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Hot water treatment in combination with calcium ascorbate dips increases bioactive compounds and helps to maintain fresh-cut apple quality
Postharvest Biology and Technology 110 (2015) 158–165
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Postharvest Biology and Technology journal homepage: www.elsevier.com/locate/postharvbio
Hot water treatment in combination with calcium ascorbate dips increases bioactive compounds and helps to maintain fresh-cut apple quality Encarna Aguayoa,* , Cecilia Requejo-Jackmanb , Roger Stanleyc , Allan Woolfb a Postharvest and Refrigeration Group. Department of Food Engineering, Technical University of Cartagena. Paseo Alfonso XIII 48, 30203 Cartagena, Murcia, Spain b The New Zealand Institute for Plant & Food Research Ltd, Private Bag 92169, Auckland, New Zealand c Centre for Food Innovation, University of Tasmania, Locked Bag 1370, Launceston, TAS 7250, Australia
A R T I C L E I N F O
A B S T R A C T
Article history: Received 23 October 2014 Received in revised form 1 July 2015 Accepted 3 July 2015 Available online xxx
Fresh-cut ‘Braeburn’ apple slices were dipped into cold water (4 C for 2 min) or hot water (HWT, 48 C or 55 C for 2 min) followed by dips into 0 or 6% w/v aqueous calcium ascorbate (CaAsc, 2 min, 0 C) and stored in air up to 28 d at 4 C. Microbial counts, changes in browning and sensory acceptance were determined to indicate changes in quality. Changes in antioxidant levels were measured using free radical scavenging activity (DPPH), reducing activity (FRAP), ascorbic acid content (AA) and polyphenolic content (by HPLC). CaAsc dips had a strong impact reducing the browning through increasing the flesh luminosity and hue angle. 6% CaAsc in fresh-cut apples extended the overall acceptability from less than 7 d to 14 d. Immediately after CaAsc treatment, AA content was 5 fold higher (0.25–1.25 g kg 1) than those not dipped into CaAsc. However, the combination of HWT treatments and CaAsc dips led to seven fold increased levels of AA inside the apple tissue (0.25–1.85 g kg 1) and consequently increased the antioxidant activity. HWT did not increase the AA content when not combined with CaAsc dips. The HWT CaAsc dip extended the overall acceptability to 21 d compared to 14 d for samples not heated but dipped into CaAsc. Shelf life was ultimately limited by sensory quality. At day 28, total plate counts were reduced from 5.3 log cfu/g (untreated slices) to 4.6 log cfu/g in the 6% CaAsc dips and further to 3.9 log cfu/g with the combination of HWT and CaAsc dip. Changes in the content of phenolic compounds with time, HWT and CaAsc dip were generally not significant except for slightly increased quercetin and phloridzin levels and decreased p-coumaric and procyanidins over time. The combination of HWT at 48 C for 2 min followed by 6% CaAsc dip would be best for preserving the eating quality of apple slices. ã 2015 Elsevier B.V. All rights reserved.
Keywords: Minimally processed Apple slices Heat treatment Shelf life Phenolic compounds Vitamin C Ascorbic acid Antioxidant
1. Introduction Apples are one of the most widely consumed fruits (FAOSTAT, 2011) and are a good source of phytochemicals (Boyer and Liu, 2004). Epidemiological studies have linked the consumption of apples with reduced risk of some cancers, cardiovascular disease, asthma, and diabetes (Boyer and Liu, 2004). One way to increase fresh fruit consumption is processing fruit into fresh-cut product to be sold as convenient single servings. However, fresh-cut processing results in major tissue disruption of surface cells and injury stress of underlying tissues (Toivonen, 2004). The main problem for fresh-cut apple is oxidation caused by polyphenol
* Corresponding author. E-mail address: [email protected] (E. Aguayo). http://dx.doi.org/10.1016/j.postharvbio.2015.07.001 0925-5214/ ã 2015 Elsevier B.V. All rights reserved.
oxidase (PPO) that exists in particularly high amounts in apple (Whitaker, 1972). The resulting browning makes the product unsuitable to the consumer. Ranges of treatments have been applied to extend the shelf life of fresh-cut apples, mainly dipping in solutions of a wide range of anti-browning agents. Ascorbic acid, oxalic acid, oxalacetic acid, kojic acid, erythorbic acid, citric acid, and/or calcium, cysteine, 4-hexylresorcinol have all been examined at different concentrations (Son et al., 2001; Rojas-Grau et al., 2006; Tortoe et al., 2007). Among the aforementioned methods, the use of calcium ascorbate (CaAsc) has been found to be the most effective anti-browning agent and can be marketed as a minimal chemical input. Its application increases the antioxidant status and extends shelf life of apple slices (Aguayo et al., 2010). On the other hand, several methods, including heat shock, have been used to inhibit polyphenol oxidase activity since this reduces the rate of the polymerisation step of browning reactions (Kwak and Lim,
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2005; Barbagallo et al., 2012). Hot water treatment (HWT) is an effective physical treatment, free of chemical residues, and readily applicable in the fresh-cut industry during the washing process (Kim et al., 1993; Abreu et al., 2003). Previous studies have shown that HWT is sufficiently effective to maintain product quality for fresh-cut products such as lettuce (Murata et al., 2004; Moreira et al., 2006), rocket (Koukounaras et al., 2009), spinach (Gómez et al., 2008; Glowacz et al., 2013), eggplants (Barbagallo et al., 2012), and onions (Siddiq et al., 2013). The use of non-chemical technologies that can improve product quality response by additional interactions could improve the effectiveness of CaAsc in the fresh-cut apple industry. This study sought to determine whether HWT could be recommended as a method in combination with CaAsc dips to help to maintain the sensory quality of fresh-cut apples. 2. Materials and methods 2.1. Raw material New Zealand grown ‘Mahana Red’ apples (Malus domestica Borkh., a sport of ‘Braeburn’) were sourced from the refrigerated storage of a commercial supermarket. The apples had been stored for up to 6 months under controlled atmosphere (2 kPa O2 plus 1 kPa CO2) at 0 C. Apples boxes were transported to the laboratory and stored at 0 C for 12 h. The boxes were opened in a food-grade processing room (10 C) and the fruit sorted to remove those damaged or with significant variation in background colour. Whole apple surfaces were washed by dipping in cold water (4 C) with 5 mg L 1 chlorine dioxide (OxineTM, Australasia Marketing Pty Ltd. Sydney, NSW, Australia) for 10 min. The apples were then manually cored with a metal corer and cut into 8 wedge slices using a handheld knife. All the slices were dipped into cold water (4 C) with 2 mg L 1 chlorine dioxide for 2 min, and the slices drained. Slices for HWT were dipped in hot water (48 C or 55 C) for 2 min. A stainless water-bath (Grant 40 L, with a Grant temperature controller units (0.1 C, 1.4 kW heater model GRAVF, U.K.) with continuous hot water recirculation and stirring was used to maintain the relevant temperature. All apple slices (heated or not) were then dipped for 2 min into 0% (control) or 6% CaAsc solution (w/w; 99.9% purity, Wolf Canyon Asia Pacific Ltd) and drained. This solution was made using water at 0 C that had been pre-treated with 2 mg L 1 chlorine dioxide. Apple skins were not removed prior to treatment, as apple slices are currently marketed with skin intact. For each treatment, apple slices were randomised across packages of 15 apple slices (350 20 g) per aluminium bag (25 cm 18 cm, 80 mm thickness, Caspak, New Zealand). To maintain nearly ambient oxygen concentration in the bags, two 5-mm holes were punched through both sides of each bag. Three replicate bags per treatment were stored at 4 C per storage duration: 7, 14, 21 and 28 d. Measures of antioxidant activity were also measured on day 0 (immediately after treatment). 2.2. Parameter evaluations 2.2.1. Colour measurement The surface colour of the apple flesh was determined on three equidistant points in each apple slice cut surface with a Minolta chromameter (D65 light source, Minolta Camera Co., Osaka, Japan). The results were expressed as CIELAB (L*a*b*) colour space. L* defines the lightness and a* and b* define the red-greenness and blue-yellowness, respectively. The flesh colour was also measured and expressed as hue angle (h = arctangent [(b*) (a*) 1]) and chroma (C* = [(a*)2 + (b*)2]1/2). Fifteen slices per treatment (5 slices per replicate) were measured.
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2.2.2. Sensory evaluation A panel of five people was trained to recognise and score the quality attributes of the treated apple slices. All assessments were compared to freshly cut slices. Appearance, taste and texture were scored on a nine-point scale where 1 = complete lacking or soft, to 9 = fully characteristic of fresh. A similar scale, where 1 = inedible, 3 = poor, 5 = fair (limit of marketability), 7 = good and 9 = excellent was used for the evaluation of the overall acceptability. 2.2.3. Microbial analyses After 28 d storage, microbial growth on the slices was determined by a certified food analysis laboratory (AgriQuality, Auckland, New Zealand). Only samples dipped into 6% CaAsc and both controls (0 and 6% CaAsc) from the HWT and control were analyzed. From each of five slices, 10 g samples were blended with 90 mL of sterile peptone buffered water (Merck Darmstadt, Germany) for 1 min in a sterile stomacher bag (Model 400 Bags 6141, London, UK) using a Masticator (Colwort Stomacher 400 Lab, Seward Medical, London, UK). Appropriate dilutions were prepared. Plate Count Agar medium (PCA, Merck) was used for TPC and Rose Bengal agar medium (Merck) for the yeast and mould counts. Incubation conditions were 30 C for 48 h for TPC, and 22 C for 5 d for yeasts and moulds, respectively. Microbial counts were reported as log 10 colony forming units per gram of sample (log cfu g 1). 2.2.4. Chemical measurements Fruit pieces were flash frozen in liquid nitrogen and stored at 80 C for a maximum of 2 months. To ensure uniformity, frozen samples (200 g) were either homogenised in 100 mL of distilled water in a commercial blender (Sunbeam Model PB7600, Type 504, 230–240 V, Sydney, Australia) to produce a juice extract for antioxidant activity analysis, or 150 g was ground to a fine powder in a Cryomill in liquid nitrogen for ascorbic acid content (AA) analysis. 2.2.4.1. Antioxidant activity. Two assays were used to measure the antioxidant activity; 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP) assays. All the material, method and equipment has been previously reported (Aguayo et al., 2010). All the antioxidant assays were carried out in triplicate. Calibration curves were made for each assay using Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) and AA as standards. The antioxidant activity (DPPH, FRAP assay) was expressed as Trolox and AA equivalent antioxidant activity per kg fresh weight of apple tissue. 2.2.4.2. Ascorbic acid evaluation. The method was adapted from Rassam and Laing (2005). A 0.2 g sample of powdered apple tissue was suspended in 1 mL of 6% metaphosphoric acid, 2 mM EDTA and 1% PVPP containing 4 mM TCEP (tris[2-carboxyethyl] phosphine hydrochloride). The slurry was vortexed for 20 s, and incubated in a heating block for 2 h at 40 C to ensure full reduction of any dehydroascorbate. The extract was clarified by centrifugation at 4 C for 10 min, and 20 mL of the supernatant was injected into a 7.8 300 mm Aminex HPX-87H HPLC column (Bio-Rad, Merck, Darmstadt, Germany). The column was run with 2.8 mM H2SO4 as the mobile phase, at a flow rate of 0.01 mL s 1. The amount of AA was detected using a Waters 996 photodiode-array detector (Milford, MA, USA) set at 245.5 nm for absorbance using AA (Sigma, St. Louis, MO) as a standard. The peak was authenticated as AA by showing that it was completely degraded by ascorbate oxidase at pH 5.5. 2.2.4.3. Phenolic compounds evaluation. One mL of apple juice extract was combined with 0.25 mL of methanol and HCl
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(90:10 v/v) and vortexed for 20 s. As mentioned, all the details have previously been reported (Aguayo et al., 2010). Chromatograms were recorded at 280 and 340 nm at a flow rate of 0.02 mL s 1. Peak assignment was performed by comparison of the retention times and UV spectra with those of reference compounds and by mass spectrometric analyses. Sample injection volume was 10 mL. UV– vis detection was by absorbance at 200–600 nm.
Table 2 Effect of CaAsc dip concentration (0 or 6%) on L*, h, texture and taste of apple slices (P = 0.05). Slices were packaged in air and stored up to 28 d at 4 C. % CaAs
L*
0 6
73.0 76.2
93.0 100.7
h
Texture
Taste
6.0 7.0
5.0 6.2
2.3. Statistical analysis A randomised design with three replicates per treatment was used where each bag constituted a replicate. To determine the effect of storage time, HWT and CaAsc on each dependent variable, a three-way analysis of variance (ANOVA, P 0.05) was carried out (Statgraphic Plus, version 5.1, 2001, Manugistic Inc., Rockville, MD, USA). Mean values were compared by LSD (least significant difference test) when significant differences among treatments and interactions between factors was found. 3. Results Table 1 shows ANOVA results of time of storage, hot water treatment and CaAsc dips (0 or 6%) factors and their interaction on apple slices. 3.1. Colour CaAsc dips had a strong immediate impact increasing L* and hue angle ( h) (Table 2) and reducing the chroma of apple slices (Fig. 1). This indicated a lighter, slightly greener colour with less saturation in the apple flesh. In contrast, the HWT reduced the luminosity of apple slices (Table 3) and slices that were not heated showed the higher levels of L* and h and decreased chroma. After 28 d of storage h was slightly reduced from 97 to 96 without significant changes in the L* parameter. 3.2. Sensory changes The appearance of slices was improved by dipping in CaAsc solutions. Immediately after treatment, the average appearance rating increased from 6 points for the non CaAsc treatment to 7.8–8 points for CaAsc treatments (Fig. 2). After 7 d, HWT significantly improved the retention of appearance of apple slices treated with CaAsc. The appearance decreased with storage time in all treatments. Slices not treated in CaAsc decreased in appearance to below marketability limit before 7 d of storage. The CaAsc also improved the texture and the taste of the apple slices (Table 2) and the time of storage reduced both parameters and aroma (Table 4). During storage time, all treatments kept texture levels above the
Fig. 1. Chroma of apple slices that were cold water (4 C, 2 min) or hot water treated (48 C or 55 C, 2 min), then either dipped in 6% CaAsc or water. Slices were packaged in bags at ambient atmosphere, and stored up to 28 d at 4 C. Solid symbols and lines indicate slices were treated with 6% CaAsc, while empty symbols and dashed lines indicated slices dipped in water only (0% CaAsc). LSDs of significant effect (P = 0.05): LSDTimeHWTCaAsc dips = 1.23.
Table 3 Effect of hot water treatment on luminosity (L*) and h of apple slices that were cold water (4 C, 2 min) or plus hot water treated (48 C or 55 C, 2 min). Slices were packaged in air and stored up to 28 d at 4 C. Letters differentiate values compared by the least significant difference (LSD) at P = 0.05. Treatment
L*
4 C/2 min 48 C/2 min 55 C/2 min
76.2 a 73.7 b 73.8 b
97.3 a 97.2 a 96.1 b
h
limit of marketability except the slices heated at 55 C and not dipped in CaAsc, which fell below marketability at 28 d (data not shown). At day 21, the taste from slices not treated with CaAsc fell down under the limit of marketability. At the end of the 28 d storage period, the taste and a mouldy aroma from all treatments qualified the apple slices as not commercially acceptable (Table 4).
Table 1 Statistical analysis of parameters studied: time of storage (T, 28 d packaged in air at 4 C), hot water treatment (HWTs, 48 C or 55 C for 2 min or not heated) and CaAsc dips (0 or 6%) and their interaction for apple slices through analysis of variance.
L* h Chroma Appearance Texture Taste Aroma Overall acceptability DPPH FRAP Ascorbic acid
T
HWT
CaAsc
T x HWT
T x CaAsc
HWT x CaAsc
T x HWT x CaAsc
– ** – *** *** *** *** *** *** *** ***
*** ** *** – – – – – *** *** ***
*** *** *** *** *** *** – *** *** *** ***
– – ** – – – – * * *** –
– – * *** – – – *** *** *** ***
– – – ** – – – – *** *** ***
– – * – – – – – * *** –
Dash represents non-significance difference at P > 0.05 and *, **, *** significant difference at P < 0.05; 0.01 and 0.001, respectively.
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Taking into account the combination of sensory parameters such as appearance, texture, taste and aroma, the overall acceptability score was found to be the highest for CaAsc treatments extending the shelf life up to 14 d (Fig. 3). In contrast, slices not treated with CaAsc had a very short shelf life (<7 d). From 14 to 28 d of storage, the use of HWT before CaAsc dips was a significant factor in the overall acceptability changes. The use of this combination helped to extend the shelf life from 14 d (slices not heated) up to 21 d (slices heated). 3.3. Microbial counts
Fig. 2. Appearance of apple slices that were cold water (4 C, 2 min) or hot water treated (48 C or 55 C, 2 min), then either dipped in 6% CaAsc or water. Slices were packaged in bags at ambient atmosphere, and stored up to 28 d at 4 C. Solid symbols and lines indicate slices were treated with 6% CaAsc, while empty symbols and dashed lines indicated slices dipped in water only (0% CaAsc). Data based on hedonic scale where 1 = unusable, 3 = poor, 5 = fair (limit of marketability), 7 = good and 9 = excellent. LSDs of significant effect (P = 0.05): LSDTimeCaAsc dips = 0.59. LSDHWTCaAsc dips = 0.46.
Table 4 Effect of days of storage on h, texture, taste and aroma of apple slices stored in air up to 28 d at 4 C. Letters differentiate values compared by the least significant difference (LSD) at P = 0.05. Days of storage
0 7 14 21 28
97.3 a 97.6 a 96.8 ab 96.8 ab 95.9 b
h
Texture
Taste
Aroma
7.3 a 7.2 a 6.5 b 6.2 b 5.2 c
7.7 a 6.5 b 5.9 bc 5.3 c 2.6 d
6.4 a 5.3 b 5.1 b 5.3 b 1.9 c
At day 28, non heated apple slices dipped in CaAsc had microbial TPC counts of 4.6 log cfu/g compared to 5.3 log cfu/g for untreated slices. However, the biggest reduction to 3.9 log cfu/g was obtained with the combination of HWT and CaAsc dips (Table 5). Yeast and mould growth was higher than TPC and the reduction obtained using CaAsc alone or in combination with HWT was lower. HWT using 48 C instead of 55 C provided a better microbial quality. 3.4. Antioxidant activity Antioxidant activity was measured using the free radical scavenging activity (DPPH) and the ferric reducing ability (FRAP) assays. In both cases, the values, calculated as AA equivalents, were very similar to those found for Trolox equivalents (data not shown) and thus, results are only presented as AA equivalents. The DPPH assay of antioxidant activity of untreated apples slices on day 0 was 0.25 g kg 1. This level increased between 1.8 and 2.4 g kg 1 with slices dipped in CaAsc solution (Fig. 4) obtaining the highest concentration when both HWT were used. The slices dipped in HWT at 55 C for 2 min and 6% of CaAsc had nearly 10 times more antioxidant activity than the control (0% CaAsc, not heated) on day 0 of storage. However, HWT did not affect the relatively low antioxidant activity levels of slices when they were not dipped into CaAsc. Independently of type of HWT, DPPH decreased with time of storage and the proportional decrease was greater in apple slices dipped in CaAsc than those not dipped (70% vs 30% lost) although the level of antioxidant was higher in the CaAsc treated slices. The greatest reduction was found before 7 d of storage. The FRAP measure followed similar patterns to that of the DPPH assay including the effect of dipping in CaAsc and storage time. The FRAP assay for control slices at day 0 showed an antioxidant activity of 0.99 g kg 1 (Fig. 5) which was 4-fold higher than the value obtained by the DPPH assay (0.25 g kg 1; Fig. 4). Treatment with 6% CaAsc resulted in higher activity as measured by FRAP as that of DPPH and during the first 21 d, both HWT provided a similar increase on antioxidant compared to non-heated sample. In order to maintain an antioxidant activity greater than the level in the original apple slice (control), a heat treatment (48 or 55 C) Table 5 Microbial counts (mean log cfu g 1) at day 28 d of apple slices that were cold water (4 C, 2 min) or hot water treated (HWT, 48 C or 55 C, 2 min), then either dipped in 6% CaAsc or water. Slices were packaged in bags at ambient atmosphere and stored at 4 C. Letters differentiate values compared by the least significant difference (LSD) at P = 0.05.
Fig. 3. Overall acceptability of apple slices that were cold water (4 C, 2 min) or hot water treated (48 C or 55 C, 2 min), then either dipped in 6% CaAsc or water. Slices were packaged in bags at ambient atmosphere, and stored up to 28 d at 4 C. Solid symbols and lines indicate slices were treated with 6% CaAsc, while empty symbols and dashed lines indicated slices dipped in water only (0% CaAsc). Data based on hedonic scale where 1 = unusable, 3 = poor, 5 = fair (limit of marketability), 7 = good and 9 = excellent. LSDs of significant effect (P = 0.05): LSDTimeHWT = 0.67. LSDTimeCaAsc dips = 0.54.
HWT
% CaAsc
28 d at 4 C Total plate count
Yeasts and moulds
4 C/2 min
0% 6%
5.3z a 4.6 b
5.9 a 5.4 a
48 C/2 min 55 C/2 min
6% 6%
3.6 c 3.9 c
4.4 b 4.7 b
z
Data are the means of 3 replicates.
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2.0 Not heated 48º C/2 min 55 ºC/2 min
Ascorbic acid content (g/kg)
1.8 1.6 1.4 1.2 1.0 0.8
LSD Heat treatment x CaAsc
0.6 0.4
LSD Time x CaAsc
0.2 0.0 0
7
14
21
28
Storage du ration (days at 4 ºC) Fig. 4. Antioxidant activity (DDPH as ascorbic acid) of apple slices that were cold water (4 C, 2 min) or hot water treated (48 C or 55 C, 2 min), then either dipped in 6% CaAsc or water. Slices were packaged in bags at ambient atmosphere, and stored up to 28 d at 4 C. Solid symbols and lines indicate slices were treated with 6% CaAsc, while empty symbols and dashed lines indicated slices dipped in water only (0% CaAsc). LSDs of significant effect (P = 0.05): LSDTimeHWTCaAsc dips = 0.16.
Fig. 6. Ascorbic acid content of apple slices that were cold water (4 C, 2 min) or hot water treated (48 C or 55 C, 2 min), then either dipped in 6% CaAsc or water. Slices were packaged in bags at ambient atmosphere, and stored up to 28 d at 4 C. Solid symbols and lines indicate slices were treated with 6% CaAsc, while empty symbols and dashed lines indicated slices dipped in water only (0% CaAsc). LSDs of significant effect (P = 0.05): LSDTimeCaAsc dips = 0.18. LSDHWTCaAsc dips = 0.14.
3.6. Phenolic compounds
Fig. 5. Antioxidant activity (FRAP as ascorbic acid) of apple slices that were cold water (4 C, 2 min) or hot water treated (48 C or 55 C, 2 min), then either dipped in 6% CaAsc or water. Slices were packaged in bags at ambient atmosphere, and stored up to 28 d at 4 C. Solid symbols and lines indicate slices were treated with 6% CaAsc, while empty symbols and dashed lines indicated slices dipped in water only (0% CaAsc). LSDs of significant effect (P = 0.05): LSDTimeHWTCaAsc dips = 0.21.
combined with dipping in 6% CaAsc was required. This treatment provided a remaining antioxidant activity of about 1.5 g kg 1 at day 21. 3.5. Ascorbic acid The initial AA content of untreated apple slices was of 0.25 g kg 1. Immediately after CaAsc treatment, AA content was 5–7-fold higher (1.3–1.9 g kg 1) than those not dipped into CaAsc (Fig. 6). HWT helped to increase the ascorbic acid absorption from CaAsc dips. These increases were an average of 34% using 48 C and about 25% with 55 C. The antioxidant activity measurement showed that HWT did not increase the retention of vitamin C when the treatment was not combined with CaAsc dips. The pattern of decrease with storage time followed the same trends as those of the FRAP and DPPH activities, indicating that these changes were the result of loss of AA content.
The total phenolic content in control (untreated) slices prior to storage was 0.61 g kg 1 (Table 6). During storage the total phenolics decreased about 20%, and ranged after 28 d from 449 to 516 mg kg 1. Interaction of time, heat treatment and CaAsc had a significant effect on the total phenolic changes although the response varied with the type of individual phenolic (Table 6). Chlorogenic and epicatechin levels only were significantly affected by time of storage. Chlorogenic decreased from day 14 and epicatechin suffered a sharp decline at day 7 remaining stable until the end of storage. Quercetin concentration increased using CaAsc. During the first 21 d, the use of HWT also increased quercetin levels although no differences were found at the end of storage when HWT was used. A treatment of 55 C for 2 min increased the phloridzin levels. In contrast HWT decreased p-coumaric and procyanidins (B1, B2, C1) levels. Dipping into CaAsc further reduced coumaric content although the procyanidins levels only reduced in apple slices not heated and the phloridzin content decreased in slices not heated or heated 48 C for 2 min.
4. Discussion 4.1. Shelf life This research has shown that use of a 6% calcium ascorbate dip in fresh-cut apples extended the shelf life from <7 d in untreated slices to 14 d when packaged in air and stored at 4 C. The factor that ultimately limited the shelf life was the sensory quality. The mechanism for the increased shelf life related to use of HWT treatments (48 C or 55 C, 2 min) before CaAsc dips which lead to increased absorption of ascorbic acid into the apple tissue, enhancing the ascorbic acid content and consequently the antioxidant activity as measured by DPPH and FRAP values. The HWT enabled further extension of the shelf life (appearance) from 14 d (samples not heated dipped in CaAsc) to 21 d. In general, there was no significant difference between HWT at 48 C and 55 C and therefore a HWT of 48 C for 2 min should be enough to achieve the benefit of extra shelf life.
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Table 6 Main phenolic compounds (mg kg 1) of apple slices that were cold water (4 C, 2 min) or hot water treated (HWT, 48 C or 55 C, 2 min), then either dipped in 0 or 6% CaAsc. Slices were packaged in bags at ambient atmosphere, and stored up to 28 d at 4 C. Storage time
HWT
CaAsc treatment
Chlorogenic
Quercetin derivatives
Epicatechin
Coumaric acid
Procyanidins (B1, B2, C1)
Phloridzin
Total phenol
0d
4 C
0% 6% 0% 6% 0% 6%
81.6a 81.1 85.9 81.8 89.3 81.9
10.2 12.0 15.4 17.0 16.3 18.6
51.5 49.5 52.7 51.1 47.4 51.4
80.1 77.8 69.4 68.9 75.2 70.7
194.3 192.1 185.1 182.2 181.7 185.6
176.6 160.9 174.0 155.5 175.5 160.3
610.7 573.4 589.1 526.1 585.3 562.4
0% 6% 0% 6% 0% 6%
80.1 78.0 78.2 80.9 83.8 82.9
13.1 14.0 17.3 21.0 18.9 20.3
42.5 39.2 40.3 40.0 41.9 42.3
66.1 66.3 67.0 70.2 68.3 70.4
188.2 163.6 167.1 170.7 168.5 179.4
119.7 122.5 122.7 119.0 127.3 134.5
486.7 466.6 476.1 478.7 508.7 518.1
0% 6% 0% 6% 0% 6%
79.0 78.0 77.5 80.2 73.6 82.5
11.0 12.2 18.1 18.3 15.9 21.0
40.4 38.4 37.8 40.5 40.0 40.4
69.3 68.5 68.9 65.6 64.9 71.3
179.2 147.4 156.2 163.3 161.7 161.0
121.6 116.0 114.5 120.9 118.6 126.6
472.5 458.4 458.6 482.8 470.2 482.9
0% 6% 0% 6% 0% 6%
81.2 80.1 76.9 80.5 73.5 79.7
16.1 15.3 15.4 18.0 18.3 18.9
40.0 37.3 38.3 40.3 38.8 39.0
72.8 65.2 68.4 61.9 64.0 64.3
161.5 151.6 152.8 157.3 155.8 153.2
118.8 108.2 110.9 108.5 119.6 120.6
534.2 441.7 454.5 481.6 466.8 472.6
0% 6% 0% 6% 0% 6%
81.5 79.2 77.7 80.2 70.8 80.1
15.3 16.1 19.9 22.1 15.3 16.5
41.2 38.5 38.5 38.2 35.0 41.2
70.2 62.4 63.2 53.5 65.6 60.9
165.5 158.6 145.6 155.1 152.1 151.5
111.9 100.3 108.3 105.6 114.2 119.4
507.6 476.3 477.6 469.4 449.4 516.2
(3.1) NS NS NS NS NS NS
(1.2) (0.9) (0.8) (2.1) NS NS NS
(1.4) NS NS NS NS (1.5) NS
(2.0) (1.6) (1.3) (3.5) (2.9) (2.2) NS
(5.3) (4.1) NS NS (5.8) NS NS
(5.4) (4.2) (3.4) NS (7.7) NS NS
(15.1) (11.7) NS NS (21.3) (16.5) (36.9)
48 C 55 C
7d
4 C
48 C 55 C
14 d
4 C
48 C 55 C
21 d
4 C
48 C 55 C
28 d
4 C
48 C 55 C
Time HWT % CaAsc Time * HWT Time * CaAsc HWT * CaAsc Time * HWT * CaAsc a
Values are means (n = 3); NS, not significant. LSD values are in brackets at P < 0.05. T: Time. HWT: Water treatment. CaAsc: Calcium ascorbate.
4.2. Colour The increases in L* and h values using 6% of CaAsc dips indicates that it is an effective way to reduce browning in accordance with prior research (Son et al., 2001; Rojas-Grau et al., 2006; Tortoe et al., 2007). AA reduces browning incidence by reducing o-quinones back to phenolic compounds prior to polymerization and subsequent formation of coloured pigments (McEvily et al., 1992). Additionally, a formulation containing calcium and ascorbic acid acts partly to prevent cell and membrane breakdown (Toivonen and Brummell, 2008) and consequently reduces the release of PPO or its substrates thus improving the colour preservation of fresh-cut product (Pérez-Cabrera et al., 2011). Others researchers have found that dipping treatments with Ca ascorbate and heat shock (60 C for 1 min) caused PPO inhibition and were less affected by discolouration (Barbagallo et al., 2012). In the present experiment, we did not measure the levels of PPO enzyme but the use of heat treatment only resulted in a slight reduction in L* and h values and increased chroma when 55 C HWT was used. The combination of HWT and CaAsc maintained the colour of fresh-cut apple with significantly less browning than with the HWT without CaAsc. Djioua et al. (2009) also found greater percentage of L* loss in mango slices when whole mangoes were subjected to HWT (46 C/75 min and 50 C/30 min). This
could indicate that 55 C for 2 min induces a superficial damage in the apple flesh thus slightly reducing the lightless and colour. For example, Glowacz et al. (2013) reported that HWT at 50 C for 120 s resulted in the greatest membrane damage (20%) compared to spinach leaves treated at 40 C up to 120 s, 45 C up to 60 s and 50 C for 30 s. In contrast to our results, Abreu et al. (2003) and Koukounaras et al. (2008) found that in fresh-cut pear and peaches, respectively, that mild heat pre-treatments were effective in avoiding the cut surface browning in fresh-cut fruits. No difference was observed in PPO activity for the control and for heated peach slices (Koukounaras et al., 2008). Glowacz et al. (2013) also found that a chroma increased in spinach leaves with increasing temperature of the treatments (40 C vs 45 C vs 50 C). The hue angle decreased with time of storage mainly because the effectiveness of ascorbic acid in anti-browning is temporary. The enzymatic browning can re-generate after the ascorbic acid from the CaAsc or from the proper apple tissue has been completely reduced to dehydroascorbic acid. 4.3. Sensory changes Appearance and overall acceptability was strongly improved by dipping in CaAsc solutions since slices not treated with CaAsc had a very short shelf life (<7 d). However, the best treatments were the
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combination of HWT (48 or 55 C) with CaAsc dips, which extended the shelf life from 14 d (slices not heated) up to 21 d (heated slices). The ultimate shelf life limitation was related to sensory quality more than microbiological growth. Texture and taste from apple slices was also improved with CaAsc dips. HWT had no significant effect on these changes. Dea et al. (2010) also found there was no effect of heat treatment on the firmness of mango slices when whole mango were immersed in hot water (46 C for 90 or 75 min). Softness and deformation of stored oranges neither were influenced by HWT treatments (52 C for 180 s and at 56 C for 20 s) (Strano et al., 2014). The calcium present in CaAsc salt may be the main effect influence on the retention of texture. However, many researchers have reported good results in maintaining or improving texture when a combination of HWT and calcium dipping is used (Aguayo et al., 2008; Silveira et al., 2011) generally explained in terms of pectin esterase (PE) activation, cleaving the methoxyl groups from methylated galacturonic acid residues in pectin (Belitz and Grosch, 1986) which contain newly available carboxyl groups. The effect of HWT on firmness maintenance during storage has been reported for fresh-cut apple, pear, and melon (Kim et al., 1993; Abreu et al., 2003; Silveira et al., 2011). In our experiment, HWT had no a negative effect on retention of texture (as measured subjectively) although HWT can inhibit the synthesis of cell wall hydrolytic enzymes in whole apple fruit (Lurie et al., 1998). The improvement on taste using CaAsc was linked to the browning reduction activity of ascorbate temporarily avoiding the oxidation of phenolic compounds and keeping the endogenous organic acid in apple slices at levels, which provide a good taste. In apple juice, Komthong et al. (2007) found that using AA increased the two aldehydes, hexanal and trans-2-hexenal about 4 and 5-fold obtaining a positive green odour of apple juice. All sensory parameters decreased with time of storage as consequence of the natural oxidative processes which are particularly high in fresh-cut products. This quality loss with increased storage time was reduced by the combination of HWTs and CaAsc dips. 4.4. Microbial counts Many authors have shown application of calcium to fresh-cut fruit also reduces microbial growth, due to calcium increasing the rigidity of the cell wall and resistance to microbial enzymes. CaAsc treatment, in combination with HWT, very efficiently reduced the microbial growth. However, the use of high temperature HWT could damage apple tissue and affect shelf life. Heat damage has been previously reported in lettuce using mild heat treatment (50 C, 2 min), spinach leaves using HWT upper than 45 C for 60 s or in whole apples using rinsing at 65 C for 20 s (Moreira et al., 2006; Maxin et al., 2012; Glowacz et al., 2013). 4.5. Antioxidant activity (DDPH, FRAP) and ascorbic acid content The use of CaAsc dips increased the AA content and consequently the antioxidant activity as measured by DPPH and FRAP. This response was increased when dips were combined with HWT. This increase is most likely due to the thermal effects of gas expansion in the hot water such that solutions are pulled into the tissue due to contraction of the gas space with the cooler calcium ascorbate dip treatment. Alternatively, high temperature could enhance the diffusion rate of ascorbic acid and increase the solubility of ascorbic in water and apple tissue. This fact has been demonstrated with calcium salt as CaCl2 where hot water treatment (60 C, 1 min) had a significantly higher internal and external free calcium concentration suggesting a possible enhanced diffusion at higher temperatures (Aguayo et al., 2008).
In samples with no added CaAsc, no significant difference in AA content or antioxidant activity changes was observed between heated and unheated apples slices. In support, others have found that HWT had no effect on AA content in fresh-cut lettuce (Murata et al., 2004), spinach (Gómez et al., 2008; Glowacz et al., 2013), mango slices (Dea et al., 2010), rocket leaves (Koukounaras et al., 2009) or fresh-cut onions (Siddiq et al., 2013). In this experiment, the AA content and antioxidant capacity decreased with time of storage particularly in treatments with highs level of ascorbic acid (HWT plus CaAsc dip). In previous work, Aguayo et al. (2010) found that apple slices with lower antioxidant activity and ascorbic acid levels seemed to be more proportionately stable than those with initial very high antioxidant activity. Kalt et al. (1999) showed that in vegetable cells most of the AA is located in the vacuole (a very low pH environment), together with phenolic flavonoids. As Cocci et al. (2006) reported, it is probable that exogenous AA in the dipped slices diffused into the apple tissue and not being located within vacuoles was therefore more exposed to oxygen and endogenous oxidases and thus more rapidly oxidized. Exogenous antioxidant treatments should be able to interfere with senescence-related oxidation reactions. Aguayo et al. (2010) found that there is an efficient level of antioxidant content which decreases or even neutralizes the active oxygen species (AOS), resulting in prevention of induced senescence and thus, extension of shelf life. The antioxidant levels, as measured by FRAP, were related to shelf life and it appears that a minimal antioxidant of about 2 g kg 1 may be needed level to maintain shelf life in ‘Braeburn’ apple slices. In the present experiment, the longest residual minimum level of antioxidant was obtained through the combination of hot water and 6% CaAsc. These apple slices, stored in air at 4 C, achieved the longest shelf life being scored above the limit of marketability at day 21. 4.6. Phenolic compounds Exogenous CaAsc dips had small but significant effects on the reduction of p-coumaric and procyanidins and phloridzin. The decrease could be a consequence of formation of a complex of ascorbic acid from CaAsc with natural products of coumaric acid (Kesinger and Stevens, 2009). The use of HWT increased the quercetin and phloridzin levels but decreased coumaric and procyanidins (B1, B2, C1) levels. HWT has been proposed to reduce browning in fresh-cut tissues because of the demonstrated effect on delaying the wound-induced production of PAL (Saltveit, 2000). In contrast, some researchers have reported that HWT enhanced some enzyme activities associated with biosynthesis of phenolic compounds in mandarins (Ghasemnezhad et al., 2008), muskmelon fruit (Yuan et al., 2013) or peach (Spadoni et al., 2014). In our experiment, the use of HWT did not provide a same response in all the phenolic compounds. This could be attributed to differences in the metabolism of different phenolic compounds by the apple cells. Heat could have enhanced the phenolic extraction in apple slices. Gerard and Roberts (2004) found that increasing temperature during pressing from 40 C to 70 C allows increasing flavonoid content (50%) in apple juice. However, some phenolics such as coumaric and procyanidin show lower stability (or higher susceptibility) to thermal degradation, autoxidation or breakdown (Ioannou et al., 2012), whilst others (such as quercetin) are more stable upon heating (Odriozola-Serrano et al., 2008). In general, heat treatments are effective at stopping or at least delaying “active” physiological processes (Woolf et al., 2012). However, in our experiment, the beneficial effects of HWTs was most likely due to the physical thermal effects of moving from high to low temperature such that solutions are pulled into the tissue due to gas space contraction with the cooler calcium ascorbate dip
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treatment. HWTs could enhance diffusion rate of ascorbic acid and increase the solubility of ascorbic in water and apple tissue increasing vitamin C, antioxidant capacity, improving appearance and extending the shelf life based on overall acceptability. 5. Conclusions CaAsc dips (6%) had a strong impact reducing the browning and therefore, extending the overall acceptability to 14 d. This treatment increased the antioxidant content 5 fold which was further increased with the combination of HWT that resulted in an increased absorption of ascorbic acid in the apple tissue. The combination of treatments helped to maintain fresh-cut apple quality, in particular, the sensory taste and aroma extending the shelf life from 14 d to up to 21 d at 4 C. HWT for 2 min at 48 C was preferable to 55 C with no significant benefit of the higher temperature that would also have increased energy cost and could induce a superficial damage in the apple flesh. Acknowledgements Encarna Aguayo thanks the Technical University of Cartagena for supporting her sabbatical stay at the New Zealand Institute for Plant & Food Research (Auckland). Thanks to Reginald Wibisono for his valuable assistance in the laboratory, and David Stevenson and Janine Cooney for their knowledge on the phenolic compound identification. References Abreu, M., Beirao-da-Costa, S., Goncalves, E.M., Beirao-da-Costa, M.L., MoldaoMartins, M., 2003. Use of mild heat pre-treatments for quality retention of fresh-cut ‘Rocha’ pear. Postharvest Biol. Technol. 30, 153–160. Aguayo, E., Escalona, V.H., Artés, F., 2008. Effect of hot water treatment and various calcium salts on quality of fresh-cut ‘Amarillo’ melon. Postharvest Biol. Technol. 47, 397–406. Aguayo, E., Requejo-Jackman, C., Stanley, R., Woolf, R., 2010. Effects of calcium ascorbate treatments and storage atmosphere on antioxidant activity and quality of fresh-cut apple slices. Postharvest Biol. Technol. 57, 52–60. Barbagallo, R.N., Chisari, M., Caputa, G., 2012. Effects of calcium citrate and ascorbate as inhibitors of browning and softening in minimally processed ‘Birgah’ eggplants. Postharvest Biol. Technol. 73, 107–114. Belitz, H.D., Grosch, W., 1986. Enzymes, Food Chemistry. second ed. Springer-Verlag, New-York, pp. 155–201. Boyer, J., Liu, R.H., 2004. Apple phytochemicals and their health benefits. Nutr. J. 3, 1–15. Cocci, E., Rocculi, P., Romani, S., Rosa, M.D., 2006. Changes in nutritional properties of minimally processed apples during storage. Postharvest Biol. Technol. 39, 265–271. Dea, S., Brecht, J.K., Nunes, M.C.N., Baldwin, E.A., 2010. Quality of fresh-cut ‘Kent’ mango slices prepared from hot water or non-hot water-treated fruit. Postharvest Biol. Technol 56, 171–180. Djioua, T., Charles, F., Lopez-Lauri, F., Filgueiras, H., Coudret, A., Freire, M., DucampCollind, M.N., Sallanon, H., 2009. Improving the storage of minimally processed mangoes (Mangifera indica L.) by hot water treatments. Postharvest Biol. Technol. 52, 221–226. FAOSTAT, 2011. http://faostat.fao.org/site/339/default.aspx. Gerard, K.A., Roberts, J.S., 2004. Microwave heating of apple mash to improve juice yield and quality. Lebensm–Wiss Technol. 37, 551–557. Ghasemnezhad, M., Marsh, K., Shilton, R., Babalar, M., Woolf, A., 2008. Effect of hot water treatments on chilling injury and heat damage in ‘satsuma’ mandarins: antioxidant enzymes and vacuolar ATPase, and pyrophosphatase. Postharvest Biol. Technol. 48, 364–371. Gómez, F., Fernandez, L., Gergoff, G., Guiamet, J.J., Chaves, A., Bartoli, C.G., 2008. Heat shock increases mitochondrial H2O2 production and extends postharvest life of spinach leaves. Postharvest Biol. Technol. 49, 229–234. Glowacz, M., Mogren, L.M., Reade, J.P.H., Cobb, A.H., Monaghan, J.M., 2013. Can hot water treatments enhance or maintain postharvest quality of spinach leaves. Postharvest Biol. Technol. 81, 23–28.
165
Ioannou, I., Hafsa, I., Hamdi, S., Charbonnel, C., Ghoul, M., 2012. Review of the effects of food processing and formulation on flavonol and anthocyanin behavior. J. Food Eng. 111, 208–217. Kalt, W., Forney, C.F., Martin, A., Prior, R.L., 1999. Antioxidant capacity, vitamin C, phenolics, and anthocyanins after fresh storage of small fruits. J. Agric. Food Chem. 47, 4638–4644. Kesinger, N.G., Stevens, J.F., 2009. Covalent interaction of ascorbic acid with natural products. Phytochemistry 70, 1930–1939. Kim, D.M., Smith, N.L., Lee, C.Y., 1993. Apple cultivar variations in response to heat treatment and minimal processing. J. Food Sci. 58, 1111–1124. Komthong, P., Igura, N., Shimoda, M., 2007. Effect of ascorbic acid on the odours of cloudy apple juice. Food Chem. 100, 1342–1349. Koukounaras, A., Siomos, A.S., Sfakiotakis, E., 2009. Impact of heat treatment on ethylene production and yellowing of modified atmosphere packaged rocket leaves. Postharvest Biol. Technol. 54, 172–176. Koukounaras, A., Siomos, A.S., Sfakiotakis, E., 2008. The effect of heat treatment on quality retention of fresh-cut peach. Postharvest Biol. Technol. 48, 30–36. Kwak, E.J., Lim, S.I., 2005. Inhibition of browning by antibrowning agents and phenolic acids or cinnamic acid in the glucose–lysine model. J. Sci. Food Agr. 85, 1337–1342. Lurie, S., Fallik, E., Klein, J.D., Kozar, E., Kovacs, K., 1998. Postharvest heat treatment of apples to control San Jose scale (Quadraspidotiotus perniciosus Comstock) and blue mold (Penicillium expansum Link) and maintain fruit firmness. J. Am. Soc. Hortic. Sci. 123, 110–114. Maxin, P., Weber, R.W.S., Pedersen, H.L., Williams, M., 2012. Control of a wide range of storage rots in naturally infected apples by hot-water dipping and rinsing. Postharvest Biol. Technol. 70, 25–31. McEvily, A.J., Iyengar, R., Otwell, W.S., 1992. Inhibition of enzymatic browning in foods and beverages. Food Sci. Nutr. 32, 253–273. Moreira, M.D.R., Ponce, A.G., Del Valle, C.E., Roura, S.I., 2006. Ascorbic acid retention, microbial growth, and sensory acceptability of lettuce leaves subjected to mild heat shocks. J. Food Sci. 71, 188–192. Murata, M., Tanaka, E., Minoura, E., Homma, S., 2004. Quality of cut lettuce treated by heat shock: prevention of enzymatic browning, repression of phenylalanine ammonia–lyase activity, and improvement on sensory evaluation during storage. Biosci. Biotechnol. Biochem. 68, 501–507. Odriozola-Serrano, I., Soliva-Fortuny, R., Martin-Belloso, O., 2008. Phenolic acids, flavonoids, vitamin C and antioxidant capacity of strawberry juices processed by high-intensity pulsed electric fields or heat treatments. Eur. Food Res. Technol. 228, 239–248. Pérez-Cabrera, L., Chafer, M., Chiralt, A., González-Martínez, C., 2011. Effectiveness of antibrowning agents applied by vacuum impregnation on minimally processed pear. LWT, Food Sci. Technol. 44, 2273–2280. Rassam, M., Laing, W., 2005. Variation in AA and oxalate levels in the fruit of Actinidia chinensis tissues and genotypes. J. Agric. Food Chem. 53, 2322–2326. Rojas-Grau, M.A., Sobrino López, A., Tapia, M.S., Martin Belloso, O., 2006. Browning inhibition in fresh-cut Fuji apple slices by natural antibrowning agents. J. Food Sci. 71, 559–565. Saltveit, M.E., 2000. Wound induced changes in phenolic metabolism and tissue browning are altered by heat shock. Postharvest Biol. Technol. 21, 61–69. Siddiq, M., Roidoung, S., Sogi, D.S., Dolan, K.D., 2013. Total phenolics, antioxidant properties and quality of fresh-cut onions (Allium cepa L.) treated with mildheat. Food Chem. 136, 803–806. Silveira, A.C., Aguayo, E., Chisari, M., Artés, F., 2011. Calcium salts and heat treatment for quality retention of fresh-cut ‘Galia’ melon. Postharvest Biol. Technol. 62, 77–84. Son, S.M., Moon, L.D., Lee, C.Y., 2001. Inhibitory effects of various antibrowning agents on apples slices. Food Chem. 73, 23–30. Spadoni, A., Guidarelli, M., Sanzani, S.M., Ippolito, A., Mari, M., 2014. Influence of hot water treatment on brown rot of peach and rapid fruit response to heat stress. Postharvest Biol. Technol. 94, 66–73. Strano, M.C., Calandra, M., Aloisi, V., Rapisarda, P., Strano, T., Ruberto, G., 2014. Hot water dipping treatments on Tarocco orange fruit and their effects on peel essential oil. Postharvest Biol. Technol. 94, 26–34. Toivonen, P.M.A., 2004. Postharvest storage procedures and oxidative stress. HortScience 39, 938–942. Toivonen, P.M.A., Brummell, D.A., 2008. Biochemical bases of appearance and texture changes in fresh-cut fruit and vegetables. Postharvest Biol. Technol. 48, 1–14. Tortoe, C., Orchard, J., Beezer, A., 2007. Prevention of enzymatic browning of apple cylinders using different solutions. Intern. J. Food Sci. Technol. 42, 1475–1481. Whitaker, J., 1972. Principles of Enzymology for the Food Sciences. M. Dekker, New York, pp. 571–577. Woolf, A.B., Combes, S., Petley, M., Olsson, S.R., Wohlers, M., Jackman, R.C., 2012. Hot water treatments reduce leaf yellowing and extend vase life of Asiatic hybrid lilies. Postharvest Biol. Technol. 64, 9–18. Yuan, L., Bi, Y., Ge, Y., Wang, Y., Liu, Y., Li, G., 2013. Postharvest hot water dipping reduces decay by inducing disease resistance and maintaining firmness in muskmelon (Cucumis melo L.) fruit. Sci. Hortic. 161, 101–110.
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Hot water treatment in combination with calcium ascorbate dips increases bioactive compounds and helps to maintain fresh-cut apple quality Encarna Aguayoa,* , Cecilia Requejo-Jackmanb , Roger Stanleyc , Allan Woolfb a Postharvest and Refrigeration Group. Department of Food Engineering, Technical University of Cartagena. Paseo Alfonso XIII 48, 30203 Cartagena, Murcia, Spain b The New Zealand Institute for Plant & Food Research Ltd, Private Bag 92169, Auckland, New Zealand c Centre for Food Innovation, University of Tasmania, Locked Bag 1370, Launceston, TAS 7250, Australia
A R T I C L E I N F O
A B S T R A C T
Article history: Received 23 October 2014 Received in revised form 1 July 2015 Accepted 3 July 2015 Available online xxx
Fresh-cut ‘Braeburn’ apple slices were dipped into cold water (4 C for 2 min) or hot water (HWT, 48 C or 55 C for 2 min) followed by dips into 0 or 6% w/v aqueous calcium ascorbate (CaAsc, 2 min, 0 C) and stored in air up to 28 d at 4 C. Microbial counts, changes in browning and sensory acceptance were determined to indicate changes in quality. Changes in antioxidant levels were measured using free radical scavenging activity (DPPH), reducing activity (FRAP), ascorbic acid content (AA) and polyphenolic content (by HPLC). CaAsc dips had a strong impact reducing the browning through increasing the flesh luminosity and hue angle. 6% CaAsc in fresh-cut apples extended the overall acceptability from less than 7 d to 14 d. Immediately after CaAsc treatment, AA content was 5 fold higher (0.25–1.25 g kg 1) than those not dipped into CaAsc. However, the combination of HWT treatments and CaAsc dips led to seven fold increased levels of AA inside the apple tissue (0.25–1.85 g kg 1) and consequently increased the antioxidant activity. HWT did not increase the AA content when not combined with CaAsc dips. The HWT CaAsc dip extended the overall acceptability to 21 d compared to 14 d for samples not heated but dipped into CaAsc. Shelf life was ultimately limited by sensory quality. At day 28, total plate counts were reduced from 5.3 log cfu/g (untreated slices) to 4.6 log cfu/g in the 6% CaAsc dips and further to 3.9 log cfu/g with the combination of HWT and CaAsc dip. Changes in the content of phenolic compounds with time, HWT and CaAsc dip were generally not significant except for slightly increased quercetin and phloridzin levels and decreased p-coumaric and procyanidins over time. The combination of HWT at 48 C for 2 min followed by 6% CaAsc dip would be best for preserving the eating quality of apple slices. ã 2015 Elsevier B.V. All rights reserved.
Keywords: Minimally processed Apple slices Heat treatment Shelf life Phenolic compounds Vitamin C Ascorbic acid Antioxidant
1. Introduction Apples are one of the most widely consumed fruits (FAOSTAT, 2011) and are a good source of phytochemicals (Boyer and Liu, 2004). Epidemiological studies have linked the consumption of apples with reduced risk of some cancers, cardiovascular disease, asthma, and diabetes (Boyer and Liu, 2004). One way to increase fresh fruit consumption is processing fruit into fresh-cut product to be sold as convenient single servings. However, fresh-cut processing results in major tissue disruption of surface cells and injury stress of underlying tissues (Toivonen, 2004). The main problem for fresh-cut apple is oxidation caused by polyphenol
* Corresponding author. E-mail address: [email protected] (E. Aguayo). http://dx.doi.org/10.1016/j.postharvbio.2015.07.001 0925-5214/ ã 2015 Elsevier B.V. All rights reserved.
oxidase (PPO) that exists in particularly high amounts in apple (Whitaker, 1972). The resulting browning makes the product unsuitable to the consumer. Ranges of treatments have been applied to extend the shelf life of fresh-cut apples, mainly dipping in solutions of a wide range of anti-browning agents. Ascorbic acid, oxalic acid, oxalacetic acid, kojic acid, erythorbic acid, citric acid, and/or calcium, cysteine, 4-hexylresorcinol have all been examined at different concentrations (Son et al., 2001; Rojas-Grau et al., 2006; Tortoe et al., 2007). Among the aforementioned methods, the use of calcium ascorbate (CaAsc) has been found to be the most effective anti-browning agent and can be marketed as a minimal chemical input. Its application increases the antioxidant status and extends shelf life of apple slices (Aguayo et al., 2010). On the other hand, several methods, including heat shock, have been used to inhibit polyphenol oxidase activity since this reduces the rate of the polymerisation step of browning reactions (Kwak and Lim,
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2005; Barbagallo et al., 2012). Hot water treatment (HWT) is an effective physical treatment, free of chemical residues, and readily applicable in the fresh-cut industry during the washing process (Kim et al., 1993; Abreu et al., 2003). Previous studies have shown that HWT is sufficiently effective to maintain product quality for fresh-cut products such as lettuce (Murata et al., 2004; Moreira et al., 2006), rocket (Koukounaras et al., 2009), spinach (Gómez et al., 2008; Glowacz et al., 2013), eggplants (Barbagallo et al., 2012), and onions (Siddiq et al., 2013). The use of non-chemical technologies that can improve product quality response by additional interactions could improve the effectiveness of CaAsc in the fresh-cut apple industry. This study sought to determine whether HWT could be recommended as a method in combination with CaAsc dips to help to maintain the sensory quality of fresh-cut apples. 2. Materials and methods 2.1. Raw material New Zealand grown ‘Mahana Red’ apples (Malus domestica Borkh., a sport of ‘Braeburn’) were sourced from the refrigerated storage of a commercial supermarket. The apples had been stored for up to 6 months under controlled atmosphere (2 kPa O2 plus 1 kPa CO2) at 0 C. Apples boxes were transported to the laboratory and stored at 0 C for 12 h. The boxes were opened in a food-grade processing room (10 C) and the fruit sorted to remove those damaged or with significant variation in background colour. Whole apple surfaces were washed by dipping in cold water (4 C) with 5 mg L 1 chlorine dioxide (OxineTM, Australasia Marketing Pty Ltd. Sydney, NSW, Australia) for 10 min. The apples were then manually cored with a metal corer and cut into 8 wedge slices using a handheld knife. All the slices were dipped into cold water (4 C) with 2 mg L 1 chlorine dioxide for 2 min, and the slices drained. Slices for HWT were dipped in hot water (48 C or 55 C) for 2 min. A stainless water-bath (Grant 40 L, with a Grant temperature controller units (0.1 C, 1.4 kW heater model GRAVF, U.K.) with continuous hot water recirculation and stirring was used to maintain the relevant temperature. All apple slices (heated or not) were then dipped for 2 min into 0% (control) or 6% CaAsc solution (w/w; 99.9% purity, Wolf Canyon Asia Pacific Ltd) and drained. This solution was made using water at 0 C that had been pre-treated with 2 mg L 1 chlorine dioxide. Apple skins were not removed prior to treatment, as apple slices are currently marketed with skin intact. For each treatment, apple slices were randomised across packages of 15 apple slices (350 20 g) per aluminium bag (25 cm 18 cm, 80 mm thickness, Caspak, New Zealand). To maintain nearly ambient oxygen concentration in the bags, two 5-mm holes were punched through both sides of each bag. Three replicate bags per treatment were stored at 4 C per storage duration: 7, 14, 21 and 28 d. Measures of antioxidant activity were also measured on day 0 (immediately after treatment). 2.2. Parameter evaluations 2.2.1. Colour measurement The surface colour of the apple flesh was determined on three equidistant points in each apple slice cut surface with a Minolta chromameter (D65 light source, Minolta Camera Co., Osaka, Japan). The results were expressed as CIELAB (L*a*b*) colour space. L* defines the lightness and a* and b* define the red-greenness and blue-yellowness, respectively. The flesh colour was also measured and expressed as hue angle (h = arctangent [(b*) (a*) 1]) and chroma (C* = [(a*)2 + (b*)2]1/2). Fifteen slices per treatment (5 slices per replicate) were measured.
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2.2.2. Sensory evaluation A panel of five people was trained to recognise and score the quality attributes of the treated apple slices. All assessments were compared to freshly cut slices. Appearance, taste and texture were scored on a nine-point scale where 1 = complete lacking or soft, to 9 = fully characteristic of fresh. A similar scale, where 1 = inedible, 3 = poor, 5 = fair (limit of marketability), 7 = good and 9 = excellent was used for the evaluation of the overall acceptability. 2.2.3. Microbial analyses After 28 d storage, microbial growth on the slices was determined by a certified food analysis laboratory (AgriQuality, Auckland, New Zealand). Only samples dipped into 6% CaAsc and both controls (0 and 6% CaAsc) from the HWT and control were analyzed. From each of five slices, 10 g samples were blended with 90 mL of sterile peptone buffered water (Merck Darmstadt, Germany) for 1 min in a sterile stomacher bag (Model 400 Bags 6141, London, UK) using a Masticator (Colwort Stomacher 400 Lab, Seward Medical, London, UK). Appropriate dilutions were prepared. Plate Count Agar medium (PCA, Merck) was used for TPC and Rose Bengal agar medium (Merck) for the yeast and mould counts. Incubation conditions were 30 C for 48 h for TPC, and 22 C for 5 d for yeasts and moulds, respectively. Microbial counts were reported as log 10 colony forming units per gram of sample (log cfu g 1). 2.2.4. Chemical measurements Fruit pieces were flash frozen in liquid nitrogen and stored at 80 C for a maximum of 2 months. To ensure uniformity, frozen samples (200 g) were either homogenised in 100 mL of distilled water in a commercial blender (Sunbeam Model PB7600, Type 504, 230–240 V, Sydney, Australia) to produce a juice extract for antioxidant activity analysis, or 150 g was ground to a fine powder in a Cryomill in liquid nitrogen for ascorbic acid content (AA) analysis. 2.2.4.1. Antioxidant activity. Two assays were used to measure the antioxidant activity; 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP) assays. All the material, method and equipment has been previously reported (Aguayo et al., 2010). All the antioxidant assays were carried out in triplicate. Calibration curves were made for each assay using Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) and AA as standards. The antioxidant activity (DPPH, FRAP assay) was expressed as Trolox and AA equivalent antioxidant activity per kg fresh weight of apple tissue. 2.2.4.2. Ascorbic acid evaluation. The method was adapted from Rassam and Laing (2005). A 0.2 g sample of powdered apple tissue was suspended in 1 mL of 6% metaphosphoric acid, 2 mM EDTA and 1% PVPP containing 4 mM TCEP (tris[2-carboxyethyl] phosphine hydrochloride). The slurry was vortexed for 20 s, and incubated in a heating block for 2 h at 40 C to ensure full reduction of any dehydroascorbate. The extract was clarified by centrifugation at 4 C for 10 min, and 20 mL of the supernatant was injected into a 7.8 300 mm Aminex HPX-87H HPLC column (Bio-Rad, Merck, Darmstadt, Germany). The column was run with 2.8 mM H2SO4 as the mobile phase, at a flow rate of 0.01 mL s 1. The amount of AA was detected using a Waters 996 photodiode-array detector (Milford, MA, USA) set at 245.5 nm for absorbance using AA (Sigma, St. Louis, MO) as a standard. The peak was authenticated as AA by showing that it was completely degraded by ascorbate oxidase at pH 5.5. 2.2.4.3. Phenolic compounds evaluation. One mL of apple juice extract was combined with 0.25 mL of methanol and HCl
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(90:10 v/v) and vortexed for 20 s. As mentioned, all the details have previously been reported (Aguayo et al., 2010). Chromatograms were recorded at 280 and 340 nm at a flow rate of 0.02 mL s 1. Peak assignment was performed by comparison of the retention times and UV spectra with those of reference compounds and by mass spectrometric analyses. Sample injection volume was 10 mL. UV– vis detection was by absorbance at 200–600 nm.
Table 2 Effect of CaAsc dip concentration (0 or 6%) on L*, h, texture and taste of apple slices (P = 0.05). Slices were packaged in air and stored up to 28 d at 4 C. % CaAs
L*
0 6
73.0 76.2
93.0 100.7
h
Texture
Taste
6.0 7.0
5.0 6.2
2.3. Statistical analysis A randomised design with three replicates per treatment was used where each bag constituted a replicate. To determine the effect of storage time, HWT and CaAsc on each dependent variable, a three-way analysis of variance (ANOVA, P 0.05) was carried out (Statgraphic Plus, version 5.1, 2001, Manugistic Inc., Rockville, MD, USA). Mean values were compared by LSD (least significant difference test) when significant differences among treatments and interactions between factors was found. 3. Results Table 1 shows ANOVA results of time of storage, hot water treatment and CaAsc dips (0 or 6%) factors and their interaction on apple slices. 3.1. Colour CaAsc dips had a strong immediate impact increasing L* and hue angle ( h) (Table 2) and reducing the chroma of apple slices (Fig. 1). This indicated a lighter, slightly greener colour with less saturation in the apple flesh. In contrast, the HWT reduced the luminosity of apple slices (Table 3) and slices that were not heated showed the higher levels of L* and h and decreased chroma. After 28 d of storage h was slightly reduced from 97 to 96 without significant changes in the L* parameter. 3.2. Sensory changes The appearance of slices was improved by dipping in CaAsc solutions. Immediately after treatment, the average appearance rating increased from 6 points for the non CaAsc treatment to 7.8–8 points for CaAsc treatments (Fig. 2). After 7 d, HWT significantly improved the retention of appearance of apple slices treated with CaAsc. The appearance decreased with storage time in all treatments. Slices not treated in CaAsc decreased in appearance to below marketability limit before 7 d of storage. The CaAsc also improved the texture and the taste of the apple slices (Table 2) and the time of storage reduced both parameters and aroma (Table 4). During storage time, all treatments kept texture levels above the
Fig. 1. Chroma of apple slices that were cold water (4 C, 2 min) or hot water treated (48 C or 55 C, 2 min), then either dipped in 6% CaAsc or water. Slices were packaged in bags at ambient atmosphere, and stored up to 28 d at 4 C. Solid symbols and lines indicate slices were treated with 6% CaAsc, while empty symbols and dashed lines indicated slices dipped in water only (0% CaAsc). LSDs of significant effect (P = 0.05): LSDTimeHWTCaAsc dips = 1.23.
Table 3 Effect of hot water treatment on luminosity (L*) and h of apple slices that were cold water (4 C, 2 min) or plus hot water treated (48 C or 55 C, 2 min). Slices were packaged in air and stored up to 28 d at 4 C. Letters differentiate values compared by the least significant difference (LSD) at P = 0.05. Treatment
L*
4 C/2 min 48 C/2 min 55 C/2 min
76.2 a 73.7 b 73.8 b
97.3 a 97.2 a 96.1 b
h
limit of marketability except the slices heated at 55 C and not dipped in CaAsc, which fell below marketability at 28 d (data not shown). At day 21, the taste from slices not treated with CaAsc fell down under the limit of marketability. At the end of the 28 d storage period, the taste and a mouldy aroma from all treatments qualified the apple slices as not commercially acceptable (Table 4).
Table 1 Statistical analysis of parameters studied: time of storage (T, 28 d packaged in air at 4 C), hot water treatment (HWTs, 48 C or 55 C for 2 min or not heated) and CaAsc dips (0 or 6%) and their interaction for apple slices through analysis of variance.
L* h Chroma Appearance Texture Taste Aroma Overall acceptability DPPH FRAP Ascorbic acid
T
HWT
CaAsc
T x HWT
T x CaAsc
HWT x CaAsc
T x HWT x CaAsc
– ** – *** *** *** *** *** *** *** ***
*** ** *** – – – – – *** *** ***
*** *** *** *** *** *** – *** *** *** ***
– – ** – – – – * * *** –
– – * *** – – – *** *** *** ***
– – – ** – – – – *** *** ***
– – * – – – – – * *** –
Dash represents non-significance difference at P > 0.05 and *, **, *** significant difference at P < 0.05; 0.01 and 0.001, respectively.
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Taking into account the combination of sensory parameters such as appearance, texture, taste and aroma, the overall acceptability score was found to be the highest for CaAsc treatments extending the shelf life up to 14 d (Fig. 3). In contrast, slices not treated with CaAsc had a very short shelf life (<7 d). From 14 to 28 d of storage, the use of HWT before CaAsc dips was a significant factor in the overall acceptability changes. The use of this combination helped to extend the shelf life from 14 d (slices not heated) up to 21 d (slices heated). 3.3. Microbial counts
Fig. 2. Appearance of apple slices that were cold water (4 C, 2 min) or hot water treated (48 C or 55 C, 2 min), then either dipped in 6% CaAsc or water. Slices were packaged in bags at ambient atmosphere, and stored up to 28 d at 4 C. Solid symbols and lines indicate slices were treated with 6% CaAsc, while empty symbols and dashed lines indicated slices dipped in water only (0% CaAsc). Data based on hedonic scale where 1 = unusable, 3 = poor, 5 = fair (limit of marketability), 7 = good and 9 = excellent. LSDs of significant effect (P = 0.05): LSDTimeCaAsc dips = 0.59. LSDHWTCaAsc dips = 0.46.
Table 4 Effect of days of storage on h, texture, taste and aroma of apple slices stored in air up to 28 d at 4 C. Letters differentiate values compared by the least significant difference (LSD) at P = 0.05. Days of storage
0 7 14 21 28
97.3 a 97.6 a 96.8 ab 96.8 ab 95.9 b
h
Texture
Taste
Aroma
7.3 a 7.2 a 6.5 b 6.2 b 5.2 c
7.7 a 6.5 b 5.9 bc 5.3 c 2.6 d
6.4 a 5.3 b 5.1 b 5.3 b 1.9 c
At day 28, non heated apple slices dipped in CaAsc had microbial TPC counts of 4.6 log cfu/g compared to 5.3 log cfu/g for untreated slices. However, the biggest reduction to 3.9 log cfu/g was obtained with the combination of HWT and CaAsc dips (Table 5). Yeast and mould growth was higher than TPC and the reduction obtained using CaAsc alone or in combination with HWT was lower. HWT using 48 C instead of 55 C provided a better microbial quality. 3.4. Antioxidant activity Antioxidant activity was measured using the free radical scavenging activity (DPPH) and the ferric reducing ability (FRAP) assays. In both cases, the values, calculated as AA equivalents, were very similar to those found for Trolox equivalents (data not shown) and thus, results are only presented as AA equivalents. The DPPH assay of antioxidant activity of untreated apples slices on day 0 was 0.25 g kg 1. This level increased between 1.8 and 2.4 g kg 1 with slices dipped in CaAsc solution (Fig. 4) obtaining the highest concentration when both HWT were used. The slices dipped in HWT at 55 C for 2 min and 6% of CaAsc had nearly 10 times more antioxidant activity than the control (0% CaAsc, not heated) on day 0 of storage. However, HWT did not affect the relatively low antioxidant activity levels of slices when they were not dipped into CaAsc. Independently of type of HWT, DPPH decreased with time of storage and the proportional decrease was greater in apple slices dipped in CaAsc than those not dipped (70% vs 30% lost) although the level of antioxidant was higher in the CaAsc treated slices. The greatest reduction was found before 7 d of storage. The FRAP measure followed similar patterns to that of the DPPH assay including the effect of dipping in CaAsc and storage time. The FRAP assay for control slices at day 0 showed an antioxidant activity of 0.99 g kg 1 (Fig. 5) which was 4-fold higher than the value obtained by the DPPH assay (0.25 g kg 1; Fig. 4). Treatment with 6% CaAsc resulted in higher activity as measured by FRAP as that of DPPH and during the first 21 d, both HWT provided a similar increase on antioxidant compared to non-heated sample. In order to maintain an antioxidant activity greater than the level in the original apple slice (control), a heat treatment (48 or 55 C) Table 5 Microbial counts (mean log cfu g 1) at day 28 d of apple slices that were cold water (4 C, 2 min) or hot water treated (HWT, 48 C or 55 C, 2 min), then either dipped in 6% CaAsc or water. Slices were packaged in bags at ambient atmosphere and stored at 4 C. Letters differentiate values compared by the least significant difference (LSD) at P = 0.05.
Fig. 3. Overall acceptability of apple slices that were cold water (4 C, 2 min) or hot water treated (48 C or 55 C, 2 min), then either dipped in 6% CaAsc or water. Slices were packaged in bags at ambient atmosphere, and stored up to 28 d at 4 C. Solid symbols and lines indicate slices were treated with 6% CaAsc, while empty symbols and dashed lines indicated slices dipped in water only (0% CaAsc). Data based on hedonic scale where 1 = unusable, 3 = poor, 5 = fair (limit of marketability), 7 = good and 9 = excellent. LSDs of significant effect (P = 0.05): LSDTimeHWT = 0.67. LSDTimeCaAsc dips = 0.54.
HWT
% CaAsc
28 d at 4 C Total plate count
Yeasts and moulds
4 C/2 min
0% 6%
5.3z a 4.6 b
5.9 a 5.4 a
48 C/2 min 55 C/2 min
6% 6%
3.6 c 3.9 c
4.4 b 4.7 b
z
Data are the means of 3 replicates.
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2.0 Not heated 48º C/2 min 55 ºC/2 min
Ascorbic acid content (g/kg)
1.8 1.6 1.4 1.2 1.0 0.8
LSD Heat treatment x CaAsc
0.6 0.4
LSD Time x CaAsc
0.2 0.0 0
7
14
21
28
Storage du ration (days at 4 ºC) Fig. 4. Antioxidant activity (DDPH as ascorbic acid) of apple slices that were cold water (4 C, 2 min) or hot water treated (48 C or 55 C, 2 min), then either dipped in 6% CaAsc or water. Slices were packaged in bags at ambient atmosphere, and stored up to 28 d at 4 C. Solid symbols and lines indicate slices were treated with 6% CaAsc, while empty symbols and dashed lines indicated slices dipped in water only (0% CaAsc). LSDs of significant effect (P = 0.05): LSDTimeHWTCaAsc dips = 0.16.
Fig. 6. Ascorbic acid content of apple slices that were cold water (4 C, 2 min) or hot water treated (48 C or 55 C, 2 min), then either dipped in 6% CaAsc or water. Slices were packaged in bags at ambient atmosphere, and stored up to 28 d at 4 C. Solid symbols and lines indicate slices were treated with 6% CaAsc, while empty symbols and dashed lines indicated slices dipped in water only (0% CaAsc). LSDs of significant effect (P = 0.05): LSDTimeCaAsc dips = 0.18. LSDHWTCaAsc dips = 0.14.
3.6. Phenolic compounds
Fig. 5. Antioxidant activity (FRAP as ascorbic acid) of apple slices that were cold water (4 C, 2 min) or hot water treated (48 C or 55 C, 2 min), then either dipped in 6% CaAsc or water. Slices were packaged in bags at ambient atmosphere, and stored up to 28 d at 4 C. Solid symbols and lines indicate slices were treated with 6% CaAsc, while empty symbols and dashed lines indicated slices dipped in water only (0% CaAsc). LSDs of significant effect (P = 0.05): LSDTimeHWTCaAsc dips = 0.21.
combined with dipping in 6% CaAsc was required. This treatment provided a remaining antioxidant activity of about 1.5 g kg 1 at day 21. 3.5. Ascorbic acid The initial AA content of untreated apple slices was of 0.25 g kg 1. Immediately after CaAsc treatment, AA content was 5–7-fold higher (1.3–1.9 g kg 1) than those not dipped into CaAsc (Fig. 6). HWT helped to increase the ascorbic acid absorption from CaAsc dips. These increases were an average of 34% using 48 C and about 25% with 55 C. The antioxidant activity measurement showed that HWT did not increase the retention of vitamin C when the treatment was not combined with CaAsc dips. The pattern of decrease with storage time followed the same trends as those of the FRAP and DPPH activities, indicating that these changes were the result of loss of AA content.
The total phenolic content in control (untreated) slices prior to storage was 0.61 g kg 1 (Table 6). During storage the total phenolics decreased about 20%, and ranged after 28 d from 449 to 516 mg kg 1. Interaction of time, heat treatment and CaAsc had a significant effect on the total phenolic changes although the response varied with the type of individual phenolic (Table 6). Chlorogenic and epicatechin levels only were significantly affected by time of storage. Chlorogenic decreased from day 14 and epicatechin suffered a sharp decline at day 7 remaining stable until the end of storage. Quercetin concentration increased using CaAsc. During the first 21 d, the use of HWT also increased quercetin levels although no differences were found at the end of storage when HWT was used. A treatment of 55 C for 2 min increased the phloridzin levels. In contrast HWT decreased p-coumaric and procyanidins (B1, B2, C1) levels. Dipping into CaAsc further reduced coumaric content although the procyanidins levels only reduced in apple slices not heated and the phloridzin content decreased in slices not heated or heated 48 C for 2 min.
4. Discussion 4.1. Shelf life This research has shown that use of a 6% calcium ascorbate dip in fresh-cut apples extended the shelf life from <7 d in untreated slices to 14 d when packaged in air and stored at 4 C. The factor that ultimately limited the shelf life was the sensory quality. The mechanism for the increased shelf life related to use of HWT treatments (48 C or 55 C, 2 min) before CaAsc dips which lead to increased absorption of ascorbic acid into the apple tissue, enhancing the ascorbic acid content and consequently the antioxidant activity as measured by DPPH and FRAP values. The HWT enabled further extension of the shelf life (appearance) from 14 d (samples not heated dipped in CaAsc) to 21 d. In general, there was no significant difference between HWT at 48 C and 55 C and therefore a HWT of 48 C for 2 min should be enough to achieve the benefit of extra shelf life.
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Table 6 Main phenolic compounds (mg kg 1) of apple slices that were cold water (4 C, 2 min) or hot water treated (HWT, 48 C or 55 C, 2 min), then either dipped in 0 or 6% CaAsc. Slices were packaged in bags at ambient atmosphere, and stored up to 28 d at 4 C. Storage time
HWT
CaAsc treatment
Chlorogenic
Quercetin derivatives
Epicatechin
Coumaric acid
Procyanidins (B1, B2, C1)
Phloridzin
Total phenol
0d
4 C
0% 6% 0% 6% 0% 6%
81.6a 81.1 85.9 81.8 89.3 81.9
10.2 12.0 15.4 17.0 16.3 18.6
51.5 49.5 52.7 51.1 47.4 51.4
80.1 77.8 69.4 68.9 75.2 70.7
194.3 192.1 185.1 182.2 181.7 185.6
176.6 160.9 174.0 155.5 175.5 160.3
610.7 573.4 589.1 526.1 585.3 562.4
0% 6% 0% 6% 0% 6%
80.1 78.0 78.2 80.9 83.8 82.9
13.1 14.0 17.3 21.0 18.9 20.3
42.5 39.2 40.3 40.0 41.9 42.3
66.1 66.3 67.0 70.2 68.3 70.4
188.2 163.6 167.1 170.7 168.5 179.4
119.7 122.5 122.7 119.0 127.3 134.5
486.7 466.6 476.1 478.7 508.7 518.1
0% 6% 0% 6% 0% 6%
79.0 78.0 77.5 80.2 73.6 82.5
11.0 12.2 18.1 18.3 15.9 21.0
40.4 38.4 37.8 40.5 40.0 40.4
69.3 68.5 68.9 65.6 64.9 71.3
179.2 147.4 156.2 163.3 161.7 161.0
121.6 116.0 114.5 120.9 118.6 126.6
472.5 458.4 458.6 482.8 470.2 482.9
0% 6% 0% 6% 0% 6%
81.2 80.1 76.9 80.5 73.5 79.7
16.1 15.3 15.4 18.0 18.3 18.9
40.0 37.3 38.3 40.3 38.8 39.0
72.8 65.2 68.4 61.9 64.0 64.3
161.5 151.6 152.8 157.3 155.8 153.2
118.8 108.2 110.9 108.5 119.6 120.6
534.2 441.7 454.5 481.6 466.8 472.6
0% 6% 0% 6% 0% 6%
81.5 79.2 77.7 80.2 70.8 80.1
15.3 16.1 19.9 22.1 15.3 16.5
41.2 38.5 38.5 38.2 35.0 41.2
70.2 62.4 63.2 53.5 65.6 60.9
165.5 158.6 145.6 155.1 152.1 151.5
111.9 100.3 108.3 105.6 114.2 119.4
507.6 476.3 477.6 469.4 449.4 516.2
(3.1) NS NS NS NS NS NS
(1.2) (0.9) (0.8) (2.1) NS NS NS
(1.4) NS NS NS NS (1.5) NS
(2.0) (1.6) (1.3) (3.5) (2.9) (2.2) NS
(5.3) (4.1) NS NS (5.8) NS NS
(5.4) (4.2) (3.4) NS (7.7) NS NS
(15.1) (11.7) NS NS (21.3) (16.5) (36.9)
48 C 55 C
7d
4 C
48 C 55 C
14 d
4 C
48 C 55 C
21 d
4 C
48 C 55 C
28 d
4 C
48 C 55 C
Time HWT % CaAsc Time * HWT Time * CaAsc HWT * CaAsc Time * HWT * CaAsc a
Values are means (n = 3); NS, not significant. LSD values are in brackets at P < 0.05. T: Time. HWT: Water treatment. CaAsc: Calcium ascorbate.
4.2. Colour The increases in L* and h values using 6% of CaAsc dips indicates that it is an effective way to reduce browning in accordance with prior research (Son et al., 2001; Rojas-Grau et al., 2006; Tortoe et al., 2007). AA reduces browning incidence by reducing o-quinones back to phenolic compounds prior to polymerization and subsequent formation of coloured pigments (McEvily et al., 1992). Additionally, a formulation containing calcium and ascorbic acid acts partly to prevent cell and membrane breakdown (Toivonen and Brummell, 2008) and consequently reduces the release of PPO or its substrates thus improving the colour preservation of fresh-cut product (Pérez-Cabrera et al., 2011). Others researchers have found that dipping treatments with Ca ascorbate and heat shock (60 C for 1 min) caused PPO inhibition and were less affected by discolouration (Barbagallo et al., 2012). In the present experiment, we did not measure the levels of PPO enzyme but the use of heat treatment only resulted in a slight reduction in L* and h values and increased chroma when 55 C HWT was used. The combination of HWT and CaAsc maintained the colour of fresh-cut apple with significantly less browning than with the HWT without CaAsc. Djioua et al. (2009) also found greater percentage of L* loss in mango slices when whole mangoes were subjected to HWT (46 C/75 min and 50 C/30 min). This
could indicate that 55 C for 2 min induces a superficial damage in the apple flesh thus slightly reducing the lightless and colour. For example, Glowacz et al. (2013) reported that HWT at 50 C for 120 s resulted in the greatest membrane damage (20%) compared to spinach leaves treated at 40 C up to 120 s, 45 C up to 60 s and 50 C for 30 s. In contrast to our results, Abreu et al. (2003) and Koukounaras et al. (2008) found that in fresh-cut pear and peaches, respectively, that mild heat pre-treatments were effective in avoiding the cut surface browning in fresh-cut fruits. No difference was observed in PPO activity for the control and for heated peach slices (Koukounaras et al., 2008). Glowacz et al. (2013) also found that a chroma increased in spinach leaves with increasing temperature of the treatments (40 C vs 45 C vs 50 C). The hue angle decreased with time of storage mainly because the effectiveness of ascorbic acid in anti-browning is temporary. The enzymatic browning can re-generate after the ascorbic acid from the CaAsc or from the proper apple tissue has been completely reduced to dehydroascorbic acid. 4.3. Sensory changes Appearance and overall acceptability was strongly improved by dipping in CaAsc solutions since slices not treated with CaAsc had a very short shelf life (<7 d). However, the best treatments were the
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combination of HWT (48 or 55 C) with CaAsc dips, which extended the shelf life from 14 d (slices not heated) up to 21 d (heated slices). The ultimate shelf life limitation was related to sensory quality more than microbiological growth. Texture and taste from apple slices was also improved with CaAsc dips. HWT had no significant effect on these changes. Dea et al. (2010) also found there was no effect of heat treatment on the firmness of mango slices when whole mango were immersed in hot water (46 C for 90 or 75 min). Softness and deformation of stored oranges neither were influenced by HWT treatments (52 C for 180 s and at 56 C for 20 s) (Strano et al., 2014). The calcium present in CaAsc salt may be the main effect influence on the retention of texture. However, many researchers have reported good results in maintaining or improving texture when a combination of HWT and calcium dipping is used (Aguayo et al., 2008; Silveira et al., 2011) generally explained in terms of pectin esterase (PE) activation, cleaving the methoxyl groups from methylated galacturonic acid residues in pectin (Belitz and Grosch, 1986) which contain newly available carboxyl groups. The effect of HWT on firmness maintenance during storage has been reported for fresh-cut apple, pear, and melon (Kim et al., 1993; Abreu et al., 2003; Silveira et al., 2011). In our experiment, HWT had no a negative effect on retention of texture (as measured subjectively) although HWT can inhibit the synthesis of cell wall hydrolytic enzymes in whole apple fruit (Lurie et al., 1998). The improvement on taste using CaAsc was linked to the browning reduction activity of ascorbate temporarily avoiding the oxidation of phenolic compounds and keeping the endogenous organic acid in apple slices at levels, which provide a good taste. In apple juice, Komthong et al. (2007) found that using AA increased the two aldehydes, hexanal and trans-2-hexenal about 4 and 5-fold obtaining a positive green odour of apple juice. All sensory parameters decreased with time of storage as consequence of the natural oxidative processes which are particularly high in fresh-cut products. This quality loss with increased storage time was reduced by the combination of HWTs and CaAsc dips. 4.4. Microbial counts Many authors have shown application of calcium to fresh-cut fruit also reduces microbial growth, due to calcium increasing the rigidity of the cell wall and resistance to microbial enzymes. CaAsc treatment, in combination with HWT, very efficiently reduced the microbial growth. However, the use of high temperature HWT could damage apple tissue and affect shelf life. Heat damage has been previously reported in lettuce using mild heat treatment (50 C, 2 min), spinach leaves using HWT upper than 45 C for 60 s or in whole apples using rinsing at 65 C for 20 s (Moreira et al., 2006; Maxin et al., 2012; Glowacz et al., 2013). 4.5. Antioxidant activity (DDPH, FRAP) and ascorbic acid content The use of CaAsc dips increased the AA content and consequently the antioxidant activity as measured by DPPH and FRAP. This response was increased when dips were combined with HWT. This increase is most likely due to the thermal effects of gas expansion in the hot water such that solutions are pulled into the tissue due to contraction of the gas space with the cooler calcium ascorbate dip treatment. Alternatively, high temperature could enhance the diffusion rate of ascorbic acid and increase the solubility of ascorbic in water and apple tissue. This fact has been demonstrated with calcium salt as CaCl2 where hot water treatment (60 C, 1 min) had a significantly higher internal and external free calcium concentration suggesting a possible enhanced diffusion at higher temperatures (Aguayo et al., 2008).
In samples with no added CaAsc, no significant difference in AA content or antioxidant activity changes was observed between heated and unheated apples slices. In support, others have found that HWT had no effect on AA content in fresh-cut lettuce (Murata et al., 2004), spinach (Gómez et al., 2008; Glowacz et al., 2013), mango slices (Dea et al., 2010), rocket leaves (Koukounaras et al., 2009) or fresh-cut onions (Siddiq et al., 2013). In this experiment, the AA content and antioxidant capacity decreased with time of storage particularly in treatments with highs level of ascorbic acid (HWT plus CaAsc dip). In previous work, Aguayo et al. (2010) found that apple slices with lower antioxidant activity and ascorbic acid levels seemed to be more proportionately stable than those with initial very high antioxidant activity. Kalt et al. (1999) showed that in vegetable cells most of the AA is located in the vacuole (a very low pH environment), together with phenolic flavonoids. As Cocci et al. (2006) reported, it is probable that exogenous AA in the dipped slices diffused into the apple tissue and not being located within vacuoles was therefore more exposed to oxygen and endogenous oxidases and thus more rapidly oxidized. Exogenous antioxidant treatments should be able to interfere with senescence-related oxidation reactions. Aguayo et al. (2010) found that there is an efficient level of antioxidant content which decreases or even neutralizes the active oxygen species (AOS), resulting in prevention of induced senescence and thus, extension of shelf life. The antioxidant levels, as measured by FRAP, were related to shelf life and it appears that a minimal antioxidant of about 2 g kg 1 may be needed level to maintain shelf life in ‘Braeburn’ apple slices. In the present experiment, the longest residual minimum level of antioxidant was obtained through the combination of hot water and 6% CaAsc. These apple slices, stored in air at 4 C, achieved the longest shelf life being scored above the limit of marketability at day 21. 4.6. Phenolic compounds Exogenous CaAsc dips had small but significant effects on the reduction of p-coumaric and procyanidins and phloridzin. The decrease could be a consequence of formation of a complex of ascorbic acid from CaAsc with natural products of coumaric acid (Kesinger and Stevens, 2009). The use of HWT increased the quercetin and phloridzin levels but decreased coumaric and procyanidins (B1, B2, C1) levels. HWT has been proposed to reduce browning in fresh-cut tissues because of the demonstrated effect on delaying the wound-induced production of PAL (Saltveit, 2000). In contrast, some researchers have reported that HWT enhanced some enzyme activities associated with biosynthesis of phenolic compounds in mandarins (Ghasemnezhad et al., 2008), muskmelon fruit (Yuan et al., 2013) or peach (Spadoni et al., 2014). In our experiment, the use of HWT did not provide a same response in all the phenolic compounds. This could be attributed to differences in the metabolism of different phenolic compounds by the apple cells. Heat could have enhanced the phenolic extraction in apple slices. Gerard and Roberts (2004) found that increasing temperature during pressing from 40 C to 70 C allows increasing flavonoid content (50%) in apple juice. However, some phenolics such as coumaric and procyanidin show lower stability (or higher susceptibility) to thermal degradation, autoxidation or breakdown (Ioannou et al., 2012), whilst others (such as quercetin) are more stable upon heating (Odriozola-Serrano et al., 2008). In general, heat treatments are effective at stopping or at least delaying “active” physiological processes (Woolf et al., 2012). However, in our experiment, the beneficial effects of HWTs was most likely due to the physical thermal effects of moving from high to low temperature such that solutions are pulled into the tissue due to gas space contraction with the cooler calcium ascorbate dip
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treatment. HWTs could enhance diffusion rate of ascorbic acid and increase the solubility of ascorbic in water and apple tissue increasing vitamin C, antioxidant capacity, improving appearance and extending the shelf life based on overall acceptability. 5. Conclusions CaAsc dips (6%) had a strong impact reducing the browning and therefore, extending the overall acceptability to 14 d. This treatment increased the antioxidant content 5 fold which was further increased with the combination of HWT that resulted in an increased absorption of ascorbic acid in the apple tissue. The combination of treatments helped to maintain fresh-cut apple quality, in particular, the sensory taste and aroma extending the shelf life from 14 d to up to 21 d at 4 C. HWT for 2 min at 48 C was preferable to 55 C with no significant benefit of the higher temperature that would also have increased energy cost and could induce a superficial damage in the apple flesh. Acknowledgements Encarna Aguayo thanks the Technical University of Cartagena for supporting her sabbatical stay at the New Zealand Institute for Plant & Food Research (Auckland). Thanks to Reginald Wibisono for his valuable assistance in the laboratory, and David Stevenson and Janine Cooney for their knowledge on the phenolic compound identification. References Abreu, M., Beirao-da-Costa, S., Goncalves, E.M., Beirao-da-Costa, M.L., MoldaoMartins, M., 2003. Use of mild heat pre-treatments for quality retention of fresh-cut ‘Rocha’ pear. Postharvest Biol. Technol. 30, 153–160. Aguayo, E., Escalona, V.H., Artés, F., 2008. Effect of hot water treatment and various calcium salts on quality of fresh-cut ‘Amarillo’ melon. Postharvest Biol. Technol. 47, 397–406. Aguayo, E., Requejo-Jackman, C., Stanley, R., Woolf, R., 2010. Effects of calcium ascorbate treatments and storage atmosphere on antioxidant activity and quality of fresh-cut apple slices. Postharvest Biol. Technol. 57, 52–60. Barbagallo, R.N., Chisari, M., Caputa, G., 2012. Effects of calcium citrate and ascorbate as inhibitors of browning and softening in minimally processed ‘Birgah’ eggplants. Postharvest Biol. Technol. 73, 107–114. Belitz, H.D., Grosch, W., 1986. Enzymes, Food Chemistry. second ed. Springer-Verlag, New-York, pp. 155–201. Boyer, J., Liu, R.H., 2004. Apple phytochemicals and their health benefits. Nutr. J. 3, 1–15. Cocci, E., Rocculi, P., Romani, S., Rosa, M.D., 2006. Changes in nutritional properties of minimally processed apples during storage. Postharvest Biol. Technol. 39, 265–271. Dea, S., Brecht, J.K., Nunes, M.C.N., Baldwin, E.A., 2010. Quality of fresh-cut ‘Kent’ mango slices prepared from hot water or non-hot water-treated fruit. Postharvest Biol. Technol 56, 171–180. Djioua, T., Charles, F., Lopez-Lauri, F., Filgueiras, H., Coudret, A., Freire, M., DucampCollind, M.N., Sallanon, H., 2009. Improving the storage of minimally processed mangoes (Mangifera indica L.) by hot water treatments. Postharvest Biol. Technol. 52, 221–226. FAOSTAT, 2011. http://faostat.fao.org/site/339/default.aspx. Gerard, K.A., Roberts, J.S., 2004. Microwave heating of apple mash to improve juice yield and quality. Lebensm–Wiss Technol. 37, 551–557. Ghasemnezhad, M., Marsh, K., Shilton, R., Babalar, M., Woolf, A., 2008. Effect of hot water treatments on chilling injury and heat damage in ‘satsuma’ mandarins: antioxidant enzymes and vacuolar ATPase, and pyrophosphatase. Postharvest Biol. Technol. 48, 364–371. Gómez, F., Fernandez, L., Gergoff, G., Guiamet, J.J., Chaves, A., Bartoli, C.G., 2008. Heat shock increases mitochondrial H2O2 production and extends postharvest life of spinach leaves. Postharvest Biol. Technol. 49, 229–234. Glowacz, M., Mogren, L.M., Reade, J.P.H., Cobb, A.H., Monaghan, J.M., 2013. Can hot water treatments enhance or maintain postharvest quality of spinach leaves. Postharvest Biol. Technol. 81, 23–28.
165
Ioannou, I., Hafsa, I., Hamdi, S., Charbonnel, C., Ghoul, M., 2012. Review of the effects of food processing and formulation on flavonol and anthocyanin behavior. J. Food Eng. 111, 208–217. Kalt, W., Forney, C.F., Martin, A., Prior, R.L., 1999. Antioxidant capacity, vitamin C, phenolics, and anthocyanins after fresh storage of small fruits. J. Agric. Food Chem. 47, 4638–4644. Kesinger, N.G., Stevens, J.F., 2009. Covalent interaction of ascorbic acid with natural products. Phytochemistry 70, 1930–1939. Kim, D.M., Smith, N.L., Lee, C.Y., 1993. Apple cultivar variations in response to heat treatment and minimal processing. J. Food Sci. 58, 1111–1124. Komthong, P., Igura, N., Shimoda, M., 2007. Effect of ascorbic acid on the odours of cloudy apple juice. Food Chem. 100, 1342–1349. Koukounaras, A., Siomos, A.S., Sfakiotakis, E., 2009. Impact of heat treatment on ethylene production and yellowing of modified atmosphere packaged rocket leaves. Postharvest Biol. Technol. 54, 172–176. Koukounaras, A., Siomos, A.S., Sfakiotakis, E., 2008. The effect of heat treatment on quality retention of fresh-cut peach. Postharvest Biol. Technol. 48, 30–36. Kwak, E.J., Lim, S.I., 2005. Inhibition of browning by antibrowning agents and phenolic acids or cinnamic acid in the glucose–lysine model. J. Sci. Food Agr. 85, 1337–1342. Lurie, S., Fallik, E., Klein, J.D., Kozar, E., Kovacs, K., 1998. Postharvest heat treatment of apples to control San Jose scale (Quadraspidotiotus perniciosus Comstock) and blue mold (Penicillium expansum Link) and maintain fruit firmness. J. Am. Soc. Hortic. Sci. 123, 110–114. Maxin, P., Weber, R.W.S., Pedersen, H.L., Williams, M., 2012. Control of a wide range of storage rots in naturally infected apples by hot-water dipping and rinsing. Postharvest Biol. Technol. 70, 25–31. McEvily, A.J., Iyengar, R., Otwell, W.S., 1992. Inhibition of enzymatic browning in foods and beverages. Food Sci. Nutr. 32, 253–273. Moreira, M.D.R., Ponce, A.G., Del Valle, C.E., Roura, S.I., 2006. Ascorbic acid retention, microbial growth, and sensory acceptability of lettuce leaves subjected to mild heat shocks. J. Food Sci. 71, 188–192. Murata, M., Tanaka, E., Minoura, E., Homma, S., 2004. Quality of cut lettuce treated by heat shock: prevention of enzymatic browning, repression of phenylalanine ammonia–lyase activity, and improvement on sensory evaluation during storage. Biosci. Biotechnol. Biochem. 68, 501–507. Odriozola-Serrano, I., Soliva-Fortuny, R., Martin-Belloso, O., 2008. Phenolic acids, flavonoids, vitamin C and antioxidant capacity of strawberry juices processed by high-intensity pulsed electric fields or heat treatments. Eur. Food Res. Technol. 228, 239–248. Pérez-Cabrera, L., Chafer, M., Chiralt, A., González-Martínez, C., 2011. Effectiveness of antibrowning agents applied by vacuum impregnation on minimally processed pear. LWT, Food Sci. Technol. 44, 2273–2280. Rassam, M., Laing, W., 2005. Variation in AA and oxalate levels in the fruit of Actinidia chinensis tissues and genotypes. J. Agric. Food Chem. 53, 2322–2326. Rojas-Grau, M.A., Sobrino López, A., Tapia, M.S., Martin Belloso, O., 2006. Browning inhibition in fresh-cut Fuji apple slices by natural antibrowning agents. J. Food Sci. 71, 559–565. Saltveit, M.E., 2000. Wound induced changes in phenolic metabolism and tissue browning are altered by heat shock. Postharvest Biol. Technol. 21, 61–69. Siddiq, M., Roidoung, S., Sogi, D.S., Dolan, K.D., 2013. Total phenolics, antioxidant properties and quality of fresh-cut onions (Allium cepa L.) treated with mildheat. Food Chem. 136, 803–806. Silveira, A.C., Aguayo, E., Chisari, M., Artés, F., 2011. Calcium salts and heat treatment for quality retention of fresh-cut ‘Galia’ melon. Postharvest Biol. Technol. 62, 77–84. Son, S.M., Moon, L.D., Lee, C.Y., 2001. Inhibitory effects of various antibrowning agents on apples slices. Food Chem. 73, 23–30. Spadoni, A., Guidarelli, M., Sanzani, S.M., Ippolito, A., Mari, M., 2014. Influence of hot water treatment on brown rot of peach and rapid fruit response to heat stress. Postharvest Biol. Technol. 94, 66–73. Strano, M.C., Calandra, M., Aloisi, V., Rapisarda, P., Strano, T., Ruberto, G., 2014. Hot water dipping treatments on Tarocco orange fruit and their effects on peel essential oil. Postharvest Biol. Technol. 94, 26–34. Toivonen, P.M.A., 2004. Postharvest storage procedures and oxidative stress. HortScience 39, 938–942. Toivonen, P.M.A., Brummell, D.A., 2008. Biochemical bases of appearance and texture changes in fresh-cut fruit and vegetables. Postharvest Biol. Technol. 48, 1–14. Tortoe, C., Orchard, J., Beezer, A., 2007. Prevention of enzymatic browning of apple cylinders using different solutions. Intern. J. Food Sci. Technol. 42, 1475–1481. Whitaker, J., 1972. Principles of Enzymology for the Food Sciences. M. Dekker, New York, pp. 571–577. Woolf, A.B., Combes, S., Petley, M., Olsson, S.R., Wohlers, M., Jackman, R.C., 2012. Hot water treatments reduce leaf yellowing and extend vase life of Asiatic hybrid lilies. Postharvest Biol. Technol. 64, 9–18. Yuan, L., Bi, Y., Ge, Y., Wang, Y., Liu, Y., Li, G., 2013. Postharvest hot water dipping reduces decay by inducing disease resistance and maintaining firmness in muskmelon (Cucumis melo L.) fruit. Sci. Hortic. 161, 101–110.