Research Article Received: 26 November 2013
Revised: 3 February 2014
Accepted article published: 10 February 2014
Published online in Wiley Online Library: 24 March 2014
(wileyonlinelibrary.com) DOI 10.1002/jsfa.6610
Response of Pink Lady® apples to post-harvest application of 1-methylcyclopropene as a function of applied dose, maturity at harvest, storage time and controlled atmosphere storage Emiliano Cocci,a* Giampiero Sacchetti,b Pietro Rocculia,c and Marco Dalla Rosaa,c Abstract BACKGROUND: 1-Methylcyclopropene (1-MCP) is an inhibitor of ethylene reception used in post-harvest treatments to delay fruit ripening. Several factors affect the efficacy of 1-MCP treatments. The effect of a post-harvest treatment with 1-MCP on the quality of Pink Lady® apples as a function of 1-MCP dose, storage time and maturity at harvest was investigated. 1-MCP treatment was further tested in combination with controlled atmosphere (CA) storage. RESULTS: 1-MCP limited fruit respiration and softening during storage and was more effective on partially matured fruits and at prolonged storage times. The delaying of 1-MCP on the increase of ripening index was greater on matured fruits at prolonged storage times. The combination of 1-MCP and CA treatments positively affected quality indices of mature apples during 6 months of storage and 7 days of commercial life, with 1-MCP being more effective than CA. 1-MCP and CA showed positive combined effects on firmness and ripening index after 6 months of storage, and on firmness and CO2 production after a further 7 days of commercial life. CONCLUSION: By knowing fruit maturity at harvest and expected storage time it is possible to choose the most suitable 1-MCP dose to meet the market requirements by applying a simple polynomial model. © 2014 Society of Chemical Industry Keywords: Pink Lady® apples; methylcyclopropene; controlled atmosphere; ripening; firmness; sensory analysis
INTRODUCTION
J Sci Food Agric 2014; 94: 2691–2698
∗
Correspondence to: Emiliano Cocci, CIRI Agroalimentare, Campus Scienze degli Alimenti, Via Ravennate 1020, 47521 Cesena (FC) Italy. E-mail: [email protected]
a CIRI Agroalimentare, Campus Scienze degli Alimenti, Via Ravennate 1020, 47521, Cesena, Italy b Faculty of Bioscience and Technology for Food, Agriculture and Environment, University of Teramo, Via C.R. Lerici 1, 64023, Teramo, Italy c Department of Agri-Food Science and Technology – DISTAL, University of Bologna, Campus of Food Science, Piazza Goidanich 60, 47023, Cesena, Italy
www.soci.org
© 2014 Society of Chemical Industry
2691
Maintaining post-harvest quality of apples during storage and distribution is one of the major challenges facing the fruit industry. Fruit and vegetables are living commodities and their respiration and ripening rate are important factors for their quality maintenance.1 In fruit storage, management techniques used to minimise the effects of ethylene include low O2 and high CO2 atmospheres, as well as reduced temperature. 1-Methylcyclopropene (1-MCP) is an inhibitor of ethylene reception in plant tissues thought to act by binding irreversibly to ethylene receptors.2 Its availability, as a stable complexed formulation that releases 1-MCP when dissolved in water, has led to a surge of research and commercial interest around the world. 1-MCP is marketed as EthylBloc™ (Floralife Inc., Walterboro, SC, USA) and SmartFresh™ (AgroFresh Inc., Yakima, WA, USA). The post-harvest gaseous application of 1-MCP has been shown to reduce ethylene production and respiration, to maintain many quality characteristics of apple, such as fruit firmness and acidity retention, and to reduce superficial scald development, peel
greasiness and various chilling-related disorders.3 – 5 Important factors which govern efficacy of 1-MCP treatment are: (1) active concentration, treatment time and temperature; (2) type of fruit and maturity stage at the time of treatment; and (3) treatment frequency, because, although 1-MCP binding to the ethylene receptor is essentially irreversible, the inhibition of the effects of ethylene may be overcome by the production of new receptors.6,7 Under commercial conditions, handling practices need to take into
www.soci.org account many other factors, which also include the desired effects on quality, because for many products, partial control of ripening is more desirable than total control.8,9 ‘Pink Lady®’ apples originated from a cross between ‘Lady Williams’ and ‘Golden Delicious’.10 Since 1990, this cultivar has been extensively cultivated in the main apple-producing areas of the world, because of its excellent sensory attributes: it is firm, has fine dense flesh, is crisp and juicy, provides excellent flavour, and has a high sugar–acid balance.11 To the authors’ knowledge only a few studies have investigated the effects of 1-MCP on the post-harvest quality of Pink Lady® apples. In particular, De Castro et al.12 found that 1-MCP (1 mL L−1 ) conserved fruit quality in air as well as a controlled atmosphere (CA) during 4 months of storage. These authors included in the experimentation different maturity stages but they used only one of them to study the effects of 1-MCP on apple quality. Other authors13 reported that the 1-MCP-treated apple (0.625 mL L−1 ) were firmer than untreated fruit. In this paper the authors used a multi-phase experiment by considering four factors (orchard, 1-MCP treatment, storage and export) but they did not investigate the relationship among different factors involved in 1-MCP effectiveness. Therefore, the aim of the present work was to study the effect of a semi-commercial post-harvest treatment with 1-MCP on the quality of Pink Lady® apple, as a function of 1-MCP dose, fruit maturity stage and storage time, by a multi-response approach based on factorial design, in order to investigate the individual and combined effect of each factor. Finally, the individual and combined effect of post-harvest 1-MCP and controlled atmosphere treatments was studied by keeping all the factors at constant level.
Table 1. Coding and levels of independent variables used in the experimental design Variable Trial no. 1 2 3 4 5 6 7 8 9 10 11
1-MCP (ppb) 325 975 325 975 325 975 325 975 650 650 650
Storage time (months) 2 2 6 6 2 2 6 6 4 4 4
Coded Maturity at harvesta 4 4 4 4 8 8 8 8 6 6 6
x1
x2
x3
−1 1 −1 1 −1 1 −1 1 0 0 0
−1 −1 1 1 −1 −1 1 1 0 0 0
−1 −1 −1 −1 1 1 1 1 0 0 0
a Maturity at harvest was measured as starch index, on a scale of 1–10. 1-MCP, 1-methylcyclopropene.
It was assumed that a mathematical function [Eqn (1)] exists for the response variable Y (quality parameters) in terms of three independent process variables X 1 (1-MCP concentration), X 2 (ripening stage at harvest) and X 3 (storage time), with all other factors unaltered: ) ( (1) Y = 𝜑 X1 , X2 , X3 To approximate the function 𝜑 a first-order polynomial of three variables was used:
MATERIALS AND METHODS Plant material Pink Lady® apples were harvested in early morning from ten 40-tree plots in the same orchard near Cesena (Italy). Thirty apples were randomly collected from orchard trees weekly starting from the first week of October; the progress of starch conversion to sugars (on a scale of 1–10) was followed over time.14 Starch index reached 3.90 ± 0.34 on 3 November, 5.95 ± 0.30 on the 15 November and 8.05 ± 0.20 on the 2 December 2006. The three different maturity stages, selected on the basis of the starch index, were chosen according to a commercial maturity chart and kept equidistant among them. When apples reached the established maturity stages they were harvested selected on the basis of visual colour, in accordance with the Association Pink Lady® Europe: fruit diameter > 70 mm; 50% of diffuse pink or 30% intense pink; background colour (changing between green and yellow) and absence of defects.
2692
Experimental design Effects of 1-MCP treatment on the quality of Pink Lady® apples as a function of fruit maturity stage, 1-MCP dose and storage time For this experiment, 1100 kg of apple fruits were used (100 kg for each trial). An incomplete three-level three-factor design was adopted, using a three-factor constrained experimental region. The three independent variable levels were coded as −1 (lowest level), 0 (intermediate level) and +1 (highest level). Their actual values (Xi ), chosen from preliminary studies and from literature and the corresponding coded value (xi ) are given in Table 1. The design consisted of 11 experimental trials, which included three replications of the central point (X 1 = X 2 = X 3 = 0).
wileyonlinelibrary.com/jsfa
E Cocci et al.
Y = 𝛽0 + 𝛽1 X1 + 𝛽2 X2 + 𝛽3 X3 + 𝛽12 X1 X2 + 𝛽23 X3 X2 + 𝛽13 X1 X3
(2)
where Y is the response (quality parameters), xi are the coded independent variables (linearly related to Xi ), and 𝛽 0 , 𝛽 i , and 𝛽 ij are the regression coefficients. Response surface methodology was used to graphically represent the mathematical functions. Effect of 1-MCP treatment and controlled atmosphere storage on fruit quality For this further experiment, 200 kg of fruits have been used (100 kg untreated and 100 kg treated apples). Actually, in order to study the effects of 1-MCP treatment in combination with the CAs, apples of an intermediate maturity stage (Table 2) were left untreated or were treated with 1-MCP (650 nL L−1 ). Fifty kilograms of both treated and untreated fruit were stored in a regular atmosphere (RA) or in CA [1.8% O2 , 1.4% CO2 at 2.0 ∘ C and 95% relative humidity (RH)]. The CA conditions were chosen according to the industrial practices and are within the range reported by Lopez et al.11 Fruit analyses were performed after 6 months of storage at 2 ∘ C plus a period of 7 days at 20 ∘ C and 65% RH, to simulate fruit commercial life. Treatment with 1-MCP For each treatment run, 100 kg of apples were placed into a commercial plastic bin (1 m3 volume) and allowed to equilibrate at 2 ∘ C overnight. The bins were inserted in sealed air-tight plastic bag and exposed to a specific 1-MCP concentration (325, 650 or 975 nL L−1 )
© 2014 Society of Chemical Industry
J Sci Food Agric 2014; 94: 2691–2698
Response of Pink Lady apples to 1-MCP and controlled atmosphere
www.soci.org
Table 2. Quality characteristics (mean ± SD) of Pink Lady® apples at different maturity levels at harvest
Maturity Partially mature Mature Fully mature
Starch indexa 3.90 ± 0.34 5.95 ± 0.35 8.05 ± 0.29
Firmness (N) 87.7 ± 0.7 84.3 ± 0.8 78.7 ± 0.6
Soluble solids content (%) 11.7 ± 0.6 13.9 ± 0.1 14.2 ± 0.1
Titratable acidityb (mg 100 g−1 FW) 0.71 ± 0.01 0.67 ± 0.02 0.65 ± 0.01
Ripening indexc 6.5 ± 0.1 20.8 ± 0.1 21.8 ± 0.6
CO2 production (mL kg−1 h−1 ) 7.89 ± 0.17 11.71 ± 2.34 15.14 ± 0.56
Ethylene production (μL kg−1 h−1 ) 0.43 ± 0.01 0.54 ± 0.03 0.57 ± 0.01
a On a scale of 1–10. b Calculated as mg of malic acid. c Soluble solids content/titrable acidity.
(SmartFresh™; Agrofresh) for 24 h at 2 ∘ C. The concentration of 1-MCP was calculated according to the % active ingredient and release from the SmartFresh™ powder into the free head-space of sealed bags, by using an electric fan provided by Agrofresh. Starch index Starch index was determined in 30 apples by dipping cross-sectional fruit halves in an iodine solution (40 g KI + 10 g I2 L−1 ) for 30 s; starch hydrolysis was rated using a scale of 1–10. To improve accuracy and precision of the starch index scores, a methodology based on image analysis was used to calculate the ratio of blue-stained to non-stained areas of iodine-treated apples, as proposed by Peirs et al.14 To calculate the percentage of dark-starch area in sample cross-sectional area, a calibration curve was built by using the images of a French 10-point radial type pictorial scale, where 1 = 0% loss of starch and 10 = 100% loss [CTIFL 2002 (http://www.fruits-et-legumes. net/revue-en-ligne/point-sur/fich-pdf/code-amidon.pdf)]. Images were captured using an image acquisition system similar to that developed by Mendoza and Aguilera.15 Samples were illuminated using two parallel lamps (mod. TL-D Deluxe, Natural Daylight, 18 W/965; Philips, New York, USA) with a colour temperature of 6500 K (D65 ) and a colour-rendering index close to 90%. Four fluorescent tubes (60 cm long) were situated 35 cm above the sample and at an angle of 45∘ to it. A colour digital camera (Canon PowerShot A70) was located vertically over the sample at a distance of 12.5 cm. The angle between the camera lens and the lighting source axis was approximately 45∘ . The lamps and the colour digital camera were kept in a black box to exclude light. Recorded images were isolated from the background and processed using the software Photoshop® v. 6.0 (Adobe System Incorporated, San Jose, CA, USA) (Fig. 1B). After conversion in grey scale (16 BPP) and elimination of seed area (Fig. 1A), images were evaluated with advance Image Analysis Software (Image Pro-Plus® v. 6.2 Media Cybernetics, USA) for automatic calculation of the ratio of non-stained to blue-stained area within fruit flesh using a grey-scale threshold value (13 575 BPP) (Fig. 1B). Reference charts were analysed using the same technique. For each maturity stage at harvest, 60 images of 60 apple halves were acquired and analysed.
J Sci Food Agric 2014; 94: 2691–2698
III S/L (Witt-Gasetechnik, Witten, Germany). The apparatus has a paramagnetic sensor for O2 and a mini-infrared spectrophotometer for detection of CO2 . The instrument was calibrated with O2 and CO2 air percentages. Free headspace volume (estimated by water displacement) was measured after gas measurement. Data were expressed as mL CO2 kg−1 h−1 . Measurements of respiration rate were made using three replicate groups of four fruit per treatment. Ethylene production rate Four fruit were weighed and sealed into 2.5-L air-tight respiration glass jars identical to those used for respiration rate determination. Gas samples (1 mL) were withdrawn from the headspace volume of jars. Ethylene was quantified using a gas chromatograph model Clarus 500 (Perkin–Elmer Corporation, Wellesley, MA, USA), equipped with a flame ionisation detector and an Elite QPlot column (30 m × 0.32 mm i.d.) at 80 ∘ C, using helium as a carrier gas (50.3 cm s−1 ). Injector and detector temperature were 120 and 155 ∘ C respectively. Three independent ethylene samples per chamber were taken and results were expressed as a mean (mL kg−1 h−1 ). Ethylene samples of known concentration (1 mL L−1 ) were routinely used for equipment calibration. Ripening index The ratio between soluble solids content (SSC) and titratable acidity (TA) was used as the ripening index (Sweeney et al.16 ). SSC was measured on the juice obtained from 20 homogenised fruit per sample per sampling time, by using a digital refractometer model PR1 (Atago, Tokyo, Japan). TA was determined by titration with 0.1 mol L−1 NaOH to pH 8.117 of the juice obtained from 20 homogenised fruit per sample per sampling time.
© 2014 Society of Chemical Industry
wileyonlinelibrary.com/jsfa
2693
Respiration rate Four fruit were weighed and sealed into 2.5-L air-tight respiration glass jars. CO2 was allowed to accumulate to 0.1–0.3% at 20 ∘ C; this took from 30 min to 2 h, depending on ripening stage. Concentration of CO2 in the headspace of each jar was determined by rapid gas analysis, using an O2 /CO2 gas analyser mod. MFA
Figure 1. Iodine-treated Pink Lady® halves after conversion in grey scale (16 BPP) and elimination of seed area (A), and virtually coloured (B) on the basis of grey-scale pixels values (threshold, 13 575 BPP).
www.soci.org Fruit firmness Penetration tests were carried out using an 11 mm diameter probe on opposite sides (blush and green) on 20 fruits flesh after peel removal. A texture analyser model HD500 (Stable Micro Systems, Guildford, UK) was used and the maximum force measured during the test was considered as firmness (in newtons). Sensory evaluation A quantitative descriptive analysis was carried out by a panel of eight trained assessors. Assessors were selected and trained according to the international standards ISO8586-1:199318 and ISO8586-2:1994.19 The analysis was carried out in individual booths located in a laboratory designed according to ISO8589:1988.20 Thirty apples per sample (treatment) were used for sensory analysis. Samples were kept in a room at 20 ∘ C overnight after removal from storage chambers before sensory evaluation. Each fruit was divided into four pieces prior to sensory evaluation. Each sample was identified by a random three-digit code. The order in which apples were presented to each judge was randomised. Judges were free to test samples as often as they desired. Mineral water was used as a palate cleaner between samples. Eleven attributes were selected by assessors in preliminary meetings: whole fruit odour intensity, cut fruit odour intensity, sweetness, sourness, apple flavour intensity, fermented flavour, off-flavour, crispness, firmness, juiciness and mealiness. Judges were asked to rate the intensity of each attribute on a structured scale of 1–9. All evaluations were conducted in individual booths under white illumination (D65 illuminant) and at room temperature. Sensory evaluations were repeated twice: in the morning and in the afternoon. Statistical analysis Results were reported as average values and standard deviations (SDs) calculated from different analytical repetitions. ANOVA was carried out to test the significance of the effects of experimental factors and comparison between means was carried out by the least significant difference (LSD) test. Multiple regression analysis was carried out by using response variables as dependent variables and the individual and interactive effects of the factors of the experimental design as independent variables. Multiple regression analysis was carried out using a backward stepwise procedure; for this purpose the F to enter was set in order to include in the model only the variables significant at a value of P < 0.05. Adequacy of fitted models was verified by using the adjusted determination coefficient (R2 adj. ), the associated P value, the Fisher’s F value and the standard error of estimate. Response surfaces on three-dimensional plots were calculated from the best-fit models by setting one factor at its intermediate level. All the statistical analyses were carried out using the Statistica v. 6.0 (Statsoft, Tulsa, OK, USA) software
RESULTS AND DISCUSSION
2694
Fruit maturity at harvest At harvest quality parameters measured on the Pink Lady® apple fruits are shown in Table 2. The starch index of the intermediate harvest was equidistance through independent variable levels; this target was required to ensure that the experimental design was valid. As a consequence of the different physiological states of fruit at harvest, firmness, soluble solids content, titratable acidity,
wileyonlinelibrary.com/jsfa
E Cocci et al.
Table 3. Values of the first order polynomial regression coefficients representing the relationship between firmness, ripening index and CO2 production and the independent variables Coefficient 𝛽0 𝛽1 𝛽2 𝛽3 𝛽 12 𝛽 23 𝛽 13 R2 adj P-level SE
Firmness (N) 85.9341 0.01890 −2.23927 NS 0.00207 −0.32593 −0.00278 0.958 < 0.0001 1.8720
Ripening indexa
CO2 production (mL kg−1 h−1 )
4.916490 0.001688 2.320445 2.993021 −0.000709 0.127765 −0.001096 0.997 < 0.0001 0.41902
−0.952460 NS 2.977988 1.189331 −0.001191 −0.270917 0.000231 0.914 < 0.0001 0.619
a Soluble solids content/titrable acidity. NS, not significant; R2 adj , adjusted R2 ; SE, standard error.
ethylene and carbon dioxide production rate were different and consistent with the starch index, confirming that this parameter is an appropriate tool to describe maturity at harvest for Pink Lady® apples.12 Effect of 1-MCP treatment on firmness The regression model calculated for firmness (Table 3) showed a high adjusted R2 value of 0.958 with a standard error of estimation of 1.87; thus the proposed model adequately described firmness values as a function of the selected variables. Firmness was influenced by all the considered variables at high probability level (P ≤ 0.001) except maturity at harvest. The response surface describing firmness after 4 months of storage (central value) as a function of 1-MCP concentration and starch index at harvest indicates (Fig. 2A) that 1-MCP concentration and fruit maturity stage had a negative combined effect on firmness in accordance with previous research in which more mature fruit usually were less affected by 1-MCP treatment.7 After 6 months of storage, the firmest fruit were those that were partially mature at harvest and treated with the highest 1-MCP concentration. The same fruit, after 4 months of storage, had a firmness value of about 87 N, which is similar to the initial value of 87.7 N (Table 2). This suggests that, in these semi-commercial conditions, fruit softening was completely blocked; this agrees with De Castro et al.12 who found no softening of Pink Lady® apples during 4 months of refrigerated air storage following treatment with 1 mL L−1 1-MCP. At the intermediate level of maturity 1-MCP was more effective in counteracting firmness loss during prolonged storage (Fig. 2B). Treatment with 975 nL L−1 1-MCP strongly delayed softening, from 84.3 N to 76 N after 6 months of storage. In contrast the lowest 1-MCP concentration led to a more rapid softening (84.3 N to 65 N) than in fruit exposed to higher 1-MCP concentrations, but it was still less (45.2 N) than that of the untreated apples (Table 4). Thus is possible that 1-MCP treatment may be used to delay undesired softening effects in fruit that is highly mature at harvest and meet the market minimum out-turn requirement of about 65–70 N (Burmeister et al., http://www.pinkladyapples. com/Technical/docs/Storage.pdf).13 After 6 months of storage apples in the present study that were fully mature at harvest had a mean firmness of 65 N when treated with 975 nL L−1 .
© 2014 Society of Chemical Industry
J Sci Food Agric 2014; 94: 2691–2698
Response of Pink Lady apples to 1-MCP and controlled atmosphere
www.soci.org
Figure 2. Response surface for the effect of 1-MCP dose and maturity at harvest (A) or storage time (B) on the firmness of Pink Lady® apples after 4 months of storage at 2 ∘ C (A), or mature at harvest (starch index, 5.95 ± 0.35) (B). Table 4. Quality indices of Pink Lady® fruits untreated and treated with 1-methylcyclopropene (1-MCP), stored in air (RA) and in a controlled atmosphere (CA) for 6 months (2 ∘ C) and submitted to a commercial life simulation at 20 ∘ C for 7 days (6 months + 7 days) Sample and effects Sample Untreated + RA Untreated + CA 1-MCP + RA 1-MCP + CA Effects 1-MCP CA 1-MCP × CA
Firmness (N) 6 months
45.27d 54.97c 62.21b 68.40a
CO2 production (mL kg−1 h−1 )
Ripening index
6 months + 7 days
46.22d 57.20c 56.98c 68.89a
∗∗∗
∗∗
∗∗
∗∗
∗
∗∗
6 months
6 months + 7 days
41.6c 42.0c 39.9c 37.2d
48.2a 46.2b 45.5b 45.0b ∗
NS NS
NS NS
∗
6 months
17.5a 19.6a 11.7c 13.8b
6 months + 7 days
17.8a 14.4b 14.1b 11.9c
∗∗
∗
NS NS
∗ ∗∗
Results are given as mean values, mean comparison and ANOVA results. Ripening index is measured as soluble solids content/titratable acidity. Mean values of the same index marked with different letters are significantly different at P < 0.05 level. Significance of effects as determined by ANOVA analysis: ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001. NS, not significant.
J Sci Food Agric 2014; 94: 2691–2698
After 6 months of storage fruit harvested at the intermediate maturity stage and treated with the highest dose of 1-MCP showed a ripening index of 32.5, whilst the untreated fruit registered a value of 41.6 (Table 4). A separate analysis of SSC and TA as a function of the factor variables (data not reported), indicated that 1-MCP did not affect SSC but strongly inhibited loss of acidity. Thus, the main effect of 1-MCP on the ripening index was to delay the physiological evolution of acidity. Fan et al.21 found that 1-MCP treatment maintained TA in ‘Red Delicious’, ‘Granny Smith’, ‘Fuji’, ‘Jonagold’, ‘Ginger Gold’ and ‘Gala’ apples during storage, and also Watkins et al.6 found that TA of ‘Law Rome’, ‘Delicious’, ‘Empire’ and ‘McIntosh’ was always higher in 1-MCP-treated than control fruit during air storage. Contrasting results were reported regarding the effect of 1-MCP treatment on soluble solids content during storage. Fan and Mattheis3 reported that soluble solids were higher in 1-MCP-treated apples but other authors4 reported that SCC was unaffected by 1-MCP. Watkins et al.6 found differences in the responses of apple cultivars to 1-MCP treatments; ‘McIntosh’ and ‘Law Rome’ fruit treated with 1-MCP had lower SCC than untreated
© 2014 Society of Chemical Industry
wileyonlinelibrary.com/jsfa
2695
Effect of 1-MCP treatment on ripening index All experimental factors and their interactions significantly influenced the ripening index at a probability ≤ 0.001 and the regression model described the evolution of ripening index with a high adjusted R2 value of 0.997, and a low standard error to estimate of 0.42 (Table 3). Regression coefficients of the model indicate that the most important factor contributing to an increase in the ripening index is maturity at harvest followed by storage time and 1-MCP concentration (Table 3). Although ripening index was positively affected by 1-MCP, the regression coefficient is very low, and thus the influence of 1-MCP concentration is negligible in practical terms; this conclusion is also supported by data presented in Table 4. Under the experimental conditions tested in this study, 1-MCP showed a negative combined effect with the other factors on ripening index. This demonstrates a higher effectiveness of the 1-MCP treatments by showing that they were very effective in delaying ripening fruits that were fully mature at harvest (contrary to that observed for firmness) and after prolonged storage, similarly to that observed for firmness (Fig. 3A and B).
www.soci.org
E Cocci et al.
Figure 3. Response surface for the effect of 1-MCP dose and maturity at harvest (A) or storage time (B) on the ripening index of Pink Lady® apples after 4 months of storage at 2 ∘ C (A), or mature at harvest (starch index, 5.95 ± 0.35) (B).
fruit whilst ‘Delicious’ and ‘Empire’ had higher SCC; thus these differences may be cultivar dependent. Effect of 1-MCP treatment on CO2 production The regression model for CO2 production showed a high adjusted R2 value of 0.915 with a standard error to estimate of 0.62 (Table 3), therefore the proposed model was adequate to describe carbon dioxide production as function of the selected factor variables. This parameter was significantly influenced by all factors (P < 0.001) except 1-MCP where the individual effect was not significant, but it did show significant combined effects with maturity stage at harvest and storage time. The response surface describing CO2 production after 4 months of storage (central value) as a function of 1-MCP concentration and maturity at harvest indicate that 1-MCP had the greatest influence on delaying of CO2 production in partially mature fruits (Fig. 4A) similarly to what was observed for firmness. In fruit harvested at the intermediate maturity 1-MCP dose had little influence on CO2 production at the beginning of storage but the negative combined effect of 1-MCP dose and storage time on the response variable resulted in a higher effectiveness of the 1-MCP treatment after prolonged storage (Fig. 4B). Thus, CO2 production of mature Pink Lady® apples did not change significantly during storage for 6 months after treatment with the highest 1-MCP concentration (975 nL L−1 ) while it showed a dramatic increase after treatment with the lowest concentration (325 nL L−1 ).
2696
Effect of 1-MCP treatment and controlled atmosphere on fruit quality Instrumental quality measurements of fruit with or without 1-MCP treatment and stored for 6 months in RA or CA at 2 ∘ C varied according to treatment (Table 4). Untreated fruit softened and lost about 50% of initial post-harvest firmness (83.3 N) during air storage. Both CA storage and 1-MCP significantly delayed softening during storage with the latter more efficient than the former. Moreover a positive effect on firmness retention was obtained by combining CA storage and 1-MCP treatment (650 nL L−1 ). De Castro et al.12 found no interaction among CA and 1-MCP treatments on firmness of Pink Lady® stored for 4 months, but in that study experimental conditions (1-MCP concentration, storage time) were different from those tested here. The 6 months storage
wileyonlinelibrary.com/jsfa
time at 2 ∘ C resulted in an increase of ripening index and CO2 production, the latter was increased by CA and limited by 1-MCP whilst the former was limited by the combined effect of 1-MCP and CA storage. Following 7 days commercial shelf life, an acceleration of metabolic reactions (in terms of firmness loss, increase of ripening index and CO2 production) was observed in both treated (1-MCP or/and CA) and untreated samples. At the end of the shelf life simulation CA and 1-MCP treated samples showed better quality retention than untreated samples for all the parameters considered, and CA and 1-MCP showed a positive combined effects on firmness and CO2 production with the combination of CA storage and 1-MCP treatment being the most effective in maintaining firmness. Sensory analysis (Table 5) indicated that 1-MCP treatment did not affect sweetness or whole fruit odour but depleted cut fruit odour. In contrast, the 1-MCP treated sample was more sour, aromatic, crisp and firm and showed lower values of mealiness and fermented odour than untreated sample. Similar effects for sourness, firmness and crispness were achieved by using CA storage alone. A positive combined effect of CA storage and 1-MCP treatment was observed on apple firmness. The good correlation among the firmness, titratable acidity and soluble solids content values by instrumental and sensory evaluation (R2 > 0.90) confirms the judges’ reliability. Apples treated with 1-MCP and stored in RA received the highest score in overall assessment after sensory evaluation. The parameters having most influence on acceptability of Pink Lady® apple were SSC, TA and aroma volatile profile rather than the firmness.11 On the basis of these findings suggested that even if CA storage better preserved fruit texture, with positive implications on fruit quality maintenance, it is not strictly necessary to meet the needs of the final consumer.
CONCLUSION 1-MCP treatment positively affected the retention of quality of Pink Lady® apples and its effect could be improved in combination with other sorting and processing variables (fruit maturity, storage time and controlled atmosphere storage). The ripening-associated quality changes were differently influenced by 1-MCP treatments, thus suggesting a specific effect of this compound on different metabolic pathways.
© 2014 Society of Chemical Industry
J Sci Food Agric 2014; 94: 2691–2698
Response of Pink Lady apples to 1-MCP and controlled atmosphere
www.soci.org
Figure 4. Response surface for the effect of 1-MCP dose and maturity at harvest (A) or storage time (B) on CO2 production of Pink Lady® apples after 4 months of storage at 2 ∘ C (A), or mature at harvest (starch index, 5.95 ± 0.35) (B). Table 5. Results (median value and ANOVA) of the sensory evaluation of Pink Lady® apples treated with 1-methylcyclopropene (1-MCP) and different storage atmospheres (RA or CA) after 6 months of storage at 2 ∘ C Samples Sensory attribute Whole fruit odour Cut fruit odour Sweetness Sourness Apple flavour Fermented flavour Off-flavour Crispness Firmness Juiciness Mealiness
Untreated + RA 5.0a 5.0a 5.5a 2.0b 4.0b 3.0a 1.5a 4.5b 4.0d 5.0b 4.5a
Untreated + CA
Effects 1-MCP + RA
5.0a 5.0ab 5.0a 4.5a 4.5ab 2.0b 2.0a 6.5a 5.0c 5.5ab 3.0b
1-MCP + CA
5.5a 4.0b 5.5a 5.5a 5.0a 2.0b 1.0a 7.0a 6.0b 5.5ab 2.5b
5.0a 4.0ab 5.0a 5.0a 4.0b 2.0b 1.5a 6.5a 7.0a 6.0a 2.0b
1-MCP NS ∗
NS
CA NS NS NS
∗∗
∗
∗
NS
∗
∗
NS
NS
1-MCP × CA NS NS NS NS NS NS NS NS
∗∗
∗
∗∗∗
∗
∗
NS
NS NS
NS ∗∗∗
∗∗
Mean values for the same attribute marked with different letters are significantly different at P < 0.05 level. Significance of effects as determined by ANOVA analysis: ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001. CA, controlled atmosphere; RA, stored in air; NS, not significant.
Results obtained in this study have commercial implications by indicating that treatment with 1-MCP at different concentrations can be optimised depending on fruit maturity and expected storage time to predict fruit quality modifications during storage to meet market/consumer needs. These considerations are very important for Pink Lady® apples because the quality of these fruits is mostly assessed by colour at harvest which, in turn, depends on weather conditions that determine the pace of red blush development and, consequently, maturity of the fruit at commercial harvest. Use of the full red blush as a ripening index can be a limiting factor for fruit quality because it becomes over-mature when left on the tree for too long and late-harvested fruit showed increased greasiness and internal browning.12
REFERENCES
J Sci Food Agric 2014; 94: 2691–2698
© 2014 Society of Chemical Industry
wileyonlinelibrary.com/jsfa
2697
1 Aked J, Maintaining the post-harvest quality of fruits and vegetables, in Fruit and Vegetable Processing. Improving quality, ed. by Jongen W. Woodhead Publishing, Cambridge, UK, pp. 119–149 (2002).
2 Watkins CB, 1-Methylcyclopropene (1-MCP) based technologies for storage and shelf-life extension. Int J Postharvest Technol Innov 1:62–68 (2006). 3 Fan X and Mattheis JP, Impact of 1-methylcyclopropene and methyl jasmonate on apple volatile production. J Agric Food Chem 47:2847–2853 (1999). 4 Rupasinghe HPV, Murr DP, Paliyath G and Skog L, Inhibitory effect of 1-MCP on ripening and superficial scald development in ‘McIntosh’ and ‘Delicious’ apples. J Hort Sci Biotechnol 75:271–276 (2000). 5 Watkins CB and Nock JF, Effects of delays between harvest and 1-methylcyclopropene treatment, and temperature during treatment, on ripening of air-stored and controlled-atmosphere-stored apples. HortScience 40:2096–2101 (2005). 6 Watkins CB, Nock JF and Whitaker BD, Responses of early, mid and late season apple cultivars to postharvest application of 1-methylcyclopropene (1-MCP) under air and controlled atmosphere storage conditions. Postharvest Biol Technol 19:17–32 (2000). 7 Mir NA, Curell E, Khan N, Whitaker M and Beaudry RM, Harvest maturity, storage temperature, and 1-MCP application frequency alter firmness retention and chlorophyll fluorescence of ‘Redchief Delicious’ apples. J Am Soc Hort Sci 126:618–624 (2001).
www.soci.org 8 Vallejo F and Beaudry R, Depletion of 1-MCP by ‘non-target’ materials from fruit storage facilities. Postharvest Biol Technol 40:177–182 (2006). 9 Huber D, Suppression of ethylene responses through application of 1-methylcyclopropene: A powerful tool for elucidating ripening and senescence mechanisms in climateric and nonclimateric fruits and vegetables. Hortscience 43:106–111 (2008). 10 Cripps JEL, Richards LA and Mairata AM, ‘Pink Lady’ apple. HortScience 28:1057 (1993). 11 Lopez ML, Villatoro C, Fuentes T, Graell J and Echeverria G, Volatile compounds, quality parameter and consumer acceptance of “Pink Lady®” apples stored in different conditions. Postharvest Biol Technol 43:55–66 (2006). 12 De Castro E, Biasi WV and Mitcham EJ, Quality of Pink Lady apples in relation to maturity at harvest, prestorage treatments, and controlled atmosphere during storage. HortScience 42:605–610 (2007). 13 Wilkinson RI, Frisina C, Partington DL, Franz PR, Brien CJ, Thomson F, et al., Effects of 1-methylcyclopropene on firmness and flesh browning in Pink LadyTM apples. J Hort Sci Biotechnol 83:165–170 (2008). 14 Peirs A, Scheerlinck N, Berna Perez A, Jancsok P and Nicolai BM, Uncertainty analysis and modelling of the starch index during
15 16 17 18
19 20 21
E Cocci et al.
apple fruit maturation. Postharvest Biol Technol 26:199–207 (2002). Mendoza F and Aguilera JM, Application of image analysis for classification of ripening bananas. J Food Sci 69:E471–E477 (2004). Sweeney JP, Chapman VJ and Heoner PA, Sugar, acid and flavor in fresh fruit. J Am Diet Assoc 57:432–435 (1970). Association of Official Analytical Chemists, Official Methods of Analysis of the Association of Official Analytical Chemists, 16th edition. AOAC, Washington DC (1995). International Organization for Standardization, Sensory analysis – General guidance for the selection, training and monitoring of assessors – Part 1: Selected assessors, ISO8586-1:1993. ISO, Geneva (1993). International Organization for Standardization, Sensory analysis – General guidance for the selection, training and monitoring of assessor – Part 2: Experts, ISO8586-2:1994. ISO, Geneva (1994). International Organization for Standardization, Sensory analysis – General guidance for the design of test rooms, ISO8589:1988. ISO, Geneva (1988). Fan X, Mattheis JP and Blankenship SM, Development of apple superficial scald, soft scald, core flush, and greasiness is reduced by MCP. J Agric Food Chem 47:3063–3068 (1999).
2698 wileyonlinelibrary.com/jsfa
© 2014 Society of Chemical Industry
J Sci Food Agric 2014; 94: 2691–2698
Revised: 3 February 2014
Accepted article published: 10 February 2014
Published online in Wiley Online Library: 24 March 2014
(wileyonlinelibrary.com) DOI 10.1002/jsfa.6610
Response of Pink Lady® apples to post-harvest application of 1-methylcyclopropene as a function of applied dose, maturity at harvest, storage time and controlled atmosphere storage Emiliano Cocci,a* Giampiero Sacchetti,b Pietro Rocculia,c and Marco Dalla Rosaa,c Abstract BACKGROUND: 1-Methylcyclopropene (1-MCP) is an inhibitor of ethylene reception used in post-harvest treatments to delay fruit ripening. Several factors affect the efficacy of 1-MCP treatments. The effect of a post-harvest treatment with 1-MCP on the quality of Pink Lady® apples as a function of 1-MCP dose, storage time and maturity at harvest was investigated. 1-MCP treatment was further tested in combination with controlled atmosphere (CA) storage. RESULTS: 1-MCP limited fruit respiration and softening during storage and was more effective on partially matured fruits and at prolonged storage times. The delaying of 1-MCP on the increase of ripening index was greater on matured fruits at prolonged storage times. The combination of 1-MCP and CA treatments positively affected quality indices of mature apples during 6 months of storage and 7 days of commercial life, with 1-MCP being more effective than CA. 1-MCP and CA showed positive combined effects on firmness and ripening index after 6 months of storage, and on firmness and CO2 production after a further 7 days of commercial life. CONCLUSION: By knowing fruit maturity at harvest and expected storage time it is possible to choose the most suitable 1-MCP dose to meet the market requirements by applying a simple polynomial model. © 2014 Society of Chemical Industry Keywords: Pink Lady® apples; methylcyclopropene; controlled atmosphere; ripening; firmness; sensory analysis
INTRODUCTION
J Sci Food Agric 2014; 94: 2691–2698
∗
Correspondence to: Emiliano Cocci, CIRI Agroalimentare, Campus Scienze degli Alimenti, Via Ravennate 1020, 47521 Cesena (FC) Italy. E-mail: [email protected]
a CIRI Agroalimentare, Campus Scienze degli Alimenti, Via Ravennate 1020, 47521, Cesena, Italy b Faculty of Bioscience and Technology for Food, Agriculture and Environment, University of Teramo, Via C.R. Lerici 1, 64023, Teramo, Italy c Department of Agri-Food Science and Technology – DISTAL, University of Bologna, Campus of Food Science, Piazza Goidanich 60, 47023, Cesena, Italy
www.soci.org
© 2014 Society of Chemical Industry
2691
Maintaining post-harvest quality of apples during storage and distribution is one of the major challenges facing the fruit industry. Fruit and vegetables are living commodities and their respiration and ripening rate are important factors for their quality maintenance.1 In fruit storage, management techniques used to minimise the effects of ethylene include low O2 and high CO2 atmospheres, as well as reduced temperature. 1-Methylcyclopropene (1-MCP) is an inhibitor of ethylene reception in plant tissues thought to act by binding irreversibly to ethylene receptors.2 Its availability, as a stable complexed formulation that releases 1-MCP when dissolved in water, has led to a surge of research and commercial interest around the world. 1-MCP is marketed as EthylBloc™ (Floralife Inc., Walterboro, SC, USA) and SmartFresh™ (AgroFresh Inc., Yakima, WA, USA). The post-harvest gaseous application of 1-MCP has been shown to reduce ethylene production and respiration, to maintain many quality characteristics of apple, such as fruit firmness and acidity retention, and to reduce superficial scald development, peel
greasiness and various chilling-related disorders.3 – 5 Important factors which govern efficacy of 1-MCP treatment are: (1) active concentration, treatment time and temperature; (2) type of fruit and maturity stage at the time of treatment; and (3) treatment frequency, because, although 1-MCP binding to the ethylene receptor is essentially irreversible, the inhibition of the effects of ethylene may be overcome by the production of new receptors.6,7 Under commercial conditions, handling practices need to take into
www.soci.org account many other factors, which also include the desired effects on quality, because for many products, partial control of ripening is more desirable than total control.8,9 ‘Pink Lady®’ apples originated from a cross between ‘Lady Williams’ and ‘Golden Delicious’.10 Since 1990, this cultivar has been extensively cultivated in the main apple-producing areas of the world, because of its excellent sensory attributes: it is firm, has fine dense flesh, is crisp and juicy, provides excellent flavour, and has a high sugar–acid balance.11 To the authors’ knowledge only a few studies have investigated the effects of 1-MCP on the post-harvest quality of Pink Lady® apples. In particular, De Castro et al.12 found that 1-MCP (1 mL L−1 ) conserved fruit quality in air as well as a controlled atmosphere (CA) during 4 months of storage. These authors included in the experimentation different maturity stages but they used only one of them to study the effects of 1-MCP on apple quality. Other authors13 reported that the 1-MCP-treated apple (0.625 mL L−1 ) were firmer than untreated fruit. In this paper the authors used a multi-phase experiment by considering four factors (orchard, 1-MCP treatment, storage and export) but they did not investigate the relationship among different factors involved in 1-MCP effectiveness. Therefore, the aim of the present work was to study the effect of a semi-commercial post-harvest treatment with 1-MCP on the quality of Pink Lady® apple, as a function of 1-MCP dose, fruit maturity stage and storage time, by a multi-response approach based on factorial design, in order to investigate the individual and combined effect of each factor. Finally, the individual and combined effect of post-harvest 1-MCP and controlled atmosphere treatments was studied by keeping all the factors at constant level.
Table 1. Coding and levels of independent variables used in the experimental design Variable Trial no. 1 2 3 4 5 6 7 8 9 10 11
1-MCP (ppb) 325 975 325 975 325 975 325 975 650 650 650
Storage time (months) 2 2 6 6 2 2 6 6 4 4 4
Coded Maturity at harvesta 4 4 4 4 8 8 8 8 6 6 6
x1
x2
x3
−1 1 −1 1 −1 1 −1 1 0 0 0
−1 −1 1 1 −1 −1 1 1 0 0 0
−1 −1 −1 −1 1 1 1 1 0 0 0
a Maturity at harvest was measured as starch index, on a scale of 1–10. 1-MCP, 1-methylcyclopropene.
It was assumed that a mathematical function [Eqn (1)] exists for the response variable Y (quality parameters) in terms of three independent process variables X 1 (1-MCP concentration), X 2 (ripening stage at harvest) and X 3 (storage time), with all other factors unaltered: ) ( (1) Y = 𝜑 X1 , X2 , X3 To approximate the function 𝜑 a first-order polynomial of three variables was used:
MATERIALS AND METHODS Plant material Pink Lady® apples were harvested in early morning from ten 40-tree plots in the same orchard near Cesena (Italy). Thirty apples were randomly collected from orchard trees weekly starting from the first week of October; the progress of starch conversion to sugars (on a scale of 1–10) was followed over time.14 Starch index reached 3.90 ± 0.34 on 3 November, 5.95 ± 0.30 on the 15 November and 8.05 ± 0.20 on the 2 December 2006. The three different maturity stages, selected on the basis of the starch index, were chosen according to a commercial maturity chart and kept equidistant among them. When apples reached the established maturity stages they were harvested selected on the basis of visual colour, in accordance with the Association Pink Lady® Europe: fruit diameter > 70 mm; 50% of diffuse pink or 30% intense pink; background colour (changing between green and yellow) and absence of defects.
2692
Experimental design Effects of 1-MCP treatment on the quality of Pink Lady® apples as a function of fruit maturity stage, 1-MCP dose and storage time For this experiment, 1100 kg of apple fruits were used (100 kg for each trial). An incomplete three-level three-factor design was adopted, using a three-factor constrained experimental region. The three independent variable levels were coded as −1 (lowest level), 0 (intermediate level) and +1 (highest level). Their actual values (Xi ), chosen from preliminary studies and from literature and the corresponding coded value (xi ) are given in Table 1. The design consisted of 11 experimental trials, which included three replications of the central point (X 1 = X 2 = X 3 = 0).
wileyonlinelibrary.com/jsfa
E Cocci et al.
Y = 𝛽0 + 𝛽1 X1 + 𝛽2 X2 + 𝛽3 X3 + 𝛽12 X1 X2 + 𝛽23 X3 X2 + 𝛽13 X1 X3
(2)
where Y is the response (quality parameters), xi are the coded independent variables (linearly related to Xi ), and 𝛽 0 , 𝛽 i , and 𝛽 ij are the regression coefficients. Response surface methodology was used to graphically represent the mathematical functions. Effect of 1-MCP treatment and controlled atmosphere storage on fruit quality For this further experiment, 200 kg of fruits have been used (100 kg untreated and 100 kg treated apples). Actually, in order to study the effects of 1-MCP treatment in combination with the CAs, apples of an intermediate maturity stage (Table 2) were left untreated or were treated with 1-MCP (650 nL L−1 ). Fifty kilograms of both treated and untreated fruit were stored in a regular atmosphere (RA) or in CA [1.8% O2 , 1.4% CO2 at 2.0 ∘ C and 95% relative humidity (RH)]. The CA conditions were chosen according to the industrial practices and are within the range reported by Lopez et al.11 Fruit analyses were performed after 6 months of storage at 2 ∘ C plus a period of 7 days at 20 ∘ C and 65% RH, to simulate fruit commercial life. Treatment with 1-MCP For each treatment run, 100 kg of apples were placed into a commercial plastic bin (1 m3 volume) and allowed to equilibrate at 2 ∘ C overnight. The bins were inserted in sealed air-tight plastic bag and exposed to a specific 1-MCP concentration (325, 650 or 975 nL L−1 )
© 2014 Society of Chemical Industry
J Sci Food Agric 2014; 94: 2691–2698
Response of Pink Lady apples to 1-MCP and controlled atmosphere
www.soci.org
Table 2. Quality characteristics (mean ± SD) of Pink Lady® apples at different maturity levels at harvest
Maturity Partially mature Mature Fully mature
Starch indexa 3.90 ± 0.34 5.95 ± 0.35 8.05 ± 0.29
Firmness (N) 87.7 ± 0.7 84.3 ± 0.8 78.7 ± 0.6
Soluble solids content (%) 11.7 ± 0.6 13.9 ± 0.1 14.2 ± 0.1
Titratable acidityb (mg 100 g−1 FW) 0.71 ± 0.01 0.67 ± 0.02 0.65 ± 0.01
Ripening indexc 6.5 ± 0.1 20.8 ± 0.1 21.8 ± 0.6
CO2 production (mL kg−1 h−1 ) 7.89 ± 0.17 11.71 ± 2.34 15.14 ± 0.56
Ethylene production (μL kg−1 h−1 ) 0.43 ± 0.01 0.54 ± 0.03 0.57 ± 0.01
a On a scale of 1–10. b Calculated as mg of malic acid. c Soluble solids content/titrable acidity.
(SmartFresh™; Agrofresh) for 24 h at 2 ∘ C. The concentration of 1-MCP was calculated according to the % active ingredient and release from the SmartFresh™ powder into the free head-space of sealed bags, by using an electric fan provided by Agrofresh. Starch index Starch index was determined in 30 apples by dipping cross-sectional fruit halves in an iodine solution (40 g KI + 10 g I2 L−1 ) for 30 s; starch hydrolysis was rated using a scale of 1–10. To improve accuracy and precision of the starch index scores, a methodology based on image analysis was used to calculate the ratio of blue-stained to non-stained areas of iodine-treated apples, as proposed by Peirs et al.14 To calculate the percentage of dark-starch area in sample cross-sectional area, a calibration curve was built by using the images of a French 10-point radial type pictorial scale, where 1 = 0% loss of starch and 10 = 100% loss [CTIFL 2002 (http://www.fruits-et-legumes. net/revue-en-ligne/point-sur/fich-pdf/code-amidon.pdf)]. Images were captured using an image acquisition system similar to that developed by Mendoza and Aguilera.15 Samples were illuminated using two parallel lamps (mod. TL-D Deluxe, Natural Daylight, 18 W/965; Philips, New York, USA) with a colour temperature of 6500 K (D65 ) and a colour-rendering index close to 90%. Four fluorescent tubes (60 cm long) were situated 35 cm above the sample and at an angle of 45∘ to it. A colour digital camera (Canon PowerShot A70) was located vertically over the sample at a distance of 12.5 cm. The angle between the camera lens and the lighting source axis was approximately 45∘ . The lamps and the colour digital camera were kept in a black box to exclude light. Recorded images were isolated from the background and processed using the software Photoshop® v. 6.0 (Adobe System Incorporated, San Jose, CA, USA) (Fig. 1B). After conversion in grey scale (16 BPP) and elimination of seed area (Fig. 1A), images were evaluated with advance Image Analysis Software (Image Pro-Plus® v. 6.2 Media Cybernetics, USA) for automatic calculation of the ratio of non-stained to blue-stained area within fruit flesh using a grey-scale threshold value (13 575 BPP) (Fig. 1B). Reference charts were analysed using the same technique. For each maturity stage at harvest, 60 images of 60 apple halves were acquired and analysed.
J Sci Food Agric 2014; 94: 2691–2698
III S/L (Witt-Gasetechnik, Witten, Germany). The apparatus has a paramagnetic sensor for O2 and a mini-infrared spectrophotometer for detection of CO2 . The instrument was calibrated with O2 and CO2 air percentages. Free headspace volume (estimated by water displacement) was measured after gas measurement. Data were expressed as mL CO2 kg−1 h−1 . Measurements of respiration rate were made using three replicate groups of four fruit per treatment. Ethylene production rate Four fruit were weighed and sealed into 2.5-L air-tight respiration glass jars identical to those used for respiration rate determination. Gas samples (1 mL) were withdrawn from the headspace volume of jars. Ethylene was quantified using a gas chromatograph model Clarus 500 (Perkin–Elmer Corporation, Wellesley, MA, USA), equipped with a flame ionisation detector and an Elite QPlot column (30 m × 0.32 mm i.d.) at 80 ∘ C, using helium as a carrier gas (50.3 cm s−1 ). Injector and detector temperature were 120 and 155 ∘ C respectively. Three independent ethylene samples per chamber were taken and results were expressed as a mean (mL kg−1 h−1 ). Ethylene samples of known concentration (1 mL L−1 ) were routinely used for equipment calibration. Ripening index The ratio between soluble solids content (SSC) and titratable acidity (TA) was used as the ripening index (Sweeney et al.16 ). SSC was measured on the juice obtained from 20 homogenised fruit per sample per sampling time, by using a digital refractometer model PR1 (Atago, Tokyo, Japan). TA was determined by titration with 0.1 mol L−1 NaOH to pH 8.117 of the juice obtained from 20 homogenised fruit per sample per sampling time.
© 2014 Society of Chemical Industry
wileyonlinelibrary.com/jsfa
2693
Respiration rate Four fruit were weighed and sealed into 2.5-L air-tight respiration glass jars. CO2 was allowed to accumulate to 0.1–0.3% at 20 ∘ C; this took from 30 min to 2 h, depending on ripening stage. Concentration of CO2 in the headspace of each jar was determined by rapid gas analysis, using an O2 /CO2 gas analyser mod. MFA
Figure 1. Iodine-treated Pink Lady® halves after conversion in grey scale (16 BPP) and elimination of seed area (A), and virtually coloured (B) on the basis of grey-scale pixels values (threshold, 13 575 BPP).
www.soci.org Fruit firmness Penetration tests were carried out using an 11 mm diameter probe on opposite sides (blush and green) on 20 fruits flesh after peel removal. A texture analyser model HD500 (Stable Micro Systems, Guildford, UK) was used and the maximum force measured during the test was considered as firmness (in newtons). Sensory evaluation A quantitative descriptive analysis was carried out by a panel of eight trained assessors. Assessors were selected and trained according to the international standards ISO8586-1:199318 and ISO8586-2:1994.19 The analysis was carried out in individual booths located in a laboratory designed according to ISO8589:1988.20 Thirty apples per sample (treatment) were used for sensory analysis. Samples were kept in a room at 20 ∘ C overnight after removal from storage chambers before sensory evaluation. Each fruit was divided into four pieces prior to sensory evaluation. Each sample was identified by a random three-digit code. The order in which apples were presented to each judge was randomised. Judges were free to test samples as often as they desired. Mineral water was used as a palate cleaner between samples. Eleven attributes were selected by assessors in preliminary meetings: whole fruit odour intensity, cut fruit odour intensity, sweetness, sourness, apple flavour intensity, fermented flavour, off-flavour, crispness, firmness, juiciness and mealiness. Judges were asked to rate the intensity of each attribute on a structured scale of 1–9. All evaluations were conducted in individual booths under white illumination (D65 illuminant) and at room temperature. Sensory evaluations were repeated twice: in the morning and in the afternoon. Statistical analysis Results were reported as average values and standard deviations (SDs) calculated from different analytical repetitions. ANOVA was carried out to test the significance of the effects of experimental factors and comparison between means was carried out by the least significant difference (LSD) test. Multiple regression analysis was carried out by using response variables as dependent variables and the individual and interactive effects of the factors of the experimental design as independent variables. Multiple regression analysis was carried out using a backward stepwise procedure; for this purpose the F to enter was set in order to include in the model only the variables significant at a value of P < 0.05. Adequacy of fitted models was verified by using the adjusted determination coefficient (R2 adj. ), the associated P value, the Fisher’s F value and the standard error of estimate. Response surfaces on three-dimensional plots were calculated from the best-fit models by setting one factor at its intermediate level. All the statistical analyses were carried out using the Statistica v. 6.0 (Statsoft, Tulsa, OK, USA) software
RESULTS AND DISCUSSION
2694
Fruit maturity at harvest At harvest quality parameters measured on the Pink Lady® apple fruits are shown in Table 2. The starch index of the intermediate harvest was equidistance through independent variable levels; this target was required to ensure that the experimental design was valid. As a consequence of the different physiological states of fruit at harvest, firmness, soluble solids content, titratable acidity,
wileyonlinelibrary.com/jsfa
E Cocci et al.
Table 3. Values of the first order polynomial regression coefficients representing the relationship between firmness, ripening index and CO2 production and the independent variables Coefficient 𝛽0 𝛽1 𝛽2 𝛽3 𝛽 12 𝛽 23 𝛽 13 R2 adj P-level SE
Firmness (N) 85.9341 0.01890 −2.23927 NS 0.00207 −0.32593 −0.00278 0.958 < 0.0001 1.8720
Ripening indexa
CO2 production (mL kg−1 h−1 )
4.916490 0.001688 2.320445 2.993021 −0.000709 0.127765 −0.001096 0.997 < 0.0001 0.41902
−0.952460 NS 2.977988 1.189331 −0.001191 −0.270917 0.000231 0.914 < 0.0001 0.619
a Soluble solids content/titrable acidity. NS, not significant; R2 adj , adjusted R2 ; SE, standard error.
ethylene and carbon dioxide production rate were different and consistent with the starch index, confirming that this parameter is an appropriate tool to describe maturity at harvest for Pink Lady® apples.12 Effect of 1-MCP treatment on firmness The regression model calculated for firmness (Table 3) showed a high adjusted R2 value of 0.958 with a standard error of estimation of 1.87; thus the proposed model adequately described firmness values as a function of the selected variables. Firmness was influenced by all the considered variables at high probability level (P ≤ 0.001) except maturity at harvest. The response surface describing firmness after 4 months of storage (central value) as a function of 1-MCP concentration and starch index at harvest indicates (Fig. 2A) that 1-MCP concentration and fruit maturity stage had a negative combined effect on firmness in accordance with previous research in which more mature fruit usually were less affected by 1-MCP treatment.7 After 6 months of storage, the firmest fruit were those that were partially mature at harvest and treated with the highest 1-MCP concentration. The same fruit, after 4 months of storage, had a firmness value of about 87 N, which is similar to the initial value of 87.7 N (Table 2). This suggests that, in these semi-commercial conditions, fruit softening was completely blocked; this agrees with De Castro et al.12 who found no softening of Pink Lady® apples during 4 months of refrigerated air storage following treatment with 1 mL L−1 1-MCP. At the intermediate level of maturity 1-MCP was more effective in counteracting firmness loss during prolonged storage (Fig. 2B). Treatment with 975 nL L−1 1-MCP strongly delayed softening, from 84.3 N to 76 N after 6 months of storage. In contrast the lowest 1-MCP concentration led to a more rapid softening (84.3 N to 65 N) than in fruit exposed to higher 1-MCP concentrations, but it was still less (45.2 N) than that of the untreated apples (Table 4). Thus is possible that 1-MCP treatment may be used to delay undesired softening effects in fruit that is highly mature at harvest and meet the market minimum out-turn requirement of about 65–70 N (Burmeister et al., http://www.pinkladyapples. com/Technical/docs/Storage.pdf).13 After 6 months of storage apples in the present study that were fully mature at harvest had a mean firmness of 65 N when treated with 975 nL L−1 .
© 2014 Society of Chemical Industry
J Sci Food Agric 2014; 94: 2691–2698
Response of Pink Lady apples to 1-MCP and controlled atmosphere
www.soci.org
Figure 2. Response surface for the effect of 1-MCP dose and maturity at harvest (A) or storage time (B) on the firmness of Pink Lady® apples after 4 months of storage at 2 ∘ C (A), or mature at harvest (starch index, 5.95 ± 0.35) (B). Table 4. Quality indices of Pink Lady® fruits untreated and treated with 1-methylcyclopropene (1-MCP), stored in air (RA) and in a controlled atmosphere (CA) for 6 months (2 ∘ C) and submitted to a commercial life simulation at 20 ∘ C for 7 days (6 months + 7 days) Sample and effects Sample Untreated + RA Untreated + CA 1-MCP + RA 1-MCP + CA Effects 1-MCP CA 1-MCP × CA
Firmness (N) 6 months
45.27d 54.97c 62.21b 68.40a
CO2 production (mL kg−1 h−1 )
Ripening index
6 months + 7 days
46.22d 57.20c 56.98c 68.89a
∗∗∗
∗∗
∗∗
∗∗
∗
∗∗
6 months
6 months + 7 days
41.6c 42.0c 39.9c 37.2d
48.2a 46.2b 45.5b 45.0b ∗
NS NS
NS NS
∗
6 months
17.5a 19.6a 11.7c 13.8b
6 months + 7 days
17.8a 14.4b 14.1b 11.9c
∗∗
∗
NS NS
∗ ∗∗
Results are given as mean values, mean comparison and ANOVA results. Ripening index is measured as soluble solids content/titratable acidity. Mean values of the same index marked with different letters are significantly different at P < 0.05 level. Significance of effects as determined by ANOVA analysis: ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001. NS, not significant.
J Sci Food Agric 2014; 94: 2691–2698
After 6 months of storage fruit harvested at the intermediate maturity stage and treated with the highest dose of 1-MCP showed a ripening index of 32.5, whilst the untreated fruit registered a value of 41.6 (Table 4). A separate analysis of SSC and TA as a function of the factor variables (data not reported), indicated that 1-MCP did not affect SSC but strongly inhibited loss of acidity. Thus, the main effect of 1-MCP on the ripening index was to delay the physiological evolution of acidity. Fan et al.21 found that 1-MCP treatment maintained TA in ‘Red Delicious’, ‘Granny Smith’, ‘Fuji’, ‘Jonagold’, ‘Ginger Gold’ and ‘Gala’ apples during storage, and also Watkins et al.6 found that TA of ‘Law Rome’, ‘Delicious’, ‘Empire’ and ‘McIntosh’ was always higher in 1-MCP-treated than control fruit during air storage. Contrasting results were reported regarding the effect of 1-MCP treatment on soluble solids content during storage. Fan and Mattheis3 reported that soluble solids were higher in 1-MCP-treated apples but other authors4 reported that SCC was unaffected by 1-MCP. Watkins et al.6 found differences in the responses of apple cultivars to 1-MCP treatments; ‘McIntosh’ and ‘Law Rome’ fruit treated with 1-MCP had lower SCC than untreated
© 2014 Society of Chemical Industry
wileyonlinelibrary.com/jsfa
2695
Effect of 1-MCP treatment on ripening index All experimental factors and their interactions significantly influenced the ripening index at a probability ≤ 0.001 and the regression model described the evolution of ripening index with a high adjusted R2 value of 0.997, and a low standard error to estimate of 0.42 (Table 3). Regression coefficients of the model indicate that the most important factor contributing to an increase in the ripening index is maturity at harvest followed by storage time and 1-MCP concentration (Table 3). Although ripening index was positively affected by 1-MCP, the regression coefficient is very low, and thus the influence of 1-MCP concentration is negligible in practical terms; this conclusion is also supported by data presented in Table 4. Under the experimental conditions tested in this study, 1-MCP showed a negative combined effect with the other factors on ripening index. This demonstrates a higher effectiveness of the 1-MCP treatments by showing that they were very effective in delaying ripening fruits that were fully mature at harvest (contrary to that observed for firmness) and after prolonged storage, similarly to that observed for firmness (Fig. 3A and B).
www.soci.org
E Cocci et al.
Figure 3. Response surface for the effect of 1-MCP dose and maturity at harvest (A) or storage time (B) on the ripening index of Pink Lady® apples after 4 months of storage at 2 ∘ C (A), or mature at harvest (starch index, 5.95 ± 0.35) (B).
fruit whilst ‘Delicious’ and ‘Empire’ had higher SCC; thus these differences may be cultivar dependent. Effect of 1-MCP treatment on CO2 production The regression model for CO2 production showed a high adjusted R2 value of 0.915 with a standard error to estimate of 0.62 (Table 3), therefore the proposed model was adequate to describe carbon dioxide production as function of the selected factor variables. This parameter was significantly influenced by all factors (P < 0.001) except 1-MCP where the individual effect was not significant, but it did show significant combined effects with maturity stage at harvest and storage time. The response surface describing CO2 production after 4 months of storage (central value) as a function of 1-MCP concentration and maturity at harvest indicate that 1-MCP had the greatest influence on delaying of CO2 production in partially mature fruits (Fig. 4A) similarly to what was observed for firmness. In fruit harvested at the intermediate maturity 1-MCP dose had little influence on CO2 production at the beginning of storage but the negative combined effect of 1-MCP dose and storage time on the response variable resulted in a higher effectiveness of the 1-MCP treatment after prolonged storage (Fig. 4B). Thus, CO2 production of mature Pink Lady® apples did not change significantly during storage for 6 months after treatment with the highest 1-MCP concentration (975 nL L−1 ) while it showed a dramatic increase after treatment with the lowest concentration (325 nL L−1 ).
2696
Effect of 1-MCP treatment and controlled atmosphere on fruit quality Instrumental quality measurements of fruit with or without 1-MCP treatment and stored for 6 months in RA or CA at 2 ∘ C varied according to treatment (Table 4). Untreated fruit softened and lost about 50% of initial post-harvest firmness (83.3 N) during air storage. Both CA storage and 1-MCP significantly delayed softening during storage with the latter more efficient than the former. Moreover a positive effect on firmness retention was obtained by combining CA storage and 1-MCP treatment (650 nL L−1 ). De Castro et al.12 found no interaction among CA and 1-MCP treatments on firmness of Pink Lady® stored for 4 months, but in that study experimental conditions (1-MCP concentration, storage time) were different from those tested here. The 6 months storage
wileyonlinelibrary.com/jsfa
time at 2 ∘ C resulted in an increase of ripening index and CO2 production, the latter was increased by CA and limited by 1-MCP whilst the former was limited by the combined effect of 1-MCP and CA storage. Following 7 days commercial shelf life, an acceleration of metabolic reactions (in terms of firmness loss, increase of ripening index and CO2 production) was observed in both treated (1-MCP or/and CA) and untreated samples. At the end of the shelf life simulation CA and 1-MCP treated samples showed better quality retention than untreated samples for all the parameters considered, and CA and 1-MCP showed a positive combined effects on firmness and CO2 production with the combination of CA storage and 1-MCP treatment being the most effective in maintaining firmness. Sensory analysis (Table 5) indicated that 1-MCP treatment did not affect sweetness or whole fruit odour but depleted cut fruit odour. In contrast, the 1-MCP treated sample was more sour, aromatic, crisp and firm and showed lower values of mealiness and fermented odour than untreated sample. Similar effects for sourness, firmness and crispness were achieved by using CA storage alone. A positive combined effect of CA storage and 1-MCP treatment was observed on apple firmness. The good correlation among the firmness, titratable acidity and soluble solids content values by instrumental and sensory evaluation (R2 > 0.90) confirms the judges’ reliability. Apples treated with 1-MCP and stored in RA received the highest score in overall assessment after sensory evaluation. The parameters having most influence on acceptability of Pink Lady® apple were SSC, TA and aroma volatile profile rather than the firmness.11 On the basis of these findings suggested that even if CA storage better preserved fruit texture, with positive implications on fruit quality maintenance, it is not strictly necessary to meet the needs of the final consumer.
CONCLUSION 1-MCP treatment positively affected the retention of quality of Pink Lady® apples and its effect could be improved in combination with other sorting and processing variables (fruit maturity, storage time and controlled atmosphere storage). The ripening-associated quality changes were differently influenced by 1-MCP treatments, thus suggesting a specific effect of this compound on different metabolic pathways.
© 2014 Society of Chemical Industry
J Sci Food Agric 2014; 94: 2691–2698
Response of Pink Lady apples to 1-MCP and controlled atmosphere
www.soci.org
Figure 4. Response surface for the effect of 1-MCP dose and maturity at harvest (A) or storage time (B) on CO2 production of Pink Lady® apples after 4 months of storage at 2 ∘ C (A), or mature at harvest (starch index, 5.95 ± 0.35) (B). Table 5. Results (median value and ANOVA) of the sensory evaluation of Pink Lady® apples treated with 1-methylcyclopropene (1-MCP) and different storage atmospheres (RA or CA) after 6 months of storage at 2 ∘ C Samples Sensory attribute Whole fruit odour Cut fruit odour Sweetness Sourness Apple flavour Fermented flavour Off-flavour Crispness Firmness Juiciness Mealiness
Untreated + RA 5.0a 5.0a 5.5a 2.0b 4.0b 3.0a 1.5a 4.5b 4.0d 5.0b 4.5a
Untreated + CA
Effects 1-MCP + RA
5.0a 5.0ab 5.0a 4.5a 4.5ab 2.0b 2.0a 6.5a 5.0c 5.5ab 3.0b
1-MCP + CA
5.5a 4.0b 5.5a 5.5a 5.0a 2.0b 1.0a 7.0a 6.0b 5.5ab 2.5b
5.0a 4.0ab 5.0a 5.0a 4.0b 2.0b 1.5a 6.5a 7.0a 6.0a 2.0b
1-MCP NS ∗
NS
CA NS NS NS
∗∗
∗
∗
NS
∗
∗
NS
NS
1-MCP × CA NS NS NS NS NS NS NS NS
∗∗
∗
∗∗∗
∗
∗
NS
NS NS
NS ∗∗∗
∗∗
Mean values for the same attribute marked with different letters are significantly different at P < 0.05 level. Significance of effects as determined by ANOVA analysis: ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001. CA, controlled atmosphere; RA, stored in air; NS, not significant.
Results obtained in this study have commercial implications by indicating that treatment with 1-MCP at different concentrations can be optimised depending on fruit maturity and expected storage time to predict fruit quality modifications during storage to meet market/consumer needs. These considerations are very important for Pink Lady® apples because the quality of these fruits is mostly assessed by colour at harvest which, in turn, depends on weather conditions that determine the pace of red blush development and, consequently, maturity of the fruit at commercial harvest. Use of the full red blush as a ripening index can be a limiting factor for fruit quality because it becomes over-mature when left on the tree for too long and late-harvested fruit showed increased greasiness and internal browning.12
REFERENCES
J Sci Food Agric 2014; 94: 2691–2698
© 2014 Society of Chemical Industry
wileyonlinelibrary.com/jsfa
2697
1 Aked J, Maintaining the post-harvest quality of fruits and vegetables, in Fruit and Vegetable Processing. Improving quality, ed. by Jongen W. Woodhead Publishing, Cambridge, UK, pp. 119–149 (2002).
2 Watkins CB, 1-Methylcyclopropene (1-MCP) based technologies for storage and shelf-life extension. Int J Postharvest Technol Innov 1:62–68 (2006). 3 Fan X and Mattheis JP, Impact of 1-methylcyclopropene and methyl jasmonate on apple volatile production. J Agric Food Chem 47:2847–2853 (1999). 4 Rupasinghe HPV, Murr DP, Paliyath G and Skog L, Inhibitory effect of 1-MCP on ripening and superficial scald development in ‘McIntosh’ and ‘Delicious’ apples. J Hort Sci Biotechnol 75:271–276 (2000). 5 Watkins CB and Nock JF, Effects of delays between harvest and 1-methylcyclopropene treatment, and temperature during treatment, on ripening of air-stored and controlled-atmosphere-stored apples. HortScience 40:2096–2101 (2005). 6 Watkins CB, Nock JF and Whitaker BD, Responses of early, mid and late season apple cultivars to postharvest application of 1-methylcyclopropene (1-MCP) under air and controlled atmosphere storage conditions. Postharvest Biol Technol 19:17–32 (2000). 7 Mir NA, Curell E, Khan N, Whitaker M and Beaudry RM, Harvest maturity, storage temperature, and 1-MCP application frequency alter firmness retention and chlorophyll fluorescence of ‘Redchief Delicious’ apples. J Am Soc Hort Sci 126:618–624 (2001).
www.soci.org 8 Vallejo F and Beaudry R, Depletion of 1-MCP by ‘non-target’ materials from fruit storage facilities. Postharvest Biol Technol 40:177–182 (2006). 9 Huber D, Suppression of ethylene responses through application of 1-methylcyclopropene: A powerful tool for elucidating ripening and senescence mechanisms in climateric and nonclimateric fruits and vegetables. Hortscience 43:106–111 (2008). 10 Cripps JEL, Richards LA and Mairata AM, ‘Pink Lady’ apple. HortScience 28:1057 (1993). 11 Lopez ML, Villatoro C, Fuentes T, Graell J and Echeverria G, Volatile compounds, quality parameter and consumer acceptance of “Pink Lady®” apples stored in different conditions. Postharvest Biol Technol 43:55–66 (2006). 12 De Castro E, Biasi WV and Mitcham EJ, Quality of Pink Lady apples in relation to maturity at harvest, prestorage treatments, and controlled atmosphere during storage. HortScience 42:605–610 (2007). 13 Wilkinson RI, Frisina C, Partington DL, Franz PR, Brien CJ, Thomson F, et al., Effects of 1-methylcyclopropene on firmness and flesh browning in Pink LadyTM apples. J Hort Sci Biotechnol 83:165–170 (2008). 14 Peirs A, Scheerlinck N, Berna Perez A, Jancsok P and Nicolai BM, Uncertainty analysis and modelling of the starch index during
15 16 17 18
19 20 21
E Cocci et al.
apple fruit maturation. Postharvest Biol Technol 26:199–207 (2002). Mendoza F and Aguilera JM, Application of image analysis for classification of ripening bananas. J Food Sci 69:E471–E477 (2004). Sweeney JP, Chapman VJ and Heoner PA, Sugar, acid and flavor in fresh fruit. J Am Diet Assoc 57:432–435 (1970). Association of Official Analytical Chemists, Official Methods of Analysis of the Association of Official Analytical Chemists, 16th edition. AOAC, Washington DC (1995). International Organization for Standardization, Sensory analysis – General guidance for the selection, training and monitoring of assessors – Part 1: Selected assessors, ISO8586-1:1993. ISO, Geneva (1993). International Organization for Standardization, Sensory analysis – General guidance for the selection, training and monitoring of assessor – Part 2: Experts, ISO8586-2:1994. ISO, Geneva (1994). International Organization for Standardization, Sensory analysis – General guidance for the design of test rooms, ISO8589:1988. ISO, Geneva (1988). Fan X, Mattheis JP and Blankenship SM, Development of apple superficial scald, soft scald, core flush, and greasiness is reduced by MCP. J Agric Food Chem 47:3063–3068 (1999).
2698 wileyonlinelibrary.com/jsfa
© 2014 Society of Chemical Industry
J Sci Food Agric 2014; 94: 2691–2698
