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Effect of 4‐Hexylresorcinol and Sodium Metabisulphite on Spoilage and

Melanosis Inhibition in Xiphopenaeus kroyeri Shrimps

Article in Journal of Food Processing and Preservation · August 2016

DOI: 10.1111/jfpp.12943

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 Journal of Food Processing and Preservation ISSN 1745-4549

EFFECT OF 4-HEXYLRESORCINOL AND SODIUM

METABISULPHITE ON SPOILAGE AND MELANOSIS
INHIBITION IN XIPHOPENAEUS KROYERI SHRIMPS
~ 1, DANIEL VAZQUEZ-S
JULIANA ANTUNES GALVAO  
ANCHEZ, VIVIANE ANGELI YOKOYAMA,
LUCIANA KIMIE SAVAY-DA-SILVA, SOLANGE GUIDOLIN CANNIATTI BRAZACA and MARILIA OETTERER
Department of Agri-Food Industry, Food and Nutrition, “Luiz de Queiroz” College of Agriculture (ESALQ), University of S~
ao Paulo (USP)

1
Corresponding author. ABSTRACT
TEL: 1 55 19 34294150;
FAX: 1 55 19 34294288; Nutritional value of Xiphopenaeus kroyeri as well as the effect of 4-hexylresorcinol
EMAIL: jugalvao@usp.br and sodium metabisulfite on its spoilage and melanosis formation were deter-
mined. Data indicated that X. kroyeri is an interesting source of proteins and
Received for Publication October 29, 2015
essential minerals, but with low lipid and energy content. The discard of the ceph-
Accepted for Publication February 12, 2016
alothorax allowed to reduce significantly (P < 0.05) both the deterioration of
doi:10.1111/jfpp.12943 physicochemical (i.e., TVB-N, TMA-N, NPN and pH) and microbial properties
(i.e., mesophilic and psychrotrophic counts) in shrimps. The immersion in
sodium metabisulfite also decreased significantly (P < 0.05) both the physico-
chemical and microbial deterioration of X. kroyeri. Nevertheless, treatments with
4-hexylresorcinol showed a higher efficacy to control the production of TVB-N,
TMA-N, NPN and blackspots, as well as it avoids the presence of residual sulfite
in shrimps. In addition, internal processes seemed to have a higher effect on the
deterioration of X. kroyeri than the microbial development.

PRACTICAL APPLICATIONS
Xiphopenaeus kroyeri is the most caught and commercialized shrimp in Brazil,
but its market value and consumer acceptability is drastically reduced by the
appearance of melanosis. This study focused on 4-hexylresorcinol because it is
less hazardous for the health of consumers and more environmentally friendly
than sodium metabisulfite. The data obtained in this study demonstrated that the
discard of the cephalothorax combined with the immersion in 4-hexylresorcinol
resulted in a highly effective treatment to slow down the deterioration and mela-
nosis formation of chilled X. kroyeri shrimps.

INTRODUCTION
Shrimps are the most important traded crustacean world-
wide, with 3.4 million of tons caught and 12.4 billions of
dollars reached in 2012 (FAO 2014). In Brazil, catches of X.
kroyeri shrimps are particularly relevant (MPA 2013), being
Ubatuba – situated in the Northern coast of the state of S~ao
Paulo – one of the main harbor of unloaded. However,
the appearance of melanosis (or blackspot) during storage
drastically reduces their market value and consumer accept-
ability (Gonçalves and Oliveira 2016). A blackspot is formed
by the enzymatic complex polyphenoloxidase, which oxi-
dizes phenols to quinones in the presence of molecular oxy-
gen (Zamorano et al. 2009; Nirmal and Benjakul 2010). The
refrigeration of shrimps or their storage on ice can slow
down this process, but not inactivate it. Therefore, addition
of chemicals is necessary to prevent melanosis in shrimps.
Sodium metabisulfite is the chemical most widely used to
control the melanosis of shrimps during the storage. It acts by
inactivating the enzyme polyphenoloxidase and combining
with quinones to prevent their polymerization in pigmented
compounds (Ferrer et al. 1989). Nonetheless, the treatment
of food products with sodium metabisulfite can cause
anaphylactic reactions to sulfite-sensitive individuals and
bronchoconstriction to asthmatic patients (Vally et al. 2009).

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4-HEXYLRESORCINOL ON DETERIORATION OF X. KROYERI
TABLE 1. TREATMENTS WITH ANTIMELANOSICS APPLIED TO SHRIMP
SAMPLES
Whole Beheaded
Treatments shrimp (W) shrimp (B)
Control (C) WC BC
0.01% of 4-Hexylresorcinol (H1) WH1 BH1
0.1% of 4-Hexylresorcinol (H2) WH2 BH2
1.25 % of Sodium Metabisulfite (S1) WS1 BS1
2.5 % of Sodium Metabisulfite (S2) WS2 BS2
Moreover, this chemical produces an alkaline pollution of the
environment that kills several aquatic species (Carvalho et al.
2011).
Alternatively to sodium metabisulfite, some studies demon-
strated the effectiveness of 4-hexylresorcinol (C12H12O2, CAS
Reg. n. 136-77-6) to prevent melanosis in several shrimps spe-
cies (Guandalini et al. 1998; Montero et al. 2001, 2004, 2006;
Thepnuan and Visessanguan 2008), but none was done with
X. kroyeri shrimps. This compound is considered Generally
Recognized As Safe (GRAS) in many countries (Australia, Bra-
zil, Canada, The United States, etc.), although the European
Union requires that residues in crustacean meat do not exceed
2 mg/kg (EFSA 2014). 4-hexylresorcinol also inhibits effectively
the activity of polyphenoloxidase produced by shrimps
after catch, but it is thermostable and functional at low concen-
trations in contrast to sodium metabisulfite (McEvily et al.
1991).
The present study was therefore aimed to evaluate com-
paratively the effect of the antimelanosics sodium metabi-
sulfite and 4-hexylresorcinol on the physicochemical and
microbiological quality of chilled X. kroyeri shrimps caught
in Ubatuba. In addition, their proximate and mineral com-
positions were also evaluated to emphasize their nutritional
value.
MATERIAL AND METHODS
Sampling
A total of 135 kg of X. kroyeri shrimp weighing 5.91 6 2.34 g
and measuring 9.45 6 1.73 cm were caught in Ubatuba
(23 8260 1300 S–45 8040 0800 W) and maintained in ice until
unloaded.
Treatment with Antimelanosics
Three batches of approximately 9 kg of shrimp were exposed
for 5 min to either sodium metabisulfite (97%, Kyma, Ameri-
cana, Brazil) or 4-hexylresorcinol (99%, Sigma-Aldrich, San
Luis) in 12 L solution of cold water (5C) added with
0.02 mL of sodium hypochlorite (1%, Kyma). Two concentra-
tions of each antimelanosic were tested: 1.25% (S1) and 2.5%
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J.A. GALVAO
(S2) of sodium metabisulfite, and 0.01% (H1) and 0.1%
(H2) of 4-hexylresorcinol. Three control (C) batches without
antimelanosics were also included. All samples were subse-
quently washed in chlorinated water, drained and separated
in whole (W) and beheaded (B) shrimps (i.e., without the
cephalothorax). Samples kept in ice were immediately trans-
ported to the processing plant in the ESALQ-USP (Piraci-
caba, Brazil). Table 1 shows nomenclature of treatments
applied to shrimps.
Packing and Storage
On the same day of catch, shrimps were packed in polystyrene
trays covered with multilayer plastic film EVOH, which were
properly labeled. Samples were then stored at 0C for 12 days.
Nutritional Analyses
Three independent measurements for each batch of nontreated
X. kroyeri shrimps were performed in the first 24 h after the
catch.
Determination of Proximate Composition. Proximate
composition of was determined following AOAC methods
(Horwitz and Latimer 2005), with slight modifications.
Both edible parts (meat) and waste (shell, gonads and stom-
ach contents) were used. Moisture content was assessed by
oven-drying 10 g of sample at 105C until they reach a con-
stant weight. The lipid fraction was quantified in 2 g of sam-
ple by Soxhlet extraction, using hexane (Labsynth Ltd.,
Diadema, Brazil) as a solvent. The content of crude protein
was first determined in terms of content of total nitrogen by
Micro-Kjeldahl procedure, and then converted using the
factor 6.25. The ash content was obtained by incineration of
2 g of sample in a muffle furnace at 550C until reach a con-
stant weight. The carbohydrate content was calculated by
weight difference, whereas the energy value was determined
using the conversion values proposed by the Anvisa (2003)
for lipids (9), proteins and carbohydrates (4). All measure-
ments were done in triplicate for each batch.
Determination of Mineral Composition. Composition
in minerals (P, K, Na, Ca, Mg, Fe, Zn, Cu, Mn and S) of
X. kroyeri shrimps was determined following Sarruge and
Haag (1974) methodology and using nitric acid (Labsynth
Ltd., Diadema, Brazil). Absorbance was measured by atomic
emission spectrometry in a Perkin Elmer 3110 Spectrometer
(Norwalk, The United States).
Physicochemical Analyses
Three independent experiments for each analysis were
performed after 1, 4, 8 and 12 days of storage, except the
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 ~ ET AL.
J.A. GALVAO
evaluation of melanosis and the colorimetric analysis, which
were determined after 1, 2, 4, 6, 8, 10 and 12 days.
Total Volatile Base Nitrogen (TVB-N). TVB-N was
determined in triplicate for each experiment following the
modified method of Savay-da-Silva et al. (2008). About 50 g
of shrimp muscle per treatment mixed with 150 mL of 5%
trichloroacetic acid (TCA, Merck, Darmstadt, Germany)
were homogenized for 1 min and then vacuum filtered.
Aliquots of 10 mL of the precipitated protein nitrogen were
added to 20 mL of distilled water and 2 g of MgO (96%,
Labsynth Ltd., Diadema, Brazil) and subsequently steam
distilled using a Kjeldahl distillation unit TE 036/1
(TECNAL, Piracicaba, Brazil). The volatile base compo-
nents were absorbed by 20 mL of 4% boric acid (LabImpex
Ltd., Diadema, Brazil) with five drops of mixed indicator
[0.66 g/L methyl red and 0.33 g/L bromocresol green
(Labsynth Ltd.) diluted in 70% (v/v) ethanol (Neon Com-
mercial Ltd., S~ao Paulo, Brazil)] and immediately assessed
by titration with 0.01 N sulfuric acid [Quimica Moderna
Ltd., Barueri, Brazil].
Trimethylamine Nitrogen (TMA-N). TMA-N was
assessed in duplicate for each independent assay following an
optimized protocol based on standard methodology (Hor-
witz and Latimer 2005). About 50 g of shrimp muscle per
treatment mixed with 150 mL of 5% TCA were homogenized
for 1 min and then vacuum filtered. Aliquots of 10 mL of the
precipitated protein nitrogen were added to 10 mL of distilled
water, 10 mL of 35% formaldehyde (Sigma-Aldrich) and 2 g
of MgO and subsequently steam distilled. The trimethyl-
amine components were absorbed by 20 mL of 4% boric acid
with five drops of mixed indicator and immediately deter-
mined by titration with 0.01 N HCl (LabImpex Ltd).
Nonprotein Nitrogen (NPN). NPN was assessed in 20%
TCA extracts by using the official Micro-Kjeldahl method
(Horwitz and Latimer 2005). Three replicates for each
experiment were included.
pH. The pH was measured in triplicate for each assay using
a digital pH meter TEC-2 (TECNAL), following the recom-
mendations of Pregnolato and Pregnolato (1985). About
10 g of shrimp muscle per treatment mixed with 10 mL of
distilled water were homogenized for 1 min just before each
pH measurement.
Residual Sulfite. Residual sulfite was assessed in samples
exposed to sodium metabisulfite, following an optimized
Monier–Williams method (Hillery et al. 1989). About 50 g of
shrimp muscle per treatment mixed with 95 mL of Milli-Q
water and 5 mL of ethanol were homogenized for 1 min. After-
ward, samples were added to 400 mL of Milli-Q water and
Journal of Food Processing and Preservation 2017; 41: e12943; V
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4-HEXYLRESORCINOL ON DETERIORATION OF X. KROYERI
90 mL of 4 N HCl and distilled under 200 mL/min N2 flux.
Residual sulfite was absorbed by 30 mL of 3% H2O2 (Labsynth
Ltd) with three drops of methyl red and immediately titrated
with 0.01 N NaOH. Two replicates were included for each of
the three independent experiments performed.
Visual Evaluation of Melanosis and Colorimetric
Analysis. Melanosis score and colorimetric properties
were only assessed in whole shrimps, because the enzyme
polyphenoloxidase is mainly synthesized in the cephalo-
thorax (Rotllant et al. 2002).
The development of melanosis was visually evaluated by
eight trained panellists. A total of 17 whole shrimps per
treatment and day were examined in each independent
assay. The degree of melanosis was classified as (1) absent,
(2) moderate (up to 30% of shrimp surface affected), (3)
severe (30–70% of shrimp surface affected) and (4) extreme
(70–100% of shrimp surface affected), following the Mon-
tero et al. (2004) scale.
In addition, the color of the cephalothorax and the abdo-
men of shrimps previously evaluated by the panellists were
measured in triplicate by direct application on the body sur-
face using a CR-400 Minolta Chroma Meter (Konica Min-
olta, Chiyoda, Japan). Color was assessed in terms of
lightness [L*, black (0) to white (100)], redness [a*, green
(2) to red (1)] and yellowness [b*, blue (2) to yellow (1)]
using a CIE L*a*b* system.
Microbiological Assays
The microbial analysis was carried out following the inter-
national official methods of the International Organization
for Standardization (ISO) for total mesophilic count (ISO
4833:2003), total psychrotrophic count (ISO 17410:2001),
total and thermotolerant coliforms (ISO 4831:2006), Salmo-
nella spp. (ISO 6579:2002) and S. aureus (ISO 6888-1:1999/
Amd 1:2003 and ISO 6888-2:1999/Amd 1:2003). Bacterial
counts were expressed as log CFU/g, except in coliforms
which were expressed as log MPN/g.
Statistical Analysis
Experimental results were statistically analyzed with the
software packages Microsoft Excel 2013 and IBM SPSS 19.0.
Statistical significance analysis was carried out using a one-
way ANOVA. Homogeneity of variances was examined by a
post-hoc least significant difference (LSD) test. Otherwise, a
Dunnett’s T3 test was performed. An independent-samples
Student’s t-test was also done to determine if there were sta-
tistical differences between pairs of treatments. A bivariate
correlation analysis was conducted using Pearson’s correla-
tion coefficient to measure the strength of the linear
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J.A. GALVAO

TABLE 2. PROXIMATE COMPOSITION (g/100 g) AND ENERGY VALUES TABLE 3. MINERAL COMPOSITION (mg/100 g) OF WHOLE (W) AND
(kJ/100 g) OF WHOLE (W) AND BEHEADED (B) SHRIMPS NONTREATED BEHEADED (B) SHRIMPS NONTREATED WITH ANTIMELANOSICS (C)
WITH ANTIMELANOSICS (C)
Mineral WC BC RDI*
Mineral WC BC a b
P 284.67 6 12.04 204.11 6 13.48 700
Moisture 78.18 6 1.43a 80.89 6 1.29a K 210.94 6 16.45a 137.06 6 23.35b 2
Ash 3.39 6 0.32a 2.45 6 0.22b Na 495.78 6 44.18a 365.33 6 49.14b 2
Proteins 13.98 6 0.46a 14.20 6 0.34a Ca 82.03 6 5.77a 47.83 6 4.05b 1,000
Lipids 1.05 6 0.16a 0.37 6 0.06b Mg 55.00 6 6.12a 35.00 6 4.88b 260
Carbohydrates 3.40 6 1.15a 2.09 6 0.85a Fe 5.68 6 0.67a 2.90 6 0.13b 14
Energy value 330.39 6 22.96a 286.45 6 19.15b Zn 1.90 6 0.07a 1.18 6 0.03b 7
Cu 1.22 6 0.06a 0.39 6 0.03b 0.9
Superscript letters indicate significant (P < 0.05) differences between
Mn 0.06 6 0.00a 0.00b 2.3
whole and beheaded shrimps.
S 0.26 6 0.03a 0.26 6 0.01a 2

* Reference Daily Intake (mg) for each mineral established by the

relationship between two variables. Statistical significance was
Anvisa 2005.
accepted at a confidence level greater than 95% (P < 0.05). Superscript letters indicate significant (P < 0.05) differences between
whole and beheaded shrimps.

RESULTS AND DISCUSSION

Nutritional Properties of X. kroyeri Shrimps
Proximate Composition. Proximate composition of X.
kroyeri shrimps obtained in this study (Table 2) was in
concordance with reference values provided by the USDA
(2015) for Penaeidae shrimps, with slight differences. How-
ever, it is known that there is a great variability between
shrimp species due to differences in size, nutrition, sexual
stage and environmental conditions, among others (Ogawa
and Maia 1999). As expected, the discard of the cephalo-
thorax produced significant (P < 0.05) differences in the
nutritional value of shrimps, excluding the protein and car-
bohydrate content.
Among shrimp species caught in Brazil, X. kroyeri showed
a similar lipid content than Penaeus brasiliensis and P. schi-
mitti (Bragagnolo and Rodrıguez-Amaya 2001; Pedrosa and
Cozzolino 2001), but lower than Farfantapenaeus brasiliensis
(Cabrera et al. 2005) and Macrobrachium amazonicum (Fur-
uya et al. 2006). Interestingly, the lipid content of X. kroyeri
was also lower than fish species such as sardine, bluefish,
tuna, among others (Visentainer et al. 2007). Meanwhile, its
protein content was higher than that of Penaeus brasiliensis
(Pedrosa and Cozzolino 2001), the second specie most
caught in Brazil (MPA 2013). Moreover, X. kroyeri shrimps
also represent an appropriate protein alternative to meat
products, e.g., as they had less lipid content than chicken
and beef (Almeida et al. 2006).
Mineral Composition. As shown in Table 3, the con-
sumption of X. kroyeri shrimps provides a high percentage
of the reference daily intake for minerals (Anvisa 2005).
Nonetheless, a considerable proportion of these minerals
are present in the cephalothorax, as indicated the significant
(P < 0.05) differences between whole and beheaded
shrimps. Among minerals detected in X. kroyeri, Na was the
most abundant, followed by P, K, Ca and Mg. Amounts of
these minerals were higher than those obtained by Oksuz
et al. (2009) in Penaeus longinostris and P. martia shrimps.
Other minerals such as Fe, Zn and Cu were also detected in
X. kroyeri, but their concentrations were significantly
(P < 0.05) lower than aforementioned minerals. Neverthe-
less, X. kroyeri shrimps contained a higher amount of Fe, Zn
and Cu than Penaeus brasiliensis, P. longinostris and P. martia
shrimps, and even higher than Panulirus argus lobsters
(Pedrosa and Cozzolino 2001; Oksuz et al. 2009). Particu-
larly high was the amount of Cu in whole shrimps, reaching
values significantly (P < 0.05) higher than the Reference
Daily Intake value for this mineral (Anvisa 2005). Cu is a co-
factor for the enzymatic reaction of polyphenoloxidase,
which generates blackspots (Gonçalves and Oliveira 2016).
Therefore, the discard of the cephalothorax is recommended
to reduce the concentration of this mineral in X. kroyeri
shrimps. Traces of S and, in the cephalothorax, Mn were
also noticed in X. kroyeri shrimps.
Effect of Antimelanosics on the
Physicochemical Quality of Shrimps
Production of Total Volatile Base Nitrogen (TVB-
N). According to Ogawa and Maia (1999), TVB-N values of
5–10 mg N/100 g represent an excellent state of freshness in
fish, 15–25 mg N/100 g are considered a reasonable state of
freshness, whereas 30–40 mg N/100 g indicate an advanced
state of deterioration. In fact, the Brazilian legislation estab-
lished a limit of TVB-N of 30 mg N/100 g for fish sales
(MAPA 1997a). As shown in Fig. 1a, chilled X. kroyeri
shrimps exceeded this limit after approximately7 days. The
discard of the cephalothorax significantly (P < 0.05)
increased this time, reaching TVB-N values higher than
30 mg N/100 g after 8 days. Cephalothorax contains most of

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J.A. GALVAO 4-HEXYLRESORCINOL ON DETERIORATION OF X. KROYERI

FIG. 1. (a–c) TVB-N (a), TMA-N (b) AND NPN (c) OF WHOLE (W) AND BEHEADED SHRIMPS (B) NONTREATED (C) AND TREATED WITH 0.01 AND
0.1% OF 4-HEXYLRESORCINOL (H1 AND H2) AND 1.25 AND 2.5% OF SODIUM METABISULFITE (S1 AND S2) AFTER 1, 4, 8 AND 12 DAYS

digestive organs in shrimps, where TVB-N constituents are
generated by endogenous and/or microbial proteolysis, so
its discard is recommended to keep the state of freshness for
a longer time. In fact, a positive correlation between TVB-N
and counts of mesophilic (r 5 0.941, P < 0.01) and psychro-
trophic bacteria (r 5 0.983, P < 0.01) was found in whole
shrimps.
Treatments with sodium metabisulfite reduced signifi-
cantly (P < 0.05) the production of TVB-N, except with the
application of 1.25% of sodium metabisulfite in whole
shrimps after 12 days in storage. Thus, chilled X. kroyeri
shrimps exceeded the legal limit after 10–11 days approxi-
mately. However, the state of freshness of whole and
beheaded shrimps were lost after only 2 and 4 days of stor-
age, respectively. Nirmal and Benjakul (2009) also reported
a TVB-N significantly (P < 0.05) lower in chilled Litope-
naeus vannamei shrimps soaked in 1.25% sodium metabi-
sulfite than in nontreated shrimps, but in both cases the
state of freshness was maintained during the 10 days
studied.

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4-HEXYLRESORCINOL ON DETERIORATION OF X. KROYERI
Meanwhile, both treatments with 4-hexylresorcinol were
significantly (P < 0.05) more effective than treatments with
sodium metabisulfite. In fact, the legal limit for TVB-N was
not exceeded by any sample during the 12 days of storage. In
addition, combination of treatments with 4-hexylresorcinol
and the discard of the cephalothorax allowed to maintain
the state of freshness in chilled X. kroyeri shrimps for 10–12
days. Thepnuan et al. (2008) observed that treatments com-
bining 4-hexylresorcinol and pyrophosphate also maintain
the state of freshness for 12 days both in whole L. vannamei
shrimps packaged in modified atmosphere, but they used
higher concentrations of antimelanosic (0.25%) and a lon-
ger time of exposure (15 min).
Production of Trimethylamine Nitrogen (TMA-
N). As shown in Fig. 1b, none of the samples exceed the
amount of TMA-N allowed by the Brazilian authorities for
fish sales (i.e., 4 mg N/100 g) (MAPA 1997b). Similarly to
TVB-N results, TMA-N of whole shrimps was also signifi-
cantly (P < 0.05) higher than in beheaded shrimps (1.9 mg
and 1.7 mg N/100 g, respectively after 12 days). In contrast,
Draetta et al. (1986) obtained a TMA-N of 4.74 mg N/100 g
after 12 days in chilled X. kroyeri shrimps. This difference in
TMA-N could be explained by a higher time of exposure of
shrimps to nonrefrigerated conditions after catch than that
indicated in this study.
Treatments with sodium metabisulfite reduced signifi-
cantly (P < 0.05) the production of TMA-N both in whole
and beheaded shrimps (1.5 and 1.2 mg N/100 g, respectively,
after 12 days). However, the treatment of crustaceans with
sodium metabisulfite can caused the degradation of trime-
thylamine oxide, forming prejudicial substances for the
human health such as formaldehyde (Yamanaka et al. 1977;
Yoshida and Imaida 1980; Cintra et al. 1999).
Meanwhile, the application of 4-hexylresorcinol was sig-
nificantly (P < 0.05) more effective than sodium metabisul-
fite (0.7 mg and 0.5 mg N/100 g, respectively, after 12 days),
probable as a consequence of its more stable chemical
nature. Thepnuan et al. (2008) observed a similar effect in
whole L. vannamei shrimps, but treated with 0.25% of
4-hexylresorcinol and 2% of pyrophosphate for 15 min and
stored for 12 days in packages with modified atmosphere.
A positive correlation between TVB-N and TMA-N was
found in this study both in whole (r 5 0.936, P < 0.01) and
beheaded shrimps (r 5 0.866, P < 0.01). Ruiz-Capillas and
Moral (2001) also found a positive correlation (r 5 0.99,
P < 0.01) between TVB-N and TMA-N in Merluccius
merluccius L. and recommended these indexes to evaluate
the quality of seafood species. Nonetheless, Orban et al.
(2011) indicated that TVB-N and TMA-N are affected by
the size of individuals. Thus, only shrimps of 9.45 6 1.73 cm
were analyzed in this study.
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J.A. GALVAO
TMA-N was also positively correlated with counts of
mesophilic bacteria both in whole (r 5 0.924, P < 0.01) and
beheaded shrimps (r 5 0.907, P < 0.01). Similarly, a positive
correlation between TMA-N and counts of psychrotrophic
microorganisms was found in whole (r 5 0.906, P < 0.01)
and beheaded shrimps (r 5 0.869, P < 0.01). Therefore,
these results are in concordance with that obtained by
Mendes et al. (2005) in Parapenaeus longirostris shrimps and
they advised that microbial population contaminating
shrimps are one of the main responsible to cause the pro-
duction of TMA-N.
Production of Nonprotein Nitrogen (NPN). The
aroma in seafood is mainly influenced by NPN constituents
such as free amino acids, peptides and nucleotides (Sikorski
et al. 1990). Microorganisms and endogenous enzymes act
firstly against these NPN constituents, leading to a gradual
loss of freshness (Contreras-Guzman 1994). A decrease in
the concentration of NPN was observed for 12 days of stor-
age (Fig. 1c), being significantly (P < 0.05) higher in whole
shrimps than in beheaded shrimps (400 and 356 mg
N/100 g, respectively). Draetta et al. (1986) even reported a
higher reduction in NPN concentration (550 mg N/100 g
after 12 days) of whole X. kroyeri shrimps stored in ice. This
reduction is probably due to the lixiviation and/or high for-
mation of volatile compounds during storage. In fact, Akin-
tola and Bakare (2013) observed that NPN decreased only
in Macrobrachium vollenhovenii prawns with direct contact
with ice. In contrast, Kirschnik and Viegas (2004) reported a
significant (P < 0.05) increase in NPN concentrations of
Macrobrachium rosenbergii prawns stored with and without
direct contact with ice for 10 days. They explained that this
increase could be due to the hydrolysis of proteins by endog-
enous and bacterial enzymes. However, a negative correla-
tion between NPN and counts of mesophilic bacteria was
found in this study both in whole (r 5 20.919, P < 0.01)
and beheaded shrimps (r 5 20.969, P < 0.01). Similarly,
NPN was also negatively correlated with counts of psychro-
trophic microorganisms detected in whole (r 5 20.952,
P < 0.01) and beheaded shrimps (r 5 20.951, P < 0.01).
These results, therefore, suggested that differences in the
concentration of NPN constituents are dependent on the
type of metabolism of the predominant bacteria in shrimps.
Meanwhile, the application of antimelanosics 2 particu-
larly, 4-hexylresorcinol 2 produced a significantly (P <
0.05) lower decrease in NPN values after 12 days in compar-
ison with nontreated shrimps. Again, this reduction was sig-
nificantly (P < 0.05) higher in whole shrimps (359, 311, 401
and 399 mg N/100 g for WH1, WH2, WS1 and WS2, respec-
tively) than in beheaded shrimps (348, 241, 386 and 363 mg
N/100 g for BH1, BH2, BS1 and BS2, respectively). These
results suggested that antimelanosics are also able to inhibit
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 ~ ET AL.
J.A. GALVAO 4-HEXYLRESORCINOL ON DETERIORATION OF X. KROYERI

TABLE 4. RESIDUAL SULFITE (mg SO2/100 g) IN WHOLE (W) AND shrimps after 8 and 12 days. Nevertheless, the amount of
BEHEADED (B) SHRIMPS NONTREATED (C) AND TREATED WITH 1.25 residual sulfite detected was lower than the European limits.
AND 2.5% OF SODIUM METABISULFITE (S1 AND S2) AFTER 1, 4, 8 AND
Similar results were reported by Rotllant et al. (2002) in Aris-
12 DAYS
teus antennatus shrimps treated with 1.6 and 2.4% sodium
Days in storage metabisulfite for 5 min. In contrast, G omez-Guillen et al.
Treatment 1 4 8 12 (2005) showed amounts of 15–30 mg SO2/100 g in P. longir-
ostris shrimps, because they were treated with 2.5–5% of
WC 0:0160:00aC 0:0160:00aC 0:0160:00aC 0:0160:00aC
WS1 4:9160:34aB 4:7360:16aB 4:4060:26aC 3:9260:20bC sodium metabisulfite for 1 h. These results demonstrated the
WS2 12:8760:36aA 11:4760:21bA 11:4660:31bA 10:9160:43bA importance of optimized the treatments applied to shrimps
BC 0:0160:00aC 0:0160:00aC 0:0160:00aC 0:0160:00aC (i.e., time of exposure, concentration) to avoid an excess of
BS1 5:1460:34aB 4:4560:27bB 3:9360:25bC 3:3060:30cD residual sulfite. In fact, several studies with commercialized
BS2 12:7560:41aA 11:6660:31bA 9:6760:14cB 9:0460:33dB shrimps confirmed the inadequate use of sodium metabisul-
Superscript letters indicate statistically significant differences (P < 0.05) fite in seafood markets, being detected amounts of residual
between the days of storage, whereas subscript letters denote significant sulfite up to the legal limits (Hardisson et al. 2002; Erkan
differences (P < 0.05) between treatments. et al. 2007). Recently, Andrade et al. (2015) determined that
10 mg of residual SO2/100 g were reached in L. vannamei
the activity of proteolytic enzymes and/or reduce the micro- shrimps immersed in 3% sodium metabisulfite shrimp for
bial population in shrimps. 13 min, so lower concentrations and/or time of exposure are
recommended.
Variation of pH. Brazilian legislation limited the pH of Amounts of residual sulfite decreased significantly
seafood in less than 6.8 (MAPA 2008), although some stud- (P < 0.05) with the time of storage, probably due to lixiviation
ies suggested pH 5 7.8 to be the critical margin for accept- in melting ice. The discard of the cephalothorax did not gener-
ability of prawns and shrimps (Chung and Lain 1979; ated significant differences in the amount of residual sulfite
Mendes et al. 2002). In this study, the pH of samples after 1 and 4 days at similar concentrations of sodium metabi-
increased significantly (P < 0.05) during the 12 days of stor- sulfite. After 8 days, residual sulfite was significantly (P < 0.05)
age, from pH  6.6 at day 1 to pH  7.3 after 12 days. Thus, lower in beheaded shrimps than in whole shrimps treated with
although the legal limit was exceed after 4 days in storage, 2.5% of sodium metabisulfite. Residual sulfite was also signifi-
pH was lower than 7.8 in all cases. Meanwhile, no significant cantly (P < 0.05) lower in beheaded shrimps than in whole
differences were detected at each day of storage between pH shrimps stored for 12 days, but in this case for both treatments.
of whole and beheaded shrimps and between pH of treat-
ments tested. In contrast, Nirmal and Benjakul (2009) Effect on the Melanosis and Color of
reported significant (P < 0.05) differences between pH of Shrimps. Melanosis in non-treated shrimps became unac-
chilled L. vannamei shrimps treated with 1.25% sodium ceptable (score > 2.5) after 2 days in storage (Fig. 2). The
metabisulfite and nontreated shrimps after 4 days in storage.
Some authors stated that biochemical alterations such as an
increase in TVB-N generate this increase of pH in shrimp
muscles (Basavakumar et al. 1998; Vongsawasdi and Noom-
horm 2000; Kirschnik and Viegas 2004). Results obtained in
this study were in concordance, being found a positive cor-
relation between these parameters both in whole (r 5 0.867,
P < 0.01) and beheaded shrimps (r 5 0.888, P < 0.01).

Amount of Residual Sulfite in Samples Treated

With Sodium Metabisulfite. According to Brazilian
legislation (MS 1998), edible shrimp muscle cannot contain
amounts higher than 10 mg of residual SO2/100 g. Mean-
while, European legislation established the limit in 15–30 mg
of residual SO2/100 g of shrimp, depending on the size of
individuals tested (EFFA 1995). As shown in Table 4, the
amount of residual sulfite in shrimps treated with 1.25% FIG. 2. MELANOSIS SCORE OF WHOLE SHRIMPS (W) NONTREATED (C)
sodium metabisulfite was lower than the Brazilian limit. In AND TREATED WITH 0.01 AND 0.1% OF 4-HEXYLRESORCINOL (H1
contrast, shrimps treated with 2.5% sodium metabisulfite AND H2) AND 1.25 AND 2.5% OF SODIUM METABISULFITE (S1 AND
exceeded this limit for residual sulfite, except in beheaded S2) AFTER 1, 2, 4, 6, 8, 10 AND 12 DAYS

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4-HEXYLRESORCINOL ON DETERIORATION OF X. KROYERI ~ ET AL.
J.A. GALVAO

FIG. 3. (a–c) a* (a), b* (b) AND L* (c) OF CEPHALOTHORAX AND ABDOMEN OF WHOLE SHRIMPS (W) NONTREATED (C) AND TREATED WITH 0.01 AND
0.1% OF 4-HEXYLRESORCINOL (H1 AND H2) AND 1.25 AND 2.5% OF SODIUM METABISULFITE (S1 AND S2) AFTER 1, 2, 4, 6, 8, 10 AND 12 DAYS

application of sodium metabisulfite slowed down signifi-
cantly (P < 0.05) the melanosis of shrimps, being acceptable
until 4–5 days. However, no significant differences were sub-
sequently observed with nontreated shrimps, showing all of
them a severe degree of melanosis. G omez-Guillen et al.
(2005) also observed an unacceptable level of melanosis
after 4–6 days in P. longirostris shrimps treated with 1.25–
2.5% of sodium metabisulfite, but immersed for 1 h. Mean-
while, Nirmal and Benjakul (2009) did not observe any dif-
ferences between nontreated Litopenaeus vannamei shrimps
and those treated with 1.25% sodium metabisulfite for
1 min, but melanosis was unacceptable after 6 days.
In contrast, 4-hexylresorcinol showed a higher efficacy
than sodium metabisulfite, reducing melanosis significantly
(P < 0.05) during the 12 days studied. Treatments with
0.01% of 4-hexylresorcinol maintained the degree of mela-
nosis acceptable for 6 days, whereas melanosis of shrimps
treated with 0.1% of 4-hexylresorcinol was acceptable for
8–12 days. Similarly, Guandalini et al. (1998) reported that
0.01% of 4-hexylresorcinol controlled melanosis for 7 days
in P. longirostris shrimps. An acceptable degree of melanosis
was also reported by Thepnuan et al. (2008) in whole
L. vannamei shrimps after 12 days, but treated with 0.25%
of 4-hexylresorcinol and 2% of pyrophosphate for 15 min

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J.A. GALVAO 4-HEXYLRESORCINOL ON DETERIORATION OF X. KROYERI

FIG. 4. (a–e) TOTAL

MESOPHILIC COUNT (A),
TOTAL PSYCHROTROPHIC
COUNT (B), TOTAL COLIFORMS
(C), THERMOTOLERANT
COLIFORMS (D) AND S.
AUREUS (E) DETECTED IN
WHOLE (W) AND BEHEADED
SHRIMPS (B) NONTREATED (C)
AND TREATED WITH 0.01 AND
0.1% OF 4-HEXYLRESORCINOL
(H1 AND H2) AND 1.25 AND
2.5% OF SODIUM
METABISULFITE (S1 AND S2)
AFTER 1, 4, 8 AND 12 DAYS

and packaged in modified atmosphere. Meanwhile,
Montero et al. (2006) used a higher dipping time (1 h) in
0.01–0.5% of 4-hexylresorcinol to achieve similar results to
this study in P. longirostris shrimps. Interestingly, 4-hexyl
resorcinol combined with ascorbic acid, citric acid and
potassium sorbate showed a synergistic effect that delayed
the development of blackspots in Panaeus aztecus shrimps
for 30 days (Pardio et al. 2011). However, excessive amounts
of ascorbic acid can change both the color and taste of
shrimps (Gonçalves and Oliveira 2016). Alternatively, Varlik
et al. (2014) proposed the combination of 4-hexyl resorcinol
with chitosan, citric acid and rosemary extract, which
significantly (P < 0.05) reduced melanosis in P. longirostris
shrimps.

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4-HEXYLRESORCINOL ON DETERIORATION OF X. KROYERI
As shown in Fig. 3a, a* of shrimps increased with the time
of storage, reaching higher values in the cephalothorax than
in the abdomen. Interestingly, this tendency of redness in
the cephalothorax seems to occur simultaneously with mel-
anosis. Application of sodium metabisulfite was able to
reduce significantly (P < 0.05) a* of shrimps, with similar
effects on cephalothorax and abdomen after 12 days. Never-
theless, 4-hexylresorcinol showed a significantly (P < 0.05)
higher effect than sodium metabisulfite, above all in the
cephalothorax, where a slight increase of a* were observed
for 12 days. In contrast, Varlik et al. (2014) observed that the
application of 2,500 mg/L of sodium metabisulfite or 50 mg/
L of 4-hexylresorcinol had a similar effect on a* of chilled P.
longirostris shrimps stored for 6 days. However, this lack of
statistical differences was probably due to the use of shelled
shrimps. Thus, data obtained in this study demonstrated
that the application of antimelanosics (particularly 4-hexyl-
resorcinol) preserved the initial redness for a longer time.
Melanosis probably caused the significant (P < 0.05)
decrease in b* value of the cephalothorax during storage (Fig.
3b). The application of sodium metabisulfite considerably
reduced this diminution, whereas 4-hexylresorcinol pro-
duced in the cephalothorax a slight increase in b*. In contrast,
b* was significantly (P < 0.05) increased in the abdomen,
although antimelanosics moderated this tendency, particu-
larly in shrimps treated with sodium metabisulfite. Varlik
et al. (2014) did not also found significant differences in b*
between these two antimelanosics in chilled P. longirostris
shrimps as a consequence of the use of shelled shrimps.
The L* of shrimps decreased with the time of storage, reach-
ing lower values in the cephalothorax than in the abdomen
(Fig. 3c). In fact, L* of the cephalothorax was highly reduced
during the first 4 days due to the formation of numerous
blackspots. Thus, the lower the value for L*, the more
advanced was the melanosis. This decrease of L* was signifi-
cantly (P < 0.05) diminished by treatments with sodium meta-
bisulfite, above all at a concentration of 2.5%. Several authors
indicated that sulfites are able to bleach the blackspots (McE-
vily et al. 1991; Rotllant et al. 2002), increasing the L* of
shrimps. However, slight differences in L* were observed
initially between nontreated and sulfite-treated shrimps.
Meanwhile, the application of 0.01% of 4-hexylresorcinol
showed a significantly (P < 0.05) higher effect on the decrease
of L* than 2.5% of sodium metabisulfite, both in the cephalo-
thorax and abdomen. Interestingly, an increase in the concen-
tration of 4-hexylresorcinol allowed the significantly
(P < 0.05) high L* of the cephalothorax to be maintained dur-
ing the 12 days of storage in comparison with the other treat-
ments. Varlik et al. (2014) also observed that the application of
50 mg/L of 4-hexylresorcinol had a significantly (P < 0.05)
higher effect on the decrease of L* of shelled P. longirostris
shrimps than 2,500 mg/L of sodium metabisulfite under
chilled conditions.
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J.A. GALVAO
Effect of Antimelanosics on the
Microbiological Quality of Shrimps
Although Brazilian legislation does not establish limits for
mesophilic and psychrotrophic microorganisms, the ICMSF
(1986) stated that counts over 7 log CFU/g reduce consider-
ably the shelf-life of seafood. In this study, the growth both
of mesophilic and psychrotrophic microorganisms signifi-
cantly (P < 0.05) increased during the 12 days of storage
(Fig. 4a,b). However, although initial counts were rather
similar, cold conditions applied to samples permitted a
faster growth of psychrotrophic bacteria. Thus, counts over
7 log CFU/g were only reached by psychrotrophic bacteria
after 12 days, whereas mesophilic bacteria achieved 5 log
CFU/g. A slower growth of psychrotrophic bacteria was
found by Kirschnik and Viegas (2004) in Macrobrachium
rosenbergii after 10 days of storage, probably because it is a
tropical shrimp specie with a predominant mesophilic pop-
ulation, which requires a higher time of adaptation to refrig-
eration. The discard of the cephalothorax allowed the
bacterial population of these shrimps to be maintained sig-
nificantly (P < 0.05) lower. Data obtained in this study
therefore indicated that psychrotrophic bacteria were the
main organisms responsible to cause the spoilage of shrimps
during chilled storage. As aforementioned, bacterial growth
were related to the decrease in NPN and increase in TVB-N
and TMA-N, since bacteria are able to transform NPN into
TVB-N and TMA-N, among other compounds.
The application of sodium metabisulfite produced a sig-
nificant (P < 0.05) decrease in the number of mesophilic
and psychrotrophic microorganisms. Previous studies also
detected an antimicrobial effect of this antimelanosic when
it was applied to other seafood (G oes et al. 2006; Omojowo
et al. 2009). However, the mode of action of sodium metabi-
sulfite (i.e., oxygen scavenging) can favor the growth of
anaerobic pathogens such as Vibrio spp. (Laurila et al. 1998;
Goes et al. 2006). In contrast, 4-hexylresorcinol had no anti-
microbial effect on X. kroyeri shrimps, being in concordance
with previous works (Lopez-caballero et al. 2000; EC 2003).
According to Brazilian legislation (Anvisa 2008), chilled
shrimps cannot contain Salmonella spp. in 25 g of product and
levels of coagulase-positive staphylococci and thermotolerant
coliforms over 3 and 2.7 log CFU/g, respectively. In this study,
Salmonella spp. was absent in all samples tested. Meanwhile,
cold storage conditions maintained the level of coliforms at
approximately 0.5 log MPN/g (Fig. 4c), with most of them
(90%) thermotolerant coliforms (Fig. 4d). Similarly, counts
of S. aureus lower than 1 log CFU/g were noticed on all days
(Fig. 4e). Nonetheless, no significant differences were detected
between shrimps treated with sodium metabisulfite and non-
treated shrimps both in the number of coliforms and S. aureus,
probably due to the low counts detected. Nevertheless, Omo-
jowo et al. (2009) demonstrated that sodium metabisulfite had
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 ~ ET AL.
J.A. GALVAO
antimicrobial activity against these pathogenic bacteria. More-
over, the discard of the cephalothorax only allowed a signifi-
cant (P < 0.05) reduction of the number of S. aureus
contaminating shrimps.
CONCLUSIONS
Data obtained in the present study demonstrated the nutri-
tional value of X. kroyeri shrimps, which could be consid-
ered as an interesting source of proteins and essential
minerals (particularly P, Mg, Fe, Zn and Cu), with low lipid
and energy content. Results also indicated that the discard
of the cephalothorax slows down the deterioration of X.
kroyeri shrimps. The use of beheaded shrimps is therefore
recommended to increase the freshness in chilled products.
The immersion of shrimps in sodium metabisulfite and 4-
hexylresorcinol solutions was also found effective to
decrease both the spoilage and melanosis formation in X.
kroyeri shrimps. In fact, only the pH of shrimps during
chilled storage was not affected by these treatments. Among
them, only sodium metabisulfite showed antimicrobial
properties against mesophilic and psychrotrophic bacteria.
Nevertheless, a higher control on the production of TVB-N,
TMA-N, NPN and blackspots was observed in shrimps
treated with 4-hexylresorcinol. Therefore, although physico-
chemical deterioration of chilled shrimps (i.e., TVB-N,
TMA-N, NPN and pH) was correlated with their microbio-
logical quality (i.e., mesophilic and psychrotrophic counts),
internal processes seemed to have a higher effect. Moreover,
the use of 4-hexylresorcinol avoids the presence of residual
sulfite in shrimps, increasing the safety of products and
reducing the environmental impact.
ACKNOWLEDGMENTS
This work was financially supported by the National Coun-
cil for Scientific and Technological Development (CNPq
479071/2004-7). Author Viviane Angeli Yokoyama was sup-
ported by a CAPES research grant.
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