CYTA - Journal of Food

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EVOLUTION OF NITRATES AND NITRITES RESIDUAL

LEVELS DURING THE MANUFACTURE AND STORAGE
OF HAM BY A FIA METHODOLOGY EVOLUCIÓN DE
LAS CONCENTRACIONES DE NITRATOS Y NITRITOS
RESIDUALES EN LA FABRICACIÓN Y ALMACENA JE
DE JAMÓN UTILIZANDO UNA METOLOGÍA FIA
EVOLUCIÓN DAS CONCENTRACIÓNS DE NITRATOS
E NITRITOS RESIDUALES NA FABRICACIÓN E
ALMACENAXE DE XAMÓN UTILIZANDO UNHA
METOLOGÍA FIA

I. M. P. L. V. O. Ferreira , N. S. Tavares & M. A. Ferreira

To cite this article: I. M. P. L. V. O. Ferreira , N. S. Tavares & M. A. Ferreira (1999) EVOLUTION

OF NITRATES AND NITRITES RESIDUAL LEVELS DURING THE MANUFACTURE AND
STORAGE OF HAM BY A FIA METHODOLOGY EVOLUCIÓN DE LAS CONCENTRACIONES
DE NITRATOS Y NITRITOS RESIDUALES EN LA FABRICACIÓN Y ALMACENAJE DE JAMÓN
UTILIZANDO UNA METOLOGÍA FIA EVOLUCIÓN DAS CONCENTRACIÓNS DE NITRATOS E
NITRITOS RESIDUALES NA FABRICACIÓN E ALMACENAXE DE XAMÓN UTILIZANDO UNHA
METOLOGÍA FIA, CYTA - Journal of Food, 2:4, 192-196, DOI: 10.1080/11358129909487601

To link to this article: https://doi.org/10.1080/11358129909487601

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Cienc. Tecnol. Aliment. Vol. 2, No. 4, pp. 192-196, 1999
Copyright 1999 Asociación de Licenciados en Ciencia y Tecnología de los Alimentos de Galicia (ALTAGA). ISSN 1135-8122

EVOLUTION OF NITRATES AND NITRITES RESIDUAL LEVELS DURING

THE MANUFACTURE AND STORAGE OF HAM BY A FIA
METHODOLOGY

EVOLUCIÓN DE LAS CONCENTRACIONES DE NITRATOS Y NITRITOS RESIDUALES EN LA

FABRICACIÓN Y ALMACENAJE DE JAMÓN UTILIZANDO UNA METOLOGÍA FIA

EVOLUCIÓN DAS CONCENTRACIÓNS DE NITRATOS E NITRITOS RESIDUALES NA FABRICACIÓN E

ALMACENAXE DE XAMÓN UTILIZANDO UNHA METOLOGÍA FIA

Ferreira, I. M. P. L. V. O.; Tavares, N. S.; Ferreira, M. A.

CEQUP / Laboratório de Bromatologia

Faculdade de Farmácia - Universidade do Porto
R. Aníbal Cunha, 164, 4050 Porto, Portugal
E-mail: bromato@pp.up.pt

Recibido: 26 Mayo 1999; recibida versión revisada: 17 Octubre 1999; aceptado: 17 Octubre 1999
Received: 26 May 1999; revised version received: 17 October 1999; accepted: 17 October 1999

Abstract
A study of the profiles of nitrates and nitrites during the different steps of manufacture of ham and during its
storage is presented. This monitorization included analyses of the meat, meat after injection of brine and massage, after
cooking and monthly during its shelf-life (3 months). An automated flow injection analytical method with
spectrophotometric detection was used. The manifold allowed simultaneous determination of nitrates and nitrites with
high sampling rate and economy of reagents. The levels of nitrates and nitrites diminished during the processing of ham
and during storage. The results obtained were statistically analysed.

Keywords: FIA, nitrates, nitrites, ham.

Resumen
Se presenta un estudio de los perfiles de nitratos y nitritos durante las diferentes etapas del procesamiento y
almacenamiento del jamón. La automatización incluyó análisis mensuales de la carne, de la carne inyectada con salmuera,
de la carne despues del masaje y de la carne cocinada durante sus periodos de caducidade (3 meses). Fue usado un método
analítico monitorizado de inyección de flujo (FIA) con detección espectofotométrica. La instalación permitió
simultaneamente la determinación de nitratos y nitritos con una alta tasa de muestreo y un uso económico de reactivos.
El nivel de nitratos y nitritos disminuyó durante el procesamiento del jamón y durante el almacenamiento. Los resultados
obtenidos fueron analizados estadísticamente.

Palabras clave: FIA, nitratos, nitritos, jamón.

Resumo
Presenta-se un estudo dos perfiles de nitratos e nitritos durante as diferentes etapas do procesamento e almacenaxe
do xamón. A automatización incluíu análisis mensuais da carne, da carne inxectada con salmuera, da carne despois del
masaje e da carne cocinada durante sus periodos de caducidade (3 meses). Usouse un método analítico monitorizado de
inxección de fluxo (FIA) con detección espectofotométrica. A instalación permitió simultaneamente a determinación de
nitratos e nitritos con unha alta tasa de muestreo e un uso económico de reactivos. O nivel de nitratos e nitritos disminuiu
durante o procesamento do xamón e durante a almacenaxe. Os resultados obtidos foron analizados estadísticamente.

Palabras llave: FIA, nitratos, nitritos, Xamón.

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ALTAGA ©1999 Ferreira et al.: Evolution of nitrates and nitrites redidual levels ...
INTRODUCTION
Ham is a processed meat product widely used in
our diet, as are other ready-to-consume foods. The
manufacture of ham involve several main steps, such as,
removal of bones from pork meat, injection of brine,
massage, molding, pressing, cooking, packaging and
storage. Due to the considerable perishability of this
product, it is usually labelled with 3 months as a maximum
consumption period.
The distribution of brine in previously prepared
pork meat is obtained by a multiple injection system.
Subsequently, the meat is massaged, an operation that is
important to assure an uniform distribution of brine up
to a limit of 20%. After, molding and pressing the meat
is cooked at 76°C, cooled and packed.
The main ingredients for curing the pork meat and
contained in the injected brine are salt and nitrite, but
other ingredients are added to accelerate the curing, to
stabilise the colour (nitrates, nitrites, eritorbate, citrate)
and to change the aroma and texture (phosphates,
dextrose, salt, aromas, Soya protein).
Among these ingredients the use of sodium and
potassium salts of nitrate and nitrite play a multi-faceted
role, because of their ability to give a particular colour
and taste to the meat as well as their preservative effect,
which provides specific protection against Clostridium
botulinum, a pathogenic micro-organism, responsible for
numerous food poisonings. The colour results from the
nitrosation of the heme in myoglobin to form
nitrosohemochrome. It also stabilizes the flavour of stored
meats by preventing the formation of the undesirable
products of oxidation (Cassens, 1995 and 1997), but meat
is an extremely complex and variable system that offers
an enormous number of constituents with capability for
nitrosation. Consequently, it is possible to obtain a great
variety of nitrosated compounds. Among them, the more
unstable ones can undergo different kinds of chemical
reactions (transnitrosation, oxidation, reduction and
hydrolysis). Endogenous or added reductants (such as
sodium ascorbate or eritorbate) play an important role.
In 1970 the use of these salts as additives in meat
was seriously questioned due to the possibility of their
involvement in the formation of N - nitroso compounds
which are considered carcinogenic and teratogenic in
animals (WHO, 1978).
Nitrate, per se, has a very low acute toxicity, but it
may be reduced to nitrite in the gastrointestinal tract.
Nitrite is the critical component, because it reacts with,
or is bound to, various chemical components of meat and
forms the N-nitroso compounds. An additional toxic effect
of nitrite is metahemoglobinemia (Report, 1996).
To reduce risks, substantial changes were made in
meat industry which resulted in a greater control of
manufacturing processes. Considerations based on the
193
ratio benefits/risks, still enable the use of these additives,
but in lower concentrations (Cassens, 1995).
The objective of this study was to contribute to
clarify the evolution of the levels of nitrate and nitrite
during the manufacture of ham and during its storage.
The official methods used for the quantification
of nitrate and nitrite (International Standard Organisation,
ISO 2019 and ISO 3001, 1975), recommend
spectrophotometric analytical methodologies, that involve
a high number of reagents, handling and consumption
which in turn cause considerable costs. For the
determination of these salts a very accurate, rapid and
precise methodology by FIA has been used (Ferreira et
al., 1996).
The manifold enables simultaneous determination
of nitrate and nitrite, with spectrophotometric detection.
Thus, it is an alternative method of analysis to perform
the conventional instrumental methods automatically,
with considerable time saving.
MATERIALS AND METHODS
Reagents and chemicals
All reagents were analytical grade and de-ionised
water had a specific conductivity lower than 0.1 mS cm-1.
N-(1-naftyl)ethylenediammonium dichloride,
phosphoric acid and sulphanilamide were obtained from
Sigma Chemical Company.
Apparatus
The absorbance was measured with a Jenway 6105
UV/VIS spectrophotometer, equipped with an 8 µl Hellma
type 178.713 flow cell, connected to a Gilson recorder.
An Ismatec model S 820 peristaltic pump was used in the
FIA system to propel the samples. The insertion of samples
and standards into the system was carried out with a
Rheodyne 5020 six port valve. Omnifit Teflon tubings
(0.8 and 0.3 mm i.d.), Gilson end fittings and connectors
were used to connect the different components of the
manifold.
FIA manifold
The FIA manifold used in the simultaneous
biparametric determination of nitrite and nitrate contained
a cadmium column applied in the reduction of nitrate to
nitrite what has already been described (Ferreira et al.,
1996).
The manifold consists in splitting the sample plug
after injection with a subsequent confluence of the flow
before reaching the spectrophotometric detector. This
enables the reduction of nitrate to nitrite in part of the
sample plug, due to an on-line copper-cadmium reductor
column. Each channel has a different residence time and
therefore two peaks are obtained which correspond to
either nitrite or nitrite plus nitrate. Spectrophotometric
detection of either peak is performed after diazotization -
Cienc. Tecnol. Aliment. Vol. 2, No. 4, pp. 192-196, 1999
Vi = 100 µl
H2 O
Q1
L1 = 30
R1 Q2
R2 Q3
Figure 1. FIA manifold used in the simultaneous multiparametric determination of nitrite and nitrate. Vi - injection volume; B - peristaltic pump; Li -
length of the reaction coils (cm); Qi - flow (ml min -1), Q1 = 3.0, Q2 = 0.5, Q3 =1.0; CR - cadmium- copper reductor column; D - spectrophotometric
detector; X and Y - confluences; R1 - a solution containing 100 g ammonium chloride, 20 g sodium tetraborate, 1 g Na2EDTA in 1 L; R2 - a solution
containing 20 g sulphanilamide, 1 g N-(1-naftyl)ethylenediammonium dichloride and 100ml of 80 % phosphoric acid diluted to 1L with water; W -
waste; R - recorder.
coupling reaction. This method allowed the simultaneous
determination of nitrite and nitrate present in different
concentration ranges in the same sample using an unique
detector and enabled the analysis to be performed within
a linear response range from 0.02 to 2 ppm for NO2-N
and from 0.1 to 5 ppm for NO3-N, with a sampling rate
of 40 samples per hour, which corresponded to 80
determinations per hour.
Calibration and quantification
Standard solutions of nitrite (1 ml = 1000 mg NO2
- N) and nitrate (1 ml = 1000 mg NO3 - N) were prepared
by dissolution of carefully weighed 4.92 g and 6.07 g of
NaNO 2 and NaNO 3 (dried for 1 hour at 100º C),
respectively, and subsequent dilution to 1000 ml. The
nitrite solution was standardized against a 0.1 N
permanganate solution. All referred solutions were treated
with some drops of chloroform to prevent the development
of microorganisms and were stored in a refrigerator.
Working standard solutions containing nitrite and nitrate
were prepared by appropriate dilution, in the following
way: standards of 0.02; 0.03; 0.05; 0.1; 0.5; ppm of NO2
- N and standards of 0.1; 0.5; 1.0; 2.0; 3.0 ppm of NO3 -
N mixed with 0.05 ppm of NO2 - N. All analyses were
performed in duplicate.
Sampling
This study was divided in two parts. In the first
part, the determinations were performed in duplicate on
three samples randomly taken from each step of the ham
manufacturing process: meat, meat after injection of brine
and massage and after cooking.
In the second part of this study, four ready-to-
consume hams (with an average weight 3.11 ± 0.13)
were divided in 3 portions, each portion was vacuum-
packed, and stored at temperatures below 4ºC. The
determinations of nitrates and nitrites were performed
monthly, during the shelf-life of the product (3 months:
June, July, August).
ISSN 1135-8122 ©1999 ALTAGA
CR L4 = 50
L5 = 100
D R
x L2 = 20 Y
L3 = 8
W
Sample preparation
Samples were pre-treated by homogeneization
according to the process recommended by the
International Standard Organisation (ISO 2019 and ISO
3001, 1975). Samples were extracted with hot water and
then purified by protein precipitation with Carrez I
(potassiumhexacyanoferrate(II)) and Carrez II (zinc
acetate) followed by filtration.
Statistical analysis
Data are represented as the mean ± standard
deviation. The units used are mg/kg of dry product
expressed as KNO3 for nitrates and NaNO2 for nitrites.
Analysis of variance was used to determine the
evolution of nitrate and nitrite levels during the
manufacture and storage of ham. Fisher's protected least
significant difference t-test (PLSD) at the 5% significance
level was applied to all experimental results to assess
intrapair significant differences. All statistical analyses
were done with the StatviewTM 4.0 statistical package
(Abacus concepts, Berkeley CA 94 704-1038, USA).
Proximate analysis
Moisture content was analysed by the method
outlined in the AOAC norm (AOAC, 1990).
RESULTS AND DISCUSSION
Under optimal conditions for nitrate and nitrite
analyses, the standards and extracts were injected directly
into the FIA system. The analytical methodology used
proved to be quite adequate for the simultaneous
quantification of nitrate and nitrite in ham (Ferreira et
al., 1996).
The detection limit, calculated as the concentration
corresponding to three times the SD of the background
noise, obtained with 10 determinations was 0.01 ppm of
NO2 - N and 0.1 ppm of NO3 - N.
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ALTAGA ©1999 Ferreira et al.: Evolution of nitrates and nitrites redidual levels ...

Table 1.- Contents of nitrate and nitrite obtained on each step of the manufacturing of the ham.

Samples NaNO 2 KNO 3

(mg/kg) (mg/kg)
Meat N.D a N.D a
b
Meat after massage 67.89 + 4.32 96.3 + 8.31 b
c
Ham after cooking 36.03 + 1.36 93.39 + 3.21 b

Values are expressed as mean ± standard deviation of six determinations. N.D.: Not detected.
a,b,c, -Means in columns without common superscripts are significantly different (p<0.05).

Table 2.- Contents of nitrate and nitrite obtained monthly during the validity of the ham.

Time NaNO2 KNO3

(mg/kg) (mg/kg)
Ham after cooking ( month 0 ) 36.03 + 1.36a 93.39 + 3.21a
After 1st month 3.82 + 0.60b 81.71 + 1.99b
nd c
After 2 month 2.24 + 0.11 78.11 + 2.76b
After 3rd month 1.82 + 0.025c 72.87 + 3.27c

Values are expressed as mean ± standard deviation of eight determinations.

a,b,c, -Means in columns without common superscripts are significantly different (p<0.05).

The analytical results obtained for nitrate and
nitrite contents in the main steps of the manufacture of
the ham are reported in Table 1.
The data pertaining to the nitrate and nitrite
contents indicated significant differences in concentration
during the manufacturing.
Nitrates and nitrites were not detected in the meat
used to prepare the ham. However, after injection of brine
different profiles of nitrate and nitrite contents were
obtained.
Over the manufacturing process period nitrate,
which ranged from 96.3 ± 8.31 to 93.39 ± 3.2 mg of
KNO3/kg of dry product, was always present at higher
concentrations than nitrite and showed a gradual decline
during the manufacture. This decrease can be explained
by the conversion of nitrate to nitrite by enzymes or/and
bacteria contained in meat and by aluminium utensils used
as suggested by Mohri (1993).
On the other hand, nitrite concentrations ranged
from 67.89 ± 4.32 to 36.03 ± 1.36 mg of NaNO2/kg of
dry product.
The decrease of nitrite content after cooking was
very prominent. This may be explained by the fact that
nitrite is converted to nitrosating agents which
subsequently react with amines and amides of the meat
during processing to form N - nitroso compounds. These
reactions are sped up by the cooking temperatures.
In contrast, the nitrate decrease was not significant
(p>0.05), probably because the nitrate reductase activity
was partly destroyed during cooking (temperature of 76ºC
was used), thereby diminishing further conversion of
nitrate to nitrite (Mohri, 1993)
The analytical results obtained for nitrate and
nitrite contents during the storage of the ham are reported
in Table 2. A large variability in nitrate and nitrite
contents was also noticed during the storage of the ham.
The general trend observed was a decrease of the
nitrate and nitrite contents over the 3 months storage
(Table 2). The nitrate content showed a moderate, yet
significant (p<0.001), overall decrease between the range
of mean values of 93.39 ± 3.2 to 72.87 ± 3.26 mg of
KNO3/kg of dry product. In contrast, a sharp decline (95
% decrease, mean values ranged from 36.03 ± 1.36 to
1.82 ± 0.025 mg of NaNO2/kg of dry product was observed
among the nitrite content as reflected by the high order
of magnitude of the F value (F = 2003.96 against F= 37.19
the nitrate counterpart).
In terms of rate of degradation (calculated as the
ratio of the difference between mean values at each
sampling date and the respective time period in days),
both nitrate and nitrite contents, showed a sharper
decrease during the first month of storage. However, it
was much more accentuated for nitrite levels (1.04 mg
NaNO 2 /kg dry product /day) than for the nitrate
counterpart (0.38 mg NaNO3/kg dry product /day). The
2nd and 3rd months of storage were characterised by slower

195
Cienc. Tecnol. Aliment. Vol. 2, No. 4, pp. 192-196, 1999
rates of degradation for both nitrate (0.12 mg NaNO3/kg
dry product /day) and nitrite (0.05 mg NaNO2/kg dry
product /day) levels but differences were also observed.
A 3-fold reduction in the rate of degradation of nitrate
levels observed during the second month of storage
contrasted with the 21-fold reduction calculated for the
nitrite levels. Furthermore, the third month of storage
reported a similar rate of degradation (0.17 mg NaNO3/
kg dry product /day) for the nitrate levels, whereas a
further 5-fold reduction (0.01 mg NaNO2/kg dry product
/day) was observed for the nitrite levels.
This behaviour may be explained by the fact that
during storage of ham, nitrate was still converted to
nitrite (acting as a reserve of nitrite) and residual nitrite
still interacted with other meat compounds. It is
interesting to note that in spite of the acceptable sensory
properties of the ham upon storage (taste, colour and
texture) the very low nitrite levels observed after the
first month of storage may lessen the protective effects
against botulinism, from this point onwards until the
end of shelf-life. It is known that the protection against
Clostridium botulinum depends on the concentration
of added salts as well as hygienic aspects, pH, water
activity and storage temperature. It is, therefore,
important to ensure the hygienic conditions obtained
by the HACCP (Hazards Analyses Critical Control
Point) methodology and maintain low temperatures
during the storage (0 - 4ºC).
196
ISSN 1135-8122 ©1999 ALTAGA
Acknowledgements
Seara, Indústria de Carnes, Lda.
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