1. Nitrite and Meat Curing Cured meats, i.e. ham, bacon, frankfurters etc. are highly distinctive and unique -curing process creates distinctive properties that cannot be duplicated -dramatic quality, shelf life and safety improvement thereby providing highly effective preservative -duplication of these properties is not straightforward and is difficult to achieve

2. The net effect of curing involves a very complex series of reactions between curing ingredients and the meat components -many are well understood, many are not To further complicate matters, some chemical reactions that involve curing ingredients are potentially harmful, while some of the curing agents have more recently been suggested as positive contributors to human health

3. The Saga of Sodium Nitrite Nitrite and nitrate have been used for some 5000 years or more to cure and preserve meat, mostly as a salt contaminant (saltpeter=nitrate) 1891 – first publication showing that nitrate-to-nitrite conversion by bacteria was the source of preservative effects 1899 – first suggestion of nitrite as the source of cured meat color 1901 – discovery that nitric oxide from nitrite was source of cured meat color 1925 – first regulatory (USDA) limits for nitrate and nitrite were established

4. 1920’s-1950’s – Industry and university researchers refined curing processes - role of nitrite in cured meat clarified -potent antimicrobial agent -powerful antioxidant -distinctive cured meat flavor -unique, attractive color that is stable

5. 1956 – a problem emerges…; first report of carcinogenic nitrosamines found in fish meal cured with very high nitrite concentrations 1960’s – intensive studies of the relationship between nitrite, nitrosamines and human cancer… …nitrosamines were found in some cured meats …the perceptions of cured meats as a contributor to human cancer became firmly entrenched

6. Mid 1970’s – Extensive research in meat curing showed that nitrosamines were not an issue with proper control of nitrite concentrations - use of reductants and improved understanding of time/temperature relationships in meat curing reduced residual nitrite to very low concentrations and eliminated nitrosamines

7. Late 1970’s – discovery of endogenous nitrate/nitrite in the human intestine and in saliva begins to suggest a biological role for these compounds -leads to discovery of nitric oxide as a very important biological molecule -beginning of the turnaround in scientific perceptions of nitrite

8. 1986 – Nitric oxide found to be “the body’s messenger” resulting in explosion of research in biology and physiology

9. Nitric oxide… -regulates blood pressure and blood flow -provides neurotransmission/brain function -kills bacteria and other immune system challengers -promotes healing (also provides critical role in the function of the drug Seldinafil )

10. 2002 – Importance of nitric oxide for blood transfusions suggested -25% greater heart attacks/strokes observed following transfusions - collected blood loses NO within hours of collection to 30% of initial after one day; 17% after 7 days and virtually none at 42 days - work with dogs showed decreased heart attacks with NO-treated transfusions Reynolds et al., Proc. Nat. Acad. Sci. 104:17058(2007).

11. 2005 – Protective role of nitric oxide in endothelial function, cardiovascular disease and heart attacks suggested - first reports of nitrite as major storage form of NO in blood and tissue - role for dietary nitrate and nitrite suggested for cardiovascular health

13. Source of nitric oxide (NO) in biology is nitric oxide synthase (NOS) conversion of arginine to citrulline L-arginine + O2 → L-citrulline + NO + H2O Nitrite serves as a low-oxygen source of NO thus nitrite may be a critical protective component for increasing blood flow when oxygen is limited Current questions – role of blood and tissue nitrite concentrations? – role of dietary nitrite?

15. Dietary nitrite in mice Insufficiency of dietary nitrite (7 days) decreased steady state nitric oxide and increased injury from heart attacks Dietary supplementation with nitrite restored NO homeostasis within 7 days, reversed injury and improved survival from heart attacks Dietary nitrite intake can restore NO homeostasis and blood and tissue nitrite to a greater extent (emphasis mine) than change in NOS activity Dietary supplementation reduced plaque formation in genetically modified, atherosclerotic-susceptible mice Bryan et al., Proc. Nat. Acad. Sci. 104:19144(2007)

16. “Most provocatively, these studies suggest that the cardioprotective effects of leafy green vegetables could derive from nitrite, in addition to the often cited antioxidant effects of these food groups.” Gladwin et al. Nat. Chem. Biol. 1:308-314(2005) “The solution to these problems should…lead to the potential consideration of nitrite as an “essential nutrient”…” Gladwin et al. Nat. Chem. Biol. 1:308- 314(2005)

17. “Could it be that the stringent regulations on nitrite/nitrate in drinking water and in foods contribute to the contemporary diseases of today due to inadequate nitrite or nitrate in the diet and NOS activity unable to supply enough nitrite to maintain cellular homeostasis?” Bryan, Free Rad. Biol. and Med. 41:691-701(2006) “Therefore, nitrite and nitrate may serve as essential nutrients for optimal cardiovascular health and may provide a treatment modality for cardiovascular disease.” Bryan et al., Proc. Nat. Acad. Sci. Early Ed. (2007). www.pnas.org/cgi/doi/10.1073/pnas.0706579104

18. Nitrite: Has profound effects on human physiology Dietary intake may be protective and maintain/restore endothelial function Dietary supplementation may inhibit onset and progression of atherosclerosis Dietary levels of nitrite and nitrate should be reconsidered for optimal health Increasing steady state NO x concentrations is a natural physiological response in humans and highlights the physiological justification and safety of such intervention from Bryan, MIRC (2007)

19. Nitrite… …derives it’s many advantages in cured meats from the unique chemistry of nitric oxide. …participates in many different chemical reactions during meat curing involving a variety of meat components, but most, if not all, of those reactions produce nitric oxide …is still not completely understood in some aspects of meat curing and several issues and questions concerning cured meats remain

20. To understand the issues… …it is necessary to understand and appreciate the complexity of conventionally cured meats

21. “Cured” meat Characterized by addition of nitrate and/or nitrite (and salt) – unequivocally defines cured meat (IMHO) – “uncured” otherwise -USDA-FSIS has ruled that any product that has not had nitrate or nitrite added is uncured, however, the USDA recognizes salted (only) products as cured also

22. Nitrate (NO 3 - ) Insignificant by itself Contributes cured meat properties only after reduction to nitrite Reduction is not easily done by chemical means in meat systems; typically requiring a bacterial culture with nitrate reductase activity May be important in dried products (hard salami, hams, etc.)

23. For cured products… “…heated within hours after adding the curing agent, nitrate is useless and superfluous.” Karl Honikel, Federal Research Center for Nutrition and Food, Kulmbach, Germany. (Encyclopedia of Meat Sciences, 2004, p. 196.)

24. Nitrite (NO 2 - ) Highly reactive compound Combines with many different substrates in meat products to provide several highly useful functions = Magic! Proportional distribution among reactions is likely dependent on product conditions Understanding nitrite complexity = understanding the issues associated with conventional curing, “natural” curing and the negative consumer image of nitrite and cured meat

25. Nitrite reactions in meats Hydrogen ions Reductants Meat pigments Salt Proteins Radicals/pro-oxidants Bacterial cells/cell walls/cell components Flavor precursors Lipids

26. Most obvious effect of nitrite Color development/change (most often desireable, sometimes not) - addition of nitrite directly affects myoglobin – necessary prerequisite for desirable cured color is nitric oxide (NO) – several reactive intermediates play critical roles for desirable color development by producing nitric oxide

27. Hydrogen ions 1. Acidic conditions (hydrogen ions) NO 2 - + H + → HNO 2 (nitrous acid) 2HNO 2 → N 2 O 3 + H 2 O (highly reactive intermediate) N 2 O 3 → NO 2 + NO (nitrogen dioxide + nitric oxide) (2 NO 2 + H 2 O → HNO 3 + HNO 2 ) (Note: nitrate + nitrous acid)

28. Effects of salt, nitrite and 0.2% GDL on residual nitrite, pH and cured color Treatments Residual nitrite % cured pigment pH Nitrite 75 ppm 20.6 b 60.3 b 5.89 b 156 ppm 46.7 a 62.3 a 5.93 a Salt ( with 75 ppm nitrite) 0% 35.4 a 59.8 b 5.79 e 0.5% 34.9 60.6 b 5.87 d 1.0% 35.4 a 60.8 b 5.91 c 2.0% 32.5 b 61.1 b 5.96 b 4.0% 26.0 c 64.2 a 6.02 a GDL (156 ppm nitrite) 0% 40.0 a 58.7 b 6.03 a 0.2% 26.7 b 63.9 a 5.79 b

29. 2. Reductants – Natural reductants (NAD, cysteine, cytochromes) – Ascorbate/erythorbate N 2 O 3 + 2 H-Asc ↔ 2 dehydro-Asc + H 2 O + 2 NO

30. 3. Myoglobin Mb-Fe +2 + NO 2 - → MMb-Fe +3 + NO + OH - (purple) (brown) reductant MMb-Fe +3 + NO →MMb-Fe +3 –NO →Mb-Fe +2 –NO (brown) (brown) (red)

31. Nitrite concentration effects on raw meat color 0 ppm 0.5 ppm 1.0 ppm 2.5 ppm 5.0 ppm 10.0 ppm 15.0 ppm 20.0 ppm 25.0 ppm 50.0 ppm Sebranek and Turner, 2002. Meat Process. 41(9):52-55.

32. 4.Salt (NaCl) HNO 2 + H + + Cl - ↔ NOCl + H 2 O (highly reactive) NOCl + Mb-Fe +2 → Mb-Fe +2 – NO + Cl - (purple) (red)

33. NaCl and ascorbate effects on measurable nitrite in cooked meat Salt (mM) Ascorbate Nitrite (% of initial) Fox et al., 1981. J. Assoc. Off. Analyt. Chem. 64:1397-1402.

34. 5.Proteins/sulfhydryl groups R-SH + 2 HNO 2 → R-S-S-R + 2 H 2 O + 2 NO sulfhydryl disulfide -besides generating nitric oxide, this reaction has been suggested as a contributor to increased firmness on cured meat by crosslinking proteins with the disulfide bond

35. In addition to cured color development from the various sources of nitric oxide Nitrite is strongly inhibitory of anaerobic bacteria, particularly Clostridium botulinum – dependent on pH, salt, reductants, iron content Interestingly, these are many of the same factors that favor nitrous acid and nitric oxide production

36. Is ingoing or residual nitrite most important to bacterial control? USDA requires minimum of 120 ppm ingoing for cured products that are refrigerated Research suggests that residual nitrite is critical to antibotulinal effects Most commercial cured meats at retail have less than 15 ppm residual nitrite Probably…a certain minimum ingoing amount is necessary to achieve initial inhibition, given competitive chemical reactions, and a certain residual amount is necessary for sustained inhibition but the the amounts of each are unclear and probably dependent on several other inherent conditions in the product

37. Nitrite provides potent antioxidant effectiveness Stabilizes heme iron Chelates metal ions and radicals Reacts with unsaturated lipids

38. Morrissey and Tichivangana, 1985. Meat Sci. 14: 175-190. Fish, chicken, pork and beef -measured 48 hours post-cook

39. Role in cured flavor? Over 100 volatiles identified in cured ham Not well understood -part of antioxidant effect? -more reaction products of nitrite/nitric oxide?

40. Nitrite safety issues Toxicity -highly toxic (~70 mg/kg body weight, i.e. 4-5 gram dose) -inconsequential in normal cured meat applications because of extremely low concentration Nitrosamines issue - secondary amines (-NH) may react with nitrite (or nitrous acid (nitrosating intermediates) to form -N-NO (nitrosamine) -most are definite carcinogens, some are quite potent - epidemiological studies based on nitrosamine hypothesis “Natural” curing -significantly reduced nitrite concentration, indistinguishable appearance

41. There are a number of processed meat products, mostly natural and organic cured meats, being introduced in the market that are labeled “Uncured” and “No nitrites or nitrates added”.

42. Ingredients: Organic grass-fed beef, water, sea salt, organic spices, organic garlic powder, organic paprika, celery powder, organic onion, lactic acid starter culture

47. Even though labeled “uncured”, almost all of these products display all the typical cured meat properties that are derived from addition of sodium nitrite, and that can be achieved ONLY with sodium nitrite… Further, chemical analyses of the products confirm the presence of nitrite and nitrate. Thus…the oxymoron in labeling; uncured = cured

48. Why the oxymoron…? …why is “Uncured” and “No nitrites or nitrates added” used on the label of products that are unequivocally cured in terms of both product properties and the presence of curing agents?

49. USDA has required that uncured versions of “typical” cured meats be “… labeled with such standard name when immediately preceded with the term “Uncured” in the same size and style of lettering as the rest of such standard name…” Code of Federal regulations 9 CFR 319.2 (2006)

50. “Natural” (processed meats) definition: “…does not contain any artificial flavor or flavoring, coloring ingredient, or chemical preservative…” (USDA Food Standards and Labeling Policy Book, 2005)

51. “Organic” Defined and controlled by Organic Foods Production Act (1990) -established National Organic Program Standards -National List of Allowed and Prohibited Substances -nitrite, nitrate not permitted

52. Thus, nitrite or nitrate, by themselves, cannot be directly added to any natural or organic processed meat product…and…any “typically” cured meat product that does not have added nitrite or nitrate must be labeled “uncured”.

53. Critical point: Cured meats, i.e. ham, bacon, frankfurters are highly distinctive and unique -nitrite/nitrate create distinctive cured meat properties that cannot be duplicated -dramatic quality, shelf life and safety improvement that consumers expect for these products

54. Alternative Production Methods Two types of “Uncured” Meat Products – No intention of replacing nitrite – Intention of replacing nitrite with natural sources of nitrite -Nitrite between 0.9ppm to 9.2ppm -Nitrite introduced indirectly as component of other ingredients

55. Manufacture of cured product without added nitrite/nitrate -utilize natural sources of nitrate -0.2%-0.4% celery juice/powder (natural flavor, celery powder) plus nitrate reducing culture -hold at 30C-37C - time/temperature considerations (franks preheated at 37C for 30 min. result in acceptable color) -or…utilize pre-converted celery juice/powder as a natural source of nitrite

56. Examples of commercially available ingredients for natural curing -VegStable™ 502 * (celery powder, sea salt)-30,000- 33,000 ppm nitrate; recommended use level of 0.2% to 0.4% -VegStable™ 504 * (celery powder, sea salt)-10,000- 12,000 ppm nitrite (pre-converted, starter and holding time not required); recommended use level of 0.2% to 0.4% *from Florida Food Products, Inc.

57. Concerns for cured products without added nitrite/nitrate -Variable nitrate sources -Nitrate reduction rate/consistency -Nitrite concentration produced-both extremes -issues will depend on product properties -Shift in nitrite dispersion -will rate of production affect distribution? -Product quality/consistency -Microbiological impacts -ingoing vs. residual nitrite?

59. Clostridium botulinum inoculations Time until botulism toxin was detected during vacuum-packed storage of inoculated frankfurters at various storage temperatures Inoculated frankfurters Products stored at: 22ºC 10ºC 4ºC cured control (156 ppm) 14 days none/84 days none/84 days uncured control (no nitrite) 5 days 14 days 63 days natural cure 5 days 56 days none/84 days natural cure-VS 507 5 days none/84 days none/84 days natural cure-VD 55 none/84 days none/84 days none/84 days -data courtesy of B. Wanless and K. Glass

60. Clostridium botulinum Time until botulism toxin was detected during vacuum-packed storage of inoculated ham at various storage temperatures Inoculated ham Products stored at: 22ºC 10ºC 4ºC cured control (156 ppm) none/28 days none/84 days none/140 days uncured control (no nitrite) 3 days 14 days none/140 days natural cure 5 days 21 days none/140 days natural cure-VS 507 5 days none/84 days none/140 days natural cure-VD 55 21 days none/84 days none/140 days -data courtesy of B. Wanless and K. Glass

61. Formulated nitrite Conventional cure 156 ppm Natural cure 504 (converted) 30 ppm Residual nitrite after cooking (ppm) frankfurters ham Cured control 115 142 Natural cure 43 36 Natural cure-VG517 30 38 Natural cure-VD55 29 37 -data courtesy of B. Wanless and K. Glass

62. It appears that natural and organic “uncured” RTE processed meat products will permit greater growth of L. monocytogenes and Cl. perfringens and earlier toxin production by Cl. botulinum than conventionally cured products. Some natural antimicrobials provide potentially suitable adjuncts for improved safety.

63. Despite the recent good news, negative past perceptions of nitrite persist Epidemiological studies continue to report associations between nitrite consumption and various forms of human cancer One of the most significant was a very large summary released on Halloween, 2007.

66. “The evidence that red meats and processed meats are a cause of colorectal cancer is convincing” (emphasis mine) “…nitrite, from processed meats… may be involved in carcinogenesis… Dietary nitrates and nitrites are probable human carcinogens because they are converted in the body to N-nitroso compounds” WCRF/AICR Report, 2007

68. It is critical to remember: These conclusions are based on epidemiology A causative link for meat consumption is still missing -NIH report (2001) found no relationship between nitrite consumption and cancer. On the Toxicology and Carcinogenesis Studies of Sodium Nitrite. 2001. U.S. Dept. of Health and Human Services, 276 p. - review and report in 2007 by Exponent, an independent consulting firm found no relationship

69. Even if nitrite is assumed to be a risk for carcinogenesis, less than 5% of human exposure to nitrite and nitrate come from processed meat There is now evidence of some very positive health effects of dietary nitrite. Meat may well be associated with diets high in fat from other sources, high in caloric content from other sources and many other confounding factors (body weight, exercise, smoking, alcohol consumption, stress, etc.)

70. “…there is an easy solution to every human problem… … neat, plausible, and wrong.” - H. L. Mencken Writer, editor, social critic (1880-1956)