Nitrite Residues of Bouillons Marketed in Turkey Summary
Nitrite Residues of Bouillons Marketed in Turkey
Bouillons are dehydrated broths formed into a small cube or granules that can be prepared from bovines, chicken, duck or lamb. Adding nitrite to meat is a common process that helps the delaying of botulinal toxin development. It also stabilizes the red meat color and retards the development of oxidative rancidity, off-odors and off-flavors during storage. However, nitrite remaining in cured meat, known as “residual nitrite”, is suspected as also being a health risk. Given that nitrate and nitrite addition to meats is legal, there is need to detect the nitrite content of meat and meat-based products. This study was aimed to detect whether there was any total nitrite content (nitrite contamination and residual nitrite) in bouillons and to estimate possible toxicological risks. For this purpose, 20 bouillon samples (bovine- and chicken- based), in cube or granule form, were randomly collected from markets in Ankara, Turkey. For the detection of nitrite contamination of the samples, Griess method was used with slight modifications. The nitrite in bovine- and chicken-based bouillon samples was obtained as 3.288±1.145 µg/g and 2.622±0.828 µg/g, respectively. It can be concluded that bouillons and other processed meat products should be consumed attentively because of the detrimental effects of the nitrite on the general population and particularly in vulnerable groups such as young children and the elderly.
Key Words: Bouillon, nitrite, food toxicity.
Received : 08.10.2007 Revised : 19.02.2008 Accepted : 26.02.2008
INTRODUCTION
Bouillons, which are convenient to use and cheap, are dehydrated broths formed into a small cubes or granules. Other than bovines, bouillons can be pre- pared from chicken, duck or lamb. Bouillons were first used during World War I because of the high price of meat and the ease of preparation. They are used in cooking in different cuisines to enhance the taste of the meals. Bouillons are used as soups in
developing countries because of their cheap price. In the meat industry, sodium and potassium nitrites in combination with salt are used for the delay of botu- linal toxin development1. According to the Turkish regulations, the nitrite limit was set as 150 mg/kg (as sodium salt) in meat products2.
Nitrites are also used as color fixatives to stabilize
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red meat color and they retard the development of oxidative rancidity and off-odors and off-flavors during storage. It is very likely that meat-based prod- ucts may also contain nitrite as a food additive3-5. Water and vegetables are substantial sources of nitrite, and <5% of daily nitrite intake comes from cured meats (6-8). Apart from exposure from food and water, humans are also exposed to nitrite from the in vivo conversion of nitrate to nitrite, as nitrites can be formed endogenously from the reduction of nitrates by the microflora in the saliva and in the gastrointes- tinal tract3,9-12.
Nitrite has been implicated in a variety of long-term adverse effects and has been of interest to public health providers and governmental regulators for the last 40 years9. The Joint Food and Agriculture Orga- nization (FAO)/World Health Organization (WHO) Expert Committee on Food Additives (JECFA) and Scientific Committee on Food (SCF) have proposed an acceptable daily intake (ADI) for nitrite of 0-0.07 mg/kg body weight while the Environmental Protec- tion Agency (EPA) has set a reference dose (RfD) of 0.10 mg nitrite nitrogen per kg/body weight/day (equivalent to 0.33 mg nitrite ion/kg body weight/day)13. Nitrates or nitrites (ingested, under conditions that result in endogenous nitrosation) are Group 2A carcinogens (probably carcinogenic to humans)14. Turkish Food Codex in “Food Additives Other than Colors and Sweeteners Announcement (Announcement No:2003/44)” stated that non-heated, cured, dried meat products could have 150 mg/kg sodium nitrite as an indicative ingoing amount and 50 mg/kg sodium nitrite as a residual amount; other cured meat products and canned meat products could have 150 mg/kg sodium nitrite as an indicative ingo- ing amount and 100 mg/kg sodium nitrite as a resid- ual amount; and cured bacons could have 175 mg/kg sodium nitrite as a residual amount as maximum levels, as also expressed in the European Parliament and Council Directive 95/2/EC of 20 February 199515,16. According to Turkish Food Codex and EC Directive 95/2/EC, bouillons could have a maximum 100 mg/kg sodium nitrite as a residual amount.
The present study was aimed to assess nitrite residues
in commonly consumed and commercially available bouillons and to evaluate the possible health risk.
MATERIALS and METHODS Sample Collection
A total of 20 bouillons (cubes or granules), which are distributed throughout the country, were randomly collected from different markets in Ankara, the capital of Turkey, in the winter of 2007. The collected samples were of different brands within the groups and the limited number of samples is due to the limited number of different brands of bouillons in Turkey.
The analyzed samples included bovine-based (n=10) or chicken-based (n=10).
Chemicals
All chemicals used in this study were analytical grade (Sigma Co., St. Louis, MO, USA and Merck Co., Darm- stadt, Germany). Ultra-high pure distilled water was used in the analytical work.
Standards. Stock nitrite solution was prepared by dissolving 50 mg sodium nitrite per dl of ammonium hydrochloride buffer (prepared from HCl and ammo- nia; pH=9.7). A working solution containing 5 µg/ml sodium nitrite was prepared daily by proper volume of stock solution. Standard solutions containing 0.010, 0.025, 0.050, 0.40, 0.60, 1.00 µg/ml sodium nitrite were obtained with dilution.
Extraction Procedure. The procedure was applied as in our last study on baby foods with slight modifications17. Briefly, 10 g of each sample was used for the nitrite analysis. 40 ml hot water was added on the sample and blended for 5 min in a blender.
The mixture was later heated to 100ºC for the preven- tion of ascorbic acid interference. The mixture was transferred to a volumetric glass and 50 ml hot water and 12 ml sodium hydroxide (2% w/v in water) was added and blended again for another 10 mins. Later, 10 ml of zinc sulfate (10% w/v in water) was added and the mixture was mixed for 5 min. The next step was to add 5 ml sodium hydroxide and the mixture
was blended for another 5 min. Water (83 ml) was added and mixed for 5 min. The last volume was 200 ml. The mixture was filtered through Nuche volumet- ric glass and then through filtered paper (preferably Whatman No. 1) until the filtrate was completely clear.
Determination of Nitrite. The Griess method was employed with a slight modification. The principle of the method was based on the reaction between nitrites and sulfanilic acid (1% w/v in 30% acetic acid) and Marshall’s reagent to produce a dye18. The absorbance can be measured at 550 nm.
In order to detect the nitrite level in the samples, the clear 1 ml filtrate was used. Ammonium hydrochloride buffer (0.9 ml) and 5 ml of 60% acetic acid were added on the filtrate tubes. Later, 0.5 ml of sulfanilic acid and 0.5 ml of Marshall’s reagent (N-(1-naphtyl) eth- ylenediamine hydrochloride; 0.1% w/v in 60% acetic acid) was added, then diluted to 5 ml by water and the mixture was vortexed for 30 sec. The mixture was left in the dark for 25 min. The same procedure was applied to sodium nitrite standard solutions and the absorbance was detected at 550 nm by using a spec- trophotometer (Shimadzu 160 UV, Japan).
Statistical Analysis
All results were expressed as mean ± standard error of mean (SEM). Comparison between two indepen- dent groups was made by the Mann-Whitney U test.
Values of p<0.05 were considered statistically signif- icant.
RESULTS
It was observed that limit of detection of the method (MDL) was 0.010 µg/ml. A linear correlation was found between absorbance and the concentration of nitrite standards (0.025-1.00 µg/ml). The recovery average was 98.82 % ± 0.25 for supplementation with 0.50 µg/ml of nitrite. At 0.50 µg/ml concentration of nitrite, the variation coefficients of within - and between-run precisions were 0.25 and 0.71, respec- tively. All the procedures in the detection of MDL
and recovery values were stated in our previous study17.
The mean nitrite concentration of the samples was 2.955 ± 0.692 µg/g with a maximum level of 10.53 µg/g. Values were 3.288 ± 1.145 µg/g and 2.622 ± 0.828 µg/g in bovine- and chicken-based bouillons, respectively. The results were summarized in Figure 1. After a simple comparison, it was observed that the bovine-based bouillon group had 20% higher nitrite contamination than the chicken-based samples.
No statistically significant difference between bovine- based and chicken-based groups was determined (p=0.796).
DISCUSSION
Since the beginning of the 20th century, nitrites and other nitrogenous compounds have been used exten- sively as fertilizers, and this change in the pattern of agricultural practice has caused an accumulation of nitrates and nitrites in the environment3,6. Nitrites are thought to be detrimental to human health19.
The reduction of nitrite to nitric oxide (NO) by bacteria and enzymatic reactions in the presence of muscle myoglobin forms iron-nitrosyl myoglobin, which gives the red color to meat and meat-based products20. However, nitrite remaining in cured meat, known as
Figure 1. Comparison of the nitrite levels between bovine- and chicken-based bouillon samples. Results of all samples were also shown.
Data were expressed as mean±SEM.
“residual nitrite”, is suspected as also being a health risk21. The oxidation of hemoglobin by nitrites causes the formation of methemoglobin, in which the ferrous iron is oxidized to ferric ion. Methemoglobin is not capable of binding molecular oxygen molecule11,22. Furthermore, ther e is thought to be an association between gastric cancer and the endogenous formation of mutagenic N-nitroso compounds, from secondary amines and nitrite by the catalyzation of thiocyanate, which is found in human saliva and the stomach23. Other than endogenous formation, N-nitroso com- pounds can be formed by heating or frying meat or meat-based products. Though there is no direct evi- dence for humans, scientists assume that N-nitroso compounds rather than nitrites are carcinogenic in man24. Therefore, diet can be the major determinant of risk. The feeding regimen is the most important issue in human carcinogenesis after cigarette smoking and diseases25.
The risk assessment for nitrite should include an assessment of exposure to both nitrite and nitrate, because of the conversion process of nitrate to nitrite.
It was also suggested that either the ADI is inappro- priate or needs to be re-evaluated for those individuals who have a high rate of conversion of nitrate to nitrite9.
According to the results of the present study, bovine- based bouillons contain a mean 3.288 µg/g and chick- en-based bouillons 2.622 µg/g nitrite. If a 70 kg person drinks soup containing 10 g (one cube) of bouillon, he will intake 32.88 µg nitrite from bovine-based bouillons and 26.22 µg from chicken-based bouillons, both values according to the mean concentration. If a 70 kg person drinks soup containing 10 g (one cube) of bouillon, he will intake 102.6 µg nitrite from bovine- based bouillons and 66.78 µg from chicken-based bouillons, both values according to the highest con- centration nitrite containing bouillon within the group.
The ADI is 0-0.07 mg/kg body weight and totally a maximum of 4.9 mg of nitrite for a 70 kg person.
Therefore, the intake of nitrite from bouillons will be below the limits given by foreign and Turkish regu- latory authorities. On the other hand, we must con- sider the overall intake of the organism in one day,
as other than bouillons, water, vegetables and other cured-meat products may have nitrite contamination.
Reinik et al.24 reported nitrite levels in 189 Estonian- origin meat products produced by 21 different com- panies in 2000-2001, and later the group studied 126 meat products from 16 different companies in 2003- 2004 by high performance liquid chromatography method. They found that cooked sausages contained 5-61 µg/g nitrite, smoked sausages contained 5-75 µg/g nitrite and hams contained 5-100 µg/g nitrite through 2000-2004. Ellen et al.26 analyzed a total of 140 samples of 16 kinds of cured meats for contents of residual nitrite and N-nitrosamines. Nitrite was determined by reaction with colorimetric measure- ment. In six samples, no nitrite was detectable (less than 1 µg/g nitrite); the remaining samples contained 1-140 µg/g nitrite, with median value 6.8 µg/g nitrite.
Therefore, bouillons contain less nitrite compared to sausages and hams.
There are a limited number of studies on nitrite levels of different foodstuff in our country. Türkdo¤an et al.27 found that herb-enriched cheese contained 4.14±0.50 µg/g nitrite, while bread baked by wood fire contained 0.82±0.22 µg/g nitrite and bread baked by animal manure contained 3.02±0.19 µg/g nitrite in Van city of Turkey. Their findings suggested that a nitrite- and nitrate-rich diet can significantly affect the development of endemic upper gastrointestinal cancers in the city.
Aside from nitrite contamination, bouillons pose other risks: Commercial bouillon concentrates are prepared by thermal treatment. This could cause heterocyclic amine formation, which is highly mutagenic. The presence of other ingredients in bouillons such as flavors, spices and food additives can contribute to heterocyclic amine formation and increase the risk28,29. We can conclude that nitrite contamination should be monitored routinely in cured meat and cured meat products for the safety of the general population.
Ultimately , surveillance should be continuous and widespread, and must be conducted by government and related authorities.
REFERENCES
1. Walker R. Nitrates, nitrites and N-nitroso com- pounds: a review of the occurrence in food and diet and the toxicological implications, Food Addit Contam, 7, 717-768, 1990.
2. Official Gazette of Turkish Republic. 22 December 25324, Report number 2003/44, 2003.
3. Chow CK, Hong CB. Dietary vitamin E and sele- nium and the toxicity of nitrite and nitrate, Toxi- cology, 180, 195-207, 2002.
4. Zanardi E, Dazzi G, Madarena G, Chizzolini R.
Comparative study on nitrite and nitrate ions determination, Ann Fac Med, 12, 79-86, 2002.
5. Christiansen LN, Johnston RW, Kauter DA, Howard, JW, Aunan WJ. Effect of nitrite and nitrate on toxin production by Clostridium botu- linum and nitrosamine formation in perishable canned comminuted cured meat, Appl Microbiol, 25, 357-362, 1973.
6. Susin J, Kmecl V, Gregorcic A. A survey of nitrate and nitrite content of fruit and vegetables grown in Slovenia during 1996-2002, Food Addit Contam, 23, 385-390, 2006.
7. Tamme T, Reinik M, Roasto M, Juhkam K, Tenno T, Kiis A. Nitrates and nitrites in vegetables and vegetable-based products and their intakes by Estonian population, Food Add Contam, 23, 355- 361, 2006.
8. Ellen G, Egmond E, Sahertian ET. N-nitrosamines and residual nitrite in cured meats from the Dutch market, Z Lebensm Unters Forsch, 182, 14-18, 1986.
9. Thompson BM, Nokes CJ, Cressy PJ. Intake and risk assessment of nitrate and nitrite from New Zealand foods and drinking water, Food Addit Contam, 24, 113-121, 2007.
10. Vermeer IT, van Maanen JM. Nitrate exposure and the endogenous formation of carcinogenic nitrosamines in humans, Rev Environ Health, 16, 105-116, 2001.
11. Bruning, CS, Kaneene JB. The effects of nitrate, nitrite and N-nitroso compounds on human health: a review, Vet Hum Toxicol, 5, 521-538, 1993.
12. McMullen, SE. Casanova JA, Gross LK, Schenck FJ. Ion chromatographic determination of nitrate and nitrite in vegetable and fruit baby foods, J AOAC Int, 88, 1793-1796, 2005.
13. Mensinga TT, Speijers GJA, Meulenbelt J. Health implications of exposure to environmental nitrog- enous compounds, Toxicol Rev, 22, 41–51, 2003.
14. IARC Monographs. Volume 94. Lyon, IARC, France, 1989.
15. Türk G›da Kodeksi. Renklendiriciler ve Tadland›r›c›lar D›fl›ndaki G›da Katk› Maddeleri Tebli¤i. Tebli¤ No: 2003/44.
16. European Parliament and Council Directive 95/2/EC of 20 February 1995 on Food Additives Other than Colours and Sweeteners. Also Avail- able at: http://ec.europa.eu/food/fs/sfp/ ad- dit_flavor/flav11_en.pdf.
17. Erkeko¤lu P, Baydar T. Evaluation of nitrite con- tamination in baby foods and infant formulas marketed in Turkey, Int J Food Sci Nut. Ahead of print (DOI: 10.1080/09637480701690311).
18. Griess P. Bemerkungen zu der Abhandlung der HH. Weselksy und Benedikt “Ueber einige Azoverbindungen”, Ber Dtsch Chem Ges, 12, 426- 428, 1879.
19. Malmauret L, Parent-Massin D, Hardy JL, Verger P. Contaminants in organic and conventional foodstuffs in France, Food Addit Contam, 19, 524- 532, 2002.
20. Gladwin MT. Haldane, hot dogs, halitosis, and hypoxic vasodilatation: the emerging biology of the nitrite anion, J Clin Invest, 113, 19-21, 2004.
21. Blot WJ, Henderson BE, Boice JD. Childhood cancer in relation to cured meat intake: review of the epidemiological evidence, Nutr Cancer, 34, 111-118, 1999.
22. Umbreit J. Methemoglobin--it's not just blue: a concise review, Am J Hematol, 82, 134-144, 2007.
23. Gr eer FR, Shannon M. Infant methemoglobinemia:
the role of dietary nitrate in food and water, Pediatrics, 116, 784-786, 2005.
24. Reinik M, Tamme T, Roasto M, Juhkam K, Jurt- senko S, Tenno T, Kiis A. Nitrites, nitrates and N- nitrosamines in Estonian cured meat products:
intake by Estonian children and adolescents, Food Add Contam, 22, 1098-1105, 2005.
25. Sugimura T. Nutrition and dietary carcinogenesis, Carcinogenesis, 21, 387-395, 2000.
26. Ellen G, Egmond E, Sahertian ET. N-nitrosamines and residual nitrite in cured meats from the Dutch
market, Z Lebensm Unters Forsch, 182 (1), 14-18, 1986.
27. Türkdo¤an MK, Testereci H, Akman N, Kahraman T, Kara K, Tuncer I, Uygan I. Dietary nitrate and nitrite levels in an endemic upper gastrointestinal (esophageal and gastric) cancer region of Turkey, Turk J Gastroenterol, 14, 50-53, 2003.
28. Solyakov A, Skog K, Jagerstad M. Heterocyclic amines in process flavours, process flavour ingre- dients, bouillon concentrates and a pan residue, Food Chem Toxicol, 37, 1-11, 1999.
29. Robbana-Barnat S, Rabache M, Rialland E, Fradin J. Heterocyclic amines: occurrence and prevention in cooked food, Environ Health Perspec., 104, 280- 288, 1996.
