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Encapsulation of protein nanoparticles within alginate microparticles: Impact of pH and ionic strength on functional performance
Accepted Manuscript Encapsulation of protein nanoparticles within alginate microparticles: Impact of pH and ionic strength on functional performance Liqiang Zou, Zipei Zhang, Ruojie Zhang, Wei Liu, Chengmei Liu, Hang Xiao, David Julian McClements PII:
S0260-8774(16)30010-3
DOI:
10.1016/j.jfoodeng.2016.01.010
Reference:
JFOE 8448
To appear in:
Journal of Food Engineering
Received Date: 2 December 2015 Revised Date:
7 January 2016
Accepted Date: 14 January 2016
Please cite this article as: Zou, L., Zhang, Z., Zhang, R., Liu, W., Liu, C., Xiao, H., Julian McClements, D., Encapsulation of protein nanoparticles within alginate microparticles: Impact of pH and ionic strength on functional performance, Journal of Food Engineering (2016), doi: 10.1016/j.jfoodeng.2016.01.010. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Encapsulation of protein nanoparticles within alginate
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microparticles: Impact of pH and ionic strength on
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functional performance
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Liqiang Zoua1, Zipei Zhangb1, Ruojie Zhangb, Wei Liua*, Chengmei Liua, Hang
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Xiaob, David Julian McClementsb,c*
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a
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Nanchang, No. 235 Nanjing East Road, Nanchang 330047, Jiangxi, China
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b
Department of Food Science, University of Massachusetts, Amherst, MA 01003
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Department of Biochemistry, Faculty of Science, King Abdulaziz University, P. O.
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Box 80203 Jeddah 21589 Saudi Arabia
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State Key Laboratory of Food Science and Technology, Nanchang University,
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* corresponding author:
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Wei Liu, State Key Laboratory of Food Science and Technology, Nanchang
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University, Nanchang, 330047, Jiangxi, China Tel: + 86 791 88305872x8106. Fax:
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+86 791 88334509. E-mail:
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[email protected].
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These authors contributed equally to this manuscript.
David Julian McClements, Department of Food Science, University of Massachusetts,
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Amherst, MA 01003, USA Tel: (413) 545-1019. Fax: (413) 545-1262. E-mail:
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[email protected].
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Journal: Journal of Food Engineering
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Submitted: December 2015
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Abstract Incorporation of bioactive proteins into functional foods is often challenging due to their instability to aggregation, sedimentation, or hydrolysis.
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core-shell protein nanoparticles, consisting of a zein core and a whey protein shell,
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were fabricated by antisolvent precipitation.
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incorporated into biopolymer microgels fabricated by electrostatic complexation of
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casein and alginate.
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5.5), but released at higher pH (6 to 7) due to microgel dissociation promoted by
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electrostatic repulsion between anionic casein and alginate.
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useful for retaining and protecting protein nanoparticles within acidic environments
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(e.g., stomach), but releasing them under neutral environments (e.g., small intestine).
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Protein nanoparticles were retained within microgels over a wide range of ionic
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strengths (0 to 2 M NaCl, pH 5).
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microgels may improve their pH and salt stability in functional foods.
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These microgels may be
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Protein nanoparticle encapsulation within
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Keywords: zein nanoparticle; hydrogel particle; stability; delivery system.
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The protein nanoparticles were then
Protein nanoparticles were retained in microgels at low pH (3 to
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In this study,
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1. Introduction There is growing interest in the utilization of proteins and peptides (referred to collectively as “polypeptides” for convenience) as functional ingredients in foods
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because of their beneficial health effects, such as antioxidant, antimicrobial, and
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anti-hypertension activities (Hernandez-Ledesma et al., 2011; Samaranayaka and
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Li-Chan, 2011; Sarmadi and Ismail, 2010; Udenigwe and Aluko, 2012).
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the activity of polypeptides depends on their three-dimensional structures and specific
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amino acid sequences (Udenigwe and Aluko, 2012).
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therefore be altered in food products during manufacture, storage, or transportation,
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due to changes in solution or environmental conditions that alter protein structure,
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such as pH, ionic strength, ingredient interactions, or temperature (Hettiarachchy et al.,
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2012).
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through the gastrointestinal tract (GIT) because they are exposed to digestive enzymes
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(proteases and peptidases) and environmental conditions (pH, ionic strength, and
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ingredient interactions) that may alter their structure (Mohan et al., 2015).
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the GIT, digestive enzymes and highly acidic gastric conditions may hydrolyze
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polypeptide chains at particular bond locations, thereby generating new peptides
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(Moreno, 2007).
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of an ingested polypeptide, or it may be desirable if it leads to the generation of new
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peptides with improved bioactivity. Consequently, it is often important to design food
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matrices that can control the gastrointestinal fate of polypeptides within foods and
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within the GIT so as to improve their bioactivity profiles (Mohan et al., 2015; Zhang
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et al., 2015b).
Typically,
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Polypeptide activity may
In addition, their activity may be altered after they are ingested and pass
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Within
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This process may be undesirable if it leads to loss of the bioactivity
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The encapsulation of polypeptides within colloidal delivery systems offers a
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potentially effective means of controlling their stability both in food products and
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within the GIT after ingestion (Mohan et al., 2015; Sagalowicz and Leser, 2010;
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Zhang et al., 2015b).
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within a colloidal particle that is fabricated from food-grade ingredients, such as lipids,
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carbohydrates, proteins, surfactants, or minerals (McClements, 2014).
For food applications, polypeptides are typically trapped
Polypeptides 3
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may be hydrophilic, hydrophobic, or amphiphilic depending on their amino acid
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composition and structural organization (Udenigwe and Aluko, 2012).
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an appropriate encapsulation material and structure must be selected for the particular
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polypeptide involved.
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explored for their potential to encapsulate polypeptides and other bioactive
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components, including microemulsions, nanoemulsions, emulsions, solid lipid
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nanoparticles, multiple emulsions, biopolymer particles, and microgels (Du and
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Stenzel, 2014; du Plessis et al., 2014; McClements, 2014; Mohan et al., 2015).
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of these delivery systems has advantages and disadvantages in terms of its ease of
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preparation, storage, and handling, cost, stability characteristics, encapsulation
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efficiency, loading capacity, and food matrix compatibility.
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Numerous kinds of colloidal delivery systems have been
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Each
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Consequently,
Previously, complex coacervation has been used by our group to develop
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hydrogel particles containing encapsulated lipid droplets (Li and McClements, 2011;
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Zhang et al., 2015a; Zhang et al., 2015b).
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number of potential applications in the food industry, including improving physical or
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chemical stability, targeted delivery, and regulation of lipid digestion and satiety
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(Zhang et al., 2015c). In the current study, we investigated the possibility of
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encapsulating protein nanoparticles within polysaccharide-based hydrogel particles
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(“microgels”), and studied the influence of solution and environmental conditions on
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their properties.
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solution conditions (pH and ionic strength) on the stability and properties of the
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protein nanoparticle-loaded microgels.
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their potential application in food products, and their potential behavior within the
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gastrointestinal tract.
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2. Materials and methods
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2.1. Materials
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These filled hydrogel particles have a
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The main focus of this study was to examine the influence of
This information is useful for establishing
Zein (Lot# SLBD5665V) and sodium alginate (Lot 50K0180) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Sodium caseinate powder was obtained
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ACCEPTED MANUSCRIPT from the American Casein Company (MP Biomedicals LLC). Whey protein isolate
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(WPI) powder was obtained from Davisco Foods International Inc. (Le Sueur, MN,
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USA). Alginic acid (sodium salt) (Lot# 180947) was purchased from the Sigma
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Chemical Company (St. Louis, MO). All other chemicals were of analytical grade.
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Double distilled water was used to prepare all solutions and colloidal suspensions.
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2.2. Solution preparation
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A weighed amount (2.64 g) of zein powder was added to 100 mL ethanol solution (80% v/v), and stirred at 500 rpm (IKA R05, Werke, GmbH) for 1 h, and then filtered
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with filter paper (Fisher Science, P5). WPI (0.25% w/v) was solubilized in phosphate
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buffer solution (10 mM, pH 6.5). 2% (w/w) sodium caseinate and 2% (w/w) alginate
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were prepared separately in 10 mM phosphate buffer at pH 7 and stirred until fully
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dissolved.
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2.3. Fabrication of Colloidal Particles
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2.3.1. Protein nanoparticles preparation
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Protein nanoparticles were fabricated from zein using an antisolvent precipitation
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method. Initially, zein (26.4 mg/mL) was dissolved in ethanol solution (80% v/v).
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Then, 25 mL of aqueous ethanol solution was rapidly injected into 75 ml of whey
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protein solution (0.25% WPI, PBS, pH 6.5) that was continuously stirred at 1200 rpm
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using a magnetic stirrer (IKA R05, Werke, GmbH). The resulting colloidal dispersion
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was then stirred for another 30 min at the same speed. The ethanol remaining in the
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final colloidal dispersions (around 16% v/v) was evaporated at 40 °C using a rotary
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evaporator (Rotavapor R110, Büchi Corp., Switzerland), and the same volume of pH
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6.5 PBS was added to compensate for the lost ethanol.
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2.3.2. Unfilled hydrogel particle preparation
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2 M sodium hydroxide was used to adjust caseinate (2%) and alginate (2%)
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solutions to pH 7. Then these two stock solutions and phosphate buffer were mixed
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together at different volume ratios under continuous stirring to form final
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compositions of 0.33% sodium caseinate/1.33% alginate (mass ratio 1:4). The 5
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mixtures were then acidified to pH 5 using 1 M citric acid at a rate of 1 drop/10 s with
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continuous stirring at 500 rpm to promote complex formation.
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2.3.3. Filled hydrogel particle preparation
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2 M sodium hydroxide was used to adjust caseinate solutions, alginate solutions, and protein nanoparticle dispersions to pH 7. After pH adjustment, 6.6 mg/mL protein
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nanoparticle dispersion and 2% sodium caseinate solution were mixed together at a
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1:1 volume ratio. Then, this system was mixed (500 rpm) with 2% alginate solution at
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a volume ratio of 1:2.
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citric acid with continuous stirring at 500 rpm to promote complex formation. The
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final composition of the resulting system was 1.1 mg/mL protein nanoparticles, 0.33%
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sodium caseinate, and 1.33% alginate.
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2.4. ζ-potential measurements
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Finally, the resulting mixture was acidified to pH 5 using 1 M
The electrical charge (ζ-potential) of biopolymers and colloidal particles was
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measured at different pH values (3.0–7.0) using a particle electrophoresis instrument
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(Zetasizer Nano ZA series, Malvern Instruments Ltd. Worcestershire, UK). Samples
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were diluted using 10 mM phosphate buffer (at the same pH as the sample) prior to
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analysis to avoid multiple scattering effects. All measurements were made on at least
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two freshly prepared samples and each sample was measured in duplicate.
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2.5. Particle size analysis
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The particle size distribution was measured using a static light scattering
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instrument (Mastersizer 2000, Malvern Instruments, Worcestershire, United
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Kingdom). This instrument infers the size of the particles from measurements of their
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angular scattering pattern. All measurements were made on at least two freshly
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prepared samples. Samples were diluted in 10 mM phosphate buffer (pH 3.0–7.0) by
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adding small aliquots into a measurement chamber. Particle size measurements were
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reported as surface-weighted mean diameters (d32).
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2.6. Microstructural analysis The microstructure of the colloidal dispersions was characterized using confocal
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scanning fluorescence microscopy (Nikon D-Eclipse C1 80i, Nikon, Melville, NY,
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U.S.).
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thiocyanate isomer I (FITC) solution (1 mg/mL dimethyl sulfoxide) by adding 0.1 mL
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of FITC dye solution to 2 mL of sample. All images were captured with a 10×
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eyepiece and a 60× objective lens (oil immersion). The microstructure images for
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confocal microscopy were digitally acquired and then analyzed using image analysis
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software (NIS-Elements, Nikon, Melville, NY).
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2.7. Stability of environmental conditions
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Prior to analysis, the protein phase of the samples was dyed with fluorescein
The particle size and microstructure of the colloidal dispersions were determined after they were exposed to various environmental conditions.
Effect of pH: Freshly prepared colloidal dispersions were mixed with equal volumes of 10 mM phosphate buffer with pH values ranging from 3.0 to 7.0. The
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samples were then adjusted to the desired pH values with 1 mol/L NaOH or HCl.
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Effect of salt: Protein nanoparticles were mixed with equal volumes of
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phosphate buffer at pH 6.5 containing sodium chloride (50–400 mM NaCl). While,
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nanoparticle-loaded microgels were mixed with equal volumes of phosphate buffer at
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pH 5.0 containing sodium chloride (0.5–2 M NaCl). If necessary, the samples were
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then adjusted to pH 6.5 or pH 5.0 with 1 mol/L NaOH or HCl.
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2.8. Statistical analysis
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All experiments were carried out on two or three freshly prepared samples. The
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results are expressed as means ± standard deviations (SD). Data were subjected to
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statistical analysis using SPSS software (version 18.0). Means were subject to
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Duncan's test and a P-value of <0.05 was considered statistically significant.
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3. Results and discussion
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3.1. Properties of initial particles
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3.1.1. Protein nanoparticles
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Initially, we characterized the properties of the core-shell protein nanoparticles produced using the antisolvent precipitation method (pH 6.5).
Based on the
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fabrication method used these protein nanoparticles should consist of a core of
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hydrophobic zein molecules and a shell of amphiphilic whey protein molecules (Chen
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and Zhong, 2015).
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diameters (d32 = 260 nm) (Table 1), and a monomodal particle size distribution with
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the majority of particles being between 100 and 1000 nm (Figure 1a).
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microscopy images indicated that the particles were uniformly distributed throughout
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the sample, indicating that they were stable to extensive aggregation (Figure 2b).
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Visual images of the protein nanoparticle suspensions indicated that they had a
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uniform milky white appearance, supporting the observation that they were stable to
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aggregation and sedimentation (Figure 2a). The optical opacity of the colloidal
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suspensions can be attributed to the fact that the protein particles had dimensions (260
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nm) that were fairly similar to the wavelength of visible light (380 to 780 nm) and
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therefore they scattered light strongly (McClements, 2002).
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nanoparticles had a relatively high negative charge (ζ = - 45 mV) at pH 6.5 (Table 1)
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due to the fact that this pH is above the isoelectric point of the protein nanoparticles.
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Interestingly, the electrical characteristics (ζ-potential versus pH profile) of the
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protein nanoparticles (which consisted of a zein core and a whey protein shell) were
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very similar to those of a solution of whey protein molecules (Figure 3), which
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suggests that the proteins at the surfaces of the nanoparticles dominated their overall
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electrical characteristics.
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has been reported to be around pH 6.0 (Patel et al., 2010), which is considerably
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higher than the value of pH 4.5 measured for the whey protein-coated zein
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nanoparticles used in our study (Figure 3), again suggesting that it is the adsorbed
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whey protein layer that dominates the overall electrical characteristics.
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Confocal
The protein
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The protein nanoparticles formed had relatively small mean
In addition, the isoelectric point of pure zein nanoparticles
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3.1.2. Unfilled and filled microgels The properties of the microgels formed by the electrostatic complexation method
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were characterized in the absence and presence of the protein nanoparticles (pH 5.0).
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The unfilled and filled microgels had mean particle diameters (d32) around 4.4 and 6.0
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µm respectively (Table 1), and had monomodal particle size distributions (Figure 1a).
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Confocal fluorescence microscopy images indicated that the microgels in both
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systems had a spheroid shape, and were non-aggregated (Figure 1b). Electrophoresis
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measurements indicated that the unfilled and filled microgels had a relatively high
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negative charge (ζ = -73 and -69 mV respectively) (Table 1), which can be attributed
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to the fact that they were constructed from an anionic polysaccharide (alginate).
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Visual observation of the filled microgel suspensions indicated that they had a
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uniform turbid appearance (Figure 2b). In summary, the presence of the protein
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nanoparticles led to an increase in the dimensions of the microgels formed by
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electrostatic complexation, but had little impact on their electrical characteristics.
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3.2. Influence of pH on stability
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Colloidal delivery systems utilized within foods and beverages may experience
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appreciably different pH environments depending on the nature of the product.
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addition, they may be exposed to considerable variations in pH as they pass through
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the human gastrointestinal tract after ingestion.
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understand the influence of pH on the properties of any colloidal delivery system
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intended for use in commercial food and beverage products.
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3.2.1 Protein-nanoparticles
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In
Consequently, it is useful to
Initially, we examined the influence of pH on the physicochemical properties of
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protein nanoparticle suspensions.
Visual observation of the suspensions indicated
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that their appearance was highly dependent on pH (Figure 2a).
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pH values (6.5 to 7) the suspensions had a uniform white appearance.
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intermediate pH values (5.5. to 4.0), a layer of white sediment was observed at the
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bottom of the test tubes with a clear or slightly turbid layer above.
At relatively high At
At relatively low
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pH values (3 to 3.5), a slightly turbid suspension that appeared uniform throughout
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was observed.
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electrostatic interactions between the protein nanoparticles.
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(ζ) on the protein nanoparticles went from highly negative at high pH values (e.g., -32
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mV at pH 7) to highly positive at low pH values (e.g., +24 mV at pH 3), with a point
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of zero charge around pH 4.5 (Figure 3).
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electrostatic repulsion between the protein nanoparticles at low and high pH values,
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but only a weak one at intermediate pH values.
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attractive forces between the protein nanoparticles (such as van der Waals) were
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strongly enough to overcome the repulsive forces (such as electrostatic and steric) at
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intermediate pH values, leading to aggregation and sedimentation (McClements,
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2014).
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influence of pH on the particle size and microstructure of the protein nanoparticle
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suspensions (Figures 4, 5 and S1).
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relatively small (d32 < 1 µm), had a monomodal distribution, and were evenly
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distributed throughout the sample.
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particle size was relatively big (d32 > 10 µm) and large irregular shaped aggregates
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were observed in the microscopy images. At low pH values (3 to 3.5), there was an
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appreciable increase in particle size detected by light scattering, and also evidence of
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some large aggregates in the microscopy images (Figures 4, 5 and S1).
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possible that these aggregates formed as the suspensions were moved from high pH to
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low pH, and were not fully broken down under acidic conditions. The reason for the
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difference in appearance of the protein nanoparticle suspensions at low and high pH
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values also indicated that the nature of the particles present had changed due to an
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alteration in the strength of the electrostatic interactions in the system.
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3.2.2 Filled microgels
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This kind of behavior can be attributed to the influence of pH on the
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The electrical potential
Consequently, there would be a strong
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It is therefore likely that the
The tendency for aggregation to occur is highlighted in measurements of the
At high pH values (6.5 to 7), the particles were
It is
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At intermediate pH values (pH 5.5. to 4), the
We then examined the influence of pH on the properties of the
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protein-nanoparticle filled microgels. Visual observation of the microgel suspensions
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indicated that they remained stable to creaming or sedimentation across the entire pH
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range (Figure 2b). Thus, the rapid sedimentation of the free protein nanoparticles that
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was observed at intermediate pH values was not observed when they were
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encapsulated within filled microgels.
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was a strong electrostatic repulsion between the microgels at all pH values where they
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remained intact.
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microgel samples had a relatively high negative charge from pH 3 to 7 (Figure 3).
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This high negative charge can mainly be attributed to the presence of relatively high
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levels of anionic alginate molecules within the microgels.
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measurements indicated that the dimensions of the microgels remained relatively
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constant from pH 3 to 6, with a monomodal particle size distribution (Figures 4 and
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S1b).
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a population of particles around 180 nm and another population around 4 µm. We
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hypothesize that the microgels dissociated at these higher pH values because the
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alginate and caseinate molecules both had strong negative charges and so there was a
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strong electrostatic repulsion between them.
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released the protein nanoparticles, which would account for the population of small
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particles observed in the particle size distributions at these pH values (Figure S1b).
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This observation was supported by the confocal microscopy images of the influence
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of pH on the microstructure of the system (Figure 5b).
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remained as discrete intact particles, i.e., the images showed discrete regions of high
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fluorescence intensity (protein-rich particles) surrounded by a black (protein-free)
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background.
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of the microgels, i.e., the images showed discrete regions of high fluorescence
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intensity (protein-rich particles) surrounded by a green (protein-rich) background. At
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pH 7, it appeared that the microgels had almost completely disintegrated and the
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protein was evenly distributed throughout the system.
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hypothesize that the protein nanoparticles had been released from the microgels and
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formed a stable colloidal dispersion.
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population of small particles was observed in the light scattering data (Figure S1b),
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the overall systems appeared uniformly turbid (Figure 2b), and the protein was
The most likely reason for this is that there
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Indeed, the electrophoresis measurements indicated that the
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The light scattering
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However, the particle size distribution became bimodal at pH 6.5 and 7, with
At pH 3 to 5, the microgels
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Consequently, the microgels may have
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At pH 5.5, there was evidence that some of the protein had leaked out
In these systems, we
This hypothesis is based on the fact that a
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evenly spread throughout the samples (Figure 6).
It should be noted that the
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microgels also contained protein (caseinate) and this may also have been released
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with the protein nanoparticles when they dissociated.
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3.3. Influence of ionic strength on stability Colloidal delivery systems may be used in foods and beverages that contain
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different types and amounts of salts in the aqueous phase, or they may be exposed to
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different ionic environments as they pass through the gastrointestinal tract.
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Consequently, it is important to understand the influence of ionic strength on the
304
properties of colloidal delivery systems intended for utilization within commercial
305
food and beverage products.
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3.3.1 Protein-nanoparticles
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Initially, the influence of ionic strength on the stability of the protein nanoparticles was examined by adding different amounts of salt to them (pH 6.5).
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This pH was selected because in the absence of salt, the protein nanoparticles
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appeared stable to aggregation and sedimentation as demonstrated by the relatively
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small particle diameter and visual absence of phase separation (Figures 6a and 7). As
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discussed earlier, this effect can be attributed to a strong electrostatic repulsion
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between the protein nanoparticles due to their relatively high ζ-potential at this pH
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(Figure 3).
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sedimentation after addition of 50 mM NaCl, which suggests that the electrostatic
316
repulsion was still strong enough to overcome any attractive colloidal interactions.
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However, after addition of 100 mM NaCl, there was evidence of an increase in
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particle size (Figures 7 and S2a) indicating some aggregation occurred, which would
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account for the increase in opacity of the suspensions (Figure 6a).
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200 mM NaCl or higher, there was a further increase in particle size and evidence of
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rapid sedimentation.
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salt screened the negative charge on the protein nanoparticle surfaces, thereby
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reducing the magnitude and range of the electrostatic repulsion.
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The suspensions remained stable to particle aggregation and
After addition of
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In this case, the positively charged counter ions (Na ) from the
The confocal
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fluorescence microscopy (Figure 8) and particle size distribution measurements
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(Figure S2a) clearly showed evidence of a population of large aggregates within the
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protein-nanoparticle suspensions at high salt concentrations.
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protein nanoparticles were not very stable to salt addition due to the fact that
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electrostatic repulsion played a major role in preventing their aggregation.
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3.3.2 Filled microgels
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In summary, the
The salt-stability of the filled microgels was very different from that of the free
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protein nanoparticles. In this case, increasing amounts of salt (0 to 2 M NaCl) were
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added to microgel suspensions at pH 5. This pH value was selected because the
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microgels remained intact under these conditions, i.e., they did not dissociate or
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aggregate.
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homogeneous (Figure 6b), which suggested that they were stable to particle
336
aggregation and gravitational separation.
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measurements (Figure S2b) and the microscopy images (Figure 8b) indicated that
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the microgels remained as discrete particles.
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(protein) was only relatively high within the microgels suggested that the
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protein-nanoparticles were retained within them at all salt levels. Thus, salt addition
341
did not promote dissociation or aggregation of the filled microgels, which may be an
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important attribute for application of these microgels in certain types of food and
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beverage products. For example, they may be useful for delivering proteins in
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products such as dressings, sauces, and meat products that have relatively high salt
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contents.
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4. Conclusions
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At all salt levels used, the microgel suspensions appeared to be
In addition, the light scattering
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The fact that the fluorescence intensity
This study has shown that protein nanoparticles can be successfully incorporated
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into biopolymer microgels formed from electrostatic complexation of casein and
349
alginate.
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pH values from 3 to 5.5, but were released at higher pH values due to dissociation of
351
the casein-alginate complexes.
The protein nanoparticles appeared to be retained within the microgels at
At higher pH values both the casein and alginate are
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negatively charged, which causes them to dissociate and release the encapsulated
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protein nanoparticles.
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nanoparticles within acidic environments (such as the stomach), but release them
355
under neutral environments (such as the small intestine).
356
were retained within the microgels at all salt levels studied (0 to 2 M, pH 5), which
357
suggests that the attraction between the biopolymers that made up the microgels was
358
strong enough to resist high ionic strengths.
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encapsulation of protein nanoparticles within microgels may be a viable method of
360
improving their stability to pH and ionic strength within foods, and possibly of
361
controlling their fate within the gastrointestinal tract. Nevertheless, further studies are
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required to monitor the potential gastrointestinal fate of the filled microgels under
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simulated or real gastrointestinal conditions.
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5. Acknowledgements
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The protein nanoparticles
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Overall, our results suggest that
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This phenomenon may be useful for retaining protein
This material was partly based upon work supported by the USDA, NRI Grants (2013-03795 and 2014-67021).
We also thank the National Aero and Space
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Administration (NASA) for partial funding of this research (NNX14AP32G). This
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project was also partly funded by the Deanship of Scientific Research (DSR), King
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Abdulaziz University, Jeddah, under grant numbers 87-130-35-HiCi. The authors,
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therefore, acknowledge with thanks DSR technical and financial support.
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6. References
Chen, H., Zhong, Q., (2015). A novel method of preparing stable zein nanoparticle dispersions for
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encapsulation of peppermint oil. Food Hydrocolloids 43, 593-602. Du, A.W., Stenzel, M.H., (2014). Drug Carriers for the Delivery of Therapeutic Peptides. Biomacromolecules 15(4), 1097-1114.
du Plessis, L.H., Marais, E.B., Mohammed, F., Kotze, A.F., (2014). Applications of Lipid based Formulation Technologies in the Delivery of Biotechnology-based Therapeutics. Current Pharmaceutical Biotechnology 15(7), 659-672.
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Hernandez-Ledesma, B., Contreras, M.D., Recio, I., (2011). Antihypertensive peptides: Production,
bioavailability and incorporation into foods. Advances in Colloid and Interface Science 165(1), 23-35. Hettiarachchy, N., Sato, K., Marshall, M., Kannan, A., (2012). Food Proteins and Peptides: Chemistry,
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Functionality, Interactions, and Commercialization. CRC Press, Boca Raton. Li, Y., McClements, D.J., (2011). Controlling lipid digestion by encapsulation of protein-stabilized lipid droplets within alginate-chitosan complex coacervates. Food Hydrocolloids 25(5), 1025-1033. McClements, D.J., (2002). Theoretical prediction of emulsion color. Advances in Colloid and Interface Science 97(1-3), 63-89.
McClements, D.J., (2014). Nanoparticle-and microparticle-based delivery systems: Encapsulation, protection and release of active compounds. CRC PRess, Boca Raton.
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Mohan, A., Rajendran, S., He, Q.S., Bazinet, L., Udenigwe, C.C., (2015). Encapsulation of food protein hydrolysates and peptides: a review. Rsc Advances 5(97), 79270-79278. Moreno, F.J., (2007). Gastrointestinal digestion of food allergens: Effect on their allergenicity. Biomedicine & Pharmacotherapy 61(1), 50-60.
Patel, A.R., Bouwens, E.C.M., Velikov, K.P., (2010). Sodium Caseinate Stabilized Zein Colloidal
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Particles. Journal of Agricultural and Food Chemistry 58(23), 12497-12503. Sagalowicz, L., Leser, M.E., (2010). Delivery systems for liquid food products. Current Opinion in Colloid & Interface Science 15(1-2), 61-72.
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Samaranayaka, A.G.P., Li-Chan, E.C.Y., (2011). Food-derived peptidic antioxidants: A review of their production, assessment, and potential applications. Journal of Functional Foods 3(4), 229-254.
Sarmadi, B.H., Ismail, A., (2010). Antioxidative peptides from food proteins: A review. Peptides 31(10), 1949-1956.
Udenigwe, C.C., Aluko, R.E., (2012). Food Protein-Derived Bioactive Peptides: Production, Processing, and Potential Health Benefits. Journal of Food Science 77(1), R11-R24.
Zhang, Z., Zhang, R., Decker, E.A., McClements, D.J., (2015a). Development of food-grade filled hydrogels for oral delivery of lipophilic active ingredients: pH-triggered release. Food Hydrocolloids 44, 345-352. Zhang, Z., Zhang, R., Tong, Q., Decker, E.A., McClements, D.J., (2015b). Food-grade filled hydrogels for oral delivery of lipophilic active ingredients: Temperature-triggered release microgels. Food Research International 69, 274-280. Zhang, Z.P., Zhang, R.J., Chen, L., Tong, Q.Y., McClements, D.J., (2015c). Designing hydrogel particles for controlled or targeted release of lipophilic bioactive agents in the gastrointestinal tract.
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Figure Captions
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Fig. 1 (a). Particle size distributions of protein (zein-WPI) nanoparticles, unfilled hydrogels, and protein nanoparticle filled hydrogels. Fig. 1 (b). Confocal micrographs of protein nanoparticles, unfilled hydrogel particles and protein nanoparticle filled hydrogel particles. Fig. 2 (a). Appearance of protein (zein-WPI) nanoparticles dispersed in different pH solutions. Extensive particle aggregation and sedimentation occurred from pH 5.5 to 4.0. Fig. 2 (b). Appearance of protein nanoparticle-filled hydrogels dispersed in different pH solutions. Fig. 3. The pH-dependence of the ζ-potential of whey protein molecules (WPI) in solution, protein (zein-WPI) nanoparticles, unfilled hydrogels, and protein nanoparticle filled hydrogels. Fig 4. Effect of pH on mean particle diameters (d32) of protein nanoparticles and filled hydrogel particles. Different letters mean significant differences (p < 0.05) of the particle diameter at different pH values (for each type of particle). Fig. 5 (a). Confocal fluorescence micrographs of protein nanoparticles after incubation at different pH values. Fig. 5 (b). Confocal fluorescence micrographs of protein nanoparticle-filled hydrogel particles after incubation at different pH values. Fig. 6 (a) Appearance of protein nanoparticle suspensions at pH 6.5 after incubation in different ionic conditions (from 50 to 400 mM NaCl); Fig. 6 (b) Appearance of protein nanoparticle-filled hydrogel particle suspensions at pH 5 after incubation in different ionic conditions (from 0.0 to 2 M NaCl). Fig 7. Effect of ionic strength onmean particle diameter (d32) of suspensions of protein nanoparticles and filled hydrogel particles. Different letters mean significant differences (p < 0.05) of the particle diameter at different ionic concentration. Fig. 8 (a). The confocal micrographs of protein nanoparticles at pH 6.5 after exposure to different ionic conditions (from 50 to 400mM). Fig. 8 (b). The confocal micrographs of filled hydrogel particles at pH 5.0 after exposure to different ionic conditions (from 0.5 to 2.0 M NaCl).
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Supporting Information
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Fig. S1 (a). The pH-dependence of the particle size distribution of protein nanoparticles. Fig. S1 (b). The pH-dependence of the particle size distribution of protein nanoparticle-filled hydrogel particles. Fig. S2 (a). The particle size distribution of protein nanoparticles at pH 6.5 after exposure to different ionic conditions (from 50 to 400 mM NaCl). Fig. S2 (b). The particle size distribution of filled hydrogel particles at pH 5.0 after exposure to different ionic conditions (from 0.5 to 2.0 M NaCl).
ACCEPTED MANUSCRIPT Table 1. Particle size and ζ-potential of different systems: protein nanoparticles; unfilled hydrogels particles; and protein nanoparticle filled hydrogels particles.
Systems
D43 (µm)
D32 (µm)
Uniformity
Protein nanoparticles
0.31±0.01a
0.26±0.02 a
0.36±0.03 a
-44.8±0.7 b
Unfilled hydrogel particles
4.41±0.25 b 3.77±0.12 b 0.35±0.03 a
-73.4±2.1 a
Filled hydrogel particles
6.01±0.70 c
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ζ-potential (mV)
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Filled hydrogel
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Protein nanoparticles
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Fig. 1 (b). Confocal micrographs of protein nanoparticles, unfilled hydrogel particles and protein nanoparticle filled hydrogel particles.
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Extensive particle aggregation and sedimentation occurred from pH 5.5 to 4.0.
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Fig. 2 (b). Appearance of protein nanoparticle-filled hydrogels dispersed in different pH
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Fig. 3. The pH-dependence of the ζ-potential of whey protein molecules (WPI) in
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Manuscript title: Encapsulation of protein nanoparticles within alginate microparticles: Impact of pH and ionic strength on functional performance, by Zou et al Highlights
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Core-shell protein nanoparticles were prepared by antisolvent precipitation Protein nanoparticles were encapsulated within biopolymer microgels The microgels were fabricated by electrostatic complexation of casein and alginate Encapsulated protein nanoparticles had a wider range of pH and salt stability than free ones
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• • • •
S0260-8774(16)30010-3
DOI:
10.1016/j.jfoodeng.2016.01.010
Reference:
JFOE 8448
To appear in:
Journal of Food Engineering
Received Date: 2 December 2015 Revised Date:
7 January 2016
Accepted Date: 14 January 2016
Please cite this article as: Zou, L., Zhang, Z., Zhang, R., Liu, W., Liu, C., Xiao, H., Julian McClements, D., Encapsulation of protein nanoparticles within alginate microparticles: Impact of pH and ionic strength on functional performance, Journal of Food Engineering (2016), doi: 10.1016/j.jfoodeng.2016.01.010. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Encapsulation of protein nanoparticles within alginate
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microparticles: Impact of pH and ionic strength on
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functional performance
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Liqiang Zoua1, Zipei Zhangb1, Ruojie Zhangb, Wei Liua*, Chengmei Liua, Hang
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Xiaob, David Julian McClementsb,c*
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a
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Nanchang, No. 235 Nanjing East Road, Nanchang 330047, Jiangxi, China
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b
Department of Food Science, University of Massachusetts, Amherst, MA 01003
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c
Department of Biochemistry, Faculty of Science, King Abdulaziz University, P. O.
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Box 80203 Jeddah 21589 Saudi Arabia
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State Key Laboratory of Food Science and Technology, Nanchang University,
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1
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* corresponding author:
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Wei Liu, State Key Laboratory of Food Science and Technology, Nanchang
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University, Nanchang, 330047, Jiangxi, China Tel: + 86 791 88305872x8106. Fax:
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+86 791 88334509. E-mail:
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[email protected].
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These authors contributed equally to this manuscript.
David Julian McClements, Department of Food Science, University of Massachusetts,
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Amherst, MA 01003, USA Tel: (413) 545-1019. Fax: (413) 545-1262. E-mail:
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[email protected].
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Journal: Journal of Food Engineering
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Submitted: December 2015
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Abstract Incorporation of bioactive proteins into functional foods is often challenging due to their instability to aggregation, sedimentation, or hydrolysis.
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core-shell protein nanoparticles, consisting of a zein core and a whey protein shell,
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were fabricated by antisolvent precipitation.
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incorporated into biopolymer microgels fabricated by electrostatic complexation of
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casein and alginate.
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5.5), but released at higher pH (6 to 7) due to microgel dissociation promoted by
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electrostatic repulsion between anionic casein and alginate.
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useful for retaining and protecting protein nanoparticles within acidic environments
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(e.g., stomach), but releasing them under neutral environments (e.g., small intestine).
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Protein nanoparticles were retained within microgels over a wide range of ionic
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strengths (0 to 2 M NaCl, pH 5).
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microgels may improve their pH and salt stability in functional foods.
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These microgels may be
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Protein nanoparticle encapsulation within
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Keywords: zein nanoparticle; hydrogel particle; stability; delivery system.
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The protein nanoparticles were then
Protein nanoparticles were retained in microgels at low pH (3 to
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In this study,
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1. Introduction There is growing interest in the utilization of proteins and peptides (referred to collectively as “polypeptides” for convenience) as functional ingredients in foods
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because of their beneficial health effects, such as antioxidant, antimicrobial, and
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anti-hypertension activities (Hernandez-Ledesma et al., 2011; Samaranayaka and
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Li-Chan, 2011; Sarmadi and Ismail, 2010; Udenigwe and Aluko, 2012).
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the activity of polypeptides depends on their three-dimensional structures and specific
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amino acid sequences (Udenigwe and Aluko, 2012).
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therefore be altered in food products during manufacture, storage, or transportation,
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due to changes in solution or environmental conditions that alter protein structure,
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such as pH, ionic strength, ingredient interactions, or temperature (Hettiarachchy et al.,
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2012).
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through the gastrointestinal tract (GIT) because they are exposed to digestive enzymes
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(proteases and peptidases) and environmental conditions (pH, ionic strength, and
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ingredient interactions) that may alter their structure (Mohan et al., 2015).
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the GIT, digestive enzymes and highly acidic gastric conditions may hydrolyze
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polypeptide chains at particular bond locations, thereby generating new peptides
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(Moreno, 2007).
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of an ingested polypeptide, or it may be desirable if it leads to the generation of new
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peptides with improved bioactivity. Consequently, it is often important to design food
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matrices that can control the gastrointestinal fate of polypeptides within foods and
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within the GIT so as to improve their bioactivity profiles (Mohan et al., 2015; Zhang
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et al., 2015b).
Typically,
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Polypeptide activity may
In addition, their activity may be altered after they are ingested and pass
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This process may be undesirable if it leads to loss of the bioactivity
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The encapsulation of polypeptides within colloidal delivery systems offers a
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potentially effective means of controlling their stability both in food products and
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within the GIT after ingestion (Mohan et al., 2015; Sagalowicz and Leser, 2010;
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Zhang et al., 2015b).
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within a colloidal particle that is fabricated from food-grade ingredients, such as lipids,
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carbohydrates, proteins, surfactants, or minerals (McClements, 2014).
For food applications, polypeptides are typically trapped
Polypeptides 3
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may be hydrophilic, hydrophobic, or amphiphilic depending on their amino acid
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composition and structural organization (Udenigwe and Aluko, 2012).
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an appropriate encapsulation material and structure must be selected for the particular
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polypeptide involved.
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explored for their potential to encapsulate polypeptides and other bioactive
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components, including microemulsions, nanoemulsions, emulsions, solid lipid
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nanoparticles, multiple emulsions, biopolymer particles, and microgels (Du and
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Stenzel, 2014; du Plessis et al., 2014; McClements, 2014; Mohan et al., 2015).
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of these delivery systems has advantages and disadvantages in terms of its ease of
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preparation, storage, and handling, cost, stability characteristics, encapsulation
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efficiency, loading capacity, and food matrix compatibility.
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Numerous kinds of colloidal delivery systems have been
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Consequently,
Previously, complex coacervation has been used by our group to develop
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hydrogel particles containing encapsulated lipid droplets (Li and McClements, 2011;
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Zhang et al., 2015a; Zhang et al., 2015b).
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number of potential applications in the food industry, including improving physical or
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chemical stability, targeted delivery, and regulation of lipid digestion and satiety
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(Zhang et al., 2015c). In the current study, we investigated the possibility of
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encapsulating protein nanoparticles within polysaccharide-based hydrogel particles
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(“microgels”), and studied the influence of solution and environmental conditions on
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their properties.
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solution conditions (pH and ionic strength) on the stability and properties of the
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protein nanoparticle-loaded microgels.
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their potential application in food products, and their potential behavior within the
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gastrointestinal tract.
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2. Materials and methods
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2.1. Materials
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These filled hydrogel particles have a
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The main focus of this study was to examine the influence of
This information is useful for establishing
Zein (Lot# SLBD5665V) and sodium alginate (Lot 50K0180) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Sodium caseinate powder was obtained
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ACCEPTED MANUSCRIPT from the American Casein Company (MP Biomedicals LLC). Whey protein isolate
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(WPI) powder was obtained from Davisco Foods International Inc. (Le Sueur, MN,
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USA). Alginic acid (sodium salt) (Lot# 180947) was purchased from the Sigma
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Chemical Company (St. Louis, MO). All other chemicals were of analytical grade.
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Double distilled water was used to prepare all solutions and colloidal suspensions.
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2.2. Solution preparation
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A weighed amount (2.64 g) of zein powder was added to 100 mL ethanol solution (80% v/v), and stirred at 500 rpm (IKA R05, Werke, GmbH) for 1 h, and then filtered
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with filter paper (Fisher Science, P5). WPI (0.25% w/v) was solubilized in phosphate
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buffer solution (10 mM, pH 6.5). 2% (w/w) sodium caseinate and 2% (w/w) alginate
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were prepared separately in 10 mM phosphate buffer at pH 7 and stirred until fully
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dissolved.
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2.3. Fabrication of Colloidal Particles
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2.3.1. Protein nanoparticles preparation
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Protein nanoparticles were fabricated from zein using an antisolvent precipitation
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method. Initially, zein (26.4 mg/mL) was dissolved in ethanol solution (80% v/v).
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Then, 25 mL of aqueous ethanol solution was rapidly injected into 75 ml of whey
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protein solution (0.25% WPI, PBS, pH 6.5) that was continuously stirred at 1200 rpm
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using a magnetic stirrer (IKA R05, Werke, GmbH). The resulting colloidal dispersion
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was then stirred for another 30 min at the same speed. The ethanol remaining in the
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final colloidal dispersions (around 16% v/v) was evaporated at 40 °C using a rotary
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evaporator (Rotavapor R110, Büchi Corp., Switzerland), and the same volume of pH
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6.5 PBS was added to compensate for the lost ethanol.
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2.3.2. Unfilled hydrogel particle preparation
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2 M sodium hydroxide was used to adjust caseinate (2%) and alginate (2%)
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solutions to pH 7. Then these two stock solutions and phosphate buffer were mixed
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together at different volume ratios under continuous stirring to form final
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compositions of 0.33% sodium caseinate/1.33% alginate (mass ratio 1:4). The 5
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mixtures were then acidified to pH 5 using 1 M citric acid at a rate of 1 drop/10 s with
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continuous stirring at 500 rpm to promote complex formation.
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2.3.3. Filled hydrogel particle preparation
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2 M sodium hydroxide was used to adjust caseinate solutions, alginate solutions, and protein nanoparticle dispersions to pH 7. After pH adjustment, 6.6 mg/mL protein
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nanoparticle dispersion and 2% sodium caseinate solution were mixed together at a
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1:1 volume ratio. Then, this system was mixed (500 rpm) with 2% alginate solution at
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a volume ratio of 1:2.
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citric acid with continuous stirring at 500 rpm to promote complex formation. The
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final composition of the resulting system was 1.1 mg/mL protein nanoparticles, 0.33%
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sodium caseinate, and 1.33% alginate.
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2.4. ζ-potential measurements
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Finally, the resulting mixture was acidified to pH 5 using 1 M
The electrical charge (ζ-potential) of biopolymers and colloidal particles was
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measured at different pH values (3.0–7.0) using a particle electrophoresis instrument
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(Zetasizer Nano ZA series, Malvern Instruments Ltd. Worcestershire, UK). Samples
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were diluted using 10 mM phosphate buffer (at the same pH as the sample) prior to
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analysis to avoid multiple scattering effects. All measurements were made on at least
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two freshly prepared samples and each sample was measured in duplicate.
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2.5. Particle size analysis
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instrument (Mastersizer 2000, Malvern Instruments, Worcestershire, United
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Kingdom). This instrument infers the size of the particles from measurements of their
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angular scattering pattern. All measurements were made on at least two freshly
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prepared samples. Samples were diluted in 10 mM phosphate buffer (pH 3.0–7.0) by
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adding small aliquots into a measurement chamber. Particle size measurements were
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reported as surface-weighted mean diameters (d32).
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2.6. Microstructural analysis The microstructure of the colloidal dispersions was characterized using confocal
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scanning fluorescence microscopy (Nikon D-Eclipse C1 80i, Nikon, Melville, NY,
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U.S.).
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thiocyanate isomer I (FITC) solution (1 mg/mL dimethyl sulfoxide) by adding 0.1 mL
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of FITC dye solution to 2 mL of sample. All images were captured with a 10×
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eyepiece and a 60× objective lens (oil immersion). The microstructure images for
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confocal microscopy were digitally acquired and then analyzed using image analysis
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software (NIS-Elements, Nikon, Melville, NY).
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2.7. Stability of environmental conditions
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Prior to analysis, the protein phase of the samples was dyed with fluorescein
The particle size and microstructure of the colloidal dispersions were determined after they were exposed to various environmental conditions.
Effect of pH: Freshly prepared colloidal dispersions were mixed with equal volumes of 10 mM phosphate buffer with pH values ranging from 3.0 to 7.0. The
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samples were then adjusted to the desired pH values with 1 mol/L NaOH or HCl.
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Effect of salt: Protein nanoparticles were mixed with equal volumes of
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phosphate buffer at pH 6.5 containing sodium chloride (50–400 mM NaCl). While,
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nanoparticle-loaded microgels were mixed with equal volumes of phosphate buffer at
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pH 5.0 containing sodium chloride (0.5–2 M NaCl). If necessary, the samples were
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then adjusted to pH 6.5 or pH 5.0 with 1 mol/L NaOH or HCl.
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2.8. Statistical analysis
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All experiments were carried out on two or three freshly prepared samples. The
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results are expressed as means ± standard deviations (SD). Data were subjected to
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statistical analysis using SPSS software (version 18.0). Means were subject to
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Duncan's test and a P-value of <0.05 was considered statistically significant.
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3. Results and discussion
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3.1. Properties of initial particles
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3.1.1. Protein nanoparticles
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Initially, we characterized the properties of the core-shell protein nanoparticles produced using the antisolvent precipitation method (pH 6.5).
Based on the
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fabrication method used these protein nanoparticles should consist of a core of
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hydrophobic zein molecules and a shell of amphiphilic whey protein molecules (Chen
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and Zhong, 2015).
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diameters (d32 = 260 nm) (Table 1), and a monomodal particle size distribution with
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the majority of particles being between 100 and 1000 nm (Figure 1a).
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microscopy images indicated that the particles were uniformly distributed throughout
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the sample, indicating that they were stable to extensive aggregation (Figure 2b).
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Visual images of the protein nanoparticle suspensions indicated that they had a
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uniform milky white appearance, supporting the observation that they were stable to
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aggregation and sedimentation (Figure 2a). The optical opacity of the colloidal
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suspensions can be attributed to the fact that the protein particles had dimensions (260
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nm) that were fairly similar to the wavelength of visible light (380 to 780 nm) and
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therefore they scattered light strongly (McClements, 2002).
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nanoparticles had a relatively high negative charge (ζ = - 45 mV) at pH 6.5 (Table 1)
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due to the fact that this pH is above the isoelectric point of the protein nanoparticles.
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Interestingly, the electrical characteristics (ζ-potential versus pH profile) of the
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protein nanoparticles (which consisted of a zein core and a whey protein shell) were
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very similar to those of a solution of whey protein molecules (Figure 3), which
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suggests that the proteins at the surfaces of the nanoparticles dominated their overall
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electrical characteristics.
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has been reported to be around pH 6.0 (Patel et al., 2010), which is considerably
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higher than the value of pH 4.5 measured for the whey protein-coated zein
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nanoparticles used in our study (Figure 3), again suggesting that it is the adsorbed
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whey protein layer that dominates the overall electrical characteristics.
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Confocal
The protein
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The protein nanoparticles formed had relatively small mean
In addition, the isoelectric point of pure zein nanoparticles
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3.1.2. Unfilled and filled microgels The properties of the microgels formed by the electrostatic complexation method
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were characterized in the absence and presence of the protein nanoparticles (pH 5.0).
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The unfilled and filled microgels had mean particle diameters (d32) around 4.4 and 6.0
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µm respectively (Table 1), and had monomodal particle size distributions (Figure 1a).
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Confocal fluorescence microscopy images indicated that the microgels in both
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systems had a spheroid shape, and were non-aggregated (Figure 1b). Electrophoresis
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measurements indicated that the unfilled and filled microgels had a relatively high
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negative charge (ζ = -73 and -69 mV respectively) (Table 1), which can be attributed
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to the fact that they were constructed from an anionic polysaccharide (alginate).
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Visual observation of the filled microgel suspensions indicated that they had a
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uniform turbid appearance (Figure 2b). In summary, the presence of the protein
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nanoparticles led to an increase in the dimensions of the microgels formed by
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electrostatic complexation, but had little impact on their electrical characteristics.
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3.2. Influence of pH on stability
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appreciably different pH environments depending on the nature of the product.
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addition, they may be exposed to considerable variations in pH as they pass through
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the human gastrointestinal tract after ingestion.
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understand the influence of pH on the properties of any colloidal delivery system
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intended for use in commercial food and beverage products.
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3.2.1 Protein-nanoparticles
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In
Consequently, it is useful to
Initially, we examined the influence of pH on the physicochemical properties of
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protein nanoparticle suspensions.
Visual observation of the suspensions indicated
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that their appearance was highly dependent on pH (Figure 2a).
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pH values (6.5 to 7) the suspensions had a uniform white appearance.
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intermediate pH values (5.5. to 4.0), a layer of white sediment was observed at the
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bottom of the test tubes with a clear or slightly turbid layer above.
At relatively high At
At relatively low
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pH values (3 to 3.5), a slightly turbid suspension that appeared uniform throughout
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was observed.
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electrostatic interactions between the protein nanoparticles.
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(ζ) on the protein nanoparticles went from highly negative at high pH values (e.g., -32
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mV at pH 7) to highly positive at low pH values (e.g., +24 mV at pH 3), with a point
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of zero charge around pH 4.5 (Figure 3).
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electrostatic repulsion between the protein nanoparticles at low and high pH values,
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but only a weak one at intermediate pH values.
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attractive forces between the protein nanoparticles (such as van der Waals) were
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strongly enough to overcome the repulsive forces (such as electrostatic and steric) at
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intermediate pH values, leading to aggregation and sedimentation (McClements,
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2014).
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influence of pH on the particle size and microstructure of the protein nanoparticle
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suspensions (Figures 4, 5 and S1).
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relatively small (d32 < 1 µm), had a monomodal distribution, and were evenly
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distributed throughout the sample.
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particle size was relatively big (d32 > 10 µm) and large irregular shaped aggregates
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were observed in the microscopy images. At low pH values (3 to 3.5), there was an
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appreciable increase in particle size detected by light scattering, and also evidence of
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some large aggregates in the microscopy images (Figures 4, 5 and S1).
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possible that these aggregates formed as the suspensions were moved from high pH to
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low pH, and were not fully broken down under acidic conditions. The reason for the
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difference in appearance of the protein nanoparticle suspensions at low and high pH
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values also indicated that the nature of the particles present had changed due to an
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alteration in the strength of the electrostatic interactions in the system.
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3.2.2 Filled microgels
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This kind of behavior can be attributed to the influence of pH on the
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The electrical potential
Consequently, there would be a strong
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It is therefore likely that the
The tendency for aggregation to occur is highlighted in measurements of the
At high pH values (6.5 to 7), the particles were
It is
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At intermediate pH values (pH 5.5. to 4), the
We then examined the influence of pH on the properties of the
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protein-nanoparticle filled microgels. Visual observation of the microgel suspensions
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indicated that they remained stable to creaming or sedimentation across the entire pH
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range (Figure 2b). Thus, the rapid sedimentation of the free protein nanoparticles that
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was observed at intermediate pH values was not observed when they were
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encapsulated within filled microgels.
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was a strong electrostatic repulsion between the microgels at all pH values where they
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remained intact.
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microgel samples had a relatively high negative charge from pH 3 to 7 (Figure 3).
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This high negative charge can mainly be attributed to the presence of relatively high
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levels of anionic alginate molecules within the microgels.
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measurements indicated that the dimensions of the microgels remained relatively
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constant from pH 3 to 6, with a monomodal particle size distribution (Figures 4 and
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S1b).
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a population of particles around 180 nm and another population around 4 µm. We
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hypothesize that the microgels dissociated at these higher pH values because the
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alginate and caseinate molecules both had strong negative charges and so there was a
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strong electrostatic repulsion between them.
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released the protein nanoparticles, which would account for the population of small
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particles observed in the particle size distributions at these pH values (Figure S1b).
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This observation was supported by the confocal microscopy images of the influence
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of pH on the microstructure of the system (Figure 5b).
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remained as discrete intact particles, i.e., the images showed discrete regions of high
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fluorescence intensity (protein-rich particles) surrounded by a black (protein-free)
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background.
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of the microgels, i.e., the images showed discrete regions of high fluorescence
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intensity (protein-rich particles) surrounded by a green (protein-rich) background. At
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pH 7, it appeared that the microgels had almost completely disintegrated and the
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protein was evenly distributed throughout the system.
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hypothesize that the protein nanoparticles had been released from the microgels and
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formed a stable colloidal dispersion.
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population of small particles was observed in the light scattering data (Figure S1b),
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the overall systems appeared uniformly turbid (Figure 2b), and the protein was
The most likely reason for this is that there
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Indeed, the electrophoresis measurements indicated that the
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The light scattering
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However, the particle size distribution became bimodal at pH 6.5 and 7, with
At pH 3 to 5, the microgels
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At pH 5.5, there was evidence that some of the protein had leaked out
In these systems, we
This hypothesis is based on the fact that a
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evenly spread throughout the samples (Figure 6).
It should be noted that the
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microgels also contained protein (caseinate) and this may also have been released
298
with the protein nanoparticles when they dissociated.
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3.3. Influence of ionic strength on stability Colloidal delivery systems may be used in foods and beverages that contain
301
different types and amounts of salts in the aqueous phase, or they may be exposed to
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different ionic environments as they pass through the gastrointestinal tract.
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Consequently, it is important to understand the influence of ionic strength on the
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properties of colloidal delivery systems intended for utilization within commercial
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food and beverage products.
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3.3.1 Protein-nanoparticles
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Initially, the influence of ionic strength on the stability of the protein nanoparticles was examined by adding different amounts of salt to them (pH 6.5).
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This pH was selected because in the absence of salt, the protein nanoparticles
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appeared stable to aggregation and sedimentation as demonstrated by the relatively
311
small particle diameter and visual absence of phase separation (Figures 6a and 7). As
312
discussed earlier, this effect can be attributed to a strong electrostatic repulsion
313
between the protein nanoparticles due to their relatively high ζ-potential at this pH
314
(Figure 3).
315
sedimentation after addition of 50 mM NaCl, which suggests that the electrostatic
316
repulsion was still strong enough to overcome any attractive colloidal interactions.
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However, after addition of 100 mM NaCl, there was evidence of an increase in
318
particle size (Figures 7 and S2a) indicating some aggregation occurred, which would
319
account for the increase in opacity of the suspensions (Figure 6a).
320
200 mM NaCl or higher, there was a further increase in particle size and evidence of
321
rapid sedimentation.
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salt screened the negative charge on the protein nanoparticle surfaces, thereby
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reducing the magnitude and range of the electrostatic repulsion.
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The suspensions remained stable to particle aggregation and
After addition of
+
In this case, the positively charged counter ions (Na ) from the
The confocal
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fluorescence microscopy (Figure 8) and particle size distribution measurements
325
(Figure S2a) clearly showed evidence of a population of large aggregates within the
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protein-nanoparticle suspensions at high salt concentrations.
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protein nanoparticles were not very stable to salt addition due to the fact that
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electrostatic repulsion played a major role in preventing their aggregation.
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3.3.2 Filled microgels
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In summary, the
The salt-stability of the filled microgels was very different from that of the free
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protein nanoparticles. In this case, increasing amounts of salt (0 to 2 M NaCl) were
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added to microgel suspensions at pH 5. This pH value was selected because the
333
microgels remained intact under these conditions, i.e., they did not dissociate or
334
aggregate.
335
homogeneous (Figure 6b), which suggested that they were stable to particle
336
aggregation and gravitational separation.
337
measurements (Figure S2b) and the microscopy images (Figure 8b) indicated that
338
the microgels remained as discrete particles.
339
(protein) was only relatively high within the microgels suggested that the
340
protein-nanoparticles were retained within them at all salt levels. Thus, salt addition
341
did not promote dissociation or aggregation of the filled microgels, which may be an
342
important attribute for application of these microgels in certain types of food and
343
beverage products. For example, they may be useful for delivering proteins in
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products such as dressings, sauces, and meat products that have relatively high salt
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contents.
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4. Conclusions
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At all salt levels used, the microgel suspensions appeared to be
In addition, the light scattering
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The fact that the fluorescence intensity
This study has shown that protein nanoparticles can be successfully incorporated
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into biopolymer microgels formed from electrostatic complexation of casein and
349
alginate.
350
pH values from 3 to 5.5, but were released at higher pH values due to dissociation of
351
the casein-alginate complexes.
The protein nanoparticles appeared to be retained within the microgels at
At higher pH values both the casein and alginate are
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negatively charged, which causes them to dissociate and release the encapsulated
353
protein nanoparticles.
354
nanoparticles within acidic environments (such as the stomach), but release them
355
under neutral environments (such as the small intestine).
356
were retained within the microgels at all salt levels studied (0 to 2 M, pH 5), which
357
suggests that the attraction between the biopolymers that made up the microgels was
358
strong enough to resist high ionic strengths.
359
encapsulation of protein nanoparticles within microgels may be a viable method of
360
improving their stability to pH and ionic strength within foods, and possibly of
361
controlling their fate within the gastrointestinal tract. Nevertheless, further studies are
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required to monitor the potential gastrointestinal fate of the filled microgels under
363
simulated or real gastrointestinal conditions.
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5. Acknowledgements
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The protein nanoparticles
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Overall, our results suggest that
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This phenomenon may be useful for retaining protein
This material was partly based upon work supported by the USDA, NRI Grants (2013-03795 and 2014-67021).
We also thank the National Aero and Space
367
Administration (NASA) for partial funding of this research (NNX14AP32G). This
368
project was also partly funded by the Deanship of Scientific Research (DSR), King
369
Abdulaziz University, Jeddah, under grant numbers 87-130-35-HiCi. The authors,
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therefore, acknowledge with thanks DSR technical and financial support.
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6. References
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encapsulation of peppermint oil. Food Hydrocolloids 43, 593-602. Du, A.W., Stenzel, M.H., (2014). Drug Carriers for the Delivery of Therapeutic Peptides. Biomacromolecules 15(4), 1097-1114.
du Plessis, L.H., Marais, E.B., Mohammed, F., Kotze, A.F., (2014). Applications of Lipid based Formulation Technologies in the Delivery of Biotechnology-based Therapeutics. Current Pharmaceutical Biotechnology 15(7), 659-672.
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Hernandez-Ledesma, B., Contreras, M.D., Recio, I., (2011). Antihypertensive peptides: Production,
bioavailability and incorporation into foods. Advances in Colloid and Interface Science 165(1), 23-35. Hettiarachchy, N., Sato, K., Marshall, M., Kannan, A., (2012). Food Proteins and Peptides: Chemistry,
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Functionality, Interactions, and Commercialization. CRC Press, Boca Raton. Li, Y., McClements, D.J., (2011). Controlling lipid digestion by encapsulation of protein-stabilized lipid droplets within alginate-chitosan complex coacervates. Food Hydrocolloids 25(5), 1025-1033. McClements, D.J., (2002). Theoretical prediction of emulsion color. Advances in Colloid and Interface Science 97(1-3), 63-89.
McClements, D.J., (2014). Nanoparticle-and microparticle-based delivery systems: Encapsulation, protection and release of active compounds. CRC PRess, Boca Raton.
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Mohan, A., Rajendran, S., He, Q.S., Bazinet, L., Udenigwe, C.C., (2015). Encapsulation of food protein hydrolysates and peptides: a review. Rsc Advances 5(97), 79270-79278. Moreno, F.J., (2007). Gastrointestinal digestion of food allergens: Effect on their allergenicity. Biomedicine & Pharmacotherapy 61(1), 50-60.
Patel, A.R., Bouwens, E.C.M., Velikov, K.P., (2010). Sodium Caseinate Stabilized Zein Colloidal
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Particles. Journal of Agricultural and Food Chemistry 58(23), 12497-12503. Sagalowicz, L., Leser, M.E., (2010). Delivery systems for liquid food products. Current Opinion in Colloid & Interface Science 15(1-2), 61-72.
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Samaranayaka, A.G.P., Li-Chan, E.C.Y., (2011). Food-derived peptidic antioxidants: A review of their production, assessment, and potential applications. Journal of Functional Foods 3(4), 229-254.
Sarmadi, B.H., Ismail, A., (2010). Antioxidative peptides from food proteins: A review. Peptides 31(10), 1949-1956.
Udenigwe, C.C., Aluko, R.E., (2012). Food Protein-Derived Bioactive Peptides: Production, Processing, and Potential Health Benefits. Journal of Food Science 77(1), R11-R24.
Zhang, Z., Zhang, R., Decker, E.A., McClements, D.J., (2015a). Development of food-grade filled hydrogels for oral delivery of lipophilic active ingredients: pH-triggered release. Food Hydrocolloids 44, 345-352. Zhang, Z., Zhang, R., Tong, Q., Decker, E.A., McClements, D.J., (2015b). Food-grade filled hydrogels for oral delivery of lipophilic active ingredients: Temperature-triggered release microgels. Food Research International 69, 274-280. Zhang, Z.P., Zhang, R.J., Chen, L., Tong, Q.Y., McClements, D.J., (2015c). Designing hydrogel particles for controlled or targeted release of lipophilic bioactive agents in the gastrointestinal tract.
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European Polymer Journal 72, 698-716.
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Figure Captions
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Fig. 1 (a). Particle size distributions of protein (zein-WPI) nanoparticles, unfilled hydrogels, and protein nanoparticle filled hydrogels. Fig. 1 (b). Confocal micrographs of protein nanoparticles, unfilled hydrogel particles and protein nanoparticle filled hydrogel particles. Fig. 2 (a). Appearance of protein (zein-WPI) nanoparticles dispersed in different pH solutions. Extensive particle aggregation and sedimentation occurred from pH 5.5 to 4.0. Fig. 2 (b). Appearance of protein nanoparticle-filled hydrogels dispersed in different pH solutions. Fig. 3. The pH-dependence of the ζ-potential of whey protein molecules (WPI) in solution, protein (zein-WPI) nanoparticles, unfilled hydrogels, and protein nanoparticle filled hydrogels. Fig 4. Effect of pH on mean particle diameters (d32) of protein nanoparticles and filled hydrogel particles. Different letters mean significant differences (p < 0.05) of the particle diameter at different pH values (for each type of particle). Fig. 5 (a). Confocal fluorescence micrographs of protein nanoparticles after incubation at different pH values. Fig. 5 (b). Confocal fluorescence micrographs of protein nanoparticle-filled hydrogel particles after incubation at different pH values. Fig. 6 (a) Appearance of protein nanoparticle suspensions at pH 6.5 after incubation in different ionic conditions (from 50 to 400 mM NaCl); Fig. 6 (b) Appearance of protein nanoparticle-filled hydrogel particle suspensions at pH 5 after incubation in different ionic conditions (from 0.0 to 2 M NaCl). Fig 7. Effect of ionic strength onmean particle diameter (d32) of suspensions of protein nanoparticles and filled hydrogel particles. Different letters mean significant differences (p < 0.05) of the particle diameter at different ionic concentration. Fig. 8 (a). The confocal micrographs of protein nanoparticles at pH 6.5 after exposure to different ionic conditions (from 50 to 400mM). Fig. 8 (b). The confocal micrographs of filled hydrogel particles at pH 5.0 after exposure to different ionic conditions (from 0.5 to 2.0 M NaCl).
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Supporting Information
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Fig. S1 (a). The pH-dependence of the particle size distribution of protein nanoparticles. Fig. S1 (b). The pH-dependence of the particle size distribution of protein nanoparticle-filled hydrogel particles. Fig. S2 (a). The particle size distribution of protein nanoparticles at pH 6.5 after exposure to different ionic conditions (from 50 to 400 mM NaCl). Fig. S2 (b). The particle size distribution of filled hydrogel particles at pH 5.0 after exposure to different ionic conditions (from 0.5 to 2.0 M NaCl).
ACCEPTED MANUSCRIPT Table 1. Particle size and ζ-potential of different systems: protein nanoparticles; unfilled hydrogels particles; and protein nanoparticle filled hydrogels particles.
Systems
D43 (µm)
D32 (µm)
Uniformity
Protein nanoparticles
0.31±0.01a
0.26±0.02 a
0.36±0.03 a
-44.8±0.7 b
Unfilled hydrogel particles
4.41±0.25 b 3.77±0.12 b 0.35±0.03 a
-73.4±2.1 a
Filled hydrogel particles
6.01±0.70 c
-69.2±3.5 a
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0.38±0.05 a
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5.02±0.64 c
ζ-potential (mV)
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Filled hydrogel
35
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Unfilled hydrogel Protein nanoparticles
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25 20 15 10 5 0.1
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1
10
100
Particle Diameter (µm)
Fig. 1 (a). Particle size distributions of protein (zein-WPI) nanoparticles, unfilled
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Protein nanoparticles
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b
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Unfilled hydrogels
Filled hydrogels
Fig. 1 (b). Confocal micrographs of protein nanoparticles, unfilled hydrogel particles and protein nanoparticle filled hydrogel particles.
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Extensive particle aggregation and sedimentation occurred from pH 5.5 to 4.0.
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solutions.
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Fig. 2 (a). Appearance of protein (zein-WPI) nanoparticles dispersed in different pH
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solutions.
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Fig. 2 (b). Appearance of protein nanoparticle-filled hydrogels dispersed in different pH
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30 WPI Protein nPs
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Unfilled hydrogels Filled Hydrogels
4
5
-20 -30 -40 -50
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ζ-pontential (mv)
0 -10
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pH
Fig. 3. The pH-dependence of the ζ-potential of whey protein molecules (WPI) in
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solution, protein (zein-WPI) nanoparticles, unfilled hydrogels, and protein nanoparticle filled hydrogels.
ACCEPTED MANUSCRIPT 1000 Protein nanoparticles
Filled hydrogel particles
c b
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Fig 4. Effect of pH on mean particle diameters (d32) of protein nanoparticles and filled
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different pH values.
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particles after incubation at different pH values.
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different ionic conditions (from 50 to 400 mM NaCl);
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Protein nanoparticles
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Fig 7. Effect of ionic strength onmean particle diameter (d32) of suspensions of protein
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pH = 5.5
pH = 6.0
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Fig. S1 (a). The pH-dependence of the particle size distribution of protein nanoparticles.
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Fig. S1 (b). The pH-dependence of the particle size distribution of protein nanoparticle-filled hydrogel particles.
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Fig. S2 (a). The particle size distribution of protein nanoparticles at pH 6.5 after exposure
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Fig. S2 (b). The particle size distribution of filled hydrogel particles at pH 5.0 after
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Manuscript title: Encapsulation of protein nanoparticles within alginate microparticles: Impact of pH and ionic strength on functional performance, by Zou et al Highlights
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Core-shell protein nanoparticles were prepared by antisolvent precipitation Protein nanoparticles were encapsulated within biopolymer microgels The microgels were fabricated by electrostatic complexation of casein and alginate Encapsulated protein nanoparticles had a wider range of pH and salt stability than free ones
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• • • •