effect of different storage conditions on n-nitrosamine content in ...

Bull. Vet. Inst. Pulawy 46, 317-324, 2002EFFECT OF DIFFERENT STORAGE CONDITIONSON N-NITROSAMINE CONTENT IN POLISH EDIBLEOFFALS PROCESSED MEAT PRODUCTSKATARZYNA DOMAŃSKA AND BOGDAN KOWALSKI*Department <strong>of</strong> Poultry Diseases,* Laboratory <strong>of</strong> Radiological Protection and Isotopic Research,National Veterinary Research Institute,24-100 Pulawy, Polande-mail: domanska@piwet.pulawy.plThe levels <strong>of</strong> volatile N-nitrosamines were determined in 18 samples <strong>of</strong> edible <strong>of</strong>falsprocessed meat products fresh and stored in <strong>different</strong> <strong>conditions</strong> e.g. 72 h at 4-8 0 C and 72 h at 4-8 0 C plus 24 h at 22-26 0 C. The analyses were carried out by gas chromatography (GC) coupledwith thermal energy analyser (TEA). Fresh samples sporadically contained low amounts <strong>of</strong> N-nitrosodimethylamine (NDMA). Storage for 72 h at 4-8 0 C influenced the appearance <strong>of</strong> NDMAand additional N-nitroso compounds. In almost all samples NDMA and N-nitrosopiperidine(NPIP) at the concentrations <strong>of</strong> 2.8 µg/kg and 4.2 µg/kg, respectively, were found. Few samplescontained low amounts <strong>of</strong> N-nitrosodibutylamine (NDBA) and N-nitrosomorpholine (NMOR).Additional <strong>storage</strong> for 24 h at 22-26 0 C induced further changes <strong>of</strong> N-nitrosamines occurrence. Aspreviously, almost in all meat products NDMA and NPIP at the concentrations 1.1 µg/kg and 4.6µg/kg, respectively, were found. In a few samples traces <strong>of</strong> NDBA were detected. No samplecontained NMOR but in three samples N-nitrosopyrrolidine (NPYR) was present.Key words: N-nitrosamines, analysis, meat products, <strong>storage</strong> <strong>conditions</strong>.N-nitroso compounds are potent carcinogens that can induce tumours invarious animal species at a variety <strong>of</strong> sites (8, 26). The acute toxicity <strong>of</strong> nitrosamines inhumans was demonstrated for the first time in 1954 by Barnes and Magee (3). Over300 N-nitroso compounds have been investigated for carcinogenicity: 86% <strong>of</strong> 232nitrosamines and 91% <strong>of</strong> 100 nitrosamides studied showed carcinogenic activity inexperimental animals (20). Humans are exposed to nitrosamines both from diet andother environmental sources as well as from endogenous synthesis within the body (4,15). These compounds are formed in food in the reaction <strong>of</strong> nitrosating agents (nitrates,nitrites, oxides <strong>of</strong> nitrogen) added as preservatives and primary, secondary and tertiaryamines and amides, proteins, peptides and amino acids during processing andpreservation (26).Many studies were carried out to determine levels <strong>of</strong> volatile nitrosamines infood and beverages. The most commonly encountered volatile nitrosamines in curedmeats were N-nitrosodimethylamine (NDMA), N-nitrosopiperidine (NPIP) and N-nitrosopyrrolidine (NPYR). Other foods and beverages that contained detectable levels

318<strong>of</strong> these nitrosamines included cheese, beer and certain milk products (10). In a surveyconducted in France, Biaudet et al. (5) analyzed 556 food samples. NDMA was foundin 427 samples (68%) but only traces were noted in meat products (0.04-0.46 µg/kg)and the highest level was observed in fish (13.4 µg/kg). In a food survey conducted inGermany, NDMA was detected in 31.5% <strong>of</strong> analyzed samples. The major dietarysources <strong>of</strong> this compound included cooked meat products (0.2-2.5 µg/kg), cooked fish(0.5-8.0 µg/kg) and spices (0.1-1.4 µg/kg). The other volatile N-nitrosamines, NPIPand NPYR were detected at very high concentration up to 23.0 µg/kg and 29.0 µg/kg,respectively, in ground pepper (25). The monitoring studies undertaken to determinethe level <strong>of</strong> N-nitrosamines in Polish food revealed that 71% cured and 61%pasteurized pork hams contained NDMA in the range <strong>of</strong> 0.1-0.5 µg/kg; also N-nitrosodiethylamine (NDEA) (0.3-1.6 µg/kg), N-nitrosodibutylamine (NDBA) (1.7-7.5µg/kg) and NPIP (1.4-2.2 µg/kg) occasionally were found (28). Kowalski et al. (13)reported NDMA in 63.3% <strong>of</strong> 330 analyzed processed poultry products with the meanconcentration <strong>of</strong> 1.57 µg/kg and also sporadically NDBA and N-nitrosomorpholine(NMOR). Similar situation was observed in the survey <strong>of</strong> edible <strong>of</strong>fals processed meatproducts: 67.6% samples contained NDMA with the mean concentration <strong>of</strong> 1.39 µg/kg.Analysis <strong>of</strong> 280 samples <strong>of</strong> tinned food revealed that 46.1% contained NDMA and/orNPIP in the range <strong>of</strong> 0.07-4.5 µg/kg.Due to tradition and consumption habits, the edible <strong>of</strong>fals processed meatproducts (such as “kaszanka” – blood sausage, “salceson” - headcheese, “pasztetowa” –liver sausage etc. (19)) are produced and consumed in large amounts in Poland. Verylimited reports on the occurrence <strong>of</strong> N-nitrosamines in such kind <strong>of</strong> meat products areavailable from other countries. Results <strong>of</strong> previous tentative studies on N-nitrosaminesoccurrence in Polish edible <strong>of</strong>fals processed meat products suggested that due tospecific recipes <strong>of</strong> production and very short shelf-life, <strong>storage</strong> <strong>conditions</strong> influence theappearance <strong>of</strong> N-nitrosamines in these products. The objective <strong>of</strong> this work was toexamine the <strong>effect</strong> <strong>of</strong> <strong>different</strong> <strong>storage</strong> <strong>conditions</strong> <strong>of</strong> these products on the content andformation <strong>of</strong> volatile N-nitrosamines.Material and MethodsSamples. Eighteen samples <strong>of</strong> <strong>different</strong> edible <strong>of</strong>fals processed meat products(“salceson”, “pasztetowa”, “kaszanka”) were taken from meat factories, just afterfinished production cycle, and transported in refrigerated containers to the laboratoryfor analysis. Samples, each about 1 kg, were divided into three groups. First one wasthe “fresh” samples, second and third were wrapped in aluminum foil and stored in<strong>different</strong> <strong>conditions</strong>. Second group <strong>of</strong> samples was stored for 72 h at 4-8 0 C and thethird group was stored for 72 h at 4-8 0 C and additionally for 24 h at room temperature(RT). On each sampling date the probes were frozen at –35 0 C and later the N-nitrosamines levels were determined.Volatile nitrosamine analysis. Volatile nitrosamines were extracted by lowtemperature vacuum distillation according to method recommended by the Food Safetyand Inspection Service (FSIS) (7). The distillate extracts were quantitatively analyzedon a gas chromatograph (GC, Varian, model 1440) interfaced with a thermal energyanalyzer (TEA, model 502A, Thermo Electron Corporation, Waltham, MA).Identification and quantification <strong>of</strong> the nitrosamines were carried out by analysis <strong>of</strong>

319known amounts <strong>of</strong> nitrosamine standards containing N-nitrosodimethylamine(NDMA), N-nitrosodiethylamine (NDEA), N-nitrosodipropylamine (NDPA), N-nitrosodibuthylamine (NDBA), N-nitrosopiperidine (NPIP), N-nitrosopyrrolidine(NPYR), N-nitrosomorpholine (NMOR) (certified standards from Chem-Services,which is also supplier <strong>of</strong> N-nitrosamine standards for the Food and DrugAdministration -FDA). N-nitrosodiisopropylamine (NDiPA) added to the samplesbefore extraction was used as the internal standard. GC-TEA <strong>conditions</strong> were asfollows: column: 2.7 m x 3 mm i.d. packed with 15% Carbowax 20 M-TPA on aChrom W-HP 80/100 mesh; column temperature: 170 0 C; injection port temperature:200 0 C; carrier gas: He at 25-30ml/min.; TEA furnace temperature: 475 0 C; vacuum: 0.3Torr, velocity <strong>of</strong> oxygen: 15-20 ml/min. The method enables the identification andsimultaneous quantification <strong>of</strong> seven nitrosamines at the level above 0.04-0.2 µg/kgdepending on analyzed material and compound. Recoveries were at the level <strong>of</strong> 76-98%. Direct confirmation <strong>of</strong> analyzed compounds was performed by gaschromatography coupled with mass spectrometry (GC: model 5970B, Hewlett-Packard;MSD: Electron Impact, EI, 70eV) according to method recommended by FSIS (7).Statistical calculations. Results are expressed as mean ± SD <strong>of</strong> N-nitrosamines concentrations (µg/kg). The Wilcoxon test was used to conduct statisticalanalysis. Differences between mean values were considered significant at P-value

320A B CNDMANDMAinternal standardNPIPA B CNDMANPIPNDBANDBANPIPA B CNDMANPIPNDMANPIPFig.1. GC-TEA chromatograms <strong>of</strong> three edible <strong>of</strong>fals processed meat productsanalyzed A – on purchase day, B – after <strong>storage</strong> for 72 h at 4-8 0 C, C – after <strong>storage</strong> for72 h at 4-8 0 C and 24 h at room temperature.

321Table 1Mean concentrations (µg/kg) <strong>of</strong> seven volatile N-nitrosaminesin studied meat products stored in <strong>different</strong> <strong>conditions</strong> (mean <strong>of</strong> 18 samples)purchaseday72 hat 4-8 0 Cplus 24 hat 22-26 0 CNDMA NDEA NDPA NDBA NPIP NPYR NMOR TN0.02 - - - - - - 0.022.8 1 - - 0.5 4.2 - 0.4 7.9 11.1 1, 2 - - 0.8 4.6 1.8 - 8.3 1NDMA - N-nitrosodimethylamine, NDEA – N-nitrosodiethylamine, NDPA – N-nitrosodipropylamine, NDBA – N-nitrosodibutylamine, NPIP – N-nitrosopiperidine,NPYR – N-nitrosopirrolidine, NMOR – N-nitrosomorpholine, TN – total N-nitrosamines1 – significantly <strong>different</strong> at P

322<strong>conditions</strong>. There are several factors which may influence the appearance <strong>of</strong>nitrosamines. Starting materials for nitrosamine formation are nitrate, nitrite andprimary, secondary and tertiary amines and amides, proteins, peptides and amino acidsor precursors <strong>of</strong> these, which are transformed into nitrosamine precursors by microbialaction (26). It seems that all these compounds could be present in analyzed meatproducts.Nitrate and nitrite are used as preservatives in meat products. These curingagents develop and stabilize the colour associated with cured meats, enhance the curedflavour and act as antioxidants. Nitrite is also antimicrobial agent that inhibitsClostridium botulinum growth and toxin production. Among studied meat productsonly “salceson” contains the greatest amount <strong>of</strong> salted compounds, other contain theirlower amounts or non-salted materials. On the other hand, all these products containingredients <strong>of</strong> plant origin (garlic, onion, paprika, semolina, buckwheat groats, andherb). There are many published papers that provide evidence for the presence <strong>of</strong>nitrate in plant foods (6, 11, 16). The other source <strong>of</strong> nitrate and nitrite in theseproducts could be water used during their preparation. Vegetables, grains and meatscan be rinsed and cooked in water. The amount <strong>of</strong> nitrate/nitrite available from watertaken in <strong>different</strong> places differs considerably depending on water source (17).Nitrosamine formation is also dependent on the presence <strong>of</strong> amines, amides,proteins, peptides and amino acids. The biogenic amines in <strong>different</strong> types <strong>of</strong> meats andmeat products were found in a great variability <strong>of</strong> concentrations (1). They areproduced by the action <strong>of</strong> microbial decarboxylases on free amino acids. The mainfactors affecting formation <strong>of</strong> the biogenic amines in foods are presence <strong>of</strong>microorganisms and <strong>storage</strong> <strong>of</strong> foods in <strong>conditions</strong> enabling their growth (23). Silla-Santos (23) reported that the content <strong>of</strong> amines could increase during the <strong>storage</strong>. It wasshown that NDMA could be formed from such amino acid as glycine, NPIP from suchamine as cadaverine or piperidine and NPYR from spermine and spermidine also fromamino acid proline (12, 14, 24, 27). Other sources <strong>of</strong> precursors <strong>of</strong> nitrosamines as wellas performed nitrosamines could be <strong>different</strong> additives occurring in food as spices,vegetables, and herbs (25).Nitrosamines can be formed as the result <strong>of</strong> chemical reaction and alsomicrobial action. The analyzed meat products are made <strong>of</strong> compounds which could becontaminated with some microorganisms (gastric walls, tongue, blood, as well asspices, herbs and cereals), contain low levels <strong>of</strong> added preservatives (nitrate or nitrite)and are processed in rather low temperatures (brewing at 80-85 0 C during 50-130 min,and sometimes smoking at 30 0 C during 30-40 min). All these cause that their shelf-lifeis rather short. They could contain some microbial load just on the purchase day. The<strong>storage</strong> <strong>conditions</strong> applied in our experiment additionally influenced the number <strong>of</strong>microorganisms, especially <strong>storage</strong> for 24 h at rt. Ayanaba reported thatmicroorganisms could take part in N-nitrosamines formation in <strong>different</strong> ways: bynitrates reduction to nitrites, degradation <strong>of</strong> proteins to amines and amino acids,production <strong>of</strong> enzymes carrying nitrosation and formation <strong>of</strong> pH suitable for nitrosation(2). Some strains <strong>of</strong> Lactobacillus casei showed a biocatalytic <strong>effect</strong> on the formation<strong>of</strong> nitrosamines, especially the extract <strong>of</strong> their intracellular enzymes (21). Other genus<strong>of</strong> bacteria, like coliforms, Clostridium or Pseudomonas could also synthetizenitrosamines (2, 9, 22).It is known that nitrosation reactions can be influenced by inhibitors (vitaminC and E) or catalysts (metal ions, nucleophilic anions as Cl - or I - , carbonyl compounds)(26). The presence <strong>of</strong> such substances in studied products could not be ruled out. The

323formation <strong>of</strong> N-nitrosamines in foods is a complex process and a large diversity <strong>of</strong>substances could influence nitrosation reaction.The changes <strong>of</strong> N-nitrosamines concentrations observed in our experimentsafter applied <strong>different</strong> <strong>storage</strong> <strong>conditions</strong> seem to be the result <strong>of</strong> chemical reactionsbetween precursors <strong>of</strong> nitrosamines present or formed in edible <strong>of</strong>fals processed meatproducts. However, in the case <strong>of</strong> <strong>storage</strong> at RT the changes in N-nitrosamines levelscould be more the result <strong>of</strong> microbial actions.Acknowledgement: Authors gratefully acknowledge A. Mazurek and H.Dudek for excellent technical assistance.References1. Ansorena D., Montel M. C., Rokka M., Talon R., Eerola S., Rizzo A., RaemaekersM., Demeyer D.: Analysis <strong>of</strong> bioobenic amines in nothern and southern Europeansausages and role <strong>of</strong> flora in amine production. Meat Science, 2002, 61, 141-147.2. Ayanaba A., Alexander M.: Microbial formation <strong>of</strong> nitrosamines in vitro. App.Mirobiol., 1973, 25, 862-868.3. Barnes J.M., Magee P. N.: Toxic properties <strong>of</strong> dimethylnitrosamine. Brit. J. Ind.Med., 1954, 11, 167-174.4. Bartsch H., Spiegelhalder B.: Environmental exposure to N-nitroso compounds(NNOC) and precursors: an overview. Europ. J. Cancer Prev., 1996, 5, 11-17.5. Biaudet H., Mavelle T., Debry G.: Mean daily intake <strong>of</strong> N-nitrosodimethylaminefrom foods and beverages in France in 1987-1992. Food Chem. Toxicol., 1994,32, 417-421.6. Borawska M., Omieljaniuk N., Rostkowski J., Otlog T., Hamid F.: Value <strong>of</strong>nitrates and nitrites in selected vegetables and potatoes sold in the marketplace <strong>of</strong>Bialystok in the years 1991-1992. Roczniki PZH, 1994, 45, 89-96.7. Food Safety and Inspection Service. NTR2. Determination <strong>of</strong> Nitrosamines by theLow Temperature Vacuum Distillation Procedure, Chemistry LaboratoryGuidebook Residue Chemistry, USDA-FSIS, 1991.8. Forman D.: Dietary exposure to N-nitroso compounds and the risk <strong>of</strong> humancancer. Cancer Surv., 1987, 6, 719-738.9. Hawksworth G.M., Hill M. J.: Bacteria and N-nitrosation <strong>of</strong> secondary amines.Brit. J. Cancer, 1971, 25, 520-526.10. Hecht S.S.: Approaches to cancer prevention based on an understanding <strong>of</strong> N-nitrosamine carcinogenesis. P.S.E.B.M., 1997, 216, 181-191.11. Hunt J., Turner M. K.: A survey <strong>of</strong> nitrite concentrations in retail fresh vegetables.Food Additives and Contam., 1994, 11, 327-332.12. Juszkiewicz T.: Żródła nitrozoamin w żywności pochodzenia zwierzęcego.Medycyna Wet., 1978, 34, 412-417.13. Kowalski B., Domańska K., Rachubik J.: Badania lotnych N-nitrozoamin wkrajowych przetworach mięsnych. Raport z badań monitorowych nad jakościągleb, roślin, produktów rolniczych i spożywczych w 1998 roku. MRiRWWarszawa 1999, 142-146.14. Maga J.A.: Amines in foods. Crit. Rev. Food Sci., 1978, 10, 373-380.15. Mirvish S.S.: Role <strong>of</strong> N-nitroso compounds (NOC) and N-nitrosation in etiology<strong>of</strong> gastric, esophageal, nasopharyngeal and bladder cancer and contribution to

324cancer <strong>of</strong> known exposures to NOC. Cancer Lett., 1995, 93, 17-48.16. Nabrzyski M., Gajewska R.: The content <strong>of</strong> nitrates and nitrites in fruits,vegetables and other foodstuffs. Roczniki PZH, 1994, 45, 167-180.17. Pennington J.A.T.: Dietary exposure models for nitrates and nitrites. Food Control,1998, 9, 385-395.18. Polska Norma., Meat products – Sausages, PN-A-82007, 1996.19. Polska Norma., Meat and meat products – Terminology, PN-A-82023, 200020. Preussmann R., Stewart B. W.: N-nitroso carcinogens. In: Chemical Carcinogens.Vol. 2 ACS Monograph 182. Ed. C.E. Searle., 1984, American Chemical Society,Washington, DC, 643-828.21. Przybyłowski P.: Studia nad rolą wybranych szczepów bakterii fermentacjimlekowej w przemianach azotanów i syntezie N-nitrozoamin. Zeszyty NaukoweART w Olsztynie, 1984, 9, 3-59.22. Przybyłowski P., Kisza J., Karłowski K., Sajko W., Urbańska J., Janicka B.:Badania występowania azotanów i produktów ich przemian w mleku i wyrobachmleczarskich. Roczniki PZH, 1987, 38, 214-218.23. Silla-Santos M.H.: Biogenic amines: their importance in foods. Intern. J. FoodMicrobiol., 1996, 29, 213-231.24. Smith T.A.: Amines in food. Food Chem., 1980, 6, 169-200.25. Tricker A.R., Pfundstein B., Theobald E., Preussmann R., Spiegelhalder B.: Meandaily intake <strong>of</strong> volatile N-nitrosamines from foods and beverages in WestGermany in 1989-1990. Food Chem. Toxic., 1991, 29, 729-732.26. Tricker A.R., Preussmann R.: Carcinogenic N-nitrosamines in the diet: occurrence,formation, mechanism and carcinogenic potential. Mutat. Res., 1991, 259, 277-289.27. Warthesen J.J., Scanlan R. A., Bills D. D., Libbey L. M.: Formation <strong>of</strong>heterocyclic N-nitrosamines from the reaction <strong>of</strong> nitrite and selected primarydiamines and amino acids. J. Agric. Food Chem., 1975, 23, 898-902.28. Żmudzki J., Niewiadowska A., Kowalski B., Szkoda J., Semeniuk S.: Pozostałościchemiczne w szynkach wieprzowych. Medycyna Wet., 1994, 50, 623-625.