Food Chemistry 188 (2015) 62–70

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Food Chemistry

journal homepage: www.elsevier .com/locate / foodchem

Dynamic controlled atmosphere and ultralow oxygen storageon ‘Gala’ mutants quality maintenance

http://dx.doi.org/10.1016/j.foodchem.2015.04.1280308-8146/� 2015 Elsevier Ltd. All rights reserved.

⇑ Corresponding author.E-mail addresses: fthewes@yahoo.com.br (F.R. Thewes), vanderleiboth@yahoo.

com.br (V. Both), auribrackmann@gmail.com (A. Brackmann), anweba@gmail.com(A. Weber), rogerio_anese@yahoo.com.br (R. de Oliveira Anese).

Fabio Rodrigo Thewes a,⇑, Vanderlei Both a, Auri Brackmann a, Anderson Weber b,Rogerio de Oliveira Anese a

a Department of Plant Science, Postharvest Research Center, Federal University of Santa Maria, Roraima Avenue 1000, Camobi, Santa Maria 97105-900, RS, Brazilb Federal University of Southern Frontier, BR-158, km 405, Laranjeiras do Sul 85301-970, PR, Brazil

a r t i c l e i n f o

Article history:Received 17 January 2015Received in revised form 27 March 2015Accepted 27 April 2015Available online 28 April 2015

Keywords:Oxygen loweringEthanolAcetaldehydePrincipal component analysis

a b s t r a c t

The aim of the present work was to compare the effect of ultralow oxygen (ULO) with dynamic controlledatmosphere (DCA) and controlled atmosphere (CA) on the post storage quality of ‘Royal Gala’ and ‘Galaxy’apples after long-term storage. Two experiments were carried out with ‘Royal Gala’ and ‘Galaxy’ apples,in the years 2012 and 2013, respectively. A higher internal ethylene concentration was observed in fruitsstored under CA; intermediate concentration in fruits under ULO; and the lowest by fruits stored underDCA-CF (DCA based on chlorophyll fluorescence). Flesh firmness was higher in fruits stored under DCA-CFand ULO differing from CA, in the year 2012, but in 2013 fruits stored under ULO showed the highest fleshfirmness, differing from CA fruits. DCA-CF is efficient in quality maintenance of ‘Royal Gala’ and ‘Galaxy’apples. Both ‘Gala’ mutants stored under ULO show a similar quality maintenance to those stored underDCA-CF.

� 2015 Elsevier Ltd. All rights reserved.

1. Introduction

Apples are one of the most important fruit produced worldwide.Their production is concentrated in temperate regions, such as thesouthern states of Brazil, where apple production is mainly limitedto two cultivar groups: Gala and Fuji and its respective mutants.Among ‘Gala’ mutants, the most cultivated are the ‘Royal Gala’and ‘Galaxy’ due to its better red skin color index (Brackmann,Weber, Pavanello, Both, & Sestari, 2009; Brackmann, Weber,Pinto, Neuwald, & Steffens, 2008), high succulence and a good sol-uble solid and acidity balance. However, these two ‘Gala’ mutantshave a short harvest period and major part of this production mustbe stored for a long time.

Nowadays, the majority of apples are stored under controlledatmosphere (CA) (Brackmann et al., 2010; Weber et al., 2012,2013). In this storage method, the oxygen levels are seated andmaintained at 1.0 up to 1.5 kPa throughout the storage, indepen-dently of fruit metabolism (Brackmann et al., 2008). The oxygenlevels, used during CA storage, stay far above the anaerobic com-pensation point (ACP), the point where O2 uptake and CO2

production is minimal (Boersig, Kader, & Romani, 1988). As such,the fruits did not stay at a minimum respiration rate during thestorage, resulting in quality loss. Thus, it is important to developtechnologies allowing for the reduction of oxygen levels at mini-mum tolerated by fruits, in order to reduce the respiration andextend storage life without significant quality losses.

The ACP has a close relation with the lowest oxygen limit (LOL)tolerated by fruits. Therefore, to reduce the oxygen to very lowlevels (0.3 up 0.4 kPa), it is necessary to employ a technique formonitoring the LOL during the entire storage period. One method-ology used to determinate the LOL throughout the storage period isbased on chlorophyll fluorescence (DeEll, van Kooten, Prange, &Murr, 1999; Prange et al., 2007; Wright, DeLong, Harrison,Gunawarena & Prange, 2010; Wright, DeLong, Gunawarena &Prange, 2012). In this methodology, a sensor monitors the chloro-phyll fluorescence. When the oxygen levels are decreased belowthe LOL, the chlorophylls present in fruit skin, emit a fluorescence,that is captured by the sensor and directed to the software, indicat-ing the oxygen is below the LOL and must be increased. Thus, thisstorage method allows the reduction of oxygen levels, as low as thefruit metabolism can tolerate. During storage with this methodol-ogy, the oxygen levels are changed according fruit metabolism,especially during the onset of storage (Raffo, Kelderer, Paoletti &Zanella, 2009), resulting in a dynamic controlled atmosphere(DCA-CF).

F.R. Thewes et al. / Food Chemistry 188 (2015) 62–70 63

Another technology employed to improve storage is the use ofultralow oxygen levels (ULO), where the oxygen is reduced atlevels below 1.0 kPa O2, but above the LOL (Both, Brackmann,Thewes, Ferreira, & Wagner, 2014; Brackmann et al., 2013;Weber et al., 2013). Fruits stored under the ULO (Zanella, 2003)or ILOs (initial low oxygen stress) showed better post storage qual-ity, due to the lower superficial scald, higher flesh firmness andgreener skin color (Sabban-Amin, Feygenberg, Belausov, & Pesis,2011). A benefit of this storage technology is that it does notrequire any additional equipment for the storage vessels, reducingthe storage cost. However, there is a lack of studies in the literaturecomparing ULO storage with DCA-CF, increasing the necessity ofresearch comparing its effect on quality after storage.

In this context, the aim of this work was to compare the effect ofDCA-CF with ULO and CA on the post storage quality of ‘Royal Gala’and ‘Galaxy’ apples after long-term storage.

2. Material and methods

2.1. Localization, plant material and treatments application

The experiments were carried out over two years in thepostharvest research center of the Federal University of SantaMaria, Brazil. In the first year (2012) the plant material composedof ‘Royal Gala’ apples and in the second year (2013) ‘Galaxy’ appleswere used as the plant material. In the two years, fruits were har-vested in a commercial orchard, located in Vacaria, RS, Brazil. Rightafter harvest, fruits were transported to the postharvest researchcenter and experimental samples were performed, with 25 fruitseach. The treatments tested in the first year were: (1) controlledatmosphere storage (CA) with 1.2 kPa O2 + 2.0 kPa CO2; (2)dynamic controlled atmosphere by chlorophyll fluorescence +1.2 kPa CO2 (DCA-CF) and (3) ultralow oxygen storage with0.4 kPa O2 + 1.2 kPa CO2 (ULO). On the second year, we tested afourth treatment composed by initial low oxygen stress (ILOs) +ULO, in addition to those previously mentioned. The ILOs wereapplied during one day (0.05 kPa of O2). Each treatment was com-posed of four replicates, of 25 fruits each. Fruits were maintained attemperature of 1.0 �C during the storage period. The relativehumidity was 94 ± 1%.

2.2. Controlled atmosphere setup and maintenance

In order to obtain the desired atmospheric condition the CAchambers (180 l) were flushed with nitrogen until they reachedthe pre-established oxygen level. This procedure was carried out,aiming to reduce fruit metabolism as fast as possible. The carbondioxide level was obtained by accumulation in the storage cham-ber due to fruit respiration.

During the storage time, the oxygen and carbon dioxide levelswere determined and corrected daily with the aid of an automaticCA control system. The equipment compared the oxygen and car-bon dioxide levels to a set point. If the oxygen level was lower thanthe set point, O2 was injected up to the desired concentration. Thesame was used for carbon dioxide correction. Generally the CO2

level was above the desired concentration and the excess wasautomatically absorbed with a lime scrubber. In the DCA-CF theoxygen partial pressure was monitored and corrected accordingto the methodology proposed by Prange et al. (2007) and main-tained between 0.35 and 0.45 kPa.

2.3. Fruit quality evaluation

After nine months of storage plus seven days of shelf life, fruitswere submitted to quality analysis. The quality analysis were

performed in four replications of 25 fruits each. The followingphysical, chemical and biochemical analysis were carried out.

2.3.1. Ethylene production: evaluated by gas chromatographyApproximately 1.5 kg of fruits were put into 5 l hermetically

closed containers for about 2 h. Thereafter, two samples of 1 mlwere taken from the container and injected into a gas chro-matograph (Varian�, model Star 3400CX), according methodologyproposed by Both, Brackmann, Weber, Anese, and Thewes (2014).Results were expressed in ll C2H4 kg�1 h�1.

2.3.2. Respiration rate: evaluated by CO2 productionFruits from the same container used for ethylene production

were measured for CO2 production, by measuring the circulationof air throughout using a gas analyzer (Schele�) which quantifiesthe CO2 concentration inside the container. Data are presented inml CO2 kg�1 h�1.

2.3.3. Internal ethylene concentration (IEC): evaluated by withdrawingthe internal air of fruits

Measurements were carried out according to the methodologyproposed by Mannapperuma, Singh, and Montero (1991). Fromthe internal air, two samples were injected into the same gas chro-matograph as used for ethylene production quantification. Resultsexpressed in ll l�1.

2.3.4. Internal carbon dioxide concentration (ICO2)Two samples, of the same air extracted for IEC, were injected

into a Dani� (Dani Instruments Spa., Viale Brianza, ColognoMonzese, Italygas) chromatograph that quantified the CO2,equipped with Carboxen� capillary column. The temperature ofthe injector, column and detector were 180, 90 and 230 �C, respec-tively. Results expressed in ml 100 ml�1.

2.3.5. Titratable acidityTitratable acidity was evaluated by the titration of 10 ml of

juice, diluted in 100 ml distillated water with a 0.1 N NaOH solu-tion up to pH 8.1. Data are expressed in meq 100 ml�1.

2.3.6. Soluble solidsSoluble solids were determined by refractometry of the juice

with automatic temperature correction. Data are expressed in�Brix.

2.3.7. ACC oxidase enzyme activityThis was evaluated according to the methodology proposed by

Bufler (1986) and the results are expressed in nl C2H4 g�1 h�1.

2.3.8. CO2 of cellular juiceEvaluated by stowage, 10 ml of juice was placed inside a 20 ml

vial and immediately hermetically closed and heated to 70 �C for30 min. Thereafter two samples of 1 ml from the vials headspacewere injected into the same gas chromatograph as used for ICO2

evaluation. Results are expressed in ml 100 ml�1.

2.3.9. Acetaldehyde; ethanol; ethyl acetateEvaluated according to Saquet and Streif (2008) with modifica-

tions, where 10 ml of juice were stowed in a 20 ml vial flask andinstantaneously sealed, and warmed up to 70 �C for 30 min.0.1 ml of headspace gas was then injected into a Dani (DaniIntruments Spa., Viale Brianza, Cologno Monzese, Italygas) gaschromatograph fitted with a DN-WAX column at 60 �C and a flameionization detector (FID) at 250 �C. Concentrations were calculatedfrom the peak area using standard solutions of the respective com-pounds, with results expressed in ll l�1.

64 F.R. Thewes et al. / Food Chemistry 188 (2015) 62–70

2.3.10. Decay incidence and pulp crackingThis was evaluated by counting the fruits that present any sign

of decay and cracking. Thereafter the percentage of fruit withdecay and cracking was calculated taking into account the totalnumber of fruits for each sample (25 fruits). Results are expressedin percentage of decayed and cracked fruits.

2.3.11. Flesh breakdown and mealinessThis was evaluated by slicing the fruits on the equatorial region

and visualization of any browning region and mealy pulp. Resultsare expressed as percentage of total fruits.

2.3.12. Healthy fruitsThis was quantified by the total number of fruits in each sample

(25 fruits) minus the fruits with decay incidence, pulp cracking,flesh breakdown and mealiness. Thereafter, the number of healthyfruits was divided to the total number of fruits in the sample, andthis result multiplied by 100 to obtain the percentage of healthyfruits.

2.3.13. Flesh firmnessThis was evaluated by the insertion of a 11 mm tip penetrome-

ter inside the flesh (on 2 opposite sides of the fruit), wherethe skin was previously removed. Results are expressed inNewton (N).

2.4. Statistical analysis

Results were submitted to analysis of variance (ANOVA) at 5% oferror probability. Data that showed a significant difference byANOVA were subjected to a Principal Component Analysis (PCA).Before the PCA the data matrix was auto scaled for each variablein order to obtain the same weight for all variables (mean = 0and variance = 1). When the variance analysis was significant(p < 0.05), means were also compared by Tukey’s test at 5% of errorprobability. Data expressed in percentage were transformed by theformula arc.sin

ffiffiffiffiffiffiffiffiffiffiffiffiffiffix=100

pbefore variance analysis.

3. Results and discussion

In order to better visualize the effect of treatments, a multivari-ate analysis (PCA) was carried out (Figs. 1 and 2). In the two yearsevaluated, the principal component 1 (PC I) showed fruits storedunder CA had a distinct behavior when compared to fruits storedunder ULO and DCA-CF (Fig. 1A) and ILOs + ULO (Fig. 2A).Another work comparing CA with ULO also found discriminationbetween treatments using PCI in ‘Royal Gala’ apples (Both,Brackmann, Thewes, et al., 2014). These results show loweringoxygen levels significantly improved the quality maintenance of‘Royal Gala’ apples. Concerning the principal component 2 (PC II),fruits stored under DCA-CF showed a different pattern, comparedto fruits stored under the other conditions, independent of theyear. A noteworthy fact is that 99.99% of the overall variable vari-ation could be explained by the PCA in the first year (Fig. 1A). In thesecond year, the PC I and PC II together explained for 93.52% of theoverall variation of variables evaluated (Fig. 2A). These resultsshow that nearly all the variation of the variables are explainedby the two major components. Both, Brackmann, Thewes, et al.(2014), working with ‘Royal Gala’ apples stored under ULO, found54.35% of the overall variation by the PC I and PC II, showing thatin our research, the PCA was efficient at detecting the differenceamong the treatments tested in the two cultivars.

Concerning the variables, the PCA also showed a good discrim-ination among the characteristics evaluated (Figs. 1B and 2B). In2012, fruits stored under ULO and DCA-CF showed high flesh

firmness, healthy fruit amount, ethanol concentration and ICO2

(Fig. 1B). However, the amount of healthy fruit was more relatedto storage under ULO and the ethanol and ICO2 to the DCA-CF, withflesh firmness showing an intermediate response (Fig. 2B). Theseresults show that the DCA-CF and ULO has a different responsein PCII, but, independently of when fruits were stored under ULOor DCA-CF, a higher quality is maintained compared to CA. The ele-vated ethylene production, IEC, CO2 concentration of cellular juiceand respiration rate in fruits stored under CA resulted in high inci-dence of physiological disorders, such as flesh browning and meali-ness. Analyzing the fruits stored in 2013, a high flesh firmness,ethanol concentration and respiration rate upon chamber openingwas related to fruits stored under ULO and ILOs + ULO (Fig. 2B).High decay incidence and pulp cracking was correlated to fruitsstored under DCA-CF. On the other hand, fruits stored under CAshow high respiration rates, IEC, ICO2, ethylene production, leadingto elevated flesh breakdown and mealiness incidence. Examiningthe two years, a close relation among incidence of physiologicaldisorders and ethylene production, respiration rate and ICO2 wasverified (Figs. 1B and 2B).

The main way to control fruit ripening is to regulate ethyleneproduction. Fruits in CA storage showed the highest ethylene pro-duction during shelf life in both years (Fig. 3A and B). The higherethylene production by these fruits has a relation with elevatedoxygen levels adopted during CA storage (1.2 kPa). This behaviorwas also found by other researchers comparing ULO and CA(Both, Brackmann, Thewes, et al., 2014; Both, Brackmann, Weber,et al., 2014; Brackmann et al., 2013; Weber et al., 2013). In thetwo years, fruits stored under DCA-CF and ULO did not differ oversix days of shelf life, which indicate that ULO has the same effect asDCA-CF on ethylene production. The lower ethylene production byfruits stored under ULO and DCA-CF may be due to lower ACC oxi-dase enzyme activity, as these two parameters show a similarresponse in PCA (Fig. 1B).

Higher ethylene production generally induces an elevated res-piration rate (Pre-Aymard, Weksler, & Lurie, 2003). In 2012, fruitsstored in CA showed a higher respiration rate upon chamber open-ing, differing only from DCA-CF (Fig. 3C). After two days of shelflife, fruits stored under DCA-CF also showed the lowest respirationrate; fruits stored under ULO an intermediate; and CA showed thehighest respiration rate. However, after four and six days of shelflife, fruits stored under DCA-CF and ULO did not show any signifi-cant difference, but statistically lower compared to CA fruits. In2013, fruits stored under ULO showed the highest respiration rateupon chamber opening (Fig. 3D), but after two days of shelf life, CAfruits presented a higher respiration in relation to all other treat-ments. Similarly to 2012, after four days of shelf life, fruits ofDCA-CF showed the lowest respiration rate showing significant dif-ference only from CA. However, after six days of shelf life there wasno difference among treatments. The lower respiration rate, in allthe shelf life days by ULO and DCA-CF fruits, proved that thesetreatments reduced fruits metabolism during shelf life period, asrespiration is a key indicator of fruit metabolism (Steffens,Brackmann, Pinto, & Eisermann, 2007). Differently to ethylene pro-duction, in some case DCA-CF decreased the respiration rate morethan to ULO, showing more efficiency on metabolism reduction.According to a previous report, oxygen level reduction significantlydecrease the respiration rate (Both, Brackmann, Thewes, et al.,2014; Brackmann et al., 2013; Weber et al., 2013). Another articlesuggests that the ILOs significantly increase the respiration rate ofapples after storage (Brackmann et al., 2013), disagreeing with thepresent study, where the ILOs before ULO storage did not increasethe respiration rate in any days of shelf life (Fig. 3D). This differ-ence is probably due to the higher period of ILOs adopted byBrackmann et al. (2013), 14 days at 0.3 kPa O2 in relation to1 day at 0.05 kPa O2 in our research.

CA

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Fig. 1. (A) Scores (treatments) and (B) loadings (variables) plots showing the two major principal components of ‘Royal Gala’ apples stored under ultralow oxygen levels anddynamic controlled atmosphere (DCA) during 9 months storage plus 7 days of shelf life. Flesh breakdown (FB), mealiness, healthy fruits (HF), flesh firmness (Firmness),carbon dioxide concentration in cellular juice (CO2 Juice), internal ethylene concentration (IEC), internal carbon dioxide concentration (ICO2) and, ACC oxidase enzymeactivity (ACC), acetaldehyde (Acet.), ethanol (EtOH), ethylene production (Eth. 0, 2, 4, 6) and respiration rate (Resp. 0, 2, 4, 6).

F.R. Thewes et al. / Food Chemistry 188 (2015) 62–70 65

In order to better understand the ethylene production andaction, the internal ethylene concentration was measured. Rightafter chamber opening, in the year 2012, a higher IEC was observedin fruits under CA; an intermediate concentration in fruits underULO and the lowest by fruits stored under DCA-CF (Table 1).However, in the year 2013, a lower IEC was measured in fruitsstored under ULO and ILOs + ULO, an intermediate by DCA-CF,and the highest concentration by fruits under CA. The differentresponse to the treatments, in the two years, could be related tothe cultivars used, ‘Royal Gala’ in 2012 and ‘Galaxy’ in 2013.However, after 7 days of shelf life, the response to the treatmentswas similar in the two years, where CA stored fruits showed thehighest IEC concentration, which is related to the high ethyleneproduction rate by these fruits (Figs. 1 and 2). Probably, the higherIEC by fruits stored under CA has a relation with the advancedripening stage, shown by the lower flesh firmness and high meali-ness (Table 3), as they have a similar response to the treatments(Fig. 1). The higher IEC by fruits stored under CA may be relatedto a lower gas diffusion rate by these fruits, as the IEC and gas dif-fusion rate has an inverse correlation (Brackmann, Thewes, Anese,& Both, 2014).

Internal carbon dioxide concentration can be related to fruitphysiological disorders, such as flesh browning (Castro, Barrett,

Jobling, & Mitcham, 2008; Castro et al., 2007). In 2012, upon cham-ber opening, fruits stored under ULO showed the highest ICO2, butin 2013 there was no difference among the treatments (Table 1). Inthe first year (2012), after seven days of shelf life, fruits storedunder DCA-CF showed the lowest ICO2. Nevertheless, in 2013,fruits stored under DCA-CF showed the highest ICO2, differing fromULO and ILOs + ULO. Brackmann et al. (2014) attribute the higherICO2 to a lower gas diffusion rate, which leads to a higher CO2 con-centration in the internal fruit atmosphere. The concentration ofCO2 inside the fruit atmosphere may be a result of higher CO2 con-centration in the cellular juice of fruits stored under CA (Table 2).Perhaps, the high CO2 concentration in cellular juice causes dam-age to the cells, resulting in physiological disorders as showed bythe PCA (Figs. 1 and 2). The high physiological incidence changesthe gas diffusion rate, which hinders CO2 diffusion from inside tooutside the fruits and thereby increases ICO2.

Titratable acidity and soluble solids, after seven days of shelflife, did not show any significant difference among the treatmentsover the two years (Table 1). On the other hand, the ACC oxidaseenzyme activity was significantly affected by the treatments inthe two years (Table 2). ‘Royal Gala’ apples stored under CAshowed the highest ACC oxidase enzyme activity, fruits storedunder DCA-CF showed an intermediary activity and ULO the

CA

DCA-CF

ILOS + ULO

ULO

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.07%

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PCI (62.45%)

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B

Fig. 2. (A) Scores (treatments) and (B) loadings (variables) plots showing the two major principal components of ‘Galaxy’ apples stored under ultralow oxygen levels anddynamic controlled atmosphere (DCA) during 9 months storage plus 7 days of shelf life. Pulp cracking (Cracking), decay incidence (Decay), flesh breakdown (FB), mealiness,flesh firmness (Firmness), carbon dioxide concentration in cellular juice (CO2 Juice), internal ethylene concentration (IEC), internal carbon dioxide concentration (ICO2) and,ACC oxidase enzyme activity (ACC), ethanol (EtOH), ethylene production (Eth. 0, 2, 4, 6) and respiration rate (Resp. 0, 2, 4, 6).

66 F.R. Thewes et al. / Food Chemistry 188 (2015) 62–70

lowest. Another research with ‘Royal Gala’ apples also found lowerACC oxidase enzyme activity in fruits stored under ULO, comparedto CA (Both, Brackmann, Weber, et al., 2014). The lower ACC oxi-dase activity may be related to the higher ethanol concentrationin this treatment group over the two years. Ethanol and ACC oxi-dase also stay in opposite quadrants of the PCA, showing the higherthe ethanol concentration, the lower ACC oxidase enzyme activity(Figs. 1 and 2). Recent studies also found significant ACC oxidaseenzyme expression and activity reduction by exogenous ethanolapplication in broccoli florets (Asoda, Terai, Kato, & Suzuki, 2009)and oriental sweet melons (Jin, Lv, Liu, Qi, & Bai, 2013).

The first indicator of fermentative metabolism is the accumula-tion of acetaldehyde, ethanol and ethyl esters (Pesis, 2005). Amongthese, the most harmful is acetaldehyde, thus it is rapidly trans-formed into ethanol by the enzyme alcohol dehydrogenase (ADH)(Lee, Rudell, Davies, & Watkins, 2012; Pesis, 2005). According toTable 2, in 2012, a higher acetaldehyde concentration was accumu-lated by fruits stored under CA, differing only from DCA-CF, whichshowed the lowest acetaldehyde concentration after 7 days of shelflife. Perhaps, the high acetaldehyde concentration has a relationwith physiological disorders, such as mealiness (Fig. 1). However,in 2013, there was no significant difference among the treatments

for acetaldehyde concentrations (Table 2). According to earlierpapers, lowering the oxygen levels down to 0.5 kPa did notincrease the acetaldehyde concentration in ‘Golden Delicious’(López, Lavilla, Recasens, Graell, & Vendrell, 2000) and ‘RoyalGala’ apples (Both, Brackmann, Thewes, et al., 2014).

Higher ethanol production was verified in fruits stored underULO, differing in all treatments in 2012, but in 2013, only differingfrom fruits stored under CA (Table 2). A remarkable fact is fruitsstored under CA have a higher acetaldehyde concentration, butdid not show a significant increase in ethanol. Ethanol concentra-tion in apple pulp can result in benefits to quality maintenance,but this effect on quality is concentration dependent (Pesis,2005). In sweet oriental melons, the ethanol application reducedACC oxidase enzyme activity and IEC, and increased ester produc-tion, especially ethyl esters (Jin et al., 2013; Liu et al., 2012). In thepresent work, is ascertained that the ethanol produced by fruits,because of O2 lowering, has a similar effect on its application, espe-cially on ACC oxidase enzyme activity, IEC and flesh firmness main-tenance (Tables 1–3). These results are also confirmed by the PCAamong these variables (Fig. 2B). Another work, with sweet cherries,also found significantly better quality maintenance by ethanolapplication (Bai, Plotto, Spotts, & Rattanapan1, 2011).

0.0

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Fig. 3. Ethylene production (A and B) and respiration rate (C and D) of ‘Royal Gala’ apples (year 2012) and ‘Galaxy’ apples (year 2013) stored under ultralow oxygen levels anddynamic controlled atmosphere (DCA-CF) during 9 months storage plus 6 days of shelf life. CA: controlled atmosphere storage (1.2 kPa O2 + 2.0 kPa CO2); CF: dynamiccontrolled atmosphere based on the chlorophyll fluorescence; ULO: storage under ultralow oxygen level (0.4 kPa O2 + 1.2 kPa CO2); ILOs + ULO: initial low oxygen stress (1 dayat 0.05 kPa O2) followed by storage under ULO. *Means followed by equals letters, at the same year and variable, do not differ by Tukey’s test at 5% of error probability (p < 0.05).

Table 1Internal ethylene concentration (IEC), internal carbon dioxide concentration (ICO2), titratable acidity and soluble solids of ‘Royal Gala’ apples (year 2012) and ‘Galaxy’ apples (year2013) stored under ultralow oxygen levels and dynamic controlled atmosphere (DCA) during 9 months storage plus 7 days of shelf life.

Treatments IEC (ll l-1) ICO2 (ml 100 ml�1) Titratable acidity (mEq 100 ml�1) Soluble solids (�Brix)

Chamber opening 7 days shelf life Chamber opening 7 days shelf life

Year 2012 – ‘Royal Gala’At harvesta – – – – 4.85 11.6CAb 3.71 ± 0.20a* 37.8 ± 1.12a 1.85 ± 0.12b 2.41 ± 0.25a 3.81 ± 0.05ns 12.15 ± 0.30ns

DCA-CFc 0.23 ± 0.03c 0.97 ± 0.38b 1.79 ± 0.10b 1.87 ± 0.22b 3.95 ± 0.09 12.10 ± 0.12ULOd 1.02 ± 0.05b 0.15 ± 0.05b 3.08 ± 0.48a 2.35 ± 0.24a 3.78 ± 0.14 12.45 ± 0.21Mean 1.65 12.96 2.24 2.21 3.85 12.23

Year 2013 – ‘Galaxy’At harvest a 1.45 4.05 5.22 11.4CAb 19.5 ± 1.63a 66.73 ± 18.30a 3.81 ± 0.49ns 2.21 ± 0.86ab 4.17 ± 0.23ns 12.65 ± 0.10ns

DCA-CFc 6.96 ± 2.51b 12.91 ± 5.80b 3.50 ± 0.45 3.25 ± 0.23a 4.10 ± 0.18 12.65 ± 0.19ULOd 1.71 ± 1.07c 8.30 ± 6.56b 2.99 ± 0.81 1.74 ± 0.53b 4.18 ± 0.06 12.45 ± 0.25ILOs + ULOe 1.14 ± 0.75c 7.67 ± 3.95b 3.11 ± 0.44 1.59 ± 0.79b 4.01 ± 0.08 12.35 ± 0.30Mean 7.31 23.90 3.35 2.20 4.11 12.53

a At harvest: initial analysis, carried out before the storage (mean of three replicates).b CA: controlled atmosphere storage (1.2 kPa O2 + 2.0 kPa CO2).c DCA-CF: dynamic controlled atmosphere based on the chlorophyll fluorescence.d ULO: storage under ultralow oxygen level (0.4 kPa O2 + 1.2 kPa CO2).e ILOs + ULO: initial low oxygen stress (1 day at 0.05 kPa O2) followed by storage under ULO.* Means followed by different letters lowercase in the columns, at the same year, differ by Tukey’s test at 5% of error probability (p < 0.05). ns: no significant difference

(p > 0.05).

F.R. Thewes et al. / Food Chemistry 188 (2015) 62–70 67

Also, with the increase in ethanol concentration, in fruit flesh,generally an increase in ethyl ester production (Jin et al., 2013),especially ethyl acetate, is also observed. The linkage of ethanolto an acetyl, by the alcohol acyltransferase (AAT), produces ethylacetate (Defilippi, Kader, & Dandekar, 2005). In both 2012 and2013 the treatments did not effect the ethyl acetate productionby the two ‘Gala’ mutants (Table 2). Both, Brackmann, Thewes,

et al. (2014), working with ‘Royal Gala’ apples, also did not find asignificant increase of ethyl acetate production by fruits storedunder different ULO conditions. However, these same authors,found a significant reduction of another ethyl ester (ethylhexanoate), by fruits stored under ULO (0.5 kPa O2) in relation toCA (1.0 kPa O2). Therefore, lowering the oxygen levels down to0.4–0.5 kPa did not start ethyl acetate formation in ‘Gala’ mutants.

Table 2ACC oxidase enzyme activity, carbon dioxide concentration in cellular juice (CO2 of cellular juice), acetaldehyde, ethanol and ethyl acetate of ‘Royal Gala’ apples (year 2012) and‘Galaxy’ apples (year 2013) stored under ultralow oxygen levels and dynamic controlled atmosphere (DCA) during 9 months storage plus 7 days of shelf life.

Treatments ACC oxidase (gL C2H4 g�1 h�1) CO2 of cellular juice (ml 100 ml�1) Acetaldehyde (ll l�1) Ethanol (ll l�1) Ethyl acetate (ll l�1)

Year 2012 – ‘Royal Gala’At harvest a 24.40 – – – –CAb 285.5 ± 17.56a* 2.30 ± 0.14a 6.85 ± 0.84a 48.25 ± 8.80b 0.21 ± 0.06ns

DCA-CFc 76.32 ± 19.87b 1.32 ± 0.06c 4.74 ± 0.88b 42.96 ± 10.81b 0.26 ± 0.22ULOd 38.21 ± 11.16c 1.64 ± 0.06b 5.80 ± 1.29ab 88.85 ± 17.96a 0.38 ± 0.17Mean 133.36 1.75 5.80 60.02 0.28

Year 2013 – ‘Galaxy’At harvest a 27.86 – – – –CAb 196.4 ± 7.42a 3.94 ± 0.84a 5.07 ± 0.19ns 48.21 ± 3.72b 1.29 ± 0.15ns

DCA-CFc 87.96 ± 15.25b 0.60 ± 0.09b 5.16 ± 1.04 53.20 ± 8.08ab 1.12 ± 0.17ULOd 77.52 ± 9.72b 1.01 ± 0.34b 5.23 ± 0.74 67.91 ± 10.51a 0.97 ± 0.26ILOs + ULOe 79.51 ± 2.31b 0.96 ± 0.16b 5.08 ± 0.58 55.75 ± 5.66ab 0.88 ± 0.35Mean 110.34 1.63 5.13 56.27 1.06

a At harvest: initial analysis, took place before the storage (mean of three replicates).b CA: controlled atmosphere storage (1.2 kPa O2 + 2.0 kPa CO2).c DCA-CF: dynamic controlled atmosphere based on the chlorophyll fluorescence.d ULO: storage under ultralow oxygen level (0.4 kPa O2 + 1.2 kPa CO2).e ILOs + ULO: initial low oxygen stress (1 day at 0.05 kPa O2) followed by storage under ULO.* Means followed by different letters lowercase in the columns, at the same year, differ by Tukey’s test at 5% of error probability (p < 0.05). ns: no significant difference

(p > 0.05).

Table 3Decay incidence, pulp cracking, flesh breakdown, mealiness, healthy fruits and flesh firmness of ‘Royal Gala’ apples (year 2012) and ‘Galaxy’ apples (year 2013) stored underultralow oxygen levels and dynamic controlled atmosphere (DCA) during 9 months storage plus 7 days of shelf life.

Treatments Decay (%) Pulp cracking (%) Flesh breakdown (%) Mealiness (%) Healthy fruits (%) Flesh firmness (N)

Year 2012 – ‘Royal Gala’At harvesta 0.0 0.0 0.0 0.0 100 83.8CAb 0.0 ± 0.0ns 0.0 ± 0.0ns 15.6 ± 8.0a* 26.7 ± 3.8a 72.2 ± 2.8b 64.8 ± 1.2bDCA-CFc 2.4 ± 1.9 1.2 ± 2.4 4.74 ± 1.8b 12.5 ± 5.4b 85.7 ± 7.7a 74.1 ± 2.2aULOd 2.9 ± 3.6 1.0 ± 2.0 9.62 ± 6.6ab 17.3 ± 5.0ab 79.9 ± 3.7ab 76.1 ± 2.1aMean 1.76 0.7 9.99 18.8 79.2 71.6

Year 2013 – ‘Galaxy’At harvesta 0.0 0.0 0.0 0.0 100 78.6CAb 4.86 ± 2.6 b 0.00 ± 0.0b 13.3 ± 3.6a 24.5 ± 4.7ab 70.8 ± 7.3ns 60.9 ± 1.8bDCA-CFc 20.1 ± 6.9a 5.56 ± 3.9a 2.78 ± 2.3b 28.0 ± 5.0a 62.5 ± 4.8 64.1 ± 1.3abULOd 8.81 ± 1.6b 2.88 ± 1.4ab 9.70 ± 6.6ab 16.3 ± 4.3b 73.6 ± 8.3 65.4 ± 0.8aILOs + ULOe 7.29 ± 4.2b 0.74 ± 2.2ab 18.5 ± 3.9a 17.1 ± 1.9ab 75.8 ± 8.6 66.0 ± 2.3aMean 10.7 2.29 11.1 21.5 70.7 64.1

a At harvest: initial analysis, took place before the storage (mean of three replicates).b CA: controlled atmosphere storage (1.2 kPa O2 + 2.0 kPa CO2).c DCA-CF: dynamic controlled atmosphere based on the chlorophyll fluorescence.d ULO: storage under ultralow oxygen level (0.4 kPa O 2 + 1.2 kPa CO2).e ILOs + ULO: initial low oxygen stress (1 day at 0.05 kPa O2) followed by storage under ULO.* Means followed by different letters lowercase in the columns, at the same year, differ by Tukey’s test at 5% of error probability (p < 0.05). ns: no significant difference

(p > 0.05).

68 F.R. Thewes et al. / Food Chemistry 188 (2015) 62–70

Decay incidence and pulp cracking are important causes of loss ofquality. In 2012, there was no difference among the treatments, butin 2013, a higher decay and fruit cracking incidence was observed inDCA-CF in relation to all other treatments (Table 3). Perhaps, thepulp cracking by fruits stored under DCA-CF increased the fungalincidence in these fruits. The higher decay incidence by fruits ofthese treatments may be a result of too long a time under the initiallow oxygen (near to 0.00 kPa) conditions, until chlorophyll fluores-cence emission spikes. Early researchers also found a significantincrease in decay incidence by ILOs application in ‘Royal Gala’ apples(Both, Brackmann, Weber, et al., 2014; Brackmann et al., 2013).

One of the main internal disorders is flesh breakdown, which ischaracterized as a browning of fruit tissue. This browning incidencegenerally increased due to low energy production by fruit storageunder low oxygen levels (Ho, Verboven, Verlinden, Schenk, Nicolaï,2013). In 2012, fruits stored under CA showed the highest fleshbreakdown, differing only from DCA-CF (Table 3). Nevertheless,there was no significant difference between DCA-CF and ULO forflesh breakdown. In the second year, a higher flesh breakdown was

observed in fruits stored under CA and ILOs + ULO, showing differ-ences from DCA-CF. Both, Brackmann, Weber, et al. (2014) did notfind a significant increase in flesh breakdown by ILOs before ULOin ‘Royal Gala’ apples, but in absolute terms, ILOs increased the phys-iological disorder. Ethylene production and IEC have a correlationwith flesh breakdown (Figs. 1B and 2B). Perhaps, the higher ethyleneproduction and IEC, shown by fruits stored under CA, induce cell walldegrading enzymes (Payasi, Mishra, Chaves, & Singh, 2009;Prassana, Prabha & Tharanathan, 2007), easing cell membranedisruption, putting polyphenol and enzymes in contact, whichresults in flesh browning. On the other hand, the elevated fleshbreakdown observed in CA fruits could be a result of the higherCO2 concentration in cellular juice (Table 2), as confirmed by PCA(Figs. 1B and 2B). These results are in agreement with those pub-lished by other researchers, which also attributed flesh breakdownto CO2 concentration (Castro et al., 2007, 2008).

Mealiness is an important physiological disorder related toadvance ripening stages of fruits, as fruit ripeness increases pro-portionally to its incidence (Both, Brackmann, Weber, et al.,

F.R. Thewes et al. / Food Chemistry 188 (2015) 62–70 69

2014). Therefore, fruits stored under CA were in an advance ripen-ing stage, shown by the high mealiness incidence (Table 3). Thelowest mealiness incidence was verified in fruits stored underDCA-CF, but there was no difference compared to fruits storedunder ULO. Nevertheless, in 2013, fruits stored under ULO showedthe lowest mealiness incidence, differing only from fruits storedunder DCA-CF. However, fruits stored under CA and ILOs + ULOalso showed a high mealiness incidence (Table 3). The high meali-ness incidence, verified in fruits stored under CA, may be related tothe higher IEC and ethylene production by these fruits(Figs. 1B and 2B). Moreover, the ethylene is responsible for startingsome enzymatic activities, such as pectin methylesterase, whichdegrade pectin from the middle layer of the cell wall (Nishiyamaet al., 2007; Payasi et al., 2009). With the degradation of pectinfruit cells lose their adhesion, leading to pulp with a mealy aspectand without succulence (Brackmann et al., 2014).

The healthy fruit amount is the product that can be commer-cialized without significant internal and external quality lossesfor consumers. In 2012, a higher healthy fruit percentage was ver-ified in DCA-CF, without differing from ULO (Table 3). The higherhealthy fruits may be related to a lower ethylene production, lowerCO2 in cellular juice and respiration rate (Fig. 1B). The higher meta-bolism, shown by fruits stored under CA, is related to fruits withhigher physiological disorders (Table 3) resulting in lower amountsof healthy fruits. However, in 2013, the higher metabolism shownin fruits stored under CA did not result in a decrease of healthyfruit amount (Table 3). Brackmann et al. (2013) and Both,Brackmann, Weber, et al. (2014) also found a significant increaseof healthy fruits by lowering oxygen down to 0.6 and 0.5 kPa,respectively, in ‘Royal Gala’ apples.

Flesh firmness was higher in fruits stored under DCA-CF andULO, differing from CA, in 2012 (Table 3). A noteworthy fact is thatthe fruits stored under ULO and DCA-CF did not show difference.Previously researchers also found a higher flesh firmness in applesstored under DCA-CF in relation to CA (Lafer, 2008; Watkins, 2008;Zanella, Cazzanelli, & Rossi, 2008). This result is also confirmed in2013, where fruits stored under ULO and ILOs + ULO showed thehighest flesh firmness, differing from CA fruits. The higher fleshfirmness, by fruits stored under ULO and ILOs + ULO, may berelated to the high ethanol production of these fruits, as evidencedin Fig. 3B. Similar results were obtained in sweet cherries (Bai et al.,2011) and melons (Liu et al., 2012). Perhaps, the high ethanol pro-duction by these fruits, led to a lower IEC and ethylene production,decreasing cell wall enzyme activities (Nishiyama et al., 2007;Payasi et al., 2009; Goulao & Oliveira, 2008) which maintainedhigher flesh firmness.

4. Conclusion

The storage of ‘Gala’ mutants (‘Royal Gala’ and ‘Galaxy’) in adynamic controlled atmosphere, based on chlorophyll fluores-cence, is efficient in quality maintenance after long-term storage.‘Gala’ mutants stored under ULO show similar quality maintenanceto those stored under DCA-CF.

Lowering the oxygen partial pressures down to 0.4 kPaincreases ethanol concentration in flesh tissue, but at a level thatdo not increase the physiological disorders and reduce the ACCoxidase enzyme activity and ethylene production by fruits.

Acknowledgements

To Conselho Nacional de Desenvolvimento Científico eTecnológico (CNPq) and Coordenação de Aperfeiçoamento dePessoal de Nível Superior (CAPES), for financial support.

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