MEAT

SCIENCE
Meat Science 72 (2006) 672679
www.elsevier.com/locate/meatsci

Zn-porphyrin formation in cured meat products: Eect of added

salt and nitrite
Christina E. Adamsen, Jens K.S. Mller, Kristoer Laursen, Karsten Olsen, Leif H. Skibsted

Food Chemistry, Department of Food Science, The Royal Veterinary and Agricultural University, Rolighedsvej 30, 4th. DK-1958 Frederiksberg C, Denmark
Received 26 April 2005; received in revised form 16 September 2005; accepted 29 September 2005

Abstract
Zn-porphyrin (Zn-pp) was quantied by uorescence spectroscopy in the cured and dry cured meat products: Parma ham, Iberian
ham, dry-cured ham with added nitrite, cooked ham with added nitrite, raw ham meat, raw bacon and Karree-Speck. The highest
amount of Zn-pp was found in dry-cured Parma ham and Iberian ham, while the use of nitrite as curing agent was found to inhibit completely the formation of Zn-pp in meat products. A positive correlation between both Zn content and Fe content and the logarithmic
transformed Zn-pp content (measured as uorescence intensity I) was found for the dierent cured and dry cured meat products, with
correlation coecients of 0.79 (p < 0.001) and 0.71 (p < 0.01), respectively. Log I correlates best with the Zn content, indicating that the
formation of Zn-pp is proportional to the Zn content. A model system with vacuum packed pork in brine with dierent added levels of
sodium chloride with or without nitrite and Zn acetate was investigated in order to further elucidate the mechanism of Zn-pp formation.
Zn-pp increased with time (up to 42 days investigated) in non-cured meat and for meat cured solely with NaCl lower than 9%. Addition
of nitrite or Zn(II) in the curing brine was found to inhibit formation of Zn-pp conrming the observations from the various cured meat
products. It is suggested that a chloride anion assisted dissociation of iron from myoglobin could be rate-determining for Zn-pp formation in meat products.

2005 Elsevier Ltd. All rights reserved.
Keywords: Dry cured meat products; Colour; Zn-protoporphyrin; Nitrite content; Salt content

1. Introduction
Visual appearance is of vital importance for the quality
of most food products, and especially the colour aects
consumers when they evaluate freshness and quality of
meat and meat products. The colour of meat products is
determined by a combination of dierent factors including
moisture and fat content, but more important is the chemical form and concentration of the hemoproteins, especially
that of myoglobin (Mb) (Fox, 1966; Lawrie, 1991; Ledward, 1992). Mb is aected by processing parameters
including heat treatment and the use of nitrite/nitrate in
meat curing is of particularly interest together with the
packing method used for the product, because compounds
*

Corresponding author. Tel.: +45 35283221; fax: +45 35283344.

E-mail address: ls@kvl.dk (L.H. Skibsted).

0309-1740/$ - see front matter
2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.meatsci.2005.09.017

formed by reduction of nitrite or nitrate react with Mb

forming the pink-coloured pigment, nitrosylmyoglobin
(MbFe(II)NO) (Fox, 1966; Mller & Skibsted, 2002). In
products with added nitrite or nitrate the complex
MbFe(II)NO is the main contributor to the characteristic
colour, e.g. in dry-cured ham, brine-cured meat products
and cooked cured ham. Cured meats and meat products
without nitrite/nitrate addition will normally attain a dull
brown colour in raw products or a grey colour in heated
products, which inuences consumer acceptance negatively. Also safety aspects of adding nitrite/nitrate as an
ingredient to meat have been discussed over the past decades (Cassens, 1990), and topics such as microbiological
safety versus human health concerns have to be considered
and analyzed.
The traditional dry-cured ham from Northern Italy,
Parma ham, is made from pork legs and only sodium

C.E. Adamsen et al. / Meat Science 72 (2006) 672679

chloride in the form of sea salt from the Mediterranean Sea

is added, and notably the product obtains a stable red colour without any addition of nitrite or nitrate (Parolari,
1996). Theories and hypotheses regarding the chemical nature and identity of the Parma ham pigment are numerous
including suggestions for the reaction pathway for their
formation. Virgili et al. (1999), thus, suggested that lowmolecular weight compounds containing electron donating
atoms, formed during maturation of Parma ham, in particular basic peptides or amino acids resulting from an extensive proteolysis, may play a role as Fe ligands in Mb.
Morita, Niu, Sakata, and Nagata (1996) claimed that the
Mb derivative present in matured Parma ham was a nitrosylated Mb formed by microbial activity in the ham during
the prolonged processing. Other studies showed that the
colour or the change of colour in meat and meat products
may depend on microbial growth (Kalchayanand, Ray,
Field, & Johnson, 1989; Arihara et al., 1993). However, it
seems highly speculative that the pigment in Parma ham
is formed solely by microbial action, since microorganisms
are mainly found at the surface of the meat during the process while a uniform colour is developed throughout the
whole ham. The colour of Parma ham has also been suspected to be due to contamination with nitrate from the
sea salt, but the total amount of nitrate from the salt has
been found negligible (Sakata, 2000). Mller, Adamsen,
and Skibsted (2003) have by electron spin resonance spectroscopy shown that the Parma ham pigment is dierent
from MbFe(II)NO and is not a nitric oxide complex.
Morita et al. (1996) found that the pigment of Parma
ham is easily extracted with 75% acetone/water solution
and that it is a dierent Mb derivative not known from
other meat and meat products. Recently, both Mller
et al. (2003) and Parolari et al. (2003) further showed that
both a lipophilic (75% acetone) and a hydrophilic pigment
(phosphate buer pH = 6.00) can be extracted from Parma
ham through processing. Both pigments exhibit unique
UVVis spectral features dierent from those of other
well-characterized Mb derivatives, and during processing
the lipophilic pigment becomes predominant.
The lipophilic pigment extracted from Parma ham has
more recently been identied as Zn-protoporphyrin IX
(Zn-pp), believed to originate from Mb in which Fe has
been substituted by Zn and the heme separated from the
native heme-protein (Wakamatsu, Nishimura, & Hattori,
2004a). Another study from Wakamatsu, Okui, Ikeda,
Nishimura, and Hattori (2004b) showed, using a model
system, that anaerobic conditions favour formation of
Zn-pp, and that endogenous enzymes as well as microorganisms may be involved in the formation of Zn-pp. Three
possible substitution patterns may accordingly be suggested: (i) a non-enzymatic reaction in which Zn(II) substitutes Fe(II) under anaerobic condition with concomitant
dissociation of the heme; (ii) a bacterial enzymatic reaction
where bacterial growths naturally degrade the meat proteins including the pigment, or (iii) an enzymatic reaction
where an endogenous ferrochelatase interchanges the two

673

metals. More knowledge about the chemical nature and

mechanisms of formation of the stable red pigment in
Parma ham is of general interest, since such knowledge presents future prospects for manufacturing cured meat products with a desirable and stable red colour without the use
of nitrite/nitrate.
The present work is part of a project investigating the
formation of stable red pigments in Parma ham, with the
aim of using such information for designing processes for
manufacturing other meat products without nitrite or
nitrate. The objective of the current study was to determine
the Zn-pp content in dierent types of meat products produced with variations in curing and processing technology,
in order to obtain more information about how the processing parameters aect the formation of Zn-pp. Moreover, a model system with vacuum packed pork cured at
dierent levels of sodium chloride with and without nitrite
and added zinc acetate was established in order to investigate how varying combinations of sodium chloride, nitrite
and Zn aect the formation of Zn-pp. The sodium nitrite
concentration used for the brine was as high as 1% (w/w)
which notably is far beyond the legal permission. However,
the aim was solely to investigate the possible mechanism of
nitrite on formation of Zn-pp.
2. Materials and methods
2.1. Chemicals
Sulphuric acid (9597%), hydrochloric acid (37%),
sodium chloride, sodium nitrite, silver nitrate, nitric acid
(65%), ammonium iron(III) sulphate, potassium thiocyanate and hydrogen peroxide (30%) were all of analytical
grade and obtained from Merck (Darmstadt, Germany).
The Fe standard (1 mg/l, Titrisol) and the Zn standard
(1 mg/l, Titrisol) were obtained from Merck (Darmstadt,
Germany). Acetone and methanol were obtained from
Lab-Scan (Dublin, Ireland). MES (Z-[N-morpholino]ethanesulfonic acid), zinc acetate dihydrate and riboavin were all obtained from Sigma-Aldrich Chemie
GmbH (Steinheim, Germany). Certied reference material,
bovine muscle CRM148, was obtained from The Institute
for Reference Materials and Measurements (Geel, Belgium). All other chemicals were of analytical grade and
used without further purication. Water was puried
through a Millipore Q-Plus unit (Millipore, Bedford,
Mass., USA).
2.2. Cured meat products
Seven dierent types of cured meat products were
included in this study. Fully matured Parma ham (12
months manufacturing time) was obtained from a local
producer in Parma through Stazione Sperimentale per
IIndustria delle Conserve Alimentari, Parma, Italy. Fully
matured Iberian ham (30 months manufacturing time)
was obtained from Senorio de Montanera S.L. Criadores

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C.E. Adamsen et al. / Meat Science 72 (2006) 672679

de Cerdo Iberico Asociados, Badajoz, Spain. Dry-cured

ham with added nitrite (3 months manufacturing time)
and cooked, nitrite-cured ham was obtained from Tulip
International (Viby J., Danmark). Raw meat from the
ham region of the pig and raw bacon salted with nitrite
as standard Danish products were obtained locally. A special dry-cured product, Karree-Speck (3 months manufacturing time), locally produced in Austria was obtained
from a local shop in Zillertal, Austria. From all seven types
of meat products a slice, without visible fat, selected as typical for the product was nely chopped and used for further
analysis.

of 0.8 g cm
3 d
1 (at 23 C and 85% RH) and were

vacuum packed (Electronic VacMit, Duggendorf, Germany). The vacuum packed meat in brine was stored at
5 C in darkness in a refrigerator for two days. After curing, the meat pieces were drained for of excess brine and
vacuum packed in 30 lm PA/100 lm PE laminated bags
(as described above). The meat was then stored at 5 C in
darkness in a refrigerator for 42 days. On day 0, 7, 14
and 42, randomly selected samples (n = 3) of each meat
sample were analysed.

2.3. Meat model system

2.4.1. Water content analysis

Two grams of nely chopped meat were placed on an
aluminium tray (with known weight) and the precise weight
was noted. The samples were dried in a thermo cabinet
(WTB Binder GmbH, Tuttingen, Germany) at 104 C in
4 h. Immediately after drying the samples were placed in
a desicator and after 15 min, the samples were weighted
and the water content was calculated.

For the meat model systems Semimembranosus (SM)

muscles were used. The meat was obtained from The Danish Meat Trade College through the Danish Meat Research
Institute, Roskilde, Denmark. The muscles were from 6 different animals (total 12 SM muscles) originating from the
same producer. The animals had been treated equally
under production, during the slaughtering process and
after slaughter. The muscles were removed from the animal
just after slaughter and immediately packed in vacuum
bags and stored at 2 C until used. All the animals had a
pH value post-mortem between 5.5 and 5.7.
The model experiment was carried out as two separate
experiments. The rst experiment included 5 dierent condition and the second experiment included 4 dierent condition, and all 9 curing condition are described in Table 1.
For each experiment, 6 SM muscles were cut in to cubes
of approximately 2 2 2 cm and mixed. The meat was
then divided into the curing-groups, as described above.
The curing was carried out as brine salting. Equal quantity
of meat and brine were added to a 13 20 cm pouches of
laminated packaging material consisting of 30 lm polyamide (PA)/100 lm polyethylene (PE) (SFK Meat system,
Denmark) with an oxygen transmission rate of 21 cm3
m
2 d
1 bar
1 (at 23 C and 75% RH), a carbon dioxide transmission rate of 97 cm3 m
2 d
1 bar
1 (at
23 C and 75% RH), and a watervapour transmission rate
Table 1
The sodium chloride content (w/w), sodium nitrite content (w/w) and zinc
acetate content (w/w) of the brine for the nine meat curing systems
Number

Sodium chloride
(w/w)

Sodium nitrite
(w/w)

Zn-acetate
(w/w)

1 Controla
2
3
4
5
6
7
8
9

b
15%
25%
15%
25%
15%
25%
15%
25%

1%
1%

1%
1%

0.6%
0.6%
0.6%
0.6%

a
b

Meat without curing.

No addition.

2.4. Chemical analysis

2.4.2. Salt content

The chloride was quantied as gram/100 g meat in the
meat sample. Ten gram of homogenized meat sample was
transfer into a 500 ml conical ask and 200 ml of boiling
milli-Q-water was added. The sample was mixed and
cooled to room temperature. The meat solutions were ltered through an S&S lter 5892 (Scheicher & Schuell
GmbH, Germany). Ten milliliters of the ltrate was mixed
with 10 ml a 0.1 M silver nitrate solution, 1.5 ml of a 65%
nitric acid and 0.5 ml ammonium iron(III) sulfate solution
(5 g ammonium iron(III) sulfate dissolved in a 10% (w/w)
nitric acid solution). The solution was boiled for 5 min
and cooled to room temperature, ltrated through a S&S
lter 5892 and titrated with 0.05 M potassium thiocyanate
solution.
2.4.3. Water activity
Water activity was measured using a CX-2 water activity
analyzer (AQUA LAB, Pullman W.A., USA). All samples
were chopped with a knife very ne and the analysis was
performed in triplicate.
2.4.4. Nitrite content
The nitrite content was measured by an NOx biosensor
rhus, Denmark) connected to a bio(
Unisense A/S, A
chamber with nitrite reductase activity. The biosensor
was calibrated by two solutions, one containing 1.0 mM
sodium nitrite with 1% NaCl and another 0.1 mM sodium
nitrite with 1% NaCl both dissolved in a 50 mM MES
buer (pH = 5.5). The nitrite contents in the meat samples
were measured in meat slurries. The meat slurries were
prepared by homogenization of the dierent ham or meat
samples with a 50 mM MES-buer (pH = 5.5), thus the
nal meat slurry had a salt content of approximately 1%.
Each sample was measured in triplicate.

C.E. Adamsen et al. / Meat Science 72 (2006) 672679

2.4.5. Colour measurements

The colour of the cured meat pieces was measured with
a Color-guide 45/0 (BYK-Gardner GmbH, Geretsried,
Germany) using the L*, a*, b* coordinates (CIE L*a*b*
colour system). Red colour was expressed as the a*-value;
the higher the a*-value the more red was the sample.
Approximately 10 g of each meat type was chopped very
ne using a mincing machine (KRUPS, Speedy Pro plus,
Groupe SEB, RCS Lyon, France) and placed between
two glass Petri-dishes, and the colour was measured though
the glass on three randomly selected locations of the
spread-out mince. For the dierent ham types the colour
was measured once. In the experiment with meat curing
the colour was measured on day 0, 7, 14 and 42.
2.4.6. Isolation of Zn-pp from ham
Zn-pp from the ham/meat was isolated as described by
Wakamatsu et al. (2004a) with minor modication. After
homogenizing the meat in distilled water, the homogenate
was centrifuged at 3000 rpm for 10 min at 4 C and ltered.
2.4.7. Fluorescence-spectroscopy
All measurements of uorescence emission intensities
(I) and spectra were made using an Aminco Bowman series 2 luminescence spectrometer (SLM-Aminco, Urbana,
IL, USA). All spectra were recorded at 25 C. Samples
were excited at 420 nm and the emission spectra were measured from 500 to 700 nm with 1 nm intervals and the
intensity of the emission maximum at 588590 nm was
monitored relative to a riboavin standard (1 mg/ml in
75% acetone/water-solution equals to 1). The index for
uorescence emission intensities was calculated from:
I fl

flsample
flriboflavin

ex420 nm;em589 nm

1 mg=ml ex420 nm;em589 nm

 M
1
dry

matter

where Mdry matter is the dry matter content of the sample in

gram.
Only solvents stored in glass containers were used and
throughout the analytical procedure, only glassware was
used. Fluorescence was measured in quartz cuvettes with
teon stoppers. For the seven dierent ham types the uorescence emission intensities were measured three times for
each ham types. In the curing experiment the uorescence
emission intensities were measured on day 0, 7, 14 and
42. All measurements were carried out three times on each
meat sample.
2.4.8. Total Fe and Zn content
The total Fe and Zn content was quantied using atomic
absorption spectrophotometry (AAS) based on the method
of the Danish Standardization Board (Dansk Standard 259,
Copenhagen, Denmark 1982). Samples (0.500 0.004 g) of
freeze-dried meat from the seven dierent types of meat
products (Parma ham, Iberian ham, dry-cured ham with
nitrite, raw bacon with nitrite, raw ham meat, KaareeSpeck with nitrite, and cooked ham with nitrite) and a
freeze-dried SM sample from 6 dierent animals were

675

weighed into vessels (Teon) (special for heating in a

microwave oven, CEA Advanced composite vessel assemblies, CEM Innovators in microwave technology, CEM
Matthews, USA), and 7 ml of concentrated nitric acid
was added. The mixture was heated in a microwave oven
(MES-81 D, CEM Matthews, USA) for 25 min (5 min at
full micro-wave power, 10 min at 75% of full micro-wave
power and 10 min at full micro-wave power). After heating, 5 drops of H2O2 (30%) were added to each vessel
and the vessels were left at room temperature overnight.
The samples were quantitatively transferred to 10 ml volumetric asks with Milli-Q water. After 1:10 dilution the
solution was analysed for iron using AAS (Atomic Absorption Spectrometer 3300, Perkin-Elmer with an Iron-lamp,
Intensitron lamp, Perkin-Elmer, Wellesley, MA 024814078, USA) using a standard curve based on an iron standard (Titrisol). These solutions were analysed for zinc using
an AAS (Atomic Absorption Spectrometer 3300, with a
Zinc-lamp, Intensitron lamp, Perkin-Elmer, Wellesley,
MA 02481-4078, USA) using a standard curve based on
a zinc standard (Titrisol). The AAS method was controlled
with a standard certied reference material (Bovine muscle,
Commission of the European communities, Community
bureau of reference BCR, Reference material no. 184).
The presented values are means of duplicate measurements.
2.5. Data analysis
Average values of the Fe content, Zn content, water
activity, water content, salt content and the initial colour
and I were compared by Students t-test and the correlations between pairs of variables were calculated as Pearsons correlation coecients. The formation of Zn-pp
measured as I in the meat curing experiment were analyzed by a two-way analysis of variance, including as main
factors curing method and storage time. All the statistical
analyses were done in the Analyst application within the
SAS system statistical software 8.02 (SAS Institute, Cary,
NC, USA).
3. Results and discussion
Zinc porphyrin has now been demonstrated to be the
main pigment in Parma ham and, in order to obtain more
knowledge about how processing parameters aect the formation of Zn-pp in meat products, Parma ham, Iberian
ham, dry-cured ham with nitrite, raw bacon with nitrite,
Kaaree-Speck with nitrite, cooked ham with nitrite and
raw ham meat were compared with respect to their Zn-pp
content. The results obtained for Fe content, Zn content,
water activity, water content, salt content, the initial colour
and the uorescence intensity I of the extract are shown in
Table 2. Due to large variation in origin and processing,
the seven ham/meat types were rather dierent in composition and initial colour (a*-value). Especially, Fe and Zn
contents were found to be higher for Parma ham and
Iberian ham compared to other meat products. For the

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C.E. Adamsen et al. / Meat Science 72 (2006) 672679

Table 2
Content of Fe (lg/g dry matter), Zn (lg/g dry matter), water activity, water content (g water/100 g meat), salt content (g NaCl/ 100 g meat), initial colour
(a*value) and uorescence intensity I (kex = 420 nm, kem = 590 nm, relative to a 1 mg/ml riboavin standard) of Zn-porphyrin in the extract of dierent
ham types, produced by varying process (dry-cured, brine-cured, cooked and raw) and with or without nitrite added
Type of ham

Fe (Fe lg/
g dry matter)

Zn (Zn lg/
g dry matter)

aw

Water (g water/
100 g meat)

Salt (g NaCl/
100 g meat)

Colour
(*a-value)

Dry-cured Parma ham

Dry-cured Iberian ham
Dry-cured ham with nitrite
Raw bacon with nitrite
Raw ham meat
Karree-Speck with nitrite
Cooked ham with nitrite

35.4 1.6
50.8 8.4
28.3 2.1
30.2 1.6
27.1 1.6
29.2 1.3
27.5 1.0

107.2 2.9
110.6 6.6
92.5 0.1
110.6 8.4
72.2 1.4
83.2 4.2
68.3 2.9

0.915 0.001
0.872 0.001
0.901 0.001
0.947 0.002
0.994 0.003
0.896 0.004
0.967 0.001

57.5 1.2
44.0 0.5
58.0 0.8
69.1 0.05
73.3 0.8
54.1 2.5
74.2 0.6

4.1 0.2
5.9 0.2
7.7 0.1
3.8 0.4
ND
6.1 0.1
3.8 0.2

5.1 0.04
9.8 0.5
13.4 0.7
7.4 0.2
9.3 0.4
12.2 0.03
9.9 0.4

39 7
25 4
0.51 0.05
0.25 0.02
0.13 0.02
0.06 0.04
0.005 0.002

ND = non detectable.
Values are the means standard deviations.

initial colour, it was observed that the addition of nitrite

leads to higher a*-values (more red colour) as expected,
since nitrite is known to contribute to the colour formation
in cured meat products (Mller & Skibsted, 2002).
Fig. 1 shows uorescence spectra of the red pigment
extracted with 75% acetone/water solution as described
by Wakamatsu et al. (2004a) from dry-cured ham produced without nitrite/nitrate (Fig. 1A), and of extracts
from the ve other meat products cured with nitrite and
of extract from raw ham (Fig. 1B). The uorescence spectra
obtained using excitation at 420 nm of extracts from the
seven dierent types of ham/meat, all show two emission
maxima, a strong band at 588590 nm and a weak band
at 640642 nm (Fig. 1), respectively, and the spectra clearly
dier in intensity. The uorescence spectra from all seven
types of ham/meat show the characteristic spectrum of
Zn-pp (the uorescence emission maximum of Zn-pp is
583589 nm) comparable with the ndings for extracts
from Parma ham and with the uorescence spectrum of

Fluorescence Intensity (EX 420 nm)

400

300
200
100
0
500

550

600

650

700

4
3
2
1
0
500

550

600

650

puried Zn-pp as reported by Wakamatsu et al. (2004a).

Mean values of the uorescence emission intensities at
589 nm, I, are reported in Table 2, and the intensities
clearly dier. The highest I is observed for Parma ham
and Iberian ham, which are signicantly higher (p < 0.001)
than dry-cured ham produced with nitrite, raw bacon produced with nitrite, raw ham meat, Karree-speck and
cooked ham produced with nitrite (Table 2).
The possible relationship between I of the meat products extracts and the physiochemical parameters measured
for the dierent ham/meat products (Fe, Zn, salt and water
content; plus aw, and initial colour) has been assessed by
analysis of Pearsons correlations. The uorescence intensities are logarithmically transformed (log I) prior to correlation analysis in order to obtain a normal distribution. The
Pearsons correlation coecients (Table 3) show that both
Zn (p < 0.001) and Fe (p < 0.01) content are positively correlated to log I, with values of 0.79 and 0.71, respectively.
Fig. 2 shows scatter plots for log I as a function of the Zn
content and Fe content. It can be observed that the log I
correlates much better with the Zn content compared to
Fe content indicating that the formation of Zn-pp is logarithmically proportional to the Zn content in the range
investigated. In contrast, a negative correlation between
log I and either aw (p < 0.05) or water content (p < 0.05)
can be observed (r =
0.56 and
0.61, respectively), in

700

Wavelength (nm)
Fig. 1. Fluorescence spectra of red pigment extracted with 75% acetone/
water solution from Parma ham (full line), Iberian ham (broken line) (A),
dry-cured ham with nitrite (dotted line), raw bacon with nitrite (dot-dash
line), raw ham meat (dot-dot-dash line), Kaaree-Speck with nitrite (full line)
and cooked ham with nitrite (broken line) (B). All spectra were recorded at
25 C. Samples were excitated at 420 nm and the emission spectra were
measured from 500 to 700 nm.

Table 3
Correlation coecients of composition Fe (lg/g dry matter), Zn (lg/g dry
matter), water activity, water content (g water/100 g meat), salt content (g
NaCl/100 g meat) and initial colour (a*value) and log I for the 7 dierent
ham types (see Table 2)
Variable

Fe

Zn
aw
Water content
Salt content
Colour
Log I

0.58*

0.61*
0.60*

0.49

0.54
0.94***
0.26
0.36

0.86***
0.75**
0.58*
0.39

0.66**
0.70** 0.42
0.71**
0.79***
0.56*

0.61* 0.19

*
**
***

Signicant p < 0.05.

Signicant p < 0.01.
Signicant p < 0.001.

Zn

aw

Water Salt
Colour
content content

0.65*

C.E. Adamsen et al. / Meat Science 72 (2006) 672679

-1

-2

-2

-3

-3
80

100

120

Zinc content
(g/g dry matter)

Corr. 0.71

-1

60

Corr. 0.79

LogI fl

LogI fl

20

30

40

50

60

Iron content
(g/g dry matter)

Fig. 2. Scatter plot for log I as a function of the Zn content (lg/g dry
matter) (A) and Fe content (lg/g dry matter) (B) for the 7 dierent ham
types, with varying process (dry-cured, brine-cured, cooked and raw) and
use of additive (with/without nitrite). The correlations coecient are given
in the gure.

agreement with the observed higher values for I in drycured products (higher concentration of Zn-pp) compared
to other types of meat/meat products. A positive correlation between log I and surface colour measured as a*-value
(p < 0.05; r = 0.65) was found, which is in agreement with
the recent suggestion that Zn-pp is the principal pigment
in dry-cured ham produced without nitrite or nitrate (Wakamatsu et al., 2004a). However, no signicant correlation
between log I and the salt content was found, despite the
fact that the salt content is much higher for dry-cured meat
products compared to brine-cured bacon and ham.
A clear correlation between aw, water content, and salt
content was observed (Table 3), which is expected due to
the interrelationship between these parameters. Also a neg-

677

ative correlation between surface colour and aw (p < 0.01)

or water content (p < 0.01) (r =
0.56 and
0.70, respectively) was found conrming that meat products with
reduced water content and low aw contain a higher concentration of pigments. A weaker correlation (p < 0.05) was
observed between Fe, Zn and aw, and between Fe content
and surface colour (p < 0.05), indicating also that Fe content, possibly through a higher pigment concentration,
has an eect on the observed colour of the meat/meat products, although the relationship is less clear.
The total processing time seems to have a positive eect
on the formation of the Zn-pp. The dry-cured hams, Parma
ham and Iberian ham (manufacturing time > 12 months)
both have much higher I than all other types of meat
products considered, which in general all have much
shorter processing times <3 months.
Besides the long maturation time involved in production
of Parma ham and Iberian ham other processing parameters could favour the formation of Zn-pp and a meat model
system was established in order to investigate the eect of
other such parameters and to understand possible reaction
mechanisms. The average Fe and Zn content in the SM
muscles used as the model system was 31.1 lg/g dry matter
and 76.4 lg/g dry matter, respectively, and the average
post-mortem pH was between 5.55.7. Nine recipies for
curing were used, and the meat was characterized by measuring aw, water content, salt content, initial colour (a*value) and nitrite content, and the results are shown in
Table 4. From Table 4 it can be seen, that meat cured with
15% (w/w) brine reached a salt content of approximately
6.4 g NaCl/100 g meat, while meat cured with 25% (w/w)
brine had a salt content of approximately 9.3 g NaCl/
100 g meat and the corresponding aw of these two cured
meat model systems were 0.946 and 0.905, respectively.
Brine with 1% (w/w) sodium nitrite resulted in a nitrite
content of approximately 1 mg NO

2 =g meat. The initial

Table 4
Water activity aw, water content (g water/100 g meat), salt content (g NaCl/ 100 g meat) and nitrite content (mg NO

2 =g meat) found for meat cured in
dierent brines followed 42 day of curing
Meat model systems

aw

Water content
(g water/ 100 g meat)

Salt content
(g NaCl/100 g meat)

Colour
(*a-value)

Nitrite content
(mg NO

2 =g meat)

Meat with out curing (control)

Meat cured with 15% (w/w) brine
Meat cured with 25% (w/w) brine
Meat cured with 15% (w/w) brine
plus 1% (w/w) sodium nitrite
Meat cured with 25% (w/w) brine
plus 1% (w/w) sodium nitrite
Meat cured with 15% (w/w) brine
plus 0.6% (w/w) Zn-acetate
Meat cured with 25% (w/w) brine
plus 0.6% (w/w) Zn-acetate
Meat cured with 15% (w/w) brine
plus 0.6% (w/w) Zn-acetate
and 1% (w/w) sodium nitrite
Meat cured with 25% (w/w) brine
plus 0.6% (w/w) Zn-acetate
and 1% (w/w) sodium nitrite

0.984 0.001
0.934 0.002
0.895 0.002
0.945 0.002

74.8 0.07
73.6 0.13
67.7 0.26
73.4 0.48

ND
6.7 0.04
9.2 0.04
6.4 0.05

0.12 0.65
0.44 0.09
0.71 0.27
4.90 0.31

ND
ND
ND
1.1 0.28

0.900 0.002

69.1 0.14

9.1 0.06

6.42 0.11

1.0 0.13

0.951 0.002

72.1 0.47

6.8 0.21

0.39 0.06

ND

0.912 0.001

67.0 0.91

9.8 0.08

0.24 0.08

ND

0.953 0.001

72.5 0.38

5.6 0.21

6.28 0.26

1.1 0.04

0.913 0.004

67.8 0.14

9.0 0.11

6.31 0.27

0.9 0.08

ND = non detectable.

678

C.E. Adamsen et al. / Meat Science 72 (2006) 672679

colour (Table 4) of meat cured with nitrite was more red as

evidenced by higher a*-values as expected from the reaction of nitrite with meat products.
Formation of Zn-pp was followed by uorescence spectroscopy, and as may be seen from Fig. 3, uorescence
intensity I (kex = 420 nm, kem = 590 nm, relative to a
1 mg/ml riboavin standard) for extracts of the meat
exposed to the nine dierent curing conditions at 5 C for
42 days varied considerably. I was measured at days 0,
7, 14 and 42 and as further seen from Fig. 3A, Zn-pp (I)
was formed and the amount increased with time for raw
meat (control), meat cured in 15% (w/w) brine and meat
cured in 25% (w/w) brine. The concentration of Zn-pp
reached a maximum after 14 day of storage followed by a
decrease in the Zn-pp content for raw meat (control), meat
cured in 15% (w/w) brine and meat cured in 25% (w/w)
brine. The formation of Zn-pp was more pronounced for
raw meat (control) and meat cured in 15% (w/w) brine.
For meat cured in 25% (w/w) brine, the formation of
Zn-pp was signicantly lower compared with raw meat
(control) and meat cured in 15% (w/w) brine. For meat
cured with nitrite and 15% (w/w) salt brine or nitrite and
25% (w/w) brine, zinc acetate and 15% (w/w) brine or
25% (w/w) brine, nitrite and zinc acetate and 15% (w/w)
brine or 25% (w/w) brine, no formation of Zn-pp was
observed through the curing period.

I fl

1.2

1.2

1.0

1.0

0.8

0.8

0.6

0.6

0.4

0.4

0.2

0.2

0.0

Acknowledgements

0.0
0

20

10

30

40

1.2

50

C
2+

1.0

I fl

B
+ NO2

+ Zn

20

10

30

40

50

1.2

1.0

+ NO and Zn

0.8

0.8

0.6

0.6

0.4

0.4

0.2

0.2

2+

0.0

0.0
0

10

20

30

Time (Day)

40

50

The change in red colour measured as a*-value was also

followed on days 0, 7, 14 and 42, but no correlation between
the formation of Zn-pp and the change in the red colour was
obtained (data not shown). Only the well known eect on
red colour from curing with nitrite was observed.
The observation that Zn(II), added as zinc acetate, had
no eect on the formation of Zn-pp was remarkable, since
it suggests that the displacement of Fe from Mb is not a
bimolecular substitution process. The rate determining step
may rather be a dissociation of Fe(II) from Mb under nonoxidizing condition followed by a fast binding of Zn (II).
In conclusion, our results show that Zn-pp is present
not only in Parma ham, but also in other meat products
although in a lower concentration. In addition, our experiments have demonstrated that the use of nitrite as a curing ingredient inhibits the formation of Zn-pp. This
inhibition was demonstrated for dierent cured and dry
cured meat products and the eect was conrmed in a
meat model system, although the content of nitrite in
the model system was far beyond what is legally permitted
and much higher than in real meat products. It seems,
however, not possible to identify the fundamental mechanism behind formation of Zn-pp in meat products. Three
possible mechanisms for the metal substation are: (i) a
non-enzymatic enzymatic reaction driven by binding of
iron in the high chloride meat matrix, (ii) a bacterial enzymatic reaction or (iii) an endogenous enzymatic reaction.
A more detailed understanding of the chemistry and the
mechanism behind the substitution between Fe and Zn
will, however, have to await a kinetic study of salt-driven
dissociation of Fe from myoglobin and the reaction
between nitrite and myoglobin, which seems to inhibit
the dissociation of Fe from myoglobin.

10

20

30

40

50

Time (Day)

Fig. 3. Changes in uorescence intensity I (kex = 420 nm, kem = 590 nm,
relative to a 1 mg/ml riboavin standard) for Zn-pp in extract of: (A)
Meat without curing (control) (square), meat cured with 15% (w/w) brine
(circle) and meat cured with 25% (w/w) brine (triangle). (B) Meat cured
with 15% (w/w) brine plus 1% (w/w) sodium nitrite (circle) and meat cured
with 25% (w/w) brine plus 1% (w/w) sodium nitrite (triangle). (C) Meat
cured with 15% (w/w) brine plus 0.6% (w/w) zinc acetate (open circle) and
meat cured with 25% (w/w) brine plus 0.6% (w/w) zinc acetate (open
triangle). (D) Meat cured with 15% (w/w) brine plus 0.6% (w/w) zinc
acetate and 1%(w/w) sodium nitrite (open circle) and meat cured with 25%
(w/w) brine plus 0.6% (w/w) zinc acetate and 1% (w/w) sodium nitrite
(open triangle) during curing. Values are means of three independent
measurements standard deviations, indicated by bars.

The present study was partly funded by the Research

School for Organic Agriculture and Food Systems (SOAR)
and the Norma and Frode S. Jacobsens Fond, Copenhagen,
Denmark. We would like to thank Dr. Giovanni Parolari,
Stazione Sperimentale per lIndustria delle Conserve Alimentari, Parma, Italy, for the kind donation of Parma
ham samples, Dr. Ana Isabel Andres Nieto, the University
of Extremadura, Spain, for the arranging the contact to the
producer of Iberian ham, Puro Iberico Artesana, S.L., Badajoz, Spain and Bent Olesen, Tulip, Vejle, Denmark, for the
kind donation of nitrite based dry-cured ham samples.
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