INTRODUCTION
To reduce the adverse effects of red meat and
artificial additives on human health, one can reduce the
consumption of red meat or processed meat products.
However, studying possible ways to reduce the harmful
effects might be as effective as limiting consumption
levels. Nitrite/nitrate salts are usually added to meat
products to guarantee their safety, since these salts
inhibit the growth of Clostridium botulinum and prevent
heat-resistant spores from producing toxins. Moreover,
nitrite/nitrate salts improve the color, flavor, and aroma
of the finished product and postpone lipid oxidation
processes [1].
In spite of the numerous advantages, an excessive
intake of nitrite can produce adverse effects on human
health. Nitrite can be transformed to a nitrosating agent
(NO+), which reacts with biogenic amines and creates
carcinogenic N-nitrosamines. Bacterial and meat
Research Article DOI: http://doi.org/10.21603/2308-4057-2020-1-107-114
Open Access Available online at http://jfrm.ru/en/
Volatile N-nitrosamine, residual nitrite, and ascorbic acid levels
in sausages during storage
Houra Ramezani, Khadijeh Abhari , Zahra Pilevar , Hedayat Hosseini* ,
Abdorreza Mohammadi**
Food Sciences and Technology Department, National Nutrition and Food Technology Research Institute, Faculty
of Nutrition Sciences and Food Technology, Shahid Beheshti University of Medical Sciences, Tehran, Iran
* e-mail: hedayat@sbmu.ac.ir
** e-mail: ab.mohammadi@sbmu.ac.ir
Received September 01, 2019; Accepted in revised form November 13, 2019; Published February 25, 2020
Abstract:
Introduction. The increasing global consumption of processed meat products has led to certain concerns. For instance, processed
meat products are known to contain carcinogen precursor compounds, thus creating the risk of chronic diseases. The present study
was performed to estimate the food safety status of processed meat products available in Iran and evaluate the related effective
factors.
Study objects and methods. 140 samples of seven most popular commercial types of cooked sausages were obtained from four
major meat factories (A, B, C and D) in 140 samples were collected from seven most popular commercial types of cooked sausages
as follows: beef salami 90%, chicken salami 90%, dry cured sausage 70%, dry cured salami 60%, beef sausages 55%, chicken
sausages 55% and Frankfurt sausage 40% (n = 5) from four major meat factories (A, B, C and D) in Tehran. The samples were
screened for residual nitrite, ascorbic acid, and nitrosamine contents on days 0, 7, 14, 21, and 28. The results indicated that products
from meat factory B had lower residual nitrite content in the samples with high content of meat. Beef salami (90% of meat) and
Frankfurt sausage (40% of meat) contained the lowest and highest amounts of residual nitrite on day 0 – 73.99 and 177.42 mg of
nitrite per 1 kg of meat, respectively.
Results and discussion. Beef salami contained 90% of meat, chicken salami – 90%, dry cured sausage –70%, dry cured salami –
60%, beef sausages – 55%, chicken sausages – 55%, and Frankfurt sausage – 40% (n = 5). Nitrite reduction rates in sausages with
a smaller diameter, e.g. Frankfurt sausage, were significantly lower (P < 0.05), compared to salami samples. The difference can be
explained by the shorter cooking time. Nitrosamine formation increased during refrigerated storage; however, it was not significant
in all samples. During refrigerated storage, nitrosamine formation depended on the level of added nitrite, the amount of residual
nitrite, ascorbic acid, pH, and cooking temperature. Ascorbic acid content decreased significantly (P < 0.05) during refrigerated
storage.
Conclusion. The findings demonstrate significant correlation between the meat content, cooking time, nitrite content, and
nitrosamine formation.
Keywords: Meat industry, processed meat, meat products safety, carcinogenic agents, preservation
Funding: This research was financially supported by National Nutrition and Food Technology Research Institute (NNFTRI)
of Iran.
Please cite this article in press as: Ramezani H, Abhari K, Pilevar Z, Hosseini H, Mohammadi A. Volatile N-nitrosamine,
residual nitrite, and ascorbic acid levels in sausages during storage. Foods and Raw Materials. 2020;8(1):107–114.
DOI: http://doi.org/10.21603/2308-4057-2020-1-107-114.
Copyright © 2020, Ramezani et al. This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International
License (http://creativecommons.org/licenses/by/4.0/), allowing third parties to copy and redistribute the material in any medium or format and to remix,
transform, and build upon the material for any purpose, even commercially, provided the original work is properly cited and states its license.
Foods and Raw Materials, 2020, vol. 8, no. 1
E-ISSN 2310-9599
ISSN 2308-4057
108
Ramezani H. et al. Foods and Raw Materials, 2020, vol. 8, no. 1, pp. 107–114
enzymes cause decarboxylation of free amino acids,
which leads to the formation of biogenic amines. Several
intrinsic and extrinsic factors, e.g. salt content, can also
affect the biogenic amine formation level [2–4].
In meat and meat products, carcinogenic
N-nitrosamines are usually formed under acidic
conditions caused by the electrophilic reaction
between a nitrosating agent, e.g. nitrite or nitrous
acid, and secondary/tertiary amines that result from
protein and lipid degradation [5]. The mechanism
of nitrosamine formation in meat products has been
thoroughly studied. Nitrosamines with generic
chemical structure of R2NN = O are produced under
certain conditions of low pH, high temperature, and
presence of some reducing agents associated with
processing and composition of a particular meat
product [6]. There are several hurdle technologies
that can reduce the concentration of nitrite required
to inhibit bacterial growth, e.g. ascorbic/erythorbic
(isoascorbic) acid and essential oils, certain processing
conditions, or various non-thermal methods [1, 7].
Exposure level of N-nitrosodimethylamine (NDMA)
compound via consumption of food and beverages was
estimated to be 0.09 and 0.1 μg/day in the Netherlands
and Germany, respectively [8, 9]. The level of these
compounds in nitrite-preserved meat products varies
significantly. It depends on the ingoing volume of nitrite,
meat quality, and fat content, as well as on processing,
ripening, and storing conditions. There have been many
reports on nitrosamines detected in processed meat
products [10, 11].
The research objective was to examine the safety of
emulsion-type cooked sausages available on the Iran
markets by assessing the contents of residual nitrite,
ascorbic acid, and nitrosamine. In addition, we also
studied the effects of processing conditions and related
factors on these compounds to provide information for
health professionals and food manufacturers.
STUDY OBJECTS AND METHODS
Samples. The samples were collected from four
major meat factories in Tehran (A, B, C, and D) out
of the total of 189 meat factories in Iran. Seven most
popular commercial types of cooked sausages were
randomly purchased. Seven most popular commercial
types of cooked sausages were randomly purchased as
follows: beef salami 90%, chicken salami 90%, dry cured
sausage 70%, dry cured salami 60%, beef sausages 55%,
chicken sausages 55% and Frankfurt sausage 40%.
Five samples (2 kg) from each type were examined on
days 0, 7, 14, 21, and 28 of storage. The samples contained
beef (15% fat) orchicken (10% fat), water, oil, sodium
caseinate, sodium polyphosphate, garlic, salt, wheat
flour starch, spices, gluten, natural flavorings (paprika,
curcumin, ginger, and cinnamon), ascorbic acid, and
sodium nitrite. The samples were immediately transferred
to the laboratory and kept refrigerated until tested.
Residual nitrite determination. The nitrite content
was evaluated during 28 days on days 0, 7, 14, 21, and
28 using slightly modified calorimetric method of AOAC
method no. 973.31 [12]. The samples were cut into pieces
and homogenized. An aliquot of about 2.5 g of minced
sausage was added to 5 mL of saturated borax and
25 mL of deionized water (> 70°C) in Falcon tubes.
After stirring and cooling in room temperature, 1 mL of
each Carrez solution (I and II) was added to each sample
and adjusted to the volume of 50 mL. Carrez solution
I was prepared by dissolving 10.6 g of ferrocyanide
in distilled water (100 mL) according to the method
introduced by Ramezani et al. [13]. Carrez solution
II was also made by mixing 21.9 g of zinc acetate
with acetic acid (3 mL) and adjusted to the volume of
100 mL using distilled water. After centrifugation at
4000 rpm for 5 min (HeltichRotorfix 32A), 25 mL of
supernatant was transferred to a 100 mL tube. Ten ml
of sulfanilamide reagent and 6 mL of dilute HCl were
added to the supernatants and kept in the dark for 5 min.
Then, 2 mL of naphtyl-ethylenadiamine solution was
added to obtain high intensity azo dyes. The samples
were kept in the dark for 10 min to achieve complete
reactions. Absorbance was measured at 538 nm.
Ascorbic acid determination. To determine the
ascorbic acid content, 2 g of minced sausage was mixed
with 10 mL of meta-phosphoric acid solution (3%),
tertiary butylhydroquinone (TBHQ) (0.1%), and acetic
acid (8%). After that, they were centrifuged at 4000 rpm
for 10 min. After filtration, 20 μL of supernatant solution
was injected to a Cecil CE-4900 high-performance
liquid chromatograph coupled to a UV-vis detector
(HPLC-UV/VIS, Cambridge, England). The analytical
HPLC was equipped with two CE-4100 pumps, vacuum
degasser, six port valves (Rheodyne, USA), mixing
chamber, multiple solvent delivery unit, and an ODS
column (250 mm·4 I.D., 5 μm). The mobile phase
consisted of acetonitrile and sodium phosphate buffer
solution (50:50). The flow rate was 1.2 mL·min–1 at room
temperature [14].
N-nitrosamine determination. The experiment
evaluated seven volatile nitrosamines in the popular
cooked sausages from four major Iran meat factories.
The nitrosamines included N-nitrosodimethylamine
(NDMA), N-nitrosodiethylamine (NDEA), N-nitrosomorpholine
(NMOR), N-nitrosopyrrolidine (NPYR),
N-nitrosopiperidine (NPIP), N-nitrosodi-n-butylamine
(NDBA), and N-nitrosodiphenylamine (NDPheA). The
presence of nitrosamine was determined and quantified
according to the method described in our previous
study [13]. The method presupposed using microwaveassisted
extraction coupled with dispersive liquid–
liquid micro extraction (DLLME) followed by gas
chromatography–mass spectrometry (GC-MS).
Statistical analysis. All experiments were carried
out in triplicate. One-way ANOVA was performed to
determine significant differences. Duncan’s multiple
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Ramezani H. et al. Foods and Raw Materials, 2020, vol. 8, no. 1, pp. 107–114
range test was used to define the differences of mean
value. Data analysis was performed using SPSS
version 21 (SPSS Inc., Chicago, IL, USA); P < 0.05 was
considered statistically significant.
RESULTS AND DISCUSSION
The present research featured samples from seven
most popular types of sausages in Iran. The selected
sausages differed in the amount of nitrite, ascorbic
acid, and nitrosamine. This difference is associated
with the variations in concentration of added nitrate
and nitrite salts (sodium and potassium), storage
conditions, and different pH values [15, 16]. Following
the EU legislations, 2006/52/EC directive limited the
usage of nitrite to 150 mg per 1 kg of meat. However,
the national laws in Iran are more restricting. Iranian
provisions allow for maximum 120 mg of nitrite per
1 kg of meat (ppm) in sausages [17]. Table 1 displays the
residual nitrite, ascorbic acid, and nitrosamine contents
in the meat samples under study during storage.
Figure 1 illustrates the results of nitrite content
changes (mg/kg meat) in the meat samples obtained
from factory B as representative of all the four meat
factories.
As it was expected, the amount of nitrite residue
in meat products of all four meat factories decreased
during 28 days of refrigerated storage. The increase
in meat content increased the reduction rate of sodium
nitrite in the products. In other words, lower residual
nitrite content was detected in high content meat
samples. The highest reduction rate of nitrite content
was observed in chicken and beef salami (90% of meat).
On the first day of refrigerated storage, the Frankfurt
sausage (40%) and both beef and chicken sausages (55%)
contained 80 μg/kg of nitrite.
The detected amount exceeded the level permitted
by Iranian laws as defined by the Institute of Standard
and Industrial Research of Iran (ISIRI). The maximum
permissible burden of nitrite is 66 and 63 μg/kg for
processed meat products with 40% and 55% of meat
content, respectivelyI. The concentration of nitrite was
higher than the expected levels even after 28 days of
storage. However, the residual nitrite content was not
exactly equal to the initial added nitrite. First, it was
partly degraded by heating process. Second, it decreased
when ascorbic acid was applied to the meat product
during the heating process to accelerate conversion of
nitrite to nitric oxide.
The nitrite content varied due to interaction with
heme-containing components, non-heme proteins,
and fat tissues, conversion to nitrate, production of
such gases as N2, CO2, and NO, and nitrosamines [18].
Therefore, residual nitrite was associated with meat
content due to different contents of myoglobin [19]. As a
reactive agent, nitrite converts to nitrite oxide and forms
I ISIRI 932. Test method for determination of nitrite in meat and meat
products (reference method). Iran: Iran Institute of Standards and
Industrial Research; 2014.
nitrite-heme-nitrosomyoglobin complex with myoglobin,
thus producing nitrosomyochromogen. The latter is
responsible for the characteristic bright pink color of
cured meat products [20].
Hence, higher content of myoglobin results in lower
nitrite content in the final meat product. The results
obtained by statistical analysis indicate that residual
nitrite in Frankfurt sausage (40%) was significantly
higher (P < 0.05), whereas in beef salami (90%) it was
at its lowest. No significant differences were observed in
other sausage and salami products (P > 0.05).
Ascorbic acid, or ascorbate, is applied to meat
products as an additive with high water solubility for
three major reasons. First, the nitrosomyoglobin-forming
reduction of nitrite to nitric oxide produces the required
color. Second, the antioxidative activity of ascorbic acid
slows down oxidation of pigments and lipids, which
results in color and flavor stability. Third, residual nitrite
decreases due to binding to nitrite in heated samples [21].
Ascorbate proved effective in nitrosamine inhibition.
This quality is associated with rapid reactions of
ascorbate with nitric oxide compared to nitrosating
agents, e.g. amines. Therefore, nitrosamines are formed
when the reaction rate constant of ascorbate is not
much larger than amines [16]. Figure 2 and Table 1
demonstrate that ascorbic acid content of samples
decreased significantly (P < 0.05) during refrigerated
storage.
Meat products with higher meat content exhibited
lower ascorbic acid reduction rate, which was due to
lower residual nitrite content. Ascorbic acid degradation
in meat products could be related to oxygen content,
temperature, light, water activity, presence of metal
ions, e.g. copper and ferric iron, and storage time
[22–24]. Degradation of ascorbic acid is increased in
acidic conditions (pH = 3.3–5.5). In the current study,
Figure 1 Residual nitrite content (mg/kg meat) in sausages
with different meat content from factory B on days 0, 7, 14, 21,
and 28
0
50
100
150
200
0 7 14 21 28
Nitrite, ppm
Time, day
Beef salami 90% Chicken salami 90%
Dry cured salami 60% Beef sausage 55%
Chicken sausage 55% Dry cured sausage 70%
Frankfurt sausage 40%
90
140
190
240
0 Ascorbic acid, ppb
Beef salami Dry cured Chicken Frankfurt 0
10
20
30
40
50
0 7 14 21 28
Nitrosamine, ppb
Time, day
Beef salami 90% Chicken salami 90%
Dry cured salami 60% Beef sausage 55%
Chicken sausage 55% Dry cured sausage 70%
Frankfurt sausage 40%
0
20
40
60
80
100
0 Nitrite, ppm
Plant A (1)
(2)
(3)
(1)
(2)
(3)
(4)
(5)
(6)
(7)
(5)
(7)
(6)
(4)
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Table 1. Residual nitrite, ascorbic acid and nitrosamine contents in sausages produced by four major meat factories during storage
Factory A Day 0 Day 7 Day 14 Day 21 Day 28
Beef salami Nitrite, mg/kg 67.91 ± 0.605a 59.95 ± 0.360b 46.06 ± 0.326c 27.75 ± 0.119d 16.07 ± 0.294e
(90% of meat) Nitrosamine, ng/g 5.43 ± 0.004a 6.71 ± 2.887b 13.44 ± 0.033c 17.25 ± 0.563d 22.01 ± 0.038e
Ascorbic acid, ng/g 214.08 ± 0.262a 216.81 ± 0.026a 194.98 ± 0.018b 171.95 ± 0.017c 167.82 ± 0.026c
Chicken salami Nitrite, mg/kg 73.20 ± 0.376a 62.90 ± 0.134b 51.72 ± 0.226c 33.00 ± 0.156d 17.75 ± 0.356e
(90% of meat) Nitrosamine, ng/g 6.30 ± 0.135a 10.91 ± 0.173b 16.47 ± 0.610c 18.68 ± 0.856c 22.44 ± 0.616d
Ascorbic acid, ng/g 175.39 ± 0.039a 168.53 ± 0.003a 159.21 ± 0.027b 140.84 ± 0.039c 134.40 ± 0.021d
Dry cured salami Nitrite, mg/kg 90.93 ± 1.036a 85.00 ± 0.457b 72.64 ± 0.678c 61.36 ± 0.719d 36.73 ± 0.381e
(60% of meat) Nitrosamine, ng/g 6.93 ± 0.0561a 14.77 ± 0.037b 18.03 ± 0.060c 21.82 ± 0.518d 24.67 ± 0.091e
Ascorbic acid, ng/g 185.86 ± 0.021a 180.79 ± 0.003b 172.05 ± 0.062c 168.86 ± 0.683d 162.35 ± 0.706d
Beef sausage Nitrite, mg/kg 181.80 ± 0.274a 137.28 ± 0.760b 108.10 ± 0.716c 83.97 ± 0.080d 70.78 ± 0.387e
(55% of meat) Nitrosamine, ng/g 14.47 ± 0.399a 24.80 ± 1.06b 32.31 ± 0.389c 33.60 ± 0.566c 42.52 ± 1.077d
Ascorbic acid, ng/g 198.22 ± 0.149a 203.91 ± 0.057a 196.78 ± 0.031a 182.49 ± 0.033b 175.88 ± 0.018c
Chicken sausage Nitrite, mg/kg 187.42 ± 0.315a 145.40 ± 0.447b 114.00 ± 0.151c 91.21 ± 1.279d 76.29 ± 0.390e
(55% of meat) Nitrosamine, ng/g 7.71 ± 0.075a 25.96 ± 0.1063b 33.32 ± 0.053c 34.29 ± 0.067c 41.91 ± 0.0167d
Ascorbic acid, ng/g 165.27 ± 0.009a 163.34 ± 0.009a 161.99 ± 0.024a 158.26 ± 0.072b 149.46 ± 0.031c
Dry cured sausage Nitrite, mg/kg 82.11 ± 0.757a 69.78 ± 0.758b 54.94 ± 0.115c 46.35 ± 0.314d 39.79 ± 1.206e
(70% of meat) Nitrosamine, ng/g 14.32 ± 0.052a 15.36 ± 0.012a 17.86 ± 0.083ab 20.81 ± 0.765b 21.55 ± 0.045b
Ascorbic acid, ng/g 129.67 ± 0.416a 120.55 ± 0.007ab 117.99 ± 0.033b 106.79 ± 0.039c 105.82 ± 0.705c
Factory B Day 0 Day 7 Day 14 Day 21 Day 28
Beef salami Nitrite, mg/kg 73.99 ± 0.011a 54.53 ± 0.130b 48.76 ± 0.649c 32.60 ± 0.626d 30.20 ± 0.598d
(90% of meat) Nitrosamine, ng/g 3.06 ± 0.058a 5.13 ± 0.204ab 7.05 ± 0.284b 13.40 ± 0.122c 14.18 ± 0.1054c
Ascorbic acid, ng/g 194.99 ± 0.016a 192.30 ± 0.021a 165.00 ± 0.699b 144.62 ± 0.004c 121.60 ± 0.060d
Chicken salami Nitrite, mg/kg 77.55 ± 0.556a 72.12 ± 0.505a 55.24 ± 0.247b 44.41 ± 0.830c 31.62 ± 0.347d
(90% of meat) Nitrosamine, ng/g 4.85 ± 0.045a 4.83 ± 0.018a 6.43 ± 0.09a 11.43 ± 0.065b 14.81 ± 0.031b
Ascorbic acid, ng/g 220.79 ± 0.022a 192.84 ± 0.554b 168.95 ± 0.134c 164.89 ± 0.025c 134.07 ± 0.492d
Dry cured salami Nitrite, mg/kg 101.79 ± 0.290a 94.85 ± 0.362a 76.18 ± 0.359b 63.30 ± 0.244c 49.24 ± 0.396d
(60% of meat) Nitrosamine, ng/g 11.93 ± 0.081a 15.20 ± 0.256ab 15.32 ± 0.298ab 21.68 ± 0.307b 28.38 ± 0.575c
Ascorbic acid, ng/g 201.24 ± 0.127a 188.65 ± 0.038b 172.34 ± 0.401c 168.01 ± 0.002d 145.69 ± 0.018e
Beef sausage Nitrite, mg/kg 156.45 ± 0.774a 128.46 ± 0.637b 95.54 ± 0.323c 87.82 ± 0.296d 65.26 ± 0.971e
(55% of meat) 37.73 ± 0.059d Nitrosamine, ng/g 13.21 ± 0.057a 14.94 ± 0.04a 23.64 ± 0.075b 29.54 ± 0.94c
Ascorbic acid, ng/g 200.90 ± 0.132a 193.71 ± 0.015ab 187.60 ± 0.345b 183.60 ± 0.031b 174.74 ± 0.297c
Chicken sausage Nitrite, mg/kg 149.47 ± 0.197a 126.70 ± 0.516b 98.98 ± 0.708c 86.83 ± 0.681d 71.21 ± 0.192e
(55% of meat) Nitrosamine, ng/g 7.85 ± 0.118a 14.70 ± 0.077b 24.73 ± 0.053c 29.77 ± 2.93d 37.99 ± 0.133e
Ascorbic acid, ng/g 181.71 ± 0.014a 160.27 ± 0.020b 138.63 ± 0.013c 121.13 ± 0.003d 104.42 ± 0.005e
Dry cured sausage Nitrite, mg/kg 106.62 ± 0.277a 87.71 ± 0.850b 79.26 ± 0.211c 65.21 ± 0.306d 57.90 ± 0.226e
(70% of meat) Nitrosamine, ng/g 8.18 ± 0.202a 9.71 ± 0.469a 15.22 ± 0.085b 25.88 ± 0.1589c 33.16 ± 0.622d
Ascorbic acid, ng/g 197.86 ± 0.215a 156.10 ± 0.021b 131.45 ± 0.073c 127.63 ± 0.015d 126.30 ± 0.007d
Frankfurt sausage Nitrite, mg/kg 177.42 ± 0.677a 141.07 ± 0.075b 103.64 ± 0.280c 88.45 ± 0.662d 78.61 ± 0.710e
(40% of meat) Nitrosamine, ng/g 14.34 ± 0.05a 16.12 ± 0.6746b 27.20 ± 0.116c 34.97 ± 0.202c 45.06 ± 0.229d
Ascorbic acid, ng/g 192.57 ± 0.062a 187.08 ± 0.032b 169.47 ± 0.057c 155.29 ± 0.008d 137.55 ± 0.006e
Factory C Day 0 Day 7 Day 14 Day 21 Day 28
Beef salami Nitrite, mg/kg 59.08 ± 0.199a 35.41 ± 0.347b 25.26 ± 0.952c 18.25 ± 0.297d 8.42 ± 0.088e
(90% of meat) Nitrosamine, ng/g 2.65 ± 0.0123a 8.37 ± 0.058b 15.51 ± 0.021c 19.27 ± 0.057c 33.83 ± 0.123d
Ascorbic acid, ng/g 232.27 ± 0.0321a 187.13 ± 0.060b 135.76 ± 0.025c 122.71 ± 0.031cd 108.52 ± 0.026d
Chicken salami Nitrite, mg/kg 62.90 ± 0.220a 50.30 ± 0.589b 36.67 ± 0.347c 19.66 ± 0.714d 9.26 ± 0.076d
(90% of meat) Nitrosamine, ng/g 2.78 ± 0.071a 9.68 ± 0.078b 14.47 ± 0.064c 18.59 ± 0.083d 25.34 ± 0.117e
Ascorbic acid, ng/g 162.54 ± 0.055a 155.95 ± 0.086b 121.19 ± 0.060c 103.93 ± 0.112d 92.68 ± 0.049e
Dry cured salami Nitrite, mg/kg 69.96 ± 0.912a 65.00 ± 0.721a 50.22 ± 0.977ab 36.57 ± 0.386b 19.17 ± 0.448c
(60% of meat) Nitrosamine, ng/g 3.51 ± 0.007a 11.91 ± 0.042b 19.61 ± 0.047c 22.57 ± 0.052d 23.76 ± 0.052d
Ascorbic acid, ng/g 248.29 ± 0.004a 213.54 ± 0.630b 198.32 ± 0.016b 165.78 ± 0.032c 119.79 ± 0.296d
Beef sausage Nitrite, mg/kg 107.41 ± 0.682a 92.93 ± 0.114ab 85.60 ± 1.314b 64.39 ± 0.142c 31.88 ± 0.397d
(55% of meat) Nitrosamine, ng/g 8.61 ± 0.157a 17.01 ± 0.05b 20.51 ± 0.547b 34.51 ± 0.068c 44.20 ± 0.051d
Ascorbic acid, ng/g 200.69 ± 0.45a 196.17 ± 0.057a 189.30 ± 0.047ab 175.08 ± 0.107b 142.58 ± 0.117c
Chicken sausage Nitrite, mg/kg 116.20 ± 0.725a 93.36 ± 0.354ab 81.96 ± 0.279b 55.90 ± 0.374c 47.44 ± 0.658d
(55% of meat) Nitrosamine, ng/g 9.85 ± 0.011a 16.19 ± 0.145ab 20.51 ± 0.547b 35.51 ± 0.068c 44.20 ± 0.051d
Ascorbic acid, ng/g 199.63 ± 0.588a 184.59 ± 0.068b 165.67 ± 1.013c 102.61 ± 0.173d 79.79 ± 0.033e
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Dry cured sausage Nitrite, mg/kg 84.48 ± 0.188a 71.59 ± 0.763b 57.72 ± 0.274c 50.18 ± 1.247c 27.29 ± 0.242d
(70% of meat) Nitrosamine, ng/g 10.05 ± 0.033a 10.35 ± 0.048a 17.68 ± 0.031b 20.81 ± 0.080c 23.44 ± 0.037d
Ascorbic acid, ng/g 149.83 ± 0.017a 114.28 ± 0.177b 103.91 ± 0.122c 89.73 ± 0.145d 71.32 ± 0.035e
Frankfurt sausage Nitrite, mg/kg 135.79 ± 1.314a 98.41 ± 1.405b 82.62 ± 0.592c 80.51 ± 0.754c 52.84 ± 0.489d
(40% of meat) Nitrosamine, ng/g 10.05 ± 0.087a 17.95 ± 0.086b 24.09 ± 0.105c 37.80 ± 0.065d 41.32 ± 0.090e
Ascorbic acid, ng/g 141.12 ± 0.059a 138.09 ± 0.012a 102.27 ± 0.027b 91.43 ± 0.068b 74.10 ± 0.707c
Factory D Day 0 Day 7 Day 14 Day 28
Beef salami Nitrite, mg/kg 96.65 ± 0.646a 75.59 ± 0.754b 59.11 ± 1.413c 52.95 ± 1.361cd 47.94 ± 0.628d
(90% of meat) Nitrosamine, ng/g 28.96 ± 0.026a 34.52 ± 0.042ab 39.02 ± 0.054b 44.38 ± 0.048bc 49.52 ± 0.077c
Ascorbic acid, ng/g 233.43 ± 0.202a 195.42 ± 0.054b 163.34 ± 0.095c 157.90 ± 0.158c 132.90 ± 0.167d
Dry cured salami Nitrite, mg/kg 101.88 ± 0.611a 90.53 ± 0.437a 78.70 ± 0.650b 66.24 ± 0.271c 53.22 ± 0.917d
(60% of meat) Nitrosamine, ng/g 31.53 ± 0.017a 35.12 ± 0.051ab 39.81 ± 0.029b 44.46 ± 0.036c 48.46 ± 0.036c
Ascorbic acid, ng/g 169.07 ± 0.115a 157.47 ± 0.032b 114.25 ± 0.174c 103.32 ± 0.151d 89.92 ± 0.341e
Beef sausage Nitrite, mg/kg 114.24 ± 0.862a 99.66 ± 0.920ab 86.06 ± 0.526b 73.10 ± 1.452c 61.39 ± 0.122d
(55% of meat) Nitrosamine, ng/g 38.02 ± 0.031a 74.74 ± 0.136b 94.90 ± 0.021c 124.58 ± 0.033d 131.558 ± 0.063d
Ascorbic acid, ng/g 198.59 ± 0.035a 104.30 ± 0.240b 54.95 ± 0.079c 35.61 ± 0.162d 29.11 ± 0.452d
Chicken sausage Nitrite, mg/kg 143.09 ± 0.787a 127.56 ± 0.488b 98.24 ± 0.196c 72.37 ± 0.808d 55.95 ± 0.069e
(55% of meat) Nitrosamine, ng/g 38.02 ± 0.562a 68.88 ± 0.125b 70.90 ± 0.152b 85.94 ± 0.099c 89.91 ± 0.059c
Ascorbic acid, ng/g 201.10 ± 0.351a 173.31 ± 0.494b 138.33 ± 0.442c 90.33 ± 0.953d 68.82 ± 0.721d
Frankfurt sausage Nitrite, mg/kg 150.45 ± 1.003a 135.95 ± 0.765b 109.52 ± 1.216c 97.55 ± 0.084d 80.41 ± 0.784e
(40% of meat) Nitrosamine, ng/g 12.91 ± 0.085a 38.73 ± 0.039b 100.42 ± 0.093c 153.60 ± 0.253d 178.60 ± 0.293e
Ascorbic acid, ng/g 282.14 ± 0.310a 181.60 ± 0.025b 170.03 ± 0.169c 117.97 ± 0.037d 75.22 ± 0.897e
Different letters in the same row during storage within the same section (i.e. factories A-D) indicate a significant difference (P < 0.05)
Continuation of the table 1
Figure 2 Ascorbic acid content (ng/kg meat) in sausages
with different meat content from factory B on days 0, 7, 14, 21,
and 28
28
salami 90%
sausage 55%
sausage 70%
90
140
190
240
0 7 14 21 28
Ascorbic acid, ppb
Time, day
Beef salami 90% Chicken salami 90%
Dry cured salami 60% Beef sausage 55%
Chicken sausage 55% Dry cured sausage 70%
Frankfurt sausage 40%
28
y = 10.951x + 80.875
R² = 0.9423
y = 16.803x + 90.748
y = 21.596x + 121.65
R² = 0.8989
40
60
80
100
Nitrite, ppm
Figure 3 Nitrosamine content (ng/kg of meat) in sausages
with different meat content from factory B on days 0, 7, 14, 21,
and 28
0
50
100
150
200
0 7 14 21 28
Nitrite, ppm
Time, day
Beef salami 90% Chicken salami 90%
Dry cured salami 60% Beef sausage 55%
Chicken sausage 55% Dry cured sausage 70%
Frankfurt sausage 40%
90
140
190
240
0 Ascorbic acid, ppb
Beef salami Dry cured Chicken Frankfurt 0
10
20
30
40
50
0 7 14 21 28
Nitrosamine, ppb
Time, day
Beef salami 90% Chicken salami 90%
Dry cured salami 60% Beef sausage 55%
Chicken sausage 55% Dry cured sausage 70%
Frankfurt sausage 40%
0
20
40
60
80
100
0 Nitrite, ppm
Plant pH values were in the range of 5–6 in all samples.
Therefore, ascorbic acid degradation might have been
due to the relative acidic condition of meat products and
heating process.
The obtained results showed an increase in
nitrosamine content of samples during refrigerated
storage. However, it was not significant in all samples
(Fig. 3).
The nitrite reduction rates in the sausages with a
smaller diameter, e.g. Frankfurt sausage, appeared
significantly lower (P < 0.05) than in the samples with
a bigger diameter, e.g. salami. This difference can be
explained by the longer cooking time for salami (5–6 h)
compared to Frankfurt sausages (3–4 h), which is
associated with a higher reduction rate of residual
nitrite. According to the results, the total volatile
nitrosamine level of Iranian meat products with
lower meat contents was generally higher than
that of samples with high meat contents. The
nitrosamine content in meat products was associated
with the added nitrite and the residual nitrite [25].
The residual nitrite is a reactive agent that can be
reduced by heating treatment or exposure to such meat
components as proteins, lipids, and pigments [26, 27].
(1)
(2)
(3)
(5)
(7)
(6)
(4)
(5)
(6)
(7)
(1)
(2)
(3)
(4)
(5)
(6)
(7)
(1)
(2)
(3)
(4)
(3) (1) (2)
(5)
(7)
(6)
(4)
112
Ramezani H. et al. Foods and Raw Materials, 2020, vol. 8, no. 1, pp. 107–114
y = 10.951x + 80.875
R² = 0.9423
y = 16.803x + 90.748
y = 13.533x + 68.202 R² = 0.9527
R² = 0.9605
y = 21.596x + 121.65
R² = 0.8989
0
20
40
60
80
100
0 7 14 21 28
Nitrite, ppm
Time, day
Plant A Plant B Plant C Plant D
Figure 4 shows that there was a significant
correlation between the amounts of added nitrite and the
nitrosamine contents in beef salami (90%) samples from
all four meat factories.
In this study, the correlation factor was 0.9, which
asserts the effects of added nitrite and also residual
nitrite contents on nitrosamine formation in the
processed meat product. Therefore, the products with
lower meat content showed higher residual nitrite and,
consequently, a greater nitrosamine formation. In meat
products, presence of nitrosating agents increases the
concentration of nitrosamine, whether grouped in NO2
related agents, e.g. N2O4, or grouped in nitrous acid
derivatives, e.g. N2O3 and HNO2 [28].
There are several suggested strategies to reduce
nitrosamine formation in meat products to improve their
healthy status and safety. The present research clarified
that the levels of nitrosamines in the samples depended
on the amount of residual nitrite, ascorbic acid, pH, and
cooking temperature. Higher levels of residual nitrite
were detected in the samples with a lower amount of
meat, compared to those with a higher amount of meat.
CONCLUSION
In the current study, the residual nitrite, ascorbic
acid, and nitrosamine contents of seven most popular
Iranian processed meat products, namely sausages
with different amounts of meat were evaluated to
monitor the safety status of the meat industry. The
samples contained various concentrations of nitrite and
nitrosamine, which were above the permitted standard
level. Several factors were found to affect the residual
nitrite and nitrosamine contents, the meat content being
a significant variable. Nitrite interacted with hemecontaining
components, non-heme proteins, and fat
tissues, thus conversed to nitrate and nitrosamine.
Therefore, the contents of residual nitrite and,
consequently, nitrosamine in products with lower meat
content were significantly higher. A longer cooking
time also decreased residual nitrite and nitrosamine
concentration.
Further research is needed to identify new
substances that could replace nitrites, as well as factors
that reduce the required amount of nitrite in meat
products.
CONTRIBUTION
The authors were equally involved in writing the
manuscript and are equally responsible for plagiarism.
CONFLICT OF INTEREST
The authors declare that there is no conflict
of interest regarding the publication of this article.
ACKNOWLEDGEMENTS
We would like to thank National Nutrition and Food
Technology Research Institute for the financial support.

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