Meat Science 93 (2013) 282–286

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Kinetics of nitrite evaluated in a meat product

G. Barbieri ⁎, M. Bergamaschi, Ge. Barbieri, M. FranceschiniStazione Sperimentale per l'Industria delle Conserve Alimentari, 43100 Parma, Italy

⁎ Corresponding author.E-mail address: giampiero.barbieri@ssica.it (G. Barb

0309-1740/$ – see front matter © 2012 Elsevier Ltd. Allhttp://dx.doi.org/10.1016/j.meatsci.2012.09.003

a b s t r a c t

a r t i c l e i n f o

Article history:Received 27 February 2012Received in revised form 31 August 2012Accepted 3 September 2012

Keywords:NitriteKineticsCooked meat

The evaluation of the efficiency with which the reactions involving nitrite proceed in mortadella and of theeffect exercised on their kinetics by some variables (ingoing amount of sodium nitrite and temperature) isthe purpose of this work. Kinetics parameters were calculated at each level of nitrite added (40, 70, 100and 150 mg/kg) and at five temperature (55°, 60°, 65°, 70° and 72 °C). While the colour formation reactionis favoured by low activation energy, it becomes crucial to enable nitrite to proceed according to direct reduc-tion thus preventing an increase in nitrate concentration as well as an excess of nitric oxide in the product.Kinetics data suggest that this scope is performed when the product achieves the temperature of 65 °C asfast as possible with an ingoing amount of sodium nitrite of 70 mg/kg.

© 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Sodium nitrite is an additive used widely in meat products. It is in-volved in a number of functions: 1) it controls the development ofsome pathogenic species (Duffy, Vanderlinde, & Grau, 1994; Glass,McDonnell, Rassel, & Zierke, 2007; Yetim, Kayacier, Kesmen, &Sagdic, 2006); 2) it develops the typical pink colour of cooked ham(Cornforth & Jayasingh, 2004; Lawrie, 1998); 3) it contributes to fla-vour formation (Guillard, Goubet, Salles, Le Quéré, & Vendeuvre,1998); and 4) it exerts an antioxidant action against fats (Gatellier,Lessire, Hermier, Maaroufi, & Renerre, 2003; Han & Yamauchi,2000). On the other hand, nitrite also plays a substantial role in for-mation of the carcinogenic N-nitrosamine in meat, especially underthe process conditions applied in the meat processing industry(Juncher et al., 2000; Lijinsky, 1999; Ward et al., 2007).

Numerous studies have been carried out to replace nitrite in meatproducts (Dineen et al., 2000; Kawahara, Nakamura, Sakagami, &Suzuki, 2006; Pegg & Shahidi, 1997; Shahidi & Pegg, 1995; Sørheimet al., 2006; Viuda-Martos et al., 2009), but so far none of the alterna-tives found are as effective in colour formation or bacteriostatic actionon pathogenic species such as listeria and clostridium (Lucke, 2008).Reducing the use of nitrites has also been the subject of studies(Hammer, 1998) by European legislators (Dir 2006/52/CE; Dec UE2010/561).

Mortadella is a product made with finely ground pork meat (shoul-ders and trimmings from other cuts) combined with diced pork fattaken from the throat or belly, which is stuffed into natural casing or,

ieri).

rights reserved.

more frequently, synthetic casing. This product requires a long cookingtime in a dry air oven, during which numerous chemical reactions cantake place between the various meat components and additives(Cornforth & Jayasingh, 2004; Fox, 1966; Sebranek & Fox, 1985). The re-actions involving nitrite can be classified, for the sake of simplicity, asconcurrent reactions that consume it (dismutation and reduction),and produce, among other compounds, nitric oxide (NO). Nitric oxide,in turn, also reacts in various ways, concurrent with one another. Atthe pH of meat, generally 5.65 up to 5.85, nitrite is mostly present inthe dissociated form, nevertheless, the reactivity of undissociatedforms leads to the formation of several intermediates, which are unsta-ble and therefore difficult to determine, especially in a real systemsubjected to thermal treatment. Nitrite consumption can be due to theaction of reducing substances endogenous to the meat, e.g. sulphur-containing amino acids (cysteine), or added ones such as ascorbate. Inaddition, a nitrite dismutation reaction also results in nitrate formation.The consumption of NO occurs by a reaction with both the denaturedpigment of meat and with some substrates present in the mixture,such as the biochemical cellular systems ofmicroorganisms, which pre-vents their growth and preserves themeat product from amicrobiolog-ical point of view (Reddy, Lancaster, & Cornforth, 1983). Therefore, theamount of NO developed is an important parameter when evaluatingthe effect of added nitrite on the microbiological shelf-life of the prod-uct, with the aim of minimizing excess. In addition to reduce the nitriteaddition, it is also important to keep the residual of nitrate and nitriteresidues as low as possible. So far, the kinetics of nitrite has been studiedonly on model systems (Fox et al., 1994; Geileskey et al., 1998).

The purpose of this workwas to study the kinetics of nitrite decreas-ing by following the evolution of analytically measurable chemical spe-cies (NO3, NO2, total pigments, and nitric oxide pigments) and calculatethe content of NO through stoichiometry of the reactions consideredduring the production of mortadella in an industrial plant. In addition,we evaluated the efficiency with which, in a real system, important

283G. Barbieri et al. / Meat Science 93 (2013) 282–286

and technologically useful reactions proceeded and determined theeffects of some variables (ingoing amount of sodium nitrite andtemperature).

2. Materials and methods

The evolution of the nitrite/nitrate/nitric oxide/nitric oxide haemcomplex was studied experimentally on mortadella mixtures preparedin pilot plant, under stationary temperature conditions obtained in a dryair oven normally used for industrial production of mortadella. The rawmixture consisted of about 62% of moisture, 17% of proteins and 20% offat.

The evolution of the system was followed by taking a sample on asuperficial layer when the temperature was balanced with the tem-perature of the oven.

2.1. Manufacturing of mortadella

The oven was set to five different cooking temperatures: 55°, 60°,65, 70°, and 72 °C. In these stationary conditions, we cooked morta-della mixtures with a variable content of added sodium nitrite (40,70, 100, and 150 mg/kg), while the percentage of sodium chloride(2%) and sodium ascorbate (0.05%) was kept constant. Superficialsamples were collected at set times according to the experimentalplan described in Fig. 1. The samples were immediately cooleddown to stop the development of further reactions. Each samplewas ground: one aliquot was used for extracting and determiningnitric oxide pigments, while the other aliquot was analyzed for nitriteand nitrate.

2.2. Nitrite and nitrate determination

Ion suppression chromatography using the SSICA modified meth-od was used to measure nitrite and nitrate contents in all samples col-lected (Pizza et al., 2007).

2.3. Nitric oxide-haem pigments measurement

The total pigments and nitric oxide-haem pigments were deter-mined by spectrophotometry following the method described byHornsey (1956). The content was expressed as milligrams of haematinand NO-haematin per 1 kg of sample (mg/kg).

Fig. 1. Experimental design.

2.4. Kinetics study

Overall, the reactions of the chemical species involved wereexpected to be as follows, as mentioned in the literature (Honikel,2008; Sebranek & Fox, 1985):

3NO−2 þ 2Hþ→2NO þ NO

−3 þ H2O ð1Þ

NO−2 þ Red→NO þ Ox ð2Þ

XNO þ Pig→NOP ð3Þ

XNO þ Subs→ ð4Þ

where: Red and Ox are the reduced and oxidized form of the samemolecule, Pig is total pigments an NOP is nitric-oxide pigments.

Although some of these are actually multiple stage reactions, thisstudy looked at the overall reaction. It was not possible to use sophisti-cated sampling methods in this “real product” system; and measure-ments were limited to the most stable chemical species: nitrite,nitrate, total pigments, and nitric oxide pigments, while the extremelyunstable NO species was determined by stoichiometry of the above re-actions through concentrations of the other chemical species. We mea-sured the overall disappearance rate of the nitrite added, including itsparticipation in two concurrent reactions: dismutation (reaction 1)and direct reduction (reaction 2).

From the content values (obtained from two replications) at dif-ferent times, we calculated the reaction rate for each experiment.

Data were processed using SPSS 14.1 software.

3. Results and discussion

The logarithm of residual nitrite content, consumed overall by re-actions 1 and 2, showed a linear pattern over time:

ln NO2−½ � ¼ KTOTt; and the reaction rate is rTOT ¼ d NO−

2½ �dt

� �TOT

¼ KTOT NO−2½ �1

where rTOT overall rate of nitrite decreasing,KTOT constant rate of nitrite decreasing.

The content of nitrite involved in the dismutation (reaction 1),evaluated from the experimental determination of nitrate content,also decreased logarithmically as the reaction progressed. Therefore,the kinetics of reaction 1 and of the overall reaction of nitrite disap-pearance could be assumed to approximate first-order with respectto NO2

−, although the dismutation reaction involves three nitrite mol-ecules. Since reducer species (such ascorbate) are not a limiting fac-tor, we could also assume that nitrite reduction (reaction 2)followed first-order kinetics.

The overall rate with which nitrite is consumed is given by thesum of the rates of the single dismutation and reduction reactions.Therefore, the rate with which nitrite is consumed by reduction(reaction 2) is calculated as the difference: rr=rTOT−rd where rr israte of reaction 2) and rd is the rate of reaction 1).

The kinetics equations corresponding to the dismutation (d) andreduction (r) reactions are as follows:

rd ¼ Kd NO2−½ �n ¼ −1=3

d NO−2½ �

dt

� �d¼ d NO−

3½ �dt

¼ 12

d NO½ �dt

� �d

Table 3Rate of formation of free nitric oxide (μmol/min) (calculated) at different temperaturesand added sodium nitrite contents.

40 mg/kg 70 mg/kg 100 mg/kg 150 mg/kg

55 °C – 0.4 2.2 4.960 °C – 2.3 3.0 n.d.65 °C – 6.2 6.5 8.370 °C 0.4 5.6 10.3 4.572 °C 1.1 5.1 6.8 9.2

Table 1Reaction rate of nitrate formation (μmol/min) for reaction 1, at different temperaturesand added nitrite contents.

40 mg/kg 70 mg/kg 100 mg/kg 150 mg/kg

55 °C 0.6 n.d. 0.2 0.760 °C 0.2 0.6 0.5 0.165 °C 0.7 1.8 0.5 0.470 °C 0.6 0.2 1.9 3.672 °C 0.6 0.5 0.9 1.1

284 G. Barbieri et al. / Meat Science 93 (2013) 282–286

therefore

− d NO−2½ �

dt

� �d¼ 3Kd NO

−2½ �n

where− d NO−2½ �

dt

h idis the rate of nitrite disappearance due to reaction 1),

d NO½ �dt

h idis the rate of nitric oxide formation due to 1), d NO−

3½ �dt

h iis the rate

of nitrate formation due to 1) and Kd is the constant rate of reaction 1).

rr ¼ Kr NO2−½ �m ¼ − d NO−

2½ �dt

� �r¼ d NO½ �

dt

� �r

− d NO−2½ �

dt

h iris the rate of nitrite disappearance due to 2), d NO½ �

dt

h iris the

rate of nitric oxide formation due to 2) and Kr is the constant rateof reaction 2).n≅m≅1, therefore, 3Kd+Kr=KTOT and the overall

rate of nitrite disappearance is given by− d NO−2½ �

dt

h iTOT ¼ KTOT NO−

2½ �;which integrated over the time interval 0, t gives

NO2−½ � ¼ NO2

−½ �ine−KTOTt ð1Þ

where [NO2−]in is the ingoing amount of nitrite.

For nitrate ion,d NO−

3½ �dt ¼ Kd NO2

−½ � by substitution, we haved NO−

3½ �dt =

Kd[NO2−]in e−KTOTt , which integrated over the time interval 0, t and

without taking into account the content of nitrate already present inthe meat at time zero [NO3

−]in=0, gives

NO3−½ � ¼ Kd

KTOTNO2

−½ �in 1−e−KTOTt� �

ð2Þ

For nitric oxide, d NO½ �dt ¼ 2Kd NO2

−½ � þ Kr NO2−½ �, by substitutionwe have

d NO½ �dt ¼ 2Kd þ Krð Þ NO2

−½ �ine−KTOTt which integrated for [NO]in=0 over thetime interval 0, t gives

NO½ � ¼ 2Kd þ Kr

KTOTNO2

−½ �in 1−e−KTOTt� �

ð3Þ

This expression, obtained from the kinetic equations, enabled usto simulate the progress of nitric oxide content over time using the ki-netic parameters derived experimentally.

At time zero, the expression on the right of the equation is equal tozero, and the initial NO content is also zero; for t→∞, the exponentialtends to zero and, therefore, NO½ � ¼ 2Kdþ Kr

KTOTNO2

−½ �inb NO2−½ �in because

some of the nitrite forms nitrate.

Table 2Reaction rate of nitrite disappearance (μmol/min) (reactions 1 and 2) at different tem-peratures and added sodium nitrite contents.

40 mg/kg 70 mg/kg 100 mg/kg 150 mg/kg

55 °C 1.1 0.6 2.3 4.760 °C 0.6 1.3 2.3 3.365 °C 0.8 7.9 5.1 3.670 °C 1.3 5.1 6.9 4.172 °C 1.4 2.4 3.8 3.9

As already mentioned, having approximated the reactions thatconsume nitrite to first-order reactions, we could calculate the Kr ofnitrite reduction as the difference between the overall KTOT and theKd of the single dismutation reaction.

Some nitric oxide was consumed immediately by the pigment. Ex-perimentally, NO consumption by the pigment proceededmore rapidlyand was completed in the first 60–100 min. Thus, this consumption ofnitric oxide was subtracted in the initial part and later on becamenegligible.

The haem nitrosation reaction followed a fractional order (1/2) ki-netics model with respect to the content of non-nitrosated pigment,while it was not affected by the NO content (calculated from the pre-vious reactions). This was explained by the great affinity of NO for thehaem molecule: nitric oxide reacted immediately, at these contents,with the pigment as soon as it became available to the reaction. In ad-dition, nitrosopigment was measured at lower temperatures (55 °C)and at smaller ingoing amount of added nitrite (40 mg/kg).

The rates of reactions obtained by plotting reagent contents vs.time, at different temperatures and added nitrite contents, areshown in Tables 1 and 2, for nitrate formation and nitrite decreasingrespectively. The difference in reaction rate of nitrosohaem formationdue to ingoing amount of nitrite is very low: 0.8, 0.9, 1.3, 1.5 and1.4 μmol/min increasing the temperature up to 72 °C. Table 3 showsan estimate of the rate of nitric oxide formation, which we then as-sumed to react according to generic reaction 4, and the data wereevaluated based on the excess of NO produced by reactions 1 and 2with respect to the consumption of reaction 3. From the reactionrates at the different content, we calculated the Kr values and finallythe activation energies according to Arrhenius (Table 4).

The reaction of dismutation 1 was kinetically favoured (due to thelow activation energy) compared to reaction 2, at least at lower tem-peratures, and was the main mode of nitrite disappearance in the firststep of cooking, when the pH value was still low. Under these condi-tions in the early cooking phases, the reduced amount of NO forma-tion was immediately bound by the pigment according to a kineticconstant that was already high, even at low temperatures. At 55 °Cand with an addition of 70 mg/kg, nitric oxide pigment is sufficientto detect the pink colour. Its formation was favoured by low activa-tion energy. Therefore, the formation of nitric oxide pigment is nota limiting factor for the technological conditions of production as itoccurs in the early phases of heating even with small additions ofnitrite.

The minimum content of nitrite ion needed for microbiologicalcontrol has been determined previously (EFSA Journal, 2004) to be50 mg/kg, which corresponds to 32 mg/kg of nitric oxide. At low tem-peratures, nitrite reduction is hampered due to its high energy of ac-tivation, and the NO contents required to carry out bacteriostatic

Table 4Energy of activation of nitrite dismutation and reduction reactions, and nitric oxidepigment formation.

Reaction Ea (kj/mol)

Nitrite dismutation reaction 33.4Direct nitrite reduction reaction 72.1Nitric oxide haem formation reaction 23.9

Fig. 2. Amount of excess NO formed at 65 °C and 100 mg/kg of added nitrite. Contentsdetermined from experimental data (red squares); interpolating kinetic equation(solid line).

285G. Barbieri et al. / Meat Science 93 (2013) 282–286

activity are not reached. Conversely, as the temperature increases, rrincreases and reduction through reaction 2 becomes predominant,partly due to the increase of pH during cooking, in fact the pH ofmeat mixture was 5.75–5.80, while mortadella had a greater value(6.2).

At low temperatures, even long cooking times will probably be in-sufficient to provide the instantaneous amount needed for effectivebacteriostatic action. However, between 65 °C and 70 °C the increasein the rate of reaction 2 enabled the formation of a sufficient nitricoxide.

The dismutation reaction was scarcely affected by the temperatureeffect. Therefore, at the beginning of cooking when the dismutation re-action was also facilitated by a low pH value, the reaction proceededfaster and the greatest production of nitrate occurred in this phase.As cooking continued and the temperature increased, reaction 2proceeded at a faster rate and nitrite was consumed by direct reductionby the reducers present in the system (ascorbate and meat reducers).

From kinetics Eqs. (1)–(3) we obtained graphs for the patterns ofthe four chemical species by replacing the values of the rate constantsobtained experimentally. Fig. 2 shows values for the NO content at65 °C with 100 mg/kg of added nitrite. The split in the curve after60 min takes into account the ceased consumption of NO by nitrosatepigments and the consequent increase in its content. From the graph,it can be observed that there is a good fit between the curve describedby kinetic Eq. (3) of NO formation at 65 °C with 100 mg/kg of addednitrite and the points calculated from experimental data. The graph inFig. 3 shows the agreement between the experimental points for

Fig. 3. Amount of nitrite at 65 °C and 100 mg/kg of added nitrite. Contents determinedfrom experimental data (red squares) ; interpolating kinetic equation (solid line).

nitrite content and the concentration described by its kineticEq. (1). The fit between the estimated kinetic curves and experimen-tal data (or values derived from them such as for nitric oxide) is good,the R square being 0.941 for nitrite decreasing, 0.950 for nitric oxideformation and 0.946 for nitrate formation (Pb0.01).

The evolution over time of a system that is stationarywith respect totemperature has been described. During the actual cooking phase, theproduct followed a continuously increasing thermal process. Nonethe-less, it is useful to be able to define at what temperatures and nitriteconcentrations it is possible to achieve the following technological ob-jectives: lower nitrate content, reduction (or disappearance) of nitriteion, sufficient amount of NO, andmaximum formation of nitrosated pig-ment. The values for each chemical species will need to be integratedinto the temperature interval and times required by the cooking proto-col of mortadella. During the cooking of mortadella, every circular ringof the product will be in the above-described conditions for an intervalof time that depends on the heating method used.

4. Conclusions

Some of the variables in the production process of mortadella havebeen tested for colour optimization, achievement of microbiological ac-tivity, and reduction of nitrogen compound residues that are potentiallyharmful for consumers' health. The pattern of the reactions involved inthese processes suggests that the endpoint temperature of the cookingprocess should not be lower than 65 °C, maintained for a sufficientamount of time, and the nitrite content should range around 70 mg/kg.While the colour formation reaction is favoured by low activation ener-gy, it becomes crucial to enable nitrite to proceed according to the directreduction reaction, thus preventing an increase of nitrate content in theproduct. However, since dismutation is favoured at lower temperaturescompared to direct reduction, optimizationwill need to take into accountthe two different requirements: rapidly reaching temperatures above65 °C to reduce the progression of dismutation and avoiding excessivelyhigh endpoint temperatures to prevent an excess of nitric oxide. Thisposes a complex problem in terms of minimum time during whicheach internal section of the product must be kept at a set range of tem-peratures in the initial cooking phases. While 70 mg/kg of nitrite is a re-alistic intake amount, the fast increasing of temperature in the first stepis far from the current practical and is difficult to achieve, above all inlarge size products.

This work is to be followed up by the evaluation of nitrogen com-pound contents during the shelf life of mortadella as a function of thevariables examined in this paper.

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