Postharvest physiological responses of pomegranate fruit(cv.Wonderful)to exogenous putrescine treatment and effects on physico-chemical and phytochemical properties

Olaniyi Amos Fawole,Julian Atukuri,Erahiema Arendse,Umezuruike Oia Oparaa,,*

a Postharvest Technology Research Laboratory,South African Research Chair in Postharvest Technology,Department of Horticultural Science,Faculty of AgriSciences,Stellenbosch University,Private Bag X1,Stellenbosch,7602,South Africa

b Postharvest Technology Research Laboratory,South African Research Chair in Postharvest Technology,Department of Food Science,Faculty of AgriSciences,Stellenbosch University,Private Bag X1,Stellenbosch,7602,South Africa

c Department of Botany and Plant Biotechnology,University of Johannesburg,P.O.Box 524,Auckland Park 2006,Johannesburg,South Africa

ABSTRACT Pomegranate fruit(cv.Wonderful)were treated with putrescine(1,2 and 3 mmol/L)before storage for 4 months at 5°C and 95%RH and the effects on postharvest life and quality attributes were studied.Results showed that incidence of physiological disorders such as external decay,husk scald,chilling injury and aril browning increased with progressive storage but treating pomegranate fruit with putrescine reduced incidence of most disorders.Control fruit had higher levels of external decay (1.72%-33.26%),chilling injury (10.53%-38.77%) and scalding (15.04%-100%) with less attractive color during 4 month storage.Variations were observed on other fruit quality parameters although treatment with putrescine at 2 and 3 mmol/L concentration reduced changes in color,total soluble solid,Titratable acidity and ascorbic acid.Sensory parameters were best preserved in fruit treated with 2 mmol/L concentration of putrescine with respect to juiciness and crispness.Treatment of pomegranate fruit with putrescine resulted in improved storability and fruit quality during storage.Therefore,for short term storage,2 mmol/L concentration of putrescine could be recommended for maintaining fruit quality especially in the first two months of storage.However,for longer storage period,a higher concentration is recommended,as 3 mmol/L concentration was the most effective in alleviating disorders and maintaining physico-chemical parameters and sensory attributes during storage in this study.

Keywords:Decay Chilling injury Phytochemical Sensory properties Principal component analysis

1.Introduction

The explosion of interest in pomegranate fruit and rapid increase in global production and consumption has been credited to its health benefiting properties which in turn are mainly attributed to the content of phytochemicals with high antioxidant activity and functional properties[1-4].Pomegranate fruit production in South Africa has seen tremendous growth over the years with 40%and 56%increase in production in 2014 and 2015,respectively,and 31%rise in total exports in 2015[5].The fruit is classified as non-climacteric due to its low respiration,ethylene production rates and the fact that the fruit does not continue ripening off the tree[3,6-8].Despite its non-climacteric nature,pomegranate fruit has short shelf life when stored at ambient temperature [3,9].The factors that contribute to the short shelf life of this fruit include rapid weight loss,incidence of fungal decay and internal browning[3].Cold storage is commonly used to slow down these processes and extend the storability and shelf life of the fruit,with a recommended optimal storage temperature of 5°C.However,chilling injury(CI)occurs at 5°C or lower during prolonged cold storage.Chilling injury is characterized by peel browning,husk scald,aril browning and susceptibility to decay among others [7,9,10].This condition limits consumer acceptability,ultimately resulting in economic loss to producers and exporters.In order to mitigate losses caused by these physiological disorders and prolong storage of pomegranate,a number of physical and chemical treatments have been employed[11].For example,heat treatments such as intermittent warming,hot water and hot air treatments have been studied for commercial application for extending fruit storage life[9,12,13].However,due to the presence of numerous micro-openings on pomegranate surfaces[3,6,14],moisture loss becomes problematic in application of heat treatments as the fruit loses moisture rapidly and become unappealing and unmarketable due to excessive shrivel and other skin defects such as browning.

Chilling injury development during low temperature storage of pomegranate fruit involves phase transition of membrane lipids which induces injurious effects on the tissue.Kramer et al.[15]observed accumulation of putrescine during exposure of apple to chilling stress and proposed that polyamines (PAs) may be involved in reducing chilling injury.PAs are a group of positively charged low molecular weight aliphatic amines that are present in living organisms and have been implicated in a number of biological processes like plant growth,development and responseto stress[16].The commonpolyamines include putrescine(diamine),spermidine (triamine) and spermine (tetramine) [17].Other uncommon polyamines such as homospermidine,1,3-diaminopropane,cadavarine and canavalmine have also been detected in biological systems of plants,animals,algae and bacteria[17].Concentration of polyamines in cells is regulated by their biosynthesis,breakdown,translocation and conjugation with different compounds[17].In nature,PAs often occur as free molecular bases and have been reported to bind with negatively charged phospholipids or other anionic sites on membranes.Thus,PAs affect membrane fluidity and indirectly modulate the activities of membrane-associated enzymes [18].Saftner and Baldi [19]reported that polyamines retarded fruit ripening in tomato and their levels decreased with ripening in most cultivars.Based on the findings,the authors suggested that free polyamines are endogenous anti-senescence agents.Treating fruit with polyamines has been reported to increase fruit firmness in apples [20],tomatoes[21],pomegranate[7]and lemons[22].These effects of polyamines are associated with their anti-ethylene property because exogenous polyamines have been shown to inhibit ethylene production and activityin vitro[23].Application of polyamines can inhibit ethylene biosynthesis by competing with ethylene for the common precursorS-adenosyl methionine [16,24].Exogenous application of polyamines impart other beneficial effects such as delayed color changes,reduced susceptibility to mechanical damage and chilling injury,and increased shelf life of both climacteric and nonclimacteric fruit[25,26].

The successful broad-spectrum benefits of polyamines such as putrescine in alleviating incidence of physiological disorders and maintaining fruit quality during storage has been widely studied [7,9,27].For pomegranates the application of polyamines such as putrescine and spermidine have shown to improve fruit quality and extend the shelf life of pomegranates[4,7,9,27].However,the response of fruit to postharvest chemical treatments is dependent on a number of factors such as cultivar,concentration used,mode of application,agro-climactic regions among others.Although the recent years have seen rapid growth of the South African pomegranate industry[28],postharvest losses of the fruit are still high and application of healthier chemical alternatives is limited in the industry.Limited published research have been reported on evaluating the physiological response,physicochemical,phytochemical and sensory properties and even fewer studies have been accomplished in evaluating the application of exogenous putrescine on Wonderful pomegranates.Therefore,the objective of this study was to investigate the exogenous application of putrescine on the physiological response of pomegranate fruit(cv.Wonderful)and study its effects on the physico-chemical,phytochemical and sensory properties of Wonderful pomegranates grown within South Africa.

2.Material and methods

2.1.Plant material

Pomegranate fruit(cv.Wonderful)were procured during commercial harvest period from Heinrich F.R.Schaefer(HFR)properties farm in Western Cape (33o44′26.185′′S 18o44′41.193′′E),South Africa.Fruits were transported in a ventilated vehicle to the Postharvest Technology Research Laboratory at Stellenbosch University and immediately sorted for presence of physical damage such as cracks,sunburn,decay and bruises.Fruit were equilibrated overnight at ambient room temperature((20±2)°C)prior to treatment.

2.2.Treatments

Fruit were divided into four treatment groups of 108 fruit per group.Fruit were dipped for 2 min in a solution in 15 L of putrescine solution containing 2% Tween-20.A dipping time of 2 min was selected based on preliminary studies in which different dipping times(2,5 and 8 min)were tested,and 2 min was the most effective.Treatments included;(1):Immersion in water(control);(2)Immersion in 1 mmol/L putrescine for 2 min;(3)Immersion in 2 mmol/L putrescine for 2 min; (4) Immersion in 3 mmol/L putrescine for 2 min.After immersion,fruit surface was thoroughly dried at ambient room condition ((20±2)°C and 65% ± 2% RH) for 12 h before storage.

2.3.Storage

Fruit were packed inside standard open top ventilated cartons(dimensions:0.4 m long,0.3 m wide and 0.133 m high) used for commercial postharvest handling of pomegranates.All the treatment groups were stored at 5°C and 95% relative humidity for 4 months.Temperature and relative humidity(%RH)inside the cold room were recorded daily throughout the storage period using Tiny Tag TV-4500 data loggers(Gemini Data Logger,Sussex,UK).At the end of cold storage,a batch of fruit(n=30)was placed at 20°C and 65%-70%RH for a further 4-day period in order to simulate a reasonable retail sale period.Fruit were thereafter analyzed for incidence of physiological disorders,physico-chemical,sensory and phytochemical properties.Measurements were carried out on a monthly basis.

2.4.Fruit respiration rate and physiological disorders

2.4.1.Respiration rate

Fruit respiration was determined using a closed system as described by Caleb et al.[29].In 5 replicates,two fruit were placed in a glass jar containing a rubber septum.The jar was sealed hermetically with vaseline to ensure a vacuum seal.Fruit were incubated for 2 h at 20°C then gas composition inside each glass jar was measured using a calibrated O2/CO2analyzer (Checkmate 3,PBI Dansensor,Ringstead,Denmark).Carbon dioxide production was determined and results presented as mean±S.E (mL CO2/kg·h) of five determinations.

2.4.2.Weight loss

Ten randomly selected fruit per treatment were used for this purpose.Fruit were weighed individually at monthly intervals during storage using an electronic scale(Mettler,Toledo,Switzerland,0.0001 g accuracy).Cumulative weight loss of each fruit was calculated as:WhereWis the weight loss (%) of fruit;Wo (g) is the weight of fruit at the beginning of storage;Wi(g)is the weight of fruit at the storage time(monthly).

2.4.3.Evaluation of fruit decay

Fruit decay incidence was visually assessed as total rots.Fruit with external decay appearance were counted and discarded.Percentage of discarded fruit was calculated using the formula;

2.4.4.Evaluation of chilling injury,husk scald and aril browning

Incidences of chilling injury,husk scald and aril browning were visually assessed monthly per treatment.The severity of disorders were assessed using a four level scale as described by Fawole and Opara[3];where 0=none(no symptom),1=trace(1%-25%),2 = slight (26%-50%),3 = moderate (51%-75%) and 4 = severe(76%-100%).

A physiological disorder index was calculated by multiplying the scores of severity by the number of fruit affected and dividing by the total number of fruit assessed[3,12]:

2.5.Measurement of color and textural attributes

2.5.1.Whole fruit and aril color

Color parameters in CIELAB coordinates (L*,a*,b*) were measured using a Chroma meter(CR-400,Minolta Corp,Osaka,Japan).Ten fruit per treatment were used to monitor change in external color by measuring peel color at two opposite spots on individual fruit,while aril color was determined by placing the arils in a colorless glass Petri dish.Color intensity or Chroma(C*)and hue angle(h°)were calculated using the Eqs.(5)and(6)[3].

Furthermore,total color difference(TCD)between the external peel and internal arils components was calculated as;

WhereL*0,a*0andb*0are the color parameters of the peel (reference value),whileL*,a* andb* are the color values of the aril[30].Measurements were done monthly and results presented as mean±S.E.of determinations obtained.

2.5.2.Fruit puncture resistance

Fruit puncture resistance was measured using a fruit texture analyzer (GüSS-FTA,model GS,South Africa).A 5 mm cylindrical probe was programmed to puncture 8.9 mm into the fruit at the speed of 10 mm/s on a steel test platform with the stem calyx axis parallel to the platform.Tests were performed in duplicate on the equilateral region of 10 individual fruit.Puncture resistance was determined as the peak force required to puncture the fruit surface and presented as mean±S.E.

2.5.3.Aril hardness

Aril compression test was performed using a texture profile analyzer XT Plus (Stable MicroSystem Ltd.,Godalming,UK)equipped with a 35 mm diameter cylindrical compression probe.Compression test was performed on individual arils with the following operating conditions:pre-test speed 1.5 mm/s,probe test speed 1 mm/s,post-test speed 10.0 mm/s,compression force 10 N.and compression distance 10 mm[28].Aril compression force(N)was captured on Exponent v.4 software (Stable MicroSystem Ltd.,Godalming,UK).At each storage interval,tests were done using 20 arils extracted from 10 randomly selected fruit for each treatment and results presented as mean±S.E of 20 determinations.

2.6.Measurements of chemical attributes

2.6.1.Titratable acidity,total soluble solids and pH

Titratable acidity (TA) was measured by diluting 2 mL of fresh juice with 70 mL of distilled water and titrated with 0.1 M NaOH to an end point of pH 8.2 using a Metrohm 862 compact titrosampler(Herisua,Switzerland).The results were expressed as percentage of citric acid (%CA).Total soluble solid (TSS,°Brix) was measured using a digital refractometer(Atago,Tokyo,Japan)calibrated with distilled water.The pH values were determined at room temperature using a calibrated pH meter(Crison,Model 00924,Barcelona,Spain).BrimA,a criterion for consumer acceptance of fruit juice was expressed as Eq.(8).

wherekis the tongue’s sensitivity index (k=2 for pomegranate)[32].All measurements were made on 10 individual fruit juice samples for each treatment and results were presented as mean±S.E.

2.7.Phytochemical analyses

2.7.1.Ascorbic acid content

Ascorbic acid content was determined colorimetrically in triplicate using the method described by Barros et al.[31]with some modifications Fawole et al.[32].Pomegranate juice was extracted with 1% metaphosphoric acid (MPA) (0.5 mL of PJ to 14.5 mL of 1% MPA).Mixture was vortexed,sonicated for 3 min in cold water and centrifuged at 5000 r/min for 10 min at 4°C.The supernatant was carefully transferred into a clean tube without disturbing the sediments at the bottom of centrifuge tubes.Approximately,1 mL of the supernatant was mixed with 9 mL of 2,6-dichlorophenolindophenol(dye),shaken to mix and incubated in the dark for 10 min.After incubation,the absorbance of samples was measured at 515 nm against a blank.Ascorbic acid content of each sample was calculated on the basis of the calibration curve of standardL-ascorbic acid.Results were expressed as milligram of ascorbic acid per a hundred millilitres of crude pomegranate juice(PJ)(mg AA/100 mL).

2.7.2.Determination of total phenolic content

Folin-Ciocalteu (Folin C) method as described by Makkar et al.[33]was used to determine the total phenolic concentration in triplicate.Pomegranate juice(1 mL)was extracted with 50%methanol in a centrifuge tube.The mixture was sonicated in cold water for 10 min and there after centrifuged to prevent interference of particles during measurement of absorbance.About 50 μL of diluted aqueous methanolic juice extracts in the test tube was mixed with 450 μL of 50% methanol.500 μL of the Folin C reagent was added to the extract mixture and after 2 min,2.5 mL of sodium carbonate solution was added.The tubes containing solution mixture were vortexed,and incubated in a dark chamber for 40 min at room temperature (20°C).The absorbance of the solution mixture was measured at 725 nm using an UV-vis spectrophotometer(Thermo Fisher Scientific,Madson,USA).The results were presented as the mean of duplicate analyses and expressed as milligrams of gallic acid equivalent per 100 mL of crude PJ(mg GAE/100 mL).

2.8.Sensory attributes

Sensory evaluation was carried out using a trained panel of 6 members of the Postharvest Technology Research Group at Stellenbosch University who are familiar with the characteristic taste of pomegranate fruit and regular consumers [34,35].Panelists received further orientation on pomegranate attributes [36].Sensory evaluation was carried out on arils (10 g) served at 21°C on Petri dishes randomly coded [28].The descriptive test required panelists to rate the intensity of the attributes on a scale of 0-4(0=none,1=slight,2=moderate,3=much,4=very much).The descriptive attributes evaluated for the study included sweet taste,sour taste,crispness,astringency,off flavor,juiciness,grittiness and hardness.Sensory evaluation was not carried out beyond 3 months of storage due to decay and limited sample size.

3.Statistical analysis

Statistical analysis was carried out using Statistica software(Statistica version 14.0,StatSoft Inc.,Tulsa,USA).Data was subjected to factorial analysis of variance (ANOVA) at 95% confidence interval.Main effects (putrescine concentration and storage duration) and their interaction effects(concentration*storage duration)were also assessed.Post-hoc test (Duncan’s Multiple Range Test) was used to test for statistical significance such that observed differences atP＜0.05 were considered significant.Principal component analysis(PCA)was carried out using XLSTAT software version 2012.04.1(Addinsoft,France).

4.Results and discussion

4.1.Physiological response

4.1.1.Fruit respiration rate

Fruit response in terms of respiration rate showed that it was majorly dependent on storage duration(Fig.1A).After one month of storage,results showed a decrease in respiration rate from harvest,with no significant difference (P＞0.05) amongst putrescine concentrations.The second month of storage was,however,characterised by a 1.5 to 2-fold increase in respiration rate both in control and fruit treated with putrescine.This was followed by slight increases in fruit respiration rate after the third month of storage when putrescine concentration influenced fruit respiration rate,with fruit treated with 2 mmol/L putrescine having the highest respiration rate (22.49 mL CO2/kg·h) compared with control with 18.16 mL CO2/kg·h.At the end of storage,fruit respiration rate declined slightly with no significant differences amongst the treatment concentrations(Fig.1A).Respiration rate is a good indicator of physiological activity as it affects other quality attributes during storage of fruit.The observed increase in respiration rate of fruit during storage could be an indication of increase in stress including presence of physiological disorders [3].This would also indicate depletion of respiratory substrates such as sugars and organic acids,and concomitant accelerated senescence process[37].Barman et al.[7]also observed increased respiration rate with advancement of storage for ‘Mridula’ pomegranate stored for 60 days at 3°C.However,lower respiration rate was reported for fruit treated with a combination of putrescine+carnauba wax compared to sole treatment with putrescine.The authors attributed this to the anti-senescence and barrier properties of putrescine and carnauba wax respectively[7].

Fig.1.Respiration (A) and cumulative weight loss (B) of control and putrescine treated pomegranate fruit during storage for 4 months at 5°C and additional 4 days at 20°C.Mean and standard error(SE)of the mean presented.------Respiration rate at harvest.

4.1.2.Weight loss

Weight is an important quality parameter as produce price is often determined on weight basis;therefore,it is crucial to ensure low weight loss during storage of fresh produce.Percentage fruit weight loss after the first month of storage was below 10%,with no significant differences(P＞0.05)among treatments(Fig.1B).Fruit continued to lose weight with prolonged storage duration regardless of treatment concentration.About 1.7 and 2.3-fold increase in weight loss was observed after the second and third months,respectively.However,putrescine concentration slightly but significantly(P＜0.05)influenced fruit weight loss after the fourth month of storage,where the highest weight loss of 24.61% was observed in fruit treated with 1 mmol/L while those treated with 3 mmol/L had the least weight loss(21.49%)(Fig.1B).This could possibly be because putrescine at 3 mmol/L was high enough to maintain cell membrane integrity.Treating fruit with putrescine was shown to reduce weight loss through consolidation of the cell integrity and permeability of the tissues,changes in biophysical properties of the fruit and ameliorating chilling injury[7,38].As indicated by the results of factorial analysis,storage duration (as opposed to concentration) played a significant (P＜0.0001) role on fruit weight loss.High porosity of the pomegranate peel is responsible for the high fruit susceptibility to weight loss due to increased potential for free water vapor movement from the peel[3,10].The increase in fruit weight loss with storage could be attributed in part to the observed increase in fruit respiration rate with storage duration(Fig.1B).Similar findings were reported by Serrano et al.[39]who observed increase in weight loss with storage of four plum cultivars,although increases were lower in putrescine-treated plums from day 7 until the end of the storage period.However,Khosroshahi et al.[17]reported that putrescine had no significant effect on weight loss of‘Selva’strawberry during storage at 5°C for 13 d.

Fig.2.Effect of putrescine on physiological disorders on pomegranate fruit during storage for 4 months at 5°C and additional 4 days at 20°C.External decay (A),internal decay(B),aril browning(C).

4.1.3.External and internal decay incidence

Decay is one of the major challenges faced during storage of pomegranate fruit.Incidence of external fruit decay was low after the one month of storage,affecting less than 10% of the fruit regardless of treatment concentration (Fig.2A).External decay incidence was however apparent after two months of storage,although the incidence remained below 10% for all treatments except in fruit treated with 3 mmol/L putrescine.In terms of the efficacy of the treatment and concentration thereof,after the third and fourth month of storage,it was clearly observed that treating fruit with putrescine minimized external fruit decay than in control in which fruit decay exceeded 30% by the end of storage.In addition,2 mmol/L putrescine treatment showed a better result with the lowest decay incidence at the end of the storage period(Fig.2A).Treating fruit with putrescine has been reported to reduce decay in fruits like mango [38],strawberry [17],among others,and this effect has been attributed to the protective function of putrescine.Exogenous application of putrescine was shown to have anti-pathogenic effect on strawberry[17].Polyamines conjugated to phenolic compounds and hydroxycinamic acid amines have been shown to accumulate in cells in interactions between plants and a variety of pathogens[40].

Internal decay in pomegranates has been attributed mainly to heart rot(black heart),a pre-harvest disease caused byAspergillus nigerandAlternariaspp.,characterized by a mass of black arils[41].The outer peel and the hard rind of infected fruit retain their healthy appearance but when opened,brown (soft) to black (dry) rot of the arils is observed[42,43].No internal decay was observed after the first month of storage except for fruit treated with 2 mmol/L putrescine which had 10% internal decay (Fig.2B).Interestingly,after the second month of storage,fruit treated with 2 and 3 mmol/L showed 10% decay while those treated with 1 mmol/L and control had no internal decay.This was unexpected because treatment with putrescine reduced external fruit decay(Fig.3A)and therefore treated fruit were expected to show less internal decay.After the third and fourth months of storage,internal decay increased with fruit treated with 2 mmol/L having the highest internal decay(20%)while those treated with 1 mmol/L showed no internal decay incidence(Fig.2B).It is noteworthy that there is no effect in terms of the efficacy of putrescine in preventing internal fruit decay.This is however not surprising since aril decay,due to heart rot occurs from infection of fruit in the orchard during flowering [43,44].Therefore,the observed decay incidence could in fact,be due to inherent fruit condition at harvest.Ezra et al.[43]showed that development of heart rot occurs when a spore(Alternariaspp.)penetrates the pistil of an open flower and into the tunnel and then into the loculus,where it remains latent until the ripening fruit can support its growth.In apple (cv.Red Delicious),core rot (mainly associated withA.alternata) was found to develop during,rather than prior to fruit development [45].Core rot is characterized by dark brown tissue that appears dry and corky within loculi and contains air pockets when it penetrates the fruit mesoderm [45].Postharvest treatment of pomegranate fruit may probably have no effect on internal decay of pomegranate fruit; therefore,internal decay could possibly best be prevented by ensuring good agricultural practices and application of pre-harvest treatments.Arendse[41]also observed increased severity of internal decay with prolonged storage and temperature of‘Wonderful’pomegranate fruit.Our results were however lower than those reported by Fawole and Opara[3],who observed severe to extremely severe aril decay for‘Bhagwa’ and ‘Ruby’ pomegranates stored for 16 weeks at 5 and 7°C.This could probably be due to differences in cultivars.

4.1.4.Aril browning

Severity of aril browning increased with prolonged storage regardless of putrescine treatment.The first month of storage was characterised by none to trace levels of aril browning (Fig.2C).However,after the second month of storage,aril browning became more apparent in fruit treated with putrescine(regardless of concentration)than in control fruit,albeit in trace to slight severity.Aril browning appearance further increased after the third and fourth months of storage to above slight and moderate,respectively,in treated samples.Visual appearance is essential especially in the pomegranate fresh-cut industry where pomegranate fruit is minimally processed into ready to eat arils.The color of arils influences consumer choice Pathare and Opara [30]and arils with browning above moderate are deemed unmarketable.Overall,external application of putrescine as postharvest treatment of pomegranate fruit did not reduce aril browning in our study.This is possibly because putrescine was exogenously applied on fruit surface and therefore had no influence on the internal fractions of the fruit.Similar results were observed by Arendse [41]who reported that aril browning after four months of storage was between moderate to severe for pomegranate(cv.Wonderful).On the other hand,Fawole and Opara[3]observed severe to extremely severe aril browning after four months of storage of ‘Bhagwa’ and ‘Ruby’ pomegranates at 5°C.The reported disparities could be due to differences in cultivars,regions and micro-climates.

Fig.3.Effect of putrescine on chilling injury incidence (A),injury index (B),husk scald incidence (C) and husk scald severity (D) on pomegranate fruit during storage for 4 months at 5°C and additional 4 days at 20°C.

4.1.5.Chilling injury incidence and severity

Chilling injury is a physiological disorder that affects chillsensitive fruits stored at low temperatures.Chilling injury incidence increased with progressive storage of pomegranate fruit irrespective of treatment.The incidence of chilling injury was low after one month of storage with fruit treated with 1 mmol/L putrescine showing the highest (16.35%) while fruit treated with 3 mmol/L had the lowest(8.08%)incidence(Fig.3A).A similar trend with treatments was observed after the second month of storage but more fruit became chill-injured,with fruit treated with 1 and 3 mmol/L putrescine having the highest and lowest chilling injury incidences,respectively.Although the number of chill-injured fruit increased with prolonged storage,regardless of treatments,the higher concentrations(2 and 3 mmol/L)were effective in minimizing incidence of chilling injury when fruit were stored beyond 2 months (Fig.3A).In addition,treatment of fruit with the highest concentration of putrescine (3 mmol/L) evidently maintained the lowest chilling injury incidence throughout the storage period.This could be attributed to the ability of putrescine to enhance cold acclimation.It was important to assess the severity of chilling injury for fruit marketability.Chilling injury severity was well below trace level throughout the storage period (Fig.3B) despite high incidence,suggesting that the fruit could be deemed marketable.With regard to treatments,the severity of chilling injury was again lowest in fruit treated with 3 mmol/L throughout the storage period.During chilling conditions in plant tissues,cell membrane lipids undergo changes in physical state from liquid-crystalline to solidgel state,which lead to an increase in membrane permeability and ion leakage[46].When polyamines(e.g.putrescine,spermidine and spermine) are exogenously applied,they induce cold acclimation,which lead to maintenance of membrane fluidity at low temperatures and thus responsible for reducing electrolyte leakage and skin browning[7]thereby reducing the chilling injury symptoms.Mirdehghan et al.[9]reported that chilling injury developed from the first sampling date but application of putrescine and spermidine significantly reduced skin browning of pomegranate fruit(cv.Mollar de Elche) stored at 2°C for 60 days.Similarly,application of putrescine,either alone or in combination with carnauba wax significantly reduced chilling injury and skin browning of‘Mridula’pomegranate after storage for 60 days at 3°C[7].

4.1.6.Husk scald incidence and severity

Husk scald is a physiological disorder faced during prolonged storage of pomegranate fruit.It appears as superficial browningthat develops from the stem end of the fruit and does not affect the internal quality.However,it affects the visual quality and marketability of fruit and also increases susceptibility of fruit to decay[47].Husk scald was a major problem observed during long term storage of pomegranate fruit in this current study.The incidence and severity of husk scald were low after the first two months of cold storage and subsequent shelf life condition regardless of treatments(Fig.3C,D).At below trace level,less than 20%of the fruit were affected by scald incidence in all treatments with the exception of fruit treated with 1 mmol/L putrescine after the second month (Fig.3C).However,there were marked increases in scald incidence and severity after the third month of storage,with further exponential increases at the end of the storage period in all treatments.All the remaining fruit developed scald with above moderate severity after the fourth month of storage.Husk scald in pomegranate has been attributed to enzymatic oxidation ofo-dihydroxyphenols which involves breakdown of phenolic compounds in the fruit peel [47,48].Although putrescine is reported to have antioxidant properties[7],this could probably not have been strong enough to prevent scalding as all the remaining fruit developed scald by the end of storage and a further shelf life irrespective of treatment.Furthermore,scalding is an oxidative process and can be minimized or controlled in the presence of low oxygen environment [48].In the current study,fruit were stored under regular atmosphere,with no alteration in atmospheric oxygen more so that the treatment was not a coating.This suggests that there was enough oxygen supply for enzymatic oxidation hence rendering putrescine ineffective in controlling scalding in pomegranate.

Fig.4.Changes in colour parameters of pomegranate fruit peel and arils during storage for 4 months at 5°C and additional 4 days at 20°C.Each bar represents mean and error bars denote standard error(SE)of the mean.Bars followed by different letters are significantly different at P ＜0.05 according to Duncan’s multiple range test.—represents investigated parameters at harvest.

4.1.7.Peel and aril color attributes

4.1.7.1.External appearance (peel).Fruit color is a vital attribute that influences consumer choice and produce purchasability [30].This current study revealed that changes in fruit peel color parameters were influenced by the concentration of putrescine applied and storage duration (Fig.4).In general,fruit peel redness (a*)and color intensity (C*) decreased gradually throughout the storage duration.This was concomitant with increase in hue angle,suggesting color loss of pomegranate peel during storage (Fig.4).Fruit redness and color intensity did not change significantly among treatments for the first two months of storage except for fruit treated with 1 mmol/L putrescine which had the least peel redness (34.92±1.56).However after the third month,control fruit had the highest peel redness and color intensity whereas there was no significant difference among treated samples,and by the end of the storage period(month 4),no differences were observed irrespective of treatment (Fig.4).The decrease in peel color during storage could be due to peel browning which was evidenced by development of husk scald.As a result,peel hue angle(h°)increased with prolonged storage but there were no significant differences among all treatments throughout the storage duration(Fig.4).Hue angle has been shown to increase with a decrease in red color of pomegranate as it measures color purity (deviation from saturation).Therefore the increase in hue could be due to the decrease in peel redness with progressive storage.Arendse[41]reported initial increase in pomegranate peel color during the first three months of storage although external appearance deteriorated up till the end of storage (5 months at 5 and 10°C).Untreated mango fruit (cv.Langra) retained higher values ofa* andb* during storage while fruit treated with 2 mmol/L of putrescine recorded minimum values[38].The authors reported that polyamines may retard chlorophyll degradation in skin tissues by inhibiting peroxidase activity.

4.1.7.2.Internal appearance(aril).Aril color is important as it influences consumer choice during purchase of minimally processed pomegranate arils.Changes were observed when fruit were stored for an additional 4 days at 20°C.Redness of fruit arils(a*)increased slightly with storage and well above the aril color at harvest.Factorial analysis showed that changes in aril redness and color intensity were predominantly influenced by storage duration(a*＜0.0001;C*＜0.0001).Again,there was no significant difference among treatments throughout the storage duration (Fig.4).The increase in aril redness is associated with anthocyanin biosynthesis which has been reported to occur during cold storage of pomegranate fruit[41,49].At the start and end of storage,control samples had higher aril redness compared to treated samples.Putrescine has been shown to prevent color development during storage[7,38,50].It is possible that it could have prevented or retarded the rate of anthocyanin biosynthesis among treated samples.However the changes were influenced by the storage time and not concentration.Decline in hue angle (h°) further buttressed this phenomenon,indicating improved color development in all treatments throughout the storage period (Fig.4).Fawole and Opara [3]also reported relatively stable aril color of ‘Bhagwa’ pomegranate during storage 5°C for 16 weeks.Total color difference (TCD) declined generally during storage of fruit with no difference between the first two months of storage (Fig.4).After the third month of storage,control fruit had the highest TCD while fruit treated with 1 mmol/L putrescine had the lowest values.A further decrease was observed at month 4 with fruit treated with 3 mmol/L putrescine showing the highest total color difference (Fig.4).Treating fruit with 3 mmol/L generally maintained a relatively stable TCD throughout storage.TCD showed a disparity in the color between the peel and aril.Given the importance of aril and juice redness,lower TCD is desired in the initial stages of storage as it is an indication of smaller differences in peel and aril redness.However,with prolonged storage,higher TCD is desired especially due to the fact that the fruit peel develops physiological disorders such as husk scald and chilling injury.Similar ranges of TCD were observed by Fawole [51]for‘Arakta’,‘Bhagwa’,‘Ganesh’,‘Mollar de Elche’and‘Wonderful’cultivars of pomegranate.However,Al-Said et al.[52]reported higher ranges of TCD (50-60) of four cultivars of pomegranate grown in the Sultanate of Oman.

4.2.Physico-textural properties

4.2.1.Juice colour

Juice color is an important quality attribute especially in the juice processing sector as it influences consumer appeal and preference.Juice color absorbance remained fairly unchanged during storage of pomegranate fruit with no significant (P＜ 0.05)changes among treatments,with significant interaction(P=0.0125)between the main factors (duration and concentration) (Table1).The changes in juice color relate to the relatively stable aril redness that was observed during storage of fruit (Fig.6).This could be because putrescine was applied exogenously and therefore no direct significant effect on the internal components of fruit.According to Shulman [53],pomegranate juice color absorbance is an indication of anthocyanins which are light-absorbing plant-based pigments.This suggests no significant changes in light-absorbing anthocyanins during storage of pomegranate fruit.No changes were similarly observed in juice color of‘Wonderful’pomegranate fruit stored for up to 10 weeks at 0 and 5°C [10].Nanda et al.[54]also reported no statistical differences in juice color of film wrapped,skin coated and control pomegranate(cv.Ganesh)stored at 8 and 15°C for 10 weeks.

4.2.2.Fruit puncture resistance(firmness)

Fruit puncture force decreased from values at harvest but did not change significantly after the first two months of storage(Table1).Slight variations were however existent in the last two months of storage with fruit treated with 2 mmol/L having the highest puncture resistance ((11.92±0.40)N) after the last month of storage,15.10% higher than control fruit and the changes were influenced by the interaction between the putrescine concentrations and storage duration (Table1).It is possible that putrescine could have maintained the integrity of the fruit peel.Putrescine has been reported to improve fruit firmness of pomegranates [7]and plums [55]due to its ability to cross link with the pectic substances in the cell wall hence preventing access of cell wall degrading enzymes like polygacturonase,pectinesterase and pectimethylesterase,thereby reducing softening during storage.The beneficial effect of putrescine was also reported by Barman et al.[7]who found 33%higher fruit firmness in pomegranate fruit (cv.Mridula)treated with putrescine+carnauba wax compared to control.

4.2.3.Aril hardness

Variations were observed in aril hardness during the current study.Aril hardness of treated fruit fluctuated during storage with increase in the last month of storage(Table1).Untreated fruit on the other hand increased after the third month but thereafter decreased by 3.2% after the last month of storage.Control fruit significantly had lower aril hardness compared to treatments,with 8.21%lower hardness at the end of the storage duration.However,no significant changes (P＞0.05) were observed among treated fruit after storage for 4 months and the changes were influenced by interaction between the factors(Table1).Decrease in aril hardness is due to loss in cell wall integrity of pomegranate arils[41,56].Loss of cell wall integrity could in turn be due to senescence which progresses with storage [41].Treatment of fruit with putrescine maintained aril hardness during storage(except at month 3),as all treatments had higher aril hardness compared to control samples.Arendse[41]found that aril hardness did not differ with storage temperature but decreased with extended storage of‘Wonderful’pomegranate fruit at 5,7.5 and 10°C for 5 months.

4.3.Chemical attributes

4.3.1.pH

The pH of pomegranate juice determines its sour taste[57].pH of pomegranate juice was characterized as acidic (low below pH 4) and increased as storage progressed for most treatments with some decreases (for concentration 2 and 3 mmol/L) (Table2).The increase could be explained by the initial decrease in titratable

acidity as the two are inversely proportional.Slight differences were observed in pH with samples treated with 2 mmol/L having the highest and lowest pH in the first two and last two months of storage respectively.The interaction between storage duration and putrescine concentration played a significant role in the pH of stored fruit (P＜0.0001) (Table2).The increase in pH with storage could be due to utilization of organic acids evidenced by the general reduction in titratable acidity with storage.Similar results were reported for pomegranate fruit(cv.Mollar de Elche)that had undergone curing and intermittent warming prior to storage at 2°C for 90 days[58].For pomegranate fruit(cv.Ruby,)Fawole and Opara[3]also reported increase in juice pH with storage at 5°C,reaching a maximum value of 3.96 after 16 weeks of storage.Contrary to our results,Mirdehghan et al.[9]observed that pomegranate fruit(cv.Mollar de Elche)decrease in pH(acidity)in both control and fruit treated with putrescine stored at 2°C for 60 days storage.

Table1 Changes in juice colour,fruit puncture and aril hardness of pomegranate fruit treated with putrescine during storage for 4 months at 5°C and additional 4 days at 20°C.

Table2 Chemical attributes of pomegranate fruit treated with putrescine during storage for 4 months at 5°C and additional 4 days at 20°C.

4.3.2.Titratable acidity(TA)

Titratable acidity decreased with storage except at month 3 where all treatments showed increases(Table2).Similarly,no differences were observed during storage with the exception of month 3 where some variations existed with the control samples showing the highest TA (2.24±0.04).This could most likely be due to concentration of acids from weight loss in control fruit.Storage duration significantly(P＜0.0001)influenced the changes in titratable acidity during storage(Table2).Organic acids(which are the main contributors to titratable acidity)have been reported to be the major substrates for pomegranate respiration during storage[3,6].Interestingly,respiration rate of fruit also prominently increased after the third month of storage (Fig.1A) and this could explain the major decrease in titratable acidity by the end of storage.These results are similar to Arendse [41]who also reported decrease in titratable acidity during storage of pomegranate fruit(cv.Wonderful) at 5 and 7.5°C for 5 months.Several authors have reported decreases in TA during storage of fruit.Decrease in acidity was observed for mango(cv.Langra)treated with putrescine and stored at 13°C for 4 weeks[38].After treating four cultivars of plum with putrescine,Serrano et al.[39]found decreased TA during storage at 20°C for nine d.Barman et al.[7]also reported decrease in TA of pomegranate fruit(cv.Mridula)treated with putrescine and stored for 60 days at 3°C.Conversely,increase in TA levels during storage has previously been reported by Gil et al.[49]for the Spanish‘Mollar de Elche’cultivar.

4.3.3.Total soluble solids(TSS)

TSS varied during storage of fruit during this study.TSS decreased from harvest,increased after the second month for some treatments and then finally decreased until end of storage.Fruit treated with 3 mmol/L putrescine had the highest TSS after the first and third months (15.65±0.27 and 15.73±0.19,respectively) of storage while no significant differences (P＞0.05) were observed after the second and last month of storage (Table2).The initial increase in TSS could be due to initial concentration of sugars due to loss of moisture whereas the subsequent decrease thereafter could be due to utilization of sugars in fruit metabolic processes[3].Although organic acids have been reported to be the major substrates of pomegranate respiration during storage,the decrease in TSS could be due to utilization of sugars in other metabolic processes with the storage duration showing a significant effect (P＜0.0001) (Table2) on TSS of pomegranate fruit.The changes with time could be due to senescence or increased metabolism with progressive storage.Our findings are in agreement with Fawole and Opara [3]who reported decrease in TSS of ‘Bhagwa’ and ‘Ruby’pomegranate with storage duration.On the contrary however,Arendse [41]reported significant increase in TSS of pomegranate(cv.Wonderful)stored at 5,7.5 and 10°C for 5 months.Ramezanian and Rahemi [59]reported that hot water had no significant effect on TSS while treating fruit with 4% calcium chloride +1 mmol/L spermidine decreased TSS of ‘Malas Yazdi’ pomegranate after 4.5 months of storage at 2°C.Mirdehghan et al.[9]observed no significant effect of putrescine and spermidine on the TSS of pomegranate(cv.Mollar de Elche) stored at 2°C for 60 days.The application of putrescine have been reported to affect TSS in a variety of other fruits for instance,decreases in TSS was observed in strawberry fruits and Aloe vera [50].While Khan et al.[60]reported that‘Angelino’ plum fruit treated with putrescine stored at low temperature (0°C) exhibited lower soluble solid content.Conversely,increase in TSS with storage duration was reported by Jawandha et al.[38]for ‘Langra’ mango fruit treated with putrescine and stored at 13°C for four weeks.

4.3.4.TSS/TA ratio

The taste of pomegranate is determined mainly by juice TSS level and the ratio between the TSS and TA[57].TSS/TA ratio influences the flavor of products and it measures that balance between the acids and sugars in produce [3].As a result of the changes in TSS and TA,fluctuations were observed in the TSS/TA during storage.There was an initial decline in TSS/TA ratio from harvest and thereafter an increase observed in all treatments after 2 months of storage (Table2).A decrease was observed after the third month followed by about 38.47%increase after 4 months.However no significant differences(P＞0.05)were observed in the TSS/TA during the entire storage duration and the changes were influenced by the storage duration.The increase in TSS/TA ratio could be due to the observed decrease in TA and slight increases in TSS values during storage which then results in higher TSS/TA ratio.The TSS/TA ratio level has been attributed mainly to breakdown of starch into water,soluble sugars,sucrose and glucose[50].The findings in this study are similar to the work by Zafari et al.[50]who observed increase in TSS/TA ratio of strawberry fruit(cv.Kamarosa)treated with putrescine and Aloe vera.Arendse[41]also reported increase in TSS/TA value during storage of pomegranate fruit(cv.Wonderful)at different temperatures (5,7.5 and 10°C).Similarly,Fawole and Opara[3]observed significant increases in TSS/TA ratios of‘Bhagwa’and‘Ruby’pomegranate stored for 16 weeks at 5,7 and 10°C from 9.98 at harvest to a maximum of 13.12 after storage.

4.3.5.BrimA

Jordan et al.[61]proposed a new index (“BrimA”) based on the TSS/TA ratio to determine acceptability of juices,but adding a tongue’s sensitivity index (“k”) as well.This index,k,takes into account that the tongue has higher sensitivity to acid than to sugar,and has values normally from 2 to 10 depending on the fruit.The authors proposed BrimA as a more sensitive predictor of consumer acceptability in different fruits.During the study,BrimA initially decreased from harvest with no significant differences among treatments except for control samples that had the lowest values(9.76±0.60)(Table2).It there after increased after the second month,decreased after the third and finally increased by 5.9%after the fourth month of storage with significant differences.The changes in BrimA were significantly affected by storage duration(P＜0.0001)(Table2).The changes in TSS and TA resulted in significant decreases in BrimA due to storage duration.BrimA decreased in all treatments during storage with the exception of month 2 where increases were observed although no significant differences existed among treatments (Table2).Overall,treating fruit with 3 mmol/L resulted in the best BrimA compared to other treatments.Similar decrease in BrimA was reported by Fawole and Opara [3]for pomegranate(cv.Ruby and Bhagwa)stored at 5,7 and 10°C for 16 weeks.Arendse [41]reported an increase in BrimA from 10.64 at harvest to 14.33,13.62,12.96,and 12.30 during storage at 5,7.5,10 and 21°C,respectively,for‘Wonderful’pomegranate.

Fig.5.Changes in ascorbic acid concentration(A)and total phenolic concentration(B) of pomegranate fruit during storage for 4 months at 5°C and additional 4 days at 20°C.Each bar represents mean and error bars denote standard error(SE)of the mean.Bars followed by different letters are significantly different at P ＜0.05 according to Duncan’s multiple range test.-Total phenolic and ascorbic acid concentration at harvest.

4.4.Phytochemical attributes

4.4.1.Ascorbic acid(AA)content

Ascorbic acid content varied slightly throughout storage of pomegranate fruit,somewhat above values at harvest.After the first month of storage,no differences existed among samples with the exception of control samples that showed lower ascorbic acid content (Fig.5A).Slight variations were observed after 2 and 3 months of storage with samples treated with 2 mmol/L showing the lowest AA content((100.2±1.2)and(100.0±2.2)mg AA/100 mL,respectively).After storage for 4 months,no differences existed among treated fruit while control fruit still maintained slightly higher ascorbic acid concentration (Fig.5A).The changes in AA content were significantly driven by the interaction between concentration and storage duration.This is similar to other studies by Sayyari et al.[62],who observed that AA content was generally maintained when ‘Mollar de Elche’ pomegranate fruit were treated with oxalic acid.Barman et al.[4]reported a declining trend in the AA content during storage of ‘Mridula’ pomegranate fruit although the decline was more pronounced in control as compared to fruit treated with putrescine.Strawberry fruit (cv.Kamarosa)treated with putrescine showed significant differences in ascorbic acid content with putrescine level of 1.8 mmol/L showing best results[50].

4.4.2.Total phenolic content(TPC)

Total phenolic content generally decreased with progressive storage,lower than values at harvest.After one month of storage,no significant differences(P＞0.05)existed among treatments with the exception of control fruit which showed significantly higher phenolic content((233.30±8.58)mg GAE/100 mL)(Fig.5B).This could be as a result of concentration due to weight loss.As TPC of control fruit declined after 2 months,TPC of treated fruit showed increases with fruit treated with 2 mmol/L having 29.7%higher TPC than control.The initial increase in phenolic content at month 2 for treated fruit could be attributed to initial concentration of anthocyanins as a result anthocyanin biosynthesis [3].TPC then gradually decreased during the last two months of storage with no significant differences in all treatments (Fig.5B) and interaction between the factors was significant (P= 0.0001).The subsequent decrease in phenolic content could be due to breakdown of phenolic compounds as a result of enzymatic activities[3,63].Similar results were obtained by Fawole and Opara[3]who also reported decrease in phenolic content with storage for‘Ruby’pomegranate.The decrease in phenolic content with storage can be attributed to decline in phenolic concentration resulting from enzymatic activities taking place in the fruit as reported in rowanberries and pomegranate fruits[3,64].However,this is contrary to studies by Arendse et al.[63]who observed increases in phenolic content during storage of pomegranate fruit and attributed this to accumulation of anthocyanins.

4.5.Sensory properties

To evaluate quality of fruits and vegetables,sensory attributes such as appearance,aroma,texture and color are some of the vital criteria used by a consumer[65].‘Wonderful’pomegranate is characterized mainly by sour over sweet taste[36].Sensory attributes of pomegranate fruit changed during storage.After the first month of storage,higher sweet taste was perceived in control fruit(nontreated) compared to treated fruit (Fig.6 A) and this could be related to the higher TSS values that were observed in control fruit.Sour taste was scored higher in fruit treated with 1 and 2 mmol/L putrescine,while off flavor was generally low in all treatments with scores of 0.2-0.4.Astringency,responsible for the tartness taste in pomegranate especially in the sweet-sour and sour cultivars,was highest in samples treated with 1 mmol/L putrescine.Crispness of arils was generally maintained among all treatments while juiciness was highest in control samples.Grittiness and hardness were scored higher for samples treated with 3 mmol/L putrescine(Fig.6A).In general,at the first month of storage samples treated with 3 mmol/L putrescine had the low acidity and sweetness.While control fruit had better sensory quality after one month of storage.Jawandha et al.[38]also reported that initially after one week of storage of mango (cv.Langra),highest palatability rating was recorded in untreated (control) fruit compared to fruit that had been treated with putrescine.

After storage of fruit for two months,prominent differences were observed in the sensory attributes,with control and fruit treated with 2 mmol/L showing higher scores while fruit treated with 1 and 3 mmol/L had lower scores (Fig.6B).As shown by the radar plot (Fig.6B),control and fruit treated with 2 mmol/L putrescine had better sensory attributes with regards to sweetness,juiciness and crispness compared to the other treatments.Pomegranate fruit (cv.Mridula) treated with putrescine and carnauba wax was reported to have higher sensory scores than control with regard to color,aroma,taste,juiciness and aril firmness after 60 days storage for storage at 5°C[4].

Moreover,at the end of three months control fruit were scored the highest for aril sweet taste while sour taste was more prominent in fruit treated with 3 mmol/L putrescine.Off flavor increased with storage although fruit with 2 mmol/L putrescine had the lowest scores (Fig.6C).Astringency was also low in all treatments,even lower than the previous months of storage.This could possibly be attributed to the decrease in T.A and increase in pH among all treatments as storage progressed(Table2).This is because organic acids(especially tartaric acid)which are responsible for astringency are utilized for metabolism during storage of pomegranate.Fawole and Opara[28]also reported low astringency and alcohol taste for‘Ruby’pomegranate fruit.Crispness and grittiness were more pronounced in fruit treated with 2 mmol/L while juiciness was higher in fruit treated with 1 mmol/L putrescine.Additionally,hardness was best maintained in fruit treated with 3 mmol/L putrescine(Fig.6B).Comparing all treatments at all storage durations,fruit treated with 2 mmol/L putrescine generally had higher sensory attribute ratings compared to other concentration.Jawandha et al.[38]reported highest palatability rating in mango fruit (cv.Langra)treated with putrescine of 2 mmol/L as fruit were still in very good quality after 3 weeks of storage.Similarly,‘Selva’strawberry fruit treated with putrescine had better quality in terms of flesh firmness,appearance,color change and taste especially when fruit were treated with 2 mmol/L putrescine[17].Treating pomegranate(Mridula)with putrescine and carnauba wax resulted in higher sensory scores compared to control after 60 days of storage at 2 and 5°C[4].

Fig.6.Radar plot showing averaged sensory scores of pomegranate fruit treated with putrescine during storage for 3 months at 5°C and additional 4 days at 20°C.The plot represents storage at month 1(A),month 2(B)and month 3(C).

4.6.Principal component analysis

To obtain a broad view on changes that occurred during fruit storage,the whole data set was subjected to principal component analysis(PCA).An Eigenvalue measures the significance of a factor,with Eigenvalues ≥1 considered significant.Therefore the highest eigenvalues are the most significant[66].The total variability was explained by 11 factors (F1-F11),with the first two factors of the PCA showing moderate correlation of 50.04%(Fig.7).The first factor (F1) was responsible for 31.14% of the total variation,while the second factor(F2)explained 18.61%of total variation,indicating that the maximum possible variation during fruit storage was explained by the F1 (Fig.8A).Positive scores on F1 corresponded with long storage duration (2-3 months).Short term storage (1 month) had high negative scores along F1 while fruit stored for 2 months had low positive scores (Fig.8A).Negatives scores along F1 corresponded with peel color,fruit firmness,juice color,aril hardness,grittiness,TSS,ascorbic acid,TPC and astringency.The peel color indicated that fruit had better peel appearance during short storage duration but decreased with prolonged storage which can be related to development of physiological disorders especially husk scald.Fruit stored for short duration (1 month) were associated with ascorbic acid,TPC and astringency.The contribution of phenolics to fruit astringency has been previously reported [67].As storage progressed,there was a shift from left to right along F1(Fig.8B) with increase in aril redness (a*),sweet taste,crispness and juiciness.This gives a clear indication that fruit stored for short duration(1 month)could clearly be distinguished from fruit stored for long duration (3 months).The increase in aril color is associated with increased anthocyanin biosynthesis while the increase in sweet taste could be as a result of concentration of sugars due to weight loss with progressive storage of fruit[28].A strong positive relationship between TSS/TA ratio and BrimA was indicated by the short distance between the two attributes on the PCA(Fig.8B)while a strong negative relationship existed between TA and TSS/TA ratio.A general view of the PCA showed that short term storage of fruit was associated with grittiness,hardness,fruit firmness,TSS,TA,ascorbic acid,TPC and astringency among others while long term storage resulted in fruit with better aril color,juiciness,sweet taste and crispiness.The results indicate that storage duration,instead of other factors(concentration)contributed to the distinction of sensory and instrumental attributes during the study.This suggests the importance of storage duration in postharvest trials.Arendse[41]also reported such observation,where storage duration rather than temperatures(5,7.5 and 10°C)influenced the storage quality of‘Wonderful’pomegranate fruit for up to 4 months of storage.

Fig.7.Scree plot of variance explained by each factor of the principal components.

Fig.8.Principal component analysis showing variables(A)and observations(B)for‘Wonderful’ pomegranate fruit using instrumental and sensory attributes of fruit stored for 3 months at 5°C and additional 4 days at 20°C.

5.Conclusions

Exogenous application of putrescine on pomegranate fruit,especially at higher concentrations (2 and 3 mmol/L) reduced the incidence of physiological disorders particularly fruit decay,chilling injury severity with effects more prominent during the last months of storage.In addition,these concentrations resulted in lower husk scald after storage for the first 3 months although 100%of the fruit developed scald at the end of storage.Husk scald was the major physiological disorder during storage of pomegranate fruit after the chemical treatments during the study.Since scalding is an enzymatic oxidative process,this highlights the significance of physical treatments as they have been shown to reduce scalding by providing a barrier to oxygen supply.Therefore treating pomegranate fruit with putrescine reduces physiological disorders but only for shorter storage time(2-3 months).

Treating fruit with putrescine also reduced changes in physicochemical properties like color through reduction of anthocyanin biosynthesis.Despite control fruit having more intense aril red color,treated fruit had adequate aril color and the additional advantage of reducing physiological disorders and decay.From the sensory point of view,treating fruit with 2 mmol/L putrescine maintained and in some cases improved the sensory properties of fruit especially after 2 and 3 months of storage.With regard to phytochemicals,treating fruit with 1 mmol/L putrescine concentration maintained the highest phenolic content while 3 mmol/L putrescine concentration reduced changes in ascorbic acid maintaining relatively constant amounts throughout storage.In conclusion,treating pomegranate fruit with 2 and 3 mmol/L concentration of putrescine would be recommended to improve postharvest quality of fruit.However,further research is required to combine the benefits of chemical treatment together with physical treatments if the maximum potential of healthier alternative chemicals is to be realized since chemical treatments alone are not substantial enough to cater for all the quality parameters of the fruit.

Declaration of Competing Interest

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

Acknowledgements

This work is based on the research supported in part by the National Research Foundation of South Africa Grant Numbers:64813 and the Foundation for Food and Agriculture Research FFAR Grant Numbers:DFs-18-0000000008.The opinions,findings and conclusions or recommendations expressed are those of the authors alone,and the funders accept no liability whatsoever in this regard.