Journal of Food Engineering 45 (2000) 11±16

www.elsevier.com/locate/jfoodeng

E€ects of high isostatic pressure on mushrooms

Ariette M. Matser *, Elaine R. Knott 1, Paul G.M. Teunissen, Paul V. Bartels
Agrotechnological Research Institute (ATO-DLO), P.O. Box 17, 6700 AA, Wageningen, Netherlands

Abstract
The possibility of high isostatic pressure treatment as an alternative to conventional blanching of mushrooms was investigated.
An important condition for successful application is a sucient inactivation of the browning enzyme polyphenoloxidase. The
experiments showed that this enzyme is very pressure-resistant in mushrooms. The activity increased after pressure treatments at
600 MPa. Higher pressures up to 950 MPa are necessary for inactivation. The e€ects on texture, colour and yield of mushrooms
were evaluated at these high pressures. If fresh mushrooms are used, pressure treatment results in a dark brown colour. However, if
mushrooms are evacuated before pressure treatment, the colour expressed in L, a* and b* values is comparable with conventionally
blanched mushrooms. The sti€ness of the pressurised mushrooms is larger than that of blanched mushrooms. The yield is roughly
the same for the conventional blanched mushrooms and the pressurised mushrooms. Ó 2000 Elsevier Science Ltd. All rights
reserved.

Keywords: High pressure; Mushroom; Polyphenoloxidase; Texture; Colour

1. Introduction ated the e€ect of pressure on several aspects of the one

High isostatic pressure can be used as a non-thermal
technique for conservation and preparation of food
products. Due to the isostatic nature of the process the
pressure is applied uniformly and immediately on the
products resulting in changes in, for example, proteins,
polysaccharides and lipids. High pressure can inactivate
micro-organisms and enzymes without large e€ects on
small molecules such as ¯avours, and vitamins. For
evaluating the e€ects of pressure on food components, it
is important to consider the in¯uence of the conditions
in food like pH and chemical composition. As Weemaes
(1998) showed, pressure inactivation of polyphenoloxi-
dase can be largely increased at lower pH, and by
components such as sodium chloride and EDTA.
Therefore, results obtained in bu€er solutions are not
always applicable to food systems. It is also important
to compare the e€ects of high pressure on di€erent
characteristics of a product. Pressure conditions, which
inactivate enzymes or micro-organisms, can be con-
¯icting with conditions necessary for preservation of
colour and texture. For this study we therefore evalu-
* jars, these jars are sterilised to inactivate the micro-or-
Corresponding author. Tel.: +31-317-475119; fax: +31-317-475347.
E-mail address: a.m.matser@ato.dlo.nl (A.M. Matser).
1
Present address: Unigate PLC, Malton Foods, Parliament street,
Norton, Malton, North Yorkshire YO17 9HG, UK.
0260-8774/00/$ - see front matter Ó 2000 Elsevier Science Ltd. All rights reserved.
PII: S 0 2 6 0 - 8 7 7 4 ( 0 0 ) 0 0 0 3 5 - 2
product, mushrooms, and compared this with a con-
ventional process to evaluate the possibilities for high
pressure as an alternative process resulting in a higher
quality.
In the Netherlands, about two third of the annual
production of mushrooms (220 ´ 106 kg) is preserved.
Common mushrooms (Agaricus bisporus) are a relatively
expensive product compared to other vegetable prod-
ucts. Therefore, the cost of high pressure processing is
relatively low compared to the overall price of the
product. If high pressure results in a higher quality of
the product it can be an interesting alternative. The
conventional conservation process of mushrooms con-
sists of several steps. After washing, the mushrooms are
evacuated. This is a vacuum hydration leading to a re-
placement of the air in the mushrooms with the evacu-
ation ¯uid, which is usually water containing citric acid
and sodiumsulphite. This evacuation results in a better,
lighter colour of the product and less shrinkage and
water loss in the subsequent heating stages. The evacu-
ated mushrooms are blanched for inactivation of en-
zymes and to shrink the tissue so this will not occur
during sterilisation. After ®lling the mushrooms in glass
ganisms. Both the blanched and the sterilised mush-
rooms are sold. Important aspects of the process are the
quality and the yield of the mushrooms expressed in
12 A.M. Matser et al. / Journal of Food Engineering 45 (2000) 11±16
colour, taste and texture (Jasinki, Stemberger, Walsh
& Kilara, 1984; McCord & Kilara, 1983). For high
pressure to be a successful conservation process, it has
to result in inactivation of the main enzyme responsible
for browning of the mushrooms, polyphenoloxidase.
Weemaes (1998) describes the mechanism of poly-
phenoloxidase catalysed browning. Polyphenoloxidase
is a copper-containing enzyme that catalyses browning
reactions of phenols oxidised to o-quinones. These qui-
nones react further to brown pigments. It is located in-
tracellularly for plants but extracellularly for
mushrooms. Polyphenoloxidases can exist in a latent or
inactive state. Activation occurs as a result of ageing or
treatments like acid or base shock, mild heating, pres-
surisation or freezing/thawing. Activation can be re-
versible or irreversible. Possible mechanisms are
cleavage of the covalent bond between enzyme and
membrane for plant polyphenoloxidases, conforma-
tional changes or removal of inhibitors. Oxygen avail-
ability, pH and concentration of inhibitors control the
activity of the enzyme. Mushrooms polyphenoloxidase
is a multi-subunit enzyme with probably four subunits.
The enzyme exists in multiple forms called isozymes
which have a di€erent sensitivity to temperature, pH
and pressure (Weemaes, 1998). Several authors investi-
gated the e€ect of pressure on inactivation of polyphe-
noloxidases from di€erent origins. For complete
inactivation at room temperature, relatively high pres-
sures are necessary of more than 600 MPa (Castellari,
Matricardi, Arfelli, Rovere & Amati, 1997; Gomes &
Ledward, 1996; Seyderhelm, Boguslawski, Michaelis &
Knorr, 1996; Weemaes, 1998). Asaka and Hayashi, 1991
and Asaka, Oayama, Nakanishi and Hayashi, 1994,
however, described that polyphenoloxidase in a cell free
extract from pears can be activated by pressure treat-
ments of 400±600 MPa, contrary to polyphenoloxidase
from apple, banana, sweet potato, pea leaves, spinach,
lettuce and celery. Hernandez and Cano (1998) and
Butz, Koller, Tauscher and Wolf (1994) described acti-
vation of polyphenoloxidase in tomato puree and
onions respectively, followed by an (incomplete) inacti-
vation at higher pressures.
The objective of this study was to evaluate the e€ects
of high isostatic pressure on polyphenoloxidase activity,
colour, texture and yield of mushrooms and to compare
these e€ects to conventional processing.
2. Materials and methods
2.1. Materials
Mushrooms (A. bisporus) were obtained from a local
grocery and used within 24 h for the experiments. For all
experiments, mushrooms were tested whole.
2.2. Evacuation
Mushrooms were evacuated according to a standard
procedure. The mushrooms were brought under vacuum
(20±40 mbar) during 5 min after which the evacuation
¯uid was sucked into the vacuum vessel. The vacuum
was held for 3 min. Finally the vacuum was released and
mushrooms were removed from the vessel. Unless stated
otherwise, tap water was used as evacuation ¯uid. Some
experiments were done with 50 g sodiumsulphite and
100 g citric acid in 100 l of tap water.
2.3. Treatment
Fresh or evacuated mushrooms were vacuum packed
in polyethylene bags. Pressure treatment was done in a
Resato high pressure apparatus (volume 180 ml, Resato,
Roden, Netherlands) at 200±1000 MPa. The computer
controlled pressure build up rate was 10±15 MPa/s.
Time for pressure treatment was 5 min, pressure increase
and decrease times not included. Experiments were done
at room temperature, due to the compression of the
pressure ¯uid (glycol, Resato) an initial temperature rise
of 10±15°C was measured with a thermocouple inside
the pressure vessel.
Fresh or evacuated mushrooms were blanched for 1,
5 or 10 min in boiling tap water.
2.4. Analysis
The weight of the mushrooms was measured before
and after the treatments after 5 min drainage in a sieve.
For determination of the activity of polyphenoloxi-
dase, the mushrooms were homogenised with sodium-
phosphate bu€er (pH 6.5). After centrifugation, the
activity was measured in the extract with the catechol
method as described by Cano, Hernandez and De Ancos
(1997). The appearance of colour compounds resulting
from the enzymatic oxidation of catechol was measured
using a spectrophotometer at 420 nm. The activity is
expressed as the change in adsorbance per minute per
gram of fresh weight.
The texture of the mushrooms was measured with a
Texture Analyser (Stable Micro Systems, Godalming,
UK). After removal of the stem of the mushroom, the
cap was compressed with a probe (diameter 10 mm) at
0.8 mm/s. The peakstress at 30% compression and the
slope of the linear part of a stress/strain curve (sti€ness)
were calculated.
The colour of the mushrooms was measured with a
Minolta Chroma meter within 10 min after opening of
the plastic bags with the mushrooms. The L*a*b colour
space was used for determining the colour, with L rep-
resenting the lightness, +a* the red direction, )a* the
green direction, +b* the yellow direction, and )b* the
blue direction.
 A.M. Matser et al. / Journal of Food Engineering 45 (2000) 11±16 13

There can be large di€erences between batches of
mushrooms for texture, colour and enzyme activity. We
therefore compared the results of the treated mush-
rooms with fresh mushrooms from the same batch.
Weight, texture and enzyme results are expressed as a
percentage of the values of fresh mushrooms.
3. Results and discussion
3.1. PPO-activity
The in¯uence of blanching and pressurisation on the
activity of polyphenoloxidase is presented in Tables 1
and 2. Experiments were done on di€erent days and
consequently with di€erent batches of mushrooms. The
activity of polyphenoloxidase increases upon storage of
the mushrooms after harvest as well as the ratio between
active and latent enzyme (Murr & Morris, 1975). We
therefore calculated the activity of polyphenoloxidase as
a percentage of the amount in fresh mushrooms of the
same batch and did not average the results from di€er-
ent batches.
Evacuation resulted in a small decrease in activity of
10±20%. Blanching caused a large reduction in activity.
As shown in Table 1, blanching for 1 min is insucient
to completely inactivate the enzyme. Longer blanching
times resulted in an almost complete reduction of the
Table 1
Polyphenoloxidase activity after evacuation and blanching of mush-
rooms as a percentage of the activity in fresh mushrooms. Values in
one column are means of experiments with one batch of mushroomsa
Treatment Fresh Evacuated
Experiment 1
Evacuation 80.9a
b
1 min blanching 54.3 34.0c
5 min blanching 3.2e;f 7.8e
10 min blanching 2.0f 13.0e
a
Values with di€erent superscripts di€er signi®cantly (P < 0.05).
activity of the enzyme. These results are comparable
with the results of McCord and Kilara (1983). Evacu-
ated and non-evacuated mushrooms showed compara-
ble reduction of polyphenoloxidase activity. There is a
di€erence in the reduction of the activity between the
two evacuated batches of mushrooms. Probably this can
be explained by a di€erence in initial activity of 0.104
and 0.447 Dadsorbance/min/g for experiments 1 and 2,
respectively. A correction is made for this by expressing
the activity as a percentage of those in fresh. However,
as Murr and Morris (1975) described, there can be also a
di€erence in proportion of latent and active enzyme and
therefore a di€erence in e€ect of blanching.
Pressurisation has a large in¯uence on the activity of
polyphenoloxidase. At pressures of 600 MPa an increase
in the activity is measured. This is probably due to a
transition from the latent to the active state of the en-
zyme. As described in Section 1, polyphenoloxidase in
mushrooms is located extracellularly and not bound to
the membranes. Therefore, the presumable mechanism
of activation will be changes in conformation of the
enzyme. Higher pressures resulted in an inactivation of
the enzyme as a result of denaturation. However, even at
950 MPa, there is still a small residual activity. For
complete inactivation of polyphenoloxidase it is there-
fore necessary to perform the high pressure treatment at
longer holding times and/or temperatures above 50±
60°C as described by Weemaes (1998). There is no sig-
ni®cant di€erence in the residual activity between evac-
uated and non-evacuated mushrooms.
Protein denaturation and thus enzyme inactivation
by high isostatic pressure can be reversible or irrevers-
ible (Hendrickx, Ludikhuyze, Van den Broeck &
Weemaes, 1998). We therefore measured the enzyme
activity as a function of the time after pressure treatment
Experiment 2 (Fig. 1). There are no signi®cant changes in the activity
of polyphenoloxidase in 48 h after pressure treatment.
91.3a
41.1d
0.3f
0.4f

Table 2
Polyphenoloxidase activity after evacuation and pressurization of
mushrooms as a percentage of the activity in fresh mushrooms. Values
in one column are means of experiments with one batch of mush-
roomsa

Treatment Fresh Evacuated

Experiment 1 Experiment 2

Evacuation 89.8a
b c
600 MPa 152.6 187.5 197.0c
800 MPa 24.6d;g 36.6d 55.2e Fig. 1. Polyphenoloxidase activity as a function of time after pressure
950 MPa 2.8f 3.2f 13.4f;g treatment expressed as a percentage of the initial activity in fresh
mushrooms. Mushrooms were not evacuated. After pressure treat-
a
Values with di€erent superscripts di€er signi®cantly (P < 0.05). ment, the homogenised mushrooms were stored at 4°C.
14 A.M. Matser et al. / Journal of Food Engineering 45 (2000) 11±16

We concluded that the inactivation of the enzyme is ir-
reversible for the conditions used in these experiments.
The mushroom industry uses water with citric acid
and sodiumsulphite as evacuation ¯uid instead of water,
which we used. From the results of Weemaes (1998), it
can be expected that due to the lower pH, inactivation of
polyphenoloxidase will be larger under these conditions.
3.2. Colour
The colour of mushrooms after processing is strongly
in¯uenced by the activity of polyphenoloxidase. Table 3
shows the colour of fresh and evacuated mushrooms
after blanching or pressurisation. The least signi®cant
di€erence for the L-values is 8.8 (P < 0.05). The blan-
ched mushrooms have colour values that are only
slightly di€erent from the fresh ones. Evacuation with
water resulted in a signi®cant reduction of the lightness
of the mushrooms. When these evacuated mushrooms
were blanched, the lightness was signi®cantly lower than
the fresh mushrooms and the blanched fresh mush-
rooms. However, the colour is comparable with the
evacuated mushrooms. It can therefore be concluded
that blanching did not result in signi®cant changes in the
colour compared to the mushrooms prior to blanching.
When fresh mushrooms were pressurised, the light-
ness reduced signi®cantly compared to blanched. Also,
the a and b values increased, representing a brown col-
our. Pressure treatment of 600 or 800 MPa of non-
evacuated mushrooms resulted in a dark brown colour.
The highest pressure used, 950 MPa, gave a slightly
better colour, but still an intense brown. This browning
can be explained by several factors. First, as shown in
Table 2, pressurisation resulted in an increase of the
activity of polyphenoloxidase at 600 MPa. At higher
pressures there is a large reduction of activity, however
this did not result in less browning. This is probably
caused by an increase in the membrane permeability due
to the pressurisation. High isostatic pressure results in
crystallisation of phospholipids in cell membranes, re-
sulting in damage of the membrane. Due to this in-
creased permeability, the extracellularly located
polyphenoloxidase can react with the phenols and con-
sequently causes an increase in browning during pres-
sure treatment. If mushrooms are evacuated before
pressurisation, the browning is less intense compared to
non-evacuated mushrooms. During the evacuation
process, the air in the vacuoles is replaced by water re-
sulting in a large reduction in the concentration of ox-
ygen, which is necessary for the enzymatic reaction. In
industry, mushrooms are evacuated with water con-
taining citric acid and sodiumsulphite. Table 3 shows
that this resulted in a colour that is comparable with the
fresh blanched mushrooms. Compared to evacuation
with water, the latter process results in a lowering of the
pH. The low pH enhanced the inactivation rate at high
pressure and resulted in less browning.
3.3. Texture
The texture of the cap of the mushrooms is expressed
as the sti€ness or modulus, which is the slope of the
linear part of a stress strain curve. The results are shown
in Fig. 2 (blanching) and Fig. 3 (pressure treatment). We
also measured the stress at 40% compression (results not
shown). These results did not lead to other conclusions
than the sti€ness. Evacuation resulted in a small de-
crease in the sti€ness compared to fresh ones. Blanching
clearly reduced the sti€ness of the mushrooms. The
shortest blanching time, of 1 min, gave only a slight
reduction of the sti€ness. However, this blanching
treatment was insucient to inactivate polyphenoloxi-
dase as can be seen in Table 1. There are no signi®cant
di€erences between the texture of evacuated and non-
evacuated mushrooms after blanching. Pressure also
reduced the texture of the mushrooms compared to

Table 3
Colour of fresh and evacuated mushrooms after several treatments
expressed in L, a* and b* values. Values are means of 3±6 experiments

Treatment Fresh Evacuated

L a* b* L a* b*

None 90.7 0.8 14.5 67.5 )1.0 10.3

1 min blanching 88.2 1.2 13.7 69.7 )1.7 12.6
5 min blanching 79.7 )0.6 15.3 68.7 )0.7 17.3
10 min blanching 75.3 1.1 17.6 68.2 )0.8 16.6
600 MPa 53.0 15.5 29.2 58.0 10.5 25.8
800 MPa 50.1 14.3 24.4 63.1 6.8 21.7
950 MPa 55.7 9.4 23.7 62.3 5.2 18.0
600 MPaa 74.8 )0.1 18.8
800 MPaa 76.5 )2.2 12.4
950 MPaa 76.4 )1.4 16.6
Fig. 2. Sti€ness of evacuated and fresh mushrooms after blanching
a
Evacuation with citric acid and sodiumsulphite instead of water. expressed as a percentage of the sti€ness of fresh mushrooms.
 A.M. Matser et al. / Journal of Food Engineering 45 (2000) 11±16
Fig. 3. Sti€ness of evacuated and fresh mushrooms after pressure
treatment expressed as percentage of the sti€ness of fresh mushrooms.
fresh mushrooms. However, as can be concluded from a
comparison of Figs. 2 and 3, the pressurised mushrooms
have a higher sti€ness than the blanched mushrooms.
So, pressure resulted in less loss of texture compared to
blanching. The texture after cooking was not measured.
Stute, Eshtiagi, Bogusiawski and Knorr (1996) de-
scribed that the improved hardness of pressure-treated
vegetables could be due to enzymatic demethylation of
pectins followed by the formation of bridge bounds.
3.4. Yield
During heat treatment of mushrooms, water is lost
which results in changes of texture and has a large in-
¯uence on the yield of the process. We measured the
in¯uence of high pressure and blanching on the water
loss of mushrooms as shown in Tables 4 and 5. The
vacuum hydration during the evacuation procedure
gave a large increase in the weight of the mushrooms
due to the replacement of air by water. This additional
water was lost during the blanching of the mushrooms.
Only after 1 min blanching the weight of the mushrooms
was larger than the fresh weight. However, this
blanching time was insucient to completely inactivate
the polyphenoloxidase. Blanching times of 5 and 10 min
resulted in a water loss of roughly 20%. There is no
Table 4
Weight after evacuation, blanching or pressure treatment of mush-
rooms as a percentage of the weight of fresh mushrooms. Values in one
column are means of experiments with one batch of mushrooms
Treatment Fresh Evacuated
Experiment 1 Experiment 2
Evacuation 177.0 ‹ 5.1 153.3 ‹ 3.8
1 min blanching 94.6 ‹ 0.4 134.0 ‹ 3.7 127.1 ‹ 4.9
5 min blanching 79.3 ‹ 3.0 79.0 ‹ 3.3 84.2 ‹ 1.8
10 min blanching 81.6 ‹ 4.8 77.2 ‹ 1.6 75.0 ‹ 0.9
15
Table 5
Weight after evacuation and pressurization of mushrooms as a per-
centage of the weight of fresh mushrooms. Values in one column are
means of experiments with one batch of mushrooms
Treatment Fresh Evacuated
Experiment 1 Experiment 2
Evacuation 181.5 ‹ 3.8
600 MPa 67.0 ‹ 4.5 72.4 ‹ 3.0 86.3 ‹ 4.8
800 MPa 68.5 ‹ 2.8 71.3 ‹ 1.0 80.5 ‹ 2.4
950 MPa 80.5 ‹ 4.3 69.4 ‹ 0.0 88.7 ‹ 14.0
signi®cant in¯uence of the evacuation prior to blanch-
ing, on the resulting weight.
Pressurisation also resulted in a weight loss of
the mushrooms. Evacuation had a positive e€ect on the
retention of water after pressurisation. The weight of
the evacuated mushrooms was higher after pressurisa-
tion than that of the non-evacuated mushrooms. After
evacuation, a blanching or pressurisation procedure,
which is sucient for the inactivation of polypheno-
loxidase, resulted in the same water loss.
It must be noted that this water loss has an in¯uence
on the calculated polyphenoloxidase activity. This ac-
tivity is measured as the change in adsorbance per
minute per gram of fresh weight of the mushroom. Due
to the evacuation procedure the concentration of the
enzyme is diluted. If the activity is calculated per gram
of evacuated mushroom, the result is a much lower
concentration compared to fresh suggesting a part in-
activation but this decrease in concentration is only
caused by dilution. We therefore calculated the activity
with respect to the weight of the mushrooms before
evacuation. Due to blanching or pressurisation, the
weight of the mushrooms is decreased. We assume that
the concentration of enzyme in the leakage ¯uid is equal
to that of the mushroom tissue, therefore, calculations
were done with the weight of fresh mushroom.
4. Conclusion
The aim of this study was to evaluate the possibilities
for high pressure as an alternative to the conventional
blanching of mushrooms. An important condition for its
successful application is a sucient inactivation of the
enzyme polyphenoloxidase. These experiments showed
that polyphenoloxidase in mushrooms is a very stable
enzyme. Pressures up to 950 MPa are necessary for in-
activation and therefore, the e€ects on texture, colour
and yield have to be evaluated at this high pressure. If
fresh mushrooms are used, such a pressure treatment
results in a strong discoloured product. However, if
mushrooms are evacuated before pressure treatment, the
colour after pressurisation is comparable with the
16 A.M. Matser et al. / Journal of Food Engineering 45 (2000) 11±16
conventionally blanched mushrooms. The texture of
pressurised mushrooms is slightly better than that of
blanched mushrooms and the yield is roughly the same
as for conventionally processed mushrooms.
We concluded that pressure treatment has the same
net e€ect as blanching on the enzyme activity. Both
processes cause irreversible enzyme inactivation. The
pressures needed to achieve this enzyme inactivation
also lead to a slightly ®rmer product. The product yield
and colour were comparable to the heat blanched
products.
Acknowledgements
The authors wish to thank Stephanie Fourmantrouw
for performing part of the experiments and the ®nancial
support of the Dutch department of Economic A€airs.
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