Vitamin D3 and phytosterols affect the properties of polyglycerol polyricinoleate (PGPR) and protein interfaces

Accepted Manuscript Vitamin D3 and phytosterols affect the properties of polyglycerol polyricinoleate (PGPR) and protein interfaces Jonathan Andrade, Milena Corredig PII:

S0268-005X(15)30107-7

DOI:

10.1016/j.foodhyd.2015.10.001

Reference:

FOOHYD 3155

To appear in:

Food Hydrocolloids

Received Date: 26 April 2015 Revised Date:

26 September 2015

Accepted Date: 1 October 2015

Please cite this article as: Andrade, J., Corredig, M., Vitamin D3 and phytosterols affect the properties of polyglycerol polyricinoleate (PGPR) and protein interfaces, Food Hydrocolloids (2015), doi: 10.1016/ j.foodhyd.2015.10.001. 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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MgCl2

MgCl2/ -sit

MgCl2/Vit.D3

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Vitamin D3 and phytosterols affect the properties of polyglycerol polyricinoleate (PGPR)

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and protein interfaces.

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Jonathan Andrade and Milena Corredig

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Department of Food Science, University of Guelph, Guelph, Ontario, Canada N1G 2W5

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Corresponding author:

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Milena Corredig

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Food Science Department,

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University of Guelph

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[email protected]

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+1 5198244120 ext. 58132

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Fax 5198246631

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Abstract

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The present work tested the effect of addition of hydrophobic compounds at interfaces

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containing polyglycerol polyricinoleate (PGPR) and β-lactoglobulin or sodium caseinate, using

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drop tensiometry. Vitamin D3 or phytosterols were also tested on a model double emulsion

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stabilized with PGPR and sodium caseinate. Emulsions were prepared using a high pressure

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homogenizer, and changes in particle size were followed using light scattering. The

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encapsulation efficiency of the double emulsions was estimated by adding Mg2+and measuring

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its release from the inner droplet. PGPR dominated the oil-water interfacial properties. In the

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presence of proteins there was a decrease of the interfacial tension, with little changes in the

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viscoelastic properties. The presence of vitamin D3 and phytosterols further affected the

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interfacial properties. Double emulsions were then prepared with 2% PGPR. While control

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emulsions showed limited stability with an increase in the particle size after one week of storage,

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emulsions containing 0.05% (w/w) of vitamin D3 or phytosterol resulted in better stability over

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the storage period. Results suggested that vitamin D3 and phytosterol molecules may interact

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with the emulsifiers at the interface, affecting the physico-chemical properties, and possibly their

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release during digestion.

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Keywords: PGPR, hydrophobic bioactives, drop tensiometry, double emulsion, stability

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Abbreviations

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β-lg: β-lactoglobulin

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β-sit: β-sitosterol

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DWS: Diffusing wave spectroscopy

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NaCas: sodium caseinate

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PGPR: polyglycerol polyricinoleate

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PS: phytosterols

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Vit. D3: vitamin D3

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W1/O: water-in-oil

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W1/O/W2: water-in-oil-in-water

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1. Introduction

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Double emulsions have gained attention in the past years, because of their increased interest in

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cosmetic, pharmaceutical and food applications. A water-in-oil-in-water emulsion (W1/O/W2)

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consists of water-in-oil (W1/O) droplets dispersed in a secondary aqueous phase (W2). It is

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possible to carry within this system, both hydrophilic and hydrophobic substances. Different

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phases can protect bioactives or nutrients from unwanted reactions (Dickinson, 2011;

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McClements, 2011). Double emulsions continue to be focus of basic research, as the interactions

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occurring at the interface between the various components in the mixtures are yet to be fully

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understood.

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A stable primary W1/O emulsion is essential to obtain a double emulsion system applicable to

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the food industry. Efforts have been carried out to improve steric repulsion at the interface of the

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inner droplets (Bouyer et al., 2011; Garti, Aserin, Tiunova, & Binyamin, 1999; Lutz, Aserin,

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Wicker, & Garti, 2009; Muschiolik et al., 2006; Su, Flanagan, Hemar, & Singh, 2006).

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Polyglycerol polyricinoleate (PGPR), a synthetic emulsifier, is the most common molecule

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employed to stabilize primary W1/O emulsion droplets. Albeit it has gained GRAS status

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(generally recognized as safe) from FDA (Food and Drug Administration, 2006), the facts of

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being synthetic and having labeling requirements make its reduction or removal from the

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formulations desirable (Márquez, Medrano, Panizzolo, & Wagner, 2010). Mixing emulsifiers has

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been shown as a good strategy to decrease PGPR in W1/O/W2 emulsions, and synergistic effects

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have been found, for example, between sodium caseinate and PGPR (Su et al., 2006). The

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interactions between PGPR with other emulsifiers may improve stability of the inner phase and

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enhance the entrapment efficiency of the emulsion (Gülseren & Corredig, 2012, 2014).

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Other hydrophobic compounds present in the oil may also affect the interface. When bioactive

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compounds are added, their release during digestion may depend on their interaction with the

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interface. A recent in-vitro digestion study of oil-in-water emulsions loaded with hydrophobic

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molecules showed differences in the transfer of the bioactives to the aqueous phase in the

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absence of lipolysis, suggesting partitioning of the molecules at the interface depending on their

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structure. For example, vitamin D3 and phytosterols were transferred into mixed micelles in

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absence of lipolysis to a higher extent (around 30%) compared to β-carotene and coenzyme Q10

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(only about 2%) (Malaki Nik, Corredig, Wright, & Nik, 2011). The authors suggested that

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phytosterols and vitamin D3 would be preferentially closer to the interface of the oil droplet,

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while β-carotene and coenzyme Q10 would place towards the core.

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Interactions between hydrophobic bioactives and emulsifiers may occur, and affect the properties

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of the oil-water interface. In this study, it was hypothesized that if interactions occur, interfacial

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properties will change, as well as the physico-chemical properties of W1/O as well as W1/O/W2

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emulsions. To test this hypothesis vitamin D3 and phytosterols were used as model bioactives,

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and interactions at the interfaces were tested using mixed interfaces containing PGPR and milk

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proteins. Furthermore, the physico-chemical properties of W1/O and W1/O/W2 emulsions

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stabilized with PGPR and sodium caseinate, and containing vitamin D3 and phytosterols were

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studied.

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2. Material and Methods

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2.1. Materials

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Soybean oil (S7381, Sigma-Aldrich, St. Louis, MO, USA), was used as oil phase. In case of

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tensiometry measurements, the oil was pre-treated with Florisil® (46385, Sigma-Aldrich)

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(Gülseren & Corredig, 2012). In brief, oil (45g) was mixed with 4.5 g Florisil using a shaking

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plate (60 rpm for 2 h). The adsorbent was then filtered (Whatman No:1, cellulose filter, Fisher

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Scientific, Fair Lawn, NY, USA). This process was carried out three times and during the final

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step, two filter papers were used. Emulsions were prepared using MilliQ water, while

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tensiometry experiments were carried out using HPLC grade water (Fisher Scientific). Synthetic

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emulsifier polyglycerol polyricinoleate (PGPR 4150) was obtained by Palsgaard (Juelsminde,

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Denmark) which stated the content of minimum 75% di-, tri- and tetraglycerols with a maximum

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of 10% of heptaglycerol or higher. Phytosterols (β-sitosterol (β-sit) as main compound – 85451)

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and vitamin D3 (VitD3) (Cholecalciferol – C9756) as well β-lactoglobulin (β-lg) (L0130), were

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all obtained by Sigma-Aldrich, while sodium caseinate (NaCas) (NaCas 180) was purchased

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from Fonterra Inc. (Rosemont, IL, USA). Magnesium chloride hexahydrate (MgCl2.6H2O –

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BP214) and sodium chloride (NaCl – S271) were obtained by Fisher Scientific.

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2.2. Drop shape tensiometry

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The interfacial tension and dilational elasticity modulus of a soybean oil-water interface was

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measured using drop shape tensiometry (Tracker, IT Concept, Longessaigne, France) at room

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temperature. The aqueous drop (6 µl), was delivered by a syringe into an optical glass cuvette

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containing the oil phase. A video image was obtained with a CCD camera and processed using

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the Young-Laplace equation, while monitoring its shape. The interfacial tension was recorded

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over time (Benjamins, Cagna, & Lucassen-Reynders, 1996; Fainerman, Lylyk, Makievski, &

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Miller, 2004; Wang & Narsimhan, 2005). After an equilibration period of at least 3 h, the

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equilibrium interfacial tension (γ) was extrapolated, with results not significantly different from

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overnight equilibration experiments (Dopierala et al., 2011). After 3 h oscillatory changes of

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volume/surface area of the drop were performed with the strain amplitude kept constant at 10%

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(∆A/A=0.1, where A is the droplet surface area) and with harmonic expansion and dilation

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cycles ranging 5 to 100 mHz of frequency. The surface dilational modulus was obtained based

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on the following equation:

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ESD= dγ/(d ln A)

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2.3. Emulsions Preparation

(Eq. 1)

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Water-in-oil emulsions (primary emulsions for the W1/O/W2) were prepared by mixing 30%

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(w/w) of aqueous and 70% (w/w) oil phases. The internal aqueous phase consisted of 0.5%

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(w/w) sodium caseinate and 0.1 M NaCl. MgCl2 (0.1 M) was also added to the internal aqueous

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phase in selected systems to follow its encapsulation efficiency in W1/O/W2. The oil phase

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contained 2% (w/w) PGPR with or without 0.05% (w/w) of bioactives (vitamin D3 or

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phytosterols) in soybean oil. The oil and the water were pre-mixed using an ultra-turrax

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(PowerGen 125, Fisher Scientific) for 1 min, and this emulsion was then circulated through a

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microfluidizer (Microfluidcs, Newton, MA, USA) at 65 MPa for 5 min to form the final W1/O

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emulsion, time equivalent of 10 passes of the sample volume of 30 mL.

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A dispersion of 10% (w/w) primary emulsion (W1/O emulsion) was then mixed in 2% (w/w)

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sodium caseinate (NaCas) solution (prepared with MilliQ water) to obtain a W1/O/W2 emulsion

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with final ratio of 3:7:90. To avoid multimodal distribution, small oil globules and possible

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disruption of the inner aqueous droplets, which would impact on encapsulation of Mg2+ ions, a

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gentler homogenization step was necessary. After optimization, the second emulsification step

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was carried by passing the formulation once through the homogenizer (EmulsiFlex C5, Avestin,

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Ottawa, Canada) at approximately 3.5 MPa.

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2.4. Emulsion Characterization

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Diffusing wave spectroscopy (DWS) measurements were carried out to obtain the average

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droplet radius of primary W1/O emulsions. The sample was transferred to a 10 mm optical glass

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cuvette (Hellma Canada Limited, Concord, Canada) and placed in a 25ºC water bath, which was

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illuminated by a 350 mW solid-state laser (532 nm) (Coherent, Santa Clara, Ca, USA).

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Transmitted scattered light was collected by a single fibre optic that was then bifurcated and fed

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to two matched photomultipliers (HC120-03, Hamamatsu, Loveland, OH) and a correlator

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(FLEX2K- 12x2, Bridgewater, NJ). Correlation functions and transmitted light intensity was

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collected for 2 min. Data was analyzed using DWS-Fit (Mediavention Inc., Guelph, ON,

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Canada). The principles of DWS have been published elsewhere(Horne & Davidson, 1993;

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Weitz, Zhu, Durian, Gang, & Pine, 1993).

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Droplet sizes distributions of the double emulsions were measured using integrated light

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scattering (Malvern Mastersizer 2000S, Malvern Instruments Inc, Westborough, MA). A small

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volume of sample was diluted in distilled water present in the measuring cell and kept under

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stirring at room temperature. Refractive indices used were 1.33 for the dispersant (water) and

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1.473 for the soy oil phase. We assumed that the refractive index of the oil droplet was not

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affected by the presence of the internal aqueous droplets (Bonnet et al., 2010; Pawlik, Cox, &

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Norton, 2010).

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The osmolalities of internal and external aqueous phases, as well the water used to disperse the

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samples, were measured using a vapour pressure osmometer (VAPRO 5520, Wescor Inc, Logan,

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Utah, USA) and standard calibration solutions of 100, 290 and 1000 mmol/kg, to estimate the

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osmotic pressure gradient between them.

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2.5. W/O/W Encapsulation efficiency

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To determine the integrity of the primary emulsion in the double emulsion, the encapsulation

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efficiency MgCl2 was measured. Freshly made double emulsions were loaded with 100 mM

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MgCl2 and then the amount of magnesium released was measured in the outer phase. The double

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emulsions were centrifuged at 16000 g for 30 min at room temperature (Eppendorf Centrifuge

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5415 D). This centrifugation parameters caused a very clear separation of the subnatant and a

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cream phase. A more intense approach would not be suitable to the holding capacity once it

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would disrupt the droplets. It is well known that centrifugation speed takes an important role in

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the internal droplet disruption, as well the oil content and PGPR concentration (Hayati, Che

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Man, Tan, & Aini, 2007; Maisuthisakul, 2011). After separation of cream phase, the subnatant

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aqueous phase was filtered with 0.22 µm Millex-GV syringe filter (Millipore, Bedford, MA,

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USA) and samples were subjected to ion chromatography analysis (861 Advanced Compact IC –

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Metrom Ion Analysis, Metrohm Ltd., Herisau, Switzerland) after diluting 20 times in acceptor

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solution (2mM HNO3). The eluent solution used was 0.7 mM dipicolinic acid and 1.7 mM

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HNO3. The unit consists of an injection valve, high pressure pump, peristaltic pump and

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conductivity detector, all controlled by the 838-advanced sample processor (IC Net 2.3,

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Metrohm Ltd.). The 833 IC liquid handling dialysis unit with a 2-channel peristaltic pump and

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dialysis cell containing a 0.2 µm cellulose acetate membrane (Fisher Scientific) prepared the

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samples in-line. A standard curve was prepared with magnesium standard for IC TraceCERT

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(1,000 mg/L of Mg2+ in HNO3, Fluka, Sigma, Steinheim, Germany) by diluting it in range

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between 1 to 10 mg/L and injected before the samples. The percentage of encapsulated material

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on the inner water droplet was calculated as follows.

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Eefficiency = [( Initialinner aq.phase – Detectedexternal after centrifugation ) / ( Initialinner aq.phase )] × 100 (Eq. 2)

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2.6 Statistical analysis

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When analysed pairwise, data were tested with Student’s T-test. The differences in groups mean

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values were submitted to analysis of variance (ANOVA). TukeyHSD test was carried out when

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significant differences among groups were obtained. All statistical tests were done with 95% of

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significance. The statistical analysis was performed using the free software [R] (www.r-

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project.org) and Addinsoft XLStat extension for MS Excel.

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3. Results and Discussion

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The properties of a water-soybean oil interface were measured using drop shape tensiometry.

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Figure 1 illustrates the interfacial tension near equilibrium, after 3 h, for PGPR, sodium caseinate

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and β-lactoglobulin (β-lg) interfaces. In the absence of surfactants, the interfacial tension at

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equilibrium of the oil water interface was 31 ± 1 mN.m-1, in agreement with previous studies

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(Gaonkar, 1989; Gülseren & Corredig, 2012). Solutions containing 0.008% (w/v) of synthetic

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emulsifier solubilised in soybean oil and aqueous solutions of 0.001% (w/v) of each protein were

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used. These concentrations were chosen based on previous literature data (Gülseren & Corredig,

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2012) to be below complete saturation of the interface by a single emulsifier. The authors

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reported a wide range of concentrations of PGPR and their correspondent equilibrium interfacial

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tension value. When used higher concentrations, a plateau value is obtained. With concentrations

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below 0.010% of PGPR it was possible to observe a further decrease on equilibrium interfacial

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value when proteins were present. In this study, with the chosen concentrations was possible to

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observe changes promoted by the proteins and the hydrophobic compounds.

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Both proteins and PGPR decreased the equilibrium interfacial tension when compared to the oil-

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water interface (Figure 1). Milk proteins are widely known for their ability to adsorb at oil water

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interfaces and decrease interfacial tension. When comparing to water-soybean oil values, NaCas

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and β-lg caused a decrease in the interfacial tension. PGPR dispersed in the oil promoted a

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slightly lower decrease in equilibrium interfacial tension compared to the proteins (Figure 1).

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These findings were in full agreement with previous studies of β-lg and NaCas at sunflower oil

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interfaces (Ogden & Rosenthal, 1997; Wüstneck, Moser, & Muschiolik, 1999). The presence of

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PGPR and proteins caused further decrease of the interfacial tension, suggesting that both

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molecules (protein and PGPR) were present at the interface.

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Dilational viscoelasticity was also measured applying a strain amplitude of 10% (∆A/A = 0.1)

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and oscillatory frequencies between 5 to 100 mHz (Table 1). In all systems studied, a similar

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pattern was observed: elasticity values were nearly constant at low frequency, reaching a

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maximum at 50 mHz. The interface containing β-lg showed higher elastic modulus than those

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obtained with the synthetic emulsifier. Nonetheless, in all cases, in the presence of PGPR,

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elasticity measurements revealed that the mixed interfaces were dominated by PGPR, as reported

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in the literature (Gülseren & Corredig, 2012). These results further explained the observation

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that the presence of NaCas in the inner phase of W1/O/W2 emulsions causes an improvement in

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the overall stability of the emulsions (Su et al., 2006). It was then suggested that proteins and

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other macromolecules adsorbed at the interface may act as a barrier to the release of bioactives

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entrapped within the emulsion droplets (Benichou, Aserin, & Garti, 2004).

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3.1. Influence of bioactives at W/O interfaces

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Figure 2 illustrates the effect of 0.005% (w/v) vitamin D3 or β-sitosterol to the oil phase. When

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these compounds were added in isolation or in the case of an interface dominated by β-lg, no

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significant changes in interfacial tension were observed.

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While the presence of PGPR in the oil showed a value of interfacial tension of the water droplet

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at equilibrium of about 20 ± 1 mN.m-1, the addition of the bioactive molecules β-sitosterol and

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vitamin D3, caused a smaller decrease in the interfacial tension. The highest value of interfacial

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tension was observed when vitamin D3 was present (28 ± 1 mN.m-1). A similar pattern of

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significant changes were observed with interfaces containing mixtures of PGPR and sodium

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caseinate and sodium caseinate alone. These results would suggest an effect of phytosterols and

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vitamin D3 on the adsorption behaviour of the emulsifiers.

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The viscoelastic properties of the mixed interfaces were also measured, and the presence of the

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bioactive molecules did not show statistically significant differences, apart from when only

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PGPR and vitamin D3 were present at the interface. In this case, the elastic modulus measured

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at 10 and 50 mHz showed an increase (with values of 38 and 60 mN.m-1, respectively) compared

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to when PGPR was measured in isolation.

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3.2. Effect of bioactive molecules on W1/O and W1/O/W2

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High pressure homogenization was used to prepare 30% water-in-oil emulsions. The oil phase

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contained 2% PGPR and 0.05% vitamin D3 or β-sitosterol when was the case. The internal water

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phase contained 0.1 M NaCl, and 0.1 M MgCl2. The double emulsions were stabilized by 2%

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NaCas. Dynamic light scattering measurements under concentrated conditions (DWS) were

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carried out in fresh made emulsions and after 1 week of storage at 4ºC (Table 2), with all fresh

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primary emulsions showing < 300 nm in radius.

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Statistical analysis revealed that control samples and those carrying only the hydrophilic

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bioactive had a significant (p<0.05) increase in the apparent radius. PGPR is known for its

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capacity of producing interfaces with very strong steric repulsion between the droplets, reducing

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droplet coalescence (Dedinaite & Campbell, 2000; Knoth, Scherze, & Muschiolik, 2005). The

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amount of emulsifier used in this work was sufficient to create small emulsion droplets, but not

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to ensure long term stability of the emulsion droplets. Using 2% or less PGPR, Su et al. (2006)

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reported complete phase separation of the W/O emulsions. In the presence of either vitamin D3

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or β-sitosterol, the emulsions showed constant apparent radius after 1 week of storage,

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suggesting their effect on the stability of the W1/O emulsion droplets for the period studied. It is

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important to note that among all samples studied, no phase separation was observed after 1 week

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of storage.

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The freshly made primary emulsions were re-dispersed in 2% NaCas, and re-homogenized to

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obtain W1/O/W2 (3:7:90) double emulsions. Particle size distribution was measured by integrated

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light scattering. It is recommended to dilute the sample in aqueous solution with similar osmotic

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pressure of the external aqueous phase, to avoid water transfer fenomena (Herzi, Essafi,

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Bellagha, & Leal-Calderon, 2014). In this case, for the light scattering measurements, we diluted

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our samples extensively in water due the fact of both external aqueous phase and the water used

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resulted in closer values, below the accuracy limit of the osmometer (19 ± 1 and 9 ± 1 mmol/Kg,

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respectively). The osmolalities obtained for the internal aqueous phase of control samples

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(without MgCl2) and the internal aqueous phase of samples containing MgCl2 were: 185 ± 3

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mmol/Kg and 745 ± 5 mmol/Kg, respectively. Figure 3 and table 3 summarize the changes in

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average diameter after 1 week storage at 4ºC. All fresh samples showed a monomodal

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distribution of droplet sizes. Due to their large particle size, the double emulsion systems showed

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rapid creaming, resulting in a heterogeneous system in approximately 30 min. However,

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creaming was fully reversible after mixing. After 1 week of storage, control emulsions and those

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containing 0.1M MgCl2 showed extensive coalescence. NaCl was found to increase interfacial

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tension of water/n-heptane interface in with increments on concentration, however, with very

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little change on surface pressure (Lima, Melo, Baptista, & Paredes, 2013). NaCl and other salts

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are known for having negative surface tension, causing an increase on interfacial tension of the

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solution (McClements, 2005). The presence of the salts might have been influencing the short

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term stability of the droplets more on ionic strength difference between the internal and external

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aqueous phases than with possible influence on interfacial tension. In addition, even with

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presence of the salts in the internal phase, the samples containing β-sitosterol and vitamin D3

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still showed a monomodal distribution of sizes, with no statistical difference after 1 week,

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consistent with the improved short term stability of the inner droplets shown in Table 2.

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Fresh formulations resulted in high values of encapsulation of MgCl2 with statistical changes

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observed (p<0.05). The values are shown in table 3: the highest value was obtained on samples

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without hydrophobic compounds, 96.3 ± 0.3%, while samples containing β-sitosterol or vitamin

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D3 showed smaller. As observed on drop tensiometry results, the supplementation of the oil

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phase with hydrophobic bioactives promoted an impact on interfacial properties of the system,

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especially in case of vitamin D3, which is a more hydrophobic molecule. When studied in a

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double emulsion system, although the possible interactions with the emulsifiers at the interface

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might promote a slightly lower ability to entrap the ions, it kept the emulsion stable for longer.

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4. Conclusions

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This research demonstrated that when hydrophobic molecules such as vitamin D3 or β-sitosterol

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are added to an emulsion, they affect the physico-chemical properties of the interface.

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hydrophobic molecules caused an increase in the interfacial tension at mixed interfaces,

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demonstrating that they affected the adsorption properties of the emulsifiers used in this study.

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Furthermore, their addition to a water-in-oil or water-in-oil-in-water emulsion showed no

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changes in particle size after 1 week of storage, in contrast to control emulsions. These effects

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on physical properties of the emulsion may indicate interactions of the bioactive molecules with

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the interface and/or with the emulsifiers, ultimately affecting their release behavior during

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digestion.

These

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Acknowledgments

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This work was funded Natural Science and Engineering council of Canada through the Canada

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Research Chair program and the discovery grant program. Coordenação de Aperfeiçoamento de

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Pessoal de Nível Superior – CAPES Foundation (Ministry of Education of Brazil, Brazilia-DF) is

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also thanked for the scholarship of JA (process 9410-13-9).

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Benjamins, J., Cagna, A., & Lucassen-Reynders, E. H. (1996). Viscoelastic properties of triacylglycerol/water interfaces covered by proteins. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 114, 245–254.

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Table 1.

5 10 50 100

25 ± 5ab 24 ± 2ab 35 ± 3a 21 ± 2a

PGPR / NaCas 20 ± 3b 20 ± 1b 28 ± 1a 22 ± 3a

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PGPR

Elasticity Modulus (mN.m-1) PGPR / β-lg NaCas β-lg 34 ± 3a 24 ± 3ab 25 ± 3ab 36 ± 3a 24 ± 4ab 28 ± 4ab 43 ± 7a 39 ± 22a 35 ± 5a 37 ± 3a 30 ± 16a 29 ± 6a

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Frequency (mHz)

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Table 2.

1 week (nm) 394 ± 16b 249 ± 37b 259 ± 16a 268 ± 52a

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Fresh (nm) 292 ± 8a 200 ± 4a 240 ± 43a 276 ± 22a

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Sample Control - without bioactive MgCl2 MgCl2 / β-sit MgCl2 / VitD3

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Table 3.

Sample

d3,2 (µm) Fresh 1 week

EE (%)

2.2 ± 0.2a

4.9 ± 0.0b

---------

MgCl2

5.2 ± 0.1a

1.2 ± 0.1b

96.3 ± 0.3x

MgCl2 / β-sit

5.5 ± 0.1a

5.5 ± 0.1a

92.3 ± 1.5xy

MgCl2 / VitD3

5.7 ± 0.3a

5.6 ± 0.4a

90.9 ± 2.6y

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Control - without bioactive

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Highlights Bioactive molecules affected PGPR ability to decrease interfacial tension.

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Bioactive molecules affected Sodium Caseinate ability to decrease interfacial tension

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Vitamin D3 and Phytosterols improved stability of W/O emulsions.

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Vitamin D3 and Phytosterols improved stability of W1/O/W2 emulsions.

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