at the air-water interface under neutral pH conditions.20 In addition to water soluble proteins, non-covalent interaction between PGA and zein led to the formation of zein-PGA binary complexes at both pH 4.0 21 and pH 7.0.22 PGA contains two 2-hydroxyprop-1-yl primary ester groups and 1-hydroxyprop-2-yl secondary ester groups.23 Hadef et al.

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found the formation of amide bonds between free amine groups of

human serum albumin and ester groups of PGA at alkaline conditions. Mohamed & Stainsby23 revealed that under alkaline conditions, hydrolysis of the ester groups in PGA took place, which resulted in the release of alginic acid and fragments of the starting materials. Based on these findings, a hypothesis is proposed in the present work that zein nanoparticles may be produced by adding PGA into aqueous alkaline zein solutions with a high pH (>11.3) because alginic acid


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released by alkaline hydrolysis of PGA can also lower pH and may improve homogeneity of proton introduction and therefore uniformity of zein nanoparticle dimension. Additionally, dispersions with zein nanoparticles prepared by the pH-cycle method using HCl were unstable, while the addition of NaCas to form composite nanoparticles resulted in the improved storage stability of zein dispersions.12 Therefore, one objective of the present work was to investigate the effect of PGA alkaline hydrolysis on the formation and characteristics of zeinNaCas nanocomplexes. Two different sequences of PGA addition were studied: with premixed zein and NaCas or with zein alone first followed by NaCas, which may affect the interaction and structure of ternary biopolymer nanocomplexes. The second objective of the present study was to investigate the possibility of ternary nanocomplexes stabilizing emulsions. Pickering emulsions, known to be stabilized by solid particles instead of molecular surfactants,25 have gained much interest in recent years due to the resistance to coalescence because of the irreversible adsorption of solid particles on the oil-water interface.26 Zein nanoparticles prepared by the ASP have been studied to stabilize Pickering emulsions,27 as well as composite particles of zein and NaCas,28 chitosan,29 stearate,30 and tannic acid.31 These emulsions can be studied as delivery systems of bioactive compounds as the oil phase serves as a solvent of lipophilic bioactive compounds.32,33 Compared to the previous reports, zein-NaCas-PGA ternary nanocomplexes prepared by the pHcycle method may have unique properties as natural interfacial stabilizers for Pickering emulsions when compared with binary complexes. Findings from this work may provide a promising novel strategy to produce nanoparticles and a new insight to design relevant food and consumer products.


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EXPERIMENTAL SECTION Materials Zein was purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). NaCas was purchased from American Casein Company (Burlington, NJ, USA). PGA (NLS-K) with a 87.9% degree of esterifying carboxyl groups and a molecular weight of 5.6 ×106 Da determined by DAWN EOS was kindly provided by Hanjun Sugar Industry Co. Ltd. (Shanghai, China). Soybean oil (Wesson®, ConAgra Brands, Chicago, IL, USA) was obtained from a supermarket in Knoxville, TN, USA. Other chemicals were obtained from Fisher Scientific (Pittsburgh, PA, USA). Preparation of Zein-NaCas-PGA Ternary Nanocomplexes Two different PGA incorporation sequences were studied to prepare zein-NaCas-PGA ternary nanocomplexes at room temperature (RT, about 21 °C). In the Method 1 (M1), zein (1.0 g) and NaCas (0.25, 0.5, 1.0, 1.5 and 2.0 g) were mixed with 100 mL distilled water with pH adjusted to 12.5 using 1.0 M NaOH and stirred at 700 rpm for 30 min on a magnetic stir-plate until no visible particulates, followed by dissolving PGA (0.25, 0.5, 1.0, 1.5 and 2.0 g) by stirring at 900 rpm for 30 min until completely dissolved. The pH of final colloidal dispersions was measured with an AE150 benchtop pH meter (Fisher Scientific). In the Method 2 (M2), zein (1.0 g) was mixed with 100 mL distilled water with pH adjusted to 12.5 using 1.0 M NaOH and stirred at 700 rpm for 30 min, followed by dissolving different amounts of PGA (0.25, 0.5, 1.0, 1.5 and 2.0 g) as above. Afterwards, NaCas (0.25, 0.5, 1.0, 1.5 and 2.0 g) was mixed with the zein-PGA mixture by stirring at 900 rpm for 40 min on a magnetic stir-plate. Liquid dispersions were stored in a refrigerator at 4 °C for further analysis or were frozen and freeze-dried for 48 h


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using an AdVantage Plus EL-85 benchtop freeze dryer (VirTis, SP Scientific, Gardiner, NY, USA) to obtain powders for further characterization. The prepared ternary nanocomplexes were named as M1-zein-NaCasx:y-PGA or M2-zeinNaCasx:y-PGA, with the subscripts representing the zein:NaCas mass ratio. For the control sample without NaCas, it was termed as zein-PGA. Dimension and Zeta-Potential of Dispersions The hydrodynamic diameter (Dh), polydispersity index (PDI), and zeta-potential of freshly prepared dispersions were determined using a combined dynamic light scattering (DLS) and particle electrophoresis instrument (Zetasizer Nano-ZS90, Malvern Instruments Ltd., Worcestershire, UK) according to our previous study.22 Dispersions were diluted 10 times with distilled water (pH 7.5) before measurement to avoid multiple particle effects. The Dh was calculated using the Stokes-Einstein equation. The zeta-potential of dispersions was obtained using the Smoluchowski model through electrophoretic mobility measurements performed in a capillary electrophoresis device inserted into the DLS instrument. All measurements were carried out at RT and each sample was analyzed in triplicate. Turbidity Sample turbidity was evaluated according to our previous study34 by measuring absorbance at 500 nm (A500). The instrument was a UV-vis spectrophotometer (model Evolution 201, Thermo Scientific, Waltham, MA, USA) with a 1 cm light path-length.


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Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) The morphology of the lyophilized samples was observed using FIB-SEM (Zeiss Auriga, Jena, Germany) at an acceleration voltage of 5.0 kV. Prior to the observation, a thin layer (45 mV. As zein has an isoelectric point at pH 6.2-6.5

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and is cationic at the dispersion pH of 9.0 (Figure

1A), the negative zeta-potential of the dispersion resulted from anionic PGA that has a higher extent of protonation at a lower pH (with a higher amount of PGA used in sample preparation), which results in a lower zeta-potential magnitude (Figure 1E) and therefore a larger effective volume (Dh) of particles (Figure 1C). At 0.25% and 0.5% w/v PGA, the Dh followed the order of zein-PGA < M1-zein-NaCas1:1PGA < M2-zein-NaCas1:1-PGA (P < 0.05), while zein-PGA and M2-zein-NaCas1:1-PGA had bigger Dh than M1-zein-NaCas1:1-PGA (P < 0.05) at 1.0% and higher PGA (Figure 1C). The data


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suggests the M1-zein-NaCas1:1-PGA dispersion, with the smallest size, may have a dimension closest to the Dh of PGA and therefore a less extent of depletion flocculation corresponding to the absence of precipitation (Figure 1F), as discussed in the previous section. The measured “Dh” of PGA alone dispersions significantly (P < 0.05) increased from 493.2 ± 5.0 nm to 1055.6 ± 71.7 nm with an increase in PGA concentration from 0.25% to 0.5%, which reached to 5210 ± 586.9 nm at 2.0% w/v PGA (Figure 1D). This observation likely results from the overlap of PGA chains that can self-assemble as multi-biopolymer structures. Compared to pH 7.5 dispersions with 0.5% PGA alone (Figure 1D), all dispersions with zein had a smaller Dh, which indicates the formation of binary zein-PGA and ternary zein-NaCas-PGA nanocomplexes. This was further confirmed with particulates observed in SEM that showed bigger dimensions of ternary than binary nanocomplexes (Figure 2), further suggesting the formation of ternary complexes. Additionally, the bigger particles of M2-zein-NaCas1:1-PGA than M1-zein-NaCas1:1PGA suggest the adsorption of NaCas on initially formed binary complexes,44 different from the involvement of all three biopolymers simultaneously to form nanocomplexes in M1 (Figure 3). In our recent study, 22 the Dh of zein-PGA binary complexes (1239.3 nm) prepared with 1.0% zein and 0.5% w/v PGA using the ASP method at pH 7.0 was much bigger than that of the present study (Figure 1C), which can result from both the alkaline hydrolysis of PGA and the uniformity of particle growth due to homogeneous distribution of protons to induce zein precipitation in this study, as discussed previously. The zein-PGA dispersions with 0.25% and 0.50% w/v PGA had a Dh of 76.9±1.8 and 120.8±1.5 nm, respectively.4 which was similar to the Dh of zein-PGA binary nanocomplexes. In another recent study, the respective Dh of zeinNaCas1:1 binary nanocomplexes at pH 7.5 prepared by GDL and HCl acidification was 73.1 ± 0.49 and 105.2 ± 1.2 nm,15 which was smaller than that of M1-zein-NaCas1:1-PGA (Figure 1C),


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indicating the formation of ternary nanocomplexes. Therefore, the present study suggests the feasibility of the pH-cycle method based on hydrolysis of PGA to produce nanoparticles. Overall, the pH of dispersions was around 7.5 at the PGA level of 0.5% (w/v) despite of two different incorporating sequences of PGA, and the dispersions exhibited a relatively small Dh, low turbidity, high zeta-potential, and good stability (Figure 1). Therefore, 0.5% w/v PGA was selected for further experiments. Effect of zein-NaCas mass ratio on dispersion turbidity and complex structures The mass ratios of zein:NaCas (4:1, 2:1, 1:1, 2:3 and 1:2) were studied at a PGA level of 0.5% w/v and 1.0% w/v zein. The A500 of fresh dispersions prepared by M1 and M2 is shown in Figure 2A. The NaCas addition initially lowered dispersion turbidity until no further reduction (P > 0.05) when the NaCas concentration exceeded that of zein, which was in good agreement with our previous reports with two biopolymers.

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For zein-NaCas-PGA ternary nanocomplex

dispersions, those prepared by M1 exhibited a much higher turbidity than those of M2. With an increase in NaCas content, particles in freeze-dried samples became less spherical, less discrete, and bigger. As PGA alone exhibited a fine filamentous structure,

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particulate

structures of samples confirmed nanocomplex formation. Less discrete particles can result from excess free biopolymers such as NaCas depositing on particulates during freeze-drying. Overall, particles formed with M1 were more populous, more spherical, smaller, and more discrete than those of M2, verifying most NaCas was involved in nanocomplex formation in M1 but adsorbed on binary nanocomplexes in M2. As dispersion turbidity can be contributed by particle dimension, light diffraction properties, and volume fraction, more turbid samples of dispersions


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prepared with M1 than M2 (Figure 2A) can be contributed by more abundant but smaller particles of M1. Physical stability of dispersions To evaluate physical stability of M1 dispersions, the size distribution and zeta-potential of dispersions are compared in Figure 4 after storage at 4 °C for 1 and 30 days. The particle size distribution of zein-PGA binary nanocomplexes was monomodal, which was right-shifted toward the larger particle size after storage, indicating that aggregation of nanocomplexes took place. The size distribution of M1-zein-NaCas-PGA ternary nanocomplexes on day 1 was much narrower than that of zein-PGA binary nanocomposites. At a lower level of NaCas (zein:NaCas mass ratios of 4:1, 2:1, and 1:1), the particle size distributions of M1-zein-NaCas-PGA ternary nanocomplexes were monomodal (Figure 4B, C and D), which also right-shifted after 30-day storage. At zein-NaCas mass ratios of 2:3 and 1:2, the distribution was multimodal, and the rightshift was less (Figure 4E and F). The smaller group of particles likely are excess NaCas.45 The monomodal distribution of particle size further indicates the involvement of all biopolymers in nanocomplexes at a zein:NaCas mass ratio from 1:0 to 1:1. Besides, no significant changes of PDI of dispersions at pH 7.5 after 30 day storage were observed (supporting information Figure S1), especially for the M1-zein-NaCas1:1-PGA dispersion, indicating the significance of NaCas in formation of stable nanocomplexes. The zeta-potential of dispersions at pH 7.5 after 1 and 30 day storage is presented in Figure 4G. Most samples showed a decrease of negative zeta-potential magnitude after storage, and the reduction became less significant at a higher amount of NaCas. The trend could result from increases in particle size (Figure 4 A-F) that also showed less significant changes with increasing


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NaCas concentration, as bigger particles with similar surface structures have a smaller electrophoretic mobility.

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Furthermore, as a higher magnitude of zeta-potential can result in

better stability against particle aggregation, but this expectation is opposite to the particle size change trend, the better particle size stability of dispersions with a higher amount of NaCas (with a smaller magnitude of zeta-potential) indicates that NaCas on particle surface provides steric repulsion against particle aggregation.12 Complex structures studied with fluorescence and FTIR spectroscopy Fluorescence spectroscopy is commonly used to detect the fluorescence property of tryptophan (Trp) residues, which are sensitive to the folding and unfolding of proteins,47 and therefore is used to characterize conformational changes of proteins. Fluorescence spectra of M1 dispersions are shown in Figure 5A. Zein exhibited a typical fluorescence emission peak at 286 nm after being excited at 280 nm, which is consistent with our recent study.15 It should be mentioned that the fluorescence intensity of zein in the presence of PGA was much lower than that of zein-only dispersion prepared with GDL but much higher than that of zein-only dispersion prepared with HCl.15 The results indicate different acidification methods in the pHcycle to precipitate zein can result in different conformations of zein. No distinct shift in the wavelength of maximum emission was observed but the fluorescence intensity of zein was significantly (P < 0.05) decreased in the presence of NaCas at different concentrations, indicating the conformational changes of zein and complex formation with the other two biopolymers.48 Similar observations were reported for λ-carrageenan/whey protein concentrate49 and β-lactoglobulin/chitosan complexes.50,51 The higher fluorescence intensity of the M1-zeinNaCas1:2-PGA dispersion than those with zein:NaCas mass ratios of 1:1 and 2:3 can result from excess NaCas with Trp residues.


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FTIR is applied to study potential interactions among zein, PGA, and NaCas. As shown in Figure 5B, individual zein and PGA exhibited a broad characteristic peak at 3289.0 and 3362.9 cm-1, respectively, which is attributed by hydroxy stretching vibration.52 No obvious shift of the characteristic peak of zein at 3362.9 cm-1 was observed after PGA was added to zein, indicating that insignificant hydrogen bonds were formed between PGA and zein. However, the combination of zein, PGA, and NaCas induced a slight shift of hydrogen bands, and the peaks were located at 3287.1, 3287.7, 3284.9, 3285.0, and 3286.8 cm-1 in the spectra of M1-zeinNaCas-PGA nanocomplexes with zein:NaCas mass ratios of 4:1, 2:1, 1:1, 2:3, and 1:2, respectively (Figure 5C). The findings revealed that hydrogen bonds may be an additional mechanism responsible for formation of ternary complexes, as proposed for pectin-gelatin53 and soy protein-agar54 complexes. Additionally, PGA had a peak at 1736.7 cm-1, and this peak became weakened for the zeinPGA and ternary complexes and slightly shifted. The band at 1800-1650 cm-1 is mostly contributed by carbonyl groups.55 As partial alkaline hydrolysis of PGA leads to the release of alginic acid and therefore a higher amount of carbonyl groups, the weaking of this FTIR band indicates the binding between carbonyl group and cationic amine groups of proteins,56 signifying the possible involvement of electrostatic attraction in complex formation. The band of 1500-1700 cm-1 corresponds to amide I (1600-1700 cm-1) and amide II groups (1400-1500 cm-1), which is due to stretching vibrations of C-O groups and the bending vibration of N-H groups, respectively.57 The amide I and amide II bands of zein were observed at 1644.4 and 1515.9 cm-1, respectively. There was no obvious change of amide I band after PGA and NaCas addition, but the amide II band of zein was significantly shifted to 1535.2 cm-1. These findings were indicative of hydrophobic interactions between zein, with more than 50% of


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hydrophobic amino acids,2 and the propylene glycol groups of PGA16 and hydrophobic amino acid residues of NaCas.58 The bands at 750-1300 cm-1 correspond to the C-O stretching vibrations, and peaks at around 1045 cm-1 are associated with the stretching of C-O linkage in C1 and C3.54 PGA alone showed a sharp peak at 1032.0 cm-1, which was slightly shifted to 1035.8 cm-1 for zein-PGA binary complexes. The blue shift was more significant for ternary nanocomplexes with a higher NaCas concentration, to 1036.5, 1038.4, 1039.6, 1042.9, and 1079.6 cm-1 at mass ratios of 4:1, 2:1, 1:1, 2:3, and 1:2, respectively. The results indicate the non-covalent complexation between PGA and both zein and NaCas. Thermal properties studied with DSC DSC was carried out to characterize thermal stability of samples. The smooth thermograms (Figure 6) indicate the inherent amorphous structures of zein and binary and ternary complexes. 12

Native zein and PGA presented an endothermic peak at 90.2 and 105.1 °C, respectively, which

was shifted to an intermediate temperature of 99.7 and 102.4 °C for the binary and M1-zeinNaCas1:1-PGA ternary complexes, respectively. The broad endothermic peak observed at around 100 °C is attributed to removing water in the specimen, leading to the degradation of the biopolymer network.59 The results indicated that the addition of PGA and NaCas lowered the mobility of zein chains, resulting in an increased activation energy to evaporate water molecules and thermal stability of zein. A second exothermic peak between 150 and 200 °C suggests the glass transition of samples.60 Native zein and PGA showed a glass transition temperature (Tg) of 154.1 and 153.2 °C, respectively, which almost disappeared for the binary and M1-zein-NaCas1:1-PGA ternary


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nanocomplexes. This observation can result from plasticization of polymer chains by associated more flexible polymers.61 Function of complexes for preparing Pickering emulsions Zein-PGA binary and M1-zein-NaCas1:1-PGA ternary nanocomplexes were used to prepare emulsions. As shown in Figure 7A, the emulsion prepared with binary nanocomplexes at an oil volume fraction of 40% showed macroscopic phase separation. The top emulsion phase was uniformly dispersed in water, indicating the formation of an O/W emulsion. After inverting the sample, the emulsion phase did not slip, indicating a self-standing gel. The CLSM data showed individual purple/pink droplets that reflect blue fluorescence of stained proteins and red fluorescence of stained oil, suggesting the adsorption of complexes on oil droplets and therefore the formation of a Pickering emulsion.25 As oil volume fraction increased to 50%-80%, phase separation and free oil were observed, indicating the emulsifying capacity of binary complexes had been exceeded. In comparison, the emulsions prepared with ternary nanocomplexes did not show free oil at an oil volume fraction of 40-80% (Figure 7B) and were verified to be O/W emulsions. Increasing the oil volume fraction to 81% resulted in an inhomogeneous sample (Figure 7B) verified to be a W/O emulsion, indicating the occurrence of phase inversion between 80 and 81% v/v oil.62 The better emulsifying capacity of ternary than binary complexes can result from the excellent emulsifying activity of NaCas.63 CLSM data showed individual oil droplets coated with complexes and packed closely (Figure 8), verifying the formation of Pickering emulsions. The non-sphericity, deformability, and heterogeneity of oil droplets (Figure 8) can result in a dispersed phase volume fraction higher than 74% of a colloidal dispersion with monodispersed


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particles in hexagonal arrangement.

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When laid on bench top, an emulsion prepared with a

higher volume fraction of oil spread to a smaller area (Figure 7B), indicating the more solid-like properties, and no free oil was observed for all samples indicating the relative strength of emulsion droplet structures. The solid-like properties were verified in rheology (Figure 9) that overall showed stronger moduli at a higher oil volume fraction, as expected for colloidal dispersions. 65 A higher frequency-dependence of emulsions with a higher oil volume fraction is expected due to deformability of oil droplets. These properties indicate the extraordinary properties of nanocomplexes in emulsifying oils as high internal phase emulsions with as much as 80% v/v oil or a complex: oil mass ratio as small as 1:30, which compares well with the literature. Pickering emulsions with 27% v/v oil were reported for zein/gellan gum complexes at a complex:oil mass ratio of 1:2.8.

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Another

study on zein/chitosan complexes reported Pickering emulsions with 50% v/v oil at a complex:oil mass ratio of 1:8.8.67 Therefore, zein-NaCas-PGA ternary nanocomplexes in this study exhibited superiority in stabilizing Pickering emulsions. CONCLUSIONS The partial alkaline hydrolysis of PGA was proved to be a feasible way to lower pH to induce simultaneous precipitation of zein and self-assembly of binary nanocomlexes or ternary complexes with additional NaCas. The sequence of NaCas addition impacted the structures of nanocomplexes. Addition of NaCas in alkaline solution with zein and PGA resulted in nanocomplexes involving with three biopolymers in the matrix, contrasting with adsorption of NaCas on surface of initially formed zein-PGA binary complexes that increased particle size and decreased dispersion stability. Formation of nanocomplexes involved with hydrophobic and


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electrostatic attractions, as well as hydrogen bonds. Nanocomplex structures were further tunable by the amount of NaCas. The M1-zein-NaCas1:1-PGA ternary nanocomplexes were proved to be an excellent stabilizer to prepare high internal phase Pickering emulsions. Such Pickering emulsions exhibited a mayonnaise-like appearance and gel-like structures, suggesting their potential application for texture modification and the development of delivery systems for bioactive compounds in the food and consumer products industries. These findings suggest that PGA can be used an approach to possibly scale-up the pH-cycle method without using any organic solvents and tedious titration process to produce zein-based nanoparticles with unique functional properties.


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Supporting Information The supporting information is available free of charge on the ACS Publication website at DOI: Polymer dispersity index (PDI) (Figure S1).

AUTHOR INFORMATION Corresponding Author *1E-mail: [email protected] *2E-mail: [email protected] Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENT Financial support from the National Natural Science Foundation of China (No. 31371835) is gratefully acknowledged. This work was also partially funded by the University of Tennessee Institute of Agriculture and the USDA NIFA Hatch Project 223984. The authors thank Dr. Scott Lenaghan for the training and use of the CLSM.


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Zein-PGA M1 Zein-NaCas1:1-PGA

A 14

B

Zein-PGA M1 Zein-NaCas1:1-PGA

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-15

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ABa Ba zein-PGA M1 zein-NaCas1:1-PGA M2 zein-NaCas1:1-PGA

Figure 1. Equilibrium pH of dispersions with 1% w/v zein, 1% w/v NaCas, and 0.25-2.0% w/v PGA (A) and the corresponding absorbance at 500 nm (A500, B), mean hydrodynamic diameter (Dh) of zein-based complex dispersions (C) and PGA alone dispersions diluted to the same levels at pH 7.5 (D), zeta-potential (E), and the appearance (F) of fresh dispersions. The dispersions were prepared by adding PGA to zein-NaCas mixture at pH 12.5 (method 1 – M1) or adding PGA to zein solution at pH 12.5 followed by dissolving with NaCas (method 2 – M2). Control zein-PGA dispersions were similarly prepared without NaCas. Different uppercase letters in plots C, D, and E represent significant difference (P  0.05) among the dispersions prepared with the same method, and different lowercase letters in plots C and E represent significant difference (P  0.05) among three kinds of dispersions with the same lever of PGA.


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A

Figure 2. Absorbance at 500 nm (A500) (A) and SEM images (B) of dispersions prepared with 0.5% w/v PGA, 1% w/v zein, and NaCas at a zein:NaCas mass ratio from 1:0 to 1:2. The dispersions were prepared by adding PGA to zein-NaCas mixture at pH 12.5 (method 1 – M1) or adding PGA to zein solution at pH 12.5 followed by dissolving with NaCas (method 2 – M2). Scale bar= 1 μm.


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A

+

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zein-PGA binary nocomplex

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+

pH 12.5 to 7.5

+ NaCas

mixture of zein and PGA

zein-PGA-NaCas ternary nanocomplex

zein

sodium caseinate (NaCas)

propylene glycol alginate (PGA)

precipitated zein

Figure 3. Proposed self-assembled structures during acidification due to partial alkaline hydrolysis of PGA to lower pH to induce precipitation of zein to form zein-PGA binary nanocomplexes (A), and zein-NaCas-PGA ternary nanocomplexes prepared by method 1 (B) and method 2 (C).


Langmuir

A

1d 30d

zein-PGA

20

Intensity (%)

18

Intensity (%)

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-10

zein

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1:1

2:3

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-20

Bb

-30

Ab -40

Ab

Ab Ba

Ca Aa

Ca

Ba

Ba Ca 1d 30d

Aa

Figure 4. Size distribution (A-F) and zeta-potential (G) of dispersions prepared with 0.5% w/v PGA, 1% w/v zein, and NaCas at a zein:NaCas mass ratio from 1:0 to 1:2 using method 1, after 4 °C storage for 1 and 30 days. Different uppercase letters in plot G represent significant difference (P  0.05) among the dispersions with different mass ratios of zein to NaCas, while different lowercase letters in plot G represent significant difference (P  0.05) among the same dispersion after storage for 1 and 30 days.


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A

zein-PGA zein-NaCas4:1-PGA

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zein-NaCas2:1-PGA

800

zein-NaCas1:1-PGA zein-NaCas2:3-PGA

Intensity

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2000

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1038.4

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Figure 5. Fluorescence (A) and FTIR (B, C) spectra of zein, PGA, zein-PGA binary nanocomplexes, and M1-zein-NaCas-PGA ternary nanocomplexes with a zein:NaCas mass ratio from 1:2 to 4:1.


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Heat flow (w/g) Exo

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0.00

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Figure 6. DSC thermograms of zein, PGA, zein-PGA binary nanocomplexes, and M1-zein-NaCas1:1-PGA ternary nanocomplexes.


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Figure 7. Photographs of gel-like emulsions stabilized by zein-PGA binary nanocomplexes (A) and M1-zein-NaCas1:1-PGA ternary nanocomplexes (B) at oil volume fractions of 40%, 50%, 60%, 70%, 80%, and 81%. The photographs were taken 7 days after storage at 21 C.


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Oil volume fractions (v/v)

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60%

70%

80%

Figure 8. CLSM images of emulsions stabilized by M1-zein-NaCas1:1-PGA ternary nanocomplexes at oil volume fractions of 50%, 60%, 70%, and 80% after storage at 21 C for 1 day.


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1000

A

1000

G G

50% oil

800

700

700

600 500 400 300

600 500 400 300

200

200

100

100

0 0.1

1000

1 10 Frequency (rad/s)

0 0.1

100

1

10

100

Frequency (rad/s)

G G

C 70% oil

900

G G

B 60% oil

900

800

G and G(Pa)

G and G (Pa)

900

1000

800

G G

D 80% oil

800

G and G (Pa)

700

Gand G (Pa)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

600 500 400 300 200

600

400

200

100 0 0.1

0

1 10 Frequency (rad/s)

100

0.1

1

10

Frequency (rad/s)

100

Figure 9. Mechanical spectra at 21 °C for emulsions stabilized by M1-zein-NaCas1:1-PGA ternary nanocomplexes at oil volume fractions of 50% (A), 60% (B), 70% (C), and 80% (D).


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

For Table of Contents Use Only

Ternary Biopolymer Nanocomplexes of zein,sodium caseinate and Propylene Glycol Alginate Prepared by a pH-Cycle Method to Stabilize High Internal Phase Pickering Emulsions Cuixia Sun1,2, Yanxiang Gao*1, Qixin Zhong*2 1

Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing Laboratory

for Food Quality and Safety, Beijing Key Laboratory of Functional Food from Plant Resources, College of Food Science & Nutritional Engineering, China Agricultural University, Beijing 100083, China . 2

Department of Food Science, University of Tennessee, Knoxville, TN 37996, USA.

+

pH 12.5 to 7.5

+

mixture of zein and NaCas

PGA zein-NaCas-PGA ternary nanocomplex SEM

CLSM

Pickering emulsions zein sodium caseinate (NaCas) propylene glycol alginate precipitated zein (PGA)


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Properties of Ternary Biopolymer Nanocomplexes ... - ACS Publications

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