Influence of electrostatic interactions on the formation and stability of multilayer fish oil-in-water emulsions stabilized by whey protein-xanthan-locust bean complexes

Journal Pre-proof Influence of electrostatic interactions on the formation and stability of multilayer fish oil-in-water emulsions stabilized by whey protein-xanthan-locust bean complexes Kristen Griffin, Hanna Khouryieh PII:

S0260-8774(19)30536-9

DOI:

https://doi.org/10.1016/j.jfoodeng.2019.109893

Reference:

JFOE 109893

To appear in:

Journal of Food Engineering

Received Date: 21 September 2019 Revised Date:

18 December 2019

Accepted Date: 21 December 2019

Please cite this article as: Griffin, K., Khouryieh, H., Influence of electrostatic interactions on the formation and stability of multilayer fish oil-in-water emulsions stabilized by whey proteinxanthan-locust bean complexes, Journal of Food Engineering (2020), doi: https://doi.org/10.1016/ j.jfoodeng.2019.109893. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2019 Published by Elsevier Ltd.

1

Influence of Electrostatic Interactions on the Formation and Stability of Multilayer Fish

2

Oil-in-water Emulsions Stabilized by Whey Protein-Xanthan-Locust Bean Complexes

3 4

Kristen Griffina, Hanna Khouryiehb*

5

a

Department of Chemistry, Western Kentucky University, Bowling Green, KY, USA

6

b

School of Engineering & Applied Sciences, Western Kentucky University, Bowling Green, KY,

7

USA

8

*

Corresponding author: Tel.: +1 (270) 745 4126; email: [email protected]

9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

1

25 26

ABSTRACT The purpose of this research was to investigate the impact of electrostatic interactions on

27

the stability of multilayered fish oil-in-water (O/W) emulsions stabilized by whey protein isolate

28

(WPI)-xanthan (XG)-locust bean gum (LBG) complexes. Emulsions were prepared using the

29

layer-by-layer deposition technique with salt concentrations (0, 5, and 50 mM NaCl) at pH below

30

(pH 3) and above (pH 7) the isoelectric point of WPI. Results indicated that zeta potential at pH

31

3 resulted in positive values, whereas at pH 7 resulted in negative values, with the magnitude of

32

the ζ-potentials increasing as the NaCl concentration increased. NaCl did not have any major

33

impact on the particle size of the emulsions. XG emulsions had the highest viscosity at pH 3

34

regardless of time, though XG-LBG emulsions showed a significant increase at 0 and 5 mM

35

NaCl over time. XG emulsions at pH 3 showed the highest viscosity at every salt concentration.

36

At pH 7, XG-LBG emulsions had the highest viscosity results, yet decreased over time,

37

indicating the negative salt effect the synergistic interaction between XG and LBG. With 0 mM

38

and 5 mM NaCl at pH 7, XG-LBG emulsions had the highest creaming stability; while with 50

39

mM NaCl, XG emulsions had the highest creaming stability. For both the primary and secondary

40

lipid oxidation tests, XG-LBG emulsions had the highest oxidative stability at every salt

41

concentration at pH 7. These results have important implications in the design of biopolymer-

42

based delivery systems for microencapsulating omega-3 polyunsaturated fatty acids for use in

43

functional foods.

44 45 46 47 48 49 50

2

51 52

1. Introduction Omega-3 polyunsaturated fatty acids (PUFA) are found in several plant and animal oils

53

(e.g., fish oil), and are crucial in human growth and development throughout the life cycle as an

54

important component of essentially all cell membranes (Ellulu, Khaza’ai, Abed, Rahmat, Ismail,

55

& Ranneh, 2015; Simopoulos, 1991). As these fatty acids are not synthesized by the body, it is

56

imperative that they are obtained through diet; however, it is difficult to include fish oils into

57

food as they are highly susceptible to oxidation. Oxidation causes loss of fat-soluble vitamins,

58

generation of off-flavors, palatability problems, and even production of toxins that cause

59

foodborne illness (Arab-Tehrany et al., 2012; Shantha & Decker, 1994). Emulsions are

60

commonly used in the food and beverage industry as delivery systems to protect and control the

61

release of bioactive components (McClements, Decker, & Weiss, 2007). However, emulsions are

62

thermodynamically unstable systems that break down over time through flocculation and

63

coalescence, leading to creaming during storage. Successful development of delivery systems

64

with omega-3 fatty acids depends on the properties of emulsion (e.g., droplet size, viscosity), the

65

effect of environmental stresses (e.g., salt, pH), and the interaction between oil droplets and

66

surface-active components.

67

The kinetic stability of emulsions can be enhanced using stabilizers such as emulsifiers

68

and texture modifiers. Emulsion stability can be enhanced using multilayer adsorption at the

69

interface. The formation of multilayer oil-in-water emulsions, utilizing the electrostatic layer-by-

70

layer (LBL) deposition technique, consists of a multistep procedure where an oil and aqueous

71

phase are first homogenized in the presence of a charged emulsifier (e.g., protein), after which

72

consecutive layers of oppositely charged polyelectrolytes (e.g., polysaccharides) are added so

73

that it adsorbs to the protein-coated oil droplets (Guzey & McClements, 2007). Many studies

3

74

reported that multilayer emulsions provide better stability than conventional emulsions against

75

environmental stresses such as pH, ionic strength, heating, and freezing (Guzey & McClements,

76

2006; Aoki, Decker, McClements, 2005; Ogawa, Decker, & McClements, 2004).

77

Mixtures of different proteins and polysaccharides are usually used to fabricate

78

biopolymer-based delivery systems. In the current study, stabilized multilayer emulsions were

79

created as delivery systems for the fish oil. Whey protein isolate (WPI) is a surface-active protein

80

product containing mainly α-lactalbumin and β-lactoglobulin. These proteins contain functional

81

groups that allow WPI-stabilized emulsions to act as an antioxidant system by scavenging free

82

radicals, thereby inhibiting lipid oxidation (Berton-Carabin, Ropers, & Genot, 2014; Sun,

83

Gunasekaran, & Richards, 2007; Elias, McClements, & Decker, 2005; Gordon, 2001). At pH 3,

84

below its isoelectric point (pI) of pH 4.7- 5.2 (Charoen, Jangchud, Jangchud, Harnsilawat,

85

Naivikul, & McClements, 2011; Demetriades, Coupland, & McClements, 1997), WPI has a very

86

net positive charge; while at the isoelectric point, the protein has a net charge of zero and the

87

potential for protein-protein interaction is at its highest point. As the pH is increased up to pH 7,

88

the net charge on the protein will shift to a negative charge. Xanthan gum (XG) is an anionic

89

polysaccharide that displays different pH-dependent interactions with the WPI. At a low pH, XG

90

and WPI form complexes that prevent coalescence of the oil droplets; alternatively, at a high pH

91

the biopolymers repel one another. Locust bean gum (LBG), a non-ionic galactomannan, is not

92

influenced by variations in pH value and salt concentration. It does provide enhanced viscosity to

93

emulsions containing other polysaccharides, such as XG or carrageenan (Barak & Mudgil, 2014;

94

Goycoolea, Morris, & Gidley, 1995). XG and LBG have a synergistic relationship, which has

95

been extensively researched (Owens, Griffin, Khouryieh, & Williams, 2018; Khouryieh, Puli,

96

Williams, & Aramouni, 2015; Bresolin et al., 1997; Tako, Asato, & Nakamura, 1984). A

4

97

synergistic interaction occurs between XG and LBG, which results in substantially enhanced

98

viscosity or gelation. This phenomenon is due to the intermolecular binding that may occur

99

between the sidechains of XG in the helical form and the backbone of the LBG (Khouryieh,

100

Herald, Aramouni, & Alavi, 2007). When added to the emulsion, these two polysaccharides

101

synergism has been shown to increase the adsorption of the WPI to the oil droplet at the O/W

102

interface, and thus, inhibiting coalescence and creaming (Khouryieh et al., 2015).

103

Various studies have investigated the antioxidative effect of salt on O/W emulsions

104

containing different oils, polysaccharides, and transition metal ions (Berton-Carabin et al., 2014;

105

Nielsen, Horn, & Jacobsen, 2013; Laguerre, Lecomte, & Villeneuve, 2007; McClements, 2004;

106

McClements & Decker, 2000). Positively charged salt ions (e.g. sodium or calcium) aid the

107

protein and polysaccharide(s) in repelling positively charged transition metals that may come

108

near the oil droplet interface; thus, the positively charged salt ion benefits the oxidative stability

109

of the emulsion (Nielsen et al., 2013). In this study, multilayered oil-in-water emulsions

110

containing menhaden droplets were fabricated. The main objective of this research was to study

111

the influence of electrostatic interactions on the formation and stability of multilayer oil-in-water

112

emulsions stabilized by WPI-XG-LBG complexes. The impact of adding XG and LBG on the

113

stability of WPI-coated O/W emulsions as function of salt was investigated to determine whether

114

WPI-stabilized emulsions containing XG-LBG mixtures would provide better physical and

115

oxidative stability than emulsions containing either XG or LBG alone. The effect of NaCl salt

116

content (0, 5, and 50 mM) at pH below (pH 3) and above (pH 7) the isoelectric point of WPI was

117

studied.

118 119

5

120 121

2. Materials and Methods

122

2.1. Materials

123

Menhaden oil (14:0 Myristic acid 6-9%, 16:0 Palmitic acid 15-20%, 16:1 palmitoelic acid

124

9-14%, 18:1 oleic acid 5-12%, 18:2 linoleic acid <3, 20:4 arachidonic acid <3%, 18:4

125

octadecatetraenoic acid 2-4%, 20:5 eocosapentanoic acid 10-15% and 22:6 docosahexaenoic acid

126

8-15%, as provided by the manufacturer), xanthan gum, locust bean gum, and sodium azide were

127

purchased from Sigma-Aldrich Co. (St Louis, MO, USA). Sodium Chloride was purchased from

128

Thermo Fisher Scientific (Waltham, MA, USA), and whey protein isolate was purchased from

129

Davisco Food International, Inc. in the form of BiPro. All other reagents and chemicals used

130

were of analytical grade. Deionized water was used to produce the emulsions.

131 132 133

2.2. Preparation of emulsions Stock solutions of WPI (10% w/v), XG (2%), LBG (2%), and each respective salt

134

solution were prepared fresh for every set of testing, and allowed to stir for up to 10 h on a

135

magnetic stirrer before use. Both XG and LBG solutions were stirred on low heat (~45°C) to

136

ensure complete dispersion and hydration. A sodium azide (0.04%) was added to prevent the

137

growth of bacteria.

138

Oil-in-water primary emulsions were prepared by homogenizing menhaden oil into WPI

139

aqueous solutions for 3 min using a Fisher Scientific PowerGen 500 homogenizer at 30,000 rpm.

140

Then secondary emulsions were created by adding the respective XG, LBG, or XG-LBG

141

solutions to the primary emulsions and homogenizing for another 3 min. The pH of the

142

secondary emulsions was then adjusted by using 1M HCl and 1M NaOH to either pH 3 or pH 7.

6

143

The pH of the emulsions before adjustment was in the range of 6.6 - 6.7. Sodium chloride

144

solution at 0 mM, 5 mM, and 50 mM was then added to the samples of emulsions, and the

145

emulsions were further homogenized for an additional 1 min. The lowering of the pH before the

146

addition of the salt allows electrostatic interaction between the xanthan gum and the whey

147

protein adsorbed oil droplets, and increases the stability of the emulsion as proposed by Guzey &

148

McClements (2007). The final composition of the emulsions was 10% (v/v) menhaden oil, 2%

149

(w/v) WPI, and 0.2% (w/v) of a XG-LBG mixture at a 1:1 ratio, XG, or LBG. Oil-in-water

150

emulsions containing only WPI were served as the study control. The final emulsions were then

151

stored in glass vials in the dark at ambient temperature to prevent oxidation occurring outside the

152

parameters of the experiment.

153 154 155

2.3. Zeta potential measurements The ζ-potential of the emulsions was determined using the Zetasizer Nano Z instrument

156

(Malvern Instruments, Ltd., Worcestershire, UK). Aliquots from the cream layer of the emulsion

157

samples were diluted by a factor of 100 to allow accurate and precise readings of the ζ-potentials.

158

The folded capillary cells in which the samples were measured were rinsed with methanol and

159

deionized water before use. To reduce the accumulation of air bubbles within in the capillary

160

cells, the diluted emulsion samples were injected upside down and tapped to dislodge any air

161

bubbles. Every emulsion type was measured in duplicate, with each duplicate receiving three

162

measurements. All ζ-potential measurements were made at day 1 and after 2 weeks.

163 164

2.4. Particle size measurements

7

165

Malvern Mastersizer hydro 2000MU (Malvern Instruments, Ltd., Worcestershire, UK)

166

was used to determine the oil droplet size. The cream layer of the emulsion samples was added to

167

800mL of deionized water until the obscuration range was within 10-20%. The dilution of the

168

sample occurred to prevent multiple scattering effects. The refractive indices for the oil droplet

169

and the deionized water were set to 1.46 and 1.33, respectively. Every emulsion sample was

170

measured in duplicates with three measurements for each duplicate. The surface weighted mean

171

(D [3,2]) of the oil droplets was gathered to represent the particle size. All particle size

172

measurements were made at day 1 and after 2 weeks.

173 174 175

2.5. Viscosity measurements A Discovery Hybrid Rheometer (Model HR-2, TA Instrument, New Castle, DE, USA)

176

was used for the emulsions rheological measurements. A 40 mm diameter parallel plate

177

geometry with a gap of 1000 µm was used for all measurements. Viscosity measurements were

178

performed at 25 °C with shear rates from 0 to 100 s-1. Trios v3.0 software controlled the

179

rheometer and was used for data collection and analysis. Measurements were performed at day 1

180

and after 2 weeks.

181 182 183

2.6. Creaming stability measurements Emulsion samples were pipetted into 15-mL vials and stored upright in a dark area at

184

room temperature to prevent undue lipid oxidation. The creaming index (CI) was measured using

185

a precise ruler every hour in centimeters for the first 6 h after preparation and then every 24 h for

186

2 weeks. Over time, due to thermodynamic instability, the emulsions separate into a top oil (or

187

cream) layer and a bottom serum layer. It can be defined by the equation:

8

188

CI (%) = Hs/HT x 100%, where Hs represents the serum height and HT represents the overall

189

height of the emulsion. Measuring the creaming index can help determine the amount of

190

aggregation within the emulsion, as an increased level of aggregation leads to larger flocs and

191

faster creaming.

192 193 194

2.7. Microstructural characterization Differential interference contrast (DIC) microscopy (Zeiss Axioplan 2 optical imaging)

195

was used to determine the microstructure of the cream layer of the emulsions. Ten microliters of

196

sample were placed on a slide and covered by a 18mm x 18mm coverslip. A droplet of

197

immersion oil was placed on top of the coverslip to allow better resolution due its differing

198

refractive index. The magnification used was 40x and all images were captured using Axiovision

199

2.0 software.

200 201 202

2.8. Lipid oxidation measurements To determine the oxidative stability, fresh emulsions were stored in screw-cap glass vials

203

in the dark at ambient temperature for up to eight weeks. The oxidative stability of the

204

emulsions was determined by measuring the primary (lipid hydroperoxides) and secondary

205

(thiobarbituric acid reactive substances (TBARS)) lipid oxidation product concentrations.

206

2.8.1 Primary lipid oxidation products

207

Lipid hydroperoxide concentrations were determined by the adapted method of Elias et

208

al. (2005). The lipid hydroperoxides were measured by mixing 100 mg of the emulsion sample

209

with 4.9 mL of 2:1 v/v isooctane/1-butanol in a test tube. Fifty microliters of ferrous iron

210

solution and 20 μL of ammonium thiocyanate were then added to the mixture. The ferrous iron

9

211

solution was prepared by mixing 0.132 M BaCl2 and 0.144 M FeSO4 in equal amounts. The test

212

tubes were then vortexed for 10 s and let rest in the dark for 20 min, after which the absorbances

213

of the samples were measured at 510 nm with a Shimadzu UV-1601 UV-vis spectrophotometer.

214

The standard curve for determining the lipid hydroperoxides was prepared using cumene

215

hydroperoxide.

216

2.8.2 Secondary lipid oxidation products

217

TBARS test was used to determine the secondary lipid oxidation products according an

218

adapted method of Elias et al. (2005). A 10 mg of emulsion samples were placed in test tubes

219

and mixed with 2.0 mL of TBA reagent, which is comprised of 15% w/v trichloroacetic acid,

220

0.375% w/v thiobarbituric acid, and 10.2 M HCl dissolved in 1-butanol. The samples were

221

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

222

15 min at 1000 g. After 10 min, the absorbance of the samples was measured at 532 nm with a

223

Shimadzu UV-1601 UV-vis spectrophotometer. The TBARS concentrations were determined by

224

using a standard curve prepared using 1,1,3,3-tetraethoxypropane.

225 226 227

2.9. Statistical analysis The experimental data was analyzed using version 9.3.1 SAS statistical software (SAS

228

Institute Inc., Carry, NC, USA). Two-way analysis of variance (ANOVA) procedure with

229

bonferroni’s post-hoc comparisons tests was used for determining statistical differences between

230

the emulsion samples. The level of significance was set at P < 0.05. At least duplicate

231

determinations of each emulsion sample were examined, and the results were reported as the

232

mean ± standard deviation (SD) of the measurements.

233

10

234

3. Results and discussion

235

3.1. Effect of ionic strength and pH on droplet charge

236

Regardless of the NaCl concentrations, all emulsions at pH 3 resulted in positive ζ-

237

potentials, while all emulsions at pH 7 demonstrated negative ζ-potential values (Fig. 1). The

238

positive ζ-potential values indicate the dominance of the WPI at the oil-water interface because

239

the surface charge values mirror the positive net charge of the WPI, resulting from pH 3 being

240

below the pI of the WPI. Similar results were reported by Long, Zhao, Liu, Kuang, Xu & Zhao

241

(2013). The reason for WPI dominating the ζ-potential in emulsions containing the anionic XG at

242

pH 3 could be due to the XG and WPI interacting mostly in the continuous phase rather than at

243

the oil-water interface (Long et al., 2013). The negative ζ-potential values at pH 7 can be

244

expected because all emulsions contain the negatively charged WPI due to the pH being above

245

the pI of the WPI. Sun et al. (2007) found that negatively charged WPI chains can adsorb

246

strongly to the oil droplet surfaces, influencing the overall charge of the droplets. Since all

247

emulsions contain the WPI and an anionic XG or neutrally LBG charged polysaccharide, the net

248

charge of all droplets was negative at pH 7, which agrees with the Sun et al. (2007) study. The

249

large ζ-potential absolute values at pH 7 suggest large amounts of repulsion between both the

250

WPI and the added polysaccharides, and therefore displaying increased stability. XG-containing

251

emulsions displayed the highest absolute values due to both XG and WPI being anionic at

252

neutral pH. At this pH, the XG dominates the surface charge (Grigoriev & Miller, 2009).

253

The small increase in ζ-potential as NaCl is added at pH 7 could be explained by

254

electrostatic screening diminishing any residual repulsions between the negatively charged WPI

255

and XG. The salt ions, both positive [Na+] and negative [Cl-] ones, arrange themselves around

256

the negatively charged WPI and XG particles. The NaCl ions tend to form a cloud of ions around

11

257

the particles, and this results in the net effect of screening or weakening, the net negative charge

258

of the particles. This contributes to the increased adsorption of the XG onto the surface of WPI-

259

coated droplets. The increased adsorption at the interface further stabilizes the oil droplets. A

260

similar occurrence was seen in a study observing interactions between ɩ-carrageenan and β-

261

lactoglobulin (Gu, Decker, & McClements, 2004). The NaCl also contributes to a larger

262

counterion cloud surrounding the individual protein-coated oil droplets; considering all droplets

263

in one emulsion are similarly charged, the counterion clouds will contain the same charge.

264

Therefore, any droplets approaching one another will not interact and cannot alter the surface

265

charge of a neighboring droplet (McClements, 2015).

266

Emulsions containing the XG-LBG mixtures at pH 3 had the lowest ζ-potential values

267

among the rest of polysaccharides. Also, XG-LBG emulsions at pH 3 displayed higher ζ-

268

potential values as the NaCl concentration increased, indicating more repulsion between the

269

polysaccharides and WPI-coated droplets and higher stability with more NaCl present. The WPI

270

in the emulsions remained adsorbed to the oil droplets and dominated the surface charge while

271

the NaCl produced an electrostatic screening for the XG-LBG emulsions to interact with the

272

cationic WPI and thereby stabilize the droplets. XG emulsions after 2 weeks of storage resulted

273

in slightly greater ζ-potential absolute values with greater NaCl concentrations. This is due to the

274

NaCl reducing any remaining electrostatic barriers between XG and WPI-coated droplets at pH

275

3, encouraging the oppositely charged polymers to interact strongly and stabilize the oil droplet.

276

In general, all emulsions at pH 7 had similar ζ-potential values regardless of the NaCl content at

277

both day 1 and 14. This indicates that NaCl did not have much of an impact on the stability of

278

the emulsions at neutral pH.

279

12

280 281

3.2. Effect of ionic strength and pH on droplet aggregation The mean particle sizes of the emulsions with varying NaCl concentrations were

282

measured at pH 3 and pH 7 over 2 weeks of storage (Table 1). With no salt added at pH 3,

283

emulsions containing XG had the largest particle size at 9 µm. Emulsions containing XG-LBG

284

had smaller particle sizes of 5 µm, and LBG and WPI emulsions consistently remained the

285

smallest. Within this trend, XG-LBG emulsions had significantly (p < 0.05) larger particle sizes

286

at 0 mM NaCl than at 5 and 50 mM NaCl on day 1; while LBG emulsions had significantly (p <

287

0.05) larger particle sizes at 50 mM NaCl on day 1 and significantly increased at every salt

288

concentration after 2 weeks. At every salt concentration at pH 7, the particle sizes of every

289

emulsion remained small (d32 ≈ 2- 3 µm). The particle size of emulsions containing XG-LBG

290

complexes at 0 mM NaCl had significantly (p < 0.05) increased after two weeks, and was the

291

largest particle size at pH 7. LBG and WPI emulsions resulted in the smallest particle sizes, but

292

the microstructure (Fig. 2) and creaming index (Fig. 3) of these emulsions illustrates the

293

instability of the emulsions. These emulsions were fully flocculated at all salt concentrations and

294

throughout the two-week storage period.

295

The larger particle size at pH 3 indicates that the pH value below the pI of the WPI

296

influenced aggregation to take place between the positively charged WPI and the negatively

297

charged XG. However, since salt was added after the pH was adjusted, the additional

298

homogenization may have broken up the aggregated clumps, which would account a smaller

299

particle size than expected (< 10 µm). Thus, an additional study (data not shown) at pH 3 was

300

conducted by adding the salt to the emulsion before the pH adjustment so that the aggregation

301

taking place would remain undisturbed. This experiment did result in significantly higher particle

302

sizes for both XG and XG-LBG emulsions, with the average between salt concentrations

13

303

occurring around 290 µm and 25 µm respectively, while droplets in LBG and WPI emulsions

304

remained small (2-3 µm). Guzey & McClements (2007) concluded that after an anionic

305

polysaccharide was able to adsorb to the droplets, the addition of salt resulted in greater stability

306

of the overall emulsion. Electrolytes affect the adsorption of the protein and polysaccharides onto

307

the oil droplet by influencing the magnitude of the charges found on the protein-coated oil

308

droplets and biopolymers, ultimately affecting the electrostatic interactions between the

309

stabilizers in the aqueous phase.

310

At all three NaCl concentrations, emulsions prepared at pH 7 displayed low particle

311

diameters without significant (p > 0.05) differences between day 1 and 14, suggesting that there

312

was minimal electrostatic attraction between the WPI-coated oil droplets and the

313

polysaccharides. McClements (2004) found that the particle size of emulsions with 0-200 mM

314

NaCl remained very small and did not have any significant changes until around 300 mM NaCl.

315

This indicates that the NaCl concentrations in our study did not have a significant effect on the

316

particle size of the emulsions, which is also similar to the results found by Cui, Cho,

317

McClements, Decker, & Park (2016).

318 319 320

3.3. Effect on viscosity of the emulsions At pH 3, emulsions containing XG had the highest viscosity, followed by XG-LBG

321

emulsions (Table 2). Both XG emulsions and XG-LBG emulsions experienced a large increase

322

in viscosity over time, significantly (p < 0.05) at 0 and 5 mM NaCl for XG-LBG emulsions and

323

at 5 mM NaCl for XG emulsions. On the other hand, both LBG and WPI emulsions decreased

324

over time, significantly (p < 0.05) for LBG at 0 and 50 mM NaCl. Among the salt levels of the

325

same gum type, XG-LBG emulsions viscosity significantly (p < 0.05) increased as the salt was

14

326

increased to 50 mM NaCl on day 1, and continued this trend after two weeks leading to XG-LBG

327

emulsion’s viscosity being highest at 50 mM NaCl. On the contrary, this trend was flipped at pH

328

7 for XG-LBG emulsions, as the viscosity significantly (p < 0.05) decreased from 0 mM to 50

329

mM NaCl on day 1, resulting in XG-LBG emulsions at 0 mM with the highest viscosity and the

330

highest stability overall at this pH. The higher viscosities at pH 7 are likely due to the strong

331

synergistic interaction between XG and LBG in XG-LBG-containing emulsions. This stabilizing

332

method can be seen in the emulsion microstructure (Fig. 2); the oil droplets retain their

333

individual integrities but have formed stabilized flocs. This is in contrast to the WPI and LBG

334

emulsions where the droplets are fully flocculated and larger in size.

335

Overall, XG emulsions at pH 3 showed the highest viscosities at every salt concentration.

336

The opposite charges increase the interaction between the the protein and polysaccharide,

337

leading to a more aggregated and viscous solution (Demetriades et al., 1997). The positively

338

charged WPI adsorbs to the oil droplet, repelling other positively charged substances; the

339

negatively charged XG will then be able to induce bridging flocculation. This phenomenon

340

occurs when a polysaccharide binds to WPI molecules on two different oil droplets, creating a

341

bridge between the two droplets (Mandala, Savvas, & Kostaropoulos, 2004; Blijdenstein, van

342

Winden, van Vliet, van der Linden, & van Aken, 2004). In the emulsion-making process, the salt

343

was added after the adsorption of gum molecules onto the surfaces of WPI-coated droplets,

344

which gave the XG time to create the bridge between droplets. This XG bridging would then be

345

advanced as the salt would promote association between protein-coated oil droplets because of

346

its electrostatic screening effect, essentially further increasing the viscosity.

347 348

The addition of salt has been shown to transition the structure of XG from a disordered, non-helical conformation to an ordered, helical conformation (Khouryieh, Herald, Aramouni, &

15

349

Alavi, 2007). The addition of salt to a solution at 25°C collapses the extended side chains of

350

xanthan gum via electrostatic screening, forcing the transition of the xanthan backbone from

351

disordered to ordered structure and therefore reducing viscosity of the solution (Khouryieh et al.,

352

2007). If the salt was added before the XG was given time to interact with the WPI-coated

353

droplets, then the side chains would collapse and the viscosity would be reduced; hence, the

354

order of emulsion formation is important in the overall stability of the emulsions.

355

In the absence of salt (0 mM NaCl) at pH 7, XG-LBG emulsions had the highest

356

viscosity. This is probably due to the lack of salt allows the XG-LBG synergistic interaction to

357

remain strong with no electrostatic interference. When the salt is added, it changes the

358

conformation of XG to ordered and thereby decreases the interaction with the LBG (Khouryieh

359

et al., 2007). The XG-LBG emulsions did not result in higher viscosity values than the XG

360

emulsions at pH 3, because the stabilizing effect of the XG bridging is stronger than the XG-

361

LBG synergistic relationship.

362 363 364

3.4. Effect on creaming stability The creaming stability of the emulsions was determined by measuring the creaming index

365

over 2-week period (Fig. 3). The creaming profile of the emulsions with different salt

366

concentrations was also established. The changes in salt concentration affected emulsions

367

containing XG-LBG or XG; however, it showed to have a limited effect on those containing only

368

LBG or WPI. With 0 mM NaCl at pH 3, emulsions containing XG alone were the most stable

369

among all gum types. With 0 mM NaCl at pH 3, all the emulsions displayed some creaming and

370

phase separation within the first 3 hours and were almost fully creamed within 48 hours. The

371

turbidity of the serum phase increased in the order of XG emulsions, LBG emulsions, XG-LBG

16

372

emulsions, to WPI emulsions. XG containing emulsions were generally less turbid than those

373

lacking XG due to the complexation between XG and WPI. Therefore, most of the

374

polysaccharides and WPI were in the cream layer.

375

At pH 7, all the emulsions, excluding XG-LBG emulsions, displayed some creaming and

376

phase separation within the first 3 hours. XG emulsions remained the least turbid out of all the

377

gum types while LBG and WPI remained consistent in their turbidity. These trends are observed

378

at every salt concentration. XG-LBG emulsions at pH 7 were the most stable at 0 and 5 mM

379

NaCl. This can be related to the higher viscosity in the continuous phase because of the strong

380

synergistic effect between XG and LBG in the absence of salt content (Khouryieh et al., 2007).

381

The increased creaming stability with no salt added is supported by Ogawa, Decker, and

382

McClements (2004), who found that salt induces greater screening between charged particles and

383

thereby induces flocculation and potentially coalescence.

384

With 5 mM NaCl at pH 3, XG emulsions and XG-LBG emulsions were equally the most

385

stable against creaming. XG-LBG emulsions were the most stable until approximately 216 hours

386

where it is slightly surpassed by XG emulsions. The addition of low NaCl content most likely

387

reduced some of electrostatic barriers between the XG and WPI, thereby allowing the XG to

388

create stronger bridges between the WPI-coated droplets. The stronger interactions would cause

389

a slower separation of phases. The addition of low NaCl content did not have the same effect on

390

the XG-LBG containing emulsions. The LBG already diminishes the ability of XG to form

391

bridges with the WPI, and the addition of salt further detracts from this capability by collapsing

392

the remaining side chains that are not interacting with the WPI. Due to the lack of XG-bridging

393

occurring in the XG-LBG emulsions, the viscosity is lower at pH 3 and therefore the creaming

394

stability is lesser as well. At pH 7, XG-LBG emulsions were more stable than XG emulsions

17

395

with no creaming at all. This is due to the NaCl content (5 mM) is no not high enough to

396

overcome the resulting high viscosity from the synergistic interaction between XG and LBG.

397

At 50 mM NaCl, XG-LBG emulsions became of equal turbidity with emulsions

398

containing XG. With increasing salt concentrations, XG and XG-LBG emulsions became

399

gradually less turbid, which resulted in the clarity of the 50 mM salt concentration emulsions

400

after 336 hours. Adding high NaCl concentration to the emulsion, thereby increasing the amount

401

of electrolyte in the aqueous phase, reduces the electrostatic interactions between the protein-

402

coated oil droplets. The droplets can then come into contact with one another and flocculate in

403

the cream layer, resulting in a clear serum phase. This can be seen in the microstructure of the

404

emulsions (Fig. 2). As the salt concentration increases, the individual droplets become more

405

flocculated in emulsions stabilized by XG and the mixture of XG-LBG. Emulsions stabilized by

406

LBG and WPI flocculated at every salt concentration.

407

The creaming index indicates that XG-LBG emulsions with 0 and 5 mM NaCl at pH 7

408

were significantly more stable than the other emulsions. Conversely, 50 mM NaCl XG emulsions

409

were the most stable overall. As detailed by McClements (2015) and Demetriades et al. (1997),

410

after adding to the emulsion, the salt reduces the electrostatic interactions between the protein-

411

coated oil droplets. The screening of electrostatic interactions between charged emulsion droplets

412

is due to the attraction of counter ions to the surface of the droplets. When adding abundant salt,

413

the screening could hinder the repulsive forces between the droplets, allowing them to collide

414

with one another, leading to increased flocculation and therefore decreased physicochemical

415

stability. This is also supported by the magnitude and range of the electrostatic repulsion

416

between two droplets decreases as the ionic strength of the surrounding solution increases

417

because of the electrostatic screening.

18

418 419 420

3.5. Effect on lipid oxidation Lipid hydroperoxide concentrations of emulsions at 0 mM, 5 mM, and 50 mM NaCl were

421

measured over a function of storage time at both pH 3 and 7 (Fig. 4). At pH 3, all the gum types

422

remain relatively close together, a trend that is seen at every salt concentration at this pH.

423

Whereas, at pH 7, XG-LBG emulsions had the lowest lipid hydroperoxide concentrations at all

424

NaCl concentrations, indicating the highest stability. This is possibly related to the enhanced

425

viscosity of continuous phase due to the synergistic interaction between XG and LBG gums. The

426

increased viscosity due to XG-LBG interaction may have enhanced the oxidative stability of

427

WPI-coated droplets at pH 7 by forming a secondary thick layer around the WPI-coated droplets

428

through WPI-XG-LBG complex. XG emulsions had the second-most stable results, and the LBG

429

and WPI emulsions displayed almost identical results. Amongst the three salt concentrations,

430

however, emulsions with 0 mM NaCl had the best oxidative stability. With 50 mM NaCl, all four

431

emulsions at both pH values displayed results that were closer together than the results observed

432

at 0 and 5 mM NaCl. For pH 3, this makes it difficult to decisively decide which emulsion had

433

the best results. Conversely, at pH 7 the XG-LBG emulsions still had the lowest lipid

434

hydroperoxide concentration, although this concentration was still higher than the lesser salt

435

concentrations. XG emulsions had the second-most stable results, and the LBG and WPI

436

emulsions displayed almost identical results indicating that XG-LBG emulsions are the most

437

positively affected emulsion type at a high ionic strength. It can be seen that as the concentration

438

of NaCl increases, the difference between XG-LBG emulsion and the other gum emulsions

439

decreases. This is because increasing amounts of NaCl change the conformation of xanthan

440

gum’s structure from disordered to ordered state by collapsing its side chains due to electrostatic

19

441

screening. As previously stated, XG can interact strongly with LBG when in its disordered state.

442

This disordered state is changed to “ordered” at NaCl concentrations above 10 mM, indicating a

443

loss of XG-LBG synergistic interactions at these salt concentrations (Khouryieh et al., 2007).

444

This decrease in the synergistic interaction between XG and LBG decreases the viscosity of the

445

emulsion reducing the physical and, thus, the chemical stability by further decreasing the

446

electrostatic repulsions between the oil droplets and decreasing the secondary thick layer of XG-

447

LBG around the WPI-coated droplets. In the presence of added iron, NaCl has been shown to

448

slightly increase the rate of lipid oxidation (Mei, McClements, Wu, & Decker, 1998a; Mei,

449

Decker, & McClements, 1998b). This prooxidant effect could be because of NaCl’s capability to

450

increase the catalytic activity of iron (Osinchak, Hultin, Zajicek, Kelleher, & Huang, 1992).

451

Thiobarbituric acid reactive substances (TBARS) concentrations are an indication of the

452

secondary by products produced during the termination phase of lipid oxidation. The

453

concentration trendline of emulsions at both pH values increases sharply until around weeks 2 or

454

3, levels off until about week 6, and decreases slightly until week 8 (Fig. 5). The TBARS assay

455

measures lipid peroxidation end-product malondialdehyde (MDA) generated from the

456

decomposition of lipid hydroperoxides. This slight decrease in TBARS at week 8 could be

457

related to the reduction of MDA due to the decomposition of secondary lipid oxidation products.

458

With 0 or 5 mM NaCl at pH 3, XG emulsions had the lowest TBARS concentration and

459

therefore the highest oxidative stability. This result is switched to XG-LBG emulsions for pH 7,

460

which can be explained by the loss of XG-WPI interaction at this pH as well as the increased

461

synergism seen between the XG and LBG at the neutral pH value. At 5 mM NaCl and pH 3, the

462

addition of salt most likely begins to disrupt the disordered conformation of the XG, as explained

463

before, which detracts from the ability of XG to form stabilizing XG-bridges. At pH 7, the values

20

464

for XG-LBG continued to be isolated from the other emulsions, and the small amount of salt

465

begins to disrupt the interaction between the XG and LBG, but not enough to fully override the

466

strong synergism between them.

467

At 50 mM NaCl, TBARS concentrations were closer to one another than they were at

468

both 0 and 5 mM for both pH values. At pH 3, XG emulsions relatively had the best oxidative

469

stability, whereas at pH 7, the XG-LBG emulsions remained the most stable with the lowest

470

TBARS concentrations. The reason behind XG emulsions having better stability at pH 3, while

471

XG-LBG emulsions are more stable at pH 7, is due to the pI occurring around 4.8 (Demetriades

472

et al., 1997). At pH 3, the WPI switches to a positive charge leading to a protein-polysaccharide

473

interaction with the anionic XG. XG has also been shown to chelate transition metal ions at

474

negatively charged sites, thus averting the close contact with the lipid phase and inhibiting lipid

475

oxidation (Shimada, Fujikawa, Yahara, & Nakamura, 1992). These factors are more beneficial

476

than the synergistic relationship between XG-LBG at pH 3, especially as the synergistic

477

relationship decreases with the increase in salt.

478 479

4. Conclusion

480

The results indicated that the addition of NaCl had a significant impact on the physical

481

and chemical stabilities of the O/W emulsions depending on the type of biopolymer and pH of

482

the emulsion. The addition of NaCl affected the physical stability of WPI-stabilized emulsions

483

containing XG-LBG mixtures or XG alone; however, it had a limited effect on those containing

484

only LBG or WPI. With 0 mM NaCl at pH 3, emulsions containing XG alone were the most

485

stable among all gum types. At pH 7, WPI-stabilized emulsions containing XG-LBG mixtures

486

with 0 and 5 mM NaCl were significantly more stable than the other emulsions, while XG

21

487

containing emulsions with 50 mM NaCl were the most stable overall. The lipid oxidation results

488

indicated that the NaCl level had a very little effect on the oxidative stability of the emulsions

489

containing XG-LBG mixtures at every salt concentration, and hence, WPI-stabilized emulsions

490

containing XG-LBG mixtures had provided better oxidative stability than emulsions containing

491

either XG or LBG alone. This study suggests that a multilayer system consisting of WPI-XG-

492

LBG layers could be used as a delivery system for microencapsulating omega-3 polyunsaturated

493

fatty acids for use in functional foods.

494 495 496

Acknowledgment This research was supported by grants from the United States Department of

497

Agriculture’s National Institute of Food and Agriculture (Grant #. 11281827) and Kentucky NSF

498

EPSCoR (No. 1355438).

499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518

22

519

References

520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563

1. Aoki, T., Decker, E. A., & McClements, D. J. (2005). Influence of environmental stresses of on stability of O/W emulsions containing droplets stabilized by multilayered membranes produced by a layer-by-layer electrostatic deposition technique. Food Hydrocolloids, 19, 209-220. 2. Arab-Tehrany, E., Jacquot, M., Gaiani, C., Imran, M., Desobry, S., & Linder, M. (2012). Beneficial effects and oxidative stability of omega-3 long-chain polyunsaturated fatty acids. Trends in Food Science & Technology, 25, 24-33. 3. Barak, S. & Mudgil, D. (2014). Locust bean gum: Processing, properties and food applications—a review. International Journal of Biological Macromolecules, 66, 74-80. 4. Berton-Carabin, C., Ropers, M., & Genot, C. (2014). Lipid oxidation in oil-in-water emulsions: Involvement of the interfacial layer. Comprehensive Reviews in Food Science & Food Safety, 13(5), 945-977.

5. Blijdenstein, T., van Winden, A., van Vliet, T., van der Linden, E., & van Aken, G. (2004). Serum separation and structure of depletion- and bridging- flocculated emulsions: A comparison. Colloids and Surfaces A, 245, 41-48. 6. Bresolin, T. M. B., Sander, P. C., Reicher, F., Sierakowski, M. R., Rinaudo, M., & Ganter, J. L. M. S. (1997). Viscometric studies on xanthan and galactomannan systems. Carbohydrate Polymers, 33, 131-138.

7. Charoen, R., Jangchud, A., Jangchud, K., Harnsilawat, T., Naivikul, O., & McClements, D. J. (2011). Influence of biopolymer emulsifier type on the formation and stability of rice bran oil-in-water emulsions: Whey protein, gum Arabic, and modified starch. Journal of Food Science, 76(1), E165-E172. 8. Cui, L., Cho, H. T., McClements, D. J., Decker, E. A., & Park, Y. (2016). Effects of salts on oxidative stability of lipids in Tween-20 stabilized oil-in-water emulsions. Food chemistry, 197, 1130-1135. 9. Demetriades, K., Coupland, J. N., & McClements, D. J. (1997). Physical properties of whey protein stabilized emulsions as related to pH and NaCl. Journal of Food Science, 62(2), 346. 10. Elias, R., McClements, D. J., & Decker, E. (2005). Antioxidant activity of cysteine, tryptophan, and methionine residues in continuous phase β-lactoglobulin in oil-in-water emulsions. Journal of Agricultural and Food Chemistry, 53, 10248-10253. 11. Ellulu, M. S., Khaza’ai, H., Abed, Y., Rahmat, A., Ismail, P., & Ranneh, Y. (2015). Role of fish oil in human health and possible mechanism to reduce inflammation. Inflammopharmacology, 23, 79-89.

23

564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608

12. Gordon, M. H. (2001). The development of oxidative rancidity in foods. Antioxidants in food: Practical applications. CRC Press. 13. Goycoolea, F. M., Morris, E. R., & Gidley, M. J. (1995). Viscosity of galactomannans at alkaline and neutral pH: evidence of ‘hyperentanglement’ in solution. Carbohydrate Polymers, 27(1), 69-71. 14. Grigoriev, D. O. & Miller, R. (2009). Mono- and multilayer covered drops as carriers. Current Opinion in Colloid & Interface Science, 14, 48-59. 15. Gu, Y. S., Decker, E. A., & McClements, D. J. (2004). Influence of pH and ι-carrageenan concentration on physicochemical properties and stability of β-lactoglobulin-stabilized oil-inwater emulsions. Journal of Agricultural and Food Chemistry, 52(11), 3626-3632. 16. Guzey, D., & McClements, D. J., (2006). Formation, stability, and properties of multilayer emulsions for application in the food industry. Advances in Colloid and Interface Science, 128-130, 227-248. 17. Guzey, D., & McClements, D. J. (2007). Impact of electrostatic interactions on formation and stability of emulsions containing oil droplets coated by β-lactoglobulin-pectin complexes. Journal of Agricultural and Food Chemistry, 55(2), 475-485. 18. Khouryieh, H. A., Herald, T. J., Aramouni, F., & Alavi, S. (2007). Intrinsic viscosity and viscoelastic properties of xanthan/guar mixtures in dilute solutions: Effect of salt concentration on the polymer interactions. Food Research International, 40(7), 883-893. 19. Khouryieh, H., Puli, G., Williams, K., & Aramouni, F. (2015). Effects of xanthan–locust bean gum mixtures on the physicochemical properties and oxidative stability of whey protein stabilised oil-in-water emulsions. Food chemistry, 167, 340-348. 20. Laguerre, M., Lecomte, J., & Villeneuve, P. (2007). Evaluation of the ability of antioxidants to counteract lipid oxidation: Existing methods, new trends and challenges. Progress in Lipid Research, 46(5), 244-282. 21. Long, Z., Zhao, Q., Liu, T., Kuang, W., Xu, J., & Zhao, M. (2013). Influence of xanthan gum on physical characteristics of sodium caseinate solutions and emulsions. Food Hydrocolloids, 32(1), 123-129. 22. Mandala, I. G., Savvas, T. P., & Kostaropoulos, A. E. (2004). Xanthan and locust bean gum influence of the rheology and structure of white model-sauce. Journal of Food Engineering, 64, 335-342. 23. McClements, D. J. (2004). Protein-stabilized emulsions. Current opinion in colloid & interface science, 9(5), 305-313.

24

609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653

24. McClements, D. J. (2015). Food emulsions: principles, practice, and techniques. CRC Series in Contemporary Food Science. 25. McClements, D. J., & Decker, E. A. (2000). Lipid oxidation in oil‐in‐water emulsions: Impact of molecular environment on chemical reactions in heterogeneous food systems. Journal of Food Science, 65(8), 1270-1282. 26. McClements, D. J., Decker, E. A., & Weiss, J. (2007). Emulsion-based delivery systems for lipophilic bioactive components. Journal of Food Science, 72(8), R109-R124.

27. Mei, L. Y., McClements, D.J., Wu, J. N., & Decker, E. A. (1998a). Iron-catalyzed lipid oxidation in emulsion as affected by surfactant, pH and NaCl. Food Chemistry, 61, 307–312. 28. Mei, L. Y., Decker, E. A., & McClements, D. J. (1998b). Evidence of iron association with emulsion droplets and its impact on lipid oxidation. Journal of Agricultural and Food Chemistry, 46, 5072–5077. 29. Nielsen, N. S., Horn, A. F., & Jacobsen, C. (2013). Effect of emulsifier type, pH and iron on oxidative stability of 5% fish oil‐in‐water emulsions. European Journal of Lipid Science and Technology, 115(8), 874-889. 30. Ogawa, S., Decker, E. A., & McClements, D. J. (2004). Production and characterization of O/W emulsions containing droplets stabilized by lecithin-chitosan-pectin multilayered membranes. Journal of Agricultural and Food Chemistry, 52, 3595-3600. 31. Osinchak, J. E., Hultin, H. O., Zajicek, O. T., Kelleher, S. D., & Huang, C. (1992). Effect of NaCl on catalysis of lipid oxidation by the soluble fraction of fish muscle. Free Radical Biology and Medicine, 12, 35–41. 32. Owens, C., Griffin, K., Khouryieh, H., & Williams, K. (2018). Creaming and Oxidative Stability of Fish Oil-in-water Emulsions Stabilized by Whey Protein-Xanthan-Locust Bean Complexes: Impact of pH. Food Chemistry, 239, 314-322. 33. Shantha, N. C., & Decker, E. A. (1994). Rapid, sensitive, iron-based spectrophotometric methods for determination of peroxide values of food lipids. Journal of AOAC International, 77(2), 421-424. 34. Shimada, K., Fujikawa, K., Yahara, K., & Nakamura, T. (1992). Antioxidative properties of xanthan on the autoxidation of soybean oil in emulsion. Journal of Agricultural and Food Chemistry, 40, 945–948. 35. Simopoulos, A. P. (1991). Omega-3 fatty acids in health and disease and in growth and development. The American journal of clinical nutrition, 54(3), 438-463.

25

654 655 656 657 658 659 660

36. Sun, C., Gunasekaran, S., & Richards, M. P. (2007). Effect of xanthan gum on physicochemical properties of whey protein isolate stabilized oil-in-water emulsions. Food Hydrocolloids, 21(4), 555-564. 37. Tako, M., Asato, A., & Nakamura, S. (1984). Rheological aspects of the intermolecular

interaction between xanthan and locust bean gum in aqueous media. Agricultural and Biological Chemistry, 48(12), 2995-3000.

661 662

26

Table 1. Effect of ionic strength and pH on the particle size of fish O/W emulsions stabilized by WPI-XG-LBG complexes as a function of storage time. pH 3 NaCl(mM)

XG-LBG

Day 1 XG

Day 14 LBG

WPI

XG-LBG

XG

LBG

WPI

0

5.0±0.05Ab

8.7±0.3Aa

2.5±0.07Aa

3.5±0.9Aa

5.1±0.3Aa

8.3±0.01Aa

2.8±0.04Ba

2.9±0.06Aa

5

4.3±0.04Aa

8.6±0.02Aa

2.5±0.02Aa

3.7±0.0Aa

4.7±0.06Ba

8.0±0.3Aa

2.7±0.01Ba

3.1±0.9Aa

50

4.2±0.2Aa

8.6±0.1Aa

3.5±0.2Ab

3.1±0.4Aa

4.6±0.1Aa

8.4±0.1Aa

14.0±3.1Bb

2.9±0.2Aa

pH 7 NaCl(mM)

XG-LBG

Day 1 XG

Day 14 LBG

WPI

XG-LBG

XG

LBG

WPI

0

2.2±0.0Aa

2.2±0.02Aa

2.5±0.4Aa

3.1±0.4Aa

5.1±4.1Aa

2.2±0.02Aa

2.3±0.01Aa

2.7±0.5Aa

5

2.1±0.0Aa

2.3±0.01Aa

2.4±0.2Aa

3.1±0.3Aa

2.3±0.2Aa

2.4±0.0Bb

2.5±0.02Ab

3.0±0.06Aa

50

2.3±0.03Ab

2.9±0.3Aa

2.5±0.1Aa

2.9±0.4Aa

2.4±0.1Aa

2.5±0.0Ac

2.5±0.02Ab

3.3±0.2Aa

ABC = different letters indicate a significant (P < 0.05) difference between day1 and day 14

for the same gum type and salt concentration. abc = different letters in columns indicate significant differences (P < 0.05) among the salt levels of the same gum type.

Table 2. Effect of ionic strength and pH on the viscosity (Pa.s) of fish O/W emulsions stabilized by WPIXG-LBG complexes as a function of storage time at shear rate of 10s-1. pH 3 NaCl(mM) XG-LBG 0.0205± 0 0.0017Aa

Day 1 XG 0.2760± 0.1410Aa

LBG 0.0529± 0.0039Aa

WPI 0.0390± 0.0091Aa

XG-LBG 0.2900± 0.0614Ba

XG 0.6820± 0.2550Aa

Day 14 LBG 0.0255± 0.0021Ba

WPI 0.0291± 0.0036Aa

5

0.0311± 0.0045Aa

0.4880± 0.0382Aa

0.0807± 0.0219Aa

0.0457± 0.0129Aa

0.3570± 0.0985Ba

0.9690± 0.0589Ba

0.0308± 0.0010Aa

0.0021± 0.0071Aa

50

0.1540± 0.0387Ab

0.3840± 0.0982Aa

0.0992± 0.0150Aa

0.0227± 0.0087Aa

0.5060± 0.1540Aa

0.9920± 0.3600Aa

0.0460± 0.0071Ba

0.0126± 0.0003Aa

XG-LBG 0.5070± 0.0377Aa

Day 1 XG 0.1180± 0.0032Aa

LBG 0.0486± 0.0112Aa

WPI 0.0274± 0.0118Aa

XG-LBG 0.2980± 0.0073Ba

XG 0.1280± 0.0015Aa

Day 14 LBG 0.0238± 0.0026Aa

WPI 0.0133± 0.0007Aa

5

0.3030± 0.0099Ab

0.1230± 0.0061Aa

0.0618± 0.0095Aa

0.0146± 0.0012Aa

0.3090± 0.1600Aa

0.1220± 0.0006Aa

0.0143± 0.0012Ba

0.0189± 0.0007Ba

50

0.1700± 0.0289Ac

0.1170± 0.0072Aa

0.0587± 0.0010Aa

0.0250± 0.0010Aa

0.2640± 0.0552Aa

0.1070± 0.0050Ab

0.0143± 0.0039Ba

0.0162± 0.0052Aa

pH 7 NaCl(mM) 0

ABC = different letters indicate a significant (P < 0.05) difference between day1 and day 14

for the same gum type and salt concentration. abc = different letters in columns indicate significant differences (P < 0.05) among the salt levels

of the same gum type.

Zeta Potential (mV)

Day 1

Day 14

25 20

c

15 10 5

a a

b

ab

b

a

0 -5 LBG

XG

Day 14

-20

b -40

c

a ac

c

a

b a

-60

XG-LBG

WPI

LBG

XG

WPI

5 mM NaCl

5 mM NaCl

0

25

-10

20

Zeta Potential (mV)

Zeta Potential (mV)

Day 1

-80 XG-LBG

-20

c

15

b

10

c

-30

c

-40

d

a a XG

LBG

a ab

b a

-60 -70

0 XG-LBG

c b

a ab

-50

b 5

0 mM NaCl

0

Zeta Potential (mV)

0 mM NaCl

30

XG-LBG

WPI

LBG

XG

WPI

50 mM NaCl c b

20 15

b

b

b

10

a 5

a a

0 XG-LBG

XG

50 mM NaCl

0

Zeta Potential (mV)

Zeta Potential (mV)

25 -10 -20 -30 -40

a

-50 -60

a

a

b

a b

a c

-70 LBG

Gum Type

WPI

XG-LBG

XG

LBG

WPI

Gum Type

Figure 1. Effect of NaCl addition on the ζ-potential of fish O/W emulsions stabilized by WPI-XG-LBG complexes at pH 3 (left) and pH 7 (right). A WPI-whey protein isolate, LBG-locust bean gum, XG-xanthan gum, XG-LBG- xanthan-locust bean gum. abcd = different letters indicate a significant (P < 0.05) difference between gums for the same pH and salt

concentration.

pH3

pH7

(A)

i.

ii.

iii.

iv.

(B)

i.

ii.

iii.

iv.

i.

ii.

iii.

iv.

(C)

Figure 2. Microstructure of O/W emulsions stabilized by WPI-XG-LBG complexes (A) 0 mM NaCl, (B) 5 mM NaCl, (C) 50 mM NaCl. i. Optical microscope images scale crosspends to 20 µM. XG-LBG- Xanthan-Locust Bean, ii. XG- xanthan gum, iii. LBGLocust bean gum iv. WPI-Whey protein isolate.

pH 3

pH 7

0 mM NaCl

0 mM NaCl

100 90 80 70 60 50 40 30 20 10 0

Creaming Index (%)

Creaming Index (%)

100 90 80 70 60 50 40 30 20 10 0 0

100

200

300

5 mM NaCl

100

200

300

5 mM NaCl

100 90 80 70 60 50 40 30 20 10 0

Creaming Index (%)

Creaming Index (%)

100 90 80 70 60 50 40 30 20 10 0

0

0

100

200

300

0

100

200

300

50 mM NaCl

50 mM NaCl

100 90 80 70 60 50 40 30 20 10 0

Creaming Index (%)

Creaming Index (%)

100 90 80 70 60 50 40 30 20 10 0 0

100

200

0

300

100

200

Storage time (hr)

Storage time (hr)

300

Figure 3. Effect of NaCl on the creaming index of fish O/W emulsions stabilized by WPI-XGLBG complexes at pH 3 and 7. -whey protein isolate -locust bean gum -xanthan gum -xanthan-locust bean gum.

pH 3

pH 7

0 mM NaCl

0 mM NaCl 0.2

LHP (mmol/kg)

LHP (mmol/kg)

0.2 0.15 0.1 0.05 0

0.15 0.1 0.05 0

0

2

4

6

8

0

2

4

6

8

5 mM NaCl

5 mM NaCl

0.2

LHP (mmol/kg)

LHP (mmol/kg)

0.2 0.15 0.1 0.05 0

0.15 0.1 0.05 0

0

2

4

6

8

0

4

6

8

6

8

50 mM NaCl

50 mM NaCl 0.2

LHP (mmol/kg)

0.2

LHP (mmol/kg)

2

0.15 0.1 0.05 0

0.15 0.1 0.05 0

0

2

4

Storage Time (Weeks)

6

8

0

2

4

Storage Time (Weeks)

Figure 4. Effect of NaCl on the lipid hydroperoxide (LHP) concentrations of fish O/W emulsions stabilized by WPI-XG-LBG complexes at pH 3 and 7. Lipid hydroperoxides in menhaden oil (no added WPI, XG, LBG) was 0.0391 mmol/kg oil. -whey protein isolate -locust bean gum -xanthan gum -xanthan-locust bean gum.

pH 3

pH 7

0 mM NaCl

0 mM NaCl 50

TBARS (mmol/kg)

TBARS (mmol/kg)

50 40 30 20 10 0 2

4

6

20 10

8

0

5 mM NaCl TBARS (mmol/kg)

40 30 20 10 0

2

4

6

8

6

8

6

8

5 mM NaCl

50

50

TBARS (mmol/kg)

30

0 0

40 30 20 10 0

0

2

4

6

8

0

50 mM NaCl TBARS (mmol/kg)

40 30 20 10 0

2

4

50 mM NaCl

50

50

TBARS (mmol/kg)

40

40 30 20 10 0

0

2

4

Storage Time (Weeks)

6

8

0

2

4

Storage Time (Weeks)

Figure 5. TBARS concnetrations of fish O/W emulsions stabilized by WPI-XG-LBG complexes as a function of NaCl and pH. TBARS concentrations in menhaden oil (no added WPI, XG, LBG) was 1.5633 mmol/kg oil -whey protein isolate -locust bean gum -xanthan gum -xanthan-locust bean gum.

Highlights 1. The effect of NaCl on the stability of O/W emulsions stabilized by WPI-XG-LBG layers was investigated. 2. XG-LBG had the highest creaming stability at 0mM and 5mM NaCl at pH 7; while XG had the highest at 50mM NaCl. 3. XG-LBG emulsions had the highest oxidative stability at every NaCl concentration at pH 7.

Author Contributions Section Kristen Griffin carried out the experiments and took the lead in writing the manuscript. Hanna Khouryieh conceived and planned the experiments, supervised the project, conducted the statistical analysis, contributed to the interpretation of the results and final manuscript.

Conflict of Interest and Authorship Conformation Form Please check the following as appropriate: o
All authors have participated in (a) conception and design, or analysis and interpretation of the data; (b) drafting the article or revising it critically for important intellectual content; and (c) approval of the final version. o


This manuscript has not been submitted to, nor is under review at, another journal or other publishing venue.

o


The authors have no affiliation with any organization with a direct or indirect financial interest in the subject matter discussed in the manuscript

o

The following authors have affiliations with organizations with direct or indirect financial interest in the subject matter discussed in the manuscript:

Author’s name

Affiliation