Physical and Oxidative Stability of Flaxseed Oil-in-Water Emulsions

May 23, 2017 - This study evaluated the impact of sunflower phospholipid type on the formation and stability of flaxseed oil-in-water emulsions. Two s...
0 downloads 0 Views 2MB Size
Article

Physical and oxidative stability of flaxseed oil-in-water emulsions fabricated from sunflower lecithins: Impact of blending lecithins with different phospholipid profiles Li Liang, Fang Chen, Xing-Guo Wang, Qingzhe Jin, Eric A Decker, and David Julian McClements J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 23 May 2017 Downloaded from http://pubs.acs.org on May 26, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 38

Journal of Agricultural and Food Chemistry

Physical and oxidative stability of flaxseed oil-in-water emulsions fabricated from sunflower lecithins: Impact of blending lecithins with different phospholipid profiles Li Liang a,b, Fang Chen b,c, Xingguo Wang a, Qingzhe Jin a, *, Eric Andrew Decker b

a

, David Julian McClements b, *

State Key Laboratory of Food Science and Technology, Synergetic Innovation Center of Food

Safety and Nutrition, School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122 China b

Department of Food Science, University of Massachusetts Amherst, Amherst, MA 01003, USA

c

School of Public Health, Nanchang University, Nanchang, Jiangxi 330006, China

* Corresponding authors E-mail addresses: [email protected] (Q. Jin), [email protected] (D.J. McClements).

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

1

ABSTRACT

2

There is great interest in the formulation of plant-based foods enriched with

3

nutrients that promote health, such as polyunsaturated fatty acids. This study evaluated

4

the impact of sunflower phospholipid type on the formation and stability of flaxseed

5

oil-in-water emulsions. Two sunflower lecithins (Sunlipon 50 and 90) with different

6

phosphatidylcholine (PC) levels (59 and 90%, respectively) were used in varying ratios

7

to form emulsions. Emulsion droplet size, charge, appearance, microstructure, and

8

oxidation were measured during storage at 55 °C in the dark. The physical and chemical

9

stability increased as the PC content of the lecithin blends decreased. The oxidative

10

stability of emulsions formulated using Sunlipon 50 was better than emulsions

11

formulated using synthetic surfactants (SDS or Tween 20). The results are interpreted

12

in terms of the impact of emulsifier type on the colloidal interactions between oil

13

droplets, and the molecular interactions between pro-oxidants and oil droplet surfaces.

14 15

Keywords: sunflower phospholipids; flaxseed oil; emulsion; physical stability;

16

oxidative stability; delivery

17 18 19 20 21 22 23 24

ACS Paragon Plus Environment

Page 2 of 38

Page 3 of 38

Journal of Agricultural and Food Chemistry

25

INTRODUCTION

26

Epidemiological and animal studies suggest that consuming a sufficiently high

27

level of w-3 polyunsaturated fatty acids (ω-3 PUFAs) may prevent the development and

28

progression of a number of chronic diseases. As a result, consumers in many developed

29

countries are being advised to increase the level of ω-3 PUFAs consumed. Flaxseed oil,

30

which principally contains α-linolenic acid (ALA), and fish oil, which principally

31

contains eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are two major

32

sources of long-chain ω-3 PUFAs in the human diet. There is strong scientific evidence

33

from human trials that ω-3 PUFAs from fish or fish oil supplements have a beneficial

34

cardio-protective effect 1, 2. However, the widespread consumption of fish oil sources is

35

often limited because of their fishy taste, smell, toxin content, allergies, high cost, and

36

tendency to cause eructation (“burping”). Moreover, ω-3 PUFAs obtained from fish are

37

unsuitable for consumption by vegetarians, vegans and certain religious groups. ALA is

38

a natural precursor of EPA and DHA that may be partially converted into these forms

39

within the human body after ingestion 3-5. Consequently, flaxseed oil, which is rich in

40

ALA, can be used as a natural source of plant-based ω-3 PUFAs in functional foods and

41

supplements 6. Clinical and animal experiments suggest that flaxseed oil has potential

42

health benefits, such as reduction in cardiovascular, atherosclerosis, diabetes, cancer,

43

arthritis, osteoporosis, autoimmune and neurological disorders 6-9.

44

The numerous potential health benefits of ω-3 PUFAs, combined with the current

45

low levels of consumption 1, 2, have meant that increasing numbers of functional foods

46

fortified with ω-3 PUFA are being developed in both the United States and

47

Europe 10. Nevertheless, the successful development of functional foods enriched with

48

ω-3 PUFAs is challenging due to their high susceptibility to lipid oxidation 11. For this

49

reason, encapsulation technologies, such as those based on emulsions, are being

50

developed so that they can be successfully incorporated into foods 12, 13. The success of

51

this approach is highly dependent on the food system involved, and each encapsulation

52

system must be carefully designed for a particular food product. Previous research has

53

ACS Paragon shown that encapsulation systems can be Plus usedEnvironment to improve the oxidative stability of ω-

Journal of Agricultural and Food Chemistry

Page 4 of 38

54

3 PUFAs in milk 14, yogurt 15, cheese 16, mayonnaise 17, meat products 18, and energy

55

bars 19.

56

In emulsions, the interface between the oil and the aqueous phase is the place of

57

contact between lipids and hydrophilic pro-oxidative components (such as transition

58

metal ions, photosensitizers, and enzymes), and therefore it plays a major role in lipid

59

oxidation in emulsions 20, 21. For ω-3 PUFAs delivery systems, it is therefore important

60

to select an emulsifier that produces an interfacial layer around the oil droplets that

61

provides good physical and chemical stability 21, 22. As consumers become more label

62

conscious, there is a movement away from synthetic ingredients toward more natural

63

ingredients 23. Many kinds of natural emulsifiers are available for utilization in foods,

64

including proteins, polysaccharides, and phospholipids 24, 25. Phospholipids act as good

65

emulsifiers because they contain a lipophilic part (two fatty acid groups) and

66

a hydrophilic part (phosphoric based esters) on the same molecule. Consequently, they

67

can adsorb to oil droplet surfaces, reduce the interfacial tension (thereby facilitating

68

emulsion formation), and generate repulsive interactions (thereby enhancing emulsion

69

stability). For phospholipids, electrostatic repulsion is usually the most important

70

repulsive interaction opposing droplet flocculation, but steric repulsion plays an

71

important role in inhibiting droplet coalescence 25. Phospholipids from various sources

72

have been shown to have good antioxidant properties, such as those derived from milk

73

26

74

properties of phospholipids have mainly been attributed to their ability to chelate

75

transition metal ions, scavenge free radicals, or act synergistically with tocopherols 31,

76

32

77

natural emulsifiers for the formulation of delivery systems for ω-3 PUFAs.

, egg yolk

27

, marine species

28

, soybean

29

and sunflower

30

. The antioxidant

. These physical and chemical attributes make phospholipids particularly promising

78

The main objective of the current study is to investigate the possibility of forming

79

both physically and chemically stable flaxseed oil-in-water emulsions using sunflower

80

phospholipids. In our previous research, we showed that emulsions could be prepared

81

from sunflower lecithins using both low-energy and high-energy homogenization

82

methods

83

phosphatidylcholine (PC) contents was investigated for their ability to form and

33, 34

. In these studies, a series of sunflower lecithins with different ACS Paragon Plus Environment

Page 5 of 38

Journal of Agricultural and Food Chemistry

84

stabilize emulsions, and it was shown that they behaved differently depending on their

85

phospholipid composition. In the current study, we investigated the influence of using

86

a blend of two sunflower lecithins with different PC contents on the physical and

87

chemical stability of flaxseed oil-in-water emulsions. Additionally, emulsions were

88

prepared using two other surfactants, Tween 20 (non-ionic) and SDS (anionic), so that

89

direct comparisons could be drawn between the performance of natural zwitterionic

90

phospholipids and synthetic nonionic or anionic surfactants.

91

MATERIALS AND METHODS

92

Materials. Flaxseed oil was purchased from a local grocery store and used

93

without further purification (AAK Ltd., England, UK). Two phospholipid ingredients

94

derived from sunflower oil were kindly donated by Perimondo (New York, NY, USA).

95

Polyoxyethylene-20-sorbitan monolaurate (Tween 20), sodium dodecyl sulfate (SDS)

96

and Nile Red were purchased from the Sigma-Aldrich Co. (St. Louis, MO). All other

97

reagents were of analytical or chromatographic grade. Double-distilled water (Milli-Q)

98

was used for the preparation of all solutions.

99

Characterization of the Phospholipids.

Phospholipid, fatty acid

100

compositions, peroxide value and tocopherol content were provided by the

101

manufacturer (Table 1), and metal ion content were determined according to methods

102

reported previously 35.

103

Free Radical Scavenging Assays. The free radical scavenging ability of

104

the phospholipids was determined using 2,2-diphenyl-1-picrylhydrazyl (DPPH) as

105

described previously with some modifications 36. Eight percent (w/v) solutions of the

106

phospholipids in methanol were prepared and then diluted to obtain a range of

107

phospholipid concentrations. Then, 0.1 mL of each phospholipid solution was added to

108

3.9 mL of methanolic DPPH solution (60 µM). The loss of DPPH was then determined

109

by measuring the reduction in the absorption of light at 515 nm using a UV-visible

110

spectrophotometer (Ultraspec 3000 pro, Biochrom Ltd., Cambridge, UK) every 15 min ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

111

until the reaction reached completion. For the blank, 0.1 mL of methanol was used

112

instead of the sample. The percentage of remaining DPPH was calculated according to

113

a method described previously 37. The concentration of the test compound needed to

114

decrease the DPPH concentration by 50% was calculated and expressed as the EC50.

115

Emulsion Preparation. Oil-in-water emulsions were prepared by

116

homogenizing 10 wt% oil phase (flaxseed oil) with 90 wt% aqueous phase. The

117

aqueous phase consisted of emulsifier (2 wt% Sunlipon 50, 2 wt% Sunlipon 90, 1 wt%

118

Tween 20, or 1wt% SDS) and phosphate buffer solution (5 mM, pH 7.0). A lower level

119

of the synthetic surfactants was utilized than the phospholipids because they are more

120

effective emulsifiers. Sodium azide (0.02% w/w) was added as an antimicrobial

121

preservative. Aqueous phases with different mass ratios of Sunlipon 50 and 90 were

122

prepared (0 to 100%), while keeping the total emulsifier concentrated fixed at 2 wt%.

123

Aqueous phases containing the lecithin ingredients had to be sonicated to evenly

124

disperse the phospholipids prior to making the emulsions. The sonication conditions

125

used were 20 cycles at an amplitude of 70% and pulse length of 5 s on, followed by 3 s

126

off. The emulsifier solutions were incubated in an ice bath during sonication to prevent

127

large temperature increases. Emulsions were prepared by blending the oil and aqueous

128

phases together using a high-speed blender for 2 min (M133/1281-0, Biospec Products,

129

Inc., ESGC, Switzerland), and then passing them through a high-pressure homogenizer

130

(M110Y, Microfluidics, Newton, MA) with a 75 µm interaction chamber (F20Y) at a

131

pressure of 12,000 psi three times.

132

Storage Experiment. Immediately after preparation, emulsions samples (2

133

mL) were placed in glass tubes and then sealed with plastic caps and parafilm. The test

134

tubes were then stored at 55 oC in the dark to determine their long-term physical and

135

chemical stability. Periodically, samples were analyzed for particle size, ζ-potential,

136

microstructure, and lipid oxidation over a 28-day period. Prior to particle size analysis,

137

the test tubes were vortexed for 30 seconds to ensure the samples were homogeneous.

138

Separate test tubes containing 10 mL of samples were prepared to observe changes in

139

their visual appearance throughout storagePlus (without vortexing). ACS Paragon Environment

Page 6 of 38

Page 7 of 38

Journal of Agricultural and Food Chemistry

140

Droplet Size and Charge. The particle size distribution of the emulsions

141

was determined using a static light scattering instrument (Mastersizer 2000, Malvern

142

Instruments Ltd., Malvern, Worcestershire, UK). Samples were diluted in aqueous

143

buffer solutions to avoid multiple scattering effects, and then stirred (1200 rpm) to

144

ensure homogeneity. The refractive indices of phosphate buffer solution and flaxseed

145

oil were taken to be 1.330 and 1.474, respectively. The droplet diameter of each sample

146

was represented as the surface-weighted mean diameter (d32), which was calculated

147

from the full particle size distribution.

148

The electrical surface potential (ζ-potential) of the particles in these samples was

149

measured using a particle electrophoresis instrument based on light scattering (Nano-

150

ZS, Malvern Instruments Ltd.). Samples were diluted with buffer solution (5 mM, pH

151

7.0) prior to measurements to avoid multiple scattering effects.

152

Microstructure Analysis. A confocal scanning laser microscope with a 60

153

× objective lens (oil immersion) and 10× eyepiece (Nikon D-Eclipse C1 80i, Nikon,

154

Melville, NY, US.) was used to determine the microstructure of the emulsions. Prior to

155

analysis, the samples were dyed with a 1 mg Nile red/mL ethanol solution to highlight

156

the location of the oil phase. The excitation and emission spectra for Nile red were 543

157

nm and 605 nm, respectively. A small aliquot of emulsions was placed on a microscope

158

slide and covered with a cover slip prior to visualization. All microstructure images for

159

confocal microscopy were taken and processed using image analysis software (EZ-CS1

160

version 3.8, Nikon).

161

Lipid Oxidation Studies. Each emulsion (2 mL) was placed in a 10-mL glass

162

test tube and then sealed to ensure that it was airtight. Sample vials were then incubated

163

at 55 oC in the dark. Lipid oxidation was monitored by measuring the formation of

164

hydroperoxides and thiobarbituric acid reactive substances (TBARS) during storage.

165

Lipid hydroperoxides were determined according to a method described previously 38.

166

Emulsions (0.3 mL) were mixed with 1.5 mL of isooctane/2-propanol solution (3:1 v/v)

167

and vortexed (10 s, 3 times). The mixed solution was then centrifuged at 3,400 g for 10

168

min (Centrific centrifuge, Thermo Fisher Scientific Inc., Fairlawn, NJ, USA). The upper

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 8 of 38

169

organic layer (200 µL) was mixed with 2.8 mL of methanol/butanol solution (2:1, v/v),

170

followed by the addition of 15 µL of 3.94 M ammonium thiocyanate and 15 µL of Fe2+

171

solution. The Fe2+ solution was prepared freshly from the supernatant of a mixture of

172

equal amounts of 0.132 M BaCl2 in 0.4 M HCl and 0.144 M FeSO4. The solution was

173

vortexed and then held for 20 min at room temperature, and the absorbance was

174

measured at 510 nm in a UV−visible spectrophotometer (Genesys 20, Thermo Fisher

175

Scientific Inc., Waltham, MA, USA). Hydroperoxide concentrations were determined

176

using a standard curve prepared using cumene hydroperoxide.

177

TBARS were measured according to a method described previously

39

. Briefly,

178

1.0 mL of emulsion was combined with 2.0 mL of TBA (thiobarbituric acid) solution

179

(prepared by mixing 15 g of trichloroacetic acid, 0.375 g of TBA, 1.76 mL of 12N HCl,

180

and 82.9 mL of H2O) in test tubes, and then placed in a boiling water bath for 15 min.

181

The tubes were then cooled to room temperature for 10 min and then centrifuged (2000

182

rpm) for 15 min. The absorbance of the samples was measured at 532 nm, and the

183

concentration of TBARS formed were calculated from a standard curve prepared using

184

1,1,3,3-tetraethoxypropane.

185 186

The initial oxidation rate was determined from the slope of plots of hydroperoxides and TBARS versus time during storage, using only the data for the first three days.

187

Statistical Analysis. All analysis was performed on two samples and repeated

188

at least twice per sample. Results are reported as means and standard deviations of these

189

measurements. The means among treatments were analyzed by a one-way ANOVA

190

followed by post-hoc Duncan test for the initial oxidation rate, and the paired-samples

191

t-test was used for evaluation of the change in particle diameter and ζ-potential before

192

and after storage. The means values of the EC 50 of the two lecithin ingredients were

193

compared by Independent-Samples t Test. Statistical significance was set as p < 0.05

194

(SPSS, IBM Corporation, Armonk, NY, USA).

195

RESULTS AND DISCUSSION

196

In our previous study, ACS it wasParagon shown Plus that both Sunlipon 50 and Sunlipon 90 could Environment

Page 9 of 38

Journal of Agricultural and Food Chemistry

197

form emulsions containing small droplets, but that the electrical charge on the droplets

198

was appreciably different 33. At neutral pH, the ζ-potential of the oil droplets was highly

199

negative when they were coated by Sunlipon 50, but close to zero when they were

200

coated by Sunlipon 90. We hypothesized that differences in the interfacial properties

201

of the oil droplets would lead to differences in their functional performances. For the

202

sake of comparison, we also prepared emulsions stabilized by two synthetic small

203

molecule surfactants: SDS (anionic) and Tween 20 (non-ionic).

204

Characteristics of Sunflower Phospholipids. The nature of the

205

surfactants used to coat the droplets in oil-in-water emulsions is known to have an

206

appreciable impact on their physical and oxidative stabilities

207

report the phospholipid, fatty acid, and peroxide contents of both the sunflower lecithins

208

used as emulsifiers in this study (Table 1). The major phospholipids in sunflower oil

209

are phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol

210

(PI), and phosphatidic acid (PA)

211

phospholipids used in this study are summarized in Fig. 1. The nature of the polar head

212

group on phospholipids is known to impact their ability to form and stabilize emulsions

213

41, 42

214

of PC they contained. Sunlipon 50 contain around 58% PC, 5% PE and minor amounts

215

of other phospholipids, while Sunlipon 90 contained around 90% PC.

40

20

. For this reason, we

. The molecular structure of the different

. The two sunflower lecithins used in our study mainly differed in the percentage

216

Palmitic acid (16:0), stearic acid (18:0), oleic acid (18:1) and linoleic acid (18:2)

217

were the major fatty acids in both Sunlipon 50 and 90 (Table 1), which is consistent

218

with the values reported in the literature 43-45. The major fatty acids in Sunlipon 50 were

219

similar to those in Sunlipon 90, with linoleic acid being the most abundant fatty acid,

220

accounting for more than half of the total. Oleic acid was the second most abundant

221

fatty acid, followed by palmitic and stearic acids. The fatty acid chain length of

222

surfactants influences the hydrophilic–lipophilic balance (HLB), which influences their

223

emulsifying ability

224

phospholipids was calculated using the following equation:

225

Swi ´ ni L ACS = Paragon Plus Environment w

46

. The average fatty acid chain length of the two sunflower

Journal of Agricultural and Food Chemistry

226

Page 10 of 38

(1)

227

Here, ni is the number of carbon atoms in the fatty acid chain, w is the total weight

228

of the fatty acids, and wi is the weight fraction of the individual fatty acids. According

229

to this equation, the average fatty acid chain length of the Sunlipon 50 and 90 were

230

fairly similar, being 17.7 and 17.8 carbons, respectively.

231

The rate of lipid oxidation in emulsions is known to be strongly influenced by the

232

presence of any pro-oxidant components, such as transition metals (iron and copper)

233

and peroxides, which may be contaminants in commercial emulsifiers

234

reason, the levels of these pro-oxidants in the two sunflower lecithins used in this study

235

were compared (Table 1). The two lecithins had similar peroxide values, and therefore

236

the effect of peroxides on lipid oxidation would be expected to be fairly similar. The

237

concentrations of iron and copper were higher in Sunlipon 50 than in Sunlipon 90. In

238

particular, the copper content of Sunlipon 50 was about four times higher than that of

239

Sunlipon 90, which may impact lipid oxidation, since transition metals are known to be

240

highly effective pro-oxidants in emulsions.

20

. For this

241

The total tocopherol content of the two lecithin products was also compared

242

because studies have shown that tocopherol radicals may be regenerated in the presence

243

of phospholipids

244

reaction products 50. The tocopherol content of Sunlipon 90 was about 2.5 times higher

245

than that of Sunlipon 50. Consequently, it is possible that differences in tocopherol

246

levels could impact oxidative stability. It should be noted that the commercial lecithin

247

ingredients used in this study contained a significant fraction of unknown minor

248

constituents (Table 1), which could also have impacted their ability to alter the physical

249

and chemical stability of emulsions. The ingredient manufacturer suggested that these

250

components were probably glycolipids, which can act as effective emulsifiers.

251

47-49

and that tocopherols may promote the formation of Maillard

Antiradical Activity of Phospholipids. The free radical scavenging capacity of a 51, 52

252

food component is an important indication of its antioxidant properties

253

decrease in DPPH concentration was therefore measured over time after adding

254

different levels of the two sunflower lecithins to the test system (Fig. 2). The DPPH

255

levels decreased rapidly during the first few minutes, but then decreased more slowly

ACS Paragon Plus Environment

. The

Page 11 of 38

Journal of Agricultural and Food Chemistry

256

at longer times until they attained a fairly constant value. The magnitude of the decrease

257

in DPPH levels increased with increasing lecithin concentration for both Sunlipon 50

258

and 90. However, Sunlipon 50 clearly had a higher radical scavenging capacity than

259

Sunlipon 90. Indeed, the EC50 values for Sunlipon 50 and 90 were calculated from the

260

data shown in Fig. 2 as 1.69 ± 0.24 and 89.6 ± 1.5 mg/mL, respectively, which were

261

significantly different (p < 0.05). In principle, the greater antioxidant activity of the

262

Sunlipon 50 may have been due to a number of factors, such as differences in tocopherol

263

levels or phospholipid head-group type 41, 42. The tocopherol levels of the Sunlipon 50

264

were actually lower than those of the Sunlipon 90 (Table 1), which suggests that some

265

other factor must have been important. It is possible that there were other components

266

within the commercial sunflower lecithin ingredients that could act as pro- or anti-

267

oxidants, but more detailed analysis of the composition of the ingredients would be

268

required to establish this.

269

Physicochemical Properties and Stability of Emulsions. In the

270

absence of oil, a sediment was observed at the bottom of the test tubes when both

271

sunflower lecithins were dispersed in aqueous buffer solutions, particularly for the

272

solutions containing Sunlipon 90 (data not shown). This result may be due to the fact

273

that phospholipids form dense colloidal structures in aqueous solutions, such as vesicles

274

and liquid crystals, that sediment due to gravitational forces 53. Previously, it has been

275

reported that the ζ-potential of the colloidal particles in aqueous solutions containing

276

Sunlipon 90 and Sunlipon 50 were about +1.7 ± 0.3 mV and -24.0 ± 0.3 mV,

277

respectively, which was attributed to differences in the charge characteristics of the

278

phospholipid head groups

279

phospholipids, such as PA, PI, phosphatidylglycerol (PG) and acyl-PE, than Sunlipon

280

90 (Fig. 1). Consequently, the greater amount of sediment observed in the Sunlipon 90

281

solutions may have been because there was a relatively weak electrostatic repulsion

282

between the colloidal structures in this system, which led to aggregation and

283

sedimentation. The presence of these colloidal structures prompted us to sonicate the

284

aqueous phases prior to mixing them with the oil phases so as to evenly distribute the

33

. Sunlipon 50 contains more negatively charged

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

285

lecithin and to avoid blocking the narrow channels in the microfluidizer used to

286

fabricate the emulsions.

287

In a previous study, we investigated the possibility of fabricating nanoemulsions

288

using sunflower phospholipids 33. The results of this study indicated that phospholipid

289

type had a pronounced influence on the electrical characteristics of the droplets.

290

Droplets made with Sunlipon 50 had a high negative charge, whereas those made with

291

Sunlipon 90 had a small positive charge. It is well known that the electrical charge on

292

oil droplets may influence the physicochemical stability and functional performance of

293

emulsions

294

electrostatic repulsion between oil droplets, which can inhibit droplet aggregation. On

295

the other hand, a strong negative surface potential can promote lipid oxidation by

296

attracting cationic transition metals to the droplet surfaces 55. For this reason, the impact

297

of using varying ratios of Sunlipon 50 and 90 (0 to 100 wt%) at a constant overall

298

emulsifier level (2 wt%) was examined. It was postulated that it may be possible to

299

form emulsions with improved physical and chemical stability by using combinations

300

of lecithins, rather than individual lecithins. Two synthetic surfactants (nonionic Tween

301

20 and anionic SDS) were used as controls to highlight the potential advantages or

302

disadvantages of using natural surfactants (sunflower lecithins) for this purpose.

54

. A strongly positive or negative surface potential leads to a strong

303

Particle size and electrical charge. The ζ-potential of the initial emulsions

304

stabilized with sunflower lecithins became more highly negatively charged as the

305

Sunlipon 50 concentration increased (Fig. 3a), which can be attributed to differences in

306

the phospholipid compositions of the two ingredients (Table 1). PC and PE both have

307

no net charge at neutral pH, whereas PA, PI, PG and acyl-PE are negatively charged 56.

308

Consequently, it is possible that the negative charge on the oil droplets arises from the

309

presence of these anionic phospholipids. There is a higher level of these anionic

310

phospholipids in the Sunlipon 50 (Table 1), which may account for the higher negative

311

charge on the droplets in the emulsions formed using this type of sunflower lecithin.

312

Interestingly, the droplets coated by Sunlipon 90 had a reasonably high negative charge

313

in this study (-26.3 mV), whereas they had a slight positive charge (+1.7 mV) in our

314

previous study

33

ACS Paragon Plus Environment

. This phenomenon may be due to differences in oil type and

Page 12 of 38

Page 13 of 38

Journal of Agricultural and Food Chemistry

315

concentration, emulsifier levels, and aqueous phase composition in the two studies. For

316

example, flaxseed oil was used in the current study, whereas a fish oil was used in the

317

previous study. As expected, the droplets coated with SDS had the highest negative

318

surface potential (-89.3 mV), which can be attributed to the presence of the sulfate (-

319

SO4-) head group on this anionic surfactant 30. The droplets coated with Tween 20 also

320

had a relatively high negative surface potential (-39.1 mV), despite the fact that it is

321

supposed to be a non-ionic surfactant. This phenomenon has also been reported by

322

other researchers 57, 58, and has been attributed to anionic impurities in the surfactants

323

or oils used (such as fatty acids) or the preferential adsorption of hydroxyl ions from

324

water

325

negative after the emulsions were stored for 28-days (Fig. 3a), which suggests that there

326

was some change in interfacial composition during storage. This effect could have

327

occurred due to chemical degradation of the oils or surfactants during storage leading

328

to the generation of anionic reaction species, such as short chain organic acids. The

329

chemical degradation of either the surfactant during storage would be undesirable for

330

commercial applications due to the generation of off-flavors, or the loss of ingredient

331

functionality. In future studies, it would therefore be interesting to monitor the change

332

in the chemical structure of the surfactants over time in more detail.

59

. With the exception of the SDS system, the surface potential became more

333

The mean particle diameter of the emulsions decreased appreciably as the

334

proportion of Sunlipon 50 in the emulsifier phase (Sunlipon 50 and 90) increased from

335

0 to 25%, e.g., d32 was 1940 nm and 246 nm at 0% and 25% Sunlipon 50, respectively

336

(Fig. 3b). The most appreciable decrease in droplet diameter occurred from 0 to 25%

337

Sunlipon 50, with no significant change occurring at higher levels. This result shows

338

that relatively small oil droplets can be formed with a range of different interfacial

339

compositions by using the mixed sunflower lecithin ingredients. Nevertheless, the

340

mean droplet diameter produced using the sunflower lecithin was appreciably higher

341

than that obtained using SDS (129 nm). This effect can probably be attributed to the

342

fact that the SDS facilitates droplet disruption and inhibits droplet coalescence more

343

effectively than the phospholipids.

344

ACS Paragon Plus Environment

There were appreciable changes in the mean particle diameter and particle size

Journal of Agricultural and Food Chemistry

345

distribution of some of the emulsions after 28-days storage (Fig. 3), which suggested

346

that they were unstable to droplet aggregation. In particular, the size of the particles in

347

emulsions containing low levels of Sunlipon 50 (0, 25 or 50%) increased appreciably

348

after storage (Figs. 3c and 3d). This effect may have been due to the relatively low z-

349

potential on these droplets leading to droplet aggregation because of the relatively weak

350

electrostatic repulsion between the droplets. Conversely, the size of the particles in the

351

emulsions containing high levels of Sunlipon 50 (75 and 100%) did not change

352

appreciably after storage (Figs. 3c and 3d). Presumably, the strong electrostatic

353

repulsion in these systems prevented the oil droplets from coming into close proximity

354

60

355

emulsions containing Tween 20 or SDS (Fig. 3). The Tween 20 emulsion was highly

356

unstable to droplet aggregation, whereas the SDS emulsion was highly stable.

357

Emulsions stabilized by Tweens are known to be unstable to aggregation when stored

358

at elevated temperatures because dehydration of their hydrophilic head groups leads to

359

a change in the optimum curvature of the surfactant monolayer and a reduction in the

360

steric repulsion

361

increase as the temperature approaches the phase inversion temperature (PIT) of the

362

surfactant-oil-water system used.

. There was also an appreciable difference between the storage stabilities of the

61

. The rate of droplet coalescence due to this mechanism tends to

363

Visual Appearance and Microstructure. The visual appearance and microstructure

364

of the emulsions was recorded initially and after 28-days storage (Fig. 4a). Initially, a

365

thin cream layer was observed on top of some of the sunflower lecithin stabilized

366

emulsions, with the thickness of the cream layer decreasing with increasing Sunlipon

367

50 ratio. This effect can be attributed to the relatively large initial droplet sizes of the

368

emulsions containing high levels of Sunlipon 90 (Fig. 3). No creaming was observed

369

in the initial emulsions stabilized by either Tween 20 or SDS, which is due to the

370

relatively small size of the droplets they contained. After 28-days storage, an oil layer

371

was visible on top of the emulsions stabilized by sunflower lecithins when they

372

contained less than 50% Sunlipon 50, which suggested that droplet coalescence and

373

oiling-off had occurred within these systems. At higher Sunlipon 50 levels, only a

374

cream layer was observed, which suggested that some droplet creaming had occurred,

ACS Paragon Plus Environment

Page 14 of 38

Page 15 of 38

Journal of Agricultural and Food Chemistry

375

but that the droplets were relatively stable to oiling-off. Creaming may have occurred

376

in these emulsions because some of the oil droplets were relatively large 62. In contrast,

377

the emulsions stabilized with Tween 20 or SDS retained a whitish color throughout

378

storage, although a thin layer of oil was observed on top of the emulsions after 28-days

379

storage suggesting that a limited amount of coalescence and oiling-off had occurred.

380

Surprisingly, the visual appearance of the emulsions after 28-days storage depended on

381

the amount of emulsion placed in the test tubes (Fig. 4). The emulsions appeared to

382

undergo more phase separation when a smaller volume was placed in the test tube,

383

particularly for the Tween 20 system. This surprising result may have been due to

384

differences in the amount of oxygen that diffused into the emulsions, which impacted

385

their physicochemical stability 20. The volume ratio of headspace oxygen to emulsion

386

volume was calculated to be 0.32 and 1.18 for the large and small test tubes,

387

respectively. One would therefore expect more oxygen to diffuse into the emulsions in

388

the smaller test tubes, and therefore they would be more susceptible to this effect. This

389

interesting phenomenon certainly deserves more attention in future studies.

390

Emulsion microstructures were determined by confocal laser scanning microscopy

391

before and after they were stored at 55 °C for 28-days (Fig. 4b). Initially, all of the

392

emulsions contained relatively small oil droplets (stained red) dispersed throughout the

393

aqueous phase (black). After 28-days storage, there were appreciable changes in the

394

microstructure of the emulsions, which depended strongly on the ratio of Sunlipon 50

395

to 90 in the systems. For the emulsions containing 0 or 25% Sunlipon 50, phase

396

inversion had occurred, with evidence of some water droplets (black) dispersed in an

397

oily continuous phase (stained red). For the emulsions containing 50% and 75%

398

Sunlipon 50, the system was still an oil-in-water emulsion but the oil droplets were

399

larger than the initial ones, indicating that some coalescence had occurred. There were

400

no appreciable changes in the microstructures of the emulsions containing only

401

Sunlipon 50 or SDS, which suggested that these systems were relatively stable to

402

droplet coalescence during storage. On the other hand, marked phase separation was

403

observed in the emulsions stabilized with Tween 20, suggesting they were highly

404

unstable to droplet coalescence and oiling-off. As mentioned earlier, this effect can be

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

405

attributed to the dehydration of the surfactant head-groups that occurs when the

406

temperature is increased towards the PIT of the system 61.

407

The fact that the water-in-oil emulsions and very large oil particles observed by

408

confocal microscopy in many of the samples (Fig. 4b) were not observed by static light

409

scattering (Fig. 3) may be due to differences in the sampling procedures used for these

410

two analytical techniques. A highly diluted and stirred emulsion was analyzed for the

411

light scattering measurements, but a non-diluted emulsion was directly placed on the

412

microscope slide for the microscopy measurements. Consequently, some of the free oil

413

or large oil droplets in the emulsions may have been broken down during the sample

414

preparation procedure used for the light scattering measurements, and some of the very

415

large droplets may have creamed to the top of the sample within the instrument and

416

therefore not been analyzed

417

light scattering measurements and microscopy measurements for this type of complex

418

colloidal system.

63

. These results highlight the importance of combining

419

Oxidative Stability

420

Influence of single emulsifier type. Initially, we examined the influence of the

421

different types of individual emulsifier on lipid oxidation. Flaxseed oil emulsions were

422

prepared using the four different emulsifiers studied: Sunlipon 50 (2%), Sunlipon 90

423

(2%), Tween 20 (1%), and SDS (1%). The formation of lipid oxidation primary

424

products (hydroperoxides) and secondary products (TBARS) was then monitored

425

during storage at 55 oC. In all samples, there was an increase in the concentration of

426

hydroperoxides and TBARS detected during storage (Figs. 5a, b). The relative impact

427

of emulsifier type on the lipid oxidation rate was compared by plotting the initial

428

oxidation rate (linear slope of the curve) based on the first 3-days of storage (R2 > 0.96)

429

(Fig. 5c). The rate of lipid oxidation clearly depended on emulsifier type, decreasing in

430

the following order: Sunlipon 90 > Tween 20≈SDS > Sunlipon 50 (for

431

hydroperoxides) and Sunlipon 90 > Tween 20>SDS > Sunlipon 50 (for TBARS)

432

(p