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Evaluation of the oxidative stability of bakery products by OXITEST method


and sensory analysis

Article  in  European Food Research and Technology · December 2016


DOI: 10.1007/s00217-016-2831-9

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Eur Food Res Technol
DOI 10.1007/s00217-016-2831-9

ORIGINAL PAPER

Evaluation of the oxidative stability of bakery products


by OXITEST method and sensory analysis
Marisa Carmela Caruso1 · Fernanda Galgano1 · Maria Antonietta Colangelo1 ·
Nicola Condelli1 · Teresa Scarpa1 · Roberta Tolve1 · Fabio Favati2 

Received: 5 September 2016 / Revised: 26 October 2016 / Accepted: 2 December 2016


© Springer-Verlag Berlin Heidelberg 2016

Abstract  The lipid oxidation is considered the main cause method for oxidative stability studies; anyway, a further
of the deterioration of the food products with a high fat research will be needed to confirm these results.
content. Lipid oxidation is associated with the formation of
off-odor and off-flavor that compromise the acceptability of Keywords  Bakery products · Shelf life · Sensory analysis ·
the products; moreover, this reaction affects the nutritional OXITEST · Oxidative stability
quality of foods, in particular the fatty acids content. In
this work the stability of bakery products with a high fat
content was monitored over a 12 months of storage, con- Introduction
sidering the lipid oxidation as the limiting factor for the
shelf-life products. For this study the OXITEST has been The modern food industry tends to satisfy the needs of
used as innovative and promising instrument. The OXIT- consumers who demand products of high quality also for
EST reactor was used to monitor the oxidative stability of a long time period. The maintenance of high levels of sen-
four different types of bakery products. The stability of the sory quality associated with the freshness of the product
products was also monitored with the sensory and chemi- has also been recommended [1]. For semi-perishable prod-
cal analyses in order to support the instrumental analysis. ucts the evaluation of the sensory characteristics is essential
The sensory analysis was useful to evaluate the perceptible for monitoring the evolution of the parameters which influ-
changes occurring on the products during storage. Moreo- ence the acceptability; in fact, the sensory characteristics of
ver, with the OXITEST an accelerated shelf-life test to pre- foods (excluding wine and cheese) change negatively dur-
dict the shelf life of each product has been performed. By ing storage, causing variations of the product and forma-
physical–chemical data obtained during experimentation it tion of unwanted aromatic compounds. The use of a panel
was found that the products have a different stability to the of tasters is useful to define the shelf life of semi-perishable
oxidation reaction, depending on the type of fats used and foods, but represents an expensive solution for the industry,
on the composition of the products. The data obtained from in terms of both time and money [1]. Regardless of the type
the OXITEST were correlated with the sensory data. Fur- of product analyzed, the shelf life is defined as the period
thermore, the OXITEST instrument can be utilized as rapid of time during which slight changes occur in the product
without compromising the safety and acceptability. The
shelf life of food is influenced by many factors, intrinsic
or extrinsic to the product [2]. Among the intrinsic factors
* Fernanda Galgano there are: levels of water activity (aw), pH and total acidity,
fernanda.galgano@unibas.it redox potential, nutrients, endogenous microflora, the pres-
1 ence of substances with antimicrobial action, type of raw
School of Agricultural, Forestry, Food and Environmental
Sciences, University of Basilicata, Viale dell’Ateneo Lucano materials and the formulation of the product. In addition to
10, 85100 Potenza, Italy these factors, extrinsic factors can trigger and/or accelerate
2
Department of Biotechnology, University of Verona, Strada the reactions of degradation of the food. Among the extrin-
Le Grazie 15, 37134 Verona, Italy sic factors there are: time–temperature combination of

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treatments during the production process, moisture (RH%) stress by increasing one or more oxidative factors. Among
and temperature of the environments of production, storage these latter, OXITEST is an innovative instrument used for
and distribution, microbiological pollution occurred in one oxidative stability studies of fat foods based on the moni-
or more stages of production, type of packaging, handling toring of pressure over a period of time in analytical cham-
by the consumer [3]. Consequently, the shelf life of a prod- bers where the sample was submitted at high O2 pressure
uct is the result of a synergistic effect between intrinsic and and high temperature. Because the OXITEST is a recent
extrinsic factors which must be taken into account [2]. Bak- instrument, at the moment there are still few studies about
ery products belong to the class of semi-perishable prod- its use [9–12]. In particular, to our knowledge, the literature
ucts. The term “bakery products” is referred to a wide range focused on the use of this instrument for analyzing pro-
of products used in most countries of the world, including cessed foods, such as bakery products, is scarce [13].
bread, biscuits, cakes, pies, breadsticks, crackers. The flour, The purpose of this work was to carry out a study of sta-
obtained from the Triticum aestivum ssp vulgare L., is the bility of bakery products with a high fat content, consid-
main ingredient of bakery products. Bakery products con- ering the lipid oxidation reaction as the limiting factor for
tain also fat ingredients, often in significant amount. The their shelf life. The evolution of the products was followed
addition of fats in bakery products influences the softness, by determining chemical parameters notoriously associated
the flavor, the texture and the alveolation of the product [4]. with fat quality (acidity, peroxides value, p-anisidine value,
The bakery products undergo phenomena of deterioration ultraviolet spectrophotometric analysis). The OXITEST
during their shelf life, and precisely [5]: reactor was used to monitor the oxidative stability of four
different types of bakery products. Furthermore, OXITEST
1. physical deterioration (moisture loss, staling) instrument test for accelerated oxidation stability has been
2. chemical changes (rancidity) performed to predict the shelf life of each product, both on
3. microbiological deterioration (growth of yeasts, molds raw materials and on finished products, without the need
or bacteria) for any preliminary fat separation. To support the chemi-
cal data, sensory analysis of bakery products by a panel
One of the most serious problems affecting the bakery of trained judges during 12 months of storage was also
goods is the staling. The staling is defined as the process conducted.
of deterioration, which occurs in bread or other products,
during the post baking, negatively affecting the sensory
characteristics of the product. Literature information indi- Materials and methods
cates that properties and molecular modifications of starch
are mainly responsible for the development of firmness Sampling
and the perception of staling [3]. However, staling evolu-
tion is also affected by other factors, such as migration of The analyses were conducted on four types of baked prod-
moisture, temperature, gluten and supplemental ingredients ucts, three salty and one sweet, having activity water rang-
[6]. The staling occurs primarily in all bakery goods with ing from 0.112 to 0.198, and with a different fat content.
high humidity (breads and pastries). Also microbiological The two different fats used for their preparation (palm oil
deterioration is typical of high aw products, while the dried and olive oil) were also analyzed. The samples were packed
ones are not affected by this problem. in small bag by using a OPP-metallized laminated film and
The products with lower humidity and with a high fat provided by an industry of the district Lucano-Pugliese
content, such as cookies, crackers and breadsticks, are par- (Matera, South of Italy). In Table 1 is illustrated the com-
ticularly susceptible to lipid oxidation. For these products position of the analyzed products.
lipid oxidation is considered the main cause of deterioration
[7]. The consequence of lipid oxidation is the formation of Chemical analyses
off-odor and off-flavor that compromise the acceptability
and also affects the nutritional quality of the products. In The products were analyzed at regular intervals of time,
fact, one of the main effects of lipid oxidation is the reduc- corresponding to the day of production and after 4, 8 and
tion in the content in essential fatty acids. Furthermore, on 12 months and indicated, respectively, as T0, T1, T2, T3.
animals a toxic effect of primary and secondary products of During the storage period the bakery products have been
oxidation has been observed [8]. maintained in the original package in a cool and dry place,
Traditional methods detected the oxidation stability of at a temperature of 20–25 °C. Raw materials (olive and
foods by evaluating the quantity of primary and secondary palm oil) from same lot used in bakery production and the
products of oxidation. Accelerated methods are new ana- fat extracted from the products at each time of analysis
lytical techniques based on the application of an oxidative were stored in refrigerator at 14 °C. The fat was extracted

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Table 1  Composition of the samples


Product Ingredients

Crackers with olives Wheat flour, palm oil, olive oil, sea salt, yeast. Total fat 15.6%, saturated fat 5.9%
Taralli with hot chili pepper Wheat flour, palm oil, olive oil, sea salt, hot chili pepper, ammonium bicarbonate. Total fat 24.6%, saturated fat
11.9%
Breadsticks with olive oil Wheat flour, olive oil, sea salt, yeast. Total fat 13.3%, saturated fat 3.33%
Biscuits with lemon Wheat flour, sugar, palm oil, eggs, leavening agents: ammonium bicarbonate, sodium bicarbonate, natural aromas,
emulsifier: soy lecithin. Total fat 16.5%, saturated fat 8.2%

with Soxhlet method by using petroleum ether as solvent. to recognize different intensities of 13 sensory attributes
On the lipid fraction a spectrophotometric analysis in the (AROMA: oxidized aroma, cardboard aroma; TASTE:
ultraviolet (∆K) was performed [14]; free acidity [15]; per- sweet, salty, sour, bitter; FLAVOR: oxidized flavor, card-
oxide value (PV) [15]; p-anisidine value (p-AnV), accord- board flavor; TEXTURE: hardness, crispness, friability;
ing to the AOCS Official Method Cd 18-90 [16]. The level TACTILE SENSATIONS: feeling fat palate, feeling dry
of total oxidation of bakery products and raw materials was palate), obtained from the bibliography [17–21]. Moreover,
expressed as TOTOX value (TOTOX = 2PV + p-AnV). All to help judges to recognize the attributes of interest were
the analyses were made in triplicate. All the chemical rea- created appropriate reference standards. In particular, as
gents were purchased from Sigma-Aldrich (Milan, Italy). a standard for the crispness were used chips, crackers for
friability, shortbread biscuits for hardness. Wet cardboard
Measure of the oxidative stability by OXITEST method has been used as a standard for the cardboard aroma. The
standard for oxidized aroma and flavor was obtained by
The analysis with OXITEST (VELP, Usmate, MB, Italy) brushing the cookies with a mixture of olive and palm oil
was performed on raw materials (olive and palm oil, 10 g) previously oxidized with OXITEST (T 90 °C, oxygen pres-
and on bakery products (30 g of grounded product). The sure 6 bar) diluted with fresh oil. All the analyses were
products were ground and analyzed by OXITEST for made in triplicate and repeated at each time (T0, T1, T2, T3).
oxidative stability. Each accelerated oxidation test was
repeated using two different reactors, for a total of four Data analysis
analytical replies (each reactor is fitted with two separate
oxidation chambers), under over-pressure of pure oxy- In order to evaluate the significance of differences between
gen (0.6 MPa, degree 5.0) and constant high temperature the storage time and the samples, least significance differ-
(90 °C). The OXITEST response is the induction period ence (LSD) test was conducted. The analysis of variance
(IP), expressed as “stability time” before fat oxidation and (ANOVA) was also performed. Sensory data were acquired
corresponding to a drop of O2 pressure due to the consump- and processed with FIZZ software (ver. 2.47, Biosystèmes,
tion of oxygen by the sample. The IP value is automati- Couternon, France).
cally calculated from oxidation curve by graphical method
(two tangent methods) by using the OXISoft™ program
included in the instrument. Results and discussion
In addition, an accelerated shelf-life test for bakery
products has been performed at three different temperatures Chemical analyses
(80, 90 and 100 °C or 90, 100 and 110 °C) and the data
were analyzed using the OXISoft™ program. With this The measurements made immediately after production
procedure it is possible to extrapolate and estimate the oxi- and packaging of the samples allowed to describe the bak-
dation stability of samples at room temperature, in case of ery products in the initial phase of the trial, highlighting
linear dependence with the temperature. the differences in terms of physicochemical and sensory
parameters. Peroxide value of the fats extracted was in the
Sensory analysis range 2.33–27.42 meq O2/kg (Table 2). The highest value
was observed for the fat extracted from the crackers with
The sensory evaluation has been conducted on the bakery olives, while the lowest one for cookies with lemon. Per-
products. It was used a panel of eight judges (six females oxide value of the raw materials was of 6.14 meq O2/kg
and two males), aged between 24 and 38 years, recruited in for olive oil, whereas a lower value, equal to 0.94 meq O2/
the University of Basilicata. The judges have been trained kg, was observed for palm oil, notoriously more stable to

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Table 2  Range values of analytical parameters throughout the storage time (12 months)


Chemical parameters Crackers with olives p Taralli with hot chili p Breadsticks with olive p Biscuits with lemon p
pepper oil

Acidity (%) 0.34–0.36 NS 0.12–0.23 *** 0.36–0.39 NS 0.23–0.30 NS


Peroxide value (PV) 9.62–27.42 ** 4.16–16.23 ** 8.14–22.63 ** 2.33–12.59 **
(meq O2/kg)
p-Anisidine value 5.22–6.11 * 6.83–10.97 ** 5.89–9.64 * 7.03–11.82 *
(p-AnV)
∆K 0.15–0.19 NS 0.13–0.15 NS 0.13–0.16 NS 0.19–0.19 NS
Induction period (IP) 1171.00–610.50 ** 2444.50–2210.50 NS 2210.50–971.00 * 8664.50–8318.50 *
(min)

* p < 0.05; ** p < 0.01; *** p < 0.001

Fig. 1  TOTOX of bakery prod-


ucts immediately after produc- 25
tion (T0) and raw materials

20
TOTOX value

15

10

0
Crackers with Breadsticks Taralli with Biscuits with Olive oil Palm oil
olives with olive oil hot chili lemon
pepper

lipid oxidation. Figure 1 shows the TOTOX value of the fat contains both olive oil and palm oil, and in addition, it
extracted from the bakery products and raw materials at T0 contains hot chili pepper. The hot chili pepper may have
time. From TOTOX data it was clear that the biscuits with played an important role in preventing the formation of
lemon and taralli with hot chili pepper were characterized peroxides. This statement seems confirmed by the results
by a lowest level of oxidation. These differences among the obtained from Bignardi et al. [25]; Gouveia et al. [26], who
products are attributable not only to the type of fat used, but reported the antioxidant power of different species of chili
also to the presence of ingredients which may have played peppers of the genus Capsicum (annum and frutescens)
a role in inhibiting or promote the reaction of lipid oxida- added to edible oils. Moreover, these authors reported that
tion. For example, the biscuits with lemon contain only the chili pepper acts as scavenger of peroxides. In addition,
palm oil, known to be more stable than other fats in oxida- the study of Si et al. [27] recognized in the capsaicin the
tion reactions [22]. In addition, the sample contains lemon; molecule responsible of the antioxidant power of hot chili
scientific studies showed that the lemon has an antioxidant pepper. Cavazza et al. [28] reported that red chili pepper
activity, protecting the lipids during reactions of oxidation is an excellent source of bioactive compounds useful for
and influencing the formation of primary compounds, such protecting olive oil from spoilage, improving the oxidative
as peroxides [23, 24]. Consequently, it can be assumed that stability during the oil storage. All samples were also ana-
in this sample the synergistic effect of the type of fat and lyzed by OXITEST instrument. At the first time the bak-
antioxidant activity exerted by lemon contributed to pro- ery products showed IP values very high for all samples. In
tect the product from the oxidation. Also taralli with hot particular, IP values confirmed a greater oxidation stability
chili pepper stood out for a high oxidation stability, espe- of lemon biscuits and taralli with hot chili pepper (Fig. 2).
cially for a low peroxides value. The sample in this case These values seem to confirm the initial chemical data.

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Sample IP [h:min]

Biscuits with lemon 146:51

Taralls with hot chilli 42:33


pepper
Breadscks with olive 39:43
oil
Crackers with olives 20:29

Fig. 2  IP values of the samples at T0 (after production)

Evolution of chemical parameters observed that the evolution of the parameters was different
for each product.
The evolution of the physical–chemical and sensory param- Mora et al. [30] found a good correlation level
eters of the products was followed for 12 months by per- between the IP value and peroxides value of a soybean
forming the analysis to four times (T0, T1, T2 and T3). The oil (R2  = 0.98); conversely, in this work no correlation
results are summarized in Table 2. As regards taralli with between these indices was found.
hot chili pepper it should be said that in the analysis phase,
in particular during the titration to measure the number of Evolution of the sensory parameters
peroxides, some problems have been observed in determin-
ing the change of color. The fat extracted from the sam- Although the chemical analyses were useful to describe
ple was intensely colored red-orange, probably due to the the evolution of a food product during storage time, only
presence of hot chili pepper. The main difficulty was to get by sensory analysis it is possible to determine whether a
the colorless, as required by the procedure of the titration. product continues to have the approval of the consumer.
It was assumed that during the fat extraction it has been The analysis of variance on sensory data showed a signifi-
extracted also coloring substances (probably carotenoids cant effect of time on the sample “crackers with olives”
content in hot chili pepper) that made it difficult to identify for many of the attributes. The only attribute for which
the end point of the titration. Instead for p-anisidine value we observed no significant change is the “acid” attribute
is believed that the intense staining of the sample, and par- (p > 0.05). In particular, as reported in Fig. 3, the attrib-
ticularly the presence of carotenoids, has interfered with utes oxidized flavor and aroma, cardboard aroma and flavor
the spectrophotometric analysis. In fact, Shahidi et al. [29] were perceived with an increasing intensity over time. The
reported that p-anisidine test is based on a reaction between analysis of the sensory parameters associated with the lipid
the aldehydes and p-anisidine that leads to the formation oxidation (oxidation aroma and flavor, cardboard aroma
of compounds of yellow color absorbing at 350 nm. In the and flavor) showed that the judges already after 4 months
case of taralli with hot chili pepper, the intense staining of perceived significant changes of the product, which became
the sample may have masked the yellow compounds, dis- more evident after 12 months of storage. In addition, the
torting the final result. However, the data of acidity, ΔK and deterioration of the product is also evidenced by a loss of
IP (Table 2), showed that the sample had a good stability to crispness and friability. For this sample it was observed
oxidation. Moreover, the data of sensory analysis helped to a correlation between the oxidized flavor and p-AnV
describe the evolution of the sample. Furthermore, it was recorded at different times (R2  = 0.517) and between the

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Fig. 3  Spider plot of sensory oxidized


analysis of crackers with aroma***
olives. *p < 0.05; **p < 0.01; 70 cardboard
***p < 0.001 dry palate*
60 aroma***
50
fat palate*** 40 sweetness*
30 T0 (after production)
20
friability*** 10 salty*** T1 (after 4 months)
0
T2 (after 8 months)
crispness*** acid NS T3 (after 12 months)

hardness*** bitter**
cardboard oxidized
flavour*** flavour***

Fig. 4  Spider plot of sensory oxidized aroma*


analysis of taralli with hot chilli 70 cardboard
dry palate***
pepper. *p < 0.05; **p < 0.01; 60 aroma**
***p < 0.001 50
fat palate*** 40 sweetness NS
30 T0 (after production)
20
friability*** 10 salty*** T1 (after 4 months)
0
T2 (after 8 months)
crispness** acid*** T3 (after 12 months)

hardness*** bitter***
cardboard oxidized flavour
flavour*** NS

oxidized aroma and the p-AnV (R2  = 0.814). In fact, the significant changes occurred for the attributes cardboard
secondary products detected with p-anisidine test are the aroma (p  = 0.0005), sweetness (p  = 0.0139), cardboard
same products responsible for sensory characteristics that flavor (p  = 0.0002), hardness (p  = 0.0001), crispness
assume an oxidized product (rancid odor and taste). (p = 0.0010) and friability (p = 0.0001). The judges who
The analysis of variance of sensory data of taralli with performed the sensory analysis have perceived high levels
hot chili pepper showed a significant change of all attrib- of cardboard flavor and aroma after just 4 months. In the
utes, excluding the attributes of sweetness and oxidized following months the intensity of the attributes increased
flavor. As also reported in Fig. 4 it was observed that as but with a smaller gap than that observed between T0 and
the judges of the panel have not perceived any change in T1. It was found a positive correlation between the card-
the oxidized flavor they perceived a growing intensity of board flavor and IP (R2 = 0.57); furthermore, also a corre-
cardboard aroma and flavor. After 8 months (T3) the judges lation between the oxidized flavor and the values of p-ani-
perceived a lower hardness, crunchiness and crispness of sidine was found (R2  = 0.53) and between the oxidized
the sample, probably caused by progressive aging of the aroma and IP (R2 = 0.65).
product. For this sample it was found a positive correlation For the biscuits with lemon, the time has not had a sig-
between the oxidized flavor and IP values (R2 = 0.50), also nificant effect on 6 of the 13 parameters considered, specifi-
between the oxidized aroma and the IP index (R2 = 0.97). cally nonsignificant changes were observed for the oxidized
Consequently, both the physicochemical and sensory data aroma and cardboard aroma, for attributes acid and bitter,
confirm of a good stability of the product to oxidation for the cardboard flavor and the crispness. The parameter
reaction. “oxidized flavor” varied significantly during storage time
For most of the attributes analyzed for the bread- (p < 0.001), and the more evident variation was observed
sticks with olive oil, the analysis of variance showed no after 8 months (time T3) (Fig. 6). For this sample a posi-
significant change of the attributes (Fig. 5). However, tive correlation between the oxidized flavor and p-anisidine

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Fig. 5  Spider plot of sensory oxidized aroma


analysis of breadsticks with NS
olive oil. *p < 0.05; **p < 0.01; 70 cardboard
***p < 0.001 dry palate NS
60 aroma***
50
fat palate NS 40 sweetness*
30
20 T0 (after production)
friability*** 10 salty NS T1 (after 4 months)
0
T2 (after 8 months)

crispness*** acid NS T3 (after 12 months)

hardness*** bitter NS
cardboard oxidized flavour
flavour*** NS

Fig. 6  Spider plot of sen- oxidized aroma


sory analysis of biscuits with NS
lemon. *p < 0.05; **p < 0.01; 70 cardboard
dry palate NS
***p < 0.001 60 aroma NS
50
fat palate* 40 sweetness***
30 T0 (after production)
20
friability*** 10 salty* T1 (after 4 months)
0
T2 (after 8 months)
crispness NS acid NS T3 (after 12 months)

hardness*** bitter NS
cardboard oxidized
flavour NS flavour***

(R2 = 0.82) was observed. Anyway, the maximum oxidized reaction limiting the shelf life of the products. The test
flavor intensity at the end of storage was lower than that was conducted at elevated temperatures and pressures of
perceived for the crackers with olives. Furthermore, the O2. Three runs of analysis were made for each sample at
judges perceived with less intensity the crispness attribute, three different temperatures, ranging from 80 to 110 °C and
friability and hardness, which are affected by aging of the at pressure of 6 bar. In particular, the test was performed
product and by moisture absorption phenomena. Although at 80, 90 and 100 °C, except for biscuits with lemon, for
the panel perceived a significant change in the flavor of oxi- which 90, 100 and 110 °C were selected. In fact, for this
dized, it was possible to note that there were no significant latter sample, the IP previously evaluated at 90 °C (Table 2)
changes in oxidized aroma. Furthermore, for this sample was very high and the temperature of 80 °C would have
it was noted that the IP values confirmed a good stability required a very long time of analysis.
of the biscuit. However, the judges perceived significantly The shelf life reported on the packing of each sample
the oxidation evolution. This highlighted the inability of was about 12 months; therefore, the results obtained with
machines to capture some aspects of the oxidation reaction, the OXITEST were compared with that indicated on the
which would be perceived by humans. pack. In Table 3 are reported in detail the temperatures used
and the results obtained. It was noted that for the sample
Accelerated shelf‑life test biscuits with lemon was estimated a shelf life longer than
that indicated by the manufacturer. For taralli and bread-
An accelerated shelf-life test was conducted with the sticks was calculated a shelf life that is closer, than the
OXITEST, considering the lipids oxidation reaction as the other samples, to what indicated by the manufacturer. On

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Table 3  Results of accelerated shelf-life test conducted with OXITEST

Samples Temperature (°C) Estimated shelf life at 20 °C Estimated shelf


(months) life at 25 °C

Biscuits with lemon 90 100 110 22 16


Taralli with hot chili pepper 80 90 100 16 11
Breadsticks with olive oil 80 90 100 15 10
Crackers with olives 80 90 100 5 3

the contrary, for the sample crackers with olives a reduced Compliance with ethical standards 
shelf-life was estimated, compared with the shelf-life indi-
cated on the pack. Conflict of interest  The authors declare no conflict of interest.

Compliance with ethics requirements  This article does not contain


Conclusions any studies with human or animal subjects.

The studied bakery products showed a different stabil-


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Acknowledgements The authors would like to express apprecia- Souza NE, Visentainer JV, Gomes STM, Matsushita M (2014)
tion to Laurieri s.r.l. industry for supplying the samples. The authors Analysis of carotenoids, a-tocopherol, sterols and phenolic com-
would also like to thank VELP Scientifica (Usmate, MB, Italy) for pounds from white bread enriched with Chia (Salvia hispanica
lending the OXITEST instrument and Dr. Stefania Corti for the tech- L.) seeds and carrot (Daucus carota L.) leaves. J Braz Chem Soc
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