Ultrasonic Removal of Mucilage for Pressurized Liquid Extraction of

Mar 7, 2017 - Natalia Castejón, Pilar Luna, and Francisco J. Señoráns*. Healthy-Lipids Group, Sección Departamental de Ciencias de la Alimentación, ...
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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

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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.

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Journal of Agricultural and Food Chemistry

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

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Rich Oil from Chia Seeds (Salvia hispanica L.)

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Natalia Castejón, Pilar Luna and Francisco J. Señoráns*

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

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

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

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ABSTRACT

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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assisted extraction, Salvia hispanica, soluble fiber

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INTRODUCTION

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

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

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

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

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cardiovascular diseases, prevention of nervous system problems and decrease

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

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

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

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

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

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

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final products.

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

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

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

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

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

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

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

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functional characteristics have gradually increased the importance of chia crop,

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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conditions to the oil.15

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Modern extraction techniques, such as pressurized liquid extraction based on

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

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

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

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

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

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

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

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extraction technique using common solvents at elevated pressures and temperatures

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

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

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

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conditions.28

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

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

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

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

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

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

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

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industry.

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MATERIALS AND METHODS

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

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

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

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

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

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carbonate, sodium hydroxide and potassium hydroxide were purchased from Panreac

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

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

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

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

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

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USA).

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

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

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

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

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

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

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

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

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

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

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

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for 8h.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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acid present in the sample.

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Tocopherol and tocotrienols analysis by HPLC. Tocopherols and tocotrienols

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

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

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

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

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

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

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δ-tocopherol standards. 7 ACS Paragon Plus Environment

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RESULTS AND DISCUSSION

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Pressurized liquid extraction of chia seed oil

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Chia seeds were extracted with several solvents using Pressurized Liquid Extraction

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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thermal degradation of the sample.

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

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

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

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

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

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

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

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

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

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

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

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pretreatment for chia oil extraction with polar solvents.

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Considering that the extraction yield calculated using ethanol included important

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

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

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

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

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

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

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

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

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

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than Soxhlet method.

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

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

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

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

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

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degradation reactions.

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Mucilage extraction

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Mucilage extraction was initially performed following the methods described in the

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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methods with extreme pHs.

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

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

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

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

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

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

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

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

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

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

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later oil extraction.

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

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

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

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

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

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

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

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

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

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

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

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

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

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deterioration of chia seed.

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

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

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

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

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

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

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

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

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

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

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

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

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without damage to the seeds.

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

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

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

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

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

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

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

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achieving good results for the extraction of chia seeds.

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Pressurized liquid extraction of oil using pretreated chia seeds

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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Determination of fatty acids in chia oil

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

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

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

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

307

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

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

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

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

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

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

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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

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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

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

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

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

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

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

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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

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

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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

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

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extraction, oil extraction technique, solvent or temperature used.

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Tocopherols and tocotrienols analysis

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

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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

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

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

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

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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

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Journal of Agricultural and Food Chemistry

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

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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

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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.

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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.

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10.00

18:1 c9 18:1 c11

18:0

16:0

18:2 n-6

18:3 n-3 (ALA)

Journal of Agricultural and Food Chemistry

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.

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Journal of Agricultural and Food Chemistry

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

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338x190mm (300 x 300 DPI)

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