Abstract

Preserving quality of crustaceans requires efficient modified

atmosphere packaging (MAP). Effects of CO (20%–80%) on quality
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and melanosis in chilled Pacific white shrimp (Litopenaeus

vannamei) under O concentrations of 5 and 15% were investigated.
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Increased headspace CO and lipid oxidation contributed to loss of

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firmness due to protein degradation. Image analysis indicated

increased degree of melanosis at higher O concentration. High
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CO above 60% and 5% O MAP effectively prevented melanosis

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(below 10% for 12 days) and formation of trimethylamine (TMA)

concurrent with limited total viable count (TVC), Enterobacteriaceae
and reduced conversion of pro-polyphenoloxidase into
polyphenoloxidase accelerated shell browning. Linear correlation was
found between TVC-melanosis and TVC-firmness for all MAP
mixtures, while CO gave higher efficacy to inhibit melanosis at low
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O MAP. Increased CO linearly prevented melanosis and TMA

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formation, whereas decreased O concentration gave higher efficacy of

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CO indicated synergistic effects on melanosis and TMA inhibition.

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Introduction
Pacific white shrimp (Litopenaeus vannamei) is one of the most
widely cultivated species in the world. They were originally caught in
the Pacific Ocean but are now farmed commercially as an
economically important crustacean resource in Asian countries and
South America. Both chilled and frozen products are exported to the
main global markets of the United States of America, the European
Union and Japan. Microbial contamination due to handling and
processing, and natural microflora accelerates quality changes in
chilled products which reduce shelf-life. Deterioration is mainly
caused by spoilage bacteria and intrinsic enzymes. This results in loss
of organoleptic quality together with discoloration and consumption
safety (Qian et al., 2016).
Dark pigmentation or so-called ‘melanosis’ indicates major
deterioration in quality that occurs along swimmerets, head, tail and
shells of shrimp, crab and other crustaceans e.g. Parapenaeus
longirostris, Penaeus duorarum, Penaeus striferus, Penaeus
japonicus, Charybdis japonica, Nephrops norvegicus, Homarus
japonicas and Litopenaeus vannamei. Melanosis involves oxidative
enzymatic browning which adversely causes discoloration in
crustacean products. Phenol oxidase (EC 1.14.18.1), known as
tyrosinase, phenolase and polyphenol oxidase (PPO) catalyzes the
hydroxylation of tyrosine to o-dihydroxyphenylalanine (DOPA), while
further oxidation of DOPA and other o-phenols to o-quinones
produces brown melanin pigments or polymerization with protein
forming cross-linked polymers (Benjakul et al., 2006, Friedman,
1996, Martínez-Alvarez et al., 2005). PPO enzyme occurs naturally
under the shells of shrimp and other crustaceans, while pro-PPO is
presented as zymogens (Gonçalves & de Oliveira, 2016).
Melanosis is harmless; however, consumers reject the products due
to their unsatisfactory appearance. For crustaceans, particularly
shrimp, melanosis usually occurs shortly after harvest and is more
important as a limiting factor for shelf-life than microorganisms
(Martínez-Alvarez et al., 2005). Chemical treatments, particularly
with sulfites, are widely used to inhibit melanosis in chilled seafood.
However, sulfite compounds adversely impact consumer safety
through asthma and allergies. Thus, several investigations on
alternative treatment for melanosis inhibition have involved the use
of natural substances to trap the color intermediates or reduce the o-
quinones to colorless compounds (Benjakul et al., 2006, Bono et al.,
2012, Gonçalves and de Oliveira, 2016, Martínez-Alvarez et al.,
2005). The extent of melanosis has been reported by color
measurement and whiteness index (Qian et al., 2016), evaluation by
trained panels (Bono et al., 2016, Martínez-Alvarez et al., 2005,
Thepnuan et al., 2008), and evaluation of organoleptic quality using
untrained panels (Qian, Yang, Xie, Xiong, & Gao, 2013). Here, we
evaluated the degree of melanosis using image analysis to directly
reflect the total area of visual dark pigmentation.
Modified atmosphere packaging (MAP) can substantially extend the
shelf-life of seafood by inhibiting microbial growth and lipid
oxidation. Carbon dioxide (CO ) plays a major role in microbial
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reduction and improves the shelf-life of various seafood products by

increasing both the lag phase and generation time for spoilage
microorganisms (Daniels, Krishnamurthi, & Rizvi, 1985). Moreover,
previous studies showed that MAP retarded melanosis in Pacific
white shrimp using CO :O :N of 80:10:10 and 80:20:0 (Thepnuan et
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al., 2008), 80:5:15, 80:10:10 and 80:20:0 (Qian et al., 2013), 95:5:0,
80:5:15 and 65:5:30 (Qian et al., 2016), 53:7:40 in pink shrimp
(Martínez-Alvarez et al., 2005), and 0:0:100 and 50:0:50 in giant red
shrimp (Bono et al., 2016). These results showed that certain
amounts of CO and O delayed melanosis formation. However, no
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systematic investigation has determined the correlation between gas

conditions and extent of melanosis. Lack of investigation indicated
minimum or exact CO concentration that gives efficacy for melanosis
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inhibition. Moreover, the roles of CO and O as inhibitors of

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melanosis require further detailed investigation to design an efficient

MAP system.
Therefore, the objective of this research was to investigate the effects
of CO on quality and melanosis of Pacific white shrimp at different
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O concentrations of 15% and 5%. Moreover, the correlation between

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gas concentrations and melanosis was also investigated to design

optimal MAP conditions for Pacific white shrimp. It was hypothesized
that certain level of CO in MAP limited microbial growth which
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subsequently retarded melanosis.

Section snippets

Sample preparation and modified atmosphere packaging

The antennae of Pacific white shrimp (50–55 pieces/kg, from
Mahachai Farm, Thailand) were removed and the shrimp were
prepared on the day of purchase by soaking in cold saline solution
(4% w/w) for 3 min and gently draining on a sieve at 4 °C for 3 min to
remove leftover liquid on their surfaces.
Modified atmosphere packaging was performed in hermetically
sealed glass bottles (124 mm height × 81.5 mm diameter). A gas-to-
product ratio (volume/weight) of 6.3:1 was applied for shrimp packed
under

Headspace gas analysis and CO solubility 2

Headspace analysis revealed change of gas components during

storage (Fig. 1). O concentration decreased by 1% to 5% in the first
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6 days in all systems, indicating that O reduction was independent of

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CO concentration. Less than 1% O was found in all packages on day

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12, except for CO :O :N of 80:5:15 system (~2%), due to microbial

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growth and enzymatic reactions leading to O consumption. Several

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oxidative enzymes made use of O in the headspace to initiate

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biochemical reactions. High level

Conclusions
Increased CO from 20% to 80% and decreased O from 15% to 5%
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effectively reduced microbial growth and hence inhibited melanosis.

Decreased O concurrent with increased CO indicated aerobic
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respiration due to microbial growth at the early storage stage.

Approximately 4% decrease of O was found on day 6 for shrimp
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stored at 4 °C in both O conditions. CO at and above 40% effectively

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reduced TVC and Enterobacteriaceae but showed less effectiveness

on psychrophiles. Increased microbial growth

CRediT authorship contribution statement

Netchanok Kimbuathong: Conceptualization, Methodology,
Writing - original draft. Pattarin Leelaphiwat: Writing - review &
editing. Nathdanai Harnkarnsujarit: Conceptualization,
Methodology, Supervision, Writing - original draft, Writing - review
& editing.

Declaration of Competing Interest

The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to
influence the work reported in this paper.

Acknowledgements
This work was supported by Department of Packaging and Materials
Technology, Faculty of Agro-Industry, Kasetsart
University and Kasetsart University Research and Development
Institute (KURDI).