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Journal of Food Science and Technology logoLink to Journal of Food Science and Technology
. 2021 Apr 1;59(3):859–868. doi: 10.1007/s13197-021-05081-w

Effects of Annona muricata extraction on inhibition of polyphenoloxidase and microbiology quality of Macrobrachium rosenbergii

Amalina Ibrahim 1, Muhamad Syazlie Che Ibrahim 2, Kamariah Bakar 3, Jamilah Bakar 4, Mhd Ikhwanuddin 1, Nurul Ulfah Karim 1,✉
PMCID: PMC8814132  PMID: 35153318

Abstract

Giant freshwater prawn (Macrobrachium rosenbergii) is one of the important aquaculture species and quickly expanding in many countries. High demand and mass commercialization on M. rosenbergii regulating 18% of the international seafood business. Seafood products contend with various level across the supply chains and time to reach the consumers depending upon the marketing and delivery channels after harvesting. Therefore, these may cause biodeterioration such as melanosis (dark pigmentation) and microbial changes that limit the shelf life. This studies reveal the antioxidant properties from Annona muricata leaves extract and their effectiveness in inhibiting the polyphenoloxidase (PPO) activity and delaying the bacterial accumulation during 20 days of chilled storage. Five metabolites including coumarins, flavonoid, 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 191.24 ± 0.03 mgGAEg−1 and 1777.48 ± 1.08 mgQEg−1, respectively. Sixteen percent (16%) of A. muricata leaf extract effectively inhibit 82.41% PPO. Furthermore, 15% of A. muricata leaves extracts showed a significant reduced (p < 0.05) in total bacteria count during 20 days of chilled storage of M. rosenbergii. These conclude that the present of listed secondary metabolites and at approximately ~ 15–16% of A. muricata leaves extracts were effectively inhibiting the melanosis and prolong the shelf life for up to 8 days of M. rosenbergii stored at chilled condition. Therefore, A. muricata leaves extract is potential used as natural preservative agent in obtaining high quality seafood products.

Keywords: Annona muricata, Macrobrachium rosenbergii, Polyphenoloxidase, Melanosis, Microbiology quality

Introduction

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 and Bangladesh which contributes approximately of 82% followed by South America (16%) and the rest comes from Saudi Arabia, Madagascar and Australia (Alam 2016). High demand and mass commercialization on M. rosenbergii regulating 18% of the international seafood business.

Crustacean are highly perishable with a limited shelf-life, mainly associated with melanosis (discolouration) and microbial spoilage (Gokoglu et al. 2008). Melanosis is triggers by a biochemical mechanisms which polyphenol oxidase (PPO) catalyses the hydroxylation to the o-position adjacent to an existing hydroxyl group by using phenols and oxygen as the substrate. The second reaction is the oxidation of the diphenol to o-benzoquinones (Kim et al. 2000). This is followed by non-enzymatic polymerisation of the quinones, giving rise to dark pigments. PPO are characterised by tyrosinase, catechol oxidase, monophenol oxidase, o-diphenol oxidase and others (according to the substrate) activities. This melanosis phenomenon starts in the cephalothorax carapace, followed by the abdominal; where the cuticle segments are articulated and where the 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 have a shorter shelf-life, usually between 3–6 days.

Besides the discolouration, M. rosenbergii faced deterioration due to the spoilage bacteria activity (Chinivasagam et al. 1998). Previously, Chinivasagam et al. (1996) found bacteria such as Pseudomonas fragi and Shewanella putrefaciens isolated from giant freshwater prawns at various storage conditions and regions. Masuda et al. (2012) emphasised that phenoloxidase is tightly regulated by the enzyme cascade that is triggered by the presence components of microbial cell wall such as β-1,3-glucans, lipopholysaccarides and peptideglycan. Pseudomonas and Enterobacteriaceae produce biogenic amine, indole and putrescine that may leads to the deterioration of giant freshwater prawn quality, changing the odours, flavours, gas accumulation and consistency of colour and appearance (Sheikh et al. 2018).

In order to avoid great economic losses, deteriorative process must be controlled and/or eliminated. Antioxidant preserve food by retarding rancidity or discoloration by interfering with oxidative processes that generate free radicals, chelating metals and also acting as singlet oxygen scavengers. However, the use of sulphites as melanosis inhibitors are frequently linked to allergic reactions and asthmatic attack in human. Therefore, the current trends in food processing focusing on the use of natural compounds is as a result of arising health related issue and increasing awareness among the consumers. Natural antioxidant is primarily plant phenolic compounds such as tocopherols, flavonoid compounds and others. Recent studies by Coria-Tellez et al. (2018) documented that Annona muricata L. have 212 bioactive compounds. A. muricata contains secondary metabolites such as phenols, tannin, flavonoids, terpenoid and many more (Daud et al. 2016). Studies by León-Fernández et al. (2017) documented that total phenolic content (TPC) of soursop leaves were recorded at 3.24–3.95 g 100 g−1 dried weight. Gavamukulya et al. (2014) added soursop leaves extract has a high concentration of TPC (372.92 ± 0.15 µg GAE mg−1).

The objectives of these studies are to identify the phytochemicals compounds and antioxidant properties as well as to determine the inhibitory effects of A. muricata extraction towards melanosis development and microbial activity. The leaves extraction of A. muricata might be a potential natural antioxidant and antibacterial that beneficial to inhibit or slow down the biodeterioration in M. rosenbergii.

Materials and methods

Chemicals

Folin-ciocalteu reagent, sodium phosphate dibasic, sodium nitrite and methanol were obtained from Merck (Darmstadt, Germany). Gallic acid, quercetin, Bradford reagent, L-DOPA, trolox and bovine serum albumin were obtained from Sigma Aldrich (Subang Jaya, Malaysia). 2, 2 diphenl-1-picryhydrazyl 95% were purchased from Alfa Aeser (Thermo Fisher, United Kingdom). All chemical were analytical grade.

Sample collection and preparation of Annona muricata

Leaves of A. muricata with homogenous treatment were collected from plantation at Kedah, Malaysia. The leaves were brought back to laboratory, rinsed with water before dry shade at room temperature for 1 week. The leaves were pulverized into powder by using Waring blender (Waring Commercial, Malaysia). The powder was sieved with 500 µm mesh size before kept in polyethylene bags. 30 g of A. muricata powder was mixed with 200 ml solvent (6:4) methanol-distilled water. The mixture was shaken at room temperature for 24 h with Stuart Orbital Shaker (Fisher Scientific, Malaysia) followed by filtration with Whatman filter paper number 1. Mixture was dried using rotary evaporator R-210 (Buchi, Switzerland) to remove excessive solvent and the obtained extract was kept in container.

Phytochemical screening of Annona muricata.

Phytochemical screening was carried out to qualitatively identify the presence of secondary metabolites which are; coumarins, flavonoid, glycoside, saponin, terpenoid and steroid in the crude leaves extract of A. muricata based on method by Daud et al. (2016).

Determination of functional group with Fourier-transform infrared (FTIR) spectroscopy

Functional group of methanolic extract of A. muricata leaves was determined with FTIR spectroscopy (Thermo Scientific, Malaysia) by referring to method by Igci et al. (2017). A small amount of crude sample was placed directly on the germanium piece of the infrared spectrophotometer in single bounce attenuated total reflection (ATR) mode. All the data collection and analysis were performed using origin pro version 2016. Physiochemical observation using FTIR spectroscopy by bond vibration and stretching give a brief features composition of leaf constituents base on functional group. The functional group of leaves components was separated based on its peak ratio.

Determination of metabolites in Annona muricata by Liquid Chromatography Mass Spectrometry (LCMS)

Annona muricata leaves were subjected to High Resolution Liquid Chromatography Tandem Mass Spectrometry (LCMS-IT-TOF) (Shimadzu, Japan) to identify the compounds present in the samples. The column used was Agilent C18 RP column (Agilent, United States). Samples were diluted with 50% methanol until concentration of 1 ppm is achieved before filtered using 0.2 µm filter syringe into vials and labelled accordingly. Solvents used were methanol HPLC grade and distilled water. 5 µL of samples was injected with the flow rate of 0.2 ml min−1, running time of 50 min. Chromatogram was analysed by LCMS Post Run Analysis system, with ‘formula predictor’ tool system to generate molecular formula and molecular weight. Online database was used as a reference to suggest possible compound present in the leaves.

Antioxidant properties of Annona muricata leaves extract

Total phenol content (TPC) was determined by Folin-ciocalteu method as described by Kamtekar et al. (2014). 1 ml of sample was added into 5 ml distilled water and 0.5 ml Folin-ciocalteu reagent. The mixture was left to stand for five minutes before the addition of 1.5 ml of 20% sodium carbonate. The mixture was made up to 10 ml with distilled water and incubated for two hours at room temperature. Absorbance reading were taken at 750 nm with UV-1800 spectrophotometer (Shimadzu, Malaysia). Gallic acid was used as a standard and the result is expressed as mg of gallic acid equivalent (GAE) g−1 dry mass.

Total flavonoid content (TFC) in A. muricata leaves extract was determined as described by Kamtekar et al. (2014). 1 ml of sample was added into 4 ml of distilled water and 0.3 ml of 5% sodium nitrite. The mixture was left to stand for 5 min before addition of 0.3 ml of 10% aluminium chloride. 2 ml of 1 M sodium hydroxide was added after 1 min. Volume was made up to 10 ml with distilled water and absorbance reading were taken at 510 nm using UV-1800 spectrophotometer (Shimadzu, Malaysia). Quercetin was used as standard and the result was expressed as mg of quercetin equivalent (QE) g−1 dry weight.

Assay of radical scavenging activity of A. muricata extract was conducted according to Gavamukulya et al. (2014) with some modification. 0.5 ml of extract was added with 2.5 ml DPPH solution. The mixture was incubated for 30 min at room temperature in dark condition. The absorbance were taken at 517 nm using UV-1800 spectrophotometer (Shimadzu, Malaysia). Trolox was used as control and the result was expressed as mg trolox equivalent (TE) g−1 sample.

FRAP assay was carried out according to method by Benzie et al. (1996). FRAP reagent containing (25 ml of 30 mM acetate buffer pH 3.6, 2.5 ml of 10 mM TPTZ in 40 mM HCl and 2.5 ml of 20 mM ferric chloride hexahydrate) were heated in Memmert waterbath (Thermo Fisher, Malaysia) at 37 °C before being added with 30 µl A. muricata extract and 90 µl of distilled water. The absorbance reading were taken by UV-1800 spectrophotometer (Shimadzu, Malaysia) at 593 nm. Trolox was used as control and the result was expressed as mg trolox equivalent (TE) g−1 sample.

Giant freshwater prawn preparation.

Giant freshwater prawn (Macrobrachium rosenbergii) at size of 20–25 prawn kg−1 were brought alive from aquaculture farm at Balok, Pahang. All samples were transported in tank filled in cold water and oxygen supply before transferred to the acclimatization tank at Universiti Malaysia Terengganu. All samples were immersed in ice at prawn:ice ratio of 1:2 (w/w) before further experiment in conjunction to reduce nerve function and metabolic activity. Prior to extraction, the prawns were rinsed with cold water. Cephalothorax of the prawns were removed manually and poured with liquid nitrogen. The samples were then powdered into powder.

Extraction of polyphenol oxidase (PPO) from cephalothorax of giant freshwater prawn.

Polyphenol oxidase (PPO) enzyme extraction was carried out according to method by Nirmal et al. (2009b) with slight modifications. 50 g of powdered cephalothorax was mixed with 150 ml extracting buffer (0.05 M sodium phosphate buffer, pH 7.2, containing 0.2% Brij-35 and NaCl). The mixture was homogenized for 2 min and stirred for 30 min. The homogenate was centrifuged using refrigerated centrifuge (Hitachi, Japan) at 10,000 rpm for 30 min at 4 °C. Ammonium sulfate was added into the supernatant to achieve 40% saturation. All samples were left to stand for 30 min prior to centrifugation at 9000 rpm for 30 min at 4 °C. Precipitate was collected. The minimum amount of 0.05 M sodium phosphate buffer, pH 7.2 were used to dilute the precipitate. Diluted precipitate was dialysed with 15 volume of the sample with 0.05 M sodium phosphate buffer, pH 7.2 with three changes every two 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 extracts’.

Kinetic activity of polyphenol oxidase (PPO).

Activity of polyphenol oxidase (PPO) enzyme in Macrobrachium rosenbergii was determined according to Nirmal et al. (2009a).

Inhibition of polyphenol oxidase (PPO) by Annona muricata extract.

Study of inhibition properties of Annona muricata extract on enzyme polyphenol oxidase (PPO) was carried out according to method Nirmal et al. (2012) with some modifications. An assay was prepared consisting of 100 µL crude PPO extract, 100 µL A. muricata extract (0.2, 0.5, 1.0, 2.0, 4.0, 8.0 and 16.0%), 400 µL 0.05 M sodium phosphate buffer pH 6.0 and 600 µL 15 mM L-DOPA as a substrate. Upon preparing the assay, all samples were left at room temperature for 30 min before being heated at 40 °C in waterbath. The incubated samples were then monitored for 3 min using UV-1800 Spectrophotometer (Shimadzu, Malaysia).

Microbiology analysis

Macrobrachium rosenbergii were subjected to iced-killed method and cleaned under cold water. Samples were randomly put into containers containing five different treatments; 10, 15 and 20% A. muricata extract and 1.25% SMS for 10 min at 4 °C. Controls were left without coating. All samples were superchilled in blast freezer (Irinox Blast Freezer, USA) for 5 min before kept in polyethylene bag and stored in the cold storage at 4 °C for 20 days. Microbiology quality analysis were done at every 4 days interval within 20 days of chilled storage. The analysis were done according to method described by Karim et al. (2011).

Statistical analysis

All the experiments were carried out in triplicate and the data were presented as mean ± standard deviation. Analysis of variance was done by using IBM SPSS Statistic Software Version 20 (IBM Corporation, New York). The estimation shelf life of each treatment were fitted as the response curve with microbiology data. The microbial shelf life was taken as the time to reach 107 CFU g−1, as recommended by International Commission on Microbiology Specification for Food (ICMSF 1986).

Results and discussion

Phytochemical screening, functional group and metabolite profiling of Annona muricata

Phytochemical screening found five secondary metabolites; coumarins, flavonoids, glycoside, terpenoids and steroids give a positive result, indicating their presence in the leaves (Table 1). However, fatty acid and saponin showed a negative result (Table 1). A study from Daud et al. (2016) reported nine metabolites including coumarins, flavonoids, glycoside, terpenoid, steroids, alkaloids, fatty acid, phlobatannin and phenolic compounds were found in A.muricata leaves extract. Meanwhile, Usunobun et al. (2015) listed six secondary metabolites; flavonoids, alkaloids, cardiac glycoside, tannins, triterpenoid and saponin from A. muricata leaves extract.

Table 1.

Phytochemical screening of secondary metabolites in Annona muricata

Secondary metabolite Presence
Coumarins  + 
Fatty acid −
Flavonoid  + 
Glycoside  + 
Saponin −
Terpenoid  + 
Steroid  + 
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Fourier Transfrom Infrared Spectroscopy (FTIR) provides the functional group present in A. muricata leaves extract. These were presented by an interaction between infrared radiation and the secondary metabolites at a particular wavenumber and time. Infrared spectrum by using attenuated total reflectance (ATR) technique revealed eight functional groups; aromatic group (511.14 cm−1), ester group (1055.06 cm−1), ether group (1278.81 cm−1), alkane group (1394.53 cm−1), alkene group (1625.99 cm−1), alkane group (2752.42 cm−1), carboxylic acid group (2922.16 cm−1) and hydroxyl group (3327.21 cm cm−1 from A. muricata leaves extract (Fig. 1). Similarly, Daud et al. (2016) listed five functional groups OH groups (3262.75 cm−1), CH2 and CH alkenes (2936.15 cm−1), CH3 alkane (1394.17 cm−1) and COC ester (1261. 00 cm−1) that were an agreement to current studies. The broad alcohol/phenol O–H streching indicated the probablity of high phenolic content in A. muricata leaves extraction.

Fig. 1.

Fig. 1

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The infrared spectrum of Annona muricata in three conditions. Dry leaves are leaves sample in powder form, aqueous are A. muricata leaves in aqueous extract and methanol is A. muricata leaves in methanol extract

Chromatographic analysis using LCMS have revealed the presence of 14 secondary metabolites in A. muricata leaves (Table 2). Coria-Tellez et al. (2018) listed 120 acetoginins were found from various part of A. muricata including seed, root, stem, pulp and leaf. These studies highlighted a presence of anomuricine of acetoginin from A. muricata leaves extract. Kim et al. (2000) suggested anomuricine extracted from leaves is a type of mono tetrahydrofurans (THF) and 5OH (hydroxyl) which capable for cytotoxic activity. Alali et al. (1999) added that acetoginin are main bioactive compound of Annonaceae family. Furthermore, Coria-Tellez et al. (2018) listed 37 phenolic compound found in A. muricata extract. However, present studies showed that quercetin, morin, caffiec acid and coumaric acids of phenolic compound were abundance in methanolic extraction of A. muricata (Table 2). Both caffiec acid and coumaric acid are subclasses of cinnamic acid of polyphenols (Sakakibara et al. 2003). Meanwhile Nawwar et al. (2012) added that quercetin is an important phenolic compound found in A. muricata leaves. Interestingly, a previous study from Correa-Gordilla et al. (2012) found morin from the pulp of A. muricata. George et al. (2015) described phenolic compound is an important phytochemical for antioxidant activity. Other compounds such as tocopherol α has also been identified in present studies. Tocopherol α is categorised in carotenoid group that have antioxidant effects (Vijayameena et al. 2013; Correa-Gordillo et al. 2012).

Table 2.

Secondary metabolites based on the predicted molecular information of LCMS analysis

Compound Molecular formula Molecular weight (measured) Molecular weight (predicted) Retention time (min) Abundance Score
Coumaric acid C9H8O3 166.1067 164.1600 1.20 2.0 × 105 67.20
Caffeic acid C9H8O4 185.1396 180.1600 1.40 3.0 × 105 58.98
Annoionol C13H26O3 230.2775 230.3400 16.70 6.5 × 105 78.26
4-Nitrobenzoic acid, C11H10N2O3 236.1027 236.1021 10.03 1.8 × 106 72.51
1,4,Dibutylisoquinoline C17H23N 241.0999 241.1003 13.00 1.1 × 106 58.28
Morin C15H10O7 302.1978 302.2300 14.00 5.0 × 104 67.20
Taxifolin C15H12O7 304.3372 304.2500 16.12 3.0 × 105 78.62
Anomuricine C19H23NO4 329.3574 329.4000 16.06 1.5 × 105 73.85
Feruloyquinic acid C17H20O9 362.2876 368.3000 18.84 2.0 × 105 88.54
Tocopherol alpha C28H48O2 415.2591 416.7000 22.23 1.0 × 105 74.03
Homoorientin C21H20O11 443.2911 448.4000 25.87 3.0 × 105 94.48
Quercetin 3-oglucoside C21H20O12 465.2751 464.4000 26.03 5.0 × 105 72.51
Corepoxylone C35H60O5 561.2048 560.8000 27.27 2.0 × 105 84.65
Corossolin C35H64O6 581.4422 580.9000 27.50 2.0 × 105 67.91
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Total phenolic content, total flavonoid content and antioxidant properties

Total phenol content (TPC) in the leaves of A. muricata was recorded at 191.24 ± 0.03 mg gallic acid equivalent (GAE) g−1 sample (Table 3). Recently Gyesi et al. (2019) reported that TPC were found at 4.38 ± 0.42 g GAE 100 g−1 sample and these showed comparatively higher than the present studies. Previous studies by Gavamukulya et al. (2014) documented that TPC were found lower than the present study which are 372.92 µg ml−1. George et al. (2015) documented the amount of TPC were recorded at 8.31 ± 0.31 µg GAE. According to Daud et al. (2016), TPC was at 24.39 ± 0.001% from the total content of primary and secondary metabolites in the A. muricata leaves. The intraspecific differences of TPC in the leaves of A. muricata could be attributed to several factors such as the origin of the plant, harvest method, storage time, extraction method and the suitability of solvent used (Siqueira et al. 2015). Phenols are known to be better diluted in methanol solvent due to the preference for slightly polar condition (Siqueira et al., 2015). The total flavonoid content in methanol extracts of A. muricata were found to be 1777.47 ± 1.08 mg quercetin equivalent (QE) g−1 (Table 3). The result is in agreement to Daud et al. (2016) which revealed TFC were found at 21.49 ± 0.001%.

Table 3.

Value of phenolic and flavonoid content, DPPH and FRAP assay of methanolic A.muricata leaves extract

Parameter Value
Total phenol content (mg GAE g−1) 191.24 ± 0.03
Total flavonoid content (mg QE g−1) 1777.47 ± 1.08
DPPH assay (mg TE g−1) 1.296 ± 0.04
FRAP assay (mg TE ml−1) 0.742 ± 0.02
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aAll experiments are done in triplicate

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 µg ml−1 A. muricata 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 leaves was recorded at 0.742 ± 0.02 mg trolox equivalent (TE) ml−1. Similar result was reported by Justino et al. (2018) which the FRAP assay were at 705 ± 35 μmol TE g−1. In addition, Najmuddin et al. (2017) described the reducing ability of antioxidant in A. muricata leaves extract to reduce Fe3+ to Fe2+ were at 15.55 µM Fe2+ g−1.

Inhibition of polyphenol oxidase (PPO) by Annona muricata extract

Sixteen percent (16%) A. muricata extract showed a significant (p < 0.05) effective to inhibit the highest level of PPO activity (82.41%) (Fig. 2). A. muricata leaves containing phenolic compound inhibits the PPO by competing with the substrate naturally presence in giant freshwater 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 for phenolic compound to inhibit the activity of polyphenol oxidase. Phenols such as 4-hexylresorcinol interact with PPO during formation of diphenols and quinone, rendering the catalysing activity of PPO (Lambrecht 1995). 4-hexylresorcinol is known to have longer preservation period, lower microbial growth and higher sensory score than the conventional sulphite-derivatives (Gomez-Guillen et al. 2007). Phenolic-derivatives including kojic acid and comic acid can act as copper-chelating agent by combining with hydroxyl group at the active site of enzyme. Kojic acid may not directly inhibiting PPO activity, rather they capturing the oxygen in the medium during oxidation of o-dihydroxyphenols and trihydroxyphenols by PPO, inhibiting the formation of black-coloured compound, melanin (Chen et al. 1991).

Fig. 2.

Fig. 2

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Inhibition of polyphenoloxidase by Annona muricata leaves extract experiment was conducted in triplicate.

Microbiology analysis of M. rosenbergii treated with A. muricata extraction

The total bacteria count showed no significant (p > 0.05) different between the controls and M. rosenbergii coated with 10% of A. muricata extraction at storage of day 4 until day 16 (Table 4). Interestingly, treatment at 15% of A. muricata extract effectively reduced (p < 0.05) bacteria accumulation compared to M. rosenbergii coated with 20% of A. muricata extraction during same storage period. Similar trend were found for M. rosenbergii coated with SMS. However, all treatments showed unacceptable limit for consumption (log10 8 CFU g−1) after 16th day of storage. Pseudomonas sp showed a gradual increase of growth during 12 days of storage in all samples (Table 4). However, a rapid increased were observed after day 16th of chilled storage. M. rosenbergii coated with 20% of A. muricata extraction showed a significant lower (p < 0.05) Pseudomonas count at initial day until day 8th compared to the controls. In addition, Enterobacteriaceae count of M. rosenbergii coated with 15% A. muricata extract were effectively reduced (p < 0.05) compared all other treatments at day 8th and 12th of chilled storage (Table 4).

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 –
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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

Chivinisagam et al. (1998) stated Pseudomonas are capable to produce melanosis in tyrosine. Treatment at 15% of A. muricata extraction reduced the Pseudomonas count. The present study suggest that 15% of A. muricata extraction are capable to slowdown the growth of Pseudomonas and thus delaying the formation of melanosis. Radji et al. (2015) reported that the synergism of flavonoids, steroids and alkaloids of A. muricata extracts gives antimicrobial bioactivity. Roger et al. (2015) emphasised alkaloids able to bind the DNA of microorganisms and disrupt the RNA synthesis. Furthermore, Mohanty et al. (2008) added that alkoloids able to inhibit glycosidase and this may consequence of antimicrobial activity. Radji et al. (2015) stated that flavonoid able to inhibit cytoplasmic membrane function and DNA synthesis, where by quercetin binds to GyrB subunit of E. coli DNA gyrase and inhibits the ATPase activity. In addition, polyphenol able to bind the membrane protein with microbial enzymes and inhibit and change their functions.

A further investigation on the shelf life prediction of the M. rosenbergii based on the microbiology analysis showed that the untreated samples are unacceptable to consume at day 8th of chilled storage (Table 4). Similar result were recorded for M. rosenbergii coated with SMS and 10% of A. muricata extract. Samples coated with 15% of A. muricata had a prolong shelf life up to 9 days of chilled storage (Table 4).

Conclusion

The presence of coumarins, flavonoids, glycoside, terpenoids and steroids in A. muricata leaves extracts gives cytotoxic and antioxidant activity. In addition, a high phenolic and flavonoid content at 191.24 ± 0.03 mg GAE g−1 and 1777.47 ± 1.08 mg QE g−1 respectively were found from leaves extraction of A. muricata. Furthermore, 16% of A. muricata leaves extract effectively to inhibit 82.41% polyphenoloxidase of the cephalothorax of M. rosenbergii. Beside, 15% of A. muricata extract showed a significant reduced in total bacteria count of M. rosenbergii. A. muricata leaves extract at approximately ~ 15–16% contribute to delay the melonosis and shelf life extension for M. rosenbergii stored at chilled condition.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (XLSX 33 kb) (33.1KB, xlsx)

Acknowledgements

The authors declare that (i) the work described has not been published before (except in the form of an abstract, a published lecture or academic thesis), (ii) this study is not under consideration for publication elsewhere, (iii) the submission to JFST publication has been approved by all authors as well as the responsible authorities (iv) if accepted, it will not be published elsewhere in the same form, in English or in any other language, including electronically without the written consent of the copyright holder and (v) JFST will not be held legally responsible should there be any claims for compensation or dispute on authorship.

Authors Contributions

Nurul Ulfah Karim: Conceptualization, Methodology, Writing-Review and Editing, Supervision. Amalina Ibrahim, Muhamad Syazlie Che Ibrahim and Kamariah Bakar: Investigation, Writing-Original Draft. Jamilah Bakar, Mhd Ikhwanuddin: Supervision.

Funding

This research was funded by The Ministry of Higher Education Malaysia (MOHE) under Fundamental Research Grant Scheme (FRGS/1/2017) [VOT 59483].

Code availability

Not Applicable.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

Consent for publication

Not Applicable.

Availability of data and material

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Ethics approval

Not Applicable.

Consent to participate

Not Applicable.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13197-021-05081-w.

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Associated Data

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Supplementary Materials

Supplementary file1 (XLSX 33 kb) (33.1KB, xlsx)

Data Availability Statement

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