Stability properties of different fenugreek galactomannans in emulsions prepared by high-shear and ultrasonic method

Accepted Manuscript Stability properties of different fenugreek galactomannans in emulsions prepared by high-shear and ultrasonic method O. Kaltsa, S. Yanniotis, I. Mandala PII:

S0268-005X(15)30034-5

DOI:

10.1016/j.foodhyd.2015.07.024

Reference:

FOOHYD 3080

To appear in:

Food Hydrocolloids

Received Date: 26 February 2015 Revised Date:

20 July 2015

Accepted Date: 24 July 2015

Please cite this article as: Kaltsa, O., Yanniotis, S., Mandala, I., Stability properties of different fenugreek galactomannans in emulsions prepared by high-shear and ultrasonic method, Food Hydrocolloids (2015), doi: 10.1016/j.foodhyd.2015.07.024. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT 1

Stability properties of different fenugreek galactomannans in emulsions prepared

2

by high-shear and ultrasonic method O. Kaltsa, S. Yanniotis, I. Mandala

4

Agricultural University of Athens, Dept. Food Science & Human Nutrition, Athens,

5

Greece

RI PT

3

6

Abstract

7

In this research we studied the emulsion stabilizing properties of different fenugreek

8

galactomannan gums, types (FGA, FGB, FGH) in comparison to common stabilizers,

9

guar and locust bean gum (GG and LBG).

SC

The fenugreek gums differ at

galactomannan (FGH>FGA>FGB) and protein content (reverse order), as well as at

11

odor, varying from odorless (FGH) to intense one (FGB). Emulsions were prepared

12

by high speed (HS) and ultrasonic (US) homogenization method and creaming was

13

evaluated during 10-day cold storage. Gum type at 0.25 % concentration did not

14

improve the stability of samples prepared by both methods, thus the serum index was

15

high, ranging from 25 % to 39 %. The stability was enhanced at 0.5 % gum, mainly

16

attributed to viscosity increase. Particularly, the serum index varied between 3.5 to

17

20.7 % for ultrasonically treated samples, while for those treated by HS ranged from

18

4.9 to 35 %.

19

US treatment was able to reduce the average droplet size of emulsions by a factor ~

20

2.7 in average. In the presence of galactomannans, the smallest droplets according to

21

D50 were around 1.48 µm. However, the viscosity loss observed in US emulsions due

22

to polysaccharide degradation ranged between 77 – 91 % in 1 %wt in gum solutions,

23

resulting in relatively poor stability enhancement observed in US emulsions. Overall,

24

the best results regarding stability against creaming were obtained for emulsions

25

containing 0.5 %wt FGH, but the lowest droplet size was achieved in the absence of

26

stabilizers.

28

TE D

EP

AC C

27

M AN U

10

Keywords: Fenugreek gum, stabilizer, ultrasonic emulsification, stability

1

ACCEPTED MANUSCRIPT 1. Introduction

30

Within the last few years numerous polysaccharide extracts have been proposed as

31

possible thickening and stabilizing agents including mesquite gum, cordial gum,

32

lepidium perfoliatum gum, psylium gum, angum gum, peach gum, durian seed gum

33

etc (Lopez-Franco et al., 2013; Haq et al., 2013; Soleimanpour et al., 2013, Alftren et

34

al., 2012; Gharibzahedi et al., 2013; Jindal et al., 2013; Qian et al., 2011; Ghorbani

35

Gorji et al., 2014).

36

This increased interest arises from the fact that commonly used food polysaccharides

37

like guar gum are used in non-food applications (well drilling, textile, paper, paint,

38

cement, cosmetic, food, pharmaceutical etc), mainly in petroleum refining and

39

pharmaceuticals (Vaughn et al., 2013; Prajapati et al., 2013). Along with the decrease

40

of global production this has resulted in price fluctuations, price increase and severe

41

supply shortage issues have arisen (Bahamdan et al., 2006; Barati et al., 2011; Anon,

42

2012).

43

Fenugreek (Trigonella foenum-graecum L.) is a legume grown annually in India,

44

Ethiopia, Egypt and Turkey in northern Africa, the Mediterranean, Western Asia,

45

northern India and Canada. The seeds are generally used as a spice but also as a

46

remedy for their medicinal properties. They contain 45% to 60% carbohydrate

47

(mainly galactomannan), 6-10% lipids, and 20-30% protein, 5 - 6% saponins and 2-3

48

% alkaloid compounds. (Rao et al., 1996; Raghuram et al., 1994).

49

As a spice, fenugreek not only provides a characteristic flavor like artificial maple

50

syrup or rum in products, but adds nutritive value to foodstuffs as well

51

(Shankaracharya et al., 1973). Fenugreek galactomannan consists of a (1 → 4)-β-D-

52

mannan backbone to which single α-D-galactopyranosyl groups are attached at the O-

53

6 position of the D-mannopyranosyl residues and the galactose/mannose ratio ranges

54

between 1.00:1.02 to 1.00:1.14 (Brummer et al., 2003). Cholesterol lowering, anti-

55

inflammatory and anti-diabetic properties have been associated to components

56

abundant in fenugreek seed, gums and leaves (Acharya et al., 2008; Im and Maliakel,

57

2008).

58

Fenugreek gum antidiabetic properties were initially attributed to its high fiber

59

content that slowed down the absorption of glucose and lipids in the intestine. Its

60

hypoglycemic effect has been demonstrated in vivo for both animals and humans

61

(Ribes et al., 1986; Amin et al., 1988; Madar et al., 1988; Sharma and Raghuram,

62

1990; Hannan et al., 2007). The addition of FG caused a dose response reduction in

AC C

EP

TE D

M AN U

SC

RI PT

29

2

ACCEPTED MANUSCRIPT glucose liberated from breads (Roberts et al., 2012). More recently it was revealed

64

that other minor seed components such as 4-hydroxy-isoleucine and steroidal

65

saponins, may also act synergistically in inhibiting glucose absorption and promoting

66

pancreatic functions (Hamden et al., 2010).

67

According to another research, the optimal galactomannan content to decrease the rate

68

of glucose uptake in the small intestine was establisted at 0.35% (wt/wt), since higher

69

concentrations of fiber did not further influence the diffusion of glucose (Srichamroen

70

et al., 2009).

71

Within the last decade, the use of fenugreek gum has been increased in the food

72

industry based on its thickening, stabilizing, and emulsifying properties in many food

73

products (Işıklı and Karababa, 2005). Despite aforementioned health promoting

74

properties, one of the major disadvantages when using it in food products is the

75

unpleasant flavor it imparts, not only when added but also after being consumed. Bad

76

taste in meat and milk products after ingestion by animals, or strong odor in human’s

77

sweat and urine (misdiagnosed as the “maple syrup” urine disease) have been reported

78

(Bartley et al., 1981; Korman et al., 2001; Mazza et al., 2002; Sewell et al., 1999).

79

Additionally, it has been found that supplementation with fenugreek flour up to 20%

80

in biscuits had a negative effect on their sensory characteristics. More specifically, the

81

sensory score regarding the taste of the biscuits was reduced from 7.25 to 2.78 (Hooda

82

and Jood, 2005).

83

The aim of this work was to compare the stabilizing properties of different fenugreek

84

gum types with commonly used polysaccharides, namely guar and locust bean gum.

85

Emulsions were prepared with two different methods, high speed and ultrasonic (US)

86

homogenization and the stability of the prepared emulsions was compared.

87

Considering that US technology is increasingly being up-scaled as its use is both time

88

and power saving, thus making it a cost-effective emulsion formation technique, the

89

interest in its improvement is high. Despite the above advantages referred a major

90

restriction on US technology arises due to polymer degradation occurring during

91

sonication. Even though the effect of sonication on rheological properties has been

92

studied for several common stabilizers such as galactomannans and xanthan (Kaltsa et

93

al., 2014; Kaltsa et al. 2013; Tiwari et al., 2010), starches (Jambrak et al., 2010;

94

Chung et al., 2002) there have been limited data concerning fenugreek gum role in an

95

emulsion (Huang et al., 2001; Sav et al., 2013) and no data in emulsions formulation

96

through US to our best knowledge.

AC C

EP

TE D

M AN U

SC

RI PT

63

3

ACCEPTED MANUSCRIPT 2. Materials and methods

98

Whey protein isolate (WPI) Lacprodan DI-9224 was kindly provided by Arla (Arla

99

Foods Ingredients, Amba-Denmark). The composition of WPI powder in protein as

100

stated by the manufacturer was protein 92 ± 2 %, and maximum amounts of fat 0.2 %,

101

ash 4.5 % and lactose 0.2 %. Fenugreek gum types A, B and H were a kind gift from

102

Airgreen (Air Green Co., Ltd, Japan). Guar gum (GG) and locust bean gum (LBG)

103

were bought from Sigma (St. Louis, MO, USA). Virgin olive oil Altis (Elais Unilever,

104

Greece) was purchased from a local store. Citric acid, phosphate and sodium azide

105

were purchased from Fluka (Fluka Chemie AG, Buchs, Switzerland).

106

2.1 Gum characterization

107

The moisture content of gum powders (dry/wet basis) was determined by AACC 44-

108

16 Official method (105oC, 2 hours). Total nitrogen contents were determined by

109

Dumas method using a flash EA 1112 NC analyzer (Thermo Fisher Scientific Inc.,

110

Waltham, Ma, USA) and 6.25 was used as protein conversion factor (Koocheki et al.,

111

2009).

112

For oil-holding capacity (OHC) determination, 5 g of olive oil was mixed with 0.25 g

113

gum. The mixture was centrifuged at 4000 rpm for 5 min after vortexing for 30 s and

114

left undisturbed for 5 min. The oil layer was carefully separated from the top of the

115

tube using a syringe. The difference between the weight of oil added and the weight

116

of oil separated at the top of the tube was calculated as a percentage to give oil-

117

holding capacity.

118

2.2 Emulsion preparation

119

Whey protein stock solutions 10 %wt were prepared by agitation with a magnetic

120

stirrer for 90 min, by mixing appropriate volumes of citric acid 0.1 M and phosphate

121

(Na2HPO4) 0.2 M solutions at pH 3.8, typical for products such as salad dressings or

122

mayonnaise. Galactomannans’ solutions 1 %wt were prepared also at pH 3.8 by hot

123

stirring in a water bath (90 oC, 90 min). Thereafter, solutions were kept overnight at 5

124

o

125

solutions as an antibacterial agent. Coarse emulsions contained 2.7 %wt WPI, 20 %wt

126

olive oil and 0.25 or 0.5 %wt of different galactomannans solutions. A total of 50 g

127

were prepared by mixing appropriate amounts of the aqueous stock solutions and oil

128

with a high-shear device Ultraturrax T25 device (IKA Werke, Staufen, Germany) at

129

13.500 rpm for 2 min. Small adjustments on emulsion pH were made with a few

130

drops of HCl 1M if required. 40 ml of the coarse emulsion were placed in a glass

AC C

EP

TE D

M AN U

SC

RI PT

97

C to ensure complete hydration. Sodium azide 0.02 %wt was added to the aqueous

4

ACCEPTED MANUSCRIPT beaker (38 mm internal diameter) covered by ice to prevent overheating. Secondary

132

emulsions were produced following a batch-type sonication approach. The ultrasonic

133

tip of a 13 mm diameter cylindrical titanium probe (VS 70T) was immersed in the

134

centre of the glass beaker at a repeatable depth of 1 cm and different sonication

135

treatments were applied generated from an ultrasonic device (Sonopuls 3200,

136

Bandelin Gmbh & Co, Berlin, Germany) operating at constant frequency of 20 kHz.

137

The sonication device operates by controlling either amplitude (100 % amplitude

138

corresponding to 170 µmss for the specific probe used) or power (150 W nominal

139

maximum power). It also has the ability to display or monitor the energy input (kJ) in

140

the sample during sonication. The selection of emulsification conditions was based on

141

our previous findings (Kaltsa et al., 2013). The application of 70 %-3 min+90 %-1

142

min sonication treatment (referred as method B) was shown to result in emulsion

143

formation with sub-micrometer size (D50 ~ 600-800 nm) at 0.5 % galactomannan gum

144

concentration (pH 7.0). The volume of sample processed was reduced though to 40

145

ml, which theoretically could lead to further droplet size reduction, preferably within

146

the nanoscale range.

147

2.3 Light microscopy

148

Emulsion structure was observed in freshly prepared samples with a conventional

149

optical microscope (Kruss Optronik, Germany) with a 40x magnification and several

150

micrographs were obtained per sample from the center area of each slide. The

151

emulsions were only slightly diluted with the same buffer (pH 3.8) in order not to

152

disturb their initial structure. Several photos were taken from random sample

153

positions. The micrographs were recorded using a camera (SONY, Hyper HAD,

154

CCD-Iris) connected to a computer.

155

2.4 Droplet size evaluation

156

Oil droplet size measurements were performed on a Bruker Minispec NMR

157

spectrometer (Bruker Optik GmbH, Ettlingen, Germany) equipped with a Bruker ND

158

2176 probe at 20oC. Measurements were performed in freshly prepared samples.

159

Droplet size measurements were carried out in duplicate and results are expressed by

160

the average volume weighted diameter D50 and cumulative diameters D2.5 and D97.5

161

(Kaltsa et al., 2013).

162

2.5 Viscosity measurements

163

Viscosity measurements of different gums were conducted with a controlled stress

164

SR-5 rheometer (Universal Stress Rheometer/Rheometrics Scientific, Inc., NJ) with

AC C

EP

TE D

M AN U

SC

RI PT

131

5

ACCEPTED MANUSCRIPT plate-plate geometry and 0.25mm gap. The diameter of the upper plate was 20 mm.

166

The temperature was constant (25 ±0.2oC) by circulating water from a constant

167

temperature circulator. Steady-state flow curves were obtained at shear rates between

168

0.1 and 500 s-1 to avoid slippage of 1 %wt gum solutions that had been subjected to

169

various sonication treatments (70 % amplitude for 1, 2, 3 min and as in method B). In

170

the case of gum solutions, a higher concentration of 1 %wt instead of 0.5 %wt was

171

selected in order to better fit the precision of the rheometer geometry. The viscosity of

172

1 % wt gum solutions was calculated at a low shear rate (10 s-1), mean values of three

173

differently prepared (Kaltsa et al, 2013). Consistency (k) and flow behavior (n)

174

indices were calculated according to the Power law model (Kaltsa et al., 2014).

175

Viscosity measurements were performed only in emulsions containing 0.5%wt of

176

various gums that were the most stable, since the emulsions containing 0.25 %wt

177

stabilizer became phase separated a few minutes after preparation. A hybrid

178

rheometer DHR (TA Instruments, New Castle, US) equipped with plate-plate

179

geometry (upper plate diameter 50 mm, gap 1 mm) was used to obtain flow curves

180

between 0.1-1000 s-1 at 25 ±0.01oC by circulating water from a peltier. The total

181

measurement time was 900 s. All measurements were performed at least in triplicate

182

and data reported as average and standard deviations. Flow curves were fitted to

183

power law model and consistency (k) and flow behavior indices (n) were calculated.

184

2.6 Storage stability

185

Emulsion stability was evaluated by obtaining daily acquisitions of back scattering

186

profiles with a vertical scan analyzer Turbiscan MA 2000 (Formulaction, Toulouse,

187

France) during 10 days of storage at 5oC. The procedure was replicated at least three

188

times. Emulsion stability due to clarification at the bottom of the tube is expressed as

189

serum index and was calculated as in Kaltsa et al. (2013).

190

Briefly, the height of serum phase separation was defined as the height at 50% loss of

191

the initial BS (H50%) and it was expressed as a percentage of the total height of

192

emulsion % Serum = (H50% / HT) (Jourdain et al., 2008; Zinoviadou et al., 2012).

193

The initial BS intensity (t = 0) is a value related to the size and density of particles in

194

emulsions; BS enhances as the increase of the number of particles, and decreases as a

195

function of the size of individual droplets and/or presence of flocs (Palazolo et al.

196

2004, 2005). It has to be noted though that according to Mie’s law, the BS intensity

197

decreases when the droplet size increases only for particles with diameter larger than

AC C

EP

TE D

M AN U

SC

RI PT

165

6

ACCEPTED MANUSCRIPT 0.5 µm (Mengual et al., 1999a,b; Buron et al., 2004; Pizzino et al., 2009).2.7

199

Statistical analysis

200

Statistical analysis of the results was performed with Statgraphics Centurion XV

201

(Statgraphics, Rockville, MD, USA) and the LSD was applied in order to compare the

202

mean values of emulsions and gum solutions properties at 95 % level of confidence.

203

3. Results and discussion

204

3.1 Oil holding capacity

205

In the present study the oil-holding capacity (OHC) and protein contents of the

206

different galactomannan gums, as seen in Table 1, varied between 47.37 for FGA up

207

to 138.02 g/100g for LBG. Values from 43.7 to 128.8 (g oil/100 g gum) have been

208

reported for Durian seed gum (Mirhosseini and Amid, 2012) and

209

(81–114 g oil/100 g) (Galla and Dubasi, 2010). The mechanism of fat/oil absorption

210

capacity was explained by Kinsella (1979) as a physical entrapment of oil by the non-

211

polar chains of protein, but it could also be due to the presence of non-polar amino

212

acids (Lazos, 1992). Among fenugreek fractions FGB presented the highest OHC

213

followed by FGH and FGA. FGB and GG contained the same amount of protein

214

(3.92 %). However, at dry basis, GG contained the highest protein content and

215

presented a slightly higher OHC. LBG having the greatest protein content presented

216

the highest OHC. Results obtained in this study indicate that the OHC of the various

217

gums does not necessarily correlate to their protein content. Other factors such as

218

surface area, porosity and capillary attraction of the fiber have also been reported to

219

affect oil absorption capacity as well (Chau et al., 1997; Chau and Huang, 2003).

220

3.2 Effect of ultrasonication on viscosity of gum solutions

221

Ultrasonic irradiation and other high shear treatments such as high pressure

222

homogenization have been reported to influence the flow behavior of hydrocolloid

223

dispersions due to reduction of their molecular weight via “mechanochemical”

224

depolymerization reactions (further explained in Kaltsa et al., 2014).

225

Apparent viscosities as a function of shear rate for fenugreek, guar and locust bean

226

gum dispersions are shown in Fig. 1 and Power law consistency and flow behavior

227

indices of untreated solutions are summarized in Table 2. Information is provided

228

only for untreated solutions, since solutions sonicated by method B exhibited

229

Newtonian behavior. The flow curves of untreated samples exhibit shear thinning

230

behavior and high low shear viscosities. The application of ultrasonication led to a

for karaya gum

AC C

EP

TE D

M AN U

SC

RI PT

198

7

ACCEPTED MANUSCRIPT sharp, approximately two-fold, decrease of the apparent viscosity at 10 s-1 rate (Fig.

232

2) at the lowest energy level input (70%-1 min). The temperature did not remain

233

constant during sonication and varied from 25 to 47 oC, and a temperature increase

234

could lead to increase of scission reaction (Prajapat and Gogate, 2015). Thus, this

235

figure mainly serves for comparing the various gum types per single treatment

236

without taking into account any temperature effect.

237

Extended sonication processing >3 min resulted in marginal viscosity decrease for all

238

gums examined. According to Ansari et al. (2013) the application of sonication for 3

239

min lead to reduction of guar gum molecular weight by 50%, as estimated by

240

viscometric measurements. When sonication is applied, the decrease in molecular

241

weight reduces the number per chain of hydrogen bonding sites leading, at the same

242

time, to a reduction of the intermolecular hydrogen bonding between polymer

243

molecules, that is, the attractions between guar chains (Cheng et al., 2002).

244

In general the consistency index k of the untreated fenugreek dispersions followed the

245

trend FGH>FGA>FGB, which is in accordance with their galactomannan content as

246

seen in Table 1. Among all stabilizers investigated FGH, GG and LGB exhibited the

247

highest viscosity values in this order but this trend was maintained throughout the

248

various ultrasonication treatments.

249

Sav et al. (2013) found that dispersions of 1% gum showed a shear thinning

250

pseudoplastic behavior and exhibited apparent viscosities in the order of

251

GG>XG>LBG>HPFG, where HPFG was a highly purified fenugreek gum. Moreover,

252

mechanical spectra of LBG, GG and FG with comparable molecular weights were

253

similar in shape, but with slightly smaller moduli values for fenugreek gum (Prajapati

254

et al., 2013; Brummer et al., 2003).

255

In our case different FG dispersions could present differences according to their

256

purification and consequently their galactomannan content as mentioned above.

SC

M AN U

TE D

EP

AC C

257

RI PT

231

258

3.3 Emulsion droplet size

259

In Table 3 the size properties of HS and US emulsions containing 0 (control), 0.25

260

and 0.5 %wt galactomannans are summarized. The size of the emulsion droplets was

261

affected by gum concentration. In the absence of stabilizer HS emulsions presented

262

the greatest droplet, size whereas gum addition resulted in finer emulsions. The

263

decrease in droplet size upon gum concentration increase has been attributed to the 8

ACCEPTED MANUSCRIPT viscosity increase of the continuous phase and consequent droplet immobilization,

265

hence living more time for the protein to adsorb and stabilize the droplet (Kiosseoglou

266

and Doxastakis, 1988; Makri and Doxastakis, 2006a; Tsaliki et al., 2004). Moreover,

267

the droplet deformation can be described by Weber number (We=Gηr/2γ). It increases

268

with increase in the Weber number, which is proportional to the external stress Gη

269

(where G is the velocity gradient and η is the viscosity). Additionally, a reduction in

270

droplet size is achieved by reducing the interfacial tension γ (Tadros et al., 2004).

271

Τhus, viscosity increase to certain levels can be beneficial regarding droplet size

272

decrease.

273

Finer oil droplets were formed when sonication was applied, resulting in micron-sized

274

particles at 0.5 %wt gum, with slight differences in between them. However, control

275

samples presented the lowest droplet size (~1.2 µm). US impact was observed for all

276

emulsions prepared, but in FGB and FGA emulsions there was no concentration

277

effect. The viscosity of these gums is low compared to all other ones and is extremely

278

reduced by ultrasonication (Figure 1 and 2). Thus, concentration effects on droplet

279

size, using US, are hardly shown.

280

In our previous findings the smallest droplet size (D50 = 0.651 µm) was observed in

281

model emulsions prepared with LBG at 0.5% wt at pH 7 after 4 min of sonication

282

(Kaltsa et al., 2013). Ultrasonication intensity increase, at these experimental

283

conditions, was more efficient in decreasing the polydispersity of the emulsions rather

284

than remarkably minimizing the size of the smaller droplets in the system.

285

In this study, droplet size in the presence of gums was found to be around 1.4 µm,

286

greater than that found in the previous study, even though the volume size of the

287

sample processed was reduced from 100 ml to 40 ml. Moreover, droplet size did not

288

depend on hydrocolloid type used (p<0.05).

289

emulsifying ability of whey protein was lower at pH ~ 4. At neutral pH values, oil

290

droplets possess a negative charge and are strongly repelled. On the contrary at pH

291

values near pI the surface charge diminishes and repulsive forces are weakened,

292

leading to enhanced attraction of the droplets. It is the charge and magnitude of the

293

ionisable groups of protein on oil droplet that affect the formation and stability of

294

emulsions, as a highly charged interface induces the electrostatic repulsion between

295

droplets (Guzey & McClements, 2007).

AC C

EP

TE D

M AN U

SC

RI PT

264

A possible explanation is that the

9

ACCEPTED MANUSCRIPT Another interesting aspect considering droplet size is the protein amount of the gums.

297

Fenugreek like all other galatomannans does not carry any electrostatic charge to

298

interact with the positively charged WPI on droplet interface. However, proteinaceous

299

moieties at concentration as low as 0.1 wt % in fenugreek gums may impart an

300

amphiphilic property to the gum which enables it to act somehow like an

301

emulsifier (Brummer et al., 2003; Youssef et al., 2009). The same researchers also

302

reported that reducing protein content reduced the surface activity of purified

303

fenugreek gum compared to unpurified gum.

304

Gaonkar (1991) also found that purified guar or locust bean gum showed no surface

305

activity. This could justify the fact that fractions of higher protein content like FGB

306

and FGA resulted in lower droplet size than FGH. However, GG and LBG that also

307

had high protein content did not result in low droplet size emulsions. Comparing also

308

FGB and GG of approximately the same protein content, FGB resulted in lower

309

droplet size (p<0.05) in all cases. This suggests that FGB can be a more effective

310

emulsifier than other galactomannans.

311

According to Sav et al. (2013) the addition of FG successively formed emulsions. FG

312

formed a thick film between adjacent droplets that stabilized the emulsions against

313

flocculation and prevented coalescence by steric stabilization owing to its high

314

surface and interfacial activity and zeta potential values compared to GG and LBG.

315

The proteins of the gums also have another particular role due to their denaturation.

316

Specifically, in our case, it is most likely that these protein moieties are denaturated

317

during gum solution preparation (90 oC, 90 min) and previous gum isolation process.

318

Fenugreek seed proteins are mainly composed of S-like globulins and vicilins (Kruse

319

Fæste et al., 2010). Tang (2008), who studied the denaturation of vicilin fractions by

320

means of DSC (Differential Scanning Calorimetry), found that their denaturation

321

temperature peak lays between 85-90 oC.

322

emulsifying properties of proteins is considered ambiguous. Moderate heating for 5

323

min at 90oC was beneficial regarding emulsifying properties of soy whey isolate due

324

to denaturation of 7S and 11S globulins (Palazolo et al., 2004; Mitidieri and Wagner,

325

2002). However, extended thermal treatment (95 oC - 15 min) decreased their

326

emulsifying properties by shifting the average droplet size to higher values, assigned

327

to denaturation of both β-conglycinin and glycinin causing formation of aggregates

328

adsorbing at the interface. As a consequence more protein was necessary to obtain

329

stable emulsions (Keerati-u-rai and Corredig, 2009). Nevertheless, fenugreek gum

AC C

EP

TE D

M AN U

SC

RI PT

296

The role of thermal denaturation on

10

ACCEPTED MANUSCRIPT derived proteins represent a small proportion (maximum concentration 0.02 % in the

331

total emulsion formulation) in comparison to whey protein. However, it seems that

332

they contribute in some extent to emulsification, as FGH, a gum without any protein,

333

gives always at 0.25% wt larger oil droplets (p<0.05).

334

This is in accordance to Brummer at al. (2003), who found that the purified fenugreek

335

gum demonstrated less surface activity compared to the unpurified gum and to

336

Dickinson (2003) saying that the apparent surface activity of gum is attributed to the

337

presence of small amounts of protein impurities.

338

3.4 Emulsion microstructure

339

As seen in Fig. 3 emulsions were highly susceptible to flocculation regardless of gum

340

concentration or method applied (for presentation reasons only FGH samples are

341

displayed). In our case, the concentration of protein is considerably above that

342

required for saturation monolayer coverage (Dickinson, 1999), Therefore it is more

343

likely that the main drive of droplet aggregation would be depletion phenomena

344

induced by unabsorbed protein and/or polysaccharide.

345

3.5 Effect of ultrasonication on viscosity properties of emulsions

346

Viscosity measurements were performed only for emulsions at 0.5 %wt gum

347

concentration, which were stable for at least 24 hours. Data concerning control

348

samples are also shown for comparison reasons, revealing less thickened and

349

pseudoplastic behavior. The viscosity of HS emulsions containing different gums

350

followed the same trend as the pure gums solutions (FGH>GG>LBG>FGA>FGB),

351

indicating that the emulsion viscosity is mainly governed by the viscosity of the

352

continuous phase. It can be denoted (Fig. 4, Table 4) that ultrasonication caused a

353

great decrease in emulsion viscosity and flow curve shape changed, so that

354

differences among samples were not distinguishable (p<0.05). Samples became less

355

pseudoplastic as flow index values were higher than those found in HS treatment

356

(Table 4) without significant differences among them (p<0.05). Even though droplet

357

size reduction may lead to higher emulsion viscosity via increase in droplet packing

358

and subsequent immobilization throughout the emulsion matrix, this was not observed

359

in our samples. The continuous phase viscosity was in all cases very low and any

360

increase by droplet packing was not able to increase it considerably.

AC C

EP

TE D

M AN U

SC

RI PT

330

361 362

3.6 Stability of emulsions prepared by HS and US emulsification 11

ACCEPTED MANUSCRIPT In Fig. 5 a,b the evolution of serum indices during storage of HS emulsions are

364

depicted. Control samples were the least stable exhibiting faster destabilization kinetic

365

owed to the combined effect of highest droplet size and lowest consistency values.

366

Samples containing 0.25 % galactomannan gum became quickly very unstable and the

367

serum index at day 10 of storage ranged between 37.2 to 40.1 %, without any

368

significant difference among samples (p<0.05). However, it should be noticed that the

369

kinetics of the serum index followed the opposite order of the viscosity of the gums,

370

hence FGB>FGA>LBG>GG>FGH. Thus, emulsions containing FGB had the

371

smallest droplet size (D50 = 4.06 µm) compared to all other emulsions, but they

372

destabilized faster, assuming that creaming is majorly governed by the continuous

373

phase viscosity. The same phenomenon was observed in emulsions containing FGB

374

0.25 % prepared by ultrasonication, for which D50 = 1.38 µm and SI = 30.7 % (Fig.

375

5c). Overall, the stability of emulsions was increased for samples by applying

376

sonication treatment, since the SI was reduced and ranged from 24.9 to 33.4 %.

377

Nevertheless, the above improvement occurs due to the reduction of the average

378

droplet size, taking into account the severe concurrent continuous phase viscosity

379

reduction during sonication.

380

As expected, the stability of the emulsions was significantly increased upon increase

381

of gum concentration at 0.5 %wt (Fig. 5d) as a result of the combined effect of droplet

382

size reduction (as discussed above) and the increased viscosity of the aqueous phase.

383

High-molecular weight polysaccharides keep the droplets apart after formation and

384

thus, protect them against creaming, flocculation and coalescence (Akthar and

385

Dickinson, 2003). Moreover stability differences among samples are more

386

pronounced at the end of the storage. FGH presented the highest stability against

387

creaming (SI = 4.9 %), whereas a limited improvement was assessed for FGB

388

samples, for which SI = 35.0 %.

389

In contrast to 0.25 %wt HS emulsions, a lag time period was observed up to 5 days

390

for GG and FGH emulsions before they became phase separated. It is also interesting

391

to note that the same trend of stability kinetics was maintained as in the case of 0.25

392

%wt HS emulsions. In particular, the SI of 0.5 %wt HS emulsions containing gums

393

followed the order FGB>FGA≥LBG>GG>FGH, also in accordance to the opposite of

394

viscosity order of the gums.

395

beneficial regarding the stability against creaming in all cases of 0.5 %wt emulsions

396

(Fig. 5d). For US emulsions containing gum, the SI ranged between 3.5 to 20.7 %,

AC C

EP

TE D

M AN U

SC

RI PT

363

The overall effect of ultrasonic application was

12

ACCEPTED MANUSCRIPT resulting in a total reduction of the SI by 54.3, 40.8, 12.2, 58.3, and 47.1 % for FGA,

398

FGB, FGH, GG and LBG respectively, in comparison to their HS counterparts.

399

Despite that the droplet size (as shown previously) was not affected by concentration

400

increase in FGB emulsions, the stability of 0.5 %wt samples was improved as

401

evidenced by slower serum formation kinetic. Noticeably, the stability of US control

402

samples was higher than those containing 0.25 % gum as well as the one containing

403

0.5 %wt FGB, probably attributed to their smaller particle size.

404

Huang et al. (2001) who compared the stability of emulsions containing different

405

hydrocolloids have shown that long term stability without phase separation could be

406

achieved upon use of higher concentration of fenugreek, guar and locust gums (1-1.5

407

%wt), attributed to droplet movement restriction caused by continuous phase viscosity

408

increase. More recently, different studies (Perrechil & Cunha, 2010; Farshchi et al.,

409

2013; Chung et al., 2013) have shown that a concentration of 0.6-0.8 %wt locust gum

410

was needed to stabilize via viscosity enhancement mechanism emulsions more

411

susceptible to creaming due to pH (close to the protein’s isoelectric point) or low oil

412

concentration. Similarly, (Neirynck et al., 2007), guar gum addition at 1 %wt level

413

could effectively stabilize emulsions containing 0.3% sodium caseinate at pH 6.5.

414

4 Conclusions

415

The droplet size, viscosity and stability of emulsions were affected by galactomannan

416

type and concentration as well as emulsification method used for preparation. The

417

droplet size was reduced in respect with the protein amount of FGs, whereas stability

418

depended on their viscosity. In total, at the experimental conditions used, unpurified

419

FG, with proteins (FGB), was more efficient in reducing the droplet size compared to

420

LBG or GG in most of the cases, whereas purified FG type (FGH) with the highest

421

galactomannan content and viscosity resulted in the formation of the most stable

422

emulsions. Concluding, FGs can be used as stabilizers in emulsions, replacing GG or

423

LBG successfully. Increase in their concentration might have a positive contribution

424

to droplet size reduction, due to further absorption on droplet surface, and to stability

425

improvement, via viscosity increase. Alternative methods of preparation e.g. pre-

426

emulsification of oil-emulsifier blend and dilution with stabilizer solution should be

427

examined.

428

Acknowledgements

429

This research has been co-financed by the European Union (European Social Fund –

430

ESF) and Greek national funds through the Operational Program "Education and

AC C

EP

TE D

M AN U

SC

RI PT

397

13

ACCEPTED MANUSCRIPT Lifelong Learning" of the National Strategic Reference Framework (NSRF) -

432

Research Funding Program: Heracleitus II. Investing in knowledge society through

433

the European Social Fund.

434

The authors would like to thank Arla Foods Hellas for kindly donating whey protein

435

isolates, and Air Green Co for fenugreek gum powders. We gratefully acknowledge

436

Alexandra Kiskini for performing the protein measurements at Wageningen

437

University.

RI PT

431

438

References

440

AACC International (2004). Approved Methods of Analysis. AACC Methods 44-16.

441

St. Paul, MN: AACC International.

442

Acharya, S.N., Thomas, J.E. & Basu, S.K. (2008). Fenugreek, an alternative crop for

443

semiarid regions of North America. Crop Science, 48, 841-853.

444

Akthar, M., & Dickinson, E. (2003). Emulsifying properties of whey protein-dextran

445

conjugates at low pH and different salt concentrations. Colloids and Surfaces B:

446

Biointerfaces, 31, 125–132.

447

Alftrén, J., Peñarrieta, J.M., Bergenståhl, B., & Nilsson, L. (2012). Comparison of

448

molecular and emulsifying properties of gum arabic and mesquite gum using

449

asymmetrical flow field-flow fractionation. Food Hydrocolloids, 26(1), 54-62.

450

Amin, R., Abdul-Ghani, A.S., & Suleiman, M.S. (1988). Effect of fenugreek and

451

lupin seeds on the development of experimental diabetes in rats. Planta Medica, 54,

452

286–290.

453

Anon., (2012b). Guar Gum Up Nearly 20×: Another Sticky Commodity Situation,

454

Available from: http://www.spendmatters.com/index.cfm/2012/4/9/Guar-Gum-

455

Ansari, S.A., Matricardi, P., Cencetti, C., Di Meo, C., Carafa, M., Mazzuca,

456

C., Palleschi, A., Capitani, D., Alhaique, F., & T. Coviello. (2013). Sonication-Based

457

Improvement of the Physicochemical Properties of Guar Gum as a Potential Substrate

458

for Modified Drug Delivery Systems. BioMed Research International, 2013, 11

459

pages.

460

Bahamdan, A., & Daly, W.H. (2006). Poly(oxyalkylene) grafts to guar gum with

461

applications in hydraulic fracturing fluids. Polymers Advanced Technology 17, 679–

462

681.

463

Barati, R., Johnson, S.J., McCool, S., Green, D.W., Willhite, G.P., Liang, J.-T.

464

(2011). Fracturing fluid cleanup by controlled release of enzymes from

AC C

EP

TE D

M AN U

SC

439

14

ACCEPTED MANUSCRIPT polyelectrolyte complex nanoparticles. Journal of Applied Polymer Science, 121,

466

1292–1298.

467

Bartley, G.B., Hiltry, M.D., Andreson, B.D., Clairemont, A.C., & Maschke, S.P.

468

(1981). “Maple-syrup” urine odor due to fenugreek ingestion. The New England

469

Journal of Medicine, 305, 467.

470

Brummer, Y., Cui, W., & Wang Q. (2003). Extraction, purification and

471

physicochemical characterization of fenugreek gum. Food Hydrocolloids, 17, 229–

472

236.

473

Buron, H., Mengual, O., Meunier, G., Cayre, I., & Snabre, P. (2004). Optical

474

characterizationof concentrated dispersions: applications to laboratory analyses and

475

on-line process monitoring and control. Polymer International, 53, 1205–1209.

476

Chau, C.F., Cheung, P.C.K., & Wong, Y.S. (1997). Functional properties of protein

477

concentrates from three Chinese indigenous legume seeds. Journal of Agricultural

478

and Food Chemistry, 45(7), 2500–2503.

479

Chau, C.F., & Huang, Y.L. (2003). Comparison of the chemical composition and

480

physicochemical properties of different fibres prepared from the peel of Citrus

481

sinensis L. Cv. Liucheng. Journal of Agricultural Food Chemistry, 51, 2615–2618.

482

Cheng, Y., Brown, K.M., & Prud'homme, R.K. (2002). Characterization and

483

intermolecular interactions of hydroxypropyl guar solutions. Biomacromolecules,

484

3(3), 456–461.

485

Chung, K.M., Moon, T.W., Kim, H., & Chun, J.K. (2002). Physicochemical

486

properties of sonicatedmung bean, potato, and rice starches. Cereal Chemistry, 79,

487

631–633.

488

Chung, C., Degner, B., & McClements, D.J. (2013). Designing reduced-fat food

489

emulsions: Locust bean gum–fat droplet interactions. Food Hydrocolloids, 32(2),

490

263–270.

491

Dickinson, E. (2003). Hydrocolloids at interfaces and the influence on the properties

492

of dispersed systems. Food Hydrocolloids, 17(1), 25-39

493

Farshchi, A., Ettelaie, R., & Holmes, M. (2013). Influence of pH value and locust

494

bean gum concentration on the stability of sodium caseinate-stabilized emulsions.

495

Food Hydrocolloids, 32(2), 402–411.

496

Galla, N.R., & Dubasi, G.R. (2010). Chemical and functional characterization of gum

497

karaya (Sterculia urens L.) seed meal. Food Hydrocolloids, 24, 479–485.

AC C

EP

TE D

M AN U

SC

RI PT

465

15

ACCEPTED MANUSCRIPT Gaonkar, A.G. (1991). Surface and interfacial activities and emulsion characteristics

499

of some food hydrocolloids. Food Hydrocolloids, 5, 329–337.

500

Gharibzahedi, S.M.T., Razavi, S.H., & Mousavi, S.M. (2013). Psyllium husk gum:

501

An attractive carbohydrate biopolymer for the production of stable canthaxanthin

502

emulsions. Carbohydrate Polymers, 92(2), 2002-2011.

503

Gorji, E.G., & Mohammadifar, M.A. (2014). Characterisation of gum tragacanth

504

(Astragalus gossypinus)/ sodium caseinate complex coacervation as a function of pH

505

in an aqueous medium. Food Hydrocolloids, 34, 161-168.

506

Guzey, D. & McClements, D. J. (2007). Impact of electrostatic interactions on

507

formation and stability of emulsions containing oil droplets coated by β-lactoglobulin-

508

pectin complexes. Journal of Agricultural and Food Chemistry, 55, 475-485.

509

Hamden, K., Jaouadi, B., Salami, T., Carreau, S., Bejar, S., and Elfeki, A. (2010).

510

Modulatory effect of fenugreek saponins on the activities of intestinal and hepatic

511

disaccharidase and glycogen and liver function of diabetic rats. Biotechnology and

512

Bioprocess Engineering, 15(5), 745-753.

513

Hannan J.M.A., Ali L., Rokeya B., Khaleque J., Akhter M., Flatt P.R., & et al. (2007).

514

Soluble dietary fibre fraction of Trigonella foenum-graecum (fenugreek) seed

515

improves glucose homeostasis in animal models of type 1 and type 2 diabetes by

516

delaying carbohydrate digestion and absorption, and enhancing insulin action. British

517

Journal of Nutrition, 97, 514–521.

518

Haq, M.A., Alam M.J., & Hasnain A. (2013). Gum Cordia: A novel edible coating to

519

increase the shelf life of Chilgoza (Pinus gerardiana). LWT - Food Science and

520

Technology, 50(1), 306-31.

521

Hooda, S., & Jood, S. (2005). Organoleptic and nutritional evaluation of wheat

522

biscuits supplemented with untreated and treated fenugreek flour. Food Chemistry,

523

90(3), 427–435.

524

Huang, X., Kakuda, Y., & Cui, W. (2001). Hydrocolloids in emulsions: particle size

525

distribution and interfacial activity. Food Hydrocolloids, 15, 533–542.

526

Im, K.K. and Maliakel, B. (2008). Fenugreek dietary fibre a novel class of functional

527

food ingredient. Agrofood industry hi-tech, 19, 18-21.

528

Işıklı, N.D., & Karababa, E. (2005). Rheological characterization of fenugreek paste

529

(çemen). Journal of Food Engineering, 69, 185–190.

AC C

EP

TE D

M AN U

SC

RI PT

498

16

ACCEPTED MANUSCRIPT Jambrak, A.R., Herceg, Z., Subaric, D., Babic, J., Brncic, M., Brncic, S.R., Bosiljkov,

531

T., Cvek, D., Tripalo, B., & Gelo, J. (2010). Ultrasound effect on physical properties

532

of corn starch Carbohydrate Polymers, 79, 91–100.

533

Jindal, M., Rana, V., Kumar V., Singh, R.S., Kennedy, J.F., & Tiwary, A.K. (2013).

534

Sulfation of Aegle marmelos gum: Synthesis, physico-chemical and functional

535

characterization. Carbohydrate Polymers, 92(2), 1660-1668.

536

Jourdain, L., Leser, M.E., Schmitt, C., Michel, M., & Dickinson, E. (2008). Stability

537

of emulsions containing sodium caseinate and dextran sulfate: Relationship to

538

complexation in solution. Food Hydrocolloids, 22(4), 647-659.

539

Kaltsa, O, Michon, C, Yanniotis, S, & Mandala, I. (2013). Ultrasonic energy input

540

influence οn the production of sub-micron o/w emulsions containing whey protein

541

and common stabilizers. Ultrasonics Sonochemistry, 20(3), 881-891.

542

Kaltsa, O., Gatsi, I.,

543

Ultrasonication Parameters on Physical Characteristics of Olive Oil Model

544

Emulsions Containing Xanthan. Food and Bioprocess Technology, 7(7), 2038-2049.

545

Keerati-u-rai, M., & Corredig, M. (2009). Heat-induced changes in oil-in-water

546

emulsions stabilized with soy protein isolate. Food Hydrocolloids, 23 (8), 2141–2148

547

Kinsella, J.E. (1979. Functional properties of soy protein. Journal of American Oil

548

Chemists Society, 56, 242–249.

549

Kiosseoglou, V., & Doxastakis G. (1988). The emulsification properties of soybean

550

protein in the presence of polysaccharides. LWT - Food Science and Technology, 21,

551

33–35.

552

Koocheki, A., Kadkhodaee, R.,

553

(2009). Influence of Alyssum homolocarpum seed gum on the stability and flow

554

properties of O/W emulsion prepared by high intensity ultrasound. Food

555

Hydrocolloids, 23(8), 2416–2424.

556

Koocheki, A., & Kadkhodaee, R. (2011). Effect of Alyssum homolocarpum seed gum,

557

Tween 80 and NaCl on droplets characteristics, flow properties and physical stability

558

of ultrasonically prepared corn oil-in-water emulsions. Food Hydrocolloids, 25(5),

559

1149–1157.

560

Korman, S.H., Cohen, E., & Preminger, A. (2001). Pseudo-maple syrup urine disease

561

due to maternal prenatal ingestion of fenugreek. Journal of Paediatrics and Child

562

Health, 37, 403–404.

SC

RI PT

530

(2014). Influence of

Mortazavi, S.A., Shahidi, F., & Taherian, A.R.

AC C

EP

TE D

M AN U

Yanniotis, S., & Mandala, I.

17

ACCEPTED MANUSCRIPT Kruse Fæstea, C., Christians, U., Egaas, E., & Jonscher, K.R. (2010). Characterization

564

of potential allergens in fenugreek (Trigonella foenum-graecum) using patient sera

565

and MS-based proteomic analysis. Journal of Proteomics, 73 (7), 1321–1333.

566

Lazos, E.S. (1992). Certain functional properties of defatted pumpkin seed flour.

567

Plant Foods for Human Nutrition, 42, 257–273.

568

López-Franco, Y.L., Cervantes-Montaño, C.I., Martínez-Robinson, K.G., Lizardi-

569

Mendoza, J., & Robles-Ozuna, L.E. (2013). Physicochemical characterization and

570

functional properties of galactomannans from mesquite seeds (Prosopis spp.). Food

571

Hydrocolloids, 30(2), 656-660.

572

Madar, Z., Abel, R., Samish, S., & Arad, J. (1988). Glucose-lowering effect of

573

fenugreek in non-insulin dependent diabetes. European Journal of Clinical Nutrition,

574

42, 51–54.

575

Makri, E., & Doxastakis, G. (2006). Study of emulsions and foams stabilized

576

with Phaseolus vulgaris or Phaseolus coccineus with the addition of xanthan gum or

577

NaCl. Journal of the Science Food and Agriculture, 86(12), 1863-1870.

578

Mazza, G., Di Tommaso, D., & Foti, S. (2002). Volatile constituents of sicilian

579

fenugreek (Trigonella foenum-graceumL.) seeds. Sciences des Aliments, 22 (), pp.

580

249–264.

581

Mengual, O., Meunier, G., Cayré, I., Puech, K, & Snabre, P. (1999a). TURBISCAN

582

MA 2000: multiple light scattering measurement for concentrated emulsion and

583

suspension instability analysis. Talanta, 50(2), 445-56.

584

Mengual, O., Meunier, G., Cayre, I., Puech, K., & Snabre, P. (1999b).

585

Characterisation of instability of concentrated dispersions by a new optical analyser:

586

the TURBISCAN MA 1000. Colloids and Surfaces A: Physicochemical and

587

Engineering Aspects, 152(1), 111-123.

588

Mirhosseini, H. & Amid, B.T. (2012). Influence of Chemical Extraction Conditions

589

on the Physicochemical and Functional Properties of Polysaccharide Gum from

590

Durian (Durio zibethinus) Seed. Molecules, 17(6), 6465-6480.

591

Mitidieri, F.E., Wagner, J.R. (2002). Coalescence of o/w emulsions stabilized by

592

whey and isolate soybean proteins. Influence of thermal denaturation, salt addition

593

and competitive interfacial adsorption. Food Research International, 35(6), 547–557.

594

Neirynck, N., Van lent, K., Dewettinck, P., & Van der Meeren, P. (2007). Influence of

595

pH and biopolymer ratio on sodium caseinate-guar gum interactions in aqueous

596

solutions and in O/W emulsions. Food Hydrocolloids, 21, 862–869.

AC C

EP

TE D

M AN U

SC

RI PT

563

18

ACCEPTED MANUSCRIPT Palazolo, G.G., Sorgentini, D.A, & Wagner, J.R. (2004). Emulsifying properties

598

and surface behavior of native and denatured whey soy proteins in comparison

599

with other proteins. Creaming stability of oil-in-water emulsions. Journal of the

600

American Oil Chemists' Society, 81(7), 625-632.

601

Palazolo, G.G., Sorgentini, D.A., & Wagner, J.R. (2005). Coalescence and

602

flocculation in o/w emulsions of native and denatured whey soy proteins in

603

comparison with soy protein isolates Food Hydrocolloids, 19(3), 595-604.

604

Pizzino, A., Catte´, M., Van Hecke, E., Salager, J.L., & Aubry, J.M. (2009). On-line

605

backscattering tracking of the transitional phase inversion of emulsions. Colloids Surf.

606

A: Physicochemical and Engineering Aspects, 338, 148–154.

607

Perrechil, F.A., & Cunha, R.L. (2010). Oil-in-water emulsions stabilized by sodium

608

caseinate: influence of pH, high-pressure homogenization and locust bean gum

609

addition. Journal of Food Engineering, 97, 441–448.

610

Prajapati, V.D.,

611

Naikwadi, N.N., & Variya, B.C. (2013). Galactomannan: A versatile biodegradable

612

seed polysaccharide. International Journal of Biological Macromolecules, 60, 83-92.

613

Prajapat, A.L., & Gogate, P.R. (2015). Depolymerization of guar gum solution using

614

different approaches based on ultrasound and microwave irradiations. Chemical

615

Engineering and Processing: Process Intensification, 88, 1-9.

616

Qian, H.F., Cui, S.W., Wang, Q., Wang, C., & Zhou, H.M. (2011). Fractionation and

617

physicochemical

618

Hydrocolloids, 25(5), 1285-1290.

619

Raghuram, T.C., Sharma, R.D., & Sivakumar, B. (1994). Effect of fenugreek seeds on

620

intravenous glucose disposition in non-insulin dependent diabetic patients.

621

Phytotherapy Research, 8, 83–86.

622

Rao, P.U., Sesikeran, B., Rao, P.S., Naidu, A.N., Rao, V.V., &. Ramacjandran, E.P.

623

(1996). Short-term nutritional and safety evaluation of fenugreek. Nutrition Research,

624

16, 1495–1505.

625

Ribes, G., Sauvaire, Y., Da Costa, C., Baccou, J.C., & Loubatieres-Mariani, M.M.

626

(1986). Antidiabetic effects of subfractions from fenugreek seeds in diabetic dogs.

627

Proceedings of the Society for Experimental Biology and Medicine, 182, 159–166.

628

Roberts, K.T., Cui, S.W., Chang, Y.H., Ng, P.K.W., & Graham T. (2012). The

629

influence of fenugreek gum and extrusion modified fenugreek gum on bread. Food

630

Hydrocolloids, 26(2), 350-358.

M AN U

SC

RI PT

597

TE D

Jani, G.K., Moradiya, N.G., Randeria, N.P., Nagar, B.J.,

of

peach

gum polysaccharides.

Food

AC C

EP

characterization

19

ACCEPTED MANUSCRIPT Sav A.R., Meer T.A., Fule R.A., & Amin P.D. (2013). Investigational Studies on

632

Highly Purified Fenugreek Gum as Emulsifying Agent. Journal of Dispersion Science

633

and Technology, 34(5), 657-662.

634

Sewell, A.C., Mosandl, A., & Bohles H. (1999). False diagnosis of maple syrup urine

635

disease owing to ingestion of herbal tea. Massachusetts Medical Society, 341, 769.

636

Shankaracharya, N.B., Anandaraman, S., & Natarajan, C.P. (1973). Chemical

637

composition of raw and roasted fenugreek seeds. Journal of Food Science &

638

Technology, 10, 179–18.

639

Sharma, R., & Raghuram, T.C. (1990). Hypoglycemic effect of fenugreek seeds in

640

non-insulin dependent diabetic subjects. Nutrition Research, 10, 731–739.

641

Soleimanpour, M., Koocheki, & A., Kadkhodaee, R. (2013). Effect of Lepidium

642

perfoliatum seed gum addition on whey protein concentrate stanilized emulsions

643

stored at cold and ambient temperature. Food Hydrocolloids, 30(1), 292-301.

644

Srinivasan, K. 2006. Fenugreek (Trigonella foenum-graccum): a review of health

645

beneficial physiological effects. Food Reviews International, 22, 203–224.

646

Srichamroen, A., Thomson, A.B.R., Field, C.J., & Basu, T.K. (2009). In vitro

647

intestinal glucose uptake is inhibited by galactomannan from Canadian fenugreek

648

seed (Trigonella foenum graecum L) in genetically lean and obese rats. Nutrition

649

Research, 29(1), 49–54.

650

Tadros, T., Izquierdo, P., Esquenca, J. & Solans, C. (2004). Formation and stability of

651

nano-emulsions. Advances in Colloid and Interface Science, 108-109, 303-318

652

Tang, C.T. (2008). Thermal denaturation and gelation of vicilin rich protein isolates

653

from Phaseolus legumes: A comparative study. LWT - Food Science and Technology,

654

41, 1380-1388.

655

Tiwari, B.K., Muthukumarappan, K., O'Donnell, C.P. & Cullen, P.J. (2010).

656

Rheological Properties of Sonicated Guar, Xanthan and Pectin Dispersions.

657

International Journal of Food Properties, 13(2), 223-233.

658

Tsaliki, E., Pegiadou, S., & Doxastakis, G. (2004). Evaluation of the emulsifying

659

properties of cottonseed protein isolates. Food Hydrocolloids, 18, 631–637.

660

Vaughn, S.F., Kenar, J.A., Felker, F.C., Berhow, M.A., Cermak, S.C., Evangelista,

661

R.L, Fanta, G.F., Behle, R.W., & Lee E. (2013). Evaluation of alternatives to guar

662

gum as tackifiers for hydromulch and as clumping agents for biodegradable cat litter.

663

Industrial Crops & Products, 43, 798-801.

AC C

EP

TE D

M AN U

SC

RI PT

631

20

ACCEPTED MANUSCRIPT Youssef, M.K., Wang, Q., Cui, S.W., & Barbut, S. (2009). Purification and partial

665

physicochemical characteristics of protein free fenugreek gums. Food Hydrocolloids,

666

23, 2049–2053.

667

Zinoviadou, K.G., Scholten, E., Moschakis, T., & Biliaderis, C.G. (2012). Properties

668

of emulsions stabilised by sodium caseinate–chitosan complexes. International Dairy

669

Journal, 26(1), 94-101.

RI PT

664

AC C

EP

TE D

M AN U

SC

670

21

ACCEPTED MANUSCRIPT 671

Figure 1

672

a Utreated 70%-1 min

2

70%-2 min 70%-3 min

70%-3 min + 90%-1 min 0 10 Rate (s-1)

100

SC

1

673

M AN U

b

Untreated

70%-1 min

2

70%-2 min 70%-3 min

70%-3 min + 90%-1 min

0

10 Rate (s-1)

100

AC C

EP

1

TE D

Apparent viscosity (Pa-s)

4

674

RI PT

Αpparent viscosity (Pa-s)

4

22

ACCEPTED MANUSCRIPT

c

14 12 10

Untreated

8

70%-1 min

6

70%-2 min

4

70%-3 min

2

70%-3 min + 90%-1 min

RI PT

Apparent viscosity (Pa-s)

16

0 1

10 100 -1 Rate (s )

675

SC

d

14 12

Untreated

10

70%-1 min

8

70%-2 min

6 4

70%-3 min

2

70%-3 min +90%-1 min

0 1

10 Rate (s-1)

100

TE D

676 16 14 12 10 6 4

e Untreated 70%-1min

EP

8

AC C

Apparent viscosity (Pa-s)

M AN U

Aapparent viscosity (Pa-s)

16

70%-2min 70%-3min

2

70%-3min +90%-1min

0

1

677

10 Rate (s-1)

100

678 679

23

ACCEPTED MANUSCRIPT 680

Figure 2 FGA

6

FGB

FGH

GG

LBG

b b

4 3 2

c a

b b a

b

a

1

b a

b

a a

b ab b

ab a

c bc bc

M AN U

SC

0

a ab

RI PT

Apparent viscosity 10 s-1 (Pa-s)

c

5

Treatment type

681

685 686 687 688 689 690 691

EP

684

AC C

683

TE D

682

692 693 694 695 24

ACCEPTED MANUSCRIPT 696 697 698

Figure 3 FGH concentration (%wt) 0.25

0.5

HS method

B

C

US method

F

EP AC C

699

E

TE D

D

M AN U

SC

A

RI PT

0

25

ACCEPTED MANUSCRIPT 700

Figure 4 Control

FGA

FGB

FGH

GG

1

10 Shear rate ( s-1)

LBG

RI PT

1

0.1

0.01

SC

Apparent visosity (Pa-s)

10

0.1

100

1000

M AN U

0.001

701

(a)

702

Control

FGA

FGH

GG

LBG

TE D

1

EP

0.1

0.01

AC C

Apparent viscosity (Pa-s)

10

FGB

0.001

0.1

703 704

1

10 Shear rate (s-1)

100

1000

(b)

705

26

ACCEPTED MANUSCRIPT 706

Figure 5 Control

FGA

FGB

FGH

GG

LBG

50 d cd

30

RI PT

% Serum

40

20

0 0

2

4

8

M AN U

707

6 Storage days

SC

10

10

12

(a)

708 50

FGA

FGB

GG

LBG

TE D

e*

30

20

d*

c*

EP

% Serum

40

FGH

AC C

10

bc* a*

0

0

709 710

2

4

6 Storage days

8

10

12

(b)

711

27

ACCEPTED MANUSCRIPT Control

FGA

FGB

FGH

GG

LBG

50

c c b b

30 20 10

RI PT

% Serum

40

a

0 2

4

6 Storage days

M AN U

712

8

10

12

SC

0

(c)

713 714

FGA

FGB

40

LBG

30 20

d*

EP

% Serum

GG

TE D

50

FGH

AC C

10

bc* ab* a*

0

0

715 716

2

4

6 Storage days

8

10

12

(d)

717

28

ACCEPTED MANUSCRIPT 719

Table 1 Gum type

Galactomannan

Protein

Moisture

OHC

Odor

content*(%wt)

(%wt)

content

(g/100 g gum)

intensity**

47.37e ± 2.50

+

(%wt)

FGB FGH GG LBG

80.7

1.97 ± 0.01

61.2

3.92 ± 0.10

88.7

0.24 ± 0.02

np***

3.92 ± 0.03

np***

6.43 ± 0.00

10.38 ± 0.06 7.95 ± 0.2 11.3 ± 0.06

c

+++

d

-

b

70.97 ± 3.17 61.54 ± 0.56

RI PT

FGA

10.25 ± 0.03

77.59 ± 1.89

-

11.99 ± 0.14

a

-

138.02 ± 3.30

Samples with different letters differ significantly (p<0.05).

721

*As stated by the manufacturer/supplier.

722

**Where (+) indicates slight odor, (+++) intense odor and (-) odorless gum powder.

723

*** Not stated.

M AN U

SC

720

AC C

EP

TE D

724

30

ACCEPTED MANUSCRIPT

726

Table 2 Gum type

k (Pa-sn)

n (-)

FGA

4.356(± 0.473)

0.523(± 0.069)

FGB

2.546(± 0.1.299)

0.642(±0.078)

FGH

18.195(± 1.408)

0.333(± 0.007)

GG

14.183(± 1.221)

0.420(± 0.033)

LBG

11.060 (±2.190

0.543 ±0.083

R2 range between 0.98-0.999.

727

SC

728 729

M AN U

730 731 732

737 738 739 740

EP

736

AC C

735

TE D

733 734

RI PT

725

741 742 743 744 31

ACCEPTED MANUSCRIPT

Table 3 HS method D97.5 i

8.50

10.79

(± 0.61)

(± 0.54)

D2.5

D50

0.44

(± 0.40)

(± 0.05)

FGH

GG

LBG

4.41

(± 0.06)

6.36

3.04

(±0.12)

(± 0.35)

(± 0.09)

(± 0.04)

2.47

4.06 e

6.76

2.62

3.77 b*

(± 0.37)

(± 0.03)

(± 0.87)

(± 0.16)

(± 0.11)

e

3.18

6.45

(± 0.11)

(± 0.06) g

3.11

5.68

(± 0.06)

(± 0.08) f

3.33

4.53

(± 0.27)

(± 0.07)

8.26

3.00

(± 0.89)

(± 0.09)

5.27

7.31

2.88

(± 0.41)

(± 0.10)

6.19

3.09

(± 0.68)

(± 0.27)

746 Samples with different letters differ significantly (p<0.05).

e*

(± 0.09) 4.42

d*

(± 0.05) 4.22

cd*

(± 0.05)

1.24

D97.5

a

(± 0.05)

D2.5

5.52

0.62

1.46

D97.5

3.50

(± 0.11)

0.25 % b

D50

0.5 % 4.06

0.55

1.45 a*

3.37

(± 0.30)

(±0.08)

(± 0.06)

(±0.16)

(±0.07)

(± 0.07)

(±0.22)

5.42

0.55

1.38 b

3.81

0.51

1.38 a*

3.91

(± 0.12

(±0.02)

(± 0.06)

(±0.13)

(±0.04)

(± 0.13)

(±0.15)

c

7.35

0.65

1.78

(± 0.14)

(±0.05)

(± 0.17)

6.25

0.89

(± 0.11)

(±0.16)

2.04

cd

(± 0.09) d

5.78

0.90

2.15

(± 0.70)

(±0.09)

(± 0.19)

3.72

0.68

(±0.23)

(±0.04)

4.18

0.62

(±0.22)

(±0.06)

3.99

0.58

(±0.24)

(±0.05)

1.48

a*

(± 0.07) 1.46

a*

(± 0.08) 1.56

a*

(± 0.05)

3.58 (±0.24) 3.60 (±0.16) 3.66 (±0.12)

AC C

747

4.09

c*

TE D

FGB

3.01

0.5 %

D50

EP

FGA

D2.5

13.7

0.25 % ef

D97.5

SC

Control

D50

US method

M AN U

D2.5

RI PT

745

32

ACCEPTED MANUSCRIPT 748

Table 4 HS method Gum type Control

k (Pa-sn) a

0.022 (± 0.006)

n (-) f

0.781 (± 0.049) c

k (Pa-sn)

n (-)

a

0.793 (± 0.017)

b

0.027 (± 0.004)

f

FGA

2.745 (± 0.397)

0.511 (± 0.028)

0.570 (±0.053)

0.647e(±0.017)

FGB

1.856c(± 0.943)

0.545d(±0.064)

0.720b(±0.052)

0.652e(±0.013)

FGH

5.469f(± 0.611)

0.403a(± 0.017)

0.654b(±0.092)

0.630e(±0.006)

GG

4.338e(± 0.274)

0.451b(± 0.021)

0.571b(±0.053)

0.651e(±0.005)

LBG

3.902e ±0.137

0.531cd(± 0.008)

1.063bc(±0.072)

0.610e(±0.010)

Samples with different letters per property differ significantly (p<0.05).

750

R2 range between 0.992-0.999.

SC

749

RI PT

d

US method

AC C

EP

TE D

M AN U

751

33

ACCEPTED MANUSCRIPT Caption of Figures Figure 1. Apparent viscosity flow curves of 1 %wt gum solutions (a) FGA, (b) FGB, (c) FGH, (d) GG and (e) LBG, as affected by different sonication treatments.

RI PT

Figure 2. Apparent viscosity of 1 %wt gum solutions (at 10 s-1 rate) as affected by sonication treatment. Figure 3. Light microscopy photos of freshly prepared emulsions containing various amounts of FGH gum, prepared by HS (A, B, C) and US method (D, E, F).

Figure 4. Apparent viscosity flow curves of emulsions containing 0.5 %wt

SC

galactomannans prepared by a) HS and b) US method.

Figure 5. Stability of emulsions during storage at 5 oC prepared by HS method a) 0

%wt gum, and d) 0.5 %wt gum.

Caption of Tables

M AN U

(Control) and 0.25 %wt gum, b) 0.5 %wt gum and US method c) 0 (Control) and 0.25

galactomannan gums.

TE D

Table 1. Partial chemical composition, OHC and odor intensity of various

Table 2. Consistency (k) and flow behavior (n) values of untreated 1 %wt gum

EP

dispersions.

Table 3. Particle size cumulative diameters (D2.5, D50 and D97.5) of emulsions

AC C

prepared by HS and US method. Table 4. Consistency (k) and flow behavior (n) values of emulsions containing 0.5 %wt galactomannans prepared by HS and US method.

ACCEPTED MANUSCRIPT Highlights Fenugreek gum fractions present different droplet size and stabilizing effects

•

Stability was improved by ultrasonication only in more viscous samples

•

Fenugreek gum can replace other, common stabilizers

AC C

EP

TE D

M AN U

SC

RI PT

•