Lebensm.-Wiss. u.-Technol.

, 28, 528–538 (1995)

Changes in Colour and Myoglobin of Minced Beef Meat

Due to High Pressure Processing
Anne Carlez, Teresa Veciana-Nogues and Jean-Claude Cheftel*

A. Carlez, T. Veciana-Nogues, J.-C. Cheftel: Unité de Biochimie et Technologie Alimentaires, Centre de Génie
Biologique et Sciences des Aliments, Université de Montpellier II, F-34095 Montpellier Cedex 5 (France)

(Received October 4, 1994; accepted March 1, 1995)

Minced beef meat was packaged under vacuum, air or oxygen, and pressurized at 10 °C for 10 min. L* colour values increased
significantly in the range 200–350 MPa, the meat becoming pink, while a* values decreased at 400–500 MPa, the meat becoming
grey-brown. Simultaneously, total extractible myoglobin decreased in the range 200–500 MPa, while the proportion of
metmyoglobin increased at the expense of oxymyoglobin at 400–500 MPa. Pressurization did not significantly increase the
extractibility of heme iron by an acid solution. Packaging of meat under vacuum with an oxygen scavenger partly protected meat
colour, since samples processed at 400 MPa became pink, without any change in a* value or metmyoglobin content. Blending
chilled minced meat with NaNO2 (and NaCl) 18 h before processing at 350–500 MPa afforded a similar protection. Cysteine,
ascorbic acid, nicotinamide or nicotinic acid had no protective effects. Meat discoloration through pressure processing may result
from (1) a ‘whitening effect’ in the range 200–350 MPa, due to globin denaturation and/or to heme displacement or release, and
(2) oxidation of ferrous myoglobin to ferric metmyoglobin, at or above 400 MPa. Only the latter phenomenon is prevented by
total oxygen removal or prior formation of nitrosomyoglobin.

©1995 Academic Press Limited

Introduction to ferric oxidation, while the addition of nitrites

The importance of meat colour in consumer acceptance
is well known. The intensity of muscle visual colour is
due to its total myoglobin content. Different colours
observed in meat are largely related to the relative
proportions of oxymyoglobin (bright red), myoglobin
(dark red), and metmyoglobin (grey-brown) (1, 2).
The stability of meat colour is determined by the
maintenance of myoglobin in its ferrous oxygenated
form. Oxidation of myoglobin to ferric (Fe3 + ) metmyo-
globin is undesirable and takes place progressively
during the refrigerated storage of fresh meat. Factors
that increase the rate of myoglobin oxidation and of
meat discolouration include the partial pressure of
oxygen, the rate of oxygen consumption by tissues, the
concentration of multivalent metal ions, the tem-
perature, the light, the pH and the meat microflora (2,
3).
Mincing exposes more meat surface to air and to
microbial contaminants, decreases the meat content of
endogenous intracellular reducing agents, and leads to
faster colour changes (4). Various industrial processes,
applied to prepare meat products with a longer shelf-
life than fresh meat, produce irreversible changes in
myoglobin structure and in colour. For instance, ther-
mal processing denatures globin and enhances ferrous
*To whom correspondence should be addressed, present address:
Unitat de Nutriciò i Bromatologia, Facultat de Farmàcia, Universitat
de Barcelona, E-08028 Barcelona, Spain.
0023-6438/95/050528 + 11 $12.00/0
stabilizes the ferrous form and the red colour through
reaction with the sixth coordinate position of the iron
atom.
The behaviour of myoglobin under pressure has not
been extensively studied. Ooi (5) observed no myoglo-
bin denaturation before 500 MPa at 10 °C, or before
570 MPa at 20 °C. In contrast, Taniguchi and Takeda
(6), using FT-IR observed an extensive denaturation of
myoglobin at 350 MPa and 30 °C with a conversion of
secondary α-helix and ß-sheet structures into a random
coil conformation. Similar observations were made by
Zipp et al. (7) and Zipp and Kauzmann (8) by
spectrophotometry in the visible and the Soret bands.
They did not find any modification of metmyoglobin in
solution at 20 °C below 225 MPa. At higher pressures
(250–500 MPa), the protein went through a transition
phase where spectrum modifications were observed:
the peak close to 620 nm progressively disappeared,
while another peak appeared at about 540 nm; the
spectrum in the Soret region shifted to higher wave-
lengths. At each pressure level, a different equilibrium
spectrum was reached. At 550 MPa, the denaturation
was rapid, complete and no further changes were seen
at higher pressures. The spectrum suggested that the
ligand in the 6th coordination position (water for
metmyoglobin) was replaced under pressure by the
imidazole group of a histidine residue (8). According to
these investigations, denaturation of metmyoglobin
under pressure was similar to denaturation by heat,
©1995 Academic Press Limited

528

acid or urea, and was partly due to the rupture of
hydrophobic interactions. Pressure denaturation varied
as a function of temperature and pH. At pH 6,
complete denaturation was reached at 450 MPa and 20
°C, or at 350 MPa and 10 °C. Jonas and Jonas (9) in
their recent general review, reported that around
100–200 MPa, near pH 7, and 20 °C, subtle structural
modifications were observed, such as compression of
the heme cavity and reorientation of the heme.
The previous results were obtained from protein
solutions, and cannot easily be extrapolated to more
complex meat systems. Shigehisa et al. (10) observed a
decrease in the L* and a* colour values of pork muscle
slurries pressurized from 100 to 600 MPa. A similar
decrease in a* value was noted in beef meat by Nose et
al. (11). Carlez et al. (12, 13) reported that the colour of
minced beef meat changed markedly when the latter
was processed at high pressure, especially above 300
MPa. Murakami et al. (14) reported a marked increase
in the proportion of metmyoglobin in red tuna meat
pressurized at 300 MPa. Pressurization at or above 400
MPa was found to inactivate microorganisms, and to
extend refrigerated shelf-life, and also to induce gelat-
ion of myofibrillar proteins. High pressure processing
would be useful for meat preservation and restructur-
ing, provided that the red colour could be maintained.
The objectives of the present study were to investigate
the effects of high pressure processing of minced meat
from lean beef muscle in terms of meat colour and
contents of myoglobin, oxymyoglobin and metmyoglo-
bin. The influence of various parameters, such as
pressure level, packaging atmosphere, duration of
pressure application, mincing, addition of different
antioxidants or stabilizers, and chilled storage after
pressure processing, was also assessed.
Materials and Methods
Preparation of meat samples
Lean beef muscle (Semimembranosus) was bought
from a single local supermarket. The time post-mortem
was not known. The pH was in the range 5.57 ± 0.06.
For each series of experiments, about 1 kg of beef
muscle was trimmed with a stainless steel knife. The
meat was minced with a food mixer (Moulinette ‘S’,
Caen, France) at room temperature for 20 s to a particle
size of 3–5 mm. Thirty gram portions were packaged in
aluminium-polyester pouches (approximately 120 3
160 mm, Bernhardt, Boulogne-sur-Mer). In most cases,
pouches contained about 100 mL air before heat
sealing. In other cases, vacuum was applied to the
pouches, with or without subsequent introduction of
about 100 mL of pure O2 (Aligal 3, O2 > 99.5 %, l’Air
Liquide, Paris La Défense) at a pressure slightly below
atmospheric pressure, before heat-sealing. For one
series of experiments, an O2 scavenger (Ageless FX
100, Mitsubishi France S.A., Paris) was introduced into
529
lwt/vol. 28 (1995) No. 5
the pouch just before vacuum application and heat-
sealing.
Samples were pressure-processed immediately after
packaging, unless otherwise stated. For experiments
under different gas atmospheres, and for chilled storage
experiments (under O2), samples were kept at 5 °C for
18 h between packaging and pressure processing.
For experiments at different meat depths, a piece of
about 500 g of Semimembranosus was cut into cubes of
30–40 mm3. The cubes were packaged in air, before
pressurization, as described above. After pressuriza-
tion, the cubes were cut into half-cubes and exposed to
air for 5–10 min at room temperature before their L* a*
b* values were determined.
For some experiments, a given amount of one of the
following compounds (mainly antioxidants) was dis-
solved in 5 to 10 mL of deionized water and mixed with
100 g of minced meat using a Stomacher 400 blender
(Prolabo, Paris, France) for 3 3 30 s at room
temperature. Nicotinic acid and nicotinamide were
from Sigma (St Louis, MO, U.S.A.) while ascorbic acid,
cysteine, NaCl and sodium nitrite were from Merck
(Darmstadt, Germany). Before pressure processing,
the mixes were kept at 5 °C for 18 h (in air for nicotinic
acid or nicotinamide, or under vacuum for nitrites +
NaCl). The mixes containing ascorbic acid or cysteine
were kept at 5 °C in air for 1 h only.
High pressure processing
Pressure processing was carried out by placing the
meat-containing pouches in water in the 1 L hydrostatic
pressure unit from ACB (Ateliers et Chantiers de
Bretagne, Nantes, France), as previously described (12,
13). The rate of pressurization was close to 120 MPa/
min and the rate of pressure release was close to 400
MPa/ min. The temperature of pressure-transmitting
water was 10 °C initially, increased to 15 °C as the
maximum pressure was reached, and equilibrated to 10
°C within about 2 min. Samples were processed at a
constant pressure (set between 100 and 500 MPa) for 2
to 30 min, usually 10 min. After pressure release,
samples were kept at 5 °C for 1–2 h in the pouches
before analysis. For chilled storage experiments, meat
samples were prepared as above, packaged in pouches
under O2, kept at 5 °C for 18 h, pressurized and then
stored in the closed pouches at 5 °C for 12 d.
Experiments with minced meat packaged under O2, air
or vacuum (without O2 scavenger) were carried out five
times at each pressure level tested (control, 350, 400,
450 and 500 MPa). The experiments under vacuum with
O2 scavenger were carried out three times in the same
conditions.
In all cases, colour measurements on pressurized meat
samples were carried out within 10 min of pouch
opening, in order to prevent drying of the meat surface
and the resulting changes in colour.
lwt/vol. 28 (1995) No. 5
Determination of total myoglobin and of related
pigments
The determination of the total myoglobin content, and
of the proportions of oxymyoglobin, myoglobin, and
metmyoglobin in meat, is based on the different
absorbance spectra of these three molecules when in
solution. Absorbance measurements are carried out
after pigment extraction from meat samples. This
method measures the heme pigment content of the
total meat sample, in contrast to reflectance methods
which indicate the pigment content at the meat
surface.
Two grams of minced meat samples were homogenized
with 20 mL of 0.04 mol/L Na/K phosphate buffer, pH
6.8 (as described in 15), using an Ultraturrax T25
homogenizer (Janke & Kunkel, Staufen, Germany) at
10,800 rpm and room temperature for 20 s. After
standing for 1 h in an iced bath, the homogenate was
centrifuged at 10,000 3 g and 10–15 °C for 30 min. The
supernatant was filtered through Whatman no. 1 paper
and the volume was brought up to 25 mL with the same
phosphate buffer. Turbidity was avoided maintaining a
low temperature, and, if necessary, by additional
filtration on a 0.25 µm Millipore filter before spec-
trophotometry. Absorbance of the clear supernatant
was measured at 525, 545, 565 and 572 nm on a Unicam
UV2 spectrophotometer (Cambridge, U.K.). The total
myoglobin content and the proportion of myoglobin,
oxymyoglobin and metmyoglobin were calculated
according to Krzywicki (16), using the following
equations:
A525
Total myoglobin (mmol/L) = Eqn [1]
MAC525
% deoxyMb = (0.369 R1 + 1.140 R2 – 0.941 R3 + 0.015)
3 100
% MbO2 = (0.882 R1 – 1.267 R2 + 0.809 R3 – 0.361) 3
100
% MetMb = (–2.541 R1 + 0.777 R2 + 0.800 R3 + 1.098)
3 100
Where MAC525 ( = 7.6) is the millimolar absorbance
coefficient of myoglobin at the isobestic point (525 nm)
of its three forms, and R1, R2, R3 are respectively the
absorbance ratio A572/A525, A565/A525, A545/A525.
Krzywicki (16) proposed another equation for total
myoglobin, based on the added absorbance of the three
myoglobin forms:
Total myoglobin (mM/L) = –0.166 A572 + 0.086 A565 +
0.088 A545 + 0.099 A525 Eqn [2]
Because this equation is based on measurements at
four different wavelengths (rather than only at 525 nm),
it minimizes errors due to absorbance coefficients. It
was therefore utilized in the present study. It was also
checked that the (A525/ Mb tot) ratio was close to 7.6.
530
Colour analysis
Control or pressurized minced meat samples were
slightly compressed and flattened with a Petri dish. The
L*, a*, b* colour values were determined using a
Minolta Chromameter II (Minolta, Tokyo, Japan) in the
1976 CIELAB system. Measurements were done in
relation to ‘C illuminant’, and the light reached the
meat surface through a polyvinylstyrene Petri dish of 1
mm thickness. The Chromameter was calibrated before
each series of measurements using a white ceramic
plate. It was checked that a* and b* values of the
ceramic plate were not modified by all Petri dishes
used, while the L* value was systematically reduced by
1.5 unit. Each measurement was repeated at least six
times.
pH measurements
Four grams of control or pressurized minced meat
sample were blended with 20 mL of deionized and
microfiltered water for 10 minutes using a magnetic
stirrer. The pH of the resultant suspension was meas-
ured with a glass electrode on a Metrohm 632 pH meter
(Metrohm S.A., Herisau, Switzerland) pH-meter.
Measurements were always carried out in triplicate.
Determination of nonheme iron
Nonheme iron was extracted according to Schricker et
al. (17). Three grams of control or pressurized minced
meat sample were mixed with 15 mL of a solution 3
mol/L in HCl and 1.22 mol/L in trichloracetic acid. The
samples were then incubated at 65 °C for 20 h in glass
tubes closed with glass marbles. The iron content of the
clear supernatants was determined by IC PAES (induc-
tively coupled plasma atomic emission spectrometry,
C.E.A, C.E. Saclay, Gif sur Yvette, France). The
standard deviation of the method is close to 2%.
Measurements were carried out in duplicate on the
same meat extract.
Results and Discussion
Variability of the myoglobin content and colour of
meat samples
The variability of the determination of myoglobin and
related pigments was first assessed using nonpressur-
ized minced meat from Semimembranosus muscle of
one animal (Table 1). The Relative Standard Deviation
(RSD) of 2.48% for total myoglobin is quite acceptable.
The very high RSD values for metmyoglobin and for
myoglobin are partly explained by the small propor-
tions of these two pigments (in the same muscle
sample).
The variability of L* a* b* colour parameters was
determined at different surface positions of a sample
 lwt/vol. 28 (1995) No. 5

Table 1 Mean values (x̄), standard deviations (sx) and relative standard deviation (RSD) of myoglobin contents and colour
values of minced meat from a single bovine Semimembranosus muscle
Total Proportions of the three myoglobin formsb Colour valuesc
myoglobina
(mg/g) Mb (%) MbO2 (%) MetMb (%) L* a* b*
x̄ 8.88 13.2 70.0 8.0 38.5 18.6 10.3
sx 0.22 3.2 3.6 3.2 0.6 1.2 0.5
RSD (%) 2.48 24.2 5.1 40.0 1.6 6.4 4.8
a Total myoglobin in a pH 6.8 meat extract as determined according to Krzywicki (16). Mean of eight measurements on
different meat extracts from a single muscle sample.
b Relative proportions of myoglobin, oxymyoglobin and metmyoglobin in a pH 6.8 meat extract as determined according to
Krzywicki (16). Mean of eight measurements on different meat extracts from the same muscle sample as for total myoglobin
determination.
c Colour values determined by reflectance using a Minolta chromameter. Mean of eight measurements on minced meat from a
single muscle sample (different from that used for myoglobin determination).

(20 g) of minced meat from another single Semi-
membranosus muscle (Table 1). The maximum RSD
(6.4%) was observed for a* values.
Determinations were also carried out on minced meat
samples from 21 distinct purchases (Semimembranosus
muscle). The total myoglobin content of the meat
samples ranged from 3.7 to 7.8 mg/g, with a mean value
of 5.8 ± 1.4. Oxymyoglobin was the main myoglobin
form (73.9% ± 9.0), as can be expected for meat
minced in the presence of air (24). The proportions of
myoglobin (9.6% ± 4.9) and of metmyoglobin (8.7% ±
7.4) were lower and displayed high variability.
The L* values of the 21 Semimembranosus meat
samples ranged from 34.1 to 41.8 (mean value = 38.3).
a* and b* values ranged respectively from 11.3 to 21.6
and from 6.0 to 13.4. The variability of L*, a* and b*
values, expressed as RSD, were 8.3%, 16.3% and
15.8%, respectively. Although meat colour may be
affected by numerous factors, the colour variability was,
in general, lower than that in myoglobin content.
The observed variability is not surprising for commer-
cial meat samples. There was no control of the breed,
sex, or age of the animal, the time between slaughter
and meat purchase, nor of the conditions of meat
preparation, storage and distribution. Furthermore,
differences occur in fibre metabolic type within a
muscle, although it was attempted to select homoge-
neous muscle parts.
For these reasons, most series of experiments were
Control minced meat samples were red. The colour of
pressure processed samples was paler (pink) above 200
MPa and became grey-brown above 400 MPa. L*, a*
and b* values are shown in Fig. 1. L* values increased
from 200 to 350 MPa and a* values decreased with
increasing pressure, specially above 400 MPa, while b*
values remained constant. These results are in agree-
ment with those previously reported by Carlez et al.
(12) for minced beef meat pressurized at 20 °C : no
colour change was observed below 150–200 MPa, L*
increased from 150 to 400 MPa, and a* decreased above
350 MPa. Discoloration of meat under pressure appears
to result both from ‘whitening’ (200–350 MPa), and
from a loss of red colour (400–500 MPa). Critical
pressure thresholds could be due to different phenom-
ena. These are discussed later.
Similar L* and a* changes were observed by Shigehisa
et al. (10) for pressure-processed pork muscle homoge-
nates. L* values started to increase between 100
(L* = 66.2) and 200 MPa, to reach a maximum value
between 300 and 400 MPa (L* = 76.1). No further
increase was observed even at 600 MPa. The decrease
60
50
L* a* b* colour values

carried out on minced meat from a single muscle.

40
Within each series, the colour values and pigment
contents of pressurized samples were compared to the
corresponding nonpressurized minced meat control. 30
The pH value of minced meat displayed a low
variability (RSD = 1.08 %). The mean pH value of 5.57 20
was a low value normal for beef meat (18).
10

Effects of pressure processing on the colour and the

myoglobin content of minced meat 0 100 200 300 400 500
Pressure (MPa)

L* a* b* values. Minced meat samples packaged in air
were processed at 250, 300, 350, 400, 450 and 500 MPa,
at 10 °C, for 10 min. Assays at each pressure value were
carried out in duplicate.
Fig. 1. L* (-j-), a* (-s-), and b* (- + -) colour values of
minced meat pressurized at 10 °C for 10 min in air. Results
are the means (with sx) of at least six measurements on two
distinct meat samples (single Semimembranosus muscle)
processed independently

531
lwt/vol. 28 (1995) No. 5
in a* value was moderate between 100 (a* = 5.8) and
200 MPa and progressed until 600 MPa (a* = 0.2). No
explanation was given for these phenomena, but white
and hard coagulates were observed above 300 MPa.
Other investigators noted an increase in L* value (tuna
flesh, 14) and a decrease in a* value (beef meat, 11)
under pressure, without interpretation.
In the present study, variability was observed among
different experiments carried out at 450 MPa with nine
different Semimembranosus muscles. The decrease in
a* value varied from 3.8 to 9.4 (mean ∆a* = 6.0 ± 2.0),
the increase in L* value from 8.1 to 14.4 (mean
∆L* = 11.5 ± 2.3). These results confirm the necessity of
controls for each series of experiments with different
meat samples.
Content of myoglobin and of related pigments. The
absorbance spectra of myoglobin extracted (with a pH
6.8 sodium phosphate buffer) from control and pressur-
ized minced meat samples are shown in Fig. 2. From 0.1
to 200 MPa, the spectra remained unchanged. From 200
to 400 MPa, the intensity of absorbance decreased with
increasing pressure, without extensive modification of
the shape of spectra. This reflects a decrease in the total
amount of myoglobin (probably due to reduced total
myoglobin extractibility), without significant changes in
the proportions of myoglobin, oxymyoglobin and met-
myoglobin. At 500 MPa, the total myoglobin content,
calculated according to Krzywicki (16), further
decreased to 3.2 ± 0.3 mg/g (from an initial content in
control samples of 4.8 ± 0.4 mg/g). Simultaneously, the
shape of the spectrum was markedly changed, with a
decrease in absorbance at 540 and 580 nm (character-
istic of oxymyoglobin) and an increase at 505 and 630
nm (characteristic of metmyoglobin).
0.3
Control
200 MPa
250 MPa
300 MPa
400 MPa
0.2
Absorbance
500 MPa
0.1
0 total Mb on one hand, and for a*, MbO2 and MetMb on
450 500 550 600 650
Wavelength (nm)
Fig. 2. Visible spectra of phosphate buffer (pH 6.8) extracts of
minced meat from Semimembranosus pressurized at 0.1, 200,
250, 300, 400 or 500 MPa and 10 °C for 10 min in air
100
Proportions of the three
80
myoglobin forms (%)
60
40
20
0
Control 200 250 300 350 400 450 500
Pressure (MPa)
A
C G
Fig. 3. Relative proportions of myoglobin ( ); oxymyoglobin
( ); and metmyoglobin ( ) in minced meat pressurized at 10
°C for 10 min in air. Results are the means of measurements
on two distinct meat samples (from the Semimembranosus
muscle of Fig. 1) processed independently
There is no published study concerning the myoglobin
extractibility of pressurized meat samples. However,
the extractibility of myoglobin is known to decrease
when meat samples are subjected to increasing tem-
perature (19) or to denaturing agents. This suggests that
partial denaturation of myoglobin also occurred under
pressure, specially above 300 MPa.
The relative proportions of the three myoglobin forms
were also modified by pressure processing (Fig. 3). The
proportion of myoglobin in pressurized meat samples
was similar to that in fresh minced meat. In contrast,
the proportion of oxymyoglobin sharply decreased
above 400 MPa. The proportion of metmyoglobin
started to increase at the ‘critical’ pressure of 400 MPa,
simultaneously to a decrease in a* value. Murakami et
al. (14) determined the proportion of metmyoglobin in
pressure-processed red tuna flesh, using the A540/A503
method. The proportion of metmyoglobin did not
increase at 150 MPa, increased drastically at 300 MPa
from 37.7% (control) to 73.1%, and remained at the
same level at 500 MPa. These results confirm that the
colour change in pressurized minced meat is partly due
to the oxidation of oxymyoglobin to metmyoglobin.
Three phenomena could induce the colour changes
observed as a result of pressure processing: (1) dena-
turation of the globin moiety of the molecule with
possible displacement or separation of the heme from
the globin; (2) modification of the porphyrin ring with a
release of the iron atom (iron release could facilitate its
oxidation and could also be related to globin denatura-
tion); (3) oxidation of ferrous oxymyoglobin (but
apparently not of the small amounts of myoglobin, see
Fig. 3) into ferric metmyoglobin. These phenomena
may also occur simultaneously or sequentially. The
observation of different pressure thresholds for L* and
700 the other hand, supports the existence of at least two
distinct mechanisms. However such mechanisms may
invalidate Krzywicki’s spectrophotometric method (16)
for the evaluation of reduced, oxy- and met-
myoglobins.
532

The pH value of minced meat increased from 5.55
(control sample) to 5.77–5.79 (after processing at 400 or
500 MPa).
Content of nonheme iron. Minced meat samples proc-
essed in air at 350, 400, 450 and 500 MPa (10 °C, 10
min) were extracted with an HCl/ trichloroacetic acid
solution in order to determine the content in nonheme
Table 2 Nonheme iron content of minced meat from a
single Semimembranosus muscle, after pressure processing at
10 °C for 10 min
Sample Nonheme irona (mg/kg of meat)
Control 4.2–4.2
350 MPa 3.7–3.9
400 MPa 4.5–4.5
450 MPa 4.4–4.6
500 MPa 5.3–5.5
a colour. The b* value (yellowness) is high, and this
Determined by inductively coupled plasma atomic emission
spectrometry after extraction with an acid solution according
to Schricker et al. (17)
iron. Results are given in Table 2. In the control
(nonpressurized) meat sample, the nonheme iron con-
tent was equal to 4.2 mg/kg of meat. A moderate
increase in nonheme iron was observed after processing
at 500 MPa but not at 350, 400 or 450 MPa. This may
indicate that opening of the porphyrin ring of myoglo-
bin occurred to a small extent at 500 MPa, thus
releasing some ‘nonheme’ iron. The total iron content
of the minced meat from Semimembranosus was not
determined. Schricker et al. (17) found that the total
iron content of beef muscles ranged from 23.4 ± 4.5
mg/kg (Longissimus dorsi) to 28.7 ± 6.6 mg/kg (Gluteus
medius), but Semimembranosus was not determined.
Nutritional tables indicate iron contents of 21 to 35 mg/
kg for a wide variety of lean beef cuts. It therefore
appears that processing at 500 MPa did not release a
significant proportion of heme iron.
Effect of the duration of pressure processing. Minced
meat was processed in air at 350 and at 400 MPa (10 °C)
for 2, 5, 10, 20 and 30 min. The L*, a* and b* values, and
the different myoglobin forms were determined. No
linear relation was observed between the duration of
pressure processing and any of the colour parameters
or myoglobin contents (data not shown). At both
pressure tested, the following changes were observed
from 2 to 10 min of processing: L* increased, a*
decreased, the total myoglobin content decreased, the
proportion of metmyoglobin increased and that of
oxymyoglobin decreased. After 10 min of pressure
processing, no further changes were observed. The
subsequent pressurization experiments were carried
out for 10 min.
The pH increased to its final value after only 2 min of
pressurization.
Effect of chilled storage after pressure processing.
Minced meat was packaged under oxygen, kept at 5 °C
533
lwt/vol. 28 (1995) No. 5
for 18 h, processed at 350 or 500 MPa (10 °C, 10 min),
and then stored at about 5 °C for up to 12 d.
The control sample displayed a rapid (between 2 and 5
d of storage) decrease in a* value and a progressive
decrease in b* value, and turned into a brown-red
colour (Fig. 4a and b). This may correspond to a partial
oxidation of oxymyoglobin into metmyoglobin. It is
likely that bacterial growth took place at 5 °C without
delay in this nonpressurized meat sample (12, 13) and
decreased the partial pressure of oxygen, thus enhanc-
ing the formation of metmyoglobin. Interactions
between phospholipids and myoglobin taking place in
minced meat may also enhance this oxidation (1).
The L* values of pressurized samples were above that
of the control sample, as already observed, and did not
change during storage. The a* and b* value of the
sample processed at 500 MPa also remained constant
(Fig. 4a and b), corresponding to a stable grey-brown
parameter therefore seems to be involved in the grey-
brown colour (together with a low a* value). The meat
sample processed at 350 MPa was pink, and pro-
gressively lost its red colour during storage to turn grey-
brown with final colour values close to those of the 500
MPa sample (as shown in Fig. 4a, where final a* values
20 (a)
16
a* colour value
12
8
4
0 2 4 6 8 10 12 14
20 (b)
16
b* colour value
12
8
4
0 2 4 6 8 10 12 14
Time (d)
Fig. 4. Changes in a* (a) and b* (b) colour values during the
chilled storage (5 °C) of nonpressurized minced meat (-s-),
and of minced meat pressurized at 350 (-d-) or 500 MPa (-e-)
at 10 °C for 10 min in oxygen. Meat samples were packaged
in oxygen and kept at 5 °C for 18 h before pressurization.
Results are the means (with standard deviation) of at least six
measurements on the same Semimembranosus meat sample
lwt/vol. 28 (1995) No. 5
are both very low). The following explanation can be
given: processing at 500 MPa caused an immediate,
complete and stable modification of myoglobin, but
processing at 350 MPa only caused a partial modifica-
tion. The residual myoglobin, still pink after pressuriza-
tion, was progressively further oxidized during chilled
storage of minced meat.
Colour changes at different meat depths. Experiments
were carried out on meat cubes (30 to 40 mm side) from
Semimembranosus muscle. These were packed in air
just before pressure treatment, processed at various
pressure levels (10 °C, 10 min), and kept at about 5 °C
for 1–2 h before pouch opening and colour measure-
ments. L*, a* and b* values were measured at meat
surface, in the centre of cubes, and in an intermediate
zone. After processing at 350 MPa, the intermediate
zone became grey-brown, while the cube centre and the
meat surface became pink (as reflected by a* values,
Fig. 5). After processing at 400, 450 or 500 MPa, the
intermediate grey-brown zone extended towards the
meat surface, while the central pink zone extended
from the centre. The meat surface of the 500 MPa
sample became completely grey-brown. Changes in L*
values were identical at different depths (data not
shown), increasing at 200 MPa, and reaching a plateau
at 350 MPa, as found previously for minced meat (Fig.
1).
In previous experiments with minced meat (processed
in polyvinylidene chloride tubing of diameter = 32 mm
and height = 40 mm) from Rectus femoris (12), a grey-
brown zone was observed in the central part of the
meat after processing at 300–350 MPa and 20 °C for 20
min, while the meat surface became pink. After
processing at 500 MPa, the whole minced meat became
grey-brown from centre to surface.
These results can be interpreted as follows. In the case
of meat cubes, a decreasing pO2 gradient must be
present from the meat surface (in contact with air) to
20
16
a* colour value
12
8
4 vacuum without oxygen scavenger) (data not shown).
0 100 200 300 400 500
Pressure (MPa)
Fig. 5. Changes in a* colour values at the surface (-s-), at an
intermediate position (-j-) or at the centre (-e-) of meat
cubes after pressurization at 10 °C for 10 min in air. Results
are the means (with sx) of at least six measurements on the
same Semimembranosus meat sample
534
the meat centre (where the oxygen concentration is
probably very low). It is known that oxidation of
myoglobin to metmyoglobin is enhanced at a pO2 of 7
mm Hg, and occurs first under the meat surface (20).
This may explain why a grey off-colour is formed in the
intermediate zone after processing at 350 MPa. At 500
MPa, even oxymyoglobin at the meat surface is
apparently oxidized into metmyoglobin. Myoglobin at
the meat centre is apparently not oxidized, probably
because of the low oxygen concentration, and became
pink, possibly due to the denaturation of globin and/or
myofibrillar proteins. The larger central pink zone at
500 MPa is difficult to explain, since it implies some
reversibility in colour change. It also appears to indicate
that higher pressure levels do not enhance oxygen
diffusion from the meat surface to the meat centre.
In the case of minced meat, mincing has introduced
some oxygen throughout the product, so that myoglo-
bin present at the centre of minced meat is oxidized
under pressure. The higher level of oxygen present at
the surface of minced meat may delay oxidation.
Overall, these results also suggest (1) that oxygenation
of myoglobin into oxymyoglobin does not protect from
pressure-induced oxidation into metmyoglobin (it may
only delay this oxidation); (2) that oxygen exclusion
may protect myoglobin from this pressure-induced
oxidation. The resulting anaerobic conditions may also
enhance metmyoglobin reductase activity.
It was therefore of interest to carry out high pressure
experiments using different packaging atmospheres.
Effects of packaging atmosphere (air, oxygen, vacuum
with or without oxygen scavenger)
The L*, a* and b* values, total myoglobin content and
pH of the nonpackaged, nonpressurized minced meat
(from four distinct muscles) used in these experiments
are included in the 95 % confidence intervals
(mean ± 2SD) calculated from previous meat variabil-
ity study, although it was necessary to use a distinct
muscle for each gas atmosphere.
L* a* b* values. Meat samples were packaged under air,
oxygen or vacuum (with or without oxygen scavenger),
kept at 5 °C for 18 h, and pressure processed at 350 to
500 MPa (10 °C, 10 min). Sealed pouches were kept at
5 °C for 1–2 h before opening and measurement of
colour values. L* values increased from 30–35 to 40–45
after processing at any of the pressure levels studied
and in all gas atmospheres tested (data not shown). b*
values remained constant or increased slightly (under
a* values are shown in Fig. 6. The initial values (before
pressurization) are variable because of the different
muscles, and of the 18 h period at 5 °C in the
corresponding atmosphere (a* decreased under vac-
uum without oxygen scavenger). Pressure processing at
or above 400 MPa caused a marked decrease in a*
value for samples packaged in air or oxygen. When
pressurization was carried out under vacuum in the
 lwt/vol. 28 (1995) No. 5

presence of an oxygen scavenger, the a* values pink, while those processed (at 450 and 500 MPa) in
remained constant at a high level (ø 15). Samples thus oxygen, air or vacuum without O2 scavenger became
processed in the complete absence of oxygen became grey-brown. It therefore appears that no myoglobin
oxidation takes place in the absence of oxygen, but that
meat ‘whitening’ (probably due to pressure denatura-
20 tion of globin) is not prevented.

Myoglobin and related forms. The total myoglobin

15 content of the meat samples kept for 18 h at 5 °C under
the four packaging atmospheres ranged from 6.7 to 7.6
a* colour value

mg/g of meat. This myoglobin content decreased after

10
5
0 100
Fig. 6. Changes in a* colour values of minced meat from
Semimembranosus packaged under different atmospheres:
oxygen (.....), air (----), vacuum ( — ), or vacuum with O2
scavenger ( — - — -), then pressurized at 10 °C for 10 min.
Results are the means (with sx) of six measurements on each
one of five distinct meat samples processed independently
(three distinct meat samples in the case of experiments with
O2 scavenger). A different muscle was used for each
atmosphere. Meat samples were kept at 5 °C for 18 h in their
respective packaging atmospheres before pressurization
pressure processing, the greatest decrease being
observed after processing at 500 MPa (23% decrease
under vacuum, 31% in oxygen, 34% in air, 35% under
vacuum + O2 scavenger). The decrease in total myoglo-
bin is not clearly related to the type of packaging
atmosphere nor to the change in colour or in a* value.
It is probably due to a partial globin denaturation and
200 300 400 500 to the resulting decrease in extractability.
Pressure (MPa) The relative proportions of myoglobin, oxymyoglobin
and metmyoglobin are shown in Fig. 7. In oxygen or in
air (Fig. 7a and b), the proportions of metmyoglobin
reached 49 or 55% respectively, after processing at 500
MPa. Under vacuum (without O2 scavenger) (Fig. 7c),
oxidation of myoglobin into metmyoglobin reached a
maximum, the proportion of metmyoglobin reaching
39% at 400 MPa and 55% at 500 MPa. In the total
absence of oxygen (Fig. 7d), the proportion of myoglo-
bin was significant (12–17%), and the proportion of

(a) (b)
100 100
Proportions of three forms

80 80
of myoglobin (%)

60 60

40 40

20 20

0 0
Control 350 400 450 500 Control 350 400 450 500
Pressure (MPa) Pressure (MPa)

(c) (d)
100 100
Proportions of three forms

80 80
of myoglobin (%)

60 60

40 40

20 20

0 0
Control 350 400 450 500 Control 350 400 450 500
Pressure (MPa) Pressure (MPa)
A
Fig. 7. Relative proportions of myoglobin ( ), oxymyoglobin ( ), and metmyoglobin ( ) of minced meat from C G
Semimembranosus packaged in oxygen (a), air (b), vacuum (c) or vacuum plus oxygen scavenger (d), then pressurized at 350,
400, 450 or 500 MPa and 10 °C for 10 min. Results are the means of measurements on five distinct meat samples processed
independently (three distinct meat samples in the case of experiments with O2 scavenger). A different muscle was used for each
atmosphere. Meat samples were kept at 5 °C for 18 h in their respective packaging atmospheres before pressurization

535
lwt/vol. 28 (1995) No. 5
metmyoglobin formed remained below 16%, even after
processing at 500 MPa.
It can be concluded from these results that oxygen does
not protect myoglobin significantly against pressure-
induced ( ≥ 400 MPa) oxidation to the ferric form.
Packaging under vacuum does not appear to remove
oxygen efficiently from minced meat, since samples
with low a* values and high metmyoglobin contents
were observed both before and after pressurization.
The total absence of oxygen does result in effective
protection, since after processing at high pressure (500
MPa), a pink colour was formed, the a* value remained
constant, and the formation of metmyoglobin remained
moderate. However, as already mentioned, none of the
packaging atmospheres provides protection against
meat ‘whitening’. This last phenomenon takes place at
pressures above 200 MPa, reduces the content of
extractible myoglobin, causes a colour change from red
to pink, and probably results from the denaturation of
globin.
Effect of chemical additives
Reducing agents. The addition of cysteine (200 or 1000
mg/kg of minced meat, packaged in air) 1 h before
processing at 450 MPa (10 °C — 10 min) had no
significant effect on the visual colour, L*, a* and b*
values, the myoglobin content or the proportions of
related pigments (compared with the pressurized
control).
The addition of ascorbic acid (250 or 1000 mg/kg of
minced meat, packaged in air), 1 h before processing at
450 MPa (10 °C, 10 min) induced an additional
decrease in a* value and increase in MetMb (as
compared to the pressurized control) (data not shown).
The latter effect was more marked at 1000 mg/kg, while
the meat pH before pressurization decreased by 0.2
unit.
Several authors (4, 21, 22) found that ascorbic acid was
effective in delaying the oxidation of myoglobin.
Govindarajan et al. (21) indicated that ascorbic acid did
not affect the initial slow oxidation step but extended
the lag time before the rapid oxidation step began.
Mitsumoto et al. (4) suggested that ascorbic acid was
active mainly in association with α-tocopherol present
in the muscle. The same authors indicated that ascorbic
acid could act as a prooxidant at high concentrations
(5000 p.p.m.), or in the presence of metal ions, such as
bound or free iron. Greene et al. (22) found a similar
result about possible prooxidant properties of ascorbic
acid and proposed the use of a combination of additives
(butylhydroxyanisole or propylgallate).
Sodium nitrite. This additive was used at 100 and 200
mg/kg of minced meat, always in association with 10 g
NaCl/kg of minced meat. The mix was blended and kept
under vacuum at 5 °C for 18 h to allow the formation of
nitrosomyoglobin. A dark red colour was formed
(L* = 36.7, a* = 16.3, b* = 8.0). Pressure processing (10
°C, 10 min) lead to increased L* values, but did not
536
60
50
L a b colour values
40
30
*
*
20
*
10
0 100 200 300 400 500
Pressure (MPa)
Fig. 8. L* (-j-), a* (-s-), and b* (- + -) colour values of
minced meat mixed with 200 mg NaNO2 and 10 g NaCl per kg
meat, packaged under vacuum, kept at 5 °C for 18 h, then
pressurized at 10 °C for 10 min. Results are the means (with
sx) of at least six measurements on each one of two distinct
meat samples (single Semimembranosus muscle) processed
independently
induce any change in a* and b* values whatever the
pressure level or the nitrite concentration (Fig. 8). The
colour of all pressurized samples was pink, close to that
of cooked ham. The absorbance spectra of meat
extracts (pressurized or not) were very close to one
another but very different from those of meat extracts
without nitrite. These results support the hypothesis
that colour changes resulting from pressure processing
are caused by two or three different mechanisms. In the
presence of nitrite, the prevalent nitrosomyoglobin is
protected against oxidation into the ferric form. The
‘whitening’ effect, however is not prevented. Similar
observations were made by Suzuki et al. (23) with
Wiener sausage processed at 400 or 600 MPa (10–40 °C,
10–30 min): L* values increased by 5 units, while a* and
b* values remained constant.
Nicotinic acid and nicotinamide. These two substances
were tested at 244, 610 and 1220 mg/kg minced meat,
packaged in air. The meat with additive was kept at 5 °C
for 18 h before processing at 450 MPa and 10 °C for 10
min.
Compared with meat pressurized without additive,
pressurized minced meat containing nicotinamide dis-
played slightly lower a* values and slightly higher b*
values, both proportional to the nicotinamide concen-
tration. Nicotinamide at all concentrations tested did
not affect meat pH nor the total myoglobin content, but
caused a decrease in the proportion of oxymyoglobin
(from an initial 50 to a final 33%) and an increase in the
proportion of metmyoglobin (from 22 to 41%).
The addition of nicotinic acid significantly decreased
the pH of pressurized meat (pH 4.8, 5.3, 5.4 and 5.7 at
respectively 1220, 610, 244 and 0 mg of nicotinic acid/
kg of meat). This pH drop was probably responsible for
the notable effects of nicotinic acid on the colour
parameters of pressurized meat. As compared to meat

pressurized without nicotinic acid, a* values were lower
and b* values higher, in proportion to the concentra-
tion of nicotinic acid. The L* values were similar to that
of the pressurized meat control, except at 1220 mg
nicotinic acid/kg where the L* value was lower.
With increasing nicotinic acid, pressurized meat
extracts contained less total myoglobin (contents from
7.2 for the initial fresh minced meat to 1.8 mg/g for the
pressurized meat containing 1220 mg/kg nicotinic acid)
and markedly higher proportions of myoglobin and of
metmyoglobin, together with much lower proportions
of oxymyoglobin. However, Krzywicki’s (16) method of
analysis cannot remain valid when nicotinamide or
nicotinic acid are present, since a yellow colour
develops.
Samples pressurized with nicotinamide or nicotinic acid
were grey but slightly more yellow than control
pressurized meat.
It is likely that the above results are mainly caused by
the pH drop due to nicotinic acid: this pH drop would
enhance globin denaturation and insolubilization, plus
myoglobin oxidation into metmyoglobin. According to
Renerre (1), myoglobin undergoes denaturation below
pH 5, and a low pH also reduces the stability constant
for the heme–globin linkage, porphyrin being released
from globin below pH 5.
In practice, nicotinic acid or nicotinamide were not
useful against pressure-induced colour changes.
Renerre (1) explained that nicotinamide acted on
colour stability by its protective effect on NAD (that
played a role in metmyoglobin reduction). But Govin-
darajan et al. (21) also observed that nicotinamide did
not prevent the formation of metmyoglobin during
chilled storage of ground beef meat.
Conclusion
The discoloration of red meat (whole or minced) taking
place at high pressure appears to be a complex
phenomenon that depends on at least two mechanisms.
At or above 200 MPa, a ‘whitening’ effect is observed,
the red colour of beef meat turning into a much paler
pink colour. Simultaneously, L* values increase mark-
edly (Fig. 1). Such an effect was also observed with red
tuna flesh at lower pressure levels (50 to 150 MPa, 0 °C,
15 min) (14) and with pork muscle homogenates at 200
MPa (10).
Here this ‘whitening’ effect is tentatively attributed to
the pressure-induced denaturation of the globin moiety
of myoglobin. Extracts from minced meat pressurized
at 200 or 300 MPa contain less total myoglobin, as
determined by spectrophotometry (Fig. 2), and it is not
known whether this is due to pressure-induced insolu-
bilization of myoglobin (possibly through enhanced
aggregation of myofibrillar proteins), and/or to the
reduced absorbance of still soluble but denatured
myoglobin, or still to the displacement, detachment or
opening of the porphyrin ring of some myoglobin
molecules. The relative proportions of reduced, oxy- or
metmyoglobin remain unchanged (Fig. 3).
537
lwt/vol. 28 (1995) No. 5
When minced meat is subjected to higher pressure
levels ( ≥ 400 MPa), its colour also becomes much paler
than initially (with a high L* value, reaching a
maximum at 350 MPa), but the red or pink hue changes
(or is masked) into a pale grey-brown colour. In
parallel, the a* value decreases markedly (Fig. 1). The
total myoglobin content of meat extracts further
decreases (Fig. 2), and metmyoglobin is formed at the
expense of oxymyoglobin (Fig. 3). The latter trans-
formation was observed also in red tuna flesh, but took
place at lower pressure (from 150 to 300 MPa, at 0 °C
for 15 min) (14). In the present study, pressures of 400
MPa or above promote the partial oxidation of ferrous
myoglobin into ferric metmyoglobin, together with the
possible denaturation of globin.
Two experimental findings support this oxidation step:
(1) a vacuum atmosphere associated to an oxygen
scavenger, i.e. a complete absence of free oxygen,
protects against pressure-induced oxidation into met-
myoglobin (no loss of pink colour, no decrease in a*
value, small proportion of metmyoglobin) (Figs 6 and
7); (2) nitrites, i.e. formation of nitrosomyoglobin,
afford a similar protection (no loss of pink colour, no
decrease in a* value) (Fig. 8). In contrast, an oxygen
atmosphere does not protect against oxymyoglobin
oxidation (Figs 6 and 7). These two findings provide
some practical means to minimize the discoloration of
whole or minced meat during pressure-induced pas-
teurization or restructuring. However, oxygen removal
or the presence of nitrites does not protect, at least at
350–500 MPa, against the ‘whitening’ effect tentatively
attributed to globin denaturation.
Both degradation mechanisms appear to depend more
on critical pressure thresholds than on time, since they
took place already 2 to 5 min after the target pressure
(350 or 400 MPa) was reached.
A third degradation mechanism (namely heme detach-
ment or opening) was not ruled out, although process-
ing minced meat at 350 to 500 MPa (10 °C, 10 min) did
not enhance markedly the extraction of ‘nonheme’ iron
by an acid solution (Table 2). The grey-brown colour of
minced meat processed at 450 or 500 MPa suggests that
not only metmyoglobin but also other myoglobin-
derived pigments could be present. Absorbance spectra
of meat extracts, however, did not permit any further
conclusion on this point. Globin denaturation (or
changes in the heme ring) may enhance oxidation into
ferric metmyoglobin (or related pigments). Other
experiments reported in the present study (colour
changes at different meat depths; colour evolution of
pressurized minced meat during further chilled storage)
do not appear to contradict the previous hypotheses
concerning meat colour changes caused by pressure
processing.
It is known that high pressure dissociates (at least
temporarily) ions pairs (Gross and Jaenicke, 24). This
induces a decrease in pH, a disruption of electrostatic
interactions, and thus probably a partial protein dena-
turation and/or displacement of the heme group. The
decrease in pH may also enhance heme oxidation to the
ferric state. The effects of pressure on myofibrillar
lwt/vol. 28 (1995) No. 5
proteins are not known with respect to the resulting
changes in meat colour. Finally pressure may affect the
activity of myoglobin-oxidizing and metmyoglobin-
reducing enzymes or factors present in muscle.
Possible interactions under pressure of meat samples
and chemicals from packaging materials were not
considered in this study. However, experiments carried
out with two different packaging materials (poly-
vinylidene chloride tubing and polyester-aluminium
pouches) led to similar colour changes.
Pressure experiments with solutions of myoglobin may
help to discriminate between the various discoloration
mechanisms. The complete stabilization of the colour of
red meat during pressure processing is a difficult task,
although pressure application in the frozen state
appears to afford some protection.
Acknowledgements
This study was supported by a grant (No 91 G 0574)
from the French Ministry of Research and Technology,
Programme Aliment 2002. We wish to thank M. Loı̈c Le
Doussal for his participation to some experiments, and
M. Xavier Costesec from Mitsubishi France S.A. for
supplying the FX-100 oxygen scavenger.
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