Physical and mechanical changes in strawberry fruit after
high carbon dioxide treatments
F. Roger Harker
a,* , H. John Elgar
a, Christopher B. Watkins
b,
Phillipa J. Jackson
a, Ian C. Hallett
aaThe Horticulture and Food Research Institute of New Zealand Ltd., Mt.Albert Research Centre,Pri6ate Bag92169,
Auckland,New Zealand
bDepartment of Fruit and Vegetable Science,Cornell Uni6ersity,Ithaca,NY14853,USA
Received 20 August 1999; accepted 15 February 2000
Abstract
‘Pajaro’ strawberry (Frageria x ananassa Duch.) fruit were exposed to 5 – 40% CO2 for 0 – 3 days, followed by
normal cold storage at 0°C for up to 3 weeks. Strawberry fruit were firmer in air storage at 0°C than at harvest. Firmness was further enhanced by CO2treatments. Adhesion between cells was measured by the application of tensile
tests to plugs of tissue, followed by the examination of fracture surfaces using low temperature scanning electron microscopy. These tests indicated that cell-to-cell adhesion increased by 60% as a result of CO2treatments. However,
there were no differences in the density, electrolyte leakage, propensity for cells to rupture in hypertonic solutions, water potential, osmotic potential or turgor of CO2-treated and control fruit. Electrical impedance spectroscopy was
used to assess changes in the electrical resistance of the apoplast and symplast. Carbon dioxide treatments reduced the resistance of the apoplast (resistance at 50 Hz) below that of control fruit, but did not affect the resistance of the symplast (resistance at 1 MHz). This result suggests that concentrations of H+and HCO
3
−increased in the apoplast,
although no change was detected in the symplast. We speculate that the mechanism for CO2-induced firmness
enhancement in strawberry is due to changes in the pH of the apoplast. Such changes in pH may promote the precipitation of soluble pectins and thus enhance cell-to-cell bonding in strawberry fruit. © 2000 Elsevier Science B.V. All rights reserved.
Keywords:Fragaria×ananassaDuch.; Strawberries; Firmness; Texture; Storage; High CO2
www.elsevier.com/locate/postharvbio
1. Introduction
Carbon dioxide-treated strawberry fruit are firmer than air-stored fruit and have lower suscep-tibility to decay, resulting in extension of posthar-vest life (Harris and Harvey, 1973; El-Kazzaz et al., 1983). Moreover, firmness of strawberries can * Corresponding author. Tel.:+64-9-8154200; fax: +
64-9-8154202.
E-mail address:[email protected] (F.R. Harker)
increase, rather than simply be maintained, when fruit are exposed to high levels of CO2during cold
storage (Ptocharski, 1982; Smith, 1992; Smith and Skog, 1992; Larsen and Watkins, 1995). This is in addition to the firmness enhancement observed when strawberry fruit are held at low tempera-tures (Ourecky and Bourne, 1968; Larsen and Watkins, 1995; Watkins et al., 1999). CO2
-in-duced firmness increases occur in most cultivars (Smith and Skog, 1992; Watkins et al., 1999), and is influenced by berry maturity (Goto et al., 1995) as well as treatment temperatures (Smith, 1992).
While these studies have characterised the influ-ence of CO2 on strawberry firmness, the
mecha-nism responsible for CO2-induced firming of
strawberry tissue is unknown. Cell wall analyses by Ptocharski (1982) and Goto et al. (1995) demonstrated that CO2 treatment reduced the
amount of pectin extracted in the water-soluble phase and increased the amount of pectin ex-tracted in the ammonium-soluble phase and/or hexametaphosphate phase. Similarly, Siriphanich (1998) found that CO2 treatment reduced the
amount of pectin extracted in a water-soluble fraction and increased the amount of pectin ex-tracted in a CDTA fraction. However, Smith (1992) found that the amounts of pectin, and the fractions they were extracted in, were similar in both CO2- and air-treated strawberry. Generally,
the hardness of fruit is related to a number of other cellular characteristics, including adhesion between neighbouring cells, cell fragility, and in-ternal turgor pressure (Harker and Hallett, 1992, 1994; Harker and Sutherland, 1993). Here, our objective was to investigate the influence of CO2
on firmness of ‘Pajaro’ strawberries both at har-vest and after subsequent air storage, and to examine its effect on cellular and tissue characteristics.
2. Materials and methods
2.1. Fruit source
Strawberry fruit (FragariaxananassaDuch. cv. Pajaro) at pink and red maturity stages were obtained from commercial growers in the
Auck-land area during November 1993 to January 1994. Fruit were freshly harvested in the early morning and transported to the laboratory within two hours. Fruit were graded for uniformity of colour and size, and damaged berries were removed.
2.2. Controlled atmosphere treatments
The fruit were divided into 30-fruit replicate samples, and stored at 0°C in 27-l flow-through chambers, each chamber holding three replicates. Precision needle valves were used to mix CO2, air
and N2to produce the required atmospheres. The
gas mixtures were humidified by bubbling the gas through water at 0°C, and passed through each chamber at a flow rate of 200 ml min−1. In each
trial, control samples of fruit were also held in flow-through chambers with humidified air as the atmosphere. The experiments were carried out using fruit of both maturity stages. There were three replicates of 30 fruit for each treatment, and each experiment was repeated.
The following experiments were carried out: 1. Fruit were treated with air, 10, 20 or 40% CO2
for 0, 1, 2 or 3 days at 0°C. Fruit were assessed immediately after treatment.
2. Fruit were treated with air, 5, 10 or 20% CO2
for 2 days at 0°C. Upon removal from CA, the fruit were covered with unsealed plastic bags to reduce water loss, and held in air at 0°C. Fruit were assessed immediately after removal from the CO2treatments and after storage for
7, 14 and 21 days at 0°C.
2.3. Quality assessments
Fruit firmness was assessed on cold fruit using an Imada penetrometer (PSM-10, Imada, Toy-ohashi, Japan) fitted with a flat round 13.16 mm diameter head. The head was pushed into the strawberry flesh through the skin to the depth of the head (5 mm).
2.4. Texture and electron microscopy studies
measurements were undertaken using an Instron materials testing machine (Model 4301; Instron, Canton, Mass., USA), and 8 individual fruit per treatment were selected for each of the three tests. Firstly, the firmness of individual cold strawber-ries was measured by driving a 13.16 mm diame-ter probe with a flat tip into the flesh at a speed of 240 mm min−1. Secondly, a radial profile of
firmness through the skin, flesh and core tissue was obtained by driving a razor blade through a block of tissue at 15 mm min−1
(Vincent, 1990). Thirdly, the tensile strength of strawberry tissue was measured by attaching plugs of tissue to plates and pulling them apart, as described in Harker and Sutherland (1993). Following tissue failure, the fracture surface was examined using low temperature scanning electron microscopy, as described by Harker and Hallett (1992, 1994).
2.5. Fruit density studies
Density of individual fruit was determined by dividing fruit weight (90.001 g) by fruit volume (90.001 ml). Volume was determined by the Archimedes principle.
2.6. Electrolyte leakage and cell rupture
Cell fragility was determined by incubating tis-sue discs in a range of hypotonic mannitol solu-tions as described previously (Harker and Hallett, 1992, 1994; Harker and Sutherland, 1993). Essen-tially the method aims to identify the solution osmolarity that causes cells to rupture and disc weight to decline. Cell rupture was confirmed by measurements of electrolyte leakage using a con-ductivity meter (Konductimeter CG875, Schott Geraete, Germany).
2.7. Electrical impedance measurements
The electrical impedance of whole strawberry fruit was measured using a precision LCR meter (model HP 4284A; Hewlett-Packard, Hyogo, Japan). Two silver/silver chloride (Ag/AgCl) elec-trodes (model EP2, World Precision Instruments, Sarasota, Florida, USA) were impaled into the flesh at locations 2 cm apart, and alternating
current at frequencies between 50 Hz and 1 MHz were passed through the fruit, as described earlier (Harker and Maindonald, 1994). Electrode resis-tance usually associated with impedance measure-ments in fruit (Harker and Dunlop, 1994) is not a problem when Ag/AgCl electrodes are used. Thus, the resistive and reactive components of impedance generated by the LCR meter were used without any need for modification (Harker and Maindonald, 1994).
2.8. Water relations
Water potential and osmotic pressure of pink strawberry was measured for ten fruit (five from high CO2 treatment and five from air treatment)
using a Westcor 5500 vapor pressure osmometer (Westcor, Logan, Utah, USA). Water potential was measured using discs (7 mm diameter×3 mm thick) excised from individual fruit with juice blotted from the surface. The discs were left for 5 min before vapour pressure was measured. Juice was extracted from fruit by microfuging freeze-thawed tissue for 5 min at 12 000 rpm. A 10 ml
sample of juice was collected from the microfuge tube and the osmotic pressure measured using the osmometer. Turgor pressure was calculated using the formula P=c+p, where P is turgor, c is water potential, and p is osmotic pressure (To-mos, 1988; note p is a positive value).
2.9. Data analysis
Data were either tested by analysis of variance, and LSDs used to separate significant effects at the 5% level, or by the Wilcoxon rank-sum test.
3. Results and discussion
3.1. Influences of low temperature and CO2
treatment
cowork-ers speculated that increases in the firmness of stonefruit were due to a change in the behaviour of the pectin (perhaps increasing viscosity) since pectin structure/chemistry did not change (Werner and Frenkel, 1978; Werner et al., 1978).
The firmness of strawberries was further en-hanced by CO2 treatment (Table 1). Fruit
firm-ness increased as the CO2concentration increased
within each treatment period, but within each CO2treatment the firmness increase generally was
small after one day. In the second experiment, fruit were given a 2-day CO2 treatment and then
transferred to air storage for up to 19 days (total
of 21 days at 0°C). The firmness of berries re-mained higher than harvest firmness values throughout the 21 day period of storage in air at 0°C for both control (air-treated) and CO2-treated
fruit (Table 2). In particular, the CO2-induced
increase in firmness did not reverse when the fruit were returned to air, as long as the strawberries were held at 0°C (Table 2). Gains in firmness were greater for pink compared to red berries, and with increasing CO2 concentration. However, within 3
days at 20°C strawberries from all treatments softened rapidly and no residual effect of CO2
-en-hanced firming was detectable (data not shown).
Table 1
Firmness (N) of pink- and red-maturity grades of ‘Pajaro’ strawberry fruit immediately after treatment with air, 10, 20 or 40% CO2
for 1–3 days at 0°C (LSD=2.0)
Treatment duration (days) CO2treatment (%)
Maturity grade
Air 10 20 40
21.9
Pink 0
1 29.8 33.0 36.7 39.8
27.6 33.7 38.1
2 40.9
3 30.0 36.4 38.2 40.6
0 17.4
Red
24.0 26.5 29.4
1 32.4
2 24.2 29.4 31.5 34.9
3 25.6 29.8 33.5 33.5
Table 2
Firmness (N) of pink- and red-maturity grades of ‘Pajaro’ strawberry fruit treated with air, 5, 10 or 20% CO2for 2 days at 0°C,
and assessed immediately after treatment and after total storage of 7, 14 and 21 days at 0°C (LSD=3.0)
Duration (Days) CO2treatment (%)
Maturity
Grade CO2 Air Total Air 5 10 20
0 0 21.8
Pink 0
2 0 2 30.9 30.9 34.4 36.0
5 7 38.1 42.9 40.8 47.2
2
14 36.5 41.4 44.1
2 12 49.4
21 37.6 36.4
19 40.9
2 46.3
0
Red 0 0 21.1
41.3 36.5
30.8 26.4
2 0
2
5 7 31.9 33.3
2 35.6 38.8
12 14 31.1 34.6 36.6 39.3
2
37.6 32.1
27.7 32.8
21
Table 3
Physiological properties of air- and CO2-treated ‘Pajaro’
strawberry fruit. Pink fruit were exposed to 20% CO2or air
for 2 days at 0°Ca
Air-treated CO2-treated
Texture measurements(n=8)
26.0b
Firmness by puncture (N) 19.1a
0.73b
0.45a
Tensile strength (N)
Density(n=8)
Density (g ml-1) 1.043a 1.045a
Water relations measurements(n=5)
−1.24a
−1.24a
Water potential (MPa)
Osmotic pressure (MPa) 0.93a 0.95a
0.28a
0.32a
Turgor pressure (MPa)
Impedance characteristics(n=10)
Resistance at 50 Hz 24 785a 21 695b
(ohms)
Resistance at 1 MHz 747a 751a
(ohms)
aData followed by different letters within a row are
signifi-cantly different at the 5% level by the Wilcoxon rank-sum test.
cell-to-cell contact. Vincent (1989) developed a model that related tissue density to the degree of cell-to-cell contact in apples. In the present study, the density of air- and CO2-treated strawberries
was similar (Table 3). This suggests that differ-ences in the tensile strength of tissue were due to the condition of the middle lamella pectin rather than any difference in the degree of cell-to-cell contact. Microscopy studies show that the extent of cell-to-cell contact is very limited in ripening strawberry (Redgwell et al., 1997).
In the present study it was clear that the strength of the cell wall had little influence on tissue strength, since cell-to-cell debonding was the predominant mechanism of failure. Attempts to determine differences in cell wall strength (cell fragility; see Section 2.6) of CO2- and air-treated
Fig. 1. Fracture surfaces of blocks of strawberry tissue follow-ing tensile tests observed usfollow-ing low temperature scannfollow-ing electron microscopy. (A) CO2-treated fruit, and (B) air-treated
fruit. Most cells in both samples have pulled apart without fracturing. Arrows indicate the presence of juice on the surface of cells. Bar=1 mm.
3.2. Physiological and mechanical properties of
cells
The mechanical properties of fruit are a func-tion of cell wall strength, cell-to-cell adhesion, cell packing and the internal pressure or turgor of cells (Harker et al., 1997). Instrumental measure-ments of tissue strength confirmed manual pen-etrometer measurements showing that fruit from CO2-treatments were firmer than air-treated fruit
(Table 3). Examination of fracture surfaces fol-lowing tensile testing indicated that the primary mode of tissue failure was cell-to-cell debonding in both air- and CO2-treated fruit, and that no cell
breakage occurred (Fig. 1). Radial profiles of tissue strength were unable to differentiate be-tween CO2- and air-treated fruit (Fig. 2),
suggest-ing that while resistance to cuttsuggest-ing with a razor blade differed between skin, flesh and core tissues, the method was not sensitive enough to detect treatment effects. However, there is no reason to expect that the influence of CO2 was associated
with any specific tissue zones.
Fig. 2. Force-distance curves for air- and CO2-treated
straw-berries. Curves represent the average profile (n=8) generated as a razor blade, attached to an Instron, cut a radial cross-sec-tion through the skin, flesh and core tissues in a block of excised strawberry. Note that the curves are offset from each other due to differences in fruit diameter.
middle lamella pectins. Cell wall analyses indicate that CO2 treatment decreases the proportion of
pectin that is water-soluble, and increases the proportion that is ionically bonded into the cell wall matrix (Ptocharski, 1982; Goto et al., 1995; Siriphanich, 1998). These cell wall studies suggest that CO2increases the amount of structural pectin
associated with the cell wall, while the present study indicates that this pectin promotes cell-to-cell adhesion.
3.3. Electrical impedance of strawberry tissue
Physical changes that occur during CO2
treat-ment were also characterised using electrical impedance measurements. Generally, low fre-quency measurements (e.g. 50 Hz) can be used to determine the resistance of the apoplast, while high frequency measurements (e.g. 1 MHz) deter-mine the resistance of the entire tissue, but are largely a measure of the symplastic resistance (Cole, 1972; Harker and Maindonald, 1994). In the present study, the resistance at 50 Hz (apo-plast) was lower in CO2- than air-treated fruit, but
the resistance at 1MHz (symplast) was not signifi-cantly different (Table 3). This reflected an in-crease in electrolytes within the extracellular solution. The electrolytes most likely to have low-ered tissue resistance were the hydrogen (H+) and bicarbonate (HCO3
−) ions that formed as CO
2
became dissolved in the cell contents, since: CO2+H2OlH2CO3lH
++HCO
3
−(Bown, 1985).
3.4. Possible mechanisms for CO2 enhancement of
strawberry firmness
Compared with other fruit, strawberries contain high levels of soluble pectin (Redgwell et al., 1997). These pectins may be modified during CO2
-treatment and result in firmness enhancement. Carbon dioxide treatments have caused both in-creases (Irving and Honnor, 1994; Holcroft and Kader, 1999) and decreases (Siriphanich and Kader, 1986; Lange and Kader, 1997) in the intracellular pH of horticultural products. Both H+and HCO
3
- produced by solubilisation of CO 2
could influence pH (Lucas, 1979; Bown, 1985). strawberries were unsuccessful, since cells from
both treatments tolerated incubation in hypotonic solutions without rupturing (data not presented). Another factor that can influence the strength of fruit tissue is the turgor or pressure of the cells. The mechanical strength of fruit tissue is largely hydrostatic, and in the absence of cell turgor, tissues have reduced load-bearing capacity (Harker et al., 1997). A recent study on carrot demonstrated that loss of tissue firmness and tur-gor was linearly related (Greve et al., 1994). Thus, it was important to characterise the influence of CO2 treatment on turgor of strawberry cells. A
psychrometric approach was used to measure wa-ter potential of tissue discs and osmotic pressure of juice, and these values were used to calculate turgor. However, there were no significant differ-ences in water potential, osmotic pressure or tur-gor of air- or CO2-treated strawberries (Table 3).
Collectively, our results identify that CO2
treat-ment enhances the strength of cell-to-cell bonds in strawberry fruit. Tissue failure during tensile tests occurred as a result of cell-to-cell debonding (Fig. 1), and tissue strength was 0.45 and 0.73 N for air- and CO2-treated strawberries, respectively
(Table 3). This suggests that the strength of cell-to-cell bonding was 60% higher in CO2-treated
While the presence of H+ clearly decreases pH, the influence of HCO3
− is more uncertain. In
aquatic algae and plants it has been shown that the uptake of HCO3
− into cells leads to an
in-crease in the pH of the apoplast (Lucas, 1979; Laing and Browse, 1985). Lucas (1979) suggests that the data for Chara corallina supports the presence of an OH− efflux rather than an H+ influx transport system that is associated with the uptake of HCO3−. While it is speculative to
sug-gest that such transmembrane transport systems can operate in strawberries in cold storage, a number of studies have found that the pH of strawberry (measured on extracted juice) increase as a result of CO2 treatment (Ke et al., 1991;
Holcroft and Kader, 1999). However, it should be noted that while extracellular pH will be influ-enced by active transmembrane H+ and/or OH− transport (Sentenac and Grignon, 1987), the in-tracellular pH of strawberry is predominantly due to the buffering capacity of organic acids (Hol-croft and Kader, 1999).
It is possible that the increase in strength of middle lammella pectins in CO2-treated
straw-berry could be explained by either an increase or a decrease in apoplastic pH. Acidification may mask the negative charged carboxyl groups that are responsible for repulsion of neighbouring pectin molecules (Shomer et al., 1991), and/or induce gelation of soluble pectins (Suggett, 1975). However, acid-induced gelation of pectin occurs at pH levels lower than 3.5 (Keller, 1984), which is much lower than might be expected to occur in the plant apoplast (Sentenac and Grignon, 1987). Alternatively, any increase in the pH of the apo-plast will favour Ca2+ rather than H+ as the ion species that binds to negatively charged carboxyl groups of the cell wall. Calcium is known to promote linking of neighbouring pectin polymers through the egg-box model (Demarty et al., 1984). Indeed, Barnes and Patchett (1976) suggest that during strawberry ripening, the flesh softens as a result of the loss of such calcium cross-links. It is perhaps intuitive to expect that Ca2+ binding will be a key part of the mechanism for firmness enhancement. We speculate that this may be pos-sible if HCO3− uptake into strawberry cells
stimu-lates either OH− efflux or H+ influx.
Interestingly, the influence of CO2 was not
re-versible when fruit held at 0°C were transferred from CO2-treatment to air (Table 2). This
sug-gests that once the soluble pectins have been bound into the cell wall, they are not released when HCO3− levels and apoplastic pH return to
normal.
In summary, the results from this study indicate that CO2 treatments enhance firmness of
straw-berry in a non-reversible manner; firmness-en-hancement is due to an increase in the strength of cell-to-cell bonding; and changes in conductivity of the apoplast, but not of the symplast, occur during CO2 treatment. It is possible to speculate
that the mechanism for CO2enhancement of
firm-ness is associated with changes in the pH of the apoplast. However, further research is needed to confirm that extracellular pH does change as a result of CO2treatment, and that such changes in
pH influence the firmness of strawberry.
Acknowledgements
This research was supported by the New Zealand Foundation for Research, Science and Technology (Contract CO6532) and The Auck-land Strawberry Growers’ Association Inc. We thank Keith Fuller, John Garelja and Derek Perry for providing fruit and helpful advice; and Rosie Schroeder for discussions on strawberry cell wall structure and chemistry.
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