Ascorbic acid and tissue browning in pears (
Pyrus communis
L. cvs Rocha and Conference) under controlled atmosphere
conditions
R.H. Veltman * , R.M. Kho, A.C.R. van Schaik, M.G. Sanders,
J. Oosterhaven
Agro-Technological Research Institute(ATO),Bornsesteeg59,PO Box17,6700AA,Wageningen,The Netherlands Received 17 December 1998; accepted 6 March 2000
Abstract
The relationships between storage gas composition and ascorbic acid (AA) levels, and between AA levels and the development of internal browning, were studied in ‘Conference’ and ‘Rocha’ pears (Pyrus communis L.). In both cultivars, AA levels declined under (browning-inducing) controlled atmosphere (CA) conditions, i.e. lowered O2and
enhanced CO2 concentrations. Browning was initiated in a cultivar-dependent manner when an AA level threshold
was passed. Several models describing the relation between AA levels and browning were fitted to estimate these AA thresholds. The thresholds depend on cultivar, picking date and growing location, varying between 2 and 6 mg 100 g FW−1. AA losses in both cultivars exceeded 50% of the initial values. In ‘Conference’ fruit, most AA was lost when
fruits were transferred to CA. In ‘Rocha’ fruit, AA was lost mainly during long-term CA. © 2000 Elsevier Science B.V. All rights reserved.
Keywords:Ascorbic acid; Brown core; CA storage; Pear;Pyrus communis; Vitamin C
www.elsevier.com/locate/postharvbio
1. Introduction
Ascorbic acid (AA) is an antioxidant, which acts against reactive O2 species in concert with
other antioxidants such as glutathione and a
-to-copherol, in a system referred to as the ascor-bate – glutathione cycle (Jime´nez et al., 1997). AA
may also be involved in the conversion of 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene (Smith et al., 1992), and in photosynthe-sis, where it removes hydrogen peroxide formed during photoreduction in PSI (Smirnoff, 1996).
AA is not only important during the life of plants and during storage or shelf-life of fruits, but also for human health. In this regard, AA is a quality parameter of fruits, and should be kept at an appropriate level. AA levels, however, tend to decrease during storage and processing of fruits and vegetables.
* Corresponding author. Tel.:+31-317-475000; fax: + 31-317-475347.
E-mail address:[email protected] (R.H. Veltman)
Generally AA levels in various apple cultivars are lower under ultra low oxygen (ULO) condi-tions than under air in cold storage (Haffner et al., 1997). Levels slowly decrease both under CA and during air storage in ‘Boskoop’ and ‘Golden Delicious’ apples (Gerber, 1958; Bohling and Hansen, 1985), and they slowly decline in pota-toes during storage by as much as 1/6 of the original amount after 8 or 9 months (Nobile and Woodhill, 1981).
In pears, a relationship was found between AA content and the susceptibility to browning during experimental storage under various brown core-inducing conditions (Veltman et al., 1999). ‘Con-ference’ pears tend to develop tissue disorders, like brown core, when AA levels drop below a certain value.
In this paper, we tested and expanded on these results. Pears were monitored in a static system, and gas conditions and storage temperatures were comparable to those in commercial practice. AA levels and the susceptibility towards browning in ‘Conference’ and ‘Rocha’ pears were monitored during CA storage throughout the season. AA and browning values are discussed in relation to storage conditions, harvest date and growing location.
2. Materials and methods
2.1. Plant material
‘Conference’ pears (Pyrus communis L.) were harvested (grower 1) in The Netherlands (province Gelderland) on September 3, 10, 16, 24 and October 1, 1997, referred to as picks 1, 2, 3, 4 and 5, respectively. Pick 2 was the optimal harvest date for CA storage (based on firmness of the fruits). ‘Rocha’ pears were picked on August 2, 1997 in Portugal at two locations, Santos (grower 2) and Picarra (grower 3).
2.2. Storage and treatment
‘Conference’ pears were precooled at the stor-age temperature (−1°C) for 1 week before CA was applied. For bulk storage, ‘Conference’ pears
were stored in crates (about 100 per crate) in a static system. Eight crates were placed in a 650-l container with a water lock, at −1°C, 2%90.1 O2 and B0.790.1% CO2 (standard CA
condi-tions for ‘Conference’ fruit) or 390.1% CO2.
Relative humidity in the containers was 97 – 99%. For short-term experiments (Fig. 4), ‘Confer-ence’ pears were kept in 70-l tanks connected to a flow-through system, at 10°C to maintain a rea-sonable rate of metabolism, and under a series of O2 concentrations (1, 2, 7 and 21%) with or
without 1090.1% CO2. Relative humidity in the
containers was 97 – 99%. N2, O2 and CO2 were
mixed by using Brooks 5850 TR series mass flow controllers, the flow rate being 500 ml min−1
(Peppelenbos and Jeksrud, 1998). Before the start of the flow-through experiment the pears (pick 2) were stored for 7 months in the static system under standard CA conditions.
‘Rocha’ pears were transported in 3 days to The Netherlands by truck under cooled condi-tions (2 – 3°C). Before transportation, pears were precooled at −0.5°C. Altogether ‘Rocha’ pears had a 1-month cooling period between harvest and application of CA. Bulk storage of ‘Rocha’ pears took place in the same static system as ‘Conference’ pears at −0.5°C, under 190.1% or 390.1% O2 combined with B0.7% or 390.1%
CO2. The standard CA condition for ‘Rocha’
pears was 3% O2 and B0.7% CO2. ‘Rocha’ pears
were also stored under control conditions (21% O2
and B0.7% CO2) in the same static system.
For every harvest date (‘Conference’), grower (‘Rocha’), storage condition, and time (both cvs) two samples of five pears each were taken. From each sample internal browning (Section 2.3) and AA content (Section 2.4) were determined. Deter-minations for ‘Conference’ pears were performed at t=0, after 100 days, and after 200 days. ‘Rocha’ pears were judged at t=0, and after 60, 100, 150, 200, and 270 days after CA had been applied.
2.3. Browning index
di-vided into four classes: no browning (0), slight browning (I, B30% of the area), moderate browning (II, about 30 – 70% of the area) and severe browning (III, \70% of the area, with only the cortex fraction just underneath the peel not showing browning). The browning index is the sum of the browning-score of the five pears divided by 15, and multiplied by 100%, and I, II, and III refer to the number of pears in the various browning classes. A browning index value of 0% means no browning; 100% means maximal browning.
Browning index=100%· I+2II+3III 3(O+I+II+III)
2.4. AA measurements
A Waters liquid chromatography system (HPLC) was used: a pump, model 600E, with a Waters 486 tuneable absorbance detector (251 nm), and a Symmetry C-18, 3.9×150 mm column, particle size 5 mm, with a Sentry Guard
Column C-18 (Waters). Measurements were per-formed at 25°C. Mobile phase: 2.5-g tetrabuty-lammoniumhydrogensulfate (z.s. Merck 818858) and 55 ml methanol (p.a. Merck 6009) dissolved in 942.5 g Milli-Q water (Millipore) (Keijbets and Ebbenhorst-Seller, 1990). Before use, the eluent was filtered and degassed using a 0.45 mm
Mil-lipore filter (HVLP 04700). The flow rate was kept at 1 ml min−1. Analyses were completed within
5.5 min, including a post-column elution time of about 1 min.
As a standard, 177 mM AA (Sigma) prepared
with Milli-Q water was used (stock I). A total of 1-ml of stock I was diluted in 39 ml Milli-Q water with 5 ml 9.5% w/v oxalic acid (Merck 100495) and 5 ml methanol v/v (stock II). A dilution series from stock II was stored on ice and kept in the dark until injection. Stocks were prepared fresh every day.
Fruits were peeled, and cut length-wise. With a corkborer (diameter 17 mm) samples were taken from the cortex tissue just above the core tissue, at the stalk side, and just adjacent to the middle vein. Samples of five pears were mixed, immedi-ately frozen in liquid nitrogen, and crushed in a
kitchen mixer. Ten grams of sample were diluted with 5 ml 9.5% w/v oxalic acid, 5-ml methanol v/v and 30 ml Milli-Q water. The mixture was directly homogenised with an Ultra Turrax mixer and filtered through fluted paper (Schleicher & Schull 595(1/2)). Filtration steps were performed at 5°C in the dark. The filtrate was passed through a unit consisting of a 0.45 mm sterile filter and an
acti-vated Sep-Pak C18 cartridge (Waters), and was directly injected in a manual-injector system with a 20 ml loop. HPLC measurements were
per-formed directly after the extraction procedure. Results were analysed by means of the Millen-nium HPLC manager (Waters).
2.5. Firmness
Firmness of the ‘Rocha’ pears during storage was monitored with a penetrometer (plunger di-ameter 11 mm) placed in a drill stand, using 20 fruits per sample. Values are expressed as Newtons.
2.6. Statistical analysis
Statistical analyses were done with the ANOVA and FITNONLINEAR directives of the computer program Genstat (Genstat 5 Committee, 1993). Average values are accompanied by standard er-rors in brackets.
3. Results
The AA level of ‘Conference’ pear cortex tissue was 7.21 mg 100 g FW−1on average att=0 (all
picks combined). AA levels strongly declined in the period between harvest and 100 days of stor-age with 61, 63, 71, 76, and 73% on averstor-age (92%) for pick 1 to 5, respectively. On average, losses were 17% (91%) higher with enhanced CO2 (3%) compared with standard CA storage.
CA 7 days after harvest, higher AA levels were retained for more than a month (data not shown). No AA determinations were done after 1 month because the pears were ripe and had lost much water.
In ‘Rocha’ pears, no decrease in AA levels of more than 35% was seen after 100 days in CA. As distinct from ‘Conference’ pears, where losses seem to have taken place just after harvest, AA losses in ‘Rocha’ took place over the whole stor-age period. After 250 days in standard CA and control conditions, levels were reduced by about 60% (Fig. 1(d and e)), while after 270 days, some incidence of browning was seen under both conditions.
Before CA storage, just after the pears arrived in The Netherlands, the initial mean AA concen-tration was 8.22 (90.29) mg 100 g FW−1 for
pears from grower 2 and 6.11 (90.24) mg 100 g FW−1 for pears from grower 3. In Fig. 1, these
(initial) values are taken as 100%, and AA values from both growers were taken together. The crease in AA levels during storage could be de-scribed by the sigmoid function of Eq. (1). The percentage variance accounted for (adjustedR2
) is 81%.
AA%= 100%
1+ebij·(storage time−gij) (1)
AA% is the AA concentration as a percentage of its initial value, the storage time is in days, and bij and gij are the slope parameter and inflection
point respectively, both estimated for each O2and
CO2 combination (Table 1). Higher CO2
concen-trations (3%, Fig. 1(a and b)) lead to flatter slopes (b) (Table 1). However, at 3% CO2, the loss of
AA during the first 60 days seemed higher than predicted from the line fitted through the data points according to Eq. (1) (Fig. 1). The pears under 3% CO2 lost more AA during the first 60
days than pears stored under B0.7% CO2. The
inflection point (g, the middle of the symmetrical sigmoid) was earlier with 1% O2 than with 3%.
The effect of the O2 concentration on the slope
was not significant, nor was the effect of CO2 on
the inflection point.
The firmness of ‘Rocha’ pears remained the same during the storage period, which indicates
that, as with ‘Conference’ pears (Veltman et al., 1999), the decrease in AA was not related to ripening. The initial firmness value was 1.2 N on average, while after 250 days values were still between 1.0 and 1.1 N. This decrease was not significant. Differences in firmness between ‘Rocha’ pears from the two growers and pears stored at the different conditions were also not significant.
Browning in ‘Conference’ pears was only found when fruits were picked after the optimal CA picking date (pick 2) (Fig. 2), independent of the CO2 concentration during storage. In Eq. (2),
internal browning of ‘Conference’ pears is de-scribed as a function of AA for different picking times (Fig. 2). The percentage variance accounted for (adjusted R2) is 84%.
Browning= ai
1+eb·(AA−gi) (2)
In Eq. (2), AA is given in mg 100 g FW−1, a i
is an estimated maximum browning percentage depending on picking time, b is a slope parame-ter, and gi is the point of inflection of the curve
depending on picking time (Table 2). For all harvest dates, b was estimated to be 3.28 (9 0.72).
AA levels in pears from pick 1 and 2 were always higher than 1.1 mg 100 g FW−1 (Fig. 2),
while no internal browning was found. When pears were harvested later (pick 3, 4 and 5), the estimated maximum increased (ai, Table 2). The
inflection point, however, decreased (gi, Table 2).
According to the browning model (Eq. (2) and Table 2), an AA limit can be recognised below which internal browning developed (Fig. 2). The AA concentration (mg 100 g FW−1) at which
browning exceeded the 5% level was 2.32 (90.31) for pick 3, 2.53 (90.24) for pick 4, and 2.15 (90.21) for pick 5. Pears of pick 1 and 2 didn’t turn brown under the conditions applied in these experiments. However, after a longer period of storage or at conditions that induce internal disor-ders, these fruits showed internal browning as well (data not shown).
which browning developed, strongly depended on the harvest date.
The first incidence of flesh browning in ‘Rocha’ pears was seen after 150 days of storage at en-hanced CO2 and/or lowered O2 concentrations.
AA concentrations were generally higher in pears from grower 2, and these concentrations were clearly more reduced by browning-inducing stor-age conditions (low O2, elevated CO2, Fig. 1),
than in pears from grower 3. Absolute AA levels
Fig. 1. Dependence of AA in ‘Rocha’ pears on storage time and storage conditions; lines fitted according to Eq. (1) (Table 1). Pears were stored at 1, 3, 21% O2combined with B0.7 or 3% CO2. Every data point ( ) is an average of a mixed sample of five fruits.
Table 1
Fitted parameters of Eq. (1) for ‘Rocha’ pears, stored in various conditions
CO2 O2
3%
1% 21%
b=0.0067
b=0.0081
3% –
(90.0014) (90.0014)
g=239 (920)
g=182 (913) –
b=0.0119
B0.7% b=0.0129 b=0.0208 (90.0028) (90.0020)
(90.0016)
g=196 (99) g=233 (911) g=215 (97)
Table 2
Fitted parameters of Eq. (2) with standard errors for ‘Confer-ence’ pears at various dates and stored in standard CA condi-tions (B0.7% CO2) and under enhanced (3%) CO2; data from
both storage conditions were combineda
Pick time ai(%) gI(mg 100 g FW−1)
1 0 (92.9) –
2 0 (92.9) –
3 45 (910.9) 1.69 (90.27) 1.70 (90.12) 82 (97.3)
4
93 (96.4) 1.27 (90.10) 5
aFor all picking times, b (the slope parameter) was
esti-mated to be 3.28 (90.72).
Fig. 2. Dependence of internal browning of ‘Conference’ pears on AA and harvest date. Every data point is an average of a mixed sample of five fruits. Data from pears stored in the standard CA conditions (B0.7% CO2) and under enhanced
CO2 (3%) were combined in this graph. Lines were fitted
according to the model formulated in Eq. (2) with the esti-mated parameters as described in Table 2 for picks 3, 4 and 5. The adjustedR2of the model is 84%.
Fig. 3. Dependence of internal browning of ‘Rocha’ pears on AA and grower. Every data point is an average of a mixed sample of five fruits. Data from pears stored in the five conditions described in Section 2 were combined in this graph. Lines were fitted according to the model formulated in Eq. (2) with the estimated parameters as described in Table 3. The adjustedR2of the model is 46%.
in fruits from grower 3 were less affected by storage conditions, although, calculated as per-centage AA, losses were comparable for both growers. The observed browning in fruits of grower 3 was limited after 270 days.
In Fig. 3, internal browning of ‘Rocha’ pears as a function of AA and growing location is de-scribed. The percentage variance accounted for (adjusted R2
), by the sigmoid given in Eq. (2) is 46%.
Table 3
Fitted parameters of Eq. (2) with standard errors for ‘Rocha’ pears stored in five different conditionsa
Grower ai(%) gi (mg 100 g FW−1)
2 42.6 (96.1) 4.61 (90.34) 28.5 (913.0) 3.15 (90.71) 3
aFor both growing locations,b (the slope parameter) was
Fig. 4. AA levels in ‘Conference’ pears after 5 days of storage in a flow-through system. Fruits were stored in the following conditions: 1, 2, 7 and 21% O2combined with 0 or 10% CO2.
From every condition, two mixed samples of five pears were taken. Differences between CO2 concentrations were
signifi-cant at the 5% level, those between O2 concentrations were
not. Values are expressed as mg 100 g FW−1.
start of CA storage of ‘Rocha’ pears might retain higher AA levels. In commercial practice, in The Netherlands, ‘Conference’ pears are precooled for about 20 days before CA storage is started. Core browning in pears can be avoided to a large extent by this method (Veltman et al., 1999).
In ‘Conference’ pears, AA losses were around 70% in the period between harvest and after 100 days standard CA storage. It seems that the main AA decline takes place when fruits are brought under CA. Pears can lose AA in a relatively short time after this event, depending on conditions applied (Fig. 4). After CA has been applied, fur-ther losses are minimal. This is in contrast with ‘Rocha’ pears, where losses take place under CA, while AA differences between standard CA and control conditions are rather small.
AA losses during storage of fruits and vegeta-bles have often been reported. In potatoes, for example, losses vary from 21 to 60%, when tubers are stored for 8 months at 5 – 6°C, depending on the cultivar (Keijbets and Ebbenhorst-Seller, 1990). AA levels decreased more quickly in apples of five different apple cultivars stored under CA compared to storage under low temperatures without CA (Haffner et al., 1997). On the other hand, vitamin C in asparagus, peas, snap beans, spinach, broccoli and Brussels sprouts was lost more quickly in air than at low O2concentrations,
and losses were dependent on storage temperature (Platenius and Brown-Jones, 1944). Data on vita-min C levels during storage in modified atmo-sphere packaging (MAP) or CA, however, are relatively rare.
In ‘Conference’ pears, AA levels were reduced by CO2, both during short-term (5 days) and
long-term (storage period) experiments. In ‘Rocha’ pears, AA losses are more complicated. During the beginning (first 50 days) of storage under CO2, levels seemed to decrease faster.
How-ever, the rate of loss after 50 days was lower than in storage without CO2. AA levels also clearly
decreased under lowered O2concentrations during
storage of ‘Conference’ pears (results not shown). In both pear cultivars, enhanced CO2and lowered
O2 concentrations induced browning as well.
Comparable results were found by Agar et al. (1997) in several berry fruits. A rapid decrease in Both,ai, the maximal browning percentage and
gi, the point of inflection of the curve, depended
on growing location (Table 3). For both loca-tions, b was estimated to be 1.74 (90.75).
According to the browning model (Eq. (2) and Table 3), the AA concentration (mg 100 g FW−1)
at which browning exceeded the 5% level was 5.77 (90.71) for grower 2 and 4.04 (91.24) for grower 3.
At higher temperatures, AA levels in ‘Confer-ence’ pears were affected by gas conditions over a very short term (5 days). Fig. 4 shows these levels in pears held in a flow-through system at four O2
concentrations with (10%) or without (0%) CO2.
At 10% CO2, AA was 0.47 (90.09) mg 100g
FW−1 lower compared to 0% CO
2. Effects of O2
concentrations were not significant at the 5% level after five days.
4. Discussion
AA was found in strawberries, when stored in 15% CO2, and the highest concentrations were
found in strawberries stored under air. Compara-ble results were found in raspberries, red and black currants and blackberries. In pomegranates, AA decreased with increasing CO2concentrations
(Ku¨pper et al., 1995).
Veltman et al. (1999) found that beneath a certain threshold of AA, pears always show browning, and that AA could be used as an indicator for this disorder. It also became clear that CO2 lowers AA levels before brown core is
developed. From the results in this paper it seems that the harvest date is a complicating factor (Fig. 2). Conference pears picked after the optimal date for CA storage showed browning already at rela-tively high AA levels. From these data it seems very likely that, at a physiological level, there is no direct relation between AA and browning, but that at least a third factor is involved. The effect of this third factor is strongly dependent on the harvest date.
For ‘Rocha’ pears grown in Portugal both the overall AA levels and the AA-threshold for browning were clearly higher (Fig. 3). In an ear-lier paper (Veltman et al., 1999) we concluded that AA levels decreased when pears are stored at brown-inducing gas conditions (lowered O2 or
elevated CO2), and, after this, browning is
in-duced when a certain threshold is reached. The data presented in this paper show the same trend for this cultivar.
AA is an important antioxidant in the plant cell, while browning is an oxidation reaction. However, little is known about the reason why AA concentrations are reduced by enhanced CO2
and lowered O2. It can be concluded that storage
conditions, storage duration and picking date have a clear influence on AA levels in ‘Rocha’ and ‘Conference’ pears. AA levels can probably act as a quality parameter, being an indicator for the development of brown core depending on the picking date. These levels can give information about the quality of fruit during storage. Sec-ondly, AA is an essential substance in the human diet, and therefore one of the reasons fruit is consumed. Suboptimal storage conditions, such as enhanced CO2 or lowered O2 concentrations, can
result in a diminished food value. This research emphasises that gas conditions should also be optimised for AA contents.
Acknowledgements
The authors would like to thank J.A. Verschoor and T.R. Lammers for technical assistance, R.E. Schouten, H.S.M. de Vries, H.W. Peppelenbos and L.H.W. van der Plas for reading the manuscript and M.L. Avelar for information about ‘Rocha’ pear storage. This research was partially financed by Frutus, Campotec S.A. and Cooperative Agricola dos Fruticultores do Cadaval.
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