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The influence of Opuntia ficus-indica mucilage edible coating on the quality of ‘Hayward’ kiwifruit slices
Postharvest Biology and Technology 120 (2016) 45–51
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The influence of Opuntia ficus-indica mucilage edible coating on the quality of ‘Hayward’ kiwifruit slices A. Allegra* , P. Inglese, G. Sortino, L. Settanni, A. Todaro, G. Liguori Department of Agriculture and Forestry Sciences, Università degli Studi di Palermo, Viale delle Scienze ed. 4 ingresso, H-90128 Palermo, Italy
A R T I C L E I N F O
Article history: Received 19 March 2016 Received in revised form 18 May 2016 Accepted 24 May 2016 Available online xxx Keywords: Actinidia deliciosa Ascorbic acid Pectin Microbial spoilage Fresh-cut Flavor score
A B S T R A C T
The aim of this work was to study the effect of mucilage edible coating extracted from Opuntia ficus-indica (OFI) on the quality and shelf life maintenance of packaged kiwifruit slices. OFI mucilage alone or added with TWEEN1 20 were applied on kiwifruit fresh cut surfaces. After treatments, kiwifruit samples were stored under passive atmosphere at 5 1 C for 3, 5, 7 and 12 days. At each storage period, visual quality and flavor score, pectin content, ascorbic acid and the microbiological characteristics were measured together with CO2 and O2 content in the packages. Kiwifruit slices coated only with mucilage or with mucilage plus Tween 20, showed a significant higher firmness and a lower weight loss than untreated slices, until 5 d of shelf life. No further differences in weight loss occurred after 7 d of shelf life, while slices treated only with mucilage retained the highest firmness until the end of the shelf life period (12 d). OFI mucilage alone had significant beneficial effects on the visual and flavor score of the kiwifruit slices, throughout the shelf life period. The treatment with Tween 20 did not affect the flavor of the kiwifruit slices, compared with untreated fruit. Although mucilage and partly tween 20 addition increased microbial growth, especially of yeasts, their levels were still below the threshold for yeast spoilage at the end of the monitoring period. Hence, the results showed that mucilage coating applied to kiwifruit minimally processed fruit improved the quality of stored fresh-cut kiwifruits. ã 2016 Elsevier B.V. All rights reserved.
1. Introduction The wide success of kiwifruit (Actinidia deliciosa) is largely due to the bright color of the flesh that, together with its flavor and high nutraceutical value (high vitamin C content), represents the most important fruit attributes. Indeed, kiwifruit slices are largely used in fruit salads or in confectionery where they are the only greenfleshed fruit used. However, peeling and slicing involved in minimal processing can cause physical damage and increase ethylene production and respiration of kiwifruit. Furthermore, the disruption of the fruit cells caused by the cut, frees the cellular content and promotes the microbial development (Garcia and Barrett, 2002). These phenomena might result in flesh softening and a shorted shelf life of minimally processed kiwifruits (Agar et al., 1999). Refrigeration (2 C and >90% RH) associated with CaCl2 or calcium lactate treatment may prolong the shelf life of minimally processed kiwifruit slices up to 9–12 d (Agar et al., 1999) while the use of edible coating based on Aloe vera combined with packaging
* Corresponding author. E-mail address: [email protected] (A. Allegra). http://dx.doi.org/10.1016/j.postharvbio.2016.05.011 0925-5214/ã 2016 Elsevier B.V. All rights reserved.
under passive modified atmosphere packaging (MAP) and low temperature (2 1 C) reduces fruit respiration rates and microbial spoilage of kiwifruit, during 7 d of storage (Benitez et al., 2013). Active coatings under passive MAP also inhibit microbial activity and reduce respiration, while the combination of MAP and active alginate-based coatings delaye quality loss and microbial spoilage of kiwifruit minimally processed slices (Mastromatteo et al., 2011). As a matter of fact, the use of edible coatings is growing, due to their multiple uses for extending fruit shelf life and also as carriers for several food additives (Mastromatteo et al., 2011). Most of the edible coatings are based on carbohydrates, proteins or lipids which can be combined (Oms-Oliu et al., 2008; Valero et al., 2013), such as casein (Ponce et al., 2008) and its derivatives (Fabra et al., 2009), guar gum, cellulose ethyl (Shrestha et al., 2003), gelatin supplemented with glycerol, sucrose and sorbitol as plasticizers (Arvanitoyannis et al., 1997; Sobral et al., 2001), pectin (Maftoonazad et al., 2007), cassava starch (Kechichian et al., 2010), wheat gluten (Tanada-Palmu and Grosso, 2005) and mixtures of sodium alginate and pectin, with the addition of CaCl2 (Da Silva et al., 2009). The mucilage obtained from cladodes of cactus pear (Opuntia ficus-indica) has a highly branched complex polymeric structure of carbohydrate nature, (Medina-Torres et al., 2000;
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Matsuhiro et al., 2006) and contains varying proportions of Larabinose, D-galactose, L-rhamnose and D-xylose (Sáenz et al., 1992; Sepùlveda et al., 2007; Goycoolea and Cárdenas, 2004). The rheological characteristics of O. ficus-indica mucilage makes it interesting for the production of edible coatings with a high nutraceutical value. The mucilage from cladodes of O. ficus-indica forms an edible coating on the fruit surface that makes the treated product shiny. So far, this mucilage has had several applications in fruit preservation. A combination of O. ficus-indica mucilage with citric acid and sodium bisulfite at high concentrations decreases browning of banana (Musa cavendish) slices during drying (Aquino et al., 2009). Treatment with O. ficus-indica mucilage applied on fresh strawberries reduces fruit weight, color and firmness loss, fruit respiration rate, and fungal infection (Del-Valle et al., 2005). However, to the best of our knowledge, this polymer has been never applied on minimally processed fruit. The efficacy of two different O. ficus-indica mucilage-based coating formulations to prolong the shelf life of kiwifruit slices was evaluated. To assess the influence of the treatments, weight loss, softening, sensory quality and microbial load of kiwi fruit were monitored during the storage. 2. Materials and methods 2.1. Samples preparation ‘Hayward’ kiwifruit (Actinidia deliciosa) were purchased from a retail market (Simply, Palermo, Italy). Fruit were uniform in terms of fresh weight (98 4.8 g), total soluble solid content (13.3 1.5%) and firmness (30.5 3.5 N), analyzed on a sample of 30 fruit. Kiwifruit were stored at 1 0.5 C (RH = 85%) for 24 h. After storage, fruit were dipped in chlorinated water (100 mL 1 of free chlorine) for 6 min. Damaged fruits (bruised or showing other physical decays) were removed, and a total of 300 fruits were processed. Cactus pear (O. ficus-indica, (L.) Mill.) cladodes were cut and cubed (2 cm3). To extract the mucilage, cladodes were crushed in a blender (Moulinex) with rotating knifes, homogenized with distilled water in the ratio 1:1.5 (w/v) at 20 C. The solution was maintained at 40 C for 90 min and centrifuged (model CS6R) at 1450g 20 min. The supernatant was boiled to half the initial volume and ethanol (99% v/v) was added in the ratio 1:2 (Sáenz et al., 1992). Afterwards, the solution was stored at 4 1.0 C for 48 h to allow a better aggregation of the mucilage. The last phase involved the elimination of the supernatant and soaking of the pure mucilage. Kiwifruits were peeled using an automatic machine (Agrimat, Maxistreap, Italy) and cut into slices with a semiautomatic machine (Sgorbati, Italy). Slices were 2.2 0.2 cm thick and 5.4 1.2 cm width; L* was 48 2.1, a* was 8.2 1.2 and b* was 25.4 3; total solid soluble content (TSS) was 13.1 2.5% and titratable acidity (TA) was 1.4 0.3%. Fresh-cut slices were dipped in the (OFI) coating solution for 60 s; the excess coating was drained and the coated slices were dried with a forced-air dryer (20 C) for 10 min. The coating treatments consisted of: a) 30 g of pure mucilage extract, 500 mL distilled water and 50 mL glycerol as a plasticizer (MC); b) 30 g of mucilage extract, 500 mL distilled water, 50 mL glycerol added with 2 mL Tween 20 (TW). The control treatment (CTR) wereslices dipped in distilled water. About 95 1.1 g of kiwifruit slices were packed in polyethylene terephthalate (PET) packages and sealed with a composite film (PP-PET), 64 mm, O2 permeability = 5.30 10 8 mL m 2 s 1 Pa 1. Packages were stored at 5 0.5 C and 90% relative humidity (RH) for 12 d. Chemical, physical and microbiological parameters were analyzed, at the beginning of the experiment (after coating/dipping = day 0) and at 3, 5, 7, and 12 d after storage, on six slices per replicate for
treatment (3 treatments combinations 5 time of storage 6 replicates = 90 box). 2.2. Chemical and physical analysis 2.2.1. Firmness Firmness was evaluated with a puncture test on kiwifruit slices flesh using a TA-XT Plus texture analyzer (Stable Micro Systems) equipped with a 50 N load cell of. Firmness measurements were taken as the median force value obtained during the test with a stainless steel probe with 4 mm diameter penetrating the fruit 4 mm, at 1 mm/s. Average values were calculated from the results of at least 6 measurements in different slices for each sample. Measures were taken in fruit outer pericarp (green flesh) where the fast rate of softening compromises fruit quality. 2.2.2. Weight loss Weight of individual bags was recorded immediately after the treatment (day 0) and at the different sampling times (3, 5, 7, and 12 days during storage). Weight loss was expressed as the percentage reduction with respect to initial time, using the following equation: % Weight loss: [(Initial fruit bag weight 100]/Initial fruit bag weight
Final fruit bag weight)
2.2.3. Total soluble solids content and titratable acidity The concentration of total soluble solids (TSS expressed as%) determined from the juice of four slices from each tray using a digital refractometer (model PR-101, Atago, Co., Tokyo, Japan) at 20 C. Titratable acidity (expressed as% citric acid) was determined by titration of 10 mL of juice with 0.1 M NaOH to an endpoint of pH 8.1. 2.2.4. Sensory evaluation 2.2.4.1. Visual appearance score. To measure the effect of cold storage on kiwifruit sensory traits at each storage time (0, 3, 5, 7 and 12 d), six slices, used as single replicates for each treatment (MC, TW and CTR), were scored by each of a six judges trained panel that generated a list of descriptors in a few preliminary meetings. Visual appearance score resulted from the medium value of color, visible structural integrity and visual appearance (Allegra et al., 2015a). The different descriptors were quantified using a subjective 5–1 rating scale with 5 = very good, 4 = good, 3 = sufficient (limit of marketability), 2 = poor (limit of usability) and 1 = very poor (inedible). 2.2.4.2. Flavor score. To measure the effect of cold storage on slices fruit flavor traits at each storage time, six slices, used as single replicates for treatment (MC, TW and CTR), were scored by six trained judges, using a subjective 5–1 rating scale with 5 = very high, 4 = high, 3 = sufficient (limit of marketability), 2 = low and 1 = none. 2.2.5. Package O2 and CO2 analysis CO2 and O2 levels (kPa) were measured on each package at the beginning of each experiment and after 3, 5, 7, 12 days of storage, using a PBI Dansensor Checkpoint O2 and CO2 analyzer (Topac, Hingham, MS, USA) with zirconium and infrared detectors, respectively. 2.2.6. Ascorbic acid Ascorbic acid concentration was determined according to Rapisarda and Intelisano (1996) by high performance liquid
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chromatography (HPLC) (Perkin Elmer, Australia) using an injector (Rheodyne with 20 mL loop), a photodiode detector, a Knauer Eurospher II 100-5 C18 column 250 mm 4.6 mm I.D. (Berlin, Germany) and a similarly packed pre-column. The elution was performed with a buffer solution consisting of KH2PO4/H3PO4 at pH 2.3, at a flow rate of 1 mL min 1, the wavelength was at 260 nm. An aliquot of freshly prepared pulp (5 g) was homogenized with 25 mL of 3% metaphosphoric acid, centrifuged, filtered through a 0.45 mm Whatman Puradisc and HPLC injected. The concentration of ascorbic acid was calculated from the experimental peak area by analytical interpolation in a standard calibration curve and was expressed as mg 100 g 1 of fresh weight. 2.2.7. Microbiological analysis Fruit samples and mucilage were microbiologically investigated for total mesophilic microorganisms (TMM) and the undesired (spoilage and/or pathogenic) microbial groups. The fruits (25 g) and the mucilage (10 mL) were collected separately suspended in Ringer’s solution (Sigma-Aldrich, Milan, Italy) to a ratio 1:10 (fruit: diluent), homogenized for 2 min at the highest speed with a stomacher (BagMixer1 400, Interscience, Saint Nom, France) and serially diluted. The cell suspensions were inoculated as follows: TMM on plate count agar (PCA), incubated at 30 C for 72 h; members of the Enterobacteriaceae family on double-layered violet red bile glucose agar (VRBGA), incubated at 37 C for 24 h; Pseudomonas on Pseudomonas agar base (PAB) supplemented with 10 cetrimide fucidin, incubated at 25 C for 48 h; yeasts on yeast potato dextrose (YPD) agar, incubated at 25 C for 48 h. All media and supplements were purchased from Oxoid (Milan, Italy). Plate Count were carried out in duplicate for each trial. 2.2.8. Pectin analysis The pectic substances from fruits were extracted according to the methods described by James and Rouse (1952) and Rouse and Atkins (1955). Frozen slices were thawed at 4 C for 24 h; about 20 g were weighed into a plastic cup, homogenized at low speed with an Ultra-Turrax tissuemizer for 2 min. The tissue sample (5 g) was weighed into a 50 mL round-bottom plastic centrifuge tube. Hot 100% ethanol (30 mL) was added into the tubes. The samples were stirred thoroughly with a glass rod, heated in a boiling water bath for 10 min, cooled and centrifuged at 14,500g for 10 min. The alcoholic supernatant was decanted and discarded. The precipitate was extracted with 30 mL 100% ethanol, centrifuged and the supernatant was discarded. The residue was transferred from the centrifuge tube to a 57 mm aluminum weighing dish (Fisher Scientific). Samples were dried for 24 h in a conventional oven at 35 C, weighed and ground in a mortar with a pestle. The precipitate from the alcohol solution was designated as alcoholinsoluble solids (AIS). Dried AlS (80 mg) was weighed into a 50 mL centrifuge tube. Distilled water (20 mL) was added and samples were stirred with a glass rod for 1 min. Samples were centrifuged at 14,500g for 10 min, filtered through Whatman No. I filter paper to obtain water-soluble pectin (WSP). The extraction procedure was repeated once. The supernatants were collected and combined in a 100 mL volumetric flask. Distilled water was added to dilute tube extract solution to volume. The residue was dispersed in 20 mL of an aqueous solution containing 0.25% ammonium oxalate and 0.25% oxalic acid and stirred with a glass rod for 1 min. Samples were refluxed in a boiling water bath for l h, centrifuged at 14,500g for 10 min and filtered through Whatman No. I fìlter paper to obtain oxalatesoluble pectin (OSP). The oxalate extraction was repeated once. Supernatants were collected and diluted to 100 mL with distilled water in a volumetric flask.
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Total pectin (TP) was extracted by the method of Ahmed and Labavitch (1978). Dried AIS (5 mg) was weighed into a 30 mL beaker containing a magnetic stir bar. Concentrated sulfuric acid (2 mL) was added to the becker, and the mixture was swirled gently. The beaker was placed on a stir plate and stirred gently and 0.5 mL of distilled water were added dropwise. Stirring continued for 5 min; an additional aliquot of 0.5 mL distilled water was added dropwise. Stirring continued furtherly for approximately 30 min until dissolution of AIS was complete. The dissolved sample was filtered through glass wool into a 25 mL volumetric flask. Each beaker was rinsed several times with distilled water, combined in a 25 mL flask, and diluted to volume. The solution was filtered through glass wool again before use. The difference between TP and the sum of WSP and OSP was used to determine the amount of non-extractable pectin (NXP), which was the protopectin fraction. TP, WSP and OSP extractions were completed in duplicate for all samples. All pectin extracts were stored at 4 C for 12 h before analysis. Extract pectin contents were analyzed by the m-hydroxydiphenyl method (Kintner and Buren, 1982). The extract from each sample (1 mL) was pipetted into a 16 150 mm test tube. Six milliliters of sulfuric acid tetraborate solution (0.0125 M sodium tetraborate in concentrated sulphuric acid) was added to each of the tubes in an ice water bath and mixed intermittently using a Vortex mixer at moderate speed to assure complete mixing. Duplicate samples were prepared for each pectin measurement with a corresponding blank. Tubes were heated in a boiling water bath for 5 min and immediately placed in ice water to cool. To duplicate tubes, 0.1 mL aliquot of 0.15% m-hydroxydiphenyl was added to develop color. To the blank tube, 0.1 mL 0.5% sodium hydroxide was added. All samples and blanks were mixed using the Vortex mixer and allowed to stand 15 min at room temperature. The absorbance of the samples following chromogen formation was measured at 520 nm using a spectrophotometer (Beckman DU 640-Canada). Galacturonic acid was used as a standard. A solution consisting of 1 mL distilled water, 6 mL sulfuric acid/tetraborate and 0.1 mL 0.5% sodium hydroxide was used as reagent blank. The determination of the pectin was done according to Yu et al. (1996) 2.2.9. Statistical analysis The experimental design consisted of two coating treatments and the untreated control, with observations made at 0, 3, 5, 7, 12 days after coating. Analysis of variance ANOVA (Systat 13.0 for Windows was used as statistical software) was performed and mean values were compared with Tukey’s test during storage among treatments, for each storage time and for each treatment. Differences were considered to be significant when the p < 0.05. 3. Results and discussion 3.1. Firmness, TSS, TA, weight loss and pectin content Kiwifruit slice firmness changed with sampling time and treatment. Untreated kiwifruit slices showed a sharp, significant (P 0.05) decrease in firmness soon after placement in storage and no significant further changes occurred during the next 9 d (Fig. 1). Slices coated with OFI mucilage + glycerol (MC) showed no significant softening during the first 5 d of storage, and a significant (P 0.05) decrease during the next 7 d. Kiwifruit slices coated with OFI mucilage + glycerol + Tween (TW) showed a continuous and significant decrease throughout the storage period (P 0.05). Differences among treatments appeared 3 d after storage when untreated slices had the lowest firmness, while no differences occurred between MC and TW coated slices (Fig. 1). TW slices and untreated ones had the same firmness values at 7 and 12 d after storage. MC coated slices had the highest firmness throughout the
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Fig. 1. Firmness of fruit slices of Actinidia deliciosa (Liang, Ferguson) coated with O. ficus-indica (OFI) mucilage + glycerol (MC), or OFI mucilage + glycerol + Tween 20 (TW) or not treated (CTR), just after being coated (0) and stored for 3, 5, 7, 12 d at 5 C. Different letters indicate significant differences between treatments at each sampling date. Tukeys’ significant test was applied at P 0.05. Data are means S.E. (n = 6).
storage period. O. ficus-indica coatings did not affect kiwifruit slice TSS content and TA during the storage (data not shown). The hydrophilic character of OFI coating could act as a barrier to water transfer, retarding dehydration and maintaining firmness during fruit shelf life. In fact, untreated fruit reached the maximum weight loss 3 d after the packaging, with no further increases in the next 9 days. During the first 5 d of storage both coated treatments showed the lowest weight loss (Fig. 2). However, the weight loss of MC and TW coated slices significantly (P 0.05) increased after one week of storage, though MC slices had the lowest weight loss. At the last sampling time no differences occurred between coated and untreated slices (Fig. 2). In general, fruit softening is associated with disassembly of the primary cell wall, with solubilization and depolymerization of pectins (Alzamora et al., 2000; Brummell et al., 2004). In kiwifruit, the loss of galactose residues occurs from the insoluble residue (Redgwell et al., 1997). After harvest, kiwifruit go through three distinct softening phases that are temporally well separated. Pectin retained in the cell wall starts to “soften” during ripening; this process clearly precedes both pectin solubilisation and depolymerisation (Schroder and Atkinson, 2006). Degradation of solubilized pectin and loss of middle lamellae in kiwifruit, are processes that are initiated in the second softening phase, but peak in the last softening phase, where cell wall disintegration is completed (Schroder and Atkinson, 2006). In our study, TW slices showed the highest total pectin content throughout the storage period (Fig. 3).
Fig. 2. Fresh weight loss of fruit slices of Actinidia deliciosa (Liang, Ferguson) coated with O. ficus-indica (OFI) mucilage + glycerol (MC), or OFI mucilage + glycerol + Tween 20 (TW) or not treated (CTR), just after being coated (0) and stored for 3, 5, 7, 12 d at 5 C. Different letters indicate significant differences between treatments at each sampling date. Tukeys’ significant test was applied at P 0.05. Data are means S.E. (n = 6).
Fig. 3. Total pectin (TP) of fruit slices of Actinidia deliciosa (Liang, Ferguson) coated with O. ficus-indica (OFI) mucilage + glycerol (MC), or OFI mucilage + glycerol + Tween 20 (TW) or not treated (CTR), just after being coated (0) and stored for 3, 5, 7, 12 d at 5 C. Different letters indicate significant differences between sampling dates for each treatment. Tukeys’ significant test was applied at P 0.05. Data are means S.E. (n = 3).
Water soluble pectin (WSP) significantly increased after one week of storage only in TW slices (Fig. 4). On the other hand, protopectin, (NXP) significantly decreased only in untreated slices from 5 days after storage onwards. This trends corresponded to the loss of firmness of CTR kiwifruit slices during storage (Fig. 1). Similar results have been reported for peaches, in which the water-soluble pectin content tends to increase during storage at different temperatures (Zhang et al., 2010) and in kiwifruit where the decrease in firmness was closely related to the soluble pectin content increase during storage (Xu et al., 2001). 3.2. Head spaces gas composition, ascorbic acid content The polysaccharide content in OFI mucilage can be used to modify the internal atmosphere of fruits, delaying senescence (Rojas-Graü et al., 2009). Edible coatings create a passive modified atmosphere, which can influence changes in minimally processed fruits (Oms-Oliu et al., 2008). Kiwifruit after cutting and packaging in atmosphere passive, have altered the metabolism showing a progressive increase of CO2 and decrease in O2. In this study, the O2 (kPa) content inside packages showed a significant lower value for the bags containing TW slices, by day 3 of storage but, no significant differences were found (Fig. 5a and b). The increase of CO2 accumulation was observed after 3 d of storage in all sample slices coated with OFI mucilage. The bags containing control slices showed an accumulation of 15 kPa CO2 after 5 d of storage (Fig. 5a and b). The reduction of O2 consumption and CO2 production in package headspace atmospheres was observed also in kiwifruit coated with A. vera (Benitez et al., 2013), alginate (Mastromatteo et al., 2011) or a CaCl2 solution (Beirão-da-Costa et al., 2014). The initial ascorbic acid content in the fresh cut kiwifruit slices analyzed in this study are in agreement with the values reported for ripe ‘Hayward’ kiwifruits (Gil et al., 2006; Tavarini et al., 2008). During storage, a progressive degradation of ascorbic acid in the coated and untreated slices was observed (Barberis et al., 2012). The ascorbic acid content of untreated, MC and TW minimally processed kiwifruit slices decreased by 49%, 46% and 42%, respectively, after 12 d of storage (Fig. 6). A sharp decrease occurred soon after 3 d of storage in untreated (35%) and in MC (28%) kiwifruit slices, while TW slices kept the initial values (Fig. 6). Significant differences among treatments occurred only during the first 3 d of storage, when untreated kiwifruit slices showed the lowest ascorbic acid content and TW coated slices the highest. However no difference between TW and MC occurred by day 5, when untreated slices had the lowest ascorbic acid content (Fig. 6)
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Fig. 4. Water soluble pectin (WSP) and non extractable pectin (NXP) of fruit slices of Actinidia deliciosa (Liang, Ferguson) coated with O. ficus-indica (OFI) mucilage + glycerol (MC), or OFI mucilage + glycerol + Tween 20 (TW) or not treated (CTR), just after being coated (0) and stored for 3, 5, 7, 12 d at 5 C. Different letters indicate significant differences between treatments at each sampling date. Tukeys’ significant test was applied at P 0.05. Data are means S.E. (n = 3).
Fig. 5. Oxygen (A) and Carbon dioxide (B) content (kPa) inside packages with fruit slices of Actinidia deliciosa (Liang, Ferguson) coated with O. ficus-indica (OFI) mucilage + glycerol (MC), or OFI mucilage + glycerol + Tween 20 (TW) or not treated (CTR), just after being coated (0) and stored for 3, 5, 7, 12 d at 5 C. Different letters indicate significant differences between treatments at each sampling date. Tukeys’ significant test was applied at P 0.05. Data are means S.E (n = 6).
3.3. Visual appearance and flavor score
Fig. 6. Ascorbic acid of fruit slices of Actinidia deliciosa (Liang, Ferguson) coated with O. ficus-indica (OFI) mucilage + glycerol (MC), or OFI mucilage + glycerol + Tween 20 (TW) or not treated (CTR), just after being coated (0) and stored for 3, 5, 7, 12 d at 5 C. Different letters indicate significant differences between treatments at each sampling date. Tukeys’ significant test was applied at P 0.05. Data are means S.E (n = 6).
The sensory evaluation revealed that MC and TW slices maintained visual quality score after 3 d of storage. Untreated kiwifruit slices had the lowest visual quality scores during the first 5 d, when no significant differences occurred between MC and TW treatments (Fig. 7a). The last day of storage, TW slices showed a significant decrease of the visual quality score and were similar to the untreated samples; at this stage MC slices kept the highest score among treatments (Fig. 7a). Considering both the visual quality and the flavor score, MC slices kept their marketability during 9 days of storage, while untreated kiwifruit slices were not marketable 7 d after storage in terms of flavor (Fig. 7b) and reached their marketability limit in terms of visual score 5 d after storage. TW slices were marketable, in terms of flavor, until 5 d after storage, while in terms of visual quality they kept good values until 7 d of storage (Fig. 7a and b). Sensory analysis of the fruit treated with mucilage maintained the characteristics of flavor of the kiwifruits, conversely coating based with Aloe vera showed bitters during consumer evaluation (Benitez et al., 2013).
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Fig. 7. Visual quality (A) and flavor score (B) of fruit slices of Actinidia deliciosa (Liang, Ferguson) coated with O. ficus-indica (OFI) mucilage + glycerol (MC), or OFI mucilage + glycerol + Tween 20 (TW) or not treated (CTR), just after being coated (0) and stored for 3, 5, 7, 12 d at 5 C. Different letters indicate significant differences between treatments at each sampling date. Tukeys’ significant test was applied at P 0.05. Data are means (n = 6).
3.4. Microbiological analysis The microbiological analyses of the mucilage did not evidence the presence of any of the microbial groups object of investigation. Differences among treatments appeared at 3 and 5 d after storage when MC and TW treatment had a lower TMM concentration than untreated slices. No further differences among treatments occurred at 7 and 12 d of storage (Fig. 8a). Members of the Enterobacteriaceae family were below the detection limit during the entire period of analysis (Allegra et al., 2015b). Pseudomonads (Fig. 8b) did not show significant differences in all sample during 5 d of storage while significant increases in this population
occurred in MC, showing a load of 1.77 Log CFU g 1 after 7 d. A similar trend was observed also for yeasts (Fig. 8c) for which the highest increase in concentration was registered on MC slices during the first 3 d. Differences between the coated slices (MC and TW) and control treatments were found after 7 d of storage. However, the cell densities of Pseudomonads and yeasts were not able to determine a microbial decay of the sliced kiwifruits. The levels of yeasts never exceeded those of TMM, indicating that the highest microbial values found in treatments MC and TW are due to yeast development. This is due to the fact that yeasts are more resistant than bacteria to the acidic conditions (Smelt, 1998) such as those found on the kiwifruit surface.
Fig. 8. Total mesophilic (A), Pseudomonas (Enterobacteriaceae) (B) and Yeast content (C) in fruit slices of Actinidia deliciosa (Liang, Ferguson) coated with O. ficus-indica (OFI) mucilage + glycerol (MC), or OFI mucilage + glycerol + Tween 20 (TW) or not treated (CTR), just after being coated (0) and stored for 3, 5, 7, 12 d at 5 C. Different letters indicate significant differences between treatments at each sampling date. Tukeys’ significant test was applied at P 0.05. Data are means S.E (n = 3).
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4. Conclusions This work reported the efficacy of OFI edible coating on fresh cut kiwifruit slices. The results showed the positive effects of mucilage on the maintenance of firmness, ascorbic acid and pectin content, visual quality and flavor score of the fruits during storage a 5 C. Furthermore, though mucilage and Tween 20 determined an increase of microbial growth, particularly that of yeasts, their final levels were below 6.0 Log CFU g 1 which, according to Thomas et al. (2004), is regarded as the threshold for yeast spoilage. This work also demonstrated that the treatment without Tween 20, was more effective in terms of quality parameters in kiwifruits slices. References Agar, I.T., Massantini, R., Hess-Pierce, B., Kader, A.A., 1999. Postharvest CO2 and ethylene production and quality maintenance of fresh-cut kiwifruit slices. J. Food Sci. 64 (3), 433–440. Ahmed, A.E.R., Labavitch, J.M., 1978. A simplified method for accurate determination of cell wall uronide content. J. Food Biochem. 1 (4), 361–365. Allegra, A., Barone, E., Inglese, P., Todaro, A., Sortino, G., 2015a. Variability of sensory profile and quality characteristics for ‘Pesca di Bivona’ and ‘Pesca di Leonforte’peach (Prunus persica Batsch) fresh-cut slices during storage. Postharvest Biol. Technol. 110, 61–69. Allegra, A., Sortino, G., Miciletta, G., Riotto, M., Fasciana, T., Inglese, P., 2015b. The influence of harvest period and fruit ripeness at harvest on minimally processed cactus pears (Opuntia ficus-indica L.) Mill. stored under passive atmosphere. Postharvest Biol. Technol. 104, 57–62. Alzamora, S.M., Tapia, M.S., Lopez-Malo, A., 2000. Minimally Processed Fruits and Vegetables. Aspen Publisher Inc, USA, Gaithersburg, Maryland. Aquino, L.V., Rodríguez, J., Méndez, L.L., Torres, K.F., 2009. Inhibición del oscurecimiento con mucílago de nopal (Opuntia ficus-indica) en el secado se plátano roatán. Inf. Tecnol. 20 (4), 15–20. Arvanitoyannis, I., Kolokuris, I., Nakayama, A., Yamamoto, N., Aiba, S.I., 1997. Physico-chemical studies of chitosan-poly (vinyl alcohol) blends plasticized with sorbitol and sucrose. Carbohydr. Polym. 34 (1), 9–19. Barberis, A., Fadda, A., Schirra, M., Bazzu, G., Serra, P.A., 2012. Detection of postharvest changes of ascorbic acid in fresh-cut melon, kiwi, and pineapple, by using a low cost telemetric system. Food Chem. 135 (3), 1555–1562. Beirão-da-Costa, S., Empis, J., Moldão-Martins, M., 2014. Fresh-cut kiwifruit structure and firmness as affected by heat pre-treatments and post-cut calcium dips. Food Bioprocess Technol. 7 (4), 1128–1136. doi:http://dx.doi.org/10.1007/ s11947-013-1151-3. Benitez, S., Achaerandio, I., Sepulcre, F., Pujola, M., 2013. Aloe vera based edible coatings improve the quality of minimally processed ‘Hayward’ kiwifruit. Postharvest Biol. Technol. 81, 29–36. Brummell, D.A., Dal Cin, V., Crisosto, C.H., Labavitch, J.M., 2004. Cell wall metabolism during maturation, ripening and senescence of peach fruit. J. Exp. Bot. 55 (405), 2029–2039. doi:http://dx.doi.org/10.1093/jxb/erh227. Da Silva, M.A., Bierhalz, A.C.K., Kieckbusch, T.G., 2009. Alginate and pectin composite films crosslinked with Ca 2+ ions: effect of the plasticizer concentration. Carbohydr. Polym. 77 (4), 736–742. Del-Valle, V., Hernández-Muñoz, P., Guarda, A., Galotto, M.J., 2005. Development of a cactus-mucilage edible coating (Opuntia ficus-indica) and its application to extend strawberry (Fragaria ananassa) shelf-life. Food Chem. 91 (4), 751–756. Fabra, M.J., Talens, P., Moraga, G., Martínez-Navarrete, N., 2009. Sorption isotherm and state diagram of grapefruit as a tool to improve product processing and stability. J. Food Eng. 93 (1), 52–58. Garcia, E., Barrett, D.M., 2002. Preservative treatments for fresh-cut fruits and vegetables. In: Lamikanra, O. (Ed.), Fresh-cut Fruits and Vegetables: Science, Technology and Market. CRC, Florida, pp. 274–310. doi:http://dx.doi.org/ 10.1201/9781420031874.ch9. Gil, M.I., Aguayo, E., Kader, A.A., 2006. Quality changes and nutrient retention in fresh-cut versus whole fruits during storage. J. Agric. Food Chem. 54 (12), 4284– 4296.
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Goycoolea, F., Cárdenas, A., 2004. Pectins from Opuntia spp.: a short review. J. Prof. Assoc. Cactus Dev. 5, 17–29. Kechichian, V., Ditchfield, C., Veiga-Santos, P., Tadini, C.C., 2010. Natural antimicrobial ingredients incorporated in biodegradable films based on cassava starch. LWT-Food Sci. Technol. 43 (7), 1088–1094. Kintner, P.K., Buren, J.P., 1982. Carbohydrate interference and its correction in pectin analysis using the m-Hydroxydiphenyl method. J. Food Sci. 47 (3), 756–759. Maftoonazad, N., Ramaswamy, H.S., Marcotte, M., 2007. Evaluation of factors affecting barrier, mechanical and optical properties of pectin-based films using response surface methodology. J. Food Process Eng. 30 (5), 539–563. Mastromatteo, M., Mastromatteo, M., Conte, A., Del Nobile, M.A., 2011. Combined effect of active coating and MAP to prolong the shelf life of minimally processed kiwifruit (Actinidia deliciosa cv. Hayward). Food Res. Int. 44 (5), 1224–1230. Matsuhiro, B., Lillo, L.E., Sáenz, C., Urzúa, C.C., Zárate, O., 2006. Chemical characterization of the mucilage from fruits of Opuntia ficus-indica. Carbohydr. Polym. 63 (2), 263–267. Medina-Torres, L., Brito-De La Fuente, E., Torrestiana-Sanchez, B., Katthain, R., 2000. Rheological properties of the mucilage gum (Opuntia ficus-indica). Food Hydrocolloids 14 (5), 417–424. Oms-Oliu, G., Soliva-Fortuny, R., Martín-Belloso, O., 2008. Edible coatings with antibrowning agents to maintain sensory quality and antioxidant properties of fresh-cut pears. Postharvest Biol. Technol. 50 (1), 87–94. Ponce, A.G., Roura, S.I., Del Valle, C.E., Moreira, M.R., 2008. Antimicrobial and antioxidant activities of edible coatings enriched with natural plant extracts: in vitro and in vivo studies. Postharvest Biol. Technol. 49, 294–300. Redgwell, R.J., Fischer, M., Kendal, E., MacRae, E.A., 1997. Galactose loss and fruit ripening: high-molecular-weight arabinogalactans in the pectic polysaccharides of fruit cell walls. Planta 203 (2), 174–181. Rojas-Graü, M.A., Soliva-Fortuny, R., Martín-Belloso, O., 2009. Edible coatings to incorporate active ingredients to fresh-cut fruits: a review. Trends Food Sci. Technol. 20 (10), 438–447. Sáenz, C., Vásquez, M., Trumper, S., Fluxá, C., 1992. Extractión y composición quı’mica del mucı’lago de tuna (Opuntia ficus-indica). Proceedings II Congreso Internacional De Tuna Y Cochinilla, Mancano le pagine, pp. 93–96. Schroder, R., Atkinson, R.G., 2006. Kiwifruit cell walls: towards an understanding of softening. N. Z. J. For. Sci. 36 (1), 112–129. Sepùlveda, E., Sàenz, C., Aliaga, E., Aceituno, C., 2007. Extration and characterization of mucilage in Opuntia spp. J. Arid Environ. 68, 534–545. Shrestha, A.K., Arcot, J., Paterson, J.L., 2003. Edible coating materials their properties and use in the fortification of rice with folic acid. Food Res. Int. 36 (9), 921–928. Smelt, J.P.P.M., 1998. Recent advances in the microbiology of high pressure processing. Trends Food Sci. Technol. 9, 152–158. doi:http://dx.doi.org/10.1016/ S0924-2244(98)00030-2. Sobral, P.D.A., Menegalli, F.C., Hubinger, M.D., Roques, M.A., 2001. Mechanical, water vapor barrier and thermal properties of gelatin based edible films. Food Hydrocolloids 15 (4), 423–432. Tanada-Palmu, P.S., Grosso, C.R., 2005. Effect of edible wheat gluten-based films and coatings on refrigerated strawberry (Fragaria ananassa) quality. Posthavest. Biol. Technol. 36 (2), 199–208. Tavarini, S., Degl’Innocenti, E., Remorini, D., Massai, R., Guidi, L., 2008. Antioxidant capacity, ascorbic acid, total phenols and carotenoids changes during harvest and after storage of Hayward kiwifruit. Food Chem. 107 (1), 282–288. Thomas, L.V., Ingrama, R.E., Yub, S., Delves-Broughtona, J., 2004. Investigation of the effectiveness of Ascopyrone P as a food preservative. Int. J. Food Microbiol. 93, 319–323. Valero, D., Huertas, M., Díaz-Mula, P., Zapata, J., Guillén, F., Martínez-Romero, D., Castillo, S., Serrano, M., 2013. Effects of alginate edible coating on preserving fruit quality in four plum cultivars during postharvest storage. Posthavest Biol. Technol. 77, 1–6. Xu, S., Chen, X., Sun, D., 2001. Preservation of kiwifruit coated with an edible film at ambient temperature. J. Food Eng. 50, 211–216. Yu, L., Reitmeier, C.A., Love, M.H., 1996. Strawberry texture and pectin content as affected by electron beam irradiation. J. Food Sci. 61 (4), 844–846. Zhang, L., Chen, F., Yang, H., Sun, X., Liu, H., Gong, X., Ding, C., 2010. Changes in firmness, pectin content and nanostructure of two crisp peach cultivars after storage. LWT-Food Sci. Technol. 43 (1), 26–32.
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Postharvest Biology and Technology journal homepage: www.elsevier.com/locate/postharvbio
The influence of Opuntia ficus-indica mucilage edible coating on the quality of ‘Hayward’ kiwifruit slices A. Allegra* , P. Inglese, G. Sortino, L. Settanni, A. Todaro, G. Liguori Department of Agriculture and Forestry Sciences, Università degli Studi di Palermo, Viale delle Scienze ed. 4 ingresso, H-90128 Palermo, Italy
A R T I C L E I N F O
Article history: Received 19 March 2016 Received in revised form 18 May 2016 Accepted 24 May 2016 Available online xxx Keywords: Actinidia deliciosa Ascorbic acid Pectin Microbial spoilage Fresh-cut Flavor score
A B S T R A C T
The aim of this work was to study the effect of mucilage edible coating extracted from Opuntia ficus-indica (OFI) on the quality and shelf life maintenance of packaged kiwifruit slices. OFI mucilage alone or added with TWEEN1 20 were applied on kiwifruit fresh cut surfaces. After treatments, kiwifruit samples were stored under passive atmosphere at 5 1 C for 3, 5, 7 and 12 days. At each storage period, visual quality and flavor score, pectin content, ascorbic acid and the microbiological characteristics were measured together with CO2 and O2 content in the packages. Kiwifruit slices coated only with mucilage or with mucilage plus Tween 20, showed a significant higher firmness and a lower weight loss than untreated slices, until 5 d of shelf life. No further differences in weight loss occurred after 7 d of shelf life, while slices treated only with mucilage retained the highest firmness until the end of the shelf life period (12 d). OFI mucilage alone had significant beneficial effects on the visual and flavor score of the kiwifruit slices, throughout the shelf life period. The treatment with Tween 20 did not affect the flavor of the kiwifruit slices, compared with untreated fruit. Although mucilage and partly tween 20 addition increased microbial growth, especially of yeasts, their levels were still below the threshold for yeast spoilage at the end of the monitoring period. Hence, the results showed that mucilage coating applied to kiwifruit minimally processed fruit improved the quality of stored fresh-cut kiwifruits. ã 2016 Elsevier B.V. All rights reserved.
1. Introduction The wide success of kiwifruit (Actinidia deliciosa) is largely due to the bright color of the flesh that, together with its flavor and high nutraceutical value (high vitamin C content), represents the most important fruit attributes. Indeed, kiwifruit slices are largely used in fruit salads or in confectionery where they are the only greenfleshed fruit used. However, peeling and slicing involved in minimal processing can cause physical damage and increase ethylene production and respiration of kiwifruit. Furthermore, the disruption of the fruit cells caused by the cut, frees the cellular content and promotes the microbial development (Garcia and Barrett, 2002). These phenomena might result in flesh softening and a shorted shelf life of minimally processed kiwifruits (Agar et al., 1999). Refrigeration (2 C and >90% RH) associated with CaCl2 or calcium lactate treatment may prolong the shelf life of minimally processed kiwifruit slices up to 9–12 d (Agar et al., 1999) while the use of edible coating based on Aloe vera combined with packaging
* Corresponding author. E-mail address: [email protected] (A. Allegra). http://dx.doi.org/10.1016/j.postharvbio.2016.05.011 0925-5214/ã 2016 Elsevier B.V. All rights reserved.
under passive modified atmosphere packaging (MAP) and low temperature (2 1 C) reduces fruit respiration rates and microbial spoilage of kiwifruit, during 7 d of storage (Benitez et al., 2013). Active coatings under passive MAP also inhibit microbial activity and reduce respiration, while the combination of MAP and active alginate-based coatings delaye quality loss and microbial spoilage of kiwifruit minimally processed slices (Mastromatteo et al., 2011). As a matter of fact, the use of edible coatings is growing, due to their multiple uses for extending fruit shelf life and also as carriers for several food additives (Mastromatteo et al., 2011). Most of the edible coatings are based on carbohydrates, proteins or lipids which can be combined (Oms-Oliu et al., 2008; Valero et al., 2013), such as casein (Ponce et al., 2008) and its derivatives (Fabra et al., 2009), guar gum, cellulose ethyl (Shrestha et al., 2003), gelatin supplemented with glycerol, sucrose and sorbitol as plasticizers (Arvanitoyannis et al., 1997; Sobral et al., 2001), pectin (Maftoonazad et al., 2007), cassava starch (Kechichian et al., 2010), wheat gluten (Tanada-Palmu and Grosso, 2005) and mixtures of sodium alginate and pectin, with the addition of CaCl2 (Da Silva et al., 2009). The mucilage obtained from cladodes of cactus pear (Opuntia ficus-indica) has a highly branched complex polymeric structure of carbohydrate nature, (Medina-Torres et al., 2000;
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Matsuhiro et al., 2006) and contains varying proportions of Larabinose, D-galactose, L-rhamnose and D-xylose (Sáenz et al., 1992; Sepùlveda et al., 2007; Goycoolea and Cárdenas, 2004). The rheological characteristics of O. ficus-indica mucilage makes it interesting for the production of edible coatings with a high nutraceutical value. The mucilage from cladodes of O. ficus-indica forms an edible coating on the fruit surface that makes the treated product shiny. So far, this mucilage has had several applications in fruit preservation. A combination of O. ficus-indica mucilage with citric acid and sodium bisulfite at high concentrations decreases browning of banana (Musa cavendish) slices during drying (Aquino et al., 2009). Treatment with O. ficus-indica mucilage applied on fresh strawberries reduces fruit weight, color and firmness loss, fruit respiration rate, and fungal infection (Del-Valle et al., 2005). However, to the best of our knowledge, this polymer has been never applied on minimally processed fruit. The efficacy of two different O. ficus-indica mucilage-based coating formulations to prolong the shelf life of kiwifruit slices was evaluated. To assess the influence of the treatments, weight loss, softening, sensory quality and microbial load of kiwi fruit were monitored during the storage. 2. Materials and methods 2.1. Samples preparation ‘Hayward’ kiwifruit (Actinidia deliciosa) were purchased from a retail market (Simply, Palermo, Italy). Fruit were uniform in terms of fresh weight (98 4.8 g), total soluble solid content (13.3 1.5%) and firmness (30.5 3.5 N), analyzed on a sample of 30 fruit. Kiwifruit were stored at 1 0.5 C (RH = 85%) for 24 h. After storage, fruit were dipped in chlorinated water (100 mL 1 of free chlorine) for 6 min. Damaged fruits (bruised or showing other physical decays) were removed, and a total of 300 fruits were processed. Cactus pear (O. ficus-indica, (L.) Mill.) cladodes were cut and cubed (2 cm3). To extract the mucilage, cladodes were crushed in a blender (Moulinex) with rotating knifes, homogenized with distilled water in the ratio 1:1.5 (w/v) at 20 C. The solution was maintained at 40 C for 90 min and centrifuged (model CS6R) at 1450g 20 min. The supernatant was boiled to half the initial volume and ethanol (99% v/v) was added in the ratio 1:2 (Sáenz et al., 1992). Afterwards, the solution was stored at 4 1.0 C for 48 h to allow a better aggregation of the mucilage. The last phase involved the elimination of the supernatant and soaking of the pure mucilage. Kiwifruits were peeled using an automatic machine (Agrimat, Maxistreap, Italy) and cut into slices with a semiautomatic machine (Sgorbati, Italy). Slices were 2.2 0.2 cm thick and 5.4 1.2 cm width; L* was 48 2.1, a* was 8.2 1.2 and b* was 25.4 3; total solid soluble content (TSS) was 13.1 2.5% and titratable acidity (TA) was 1.4 0.3%. Fresh-cut slices were dipped in the (OFI) coating solution for 60 s; the excess coating was drained and the coated slices were dried with a forced-air dryer (20 C) for 10 min. The coating treatments consisted of: a) 30 g of pure mucilage extract, 500 mL distilled water and 50 mL glycerol as a plasticizer (MC); b) 30 g of mucilage extract, 500 mL distilled water, 50 mL glycerol added with 2 mL Tween 20 (TW). The control treatment (CTR) wereslices dipped in distilled water. About 95 1.1 g of kiwifruit slices were packed in polyethylene terephthalate (PET) packages and sealed with a composite film (PP-PET), 64 mm, O2 permeability = 5.30 10 8 mL m 2 s 1 Pa 1. Packages were stored at 5 0.5 C and 90% relative humidity (RH) for 12 d. Chemical, physical and microbiological parameters were analyzed, at the beginning of the experiment (after coating/dipping = day 0) and at 3, 5, 7, and 12 d after storage, on six slices per replicate for
treatment (3 treatments combinations 5 time of storage 6 replicates = 90 box). 2.2. Chemical and physical analysis 2.2.1. Firmness Firmness was evaluated with a puncture test on kiwifruit slices flesh using a TA-XT Plus texture analyzer (Stable Micro Systems) equipped with a 50 N load cell of. Firmness measurements were taken as the median force value obtained during the test with a stainless steel probe with 4 mm diameter penetrating the fruit 4 mm, at 1 mm/s. Average values were calculated from the results of at least 6 measurements in different slices for each sample. Measures were taken in fruit outer pericarp (green flesh) where the fast rate of softening compromises fruit quality. 2.2.2. Weight loss Weight of individual bags was recorded immediately after the treatment (day 0) and at the different sampling times (3, 5, 7, and 12 days during storage). Weight loss was expressed as the percentage reduction with respect to initial time, using the following equation: % Weight loss: [(Initial fruit bag weight 100]/Initial fruit bag weight
Final fruit bag weight)
2.2.3. Total soluble solids content and titratable acidity The concentration of total soluble solids (TSS expressed as%) determined from the juice of four slices from each tray using a digital refractometer (model PR-101, Atago, Co., Tokyo, Japan) at 20 C. Titratable acidity (expressed as% citric acid) was determined by titration of 10 mL of juice with 0.1 M NaOH to an endpoint of pH 8.1. 2.2.4. Sensory evaluation 2.2.4.1. Visual appearance score. To measure the effect of cold storage on kiwifruit sensory traits at each storage time (0, 3, 5, 7 and 12 d), six slices, used as single replicates for each treatment (MC, TW and CTR), were scored by each of a six judges trained panel that generated a list of descriptors in a few preliminary meetings. Visual appearance score resulted from the medium value of color, visible structural integrity and visual appearance (Allegra et al., 2015a). The different descriptors were quantified using a subjective 5–1 rating scale with 5 = very good, 4 = good, 3 = sufficient (limit of marketability), 2 = poor (limit of usability) and 1 = very poor (inedible). 2.2.4.2. Flavor score. To measure the effect of cold storage on slices fruit flavor traits at each storage time, six slices, used as single replicates for treatment (MC, TW and CTR), were scored by six trained judges, using a subjective 5–1 rating scale with 5 = very high, 4 = high, 3 = sufficient (limit of marketability), 2 = low and 1 = none. 2.2.5. Package O2 and CO2 analysis CO2 and O2 levels (kPa) were measured on each package at the beginning of each experiment and after 3, 5, 7, 12 days of storage, using a PBI Dansensor Checkpoint O2 and CO2 analyzer (Topac, Hingham, MS, USA) with zirconium and infrared detectors, respectively. 2.2.6. Ascorbic acid Ascorbic acid concentration was determined according to Rapisarda and Intelisano (1996) by high performance liquid
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chromatography (HPLC) (Perkin Elmer, Australia) using an injector (Rheodyne with 20 mL loop), a photodiode detector, a Knauer Eurospher II 100-5 C18 column 250 mm 4.6 mm I.D. (Berlin, Germany) and a similarly packed pre-column. The elution was performed with a buffer solution consisting of KH2PO4/H3PO4 at pH 2.3, at a flow rate of 1 mL min 1, the wavelength was at 260 nm. An aliquot of freshly prepared pulp (5 g) was homogenized with 25 mL of 3% metaphosphoric acid, centrifuged, filtered through a 0.45 mm Whatman Puradisc and HPLC injected. The concentration of ascorbic acid was calculated from the experimental peak area by analytical interpolation in a standard calibration curve and was expressed as mg 100 g 1 of fresh weight. 2.2.7. Microbiological analysis Fruit samples and mucilage were microbiologically investigated for total mesophilic microorganisms (TMM) and the undesired (spoilage and/or pathogenic) microbial groups. The fruits (25 g) and the mucilage (10 mL) were collected separately suspended in Ringer’s solution (Sigma-Aldrich, Milan, Italy) to a ratio 1:10 (fruit: diluent), homogenized for 2 min at the highest speed with a stomacher (BagMixer1 400, Interscience, Saint Nom, France) and serially diluted. The cell suspensions were inoculated as follows: TMM on plate count agar (PCA), incubated at 30 C for 72 h; members of the Enterobacteriaceae family on double-layered violet red bile glucose agar (VRBGA), incubated at 37 C for 24 h; Pseudomonas on Pseudomonas agar base (PAB) supplemented with 10 cetrimide fucidin, incubated at 25 C for 48 h; yeasts on yeast potato dextrose (YPD) agar, incubated at 25 C for 48 h. All media and supplements were purchased from Oxoid (Milan, Italy). Plate Count were carried out in duplicate for each trial. 2.2.8. Pectin analysis The pectic substances from fruits were extracted according to the methods described by James and Rouse (1952) and Rouse and Atkins (1955). Frozen slices were thawed at 4 C for 24 h; about 20 g were weighed into a plastic cup, homogenized at low speed with an Ultra-Turrax tissuemizer for 2 min. The tissue sample (5 g) was weighed into a 50 mL round-bottom plastic centrifuge tube. Hot 100% ethanol (30 mL) was added into the tubes. The samples were stirred thoroughly with a glass rod, heated in a boiling water bath for 10 min, cooled and centrifuged at 14,500g for 10 min. The alcoholic supernatant was decanted and discarded. The precipitate was extracted with 30 mL 100% ethanol, centrifuged and the supernatant was discarded. The residue was transferred from the centrifuge tube to a 57 mm aluminum weighing dish (Fisher Scientific). Samples were dried for 24 h in a conventional oven at 35 C, weighed and ground in a mortar with a pestle. The precipitate from the alcohol solution was designated as alcoholinsoluble solids (AIS). Dried AlS (80 mg) was weighed into a 50 mL centrifuge tube. Distilled water (20 mL) was added and samples were stirred with a glass rod for 1 min. Samples were centrifuged at 14,500g for 10 min, filtered through Whatman No. I filter paper to obtain water-soluble pectin (WSP). The extraction procedure was repeated once. The supernatants were collected and combined in a 100 mL volumetric flask. Distilled water was added to dilute tube extract solution to volume. The residue was dispersed in 20 mL of an aqueous solution containing 0.25% ammonium oxalate and 0.25% oxalic acid and stirred with a glass rod for 1 min. Samples were refluxed in a boiling water bath for l h, centrifuged at 14,500g for 10 min and filtered through Whatman No. I fìlter paper to obtain oxalatesoluble pectin (OSP). The oxalate extraction was repeated once. Supernatants were collected and diluted to 100 mL with distilled water in a volumetric flask.
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Total pectin (TP) was extracted by the method of Ahmed and Labavitch (1978). Dried AIS (5 mg) was weighed into a 30 mL beaker containing a magnetic stir bar. Concentrated sulfuric acid (2 mL) was added to the becker, and the mixture was swirled gently. The beaker was placed on a stir plate and stirred gently and 0.5 mL of distilled water were added dropwise. Stirring continued for 5 min; an additional aliquot of 0.5 mL distilled water was added dropwise. Stirring continued furtherly for approximately 30 min until dissolution of AIS was complete. The dissolved sample was filtered through glass wool into a 25 mL volumetric flask. Each beaker was rinsed several times with distilled water, combined in a 25 mL flask, and diluted to volume. The solution was filtered through glass wool again before use. The difference between TP and the sum of WSP and OSP was used to determine the amount of non-extractable pectin (NXP), which was the protopectin fraction. TP, WSP and OSP extractions were completed in duplicate for all samples. All pectin extracts were stored at 4 C for 12 h before analysis. Extract pectin contents were analyzed by the m-hydroxydiphenyl method (Kintner and Buren, 1982). The extract from each sample (1 mL) was pipetted into a 16 150 mm test tube. Six milliliters of sulfuric acid tetraborate solution (0.0125 M sodium tetraborate in concentrated sulphuric acid) was added to each of the tubes in an ice water bath and mixed intermittently using a Vortex mixer at moderate speed to assure complete mixing. Duplicate samples were prepared for each pectin measurement with a corresponding blank. Tubes were heated in a boiling water bath for 5 min and immediately placed in ice water to cool. To duplicate tubes, 0.1 mL aliquot of 0.15% m-hydroxydiphenyl was added to develop color. To the blank tube, 0.1 mL 0.5% sodium hydroxide was added. All samples and blanks were mixed using the Vortex mixer and allowed to stand 15 min at room temperature. The absorbance of the samples following chromogen formation was measured at 520 nm using a spectrophotometer (Beckman DU 640-Canada). Galacturonic acid was used as a standard. A solution consisting of 1 mL distilled water, 6 mL sulfuric acid/tetraborate and 0.1 mL 0.5% sodium hydroxide was used as reagent blank. The determination of the pectin was done according to Yu et al. (1996) 2.2.9. Statistical analysis The experimental design consisted of two coating treatments and the untreated control, with observations made at 0, 3, 5, 7, 12 days after coating. Analysis of variance ANOVA (Systat 13.0 for Windows was used as statistical software) was performed and mean values were compared with Tukey’s test during storage among treatments, for each storage time and for each treatment. Differences were considered to be significant when the p < 0.05. 3. Results and discussion 3.1. Firmness, TSS, TA, weight loss and pectin content Kiwifruit slice firmness changed with sampling time and treatment. Untreated kiwifruit slices showed a sharp, significant (P 0.05) decrease in firmness soon after placement in storage and no significant further changes occurred during the next 9 d (Fig. 1). Slices coated with OFI mucilage + glycerol (MC) showed no significant softening during the first 5 d of storage, and a significant (P 0.05) decrease during the next 7 d. Kiwifruit slices coated with OFI mucilage + glycerol + Tween (TW) showed a continuous and significant decrease throughout the storage period (P 0.05). Differences among treatments appeared 3 d after storage when untreated slices had the lowest firmness, while no differences occurred between MC and TW coated slices (Fig. 1). TW slices and untreated ones had the same firmness values at 7 and 12 d after storage. MC coated slices had the highest firmness throughout the
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Fig. 1. Firmness of fruit slices of Actinidia deliciosa (Liang, Ferguson) coated with O. ficus-indica (OFI) mucilage + glycerol (MC), or OFI mucilage + glycerol + Tween 20 (TW) or not treated (CTR), just after being coated (0) and stored for 3, 5, 7, 12 d at 5 C. Different letters indicate significant differences between treatments at each sampling date. Tukeys’ significant test was applied at P 0.05. Data are means S.E. (n = 6).
storage period. O. ficus-indica coatings did not affect kiwifruit slice TSS content and TA during the storage (data not shown). The hydrophilic character of OFI coating could act as a barrier to water transfer, retarding dehydration and maintaining firmness during fruit shelf life. In fact, untreated fruit reached the maximum weight loss 3 d after the packaging, with no further increases in the next 9 days. During the first 5 d of storage both coated treatments showed the lowest weight loss (Fig. 2). However, the weight loss of MC and TW coated slices significantly (P 0.05) increased after one week of storage, though MC slices had the lowest weight loss. At the last sampling time no differences occurred between coated and untreated slices (Fig. 2). In general, fruit softening is associated with disassembly of the primary cell wall, with solubilization and depolymerization of pectins (Alzamora et al., 2000; Brummell et al., 2004). In kiwifruit, the loss of galactose residues occurs from the insoluble residue (Redgwell et al., 1997). After harvest, kiwifruit go through three distinct softening phases that are temporally well separated. Pectin retained in the cell wall starts to “soften” during ripening; this process clearly precedes both pectin solubilisation and depolymerisation (Schroder and Atkinson, 2006). Degradation of solubilized pectin and loss of middle lamellae in kiwifruit, are processes that are initiated in the second softening phase, but peak in the last softening phase, where cell wall disintegration is completed (Schroder and Atkinson, 2006). In our study, TW slices showed the highest total pectin content throughout the storage period (Fig. 3).
Fig. 2. Fresh weight loss of fruit slices of Actinidia deliciosa (Liang, Ferguson) coated with O. ficus-indica (OFI) mucilage + glycerol (MC), or OFI mucilage + glycerol + Tween 20 (TW) or not treated (CTR), just after being coated (0) and stored for 3, 5, 7, 12 d at 5 C. Different letters indicate significant differences between treatments at each sampling date. Tukeys’ significant test was applied at P 0.05. Data are means S.E. (n = 6).
Fig. 3. Total pectin (TP) of fruit slices of Actinidia deliciosa (Liang, Ferguson) coated with O. ficus-indica (OFI) mucilage + glycerol (MC), or OFI mucilage + glycerol + Tween 20 (TW) or not treated (CTR), just after being coated (0) and stored for 3, 5, 7, 12 d at 5 C. Different letters indicate significant differences between sampling dates for each treatment. Tukeys’ significant test was applied at P 0.05. Data are means S.E. (n = 3).
Water soluble pectin (WSP) significantly increased after one week of storage only in TW slices (Fig. 4). On the other hand, protopectin, (NXP) significantly decreased only in untreated slices from 5 days after storage onwards. This trends corresponded to the loss of firmness of CTR kiwifruit slices during storage (Fig. 1). Similar results have been reported for peaches, in which the water-soluble pectin content tends to increase during storage at different temperatures (Zhang et al., 2010) and in kiwifruit where the decrease in firmness was closely related to the soluble pectin content increase during storage (Xu et al., 2001). 3.2. Head spaces gas composition, ascorbic acid content The polysaccharide content in OFI mucilage can be used to modify the internal atmosphere of fruits, delaying senescence (Rojas-Graü et al., 2009). Edible coatings create a passive modified atmosphere, which can influence changes in minimally processed fruits (Oms-Oliu et al., 2008). Kiwifruit after cutting and packaging in atmosphere passive, have altered the metabolism showing a progressive increase of CO2 and decrease in O2. In this study, the O2 (kPa) content inside packages showed a significant lower value for the bags containing TW slices, by day 3 of storage but, no significant differences were found (Fig. 5a and b). The increase of CO2 accumulation was observed after 3 d of storage in all sample slices coated with OFI mucilage. The bags containing control slices showed an accumulation of 15 kPa CO2 after 5 d of storage (Fig. 5a and b). The reduction of O2 consumption and CO2 production in package headspace atmospheres was observed also in kiwifruit coated with A. vera (Benitez et al., 2013), alginate (Mastromatteo et al., 2011) or a CaCl2 solution (Beirão-da-Costa et al., 2014). The initial ascorbic acid content in the fresh cut kiwifruit slices analyzed in this study are in agreement with the values reported for ripe ‘Hayward’ kiwifruits (Gil et al., 2006; Tavarini et al., 2008). During storage, a progressive degradation of ascorbic acid in the coated and untreated slices was observed (Barberis et al., 2012). The ascorbic acid content of untreated, MC and TW minimally processed kiwifruit slices decreased by 49%, 46% and 42%, respectively, after 12 d of storage (Fig. 6). A sharp decrease occurred soon after 3 d of storage in untreated (35%) and in MC (28%) kiwifruit slices, while TW slices kept the initial values (Fig. 6). Significant differences among treatments occurred only during the first 3 d of storage, when untreated kiwifruit slices showed the lowest ascorbic acid content and TW coated slices the highest. However no difference between TW and MC occurred by day 5, when untreated slices had the lowest ascorbic acid content (Fig. 6)
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Fig. 4. Water soluble pectin (WSP) and non extractable pectin (NXP) of fruit slices of Actinidia deliciosa (Liang, Ferguson) coated with O. ficus-indica (OFI) mucilage + glycerol (MC), or OFI mucilage + glycerol + Tween 20 (TW) or not treated (CTR), just after being coated (0) and stored for 3, 5, 7, 12 d at 5 C. Different letters indicate significant differences between treatments at each sampling date. Tukeys’ significant test was applied at P 0.05. Data are means S.E. (n = 3).
Fig. 5. Oxygen (A) and Carbon dioxide (B) content (kPa) inside packages with fruit slices of Actinidia deliciosa (Liang, Ferguson) coated with O. ficus-indica (OFI) mucilage + glycerol (MC), or OFI mucilage + glycerol + Tween 20 (TW) or not treated (CTR), just after being coated (0) and stored for 3, 5, 7, 12 d at 5 C. Different letters indicate significant differences between treatments at each sampling date. Tukeys’ significant test was applied at P 0.05. Data are means S.E (n = 6).
3.3. Visual appearance and flavor score
Fig. 6. Ascorbic acid of fruit slices of Actinidia deliciosa (Liang, Ferguson) coated with O. ficus-indica (OFI) mucilage + glycerol (MC), or OFI mucilage + glycerol + Tween 20 (TW) or not treated (CTR), just after being coated (0) and stored for 3, 5, 7, 12 d at 5 C. Different letters indicate significant differences between treatments at each sampling date. Tukeys’ significant test was applied at P 0.05. Data are means S.E (n = 6).
The sensory evaluation revealed that MC and TW slices maintained visual quality score after 3 d of storage. Untreated kiwifruit slices had the lowest visual quality scores during the first 5 d, when no significant differences occurred between MC and TW treatments (Fig. 7a). The last day of storage, TW slices showed a significant decrease of the visual quality score and were similar to the untreated samples; at this stage MC slices kept the highest score among treatments (Fig. 7a). Considering both the visual quality and the flavor score, MC slices kept their marketability during 9 days of storage, while untreated kiwifruit slices were not marketable 7 d after storage in terms of flavor (Fig. 7b) and reached their marketability limit in terms of visual score 5 d after storage. TW slices were marketable, in terms of flavor, until 5 d after storage, while in terms of visual quality they kept good values until 7 d of storage (Fig. 7a and b). Sensory analysis of the fruit treated with mucilage maintained the characteristics of flavor of the kiwifruits, conversely coating based with Aloe vera showed bitters during consumer evaluation (Benitez et al., 2013).
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Fig. 7. Visual quality (A) and flavor score (B) of fruit slices of Actinidia deliciosa (Liang, Ferguson) coated with O. ficus-indica (OFI) mucilage + glycerol (MC), or OFI mucilage + glycerol + Tween 20 (TW) or not treated (CTR), just after being coated (0) and stored for 3, 5, 7, 12 d at 5 C. Different letters indicate significant differences between treatments at each sampling date. Tukeys’ significant test was applied at P 0.05. Data are means (n = 6).
3.4. Microbiological analysis The microbiological analyses of the mucilage did not evidence the presence of any of the microbial groups object of investigation. Differences among treatments appeared at 3 and 5 d after storage when MC and TW treatment had a lower TMM concentration than untreated slices. No further differences among treatments occurred at 7 and 12 d of storage (Fig. 8a). Members of the Enterobacteriaceae family were below the detection limit during the entire period of analysis (Allegra et al., 2015b). Pseudomonads (Fig. 8b) did not show significant differences in all sample during 5 d of storage while significant increases in this population
occurred in MC, showing a load of 1.77 Log CFU g 1 after 7 d. A similar trend was observed also for yeasts (Fig. 8c) for which the highest increase in concentration was registered on MC slices during the first 3 d. Differences between the coated slices (MC and TW) and control treatments were found after 7 d of storage. However, the cell densities of Pseudomonads and yeasts were not able to determine a microbial decay of the sliced kiwifruits. The levels of yeasts never exceeded those of TMM, indicating that the highest microbial values found in treatments MC and TW are due to yeast development. This is due to the fact that yeasts are more resistant than bacteria to the acidic conditions (Smelt, 1998) such as those found on the kiwifruit surface.
Fig. 8. Total mesophilic (A), Pseudomonas (Enterobacteriaceae) (B) and Yeast content (C) in fruit slices of Actinidia deliciosa (Liang, Ferguson) coated with O. ficus-indica (OFI) mucilage + glycerol (MC), or OFI mucilage + glycerol + Tween 20 (TW) or not treated (CTR), just after being coated (0) and stored for 3, 5, 7, 12 d at 5 C. Different letters indicate significant differences between treatments at each sampling date. Tukeys’ significant test was applied at P 0.05. Data are means S.E (n = 3).
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