Food Chemistry 403 (2023) 134347

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

The impacts of pink pepper (Schinus terebinthifolius Raddi) on fatty acids

and cholesterol oxides formation in canned sardines during
thermal processing
Carla Fernanda Targueta Barreira a, Vanessa Sales de Oliveira a, Davy William Hidalgo Chávez a,
Ormindo Domingues Gamallo a, Rosane Nora Castro b, Pedro Côrrea Damasceno Júnior c,
Alexandra Christine Helena Frankland Sawaya d, Micheli da Silva Ferreira e,
Geni Rodrigues Sampaio f, Elizabeth Aparecida Ferraz da Silva Torres f, *, Tatiana Saldanha a
a
Department of Food Technology, Institute of Technology, University Federal Rural of Rio de Janeiro, Rodovia Br 465, Seropédica, RJ 23890-000, Brazil
b
Department of Chemistry, Institute of Chemistry, University Federal Rural of Rio de Janeiro, Rodovia Br 465, Seropédica, RJ 23890-000, Brazil
c
Department of Phytotechny, Institute of Agronomy, University Federal Rural of Rio de Janeiro, Rodovia Br 465, Seropédica, RJ 23890-000, Brazil
d
Faculty of Pharmaceutical Science, University of Campinas, UNICAMP, Campinas, SP, Brazil
e
Department of Food Technology, Faculty of Veterinary, University Federal Fluminense, UFF, Niterói, RJ, Brazil
f
Department of Nutrition, School of Public Health, University of São Paulo (USP), Ave. Dr. Arnaldo, 715 São Paulo, SP 01246–904, Brazil

A R T I C L E I N F O A B S T R A C T

Keywords: The effect of adding pink pepper (Schinus terebinthifolius Raddi) fruits during sardines canning to minimize
Sardina pilchardus cholesterol oxidation were investigated. Canning resulted in an exchange of fatty acids, cholesterol and
Canned sardines cholesterol oxides between fish muscle and liquid medium (soybean oil). It also induced lipid oxidation, which
Cholesterol oxides
was demonstrated by the degradation of fatty acids and the formation of cholesterol oxides. Cholesterol oxides
Natural antioxidants
Bioactive compounds
increased from 39.53 ± 2.14 μg/g (raw sardines) to 116.04 ± 0.78 μg/g (control sardines) after canning.
However, lower levels were found in samples with pink pepper. Additionally, chromatographic analyses showed
the migration of compounds (phenolic acids, flavonoids, tannins, terpenes) from pink pepper to sardines, indi­
cating the constituents that could have contributed to its antioxidant properties. Thus, pink pepper may be
highlighted as a suitable additive to reduce the intake of cholesterol oxides, minimizing the loss of nutritional
quality in canned fish.

1. Introduction in the world economy due to the expressive amount captured. It is

widely used by Brazilian canning industries and the national supply has
The lack of time to prepare meals has resulted in a growing demand struggled to follow the growing expansion of canned products (FAO,
for ready-to-eat products such as canned sardines (Barbosa, Trigo, 2018). Thus, in 2019, the Brazilian government reduced the import
Campos, & Aubourg, 2019; Gómez-Limia, Cobas, Franco, & Martínez- tariffs to protect the local species and attend to market needs (MAPA,
Suárez, 2020). Fish and fish products are valuable sources of n-3 poly­ 2020).
unsaturated fatty acids (PUFAs), mainly eicosapentaenoic (EPA, C20:5) Canned sardines present the advantages of preservation and conve­
and docosahexaenoic acids (DHA, C22:6), which present several bio­ nience; however, canning involves industrial steps (e.g. chilling,
logical activities (Djuricic & Calder, 2021). Therefore, canning is a freezing, cooking, sterilization, storage) that may degrade essential fatty
preservative technique that allows increased access to fish, supporting a acids and cause the formation of harmful compounds, such as choles­
crucial role in human nutrition (Barbosa et al., 2019; Gómez-Limia et al., terol oxides (Dantas et al., 2021).
2020). Besides PUFAs, sardines also contain high levels of cholesterol,
Sardina pilchardus, commonly known as European sardine, stands out which is prone to oxidation when exposed to factors such as heating (de

* Corresponding author at: Department of Nutrition, School of Public Health, University of São Paulo (USP), Ave. Dr. Arnaldo, 715, São Paulo, SP 01246–904,
Brazil.
E-mail address: eatorres@usp.br (E.A.F.S. Torres).

https://doi.org/10.1016/j.foodchem.2022.134347
Received 28 January 2022; Received in revised form 8 September 2022; Accepted 17 September 2022
Available online 20 September 2022
0308-8146/© 2022 Elsevier Ltd. All rights reserved.
C.F.T. Barreira et al. Food Chemistry 403 (2023) 134347

Carvalho et al., 2021; de Oliveira et al., 2020a). Since canned sardines (São Paulo, Brazil). Undecanoic methyl ester was purchased from Sigma
are submitted to high temperatures during cooking and sterilization, Chemical Co. (St. Louis, MO, USA) and the standard fatty acid mixtures
they may be considered sources of exogenous cholesterol oxidation were from Supelco TM 37 (FAME Mix 18919, Bellefonte, Pa., USA).
products (COPs) (Dantas et al., 2021), which have been related to the Cholesterol and cholesterol oxides were achieved from Sigma-Aldrich
development of several diseases, as a consequence of their cytotoxic, (St. Louis, MO, USA). Solvents used during the chromatographic ana­
atherogenic, neurodegenerative, inflammatory, and carcinogenic effects lyses were obtained as follows: formic acid (Synth, São Paulo, Brazil),
(Garcia-Llatas, Mercatante, López-García, & Rodríguez-Estrada, 2021; hexane (Scharlau, Barcelone, Spain), methanol, and 2-propanol (Merck,
Liu et al., 2022). Darmstadt, Germany).
Therefore, strategies to control COPs formation during canned fish
processing are of great importance. Synthetic antioxidants are widely 2.2. Plant material
applied by the food industry; however, their intake has become a public
health concern due to their toxic and carcinogenic effects. As a result, Fresh pink pepper samples were donated by the Institute of
increasing interest in natural sources of antioxidant compounds has Agronomy of the University Federal Rural of Rio de Janeiro (UFRRJ),
been reported (de Carvalho et al., 2021; de Oliveira et al., 2020a, Seropédica, Rio de Janeiro, Brazil, in May 2020. The genotype used was
2020b). the UFRRJ Aro200-F, which is characterized as a female plant of the
Gómez-Limia et al. (2021) investigated the effects of sunflower oil, species Schinus terebinthifolius Raddi selected for fruit production. The
olive oil, and spiced olive oil on the oxidation processes and antioxidant selection was performed by the Genetic Improvement Program of the
capacity of canned eels, revealing the greater protection of sunflower Laboratory of Cytogenetics and Molecular Biology of Plants of the
and spiced olive oil against lipid oxidation after one year of storage. Department of Plant Science at UFRRJ.
Aqueous algae extracts (Barbosa et al., 2019) and waste liquor obtained The ripe fruit were manually harvested, selected, washed, and dried
from octopus (Octopus vulgaris) cooking (Malga, Trigo, Martínez, & in a ventilated oven (Solab, São Paulo, Brazil) at 40 ◦ C for 48 h to remove
Aubourg, 2022) preserved Atlantic Chub mackerel (Scomber colias) the excess moisture. After drying, they were stored in glass containers,
against lipid oxidation during the canning process. which were placed in a desiccator protected from light at room tem­
Pink pepper (Schinus terebinthifolius Raddi) fruit has emerged as a perature (25 ◦ C), until analyses or their immediate use by the food
sophisticated condiment. Additionally, studies have described its anti­ industry.
oxidant, antidiabetic, anti-inflammatory, and antimicrobial properties
(de Oliveira et al., 2020b; Gomes et al., 2020). Apart from presenting 2.3. Pink pepper analyses
health-promoting effects, pink pepper contains antioxidant compounds
(e.g. phenolic acids, flavonoids, anthocyanins, tannins) that may hinder 2.3.1. Preparation of extract
lipid oxidation, showing its potential to act as a natural antioxidant (de Ground pink pepper fruit (3 g) were added to 20 mL of an ethanol/
Oliveira et al. 2020a, 2020b; Feuereisen, Zimmermann, Schulze- water solution (70:30%, v/v). The mixture was sonicated in a sonic bath
Kaysers, & Schieber, 2017; Gomes et al., 2020). (40 kHz) (Elmasonic P, Elma Schmidbauer GmbH, Singen, Germany) for
A recent study performed by Dantas et al. (2021) determined the 30 min and centrifuged (NI 1813, Nova Instruments, São Paulo, Brazil)
content of cholesterol oxides in commercial samples of canned tuna from at 18,000g for 5 min. The procedures were conducted at room temper­
the Brazilian market, revealing canned fish as a potential source of di­ ature (25 ◦ C). Then, the supernatant was transferred to a volumetric
etary COPs. Although canned sardines are among the most consumed amber flask of 50 mL and made up to volume with the extracting
fishery products worldwide, there are no available studies on the use of solution.
natural antioxidants to minimize oxidative processes in these foodstuffs,
regarding COPs formation and natural sources with great sensory po­ 2.3.2. In vitro antioxidant capacity
tential such as pink pepper. DPPH, oxygen radical absorbance capacity (ORAC), and β-Carotene/
As pink pepper has shown promising antioxidant potential and one of linoleic acid assays were carried out as described by de Oliveira et al.
the main challenges of the food industry is to meet consumers’ demand (2020b). For DPPH, the absorbance was measured at 517 nm and the
for more natural products with high nutritional value, quality and DPPH radical scavenging activity was calculated as follows: %I DPPH =
safety, the objective of this study was to evaluate the effectiveness of (A0 − A)/A0) × 100 (Eq. 1), where A0 and A are the absorbances of the
adding pink pepper as an alternative strategy to minimize the degra­ control (DPPH) and sample, respectively.
dation of fatty acids and the formation of cholesterol oxides during The ORAC assay was performed using Trolox as a control standard
canned sardine processing. Therefore, pink pepper fruit (0.25, 0.5, 0.75, and a curve of relative fluorescence intensity to calculate the results,
and 1.0%) were added to the commercial liquid medium (soybean oil) which were expressed as µM Trolox Equivalent (TE)/g.
during canning. Fatty acids degradation and cholesterol oxides forma­ Regarding the β-Carotene/linoleic acid assay, readings were
tion were considered. Furthermore, ultra-high performance liquid measured every 15 min at 50 ◦ C at 470 nm. BHT was used as control. The
chromatography/electrospray ionization mass spectrometry (UHPLC- percentage of oxidation inhibition (%I) was calculated as shown in the
ESI-MS) analyses were carried out to determine the possible migration of following equations (2, 3, and 4), where Ac and As are the absorbances
bioactive compounds present in pink pepper to the fish muscle and of the control and samples, respectively:
liquid medium, indicating pink pepper’s constituents that may have
presented antioxidant properties. Ac = Initial absorbance − final absorbance

As = Initial absorbance − final absorbance

2. Materials and methods
%I = (Ac − − As/ Ac) × 100 (4)
2.1. Standards, reagents, and solvents
The procedures reported by Thaipong, Boonprakob, Crosby,
TPTZ (2,4,6-tris(2-pyridyl)-s-triazine), DPPH (1,1-diphenyl-2-pic­ Cisneros-Zevallos, and Byrne (2006) were applied for ferric reducing
rylhydrazyl), Trolox, linoleic acid, Tween-40, β-carotene, BHT (butyl­ antioxidant power (FRAP) assay. The FRAP reagent was prepared by
ated hydroxytoluene), and sodium methoxide were obtained from mixing 300 mM sodium acetate buffer (pH 3.6), 10 mM TPTZ (2,4,6-tris
Sigma-Aldrich (St. Louis, MO, USA). AAPH (2,2′ -azobis (2-amidinopro­ (2-pyridyl)-s-triazine) solution in 40 mM hydrochloric acid, and 20 mM
pane) dihydrochloride) and fluorescein were acquired from Merck ferric chloride (10:1:1, v/v/v). The absorbance was measured at 595 nm
(Darmstadt, Germany). All other chemicals were acquired from Vetec and the results were expressed as µmol Trolox equivalent (TE)/g.

2
C.F.T. Barreira et al. Food Chemistry 403 (2023) 134347

2.4. Sample preparation and canning process CP-SIL 88 (100 mx 0.25 mm id, thickness of 0.20 μm film) (Chrompack,
Middelburg, Netherlands) and flame ionization detector. Chromato­
The experiment was performed using approximately 10 kg of sar­ graphic conditions were as described by de Oliveira, et al. (2020a), using
dines (Sardina pilchardus) donated by Nova Piracema (São Gonçalo, Rio hydrogen as the carrier gas (1 mL/min) and nitrogen as the make-up gas
de Janeiro, Brazil) in July 2020. Regarding pink pepper (PP), the whole (30 mL / min). Regarding the qualitative analysis, the identification of
fruit, which were previously dried as described in Section 2.2, were chromatographic peaks of samples was determined by comparison of the
added directly to the liquid medium (soybean oil). The canned sardines retention times of FAME (fatty acid methyl ester) standards (Supelco TM
were produced by the Nova Piracema industry according to five treat­ 37, FAME Mix 18919, Bellefonte, Pa., USA). Quantification was per­
ments. The control samples (also named 0.0% PP) include canned formed by internal standardization with the undecanoic methyl ester as
samples with no addition of antioxidants (cans containing only the standard.
sardine and the liquid medium). For pink pepper addition, four treat­
ments were considered, where the fruit were added at concentrations of 2.5.3. Determination of cholesterol and cholesterol oxides of sardines and
0.25, 0.5, 0.75, and 1.0% (named 0.25% PP, 0.5% PP, 0.75% PP, and liquid medium
1.0% PP, respectively). The addition of pink pepper was calculated ac­ Cholesterol and cholesterol oxides were obtained simultaneously by
cording to the average net weight of canned sardines (125 ± 0.5 g), direct saponification of samples. The method described by de Oliveira
including fish muscle and liquid medium. et al. (2020a) was used for the liquid medium, while analyses of sardines
Since there are no studies in the literature reporting the addition of were conducted according to de Carvalho et al. (2021).
fruit such as pink pepper in canned fish, these percentages were deter­ Identification and quantification were performed by High-
mined according to de Oliveira et al. (2020a), who reported the effec­ Performance Liquid Chromatography-Mass Spectrometry on a HPLC-
tiveness of pink pepper (0.2 and 0.5%) as a natural antioxidant in a MS (Shimadzu LCMS-2020, Tokyo, Japan), equipped with an Hypersil
model system containing sardine oil during heating. Cyano column (250 mm × 4.6 mm) (Thermo Scientific, Massachusetts,
The sardine samples (84 ± 0.5 g) were manually placed in rectan­ USA) and a Photodiode Array Detector (PDA) (SPD-M20A, Shimadzu,
gular cans and, then, pink pepper fruit were added according to the Tokyo, Japan). The analyses were performed following the conditions
percentage determined for each treatment. The cans were directed to the established by de Oliveira et al. (2020a): pump flow of 1 mL/min, col­
production line and received the liquid medium (41 ± 0.5 g soybean umn temperature at 35 ◦ C, n-hexane:2-propanol (97:3, v/v) as the
oil). Then, they were hermetically sealed, washed and sterilized (com­ mobile-phase. MS detection was carried out in the positive ion mode
mercial sterilization at 126 ◦ C for 40 min, f0 value = ≥ 16 min). Once the with Atmospheric Pressure Chemical Ionization (APCI) and the chro­
heating time was completed, the cans were cooled to achieve a final matograms were obtained in the selective ion monitoring (SIM) mode.
temperature varying from 25 to 30 ◦ C. Cholesterol and cholesterol oxides were determined by comparison of
The canned sardines were produced following the standard proced­ retention times of peaks in samples with those of reference standards
ures applied by the industry, where the cans are submitted to a step and by m/z. External standardization using standard calibration curves
called quarantine. During the quarantine, the canned sardines are stored was used for quantification.
for 10 days at 37 ◦ C, with a daily check of cans, and only samples that do
not present defects can be destined to the consumer market at the end of 2.6. Identification of bioactive compounds by UHPLC-ESI-MS analyses
the quarantine period. Thus, after the quarantine (10 days), the cans
were sent to UFRRJ for analysis, which started after two days of storage Chromatographic analyses of pink pepper, sardines, and liquid me­
at room temperature (25 ± 1 ◦ C). diums were conducted with an UHPLC Acquity chromatograph (Waters,
Fifteen cans were produced for each treatment (control, 0.25% PP, Milford, MA, USA) coupled with a triple quadrupole (TQD) Acquity mass
0.5% PP, 0.75% PP, and 1.0% PP), totaling seventy-five samples, and spectrometer (Micromass-Waters), with Electrospray Ionization (ESI) in
twelve cans of each treatment were used. The cans were opened and the the negative and positive ion modes. The analyses were performed
liquid part was carefully drained off gravimetrically. Then, the liquid following the conditions reported by de Oliveira et al. (2020a), with data
mediums obtained from these cans were mixed, filtered through filter acquisition between m/z 100 and 900. MS/MS of selected peaks were
paper and stored in sterile amber flasks at 4 ◦ C until analyses. The sar­ acquired via collision-induced dissociation (CID) with collision energy
dines were ground using in a domestic processor (Cadence, São Paulo, of 30 V. For chromatographic separation, a C18 BEH Waters Acquity
Brazil) to obtain a single and homogenous mass and lyophilized (Model (2.1 mm × 50 mm × 1.7 μm) column at 30 ◦ C was used. Mobile phases A
L101, Liotop, São Paulo, Brazil) to obtain the results on dry basis. These (0.1% formic acid) and B (methanol) were applied (0.2 mL/min) with a
procedures were carried out separately for each treatment. linear gradient starting at 5% B and increasing to 100% methanol in 7.5
Additionally, one lot containing twelve sardines (previously evis­ min, before holding until 9 min, then returning to the initial conditions,
cerated, without head and tail, as used in the industry) was transported followed by column re-equilibration until 10 min. Compounds were
in a refrigerated truck to UFRRJ. Once at the laboratory, the sardines putatively identified by comparison of their precursor and product ions
were also ground and lyophilized. Then, convenient aliquots were taken with data from literature.
for analyses, corresponding to raw samples. Moreover, aliquots of the
liquid medium before canning were also taken for analysis. All analyses 2.7. Statistical analysis
were performed in triplicate.
The experiment was conducted using a 2 × 5 factorial design: factor
2.5. Canned fish and liquid medium analyses A was the type of matrix (a1: sardine and a2: liquid medium) and factor
B was the percentage of pink pepper addition (b1: 0.0%; b2: 0.25%; b3:
2.5.1. Moisture and total lipids of sardines 0.5%; b4: 0.75%, and b5: 1.0%). Besides, there were two additional
Moisture and total lipids were determined according to AOAC (2006) treatments, which were the liquid medium and sardine without any
following methods 950.46 and 991.36, respectively. addition or treatment (before canning).
The two-way ANOVA (Analysis of Variance) test was used, followed
2.5.2. Determination of fatty acids of sardines and liquid medium by the multiple mean comparison test of Tukey, where significant dif­
Lipids were converted into methyl esters by transesterification with ferences were detected. The Dunnett test was used to compare: 1) con­
sodium methoxide (de Oliveira et al., 2020a). Then, the methyl esters trol liquid medium samples against liquid medium samples treated with
were analyzed using a gas chromatograph (Shimadzu GC 2010, Tokyo, pink pepper, 2) control sardine samples against sardines treated with
Japan) with injector in split mode (1:50), capillary column of fused silica pink pepper, 3) canned liquid medium samples against liquid medium

3
C.F.T. Barreira et al. Food Chemistry 403 (2023) 134347

samples evaluated before canning, and 4) canned sardine samples 3.2. Bioactive compounds from pink pepper extract determined by
against sardines evaluated before canning (raw). Principal Component UHPLC-ESI-MS
Analysis (PCA) and Hierarchical Clustering of Principal Components
(HCPC) were used for multivariate statistics. PCA was performed after Table 1 presents the proposed identification of bioactive compounds
data standardization to avoid the influence of different units and orders from pink pepper extract. Twelve compounds were identified, including
of magnitude. The HCPC was carried out to confirm the sample groups organic acids, phenolic acids, flavonoids, anthocyanins, gallotannins,
suggested by PCA. Finally, Pearson’s correlation test was applied to and terpenes. Most of them were detected in the negative ion mode;
evaluate fatty acids, cholesterol and COPs correlations according to however, three compounds were found in the positive ion mode (Fig. 1).
Teles et al. (2019). In the negative ion mode, organic acids such as malic and citric acids
were determined as m/z 133 and 191, respectively (Fernández-Fernán­
3. Results and discussion dez et al., 2010). Representing the class of phenolic acids, gallic acid (m/
z 169) was found in pink pepper extract, as reported by Gomes et al.
3.1. In vitro antioxidant capacity of pink pepper extract (2020). A gallotannin was detected as m/z 539, in agreement with other
authors (de Oliveira et al., 2020a, 2020b; Feuereisen et al., 2017).
The DPPH method showed inhibition of 95.03 ± 0.07%, which is in The flavonoids agathisflavone (m/z 537) and tetrahydroamento­
accordance with a study carried out by Bernardes et al. (2014) (95.60%). flavone (m/z 541) were also identified in pink pepper extract by their
In contrast, a lower percentage was found by de Oliveira et al. (2020b) MS/MS fragmentation in comparison to the literature (de Oliveira et al.,
(42.68%). 2020a, 2020b; Gomes et al., 2020). In addition, a triterpene acid was
The ORAC value was 77.01 ± 3.70 μM TE/g. Values ranging from found with m/z 469, as well as the terpenes masticadienoic acid (m/z
43.40 μM TE/g (de Oliveira et al., 2020b) to 158.24 μM TE/g (Serrano- 453) and 3α-hydroxymasticadienolic (schinol, m/z 455) (de Oliveira
León et al., 2018) were reported. Regarding the β-carotene/linoleic acid et al., 2020a; Gomes et al., 2020).
assay, 11.46% of oxidative inhibition was determined, which was lower Anthocyanins like cyanidin (m/z 287) and 7-O-methylpelargonidin
than the result described by de Oliveira et al. (2020b) (61.41%). 3-O-galactoside (m/z 447) were determined in the positive ion mode
The FRAP assay showed 58.51 ± 1.33 μmol TE/g, while Oliveira (de Oliveira et al., 2020a, 2020b; Feuereisen et al., 2017; Koh, Youn, &
et al. (2020) determined 488.60 μmol TE/g. Pink pepper extracts eval­ Kim, 2014), while the biflavonoid hinokiflavone was detected as m/z
uated by other authors also showed antioxidant potential by the FRAP 539 (de Oliveira et al., 2020a, 2020b; Feuereisen et al., 2017).
assay; however, the results were expressed in different units, making it The bioactive compounds putatively identified in pink pepper
difficult to compare (Russo, Balistreri, Tapanes-Castillo, & Pina, 2017). extract highlight this fruit not only as a flavor enhancer but also as a
Plant extracts contain a wide range of bioactive compounds with health-promoting food additive. Biflavonoids, such as agathisflavone,
different chemical structures and antioxidant characteristics. The use of tetrahydroamentoflavone, and hinokiflavone, have shown potential as
a single assay does not provide accurate results. Thus, methods based on therapeutic agents against microbial diseases (e.g. influenza, hepatitis)
distinct mechanisms were performed for complementary insights (Gul­ (Menezes & Campos, 2021). Santos et al. (2020) reported the neuro­
cin, 2020). Although the antioxidant capacity of pink pepper is well protection of agathisflavone in a Parkinson’s disease study model. The
documented, it is worth characterizing each studied sample since the role of terpenes in treating cardiovascular and metabolic diseases was
level of antioxidant compounds is highly influenced by factors such as summarized in a recent review (Oliveira, Ribeiro, Rezende, & Fraga-
origin, harvest time, genotype, climatic conditions, maturity and others, Silva, 2021).
affecting the extract‘s antioxidant potential. Moreover, the extraction The biological activities presented above are mainly related to the
effectiveness is directly impacted by the parameters and conditions used antioxidant properties of these compounds. Besides providing extra
(e.g. temperature, solvent type, solvent ratio, extracting time, method) nutritional value, the use of pink pepper as a condiment may also
(Gulcin, 2020; Russo et al., 2017). represent an alternative to control lipid oxidation in food (de Oliveira
et al., 2020a, 2020b; Serrano-León et al., 2018). Pink pepper contains
antioxidant constituents, such as phenolic compounds, which are

Table 1
Proposed identification of bioactive compounds of pink pepper extract by UHPLC-ESI-MS.
ESI ion RT Precursor ion (m/ Main fragments Proposed identification Reference
mode (min) z) (m/z)

– 0.803 133 132.7; 131.8; 71.0 Malic acid Fernández-Fernández et al. (2010)
– 0.812 169 168.9; 125.0 Gallic acid Gomes et al. (2020)
– 0.878 191 191; 111.2; 87.0; 85.1 Citric acid Fernández-Fernández et al. (2010)
+ 0.991 447 447; 285.2 7-O-methylpelargonidin 3-O- De Oliveira et al. (2020a, 2020b)
galactoside Feuereisen et al. (2017)
+ 1.172 287 287 Cyanidin Koh, Youn, & Kim (2014)
+ 2.315 539 539 Hinokiflavone De Oliveira et al. (2020a, 2020b)
Feuereisen et al. (2017)
– 2.534 539 539; 413.2; 387.4; 386.5; 319.2; Gallotannin De Oliveira et al. (2020a, 2020b)
150.9 Feuereisen et al. (2017)
– 2.630 541 415.1; 389.2 Tetrahydroamentoflavone De Oliveira et al. (2020a, 2020b)
Gomes et al. (2020)
– 5.492 537 537 Agathisflavone De Oliveira et al. (2020a, 2020b)
Gomes et al. (2020)
– 5.711 469 469 Triterpene acid De Oliveira et al. (2020a)Gomes et al.
(2020)
– 7.351 455 455 3α-hydroxymasticadienolic De Oliveira et al. (2020a)Gomes et al.
(schinol) (2020)
– 7.895 453 453 Masticadienoic acid De Oliveira et al. (2020a)Gomes et al.
(2020)

*RT = retention time.

4
C.F.T. Barreira et al. Food Chemistry 403 (2023) 134347

capable of donating hydrogen atoms to lipid radicals that initiate

oxidative processes (Gulcin, 2020).
Farhoosh and Nyström (2018) evaluated the antioxidant effect of
gallic acid in sunflower oil showing that this natural phenolic compound
was more effective than the synthetic antioxidant TBHQ (tert-butylhy­
droquinone) in preventing oxidation at a high temperature (120 ◦ C).
Gallic acid enhanced the antioxidant activity of chitosan films in a study
performed by Zarandona, Puertas, Dueñas, Guerrero, and de la Caba
(2020), suggesting their suitability as active food packing. Therefore,
regarding the increasing relevance of pink pepper to the food industry as
a functional ingredient and natural antioxidant, its effects against the
impacts of canning on the lipid fraction of sardines were investigated.

3.3. Sardines

3.3.1. Moisture and total lipid contents of canned sardine samples

The moisture level of raw sardines was 68.73 ± 0.43 g/100 g.
However, a lower content (p < 0.05) of 62.73 ± 0.61 g/100 g was
assessed in canned control samples. The addition of pink pepper to
canned sardines did not promote changes in the moisture level for most
samples (p > 0.05), with values ranging from 62.49 ± 0.16 to 63.99 ±
0.22 g/100 g (Supplementary material Table 1). It indicates that mois­
ture was only affected by the canning process (p < 0.05), which can be
explained by the denaturation and decrease of water-holding capacity of
fish proteins due to the thermal treatment, resulting in water loss from
the fish muscle to the liquid medium (Gómez-Limia et al., 2020).
Water loss due to canning was also determined by Domiszewski
(2021) in mackerel (Scomber scombrus), herring (Clupea harengus) and
sprat (Sprattus sprattus). The authors evaluated fish conserved in tomato
sauce and oil, showing that the decrease of water content depends on the
fish species, with reductions ranging from 8.41% (mackerel) to 15.14%
(sprat). According to Malga et al. (2022), the canning process also
reduced the moisture content of Scomber colias.
Regarding lipid contents, 16.84 ± 0.72 and 16.23 ± 0.70 g /100 g
(dry basis) were found in raw and control samples, respectively. In
samples with pink pepper, the contents varied from 15.53 ± 0.54 to
17.08 ± 0.63 g/100 g (Supplementary material Table 1). These results
showed that canning did not significantly affect the lipid contents (p >
0.05).
Previous studies have demonstrated higher levels of lipids in fish
canned with oil compared to fresh fish, which is attributed to the oil
absorption by the fish muscle (Dantas et al., 2021; Domiszewski, 2021;
Gómez-Limia et al., 2020). However, this trend was not observed in the
present study. Although sardine samples from the same lot were used
during the experiment, the fish composition is influenced by factors such
as environmental conditions, feeding, age, and sex (Bandarra, Marçalo,
Cordeiro, & Pousão-Ferreira, 2018). In addition, variable findings were
reported by Domiszewski (2021), who found lower and higher contents
of lipids in herring and mackerel in oil after canning, respectively, while
lipid contents did not change for canned sprat (p > 0.05).

3.3.2. Fatty acid composition of canned sardines and liquid mediums

The main fatty acids found in raw sardines were: palmitic (C16:0,
20.58 ± 1.98 g/100 g oil), oleic (C18:1n9c, 17.88 ± 0.88 g/100 g oil),
eicosapentaenoic (EPA, C20:5n3, 11.95 ± 0.28 g/100 g oil), palmitoleic
(C16:1, 11.30 ± 0.81 g/100 g oil), and myristic acids (C14:0, 8.91 ±
0.59 g/100 g oil) (Table 2), which is in agreement with the fatty acid
profile of sardines described by Bandarra et al. (2018) and de Carvalho
et al. (2021). Regarding the groups of fatty acids, the sum fatty acid
contents increased in the following order: polyunsaturated (PUFA)
(31.85 ± 0.48 g/100 g oil) < monounsaturated (MUFA) (32.14 ± 1.44
Fig. 1. MS/MS spectra in the negative and positive ion mode of compounds
g/100 g oil) < saturated (SFA) (37.33 ± 2.90 g/100 g oil). Additionally,
identified in pink pepper extract. (For interpretation of the references to color
in this figure legend, the reader is referred to the web version of this article.) the sum of EPA and DHA was 18.37 ± 0.42 g/100 g oil.
The liquid medium evaluated before canning presented linoleic
(C18:2n6c, 48.07 ± 0.69 g/100 g oil), oleic (C18:1n9c, 27.61 ± 0.02 g/
100 g oil) and palmitic acids (C16:0, 9.53 ± 0.41 g/100 g oil) as the

5
C.F.T. Barreira et al. Food Chemistry 403 (2023) 134347

Table 2 Table 2 (continued )

Fatty acid composition (g/100 g oil) of sardine and liquid medium samples Sardines
before and after canning (control and with pink pepper at 0.25, 0.5, 0.75, and
1.0%). Before After canning
canning
Sardines
Fatty Raw Control 0.25% 0.5% PP 0.75% 1.0% PP
Before After canning acid (0.0% PP PP
canning PP)
Fatty Raw Control 0.25% 0.5% PP 0.75% 1.0% PP 15.92 16.34
acid (0.0% PP PP ± ± 0.48A;
PP) 0.70a;β a;β
∑
C14:0 8.91 ± 5.02 ± 5.14 ± 4.99 ± 5.72 ± 5.85 ± ω6 8.22 ± 22.82 22.36 23.59 ± 23.21 ± 23.43 ±
0.59 0.30a;β 0.10B;a;β 0.17B;a;β 0.09A; 0.31A; 0.43 ± ± 0.14A; 0.38A;b;β 0.32A;b;β 0.44A;b;β
a;β;α a;β;α 0.80a;β b;β

C16:0 20.58 14.35 15.90 15.85 ± 15.86 ± 16.18 ± ω3/ω6 2.88 ± 0.70 ± 0.73 ± 0.68 ± 0.7 ± 0.71 ±
± 1.98 ± ± 0.23A; 1.01A;a;β 1.09A;a;β 0.41A;a;β 0.15 0.05a;β 0.02A;a;β 0.05A;a;β 0.04A;a;β 0.04A;a;β
∑
1.00a;β a;β Trans – 0.77 ± 0.83 ± 0.75 ± 0.57 ± 0.52 ±
C16:1cis 11.30 4.43 ± 4.82 ± 4.49 ± 6.17 ± 5.70 ± 0.05a 0.05A;a 0.05A;a 0.09B;a;α 0.04B;a;α
± 0.81 0.59a;β 0.09BC; 0.44C;a;β 0.22A; 0.40AB; EPA + 18.37 12.05 12.55 13.23 ± 13.70 ± 14.07 ±
a;β a;β;α a;β;α DHA ± 0.42 ± ± 0.22A; 1.17A;a;β 1.00A;a;β 1.16A;a;β
C18:0 5.88 ± 4.20 ± 4.19 ± 3.63 ± 4.02 ± 4.06 ± 0.76a;β a;β

0.51 0.76a;β 0.38A;a;β 0.46A;a;β 0.63A;a;β 0.51A;a;β

C18:1n9t – 0.31 ± 0.32 ± 0.38 ± 0.37 ± 0.34 ± Liquid medium
0.02A 0.01A 0.03A 0.07A 0.05A
C18:1n9c 17.88 18.69 18.97 19.59 ± 19.83 ± 20.11 ± Before After canning
± 0.88 ± 1.26b ± 0.45A; 0.81A;b 1.56A;b 0.38A;b canning
b
Fatty Raw Control 0.25% 0.5% PP 0.75% 1.0% PP
C18:2n6t – 0.46 ± 0.51 ± 0.37 ± 0.20 ± 0.18 ± acid (0.0% PP PP
0.03a 0.05A;a 0.03B;a;α 0.04C;b;α 0.03C;b;α PP)
C18:2n6c 4.80 ± 20.93 20.50 22.06 ± 21.97 ± 22.00 ±
0.47 ± ± 0.29B; 0.19A; 0.27A;b;β 0.48A;b;β C14:0 – 0.36 ± 0.46 ± 0.66 ± 1.06 ± 1.71 ±
0.80b;β b;β b;α;β 0.03b 0.06CD;b 0.12C;b;γ 0.18B;b;γ 0.07A;b;γ
C18:3n6 0.56 ± 0.20 ± 0.22 ± 0.22 ± 0.24 ± 0.24 ± C16:0 9.53 ± 10.25 10.46 10.95 ± 11.32 ± 12.18 ±
0.04 0.03a;β 0.00A;b;β 0.02A;b;β 0.01A; 0.02A; 0.41 ± 0.62b ± 0.07B; 0.91AB; 0.81AB; 0.02A;
b;β;α b;β;α b b;δ b;δ b;δ;γ

C20:1n9 1.84 ± 0.47 ± 0.49 ± 0.89 ± 1.19 ± 1.24 ± C16:1 cis – 0.55 ± 0.55 ± 0.79 ± 1.64 ± 2.33 ±
0.09 0.04a;β 0.02C;a;β 0.04B; 0.01A; 0.09A; 0.01b 0.06D;b 0.07C;b;γ 0.13B;b;γ 0.05A;b;γ
a;β;α a;β;α a;β;α C18:0 4.25 ± 3.37 ± 3.44 ± 3.45 ± 3.60 ± 3.74 ±
C18:3n3 2.82 ± 2.39 ± 2.26 ± 2.1 ± 2.29 ± 2.30 ± 0.73 0.13a 0.46A;a 0.08A;a 0.32A;a 0.03A;a
0.03 0.21b 0.28A;b 0.12A;b;β 0.24A;b;β 0.11A;b;β C18:1 n9t – – – – – –
C20:2n6 0.36 ± 0.15 ± 0.17 ± 0.22 ± 0.24 ± 0.17 ± C18:1 27.61 20.94 22.55 22.60 ± 22.74 ± 22.16 ±
0.00 0.03β 0.03AB;β 0.05AB;β 0.01A;β;α 0.02AB;β n9c ± 0.02 ± ± 0.34AB; 0.10A; 0.56AB;
C20:3n6 1.70 ± 0.75 ± 0.63 ± 0.23 ± 0.23 ± 0.39 ± 0.61a;δ 1.08AB; a;δ;γ a;δ;γ a;δ
a;δ;γ
0.24 0.06β 0.11A;β 0.03B;β;α 0.04B;β;α 0.06B;β;α
C22:1n9 0.69 ± 0.36 ± 0.33 ± 0.39 ± 0.35 ± 0.38 ± C18:2 n6t – 0.42 ± 0.40 ± 0.33 ± 0.30 ± 0.28 ±
0.00 0.02a;β 0.06A;a;β 0.08A;a;β 0.04A;a;β 0.08A;a;β 0.06a 0.03AB;b 0.05AB;a 0.04B;a;γ 0.02B;a;γ
C20:3n3 2.45 ± 1.48 ± 1.53 ± 0.83 ± 0.26 ± 0.38 ± C18:2 48.07 30.23 39.62 40.65 ± 38.16 ± 38.73 ±
0.19 0.09a;β 0.01A;a;β 0.04B; 0.03C; 0.07C; n6c ± 0.69 ± ± 2.22A; 2.20A; 2.16AB; 1.14A;
a;β;α b;β;α b;β;α 5.66a;δ a;δ;γ a;δ;γ a;δ;γ a;δ;γ

C20:4n6 0.81 ± 0.33 ± 0.33 ± 0.49 ± 0.34 ± 0.46 ± C18:3 n6 – 0.28 ± 0.31 ± 0.29 ± 0.32 ± 0.31 ±
0.07 0.06a;β 0.07B;b;β 0.08A; 0.03B;b;β 0.05AB; 0.04a 0.01A;a 0.01A;a 0.04A;a 0.02A;a
a;β;α a;β;α C20:1n9 – 0.45 ± 0.50 ± 0.61 ± 0.60 ± 0.60 ±
C24:0 1.96 ± 0.82 ± 0.81 ± 0.86 ± 0.88 ± 0.79 ± 0.00a 0.04B;a 0.04A;b;γ 0.03A;b;γ 0.01A;b;γ
0.06 0.01a;β 0.03A;a;β 0.05A;a;β 0.10A;a;β 0.10A;b;β C18:3 n3 3.19 ± 4.43 ± 4.65 ± 4.63 ± 4.86 ± 4.85 ±
C20:5n3 11.95 8.06 ± 8.51 ± 9.18 ± 9.63 ± 9.98 ± 0.17 0.15a; δ 0.27A;a; 0.09A;a; δ 0.17A;a;γ; 0.00A;a;γ;
± 0.28 0.76a;β 0.27A;a;β 0.98A;a;β 1.04A;a;β 0.85A;a;β
δ δ δ

C24:1n9 0.43 ± 0.17 ± 0.16 ± 0.33 ± 0.43 ± 0.43 ± C20:2n6 – – – – – –

0.04 0.01β 0.00B;β 0.04A;β;α 0.07A;α 0.07A;α C20:3n6 – – – – – –
C22:6n3 6.41 ± 3.98 ± 4.04 ± 4.05 ± 4.07 ± 4.09 ± C22:1n9 – 0.29 ± 0.30 ± 0.24 ± 0.28 ± 0.33 ±
0.34 0.25a;β 0.36A;a;β 0.20A;a;β 0.09A;a;β 0.45A;a;β 0.01b 0.05A;a 0.04A;b 0.03A;a 0.02A;a
∑
FA 80.75 12.05 12.55 13.23 ± 13.70 ± 14.07 ± C20:3n3 – 0.41 ± 0.56 ± 0.69 ± 0.76 ± 1.02 ±
± 2.55 ± ± 0.22A; 1.17A;a;β 1.00A;a;β 1.16A;a;β 0.03b 0.07C;b;γ 0.01BC; 0.03B;a;γ 0.07A;a;γ
b;γ
0.76a;β a;β
∑
SFA 37.33 24.40 26.04 25.33 ± 26.49 ± 26.89 ± C20:4 n6 – 0.41 ± 0.45 ± 0.33 ± 0.51 ± 0.38 ±
± 2.90 ± ± 0.65A; 0.96A;a;β 1.18A;a;β 1.29A;a;β 0.04a 0.05AB;a 0.05B;b 0.05A;a 0.06AB;a
1.44a;β a;β C24:0 – 0.50 ± 0.59 ± 0.84 ± 1.02 ± 1.17 ±
0.07b 0.02C;b 0.09B;a;γ 0.02AB; 0.11A;a;γ
∑
MUFA 32.14 24.43 25.09 26.07 ± 28.35 ± 28.21 ±
a;γ
± 1.44 ± ± 0.40B; 1.20AB; 1.35A; 0.86A;
0.78a;β a;β a;β a;β;α a;β;α C20:5 n3 – 1.00 ± 1.23 ± 1.49 ± 1.93 ± 2.09 ±
0.06b 0.07BC;b 0.23B;b;γ 0.02A;b;γ 0.10A;b;γ
∑
PUFA 31.85 38.74 38.70 39.75 ± 39.46 ± 40.18 ±
± 0.48 ± ± 0.56A; 0.91A;b;β 0.80A;b;β 1.40A;b;β C24:1n9 –
0.77a;β b;β C22:6 n3 – 0.88 ± 1.07 ± 1.38 ± 1.38 ± 1.71 ±
0.05b 0.21BC;b 0.10B;b;γ 0.10B;b;γ 0.00A;b;γ
∑
UFA 64.00 63.17 63.80 65.82 ± 67.81 ± 68.39 ± ∑
± 1.82 ± 1.23a ± 0.78B; 1.94AB;b 0.55A;b;α 1.06A; FA 92.66 74.77 87.13 89.72 ± 90.49 ± 93.61 ±
b b;β;α ± 1.80 ± ± 3.08A; 2.76A;a;γ 1.77A;a;γ 0.88A;a;γ
5.64a;δ a;γ
∑
ω3 23.63 16.16 ± 16.25 ± 16.75 ± ∑
± 0.23 1.09A;a;β 0.82A;a;β 1.09A;a;β SFA
(continued on next page)

6
C.F.T. Barreira et al. Food Chemistry 403 (2023) 134347

Table 2 (continued ) during canning may accelerate the chain reaction of unsaturated fatty
Sardines acids during oxidation (Dantas et al., 2021; Mariutti & Bragagnolo,
2017).
Before After canning
canning
Domiszewski (2021) studied canned mackerel (Scomber scombrus)
and showed that the canning process carried out with oil resulted in
Fatty Raw Control 0.25% 0.5% PP 0.75% 1.0% PP
lower contents of SFAs and higher levels of PUFAs in fish muscle, while
acid (0.0% PP PP
PP) amounts of MUFAs were maintained. Malga et al. (2022) did not observe
changes in SFAs and MUFAs after canning when studying mackerel
13.79 14.48 14.94 15.89 ± 17.00 ± 18.80 ±
± 1.13 ± 0.61b ± 0.42C; 1.19BC; 0.68AB; 0.10A;
(Scomber colias) canned with distilled water. As shown above, variable
b b;δ b;δ;γ b;δ;γ findings have been reported concerning changes in fatty acids during
∑
MUFA 27.61 22.23 23.90 24.25 ± 25.26 ± 25.43 ± canning, which may be due to factors such as fish species, processing
± 0.02 ± ± 0.97A; 0.36A; 0.05A; 0.59A; parameters and liquid medium used. On the other hand, the content of
0.60b;δ a;δ;γ a;δ;γ b;δ;γ b;δ;γ
∑ PUFAs in control sardines increased after canning. Since linoleic acid
PUFA 51.26 38.06 48.29 49.58 ± 48.22 ± 49.38 ±
± 0.83 ± ± 2.32A; 2.21A;a;γ 2.26A;a;γ 1.09A;a;γ was the major contributor to the higher amount of PUFAs determined
5.55a;δ a;γ after canning, it indicates the absorption of fatty acids from the liquid
∑
UFA 78.88 60.29 72.19 73.83 ± 73.49 ± 74.81 ± medium by the fish muscle, as reported in previous studies (Dantas et al.,
± 0.82 ± ± 2.68A; 1.92A;a;γ 2.23A;a;γ 0.79A;a;γ 2021; Domiszewski, 2021; Gómez-Limia et al., 2020; Mesías, Holgado,
5.08a;δ a;δ;γ
∑
ω3 3.19 ± 6.72 ± 7.52 ± 7.97 ± 8.93 ± 9.67 ±
Sevenich, Briand, Márquez Ruiz, & Morales, 2015).
0.17 0.08b;δ 0.17C; 0.22C; 0.26B; 0.06A; Mesías et al. (2015) studied canned tunas conserved in different
∑
b;δ;γ b;δ;γ b;δ;γ b;δ;γ
liquid mediums (brine, sunflower oil, and olive oil) and assessed higher
ω6 48.07 31.34 40.77 41.61 ± 39.30 ± 39.71 ± levels of linoleic acid in tuna in sunflower oil, which presents linoleic
± 0.69 ± 2.20A; 2.12A;a;γ 2.09AB; 1.16A;
acid as its main fatty acid. Dantas et al. (2021) evaluated different
±
5.62a;δ a;δ;γ a;δ;γ a;δ;γ

ω3/ω6 0.07 ± 0.22 ± 0.185 0.19 ± 0.23 ± 0.24 ± brands of tuna canned in brine and soybean oil and found higher con­
0.00 0.04b;δ ± 0.01B; 0.01B;b;δ 0.01AB; 0.01A;b;δ tents of linoleic acid in tuna canned in oil (from 39.03 to 46.49 g/ 100 g
∑
b;δ b;δ
oil) than in brine (from 1.62 to 1.72 g/100 g oil), revealing the migration
trans – 0.42 ± 0.40 ± 0.33 ± 0.30 ± 0.28 ± of linoleic acid from the oil to the fish muscle.
0.06b 0.03AB;b 0.05AB;b 0.04B;b;γ 0.02B;b;γ
∑
EPA + – 1.88 ± 2.31 ± 2.66 ± 3.31 ± 3.80 ±
Regarding the effects of canning in the liquid medium, changes in the
DHA 0.11b 0.25C;b;γ 0.15C;b;γ 0.12B;b;γ 0.10A;b;γ levels of MUFAs and PUFAs were observed, which decreased by
approximately 19% and 26%, respectively (p < 0.05). This may be
*PP = pink pepper; FA = fatty acid; SFA = saturated fatty acid; MUFA =
attributed to both the degradation and migration of fatty acids. Canning
monounsaturated fatty acid; PUFA = polyunsaturated fatty acid; UFA = unsat­
urated fatty acid; EPA = eicosapentaenoic acid; DHA = docosahexaenoic acid.
reduced the amounts of MUFAs in sardines and liquid medium, indi­
Values represent means ± standard deviation in triplicates. Different capital cating a degradative processes. On the contrary, the content of PUFAs
letters in the same row indicate significant differences among samples with increased in sardines and decreased in the liquid medium, highlighting
different concentrations of pink pepper for the same type of material (sardine or the migration.
liquid medium) by the Tukey test. Different lowercase letters in the same column Fatty acids such as EPA and DHA, which are typical of fish species,
indicate significant differences among samples of distinct type of samples were detected in the liquid medium only after canning, which suggests
(sardine or liquid medium) at the same concentration of pink pepper, including their migration. Thus, the loss of sardine fatty acids after canning is not
control (0.0% PP), by the t Student test. “α” indicates significant differences in only due to oxidative processes but migration, as well. A similar trend
comparison with “Control” by the Dunnett test for sardine samples. “β” indicates was observed by Domiszewski (2021), who evidenced the migration of
significant differences in comparison with “Raw” by the Dunnett test for sardine
fish lipids by the EPA and DHA content of liquid mediums (oil and to­
samples. “γ” indicates significant differences in comparison with “Control” by
mato sauce) in canned fish, which could only come from fish muscle.
the Dunnett test for liquid medium samples. “δ” indicates significant differences
in comparison with “Raw” by the Dunnett test for liquid medium samples. Therefore, canning may compromise the contents of EPA and DHA in
canned fish, reducing its nutritional value since these essential fatty
acids are known for their potential to reduce the risks of various in­
predominant fatty acids, which were also found at high levels in soybean
flammatory, metabolic, and neurologic disorders (Djuricic & Calder,
oil by other authors (Kozłowska & Gruczyńska, 2018).
2021).
The canning process significantly modified the concentration of fatty
In samples with pink pepper, the levels of SFAs, MUFAs, and PUFAs
acids in control sardines (p < 0.05). Thus, the contents of SFAs and
in sardines varied as follows: from 25.33 ± 0.96 to 26.89 ± 1.29 g/100 g
MUFAs decreased to 24.40 ± 1.44 and 24.43 ± 0.78 g/100 g oil,
oil (SFAs), from 25.09 ± 0.40 to 28.35 ± 1.35 g/100 g oil (MUFAs), and
respectively, while the amount of PUFAs increased to 38.74 ± 0.77 g/
from 38.70 ± 0.56 to 40.18 ± 1.40 g/100 g oil (PUFAs). The results
100 g oil. Moreover, the sum of EPA and DHA reduced (p < 0.05) from
obtained for samples treated with pink pepper were not statistically
18.37 ± 0.42 (raw sardines) to 12.05 ± 0.76 g/100 g oil (control canned
different from those found in control samples for SFAs and PUFAs (p >
sardines). Previous studies have also reported lower contents of fish fatty
0.05). However, higher contents of MUFAs (p < 0.05) were found in
acids after canning (Dantas et al., 2021; Naseri, Rezaei, Moieni, Hos­
samples with 0.5, 0.75, and 1.0% pink pepper compared to control
seini, & Eskandari, 2011; Naseri & Rezaei, 2012). Reductions of 23.26%
samples. Regarding the sum of EPA and DHA, the addition of pink
(SFAs) and 24.49% (MUFAs) were observed in canned silver carp after
pepper did not affect the results compared to control samples (p > 0.05).
processing (Naseri et al., 2011). Naseri and Rezaei (2012) showed that
In liquid mediums treated with pink pepper, higher amounts of SFAs
canning decreased the sum of EPA and DHA from 24.84 (raw fish) to
(14.94 ± 0.42 – 18.80 ± 0.10 g/100 g oil), MUFAs (23.90 ± 0.97–25.43
13.01 g/100 g (fish canned in oil) in canned sprat (Clupeonella
± 0.59 g/100 g oil), and PUFAs (48.22 ± 2.26–49.58 ± 2.21 g/100 g oil)
cultriventris).
were found in comparison to control (p < 0.05). These results suggest
Results reported by Dantas et al. (2021) suggested that oxidative
the protective effect of pink pepper. For PUFAs, the level of linoleic acid,
reactions caused the degradation of fatty acids in canned tuna. In fact,
which was the most relevant PUFA, in the liquid medium of samples
lipid oxidation seems to be strongly linked to canning. Autoxidation
with pink pepper increased in comparison to control samples. However,
occurs via a free radical mechanism, which initiates with hydrogen
the concentration of linoleic acid did not reduce in sardines, showing
abstraction and free radical formation. Thus, since heating reduces the
that the addition of pink pepper did not influence the migration. Thus,
activation energy required for initiation, high temperatures applied
the higher amounts of PUFAs found in the liquid mediums containing

7
C.F.T. Barreira et al. Food Chemistry 403 (2023) 134347

pink pepper may be linked to the antioxidant effect promoted by the Table 3
fruit. Moreover, the sum of EPA and DHA also showed higher values in Cholesterol (mg/100 g, dry basis) and cholesterol oxides (μg/g, dry basis) con­
samples containing the natural antioxidant (2.31 ± 0.25–3.80 ± 0.10 g/ tents of sardine and liquid medium samples before and after canning (control
100 g oil) than in control (1.88 ± 0.11 g/100 g oil). and with pink pepper at 0.25, 0.5, 0.75, and 1.0%).
The liquid medium applied in canning directly influences lipid Sardine
oxidation by providing pro-oxidant or antioxidant environments Before After canning
depending on its composition (Atitallah et al., 2019; Barbosa et al., canning
2019; Dantas et al., 2021; Gómez-Limia et al., 2020; Medina, Sacchi, COPs Raw Control 0.25% 0.5% 0.75% 1.0% PP
Biondi, Aubourg, & Paolillo, 1998). The liquid medium (soybean oil) (0.0% PP PP PP
used can be oxidized more rapidly than esterified fatty acids and act as PP)
pro-oxidants. A number of substances that can be present in in natura 25-OH – 38.55 31.25 23.97 6.07 ± –
vegetable oils may contribute to changes in color, taste, and aroma, as ± 1.22b ± ± 0.05D;
well as restrict their application and reduce their shelf life. Among these 0.36B; 1.19C; b;α
b;α b;α
substances are free fatty acids, phospholipids, carbohydrates, and pro­
5,6α-OH 14.98 16.24 15.98 15.65 16.12 15.87 ±
teins (as well as their degradation by-products), water, chlorophylls, ± 0.90 ± ± ± ± 0.14A;a;β
carotenoids, and fatty acid oxidation products (Nawar, 1996). In this 0.26a;β 0.28A; 0.51A;a 0.15A;a
study, it was not possible to measure the free fatty acids. However, the a;β

free fatty acid content cannot be used alone as an evaluation parameter 5,6 β-OH 3.24 ± 8.41 ± 8.56 ± 8.14 ± 7.22 ± 6.94 ±
0.50 0.30a;β 0.2A;a;β 0.34A; 0.23A; 1.32A;a;β
but must be associated with other quality indicators, because free fatty a;β a;β
acids may undergo oxidation processes, generating secondary products 7-keto – 1.91 ± 1.70 ± 1.92 ± 1.91 ± 1.55 ±
that are also deleterious to the oil’s quality. Thus, enhancing the liquid 0.01b 0.06AB;b 0.02A;b 0.01A;b 0.20B;b;α
medium with sources of natural antioxidant compounds is a promising 7α-OH 15.16 22.08 18.95 16.55 18.36 17.58 ±
strategy to control or retard oxidative processes. ± 0.49 ± ± ± ± 1.22B; 0.88B;α;β
0.88b;β 1.21B; 0.59B; b;α;β
Barbosa et al. (2019) described the protective effect of adding algae b;α;β b;α;β

extracts, which contain chemical constituents with potential antioxidant 7β-OH 6.14 ± 28.86 16.81 23.10 9.78 ± 19.16 ±
properties, to the liquid medium on the lipid fraction of canned chub 1.21 ± ± ± 0.33E; 0.98C;α;β
mackerel (Scomber colias). According to Atitallah et al. (2019), 0.79a;β 0.21D; 1.27B; a;α;β
a;α;β a;α;β
microalgae-fortified canned fish burgers showed higher DPPH scav­
Total COPs 39.53 116.04 93.26 89.33 59.46 61.11 ±
enging activities (from 66 to 98%) than burgers not containing micro­ ± 2.14 ± ± ± ± 1.42D;
algae (40%). The authors also determined pigments such as carotenoids 0.78b;β 1.99B; 1.02C; 0.87D; b;α;β

in microalgae, associating them with the increased antioxidant potential b;α;β b;α;β b;α;β

of burgers treated with the natural antioxidant. Moreover, Aubourg, Cholesterol 143.02 85.77 90.52 93.11 96.45 102.76
± 10.69 ± 0.13A;
Trigo, Martínez and Rodríguez (2020) reported that a packaging system
± ± ± ±
5.07a;β 1.27BC; 0.70B; 0.17AB; a;α;β

including a macroalgae extract in the liquid medium enhanced the a;β a;α;β a;α;β

quality of a canned under-valued fish species regarding oxidative

processes. Liquid medium

Before After canning

3.3.3. Cholesterol and cholesterol oxides contents of canned sardines and canning
liquid mediums
COPs Raw Control 0.25% 0.5% 0.75% 1.0% PP
The raw sardines had a cholesterol level of 143.02 ± 10.69 mg/100 g
(0.0% PP PP PP
(dry basis) (Table 3), which was lower than the one determined by de PP)
Carvalho et al. (2021) (323 mg/100 g). Varied values may be attributed
25-OH – 110.89 110.67 106.13 96.15 99.85 ±
to environmental conditions and genetic aspects (Bandarra et al., 2018). ± 0.10a ± ± ± 0.05C;γ
In contrast, the liquid medium evaluated before canning did not present 2.95A;a 0.01B;a;γ 0.01D;
detectable cholesterol contents. a;γ

After canning, the amount of cholesterol in sardines reduced from 5,6α-OH – 5.24 ± 5.02 ± 5.11 ± 4.38 ± 4.34 ±
0.33b 0.26AB;b 0.09AB;b 0.01B; 0.55B;b;γ
143.02 ± 10.69 (raw sardine) to 85.77 ± 5.07 mg/100 g, which corre­ b;γ
sponds to a degradation of 40.03% (p < 0.05) (Table 3). Cholesterol is a 5,6 β-OH – 5.04 ± 4.78 ± 4.95 ± 3.31 ± 2.16 ±
crucial component of several biological processes that may be highly 0.31b 0.02A;b 0.14A;b 0.45B; 0.26C;b;γ
affected during thermal processing (de Carvalho et al., 2021; de Oliveira b;γ

et al., 2020a). High temperatures induce cholesterol degradation by 7-keto – 26.41 26.93 24.75 23.15 25.94 ±
± 0.54a 0.19AB;a
oxidation and polymerization, resulting in the formation of cholesterol
± ± ±
1.33A;a 0.24BC; 0.37C;a;γ
oxides, ketones, aldehydes, hydrocarbons, alcohols, and volatile organic a;γ

acids (Derewiaka & Molińska, 2015). 7α-OH – 60.86 59.47 54.67 30.00 –
The liquid medium was also evaluated after canning, showing a ± 0.78a ± ± ±
2.10A;a 0.11B;a;γ 1.53C;a;γ
cholesterol content of 32.41 ± 6.19 mg/100 g (Table 3), which revealed
7β-OH – 9.85 ± 9.28 ± 10.72 7.89 ± –
the migration of cholesterol from sardine muscle to the liquid medium. 0.76b 0.11B;b ± 0.01C;
This finding is in agreement with Dantas et al. (2021), who evaluated 0.20A;b b;γ

canned tuna conserved in brine and soybean oil and found cholesterol in Total COPs – 218.28 216.16 206.33 164.88 132.30
the liquid mediums. Thus, lower levels of cholesterol assessed in sar­ ± 1.81a ± ± ± ± 0.95D;
3.48A;a 0.45B;a;γ 1.82C;a;γ a;γ
dines after canning may be attributed to both cholesterol degradation Cholesterol – 32.41 47.09 48.94 44.28 40.14 ±
and migration to the liquid medium. ± 6.19b ± ± ± 1.06AB;b
In sardine samples canned with pink pepper, cholesterol contents 3.61A; 9.46A; 0.79AB;b
b;γ b;γ
varied from 90.52 ± 1.27 to 102.76 ± 0.13 mg/100 g, where the highest
amounts were found when 0.75 and 1.0% of pink pepper were applied.
For the liquid mediums, contents from 40.14 ± 1.06 to 48.94 ± 9.46

8
C.F.T. Barreira et al. Food Chemistry 403 (2023) 134347

PP = pink pepper. 5,6α-EP (5,6α-epoxycholesterol), 5,6β-EP (5,6β-epox­ > 0.05), which was the highest concentration applied. Therefore, the
ycholesterol), 7-keto (7-ketocholesterol), 7β-OH (7β-hydroxycholesterol), 7α- higher the concentration of pink pepper used the higher its effectiveness
OH (7α-hydroxycholesterol), 25-OH (25-hydroxycholesterol). Values represent against cholesterol oxidation.
means ± standard deviation in triplicates. Different capital letters in the same As observed for control samples, 25-OH was the main COP deter­
row indicate significant differences among samples with different concentra­
mined in samples containing pink pepper, with contents ranging from
tions of pink pepper for the same type of material (sardine or liquid medium) by
96.15 ± 0.01 to 110.67 ± 2.95 μg/g. 7α-OH and 7β-OH were found in
the Tukey test. Different lowercase letters in the same column indicate signifi­
cant differences among samples of distinct type of samples (sardine or liquid
samples with pink pepper; however, at the treatment with 1.0%, they
medium) at the same concentration of pink pepper, including control (0.0% PP), were not found at detectable levels. The other cholesterol oxides varied
by the t Student test. “α” indicates significant differences in comparison with as follows: 7k-OH (from 23.15 ± 0.37 to 26.93 ± 1.33 μg/g), 5,6α-EP
“Control” by the Dunnett test for sardine samples. “β” indicates significant dif­ (from 4.34 ± 0.55 to 5.11 ± 0.09 μg/g) and 5,6β-EP (from 2.16 ± 0.26
ferences in comparison with “Raw” by the Dunnett test for sardine samples. “γ” to 2.16 ± 0.26 μg/g).
indicates significant differences in comparison with “Control” by the Dunnett For the liquid mediums, the amounts of total COPs in samples treated
test for liquid medium samples. “δ” indicates significant differences in com­ with pink pepper varied from 216.16 ± 3.48 to 132.30 ± 0.95 μg/g.
parison with “Raw” by the Dunnett test for liquid medium samples. These values were lower (p < 0.05) than the one determined in the
control sample (218.28 ± 1.81 μg/g), except in samples treated with
mg/100 g were assessed (Table 3). 0.25% pepper. The addition of pink pepper reduced COPs formation,
The following COPs were identified in raw sardines: 7α-OH (7α- which reflected in their amount in the liquid medium.
hydroxycholesterol), 7β-OH (7β-hydroxycholesterol), 5,6α-EP (5,6α- Besides the high temperatures applied during cooking and steriliza­
epoxycholesterol), and 5,6β-EP (5,6β-epoxycholesterol) (Table 3). The tion, other aspects must be considered regarding the COPs formation
main COP was 7α-OH (15.16 ± 0.49 μg/g, dry basis), followed by 5,6α- during canning. Heavy metals are strong inducers of oxidation. Thus,
EP (14.98 ± 0.90 μg/g) and 7β-OH (6.14 ± 1.21 μg/g). Other authors metals, which are present in both fish and cans, contribute to oxidative
also found cholesterol oxides in raw sardines, attributing them to reactions (Eboh, Mepba, & Ekpo, 2006). The pressure applied in the
enzymatic processes of fish metabolism (de Carvalho et al., 2021). COPs retort system promotes the release of water and/or metal ions from
were found in the liquid medium after canning, indicating their migra­ hemoprotein, increasing the rate of lipid oxidation in fish muscle
tion from the sardine muscle. The total amount of COPs in the liquid (Mesías et al., 2015). Moreover, pro-oxidants, such as salt and PUFAs,
medium was 218.28 ± 1.81 μg/g (control), with higher levels of 25-OH present in the liquid medium, cannot be ignored (Dantas et al., 2021).
(25-hydroxycholesterol, 110.89 ± 0.10 μg/g) and 7α-OH (60.86 ± 0.78 The results demonstrated that canned sardines are exogenous sour­
μg/g). ces of COPs, which play a role in the development of numerous diseases
Canning induced the formation of COPs in sardines without pink (Garcia-Llatas et al., 2021; Liu et al., 2022). Therefore, due to the risks
pepper. The total content of COPs increased from 39.53 ± 2.14 μg/g caused by the intake of these oxidized products, their formation must be
(raw sardines) to 116.04 ± 0.78 μg/g (control sardines) (p < 0.05). In avoided. However, the ingestion of synthetic antioxidants has been a
addition to the cholesterol oxides found in raw sardines, 25-OH and 7- subject of increasing concern. Thus, as shown in this study, COPs for­
keto (7-ketocholesterol) were detected in control samples after pro­ mation in canned sardines may be reduced by incorporating natural
cessing. Moreover, 25-OH was identified as the predominant COP (38.55 sources of antioxidants such as pink pepper during canning.
± 1.22 μg/g). In fact, C25 is a highly reactive site of cholesterol side
chain. The double bond between carbons 5 and 6 enables allylic 3.4. Migration of bioactive compounds from pink pepper during canning
hydrogen abstraction at C7 by reducing the activation energy required.
Thus, 7-keto, 7α-OH, and 7β-OH are formed from C7. In the present Twelve compounds were identified in pink pepper (Section 3.2);
study, high levels of 7β-OH and 7α-OH were found in control sardine however, not all of them were detected in sardine and liquid medium
samples after canning, which increased from 6.14 ± 1.21 (raw) to 28.86 samples (Table 4).
± 0.79 μg/g and from 15.16 ± 0.49 (raw) to 22.08 ± 0.88 μg/g, As expected, both sardine and liquid medium control samples did not
respectively. In contrast, a lower level of 7-keto was formed (1.91 ± present pink pepper constituents. Six compounds (malic acid, gallic acid,
0.01 μg/g). Epoxidation leads to 5,6α- and 5,6β-epoxycholesterol, which citric acid, gallotanin, 3α-hydroxymasticadienolic, masticadienoic acid)
were also formed during canning. The content of 5,6α-EP increased from from pink pepper were detected in sardines canned with the fruit,
14.98 ± 0.90 (raw) to 16.24 ± 0.26 μg/g, whereas 5,6β-EP increased regardless of the level added. Hinokiflavone and the triterpene acid were
from 3.24 ± 0.50 (raw) to 8.41 ± 0.30 μg/g. observed only in samples containing 1.0% of pink pepper, suggesting
Studies have shown the presence of cholesterol oxides in canned fish. that the contents present in the other treatments were not enough to
Zunin, Boggia, and Evangelisti (2001) determined total COPs levels from indicate a migration. Moreover, agathisflavone was determined at all
37.7 to 328.9 μg/g in tuna canned in brine, while Dantas et al. (2021) levels of addition, except at 0.25%.
reported values from 933.14 to 1914.23 μg/g and from 698.24 to Regarding the liquid mediums, eight compounds from pink pepper
1167.88 μg/g in tuna canned in brine and soybean oil, respectively. In were found (malic acid, gallic acid, citric acid, hinokiflavone, gallotanin,
addition, Dantas et al. (2021) also quantified COPs in the liquid 3α-hydroxymasticadienolic, agathisflavone, masticadienoic acid) when
mediums. 1.0% was added. However, only malic and citric acid were detected in
Cholesterol oxidation in food is a complex process similar to the the treatment with 0.75%. Cyanidin, 7-O-methylpelargonidin 3-O-
oxidation of unsaturated fatty acids. Moreover, it is influenced by galactoside, and tetrahydroamentoflavone did not migrate from the
various factors (e.g. temperature, length of exposure to heat, oxygen, fruit.
light, initial concentration of cholesterol, pH, presence of metals and The migration of bioactive compounds was more prominent in
salt) and may occur through different mechanisms, leading to the for­ sardine samples. The compounds that migrated to the sardines also
mation of different COPs at varied levels (Derewiaka & Molińska, 2015; migrated to the liquid medium, except for the triterpene acid.
Mariutti & Bragagnolo, 2017; Smith, 1987). Conversely, in the liquid medium, this migration was noticed only in
While control sardines contained 116.04 ± 0.78 of total COPs, samples containing 1.0% of pepper. High temperatures applied during
samples with pink pepper presented contents ranging from 59.46 ± 0.87 canning may degrade fish proteins, compromising their structural
to 93.26 ± 1.99 μg/g (p < 0.05). The lowest value of total COPs was integrity and, consequently, facilitating the diffusion of compounds to
determined in samples treated with 0.75% of pink pepper (59.46 ± 0.87 the muscle. Thus, even when lower concentrations of pink pepper were
μg/g). However, this result was not significantly different from the one added to samples, the migration occurred in sardine muscles.
observed when pink pepper was added at 1.0% (61.11 ± 1.42 μg/g) (p It is also important to consider the hydrophilicity/lipophilicity of the

9
C.F.T. Barreira et al. Food Chemistry 403 (2023) 134347

Table 4
Migration of bioactive compounds from pink pepper to sardine and liquid medium.
Canned samples

Sardine Liquid medium

Bioactive Pink pepper Control 0.25% PP 0.5% PP 0.75% PP 1.0% PP Control 0.25% PP 0.5% PP 0.75% PP 1.0% PP
compound (PP)

Malic acid x – x x x x – – – x x
(m/z 133)
Gallic acid x – x x x x – – – – x
(m/z 169)
Citric acid x – x x x x – – – x x
(m/z 191)
7-O-methylpelargonidin x – – – – – – – – – –
3-O-galactoside
(m/z 447)
Cianidin x – – – – – – – – – –
(m/z 287)
Hinokiflavone x – – – – x – – – – x
(m/z 133)
Gallotanin x – x x x x – – – – x
(m/z 133)
Tetrahydroamentoflavone (m/z 541) x – – – – – – – – – –
3α-hydroxymasticadienolic x – x x x x – – – – x
(schinol)
(m/z 455)
Agathisflavone x – – x x x – – – – x
(m/z 537)
Triterpene acid x – – – – x – – – – –
(m/z 469)
Masticadienoic acid x – x x x x – – – – x
(m/z 453)

PP = pink pepper. “X” indicates presence of the bioactive compound in the corresponding sample. “–” indicates absence of the bioactive compound in the corre­
sponding sample.

compounds. In general, hydrophilic compounds such as phenolic acids considered adequate to explain the variance of experimental data. The
are virtually oil-insoluble and will mostly distribute between the sardines canned with 0.75 and 1.0% of pink pepper (Fig. 2a) were
aqueous and interfacial regions of a food system, while hydrophobic characterized by higher levels of cholesterol (Fig. 2b), suggesting the
antioxidants are water-insoluble and will primarily distribute between protective effect of pink pepper against cholesterol degradation.
the oil and interfacial regions (Freiría-Gándara, Losada-Barreiro, Paiva- Regarding correlations (Fig. 2c), PUFAs presented a strong positive
Martins, & Bravo-Díaz, 2018). However, due to the variety of chemical correlation with Σn6 (0.96), which may be mainly attributed to the high
structures of naturally occurring compounds, their hydrophilic and contents of linoleic acid (C18:2n6c) present in the liquid medium that
lipophilic nature cannot be determined unambiguously. also migrated to sardines. A strong positive correlation was also
In addition, the distribution of compounds in a medium also depends observed for Σn3 vs the sum of EPA and DHA (0.98). Total COPs showed
on the specific environmental conditions (Freiría-Gándara et al., 2018). a moderate positive correlation with 7-keto (0.88) and a negative cor­
Diverse molecules of a mixture of antioxidant compounds may interact relation with the sum of EPA and DHA (-0.61).
with the food constituents in different ways. Thus, further studies must Fig. 2d shows four groups formed according to their similarities. The
be conducted to elucidate the behavior of each compound in a food first group presents only the liquid medium evaluated before canning
system like canned fish. (LM_bc), indicating the distinct composition of this sample (e.g. it did
Nevertheless, most of the bioactive compounds found in pink pepper not contain cholesterol and COPs). The liquid medium samples evalu­
belong to hydrophilic groups. Therefore, since lipid oxidation occurs in ated after canning without pink pepper (LM_control) and treated with
the water-muscle interface due to the high concentration of oxygen and the natural antioxidant (LM_0.25, LM_0.5, LM_0.75; LM_1.0) formed the
pro-oxidants, the antioxidant effect of pink pepper may be related to the second group. The third group is composed of sardine samples before
migration of its hydrophilic compounds to this region. canning (S_bc, raw sardines), while the fourth one is formed by canned
According to Medina et al. (1998), tuna canned in vegetable oils sardines (S_control, S_0.25, S_0.5, S_0.75, S_1.0).
showed lower oxidation rates than tuna in brine, which was attributed to In addition, the samples may be divided into seven subgroups (one
the migration of hydrophilic phenols from the oils to the water-muscle per color in Fig. 2d). The existence of different groups for each type of
interface. Additionally, they also observed interactions between pro­ sample (sardine and liquid medium) containing just the sample evalu­
teins from tuna and phenolic compounds, pointing out the importance of ated before canning demonstrates the great influence of canning on the
evaluating the antioxidant properties of the complexes formed. lipid composition of samples. Moreover, in the group formed by canned
Regarding the compounds that did not migrate from pink pepper, sardines, samples with 0.75 and 1.0% of pink pepper can be separated
various aspects may impair their diffusion (e.g. molecular weight, sol­ into a subgroup, suggesting the effects of adding pink pepper at these
ubility, polarity, the molecular arrangement of structures, steric concentrations.
impediment), suggesting further studies to better understand the
mechanisms involved in their migration. 4. Conclusions

3.5. Principal component analysis This study showed that canning induced lipid oxidation in canned
sardines, which was proved by the degradation of fatty acids and the
The first two principal components (PCs) explain ~87% of the data formation of COPs. Moreover, the results revealed an exchange of fatty
variability (Dim 1: 67.8%, Dim 2: 19.3%) (Fig. 2a and b), which could be acids, cholesterol, and COPs between fish muscle and liquid medium.

10
C.F.T. Barreira et al. Food Chemistry 403 (2023) 134347

Fig. 2. Multivariate analyses. Principal component analyses for treatments (a) and variables (b). Correlogram, where negative or positive correlation are represented
by red or blue numbers, respectively (c). Hierarchical clustering from principal components (d). LM_bc = liquid medium before canning; LM_control = control liquid
medium after canning; LM_0.25, LM_0.5, LM_0.75, LM_1.0 = liquid medium after canning with 0.25, 0.5, 0.75, and 1.0% of pink pepper, respectively; S_bc = sardine
before canning; S_control = control canned sardine; S_0.25, S_0.5, S_0.75, S_1.0 = canned sardine with 0.25, 0.5, 0.75, and 1.0% of pink pepper, respectively. (For
interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

The liquid medium presented a strong influence on the fatty acid profile COPs. However, further studies concerning the parameters for pink
of sardines, which was demonstrated by the high levels of linoleic acid pepper addition (e.g. levels of addition, ground or whole fruit) to
found in canned sardines. The canning process induced the formation of improve its effectiveness and sensorial acceptance must be conducted to
COPs; however, the addition of pink pepper minimized cholesterol support its use by the canning industry.
oxidation. The protective potential of pink pepper may be linked to the
antioxidant capacity of its extract and its bioactive compounds. UHPLC- Author statement
ESI-MS analyses showed that some constituents of pink pepper migrated
to the sardine, suggesting their role against cholesterol oxidation. Thus, Carla Fernanda Targueta Barreira undertook almost most of the
considering the efforts made by the food industries to produce safe and experimental work presented in this paper. Vanessa Sales de Oliveira
quality products which attend to consumers’ demand for food free of designed, supervised, and organized the study. Davy William Hidalgo
synthetic additives, this study highlights pink pepper as a promising Chávez did the statistical analysis. Ormindo Domingues Gamallo and
natural antioxidant in canned fish, contributing to the maintenance of Rosane Nora Castro were performance the cholesterol oxides analyses.
the nutritional quality of these products and reducing the intake of Pedro Côrrea Damasceno Júnior was responsible for processing the

11
C.F.T. Barreira et al. Food Chemistry 403 (2023) 134347

samples and interpreted the results. Alexandra Christine Helena Frank­ Djuricic, I., & Calder, P. C. (2021). Beneficial outcomes of omega-6 and omega-3
polyunsaturated fatty acids on human health: An update for 2021. Nutrients, 13(7),
land Sawaya was responsible for the mass spectrometry analyses.
2421.
Micheli da Silva Ferreira was responsible for processing the samples and Domiszewski, Z. (2021). Effect of sterilization on true retention rate of eicosapentaenoic
interpreted the results. Geni Rodrigues Sampaio and Elizabeth Apar­ and docosahexaenoic acid content in mackerel (Scomber scombrus), herring (Clupea
ecida Ferraz da Silva Torres supervised and organized the study. Tatiana harengus), and sprat (Sprattus sprattus) canned products. Journal of Food Processing
and Preservation, 45(5), e15461.
Saldanha interpreted the results and drafted the manuscript with help Eboh, L., Mepba, H. D., & Ekpo, M. B. (2006). Heavy metal contaminants and processing
from the other authors. effects on the composition, storage stability and fatty acid profiles of five common
commercially available fish species in Oron Local Government, Nigeria. Food
Chemistry, 97(3), 490–497.
Declaration of Competing Interest FAO (2018). Food and Agriculture Organization of the United Nations. Fisheries and
aquaculture statistics. FAO Fisheries and Aquaculture Department. Rome.
Farhoosh, R., & Nyström, L. (2018). Antioxidant potency of gallic acid, methyl gallate
The authors declare that they have no known competing financial and their combinations in sunflower oil triacylglycerols at high temperature. Food
interests or personal relationships that could have appeared to influence Chemistry, 244, 29–35.
the work reported in this paper. Fernández-Fernández, R., López-Martínez, J. C., Romero-González, R., Martínez-
Vidal, J. L., Flores, M. I. A., & Frenich, A. G. (2010). Simple LC–MS determination of
citric and malic acids in fruits and vegetables. Chromatographia, 72(1), 55–62.
Data availability Feuereisen, M. M., Zimmermann, B. F., Schulze-Kaysers, N., & Schieber, A. (2017).
Differentiation of Brazilian peppertree (Schinus terebinthifolius Raddi) and Peruvian
peppertree (Schinus molle L.) fruits by UHPLC–UV–MS analysis of their anthocyanin
Data will be made available on request. and biflavonoid profiles. Journal of Agricultural and Food Chemistry, 65(26),
5330–5338.
Acknowledgements Freiría-Gándara, J., Losada-Barreiro, S., Paiva-Martins, F., & Bravo-Díaz, C. (2018).
Enhancement of the antioxidant efficiency of gallic acid derivatives in intact fish oil-
in-water emulsions through optimization of their interfacial concentrations. Food &
The authors thank the Natl. Brazilian Research Foundations Function, 9(8), 4429–4442.
(CAPES) for the scholarship and Rio de Janeiro Research Foundation for Garcia-Llatas, G., Mercatante, D., López-García, G., & Rodriguez-Estrada, M. T. (2021).
Oxysterols—how much do we know about food occurrence, dietary intake and
the financial support (FAPERJ, Project E-26/211.573/2021). The au­ absorption? Current Opinion in Food Science, 41, 231–239.
thors would like to thank the Industry Nova Piracema (São Gonçalo, Rio Gomes, R. B. A., de Souza, E. S., Barraqui, N. S. G., Tosta, C. L., Nunes, A. P. F.,
de Janeiro, Brazil) and its responsible technician, the veterinarian Schuenck, R. P., … Kuster, R. M. (2020). Residues from the Brazilian pepper tree
(Schinus terebinthifolia Raddi) processing industry: Chemical profile and
Claudia Fabiana Augusto Magalhães, for their support for the samples antimicrobial activity of extracts against hospital bacteria. Industrial Crops and
production. Products, 143, Article 111430.
Gómez-Limia, L., Cobas, N., Franco, I., & Martínez-Suárez, S. (2020). Fatty acid profiles
and lipid quality indices in canned European eels: Effects of processing steps, filling
Appendix A. Supplementary data medium and storage. Food Research International, 136, Article 109601.
Gómez-Limia, L., Sanmartín, N. M., Carballo, J., Domínguez, R., Lorenzo, J. M., &
Supplementary data to this article can be found online at https://doi. Martínez, S. (2021). Oxidative stability and antioxidant activity in canned eels:
Effect of processing and filling medium. Foods, 10(4), 790.
org/10.1016/j.foodchem.2022.134347.
Gulcin, İ. (2020). Antioxidants and antioxidant methods: An updated overview. Archives
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