Innovative Food Science and Emerging Technologies 61 (2020) 102326

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Innovative Food Science and Emerging Technologies

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

High hydrostatic pressure treatment effects on selected tissue properties of T

fresh horticultural products
⁎
G. Rux, R. Gelewsky, O. Schlüter, W.B. Herppich
Leibniz Institute for Agricultural Engineering and Bioeconomy (ATB), Department of Horticultural Engineering, Max-Eyth-Allee 100, 14469, Potsdam, Germany

A R T I C LE I N FO A B S T R A C T

Keywords: High hydrostatic pressure (HHP) treatments affect the integrity and function of biomembranes and proteins of
Nonthermal processing fresh produces. Initially, pressure affects membrane permeability, directly linked to a reversible decline in cell
Postharvest physiology turgor. Reversibility reflects the restoration of intracellular solute gradients by re-established membrane semi-
Postharvest processing permeability and functionality of membrane-bound proteins. Above a certain threshold, pressure effects become
Cell pressure probe
irreversible resulting in tissue damage. Combining cell pressure probe and tissue impedance measurements al-
Produce quality
lows differentiation between reversible and irreversible effects. Thus, these parameters were comparatively
investigated in both fresh red cabbage leaves and in radish tubers, species-specifically exposed to various pre-
determined relevant HHP conditions (red cabbage: 150–200 MPa at 35–55 °C for 5–20 min; radish:
100–200 MPa, at 20–40 °C for 5–10 min). These specific treatments facilitated the evaluation of the transition
between reversible changes in membrane and protein properties and irreversible damages. While turgor declined
immediately after HHP treatments, tissue damage occurred only delayed. The two products differed in their
responses to various HHP conditions. Generally, HHP effects were limited and reversible at (and certainly also
below) 100 MPa but were amplified with intensity of pressure treatments.
Industrial relevance: Increasing food security aspects but also growing concerns against conventional thermal
treatments demand the application of novel and gentle nonthermal hygienisation techniques such as high hy-
drostatic pressure (HHP) also for the production of fresh ready-to-eat salads. However, knowledge about po-
tential impacts of different HHP ranges on metabolic functionality and freshness of fruits and vegetables, and
thus produce quality, is still only sketchy.
This study comprehensively differentiated the reversible and irreversible effects of processing conditions on
the physiological and structural basis of HHP effects on fresh red cabbage leaves and radish tubers. It thus
provides valuable information on the absolute limits of the applicability of HHP for safety of fresh produces. The
fact that pressure treatments above 150 MPa always resulted in irreversible tissue damage indicated that even
short-term HHP treatments at room temperature are not suitable for the gentle sanitation of perishable fresh
produces but may probably be useful as quarantine measure against parasites and viruses.

1. Introduction
The high hydrostatic pressure technique (HHP; Knorr, 1993;
Rastogi, Raghavarao, Balasubramaniam, Niranjan, & Knorr, 2007;
Knorr et al., 2011; Huang, Wu, Lu, Shyu, & Wang, 2017) gently but
effectively inactivates microorganisms in various food systems
(Bermúdez-Aquirre & Barbosa-Cánovas, 2011; Georget et al., 2015;
Krebbers, Matser, Koets, Bartels, & Van den Berg, 2002). Although HHP
may also retain flavour and quality-related components (Ludikhuyze &
Hendrickx, 2002; Vervoort et al., 2012), it may, nevertheless, affect
functionality of protein such as enzymes (Indrawati, Ludikhuyze, van
Loey, & Hendrickx, 2000; Knockaert et al., 2011) and the overall tissue
structure of the produce (Gonzalez & Barrett, 2010; Knorr, Heinz, &
Buckow, 2006). Although very promising (Husband et al., 2011), the
meaningful application of HHP for perishable horticultural fresh
“living” produce has recently been questioned (Rux, Gelewsky,
Schlüter, & Herppich, 2019; Rux, Schlüter, Geyer, & Herppich, 2017;
Schlüter, Foerster, Geyer, Knorr, & Herppich, 2009).
In this context, Rux et al. (2019) summarised the increasing current
knowledge on effects of HHP treatments on the functional structures
and the metabolic activity of fruits and vegetables (Marigheto, Vial,
Wright, & Hills, 2004; Schlüter et al., 2009; Trejo Araya et al., 2007;
Vargas-Ortiz et al., 2013). Due to the high diversity in form, function
and sensitivity of the various fruits and vegetables, HHP highly

⁎
Corresponding author.
E-mail address: wherppich@atb-potsdam.de (W.B. Herppich).

https://doi.org/10.1016/j.ifset.2020.102326
Received 30 October 2019; Received in revised form 28 February 2020; Accepted 4 March 2020
Available online 07 March 2020
1466-8564/ © 2020 Elsevier Ltd. All rights reserved.
G. Rux, et al.
specifically and differentially affects the physiological activity of fresh
produce. Despite this pronounced variability, integrity and function of
biomembranes have been identified as the initial targets of various
parameters of HHP treatments, partially reversibly affecting their per-
meability (Rux et al., 2017, 2019; Schlüter et al., 2009). Only if the
function of relevant membrane-associated proteins is disturbed at HHP
higher than 150 MPa, the pressure-induced changes become irrever-
sible (Rux et al., 2019).
Variations of cell membrane permeability are directly related to that
of cell turgor, because turgor can only be maintained when cell mem-
branes and associated proteins are fully functional (von Willert,
Matyssek, & Herppich, 1995). If HHP treatment causes serious pertur-
bations of the membrane semi-permeability, this finally results in the
loss of turgor.
Despite its importance, evaluations of HHP effects on fresh vege-
table tissue by direct cell turgor measurements with the cell pressure
probe (Hüsken, Steudle, & Zimmerman, 1978) were limited. Rux et al.
(2017) analysed the impact of a range of HHP treatments (150 MPa to
250 MPa for 5 to 20 min at 35 °C to 55 °C) on the turgor of red cabbage
leaf tissue. In addition, responses of cell turgor, tissue colour and
firmness to HHP (100 MPa to 200 MPa for 5 to 10 min at 20 °C to 40 °C)
were determined in fresh radish tuber tissues (Rux et al., 2019). These
studies clearly indicate that pressure intensities, and the duration and
temperature of HHP treatments directly and interactively affected cell
turgor. Both products showed similar but nevertheless species-specific
critical thresholds of the HHP treatment parameters for irreversible
changes, ranging from 150 MPa and 45 °C, and turgor of 0.3 MPa for
red cabbage leaf tissue to 100 MPa at 40 °C up to 10 min and 0.1 MPa
for the thin-walled parenchymatous radish tubers. Reduction of turgor
was also accompanied by changes in stiffness and skin colour. It was
suggested that HHP finally induced irreversible changes in the semi-
permeability of membranes due to membrane damages and con-
comitant denaturation of membrane-bound proteins (Rux et al., 2019).
Reversibility of changes beneath critical thresholds may reflect the re-
storation of intracellular solute gradients by functioning membrane-
bound ion channels and pumps, and transporter proteins.
In general, cell membrane damage and the, thus, induced leakage of
electrolytes from the symplast into the apoplast finally increase tissue
conductivity, which, in turn, can be evaluated by impedance mea-
surements (Angersbach, Heinz, & Knorr, 2002). Impedance measure-
ments were used to analyse the degree of cell-damaging due to high
voltage pulses or freeze-thaw processes (Ade-Omowaye, Angersbach,
Eshtiaghi, & Knorr, 2001; Ade-Omowaye, Rastogi, Angersbach, &
Knorr, 2003; Angersbach, Heinz, & Knorr, 1999; Angersbach et al.,
2002; Guderjan, Töpfl, Angersbach, & Knorr, 2006; Luscher, Schlüter, &
Knorr, 2005), and viral infections or frost damage (Greenham, Helms, &
Müller, 1978; Zhang & Willison, 1991) in various fresh products. Ac-
cording to Angersbach et al. (2002), evaluating changes in the specific
conductivity of tissue samples enables estimating the functional state of
cell membranes and, thus, the degree of cell destruction. Due to their
insulating properties, intact membranes influence the conductivity of
the entire symplast and, thus, the integrity of the cell membrane system
can be analysed (Angersbach et al., 2002). Conductivity changes are
particularly characteristic in the range from 1 kHz to 100 MHz and
referred to as β-dispersion (Schwan, 1968). In the low-frequency range,
conductivity increases as a function of the destruction of the membrane,
until they obtain similar values than those in the high-frequency range
at completely destructed membranes (Angersbach et al., 1999, 2002).
Consequently, the combined application of cell pressure probe and
impedance measurements allows the direct comprehensive differ-
entiation of reversible and irreversible effects of high-pressure treat-
ments on the physiological activity of fresh living plant organs. Thus,
changes in cell turgor, the latter based on recalculated data originally
presented in Rux et al. (2017, 2019), and the degree of cell destruction
were comparatively investigated in fresh red cabbage (Brassica oleracea
L. convar. capitata (L.) Alef. var. rubra DC.) leaves and in radish
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Innovative Food Science and Emerging Technologies 61 (2020) 102326
(Raphanus sativus L. var. sativus ‘Donar’) tubers, species-specifically
exposed to various HHP ranges (red cabbage: 150–200 MPa at 35–55 °C
for 5–20 min; radish: 100–200 MPa, at 20–40 °C for 5–10 min; Rux
et al., 2017, 2019). This data combined with the new results published
in this paper yield to directly identify the relevant underlying metabolic
processes by comparing the respective dynamics of turgor losses and
cell destruction at the different parameter combinations. This will fur-
ther advance the understanding of the mechanisms behind high hy-
drostatic pressure-related variations in the integrity of membrane and
functions of relevant proteins.
2. Materials and methods
2.1. Material
For the experiments, red radish (Raphanus sativus L. var. sativus;
‘Donar’) tubers and red cabbage (Brassica oleracea L. convar. capitata
(L.) Alef. var. rubra DC.) leaves were used. Three mature heads of red
cabbage that may have been stored at the producer for approx. four
months were purchased at a wholesaler (Frucht Express GmbH, Groß
Kreutz, Germany), transported to the laboratory and stored in water
vapour-saturated atmosphere at 7 °C for the duration of the experi-
ments. Small radish plants were grown to commercial maturity in a
Weisshaar climate chamber (Bad Salzuflen, Germany) at Tday/night of
15/12 °C and rHday/night 40/85 %. Mean photosynthetically active
photon fluence rates (PFR) were set to approx. 250 μmol m−2 s−1
(high-pressure sodium lamps, SON-T AGRO 400, Philips, Hamburg,
Germany) for a 14 h photoperiod.
One day before the study, one mature leaf was removed from each
cabbage head and radish tubers of uniform size (diameter approx. 3 cm)
were selected and their leaves detached. Both produces were gently
washed, dabbed dry and stored in a box in the water vapour-saturated
atmosphere at approx. 20 °C. Immediately before the high-pressure
treatment, a total of 16 discs (d = 17.5 mm; 5–6 per cabbage head)
were punched out from intercostal areas of a cabbage leaf with a cork
borer, carefully avoiding any vein. Immediately before the high-pres-
sure treatment, 4 cabbage leaf discs or 2 radishes (a total of 6 tubers)
each were carefully inserted into a sample bag (Whirle Pak B00679,
Nasco, Fort Atkinson, USA), which was hermetically sealed after re-
moving the air. These samples were subjected to high-pressure treat-
ments.
After treatments, samples were placed into Petri dishes, lined with
water-soaked (deionised water) paper tissue to prevent dehydration and
kept in a temperature-controlled room at 20.0 ± 0.3 °C during the
subsequent investigations. To differentiate short-term disturbances of
plant physiology, and biochemical and biophysical processes from ir-
reversible damages, cell turgor and impedance were repeatedly mea-
sured over a period of 24 h. To evaluate the influence of storage on
overall plant performance and the biological variability of physiological
properties, the measurements were repeated several times with un-
treated fresh plant material.
2.2. High-pressure processing
Following Rux et al. (2017, 2019), the high-pressure experiments
were carried out in a custom-built high-pressure device (Uhde High-
Pressure Technologies GmbH, Hagen, Germany), which had a max-
imum design pressure of 360 MPa (Table 1). It is assembled from a
stainless steel vessel (volume: 600 mL; internal diameter: 56 mm;
height: 250 mm) and an upper plug with the grommets for pressure
release valve, two temperature sensors (Pt-100, thermal resolution
better than 0.1 K in the range used) and a pressure transducer (HP28,
Intersonde Ltd., Watford, England). The temperature near the sample
and the pressure were recorded for each pressure treatment (for detail,
Fig. S1). A high-pressure reciprocating pump (DSXHW, Haskel Ltd.,
California, USA) pumped the pressure medium (Type 6165 silicone oil,
G. Rux, et al. Innovative Food Science and Emerging Technologies 61 (2020) 102326

Table 1
P-T-time combinations used in HHP treatment of red cabbage and radish tubers.
Produce Holding time (min) Treatment temperatures (°C) at the specific HHP

100 MPa 150 MPa 175 MPa 200 MPa

Red cabbage 5 – 35 35 35
10 – 35/45/55 35 35/45/55
15 – 35 35 35
20 – – 35 –
Radish tubers 5 20/30/40 20/30/40 – 20/30/40
10 20/30/40 20/30/40 – 20/30/40

Huber, Offenburg, Germany) into the vessel from a reservoir. To facil- the results of these five cells per sample averaged. The averages of the
itate temperature regulation during pressurisation, flexible tubes were three discs were used for further analyses. The hydrostatic pressures of
coiled around the vessel and connected to a thermal bath (Haake, tissue cells (turgor, P) were measured before and immediately after
Karlsruhe, Germany). This operated with deionised water, which was high-pressure treatments, and after subsequent storage for 4 h and 24 h.
tempered in the range from 20 °C to 55 °C to guarantee the specific To directly compare the results with impedance results, the turgor
targeted constant treatment conditions (Table 1). values were converted into relative cell turgor (Prel) as
Before the treatment, the sample bag was introduced into a Teflon
Pt ∗ 100
cylinder, filled with deionised water. The Teflon cylinder was fixed to Prel = ,
Pi
the upper plug of the high-pressure vessel before closing the unit. A
moving piston at the bottom of the Teflon cylinder allowed pressure- where Pi is the turgor of the untreated sample and Pt is the turgor of the
related compression without deformation. By pre-tempering (Table 2) respective treated sample, thus, 100 % represents the initial value.
the filled cylinder for about 45 min, the targeted temperature was ob-
tained due to quasi-adiabatic heating during the pressure built-up phase 2.4. Impedance measurements
(Knoerzer, Buckow, & Versteeg, 2010) and remained approximately
constant during applied treatment durations. The specific temperatures In case of red cabbage leaves, each of four discs were cut into ap-
for pre-tempering were determined in preliminary experiments. Tem- proximately 1 mm3 cubes with a razor blade, carefully mixed and
perature and pressure inside the Teflon cylinder were recorded near the transferred into a PVC sleeve (approx. 9.6 mm inner diameter). On one
sample for each pressure treatment. side, the PVC sleeve was closed by a cylindrical electrode (d = 9.6 mm)
optimised for the analysis of biological material. After filling, another
electrode was pushed into the sleeve from the opposite side. The
2.3. Cell turgor measurements
sample-filled space between the electrodes was approx. 7 mm wide. For
radish tubers, one cylindrical tissue segment was extracted with a cork
Following Martin, Rux, and Herppich (2013), the turgor of in-
borer (d = 8.9 mm); from the middle of the segment, a 7 mm long piece
dividual cells was measured with a cell pressure probe (Hüsken et al.,
was cut with a razor blade and placed into the PVC sleeve, which was
1978), provided by E. Steudle (Bayreuth, Germany), and which was
then closed by the two electrodes. Both sample materials were fixed by
equipped with a 24PC series pressure sensor (Honeywell Inc., Freeport,
gently pressing the electrodes into the sleeve. Then, the sleeve was
USA). From each of 3 of the 6 radish tubers treated, one cylindrical core
inserted into the SigmaCheck impedance measurement device
segment was extracted with a cork borer (17.5 mm); from the centre of
(Biotronix, Hennigsdorf, Germany; Fig. 1), which measures the im-
each of these segments, a single 2 mm thick disc was cut with a razor
pedance over 14 frequency channels ranging from 1.38 kHz to
blade.
11.2 MHz (Table 3). Three individual measurements for each sample
For measurements, discs of cabbage leaves or radish tissue were
were performed.
placed in the indentation of a custom-made sample holder on a preci-
Each frequency channel was measured in triplicate. The signals
sion microscope stage and positioned under the microscope (both Leitz,
Wetzlar, Germany) so that both the tissue cells and the microcapillary
of the cell pressure probe could be clearly viewed at magnifications of
40× or 100× (Rux et al., 2019). The tip of the silicone oil-filled glass
microcapillary was inserted into a single cell of the discs under view
using a micromanipulator (Leitz, Wetzlar, Germany). For each of three
discs, five individual cells were measured once per sampling times and

Table 2
Temperatures used to pre-tempering the filled cylinder (45 min) to obtain the
targeted treatment temperatures at the specific applied pressures after quasi-
adiabatic heating during the pressure built-up phase (holding phase). The
specific temperatures were determined in preliminary experiments.
Treatment Used pre-tempering temperatures (°C) at the specific HHP
temperature (°C)
100 MPa 150 MPa 175 MPa 200 MPa

20 14.8 13.5 – 10.6

30 24.4 21.8 – 20.9
35 – 27.4 26.5 25.7
40 34.7 32.9 – 30.4
45 – 40.0 – 37.6
55 – 47.1 – 44.7
Fig. 1. Impedance measurement setup - schematic representation (Biotronix).

3
G. Rux, et al. Innovative Food Science and Emerging Technologies 61 (2020) 102326

Table 3 3. Results
Measuring frequencies during impedance measurement.
Channel Frequency [kHz] Channel Frequency [kHz] 3.1. Interactive effects of duration and extent of HHP treatment, and
temperature on turgor
1 1.4 8 175
2 2.7 9 351
The recalculated results of turgor measurements, originally pre-
3 5.5 10 703
4 11 11 1400
sented by Rux et al., (2017, 2019), are summarized in this section, to
5 22 12 2800 allow a clear and profound identification of the relevant underlying
6 44 13 5600 metabolic processes by comprehensively comparing the different dy-
7 88 14 11,200 namics of turgor losses and cell destruction at the different parameter
combinations. Mean (n = 3) turgor of fresh untreated red cabbage
leaves and radish tubers was 0.59 ± 0.02 MPa and 0.27 ± 0.02 MPa,
were averaged and converted into the specific electrical conductivity σ
respectively. Irrespective of the produce, HHP treatments generally
[μS]. The cell destruction rate was calculated from the results of the
induced an immediate drop of mean turgor as revealed by the reduced
frequency channels of 5.5 kHz and 1.4 MHz. The values of the other
relative turgor at t = 0 h (Figs. 2 to 5). This decline was always sig-
frequency channels were used to control the measurement. The cell
nificantly amplified by the increase in treatment temperature and
disruption index (CDI) was calculated (Angersbach et al., 1999) as
duration.
σi
For red cabbage, only cell turgor of samples treated with 150 MPa at
⎛ hs ⎞ ∗ σls − σli 35 °C recovered within 24 h (Fig. 2A). Temperatures of 55 °C always
σh
CDI = ⎝ ⎠ i , resulted in immediately, completely and irreversibly dropped turgor
σh − σli
(Fig. 2). After treatments with 200 MPa, relative turgor rapidly de-
creased below 50 % and progressively declined further (Figs. 2B, 3).
where the superscripts/subscripts i indicates intact samples, s treated
Radish tubers are significantly more sensitive to HHP treatments
samples, h the high (1.4 MHz) and l the low frequency (5.5 kHz). Values
than red cabbage leaves. Nevertheless, short-term (5 min) HHP appli-
for the intact tissue were obtained in triplicate on reference samples.
cations of 100 MPa at moderate temperatures (< 30 °C) can be assumed
For direct comparison with relative turgor, the CDI was converted into
as gently for tubers. Here, turgor did not drop below 30 % immediately
relative cell intactness (RCI), where the value of 100 % represents the
after treatment and also partially retained the ability to recover within
initial state of the intact tissue (RCI = 100 – CDI ∗ 100). RCI is a
24 h (Fig. 5). All other HHP treatment conditions, in particular at
sensitive index for the evaluation of HHP effects.
200 MPa, caused pronounced and irreversible turgor losses.

2.5. Statistical analysis 3.2. Influence of HHP treatment on tissue impedance

One disc of each of 3 radish tubers or cabbage leaves per treatment
was measured (n = 3 per treatment); for each disc, the turgor of five
single cells was measured and the results averaged. Impedance on
radish tuber tissue was measured on each of 3 intact cylindrical core
segment per treatment (n = 3 per treatment) in triplicate. For red
cabbage leaves, three measurements per treatment (n = 3 per treat-
ment) were conducted on four of 12 slices each in triplicate. All data
were statistically analysed (ANOVA) with WinSTAT (R. Fitch Software,
Staufen, Germany). Significance of differences between means was
analysed by Duncan's multiple range test (p < 0.05).
In contrast to turgor, relative tissue impedance (RCI) is generally
much less responsive to HHP treatments (Figs. 2–5). In red cabbage
leaves, RCI did not immediately decrease after treatments with
150 MPa at 35 °C for up to 15 min (Fig. 3A, B) or with 175 MPa for up
to 5 min (Fig. 5). All treatments above these thresholds immediately
reduced RCI by approx. 40 % (Figs. 2–4). Long-term treatments at high
pressures and high temperatures significantly amplified this drop and
led to a continuous and irreversible decrease in RCI. Overall, relative
membrane damages ranged from 12 % (RCI = 88 %) to 100 %
(RCI = 0 %). Clearly, treatments at 55 °C or HHP applications longer
than 15 min had the highest impacts on RCI.
In radish tubers, RCI never decreased immediately after HHP
treatment at pressures up to 150 MPa, regardless of treatment tem-
perature and duration (Fig. 5). At 200 MPa, RCI maximally declined by

Fig. 2. Interactive effects of HPP treatment pressure

(A: 150 MPa; B: 200 MPa) and pressure holding
temperature (35 °C, grey; 45 °C, diagonally hatched;
55 °C, black) applied for 10 min on percentage turgor
(based on specific data sets recalculated from Rux
et al., 2019) and RCI of red cabbage leaves. Turgor (5
individual cells of each of 3 tissue discs per treat-
ment) and RCI (3 repeated measurements each of 3
tissue samples per treatment) was measured before
(dashed line = 100 %) and directly after HHP
treatments, and after approx. 4 h (just turgor) and
24 h of recovery at 20 °C in water vapour saturated
atmosphere in darkness. Given are means ( ± SD,
n = 3); different letters indicate significant differ-
ences between means.

4
G. Rux, et al.
Fig. 3. (A–C) Interactive effects of pressure holding time (A: 5 min; B: 10 min;
C: 15 min) and HHP (100 MPa, solid grey; 150 MPa, diagonally hatched;
200 MPa, solid black) applied at 35 °C on percentage turgor (based on specific
data sets recalculated from Rux et al., 2019) and RCI of red cabbage leaves.
Turgor (5 individual cells of each of 3 tissue discs per treatment) and RCI (3
repeated measurements each of 3 tissue samples per treatment) was measured
before (dashed line = 100 %) and directly after HHP treatments, and after
approx. 4 h (just turgor) and 24 h of recovery at 20 °C in water vapour saturated
atmosphere in darkness. Given are means ( ± SD, n = 3); different letters in-
dicate significant differences between means.
25 % immediately after treatment (Fig. 5). If treated with HHP above
100 MPa, RCI always continued to decline during 24 h of storage. In
contrast to treatment duration, temperature exhibited no clear cut
5
Innovative Food Science and Emerging Technologies 61 (2020) 102326
Fig. 4. Interactive effects of pressure holding time (5 min, solid grey; 10 min,
diagonally hatched; 15 min, horizontally hatched; 20 min, solid black) applied
at 175 MPa and 35 °C on percentage turgor (based on specific data sets re-
calculated from Rux et al., 2019) and RCI of red cabbage leaves. Turgor (5
individual cells of each of 3 tissue discs per treatment) and RCI (3 repeated
measurements each of 3 tissue samples per treatment) was measured before
(dashed line = 100 %) and directly after HHP treatments, and after approx. 4 h
(just turgor) and 24 h of recovery at 20 °C in water vapour saturated atmo-
sphere in darkness. Given are means ( ± SD, n = 3); different letters indicate
significant differences between means.
effects on RCI (Fig. 5).
3.3. Correlation analyses of HHP effects on cell turgor and tissue impedance
The correlation analyses of HHP effects on cell turgor and tissue
impedance revealed a distinct two-phase response of both parameters
(Fig. 6), which, in addition, differed between red cabbage leaf
(Fig. 6A,C) and radish tuber tissues (Fig. 6B,D). Immediately after
treatments, cell turgor and tissue impedance were linearly correlated,
yielding significant correlation for red cabbage leaves with a coefficient
of determination (R2) of 0.75 (Fig. 6A), while no correlation
(R2 = 0.17) was apparent for radish tubers (Fig. 6B).
After 24 h of storage, however, the correlation between both
parameters became non-linear and R2 increased to 0.90 for red cabbage
(Fig. 6C), and to 0.59 (Fig. 6D) for radish tubers. In red cabbage leaf
tissues, the gradual changes indicated three discrete response stages
with concomitant decline of both turgor and tissue integrity (cluster I,
RCI 80 % – 100 %, Prel > 50 %; cluster II, RCI 25 % – 80 %, Prel 10 % –
20 %; cluster III, RCI 0 % – 25 %, Prel ≈ 0).
On the other hand, radish tubers seemed to respond in an all-or-
none reaction (Fig. 6D) in particular related to the degree of turgor loss
(cluster I, RCI 80 % – 100 %, Prel > 50 %; cluster II, RCI: 30 % – 50 %,
Prel ≈ 0 – 30 %). Nevertheless, exclusively tubers treated at 100 MPa
contributed to clustering I. Interestingly, increasing treatment tem-
perature and pressure holding time at this HHP only led to a reduction
of turgor but did not affect the tissue integrity. Even after the harshest
treatments considered, radish tuber tissue was not completely damaged
but retained a reasonable level of relative cell integrity yet at total
turgor loss at the highest temperature.
4. Discussion
Although both radish tubers and red cabbage leaves are very sus-
ceptible to high hydrostatic pressures, the latter is pronouncedly more
resistant. Nevertheless, only short-term applications at moderate HHP
G. Rux, et al.
(< 150 MPa) and temperatures (< 40 °C) only partially and reversibly
affected the metabolic activity of treated tissues. This has also been
previously shown for the photosynthetic capacity of lambs lettuce
(Schlüter et al., 2009) leaves and for single-cell pressure potential in red
cabbage leaves (Rux et al., 2017) and in radish tubers (Rux et al., 2019).
Both for active photosynthesis and for a positive turgor, functional
subcellular compartmentation and the resulting gradients of relevant
metabolites and ions are inevitable prerequisites (e.g., Buchanan,
Gruissem, & Jones, 2000; von Willert et al., 1995). This, however, re-
quires intact semipermeable cell membrane systems and operational
membrane-associated proteins such as solute channels or pumps. Any
reversibility of HHP induced disturbance of cell homeostasis as ob-
served at conditions below the above-mentioned thresholds may thus
indicate temporally increased membrane permeability and/or reduced
activity of separating proteins, most probably due to direct physical
high-pressure effects on the respective cell components (Kato &
Hayashi, 1999). This facilitates the leakage of solutes either across the
thylakoid membrane or the tonoplast or even across the plasmalemma
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Innovative Food Science and Emerging Technologies 61 (2020) 102326
Fig. 5. Interactive effects of pressure holding tem-
perature (A + D: 20 °C; B + E: 30 °C; C + F: 40 °C)
and HHP (100 MPa, solid grey; 150 MPa, hatched;
200 MPa, solid black) applied for 5 min (A-C) or
10 min (D-F) on percentage turgor (based on specific
data sets recalculated from Rux et al., 2019) and RCI
of radish tubers. Turgor (5 individual cells of each of
3 tissue discs per treatment) and RCI (3 repeated
measurements of each of 3 tissue samples per treat-
ment) was measured before (dashed line = 100 %)
and directly after HHP treatments, and after approx.
4 h (turgor) and 24 h of recovery at 20 °C in water
vapour saturated atmosphere in darkness. Given are
means ( ± SD, n = 3); different letters indicate sig-
nificant differences between means.
out of the symplast into the apoplast (Otero & Préstamo, 2009;
Vázquez-Gutiérrez, Hernández-Carrión, Quiles, Hernando, & Pérez-
Munuera, 2012). After pressure release, these physical effects dis-
appear, the “normal” semi-permeability returns and the pumps and
channels re-establish the original gradients (Kato & Hayashi, 1999).
Only above the respective critical thresholds, changes such as turgor
loss (Rux et al., 2017, 2019) became irreversible. Only then, the high
hydrostatic pressures completely disturbs membrane integrity and
functionality (Indrawati et al., 2000), e.g. by phase transitions of the
biomembranes phospholipid bilayer (Haltia & Freire, 1994; Maheswari,
Joshi, Saha, Nagarajan, & Gambhir, 1999), separation of membrane-
bound proteins (Kato & Hayashi, 1999), and/or the destruction of
membrane-associated and dissolved proteins (Angersbach et al., 2002).
By concomitantly assessing the dynamics of both turgor changes
with the cell pressure probe and the degree of cell destruction by im-
pedance analyses (Angersbach et al., 1999), the transition between the
reversible changes in membrane and protein properties, and the irre-
versible damages could be experimentally evaluated. The turgor, as
G. Rux, et al.
measured within single cells, immediately declined after treatment due
to the direct impairment of cell membrane function, whereas electrolyte
leakage measured outside cells by changes in impedance were only
detectable after pronounced apoplastic diffusion (Gonzalez & Barrett,
2010).
After application of HHP above the critical threshold level, the
disturbing and/or damaging effects of this treatment on membranes
lasted for hours after the pressure release, although this obviously only
started with some delay. Angersbach et al. (2002) also found a close
and positive correlation between continuous membrane disintegration
and the duration of storage after high-pressure treatments. In addition,
this group obtained similar results after the application of high voltage
pulse on potato tissue. Assuming that membrane-associated proteins in
the biomembrane were solubilized during treatments but not after-
wards, Angersbach et al. (2002) suggested the occurrence of secondary
post-treatment effects, which may explain the gradual increase in
membrane permeability. This may be due to localized cell membrane
damage or increased enzyme activities of unimpaired enzymes. The first
describes local damage of the membrane matrix initiated by the pres-
surisation (and pulsed electric fields) and resulting from phase transi-
tion of the phospholipid or from denaturation of membrane-associated
proteins.
Consequently, reversible and irreversible effects clearly differentiate
only after 24 h of storage. In particular, for the red cabbage, the clus-
tering of the respective data points observed in Fig. 6 may reflect the
different stages of HHP effects. The first cluster (cluster I) indicates
moderately disturbed systems by the marginally decreased cell turgor
(> 60 %) and high tissue integrity. The cell membranes are only tem-
porarily affected but not permanently damaged and cell turgor can
recover. In contrast, cluster III is characterized by more or less complete
turgor losses and pronounced cell destruction (RCI: 0 % – 25 %). The
cell membranes systems, i.e. phospholipid bilayers and intrinsic trans-
port proteins, are drastically and irreversibly damaged. The
7
Innovative Food Science and Emerging Technologies 61 (2020) 102326
Fig. 6. Correlation between relative turgor (based on
specific data sets recalculated from Rux et al., 2017,
2019) and RCI of HHP treated (A + C) red cabbage
leaves and (B + D) radish tubers. Best-fit analyses
indicated a linear (A) and a non-linear (C) relation-
ships for the red cabbage but not for radishes data (B,
D). The respective best fits are highlighted by solid
dark lines for cabbage (A,C) and by dotted lines for
radish. Symbols indicate the treatment pressure
(100 MPa, circles; 150 MPa, squares; 175 MPa, tri-
angles; 200 MPa, diamonds), colours the pressure
holding temperature (green/light grey; 45 °C, blue/
medium grey; 55 °C, red/dark grey for red cabbage
leaves and 20 °C, green/light grey; 30 °C, blue/
medium grey; 40 °C, red/dark grey for radish tubers)
and different numbers indicate the pressure holding
times (min). Results of turgor (measured in 5 in-
dividual cells of each of 3 tissue discs per treatment)
and RCI (3 repeated measurements each of 3 tissue
samples per treatment) obtained directly after HHP
treatments (A + B) and after approx. 24 h of storage
(C + D) at 20 °C in water vapour saturated atmo-
sphere and darkness. Solid (red) lines (A, C) indicate
the respective response zones (for details, please see
text).
intermediate cluster (cluster II) may indicate the temporary impairment
of membrane properties and the beginning denaturation of membrane-
bound or membrane-spanning ion pumps. Due to the latter, the elec-
trolytes gradients could not re-establish after pressure release but tissue
damage did not further increase.
By applying nuclear magnetic resonance techniques, Marigheto
et al. (2004) and Otero and Préstamo (2009) also found minor dis-
turbance of water distribution in the extreme susceptible strawberry
fruit after application of 100 MPa but only increasing HHP to 200 MPa
induced serious tissue damage. Similarly, Kurenda, Zdunek, Schlüter,
and Herppich (2014) reported pronounced and complete tissue
browning of apples treated with high hydrostatic pressure above
100 MPa but only minor effects in fruit subjected to this threshold
pressure. In addition, Vázquez-Gutiérrez et al. (2012) reported cell wall
disruption in fresh Persimmons after treatment of 200 MPa.
There are clear differences in the long-term HHP responses of the
two products. Red cabbage leaf tissue showed a more or less pro-
nounced three stages decline of both turgor and relative cell integrity
after 24 h of storage. In contrast, the thin-walled parenchymatous
radish tuber tissue retained a certain level of cell integrity after 24 h of
storage but turgor could not be re-established after HHP treatments
above the critical treatment thresholds. The fact that below these limits,
no tissue damage was observed at all reflected results on potato tissue
(Angersbach et al., 2002). These authors, and others (Kurenda et al.,
2014; Marigheto et al., 2004), also found that tissue damage occurred
only at HHP beyond 100 MPa.
At 100 MPa (and below), HHP only influences the permeability of
the membranes. This may lead to a partial electrolyte leakage out of the
symplast and, thus, a reduction of the osmotic gradient over the
membranes, directly reducing the cell turgor (Rux et al., 2017, 2019).
This partially reversible effect is the more pronounced, the longer the
pressure holding time and the higher the treatment temperature. The
fact that these changes are partially reversible (cf. Fig. 6B,D) also shows
G. Rux, et al.
that membrane intrinsic proteins are still functional after this moderate
HHP treatment and not solubilised (Angersbach et al., 2002) by this
pressure. On the other hand, it might also be that the HHP-enhanced
membrane permeability (Kato & Hayashi, 1999) could potentially not
fully recover after depressurization, preventing the complete re-estab-
lishing of the osmotic gradient and, thus, of turgor.
The higher stability of red cabbage leaf tissue compared to that of
radish tubers may primarily be explained by the pronounced structural
differences between fully mature leaf tissue with well-developed cell
walls and the thin-walled parenchymatous water storage tuber tissue.
Stronger cell walls may provide a certain mechanical stabilisation effect
to stressed biomembranes (Novaković, Guo, Bacic, Sampathkumar, &
Johnson, 2018; Tenhaken, 2015). On the other hand, Angersbach et al.
(2002) highlighted the protective effects solved sugars may have on
biomembranes. In this context, sugar concentrations in red cabbage leaf
cells are much higher than in those of radish tubers (De Belie, Herppich,
& De Baerdemaeker, 2000; Landahl, Herppich, Herold, Geyer, & De
Baerdemaeker, 2004).
5. Conclusions
Intensity, duration and temperature of HHP treatments inter-
actively, pronouncedly and directly affected cell turgor and tissue in-
tegrity of fresh radish tubers and red cabbage leaves. Only beyond
species-specific critical thresholds of HHP parameters but never at or
below 100 MPa, tissue damage became serious and, thus, turgor losses
irreversible. Both occurred at any HHP treatment with pressures above
150 MPa and at temperatures higher than 45 °C. Both high hydrostatic
pressure and temperature have the same primary targets, which can be
associated with the functionality of the phospholipid biomembrane
leading to a pronounced increase in permeability. At (or below)
100 MPa, these effects were limited and reversible, as indicated by the
partial restoration of the cell turgor. At this pressure limit, no tissue
damage occurred. In addition, the ability to re-establish turgor also
proves the action and thus intactness of membrane-bound or mem-
brane-spanning ion pumps or ion channels. Increasing HHP above
100 MPa intensified turgor loss and its irreversibility and resulted in
tissue damages. These effects were amplified with the intensity and the
duration of pressure treatments. The thin-walled parenchymatous
radish tuber tissue seemed more susceptible to high HHP than the
strong mature red cabbage leaf tissue with well-developed cell walls.
Nevertheless, only in cabbage leaves, complete tissue damage was
measured after 24 h of storage after HHP treatments.
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.ifset.2020.102326.
CRediT author statement
Guido Rux: Methodology, Investigation, Formal analysis, Data
Curation, Visualization, Writing - Original draft preparation, Writing -
Review & Editing.
Ronja Gelewsky: Investigation, Formal analysis.
Oliver Schlüter: Conceptualization, Resources, Methodology,
Validation, Writing - Review & Editing, Supervision.
Werner B. Herppich: Conceptualization, Resources, Methodology,
Validation, Visualization, Writing - Original draft preparation, Writing-
Reviewing and Editing, Supervision.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
8
Innovative Food Science and Emerging Technologies 61 (2020) 102326
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