Food Control 54 (2015) 31e38

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Food Control
journal homepage: www.elsevier.com/locate/foodcont

Tocopherol and tocotrienol analysis as a tool to discriminate different

fat ingredients in bakery products
R. Mignogna, A. Fratianni*, S. Niro, G. Panfili
 degli Studi del Molise, Via De Sanctis, 86100, Campobasso, Italy
Dipartimento Agricoltura, Ambiente e Alimenti (DiAAA), Universita

a r t i c l e i n f o a b s t r a c t

Article history: Fats are widely used in the food industry as ingredients in many processed foods, in particular, in bakery
Received 2 August 2014 products, they cover an important technological and sensorial role. With the introduction of new EU
Received in revised form Regulation on Food Information (1169/2011) fats, in particular refined vegetable oils, should be clearly
20 January 2015
identified. Tocopherols and tocotrienols are eight different compounds with a variable qualitative/
Accepted 21 January 2015
quantitative content in different fats. In this work, the content of tocopherols and tocotrienols of 38
Available online 31 January 2015
commercial bakery products, such as different type of biscuits, croissants and sandwiches loaves,
available on the Italian market in 2013e2014, was evaluated. Significant differences were found in the
Keywords:
Tocopherol
tocol profile of analysed samples. Moreover, the tocol profile reflected the tocol composition of the
Tocotrienol specified fat used as ingredient, thus giving the possibility to use tocols as a tool to verify the information
Vegetable oil given in food labelling. Regarding vitamin E activity, expressed as Tocopherol Equivalent (T.E.), the 68% of
Bakery product the analysed bakery products can be declared in label as a source of vitamin E, in particular, all biscuits
Fat made with vegetable oils and croissants, while no analysed sandwich loaves provided the recommended
allowance.
© 2015 Elsevier Ltd. All rights reserved.

1. Introduction
Bakery products, like biscuits, snacks, croissants and sandwich
loaves, are very popular among people, in particular kids. They
contain several ingredients, sugars, cereals and/or pseudocereals,
eggs, cocoa, vegetable and animal fats, most of which have
bioactive compounds with nutritional and health effects, like
vitamin E and A (carotenoids and retinols). Traditionally, in the
production of bakery foods, animal fats, like butter and lard, are
often employed, together with the use of different vegetable oils
and fats, such as corn oil, sunflower oil, olive oil, palm oil or hy-
drogenated fats or their blends. The quality of baked products is
affected by lipids in many ways, including air incorporation,
lubrication, heat transfer, tenderness and texture, moisture, mouth
feel, flavour and shelf life (Ghotra, Dyal, & Narine, 2002; Rogers,
2004; Stauffer, 1998). To date, the generic term ‘vegetable oils’
has been used in the labelling of food containing oil blends. Hy-
drogenated fats, with a high melting point, have been used for a
long time in the production of bakery foods, as a replacement for
* Corresponding author. Tel.: þ39 874 404875; fax: þ39 874 404652.
E-mail address: fratianni@unimol.it (A. Fratianni).
those of animal origin. These fats are rich in trans fatty acids (TFA)
(Kumari, Jeyarani, Soumya, & Indrani, 2011) that several studies
(Albuquerque, Costa, Castilho, & Sanches-Silva, 2011; Aranceta &
Perez-Rodrigo, 2012; Tarrago-Trani, Phillips, Lemar, & Holden,
2006) associate with an undesirable effect on serum lipid profiles,
and thus with an increase of the risk of cardiovascular diseases. In
line with this, recommendations for trans fatty acids intake advice
to limit them to less than 1% of overall energy intake (Aranceta &
Perez-Rodrigo, 2012) and the European Food Safety Agency and
the Food and Drug Administration require that trans fats should be
listed on the nutrition labelling (EFSA, 2010; FDA, 2010). Therefore,
food industry has increased efforts in order to reduce TFA amounts
in food, through the application of fractionation, interesterification
and blending, as alternatives to hydrogenation (Li et al., 2011). In
this context, palm oil, a physically saturated fat with desirable
physical properties, free from trans fatty acids (Wong &
Radhakrishnan, 2012), became very popular. It is estimated that
7 in 10 products on UK supermarket shelves, from margarines and
spreads to cooking oils, and from chocolates and pastries to ice
creams and biscuits, contain palm oil (Bottriell & Judd, 2011).
Regarding Italian products, a recent 2014 survey made over
different commercial bakery products reports that, among those
containing palm oil, its presence in the label is not often declared

http://dx.doi.org/10.1016/j.foodcont.2015.01.032
0956-7135/© 2015 Elsevier Ltd. All rights reserved.
32 R. Mignogna et al. / Food Control 54 (2015) 31e38
(Altroconsumo, 2014). Few studies have investigated the effects of
palm oil on human health: its negative role is related to its high
content in saturated fatty acids, in particular palmitic acid, which
in turn have been associated with an increase in the risk of cor-
onary heart diseases and some tumours (Fattore & Fanelli, 2013).
Recent investigations seem to reduce the negative role of satu-
rated fatty acids on cardiovascular disease and new lines of
research would be necessary to investigate the effects of single
nutrients present in combination in palm oil, like oleic acid and
antioxidant compounds (Fattore & Fanelli, 2013). In fact, palm oil,
like other vegetable oils, is the major source of tocols, but sub-
stantial amounts of these compounds are also reported in different
ingredients of bakery products, such as most cereal flours (Hussain
et al., 2012; Panfili, Fratianni, Di Criscio, & Marconi, 2008; Panfili,
Fratianni, & Irano, 2003), eggs (Caboni et al., 2005; Fratianni, Di
Criscio, Mignogna, & Panfili, 2012), milk and dairy products
(Gambelli, Manzi, Panfili, Vivanti, & Pizzoferrato, 1999; Manzi,
Panfili, & Pizzoferrato, 1996; Panfili, Manzi, & Pizzoferrato, 1994)
and cocoa (Bonvehi, Coll, & Rius, 2000). Tocols, summarized under
the term vitamin E, are naturally occurring antioxidants, well
recognized for their bioactivity and antioxidant properties (Laus
et al., 2012; Packer, Weber & Rimbach, 2001; Tiwari & Cummins,
2009). Vitamin E is a generic term indicating a group of struc-
turally related compounds comprising two vitamers, i.e. tocoph-
erols and tocotrienols, differing only in the saturation state of the
isoprenoid side chain. Tocols occur in eight forms: a-tocopherol
(a-T), b-tocopherol (b-T), g-tocopherol (g-T), and d-tocopherol (d-
T) and a-tocotrienol (a-T3), b-tocotrienol (b-T3), g-tocotrienol (g-
T3), and d-tocotrienol (d-T3). The potential health benefits of
tocols for humans have been the subject of several reviews that
have analysed clinical, animal, and “in vitro” evidence for their
biological activity (Tiwari & Cummins, 2009). Several studies
report that tocotrienols may have more potent antioxidant and
anticancer effects than tocopherols (Aggarwal, Sundaram, Prasad,
& Kannappan, 2010). Studies also report that tocotrienols
possess lipid-lowering, anti-atherogenic, blood-pressure lowering,
anti-diabetic, neuroprotective, and anti-inflammatory effects
(Wong & Radhakrishnan, 2012). With the introduction of new EU
legislation on Food Information” (Regulation EU 1169/2011),
vegetable oil labelling as an ingredient in other foods is changing,
since refined oils of vegetable origin have to be grouped, together
in the list of ingredients, under the designation ‘vegetable oils or
fats’, immediately followed by a list of indications of specific
vegetable origin and by the phrase ‘in varying proportions’
(Regulation EU 1169/2011). To support regulators, food control
agencies and companies, and to provide meaningful information
to consumers, an analytical methodology should be necessary in
order to identify the nature of vegetable oils in a vegetable oil
blend and in foods (Osorio, Haughey, Elliott, & Koidis, 2013).
Several authors have suggested the utilization of tocopherols as
tracers for the identification, differentiation and evaluation of the
quality of vegetable oils (Aparicio & Aparicio-Ruiz, 2000; Cerre-
tani, Lerma-García, Herrero-Martínez, Gallina-Toschi, & Simo-
Alfonso, 2010; Dionisi, Prodolliet, & Tagliaferri, 1995; Manzi, Pan-
fili, Esti, & Pizzoferrato, 1998) and, in particular, in order to aid in
the distinction of vegetable oils, tocopherol analysis is often
considered complementary to triacylglycerol, fatty acid and sterol
determinations, spectroscopic, chemometric (Popescu et al., 2015)
and DNA analysis (He et al., 2013) and differential scanning
calorimeter-DSC (Fasina, Craig-Schmidt, Colleya, & Hallman, 2008;
Li et al., 2011; Osorio et al., 2013). The same investigation could be
useful in discriminating different oil/fat addition in different
products. In the light of this evidence, and in the lack of literature
data, the aim of this work was to evaluate the qualitative/quan-
titative content of tocopherols and tocotrienols in different
commercially bakery products and to consider the possibility to
use them as a tool to verify the information on the different fat
ingredients used in food labelling.
2. Materials and methods
2.1. Chemicals
a-, b-, g- and d-tocopherol standards were from Merck (Darm-
stadt, Germany); a-, b-, g- and d-tocotrienol standards were ob-
tained as reported in Panfili et al., (2003). a-Tocomonoenol (a-T1)
was tentatively identified on the basis of its retention time and by
comparison to literature data (Ng, Choo, Ma, Chuah, & Ali, 2004;
Puah, Choo, Ma, & Chuah, 2007). All other reagents were of
analytical or HPLC grade and were purchased from Carlo Erba
(Milano, Italy).
2.2. Sample and sampling
Thirty eight commercially available bakery products were ob-
tained from different local markets in 2013e2014. The choice was
made considering the most popular samples on the market and the
variability in their composition. In particular, the fats used in their
production and declared in label were taken into account. Dry
biscuits were investigated and samples were all without filling.
Each sample was made of at least 5e6 whole subsamples that were
grounded with a refrigerated IKA A10 laboratory mill (Staufen,
Germany), carefully mixed and stored at 20  C until analysis. Each
sample was analysed in triplicate. Table 1 reports the list of prod-
ucts with the ingredients that contain tocols, classified as samples
made with animal fats (1e8), samples made with specified vege-
table oils (9e23), samples made with unspecified vegetable oils
(24e38).
2.3. Chemical analysis
Moisture and fats were determined using an ICC standard pro-
cedure (ICC, 1995), Method 110/1 and Method 136, respectively.
Briefly, moisture was determined by measuring the weight loss
after heating the sample in a vessel at a temperature of 130  C. Fats
were calculated after hydrolysis of the sample by hydrochloric acid
in the presence of ethanol and formic acid, extraction of the fat by
hexane, removal of the solvent and weighing the residue obtained.
2.4. Tocol analysis
Tocols were extracted, after saponification, and determined as
reported by Panfili et al. (2003), as follows: 0.5e1 g of sample (0.5 g
for biscuits and croissants, 1 g for sandwich loaves) was saponified,
under nitrogen, in a screw-capped tube with 2 mL of potassium
hydroxide (600 g/L), 2 mL of ethanol (95%), 2 mL of sodium chloride
(10 g/L), and 5 mL of ethanolic pyrogallol (60 g/L), added as anti-
oxidant. The tubes were placed in a 70  C water bath and mixed
every 5e10 min during saponification. After alkaline digestion at
70  C for 45 min, the tubes were cooled in an ice bath, and 15 mL of
sodium chloride (10 g/L) were added. The suspension was then
extracted twice with a 15 mL portion of n-hexane/ethyl acetate (9:1
v/v). The organic layer was collected and evaporated to dryness,
and the dry residue was dissolved in 2 mL of isopropyl alcohol (1%)
in n-hexane. Extracted samples were analysed by high-
performance liquid chromatography, under normal phase condi-
tions, using a 250  4.6 mm i.d., 5 mm particle size, Kromasil Phe-
nomenex Si column (Torrance, CA, USA). An HPLC Dionex
(Sunnyvale, CA, USA) analytical system, consisting in a U3000
pumps and a 50 ml injector loop (Rheodyne, Cotati, USA), was used.
 R. Mignogna et al. / Food Control 54 (2015) 31e38 33

Fluorometric detection of all peaks was performed at an excitation

Table 1
List of analysed products and their ingredients that contain tocols.
wavelength of 290 nm and an emission wavelength of 330 nm, by
means of an RF 2000 spectrofluorimeter (Dionex, Sunnyvale, USA).
Code Product Ingredients that have tocols Fat The mobile phase was n-hexane/ethyl acetate/acetic acid
content
(%)
(97.3:1.8:0.9 v/v/v), at a flow rate of 1.6 mL/min (Fratianni, Caboni,
Irano, & Panfili, 2002; Panfili et al., 2003). The mobile phases were
1 Biscuit Wheat flour, butter, milk, malt extract 12.2
previously degassed by sonication for 10 min. After every 10 in-
2 Biscuit Wheat flour, butter, malt extract, milk 12.6
powder jections the column was reactivated with a solution of 10% iso-
3 Biscuit Wheat flour, butter, eggs, milk 11.8 propyl alcohol in n-hexane (v/v). Compounds were identified by
4 Biscuit Whole meal flour, wheat flour, butter, eggs, 13.9 comparison of their retention times (RT) with those of known
milk powder, malt extract available standard solutions and quantified on the basis of cali-
5 Biscuit Wheat flour, eggs, lard, milk 18.0
6 Sandwich loaf Wheat flour, lard, barley malt-flour, milk 5.3
bration curves of standard solutions. The concentration range was
powder 5e25 mg/ml for every tocol standard. a-Tocomonoenol (a-T1) was
7 Sandwich loaf Wheat flour, lard, malt extract 5.3 quantified using the calibration curve of a-tocopherol standard
8 Sandwich loaf Wheat flour, lard, malt extract 5.0 solutions. Data were stored and processed by a Dionex Chromeleon
9 Biscuit Wheat flour, sunflower oil, barley flakes, 8.1
Version 6.6 chromatography system (Sunnyvale, CA). The limit of
extruded cereal food (barley fibre, rice flour,
oat flour, powdered malt extract), barley detection (LOD) and limit of quantification (LOQ) were estimated
flour according to the IUPAC (International Union of Pure and Applied
10 Biscuit Whole spelt flour, corn malt, sunflower oil, 13.8 Chemistry) definitions. Vitamin E activity was expressed as
rice flour Tocopherol Equivalent (T.E.) (mg/100 g product), calculated as re-
11 Biscuit Wheat flour, corn oil, wheat bran, eggs, 14.7
barley malt-flour
ported by Sheppard, Pennington, and Weihrauch (1993). T.E. was
12 Sandwich loaf Wheat flour, olive oil, barley malt-flour 4.0 also expressed per piece, considering an average piece from 5 g to
13 Biscuit Wheat flour, extra virgin olive oil, eggs 18.0 15 g, for biscuits, 40 g for a croissant and 18 g for a sandwich loaf.
14 Biscuit Wheat flour, extra virgin olive oil, cocoa 17.1
powder
2.5. Statistical analysis
15 Biscuit Spelta flour, spelta flakes, extra virgin olive 15.6
oil
16 Biscuit Wheat flour, extra virgin olive oil 8.9 Data were reported as mean and standard deviation of three
17 Sandwich loaf Wheat flour, extra virgin olive oil, barley 4.3 different determinations for each sample, expressed on fat basis. An
malt-flour ANOVA was applied to the data. Least significant differences were
18 Croissant Wheat flour, vegetable oils (palm oil, 21.4
sunflower oil, coconut oil), eggs, skimmed
obtained using a least significant difference test (P < 0.05). The
milk powder Wilks' Lambda index was computed to determine the possibility to
19 Croissant Wheat flour, margarine (palm oil, rapeseed 20.8 use tocol profile for discriminating fat ingredients. Linear
oil), eggs Discriminant Analysis (LDA) was then applied using the selected
20 Biscuit Wheat flour, margarine (refined palm oil, 12.0
tocol variables to explore the possibility of classifying the samples
fractionated palm oil), milk
21 Biscuit Wheat flour, non-hydrogenated vegetable 12.2 according to the oil-fat used in their preparation. Statistical analysis
oil (palm oil), milk, whole milk powder, was performed using an SPSS version 13.0 for Windows (SPSS Inc.,
malt extract Chicago, IL, USA).
22 Biscuit Wheat flour, palm oil, eggs, milk 17.0
23 Biscuit Wheat flour, non-hydrogenated palm oil, 16.9
eggs, milk
3. Results and discussion
24 Biscuit Wheat flour, non-hydrogenated vegetable 10.1
oil, skimmed milk powder 3.1. Tocopherol and tocotrienol content of bakery products
25 Biscuit Whole wheat flour, cereals (wheat flour, 12.2
barley flour, rice flour, rye flour, oat flour),
Table 1 shows data about fat content, which, for biscuits, went
non-hydrogenated vegetable oil
26 Biscuit Wheat flour, vegetable oil, skimmed milk 11.7 from 11% to 22% and, for croissants, from 21% to 24%. A lower
powder content, about 5%, was found for sandwich loaves. The HPLC
27 Biscuit Wheat flour, vegetable oil, eggs, milk 17.1 chromatograms of tocols in biscuits made with palm oil and sun-
28 Biscuit Whole wheat, cereals flours (oat, barley, 18.2 flower oil are shown in Fig. 1. Some of the analytical performances
rye, corn, rice), vegetable oil, eggs
of the method have been already reported in the original paper
29 Biscuit Wheat flour, non-hydrogenated vegetable 19.5
oil, fresh eggs (Panfili et al., 2003). In particular, the detection limit values (LOD)
30 Croissant Wheat flour, non-hydrogenated vegetable 22.7 were 2 ng/ml (a-tocopherol, g-tocopherol, d-tocopherol, and d-
oil, fresh eggs, powered skim milk tocotrienol), 4 ng/ml (b-tocopherol and b-tocotrienol), and 6 ng/ml
31 Sandwich loaf Wheat flour, vegetable oil, wheat malt 4.1
(a-tocotrienol and g-tocotrienol). The quantification limit values
extract
32 Sandwich loaf Semolina, vegetable oil, barley malt-flour 4.0 (LOQ) were from 6 ng/ml for a-tocopherol to 18 ng/ml for a-toco-
33 Biscuit Wheat flour, whole wheat flour, non- 19.0 trienol. The tocol content in the investigated samples is reported in
hydrogenated vegetable oil, eggs, malt Tables 2e4. Values are given as single forms of tocopherols and
extract, whole milk powder, oat flakes tocotrienols (Table 2a, 3a, 4a), as the sum of tocopherols (T), the
34 Biscuit Oat flakes, wheat flour, non-hydrogenated 18.3
sum of tocotrienols (T3) and as total tocols (T þ T3) (Table 2b, 3b,
vegetable oil, cereals (corn, barley, rice,
malt extract), whole wheat flour, malt 4b). Moreover, the amount of another compound is reported,
extract which was tentatively identified as a-tocomonoenol (a-T1). This
35 Biscuit Oat flakes, whole wheat flours, non- 18.2 compound is typical of palm oil, as also found in different analysis
hydrogenated vegetable oil
of ours on tocols of palm oil (data not shown). All samples were
36 Croissant Wheat flour, margarine (non- 24.3
hydrogenated vegetable oil), eggs, milk
grouped according to the type of fat declared in label, so that they
37 Biscuit Wheat flour, margarine, eggs 18.1 were classified as products made with animal fats (Table 2), prod-
38 Biscuit Wheat flour, margarine, eggs 21.7 ucts made with specified vegetables oils (Table 3) and products
made with unspecified vegetable oils (Table 4). The bakery
34 R. Mignogna et al. / Food Control 54 (2015) 31e38

Fig. 1. Typical chromatograms of a palm oil (A) and a sunflower oil (B).

products with the highest content of total tocols were those made et al., 2002; Panfili et al., 2003). a-Tocopherol and traces of other
with vegetable oils, both specified and unspecified, while those tocols are instead characteristics of butter. Some samples (3, 4 and
made with animal fats had the lowest content. In particular, the 5) also contained g-T, which was probably given by eggs, in which
total tocol content ranged from 83.2 mg/kg fat basis of a biscuit this tocopherol is present in significant amounts (Caboni et al.,
made with lard (sample 5), to 991.3 mg/kg fat basis of a biscuit 2005; Fratianni et al., 2012). The bakery products made with
made with corn oil (sample 11). The coefficient of variability (CV%) specified vegetable oils (9e23) had, by average, higher tocol con-
of total tocols went from 0.1% to 7.9% (Table 2b, 3b, 4b). In all tent than those made with animal fats (Table 3b). In particular, in
samples made with animal fats (Table 2a), either butter or lard samples made with sunflower oil (samples 9, 10) and olive and
(1e8) the principal tocol was b-T3, which was mainly provided by extra virgin olive oil (samples 12, 13, 14, 15, 16, 17), a-T and b-T3
flour where this tocol is the most representative isomer (Fratianni were the most representative tocols, while low or undetectable
amounts of g-T3 were found (Table 3a). In these products a-
tocopherol was provided by extra virgin oil (Cerretani et al., 2010;
Table 2a Schwartz, Ollilainen, Piironen, & Lampi, 2008), while b-toco-
Tocopherols and tocotrienols in bakery products made with animal fats (mg/Kg fat
trienol by flour. In all these samples, the ratio T3/T was lower than 1
basis).
(Table 3b), as also observed by other authors in similar oils (Bonvehi
Code Tocopherols (T) Tocotrienols (T3) a-T1 et al., 2000; Schwartz et al., 2008). In a biscuit made with corn oil
a-T b-T g-T a-T3 b-T3 g-T3 d-T3 (sample 11), the tocol at the highest amounts was g-T3, followed by
1 36.3 (1.6)a 14.1 (1.6) n.d.b 5.9 (0.6) 129.1 (4.4) n.d. n.d. n.d. a-T; this result reflects the tocol distribution of this vegetable oil
2 39.1 (0.1) 20.5 (2.3) n.d. 8.0 (0.4) 114.5 (2.6) n.d. n.d. n.d. (Bonvehi et al., 2000; Schwartz et al., 2008). Samples from 18 to 23
3 42.0 (0.6) 13.4 (0.1) 7.0 (0.6) 7.8 (0.6) 81.6 (0.4) n.d. n.d. n.d. included all bakery products labelled as made with palm oil. In
4 75.8 (5.3) 6.2 (1.0) 3.2 (0.6) 14.7 (2.9) 100.8 (7.5) n.d. n.d. n.d. addition, other oils, like sunflower and coconut oil and rapeseed oil,
5 25.6 (1.6) 7.3 (0.4) 2.8 (0.0) 3.6 (0.4) 43.9 (3.6) n.d. n.d. n.d.
were present in samples 18 and 19, respectively. In all these
6 28.9 (3.6) 25.8 (2.7) n.d. 6.1 (1.5) 174.7 (12.1) n.d. n.d. n.d.
7 45.1 (4.4) 19.8 (1.5) n.d. 19.6 (1.4) 216.7 (14.0) n.d. n.d. n.d. products, total tocol content ranged from about 490 mg/kg to about
8 46.8 (2.2) 22.4 (1.2) nd 15.7 (1.0) 186.8 (8.3) n.d. n.d. n.d. 890 mg/kg of fat basis (Table 3b). g-T3, a-T3 and a-T were the most
a
Standard deviation (S.D.). representative tocols; the presence of d-T3 was also observed, as
b
Not detectable. well as that of b-T and of b-T3 (Table 3a). This tocol profile reflected
the composition of vitamin E in palm oil, which is characterized by
the presence of g-T3, a-Τ3, a-Τ and d-T3 (Bonvehi et al., 2000; Ng
Table 2b et al., 2004). Moreover, in all samples, the presence of another
Total tocopherols (T), tocotrienols (T3) and total tocols in bakery products made with
animal fats (mg/Kg fat basis).
peak, tentatively identified as a-tocomonoenol (a-T1), was reported
(Table 3a). This tocol accounted for 2e4% of total tocols in these
Code Tot T Tot T3 Total tocols T3/T bakery products. All samples made with palm oil showed a ratio T3/
T þ T3 CV % T higher than 1 (Table 3b); the same ratio was observed in different
1 50.4 (3.2)a 135.0 (2.5) 185.4 3.1 2.7 analysis of our regarding the tocols of palm oil (data not shown).
2 59.6 (5.2) 122.5 (1.5) 182.1 4.7 2.1 Samples made with unspecified vegetable oils, from 24 to 38,
3 62.4 (1.4) 89.4 (0.7) 151.8 0.2 1.4 provided the highest tocol amount, ranging from about 407 to
4 85.2 (6.0.) 115.5 (8.5) 200.7 5.1 1.4
900 mg/kg of fat basis (Table 4b). In most of these products, toco-
5 35.7 (2.0) 47.5 (2.0) 83.2 4.8 1.3
6 54.7 (6.3) 180.8 (11.2) 235.4 6.1 3.3
trienols were the most representative, so that the T3/T ratio was
7 64.9 (5.0) 236.3 (16.1) 301.2 6.1 3.6 higher than 1, with the only exception of samples made with
8 71.0 (3.2) 202.5 (4.7) 273.5 2.8 2.9 margarine (36 and 37), in which T3/T ratio was about 1. In all
a
Standard deviation (S.D.). samples of Table 4a high amounts of g-T3, a-T3 and a-T were found,
 R. Mignogna et al. / Food Control 54 (2015) 31e38 35

Table 3a
Tocopherols and tocotrienols in bakery products made with specified vegetable oils (mg/Kg fat basis).

Code Tocopherols (T) Tocotrienols (T3) d-T3 a-T1

a-T b-T g-T a-T3 b-T3 g-T3
9 494.4 (26.8)a 26.9 (0.2) 27.6 (0.8) 61.3 (2.9) 59.7 (2.2) 14.7 (2.1) n.d.b n.d.
10 410.0 (2.4) 40.4 (1.4) 39.0 (1.1) 6.8 (0.0) 63.6 (2.3) n.d. n.d. n.d.
11 208.3 (6.4) n.d. 668.9 (13.2) 20.9 (0.2) 81.3 (6.6) 11.9 (1.2) 17.5 (1.2) n.d.
12 79.1 (4.6) 28.9 (3.1) 27.3 (1.0) 6.9 (0.1) 158.4 (12.9) n.d. n.d. n.d.
13 258.3 (4.7) 12.1 (0.7) 16.5 (1.9) 8.9 (0.3) 52.2 (1.2) 3.7 (0.2) n.d. n.d.
14 199.7 (15.2) 4.0 (0.3) 27.8 (6.9) 2.9 (0.6) 44.8 (7.0) n.d. n.d. n.d.
15 254.6 (2.0) 21.9 (2.1) 15.4 (0.1) 14.8 (0.4) 91.6 (0.1) n.d. n.d. n.d.
16 186.6 (4.9) 38.1 (1.8) 22.0 (2.8) 6.4 (0.6) 91.1 (0.3) n.d. n.d. n.d.
17 200.9 (7.3) 35.7 (0.8) 14.7 (1.8) 17.8 (0.7) 222.1 (5.9) n.d. n.d. n.d.
18 202.9 (2.1) 12.8 (1.8) 13.9 (1.1) 105.1 (1.5) 44.3 (2.6) 148.0 (2.3) 29.8 (1.3) 20.8 (0.2)
19 181.3 (14.5) 5.8 (0.3) 25.2 (0.2) 175.1 (1.4) 52.4 (4.0) 233.8 (20.1) 39.8 (7.4) 24.2 (1.1)
20 110.4 (3.0) 10.3 (0.4) n.d. 103.8 (3.5) 78.6 (1.1) 155.1 (0.6) 31.7 (1.1) 11.5 (0.1)
21 170.0 (6.4) 14.5 (1.1) n.d. 166.9 (7.0) 106.9 (6.0) 211.5 (7.5) 42.7 (0.2) 18.9 (0.7)
22 217.1 (0.7) 13.0 (0.3) 3.0 (0.4) 250.1 (2.1) 88.5 (3.0) 271.2 (3.6) 46.2 (2.4) 27.0 (0.2)
23 188.3 (16.1) 13.7 (3.6) 8.7 (2.0) 192.4 (16.5) 75.6 (5.2) 217.7 (15.5) 38.1 (2.7) 22.3 (2.8)
a
Standard deviation (S.D.).
b
Not detectable.

together with the presence of d-T3 and, in particular, of a-T1. These
results are consistent with in the use of palm oil in the realization of
these products and in the inclusion of palm oil under the generic
term “non-hydrogenated vegetable oils”.
3.2. Classification of bakery samples according to fat ingredients
based on tocol profile
The LDA was used for the categorization of samples. The
grouping variables were all the 38 analysed samples (grouped from
1 to 9), the independent variables were all tocol compounds and
T3/T ratio. Wilks' Lambda ¼ 0.000 (approximate F ¼ 37.415
p ¼ 0.000 < 0.05) indicated the ability of tocols to discriminate fat
ingredients among groups. Five variables: a-T, a-T1, g-T, d-T3 and
T3/T were found to be significant (p < 0.05) for the discrimination
of fat ingredients. Other tocol compounds, typical of cereals, such as
a-T3 and b-T3, were not found to be significantly discriminant due
to the prevalence of a similar cereal matrix for all samples. The
selected variables were subjected to Linear Discriminant Analysis
(LDA). Results showed that two statistically significant discriminant
functions were formed (Wilks' Lambda ¼ 0.000, X2 ¼ 348.698,
df ¼ 40, p ¼ 0.000 < 0.05 for the first function, and Wilks'
Lambda ¼ 0.003, X2 ¼ 175.801, df ¼ 28, p ¼ 0.000 < 0.05 for the
second, respectively). The first discriminant function accounted for
91.7% of total variance, while the second accounted for 6%. Both
accounted for 97.7% of total variance. In Fig. 2 it is shown that fat
ingredients are well differentiated among bakery samples. In
particular, samples made with animal fats, such as butter and lard
(1 and 2, respectively), were located in a well-defined area in LDA.
In these products cereal tocols were relevant. Samples grouped as 3
and 4 were characterized by the predominance of tocols on toco-
trienols; this composition reflected the presence of specified
vegetable oils, such as sunflower oil (3) and corn oil (4). Corn oil is
characterized for high amounts of g-T. The first discriminant
function fully differentiated this fat ingredient which, therefore,
greatly separated from the others (not reported in figure). Samples
made with extra virgin oil (6) were located in a well-defined area,
while sample labelled as made with olive oil (5), produced by
mixing refined olive oil and virgin olive oil, didn't resemble the
original composition of extra virgin oil and therefore was located in
a different area. The second discriminant function differentiated
samples made with palm oil (7), unspecified vegetable oils (8) and
margarine (9) from samples made with extra virgin olive oil and
sunflower oil.

3.3. Vitamin E activity

Table 3b Fig. 3 reports values of vitamin E activity provided by 100 g of

Total tocopherols (T), total tocotrienols (T3) and total tocols in bakery products made
bakery product, expressed as Tocopherol Equivalent (TE) (mg/100 g
with specified vegetable oils (mg/Kg fat basis).
product). Samples were grouped into biscuits, croissants and
Code Tot T Tot T3 Total tocols T3/T sandwich loaves. Biscuits have been further grouped in biscuits
T þ T3 CV % made without eggs and those made with eggs, since this ingredient
9 548.9 (25.9)a 135.7 (2.5) 684.6 4.2 0.2 is a good source of a-tocopherol, which is the compound that
10 489.4 (2.1) 70.4 (1.2) 559.8 0.6 0.1 contributes to the highest vitamin E value. On the whole, samples
11 877.2 (18.6) 114.1 (3.2) 991.3 2.2 0.1 made with animal fats (from 1 to 8) had the lowest TE values,
12 135.3 (10.7) 165.3 (6.5) 300.6 5.8 1.2 resembling the composition of the used fats. Among biscuits made
13 286.9 (7.3) 64.8 (0.9) 351.7 2.3 0.2
14 231.7 (17.2) 47.7 (3.8) 279.4 7.9 0.2
with vegetable fats, by average, those made with eggs had the
15 291.9 (6.6) 106.4 (0.1) 398.3 1.7 0.4 higher TE values, even if, among those without eggs, samples made
16 246.7 (9.5) 97.5 (0.5) 344.2 2.6 0.4 with sunflower oil (9 and 10), had the highest values. Croissants
17 251.3 (9.8) 239.9 (3.3) 491.3 2.7 0.9 showed similar values of vitamin E activity, while sandwich loaves
18 229.6 (1.4) 327.2 (0.6) 556.8 0.1 1.4
reported the lowest values, also related to the lowest fat content
19 212.3 (15.7) 501.1 (0.9) 713.4 3.4 2.4
20 120.7 (2.6) 369.2 (2.3) 489.9 1.2 3.1 (Table 1). Taking into account the Recommended Daily Allowance
21 193.6 (7.5) 528.0 (5.8) 721.6 1.8 2.7 (RDA) of vitamin E of 12 mg (Regulation EU 1169/2011), 68% of the
22 233.1 (0.6) 656.0 (2.6) 889.1 0.4 2.8 analysed bakery products can be declared in label as a source of
23 210.7 (18.2) 523.8 (15.7) 734.5 4.5 2.5 vitamin E. In particular, all biscuits, except those made with butter
a
Standard deviation (S.D.). and lard, and croissants were sources of this vitamin. No analysed
36 R. Mignogna et al. / Food Control 54 (2015) 31e38

Table 4a
Tocopherols and tocotrienols in bakery products made with unspecified vegetable oils (mg/Kg fat basis).

Code Tocopherols (T) Tocotrienols (T3) a-T1

a-T b-T g-T a-T3 b-T3 g-T3 d-T3
24 134.8 (0.4)a 20.0 (2.5) n.d.b 125.5 (11.0) 115.9 (6.8) 181.9 (13.3) 37.3 (0.3) 17.5 (2.8)
25 155.6 (10.6) 15.1 (0.8) 13.3 (2.2) 131.4 (9.6) 123.5 (8.3) 175.7 (13.0) 33.1 (3.7) 15.0 (0.3)
26 139.4 (1.9) 16.7 (0.3) n.d. 129.2 (0.0) 141.8 (11.0) 192.9 (9.8) 41.1 (2.6) 19.6 (0.3)
27 195.8 (4.7) 11.0 (0.4) 8.6 (0.1) 238.2 (4.7) 79.6 (1.5) 236.1 (4.6) 42.0 (1.3) 18.5 (0.6)
28 155.1 (12.1) 14.6 (4.3) 8.9 (1.4) 162.9 (13.0) 59.1 (7.0) 193.2 (14.2) 32.2 (4.4) 16.1 (1.5)
29 189.1 (4.0) 11.4 (2.2) 4.2 (0.0) 208.2 (2.8) 69.2 (0.5) 214.5 (2.7) 36.5 (0.2) 21.2 (0.5)
30 151.1 (0.3) 9.9 (0.8) 14.0 (0.6) 92.1 (3.1) 46.4 (2.1) 142.7 (2.1) 30.4 (0.6) 16.4 (1.4)
31 198.7 (14.7) 50.5 (4.2) n.d. 187.6 (18.2) 209.5 (16.6) 181.9 (16.0) 32.1 (5.3) 14.3 (0.5)
32 184.8 (1.8) 18.7 (0.8) 12.9 (0.7) 186.2 (0.5) 276.2 (0.5) 185.6 (2.6) 35.7 (2.4) 20.2 (0.4)
33 122.0 (5.0) 12.6 (1.1) 4.8 (0.1) 115.9 (5.6) 43.6 (3.9) 71.0 (4.7) 36.9 (4.8) 18.5 (3.6)
34 121.2 (1.6) 11.3 (1.6) 4.2 (0.1) 126.9 (5.1) 35.3 (1.0) 66.5 (3.2) 62.4 (3.4) 14.3 (0.7)
35 154.7 (7.9) 17.5 (0.7) 3.2 (0.3) 161.6 (7.8) 43.8 (2.6) 89.7 (5.0) 67.1 (5.8) 22.0 (2.1)
36 214.7 (10.2) 10.5 (0.4) 14.1 (0.1) 98.1 (4.7) 37.4 (0.9) 118.1 (0.6) 21.5 (0.3) 21.6 (0.6)
37 138.0 (12.1) 6.7 (0.3) 76.2 (8.2) 72.4 (7.6) 45.4 (5.0) 90.7 (9.5) 16.6 (2.1) 7.7 (0.7)
38 186.9 (10.5) 8.5 (0.1) 7.0 (4.1) 201.4 (7.0) 57.0 (2.0) 231.7 (9.1) 44.7 (7.8) 22.3 (0.5)
a
Standard deviation (S.D.).
b
Not detectable.

Table 4b
Total tocopherols (T), total tocotrienols (T3) and total tocols in bakery products made
with unspecified vegetable oils (mg/Kg fat basis).
sandwich loaves provided the recommended allowance. When
Code Tot T Tot T3 Total tocols T3/T expressing the T.E. values for portion of bakery product, only
T þ T3 CV % croissants covered 15% of RDA (data not shown).
24 154.8 (2.9)a 460.6 (16.4) 615.4 3.0 3.0
25 184.0 (11.9) 463.7 (10.5) 647.7 3.4 2.5
26 156.1 (2.2) 505.0 (3.5) 661.1 0.9 3.2 4. Conclusions
27 215.4 (4.3) 595.8 (12.1) 811.2 2.0 2.8
28 178.6 (15.0) 447.4 (12.1) 626.0 3.2 2.5
29 204.7 (6.2) 528.4 (4.9) 733.1 1.5 2.6
Literature data report the utilization of tocols as tracers for
30 175.0 (1.7) 311.8 (10.8) 486.8 1.9 1.8 the identification, differentiation and evaluation of the quality of
31 249.2 (18.3) 611.1 (17.4) 860.3 2.3 2.5 vegetable oils. Results of our work show that the use of tocol
32 216.4 (3.4) 683.7 (1.0) 900.1 0.5 3.2 profile could be extended also to the evaluation of food products
33 139.4 (13.3) 267.5 (6.3) 406.9 1.7 1.9
with added fats and can be used as an aid to verify the infor-
34 136.7 (0.1) 291.1 (5.2) 427.8 3.9 2.1
35 175.4 (8.3) 362.2 (9.7) 537.6 2.5 2.1 mation about the used fat/oil given in the label. Moreover, in
36 239.3 (9.9) 275.0 (3.5) 514.3 2.6 1.1 order to provide meaningful information in food labelling and
37 200.9 (14.6) 225.1 (6.3) 446.0 3.8 1.0 to prevent adulteration, it can be used, in conjunction with
38 202.4 (9.5) 534.8 (4.5) 737.2 1.9 2.6 other common used analytical methods, such as fatty acid, tri-
a
Standard deviation (S.D.). glyceride or sterol profile, as a methodology to identify the exact

Fig. 2. Linear discriminant analysis of the different fats used in bakery products based on tocol profile.
 R. Mignogna et al. / Food Control 54 (2015) 31e38
Fig. 3. Vitamin E activity of analysed bakery products, expressed as Tocopherol Equivalents (T.E.).
nature of a fat/oil, or oil blend, present alone or added to a food
product.
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