Refining of Roundup Ready® soya bean oil: Effect on the fatty acid, phytosterol and tocopherol profiles

Research Article

Refining of Roundup ReadyW soya bean oil: Effect on thefatty acid, phytosterol and tocopherol profiles

Joana Costa1, Joana S. Amaral1,2, Isabel Mafra1 and M. Beatriz P. P. Oliveira1

1 REQUIMTE, Servico de Bromatologia, Faculdade de Farmacia, Universidade do Porto, Porto, Portugal2 ESTiG, Instituto Politecnico de Braganca, Campus de Sta. Apolonia, Braganca, Portugal

In this study, genetically modified (GM) soya bean seeds with the event known as Roundup Ready1were

used for the production of soya bean oil and their fatty acid, phytosterol and tocopherol composition was

characterised. Since these compounds can be partially lost during refining, this study also aimed at

evaluating the compositional changes along the GM soya bean oil extraction and refining processes

carried out industrially. During the refining process of crude soya bean oil, neutralisation was responsible

for the major losses on phytosterols (15%). The greatest reduction of tocopherols was also caused by the

neutralisation step (20%), while the deodorisation step caused minor losses (9%). Along the refining

process, the decreases of total phytosterols and total tocopherols were 20 and 30%, respectively, which

are lower than the losses reported in other studies, reflecting the industrial improvements in preserving

beneficial health compounds in the refined oil. The results showed that the refined GM soya bean oil

presented higher contents of phytosterols (313 mg/100 g) and tocopherols (931 mg/kg) comparing to

other reports.

Keywords: Fatty acids / Genetically modified organisms / Oil refining / Phytosterols / Soya bean oil

Received: June 16, 2010 / Revised: October 14, 2010 / Accepted: November 16, 2010

DOI: 10.1002/ejlt.201000385

1 Introduction

In the last years, the production of soya bean (Glycine max L.)

has been increasing mainly due to its various uses in food,

feed and industrial applications such as biodiesel. Soya bean

is an exceptional source of high quality proteins and a source

of PUFA. The oil is also rich in vitamin E, an important class

of antioxidants that naturally occur in vegetable oils [1].

Consequently, soya bean is present in human diet mainly

as a source of protein and vegetable oil. Nowadays, soya bean

oil represents almost 28% of the world’s vegetable oil

consumption (Soy Stats 2010 – A publication of the

American Soybean Association. Accessed on 19.05.2010 –

http://www.soystats.com/2010/Default-frames.htm.) with a

considerable contribution arising from genetically modified

(GM) oilseeds. Soya bean is the most important GM crop in

the world, occupying 69.2 millions of planted hectares in

2009, which correspond to 52% of the total biotechnological

planted area (C. James, ISAAA Brief 41. 2009, accessible in

http://www.isaaa.org). In Europe, since the approval of the

glyphosate tolerant soya bean event (GTS-40-3-2) also

known as Roundup Ready1 (RR) soya bean, the oil pro-

duction from this GM oilseed has been increasing.

Asmost vegetable oils, crude soya bean oil must be refined

to remove several substances that may contribute to undesir-

able colour, taste and aroma, and therefore limiting the oil

application and narrowing its shelf-life. The refining process,

either physical or chemical, allows the elimination of those

unacceptable substances, such as phospholipids, free fatty

acids (FFAs) and pigments, which may cause a decrease in

the product quality. On the other hand, it is advantageous

that the refining process occurs with a minimum effect on the

desirable components present in oil. Some beneficial com-

pounds, such as EFA, tocopherols, phytosterols, carotenoids

and polyphenols can be partially lost during this process.

Additionally, some undesirable compounds, such as trans

FA and cyclic FA monomers, among others, can be formed

along the refining steps. Crude soya bean oil is usually sub-

mitted to a chemical refining process, which comprises the

steps of degumming, alkali refining (neutralisation), bleach-

ing and deodorisation, to obtain an odourless, bland and

Correspondence: Dr. Isabel Mafra, REQUIMTE, Servico de

Bromatologia, Faculdade de Farmacia, Universidade do Porto, Rua Anıbal

Cunha, 164, Porto 4099-030, Portugal

E-mail: [email protected]

Fax: þ351 222003977

Abbreviations: FFA, free fatty acid; GLC, gas–liquid chromatography;

GM, genetically modified; IS, internal standard; RR, Roundup Ready1

528 Eur. J. Lipid Sci. Technol. 2011, 113, 528–535

� 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.ejlst.com

stable oil, with acceptable organoleptic properties for con-

sumers [2]. Each step is specialised in the removal of distinct

groups of undesirable compounds. The degumming step

allows the removal of phospholipids and mucilaginous gums,

while neutralisation (alkali refining) is mainly directed to the

elimination of FFA [3]. The bleaching step reduces residual

FFA, salts, metals, peroxides and chlorophylls, as well as

harmful compounds, such as PAH and pesticides [3]. The

deodorisation step facilitates the nearly complete removal of

the remaining FFA and the thermal destruction of peroxides,

increasing the oxidative stability of the oil [2, 3].

Furthermore, during the deodorisation, volatile compounds,

as well as tocopherols and carotenoids, can be partially lost.

Some studies concerning the effects of the refining on

major and minor compounds of soya bean oil have been

reported. However, they have been performed several years

ago [4–6], before the first commercialisation of genetically

modified organisms (GMO). As nowadays GMOhave been a

topic of discussion among the scientific community and

consumers, and since GM crops are widely used to obtain

edible oils, the oilseeds fromRR soya bean were the subject of

the present study.

The aim of this work was to evaluate the chemical com-

position of GM soya bean seeds used industrially for the

production of oil as well as to monitor the chemical changes

along the extraction and refining processes of soya bean oil.

To accomplish this objective, the effects of each step of

refining carried out industrially on the total and individual

contents of those compounds were also determined. To our

knowledge, this is the first report regarding the effects of

industrial refining on the chemical composition of crude

oil obtained from GM soya bean.

2 Materials and methods

2.1 Sampling

The soya bean oil under study was extracted and chemically

refined at industrial scale. Representative samples were col-

lected along the main steps of the soya bean oil production

line. According to the industrial process, the first step con-

sisted on cleaning and removing impurities from the soya

bean seeds. At this point, the first sample (whole seed) con-

sisting of six random portions of 500 g was collected directly

from the industrial silo, and further assembled in a unique

homogenised sample. The following steps consisted on crack-

ing, laminating and expelling-extruding the soya bean seeds.

For each one, samples were collected, corresponding to

cracked, laminated and extruded soya bean, respectively

(Fig. 1). As the production line under study is performed

continuously, the samples were collected directly from the

industrial quality control check points. Thus, each sample

(cracked, laminated and extruded soya bean) was obtained by

collecting six portions of 200 g at regular time-intervals

during 24 h. All the portions, corresponding to each step,

were assembled to obtain representative and homogeneous

samples. The final step at the industrial soya bean oil extrac-

tion line consisted on the continuous extraction of the

extrudedmaterial with n-hexane at 608C, obtaining the crude

soya bean oil and the defatted flour, which in this specific

process is a by-product of the extraction process and con-

ducted for animal feed production.

The refining process of the crude oil begins with the

addition of phosphoric acid (1.1 L per oil ton), followed

by a neutralisation step with NaOH solution (12.7–15.5%,

depending on the acidity of crude oil) and centrifugation.

These combined operations allowed the removal of phospho-

lipids and FFA (neutralised oil sample). Subsequently, the oil

was washed with water (808C), centrifuged to eliminate the

remaining soaps (washed oil sample) and bleached with

activated clay and activated charcoal (1008C, 600 mmHg)

to clean the oil from impurities like pigments and residual

metals (bleached oil sample). The last step consisted on the

deodorisation process (2408C at 2 mbar, during �2 h), in

order to remove unpleasant odours (deodorised oil sample)

(Fig. 1). Each oil sample (crude, neutralised, washed,

bleached and deodorised oil) was obtained by collecting

six portions of 500 mL of oil at regular time-intervals during

24 h directly from the industrial soya bean oil refining line.

All portions corresponding to each step were also assembled

to obtain homogeneous and representative samples. The oil

Cracked seeds

Laminated seeds

Extruded seeds

Crude oil

Deodorised oil

Soya bean seeds

Cleaning

Cracking

Cleaving

Extrusion

Continue oil extraction with n-hexane

Degumming/ Neutralisation

Washing with H2O at 80ºC

Bleaching (activated clay and activated charcoal)

Deodorisation

Pre-

extr

actio

n R

efin

ing

Ext

ract

ion

Washed oil

Bleached oil

Neutralised oil

Figure 1. Extraction and refining steps of soya bean oil production.

Eur. J. Lipid Sci. Technol. 2011, 113, 528–535 Chemical refining of GM soya bean oil as affected by processing 529


samples were kept in dark bottles filled to the top and flushed

with nitrogen until analysis.

2.2 Reagents and standards

For the analysis of FA a standard mixture of 37 FAME

(47885-U; Supelco, Bellefonte, PA, USA) and the individual

FA isomers cis-9-trans-12-octadecadienoate (C18:2n9c12t),

trans-9-cis-12-octadecadienoate (C18:2n9t12c) and cis-11-

octadecenoate (C18:1n7) were used. For the analysis of

phytosterols, the standards cholestanol, cholesterol, campes-

terol, stigmasterol, b-sitosterol, b-sitostanol and betulin, as

internal standard (IS), were obtained from Sigma (St. Louis,

USA). Tocopherols (a, b, g and d) and tocotrienols (a, b, g

and d) were purchased from Calbiochem (La Jolla, CA,

USA). Rac-tocopherol (Tocol) (Matreya Inc., PA, USA)

was used as IS for vitamin E analysis. A stock solution of

the IS (10 mg/mL) was prepared in n-hexane, kept at �48C,

protected from light, and diluted to working solutions

(500 mg/mL) as necessary. A 10 mg/mL working solution

of butylated hydroxytoluene (BHT) (Aldrich, Madrid,

Spain) was prepared in n-hexane to be used as antioxidant.

n-Hexane HPLC-grade and 1,4-dioxane were acquired from

Merck (Darmstad, Germany) and Fluka (Madrid, Spain),

respectively. All other reagents used were of analytical grade.

2.3 Sample preparation

Soya bean seeds and pre-extraction samples were triturated

prior to automatic Soxhlet extraction (Buchi, Flawil,

Switzerland). The extraction was performed using petroleum

ether as solvent in order to extract the oil during a 3 h period,

with the addition of BHT as antioxidant.

Prior to analysis, all the oil samples were stirred at 308Cfor a complete homogenisation of all the components, treated

with anhydrous sodium sulphate and filtered.

2.4 Major nutrient analysis

The analyses of moisture, total fat, ash and protein contents

were performed in triplicate assays. Moisture was determined

(ca. 5 g test sample) using an SMO 01 IR moisture balance

(Scaltec, Goettingen, Germany) at 100 � 28C. Ash, crude

protein (N � 6.25), and total fat contents were determined

according to AOAC Official Methods [7]. Carbohydrate con-

tent was estimated by difference using the following formula:

Carbohydrate content

¼ 100%� %moisture þ %protein þ % fat þ % ashð Þ

2.5 Analysis of free fatty acids

FFAs were determined according to the NP EN ISO 660 [8]

by the dissolution of 5 g of each oil sample in an organic

solvent mixture (diethyl ether/ethanol 95%, 50:50 v/v) and

titration with NaOH solution (0.05 N) after the addition of

phenolphthalein.

2.6 Analysis of fatty acids

Fatty acids were determined by gas–liquid chromatography

with FID (GLC-FID) according to the ISO 5509 [9], which

is applied to oil samples with low FFA content (<2%).

Briefly, 50 mL of each oil sample were dissolved in isooctane,

the FA were converted to FAME by transesterification with

methanolic potassium hydroxide and mixed in vortex for

about 60 s. The reaction was stopped by neutralising the

potassium hydroxide with sodium hydrogen sulphate. The

clear layer containing the FAME was transferred to a 4 mL

vial, after proper dilution, and injected (1.2 mL with a split

ratio of 1:50) in the GLC (Chrompack CP 9001,

Middelburg, The Netherlands) equipped with a

Chrompack CP-9050 auto sampler. The temperatures of

the injector and detector were 230 and 2708C, respectively.

Separation was achieved in a 50 m � 0.25 mm id fused-silica

capillary column coated with a 0.19 mm film of CP-Sil 88

(Chrompack, Middelburg, The Netherlands). Helium was

used as carrier gas at an internal pressure of 120 kPa. The

column temperature program was 1 min at 1608C, increased

at a 48C/min rate until 2398C and then held for 10 min. The

results were expressed as gram per 100 g of oil for each FA.

Fatty acid identification was made by comparing the relative

retention times of FAME peaks with those of standards. The

analysis of all samples was performed in duplicate assays,

each one injected in triplicate.

2.7 Analysis of phytosterols

Sterol composition was determined byGLC-FID according to

the ISO 12228 [10]. Briefly, after the addition of 1.0 mL of IS

solution (betulin, 1.0 mg/mL), 250 mg of oil sample were

saponified with an ethanolic potassium hydroxide solution.

The unsaponifiable fractionwas isolated by solid-phase extrac-

tion on an aluminium oxide column. The steroid fraction was

separated by TLC using n-hexane/diethyl ether 1:1 v/v, as the

developing solvent, and methanol spray for band visualisation.

Sterols were extracted from the silica using ethanol and diethyl

ether, andwere taken to dryness. The trimethylsilyl etherswere

obtained by the addition of 1-methylimidazole andN-methyl-

N-(trimethylsilyl)-heptafluorobutyramide (MSHFBA).

The sterol profile was analysed on the same equipment

used for FA analysis using a column of 30 m � 0.25 mm id,

with a 0.25 mm film of DB-5MS (J&W Scientific, Folsom,

CA; USA). The temperatures of the injector and the detector

were both 3208C. The column temperature program con-

sisted on an increased temperature from 250 to 3008C at a

28C/min rate and then held for 12 min. Helium was used as

carrier gas at an internal pressure of 100 kPa. The injected

volume was 1.5 mL with a split ratio of 1:50.

530 J. Costa et al. Eur. J. Lipid Sci. Technol. 2011, 113, 528–535


The total sterol content was determined considering all

peaks of sterols eluted between cholesterol and D7-avenas-

terol. Identification was achieved by comparing the relative

retention times from samples with those obtained with stand-

ards. To identify the D5-avenasterol and other minor sterols

(campestanol, clerosterol, D7-stigmastenol and D7-avenas-

terol), samples of sunflower and palm oils were submitted

to the same extraction procedure and analysis. Compounds

were identified by comparison with literature [10, 11] and

with the results obtained for those samples. b-Sitostanol and

D5-avenasterol were closely eluted, and therefore, they were

quantified together as D5-avenasterol. The analysis of all

samples was done in duplicate assays, each one injected in

triplicate.

2.8 Analysis of tocopherols

For the analysis of tocopherols, the pre-step of Soxhlet extrac-

tion for solid samples previously described was carried out

overnight followed by 3 h of heat extraction according to the

described by Amaral et al. [12]. Each oil sample (100 mg) was

weighted to a 2 mL reaction tube protected from light. After

the addition of the IS solution (75 mL of tocol 500 mg/mL),

n-hexane was added to a final volume of 2 mL. All samples

were homogenised, transferred to dark injection vials and

analysed by HPLC.

The HPLC system (Jasco, Tokyo, Japan) was equipped

with a PU-980 pump, an AS-950 auto-sampler with a 10 mL

injection loop, an MD-910 multiwavelength diode-array

detector (DAD) and an FP-920 programmable fluorescence

detector. The chromatographic separation of the compounds

was achieved using an Inertsil 5 SI (250 mm � 3 mm, id)

normal phase column (Varian, Middelburg, The

Netherlands) operating at RT (�208C) and a mobile phase

mixture of n-hexane and 1,4-dioxane (95.5:4.5 v/v). Elution

was performed at a solvent flow rate of 0.7 mL/min with an

isocratic program. The effluent was monitored by a DAD

connected to a fluorescence detector set with a gain of 10,

programmed at the excitation and emission wavelengths of

290 and 330 nm, respectively. The compounds were ident-

ified by comparing their retention times and UV spectra with

standards. Quantification was performed by fluorescence

detection based on the IS method. Chromatograms were

acquired and processed using the Borwin-PDA Controller

Software (JMBS, France). The analysis of all samples was

done in duplicate assays, each one injected in triplicate.

3 Results and discussion

3.1 Content of major nutrients

The industrial samples under study were previously analysed

by PCR targeting the RR soya bean (event GTS-40-2-3),

which indicated the use of soya bean seeds at the industrial

soya bean oil production with an average proportion of 50.7%

of GM material [13].

The chemical composition of soya bean samples collected

along the pre-extraction steps (whole, cracked, laminated and

extruded seeds), including the final defatted flour, is pre-

sented in Table 1. In general, the values obtained for the

nutritional evaluation of the whole GM soya bean seeds were

in good agreement with data reported by the USDANational

Nutrient Database for Standard Reference concerning ma-

ture seeds of raw soya bean for moisture (8.54%), fat

(19.94%), protein (36.49%), ash (4.87%) and carbohydrates

(30.16%) (USDA –National Nutrient Database for Standard

Reference, 2009. Accessed on 02.02.2009 – http://www.

nal.usda.gov/fnic/foodcomp/search). The fat content, deter-

mined by Soxhlet, was initially obtained after a 3 h extraction

period with boiling petroleum ether. However, the period of

Soxhlet extraction was further increased since the extraction

was not effective for the whole and cracked seeds. The values

of fat content for whole and cracked seeds were lower than the

expected (�20%) and not in good agreement with the results

for laminated and extruded material. The results presented

on Table 2 show that after a 12 h extraction period it was

possible to maximise the extracted fat from seeds prior to

lamination, with no change observed in laminated and

extrudedmaterial. This finding demonstrates that pre-extrac-

tion steps are crucial to maximise soya bean oil extraction.

During those steps, the seeds are reduced to small particles,

which facilitate the vesicles burst, making the fat more acces-

sible for the extraction.

Table 1. Chemical composition (%) of samples collected along the pre-extraction steps of soya bean oila)

Samples Moisture Fat Protein Ash Carbohydrates

Seed 7.7 � 0.3 19.8 � 0.2 36.8 � 0.3 4.6 � 0.0 31.1 � 0.1

Cracked 7.8 � 0.3 20.5 � 0.1 36.1 � 0.9 4.8 � 0.1 30.8 � 0.4

Laminated 8.4 � 0.2 21.1 � 0.2 37.5 � 0.5 4.9 � 0.0 28.1 � 0.2

Extruded 8.2 � 0.3 20.5 � 0.3 35.3 � 0.2 4.9 � 0.1 31.1 � 0.1

Defatted flour 11.2 � 0.3 1.0 � 0.2 45.4 � 0.1 6.0 � 0.1 36.5 � 0.1

a) Values are given as mean � SD of replicate assays (n ¼ 3).



3.2 Fatty acids

As expected, the neutralisation step decreased the FFA con-

tent (from 0.77 to 0.26%) since they are removed as soaps

during this step (Table 3). Bleaching increased FFA content

because it causes some degree of TAGs hydrolysis due to the

use of activated clay at high temperatures. The deodorisation

step had also an important contribution for FFA reduction.

After this step, a final value of 0.08% was achieved, which

corresponds to almost a 90% decrease of total FFA. These

results are in good agreement with the values reported by

Jung et al. [5] for soya bean oil refining process and are in

compliance with the maximum FFA level of 0.6% imposed

by Portuguese legislation for commercial refined oils [14].

Table 3 presents the FA composition of the analysed

samples. In general, there were no substantial changes

regarding saturated, monounsaturated and polyunsaturated

FA along the pre-extraction, extraction and refining proc-

esses (from the whole grain to the deodorised soya bean oil).

However, the deodorisation step led to an increase of trans

isomers, which is probably related to the high temperatures

used during the deodorisation process, reaching a final con-

tent of 0.7% of transFA in the fully refined oil. This result is in

good agreement with other reported studies that also verified

an increase of trans FA during the deodorisation step of other

vegetable oils [3, 15]. On the other hand, the final value of

total trans FA is lower than the reported by Ferrari et al. [16]

for industrially refined soya bean oil (4.6%). As reported by

the same authors, the parameters that affect the formation of

trans isomers are the temperature and the time of heating.

Thus, the obtained low levels of trans FA were the result of

industrial optimisation of the actual deodorisation process,

minimising the formation of compounds with negative health

effects.

Table 2. Fat content of samples collected along the pre-extraction

steps after 3 and 12 h of automatic Soxhlet extraction

Sample

Fat (%)

3 h extraction 12 h extraction

Seed 16.7 19.8

Cracked 17.9 20.5

Laminated 21.1 21.1

Extruded 20.5 20.5

Table 3. Fatty acid composition of samples collected along the soya bean oil extraction and refining processes

Fatty acids

Soya bean material (%)a) Soya bean oil (%)a)

Seed Cracked Laminated Extruded Crude Neutralised Washed Bleached Deodorised

C14:0 0.11 � 0.02 0.09 � 0.01 0.11 � 0.01 0.11 � 0.02 0.11 � 0.01 0.10 � 0.01 0.10 � 0.01 0.09 � 0.01 0.09 � 0.00

C16:0 10.8 � 0.0 10.9 � 0.0 11.0 � 0.0 11.0 � 0.0 10.8 � 0.0 11.0 � 0.0 11.1 � 0.0 11.2 � 0.0 11.1 � 0.0

C16:1n7c 0.09 � 0.01 0.10 � 0.00 0.10 � 0.01 0.11 � 0.01 0.10 � 0.01 0.09 � 0.00 0.09 � 0.00 0.10 � 0.01 0.10 � 0.00

C17:0 0.10 � 0.00 0.10 � 0.01 0.11 � 0.01 0.10 � 0.01 0.09 � 0.00 0.09 � 0.00 0.09 � 0.00 0.10 � 0.01 0.10 � 0.01

C17:1n7c tr tr 0.06 � 0.01 0.06 � 0.01 0.06 � 0.01 tr 0.06 � 0.01 tr tr

C18:0 3.45 � 0.01 3.46 � 0.01 3.46 � 0.02 3.54 � 0.14 3.44 � 0.01 3.44 � 0.04 3.46 � 0.01 3.52 � 0.01 3.48 � 0.01

C18:1n9t tr tr tr tr tr tr tr tr tr

C18:1n9c 25.4 � 0.1 25.2 � 0.2 25.1 � 0.2 24.9 � 0.2 25.0 � 0.2 24.8 � 0.2 24.8 � 0.2 24.8 � 0.2 24.9 � 0.1

C18:2n6t tr tr tr 0.06 � 0.01 tr tr tr tr 0.61 � 0.01

C18:2n6cc 52.0 � 0.1 52.2 � 0.3 52.0 � 0.2 52.1 � 0.2 52.3 � 0.2 52.9 � 0.2 52.9 � 0.2 52.7 � 0.2 53.0 � 0.2

C20:0 0.36 � 0.00 0.37 � 0.00 0.37 � 0.01 0.38 � 0.02 0.37 � 0.01 0.37 � 0.01 0.37 � 0.01 0.38 � 0.00 0.37 � 0.00

C20:1n9c 0.21 � 0.00 0.21 � 0.00 0.21 � 0.00 0.24 � 0.04 0.21 � 0.01 0.21 � 0.01 0.21 � 0.00 0.21 � 0.00 0.37 � 0.01

C18:3n3c 6.59 � 0.02 6.66 � 0.02 6.60 � 0.05 6.81 � 0.32 6.71 � 0.04 6.06 � 0.04 6.03 � 0.02 6.02 � 0.03 5.03 � 0.01

C22:0 0.46 � 0.00 0.47 � 0.01 0.48 � 0.01 0.49 � 0.02 0.46 � 0.01 0.48 � 0.01 0.48 � 0.01 0.49 � 0.01 0.49 � 0.00

C22:1n9c tr 0.06 � 0.01 0.07 � 0.01 0.07 � 0.01 0.06 � 0.01 0.06 � 0.01 tr tr 0.06 � 0.00

C24:0 0.16 � 0.01 0.15 � 0.00 0.17 � 0.02 0.18 � 0.03 0.16 � 0.01 0.15 � 0.01 0.15 � 0.01 0.15 � 0.00 0.15 � 0.00

SFA 15.5 � 0.1 15.5 � 0.0 15.7 � 0.0 15.8 � 0.1 15.5 � 0.1 15.7 � 0.0 15.7 � 0.0 15.9 � 0.0 15.8 � 0.0

MUFA 25.9 � 0.1 25.6 � 0.2 25.6 � 0.3 25.4 � 0.3 25.5 � 0.1 25.3 � 0.2 25.3 � 0.2 25.3 � 0.2 25.6 � 0.1

PUFA 58.6 � 0.0 58.9 � 0.3 58.6 � 0.1 58.9 � 0.1 59.0 � 0.2 59.0 � 0.2 59.0 � 0.2 58.8 � 0.2 58.6 � 0.2

Trans FA 0.09 � 0.01 0.09 � 0.01 0.10 � 0.02 0.10 � 0.02 0.10 � 0.02 0.09 � 0.02 0.09 � 0.00 0.11 � 0.02 0.66 � 0.03

FFAb) – – – – 0.77 � 0.03 0.26 � 0.01 0.11 � 0.00 0.28 � 0.00 0.08 � 0.00

a) Values are given as mean � SD of replicate assays, that correspond to duplicate analysis for each sample, each one injected in triplicate,

except for FFA (n ¼ 3). SFA, saturated fatty acids.b) FFA, free fatty acids. tr – trace amount (<0.06 g/100 g).



3.3 Phytosterols

TAGs are the main components of oilseed crops, making up

to about 98% of the total fat content. However, minor con-

stituents such as phytosterols and Vitamin E are of great

importance since both groups of compounds have been

associated to health benefits. Phytosterols are known for their

ability to reduce blood cholesterol [17]. These compounds

are also considered technologically important because they

can improve the oxidative stability of oils, since phytosterols

with an ethylidene group in the side chain (D5 and D7-ave-

nasterols) are believed to hold anti-polymerisation properties,

thus protecting the oils from oxidation during thermal proc-

essing [18].

The phytosterol composition along the soya bean oil

production is shown in Table 4, with the individual com-

pounds expressed as percentage of total sterols and total

sterols expressed as mg/100 g of oil. In all samples, b-sitos-

terol is the most abundant sterol, followed by campesterol

and stigmasterol, with contents in close proximity to the

values established for commercial edible soya bean oil in

the Portuguese legislation [14]. The comparison of the herein

obtained results concerning GM soya bean with the same

parameters for conventional soya bean is difficult to establish

due to the scarcity of data in the literature. The obtained

values for total sterol content in the refined soya bean oil

(313 mg/100 g) are higher than those reported by Ferrari

et al. [16] and Verleyen et al. [19] with 295 and 216 mg/100 g

of refined oil, respectively, although both works do not specify

the type of soya bean seed used.

During the pre-extraction steps an increase of total phy-

tosterols (from 353 to 409 mg/100 g) was observed. This

increase was probably related to the enhanced fat extraction

after the lamination step, since the oil sample used for the

analysis of phytosterols was obtained after a 3 h Soxhlet

extraction which, as referred above (Table 2), was an

insufficient time for the complete fat extraction from the

whole and cracked soya bean seeds. Along refining, a

reduction of total sterols was observed (from 393 mg in crude

oil to 313 mg/100 g in the refined oil), being the neutralis-

ation the step with the highest contribution. During neutral-

isation, free phytosterols can be transferred by liquid–liquid

partitioning to the soapstock and, consequently, be elimi-

nated by centrifugation at the end of this step [19]. After

neutralisation, the oil was washed to remove the remaining

soaps, contributing to a slight further reduction of phytoster-

ols. Both the neutralisation and washing steps were respon-

sible for a 14.9% of total sterols reduction, which is a lower

decrease than the reported by Sleeter [6] and Verleyen et al.

[19] for soya bean oil refining (21.2 and 19.6%, respectively).

Verleyen et al. [19] studied the effect of parameters such as

the concentration and volume of NaOH solution on soya

bean crude oil submitted to a chemical refining process at

laboratory scale, concluding that the use of high NaOH

concentrations (15%) induces low losses on phytosterols

during the chemical neutralisation, while the use of low

strength solutions (2.5–7.5%) were responsible for higher

losses. Thus, the low reduction verified in the total content

of phytosterols under study could be related to the high

concentration of the NaOH solution (12.7–15.5%) used in

the neutralisation step of the industrial process.

During the bleaching step, it was observed a 2.6%

reduction on the total sterols content, possibly explained

by some degree of phytosterol adsorption onto the activated

clays and charcoal. Moreover, it was reported that some non-

polar phytosterols dehydration products can be formed

during vegetable oil bleaching, contributing to the decrease

of total sterols along the refining process [3, 19]. The last

refining step (deodorisation) is considered an aggressive

stage, due to the high temperature (2408C) and low pressure

conditions, which are believed to induce a gradual loss of

phytosterols. The results herein show that deodorisation

Table 4. Individual phytosterol proportion and total phytosterol content of samples collected along the soya bean oil extraction and refining

processes

Sterols (%)a)

Total

Cholesterol Campesterol Campestanol Stigmasterol Clerosterol b-Sitosterol D5-Avenasterol D7-Stigmasterol D7-Avenasterol mg/100 g oil

Soya bean material

Seeds 1.06 � 0.01 18.5 � 0.2 0.58 � 0.02 18.8 � 0.4 0.48 � 0.03 51.8 � 0.5 4.59 � 0.62 3.23 � 0.17 0.96 � 0.27 353 � 1

Cracked 0.55 � 0.02 19.1 � 0.1 0.60 � 0.03 19.9 � 0.1 0.55 � 0.02 51.9 � 0.1 4.30 � 0.11 2.03 � 0.13 1.09 � 0.09 379 � 20

Laminated 0.46 � 0.03 16.7 � 0.1 0.51 � 0.02 17.2 � 0.2 0.49 � 0.03 55.1 � 0.4 4.26 � 0.72 4.02 � 0.51 1.38 � 0.49 430 � 27

Extruded 0.47 � 0.01 17.0 � 0.2 0.51 � 0.02 17.5 � 0.4 0.52 � 0.01 54.2 � 0.7 4.40 � 0.60 3.66 � 0.53 1.70 � 0.11 409 � 22

Soya bean oil

Crude 0.47 � 0.03 17.4 � 0.2 0.51 � 0.01 17.6 � 0.6 0.49 � 0.04 53.8 � 0.1 4.65 � 1.04 3.91 � 0.16 1.27 � 0.42 393 � 10

Neutralised 0.55 � 0.05 15.7 � 0.0 0.48 � 0.02 17.4 � 0.2 0.52 � 0.01 53.4 � 0.5 4.73 � 0.53 5.38 � 0.03 1.89 � 0.10 343 � 16

Washed 0.66 � 0.06 15.7 � 0.1 0.53 � 0.03 17.8 � 0.1 0.54 � 0.04 54.5 � 0.7 3.43 � 0.12 5.12 � 0.54 1.63 � 0.03 334 � 1

Bleached 0.45 � 0.02 16.2 � 0.0 0.58 � 0.03 18.2 � 0.2 0.43 � 0.01 55.5 � 0.3 2.78 � 0.09 4.92 � 0.48 1.00 � 0.05 324 � 17

Deodorised 0.60 � 0.06 15.9 � 0.1 0.56 � 0.04 17.7 � 0.4 0.46 � 0.06 55.9 � 0.7 2.97 � 0.54 5.00 � 0.50 1.01 � 0.08 313 � 10

a) Values are given as mean � SD of replicate assays that correspond to duplicate analysis for each sample, each one injected in triplicate.

Individual compounds expressed as percentages of total phytosterols.



caused a slight decrease (2.9%) of total sterols when com-

pared to the 16.7% reported by Verleyen et al. [19] for the

deodorisation at 2608C. Those authors, when studying

the effect of deodorisation temperature (220–2608C) on

the physical and chemical refining of soya bean oil, verified

that the increase of temperature contributed to higher losses

of esterified and free sterols. They also reported that free

sterols are distilled during deodorisation at elevated tempera-

tures and that, at the same time, those conditions induce an

esterification reaction between free sterols and FFA, resulting

in an increase in the content of steryl esters after deodor-

isation. Since the FFA content of soya bean oil after bleaching

was much higher (0.28%) than that used by Verleyen et al.

[19] in chemical refining (0.008–0.074%), this could explain

the lower losses obtained in this study, together with the use

of lower deodorisation temperature.

The complete refining process caused a 20% reduction on

total phytosterols, which agrees with the same reduction

(18%) obtained by Ferrari et al. [16], but is lower than the

34% reported by Verleyen et al. [19].

3.4 Tocopherols

Vitamin E is a powerful antioxidant that is believed to be

involved in a diversity of physiological and biochemical func-

tions [20], mainly due to their antioxidant activity, to its

action as membrane stabilisers and as a lipidic antioxidant,

helping to prevent the polyunsaturatedmembrane lipids from

free radical attack [21]. Vitamin E also plays an important

role in the prevention of lipid oxidation processes, and its

presence is particularly important in foods with high content

of unsaturated fatty acids, which are more susceptible to

rancidity phenomena.

The contents of total and individual tocopherols along the

industrial refining are presented in Table 5. The obtained

results revealed the presence of the four tocopherols (a-, b-,

g- and d-), with g-tocopherol as the major compound

followed by d-tocopherol, while no tocotrienols were

detected, as reported by Ferrari et al. [16] for soya bean oil.

In the present work, total tocopherols decreased from

1344 mg/kg in crude oil to 942 mg/kg in refined oil, corre-

sponding to a loss of 30%. This reduction was lower than

other values (32, 36 and 41%), reported by Jung et al. [5],

Sleeter [6] and Medina-Juarez et al. [22], respectively, con-

cerning the refined soya bean oil. Tocopherols are degraded

by high temperatures, high pH and certainmetals such as iron

and copper [23], thus, under the refining process conditions

they can be easily lost. The neutralisation step caused the

greatest reduction (20%), followed by the deodorisation with

a 9% loss. The high decrease of tocopherols observed after

neutralisation is in good agreement with other reports con-

cerning the chemical refining of other vegetable oils [2]. Jung

et al. [5] and Sleeter [6] reported lower decreases (7.4 and

11.9%, respectively) after soya bean oil neutralisation,

possibly because of the use of lower concentrations of

NaOH solution than that in the industrial process under

study. This decrease can be attributed to tocopherols insta-

bility caused by the contact of oil with air and concentrated

alkali solution during this refining step [23]. The fact that the

content of tocopherols was not affected by the subsequent

step (washing) proves that the mechanism that explains toco-

pherol decrease during neutralisation step is different from

the one responsible for phytosterols decrease (elimination in

the soapstock). In other works, major losses were reported

during the deodorisation of soya bean oil (12, 20 and 29%)

[5, 6, 19]. The decrease of tocopherols during deodorisation

is reported to depend on the time, temperature, pressure and

stripping steam flow used [23]. Ideally, the deodorisation

conditions should be carefully chosen in order to guarantee

that the loss of total tocopherols does not exceed 25% [22],

which was verified in the analysed samples. The bleaching

step accounted to negligible losses (1.9%), although it has

been associated to moderate decreases of tocopherols in soya

bean oil (4.8%) [5].

Table 5. Tocopherol composition of samples collected along the soya bean oil extraction and refining processes

Tocopherols (mg/kg of oil)a)

a-Tocopherol b-Tocopherol g-Tocopherol d-Tocopherol Total

Soya bean material

Seed 74.7 � 7.6 13.0 � 0.4 700 � 21 402 � 19 1190 � 32

Cracked 67.1 � 0.1 13.0 � 0.4 739 � 7 429 � 2 1248 � 9

Laminated 77.1 � 5.9 16.1 � 1.3 799 � 46 479 � 4 1371 � 57

Extruded 74.9 � 2.6 15.3 � 0.2 798 � 15 479 � 8 1367 � 26

Soya bean oil

Crude 75.2 � 4.1 15.0 � 1.0 789 � 42 465 � 26 1344 � 26

Neutralised 83.0 � 2.2 12.8 � 0.8 659 � 18 324 � 7 1079 � 28

Washed 83.8 � 0.2 13.1 � 0.3 664 � 0 327 � 1 1088 � 2

Bleached 82.6 � 2.0 12.5 � 0.4 646 � 6 322 � 3 1063 � 12

Deodorised 77.1 � 1.5 11.1 � 0.1 576 � 14 278 � 2 942 � 18

a) Values are given as mean � SD of replicate assays that correspond to duplicate analysis for each sample, each one injected in triplicate.



Regarding the contents of individual tocopherols

(Table 5), a-tocopherol seems to stay almost unchanged

along refining, while d-tocopherol was the vitamer more

affected with a total decrease of 40%. d-Tocopherol was

substantially affected by the neutralisation step with a 30%

loss and a further 10% decrease after deodorisation, while

washing and bleaching had negligible effects. b- and g-toco-

pherol showed identical and gradual decreases along the

refining process, totalising 26 and 27%, respectively.

4 Conclusions

The results showed that the pre-extraction steps used at the

industrial soya bean oil production are crucial for maximising

the oil extraction of soya bean seeds. Several studies have

demonstrated that beneficial compounds, such as tocopher-

ols and phytosterols can be lost during oil refining. However,

the industrial conditions used along the different steps of the

chemical refining are key factors for the final contents of

these compounds in the fully refined oils. The loss of

beneficial compounds seem to be low when compared to

other works concerning soya bean oil refining, since the final

oil contained high levels of phytosterols and tocopherols.

This finding reflects the improvements made by the oil indus-

try regarding the refining parameters used, as well as an

increasing effort to obtain a final product with high nutri-

tional value.

In this work, the composition of soya bean oil produced

from RR soya bean was, as far as we know, reported for the

first time. However, more studies are needed to establish a

comparison between the chemical composition (especially

concerning beneficial compounds) of conventional and

GM soya bean seeds.

The authors wish to thank to SOVENA industry for supplying the

samples. Joana Costa is grateful to FCT PhD grant (SFRH/BD/

64523/2009) financed by POPH-QREN (subsidised by FSE and

MCTES).

The authors have declared no conflict of interest.

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Refining of Roundup Ready® soya bean oil: Effect on the fatty acid, phytosterol and tocopherol profiles