Ultrasonic Removal of Mucilage for Pressurized Liquid Extraction of

Subscriber access provided by University of Newcastle, Australia

Article

Ultrasonic Removal of Mucilage for Pressurized Liquid Extraction of Omega-3 Rich Oil from Chia Seeds (Salvia hispanica L.) Natalia Castejón, Pilar Luna, and Francisco J. Señoráns J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b05726 • Publication Date (Web): 07 Mar 2017 Downloaded from http://pubs.acs.org on March 8, 2017

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

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

Page 1 of 26

Journal of Agricultural and Food Chemistry

1

Ultrasonic Removal of Mucilage for Pressurized Liquid Extraction of Omega-3

2

Rich Oil from Chia Seeds (Salvia hispanica L.)

3

4

Natalia Castejón, Pilar Luna and Francisco J. Señoráns*

5

Healthy-Lipids Group, Sección Departamental de Ciencias de la Alimentación, Faculty

6

of Sciences, Universidad Autónoma de Madrid, 28049 Madrid, Spain

7

8 9

*Corresponding author: Francisco J. Señoráns [email protected]

10

11

12

13

14

ABSTRACT

15

Chia (Salvia hispanica L.) seeds contain an important amount of edible oil rich in

16

omega-3 fatty acids. Fast and alternative extraction techniques based on polar

17

solvents, such as ethanol or water, have become relevant for oil extraction in recent

18

years. However, chia seeds also contain a large amount of soluble fiber or mucilage,

19

which makes difficult an oil extraction process with polar solvents. For that reason, the

20

aim of this study was to develop a gentle extraction method for mucilage in order to 1 ACS Paragon Plus Environment


Page 2 of 26

21

extract chia oil with polar solvents using pressurized liquids and compare with organic

22

solvent extraction. The proposed mucilage extraction method, using an ultrasonic

23

probe and only water, was optimized at mild conditions (50 °C and sonication 3 min) to

24

guarantee the omega-3 oil quality. Chia oil extraction was performed using Pressurized

25

Liquid Extraction (PLE) with different solvents and their mixtures at five different

26

extraction temperatures (60, 90, 120, 150 and 200 °C). Optimal PLE conditions were

27

achieved with ethyl acetate or hexane at 90 °C in only ten minutes of static extraction

28

time (chia oil yield up to 30.93%). In addition, chia oils extracted with non-polar and

29

polar solvents by PLE were analyzed by gas chromatography–mass spectrometry (GC-

30

MS) to evaluate fatty acids composition at different extraction conditions. Chia oil

31

contained ∼65% of α-linolenic acid regardless of mucilage extraction method, solvent

32

or temperature used. Furthermore, tocopherols and tocotrienols were also analyzed

33

by HPLC in the extracted chia oils. The mucilage removal allowed the subsequent

34

extraction of the chia oil with polar or non-polar solvents by PLE producing chia oil

35

with the same fatty acid and tocopherol composition as traditional extraction.

36

37

Key words: α-linolenic acid, n-3 PUFA, accelerated solvent extraction, ultrasound

38

assisted extraction, Salvia hispanica, soluble fiber

39

40

INTRODUCTION

41

In recent years, there has been a growing interest and promising development of

42

unusual plants as alternative sources of vegetable edible oils. Novel plants like chia or

2 ACS Paragon Plus Environment

Page 3 of 26


43

camelina contain significant amount of oil with a good nutritional value, including

44

omega-3 fatty acids. Beneficial effects of omega-3 are related to reduce the risk of

45

cardiovascular diseases, prevention of nervous system problems and decrease

46

symptoms of inflammatory diseases, such as rheumatoid arthritis.1 Also, omega-3 fatty

47

acids play a vital role in the human physiology, especially during fetal and infant

48

growth.2 The World Health Organization (WHO) and health authorities in many

49

countries3,4 have promoted the intake of foods that contain high amounts of omega-3

50

fatty acids and an optimal ratio ω6/ω3. For that reason, food industry is looking for

51

fats and oils with specific characteristics in order to improve the lipid profile of the

52

final products.

53

Chia (Salvia hispanica L.) is an annual herbaceous plant belonging to the Lamiaceae

54

family, which is native to south of Mexico and north Guatemala5,6. In pre-Columbian

55

era, chia was one of the main crops in daily nutrition, but disappeared for centuries

56

until the middle of the 20th when it was rediscovered.7 Chia seed is considered a food

57

by the FDA (Food and Drug Administration) and hence is exempt from regulation.

58

Similarly, the European Commission authorized the placing on the market of chia seeds

59

as a novel food ingredient (2009/827/EC).8 In the last few years, the nutritional and

60

functional characteristics have gradually increased the importance of chia crop,

61

because of its oil quality, and the amount of natural antioxidants, fiber and proteins.9

62

Chia seeds contain a large amount of soluble fiber or mucilage, that it is easily

63

observed when chia seeds are soaked in water (see Figure 1), since seeds exude a

64

transparent mucilaginous gel that remains strongly attached to the coat seed.10,11

65

Mucilage is a polysaccharide with a high molecular weight, which is described as a

66

potential source of plant gum.12-14 However, mucilage also makes difficult the oil 3 ACS Paragon Plus Environment


Page 4 of 26

67

extraction process with polar solvents, such as ethanol or water. For that reason,

68

mucilage removal is necessary in order to extract chia oil with polar solvents. Methods

69

described in the literature are very aggressive for seeds and do not guarantee the oil

70

quality. In this study an alternative mucilage extraction method, using mild conditions

71

with water and probe ultrasounds, has been developed and used as a first step prior to

72

oil extraction. Lipid content of chia seeds is approximately 30%, depending on growing

73

conditions, plant location, weather circumstances and irrigation.15,16 The main

74

constituents of chia oil are triglycerides rich in polyunsaturated fatty acids (PUFA),

75

specifically, α-linolenic acid (ALA) and linoleic acid.5,17 Chia seeds contain the highest

76

known natural percentage of α-linolenic acid omega-3 (~ 60%).17 Moreover, natural

77

antioxidants are present in chia seed as tocopherols and give optimal stability

78

conditions to the oil.15

79

Modern extraction techniques, such as pressurized liquid extraction based on

80

green chemistry solvents, are being developed as an alternative to traditional methods

81

for oil extraction. The advantages of modern techniques are shorter extraction times,

82

less solvent use, full automation and greater reliability.20 Soxhlet is a traditional

83

extraction method frequently used to produce chia oil.5,21-23 However, to date there

84

are no references for chia oil extraction using PLE, while there are several references

85

to oil extractions from other seeds, plants or fruits, such as wheat germ,24 rosemary,25

86

microalgae Haematococcus pluvialis,26 marjoram and oregano,27 etc. PLE is a new

87

extraction technique using common solvents at elevated pressures and temperatures

88

that render faster extractions than traditional methods, achieving high yields.

89

Moreover, PLE allows the use of green solvents such as water, and due to the high


Page 5 of 26


90

pressure and temperatures reached, it can be used as subcritical water extraction

91

conditions.28

92

Therefore, the main objective of this study was to develop an extraction method

93

for mucilage at mild conditions, as a previous step to subsequently extract chia oil with

94

polar solvents using pressurized liquids as green solvent extraction technology, and

95

compare with organic solvent extraction. Fatty acid composition of the produced chia

96

oils at different extraction conditions were evaluated by GC-MS. Furthermore,

97

tocopherols and tocotrienols were also analyzed to evaluate the potential interest of

98

chia oil as an alternative source of omega-3 with natural tocopherols in the food

99

industry.

100

101

MATERIALS AND METHODS

102

Samples and Chemicals. Commercial chia seeds from Bolivia were purchased to

103

Dietica (Cuenca, Spain). Seeds were ground with a particle size less than 500 µm using

104

a grinder (Moulinex-A320R1 700W) and stored at 4 °C until their use. Ethyl acetate,

105

hexane and methanol were purchased from Lab Scan Analytical Sciences (Gliwice,

106

Poland). All solvents were HPLC grade. Absolute ethanol (PRS grade), sodium hydrogen

107

carbonate, sodium hydroxide and potassium hydroxide were purchased from Panreac

108

Quimica S.A (Barcelona, Spain). The water used was MilliQ grade (Millipore, USA).

109

Hydrochloric acid was purchased from Scharlau (Spain). α-tocopherol standard with

110

purity greater than 95.5% was purchased from Sigma (Sigma, St. Louis, USA) and δ-

111

tocopherol standard pure was purchased from Supelco (Bellefonte, PA, USA). Fatty



Page 6 of 26

112

acid methyl esters standard (Supelco 37 FAME Mix) was from Supelco (Bellefonte, PA,

113

USA).

114

Mucilage extraction. A pre-treatment method in order to use polar solvents for oil

115

extraction was done for chia seeds. Mucilage extraction methods were used before in

116

acid medium12 or basic medium.12,29 The mucilage extraction developed method was

117

carried out in aqueous medium at 50 °C for 2 h and sonication for 3 min at 30%

118

intensity using a Hielscher-Ultrasound Technology UP200S (Teltow, Germany). Samples

119

were centrifuged at 4000 rpm for 20 min and seeds were recovered. Seeds without

120

mucilage were ground and stored at 4 °C until their use.

121

Oil extraction. Chia oilseed extractions were carried out using Soxhlet and

122

pressurized liquid extraction, in all cases at least in duplicate.

123

Soxhlet extraction. Ground chia seeds (5.00 g) were extracted with hexane in a Soxhlet

124

apparatus by a continuous series of cycles of boiling and condensation of the solvent

125

for 8h.

126

Pressurized liquid extraction. Pressurized liquid extraction was carried out with an ASE

127

350 DIONEX (Sunnyvale, California) extractor. Oil extraction was performed using 3.00

128

g of ground chia seeds. Stainless steel extraction cells were used with a capacity of 10

129

mL. Extracts were collected under a nitrogen stream in different vials of 50 mL.

130

Solvents used were ethyl acetate, hexane ethanol and ethanol:water (50:50).

131

Temperatures used were 60, 90, 120 and 150 °C for the first three solvents and 120,

132

150 and 200 °C for ethanol:water. Static extraction time was 10 min and the solvent

133

total volume used was 20-25 mL depending on the cell temperature and pressure.

134

The samples were evaporated in a rotary evaporator (Heidolph Hei-Vap Value HB/G3,

135

Germany) under reduced pressure at 40 °C and dried under a nitrogen stream until 6 ACS Paragon Plus Environment

Page 7 of 26


136

constant weight. The oil content was determined gravimetrically and expressed as dry

137

weight percentage. Oils obtained by both extraction systems were stored in dark

138

vessels with a nitrogen atmosphere at 4 °C until their analysis.

139

Fatty acid composition of extracted chia oil. Previous to analysis on an Agilent

140

GC-MS series 5975 MSD (Palo Alto, Cal., USA), fatty acid methyl esters (FAMEs) were

141

prepared by base-catalyzed methanolysis of the glycerides (KOH in methanol). FAMEs

142

were separated using a HP 88 capillary column (100 m x 0.25 mm, i.d. 0.2 µm) (Agilent,

143

CA, USA). 1 µL sample was injected using a split ratio of 1:100. The column was held at

144

175 °C for 10 min after injection, the temperature programmed at 3 °C/min to 220 °C

145

and held for 20 minutes more. Helium was used as gas carrier, at a constant column

146

flow rate of 1.5 ml/min. The injector temperature was 250 °C and the detector

147

temperature was 230 °C. The mass spectrometer was operated at 70 eV with a mass

148

range from 30 to 400 amu. FAMEs were identified comparing their retention times and

149

the mass spectra (NIST MassSpectral Library Version 2.0) with those obtained from the

150

standards. Results were expressed as the individual relative percentage of each fatty

151

acid present in the sample.

152

Tocopherol and tocotrienols analysis by HPLC. Tocopherols and tocotrienols

153

were separated in a HPLC Agilent 1290 equipped with automatic injector and DAD

154

detector connected in series with a Evaporative Light Scattering Detector (evaporator

155

temperature, 40 °C; nebulizer temperature, 30 °C; gas flow rate, 1.2 SLM), using a ACE-

156

5 Silica de 250 x 4.6mm maintained at 25 °C. The mobile phase consisted of

157

hexane:1,4-dioxane (96:4) according to Cunha et al.30 but with a different flow. The

158

qualitative and quantitative analysis was performed at 295 nm using α-tocopherol and

159

δ-tocopherol standards. 7 ACS Paragon Plus Environment


160

RESULTS AND DISCUSSION

161

Pressurized liquid extraction of chia seed oil

Page 8 of 26

162

Chia seeds were extracted with several solvents using Pressurized Liquid Extraction

163

(PLE) and the conditions were optimized in order to achieve a fast and automated oil

164

extraction method with high yield. Oil yields obtained with hexane, ethyl acetate and

165

ethanol from chia seed by PLE at different temperatures are shown in Figure 2. As can

166

be seen, maximum oil extraction yield is achieved at temperature of 90 °C using

167

medium polar or non-polar solvents. In general, high temperature increase does not

168

lead to an extraction yield increase and, moreover, higher temperatures may cause

169

degradation reactions. Considering the less polar solvents, there are small differences

170

in the extraction efficiency using hexane or ethyl acetate at 90 °C. At lower

171

temperatures, the extraction yield is higher with hexane; however, at elevated

172

temperatures the extraction yield is enhanced with ethyl acetate. Using ethanol as

173

extraction solvent, there are larger differences in oil yield when increasing

174

temperature from 90 to 120 °C. It is not necessary to further increase the temperature

175

for optimal extraction yields; oil yield is lower at 150 °C with all tested solvents, so

176

temperatures of 90°C are sufficient and have lower energy costs and a reduced

177

thermal degradation of the sample.

178

Despite of the highest yield obtained with ethanol at 120 °C (32% ± 1) slightly higher

179

than with hexane at 90°C, it was observed that ethanol extracts were not clear and

180

homogeneous, indicating that they did not contain only oil, as proved with other tests.

181

The fact that chia seed contains a significant amount of soluble fiber in form of

182

mucilage, that may be removed with polar solvents,12 was the probable cause of this 8 ACS Paragon Plus Environment

Page 9 of 26


183

homogeneity lack observed. Chia mucilage is a polysaccharide with a high molecular

184

weight (0.8-2 x 106 Da), having a chemical structure comprising β-D-xylose, α-D-

185

glucose and acid 4-O-methyl-α-D-glucuronide,31 so it is not extracted with medium

186

polar or nonpolar solvents as hexane. Therefore, after their isolation from ethanol

187

extracts, it was concluded that the particles suspended in the oil of ethanol extracts

188

were mucilage. A method for mucilage extraction was developed as part of sample

189

pretreatment for chia oil extraction with polar solvents.

190

Considering that the extraction yield calculated using ethanol included important

191

amounts of mucilage, it cannot be considered a valid oil yield. Therefore, the optimum

192

results using pressurized liquid extraction were achieved at 90 °C with hexane as an

193

extraction solvent (oil yield 30.9% ± 0.8), similar to PLE with ethyl acetate at 90 °C (30%

194

± 1) and in the same order than previous results with Soxhlet in the bibliography. The

195

yield using Soxhlet method with hexane was 33.8 ± 0.2 in the present study, and

196

similar to the reported by Ixtaina et al. in 2011 with different chia seeds (33.6%)32 and

197

little higher that published by Ayerza in 2011 for Bolivian chia seeds (29.98%).18 The

198

result obtained in the present work using pressurized liquid hexane is also within the

199

range published by Martinez et al. in 2012 using a screw press33 and is only 2% lower

200

than Soxhlet method.

201

Comparing both techniques, time needed for the extraction was much smaller with

202

pressurized liquid extraction, since required only 10 minutes in place of 8 hours of the

203

traditional method. The pressurized liquid extraction using hexane or ethyl acetate

204

could be a competitive method for process optimization, since the reduction of



Page 10 of 26

205

extraction time carries great energy savings and a lower risk of undesirable

206

degradation reactions.

207

Mucilage extraction

208

Mucilage extraction was initially performed following the methods described in the

209

literature, using as extraction parameters pH and temperature. Marambe29 and

210

Muñoz12 extracted mucilage from flax seeds and chia seeds respectively, in basic

211

medium and acid medium for chia seeds too. These published methods were

212

developed with the aim to extract mucilage, a polysaccharide difficult to isolate and

213

separate, but not as a sample pretreatment to extract oil with polar solvents.

214

Consequently, the purpose of the methods described in the bibliography was achieving

215

the maximum possible gum yield, without concern of the remaining oilseeds. For that

216

reason, former methods are very aggressive to seeds and use extreme pH and other

217

conditions that could adversely affect latter extracted oil quality, especially dealing

218

with omega-3 PUFA. In the present work, the interest is not only related to extract the

219

maximum amount of mucilage, but also to have the least aggressive extraction

220

conditions possible, thus avoiding any alteration of the PUFA rich seed oil. Optimal

221

results were not obtained in experiments with published pretreatments. After the

222

removal of mucilage, chia seeds presented not desirable appearance considering color

223

and shape. The seed deterioration was the main reason to reject the use of these

224

methods with extreme pHs.

225

Our next experiments were to avoid the use of strong acids or extreme pHs, while

226

producing a less aggressive mucilage removal. Extraction was performed in aqueous

227

medium with stirring for two hours at 50 °C, proving that it was sufficient to remove 10 ACS Paragon Plus Environment

Page 11 of 26


228

the entire seed mucilage in spite of bibliographic results. No apparent changes in color

229

and shape were observed in chia seeds using acid and basic medium. During mucilage

230

extraction, once chia seeds are subjected to hydration, seeds are encircled by a

231

mucilage halo difficult to remove and separate from the seeds. This interaction is

232

characteristic of chia mucilage and it avoids the direct use of the seeds for obtaining

233

oil. Therefore, it was necessary to find a method that would allow to eliminate the

234

interaction between mucilage and the seed, and thereby obtain cleaning seeds for

235

later oil extraction.

236

Methods described in the literature for separating hydrated seeds and mucilage are

237

lyophilization,23 high vacuum industrial filtration34 or using a basket centrifuge35 for

238

mustard seeds. Another method described by Muñoz12 in 2012 was dry the aqueous

239

suspension of mucilage and seeds under a hot air stream, though this method was

240

rejected due to oxidative damage that could cause in the polyunsaturated fatty acids

241

of chia oil. Because of the unfeasibility of performing in our lab some of the methods

242

described above, direct centrifugation of the aqueous suspension was tested without

243

satisfactory results. The high viscosity of mucilage prevents right filtration of the

244

sample, saturating the filter pores even under vacuum. Finally, sample sonication was

245

tested both in an ultrasound bath and using an ultrasonic probe. Of the above

246

methods tested, optimum results could only be obtained with probe sonication. High-

247

power ultrasonic waves were able to eliminate the mucilage and seed interaction, so it

248

was chosen as the best method for extracting mucilage under mild conditions to avoid

249

deterioration of chia seed.



Page 12 of 26

250

The optimized parameters for the extraction method of chia seed mucilage were:

251

aqueous extraction with ratio 1:40 (seed:water), extraction time of 2 hours with

252

constant stirring at 50 °C and sonication with high intensity probe ultrasound. Optimal

253

sonication conditions among all tested, were 30% of maximum ultrasonic power for 3

254

minutes. These parameters were optimized taking into account the necessity of

255

ultrasonic waves to eliminate interaction between the mucilage and the seed, but

256

minimizing the ultrasonic power and time of treatment, to avoid the raising of sample

257

temperature caused by the ultrasound application. The temperature must be kept low

258

during mucilage removal to avoid chia oil degradation. The temperature of the sample

259

after 3 min of sonication ranged from 37-40 °C, this temperature did not exceed in any

260

case the 50 °C used in the hydration of the seed. The low temperature generated and

261

the use of neat water makes this method an alternative for mucilage extraction

262

without damage to the seeds.

263

Mucilage yield obtained was 6.52% ± 0.08. This result was in the range obtained by

264

Muñoz et al.12 (6.97% of extracted mucilage) but higher that obtained by Reyes-

265

Caudillo et al.36 and Ayerza and Coates18, 6 and 5% respectively. The main difference

266

with the method performed by Muñoz is that although mucilage extraction yields

267

obtained were similar or higher to those described in the literature, former methods

268

were more aggressive than the proposed in the present work. In subsequent

269

experiments, ultrasound pretreated seeds were used for oil extraction by PLE

270

achieving good results for the extraction of chia seeds.

271

272 12 ACS Paragon Plus Environment

Page 13 of 26

273


Pressurized liquid extraction of oil using pretreated chia seeds

274

Once optimized the mucilage extraction method, oil extractions of seeds without

275

mucilage were performed using mixtures of ethanol:water and water as extraction

276

solvents. The use of mixtures of ethanol:water produced promising results, finding a

277

50:50 ratio as the most suitable (see results shown in Table 1). At the lowest

278

temperature tested, an oil yield of 7% was obtained, and when temperature was

279

increased 30 °C to 150°C, an oil yield increase of 50% was achieved. However, there

280

were no differences when increasing temperature further from 150 °C to 200 °C. The

281

best yield was obtained at 150 °C (10.5%), without need of reaching extreme

282

temperatures of 200 °C. When we compare PLE results obtained with ethanol:water to

283

those found using organic solvents, as expected, the oil yield at 120 °C using hexane or

284

ethyl acetate, it is almost 3 times higher than obtained with the ethanol:water mixture.

285

Despite the lower oil yield, the advantages of these solvents are related to green

286

chemistry purposes and allowed to have a representative chia oil (see below and

287

Figure 3) in short time and with environmentally friendly solvents.

288

Nevertheless, when chia seeds were extracted with water using PLE (as Subcritical

289

Water Extraction) to produce chia oil, desirable results were not obtained due to

290

presence of residual seed mucilage which was also extracted at high temperature. An

291

elevated viscosity occurred when the seeds were in contact with hot water, making it

292

difficult to oil extraction with subcritical water. Thus, a second ultrasonic mucilage

293

extraction was performed at the same conditions as the first gum extraction, to

294

determine the total mucilage content of chia seeds and it was found that seeds still

295

contained appreciable quantities of mucilage. Results showed that the total mucilage



Page 14 of 26

296

content after two cycles of extraction was 10.77%, value higher than the results

297

reported in the literature to date. Further experiments are being performed in our lab

298

to develop a seed oil production method including Subcritical Water Extraction for

299

different oilseeds. The ultrasonic method proposed in this work, besides of being

300

cleaner and less aggressive for seeds, allows achieving with two extraction cycles a

301

much higher percentage of mucilage removal than reported before in the literature.

302

Determination of fatty acids in chia oil

303

Fatty acid content of all chia oil extracts were analyzed by GC-MS in order to

304

evaluate the fatty acid composition obtained with different techniques, solvents, and

305

temperatures. Figure 3 shows the representative fatty acids profile of extracted chia

306

oil which was comparable to that achieved by other investigators17, although in most

307

analysis without the detection of vaccenic acid.18,37-39

308

Table 2 shows the fatty acid composition of chia oil extracted by Soxhlet and PLE

309

with different solvents at the optimal extraction conditions. Chia oil was characterized

310

by a low percentage of saturated and monounsaturated fatty acids, while the

311

percentage of PUFA was high, around 82% of the total fatty acids. The highest fatty

312

acid composition in chia oil was ALA, having percentages between 63.6 to 65.6% of

313

total fatty acids. In addition, the percentage of ALA was compared with other oilseeds,

314

such as Camelina (Camelina sativa L.) 36%; perilla (Perilla flutescens L.) 53%, and flax

315

(Linum usitatissimum L.) 57%.40 It was confirmed that chia oil contains the highest

316

percentage of ALA of these vegetable sources rich in omega-3. Moreover, essential

317

fatty acids content (α-linolenic and linoleic acids) was between 81.9 to 83.6%. The ratio

318

ω-6/ω-3 in chia oil was from 0.27 to 0.29, still much lower than those values from most 14 ACS Paragon Plus Environment

Page 15 of 26


319

vegetable oils: borage oil (1.50), canola (2.18), soybean (7.50), wheat germ (7.94) and

320

olive (13.17).41 Therefore, the incorporation of chia oil to diet could be a great interest

321

because there are scientific evidences showing that foods with an appropriate balance

322

ω-6/ω-3 give numerous benefits for health.42

323

On the other hand, fatty acid composition of chia oil from seeds without mucilage

324

was also evaluated in order to compare and to check that the mucilage extraction

325

method did not affect to the omega-3 percentage. Figure 4 shows the percentage of

326

fatty acid composition obtained by PLE with ethanol:water (50:50) in pre-treated chia

327

seeds. The percentage of ALA was between 65.9 to 64.6%, depending on the

328

temperature used, but it was equal than obtained in non pre-treated seeds. As a result

329

of GC-MS analysis, the fatty acid profile was not changed with respect to mucilage

330

extraction, oil extraction technique, solvent or temperature used.

331

Tocopherols and tocotrienols analysis

332

Figure 5 shows tocopherol and tocotrienol profiles of chia oil obtained by Soxhlet

333

and PLE. Chia seed oil contained between 587-895 mg/kg of tocopherols depending on

334

the extraction technique, temperature and solvent used. The main tocopherol was γ-

335

tocopherol (68.3-72.7%) in the most extraction solvents studied. However, β-

336

tocopherol had not been identified in chia oil to date, but in the present work, it was

337

determined in different amounts ranged from 1.4 to 15.5 mg/kg. In this study total

338

amount of tocopherols in chia oil was significantly higher than data reported for others

339

authors32,43-46 and at comparable amount as Bodoira et al. (716.8 mg/kg).47 The total

340

tocopherols in chia oil was similar as in other oilseeds, such as camelina oil (790



Page 16 of 26

341

mg/kg), canola oil (694 mg/kg), sunflower oil (649 mg/kg) and corn oil (603 mg/kg), but

342

lower than soybean oil (1162 mg/kg).48

343

It is known that there is a positive relationship between high PUFA and tocopherols

344

content, playing a role in oxidation protect. In addition, tocotrienols had not been

345

identified in chia oil to date, but other vegetable oils contain high amounts of

346

tocotrienols, such as red palm oil contains the highest amount (585 mg/kg).49 In this

347

study, tocotrienols in extracted chia oil were identified (see Figure 5) in different

348

amounts ranging from 77 to 155 mg/kg. The quantity of chia tocotrienols, in the same

349

way that tocopherols, and in both cases, depended on the extraction technique,

350

temperature and solvent used. The main tocotrienol was α-tocotrienol with a

351

percentage around 43.3% of total tocotrienols.

352

In conclusion, chia seed oil can be extracted in a very fast and effective way using PLE

353

with traditional solvents. For the effective extraction of chia mucilage, a new method

354

using high intensity ultrasounds was developed at mild conditions with plain water as

355

solvent. The mucilage removal allowed the subsequent extraction of the chia oil with

356

polar or non-polar solvents producing a chia oil with the fatty acid composition as

357

traditional extraction.

358 359

ABBREVIATIONS USED

360

WHO, Word Health Organization; FDA, Food and Drug Administration; PUFA,

361

polyunsaturated fatty acids; ALA, α-linolenic acid; PLE, pressurized liquid extraction;

362

GC-MS, gas chromatography-mass spectrometry; FAME, fatty acid methyl esters; SFA,

363

saturated fatty acids; MUFA, monounsaturated fatty acids; 16 ACS Paragon Plus Environment

Page 17 of 26


364

365

ACKNOWLEDGEMENTS

366

Authors thank NOBO (Coruña, Spain) for its support to the Research Group in Healthy

367

Lipids at UAM. Authors thank the Spanish Ministry of Education, Culture and Sport for

368

the pre-doctoral contract (FPU 2013-01796) granted to Natalia Castejón.

369

370

371

REFERENCES

372 373

1. Galli, C.; Marangoni, F. N-3 fatty acids in the Mediterranean diet. PLEFA 2006, 75, 129-133.

374 375 376

2. Raffick A. R. Bowena and Michael T. Clandinin Maternal dietary 22: 6n-3 is more effective than 18: 3n-3 in increasing the 22: 6n-3 content in phospholipids of glial cells from neonatal rat brain. Br. J. Nutr. 2005, 93, 601-611.

377 378 379 380 381

3. European Food Safety Authority (EFSA) ALA and LA and growth and development of children - Scientific substantiation of a health claim related to α-linolenic acid and linoleic acid and growth and development of children pursuant to Article 14 of Regulation (EC) No 1924/2006 - Scientific Opinion of the Panel on Dietetic Products, Nutrition and Allergies. EFSA Journal 2008, 6, 783-n/a.

382 383 384 385 386

4. EFSA Panel on Dietetic Products,Nutrition and Allergies (NDA) Opinion on the substantiation of health claims related to alpha linolenic acid and maintenance of normal blood cholesterol concentrations (ID 493) and maintenance of normal blood pressure (ID 625) pursuant to Article 13(1) of Regulation (EC) No 1924/2006. EFSA Journal 2009, 7, 1252-n/a.

387 388

5. Ayerza, R. Oil Content and Fatty Acid Composition of Chia (Salvia hispanica L.) from Five Northwestern Locations in Argentina. J. Am. Oil Chem. Soc. 1995, 72, 1079-1081.

389 390

6. Coates, W.; Ayerza (h), R. Production potential of chia in northwestern Argentina. Ind. Crops Prod. 1996, 5, 229-233.

391 392 393

7. Valdivia-López, M.Á; Tecante, A. Chapter Two - Chia (Salvia hispanica): A Review of Native Mexican Seed and its Nutritional and Functional Properties. Adv. Food Nutr. Res. 2015, 75, 53-75. 17 ACS Paragon Plus Environment


Page 18 of 26

394 395 396

8. European Parliament and of the Council 2009/827/EC: Commission Decision of 13 October 2009 authorising the placing on the market of Chia seed (Salvia hispanica) as novel food ingredient. 2009, No 258/97, (notified under document C(2009) 7645).

397 398

9. Sandoval-Oliveros, M.; Paredes-López, O. Isolation and Characterization of Proteins from Chia Seeds (Salvia hispanica L.). J. Agric. Food Chem. 2013, 61, 193-201.

399 400 401

10. Dick, M.; Costa, T.M.H.; Gomaa, A.; Subirade, M.; Rios, A.d.O.; Flôres, S.H. Edible film production from chia seed mucilage: Effect of glycerol concentration on its physicochemical and mechanical properties. Carbohydr. Polym. 2015, 130, 198-205.

402 403

11. Capitani, M.I.; Nolasco, S.M.; Tomás, M.C. Stability of oil-in-water (O/W) emulsions with chia (Salvia hispanica L.) mucilage. Food Hydrocoll. 2016, 61, 537-546.

404 405

12. Muñoz, L.A.; Cobos, A.; Diaz, O.; Aguilera, J.M. Chia seeds: Microstructure, mucilage extraction and hydration. J. Food Eng. 2012, 108, 216-224.

406 407 408

13. Timilsena, Y.P.; Adhikari, R.; Kasapis, S.; Adhikari, B. Molecular and functional characteristics of purified gum from Australian chia seeds. Carbohydr. Polym. 2016, 136, 128-136.

409 410 411

14. Timilsena, Y.P.; Adhikari, R.; Kasapis, S.; Adhikari, B. Rheological and microstructural properties of the chia seed polysaccharide. Int. J. Biol. Macromol. 2015, 81, 991-999.

412 413 414

15. Salgado-Cruz, M.; Cedillo-López, D.; Beltran, M. Estudio de las Propiedades Funcionales de la Semilla de Chía (Salvia hispánica) y de la Fibra Dietaria Obtenida de la misma. VII Congreso Nacional de Ciencia de los Alimentos, Mexico 2005, 358-366.

415 416 417

16. Ayerza (h), R.; Coates, W. Influence of environment on growing period and yield, protein, oil and α-linolenic content of three chia (Salvia hispanica L.) selections. Ind. Crops Prod. 2009, 30, 321-324.

418 419 420

17. Ixtaina, V.Y.; Martínez, M.L.; Spotorno, V.; Mateo, C.M.; Maestri, D.M.; Diehl, B.W.K.; Nolasco, S.M.; Tomás, M.C. Characterization of chia seed oils obtained by pressing and solvent extraction. J. Food Comp. Anal. 2011, 24, 166-174.

421 422 423

18. Ayerza (h), R.; Coates, W. Protein content, oil content and fatty acid profiles as potential criteria to determine the origin of commercially grown chia (Salvia hispanica L.). Ind. Crops Prod. 2011, 34, 1366-1371.

424 425

19. Frankel, E.N. Antioxidants in lipid foods and their impact on food quality. Food Chem. 1996, 57, 51-55.

426 427 428

20. Señoráns, F.J.; Luna, P. 4.10 - Sample Preparation Techniques for the Determination of Fats in Food, In Comprehensive Sampling and Sample Preparation, Pawliszyn, J., Ed.; Academic Press: Oxford, 2012; pp. 203-211.


Page 19 of 26


429 430

21. Bushway, A., Belya Chia seed as a Source of Oil, Polysaccharide and Protein. J. Food Sci. 1981, 46, 1349-1356.

431 432

22. Coates W, A.R. Commercial production of chia in Northwestern Argentina. J. Am. Oil Chem. Soc. 1998, 75, 1417 – 1420.

433 434 435

23. Capitani,M.I., M. Nolasco, S., C. Tomás, M. Effect of Mucilage Extraction on the Functional Properties of Chia Meals, In Food Industry, 10th ed.; Muzzalupo, I., Ed.; Agricultural and Biological Sciences: 2013; pp. 5772-53171.

436 437

24. Dunford, N.T.; Zhang, M. Pressurized solvent extraction of wheat germ oil. Food Res. Int. 2003, 36, 905-909.

438 439

25. Rodríguez-Rojo, S.; Visentin, A.; Maestri, D.; Cocero, M.J. Assisted extraction of rosemary antioxidants with green solvents. J. Food Eng. 2012, 109, 98-103.

440 441 442

26. Rodríguez-Meizoso, I.; Jaime, L.; Santoyo, S.; Señoráns, F.J.; Cifuentes, A.; Ibáñez, E. Subcritical water extraction and characterization of bioactive compounds from Haematococcus pluvialis microalga. J. Pharm. Biomed. Anal. 2010, 51, 456-463.

443 444 445 446

27. Hossain, M.B.; Barry-Ryan, C.; Martin-Diana, A.B.; Brunton, N.P. Optimisation of accelerated solvent extraction of antioxidant compounds from rosemary (Rosmarinus officinalis L.), marjoram (Origanum majorana L.) and oregano (Origanum vulgare L.) using response surface methodology. Food Chem. 2011, 126, 339-346.

447 448 449

28. Ibañez, E.; Kubátová, A.; Señoráns, F.J.; Cavero, S.; Reglero, G.; Hawthorne, S.B. Subcritical Water Extraction of Antioxidant Compounds from Rosemary Plants. J. Agric. Food Chem. 2003, 51, 375-382.

450 451 452

29. Marambe, P. W. M. L. H. K. An In-vitro Investigation of Selected Biological Activities of Hydrolysed Flaxseed (Linum usitatissimum L.) Proteins. J. Am. Oil Chem. Soc. 2008, 85, 1155; 1155-1164; 1164.

453 454 455

30. Cunha, S.C.; Amaral, J.S.; Fernandes, J.O.; Oliveira, M.B. Quantification of Tocopherols and Tocotrienols in Portuguese Olive Oils Using HPLC with Three Different Detection Systems. J. Agric. Food Chem. 2006, 54, 3351-3356.

456 457

31. Lin, K.; Daniel, J.R.; Whistler, R.L. Structure of chia seed polysaccharide exudate. Carbohydr. Polym. 1994, 23, 13-18.

458 459 460

32. Ixtaina, V.Y.; Martínez, M.L.; Spotorno, V.; Mateo, C.M.; Maestri, D.M.; Diehl, B.W.K.; Nolasco, S.M.; Tomás, M.C. Characterization of chia seed oils obtained by pressing and solvent extraction. J. Food Comp. Anal. 2011, 24, 166-174.

461 462 463

33. Martínez, M.L.; Marín, M.A.; Salgado Faller, C.M.; Revol, J.; Penci, M.C.; Ribotta, P.D. Chia (Salvia hispanica L.) oil extraction: Study of processing parameters. LWT Food Sci. Tech. 2012, 47, 78-82.



Page 20 of 26

464 465 466

34. Marin, F.; Acevedo, M.; Tamez, R.; Nevero, M.; Garay, A. WO/2008/0044908 Method for obtaining mucilage from Salvia hispánica. Word International Property Organization 2008,

467 468

35. Balke, D.T.; Diosady, L.L. Rapid aqueous extraction of mucilage from whole white mustard seed. Food Res. Int. 2000, 33, 347-356.

469 470 471

36. Reyes-Caudillo, E.; Tecante, A.; Valdivia-Lopez, M. Dietary fiber content and antioxidant activity of phenolic compounds in Mexican chia (Salvia hispanica L.) seeds. Food Chem. 2008, 107,

472 473 474 475

37. Ixtaina, V.Y.; Vega, A.; Nolasco, S.M.; Tomás, M.C.; Gimeno, M.; Bárzana, E.; Tecante, A. Supercritical carbon dioxide extraction of oil from Mexican chia seed (Salvia hispanica L.): Characterization and process optimization. J. Supercrit. Fluids 2010, 55, 192-199.

476 477 478

38. Martínez, M.L.; Marín, M.A.; Salgado Faller, C.M.; Revol, J.; Penci, M.C.; Ribotta, P.D. Chia (Salvia hispanica L.) oil extraction: Study of processing parameters. LWT Food Sci. Tech. 2012, 47, 78-82.

479 480

39. Uribe, J.A.R.; Perez, J.I.N.; Kauil, H.C.; Rubio, G.R.; Alcocer, C.G. Extraction of oil from chia seeds with supercritical CO2. J. Supercrit. Fluids 2011, 56, 174-178.

481 482

40. The Seed Oil Fatty Acids Database (SOFA) URL (http://sofa.mri.bund.de/). (accessed 24.11.2016),

483 484

41. United States Department of Agriculture USDA Food Composition Databases. URL (https://ndb.nal.usda.gov/ndb/). (accessed 24.11.2016),

485 486

42. Simopoulos, A.P. The importance of the ratio of omega-6/omega-3 essential fatty acids. Biomed. Pharmacother. 2002, 56, 365-379.

487 488

43. Ciftci, O.N.; Przybylski, R.; Rudzi?ska, M. Lipid components of flax, perilla, and chia seeds. Eur. J. Lipid Sci. Technol. 2012, 114, 794-800.

489 490

44. Grompone, M.A.; Irigaray, B.; Rodríguez, D.; Sammán, N. Assessing the Oxidative Stability of Commercial Chia Oil. J. Food Sci. Eng. 2013, 3, 349-356.

491 492 493

45. Capitani, M.I.; Spotorno, V.; Nolasco, S.M.; Tomás, M.C. Physicochemical and functional characterization of by-products from chia (Salvia hispanica L.) seeds of Argentina. LWT - Food Sci. Tech. 2012, 45, 94-102.

494 495 496

46. Ixtaina, V.Y.; Nolasco, S.M.; Tomas, M.C. Oxidative stability of chia (Salvia hispanica L.) seed oil: effect of antioxidants and storage conditions. J. Am. Oil Chem. Soc. 2012, 89,


Page 21 of 26


497 498 499

47. Bodoira, R.M.; Penci, M.C.; Ribotta, P.D.; Martínez, M.L. Chia (Salvia hispanica L.) oil stability: Study of the effect of natural antioxidants. LWT - Food Sci. Tech. 2017, 75, 107-113.

500 501 502

48. Waraich, E.A.; Ahmed, Z.; Ahmad, R.; Ashraf, M.Y.; Naeem, M.S.; Rengel, Z. Camelina sativa', a climate proof crop, has high nutritive value and multiple-uses: A review. Aust. J. Crop Sci. Sep 2013, Vol. 7, No. 10, 1551-1559.

503 504 505

49. Al-Saqer, J.M.; Sidhu, J.S.; Al-Hooti, S.N.; Al-Amiri, H.A.; Al-Othman, A.; Al-Haji, L.; Ahmed, N.; Mansour, I.B.; Minal, J. Developing functional foods using red palm olein. IV. Tocopherols and tocotrienols. Food Chem. 2004, 85, 579-583.

506 507 508

FIGURE CAPTIONS

509

Figure 1. Chia seeds (a) and hydrated chia seeds (b) (water: seeds ratio 10:1). 1.44

510

grams of chia seeds in both cases.

511

Figure 2. Chia oil extraction yield obtained by PLE using different solvents and

512

temperatures.

513

Figure 3. Representative Fatty acids methyl esters profile of extracted chia oil

514

determined by GC-MS.

515

Figure 4. Fatty acid composition of chia oil from seed without mucilage, extracted with

516

PLE using ethanol:water (50:50) at different temperatures.

517

Figure 5. Tocopherol (a) and tocotrienol (b) composition of chia oil extracted with PLE

518

and Soxhlet at the optimal extraction conditions.

519

520



Page 22 of 26

TABLES Table 1. Oil extraction yield from chia seeds without mucilage obtained by PLE using ethanol:water (50:50) as extraction solvent. Oil yielda (%) 120 °C 150 °C 200 °C Etanol : water (50:50) 7.0 ± 0.6 10.5 ± 0.1 10.2 ± 0.6 a

Values are the mean ± SD of two determinations

Table 2. Fatty acid composition (as percentage of total fatty acids) of chia oil determined by GC-MS at optimal extraction conditions.

RT (min) 16:0 12.7 18:0 16.3 18:1 cis9 17.4 18:1 cis11 17.6 18:2 19.2 18:3 21.3 SFA MUFA PUFA n-6 n-3 n-6/n-3 Ratio Fatty acid

a

Soxhlet 7.19 ± 0.17 3.65 ± 0.24 5.57 ± 0.08 1.71 ± 0.05 18.26 ± 0.17 63.63 ± 0.65 10.8 7.28 81.89 18.26 63.63 0.287

PLE hexaneb 6.29 ± 0.08 3.19 ± 0.07 5.93 ± 0.17 1.01 ± 0.06 17.99 ± 0.16 65.59 ± 0.53 9.48 6.94 83.58 17.99 65.59 0.274

% Fatty acidsa PLE ethyl acetatec 6.70 ± 0.14 3.30 ± 0.01 5.87 ± 0.01 1.01 ± 0.03 18.36 ± 0.18 64.77 ± 0.35 10.0 6.88 83.12 18.36 64.77 0.283

PLE ethanold 6.62 ± 0.13 3.18 ± 0.07 5.74 ± 0.10 0.99 ± 0.02 18.27 ± 0.08 65.19 ± 0.40 9.81 6.73 83.47 18.27 65.19 0.280

PLE ethanol:watere 6.65 ± 0.33 3.24 ± 0.08 6.40 ± 0.29 1.36 ± 0.01 17.84 ± 0.15 64.51 ± 0.70 9.89 7.76 82.35 17.84 64.51 0.277

Values are the mean ± SD of two determinations

b

c

d

e

PLE hexane (T 90 °C); PLE ethyl acetate (T 120 °C); PLE ethanol (T 120 °C); PLE ethanol:water (50:50) (T 150 °C) SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids.


Page 23 of 26


FIGURE GRAPHICS

Figure 1.

35 30

Oil yield (%)

25

60 °C

20

90 °C 15

120 °C

10

150 °C

5 0

Hexane

Ethyl acetate

Ethanol

Figure 2.


Page 24 of 26

10.00

18:1 c9 18:1 c11

18:0

16:0

18:2 n-6

18:3 n-3 (ALA)


15.00

20.00

25.00

Time (min)

Figure 3.

70

120 °C

150 °C

200 °C

60

Fatty acids (%)

50 40 30 20 10 0

16:0

18:0

18:1 n-9

18:1 n-11

18:2 n-6

18:3 n-3

Figure 4.


Page 25 of 26


mg/kg oil

PLE-hexane

PLE- ethyl acetate

PLE-ethanol

PLE-ethanol:water ethanol:water

Soxhlet

1000 900 800 700 600 500 400 300 200 100 0 a-T

a-T3

b-T

g-T

b-T3

g-T3

d-T

d-T3

T total T3 total

Figure 5.

GRAPHIC FOR TABLE OF CONTENTS



338x190mm (300 x 300 DPI)

ACS Paragon Plus Environment

Page 26 of 26

Ultrasonic Removal of Mucilage for Pressurized Liquid Extraction of

Recommend Documents