Abstract
The effect of two extraction methods of pomegranate juice on itsquality and stability was evaluated. The first method consistedof separation of the seeds from fruits and centrifugation. Thesecond method consisted of squeezing fruit halves with anelectric lemon squeezer. During a period of 72 hours of coldstorage at 4°C, the juices were evaluated for thepresence of sugars, organic acids, and anthocyanins. Delphinidin3-glucoside was identified to be the major anthocyanin present atthe level of 45–69 mg/L. Among the organic acids, oxalic andtartaric acids dominated. The major sugars detected inpomegranate juice were glucose and sucrose. No significantdifferences in the content of sugars, organic acids, oranthocyanins in juices obtained through application of the twodifferent extraction methods were detected, with the exception ofthe drastic decrease of cyanidin 3,5-diglucoside level in juiceobtained by seed centrifugation. The pH did not show differencesbetween treatments. Titrable acidity and the level of sugarsexpressed as °Brix decreased after 32 and 15 hoursafter extraction, respectively, when juice was obtained bycentrifuging the seeds.
INTRODUCTION
Phenolic compounds are important components of many fruits,vegetables, and beverages contributing to their colour and sensoryproperties. Epidemiological studies have demonstrated that thecomposition of phenol-rich food retards the progression ofarteriosclerosis and reduces the incidence of heart diseases bypreventing the oxidative stress, that is, lipid peroxidation inarterial macrophages and in lipoproteins [1, 2]. Morerecently, some authors reported that anthocyanins decreasedcadmium accumulation in liver and kidney, the concentration ofbilirubin and urea in blood serum, and aspartate aminotransferaseand alanine aminotransferase [3].
Pomegranate juice is an important source of phenolic compounds,with anthocyanins being one of the most important, especially the3-glucosides and 3,5-diglucosides of delphinidin, cyanidin, andpelargonidin [4]. These components along withgallagyl-type tannins, ellagic acid derivatives, andother hydrolysable tannins could contribute in some way to theantioxidant activity of pomegranate juice [2].
“Assaria” pomegranateis a Portuguesevariety cultivated in the southern region of the country. Its edibleseeds are a favourite snack due tosweet taste and tenderness, and itsfruits are mainly used for direct consumption. However, they couldalso be used for production of fruit juices or production ofprocessed products such as jams, jellies, syrups, or carbonatedbeverages [5]. At present the damaged fruits, with cracks,cuts, or bruises in the husk, are discarded. Their application forthe production of processed food products could improve theeconomic yield of this crop.
The composition of pomegranate juice depends on cultivar type,environmental and postharvest factors, and storageand processing factors [2, 6, 7, 8, 9, 10].
Although Assaria pomegranate is the main Portuguese variety, the composition ofits juice is not yet well studied. The objective of ourresearch is to evaluate the composition of the Assariapomegranate juices obtained using two different extractionmethods and their effect on the juice quality during storageover 72 hours at 4°C.
MATERIALS AND METHODS
Fruits and treatments
Sweet pomegranates (Punica granatum cvAssaria) were harvested in a commercial orchard ineastern Algarve. Fruits were transported on the same day to thelaboratory at the University of Algarve. The damaged fruits wereremoved and the healthy fruits of uniform size and appearancewere washed and randomly distributed into groups of 10 fruits forjuice extraction.
To obtain juice, two extraction methods were applied. The firstmethod consisted of manually peeling the fruits, separating theseeds, and extracting the juice by a Phillips Electric juicecentrifuge. In the second method, fruits were cut in two halvesand the juice was immediately extracted using a Phillips Electriclemon squeezer. Each extraction was replicated 4 times.
The obtained juices were immediately stored at 4°C inthe dark. Samples were collected at 0, 5, 15, 32, 48, and 72hours after extraction. At each sampling point, the juices wereanalysed for °Brix (which is a percentage by weight ofsugar in a solution at room temperature), pH, titrable acidity,anthocyanins, sugars, and organic acids. The changes in colourwere monitored according to the Munsell Colour Chart [11].
Standards and reagents
Delphinidin 3,5-diglucoside (Dp3,5), delphinidin 3-glucoside(Dp3), cyanidin 3,5-diglucoside (Cy3,5), cyanidin 3-glucoside(Cy3), pelargonidin 3,5-diglucoside (Pg3,5), and pelargonidin3-glucoside (Pg3) standards were purchased from Apin ChemicalsLtd, UK. Methanol (HPLC gradient grade) was purchased from Sigma-Aldrich Quimica, SA(Spain). Formic, oxalic, tartaric, pyruvic, malic, ascorbic,maleic, citric, fumaric, and sulphuric acids glucose, andfructose were purchased from Riedel-de-Haën(Germany). The ultrapure water was purified withthe MilliQ system, from Millipore, USA.
Titrable acidity, pH, and °Brix
Titrable acidity was calculated as percentage of citric acid bytitrating 10 mL of the pomegranate juice with a solution ofNaOH (0.1 N) till pH 8.1. The pH was measured by a pHmeter (Crison micropH 2001—Crison Instruments, SA(Spain)). The level of sugars was measured as °Brix bya digitalrefractometer, model PRI-Atago Co LTD (Japan).
Anthocyanins
The juice sample (1 mL) was centrifuged for 2 minutes at10 000 rpm and filtered through a 0.45 μm filter(Millipore). The identification of anthocyanins was performed byHPLC with a System Gold Programmable DetectorModule 166-UV-Vis (Beckman Coulter, USA), using aLiChroCART 100 RP-18 column (25 cm × 0.4 cm i.d.; 5 μm particlesize; Merck (Germany)). The mobile phase was 5%formic acid (A) and methanol (B) in a linear gradient from 15%to 35% B at 15 minutes, followed by isocratic run until 20minutes. The flow rate was 1 mL/min. Chromatograms wererecorded at absorbance of 510 nm. The different anthocyaninswere identified by comparison of their retention times with thoseof pure standards. The concentrations of anthocyanins werecalculated from standard curves of Dp3,5, Dp3, Cy3,5, Cy3,Pg3,5, and Pg3, at four concentrations (0.01, 0.02, 0.04,0.08 mg/L). Injection volume was 20 μL using aninjector with a 20 μL loop (Rheodyne, USA).
Sugars and organic acids
To determine the content of sugars and organic acids in juice,samples (1 mL) were centrifuged for 20 minutes at13 000 rpm and filtered through a 0.45 μm filter(Millipore). The composition of sugars and acids was detectedwith an HPLC (Beckman) equipped with a Jasco (Japan)refractive index (RI) 1530 detector. The column Polyspher OA HY(30 cm × 0.65 cm i.d.; 9 μm particle size)from Merck was used at 35°C. The mobilephase consisted of 0.0025 N H2SO4 applied at aflow rate of 0.4 mL/min. The injection volume was20 μL using an injector with a 20 μL loop(Rheodyne). The different sugars and organic acids wereidentified by comparison of their retention times with those ofpure standards. The concentrations of these compounds werecalculated from standard curves of the respective sugars andorganic acids.
RESULTS AND DISCUSSION
The levels of major anthocyanins detected in pome-granate juices obtained through two different extraction methods,centrifugation of seeds or squeezing of fruit halves with anelectric lemon squeezer, and stored at 4°C over 72 hoursare presented in Figures 1 and 2. Nosignificant differences in the composition of anthocyanins weredetected among the treatments. In both cases, the mainanthocyanin was Dp3, followed by Dp3,5, Cy3,5, and Cy3.Pg3,5 and Pg3 were present in the lowest amounts. Theanthocyanins detected in our analysis of the Assaria pomegranatejuices were as identified in other cultivars [12]; however,concentrations of the individual pigments differed.
Figure 1.
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Figure 2.
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Colour is one of the most important parameters when making asensorial evaluation of food quality. No significantdifferences were observed between the colours of juices obtainedthrough various extraction methods. At the extraction time, theAssaria juice colour was noted as 53A, according to the MunsellColour Chart. The juice colour did not change during experimentaltime.
The bright colour of pomegranate fruit and juice is due toanthocyanins, so their stability through juice processing is ofmajor importance. The anthocyanin content in both extractionmethods was similar as was the respective evolution profile overtime. During the first 5 hours of storage at 4°C, aslight increase (0.3%–4%) in the amounts of each anthocyaninwas registered, followed by a decrease in the next ten hours andthen stabilisation occurred. Additionally, the fruit juiceobtained by centrifugation of seeds showed a pronounced decreaseof Cy3,5, mainly after 48 hours of storage(Figure 1). It was reported previously that inPOM Wonderful pomegranatejuices the diglucoside anthocyanins weremore stable than the monoglucosides [8], therefore the highdecrease of Cy3,5 observed in our experiment was unexpected.
The main organic acids present in the pomegranate juices wereoxalic and tartaric acids, either in juices obtained by seedcentrifugation or by squeezing of fruit halves, respectively(Figures 3 and 4). These results were quitedifferent from those reported by others [6, 13] for 11Spanish pomegranate cultivars in which citric acid was the mainorganic acid, sometimes followed closely by malic acid. Oxalicand tartaric acids were present only in small amounts. Incontrast, a great variation in the organic acid composition amongthe Turkish pomegranate cultivars has been reported[14].Citric and malic acids were predominant in the majority ofvarieties, but in some cultivars large amounts of oxalic andtartaric acids were detected. In those varieties, only one hadoxalicacid as the major organic acid.
Figure 3.
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Figure 4.
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Theevolution profiles of organic acids over time in juicesobtained through application of both methods were quite similar,with only a few exceptions. A decrease of each organic acid duringthe first 5–15 hours of cold storage was observed, followed byan increase reaching the maximal values after 32 hours of storage.At this point, the levels of oxalic acid were of 292.9 and271.1 mg/L and those of tartaric acids were of 228.9 and228.0 mg/L in juices obtained by seed centrifugation andsqueezing of fruit halves, respectively. The exceptionsreported above were malic, maleic, and citric acids in the samplesobtained by squeezing the fruits, and ascorbic acid in thoseobtained through application of both methods(Figure 4). The concentrations of malic and citricacids reached the maximum after 15 hours of storage. The highestlevel of maleic acid was observed just after juice extraction.The ascorbic acid content remained quite stable over the wholestorage period independently of the method application.
The main carbohydrates detected in the pomegranate juices wereglucose and fructose (Figures 5 and 6). Thissupports previously reported results for other pomegranatecultivars [6]. Only traces of sucrose were found andtherefore sucrose was not considered in the present work. Theamounts of glucose and fructose were quite similar in juicesobtained through application of both methods. A pronounceddecrease of total carbohydrate content of about 50% was observedin juices independently of the extraction method after 5 hours ofstorage at 4°C. It can be assumed that the decreaseof sugar and organic acids content during the first 15 hours ofstorage occurs due to de novo synthesis of anthocyanins, whoselevel increased exactly at the same time in juices obtainedthrough both methods.
Figure 5.
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Figure 6.
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Titrable acidity showed a clear decrease after 32 hours ofstorage when juice was obtained by centrifuging the seeds, thatwas less marked in the juice extracted by squeezing fruits(Figure 7). The level of sugars measured as°Brix changed over time but the main feature was thesharp decrease after 15 hours until the end of the experiment in the seed centrifugationprocedure (Figure 8). This behaviour may be relatedto the different chemical composition of juices due to thepresence of tannins as a result of rind cell damage during fruitsqueezing. The presence of tannins is the main problem whenjuices are extracted from whole fruits. As a result, a bittertaste develops that must be corrected by industrial processing[15]. In our experiment, according to panelist evaluation ofthe fresh juices, the juice obtained by fruit squeezing showed abitter taste in comparison to the sweet taste of the juiceobtained by centrifuging seeds.
Figure 7.
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Figure 8.
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The pH presented a slight increase over time, more pronouncedfrom 5 to 15 hours after extraction (Figure 9). Therewere no differences in pH related with the method used for juiceextraction. This could partially explain the relative stabilityof anthocyanins found in the juices obtained by both extractionmethods.
Figure 9.
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Both methods used for pomegranate juice extraction did notaffect the evaluated characteristics of juice quality; namely, theanthocyanins content, the juice colour, the organic acids andsugars composition, as well as the pH values. Squeezing unpeeled fruit halves is the most economical and easy method to use. Thejuice obtained by that method was more stable over time asindicated by the titrable acidity and °Brixdeterminations. The main disadvantage of the squeezing method isthe production of juice with bitter taste if additionaltreatments are applied. Besides, the bitterness could beovercome because the actual trend in juice production is theblending of several fruit juices. Additionally, the use ofAssaria pomegranate in fruit juice mixtures will bebeneficial for human health in reducing the riskfrom oxalic acid consumption [16].
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