Food Chemistry 142 (2014) 294298

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Degradation of phytosterols during storage of enriched margarines

Magdalena Rudzinska , Roman Przybylski, Erwin Wasowicz
, Poland
Poznan University of Life Sciences, Faculty of Food Science and Nutrition, Wojska Polskiego 31, 60-624 Poznan

a r t i c l e

i n f o

Article history:
Received 10 January 2013
Received in revised form 2 June 2013
Accepted 9 July 2013
Available online 17 July 2013
Keywords:
Oxidation
Margarines
Phytosterol
Storage
Gas chromatography

a b s t r a c t
Oxidative changes of phytosterols were recently studied in vegetable oils and some food products. Cholesterol-lowering properties of phytosterols and phytostanols are the main driver for formulating functional foods containing these compounds.
Margarines enriched in plant stanols were stored at two typical temperatures for up to 18 weeks. Analysed margarines contained four phytosterols: brassicasterol, campesterol, sitosterol, avenasterol and
two phytostanols: sitostanol, campestanol. The content of phytosterols and phytostanols in margarines
changed from 79 mg/g in a control sample to 63 mg/g and 55 mg/g in samples stored for 18 weeks at
4 C and 20 C, respectively. At the end of storage, contents of sitostanol decreased by 23% and 30%, while
the amounts of oxidised sterols increased by 35% and 100%, respectively, for both temperatures. 7Hydroxy derivatives dominated among all oxidised phytosterols and their content increased threefold
at the end of storage. Epoxy derivatives exhibited a maximum after 6 weeks of storage at 20 C and thereafter decreased constantly.
2013 Published by Elsevier Ltd.

1. Introduction
Phytosterols are endogenous components of all plant-origin
food ingredients. Most naturally occurring phytosterols share the
chemical structure of cholesterol; however, some have a double
bond in a side chain. Phytosterols with a double bond in their
structure are usually named unsaturated sterols while phytostanols are saturated (Fig. 1). The cholesterol-lowering properties of
phytosterols were discovered in 1951, when Peterson fed soybean
sterols to chicken as part of a high-cholesterol diet and established
that the expected increase in blood cholesterol did not occur (Peterson, 1951). Since then many studies have addressed this physiological effect in both humans and animals (Gylling et al., 2009; Lin
et al., 2010; Rasmussen et al., 2006). In the early trials, wood-derived stanols were used as bioactive compounds, followed by phytosterols and their esters, which exhibited similar lowering of
cholesterol (Gylling et al., 2009; Moreau, 2004).
Phytosterols and cholesterol share a similar chemical structure,
and undergo oxidation during food processing and storage. Many
types of cholesterol oxidation products have been found in different food products and their negative biological activities have been
extensively reviewed (Gill, Chow, & Brown, 2008; Otaegui-Arrazo-

Corresponding author. Address: Poznan University of Life Sciences, Institute of

Food Technology of Plant Origin, Wojska Polskiego 31, 60-624 Poznan, Poland. Tel.:
+48 61 8487276; fax: +48 61 8487314.
E-mail address: magdar@up.poznan.pl (M. Rudzinska).
0308-8146/$ - see front matter 2013 Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.foodchem.2013.07.041

la, Menndez-Carreo, Ansorena, & Astiasarn, 2010; Paniangvait,

King, Jones, & German, 1995).
Phytostanols, due to the lack of a double bond in the structure,
are considered to be less prone to oxidative degradation; however,
the presence of tertiary carbons makes them prone to this degradation and formation of a variety of oxidised derivatives is possible
(Smith, 1981). One of the oxidation mechanisms proposed for phytostanol degradation includes oxidation in a side-chain and generation of 3-keto compounds (Dutta, 2004). Aringer and Nordstrm
(1981) found several saturated hydroxyl-steroids and C-24 ethyl
analogues of these oxides. Soupas, Juntunen, Lampi, and Piironen
(2004) characterised sitostanol oxides, such as epimers of 7hydroxysitostanol and 7-ketositostanol, formed during heating
sitostanol at 180 C for 3 h. However, oxidative degradation of
sitostanol in rapeseed oil and tripalmitin was very slow.
Commercial spreadable fats, margarines, milk and yoghurts,
formulated with phytosterols, are available on the market, and
only a few papers have dealt with the evaluation of oxidative stability of sterols, and factors affecting the formation of oxidised sterol derivatives (Conchillo et al., 2005; Grandgirard, Martine, Joffre,
Juaneda, & Berdeaux, 2004; Johnsson & Dutta, 2006; Moreau, 2004;
Moreau, Whitaker, & Hicks, 2002; Soupas et al., 2004).
The main goal of this work was assessment of oxidative degradation of phytosterols and phytostanols during storage of margarine enriched with these compounds at 4 C and 20 C for
18 weeks.

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295

Fig. 1. Structure of typical phytosterols and stanols.

2. Materials and methods

2.1. Materials
Margarines enriched in phytosterols/phytostanols were purchased in local stores and stored on open Petri dishes in a 0.5 cm
layer at 4 and 20 C for up to 18 weeks. Samples were analysed
after 6, 12 and 18 weeks of storage; the fresh sample was used
as control.
Internal standard, 19-hydroxycholesterol, was purchased from
Steraloids (Newport, RI, USA). Solvents, 5-cholestane, and anhydrous pyridine were supplied by SigmaAldrich (St. Louis, MO,
USA). Silylation mixture containing of BSTFA (N,O-bis(trimethylsilyl) triuoroacetamide) and with 1% TMCS (trimethylchlorosilane), was received from Fluka Chemie (Buchs, Switzerland),
while SEP-PAK amino cartridges were from Waters (Milford, USA).

2.2. Determination of phytosterols/phytostanols content

Plant sterols were analysed using the procedure described by
Rudzinska, Kazus, and Wasowicz (2001). Briey, to 0.25 g of
margarines, 0.5 mg of 5-cholestane dissolved in chloroform was
added; the latter was used as internal standard. Saponication
was performed with 1 M methanolic KOH at room temperature
for 18 h. Unsaponiables were extracted with diethyl ether after
water was added to the saponication mixture. Isolated sterols
were silylated with BSTFA containing 1% TMCS and analysed
on a HewlettPackard model 6890 GC (Agilent, Wilmington,
DE, USA) equipped with a DB5 capillary column (30 m
 0.25 mm  0.25 m; J&W Scientic, Folsom, CA, USA). Separation was done isothermally at 290 C, using helium as carrier
gas at a ow rate of 1.6 ml/min. The injector and detector temperatures were set at 310 C, and samples were injected in a
split mode (1:25). Identication of phytosterols was done on a
GC/MS (Trace 2000/Finnigan-Polaris Q, Thermo Electron, San
Jose, CA, USA), using the same chromatographic conditions as
described above.
Samples from autonomous series were analysed in triplicate.

2.3.2. Transestrication
To 0.25 g of margarine in 1 mL of MTBE, 10 mg of the internal
standard (19-hydroxycholesterol) and 2 mL of sodium methoxide
(10% in methanol) were added and vortexed. The mixture was left
for 1 h at room temperature; then oxysterol fraction was extracted
with chloroform. Extract was rinsed with water and chloroform
and evaporated to dryness under a stream of nitrogen, and residue
dissolved in 250 lL of chloroform.
2.3.3. SPE fractionation
The SEP-PAK NH2 cartridge was conditioned with 10 ml of hexane and the isolated sterol fraction loaded. The column was
sequentially eluted with 10 ml of hexane, 5 ml of hexane-MTBE
(5:1; v/v) and 5 ml of hexane-MTBE (3:1; v/v) and nally with
7 ml of acetone to remove sterol oxides. From this fraction, solvent
was evaporated under a stream of nitrogen and the residue derivatized with 100 ll of anhydrous pyridine and 100 ll of BSTFA + 1%
TMCS mixture and, after 4 h at room temperature, the sample was
ready for analysis.
2.3.4. Gas chromatography/mass spectrometry
Derivatized sterol oxides were analysed on a HewlettPackard
6890 gas chromatograph equipped with an HP-5 column (50 m
 0.2 mm  0.32 lm; J&W, Folsom, CA). Samples were injected in
a splitless mode and column temperature programmed as follows:
initial temperature (of 160 C) was held for 1 min, then programmed at 40 C/min to 270 C and held for 1 min; it was further
programmed at 4 C/min to 280 C; nal temperature was held for
25 min. Helium carrier gas, at ow of 1 ml/min, was used.
Oxidised derivatives were identied using a Finnigan TRACE
2000 gas chromatograph coupled to a Finnigan Polaris Q Quadrupole Ion Trap mass spectrometer, using the same column and conditions as described above. All mass spectra were recorded using
electron impact ionisation mode at 70 eV and masses were
scanned from 100 to 650 Da. Ion source was held at 200 C, while
the injector was at 300 C. For identication of compounds, the
combination of NIST Mass Spectra Library, our laboratory library
of collected sterol data and retention data of standards were
utilised.
Samples from an autonomous series were analysed in triplicate.

2.3. Determination of phytosterol/stanols oxidation products (POPs)

2.4. Peroxide value (PV) and anisidine value (AV)
2.3.1. General
Phytosterol and phytostanol oxides were quantied and identied after sample preparation, following the procedure of Przygonski, Jelen, and Wasowicz (2000), briey described below:

For the assessment of oxidative status of stored margarines,

peroxide value and anisidine value were determined according to
AOCS methods Cd 8-53 (2003) and Cd 18-90 (1997), respectively.

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296

2.5. Residual tocopherol

Tocopherols were analysed according to AOCS Ofcial Method
Ce 8-89 (1997). Briey, oil samples (50 mg) were weighed directly
into autosampler vials and dissolved in 1 ml of hexane. The mobile
phase, 7% methyl tert-butyl ether in hexane, with a ow rate of
0.6 ml/min, was used. The uorescence detector was set for excitation at 292 nm and emission at 325 nm. For each run, 10 ll of sample was injected.
2.6. Fatty acid composition
The fatty acid composition of margarine was analysed by following AOCS Ofcial Method Ce 1h-05 (2005). The FAMEs were
separated on a TR-FAME capillary column (100 m  0.25 mm 
0.25 lm; ThermoFisher Scientic, Waltham, USA) installed in a
Trace GC Ultra gas chromatograph (Thermo Electron, Rodano,
Italy). The FAME sample, 1 ll, was injected with an AS 3000 autosampler (Thermo Electron, Rodano, Italy) into a temperature-programmed injector (PTV). Hydrogen was used as carrier gas at a
ow rate of 1.5 ml/min. Column temperature was programmed
from 70 C to 160 C at 25 C/min, held for 30 min, then further
programmed to 210 C at 3 C/min. Initial and nal temperatures
were held for 5 and 30 min, respectively. Detector temperature
was set at 250 C. Fatty acids were identied by comparison of
the retention times with those of authentic standards and the results are reported as a weight percentage of the lipid.
3. Results and discussion
3.1. The oxidative stability of margarines
Margarines enriched in phytosterols/phytostanols evaluated in
this study were of good initial quality, as indicated by low PV
and anisidine values (Table 1). Storage temperatures signicantly
affected PV; its value increased to 9.4 and 35.4 meq/kg after
12 weeks of storage at 4 and 20 C, respectively. These PV values
highly surpass the level of oxidation allowed by many quality standards. Such fast increase of PV was not observed during storage of
margarine in standard packages at 20 C, probably due to limited
exposure to oxygen (Nogala-Kaucka and Gogolewski, 2002; Zhang,
Jacobsen, Pedersen, Christensen, & Adler-Nissen, 2006).
Increase of the peroxide value was accompanied by a decreasing amount of PUFA, together with accelerated decomposition of
d- and c-tocopherols (Table 1).
3.2. Changes of phytosterol content
In margarine, four phytosterols: brassicasterol, campesterol,
sitosterol, avenasterol and two phytostanols, namely sitostanol
and campestanol, were identied. Total amount of sterols at the

Fig. 2. Phytosterol and oxysterol changes during margarine storage at different

temperatures. Sterols sum of sterols and stanols amounts; Oxysterols sum of
oxidised derivatives of sterols and stanols amounts.

beginning of the experiment was 79 mg/g, with the main components being sitostanol (58 mg/g) and campestanol (12.6 mg/g).
During storage, losses of the total sterols were observed at 20%
and 31% at the end of storage at 4 C and 20 C, respectively (Fig
2). The individual sterols disappeared in a similar manner and differences between saturated and unsaturated sterol disappearance
rates were not observed (Table 2). Soupas, Huikko, Lampi, and
Piironen (2006) have not found losses in phytosterols content
when storage was done using a microcrystalline suspension of different fats/oils at 4 C for 12 months. In enriched whole milk powder, 4.3% of phytosterols disappeared during storage at room
temperature and at 38 C for 12 months. The highest loss of sterols,
60% of the total amount disappeared, was when phytosterol-enriched milk was heated for 2 min in a microwave oven and for
15 min at 90 C(Menndez-Carreo, Ansorena, & Astiasarn, 2008).
The content of total phytosterols and stanols in analysed margarine ranged from 79 mg/g in the control sample to 63 mg/g
and 55 mg/g at the end of storage time at 4 C and 20 C, respectively (Fig. 2). During storage at higher temperature, sterols were
oxidised 1.5 times faster than at the refrigeration temperature
(4 C). Since oxidative degradation of sterols usually follows a free
radical mechanism, similar to fatty acids, the radicals from the latter and previous condition may initiate and stimulate oxidation.
(Dutta, 2004; Smith, 1981). Grandgirard et al. (2004) established
that 0.08% of stanols and sterols were oxidised in analysed spreads.
Among phytosterols, sitostanol was the most abundant, followed
by campestanol; both saturated sterols degraded at the fastest rate
under both storage conditions (Table 2). During the storage under
refrigeration and at room temperatures, sitostanol degraded 19
and 14 times faster than did the unsaturated parent sterol (Table 2).

Table 1
Properties and composition of margarines stored at different temperatures.
Samples stored at

Storage time [weeks]

Peroxide value [meq/kg]

Anisidine value

Tocopherols [lg/g]

Fatty acids [%]

SAT

MUFA

PUFA

1.1 0.1

1.8 0.1

120 9

250 10

19 1

51 2

31 2

6
12
18

1.4 0.1
9.4 0.7
21.9 1.4

1.8 0.1
2.4 0.2
3.6 0.2

108 7
101 7
99 6

246 9
218 9
194 9

18 1
19 1
20 1

52 2
53 2
53 2

30 2
28 1
27 1

6
12
18

1.5 0.1
35.4 2.1
114 4.9

1.9 0.1
2.5 0.2
6.5 0.3

107 7
92 5
82 4

244 9
146 8
118 7

19 1
19 1
20 1

52 2
54 2
54 2

29 1
27 1
25 1

Control

4 C

20 C

SFA saturated fatty acids; MUFA monounsaturated fatty acids; PUFA polyunsaturated fatty acids.

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297

Table 2
The content of phytosterols [mg/g] in margarine stored at 20 C and 4 C for 6, 12 and 18 weeks.
Phytosterols

Control

Storage (weeks)
4 C

20 C

12

18

12

18

Brassicasterol
Campesterol
Campestanol
Sitosterol
Sitostanol
Avenasterol

0.82 0.18
2.95 0.23
12.7 1.02
3.62 0.26
58.1 4.31
0.68 0.06

0.80 0.07
2.82 0.24
12.3 1.02
3.53 0.26
54.4 4.91
0.67 0.06

0.73 0.06
2.64 0.22
11.3 1.03
3.29 0.25
50.7 4.67
0.62 0.05

0.71 0.06
2.12 0.18
11.3 1.05
2.95 0.22
45.1 4.05
0.56 0.05

0.78 0.07
2.59 0.22
12.1 1.05
2.52 0.21
51.7 4.14
0.64 0.05

0.62 0.05
2.46 0.21
9.98 0.82
2.34 0.20
43.0 3.72
0.57 0.05

0.58 0.05
2.04 0.18
8.29 0.76
2.23 0.20
40.9 3.73
0.56 0.05

Total [mg/g (%)]a

Sterols
Stanols

8.07
70.8

7.82(96)
66.7(94)

7.28(90)
62.0(88)

6.34(79)
56.4(80)

6.53(81)
63.8(90)

5.99(74)
53.0(75)

5.41(67)
49.1(69)

Units used to express amount (mg/g) and in bracket percentage of the initial value.

Campestanol degraded 2 and 5 times faster than campesterol at

comparable storage temperatures; however, signicantly more
slowly than sitostanol. These degradation rates are unexpected
when chemical structure is taken into account and more unsaturated compounds ought to be more easily oxidised (Johnsson &
Dutta, 2006; Smith, 1981). Analysed margarine contained more
than 80% of unsaturated fatty acids and, in it, campestanol and
sitostanol disappeared at a faster rate than did the unsaturated
parent components. As mentioned above, similarity in free radical
oxidation mechanisms between fatty acids and sterols may indicate that radicals from fatty acids and sterols may stimulate oxidative degradation (Dutta, 2004; Smith, 1981). Brassicasterol and
avenasterol, containing an additional double bond in the side
chain, were found as the minor sterols, and their degradation rates
were at the lowest level among all assessed phytosterols (Table 2).
3.3. Changes of phytosterols/phytostanols oxidation products content
Phytosterol oxidation products were detected in all analysed
samples. The lowest level of oxides was found in fresh margarines
at 255 lg/g and its amount increased to 354 and 734 lg/g after
storage at 4 C and 20 C for 6 weeks, respectively (Fig. 2). For samples stored at 20 C, the amount of oxyphytosterols increased three
times faster than at a lower temperature (Fig. 2). Grandgirard et al.
(2004) reported 68 lg/g of oxidised sterol and stanol derivatives in
fresh spread, and Johnsson and Dutta (2006) 12 lg/g. Comparison
of published data with our results may suggest that used in formulation, phytosterols were already oxidised to a relatively high
extent.
In all samples, 7-hydroxy derivatives formed the majority of all
oxidised sterols and this change reects further transformation of
these derivatives into other compounds (Fig. 3). The content of
epoxy derivatives in fresh margarine was 71 lg/g and increased
to 333 lg/g at the 6th week of storage at 20 C. The amount of
epoxides decreased to 184 lg/g after 12 weeks and 106 lg/g after
18 weeks. At the same time, the amount of triol increased signicantly, the latter is formed from epoxides (Figs. 2 and 3). In samples stored at 4 C, the epoxides content ranged from 98 lg/g
after 6 weeks, to 89 lg/g after 12 weeks and 65 lg/g after
18 weeks. These results indicate that, at lower storage temperature, formation of hydroxides dominated over epoxides, demonstrating that lower activation energy is required for hydroxides
formation (Chien, Hsu, & Chen, 2006). Dynamic changes in the
amount of the particular group of phytosteroloxides, indicate
interconversion between them (Fig. 2).
The amount of sitosterol and sitostanol oxides was at the highest level at the sixth week of the storage at 20 C. In samples stored
at 4 C and 20 C, the amount of campesterol and campestanol oxides was higher than that of sitosterol and sitostanol derivatives

Fig. 3. Phytosterol oxidation during fortied margarine storage at different

temperatures expressed by compounds formed. Phytosterol oxides were combined
into the following chemical groups: sum of hydroxy enantiomers; sum of epoxy
enantiomers; 7-keto and triol derivatives.

after the sixth week of storage (Fig. 4). Faster formation of campesterol and campestanol derivatives during storage is affected by the
amount of these compounds in margarines. Secondly, this indicates
that these compounds have lower activation energies and are easier oxidised (Lengyel et al., 2012).
Conchillo, Cercaci, Ansorena, Rodriguez-Estrada, Lercker, & Astiasarn (2005) detected phytosterol oxidation products in commercial vegetable spreads and low-fat spreads, both enriched in
phytosterol esters. The phytosterol-enriched products exhibited
four times higher amounts of phytosterol oxidation products than
did traditional spreads. However, Garcia-Llatas et al., 2008

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M. Rudzin

Fig. 4. Formation of oxidised derivatives from sitosterol, sitostanol, campesterol

and campestanol during margarine storage. The amounts represent all detected
derivatives of: SitOX oxides of sitosterol and sitostanol; CampOX oxides of
campesterol and campestanol. Numbers 4 and 20 represent storage temperatures.

observed no signicant differences in the total amounts of sterol

oxidation products in ready-to-eat infant formulas when stored
at 25 C for 9 months. Soupas, Huikko, Lampi, and Piironen
(2007) observed that, during a pan-frying, phytosterol oxides were
formed at a very low level.
This study exhibited that during typical conditions used for food
storage and utilisation, oxidation of fatty acids in margarine was
observed, together with degradation of phytosterols and phytostanols, including formation of oxidative derivatives. According to
Smith (1981) and Dutta (2004), oxidative degradation of sterols
follows a free radical mechanism, similar to the fatty acids, and
radicals can either initiate or stimulate oxidative degradation of
both groups of compounds. Cercaci, Rodriguez-Estrada, Lercker,
and Decker (2007) suggested that oxidation in emulsion occurs
at the droplet interface. Phytosterols, as surface active compounds,
could be particularly prone to oxidation when they are incorporated in emulsions, such as margarine. The study also showed a decrease in the amount of sterol during storage of enriched
margarine without a corresponding increase in sterol oxidation
products, indicating further reaction of sterol oxidation products
with other compounds (Rudzinska, 2011).
This study has revealed the importance of storage condition on
the stability of sterols in enriched margarines, leading to the necessity to monitor sterol oxidation products due to their potential
negative health effects. Additionally, it also indicated that utilised
phytosterol and stanols, used in these formulations, are not always
of the best quality, which may further accelerate oxidative degradation in those products.
Acknowledgement
The National Science Centre is acknowledged partly for nancial support within the project 2011/03/B/NZ9/00276.
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