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Inhibition of Melanosis in Whiteleg Shrimp ( Litopenaeus vannamei ) during

Refrigerated Storage Using Extracts of Different Avocado ( Persea americana
Mill.) By-Products

Article  in  Preventive Nutrition and Food Science · June 2021

DOI: 10.3746/pnf.2021.26.2.209

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Prev. Nutr. Food Sci. 2021;26(2):209-218
https://doi.org/10.3746/pnf.2021.26.2.209
ISSN 2287-8602

Inhibition of Melanosis in Whiteleg Shrimp (Litopenaeus

vannamei) during Refrigerated Storage Using Extracts of
Different Avocado (Persea americana Mill.) By-Products

Dao Thi Anh Phan1, Trung Huu Bui1, Tram Quynh Thi Doan1, Nguyen Van Nguyen2, and Trieu Hai Ly3
1
Faculty of Chemical and Food Technology, HCMC University of Technology and Education, Ho Chi Minh 70000, Viet Nam
2
Research Center for Aquafeed Nutrition and Fishery Post-Harvest Technology (APOTEC), Ho Chi Minh 70000, Viet Nam
3
Research Center of Ginseng and Medicinal Materials, National Institute of Medicinal Materials, Ho Chi Minh 70000, Viet Nam

ABSTRACT: Melanosis in shrimp usually leads to reduction in its shelf life and quality, which causes a significant loss in
economic value of shrimp products. This study reports potential applications of nine ethanolic extracts of by-products,
i.e., peel and/or seed from three Vietnamese avocado varieties as effective inhibitors of melanosis in whiteleg shrimp. Six
out of nine shrimp samples treated with the prepared extracts (0.025%, w/v) reduced melanosis and lipid oxidation more
significantly as compared to those treated with sodium metabisulfite (SMS, 1.25%, w/v) and control groups (treated with
o
water) during 8-day storage at 4 C (P<0.05). These six extracts had mean gray values ranging from 47.0±0.7 to 57.3±
0.4% were lower than those treated with SMS (mean gray of 39.8±0.4%). The inhibition of melanosis and lipid oxidation
in shrimp for these extracts could be attributed to their high content of polyphenols [total phenolic content (TPC) from
44.5±1.1 to 144.7±1.9 mg gallic acid equivalents/g dried weight] and strong antioxidant activities [including 2,2-diphen-
yl-1-picrylhydrazyl (DPPH) radical scavenging, ferric reducing antioxidant power (FRAP), and tyrosinase enzyme inhibi-
tion]. Pearson statistical analysis showed strong correlation for melanosis inhibition to TPC and DPPH scavenging (r>
0.80) followed by tyrosinase inhibition and FRAP (r>0.50). The findings obtained from this study suggest potential uti-
lization of avocado by-product extracts as safe and cheap natural alternatives to traditional sulfites for anti-melanosis and
shelf life extension of whiteleg shrimp.

Keywords: antioxidant, avocado by-products, melanosis, tyrosinase, whiteleg shrimp

INTRODUCTION nosis development in shrimp during storage, such as

treatments with sulfite and phosphate compounds (Mar-
Whiteleg shrimp (Litopenaeus vannamei) is one of the most tínez-Álvarez et al., 2008; Galvão et al., 2017). However,
widely cultured and consumed shrimp species because the potential risks of using those chemical additives, i.e.,
of its delicacy, accounting for 80% of the global shrimp nausea, abdominal pain, vomiting, and choking have
production (Jescovitch et al., 2018). However, this high- raised awareness among customers and regulators. Con-
value shrimp has a limited shelf life, in which melanosis sequently, researchers and scientists worldwide have re-
(or black spot) formation during postmortem storage is cently paid considerable attention to natural, safe, and
problematic (Gonçalves and Oliveira, 2016), and causes effective additives (Djeridane et al., 2006; Encarnacion
substantial loss in the nutritional and economic value of et al., 2012). Natural antioxidant substances have been
these shrimp products. widely studied as substitutes to chemical additives to
Melanosis in shrimp and other crustaceans is developed prevent melanosis. Natural additives, such as tocopher-
by the oxidation of phenol substrates (such as tyrosine ols, flavonoids, cinnamic acids, and coumarins were re-
in shrimp) to quinones catalyzed by polyphenolase, which ported to exhibit potent antioxidant, antimicrobial, and
generates dark pigments of high molecular compounds polyphenoloxidase (PPO) inhibitory activities and thus,
(Sae-leaw and Benjakul, 2019). From a commercial per- they could prevent the melanosis process (Gonçalves and
spective, some methods have been used to prevent mela- de Oliveira, 2016). Plant extracts containing polyphenol-

Received 18 January 2021; Revised 27 March 2021; Accepted 12 April 2021; Published online 30 June 2021
Correspondence to Phan Thi Anh Dao, Tel: +84-902-373-656, E-mail: [email protected]
Author information: Dao Thi Anh Phan (Professor), Trung Huu Bui (Professor), Tram Quynh Thi Doan (Graduate Student)
Copyright © 2021 by The Korean Society of Food Science and Nutrition. All rights Reserved.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits
unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
210 Phan et al.

ic compounds demonstrated a significant retardation of rieties from Viet Nam, and determination of their total
melanosis formation in crustaceans, which was associated phenolic content (TPC) and antioxidant activities; (ii)
with tyrosinase inhibitory activity (Sae-leaw and Benjakul, evaluation of the capability of these prepared extracts in
2019). Nirmal and Benjakul (2012) reported that the preventing melanosis formation and lipid oxidation in
ethanolic extract of green tea containing a high amount whiteleg shrimp during 8 days storage at 4oC; and (iii)
of catechin and derivatives could significantly decrease discussion on the relationship between the inhibition on
the lipid oxidation and melanosis formation in whiteleg melanosis of these prepared extracts and their TPC val-
shrimp during cold storage. Encarnacion et al. (2012) ues and antioxidant activities. The utilization of these by-
prepared an extract of edible mushroom (Flammulina ve- products as food additives could not only reduce the neg-
lutipes) containing rich ergothioneine, which showed pos- ative impact on the environment but also expand the
itive effects on melanosis prevention in shrimps Penaeus range of avocado value-added products.
monodon and Litopenaeus vannamei. Chamuang leaf extract
and lead (Leucaena leucocephala) seed extract that were
rich in phenolic compounds effectively inhibited mela- MATERIALS AND METHODS
nosis in whiteleg shrimp (Nirmal and Benjakul, 2011;
Shiekh et al., 2019). Materials and chemicals
Recently, studies on utilizing agricultural by-products Folin-Ciocalteu reagent, iron (III) chloride hexahydrate
for food additives and preservation have been extensively (FeCl3･6H2O), malondialdehyde (MDA), sodium metabi-
investigated due to economic and environmental benefits sulfite (SMS), potassium hexacyanoferrate [K3Fe(CN)6],
(Gómez et al., 2014; Saavedra et al., 2017). Among vari- thiobarbituric acid (TBA), trichloracetic acid (TCA), gal-
ous by-products, avocado seed and peel contained great lic acid, kojic acid, 2,2-diphenyl-1-picrylhydrazyl (DPPH),
amounts of phenolic compounds, including flavanol mon- and tyrosinase were purchased from Sigma-Aldrich Co.
omers, proanthocyanidins, hydroxycinnamic acids, and (St. Louis, MO, USA). All other chemicals were of analyt-
flavonol glycosides (Kosińska et al., 2012). This would at- ical grade.
tribute to a high potential of antioxidant activities of those By-products (peels and seeds) from three popular vari-
by-products. Rodríguez-Carpena et al. (2011) showed eties of avocado, namely ‘Nuoc’, ‘Sap’, and ‘034’ were
that raw porcine patties treated with the extracts of avo- supplied from several local markets in Ho Chi Minh City,
cado peel and seed could effectively inhibit lipid oxida- Viet Nam. The samples were then dried to the moisture
tion and color deterioration during chilled storage. Sev- content of about 10% [using a gravimetric method fol-
eral studies also reported the applications of avocado by- lowed by the European Brewery Convention (2000)]. The
products as potential sources of natural antioxidants or dried samples were subsequently ground in a blender in-
preservatives (Gómez et al., 2014: Saavedra et al., 2017; to a homogenous fine pulp that was then extracted with
Gashahun and Solomon, 2018). ethanol.
It is worth mentioning that avocado peel and seed are Alive whiteleg shrimp with the size of 30∼40 shrimps/
discarded as wastes, accounting for about 20% (w/w) of kg were purchased from Tan Binh market (Ho Chi Minh
the fruit (Wang et al., 2010). Avocados are widely grown City, Viet Nam). The shrimps were immediately trans-
in Viet Nam, which has been considered as one of the ported to the laboratory for the experiments.
seven important fruits crops in the country. Consequent-
ly, a large quantity of avocado by-products is discarded Preparation of extracts
from fruit processing annually. Although the phenolic An extract of each avocado by-product was prepared us-
compounds, such as catechin, quercetin, and their deriv- ing a maceration technique (Azwanida, 2015). Dried
atives from avocado by-products could exhibit a potential powder (30 g) was soaked in 100 mL of 70% ethanol
o
in preventing melanosis on crustaceans or shrimps (Nir- (v/v) at 32 C for 24 h. The mixture was filtered to col-
mal and Benjakul, 2012; Qian et al., 2018; Tremocoldi et lect the extract solution, and the remaining residue was
al., 2018; Rosero et al., 2019), to the best of our knowl- re-extracted with ethanol twice using the same procedure.
edge, there has been no report on utilizing avocado by- Then, the mixture of three collected extracts was concen-
products as potential natural additives to prevent mela- trated using a rotary evaporator to produce a crude ex-
nosis and/or extend the shelf life of whiteleg shrimp. tract. Table 1 presents the extraction yield (E%) of the
In this study, the roles of some popular avocado by- nine prepared avocado by-product extracts. It includes
products on the retardation of melanosis and lipid oxi- three seed extracts and three peel extracts prepared from
dation in whiteleg shrimp during cold storage were eval- each variety [‘Sap’, ‘034’, and ‘Nuoc’: No. (1) to (6)], and
uated and discussed. The study aimed to obtain three three mixed extracts [No. (7) to (9)] consisting of peel
main objectives: (i) preparation of nine ethanolic extracts mixture, seed mixture, and peel-seed mixture from 3 va-
from by-products (seeds and peels) of three avocado va- rieties mixed in equal weights. Note, the extraction yield
 Preserve Shrimp by Avocado By-Products 211

Table 1. The extraction yield of nine extracts prepared from Ferric reducing antioxidant power assay: Ferric reducing anti-
by-products of three avocado varieties: ‘Sap’, ‘034’, ‘Nuoc’ oxidant power (FRAP) assay was conducted according to
Sample Extraction the method reported by Yen and Chen (1995). Different
No. Formulation/mixture
name yield (%) amounts of each extract (0.1, 0.5, and 1.0 mg) were
1 SS Sap seed 12.5 mixed with 1 mL of 2 M phosphate buffer (pH 6.6) and
2 SP Sap peel 10.0 1 mL of 1% K3Fe(CN)6. After incubating for 20 min, 1
3 OS 034 seed 10.1 mL of 10% TCA was added, blended, and centrifuged at
4 OP 034 peel 7.6 4,000 rpm for 10 min to collect the supernatant. Subse-
5 NS Nuoc seed 18.1
quently, 1 mL of the supernatant was diluted with 1 mL
6 NP Nuoc peel 7.8
7 MS Sap seed + 034 seed + Nuoc seed 13.5
of deionized water and then mixed with 0.2 mL of FeCl3
8 MP Sap peel + 034 peel + Nuoc peel 7.6 (0.1%). The absorbance was measured at 700 nm. The re-
9 MSP Mixture of all seeds and peels 9.8 ducing capacity was calculated based on the increase in
absorbance. Vitamin C was used as a positive control at
various concentrations (0.1, 0.5, and 1.0 μM).
(E%) can be calculated as in Eq. (1): Tyrosinase inhibitory assay: Tyrosinase inhibitory activity
was performed following the procedure established by
mextract Nirmal and Benjakul (2011), with slight modifications
Yield (%)= ×100 (1)
mpowder using a 96-well reader. Each 40 μL of the extract (100 μg/
mL) mixed with 20 μL of tyrosinase (100 U/mL) was in-
where mextract is the weight (g) of the dried extract resi- cubated at room temperature for 30 min. The reaction
due obtained after solvent removal, and mpowder is the was initiated with the addition of 140 μL of 0.714 mM
o
weight (g) of plant powder. L-3,4-dihydroxyphenylalanine (L-DOPA) at 45 C for 5
min. The absorbance of the mixture was then measured
Determination of TPC at 475 nm. The tyrosinase inhibitory percentage and IC50
The TPC of the prepared extracts was determined using values were calculated similarly to those of the DPPH as-
the colorimetric Folin-Ciocalteu method (Jung et al., say. Kojic acid was used as a positive control with the IC50
2008). The calibration curve was constructed using gallic value of 42.80 μg/mL.
acid as standard at a concentration range of 0 to 20 mg/L,
showing a linear equation of y=0.1027x+0.0365 (R2= Evaluation of melanosis formation and lipid peroxidation
0.9979). The TPC value was expressed as mg gallic acid inhibition in shrimp
equivalents (GAE) per gram dried weight (g DW). Each Shrimp treatment: Shrimp were divided into 11 equal parts.
experiment was performed in triplicate. Each part was immersed in either each of the extract so-
lutions (0.025%, w/v), or the SMS solution (1.25%, w/
Antioxidant activities v), or deionized water (as the control), in the ratio of 1:2
DPPH assay: The scavenging activity of DPPH radical was (shrimp/solution, w/v) at 4oC for 15 min. Subsequently,
performed using a method documented by Molyneux the treated shrimp were placed in polypropylene boxes
o
(2004). Each sample (1.5 mL) at four concentrations of and stored at 4 C for eight days. Note that the control
10, 25, 50, and 100 μg/L (in 90% ethanol) was mixed samples, known as blank samples, were performed by
with 1.5 mL of 0.1 mM DPPH solution and the mixture using deionized water as a replacement for the extract
was then incubated for 30 min under dark. The absorb- solutions.
ance of the obtained mixture and blank samples (without Melanosis evaluation: Visual images of the shrimp samples
extract) was measured at 519 nm. The inhibitory percent- during storage were taken daily. The changes in color of
age of DPPH from the tested samples was calculated as the samples were analyzed using ImageJ software (ver-
in Eq. (2): sion 1.52a, National Institutes of Health, Bethesda, MD,
USA). Relative changes in the gray value of the carapace
B were calculated as in Eq. (3) (Encarnacion et al., 2012;
I (%)=(1− )×100 (2)
A Sae-leaw and Benjakul, 2019). The decrease in the % rel-
ative change indicates an increase of melanosis formation
where A is the absorbance of the test sample, and B is the in shrimp during storage. Three shrimps were used for
absorbance of the blank sample. one treatment:
The half-maximal inhibitory concentration (IC50) was
calculated from the mean values of three measurements C×100
% Relative change=100− (3)
at the used concentration range. Gallic acid was used as D
a positive control with the IC50 of 5.62 μg/mL.
212 Phan et al.

where C is the actual gray value of the shrimp sample,

and D is the average gray value of the shrimp on the first
day.
Lipid peroxidation inhibition assay: The peroxidation prod-
ucts (TBA reactive substances, TBARS) produced during
shrimp preservation were analyzed using the procedure
established by Benjakul and Bauer (2001). Ground shrimp
(10 g) were thoroughly mixed with 10 mL of 7.5% w/v
TCA and filtered to collect the filtrate. Subsequently, the
filtrate was mixed with 0.02 M TBA solution (at the same
volume ratio) and incubated at 100oC for 15 min. The
mixture was then cooled to about 32oC, and the absorb-
ance was measured at 532 nm. The calibration curve was
constructed using MDA standard solutions at concentra- Fig. 1. Total phenolic content (TPC) of 9 avocado by-product ex-
tions from 0.01 to 0.05 μM. The obtained linear equation tracts [Sap seed (SS), Sap peel (SP), 034 seed (OS), 034 peel
2 (OP), Nuoc seed (NS), Nuoc peel (NP), seed mixture (MS), peel
was y=0.0441x+0.0148 (R =0.9993). The TBARS values mixture (MP), and peel and seed mixture (MSP)]. Values repre-
were expressed as mg MAD/kg shrimp. sent the mean±standard deviation (n=3). Different letters (a-f)
indicate significant differences (P <0.05).
Analysis method
Instrument analysis: A Hitachi UH-530 ultraviolet/visible (P<0.05). It is clearly seen that TPC values for the three
spectrophotometer (Hitachi, Ltd., Tokyo, Japan) was used mixed extracts [mixture of all seeds (MS), mixture of all
to measure the absorbance at a specific wavelength con- peels (MP), and mixture of all seeds and peels (MSP)]
ducted in the experiments for TPC, antioxidant assays, were found in the order of MSP (45.8±2.0 mg/g DW)<
and lipid peroxidation assay. Photographs of the shrimps MS (85.52±1.4 mg/g DW)< MP (86.6±3.8 mg/g DW)
during 8 days storage were taken using a Canon Eos M10 (P<0.05). The results of TPC in this study were com-
Kit Ef-M15-45 (Canon Inc., Tokyo, Japan). An ImageJ parable or even higher than those reported in a previous
software (version 1.52a, National Institutes of Health) study for other types of avocado by-product, e.g., Hass
was used to analyze the color changes for melanosis de- peel (63.5±7.2 mg/g DW), Hass seed (57.3±2.7 mg/g
termination. DW), Fuerte peel (120.3±7.8 mg/g DW), and Fuerte
Statistical analysis: Experimental data were expressed as seed (59.2±6.9 mg/g DW) (Tremocoldi et al., 2018).
mean±standard deviation (SD) and analyzed by analysis Githinji et al. (2013) showed the TPC of Fuerte seed ex-
of variance (ANOVA) with Tukey’s test (P<0.05). All tract was only 18.5±2.8 mg/g DW. The TPC of avocado
measurements were replicated three times. Statistical cal- seed and peel [29.9±1.1 (OS) and 144.7±1.9 (NP) mg
culations were carried out using a SPSS package (SPSS GAE/g DW respectively] in this study was greater than
20 for Windows Evaluation Version, IBM Corporation, that reported for blueberry (9.44±0.22 mg GAE/g DW),
Armonk, NY, USA). blackberry (5.58±0.18 mg GAE/g DW), and strawberry
(2.72±0.18 mg GAE/g DW), all of which have been
known for their high antioxidant capacity (Huang et al.,
RESULTS AND DISCUSSION 2012). A variety of phenolic compounds has been re-
ported in avocado seed and peel by-products such as, fla-
Total phenolic content vanol monomers, proanthocyanidins, hydroxycinnamic
Fig. 1 shows the TPC of the prepared extracts. All ex- acids, and flavonol glycosides, 3-O-caffeoylquinic acid,
tracts had high TPC with values ranging between 44.5± 3-O-p-coumaroylquinic acid, and procyanidin A trimers
1.1 [‘034’ seed (OS)] and 144.7±1.9 mg GAE/g DW (Kosińska et al., 2012). Tremocoldi et al. (2018) reported
[‘Nuoc’ peel (NP)] (P<0.05). For each type of avocado that procyanidin B2 and epicatechin in avocado peel and
by-product, the extract from the peel exhibited higher trans-5-O-caffeoyl-D-quinic acid, procyanidin B1, catechin,
TPC than that from the seed [‘Sap’ peel (SP)> ‘Sap’ seed and epicatechin in avocado seed played a key role in an-
(SS), ‘034’ peel (OP)> OS, and NP> ‘Nuoc’ peel (NS), tioxidant and anti-inflammatory activities. It could be
respectively]. Similar results were reported in a previous concluded that the selected avocado by-products in this
study on TPC in Hass and Fuerte avocado (Tremocoldi study were valuable sources of antioxidants, especially
et al., 2018). Specifically, the extracts of ‘Nuoc’ variety phenolic compounds.
exhibited the highest TPC values (114.7±3.5 and 144.7
±1.9 mg GAE/g DW for NS and NP, respectively), fol- Melanosis evaluation
lowed by ‘Sap’ and ‘034’ avocado by-product extracts Fig. 2 shows the melanosis scores in shrimp carapace
 Preserve Shrimp by Avocado By-Products 213

Fig. 2. Changes in mean gray values of the carapace area of whiteleg shrimp, Litopenaus vannamei treated with different extracts
[Sap seed (SS), Sap peel (SP), 034 seed (OS), 034 peel (OP), Nuoc seed (NS), Nuoc peel (NP), seed mixture (MS), peel mixture
(MP), and peel and seed mixture (MSP)] during refrigerated storage, compared with that of sodium metabisulfite (SMS) and the
control. Different uppercase letters (A-D) are related to significant differences among other storage days in the same treatment
(P <0.05). Different lowercase letters (a-g) showed significant differences among other treatments in the same storage time
(P <0.05).

treated with avocado by-product extracts (0.025%, w/v), which was comparable to the prepared extracts in this
SMS (1.25%, w/v), and the control (with water) during study after 4 days storage at similar treatments with
8 days storage at 4oC. Changes in mean gray value were mean gray values in the range of 77.10 to 81.12% for NP,
the indication of melanosis. It is also worth noting that MS, MSP, MP, NS, and SP. Overall, the results showed
high melanosis prevention level was associated with low the paramount importance of adding value to avocado by-
decreasing level in mean gray value during storage. At day products for melanosis treatment of whiteleg shrimp as
0, there were no significant differences in the gray values a cheap natural source and substituting for SMS.
(P>0.05) among the shrimp samples. However, after 4
days storage, most samples treated with the prepared ex- Lipid peroxidation inhibition
tracts (SP, NS, NP, MS, MP, and MSP) demonstrated a Fig. 3 shows changes in TBARS values of whiteleg shrimp
slight decline in the mean gray values, which showed the treated with the prepared extracts (0.025%, w/v), SMS
effective prevention of melanosis. The mean gray values (1.25%, w/v), and control (deionized water). No signifi-
then significantly decreased from day 4 to day 6 but the cant difference was observed in TBARS values among all
changes were limited the last 2 days (P<0.05). Conse- samples at day 0 (P>0.05). The TBARS values of all treat-
quently, after 8 days of storage, samples treated with the ment batches overall showed an upward trend at various
six extracts (NP, NS, SP, MS, MP, and MSP) had mean degrees (P<0.05), indicating an increase in lipid perox-
gray values ranging from 47.0±0.7 to 57.3±0.4% that idation over storage. It is noticeable that all extracts of
were significantly higher than those in SMS-treated sam- avocado by-products impeded the degree of lipid perox-
ples (the mean gray value of 39.8±0.4%), meaning that idation compared with SMS and the control groups dur-
the six extracts demonstrated better melanosis preven- ing 8 days storage (P<0.05). Among the samples, the NP
tion than SMS. Among them, NP extract showed the treatment had the smallest TBARS value of 3.3±0.1 (mg
highest efficiency of retarding melanosis in shrimp with MDA/kg), whereas the OS treatment showed the highest
the mean gray value of 57.3±0.4%. Another noticeable TBARS value of 5.5±0.1 (mg MDA/kg) at the end of the
point was that samples stored with ternary or multiple period (P<0.05). These findings indicated that extracts of
mixtures demonstrated higher mean gray values than avocado by-products could mitigate the peroxidation of
those stored with single extracts, except for NP (P<0.05). lipids in shrimp samples during storage, which was also
Other plant extracts from oyster mushroom (Llanto and reported in the literature for various plant extracts. Basiri
Encarnacion, 2018), pomegranate (Basiri et al., 2015; et al. (2015) revealed that after 10 days of refrigerated
Yuan et al., 2016a), green tea (Nirmal and Benjakul, storage, whiteleg shrimp treated with pomegranate peel
2012; Yuan et al., 2016b), and grape seed (Sun et al., extract had 30% lower TBARS values compared to the
2014) were also reported to retard melanosis. For in- control group. Similarly, Abbasvali et al. (2016) reported
stance, whiteleg shrimp treated with Pleurotus ostreatus, that aqueous saffron (Crocus sativus) tepal extract inhib-
mushroom extract recorded a relative change of about ited lipid oxidation in treated shrimp during 9 days of ic-
80% after 3 days storage (Llanto and Encarnacion, 2018), ed storage. Other authors reported a delay in the TBARS
214 Phan et al.

Fig. 3. Changes in TBARS values of whiteleg shrimp, Litopenaus vannamei treated with different extracts [Sap seed (SS), Sap
peel (SP), 034 seed (OS), 034 peel (OP), Nuoc seed (NS), Nuoc peel (NP), seed mixture (MS), peel mixture (MP), and peel and
seed mixture (MSP)] during refrigerated storage, compared with that of sodium metabisulfite (SMS) and the control. Different
uppercase letters (A-E) are related to significant differences among other storage days in the same treatment (P <0.05). Different
lowercase letters (a-h) showed significant differences among other treatments in the same storage time (P <0.05).

values in L. vannamei shrimp treated with chamuang (Gar- ing activity (IC50=63.1 μg/mL). Notably, no significant
cinia cowa Roxb.) leaf extract (Shiekh et al., 2019), a green difference (P>0.05) was observed in the DPPH scaveng-
tea extract in combination with ascorbic acid (Nirmal and ing activity among ternary, and multiple mixtures with
Benjakul, 2012), and a coat with quince seed mucilage the inhibition level ranging from 94.3±0.1% to 94.9±
and green tea extract (Noshad et al., 2017). 0.3% (IC50=5.5∼8.6 μg/mL).
At the highest concentration of 100 μg/mL, the pre-
Antioxidant activities pared extracts displayed DPPH scavenging percentage
DPPH radical scavenging assay: Fig. 4 shows the result of between 73.4±1.0 and 99.0±0.5% and IC50 values of 3.6
DPPH radical scavenging activity of the prepared extracts, to 63.1 μg/mL. It is noted that the scavenging activity of
acting in a dose-dependent manner. It is noted that, for avocado by-products in this study was significantly higher
each type of avocado, the peel extract possessed consid- than those of avocado peels and seeds in previous stud-
erably higher activity than the seed extract, which was ies. Melgar et al. (2018) showed that IC50 values calcu-
also observed for the mixtures containing peels. Among lated from a DPPH radical scavenging assay for the hy-
the extracts, at the highest concentration of 100 μg/mL, droethanolic extracts of Persea america Mill. var. Hass peel
SP exhibited the highest inhibitory activity with 97.0±0.5 and kernel were 149±5 and 220±3 μg/mL, respectively.
% DPPH scavenging activity (IC50=3.6 μg/mL), whereas Antasionasti et al. (2017) reported that the most active
OS showed the lowest activity with 73.4±1.0% scaveng- fraction of methanolic extract from avocado peel was able

Fig. 4. DPPH radical inhibitory activity of 9 avocado by-product extracts [Sap seed (SS), Sap peel (SP), 034 seed (OS), 034 peel
(OP), Nuoc seed (NS), Nuoc peel (NP), seed mixture (MS), peel mixture (MP), and peel and seed mixture (MSP)]. Different uppercase
letters (A-E) are related to significant differences among other storage days in the same treatment (P <0.05). Different lowercase
letters (a-f) showed significant differences among other treatments in the same storage time (P <0.05).
 Preserve Shrimp by Avocado By-Products 215

tract possessed the highest FRAP, while the OP extract

showed the lowest activity (P<0.05). These ferric reduc-
ing capacities could be comparable to those polyphenols
and flavonoids, such as callistephin, malvin, ID-8, sily-
christin, pelargonin, oenin, 2,4,6-trihydroxybenzaldehyde,
3,4-dihydroxy-5-methoxybenzoic acid, and arachidonoyl
dopamine (the absorbance range from 0.615 to 2.430 at
30 μg/mL) (Huyut et al., 2017). It indicates that the by-
products investigated in this study were great antioxidant
sources for potential food applications.
Tyrosinase enzyme inhibition assay: Fig. 6 shows the effects
of the prepared extracts on the tyrosinase inhibition us-
ing L-DOPA as a substrate. Like DPPH and FRAP assays,
Fig. 5. Reducing power of 9 avocado by-product extracts [Sap all the extracts illustrated positive effects on the tyrosi-
seed (SS), Sap peel (SP), 034 seed (OS), 034 peel (OP), Nuoc nase inhibition with a dose-dependent property. The lev-
seed (NS), Nuoc peel (NP), seed mixture (MS), peel mixture
(MP), and peel and seed mixture (MSP)]. Different uppercase el of inhibition at the highest concentration of 100 μg/mL
letters (A-C) are related to significant differences among other ranged from 32.6±2.1 to 65.8±2.8. Among the nine ex-
storage days in the same treatment (P <0.05). Different lower- tracts, those from ‘Nuoc’ and ‘Sap’ avocado peels and
case letters (a-f) showed significant differences among other
treatments in the same storage time (P <0.05). seeds (i.e., NP, NS, SP, and SS) exhibited better inhibi-
tion levels [I>50%, IC50 values from 48.0 to 94.6 μg/mL
(P<0.05)] as compared to the other five extracts. In ad-
to scavenge DPPH with an IC50 value of 4,221±137 μg/ dition, tyrosinase inhibition capacities of ‘Nuoc’ and ‘Sap’
mL. The high DPPH scavenging activity of these investi- avocado peel and seed extracts were similar to most of
gated extracts was consistent with their high TPC (Fig. the 50 methanolic extracts from Sudanese medicinal
2). A possible explanation was that phenolic compounds plants whose inhibition levels were from 1.58 to 84.39%
could provide hydrogen atoms or donate electrons, which at 125 μg/mL (Muddathir et al., 2017). Laksmiani et al.
quenched DPPH free radicals to stabilize diamagnetic (2020) found that the tyrosinase inhibitory activity of
molecules, leading to a decrease in the absorbance ethyl acetate extract of avocado seed possessed an IC50
(Mathew et al., 2015). value of 93.02 μg/mL, which was lower than that for NP,
Ferric reducing power assay: The antioxidant activity of the SP, and NS in this study. This high inhibitory activity to-
prepared extracts analyzed via FRAP assay was shown in wards tyrosinase of the prepared extracts could be attrib-
Fig. 5. Higher absorbance indicates stronger reducing uted to their high content of polyphenols and strong an-
power. Like the DPPH scavenging activity, the ferric re- tioxidant activity. It is likely that active antioxidant com-
ducing power of these extracts augmented as their con- pounds in these extracts could retard the initial step of
centrations increased (P<0.05). At the concentration of radical species creation and reduce the supply of oxygen
1.0 mg/mL, the absorbance of all extracts was in the during the tyrosinase reaction, leading to the inhibition
range of 0.8890±0.0085 to 2.9547±0.0025. The NP ex- of tyrosinase. Another possible reason is that some active

Fig. 6. Tyrosinase inhibitory activity of 9 avocado by-product extracts [Sap seed (SS), Sap peel (SP), 034 seed (OS), 034 peel (OP),
Nuoc seed (NS), Nuoc peel (NP), seed mixture (MS), peel mixture (MP), and peel and seed mixture (MSP)]. Different uppercase
letters (A-E) are related to significant differences among other storage days in the same treatment (P <0.05). Different lowercase
letters (a-h) showed significant differences among other treatments in the same storage time (P <0.05).
216 Phan et al.

antioxidants in these extracts could prevent the catalytic

activity of tyrosinase (Cui et al., 2018).
Melanosis in shrimp was characterized by the formation
of black spots caused by the oxidation of phenol sub-
strates (such as tyrosine) to quinones catalyzed by poly-
phenoloxidase (Gonçalves and de Oliveira, 2016; Pan et
al., 2019). Phenolic compounds could alleviate melanosis
formation in different mechanisms based on the elimina-
tion of one or more important components involved in
the enzymatic reaction, such as oxygen, copper, substrate,
and the enzyme itself. Certain polyphenols can lower PPO
activity by direct interaction with PPO through chelating
Fig. 7. Pearson’s correlation coefficient (r) of the melanosis in-
or forming hydrogen bonding and evenly donating an hibitory activity (at day 6 storage) to total phenolic content
electron to the intermediate quinones, and hence they (TPC), antioxidant activities (DPPH radical scavenging, ferric re-
could interrupt the oxidation process (Sae-leaw and Ben- ducing power (FRAP) and tyrosinase enzyme inhibition (at con-
centration of 100 μg/mL), and TBARS.
jakul, 2019). Several polyphenols have been documented
with intense PPO inhibitory activity such as catechin and
its derivatives, quercetin and its derivatives, and coumaric enzyme found in crustaceans, also plays a reasonable role
acid (Sae-leaw et al., 2017; Rosero et al., 2019). Among in melanosis formation. The lower Pearson coefficient (r)
various polyphenolic compounds found in avocado by- for tyrosinase inhibition than TPC value could be ex-
products, 30 of them belonged to organic acids, hydrox- plained through different melanosis-inhibition mecha-
ycinnamic acids, catechins, free and glycosylated flavo- nisms during cold storage of shrimp with avocado by-
noids, and dimeric and trimeric procyanidins. Catechin, product extracts. A similar relationship was found for the
epicatechin, six quercetin derivatives, four dimeric pro- FRAP data. However, the values for TBARS indicated the
cyanidins, and three trimeric procyanidins were identi- decrease in quality of shrimp, which was due to the for-
fied in the most active fractions of avocado peel and seeds mation of lipid oxidizing products (e.g., aldehydes pro-
reported in the literature (Rosero et al., 2019). Catechin duce off flavors) (Chaijan et al., 2006; Zhang et al., 2015)
and its derivatives exhibited tyrosinase inhibitory activ- This high absolute r value indicated that retardation of
ity based on the combined effects of metal chelation and both melanosis and lipid peroxidation were attributed to
the reduction of quinone (Nirmal and Benjakul, 2012). the high phenolic content in the extracts. Phenolic com-
As shown in Fig. 1 the prepared extracts of avocado by- pounds might chelate the metal pro-oxidants in shrimp
products were rich in polyphenols, which could explain muscle and that resulted in the delay of lipid oxidation in
the significant retardation of melanosis formation in shrimp (Abbasvali et al., 2016). Sai-Ut et al. (2020) re-
shrimp during storage. ported that extract of mango seed kernel possessing rich
Fig. 7 plots the correlations of the anti-melanosis of the phenolic compounds could appreciably retard the mela-
prepared extracts to their TPC values, the oxidant inhib- nosis formation and lipid oxidation process. Further stud-
itory activities and lipid peroxidation (TBARS) values at ies on the influence of phenolic profiles on melanosis pre-
day 6 storage (confidence level of 95%). The data at the vention and lipid oxidation inhibition should be investi-
6th day was chosen because it showed the most variation gated.
in the melanosis score (Fig. 2). It is clearly seen that the Overall, the present study highlights the promising ap-
melanosis inhibition on shrimp stored up to 6 days had plications of avocado by-product (seed and peel) extracts
a good correlation with the TPC and antioxidant activi- as efficient inhibitors of black spots and lipid oxidation
ties of the corresponding extracts (r=0.547∼0.857). The during refrigerated storage of whiteleg shrimp. These in-
high correlation coefficient r of 0.811 for the TPC value hibitory activities varied depending on the type of avoca-
could confirm the important role of phenolic compounds do fruits, which was due to the difference in TPC and an-
in the prepared by-product extracts on retarding mela- tioxidant power among them. Between the investigated
nosis formation in shrimp. It should be noted that the samples, the extract of ‘Nuoc’ avocado peel showed the
scavenging activity toward DPPH radical behaved in the most promising impact for shrimp preservation with its
same manner with TPC value toward melanosis inhibi- high TPC (144.7±1.9 mg GAE/g DW) and antioxidant
tory activity as the DPPH scavenging activity (r=0.857) activities (IC50 values for DPPH and PPO inhibition as-
was known to be consistent with the TPC value (Sun et says of 2.3 and 48.0 μg/mL, respectively). These proper-
al., 2014; Sae-leaw and Benjakul, 2019; Shiekh et al., ties were even better than those of the commercial SMS
2019). A moderate correlation with tyrosinase inhibitory additive at high preserving dose. Therefore, these avocado
activity (r=0.588) indicates that tyrosinase, a common by-products can be considered as safe and cheap natural
 Preserve Shrimp by Avocado By-Products 217

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Githinji P, Gitu L, Marete E, Githua M, Mugo M, Mbaka EN. Quan-
This work belongs to the project grant 67/2019/HĐ- titative analysis of total phenolic content in avocado (Persia
QPTKHCN funded by Ho Chi Minh City Department of americana) seeds in Eastern province of Kenya. 2013. 3:48-51.
Gómez FS, Sánchez SP, Iradi MGG, Azman NAM, Almajano MP.
Science and Technology, Viet Nam. The study was sup-
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