INTRODUCTIONTo reduce the adverse effects of red meat andartificial additives on human health, one can reduce theconsumption of red meat or processed meat products.However, studying possible ways to reduce the harmfuleffects might be as effective as limiting consumptionlevels. Nitrite/nitrate salts are usually added to meatproducts to guarantee their safety, since these saltsinhibit the growth of Clostridium botulinum and preventheat-resistant spores from producing toxins. Moreover,nitrite/nitrate salts improve the color, flavor, and aromaof the finished product and postpone lipid oxidationprocesses [1].In spite of the numerous advantages, an excessiveintake of nitrite can produce adverse effects on humanhealth. Nitrite can be transformed to a nitrosating agent(NO+), which reacts with biogenic amines and createscarcinogenic N-nitrosamines. Bacterial and meatResearch Article DOI: http://doi.org/10.21603/2308-4057-2020-1-107-114Open Access Available online at http://jfrm.ru/en/Volatile N-nitrosamine, residual nitrite, and ascorbic acid levelsin sausages during storageHoura Ramezani, Khadijeh Abhari , Zahra Pilevar , Hedayat Hosseini* ,Abdorreza Mohammadi**Food Sciences and Technology Department, National Nutrition and Food Technology Research Institute, Facultyof 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.irReceived September 01, 2019; Accepted in revised form November 13, 2019; Published February 25, 2020Abstract:Introduction. The increasing global consumption of processed meat products has led to certain concerns. For instance, processedmeat products are known to contain carcinogen precursor compounds, thus creating the risk of chronic diseases. The present studywas performed to estimate the food safety status of processed meat products available in Iran and evaluate the related effectivefactors.Study objects and methods. 140 samples of seven most popular commercial types of cooked sausages were obtained from fourmajor meat factories (A, B, C and D) in 140 samples were collected from seven most popular commercial types of cooked sausagesas follows: beef salami 90%, chicken salami 90%, dry cured sausage 70%, dry cured salami 60%, beef sausages 55%, chickensausages 55% and Frankfurt sausage 40% (n = 5) from four major meat factories (A, B, C and D) in Tehran. The samples werescreened for residual nitrite, ascorbic acid, and nitrosamine contents on days 0, 7, 14, 21, and 28. The results indicated that productsfrom meat factory B had lower residual nitrite content in the samples with high content of meat. Beef salami (90% of meat) andFrankfurt sausage (40% of meat) contained the lowest and highest amounts of residual nitrite on day 0 – 73.99 and 177.42 mg ofnitrite 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 witha smaller diameter, e.g. Frankfurt sausage, were significantly lower (P < 0.05), compared to salami samples. The difference can beexplained by the shorter cooking time. Nitrosamine formation increased during refrigerated storage; however, it was not significantin all samples. During refrigerated storage, nitrosamine formation depended on the level of added nitrite, the amount of residualnitrite, ascorbic acid, pH, and cooking temperature. Ascorbic acid content decreased significantly (P < 0.05) during refrigeratedstorage.Conclusion. The findings demonstrate significant correlation between the meat content, cooking time, nitrite content, andnitrosamine formation.Keywords: Meat industry, processed meat, meat products safety, carcinogenic agents, preservationFunding: 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 InternationalLicense (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. 1E-ISSN 2310-9599ISSN 2308-4057108Ramezani H. et al. Foods and Raw Materials, 2020, vol. 8, no. 1, pp. 107–114enzymes cause decarboxylation of free amino acids,which leads to the formation of biogenic amines. Severalintrinsic and extrinsic factors, e.g. salt content, can alsoaffect the biogenic amine formation level [2–4].In meat and meat products, carcinogenicN-nitrosamines are usually formed under acidicconditions caused by the electrophilic reactionbetween a nitrosating agent, e.g. nitrite or nitrousacid, and secondary/tertiary amines that result fromprotein and lipid degradation [5]. The mechanismof nitrosamine formation in meat products has beenthoroughly studied. Nitrosamines with genericchemical structure of R2NN = O are produced undercertain conditions of low pH, high temperature, andpresence of some reducing agents associated withprocessing and composition of a particular meatproduct [6]. There are several hurdle technologiesthat can reduce the concentration of nitrite requiredto inhibit bacterial growth, e.g. ascorbic/erythorbic(isoascorbic) acid and essential oils, certain processingconditions, or various non-thermal methods [1, 7].Exposure level of N-nitrosodimethylamine (NDMA)compound via consumption of food and beverages wasestimated to be 0.09 and 0.1 μg/day in the Netherlandsand Germany, respectively [8, 9]. The level of thesecompounds in nitrite-preserved meat products variessignificantly. 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 manyreports on nitrosamines detected in processed meatproducts [10, 11].The research objective was to examine the safety ofemulsion-type cooked sausages available on the Iranmarkets by assessing the contents of residual nitrite,ascorbic acid, and nitrosamine. In addition, we alsostudied the effects of processing conditions and relatedfactors on these compounds to provide information forhealth professionals and food manufacturers.STUDY OBJECTS AND METHODSSamples. The samples were collected from fourmajor meat factories in Tehran (A, B, C, and D) outof the total of 189 meat factories in Iran. Seven mostpopular commercial types of cooked sausages wererandomly purchased. Seven most popular commercialtypes of cooked sausages were randomly purchased asfollows: beef salami 90%, chicken salami 90%, dry curedsausage 70%, dry cured salami 60%, beef sausages 55%,chicken sausages 55% and Frankfurt sausage 40%.Five samples (2 kg) from each type were examined ondays 0, 7, 14, 21, and 28 of storage. The samples containedbeef (15% fat) orchicken (10% fat), water, oil, sodiumcaseinate, sodium polyphosphate, garlic, salt, wheatflour starch, spices, gluten, natural flavorings (paprika,curcumin, ginger, and cinnamon), ascorbic acid, andsodium nitrite. The samples were immediately transferredto the laboratory and kept refrigerated until tested.Residual nitrite determination. The nitrite contentwas evaluated during 28 days on days 0, 7, 14, 21, and28 using slightly modified calorimetric method of AOACmethod no. 973.31 [12]. The samples were cut into piecesand homogenized. An aliquot of about 2.5 g of mincedsausage was added to 5 mL of saturated borax and25 mL of deionized water (> 70°C) in Falcon tubes.After stirring and cooling in room temperature, 1 mL ofeach Carrez solution (I and II) was added to each sampleand adjusted to the volume of 50 mL. Carrez solutionI was prepared by dissolving 10.6 g of ferrocyanidein distilled water (100 mL) according to the methodintroduced by Ramezani et al. [13]. Carrez solutionII was also made by mixing 21.9 g of zinc acetatewith acetic acid (3 mL) and adjusted to the volume of100 mL using distilled water. After centrifugation at4000 rpm for 5 min (HeltichRotorfix 32A), 25 mL ofsupernatant was transferred to a 100 mL tube. Ten mlof sulfanilamide reagent and 6 mL of dilute HCl wereadded to the supernatants and kept in the dark for 5 min.Then, 2 mL of naphtyl-ethylenadiamine solution wasadded to obtain high intensity azo dyes. The sampleswere kept in the dark for 10 min to achieve completereactions. Absorbance was measured at 538 nm.Ascorbic acid determination. To determine theascorbic acid content, 2 g of minced sausage was mixedwith 10 mL of meta-phosphoric acid solution (3%),tertiary butylhydroquinone (TBHQ) (0.1%), and aceticacid (8%). After that, they were centrifuged at 4000 rpmfor 10 min. After filtration, 20 μL of supernatant solutionwas injected to a Cecil CE-4900 high-performanceliquid chromatograph coupled to a UV-vis detector(HPLC-UV/VIS, Cambridge, England). The analyticalHPLC was equipped with two CE-4100 pumps, vacuumdegasser, six port valves (Rheodyne, USA), mixingchamber, multiple solvent delivery unit, and an ODScolumn (250 mm·4 I.D., 5 μm). The mobile phaseconsisted of acetonitrile and sodium phosphate buffersolution (50:50). The flow rate was 1.2 mL·min–1 at roomtemperature [14].N-nitrosamine determination. The experimentevaluated seven volatile nitrosamines in the popularcooked 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). Thepresence of nitrosamine was determined and quantifiedaccording to the method described in our previousstudy [13]. The method presupposed using microwaveassistedextraction coupled with dispersive liquid–liquid micro extraction (DLLME) followed by gaschromatography–mass spectrometry (GC-MS).Statistical analysis. All experiments were carriedout in triplicate. One-way ANOVA was performed todetermine significant differences. Duncan’s multiple109Ramezani H. et al. Foods and Raw Materials, 2020, vol. 8, no. 1, pp. 107–114range test was used to define the differences of meanvalue. Data analysis was performed using SPSSversion 21 (SPSS Inc., Chicago, IL, USA); P < 0.05 wasconsidered statistically significant.RESULTS AND DISCUSSIONThe present research featured samples from sevenmost popular types of sausages in Iran. The selectedsausages differed in the amount of nitrite, ascorbicacid, and nitrosamine. This difference is associatedwith the variations in concentration of added nitrateand nitrite salts (sodium and potassium), storageconditions, and different pH values [15, 16]. Followingthe EU legislations, 2006/52/EC directive limited theusage of nitrite to 150 mg per 1 kg of meat. However,the national laws in Iran are more restricting. Iranianprovisions allow for maximum 120 mg of nitrite per1 kg of meat (ppm) in sausages [17]. Table 1 displays theresidual nitrite, ascorbic acid, and nitrosamine contentsin the meat samples under study during storage.Figure 1 illustrates the results of nitrite contentchanges (mg/kg meat) in the meat samples obtainedfrom factory B as representative of all the four meatfactories.As it was expected, the amount of nitrite residuein meat products of all four meat factories decreasedduring 28 days of refrigerated storage. The increasein meat content increased the reduction rate of sodiumnitrite in the products. In other words, lower residualnitrite content was detected in high content meatsamples. The highest reduction rate of nitrite contentwas observed in chicken and beef salami (90% of meat).On the first day of refrigerated storage, the Frankfurtsausage (40%) and both beef and chicken sausages (55%)contained 80 μg/kg of nitrite.The detected amount exceeded the level permittedby Iranian laws as defined by the Institute of Standardand Industrial Research of Iran (ISIRI). The maximumpermissible burden of nitrite is 66 and 63 μg/kg forprocessed meat products with 40% and 55% of meatcontent, respectivelyI. The concentration of nitrite washigher than the expected levels even after 28 days ofstorage. However, the residual nitrite content was notexactly equal to the initial added nitrite. First, it waspartly degraded by heating process. Second, it decreasedwhen ascorbic acid was applied to the meat productduring the heating process to accelerate conversion ofnitrite to nitric oxide.The nitrite content varied due to interaction withheme-containing components, non-heme proteins,and fat tissues, conversion to nitrate, production ofsuch gases as N2, CO2, and NO, and nitrosamines [18].Therefore, residual nitrite was associated with meatcontent due to different contents of myoglobin [19]. As areactive agent, nitrite converts to nitrite oxide and formsI ISIRI 932. Test method for determination of nitrite in meat and meatproducts (reference method). Iran: Iran Institute of Standards andIndustrial Research; 2014.nitrite-heme-nitrosomyoglobin complex with myoglobin,thus producing nitrosomyochromogen. The latter isresponsible for the characteristic bright pink color ofcured meat products [20].Hence, higher content of myoglobin results in lowernitrite content in the final meat product. The resultsobtained by statistical analysis indicate that residualnitrite in Frankfurt sausage (40%) was significantlyhigher (P < 0.05), whereas in beef salami (90%) it wasat its lowest. No significant differences were observed inother sausage and salami products (P > 0.05).Ascorbic acid, or ascorbate, is applied to meatproducts as an additive with high water solubility forthree major reasons. First, the nitrosomyoglobin-formingreduction of nitrite to nitric oxide produces the requiredcolor. Second, the antioxidative activity of ascorbic acidslows down oxidation of pigments and lipids, whichresults in color and flavor stability. Third, residual nitritedecreases due to binding to nitrite in heated samples [21].Ascorbate proved effective in nitrosamine inhibition.This quality is associated with rapid reactions ofascorbate with nitric oxide compared to nitrosatingagents, e.g. amines. Therefore, nitrosamines are formedwhen the reaction rate constant of ascorbate is notmuch larger than amines [16]. Figure 2 and Table 1demonstrate that ascorbic acid content of samplesdecreased significantly (P < 0.05) during refrigeratedstorage.Meat products with higher meat content exhibitedlower ascorbic acid reduction rate, which was due tolower residual nitrite content. Ascorbic acid degradationin meat products could be related to oxygen content,temperature, light, water activity, presence of metalions, e.g. copper and ferric iron, and storage time[22–24]. Degradation of ascorbic acid is increased inacidic conditions (pH = 3.3–5.5). In the current study,Figure 1 Residual nitrite content (mg/kg meat) in sausageswith different meat content from factory B on days 0, 7, 14, 21,and 280501001502000 7 14 21 28Nitrite, ppmTime, dayBeef salami 90% Chicken salami 90%Dry cured salami 60% Beef sausage 55%Chicken sausage 55% Dry cured sausage 70%Frankfurt sausage 40%901401902400 Ascorbic acid, ppbBeef salami Dry cured Chicken Frankfurt 010203040500 7 14 21 28Nitrosamine, ppbTime, dayBeef salami 90% Chicken salami 90%Dry cured salami 60% Beef sausage 55%Chicken sausage 55% Dry cured sausage 70%Frankfurt sausage 40%0204060801000 Nitrite, ppmPlant A (1)(2)(3)(1)(2)(3)(4)(5)(6)(7)(5)(7)(6)(4)110Ramezani H. et al. Foods and Raw Materials, 2020, vol. 8, no. 1, pp. 107–114Table 1. Residual nitrite, ascorbic acid and nitrosamine contents in sausages produced by four major meat factories during storageFactory A Day 0 Day 7 Day 14 Day 21 Day 28Beef 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 ± 0.038eAscorbic acid, ng/g 214.08 ± 0.262a 216.81 ± 0.026a 194.98 ± 0.018b 171.95 ± 0.017c 167.82 ± 0.026cChicken 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 ± 0.616dAscorbic acid, ng/g 175.39 ± 0.039a 168.53 ± 0.003a 159.21 ± 0.027b 140.84 ± 0.039c 134.40 ± 0.021dDry 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 ± 0.091eAscorbic acid, ng/g 185.86 ± 0.021a 180.79 ± 0.003b 172.05 ± 0.062c 168.86 ± 0.683d 162.35 ± 0.706dBeef 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 ± 1.077dAscorbic acid, ng/g 198.22 ± 0.149a 203.91 ± 0.057a 196.78 ± 0.031a 182.49 ± 0.033b 175.88 ± 0.018cChicken 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 ± 0.0167dAscorbic acid, ng/g 165.27 ± 0.009a 163.34 ± 0.009a 161.99 ± 0.024a 158.26 ± 0.072b 149.46 ± 0.031cDry 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 ± 0.045bAscorbic acid, ng/g 129.67 ± 0.416a 120.55 ± 0.007ab 117.99 ± 0.033b 106.79 ± 0.039c 105.82 ± 0.705cFactory B Day 0 Day 7 Day 14 Day 21 Day 28Beef 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 ± 0.1054cAscorbic acid, ng/g 194.99 ± 0.016a 192.30 ± 0.021a 165.00 ± 0.699b 144.62 ± 0.004c 121.60 ± 0.060dChicken 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 ± 0.031bAscorbic acid, ng/g 220.79 ± 0.022a 192.84 ± 0.554b 168.95 ± 0.134c 164.89 ± 0.025c 134.07 ± 0.492dDry 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 ± 0.575cAscorbic acid, ng/g 201.24 ± 0.127a 188.65 ± 0.038b 172.34 ± 0.401c 168.01 ± 0.002d 145.69 ± 0.018eBeef 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 ± 0.94cAscorbic acid, ng/g 200.90 ± 0.132a 193.71 ± 0.015ab 187.60 ± 0.345b 183.60 ± 0.031b 174.74 ± 0.297cChicken 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 ± 0.133eAscorbic acid, ng/g 181.71 ± 0.014a 160.27 ± 0.020b 138.63 ± 0.013c 121.13 ± 0.003d 104.42 ± 0.005eDry 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 ± 0.622dAscorbic acid, ng/g 197.86 ± 0.215a 156.10 ± 0.021b 131.45 ± 0.073c 127.63 ± 0.015d 126.30 ± 0.007dFrankfurt 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 ± 0.229dAscorbic acid, ng/g 192.57 ± 0.062a 187.08 ± 0.032b 169.47 ± 0.057c 155.29 ± 0.008d 137.55 ± 0.006eFactory C Day 0 Day 7 Day 14 Day 21 Day 28Beef 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 ± 0.123dAscorbic acid, ng/g 232.27 ± 0.0321a 187.13 ± 0.060b 135.76 ± 0.025c 122.71 ± 0.031cd 108.52 ± 0.026dChicken 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 ± 0.117eAscorbic acid, ng/g 162.54 ± 0.055a 155.95 ± 0.086b 121.19 ± 0.060c 103.93 ± 0.112d 92.68 ± 0.049eDry 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 ± 0.052dAscorbic acid, ng/g 248.29 ± 0.004a 213.54 ± 0.630b 198.32 ± 0.016b 165.78 ± 0.032c 119.79 ± 0.296dBeef 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 ± 0.051dAscorbic acid, ng/g 200.69 ± 0.45a 196.17 ± 0.057a 189.30 ± 0.047ab 175.08 ± 0.107b 142.58 ± 0.117cChicken 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 ± 0.051dAscorbic acid, ng/g 199.63 ± 0.588a 184.59 ± 0.068b 165.67 ± 1.013c 102.61 ± 0.173d 79.79 ± 0.033e111Ramezani H. et al. Foods and Raw Materials, 2020, vol. 8, no. 1, pp. 107–114Dry 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 ± 0.037dAscorbic acid, ng/g 149.83 ± 0.017a 114.28 ± 0.177b 103.91 ± 0.122c 89.73 ± 0.145d 71.32 ± 0.035eFrankfurt 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 ± 0.090eAscorbic acid, ng/g 141.12 ± 0.059a 138.09 ± 0.012a 102.27 ± 0.027b 91.43 ± 0.068b 74.10 ± 0.707cFactory D Day 0 Day 7 Day 14 Day 28Beef 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 ± 0.077cAscorbic acid, ng/g 233.43 ± 0.202a 195.42 ± 0.054b 163.34 ± 0.095c 157.90 ± 0.158c 132.90 ± 0.167dDry 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 ± 0.036cAscorbic acid, ng/g 169.07 ± 0.115a 157.47 ± 0.032b 114.25 ± 0.174c 103.32 ± 0.151d 89.92 ± 0.341eBeef 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 ± 0.063dAscorbic acid, ng/g 198.59 ± 0.035a 104.30 ± 0.240b 54.95 ± 0.079c 35.61 ± 0.162d 29.11 ± 0.452dChicken 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 ± 0.059cAscorbic acid, ng/g 201.10 ± 0.351a 173.31 ± 0.494b 138.33 ± 0.442c 90.33 ± 0.953d 68.82 ± 0.721dFrankfurt 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 ± 0.293eAscorbic acid, ng/g 282.14 ± 0.310a 181.60 ± 0.025b 170.03 ± 0.169c 117.97 ± 0.037d 75.22 ± 0.897eDifferent 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 1Figure 2 Ascorbic acid content (ng/kg meat) in sausageswith different meat content from factory B on days 0, 7, 14, 21,and 2828salami 90%sausage 55%sausage 70%901401902400 7 14 21 28Ascorbic acid, ppbTime, dayBeef salami 90% Chicken salami 90%Dry cured salami 60% Beef sausage 55%Chicken sausage 55% Dry cured sausage 70%Frankfurt sausage 40%28y = 10.951x + 80.875R² = 0.9423y = 16.803x + 90.748y = 21.596x + 121.65R² = 0.8989406080100Nitrite, ppmFigure 3 Nitrosamine content (ng/kg of meat) in sausageswith different meat content from factory B on days 0, 7, 14, 21,and 280501001502000 7 14 21 28Nitrite, ppmTime, dayBeef salami 90% Chicken salami 90%Dry cured salami 60% Beef sausage 55%Chicken sausage 55% Dry cured sausage 70%Frankfurt sausage 40%901401902400 Ascorbic acid, ppbBeef salami Dry cured Chicken Frankfurt 010203040500 7 14 21 28Nitrosamine, ppbTime, dayBeef salami 90% Chicken salami 90%Dry cured salami 60% Beef sausage 55%Chicken sausage 55% Dry cured sausage 70%Frankfurt sausage 40%0204060801000 Nitrite, ppmPlant pH values were in the range of 5–6 in all samples.Therefore, ascorbic acid degradation might have beendue to the relative acidic condition of meat products andheating process.The obtained results showed an increase innitrosamine content of samples during refrigeratedstorage. However, it was not significant in all samples(Fig. 3).The nitrite reduction rates in the sausages with asmaller diameter, e.g. Frankfurt sausage, appearedsignificantly lower (P < 0.05) than in the samples witha bigger diameter, e.g. salami. This difference can beexplained by the longer cooking time for salami (5–6 h)compared to Frankfurt sausages (3–4 h), which isassociated with a higher reduction rate of residualnitrite. According to the results, the total volatilenitrosamine level of Iranian meat products withlower meat contents was generally higher thanthat of samples with high meat contents. Thenitrosamine content in meat products was associatedwith the added nitrite and the residual nitrite [25].The residual nitrite is a reactive agent that can bereduced by heating treatment or exposure to such meatcomponents 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)112Ramezani H. et al. Foods and Raw Materials, 2020, vol. 8, no. 1, pp. 107–114y = 10.951x + 80.875R² = 0.9423y = 16.803x + 90.748y = 13.533x + 68.202 R² = 0.9527R² = 0.9605y = 21.596x + 121.65R² = 0.89890204060801000 7 14 21 28Nitrite, ppmTime, dayPlant A Plant B Plant C Plant DFigure 4 shows that there was a significantcorrelation between the amounts of added nitrite and thenitrosamine contents in beef salami (90%) samples fromall four meat factories.In this study, the correlation factor was 0.9, whichasserts the effects of added nitrite and also residualnitrite contents on nitrosamine formation in theprocessed meat product. Therefore, the products withlower meat content showed higher residual nitrite and,consequently, a greater nitrosamine formation. In meatproducts, presence of nitrosating agents increases theconcentration of nitrosamine, whether grouped in NO2related agents, e.g. N2O4, or grouped in nitrous acidderivatives, e.g. N2O3 and HNO2 [28].There are several suggested strategies to reducenitrosamine formation in meat products to improve theirhealthy status and safety. The present research clarifiedthat the levels of nitrosamines in the samples dependedon the amount of residual nitrite, ascorbic acid, pH, andcooking temperature. Higher levels of residual nitritewere detected in the samples with a lower amount ofmeat, compared to those with a higher amount of meat.CONCLUSIONIn the current study, the residual nitrite, ascorbicacid, and nitrosamine contents of seven most popularIranian processed meat products, namely sausageswith different amounts of meat were evaluated tomonitor the safety status of the meat industry. Thesamples contained various concentrations of nitrite andnitrosamine, which were above the permitted standardlevel. Several factors were found to affect the residualnitrite and nitrosamine contents, the meat content beinga significant variable. Nitrite interacted with hemecontainingcomponents, non-heme proteins, and fattissues, thus conversed to nitrate and nitrosamine.Therefore, the contents of residual nitrite and,consequently, nitrosamine in products with lower meatcontent were significantly higher. A longer cookingtime also decreased residual nitrite and nitrosamineconcentration.Further research is needed to identify newsubstances that could replace nitrites, as well as factorsthat reduce the required amount of nitrite in meatproducts.CONTRIBUTIONThe authors were equally involved in writing themanuscript and are equally responsible for plagiarism.CONFLICT OF INTERESTThe authors declare that there is no conflictof interest regarding the publication of this article.ACKNOWLEDGEMENTSWe would like to thank National Nutrition and FoodTechnology Research Institute for the financial support.