Fabrication of Concentrated Fish Oil Emulsions Using Dual-Channel

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Fabrication of Concentrated Fish Oil Emulsions using Dual-channel Microfluidization: Impact of Droplet Concentration on Physical Properties and Lipid Oxidation Fuguo Liu, Zhenbao Zhu, Cuicui Ma, Xiang Luo, Long Bai, Eric Andrew Decker, Yanxiang Gao, and David Julian McClements J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04413 • Publication Date (Web): 28 Nov 2016 Downloaded from http://pubs.acs.org on December 3, 2016

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Journal of Agricultural and Food Chemistry

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Fabrication of Concentrated Fish Oil Emulsions using Dual-channel Microfluidization: Impact of Droplet Concentration on Physical Properties and Lipid Oxidation

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Fuguo Liu1, 2, Zhenbao Zhu3, Cuicui Ma1, Xiang Luo2, Long Bai2, Eric Andrew

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Decker2, Yanxiang Gao1*, David Julian McClements2*

1 2

6 7

1

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Laboratory for Food Quality and Safety, Beijing Key Laboratory of Functional Food

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from Plant Resources, College of Food Science & Nutritional Engineering, China

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

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Agricultural University, Beijing 100083, China

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2

12

01003, USA

13

3

14

Technology

Department of Food Science, University of Massachusetts Amherst, Amherst, MA School of Food and Biological Engineering, Shaanxi University of Science and

15 16 17 18

Journal: Journal of Agricultural and Food Chemistry

19

Submitted: October 2015

20 21 22

*Corresponding author: Yanxiang Gao, College of Food Science & Nutritional

23

Engineering, China Agricultural University, No.17 Qinghua East Road, Haidian

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District, Beijing 100083, China; [email protected]; Phone: + 86-10-62737034. Fax: +

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86-10-62737986.

26

*Corresponding Author: David Julian McClements, Department of Food Science,

27

University

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[email protected]; Phone: 413 545 1019. Fax: 413 545 1262.

of

Massachusetts

Amherst,

Amherst,

29

1

ACS Paragon Plus Environment

MA

01003,

USA;


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ABSTRACT: Chemically unstable lipophilic bioactives, such as polyunsaturated

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lipids, often have to be encapsulated in emulsion-based delivery systems before they

32

can be incorporated into foods, supplements, and pharmaceuticals.

33

this study was to develop highly concentrated emulsion-based fish oil delivery

34

systems using natural emulsifiers.

35

a highly efficient dual-channel high-pressure microfluidizer. The impact of oil

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concentration on the formation, physical properties and oxidative stability of fish oil

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emulsions prepared using two natural emulsifiers (quillaja saponins and rhamnolipids)

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and one synthetic emulsifier (Tween-80) was examined. The mean droplet size,

39

polydispersity, and apparent viscosity of the fish oil emulsions increased with

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increasing oil content.

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levels (30 or 40 wt%) could be produced using all three emulsifiers, with

42

rhamnolipids giving the smallest droplet size (d < 160 nm). The stability of the

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emulsions to lipid oxidation increased as the oil content increased.

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stability of the emulsions also depended on the nature of the emulsifier coating the

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lipid droplets, with the oxidative stability decreasing in the following order:

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rhamnolipids > saponins ≈ Tween-80.

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be particularly effective at producing emulsions containing high concentrations of ω-3

48

rich fish oil.

The objective of

Fish oil-in-water emulsions were fabricated using

However, physically stable emulsions with high fish oil

The oxidative

These results suggest that rhamnolipids may

49 50

KEYWORDS: Natural emulsifiers; fish oil; omega-3; PUFA; emulsions;

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nanoemulsions; oil content; oxidation; dual-channel microfluidizer

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INTRODUCTION

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Fish oil is considered to be of great nutritional importance due to its high levels of ω-3

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polyunsaturated fatty acids (PUFAs), such as eicosapentaenoic acid (EPA) and

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docosahexaenoic acid (DHA) 1. Consumption of sufficiently high levels of oil sources

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rich in these types of PUFAs has been linked to beneficial health outcomes, such as

57

reduced risk of coronary heart disease, decreased hypertension, and improved brain

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function. 2, 3

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However, PUFAs are particularly vulnerable to chemical degradation in the presence

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of oxygen, which leads to the generation of undesirable off-flavors, a reduction in their

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nutritional quality, and the formation of potentially toxic reaction products.4 The

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utilization of oil-in-water emulsions to encapsulate PUFA-rich lipids is a promising

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approach to protect them against oxidation because it reduces the undesirable interactions

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between the hydrophobic lipid molecules inside the oil droplets and any hydrophilic

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pro-oxidative species in the aqueous phase, such as transition metals.5, 6 A well-designed

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emulsion-based delivery system can therefore improve the sensory quality, shelf life, and

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nutritional attributes of products containing PUFAs. The functional attributes of this type

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of delivery system can be manipulated by careful selection of ingredients and fabrication

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conditions so as to create emulsions with different droplet compositions, concentrations,

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sizes, physical states, and/or interfacial properties.7-10

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There has been growing interest in replacing synthetic ingredients with natural

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alternatives in many food applications.11 Various types of natural emulsifier have been

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utilized to form emulsion-based delivery systems for PUFA-rich oils, including

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amphiphilic proteins (whey protein isolate, soy protein isolate, and sodium caseinate),

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polysaccharides (gum arabic, beet pectin, and modified starch), and phospholipids (egg

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and soy).12-14 More recently, there has been interest in utilizing natural small molecule

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surfactants (such as saponins or rhamnolipids) because they can often be utilized at lower

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levels than proteins, polysaccharides, and phospholipids, and because they can often

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produce smaller droplets during homogenization

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may contain antioxidant functional groups that can enhance the chemical stability of

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encapsulated lipids 16-18.

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surfactants: quillaja saponins and rhamnolipids.

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isolated from the bark of an evergreen tree found in Chile (Quillaja saponaria), and have

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been shown to be highly surface active molecules

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relatively low surfactant-to-oil ratios (1:10) under appropriate homogenization

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conditions.21,

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available and legally acceptable for utilization in the food industry (Q-NaturaleTM,

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Ingredion Inc., Bridgewater, NJ, USA).

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isolated from specific microorganisms, which have also been shown to be highly surface

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active molecules 23, 24, and are also capable of forming oil-in-water emulsions. 23

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22

15

. Moreover, some natural emulsifiers

In the present study, we focused on two natural small-molecule Quillaja saponins are traditionally

19, 20

that can form stable emulsions at

Emulsifier ingredients containing quillaja saponins are commercially

Rhamnolipids are glycolipids that are typically

Although a lot of work has already been carried out to establish the major factors 10, 21, 25, 26

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affecting the chemical stability of fish oil emulsions and emulsions

, very little

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research has focused on the impact of total oil content. From a commercial standpoint, it

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would be advantageous to develop emulsion-based delivery systems with high oil

95

loadings because this could reduce production, transport, and storage costs.

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there may be additional advantages in terms of improving the oxidative stability of the

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emulsions when they are highly concentrated.

Moreover,

For example, there will be less

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water-soluble pro-oxidants present at high oil contents.

In our recent investigations, we

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found that highly concentrated oil-in-water emulsions (up to 50 wt% oil) could be

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efficiently produced using dual-channel microfluidization.27 Unlike conventional

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single-channel microfluidizers, this type of device produces emulsions directly from

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separate oil and aqueous phases.

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impinge on an aqueous phase flowing through another channel at high velocity, which

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generates intense disruptive forces that efficiently breakup and intermingle the two

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phases leading to the production of fine oil droplets.

An oil phase flowing through one channel is made to

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The purpose of the present study was therefore to investigate the possibility of

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fabricating highly concentrated fish oil emulsions from natural emulsifiers (quillaja

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saponins and rhamnolipids) using dual-channel microfluidizers. These emulsions could

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then be used as effective delivery systems for PUFA-rich oils in foods, supplements, and

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pharmaceuticals.

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MATERIALS AND METHODS

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Materials and Chemicals.

Fish oil was kindly provided by DSM Co., Ltd.

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(Columbia, USA) and stored at 4°C in the dark. Quillaja saponins (Q-Naturale® 200) was

114

kindly provided by Ingredion Inc. (Westchester, IL, USA).

115

to contain around 70% of the active component.

116

which consist of a mixture of di- and mono-rhamnolipids, were purchased from

117

Sigma-Aldrich Co., LLC. (St. Louis, MO, USA).

118

(Tween-80) and oil-soluble fluorescent dye (Nile red) were purchased from

119

Sigma-Aldrich Co., LLC.

120

analytical grade. A TBA reagent was prepared for the lipid oxidation experiments by

This ingredient was reported

Rhamnolipids (R90, purity>90%),

The synthetic non-ionic surfactant

All other reagents and solvents used in this study were of

5



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dissolving 15% (w/v) trichloroacetic acid, 0.375% (w/v) thiobarbituric acid, and 0.25 M

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hydrochloric acid with 2% BHT in ethanol solution. Double distilled water was used as

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the solvent throughout the study (Milli-Q® Integral Water Purification System, Merck

124

Millipore Corp., Darmstadt, Germany).

125

Interfacial tension measurements.

Interfacial tension versus emulsifier

126

concentration profiles were determined using a drop shape analysis instrument (DSA100,

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Krüss GmbH, Hamburg, Germany) equipped with an environmental chamber and a

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microsyringe steel needle of 0.906 mm diameter. The fish oil was used as the oil phase

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and the aqueous phases were prepared by dissolving appropriate amounts of emulsifier

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(0.0005 to 2.0 wt%) into buffer solution (sodium phosphate, 5 mM, pH 7.0). The oil

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phase was injected into the aqueous phase and the interfacial tension was determined by

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drop shape analysis after the system had reached equilibrium (at least 5 min) or the oil

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drop completely detached from the needle. Digital images were captured using the

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device’s camera function. Interfacial tension values were calculated from the shape of the

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oil drops formed using the Young–Laplace equation program supplied by the instrument

136

manufacturer.

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Emulsion preparation.

Fish oil-in-water emulsions were prepared using the

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dual-channel microfluidization method described by Bai and McClements28 with some slight

139

modifications. Emulsions were prepared with different oil phase concentrations (10 to 50

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wt%) while keeping the emulsifier-to-oil ratio fixed at 1:10. The aqueous phase contained

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emulsifier (quillaja saponins, rhamnolipids or Tween-80) and 5 mM sodium phosphate

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buffer (pH 7.0). The aqueous phase and oil phase were poured into two different glass

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reservoirs. Fine emulsions were then formed by simultaneously forcing the oil phase and

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aqueous phase through an air-driven high-pressure microfluidizer (Microfluidics

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PureNano, Newton, MA, USA) at a homogenization pressure of 13 kpsi. The final

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concentration of oil within the emulsions was controlled by manipulating the flow rates

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(mL/min) of the oil phase (fO) and water phase (fW) through the microfluidizer. The total

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flow rate of the microfluidizer was set as 500 ml/min. Because the flow rates were based

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on volume, the densities of the oil and water phases were needed to calculate the final

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emulsion composition on a weight basis. The densities of the water and oil phases were

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therefore measured using a density bottle as 1000 and 923 kg/m3, respectively. The mass

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fraction (φ) of fish oil in the final emulsions was then calculated from the following

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expressions:

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fO + fW = 500

155

! ∅ = 1 + !.!"#!

156

(1)

!

(2)

!

Measurement of droplet electrical characteristics.

The ζ-potential of the

157

emulsifier-coated droplets in the fish oil emulsions was determined using particle

158

electrophoresis (Zetasizer Nano ZS Series, Malvern Instruments, Worcestershire, UK).

159

Emulsions were diluted to a droplet concentration of approximately 0.01 wt% using a

160

buffer solution to avoid multiple scattering effects. The ζ-potential of the sample was then

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calculated by the device from measurements of the direction and velocity of droplet

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movement in the well-defined electrical field. All measurements were made on at least

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three freshly prepared samples.

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Measurement of droplet size.

The surface-weighted mean particle diameter (d32)

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and particle size distribution of the emulsions were measured using a static light

166

scattering

instrument

(Malvern

Mastersizer

2000,

Malvern

7


Instruments

Ltd.,


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Worcestershire, UK). Emulsions were diluted 20× in phosphate buffer solution prior to

168

analysis to avoid multiple scattering effects. The particle size distribution of the

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emulsions was calculated by the software in the light scattering instrument based on Mie

170

theory and the refractive indices of the oil (1.481) and water (1.330) phases. All

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measurements were carried out at 25 °C and three replicates were performed. To

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determine the width of the particle size distribution, the span was calculated from the

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following equation:

174 175 176

span =

(d90 -d10 ) d50

(3)

Where d90, d50, and d10 represent the diameters at 90%, 50%, and 10% cumulative volume, respectively, a high span value indicates a wide size distribution.

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Microstructure analysis. The microstructure analysis of emulsions was carried out at

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room temperature using fluorescence confocal laser scanning microscopy (Nikon

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D-Eclipse C1 80i, Nikon, Melville, NY). Prior to analysis, the samples were dyed with

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Nile Red (0.1%) to highlight the location of the oil phase. An excitation wavelength of

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543 nm and an emission wavelength of 605 nm was used to detect the fluorescence signal

182

from the Nile Red. The sample was gently stirred with a glass rod to form a homogenous

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mixture without creating any air bubbles. After stirring, a small amount of the sample

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was transferred onto a glass microscope slide and covered with a glass cover slip. All

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images were captured with a 10× eyepiece and a 60× objective lens (oil immersion) and

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then processed using the instruments software program (EZ- CS1 version 3.8, Nikon,

187

Melville, NY).

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Measurement of rheological properties of fish oil emulsions. The influence of the

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oil concentration on the rheological properties of the emulsions was tested using a 8


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dynamic shear rheometer (Kinexus Pro rheometer, Malvern Instruments, Ltd.,

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Worcestershire, UK) equipped with a cone and plate measurement cell (CP4/40, PL 65)

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at 25°C. The apparatus was controlled and data acquisition was performed via rSpace

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software, as supplied with the rheometer. Approximately 1.5 ml of sample was placed

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between the cone and plate and then held for 5 minutes prior to carrying out the

195

measurements to allow it to reach the measurement temperature. Both constant shearing

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rate (10 s− 1) and varied shearing rate (0.01–100 s-1) were used to measure the apparent

197

viscosities of the emulsions.

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Measurement of oxidative stability.

Peroxide value (PV): Fish oil emulsions held

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in 10 mL disposable tubes were incubated in the dark at 55 °C for 15 days, with

200

measurements being made every three days. Lipid hydroperoxides, which are primary

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oxidation products, were measured according to the method of Shantha & Decker 29 with

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some slight modifications. In brief, lipids were extracted from the fish oil emulsions by

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adding 0.3 mL sample to a 1.5 mL mixture of isooctane/2-propanol (3:1 v/v) and then

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vortexing the mixture 3 times for 10 s, followed by centrifuging at 1000 × g for 2 min.

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0.2 mL of the supernatant (top organic layer) was taken and 2.8 mL of a

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methanol:1-buthanol (3:1, v/v) solution were added, followed by 30 µL of 1:1 (v/v) 3.94

207

M ammonium thiocyanate/ferrous iron solution (solution prepared by adding equal

208

amounts of 0.132 M barium chloride and 0.144 M ferrous sulfate). After 20 min, the

209

absorbance of the solutions was measured at 510 nm using a spectrophotometer

210

(Ultraspec 3000 pro, Biochrom Ltd., Cambridge, UK). The concentration of

211

hydroperoxides was calculated as mM cumene hydroperoxide using a standard

212

calibration curve prepared with cumene hydroperoxide. All analyses were carried out in

9



213 214

triplicate. Thiobarbituric acid-reactive substances (TBARS):

Samples were held in 10 mL

215

disposable tubes at 55 °C in the dark and TBARS were measured every 3 days for 15

216

days using the method of McDonald and Hultin.30 1 mL emulsion was mixed with 2 mL

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TBA reagent (15% w/v trichloroacetic acid, and 0.25 M HCl with 2% BHT in ethanol

218

solution) and then vortexed in glass test tubes with screw caps. These tubes were then

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placed in a boiling water bath for 15 min, and then moved to a room temperature water

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bath to cool them down for 10 min. The tubes were centrifuged at 1000 × g for 15 min.

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After standing for 10 min, the absorbance of the supernatant was measured at 532 nm.

222

Concentrations of TBARS were calculated as µM using a standard curve prepared with

223

1,1,3,3-tetraethoxypropane. All analyses were carried out in triplicate.

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Statistical analysis.

All the data obtained were average values of triplicate

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determinations and mean values and standard deviations were calculated from these

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values.

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RESULTS AND DISCUSSION

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Influence of emulsifier type on interfacial properties. The interfacial properties

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of the three surfactants were characterized by measuring their equilibrium interfacial

230

tension versus emulsifier concentration profiles (Fig. 1a). When no emulsifier was

231

present in the aqueous phase, an oil drop could be formed at the tip of the needle (Fig. 1b),

232

and the interfacial tension was determined to be 22.5 ± 0.3 mN m-1. In the presence of

233

emulsifier, the interfacial tension decreased with increasing emulsifier concentration,

234

indicating that the surfactant molecules adsorbed to the fish oil–water interface. The

235

magnitude of the interfacial tension determines the ease of droplet disruption during

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homogenization, since a smaller value means that less energy is needed to breakup the oil

237

droplets

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which is indicative of the oil-water interface becoming saturated. All the emulsifiers were

239

effective at reducing the interfacial tension, but there were considerable differences in

240

their performances.

241

both the quillaja saponins and the rhamnolipids, while it was around 6.8 mN m-1 for the

242

Tween 80.

243

screening the unfavorable thermodynamic contact between the oil and water phases when

244

the interfaces were completely covered with emulsifier molecules.

245 246

31

. A fairly constant interfacial tension was reached at high emulsifier levels,

The interfacial tension at saturation was around 3.7 mN m-1 for

This suggested that the two natural emulsifiers were more effective at

More quantitative information about the interfacial properties of the three emulsifiers was obtained by calculating their surface activity (k) using the following expression:

k=

247

ΔG 1 = exp(− ads ) c1/2 RT

248

Here C1/2 is the emulsifier concentration where 50% of the adsorption sites on the oil–

249

water interface are covered by emulsifier molecules, and ΔGads is the free energy change

250

associated with the transfer of an emulsifier molecule from the aqueous phase to the

251

interface. To a rough approximation, C½ is the emulsifier concentration at which the

252

surface pressure is half its maximum value (π∞), where the surface pressure is the

253

difference between the interfacial tension in the absence and presence of emulsifier (π =

254

γ0 - γ).

255

pressure versus emulsifiers concentration values (Fig. S1, see Supplementary materials):

256

for the quillaja saponins, C½ = 0.0030 wt% (18.3 µM), k = 5464 M-1, and ΔGads= -8.61RT;

257

for the rhamnolipids, C½ =0.0077 wt% (133.0 µM), k = 7519 M-1, and ΔGads = -8.93RT;

258

for the Tween 80, C½ =0.0023 wt% (17.6 µM), k = 5688 M-1 and ΔGads = -8.65RT. The

In this study, the following parameters were calculated from the interfacial

11



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molecular weights of quillaja saponins, rhamnolipids and Tween-80 used in the

260

calculations were 1650, 578 and 428.6 g/mol, respectively, which were obtained from

261

published literature values.11, 23 These calculations suggest that the rhamnolipids had the

262

strongest affinity for the oil-water interface (i.e., highest k value and free energy change).

263

Impact of emulsifier type on droplet characteristics. The mean particle diameter,

264

particle size distribution, and electrical properties of fish oil emulsions fabricated using

265

the different emulsifiers were determined (Figs. 2 to 4). All of the emulsions had

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negatively charged droplets, but the magnitude of the ζ-potential depended on emulsifier

267

type (Fig. 2): being -70, -85 and -15 mV for quillaja saponins, rhamnolipids and

268

Tween-80, respectively.

269

quillaja saponins-coated droplets was mainly due to their anionic functional groups. The

270

hydrophilic head groups of rhamnolipids have carboxylic acid groups (pKa = 5.6) while

271

those of quillaja saponins have glucuronic acid groups (pKa ≈ 3.25), which accounts for

272

their negative charge at neutral pH.11, 32 Generally, a ζ-potential with a magnitude greater

273

than 30 mV is sufficient to prevent droplet aggregation by generating a strong

274

electrostatic repulsion between the droplets.33 The slight negative charge on the Tween

275

80-coated droplets can be attributed to the presence of anionic impurities in either the oil

276

or surfactant ingredients used to fabricate the emulsions, such as free fatty acids.

277

results suggest that electrostatic repulsion plays an important role in stabilizing droplets

278

coated by quillaja saponins or rhamnolipids, whereas steric repulsion is important for

279

droplets coated by Tween 80. For all three emulsifiers, the initial droplet concentration in

280

the emulsions did not have a major impact on their electrical characteristics (Fig. 2),

281

which suggests that the interfacial composition was fairly similar in all of the systems.

The higher negative potential of the rhamnolipids-coated and

12


These

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There was a significant effect of oil concentration on the initial size of the droplets in

283

the emulsions (Fig. 3a). At low droplet concentrations (10%), the dual-channel

284

microfluidizer produced relatively small droplets using all three emulsifiers: 0.24, 0.15

285

and 0.13 µm for quillaja saponins, Tween 80, and rhamnolipids stabilized emulsions,

286

respectively. In addition, these emulsions had narrow distributions (Fig. 3b), monomodal

287

particle size distributions (Fig. 4), and exhibited no visible separation throughout storage

288

for 1 month (Fig. S2, see Supplementary materials).

289

rhamnolipids produced the smallest oil droplets across the entire range of oil

290

concentrations studied.

291

particularly fine droplets from 10 to 40 wt% oil: d < 0.15µm (Fig. 3).

292

emulsifiers also produced relatively small droplets at lower and intermediate oil

293

concentrations (d < 0.35 µm), but were not as effective as the rhamnolipids.

294

observed differences in the ability of the three emulsifiers to produce small droplets may

295

have been due to a number of phenomena related to droplet disruption and coalescence

296

within the homogenizer; the kinetics of adsorption to the droplet surfaces; the extent of

297

the reduction in interfacial tension; and the ability to inhibit droplet coalescence 15.

298

Of the three emulsifiers tested, the

In particular, the rhamnolipids produced emulsions with The other two

The

In general, there was an increase in mean droplet diameter with increasing oil

299

concentration (Fig. 3a).

In particular, there was an appreciable increase in droplet size

300

and polydispersity when the oil concentration increased from 40 to 50% (Figs. 3a and 3b).

301

The particle size distribution measurements indicated that all the emulsions with low oil

302

concentrations (10 to 40 wt%) were monomodal, whereas the emulsions with the highest

303

oil concentration (50% wt%) were bimodal (Fig. 4).

304

polydispersity at high oil concentrations can be attributed to a greater frequency of

The increase in droplet size and

13



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droplet collisions within the homogenizer.

306

coalescence may have occurred because the droplets collided with each other before they

307

were completely covered by emulsifier molecules.34 This trend is in agreement with the

308

study of Jafari et al.

309

microfluidization increased as the oil concentration increased. These authors explained

310

their results in terms of there being insufficient emulsifier present to completely cover all

311

the oil droplets at high oil concentrations. However, in the present study, the ratio of

312

emulsifier-to-oil was kept constant (1:10) for all the emulsions, and so it is more likely

313

that the increase in droplet size observed at high oil concentrations was due to the

314

increase in droplet collision frequency.

315

was found to be the most surface active in the interfacial tension measurements (Fig. 1a)

316

was also found to be the most effective at producing small droplets during

317

homogenization (Fig. 3a).

318

emulsifiers could form stable emulsions containing small droplet sizes and high fish oil

319

contents, with the rhamnolipids being the most effective.

35

As a result, a greater extent of droplet

, who reported that the size of the oil droplets produced by

Interestingly, the emulsifier (rhamnolipids) that

Overall, these experiments show that both of the natural

320

Impact of emulsifier type on emulsion microstructure and creaming stability.

321

Emulsion microstructure was analyzed by confocal laser-scanning microscopy after they

322

were stored at 4 °C for 3 days (Fig. 5). At low oil contents (10 and 20 wt%), no large

323

particles were observed in any of the emulsions, but a few larger particles were observed

324

in the emulsions at intermediate oil levels (30 and 40 wt%).

325

surprising since the light scattering measurements did not indicate the presence of any

326

large particles at oil levels ≤ 40 wt% (Figs. 3 and 4).

327

differences in the sampling procedures associated with the two analytical techniques used.

These results were

This discrepancy may due to

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328

For the microscopy measurements, a non-diluted emulsion is placed directly on the

329

microscope slide, but for the light scattering measurements a highly diluted and stirred

330

emulsion is analyzed.

331

been some dissociation of flocculated droplets or large oil droplets may have creamed to

332

the top of the sample and not been analyzed.36 At the highest oil concentration (50 wt%),

333

all of the emulsions contained many large individual oil droplets (Fig. 5), which can be

334

attributed to droplet coalescence inside the homogenizer.35

Consequently, for the light scattering measurements there have

335

Visual observation indicated that there was extensive droplet creaming and phase

336

separation in some of the highly concentrated emulsions (Fig. 6). In the quillaja saponins

337

systems, a brownish transparent serum layer was observed at the top and a white cream

338

layer at the bottom of the emulsions containing 40 and 50 wt% oil after 15 days storage.

339

Interestingly, in the rhamnolipids systems, a dark brown layer was formed at the bottom

340

and a light brown layer at the top of the 50% fish oil emulsions after 15 days. Conversely,

341

in the Tween 80 systems, all the emulsions retained a whitish color throughout storage,

342

although a little bit of oil was observed on top of the emulsions.

343

formed in the emulsions containing the natural emulsifiers suggested that some form of

344

chemical reaction occurred that led to browning.

345

Impact of emulsifier type on emulsion rheology.

The brownish color

The rheological properties of

346

emulsions are important because they influence their processing and quality attributes.

347

For this reason, the apparent shear viscosities (at 10 s-1) and flow profiles of fish oil

348

emulsions with different oil concentrations were measured (Fig. 7). As expected, the

349

apparent shear viscosity of the emulsions increased as the oil concentration increased (Fig.

350

7a). Interestingly, the apparent viscosity of the 50 wt% oil-in-water emulsions containing

15



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351

Tween 80 was appreciably lower than that of the other two emulsifiers.

Nevertheless,

352

all of the concentrated emulsions were highly aggregated and unstable to phase

353

separation (Fig. 6), and so it is difficult to make accurate measurements.

354

The emulsions also exhibited pronounced shear thinning behavior, with the apparent

355

viscosity decreasing with increasing shear rate, especially for the more concentrated

356

emulsions (Figs. 7b-7d). The flow behavior of the different emulsions was described by

357

fitting the experimental data to the Sisko model:

358

ηa= η∞+K γn-1

359

Where η∞ is the infinite-shear rate viscosity, K is the consistency coefficient, and n is

360

flow behavior index (Table 1). There was a good correlation (R2 > 0.94) between the

361

experimental data and the Sisko model for all the emulsions. Overall, the consistency

362

coefficient increased and the flow behavior index decreased with increasing oil

363

concentration.34 These results suggest that it is possible to make emulsions with relatively

364

low viscosities at oil levels up to about 40 wt%. Above this value, the viscosity of the

365

emulsions increased steeply, which might be a disadvantage for some applications, but an

366

advantage for others.

367

Impact of emulsifier type on oxidation stability of fish oil emulsions.

In this

368

section, we examined the influence of emulsifier type and oil concentration on the

369

oxidative stability of the fish oil emulsions.

370

determined by measuring the primary (peroxide values) and secondary (TBARs) reaction

371

products formed when the fish oil emulsions were stored at 55 °C (Figs. 8a and 8b).

372

There were appreciable differences in the oxidative stabilities of the emulsions depending

373

on emulsifier type and oil concentration.

The extent of lipid oxidation was

In general, there was an increase in peroxide

16


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374

values and TBARS over time in all the emulsions indicating that lipid oxidation was

375

occurring.

376

concentration for all emulsifiers, as seen by the final amount of peroxides and TBARs

377

produced at the end of the oxidation period (when expressed per unit mass of oil phase)

378

(Figs. 8c and 8d).

379

concentrated emulsions contained less aqueous phase, and therefore less hydrophilic

380

pro-oxidants; (ii) the concentrated emulsions contained larger oil droplets, which meant

381

that the fish oil droplets had a smaller specific surface area exposed to the pro-oxidants in

382

the aqueous phase; (iii) some of the concentrated emulsions underwent phase separation

383

(oiling off), which would have further reduced the specific surface area; (iv) the

384

concentrated emulsions were more viscous, which may have led to less efficient mixing

385

of the system. A number of previous studies have shown that lipid oxidation is promoted

386

by hydrophilic pro-oxidants (such as transition metals) and may depend on specific

387

surface area (droplet size), which have been reviewed elsewhere 10, 37-39.

However, the extent of lipid oxidation decreased with increasing oil

This phenomenon may have occurred for a number of reasons: (i) the

388

In the less concentrated emulsions (≤ 30 wt%), the extent of lipid oxidation was

389

appreciably less for the rhamnolipids-coated droplets than for the other two emulsifiers.

390

Conversely, the extent of lipid oxidation was less in the quillaja saponins-coated droplets

391

in the more concentrated emulsions (40 and 50 wt% oil). In most of the systems, the

392

natural emulsifiers (quillaja saponins and rhamnolipids) were more effective antioxidants

393

than Tween 80.

394

products formed should be treated with some caution, since the reaction products usually

395

increase and then decrease as lipid oxidation progresses.40 Nevertheless, this effect was

396

not pronounced in this system.

It should be noted that comparisons of the level of final reaction

A number of previous studies have also reported the

17



Page 18 of 31

16, 41

397

effectiveness of saponins as antioxidants in oil-in-water emulsions

.

However, to

398

the authors’ knowledge, this is the first study to report the strong antioxidant activity of

399

rhamnolipids in emulsions.

400

In summary, the influence of emulsifier type and oil content on the physical properties

401

and oxidative stability of fish oil-enriched emulsions prepared using a dual-channel

402

microfluidizer was examined. The mean droplet diameter increased with increasing oil

403

content, but the droplet charge was largely independent of oil content.

404

droplet charge did depend on emulsifier type, which may be important because positively

405

charged transition methods (such as iron) may be adsorbed to negatively charged lipid

406

droplets.

407

(50 wt% oil) stabilized by the natural emulsifiers after prolonged storage at elevated

408

temperatures, which was attributed to their relatively large initial droplet size. The

409

apparent shear viscosity and shear thinning of the emulsions increased with increasing oil

410

content, with a steep rise in viscosity occurring above 40 wt% oil.

411

oxidative stability of the emulsified fish oil increased as the oil content in the emulsions

412

increased.

413

effective at inhibiting lipid oxidation than the synthetic non-ionic surfactant (Tween 80).

414

Overall, this study demonstrates that high levels of fish oil (at least 40 wt%) can be

415

encapsulated in natural surfactant-stabilized emulsions containing small anionic droplets

416

that have a relatively good stability to oxidation.

417

amount even further by examining oil contents between 40 and 50 wt%.

Nevertheless, the

Extensive phase separation was observed in the most concentrated emulsions

Interestingly, the

The natural emulsifiers (quillaja saponins and rhamnolipids) were more

It may be possible to increase this

418

18


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419


ACKNOWLEDGEMENTS

420

The research work was funded by the National Natural Science Foundation of China

421

under Grant No. 31371835 and 31671888. Fuguo Liu would like to thank the Chinese

422

Scholarship Council for support.

423

by the Cooperative State Research, Extension, Education Service, USDA, Massachusetts

424

Agricultural Experiment Station (MAS00491) and USDA, NRI Grants (2013-03795).

425

We also thank DSM for partial support of this research, and thank Jenny Tang and John

426

Krill from DSM for useful advice and discussion.

This material was partly based upon work supported

427 428

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Figure captions Fig. 1. (a) Influence of emulsifier concentration on the interfacial tension measured at the fish oil-water interface. (b) The morphology of fish oil droplets used for interfacial tension measurements, oil phase was slowly added into the aqueous phase (emulsifier in buffer solution), the upper line represents the start point for measurement, while the lower lines define the reference of drop shape based on the width of the needle. Fig. 2. The zeta-potential of quillaja saponins, rhamnolipids and Tween-80 emulsions with different concentrations of fish oil. Fig. 3. Mean droplet size and span of quillaja saponins, rhamnolipids and Tween-80 emulsions with different concentrations of fish oil. Fig. 4. Particle size distribution for emulsions containing different fish oil concentrations produced by dual-channel methods (a) quillaja saponins emulsion (b) rhamnolipids emulsion, and (c) Tween-80 emulsion. Fig. 5. Confocal micrographs of quillaja saponins, rhamnolipids and Tween-80 emulsions with different concentrations of fish oil. Fig. 6. Effect of oil concentration on appearance of quillaja saponins, rhamnolipids and Tween-80 emulsions after storage of 0, 6 and 15 days at 55°C. Fig. 7. Apparent shear viscosity (at 10 s-1 shear rate) of quillaja saponins, rhamnolipids and Tween-80 emulsions with different concentrations of fish oil (a), Flow profiles (shear viscosity versus shear rate) of quillaja saponins (b), rhamnolipids (c) and Tween-80 (d) emulsions containing different concentrations of fish oil. Fig. 8. Effect of storage time and oil concentration on hydroperoxide values (PV) and thiobarbituric acid reactive substances (TBARS) of quillaja saponins, rhamnolipids and

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Tween-80 stabilized emulsions at 55°C. (a) PV value during storage (b) TBARS during storage (c) PV value at the storage of 15 days (d) TBARS at the storage of 15 days

23



Figures

Figure. 1

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Page 25 of 31


Figure. 2

25



Figure. 3

26


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Figure. 4

27



Figure. 5

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Figure. 6

29



Figure. 7

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80 70

TBARS (nmol/g)

Quillaja Saponins

10% 20% 30%

60

40%

50

50%

40 30 20 10 0 0

2

4

6

8

10

12

Storage Time (Days)

14

16


Fabrication of Concentrated Fish Oil Emulsions Using Dual-Channel

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