Innovative Food Science and Emerging Technologies 18 (2013) 6573

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Innovative Food Science and Emerging Technologies

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

Effect of high pressure processing on avocado slices

Allan B. Woolf a,, Reginald Wibisono a, Judie Farr a, Ian Hallett a, Lena Richter b, Indrawati Oey b,
Mark Wohlers a, Jing Zhou a, Graham C. Fletcher a, Cecilia Requejo-Jackman a
a
b

The New Zealand Institute for Plant & Food Research Limited, 120 Mt Albert Road, Sandringham, Auckland 1025, New Zealand
The University of Otago, Department of Food Science, Dunedin 9054, New Zealand

a r t i c l e

i n f o

Article history:
Received 21 November 2012
Accepted 21 February 2013
Editor Proof Receive Date 28 March 2013
Keywords:
High pressure processing (HPP)
Polyphenol oxidase
Peroxidase
Respiration rate
Ethylene production
Cryo-SEM
Microstructure

a b s t r a c t
While the commercial benets of high pressure processing (HPP) on pulp products (e.g. guacamole) are known,
little information is available on HPP of whole avocado tissue. We examined HPP effects on the quality, physiology,
biochemistry and microstructure of avocado slices. Flesh colour was affected, with L (lightness) and C (chroma)
values decreasing after some treatments, although these changes were not obvious visually. HPP (>300 MPa)
dramatically reduced respiration rates and ethylene production 1 h after treatment, and particularly after 17 h
at 20 C. Treatment resulted in some peroxidase (POD) activity reduction, particularly at higher pressures,
while polyphenoloxidase (PPO) activity was generally increased. HPP (600 MPa) induced changes in the cell
wall structure, disruption of the cellular network and coalescence of oil vesicles. HPP treatment of avocado slices
provides potentially benecial reduced respiratory activity, without obvious colour changes a solid foundation
for examining shelf life quality implications.
Industrial relevance:
This work outlines underpinning science that may have commercial relevance in the future.
It is not directly relevant to HPP industries in the immediate future.
The effect of HPP on respiration rate and ethylene production is useful to industry in their understanding of
the physiology of horticultural products after HPP.
2013 Elsevier Ltd. All rights reserved.

1. Introduction
There has been an increasing consumer demand worldwide for high
quality, minimally processed fruit products that are not only convenient,
but also have fresh appearance, texture not dissimilar to that of the fresh
product, and are free from articial food additives (Palou et al., 2000).
High pressure processing (HPP) is a process that has gained signicant
traction in development of products such as avocado pulps e.g. guacamole (Torres & Velazquez, 2005). Treatment is generally carried out at
pressures around 600 MPa on avocados that have been pre-prepared
(pulped) and sealed in pouches or trays under modied atmosphere
(MA) conditions, resulting in signicant reductions (35 log) in microbial
counts (Jacobo-Velzquez & Hernndez-Brenes, 2010; Lopez-Malo, Palou,
Barbosa-Canovas, Welti-Chanes, & Swanson, 1998; Palou et al., 2000).

Corresponding author at: The New Zealand Institute for Plant & Food Research Limited,
Plant & Food Research Mt Albert, 120 Mt Albert Road, Sandringham 1025, Auckland, New
Zealand. Plant & Food Research Mt Albert, Private Bag 92169, Auckland Mail Centre 1142,
Auckland, New Zealand.
E-mail address: allan.woolf@plantandfood.co.nz (A.B. Woolf).
1466-8564/$ see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.ifset.2013.02.011

Along with reduced microbial count, HPP can inactivate enzymes while
retaining the avour and colour quality of foods (Ludikhuyze, Loey,
Indrawati, & Hendrickx, 2002).
Research into the response of avocado pulp or guacamole to HPP
has shown few changes to colour immediately after treatment, although
changes do occur during subsequent storage/shelf life (Lopez-Malo et al.,
1998; Palou et al., 2000). This is likely to be a result of partial disruption
of cellular organelles releasing enzymes such as polyphenol oxidase
(PPO) and its substrates, causing the formation of o-quinones, which
undergo further polymerization to form brown pigments (Weemaes,
Ludikhuyze, Van den Broeck, & Hendrickx, 1998). HPP treatment of
avocado pulp, puree, paste and guacamole has been shown to impart
longer shelf life and that this can be in part attributed to effects on endogenous enzyme activity (Jacobo-Velzquez & Hernndez-Brenes,
2010; Lopez-Malo et al., 1998; Ludikhuyze et al., 2002). It has been
reported that PPO activity can be signicantly reduced by HPP. For
example, the residual PPO activities of guacamole reduced to 51% after
a 5-min, and 37% after a 10-min treatment at 689 MPa (Palou et al.,
2000). Similarly, Jacobo-Velzquez and Hernndez-Brenes (2010)
reported an average residual PPO activity of 51% and lipoxygenase
(LOX) activity of 55% in avocado paste following 600 MPa for 3 min.
Respiration rate and ethylene production are important fundamental processes in plant tissues. Clearly, the rates of O2 consumption and

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CO2 production are important in packaged products (such as most HPP

products) since, for example, excessively low O2 concentrations will
lead to anaerobic responses and off-avours. Thus, to select appropriate
packaging and to understand the basic mechanisms of HPP treatment
on intact plant tissues, it is important to understand the effects of HPP
on the respiration rate of the fruit. Ethylene is also important because
of its control of ripening and senescent responses in fruit tissues and
in fresh-cut products. Despite this, there is a dearth of research into
the effects of HPP treatment on respiration and ethylene production
rates of plant materials. Preliminary work by Eggleston and Tanner
(2005) showed a reduction in respiration rate of carrot sticks treated
with HPP at 600 MPa for 2 min, with a 10-fold drop in CO2 production.
The only published study on fruit responses to HPP was by Baba, Como,
Ohtsubo, Ikeda, and Lizada (1999), who showed that in whole mume
fruit, HPP could reduce both CO2 and ethylene production (reductions
by 2-fold and 5-fold, respectively). However, they used a maximum
pressure of only 200 MPa.
While the treatment of avocado pulp/guacamole has been a commercial success, production of acceptable slices or halves (i.e. not pulped) has
been a challenge. Although there are commercial products reported
(http://avofresh.com.au/products/originals), we can nd no published
information on the effect of HPP on avocado slices or halves. Thus, we
sought to determine the responses of avocado tissue slices to a range of
HPP conditions by measuring various physiological, ultra-structure, and
biochemical responses.

2. Methodology
2.1. Fruit source and ripening
Unripe green Hass avocado fruit were harvested from three growers
in the North Island, New Zealand, during March 2011 (late maturity).
Fruit were commercially packed and transported to the Plant & Food
Research laboratory in Auckland within 4 days of harvest and held in
cool storage (5 C) for 3 weeks. Fruit were ripened using 100 L L1
ethylene for 24 h at 20 C and allowed to soften to the target fruit rmness. Fruit rmness was measured using Aweta AFS (AWETA Impact &
Acoustic Firmness System, Nootdorp, Holland), and fruit were selected
in the range of 2026 Acoustic Index (AI) (equivalent to 0.60.8 kgf
Effegi puncture 8-mm probe), or rm-ripe on the hand-rmness
scale (rating of 3.54) (White, Woolf, Hofman, & Arpaia, 2005).
2.2. Sample preparation
Firm-ripe avocados from each of the three growers were halved
longitudinally, de-stoned, and peeled and the mesocarps cut longitudinally. The ends of the tissue were removed to produce a curved
equatorial slice of the fruit esh 10 mm wide, 10 mm thick, and
40 mm long (Fig. 1). Slices from a given fruit were randomised
across treatments. Slices were vacuum packed into polyethylene
with nylon barrier bag pouches (80 120 mm, 80 m, O2 transmission

B
Green back of slice

Yellow side of slice

Control

600 MPa

Fig. 1. a) Photograph of avocado slices prepared for HPP treatment (sealed in lm that will be cut into four bags). b) Avocado slices immediately after treatment for control (left) and
600 MPa for 3 min (right). Arrows indicate the location of the two places that colour measurements were carried out: back of slice (green tissue), and the side (yellow tissue).

A.B. Woolf et al. / Innovative Food Science and Emerging Technologies 18 (2013) 6573

rate 55 mL m 2 d 1 atm 1 and CO2 transmission rate 160 mL

m 2 d 1 atm1 at 23 C at 75% relative humidity; Convex Plastics
Ltd., Auckland, NZ; Fig. 1a) and were held on ice before and after HPP
treatments.
2.3. HPP treatment
HPP treatments were carried out within 3 h of sample preparation.
HPP conditions examined were 200, 400 and 600 MPa each for 3, 6 or
10 min, and 300 and 500 MPa for 6 min only. Each HPP run was set to
one of the eleven possible pressure and time treatment combinations
and contained nine samples, three from each of the three growers. For
each of the three days, the order of the 11 treatment runs was
randomised. HPP treatment was carried out in a 2-L Food Isostatic
Press (Model: Avure 2L, Avure Technology Inc., Columbus, OH, USA).
Pressures were applied to 10 MPa above the target pressure, and typically declined by 1015 MPa during the treatment period. The rate of
pressure ramping up was 12.3 MPa s 1, and pressure release was at
110 MPa s 1. Temperature in the chamber typically increased from
22 C to 26, 29, 31, 32 and 38 C for 200, 300, 400, 500 and
600 MPa, respectively, and upon release reduced to 20 C. The control
treatment comprised avocado slices sealed in pouches in the same manner to HPP-treated slices and transported to and from the HPP machine
under the same conditions.
The experiment was replicated on three consecutive days, with
three slices from each of the three growers for each treatment on
each day. For all treatments and days, one slice from each grower
was used for assessment of respiration and ethylene production,
one for sampling for enzyme analysis and microscopy, and one for
colour measurement.
2.4. Assessments
Packaged samples were returned to ice immediately after HPP
treatment and samples were assessed within 3 h for colour changes
(lightness L, Chroma C and hue angle h), respiration rate
(CO2 production), ethylene production, and tissue samples were taken
for measurement of enzyme activity and for microscopy (see below
for details).
2.4.1. Colour
Lightness (L), chroma (C) and hue (h) colour values were measured
using a Minolta CR-300 colorimeter (Konica Minolta Sensing, Inc.,
Osaka, Japan) three times on the yellow side, and three times on the
green tissue on the back of the slices (where the skin had been
detached; see arrows on Fig. 1b).
2.4.2. Respiration and ethylene production
A slice from each treatment and grower was removed from the pouch
and carefully placed in a 69-mL container, ushed with ethylene- and
CO2-free air, sealed and left for 1 h at 20 C. Two 1-mL samples were
removed and CO2 and ethylene concentrations were measured using
gas chromatography as described elsewhere (Besada, Jackman, Olsson,
& Woolf, 2010). This measurement was repeated on the same samples
17 h after HPP treatment (holding tissue at 20 C) to determine whether
a build-up of CO2 or ethylene in the packaging before and after HPP treatment might have given false measures of respiration rate. Concentrations of CO2 and ethylene were expressed as mol kg1 s 1 and
nmol kg1 s1, respectively.
2.4.3. Microscopy (light, scanning and transmission electron microscopy)
2.4.3.1. Light and transmission electron microscopy. Avocado tissue was
cut into 10-mm diameter discs approximately 2 mm thick. Discs were
transferred into xative (2% freshly prepared formaldehyde, 2.5% glutaraldehyde in 0.1 M phosphate buffer pH 7.2) and stored at 4 C before

67

processing and observation. For light microscopy blocks of tissue around

3 4 2 mm were excised from the disc, washed three times in 0.1 M
phosphate buffer (pH 7.2) and then sectioned at 200 or 300 m using a
stainless steel blade attached to a Vibratome 1000 (Technical Products
International, St Louis, MO, USA). Sections were either stained using a
0.1% aqueous solution of toluidine blue and observed using bright eld
microscopy or were viewed without staining by differential interference
contrast microscopy using an Olympus Vanox AHT3 (Olympus Optical
Co. Ltd., Tokyo, Japan).
Material for transmission electron microscopy was washed in buffer,
post-xed in a 1% solution of osmium tetroxide in 0.1 M phosphate
buffer, dehydrated in an ethanol series and embedded in Spurr's
epoxy resin (Spurr, 1969). Samples were then sectioned at 130 nm
using a diamond knife in a Leica UCT ultramicrotome (Leica Microscopy
Systems Ltd., Heerbrugg, Switzerland), mounted on copper sample
grids and stained sequentially with a saturated solution of uranyl acetate
in 50% ethanol followed by aqueous lead citrate (Roland, 1978). Transmission electron microscopy was carried out using a JEOL JEM-1200EXII
transmission electron microscope (JEOL Tokyo, Japan) operating at an
accelerating voltage of 80 kV.
Cryo-Scanning Electron Microscopy (cryo-SEM) samples were cut
into rectangular blocks less than 10 mm on the longest dimension,
45 mm wide and approximately 2 mm thick, snap frozen using liquid
nitrogen and were kept under liquid nitrogen until analysed.
Cryo-SEM was performed using a Polaron PP2000 Cryo Transfer system (Quorum Technologies, Ringmer, UK) attached to an FEI Quanta 250
Scanning Electron Microscope (FEI, Hillsboro, OR, USA). Frozen tissue
was mounted in sample holders containing a mixture of colloidal graphite and OCT compound (Sakursa Finetek, Zoeterwoude, NL) under an
argon atmosphere and immersed in liquid nitrogen with about half
the sample protruding. Samples were transferred to the sample handling chamber of the PP2000 in liquid nitrogen then transferred under
vacuum to the PP2000 preparation stage at 150 C and the protruding
tissue fractured using a cooled metal blade or probe. Ice sublimation was
then carried out at 90 C for between 1 and 7 min to improve surface
contrast. The temperature was then returned to 150 C before the
fractured surface was sputter coated with gold/palladium (60 s)
and transferred to the cryo-SEM for observation on a stage cooled to
150 C using accelerating voltages of between 5 and 15 kV.
2.4.4. Enzyme activity
Immediately after removal from the sealed pouch, 2 g of tissue was
ash frozen in liquid nitrogen and stored at 80 C for later analysis
when frozen tissue was ground to a powder with ne homogenous
talc-like consistency using a SPEX SamplePrep cryomill (2 cycles each
of 1 min pre-cool, 1 min run at 7 collisions per s and 1 min cool;
Model 6870, Metuchen, NJ, USA).
2.4.4.1. Extraction. Enzyme extraction and assay were carried out based
on the combined procedures of Rojas-Graue, Soliva-Fortuny, and
Martin-Belloso (2008), Lopez-Malo et al. (1998) and Jacobo-Velazquez,
Ramos-Parra, and Hernandez-Brenes (2010) with minor modications.
Frozen avocado powder (250 g) was homogenised with 1 mL phosphate buffer (0.1 M, pH 6.5 pH containing 1 M NaCl) using a Polytron
(PT2100, Kinematica, Littau, Switzerland) for 15 s. The mixture was
centrifuged at 21,000 g and 4 C for 15 min. The supernatant was
then ltered using a 0.45-m lter and held in an ice water bath before
analysis.
2.4.4.2. Enzyme assays. Activity for both peroxidase (POD) and PPO was
determined using a spectrophotometer (Ultrospec 3300 Pro, GE Healthcare, Auckland, NZ). For POD (EC 1.11.1.7), the assay was based on the
oxidation reaction of guaiacol by hydrogen peroxide. The assay mixture
consisted of 1.5 mL phosphate buffer (0.1 M, pH 6.5), 0.2 mL guaiacol
(1% w/v in water), 0.1 mL H2O2 (1.5% w/v in water) and 30 L enzyme
extract. The formation of oxidised tetraguaiacol polymer was monitored

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A.B. Woolf et al. / Innovative Food Science and Emerging Technologies 18 (2013) 6573

at 20 C over 3 min by changes in absorbance at 485 nm. For PPO

(EC 1.10.3.1), the assay was based on the oxidation reaction of catechol.
The assay mixture consisted of 1 mL phosphate buffer (0.1 M, pH 6.5),
500 L catechol (0.1 M dissolved in phosphate buffer (0.1 M, pH 6.5))
and 13 L enzyme extract. The formation of oxidised catechol polymer
was monitored at 20 C over 4 min by changes in absorbance at
420 nm. In each case, substrate solution was freshly prepared before
enzyme measurements and changes in absorbance were followed using
kinetic software (Swift II, GE Healthcare, Auckland, NZ). Enzyme activity
was calculated based on the slope of the linear portion of the curve and
expressed as the change in absorbance (A485 nm or A420 nm) per
minute per g avocado tissue (fresh weight). The enzyme activity measurements were carried out in triplicate.
2.5. Statistics
Statistical analysis was carried out in SAS 9.2 using the MIXED
model procedure. A mixed model was tted to each of the fruit locations (i.e. green v. yellow tissue) and colour measure combinations
separately. Pressure, time, pressure time interaction, day and sample
order were all treated as xed effects. However, grower was treated as
a sample of the population of growers and if the experiment were repeated, a different sample of growers may have been selected. Therefore,
grower was included in the model as a random effect. The respiration
rate data sets were analysed for the same effects except that the sample
order was not included in the model. To meet the model assumptions of
normally distributed residuals with constant variance for the respiration
rate data, all statistical tests were carried out on log transformed respiration data, with tted means back transformed onto the original scale. The
ethylene measures at 1 h contained a number of zeros, so half the minimum non-zero value was added to these before the log transformation.
As the ethylene values were very low at 17 h and contained a very large
numbers of zeros; only summary means for these, without further analysis, are presented. Pair-wise comparisons involved the means for each
pressure/time combination being tested against the control. All tests
were conducted at the 5% level of signicance, with Dunnett's adjustment used to account for multiple testing in the post hoc tests.
3. Results and discussion
3.1. Colour
Based on the objective colour measurements using a chromameter,
the effect of HPP on the three colour variables progressed from strongest
to least for lightness, chroma and hue angle. Using the mixed effects
model, there was evidence that pressure but not duration had signicant
effects on tissue colour, and there was no interaction of the effects of
pressure with duration (results not shown). We examined the effect of
time between tissue preparation and HPP treatment and found that
this was not statistically signicant (results not shown).
For lightness of green and yellow tissues, analysis of differences of
least squares means showed that, with the exception of the 200 MPa
treatments, all the treatment values were signicantly lower than the
control values (P b 0.05) and this difference increased as the pressure
increased (Fig. 2a). There were no lightness differences (P b 0.05) between the pressures > 300 MPa or at duration times of 3 to 10 min.
Slices became slightly darker at 300 MPa decreasing from 57.5 to
52.5 for the outside (i.e. dark green layer) of slices, and 69.5 decreasing to 6366 for the internal yellow side of the slices.
Chroma (Fig. 2b) showed a similar trend to that of lightness for
the internal side of the slices, with reduced values at the higher pressures (> 500 MPa). However, the back of the tissue slice (dark green
tissue) showed an increase in chroma only at pressures of 200 MPa
at 3 min, no signicant difference at pressures of 300500 MPa, and
a decrease at 600 MPa for 3 and 6 min.

Although pressure had an overall signicant effect on hue angle

(h) of the back of the slices, none was signicantly different from the
control (data not shown). For measures of hue on the sides of the slices,
duration (but not pressure) had a signicant effect, but only four treatments (all 6-min treatments except at 400 and 500 MPa) resulted in an
increase in hue angle (data not shown).
Given the higher concentration of pigments in the outer layers of
avocado tissue (Ashton et al., 2006), it is perhaps surprising that the
effects of HPP on colour were greater on the inside of the slices (yellow
tissue) than the back (green tissue). A possible reason for this might be
the higher dry matter of the outer tissues (Schroeder, 1985), which
with the higher oil content might make it more tolerant to high pressure.
Although these objective measures of colour showed some statistically signicant effects of HPP treatment, the differences between treated and untreated slices were not readily discernable to the human eye
(Fig. 1b), and the product was of acceptable appearance. This is in contrast
to many other fruit, where changes immediately after HPP treatment are
readily visible, including obvious water-soaking and signicant colour
changes (Oey, Van der Plancken, Van Loey, & Hendrickx, 2008). In
avocado puree (with added NaCl and acidication), HPP treatment
did not have signicant effects on L, C, and h until after storage of
the pulp (Palou et al., 2000).
3.2. Carbon dioxide and ethylene production
High pressure processing had a large effect on the CO2 and ethylene
production of the avocado slices, but duration of treatment did not generally have as much effect as that of pressure. When measured 1 h after
HPP treatment, 200 and 300 MPa treatments had no effect on CO2 production, but 400600 MPa treatments resulted in 50% reduction compared with the control (Fig. 3a). This result is different from that found
in mume fruit, where a 200-MPa treatment reduced respiration rate to
1/4 of the untreated (Baba et al., 1999), but the authors did not examine
higher pressures. For carrot sticks, the only HPP pressure examined
(600 MPa) reduced respiration rate to 1/10th of the control when applied for 2 min, and a further 2-fold reduction after a 10-min treatment
(Eggleston & Tanner, 2005). Thus, the reductions in respiration rate observed in carrot sticks and mume fruit were generally greater than for
the avocado tissue examined here.
When control avocado slices were re-measured at 17 h after HPP
treatment, there had been a reduction in CO2 production to 30% of
the value with the 1-h measurement (dropping from 1.57 to
0.54 mol kg 1 h 1; Fig. 3a). Carbon dioxide production of fruit in
the 200 MPa treatments was again not signicantly lower than that
of the controls, but 300 and 400 MPa treatments did reduce CO2
production, and the concentrations at 500600 were very low
(0.020.05 mol kg 1 h 1). We suggest that part of the drop in
CO2 over 17 h for the control treatment reects diffusion of CO2 that
had built up in the bag around the slices between the time of sealing
and treatment, but that it also reects a reduction of respiration rate
(and therefore very minimal metabolic activity).
Ethylene production was more sensitive to HPP treatment than
CO2 production. One hour after HPP treatment, even 200 MPa reduced
ethylene production by 50% or more, and 400600 MPa reduced production to 20% of the control (Fig. 3b). Avocados appear to be less sensitive
than mume fruit, where even 200 MPa for 10 min reduced ethylene production to 5% of control (Baba et al., 1999). Similarly to CO2 production,
17 h after HPP treatment, ethylene production declined further and
there were no measurable amounts from treatments of 300600 MPa
(except for traces at 600 MPa for 3 min). This reduction in ethylene
production over time after treatment appears to reect damage to the
ethylene biosynthetic mechanisms, which are much more sensitive to
HPP treatment than the respiratory mechanisms. This is a likely result
of the ethylene biosynthetic pathway being more sensitive to environmental factors, as is found for other stress treatments in avocado, such
as heat (Woolf & Ferguson, 2000). With the control and 200 MPa for

A.B. Woolf et al. / Innovative Food Science and Emerging Technologies 18 (2013) 6573

72.5

Yellow side of slice

Green back of slice

70.0
67.5

Lightness

69

65.0

62.5
60.0
57.5

55.0

52.5

50.0

in

in

10 m

6m

in

600 MPa

35.0
32.5

Chroma

3m

10 m

6m

in

in

in
3m

400 MPa

500
6
MP min
a

200 MPa

300
6
MP min
a

in
10 m

in
6m

in
3m

Con
tr

ol

30.0

27.5

25.0

in
10 m

in
6m

in
3m

in

6
MP min
a
500

400 MPa

10 m

in
6m

in
3m

in

6m
in
a
MP
300

200 MPa

10 m

in
6m

in
3m

Con
tr

ol

22.5
0

600 MPa

HPP Treatment
Fig. 2. Colour measurements (a lightness and b chroma) of avocado slices after HPP processing at pressures of 200600 MPa for 3, 6 or 10 min. The control treatment is
non-HPP treated samples. Values represent average measured from avocados sourced from three growers treated on three consecutive days (replicates). Error bars represent standard deviation. * = signicant difference from the control at P b 0.05.

3 min treatments, there was an increase in ethylene production with

time after treatment, this probably reecting wound-induced ethylene
production, which has been shown to increase after wounding, with a
peak at 18 h (Starrett & Laties, 1991).
3.3. Microscopy (light, scanning and transmission electron microscopy)
Microscopic observations showed that identiable cells were
maintained after HPP treatments (Figs. 4, 5). Parenchyma cells contain extensive oil deposits in the form of oil droplets (vesicles),
which in untreated esh take the form of more or less spherical droplets around 10 m in diameter (Fig. 4A, C, D). Occasional oil idioblast
cells completely lled with oil can also be seen (Fig. 4A). After treatment with a pressure of 200 MPa, oil droplets maintained a similar
appearance. However, after higher pressure treatments, coalescence
of the parenchyma oil droplets into much larger droplets could be
seen in both xed tissue observed using light microscopy (Fig. 4B)
and in the cryo-SEM samples frozen within a few hours of processing,
where droplets often showed an irregular shape (Fig. 4E, F). There was
no observable change to oil contained in idioblast cells. Despite the
change in oil droplet size, the oil remained within the cell boundaries
and there was no evidence of oil between cells. Cell walls showed no
evidence of rupture. However, in a few samples cryo-SEM showed internal structural disruption of cell walls (Fig. 4E). This could be detected at
mild elevated pressure (200 MPa for 10 min) but was not seen in any

untreated tissue and in most samples the cell walls stayed more intact
(Fig. 4F). These differences may reect variation in the extent of cell
wall structure and integrity in the rm-ripe fruit. Cell walls in avocado
can undergo extensive swelling during ripening and softening
(Redgwell et al., 1997) and depending on the extent of hydrolysis, exertion of external pressure may result in increased free water not only in
the intercellular spaces, but in the wall itself, resulting in ice crystal
development between walls during subsequent freezing.
Transmission electron microscopic observations (Fig. 5) indicated that
while the internal cytoplasmic structure of the cells was disrupted after
HPP, it retained some degree of continuity and individual cell components
remained identiable. Cytoplasmic content remained more intact than
that seen in many plant cells subjected to HPP (e.g. Vazquez-Gutierrez,
Quiles, Hernando, & Perez-Munuera, 2011; Xu, 2007). These differences
may be the result of the high oil and relatively low water content of
these cells providing some protection from the damaging effects of high
pressure. Despite the micro-structural changes observed, the retention
of the overall structure of ripe avocado esh (oil contained in discrete
units delimited by a swollen cell wall) suggests that a reasonable degree
of the structural properties of avocado may be retained after HPP.
3.4. POD and PPO activity
There were signicant reductions of POD activity (P b 0.05) of up
to 50% found at higher pressures studied (500 and 600 MPa), while

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A.B. Woolf et al. / Innovative Food Science and Emerging Technologies 18 (2013) 6573

CO2 production (mol kg-1 s-1)

3.0

1 h after HPP
17 h after HPP

2.5
2.0

1.5

1.0
0.5

10 m
in

6m
in

3m
in

10 m
in

6m
in

3m
in

400 MPa

500
MP 6 min
a

200 MPa

300
MP 6 min
a

10 m
in

6m
in

3m
in

C on
trol

0.0

600 MPa

0.20

0.035
0.030
0.025
0.020
0.015
0.010

0.005

400 MPa

10 m
in

6m
in

3m
in

500
MP 6 min
a

10 m
in

6m
in

3m
in

300
MP 6 min
a

200 MPa

10 m
in

6m
in

3m
in

0.000
Con
trol

C2H4 production (nmol kg-1 s-1)

600 MPa

HPP Treatment
Fig. 3. Carbon dioxide production (a) and ethylene production (b) of avocado slices after HPP processing at pressures of 200600 MPa for 3, 6 or 10 min. Slices were measured 1 h
after HPP treatment, and 17 h after treatment (held at 20 C non-packaged). The control treatment is non-HPP treated samples. Values represent average measured from avocados
sourced from three growers treated on three consecutive days (replicates). Error bars represent standard deviation. * = signicant difference from the control at P b 0.05.

the majority of the pressures (200400 MPa) had no signicant effect

on POD activity, except for 400 MPa for 6 min (Fig. 6a). At 600 MPa,
increasing HPP duration from 3 to 10 min did not reduce POD activity
(P b 0.05). Although HPP has been observed to reduce POD in strawberry puree (Terefe, Yang, Knoerzer, Buckow, & Versteeg, 2010) and
green peas (Akyol, Alpas, & Bayindirli, 2006), it did not in peppers
(Castro et al., 2008).
In contrast to POD, higher PPO activity compared with that in untreated samples was found with increased pressures, with up to 30%
increases (P b 0.05) at 400 MPa (Fig. 6b). This phenomenon might be
explained by cell membrane breakdown due to pressurization facilitating enhanced enzyme release/extractability, which indirectly results in
an increase in the measured enzyme activity (Fig. 6b). Increasing pressure above 400 MPa resulted in a lower PPO activity compared with
that in the 400 MPa-treated samples, but similar activity to that of the
untreated samples.
Palou et al. (2000) reported PPO inactivation in avocado guacamole.
This contradictory result could be explained by the differences in the pH
of the avocado products. In the study of Palou et al. (2000), the pH of
avocado guacamole was lowered to 4.3 using citric acid, while our study
used intact slices and did not employ adjustment of the natural pH of

avocado (pH 6.26.5; Punter, unpublished data). Lopez-Malo et al.

(1998) and Weemaes et al. (1998) have shown that avocado PPO has different sensitivity towards HPP in terms of pressure and temperature.
They also found that PPO was more stable at a neutral or slightly higher
pH (pH 8) than at low pH (bpH 7). The absence of PPO inactivation by
HPP processing has also been observed in strawberry puree (Terefe et
al., 2010), but it was pressure sensitive in fresh-cut red and green peppers,
which had pH values of 4.81 and 4.65, respectively (Castro et al., 2008).
Thus, it appears that HPP processing at pressure/time combinations
normally used in industrial HPP applications is unlikely to achieve significant PPO inactivation under the natural conditions existing in avocado
slices (i.e. without adjusting the product properties, such as lowering
pH, or using a reducing agent). Therefore, the effects of HPP on avocado
slice quality during storage when combined with adjustment of product
properties (such as pH modication) should be further investigated.
4. Conclusions
This study has demonstrated that HPP has benecial effects by
inactivating respiration and ethylene production in avocado slices
(without the use of high temperature treatment), but only POD activity

A.B. Woolf et al. / Innovative Food Science and Emerging Technologies 18 (2013) 6573

71

Fig. 4. Tissue structure and oil droplet distribution in avocado esh with no treatment (A, C, D) and after HPP treatment for 6 min at 600 MPa (B, E, F). Oil droplets, apart from oil
idioblast cells, are small and relatively uniform in untreated tissue; after treatment, fewer larger droplets of variable size are present. Cells remain delimited by cell walls, although
in some cases the cell wall viewed using cryo-SEM showed internal disruption (E). Insets in E and F show details of disrupted and non-disrupted walls in different samples after
HPP. A and B. 100 m vibratome section viewed using differential interference contrast; bar = 0.1 mm. CF. cryo-SEM fractures (C minimal ice sublimation) bar = 10 m. O
oil droplets, I oil idioblast cell, arrows indicate cell walls, and arrowheads in E delimit the large irregular oil droplets.

Fig. 5. Detailed structure of avocado cytoplasm. a. Untreated tissue with easily distinguishable cell organelles and membranes. b and c. Tissue treated for 6 min at 600 MP; cytoplasm is grainier, pressed to the cell wall and contains many electron-dense spots; however, some membrane integrity is retained (arrows), all transmission electron microscopy.
Bars = 0.1 m, O oil deposit, CW cell wall.

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A.B. Woolf et al. / Innovative Food Science and Emerging Technologies 18 (2013) 6573

POD (Abs min-1 g-1 fresh weight)

2.5

2.0

1.5

1.0

0.5

PPO (Abs min-1 g-1 fresh weight)

2.5

2.0

600 MPa

10 m
in

6m
in

3m
in

10 m
in

6m
in

3m
in

400 MPa

500
MP 6 min
a

200 MPa

300
MP 6 min
a

10 m
in

6m
in

3m
in

C on
trol

0.0

1.5

1.0

0.5

400 MPa

10 m
in

6m
in

3m
in

500
MP 6 min
a

10 m
in

6m
in

3m
in

300
MP 6 min
a

200 MPa

10 m
in

6m
in

3m
in

Con
trol

0.0

600 MPa

HPP Treatment
Fig. 6. Activity of (a) guaiacol peroxidase (POD) and (b) polyphenoloxidase (PPO) in avocado slices after HPP processing at pressures of 200600 MPa for 3, 6 or 10 min. The control
treatment is non-HPP treated samples. Values represent average enzyme activity measured from avocados sourced from three growers treated on three consecutive days (replicates).
Error bars represent standard deviation. * = signicant difference from the control at P b 0.05.

moderately at high pressures and little effect on activate PPO activity.

Although ultrastructure is affected and colour changes can be measured
instrumentally, these changes are not readily visually apparent. The effects of HPP treatments (possibly combined with the use of natural food
preservatives) on shelf life is warranted.
Acknowledgement
This work was funded by the Plant & Food Research High Pressure
Processing Capability project. Thanks to Matt Punter and Di Brewster
for their assistance with enzyme work.
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