Effect of Maillard Conjugates on the Physical Stability of Zein

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Effect of Maillard conjugates on the physical stability of zein nanoparticles prepared by liquid antisolvent co-precipitation. Gabriel Davidov-Pardo, Iris J. Joye, Mauricio Espinal-Ruiz, and David Julian McClements J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b02699 • Publication Date (Web): 03 Sep 2015 Downloaded from http://pubs.acs.org on September 7, 2015

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Journal of Agricultural and Food Chemistry

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Effect of Maillard conjugates on the physical stability of zein

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nanoparticles prepared by liquid antisolvent co-precipitation.

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Gabriel Davidov-Pardo1,2, Iris J. Joye1,3,* , Mauricio Espinal-Ruiz1,4 and

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David Julian McClements1,5

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Department of Food Science, University of Massachusetts, Amherst, MA, USA

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Department of Food Technology, Ænoltec research group, Public University of Navarre,

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Campus Arrosadia s/n, Pamplona 31006, Spain.

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3

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(permanent address)

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Bogotá DC, Colombia.

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80203 Jeddah 21589 Saudi Arabia

Department of Microbial and Molecular Systems, KU Leuven, Leuven, Belgium

Departamento de Química, Facultad de Ciencias, Universidad Nacional de Colombia,

Department of Biochemistry, Faculty of Science, King Abdulaziz University, P. O. Box

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*Corresponding author: [email protected]

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Tel.: +32 16376123

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Fax.: +32 16321997

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Abstract Protein nanoparticles are often not very stable in a complex food matrix as they are

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primarily stabilized by electrostatic repulsion. In this study we envisaged the stabilization

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of zein nanoparticles through maillard conjugation reactions with polysaccharides of

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different molecular mass. Zein nanoparticles (0.5% w/v) containing resveratrol (0.025%

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w/v grape skin extract) were produced by liquid antisolvent precipitation and coated with

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Maillard conjugates (MC) of sodium caseinate and different molecular mass carbohydrates

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during particle production. Zein nanoparticles coated with conjugated polysaccharides of

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2.8, 37 and 150 kDa had diameters of 198±5, 176±6 and 180±3 nm, respectively. The

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encapsulation efficiency (≈ 83%) was not affected by conjugation, but the conjugates

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significantly improved particle stability against changes in pH (2.0 – 9.0), CaCl2 addition

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(up to 100 mM), and heat treatment (30 – 90 ˚C, 30 min). Zein nanoparticles coated by MC

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may therefore be suitable delivery systems for hydrophobic bioactive molecules in a wide

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range of commercial products.

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Keywords: Nanoparticles; resveratrol; encapsulation; antisolvent precipitation; Maillard;

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zein.

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Introduction The fabrication of zein nanoparticles by liquid antisolvent precipitation (LAS) has

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proven to be a promising way to create a delivery system for hydrophobic nutraceuticals,

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such as resveratrol 1, curcumin 2, quercetin 3, essential oils 4, bioactive compounds from

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cruciferous vegetables 5, and α-tocopherol 6. In general, encapsulation is believed to

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improve the stability, water-dispersibility, and release behavior in the gastro-intestinal tract

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of the encapsulated nutraceuticals. Using LAS, protein nanoparticles are formed by

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decreasing the quality of the solvent in which the molecules are solubilized 7. One of the

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advantages of using hydrophobic proteins, such as zein, is that the particles formed do not

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require a hardening step to stabilize them against redissolution in aqueous solutions. One of

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the main disadvantages is that they tend to aggregate in aqueous solutions due to their high

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surface hydrophobicity leading to destabilization of the delivery system 8-10.

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Nanoparticle aggregation due to hydrophobic attraction can be reduced by coating the

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particles with biopolymers that increase the electrostatic and/or steric repulsion between

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them. Particle coating can be carried out in two ways: (i) during particle formation - the

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coating biopolymer is included in the antisolvent phase (co-precipitation) 11-13 or, (ii) after

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particle formation - the biopolymer is deposited onto the particle surfaces by electrostatic

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attraction 9, 14. Sodium caseinate has previously been used to stabilize particles against

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thermal treatments and elevated ionic strength 1, 3, 11, 13. Nevertheless, the coated zein

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particles were still unstable to particle aggregation near the isoelectric point of the protein

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coating. This effect was attributed to the fact that protein coatings usually inhibit particle

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aggregation through electrostatic repulsion rather than steric repulsion 15. Near the

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isoelectric point, the net surface charge of the particles will be low and, therefore, the

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electrostatic repulsion between the particles will be limited 16. ACS Paragon Plus Environment

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Improvements in the stability of proteins and protein-coated colloidal particles have

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been achieved by employing the Maillard reaction to form conjugates with high molecular

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mass polysaccharides 17. The protein part causes the conjugate to adsorb to hydrophobic

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surfaces, whereas the polysaccharide part provides strong steric repulsion. The Maillard

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reaction is a non-enzymatic reaction that involves the condensation of the carbonyl group

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of a reducing carbohydrate with a free amino group of a protein (either a lysine residue or

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an N-terminal amino group). The Maillard reaction is initiated by the attack of the amino

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group (caseinate) on the carbonyl group (dextran), leading to the formation of an N-

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substituted glycosylamine and then to an aldimine (Schiff base). The thermolabile

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glycosylamine subsequently undergoes an Amadori rearrangement and the resulting 1-

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amino-1-deoxy-2-ketose is referred to as the Amadori rearrangement product (Figure 1).

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This initial attack triggers a series of complex chemical reactions, known as the Maillard

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reaction 18. The reaction is greatly accelerated by heat and in alkaline conditions 17. Studies

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have shown that emulsions prepared using β-lactoglobulin-dextran conjugates as

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emulsifiers were more stable during in vitro digestion and to calcium addition than

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emulsions prepared using only β-lactoglobulin 19, 20. Maillard conjugates (MC) between

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caseinate and maltodextrin have previously been used to stabilize caseinate against

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precipitation at the isoelectric point 21. Biopolymer particles fabricated using conjugates of

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whey protein isolate and maltodextrin displayed better thermal stability than particles

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produced using non-conjugated biopolymers 22.

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Resveratrol (trans-resveratrol; trans-3,5,4'-trihydroxy-stilbene), a polyphenolic

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compound that can be isolated from wine by-products 23 has been shown to have several

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potential beneficial effects on human health 24-26. Nevertheless, its utilization as a

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nutraceutical ingredient within the food industry is currently limited due to its poor water-


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solubility, low oral bioavailability, and chemical instability 27-29. There is therefore a need

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to encapsulate this bioactive component so that it can be used as a functional ingredient in

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the food and other industries.

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Based on this background, we hypothesize that the use of MC of caseinate and high

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molecular mass polysaccharides will generate enough steric repulsion to stabilize the zein

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nanoparticles created by LAS even in the presence of low or zero electrostatic repulsion.

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Therefore, the aim of the current work was to evaluate the physical stability of zein

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nanoparticles produced by LAS and co-precipitation with MC of sodium caseinate and

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polysaccharides with different molecular masses. The physical stability of these particles

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was assessed under conditions commonly encountered within the food industry, i.e.,

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thermal processing, salt addition, and pH changes. The capacity of this system to

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encapsulate a grape skin extract (GSE) rich in resveratrol, was also assessed by analyzing

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the encapsulation efficiency. The results of our study will be useful to evaluate the viability

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of creating stable delivery systems to encapsulate hydrophobic nutraceuticals to enrich a

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wide variety of food products without compromising their physical quality.

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Materials and methods

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Materials

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Resveratrol standard (purity ≥ 99%), potassium bromide (≥ 99%), calcium chloride (≥

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97%), o-phthalaldehyde (OPA, ≥ 99%), 2-mercaptoethanol, and dextran (with molecular

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masses of 37 and 150 kDa) were purchased from Sigma-Aldrich Co. (St. Louis MO, USA).

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All other chemicals, reagents, and solvents were purchased from Fisher Scientific

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(Waltham, MA, USA) and were of analytical grade unless stated otherwise. Grape skin

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extract containing 99% resveratrol was purchased from Changsha Organic Herb Inc. ACS Paragon Plus Environment

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(Changsha, China). Food grade zein (F4000) was purchased from Flo Chemicals

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(Ashburnham, MA, USA). Sodium caseinate was purchased from American Caseinate

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Company (Burlington, NJ, USA). Maltodextrin samples (with a degree of esterification of

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6.4 and 17.2 and calculated molecular masses of 1 and 2.8 kDa) were kindly provided by

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Grain Processing Corporation (Muscatine, IO, USA).

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Maillard conjugate formation

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Sodium caseinate (2.00 w/v%) and the polysaccharides (3.5 w/v%) were individually

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solubilized overnight at 5 ºC in water. The completely solubilized and hydrated samples

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were subsequently mixed in a one-to-one ratio, which led to a final sodium caseinate

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concentration of 1.00 w/v%. The mixture was spray-dried (Buchi, Switzerland ) using an

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inlet temperature of 150 ºC, a feed rate of 7.5 mL/min, a compressed air pressure of 600

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kPa, and air flow rate of 35 m3/h 22. Maillard conjugation reactions were performed by

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incubating the spray-dried mixture at 60 ºC and 76% relative humidity for 48 h 21, 30. After

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conjugation, the samples were allowed to cool down to room temperature and ground using

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a mortar and pestle. The samples were subsequently stored in a desiccator prior to their use.

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Maillard conjugate characterization

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Conjugation efficiency

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The conjugation efficiency was determined by measuring the reduction in free amino

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groups using the OPA assay (Pan et al., 2006). The OPA reagent was prepared according to

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Pan and colleagues 30. In short, 40 mg OPA (dissolved in 1.0 mL 95% ethanol), 25 mL

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0.100 M sodium tetraborate buffer (pH 9.5), 2.5 mL 20% SDS solution, and 0.10 mL 2-

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mercaptoethanol were mixed together and brought to a final volume of 50 mL. The OPA

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reagent was prepared freshly before use. After dispersing the conjugates, 0.10 mL of the ACS Paragon Plus Environment

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dispersion was mixed with 2.70 mL of OPA reagent and incubated for 1 min at room

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temperature, the absorbance at 340 nm was measured immediately. The calibration curves

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were constructed using L-leucine (0,2-5 mM) as a standard amino group containing

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compound 21, 30. The conjugation efficiency was defined as follows:

(%) = (1 −

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() )100 ()

Quantification of unreacted protein using the Lowry assay The quantification of the unreacted protein remaining in the system was assessed based

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on the methodology described by Markman and Livney 21. The samples were dissolved in

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double distilled water at concentrations of 10 mg/mL, and subsequently acidified to pH 4.6

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with HCl. The sample suspensions were centrifuged at 1000 g for 10 min and then filtrated

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(P5 filter paper, Fisher Scientific, Pittsburgh, PA). The pH of the supernatant was

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readjusted to 7.0 with NaOH. The protein content of the suspension (after pH adjustment

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and before centrifugation) and supernatant was measured by the Lowry method 31. The

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amount of protein was subsequently calculated using a calibration curve prepared using

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sodium caseinate concentrations ranging from 0.2 to 1.0 mg/mL. The conjugation yield was

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defined as follows: ! (%) = ( )100 ℎ !

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Size exclusion chromatography

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The conjugation products were analyzed by high performance size exclusion

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chromatography (1260 Infinity, Agilent Technologies, Santa Clara, CA). 100 µL of the

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sample (containing 1.0 w/v% solids) was injected and separated over a OHpak SB-806M ACS Paragon Plus Environment

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HQ column (Shodex, Showa Denko America Inc., Torrance, CA, USA) at a flow rate of 1.0

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mL/min and 20 ˚C. The mobile phase consisted of 0.200 M NaCl solution. Two detectors

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were used, a light scattering (Dawn Heleos-II, Wyatt Technology) and a refractive index

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detector (Optilab, T-rex, Wyatt Technology). The light scattering data were collected at a

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cell temperature of 30 ˚C and with a detector angle of 90˚. The cell temperature of the

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refractive index detector was set to 40 ˚C.

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Fabrication of nanoparticles

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Protein nanoparticles were fabricated by LAS as described in a previous manuscript 1.

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Zein (3.0 w/v%) was suspended in 85 v/v% ethanol by stirring for 2 h followed by

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centrifugation at 9900 g for 12 min. The supernatant was collected and after overnight

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storage at 4 ˚C, it was centrifuged (9900 g for 12 min) again and filtered using filter paper

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(P5, Fisher Scientific) to remove any precipitated material. The filtered zein solution was

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subsequently added to the antisolvent at a 1:5 ratio under continuous stirring (440 rpm) at

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room temperature. Bare zein particles were produced using water (at pH 7.0) as antisolvent,

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while coated particles were produced by co-precipitation in aqueous sodium caseinate and

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maltodextrin or dextran or conjugate solutions (pH 7.0, final protein concentration of 1.00

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w/v%). After addition, the particle suspensions were stirred for an additional 2 min. Ethanol

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was removed from the mixtures using a rotary evaporator at 30 ºC (Buchi RE 111, Flawil,

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Switzerland). For the nanoparticles loaded with resveratrol, 0.15 w/v% of GSE was added

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to the solvent phase (i.e. the initial zein solution), stirred for 30 min, and then filtered using

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Whatman paper (Nr. 42) prior to particle production. The particle suspensions, after the

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LAS procedure, hence, contained 0.5 w/v% zein and 0.025 w/v% resveratrol.


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Resveratrol quantification The resveratrol level contained in the nanoparticles was measured using a UV-VIS

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spectrophotometer (Ultrospec 3000 pro, Biochrom Ltd., Cambridge, UK) at a wavelength

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of 307 nm. The nanoparticles were dissolved in dimethyl sulfoxide prior the analysis.

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Nanoparticles without resveratrol were used as blank. Pure resveratrol was dissolved in

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dimethyl sulfoxide at 100 µg/mL and then diluted to create a calibration curve with

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concentrations ranging from 0.2 to 6.0 µg/mL.

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Characterization of nanoparticles

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Particle size and ζ-potential

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Mean particle diameter, polydispersity index and particle size distribution were

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obtained using a dynamic light scattering instrument (Zetasizer Nano ZS, Malvern

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Instruments, Malvern, UK). The ζ-potential was determined by particle electrophoresis

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using the same instrument. Samples were diluted 40 times to avoid multiple scattering

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effects and equilibrated at 25 ˚C prior to analysis.

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Encapsulation efficiency

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The encapsulation efficiency (EE,%) of the nanoparticles prepared by co-precipitation

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with the physical mixture of caseinate and maltodextrin of 2.8 kDa or dextrans of 37 and

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150 kDa, as well as, those prepared with the Maillard conjugates was determined using the

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following equation: ## (%) = (1 −

$% )100 $&

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In which ResT is the resveratrol contained in the suspension (M) and ResF is the amount of

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free resveratrol (M). The free resveratrol was determined as the amount of resveratrol that


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was collected into the filtrate receiver after being centrifuged at 1840 g for 60 min using a

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centrifugal filter (Amicon® Ultracel-10kDa 2 mL Millipore, Cork Ireland) 6. The recovery

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yield of resveratrol after passing through the filter (92%) was taken into account to correct

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the actual amount of resveratrol in the filtrate. The resveratrol contained in the

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nanoparticles and the filtrate was assessed by diluting the suspension in dimethyl sulfoxide

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Morphology

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The morphology of the conjugated and non-conjugated particles with dextran 37 kDa, was

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analyzed using transmission electron microscopy (2000FX TEM, JEOL, Tokyo, Japan) at a

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voltage of 200 kV. The sample was diluted 40 times following the same procedure as the

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particle size and ζ-potential measurements. An aliquot (5 μL) of the diluted sample was

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drop-casted onto a carbon-coated copper grid (400 mesh). The grid was then air-dried at

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room temperature before loading it into the microscope. Images of the biopolymer particles

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were recorded in randomly selected fields 33.

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Particle stability to environmental stresses

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CaCl2 Stability

prior to quantification. The suspensions without resveratrol were used as blank samples.

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After production, the particle dispersions were diluted two-fold using CaCl2 solutions

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(0.0 – 200 mM) to obtain systems with final CaCl2 concentrations ranging from 0.0 to 100

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mM. The samples were then stored for 24 h at room temperature after which the particle

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size distribution was analyzed by dynamic light scattering.

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Temperature stability

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Freshly prepared particle dispersions were diluted two-fold using NaCl solutions (600 and 1200 mM) to obtain systems with final NaCl concentrations ranging of 300 and 600 ACS Paragon Plus Environment

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mM, and then incubated in water bath set at different temperatures (30-90 °C) for 30 min.

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The samples were then allowed to cool down to 23 ˚C, stored for 24 h, after which the

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particle size distribution was analyzed by dynamic light scattering.

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pH stability

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The pH stability of freshly prepared 40–fold diluted particle dispersions was assessed

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using an automatic titrator (MPT-2, Malvern Instruments) with NaOH (0.25 M) and HCl

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(0.10 M) as the titrating base and acid. The particle size distribution and ζ-potential were

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measured from pH 9.0 to 3.0 (titrating intervals of 0.5) by dynamic light scattering and

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particle electrophoresis.

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Data analysis

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All experiments were performed at least in duplicate and the results are given as mean

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values ± standard deviation.

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Results and Discussion

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Conjugation efficiency and yield

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The conjugation efficiency of the reaction of caseinate with carbohydrates with

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molecular masses of 1.0, 2.8, 37 or 150 kDa was analyzed by quantifying the unreacted

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amino groups of the caseinate after the conjugation reaction using the OPA test (Figure 2).

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The conjugation yield was tested by studying the behavior of the non-conjugated and

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conjugated protein-carbohydrate samples at the isoelectric point of the protein (pH 4.6).

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The protein that does not precipitate at this pH is considered to be stabilized by conjugation

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to the carbohydrate. It is, however, possible that this technique underestimates the real

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conjugation yield as protein aggregates, although conjugated to dextran molecules, will ACS Paragon Plus Environment

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likely also precipitate in these conditions and, hence, be considered unconjugated. This

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technique is also a simple way to purify the conjugated from the non-conjugated proteins

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(Figure 2) 21. The molecular mass of the polysaccharide had an influence on both the

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conjugation efficiency and conjugation yield. With an increase in molecular mass of the

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polysaccharide the conjugation efficiency diminished. The lowest efficiency was reached at

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37 kDa and did not significantly change upon further increases in molecular mass (Figure

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2). Simultaneously, the increment in molecular mass of the polysaccharide substantially

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increased the conjugation yield. The conjugation of carbohydrates with a higher molecular

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mass to the proteins clearly stabilized the caseinate proteins against aggregation near the

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isoelectric point. The conjugation yield, hence, reflects two processes: the conjugation of

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the carbohydrate and the protein (conjugation efficiency) and the ability of the carbohydrate

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to stabilize the protein near its isoelectric point. Based on previous studies 21, 30, the average

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number of amino groups per caseinate molecule available to react with the reducing sugars

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of the polysaccharides is 13.6. We therefore estimated the average number of carbohydrates

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conjugated per caseinate molecule in our study to be about 9, 5, 0.7 and 0.7 for the 1.0, 2.8

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maltodextrins and 37, and 150 kDa dextrans, respectively. The reduction in the conjugation

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efficiency could be explained by an increase in the steric hindrance when longer

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polysaccharides are conjugated with a protein, reducing the accessibility of the surrounding

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unconjugated amino groups. The steric hindrance effect using dextrans of 37 and 150 kDa

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appears to be similar, since the number of carbohydrates conjugated per caseinate molecule

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was equal for both treatments. Furthermore, the molar ratio between the caseinate and the

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polysaccharides was also reduced by increasing the molecular mass of the carbohydrates,

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resulting in less reducing sugar ends to conjugate to the protein. The protein-carbohydrate

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mass ratio used was not changed between the carbohydrate samples as increasing the


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concentration of the high molecular mass dextran to keep the same molar ratio, resulted in

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hazy solutions with incomplete solubilization.

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Size exclusion chromatography confirmed the molecular mass reported by the supplier

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of the dextrans and showed that the molecular mass of the dextran molecules was not

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altered by spray-drying the dextran-caseinate mixture (Table 1). The subsequent incubation

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of the spray dried caseinate-dextran mixture resulted in the formation of large complexes

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(Table 1). It was, however, impossible to estimate the ratio of caseinate to dextran

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molecules based on these data as caseinate probably does not occur as single molecules, but

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rather as caseinate aggregates 34. The broad molecular weight distribution of the signals

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picked up for the conjugated samples could be ascribed to the presence of a variety of

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conjugation degrees between the caseinate and the dextran molecules or, more likely, to the

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aggregation or micellization of unreacted protein in the column 34, 35. Around pH 4.6, non-

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conjugated caseinate has no or a very low net charge, leading to aggregation and

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precipitation due to the loss of electrostatic repulsion between the protein chains. After

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conjugation, the stability of the caseinate-carbohydrate conjugates is believed to be driven

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by steric repulsion. As clearly shown by the conjugation yield data, the steric repulsion

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caused by the conjugation with the maltodextrin of 1.0 kDa is not enough to stabilize the

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protein solution near the isoelectric point of the protein. Increasing the molecular mass of

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the polysaccharide approximately 3 times already had a sharp effect on the stability of the

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protein by increasing the recovery yield from 10% to almost 90% at pH 4.6. Further

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increments in the molecular mass of the polysaccharide resulted in even higher yields of up

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to 93%.


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Particles characterization MC of caseinate and carbohydrates with molecular masses of 2.8, 37 and 150 kDa

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were included in the antisolvent phase to study the effect of the glycation of caseinate on

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the characteristics and stability of caseinate-coated zein nanoparticles encapsulating

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resveratrol produced by LAS. The conjugates formed with maltodextrin of 1.0 kDa were

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not used for further studies, because these polysaccharide chains were not long enough to

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create the steric repulsion needed to stabilize the caseinate at pH 4.6. The mean particle

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diameter of the resveratrol-loaded particles created with the conjugates and the non-

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conjugated physical mixture of caseinate and carbohydrates was subsequently analyzed

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(Figure 3a). All the samples presented a monomodal particle size distribution (Figure 3c).

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The mean particle diameter remained relatively stable for particles produced with the non-

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conjugated mixture of caseinate and carbohydrates. Furthermore, the diameter did not

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change significantly for the particles co-precipitated with the caseinate-maltodextrin (2.8

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kDa) conjugates versus those produced in the presence of the non-conjugated mixture of

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caseinate and maltodextrin (2.8 kDa).

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For the zein particles produced with the antisolvent mixtures containing conjugates of

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caseinate and the higher molecular mass dextrans (37 and 150 kDa), the mean particle

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diameter decreased relative to those particles co-precipitated with non-conjugated protein-

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carbohydrate mixtures. The reason for this decrease could be a reduction in the packing

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parameter and an associated increase in the curvature of the coated nanoparticles. The

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reduction is a consequence of the increase in the bulkiness of the hydrophilic domains of

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the caseinate protruding into the environment caused by the attachment of the

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polysaccharide chains 21. Another possibility to explain the reduction in size is steric

311

repulsion caused by the carbohydrate structures protruding from the particle surface which


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restrict particle growth as no other zein or caseinate molecules can approach the inner core

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of the particles. The increment of the bulkiness of the conjugates with maltodextrin with a

314

molecular mass of 2.8 kDa is not enough to decrease the packing parameter or ensure

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enough steric repulsion. Conversely, the zein particles co-precipitated with the non-

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conjugated mixture of caseinate and dextran (37 and 150 kDa) had an increased particle

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diameter compared to the ones made with 2.8 kDa maltodextrin. A slight aggregation of the

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particles caused by depletion flocculation could be responsible for the observed increase in

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mean particle diameter. The long chain polysaccharides generated a thermodynamic

320

incompatibility with the nanoparticles pushing the particles together 36. Electrophoresis

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measurements of the particles co-precipitated with the conjugates and the non-conjugated

322

mixtures of caseinate and carbohydrates indicated that the magnitude of the zeta-potential

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decreased upon conjugation (Figure 3b). The decrease, however, was greater for the

324

conjugates made with the dextrans of 37 and 150 kDa. Lesmes and McClements 19

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observed similar results with lipid droplets coated by ß-lactoglobulin-dextran MC and

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hypothesized that the reduction in the zeta-potential could be explained by two phenomena.

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Firstly, the number of ionizable groups at the particle surface is reduced due to the

328

formation of the Amadori compounds which consume free amino groups (Figure 1).

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Secondly, the decrease in the apparent zeta-potential of the nanoparticles can be due to a

330

screening effect in which the distance between the protein charges and the aqueous

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continuous phase is increased. The presence of resveratrol did not change the size and zeta

332

potential of the nanoparticles (data not shown), which can be attributed to the fact that it is

333

incorporated into the internal hydrophobic core or embedded in the biopolymer network of

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the particles. Figure 4 shows the microscopic morphology of zein nanoparticles loaded

335

with resveratrol prepared with non-conjugated and conjugated caseinate with dextran 37


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kDa. The particles have a spheroid shape and proved to be stable against aggregation. The

337

size of the conjugated particles is smaller supporting the results obtained with the dynamic

338

light scattering instrument. The surface of the non-conjugated particles appears to be

339

rougher and less even than the surface of the conjugated particles. The difference in the

340

surface of the particles can be related to the hypothesized differences in molecular

341

arrangements in the particle structure.

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Encapsulation efficiency

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The EE was measured for the particles co-precipitated with the non-conjugated mixture

344

of caseinate and the polysaccharides with molecular masses of 2.8, 37 and 150 kDa and for

345

their conjugates. The EE of the particles produced with the non-conjugated and the

346

conjugated caseinate-carbohydrate samples in the antisolvent all varied around 80 and 85%

347

without a clear tendency of increasing or decreasing EE depending on the evaluated

348

treatments (Figure 5). Therefore it was concluded that the conjugation of caseinate did not

349

negatively affect the EE. Moreover, the percentages are similar to the ones obtained

350

previously, in which resveratrol was encapsulated in zein particles stabilized by sodium

351

caseinate without the addition of carbohydrates1. In that research, it was found that

352

resveratrol could form complexes with sodium caseinate in the system that could be part of

353

the particle coating or be dispersed within the continuous phase as a complementary

354

method of resveratrol encapsulation. It is also know that polysaccharides can stabilize

355

resveratrol in aqueous medium mainly through the formation of hydrogen bonds 37. This

356

can certainly also contribute to a higher EE of resveratrol in these systems.


16

Page 17 of 39

357


Particle stability

358

The ultimate goal of the use of protein-polysaccharide conjugates to coat the zein

359

particles was to increase the stability of the final nanoparticles without compromising

360

particle functionality, e.g. EE. We therefore tested the influence of a variety of

361

environmental stresses on the stability of the particles.

362

Sodium ions were included in the aqueous phase of the nanoparticle suspensions and

363

then they were subjected to thermal treatments. The ions shield the surface charges on the

364

particles and, hence, reduce the electrostatic repulsion between them. This approach also

365

more closely matches the conditions encountered in real-world food systems. In accordance

366

with previous research on caseinate-coated zein particles 38, the mean diameter of zein

367

nanoparticles made with caseinate and maltodextrin (both non-conjugated and conjugated)

368

did not change appreciably with increasing temperature or high salt concentrations (Figure

369

6a). For both dextran samples, however, the mean diameter increased with increasing

370

temperature for the non-conjugated samples (Figure 6b&c) and presented a multimodal

371

particle size distribution indicating the presence of aggregates (data non shown). This was

372

more pronounced with increasing salt concentration and increasing molecular mass of the

373

carbohydrate moiety. In non-conjugated samples, free dextran molecules will be found in

374

the suspension which promote depletion flocculation and destabilization of the particle

375

suspensions, resulting in increased particle sizes. In this case, the free biopolymers

376

therefore act as an additional destabilization factor. Depletion flocculation occurs due to

377

steric exclusion of polymers from the particle surfaces, which leads to osmotic pressure

378

gradients that increase the attractive forces between the particles 39. Previous research

379

already indicated that the critical concentration at which depletion flocculation occurs,

380

decreases with the molecular mass of the non-adsorbed polymer 40. Larger molecules will


17


Page 18 of 39

381

be more easily excluded from the particle surfaces and their effect will therefore be more

382

pronounced at lower concentrations (assuming similar properties of the polymers). Particle

383

and biopolymer mobility will be increased by higher temperatures which will speed up the

384

flocculation process. The conjugation of dextran to caseinate molecules coating the zein

385

particles inhibited destabilization due to depletion flocculation, probably due to steric

386

repulsion and the fact that the free dextran concentration in the conjugated samples was

387

also significantly lower than in the non-conjugated samples. The mean diameter of the

388

conjugated dextran-caseinate zein nanoparticles did not grow significantly with increasing

389

temperature, even in the presence of elevated salt concentrations (Figure 6b&c) and the

390

particle size distribution remained monomodal.

391

Calcium chloride, as a divalent salt, has a strong crosslinking and destabilizing effect

392

on biopolymer-based nanoparticles 41, especially when they are only stabilized by

393

electrostatic repulsion. As outlined above, in general, protein particles are mainly stabilized

394

by electrostatic repulsion between the particles. Calcium ions can form salt bridges between

395

anionic protein particles. The increase of the steric repulsion by conjugating carbohydrates

396

to the caseinate molecules coating the zein particles is expected to increase the stability of

397

the nanoparticles. The hairy carbohydrate protrusions hamper the formation of calcium

398

bridges between particles. The particles coated with non-conjugated caseinate destabilized

399

in the presence of calcium ions (Figure 6d insert). Conjugation of the carbohydrate with

400

the lowest molecular mass, i.e. maltodextrin, to the coating caseinate, only marginally

401

stabilized the particles against bridging by calcium ions (Figure 6d). Conversely, dextran

402

stabilized the particles against aggregation at elevated calcium concentrations, which can be

403

attributed to the greater steric repulsion generated by the longer carbohydrate molecules

404

(Figure 6d).


18

Page 19 of 39

405


Protein particles are especially sensitive to pH changes. Near the isoelectric point of

406

the protein particles, the charge on the particle surface is reduced, which minimizes the

407

electrostatic repulsion between particles and promotes aggregation. Plain zein particles

408

destabilized around pH 5.0 (Figure 7a) where they showed the lowest absolute zeta

409

potential (Figure 8a). This would seriously hamper the use of these protein particles in

410

many food products. Coating of zein particles with caseinate slightly reduced the pH at

411

which the particles aggregated, i.e. to around pH 4.5 (Figure 7b-d) 38, but extensive

412

aggregation still occurred. Conjugation of a carbohydrate to the caseinate coating the zein

413

particles does not significantly affect the isoelectric point of the particles relative to the

414

values found for the non-conjugated samples (Figure 8b-d). However, it did reduce the

415

magnitude of the electrical charge on the particles below and above the isoelectric point. As

416

discussed earlier, this can be attributed to the association of carbohydrates to amino groups

417

on the proteins and to shielding of the particle surface charge by carbohydrate layer. Non-

418

conjugated particles were still destabilized near and below the isoelectric point. However,

419

when the zein particles were coated with caseinate-carbohydrate conjugates, the mean

420

particle diameter did not increase appreciably when the pH was reduced to around and

421

below the isoelectric point. Maltodextrin did not perform as efficiently as the higher

422

molecular mass dextrans. Indeed, the dextran molecules with a molecular mass of 37 kDa

423

were able to completely stabilize the particles against size increments caused by pH effects.

424

We have shown that the stability of resveratrol-fortified zein nanoparticles can be

425

improved by coating them with MC made from sodium caseinate and neutral

426

carbohydrates. The molecular mass of the carbohydrates used to form the MC had a

427

significant impact on particle stabilization, with the stability of the delivery systems

428

increasing with increasing carbohydrate molecular mass. This is an important result in view


19


Page 20 of 39

429

of the potential application of zein particles in real-world food applications. The

430

nanoparticles produced in this work were highly stable against environmental stresses that

431

might be encountered by delivery systems in real-life food processing applications, e.g.,

432

temperature, pH, and ionic strength changes.


20

Page 21 of 39


433

References

434

1.

435

biopolymer particles produced using liquid antisolvent precipitation. Part 1: preparation and

436

characterization. Food Hydrocolloids in press, 45, 309-316.

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2.

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zein-curcumin colloidal particles. Soft Matter 2010, 6, 6192-6199.

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3.

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loaded biopolymeric colloidal particles prepared by simultaneous precipitation of quercetin

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with hydrophobic protein in aqueous medium. Food Chemistry 2012, 133, 423-429.

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4.

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nanospherical particles. Journal of Agricultural and Food Chemistry 2005, 53, 4788-4792.

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5.

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indole-3-carbinol and 3,3 '-diindolylmethane in zein/carboxymethyl chitosan nanoparticles

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with controlled release property and improved stability. Food Chemistry 2013, 139, 224-

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230.

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6.

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characterization of zein/chitosan complex for encapsulation of alpha-tocopherol, and its in

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vitro controlled release study. Colloids and Surfaces B-Biointerfaces 2011, 85, 145-152.

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for use in food systems. Trends in Food Science and Technology 2013, 34, 109-123.

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pH modulated antisolvent precipitation method. Food Research International 2014, 64,

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329-335.

Davidov-Pardo, G.; Joye, I. J.; McClements, D. J., Encapsulation of resveratrol in

Patel, A.; Hu, Y. C.; Tiwari, J. K.; Velikov, K. P., Synthesis and characterisation of

Patel, A. R.; Heussen, P. C. M.; Hazekamp, J.; Drost, E.; Velikov, K. P., Quercetin

Parris, N.; Cooke, P. H.; Hicks, K. B., Encapsulation of essential oils in zein

Luo, Y. C.; Wang, T. T. Y.; Teng, Z.; Chen, P.; Sun, J. H.; Wang, Q., Encapsulation of

Luo, Y. C.; Zhang, B. C.; Whent, M.; Yu, L. L.; Wang, Q., Preparation and

Joye, I. J.; McClements, D. J., Production of nanoparticles by anti-solvent precipitation

Hu, K.; McClements, D. J., Fabrication of surfactant-stabilized zein nanoparticles: A


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Joye, I. J.; Nelis, V. A.; McClements, D. J., Gliadin-based nanoparticles: Stabilization

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by post-production polysaccharide coating. Food Hydrocolloids 2015, 43, 236-242.

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10. Zhong, Q.; Jin, M., Zein nanoparticles produced by liquid–liquid dispersion. Food

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Hydrocolloids 2009, 23, 2380-2387.

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11. Patel, A. R.; Bouwens, E. C. M.; Velikov, K. P., Sodium caseinate stabilized zein

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colloidal particles. Journal of Agricultural and Food Chemistry 2010, 58, 12497-12503.

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12. Li, K. K.; Yin, S. W.; Yin, Y. C.; Tang, C. H.; Yang, X. Q.; Wen, S. H., Preparation of

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water-soluble antimicrobial zein nanoparticles by a modified antisolvent approach and their

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characterization. Journal of Food Engineering 2013, 119, 343-352.

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13. Chen, H. Q.; Zhong, Q. X., Processes improving the dispersibility of spray-dried zein

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nanoparticles using sodium caseinate. Food Hydrocolloids 2014, 35, 358-366.

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14. Hu, K.; McClements, D. J., Fabrication of biopolymer nanoparticles by antisolvent

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precipitation and electrostatic deposition: Zein-alginate core/shell nanoparticles. Food

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Hydrocolloids 2015, 44, 101-108.

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15. McClements, D. J., Protein-stabilized emulsions. Current Opinion in Colloid &

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Interface Science 2004, 9, 305-313.

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16. Jahanshahi, M.; Babaei, Z., Protein nanoparticle: a unique system as drug delivery

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vehicles. African Journal of Biotechnology 2008, 7, 4926-4934.

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17. Oliver, C. M.; Melton, L. D.; Stanley, R. A., Creating proteins with novel functionality

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via the maillard reaction: A review. Critical Reviews in Food Science and Nutrition 2006,

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46, 337-350.

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18. Ames, J. M., Control of the Maillard reaction in food systems. Trends in Food Science

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and Technology 1990, 1, 150-154.


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19. Lesmes, U.; McClements, D. J., Controlling lipid digestibility: Response of lipid

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droplets coated by β-lactoglobulin-dextran Maillard conjugates to simulated gastrointestinal

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conditions. Food Hydrocolloids 2012, 26, 221-230.

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20. Wooster, T. J.; Augustin, M. A., β-Lactoglobulin-dextran Maillard conjugates: Their

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effect on interfacial thickness and emulsion stability. Journal of Colloid and Interface

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Science 2006, 303, 564-572.

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21. Markman, G.; Livney, Y. D., Maillard-conjugate based core-shell co-assemblies for

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nanoencapsulation of hydrophobic nutraceuticals in clear beverages. Food & Function

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2012, 3, 262-70.

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22. Shah, B.; Davidson, P. M.; Zhong, Q., Encapsulation of eugenol using Maillard-type

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conjugates to form transparent and heat stable nanoscale dispersions. LWT - Food Science

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and Technology 2012, 49, 139-148.

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23. Walle, T.; Hsieh, F.; DeLegge, M. H.; Oatis Jr, J. E.; Walle, U. K., High absorption but

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very low bioavailability of oral resveratrol in humans. Drug Metabolism and Disposition

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2004, 32, 1377-1382.

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24. Alves, N. E. G., Studies on mechanistic role of natural bioactive compounds in the

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management of obesity, an overview. Open Nutraceuticals Journal 2012, 5, 193.

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25. Catalgol, B.; Batirel, S.; Taga, Y.; Ozer, N. K., Resveratrol: French paradox revisited.

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Frontiers in Pharmacology 2012, 3 JUL.

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26. Neves, A. R.; Lucio, M.; Martins, S.; Lima, J. L. C.; Reis, S., Novel resveratrol

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nanodelivery systems based on lipid nanoparticles to enhance its oral bioavailability.

500

International Journal of Nanomedicine 2013, 8, 177-187.


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27. Hung, C.-F.; Chen, J.-K.; Liao, M.-H.; Lo, H.-M.; Fang, J.-Y., Development and

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evaluation of emulsion-liposome blends for resveratrol delivery. Journal of Nanoscience

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and Nanotechnology 2006, 6, 2950-2958.

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28. Patel, K. R.; Scott, E.; Brown, V. A.; Gescher, A. J.; Steward, W. P.; Brown, K.,

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Clinical trials of resveratrol. In Resveratrol and Health, Vang, O.; Das, D. K., Eds. 2011;

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Vol. 1215, pp 161-169.

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29. Trela, B. C.; Waterhouse, A. L., Resveratrol: Isomeric molar absorptivities and

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stability. Journal of Agricultural and Food Chemistry 1996, 44, 1253-1257.

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30. Pan, X.; Mu, M.; Hu, B.; Yao, P.; Jiang, M., Micellization of casein-graft-dextran

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copolymer prepared through Maillard reaction. Biopolymers 2006, 81, 29-38.

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31. Lowry, O. H.; Rosebrough, N. J.; Rarr, L. A.; Randall, R. J., Protein measurement with

512

the Folin phenol reagent. Journal of Biological Chemistry 1951, 193, 265-275.

513

32. Podaralla, S.; Perumal, O., Influence of formulation factors on the preparation of zein

514

nanoparticles. AAPS PharmSciTech 2012, 13, 919-927.

515

33. Arroyo-Maya, I. J.; McClements, D. J., Biopolymer nanoparticles as potential delivery

516

systems for anthocyanins: Fabrication and properties. Food Research International 2015,

517

69, 1-8.

518

34. Morris, G. A.; Sims, I. M.; Robertson, A. J.; Furneaux, R. H., Investigation into the

519

physical and chemical properties of sodium caseinate-maltodextrin glyco-conjugates. Food

520

Hydrocolloids 2004, 18, 1007-1014.

521

35. O’Regan, J.; Mulvihill, D. M., Preparation, characterisation and selected functional

522

properties of sodium caseinate–maltodextrin conjugates. Food Chemistry 2009, 115, 1257-

523

1267.


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524

36. McClements, D. J., Food Emulsions: Principles, practice, and techniques. CRC Press

525

Inc.: 2005; p 374.

526

37. Zhang, X. P.; Le, Y.; Wang, J. X.; Zhao, H.; Chen, J. F., Resveratrol nanodispersion

527

with high stability and dissolution rate. LWT-Food Science and Technology 2013, 50, 622-

528

628.

529

38. Joye, I. J.; Davidov-Pardo, G.; McClements, D. J., Encapsulation of resveratrol in

530

biopolymer particles produced using liquid antisolvent precipitation. Part 2: Stability and

531

functionality. Food Hydrocolloids 2015, 49, 127-134.

532

39. Walstra, P., Physical Chemistry of Foods. Marcel Dekker, Inc.: New York (USA),

533

2003; p 807.

534

40. Burns, J. L.; Yan, Y.; Jameson, G. J.; Biggs, S., The effect of molecular weight of

535

nonadsorbing polymer on the structure of depletion-induced flocs. Journal of Colloid and

536

Interface Science 2002, 247, 24-32.

537

41. Agboola, S. O.; Dalgleish, D. G., Calcium-induced destabilization of oil-in-water

538

emulsions stabilized by caseinate of by beta-lactoglobulin. Journal of Food Science 1995,

539

60, 399-404.

540

Note

541

Dr. Gabriel Davidov-Pardo is recipient of a post-doctoral fellowship by the Secretaría

542

de Ciencia Tecnología e Innovación del Distrito Federal (SECITI, Mexico City). Dr. Iris

543

Joye gratefully acknowledges financial support from the ‘Fonds voor Wetenschappelijk

544

Onderzoek – Vlaanderen’ (FWO, Brussels, Belgium) and from the European Commission

545

7th Framework Program (FP7-People-2011-IOF-300408). This material is partly based


25


546

upon work supported by United States Department of Agriculture, NRI Grants (2011-

547

03539, 2013-03795, 2011-67021, and 2014-67021).

Page 26 of 39

548


26

Page 27 of 39


549

Figure captions

550

Figure 1. Schematic representation of the Maillard conjugation reaction between the

551

reducing end of a sugar molecule and a free amine group of a protein. This figure only

552

shows the initial phase of the Maillard reaction until the formation of the Amadori

553

products, the further stages of the reaction are not shown.

554 555

Figure 2. Conjugation yield and efficiency of the conjugation of caseinate to

556

maltodextrin/dextran.

557 558

Figure 3. Mean particle diameter (A) and zeta-potential (B) and particle size distribution

559

(C) of zein particles loaded with grape skin extract (GSE) prepared with conjugated

560

(Maillard conjugates of sodium caseinate and dextran) and non-conjugated (physical

561

mixture of unreacted sodium caseinate and dextran) biopolymers in the antisolvent [1.0%

562

(w/v)]. The ratio of polysaccharides to sodium caseinate was 1.75 to 1.0. Zein and GSE

563

concentrations in the particle suspensions were 0.5 and 0.025% (w/v), respectively.

564 565

Figure 4. Transmission electron microscopy (TEM) images of zein particles loaded with

566

grape skin extract (GSE) prepared with (A) conjugated (Maillard conjugates of sodium

567

caseinate and dextran) and (B) non-conjugated (physical mixture of unreacted sodium

568

caseinate and dextran) biopolymers in the antisolvent [1.0% (w/v)]. The caseinate was

569

conjugated with dextran of 37 kDa. The ratio of polysaccharides to sodium caseinate was

570

1.75 to 1.0. Zein and GSE concentrations in the particle suspensions were 0.5 and 0.025%

571

(w/v), respectively.

572 ACS Paragon Plus Environment

27


Page 28 of 39

573

Figure 5. Encapsulation efficiency of zein particles loaded with grape skin extract (GSE)

574

prepared with conjugated (Maillard conjugates of sodium caseinate and dextran) and non-

575

conjugated (physical mixture of unreacted sodium caseinate and dextran) biopolymers in

576

the antisolvent [1.0% (w/v)]. The ratio of polysaccharides to sodium caseinate was 1.75 to

577

1.0. Zein and GSE concentrations in the particle suspensions were 0.5 and 0.025% (w/v),

578

respectively.

579 580

Figure 6. Effect of ions on the particle diameter of zein particles coated with non-

581

conjugated or conjugated caseinate. The particles were incubated in short high temperature

582

treatments (30 min) and/or elevated salt conditions (300 and 600 mM NaCl and 0-100 mM

583

CaCl2). The effect of the molecular mass of the conjugated carbohydrate was studied for

584

NaCl/temperature treatments [(A) maltodextrin (MD) 2.8 kDa, (B) Dextran 37 kDa, and

585

(C) Dextran 150 kDa] and CaCl2 incubation (D). Insert (6D): Appearance of precipitated

586

particles made with the physical mixture of sodium caseinate and dextran 37 kDa in the

587

presence of CaCl2. Zein and GSE concentrations in the particle suspensions were 0.5 and

588

0.025% (w/v), respectively.

589 590

Figure 7. Effect of pH on the mean diameter of plain zein nanoparticles (A) and zein

591

nanoparticles prepared with conjugated (Maillard conjugates of sodium caseinate and

592

dextran) and non-conjugated (physical mixture of unreacted sodium caseinate and dextran)

593

biopolymers in the antisolvent [1.0% (w/v)]. The effect of the molecular mass of the

594

conjugated carbohydrate was studied in function of pH [(B) maltodextrin (MD) 2.8 kDa,

595

(C) Dextran 37 kDa, and (D) Dextran 150 kDa]. Zein and GSE concentrations in the

596

particle suspensions were 0.5 and 0.025% (w/v), respectively.


28

Page 29 of 39


597 598

Figure 8. Effect of pH on the zeta-potential of plain zein nanoparticles (A) and zein

599

nanoparticles prepared with conjugated (sodium caseinate and dextran) and non-conjugated

600

(physical mixture of unreacted sodium caseinate and dextran) in the antisolvent [1.0%

601

(w/v)]. The effect of the molecular mass of the conjugated carbohydrate was studied in

602

function of pH [(B) maltodextrin (MD) 2.8 kDa, (C) Dextran 37 kDa, and (D) Dextran 150

603

kDa]. Zein and GSE concentrations in the particle suspensions were 0.5 and 0.025% (w/v),

604

respectively.


29


Page 30 of 39

Tables Table 1. Molecular mass of the dextran, non-conjugated dextran-caseinate and conjugated dextran-caseinate samples as determined by High Performance Size Exclusion Chromatography.

Dextran 37 kDa

Dextran 150 kDa

Pure dextran

32 kDa

229 kDa

Non-conjugated casein-dextran

34 kDa

231 kDa

1402 kDa and 628 kDa

1242 kDa and 682 kDa

Conjugated casein-dextran


30

Page 31 of 39


Figure graphics Figure 1. OH

Dextran

OH

Anomeric Equilibrium

O HO

OH HO

O

H

O

+

OH

H2 C

H2N

H2 C

H2 C

OH OH

Caseinate

C H2

O

OH OH HO

H

O

N H

OH

H2 C

H2 C

H2 C

C H2

OH

N-substituted glycosylamine

OH

OH OH

HO

Amadori rearrangement

H

O

N H

O

H2 C

H2 C

H2 C

OH HO O

C H2

H N

H2 C

OH

H2 C

H2 C

C H2

+

H 2O

H

Amadori rearrangement product (ARP) 1-amino-1-deoxy-2-ketose


Schiff base (aldimine)

31


Page 32 of 39

Figure 2. Conjugation efficiency

100

Conjugation yield

90

90

80

80

70

70

60

60

50

50

40

40

30

30

20

20

10

10

0

0 1

2.8

37

Conjugation yield (%)

Conjugation efficiency (%)

100

150

Carbohydrate molecular mass (kDa)


32

Page 33 of 39


Figure 3.

Mean Particle Diameter (nm)

A

250 200 150 Non-conjugated

100

Conjugated

50 0 2.8

37

150

Carbohydrate molecular mass (kDa) Carbohydrate molecular mass (kDa)

B Zeta potential (mV)

2.8

Intensity (%)

C

37

150

0 -10

Non-conjugated

-20

Conjugated

-30 -40 -50

60 50 40 30 20 10 0

Non-conjugated Conjugated

Polysaccharide MW 150 kDa

37 kDa

2.8 kDa

0.4

40

4000

Particle diameter (nm) ACS Paragon Plus Environment

33


Page 34 of 39

Figure 4

B

A

500 nm

500 nm

605


34

Page 35 of 39

606 607 608 609


Figure 5

100

Non-conjugate Conjugate

Encapsulation efficiency (%)

90 80 70 60 50 40 30 20 10 0 2.8

37

150

Carbohydrate molecular weight (KDa) 610


35


Figure 6

A 350 330 310 290 270 250 230 210 190 170 150

B Non-conjugated - 300 mM NaCl Conjugated - 300 mM NaCl Non-conjugated - 600 mM NaCl Conjugated - 600 mM NaCl



611 612

350 330 310 290 270 250 230 210 190 170 150

Non-conjugated - 300 mM NaCl conjugated - 300 mM NaCl Non-conjugated - 600 mM NaCl Conjugated - 600 mM NaCl

30 40 50 60 70 80 90

30 40 50 60 70 80 90

Temperature (ºC)

Temperature (ºC) 350 330 310 290 270 250 230 210 190 170 150

Non-conjugated - 300 mM NaCl Conjugated - 300 mM NaCl Non-conjugated - 600 mM NaCl Conjugated - 600 mM NaCl

D 750

Conjugated MD 2.8 kDa Conjugated Dextran 37 kDa Conjugated Dextran 150 kDa


C Mean Particle Diameter (nm)

Page 36 of 39

650 550

Non-conjugated Dextran 37

450 350 250 150

30 40 50 60 70 80 90

Temperature (ºC)

0 50 100 CaCl2 Concentration (mM)


36

Page 37 of 39


A

900



Figure 7.

750 600 450 300 150 0

B

900


750 600 450 300 150 0

3

4

5

6

7

8

9

3

4

5

Conjugated

750 600 450 300 150 0 4

5

6

7

8

D

900 Mean Particle Diameter (nm)


Non-conjugated

C

3

7

8

9

pH

pH

900

6


750 600 450 300 150 0

9

3

4

5

6

7

8

9

pH

pH ACS Paragon Plus Environment

37


Page 38 of 39

Figure 8.

A

B

50 40 30 20 10 0 -10 3 -20 -30 -40 -50

30 Non-conjugated

20

4

5

6

7

8

9

Zeta potential (mV)

Zeta potential (mV)

613 614

Conjugated

10 0 -10

3

4

5

6

-30

pH

D Non-conjugated

30

Non-conjugated

Conjugated

Conjugated

20

10 0 4

5

6

7

-20 -30 -40

8

9

Zeta potential (mV)

20 Zeta potential (mV)

9

-20

-50

pH

30

-50

8

-40

C

-10 3

7

10 0 -10 3

4

5

6

7

8

9

-20 -30 -40

pH

-50


pH 38

Page 39 of 39


615


39

Effect of Maillard Conjugates on the Physical Stability of Zein

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