Emulsification properties of garlic aqueous extract

Accepted Manuscript Emulsification Properties of Garlic Aqueous Extract

Ángela Bravo-Núñez, Matt Golding, Tony K. McGhie, Manuel Gómez, Lara MatíaMerino PII:

S0268-005X(18)32347-6

DOI:

10.1016/j.foodhyd.2019.02.029

Reference:

FOOHYD 4955

To appear in:

Food Hydrocolloids

Received Date:

30 November 2018

Accepted Date:

13 February 2019

Please cite this article as: Ángela Bravo-Núñez, Matt Golding, Tony K. McGhie, Manuel Gómez, Lara Matía-Merino, Emulsification Properties of Garlic Aqueous Extract, Food Hydrocolloids (2019), doi: 10.1016/j.foodhyd.2019.02.029

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ACCEPTED MANUSCRIPT 1

Emulsification Properties of Garlic Aqueous Extract

2

Ángela Bravo-Núñezb, Matt Goldinga, Tony K. McGhiec, Manuel Gómezb and Lara

3

Matía-Merinoa*

4

a School of Food and Advanced Technology. Massey University, Private Bag 11222,

5

Palmerston North, New Zealand.

6

b Food Technology Area. College of Agricultural Engineering. University of Valladolid,

7

34071 Palencia, Spain.

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c The New Zealand Institute for Plant & Food Research Limited (PFR), Private Bag

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11600, Palmerston North 4442, New Zealand

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* Corresponding author: Lara Matia-Merino

11 12 13 14 15 16 17 18 19 20 21

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Abstract

23

The aim of this study was to evaluate the emulsification properties of garlic water-soluble

24

compounds, currently non-existing in the literature. Compositional analysis along with surface

25

tension measurements were carried out on the garlic aqueous extract, followed by the evaluation

26

of 10% oil-in-water emulsions made with this extract at various concentrations. Microstructural

27

analysis—droplet size by static light scattering, and light, confocal and transmission electron

28

microscopy—along with phase separation measurements of the emulsions under storage, were

29

also investigated. Whereas surface tension decreased with increasing garlic extract concentration,

30

the lowest concentration (0.48% wt/wt) produced the smallest oil droplet size (d32=0.36 µm)

31

increasing up to d32=6.55µm at the highest extract content (6.55% wt/wt). Microstructural

32

analysis also indicated the occurrence of depletion and bridging flocculation phenomena with

33

increasing garlic extract concentrations, apart from obtaining bigger initial droplet sizes.

34

Compositional analysis revealed that the ratio of saponins:fructans:proteins could be playing a

35

key role in the droplet size distributions obtained, as confocal scanning laser microscopy and

36

transmission electron microscopy also indicated differences at the interface when changing

37

concentrations. Creaming was observed over time in all the emulsions but it was more evident

38

with increasing garlic concentration. Beside flocculation, coalescence also took place after one-

39

day storage at low and high concentrations. The underpinning reason of these phenomena can be

40

related to the coexistence of compounds of different surface-active nature (i.e. a combination of

41

saponins, proteins/peptides and fructans), and their interactions. This research brings valuable

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insights on the study of novel natural food emulsifiers from plant sources.

43 44 45

Keywords: Emulsions; Garlic Proteins; Garlic Saponins; Garlic Fructans; Surface activity

46

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1. Introduction

48

Garlic (Allium sativum) is a bulbous plant with a strong taste and smell, well known

49

for its health benefits and used for treatment of diseases since ancient times (Rivlin,

50

2001). The most significant bioactive component in garlic is allicin or diallyl thiosulphate,

51

responsible for the pungent smell and many of its medicinal properties (Macpherson et

52

al., 2005). Some of the most remarkable health benefits of garlic include: i) a reduction

53

of lipids and fibrinogen concentrations (Arreola et al., 2015; Edwards, Rocha,

54

Williamson, & Heinrich, 2015); ii) a reduction of LDL oxidation (Lau, 2006); iii) an

55

increase of fibrinolytic activity (Bordia, Verma, & Srivastava, 1998); iv) a reduction of

56

blood pressure (H. P. Wang, Yang, Qin, & Yang, 2015; Xiong et al., 2015) and v) an

57

inhibition of coronary artery calcification progression (Hom, Budoff, & Luo, 2015).

58

Beyond the study of garlic’s nutritional value, research has also been conducted

59

on the effect of pickling and fermentation on the composition and microbiological

60

stability of garlic (De Castro, Montaño, Sánchez, & Rejano, 1998; Montaño, Casado, De

61

Castro, Sánchez, & Rejano, 2004; Rejano, Sanchez, De Castro, & Montano, 1997), as

62

well as on the characterization of garlic straw residues, involving cellulose, hemicellulose

63

and/or lignin (Kallel et al., 2016; Reddy & Rhim, 2014). However, to our knowledge little

64

or no research investigating the functional properties of garlic components

65

(emulsification, foaming, gelling, etc..) has been carried out to date.

66

It is speculated that particular garlic bulb components may possess emulsifying

67

properties, noting that garlic has been traditionally used in Spain in the preparation of a

68

dressing sauce called “alioli”, which is composed primarily of olive oil, garlic and salt

69

without any addition of egg yolk (noting that “alioli” should not be confused with

70

“ajonesa”, which contains egg and therefore it is a mayonnaise-containing garlic).

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Therefore, this culinary colloid triggered our curiosity to investigate the emulsification

72

properties of garlic extract, unknown to date. Garlic bulbs are mainly composed of water and fructans (USDA, 2017). This

73 74

fructan

component

comprises

a

mixture

of

fructooligosaccharides

and

75

fructopolysaccharides (Losso & Nakai, 1997). In addition to water and fructans, other

76

minor components present in garlic bulbs are proteins and saponins (Smoczkiewiczowa,

77

Nitschke, & Wieladek, 1978); the first isolated saponin in garlic was the “furostanol

78

saponin proto-eruboside B” (Matsuura, Ushiroguchi, Itakura, Hayashi, & Fuwa, 1988),

79

and the major garlic proteins present in the bulb are two agglutinins (25 kD; ASA25), and

80

alliinase (110 kD) (Gorinstein et al., 2005). In addition to these proteins, the presence of

81

several other proteins has also been reported: high molecular weight glycoprotein

82

agglutinin (110 kD; ASA110) (Gupta & Sandhu, 1996), the antifungal protein, allivin (13

83

kD), from round-cloved garlic (H. X. Wang & Ng, 2001), and the anti-microbial protein,

84

alliumin (13 kD) (Xia & Ng, 2005).

85

Apart from the well-known surface activity of proteins used as emulsifiers in oil-

86

in-water emulsions (Norde, 2011), fructans and saponins also show surface activity. This

87

has been recently reported for fructans (Sosa-Herrera, Martínez-Padilla, Delgado-Reyes,

88

& Torres-Robledo, 2016), where the surface activity properties of mixtures of Agave

89

fructans and sodium caseinate were investigated. This suggests that garlic fructans may

90

also have some surface activity. Regarding saponins, it is well-known that due to the

91

presence of lipid-soluble aglycone and water-soluble sugar chain(s) in their structure

92

(amphiphilic nature) (Guclu-Ustundag & Mazza, 2007), they are surface-active

93

compounds with detergent, wetting, emulsifying and foaming properties. This has been

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widely reported (Ibanoglu & Ibanoglu, 2000; Mitra & Dungan, 1997; Sarnthein-Graf &

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La Mesa, 2004; Z. Wang, Gu, & Li, 2005) and currently saponins from sources such as

96

quillaja tree, oats and ginseng, attract great research interest as natural surfactants.

97

Given this background, the aim of the present work was to study the possible

98

emulsification properties of garlic aqueous extracts—potentially rich in surface-active

99

compounds such as proteins, saponins and fructans, by analysing their composition and

100

surface activity, followed by the evaluation of 10% oil-in-water emulsions made with

101

garlic extracts at various concentrations (0.48–6.55% wt/wt). Microstructural analysis—

102

droplet size by static light scattering, and light, confocal and transmission electron

103

microscopy—along with phase separation measurements of the emulsions under storage

104

were also investigated.

105

2. Materials and Methods

106

2.1.

Materials

107

Fresh peeled garlic was purchased from Marlborough Garlic LTD (Marlborough, New

108

Zealand) and frozen upon arrival. Garlic was then defrosted overnight at 4 ºC when

109

needed. The total solids content of the garlic bulbs was 32.7 ± 0.75 (wt/wt %) and water

110

content was 67.3 ± 0.75 (wt/wt %). Soya bean oil (containing antioxidant 319) was

111

purchased from Gilmours (Palmerston North, New Zealand).

112

2.2.

Methods

113

2.2.1. Extraction of garlic water-soluble compounds (GWSC)

114

The aqueous extract was prepared by blending defrosted garlic bulbs with reverse osmosis

115

(RO) water (ranging from 0.89 to 28.44 g garlic/ 100 g RO water) with a Nutri Ninja®

116

slim blender (SharkNinja Operating LLC, Needham, Massachusetts, United States of

117

America) for 1 min. The blended garlic + water mixtures were heated up to 50 ºC in a

118

water bath during 2 h under continuous stirring to favour the extraction of the garlic water-

119

soluble compounds (GWSC). Subsequently the mixtures were centrifuged for 30 min at 5

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14000 x g and at 20 ºC (Thermo Scientific Sorval RC 6+, USA). The supernatants were

121

then filtered with Whatman® qualitative filter paper, Grade 1, under vacuum conditions.

122

Remaining solid material on the filter was collected and added to the sediment (obtained

123

after centrifugation) and dried in a stove at 108 ºC for 24 h to quantify the actual

124

concentration of soluble compounds in the garlic extracts by difference, which ranged

125

between 0.24 and 6.55% wt/wt. To calculate the concentration of soluble compounds, the

126

following formula was used:

127

% 𝐺𝑊𝑆𝐶 =

𝐺𝑊 ― 𝐺𝑀 ― 𝐺𝑆 𝑊 + 𝐺𝑀

∗ 100

128

Where 𝐺𝑊 is grams of entire garlic, 𝐺𝑀 is grams of water in garlic (water in the garlic

129

bulbs), 𝐺𝑆 is grams of non-soluble garlic (remaining garlic after drying in the stove at 108

130

ºC for 24 h the solid residue after centrifugation and filtration) and W is grams of RO

131

water used to prepare the extract. Extracts were prepared at least in duplicate.

132

2.2.2. Extract characterization

133

2.2.2.1.

Quantification of soluble components

134

Protein quantification of the extracts was done by Dumas method (AOAC 968.06).

135

Saponin content was determined by the colorimetric vanillin-sulphuric acid assay,

136

following the methodology of Palniyandi, Suh and Yang (2017) with some modifications.

137

Briefly, 100 µl of each sample were made up to 0.25 ml with aqueous methanol (80%).

138

Then 0.25 ml of 8% vanillin (Sigma-Aldrich) reagent (8% vanillin in pure ethanol wt/v)

139

and 2.5 ml of 72% sulphuric acid were added to the tubes. Solutions were vortexed for

140

30 s and tubes were incubated at 63 ºC for 10 min. After incubation, they were cooled

141

down for 5 min in cold water and the absorbance was measured at 544 nm against the

142

reagent blank. Reagent blank contained 0.25 ml of aqueous methanol (80%). A standard

143

curve was prepared using various concentrations of diosgenin (Sigma-Aldrich). The

144

standard saponin solution was prepared in 80% aqueous methanol. 6

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Quantification of total carbohydrates (mainly fructans) was carried out following the

146

phenol sulphuric acid method. Briefly, 1 ml of sample/ blank, 1 ml of 5% phenol solution

147

and 5 ml of concentrated sulphuric acid were added to a tube. Tubes were then vortexed

148

and let to stand for 10 min. Samples were vortexed again and let to stand for another 30

149

min. Absorbance was measured at 480 nm. A standard curve was prepared using various

150

concentrations of glucose (Sigma-Aldrich). Extracts were prepared and measured in

151

duplicate.

152

2.2.2.2.

Identification of soluble components

153

Protein molecular weight was qualitatively determined by SDS-PAGE gel electrophoresis

154

using a Mino-PROTEAN® Cell (Bio-Rad, California, United States of America) and a 4-

155

20% Tris-HCL CriterionTM Gel (Bio-Rad, California, United States of America) at 200 V

156

for around 30 min. Tank buffer contained 25mM Tris, 192 mM Glycine and 0.1% (v/v)

157

SDS solution, and was diluted 5 times before usage. Sample buffer contained 10% (v/v)

158

of 50% Glycerol, 0.76% (v/v) of 0.5 M Tris-HCL, 0.0025% (v/v) of 0.1% Bromophenol

159

Blue and 2% (v/v) of 10% SDS. Both non-reduced and reduced electrophoresis were

160

carried out. Samples were diluted in the sample buffer five times. For the reduced

161

electrophoresis Ditiotreitol was also added to achieve a final concentration of 0.2 M.

162

Samples for reduced electrophoresis were heated in a water bath at 70 ºC for 10 min.

163

Aliquots of 5 or 10 µl of each sample were loaded into the cells. Undiluted 10 µl of

164

Precision Plus ProteinTM Unstained Standards #1610363 (Bio-Rad, California, United

165

States of America) was used as standard.

166

Identification of saponins was carried out by liquid chromatography-high resolution

167

accurate mass-mass spectrometry (LC-HRAM-MS). The system was composed of a

168

Dionex Ultimate® 3000 Rapid Separation LC and a micrOTOF QII high resolution mass

169

spectrometer (Bruker Daltonics, Bremen, Germany) fitted with an electrospray ion

7

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source. The LC column was a Luna Omega Hydro C18 100 x 2.1 mm, 1.6 µm

171

(Phenomenex, Auckland, New Zealand) and was maintained at 40 °C. The flow was 400

172

µL/min. The solvents were A = 0.2% formic acid and B = 100% acetronitrile. The

173

solvent gradient was: 5% A, 95% B, 0-0.5 min; linear gradient to 35% A, 65% B, 0.5-12

174

min; linear gradient to 35% A, 65% B, 12-20 min; composition held at 35% A, 65% B,

175

20-22 min; linear gradient to 5% A, 95% B, 20-20.2 min; to return to the initial conditions

176

before another sample injection at 25 min. The micrOTOF QII parameters for

177

polyphenolic analysis were: temperature 225 ºC; drying N2 flow 6 L/min; nebulizer N2

178

1.5 bar, endplate offset -500 V, mass range 100-1500 Da, acquired were acquired at 5

179

scans/s. Negative ion electrospray was used with a capillary voltage of +3500 V. Post-

180

acquisition internal mass calibration used sodium formate clusters with the sodium

181

formate delivered by a syringe pump at the start of each chromatographic analysis.

182

2.2.3. Dynamic surface tension

183

Surface tension of the aqueous garlic extracts was measured with a bubble pressure

184

tensiometer (SITA science t100, Dresden, Germany). Aqueous extracts were prepared as

185

detailed in 2.2.1. Surface tension as a function of bubble life time (from 0.015 to 100 s)

186

was measured at ambient temperature for the fresh extracts and after 24 and 48 h of cold

187

storage at 4 ºC. Extracts were prepared and measured in duplicate.

188

2.2.4. Emulsion preparation

189

10% (wt/wt) oil-in-water emulsions were prepared by emulsifying soy bean oil and an

190

aqueous phase containing the water soluble compounds extracted from garlic (from 0.24

191

to 6.55% wt/wt). Oil + the garlic aqueous extract mixture was heated up to 50 ºC (in order

192

to decrease oil viscosity and o/w interfacial tension to facilitate emulsification) and

193

subsequently pre-homogenized using a Silverson mixer (Silverson Machines Ltd.,

194

Chesham, U.K.). The coarse emulsions were immediately homogenised with three passes 8

ACCEPTED MANUSCRIPT 195

through a two-stage high-pressure homogenizer (APV 2000; Copenhagen, Denmark)

196

operating at pressures: 25 MPa-first stage and 5 MPa-second stage. Sodium azide solution

197

(0.02% v/v, 5 M) was added to the freshly homogenised emulsions as antimicrobial agent.

198

The emulsion prepared with 0.24% wt/wt garlic extract could not be formed as free oil

199

was detected—a sign of insufficient amount of emulsifier present in the extract, so this

200

emulsion was discharged for analysis. All emulsions were prepared and analysed in

201

duplicate.

202

2.2.5. Droplet size measurement

203

The droplet size distribution of oil-in-water emulsions was determined by laser light

204

scattering technique, using a Malvern Mastersizer MS 2000 (Malvern Instruments Ltd,

205

Worcestershire, UK). Deionised water was used as a dispersant and the relative refractive

206

index (N) of the emulsion was 1.105, i.e., the ratio of the refractive index of soy oil (1.470)

207

to that of the aqueous medium (1.33). The size of the emulsion droplets was analysed the

208

same day of preparation and on subsequent days (1st, 2nd, 3rd and 7th day)—when

209

following emulsion stability over time. Samples were kept under storage at 4 ºC, and the

210

emulsions were always mixed before sampling to obtain representative measurements.

211

Emulsions were prepared and measured in duplicate. The volume-mean droplet diameter

212

(d43) and surface-mean droplet diameter (d32) were reported.

213

2.2.6. Light microscopy of the emulsions

214

A light microscope Olympus BX53 was used to visualise the microstructure of the

215

emulsions. An aliquot portion of the emulsion sample was placed into a microscope slide.

216

A cover slip was placed on top of the well ensuring that no air bubbles were trapped

217

inside. The images were captured at 10x, 40x, and 100x magnifications.

218 219

2.2.7. Confocal scanning laser microscopy (CSLM)

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A confocal scanning laser microscope (Leica SP5 DM6000B, Leica Microsystems,

221

Heidelberg, Germany) was used to visualise the microstructure of the emulsions. This

222

microscope operated in fluorescence mode, with x10 and x40 objectives of numerical

223

aperture 0.40 and 1.25, respectively. Rhodamine B dye was used to stain the protein

224

present in the emulsion (Ex: 568 nm, 160 Em: 571-625 nm). 45 µL of the fluorescent dye

225

(0.05%) were added to a 2.5 mL emulsion sample, while stirring. An aliquot portion of

226

the stained emulsion sample was placed into a microscope slide and a cover slip was

227

placed on top of the well avoiding air bubbles being trapped inside. Samples were

228

examined in duplicates.

229

2.2.8. Transmission electron microscopy (TEM)

230

Emulsions formulated using aqueous extracts with soluble garlic concentrations of 0.95,

231

3.56 and 6.55% wt/wt were viewed using a Philips CM10 Transmission Electron

232

Microscope (Eindhoven, The Netherlands) (Camera: Morada, Olympus SIS Germany)

233

with an FEI Tecnai G 2 Spirit BioTWIN (Czech Republic) (Camera: Veleta, Olympus

234

SIS Germany). Tubes were prepared from 3% wt/wt agarose and liquid sample was

235

injected into the tube using an autopipette. The ends were sealed with agarose to form an

236

enclosed capsule and samples were placed into 3% wt/wt glutaraldehyde in 0.1 M sodium

237

cacodylate buffer (pH 7.2) for at least 24 h. Then the samples were buffer washed in 0.1

238

M sodium cacodylate (pH 7.2) 3x for 45 min each. Then, they were post-fixed in 1%

239

osmium tetroxide in 0.1 M sodium cacodylate buffer for 1 h at room temperature,

240

overnight at 4 oC, and for another hour at room temperature. Samples were buffer washed

241

again as above 3x for 45 min each and dehydrated through a graded acetone series (25%,

242

50%, 75%, 95%, 100%, 100%, 100%) for 30 min each. Samples were then put into 50:50

243

resin:acetone and placed on the stirrer overnight. This was replaced by fresh 100% resin

244

(Procure 812, ProSciTech Australia) for 8 h on the stirrer. This step was repeated four

10

ACCEPTED MANUSCRIPT 245

more times (overnight–8 h—overnight—8 h using 100% resin each time). Samples were

246

embedded in moulds with fresh resin and cured in a 60 oC oven for 48 h. Light microscope

247

sections were cut at 1 micron using a glass knife on the ultramicrotome (Leica EM UC7,

248

Germany) and heat fixed onto glass slides. The sections were stained with 0.05%

249

Toluidine Blue for approximately 12 s and viewed under the light microscope. The block

250

was then trimmed down to the selected area and cut using a Diamond Knife (Diatome,

251

Switzerland) at 100 nm. These were stretched with chloroform and mounted on a grid

252

using a Quick Coat G pen (Daido Sangyo, Japan). Grids were stained in Saturated Uranyl

253

Acetate in 50% Ethanol for 4 min, washed with 50% ethanol and MilliQ water and then

254

stained in Lead Citrate (Venable & Coggeshall, 1965) for a further 4 min. This was

255

followed by a wash in MilliQ water. Samples were then viewed under TEM.

256

2.2.9. Statistical analysis

257

Data were analysed using one-way analysis of variance (simple ANOVA). When

258

significant (p<0.05) differences were found, Fisher’s least significant differences (LSD)

259

test was used to determine the differences among means. Statistical analyses were

260

completed using Statgraphics Centurion XVI software (StatPoint Technologies Inc,

261

Warrenton, EE.UU).

262

3. Results and discussion

263 264

3.1.

Extract characterization

265

The relative amounts of protein, carbohydrates and saponins in the garlic extracts are

266

shown in Table 1. As expected, carbohydrates were the most abundant component. Very

267

likely these are fructans, a mixture of fructooligosaccharides and fructopolysaccharides

268

ranging in molecular mass from <1000 Da to ∼6800 Da corresponding to a degree of

269

polymerization (DP) as high as 38 as reported earlier (Losso & Nakai, 1997). Protein and

11

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saponin content resulted to be present at very similar level, although protein content

271

tended to be slightly higher. Other minor components were also present in the extract, but

272

were calculated by difference and not identified (most probably minerals and

273

phytomolecules, including organosulfur compounds, phenolic acids, allyl thiosulfinates,

274

flavonoids, and vitamins) (Chen, et al., 2013).

275 276

Table 1. Approximate composition of various garlic aqueous extracts Other

GWSC

Water

Proteins

Carbohydrates

Saponins

(%)

(%)

(%)

(%)

(%)

0.48

99.52

No data

0.21 ±0.03

0.06 ± 0.01

No data

0.95

99.05

0.19

0.43 ±0.09

0.13 ± 0.01

0.20

3.56

96.44

0.63

1.55 ± 0.08

0.50 ± 0.05

0.88

6.55

93.45

1.06

3.45 ± 0.75

0.90 ± 0.12

1.14

components (%)

277 278

Regarding extract component identification, the protein size distribution of the extract

279

carried out at the highest concentration of soluble compounds, 6.55% wt/wt, is shown in

280

Figure 1. SDS-PAGE gel electrophoresis of both reduced and non-reduced samples

281

revealed the presence of proteins of ~37 kDa, ~25 kDa and ~13 kDa in the extract. The

282

specific band at 25kDa is most likely the agglutinin fraction, as reported by Gorinstein et

283

al. (2005), whilst band at 13 kDa could be related to the presence of both allivin, reported

284

by Wang and Ng (2001), and alliumin, reported by Xia and Ng (2005), or a mixture of

285

both. To the best of our knowledge, garlic protein of ~37 kDa has never been reported

286

before.

12

ACCEPTED MANUSCRIPT

287 288

Figure 1. Non-reduced and reduced SDS electrophoresis of 6.55% wt/wt GWSC.

289 290

The 10 most abundant compounds identified by LC-MS (including two of the previously

291

reported saponins) are shown in Table 2. Remarkably, the most abundant compounds

292

found with these settings were peptides: Γ –Glutamyl-S-(1-propenyl)-L-cysteine, γ-

293

Glutamyl-S-allyl-L-cysteine and guanosine, in agreement with Molina-Calle, Sánchez de

294

Medina, Calderón-Santiago, Priego-Capote and Luque de Castro (2017). Rivlin, (2001)

295

had also previously found that intact garlic bulbs contain significant amounts of γ-

296

Glutamylcysteines. The presence of peptides will influence the interfacial layer as they

297

are known to be highly surface-active. Regarding saponins, four different steroidal

13

ACCEPTED MANUSCRIPT 298

saponins were identified by LC-MS: Proto-eruboside B/ iso-proto-eruboside B

299

(m/z=1259.8931, m/z=1259.6012 [M-H]), reported by Matsuura et al. (1988) and Peng

300

et al. (1996), proto-desgalactotigonin (m/z= 1213.5947 [M-H]), reported by Matsuura,

301

Ushiroguchi, Itakura and Fuwa (1989) and an unnamed saponin (m/z=1225.6006 [M-H])

302

identified firstly by Matsuura (2001). Further information on this topic has been described

303

by Lanzotti (2006). In addition, five other compounds were detected although not

304

identified. The compound detected at an m/z [M-H] of 1229.5877 may additionally be a

305

saponin. Because the conditions of the LC-MS were set to identify saponins, it has to be

306

considered that some very soluble or/and very volatile compounds were not detected.

307 308

Table 2. 10 most abundant components eluted by liquid chromatography - high resolution accurate mass – mass spectrometry Retention time (min)

Assigned compound name

Proposed elemental composition

Found m/z [M-H]-1

Mass difference (mDa)

mSigma*

Relative abundance

1.05

Guanosine

C10H13N5O5

282.0852

0.8

3.2

6

2.58

γ-Glutamyl-S-allyl-L-cysteine

C11H18N2O5S

289.0868

-0.4

8.1

3

3.23

γ -Glutamyl-S-(1-

C11H18N2O5S

289.0865

-0.2

15.3

1

propenyl)-L-cysteine 3.86

Unknown**

C14H18N2O5

293.1146

0.3

1.8

2

4.57

Unknown**

No formula

448.1206

-

-

9

4.83

Unknown**

C13H23OPS3

321.058

-0.4

16.6

5

9.8

Unknown**

C62H90N2O23

1229.5877

-1.5

12.3

7

10.31

Proto-eruboside B/ proto-isoeruboside B

C57H96O30

1259.8981

-6.7

43.9

4

11.37

Proto-desgalactotigonin

C56H94O28

1213.5947

-8.8

19.9

8

16

Unknown**

C17H26O4

293.1761

-0.3

15.5

10

309 310 311 312 313 314 315

* mSigma values are calculated from the isotope ratios expected for a given elemental composition, compared with that which is experimentally determined. Low values (<20) indicate a good fit and provide complementary evidence for assigning elemental composition. ** For unknown compounds, the best tentative formula that fit for the accurate masses and isotope ratios that were measured are shown.

14

ACCEPTED MANUSCRIPT 316 317

3.2.

Surface tension of garlic extracts

318

The interfacial activity of surfactants plays an important role in determining their ability

319

to form and stabilize emulsions and foams (Schubert & Engel, 2004). A quick way to

320

detect surface-activity, was to measure the surface tension of the aqueous extract using a

321

bubble pressure tensiometry with a bubble lifetime of 100 s (Figure 2).

322 323 324 325 326 327 328

Figure 2. Surface tensions of a range of GWSC (0.24-6.55% wt/wt). A different statistical analysis was made for each day, where all extracts were included (low case letters). Samples of the same day with the same letter(s) did not present significant differences (p > 0.05). In addition, statistical analysis was made for each concentration for the three days (capital letters). Samples of the same concentration with the same letter did not present significant differences (p > 0.05).

329 330

For the fresh extracts (day 0), a decrease of the surface tension with increasing garlic

331

concentration was observed, with no significant differences (p>0.05) among the various

332

extracts when concentration was 3.56% wt/wt or higher. This shows evidence of the

333

presence of surface-active compounds in the extracts and probably a saturation point at

334

the interface above 3.56% wt/wt concentration. In order to determine any aging effect of

335

the extract, surface tension was measured after 24 and 48 h storage. Only a statistical

336

significant (p<0.05) increase in surface tension after 1 and 2 days storage was observed

337

for the extract with 0.95% wt/wt of soluble compounds, which indicates that an ageing

15

ACCEPTED MANUSCRIPT 338

effect (or less amount of surface-active material getting adsorbed at the air-water

339

interface) may be more obvious at intermediate concentrations of GWSC. Longer periods

340

of storage would need to be tested for further conclusions, however, taking it into

341

consideration that fructans, proteins and saponins coexist in our extracts, any loss of

342

surface activity could be related to protein deterioration (Miller, Fainerman, Aksenenko,

343

Leser, & Michel, 2004) and complexation between saponins and proteins. A decrease in

344

surface activity of saponin-protein mixtures over time has been reported by Morton and

345

Murray (2001), who studied the surface tension of mixtures of saponins and proteins from

346

beet sugar, and by Wojciechowski, Kezwon, Lewandowska, and Marcinkowski (2014)

347

with quillaja bark saponins and β-caseins, although in this last study, this was only true

348

when saponin concentration was lower than 4·10−5 mol L−1. 3.3.

349

Effect of garlic concentration on emulsification properties

350

Droplet size distributions of the fresh formed emulsions are shown in Figure 3 and Table

351

3.

352 353 354

Figure 3. Particle size distribution of 10% oil-in-water emulsions made with various garlic aqueous extract concentrations.

355 356 357

16

ACCEPTED MANUSCRIPT 358

Table 3. d43 and d32 values (in µm) of 10% oil-in-water emulsions made with GWSC. GWSC

359 360

d43

d32

0.48%

0.67 ± 0.03 a

0.36 ± 0.03 a

0.72%

1.45 ± 0.08 a

0.40 ± 0.07 a

0.95%

6.07 ± 1.32 b

3.34 ± 1.71 b

1.86%

7.42 ± 3.14 bc

3.57 ± 0.99 b

3.56%

6.93 ± 0.26 bc

5.69 ± 0.03 c

4.35%

11.16 ± 0.00 cd 8.44 ± 0.00 d

5.11%

11.31 ± 0.00 cd 6.25 ± 0.00 cd

6.55%

12.47 ± 1.84 d

6.55 ± 0.09 d

*Data are expressed as means ± SD of duplicate assays. Samples in the same column with the same letter(s) did not present significant differences (p > 0.05).

361 362

Surprisingly, the smallest droplet size distributions were observed for the emulsions made

363

with the lowest garlic concentrations (0.48 and 0.72% wt/wt). Droplet size progressively

364

increased at higher concentrations of GWSC, reaching an upper value of d43 = 12.47 ±

365

1.84 µm, at the highest concentrations of proteins, fructans and saponins at 6.55% wt/wt.

366

This seems to be in contradiction with the surface tension results, as surface tension values

367

were quite high for extracts at the lowest garlic concentration. A future publication,

368

showing interfacial tension measurements (results not shown) also demonstrates a similar

369

phenomenon; higher values of interfacial tension at the lowest garlic content, proving that

370

the effect is real. Nevertheless, garlic aqueous extract heterogenic composition may have

371

an important influence on this, causing interfacial layers of variable packing density,

372

which would lead to a different degree of reduction in the surface/interfacial tension. The

373

fact that a smaller droplet size distribution was achieved with the lowest concentration

374

could be the evidence of competitive adsorption between the different surface-active

375

molecules present in the extract. Additionally, visual imaging of the emulsion made with

376

the highest concentration at 6.55% wt/wt (Figure 4A), indicates that, apart from any 17

ACCEPTED MANUSCRIPT 377

competitive adsorption, other phenomena such as depletion flocculation and/or bridging

378

flocculation could be taking place, as evidenced by the presence of strong droplet

379

aggregation.

380 381 382 383 384

Figure 4. Particle size distribution and light microscopy of 10% oil-in-water emulsions of A) Emulsion stabilised with 6.55% wt/wt GWSC. B) Emulsion stabilised with 6.55% wt/wt GWSC with serum substituted with RO water after centrifugation. C) Emulsion stabilised with 6.55% wt/wt GWSC mixed with 2% SDS solution (1:1 ratio.)

385

In order to test the type of flocculation, a fresh emulsion was centrifuged at 5000 x g for

386

10 min and the serum was substituted by RO water and observed under the microscope

387

(Figure 4B). In addition, a fresh emulsion was also observed after mixing with 2% SDS

388

solution (1:1 ratio) (Figure 4C). When substituting the serum phase by water, many of the

389

flocs observed in Figure 4A were broken up, although remaining small flocs could still

390

be detected (presumably bridged droplets). The disappearance of flocs by removing non-

391

adsorbed material from the serum phase, when substituting it by water, indicates a

392

possible depletion flocculation mechanism occurring in the unmodified emulsion. This

393

phenomenon occurs when non-adsorbing biopolymers in the aqueous phase of an

394

emulsion could cause an increase in the attractive forces between the droplets, due to an

395

osmotic effect associated with the exclusion of the biopolymers from the narrow region

396

between approaching droplets (McClements, 2000; Walstra, 2003). At very low

397

concentrations of free polymer, the entropy loss linked to particle aggregation outweighs

398

the depletion effect and the system remains stable (Figure 5A).

18

ACCEPTED MANUSCRIPT

399 400 401

Figure 5. Light microscopy of 10% oil-in-water emulsions of A) Emulsion stabilised with 0.48% wt/wt GWSC. B) Emulsion stabilised with 6.55 % wt/wt GWSC.

402 403

However, beyond certain critical polymer concentration, attractive forces are large

404

enough and reversible depletion flocculation occurs (Figure 5B), causing droplets to

405

flocculate

406

identified as responsible for this effect, including polysaccharides and proteins as well as

407

surfactant micelles, and therefore in our emulsions, both fructans and proteins, and

408

potentially saponin micelles are likely to be responsible for this. The appearance of a

409

second peak at 50 µm in Figure 4B is likely due to coalescence caused via centrifugation

410

of the emulsions when separating the serum phase. Figure 4C showed that when mixing

411

the fresh emulsion with SDS, some of the flocs also disappeared but again some remained.

412

SDS is used to displace any protein/polysaccharide adsorbed at the interface, thereby

413

breaking any floc formed by bridging phenomena, which would explain the

414

disappearance of the strong flocculation. This phenomenon occurs when a polymer

415

adsorbs on two or more emulsions droplets, and therefore droplets are interconnected by

416

a polymer bridge (Healy & La Mer, 1964). Bridging-flocculation has been shown to

417

occur in protein-stabilised emulsions containing polysaccharides such as carrageenan

418

(Dickinson & Pawlowsky, 1998). The fact that flocculation still happens with SDS added

419

reinforces the fact that depletion mechanisms are still taking place in the presence of the

(Dickinson, 2003; McClements, 2004). Several biopolymers have been

19

ACCEPTED MANUSCRIPT 420

non-adsorbed material. In order to identify the protein phase dyed with Rhodamine B,

421

CSLM images of the emulsions at four garlic extract concentrations are shown in Figure

422

6. The increasing droplet size observed with increasing GWSC, is an agreement with the

423

distributions obtained by light scattering (Figure 3) (noting that weak depletion

424

flocculated structures will be disrupted in the Mastersizer, and thus the size distribution

425

measured via light scattering is indicative of large droplets and the presence of bridging

426

flocculation). Apart from any flocculation effects, a bigger droplet size is undoubtedly

427

obtained when the amount of protein, fructans and saponins increase in the extract. There

428

is a possibility that proteins may be linked or complex to the fructans and/or saponins in

429

the native state in garlic, making the emulsification process less efficient when they tend

430

to dominate at the interface (i.e at higher concentrations of the extract). A protein

431

dominance at the interface of the emulsions is clear from the confocal images especially

432

at 0.95 and 3.55% (wt/wt), with an obvious dense matrix of protein in the continuous

433

phase as observed at 6.55% wt/wt GWSC. A future step of this research will be trying to

434

dye the fructans present in the extract to elucidate their location in the emulsion and infer

435

more on the surface activity. In addition to CSLM, TEM images are also shown (Figure

436

7) to investigate further the structure of these emulsions.

20

ACCEPTED MANUSCRIPT

437 438

Figure 6. CSLM images of 10% oil-in-water emulsions made with the indicated % GWSC.

439 21

ACCEPTED MANUSCRIPT

440 441 442 443

Figure 7. TEM images of 10% oi-in-water emulsions of A) and B) Emulsions stabilised with 0.95% wt/wt GWSC; C) and D) Emulsions stabilised with 3.56% wt/wt GWSC; and E) and F) Emulsions stabilised with 6.55% wt/wt GWSC.

444 445

Filament-like structures could be observed in the serum of all emulsions. These filaments

446

were more obvious at higher GWSC (Figure 6E). In addition, some unusual assemblies

447

were observed at the surface of droplets (Figure 6F), becoming more apparent with

448

increasing GWSC concentration. Figure 6F (arrows) also shows the apparent interfacial

449

bridging of oil droplets comprising these interfacial assemblies, which supports the

450

bridging flocculation phenomenon described above.

451

It is known that during the adsorption of mixed systems—proteins and surfactants (like

452

the saponins present here), the relative concentration of these components will have an

453

impact on the final composition at the interface, with the small molecule surfactants

454

dominating the interface at high enough concentrations (Mackie, Gunning, Wilde, &

455

Morris, 2000; Miller, Fainerman, Makievski, Kragel, Grigoriev, Kazakov & 22

ACCEPTED MANUSCRIPT 456

Sinyachenko, 2000). Most probably at low garlic concentrations, with low quantities of

457

protein and saponins (Table 1), the saponins are likely to adsorb at the interface during

458

the emulsification step, achieving very small droplet sizes (Figure 3). When increasing

459

garlic concentration, we hypothesize that proteins (or protein-fructan complexes) are

460

responsible for stabilizing oil droplets at least initially.

461

The evolution of the surface-active compounds may lead to a different interfacial

462

composition over time given the presence of fructans, proteins and saponins in the extract.

463

It is possible that some of the surface-active aggregates (likely to be of protein nature)

464

could be displaced by saponins over time. Nevertheless, Böttcher, Scampicchio and

465

Drusch (2016) reported that Quillaja saponins and β-lactoglobulins interact at the

466

interface, and that these saponins could not fully deplete proteins from the interface. The

467

authors postulated that this could be due an interaction via “complexation and competitive

468

adsorption” (Kotsmar et al., 2009) or via “orogenic displacement” (Mackie, Gunning,

469

Wilde, & Morris, 1999), as it has been shown to be a mechanism by which protein can be

470

removed from an interface by small surfactant molecules. Taking into account the ionic

471

nature of the steroidal saponins detected here (Table 2), interactions with proteins at the

472

interface are possible, regardless the degree of displacement which would need to be

473

quantified, if any, in future research. The role of significant amounts of highly surface-

474

active peptides on the adsorption dynamics of the mixed system and final interfacial layer

475

composition will also need to be determined.

476

Fructans surface activity has not been widely studied and their effect on the droplet size

477

distribution remains unclear. Sosa-Herrera et al. (2016) reported that fructans can interact

478

with sodium caseinate and reduce the surface tension relative to the surface tension of

479

caseinate alone, but that this decrease was fructan concentration dependent. They

480

hypothesised that fructans favour a change in protein conformation that consequently

23

ACCEPTED MANUSCRIPT 481

affects the fraction of the segments in contact with the surface. In our case, we have not

482

explored the presence of possible protein-fructan complexes (which could explain the big

483

initial droplet sizes obtained at high GWSC concentrations, also contributing to bridging

484

flocculation) or the surface activity of the isolated fractions, and this again, will be the

485

subject of future research.

486 487 488

3.4.

Emulsion stability over time

Droplet size evolution with time of garlic-based emulsions is shown in Figure 8.

489 490 491 492 493

Figure 8. d32 (A) and d43 (B) evolution during 7 days storage of 10% oil-in-water emulsions made with various GWSC concentrations. A different statistical analysis was applied for each concentration. Samples within the concentration with the same letter(s) did not present significant differences (p > 0.05).

494 495

The volume mean diameter (d43) increased for emulsions made with 0.48% wt/wt GWSC

496

after 2 days of storage, while for emulsions made with 0.72 and 6.55% wt/wt GWSC the

497

increase was significant (p<0.05) after only 1 day of storage. Emulsions made with 6.55%

498

wt/wt extract also showed a significant increase (p<0.05) after 7 days of storage.

499

Significant differences in d32 were observed for emulsion made with 0.72% wt/wt extract

24

ACCEPTED MANUSCRIPT 500

after 1 day of storage and for the one with 6.55% wt/wt extract after 7 days of storage. A

501

high variability in size measurements was only observed for intermediate concentrations

502

(0.95–1.86% wt/wt), probably indicative of a variable interfacial composition of

503

competitive surface-active species at these concentrations.

504

It is possible that the high degree of interfacial contact between flocculated droplets was

505

contributing to a gradual coalescence of droplets within the aggregates over the storage

506

period. Bridging and depletion flocculation effects, together with significant greater

507

droplet sizes observed at 6.55% wt/wt resulted in faster phase separation at higher

508

concentrations, as shown in Figure 9. The presence of peptides at the interface has earlier

509

been shown to lead to high degree of coalescence in emulsions (Ye, Hemar, & Singh,

510

2004) so this could also be an important factor for the instability shown at high GWSC.

511 512

Figure 9. Phase separation over time of emulsions made with GWSC (0.48–6.55% wt/wt).

513 514

In contrast, at low concentrations of GWSC (0.48% wt/wt), emulsion phase separation

515

was only visible after 7 days of storage. The greater creaming stability of this composition

516

is indicative of that the droplets in the emulsion are most likely in a non- or minimally-

517

aggregated state apart from the small original sizes achieved in these emulsions (d3,2 < 1

518

µm).

25

ACCEPTED MANUSCRIPT 519

Figure 10 shows phase separation after 1 day of storage at 4 ºC of the same emulsions

520

presented in Figure 4, prepared with 6.55% wt/wt GWSC: first sample with no

521

modification; second sample with replacement of the aqueous phase after emulsion

522

centrifugation; third sample mixed with 2% of SDS solution (1:1). It could clearly be

523

observed that phase separation of the modified samples was decreased with respect to the

524

unmodified emulsion, but not completely eliminated, thereby confirming the occurrence

525

of a combination depletion flocculation and bridging phenomena reported earlier in the

526

text.

527

528 529 530 531

Figure 10. Emulsion made with 6.55% wt/wt GWSC (Unmodified), similar emulsion with serum substituted with RO water after centrifugation (RO water) and unmodified + 2% SDS solution (1:1 ratio) (SDS).

26

ACCEPTED MANUSCRIPT 532

4. Conclusions

533

With this study we have proven the initial hypothesis that garlic contains surface active

534

components capable of forming and stabilising emulsions. At low garlic extract

535

concentrations, very small droplet sizes (d32=0.36 µm) can be obtained, however

536

emulsification using high concentrations resulted in apparent depletion flocculation and

537

bridging phenomena taking place, which had considerable impact on droplet size and

538

corresponding emulsion stability. The underpinning reason of these phenomena can be

539

related to the coexistence of compounds of different surface-active nature (i.e. a

540

combination of saponins, proteins/peptides and fructans), and their interactions. In order

541

to better understand this novel emulsifier, more research needs to be done regarding the

542

emulsifying capacity of each water soluble compound after separation, as well as the

543

extract stability in different environments (pH or temperature treatments).

544

5. Acknowledgments

545

Ángela Bravo would like to acknowledge the University of Valladolid for her pre-

546

doctoral scholarship and for traveling funding that made possible a temporal mobility to

547

Massey University (New Zealand).

548

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List of Figures

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Figure 5. Non-reduced and reduced SDS electrophoresis of 6.55% wt/wt GWSC.

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Figure 6. Surface tensions of a range of GWSC (0.24-6.55% wt/wt). A different statistical analysis was made for each day, where all extracts were included (low case letters). Samples of the same day with the same letter(s) did not present significant differences (p > 0.05). In addition, statistical analysis was made for each concentration for the three days (capital letters). Samples of the same concentration with the same letter did not present significant differences (p > 0.05).

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Figure 7. Particle size distribution of 10% oil-in-water emulsions made with various garlic aqueous extract concentrations.

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Figure 8. Particle size distribution and light microscopy of 10% oil-in-water emulsions of A) Emulsion stabilised with 6.55% wt/wt GWSC. B) Emulsion stabilised with 6.55% wt/wt GWSC with serum substituted with RO water after centrifugation. C) Emulsion stabilised with 6.55% wt/wt GWSC mixed with 2% SDS solution (1:1 ratio.)

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Figure 5. Light microscopy of 10% oil-in-water emulsions of A) Emulsion stabilised with 0.48% wt/wt GWSC. B) Emulsion stabilised with 6.55 % wt/wt GWSC.

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Figure 6. CSLM images of 10% oil-in-water emulsions made with the indicated % GWSC.

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Figure 7. TEM images of 10% oi-in-water emulsions of A) and B) Emulsions stabilised with 0.95% wt/wt GWSC; C) and D) Emulsions stabilised with 3.56% wt/wt GWSC; and E) and F) Emulsions stabilised with 6.55% wt/wt GWSC.

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Figure 8. d32 (A) and d43 (B) evolution during 7 days storage of 10% oil-in-water emulsions made with various GWSC concentrations. A different statistical analysis was applied for each concentration. Samples within the concentration with the same letter(s) did not present significant differences (p > 0.05).

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Figure 9. Phase separation over time of emulsions made with GWSC (0.48–6.55% wt/wt).

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Figure 10. Emulsion made with 6.55% wt/wt GWSC (Unmodified), similar emulsion with serum substituted with RO water after centrifugation (RO water) and unmodified + 2% SDS solution (1:1 ratio) (SDS).

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Table captions

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Table 4. Approximate composition of various garlic aqueous extracts 34

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Table 5. 10 most abundant components eluted by liquid chromatography - high resolution accurate mass – mass spectrometry

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Table 6. d43 and d32 values (in µm) of 10% oil-in-water emulsions made with GWSC.

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Highlights    

Garlic aqueous extract is a source of emulsifiers (proteins and saponins) At low garlic content emulsions with small droplet sizes are achieved (d32=0.36 µm) At higher garlic concentrations depletion and bridging flocculation phenomena occur Strong emulsion phase separation occur after one day at high garlic concentrations