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Fruit maturity affects the response of apples to heat stress
Postharvest Biology and Technology 62 (2011) 35–42
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Fruit maturity affects the response of apples to heat stress Lihua Fan ∗ , Jun Song, Charles F. Forney, Michael A. Jordan Agriculture and Agri-Food Canada, Atlantic Food and Horticulture Research Centre, 32 Main Street, Kentville, Nova Scotia B4N 1J5, Canada
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Article history: Received 23 November 2010 Accepted 18 April 2011 Keywords: Malus sylvestris var. domestica Apple maturity Heat stress Chlorophyll fluorescence Apple quality
a b s t r a c t Quality changes of apple fruit at different maturity stages in response to heat stress were investigated. ‘Jonagold’ and ‘Cortland’ apples at immature (pre-climacteric), commercial harvest maturity (CHM) and post climacteric maturity (PCM, CHM plus 4 weeks) were harvested and held at 46 ◦ C for 0, 4, 8, or 12 h. Following treatments, fruits were stored in air at 0 ◦ C and evaluated after 0, 1, 2, or 3 months. Quality indices including peel and flesh browning, firmness, titratable acidity, soluble solids, chlorophyll fluorescence (CF), and ethanol production were measured. Results indicated that different cultivars and maturities affected the fruit’s resistance to heat stress. ‘Jonagold’ was more resistant to heat stress than ‘Cortland’. Fruit at PCM were most sensitive to heat stress, followed by fruits at CHM and immature stages. When ‘Jonagold’ apples at immature and CHM stages were held at 46 ◦ C for 12 h and then stored for 3 months, flesh browning ratings were negligible compared with 1.4 or 2.9, respectively in ‘Cortland’. Flesh browning rating increased to 1.4 or 4.5 in PCM ‘Jonagold’ held at 46 ◦ C for 8 or 12 h and then stored for 3 months while it was 4.9 or 5.0, respectively, in ‘Cortland’. Heat treatment-induced flesh injury was associated with a decrease in CF. After fruit were exposure to 46 ◦ C for 12 h and then stored for 3 months, Fv/Fm was reduced by 13%, 30%, and 55% in ‘Jonagold’ at immature maturity, CHM and PCM, respectively, while it was reduced by 51%, 58% and 75%, respectively, in ‘Cortland’. Heat stress also caused a decrease in fruit titratable acidity, but had no effect on soluble solids contents. The 8 or 12 h heat treatment resulted in an increase in ethanol production, which was greatest in PCM apples. © 2011 Published by Elsevier B.V.
1. Introduction Heat treatments are being actively pursued for postharvest treatments of fresh produce. Some beneficial responses of commodities to heat treatments include the slowing of ripening of climacteric fruit and vegetables, sweetening of commodities either by increasing sugars or decreasing acidity, and prevention of storage disorders such as superficial scald on apples and chilling injury on subtropical fruits and vegetables (Lurie, 1998). Although many reports focus on the positive response of horticultural commodities to heat treatment, high temperatures with long durations may cause tissue damage and increase decay development (Klein and Lurie, 1992; Song et al., 2001; Fan et al., 2005). It is often challenging to find a time–temperature regime that will produce the desired effect of disease control or quality improvement without risk of damaging the fruit or vegetable. In our previous studies on apples, we found that heat stress at 46 ◦ C for 8 and 12 h could result in reduced fruit storage life and quality (Fan et al., 2005). Heat damage on apples can be both external and internal, appearing as peel and flesh browning. In mild to moderate heat injury, visual symptoms are often not immediately apparent, but injury develops during
∗ Corresponding author. Tel.: +1 902 6795550; fax: +1 902 6792311. E-mail address: [email protected] (L. Fan). 0925-5214/$ – see front matter © 2011 Published by Elsevier B.V. doi:10.1016/j.postharvbio.2011.04.007
subsequent storage (Fan et al., 2005). Using nondestructive indicators such as chlorophyll fluorescence (CF) or volatile emissions to identify stressed fruit and determine their potential to breakdown during storage has been reported (DeEll et al., 1995; Fan et al., 2005; Forney et al., 2000; Woolf and Laing, 1996; Song et al., 2001). However, questions remain as to how fruit maturity may affect the response of fruit to heat stress. Beaudry et al. (1993) reported that very immature fruit are unable to develop acceptable quality after harvest including the development of adequate sugars or flavor and are prone to shrivel and excessive weight loss. In contrast, over mature fruits are prone to excessive ripening and are more susceptible to bruising, flesh browning, senescent breakdown, and decay during storage. Apparently, the stage of fruit development at harvest has a significant impact on the potential quality of the stored product. So far, there have been limited reports regarding the impact of maturity on the fruit’s response to heat treatments. In order to increase our understanding of physiological responses induced by heat stress, fruit maturity and cultivar difference need to be considered. Therefore, the objectives of this study were to investigate the physiological changes of two popular apple cultivars ‘Cortland’ and ‘Jonagold’ at different maturity stages in response to heat stress by characterizing changes in CF and ethanol production as well as various fruit quality parameters; to assess the usefulness of CF and ethanol production induced by heat treatment to predict fruit flesh browning (injury) development during cold storage.
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2. Materials and methods 2.1. Apple fruit and maturity assessment Apple fruit of cultivars Cortland and Jonagold that were grown at the Atlantic Food and Horticulture Research Centre in the Annapolis Valley, Nova Scotia, Canada, were harvested at three maturities: immature, commercial harvest maturity (CHM) and post climacteric maturity (PCM, CHM plus one month). The harvest dates for ‘Cortland’ were September 22nd, October 8th and November 9th, respectively, and for ‘Jonagold’ were September 24th, October 15th and November 14th. Fruit maturity was determined based on the internal ethylene concentration and local commercial harvest dates (Lau, 1985). The internal ethylene concentration was determined to be <0.01 L L−1 for immature fruit and about 0.2 L L−1 for CHM apples. Internal ethylene concentration was determined on 10 apples. One milliliter samples of internal gas was drawn into a syringe through a needle inserted into the core cavity of each fruit and were then injected into a gas chromatograph with a flame ionization detector (Carle Instruments, Anaheim, CA) equipped with a 1.9 m × 3.2 mm (o.d.) activated alumina column with a helium carrier flow of 0.83 mL s−1 . Internal ethylene concentration was quantified using a known concentration of standard ethylene. 2.2. Heat treatment Heat treatments were conducted by placing fruit in a 46 ◦ C chamber with about 90% relative humidity for 0, 4, 8, or 12 h. The treatment times were chosen to induce varying degrees of heat stress. The 0 h fruit were used as the control. Temperature was monitored for treated fruit by placing thermocouples in the cortex of selected apples. Following heat treatment, fruit were stored in air at 0 ◦ C and evaluated after 0, 1, 2, or 3 months plus an additional 1 or 7 d at 20 ◦ C.
peak areas were normalized using the peak area of a 4 ng dodecane standard that was run on the same day of the analysis to account for any variability in detector response. 2.4. Fruit quality Samples of 10 apples of each cultivar at different maturity–heat treatment combinations were assessed at each removal. Quality was evaluated following the procedures described by Fan et al. (2005). Briefly, fruit injury was rated as peel or flesh browning using a scale of 0–5, where 0 = none, 1 = up to 20%, 2 = 21–40%, 3 = 41–60%, 4 = 61–80%, and 5 = 81–100% of the fruit peel or flesh area affected. Fruit firmness was determined using a Magness–Taylor firmness tester (type 30 lb, model 30A; Ballauf Manufacturing Co., Laurel, MD). Titratable acidity was measured with a 2 mL juice sample collected from 10 apples, and 0.1 mol L−1 NaOH was used to titrate the apple juice using a semi-automatic titrator (Multi-Dosimat E-415 titrator; Metrohm AG, Switzerland) to a phenolphthalein endpoint of pH 8.1. Titratable acidity was expressed as malic acid equivalents. Soluble solids were determined with a hand-held temperaturecompensated refractometer (Atago Co., Tokyo). 2.5. Statistical analysis The experimental design was a completely randomized 2 × 3 × 4 × 4 factorial with two cultivars (‘Jonagold’ and ‘Cortland’), three fruit maturities (immature, CHM, or PCM), four heat treatment durations (0, 4, 8, or 12 h), and four storage periods (0, 1, 2, or 3 months). For each combination, three sub-samples of four apples were used for ethanol analysis, and 10 apples were used for quality parameters and CF measurement. Data were analyzed using ANOVA, LSD, and correlation options of Genstat 5 (Genstat 5 Committee, 1993). 3. Results
2.3. Physiological responses
3.1. Chlorophyll fluorescence (Fv/Fm)
2.3.1. Chlorophyll fluorescence Chlorophyll fluorescence was determined using a modulated fluorometer (model OS-500; Opti-Science, Tyngsboro, MA) following the method of Fan et al. (2005). Three readings of Fv/Fm from each fruit were recorded and averaged, where Fo = minimal fluorescence, Fm = maximal fluorescence, and Fv = Fm − Fo. The ratio of Fv/Fm, a measure of the quantum efficiency of photosystem II was used to express the change of CF in this study.
Heat treatments of 12 h at 46 ◦ C reduced CF (Fv/Fm) substantially at all harvest maturities and in both cultivars (Fig. 1). Treatments of 8 h reduced CF at all maturities in ‘Cortland’ apples and in PCM ‘Jonagold’ fruit. The 4 h treatment had no effect on CF. CF declined during the 3 month storage period at 0 ◦ C in all fruit. CF also decreased with increasing fruit maturity. Immature fruit had the highest CF while PCM fruit had the lowest CF following heat treatment and storage. Heat treatments at 46 ◦ C for 12 h reduced Fv/Fm of immature, CHM, and PCM fruit by 51%, 58% and 75%, respectively, in ‘Cortland’ and 13%, 30%, and 55%, respectively, in ‘Jonagold’ compared to control fruit following 3 months of storage (Fig. 1). When CF was measured after 7 d at 20 ◦ C following removal from storage, Fv/Fm averaged 12% less than measurements taken 1 d after removal from storage. However, when CF was measured 7 d after the 8 and 12 h heat treatments, CF was 8% and 35% greater than that measured 1 d after treatment, respectively, suggesting some recovery from the heat stress occurred.
2.3.2. Ethanol analysis Three sub-samples of four apples each from each cultivar at different maturity–heat treatment combinations were placed in 4-L glass jars, sealed with Teflon lids, and held at 20 ◦ C. The headspace over the apples was allowed to equilibrate for 1 h under a 1.67 mL s−1 flow of purified air. A 100 mL head space sample in the jar was then collected onto a 100 mm × 6.4 mm (o.d.) glass collection tube containing 120 mg of Tenax GR 20/35 (Alltech Associates, Inc., Deerfield, IL) and 50 mg of carbosieve SIII 60/80 (Supelco Inc., Bellefonte, PA). The tubes were then stored at −80 ◦ C until analysis. Samples were analyzed on a Magnum gas chromatograph–mass spectrometer (GC–MS) system (Finnigan MAT, San Jose, CA) equipped with an LSC 2000 purge and trap concentrator and Aerotrap 6016 autosampler (Tekmar, Cincinnati) following the methods described by Forney and Jordan (1998). Mass spectra were acquired in electron ionization mode using automatic gain control within the range of m/z 30–400. Quantification of ethanol was done using the 45 m/z ion and external standards. All
3.2. Ethanol production The production of ethanol by PCM ‘Cortland’ and ‘Jonagold’ fruit was greatest in fruit held for 8 or 12 h at 46 ◦ C (Fig. 2). However, immature and CHM fruit did not show a significant increase in ethanol concentration as a result of heat treatment. In ‘Cortland’ fruit, ethanol concentration increased during storage at 0 ◦ C in both control and heat treated fruit. After 3 months of storage,
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ethanol concentrations in ‘Cortland’ fruit averaged 7.5-, 2.9-, and 7.8-fold greater than initial values in immature, CHM and PCM fruit, respectively. Storage had no significant effect on the ethanol concentration in ‘Jonagold’ fruit.
3.3. Flesh browning Severity of flesh browning increased in PCM ‘Cortland’ and ‘Jonagold’ apples exposed to 46 ◦ C for 8 or 12 h following 3 months of storage (Fig. 3). Flesh browning ratings increased to 4.9 or 5.0 following 3 months of storage in PCM ‘Cortland’ apples exposed to 46 ◦ C for 8 or 12 h, respectively, while it was 1.4 or 4.5, respectively, in ‘Jonagold’. Flesh browning of apple fruit was not significantly affected by the 4 h heat treatment regardless of cultivar or maturity stage. Overall, PCM apples were most sensitive to heat stress, followed by CHM and immature fruit. ‘Jonagold’ apples were more resistant to heat stress than ‘Cortland’. When ‘Jonagold’ apples at the immature and CHM stages were exposed to 46 ◦ C for 12 h and then stored for 3 months, flesh browning ratings were negligible compared with 1.4 and 2.9, respectively in ‘Cortland’ (Fig. 3).
3.4. Peel browning Heat treatments of 8 and 12 h caused peel browning in the PCM fruit (Fig. 4). After 3 months of storage, ratings averaged 3.1 and 4.4 in ‘Cortland’ and 1.4 and 4.2 in ‘Jonagold’ apples subjected to the 8 and 12 h heat treatments, respectively. The 4 h heat treatment had no effect on inducing peel browning. As well, no significant peel browning was induced by any of the heat treatments in the immature or CHM apples. In the PCM fruit, the development of peel browning was more apparent in ‘Jonagold’ fruit after 1 month of storage, but after 3 months severity was slightly greater in ‘Cortland’ fruit.
3.5. Fruit firmness Exposure of immature and CHM apple fruit to 46 ◦ C for 8 or 12 h maintained fruit firmness above that of the untreated controls during storage (Fig. 5). However, this maintenance of firmness was not observed in heat-treated PCM apples, and after 3 months of storage 12 h treated fruit were softer than controls. Fruit firmness declined with later harvest date and during storage at 0 ◦ C. Immature and
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CHM apples averaged 44% and 31% firmer than PCM apples during the 3 month storage period and fruit firmness declined an average of about 30% during storage (Fig. 5).
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Heat treatment (h) Fig. 2. Ethanol production of ‘Cortland’ and ‘Jonagold’ apples harvested at immature maturity, commercial harvest maturity (CHM) and post climacteric maturity (PCM) and exposed to 46 ◦ C for 0, 4, 8, or 12 h. Values are averages of measurements from fruit stored for 0, 1, 2, or 3 months plus 1 or 7 d at 20 ◦ C. The vertical bars represent the LSD at P < 0.05 for comparison of means within the figure.
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Heat treatments can have a variety of effects on fresh apple fruit depending on temperature and duration of exposure. In our study exposure of fruit to 46 ◦ C for 4 h had little effect on fruit quality, but exposures of 8 or 12 h were effective in lowering fruit acidity and reducing softening of the fruit during storage. Similar results were found in our previous study (Fan et al., 2005). Porritt and Lidster (1978) and Klein and Lurie (1990) also reported that TA declines while soluble solids concentration in apples is unaffected by heat treatment, and apples held at 38 ◦ C for 3 or 4 d were firmer than untreated fruit even after 6 months of storage at 0 ◦ C and subsequent shelf life at 20 ◦ C. However, heat treatments at 46 ◦ C for 8
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Storage time (month) Fig. 3. Flesh browning rating of ‘Cortland’ and ‘Jonagold’ apples harvested at immature maturity, commercial harvest maturity (CHM) and post climacteric maturity (PCM) and exposed to 46 ◦ C for 0, 4, 8, or 12 h following 0, 1, 2, or 3 months of storage at 0 ◦ C. Values are averages of measurements taken after 1 or 7 d at 20 ◦ C following removal from storage. Flesh browning was rated using a scale of 0–5, where 0 = none, 1 = up to 20%, 2 = 21–40%, 3 = 41–60%, 4 = 61–80%, and 5 = 81–100% of the fruit flesh area affected. The vertical bar represents the LSD at P < 0.05 for comparison of means within the figure.
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Storage time (month) Fig. 4. Peel browning rating of ‘Cortland’ and ‘Jonagold’ apples harvested at immature maturity, commercial harvest maturity (CHM) and post climacteric maturity (PCM) and exposed to 46 ◦ C for 0, 4, 8, or 12 h following 0, 1, 2, or 3 months of storage at 0 ◦ C. Values are averages of measurements taken after 1 or 7 d at 20 ◦ C following removal from storage. Peel browning was rated using a scale of 0–5, where 0 = none, 1 = up to 20%, 2 = 21–40%, 3 = 41–60%, 4 = 61–80%, and 5 = 81–100% of the fruit peel area affected. The vertical bar represents the LSD at P < 0.05 for comparison of means within the figure.
or 12 h caused flesh and peel browning in some fruit. This browning was not apparent 1 week after treatment, but rather developed during storage for 3 months at 0 ◦ C. The variable response to heat treatments among fruit appears to be dependent on numerous factors including fruit maturity and cultivar. Fruit maturity has a strong effect on fruit quality, storage life, and tolerance of fruit to heat treatments. As fruit increase in maturity, respiration, ethylene, aroma volatiles, soluble solids, and color increase, while acidity, firmness and chlorophyll content decrease (Beaudry et al., 1993). Immature fruit never achieve good eating quality, lacking sweetness and aroma, while over-mature fruit are soft, subject to bruising and more prone to breakdown during storage. Watkins et al. (2005) reported that development of disorders such as soggy breakdown and soft scald in ‘Honeycrisp’ apples is associated with later harvest dates and storage temperature. Fruit harvested in this study decreased in firmness and titratable acidity when harvest date was delayed. In addition, we found that as fruit became over mature (PCM), its tolerance to heat declined resulting in the most severe flesh and skin browning that developed during storage. These fruit were severely injured with the 8 h treatment, which was well tolerated in the immature and CHM fruit. The PCM fruit would be considered post climacteric, which is characterized by advanced senescence and loss of flesh firmness, which appears to influence the fruit’s tolerance to heat stress. To ensure a long-term storage potential of apples, it is essential that the fruit are harvested within a well defined optimal harvest period. The stage of fruit development at harvest has a significant impact on the potential quality of the stored product (Beaudry et al.,
1993). Although immature fruit are more tolerant to severe heat treatments than ripe or over ripe fruit, they have not ripened sufficiently upon removal from storage resulting in inferior organoleptic quality. Conversely, over ripe fruit become soft and mealy during storage. In this study, over ripe fruit were also most sensitive to moderate and severe heat treatments (8–12 h at 46 ◦ C) as shown by fruit tissue injury, reduced Fv/Fm and increased ethanol production. Cultivar also may have had an effect on the tolerance of fruit to heat treatments. ‘Cortland’ fruit appeared to be less tolerant than ‘Jonagold’ in this study based on flesh browning rates. Immature and CHM fruit developed flesh browning with the 12 h treatment in ‘Cortland’ but not ‘Jonagold’. Song et al. (2001) reported ‘Cortland’ and ‘Jonagold’ to be more tolerant to heat treatments than ‘McIntosh’ and ‘Northern Spy’, but that ‘Jonagold’ was the most tolerant. Fan et al. (2005) also found that flesh browning was more severe in ‘Cortland’ fruit when compared to those of ‘Jonagold’ subjected to the same heat treatments. Although untreated ‘Cortland’ fruit showed no injury during storage at 0 ◦ C, it is a chilling sensitive cultivar and the chilling stress of storage may have enhanced heat injury symptoms as suggested by Fan et al. (2005). Kim et al. (1993) compared the tolerance of eleven cultivars including ‘Cortland’ to hot water dips of 40–50 ◦ C for up to 4 h. They found the heat-stress tolerance of ‘Cortland’ to be intermediate, with ‘Rome’, ‘Monroe’ and ‘Liberty’ being very susceptive and ‘Delicious’ and ‘Golden Delicious’ being most tolerant. While apple cultivars appear to differ in their tolerance to heat, the degree of tolerance is influenced by other factors such as maturity and growing season. Tomato cultivars
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Storage time (month) Fig. 5. Firmness of ‘Cortland’ and ‘Jonagold’ apples harvested at immature maturity, commercial harvest maturity (CHM) and post climacteric maturity (PCM) and exposed to 46 ◦ C for 0, 4, 8, or 12 h following 0, 1, 2, or 3 months of storage at 0 ◦ C. Values are averages of measurements taken after 1 or 7 d at 20 ◦ C following removal from storage. The vertical bar represents the LSD at P < 0.05 for comparison of means within the figure.
Fig. 6. Titratable acidity in malic acid equivalents of juice extracted from ‘Cortland’ and ‘Jonagold’ apples harvested at immature maturity, commercial harvest maturity (CHM) and post climacteric maturity (PCM) and exposed to 46 ◦ C for 0, 4, 8, or 12 h. Values are averages of measurements from fruit stored for 0, 1, 2, or 3 months at 0 ◦ C plus 1 or 7 d at 20 ◦ C.
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Fig. 7. Chlorophyll fluorescence (Fv/Fm) values of ‘Cortland’ and ‘Jonagold’ apples harvested at immature maturity, commercial harvest maturity (CHM) and post climacteric maturity (PCM), exposed to 46 ◦ C for 0, 4, 8, or 12 h and stored for 0, 1, 2, or 3 months at 0 ◦ C plotted against flesh browning ratings. The correlation coefficient (rs ) represents that calculated using Spearman’s rank correlation.
also appeared to respond differently to heat treatments. Lurie and Sabehat (1997) reported that a heat treatment at 38 ◦ C reduced low temperature sensitivity of mature green ‘Daniella’ tomato fruit, and they could be stored for up to a month at 2 ◦ C without developing chilling injury. However, Whitaker (1994) found that heat treatments at 38 ◦ C had no benefit in ‘Rutgers’ tomato fruit suggesting that the response of fruit to heat treatment may be cultivar specific. Chlorophyll fluorescence, a sensitive indicator of the state of the photosynthetic apparatus, is often used as a nondestructive probe of heat damage (Schreiber and Berry, 1977). It is a useful indicator of several postharvest stresses in apples including exposure to low O2 or high CO2 concentrations (DeEll et al., 1995) and freezing injury (Forney et al., 2000). When apple fruit are stressed by heat, chloroplast integrity may decline resulting in a decline in Fv/Fm (Fan et al., 2005). In this study, we found that moderate and severe heat treatments reduced Fv/Fm values and they never recovered to control values indicating that the apple fruit were damaged irreversibly. This study also confirmed our previous findings that flesh browning reflected the physiological injury caused by heat stress. The severity of flesh browning over the storage period correlated with the decrease in CF (Fig. 7). Ethanol is the primary product of fermentation, which occurs when oxidative phosphorylation is disrupted, preventing the oxidation of pyruvate by the Krebs Cycle. Since oxidative phosphorylation involves electron transport through a complex of membrane bound proteins in the mitochondria, disruption of these membranes could inhibit this process. Any stress that can disrupt normal mitochondrial membrane function could induce fermentation in the plant tissue. Ethanol production by stressed plant tissues
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Fig. 8. Headspace ethanol concentrations of ‘Cortland’ and ‘Jonagold’ apples harvested at immature maturity, commercial harvest maturity (CHM) and post climacteric maturity (PCM), exposed to 46 ◦ C for 0, 4, 8, or 12 h and stored for 0, 1, 2, or 3 months at 0 ◦ C plotted against flesh browning ratings. The correlation coefficient (rs ) represents that calculated using Spearman’s rank correlation.
has been observed in previous studies (Fan et al., 2005; Kimmerer and Kozlowski, 1982). In this study, heat treatment only increased ethanol production substantially in PCM fruit, suggesting that the advanced stage of senescence may render the mitochondrial membranes more susceptible to disruption. In addition, high respiration rates associated with PCM fruit and heat stress may deplete available oxygen and induce fermentation. ‘Jonagold’ fruit showed a stronger relationship of flesh browning with ethanol production than ‘Cortland’ fruit (Fig. 8). Overmature ‘Cortland’ fruit tend to produce ethanol regardless of heat treatment, while ethanol production in ‘Jonagold’ was more strongly related to heat-induced flesh browning. Both changes in CF and in ethanol content of apple fruit have been correlated to injury induced by both heat (Fan et al., 2005; Song et al., 2001) and freezing (Forney et al., 2000) stress. Fan et al. (2005) suggested that an Fv/Fm value of <0.6 measured 1 d after heat treatment was predictive of the development of flesh browning during storage. A similar relationship was observed in this study (Fig. 7). Both ‘Cortland’ and ‘Jonagold’ apples that had Fv/Fm values of <0.6 tended to develop flesh browning ratings >1 and was most apparent after 3 months of storage. However, a large number of fruit had reduced CF while showing no sign of flesh browning. Many of these fruit were reflecting an immediate decrease in CF in response to heat before flesh browning had time to develop or the decline in CF that was observed in all fruit during storage (Fig. 1). When correlations were run comparing CF values 1 d after heat treatment with flesh browning ratings after 3 months of storage, the relationship with ‘Cortland’ improved (rs = −0.824). However, with ‘Jonagold’ there was no improvement (rs = −0.575). In all cases if fruit had Fv/Fm values >0.6 one day following heat treatments, browning
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ratings remained <1. This suggests that CF could be used to nondestructively identify fruit with an increased likelihood to develop internal browning during storage. However, additional research is needed to identify other factors that may affect CF. For apples, a headspace value of ethanol >200 mol m−3 was suggested to be predictive of irreversible tissue damage (Fan et al., 2005). In this study, this relationship was not as apparent (Fig. 8). When correlations were run comparing ethanol concentration 1 d after heat treatment with flesh browning after 3 months of storage, the relationship with ‘Cortland’ improved (rs = 0.644), but declined for ‘Jonagold’ (rs = 0.424). For ‘Jonagold’, any fruit that had ethanol headspace concentrations <100 mol kg−1 m−3 had flesh browning ratings throughout storage of <1. However, results for ‘Cortland’ were less consistent, with some fruit that had low headspace ethanol concentrations developing severe flesh browning. 5. Conclusions Maturity affected fruit resistance to heat stress. Post climacteric fruit were most sensitive to heat stress, followed by commercial harvest maturity and immature fruit based on expression of flesh browning, fruit softening and reduced Fv/Fm value. Fruit of the commercial harvest maturity benefited from the heat treatment of 8 h at 46 ◦ C through greater firmness and reduced acidity following storage, while developing no browning. In most cases, heat injury was not visible in apples immediately following treatments. There was no peel browning in immature and CHM apples while only slight peel browning were shown in PCM apples. However, severity of both peel and flesh browning increased with storage time and duration of heat stress, especially in PCM apples treated for 8 or 12 h. Flesh browning rating was negatively related to CF and positively associated with ethanol concentration. Measurements of CF soon after heat treatment could be used as a nondestructive indicator of the severity of fruit injury resulting from heat stress. A substantial decline in Fv/Fm values may indicate the potential for flesh or skin browning of the fruit during storage. Acknowledgements Contribution no. 2384 of the Atlantic Food and Horticulture Research Centre, Agriculture and Agri-Food Canada. The authors
thank the Nova Scotia Fruit Grower Association for providing funding for this research. The authors also wish to thank Dr. John DeLong and Mr. Douglas Nichols for their critical review of this manuscript. References Beaudry, R., Schwallier, P., Lennington, M., 1993. Apple maturity prediction: an extension tool to aid fruit storage decisions. HortTechnology 3 (2), 233– 239. DeEll, J.R., Prange, R.K., Murr, D.P., 1995. Chlorophyll fluorescence as an indicator of low O2 or high CO2 stress in apples during storage. HortScience 30, 1058– 1059. Fan, L., Song, J., Forney, C.F., Jordan, M.A., 2005. Ethanol production and chlorophyll fluorescence predict breakdown of heat-stressed apple fruit during cold storage. J. Am. Soc. Hort. Sci. 130 (2), 237–243. Forney, C.F., Jordan, M.A., 1998. Induction of volatile compounds in broccoli by postharvest hot water dips. J. Agric. Food Chem. 46, 5295–5301. Forney, C.F., Jordan, M.A., Nicholas, K.U.K.G., DeEll, J.R., 2000. Volatile emissions and chlorophyll fluorescence as indicators of freezing injury in apples. HortScience 35, 1283–1287. Genstat 5 Committee, 1993. Genstat 5 Release 3 Reference Manual. Clarendon Press, Oxford, UK. 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. Kimmerer, T.W., Kozlowski, T.T., 1982. Ethylene, ethane, acetaldehyde, and ethanol production by plants under stress. Plant Physiol. 69, 840–847. Klein, J.D., Lurie, S., 1990. Prestorage heat treatment as a means of improving poststorage quality of apples. J. Am. Soc. Hort. Sci. 115, 256–259. Klein, J.D., Lurie, S., 1992. Prestorage heating of apple fruit for enhanced postharvest quality: interaction of time and temperature. HortScience 27, 326– 328. Lau, O.L., 1985. Harvest indices for BC apples. Br. Columbia Orchardist 7 (7), 1A–20A. Lurie, S., 1998. Postharvest heat treatments of horticultural crops. In: Janick, J. (Ed.), Horticultural Review, vol. 22. John Wiley & Sons, Inc., New York, pp. 91– 122. Lurie, S., Sabehat, A., 1997. Prestorage temperature manipulations to reduce chilling injury in tomatoes. Postharvest Biol. Technol. 11, 57–62. Porritt, S.W., Lidster, P.D., 1978. The effect of prestorage heating on ripening and senescence of apples during cold storage. J. Am. Soc. Hort. Sci. 103, 584–587. Schreiber, U., Berry, J.A., 1977. Heat-induced changes of chlorophyll fluorescence in intact leaves correlated with damage to the photosynthetic apparatus. Planta 136, 233–238. Song, J., Fan, L., Forney, C.F., Jordan, M.A., 2001. Using volatile emissions and chlorophyll fluorescence as indicators of heat injury in apples. J. Am. Soc. Hort. Sci. 126 (6), 771–777. Watkins, C.B., Erkan, M., Nock, J.F., Beaudry, R.M., Moran, R.E., 2005. Harvest date effects on maturity, quality, and storage disorders of ‘Honeycrisp’ apples. HortScience 40 (1), 164–169. Whitaker, B.D., 1994. A reassessment of heat treatments as a means of reducing chilling injury in tomato fruit. Postharvest Biol. Technol. 4, 75–83. Woolf, A.B., Laing, W.A., 1996. Avocado fruit skin fluorescence following hot water treatments and pretreatments. J. Am. Soc. Hort. Sci. 121, 147–151.
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Postharvest Biology and Technology journal homepage: www.elsevier.com/locate/postharvbio
Fruit maturity affects the response of apples to heat stress Lihua Fan ∗ , Jun Song, Charles F. Forney, Michael A. Jordan Agriculture and Agri-Food Canada, Atlantic Food and Horticulture Research Centre, 32 Main Street, Kentville, Nova Scotia B4N 1J5, Canada
a r t i c l e
i n f o
Article history: Received 23 November 2010 Accepted 18 April 2011 Keywords: Malus sylvestris var. domestica Apple maturity Heat stress Chlorophyll fluorescence Apple quality
a b s t r a c t Quality changes of apple fruit at different maturity stages in response to heat stress were investigated. ‘Jonagold’ and ‘Cortland’ apples at immature (pre-climacteric), commercial harvest maturity (CHM) and post climacteric maturity (PCM, CHM plus 4 weeks) were harvested and held at 46 ◦ C for 0, 4, 8, or 12 h. Following treatments, fruits were stored in air at 0 ◦ C and evaluated after 0, 1, 2, or 3 months. Quality indices including peel and flesh browning, firmness, titratable acidity, soluble solids, chlorophyll fluorescence (CF), and ethanol production were measured. Results indicated that different cultivars and maturities affected the fruit’s resistance to heat stress. ‘Jonagold’ was more resistant to heat stress than ‘Cortland’. Fruit at PCM were most sensitive to heat stress, followed by fruits at CHM and immature stages. When ‘Jonagold’ apples at immature and CHM stages were held at 46 ◦ C for 12 h and then stored for 3 months, flesh browning ratings were negligible compared with 1.4 or 2.9, respectively in ‘Cortland’. Flesh browning rating increased to 1.4 or 4.5 in PCM ‘Jonagold’ held at 46 ◦ C for 8 or 12 h and then stored for 3 months while it was 4.9 or 5.0, respectively, in ‘Cortland’. Heat treatment-induced flesh injury was associated with a decrease in CF. After fruit were exposure to 46 ◦ C for 12 h and then stored for 3 months, Fv/Fm was reduced by 13%, 30%, and 55% in ‘Jonagold’ at immature maturity, CHM and PCM, respectively, while it was reduced by 51%, 58% and 75%, respectively, in ‘Cortland’. Heat stress also caused a decrease in fruit titratable acidity, but had no effect on soluble solids contents. The 8 or 12 h heat treatment resulted in an increase in ethanol production, which was greatest in PCM apples. © 2011 Published by Elsevier B.V.
1. Introduction Heat treatments are being actively pursued for postharvest treatments of fresh produce. Some beneficial responses of commodities to heat treatments include the slowing of ripening of climacteric fruit and vegetables, sweetening of commodities either by increasing sugars or decreasing acidity, and prevention of storage disorders such as superficial scald on apples and chilling injury on subtropical fruits and vegetables (Lurie, 1998). Although many reports focus on the positive response of horticultural commodities to heat treatment, high temperatures with long durations may cause tissue damage and increase decay development (Klein and Lurie, 1992; Song et al., 2001; Fan et al., 2005). It is often challenging to find a time–temperature regime that will produce the desired effect of disease control or quality improvement without risk of damaging the fruit or vegetable. In our previous studies on apples, we found that heat stress at 46 ◦ C for 8 and 12 h could result in reduced fruit storage life and quality (Fan et al., 2005). Heat damage on apples can be both external and internal, appearing as peel and flesh browning. In mild to moderate heat injury, visual symptoms are often not immediately apparent, but injury develops during
∗ Corresponding author. Tel.: +1 902 6795550; fax: +1 902 6792311. E-mail address: [email protected] (L. Fan). 0925-5214/$ – see front matter © 2011 Published by Elsevier B.V. doi:10.1016/j.postharvbio.2011.04.007
subsequent storage (Fan et al., 2005). Using nondestructive indicators such as chlorophyll fluorescence (CF) or volatile emissions to identify stressed fruit and determine their potential to breakdown during storage has been reported (DeEll et al., 1995; Fan et al., 2005; Forney et al., 2000; Woolf and Laing, 1996; Song et al., 2001). However, questions remain as to how fruit maturity may affect the response of fruit to heat stress. Beaudry et al. (1993) reported that very immature fruit are unable to develop acceptable quality after harvest including the development of adequate sugars or flavor and are prone to shrivel and excessive weight loss. In contrast, over mature fruits are prone to excessive ripening and are more susceptible to bruising, flesh browning, senescent breakdown, and decay during storage. Apparently, the stage of fruit development at harvest has a significant impact on the potential quality of the stored product. So far, there have been limited reports regarding the impact of maturity on the fruit’s response to heat treatments. In order to increase our understanding of physiological responses induced by heat stress, fruit maturity and cultivar difference need to be considered. Therefore, the objectives of this study were to investigate the physiological changes of two popular apple cultivars ‘Cortland’ and ‘Jonagold’ at different maturity stages in response to heat stress by characterizing changes in CF and ethanol production as well as various fruit quality parameters; to assess the usefulness of CF and ethanol production induced by heat treatment to predict fruit flesh browning (injury) development during cold storage.
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2. Materials and methods 2.1. Apple fruit and maturity assessment Apple fruit of cultivars Cortland and Jonagold that were grown at the Atlantic Food and Horticulture Research Centre in the Annapolis Valley, Nova Scotia, Canada, were harvested at three maturities: immature, commercial harvest maturity (CHM) and post climacteric maturity (PCM, CHM plus one month). The harvest dates for ‘Cortland’ were September 22nd, October 8th and November 9th, respectively, and for ‘Jonagold’ were September 24th, October 15th and November 14th. Fruit maturity was determined based on the internal ethylene concentration and local commercial harvest dates (Lau, 1985). The internal ethylene concentration was determined to be <0.01 L L−1 for immature fruit and about 0.2 L L−1 for CHM apples. Internal ethylene concentration was determined on 10 apples. One milliliter samples of internal gas was drawn into a syringe through a needle inserted into the core cavity of each fruit and were then injected into a gas chromatograph with a flame ionization detector (Carle Instruments, Anaheim, CA) equipped with a 1.9 m × 3.2 mm (o.d.) activated alumina column with a helium carrier flow of 0.83 mL s−1 . Internal ethylene concentration was quantified using a known concentration of standard ethylene. 2.2. Heat treatment Heat treatments were conducted by placing fruit in a 46 ◦ C chamber with about 90% relative humidity for 0, 4, 8, or 12 h. The treatment times were chosen to induce varying degrees of heat stress. The 0 h fruit were used as the control. Temperature was monitored for treated fruit by placing thermocouples in the cortex of selected apples. Following heat treatment, fruit were stored in air at 0 ◦ C and evaluated after 0, 1, 2, or 3 months plus an additional 1 or 7 d at 20 ◦ C.
peak areas were normalized using the peak area of a 4 ng dodecane standard that was run on the same day of the analysis to account for any variability in detector response. 2.4. Fruit quality Samples of 10 apples of each cultivar at different maturity–heat treatment combinations were assessed at each removal. Quality was evaluated following the procedures described by Fan et al. (2005). Briefly, fruit injury was rated as peel or flesh browning using a scale of 0–5, where 0 = none, 1 = up to 20%, 2 = 21–40%, 3 = 41–60%, 4 = 61–80%, and 5 = 81–100% of the fruit peel or flesh area affected. Fruit firmness was determined using a Magness–Taylor firmness tester (type 30 lb, model 30A; Ballauf Manufacturing Co., Laurel, MD). Titratable acidity was measured with a 2 mL juice sample collected from 10 apples, and 0.1 mol L−1 NaOH was used to titrate the apple juice using a semi-automatic titrator (Multi-Dosimat E-415 titrator; Metrohm AG, Switzerland) to a phenolphthalein endpoint of pH 8.1. Titratable acidity was expressed as malic acid equivalents. Soluble solids were determined with a hand-held temperaturecompensated refractometer (Atago Co., Tokyo). 2.5. Statistical analysis The experimental design was a completely randomized 2 × 3 × 4 × 4 factorial with two cultivars (‘Jonagold’ and ‘Cortland’), three fruit maturities (immature, CHM, or PCM), four heat treatment durations (0, 4, 8, or 12 h), and four storage periods (0, 1, 2, or 3 months). For each combination, three sub-samples of four apples were used for ethanol analysis, and 10 apples were used for quality parameters and CF measurement. Data were analyzed using ANOVA, LSD, and correlation options of Genstat 5 (Genstat 5 Committee, 1993). 3. Results
2.3. Physiological responses
3.1. Chlorophyll fluorescence (Fv/Fm)
2.3.1. Chlorophyll fluorescence Chlorophyll fluorescence was determined using a modulated fluorometer (model OS-500; Opti-Science, Tyngsboro, MA) following the method of Fan et al. (2005). Three readings of Fv/Fm from each fruit were recorded and averaged, where Fo = minimal fluorescence, Fm = maximal fluorescence, and Fv = Fm − Fo. The ratio of Fv/Fm, a measure of the quantum efficiency of photosystem II was used to express the change of CF in this study.
Heat treatments of 12 h at 46 ◦ C reduced CF (Fv/Fm) substantially at all harvest maturities and in both cultivars (Fig. 1). Treatments of 8 h reduced CF at all maturities in ‘Cortland’ apples and in PCM ‘Jonagold’ fruit. The 4 h treatment had no effect on CF. CF declined during the 3 month storage period at 0 ◦ C in all fruit. CF also decreased with increasing fruit maturity. Immature fruit had the highest CF while PCM fruit had the lowest CF following heat treatment and storage. Heat treatments at 46 ◦ C for 12 h reduced Fv/Fm of immature, CHM, and PCM fruit by 51%, 58% and 75%, respectively, in ‘Cortland’ and 13%, 30%, and 55%, respectively, in ‘Jonagold’ compared to control fruit following 3 months of storage (Fig. 1). When CF was measured after 7 d at 20 ◦ C following removal from storage, Fv/Fm averaged 12% less than measurements taken 1 d after removal from storage. However, when CF was measured 7 d after the 8 and 12 h heat treatments, CF was 8% and 35% greater than that measured 1 d after treatment, respectively, suggesting some recovery from the heat stress occurred.
2.3.2. Ethanol analysis Three sub-samples of four apples each from each cultivar at different maturity–heat treatment combinations were placed in 4-L glass jars, sealed with Teflon lids, and held at 20 ◦ C. The headspace over the apples was allowed to equilibrate for 1 h under a 1.67 mL s−1 flow of purified air. A 100 mL head space sample in the jar was then collected onto a 100 mm × 6.4 mm (o.d.) glass collection tube containing 120 mg of Tenax GR 20/35 (Alltech Associates, Inc., Deerfield, IL) and 50 mg of carbosieve SIII 60/80 (Supelco Inc., Bellefonte, PA). The tubes were then stored at −80 ◦ C until analysis. Samples were analyzed on a Magnum gas chromatograph–mass spectrometer (GC–MS) system (Finnigan MAT, San Jose, CA) equipped with an LSC 2000 purge and trap concentrator and Aerotrap 6016 autosampler (Tekmar, Cincinnati) following the methods described by Forney and Jordan (1998). Mass spectra were acquired in electron ionization mode using automatic gain control within the range of m/z 30–400. Quantification of ethanol was done using the 45 m/z ion and external standards. All
3.2. Ethanol production The production of ethanol by PCM ‘Cortland’ and ‘Jonagold’ fruit was greatest in fruit held for 8 or 12 h at 46 ◦ C (Fig. 2). However, immature and CHM fruit did not show a significant increase in ethanol concentration as a result of heat treatment. In ‘Cortland’ fruit, ethanol concentration increased during storage at 0 ◦ C in both control and heat treated fruit. After 3 months of storage,
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ethanol concentrations in ‘Cortland’ fruit averaged 7.5-, 2.9-, and 7.8-fold greater than initial values in immature, CHM and PCM fruit, respectively. Storage had no significant effect on the ethanol concentration in ‘Jonagold’ fruit.
3.3. Flesh browning Severity of flesh browning increased in PCM ‘Cortland’ and ‘Jonagold’ apples exposed to 46 ◦ C for 8 or 12 h following 3 months of storage (Fig. 3). Flesh browning ratings increased to 4.9 or 5.0 following 3 months of storage in PCM ‘Cortland’ apples exposed to 46 ◦ C for 8 or 12 h, respectively, while it was 1.4 or 4.5, respectively, in ‘Jonagold’. Flesh browning of apple fruit was not significantly affected by the 4 h heat treatment regardless of cultivar or maturity stage. Overall, PCM apples were most sensitive to heat stress, followed by CHM and immature fruit. ‘Jonagold’ apples were more resistant to heat stress than ‘Cortland’. When ‘Jonagold’ apples at the immature and CHM stages were exposed to 46 ◦ C for 12 h and then stored for 3 months, flesh browning ratings were negligible compared with 1.4 and 2.9, respectively in ‘Cortland’ (Fig. 3).
3.4. Peel browning Heat treatments of 8 and 12 h caused peel browning in the PCM fruit (Fig. 4). After 3 months of storage, ratings averaged 3.1 and 4.4 in ‘Cortland’ and 1.4 and 4.2 in ‘Jonagold’ apples subjected to the 8 and 12 h heat treatments, respectively. The 4 h heat treatment had no effect on inducing peel browning. As well, no significant peel browning was induced by any of the heat treatments in the immature or CHM apples. In the PCM fruit, the development of peel browning was more apparent in ‘Jonagold’ fruit after 1 month of storage, but after 3 months severity was slightly greater in ‘Cortland’ fruit.
3.5. Fruit firmness Exposure of immature and CHM apple fruit to 46 ◦ C for 8 or 12 h maintained fruit firmness above that of the untreated controls during storage (Fig. 5). However, this maintenance of firmness was not observed in heat-treated PCM apples, and after 3 months of storage 12 h treated fruit were softer than controls. Fruit firmness declined with later harvest date and during storage at 0 ◦ C. Immature and
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CHM apples averaged 44% and 31% firmer than PCM apples during the 3 month storage period and fruit firmness declined an average of about 30% during storage (Fig. 5).
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Heat treatment (h) Fig. 2. Ethanol production of ‘Cortland’ and ‘Jonagold’ apples harvested at immature maturity, commercial harvest maturity (CHM) and post climacteric maturity (PCM) and exposed to 46 ◦ C for 0, 4, 8, or 12 h. Values are averages of measurements from fruit stored for 0, 1, 2, or 3 months plus 1 or 7 d at 20 ◦ C. The vertical bars represent the LSD at P < 0.05 for comparison of means within the figure.
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Heat treatments can have a variety of effects on fresh apple fruit depending on temperature and duration of exposure. In our study exposure of fruit to 46 ◦ C for 4 h had little effect on fruit quality, but exposures of 8 or 12 h were effective in lowering fruit acidity and reducing softening of the fruit during storage. Similar results were found in our previous study (Fan et al., 2005). Porritt and Lidster (1978) and Klein and Lurie (1990) also reported that TA declines while soluble solids concentration in apples is unaffected by heat treatment, and apples held at 38 ◦ C for 3 or 4 d were firmer than untreated fruit even after 6 months of storage at 0 ◦ C and subsequent shelf life at 20 ◦ C. However, heat treatments at 46 ◦ C for 8
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Storage time (month) Fig. 4. Peel browning rating of ‘Cortland’ and ‘Jonagold’ apples harvested at immature maturity, commercial harvest maturity (CHM) and post climacteric maturity (PCM) and exposed to 46 ◦ C for 0, 4, 8, or 12 h following 0, 1, 2, or 3 months of storage at 0 ◦ C. Values are averages of measurements taken after 1 or 7 d at 20 ◦ C following removal from storage. Peel browning was rated using a scale of 0–5, where 0 = none, 1 = up to 20%, 2 = 21–40%, 3 = 41–60%, 4 = 61–80%, and 5 = 81–100% of the fruit peel area affected. The vertical bar represents the LSD at P < 0.05 for comparison of means within the figure.
or 12 h caused flesh and peel browning in some fruit. This browning was not apparent 1 week after treatment, but rather developed during storage for 3 months at 0 ◦ C. The variable response to heat treatments among fruit appears to be dependent on numerous factors including fruit maturity and cultivar. Fruit maturity has a strong effect on fruit quality, storage life, and tolerance of fruit to heat treatments. As fruit increase in maturity, respiration, ethylene, aroma volatiles, soluble solids, and color increase, while acidity, firmness and chlorophyll content decrease (Beaudry et al., 1993). Immature fruit never achieve good eating quality, lacking sweetness and aroma, while over-mature fruit are soft, subject to bruising and more prone to breakdown during storage. Watkins et al. (2005) reported that development of disorders such as soggy breakdown and soft scald in ‘Honeycrisp’ apples is associated with later harvest dates and storage temperature. Fruit harvested in this study decreased in firmness and titratable acidity when harvest date was delayed. In addition, we found that as fruit became over mature (PCM), its tolerance to heat declined resulting in the most severe flesh and skin browning that developed during storage. These fruit were severely injured with the 8 h treatment, which was well tolerated in the immature and CHM fruit. The PCM fruit would be considered post climacteric, which is characterized by advanced senescence and loss of flesh firmness, which appears to influence the fruit’s tolerance to heat stress. To ensure a long-term storage potential of apples, it is essential that the fruit are harvested within a well defined optimal harvest period. The stage of fruit development at harvest has a significant impact on the potential quality of the stored product (Beaudry et al.,
1993). Although immature fruit are more tolerant to severe heat treatments than ripe or over ripe fruit, they have not ripened sufficiently upon removal from storage resulting in inferior organoleptic quality. Conversely, over ripe fruit become soft and mealy during storage. In this study, over ripe fruit were also most sensitive to moderate and severe heat treatments (8–12 h at 46 ◦ C) as shown by fruit tissue injury, reduced Fv/Fm and increased ethanol production. Cultivar also may have had an effect on the tolerance of fruit to heat treatments. ‘Cortland’ fruit appeared to be less tolerant than ‘Jonagold’ in this study based on flesh browning rates. Immature and CHM fruit developed flesh browning with the 12 h treatment in ‘Cortland’ but not ‘Jonagold’. Song et al. (2001) reported ‘Cortland’ and ‘Jonagold’ to be more tolerant to heat treatments than ‘McIntosh’ and ‘Northern Spy’, but that ‘Jonagold’ was the most tolerant. Fan et al. (2005) also found that flesh browning was more severe in ‘Cortland’ fruit when compared to those of ‘Jonagold’ subjected to the same heat treatments. Although untreated ‘Cortland’ fruit showed no injury during storage at 0 ◦ C, it is a chilling sensitive cultivar and the chilling stress of storage may have enhanced heat injury symptoms as suggested by Fan et al. (2005). Kim et al. (1993) compared the tolerance of eleven cultivars including ‘Cortland’ to hot water dips of 40–50 ◦ C for up to 4 h. They found the heat-stress tolerance of ‘Cortland’ to be intermediate, with ‘Rome’, ‘Monroe’ and ‘Liberty’ being very susceptive and ‘Delicious’ and ‘Golden Delicious’ being most tolerant. While apple cultivars appear to differ in their tolerance to heat, the degree of tolerance is influenced by other factors such as maturity and growing season. Tomato cultivars
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Storage time (month) Fig. 5. Firmness of ‘Cortland’ and ‘Jonagold’ apples harvested at immature maturity, commercial harvest maturity (CHM) and post climacteric maturity (PCM) and exposed to 46 ◦ C for 0, 4, 8, or 12 h following 0, 1, 2, or 3 months of storage at 0 ◦ C. Values are averages of measurements taken after 1 or 7 d at 20 ◦ C following removal from storage. The vertical bar represents the LSD at P < 0.05 for comparison of means within the figure.
Fig. 6. Titratable acidity in malic acid equivalents of juice extracted from ‘Cortland’ and ‘Jonagold’ apples harvested at immature maturity, commercial harvest maturity (CHM) and post climacteric maturity (PCM) and exposed to 46 ◦ C for 0, 4, 8, or 12 h. Values are averages of measurements from fruit stored for 0, 1, 2, or 3 months at 0 ◦ C plus 1 or 7 d at 20 ◦ C.
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Fig. 7. Chlorophyll fluorescence (Fv/Fm) values of ‘Cortland’ and ‘Jonagold’ apples harvested at immature maturity, commercial harvest maturity (CHM) and post climacteric maturity (PCM), exposed to 46 ◦ C for 0, 4, 8, or 12 h and stored for 0, 1, 2, or 3 months at 0 ◦ C plotted against flesh browning ratings. The correlation coefficient (rs ) represents that calculated using Spearman’s rank correlation.
also appeared to respond differently to heat treatments. Lurie and Sabehat (1997) reported that a heat treatment at 38 ◦ C reduced low temperature sensitivity of mature green ‘Daniella’ tomato fruit, and they could be stored for up to a month at 2 ◦ C without developing chilling injury. However, Whitaker (1994) found that heat treatments at 38 ◦ C had no benefit in ‘Rutgers’ tomato fruit suggesting that the response of fruit to heat treatment may be cultivar specific. Chlorophyll fluorescence, a sensitive indicator of the state of the photosynthetic apparatus, is often used as a nondestructive probe of heat damage (Schreiber and Berry, 1977). It is a useful indicator of several postharvest stresses in apples including exposure to low O2 or high CO2 concentrations (DeEll et al., 1995) and freezing injury (Forney et al., 2000). When apple fruit are stressed by heat, chloroplast integrity may decline resulting in a decline in Fv/Fm (Fan et al., 2005). In this study, we found that moderate and severe heat treatments reduced Fv/Fm values and they never recovered to control values indicating that the apple fruit were damaged irreversibly. This study also confirmed our previous findings that flesh browning reflected the physiological injury caused by heat stress. The severity of flesh browning over the storage period correlated with the decrease in CF (Fig. 7). Ethanol is the primary product of fermentation, which occurs when oxidative phosphorylation is disrupted, preventing the oxidation of pyruvate by the Krebs Cycle. Since oxidative phosphorylation involves electron transport through a complex of membrane bound proteins in the mitochondria, disruption of these membranes could inhibit this process. Any stress that can disrupt normal mitochondrial membrane function could induce fermentation in the plant tissue. Ethanol production by stressed plant tissues
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Fig. 8. Headspace ethanol concentrations of ‘Cortland’ and ‘Jonagold’ apples harvested at immature maturity, commercial harvest maturity (CHM) and post climacteric maturity (PCM), exposed to 46 ◦ C for 0, 4, 8, or 12 h and stored for 0, 1, 2, or 3 months at 0 ◦ C plotted against flesh browning ratings. The correlation coefficient (rs ) represents that calculated using Spearman’s rank correlation.
has been observed in previous studies (Fan et al., 2005; Kimmerer and Kozlowski, 1982). In this study, heat treatment only increased ethanol production substantially in PCM fruit, suggesting that the advanced stage of senescence may render the mitochondrial membranes more susceptible to disruption. In addition, high respiration rates associated with PCM fruit and heat stress may deplete available oxygen and induce fermentation. ‘Jonagold’ fruit showed a stronger relationship of flesh browning with ethanol production than ‘Cortland’ fruit (Fig. 8). Overmature ‘Cortland’ fruit tend to produce ethanol regardless of heat treatment, while ethanol production in ‘Jonagold’ was more strongly related to heat-induced flesh browning. Both changes in CF and in ethanol content of apple fruit have been correlated to injury induced by both heat (Fan et al., 2005; Song et al., 2001) and freezing (Forney et al., 2000) stress. Fan et al. (2005) suggested that an Fv/Fm value of <0.6 measured 1 d after heat treatment was predictive of the development of flesh browning during storage. A similar relationship was observed in this study (Fig. 7). Both ‘Cortland’ and ‘Jonagold’ apples that had Fv/Fm values of <0.6 tended to develop flesh browning ratings >1 and was most apparent after 3 months of storage. However, a large number of fruit had reduced CF while showing no sign of flesh browning. Many of these fruit were reflecting an immediate decrease in CF in response to heat before flesh browning had time to develop or the decline in CF that was observed in all fruit during storage (Fig. 1). When correlations were run comparing CF values 1 d after heat treatment with flesh browning ratings after 3 months of storage, the relationship with ‘Cortland’ improved (rs = −0.824). However, with ‘Jonagold’ there was no improvement (rs = −0.575). In all cases if fruit had Fv/Fm values >0.6 one day following heat treatments, browning
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ratings remained <1. This suggests that CF could be used to nondestructively identify fruit with an increased likelihood to develop internal browning during storage. However, additional research is needed to identify other factors that may affect CF. For apples, a headspace value of ethanol >200 mol m−3 was suggested to be predictive of irreversible tissue damage (Fan et al., 2005). In this study, this relationship was not as apparent (Fig. 8). When correlations were run comparing ethanol concentration 1 d after heat treatment with flesh browning after 3 months of storage, the relationship with ‘Cortland’ improved (rs = 0.644), but declined for ‘Jonagold’ (rs = 0.424). For ‘Jonagold’, any fruit that had ethanol headspace concentrations <100 mol kg−1 m−3 had flesh browning ratings throughout storage of <1. However, results for ‘Cortland’ were less consistent, with some fruit that had low headspace ethanol concentrations developing severe flesh browning. 5. Conclusions Maturity affected fruit resistance to heat stress. Post climacteric fruit were most sensitive to heat stress, followed by commercial harvest maturity and immature fruit based on expression of flesh browning, fruit softening and reduced Fv/Fm value. Fruit of the commercial harvest maturity benefited from the heat treatment of 8 h at 46 ◦ C through greater firmness and reduced acidity following storage, while developing no browning. In most cases, heat injury was not visible in apples immediately following treatments. There was no peel browning in immature and CHM apples while only slight peel browning were shown in PCM apples. However, severity of both peel and flesh browning increased with storage time and duration of heat stress, especially in PCM apples treated for 8 or 12 h. Flesh browning rating was negatively related to CF and positively associated with ethanol concentration. Measurements of CF soon after heat treatment could be used as a nondestructive indicator of the severity of fruit injury resulting from heat stress. A substantial decline in Fv/Fm values may indicate the potential for flesh or skin browning of the fruit during storage. Acknowledgements Contribution no. 2384 of the Atlantic Food and Horticulture Research Centre, Agriculture and Agri-Food Canada. The authors
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