Internal atmosphere composition and skin permeance to
gases of pepper fruit
Nigel H. Banks, Sue E. Nicholson *
Food Technology Research Centre,Massey Uni6ersity,Pri6ate Bag11-222,Palmerston North,New Zealand
Received 16 December 1998; accepted 2 September 1999
Abstract
Characterisation of internal atmosphere composition offers the potential to explain variability in responses of horticultural crops to modified atmosphere treatments and to quantify permeance of fruit skins to the respiratory gases. In this paper, the theoretical basis by which fruit skin permeance can be calculated from other gas exchange variables is presented. Surface chambers close to equilibrium with the fruit’s internal atmosphere were used to monitor internal atmosphere composition of sweet pepper (Capsicum annum, cv. Reflex). Physical equilibration of chamber contents over wounded fruit surface was essentially complete in less than 4 h. However, physiological drift in internal atmosphere composition meant that substantial changes continued to develop over more extensive periods. Removal of cuticle beneath the chamber was shown to be essential for equilibration of chamber contents within physiologically meaningful periods. Samples of atmosphere removed destructively from the fruit cavity consistently contained more O2but less CO2than samples similarly removed from the fruit flesh. Levels of CO2were higher in
samples removed directly from the flesh by syringe than in those taken from surface chambers, indicating potential for an effect of the vacuum used to take direct removal samples on sample composition. Permeance of pepper cuticle to CO2was about ten times greater than that to O2(244 and 24 pmols−1m−2per Pa, respectively). Removal of
cuticle dramatically increased permeance of the fruit surface and hastened equilibration of surface chambers with the fruit’s internal atmosphere. Surface chambers adhered over fruit surface from which the cuticle has been removed would be the most reliable means to assess composition of the atmosphere in immediate contact with the cells of pepper tissue. © 2000 Elsevier Science B.V. All rights reserved.
Keywords:Carbon dioxide; Cuticle; Equilibration; Gas transfer; Oxygen; Respiration; Wounding
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1. Introduction
Much of the variability in responses of fruits and vegetables to modified atmospheres can be explained if these are considered on the basis of internal, rather than external, atmosphere compo-sition (Burton, 1982; Banks et al., 1994; Dadzie et
* Corresponding author. Tel.: +64-6-3505551.
E-mail address: [email protected] (S.E. Nichol-son)
al., 1996). Ways to characterise internal atmo-sphere composition that develops in response to environmental manipulation are therefore of po-tential value to those attempting either to explain or manage the effects of modified atmospheres on fruit and vegetable storage behaviour.
Proven methods to study internal atmosphere composition are not available for a wide range of crop types. Rajapakse et al. (1990) reported satis-factory use of surface chambers for study of inter-nal atmosphere composition in three fruit types (apple, Asian pear and nectarine). In this study, we evaluated the potential for use of such surface chambers for characterising internal atmosphere composition of sweet pepper (Capsicum annum, cv. Reflex) and, by studying the kinetics of ex-change between such chambers and the internal atmosphere, as a means of estimating skin perme-ance of the fruit to O2 and CO2. This is of
particular interest in solanaceous fruits because most of the fruit surface is devoid of pores (Blanke, 1986; Blanke and Holthe, 1997). Such fruits should therefore make ideal subjects for assessing whether fruit cuticles are differentially permeable to the two respiratory gases, as pro-posed by Ben-Yehoshua et al. (1985). Differential permeability of the cuticle to these two gases has been invoked as a means to explain the overall differential permeability of fruit skins that devel-ops after treatment with surface coatings (Banks et al., 1997). In addition, the sweet pepper has an internal cavity, the contents of which might be expected to be similar to the true internal atmo-sphere surrounding the cells within the tissue. If cavity atmosphere and flesh internal atmosphere were shown to be identical, then characterising internal atmosphere of sweet pepper, and perhaps other capsicum fruits, would be as simple as de-termining composition of samples taken from the cavity within the fruit. On the other hand, if the permeance of the cuticle on the outer fruit surface were low, this raises the interesting possibility that the internal atmosphere of the flesh may be more modified than the cavity that lies anatomically within it. In this study, we evaluated these propo-sitions through experiments that involved sam-pling of internal atmosphere from chambers close to equilibrium with the internal atmosphere of the
fruit flesh and by direct removal from both the flesh and the inner cavity.
1.1. Theoretical de6elopment
The derivation of parameters associated with equilibration of chambers of this type with the internal atmosphere with which they are in con-tact has been published previously (Banks and Kays, 1988). However, they are presented here in the new units recommended by Banks et al. (1995) for the sake of clarity.
Exchange between the chamber and the internal atmosphere of gasj(rj
ch,t
, mol s−1
) at timetafter sealing or flushing (s) can be expressed in two ways:
iis the partial pressure of gas
jbeneath the skin surface (Pa),pjch,t=partial
pres-sure of gasjinside the chamber (Pa) at timetand:
r
where R is the gas constant (8.314 m3
Pa mol−1
per K), T is the temperature (°C), Vch is the
chamber volume (m3).
Re-arranging and integrating provides:
ln(pj
where k1 is the integration constant. If pjch,t at
t=0, then k1=ln(pji), so that:
Times required for 99% equilibration of cham-ber contents (t0.99, s) with the internal atmosphere
constant (k2= −Pj%·A ch·
R· (T+273.15/Vch,
s−1)) in the function used to derive fitted curves
to plots of chamber atmosphere composition with respect to gas j (pj
ch,t) versus time after flushing
with nitrogen.
Permeance to gasjcan be estimated fromk2for
the same gas as follows:
p%j= k2,j·V
ch
Ach·R· (T+273,15) (8)
which, given the values ofVch,AchandTused in
this study for chambers over intact and wounded cuticle equated to −5.44×10−6
·k2,j and
−2.27×10−5
·k2,j, respectively.
2. Materials and methods
Two experiments were conducted at 20.49
0.32°C, 9691.7% relative humidity on freshly harvested, greenhouse-grown sweet peppers (Cap
-sicum annuum, cv. Reflex) obtained directly from
a local grower.
2.1. Experiment1.Physical and physiological equilibration time for O2 and CO2 in chambers adhered o6er wounded fruit surface
Sixty-four fruit were assigned at random to two treatments: ‘control’ (no treatment; 28 fruit) and ‘wounded’ (36 fruit). In the case of wounded fruit, a disc of outer tissues including cuticle and epider-mis (5.8 mm diameter; 0.5 mm average thickness) was removed aseptically from the surface of each fruit (time after wounding, taw
=0 h). A glass chamber (9.65 mm internal diameter, 0.996 – 1.080 ml) was adhered over the wound with Araldite (24 h curing) and the fruit left overnight for the glue to cure. Chambers on eight of the 36 fruit were supported in frames to prevent dislodging the chamber from the waxy fruit surface during re-peated sampling. Septa and water seals were then added to all chambers (see Fig. 1 in Rajapakse et
al., 1990) and silicone grease used to cover the glue seal (taw
=16 h).
Samples of flesh atmosphere were taken de-structively by direct removal (Banks, 1983) from the flesh of the pericarp wall on four fruit from each treatment at taw=16, 28, 40, 52, 64, 76, 88
and 112 h. Special care was taken to avoid con-tamination of flesh atmosphere samples with ex-ternal or fruit cavity atmospheres by filling the fruit cavity with water immediately before sam-pling and withdrawing samples of flesh atmo-sphere whilst fruit were immersed in water. Repeated samples were taken from chambers on the eight fruit in frames attaw
=16, 40, 52, 64, 76, 88 and 112 h to estimate long term trends with time. In addition, vials on these fruit were flushed with nitrogen after sampling at taw
=16 and 64 h and resampled after an additional 0, 0.5, 1, 4 and 12 h after flushing. Samples of flesh and cavity atmospheres of these fruit were taken by direct removal at taw=112 h.
2.2. Experiment 2. Physical equilibration time for O2 and CO2 le6els in chambers adhered o6er non-wounded fruit surface
Two chambers, mounted in supporting frames to facilitate repeated sampling, were adhered adja-cent to each other on the surface of each of eight peppers, one over a surface wound as described in Experiment 1 and the other over intact, non-dam-aged surface. After sealing the chambers, fruit were left for a further 48 h before sampling the contents of chambers over wounds (taw=48 h).
Chambers over intact surface were then flushed with nitrogen and their contents were sampled at
taw=52 h. All chambers were sampled attaw=60
and 72 h. Direct removal was used to take sam-ples of cavity atmosphere from each fruit (taw
=
96 h), and subsequently from the flesh of the fruit following replacement of the cavity atmosphere with water to prevent accidental contamination with contents of the cavity.
2.3. Gas analysis
Oxygen and CO2 contents were determined using
an O2 electrode (Citicell C/S type, City
Technol-ogy Ltd., London, UK) in series with a miniature infra-red CO2 transducer (Analytical
Develop-ment Company, Hoddesdon, UK) with O2-free
N2as carrier gas (flow rate 580 mm3s−1). Output
signals were analysed using integrators (Hewlett Packard, model 3394A). Commercially prepared standards, (BOC Special Gases, Wellington, NZ) were used for calibration of the gas analysers (2.05 kPa CO2, 5.1 kPa O2or 10.01 kPa CO2, 20.1
kPa O2, when held at one standard atmosphere
total pressure.
2.4. Data analysis
For experiment 1, differences between samples taken from flesh or cavity by direct removal and those from chambers held on to the fruit surface with frames were analysed separately by time (GLM procedure; SAS Institute, 1988). Values for
k2 for the equilibration of both O2 and CO2 in
chambers over wounded surface (experiment 1) and intact cuticle (experiment 2) were obtained by non-linear regression (SAS Institute, 1988) of pO2
ch,t
andpCO2 ch,t
against time using Eq. (6). Before analy-sis, data at any given time were standardised by dividing by the final values (when equilibration was complete) for these variables in chambers over wounded surface, which were taken to repre-sent internal atmosphere composition. The same general form of function as that in Eq. (6) was fitted to longer term data from experiment 2 to characterise physiological drift in internal atmo-sphere estimates with time.
3. Results
3.1. Experiment 1
Analysis of standardised data for equilibration of both O2 and CO2 in chambers adhered over
wounded areas on the fruit surface revealed con-sistent and rapid equilibration of both gases (Fig. 1A and B, respectively). Values for k2, and
corre-sponding estimates for surface permeance andt0.99
were similar for O2 and CO2. There was some
indication of downward drift in CO2 values with
longer equilibration periods (Fig. 1B).
Quasi-steady state levels of CO2 in the
cham-bers over wounded fruit surface underwent con-tinuous decline from the time when the chambers were first equilibrated with the internal atmo-sphere of the peppers (Fig. 2B). On a gross scale, levels of O2in chambers were approximately
con-stant throughout the experiment except for peri-ods of a few hours following flushing chambers with nitrogen (Fig. 2A), although the slight ap-parent upward drift in O2 values was consistent
with the decline in CO2. Neither flesh nor cavity
atmospheres were affected by wounding (data not
Fig. 1. Changes with time in atmosphere composition within chambers adhered on the surface of pepper fruit at 20.4°C following flushing with nitrogen att=0: (A) O2and (B) CO2
contents for wounded surface (experiment 1) and (C) O2and
CO2contents for intact cuticle (experiment 2). Values
Fig. 2. Long term changes with time after wounding (taw, h) in
partial pressures of (A) O2 (pO2
ch, kPa), and (B) CO
2 (pCO2
ch ,
kPa) in chambers adhered over wounded areas on the surface of pepper fruit at 20.4°C. Chambers were flushed with N2 at
16 and 64 h, denoted by arrows, and measurements made at 0, 0.5, 1, 4 and 12 h after flushing. Values represent averages of eight peppers; the line in (B) was obtained by fitting the following function to data for times after flushing greater than 2 h: pCOch2=(1.0290.078)+(1.3990.070) · (1−taw/(19.29
5.35+taw)).
Samples of flesh atmosphere taken by direct removal consistently contained less O2 and more
CO2 than samples similarly removed from the
fruit cavity. Average values for cavity values mi-nus flesh values were 1.190.12 kPa O2 and −0.6890.044 kPa CO2 (S.E.M. had 46 degrees
of freedom in both cases), with no effect of wounding or time. For the final destructive sam-pling of fruit with surface chambers, flesh samples had similar O2 contents to those removed from
chambers (data not shown). In contrast, there was a marked elevation of CO2level in direct removal
samples relative to their chamber counterparts, regardless of the level of CO2 level in the fruit
cavity, which ranged from 0.3 to 0.8 kPa. Average
Fig. 3. Changes with time in (A) O2 and (B) CO2 partial
pressures in samples of cavity, flesh and chamber atmospheres taken from in sweet pepper fruit at 20.4°C. Values represent averages for eight peppers. Chamber samples were taken re-peatedly on the same fruit. Destructive, direct removal samples from flesh and cavity atmospheres were taken on a fresh batch of fruit each time, combining data from fruit with and without surface wounding. Error bars are standard errors of means for comparison of means obtained on a single occasion).
shown). The initial downward drift of CO2 and
upward drift of O2 levels in flesh and cavity
atmospheres with time, averaged over wounded and control treatments, was similar to that in samples taken from chambers over wounded fruit surface (Fig. 3A and B). There were subsequently some apparently random deviations in the de-structive samples from the consistent overall trends detected through analysis of chamber tents. Differences between flesh and chamber con-tents became statistically significant for CO2 after
76 h (PB0.01 on each occasion) but were only significant at one measuring time for O2 (tw=88
Table 1
Values for the exponential decay constant from Eq. (6) (k2, s−1), fruit surface permeance to gases (P%j, pmol s
−1 m−2 per Pa,
calculated using Eq. (8)) and 99% equilibration time (t0.99, h; calculated using Eq. (7)) for chambers adhered to pepper fruit surface
at 20.4°C for both O2and CO2for experiments 1 and 2a
O2
Variable CO2
S.E.
Average Average S.E.
Experiment1 (wounded)
0.103×10−4 −3.77×10−4
−3.97×10−4 0.045×10−4
k2(s−1)
9001
Pj%(pmol s−1m−2per Pa) 234 8548 103
t0.99(h) 3.22 0.08 3.39 0.04
Experiment2 (intact)
k2(s−1) −4.23×10−6 0.358×10−6 −43.6×10−6 3.55×10−6
1.95 237
Pj%(pmol s−1m−2per Pa) 23.0 19.3
24 29.3
302 2.14
t0.99(h)
aPermeance values calculated for experiment 1 assume negligible contribution of intact cuticle to measured permeance. elevation of CO2 levels in flesh samples above
those taken from the chambers was 0.5190.051 kPa, whilst the elevation in CO2 level in the flesh
relative to samples from the fruit cavity was 0.7790.057 kPa. Thus, overall difference between fruit cavity and chamber contents was slight but statistically significant (0.2690.091 kPa).
3.2. Experiment2
Rates of change of gas composition were much slower for chambers over intact cuticle than for those over wounded surface in experiment 1 (Fig. 1) and corresponding k2 values were very much
smaller (Table 1). For O2, the absence of
wound-ing decreased the magnitude of k2 by 99%
whilst that for CO2 was reduced by 90%.
Val-ues fort0.99were correspondingly increased.
Equi-libration of O2 contents required about ten times
as long as equilibration of CO2. Estimates of
permeance to O2 ranged between 16 and 36 pmol
s−1 m−2 per Pa. Average permeance to O 2 was
only 10% of that to CO2; similarly, the
relation-ship between permeances to the two gases had a slope of about 10 (Fig. 4).
4. Discussion
Experiment 1 demonstrated that physical equili-bration of the gaseous contents of chambers
ad-hered over wounded areas on the fruit surface could be expected to be effectively complete within 4 h. Equilibration was much more rapid over the wounded fruit surface used in experiment 1 than when the fruit surface was left intact as in experiment 2 (Table 1). It was also much more rapid than for equilibration of similar chambers on other types of fruits and vegetables (Banks and Kays, 1988; Rajapakse et al., 1990). This can be attributed to removal of the fruit cuticle that
Fig. 4. Relationship between estimates of permeance to CO2
(P%CO
2, pmol s
−1 m−2 per Pa) and permeance to O 2 (P%O2,
pmol s−1 m−2 per Pa) of pepper cuticle at 20.4°C obtained
from fitted lines shown in Fig. 4 using Eq. (8). The fitted line was obtained by linear regression:P%CO
2=(38921.9)+(9.29
0.92) ·P%O
would be expected to comprise the principal resistance to gas exchange between the fruit’s internal atmosphere and the chamber con-tents.
In contrast to the rapid process of physical equilibration, physiological equilibration of inter-nal atmosphere composition beneath wounded tis-sue was much more extended, requiring 60 h or more. The downward trend inpCOi 2was consistent
throughout the experiment up to this time. A similar trend in the reverse direction was present for pO2
i
, although this was within the limits of measurement error for much of the experiment, given the much higher levels of O2 present within
the chambers. The increase in O2 levels would be
consistent with a settling of respiratory activity with increased time after harvest, as has previ-ously been reported in non-climacteric fruits (Biale, 1950). Comparison of direct removal sam-ples provided no substantial evidence that wound-ing affected estimates of internal atmosphere composition, though the physiological drift ob-served in contents of chamber samples would also have been consistent with this explanation. The apparently random deviations in composition of destructively-taken samples from the consis-tent trends observed in the chamber atmos-pheres presumably related to among-fruit varia-tion since cavity and flesh samples were closely correlated.
The similar trend for O2levels in both chamber
and direct removal samples indicates that both techniques are suitable for characterising pOi2 in
pepper flesh. In contrast, the difference in compo-sition between contents of chambers and direct removal samples from the fruit flesh demonstrates that care must be taken in choosing a sampling method for characterising internal CO2. The lower
level of CO2found in chamber samples relative to
flesh samples taken by direct removal cannot have developed simply because the equilibration of chamber contents was lagging behind actual levels because equilibration periods were greatly in ex-cess oft0.99and because long term CO2levels were
in decline throughout the experiment. Rather, the lower levels in chamber contents were close to true levels within the internal atmosphere of the fruit tissue and the higher levels found in direct
removal ‘flesh’ samples would have developed as a result of negative pressures involved in withdraw-ing samples from the flesh; considerable difficulty was encountered in removing adequate volumes of gas to analyse from many of the fruit. This finding is consistent with similar problems previ-ously reported in potatoes (Banks and Kays, 1988).
Permeance to oxygen of intact pepper surface was more than an order of magnitude less than values obtained for apples (Cameron and Reid, 1982; Rajapakse et al., 1992; Dadzie et al., 1996). Presumably, this is because the pepper fruit sur-face is devoid of pores (Blanke, 1986; Blanke and Holthe, 1997); the values presented here should therefore be representative of pepper fruit cuticle. Taking contribution of intact cuticle to total ex-change of chambers over wounded surface to be negligible, permeance of the wounded area to both gases was approximately 260 times the value for the intact surface cuticle in the case of O2and
26 times for CO2. Damage to the intact cuticle
therefore has the potential to contribute very sub-stantially to the overall gas exchange characteris-tics of such fruits. Such wounding would be necessary for chamber contents to become repre-sentative of the fruit’s internal atmosphere within periods relevant to physiological change within the pepper and would clearly be essential for use of chambers to monitor internal atmosphere com-position within peppers.
Interestingly, values for P%CO
2 were consistently an order of magnitude greater than those forP%O
calyx end and transfer through cuticle covering the remainder of the fruit surface could be studied separately.
Samples of internal atmosphere removed from the fruit cavity consistently had more oxygen and less carbon dioxide than those removed from the flesh, whether these were taken from chambers or by direct removal. This is consistent with the proposition that the majority of gas exchange in some solanaceous fruits occurs through the calyx (Cameron and Yang, 1982). It provides some counter-evidence to the conclusions drawn by Blanke and Holthe (1997), based on the ab-sence of pores in the fruit surface, that the majority of gas exchange of peppers occurs di-rectly through the outer cuticle. Oxygen diffusion into the flesh at a distance from the calyx could presumably occur either longitudinally through the flesh of the wall itself or via the cavity, through the inner lining of the pericarp wall. Clearly, this inner lining has some significant re-sistance to gas transfer because the depression of O2 between the cavity and the internal
atmo-sphere of the flesh was about half of the total difference between the external atmosphere and the fruit flesh.
In conclusion, we have evaluated the strengths and weaknesses of alternative approaches to char-acterising internal atmosphere composition of sweet peppers. Sampling from the inner cavity does not provide samples that are representative of the composition of the atmosphere immediately surrounding the cells of the tissue. Direct removal from the flesh was adequate for O2 but would
generally not be suitable because it tended to overestimate CO2 levels in the internal
atmo-sphere. Chambers adhered to the fruit surface could be sampled to analyse internal atmosphere composition but only if the cuticle had previously been removed to facilitate exchange between the fruit tissues and the chamber. There was no sub-stantial evidence for this wounding altering inter-nal atmosphere composition and, even with respect to other components of physiological drift, composition had become largely stable within 60 h. Such chambers could therefore be used to determine long term changes in internal atmosphere composition. Permeance of the
pep-per cuticle to CO2 was shown to be 10 times
greater than for that to O2, information which
will be useful in interpreting the effects of surface coatings on the overall permeance characteristics of fruits and vegetables.
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