LWT - Food Science and Technology 58 (2014) 63e70

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LWT - Food Science and Technology

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Effect of the addition of spices on reducing the sodium content

and increasing the antioxidant activity of margarine
Cristiane de Oliveira Lopes*, Maria de Fátima Píccolo Barcelos,
Nayane Aparecida Araujo Dias, João de Deus Souza Carneiro, Wilson César de Abreu
Universidade Federal de Lavras (UFLA), Department of Food Science, University Campus, Lavras, MG, CEP 37200-000, CP 3037, Brazil

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

Article history: Margarine contains a considerable amount of sodium and is used on breads and crackers. In this
Received 6 November 2013 context, the objective of the present study was to evaluate the market appeal and effects of adding
Received in revised form spices to margarine in terms of reducing the sodium content and increasing antioxidant activity. A
14 February 2014
questionnaire assessing market appeal was developed along with 12 margarine formulations. The
Accepted 16 February 2014
treatment varied in terms of reduced sodium levels (from 100% to 0% reduction) and the addition of
two different spice mixtures (A or B) containing green onion, garlic, marjoram, thyme, lemon, oregano
Keywords:
and/or basil. The questionnaire was given to 400 respondents, and the sensory analysis was performed
Margarine
Spices
with 62 panelists. The total phenolic content was determined using the FolineCiocalteu reagent, and
Sodium the DPPH and ABT
þ radical methods were used to quantify total antioxidant activity. Sensory analysis
Sensory analysis for the margarine formulations with added spice mixtures (A or B) indicated good acceptance, and the
Phenolic compounds chemical analysis revealed a greater than 5-fold increase in total phenolic content and a significant
increase (p<0.05) in antioxidant activity. The addition of either spice mixture to the margarine for-
mulations permitted 50% sodium reduction and considerably increased the antioxidant activity of the
product.
Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction breads and crackers as well as an ingredient in the preparation of

Sodium is an essential chemical element for the proper func-
tioning of organisms, but excessive sodium intake contributes to
the development of hypertension and cardiovascular diseases,
which are the leading causes of mortality worldwide (Laurenti,
Buchalla, & Caratin, 2000). Hypertension affects approximately
25% of the adult population, with a predicted prevalence of 60% by
2025. Although naturally present in many foods, most of the so-
dium in the diet comes from sodium compounds added during food
processing or from table salt (NaCl) (Santos, 2009; Willetti, 2001).
Margarine is an industrialized product with a high content of fat
(80%) derived from vegetable oils, soybean, sunflower or linseed,
that of soybean being the preferentially utilized. It is often used on
* Corresponding author. Universidade Federal de Lavras (UFLA), Departamento de
Ciência dos Alimentos, CP 3037, Lavras-MG, CEP 37200-000, Brazil. Tel.: þ55 (35)
3829 1406; fax: þ55 (35) 3829 1401.
E-mail addresses: lopes.co@hotmail.com, nayaneadias@yahoo.com.br (C.
deO. Lopes).
other food products (Brazil, 1997). It is offered with or without
added sodium, but margarine without sodium has low consumer
acceptance. With the goal of improving diets and promoting health
and quality of life, the Brazilian government and the leadership of
the Brazilian Association of Food Industries (Associação das
Indústrias Brasileiras de Alimentos, ABIA) agreed to set targets for
reducing the sodium content of processed foods. The agreement
indicating reduced sodium content in margarine was signed on
August 28, 2012.
Reducing the sodium content of foods is a difficult process
because the reformulated products are rejected by consumers in
most cases. However, the use of spices represents one option for the
development of food products with reduced sodium without
compromising acceptance (Dallepiane & Bós, 2007).
Spices have natural chemical constituents, such as vitamins A, E
and C as well as phenolic compounds, which contribute to anti-
oxidant capacity. Antioxidants are compounds that protect bio-
logical systems against the harmful effects of free radicals. Radicals
that are not properly neutralized can initiate the detrimental pro-
cess of oxidative stress in cells and tissues, which may lead to

http://dx.doi.org/10.1016/j.lwt.2014.02.029
0023-6438/Ó 2014 Elsevier Ltd. All rights reserved.
64 C.O. Lopes et al. / LWT - Food Science and Technology 58 (2014) 63e70
various diseases, including neurodegenerative and cardiovascular
diseases, cancer and premature aging. The acting of antioxidants
can be as interceptors of free radicals, sequestering agents of metals
and inhibitors oxidative enzymes or as cofactors of selenium
(Fanhani & Ferreira, 2006).
Because they effectively slow the oxidative degradation of lipids
and contribute to the quality and nutritional value of the foods to
which they are added, spices and their extracts are of increasing
interest to the food industry (Ozcan, Erel, & Herken, 2009).
The objective of the present study was to evaluate the market
appeal and effects of the addition of spices on the acceptance and
antioxidant activity of margarine with reduced sodium content.
2. Material and methods
2.1. Questionnaire on market appeal
A questionnaire was devised to assess the market appeal of
margarine with reduced sodium content and added spices (Table 1).
Four hundred respondents were recruited to fill out the question-
naire at four supermarkets in Lavras, Minas Gerais, Brazil at different
times without discrimination in terms of sex, age and income. The
questionnaire included questions about socioeconomic status, age,
occupation and frequency of physical activity as well as knowledge of
the existence and interest in food products with reduced salt content.
The respondents were asked whether they consumed margarine and
how frequently and whether they would be interested in buying
margarine with reduced salt content and added spices.
The questionnaire was developed and administered to assess
respondents’ interest in buying margarine with reduced salt and
added spices and to generate a profile of consumers of traditional
margarine and potential consumers of margarine with reduced salt
content and added spices.
2.2. Elaboration of the margarine formulations
To prepare the margarine formulations with reduced sodium
and added spices, margarine without sodium (Sadia brand, Brazil)
was purchased from the local market in the city of Lavras, Minas
Gerais and used as the base product for the various formulations.
According to the manufacturer, the product was 80% fat, 20% water
and sodium free.
Refined salt (Cisne brand, Brazil) and the following spices were
also obtained locally: green onion (Allium fistulosum), garlic (Allium
Table 1
Questions of the questionnaire of the concept test and respective codes for analysis of Fig. 1.
Questions
A Do you consume margarine?
B Do you buy margarine with reduced salt content (NaCl)?
C Do you buy margarine with reduced salt content (NaCl)?
D This margarine with reduced salt content (NaCl) should?
E Do you know that on market there are products with
reduced salt content (NaCl)? (Reduced Salt)
F Do you have the habit of consuming products with
reduced salt content (NaCl)?
G Do you buy margarine with reduced salt content (NaCl)
and added spices?
H You ever heard of salt light?
I Gender
J Age in years
K Monthly income
L Marital status
M Children
N Schooling
O How often do you practice physical activity
sativum), marjoram (Origanum majorana), thyme (Thymus vulgaris),
lemon (Citrus limon), oregano (Origanum vulgare) and basil (Oci-
mum basilicum) in dehydrated powder form. Twelve margarine
formulations were defined, including two controls and 10 treat-
ments. The two controls were sodium-free margarine (corre-
sponding to 100% sodium reduction) and standard margarine with
sodium (600 mg/100 g, corresponding to 0% sodium reduction).
Spices were not added to either of the controls. The 10 treatments
varied in terms of the mixture of spices added (A or B) and the
sodium content. A fixed quantity of spice mixture (1.5 g/100 g) was
used in all of the formulations, and the sodium content reduction
varied from 0% to 100% at 25% increments based on the standard
amount of 600 mg/100 g. The 12 formulations are summarized in
Table 2.
To quantity the amount of sodium to be added to the margarine
formulations, it was necessary to determine the sodium content of
commercial sodium salt (NaCl).
For sodium analysis, the extract was prepared by utilizing 0.5 g
of the sample of NaCl added of 6 mL of nitric perchloric acid (2:1),
the material was taken to the digestion block at the temperature of
250  C for 4 h. After digestion, the sample was diluted with distilled
water for concentration of 0.01 g/mL and quantification was carried
out. Quantification was performed by using a flame photometer
(Analyser, model 910) calibrated using standards at different con-
centrations. The results were expressed as g/100 g of integral
product (AOAC, 1990).
The compositions of the two spice mixtures (A and B) were
defined in pilot tests conducted for 6 (six) panelists. After several
sensorial assessments, the tasters defined, in consensus, the mix-
tures of spices which stood out the most with relation to flavor and
these ones were added to the margarines. Mixture A was 40 g/100 g
green onion (A. fistulosum), 35 g/100 g garlic (A. sativum), 20 g/100 g
marjoram (Origanum Majorana) and 5 g/100 g thyme (T. vulgaris),
and mixture B was 60 g/100 g lemon (C. limon), 15 g/100 g oregano
(O. vulgare), 20 g/100 g basil (Ocimum basilicum) and 5 g/100 g
thyme (T. vulgaris). All of the spices used were in dehydrated
powder form.
After preparation, all margarine formulations were chilled to
3  C for subsequent sensory and chemical analysis.
2.3. Sensory analysis
The tests of acceptance and purchase intent for the different
margarine formulations were conducted in individual booths in the
Coding
Yes (1) No (2)
Yes (1) No (2)
Yes (1) No (2)
Maybe I would buy/I would not buy (3)
Taste like the traditional (1), with spices (2), others (3)
Yes (1) No (2)
Yes (1) No (2)
Yes (1) Not (2)
Maybe I would buy/I would not buy (3)
Yes (1) No (2)
Male (1) Female (2)
15e25 (1), 26e35 (2), 36e50 (3), above 50 (4)
Up to R$ 622 (1), R$ 623 to R$ 3110 (2), R$ 3111 to R$ 6220 (3), above R$ 6621 (4)
Married (1), single (2), others (3)
Yes (1) No (2)
Fundamental (1), High school (2), Higher and graduate (3)
Daily (1), 5 to 3 times per week (2), 2 times per week (3), 1 time per week (4), does
not practice (5)
 C.O. Lopes et al. / LWT - Food Science and Technology 58 (2014) 63e70 65

Table 2 To summarize and graphically represent the phenolic content

Treatments and descriptions of the margarine samples with reduced sodium con- (Fig. 3) and total antioxidant activity determined using the ABTS
þ
tent and added spices.
method (Fig. 4), the samples were divided into six groups: group A
Treatments Formulations Mixture spcies added Reduced sodium (%) (control margarine without sodium or added spices), group B
C1 C100 e 100 (control margarine with sodium and no added spices), group C
C2 C0 e 0 (margarine formulation without sodium and with spice mixture A),
T1 A100 A 100 group D (margarine formulations with sodium and spice mixture
T2 A75 A 75
A), group E (margarine formulation without sodium and with spice
T3 A50 A 50
T4 A25 A 25 mixture B) and group F (margarine formulations with sodium and
T5 A0 A 0 spice mixture B).
T6 B100 B 100 All chemical analyses were performed in triplicate using the
T7 B75 B 75
extract preparation method described above, and all of the reagents
T8 B50 B 50
T9 B25 B 25
used were SigmaeAldrich brand.
T10 B0 B 0
2.5. Statistical analyses
C ¼ control, T ¼ treatment. Mixture A: green onion (40 g/100 g), garlic (35 g/100 g),
marjoram (20 g/100 g) and thyme (5 g/100 g). Mixture B: lemon (60 g/100 g),
oregano (15 g/100 g), basil (20 g/100 g) and thyme (5 g/100 g). Sodium reductions Data from the questionnaire Market Appeal of Margarine with
were based on the standard of 600 mg/100 g. Reduced Salt Content (NaCl) and Addition of Spices was statistically
analyzed using multiple correspondence analysis.
The sensory analysis scores and the results from the phenolic
sensory analysis laboratory by 62 untrained panelists. A 9-point compound and total antioxidant activity (ABTS
þ and DPPH)
hedonic scale was used for acceptance testing, ranging from 9 determination were subjected to analysis of variance followed by
(‘extremely liked’) to 1 (‘extremely disliked’), as described by the ScotteKnott average test at 5% significance.
Piggott (1984). A 5-point structured scale was used for purchase The main components of the sensory analysis average scores
intent, ranging from 5 (‘would certainly buy this product’) to 1 were graphically represented to permit condensed visualization
(‘would definitely not buy this product’). The 12 samples were and to investigate the association between the variables and
evaluated by the same 62 panelists over two sessions held on treatments. For the phenolic compound and total antioxidant ac-
consecutive days. Six samples were presented at both sessions in a tivity (DPPH and ABTS
þ) determination, Pearson correlation
balanced manner as described by Macfie and Bratchell (1989). analysis was performed using the mean values obtained.
For the margarine tasting tests, crackers produced in the UFLA All statistical tests were performed using R (R Core Development
Laboratory of Nutrition and Dietetics, consisting of only wheat, Team, 2011) except for principal component analysis, which was
water and oil, were used as vehicles. performed using Sense Maker software (Nunes & Pinheiro, 2012).
In each session, a tray containing six crackers was delivered to
the panelists. Each cracker was served with approximately 1.5 g of 3. Results and discussion
the individual margarine formulations, which were coded with
random three-digit numbers and accompanied by a glass of water 3.1. Market appeal questionnaire
to be used between samples.
Table 1 shows the market appeal questionnaire for margarine
2.4. Total phenolic compounds and antioxidant activity with reduced salt content and added spices, and the respective
encodings support the correspondence analysis presented in Fig. 1.
Extracts were prepared with 5 g of the samples in natura using As shown in Fig. 1, there was an association between re-
three different solvents and volumes: 30 mL absolute methanol, spondents who consumed margarine and those who did not
10 mL acetonitrile and 15 mL hexane. Each solvent was left in observe the sodium content of foods, the majority of whom were
contact with the sample for approximately 10 min, and the su- single 15- to 25-year-old males with no children. This group was
pernatant was subsequently removed and stored away from light. not associated with any pre-defined monthly income range in the
Using the methodology described by Longoberdi et al. (2012) with questionnaire. In contrast, the respondents who did not consume
some modifications, the solvent mixture was evaporated in a rotary margarine were mostly female and greater than 36 years old. It was
evaporator (55  C) to a volume of approximately 4 mL, filtered not possible to define a profile of consumers who would buy
through Whatman No. 2 filter paper and brought up to a final margarine with reduced sodium and added spices or a profile of
volume of 10 mL with absolute methanol. consumers who would not. Thus, this product is one of interest to
The phenolic content of the samples was quantified using the the general population, and preference is not associated with
FolineCiocalteu reagent according to the method described by particular factors, including gender, marital status, monthly in-
Waterhouse (2002) and the results were presented in mg gallic acid come, education and physical activity.
equivalents (GAE)/100 g of sample. Total antioxidant activity was The majority of respondents (62.8%) indicated they would buy
assessed using the 2,20 -azinobis-3-ethylbenzothiazoline-6-sulfonic margarine with reduced sodium, and 87.3% of these respondents
acid (ABTS
þ) method, as described by Re et al. (1999), and the 2,2- indicated they would do so because such a product would represent
diphenyl-2-picrylhydrazyl (DPPH) method, as described by Brand- a healthier alternative to traditional products. Regarding the flavor
Wiliams, Cuvelier and Berset (1995). The means of the results for of margarine with reduced sodium, 46.8% of respondents favored a
antioxidant activity by the ABTS
þ and DPPH method were taste identical to traditional margarine, 39.3% favored the flavor of
expressed in % of inhibition. The original concentration of the spices, and the remaining 13% favored a different taste. When the
extract for determination of the antioxidant activity by the DPPH respondents were asked if they would buy margarine with reduced
method was of 500 mg/mL and from this one; five dilutions were sodium and added spices, most participants (61.3%) said they would
conducted to make the determination of the regression equation buy it, and 6.8% selected the option of ‘may/may not buy’.
possible, the minimal dilution being equal to 37.5 mg/mL and the Of the 400 respondents, 74% were self-reported consumers of
maximum dilution equal to 2.5 mg/mL. margarine, and among them, 68.8% said they used the product one
66 C.O. Lopes et al. / LWT - Food Science and Technology 58 (2014) 63e70

Fig. 1. Analysis of correspondence among the 400 interviewees of the appeal questionnaire of margarine market with reduced sodium content and added spices.

or more times per day. Considering that an estimated 60%e90% of
sodium ingested comes from industrially processed foods (Sarno,
Castro, Bandoni, Bombem, & Jaime, 2009), reduced sodium con-
tent in margarine may have a positive health impact.
The proposed development of margarine with reduced sodium
content and added spices assessed in the questionnaire was moti-
vated in part by studies indicating the need for such products. Liem,
Miremadi, and Keast (2011) reported that there are few low-sodium
processed foods available on supermarket shelves, making it very
difficult for consumers to not exceed the maximum tolerable so-
dium intake of 2 g/day. These authors also suggested that the
acceptance of reduced sodium products may be more effective if
multisensory principles are applied. The optimization of flavor
characteristics and improved aroma through the use of spices, as
opposed to dietary advice alone, is one such example.
result is quite promising because it indicates that the sodium
content of margarine may be reduced by 75% and 50% when adding
spice mixtures A and B, respectively, without compromising the
taste attribute compared to traditional margarine (C0).
For overall impression, the panelists performed a general anal-
ysis of all perceived sensory attributes of the sample. In this case,
the margarine formulations A0, A25, B0, A50 and B25 scored
highest, with average scores of 7.37, 7.24, 7.08, 7.02 and 6.73,
respectively (Table 3). These were significantly higher than the
average score for the control margarine without sodium reduction
(C0, 6.73), indicating panelist preference for the margarine
Table 3
Average values of the tasters and respective coefficients of variation (CV) for the
attributes appearance, aroma, flavor, overall impression and purchase intention of
the margarines.

Formulations Coding Average Acceptance Purchase

3.2. Sensory analysis
intention
Appearance Aroma Flavor Overall
The average acceptance results of the 12 margarine formula- impression

tions in terms of appearance, aroma, flavor, overall impression and C1 C100 7.26 6.50b 4.92d 5.61d 2.61d
purchase intent are listed in Table 3. The analyses of their main C2 C0 7.05 6.60b 6.84b 6.73b 3.68b
T1 A100 7.19 7.18a 5.52c 6.16c 2.95c
components are presented in Fig. 2.
T2 A75 7.29 7.31a 6.53b 6.85b 3.74b
Acceptance for the appearance attribute did not differ (p > 0.05) T3 A50 7.19 7.37a 6.82b 7.02a 3.74b
among the formulations (Table 3), indicating that the addition of T4 A25 7.18 7.19a 7.27a 7.24a 4.03a
spice mixtures A or B did not interfere in this regard. The average T5 A0 7.08 7.29a 7.47a 7.37a 4.10a
values for this attribute ranged from 6.87 to 7.26, for B25 and C100, T6 B100 6.81 6.69b 4.95d 5.71d 2.58d
T7 B75 7.00 6.89b 5.89c 6.31c 3.23c
respectively, and corresponded to the classification ‘moderately T8 B50 6.87 6.84b 6.34b 6.66b 3.42b
liked’ on the hedonic scale where the maximum score is 9 T9 B25 6.87 6.82b 6.61b 6.73b 3.47b
(‘extremely liked’). Acceptance for the flavor attribute was signifi- T10 B0 6.90 6.90b 7.06a 7.08a 4.00a
cantly higher for all margarine formulations containing spice CV% 14.52 13.97 19.36 15.29 21.02
mixture A compared to the formulations with spice mixture B, and C100: control margarine with no sodium (100% reduction) and no spices; C0:
both spice mixtures were found to have greater acceptance than control margarine with standard sodium content (0% reduction) and no spices.
the controls. A100, A75, A50, A25 and A0: margarine with 100%, 75%, 50%, 25% and 0% reduction
of sodium, respectively, and 1.5 g/100 g of mixture A of spice. B100, B75, B50, B25
For the taste attribute, the average acceptance score of the and B0: margarine with 100%, 75%, 50%, 25% and 0% reduction of sodium, respec-
control margarine without sodium reduction (C0) did not differ tively, and 1.5 g/100 g of mixture B of spice. Means followed by the same letter in the
from those of the margarine formulations A50, A75 and B50. This same column do not differ according to the ScotteKnott test at 5% significance.
 C.O. Lopes et al. / LWT - Food Science and Technology 58 (2014) 63e70
Fig. 2. Principal component analysis of the treatments and attributes evaluated in
margarines with the addition of spices and reduction of sodium chloride. C100: control
margarine with 100% reduction of sodium and free from spices; C0: control margarine
with standard sodium content (0% reduction) and free from spices. A100, A75, A50,
A25 and A0: margarine with 100%, 75%, 50%, 25% and 0% of reduction of sodium,
respectively and addition of 1.5 g/100 g of mixture A of spices, respectively; B100, B75,
B50, B25 and B0: margarines with 100%, 75%, 50%, 25% and 0% of reduction of sodium
and addition of 1.5 g/100 g of mixture B of spices, respectively.
formulations with added spices (A or B) even with reduced sodium
content.
With 5 representing the highest possible score, the purchase
intent scores (Table 3) of margarine formulations A0, A25 and B0
were 4.10, 4.03 and 4.00, respectively, indicating a greater purchase
intent for these samples compared to the control margarine
without sodium reduction (C0, equivalent to traditional marga-
rine). Moreover, the scores of margarine formulations A50 and B50
did not differ from that of control margarine C0. This result again
highlights the commercial potential of margarine formulations
with 50% sodium reduction and the addition of spice mixture A or
B.
The lowest scores for purchase intent were observed for the
control margarine without sodium (C100) and the margarine
formulation with spice mixture B and no sodium (B100). These
findings are consistent with those of Yabuta, Cardoso, and Isosaki
(2006) that low-sodium diets have the lowest indices of adher-
ence, despite being widely cited as one of the most effective ways to
control hypertension.
Fig. 2 shows that margarine formulations with 100% sodium
reduction (C100, B100 and A100) diverged for all of the attributes
evaluated relative to the control without sodium reduction (C0).
However, margarine formulations with spice mixture A and 50% or
75% sodium reduction (A50 and A75, respectively) exhibited similar
aroma and appearance attributes. Samples A25 and A0 were most
similar to the C0 control in terms of overall impression and pur-
chase intent, with an advantage in terms of flavor. Samples B0, B25,
B50, A50 and A75 were similarly positioned in terms of flavor.
These findings indicate that the addition of spice mixtures to
margarine increased acceptance in terms of flavor, taste and overall
impression as well as purchase intent. In particular, the increased
acceptance of reduced sodium margarine formulations when spices
were added indicates that the spices represent an added flavor to
this food, as previously discussed by Dallepiane and Bós (2007),
67
who argued that adding spices to products with sodium reduction
helps ensure good acceptance.
Carraro, Machado, Espindola, Campagnol, and Pollonio (2012)
also investigated the sensory benefits of adding spices to foods by
developing mortadella with 50% sodium replacement by potassium
chloride (KCl). KCl has salting power without promoting elevated
blood pressure, and in the study, its unpleasant aftertaste was
masked with a mixture of spices containing coriander, onion, white
pepper, cardamom and Jamaican pepper. The panelists preferred
the mortadella with partial sodium substitution by KCl and added
spices over mortadella with sodium replacement by KCl without
the added spice mixture, as indicated by good acceptance of the
former in terms of the flavor attribute.
Industrial products with added spices to replace synthetic an-
tioxidants have also been found to have good sensory acceptance.
As one example, Bandyopadhyay, Chakraborty, and Raychaudhuri
(2007) developed a cottage cheese product and added turmeric
(Curcuma longa L.), coriander (Coriandrum sativum L.), curry (Mur-
raya koenigii L.), spinach (Spinacia oleracea) and amla (Emblica
officinalis) then compared the product to others containing the
synthetic antioxidants TBHQ, BHA and BHT to assess indicators of
oxidation. According to the sensory analysis, the spices did not
affect the overall acceptance of the product, and the chemical an-
alyses indicated that the samples with added spices exhibited more
healthy attributes compared to the control.
The following year, these same authors added sugar beet (Beta
vulgaris), peppermint (Mentha spicata L.) and ginger (Zingiber offi-
cinale L.) to the cottage cheese product to assess indicators of
oxidation. The products prepared with a combination of sugar beet
with ginger and peppermint and those with a combination of
peppermint and ginger had greater acceptance than the control
sample without added components (Bandyopadhyay, Chakraborty,
& Raychaudhuri, 2008).
3.3. Phenolic compounds and total antioxidant activity (ABTS
þ and
DPPH)
Fig. 3 shows the results for phenolic content in mg gallic acid
equivalents (GAE)/100 g.
A significant increase in phenolic content was observed in the
margarine formulations containing spice mixtures A and B (groups
C, D, E and F). The formulations containing spice mixture B (groups
E and F) had phenolic content levels five times greater than the
formulations without added spices (groups A and B) and one and a
half times greater than the formulations with spice mixture A
(groups C and D). The ranking of the formulations in terms of
phenolic content was A < B < C < D < E < F, with average con-
centrations of 17.1, 19.1, 56.4, 57.4, 91.9 and 92.3 mg GAE/100 g,
respectively.
The large differences in phenolic content between margarine
formulations containing spice mixture A (groups C and D) and
those containing spice mixture B (groups E and F) is most likely
attributable to the different spices and proportions used in the
mixtures. Because of the different compositions of the two mix-
tures (40 g/100 g green onion, 35 g/100 g garlic, 20 g/100 g mar-
joram and 5 g/100 g thyme in spice mixture A and 60 g/100 g
lemon, 15 g/100 g oregano, 20 g/100 g basil and 5 g/100 g thyme in
spice mixture B), it is not possible to attribute the difference in
phenolic content to one specific spice. Nevertheless, garlic and
lemon, present in spice mixtures A and B, respectively, may have
contributed to this difference, as the phenolic content of garlic is
much lower than that of lemon. Lu, Ross, Powers, Aston, and Rasco
(2011) and Nieto, Jongberg, Andersen, and Skibsted (2013) reported
phenolic content levels in garlic of 17 mg GAE/100 g and 12.8 mg
GAE/100 g, respectively. In contrast, the phenolic content of
68 C.O. Lopes et al. / LWT - Food Science and Technology 58 (2014) 63e70
Fig. 3. Analysis of phenolic compounds (mg GAE/100g) of the samples grouped in
control free from Na (A), control containing Na (B), free from Na and containing
mixture A of spices (C), containing sodium and containing mixture A of spices (D), free
from Na and containing mixture B of spices (E); containing Na and containing mixture
B of spices (F). Means followed by the same letter in the column do not differ from one
another by the ScotteKnott test at 5% of significance.
436.92 mg GAE/100 g in lemon reported by Ramful et al. (2011) is
much higher. The main phenolic compounds present in lemon are
catechin, epicatechin, vanillic acid, myricetin, kaempferol and
naringenin, which present considerable antioxidant activity
(Sommano, Caffin, & Kerven, 2013).
The use of oregano and basil exclusively in mixture B is another
possible explanation for the differences in phenolic content be-
tween the margarine formulations containing the two different
spice mixtures. The phenolic content of oregano is high, and that of
basil is considerable, with previously reported contents of 288 mg
GAE/100 g (Amarowicz et al., 2009) and 98.5 mg GAE/100 g
(Dorman & Hiltunen, 2011), respectively. The main phenolic com-
pounds of oregano are (E) beta terpineol, terpinen-4-ol and thymol
(Olmedo, Nepote, & Grosso, 2013).
Thyme has a low phenolic content, with a previously reported
value of 8.1 mg GAE/100 g dry weight (Roby, Sarhan, Selim, &
Khalel, 2013), and this spice most likely did not affect the
phenolic content results considerably because it was added to both
spice mixtures (A and B) at the same proportion (5 g/100 g).
Marjoram, present only in spice mixture A (at a proportion of
20 g/100 g), also has a low phenolic content. When quantified by
extraction with methanol, ethanol, ether and hexane, the concen-
trations observed were 5.2, 4.6, 4.5 and 3.9 mg GAE/100 g,
respectively (Roby et al., 2013). Thus, marjoram may have
contributed to the low phenolic content of the margarine formu-
lations containing spice mixture A compared to those containing
spice mixture B.
It is important to emphasize that regardless of the types and
proportions of spices used in spice mixtures A and B and the
respective contributions to the final phenolic content, both mix-
tures presented an efficient aggregation of these compounds in the
margarine formulations.
Fig. 4 shows the results for total antioxidant activity in the
margarine formulations using the ABTS
þ method, with the results
presented in mg trolox/g of sample.
A difference was observed (p  0.05) in the total antioxidant
activity of the control margarine formulations (groups A and B)
compared to the formulations containing spice mixtures A and B
(groups C, D, E and F). No significant differences were observed
between groups C and D and between groups E and F. The ranking
of the groups in terms of antioxidant activity was
A < B < C < D < E < F, with values of 28.8, 29.6, 80, 78.6, 80.7 and
81.6 mg Trolox/g, respectively. Sodium did not affect the antioxidant
Fig. 4. Analysis of the total antioxidant activity by the ABTS
þ method of the samples
grouped in a control free from Na (A), control containing sodium (B), free of Na and
containing mixture A de spices (C), contend Na and containing mixture A of spices (D),
free of Na and containing mixture B of spices (E), contend Na and containing mixture B
of spices (group F). Means followed by the same letter in the same column do not differ
from one another by the ScotteKnott test at 5% of significance.
activity of margarine, as differences were not observed between
groups A and B, between C and D and between E and F.
Spices or their respective essential oils generally have high
antioxidant activity, and researchers have conducted various
studies to assess these attributes. Seeking to prevent the rancidity
of margarine, which is lipid rich, Amarowicz et al. (2009) added
thyme, oregano and marjoram to the product. Azizkhani and Zandi
(2010) developed a similar study in margarine but instead added
essential oils from different spices. Olmedo et al. (2013) added the
essential oils of oregano and rosemary to a commercial brand
cream cheese (Nestlé, Argentina) to assess changes in the quality
parameters of the product from 7 to 35 days. They found that the
essential oils of oregano and rosemary improved oxidative and
fermentative stability and prevented lipid oxidation, development
of rancid flavor and product fermentation, all of which contributed
to prolonged shelf life.
Some of the antioxidant compounds present in spices are
linalool, a powerful antioxidant found at a concentration of 69.8 g/
100 g in basil (Nurzynska-Wierdak, Bogucka-Kocka, Kowalski, &
Borowski, 2012); thymol and carvacrol, the main antioxidant
components of thyme (Amarowicz et al., 2009) and present at
proportions of 42.89 g/100 g and 42.75 g/100 g, respectively, in
marjoram (Al-Bandak & Oreopoulou, 2007); and N-transferuloyl
methoxydopamine-30 , N-cis-feruloil-30 -methoxytyramine, N-
trans-p-coumaroyltyramine and 3,5,7-trihydroxyflavone (kaemp-
ferol), found in green onion (Seo, Cho, Moon, & Park, 2011).
Table 4 shows the results for the total antioxidant activity of the
12 margarine formulations using the DPPH radical method, the
results are expressed as percent antioxidant activity inhibition.
Spice mixtures A and B significantly increased the total antiox-
idant activity of the margarine formulations to which they were
added, and the effect was particularly evident for mixture B.
Considering that the same amount of spice mixtures A and B were
added to the formulations (1.5 g/100 g) and that antioxidant ac-
tivity is positively associated with phenolic content (Terra et al.,
2012), the greater total antioxidant activity observed for the for-
mulations containing mixture B may reflect many of the same ar-
guments presented for the differences observed in phenolic
content (Fig. 3).
It is generally difficult to compare different studies investigating
the antioxidant activity of spices due to existing variables, such as
type of extraction, plant parts and varieties used, plant origin and
spice form (e.g., in natura, dehydrated or cooked). For example,
 C.O. Lopes et al. / LWT - Food Science and Technology 58 (2014) 63e70 69

Table 4 Association of official analytical chemistry. (1990). Official methods of analysis (14th
Average values of the antioxidant activity by the DPPH method in % inhibition for 5 g ed.) (p. 1298) Washington, DC.
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