Review

Nitric oxide and quality and safety of muscle based foods

Leif H. Skibsted
Food Chemistry, Department of Food Science, Faculty of Life Science, University of Copenhagen, Rolighedsvej 30, DK-1958 Frederiksberg C, Denmark
a r t i c l e i n f o
Article history:
Received 19 November 2010
Revised 16 March 2011
Keywords:
Meat curing
Nitrosylmyoglobin
Nitric oxide
Nitroxyl
Nitrosamine
Lipid oxidation
a b s t r a c t
Preservation of meat with nitrite or nitrate has become important to mankind in controlling meat spoil-
age and in producing safe and palatable meat products with good keeping properties even at ambient
temperature. Nitric oxide was early recognised as pivotal for colour and colour stability of such meat
products. Later specic effects on microbial growth became evident, followed by an understanding of
nitric oxide as an antioxidant in processed meat, while a future recognition of nitric oxide as modulator
of transmetallisation reactions in meat seems possible. Central for all these functions of nitric oxide in
meat is the heme cavity in the meat pigment myoglobin with its facile conversions among reactive oxy-
gen and nitrogen species in certain cases assisted by curing additives such as ascorbate and with a pos-
sible involvement of nitroxyl.
2011 Published by Elsevier Inc.
Contents
Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176
Formation of nitric oxide in meat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177
Alternative to nitrite . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179
Oxidative processes in cured meats. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179
Discoloration and rancidity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180
Other aspects of nitric oxide in meat. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
Perspective. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182
Introduction
Salt has been used to preserve meat and sh since ancient
times. Salt binds water and in the amounts traditionally used for
meat and sh, water activity is lowered below the limit for growth
of most microorganisms in effect inhibiting microbial spoilage. In
the 19th century it became evident that some of the less clean
qualities of salt were better than the more pure salt in preserving
meat. As described by Honikel [1], KNO
3
known as saltpetre was
identied as the active contaminant, which in brine could be con-
verted to nitrite by some microorganisms and further turn the
salted meat attractively red [1,2]. Nitrosylmyoglobin was identi-
ed following extraction from such cured meats by Haldane [3]
as early as 1901 opening up for a gradual understanding of the
chemistry behind meat curing. Nitrosylmyoglobin, MbFe(II)NO, is
bright red and formed by reaction of nitrous acid, the conjugate
acid of nitrite, in the meat matrix with oxymyoglobin in a complex
pattern of processes still only being partly understood [4].
Nitrite now being routinely used in industrial meat curing can
be toxic and great concerns have been expressed over the years
of the exposure of individuals by intake of cured meats to the reac-
tion products of nitrite formed in meat and to any residual nitrite
still present in meat after curing [5]. The view of negative health
effects of dietary nitrate and nitrite is now less predominant [6].
However, some population segments are still in favour of at
complete ban on especially nitrite as a food additive. Nitrate is nor-
mally used for curing of meat with added starter cultures based on
bacteria with nitrate reductase activity converting nitrate to
nitrite.
During meat curing, the nitric oxide formed from nitrite reacts
with various components present in the muscle. Many of these
reaction products have been found to be important for controlling
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doi:10.1016/j.niox.2011.03.307
E-mail address: ls@life.ku.dk
Nitric Oxide 24 (2011) 176183
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lipid oxidation otherwise deteriorative for salted and cooked meat
products [2]. Research efforts have accordingly not only been di-
rected towards an understanding of the chemistry behind forma-
tion of such compounds in meat during curing but also of their
protection during any maturation period and during subsequent
storage and display in the retail trade [79].
Protection of meat products against growth of spore forming
bacteria especially Clostridium botulinum strains have been impor-
tant in relation to legislation concerning use of nitrite [5]. How-
ever, traditionally cured meat products like the Parma ham and
some Spanish hams are produced with the use of sodium chloride
alone in the form of pure sea salt. Still these dry-cured products
turn red with good colour stability and do not go rancid [10]. The
absence of nitric oxide in these stable products is astonishing,
but apparently allows chemical reactions to occur otherwise pre-
vented by nitric oxide [11,12].
Nitric oxide ties the many aspects of meat curing together
including safety and quality aspects of meat products and will
accordingly form the focal point of the present review.
Formation of nitric oxide in meat
Curing of meat is most signicantly recognised by the colour
changes as the fresh red meat or meat batter initially turns brown
and subsequently again red following addition of nitrite and salt
possibly also together with other curing additives such as ascor-
bate. In contrast to fresh meat, cured meat does not develop the
rancid and oxidised smell and taste known as warmed-over-a-
vour upon heating and especially reheating, in contrast unique a-
vours are developed as known for sausages, hams and bacon. The
improved oxidative stability of cured meat relates to the transfor-
mation of the meat pigments as evidenced by the colour changes
[2].
Nitrite (E
h
= 0.46 V) added to meat acts primarily as an oxidant:
NO

2
H
2
O e

!NO 2OH

1
especially at low pH (2). Endogeneous reductants such as NADH or
added ascorbate or erythorbate become oxidised to yield NO with a
1:2 stoechiometry:
Ascorbic acid 2HNO
2
!Dehydroascorbic acid 2NO 2H
2
O
2
Kinetic aspects are important for NO formation and while the
reactivity of nitrous acid/nitrite increases with decreasing pH, the
reactivity of ascorbic acid/ascorbate increases with increasing pH,
see Fig. 1. The reaction between the anhydrous form of nitrous
acid, N
2
O
3
, and hydrogen ascorbate has been demonstrated to be
quantitatively the most important for conditions as those prevail-
ing in the meat matrix [13,14]. For increasing salt concentration ni-
trous acid may transform into nitrosyl chloride:
HNO
2
H

Cl

!NOCl H
2
O 3
NOCl is more reactive than N
2
O
3
, although less reactive than
nitrosium acidium, H
2
NO

2
, or the kinetically equivalent nitrosium
ion, NO
+
, a strong electrophile, which, however, must be concluded
to have little if any importance during meat curing [15]. The
importance of N
2
O
3
as reactant is conrmed by the second-order
dependence for the rate of disappearance for ascorbic acid on ni-
trous acid concentration in agreement with the following reaction
sequence with formation of N
2
O
3
as rate determining [16]:
2HNO
2
!
k
2
N
2
O
3
H
2
O 4
N
2
O
3
HAsc

!
fast
reaction intermediates 5
As many as seven intermediates have been suggested for the
subsequent reactions including an unknown powerful nitrosating
agent important for nitrosation of heme proteins including the
pK
a
= 4 +/
+
H
2
O + NO
+
pK
a
= 3.22 +/ H
+
HNO
2
NO
2

+/ H
2
O
N
2
O
3
O
O
OH HO
HO
OH
O
O
OH
-
O
HO
OH
pK
a
= 4.04
+/ H
+
pK
a
= 11.34
+/ H
+
O
O
O
- -
O
HO
OH
Fig. 1. The reaction between N
2
O
3
and hydrogen ascorbate contributes most signicantly to formation of NO in meat followed by the reaction between N
2
O
3
and ascorbic
acid [13,14].
L.H. Skibsted/ Nitric Oxide 24 (2011) 176183 177
myoglobins [9]. Both 3-nitrosoascorbic acid, suggested as the ini-
tially formed of the intermediates as may be seen from Fig. 2,
and the 2,3-dinitroascorbic acid subsequently formed, have been
considered as the key intermediate for transfer of NO to myoglo-
bins, but none of these nitrous acid esters of ascorbic acid as an
endiol seem, however, to be the actual reactant [9]. The involve-
ment of thiol groups, as present in glutathione and in cystein side
chains in protein, through formation of S-nitrosothiol groups
should also be considered as possible intermediates in the transfer
of NO in meat during curing, since it now is known, that reactive
cystein side chain and their radicals are important for cross-linking
of the muscle protein myosin [19].
Three reactions seem possible for 3-nitrosoascorbate: (i) homo-
lytic cleavage to yield NO and the ascorbyl radical, (ii) hydrolysis to
yield ascorbate and nitrite as a backreaction, and (iii) after proton-
ation to form 3-nitrosoascorbic acid as shown in Fig. 2, cleavage to
yield dehydroascorbate, a common oxidation product of ascorbic
acid, and nitroxyl, HNO. These reactions are all exergonic, and for-
mation of HNO seems to dominate under physiological conditions
and possibly also in the meat matrix [20]. HNO seems, however,
not to have been considered as a reactive intermediate in meat cur-
ing [9]. HNO is the reduced form of NO; which although short-
lived, may be the long-sought nitrosating agent for metmyoglobin,
MbFe(III), operating through a reductive nitrosylation [21]:
MbFeIII HNO !MbFeIINO H

6
The life-time of HNO with respect to oxidation by O
2
or dimer-
ization to H
2
N
2
O
2
to form H
2
O and N
2
O (used as a reporter mole-
cule for HNO) seems to have been underestimated and appears
long enough for reaction with MbFe(III) [21]. Furthermore, the acid
dissociation of HNO is relatively slow due to the requirement of a
change in spin-state:
1
HNO !
3
NO

7
HNO deserves more attention in mechanistic investigations of
reactions of nitrite in meat.
The formation of N-nitrosamines in cured meat has been of con-
cern especially for frying at high temperature of cured meats like
bacon:
R
1
R
2
NHHNO
2
!R
1
R
2
NNO H
2
O 8
During meat curing at pH relevant for meat, ascorbate reacts
faster than secondary amines such as proline with the nitrosating
agent N
2
O
3
[22]. Excess ascorbate may accordingly prevent forma-
tion of potentially carcinogenic N-nitroso compounds [23]. Meat
proteins serve, however, as a reservoir for NO and nitrosating
agents, and any residual nitrite is a hidden NO generating pool
providing nitric oxide for the numerous reactions occurring during
storage and cooking of cured meats (outlined in Fig. 3), also when
ascorbate is becoming depleted.
Nitrite will oxidise myoglobin to yield metmyoglobin
(E
h
= 0.06 V), cf. the reaction of Eq. (1):
MbFeII NO

2
H
2
O !MbFeIII NO 2OH

9
and this reaction is responsible for the initial colour change from
the fresh red meat colour of MbFe(II)O
2
(in equilibrium with the
violet MbFe(II) depending on oxygen pressure) to the brown colour
of MbFe(III), which is seen during curing of meat or meat batter.
Subsequently MbFe(III) is reduced to MbFe(II) by reductants like
NADHor added ascorbate. Thermodynamical calculations show that
NO cannot reduce MbFe(III) or MbFe(III)NO [2], but HNO may. The
reduction of MbFe(III) causes a second colour transformation to
yield the characteristic red colour of cured meat, since NO binds
strongly to MbFe(II):
MbFeII NO !MbFeIINO 10
A denaturated pink form of nitrosylmyoglobin, the nitrosylhe-
mochromogen pigment, is characteristic for cooked cured meat
products such as many hams and sausages [7].
The ligand substitution reaction to yield MbFe(II)NO:
MbFeIIO
2
NO !MbFeIINO O
2
11
is of little if any importance despite a favourable equilibrium con-
stant of 10
5
[2], since a competing redox reaction is very fast
(k
2
= 3.7 10
7
l mol
1
s
1
at 25 C [24]):
MbFeIIO
2
NO H
2
O !MbFeIIIH
2
O NO

3
12
and becomes a source for nitrate in meat cured with nitrite. Nitrite
and NO may both coordinate to metmyoglobin although weakly
[25], and reduction of these iron(III) species has also been recogni-
sed as a reaction path for formation of nitrosylmyoglobin [26,27]:
Fig. 2. Intermediates in the reaction between nitrite and ascorbic acid/ascorbate in
meat added salt are reactive nitrosating agents or form NO as shown for
dinitrosoascorbic acid. The mechanism as proposed by Fox et al. [17,18] involves
a dismutation of a dimeric form of 3-nitrosoascorbic acid, which not is in agreement
with the principle of microscopic reversibility [2].
Fig. 3. NO formed by nitrite during meat curing can participate in numerous
reactions modifying proteins and pigments.
178 L.H. Skibsted / Nitric Oxide 24 (2011) 176183
MbFeIIINO
2
NO

2
!MbFeIINO NO

3
13
For MbFe(II)NO other reductants than NO have, however, to be
available, since the proposed reaction [27]:
MbFeIIINO NO H
2
O !MbFeIINO HNO
2
H

14
has been shown not to be energetically favoured even at high pH
[2]. MbFe(III)NO may, however, be more adequately described as
the imidazole p-cation radical of MbFe(II)NO (Fig. 4), and this pro-
tein radical may be reduced by electron-rich groups within the pro-
tein or by exogeneous reductants [28,29]. MbFe(II)NO is formed
with iron(II) hexacoordinated, however, NO is strongly trans-labiliz-
ing, and ESR-studies have shown that nitrosylmyoglobin in meat
easily converts to pentacoordination of iron(II). In cooked cured
meat, the pink so-called nitrosylhemochromogen pigment is a pen-
tacoordinate nitrosylprotohaem trapped inside a matrix of denatur-
ated globin for which reaction of amino acid side chains with nitrite
further may have contributed to the denaturation [28].
Alternative to nitrite
In the search of alternatives to nitrite for meat curing, nitrosyl-
heme pigments synthesized outside the meat matrix has been pro-
posed as food additives [30]. Likewise have various bacteria shown
to modify oxidised heme pigments to yield MbFe(II)O
2
or MbFe(II)-
NO been tested as starter cultures for meat products [31]. Bacterial
NO synthases (NOS) have further been isolated, and although their
biological signicance remains unclear in such unicellular organ-
isms, the relevant bacteria including strains of Lactobacillus fermen-
tum are relevant to the meat industry [32]. Nitrate reductase
activity has also been found in some Lactobacillus species used as
starter cultures by the meat industry and capable of producing
NO

2
, NO and N
2
O [33]. Notably N
2
O is known as a reporter mol-
ecule for the intermediary of HNO to be considered a potential
MbFe(III) reductant [20]. Nitrite is important for protection of meat
products against the toxin producingC. botulinum and growth of its
heat-resistant spores, and several mechanisms for the effective
growth inhibition by NO
2
-
have been considered. A heme protein
in C. botulinum displays sensitivity to NO at a femto-molar level
and may be the ultimate taget for nitrite inhibition of this patho-
genic bacterium responsible for botulism [34].
Oxidative processes in cured meats
Nitrite is a strong oxidant, ascorbate is effective as reductant in
the Fenton reaction producing hydroxyl radicals in presence of
iron, while chloride is a more unspecic prooxidant in many bio-
logical systems, still their combination when added to meat pro-
duces meat products with little lipid oxidation [1]. Various
explanations in favour of nitrite as the active antioxidant have
been offered including iron chelation and stabilization of unsatu-
rated lipids by NO

2
[35]. However, NO derived from NO

2
now
seems to be accepted as the species interfering with the free radical
intermediates in lipid oxidation in cured meat products [3638].
N
N
H
N
N
H
N
H
N
O
N
N
O
N
H
N
(-)
(+)
e
-
Nitrosylmyoglobin
(hexa-coordinated)
Fe
II
Nitrosylmyoglobin
(penta-coordinated)
Fe
II
His
93
(F8)
His
93
(F8)
(-)
His
64
(E7)
(-)
(+)
(-)
(+)
His
64
(E7)
N N
H
N
O
N
N
H
N
N
H
N
H
N
N
O
(+)
His
64
(E7)
(-)
Nitrosylmetmyoglobin
Fe
III
(+)
His
9
3
(F8)
(-)
His
93
(F8)
Nitrosylmyoglobin
radical cation
+
.
Autoreduction Fe
II
(-)
(+)
His
6
4
(E7)
Fig. 4. A reaction mechanism for formation of MbFe(II)NO from initially formed MbFe(III)NO through reduction of an intermediate p-cation radical as proposed by Bonnet
et al. [28].
L.H. Skibsted/ Nitric Oxide 24 (2011) 176183 179
During lipid oxidation NO, being a lipophilic radical with a rel-
atively long lifetime, may combine with lipid peroxyl radicals to
form non-radical addition products, in effect breaking the radical
chain processes characteristic of oxidation of unsaturated lipids
[39]. NO reacts rapidly with most radicals including the lipid de-
rived alkyl (L

), alkoxyl (LO

), and peroxyl radicals (LOO

). For reac-
tion of NO with the hydroxyl radical (HO

) and the peroxyl radical:

NO HO

!HNO
2
!H

NO

2
15
NO LOO

!LOONO !LONO
2
16
the diffusion limit is approached. The reaction of NO with the super-
oxide radical anion is also close to the diffusion limit:
NO O

2
!ONOO

!NO

3
17
and peroxynitrite as product may initiate lipid oxidation or oxidise
meat proteins or deactivate by isomerisation to nitrate [40].
MbFe(II)NO should be considered as an antioxidant buffer. For
the radical exchange reaction:
MbFeII
15
NO NO !MbFeIINO
15
NO 18
studied by isotopic labelling, the activation barrier was found to
have the low value of DH
#
= 47 kJ mol
1
indicating that regenera-
tion of active NO derived antioxidants is facile even at low temper-
ature [41]. MbFe(II)NO appears accordingly to be active as an
antioxidant both through dissociation of NO:
MbFeIINO !MbFeII NO 19
and through direct reaction with activated oxygen species [36]:
MbFeIINO LOO

!non-radical products 20
Notably, reaction of H
2
O
2
with MbFe(II)NO does not yield
hypervalent heme pigments like perferrylmyoglobin as the reac-
tion with MbFe(II)O
2
, and MbFe(II)NO is effectively deactivating
peroxides [38,42]. A comparison between the role of MbFe(II)O
2
in fresh meat with MbFe(II)NO in cured meat and their role as pro-
oxidant and antioxidant, respectively, may be seen in Fig. 5.
Discoloration and rancidity
Pigment oxidation in cured meat results in discoloration and
concomitant loss of oxidative stability of the meat lipids, since
the antioxidant buffer of MbFe(II)NO becomes depleted and more-
over the oxidation product MbFe(III), is prooxidative and a source
of hypervalent heme pigments. Protection of the red colour of
cured meats during retailing against oxidation is accordingly also
important for the avour stability and palatability of cured meats
and has been the subject of numerous studies in order to devise
improved packaging system[4345]. In contrast to the reaction be-
tween MbFe(II)O
2
and NO (Eq. (2)), the reaction between O
2
and
MbFe(II)NO is slow:
MbFeIINO O
2
!MbFeIII NO

3
21
despite the reaction products are the same for the two reactions
[4649]. Discoloration of cured meat is signicantly faster upon
light exposure compared to dark storage indicating photooxidation
of MbFe(II)NO [8]. Notably, the sensitivity to light disappears in the
absence of oxygen, and the reversible fading of the red colour of
vacuum packed ham during storage exposed to light often observed
indicates reformation upon oxygen depletion in packages of the
MbFe(II)NO initially photooxidised in agreement with a rapid inter-
change between the different pools of nitrite modied compounds,
cf. Figs. 3 and 5 [44].
Thermal oxidation of MbFe(II)NO by O
2
shows saturation kinet-
ics with a linear dependence of the rate on [O
2
] at low oxygen pres-
sure approaching a constant rate at approximately atmospheric
and higher oxygen pressure [46]. In contrast, the degree of photo-
oxidation depends linearly on oxygen pressure [46]. Both types of
reaction are rather insensitive to pH.
The thermal oxidation is best described as two consecutive
rst-order reactions of comparable rate [4750]. The initial step
in the thermal reaction depends on [O
2
] and has in air-saturated
aqueous solution the high activation barrier of DH
#
= 121 kJ mol
1
,
while the second and faster reaction step has a lower activation
barrier (DH
#
= 88 kJ mol
1
) and shows no dependence on oxygen
concentration [49]. The reaction intermediate is suggested to be
formed by ligand exchange:
MbFeIINO O
2
!fMbFeIIO
2
; NOg !MbFeIII NO

3
22
with bond breaking as rate determining in agreement with the high
value of DH
#
followed by a non-activated escape of NO into the
heme cavity and further relocation in available protein pockets
[51]. The elementary reaction by which NO dissociates may be as-
sisted by the O
2
entering the heme pocket, although the rate is very
similar to the rate of NO dissociation from MbFe(II)NO. The over-all
rate of thermal oxidation decreases with increasing pressure corre-
sponding to a positive volume of activation (DH
#
= 8 mL/mol),
which is in agreement with a dissociative mechanism for the rate-
determining ligand exchange [52].
The second reaction step is suggested to have a transition state
similar to the NO induced oxidation of MbFe(II)O
2
, the reaction of
Eq. (12), in which NO establishes contact with the O
2
coordinated
to Fe(II). For oxidation of MbFe(II)NO by O
2
, NO is approaching the
coordinated oxygen from within the heme cavity and transferring
an electron to O
2
to yield ONOO

and iron(III). The peroxynitrite

isomerizes rapidly coordinated to Fe(III) and leaves the heme cav-
ity as NO
3
-
, as depicted in Fig. 6. The negative entropy of activation
observed for this second reaction step (DS
#
64 J mol
1
K
1
)
suggest an inner-sphere electron transfer between cavity trapped
NO and coordinated O
2
[49].
The mechanism of oxidation of NO by MbFe(II)O
2
and of
MbFe(II)NO by O
2
is also relevant for myoglobin in the mammalian
muscle cell. Under normal O
2
supply, MbFe(II)O
2
becomes a nitric
O
2
.-
MbFe
III
MbFe
II
O
2
O
2
MbFe
II
H
2
O
2
O
2
Red
O
MMR
O
2
ox
NO
MbFe
III
MbFe
II
NO
H
2
O
2
red
MbFe
III
NO
ox
NO
2
-
red
red
ox
NO
3
-
.
O
2
ox
NO
MbFe
III
MbFe
II
NO
H
2
O
2
red
MbFe
III
NO
ox
NO
2
-
red
red
ox
NO
3
-
.
Fig. 5. A comparison between the prooxidative cycling of oxymyoglobin generating
superoxide radicals and the antioxidative cycling of nitrosylmyoglobin depleting
oxygen or peroxides and with regeneration of nitric oxide by various reductants.
180 L.H. Skibsted / Nitric Oxide 24 (2011) 176183
oxide oxidase ensuring rapid oxidation for control of the level of
NO (reaction of Eq. (12)). In the absence of O
2,
MbFe(II) stores NO
until O
2
again becomes available for oxidation of NO. The large rate
difference between these two reactions is simply controlled by the
ordering of binding of the two small molecules to myoglobin and is
of importance for cytoprotection [53].
For electronically excited MbFe(II)NO, an additional reaction
path opens up, as NO and MbFe(II) becomes separated followed
by fast rebinding for anaerobic conditions [50]. In the presence of
O
2
, nitric oxide rebinding was shown to be partly inhibited sug-
gesting formation of ONOO

, the nitrosyldioxyl radical, capable of

oxidising MbFe(II) to MbFe(III) and forming ONOO
-
. The appear-
ance of this reaction channel as shown in Fig. 6, is responsible
for the light sensitivity of MbFe(II)NO in the presence of O
2
. The
quantum yield is modest and shows little dependence on excita-
tion wavelength but this photooxidation is of outmost practical
importance for the meat industry [44,46,54].
Other aspects of nitric oxide in meat
Nitric oxide is concluded to be the important antioxidant in
cured meat operating through scavenging of lipid derived radicals
forming non-radical products and through deactivation of perox-
ides without forming hypervalent heme pigment otherwise capa-
ble of initiating lipid and protein oxidation [38,39]. NO has also
been found to inactivate oxidising hypervalent species like ferryl-
myoglobin by a two step mechanism [55]:
MbFeIV O NO !MbFeIIIONO !MbFeIII NO

2
23
It is unclear whether this type of reactions are of importance in
cured meat, since reaction of peroxides with MbFe(II)NO result in
formation of MbFe(III) and not MbFe(IV)=O. However, under oxida-
tive stress, any hypervalent heme formed becomes deactivated and
further, NO
2
-
is reformed.
Meat from pasture fed animals has a higher content of carote-
noids like b-carotene and lutein. Carotenoids are lipophilic and
for certain breeds, the adipose fat may even become of yellow col-
our. Reaction between NO and carotenoids in membranes is only
sparsely investigated, but NO seems to add to the conjugated poly-
ene backbone of carotenoids forming nitroxides [56,57]. Whether
such addition reactions, which has been demonstrated by Electron
Spin Resonance spectroscopy, are reversible remains unclear and it
is accordingly not known, whether carotenoids in adipose tissue or
membranes may serve as a NO reservoir like MbFe(II)NO under
aqueous conditions. The nitroxides formed are stable radicals and
may as such be active as antioxidants. Nitroxides have thus been
demonstrated to be effective scavengers of protein-derived free
radicals [58].
The development of red colour in hams cured with salt without
addition of nitrite or nitrate like the traditional Parma ham and
most variants of the Iberian hams was unexplained until recently
although various suggestions of other types of nitric oxide or ami-
no acid binding to myoglobins were offered [59]. The main chro-
mophore in Parma ham has now been identied as a zinc
complex of protoporphyrin IX [60]. This zinc complex formed by
transmetallation between myoglobins and zinc proteins during
the long maturation process of these dry-cured hams has now also
been identied at low concentrations in other meat products [12].
The mechanism is still uncertain, but it was demonstrated that the
enzyme Zn-chelatase remains active to some degree during the
long maturation process characteristic for traditional Parma ham
production [61]. Whether this or other enzymes are active in the
transmetallation process is still unclear, but nitrite added in the
curing process inhibits the formation of zinc protophorphyrin
[12]. More specically, NO has been found to decrease the forma-
tion of the Parma pigment by an unknown mechanism [62]. In con-
clusion, the absence of NO facilitates such transmetallation
reactions or the presence of NO poisons the same reaction. It
should further be noted that the transmetallation reaction appar-
ently must be coupled to another reactions, since iron binds stron-
ger to protophorphyrin than does zinc. Iron seems moreover to
become inactivated as a catalyst for lipid oxidation as Parma hams
are known for their high oxidative stability [59].
Perspective
Many cured meat products have high gastronomic value and
most countries have characteristic products highly appreciated lo-
cally or for export. In Europe for example, dry-fermented sausages
like the salamis of Southern Europe are characterised by a high le-
vel of free fatty acids formed by lipid hydrolysis, while similar
products of Northern Europe have avours more dependent on li-
pid oxidation [63]. Provided the recent recommendation of a re-
duced meat intake especially of processed meat [64], a future
scientic challenge is to understand which compounds in pro-
cessed meat are increasing the cancer risk and how to suppress
their formation. Meat curing using nitrite and nitrate has a long
tradition and used properly the resulting products is of high qual-
ity and safe. A correct use of nitrite in meat processing should al-
ways aim to reduce formation of nitrosamines and to increase
formation of nitrosylmyoglobin important as an antioxidant. Selec-
tion of meat with a good reducing capacity and appropriate low pH
from non-stressed animals is part of such a production strategy.
i
O
2
O
2
O
2
O
2
O
2
O
2
O
2
O
2
O
2
O
2
O
2
O
2
O
2
O
2
O
2
O
2
O
2
O
2
Fe
II
NO

His
93
Fe
II
NO

His
93
Fe
II
NO

His
93
Fe
II
NO

His
93
NO

O
2
Fe
II
His
93
NO

NO

O
2
Fe
II
His
93
His
93
Fe
III
NO
3
-
His
93
Fe
III
NO
3
-
NO
3
-
ii iii iv
O
2
O
2
O
2
O
2
O
2
O
2
Fe
II
NO

His
93
O
2
Fe
II
NO

His
93
Fe
II
NO

His
93
Fe
II
NO

His
93
Fe
II
NO

His
93
O
2
His
93
Fe
III
NO
3
-
His
93
Fe
III
NO
3
-
NO
3
-
O
2
O
2
O
2
O
2
O
2
O
2
O
2
O
2
O
2
O
2
O
2
O
2
O
2
O
2
O
2
O
2
O
2
O
2
O
2
O
2
O
2
O
2
O
2
O
2
O
2
O
2
O
2
O
2
O
2
O
2
O
2
O
2
O
2
photochemical
thermal
NO

Fe
II
O
O
His
93
NO

NO

Fe
II
O
O
His
93
O
2
O
2
O
2
O
2
O
2
O
2
v
vi
vii viii
ix
His
93
Fe
III
-
O
O
N
O
His
93
Fe
III
His
93
Fe
III
-
O
O
N
O
His
93
Fe
II
ONOO

His
93
Fe
II
ONOO

ONOO

His
93
Fe
III
ONOO
-
His
93
Fe
III
ONOO
-
ONOO
-
-i
Fig. 6. Mechanism of aerobic photochemical and thermal degradation of MbFe(II)NO, involving (i) hv; (i): rebinding; (ii): spin-allowed NO and O
2
combination; (iii):
electron transfer; (iv): catalytic isomerisation of peroxynitrite to nitrate; (v): O
2
binding to ferrous heme centre. Thermal degradation includes (vi) and (vii): oxygen diffusion
and ligand exchange; (viii): fast nitrosylation of MbFe(II)O
2
with formation of a ferric-peroxynitrite complex; (ix): catalytic isomerisation of peroxynitrite to nitrate. Adapted
from [50].
L.H. Skibsted/ Nitric Oxide 24 (2011) 176183 181
The role of nitric oxide is increasingly becoming understood,
and nitric oxide derived from nitrite seems important in inhibiting
lipid oxidation otherwise producing toxic lipid oxidation products.
Understood to a lesser degree is the role of hypervalent heme pig-
ment formed in meats and under their digestion and capable of ini-
tiation free radical processed involving long-lived protein radicals.
Nitric oxide counteract such processes under various physiological
conditions and possibly also in meat and during the digestion of
meat [6567]. The interaction of nitric oxide with peptide radicals
need further studies, since certain meat peptides like carnosine are
known to be important as endogenous antioxidant in muscles and
meat [68].
Acknowledgment
This research has been supported by the Danish Research Coun-
cil for Technology and Production as the grant 09-064212/FTP: No-
vel production strategy for cured meat.
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