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Extension of shelf life of chilled hake (Merluccius capensis) by high pressure/Prolongación de la

vida útil de merluza (Merluccius capensis) sometida a altas presiones conservada en refrigeración
J.L. Hurtado, P. Montero and A.J. Borderias
Food Science and Technology International 2000 6: 243
DOI: 10.1177/108201320000600307

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 Extension of shelf life of chilled hake (Merluccius capensis)
by high pressure

Prolongación de la vida útil de merluza (Merluccius capensis)

sometida a altas presiones conservada en refrigeración

J.L. Hurtado, P. Montero and A.J. Borderías

1*

Instituto del Frío (CSIC). Ciudad Universitaria s/n, Madrid 28040, Spain
Vacuum packed hake muscle subjected to 400 MPa (three 5-min cycles) at 7 °C proved to be more
stable chilled temperatures (2-3 °C), and sensorially acceptable, until 43 days of storage in com-
at

parison with the nine days for the non-pressurized hake. The lot pressurized at 400 MPa had the
appearance of cooked muscle. The microbial load was initially reduced by two log units by pressur-
ization at 400 MPa. Trimethylamine nitrogen (TMA-N) values were also very low. Pressurization had
no effect on dimethylamine nitrogen (DMA-N) production. The lot pressurized at 400 MPa showed
slower increase in drip loss from day 15 of storage. In the lot subjected to 200 MPa (three 5-min
cycles) at 7 °C, which retained the appearance of raw fish, microorganisms were one log unit lower
than in the non-pressurized sample and TMA-N was lower. In conclusion, the chilled shelf life was
prolonged by about one week in the lot pressurized at 200 MPa and about two weeks in the lot
pressurized at 400 MPa.
Keywords: hake, shelf life, chilling, high pressure, storage, vacuum packaging
La presurizaci6n increment6 notablemente la vida util del musculo de merluza almacenado en
refrigeraci6n (2-3 °C). El lote presurizado a 400 MPa (tres ciclos de cinco minutos) a 7°C result6 ser el
-

mas estable a temperaturas de refrigeraci6n, y aceptable desde el punto de vista sensorial hasta los 43
dias de almacenamiento, tiempo muy superior a los 9 dias en los que se descart6 el lote sin presurizar.
El lote presurizado tenia la apariencia de musculo cocido. La carga microbiana (ufc/g) se redujo
inicialmente en dos unidades logaritmicas con el tratamiento a 400 Mpa, asimismo se obtuvieron valores
muy bajos en nitr6geno de trimetilamina (N-TMA). La presurizad6n no tuvo ningun efecto sobre la
producci6n de nitr6geno de dimetilamina (N-DMA). El lote presurizado a 400 MPa mostr6 unos valores
de exudado por debajo de los demas lotes a partir de los 15 dias de conservaci6n. El lote presurizado a
200 MPa (tres ciclos de cinco minutos) a 7 °C, que conserv6 una apariencia de pescado crudo, present6
una reducci6n de microorganismos de una unidad logaritmica y una menor cantidad en N-TMA en
relaci6n con el lote testigo sin presurizar. En conclusi6n, los lotes presurizados a 200 MPa y 400 MPa
mostraron un incremento de la vida util de aproximadamente una y dos semanas, respectivamente.

Palabras clave: merluza, vida util, refrigeraci6n, altas presiones, almacenamiento, envasado a vacio

INTRODUCTION and has limited commercial shelf life. This practice is

a

common for most of the hake consumed in Spain, which

Countries such as Spain with fish-rich diets frequently
capture fish far from the coast. This means that when
the fish arrives it has been kept for several days in ice,
(e-mail: jborderias@if.csic.es).
is usually caught off on the Saharan Bank, the south
coast of Ireland, Chile or Namibia, and therefore chilled.
To make fresh commerce of such products viable, it is
essential to prolong their shelf life by one or two weeks.
The use of high pressure to prolong the average life
of chilled products appears to be a suitable method for
various kinds of foods, but not all experimental results
have thus far been transferred to industry. At over 200
MPa, raw fish muscle acquires a whitish hue similar to
that produced by cooking, and the effect increases as
pressure is raised (Ohshima et al., 1993). However, on

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244
the basis of experiments on other products, preliminary
trials have been carried out using several cycles of high
pressure, as this seems more effectively to reduce flora
than does continuous treatment (Hayakawa ct al., 1994,
Aleman et al., 1996, Hurtado ct al., 1998).
In the case of chilled storage of pressurized fish, the
main problems are microbiological and enzymatic. Con-
cerning the microbiological aspect, Shoji and Shaeki
(1989) experimented with tuna and squid muscle to
evaluate the reduction in microorganisms after pressur-
izing at 200 and 450 MPa for 15 min. With respect to
enzymatic deterioration, high pressure has been shown
to reduce the proteolytic activity of certain enzymes
(Ashie and Simpson, 1996; Ashie et al., 1996, 1997). How-
ever, to our knowledge no work has been done on the
effect of enzymatic DMA production in chilled vacuum
packed products, which is important considering that
vacuum packing itself causes an increase in DMA pro-
duction during chilled storage (Lundstrom et al.,1981 ).
Also, evolution of TMA has not been studied in
pres-
surized samples.
The aim of this work
was tostudy cyclic high pres-
sure treatment of vacuum packed hake muscle and sub-
sequent chilled storage (2-3 °C), with a view to obtain-
ing two types of products, one having a raw appearance
(pressurized at 200 MPa, 7 °C) which can be commer-
cialized as raw fish, and the other having a cooked ap-
pearance (pressurized at 400 MPa, 7 °C), suitable for
commercialization
as pre-cooked products.
MATERIAL AND METHODS
Raw material and high pressure treatments
The experiment was performed with hake (Merliicciiis
capensis) caught off the coast of Namibia and stored in ice
for five days after gutting, the time it took to reach the
laboratory. At the laboratory the fishes were headed and
washed, then cut into slices which were vacuum packed
in flexible bags (WIPAK/GRYSPEERT, Winnipeg,
Manitoba, Canada PAE 110K FP, oxygen permeability 3
cm3 / m2 / 24 h at 4 °C / 80% RH, and maximum cooking
temperature 115 °C) ready for high pressure treatment
and storage at chilled temperature (2.0-3.0 °C). Two dif-
ferent packs with approximately 300-350 g of muscle in
each bag were pressurized simultaneously.
Pressure was applied by a hydrostatic pressure unit
(Gec Alsthom ACB 900 HP, Type ACIP model 665,
Nantes, France). Pressures of 200 MPa (lot P1 ) and 400
MPa (lot P2) were applied in three 5-min cycles at 30 s
intervals at a temperature of 7 °C, as measured by a
thermocouple inside the vessel when the desired pres-
sure was reached. Following pressurization (not includ-
ing control, lot T), all lots were kept in a cold store in
their separate packs for the entire storage period. Peri-
odic analyses were performed during this time period.
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Analysis .
Microbiological a1Zalysis
Samples for microbiological analysis were prepared by
homogenizing 10 g of hake muscle with 90 mL of pep-
tone water (Adsa-Micro, Spain) in a Stomacher 400 ho-
mogenizer (Seward, UK); a series of decimal dilutions
were then made. Total viable counts were
performed by
pour plating on plate count agar (PCA, Oxoid, UK) and
incubating at 30 °C for three days. Counts were expressed
as the log of colony forming units per gram (cfu/g).
Samples for analysis were taken from two different packs
with approximately 300-350 g of muscle in each bag.
TMA-N and DMA-N determination
Trimethylamine nitrogen (TMA-N) and dimethylamine
nitrogen (DMA-N) were determined according to Dyer
(1945) as modified by Tozawa et al. (1971), and Dyer
and Mounsey (1945), respectively. Analyses were per-
formed in duplicate from two different packs.
Color measurements
Color measured in a colorimeter (MiniScan Ms / s-
was
4000S, HunterlaB, Hunter Associates Laboratory Inc.,
Reston, VA, USA) using a CIELab scale. The whiteness
index used was recommended by NFI (1991): whiteness
= 100-[(100-L~+~’+~]~. The viewing area of the
instrument was 8 mm and the sample size diameter is
5,5 mm. Analyses were performed in duplicate from two
different packs.
Sensorial analysis
Sensorial characteristics, general appearance, odor, and
flavor, were analyzed in samples cooked in boiling water
for 5 min in the same storage packaging. Sensorial analy-
sis was performed by a panel of five semi-trained tasters,
chosen from laboratory staff after a training sesion, using
a non-structured scale running from 10 (ideal) to 0 (poor-
est quality). To appreciate the term ’ideal’ and ’poor qual-
ity’, very fresh and unfresh fish were tested by the panel
at the same time as the samples. Lots scoring less than 4
for any sensory parameter were rejected.
Drip and cooking loss measurements
Drip loss and cooking loss were measured throughout
the storage period. Drip loss was measured as the dif-
ference between muscle weight at the outset of storage
and at the time of analysis after draining off the released
liquid. Cooking loss was measured after placing samples
in boiling water for 5 min. Cooking loss was calculated
as the difference between muscle weight before and af-
ter heat treatment following removal of exudate once
 245

the samples had cooled to room temperature. Both types
of loss were expressed as percentages. Analyses were
performed in duplicate from different packs.
Statistical alT~ljl/SIS -
Analysis of variance and regression analysis were per-
formed using an SAS program (SAS Institute Inc, Cary,
NC, USA)..
RESULTS AND DISCUSSION
Experiments at different pressures produced two types
of product: one having a cooked appearance following
pressurizing at 400 MPa (lot P2), and another having a
fresh appearance after pressurizing at 200 MPa (lot P1 ).
As noted by Ohshima et al. (1993) and by Ashie and
Simpson (1996) high pressure can induce modifications
in the appearance of fish muscle, which generally be-
comes whiter the higher the pressure. The mechanism
involved in color change is, however, still unclear even
though pressure-induced denaturation of heme com-
pounds like metmyoglobin have been shown to cause
spectral shifts in such compounds (Zipp and Kauzmann,
Immediately following pressurization at 200 and 400
MPa, the high initial microbial load of samples on re-
ception log units (cfu / g)) was 1 and 2 log units lower,
(6
respectively (Figure 1). Pressure-dependent reduction
of microorganisms has been described by other authors,
who found that inactivation increased with pressure
(Cheftel, 1995). The evolution of counts was similar in
all three lots up to 12 days of storage, but the lot pres-
surized at 400 MPa presented significantly lower val-
ues than the others, except
at day 12. All lots exhibited
latency from day 0 to day of storage probably due to
5
high pressure and vacuum, thereafter the microbial load
began to increase. The control lot (unpressurized) pre-
sented counts of over 101 cfu / g on day 9 of storage,
when it was rejected by the sensory panel. Considering
that at day 0 of storage, the hake had been five days in
ice between capture and arrival at the laboratory, these
values are very similar to those of Sumner and Gorczyca
(1981) in Merluccius capel1sis at day 14 of chilled stor-
age. When lots PI and P2 were rejected by the panel,
the counts were over 8 log units (cfu / g). According to
Olafsd6ttir et al. (1997), total aerobic microorganism
counts in the range 10’-108 cfu / g are typical for fish
products at the time of rejection. Possible hazards of the
vacuum packed products are: (i) the growth of patho-

1973; Gibson and Carey, 1977).
Immediately after pressurization both pressurized
and control lots had similar scores in general appear-
ance, odor and flavor (Table 1). During chilled storage
the pressurized lots (PI and P2) scored higher than the
control in all sensory parameters. The control lot was
rejected after nine days of storage due to low scoring in
all three parameters, whereas the pressurized lots (PI
and P2) were respectively rejected between 15 and 22,
or 36 and 43 days.
genic vegetative forms such as Clostridium botulinum,
chiefly those non-proteolytic strains (B or E) that can
produce botulin toxin without fish spoiling (Phillips,
1996); and (ii) the growth of pathogenic microorganisms
like Salmonella spp. and Listeria monocytogel1es (Anony-
mous, 1991). Proper processing will minimize these
risks, and at a storage temperature below 3 °C, as in the
present study, it is essential to prevent the growth of
Clostridium botuli1Zum (Cann, 1988; Baldratti et al., 1989;
Gibbs et al., 1994; Garren ef nl., 1995; Reddy et al., 1995)

Table 1. Sensory analysis of hake during chilled storage. T: Control. P1: Pressurized lot at 200 MPa in three 5-minute
cycles at 7 °C. P2: Pressurized lot at 400 MPa in three 5-minute cycles at 7 °C.
Tabla 1. An~lisis sensorial durante el almacenamiento de la merluza en refrigeraci6n. T: Lote control. P1: Lote
presurizado a 200 MPa en tres ciclos de 5 minutos cada una a 7 °C. P2: Lote presurizado a 400 MPa en tres ciclos
de 5 minutos cada uno a 7°C.

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246
and pathogenic microorganisms (Phillips, 1996). A num-
ber of papers has shown that pressurization at 400 MPa
effectively reduces pathogenic microorganisms
(Shigehisa et al., 1991; Styles et al., 1991; Carlez et al.,
1993, 1994; Gola et al., 1996; Simpson and Gilmour, 1997;
Smelt, 1998). Other studies have shown that the micro-
bial flora (Gram positive and Gram negative bacteria)
changes radically with the application of pressure
(Hurtado et al., 1998), and the counts of H,S producers,
lactic bacteria, Bacillus tlzermosplzacta and coliforms are
reduced, sometimes by more than five log units, by cy-
clic pressurization at 400 MPa, 7 °C (Lopez-Caballero et
al., 1999).
Initial TMA-N levels in all lots were 0.4 ± 0.08 mg /
100 g, very close to the values reported by Ruiz-Capillas
(1997) in hake (Merluccills merluccius), despite the high
initial microbial load. TMA-N increased considerably
(Figure 2) in the control from storage day 5; by day 9 it
had increased to 29 mg/ 100 g, at which point it was
rejected by the tasting panel. This figure contrasts with
the findings of other authors (Sumner and Gorczyca,
1981), who reported 4.3 mg TMA-N / 100 g on the last
day for acceptability (day 14) of vacuum packed hake
(M. capensis) during chilled storage (4-6 °C). Dalgaard
et al. (1993) reported higher values (30 mg / 100 g), closer
to those found in the present study, in vacuum packed
cod fillets stored at 0 °C for 14 days, while 50 mg / 100 g
was detected in the same product after 12 days in stor-
age at 2 °C (Jensen et al., 1980). According to Dalgaard
et al. (1993), such a large increase in TMA-N values in
the control could be due to selection of Gram negative
microorganisms (the main reducers of trimethylamine
oxide (TMAO) to TMA in anaerobic conditions), which
Figure 1. Total viable counts during chilled storage of
hake. (*) Control. (0) Pressurized batch at 200 MPa, 7
°C (P1 ). (A) Pressurized batch at 400 MPa, 7 °C (P2).
Figura 1. Microorganismos aerobios totales durante la
conservaci6n de la merluza en refrigeraci6n. (*) Control.
(M) Lote sometido a 200 MPa a 7 °C (P1); (A) Lote
sometido a 400 MPa a 7 °C (P2).
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Figure 2. Development of trimethylamine (TMA-N) in
hake during chilled storage. (*) Control. (0) Pressurized
batch at 200 MPa, 7 °C (P1 ). (A) Pressurized batch at
400 MPa, 7 °C (P2).
Figura 2. Desarrollo de la trimetilamina (N-TMA) durante
la conservaci6n de la merluza en refrigeraci6n. (*)
Control. (0) Lote sometido a 200 MPa a 7 °C (P1 ). (A)
Lote sometido a 400 MPa a 7°C (P2).
increased in number in vacuum packed samples dur-
ing chilled storage. In the pressurized lots the increase
in TMA was significantly smaller ~7:!~ 0.05). Evolution
was similar in lots PI and P2 up to storage day 9, there-
after TMA-N increased faster in PI than in P2 ~7!~ 0.05),
probably because of a higher concentration of microor-
ganisms able to convert TMAO to TMA. TMA-N
evolved more slowly the higher the pressure. This may
have been due to a pressure-induced decrease in the
load of Gram negative TMA-N producers, as has been
reported by other authors (Knorr, 1994; Cheftel, 1995).
Initial DMA-N (0.6 ± 0.07 mg / 100 g) was very close
to the initial values reported by GonzAlez Sotelo (1991)
for muscle of Merlllccills mer~licccins (0.7 mg/100 g) and
slightly lower than those found by Almadoset al. (1984):
1.1 mg /100 g of muscle in fillets of Merlllccills hubbsi.
DMA-N increased progressively (Figure 3) in all three
lots throughout chilled storage, probably favoured by
vacuum packaging. According to Lundstrom et al.
(1981), low oxygen availability caused an increase in
degradation of TMAO to DMA, reaching as much as 47
mg DMA-N / 100 g in vacuum packed fillets (Llrophysis
clmss) after seven days of storage in ice. The enzyme
that catalyzes the conversion of TMAO to DMA and FA
is TMAOase, which is located in membranes, most prob-
ably subcellular membranes. However, the investiga-
tions carried out did not clarify which subcellular or-
ganelle hosted TMAOase activity (Gill and Paulson,
1982, Joly et al., 1992). The high levels of DMA in lot P2,
besides being due to the effect of vacuum, could have
been related to a breach in these subcellular membranes
which would facilitate contact between enzyme and
substrate. Ohmori et al. (1991 ) found that pressures over
200 MPa produced a lysosomal rupture, which could

Figure 3. Development of dimethylamine (DMA-N) in
hake during chilled storage. (*) Control. (0) Pressurized
batch at 200 MPa, 7 °C (P1 ). (A) Pressurized batch at
400 MPa, 7 °C (P2).
Figura 3. Desarrollo de la dimetilamina (N-DMA) durante
la conservaci6n de la merluza en refrigeraci6n. (*)
Control. (0) Lote sometido a 200 MPa a 7 °C (P1 ). (A)
Lote sometido a 400 MPa a 7 °C (P2).
also facilitate such contact. Other studies also reveal the
possible activation of membrane-associated enzymes on
pressurization due to stabilizing of the lipid-protein
bond, although in most cases irreversible deactivation
occurs (Heremans, 1992; Kavecansky et nl.,1992).
Drip loss increased in all lots during chilled storage,
the increase was significantly (p <- 0.05) slower in the lot
pressurized at 400 MPa from day 15 of storage. There
were no significant differences in evolution between the
control and the lot pressurized at 200 MPa. The three
&dquo;&dquo;
Figure 4. Changes of drip-loss in hake during chilled
storage. (*) Control. (M) Pressurized batch at 200 MPa,
7 °C (P1 ). (A) Pressurized batch at 400 MPa, 7 °C (P2).
Figura 4. Evoluci6n del exudado durante la
conservaci6n de la merluza en refrigeraci6n. (*) Control.
(0) Lote sometido a 200 MPa a 7 °C (P1 ). (A) Lote
sometido a 400 MPa a 7 °C (P2).
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247
Figure 5. Changes of cooking-loss in hake during chilled
storage. (*) Control. (0) Pressurized batch at 200 MPa,
7 °C (P1 ). (A) Pressurized batch at 400 MPa, 7 °C (P2).
Figura 5. Evoluci6n del exudado a la cocci6n durante
la conservaci6n de la merluza en refrigeraci6n. (*)
Control. (M) Lote sometido a 200 MPa a 7 °C (P1 ). (A)
Lote sometido a 400 MPa a 7 °C (P2).
lots (including lot P2) exhibited a sharp increase of drip
loss as from storage day 5 (Figure 4). Values were high
in all lots, but this may have been due to vacuum pack-
ing (Cann ct al., 1983). Yoshioka and Yamamoto (1998)
reported that in pressurized carp fillets (100-500 MPa /
10 min/20-22 °C) there was drip loss in the range of 1-
2%, lower than in heated fillets. Other authors have re-
ported that high pressure reduced drip loss (Murakami
et al., 1992; Cheftel and Culioli, 1997). The extent and
variability of high pressure effects on proteins depends
on the pressure level. In general, reversible effects are
observed below 100-200 MPa, while irreversible effects
occur above 200 MPa (Balny and Masson, 1993). There-
fore, the lot that was pressurized at 400 MPa probably
evolved differently during chilled storage.
The evolution of cooking loss was similar in all lots
for the first 15 days of storage; thereafter, loss was high-
est in lot P2 (Figure 5). Taking drip loss and cooking
loss together, total exudate loss was similar in all lots.
Initial whiteness increased slightly in the control lot
during chilled storage (Figure 6). This is unlike the find-
ings of Gerdes and Santos-Valdez (1991), who reported
a decrease of whiteness in fish fillets during storage.
High pressure causes whiteness to increase in propor-
tion to the increase in pressure (Ohshima et al., 1993),
although in the present case the variation of whiteness
was smaller between lot PI and P2. Unlike the control,
whiteness values decreased in the pressurized lots dur-
ing storage. Although the whiteness of lot PI was more
like that of lot P2 than the control throughout chilled
storage, from a sensory standpoint lots C and PI looked
raw while P2 looked cooked.
In summary, pressurization prolonged the shelf life
of chilled hake, whether pressurized at 200 MPa and
retaining the appearance of fresh fish, or pressurized at
248
Figure 6. Changes of whiteness during chilled storage.
(*) Control. (0) Pressurized batch at 200 MPa, 7 °C
(P1 ). (A) Pressurized batch at 400 MPa, 7 °C (P2).
Figura 6. Evoluci6n de la blancura durante la
conservaci6n de la merluza en refrigeraci6n. (*) Control.
(M) Lote sometido a 200 MPa a 7 °C (P1 ). (A) Lote
sometido a 400 MPa a 7 °C (P2).
400 MPa and acquiring the appearance of cooked fish.
Pressurization at 400 MPa produced lower microbial
counts, TMA-N and drip loss throughout storage, al-
though there was a pronounced increase in DMA-N
values during this time. Although this lot had higher
microbial counts than the control at the time of rejec-
tion, the microbiological hazard presented by the
vacuum pack was minimized by the effect of high pres-
sure treatment, storage conditions and subsequent heat-
ing for consumption. From a practical point of view this
technology could be applied to high value fish like hake,
where the logistics of transport mean that the fish takes
several days to reach its destination, by which time the
microbial load is relatively high. Pressurization would
thus prolong its shelf life.
ACKNOWLEDGEMENTS
This paper was financed by Madrid region project 06G /
053 / 96 and CICyT project ALI97-0759.
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