J Food Sci Technol

https://doi.org/10.1007/s13197-021-05081-w

ORIGINAL ARTICLE

Effects of Annona muricata extraction on inhibition

of polyphenoloxidase and microbiology quality of Macrobrachium
rosenbergii
Amalina Ibrahim1 • Muhamad Syazlie Che Ibrahim2 • Kamariah Bakar3 •

Jamilah Bakar4 • Mhd Ikhwanuddin1 • Nurul Ulfah Karim1

Revised: 10 February 2021 / Accepted: 19 March 2021

Ó Association of Food Scientists & Technologists (India) 2021

Abstract Giant freshwater prawn (Macrobrachium muricata leaf extract effectively inhibit 82.41% PPO.
rosenbergii) is one of the important aquaculture species Furthermore, 15% of A. muricata leaves extracts showed a
and quickly expanding in many countries. High demand significant reduced (p \ 0.05) in total bacteria count during
and mass commercialization on M. rosenbergii regulating 20 days of chilled storage of M. rosenbergii. These con-
18% of the international seafood business. Seafood prod- clude that the present of listed secondary metabolites and at
ucts contend with various level across the supply chains approximately * 15–16% of A. muricata leaves extracts
and time to reach the consumers depending upon the were effectively inhibiting the melanosis and prolong the
marketing and delivery channels after harvesting. There- shelf life for up to 8 days of M. rosenbergii stored at
fore, these may cause biodeterioration such as melanosis chilled condition. Therefore, A. muricata leaves extract is
(dark pigmentation) and microbial changes that limit the potential used as natural preservative agent in obtaining
shelf life. This studies reveal the antioxidant properties high quality seafood products.
from Annona muricata leaves extract and their effective-
ness in inhibiting the polyphenoloxidase (PPO) activity and Keywords Annona muricata
Macrobrachium
delaying the bacterial accumulation during 20 days of rosenbergii
Polyphenoloxidase
Melanosis

chilled storage. Five metabolites including coumarins, fla- Microbiology quality
vonoid, glycoside, terpenoids and steroid compound were
found in A. muricata leaves extract. Total phenolic content
and total flavonoid content of A. muricata were recorded at Introduction
191.24 ± 0.03 mgGAEg-1 and 1777.48 ± 1.08
mgQEg-1, respectively. Sixteen percent (16%) of A. Giant freshwater prawn (Macrobrachium rosenbergii) is
one of the important aquaculture species and quickly
expanding in many countries. Major production comes
from China, Thailand, Vietnam, Indonesia, Malaysia, India
& Nurul Ulfah Karim and Bangladesh which contributes approximately of 82%
[email protected] followed by South America (16%) and the rest comes from
1
Saudi Arabia, Madagascar and Australia (Alam 2016).
Higher Institution Centre of Excellent (HICoE), Institute of
Tropical Aquaculture and Fisheries, Universiti Malaysia
High demand and mass commercialization on M. rosen-
Terengganu, Terengganu, Malaysia bergii regulating 18% of the international seafood business.
2
Faculty of Science and Marine Environment, Universiti
Crustacean are highly perishable with a limited shelf-
Malaysia Terengganu, Terengganu, Malaysia life, mainly associated with melanosis (discolouration) and
3 microbial spoilage (Gokoglu et al. 2008). Melanosis is
Institute of Marine Biotechnology, Universiti Malaysia
Terengganu, Terengganu, Malaysia triggers by a biochemical mechanisms which polyphenol
4 oxidase (PPO) catalyses the hydroxylation to the o-position
Department of Food Technology, Faculty of Food Science
and Technology, Universiti Putra Malaysia, Terengganu, adjacent to an existing hydroxyl group by using phenols
Malaysia and oxygen as the substrate. The second reaction is the

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oxidation of the diphenol to o-benzoquinones (Kim et al. The objectives of these studies are to identify the phy-
2000). This is followed by non-enzymatic polymerisation tochemicals compounds and antioxidant properties as well
of the quinones, giving rise to dark pigments. PPO are as to determine the inhibitory effects of A. muricata
characterised by tyrosinase, catechol oxidase, monophenol extraction towards melanosis development and microbial
oxidase, o-diphenol oxidase and others (according to the activity. The leaves extraction of A. muricata might be a
substrate) activities. This melanosis phenomenon starts in potential natural antioxidant and antibacterial that benefi-
the cephalothorax carapace, followed by the abdominal; cial to inhibit or slow down the biodeterioration in M.
where the cuticle segments are articulated and where the rosenbergii.
cuticle is joined to the pleopods and latter spreads to the
caudal zone (telson and uropods) (Nirmal et al. 2012).
Activity of polyphenol oxidase has caused the prawn to Materials and methods
have a shorter shelf-life, usually between 3–6 days.
Besides the discolouration, M. rosenbergii faced dete- Chemicals
rioration due to the spoilage bacteria activity (Chini-
vasagam et al. 1998). Previously, Chinivasagam et al. Folin-ciocalteu reagent, sodium phosphate dibasic, sodium
(1996) found bacteria such as Pseudomonas fragi and nitrite and methanol were obtained from Merck (Darm-
Shewanella putrefaciens isolated from giant freshwater stadt, Germany). Gallic acid, quercetin, Bradford reagent,
prawns at various storage conditions and regions. Masuda L-DOPA, trolox and bovine serum albumin were obtained
et al. (2012) emphasised that phenoloxidase is tightly from Sigma Aldrich (Subang Jaya, Malaysia). 2, 2 diphenl-
regulated by the enzyme cascade that is triggered by the 1-picryhydrazyl 95% were purchased from Alfa Aeser
presence components of microbial cell wall such as b-1,3- (Thermo Fisher, United Kingdom). All chemical were
glucans, lipopholysaccarides and peptideglycan. Pseu- analytical grade.
domonas and Enterobacteriaceae produce biogenic amine,
indole and putrescine that may leads to the deterioration of Sample collection and preparation of Annona
giant freshwater prawn quality, changing the odours, fla- muricata
vours, gas accumulation and consistency of colour and
appearance (Sheikh et al. 2018). Leaves of A. muricata with homogenous treatment were
In order to avoid great economic losses, deteriorative collected from plantation at Kedah, Malaysia. The leaves
process must be controlled and/or eliminated. Antioxidant were brought back to laboratory, rinsed with water before
preserve food by retarding rancidity or discoloration by dry shade at room temperature for 1 week. The leaves were
interfering with oxidative processes that generate free pulverized into powder by using Waring blender (Waring
radicals, chelating metals and also acting as singlet oxygen Commercial, Malaysia). The powder was sieved with
scavengers. However, the use of sulphites as melanosis 500 lm mesh size before kept in polyethylene bags. 30 g
inhibitors are frequently linked to allergic reactions and of A. muricata powder was mixed with 200 ml solvent
asthmatic attack in human. Therefore, the current trends in (6:4) methanol-distilled water. The mixture was shaken at
food processing focusing on the use of natural compounds room temperature for 24 h with Stuart Orbital Shaker
is as a result of arising health related issue and increasing (Fisher Scientific, Malaysia) followed by filtration with
awareness among the consumers. Natural antioxidant is Whatman filter paper number 1. Mixture was dried using
primarily plant phenolic compounds such as tocopherols, rotary evaporator R-210 (Buchi, Switzerland) to remove
flavonoid compounds and others. Recent studies by Coria- excessive solvent and the obtained extract was kept in
Tellez et al. (2018) documented that Annona muricata L. container.
have 212 bioactive compounds. A. muricata contains sec-
ondary metabolites such as phenols, tannin, flavonoids, Phytochemical screening of Annona muricata.
terpenoid and many more (Daud et al. 2016). Studies by
León-Fernández et al. (2017) documented that total phe- Phytochemical screening was carried out to qualitatively
nolic content (TPC) of soursop leaves were recorded at identify the presence of secondary metabolites which are;
3.24–3.95 g 100 g-1 dried weight. Gavamukulya et al. coumarins, flavonoid, glycoside, saponin, terpenoid and
(2014) added soursop leaves extract has a high concen- steroid in the crude leaves extract of A. muricata based on
tration of TPC (372.92 ± 0.15 lg GAE mg-1). method by Daud et al. (2016).

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Determination of functional group with Fourier- water and 0.3 ml of 5% sodium nitrite. The mixture was
transform infrared (FTIR) spectroscopy left to stand for 5 min before addition of 0.3 ml of 10%
aluminium chloride. 2 ml of 1 M sodium hydroxide was
Functional group of methanolic extract of A. muricata added after 1 min. Volume was made up to 10 ml with
leaves was determined with FTIR spectroscopy (Thermo distilled water and absorbance reading were taken at
Scientific, Malaysia) by referring to method by Igci et al. 510 nm using UV-1800 spectrophotometer (Shimadzu,
(2017). A small amount of crude sample was placed Malaysia). Quercetin was used as standard and the result
directly on the germanium piece of the infrared spec- was expressed as mg of quercetin equivalent (QE) g-1 dry
trophotometer in single bounce attenuated total reflection weight.
(ATR) mode. All the data collection and analysis were Assay of radical scavenging activity of A. muricata
performed using origin pro version 2016. Physiochemical extract was conducted according to Gavamukulya et al.
observation using FTIR spectroscopy by bond vibration (2014) with some modification. 0.5 ml of extract was
and stretching give a brief features composition of leaf added with 2.5 ml DPPH solution. The mixture was incu-
constituents base on functional group. The functional group bated for 30 min at room temperature in dark condition.
of leaves components was separated based on its peak ratio. The absorbance were taken at 517 nm using UV-1800
spectrophotometer (Shimadzu, Malaysia). Trolox was used
Determination of metabolites in Annona muricata as control and the result was expressed as mg trolox
by Liquid Chromatography Mass Spectrometry equivalent (TE) g-1 sample.
(LCMS) FRAP assay was carried out according to method by
Benzie et al. (1996). FRAP reagent containing (25 ml of
Annona muricata leaves were subjected to High Resolution 30 mM acetate buffer pH 3.6, 2.5 ml of 10 mM TPTZ in
Liquid Chromatography Tandem Mass Spectrometry 40 mM HCl and 2.5 ml of 20 mM ferric chloride hex-
(LCMS-IT-TOF) (Shimadzu, Japan) to identify the com- ahydrate) were heated in Memmert waterbath (Thermo
pounds present in the samples. The column used was Fisher, Malaysia) at 37 °C before being added with 30 ll
Agilent C18 RP column (Agilent, United States). Samples A. muricata extract and 90 ll of distilled water. The
were diluted with 50% methanol until concentration of absorbance reading were taken by UV-1800 spectropho-
1 ppm is achieved before filtered using 0.2 lm filter syr- tometer (Shimadzu, Malaysia) at 593 nm. Trolox was used
inge into vials and labelled accordingly. Solvents used as control and the result was expressed as mg trolox
were methanol HPLC grade and distilled water. 5 lL of equivalent (TE) g-1 sample.
samples was injected with the flow rate of 0.2 ml min-1,
running time of 50 min. Chromatogram was analysed by Giant freshwater prawn preparation.
LCMS Post Run Analysis system, with ‘formula predictor’
tool system to generate molecular formula and molecular Giant freshwater prawn (Macrobrachium rosenbergii) at
weight. Online database was used as a reference to suggest size of 20–25 prawn kg-1 were brought alive from aqua-
possible compound present in the leaves. culture farm at Balok, Pahang. All samples were trans-
ported in tank filled in cold water and oxygen supply before
Antioxidant properties of Annona muricata leaves transferred to the acclimatization tank at Universiti
extract Malaysia Terengganu. All samples were immersed in ice at
prawn:ice ratio of 1:2 (w/w) before further experiment in
Total phenol content (TPC) was determined by Folin-cio- conjunction to reduce nerve function and metabolic activ-
calteu method as described by Kamtekar et al. (2014). 1 ml ity. Prior to extraction, the prawns were rinsed with cold
of sample was added into 5 ml distilled water and 0.5 ml water. Cephalothorax of the prawns were removed manu-
Folin-ciocalteu reagent. The mixture was left to stand for ally and poured with liquid nitrogen. The samples were
five minutes before the addition of 1.5 ml of 20% sodium then powdered into powder.
carbonate. The mixture was made up to 10 ml with dis-
tilled water and incubated for two hours at room temper- Extraction of polyphenol oxidase (PPO)
ature. Absorbance reading were taken at 750 nm with UV- from cephalothorax of giant freshwater prawn.
1800 spectrophotometer (Shimadzu, Malaysia). Gallic acid
was used as a standard and the result is expressed as mg of Polyphenol oxidase (PPO) enzyme extraction was carried
gallic acid equivalent (GAE) g-1 dry mass. out according to method by Nirmal et al. (2009b) with
Total flavonoid content (TFC) in A. muricata leaves slight modifications. 50 g of powdered cephalothorax was
extract was determined as described by Kamtekar et al. mixed with 150 ml extracting buffer (0.05 M sodium
(2014). 1 ml of sample was added into 4 ml of distilled phosphate buffer, pH 7.2, containing 0.2% Brij-35 and

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NaCl). The mixture was homogenized for 2 min and stirred Statistical analysis
for 30 min. The homogenate was centrifuged using
refrigerated centrifuge (Hitachi, Japan) at 10,000 rpm for All the experiments were carried out in triplicate and the
30 min at 4 °C. Ammonium sulfate was added into the data were presented as mean ± standard deviation. Anal-
supernatant to achieve 40% saturation. All samples were ysis of variance was done by using IBM SPSS Statistic
left to stand for 30 min prior to centrifugation at 9000 rpm Software Version 20 (IBM Corporation, New York). The
for 30 min at 4 °C. Precipitate was collected. The mini- estimation shelf life of each treatment were fitted as the
mum amount of 0.05 M sodium phosphate buffer, pH 7.2 response curve with microbiology data. The microbial shelf
were used to dilute the precipitate. Diluted precipitate was life was taken as the time to reach 107 CFU g-1, as rec-
dialysed with 15 volume of the sample with 0.05 M sodium ommended by International Commission on Microbiology
phosphate buffer, pH 7.2 with three changes every two Specification for Food (ICMSF 1986).
hours and left overnight. The dialysed was centrifuged at
3,000 rpm for 30 min to remove the insoluble materials.
Supernatant was collected and labelled as ‘crude PPO Results and discussion
extracts’.
Phytochemical screening, functional group
Kinetic activity of polyphenol oxidase (PPO). and metabolite profiling of Annona muricata

Activity of polyphenol oxidase (PPO) enzyme in Macro- Phytochemical screening found five secondary metabolites;
brachium rosenbergii was determined according to Nirmal coumarins, flavonoids, glycoside, terpenoids and steroids
et al. (2009a). give a positive result, indicating their presence in the leaves
(Table 1). However, fatty acid and saponin showed a
Inhibition of polyphenol oxidase (PPO) by Annona negative result (Table 1). A study from Daud et al. (2016)
muricata extract. reported nine metabolites including coumarins, flavonoids,
glycoside, terpenoid, steroids, alkaloids, fatty acid, phlo-
Study of inhibition properties of Annona muricata extract batannin and phenolic compounds were found in A.muri-
on enzyme polyphenol oxidase (PPO) was carried out cata leaves extract. Meanwhile, Usunobun et al. (2015)
according to method Nirmal et al. (2012) with some listed six secondary metabolites; flavonoids, alkaloids,
modifications. An assay was prepared consisting of 100 lL cardiac glycoside, tannins, triterpenoid and saponin from A.
crude PPO extract, 100 lL A. muricata extract (0.2, 0.5, muricata leaves extract.
1.0, 2.0, 4.0, 8.0 and 16.0%), 400 lL 0.05 M sodium Fourier Transfrom Infrared Spectroscopy (FTIR) pro-
phosphate buffer pH 6.0 and 600 lL 15 mM L-DOPA as a vides the functional group present in A. muricata leaves
substrate. Upon preparing the assay, all samples were left extract. These were presented by an interaction between
at room temperature for 30 min before being heated at infrared radiation and the secondary metabolites at a par-
40 °C in waterbath. The incubated samples were then ticular wavenumber and time. Infrared spectrum by using
monitored for 3 min using UV-1800 Spectrophotometer attenuated total reflectance (ATR) technique revealed eight
(Shimadzu, Malaysia). functional groups; aromatic group (511.14 cm-1), ester
group (1055.06 cm-1), ether group (1278.81 cm-1), alkane
Microbiology analysis group (1394.53 cm-1), alkene group (1625.99 cm-1),

Macrobrachium rosenbergii were subjected to iced-killed

Table 1 Phytochemical screening of secondary metabolites in An-
method and cleaned under cold water. Samples were ran- nona muricata
domly put into containers containing five different treat-
Secondary metabolite Presence
ments; 10, 15 and 20% A. muricata extract and 1.25% SMS
for 10 min at 4 °C. Controls were left without coating. All Coumarins ?
samples were superchilled in blast freezer (Irinox Blast Fatty acid -
Freezer, USA) for 5 min before kept in polyethylene bag Flavonoid ?
and stored in the cold storage at 4 °C for 20 days. Micro- Glycoside ?
biology quality analysis were done at every 4 days interval Saponin -
within 20 days of chilled storage. The analysis were done Terpenoid ?
according to method described by Karim et al. (2011). Steroid ?

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alkane group (2752.42 cm-1), carboxylic acid group (2015) described phenolic compound is an important
(2922.16 cm-1) and hydroxyl group (3327.21 cm cm-1 phytochemical for antioxidant activity. Other compounds
from A. muricata leaves extract (Fig. 1). Similarly, Daud such as tocopherol a has also been identified in present
et al. (2016) listed five functional groups OH groups studies. Tocopherol a is categorised in carotenoid group
(3262.75 cm-1), CH2 and CH alkenes (2936.15 cm-1), that have antioxidant effects (Vijayameena et al. 2013;
CH3 alkane (1394.17 cm-1) and COC ester (1261. Correa-Gordillo et al. 2012).
00 cm-1) that were an agreement to current studies. The
broad alcohol/phenol O–H streching indicated the proba- Total phenolic content, total flavonoid content
blity of high phenolic content in A. muricata leaves and antioxidant properties
extraction.
Chromatographic analysis using LCMS have revealed Total phenol content (TPC) in the leaves of A. muricata
the presence of 14 secondary metabolites in A. muricata was recorded at 191.24 ± 0.03 mg gallic acid equivalent
leaves (Table 2). Coria-Tellez et al. (2018) listed 120 (GAE) g-1 sample (Table 3). Recently Gyesi et al. (2019)
acetoginins were found from various part of A. muricata reported that TPC were found at 4.38 ± 0.42 g GAE
including seed, root, stem, pulp and leaf. These studies 100 g-1 sample and these showed comparatively higher
highlighted a presence of anomuricine of acetoginin from than the present studies. Previous studies by Gavamukulya
A. muricata leaves extract. Kim et al. (2000) suggested et al. (2014) documented that TPC were found lower than
anomuricine extracted from leaves is a type of mono the present study which are 372.92 lg ml-1. George et al.
tetrahydrofurans (THF) and 5OH (hydroxyl) which capable (2015) documented the amount of TPC were recorded at
for cytotoxic activity. Alali et al. (1999) added that ace- 8.31 ± 0.31 lg GAE. According to Daud et al. (2016),
toginin are main bioactive compound of Annonaceae TPC was at 24.39 ± 0.001% from the total content of
family. Furthermore, Coria-Tellez et al. (2018) listed 37 primary and secondary metabolites in the A. muricata
phenolic compound found in A. muricata extract. However, leaves. The intraspecific differences of TPC in the leaves of
present studies showed that quercetin, morin, caffiec acid A. muricata could be attributed to several factors such as
and coumaric acids of phenolic compound were abundance the origin of the plant, harvest method, storage time,
in methanolic extraction of A. muricata (Table 2). Both extraction method and the suitability of solvent used
caffiec acid and coumaric acid are subclasses of cinnamic (Siqueira et al. 2015). Phenols are known to be better
acid of polyphenols (Sakakibara et al. 2003). Meanwhile diluted in methanol solvent due to the preference for
Nawwar et al. (2012) added that quercetin is an important slightly polar condition (Siqueira et al., 2015). The total
phenolic compound found in A. muricata leaves. Interest- flavonoid content in methanol extracts of A. muricata were
ingly, a previous study from Correa-Gordilla et al. (2012) found to be 1777.47 ± 1.08 mg quercetin equivalent (QE)
found morin from the pulp of A. muricata. George et al. g-1 (Table 3). The result is in agreement to Daud et al.
(2016) which revealed TFC were found at
21.49 ± 0.001%.
In this study, the ability of antioxidant to scavenge
DPPH radical was found to be 1.296 ± 0.04 mg Trolox
equivalent (TE) ml-1 (Table 3). Gyesi et al. (2019)
reported the IC50 value of 244.8 ± 3.2 lg ml-1 A. muri-
cata leaves extract able to scavenge 50% of DPPH radical.
This value is congruent with the high amount of radical
scavenging ability found in present study. In addition,
Daud et al. (2016); Gavamukulya et al. (2014) suggested
the concentration-dependent relationship between DPPH
free radical and the antioxidant activity of the A. muricata
extract.
FRAP assay is based on the reduction of Fe3? to Fe2?
by antioxidant compounds, thus producing Fe2?-2,4,6-
tris(2-pyridyl)-s-triazine (TPTZ) complex (Benzie et al.
1996). The ability of antioxidant compounds in A. muricata
Fig. 1 The infrared spectrum of Annona muricata in three conditions. leaves was recorded at 0.742 ± 0.02 mg trolox equivalent
Dry leaves are leaves sample in powder form, aqueous are A.
muricata leaves in aqueous extract and methanol is A. muricata leaves (TE) ml-1. Similar result was reported by Justino et al.
in methanol extract (2018) which the FRAP assay were at 705 ± 35 lmol TE

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Table 2 Secondary metabolites based on the predicted molecular information of LCMS analysis
Compound Molecular Molecular weight Molecular weight Retention time Abundance Score
formula (measured) (predicted) (min)

Coumaric acid C9H8O3 166.1067 164.1600 1.20 2.0 9 105 67.20

5
Caffeic acid C9H8O4 185.1396 180.1600 1.40 3.0 9 10 58.98
Annoionol C13H26O3 230.2775 230.3400 16.70 6.5 9 105 78.26
4-Nitrobenzoic acid, C11H10N2O3 236.1027 236.1021 10.03 1.8 9 106 72.51
1,4,Dibutylisoquinoline C17H23N 241.0999 241.1003 13.00 1.1 9 106 58.28
Morin C15H10O7 302.1978 302.2300 14.00 5.0 9 104 67.20
5
Taxifolin C15H12O7 304.3372 304.2500 16.12 3.0 9 10 78.62
Anomuricine C19H23NO4 329.3574 329.4000 16.06 1.5 9 105 73.85
Feruloyquinic acid C17H20O9 362.2876 368.3000 18.84 2.0 9 105 88.54
Tocopherol alpha C28H48O2 415.2591 416.7000 22.23 1.0 9 105 74.03
Homoorientin C21H20O11 443.2911 448.4000 25.87 3.0 9 105 94.48
5
Quercetin 3-oglucoside C21H20O12 465.2751 464.4000 26.03 5.0 9 10 72.51
Corepoxylone C35H60O5 561.2048 560.8000 27.27 2.0 9 105 84.65
Corossolin C35H64O6 581.4422 580.9000 27.50 2.0 9 105 67.91

Table 3 Value of phenolic and flavonoid content, DPPH and FRAP Inhibition of polyphenol oxidase (PPO) by Annona
assay of methanolic A.muricata leaves extract muricata extract
Parameter Value
Sixteen percent (16%) A. muricata extract showed a sig-
-1
Total phenol content (mg GAE g ) 191.24 ± 0.03 nificant (p \ 0.05) effective to inhibit the highest level of
Total flavonoid content (mg QE g-1) 1777.47 ± 1.08 PPO activity (82.41%) (Fig. 2). A. muricata leaves con-
DPPH assay (mg TE g-1) 1.296 ± 0.04 taining phenolic compound inhibits the PPO by competing
FRAP assay (mg TE ml-1) 0.742 ± 0.02 with the substrate naturally presence in giant freshwater
a
All experiments are done in triplicate prawn (Maqsood et al. 2013). This will prevent melanosis
from occurring as fast as it usually happen, therefore
slowing the rate of discoloration. There are several ways
g-1. In addition, Najmuddin et al. (2017) described the
for phenolic compound to inhibit the activity of polyphenol
reducing ability of antioxidant in A. muricata leaves extract
oxidase. Phenols such as 4-hexylresorcinol interact with
to reduce Fe3? to Fe2? were at 15.55 lM Fe2? g-1.
PPO during formation of diphenols and quinone, rendering

Fig. 2 Inhibition of a
polyphenoloxidase by Annona 100
muricata leaves extract ad
90
experiment was conducted in
triplicate. 80
Relative activity (%)

70 ac
60
50
bcd
40 bcd bcd
bc bc
30 bc
bc
bc
20
10
0
0 0.2 0.5 1.0 2.0 4.0 8.0 16 30 50 70
A. muricata extract concentration (%)

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Table 4 Bacteria count (log10 CFU g-1) of Macrobrachium rosenbergii stored at 20 days of chilled storage with treatment of control and sulphite metabisulpide (SMS), 10,15 and 20% of
Annona muricata leaves extraction and shelf life prediction in regards to the total bacteria count
Microbiology analysis Day0 Day 4 Day 8 Day 12 Day 16 Day 20 Shelf life prediction

Total bacteria. count Control 1.699 ± 0.07 bA 2.329 ± 0.11 bA 4.657 ± 0.22 bB 9.314 ± 0.44 bC 14.552 ± 0.06 aD 18.286 ± 0.02 aE 8
SMS 3.072 ± 0.10 dB 1.933 ± 0.03 aA 3.867 ± 0.06 aC 7.734 ± 0.11 aD 15.628 ± 0.02 bE 18.396 ± 0.08 aF 8
10% 1.207 ± 0.01 aA 2.414 ± 0.01 bB 4.827 ± 0.03 bC 9.655 ± 0.05 bD 14.633 ± 0.00 aE 20.735 ± 0.09 cF 8
15% 2.159 ± 0.12 cA 1.848 ± 0.02 aA 3.696 ± 0.04 aB 7.392 ± 0.07 aC 14.784 ± 0.15 aD 18.878 ± 0.11 bE 9
20% 3.122 ± 0.03 dA 2.822 ± 0.04 cA 5.681 ± 0.20 cB 9.453 ± 0.16 bC 15.886 ± 0.10 bD 20.593 ± 0.09 cE 7
Pseudomonas Control 2.655 ± 0.10 cB 2.920 ± 0.04 cB 2.450 ± 0.22 bAB 2.071 ± 0.00 aB 4.141 ± 0.01 aC 8.283 ± 0.02 aD –
SMS 2.632 ± 0.11 cA 3.559 ± 0.22 dB 2.338 ± 0.10 aA 2.230 ± 0.11 aA 4.461 ± 0.22 aC 9.236 ± 0.03 bD –
10% 2.082 ± 0.08 bB 1.367 ± 0.06 aA 2.382 ± 0.07 bBC 2.591 ± 0.03bC 5.182 ± 0.07 bD 10.365 ± 0.14 cE –
15% 2.109 ± 0.04 bA 2.623 ± 0.22 bcB 2.602 ± 0.13 bB 2.670 ± 0.02 bB 5.340 ± 0.04 bC 10.679 ± 0.07 dD –
20% 1.586 ± 0.13 aA 2.069 ± 0.01 bB 2.064 ± 0.04 bB 2.108 ± 0.00 aB 4.217 ± 0.01 aC 8.433 ± 0.01 aD –
Enterobacteriaceae Control 2.753 ± 0.15 bcA 3.716 ± 0.00 bAB 4.328 ± 0.04 bB 8.656 ± 0.08 cC 13.744 ± 0.52 aD 17.739 ± 0.26 aE –
SMS 1.772 ± 0.07 aA 2.960 ± 0.24 aB 4.247 ± 0.04 bC 8.494 ± 0.07 cD 15.761 ± 0.06 bE 18.390 ± 0.04 bF –
10% 2.176 ± 0.19 bA 3.699 ± 0.00 bB 4.792 ± 0.01 cC 9.583 ± 0.01 dD 13.123 ± 0.08 aE 18.627 ± 0.01 bF –
15% 2.879 ± 0.03 cA 4.278 ± 0.02 cC 3.426 ± 0.05 aB 6.851 ± 0.10 aD 13.703 ± 0.19 aE 20.541 ± 0.05 cF –
20% 1.739 ± 0.00 aA 3.432 ± 0.06 abB 4.851 ± 0.07 cC 7.433 ± 0.06 bD 14.865 ± 0.12 bE 20.600 ± 0.09 cF –
Different superscript (a, b, c) indicate significant difference (P \ 0.05) between treatment (controls, SMS, 10, 15 and 20% of Annona muricata leaves extraction). Different superscript (A, B, C)
indicate significant difference (P \ 0.05) between the storage days

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the catalysing activity of PPO (Lambrecht 1995). inhibits the ATPase activity. In addition, polyphenol able
4-hexylresorcinol is known to have longer preservation to bind the membrane protein with microbial enzymes and
period, lower microbial growth and higher sensory score inhibit and change their functions.
than the conventional sulphite-derivatives (Gomez-Guillen A further investigation on the shelf life prediction of the
et al. 2007). Phenolic-derivatives including kojic acid and M. rosenbergii based on the microbiology analysis showed
comic acid can act as copper-chelating agent by combining that the untreated samples are unacceptable to consume at
with hydroxyl group at the active site of enzyme. Kojic day 8th of chilled storage (Table 4). Similar result were
acid may not directly inhibiting PPO activity, rather they recorded for M. rosenbergii coated with SMS and 10% of
capturing the oxygen in the medium during oxidation of A. muricata extract. Samples coated with 15% of A.
o-dihydroxyphenols and trihydroxyphenols by PPO, muricata had a prolong shelf life up to 9 days of chilled
inhibiting the formation of black-coloured compound, storage (Table 4).
melanin (Chen et al. 1991).

Microbiology analysis of M. rosenbergii treated Conclusion

with A. muricata extraction
The presence of coumarins, flavonoids, glycoside, ter-
The total bacteria count showed no significant (p [ 0.05) penoids and steroids in A. muricata leaves extracts gives
different between the controls and M. rosenbergii coated cytotoxic and antioxidant activity. In addition, a high
with 10% of A. muricata extraction at storage of day 4 until phenolic and flavonoid content at 191.24 ± 0.03 mg GAE
day 16 (Table 4). Interestingly, treatment at 15% of A. g-1 and 1777.47 ± 1.08 mg QE g-1 respectively were
muricata extract effectively reduced (p \ 0.05) bacteria found from leaves extraction of A. muricata. Furthermore,
accumulation compared to M. rosenbergii coated with 20% 16% of A. muricata leaves extract effectively to inhibit
of A. muricata extraction during same storage period. 82.41% polyphenoloxidase of the cephalothorax of M.
Similar trend were found for M. rosenbergii coated with rosenbergii. Beside, 15% of A. muricata extract showed a
SMS. However, all treatments showed unacceptable limit significant reduced in total bacteria count of M. rosen-
for consumption (log10 8 CFU g-1) after 16th day of bergii. A. muricata leaves extract at approxi-
storage. Pseudomonas sp showed a gradual increase of mately * 15–16% contribute to delay the melonosis and
growth during 12 days of storage in all samples (Table 4). shelf life extension for M. rosenbergii stored at chilled
However, a rapid increased were observed after day 16th of condition.
chilled storage. M. rosenbergii coated with 20% of A.
muricata extraction showed a significant lower (p \ 0.05)
Supplementary Information The online version contains
Pseudomonas count at initial day until day 8th compared to supplementary material available at https://doi.org/10.1007/s13197-
the controls. In addition, Enterobacteriaceae count of M. 021-05081-w.
rosenbergii coated with 15% A. muricata extract were
effectively reduced (p \ 0.05) compared all other treat- Acknowledgements The authors declare that (i) the work described
has not been published before (except in the form of an abstract, a
ments at day 8th and 12th of chilled storage (Table 4). published lecture or academic thesis), (ii) this study is not under
Chivinisagam et al. (1998) stated Pseudomonas are consideration for publication elsewhere, (iii) the submission to JFST
capable to produce melanosis in tyrosine. Treatment at publication has been approved by all authors as well as the respon-
15% of A. muricata extraction reduced the Pseudomonas sible authorities (iv) if accepted, it will not be published elsewhere in
the same form, in English or in any other language, including elec-
count. The present study suggest that 15% of A. muricata tronically without the written consent of the copyright holder and
extraction are capable to slowdown the growth of Pseu- (v) JFST will not be held legally responsible should there be any
domonas and thus delaying the formation of melanosis. claims for compensation or dispute on authorship.
Radji et al. (2015) reported that the synergism of flavo-
noids, steroids and alkaloids of A. muricata extracts gives Authors Contributions Nurul Ulfah Karim: Conceptualization,
Methodology, Writing-Review and Editing, Supervision. Amalina
antimicrobial bioactivity. Roger et al. (2015) emphasised Ibrahim, Muhamad Syazlie Che Ibrahim and Kamariah Bakar:
alkaloids able to bind the DNA of microorganisms and Investigation, Writing-Original Draft. Jamilah Bakar, Mhd
disrupt the RNA synthesis. Furthermore, Mohanty et al. Ikhwanuddin: Supervision.
(2008) added that alkoloids able to inhibit glycosidase and
Funding This research was funded by The Ministry of Higher
this may consequence of antimicrobial activity. Radji et al.
Education Malaysia (MOHE) under Fundamental Research Grant
(2015) stated that flavonoid able to inhibit cytoplasmic Scheme (FRGS/1/2017) [VOT 59483].
membrane function and DNA synthesis, where by quer-
cetin binds to GyrB subunit of E. coli DNA gyrase and Code availability Not Applicable.

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Compliance with ethical standards Gyesi JN, Opoku R, Borquaye LS (2019) Chemical composition, total
phenolic content and antioxidant activities of the essential oils of
Conflict of interest The authors declare that they have no competing the leaves and fruit pulp of Annona muricata L. (Soursop) from
interests. Ghana. Biochem Res Int. 1–9
Igci N, Sharafi P, Demiralp DO, Demiralp CO, Yuce A, Emre SD
Consent for publication Not Applicable. (2017) Application of Fourier transform infrared spectroscopy to
biomolecular profiling of cultured fibroblast cells from Gaucher
Availability of data and material The datasets used and/or analysed disease patients: A preliminary investigation. Adv Clin Exp Med
during the current study are available from the corresponding author 26(7):1053–1061
on reasonable request. Justino AB, Miranda NC, Franco RR, Martins MM, da Silva NM,
Espindola FS (2018) Annona muricata Linn. leaf as a source of
Ethics approval Not Applicable. antioxidant compounds with in vitro antidiabetic and inhibitory
potential against a-amylase, a-glucosidase, lipase, non-enzy-
Consent to participate Not Applicable. matic glycation and lipid peroxidation. Biomed Pharmacother
100:83–92
Kamtekar S, Keer V, Patil V (2014) Estimation of phenolic content,
flavonoid content, antioxidant and alpha amylase inhibitory
activity of marketed polyherbal formulation. J Appl Pharm
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