Food Research International 34 (2001) 189±195
www.elsevier.com/locate/foodres
Contributions of cellular components to the
rheological behaviour of kiwifruit
A.M. Rojas a, L.N. Gerschenson a,b, A.G. Marangoni c,*
a
Departamento de Industrias, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires,
1428 Buenos Aires, Argentina
b
National Research Council (CONICET) Argentina
c
Department of Food Science, University of Guelph, Guelph, Canada ON N1G2W1
Received 5 May 2000; accepted 21 July 2000
Abstract
In this research work we propose an empirical model and develop a methodology for the determination of the magnitude of the
contributions of turgor pressure, cell wall and middle lamellae, to the elastic properties of a plant material within a particular tissue
type. The model system used was outer pericarp tissue from unripe and ripe kiwifruit (Actinidia deliciosa cv. Hayward). Samples
were equilibrated in a series (0±0.96 M) of polyethylene glycol 400 (PEG) solutions, and the volumes, storage (G0 ) and loss (G00 )
moduli, and the tangent of the phase angle (tan) of the tissue samples determined. Tissue specimens were also examined using cryoscanning electron microscopy (cryo-SEM) to seek support for the rheological evidence obtained. The model proposed and the
methodology applied allowed us to establish that the complex modulus (G*) of ripe and unripe raw outer pericarp kiwifruit tissue
was in¯uenced mostly by turgor pressure and cell wall rigidity. The loss of middle lamellae during ripening was accompanied by a
rigidi®cation of the cell wall. # 2001 Elsevier Science Ltd. All rights reserved.
Keywords: Cell wall; Middle lamellae; Turgor; Elasticity; Kiwifruit; Osmotic pressure
1. Introduction
The structure and chemical composition of plant
materials govern their textural characteristics including,
among others, ®rmness, crispness, mealiness and
juiciness (Ilker & Szczesniak, 1990). The mechanical
properties of fruit and vegetable tissue depend on the
mechanical properties and structural arrangement of
cell agglomerates (Frey-Wyssling, 1952; Pitt, 1982; Pitt
& Chen, 1983). The cell is the smallest structural unit
within the tissue, and textural quality lies in this cellular
`mechanical unit' whose components are cell wall, cellular membrane or plasmalemma, and middle lamellae.
Turgor pressure is the hydrostatic pressure exerted by
intracellular liquid on cellular membranes which, due to
their semipermeable properties, give rise to osmosis.
* Corresponding author. Tel.: +1-519-824-4120; fax: +1-519-8246631.
E-mail addresses: [email protected] (A.G. Marangoni),
[email protected] (A.M. Rojas).
This turgor pressure has profound e€ects on fruit and
vegetable rheological properties (Falk, Hertz, & Virgin,
1958; Jackman, Marangoni, & Stanley, 1992; Lin & Pitt,
1984; Murase, Merva, & Segerlind, 1980; Nilsson, Hertz,
& Falk, 1958; Phillip, 1958). The rigidity and perceived
crispness of fruits and vegetables has been largely
attributed to cell turgor (Bourne, 1976). Since the plasmalemma has little mechanical resistance, it is the
pressure exerted on the cell wall which accounts for the
turgor pressure-induced elasticity of cells and tissue.
The middle lamellae maintains cells within a tissue in
mechanical contact. It has been suggested that the ratio
of the mechanical resistance of the cell wall over that of
the middle lamellae determines sensory perception of
juiciness or dry, chalky granular texture during mastication (Ilker & Szczesniak, 1990).
In this research work we propose a model and
develop a methodology for the determination of the
magnitude of the contributions of turgor pressure, cell
wall and middle lamellae, to the dynamic rheological
properties of kiwifruit tissue.
0963-9969/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0963-9969(00)00151-4
190
A.M. Rojas et al. / Food Research International 34 (2001) 189±195
2. Model
Our model proposes that the elasticity of an intact
plant tissue is mostly in¯uenced by the structure and
integrity of cellular membranes, cell walls and middle
lamellae. Cellular membrane integrity will strongly
a€ect turgor pressure. In mathematical terms, the
modulus of elasticity of an intact plant tissue (GT) is
proposed to be proportional to the linear combination
of cell wall (GCW), middle lamellae (GML), and turgor
pressure (Gc) related moduli, i.e. a parallel arrangement
of springs, namely:
GT GCW ‡ GML ‡ G
…1†
Knowledge of the magnitude of these moduli would
shed light on the importance of cell wall, middle lamellae and cellular membrane structure and integrity on the
elasticity of a particular plant material. Obviously, tissue structural organization, e.g. spatial distribution of
cells within the tissue, number of cells per unit volume,
cell size and shape, as well as tissue dimensions will also
greatly in¯uence the magnitude of the macroscopic
modulus of elasticity. This tissue structure e€ect will be
di€erent for di€erent plant materials, and could also
conceivably di€er within a material. However, our
model seeks to de®ne the relative contributions of cell
walls, middle lamellae and turgor pressure towards the
elastic properties of a plant material within a particular
tissue type. The system used to test the proposed model
was kiwifruit tissue. The reader should be aware that in
this treatment we consider kiwifruit tissue as a homogenous and isotropic material. The material's response
will have a directional dependence which will in¯uence
results obtained. It is imperative thus that all materials
be tested in the same orientation within a study.
When a piece of plant material is placed in a medium
at a particular osmotic pressure, it will either shrink,
swell, or remain the same. If the osmotic pressure of the
medium surrounding the tissue is higher than the cellular osmotic potential, the material will shrink. If the
osmotic pressure of the medium surrounding the tissue
is lower than the osmotic potential of the cells, the
material will swell. If the osmotic pressure of the
medium matches that of the cells, then the volume of
the material will not change. By placing pieces of plant
tissue in media of di€erent osmotic pressures and measuring tissue volume, storage and loss moduli, and
tangent of the phase angle (tan), it is possible to determine the point at which the turgor pressure of the cells
is zero (incipient plasmolysis), the point at which the
cellular osmotic potential of the intact tissue matches
that of the surrounding medium (isotonicity), as well as
the point at which cells burst (plasmoptysis) (Stadelmann, 1966). If this is carried out using tissues at
di€erent stages of maturity, where a de®ned change in
one of the structural components has taken place (e.g.
middle lamellae dissolution), it would theoretically be
possible to determine the relative contributions of the
moduli of the three structural units towards the macroscopic elastic modulus of the tissue. As stated above, the
overall tissue elastic modulus can be considered a linear
combination of the moduli arising from the three major
structural elements present in the tissue, namely cell
walls, middle lamellae and membranes (responsible for
the existence of turgor pressure):
GT ˆ GCW ‡ GML ‡ G
At incipient plasmolysis (IP) of unripe (UR) tissue,
where turgor pressure is zero,
GIP;UR ˆ GCW ‡ GML
At incipient plasmolysis (IP) of ripe (R) tissue, where
turgor pressure is zero, and if the middle lamellae has
disappeared (GML=0),
GIP;
R
ˆ GCW
If GCW remains constant during the ripening process,
GML can then be calculated from
GML ˆ GIP;UR
GIP;R
For all cases, the contribution of turgor pressure
towards the tissue elastic modulus can be calculated
from:
Gc ˆ GT
GIP
3. Experimental
3.1. Sample preparation
Kiwifruit (Actinidia deliciosa, var. Hayward), originating in Italy, were bought at the Ontario Food Terminal at
two di€erent times. In each occasion, 9±11 Brix fruit were
selected and divided into two groups. The ®rst group was
used immediately (unripe fruit), while the other group of
fruit was allowed to ripen to 12±15 Brix, at a constant
temperature of 23 C (ripe fruit).
Kiwifruit were carefully cleaned with detergent, rinsed
and ®nally treated by immersion in a 0.1% (w/v) bleach
solution for 5 min and rinsed with distilled water. Fruit
were then carefully peeled with a surgical blade and cut
into halves along the major axis. A cylindrical specimen
of 20 mm diameter and 20 mm length was then obtained
from each half, with the aid of a cork borer. An outer
pericarp tissue sample was thus obtained with minimum
tissue damage.
A.M. Rojas et al. / Food Research International 34 (2001) 189±195
3.2. Tissue volume measurements
Relative tissue volume changes of nine excised tissue
cylinders equilibrated in bu€ered (20 mM potassium
phosphate, pH 6.8) polyethylene glycol 400 (PEG)
solutions in the range 0.00±0.96 M were determined.
The equilibration time of the tissue samples was 36 h at
12 C, after which the dimensions of the cylinders were
measured with the aid of calipers and the ®nal volume
calculated as described by Sanjin, Gerschenson, and
Rojas (1999). The volumes of cylinders of untreated
tissue were considered initial values …V0 †, and the
relative volume change which occurred upon equilibration in the di€erent PEG solutions was calculated as
…V V0 †=V0 .
3.3. Dynamic rheological tests
Treated and untreated tissue samples of outer pericarp were carefully excised with the aid of a surgical
blade and submitted to small-deformation dynamic
rheological analysis. Stress amplitude sweeps from 0.65
to 100 Pa (1 Hz) were carried out on raw and equilibrated kiwifruit in order to determine the boundaries of
linear viscoelastic behavior. Storage (G0 ) and loss
moduli (G00 ), as well as the tangent of the phase shift
angle (tan =G00 /G0 ) were determined at 20C using a
Carri-Med CSL2-500K Rheometer (TA Instruments,
England). The rheological behavior of soaked and
unsoaked outer pericarp kiwifruit tissue discs (20 mm
diameter and 3 mm height) was evaluated using a parallel plate geometry using a 2 mm shearing gap. The
upper plate had a diameter of 20 mm and sandpaper
was glued on its surface to minimize slippage during
measurements. Finally, all samples were evaluated at a
constant strain of 0.01% by carrying out frequency
sweeps between 0.1 and 10 Hz. The complex modulus
(G*) at a frequency of 5 Hz, was then calculated as:
G ˆ
G0
cos 3.4. Microscopy
Microscopic observation of tissues was performed
with a Hitachi S-570 (Tokyo, Japan) scanning electron
microscope (SEM). Samples were taken from outer
pericarp tissue of untreated kiwifruit, from samples
equilibrated in an isotonic PEG solution (0.48 M PEG),
and from plasmoptyzed (0.10 M PEG) and plasmolyzed
(0.58 M PEG) tissue samples. Tissue segments removed
with the aid of a surgical blade were placed on a ¯at
copper holder of an Emscope SP2000 Cryo Preparation
Unit (Emscope, Ashford Kent, UK). Specimens were
then rapidly plunge-frozen in liquid nitrogen slush at
191
207 C and transferred under vacuum to a cold stage in
the pre-chamber of the cryo-system. In order to examine
internal features, frozen specimens were fractured with a
pre-cooled metal blade at 170 C. The freeze fractured
surface of the frozen specimens was etched in the prechamber by raising the temperature to 95 C to remove
the surface-water ice through sublimation. The specimens were then sputter coated with 20±30 nm of gold at
130 C and transferred under vacuum into the cryochamber of the SEM. Microscopy was carried out using
an accelerating voltage of 10 or 15 kV at a stage temperature lower than 150 C.
4. Results and discussion
Fig. 1 shows changes in tissue volume with increasing
PEG concentration for unripe (A) and ripe (B) kiwifruit
samples. Relative volume changes as a function of
osmotic pressure were calculated from these data and
are shown in Fig. 1C and D. The solid symbol in Fig.
1A and B corresponds to initial raw tissue volumes.
Relative tissue volume varied in a linear fashion with
osmotic pressure in the range 0.36±0.66 M PEG for
both ripe (r2=0.99) and unripe (r2=0.92) kiwi tissue.
The slope of this line corresponds to the, so called, cell
volumetric elastic modulus (Cosgrove, 1988), which is
not a bulk modulus. Di€erences between cell volumetric
elastic modulus and bulk modulus have been previously
discussed by Cosgrove (1988). More strictly, however,
this parameter is a volumetric expansion coecient
(VEC). The VEC for unripe tissue was 2.02 MPa, while
that of ripe tissue was 3.07 MPa. A greater VEC means
that ripe tissue volume changes to a greater extent as a
function of changing external osmotic pressure than
ripe tissue volume. This e€ect could be related to structural changes within kiwi tissue upon ripening.
As can be appreciated in Fig. 1, it is somewhat dicult to judge where incipient plasmolysis occurs from
tissue volume determinations. Unripe and ripe kiwifruit
continued to lose water and shrink after incipient
plasmolysis; therefore, a continuous relative volume
decrease as a function of increasing PEG concentration
was observed. Cell bursting (plasmoptysis) occurred in
unripe and ripe kiwifruit tissues equilibrated in solutions in the range 0±0.35 M PEG.
Microscopic investigations were carried out on selected tissue-treatment combinations in order to help
understand the observed e€ects. Plasmoptyzed (0.10 M
PEG) tissue microstructure is shown in Fig. 2A. Cellular structural features were lost, and all that could be
discerned was possibly cell wall and membrane fragments which appeared dispersed in a continuous solute
network. Ice crystals form a dendritic structure in tissue
during freezing and their subsequent sublimation results
in numerous small holes (Roy, Watada, Conway, Erbe,
192
A.M. Rojas et al. / Food Research International 34 (2001) 189±195
Fig. 1. Kiwifruit tissue volume as a function of PEG concentration after equilibration for 36 h at 12 C.
& Wergin, 1994). Fig. 2B and C correspond to unripe
raw and isotonic equilibrated tissues, respectively. In
both cases, cells displayed well-de®ned cell wall boundaries and empty intercellular spaces. Ice crystals in the
cytoplasm were smaller than those in plasmoptyzed
tissue (Fig. 2A). Maybe a naturally higher sugar content
acted as a cryoprotectant (Roy et al., 1994). Separation
of the cell membrane from the cell wall was evident in
incipiently plasmolyzed outer pericarp tissue (Fig. 2D).
PEG crystal deposited as collars around the cell walls in
empty cellular spaces. This is possibly due to apoplastic
PEG transport and the fact that PEG molecules are
retained outside cell walls. Membrane, cell wall and
middle lamellae all appeared well de®ned.
Table 1
Dynamic rheological parameters for unripe kiwifruit equilibrated in polyethylene glycol 400 (PEG) solutions
PEG
concentration
(M)
Osmotic
pressurea
(MPa)
G0 (Pa)b
G00 (Pa)b
Tanb
G (Pa)b
0.10
0.19
0.22
0.36
0.40
0.42
0.45
RAW
0.48
0.50
0.53
0.55
0.58
0.61
0.64
0.66
0.71
0.96
0.237
0.450
0.521
0.853
0.948
0.995
1.066
163392212
182162718
191791867
52864927637
52145174027
48287320170
45562131749
15046232010
12982527774
13490031608
11274123125
7316313313
420504987
580906528
521129487
522368618
39090571
507812946
3214125
3915349
3561425
760053352
8573210328
704022346
61698986
303667025
220914648
241834898
197033890
137732145
8434729
120792351
106611077
115011595
7931151
10430625
0.1990.021
0.2210.021
0.1900.041
0.1440.004
0.1650.007
0.1460.004
0.1360.011
0.2010.008
0.1700.001
0.1800.006
0.1750.003
0.1890.008
0.2030.032
0.2070.02
0.2070.02
0.2210.005
0.2030.002
0.2050.003
166512196
186632825
195221755
53408727780
52846174645
48798320230
45980031749
15350032010
13169127774
13705131608
11445023125
7444913313
429004987
593406528
531999487
534878618
39880571
518412946
a
b
c
d
1.137
1.185
1.256
1.303
1.375
1.446
1.517
1.564
1.683
2.275
Osmotic pressure=MRT.
Average and standard deviations for n=6 are shown.
G* due to turgor pressure (G GIP ).
Average G for plasmoptyzed (0.10±0.19 M) and incipiently plasmolyzed (0.58±0.96 M) tissue.
G (Pa)c
(turgor)
G (Pa)d
18279
483979
478353
437875
409692
103391
81583
86943
64342
24342
0
0
0
0
0
0
I.P.
50108
A.M. Rojas et al. / Food Research International 34 (2001) 189±195
193
Fig. 2. Cryo-scanning electron micrographs of unripe outer pericarp kiwifruit tissue. (A) Plasmoptyzed tissue equilibrated in 0.1 M PEG. (B1 and
B2) Raw tissue. (C) Tissue equilibrated in an isotonic (0.48 M) PEG solution. (D1 and D2) Incipiently plasmolyzed tissue (0.58 M PEG). Arrows
point to the plasmalemma separating from the cell wall. Magni®cation bars correspond to 200 mm (A, B2), 20 mm (B1, D1), 60 mm (C, D2).
Linear viscoelastic behavior, and, therefore constant
G , G00 and tan values, was observed in the entire stress
range studied (0±100 Pa stress, 0±0.13% strain). The
0
G0 =G00 ratio was in the range 5.5±8 for treated and
untreated, ripe and unripe tissue, while moduli were
independent of frequency in the range 1±10 Hz (not
194
A.M. Rojas et al. / Food Research International 34 (2001) 189±195
Table 2
Dynamic rheological parameters for ripe kiwifruit equilibrated in polyethylene glycol 400 (PEG) solutions
PEG
concentration
(M)
Osmotic
pressurea
(MPa)
G0 (Pa)b
G00 (Pa)b
Tanb
G (Pa)b
0.10
0.19
0.22
0.36
0.40
0.42
0.45
0.48
0.50
0.53
RAW
0.55
0.58
0.61
0.64
0.66
0.71
0.96
0.237
0.450
0.521
0.853
0.948
0.995
1.066
1.137
1.185
1.256
16538295
175243111
3532011013
8820725578
25916864408
29839750847
17476754304
19280032412
14473417523
14066714469
12648724691
585476425
5050010261
888472366
586401868
5519713701
817277636
699861581
2281208
30341041
65453119
155534741
421706011
503297103
301809512
312834466
247061492
239602233
243703166
118431276
99632202
18043573
12589242
121062325
170501179
13351465
0.1380.01
0.1710.031
0.1800.033
0.1760.005
0.16540.017
0.1690.006
0.1720.003
0.1630.006
0.1720.015
0.1700.003
0.1950.012
0.2020.002
0.1970.006
0.2030.003
0.2150.009
0.2210.014
0.2090.005
0.1910.003
16695321
177913232
3593311393
8956826010
2626034527
30261451317
17735454304
19532332412
14683817523
14269314469
12882224691
597326425
5147410261
906602366
599781868
5651313701
834877636
712491581
a
b
c
d
1.303
1.375
1.446
1.517
1.564
1.683
2.275
G (Pa)c
(turgor)
G (Pa)d
17243
22150
194790
235201
109941
127910
79425
75280
61409
0
0
0
0
0
0
0
I.P.
67585
Osmotic pressure=MRT.
Average and standard deviations for n=6 are shown.
G* due to turgor pressure (G GIP ).
Average G* for plasmoptyzed (0.10±0.19 M) and incipiently plasmolyzed (0.55±0.96 M) tissue.
shown). We can, therefore, safely conclude that the
material behaved predominantly as a solid.
Complex moduli were determined on tissue equilibrated in PEG solutions at di€erent osmotic pressures.
Results for unripe tissue are presented in Table 1 while
results for ripe tissue are presented in Table 2. It was
extremely straightforward to determine the PEG concentration, or osmotic pressure, at which incipient
plasmolysis occurred. From the value of G* at incipient
plamolysis it was possible to calculate the contribution
of turgor pressure to the complex modulus of the tissue.
Also clearly de®ned was the low PEG concentration
region where plamoptysis (cell bursting) had taken
place.
With these rheological data, attempts were made to
calculate the relative contribution of cell walls, middle
lamellae and turgor pressure to the complex modulus of
the tissue. Table 3 shows results from these attempts.
The G* of kiwifruit tissue decreased upon ripening. The
relative contribution of turgor pressure towards tissue
G* was 67% for unripe tissue and 48% for ripe tissue. The decreased turgor e€ect in ripe tissue could be
due to degradation of biological membranes upon
ripening and senescence (Marangoni, Palma, & Stanley,
1996; Stanley, 1991). Losses in the integrity of plasmalemma would lead to a decrease in cellular, and therefore tissue, turgor. It was not possible to clearly
determine the contributions of cell walls and middle
lamellae to tissue G*, since the G* corresponding to cell
walls changed upon ripening. Since considerable
solubilization and degradation of pectin and xyloglucans takes place during kiwifruit ripening (Redgwell,
Melton, & Brasch, 1991, 1992), we set the middle
lamellae G* term to zero for ripe tissue. The resulting
calculated value of G* for cell walls, however, was
higher than the combined cell wall and middle lamella
G* for unripe tissue. Clearly, changes in the structure of
the cell wall during ripening had taken place. This proposal was supported in the literature by reports of
increases in the amounts of extensin-type cell wall proteins during ripening of kiwifruit (Redgwell et al., 1992).
This e€ect could potentially lead to increases in the G*
of the cell walls, and also result in a greater cell wall
extensibility. Residual values of G* after cell bursting
were similar for both ripe and unripe tissue. Even
though calculation of the exact contributions of cell
Table 3
Complex modulus of outer pericarp tissue structural componentsa
Unripe kiwifruit
Native outer pericarp tissue
Cell wall+middle lamella
Cell wall
Middle lamella
Turgor e€ect
Residual
a
Ripe kiwifruit
G (Pa)
G (Pa)
15350032010
501087291
12882224691
10339232830
182794664
6758514722
[0]
6123728747
172432140
Values represent averages and standard deviations of n ˆ 6
samples.
A.M. Rojas et al. / Food Research International 34 (2001) 189±195
walls and middle lamellae to the overall tissue G* was not
possible, the model helped shed light on the structural
changes that occur in kiwifruit tissue upon ripening.
Acknowledgements
We acknowledge the ®nancial support of the FundacioÂn Antorchas, Universidad de Buenos Aires, Consejo
Nacional de Investigaciones Cientõ®cas y TeÂcnicas de la
RepuÂblica Argentina, Agencia Nacional de Investigaciones Cientõ®cas y TecnoloÂgicas de la RepuÂblica
Argentina, Interamerican Development Bank, the Natural Sciences and Engineering Research Council of
Canada and the Ontario Ministry of Agriculture, Food
and Rural A€airs.
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